Evidence that ATP and noradrenaline are released during electrical field stimulation of the rat isolated seminal vesicle

The effects of electrical field stimulation (at 1-100 Hz with 20-25 V, supramaximal, and 0.2 ms pulse duration, for 10-20 s), on tone and contractility of rat isolated seminal vesicle was studied, in the absence and presence of specific antagonists (phentolamine, 1 microM, alpha, beta-Me-ATP, 10 microM, and verapamil, 1 microM). The aim was to provide pharmacological evidence of the release of ATP, alongside NA, during electrical stimulation of the rat seminal vesicle. Electrical field stimulation of the rat seminal vesicle produced frequency-dependent contractions. At low rates of stimulation (1-20 Hz), only small tonic contractions were observed (0.3 g tension), whereas at high rates of stimulation (80-100 Hz), only phasic contractions (2-3 g) were seen. However, at intermediate rates of stimulation (e.g. 50 Hz), a biphasic response was produced: an initial fast, twitch-like response (phasic), followed by a slow tonic contraction. The phasic contraction was blocked by the stable analogue of ATP (alpha, beta-Me-ATP), which desensitizes P2-purinoceptors. The tonic contraction is adrenergic and was blocked by phentolamine. Verapamil reduced both types of contractions in this preparation and the block was unselective on tonic versus phasic contraction. It was concluded that electrical field stimulation produced frequency-dependent contractions tonic and phasic in the rat seminal vesicle. These responses were blocked by specific antagonists, suggesting the presence of different types of neurotransmitters released during electrical stimulation of the rat seminal vesicle.     

Presence of neuropeptide Y in the rat seminal vesicle and its effects on noradrenaline- and nerve-induced contractions.

1. Immunohistochemical and functional studies have been performed to localize and determine the effects of neuropeptide Y (NPY) in the rat seminal vesicle. 2. An abundant presence of NPY-immunoreactive nerves, mainly concentrated in the smooth muscle layer of the seminal vesicle was found. Chronic 6-hydroxydopamine treatment (four doses of 50 mg kg-1 i.p. on days 1, 2, 4 and 6; rats killed one week after the last injection) led to a large reduction but not abolition of the NPY-immunoreactivity. 3. NPY (1-250 nM) did not affect the resting tone of the seminal vesicle. 4. The seminal vesicle was contracted by electrical field stimulation (EFS) and by exposure to 5 microM noradrenaline (NA). These contractions were abolished by phentolamine (1 microM). Tetrodotoxin (0.5 microM) abolished EFS-evoked contractions but did not affect NA-evoked contractions. 5. Seminal vesicles, from animals chronically-treated with reserpine (5 mg kg-1 i.p. on days 1 and 2; rats killed on day 3), were contracted by NA but not by EFS. 6. NPY (0.25-250 nM), concentration-dependently, inhibited EFS-evoked contractions by up to 70% maximum inhibition. Contractions evoked by EFS with short trains of pulses were inhibited by NPY to a greater degree than those with longer trains. 7. NPY had no significant effect on NA-evoked contractions. 8. These data provide strong evidence that the motor transmission in rat seminal vesicle is predominantly if not exclusively, adrenergic. It is further concluded that a rich NPY-containing innervation is present in the smooth muscle layer of rat seminal vesicle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that ATP does not act as a cotransmitter with noradrenaline in field-stimulated preparations of rat seminal vesicles

Field stimulation (20 pulses, 60 V, 2 ms, 5-70 Hz) of the nerve terminals within the rat seminal vesicle produced monophasic contractions which were blocked by guanethidine 5 microM and phentolamine 1 microM, and minimal in tissues from rats pretreated with reserpine, but unaffected by repeated application of alpha,beta-methylene ATP. Field stimulation at 40 Hz led to the release of ATP (0.39 +/- 0.12 pmol/mg; n = 10). However neither ATP nor alpha,beta-methylene ATP 10 microM caused contraction of the tissue. We conclude that ATP does not act as a cotransmitter in preparations of rat seminal vesicle because P2-purinoceptors appear to be absent.     

Specific binding sites for [3H]Ro 5-4864 in rat prostate and seminal vesicle.

1. The peripheral-type benzodiazepine receptor was characterized in rat prostate and seminal vesicle using [3H]Ro 5-4864 as radioligand. 2. The affinity of this radioligand for this receptor was higher in rat prostate (KD = 4.36 +/- 1.02 nM) than in seminal vesicle (KD = 8.45 +/- 1.34 nM). 3. The density of binding sites obtained in these two tissues was Bmax = 4164 +/- 873 fmol/mg in prostate and 5978 +/- 1022 fmol/mg in seminal vesicle. 4. The [3H]Ro 5-4864 binding was inhibited non-competitively by atractyloside and alpha,beta-methylene-ATP, suggesting a modulation by the ADP/ATP mitochondrial carrier. 5. Flutamide was able to displace bound [3H]Ro 5-4864 with an IC50 similar to protoporphyrin IX.     

Interactions of chloroethylclonidine with rauwolscine- and prazosin-sensitive adrenoceptors in dog saphenous vein.

1. alpha 1-Adrenoceptors have been classified pharmacologically into four subtypes (alpha 1A, alpha 1B, alpha 1C and alpha 1D) on the basis of their differential affinity for novel antagonists such as chloroethylclonidine (CEC). While CEC is considered an alpha 1B-adrenoceptor antagonist, our earlier studies revealed that it also acted like an agonist in the dog saphenous vein (DSV). The present study characterized the contraction induced by CEC in endothelium-denuded rings from DSV. 2. Concentration-response curves for CEC were constructed in the absence (EC50 value of 11.13 +/- 3.6 microM, n = 8) and presence of propranolol (beta-adrenoceptor antagonist, 30 nM), rauwolscine (alpha 2-adrenoceptor antagonist, 30 nM), prazosin (alpha 1-adrenoceptor antagonist, 30 nM) or methysergide (5HT2 antagonist, 30 nM) or both prazosin and rauwolscine. Pretreatment with methysergide (9.83 +/- 5.14 microM, n = 4) or propranolol (23.78 +/- 12.32 microM, n = 4) had no consistent effect. In the presence of rauwolscine, the concentration-response curve for CEC was significantly shifted to the right with an EC50 value of 48.82 +/- 13.2 microM (n = 8). In the presence of prazosin, the CEC concentration-response curve had an EC50 value of 29.12 +/- 6.42 microM (n = 8). Pretreatment with both prazosin and rauwolscine shifted the concentration-response curve for CEC to the right with an EC50 value of 72.67 +/- 10.69 microM (n = 8, P < 0.05). Maximum responses were significantly reduced only in tissues that were treated with both prazosin and rauwolscine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences between the third cardiac beta-adrenoceptor and the colonic beta 3-adrenoceptor in the rat.

1. The heart of several species including man contains atypical beta-adrenoceptors, in addition to coexisting beta 1- and beta 2-adrenoceptors. We now asked the question whether or not the third cardiac beta-adrenoceptor is identical to the putative beta 3-adrenoceptor. We compared the properties of the third cardiac beta-adrenoceptor with those of beta 3-adrenoceptors in isolated tissues of the rat. To study the third cardiac beta-adrenoceptor we used spontaneously beating right atria, paced left atria and paced left ventricular papillary muscles. As a likely model for putative beta 3-adrenoceptors we studied atypical beta-adrenoceptors of the colonic longitudinal muscle precontracted with 30 mM KCl. We used beta 3-adrenoceptor-selective agonists, antagonists and non-conventional partial agonists (ie high-affinity blockers of both beta 1- and beta 2-adrenoceptors know to exert also stimulant effects through beta 3-adrenoceptors). 2. The non-conventional partial agonist (-)-CGP 12177 caused positive chronotropic effects in right atria (pD2 = 7.3) and positive inotropic effects in left atria (pD2 = 7.5). The stimulant effects of (-)-CGP 12177 were resistant to blockade by 200 nM-2 microM (-)-propranolol and 3 microM ICI 118551 (a beta 2-selective antagonist) but antagonized by 1 microM (-)-bupranolol (pKB = 6.4-6.8), 3 microM CGP 20712A (a beta 1-selective antagonist) (pKB = 6.3-6.4) and 6.6 microM SR 59230A (a beta 3-selective antagonist, pKB = 5.1-5.4). 3. The non-conventional partial agonist cyanopindolol caused positive chronotropic effects in right atria (pD2 = 7.7) and positive inotropic effects in left atria (pD2 = 7.1). The stimulant effects of cyanopindolol were resistant to blockade by 200 nM (-)-propranolol but antagonized by 1 microM (-)-bupranolol (pKB = 6.8-7.1). 4. Neither (-)-CGP 12177 nor cyanopindolol caused stimulant effects in papillary muscles at concentrations between 0.2 nM and 20 microM. 5. In the presence of 200 nM (-)-propranolol the beta 3-adrenoceptor-selective agonists BRL 37344 (6 microM), SR 58611A (6 microM), ZD 2079 (60 microM) and CL 316243 (60 microM) did not cause stimulant effects or modify the potency and efficacy of the effects of (-)-CGP 12177 in right and left atria. The combination of 2 microM (-)-propranolol and 2 microM (-)-noradrenaline did not modify the chronotropic potency and efficacy of (-)-CGP 12177 compared to the potency and efficacy in the presence of 2 microM (-)-propranolol alone. 6. (-)-CGP 12177 relaxed the colon with a pD2 of 6.9 and a maximum effect of 55% compared to (-)-isoprenaline. The relaxant effects of (-)-CGP 12177 were resistant to blockade by 200 nM (-)-propranolol, 3 microM CGP 20712A, 3 microM ICI 118551 but blocked by 2 microM (-)-propranolol (pKB = 6.0), 1 microM (-)-bupranolol (pKB = 6.4) and 3 microM SR 59230A (pKB = 6.3). In the presence of 200 nM (-)-propranolol, (-)-CGP 12177 (20 microM) antagonized surmountably the relaxant effects of BRL 37344 (pKP = 7.3) (-)-noradrenaline (pKP = 7.0); and CL 316243 (pKP = 7.0). 7. Cyanopindolol in the presence of 200 nM (-)-propranolol relaxed the colon with a pD2 of 7.0 and a maximum effect of 40% compared to (-)-isoprenaline. As expected from a partial agonist, cyanopindolol antagonized the relaxant effects of both BRL 37344 and CL 316243 with a pKP = 7.6 and (-)-noradrenaline with a pKP = 7.4. 8. The following beta 3-adrenoceptor-selective agonists were potent colonic relaxants (pD2 values between parentheses): BRL 37344 (9.1), ZD 2079 (7.0), CL 316243 (9.0) and SR 58611A (8.2). The relaxant effects of these agonists were only marginally affected by 200 nM (-)-propranolol, not blocked by 3 microM CGP 20712A or 3 microM ICI 118551, and blocked by SR 59230A 3 microM (pKB = 6.9-7.5), 1 microM (-)-bupranolol (pKB = 6.2-6.4) and 2 microM (-)-propranolol (pKB = 6.3-6.5). 9...     

Effects of GABA on noradrenaline release and vasoconstriction induced by renal nerve stimulation in isolated perfused rat kidney.

We examined effects of gamma-aminobutyric acid (GABA) on vasoconstriction and noradrenaline (NA) release induced by electrical renal nerve stimulation (RNS) in the isolated pump-perfused rat kidney. RNS (1 and 2 Hz for 2.5 min each, 0.5-ms duration, supramaximal voltage) increased renal perfusion pressure (PP) and renal NA efflux. GABA (3, 10 and 100 microM) attenuated the RNS-induced increases in PP by 10-40% (P<0.01) and NA efflux by 10-30% (P<0.01). GABA did not affect exogenous NA (40 and 60 nM)-induced increases in PP. The selective GABA(B) agonist baclofen (3, 10 and 100 microM) also attenuated the RNS-induced increases in PP and NA efflux, whereas the RNS-induced responses were relatively resistant to the selective GABA(A) agonist muscimol (3, 10 and 100 microM). The selective GABA(B) antagonist 2-hydroxysaclofen (50 microM), but not the selective GABA(A) antagonist bicuculline (50 microM), abolished the inhibitory effects of GABA (10 microM) on the RNS-induced responses. The selective alpha2-adrenoceptor antagonist rauwolscine (10 nM) enhanced the RNS-induced responses. GABA (3, 10 and 100 microM) potently attenuated the RNS-induced increases in PP by 40-60% (P<0.01) and NA efflux by 20-50% (P<0.01) in the presence of rauwolscine. Prazosin (10 and 30 nM) suppressed the RNS-induced increases in PP by about 70-80%. Neither rauwolscine (10 nM) nor GABA (10 microM) suppressed the residual prazosin-resistant PP response. These results suggest that GABA suppresses sympathetic neurotransmitter release via presynaptic GABA(B) receptors, and thereby attenuates adrenergically induced vasoconstriction in the rat kidney.     

Existence of different alpha(1)-adrenoceptor subtypes in junctional and extrajunctional neurovascular regions in canine splenic arteries

The present study attempted to characterize the alpha(1)-adrenoceptor subtypes mediating vasoconstrictor responses to administered and nerve stimulation-evoked noradrenaline (NA) release in the isolated and perfused canine splenic artery. A previous study demonstrated that periarterial electrical nerve stimulation (30 s trains of pulses at a frequency of 1, 4 or 10 Hz) induced a double peaked vasoconstriction consisting of an initial transient, predominantly P2X-purinoceptor-mediated constriction followed by a prolonged, mainly alpha(1)-adrenoceptor-mediated response in the canine splenic artery. The effects of alpha(1)-adrenoceptor subtype antagonists on neuronally-mediated second peaked vasoconstrictions were analysed. BMY 7378 (10 - 100 nM), a selective alpha(1D)-adrenoceptor antagonist produced a dose-dependent inhibition of the second peak responses at all frequencies used. BMY 7378 (100 nM) reduced these responses by approximately 30%. Exposure of tissues to chloroethylclonidine (CEC, 60 microM), a selective alpha(1B)-adrenoceptor antagonist attenuated the second peak response by approximately 60%, even in the presence of BMY 7378 (100 nM). On the other hand, WB 4101 (100 nM), a selective alpha(1A)-adrenoceptor antagonist potentiated nerve-stimulation-evoked double peaked vasoconstrictions, especially at low frequencies (1 and 4 Hz). Vasoconstrictor responses to administered NA were dose-dependently antagonized by WB 4101 (10 - 100 nM), but were not significantly affected by either BMY 7378 (10 - 100 nM) or by CEC (60 microM). The present results indicate that NA released from sympathetic nerves may junctionally exert its vasoconstrictor effect via activation of postjunctional alpha(1B)- and in part alpha(1D)-adrenoceptors, whereas exogenous NA extrajunctionally activates alpha(1A)-adrenoceptors to produce its vascular action in canine splenic arteries.     

Pharmacological evaluation of the histamine H1 and 5-HT blocking properties of 2-N-(carboxamidinonormianserin) (FCC5): in-vitro studies.

Some in-vitro pharmacological effects of a novel analogue of mianserin, 2-carboxamidino-1,2,3,4,10,14b-hexahydrodibenzo (c,f) pyrazino (1,2-alpha) azepine hydrochloride (FCC5) have been studied. FCC5 was a non-competitive antagonist of both histamine-induced contractions of the guinea-pig ileum and 5-HT-induced contractions of rat fundal strips with pD'2 values of 6.13 and 5.57, respectively. The insurmountable antihistaminic effect of FCC5, 100 nM, in the guinea-pig isolated ileum was not removed by washing. FCC5, 10-100 nM, had no effect on responses to acetylcholine or barium chloride of the guinea-pig isolated ileum. In guinea-pig isolated right atria, FCC5, 1-30 microM, had no effect on H2-receptor-mediated chronotropic responses to histamine. FCC5, 10-1000 nM, had no alpha 2-adrenoceptor antagonist activity, as assessed by lack of effect on the inhibitory responses to B-HT 920 in the electrically stimulated rat isolated vas deferens. FCC5 resembles mianserin by being a potent, non-competitive antagonist at histamine H1 and 5-HT receptors, but differs from mianserin in a number of respects including having much less effect at alpha 2-adrenoceptors.     

Actions of 3-[2-phosphonomethyl[1,1-biphenyl]-3-yl]alanine (PMBA) on cloned glycine receptors

1. PMBA is a novel antagonist of strychnine-sensitive glycine receptors in the rat spinal cord, however, its mode of action is unknown. The actions of PMBA on rat glycine receptor alpha1 and alpha2 homomers in Xenopus oocytes were studied under two-electrode voltage-clamp. 2. Co-application of PMBA and glycine to both alpha1 and alpha2 homomers yielded inward currents which decayed to a steady-state. Responses rose slowly to the same steady-state amplitude following a 2 min pre-incubation in PMBA. Strychnine, but not picrotoxinin, showed similar antagonism to PMBA. The potency of PMBA was independent of membrane potential between -100 and 0 mV. 3. When tested against EC50 concentrations of glycine, PMBA was almost equally potent on alpha1 (IC50, 406+/-41 nM: Hill coefficient, 1.5+/-0.2) and alpha2 (IC50, 539+/-56 nM; Hill coefficient, 1.4+/-0.2) homomers. 4. PMBA (1-I0 microM) and strychnine (200 nM) reduced the potency of glycine and the amplitude of the maximal agonist response of alpha1 and alpha2 homomers. In 10 microM PMBA, two distinct classes of glycine response were observed on alpha2, only a single class of responses were observed on alpha1. 5. There are similarities in PMBA and strychnine antagonism, although these compounds are structurally distinct. The possibility that PMBA interacts at two binding sites which differ in alpha1 and alpha2 subunits is discussed. PMBA may provide a lead structure for novel antagonists with which to investigate structural differences in glycine receptor at alpha1 and alpha2 subunits.     

Multiple receptors for calcitonin gene-related peptide and amylin on guinea-pig ileum and vas deferens.

1. The responses of the electrically stimulated guinea-pig ileum and vas deferens to human and rat calcitonin gene-related peptide (CGRP) and amylin were investigated. 2. The inhibition of contraction of the ileum produced by human alpha CGRP was antagonized by human alpha CGRP8-37 (apparent pA2 estimated at 7.15 +/- 0.23) > human alpha CGRP19-37 (apparent pA2 estimated as 6.67 +/- 0.33) > [Tyr0]-human alpha CGRP28-37. The amylin antagonist, AC187, was three fold less potent than CGRP8-37 in antagonizing human alpha CGRP. 3. Both human beta- and rat alpha CGRP inhibited contractions of the ileum, but this was less sensitive to inhibition by CGRP8-37 than the effect of human alpha CGRP. However, CGRP19-37 was twenty times more effective in inhibiting the response to rat alpha CGRP (apparent pA2 estimated as 8.0 +/- 0.1) compared to human alpha CGRP. 4. Rat amylin inhibited contractions in about 10% of ileal preparations; this effect was not antagonized by any CGRP fragment. Human amylin had no action on this preparation. 5. Both human and rat alpha CGRP inhibited electrically stimulated contractions of the vas deferens, which were not antagonized by 3 microM CGRP8-37 or 10 microM AC187. 6. Rat amylin inhibited the stimulated contractions of the vas deferens (EC50 = 77 +/- 9 nM); human amylin was less potent (EC50 = 213 +/- 22 nM). The response to rat amylin was antagonized by 10 microM CGRP8-37 (EC50 = 242 +/- 25 nM) and 10 microM AC187 (EC50 = 610 +/- 22 nM). 7. It is concluded that human alpha CGRP relaxes the guinea-pig ileum via CGRP1-like receptors, but that human beta CGRP and rat alpha CGRP may use additional receptors. These are distinct CGRP2-like and amylin receptors on guinea-pig vas deferens.     

Neurally evoked responses of human isolated resistance arteries are mediated by both alpha 1- and alpha 2-adrenoceptors.

1. Human subcutaneous resistance arteries (internal diameter 113-626 microns) were mounted in an isometric myograph. Electrical field stimulation was applied either continuously in the form of a frequency-response curve or intermittently at 16 Hz. The magnitude of the maximum contraction induced by continuous stimulation expressed as a percentage of the response to a supramaximal concentration of noradrenaline (10 microM) was highly variable but unrelated to vessel calibre. Contractile responses to both continuous and intermittent stimulation were abolished by 1 microM tetrodotoxin. 2. Prazosin (100 nM and 1 microM, alpha 1-adrenoceptor antagonist) inhibited responses to continuous stimulation over a range of frequencies (2-8 Hz). The response to continuous stimulation at 8 Hz was inhibited by 78 +/- 6% by 1 microM prazosin. Rauwolscine (100 nM, alpha 2-adrenoceptor antagonist) had a smaller effect on contractions induced by continuous stimulation. Rauwolscine inhibited the response at 8 Hz by 36 +/- 6%. Rauwolscine at a higher concentration (1 microM) caused further inhibition of the response to continuous stimulation. Prazosin and rauwolscine in combination almost completely inhibited the response to continuous stimulation at concentrations of 1 microM. 3. Prazosin and rauwolscine inhibited responses to intermittent stimulation in a concentration-dependent manner. The IC50 for this action of prazosin was 3.7 +/- 1.6 nM and the maximum inhibition induced by 100 nM prazosin was 78 +/- 6%. The IC50 of rauwolscine was 12.0 +/- 1.3 nM and 100 nM rauwolscine caused a 86 +/- 7% reduction in the response to intermittent stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha2A- but not alpha2B/C-adrenoceptors modulate noradrenaline release in rat locus coeruleus: voltammetric data

In this study, we used subtype-selective antagonists to determine the subtype of alpha2-adrenoceptor controlling noradrenaline release in rat locus coeruleus. Noradrenaline release was measured in locus coeruleus slices using fast cyclic voltammetry at carbon fibre microelectrodes. On long stimulation trains (40 pulses, 20 Hz), the alpha2A-adrenoceptor selective antagonist BRL 44408 (2-[2H-(1-methyl-1,3-dihydroisoindole) methyl]-4,5-dihydroimidazole) at 100 nM and 1 microM significantly increased stimulated noradrenaline release, whereas the alpha2B/C-selective antagonist ARC 239 (2-[2[4-(o-methoxyphenyl)piperazin-1-yl] ethyl]-4,4dimethyl-1,3-(2H,4H)-isoquinolinedione) at 50 and 500 nM had no effect. On short stimuli (20 pulses, 200 Hz), the non-specific alpha2-adrenoceptor agonist dexmedetomidine (10 nM) significantly decreased noradrenaline release, an effect reversed by BRL 44408 (1 microM) but not by ARC 239 (500 nM). These data demonstrate that autoreceptor control of noradrenaline release in the locus coeruleus is mediated by alpha2A but not alpha2B/C-adrenoceptors.     

Alpha3beta4-nicotinic receptors mediate adrenergic nerve- and peptidergic (CGRP) nerve-dependent vasodilation induced by nicotine in rat mesenteric arteries

BACKGROUND AND PURPOSE: Previous studies demonstrated that nicotine-induced endothelium-independent vasodilation is mediated by perivascular adrenergic nerves and nerves releasing calcitonin gene-related peptide (CGRPergic nerves). We characterized the nicotinic acetylcholine (ACh) receptor subtype underlying the vasodilation in response to nicotine in rat mesenteric arteries. EXPERIMENTAL APPROACH: Rat mesenteric vascular beds without endothelium were contracted by perfusion with Krebs solution containing methoxamine and the perfusion pressure was measured with a pressure transducer. KEY RESULTS: Perfusion of nicotine (1-100 microM) for 1 min caused a concentration-dependent decrease in perfusion pressure due to vasodilation. Perfusion of (+/-)-epibatidine (1-100 nM) (non-selective agonist) or (-)-cytisine (1-100 microM) (partial agonist for nicotinic beta2 subtype and full agonist for nicotinic beta4 subtype) induced vasodilation in a concentration-dependent manner. Vasodilation induced by nicotine, (-)-cytisine- and (+/-)-epibatidine was markedly attenuated by guanethidine (5 microM) and pretreatment with capsaicin (1 microM). Mecamylamine (relatively selective antagonist for alpha3beta4 subtype), but not dihydro-beta-erythroidine (selective antagonist for alpha4beta2 subtype) or alpha-bungarotoxin (selective antagonist for alpha7 subtype), markedly inhibited nicotine-induced vasodilation. Nicotine-induced vasodilation was inhibited by methyllycaconitine at high concentrations (>1 microM), which non-selectively antagonize nicotinic receptors, while a low concentration of 10 nM, which selectively antagonizes alpha7 subtype, had no effect. (-)-Cytisine and (+/-)-epibatidine-induced vasodilation were abolished by mecamylamine. CONCLUSION AND IMPLICATIONS: These results suggest that the nicotinic alpha3beta4 receptor subtype, but not the alpha7 and alpha4beta2 subtypes, is responsible for the vasodilation in rat mesenteric arteries induced by nicotine- and nicotinic ACh receptor agonists through stimulation of adrenergic and CGRPergic perivascular nerves.     

The function of alpha- and beta-adrenoceptors of the saphenous artery in caveolin-1 knockout and wild-type mice

BACKGROUND AND PURPOSE: Adrenoceptors can associate with cardiac caveolae. To investigate the function of vascular caveolae, adrenoceptor-mediated effects were compared in the saphenous artery of caveolin-1 knockout (cav-1KO) and wild-type (WT) mice. EXPERIMENTAL APPROACH: Electronmicroscopy was used to detect caveolae. Real-Time quantitative PCR was used for adrenoceptor subtypes. Catecholamine-evoked contractions and relaxations were studied in arterial segments. KEY RESULTS: Caveolae were found in arterial smooth muscle from WT but not from cav-1KO mice. Arterial mRNA levels for the adrenoceptors alpha1A, alpha1B, alpha1D, beta1, beta2 and beta3 were similar in cav-1KO and WT. (-)-Noradrenaline contracted cav-1KO (-log EC50M=7.1) and WT (-log EC50M=7.3) arteries through prazosin-sensitive receptors. Maximum (-)-noradrenaline-evoked contractions were greater in cav-1KO than WT arteries. (-)-Isoprenaline relaxed WT arteries (-log EC50M=7.3) more potently than cav-1KO arteries (-log EC50M=6.8); the effects were antagonized partially and similarly by the beta2-selective antagonist ICI118551 (50 nM). The (-)-isoprenaline-evoked relaxation was partially antagonized by the beta1-adrenoceptor-selective antagonist CGP20712 (300 nM) in WT but not cav-1KO arteries. The beta3-adrenoceptor-selective antagonist L748337 (100 nM) partially antagonized the relaxant effects of (-)-isoprenaline in cav-1KO but not in WT arteries. BRL37344 partially relaxed arteries through beta3-adrenoceptors in cav-1KO but not WT. The relaxant effects of BRL37344 were decreased by the NO synthase inhibitor OmegaL-nitroarginine. CONCLUSIONS AND IMPLICATIONS: The function of arterial alpha1- and beta2-adrenoceptors is similar in cav-1KO and WT mice. beta1-adrenoceptor-mediated relaxation in WT is lost in cav-1KO and replaced by the appearance of beta3-adrenoceptors.     

Calcium antagonists inhibit positive chronotropic responses to alpha 1-adrenoceptor activation in rat isolated atria

Positive chronotropic responses of rat isolated atria to phenylephrine were reduced by propranolol (0.3 microM) and the residual response was further depressed by the selective alpha 1-adrenoceptor antagonist prazosin (1 nM) but not yohimbine (10 nM), confirming that a component of the response to phenylephrine was due to activation of alpha 1-adrenoceptors. When beta-adrenoceptors were blocked by propranolol, the positive chronotropic response to phenylephrine was enhanced by increasing the calcium concentration and by the calcium channel activator Bay K 8644 (0.1 microM), whereas the response was decreased by lowering the calcium concentration and by the calcium antagonists verapamil (10 nM), nifedipine (10 nM) and diltiazem (100 nM). In the presence of prazosin, when phenylephrine acts only on beta-adrenoceptors, calcium antagonists had no effect on the response. In rat isolated aortic strips in a calcium-free, high K+ (40 mM) solution, verapamil (10 nM), nifedipine (10 nM) and diltiazem (100 nM) shifted the calcium-induced contraction curves to the right, but prazosin (10 nM) had no effect, indicating that it is not a calcium antagonist. The calcium antagonists in the concentrations stated above had no effect on phenylephrine-induced contractions of rat aortic strips in normal Krebs-Henseleit solution, indicating that they did not block alpha 1-adrenoceptors in these concentrations. Taken together, these data suggest that the positive chronotropic effect of phenylephrine resulting from activation of alpha 1-adrenoceptors involves an increased influx of calcium through channels that are sensitive to organic calcium antagonists.     

Glycine receptor beta subunits play a critical role in potentiation of glycine responses by ICS-205,930

The sensitivity of various types of recombinant glycine receptors (GlyRs) to ICS-205,930 was studied by fast perfusion in Xenopus laevis oocytes. This compound has previously been shown to potentiate glycine responses in rat spinal neurons between 10 nM and 1 microM, independently of its 5-HT(3) antagonist properties. In contrast, submicromolar concentrations of ICS-205,930 failed to affect responses of homomeric GlyRs formed from human alpha1 or alpha2 subunits, and micromolar concentrations (1-20 microM) acted differentially on the two types of homomeric receptors, potentiating the responses to glycine (10-20 microM) of alpha1 homomeric GlyRs and inhibiting the responses of alpha2 homomeric GlyRs. GlyRs beta subunits markedly influenced the modulations induced by ICS-205,930. In oocytes expressing alpha1/beta or alpha2/beta heteromeric GlyRs, low concentrations of ICS-205,930 (20 nM-1 microM) induced a potentiation of glycine responses that was counteracted by an inhibitory effect at higher concentrations. Thus, GlyRs beta subunits reduce by 2 orders of magnitude the concentration range potentiating alpha1-containing GlyRs and are required for potentiation of alpha2-containing GlyRs. These results reveal a new high-affinity potentiating site on GlyRs, to which beta subunits participate. The difference in ICS sensitivity between alpha1 and alpha2 GlyRs cannot be explained by their difference in TM2 segment and extracellular domains partly conserved between glycine and 5-HT(3) receptors are probably involved in the interaction of some 5-HT(3) antagonists with GlyRs.     

Pharmacological characteristics of inhibitory action of the selective alpha1-antagonist JTH-601 on the positive inotropic effect mediated by alpha1-adrenoceptors in isolated rabbit papillary muscle

Influence of JTH-601 [N-(3-hydroxy-6-methoxy-2,4,5-trimethylbenzyl)-N-methyl-2-(4-hydroxy-2-isopropyl-5-methylphenoxy)ethylamine hemifumarate], a selective alpha1-adrenoceptor antagonist, on alpha1-mediated positive inotropic effect (PIE) was studied in isolated rabbit papillary muscle (1 Hz at 37 degrees C). JTH-601 (0.1-10 microM) shifted the concentration-response curve (CRC) for PIE of phenylephrine mediated by alpha1-adrenoceptor (with timolol at 1 microM) to the right and downward. In the presence of 100 nM WB 4101, an alpha1A antagonist, the shift to the right disappeared and JTH-601 (1-3 microM) shifted CRC for phenylephrine downward. The antagonistic action of JTH-601 was unchanged by 100 nM (+)-niguldipine, another alpha1A antagonist. Following pretreatment with 10 microM chloroethylclonidine, an alpha1B antagonist, the shift of CRC for phenylephrine to the right disappeared and JTH-601 (3-10 microM) shifted CRC downward. Antagonistic action of JTH-601 (3 microM) was unaltered by 100 nM BMY 7378, an alpha1D antagonist. JTH-601 (10 microM) had no effect on beta-mediated PIE of isoproterenol. These results indicate that JTH-601 exerts an inhibitory action on alpha1-mediated PIE through antagonism of alpha1A- and/or alpha1B-adrenoceptors in rabbit ventricular myocardium. As an alpha1 antagonist, JTH-601 is much less potent in rabbit ventricular muscle than in smooth muscle.     

Sensory nerve-mediated relaxation of guinea-pig isolated pulmonary artery: prejunctional modulation by alpha 2-adrenoceptor agonists but not sumatriptan.

1. Effects of the alpha 2-adrenoceptor agonists, UK14304 and clonidine, the 5-HT1 receptor agonist, sumatriptan and the kappa-opioid receptor agonist, GR103545, on sensory neurotransmission in histamine-contracted guinea-pig isolated pulmonary artery (GPPA) have been studied. 2. Electrical field stimulation (EFS) induced frequency-dependent relaxations of histamine-contracted GPPA, which were attenuated by tetrodotoxin and capsaicin pretreatment but not by the nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME). 3. Substance P (0.3 microM) induced relaxations which were subject to rapid tachyphylaxis. Neither the NK1 receptor antagonist, (+/-)-CP 96,345, nor desensitization to substance P had any effect of EFS-induced relaxations of histamine-contracted GPPA. 4. Calcitonin gene-related peptide (CGRP; 3 and 30 nM) induced concentration-dependent relaxations of histamine-contracted GPPA. The putative CGRP receptor antagonist, CGRP8-37 (1 microM), markedly attenuated EFS-induced relaxations as well as relaxations induced by a low concentration of CGRP. 5. Sumatriptan (0.1 and 1 microM) and the selective kappa-opioid receptor agonist, GR103545 (10 and 100 nM) had no effect on EFS-induced relaxations of histamine-contracted GPPA. In contrast, the alpha 2-adrenoceptor agonists UK14304 (1-100 nM) and clonidine (300 nM) attenuated responses to EFS, the attenuation of UK14304 (100 nM) being reversed by yohimbine (300 nM). 6. It is concluded that in GPPA, where a presynaptic inhibition of sensory neurotransmission by alpha 2-adrenoceptor activation could be shown, there was no evidence for such modulation by either sumatriptan-sensitive 5-HT1 receptors or kappa-opioid receptors.     

Pre- and post-junctional alpha-adrenoceptor-mediated responses in the rat gastric fundus in-vitro

The effects of alpha 1- and alpha 2-adrenoceptor agonists on smooth muscle tone and on cholinergic excitatory and non-adrenergic, non-cholinergic inhibitory responses to field stimulation have been investigated in the rat gastric fundus in-vitro. None of the alpha-adrenoceptor agonists tested, noradrenaline, phenylephrine, cirazoline, guanoxabenz or UK-14,304 showed any contractile effects at concentrations up to 30 microM. In preparations where tone was raised by barium (0.5-2 mM), the mixed alpha 1- and alpha 2-adrenoceptor agonist noradrenaline (0.01-10 microM), and the selective alpha 1-adrenoceptor agonists cirazoline (0.01-10 microM) and phenylephrine (0.01-10 microM) produced concentration-dependent relaxations which were antagonized by the alpha 1-adrenoceptor antagonist prazosin (0.01-1.0 microM). The selective alpha 2-adrenoceptor agonists UK-14,304 (0.03-30 microM) and guanoxabenz (0.03-30 microM), had no relaxant effects in raised tone. UK-14,304 (0.03-1.0 microM) produced a concentration-dependent inhibition of cholinergic nerve-induced responses which was antagonized by the alpha 2-adrenoceptor antagonist idazoxan (0.03-1.0 microM) but not by prazosin (0.03-1.0 microM). Noradrenaline (0.03-1.0 microM) also produced an inhibition of cholinergic nerve-induced responses which was antagonized by idazoxan (0.03-1.0 microM). A small component of the noradrenaline inhibitory effects was antagonized by prazosin (10%). Cirazoline (0.03-1.0 microM) produced a small inhibition of cholinergic nerve-induced responses which was antagonized by prazosin (0.03-1.0 microM). The prazosin-sensitive components of the inhibitory effects of noradrenaline and cirazoline occurred at concentrations which also produced post-junctional relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)