Investigation of the different adrenoceptor targets of nebivolol enantiomers in rat thoracic aorta

Background and purpose:

Nebivolol is a highly selective β₁-adrenoceptor antagonist with β₃-adrenoceptor agonist properties and is a racemate mixture of D- and L-enantiomers. However, the cellular mechanisms of the effects of each enantiomer are not yet clear and are a matter for debate. The aim of the present experiments was to determine the adrenoceptors involved in the vascular effects of D- and L-enantiomers of nebivolol in rat thoracic aorta.

Experimental approach:

Responses to nebivolol enantiomers were evaluated in rings of thoracic aorta from male Sprague-Dawley rats.

Key results:

D-nebivolol (0.1–10 µmol·L⁻¹), but not L-nebivolol, significantly shifted to the right the concentration-response curve to phenylephrine, an α₁-adrenoceptor agonist, in a concentration-dependent manner. For the following experiments, aortic rings were constricted with endothelin 1 and now both enantiomers produced an endothelium-dependent relaxation of the rings involving the nitric oxide pathway. This relaxation was not modified by 1 µmol·L⁻¹ CGP 20,712A (β₁-adrenoceptor antagonist), but significantly blunted by 7 µmol·L⁻¹ L-74,8337 (β₃-adrenoceptor antagonist). However, only the vasorelaxation induced by D-nebivolol was significantly reduced by 1 µmol·L⁻¹ ICI 118,551 (β₂-adrenoceptor antagonist).

Conclusions and implications:

Our results suggest that the nebivolol enantiomers act on different targets. D-nebivolol induced vasorelaxation by activating β₂- and β₃-adrenoceptors and antagonizing α₁-adrenoceptors. L-nebivolol induced vasorelaxation by activating only β₃-adrenoceptors in our model. Our results emphasize that nebivolol is a β₁-adrenoceptor antagonist with several important pharmacological differences from other β₁-adrenoceptor antagonists.     

No evidence for a significant non-nitrergic,hyperpolarising factor contribution to field stimulation-induced relaxation of the mouse anococcygeus

1. The aim of the study was to determine whether a nerve-derived hyperpolarizing factor (NDHF) might contribute to non-adrenergic, non-cholinergic (NANC) relaxations of the mouse anococcygeus when low concentrations of contractile agent are used to raise tone and low frequencies of field stimulation applied; such a non-nitrergic NDHF has been proposed to contribute to NANC relaxations of the rat anococcygeus and guinea-pig taenia coli.
2. Phenylephrine (0.1–100 μM) produced concentration-related contractions of the mouse isolated anococcygeus muscle; 0.2 μM phenylephrine (EC₂₆) was used to raise tone in subsequent experiments.
3. Field stimulation (0.5, 1.0 and 5.0 Hz) produced frequency-dependent relaxations of phenylephrine-induced tone. In the presence of the nitric oxide synthase inhibitor L-N^G-nitro-arginine (L-NOARG; 100 μM), the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-1-one (ODQ; 5 μM), or a combination of these two drugs, relaxations to field stimulation were abolished at all frequencies studied. Relaxations to sodium nitroprusside (0.01–5 μM) were unaffected by L-NOARG but strongly inhibited by ODQ; neither enzyme inhibitor affected relaxations to 8-Br-cyclic GMP (10 μM).
4. Nifedipine (1 μM) reduced the contractile response to 0.2 μM phenylephrine by 38%; however, it had no effect on NANC relaxations.
5. It is concluded that NANC relaxations of the mouse anococcygeus are purely nitrergic and that there is no significant contribution from a putative NDHF.

     

Investigation of the mechanisms underlying the hypophagic effects of the 5-HT and noradrenaline reuptake inhibitor,sibutramine, in the rat

1. Sibutramine is a novel 5-hydroxytryptamine (5-HT) and noradrenaline reuptake inhibitor (serotonin- noradrenaline reuptake inhibitor, SNRI) which is currently being developed as a treatment for obesity. Sibutramine has been shown to decrease food intake in the rat. In this study we have used a variety of monoamine receptor antagonists to examine the pharmacological mechanisms underlying sibutramine-induced hypophagia.
2. Individually-housed male Sprague-Dawley rats were maintained on reversed phase lighting with free access to food and water. Drugs were administered at 09 h 00 min and food intake was monitored over the following 8 h dark period.
3. Sibutramine (10 mg kg⁻¹, p.o.) produced a significant decrease in food intake during the 8 h following drug administration. This hypophagic response was fully antagonized by the α₁-adrenoceptor antagonist, prazosin (0.3 and 1 mg kg⁻¹, i.p.), and partially antagonized by the β₁-adrenoceptor antagonist, metoprolol (3 and 10 mg kg⁻¹, i.p.) and the 5-HT receptor antagonists, metergoline (non-selective; 0.3 mg kg⁻¹, i.p.); ritanserin (5-HT_2A/2C; 0.1 and 0.5 mg kg⁻¹, i.p.) and SB200646 (5-HT_2B/2C; 20 and 40 mg kg⁻¹, p.o.).
4. By contrast, the α₂-adrenoceptor antagonist, RX821002 (0.3 and 1 mg kg⁻¹, i.p.) and the β₂-adrenoceptor antagonist, ICI 118,551 (3 and 10 mg kg⁻¹, i.p.) did not reduce the decrease in food intake induced by sibutramine.
5. These results demonstrate that β₁-adrenoceptors, 5-HT_2A/2C-receptors and particularly α₁-adrenoceptors, are involved in the effects of sibutramine on food intake and are consistent with the hypothesis that sibutramine-induced hypophagia is related to its ability to inhibit the reuptake of both noradrenaline and 5-HT, with the subsequent activation of a variety of noradrenaline and 5-HT receptor systems.

     

Effects of noradrenaline and neuropeptide Y on rat mesenteric microvessel contraction

We have studied the contractile effects of the sympathetic transmitter noradrenaline and its cotransmitter neuropeptide Y (NPY) given alone and in combination on isolated rat mesenteric resistance vessels (200–300 m diameter). Noradrenaline and NPY each concentration-dependently contracted rat mesenteric microvessels (EC₅₀ 800 nM and 10 nM, respectively), but noradrenaline caused considerably greater maximal effects than NPY (14.3 mN vs. 3.5mN). A low antagonistic potency of yohimbine indicated that the response to noradrenaline did not involve ₂-adrenoceptors, and the subtype-selective antagonists 5-methylurapidil, tamsulosin and chloroethylclonidine indicated mediation via an _1A-adrenoceptor. Shallow Schild regressions for prazosin and 5-methylurapidil indicated that an ₁-adrenoceptor subtype with relatively low prazosin affinity might additionally be involved. Studies with the NPY analogues PYY, [Leu³¹, Pro³⁴]NPY and NPY_18–36 demonstrated that NPY acted via a Y₁ NPY receptor. In addition to its direct vasoconstricting effects NPY also lowered the noradrenaline EC₅₀ but did not appreciably affect maximal noradrenaline responses indicating possible potentiation. The potentiating NPY response occured with similar agonist potency as the direct contractile NPY effects and also via a Y₁ NPY receptor. The Ca²⁺ entry blocker nitrendipine (300 nM) reduced direct contractile responses to noradrenaline and NPY but did not affect the potentiation response to NPY.     

Pharmacological characterization of the presynaptic activity of Tityus serrulatus venom in the rat anococcygeus muscle.

Scorpion venoms are known to cause peripheral nerve stimulation with enhanced autonomic responses. This study, therefore, examined the effects of Tityus serrulatus venom (TSV) on adrenergic, cholinergic and nitrergic nerve fibers using the rat anococcygeus muscle. The contractile effects of TSV (1 microg/ml) and electrical field stimulation were markedly reduced by phentolamine (5 microM), prazosin (0.1 microM), guanethidine (30 microM) and tetrodotoxin (TTX, 1 microM), whereas imipramine (3 microM) enhanced these responses. The responses to tyramine (10 microM) were partially reduced by guanethidine and completely blocked by phentolamine, prazosin and imipramine. Atropine (1 microM) fully prevented carbachol (CCh, 30 microM)-induced contractions without affecting those mediated by TSV. Neostigmine significantly potentiated TSV-and ACh-evoked contractions, whereas hexamethonium had no effect. The relaxant responses induced by EFS and TSV (3 microg/ml) were completely blocked by L-NAME (100 microM), ODQ (1 microM) or TTX (1 microM). Addition of L-arginine (1 mM) reversed the effect of L-NAME. Thus, the motor and inhibitory responses of TSV in the rat anococcygeus muscle are mediated by prejunctional mechanisms dependent on Na(+) channel activation, causing the stimulation of NA and NO release from adrenergic and nitrergic nerve fibers, respectively.     

Mechanism of nitric oxide-induced contraction in the rat isolated small intestine

1. The contractile response to nitric oxide (NO) in ral ileal myenteric plexus-longitudinal muscle strips was pharmacologically analysed.
2. NO (10⁻⁷ M) induced only contraction while 10⁻⁶ M NO induced contraction followed by relaxation. Methylene blue (up to 10⁻⁴ M) did not affect the NO-induced contractions but significantly reduced the relaxation evoked by 10⁻⁶ M NO. Administration of 8-bromo-cyclic GMP (10⁻⁶–10⁻⁴ M) only induced relaxation.
3. Sodium nitroprusside (SNP; 10⁻⁷–10⁻⁵ M) induced concentration-dependent contractions per se; the contractile response to NO, administered within 10 min after SNP, was concentration-dependently reduced. The guanosine 3′:5′-cyclic monophosphate (cyclic GMP) content of the tissues was not increased during contractions with 10⁻⁸ M NO and 10⁻⁶ M SNP; it was increased by a factor of 2 during contraction with 10⁻⁷ M NO, and by a factor of 12 during relaxation with 3×10⁻⁶ M NO.
4. The NO-induced contractions were not affected by ryanodine (3×10⁻⁵ M) but were concentration-dependently reduced by nifedipine (10⁻⁸–10⁻⁷ M) and apamin (3×10⁻⁹–3×10⁻⁸ M).
5. These results suggest that cyclic GMP is not involved in the NO-induced contraction in the rat small intestine. The NO-induced contraction is related to extracellular Ca²⁺ influx through L-type Ca²⁺ channels, that might be activated in response to the closure of Ca²⁺-dependent K⁺ channels.

     

Pharmacological characterization of CGRP receptors mediating relaxation of the rat pulmonary artery and inhibition of twitch responses of the rat vas deferens

1. CGRP receptors mediating vasorelaxation of the rat isolated pulmonary artery and inhibition of contractions of the rat isolated prostatic vas deferens were investigated using CGRP agonists, homologues and the antagonist CGRP_8-37.
2. In the pulmonary artery, human (h)α-CGRP-induced relaxation of phenylephrine-evoked tone was abolished either by removal of the endothelium or by N^G-nitro-L-arginine (10⁻⁵ M). The inhibitory effect of N^G-nitro-L-arginine was stereoselectively reversed by L- but not by D-arginine (10⁻⁴ M). Thus, CGRP acts via nitric oxide released from the endothelium.
3. In the endothelium-intact artery, hα-CGRP, hβ-CGRP and human adrenomedullin (10⁻¹⁰–3×10⁻⁷ M), dose-dependently relaxed the phenylephrine-induced tone with similar potency. Compared with hα-CGRP, rat amylin was around 50 fold less potent, while [Cys(ACM^2,7)] hα-CGRP (10⁻⁷–10⁻⁴ M) was at least 3000 fold less potent. Salmon calcitonin was inactive (up to 10⁻⁴ M).
4. Human α-CGRP_8-37 (3×10⁻⁷–3×10⁻⁶ M) antagonized hα-CGRP (pA₂ 6.9, Schild plot slope 1.2±0.1) and hβ-CGRP (apparent pK_B of 7.1±0.1 for hα-CGRP_8-37 10⁻⁶ M) in the pulmonary artery. Human β-CGRP_8-37 (10⁻⁶ M) antagonized hα-CGRP responses with a similar affinity (apparent pK_B 7.1±0.1). Human adrenomedullin responses were not inhibited by hα-CGRP_8-37 (10⁻⁶ M).
5. In the prostatic vas deferens, hα-CGRP, hβ-CGRP and rat β-CGRP (10⁻¹⁰–3×10⁻⁷ M) concentration-dependently inhibited twitch responses with about equal potency, while rat amylin (10⁻⁸–10⁻⁵ M) was around 10 fold less potent and the linear analogue [Cys(ACM^2,7)] hα-CGRP was at least 3000 fold weaker. Salmon calcitonin was inactive (up to 10⁻⁴ M).
6. The antagonist effect of hα-CGRP_8-37 (10⁻⁵–3×10⁻⁵) in the vas deferens was independent of the agonist, with pA₂ values against hα-CGRP of 6.0 (slope 0.9±0.1), against hβ-CGRP of 5.8 (slope 1.1±0.1), and an apparent pK_B value of 5.8±0.1 against both rat β-CGRP and rat amylin. Human β-CGRP_8-37 (3×10⁻⁵–10⁻⁴ M) competitively antagonized hα-CGRP responses (pA₂ 5.6, slope 1.1±0.2). The inhibitory effect of hα-CGRP on noradrenaline-induced contractions in both the prostatic and epididymal vas deferens was antagonized by hα-CGRP_8-37 (pA₂ 5.8 and 5.8, slope 1.0±0.2 and 1.0±0.3, respectively).
7. The effects of hα-CGRP and hα-CGRP_8-37 in both rat pulmonary artery and vas deferens were not significantly altered by pretreatment with peptidase inhibitors (amastatin, bestatin, captopril, phosphoramidon and thiorphan, all at 10⁻⁶ M). The weak agonist activity of [Cys(ACM^2,7)] hα-CGRP in the vas deferens was not increased by peptidase inhibitors.
8. These data demonstrate that two different CGRP receptors may exist in the rat pulmonary artery and vas deferens, a CGRP₁ receptor subtype in the rat pulmonary artery (CGRP_8-37 pA₂ 6.9), while the lower affinity for CGRP_8-37 (pA₂ 6.0) in the vas deferens is consistent with a CGRP₂ receptor.

     

Effects of T-type calcium channel blockers and thalidomide on contractions of rat vas deferens

Background and purpose:

In rat vas deferens, nerve mediated-contractions to a single electrical stimulus consist of an early purinergic and a later adrenergic component with differing sensitivities to L-type calcium channel blockers. We have investigated the effects of the T-type calcium channel blockers mibefradil and (1S, 2S)-2-[2-[[3-(1H-benzimidazol-2-yl)propyl]methylamino]ethyl]-6-fluoro-1,2,3,4-tetrahydro-1-(1-methylethyl)-2-naphthalenyl cyclopropanecarboxylic dihydrochloride (NNC 55-0396) against contractions in rat vas deferens. In addition, the actions of thalidomide were examined.

Experimental approach:

Prostatic and epididymal portions of rat vas deferens were stimulated with a single electrical stimulus every 5 min, and mouse whole vas deferens was stimulated with 40 pulses at 10 Hz every 5 min.

Key results:

Both mibefradil and NNC 55-0396 (100 µM) produced inhibition of contractions of epididymal portions (42 ± 13%, n= 7, and 43 ± 4%, n= 15, of control respectively). However, both agents produced small inhibitions of responses in prostatic portions, presumably by L-type calcium channel block. Thalidomide (100 µM) inhibited contractions in epididymal (55 ± 4% of control, n= 17) but not in prostatic portions of rat vas deferens. Thalidomide (10–100 µM) also inhibited contractions in mouse vas deferens.

Conclusions and implications:

The T-type calcium channel blockers mibefradil and NNC 55-0396 block particularly the adrenoceptor-mediated, nifedipine-resistant response to nerve stimulation in rat vas deferens, and this may suggest that this component involves T-type calcium channels. In addition, thalidomide has actions that resemble those of the T-type calcium channel blockers, in that it blocks nifedipine-resistant contractions in epididymal portions.     

Role of alpha1-adrenoceptor subtypes in the effects of methylenedioxy methamphetamine (MDMA) on body temperature in the mouse

BACKGROUND AND PURPOSE: We have investigated the ability of alpha(1)-adrenoceptor antagonists to affect the hyperthermia produced by methylenedioxy methamphetamine (MDMA) in conscious mice. EXPERIMENTAL APPROACH: Mice were implanted with temperature probes under ether anaesthesia and allowed 2 weeks recovery. MDMA (20 mg kg(-1)) was administered subcutaneously 30 min after vehicle or test antagonist or combination of antagonists and effects on body temperature monitored. KEY RESULTS: Following vehicle, MDMA produced a hyperthermia, reaching a maximum increase of 1.8 degrees C at 140 min. Prazosin (0.1 mg kg(-1)) revealed an early significant hypothermia to MDMA of -1.94 degrees C. The alpha(1A)-adrenoceptor antagonist RS 100329 (0.1 mg kg(-1)), or the alpha(1D)-adrenoceptor antagonist BMY 7378 (0.5 mg kg(-1)) given alone, did not reveal a hypothermia to MDMA, but the combination of the two antagonists revealed a significant hypothermia to MDMA. The putative alpha(1B)-adrenoceptor antagonist cyclazosin (1 mg kg(-1)) also revealed a significant hypothermia to MDMA, but actions of cyclazosin at the other alpha(1)-adrenoceptor subtypes cannot be excluded. CONCLUSIONS AND IMPLICATIONS: More than one subtype of alpha(1)-adrenoceptor is involved in a component of the hyperthermic response to MDMA in mouse, probably both alpha(1A)- and alpha(1D)-adrenoceptors, and removal of this alpha(1)-adrenoceptor-mediated component reveals an initial hypothermia.     

Role of α1- and β3-adrenoceptors in the modulation by SR59230A of the effects of MDMA on body temperature in the mouse

Background and purpose:

We have investigated the ability of the β₃-adrenoceptor antagonist 1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4,-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride (SR59230A) to affect the hyperthermia produced by methylenedioxymethamphetamine (MDMA) in conscious mice and whether α₁-adrenoceptor antagonist actions are involved.

Experimental approach:

Mice were implanted with temperature probes under anaesthesia, and allowed 2 week recovery. MDMA (20 mg·kg⁻¹) was administered subcutaneously 30 min after vehicle or test antagonist and effects on body temperature monitored by telemetry.

Key results:

Following vehicle, MDMA produced a slowly developing hyperthermia, reaching a maximum increase of 1.8°C at 130 min post injection. A low concentration of SR59230A (0.5 mg·kg⁻¹) produced a small but significant attenuation of the slowly developing hyperthermia to MDMA. A high concentration of SR59230A (5 mg·kg⁻¹) revealed a significant and marked early hypothermic reaction to MDMA, an effect that was mimicked by the α₁-adrenoceptor antagonist prazosin. Functional and ligand binding studies revealed actions of SR59230A at α₁-adrenoceptors.

Conclusions and implications:

1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4,-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride in high concentrations modulates the hyperthermic actions of MDMA in mice in two ways: by blocking an early α₁-adrenoceptor-mediated component to reveal a hypothermia, and by a small attenuation of the later hyperthermic component which may possibly be β₃-adrenoceptor-mediated (this seen with the low concentration of SR59230A). Hence, the major actions of SR59230A in modulating the actions of MDMA on temperature involve α₁-adrenoceptor antagonism.     

Effects of adrenomedullin and calcitonin gene-related peptide on contractions of the rat aorta and porcine coronary artery

1. Effects of adrenomedullin and α-calcitonin gene-related peptide (CGRP) on the contractions and cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) of the rat aorta and porcine coronary artery were investigated. Characteristics of the receptors mediating the effects of adrenomedullin and α-CGRP were also investigated.
2. Adrenomedullin and α-CGRP caused a concentration-dependent relaxation in the rat aorta contracted with noradrenaline. The IC₅₀ values for adrenomedullin and α-CGRP were 2.4 nM and 4.0 nM, respectively. The relaxant effects of these peptides were abolished by removal of the endothelium and significantly attenuated by an inhibitor of nitric oxide synthase, N^G-monomethyl-L-arginine (L-NMMA, 100 μM), but not by a cyclo-oxygenase inhibitor, indomethacin (10 μM).
3. Adrenomedullin and α-CGRP increased the endothelial [Ca²⁺]_i in the rat aorta with endothelium, whereas they did not change [Ca²⁺]_i in the smooth muscle.
4. An antagonist of the CGRP₁ receptor, CGRP (8–37), antagonized the relaxant effects of α-CGRP and the β-isoform of CGRP (β-CGRP) but not those of adrenomedullin in the rat aorta.
5. In the porcine coronary artery contracted with U46619, adrenomedullin and α-CGRP caused a concentration-dependent relaxation with an IC₅₀ of 27.6 and 4.1 nM, respectively. Removal of the endothelium altered neither the IC₅₀ values nor the maximal relaxations induced by adrenomedullin or α-CGRP. When the artery was contracted with high K⁺ solution (72.7 mM), these peptides caused a small relaxation.
6. Adrenomedullin and α-CGRP increased cyclic AMP content and decreased the smooth muscle [Ca²⁺]_i in the porcine coronary artery.
7. CGRP (8–37) significantly antagonized the relaxant effects of adrenomedullin and α-CGRP in the porcine coronary artery. However, it had little effect on the relaxations induced by the β-isoform of CGRP (β-CGRP).
8. These results suggest that in the rat aorta, adrenomedullin and α-CGRP increase the endothelial [Ca²⁺]_i, activate nitric oxide synthase and release nitric oxide, without a direct inhibitory action on smooth muscle. In the porcine coronary artery, in contrast, adrenomedullin and α-CGRP directly act on smooth muscle, increase cyclic AMP content, decrease the smooth muscle [Ca²⁺]_i and inhibit contraction. The rat aortic endothelium seems to express the CGRP receptor which is sensitive to α-CGRP, β-CGRP and CGRP (8–37) and the adrenomedullin specific receptor. The porcine coronary smooth muscle, in contrast, seems to express two types of CGRP receptor; one of which is sensitive to α-CGRP, CGRP (8–37) and adrenomedullin and the other is sensitive only to β-CGRP.

     

α1L-Adrenoceptor mediation of smooth muscle contraction in rabbit bladder neck: a model for lower urinary tract tissues of man

1. The α₁-adrenoceptor population mediating contractile responses to noradrenaline (NA) in smooth muscles of the bladder neck from rabbit (RBN) has been characterized by use of quantitative receptor pharmacology.
2. Experiments with several `key'' α₁-adrenoceptor antagonists of varying subtype selectivities (RS-17053, BMY 7378, indoramin, 5-methylurapidil, prazosin, REC 15/2739, SNAP 5089, terazosin, WB 4101, tamsulosin, (+)-cyclazosin and RS-100329) were conducted. Schild regression analyses yielded affinity (mean pK_b) estimates of 7.1, 6.2, 8.6, 8.6, 8.4, 9.3, 7.0, 7.4, 8.9, 10.0, 7.1 and 9.3, respectively, although deviations from unit Schild regression slope question the robustness of data for RS-17053 and SNAP 5089.
3. The nature of antagonism by these agents and the profile of affinity determinations generated together suggest that a single α₁-adrenoceptor subtype mediates contractile responses of RBN to NA. Additional studies with phenylephrine indicated also an agonist-independence of this profile. Pharmacologically, this profile was reminiscent of that described as `α<sub>1L</sub>''-adrenoceptor, which has been shown to mediate contractions of several tissues including lower urinary tract (LUT) tissues of man. Furthermore, a similarity was noticed between the `α_1L''-adrenoceptor described here in RBN and the rabbit and human cloned α_1a-adrenoceptor (based on data from both whole cell radioligand binding at 37°C and [³H]-inositol phosphates accumulation assays), characterizations of which have been published elsewhere.
4. In conclusion, the RBN appears to provide a predictive pharmacological assay for the study of NA-induced smooth muscle contraction in LUT tissues of man.

     

Effects of dopamine on L-type Ca2+ current in single atrial and ventricular myocytes of the rat

1. The effects of dopamine on the L-type Ca²⁺ current (I_Ca,L) of both atrial and ventricular single myocytes and on the force of contraction of atrial trabeculae in rat heart were investigated.
2. Dopamine increased atrial I_Ca,L at concentrations higher than 1 μM, but had little or no effect on I_Ca,L at lower concentrations. The increase in I_Ca,L at high concentrations was reversed by propranolol and acetylcholine, but not by phentolamine. Activation and inactivation kinetics of I_Ca,L were not altered by dopamine.
3. In rat ventricular myocytes in which the D₄ receptor mRNA does not express, dopamine (20–100 μM) also increased the I_Ca,L amplitude and propranolol reversed this effect.
4. Clozapine, a potent D₄ receptor antagonist, blocked the augmenting effect of dopamine on I_Ca,L. However, this effect could be explained by β-antagonism, since clozapine also inhibited the isoprenaline effect.
5. In the atrial trabeculae, the increase in contraction by dopamine (1 to 30 μM) was reversed by 1 μM propranolol, but not by 2 μM phentolamine. Low doses of dopamine (0.01 to 0.3 μM) did not affect the contraction in the controls or during a modest stimulation of the β-adrenoceptor with 0.01 μM isoprenaline.
6. These results indicate that the positive inotropic action of dopamine is mediated through direct stimulation of the β-adrenoceptor in both atrial and ventricular myocytes. Involvement of D₄ receptor appears unlikely in the regulation of the atrial contraction.

     

Agonist pharmacology at recombinant α1A- and α1L-adrenoceptors and in lower urinary tract α1-adrenoceptors

BACKGROUND AND PURPOSE

Two distinct α₁-adrenoceptor phenotypes (α_1A and α_1L) have recently been demonstrated to originate from a single α_1A-adrenoceptor gene. Here, we examined the agonist profiles of recombinant α_1A and α_1L phenotypes and of lower urinary tract (LUT) α₁-adrenoceptors.

EXPERIMENTAL APPROACH

A series of drugs (A61603, Ro 115–1240, NS-49, MK017 and ESR1150) originally developed for stress urinary incontinence (SUI) therapy were used to stimulate recombinant α_1A- and α_1L-adrenoceptor phenotypes, and their potencies and intrinsic activity estimated from Ca²⁺ responses. Agonist-induced contractions were also examined in LUT tissues of rats and humans and in human mesenteric artery and rat tail artery.

KEY RESULTS

All the drugs were potent agonists of the α_1A-adrenoceptor compared with the α_1L-adrenoceptor phenotype. Among them, Ro 115–1240 was shown to be an α_1A-specific partial agonist that produced partial contractions through α_1A-adrenoceptors in rat prostate and tail artery, but not in the other LUT tissues and human mesenteric artery. In contrast, P-come 102 showed full agonist activity at α_1A- and α_1L-adrenoceptors, but was less selective than noradrenaline for α_1A-adrenoceptors. Like noradrenaline, P-come 102 was highly potent at inducing contractions in all of the LUT tissues tested. However, the potency and intrinsic activity of P-come 102 were significantly lower than those of noradrenaline in human mesenteric artery.

CONCLUSIONS AND IMPLICATIONS

The α_1A- and α_1L-adrenoceptor phenotypes and LUT α₁-adrenoceptors were demonstrated to have distinct agonist profiles. As adrenergic contractions in LUT are predominantly mediated through α_1L-adrenoceptors, the development of α_1L-selective agonists may provide clinically useful drugs for SUI therapy.     

Relationships between structure and vascular activity in a series of benzylisoquinolines

1. In the present work, the properties of 3-methyl isoquinoline, 3,4-dihydropapaverine, tetrahydropapaverine and tetrahydropapaveroline were compared with those of papaverine and laudanosine. The work includes functional studies on rat isolated aorta contracted with noradrenaline, caffeine or KC1, and a determination of the affinity of the compounds for α₁-adrenoceptors and calcium channel binding sites, with [^#H]-prazosin, [^#H]-nitrendipine and [^#H]-(+)-cis-diltiazem binding to rat cerebral cortical membranes. The effects of papaverine derivatives on the different molecular forms of cyclic nucleotide phosphodiesterases (PDE) isolated from bovine aorta were also determined.
2. The three papaverine derivatives show greater affinity than papaverine for the [^#H]-prazosin binding site. They are therefore more selective as inhibitors of [^#H]-prazosin binding as opposed to [^#H]-(+)-cis-diltiazem, while papaverine appears to have approximately equal affinity for both. [^#H]-nitrendipine binding was not affected by either papaverine or papaverine derivatives in concentrations up to 100 μM. 3-Methylisoquinoline had no effect on any of the binding sites assayed.
3. Contractions evoked by noradrenaline (1 μM) in rat aorta were inhibited in a concentration-dependent manner by 3,4-dihydropapaverine, tetrahydropapaverine and with a lower potency, by tetrahydropapaveroline. In Ca²⁺-free solution, tetrahydropapaverine and to a lesser extent, tetrahydropapaveroline, inhibited the noradrenaline (1 μM) evoked contraction in a concentration-dependent manner and did not modify the phasic contractile response evoked by caffeine (10 mM). This suggests that these alkaloids do not act at the intracellular level, unlike papaverine which inhibits the contractile response to caffeine and noradrenaline.
4. Inositol phosphates formation induced by noradrenaline (1 μM) in rat aorta was inhibited by tetrahydropapaverine (100 μM) and tetrahydropapaveroline (300 μM), thus suggesting that α_1D-adrenoceptors are coupled to phosphoinositide metabolism in rat aorta.
5. Unlike papaverine, which has a significant effect on all the PDE isoforms, the three alkaloids assayed did not have an inhibitory effect on the different forms of PDE isolated from bovine aorta.
6. These results provide evidence that papaverine derivatives with a partially or totally reduced isoquinoline ring have a greater affinity for α₁-adrenoceptors and a lower affinity for benzothiazepine sites in the Ca²⁺-channel than papaverine. This structural feature also implies a loss of the inhibitory activity on PDE isoforms. The planarity of the isoquinoline ring (papaverine) impairs the interaction with the α₁-adrenoceptor site and facilitates it with the Ca²⁺-channels and PDEs, whereas the more flexible tetrahydroisoquinoline ring increases the binding to α₁-adrenoceptors.

     

Inhibition of adrenergic human prostate smooth muscle contraction by the inhibitors of c-Jun N-terminal kinase, SP600125 and BI-78D3

BACKGROUND AND PURPOSE α(1) -Adrenoceptor-induced contraction of prostate smooth muscle is mediated by calcium- and Rho kinase-dependent mechanisms. In addition, other mechanisms, such as activation of c-jun N-terminal kinase (JNK) may be involved. Here, we investigated whether JNK participates in α(1)-adrenoceptor-induced contraction of human prostate smooth muscle. EXPERIMENTAL APPROACH Prostate tissue was obtained from patients undergoing radical prostatectomy. Effects of the JNK inhibitors SP600125 (50 μM) and BI-78D3 (30 μM) on contractions induced by phenylephrine, noradrenaline and electric field stimulation (EFS) were studied in myographic measurements. JNK activation by noradrenaline (30 μM) and phenylephrine (10 μM), and the effects of JNK inhibitors of c-Jun phosphorylation were assessed by Western blot analyses with phospho-specific antibodies. Expression of JNK was studied by immunohistochemistry and fluorescence double staining. KEY RESULTS The JNK inhibitors SP600125 and BI-78D3 reduced phenylephrine- and noradrenaline-induced contractions of human prostate strips. In addition, SP600125 reduced EFS-induced contraction of prostate strips. Stimulation of prostate tissue with noradrenaline or phenylephrine in vitro resulted in activation of JNK. Incubation of prostate tissue with SP600125 or BI-78D3 reduced the phosphorylation state of c-Jun. Immunohistochemical staining demonstrated the expression of JNK in smooth muscle cells of human prostate tissue. Fluorescence staining showed that α(1A)-adrenoceptors and JNK are expressed in the same cells. CONCLUSIONS AND IMPLICATIONS Activation of JNK is involved in α(1)-adrenoceptor-induced prostate smooth muscle contraction. Models of α(1)-adrenoceptor-mediated prostate smooth muscle contraction should include this JNK-dependent mechanism.     

Chronic inhibition of monoamine oxidase reduces noradrenaline release in rat vas deferens and anococcygeus muscle

Summary Rats were treated once (acute) or once daily for 21 days (chronic) with clorgyline (2 mg/kg) or nialamide (50 mg/kg). (–)Deprenyl (1 mg/kg) was given for 21 days. One day after the last injection, vas deferens and anococcygeus muscles were removed and noradrenaline stores labelled with ³H-noradrenaline. Efflux of total tritium following electrical field stimulation was decreased by both acute and chronic treatment with clorgyline and nialamide, as well as by chronic treatment with deprenyl. Total tritium release from anococcygeus muscle was reduced by both acute and chronic treatment with clorgyline. Fractionation of the effluent showed that release of both free noradrenaline and metabolites was decreased by MAO inhibitor treatment in vivo, but this effect was not reproduced by in vitro incubation of the vas deferens with clorgyline (1 M). By contrast to the effect of electrical field stimulation, release of 3Hnoradrenaline induced by veratrine was increased by chronic treatment with both clorgyline and nialamide. Release of total tritium by depolarising concentrations of KCl was also increased by chronic clorgyline treatment. These results could be explained by a proportionally greater release of tritium from a cytoplasmic compartment following veratrine and KCl than electrical stimulation, since MAO inhibition increases cytoplasmic noradrenaline levels. Alternatively, release by electrical stimulation may be affected to a greater extent by presynaptic ₂-adrenoceptors, and presynaptic receptors may be stimulated by increased synaptic levels of free noradrenaline following MAO inhibition. Send offprint requests to J. P. M. Finberg at the above address     

Influence of combined hypertension and renal failure on functional alpha(1)-adrenoceptor subtypes in the rat kidney

BACKGROUND AND PURPOSE: This study investigated whether the alpha(1)-adrenoceptor responsiveness of the renal vasculature was altered in the state of hypertension combined with renal failure. EXPERIMENTAL APPROACH: Male spontaneously hypertensive rats (SHR) received cisplatin (5 mg kg(-1) i.p.) to induce renal failure. Seven days later, the rats were anaesthetized and the reductions in renal blood flow (RBF) to electrical renal nerve stimulation (RNS) and intrarenal administration of three adrenoceptor agonists (noradrenaline, phenylephrine and methoxamine) were determined before and after amlodipine, 5-methylurapidil, chloroethylclonidine or BMY 7378. KEY RESULTS: In renal failure SHR (RFSHR), RBF and creatinine clearance were significantly reduced (approximately 70%), while urine output and fractional sodium excretion were four and twenty-fold higher, respectively, compared to SHR. Vasoconstrictions induced by RNS or the adrenoceptor agonists were greater in RFSHR than SHR, and these responses were blunted by 5-methylurapidil, BMY 7378 and amlodipine in the SHR, while chloroethylclonidine had no effect. In the RFSHR, all renal vasoconstrictions were reduced by amlodipine and BMY 7378 but 5-methylurapidil attenuated those caused by RNS, noradrenaline and methoxamine while those to phenylephrine were enhanced. Chloroethylclonidine potentiated renal vasoconstrictor responses to methoxamine and phenylephrine but not RNS or noradrenaline in RFSHR. CONCLUSIONS AND IMPLICATIONS: These findings suggest alpha(1A)- and alpha(1D)-adrenoceptors mediated the renal vasoconstrictor responses in SHR and RFSHR. In the RFSHR, other alpha(1)-adrenoceptor subtypes, for example, alpha(1B)-adrenoceptors appeared to play a greater role.     

Identification of the alpha1L-adrenoceptor in rat cerebral cortex and possible relationship between alpha1L- and alpha1A-adrenoceptors

BACKGROUND AND PURPOSE: In addition to alpha1A, alpha1B and alpha1D-adrenoceptors (ARs), putative alpha1L-ARs with a low affinity for prazosin have been proposed. The purpose of the present study was to identify the alpha1A-AR and clarify its pharmacological profile using a radioligand binding assay. EXPERIMENTAL APPROACH: Binding experiments with [3H]-silodosin and [3H]-prazosin were performed in intact tissue segments and crude membrane preparations of rat cerebral cortex. Intact tissue binding assays were also conducted in rat tail artery. KEY RESULTS: [3H]-silodosin at subnanomolar concentrations specifically bound to intact tissue segments and membrane preparations of rat cerebral cortex at the same density (approximately 150 fmol mg(-1) total tissue protein). The binding sites in intact segments consisted of alpha1A and alpha1L-ARs that had different affinities for prazosin, while the binding sites in membranes showed an alpha1A-AR-like profile having single high affinity for prazosin. [3H]-prazosin also bound at subnanomolar concentrations to alpha1A and alpha1B-ARs but not alpha1L-ARs in cerebral cortex; the binding densities being approximately 200 and 290 fmol mg(-1) protein in the segments and the membranes, respectively. In the segments of tail artery, [3H]-silodosin only recognized alpha1A-ARs, whereas [3H]-prazosin bound to alpha1A and alpha1B-ARs. CONCLUSIONS AND IMPLICATIONS: The present study clearly reveals the presence of alpha1L-ARs as a pharmacologically distinct entity from alpha1A and alpha1B-ARs in intact tissue segments of rat cerebral cortex but not tail artery. However, the alpha1L-ARs disappeared after tissue homogenization, suggesting their decomposition and/or their pharmacological profile changes to that of alpha1A-ARs.