Safety of Edhazardia aedis (Microspora: Amblyosporidae) for nontarget aquatic organisms.

The susceptibility of common nontarget aquatic organisms to the microsporidium Edhazardia aedis was investigated in the laboratory. Eight predacious species along with 9 scavengers and filter feeders were tested. The nontarget organisms were not susceptible to infection by E. aedis and there was no appreciable mortality. To measure the relative safety of E. aedis to nontarget organisms, a simple mathematical expression was employed where risk is defined as the product of the probability of exposure and the result of exposure (infection) expressed as P(e)P(i). In these laboratory tests, the probability of exposure was fixed at 1 (maximum challenge) and the probability of infection was determined to be 0. Therefore, the risk associated with release of E. aedis into the environment is considered to be negligible under these conditions. The true risk for nontarget organisms to E. aedis can only be determined by careful evaluation of controlled field studies in the natural habitat of the target host.


Host range of Clostridium bifermentans serovar. malaysia, a mosquitocidal anaerobic bacterium.

Clostridium bifermentans serovar. malaysia (C.b.m.) is toxic to mosquito larvae. In this study, we quantified its toxicity to the mosquitoes, Aedes aegypti, Ae. albopictus, Ae. caspius, Ae. detritus, Anopheles stephensi, An. gambiae, Culex pipiens and Cx. quinquefasciatus. Anopheles larvae are the most susceptible, followed by Ae. detritus and Ae. caspius, then Culex and other Aedes larvae. According to mosquito species, the LC50 varies from 7 x 10(3) to 1.3 x 10(6) cells/ml. Three concentrations (10(7), 10(6) and 10(5) cells/ml) of C.b.m., Bacillus thuringiensis var. israelensis (B.t.i.) and Bacillus sphaericus were tested on Ae. aegypti, An. stephensi and Cx. pipiens larvae in order to determine the time necessary for each concentration to kill 50 and 90% of the population. Ninety percent of the 3 mosquito populations are killed within 4-15 h by the C.b.m. concentrations. Whatever the concentrations, C.b.m. kills at least 10 times less rapidly than B.t.i. but always quicker than B. sphaericus. Bioassays of C.b.m. bacterial cells or final whole culture were not toxic to Musca domestica and Drosophila melanogaster (Diptera) as well as to Phaedon cochleariae (Coleoptera) and Spodoptera littoralis (Lepidoptera).


Factors influencing the activity of Bacillus thuringiensis var. israelensis treatments.

Environmental factors influence the effectiveness of microbial control agents in mosquito control programs. Four of these factors (water temperature, larval density, sunlight and the effect of associated filter feeders) were studied with Bacillus thuringiensis var. israelensis under laboratory and semifield conditions in Europe using different instars of Aedes vexans, Ae. aegypti and Culex pipiens. Bioassays conducted at a low temperature (5 degrees C) yielded 10-fold higher LC50 and LC90 values compared with those conducted at a high temperature (25 degrees C). The efficacy of B.t.i. decreased in a linear manner with increasing larval density. Sunlight can reduce the effectiveness of B.t.i. by several times. Competition in food intake by filter feeding Daphnia resulted in lower mortality of mosquito larvae after B.t.i. applications.


Use of an indigenous fish species, Fundulus zebrinus, in a mosquito abatement program: a field comparison with the mosquitofish, Gambusia affinis.

Studies were conducted relating mosquito production in small ponds to presence or absence of larvivorous fishes. Data collected showed that native killifish and introduced mosquitofish controlled mosquito larvae at the same level and support the use of indigenous fish species in mosquito abatement programs.


Effectiveness of aerially applied Arosurf MSF in the control of the cattail mosquito, Coquillettidia perturbans.

Arosurf MSF was applied to a Massachusetts cattail marsh at 0.5 gallons/acre (4.67 liters/hectare) to prevent emergence of Coquillettidia perturbans. One application was made by helicopter and three, later in the season, by fixed-wing aircraft. The material appeared to prevent adult emergence for about a week after the helicopter application, but due to large inter-trap variances in untreated controls the results were not statistically significant. Control was spotty with the fixed-wing application. One problem was obtaining good coverage at a site with difficult aerial access, the second was the interference to delivery of the pupicide as the emergent plant canopy developed later in the season.


Eastern equine encephalomyelitis virus and Culiseta melanura activity at the Patuxent Wildlife Research Center, 1985-90.

Mosquito population densities, virus isolations and seroconversion in sentinel quail were used to monitor eastern equine encephalomyelitis virus (EEE) activity at the Patuxent Wildlife Research Center, Laurel, Maryland, from 1985 through 1990. A dramatic increase in the number of Culiseta melanura collected in 1989, as compared with the 3 previous years, was associated with virus isolations from this species (5/75 pools; n = 542 mosquitoes) and with seroconversion in sentinel quail (4/22 birds positive). This was the first detection of EEE virus activity in this area since a 1984 EEE outbreak killed 7 whooping cranes.


Changes in local mosquito fauna following beaver (Castor canadensis) activity--an update.

Drastic reduction of populations of univoltine temporary pool mosquitoes followed impoundment of breeding areas by beavers. Mosquito populations persist at very low levels over a 10-year period with no evidence of mosquito development in the impoundment.


Analogues of somatostatin bind selectively to brain somatostatin receptor subtypes.

Somatostatin (SRIF) is a neurotransmitter that produces its multiple effects in the CNS through interactions with membrane-bound receptors. Subtypes of SRIF receptors are found in the CNS that are distinguished by their sensitivities to the cyclic hexapeptide MK-678, such that SRIF1 receptors are sensitive to MK-678 and SRIF2 receptors are insensitive to MK-678. In the present study, we further examined the selectivities of a series of structurally diverse SRIF analogues for SRIF receptor subtypes. SRIF receptors were labeled by 125I-Tyr11-SRIF, which has indistinguishable affinities for SRIF receptor subtypes. The inhibition by MK-678 was incomplete, indicating this peptide is highly selective for a subtype of SRIF receptor that we have termed the SRIF1 receptor. The binding of 125I-MK-678 to SRIF1 receptors was monophasically inhibited by SRIF, the octapeptides (such as SMS-201-995), and the hexapeptides (such as MK-678), consistent with the highly selective labeling of a subtype of SRIF receptor. In contrast, the smaller CGP-23996-like analogues did not inhibit 125I-MK-678 binding to SRIF1 receptors. The binding of 125I-CGP-23996 to SRIF receptors was inhibited by SRIF and the octapeptides with Hill coefficients of less than 1, indicating that 125I-CGP-23996 labels multiple SRIF receptor subtypes. The hexapeptides and CGP-23996-like compounds produced only partial inhibitions of 125I-CGP-23996 binding, which were additive, indicating selective interactions of these compounds with the different receptor subpopulations labeled by 125I-CGP-23996. 125I-Tyr11-SRIF binding and 125I-CGP-23996 binding to SRIF receptors were likewise only partially affected by 100 microM guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), a concentration that completely abolishes specific 125I-MK-678 binding to SRIF1 receptors. The component of 125I-CGP-23996 labeling that was sensitive to GTP gamma S was also MK-678 sensitive. Thus, two subpopulations of SRIF receptors exist in the CNS. The SRIF1 receptor is sensitive to cyclic hexapeptides such as MK-678 and to GTP gamma S but insensitive to smaller CGP-23996-like compounds. The SRIF2 receptor is sensitive to the CGP-23996-like compounds and can be selectively labeled by 125I-CGP-23996 in the presence of high concentrations of the hexapeptides or GTP gamma S because, unlike the SRIF1 receptor, the SRIF2 receptor is insensitive to these agents. The SRIF receptor subtype-selective peptide analogues will be useful in the future characterization of the functions mediated by SRIF receptor subtypes in the CNS.


Characterization of the 5-hydroxytryptamine receptor modulating the release of 5-[3H]hydroxytryptamine in slices of the human neocortex.

In the rat brain, the presynaptic 5-hydroxytryptamine (5-HT) autoreceptors located on 5-HT terminals correspond to the 5-HT1B subtype. The presence of a 5-HT receptor probably located on 5-HT nerve endings and modulating transmitter release in the human neocortex has been reported, but its detailed pharmacological characterization is not yet available. On the other hand, receptor binding and autoradiographic results indicate that the 5-HT1B receptor subtype is not present in the human brain. We, therefore, studied the modulation of the electrically evoked release of [3H]5-HT by various 5-HT receptor agonists and antagonists in preloaded slices of human neocortex obtained from 18 patients undergoing neurosurgery. The nonselective 5-HT1A/1B/1D receptor agonist 5-carboxamidotryptamine produced a potent inhibition (70% at 0.03 microM) of the electrically evoked release of [3H]5-HT which was blocked by 5-HT receptor antagonists with the following relative order of potency: methiothepin greater than metergoline = methysergide greater than propranolol. The selective 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin at 0.1 microM did not modify the electrically evoked release of [3H]5-HT. The 5-HT1A/1B receptor agonist RU 24969 was 10 times more potent at inhibiting [3H]5-HT overflow in the rat frontal cortex than in the human neocortex. The potent 5-HT1B receptor antagonist cyanopinodolol did not modify the 5-carboxamidotryptamine-induced inhibition of the electrically evoked release of [3H]5-HT in slices of the human neocortex, but produced by itself a small inhibition of [3H]5-HT overflow.(ABSTRACT TRUNCATED AT 250 WORDS)


5,5'-Dihydroxy-4,4'-bitryptamine: a potentially aberrant, neurotoxic metabolite of serotonin.

Previous investigators have detected unknown oxidized forms of 5-hydroxytryptamine (5-HT) in the CSF of Alzheimer's disease (AD) patients. Furthermore, an unidentified autoxidation product of this neurotransmitter is an inhibitor of acetylcholinesterase (AChE), an enzyme compromised in the Alzheimer brain. In this study it is demonstrated that the major product of autoxidation of 5-HT is 5,5'-dihydroxy-4,4'-bitryptamine (DHBT). Central administration of DHBT to mice at a dose of 40 micrograms (free base) evokes profound behavioral responses, which persist until the animals die (approximately 24 h). One hour after central administration of DHBT, the levels of norepinephrine, dopamine, 5-HT, and acetylcholine and their metabolites in whole brain are greatly elevated. Disturbances to the catecholaminergic and serotonergic systems were still evident shortly before the death of animals. DHBT is also shown to be a noncompetitive inhibitor of AChE in vitro. These observations suggest that if DHBT is formed as an aberrant metabolite of 5-HT in the human brain, it could potentially be neurotoxic and contribute to the neuronal degeneration and other neurochemical and neurobiochemical changes associated with AD or perhaps other neurodegenerative diseases.


Eclosion hormone stimulates cyclic GMP levels in Manduca sexta nervous tissue via arachidonic acid metabolism with little or no contribution from the production of nitric oxide.

The neuropeptide eclosion hormone acts directly on the nervous system of the tobacco hornworm, Manduca sexta, to trigger ecdysis behavior at the end of each molt. Previous studies have shown that the action of eclosion hormone is mediated via the intracellular messenger cyclic GMP. In the present study we have investigated the mechanisms involved in the eclosion hormone-stimulated increases in cyclic GMP. No stimulation of guanylate cyclase was seen in homogenized nervous tissue, suggesting that eclosion hormone does not directly stimulate a membrane-bound form of guanylate cyclase. Nitric oxide synthase inhibitors, N-methylarginine and nitroarginine, had no effect on eclosion hormone-stimulated cyclic GMP levels. By contrast, 4-bromophenacyl bromide, an inhibitor of arachidonic acid release, and nordihydroguaiaretic acid, an inhibitor of arachidonic acid metabolism, almost completely abolished the eclosion hormone-stimulated cyclic GMP increase. We hypothesize that eclosion hormone receptors are coupled to a lipase, activation of which causes the release of arachidonic acid. Either the arachidonic acid directly stimulates the soluble guanylate cyclase or further metabolism of arachidonic acid yields compounds that activate guanylate cyclase.


Activation of protein kinase C by phorbol esters and arachidonic acid required for the optimal potentiation of glutamate exocytosis.

The effects of arachidonic acid and phorbol esters in the Ca(2+)-dependent release of glutamate evoked by 4-aminopyridine (4-AP) in rat cerebrocortical synaptosomes were studied. In the absence of arachidonic acid, high concentrations (500 nM) of 4 beta-phorbol dibutyrate (4 beta-PDBu) were required to enhance the release of glutamate. However, in the presence of arachidonic acid, low concentrations of 4 beta-PDBu (1-50 nM) were effective in potentiating glutamate exocytosis. This potentiation of glutamate release by phorbol esters was not observed with the methyl ester of arachidonic acid, which does not activate protein kinase C. Moreover, pretreatment of synaptosomes with the protein kinase inhibitor staurosporine also prevented the stimulatory effect by arachidonic acid and phorbol esters. These results suggest that the activation of protein kinase C by both arachidonic acid and phorbol esters may play a role in the potentiation of glutamate exocytosis.


Effect of quinine on autoreceptor-regulated dopamine release in the rat striatum.

In vivo brain microdialysis was used to examine the role of potassium channel activation in dopamine (DA) autoreceptor function in the striatum of freely moving rats. Local application of the D2 receptor agonists quinpirole or N-0437 through the dialysis probe significantly reduced extracellular concentrations of DA. Local application of the D2 antagonist (-)-sulpiride produced significant increases in DA. Local perfusion with quinine, a K+ channel blocker, completely blocked the (-)-sulpiride-induced increases in DA but did not affect the DA agonist-induced decreases. (-)-Sulpiride completely blocked the effect of quinpirole on DA both in control and in quinine-treated animals. At the highest dose used, quinine caused a large transient increase in extracellular DA. Local application of tetrodotoxin or infusion of Mg2+ in the absence of Ca2+ did not prevent this quinine-induced transient increase in extracellular DA. These results demonstrate that DA autoreceptors in the striatum regulate DA release in awake, behaving animals. Local application of (-)-sulpiride increases DA levels by blocking the tonic activation of autoreceptors by endogenous DA. Quinine blocks the neuroleptic-induced increase in DA, perhaps by preventing the K+ channel opening that would normally accompany endogenous autoreceptor activation. The fact that exogenously applied DA receptor agonists can decrease extracellular DA levels in the presence of quinine suggests that they may be acting at extrasynaptic autoreceptors that are not tonically active in vivo. The effect of DA agonists on this site is via a DA receptor because it is blocked by (-)-sulpiride. However, this receptor does not appear to be coupled to a quinine-sensitive potassium channel.


Stimulation of delta-opioid receptors reduces the in vivo binding of the cholecystokinin (CCK)-B-selective agonist [3H]pBC 264: evidence for a physiological regulation of CCKergic systems by endogenous enkephalins.

Cholecystokinin (CCK) and enkephalins appear to be colocalized in several brain structures, and a physiological interaction between these peptides has been suggested by a large number of pharmacological studies. In this work we have shown, by in vivo binding experiments, that the endogenous enkephalins, protected from degrading enzymes by mixed inhibitors such as kelatorphan and N-[(R,S)-2-benzyl-3-[(S)-2-amino-4-methylthiobutyldithio]-1-oxo pro pyl]- L-phenylalanine benzyl ester (RB 101), a systemically active prodrug, modulate CCK release in mouse brain, leading to an overall increase in the extracellular levels of CCK. This was quantified by measuring the effects of both inhibitors on the in vivo binding of [3H]propionyl-Tyr(SO3H)-gNle-mGly-Trp-(N-Me)Nle-Asp-Phe-NH2 ([3H]pBC 264), a selective and highly potent CCK-B agonist. Thus, intracerebroventricular injection of kelatorphan produced a dose-dependent inhibition of the in vivo binding of [3H]pBC 264 with a maximal effect (40%) at 50 nmol. A similar response was observed after intravenous injection of RB 101 (40 mg/kg). The specific binding of [3H]pBC 264 was also inhibited (25%) by intravenous injection of the selective delta-opioid agonist H-Tyr-D-Cys(StBu)-Gly-Phe-Leu-Thr(OtBu)-OH (BUBUC; 2 mg/kg) but not by the mu-agonist H-Tyr-D-Ala-Gly-(N-Me)Phe-Gly-ol (5 mg/kg), suggesting a preferential involvement of delta-opioid receptors in the modulation of CCK release. This was confirmed by using the selective delta-opioid antagonist naltrindole, which prevented the inhibitory effects of BUBUC and of enkephalin-degrading enzyme inhibitors on [3H]pBC 264 binding.(ABSTRACT TRUNCATED AT 250 WORDS)


Activation of dopamine D2 receptors linked to voltage-sensitive potassium channels reduces forskolin-induced cyclic AMP formation in rat pituitary cells.

3,4-Dihydroxyphenylethylamine (dopamine) D2 receptor agonists, including BHT 920 and bromocriptine, and the potassium channel opener minoxidil share the property of hyperpolarizing the plasma membrane by activating voltage-dependent potassium channels. These drugs were tested for their ability to inhibit the cyclic AMP formation induced by forskolin either in intact or in broken pituitary cells. In contrast to bromocriptine, which was active in both experimental systems, BHT 920 and minoxidil inhibited the forskolin-induced cyclic AMP formation in intact-cell but not in broken-cell preparations. The effects of BHT 920 were (a) concentration dependent, with a calculated IC50 of 0.7 microM, (b) dopaminergic in nature, being specifically antagonized by sulpiride, (c) not additive with those induced by minoxidil, and (d) less effective in the presence of potassium channel blockers, such as 4-aminopyridine and tetraethylammonium. These data indicate that the inhibition of forskolin-induced cyclic AMP formation by BHT 920 in intact pituitary cells is not a primary consequence of receptor occupation, but a late event, possibly related to the opening of voltage-dependent potassium channels elicited by this drug through the activation of a subtype of dopamine D2 receptors uncoupled to adenylyl cyclase.


3-[(+-)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid recognizes two N-methyl-D-aspartate binding sites in rat cerebral cortex membranes.

Binding of 3-[(+-)-2-carboxypiperazin-4-yl][3H]-propyl-1-phosphonic acid ([3H]CPP), a competitive inhibitor of N-methyl-D-aspartate (NMDA), has been studied in synaptic plasma membranes from rat cerebral cortex. Computer analysis of saturation and homologous displacement isotherms deriving from these plasma membranes indicated the existence of two binding sites: a specific, saturable, high-affinity binding site with a pKD value of 7.53 +/- 0.03 (29.5 nM) and a maximum binding value (Bmax) of 2.25 +/- 0.36 pmol/mg of protein, and a low-affinity site with a KD of approximately 600 nM and a Bmax of 7.0 pmol/mg of protein. It is argued that, in the light of current literature evidence, the low-affinity binding site may represent an agonist-dependent receptor, linked to physiological processes such as neurotransmitter release and channel regulation, whereas the high-affinity binding site may be linked to an antagonist-preferred receptor, for which no function has yet been reported.


The effect of cyclohexyladenosine on the periischemic increases of hippocampal glutamate and glycine in the rabbit.

We investigated the ability of N6-cyclohexyladenosine (CHA), a potent and selective agonist of the adenosine A1 receptor, to attenuate elevations of levels of extracellular hippocampal glutamate and glycine that result from episodes of transient global cerebral ischemia (TGCI). A total of 30 New Zealand white rabbits were randomly assigned to receive 0 (n = 5), 0.1 (n = 8), 1.0 (n = 6), 10 (n = 6), or 100 (n = 5) microM CHA. The drug was dissolved in artificial CSF (vehicle) and administered via a microdialysis probe placed stereotactically into the dorsal hippocampus. A second microdialysis probe placed into the contralateral hippocampus of each animal was perfused with vehicle alone. Ten minutes of TGCI was induced by neck tourniquet inflation and deliberate hypotension from 0 to 10 min. Microdialysis samples were collected as follows: every 20 min preischemia (at -80, -60, -40, -20, and 0 min); every 5 min during ischemia and in the immediate reperfusion period (at 5, 10, 15, and 20 min); and every 20 min for the remainder of the reperfusion period (at 40, 60, and 80 min). Samples were then analyzed for their concentration of glutamate and glycine by HPLC. Following 10 min of ischemia, glutamate levels increased to a peak of 3.28 +/- 0.55 times baseline and returned to preischemic levels by 40 min, i.e., during reperfusion. Glycine concentrations increased to 5.41 +/- 0.91 times over baseline and remained elevated for the duration of the study.(ABSTRACT TRUNCATED AT 250 WORDS)


Glucose metabolism assessed with 2-deoxyglucose and the effect of glutamate in subdivisions of rat hippocampal slices.

A new approach to the study of glucose phosphorylation in brain slices is described. It is based on timed incubation with nonradioactive 2-deoxyglucose (DG), after which the tissue levels of DG and 2-deoxyglucose-6-phosphate (DG6P) are measured separately with sensitive enzymatic methods applied to specific small subregions. The smallest samples had dry weights of approximately 0.5 microgram. Direct measurements in different regions of hippocampal slices showed that within 6 min after exposure to DG, the ratios of DG to glucose in the tissue were almost the same as in the incubation medium, which simplifies the calculation of glucose phosphorylation rates and increases their reliability. Data are given for ATP, phosphocreatine, sucrose space, and K+ in specific subregions of the slices. DG6P accumulation proceeded at a constant rate for at least 10 min, even when stimulated by 10 mM glutamate in the medium. The calculated control rate of glucose phosphorylation was 2 mmol/kg (dry weight)/min. In the presence of 10 mM glutamate it was twice as great. The response to 10 mM glutamate of different regions of the slice was not uniform, ranging from 164% of control values in the molecular layer of CA1 to 256% in the stratum radiatum of CA1. There was a profound fall in phosphocreatine levels (75%) in response to 10 mM glutamate despite a 2.4-fold increase in glucose phosphorylation. Even in the presence of 1 mM glutamate, the increase in glucose phosphorylation (50%) was not great enough to prevent a significant drop in phosphocreatine content.


On the bioavailability of oral and subcutaneous 2-chloro-2'-deoxyadenosine in humans: alternative routes of administration.

The antimetabolite 2-chloro-2'-deoxyadenosine (CdA) is a promising alternative to alkylating agents for the treatment of lymphoproliferative disorders. Its use, however, is hampered by the need for intravenous (IV) administration. The aim of the present study was to determine the bioavailability of subcutaneously (SC) and orally administered CdA, and to establish an oral dose of CdA that could supersede IV administration. A previously developed high-performance liquid chromatography method was used for the determination of plasma CdA concentrations in 13 patients. Ten patients were treated on alternate days with 0.14 mg/kg/d CdA as a 2-hour IV infusion or by a SC injection. Three of these patients were also given 0.14 mg/kg CdA orally in enteric-coated capsules. Ten patients were administered CdA orally that was dissolved in phosphate-buffered saline (PBS) after treatment with 20 mg omeprazole 1 and 6 hours before the administration of CdA. The bioavailability of SC CdA was 102% +/- 28% (mean +/- SD), and the bioavailability of CdA administered in enteric-coated capsules was 19%, 24%, and 60%. In the three patients who were given 0.14 mg/kg orally dissolved in PBS, the bioavailability was 48% +/- 8%, whereas in the seven patients who received 0.28 mg/kg, the bioavailability was 55% +/- 17%. In the 10 patients who were treated with the CdA solution orally, the coefficients of variation of the areas under the curve (AUCs) after oral and IV administration were similar. Thus, oral administration did not add to the interindividual variability. We conclude that orally administered CdA can supersede IV infusion if the dose is doubled. SC administration gives a high peak concentration of short duration with an AUC identical to that of IV infusion. Thus, SC injection can also be used as an alternative to IV infusion.


Feasibility of using quinine, a potential multidrug resistance-reversing agent, in combination with mitoxantrone and cytarabine for the treatment of acute leukemia.

We demonstrated previously that sera from quinine-treated patients reversed the multidrug resistance (MDR) of a human leukemic cell line. We now report a phase I and II clinical study that examined the toxicity of the combination of quinine with mitoxantrone and cytarabine (Ara-C). Fifteen adult patients with relapsed or refractory acute leukemia were treated with quinine formiate (30 mg/kg/d in continuous intravenous (IV) infusion from day 1 through day 5 or 6) associated with Ara-C (1 g/m2 in 3-hour IV infusion twice a day for 5 days) and five increasing doses of mitoxantrone (from 8 mg/m2/d for 4 days to 12 mg/m2/d for 5 days). The main toxicity was severe myelosuppression: the mean times to leukocyte recovery (> 500/microL), granulocytes recovery (> 500/microL), and platelet count recovery (> 50,000/microL) were 23 days (range, 17 to 29 days), 30.6 days (range, 17 to 48 days), and 35.4 days (range, 14 to 75 days), respectively. The nonhematopoietic toxicity of this regimen was acceptable. Nausea and vomiting were common, but severe mucositis was observed in only two patients. Cardiotoxicity was limited to transient episodes of moderate supraventricular tachycardia and a clinically well-tolerated bradycardia. Tinnitus and vertigo were observed in 10 cases (67%), and mild hearing loss and transient increase of serum bilirubin were observed in six patients (40%). Total quinine serum levels reached a steady-state concentration between 6.4 and 18 mg/L in 24 hours. Complete remission (CR) was achieved in eight of 14 (57%) assessable patients, and partial response (PR) was achieved in two additional patients (14%). P-glycoprotein expression was detected on blast cells from five of 13 studied patients before treatment. A response was observed in all P-glycoprotein-positive cases. Quinine can be used safely as a potential reversing agent of MDR for the treatment of clinically resistant acute leukemias.


High-dose consolidation therapy with autologous stem-cell rescue in stage IV breast cancer: follow-up report.

Fifty-nine patients with newly diagnosed metastatic breast cancer were treated with induction chemotherapy followed by high-dose intensification and autologous stem-cell rescue (ASCR) to determine therapeutic efficacy. Induction consisted of cyclophosphamide, doxorubicin, vincristine, and methotrexate with leucovorin rescue (LOMAC) in 27 patients, or fluorouracil, cisplatin, doxorubicin, and cyclophosphamide (FCAP) in 32 patients. Intensification after LOMAC was cyclophosphamide and thiotepa (CyTepa) with ASCR, and after FCAP it was cyclophosphamide, thiotepa, and carmustine (BCNU) in all but eight patients who received CyTepa. Median survival from study entry for the entire group was 13.3 months. Median time to progression from reinfusion for the 45 patients who underwent intensification was 7.5 months. After LOMAC and intensification, there were 12 complete responses (CR) (nine partial responses [PRs] after induction converted to CRs). Responses after FCAP and intensification were eight CRs (two PRs after induction converted to CRs). Median time to treatment failure from reinfusion was 5.4 months for LOMAC and intensification, and was 10.5 months for FCAP and intensification. Median survival from study entry was 15.1 months for all 27 LOMAC patients and 9.3 months for all 32 FCAP patients. Median time to treatment failure from reinfusion for 11 patients who were CRs at intensification has not been reached and is more than 13 months compared with a median of 5.5 months for the 23 patients in partial remission at intensification. High-dose intensification therapy has led to increased CR rates in metastatic breast cancer. The role of such therapy in the treatment of stage IV breast cancer requires further refinement.


Phase II study and long-term follow-up of patients treated with taxol for advanced ovarian adenocarcinoma.

Based on the results of our phase I study that demonstrated the antitumor activity of taxol in a previously treated patient with ovarian cancer, a phase II study was conducted to evaluate the efficacy of taxol in patients with metastatic ovarian cancer and to evaluate further the toxicity of taxol in this group of patients. Thirty-four patients with metastatic ovarian cancer received taxol (180 to 250 mg/m2) as a 24-hour continuous infusion. A premedication regimen was used to reduce the likelihood of an acute hypersensitivity reaction. Six of 30 assessable patients demonstrated complete responses (one patient) or partial responses (five patients; 20%; 95% confidence interval [CI], 6% to 34%; range, 2 to 30 months). Additionally, one patient had a less than partial objective response (2 months), and two patients had stable disease for 6 and 15 months. Those responders had a median survival of 27 months, and the nonresponders had a median survival of 6 months (P = .0001). Myelosuppression was the most significant toxicity. Other adverse effects included alopecia and peripheral neuropathy. Taxol has significant activity in ovarian cancer and should be studied in combination with other active agents earlier in this disease.


D1-like and D2-like dopamine receptors synergistically activate rotation and c-fos expression in the dopamine-depleted striatum in a rat model of Parkinson's disease.

Selective agonists for D1-like and D2-like dopamine receptors can interact synergistically to enhance each other's actions on locomotion and behavior in experimental animals. Clinically, the combination of the D2 agonist bromocriptine with L-dopa (which has pronounced D1 effects) is a highly effective treatment for Parkinson's disease. The mechanisms underlying this important receptor interaction are poorly understood and are the subject of intense study in vitro. In rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal pathway, D1-selective (but not D2-selective) dopamine agonists produce a marked increase in expression of the immediate-early gene c-fos in the striatum ipsilateral to the 6-OHDA lesion. In the experiments reported here, we have used this in vivo model to explore the possibility that combinations of D1-selective and D2-selective agonists might have effects on c-fos transcription that are different from those exhibited by D1 or D2 agonists administered alone. We examined the effects of the D1-selective agonist SKF-38393 and the D2-selective agonist quinpirole (LY 171555) on the expression of Fos-like protein and c-fos mRNA in the caudoputamen and made parallel behavioral observations in the same animals. A low dose of SKF-38393 produced little contraversive rotation and little induction of Fos-like immunoreactivity in the striatum. A low dose of quinpirole elicited contralateral rotation but little or no induction of Fos-like immunoreactivity in the caudoputamen; there was, however, induction of Fos in the globus pallidus ipsilateral to the 6-OHDA lesion. Combination of the low dose of SKF-38393 and quinpirole produced a synergistic effect on rotation and elicited, in the dopamine-depleted caudoputamen, a striking pattern of Fos-like protein expression in which Fos-positive neurons were concentrated in striosomes and in the dorsolateral caudoputamen. Northern blot analysis showed that c-fos mRNA was expressed following combined agonist treatment but was not detectable after the single-agonist treatments. Both the contraversive rotation and the induction of Fos-like immunoreactivity were blocked by the preadministration of the D1-preferring antagonist SCH-23390 and the D2-selective antagonist raclopride in combination. Pretreatment with the glutamate NMDA receptor antagonist MK-801 also blocked the induction of Fos-like immunoreactivity, and it reversed the rotation. These findings suggest a D1/D2 synergistic mechanism that involves the participation of D1-responsive striatonigral and D2-responsive striatopallidal output pathways, and that is sensitive to glutamatergic modulation.


GABAergic inhibition of endogenous dopamine release measured in vivo with 11C-raclopride and positron emission tomography.

Extensive neuroanatomical, neurophysiological, and behavioral evidence demonstrates that GABAergic neurons inhibit endogenous dopamine release in the mammalian corpus striatum. Positron emission tomography (PET) studies in adult female baboons, using the dopamine D2-specific radiotracer 11C-raclopride, were undertaken to assess the utility of this imaging technique for measuring these dynamic interactions in vivo. 11C-raclopride binding was imaged prior to and following the administration of either gamma-vinyl-GABA (GVG), a specific suicide inhibitor of the GABA-catabolizing enzyme GABA transaminase, or lorazepam, a clinically prescribed benzodiazepine agonist. Striatal 11C-raclopride binding increased following both GVG and lorazepam administration. This increase exceeded the test/retest variability of 11C-raclopride binding observed in the same animals. These findings confirm that changes in endogenous dopamine concentrations resulting from drug-induced potentiation of GABAergic transmission can be measured with PET and 11C-raclopride. Finally, this new strategy for noninvasively evaluating the functional integrity of neurophysiologically linked transmitter systems with PET supports its use as an approach for assessing the multiple mechanisms of drug action and their consequences in the human brain.


Immunogold quantification of glutamate in two types of excitatory synapse with different firing patterns.

A quantitative immunocytochemical method was used to study the regional levels of glutamate in two types of lamprey (Ichtyomyzon unicuspis) axon, which both activate excitatory amino acid receptors, but which when active exhibit different firing patterns. Giant reticulospinal axons fire in brief bursts, while dorsal column axons, mainly belonging to cutaneous afferents, show a sustained firing at high frequency. In both types of axon, clusters of synaptic vesicles showed a strong accumulation of glutamate immunogold labeling, and the density of gold particles correlated strictly with the packing density of synaptic vesicles. The most densely packed vesicle areas had a particle density corresponding to a concentration of fixed glutamate of about 30 mM in coprocessed glutamate conjugates, suggesting an intravesicular glutamate concentration of at least 60 mM. The level of labeling in axoplasmic matrix was considerably lower, but differed significantly between the two types of axon. Dorsal column axons showed a particle density in axoplasmic matrix that was approximately four times higher than that in giant reticulospinal axons. The mitochondrial glutamate labeling was also significantly stronger in the dorsal column axons. In addition, the number of mitochondrial profiles surrounding vesicle clusters was about four times higher in dorsal column synapses than in reticulospinal synapses. Antisera to aspartate, GABA, glutamine, and homocysteate failed to produce a specific labeling of synaptic vesicle clusters in reticulospinal or dorsal column axons. In conjunction with previous demonstrations of a stimulus-induced vesicle depletion in giant reticulospinal synapses (Wickelgren et al., 1985), these results imply that glutamate is released from synaptic vesicles. The different extravesicular glutamate levels in reticulospinal axons and dorsal column axons may relate to different requirements for the refilling of synaptic vesicles in these functionally divergent neurons.


Developmental regulation of leucine-enkephalin expression in adrenal chromaffin cells by glucocorticoids and innervation.

Most catecholaminergic cells derived from the sympathoadrenal lineage of the neural crest contain one or more neuropeptides. Although a great deal is known about the development and regulation of catecholaminergic properties in these cells, relatively little is known about the developmental control of their neuropeptidergic properties. We have investigated the possible role of glucocorticoids and preganglionic innervation in the regulation of leucine-enkephalin (L-Enk) expression in cultures of embryonic and neonatal adrenal chromaffin cells and in mature chromaffin cells in vivo. Exposure of embryonic and neonatal chromaffin cells to the synthetic glucocorticoid dexamethasone increases L-Enk content. Neonatal chromaffin cells grown in medium containing elevated levels of potassium to mimic depolarization also exhibited increased L-Enk levels. The depolarization-induced increase in L-Enk was selectively inhibited by treatment with the enkephalin analog [D-Ala, d-Leu]-enkephalin to mimic the enkephalinergic component of the preganglionic innervation. Denervation of the adrenal gland in vivo resulted in a dramatic increase in L-enk expression that could be partially mimicked by selectively blocking enkephalinergic transmission with administration of the opiate receptor antagonist naloxone. Taken together with the developmental time course and pattern of L-Enk expression in vivo, our results suggest that glucocorticoids and the preganglionic innervation regulate the developmental expression of this peptide in adrenal chromaffin cells and therefore participate in the generation of the mature neurochemical phenotypes present in the adrenal medulla. Further, in adult chromaffin cells similar factors appear to regulate the expression of L-Enk, which could in turn participate in physiological responses to stress.


Neurochemical afferents controlling the activity of serotonergic neurons in the dorsal raphe nucleus: microiontophoretic studies in the awake cat.

Serotonergic (5-HT) neurons of the brainstem dorsal raphe nucleus (DRN) have been implicated in a diversity of physiological and behavioral processes in vertebrates. However, despite extensive information about the intrinsic properties and the efferent projections of this neurochemical system, little information is available regarding the afferents that control its activity. This study investigated the neurotransmitters that regulate the activity of DRN-5-HT neurons under physiologically relevant conditions, by utilizing microiontophoresis in combination with single-unit recordings in the awake, head-restrained cat. This made it possible to examine the direct effects of neurotransmitters on DRN-5-HT neuronal activity, and, through the use of specific antagonists, to study the roles of these neurotransmitter inputs during physiological conditions that influence DRN-5-HT neuronal activity. The results indicate that (1) iontophoretic application of the GABA antagonist bicuculline reversed the typical suppression of neuronal activity seen during slow wave sleep, but had no effect on maintained activity during wakefulness. The suppression of neuronal activity during REM sleep was generally unaffected by application of bicuculline. This suggests a role for a GABAergic input to DRN-5-HT neurons in controlling some aspects of their state-dependent activity. (2) Iontophoretic application of the excitatory amino acid (EAA) antagonist kynurenic acid reduced the magnitude of the neuronal response evoked by phasic auditory stimuli, but had no effect on the spontaneous activity of these neurons, suggesting a role for an EAA input to the DRN in mediating the response to phasic sensory stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)


Structural determinants of barium permeation and rectification in non-NMDA glutamate receptor channels.

A single site in recombinant glutamate receptor channels of the GluR1-GluR4 family has been previously identified as a key regulator of ion permeation. The natural amino acid at this position (arginine in GluR2 but glutamine in GluR1, GluR3, and GluR4) determines both the ability to pass outward current and the divalent cation permeability of kainate-activated receptor channels. By mutagenesis of GluR6, we demonstrated that the same site also controls the ability to pass outward current in another non-NMDA receptor family. Additional mutations at and near this site in GluR3 indicated that the position of the arginine is critical to function, that the ability to pass outward current is not necessarily linked to low barium permeability, and that the size as well as the charge of the side chain at this position influences barium permeation. These results provide evidence that this site forms part of the selectivity filter of glutamate receptor channels.


[Effects of anti-PAF agents on nasal response after allergen challenge in guinea pigs].

We previously reported that Platelet-activating factor (PAF) activities were detected in nasal lavage fluids from patients with allergic rhinitis and in ovalbumin (OA) sensitized guinea pigs after topical allergen challenge. In guinea pigs, a topical application of PAF produced an increase in nasal vascular permeability which was inhibited by a PAF-antagonist (CV3988). To define the role of PAF in allergic rhinitis, we examined the effects of anti-PAF agents (WEB2086) and anti-allergic agents (Azelastine) on nasal airway resistance (NAR) and nasal symptoms in actively sensitized guinea pigs. Guinea pigs (200-300g) were sensitized by intraperitoneal injection of OA (10 micrograms/kg) and alum (5mg/kg) three times at two week intervals and repeated inhalation of OA (1mg/min., for 3 minutes) everyday for four weeks. The NAR was measured serially for 6 hours by an apparatus using the oscillation method. NAR change was expressed as a percent ratio to the pre-challenge value. Nasal symptoms were evaluated by counting the number of sneezing discharges and scratching movements for 30 minutes. The anti-PAF agent or Azelastine were administered orally at 2-3 hours before the topical allergen challenge. We noted a biphasic increase in NAR after allergen challenge. The increase in NAR during early phase was not affected but that during late phase was significantly inhibited by the anti-PAF agent and Azelastine. The nasal symptoms were inhibited by WEB2086 and Azelastine. Our results suggest that PAF activities might play an important role in late phase NAR increase following allergen challenge.


Histocompatibility screening by molecular techniques: use of polymerase chain reaction products and heteroduplex formation.

Novel molecular approaches have recently become available allowing improved major histocompatibility complex (MHC) matching of potential allogeneic bone marrow donors and recipients. Current cellular and serological assays are hindered by aberrant cell populations and limited reagents which only detect an individuals' phenotype. Therefore, a molecular screening protocol which discriminates at the genotypic level would be advantageous. Here we describe a two-step DNA-based approach that can be applied to large-scale screening of potential donors. A primary screen, utilizing polymerase chain reaction (PCR), reduces the potential donor population, whereas a secondary or fine resolution screen uses DNA heteroduplex analysis to determine identity or non-identity at specific loci. Heteroduplex analysis generates a DNA migration pattern that is unique for alleles at a given locus, and is more sensitive than serology in discriminating among individuals. Here we demonstrate the potential feasibility of this approach by analyzing results at one MHC locus, HLA-DQ. Since this method does not rely on typing sera or viable lymphocytes, it is not subject to the variability found in the traditional methods. In contrast to traditional methods, these molecular techniques can provide the critical information needed to select a potential bone marrow donor.


A sensitive method for the detection of poly-A tails of mRNA using a biotin-labelled heteropolymer of dT:rA.

We have developed a highly sensitive non-radioactive in situ hybridization technique that enables us to study the production of mRNAs in tissues. As part of the validation procedure of our methods, we examined various methods of detecting poly-A RNA tails of mRNA. We have used three types of biotin-labelled probes complementary to poly-A sequences: a 25-mer poly-dT oligonucleotide, a polymer of dT, and a heteropolymer of dT:rA. All the probes had the same specificity of reactivity but the heteropolymer of dT:rA gave the strongest signals as visualized histochemically by the use of alkaline phosphatase as the detection enzyme. All the probes tested for poly-A detection showed reactivity. The poly-dT oligonucleotide showed a strength of signal comparable to published results. The biotinylated polymer of dT gave a stronger signal than that of the oligonucleotide, and the heteropolymer was the strongest of all. The strong signal seen with the heteropolymer probe is due to probe complexing during hybridization, in which additional binding between sense and antisense strands of the probe (i.e. poly-rA and poly-dT) amplifies the number of biotin molecules at the hybridization site; this strategy has been exploited by us as a means of visualizing low copy numbers of specific mRNAs.


Assessment of rejection in orthotopic human heart transplantation using proliferating cell nuclear antigen (PCNA) as an index of cell proliferation.

Myocardial biopsies taken during the management of cardiac transplantation were stained for proliferating cell nuclear antigen (PCNA). Counts of PCNA-positive interstitial cells were compared, in retrospect, with the reported histological grade of rejection. Biopsies without rejection had negligible numbers of PCNA-positive cells. Ascending grades of rejection were paralleled by an increase in the number of PCNA-positive cells [grade 1, 13 +/- 35 (mean +/- SD); grade 2a, 38 +/- 40; grade 2b, 91 +/- 75; grade 3, 170 +/- 78]. While highly significant, in statistical terms, the overlap in the counts between different grades means that prediction of rejection from the PCNA count alone is not feasible. Biopsies graded as 0 or 1 and which immediately preceded more severe rejection episodes showed no increase in PCNA-positive cells. The majority of PCNA-positive cells are fibroblasts, although in grade 2b and 3 rejection a small population of PCNA-positive T lymphocytes occurs. PCNA staining is also seen in cardiac myocytes immediately after transplantation, during rejection episodes, and late after transplantation in the absence of rejection. The positive PCNA staining of cardiac myocytes probably reflects DNA synthesis that occurs with the shift toward polyploidy in hypertrophy.


Correction of antigen-specific T-lymphocyte function by recombinant cytokines in children infected with human immunodeficiency virus type 1.

To determine the role of cytokines in the immunodeficiency of children infected with human immunodeficiency virus type 1 (HIV-1), we compared the antigen-specific (tetanus toxoid-induced) T-lymphocyte blastogenesis of HIV-1-infected patients with and without the addition of exogenous interleukin-1 and interleukin-2. Acquisition of in vitro antigen-specific immunologic function was seen in some patients after the addition of exogenous cytokines. The antigen-specific immunodeficiency in some HIV-1-infected children is due to defects in cytokine production rather than to an absence of antigen-specific T lymphocytes.


Phase I study of continuous-infusion soluble CD4 as a single agent and in combination with oral dideoxyinosine therapy in children with symptomatic human immunodeficiency virus infection.

To determine the safety and pharmacokinetics of recombinant soluble CD4 (sCD4) administered by continuous intravenous infusion to children with symptomatic human immunodeficiency virus type 1 infection, we conducted a phase I study at the National Cancer Institute. Three dose levels of sCD4 were evaluated: 100, 300, and 1000 micrograms/kg per day. After an initial 12 weeks of treatment with sCD4 alone, dideoxyinosine at a dose of 90 mg/m2 every 8 hours was added and subjects were observed for an additional 12 weeks. Combination therapy was continued in patients in whom it was well tolerated. In addition to toxicity and pharmacokinetic monitoring, surrogate markers of antiviral activity were evaluated. Eleven children were enrolled in the study. During the 12 weeks of treatment with sCD4 alone, and during subsequent sCD4 plus dideoxyinosine combination therapy, no significant toxic reaction attributable to sCD4 or dideoxyinosine was encountered. Low-level anti-CD4 antibodies developed in two patients. Steady-state sCD4 levels increased proportionately at higher doses. The CD4 cell counts and serum p24 antigen levels did not provide evidence of antiviral activity. We conclude that sCD4 was well tolerated at doses up to 1000 micrograms/kg per day when administered by continuous intravenous infusion; however, evidence of in vivo antiviral activity was not observed in this study.


Measurement of colonic tissue cyclosporine concentration in children with severe ulcerative colitis.

Recent studies suggest that cyclosporine may be an alternative to colectomy in children with severe ulcerative colitis who fail traditional therapy with bowel rest and high-dose corticosteroids. We report the clinical course of two such children. Patient 1 received oral cyclosporine at 15 mg/kg/day for 1 week followed by 8 mg/kg/day for 6 weeks. Clinical remission occurred within 5 days and has been sustained for 24 weeks after azathioprine was substituted for cyclosporine. Patient 2 received oral cyclosporine at 7-8 mg/kg/day for 7 days after which colectomy was performed due to lack of clinical improvement. Blood cyclosporine levels for patient 1 ranged from 95 to 406 ng/ml and for patient 2 from 36 to 65 ng/ml. Sigmoid colonic tissue cyclosporine concentrations for patients 1 and 2 were 10,058 and 3,205 ng/g, respectively. The patient who responded had significantly higher blood and colonic tissue cyclosporine concentrations than the patient who did not respond. Further studies with larger numbers of patients are needed to determine if there is a correlation between blood and colonic tissue cyclosporine concentrations and clinical response.


Comparative effects of atrial natriuretic peptide and E. coli heat-stable toxin on rat intestinal transport.

Conflicting data have been published in favor of or against a secretory effect of atrial natriuretic peptide (ANP) in the intestine. The reported effects resemble that of Escherichia coli heat-stable enterotoxin (ST). In this work the effects of ANP were studied in well established experimental systems and compared with that of ST. Both peptides induced a prompt secretion of water, Na, and Cl with no effects on K net transport in the in vivo rat perfused jejunum. The addition of ST, but not of ANP, evoked an increase of short circuit current in rat intestinal mucosa mounted in Ussing chambers. ST induced a significant increase in guanylate cyclase activity in intestinal homogenates, whereas ANP showed no effect. No binding sites for ANP were detected in basolateral or brush border membranes, nor in isolated enterocytes by a suction filtration technique. In conclusion, ANP acts as a short-lived intestinal secretagogue in the rat. Its mechanism of action is different from that of E. coli ST and appears to be indirect, since is not mediated by specific intestinal receptors and is not evident in vitro.


Pancreatoduodenectomy with preservation of the stomach and pylorus: a safe and effective alternative in children.

The time-honored surgical approach to resectable tumors of the head of the pancreas or distal common bile duct has been a Whipple operation for adults and children. In recent years, pylorus-preserving pancreatoduodenectomy has been used for adults. The postoperative physiological advantages may be enhanced in children, and for this reason the pylorus-sparing approach was used in a 15-year-old girl with a tumor of the distal common duct. The pylorus-preserving pancreatoduodenectomy was safe and effective in this patient and should be considered for children with resectable tumors of the distal common bile duct and pancreatic head.


Inhibition of adhesion of S-fimbriated Escherichia coli to epithelial cells by meconium and feces of breast-fed and formula-fed newborns: mucins are the major inhibitory component.

We investigated the ability of meconium, feces from human milk-fed (HMF) newborns, and feces from formula-fed (FF) newborns to inhibit adhesion of S-fimbriated E. coli to human buccal epithelial cells. S-fimbriae are a common property of E. coli strains causing sepsis and meningitis in neonates. Meconium had the highest content of neuraminic acid and the strongest inhibitory effect on bacterial adhesion. HMF also exerted high inhibitory activity while FF was markedly less active: To achieve inhibitory effects comparable to HMF a sixfold amount of FF was required. Glycoproteins from excretions were separated by gel chromatography. Fractions obtained were analyzed for adhesion-inhibiting activity. In all excretions analyzed, the mucin-containing fraction could be identified as the major inhibitory component. Inhibition was probably mediated by specific interaction of this fraction with S-fimbriae, as shown by binding of isolated fimbriae on Western blots after electrophoretic separation of glycoproteins. In conclusion, our data support the view that the mucin-containing fraction from meconium and human milk exerts antibacterial functions by preventing adhesin-mediated binding of pathogenic bacteria to mucosal epithelia.


Hydrolysis kinetics of phospholipids in thermally stressed intravenous lipid emulsion formulations.

A model 20% w/v emulsion, prepared with either a commercially available pharmaceutical grade soy oil or a highly purified grade of oil from the same origin and stabilized with a commercially available mixture of egg yolk phospholipids was passed through a Microfluidics homogenizer until the mean particle size fell below 500 nm diameter. Samples stored in sealed all-glass ampoules were thermally stressed over a temperature range of 5-90 degrees C and samples taken at appropriate intervals for analysis by HPLC. Hydrolysis degradation kinetics were in conformation with the Arrhenius equation. The energy of activation for phosphatidylcholine was virtually identical for emulsions prepared with either pharmaceutical or purified oil (65 and 63 kJ mol-1, respectively). For phosphatidylethanolamine itself the respective activation energies were 53 and 54 kJ mol-1, suggesting that the source of the oil used in preparing the emulsions had no significance in the degradation processes of the resulting systems.


Epi-fluorescence microscopy and image analysis used to measure diffusion coefficients in gel systems.

A method using epi-fluorescence microscopy and image analysis has been developed to follow and quantify the diffusion of fluorescent compounds through gels. Two mathematical approaches were employed to calculate diffusion coefficients. The spatial resolution provided by the fluorescence microscope allowed diffusion to be followed over very short distances; accordingly diffusion coefficients were obtained within minutes, even for slowly diffusing systems. The method was successfully applied to the diffusion of macromolecules into agar, carbopol and mucus gel systems.


Improvement of large intestinal absorption of insulin by chemical modification with palmitic acid in rats.

The intestinal absorption of 125I-labelled palmitoyl insulin was examined following administration into in-situ closed large intestinal loops of rats. When mono- and dipalmitoyl insulins (Palins-1 and Palins-2, respectively) were administered in polyoxyethylene hydrogenated castor oil (HCO 60) micellar system into intestinal loops, a marked increase in plasma radioactivity and a corresponding disappearance of residual radioactivity in the intestinal lumen were observed in the following rank order: Palins-2 greater than Palins-1 greater than native insulin. In addition, the derivatives were more stable than native insulin in the mucosal tissue homogenates of the large intestine. These results suggest that chemical modification of insulin with palmitic acid may not only increase the lipophilicity of insulin but also reduce its degradation, resulting in the increased transfer of insulin across the large intestinal mucous membrane. The linoleic acid-HCO 60 mixed micelles system did not have a significant effect on the large intestinal absorption of radioactivity associated with the lipophilic insulin analogues.


Effect of oily vehicles on absorption of mepitiostane by the lymphatic system in rats.

[14C]Mepitiostane in various vehicles was administered to the small intestine of anaesthetized rats with cannulated thoracic ducts, and the effect of lipids on lymphatic absorption was examined. The extent of lymphatic absorption was greatest when administered in triolein and sesame oil, which are triglycerides of long-chain fatty acids. Absorption in the presence of other vehicles was in the order of 10% Tween 80 aqueous solution greater than monolein greater than oleic acid approximately oleic acid/monolein (2:1 mol/mol) greater than aqueous suspension. Differences between the extents of lymphatic absorption of mepitiostane in the various formulations were not due to variation in the lymph flow but to the increased secretion of chylomicron and very low density lipoproteins. During absorption of mepitiostane from the small intestine, oil affected not only the penetration into epithelium cells and the metabolism in them, but also the partition between blood and lymph.


Effect of bile on absorption of mepitiostane by the lymphatic system in rats.

The effects of bile and site of gastrointestinal absorption on the lymphatic absorption of the highly lipophilic drug, mepitiostane were examined using thoracic duct-cannulated rats. The lymphatic absorption from the small intestine was very small in the absence of bile compared with that when bile was present. The lymphatic absorption was greatest when drug was administered to the upper small intestine with bile, was smaller for the lower regions of the small intestine, and was negligible for the stomach and the large intestine. A correlation was observed between the extent of lymphatic absorption and the secretion of chylomicron and very low density lipoproteins after administration to various regions with or without bile. The portal absorption data of mepitiostane confirmed that site specificity occurs in the partition of drug between blood and lymph.


Effects of salicylate and other enhancers on rectal absorption of erythropoietin in rats.

To develop a new treatment for patients with anaemia in which erythropoietin (EPO) can be given without injection, the effects of promoters of the rectal absorption of EPO were studied. Recombinant human (rHu) EPO (5000 units) in a dosing solution or in a rectal suppository was placed in the rectum of healthy rats and changes in serum EPO levels were monitored by an enzyme-linked immunosorbent assay. Without a promoter, rHuEPO was not absorbed. Sodium glycocholate, sodium caprate, and sodium salicylate in the solution of rHuEPO increased the absorption of rHuEPO. Sodium salicylate or sodium caprate in the suppository with rHuEPO also increased its absorption. The bioavailability of rHuEPO in a suppository containing 5% sodium salicylate compared with that by an intravenous injection was 1.2%. rHuEPO given in rectal suppositories containing sodium salicylate and inserted once a day for 6 consecutive days increased erythropoiesis in peripheral blood.


Pharmacology of nebivolol.

Nebivolol is a mixture of equal amounts of two enantiomers: SR3-nebivolol (d-nebivolol) and RS3-nebivolol (l-nebivolol). SR3-nebivolol is a potent and selective beta 1-adrenergic antagonist both in vitro and in vivo. Nebivolol acutely lowers blood pressure in spontaneously hypertensive rats and induces a slight decrease in total peripheral vascular resistance and a slight increase in cardiac output in anaesthetised dogs. These hemodynamics effects cannot be explained by beta 1-adrenergic antagonism and are largely attributable to RS3-nebivolol.


Interaction between cicletanine and the eicosanoid system in human subcutaneous resistance arteries.

In human subcutaneous resistance arteries in-vitro, the relaxation produced by the novel furopyridine compound cicletanine (30 microMs) was inhibited by 51% (P less than 0.0001) in the presence of 20 microMs of the cyclo-oxygenase inhibitor indomethacin. Maximal cicletanine-induced relaxation was reduced by both the cyclo-oxygenase inhibitor mefenamic acid and the relatively specific blocker of prostacyclin synthetase, tranylcypromine by 55% (P less than 0.0005) and 43% (P less than 0.01), respectively. The potassium channel blocker, glibenclamide (100 microMs), did not affect the relaxation produced by cicletanine but did inhibit the relaxation produced by the potassium channel blocker cromakalim (P less than 0.0001). Cromakalim-induced relaxation was inhibited in the presence of indomethacin; relaxation induced by cromakalim (30 microMs) was reduced by 22% (P less than 0.02). The relaxation produced by the hypotensive agonists sodium nitroprusside, hydrochlorothiazide, bumetanide and nicardipine was unaffected by incubation with indomethacin. The results suggest that the vascular eicosanoid system, specifically prostacyclin, may be involved in the mechanism of the acute vasodilator action of cicletanine. Although at high doses, cicletanine is a diuretic, in this model it did not act like either a thiazide or a loop diuretic. The acute vasodilator action of the thiazide diuretic, hydrochlorothiazide was a novel finding of this study. Cromakalim showed a reduced response in the presence of indomethacin suggesting an involvement of the eicosanoid system in its mechanism of action.


Tetrandrine and isotetrandrine, two bisbenzyltetrahydroisoquinoline alkaloids from Menispermaceae, with rat uterine smooth muscle relaxant activity.

The effects of two bisbenzyltetrahydroisoquinoline alkaloids, 1S,1'S tetrandrine and its isomer 1R,1'S isotetrandrine, were investigated in rat isolated uterus in order to identify the mechanism of relaxant action and to study the influence of the absolute configuration on the activity of these alkaloids. Both inhibited the uterine contraction induced by high K+, acetylcholine and oxytocin. In Ca(2+)-free medium, isotetrandrine relaxed the sustained contraction induced by oxytocin but tetrandrine did not. The relaxant effects of the alkaloids may be due to blockade of calcium influx through specific channels. Tetrandrine and isotetrandrine modify the calcium channel in a nonreversible manner whilst only isotetrandrine acts intracellularly. Tetrandrine shows a more specific relaxant activity as a calcium entry blocker.


In-vivo blood-brain barrier transport of a novel adrenocorticotropic hormone analogue, ebiratide, demonstrated by brain microdialysis and capillary depletion methods.

The transport of ebiratide, a novel adrenocorticotropic hormone (ACTH) analogue, [H-Met-(O2)-Glu-His-Phe-D-Lys-Phe-NH(CH2)8-NH2], through the blood-brain barrier was directly demonstrated in-vivo. [125I]Ebiratide (16.9 MBq mL-1) or [14C]sucrose (29.2 MBq mL-1) known to be restrictively transported through the blood-brain barrier was infused into the rat internal carotid artery at a flow rate of 50 microL min-1 for 10 min, and after 15 min infusion the distribution volume of each compound in the brain parenchyma was determined by the capillary depletion method. The distribution volume of [125I]ebiratide was 167.8 +/- 62.2 microL (g brain)-1, which was about seven times higher than that of [14C]sucrose (24.9 +/- 4.0 microL g brain)-1, indicating the uptake of ebiratide into brain parenchymal cells. During the infusion into the internal carotid artery, brain microdialysis was simultaneously performed to directly collect the brain interstitial fluid as the dialysate. Radioactivity was detected in the dialysate during the [125I]ebiratide infusion and HPLC analysis of the dialysate revealed that the intact ebiratide accounted for greater than or equal to 80% total radioactivity. The concentrations of [125I]ebiratide and [14C]sucrose in the brain interstitial fluid were estimated based on the relative recovery obtained in the in-vitro recovery study. The brain interstitial fluid/internal carotid arterial blood concentration ratio for [125I]ebiratide was determined to be 1.47 x 10(-2) +/- 0.17 x 10(-2) and was about eight times higher than that for [14C]sucrose (1.92 x 10(-3) +/- 0.36 x 10(-3)), indicating significant transport of ebiratide to the brain interstitial fluid.(ABSTRACT TRUNCATED AT 250 WORDS)


Design, synthesis and biological activity of a series of torasemide derivatives, potent blockers of the Na+ 2Cl- K+ co-transporter: in-vitro study.

Pharmacomodulation of the torasemide molecule, a loop diuretic inhibiting Na+ 2Cl- K+ co-transport in the thick ascending limb of the loop of Henlé has been performed in order to obtain new long-acting diuretics. The aim of this study was to decrease the metabolism of the drug and to slow down its rate of excretion by increasing its hydrophobicity. The present study describes the synthesis and the inhibitory potency of new torasemide derivatives in the bioassay system of the cortical thick ascending limb of rabbit. A correlation between the lipophilicity (log P') of these substances and their activity as inhibitors of the Na+ Cl- K+ co-transporter was observed. The present design led to compounds more active than torasemide. Structure-activity relationships permit us to propose an interaction model between torasemide derivatives and the Na+ 2Cl- K+ co-transport system of the cortical thick ascending limb.


Interspecies differences and scaling for the pharmacokinetics of xanthine derivatives.

Pharmacokinetic characteristics of the new xanthine bronchodilators, enprofylline and 1-methyl-3-propylxanthine (MPX), were investigated in mice, rats, guinea-pigs, rabbits and dogs. The possibility of an interspecies pharmacokinetic scale was also evaluated. The concentration of these two drugs in plasma and urine was determined by HPLC. Pharmacokinetic parameters were calculated using model-independent methods. The disappearance curves of the two drugs from plasma varied markedly among animal species. Interspecies differences in the plasma protein binding of each drug were observed for all animals in the study. Differences in the biotransformation of enprofylline and MPX were also confirmed among the various animal species: enprofylline is mainly excreted in an unchanged form in urine while MPX follows a non-renal route of elimination. In all animals, the renal clearance for enprofylline was greater than the glomerular filtration rate, indicating active tubular secretion. Significant allometric relationships were seen between the values of total body clearance and steady state volume of distribution for both total and unbound enprofylline and species body weight, but similar correlations could not be recognized for MPX. Renal clearance of enprofylline was also closely correlated with species body weight, suggesting no interspecies difference with relation to affinity and/or capacity for the active tubular secretion mechanism of enprofylline. Our findings suggest that xanthine derivatives, including enprofylline, are mainly eliminated via the kidney, and an estimate of the basic pharmacokinetics in man can be obtained from data in experimental animals.


Enhancement of the stability of thrombin by polyols: microcalorimetric studies.

Glycerol increased the transition temperature (Tm) of thrombin in a concentration-dependent fashion up to a concentration of 50% glycerol in aqueous buffer solution. Glycerol showed a comparable effect on Tm of trypsin. This effect on Tm of thrombin was not seen in the presence of excess sodium chloride (1.2 M) in aqueous buffer solution. The stabilizing effect of glycerol may be due to increased energy demand to unfold the protein molecule, as reflected by an increase in Tm. This stabilizing effect, as measured by Tm, was seen for other polyols, including sucrose, and was also dependent on the concentration of the stabilizing agent. Microcalorimetry may be used as an effective tool to screen for the protective action of compounds in enzyme stabilization studies before conducting the time-consuming and expensive stability studies of proteins in the presence of additives under different storage conditions.


Failure of tetrodotoxin to inhibit the prostaglandin-induced secretory response of rat small intestine in-vitro.

Tetrodotoxin (TTX, 10 microM) did not inhibit prostaglandin E2 (PGE2)-stimulated increases in electrical activity in intact or stripped sheets of rat small intestine, although it reduced basal electrical activity in the intact preparation. Basal and PGE2-stimulated cAMP production by enterocytes isolated from the small intestine was unaffected by TTX. Thus its reported ability to inhibit prostaglandin-induced fluid secretion in-vivo does not appear to represent a direct interaction of the neurotoxin with the mechanism of prostaglandin action at the enterocyte.


The effect of temperature and pH on the deacetylation of diamorphine in aqueous solution and in human plasma.

The effect of temperature on the kinetics of the deacetylation of diamorphine and 6-monoacetylmorphine was studied in human plasma. Diamorphine was rapidly and quantitatively degraded to 6-monoacetylmorphine with initial half-lives of 354, 18 and 3 min at temperatures of 4, 25 and 37 degrees C, respectively. Further deacetylation to morphine was not detected. In aqueous solution, diamorphine was quantitatively degraded to give 6-monoacetylmorphine as the major product and morphine as a minor product, the rate of deacetylation being dependent on temperature and pH. At pH 4.0 and 5.6 diamorphine had a half-life of greater than 14 days at all temperatures but at alkaline pH diamorphine was rapidly deacetylated. The rate of deacetylation of 6-monoacetylmorphine was consistently slower than that of diamorphine under identical conditions of pH and temperature. A method is described for the rapid stabilization and subsequent assay of diamorphine in plasma which will prevent errors in estimation of the drug due to unwanted hydrolysis.


Effect of exogenous copper on lipid peroxidation in rat hepatocytes. Possible involvement of protein kinase C.

We have investigated the direct effect of copper on malondialdehyde formation in rat isolated hepatocytes. Copper was found to decrease the cell viability with concomitant production of malondialdehyde in a time related manner. In addition the protein kinase C activator, PMA, was found to have a synergistic effect with copper on rat hepatocytes. These results indicate that protein kinase C may be important in mediating hepatotoxicity after exposure to copper.


Induction of propranolol metabolism in the Hep G2 human hepatoma cell line.

Metabolism of propranolol by the human hepatoma cell line Hep G2 was studied. Although metabolism qualitatively was similar to that in-vivo, the P450-mediated N-desisopropylation clearly predominated. Pretreatment of cells with 3-methylcholanthrene increased the activity of this pathway 14-fold, whereas phenobarbitone had no effect. This is similar to the pathway-selective inductive response observed for cigarette smoking in-vivo. As in-vivo, secondary metabolism of N-desisopropylpropranolol was extensive. This could, however, be completely blocked by 0.1 microM clorgyline, a potent MAO type A inhibitor. As in human liver microsomes, the stereochemistry of propranolol metabolism demonstrated a preference for the R(+)-enantiomer. These observations emphasize the usefulness of the Hep G2 cell line as a model of man.


Atrial natriuretic peptide inhibits the spontaneous contractions of rabbit isolated ileum.

The present study investigates the effects of atriopeptin II on spontaneous phasic contractions of rabbit isolated ileum. Atriopeptin II caused a significant and concentration-dependent decrease in ileum motor activity. This effect was mimicked by 8-Br-cGMP and it was not affected by pretreatment with tetrodotoxin. Verapamil significantly decreased ileum contractions; however, in the presence of this calcium blocker, atriopeptin II further reduced ileal motility. These findings demonstrate that atriopeptin II depresses the motility of rabbit ileum through a cGMP-dependent mechanism and suggest that neither ileal neural networks nor extracellular calcium are involved in this effect.


The pharmacokinetics of oral itraconazole in AIDS patients.

To test the hypothesis that absorption of orally administered itraconazole may be reduced in patients with the Acquired Immunodeficiency Syndrome (AIDS), 8 patients with AIDS were given two 100 mg capsules of itraconazole for 15 days. Plasma levels were measured by HPLC hourly for the first 8 h following ingestion, then at 24, 48, 72 and 96 h on days 1, 8 and 15. Peak plasma levels occurred after approximately 4 h. Mean peak plasma concentrations were 446 +/- 196 and 530 +/- 214 ng mL-1 at days 8 and 15. The area under the curve over 24 h (AUC0-24) was 2105 +/- 1241, 7679 +/- 3838 and 8748 +/- 4385 ng mL-1 h for days 1, 8 and 15, respectively. Steady-state was achieved after one week of dosing. There was no evidence of hepatotoxicity and one patient stopped itraconazole due to a rash. It may be concluded that the absorption of oral itraconazole capsules is reduced in AIDS patients, by a factor of approximately 50%, when compared with normal volunteers. AIDS patients may require higher doses of itraconazole than used in non-AIDS patients to achieve comparable plasma levels.


Disposition of asarone after intravenous administration to rabbits assessed using HPLC.

A simple and sensitive high-performance liquid chromatography (HPLC) method for the determination of asarone in rabbit plasma has been developed. Up to 0.1 mL of plasma containing asarone was deproteinated by acetonitrile, which contained an internal standard (indomethacin). The supernatant was injected into a Nucleosil 7C18 column using acetonitrile-water-triethylamine (55:45:0.1 v/v, pH 5.4-5.5, adjusted with orthophosphoric acid) as the mobile phase and UV detection at 257 nm, followed by UV spectrum identification (between 200 and 380 nm) with a photodiode array detector. The method is rapid, easily reproduced, selective and sensitive. It was applied to pharmacokinetic studies of asarone in rabbit, after 5, 10, or 20 mg kg-1 intravenous administration. Rapid distribution followed by a slower elimination phase was observed from the plasma concentration-time curve. The plasma disposition at each dose fitted well to a two-compartment open model and the terminal disposition became much slower as the dose was increased, suggesting a nonlinear dose-dependent plasma asarone disposition.


An in-vitro comparison of controlled release aminophylline tablets: Phyllocontin Continus and Pecram.

It has been suggested that two controlled release preparations containing aminophylline, Phyllocontin Continus and Pecram, are clinically equivalent and are therefore interchangeable. In this study, an in-vitro evaluation of the two preparations was completed using the British Pharmacopoeia dissolution apparatus, initially using water and then an acid/buffer medium to provide a similar pH environment to that within the gastrointestinal tract. Similar release profiles were found when water was used as the dissolution medium, with very little variation between tablets within each group. Good fits were obtained for dissolution-controlled release and diffusion-controlled release models. When the acid/buffer solution was used as the dissolution medium a reduction in the rate of release was observed with Phyllocontin. It was predicted that if this was repeated in-vivo then differences in the peak plasma levels between the two formulations would be seen, although these may be masked by the other variables encountered.


Risk of developing cytomegalovirus retinitis in persons infected with the human immunodeficiency virus.

This study examines the risk of developing cytomegalovirus (CMV) retinitis as a function of the duration and degree of CD4+ lymphocyte depletion. A retrospective analysis of 135 persons infected with the human immunodeficiency virus (HIV) was performed. Kaplan-Meier estimates for the percentage of patients developing CMV retinitis during the 27-month study period were calculated. Twenty-six patients were diagnosed as having CMV retinitis. In 14 of these patients, T cell phenotyping was done within the 3 months preceding diagnosis. The mean CD4+ lymphocyte count for these patients was 15.6 cells/mm3 (range, 2-33/mm3). At 27 months, the percentage of patients developing CMV retinitis with baseline CD4+ lymphocyte counts of 0-50, 51-100, and 101-250 cells/mm3 was 41.9%, 26.3%, and 14.7%, respectively (log-rank test, p = 0.003). The odds ratio for developing CMV retinitis for those with baseline CD4+ lymphocyte counts of 0-50 cells/mm3 compared with those with CD4+ lymphocyte counts of 101-250 cells/mm3 was 4.62 (p = 0.002). Twenty-four patients had CD4+ lymphocyte counts of < or = 50 cells/mm3 for an average of 13.1 months prior to diagnosis. Twenty-two patients had an acquired immune deficiency syndrome (AIDS)-defining illness diagnosed for an average of 18.0 months prior to the onset of retinitis. CMV retinitis is most likely to develop in patients with AIDS when the CD4+ lymphocyte count is < or = 50 cells/mm3.


Randomized, placebo-controlled, double-blind study of low-dose oral interferon-alpha in HIV-1 antibody positive patients.

One hundred forty-nine patients of private physicians in Toronto, Canada, who were positive for human immunodeficiency virus (HIV), medically stable, and had CD4 cell counts of < 700 cells/mm3 participated in a randomized, double-blind trial of placebo versus low-dose (50 U) versus high-dose (100 U) oral interferon-alpha. Treatment allocation was balanced according to baseline CD4 cell count and history of prior antiviral therapy. Patients were observed at 4 and 8 weeks for assessment of adverse events and several measures of disease status, including CD4 cell count, beta 2-microglobulin, weight, and Karnofsky score. We detected neither short-term benefits nor adverse effects from oral interferon-alpha therapy.


Nutritional status and food intake in human immunodeficiency virus infection. GI/HIV Study Group.

Nutritional status and food intake of HIV+ and HIV- homosexual men that were free from enteric pathogens were compared. Food intake (7-day weighed record), anthropometry, and D-xylose excretion were measured in 44 patients (9 HIV-, 35 HIV+). HIV+ patients were found to be thinner, based on anthropometric measurements of skinfold thickness (p < 0.05) and percentage body fat (p < 0.05), and they also tended to be lighter than the HIV- patients. No differences were observed in the arm muscle mass or the food intake of the two groups. In the HIV+ patients, regression analysis was used to correlate changes in nutritional status with progression of the disease, using CD4+ lymphocyte count as a measure of severity. A decrease in CD4 count positively correlated with a decrease in weight (r = 0.48, p < 0.01), body mass index (r = 0.41, p < 0.05), and arm muscle area (r = 0.42, p < 0.01). Energy intake (r = 0.67, p < 0.01), serum albumin (r = 0.52, p < 0.01), and D-xylose excretion (r = 0.57, p < 0.0001) also positively correlated with CD4 count. Multiple regression analysis revealed a relationship between CD4 count, weight, and energy intake, indicating that as the disease progresses, a decline in weight is seen parallel to a reduction in food intake. These data indicate that changes in body composition and nutritional status are present throughout the stages of HIV disease, though no causal relationships can be interpreted from this study. The initial changes appear to be due to loss of fat stores, as determined by anthropometry.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of polymorphs and solvates of 3-amino-1-(m-trifluoromethylphenyl)-6-methyl-1H-pyridazin-4-one.

The characterization of two polymorphs of the title compound (F2692; 1) by differential scanning calorimetry (DSC), microanalysis, proton nuclear magnetic resonance spectroscopy, thermogravimetry, thermomicroscopy, infrared spectroscopy, and X-ray diffractometry is described. Both polymorphs are crystalline, with form II being more stable at temperatures less than 160 degrees C. The thermal behavior was studied at different rates of heating, and the enthalpies of transition were calculated from DSC data. The transformation of aqueous suspensions of form I to the water-stable form II is described, and the heats of solution and intrinsic aqueous dissolution rates of both polymorphs were determined. 1 also formed solvates with dimethyl sulfoxide and 1-methyl-2-pyrrolidinone. The solvates were studied by thermogravimetry, DSC, and infrared spectroscopy.


Nicotinic receptor-evoked release of acetylcholine and somatostatin in the myenteric plexus is coupled to calcium influx via N-type calcium channels.

Entry of extracellular calcium (Ca++) via voltage-gated Ca++ channels is essential for neurotransmitter release. In this study, we examined whether nicotinic receptor-stimulated release of acetylcholine (ACh) and somatostatin (S14) are coupled to calcium influx via distinct calcium channel subtypes in the myenteric plexus. Isolated ganglia from the guinea pig ileal myenteric plexus were prepared and placed in perfusion chambers under standard conditions. The ganglionic agonist dimethylphenylpiperazinium (DMPP, 10(-6) to 10(-3) M) stimulated the release of [3H]ACh in a concentration-dependent manner. This release was blocked by hexamethonium or Ca(++)-free medium containing 1 mM EGTA and was antagonized by omega-conotoxin, a preferential N calcium channel blocker, but was not affected by nifedipine (L channel antagonist) or nickel (T calcium channel antagonist). DMPP-evoked release of somatostatin was also antagonized by omega-conotoxin, but was not affected by nifedipine or nickel. These observations indicate that neurosecretion of ACh and S14 evoked by DMPP is mediated by calcium entry via voltage-sensitive N-type Ca++ channels. To provide additional evidence that nicotinic receptor stimulation is associated with Ca++ entry via the N-type Ca++ channels, we examined the intracellular calcium [Ca++]i concentration of the myenteric plexus neurons using fura-2 microspectrofluorometry. Basal [Ca++]i of single ileal myenteric neurons was 65 +/- 5 nM. Perfusion with DMPP (10(-6) to 10(-3) M) caused a rapid, transient elevation in [Ca++]i which was abolished by Ca(++)-free medium containing 1 mM EGTA.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of [3H]YM617, R-(-)-5-[2-[[2[ethoxyring(n)-3H](o-ethoxyphenoxy)ethyl]amino]- propyl]-2-methoxybenzenesulfonamide HCl, a potent and selective alpha-1 adrenoceptor radioligand.

The binding properties of a new radioligand, R(-)-5-[2-[[2[ethoxyring(n)-3H](o- ethoxyphenoxy)ethyl]amino]propyl]-2-methoxybenzenesulfonamide++ + HCl ([3H]YM617), were studied in membranes of the rat hippocampus and spleen. [3H]YM617 rapidly associated with its binding sites in both membranes and reached steady state by 20 min at 25 degrees C. The specific binding of [3H]YM617 appeared to be saturable, and Scatchard analysis revealed a linear plot, suggesting a single population of binding sites with a dissociation constant of 0.170 +/- 0.016 nM (n = 6) in the hippocampus and 0.195 +/- 0.036 (n = 4) in the spleen. The maximal binding sites in the hippocampus and spleen were 203.0 +/- 43.2 (n = 6) and 72.4 +/- 17.0 (n = 4) fmol/mg protein, respectively. Chlorethylclonidine (10(-5) M for 10 min) treatment reduced the Bmax values of [3H]YM617 and [3H]prazosin to a similar degree in the rat hippocampus (10-15%) and spleen (40-50%). Alpha adrenoceptor agonists and antagonists competed with [3H]YM617 for binding sites in the following order: YM617 > prazosin > WB4101 > bunazosin > 5-methylurapidil > S(+)-isomer of YM617 > phentolamine > yohimbine > norepinephrine = phenylephrine > methoxamine in the hippocampus, and prazosin > YM617 > bunazosin > WB4101 > 5-methylurapidil > phentolamine > S(+)-isomer of YM617 > yohimbine > norepinephrine > phenylephrine > methoxamine in the spleen. In the hippocampus, prazosin and bunazosin produced biphasic displacement of [3H]YM617, but not [3H]prazosin binding. In contrast, only monophasic curves were obtained against either radioligand in the spleen.(ABSTRACT TRUNCATED AT 250 WORDS)


Evidence that S-nitrosothiols are responsible for the smooth muscle relaxing activity of the bovine retractor penis inhibitory factor.

Inhibitory factor (IF), an extract of the bovine retractor penis muscle, when treated with acid, becomes a vasodilator with properties similar to endothelium-derived relaxing factor (EDRF). EDRF has been proposed to be nitric oxide (NO), long known to be a potent vasodilator. Recently, biologically active IF was proposed to be NO, as well, generated by acid activation of inorganic nitrite. We compared acid-activated IF with acid-activated nitrite and found that NO formation was not sufficient to explain the properties of acid-activated IF. Endothelium-denuded rings of rabbit aorta were used to test the smooth muscle-relaxing properties of IF and nitrite. Although both IF (0.5 ml) and nitrite (1 microM) relaxed phenylephrine-contracted rabbit aorta to a similar extent after acid activation (approximately 30%), several significant differences were observed. IF was most active when acid activated by a 5-min, pH 2 step followed by neutralization; nitrite was relatively inactive when acid activated in this manner, and was most active when assayed immediately after acidification to pH 2. Purging with argon for 5 min reduced the smooth muscle-relaxing activity of 1.0 microM nitrite from 27 +/- 2 to 10 +/- 2% relaxation, whereas the activity of IF was not changed by argon purging (control, 31 +/- 2% relaxation; argon purged, 34 +/- 2% relaxation). When IF samples were assayed for nitrite content, the amount of nitrite found (0.5-5 nmol/0.5 ml sample) was not sufficient to explained the observed smooth muscle relaxing activity. Furthermore, acid-activated IF significantly stimulated cyclic GMP production by platelet-soluble guanylate cyclase from 3.2 +/- 0.2 to 12.4 +/- 0.4 pmol/min/mg protein.(ABSTRACT TRUNCATED AT 250 WORDS)


Tolerance and cross-tolerance to the activating effects of 3,4-methylenedioxymethamphetamine and a 5-hydroxytryptamine1B agonist.

Previous studies indicate that 3,4-methylenedioxymethamphetamine (MDMA) produces locomotor hyperactivity in rats by increasing the presynaptic release of serotonin (5-HT). Interactions between MDMA and 5-HT receptor antagonists suggest that the activating effects of MDMA are mediated via 5-HT1-like receptors. In order to assess the contribution of particular 5-HT receptor subtypes to the behavioral effects of MDMA, the present studies examined the development of tolerance and cross-tolerance to the behavioral effects of MDMA and selective 5-HT receptor agonists. In the first study, rats were pretreated with saline, a 5-HT1A receptor agonist (8-hydroxy-2-(di-n-propylamino)tetralin, 1.0 mg/kg), a 5-HT1B receptor agonist [5-methoyx-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole butane dioate (RU24969), 2.5 mg/kg] or a 5-HT1C/5-HT2 receptor agonist (2,5-dimethoxy-4-iodoamphetamine, 1.0 mg/kg) twice daily for 3 days. The behavioral response to S-MDMA (3.0 mg/kg) was assessed 36 hr after the last pretreatment injection. Pretreatment with RU24969 antagonized the activating effects of S-MDMA. In contrast, pretreatment with 2,5-dimethoxy-4-iodoamphetamine did not alter the response to S-MDMA, and pretreatment with 8-hydroxy-2-(di-n-propylamino)tetralin reduced the activity of both control and S-MDMA-treated animals. In the second study, rats were pretreated with saline or RS-MDMA (10 mg/kg), twice daily for 4 days. The behavioral response to saline, S-MDMA (3.0 mg/kg), RU24969 (2.5 mg/kg) or S-amphetamine (1.0 mg/kg) was assessed 36 hr after the last pretreatment injection.(ABSTRACT TRUNCATED AT 250 WORDS)


N-methyl-D-aspartic acid (NMDA) and non-NMDA receptors regulating hippocampal norepinephrine release. III. Changes in the NMDA receptor complex induced by their functional cooperation.

N-methyl-D-aspartic acid (NMDA) and non-NMDA ionotropic receptors mediating increase of norepinephrine (NE) release coexist on NE rat hippocampus axon terminals. Activation of non-NMDA receptors permits activation of NMDA receptors also in presence of Mg++ ions and induces important changes in the NMDA receptor recognition site and in its intrinsic ion channel. We have now studied the effects of this receptor-receptor interaction on the glycine site of the NMDA receptor by using two antagonists, 7-chloro-kynurenic acid and (+-)-3-amino-1-hydroxy-2-pyrrolidone (HA-966). Both the [3H]NE releases induced from rat hippocampus synaptosomes by NMDA (no Mg++ added) and by NMDA+quisqualic acid (QA), in the presence of 1.2 mM Mg++, were prevented by 7-chloro-kynurenic acid with almost identical potency (IC50 values: 0.19 and 0.39 microM, respectively). In contrast, HA-966, up to 1000 microM, was ineffective toward NMDA (no Mg++), but it blocked the effect of NMDA + QA in presence of Mg++ (IC50 = 1.1 microM). HA-966 also antagonized NMDA + QA in Mg(++)-free medium. Thus coactivation of non-NMDA and NMDA receptors seems to permit the antagonistic activity of HA-966. In the presence of Mg++, L-glutamic acid (L-Glu) enhanced [3H]NE release. The sensitivity of the L-Glu effect to various antagonists was similar to that of the effect of NMDA + QA, indicating that the NMDA receptor complex activated either by NMDA + QA or by the physiological transmitter L-Glu in presence of Mg++ ions undergoes dramatic conformational changes at the recognition site, at the ion channel as well as at the glycine site.


The role of adrenergic receptors in the initiation of vomiting and its gastrointestinal motor correlates.

We investigated the role of adrenergic receptors in the mechanisms of initiation of vomiting and its gastrointestinal (GI) motor correlates. The effects of clonidine, UK-14304, St-91, naphazoline, phenylephrine and isoproterenol were examined for their ability to initiate vomiting and its GI motor correlates. Only the alpha-2 adrenoceptor agonists UK-14304, clonidine, St-91 and naphazoline activated vomiting and its GI motor correlates. Tolerance of vomiting, but not its GI motor correlates, readily developed to all alpha-2 adrenergic receptor agonists but St-91. The responses to UK-14304 or clonidine were blocked by idazoxan, yohimbine, clonidine tolerance or high doses of phenoxybenzamine, but not by propranolol or prazosin. The responses to UK-14304 or clonidine were also blocked by fentanyl, 1-(1-naphthyl) piperazine, methysergide, SCH 22390 or scopolamine, but not by haloperidol, sulpiride, domperidone or naloxone. Adrenoceptor antagonists, clonidine tolerance or sympathetic blockade did not block vomiting or its GI motor correlates activated by apomorphine, CuSO4 or cholecystokinin-octapeptide. We concluded that alpha-2 adrenergic receptors of the chemoreceptive trigger zone can initiate vomiting and its GI motor correlates, but these receptors do not mediate vomiting induced by another chemoreceptive trigger zone stimulant, apomorphine, or stimulation of the GI tract using CuSO4. However, 5-hydroxytryptamine-2 serotonergic, muscarinic cholinergic and opiate receptors within the central nervous system participate in controlling emesis activated by alpha-2 adrenergic agonists. Peripheral adrenergic receptors do not mediate the GI motor correlates of vomiting.


Release of endogenous norepinephrine from rat hypothalamus by stimulation of N-methyl-D-aspartic acid receptors.

Release of endogenous dopamine and norepinephrine (NE) from rat hypothalamic slices superfused with Mg(++)-free medium in the presence of nomifensine and tyrosine was measured by high-performance liquid chromatography coupled to an electrochemical detector. Superfusion with L-glutamic acid or N-methyl-D-aspartic acid elicited a concentration-dependent release of NE but not of dopamine. The release of NE was transient, returning toward basal values despite the continued presence of the amino acid. Superfusion with 20 mM K+ caused a release of NE that declined at a slower rate. Mg++, DL-2-amino-5-phosphonopentanoic acid and MK-801 (D-5-methyl-10,11,dihydro-5H-dibenzo[a,d] cyclohepten-5-10-imine maleate), but not 6-cyano-7-nitroquinoxaline-2,3-dione, inhibited the L-glutamic acid-evoked release of NE. The release of NE by L-glutamic acid was virtually abolished by tetrodotoxin and by elimination of Ca++ from and inclusion of 2 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid in the superfusion medium. Repeated L-glutamic acid applications displayed a decreased response, whereas repeated exposure to 20 mM K+ did not. Exposure to L-glutamic acid in the absence of Ca++ (plus 2 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid) or in the presence of DL-2-amino-5-phosphonopentanoic acid did not reduce the effects seen on subsequent exposure to L-glutamic acid. Exposure to L-glutamic acid in the absence of Mg++ reduced the effect of a subsequent exposure to L-glutamic acid. These observations provide evidence for an indirect modulation of rat hypothalamic endogenous NE by the N-methyl-D-aspartate receptor.


Carvedilol, a new vasodilator and beta adrenoceptor antagonist, is an antioxidant and free radical scavenger.

The antioxidant effect of carvedilol, a new vasodilating, beta adrenoceptor blocker was studied and compared with five other beta blockers. Carvedilol rapidly inhibited Fe(++)-initiated lipid peroxidation, measured as thiobarbituric acid reactive substance (TBARS), in rat brain homogenate with an IC50 of 8.1 microM. Under the same conditions, the IC50 values of atenolol, pindolol propranolol, celiprolol and labetalol were over 1.0 mM. Carvedilol protected against Fe(++)-induced alpha-tocopherol depletion in rat brain homogenate with an IC50 of 17.6 microM; propranolol, celiprolol and labetalol, up to 200 microM, did not show any effect. Using dihydroxyfumarate/Fe(++)-ADP as a OH.radical generating system and 5,5-dimethyl pyrroline-N-oxide (DMPO) as a trapping agent, the characteristic DMPO-OH signals were monitored by electron paramagnetic resonance. Carvedilol dose-dependently decreased the intensity of the DMPO-OH signal, with an IC50 of 25 microM, whereas propranolol, at 500 microM, and U74500A, a 21-aminosteroid, at 100 microM, had no effect. The antioxidant effect of carvedilol mainly resides in the carbazole moiety, and the substitution of a hydroxyl group at certain positions on the phenyl ring of either carbazole or the ortho-substituted phenoxylethylamine part of carvedilol resulted in an increase in antioxidant activity. Furthermore, the protective effect of carvedilol analogs against OH.-mediated neuronal death positively correlated to their antioxidant effect. We conclude that carvedilol is a far more potent antioxidant than other commonly used beta blockers. The apparent mechanism of carvedilol's inhibition of lipid peroxidation is mainly via scavenging free radicals. This novel property of carvedilol may contribute to the known cardioprotective activity of this compound.


Comparison of tensile strength of solder joints by infrared and conventional torch technique.

Fabrication of a fixed partial denture may require a soldering step. This study compared soldering by a conventional torch procedure with an infrared soldering technique. Comparisons were made for tensile strength, porosity, and time efficiency between the two methods. No significant difference was found in ultimate tensile strength between the two types of solder joints and the nonsoldered control samples. Random samples photographed with a scanning electron microscope revealed no difference in joint porosity between the two techniques. Torch soldering took consistently less time that infrared soldering.