Adenosine A(1) receptor stimulation inhibits alpha(1)-adrenergic activation of the cardiac sarcolemmal Na(+)/H(+) exchanger.
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Sarcolemmal Na(+)/H(+) exchanger (NHE) activity is increased by stimulation of G(q) protein-coupled receptors (G(q)PCRs), but the roles of other GPCRs are largely unknown. We determined the effects of N-[(1S,trans)-2-hydroxycyclopentyl]adenosine (GR79236), a selective agonist of the G(i)PCR adenosine A(1) receptor, on sarcolemmal NHE activity in adult rat ventricular myocytes (n=8-10 per group). NHE activity was indexed by the H(+) efflux rate after intracellular acidification, measured by microepifluorescence. GR79236 alone (0.01-10 microM) had no effect on NHE activity. However, co-administration of GR79236 inhibited, in a concentration-dependent manner, the stimulation of NHE activity by the alpha(1)-adrenoceptor agonist phenylephrine (10 microM). The inhibitory effect of GR79236 (10 microM) was abolished by (1) the selective A(1) antagonist 1,3-dipropyl-8-cyclopentylxanthine (0.1 microM), confirming an A(1) receptor-mediated action, and (2) pre-treatment with pertussis toxin (5 microgram ml(-1) for 60 min), indicating a G(i) protein-mediated mechanism. Our data suggest the existence of inhibitory crosstalk between the G(i)PCR adenosine A(1) receptor and the G(q)PCR alpha(1)-adrenoceptor in the regulation of sarcolemmal NHE activity. (+info)
Endogenous circulating sympatholytic factor in orthostatic intolerance.
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Sympathotonic orthostatic hypotension (SOH) is an idiopathic syndrome characterized by tachycardia, hypotension, elevated plasma norepinephrine, and symptoms of orthostatic intolerance provoked by assumption of an upright posture. We studied a woman with severe progressive SOH with blood pressure unresponsive to the pressor effects of alpha(1)-adrenergic receptor (AR) agonists. We tested the hypothesis that a circulating factor in this patient interferes with vascular adrenergic neurotransmission. Preincubation of porcine pulmonary artery vessel rings with patient plasma produced a dose-dependent inhibition of vasoconstriction to phenylephrine in vitro, abolished vasoconstriction to direct electrical stimulation, and had no effect on nonadrenergic vasoconstrictive stimuli (endothelin-1), PGF-2alpha (or KCl). Preincubation of vessels with control plasma was devoid of these effects. SOH plasma inhibited the binding of an alpha(1)-selective antagonist radioligand ([(125)I]HEAT) to membrane fractions derived from porcine pulmonary artery vessel rings, rat liver, and cell lines selectively overexpressing human ARs of the alpha(1B) subtype but not other AR subtypes (alpha(1A) and alpha(1D)). We conclude that a factor in SOH plasma can selectively and irreversibly inhibit adrenergic ligand binding to alpha(1B) ARs. We propose that this factor contributes to a novel pathogenesis for SOH in this patient. This patient's syndrome represents a new disease entity, and her plasma may provide a unique tool for probing the selective functions of alpha(1)-ARs. (+info)
Effects of castration on contraction and alpha(1)-adrenoceptor expression in rat prostate.
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1. The prostate function is regulated by androgens and alpha-adrenergic activity. Clinically, antiandrogens and/or alpha(1)-adrenergic antagonists are commonly used to treat symptomatic prostatic hypertrophy. To elucidate the effects of androgen deprivation on prostate contractility via alpha(1)-adrenoceptor, the characteristics and expression of alpha(1)-adrenoceptors were examined in castrated rats. 2. Isolated prostate strips from intact and castrated rats were subjected to a phenylephrine stimulated contraction. Prazosin (10 nM), [(3)H]-prazosin and phenoxybenzamine (3 - 300 nM) were used for inhibition assay, receptor characterization and partial alkylation of alpha-adrenoceptor, respectively. The mRNA content of three subtypes of alpha-adrenoceptors was determined by reverse transcription combined with polymerase chain reaction (RT - PCR). 3. Contractile response to phenylephrine increased in castrated rats, which could be explained by a relative increase of the stromal component. A lowered contraction potency was also noted in castrated rats. Receptor binding assay indicated minimal changes in the affinity or density of alpha(1)-adrenoceptor. Escalating alkylation of the alpha(1)-adrenoceptor population resulted in a rightward shift in the contraction-response curves before depressing maximal contractile force, and the suppression was detected at lower doses in castrated rats. RT - PCR study confirmed the expression of three types of alpha(1)-adrenoceptor, alpha(1a), alpha(1b) and alpha(1d)-adrenoceptors, in intact rat prostate, and revealed that alpha(1a)-adrenoceptor, but not alpha(1b) or alpha(1d)-adrenoceptors, was down-regulated in castrates. 4. The results show that androgen deprivation suppressed alpha(1)-adrenergic contractility of rat prostate strips, and the suppression was associated with down-regulation of receptor reserve for the alpha(1a)-adreneroceptor population expressed in intact rat prostate. (+info)
The role of alpha(1)-adrenoceptors and 5-HT(1A) receptors in the control of the micturition reflex in male anaesthetized rats.
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1. The effects of the alpha(1)-adrenoceptor antagonists doxazosin (0.1 -- 2 mg kg(-1)), RS-100329 (alpha(1A); 0.01 -- 1 mg kg(-1)), RS-513815 (Ro 151-3815, alpha(1B); 0.3 -- 3 mg kg(-1)) and BMY 7378 (alpha(1D); 0.1 -- 1 mg kg(-1)), the 5-HT(1A) receptor agonist, 8-OH-DPAT (0.03 -- 0.3 mg kg(-1)) and antagonist WAY-100635 (0.03 -- 0.3 mg kg(-1)) were investigated (i.v.) on the 'micturition reflex' in the urethane anaesthetized male rat. 2. Reflex-evoked urethra contractions were most sensitive to the inhibitory action of RS-100329, followed by doxazosin, BMY 7378 and WAY-100635 and then RS-513815. The maximum inhibition was 66, 63, 54, 46 and 22% at doses of 0.3, 0.5, 0.3, 0.3 and 3 mg kg(-1) respectively. 3. BMY 7378 and 8-OH-DPAT decreased, while WAY-100635 increased, the pressure threshold to induce bladder contraction. WAY-100635 (0.01 mg kg(-1)) blocked the effects of BMY 7378 (1 mg kg(-1)) on bladder pressure and volume threshold. 4. Doxazosin, RS-100329 and BMY 7378 had a similar potency in inducing a fall in arterial blood pressure while WAY-100635 only caused a fall at the highest dose. 5. Therefore, reflex-evoked urethral contraction involves the activation of alpha(1A/1D)-adrenoceptors, as BMY 7378 and RS-100329 are similarly potent in attenuating this effect. The ability of WAY-100635 to attenuate this contraction may suggest that 5-HT(1A) receptors are also involved. However, as this inhibition occurred at the highest dose of WAY-100635, which also caused a fall in arterial blood pressure; this effect is considered to be due to blockade of alpha(1)-adrenoceptors not 5-HT(1A) receptors. Nevertheless the initiation of the 'micturition reflex' involves the activation of 5-HT(1A) receptors. (+info)
beta-Adrenergic stimulation synchronizes intracellular Ca(2+) release during excitation-contraction coupling in cardiac myocytes.
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To elucidate microscopic mechanisms underlying the modulation of cardiac excitation-contraction (EC) coupling by beta-adrenergic receptor (beta-AR) stimulation, we examined local Ca(2+) release function, ie, Ca(2+) spikes at individual transverse tubule-sarcoplasmic reticulum (T-SR) junctions, using confocal microscopy and our recently developed technique for release flux measurement. beta-AR stimulation by norepinephrine plus an alpha(1)-adrenergic blocker, prazosin, increased the amplitude of SR Ca(2+) release flux (J(SR)), its running integral (integralJ(SR)), and L-type Ca(2+) channel current (I(Ca)), and it shifted their bell-shaped voltage dependence leftward by approximately 10 mV, with the relative effects ranking I(Ca)> J(SR)>integralJ(SR). Confocal imaging revealed that the bell-shaped voltage dependence of SR Ca(2+) release is attributable to a graded recruitment of T-SR junctions as well as to changes in Ca(2+) spike amplitudes. beta-AR stimulation increased the fractional T-SR junctions that fired Ca(2+) spikes and augmented Ca(2+) spike amplitudes, without altering the SR Ca(2+) load, suggesting that more release units were activated synchronously among and within T-SR junctions. Moreover, beta-AR stimulation decreased the latency and temporal dispersion of Ca(2+) spike occurrence at a given voltage, delivering most of the Ca(2+) at the onset of depolarization rather than spreading it out throughout depolarization. Because the synchrony of Ca(2+) spikes affects Ca(2+) delivery per unit of time to contractile myofilaments, and because the myofilaments display a steep Ca(2+) dependence, our data suggest that synchronization of SR Ca(2+) release represents a heretofore unappreciated mechanism of beta-AR modulation of cardiac inotropy. (+info)
Phe-308 and Phe-312 in transmembrane domain 7 are major sites of alpha 1-adrenergic receptor antagonist binding. Imidazoline agonists bind like antagonists.
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Although agonist binding in adrenergic receptors is fairly well understood and involves residues located in transmembrane domains 3 through 6, there are few residues reported that are involved in antagonist binding. In fact, a major docking site for antagonists has never been reported in any G-protein coupled receptor. It has been speculated that antagonist binding is quite diverse depending upon the chemical structure of the antagonist, which can be quite different from agonists. We now report the identification of two phenylalanine residues in transmembrane domain 7 of the alpha(1a)-adrenergic receptor (Phe-312 and Phe-308) that are a major site of antagonist affinity. Mutation of either Phe-308 or Phe-312 resulted in significant losses of affinity (4-1200-fold) for the antagonists prazosin, WB4101, BMY7378, (+) niguldipine, and 5-methylurapidil, with no changes in affinity for phenethylamine-type agonists such as epinephrine, methoxamine, or phenylephrine. Interestingly, both residues are involved in the binding of all imidazoline-type agonists such as oxymetazoline, cirazoline, and clonidine, confirming previous evidence that this class of ligand binds differently than phenethylamine-type agonists and may be more antagonist-like, which may explain their partial agonist properties. In modeling these interactions with previous mutagenesis studies and using the current backbone structure of rhodopsin, we conclude that antagonist binding is docked higher in the pocket closer to the extracellular surface than agonist binding and appears skewed toward transmembrane domain 7. (+info)
Antiparkinsonian agent piribedil displays antagonist properties at native, rat, and cloned, human alpha(2)-adrenoceptors: cellular and functional characterization.
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Compared with cloned, human (h)D(2) receptors (pK(i) = 6.9), the antiparkinsonian agent piribedil showed comparable affinity for halpha(2A)- (7.1) and halpha(2C)- (7.2) adrenoceptors (ARs), whereas its affinity for halpha(2B)-ARs was less marked (6.5). At halpha(2A)- and halpha(2C)-ARs, piribedil antagonized induction of [(35)S]guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) binding by norepinephrine (NE) with pK(b) values of 6.5 and 6.9, respectively. Furthermore, Schild analysis of the actions of piribedil at halpha(2A)-ARs indicated competitive antagonism, yielding a pA(2) of 6.5. At a porcine alpha(2A)-AR-Gi1alpha-Cys351C (wild-type) fusion protein, piribedil competitively abolished (pA(2) = 6.5) GTPase activity induced by epinephrine. However, at a alpha(2A)-AR-Gi1alpha-Cys351I (mutant) fusion protein of amplified sensitivity, although still acting as a competitive antagonist (pA(2) = 6.2) of epinephrine, piribedil itself manifested weak partial agonist properties. Similarly, piribedil weakly induced mitogen-activated protein kinase phosphorylation via wild-type halpha(2A)-ARs, although attenuating its phosphorylation by NE. As demonstrated by functional [(35)S]GTPgammaS autoradiography in rats, piribedil antagonized activation by NE of alpha(2)-ARs in cortex, amygdala, and septum. Antagonist properties were also expressed in a dose-dependent enhancement of the firing rate of adrenergic neurons in locus ceruleus (0.125-4.0 mg/kg i.v.). Furthermore, piribedil (2.5-4.0 mg/kg s.c.) accelerated hippocampal NE synthesis, elevated dialysis levels of NE in hippocampus and frontal cortex, and blocked hypnotic-sedative properties of the alpha(2)-AR agonist xylazine. Finally, piribedil showed only modest affinity for rat alpha(1)-ARs (5.9) and weakly antagonized NE-induced activation of phospholipase C via halpha(1A)-ARs (pK(b) = 5.6). In conclusion, piribedil displays essentially antagonist properties at cloned, human and cerebral, rat alpha(2)-ARs. Blockade of alpha(2)-ARs may, thus, contribute to its clinical antiparkinsonian profile. (+info)
Vascular actions of MDMA involve alpha1 and alpha2-adrenoceptors in the anaesthetized rat.
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We have investigated the effects of methylenedioxymethamphetamine (MDMA, 'ecstasy'), i.v., on diastolic blood pressure (DBP) in pithed and pentobarbitone anaesthetized rats. In pithed rats, the non-selective 5-HT receptor antagonist methiothepin (0.1 mg kg(-1)) and the alpha2-adrenoceptor antagonists methoxyidazoxan and yohimbine (1 mg kg(-1)) showed significant alpha1-adrenoceptor antagonist potency, but methiothepin did not show alpha2-adrenoceptor antagonist potency. MDMA (1 and 5 mg kg(-1)) produced pressor responses which were significantly reduced by the alpha(1)-adrenoceptor antagonist prazosin (0.1 mg kg(-1)), yohimbine (1 mg kg(-1)) or methiothepin (0.1 mg kg(-1)), but not by the 5-HT2 receptor antagonist ritanserin (1 mg kg(-1)). In anaesthetized rats, antagonists revealed two phases with three components to the effects of MDMA (5 mg kg(-1)) on DBP: an initial pressor response, a later pressor component at 1 min, the sustained depressor response. Methoxyidazoxan, methiothepin or the combination ritanserin/prazosin significantly reduced the initial pressor response, although neither of the latter compounds alone had any effect. The pressor response to MDMA (5 mg kg(-1)) at 1 min was converted to a depressor response by prazosin and to a lesser extent methiothepin and methoxyidazoxan. The depressor response to MDMA (5 mg kg(-1)) was significantly reduced by methoxyidazoxan (0.1 mg kg(-1)), and by the noradrenaline re-uptake blocker cocaine 10 mg kg(-1) but not 1 mg kg(-1). However, the most marked reduction in the depressor response was produced by the combination of methoxyidazoxan and cocaine. It is concluded that the initial pressor response to MDMA (5 mg kg(-1)) in anaesthetized rats involves alpha2- and possibly alpha1-adrenoceptors and 5-HT2 receptors, the pressor component at 1 min is largely alpha1-adrenoceptor mediated, and the sustained depressor response involves alpha2-adrenoceptors. (+info)