Effects of some calcium antagonists upon the activity of common antiepileptic compounds on sound-induced seizures in DBA/2 mice.

1. Flunarizine (2.65 mumol/kg, i.p.) and nimodipine (5.25 mumol/kg, i.p.) potentiated the anticonvulsant properties of phenytoin, phenobarbital and valproate against audiogenic seizures in DBA/2 mice. 2. Diltiazem (5.25 mumol/kg, i.p.) was able to potentiate the antiseizure activity of phenytoin but was not effective against the anticonvulsant action of phenobarbital and valproate. 3. Verapamil (5.25 mumol/kg, i.p.) was unable to potentiate the anticonvulsant properties of all antiepileptic drugs studied. 4. Bay K 8644 (1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluorophenyl)-pyridine- 5-carboxylic acid), a calcium agonist at a dose of 2.65 mumol/kg, i.p., induced a reduction of anticonvulsant potency of phenytoin, phenobarbital and valproate. 5. None of the calcium antagonists used significantly increased the plasma levels of antiepileptic compounds or significantly affected the body temperature changes induced by anticonvulsant drugs. 6. It may be concluded that some calcium antagonists enhance the anticonvulsant properties of some antiepileptic drugs against audiogenic seizures. A pharmacokinetic interaction does not seem responsible for these effects.


Pathophysiology and management of phantom limb pain.

Phantom pain phenomenon is a poorly understood but relatively common sequela of limb amputation that may result in significant psychological and physical morbidity. In this review, proposed pathoneurophysiological mechanisms for the development of phantom pain are reviewed as well as psychological mechanisms that may be involved. The authors recommend an integrated approach to management of chronic phantom pain that takes into consideration the multiple factors that may contribute to its etiology.


Immunobiochemical assay for determination of nuclear steroid receptors.

Knowledge of receptor status is important for therapeutic strategies in hormone-dependent tumors. Therefore, methods specifically predicting biological response to endocrine therapy are essential. We investigated the receptor modulation of two breast tumors and one endometrial tumor in the nude mice model after injection of 20 micrograms 17 beta-estradiol. To differentiate the unoccupied and the occupied receptor sites, we used the enzyme immunoassay (EIA) for estrogen receptors (ER) under low- and high-salt conditions. With low-salt extraction we found a sharp decrease of the ER-EIA values within the first hour after estradiol treatment. This decrease continued for 24 hr until recovery to pretreatment levels occurred. In contrast, the ligand-receptor complexes tightly bound to acceptor sites on the DNA increased more than three times within 1 hr. These high levels could be measured for almost 12 hr, and then pretreatment levels were reestablished. The PgR-EIA values under low-salt conditions increased 10-fold within 12 hr, indicating an intact receptor mechanism. We conclude that this immunobiochemical method is a useful tool in determining receptor sites: those both unbound and those tightly bound to nuclear acceptors.


Molecular analysis of the zeste-white interaction reveals a promoter-proximal element essential for distant enhancer-promoter communication.

We have analyzed the eye and testis enhancers located 1 kb upstream of the transcription start site of the white gene. Both enhancers confer the corresponding tissue-specific expression on a heterologous promoter as well as on the white promoter. The eye determinant consists of multiple elements, each able to stimulate eye-specific expression. It also contains five binding sites for the zeste protein while the immediately adjacent testis element contains none. Site-directed mutation of these zeste binding sites abolishes the zeste-white interaction but does not significantly affect the eye enhancer activity, indicating that they are not important for the eye enhancer activity per se. Other zeste binding sites just upstream of the promoter are not necessary for the zeste-white interaction. We conclude that the overlap of the eye enhancer with the zeste binding sites is responsible for the zeste-white interaction and explains why this interaction affects eye but not testis expression. Sequence deletion or substitution experiments suggested that the white promoter is internal to the transcription start site; the zeste protein is not required for distant enhancer action but a 95-bp promoter-proximal sequence is essential for distant enhancer-promoter interaction. This element may serve as an anchor to stabilize formation of a loop that brings the enhancer to the vicinity of the promoter.


Malignant ovarian germ cell tumors: the experience at the Hospital de la Santa Creu i Sant Pau.

From 1979 to 1990, 33 patients with pathologically confirmed malignant ovarian germ cell tumors (MOGCT) were referred after initial surgical procedure at the Department of Oncology, Hospital de la Santa Creu i Sant Pau. The median age was 22 years (range, 10 to 39). Stage distribution was as follows: stage I, 12 patients; stage II, 6 patients; stage III, 11 patients; stage IV, 3 patients; and unstaged, 1 patient. The histologic diagnoses were 10 dysgerminomas, 4 endodermal sinus tumors, 11 immature teratomas, and 8 mixed germ cell tumors. Twenty-eight out of the thirty-three patients received postoperative chemotherapy with POMB-ACE-PAV or other platinum-containing regimens. One patient with stage IV disease failed to respond to chemotherapy and she died. Sixteen out of the twenty-eight treated patients had second-look laparotomy, which showed mature teratoma in six and persistent malignant teratoma in one patient. This last patient had complete remission with second regimen. No patient has developed recurrence. With a median follow-up of 66 months (range, 10 to 133), 32 patients (97%) are alive without evidence of disease. These data confirm that platinum-containing regimens have dramatically improved the prognosis for patients with MOGCT. This paper discusses primary chemotherapy and the role of the second-look in these patients.


[Expression and function of glycine-gated Cl- channels].

Glycine is a major inhibitory neurotransmitter in the spinal cord and brain stem. Glycine acts by increasing the Cl- permeability through activation of a specific receptor/ion channel complex consisting of a pentameric subunit assembly. Molecular cloning has disclosed the nature of receptor subunits alpha and beta. While the role of the beta subunit is still unclear, the alpha subunit functions in both ligand (agonist/antagonist) binding and ion channel formation. It has been demonstrated that there are two isoforms of the alpha subunit, alpha 1 and alpha 2. The mRNAs encoding these subunit isomers are transcribed from different genes, in spite of their structural similarity. The alpha 1 mRNA is abundant in adult spinal cord, whereas the alpha 2 mRNA is mainly expressed in developing spinal cord as well as various regions of brain tissue. The single channel properties were examined in outside-out patches excised from Xenopus oocyte membrane expressing alpha 1 or alpha 2 homomeric receptors. The mean open time of alpha 2 channels was 70-times longer than that of alpha 1 channels. The subunit switching from alpha 2 to alpha 1, and resulting shortening of channel kinetics, may ensure a rapid motor control in adult animals.


Inhibition of superoxide and nitric oxide release and protection from reoxygenation injury by Ebselen in rat Kupffer cells.

Luminol chemiluminescence in phorbolester-activated cultured rat liver Kupffer cells was strongly inhibited by the selenoorganic compound ebselen (IC50 = 2 mumol/L). Ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]one) also diminished reduction of ferricytochrome c (IC50 = 10 mumol/L), indicating a suppression of superoxide anion formation. Likewise, in lipopolysaccharide-pretreated Kupffer cells, ebselen proved to be a potent inhibitor of the conversion of oxyhemoglobin to methemoglobin (IC50 = 3 mumol/L) as a measure of nitric oxide formation. The sulfur-containing analog (2-phenyl-1,2-benzisothiazol-3[2H]one) and the ebselen derivative, methylselenobenzanilide, were inactive. These results indicate that ebselen is a potent inhibitor of NADPH oxidase in Kupffer cells, as has been reported for other macrophages and granulocytes. In addition, they suggest a novel characteristic of ebselen, namely very effective inhibition of nitric oxide synthase of macrophages. In line with its inhibitory effects on the release of reactive oxygen species by macrophages, complemented by its antioxidant properties, ebselen was potent in the prevention of reoxygenation injury of Kupffer cells (IC50 approximately 5 mumol/L).


Immunolocalization, quantitation and cellular heterogeneity of apolipoprotein B in rat hepatocytes.

Hepatocyte autofluorescence represents a major problem in immunofluorescence studies with fluorescein conjugates because of significant spectral overlap. We describe a method for immunostaining hepatocytes with R-phycoerythrin (a fluorochrome with minimal overlap with autofluorescence) with paraformaldehyde fixation and Triton X-100 permeabilization for better antibody penetration. This method produced both perinuclear (presumed Golgi apparatus) and dispersed, reticular staining (presumed endoplasmic reticulum) in rat hepatocytes in culture stained with a monoclonal antibody to rat apolipoprotein B. Treatment with brefeldin A resulted in loss of apolipoprotein B perinuclear staining and increased reticular immunofluorescence consistent with known properties of brefeldin A (inhibition of protein transport within the secretory pathway by dissolution of Golgi bodies). This suggests that apolipoprotein B epitopes are present in both Golgi bodies and endoplasmic reticulum. To demonstrate the utility of the technique for quantitative studies, static cell cytofluorometry of brefeldin A-treated cells was performed, demonstrating increases in specific immunofluorescence of apolipoprotein B corresponding closely to results estimated by monoclonal antibody radioimmunoassays of cellular homogenates. The technique was then used with flow cytometry of single-cell suspensions of control rat hepatocytes derived from immunostained primary cultures to reveal cell-to-cell heterogeneity of apolipoprotein B epitope expression manifested as apolipoprotein B-negative and positive populations. Results for brefeldin A-treated cells revealed even clearer delineation of heterogeneity as indicated by frank bimodality of the populations, along with not only higher mean apolipoprotein B levels but also a significantly higher proportion of apolipoprotein B-positive cells than in the control.


Effects of partial hepatectomy on hepatic insulinlike growth factor binding protein-1 expression.

Insulinlike growth factor binding proteins modulate the action of the insulinlike growth factors in various bioassays and may regulate the bioavailability of the insulinlike growth factors in vivo. Because the insulinlike growth factors may influence hepatic regeneration, we have examined the effect of partial hepatectomy on serum insulinlike growth factor binding proteins and on the abundance of insulinlike growth factor binding protein-1 messenger RNA in the liver. All rats were fasted before and after partial hepatectomy or sham operation to avoid the confounding effects of difference in food intake. Using a conventional protocol, 70% of the liver was removed, and groups of four or five rats were killed at different intervals after partial hepatectomy. Sham-operated rats served as controls. Pooled sera from each group of rats were analyzed by ligand blotting with 125I-insulinlike growth factor-I. Liver RNA from individual rats was analyzed by slot-blot and Northern-blot hybridization. A small decrease in the 39- to 42-kD insulinlike growth factor binding protein was apparent in sera from both the sham-operated and partial hepatectomized rats. In contrast, a dramatic increase (fivefold) in the 29-kD serum insulinlike growth factor binding protein (insulinlike growth factor binding protein-1) was apparent only in the partial hepatectomized rats. Hepatic insulinlike growth factor binding protein-1 messenger RNA abundance was significantly increased (1.99 +/- 0.18-fold; p less than 0.05) at 1 hr, reached a peak of 2.32 +/- 0.22-fold (p less than 0.01) at 3 hr after partial hepatectomy and returned to basal levels over the subsequent 6 to 12 hr.(ABSTRACT TRUNCATED AT 250 WORDS)


Cytokeratin polypeptide in gastrointestinal adenocarcinomas displaying squamous differentiation.

In the present study we investigated the cytokeratin (CK) polypeptide expression in gastric and colonic adenocarcinomas. A battery of monoclonal anti-cytokeratin-specific antibodies and anti-vimentin were used. While the majority of cases displayed simple epithelial characteristics, in three of 17 cases of gastric adenocarcinomas and in one of 20 cases of colonic adenocarcinomas, CK polypeptides 13 (54 kd) and 16 (48 kd) were occasionally detected. These CK polypeptides, characteristic of squamous nonkeratinizing epithelia, were found in cases in which no evidence of squamous differentiation could be demonstrated by histologic examination. We believe that the presence of these unique CK polypeptides points to the squamous differentiation potential of the tumor cells.


Determinant analysis and interaction studies on monoclonal antibodies to bovine IgG1 and IgG2.

Two mouse monoclonal antibodies SKb1 and SKb6 were prepared by fusion of myeloma cells with spleen cells of female Balb/c mouse immunized with a mixture of bovine IgG1 and IgG2. In radioimmunoassay, SKb1 bound specifically to IgG2 but SKb6 reacted with both IgG1 and IgG2 molecules. In the competition experiments, heavy chain isolated from bovine IgG could inhibit the binding of 125I-IgG1 and 125I-IgG2 to SKb6, while it failed to inhibit the binding of 125I-IgG2 to SKb1. The epitope reacting with SKb1 was found to be present not only on bovine IgG2 but also on goat IgG and was not present on IgG molecules isolated from the serum of rabbit, rat, sheep, horse, human and monkey. Similarly, the epitope reacting to SKb6 was found to be present on bovine IgG1 and IgG2 and also on IgG molecules isolated from goat and sheep serum but was absent in the IgG molecules isolated from the serum of rabbit, rat, horse, human and monkey. The association constants of the interactions of SKb1 with 125I-IgG2 and of SKb6 with 125I-IgG1 and 125I-IgG2, determined by Scatchard analysis, Steward-Petty plot and Sips plot, were found to be in the order of 10(8)-10(10) L/M. The association constants were determined at varying temperatures to obtain the thermodynamic parameters. The enthalpy (delta H0) and entropy (delta S0) values for the above antigen-antibody interactions were in the range of 9.15-15.96 kcal/mole and 36.96-41.15 eu/mole respectively. The heterogeneity indices for similar interactions determined by Sips equation were consistent with the expected values for binding of monoclonal antibodies with homogeneous protein determinants.


Parasite-specific T-cell responses of trypanotolerant and trypanosusceptible cattle during infection with Trypanosoma congolense.

During primary tsetse-transmitted challenge of Boran (Bos indicus) cattle with Trypanosoma congolense ILNat 3.1, a transient parasite antigen-specific T-cell proliferative response was observed in peripheral blood mononuclear cells and splenic mononuclear cells stimulated in vitro. A response was also observed with cells of N'Dama (Bos taurus) cattle, but in this case higher stimulation indices were observed and the response was maintained until the termination of the experiment at 40 days post-infection (p.i.). The highest parasite antigen-specific proliferative responses were observed at 20 days post-infection. At this time N'Dama cattle not only responded to the antigens derived from the infecting clone (ILNat 3.1), but also to antigens from a clone of a different serodeme (ILNaR 2), whereas Boran cattle only recognized antigens from the infecting clone of parasites. To determine the molecular mass of the antigenic trypanosome proteins, whole trypanosome lysates made from T. congolense ILNat 3.1 were fractionated by SDS-PAGE and transferred onto nitrocellulose membranes. The major protein bands were isolated and used directly in T-cell proliferation assays. In this instance, no differences in the antigen recognition profiles of Boran and N'Dama cattle were observed. The variable surface glycoprotein did not induce T-cell proliferation in infected cattle despite the presence of serum antibodies to this variable antigenic type.


Immunization of guinea-pigs and cattle against adult Rhipicephalus appendiculatus ticks using semipurified nymphal homogenates and adult gut homogenate.

Guinea-pigs inoculated with crude homogenate of unfed nymphs of the tick Rhipicephalus appendiculatus and with three semipurified fractions of the homogenate obtained by gel permeation chromatography, acquired a significant degree of immunity to infestation with adults of this tick. Fraction 2 induced the highest reduction (66%) in mean weight of engorged females followed by crude homogenate and fractions 1 and 3. Calves immunized with crude homogenates of unfed nymphs, fraction 2 of nymphal homogenate, and gut homogenate of unfed females also acquired immunity against adults of R. appendiculatus. The mean weight of engorged females fed on calves inoculated with nymphal fraction 2 was the lowest of all five groups of calves on which females fed. The reduction in weight (38%) was not significantly different from that observed for females fed on calves inoculated with crude nymphal homogenate (31%) or females from third infestation of adult ticks. No differences in the weight and hatchability of egg batches produced by engorged females collected from the five groups of calves were observed. Analysis of sera collected from the five groups of calves showed that the concentration of albumin, alpha-1, alpha-2 and beta-globulins fluctuated and no significant differences between the treated groups were observed. The levels of gamma-globulin increased in treated groups including the group inoculated with adjuvant only, but unlike previous reports no increase in gamma-globulin or a correlation between the level of gamma-globulin and the degree of resistance acquired were observed in calves exposed to repeated tick infestations. However, the increase in the concentration of gamma-globulin in calves inoculated with fraction 2 or crude nymphal homogenate was higher than that observed in the other groups.


Asymmetric non-precipitating antibodies in commercial hyperimmune gamma-globulin for therapeutic use.

The presence of asymmetric (non-precipitating or co-precipitating) antibodies has been studied in three commercial preparations of antitetanus gamma-globulin. It was found that 27% of the specific antibodies are of asymmetric type, a value two to three times higher than that found in normal IgG. In toxicity tests in mice with tetanus toxin, the asymmetric antibodies were 2.7 times less effective than the symmetric ones. It is concluded that the protection capacity of the tetanus gamma-globulin preparations is dependent on the ratio of symmetric to asymmetric antibodies. The high content of asymmetric antibodies is probably due to the fact that immunization is performed with the antigen in a particulate form.


Calcitonin gene-related peptide in noradrenergic transmission in rat hypothalamus.

In the present study, we examined the regulatory mechanisms of calcitonin gene-related peptide on norepinephrine release in rat hypothalamus. Calcitonin gene-related peptide inhibited the stimulation-evoked norepinephrine release from hypothalamic slices of Sprague-Dawley rats in a dose-dependent manner, although the peptide did not affect basal release of norepinephrine. The blockade of the alpha 2-adrenergic receptors by RX 781094 failed to modulate the inhibitory effects of calcitonin gene-related peptide on norepinephrine release. Pretreatment of slices with islet activating protein, a toxin that interferes with the coupling of the inhibitory receptors to adenylate cyclase, did not affect the suppression of norepinephrine release by calcitonin gene-related peptide. However, Bay K 8644, a dihydropyridine-sensitive calcium channel agonist, significantly reversed the inhibitory effects of calcitonin gene-related peptide on norepinephrine release. These results show that calcitonin gene-related peptide might inhibit norepinephrine release in rat hypothalamus, partially mediated by interactions with dihydropyridine-sensitive Ca2+ channels but not by interactions with presynaptic alpha 2-adrenergic receptors and inhibitory guanosine triphosphate binding proteins. Furthermore, the finding suggests the possible involvement of calcitonin gene-related peptide in the regulation of sympathetic nervous activity in the central nervous system.


Ganglionic blockade with tetraethylammonium in conscious rats.

The sympathetic ganglionic blocking agent tetraethylammonium has been used as a clearance marker for the measurement of renal plasma flow, but the sympathetic ganglionic blocking dose in rats is unknown. In light of differential reflex activation of sympathetic nerve activity to heart and kidney, we compared the effect of tetraethylammonium on renal nerve activity, mean arterial pressure, and heart rate. Conscious rats were infused with either vehicle (isotonic saline) or tetraethylammonium (n = 7 in both groups). Tetraethylammonium was infused cumulatively (35 minutes per dose) in the following doses: 10(-5), 10(-4), 10(-3), 10(-2), and 10(-1) g/kg body wt per hour. Doses for 15% reduction were 10(-1) for mean arterial pressure, 0.55 x 10(-1) for heart rate, and 0.055 x 10(-1) g/kg body wt per hour for renal nerve activity. Renal nerve activity was abolished at 10(-1) g/kg body wt per hour; mean arterial pressure and heart rate were unchanged at doses lower than 10(-1) g/kg body wt per hour. The lethal dose was 1 g/kg body wt per hour. No changes were observed in vehicle-treated animals. Tetraethylammonium at 10(-1) g/kg body wt per hour resulted in an attenuated increase in renal nerve activity during acetylcholine-induced reduction in mean arterial pressure, reflecting arterial baroreceptor inhibition. We conclude that renal nerve activity is 10- and 18-fold more sensitive to sympathetic ganglionic blockade than heart rate and mean arterial pressure, respectively. When tetraethylammonium is used as a clearance marker for measurement of renal plasma flow in rats, it should be administered in doses less than 10(-2) g/kg body wt per hour.


Expression and different polarity of aminopeptidase N in normal human colonic mucosa and colonic tumors.

Expression and cellular localization of brush-border enzymes (aminopeptidase N, dipeptidylpeptidase IV, lactase, maltase) in normal human colon, colonic polyps and malignant intestinal tumors were investigated with a panel of monoclonal antibodies reacting with either native or denatured proteins. The enzymes were detected on cryostat sections by indirect immunofluorescence staining, or affinity-purified and analyzed by gel electrophoresis and immunoblotting. Dipeptidylpeptidase IV, lactase and maltase were absent from all samples examined, while aminopeptidase N (APN) was detected at the basal membrane of the epithelial cells in most specimens of colon obtained from individuals free of intestinal tumors. In contrast, APN was frequently localized at the luminal membrane of the surface epithelium in large-intestinal mucosa distal to tumors, adenomas and hyperplastic polyps, and from members of hereditary colon cancer syndrome families. APN was also expressed in colonic tumors, where it was present in an apical cell membrane location in 3/23 adenomas and 14/35 adenocarcinomas examined. No correlation was found between tumor-cell invasiveness (classified by "Dukes" stage) and expression or cellular location of aminopeptidase N. Histologically, all positive tumors were moderately or well differentiated. These results suggest that aminopeptidase N is normally expressed in adult human colon, but epithelial cells in the large and small intestine differ in their ways of sorting this enzyme intracellularly and eventually inserting it into different aspects of their surface membrane, a process which may be altered at an early stage of carcinogenesis.


Increased expression of alpha IIb beta 3 integrin in subpopulations of murine melanoma cells with high lung-colonizing ability.

Four subpopulations of B16 amelanotic melanoma cells, possessing different abilities to induce platelet aggregation (TCIPA) and to form lung colonies, were isolated by centrifugal elutriation. The expression of alpha IIb beta 3, alpha v beta 3 and alpha 5 beta 1 integrins was examined in the 4 subpopulations in order to determine the relationship between integrin receptor expression and tumor-cell metastatic potential. The mRNA of alpha IIb, alpha 5, beta 1 and beta 3 was detectable in the 4 subpopulations by Northern blotting. A gradual increase in mRNAs and cell-surface immunoreactivity of the alpha IIb beta 3 receptor, but not in their gene copies, was observed from the low to the high metastatic subpopulations. The ability of tumor cells to adhere to fibronectin and subendothelial matrix (SEM) increased in parallel. In the high metastatic cells, the alpha IIb beta 3 receptors, but not the alpha 5 beta 1 receptors, were localized to focal adhesion plaques. Incubation of the high metastatic cells with alpha IIb beta 3-specific antibodies reduced their matrix adhesion, TCIPA and lung-colonizing abilities. In contrast, in the low met- astatic cells, SEM adhesion and lung-colony formation were not affected by anti-alpha IIb beta 3 antibody treatment. Incubation of either the low or the high metastatic subpopulation with an alpha 5 beta 1-specific antibody had no effect in vitro and showed a slight inhibition of lung colonization in vivo. Our results suggest that several phenotypic characteristics of the enhanced metastatic potential of B16a subpopulations may be mediated by increased expression of alpha IIb beta 3 receptors and that expression of these receptors may be regulated at the transcriptional level.


Delayed motor function and results of vestibular function tests in children with inner ear anomalies.

The relation between the results of vestibular function tests and gross motor development was examined in 4 children with inner ear anomalies. CT scans demonstrated the absence of lateral semicircular canals in both ears in all 4 cases. None responded to caloric stimulation using 40 ml of icewater. In contrast, the damped rotation test elicited per-rotatory nystagmus in all cases. Per-rotatory nystagmus was provoked in only two cases by the Bárány rotation test. Development of gross motor function, especially independent walking, was more delayed in the two children in whom the Bárány rotation test failed to elicit per-rotatory nystagmus.


On the mechanism of lipoxygenase-like action of bleomycin-iron complexes.

The mechanism of lipid peroxidation catalyzed by bleomycin (BLM)-iron (Fe) complexes has been studied in vitro using sodium linoleate as a substrate. BLM-Fe(II)-O2 and BLM-Fe(III) complexes catalyze lipid peroxidation concomitantly with singlet oxygen evolution. The results from spin trapping methods and gas chromatography-mass spectroscopy (GCMS) analyses suggest that the initial step of lipid peroxidation catalyzed by BLM-Fe complexes is similar to that of soybean lipoxygenase, viz., hydrogen abstration. However, another mechanism might be concerned in the case of BLM-Fe(II)-O2 complex. BLM-Fe complexes are also capable of enhancing singlet oxygen evolution from the hydrogen peroxide (H2O2)-hypochlorite (OCl-) system.


Detection of antibiotic-induced platelet dysfunction in whole blood using flow cytometry.

Using flow cytometry and activation-dependent monoclonal antibodies, we have developed a technique based on forward angle-light scatter (FALS) and immunofluorescence that simultaneously detects human platelet activation, secretion, and aggregation in whole blood. To detect the effects of cefotetan and latamoxef, both of which contain an N-MTT side chain, and of free N-MTT and cefoxitin, which does not contain the N-MTT side chain, on platelet activation and secretion, platelets were stained by the indirect method using a murine-produced platelet specific activation-dependent monoclonal antibody, S12, and a goat anti-mouse IgG fluorescein-conjugated antibody. S12 binds to a 140kd alpha granule membrane protein (GMP-140) that is expressed during secretion. Single parameter, 256 channel, log integrated green fluorescence histograms were generated, and negative and positive fluorescent populations were defined. Latamoxef and cefotetan reduced the number of platelets expressing S12 by more than 43%. In contrast, cefoxitin reduced the number of platelets expressing S12 by only 13.5%. The inhibition of GMP-140 expression per platelet was calculated by converting the log data to linear fluorescence intensity. Latamoxef and cefotetan inhibited expression of GMP-140 by 88% and 87% respectively. Free N-MTT inhibited its expression by 68%. In contrast cefoxitin reduced GMP-140 expression per platelet by only 45%.


Temocillin and cystic fibrosis: outcome of intravenous administration in patients infected with Pseudomonas cepacia.

Twelve courses of intravenous temocillin were given in combination with an intravenous aminoglycoside to five patients with cystic fibrosis (CF) for pulmonary exacerbations associated with Pseudomonas cepacia. All patients were infected concurrently with Pseudomonas aeruginosa in addition to P. cepacia. Improvement occurred after six of seven courses given to three patients in which temocillin was used as first-line therapy and following three of five courses given to two patients after failure of other antipseudomonal agents. All ten pre-treatment isolates of P. cepacia were resistant to aminoglycosides and eight were sensitive to temocillin. Clinical improvement was seen on both occasions in which the pre-treatment isolates were resistant to temocillin.


Effect of neuropeptides released from sensory nerves on blood flow in the rat airway microcirculation.

Stimulation of sensory nerves in the airway mucosa causes local release of the neuropeptides substance P and calcitonin gene-related peptide (CGRP). In this study we used a modification of the reference-sample microsphere technique to measure changes in regional blood flow and cardiac output distribution produced in the rat by substance P, CGRP, and capsaicin (a drug that releases endogenous neuropeptides from sensory nerves). Three sets of microspheres labeled with different radionuclides were injected into the left ventricle of anesthetized F344 rats before, immediately after, and 5 min after left ventricular injections of capsaicin, substance P, or CGRP. The reference blood sample was withdrawn from the abdominal aorta and was simultaneously replaced with 0.9% NaCl at 37 degrees C. We found that stimulation of sensory nerves with a low dose of capsaicin causes a large and selective increase in microvascular blood flow in the extrapulmonary airways. The effect of capsaicin is mimicked by systemic injection of substance P but not by CGRP, suggesting that substance P is the main agent of neurogenic vasodilation in rat airways.


Hyperserotoninemia and antiserotonin antibodies in autism and other disorders.

This study examined the linkage between elevated blood serotonin in autism and the presence of circulating autoantibodies against the serotonin 5HT1A receptor. Information was also obtained on the diagnostic and receptor specificity of these autoantibodies. Blood serotonin was measured as was inhibition of serotonin binding to human cortical membranes by antibody-rich fractions of blood from controls and from patients with childhood autism, schizophrenia, obsessive-compulsive disorder, Tourette's, and multiple sclerosis. The results showed elevated blood serotonin was not closely related to inhibition of serotonin binding by antibody-rich blood fractions. Inhibition of binding was highest for patients with multiple sclerosis and was not specific to the 5HT1A receptor as currently defined. Although inhibition was not specific to autism, the data were insufficient to establish if people with autism differed from normal controls on this measure.


Galanin decreases circulating growth hormone levels in acromegaly.

Galanin is able to elicit GH secretion in normal man. In acromegaly, circulating GH levels are elevated, and GH secretory dynamics are usually abnormal. The aim of our study was to investigate the effects of galanin on GH secretion in acromegalic subjects. Six acromegalic patients (four males and two females) and seven healthy adult subjects (five males and two females) underwent in randomized order: 1) iv infusion of 100 mL saline from 0-45 min, and 2) iv infusion of synthetic porcine galanin (0.5 mg in 100 mL saline) from 0-45 min. In normal subjects, peak GH levels after porcine galanin administration (8.2 +/- 1.9 micrograms/L) were significantly higher than after saline infusion (1.3 +/- 0.1 micrograms/L; P less than 0.05). In acromegalic patients, GH values fell from baseline (32.5 +/- 12 micrograms/L) to a mean nadir of 24.5 +/- 12.7 micrograms/L after galanin infusion. The mean change in GH values from baseline after galanin treatment in these subjects significantly differed from that observed after saline infusion from 15-90 min. Serum PRL levels were not significantly affected by galanin in either normal or acromegalic patients. Our results give the first evidence that the same dose of galanin, acting as a GH secretagogue in normal man, is, on the contrary, able to significantly inhibit GH in acromegalic patients. The cause of this paradoxical GH fall after galanin treatment in acromegaly remains to be explained. It can be hypothesized that galanin may interact at the pituitary level with its own receptors expressed by GH-secreting adenomatous cells.


Imbalanced follicle-stimulating hormone beta-subunit hormone biosynthesis in human pituitary adenomas.

Clinically nonfunctioning pituitary adenomas represent approximately 25% of all pituitary tumors. Recent studies using a number of in vitro techniques show that the majority of such tumors produce gonadotropins. Hypersecretion of uncombined gonadotropin subunits by these tumors has also been identified raising the possibility that gonadotropin biosynthetic alterations may occur in neoplastic pituitary tissue. To determine whether underlying intracellular biosynthetic alterations lead to imbalanced secretion of the gonadotropin subunits by such tumors, we investigated 1) steady state gonadotropin-subunit messenger ribonucleic acid (mRNA) levels in tumor tissue from 49 patients with clinically nonfunctioning adenomas, 2) secretion of gonadotropins in dispersed pituitary tumor cultures, and 3) serum concentrations of gonadotropins and free subunits. Northern blots of RNA extracted from surgically obtained pituitary tumor tissue were hybridized with complementary DNA probes for FSH beta, LH beta, and alpha-subunit, and quantitative analysis was done to compare alpha- and beta-subunit biosynthesis in individual tumors. Of these tumors, 47 contained sufficient RNA for Northern analysis and 77% of these tumors contained one or more of the gonadotropin-subunit mRNAs. Steady state alpha-subunit mRNA was detected in 57% of tumors, FSH beta mRNA in 49%, and LH beta in 1 (2%). We found FSH beta mRNA in excess of alpha-subunit mRNA in one-third of tumors, including 9 tumors where alpha-subunit mRNA was undetectable. In cultured cells, FSH beta was secreted in excess of alpha-subunit in 41% of tumors. For those tumors in which both mRNA and culture data were available, FSH beta mRNA and secreted subunit levels were in excess of alpha-subunit in 64% of tumors. We conclude that clinically nonfunctioning pituitary adenomas frequently synthesize excess FSH beta subunit relative to alpha-subunit. This finding is in contrast to previous data in normal pituitary or placental tissue where alpha-subunit is present in excess of beta-subunits at both the mRNA and protein levels. The free-beta-subunit hypersecretion identified in pituitary adenomas may be due to biosynthetic abnormalities intrinsic to neoplastic gonadotrophs.


Insulin regulation of insulin-like growth factor binding protein-1 in obese and nonobese humans.

Insulin is the principal regulator of hepatic insulin-like growth factor binding protein-1 (IGFBP-1) production, mediating the rapid decrease in plasma IGFBP-1 in response to nutritional intake. In this study, we defined IGFBP-1 regulation by insulin in upper and lower body obesity, conditions associated with insulin resistance and chronic hyperinsulinemia. Overnight postabsorptive IGFBP-1 levels in obese and nonobese women showed an inverse, nonlinear relationship with plasma insulin concentrations. Maximum suppression of IGFBP-1 was seen at 70-90 pmol/L plasma insulin. Both groups of obese women had mean fasting plasma insulin concentrations above this threshold level and, consequently, markedly suppressed IGFBP-1 levels. To assess the dynamics of insulin regulated IGFBP-1, 10 obese and 8 nonobese women were studied during sequential saline infusion (0-90 min), hyperinsulinemia (insulin infusion; 90-210 min) and hypoinsulinemia (somatostatin + GH infusion; 210-330 min). Insulin infusion rapidly decreased plasma IGFBP-1 levels in nonobese subjects (60% decrease in 2 h), but had little or no further suppressive effect in obese subjects. Complete insulin withdrawal resulted in a significant rise in plasma IGFBP-1 concentrations in all subjects, but the response was blunted in obese compared to nonobese groups. In contrast to plasma IGFBP-1, IGF-I concentrations did not vary during hyper- and hypoinsulinemic infusion periods and were not significantly different between groups. Basal GH levels were significantly higher in nonobese when compared to obese women, but did not change with infusions. In conclusion, low IGFBP-1 levels in obesity are related to elevated insulin levels which are, in turn, related to body fat distribution and insulin resistance. The chronically depressed levels of IGFBP-1 may promote IGF bioactivity as well as its feedback regulation of GH secretion, thus contributing to the metabolic and mitogenic consequences of obesity. In addition, our findings imply that hepatic insulin sensitivity in terms of IGFBP-1 production is preserved despite peripheral insulin resistance in obesity.


Human decidua and in vitro decidualized endometrial stromal cells at term contain immunoreactive corticotropin-releasing factor (CRF) and CRF messenger ribonucleic acid.

CRF, a hypothalamic neurohormone, has been shown to be present in several tissues outside the brain. During pregnancy, both fetal (placental trophoblast, chorion, and amnion) and maternal (decidua) intrauterine tissues contain immunoreactive CRF. A paracrine/autocrine role of CRF as a regulator of hormonogenesis in human placenta and decidua has been suggested. The expression of CRF mRNA in human decidua was demonstrated in the present study by Northern blot analysis and was found to be higher in specimens collected at term than in those collected during the first and second trimesters of gestation. Furthermore, the presence of CRF was detected immunocytochemically in cultured decidual cells isolated from term decidua as well as in endometrial stromal cells decidualized in vitro by treatment with a mixture of medroxyprogesterone acetate, estradiol, and relaxin. These results indicate that human decidua is an intrauterine extrahypothalamic source of CRF in the maternal compartment and offer new tools to explore the in vitro decidualization processes and the regulation of CRF release from decidual cells.


Neuronal excitability: voltage-dependent currents and synaptic transmission.

Neuronal membrane excitability and the synaptic connections among neurons produce behavior and cognition. The intracellular compartment of neurons is negatively charged relative to the extracellular space, and this charge, as well as current flow, is produced by ions. From the perspective of charged ions, the lipid bilayer of the neuronal membrane acts as a capacitor, and transmembrane glycoprotein pores or channels act as resistors. The open and closed states of ionic channels determine the membrane potential. At equilibrium, the lowest resistance or greatest permeability is for potassium, and the resting membrane potential is close to the equilibrium potential for potassium. When a channel is opened, permeable ions diffuse down their electrochemical gradients and the membrane potential is changed. Channels are gated (opened or closed) by voltage, neurotransmitters, and second messengers. The neuron integrates synaptic potentials produced by transmitter-gated channel activity and either generates a subthreshold potential, or a suprathreshold depolarization that generates an action potential or a burst of action potentials. Action potential generation is mediated by a large, brief sodium influx that is followed by activation of a voltage-dependent potassium eflux. The pattern of action potential firing is dependent on the interaction of a repertoire of voltage-dependent ion conductances. The action potential is the main signaling mechanism to activate synaptic transmission at axon terminals. Synaptic transmission is graded depending on the amount of calcium entering the presynaptic terminal. The number of action potentials, or the shape of the action potential, will determine the amount of calcium entering the terminal and the efficacy of synaptic transmission. Presynaptic ion channels may also be controlled by neurotransmitters or modulators and affect synaptic transmission by altering the amount of calcium influx.


The neurophysiology of glial cells.

Although glial cells occupy about half of the brain's total volume, they have been less well studied than neurons and have been the subject of endless speculation regarding their role in the brain's work. An enormous amount of new information about glial cells has become available in recent years and has led to the realization that these cells interact importantly with neurons in the context of all of the brain's main functions. In most instances, the details of these complex interactions are still being worked out. We review some of the basic physiological properties of glial cells that are important in understanding many of their determined or proposed functions.


The nigral projection to predorsal bundle cells in the superior colliculus of the rat.

Predorsal bundle cells give rise to the major efferent pathway from the superior colliculus to the premotor centers of the brainstem and spinal cord responsible for initiating orienting movements. The activity of predorsal bundle cells is profoundly influenced by an inhibitory pathway from substantia nigra pars reticulata that uses gamma aminobutyric acid (GABA) as a neurotransmitter. The present study examines the morphological basis for this influence of substantia nigra on predorsal bundle cells in the rat. In the first experiments, the laminar distributions of the nigrotectal tract terminals and the predorsal bundle cells were compared. The predorsal bundle cells were labeled by the retrograde axonal transport of horseradish peroxidase from either the decussation of the predorsal bundle or the cervical spinal cord, while the terminations of the pathway from substantia nigra pars reticulata were labeled by anterograde axonal transport from the substantia nigra. Either horseradish peroxidase, wheat germ agglutinin conjugated to horseradish peroxidase, or Phaseolus vulgaris leucoagglutinin were used as anterograde tracers. The results showed that the distributions of both the predorsal bundle cells and the nigrotectal terminals are restricted almost entirely to the intermediate grey layer and that they overlap extensively. Predorsal bundle cells varied in size. Within the areas of maximum overlap, the majority, regardless of size, was closely apposed by nigrotectal terminals. In a second series of experiments, the synaptic contacts between nigrotectal terminals and the tectospinal component of the predorsal bundle were examined in tissue in which both the terminals and the tectospinal cells were labeled for electron microscopy. In the final experiments, the distribution and fine structure of the nigrotectal terminals were compared to those of terminals that had been labeled immunocytochemically with an antibody to glutamic acid decarboxylase, the synthesizing enzyme for GABA. The results showed that nigrotectal terminals contain large numbers of mitochondria and pleomorphic vesicles, and form synaptic contacts with the somas and proximal dendrites of tectospinal cells. These synapses have modest postsynaptic densities. In both their distribution and fine structure, these terminations resemble the glutamic acid decarboxylase immunoreactive terminals that contact tectospinal cells. Taken together, these results support the view that the nigrotectal tract is an important source of GABAergic input to most, if not all, predorsal bundle cells.


The somatosensory thalamus of monkeys: cortical connections and a redefinition of nuclei in marmosets.

Thalamic connections of three subdivisions of somatosensory cortex in marmosets were determined by placing wheatgerm agglutinin conjugated with horseradish peroxidase and fluorescent dyes as tracers into electrophysiologically identified sites in S-I (area 3b), S-II, and the parietal ventral area, PV. The relation of the resulting patterns of transported label to the cytoarchitecture and cytochrome oxidase architecture of the thalamus lead to three major conclusions. 1) The region traditionally described as the ventroposterior nucleus (VP) is a composite of VP proper and parts of the ventroposterior inferior nucleus (VPi). Much of the VP region consists of groups of densely stained, closely packed neurons that project to S-I. VPi includes a ventral oval of pale, less densely packed neurons and finger-like protrusions that extend into VP proper and separate clusters of VP neurons related to different body parts. Neurons in both parts of VPi project to S-II rather than S-I. Connection patterns indicate that the proper and the embedded parts of VPi combine to form a body representation paralleling that in VP. 2) VPi also provides the major thalamic input into PV. 3) In architecture, location, and cortical connections, the region traditionally described as the anterior pulvinar (AP) of monkeys resembles the medial posterior nucleus, Pom, of other mammals and we propose that all or most of AP is homologous to Pom. AP caps VP dorsomedially, has neurons that are moderately dense in Nissl staining, and reacts moderately in CO preparations. AP neurons project to S-I, S-II, and PV in somatotopic patterns.


Direct W-like geniculate projections to the cytochrome oxidase (CO) blobs in primate visual cortex: axon morphology.

The primate lateral geniculate nucleus (LGN) is composed of large, medium, and small cells located, respectively, in magnocellular (M), parvocellular (P), and specialized layers (intercalated and S-layers in simians, koniocellular (K) layers in prosimians). Several studies have examined the physiology and connections of M and P LGN cells and have concluded that they provide separate contributions to visual perception via separate pathways. Less is known about the structure and contributions of the small LGN cells. This study examined the distribution and structure of K LGN cell axons in the cortex of the prosimian, Galago crassicaudatus. Wheat germ agglutinin conjugated to horseradish peroxidase, or Phaseonlus vulgaris leucoaglutinin, was injected into the LGN K layers to demonstrate the overall axon projection pattern and the details of individual axons, respectively. Location of axons within striate cortex was specified relative to boundaries determined by Nissl or cytochrome oxidase (CO) stains on the same or adjacent sections. Our results show that K LGN axons end as single complex arbors within one CO blob zone in layer III; they never terminate in interblob zones. These axons also emit a collateral in layer I that arborizes more broadly and spans both CO blob and interblob zones. These data, together with data on K cell physiology and intralaminar cortical connections, suggest that the LGN small cell pathway could modulate the activity of the other two pathways in striate cortex and contribute directly to visual perception.


Lateral geniculate projections to the superficial layers of visual cortex in the tree shrew.

Our recent studies of tree shrew striate cortex have focused on the organization of lateral geniculate projections to layer IV and the projections from IV to layer III. Although these pathways play an important role in determining the response properties of layer III neurons, there are additional pathways from the lateral geniculate nucleus (LGN) that terminate directly in layer III. Previous studies provided evidence that these projections originate from layers 6 and 3 of the LGN and terminate in different subdivisions of layer III. In this study we used injections of biocytin to examine the projections of layers 6 and 3 to the cortex in more detail. Consistent with earlier work, we found that LGN layer 6 projects heavily to lower IIIc, while LGN layer 3 terminates densely in layer IIIb and sparsely throughout layers IIIa-I. In addition, we found that neurons in layers 6 and 3 have collateral projections: neurons in LGN layer 6 project to the bottom of layer IVb and sparsely to I-IIIb; neurons in LGN layer 3 project sparsely to layers V and VI and to the middle of IV. These patterns of projections are significant in the light of our studies of the connections from cortical layer IV to layer III. LGN projections to the superficial layers are organized into parallel pathways that exert selective influence over different populations of neurons in layers I-III and on the layer IV neurons that supply them.


Fetuin: an acute phase protein in cattle.

Fetuin is a plasma protein present in high concentrations during fetal development in animals of the order Artiodactyla. Its role is not known. The human homologue of fetuin--alpha 2HS glycoprotein--has been shown to be a negative acute phase protein in adult plasma. In the present study, the concentration of fetuin was measured in the serum of healthy cattle (Bovis bovis) and in animals with various injuries and inflammatory disorders. The levels were decreased by 30% in pregnancy but increased up to 10-fold in some trauma cases. A significant negative correlation between the concentrations of fetuin and albumin has also been found. Thus, fetuin appears to be a positive acute phase protein in cattle.


Release of substance P-like immunoreactive material from the stomach of the rainbow trout.

The release of substance P-like immunoreactive material (SPLI) from the vascularly perfused stomach of the rainbow trout, Oncorhynchus mykiss, was studied. In most cases, SPLI was detected in the collected vascular perfusate during experimental resting conditions. Distensions of the stomach, accomplished by a water-filled intragastric balloon, produced an initial rapid relaxation of the stomach, followed by a slow further relaxation and a stimulation of contractile activity. The amount of SPLI in the vascular perfusate was significantly elevated during the distension period. Tetrodotoxin had no effect on the response to distension or on the release of SPLI during distension, indicating release from tetrodotoxin-insensitive neurons or endocrine cells. The results suggest that a substance P-like peptide may be involved in the contractile response and/or in the maintenance of muscular tone during gastric distensions in the rainbow trout. Infusion of capsaicin had no effect on the release of SPLI. However, capsaicin caused an increase in vascular flow, an effect that could be repeated on a second infusion of capsaicin, indicating that the action may not be specific to sensory neurons.


The effect of human anaphylatoxins and neutrophils on histamine release from isolated human skin mast cells.

The concept of mast cell heterogeneity has been studied extensively. Recently developed techniques to enzymatically disperse skin mast cells from human skin have shown that skin mast cells are somehow different from those of other organs such as lung and intestine. In this report, we have isolated and partially purified human skin mast cells from human neonatal foreskins by collagenase and hyaluronidase digestion. These mast cells are morphologically intact by histological, immunohistochemical and electron microscopic criteria. These human skin mast cells secrete histamine significantly (max. net histamine release, 20-30%) in a dose-related, temperature- and time-dependent fashion following stimulation with purified human C5a and C3a (over the ranges of 5 x 10(-8) M to 10(-7) M and 3 x 10(-7) M to 6 x 10(-6) M, respectively). On the other hand, interactions between human skin mast cells and other leukocytes have long been suspected of playing a very important role in cutaneous inflammation. Recently, a human neutrophil-derived histamine-releasing activity termed HRA-N was partially purified. HRA-N has been shown to cause human and rat basophil leukemia cells to degranulate. This study was also undertaken to assess the ability of HRA-N to directly induce histamine release from isolated human skin mast cells. HRA-N causes dose- and time-dependent histamine release as do human anaphylatoxins. These results suggest that HRA-N may lead to a better comprehension of allergic and inflammatory reactions and their modulation in the skin.


Distributions of interstitial cells of Cajal in stomach and colon of cat, dog, ferret, opossum, rat, guinea pig and rabbit.

Electrical slow waves vary in prominence from one location to another in stomach and colon. If interstitial cells of Cajal are involved in generation of the slow waves, they might be more abundant in regions where slow waves are prominent than in regions where slow waves are small or absent. We calculated the density of distribution of interstitial cells of Cajal in colon and stomach of cat, dog, ferret, guinea pig, opossum, rabbit, and rat, using the zinc iodide-osmic acid reaction for light microscopy. The cells in the stomach were concentrated in the myenteric plexus and in the circular muscle layer. Along the greater curvature, cells were relatively sparse in the fundus, more frequent in the corpus and densest in the antrum. Along the lesser curvature, they were denser distally than proximally in all species except guinea pig. In the colon most cells lay with an axonal network at the circular muscle-submucosal interface. The axons were sparse in the cecum but uniformly conspicuous from the ileocolonic junction to the internal and sphincter. The density of the interstitial cells of Cajal in cat, dog, opossum, and rabbit rose from a relatively low level in the cecum to a maximum in the mid-colon, declining toward the rectum. In guinea pig, rat, and ferret, the levels throughout the most proximal colon were high. Since gastric slow waves are absent from the fundus, diminutive in the body, and prominent in the antrum, the density of interstitial cells of Cajal in the stomach roughly parallels the prominence of slow waves. Colonic slow waves are most prominent in the right colon and mid-colon, and so the density distribution of interstitial cells of Cajal in the colon also roughly parallels the prominence of slow waves.


A cluster of severe postoperative bleeding following open heart surgery.

To investigate a cluster of postoperative bleeding following open heart surgery. A cohort and case/control study. Palo Alto Veterans Administration Medical Center, Palo Alto, California. Six (21.4%) of 28 patients undergoing open heart surgery who developed severe, nonsurgical, postoperative bleeding from July 1 through August 30, 1988 (outbreak period). All case-patients had chest tube drainage of greater than or equal to 1000 ml within 4 hours of surgery but did not have identifiable bleeding vessel(s) on exploration. Upon comparison of the pre-outbreak (January 1986 through June 1988) and the outbreak period, a significant increase was found in the incidence of postoperative nonsurgical bleeding (5/440 versus 6/28, p = .0006), but not of postoperative surgical bleeding (8/440 versus 0/28, p = 1.0). Of all patients undergoing open heart surgery during the outbreak period, case patients were found to be older (67.8 versus 60.6, p = .02) and to have received a larger volume of hetastarch (HES), a synthetic colloidal plasma-volume expander (mean = 19.4 ml/kg versus 14.1 ml/kg, p = .02). We conclude that the use of large volumes of HES during surgery in the elderly open heart surgery patient may increase the risk for severe, nonsurgical postoperative bleeding, probably caused by alterations of the coagulation system. As the incidence of open heart surgery increases among the elderly, surgeons and anesthesiologists should be alert to possible adverse reactions from exposures not associated with adverse reactions in younger patients.


Induction and identification of mast cells from long-term culture of mouse spleen cells without conditioned medium.

A simple method is described for the preparation of large numbers of mast cells from mouse spleen cells in vitro. Mouse spleen cells were cultured with RPMI 1640 medium supplemented with 10% FCS and 2-ME. Half of the total volume of the medium was changed every 4-5 days. Mast cell numbers increased with the culture time and reached a peak between 16 and 20 days. Using this method, 2 x 10(6) mast cells could be induced from 1 x 10(7) nucleated normal spleen cells. T cells and supernatant derived from ConA-stimulated T cells were unnecessary for mast cell induction. Phenotype analysis by FACS showed that Thy1,2, L3T4, Ly-2, Ig, B220, Asialo GM1, and WGA receptors were all negative but functional IgE receptors were positive. The granules in the cells could be stained by alcian blue but not by safranin. There was 1.632 +/- 0.024 micrograms stored histamine in 1 x 10(6) of the cells. Histamine was released from the cells in an antigen-induced and IgE-mediated process. Compound 48/80 and A23187 induced degranulation of the cells, and the mast cells were able to respond to ConA.


An improved method for the purification of basophilic granulocytes from human blood.

Studies on human basophils are hampered by the low number of basophils in peripheral blood. Here we describe the purification of human basophilic granulocytes with immunomagnetic beads to improve an already established method of purification. A 70% pure basophil suspension was incubated with monoclonal antibodies (CD2, CD14, CD16 and CD19) recognizing the contaminating cells. After incubation with magnetic beads coated with goat anti-mouse IgG, the bead-cell rosettes were removed by a magnet. In this way, the basophil purity increased to 94%. The loss of basophils during the bead procedure was about 20%. The amount of histamine per basophil and the spontaneous release of histamine during subsequent incubation of the cells was not altered by the purification procedure. The kinetics and the dose response of histamine release after the addition of anti-IgE or FMLP was also unchanged. Binding of CD63 was not altered, indicating that the purification procedure did not result in activation of the basophils. This improved method for the purification of human basophils should permit the measurement of non-basophil-specific parameters, such as changes in intracellular free Ca2+, without the problem of interference from contaminating cells.


Immunosuppressive macrolides of the type FK 506: a novel class of topical agents for treatment of skin diseases?

The immunosuppressive macrolide antibiotics FK 506 and rapamycin were tested for topical activity in experimental allergic contact dermatitis of farm pigs. This species was used because pig skin, in comparison to rodent skin, resembles human skin more closely. For comparison, cyclosporine A (CyA), which is orally but not topically active in patients with skin disease, dexamethasone, and clobetasol propionate were used. Treatment was performed twice, 30 min and 6 h after elicitation of challenge reaction. Topical application of 0.4 to 0.04% FK 506 caused a pronounced inhibition of inflammatory skin reactions of hypersensitivity to dinitrofluorobenzene. The treatment response was similar to the activity of 0.13% clobetasole. Dexamethasone (1.2%) was less active than clobetasol. In contrast, rapamycin and CyA were inactive at concentrations of 1.2 and 10%, respectively. Because the pig data on corticosteroids and cyclosporine A are in agreement with clinical findings, these studies indicate that immunosuppressive macrolides of the type FK 506 may be useful drugs for the topical treatment of human skin diseases that respond to local corticosteroids and oral treatment with cyclosporine A.


Differential expression of laminin isoforms and beta 4 integrin epitopes in the basement membrane zone of normal human skin and basal cell carcinomas.

The basement membrane zone biology of normal human skin and basal cell carcinomas was explored by indirect immunofluorescence with monoclonal antibodies recognizing five subunit polypeptides of three different laminin isoforms as well as the beta 4 integrin epitopes. The laminin antibodies were specific for A, B1, and B2 chains of classic laminin, for the M chain of merosin, or for the S chain in S-laminin. Immunostaining of normal human skin revealed a strong signal with antibodies for A, B1, and B2 chain epitopes. A weak immunosignal was detected with an anti-M chain antibody, whereas the S-chain epitopes were undetectable, even following pretreatment of sections with hyaluronidase. Thus, the laminin at the epidermal-dermal junction of normal human skin is primarily of the classic type, with some merosin molecules being present. The staining of six nodular basal cell carcinomas revealed the presence of A, B1, and B2 chain epitopes in a linear pattern, but, in contrast to normal skin, the antibody recognizing M-chain epitopes yielded a strong immunosignal, and S-chain epitopes could also be readily detected. Staining for beta 4 integrins, potential receptors for laminin, revealed a strong staining reaction in normal skin as well as in the superficial portions of the basal cell carcinoma. However, the immunofluorescence pattern in the deeper portions of the lesions was scattered and interrupted. Thus, altered composition of the basement membrane of nodular basal cell carcinomas with respect to laminin isoforms and their interactions with putative cell-surface receptors, the beta 4 integrins, may change the containment of the tumor islands, contributing to the local aggressive behavior of basal cell carcinomas.


Induction of 72-kD heat shock protein and cytoskeleton damage by cytotoxic prostaglandin delta 12-PGJ2 in transformed human epidermal cells in culture.

Cyclopentenone prostaglandins (PG) such as delta 12-PGJ2 and PGA are potent inhibitors of growth in a variety of cultured cells, including human epidermal cells. To clarify the mechanism of PG cytotoxicity in human epidermal cells, we examined the effects of delta 12-PGJ2 on the induction of a heat shock protein (HSP), and on the organization of cytoskeletons in the HSC-I-transformed human epidermal cell line. Immunoblot analysis using a monoclonal antibody specific for the 72-kD heat shock protein (HSP72) revealed that a 12-h incubation with 5 micrograms/ml of delta 12-PGJ2 induced HSP72 formation in HSC-I cells. HSP72 was also induced by heat shock treatment at 43 degrees C for 90 min. The quantity of HSP72 produced was markedly decreased by co-treatment with 1 microgram/ml of cycloheximide in delta 12-PGJ2-treated cells, and similarly reduced in HSC-I cells following heat treatment. Immunofluorescence using a monoclonal antibody to HSP72 demonstrated that HSP72 was localized mainly in the cytoplasm of HSC-I cells. Following treatment with 5 micrograms/ml of delta 12-PGJ2, however, HSP72 was found in the nucleolus as well as in the cytoplasm. The accumulation of HSP in the nucleolus was similarly prominent in HSC-I cells after treatment at 43 degrees C for 90 min. Addition of delta 12-PGJ2 to confluent HSC-1 cells resulted in the disappearance of actin filaments and the disarrangement of keratin filaments, as visualized with fluorescent-labeled phallacidine or immunofluorescence. These results suggest that the cytotoxicity of cyclopentenone PG is related to the induction of HSP72, and to cytoskeleton damage in transformed human epidermal cells in culture.


Delayed onset of epidermal differentiation in psoriasis.

In normal epidermis, as previously reported, the first signs of differentiation occur within the basal layer in a subpopulation of keratinocytes that start to express K1 and K10 "supra-basal" keratin transcripts (20-30% of the basal cells) and proteins (5-10% of the basal cells). We found that in psoriatic lesions, the basal layer was devoid of cells expressing these early differentiation markers. This was already the case at the periphery of the lesions, where epidermis, although slightly acanthotic, still completes the keratinization process. In the center of the lesions, not only the basal layer, but also several rows of suprabasal cells, were negative for keratin K10 transcripts or protein. Moreover, the upper nucleated layers of involved epidermis were also devoid of K10 keratin transcripts or proteins. In normal epidermis, as previously reported, transcripts for the "basal" K5 keratin were mainly restricted to the basal layer, whereas the protein persisted in a few suprabasal layers. We found that in psoriatic epidermis, K5 keratin transcripts persisted in several suprabasal layers up to the level where K10 keratin transcripts appeared. These data, although not contradictory with previous reports showing a reduction of K1-K10 keratins and other differentiation markers in psoriasis, demonstrate that these quantitative changes are in fact the result of major qualitative differences in the distribution of these markers in psoriatic versus normal skin. Our results indicating that the onset of differentiation is delayed in psoriasis show that, contrary to conclusions accepted so far, not only the suprabasal compartment, but also the basal one, is abnormal in psoriatic epidermis.


Antigenic analysis of Clostridium chauvoei flagella with protective and non-protective monoclonal antibodies.

Five monoclonal antibodies (mAbs) directed against the flagellin of Clostridium chauvoei were used to analyse the structural and antigenic characteristics on the bacterial flagellar surface. Immune electron microscopy showed that three protective mAbs recognized the surfaced-exposed epitopes on the flagellar filament of this bacteria. In contrast, two non-protective mAbs recognized internal epitopes of the flagellar filament. These findings have been confirmed by ELISA using mAbs absorbed with whole cells of C. chauvoei possessing flagella. Competitive binding assays showed that protective mAbs indicated reciprocal competition, while each of the non-protective mAbs had topographically distinct epitopes. Moreover, immunoblotting analysis with cyanogen-bromide-cleaved flagellin showed that protective mAbs may preferentially recognize conformational epitopes, whilst one of the non-protective mAbs may recognize a linear and conformation-independent epitope in the flagellin of C. chauvoei.


The bactericidal action of streptomycin: membrane permeabilization caused by the insertion of mistranslated proteins into the cytoplasmic membrane of Escherichia coli and subsequent caging of the antibiotic inside the cells due to degradation of these proteins.

The mechanism by which the aminoglycoside antibiotic streptomycin permeabilizes the cytoplasmic membrane of Escherichia coli cells was reinvestigated. For this purpose, the extent of streptomycin-induced K+ loss from cells growing at low external K+ concentrations was taken as a measure of membrane permeabilization. Experiments with different K(+)-uptake mutants showed that the antibiotic specifically increased the passive permeability of the cell membrane to K+ and other ions. These permeability changes were small and the membrane potential of the treated cells remained high. The membrane permeabilization was not due to a direct interaction of the antibiotic with the cell membrane, since cells that carry an rpsL mutation and synthesize proteins in a streptomycin-insensitive way did not lose K+ after the addition of the antibiotic. Due to misreading and premature termination of translation the cells synthesized aberrant proteins under the conditions where membrane permeabilization occurred. Two conditions are described under which the cells both degraded these mistranslated proteins rapidly and reaccumulated K+, lending support to the hypothesis that membrane permeabilization is due to the presence of the mistranslated proteins in the cell membrane. Evidence is presented that the irreversibility of (dihydro)streptomycin uptake by cells washed free from the antibiotic might also be due to rapid degradation of the mistranslated proteins, leading to 'caging' of the antibiotic inside the cells.


Aminoglycoside and aminocyclitol antibiotics: hygromycin B is an atypical bactericidal compound that exerts effects on cells of Escherichia coli characteristics for bacteriostatic aminocyclitols.

The effects of aminoglycoside and aminocyclitol antibiotics on intact cells of Escherichia coli were compared. The aminoglycosides streptomycin, gentamicin, kanamycin and neomycin had similar, but not identical, effects. They all caused misreading during protein synthesis, permeabilization of the cell membrane, inhibition of the initiation of DNA replication, and loss of cell viability. Cells treated with these antibiotics continued to synthesize two proteins (apparent molecular masses 72 and 60 kDa) that were not made by cells treated with the aminocyclitol hygromycin B, which did not cause misreading. Cells treated with the aminoglycosides regained their membrane tightness after residual protein synthesis in these cells had been inhibited by chloramphenicol, suggesting that under these conditions the mistranslated membrane proteins were rapidly degraded. The bacteriostatic aminocyclitols spectinomycin and kasugamycin did not cause membrane permeabilization, suggesting that these compounds do not cause misreading. Hygromycin B resembled these aminocyclitols in that it inhibited protein synthesis without causing misreading, membrane permeabilization or inhibition of initiation of DNA synthesis. However, hygromycin B also decreased cell viability. In minimal medium this lethal effect began late in comparison to the process of inhibition of protein synthesis. It is concluded that hygromycin B is an atypical bactericidal antibiotic that strongly resembles the bacteriostatic aminocyclitols spectinomycin and kasugamycin in its action.


Isolation and characterization of temperature-sensitive mutants of Streptomyces coelicolor A3(2) blocked in macromolecular synthesis.

A collection of temperature-sensitive mutants of Streptomyces coelicolor A3(2) was isolated. The majority of the mutants showed an osmotically remedial phenotype. Mutants defective in macromolecular synthesis were identified and characterized further. Four mutants were found in which DNA replication was defective, but which continued to synthesize RNA and protein at the restrictive temperature (39 degrees C). The kinetics of cessation of DNA synthesis allowed a tentative identification of slow (initiation) and fast (elongation) stop dna mutants. The inhibition of DNA replication in the four mutants was found to be reversible on returning to the permissive temperature (30 degrees C), but only after a delay of about 2 h. Three other mutants were identified which showed not only cessation of DNA replication at the restrictive temperature, but also defects in other macromolecular synthesis events.


Anti-aids agents, 6. Salaspermic acid, an anti-HIV principle from Tripterygium wilfordii, and the structure-activity correlation with its related compounds.

Salaspermic acid [1], an inhibitor of HIV reverse transcriptase and HIV replication in H9 lymphocyte cells, was isolated from the roots of Tripterygium wilfordii for the first time. The structure of 1 derived from spectral data was established unequivocally by an X-ray analysis of crystals of the monohydrate. A structure-activity correlation of 1 with ten related compounds indicated that the acetal linkage in ring A and the carboxyl group in ring E of 1 may be required for the anti-HIV activity.


Evidence of hyaluronic acid and hyaluronic acid binding sites on human corneal endothelium.

A highly specific hyaluronic acid (HA) recognizing protein (HABR) was used to study whether the human corneal endothelium is covered by HA and to quantify the amount. Tritiated high molecular weight HA was used to determine the capacity of the human endothelium to bind exogenous HA. Human corneas were obtained from keratoconus patients having corneal transplantation and from postmortem eyes. The corneas were immersed in a 4% formaldehyde solution containing 1% cetylpyridine chloride for histochemistry, frozen for biochemistry, or used for 3H-HA (Mr 3 x 10(6) binding. For the biochemical determinations, 125I-labeled HABR was used. Tritiated HA was used for the binding experiment. A specific layer of HA covering the endothelial cells of the corneal buttons was demonstrated. The biochemical analysis also revealed the presence of HA. Finally, the human endothelial cells had specific hyaluronic acid binding sites.


Acetylcholine and kinin augmentation of Cl- secretion stimulated by prostaglandin in a canine renal epithelial cell line.

1. The actions of kinins and of acetylcholine upon transepithelial ion transport in a renal-derived cultured epithelium (Madin-Darby canine kidney cells, MDCK) have been investigated. 2. In voltage-clamped epithelial layers mounted in Ussing chambers and with prior stimulation of inward short-circuit current (SCC) by 5 or 10 microM-prostaglandin E1 (PGE1), both bradykinin (1 microM) and acetylcholine (0.1 mM) stimulate an additional, but transient, inward SCC. In the absence of PGE1 minimal effects of both bradykinin and acetylcholine upon SCC are observed. The SCC response to bradykinin and acetylcholine are attenuated with prior stimulation by 10 microM-adrenaline. 3. Measurements of bradykinin and acetylcholine-stimulated inward SCC with cation and anion replacement of the bathing media and the use of the Cl channel blocker 5-nitro-2(3-phenylpropylamino)-benzoic acid (NPPB) together with bumetanide to inhibit Na(+)-K(+)-Cl- 'co-transport', are consistent with the bradykinin- and acetylcholine-stimulated SCC being the result of basal to apical Cl- secretion. 4. Bradykinin (1 microM) is capable of stimulation of inward SCC from both epithelial surfaces, whilst acetylcholine is only effective from the basolateral surface. Kallidin (lys-bradykinin) was similar in effect to bradykinin from both epithelial surfaces whereas bradykinin (1-8) was ineffective, suggesting that B2 bradykinin receptors mediate the effect of bradykinin upon SCC. Dose-response relationships show that the response to kallidin and bradykinin was of higher sensitivity for additions to the apical cell aspects. 5. The data are discussed in relation to a model for epithelial Cl- secretion, and to the mechanism of natriuresis observed with kinins and acetylcholine in vivo.


The ATP-induced inward current in mouse lacrimal acinar cells is potentiated by isoprenaline and GTP.

1. ATP activates calcium (Ca2+) influx in mouse lacrimal acinar cells in the absence of phosphoinositide hydrolysis. Extracellular ATP (1 mM) activates receptor-operated cation channels, promoting entry of Na+ and Ca2+ (inward current). This Ca2+ influx in turn activates K+ channels resulting in a delayed, outward, current component. The present study uses patch-clamp current recording techniques to investigate the role of beta-adrenoceptor mechanisms, intracellular cyclic AMP and GTP in the regulation of the ATP-induced inward currents. 2. The beta-adrenoceptor agonist, isoprenaline (1 microM), does not increase the resting membrane currents but markedly enhances the ATP-induced inward and outward currents. This effect of isoprenaline is blocked by the beta-adrenoceptor antagonist propranolol. 3. Internal application of cyclic AMP mimics the potentiating effect of isoprenaline. 100 microM-cyclic AMP increases the ATP-induced inward and outward currents to about 200% as compared to control responses. 4. Pre-treatment of the cells with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 1 mM), also results in a marked potentiation of the ATP-induced inward currents to 170% as compared to control responses. 5. The ATP-induced inward current responses are not blocked by either the removal of extracellular Ca2+ or by chelation of intracellular Ca2+ (by inclusion of 10 mM-EGTA in the recording pipette). Both protocols did however block the potentiating effect of internal cyclic AMP on the ATP-induced inward current responses. 6. Intracellular ATP (10 mM) reduces the amplitude of the inward currents evoked by external ATP application by about 60% and the currents were no longer potentiated by internal cyclic AMP. 7. Intracellular GTP or GTP-gamma-S (100 microM in the pipette solution) potentiates the current responses to ATP, increasing both the amplitude and duration of the inward currents. 8. In excised inside-out patches, with ATP in the recording pipette (i.e. external ATP), the catalytic subunit of the cyclic AMP-dependent protein kinase activated the cation channels. The effect of the catalytic subunit was readily reversible and abolished by an inhibitor of the protein kinase. 9. External ATP activates Ca2+ influx in lacrimal acinar cells by a mechanism that is distinct from that activated by phosphoinositide-coupled receptors. The effect is mediated by direct activation of cation channels in the cell surface membrane which allow for significant entry of Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)


Substance P inhibits activation of calcium-dependent potassium conductances in guinea-pig myenteric neurones.

1. Intracellular recordings were made from myenteric AH neurones of the guinea-pig ileum in vitro. Some experiments were done with a single-electrode voltage clamp to measure membrane currents. 2. Substance P (SP) applied by superfusion (10 nM-300 nM), pressure ejection (100 nM-10 microM, 760 mmHg, for 10-20 ms) or ionophoresis (1 mM, 100 nA, for 0.2 s) caused a membrane depolarization and an inward current, associated with a decrease in potassium conductance. 3. The SP-induced depolarization was abolished within 15 min by superfusion with calcium-free/high-magnesium (10 mM) solution or solutions containing cobalt, manganese or nickel at 1-3 mM. The response persisted even after 40-60 min of superfusion with calcium-free/normal-magnesium (1.2 mM) solution. In all these solutions, synaptic potentials were abolished within 5 min. 4. SP inhibited a slowly developing outward current and an outward tail current during and after a long depolarizing command pulse (2-10 s), and an outward after-current following single or multiple brief depolarizing command pulses (10-50 ms). These outward currents were suppressed in calcium-free/high-magnesium solution. 5. SP depressed both a calcium-dependent slow after-hyperpolarization following the action potential and an outward after-current preceded by a brief depolarizing command. Both the SP-induced depolarization and the SP-induced inward current were augmented when the peptide was pressure-ejected during the recovery phase of the slow after-hyperpolarization and during that of the slow outward after-current, but both of them were inhibited or almost abolished when SP was applied immediately after spike initiation or a brief depolarizing command. 6. The SP-induced response was depressed by barium (1-2 mM). The SP response was not inhibited by tetraethylammonium at low concentrations (5-10 mM), but was depressed at high concentration (20 mM). 7. Superfusion (1-10 nM) or pressure application of a calcium ionophore, A23187, inhibited or even reversed the SP depolarization and the SP-induced inward current. 8. These results indicate that SP inhibits activation of a calcium-dependent potassium conductance which contributes to both the slow after-hyperpolarization and the resting membrane potential. SP may affect the process by which calcium activates this potassium conductance.


Neuronal nicotinic acetylcholine receptor currents in phaeochromocytoma (PC12) cells: dual mechanisms of rectification.

1. Neuronal nicotinic acetylcholine receptor (nAChR) currents in PC12 cells were studied using single-channel and whole-cell gigaohm seal voltage-clamp techniques. Nicotinic AChR agonists were applied using external pipettes. 2. The single-channel conductance of neuronal nAChRs in outside-out patches under Mg(2+)-free ionic conditions was 48 pS. In the absence of internal Mg2+ single-channel currents (outside-out patches) did not rectify. 3. Whole-cell nAChR currents recorded with normal internal solution (lacking Mg2+ chelators as described below) were strongly inwardly rectifying. Current vs. voltage relations showed a sharp inflexion near 0 mV, and outward current was never observed. 4. Extensive dialysis with internal solution containing EDTA and Na2-ATP, chelators of intracellular Mg2+ (Mgi2+), relieved rectification of instantaneous nAChR currents during voltage jumps from negative potentials. The instantaneous I-V relation became linear with voltage, in agreement with the expectation from single-channel measurements made under similar ionic conditions. 5. Agonist-induced currents recorded under Mgi(2+)-free conditions relaxed towards zero current during depolarizing voltage steps. Rectification of ACh-induced currents at the steady state could be described by a Boltzmann relation, with one-half of channels available at +3.4 mV and an effective gating charge of -0.97. Channel availability was approximately 95% at the resting potential. Opening and closing relaxations under Mgi(2+)-free conditions could be fitted by single exponential functions whose time constants were weakly voltage dependent. 6. A model of rectification was constructed incorporating two voltage-dependent processes: block by Mgi2+ and intrinsic channel gating. The I-V relations predicted by this model for both normal and Mgi(2+)-free conditions were in good agreement with the experimental data. We suggest that rectification of nAChR currents in these cells is due to concurrent activity of these two voltage-dependent processes. The relative contributions of these two mechanisms are frequency dependent, with block by Mgi2+ dominant for fast events and intrinsic channel gating more important at the steady state.


A novel modulatory binding site for zinc on the GABAA receptor complex in cultured rat neurones.

1. The properties of gamma-aminobutyric acidA (GABAA) receptor-ion channel complexes and the interaction with the transition metal zinc, were studied on rat sympathetic and cerebellar neurones in dissociated culture using patch clamp recording techniques. 2. The antagonism of GABA-induced membrane currents by zinc on sympathetic neurones was subject to developmental influence. Using embryonic sympathetic neurones acutely cultured for 24-72 h, GABA responses were more depressed by zinc when compared to responses evoked on adult neurones cultured for the same period. For neurones developing in vivo, the percentage inhibition of GABA responses produced by zinc in embryonic neurones was estimated to decline by 50% after 48.2 days following birth. 3. Embryonic sympathetic neurones maintained in culture for prolonged periods (40-50 days in vitro, DIV) became less sensitive to zinc when compared to neurones cultured for shorter periods (10-20 DIV). The decrease in the zinc inhibition for neurones maintained in vitro proceeded at an apparent rate of 0.55% per day. 4. Activation of the GABA receptor by muscimol (0.2-2 microM) was also antagonized by zinc (50-100 microM). 5. Lowering the pH of the perfusing Krebs solution did not affect the inhibition of GABA responses by zinc on sympathetic neurones. 6. Modulation of the GABAA receptor by some benzodiazepines, a barbiturate, a steroid based on pregnanolone, or antagonists bicuculline and picrotoxinin, did not interfere with the antagonism exerted by zinc on sympathetic neurones. A novel binding site for zinc on the GABAA receptor is proposed. 7. Analysis of the GABA-activated current noise on sympathetic neurones revealed two kinetic components to the power spectra requiring a double Lorentzian fit. The time constant describing the fast component (tau 2, 2.1 ms) was unaffected by zinc, whereas the slow component time constant (tau 1, 21.7 ms) was slightly reduced to 17.1 ms. 8. The apparent single-channel conductance for GABA-activated ion channels was determined from the power spectra (gamma s = 22.7 pS) and also from the relationship between the mean GABA-induced inward current and the variance of the current (gamma v = 24 pS). Zinc (25-100 microM) did not affect the single-channel conductance. 9. Single GABA-activated ion channels were recorded from outside-out patches taken from the soma of large cerebellar neurones. Single GABA channels were capable of activation to multiple current amplitudes which were assessed into the following conductance levels: 8, 18, 23, 29 and 34 pS.(ABSTRACT TRUNCATED AT 400 WORDS)


Effects of sympathetic activity and galanin on cardiac vagal action in anaesthetized cats.

1. Stimulation of the right cardiac sympathetic nerve for 3 or 5 min at 16 Hz in the presence of effective beta-adrenoceptor blockade evoked prolonged attenuation of subsequent cardiac vagal action in anaesthetized cats. Concurrent sympathetic and vagal stimulation, both at 16 Hz for the same period of time as sympathetic stimulation alone, reduced or abolished the inhibition of vagal action which followed when sympathetic stimulation was given alone. 2. A series of three successive intravenous injections of galanin all caused attenuation of vagally induced slowing of the heart, but the effect of each injection was less than that of the previous one. A fourth injection of galanin given after a 45 or 60 min rest period showed return of sensitivity. 3. Sympathetic stimulation at 16 Hz for 5 min was less efficient in causing attenuation of vagal slowing when applied following the administration of exogenous galanin. This is consistent with galanin being the mediator of vagal attenuation following sympathetic stimulation.


Dihydropyridine-sensitive and omega-conotoxin-sensitive calcium channels in a mammalian neuroblastoma-glioma cell line.

1. Pharmacological and kinetic properties of high-voltage-activated (HVA) Ca2+ channel currents were studied using the whole-cell and perforated patch-clamp methods in a mouse neuroblastoma and rat glioma hybrid cell line, NG108-15, differentiated by dibutyryl cyclic AMP or by prostaglandin E1 and theophylline. 2. The HVA currents were separated into two components by use of two organic Ca2+ channel antagonists, omega-conotoxin GVIA (omega CgTX) and a dihydropyridine (DHP) compound, nifedipine. One current component, IDHP, was blocked by nifedipine (Kd = 8.2 nM) and was resistant to omega CgTX. Conversely, the other component, I omega CgTX, was irreversibly blocked by omega CgTX and was resistant to DHPs. Thus, IDHP could be studied in isolation by a short application of omega CgTX, while I omega CgTX could be studied in the presence of nifedipine. 3. The voltage for half-activation of IDHP was smaller than that of I omega CgTX by 13 mV. IDHP was activated at potentials that were subthreshold for voltage-dependent K+ currents of the cell, whereas I omega CgTX was not. 4. Time courses of activation and deactivation of IDHP were faster than those of I omega CgTX. 5. Voltage-dependent inactivation was small for both IDHP and I omega CgTX at any potential. 6. Ca(2+)-dependent inactivation of IDHP was faster and more prominent than that of I omega CgTX. The time course of the Ca(2+)-dependent inactivation of IDHP, but not I omega CgTX, was slowed as the membrane potential was made more positive between -20 and 30 mV, although amplitude of the current was increased. 7. Alkaline earth metal ions carried the two components of IHVA in the same order: Ba2+ greater than Sr2+ greater than Ca2+. 8. Metal ions blocked the two components of IHVA in the same order of potency: Gd3+ greater than La3+ greater than Cd2+ greater than Cu2+ greater than Mn2+ greater than Ni2+. 9. An alkylating agent, N-ethylmaleimide (NEM, 0.1 mM), selectively augmented IDHP by 30%. 10. During the course of cellular differentiation induced by dibutyryl cyclic AMP, IDHP appeared earlier than I omega CgTX. 11. These results indicate that two classes of Ca2+ channels contribute to the HVA currents of this cell line. The DHP-sensitive channel is more apt to generate Ca2+ spikes and Ca2+ plateau potentials than the omega CgTX-sensitive channel.


5-HT3 receptor channels in dissociated rat superior cervical ganglion neurons.

1. Whole-cell and single-channel voltage-clamp techniques were used to record the 5-HT3 receptor-mediated currents in neurons freshly dissociated from rat superior cervical ganglia. 2. Whole-cell currents elicited by brief pressure ejection of 5-HT (10 microM) reversed at -4.5 mV when extracellular and intracellular solutions mainly contained NaCl and CsCl. The peak current-voltage relation showed modest inward rectification that was fully developed within less than 2 ms of the applied voltage step. 3. With prolonged application of 5-HT (10 microM) using a fast perfusion system, the response desensitized in two phases with fast and slow time constants of 0.57 and 6.0 s at -74 mV. The time constants showed little voltage dependence; however, the relative amplitude of the two components was significantly dependent on voltage. The time course of desensitization was not affected by agents that increase the levels of intracellular cyclic AMP. 4. The relative permeability of the channel was determined from reversal potential changes. The channel passed small cations non-selectively, with permeability ratios (PX/PNa) of 0.93 and 1.24 for Cs+ and K+. The organic cations Tris and glucosamine were measurably permeant with permeability ratios of 0.19 and 0.06. Ca2+ was fairly permeant with a relative permeability of 0.55 in 20 mM solution and of 0.16 when the concentration of CaCl2 was increased to 115 mM. No permeability was detected for Cl-. 5. Fluctuation analysis of the whole-cell current revealed an apparent single-channel current of approximately 0.18 pA at -74 mV. 6. 5-HT-activated single-channel currents were recorded in excised outside-out patches. When 5-HT (10 microM) was delivered by pressure ejection, channel openings appeared rapidly with a delay of 28 ms. The unitary current was about approximately 0.80 pA at -74 mV. The channel activity induced by bath perfusion of 5-HT (0.8 microM) was significantly reduced by 100 nM of the 5-HT3 receptor-specific antagonists 3-tropanyl-3,5-dichlorobenzoate (MDL 72222) or 3-tropanyl-indole-3-carboxylate (ICS 205-930). 7. The single-channel current-voltage relation was non-linear, with moderate inward rectification similar to that of the whole-cell current. The chord conductance of the channel decreased with membrane depolarization from 14.6 pS at -104 mV to only 9.9 pS at -54 mV. Open-time distributions consisted of two components with mean time constants of 0.45 and 2.8 ms at -104 mV. Burst-length distributions were also made up of two components with time constants of 0.45 and 4.6 ms.(ABSTRACT TRUNCATED AT 400 WORDS)


Cyclic GMP-activated channels of salamander retinal rods: spatial distribution and variation of responsiveness.

1. Patch-clamp methods were used to investigate the areal density and spatial location of cyclic GMP-activated channels in the surface membrane of salamander rod outer segments. 2. The density of active channels (i.e. channels able to respond to cyclic GMP) in patches excised from outer segments was determined from the number of active channels, N, and the membrane area, A. N was estimated from the current induced by a saturating concentration of cyclic GMP, while A was estimated from the electrical capacitance of the patch. 3. In patches excised from forty-one isolated outer segments prepared in the light the active channel density varied over a remarkable range: 0.34-629 microns-2, with a mean of 166 microns-2. Density was not correlated with patch area in this or any of the conditions studied. 4. The spatial distribution of open channels on the outer segment of a transducing rod was measured by recording the local dark current at various positions with a loose-patch electrode. The apparent density of open channels varied by only about +/- 50% around the circumference of the outer segment and up and down its length. This indicates that the wide range of densities in excised patches did not result from sampling a non-uniform spatial distribution of channels. 5. Patches excised from sixteen dark-adapted whole cells with healthy appearances and saturating light responses of normal size had active channel densities of 1.1-200 microns-2, with a mean of 60 microns-2. Patches from twenty light-adapted whole cells had similar densities. Many densities from the whole cells were much lower than expected. This, and the wide variation in densities, suggests that obtaining a patch often lowered the density of active channels. The number of channels in a patch was quite stable from 1 s to 30 min after excision, ruling out progressive denaturation or adsorption of channels to the glass as a cause for this effect. 6. The mean active channel density in patches excised from whole cells was lower with calcium present in the external solution than with calcium absent (80 vs. 152 microns-2, n = 36 and 30 respectively). 7. We conclude that copies of the channel protein were present at a density of at least 650 microns-2 in the surface membrane of the outer segment and that the distribution of channels was fairly uniform on a 1 micron scale.(ABSTRACT TRUNCATED AT 400 WORDS)


Arachidonic acid and prostaglandin E2 activate small-conductance Cl- channels in the basolateral membrane of rabbit parietal cells.

1. Cl- channels in the basolateral membrane of non-stimulated parietal cells in isolated rabbit gastric glands were studied by patch-clamp and noise analysis techniques. 2. Voltage-independent whole-cell currents were recorded from parietal cells equilibrated with Cl(-)-containing solutions. Upon reducing the Cl- concentration of the basolateral bathing solution, the current-voltage curve was rapidly shifted to the right. The reversal potential changed according to changes in the equilibrium potential for Cl-. 3. A Cl- channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) inhibited whole-cell Cl- currents. The half-maximal inhibitory concentration of NPPB was 300 microM, and the inhibition was reversible (at less than or equal to 200 microM). Another Cl- channel blocker, 4-acetamido-4'-isothiocyanatostilbene-2,2'-dilsulphonic acid or diphenylamine-2-carboxylate was much less effective. 4. The power spectra of whole-cell Cl- current fluctuations contained one Lorentzian component. The single Cl- channel conductance was estimated to be 0.35 pS by the variance noise analysis, which is in agreement with the value obtained by a method of power spectrum analysis (0.29 pS). 5. The addition of arachidonic acid (10 microM) to the basolateral medium markedly increased the whole-cell Cl- current. The combined application of a cyclo-oxygenase inhibitor, indomethacin (50 microM), and a lipoxygenase inhibitor, esculetin (100 microM), increased the Cl- current, whereas the administration of a phospholipase A2 inhibitor, mepacrine (100 microM), significantly decreased the whole-cell Cl- current. 6. A sizeable increase in the whole-cell Cl- current was also induced by a metabolite of arachidonic acid, prostaglandin E2 (10 microM), but not by leukotriene B4 (5 microM) or D4 (10 microM). 7. The present study has shown that small-conductance Cl- channels are present in the basolateral membrane of rabbit parietal cells, and that the channel was functionally regulated by arachidonic acid and prostaglandin E2.


Effects of alcohols on responses evoked by inositol trisphosphate in Xenopus oocytes.

1. The effects of ethanol and other alcohols on inositol 1,4,5-trisphosphate (InsP3) signalling were studied in Xenopus oocytes by the use of flash photolysis of caged InsP3. Calcium liberation induced by InsP3 was monitored by voltage-clamp recording of Ca(2+)-activated membrane currents, and by fluorescence of the Ca2+ indicator Fluo-3. 2. Membrane current and fluorescence Ca2+ signals evoked by light flashes giving small responses were initially potentiated by bath application of ethanol (80-400 mM). However, the responses subsequently declined while ethanol was present and were strongly reduced or suppressed when it was removed. 3. These effects did not arise artifactually from changes in photolysis of caged InsP3, as similar results were seen with responses evoked by intracellular injections of InsP3. Also, the effects on the membrane current did not arise primarily through actions on the Ca(2+)-dependent Cl- channels, since currents evoked by intracellular injections of Ca2+ were little changed by ethanol. 4. Ethanol reduced the threshold level of InsP3 required to cause Ca2+ liberation. Thus, potentiation was most prominent with small responses evoked by brief light flashes, whereas the predominant effect on larger responses was inhibitory. 5. The facilitatory and inhibitory actions of ethanol persisted after removing extracellular Ca2+. 6. Intracellular injections of ethanol produced an initial inhibition of InsP3 responses, followed, in some oocytes, by a potentiation. 7. Methanol had little effect on InsP3 responses, whereas butanol and other long-chain alcohols produced strong inhibition, but little or no potentiation. 8. We conclude that extracellular application of ethanol produces a rapid potentiation of InsP3-mediated Ca2+ liberation, and a more slowly developing inhibition. The potentiation may arise through stimulation of InsP3 formation at the plasma membrane, whereas the inhibition occurs more deeply in the cell. Both actions were evident at relatively low concentrations (a few tens of millimoles per litre), and might thus be important in the behavioural effects of ethanol intoxication.