Antinociceptive and antiamnesic properties of the presynaptic cholinergic amplifier PG-9

The antinociceptive effect of 3 alpha-tropyl 2-(p-bromophenyl)propionate [(+/-)-PG-9] (10-40 mg kg-1 s.c.; 30-60 mg kg-1 p.o.; 10-30 mg kg-1 i.v.; 10-30 micrograms/mouse i.c.v.) was examined in mice, rats and guinea pigs by use of the hot-plate, abdominal-constriction, tail-flick and paw-pressure tests. (+/-)-PG-9 antinociception peaked 15 min after injection and then slowly diminished. The antinociception produced by (+/-)-PG-9 was prevented by the unselective muscarinic antagonist atropine, the M1-selective antagonists pirenzepine and dicyclomine and the acetylcholine depletor hemicholinium-3, but not by the opioid antagonist naloxone, the gamma-aminobutyric acidB antagonist 3-aminopropyl-diethoxy-methyl-phosphinic acid, the H3 agonist R-(alpha)-methylhistamine, the D2 antagonist quinpirole, the 5-hydroxytryptamine4 antagonist 2-methoxy-4-amino-5-chlorobenzoic acid 2-(diethylamino)ethyl ester hydrochloride, the 5-hydroxytryptamin1A antagonist 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine hydrobromide and the polyamines depletor reserpine. Based on these data, it can be postulated that (+/-)-PG-9 exerted an antinociceptive effect mediated by a central potentiation of cholinergic transmission. (+/-)-PG-9 (10-40 mg kg-1 i.p.) was able to prevent amnesia induced by scopolamine (1 mg kg-1 i.p.) and dicyclomine (2 mg kg-1 i.p.) in the mouse passive-avoidance test. Affinity profiles of (+/-)-PG-9 for muscarinic receptor subtypes, determined by functional studies (rabbit vas deferens for M1, guinea pig atrium for M2, guinea pig ileum for M3 and immature guinea pig uterus for putative M4), have shown an M4/M1 selectivity ratio of 10.2 that might be responsible for the antinociception and the anti-amnesic effect induced by (+/-)-PG-9 through an increase in acetylcholine extracellular levels. In the antinociceptive and antiamnesic dose range, (+/-)-PG-9 did not impair mouse performance evaluated by the rota-rod test and Animex apparatus.     

Antinociceptive profile of 3-alpha-tropanyl 2-(4-Cl-phenoxy)butyrate (SM-21) [corrected]: a novel analgesic with a presynaptic cholinergic mechanism of action

The antinociceptive effect of (+/-)-3-alpha-tropanyl 2-(4-Cl-phenoxy)butyrate [corrected] (SM-21) (10-40 mg kg(-1) s.c., 10-30 mg kg(-1) i.p., 20-60 mg kg(-1) p.o., 3-20 mg kg(-1) i.v. and 5-20 microg per mouse i.c.v.) was examined in rodents and guinea pigs by using the hot-plate, abdominal constriction, tail-flick and paw-pressure tests. The antinociception produced by (+/-)-SM-21 was prevented by atropine, pirenzepine and hemicholinium-3 but not by quinpirole, R-(alpha)-methylhistamine, [1-[2(methylsufonyl)amino]ethyl]-4-piperidinyl]methyl-5-floro++ +-2-methoxy-1H-indole-3-carboxylate hydrochloride, N6-cyclopentyladenosine, 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine hydrobromide, naloxone, 3-aminopropyl-diethoxy-methyl-phosphinic acid or reserpine. On the basis of the above data, it can be postulated that (+/-)-SM-21 exerted an antinociceptive effect mediated by a central potentiation of cholinergic transmission. Affinity profiles of (+/-)-SM-21 for muscarinic receptor subtypes, determined by functional studies (rabbit vas deferens for M1, guinea pig atrium for M2, guinea pig ileum for M3 and immature guinea pig uterus for putative M4) have shown a selectivity ratio M2/M1 of 4.6 that, although very low, might be responsible for the antinociception induced by (+/-)-SM-21 through an increase in ACh extracellular levels. In the antinociceptive dose range, (+/-)-SM-21 did not impair mouse performance evaluated by the rota-rod and hole-board tests.     

Structure, interaction and electron transfer between cytochrome b5, its E44A and/or E56A mutants and cytochrome c

Site-directed mutagenesis has been used to produce variants of a tryptic fragment of bovine liver cytochrome b5 in which Glu44 and Glu56 are mutated to alanine. The reduction potentials measured by spectroelectrochemical titration (in the presence of 1 mM (Ru(NH3)6)3+, pH 7.0 and I=0.1 M) are 4.5, 6.0, 6.0 and 7.5 mV versus the standard hydrogen electrode (SHE) for the wild-type and E44A, E56A and E44/56A mutants of cytochrome b5, respectively. A comparative two-dimensional NMR study of cytochrome b5 and its E44/56A mutant in water solution has been achieved. Resonance assignments of side-chains have been completed successfully. The NMR results suggest that the secondary structures and global folding of the E44/56A mutant remain unchanged, but the mutation of both Glu44 and Glu56 to hydrophobic alanine may lead to the two helices containing mutated residues contracting towards the heme center. The inner mobility of the Gly42 approximately Glu44 segment in cytochrome b5 may be responsible for the difference of the binding mode between Glu44 and Glu56 with cytochrome c. The binding between cytochrome c and cytochrome b5 was studied by optical difference spectra of cytochrome c and variants of cytochrome b5. The association constants (KA) for the wild-type, E44A, E56A, and E44/56A mutants of cytochrome b5 with cytochrome c, are 4.70(+/-0. 10)x10(6) M-1, 1.88(+/-0.03)x10(6) M-1, 2.70(+/-0.13)x10(6) M-1, and 1.14(+/-0.05)x10(6) M-1, respectively. This is indicative that both Glu44 and Glu56 are involved in the complex formation between cytochrome b5 and cytochrome c. The reduction of horse heart ferricytochrome c by recombinant ferrocytochrome b5 and its mutants has been studied. The rate constant of the electron transfer reaction between ferricytochrome c and wild-type ferrocytochrome b5 (1.074(+/-0.49)x10(7) M-1 s-1) is higher than those of the mutant protein E44A (8.98(+/-0.20)x10(6) M-1 s-1), E56A (8.76(+/-0. 39)x10(6) M-1 s-1), and E44/56A (8.02(+/-0.38)x10(6) M-1 s-1) at 15 degreesC, pH 7.0, I=0.35 M. The rate constants are strongly dependent on ionic strength and temperature. These studies, by means of a series of techniques, provide conclusive results that the interaction between cytochrome b5 and cytochrome c is electrostatically guided, and, more importantly, that both Glu44 and Glu56 participate in the electron transfer reaction.     

Estimation of Koc values for deuterated benzene, toluene, and ethylbenzene, and application to ground water contamination studies

Sorption partition coefficients between water and organic carbon (Koc) for deuterated benzene, toluene, and ethylbenzene have been estimated by measuring values of the octanol-water partition coefficient (Kow) and HPLC retention factors (k1), which correlate closely to values of Koc. Measured values of log Kow for non-deuterated and deuterated toluene are 2.77 (+/- 0.02) and 2.78 (+/- 0.04), respectively, indicating that within experimental error, log Koc for deuterated and non-deuterated toluene are the same. The HPLC method provides greater precision, and yields values of delta log Koc (= log Koc [deuterated]-log Koc [non-deuterated]) of -0.021 (+/- 0.001) for benzene, -0.028 (+/- 0.002) for toluene, and -0.035 (+/- 0.003) for ethylbenzene. The small values of delta log Koc demonstrates that deuterated compounds are excellent tracers for the hydrologic behavior of ground water contaminants.     

Clinical significance of electrically evoked auditory brainstem response

Electrically evoked auditory brainstem responses (EABR) were recorded in 31 postlingually deafened adults, who had recently received cochlear implants (mini-system, Cochlear Ltd). The wave consisted of three distinct positive peaks labeled P1, P2, and P3 with latency of 1.35 (+/- 0.14), 2.17 (+/- 0.18) and 4.08 (+/- 0.31) ms, respectively. The P3 threshold (EABR-T) and slope (EABR-S) were 0.9 (+/- 0.47) mA and 0.6 (+/- 0.28) muv/mA, respectively. The relationships between the EABR parameters (EABR T and -S of the P3 wave) and age, duration of deafness, promontory test and subjective response (T and C-level) were investigated. The scattergram showed a strong negative linear relationship between EABR-S and subjective T-level. This finding suggests that EABR-S is good measure of postoperative perception.     

Pharmacological activities of trimetoquinol and 1-benzyl halogen-substituted analogues on rat beta-adrenoceptor subtypes

The beta-adrenoceptor activity profile of trimetoquinol and its 1-benzyl halogen-substituted analogues was studied in rat tissues containing primarily beta 1 (atria)-, beta 2 (trachea)- and atypical beta/beta 3 (distal colon and brown adipose tissue)-adrenoceptors. Functional biological activity resided in the (-)-isomer of trimetoquinol which was 112-, 275-, 372- and 513-fold more potent than (+)-trimetoquinol in trachea, right atria, distal colon and brown adipose tissue, respectively. (+/-)-Trimetoquinol was equally or slightly less active than (-)-trimetoquinol. The 1-benzyl halogen-substituted analogues of trimetoquinol exhibited differential activation of beta-adrenoceptor subtypes. In functional assays, 3'-iodotrimetoquinol was a potent activator of all beta-adrenoceptor subtypes. 3',5'-Diiodotrimetoquinol was 10-fold more potent as an agonist in tissues containing atypical beta/beta 3-adrenoceptors than those tissues containing beta 1- and beta 2-adrenoceptor sites. Furthermore, this drug was a partial agonist as compared to (+/-)-trimetoquinol and 3'-iodotrimetoquinol on beta 1-adrenoceptors. Pharmacological properties of the compounds on rat beta 3-adrenoceptors expressed in Chinese hamster ovary (CHO) cells were consistent with results observed in functional assays. 3',5'-Diiodotrimetoquinol possessed the greatest potency for activation of adenylyl cyclase. Rank order of affinity for rat beta 3-adrenoceptor was 3'-iodotrimetoquinol = 3',5'-diiodotrimetoquinol > (+/-)-trimetoquinol > (-)-isoprenaline. These results suggest that 3',5'-diiodotrimetoquinol is a promising drug for further chemical modification in the development of selective beta 3-adrenoceptor ligands.     

Contributions of exercise, body composition, and age to bone mineral density in premenopausal women

The purpose of this cross-sectional study were to determine whether exercisers have greater bone mineral density (BMD) than nonexercisers, whether aerobic dancers have greater BMD than walkers, and to determine the contributions of energy expenditure, body composition, and dietary factors to spine and femur BMD. Measurements were obtained on 93 eumenorrheic women (walkers N = 28; aerobic dancers, N = 34; nonexercisers, N = 31) ages 25-41 yr; lumbar spine and proximal femur BMD, body composition, physical activity, and nutrient intakes. Mean height, weight, and body mass index and median age and calcium intakes were similar for the three groups. Mean (+/- SD) values of the spine, total femur, and femoral neck BMD, respectively, were: walkers (1.092 (+/- 0.098), 0.947 g.cm-2), dancers (1.070 (+/- 0.124), 0.990 (+/- 0.104), 0.908 (+/- 0.106) g.cm-2), and nonexercisers (1.020 (+/- 0.112), 0.887 (+/- 0.073), 0.792 (+/- 0.089) g.cm-2) multiple regression analyses indicated that exercise contributed to spine (P = 0.018), total femur (P =0.012), and femoral neck (P < 0.0001) BMD, whereas type of exercise (aerobic dance vs walking) did not (P > 0.05). Total femoral BMD was influenced by exercise (P = 0.012) and energy expenditure (P = 0.023), while vertebral BMD was influenced by age (P = 0.0067), body weight (P = 0.017), and exercise (P = 0.018). These findings suggest that walking and aerobic dance exercise may provide physically active premenopausal women with greater lumbar and femoral BMD than sedentary females.     

New antipsychotic agents with serotonin and dopamine antagonist properties based on a pyrrolo[2,1-b][1,3]benzothiazepine structure

The development of a synthetic approach to the novel pyrrolo[2, 1-b][1,3]benzothiazepine and its derivatives and their biological evaluation as potential antipsychotic drugs are described. In binding studies these compounds proved to be potent 5-HT2, D2, and D3 receptor ligands. The more potent benzothiazepine (+/-)-3b was resolved into its enantiomers by using HPLC techniques. In vitro testing confirmed that (-)-3b is a more potent D2 receptor ligand, maintaining high affinity for 5-HT2 receptors. In contrast, the (+)-3b enantiomer presents a 35 times higher affinity for 5-HT2 than for dopamine D2 receptors with a similar dopamine D1 receptor affinity to that of (-)-3b. Overall, (+)-3b shows an "atypical" neuroleptic binding profile, while (-)-3b has a more "classical" profile. Furthermore pharmacological and biochemical testing displayed that the novel benzothiazepine (+/-)-3b is able to increase the extracellular levels of dopamine in the rat striatum and causes a dose-related suppression of apomorphine-induced locomotor activity. At low doses (+/-)-3b does not induce catalepsy, showing atypical antipsychotic properties similar to those of olanzapine. These heterocyclic compouds represent new leads for the development of novel antipsychotic drugs with atypical properties.     

Synthesis and biological activity of conformationally restricted analogues of milnacipran: (1S, 2R)-1-phenyl-2-[(R)-1-amino-2-propynyl]-N,N- diethylcyclopropanecarboxamide is a novel class of NMDA receptor channel blocker

Conformationally restricted analogues of (+/-)-(Z)-2-aminomethyl-1-phenyl-N,N-diethylcyclopropanecarboxamide++ + [milnacipran, (+/-)-1] were designed on the basis of its characteristic cyclopropane structure and were synthesized enantioselectively to develop efficient NMDA receptor antagonists. Among these analogues, (1S,2R)-1-phenyl-2-[(R)-1-amino-2-propynyl]-N, N-diethylcyclopropanecarboxamide (2d) had one of the most potent affinities for the receptor, with a Ki value of 0.29 microM. The blockade of NMDA receptor channels expressed by Xenopus oocytes by 2d was investigated in detail, and 2d was identified as a new class of open channel blocker against this receptor.     

Characterization of the catecholamine extraneuronal uptake2 carrier in human glioma cell lines SK-MG-1 and SKI-1 in relation to (2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU) selective cytotoxicity

Transport of (2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU) and (-)-norepinephrine was investigated in SarCNU-sensitive SK-MG-1 and -resistant SKI-1 human glioma cell lines. [3H]SarCNU influx was inhibited by SarCNU, sarcosinamide, and (+/-)-epinephrine in SK-MG-1 cells with competitive inhibition observed by (+/-)-epinephrine (Ki = 140 +/- 12 microM) and (+/-)-norepinephrine (Ki = 255 +/- 41 microM). No effect on influx was detected in SKI-1 cells. [3H](-)-Norepinephrine influx was linear to 15 sec in both cell lines and temperature dependent only in SK-MG-1 cells. Influx of [3H](-)-norepinephrine was found to be saturable in SK-MG-1 (K(m) = 148 +/- 28 microM, Vmax = 1.23 +/- 0.18 pmol/microL intracellular water/sec) but not in SKI-1 cells. In SK-MG-1 cells, [3H](-)-norepinephrine influx was found to be inhibited competitively by (-)-epinephrine (Ki = 111 +/- 7 microM) and SarCNU (Ki = 1.48 +/- 0.22 mM). Ouabain and KCl were able to inhibit the [3H](-)-norepinephrine influx in SK-MG-1 cells, consistent with influx being driven by membrane potential. Several catecholamine uptake2 inhibitors were able to reduce significantly the influx of [3H](-)-norepinephrine and [3H]SarCNU with no inhibition by a catecholamine uptake1 inhibitor. These findings suggest that increased sensitivity of SK-MG-1 to SarCNU is secondary to enhanced accumulation of SarCNU mediated via the catecholamine extraneuronal uptake2 transporter, which is not detectable in SKI-1 cells. The introduction of SarCNU into clinical trials will confirm if increased uptake via the catecholamine extraneuronal uptake2 transporter will result in increased antitumor activity.     

Cardiac responses to maximal upright cycle exercise in healthy boys and men

Previous investigations have indicated that children demonstrate a lower cardiac output at a given oxygen uptake during exercise compared with adults. This study compared cardiac responses with maximal upright cycle exercise in 15 boys (mean age 10.9 yr) and 16 men (mean age 30.7 yr) to determine whether this observation reflects differences in size or age-related influences on myocardial function. Stroke volume, aortic peak velocity, and systolic ejection time were measured to peak exercise in all subjects using Doppler ultrasound techniques. No significant differences were observed in resting, submaximal, or peak mean values for these variables relative to body size between the boys and men. Average values for peak stroke index, cardiac index, and peak aortic velocity were 59 (+/-11) mL.m-2, 11.33 (+/-2.32) L.min-1.m-2, and 152 (+/-30) cm.s-1, respectively, for the boys. Respective values for the men were 61 (+/-14) mL.m-2, 11.08 (+/-2.52) L.min-1.m-2, and 144 (+/-24) cm.s-1 (P > 0.05). This study failed to demonstrate evidence of impaired cardiac responses to maximal exercise in prepubertal boys compared with that in adult males.     

Introducing a downsized, open computer system with asynchronous transfer mode in PACS and its effect on image data transfer

(+/-)-8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), (+)-8-OH-DPAT, and (-)-8-OH-DPAT produced dose-related reversals of haloperidol-induced extrapyramidal side effects (EPS) in cebus monkeys, with all compounds producing similar almost complete reversals at 0.1 mg/kg i.m. These compounds were more potent than apomorphine, which reversed haloperidol-induced EPS at 0.3, but not 0.1, mg/kg i.m. The data indicate that the reversal of haloperidol-induced EPS by (+/-)-8-OH-DPAT and its enantiomers is mediated via effects at 5-HT1A receptors, not dopamine D2 receptors. Thus, inclusion of 5-HT1A agonist activity in novel antipsychotics may reduce EPS liability.     

Stereochemistry of the biotransformation of 1-hexene and 2-methyl-1-hexene with rat liver microsomes and purified P450s of rats and humans

The epoxidation of 1-hexene (1a) and 2-methyl-1-hexene (1b), two hydrocarbons present in the ambient air as pollutants, is catalyzed by some human and rat P450 enzymes. The enantioselectivities of these processes, when the reactions were carried out using rat and human liver microsomal preparations, were modest and dependent on both P450 composition and substrate concentrations. Various P450 isoforms (rat P450 2B1 and human P450 2C10 and 2A6) catalyzed the double bond oxidation of 1a and 1b with different product enantioselectivities. In the case of 1a, a moderately enantioselective hydroxylation at the allylic C(3) with the formation of 1-hexen-3-ol (4a) by microsomes from control or preinduced rats was also observed. The oxidation of this metabolite was, in turn, catalyzed by rat liver microsomes and mainly by rat P450 2C11, leading exclusively to the formation of 1-hexen-3-one, with no double bond epoxidation being observed. The stereochemical course of the microsomal epoxide hydrolase-catalyzed hydrolysis of the epoxy alcohols, threo-(+/-)- and erythro-(+/-)-1, 2-epoxyhexan-3-ol, theoretically expected to be formed from 4a, has been investigated.     

Nonpeptide angiotensin II receptor antagonists. Synthesis and biological activity of potential prodrugs of benzimidazole-7-carboxylic acids

In order to improve the oral bioavailability (BA) of 2-butyl-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-benzimid azole - 7-carboxylic acid (3: CV-11194) and 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4- yl]methyl]-1H-benzimidazole-7-carboxylic acid (4: CV-11974), novel angiotensin II (AII) receptor antagonists, chemical modification to yield prodrugs has been examined. After selective tritylation of the tetrazole rings in 3 and 4, treatment of N-tritylated benzimidazole-7-carboxylic acids (6, 7) with a variety of alkyl halides, followed by deprotection with hydrochloric acid, afforded esters of 3 and 4. Mainly 1-(acyloxy)alkyl esters and 1-[(alkoxycarbonyl)oxy]alkyl esters, double ester derivatives, were synthesized. Their inhibitory effect on AII-induced pressor response in rats and oral BA were investigated. (Pivaloyloxy)methyl and (+/-)-1-[[(cyclohexyloxy)-carbonyl]oxy]ethyl esters of 3 and 4 showed marked increases in oral bioavailability which significantly potentiated the inhibitory effect of the parent compounds on AII-induced pressor response. Among them, (+/-)-1-[[(cyclohexyloxy)carbonyl]oxy]ethyl 2- ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-benzimida zole- 7-carboxylate (10s, TCV-116) was selected as a candidate for clinical evaluation.     

Design and synthesis of m1-selective muscarinic agonists: (R)-(-)-(Z)-1-Azabicyclo[2.2.1]heptan-3-one, O-(3-(3'-methoxyphenyl)-2-propynyl)oxime maleate (CI-1017), a functionally m1-selective muscarinic agonist

The synthesis and SAR of a series of (Z)-(+/-)-1-azabicyclo[2.2. 1]heptan-3-one, O-(3-aryl-2-propynyl)oximes are described. The biochemistry and pharmacology of 24Z (PD 142505) and its enantiomers are highlighted. 24Z is functionally an m1-selective muscarinic agonist. Efficacy and m1 selectivity reside in the R enantiomer, (R)-24Z (CI-1017).     

Diazepam and N-desmethyldiazepam concentrations in saliva, plasma and CSF

1 Salivary and plasma diazepam and nordiazepam concentrations were measured in 51 paired samples from four experimental situations. In seven of the patients CSF samples were estimated. 2 Correlation of 0.89 (P less than 0.001) was observed between salivary and plasma diazepam and 0.81 (P less than 0.001) between salivary and plasma nordiazepam. 3 Mean salivary diazepam was 1.6% (+/- 0.3%) of the plasma diazepam. It was found to vary markedly in an acute dosage study. Mean salivary nordiazepam was 2.9% (+/- 1%) of the plasma measure and was dependent on salivary flow rate. 4 CSF diazepam was in equilibrium with unbound plasma diazepam and salivary diazepam. 5 Mean protein binding of diazepam in vitro was 99.3% with no variations as a function of concentration. 6 The results suggest salivary diazepam and nordiazepam measures to be of value in epidemiological studies. However, they do not predict accurately the plasma total or unbound drug concentration from a salivary sample in an individual.     

Cyclooxygenase-2 unlike cyclooxygenase-1 is highly expressed in ovine embryos during the implantation period

In this study we investigated expression of the two isoforms of the prostaglandin-forming enzyme, cyclooxygenase-1 (Cox-1) and cyclooxygenase-2 (Cox-2), in sheep embryos. Using Western blot and immunohistochemical analyses, we demonstrated that Cox-2 was highly expressed in embryos from Day 8 to Day 17 of development whereas Cox-1 was undetectable during this time. The expression of Cox-2 was developmentally regulated. It was maximal between Days 14 and 16. There was a 30-fold increase in Cox-2 content per protein extract between Day 10 and Day 14, corresponding to a 50,000-fold increase in the whole embryo. The expression of Cox-2 declined after Day 16 to become undetectable by Day 25 of pregnancy. Cox-2 was localized in the trophoblastic cells and was not detected in the inner cell mass. The [3H]arachidonic acid metabolites synthesized by Cox-2-rich conceptuses were analyzed by HPLC after short-term embryo culture. Day 14 conceptuses released mainly cyclooxygenase metabolites and to a lesser extent lipoxygenase derivatives. Cyclooxygenase products were 6-keto-prostaglandin (PGF)1alpha 18.2% (+/- 4.2), thromboxane-B2 22.51% (+/- 15.9), PGF2alpha 21% (+/- 11), PGE2 14.5% (+/- 7.4), and PGD2 2.7% (+/- 2.6). Taken together, these results suggest an important role for the Cox-2-dependent cyclooxygenase metabolites during embryo development.     

Dopaminergic transmission between identified neurons from the mollusk, Lymnaea stagnalis

1. Dopaminergic transmission was investigated in the central nervous system (CNS) of the freshwater snail, Lymnaea stagnalis. 2. The giant pedal neuron, designated as right pedal dorsal one (RPeD1), makes chemical, monosynaptic connections with a number of identified follower cells in the CNS. Previous work has shown that RPeD1 is an interneuron and a important component of the Lymnaea respiratory central pattern generator. In this study, the hypothesis that RPeD1 uses dopamine as its neurotransmitter was tested by chromatographic, pharmacological, and electrophysiological methods. Characterization of RPeD1's transmitter pharmacology is essential to clearly understand its role in Lymnaea. 3. Earlier studies demonstrated that the soma of RPeD1 contains dopamine. This was quantitated in the present study by high-performance liquid chromatography (with electrochemical detection) of isolated RPeD1 somata and growth cones, which yielded 0.8 +/- 0.3 and 0.10 +/- 0.08 pmol of dopamine per soma and growth cone, respectively. 4. Bath or pressure application of dopamine to follower cells of RPeD1, in situ, mimicked the effects of RPeD1 stimulation. Dose-response curves were constructed for the excitatory effect of dopamine on follower cells, visceral dorsal two and three (VD2/3) (ED50 = 39 microM; Hill coefficient = 1.03), and the inhibitory effect of dopamine on follower cell, visceral dorsal four (ED50 = 33 microM; Hill coefficient = 0.92). 5. The following dopamine agonists (100 microM) were tested by bath application: 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (ADTN), apopmorphine, 2-bromo-alpha-ergocryptine, deoxyepinephrine (DE), mesulergine, (-) quinpirole, SKF 38393, and tyramine. Only the general dopamine agonists, ADTN and DE, mimicked RPeD1's effects on its follower cells. 6. When VD2/3 was isolated and plated in vitro, it maintained a depolarizing response to dopamine. This response was reduced by intracellular injection of the G-protein blocker, GDP-beta-S (2 mM in electrode). Similarly, incubation of VD2/3, in vitro for approximately 18 h, with pertussis toxin (PTX; 5 micrograms/ml), the G-protein inactivating exotoxin, also reduced the dopamine response. Injecting GDP or incubating in heat-inactivated PTX did not effect the response. 7. Several dopamine antagonists were used in an attempt to block RPeD1's synapses: chlorpromazine, ergonovine, fluphenazine, haloperidol, 6-hydroxydopamine, SCH 23390, (+/-) sulpiride, and tubocurarine. Only the D-2 dopamine receptor antagonist, (+/-) sulpiride, reversibly blocked synaptic transmission from RPeD1 to its follower cells. Both the (+) and the (-) enantiomer of sulpiride also antagonized synaptic transmission. A dose-inhibition curve for (+/-) sulpiride was constructed (IC50 = 47 microM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in extracellular nitrite and nitrate levels after inhibition of glial metabolism with fluorocitrate

The role of glial cells in nitric oxide production in the cerebellum of conscious rats was investigated with a glial selective metabolic inhibitor, fluorocitrate. The levels of nitric oxide metabolites (nitrite plus nitrate) in the dialysate following in vivo microdialysis progressively increased to more than 2-fold the basal levels during a 2-h infusion of fluorocitrate (1 mM), and the increase persisted for more than 2 h after the treatment. Pretreatment with N(G)-nitro-L-arginine methyl ester attenuated the fluorocitrate-induced increase in nitric oxide metabolite levels. None of the glutamate receptor antagonists, including D(-)-2-amino-5-phosphonopentanoic acid, 6,7-dinitroquinoxaline-2,3-dione, and (+/-)-alpha-methyl-4-carboxyphenylglycine, inhibited the fluorocitrate-induced increase. The L-arginine-induced increase was significantly reduced by fluorocitrate treatment, while N-methyl-D-aspartate, (+)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and trans-(+/-)-1-amino-(1S,3R)-cyclopentane-dicarboxylic acid increased nitric oxide metabolites levels in the fluorocitrate-treated rats, as much as in control animals. These results suggest that glial cells play an important role in modulating nitric oxide production in the cerebellum by regulating L-arginine availability.     

Beta3-adrenoceptors mediate relaxation of guinea pig taenia caecum by BRL37344A and BRL35135A

Beta-adrenoceptor-mediated relaxation of guinea pig taenia caecum was investigated by studying the effects of the beta3-adrenoceptor agonists, BRL37344A [(R*,R*)-(+/-)-4-[2'-[2-hydroxy-2-(3-chlorophenyl) ethylamino] propyl] phenoxyacetic acid sodium salt sesquihydrate] and BRL35135A [(R*,R*)-(+/-)-methyl-4-[2-[2-hydroxy-2-(3-chlorophenyl) ethylamine] propyl] phenoxyacetate hydrobromide]. BRL37344A and BRL35135A caused dose-dependent relaxation of the guinea pig taenia caecum. The concentration-response curves for BRL37344A and BRL35135A were unaffected by propranolol, ICI118551 [erythro-1-(7-methylindan-4-yloxy)-3-(isopropylamine)-but an-2-ol], atenolol, butoxamine, prazosin, yohimbine and phentolamine. Bupranolol produced shifts of the concentration-response curves for BRL37344A and BRL35135A. Schild regression analyses carried out for bupranolol against BRL37344A and BRL35135A gave pA2 values of 5.79 and 5.84, respectively. These results suggest that the relaxant response to BRL37344A and BRL35135A of the guinea pig taenia caecum is mediated by beta3-adrenoceptors.