Inhibition Of Neuronal Uptake Research Articles

Novel tacrine analogues for potential use against Alzheimer's disease: potent and selective acetylcholinesterase inhibitors and 5-HT uptake inhibitors.

Several novel analogues of tacrine have been synthesized and tested for their ability to inhibit acetylcholinesterase, butyrylcholinesterase, and neuronal uptake of 5-HT (serotonin) and noradrenaline. Changes in the size of the carbocyclic ring of tacrine produced modest potency against cholinesterase enzymes. Addition of a fourth ring resulted in compounds with marked selectivity for acetylcholinesterase (AChE) over butyrylcholinesterase (BChE): e.g. 6-amino-4,5-benzo-5H-cyclopenta[1,2-b]-quinoline (14a) had an IC50 of 0.35 microM against AChE and 3.1 microM against BChE. Some tetracyclic compounds are 100-400 times more active than tacrine as inhibitors of neuronal uptake of serotonin, in particular 13-amino-6,7-dihydro-5H-benzo-[3,4]cyclohepta[1,2-b]quinoline (18), which had an IC50 of 20 nM. These compounds would be expected to facilitate both cholinergic and monoaminergic transmission. They should be worth investigating in models of memory impairment.

Differential effects of nipecotic acid and 4,5,6,7-tetrahydroisoxazolo[4,5- c]pyridin-3-ol on extracellular γ-aminobutyrate levels in rat thalamus

Using the microdialysis technique and HPLC (high-performance liquid chromatography) determination of amino acids, the extracellular concentrations of γ-aminobutyrate (GABA), glutamate, aspartate and a number of other amino acids were determined in rat thalamus during infusion through the microdialysis tubing of the GABA transport inhibitors 4,5,6,7-tetrahydroisoxazolo[4,5- c]pyridin-3-ol (THPO) and nipecotic acid. Administration of 5.0 mM THPO led to a 200% increase in the extracellular GABA concentration. Simultaneous infusion of THPO and GABA (50 μM) increased the extracellular GABA concentration to 1200% of the basal level whereas GABA alone was found to increase the GABA level to 500%. If nipecotic acid (0.5 mM) was administered together with GABA (50 μM) the extracellular concentration of GABA was not increased further. While administration of GABA alone or GABA together with nipecotic acid had no effect on the extracellular levels of glutamate and aspartate it was found that GABA plus THPO increased the extracellular concentration of these amino acids. GABA administered alone or together with nipecotic acid or THPO led to relatively small but significant increases in the extracellular concentrations of the amino acids glycine, glutamine, serine and threonine. The results demonstrate that THPO, which preferentially inhibits glial GABA uptake and which is not a substrate for the GABA carriers, was more efficient increasing the extracellular concentration of GABA than nipocotic acid which is a substrate and an inhibitor of both neuronal and glial GABA uptake. This indicates that GABA uptake inhibitors that are not substrates for the carrier and which preferentially inhibit glial GABA uptake may constitute a group of drugs by which the efficacy of GABAergic neurotransmission may be enhanced.

GABA A receptor-mediated k +-evoked gaba release from globus pallidus—analysis using microdialysis

Presynaptic modulation of γ-aminobutyric acid (GABA) release in the globus pallidus of rat was examined using in vivo microdialysis procedures. The addition of nipecotic acid (0.5 mM), a neuronal GABA uptake inhibitor, into perfusate, resulted in an increase in the basal GABA release from the globus pallidus. GABA release from the globus pallidus was also augmented dose-dependently by the addition of KCl. Muscimol, a GABA A receptor agonist, caused a significant suppression of the high potassium (100 mM)-evoked release of GABA, and this suppressive effect of muscimol was antagonized invariably by bicuculline, a GABA A receptor antagonist. On the other hand, baclofen, a GABA B receptor agonist, did not induce any significant changes in the 100 mM KCl-evoked GABA release. Similarly, 3-aminopropylphosphonous acid, a GABA B receptor agonist, failed to suppress the GABA release induced by high (100 mM) and low (50 mM) concentrations of KCl from the globus pallidus. Furthermore, CGP 54626A, a GABA B receptor antagonist, had no significant effect on these KCl-evoked GABA releases. These results suggest that presynaptic modulation of GABA release in the globus pallidus may be mediated by the GABA A autoreceptor.

Inhibition of rat brain monoamine oxidase enzymes by fluoxetine and norfluoxetine.

Fluoxetine and its primary metabolite, norfluoxetine, are inhibitors of neuronal uptake of 5-hydroxytryptamine. While fluoxetine has also been reported to inhibit monoamine oxidase (MAO) in vitro at concentrations much lower than those measured in brain following chronic fluoxetine treatment, neurochemical profiles are not consistent with substantial MAO inhibition in vivo. In an attempt to explain this inconsistency, we have examined the interactions of fluoxetine and norfluoxetine with rat brain MAO-A and -B by a radiochemical assay method. Fluoxetine and norfluoxetine were competitive inhibitors of MAO-A in vitro, with Ki values of 76.3 microM and 90.5 microM, respectively. Both compounds were non-competitive or uncompetitive inhibitors of MAO-B in vitro. Inhibition of MAO-B was time-dependent and was very slowly reversible by dialysis. IC50 values versus metabolism of 50 microM beta-phenylethylamine were 17.8 microM (fluoxetine) and 18.5 microM (norfluoxetine). Analysis of the time-dependence of MAO-B inhibition by fluoxetine revealed that an initial competitive interaction between the enzyme and the inhibitor (Ki 245 microM) was followed by tight-binding enzyme inactivation (K(inact) 0.071 min-1). Following administration of fluoxetine (20 mg kg-1 day-1) for 7 days, the cortical concentration of fluoxetine + norfluoxetine was estimated by gas-liquid chromatography to be 700 microM. Such drug treatment reduced MAO-A activity by 23% in 1:8 (w/v) cortical homogenates, but not in 1:80 homogenates. Inhibition of MAO-B in 1:8 homogenates was modest (12%) and was not significantly reduced by homogenate dilution. The concentration of 5-hydroxyindole-3-acetic acid, measured by high pressure liquid chromatography, was reduced by 47% in cortices from drug-treated rats, while concentrations of 5-hydroxytryptamine, noradrenaline, dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid were unchanged. These results suggest that, following chronic drug administration leading to relatively high tissue concentrations of fluoxetine and norfluoxetine, inhibition of either form of MAO would be restricted by competition for the enzyme with intraneuronal amine substrates.

809-3 Desipramine Attenuates the Cardiac Sympathetic Nerve Terminal Abnormalities in Congestive Heart Failure

We have shown that cardiac norepinephrine (NE) reuptake activity and tyrosine hydroxylase (TH), a rate-limiting enzyme for NE synthesis, are reduced in congestive heart failure (CHF). To determine whether the changes of TH are caused by neurotoxic effects of NE, we administered the neuronal uptake inhibitor desipramine (DMI, 225 mg/day) to pacing-induced CHF and sham-operated (SHAM) dogs for 6 weeks. CHF was characterized by tachycardia, low aortic pressure, elevated left atrial pressure, decreased left ventricular dP/dt and reduced cardiac output. DMI produced no hemodynamic effects. The effects of OMI on left ventricular NE uptake activity (fmol/mg/15 min) and immunocytochemical TH profiles were: Group NE uptake TH SHAM (n = 12) 133 ± 9 813 ± 43 SHAM + OMI (n = 6) 49 ± 11 * 769 ± 14 CHF (n = 12) 64 ± 8 * 409 ± 42 * CHF + OMI (n = 7) 40 ± 9 * , † 642 ± 49 * , † Values are mean ± SE * p < 005 vs. SHAM: † P < 0.05 vs. CHF As expected, DMI reduced NE uptake activity in both SHAM and CHF animals, but its actions on TH differed between the two groups. While it exerted no effect on TH in SHAM animals, DMI reduced the decrease in TH that occurred in CHF. The results suggest that the cardiac sympathetic changes that occur in CHF may be caused by excessive NE, and that the neurotoxic effect of NE on TH involves the NE reuptake mechanism.

Mathematical modelling of the enteric nervous network. 4. Analysis of adrenergic transmission

Based on the model of the adrenergic neurone proposed earlier, the dynamics of nerve-pulse transmission after treatment with inhibitors of neuronal uptake and catechol- O-methyltransferase, α 1-adrenoceptor antagonists, changes in the concentration of Ca 2+ ions in the external medium, the action of tetrodotoxin (TTX) and repetitive stimulation, are analysed. The results of numerical simulation show that: the addition of drugs that inhibit neuronal uptake and catechol- O-methyltransferase cause the augmentation of noradrenaline action on post-synaptic structures and an increase in the amplitude of the generated inhibitory post-synaptic potential (IPSP); treatment with adrenergic antagonists reduces the amplitude of IPSP; decrease in the concentration of extracellular Ca 2+ ions and application of TTX abolish the post-synaptic response. All these effects are shown to be dose-dependent. The repetitive stimulation of the neurone reproduces the effects of accumulation and potentiation. Possible applications of the results obtained are discussed.

Characterization of the effect of cocaine on catecholamine uptake by pregnant myometrium

To characterize the effect of cocaine on catecholamine uptake by myometrium from pregnant women. Slices of myometrium obtained from nine women during elective cesarean delivery at term were incubated with [3H]-norepinephrine and various uptake inhibitors for 30 minutes. The radiolabeled material was extracted with perchloric acid, expressed as percent control (+/- standard error of the mean), and compared by one-factor analysis of variance and Fisher multiple range test. Myometrial uptake was inhibited by cocaine (42 +/- 9%) by neuronal (type 1) uptake inhibitors (desipramine 41 +/- 14%; N-ethylmaleimide 53 +/- 8%), and by extraneuronal (type 2) inhibitors (normetanephrine 56 +/- 19%; corticosterone 73 +/- 9%). When uptake inhibitors were used in combination with cocaine, uptake was not decreased further in the presence of neuronal inhibitors (desipramine plus cocaine 40 +/- 20%; N-ethylmaleimide plus cocaine 42 +/- 4%). However, the effect of cocaine appeared to be added to that of extraneuronal inhibitors (normetanephrine plus cocaine 25 +/- 14%; corticosterone plus cocaine 32 +/- 1%). Catecholamine uptake by myometrium in pregnant women appears to be both extraneuronal and neuronal in nature, and cocaine inhibits the neuronal portion of this uptake. This mechanism may play a role in the increased rate of premature delivery associated with cocaine abuse.

Uptake mechanisms of meta-[ 123I]iodobenzylguanidine in isolated rat heart

In order to clarify the uptake and retention mechanisms of radioiodinated meta-iodobenzylguanidine (MIBG) in heart, the kinetics of no-carrier-added [ 123I]MIBG were studied in the isolated working rat heart in interaction with pharmacologic agents. The tracer was administered in the perfusate as a 10-min pulse, followed by a 90-min washout period. Kinetic analysis of the externally monitored time-activity curves of control hearts showed avid uptake ( K i = 4.4 ± 0.7 mL/min/g), and monoexponential clearance ( k o = 0.0056 ± 0.0017 l/min), indicating a distribution volume ( V d = K i k o ) of 834 ± 214 mL/g. Blocking experiments ( n = 41) were performed with neuronal uptake (uptake-1) inhibitor desipramine (DMI; 50–100 nM) and the extraneuronal uptake (uptake-2) inhibitor N-(9-fluorenyl)- N-methyl-β-chloroethylamine (SKF550; 0.4–0.8 μM). Uptake rate was 27% reduced ( P < 0.05) by 50 nM DMI but not significantly affected by 0.4 μM SKF550. Distribution volume was 88% reduced ( P < 0.0005) by 50 nM DMI and 28% reduced ( P < 0.05) by 0.4 μM SKF550. In DMI-blocked hearts, uptake rate was dramatically decreased (−80%, P < 0.0005) by SKF550 (0.4 μM), indicating uptake-2 transport contributed predominantly to the extraneuronal uptake of the tracer. The slow uptake rate seen with concomitant inhibition of uptake-1 and uptake-2 was further decreased by addition of unlabeled MIBG (1–10 μM) in a concentration-dependent manner, yet unaffected by addition of the vesicular uptake inhibitor Ro 4–1284 (1 μM). Thus, the uptake rate of [ 123I]MIBG is primarily dependent on uptake-1 and uptake-2 activity. Other possible mechanisms of uptake such as passive diffusion in association with intracellular binding are significant only in conditions where uptake-1 and uptake-2 mechanisms are largely inhibited.

Pharmacological studies of stonefish ( Synanceja trachynis) venom

The present study was designed to examine some of the pharmacological properties of venom from the stonefish ( Synanceja trachynis), with particular reference to the presence in the venom of pain-producing/enhancing substances. Stonefish venom (1–6 μg/ml) produced concentration-dependent contractile responses in guinea-pig isolated ileum. No tachyphylaxis, or reduction in responses with time, was observed to venom (3 μg/ml) in ileum. The response to venom (3 μg/ml) was not significantly affected by the histamine antagonist mepyramine (0.5 μM), or a preceding anaphylactic response. Mecamylamine, 5HT-desensitization or EXP3174 failed to have any significant effect on responses to venom (3 μg/ml), suggesting that the venom does not have action at ganglionic nicotine, 5HT or angiotensin II receptors, respectively. Prior incubation with blood acetylcholinesterase did not significantly inhibit responses to venom (3 μg/ml). Responses to venom (3 μg/ml) were significantly inhibited by the cyclooxygenase inhibitor indomethacin (5 μM), the leukotriene D 4 receptor antagonist FPL55712 (1 μM), the thromboxane A 2 receptor antagonist GR32191B (1 μM), the muscarinic receptor antagonist atropine (10 nM) and the neurokinin-1 receptor antagonist CP96345 (0.1 μM). Venom (6 μg/ml) produced contractile responses in the rat isolated vas deferens which were abolished by the α 1-adrenoceptor antagonist prazosin (0.3 μM) and significantly potentiated by the neuronal uptake inhibitor DMI (1 μM). However, noradrenergic transmitter depletion with reserpine (5 mg/kg, i.p.) did not significantly inhibit responses to venom (6 μg/ml). Histamine fluorometric and phospholipase A 2 assays failed to detect significant quantities of either substance in the venom. These results suggest that stonefish venom may cause the release of acetylcholine, substance P, and cyclooxygenase products, or contain components which act at these receptors. The venom also appears to contain a component which is a substrate for neuronal uptake and has a direct action at α 1-adrenoceptors.

Increase in myocardial interstitial norepinephrine during a short period of coronary occlusion

The neurally intact in vivo beating hearts of cats were used to examine the influence of a short period of coronary occlusion on regional myocardial norepinephrine levels. Using a cardiac dialysis technique, dialysate norepinephrine levels were measured as an index of myocardial interstitial norepinephrine levels, and the left anterior descending coronary artery was occluded for 10 min. In anesthetized cats, 10 min occlusion increased the dialysate norepinephrine level in the ischemic region but not in the non-ischemic region. To investigate the influence of neuronal norepinephrine uptake on occlusion-induced dialysate norepinephrine response, a neuronal uptake inhibitor (desipramine) was locally administered to both regions with the dialysis perfusate. In the ischemic region, 10 min occlusion significantly increased the dialysate norepinephrine level from the control level in the presence of desipramine. We concluded that 10 min coronary occlusion was associated with an increase in the myocardial interstitial norepinephrine level in the ischemic region. This increase in the early phase of ischemia was not attributed to attenuation of neuronal norepinephrine uptake function.

Ischaemia/reperfusion enhances phenylephrine-induced contraction of rabbit aorta due to impairment of neuronal uptake.

We studied the effect of ischaemia and reperfusion on vasoconstrictor and vasodilator mechanisms. Anaesthetized rabbits were subjected to 4-h abdominal aortic occlusion and 1-h reperfusion in vivo. Segments of the abdominal (ischaemic-reperfused) and thoracic (control) aorta were then removed for in vitro studies. Ischaemia/reperfusion had no significant effect on relaxant responses to either acetylcholine (ACh:endothelium-dependent) or sodium nitroprusside (SNP:endothelium-independent). The sensitivity of the aorta to contraction by phenylephrine was significantly increased in aortic rings with or without endothelium (by 2.2- and 3.7-fold, respectively), but was not different after 4-h ischaemia without reperfusion. In contrast, responses to methoxamine, serotonin, and U46619 were not affected by ischaemia/reperfusion. Moreover, the relative increase in aortic sensitivity to phenylephrine was prevented by treatment of control and ischaemic-reperfused aortic rings with the neuronal uptake inhibitor cocaine (10(-5) M). These results suggest that after 4-h ischaemia, reperfusion damages sympathetic neuronal uptake mechanisms in rabbit aorta. As a result, phenylephrine, an agonist normally susceptible to neuronal uptake, may exert more potent contractile effects. Endothelium-dependent and endothelium-independent relaxant mechanisms in the aorta appear to be resistant to acute ischaemia and reperfusion.

Na +-Dependent High-Affinity Uptake of Choline into Cultured Fibroblasts

The acetylcholine precursor choline is transported into cholinergic neurons by a high-affinity, sodium-dependent mechanism that is selectively localized to the cholinergic nerve terminal. In addition, a low-affinity, sodium-independent choline uptake system is present in cholinergic and non-cholinergic cells which deliver choline for cell membrane anabolism. Here, we show that uptake of [ 3H]choline into cultured fibroblast cell lines exhibits high affinity (K m≤10μM), is sodium-dependent, and is blocked by hemicholinium, a classical inhibitor of neuronal high-affinity choline uptake. Our data indicate that sodium-dependent high-affinity choline transport systems are also present in non-cholinergic cells.

Maturation of catecholamine response and extraneuronal uptake in rabbit trachea.

The present study investigated whether maturational changes in extraneuronal catecholamine clearance accounted for the ontogenetic attenuation of isoproterenol responsiveness in rabbit tracheal segments. Following half-maximal contraction with acetylcholine (ACh) or KCl, tracheal ring segments (TS) isolated from newborn, 1-mo, and adult rabbits were relaxed with cumulative administration of isoproterenol. With postnatal maturation, there occurred a significant decrease in both maximal relaxation and sensitivity to isoproterenol in both ACh- and KCl-contracted TS. Inhibition of extraneuronal catecholamine uptake with methylprednisolone resulted in enhanced sensitivity to isoproterenol in 1-mo and adult, but not in newborn tracheal segments. In separate studies, extraneuronal catecholamine uptake was directly assayed in tracheal segments isolated from rabbits of similar age and pretreated with neuronal uptake and monoamine oxidase inhibitors. Maximal extraneuronal uptake of [3H]norepinephrine significantly decreased with age in tracheal tissues. In contrast, the sensitivity of the uptake process to norepinephrine increased with age. The relative catecholamine reserve markedly decreased with age in rabbit tracheal segments and was directly related to the maximal degree of relaxation elicited with the beta-adrenergic catecholamine, isoproterenol, in age-matched TS precontracted with either ACh or KCl. These findings suggest that, with maturation, extraneuronal uptake more effectively competes with beta-adrenoreceptor stimulation and, accordingly, may contribute to the ontogenetic attenuation of beta-adrenergic responsiveness in rabbit tracheal tissues.

Pharmacological study of several effects of hydralazine in the bisected rat vas deferens

We have studied several effects of hydralazine in the bisected rat vas deferens. Hydralazine produced a shift to the left of the concentration-response curve for noradrenaline, with potentiation of the maximal response in both portions of the vas deferens. In contrast it caused a shift to the right of the concentration-response curve for noradrenaline in preparations pretreated with cocaine (inhibitor of catecholamine neuronal uptake), and of the curve for methoxamine and for CaCl 2 (in depolarizing medium with K + 55 mM), in all cases with depression of the maximal response. Hydralazine enhanced the contractions induced by noradrenaline in Ca 2+-free medium, except in the presence of cocaine. It had no effect on [ 3H]noradrenaline neuronal uptake into noradrenergic neurons of the vas deferens, nor did it affect basal or K +-induced 45Ca 2+ uptake. These results suggest that hydralazine potentiates the contractions elicited by noradrenergic by a mechanism other than blockade of the neuronal uptake of this catecholamine. Our results also suggest that the inhibition by hydralazine of the contraction elicited by Ca 2+ (in Ca 2-free depolarizing high-K + 55 mM solution) and by methoxamine is not due to an action on voltage-dependent Ca 2+ channels, but may reflect an intracellular site of action.

GABA uptake and release by a mammalian cell line stably expressing a cloned rat brain GABA transporter.

In order to facilitate study of the neuronal GABA transporter and provide a convenient system for potential drug screening, we have established a CHO cell line, designated 1F9, which stably expresses the cloned GABA transporter from rat brain (GAT-1). 1F9 cells transport GABA at levels approximately 300-fold higher than untransfected CHO cells, and GABA transport in these cells has the following properties: (1) a dependence on sodium and chloride ions; (2) higher sensitivity to neuronal subtype uptake inhibitors (DABA and ACHC) than to glial subtype inhibitors (beta-alanine and THPO); and (3) Km (2.5 microM) and IC50 values for various competitive ligands that are comparable with values determined in synaptosomes and brain slices. Given the fidelity with which the 1F9 cell line expresses these characteristics of the native neuronal GABA transporter, we have used it to further address GABA transporter activity. [3H]GABA uptake by 1F9 cells is inhibited approximately 50% by the chloride transport blockers DIDS and SITS. The GABA receptor agonists muscimol and baclofen also inhibit GABA transport; however, the receptor antagonists bicuculline and phaclofen have no effect. 1F9 cells also show release of [3H]GABA release is calcium independent, and is differentially affected by changes in the ion gradient, as well as by the presence of external substrates and uptake blockers. These experiments indicate that 1F9 cells provide a convenient system for the screening of GABA transport inhibitors.

(2 S,3 S,4 R)-2-(Carboxycyclopropyl)glycine, a potent and competitive inhibitor of both glial and neuronal uptake of glutamate

The effects of several diastereoisomers of L-2-(carboxycyclopropyl)glycine (CCG) on L-glutamate uptake were compared among three different preparations, glial plasmalemmal vesicles (GPV), synaptosomes and cultured astrocytes from rat hippocampus. The (2 S,3 S,4 R)-isomer ( L-CCG-III) inhibited a Na +-dependent high-affinity L-glutamate uptake in GPV and synaptosomes in a dose dependent manner at a micromolar range. The potency was quite similar to that of L- threo-β-hydroxyaspartate in both subcellular fractions and much higher than L-aspartate-β-hydroxamate, which were known as potent inhibitors of glutamate uptake. The (2 S,3 S,4 R)-isomer ( L-CCG-IV) also inhibited the glutamate uptake in GPV and synaptosomes, but it was about 100 times less active than L-CCG-III. The (2 S,3 S,4 R)- and (2 S,3 R,4 R)-isomers ( L-CCG-I and L-CCG-II, respectively) hardly showed any inhibitory action on the glutamate uptake. Dixon plot analysis of the initial uptake rate revealed that the inhibition was in a competitive manner and the value of the inhibition constant ( K i ) was about 1 μM in both GPV and synaptosomes. L-CCG-III effectively inhibited the glutamate uptake by cultured hippocampal astrocytes as well. These results suggested that L-CCG-III inhibited the glutamate uptake in both neurones and glial cells of the mammalian central nervous system in a similar manner.

Post-natal development modulates the rat seminal vesicle sensitivity to sympathomimetic agonists.

The post-natal development-related changes in the sensitivity to sympathomimetic agonists were studied in the seminal vesicle of 40-, 60- and 120-day old rats by determining the pD2 values for adrenaline, phenylephrine and methoxamine. The age-related changes in the neuronal and extraneuronal uptake of sympathomimetic agonists were also determined. The seminal vesicle sensitivity to adrenaline, phenylephrine and methoxamine increased in sexually mature rats (60- and 120-day old) (concentration-effect curves shifted to the left and pD2 values increased) when compared with immature rats (40-day old). Cocaine, a neuronal uptake inhibitor, induced supersensitivity to adrenaline at all ages, but did not affect the sensitivity to methoxamine. The ratio between the EC50 values for adrenaline in the presence and in the absence of cocaine was reduced in tissues of mature animals. This reduction suggests a loss of function of the neuronal uptake system with sexual development. In the presence of cocaine the pD2 value for adrenaline was increased during sexual development. Thus, the age-related changes in the adrenaline sensitivity were observed even when neuronal uptake was not operative, suggesting age-related changes at the post-junctional level, which was confirmed by the increase in the seminal vesicle sensitivity to methoxamine. In the presence of the extraneuronal uptake inhibitor, corticosterone, the sensitivity to adrenaline did not change and the age-related changes persisted.(ABSTRACT TRUNCATED AT 250 WORDS)

Supersensitivity to the stimulant action of noradrenaline on human myometrium near term.

Noradrenaline (10-50 nM) and tyramine (0.05-1 mM) enhanced contractile force elicited by field stimulation of strips of myometrium from non-pregnant and pregnant women. In higher concentrations, noradrenaline produced sustained contractions. The EC50 values for noradrenaline were 0.4 microM in tissues from pregnant women and 3.1 microM in tissues from non-pregnant women; maximum responses were greater in the former tissues. In addition, the effects of noradrenaline on myometrium from pregnant women were more marked on the inner layer than on the outer layer, antagonized by the alpha 1-adrenoceptor antagonist prazosin (0.1 and 1.0 microM), and unaffected by the inhibitor of neuronal uptake, nisoxetine (0.1 microM). Taken together, these observations confirm that supersensitivity to noradrenaline develops during pregnancy and is present near term. The supersensitivity to noradrenaline at term can be attributed only in part to a decrease in its removal by the sympathetic innervation, which declines towards term, because responses to tyramine were also enhanced in tissues from pregnant women. It is possible that gap junction formation may also contribute to this supersensitivity.

Influence of angiotensin II, alpha- and beta-adrenoceptors on peripheral noradrenergic neurotransmission in canine gracilis muscle in vivo.

Interactions between angiotensin II and adrenoceptor-mediated effects on peripheral sympathetic neurotransmission were investigated in constant flow blood-perfused canine gracilis muscle in situ, without and with pretreatment by non-competitive alpha-adrenoceptor blockade. Angiotensin converting enzyme (ACE)-inhibition by benazeprilat increased nerve stimulation (2 Hz, 4 min)-evoked noradrenaline (NA) overflow (+ 21 +/- 5%) with alpha-adrenoceptors intact, but reduced NA overflow (- 18 +/- 6%) when alpha-adrenoceptors were blocked. Vasoconstrictor responses were slightly reduced by benazeprilat. Subsequent infusion of angiotensin II (Ang II, 20 and 500 ng kg-1 min-1 i.v., raising arterial concentrations from 0.6 +/- 0.2 pM to 1390 +/- 240 and 25,110 +/- 3980 pM, respectively) failed to increase NA overflow or to enhance stimulation-evoked vasoconstriction. Adrenaline (0.4 nmol kg-1 min-1 i.v.) did not change evoked NA overflow before or after benazeprilat, either with or without alpha-adrenoceptor blockade, despite high concentrations (approximately 10 nM) in arterial plasma. Following benazeprilat, propranolol reduced NA overflow (- 24 +/- 3%) only if the alpha-adrenoceptors were blocked. In conclusion, benazeprilat reduced evoked NA overflow in the presence of alpha-adrenoceptor blockade to a similar degree as previously shown in the presence of neuronal uptake inhibition in this model. However, contrasting to our previous findings, benazeprilat enhanced NA overflow and reduced the post-junctional response to nerve stimulation in the absence of alpha-adrenoceptor blockade. This could be related to bradykinin accumulation during ACE-inhibition, in addition to the reduction of Ang II generation. Our data are not compatible with facilitation of NA release by circulating Ang II even at pharmacological dose levels. Although activation of prejunctional beta-adrenoceptors may facilitate evoked NA overflow in this model, circulating adrenaline is ineffective under physiological conditions even after alpha-adrenoceptor blockade. Also, beta-adrenoceptor-mediated prejunctional effects do not seem to involve Ang II in canine skeletal muscle in vivo.

Effects of Inhibitors of Neuronal Uptake of 5-HT on Sympathetic Cardiovascular Regulation

The effect on sympathetic cardiovascular regulation of inhibition of the high-affinity neuronal uptake of 5-hydroxytryptamine (5-HT) by fluvoxamine, citalopram, and fluoxetine was studied in anesthetized rabbits. The mean arterial pressure, postganglionic renal sympathetic nerve activity, heart rate, clearance of [3H]norepinephrine from plasma and the plasma norepinephrine concentration were measured, and from the latter two parameters the spillover of norepinephrine into the blood was calculated. The effect of fluvoxamine and fluoxetine on the uptake of [3H]5-HT into platelets was also examined. Of four increasing doses of fluvoxamine (0.6, 1.7, 5, and 15 mg/kg i.v.) and citalopram (0.3, 1, 3, and 9 mg/kg i.v.), only the two highest ones decreased the renal sympathetic nerve activity, heart rate, and clearance of [3H]norepinephrine from plasma. Only the highest of four increasing doses of fluoxetine (0.2, 0.6, 1.7, and 5 mg/kg i.v.) inhibited sympathetic nerve activity. However, lower doses of fluvoxamine (0.6 and 1.7 mg/kg i.v.) and fluoxetine (0.6 and 1.7 mg/kg i.v.) already markedly inhibited the uptake of [3H]5-HT into platelets. The data indicate that selective inhibition of neuronal uptake of 5-HT has no effect on central sympathetic regulation in anesthetized rabbits. The sympathoinhibition observed at high doses of fluvoxamine and citalopram is probably due to inhibition of the neuronal uptake of norepinephrine.