Activation of protein kinase C inhibits 45Ca-accumulation in cultures of osteoblast-like cells: possible involvement of insulin-like growth factor-I.

In a previous report, we have demonstrated that exogenous insulin-like growth factor-I (IGF-I) stimulates 45Ca-accumulation into extracellular matrix in long-term cultures of osteoblast-like MC3T3-E1 cells and that 45Ca-accumulation occurs even in the cultures without exogenous IGF-I. In this study, effects of protein kinase C (PKC) on IGF-I secretion and 45Ca-accumulation into extracellular matrix were examined in 6-week cultured MC3T3-E1 cells. The MC3T3-E1 cells secreted IGF-I spontaneously. The PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA) suppressed IGF-I secretion in a dose-dependent manner. 4 alpha-Phorbol 12,13-didecanoate (4 alpha-PDD), which is inactive for PKC, had little effect on the secretion. 1-Oleoyl-2-acetylglycerol, a specific activator for PKC, also suppressed the IGF-I secretion dose dependently. H-7, a PKC inhibitor, recovered the inhibitory effect of TPA. On the other hand, TPA inhibited the 45Ca-accumulation into extracellular matrix in cultures of these cells dose dependently, whereas 4 alpha-PDD was ineffective in this capacity. The TPA-induced inhibition of 45Ca-accumulation was recovered almost to the control level by H-7. Exogenous IGF-I recovered the inhibitory effect of TPA on 45Ca-accumulation. In spite of the inhibitory effects of TPA as above, TPA had little effect on DNA synthesis in these cells. These results suggest that the activation of PKC inhibits calcification via suppression of IGF-I secretion in osteoblast-like cells.


Perspectives: bone's mechanical usage windows.

As mechanical usage (MU) of a bone changes from complete disuse towards maximal vigor, the biologic mechanisms that can adapt it to its MU tend to react predictably. Acute disuse can increase BMU (Basic Multicellular Unit, the remodeling 'packet') creations but reduces how much bone they form, to increase bone loss next to marrow. Normal usage reduces those creations to normal and tends to equalize their resorption and formation; this conserves bone. In mild overloading, BMUs still conserve existing bone, while modeling drifts can begin adding to and/or reshaping it. Severe overloading can increase microdamage alarmingly, its repair by BMUs too, and can cause woven bone formation, anarchic resorption and a regional acceleratory phenomenon. Those ranges of MU vigor can define four 'windows'. An adapted window should apply to healthy, normally active adult mammals, and a mild overload window to healthy, normally active growing ammals. The biologic responses in the pathologic window could explain among other things some total joint and internal fixation failures, some pathologic fractures and some bone healing and sports medicine problems.


Which is more osteoporosis-inducing, menopause or oophorectomy?

The present study was designed to investigate in a comparative manner whether menopause or oophorectomy (OPX) would be a more osteoporosis-inducing factor with regard to sex steroids, bone metabolism and bone mineral density (BMD), including postmenopausal subjects, OPX subjects and age- and body-size-matched premenopausal controls. Serum levels of estradiol (E2), testosterone (TS) and dehydroepiandrosterone (DHEA) were found to decrease in both the postmenopausal and OPX subjects without any significant difference between them, while serum levels of estrone (E1) and androstenedione (delta 4), which have been reported to be related to bone metabolism, were significantly lower in the OPX subjects than in the postmenopausal subjects. According to the indices representing bone formation and bone resorption, as well as the changes in serum levels of Ca-regulating hormones, bone metabolic balance seemed to be slightly more negative in the former than in the latter. However, there was no difference between these two groups of subjects with regard to BMD in lumbar vertebral spongy portion which sensitively reflects the changes in total lumbar BMD and bone metabolism and in which compressive fracture is apt to occur. This fact suggests that these two groups of subjects may be managed in the same way in clinical practice. In other terms, the menopause, a natural event in women, influences BMD as much as OPX, which is the greatest risk factor in osteoporosis, does.


Immunolocalization of alkaline phosphatase in osteoblasts and matrix vesicles of human fetal bone.

A monoclonal antibody raised against alkaline phosphatase (ALP) of human osteosarcoma was used to localize this enzyme in human fetal bone tissue. For light microscopy, the presence of alkaline phosphatase in osteoblasts and osteocytes was demonstrated by use of an avidin-biotin immunoperoxidase procedure. Electron microscopic immunolocalization was accomplished with an indirect immunoperoxidase method which revealed a concentration of the enzyme on matrix vesicle and osteoblast plasma membranes. In addition, many vesicular protrusions arising from areas of plasma membrane on the lateral surfaces of adjacent osteoblasts were strongly immunolabeled. Immunostaining for ALP was absent in vesicles which contained fine crystallites. Alkaline-phosphatase-rich matrix vesicles may play a significant role in the mineralization of the extracellular matrix.


Do co-translated product(s) of lactase-phlorizin hydrolase accumulate in the rat intestine?

The 225 kDa precursor of intestinal lactase-phlorizin hydrolase (LPH) consists of four tandemly-organized homologous domains flanked by a signal peptide at the N-end and by a transmembrane anchor at the C-end of the polypeptide chain. While the mature LPH of 130 kDa has already been shown to originate from the C-half of the precursor, no protein deriving from the N-half has been identified so far. Using monospecific antibodies raised against the mature LPH or against a recombinant protein containing the sequence of the N-end of the LPH precursor, we have searched for co-translated protein(s) of LPH in enterocytes and in the intestinal lumen of suckling rats. Since no additional protein to LPH was revealed by these antibodies, it is suggested that the polypeptide chain corresponding to the N-half of the LPH precursor undergoes rapid turnover.


Adaptational increase of liver glutathione content during long-term application of cyclosporine A may attenuate toxic side effects.

The concentrations of reduced and oxidized glutathione and of adenine nucleotides were determined in liver, kidney and heart of rats during long-term (four weeks), high-dose therapy with cyclosporine A. In liver and kidney the concentration of oxidized glutathione increased following 4 weeks-therapy suggesting increased formation of free radicals and accelerated lipid peroxidation processes. These processes may be due to an increased activity of the cytochrome P-450 system. Compensatory levels of reduced glutathione were also increased. The adaptational increase of the tissue level of reduced glutathione, presumably the response to a chronic oxidative stress, was more distinct in the liver. The liver did not lose adenine nucleotides. In contrast the kidney, after 4 weeks of cyclosporine A therapy, lost 25% of the adenine nucleotides. These findings suggest that the liver is characterized by a greater potential for effective adaptation to oxidative stress conditions compared to the kidney. These adaptations may prevent distortions of energy and nucleotide metabolism in the liver which is in agreement with the minor ultrastructural changes we have observed.


Preconditioning of heart by repeated stunning. Adaptive modification of antioxidative defense system.

Previous studies demonstrated that preconditioning of a heart by repeated stunning can reduce the cellular injury to the heart from subsequent acute ischemic insult. To examine the possible biochemical mechanism for such myocardial preservation afforded by preconditioning, swine heart was subjected to four episodes of 5 min. stunning by occluding the left anterior descending coronary artery (LAD), followed by 10 min. of reperfusion after each stunning. Heart was then made regionally ischemic for 60 min. by LAD occlusion, followed by 6 hrs. reperfusion. Control heart was perfused for 60 min., followed by 60 min. ischemia and 6 hrs. reperfusion. The results of our studies indicated the stimulation of a number of antioxidative enzymes, including Mn-superoxide dismutase (Mn-SOD), catalase, glutathione peroxidase, and glutathione reductase, after repeated stunning and reperfusion. In addition, a number of new proteins were expressed after preconditioning the heart, including some oxidative-stress related proteins and 72 kDa heat-shock protein. These results suggest that preconditioning of a heart by repeated stunning may lead to strengthening of the oxidative defense system of the heart, which is likely to play a role in myocardial preservation during subsequent ischemic and reperfusion injury.


Light and electron microscopic radioautography of DNA synthesis in the endometria of pregnant-ovariectomized mice during activation of implantation window.

Pregnant mice were ovariectomized at pre-implantation stage and exogenous nidatory estradiol was administered to evaluate the DNA synthesis of the endometrial cells during activation of uterine receptivity for blastocyst implantation. After 0, 3, 6, 12 and 18 hrs. of estradiol treatment, the animals received 3H-thymidine injection, sacrificed 1 hr. later, and the uteri were prepared for light and electron microscopic radioautography. At time 0, no labelled stromal or epithelial cells was found in the endometrium. According to the time-lapse after estradiol induction, a gradual increase of labelled stromal and endothelial cells was seen in the endometrium. The highest labeling index was observed at the antimesometrial side of the implantation sites and the lowest value was found at the interimplantation site. The cells found at mesometrial side of the implantation site showed an intermediate labeling index. Eighteen hrs. after estradiol treatment, the labelled stromal cells found near the implantation chamber resembled the morphology of decidual cells while those labelled cells localized at the interimplantation sites were similar to the fibroblast. The uterine luminal epithelial cells showed low DNA synthesis after estradiol treatment resulting in only a few labelled cells at the interimplantation sites and no labelled cells at the implantation sites. A similar labeling pattern was seen in the glandular epithelium. The distribution of labelled cells seen among the regions of pregnant endometrium under estradiol effect suggest that DNA synthesis related to uterine activation for blastocyst implantation is a focal reaction, where the luminal epithelium does nt proliferate while the stromal and endothelial cells around the conceptus increase the DNA synthesis to prepare the endometrial decidualization.


Hemostatic activity of the bovine parotid gland glycoconjugates.

The bovine parotid gland was studied by means of biochemical analyses and the glycoconjugates extracted were used to investigate the activity on the human hemostatic system. Thromboelastography was unable to reveal anticoagulant properties. Conversely, the Thrombin Time (TT) was prolonged in a statistically significant way and with dose-coupling response. Reptilase Time (RT) was affected by the highest concentration of extract suggesting that the bovine parotid glycoconjugates alter the fibrinogen polymerization.


Purification of the Atlantic salmon hepatic 21 kDa protein and classification as a high mobility group chromatin protein.

The 21 kDa protein of liver from Atlantic salmon (Salmo salar) has been purified. Hepatic nuclei were extracted with 0.75 M HClO4. The extracted proteins were fractionated using reversed phase high performance liquid chromatography. The purity of the protein was analysed by isoelectric focusing in the first, and SDS-polyacrylamide gel electrophoresis in the 2nd dimension. Isoelectric focusing separated the protein into 5 spots. In gel trypsin digestion after isoelectric focusing followed by SDS-polyacrylamide gel electrophoresis resulted in identical migration of the tryptic peptides. The amino acid composition of the 21 kDa protein was similar to that of high mobility group (HMG) proteins C and D from rainbow trout (Oncorhynchus mykiss). The N-terminal sequence of the amino acids 1-19 revealed a conserved region characteristic for HMG 14/17 proteins of mammals and avians, and their equivalents in rainbow trout. Considering the electrophoretic mobility, amino acid composition and N-terminal amino acid sequence it is concluded that the 21 kDa protein of Atlantic salmon is a member of the HMG protein family resembling the HMG D protein of rainbow trout.


Modification of proto-oncogene expression by phorbol esters in human dermal microvascular endothelial cells.

Following activation with the inflammatory mediator phorbol myristate acetate (PMA), human microvascular endothelial cells (DMEC) is olated from the human dermis (DMEC) rapidly and dramatically convert from a classical epithelioid morphology to a spindle-shaped configuration. This is accompanied by changes in the organization of gap junctions and the vimentin and actin cytoskeletons. This report describes the sequential changes in the expression of four proto-oncogenes, c-fos, c-myc, c-sis and H-ras in DMEC following PMA exposure. The synthesis of c-fos mRNA was transiently induced by PMA from a basal concentration below the limit of detection to a maximum at 60 min., declining to the unstimulated level within 2 hrs. Synthesis of c-myc mRNA declined continuously and reached 37% of control levels over 16 hrs. Expression of c-sis which encodes for the B chain of platelet-derived growth factor, also declined to 34% of the control value over 16 hrs. There was no change in the synthesis of H-ras mRNA nor of beta-actin mRNA which was used as a control. The expression of c-myc in normal DMEC was compared to a human dermal microvascular cell line transformed by SV-40 (TREND). The TREND cell line maintains a permanent spindle-shaped configuration under all growth conditions and multiplies faster than DMEC. In contrast to the non-transformed cell cultures, expression of c-myc in TREND cells was induced by PMA.(ABSTRACT TRUNCATED AT 250 WORDS)


Cassia alata and the preclinical search for therapeutic agents for the treatment of opportunistic infections in AIDS patients.

In our search for therapeutic agents from natural sources with potential for the treatment of opportunistic infections in patients afflicted with acquired immunodeficiency syndrome (AIDS), we investigated antibacterial and antifungal activities of water extracts of Cassia alata (C. alata). The extracts are traditionally used in Ivory Coast, West Africa to treat bacterial infections caused by Escherichia coli (E. coli), and fungal infections caused by Candida albicans (C. albicans) and dermatophytes. Our working hypothesis was that the extract contains active ingredient(s) which can be isolated, identified and developed into useful antibacterial/antifungal agents for the treatment of opportunistic infections in patients with AIDS. We used the broth dilution and agar dilution methods. Specifically, we focused on E. coli and C. albicans and the effectiveness of the extracts was evaluated relative to those of standard antibacterial agent chloramphenicol and antifungal agent amphotericin B. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for the water extract of C. alata against E. coli were 1.6 mg/ml and 60 mg/ml, respectively; corresponding data for chloramphenicol were 2 micrograms/ml and 10 micrograms/ml. Similarly, the MIC and minimum fungicidal concentration (MFC) for the extract against C. albicans were 0.39 mg/ml and 60 mg/ml in contrast to 0.58 micrograms/ml and 0.98 micrograms/ml for amphotericin B. From the dose-response curve plots, the extract had an IC50 of 31 mg/ml for E. coli and 28 mg/ml for C. albicans. The data suggest that C. alata extracts contain agent(s) which have therapeutic potential and might be useful if isolated and developed for the treatment of opportunistic infections of AIDS patients.


Presence of single-chain ribosome-inactivating protein-like activities in several plant species.

The present screening work was devoted to the search for new ribosome-inactivating proteins (RIPs) in 21 plant species. Eight plants proved to be very active, inhibiting protein synthesis in eukaryotic in vitro systems (rat liver, Vicia sativa and wheat germ). They fulfil the major requirements for consideration as type 1 RIPs. Also, eight plants were found to contain haemagglutinating activity of human red cells but this was not related to the simultaneous presence of RIPs.


Microanalytical characterization and surface modification of TiNi orthodontic archwires.

Orthodontic archwires (equiatomic TiNi alloy) of both used (4 weeks) and unused conditions were microanalyzed by optical and scanning electron microscopes, energy dispersive X-ray spectroscopy, and electron diffraction to characterize the surface layers. They were also subjected to immersion and polarization corrosion tests in a 0.9% NaCl aqueous solution. Based on results obtained from these analytical and experimental studies, surfaces of TiNi archwires were further electrochemically treated to etch away nickel selectively and reform the surface morphology to uniform and porous surface layers. Main conclusions were: (a) surface layers of used archwires were covered contaminants causing the discoloration, and the contaminants were identified as mainly KCl crystals, (b) surfaces of both used and unused wires were observed to be irregular features characterized by lengthy island-like structures, where nickel was selectively dissolved, (c) corrosion tests in a 0.9% NaCl aqueous solution in immersion and polarization methods indicated that by increasing temperature from 3 degrees to 60 degrees C and acidity from pH 11 to pH 3, calculated corrosion rates increased, and (d) surface layers of TiNi archwires can be electrochemically modified to selectively etch nickel away, leaving a Ti-enriched surface layer and forming a uniformly distributed porous surface that may reduce the coefficient of friction against the orthodontic brackets.


A study on elongation and knot slacking of various sutures.

The tension holding capacity of suture materials was measured by in vitro and in vivo experiments. Silk and expanded polytetrafluoroethylene sutures showed large elongation and significant knot slacking after tight tying. Although ultrahigh molecular-weight polyethylene suture had excellent creep resistance, it showed a relatively low tension holding capacity because of substantial knot slacking. On the other hand, polyester and high-strength poly(vinyl alcohol) sutures showed an excellent in vivo tension holding capacity, along with low elongation and insignificant knot slacking. Clinical performance of these sutures was briefly discussed from the mechanical point of view.


Ultrasonic velocity and attenuation measurement in kidney stones: correlation to constituents and hardness.

In this investigation, ultrasonic parameters of kidney stones are measured, in vitro, using a double probe ultrasonic-through-transmission technique. Due to the complex chemical structure of these stones, a large variation in the values of the ultrasonic parameters is found. A correlation between ultrasonic properties and variation in pathology of the renal calculi is also discussed.


A new, non-toxic, curing agent for synthetic polyolefins.

The purpose of this study was to investigate benzocyclobutene (BCB) derivatives as non-toxic curing agents to replace accelerated sulfur systems in certain synthetic biomedical materials. Prototype artificial spinal discs have been made and implanted in a few patients from a carbon black-filled copolymer of 1-hexene and 5-methyl-1,4-hexadiene (MHD), which is sulfur cured. The polymers investigated in this study were composed of 1-hexene, allyl-BCB, and MHD (or 7-methyl-1,6-octadiene, MOD). Curing was effected through reactions on BCB, forming carbon-carbon crosslinks. Preliminary cytotoxicity tests showed the material to be non-toxic, and the physical and mechanical properties of the BCB-cured materials were comparable with those of the sulfur-cured polymers.


A basic study on removal of nutrient salts in wastewater using plants (removal by mung beans; Phaseolus radiatus L).

Many studies have reported on the removal of pollutants from wastewater using aquatic plants. The water hyacinth has been the most widely used and its system is the most well established. This system however, has a few problems in practical use. The purpose of this study is to obtain basic information on a new system that can substitute for a conventional system or be used as a secondary system to assist the conventional one. We first envisioned a model of this new system and then conducted a preliminary experiment using a small experimental unit to simulate the new system. The experiment showed that mung beans were a suitable plant for our study. Their removal rate of pollutants was the highest before they developed leaves and started photosynthesis. We found that nutrients were expelled outside the plant root when nutrient concentration inside the plant tissue became too high.


Trisomy 13 in acute leukemia.

Trisomy 13 occurring as a sole cytogenetic abnormality has recently been demonstrated to have adverse prognostic significance in acute leukemia. Trisomy 13 is seen primarily in an older male population, and has been reported in treatment-associated acute leukemia and acute leukemia evolved from myelodysplastic syndromes, as well as in de novo leukemia. The 36 cases of acute leukemia with trisomy 13 reported to date include 26 AML, 6 AUL, 2 ALL and 2 mixed lineage patients. Immunophenotyping studies have demonstrated an undifferentiated phenotype or biphenotypic markers in most cases. Trisomy 13 is associated with a low complete remission rate and with brief remission duration. The role of the additional copy of chromosome 13 in the pathogenesis of these cases of acute leukemia and the gene(s) of importance on chromosome 13 are yet to be determined.


Serum soluble interleukin-2 receptor levels in hairy cell leukemia: correlation with clinical and hematological parameters and with alpha-interferon treatment.

The soluble Interleukin-2 Receptor (sIL-2R) serum levels were assessed in 42 patients with Hairy-Cell Leukemia (HCL) at diagnosis and after alpha-Interferon therapy and correlated with spleen size, peripheral hematological values, hairy cell index (HCI) and clinical response. Serum sIL-2R levels were significantly increased in all HCL patients, particularly in those with a higher HCI (> 0.50) and in non-splenectomized patients. Among the 26 HCL patients who were studied before and after 12 months of alpha-IFN treatment, 16 normalized the sIL-2R level and 10 did not. Our findings suggest that sIL-2R levels in HCL patients correlate with the splenic and bone marrow tumor burden as assessed by HCI. In addition patients with low levels of sIL-2R at diagnosis appear to have a better chance of achieving a good clinical response.


Autoantibodies in the sera of patients with lymphoma.

Sera of 84 patients with Hodgkin's disease (HD) and 55 patients with non-Hodgkin's lymphoma (NHL) were examined for the presence of autoantibodies to ssDNA, dsDNA, Poly (I), Poly (G), cardiolipin, histones, RNP. Sm, Ro (SS/A), La (SS/B) and the common anti-DNA idiotype (16/6) using an enzyme-linked immunosorbent assay (ELISA). Anti-ssDNA antibodies were detected in the sera of 20 patients with lymphoma (23.8%), more among those with NHL than HD (16 vs. 4 patients p < 0.01). Anti-RNP and anti-Sm antibodies were found in 16 (21.7%) and 14 lymphoma patients (20%) respectively, significantly more than in the controls (p < 0.05) in both antibodies). These findings remained valid following subgrouping of the patients into those with HD and NHL. With all the other autoantibodies examined no significant difference could be observed in the incidence between lymphoma patients and controls. These results differ from our previous survey carried out on sera of patients with solid tumors in whom no increased frequency of any of the autoantibodies could be determined. In view of the evidence suggesting an increased risk of lymphoma in a number of autoimmune diseases our results extend this relationship to an increased incidence of autoantibodies among patients with lymphoma.


Interleukin-6 levels in the plasma of patients with lymphoma.

We have suggested a growth stimulatory role of lymphoma cells in culture by interleukin 6. To test the hypothesis that IL-6 may have a role in vivo, the plasma of lymphoma patients was assayed for the presence of IL-6 bioactivity. A significantly greater proportion of lymphoma patients had elevated IL-6 levels (23 of 40) compared to controls (3 of 35). In follow-up, eight of 15 patients with previously elevated levels returned with decreased or undetectable levels of IL-6 post radio- or chemotherapy. This response was seen primarily in those with intermediate or high grade lymphomas. In contrast no change in IL-6 levels was seen in patients with low grade lymphoma despite measurable reductions in tumor size in both groups.


Role of splenectomy in chronic lymphocytic leukaemia with massive splenomegaly and cytopenia.

Fourteen cases of chronic lymphocytic leukaemia (CLL) and massive splenomegaly associated with anaemia and/or thrombocytopenia (stage C) with or without mechanical distress underwent splenectomy. Eleven regressed to stage A and 2 to stage B. All 6 patients with mechanical distress were relieved of their symptoms. Among 13 patients with anaemia, haemoglobin (Hb) level was normalised in 3, improved in 8 and showed no change in 2 cases. Among 13 patients with thrombocytopenia, platelet count was normalised in 10, improved in 1 and was unchanged in 2 cases. Mean Hb level increased from 89 g/l to 125 g/l and mean platelet count from 77 x 10(9)/l to 241 x 10(9)/l (p < 0.001 for both). Duration of response lasted for a mean period of 42.1 (CI 20.8-63.5) months. Mean survival after splenectomy was 44.5 months (CI 21.2-67.7). There was no operative mortality but 4 patients had significant post-operative morbidity. It is concluded that splenectomy is beneficial in advanced cases of CLL with massive splenomegaly and cytopenia.


Beta 2 microglobulin level as an indicator of prognosis in diffuse large cell lymphoma.

We report results of our investigation of prognostic factors for patients with diffuse large cell lymphoma (DLCL) who were entered on the same treatment protocol and who had known pretreatment serum beta 2 microglobulin levels. Serum beta 2 microglobulin, bone marrow involvement, performance status and lactic dehydrogenase (LDH) levels were associated with a poor prognosis in univariate analysis. However, only beta 2 microglobulin remained of prognostic significance in a multivariate analysis with statistical differences at different cut off levels. We believe that beta 2 microglobulin levels accurately separate patients into low-, intermediate- and high-risk patients. It is concluded that serum beta 2 microglobulin is the most significant prognostic factor currently available for DLCL and should be incorporated in the initial staging in order to provide a basis for designing the therapeutic approach in these cases.


Incidence of thrombocytosis in lymphomas.

Reactive thrombocytosis due to malignancies and in particular those related to lymphomas have not yet been extensively evaluated. We report data on thrombocytosis recognized in 18 out of 101 patients with lymphomas diagnosed in our department over the last 3 years. All showed high platelet counts at the time of diagnosis. The incidence of thrombocytosis seems to be more frequent in males (21.2%) than in females (14.8%) and a slightly higher frequency was found in Hodgkin's disease (21.4%) than in non Hodgkin's lymphomas (16.4%). The incidence of thrombocytosis in lymphomas seems to be similar to that seen in other malignancies and because of this we conclude that a high platelet count cannot be used to distinguish malignancies.


Role of interleukin-2 activated MHC-nonrestricted lymphocytes in antileukemia activity and therapy.

Although the application of biological therapy for solid tumors with cytokines and adoptively transferred activated lymphocytes has received substantial attention, this approach has not been actively explored in treatment of hematopoietic neoplasms. This review will address the possibilities of interleukin-2 (IL-2) and IL-2 activated natural killer (NK) cells and T cells in antileukemia reactivity and therapy. The new approaches to optimal activation and generation of oncolytic cells, selective propagation of lymphocyte subsets, and the role of adhesion molecules in antileukemia cytotoxicity will also be addressed. We trust that this article will be conducive to the development of new directions in leukemia research and treatment.


Glycosylated serum ferritin in patients with hematological malignancies before and after bone marrow transplantation.

Glycosylated and total serum ferritin levels were monitored in patients with acute leukemia and lymphoma undergoing bone marrow transplantation (BMT). Serum ferritin was high in relapsing patients and normal in most patients in complete remission (CR). In patients with an uncomplicated course, levels of ferritin increased during the first month after BMT with subsequent decrease. Three patients with lymphoma and five with acute leukemia had high serum ferritin levels despite achieving apparent complete hematological remission which was of short duration. The results were compared with groups of lymphoma patients at presentation and during remission and with healthy normal controls. In all the lymphoma patients and in 3 of the 5 leukemia patients the percent of ferritin glycosylation was normal at CR. It was low at the time of diagnosis in all patients. Thus, the percent glycosylation proved a more reliable marker for clinical remission than total serum ferritin. During follow up after BMT in uncomplicated cases, the percent of glycosylated ferritin returned to normal levels earlier than the total serum ferritin. These findings indicate that the evaluation of the amount of glycosylated ferritin may provide useful information in hematological patients in whom there is a discrepancy between high serum ferritin levels and the clinical condition.


Vincristine-resistant human leukemia cell line: new monoclonal antibodies to a 65kDa membrane protein.

A vincristine-resistant human myelomonocytic leukemic cell line (KY-VCR) was established. KY-VCR exhibited approximately a 2.5 x 10(6)-fold increase in resistance to vincristine compared to the parental cell line. KY-VCR showed a decreased uptake and, an increased efflux of vincristine, and cross-resistance to Adriamycin and Actinomycin D. The M(r) 200,000 membrane glycoprotein was overexpressed in KY-VCR. Furthermore, two antibodies, designated TO73 and TO77, preferentially reacting with KY-VCR were obtained. Enzyme linked immunosorbent study indicated that both antibodies recognized the same epitope and TO77 the wide portion. Immunoprecipitation analysis demonstrated that the antibodies recognized M(r) 65,000 membrane protein, which was distinct from overexpressed glycoprotein in KY-VCR. The induction of membrane protein identified by the antibodies may play a role in drug resistance. KY-VCR cells and two antibodies to them may be very useful for the study of drug resistance and prediction of drug efficacy.


AgNORs predictive value of prognosis in non-Hodgkin's lymphoma: comparison with flow cytometric cell cycle analysis.

Paraffin-embedded histopathologic specimens, taken before the commencement of therapy from 14 low-grade and 21 high-grade malignant lymphoma patients, and 9 normal lymph nodes were utilized to analyze six cell DNA-related parameters. The flow cytometry technique was used to determine cell-cycle G0/G1, S and G2/M phases, and silver staining to enumerate nuclear organized regions (AgNORs); nucleus surface area was determined by an image-analyzing system. The six parameters and natural logarithm of cell proportion in the S-phase (LS) were determined according to the histologic tumor type and achievement of the first complete remission (CR). All parameters except cell proportion in G1/M cycle phase differed significantly with respect to histologic cell type, but were not related to the achievement of first CR. Inasmuch as the parameters significantly correlated with each other, multivariate discriminant analysis and proportional hazard regression were applied to estimate their discriminant/predictive values with respect to tumor malignancy. AgNORs proved to be far superior in all three clinical parameters, S-phase was significantly predictive for the achievement of first CR, and LS for tumor histology type. The statistical model applied narrowed down the analysis of seven parameters to two with respect to tumor histology type (AgNORs and LS) and achievement of first CR (AgNORs and S), but only to one for overall patient survival (AgNORs). Only the model for tumor histology type discrimination was statistically significant (R2 = 0.904, p < 0.001). It appears that AgNORs may be of utmost predictive importance for the clinical outcome in NHL.


Mastocytosis and co-existent non-Hodgkin's lymphoma and myeloproliferative disorders.

Generalised mastocytosis is a rare condition characterised by the clinical features of the release of vasoactive peptides from tissue mast cells infiltrating in the reticuloendothelial tissues. The mast cell however appears to have its origin in the pluripotential bone marrow stem cell committed to a basophil and it is therefore not surprising that myeloproliferative and myelodysplastic disorders commonly co-exist or terminate the clinical phase of mastocytosis. Both abnormal proliferation and maturation of the myeloid committed cells are found. Non-Hodgkin's lymphoma can occur before and after mastocytosis becomes manifest. While this is statistically a random event the relationship between lymphokines and mast cell differentiation and proliferation raises the possibility of a benign reactive lymphoid event eventually becoming malignant.


HTLV-I induced extranodal lymphomas.

HTLV-I induced not only nodal but also primary extranodal lymphomas. In this report we describe 12 patients with HTLV-I induced extranodal T-cell lymphoma collected from the literature and our institute experience. There were 5 males and 7 female patients of middle age positive for HTLV-I antibody. The sites of primary tumor were gastrointestinal, Waldeyer's ring, skin, facial sinuses, and the pleura. All of these were histologically diffuse lymphomas and most of them were found to be a helper/inducer T-cell phenotype, showing integration of HTLV-I proviral DNA. Late leukemic changes and skin infiltration often occurred, but hypercalcemia was rare. Survival time varied from 4 to 35 months, and late organ infiltrations were common. These HTLV-I induced extranodal lymphomas were compared with HTLV-I unrelated extranodal lymphomas or HTLV-I induced nodal lymphomas (lymphoma type ATL). Between 1981 and 1990, we had 110 ATL patients and of these, 5 (4.6%) were HTLV-I induced primary extranodal lymphomas. The frequency of HTLV-I induced extranodal lymphoma might be much higher than expected because until now attention has not been paid to this entity. From the present review, it is suggested that HTLV-I could cause primary extranodal lymphoma which may have some different characteristics from other types of lymphoma. Therefore, patients with T-cell extranodal lymphomas should be investigated further for the presence of HTLV-I antibody and the tumor cells should be examined for the integration of HTLV-I proviral DNA using Southern blot analysis.(ABSTRACT TRUNCATED AT 250 WORDS)


Attempted prevention of blast crisis in chronic myeloid leukemia by the use of pulsed doses of cytarabine and lomustine. A Cancer and Leukemia Group B study.

An attempt to prevent the blast crisis in chronic myeloid leukemia by the use of pulsed doses of (cytarabine cytosine arabinoside) and lomustine was attempted as a cooperative group study by Cancer and Leukemia Group B. The basis for this study was to delay the development of blast crisis by pulsing dose of drugs known to be effective against emerging "blast" cells. The experimental arm which consisted of cytarabine and lomustine did not produce overall results superior to conventional treatment with busulfan. This was related to the non-hematologic effects of the combination which produced significant gastrointestinal toxicity leading to relatively early discontinuation of the combination. Nevertheless, the trial design allowed relatively prompt discontinuation of experimental arm and cross-over to conventional treatment with either hydrea or busulfan. No evidence existed that the use of new drug combinations in CML prejudiced the patient's chance to response to conventional chemotherapy. Thus, a role model for future trials in this disease was developed. With the development of the interferons and other experimental forms of therapy this conceptual development may be of significance.


alpha-Interferon inhibits spontaneous and induced DNA synthesis in hairy cell leukemia.

The effect of alpha-interferon (alpha-IFN) on spontaneous or induced proliferation of isolated peripheral blood mononuclear cells from 5 hairy cell leukemia patients was studied. alpha-IFN inhibited the low spontaneous proliferation of B-ly7 positive hairy cells (HCs) and also the proliferation induced by tumour necrosis factor (TNF). Interleukin-2 did not affect HCs, but induced CD4 positive T cells to proliferate, an effect which alpha-IFN antagonized. The stimulatory effect of TNF on the growth of HCs proved to be reversible and was partially blocked with either anti-TNF receptor or anti-lymphotoxin antibodies. Cellular or secreted thymidine kinase levels reflected the proliferative state of HCs in response to different in vitro treatments, as confirmed by thymidine incorporation and cell cycle studies.


Bone marrow transplantation for acute non-lymphoblastic leukemia.

Most results obtained by different study and analytic designs favor that matched allogeneic BMT is superior to chemotherapy in young adults with ANLL in first remission. The place of ABMT is more difficult to assess and requires further study both compared to chemotherapy and allogeneic BMT. Furthermore, the question of purging needs further study in a controlled fashion. For older patients the choice is more difficult. Transplant related mortality increases with age which makes ABMT an attractive alternative to allogenic BMT. However, recent advances in prophylaxis and treatment of transplant related complications such as cytomegalovirus interstitial pneumonia and veno-occlusive disease of the liver might increase long-term survival after allogeneic BMT in older patients. The role of matched unrelated donors in the treatment of ANLL is unresolved but this procedure should probably be reserved for relatively young patients in second complete remission or later.


A prospective study of the evolution of chronic lymphocytic leukaemia.

The clinical course of chronic lymphocytic leukaemia (CLL) in 126 patients has been examined over the period 1961-88. The evolution of early stages, 0-I, to late stages, III-IV, occurred frequently without an orderly progression through the intermediate stages. The tumour load as judged by lymphadenopathy and splenomegaly was similar in stages II, III and IV. It would seem that stages 0-II represent a continuous spectrum of increasing tumour load, whereas stages III and IV with tumour load of equal magnitude and suppression of haemopoiesis are likely results of a change in biological behaviour of malignant lymphocytes. Stage II(S) characterised by splenomegaly and absence of lymphadenopathy seems a distinct entity with a unique clinical course. Further evolution of CLL in these patients was characterised by progressive splenomegaly, and as this became marked the clinical course was dominated by 'hypersplenism'. Splenectomy at this late stage led to rapid relief of symptoms, progressive increase of Hb and platelets to normal levels and to a quiescent phase of CLL for a considerable period. The tumour load in stage II(S) was of the same order of magnitude as in stages II, III and IV, yet the survival was similar to that in stage 0. Survival correlated with clinical stage and age at the time of diagnosis, but showed no relation to sex. The significance of these findings is further discussed.


Recombinant alpha 2B interferon in combination with oral chemotherapy in late chronic phase chronic myeloid leukaemia.

The place of alpha interferon (IFN) therapy in the treatment of chronic myeloid leukaemia (CML) is under intensive investigation at present. It is now established that as a single agent it can provide good disease control in the chronic phase and that cytogenetic responses will occur in a minority of patients. However its impact on long term survival has been less certain. Optimal haematological and cytogenetic results have to date been seen when IFN is used in the early phase of the disease, i.e. within one year of diagnosis. We have performed a prospective single arm study on the effect on survival of the addition of low dose IFN (9 mU/week) to conventional oral chemotherapy in patients who were at a median of 19 months from the initial diagnosis at the time of study entry. Comparison of this cohort with a control group of CML patients treated with oral chemotherapy only at the same participating institutions gave an estimated 72% reduction in the risk of death as a result of IFN therapy. Median survival for the IFN group has not been reached at 43 months compared with a median survival of 33 months for the chemotherapy alone group. These results suggest that the introduction of low dose IFN at any stage in the chronic phase may produce a worthwhile improvement in survival.


A PCR-based assay for reporter gene expression.

Transient transfection is a widely used tool for the identification of cis-acting regulatory elements. These elements are detected by their effect on the expression of a reporter gene, which is quantified by measuring the reporter gene product in the form of mRNA, protein (hGH), or enzymes (CAT, luciferase). Measurements of mRNA levels have several advantages over enzyme or protein assays. However, mRNA quantification by RNase protection or S1 mapping has considerably lower signal-to-background ratio than protein assays and is therefore less sensitive. In this paper we report the development of a system that takes advantage of the polymerase chain reaction (PCR) to quantify rabbit beta-globin reporter gene expression. Cells are co-transfected with constructs whose activity is to be tested and a reference plasmid with a small deletion in the second exon of the beta-globin gene. We show that the ratio of the two amplified cDNA signals is a highly reliable measure of test gene expression. The sensitivity of this assay is at least 1000-fold higher than RNase protection.


Reliable and efficient direct sequencing of PCR-amplified double-stranded genomic DNA template.

Modified PCR amplification and direct sequencing procedures for the double-stranded genomic DNA template are described. Advantages of the approach we describe are: background artifact bands previously observed using high-molecular-weight DNA as a template were eliminated by this protocol; no gel purification or subcloning of the PCR-amplified double-stranded fragment was required prior to direct sequencing; and sequences of 300 nucleotides can be easily read even after a single loading. The successful use of the modified dideoxynucleotide chain-termination method for direct sequencing of both strands demonstrates the efficiency of this technique for removing sequencing artifacts and for producing reliable sequence data.


A method for specific amplification and PCR sequencing of individual members of multigene families: application to the study of steroid 21-hydroxylase deficiency.

Mutations at the human HLA-linked CYP21B locus are responsible for 21-hydroxylase deficiency, a recessively inherited disorder of steroidogenesis. The scope for PCR-based analysis of the CYP21B gene has been restricted by the very high sequence homology between CYP21B and a closely related pseudogene, CYP21A. Here we describe a novel PCR sequencing strategy that allows the independent amplification of the entire CYP21B coding sequence and the subsequent enzyme-mediated conversion of the PCR product to a single-stranded form for dideoxy sequencing. We have used this approach to characterize the 21-hydroxylase deficiency allele associated with HLA-B55, the most frequent HLA marker associated with a CYP21B point mutation in the British population, and also an HLA-B35 associated allele of Asian origin. Allele-specific oligonucleotide (ASO) hybridization analyses have confirmed the selective amplification of CYP21B genes and the identity of the pathological mutations. The method can be adapted to permit selective amplification and PCR sequencing of individual closely related members of other multigene families and small-copy-number repetitive DNA families.


Estrogen dependent regulation of estrogen receptor gene expression in normal mammary gland and its relationship to estrogenic sensitivity.

In the present study, we have examined the relationship between estradiol (E2)-dependent regulation of estrogen receptor (ER) gene expression in normal mammary glands and its relationship to progesterone receptor (PgR) gene expression using tissues from E2-sensitive and -insensitive states. Estradiol caused a time-dependent decrease in ER mRNA levels in E2-sensitive mammary glands reaching a maximum at approx 6 h, at which time the levels of PgR mRNA also reached a maximum. In contrast, in E2-insensitive mammary glands, there was no E2-dependent decrease in ER mRNA at all times tested. Experiments using dissociated cells revealed that although the epithelial cells of mammary glands from both E2-sensitive and -insensitive states contained ER mRNA, in the intact E2-sensitive mammary glands, it was the nonepithelial ER that was decreasing in response to E2. Since the epithelial cells of normal mammary glands are the primary target for E2-dependent PgR synthesis, our studies suggest that a positive correlation between E2-dependent PgR gene expression and E2-dependent downregulation of ER may simply be coincidental and may not bear any true biological relationship.


A modeling study of the alpha-subunit of human high-affinity receptor for immunoglobulin-E.

The extracellular portion of the alpha-subunit of human high-affinity receptor for immunoglobulin-E (IgE), which contains two immunoglobulin (Ig) domains, was modeled on the basis of sequence similarity with antibody domains of known three-dimensional structure. Each receptor domain contains 86 amino acid residues, and both domains were modeled as bilayer structures. In both domains, one layer is made up of three anti-parallel beta-strands and the other of four strands, with the two layers linked by a disulfide bridge. The two domains show significant sequence similarity with each other (22 identities) and with the homologous domains of the murine and rat high-affinity receptors for IgE and the Fc gamma receptors from various species. Two plausible modes of association of the domains were considered: In the first, the two domains were positioned end-to-end, with essentially only longitudinal interactions between them; in the second, the molecule is more bent, with more lateral interactions between the two domains. The models will be useful in the design of protein engineering studies of this and homologous receptors to delineate the site of interaction with ligand. Furthermore, they may lend themselves as possible probes in crystallographic analyses by molecular replacement techniques.


Overproduction of full-length and truncated human estrogen receptors in Escherichia coli.

The full-length human estrogen receptor (hER) as well as two overlapping peptides of hER were overproduced in Escherichia coli JM109 cells, using the inducible pIC vector system. The N-terminal receptor peptide contains the DNA-binding domain as well as the hinge region, whereas the C-terminal peptide contains the same hinge region and the hormone-binding domain. Typically, 1-6 mg of estrogen receptor (ER) peptides can be recovered from 1 L E. coli cell cultures. The majority of the overexpressed proteins are found in inclusion bodies, which allow the isolation of ER peptides in high yields and of 50-80% purity. Induction for short time periods at 10 microM inducer yielded up to 50% of the ER peptides in soluble form with full biological activity. Both the intact receptor and the C-terminal fragment specifically bound estrogens and antiestrogens, whereas ER peptides that contained the DNA-binding domain were retained on a DNA-agarose resin.


Polyclonal antibodies from rabbits and chickens against the estrogen receptor and related peptides. Use in the affinity isolation of estrogen receptors and the retrieval of chromatin fragments associating with estrogen receptors.

Polyclonal antibodies from chickens and rabbits have been prepared against polypeptides representing two regions of the human estrogen receptor (hER). The estrogen receptor (ER) peptides used as antigens were overproduced in Escherichia coli. When indicated, the antibodies were affinity purified using resins to which the antigens contained in bacterial inclusion bodies had been coupled in high yield to epoxy-activated agarose. The antibodies recognize denatured human, bovine, rat, and rabbit ER in immunoblotting experiments. Immuno-precipitation of native ER protein was readily accomplished using rabbit antisera and immobilized protein A. The chicken antibodies, available in larger quantities, were also useful for immunoisolation after coupling to agarose. With the use of these reagents, the selective retrieval of chromatin fragments from MCF-7 cells that interact with ER has been achieved.


Characterization of insulin-like growth factor receptors in human thyroid tissue.

We have characterized the binding of 125I-IGF-I and 125I-IGF-II to plasma membranes purified from human thyroid tissue. IGF binding was time- and temperature-dependent. At 4 degrees C, maximal specific binding of 125I-IGF-I was 17.3 +/- 2.5% and of 125I-IGF-II was 8.8 +/- 2.0% (mean +/- SD/60 micrograms membrane protein). 125I-IGF-I binding was inhibited completely by unlabeled IGF-I, IGF-II, insulin, and the type-I IGF receptor monoclonal antibody, alpha IR-3. 125I-IGF-II was inhibited completely by unlabeled IGF-II and nearly completely by IGF-I. 125I-IGF-II binding also was inhibited significantly by insulin, suggesting that much or all of the IGF-II was bound to the type-I IGF receptor. Scatchard analysis revealed a single class of binding sites with a Kd of 6.0 +/- 4.2 x 10(-10) M for IGF-I binding and 5.7 +/- 1.3 x 10(-10) M for IGF-II binding. IGF-I binding was inhibited by a variety of salts in a dose-dependent manner, calcium and magnesium salts being more effective than sodium or potassium salts. Affinity crosslinking of 125I-IGF-I and -II showed clear evidence only for type-I IGF receptors. Thus, a crude plasma membrane fraction of human thyroid tissue expresses predominantly type-I IGF receptors.


5-Hydroxytryptamine (5-HT) and SK&F 103829 contract canine lower esophageal sphincter smooth muscle by stimulating 5-HT2 receptors.

Addition of 5-HT or SK&F 103829 (2,3,4,5 tetrahydro-8[methyl-sulfonyl]-1 H-3-benzazepin-7-ol hydrobromide) contracts isolated strips of canine lower esophageal sphincter (LES) circular smooth muscle. 5-HT acts directly on the smooth muscle, since pretreatment with the neurotoxin TTX does not inhibit this contraction. Depletion of extracellular calcium or pretreatment with nifedipine inhibited the contraction to both 5-HT and SK&F 103829. Therefore, in this smooth muscle, the contraction produced by both 5-HT and SK&F 103829 requires extracellular calcium and is sensitive to inhibition by a voltage-dependent Ca2+ channel antagonist. In addition, with respect to 5-HT, SK&F 103829 appeared to act as a partial agonist. Receptor alkylation studies using phenoxybenzamine demonstrated no receptor reserve for the contractile response to 5-HT. Nonsurmountable antagonism of the contraction induced by 5-HT and SK&F 103289 was observed with several 5-HT2 antagonists, i.e., methysergide, ketanserin, cyproheptadine, and LY 53857. Using a method established for pseudoirreversible antagonism, the Ki values for these 5-HT2 receptor antagonists were estimated. Results suggested that both 5-HT and SK&F 103829 contract the canine LES by interacting at the same receptor site and that this receptor site has characteristics of the 5-HT2 receptor. Finally, neither bulbocapnine, domperidone, nor prazosin significantly alters the response to 5-HT or SK&F 103829. Thus, isolated strips of canine LES contain a contractile 5-HT2 receptor, and SK&F 103829 behaves as a partial agonist at this site.


Characterization of glomerular angiotensin II receptor subtypes.

Radioligand-receptor binding techniques identified two angiotensin II (ANG II) receptor subtypes in rat renal glomerular membranes. This characterization was made possible by employing two highly-specific nonpeptide ANG II antagonists: Losartan (DuP 753), which is specific to the AT1 subtype, and PD 123319, which is specific to the AT2 subtype. The majority of ANG II receptors in glomerular membranes corresponded to the AT1 subtype.


A predominant basic alpha-tubulin isoform present in prophase Xenopus oocyte decreases during meiotic maturation.

Xenopus oocytes are blocked in prophase of the first meiotic division. During the G2/M transition drastic changes occur both in the cytoskeletal organization and in the capacity of tubulin to polymerize. Posttranslational modification of tubulin isoforms might be one of the factors that control the dynamic properties of microtubules. We have therefore analysed, by two-dimensional polyacrylamide gel electrophoresis, the isotubulins purified from Xenopus oocytes, and we show that tubulin is resolved into at least four alpha-isoforms and four beta-isoforms. We have identified a basic alpha (alpha b)-tubulin isoform which is specific to prophase arrested oocyte and that progressively disappears during meiotic maturation; its decrease is initiated when the nuclear envelope breaks down and is controlled by the nucleus. Using 35S methionine labelled oocytes we demonstrate that the disappearance of the alpha b isotubulin results from both an arrest of its biosynthesis after maturation, and from posttranslational modification which induces a shift of this alpha-isoform to a more acidic pI. Moreover, in vitro experiments using 35S prelabelled tubulin purified from prophase oocytes show that metaphase extracts containing MPF activity are able to induce the acidification of the alpha b-isoform, suggesting that the observed posttranslational modification might be regulated by p34cdc2. However, the nature of this modification remains to be elucidated.


Spatial distribution of the Sm antigen in Drosophila early embryos.

Anti-Sm antibodies recognize the major small nuclear RNA-protein particles (snRNPs) involved in pre-mRNA processing. The spatial distribution of the snRNPs has been investigated in Drosophila embryos up to the cellularization stage (cycle 14), using the Y12 anti-Sm antibody. Our results show that: 1) all or most of the Sm antigen is localized in the cytoplasm of the syncytial blastoderm until the 12th cycle of division, in both the nuclear and cytoplasmic compartments at cycle 13, and then in the nuclei at cycle 14 and later. This relocalization takes place when zygotic transcriptional activation occurs; 2) at the subcellular level, the Sm antigen localizes in a speckled pattern and in foci-like structures within the nucleus of Drosophila blastoderm embryos; 3) strikingly, some nuclei of embryos at the 14th cycle appear to contain more snRNPs than others. The position of these nuclei differs from one embryo to another, and their distribution does not resemble any known developmental pattern of Drosophila embryogenesis. We propose that random differences in snRNP concentration may serve as an epigenetic signal for stochastic events occurring during development.


Attachment of Madin-Darby canine kidney cells to extracellular matrix: role of a laminin binding protein related to the 37/67 kDa laminin receptor in the development of plasma membrane polarization.

Madin-Darby canine kidney (MDCK) cells have been extensively used as a model for the study of epithelial polarization. The contacts between the cell and extra-cellular matrix (ECM) provide a signal for the polarization of apical membrane markers. In order to study the molecular basis of these contacts, MDCK cells extracts in Triton X-100 were affinity-purified on laminin, yielding polypeptides of 100-110 and 36 kDa, but only the second one could be enzymatically iodinated from the cell surface. This protein was also recognized by an antibody against the 37/67-kDa laminin/elastin family of proteins. Different polypeptides were purified by the same method on type I collagen. An antibody developed against the polypeptides purified on laminin recognized also a 67-kDa protein, blocked 125I-laminin binding to a population of high affinity (1.5 nM KD) binding sites and caused a significant decrease in cell attachment and spreading to laminin or endogenous ECM. This antibody did not interfere with MDCK cell attachment to fibronectin or collagen matrices, but still impaired cell spreading. An apical MDCK plasma membrane protein (184 kDa), fully polarized in untreated cells, was partially mispolarized after treatment with anti-36 kDa antibody. These results are consistent with a model of various ECM receptors operating together in these cells, and show an important role of a non-integrin 36-kDa laminin binding protein related to the 67-kDa laminin receptor family in cell attachment, spreading and polarization.


Subcellular localization of acid carboxypeptidase in rat liver and human fibroblasts.

The subcellular distribution of acid carboxypeptidase was investigated in rat liver, normal human skin (CRL 1501) and lung (WI-38) fibroblasts, galactosialidosis skin fibroblasts (GM 00806) and transformed lung fibroblasts (WI-38 VA 13). Results of differential and isopycnic centrifugations and osmotic activation experiments clearly indicate that the enzyme is located in lysosomes, in agreement with observations suggesting that carboxypeptidase is the protective protein of the 'Galjaard complex' which is defective in galactosialidosis.


Characterization of two new ETB selective radioligands, [125I]-BQ3020 and [125I]-[Ala1,3,11,15]ET-1 in human heart.

Two new endothelin receptor radioligands, [125I]-BQ3020 and [125I]-[Ala1,3,11,15]ET-1, were characterized in tissue sections of human right atrium and left ventricle. Both radioligands had high affinity ([125I]-BQ3020 right atrium: KD = 0.145 +/- 0.037 nM, left ventricle: KD = 0.107 +/- 0.004 nM; [125I]-[Ala1,3,11,15]ET-1 right atrium: KD = 0.239 +/- 0.036 nM, left ventricle: KD = 0.199 +/- 0.027 nM). Competition binding experiments were performed in the left ventricle. The selective ETA receptor compound BQ123 competed with low affinity against [125I]-BQ3020 (KD = 28.7 +/- 2.7 microM) and [125I]-[Ala1,3,11,15]ET-1 (KD = 28.5 +/- 4.2 microM). The selective ETB receptor compound BQ3020 competed with high affinity against [125I]-BQ3020 (KD = 40.8 +/- 6.6 pM) and [125I]-[Ala1,3,11,15]ET-1 (KD = 0.276 +/- 0.099 nM). Another selective ETB receptor compound, [Ala1,3,11,15]ET-1 also competed with high affinity against [125I]-BQ3020 (KD = 0.663 +/- 0.120 nM) and [125I]-[Ala1,3,11,15]ET-1 (KD = 0.643 +/- 0.124 nM). These results indicate that [125I]-BQ3020 and [125I]-[Ala1,3,11,15]ET-1 are selective ETB receptor radioligands. [Ala1,3,11,15]ET-1 competed with the non-selective radioligand [125I]-ET-1 in left ventricle and revealed the presence of ETA and ETB receptors in the proportions of 76:24% respectively in the human left ventricle.


Central neuropeptide Y and the sigma ligand, JO 1784, reverse corticotropin-releasing factor-induced inhibition of gastric acid secretion in rats.

1. The central interactions between the sigma ligand, JO 1784, [(+)-N-cyclopropylmethyl-N-methyl-1,4-diphenyl-1-ethylbut-3- en-1-ylamine hydrochloride], or neuropeptide Y (NPY) and corticotropin-releasing factor (CRF)-induced inhibition of gastric acid secretion were investigated in rats anaesthetized with urethane. Drugs were injected intracisternally (i.c.) or into specific hypothalamic nuclei. Gastric acid secretion was measured by the flushed technique under basal and pentagastrin (10 micrograms kg-1 h-1, i.v.) stimulated conditions. 2. Intracisternal injection of CRF (10 micrograms), bombesin (0.1 microgram) and human recombinant interleukin-1 beta (hIL-1 beta, 0.1 microgram) inhibited gastric acid response to pentagastrin by 72%, 56% and 62%, respectively. NPY (0.5 microgram) or JO 1784 (0.5 microgram) injected i.c. did not alter acid secretion but completely prevented the inhibitory effect of CRF. The antagonistic effect of NPY and JO 1784 against CRF was dose-related (0.01-0.5 microgram) and peptide-specific since NPY and JO 1784 did not alter the antisecretory action of bombesin or hIL-1 beta. 3. The putative sigma receptor antagonist, BMY 14802, (1 mg kg-1, s.c.) did not influence pentagastrin-stimulated acid secretion nor CRF-induced inhibition of gastric acid secretion; however, BMY 14802 administered s.c. 20 min before JO 1784 or NPY, abolished the antagonistic effect of both JO 1784 and NPY. 4. CRF (3 micrograms) microinjected into the hypothalamic paraventricular nucleus (PVN) and the lateral hypothalamus (LH) inhibited pentagastrin-stimulated gastric acid secretion by 61% and 51%; NPY (0.03 micrograms) or JO 1784 (0.03 micrograms) microinjected into the PVN had no effect by themselves but blocked CRF antisecretory action.There were more VPBs (220 +/- 75), a higher incidence of VT (60%) and more episodes of VT (11.5 +/- 6.0 compared to 0.7 +/- 0.3 episodes in the preconditioned dogs not given L-NAME); none of the animals survived reperfusion (incidence of VF 100%). The improvement in the severity of the degree of inhomogeneity which resulted from preconditioning was abolished by L-NAME administration.5. L-NAME itself elevated blood pressure (from 96 +/- 5 mmHg diastolic to 119 +/- 7 mmHg), reduced heart rate (from 155 +/- 7 to 144 +/- 4 beats min-') but did not change LVEDP, LVdP/dt,,,,, coronary blood flow, ST-segment elevation or the degree of inhomogeneity of conduction. When given 10 min before the prolonged coronary artery occlusion in dogs not subjected to preconditioning, L-NAME had no significant effect on the severity of arrhythmias except for more periods of VT (a mean of 11.7 +/- 4.7 episodes per dog).6. It is concluded from these studies that the generation of nitric oxide contributes to the marked antiarrhythmic effects of preconditioning in the canine myocardium, probably through elevation of cyclic GMP.


Preconditioning of the ischaemic myocardium; involvement of the L-arginine nitric oxide pathway.

1. Short periods of coronary artery occlusion protect the heart against the effects of a subsequent prolonged period of ischaemia. This phenomenon is known as preconditioning of the ischaemic myocardium. 2. In mongrel, chloralose-urethane anaesthetized open-chest dogs, within a restricted body weight range, two 5 min periods of occlusion of the anterior descending branch of the left coronary artery markedly reduced the severity of the early ischaemic arrhythmias resulting from a prolonged (25 min) occlusion of the same coronary artery starting 20 min later. Thus, the number of ventricular premature beats (VPBs) was reduced from 528 +/- 140 in controls to 78 +/- 27 in preconditioned dogs, the incidence of ventricular fibrillation (VF) was reduced from 47% to 0% and the incidence of ventricular tachycardia (VT) from 100% to 20%. ST-segment elevation recorded from electrodes within the ischaemic area, and the degree of inhomogeneity of conduction within the ischaemic area were markedly reduced in these preconditioned dogs. 3. The incidence of VF following reperfusion of the ischaemic myocardium at the end of the 25 min occlusion period was reduced in the preconditioned dogs from 100% to 60%; there was thus a 40% survival from the combined ischaemia-reperfusion insult compared with 0% in the controls. 4. NG-nitro-L-arginine methyl ester (L-NAME) an inhibitor of the L-arginine nitric oxide pathway, given in a dose of 10 mg kg-1 intravenously on two occasions, both before the initial preconditioning occlusion and then again before the prolonged occlusion, partially attenuated the protective effects of preconditioning.There were more VPBs (220 +/- 75), a higher incidence of VT (60%) and more episodes of VT (11.5 +/- 6.0 compared to 0.7 +/- 0.3 episodes in the preconditioned dogs not given L-NAME); none of the animals survived reperfusion (incidence of VF 100%). The improvement in the severity of the degree of in homogeneity which resulted from preconditioning was abolished by L-NAME administration.5. L-NAME itself elevated blood pressure (from 96 +/- 5 mmHg diastolic to 119 +/- 7 mmHg), reduced heart rate (from 155 +/- 7 to 144 +/- 4 beats min-') but did not change LVEDP, LVdP/dt,,,,, coronary blood flow, ST-segment elevation or the degree of inhomogeneity of conduction. When given 10 min before the prolonged coronary artery occlusion in dogs not subjected to preconditioning, L-NAME had no significant effect on the severity of arrhythmias except for more periods of VT (a mean of 11.7 +/- 4.7episodes per dog).6. It is concluded from these studies that the generation of nitric oxide contributes to the marked antiarrhythmic effects of preconditioning in the canine myocardium, probably through elevation of cyclic GMP.


The pivotal role of tumour necrosis factor alpha in the development of inflammatory hyperalgesia.

1. The hyperalgesic activities in rats of interleukin-1 beta (IL-1 beta), IL-6, IL-8, tumour necrosis factor alpha (TNF alpha) and carrageenin were investigated. 2. IL-6 activated the previously delineated IL-1/prostaglandin hyperalgesic pathway but not the IL-8/sympathetic mediated hyperalgesic pathway. 3. TNF alpha and carrageenin activated both pathways. 4. Antiserum neutralizing endogenous TNF alpha abolished the response to carrageenin whereas antisera neutralizing endogenous IL-1 beta, IL-6 and IL-8 each partially inhibited the response. 5. The combination of antisera neutralizing endogenous IL-1 beta + IL-8 or IL-6 + IL-8 abolished the response to carrageenin. 6. These results show that TNF alpha has an early and crucial role in the development of inflammatory hyperalgesia. 7. The delineation of the role of TNF alpha, IL-1 beta, IL-6 and IL-8 in the development of inflammatory hyperalgesia taken together with the finding that the production of these cytokines is inhibited by steroidal anti-inflammatory drugs provides a mechanism of action for these drugs in the treatment of inflammatory hyperalgesia.


Modification by hypoxia, hyperkalaemia and acidosis of the cardiac electrophysiological effects of a range of antiarrhythmic drugs.

1. The electrophysiological effects of a series of drugs with Class I antiarrhythmic activity were examined in sheep Purkinje fibres, superfused in vitro with either a normal or hypoxic, hyperkalaemic and acidotic physiological salt solution (PSS). 2. In normal sheep Purkinje fibres, lignocaine, disopyramide, nicainoprol and propranolol all significantly reduced action potential height and the maximum rate of depolarization of phase zero (MRD) and abbreviated the action potential, without modifying resting membrane potential (RMP). 3. Verapamil at the highest concentration studied, 8 microM, significantly reduced MRD with an associated slight membrane depolarization and abbreviated action potential duration measured at 50% repolarization (APD50). 4. Superfusion of sheep Purkinje fibres with a hypoxic, hyperkalaemic and acidotic PSS resulted in marked reductions in resting membrane potential, upstroke and duration of the action potential. 5. In the presence of modified PSS, lignocaine, propranolol and verapamil all reduced MRD to a greater extent than in normal PSS. The effects of nicainoprol on MRD were not affected whereas those of disopyramide were significantly attenuated. 6. Under simulated ischaemic conditions, lignocaine, propranolol and nicainoprol did not produce a concentration-dependent reduction in action potential duration whereas disopyramide and verapamil, respectively, prolonged and abbreviated both APD50 and APD90. 7. The Na+ channel blocking actions of the different subtypes of Class I antiarrhythmic agents studied, as well as their effects on action potential duration, were modified differently by simulated ischaemia.


In vitro effect of oestradiol on thymidine uptake in pulmonary vascular smooth muscle cell: role of the endothelium.

1. The effect of different concentrations of oestradiol-17 beta (3-300 nM) on [3H]-thymidine uptake was studied in segments from canine pulmonary artery, and cultures of rat pulmonary vascular smooth muscle cells (VSMC). 2. Incubation with oestradiol-17 beta for 24 h, potentiated in a concentration-dependent manner [3H]-thymidine uptake in VSMC cultures. 3. Oestradiol-17 beta potentiated thymidine uptake by pulmonary arterial segments but only when the endothelium had been removed. Autoradiography showed dense incorporation of radioactive thymidine in the vascular smooth muscle cells of the media. 4. The non-steroidal oestrogen, stilboestrol (300 nM), also significantly potentiated [3H]-thymidine uptake, in both VSMC cultures and pulmonary artery segments. Testosterone was ineffective at a similar concentration. 5. Pre-incubation of the pulmonary VSMC with the anti-oestrogen tamoxifen (1 microM) antagonized the potentiating effect of oestradiol-17 beta on [3H]-thymidine incorporation. The effect of tamoxifen was less pronounced in pulmonary arterial segments. 6. These data suggest that oestrogen may promote proliferation of pulmonary VSMC. Endothelial injury or dysfunction may be an important factor in the expression of the oestrogenic effect. 7. We speculate that plasma oestrogen may be a contributing factor to the proliferative lesion observed in certain forms of pulmonary vascular injury in women.


Effects of the PAF antagonists BN50726 and BN50739 on arrhythmogenesis and extent of necrosis during myocardial ischaemia/reperfusion in rabbits.

1. The effects of two novel platelet activating factor (PAF) antagonists BN50726 and BN50739 on arrhythmias, haemodynamics and extent of necrosis during myocardial ischaemia and reperfusion were investigated in anaesthetized rabbits subjected to coronary artery ligation. 2. BN50739 reduced heart rate prior to coronary artery occlusion (P < 0.005) but had no other significant haemodynamic effects at this time. BN50739 and BN50726 did not significantly alter heart rate or blood pressure during 30 min of ischaemia or 30 min of reperfusion, compared to control hearts. 3. BN50739 and BN50726 had no effect on the incidence of arrhythmias during ischaemia. BN50726 significantly reduced the incidence of reperfusion ventricular fibrillation compared to controls (0% v 40%, P < 0.05), and improved survival (80% v 39%, P < 0.05). Similar trends were observed with BN50739. 4. BN50726 reduced the extent of necrosis compared to control hearts (18 +/- 2% v 30 +/- 3%, P < 0.01). A similar trend was observed with BN50739. 5. These results demonstrate that PAF antagonism with BN50726 attenuates reperfusion-induced arrhythmias and preserves myocardium in the early phase of ischaemia, independently of haemodynamic effects.


Chronic antihypertensive treatment with captopril plus hydrochlorothiazide improves aortic distensibility in the spontaneously hypertensive rat.

1. Adult male spontaneously hypertensive rats (SHR) were given captopril plus hydrochlorothiazide mixed in the diet for 10 weeks. Calculated daily doses were 44 mg kg-1 per day for captopril, and 22 mg kg-1 per day for hydrochlorothiazide. Separate groups received captopril or hydrochlorothiazide alone, at similar doses, or no treatment. A final group of WKY normotensive rats received no drug. 2. Systolic arterial blood pressure, measured at regular intervals throughout the 10 weeks' period was lowered but not normalized, in groups receiving either captopril plus hydrochlorothiazide, or captopril alone, but not in the group receiving hydrochlorothiazide alone. 3. Following pentobarbitone anaesthesia, systolic arterial blood pressure, measured in the femoral artery, was found to be lower in all treated groups, but the greatest effect was observed in SHR previously treated with captopril plus hydrochlorothiazide. Aortic pulse wave velocity was also lower in treated SHR, and once again the greatest decrease was observed in the group previously treated with captopril plus hydrochlorothiazide. 4. Following pithing, systolic arterial blood pressures were similar in all SHR groups. Aortic pulse wave velocity was lower in pithed rats previously treated with captopril and hydrochlorothiazide. 5. In conclusion, antihypertensive treatment of SHR produces falls in blood pressure and pulse wave velocity, an indicator of aortic distensibility. Results in pithed rats suggest that treatment with the combination of captopril plus hydrochlorothiazide may increase aortic distensibility independently of blood pressure.


An investigation of some S-nitrosothiols, and of hydroxy-arginine, on the mouse anococcygeus.

1. The effect of five S-nitrosothiols, and of the stereoisomers of NG-hydroxy-arginine (HOARG), were investigated on the mouse anococcygeus. 2. All five S-nitrosothiols produced concentration-related (0.1-100 microM) relaxations of carbachol (50 microM)-induced tone; the order of potency was S-nitroso-L-cysteine (CYSNO) > S-nitroso-N-acetyl-D,L-penicillamine (SNAP) > S-nitrosoglutathione (GSNO) > S-nitrosocoenzyme A (CoASNO) > S-nitroso-N-acetyl-L-cysteine (NACNO). The relaxations were unaffected by the nitric oxide synthase (NOS) inhibitor, L-NG-nitro-arginine (10 microM) (L-NOARG). 3. Cold-storage of the tissue for 72 h resulted in loss of sympathetic and non-adrenergic, non-cholinergic (NANC) nerve function. NOS activity in the tissue was reduced by 97%. Despite this, relaxations induced by the S-nitrosothiols were unaffected. 4. Haemoglobin (50 microM) attenuated relaxations induced by NO and the S-nitrosothiols, although responses to 3-isobutyl-1-methyl-xanthine were unaffected. N-methyl-hydroxylamine (2 mM) which has been shown previously to produce selective inhibition of NANC and nitrovasodilator responses in this tissue, also reduced responses to all S-nitrosothiols. 5. Hydroquinone (100 microM) greatly reduced relaxations to CYSNO (by 88%) but had no effect on those to SNAP, GSNO, CoASNO or NACNO. Since hydroquinone does not reduce responses to NANC stimulation, CYSNO is unlikely to be the NANC transmitter. 6. L-HOARG by itself (up to 100 microM) had no significant effect on carbachol-induced tone or on NANC (10 Hz; 10 strain every 100 s) relaxations. However, it produced reversal of the inhibitory effects of L-NOARG (10;pM), being only slightly less potent than L-arginine. D-HOARG was without effect.L-HOARG had no effect on relaxations induced by 1.51iM NO.7. The results show that S-nitrosothiols are potent relaxants of the mouse anococcygeus; they act directly on the smooth muscle with a mechanism similar to NO and other nitrovasodilators. In addition,the results are consistent with L-HOARG being an intermediate in the biosynthesis of NO from L-arginine, although there is no evidence for it acting to stabilize NO extracellularly.


Contribution of NO and cytochrome P450 to the vasodilator effect of bradykinin in the rat kidney.

1. Inhibition of nitric oxide generation with Nw-nitro-L-arginine (nitroarginine) reduced vasodilator responses to bradykinin and acetylcholine and enhanced those to nitroprusside in the rat isolated perfused kidney, preconstricted with phenylephrine. 2. Inhibition of cyclo-oxygenase with indomethacin, decreased the vasodilator responses to bradykinin by approximately 25% without affecting those to acetylcholine or nitroprusside. 3. BW755c, a dual inhibitor of cyclo-oxygenase and lipoxygenase, reduced renal vasodilator responses to bradykinin, comparable to the effect of indomethacin suggesting an effect related to inhibition of cyclo-oxygenase rather than lipoxygenase. 4. ETYA, an inhibitor of all arachidonic acid metabolic pathways, markedly reduced vasodilator responses to bradykinin but was without effect on the renal vasodilatation induced by acetylcholine or nitroprusside. 5. Clotrimazole and 7-ethoxyresorufin, inhibitors of cytochrome P450, greatly attenuated vasodilator responses to bradykinin without affecting those to acetylcholine or nitroprusside. 6. These data suggest that the renal vasodilator response to bradykinin is subserved by arachidonic acid metabolites as well as nitric oxide, the former accounting for up to 70% of the vasodilator effect of bradykinin.


Protection by Ca2+ channel blockers (nifedipine, diltiazem and verapamil) against the toxicity of oxidized low density lipoprotein to cultured lymphoid cells.

1. Ca2+ channel blockers from 3 different chemical classes (diltiazem, verapamil and nifedipine) were compared in their ability to inhibit low density lipoprotein (LDL) oxidation and to protect cells directly against the cytotoxicity of oxidized LDL. 2. LDL oxidation promoted either by u.v. radiations or by copper ions was inhibited by nifedipine (IC50 of 10 +/- 2 and 4 +/- 0.5 mumol l-1, respectively) whereas diltiazem and verapamil were only poorly active or completely ineffective. As expected, LDL protected from oxidation by nifedipine (nifedipine/oxidized LDL) were much less cytotoxic than (unprotected) oxidized LDL (or than LDL oxidized in the presence of diltiazem or verapamil). The cytotoxic effect correlated well with the lipid peroxidation derivatives measured as the thiobarbituric acid reactive substances (TBARS) content of LDL oxidized in the presence of Ca2+ channel blockers, which suggests that the antioxidant effect of Ca2+ channel blockers protected cells indirectly from the cytotoxic effect of oxidized LDL. 3. Nifedipine also exhibited a direct cytoprotective effect against the cytotoxicity of oxidized LDL as demonstrated by incubating cells in the presence of unprotected oxidized LDL and nifedipine (IC50 of 1 +/- 0.2 mumol l-1), whereas diltiazem and verapamil did not exhibit any significant protective effect. At the concentrations used, the protective effect of nifedipine was not due to inhibition of LDL uptake by Ca2+ channel blockers. 4. The direct protective activity of nifedipine is probably unrelated to its antioxidant properties since it did not inhibit the cellular TBARS evoked by oxidized LDL taken up by the cells, in contrast to vitamin E. 5. Nifedipine (and diltiazem to a lesser extent) inhibited the slow [Ca2+]J rise induced by oxidized LDL and the subsequent cytotoxicity.6. It is proposed that relatively high concentrations of nifedipine (unlike diltiazem and verapamil)protect cultured lymphoid cells against the cytotoxicity of oxidized LDL by two different mechanisms:(i) an antioxidant effect inhibiting LDL oxidation (outside the cell); (ii) a direct cytoprotective effect (at lower concentrations), the mechanism of which is unknown. These data could explain in part the mechanism of action of nifedipine in the prevention of cellular damage potentially involved in atherogenesis.


Nitric oxide is involved in 5-HT-induced relaxations of the guinea-pig colon ascendens in vitro.

1. In the guinea-pig colon ascendens, 5-hydroxytryptamine (5-HT) induces contractions, mediated by 5-HT2, 5-HT3 and 5-HT4 receptors, and relaxations, through a 5-HT1 receptor subtype, that triggers the release of an inhibitory neurotransmitter. Nitric oxide (NO) is one of the main candidates of NANC inhibitory neurotransmission in the gut. The aim of this study was to establish whether NO is involved in 5-HT-induced relaxations of the guinea-pig colon ascendens. 2. Antagonists to block the contractile responses to 5-HT via 5-HT2, 5-HT3 and 5-HT4 receptors were present throughout the experiments and methacholine was administered to precontract the strips. Under these conditions, 5-HT concentration-dependently induced relaxations from 10 nM onwards (EC50 = 258 (172-387) nM). The relaxations were inhibited by metergoline (10 nM) and methiothepine (100 nM) and abolished by tetrodotoxin (TTX, 320 nM). Guanethidine (3.2 microM) did not affect them. 3. NG-nitro-L-arginine (L-NNA) inhibited the responses to 5-HT (IC50 = 18.7 (13.3-26.3) microM); at the highest 5-HT concentration a maximum inhibition of about 75% was observed with 320 microM L-NNA. This inhibition was reversed with L-arginine. Relaxations to glyceryl trinitrate (GTN) were not inhibited by L-NNA. 4. Haemoglobin (32 microM) inhibited the relaxations to 5-HT and GTN, but not those to isoprenaline (Iso). Methylene blue (10 microM) inhibited the relaxations to 5-HT but did not affect those caused by GTN or Iso. 5. It is concluded that 5-HT induces relaxations that involve NO.We also confirmed that 5-HT induces these relaxations via (a) 5-HT, receptor subtype(s), located on neurones.


The effect of in vivo oestrogen pretreatment on the contractile response of rat isolated detrusor muscle.

1. The effect of oestradiol pretreatment was investigated on the response of rat isolated detrusor muscle to cholinergic, electrical and 5-hydroxytryptamine (5-HT) stimulation with and without diethylstilbestrol (DES) (2 microM) in the organ bath. 2. Virgin female Wistar rats were injected subcutaneously for 8 days with oestradiol benzoate 150 micrograms kg-1. Control rats received no injections or injection only with the vehicle, ethyl oleate. 3. Detrusor muscle from treated rats showed a decreased sensitivity to acetylcholine (ACh) and carbachol-induced contractile responses. The dose-response curves to these agonists showed a 44% reduction in maximum contractile response for ACh (P < 0.001), and a 38% reduction in maximum contractile response for carbachol (P < 0.05). The addition of 2 microM DES to the bathing medium further significantly reduced the maximum contractile response by 56 and 57% of control respectively. 4. Electrically stimulated detrusor muscle from treated rats showed a significant 49% reduction in the maximum contractile response (P < 0.001). The addition of 2 microM DES to the bathing medium further significantly reduced the maximum contractile response by 66% of control. The tetrodotoxin resistant responses were smaller in pretreated rats, suggesting a reduced sensitivity of the smooth muscle to direct electrical stimulation. 5. The response to 5-HT stimulation by detrusor muscle samples from oestradiol-treated rats showed a non-significant reduction in maximum contractile response, but the addition of 2 microM DES to the bath chamber resulted in a 67% reduction in the response (P < 0.001). 6. Oestradiol pretreatment did not affect the potassium dose-response curve.7. Oestradiol pretreatment reduced the rat detrusor muscle sensitivity to the blocking effect of atropine on the response to electrical field stimulation. Pretreatment also reduced the potentiating effect of physostigmine on the same response.8. These results suggest that oestradiol pretreatment had a modulating effect on cholinergic responses.The addition of oestrogen to the tissue environment enhances this inhibitory effect.


Effects of neuropeptide Y and agonists selective for neuropeptide Y receptor sub-types on arterioles of the guinea-pig small intestine and the rat brain.

1. The actions of neuropeptide Y (NPY) and agonists selective for NPY receptor subtypes were examined on arterioles from the guinea-pig small intestine and the rat pia in order to characterize the receptors mediating the vasoconstrictor and potentiating effects of NPY. 2. A method was developed for measuring the potentiating effects of NPY in situations where it was not possible to obtain a full concentration-response relationship for the vasoconstrictor. NPY, 50 nM, had a greater potentiating effect on the guinea-pig intestinal arterioles than those from the rat pia. 3. NPY and the Y1-selective agonist, NPY[Leu31,Pro34], potentiated the constrictor responses to U46619 in both arterioles and responses to noradrenaline in the guinea-pig arterioles. There was marked desensitization of the potentiating effect, and cross-desensitization between NPY and NPY[Leu31,Pro34]. Both NPY and NPY[Leu31,Pro34] caused constriction of the rat pial arterioles but not of those from the guinea-pig intestine. 4. The Y2-selective agonist PYY(13-36) caused no potentiation or vasoconstriction and did not affect the potentiation by NPY or NPY[Leu31,Pro34]. 5. The potentiating and vasoconstrictor effects of NPY on these arterioles were mediated by Y1 receptors.


Membrane potential and current responses to neurotensin in the longitudinal muscle of the rectum of the fowl.

1. The effects of neurotensin (NT) on membrane potential and membrane current of the longitudinal smooth muscle of chicken rectum were investigated by intracellular recording and whole-cell voltage clamp. 2. NT (3 nM-1.2 microM), when applied via the bathing medium, produced a concentration-dependent membrane depolarization with an EC50 of 18 +/- 2 nM (n = 7) which was accompanied by an increase in the membrane conductance. The effect was biphasic: an initial, rapid depolarization reached a peak within 2-3 min and then declined to a lower but still elevated level which was sustained until washout. 3. Excitatory junction potentials (e.j.ps), which were non-adrenergic non-cholinergic (NANC) in nature, were decreased in amplitude and total duration in the presence of NT (0.6 microM). The depression of the e.j.p. was due mainly to the reduction of the membrane resistance. 4. When NT was applied locally by means of pressure ejection from a micropipette containing NT, some cells responded with a membrane depolarization and some failed to respond, whereas e.j.ps could invariably be elicited from all of them. 5. In single muscle cells enzymatically isolated from the muscle and dialyzed under voltage clamp at -50 mV with a CsCl-rich solution, NT (5 or 10 microM) produced an inward current. NT-induced inward currents were obtained with inclusion of 10 mM EGTA in the pipette solution and their reversal potential was around 0 mV. In cells dialyzed under voltage clamp at 0 mV with a KCl-rich solution, NT (5 microM) produced a brief outward current followed by abolition of spontaneous transient outward currents.6. The present results suggest that the membrane depolarization, which may arise from activation of non-selective cation channels, and release of calcium from internal stores produced by neurotensin are responsible for its contractile activity in the longitudinal smooth muscle of chicken rectum. Further, the depolarizing effect may provide support for the involvement of NT in the NANC transmission in this preparation.


Iron-sulphur cluster nitrosyls, a novel class of nitric oxide generator: mechanism of vasodilator action on rat isolated tail artery.

1. Two iron-sulphur cluster nitrosyls have been investigated as potential nitric oxide (NO.) donor drugs (A: tetranitrosyltetra-mu 3-sulphidotetrahedro-tetrairon; and B: heptanitrosyltri-mu 3-thioxotetraferrate(1-)). Both compounds are shown to dilate precontracted, internally-perfused rat tail arteries. 2. Bolus injections (10 microliters) of compound A or B generate two kinds of vasodilator response. Doses below a critical threshold concentration (DT) evoke transient (or T-type) responses, which resemble those seen with conventional nitrovasodilators. Doses > DT produce sustained (or S-type) responses, comprising an initial, rapid drop of pressure, followed by incomplete recovery, resulting in a plateau of reduced tone which can persist for several hours. 3. T- and S-type responses are attenuated by ferrohaemoglobin (Hb) and by methylene blue (MB), but not by inhibitors of endothelial NO. synthase. Addition of either Hb or MB to the internal perfusate can restore agonist-induced tone when administered during the plateau phase of an S-type response. Moreover, subsequent removal of Hb causes the artery to re-dilate fully. 4. We conclude that T- and S-type responses are both mediated by NO.. It is postulated that S-type responses represent the sum of two vasodilator components: a reversible component, superimposed upon a non-recoverable component. The former is attributed to free NO., preformed in solution at the time of injection; and the latter to NO. generated by gradual decomposition of a 'store' of iron-sulphur-nitrosyl complexes within the tissue. This hypothesis is supported by histochemical studies which show that both clusters accumulate in endothelial cells.


Blockade of 5-HT3 receptor-mediated currents in dissociated frog sensory neurones by benzoxazine derivative, Y-25130.

1. The effect of Y-25130, ((+-)-N-(1-azabicyclo[2.2.2]oct-3-yl)-6-chloro-4-methyl-3-oxo-3,4-dih ydr o- 2H-1,4-benzoxazine-8-carboxamide hydrochloride), a high affinity 5-hydroxytryptamine3 (5-HT3) receptor ligand, was examined on the 5-HT-induced response in dissociated frog dorsal root ganglion (DRG) neurones by use of the extremely rapid concentration-jump ('concentration-clamp') and the conventional whole-cell patch-clamp techniques. 2. 5-HT induced a rapid transient inward current associated with an increase in membrane conductance at a holding potential of -70 mV. The current amplitude increased sigmoidally as 5-HT concentration increased. The half-maximum value (Ka) and the Hill coefficient estimated from the concentration-response curve were 1.7 x 10(-5) M and 1.7, respectively. 3. The current-voltage (I-V) relationship of 5-HT-induced current (I5-HT) showed inward rectification at potentials more positive than -40 mV. The reversal potential (E5-HT) was -11 mV. The E5-HT value was unaffected by total replacement of intracellular K+ by Cs+, indicating that the 5-HT-gated channels might be large cation channels. 4. Both the activation and inactivation phases of I5-HT were single exponentials. The time constants of activation and inactivation (tau a and tau i) decreased with increasing 5-HT concentration. 5. The 5-HT response was mimicked by a selective 5-HT3 receptor agonist, 2-methyl-5-HT, but the maximum response induced was approximately 25% that of 5-HT. The 5-HT response was reversibly antagonized by the 5-HT3 receptor antagonists, ICS 205-930, metoclopramide and Y-25130, but not by a 5-HTIA receptor antagonist, spiperone, and a 5-HT2 receptor antagonist, ketanserin. The half-inhibition concentrations (IC50) were 4.9 x 10-10 M for Y-25130, 4.8 x 10-10 M for ICS 205-930 and 8.6 x 10-9 M for metoclopramide.6. Y-25130 (5 x 10-10 M) caused a rightward shift of the concentration-response curve for 5-HT while decreasing the maximum response.7. The results suggest that Y-25130 is a potent antagonist of the 5-HT3 receptor-channel complex.


Endothelin ETA and ETB receptors mediate vascular smooth muscle contraction.

1. We have investigated the receptors mediating endothelin-induced contraction of rabbit isolated jugular vein (RJV) and rat isolated thoracic aorta (RTA). 2. Endothelin-1 (ET-1) and endothelin-3 (ET-3) contracted RJV preparations with similar potency (EC50 values approximately 1 nM), whereas, ET-1 (EC50:4.5 nM) was approximately 80 fold more potent than ET-3 in contracting RTA. In addition, the ETB receptor-selective agonist [Ala1,3,11,15]ET-1 contracted RJV (EC50:2.1 nM) but not RTA. 3. The ETA receptor antagonist, BQ123, competitively antagonized (pA2 6.93) the contraction of RTA produced by ET-1, but had no effect (at 10 microM) on the contractile effects of either ET-1, ET-3 or [Ala1,3,11,15]ET-1 in RJV. 4. These data suggest that both ETA and ETB receptors can mediate vascular smooth muscle contraction.


Protection against the effects of anticholinesterases on the latencies of action potentials in mouse skeletal muscles.

1. Adult male albino mice were injected subcutaneously with an organophosphorous anticholinesterase to initiate excessive variability in the latency of indirectly elicited muscle action potentials (jitter) when assessed 5 days later. 2. Pretreatment of the mice with a single dose of pyridostigmine prevented the development of jitter after subsequent dosing with an organophosphate. 3. Treatment with one dose of pralidoxime (2PAM) prevented the development of jitter if given less than 1 h after treatment with ecothiopate, a reactivatable inhibitor of cholinesterase. Similar treatment with 2PAM after a non-reactivatable inhibitor did not prevent the development of jitter. The repeated administration of 2PAM over 12 h did ameliorate jitter. 4. Pretreatment of mice orally with alpha-tocopherol and N-acetylcysteine, known to prevent ecothiopate-induced myopathy, did not prevent the development of jitter after ecothiopate. 5. It is concluded that the development of jitter was a consequence of the inhibition of acetylcholinesterase, and although jitter did not develop acutely, the potential for the full development of jitter was achieved about 1 h after intoxication with ecothiopate. The development of jitter did not involve the generation of free radicals. Reduction of the early effects of intoxication with anticholinesterases by pyridostigmine or 2PAM prevented the development of jitter.


Inhibition of the haemodynamic effects of angiotensin II in conscious rats by AT2-receptor antagonists given after the AT1-receptor antagonist, EXP 3174.

1. Conscious, Long Evans rats (n = 10), chronically instrumented for the measurement of regional haemodynamics, were studied on 3 consecutive experimental days to assess responses to angiotensin II (AII) (125 pmol kg-1, i.v.) and noradrenaline (1 nmol kg-1, i.v.) in the absence and presence of the AT2-receptor antagonist, PD 123319 (10 mg kg-1, i.v.) (day 1), the AT1-receptor antagonist, EXP 3174 (1 mg kg-1, i.v.) (day 2), and PD 123319 (10 mg kg-1, i.v.) given 24 h after EXP 3174 (day 3). 2. In naive rats (day 1), PD 123319 did not antagonize the haemodynamic effects of AII or noradrenaline. EXP 3174 (day 2) caused a marked, prolonged blockade of the haemodynamic effects of AII but not those of noradrenaline. Twenty four h after administration of EXP 3174 (day 3) there was still significant attenuation of the haemodynamic effects of AII. However, administration of PD 123319 at this time caused a further inhibition (lasting 1 h) of the effects of AII but not those of noradrenaline. 3. An identical 3 day protocol was used in a separate group of rats (n = 6) in which the AT2-receptor antagonist, PD 123177, was given instead of PD 123319, and the results were essentially the same, i.e., PD 123177 significantly attenuated the haemodynamic effects of AII but only when given 24 h after EXP 3174.4. In a separate group of rats (n = 4), a low dose of EXP 3174 (60 pg kg-' i.v.) was given to naive rats in order to simulate the degree of inhibition of the effects of All seen after administration of AT2-receptor antagonists in animals pretreated with EXP 3174. This low dose of EXP 3174 did not produce a sustained inhibition of the effects of All and the time course of recovery of All responses was similar to that seen with PD 123319 or PD 123177 given after the high dose of EXP 3174.5. The apparent inhibition of the effects of AII by the AT2-receptor antagonists, PD 123319 and PD 123177, when these were administered 24 h after the AT,-receptor antagonist, EXP 3174, may have been due to the functional activation of AT2-receptors and/or loss of AT2-receptor antagonist selectivity,and/or the displacement of nonspecifically bound EXP 3174 by AT2-receptor antagonists. While the latter explanation seems the most likely, these results raise the possibility that nonpeptide, All-receptor antagonists that act at both AT,- and AT2-receptors may have therapeutic advantages over selective AT,-receptor antagonists.