Modulation of basal intracellular calcium by inverse agonists and phorbol myristate acetate in rat-1 fibroblasts stably expressing alpha1d-adrenoceptors.
In rat-1 fibroblasts stably expressing alpha1d-adrenoceptors BMY 7378, phentolamine, chloroethylclonidine and 5-methyl urapidil decreased basal [Ca2+]i. WB 4101 induced a very small effect on this parameter but when added before the other antagonists it blocked their effect. All these agents inhibited the action of norepinephrine. Phorbol myristate acetate also blocked the effect of norepinephrine and decreased basal [Ca2+]i. Staurosporine inhibited these effects of the phorbol ester. Our results suggest that: (1) alpha1d-adrenoceptors exhibit spontaneous ligand-independent activity, (2) BMY 7378, phentolamine, chloroethylclonidine and 5-methyl urapidil act as inverse agonists and (3) protein kinase C activation blocks spontaneous and agonist-stimulated alpha1d-adrenoceptor activity. (+info)
Neuroregulation by vasoactive intestinal peptide (VIP) of mucus secretion in ferret trachea: activation of BK(Ca) channels and inhibition of neurotransmitter release.
1. The aims of this study were to determine: (1) whether vasoactive intestinal peptide (VIP) regulates cholinergic and 'sensory-efferent' (tachykininergic) 35SO4 labelled mucus output in ferret trachea in vitro, using a VIP antibody, (2) the class of potassium (K+) channel involved in VIP-regulation of cholinergic neural secretion using glibenclamide (an ATP-sensitive K+ (K(ATP)) channel inhibitor), iberiotoxin (a large conductance calcium activated K+ (BK(ca)) channel blocker), and apamin (a small conductance K(ca) (SK(ca)) channel blocker), and (3) the effect of VIP on cholinergic neurotransmission using [3H]-choline overflow as a marker for acetylcholine (ACh) release. 2. Exogenous VIP (1 and 10 microM) alone increased 35SO4 output by up to 53% above baseline, but suppressed (by up to 80% at 1 microM) cholinergic and tachykininergic neural secretion without altering secretion induced by ACh or substance P (1 microM each). Endogenous VIP accounted for the minor increase in non-adrenergic, non-cholinergic (NANC), non-tachykininergic neural secretion, which was compatible with the secretory response of exogenous VIP. 3. Iberiotoxin (3 microM), but not apamin (1 microM) or glibenclamide (0.1 microM), reversed the inhibition by VIP (10 nM) of cholinergic neural secretion. 4. Both endogenous VIP (by use of the VIP antibody; 1:500 dilution) and exogenous VIP (0.1 microM), the latter by 34%, inhibited ACh release from cholinergic nerve terminals and this suppression was completely reversed by iberiotoxin (0.1 microM). 5. We conclude that, in ferret trachea in vitro, endogenous VIP has dual activity whereby its small direct stimulatory action on mucus secretion is secondary to its marked regulation of cholinergic and tachykininergic neurogenic mucus secretion. Regulation is via inhibition of neurotransmitter release, consequent upon opening of BK(Ca) channels. In the context of neurogenic mucus secretion, we propose that VIP joins NO as a neurotransmitter of i-NANC nerves in ferret trachea. (+info)
Assessment of cardiac sympathetic regulation by respiratory-related arterial pressure variability in the rat.
1. Mechanical ventilation evokes a corresponding arterial pressure variability (APV) which is decreased by beta-adrenoceptor antagonism. Therefore, in this study we set out to determine whether the respiratory-related APV can be used to assess cardiac sympathetic tone. 2. Computer-generated broad-band mechanical ventilation (0-3 Hz) was applied to Sprague-Dawley rats that had been anaesthetized with ketamine and paralysed with pancuronium. APV and its relationship to lung volume variability (LVV-APV) was systematically quantified with auto- or cross-spectral frequency domain analysis. 3. APV and LVV-APV transfer magnitudes between 0.5 and 1.5 Hz showed dose-dependent suppression by propranolol from 0.01 to 1 mg kg-1, while the static value of arterial pressure remained unchanged. Stroke volume variability, assessed by the use of a pulse contour method, exhibited a similar pattern of suppression by propranolol. In contrast, heart rate variability was not lowered with propranolol. 4. The effect of propranolol on respiratory-related APV persisted even in the presence of combined alpha-adrenoceptor and muscarinic receptor blockade by phentolamine and atropine. 5. The frequency range of 0.5-1.0 Hz was optimal for LVV-APV transfer magnitude to correlate with cardiac sympathetic tone. 6. We conclude that respiratory-related APV may provide a valid assessment of cardiac sympathetic regulation which is independent of parasympathetic and vascular sympathetic influences in ketamine-anaesthetized and positive pressure-ventilated rats. (+info)
Effects of vasopressin on the sympathetic contraction of rabbit ear artery during cooling.
In order to analyse the effects of arginine-vasopressin on the vascular contraction to sympathetic nerve stimulation during cooling, the isometric response of isolated, 2-mm segments of the rabbit central ear (cutaneous) artery to electrical field stimulation (1-8 Hz) was recorded at 37 and 30 degrees C. Electrical stimulation (37 degrees C) produced frequency-dependent arterial contraction, which was reduced at 30 degrees C and potentiated by vasopressin (10 pM, 100 pM and 1 nM). This potentiation was greater at 30 than at 37 degrees C and was abolished at both temperatures by the antagonist of vasopressin V1 receptors d(CH2)5 Tyr(Me)AVP (100 nM). Desmopressin (1 microM) did not affect the response to electrical stimulation. At 37 degrees C, the vasopressin-induced potentiation was abolished by the purinoceptor antagonist PPADS (30 microM), increased by phentolamine (1 microM) or prazosin (1 microM) and not modified by yohimbine (1 microM), whilst at 30 degrees C, the potentiation was reduced by phentolamine, yohimbine or PPADS, and was not modified by prazosin. The Ca2+-channel blockers, verapamil (10 microM) and NiCl2 (1 mM), abolished the potentiating effects of vasopressin at 37 degrees C whilst verapamil reduced and NiCl2 abolished this potentiation at 30 degrees C. The inhibitor of nitric oxide synthesis, L-NOARG (100 microM), or endothelium removal did not modify the potentiation by vasopressin at 37 and 30 degrees C. Vasopressin also increased the arterial contraction to the alpha2-adrenoceptor agonist BHT-920 (10 microM) and to ATP (2 mM) at 30 and 37 degrees C, but it did not modify the contraction to noradrenaline (1 microM) at either temperature. These results suggest that in cutaneous (ear) arteries, vasopressin potentiaties sympathetic vasoconstriction to a greater extent at 30 than at 37 degrees C by activating vasopressin V1 receptors and Ca2+ channels at both temperatures. At 37 degrees C, the potentiation appears related to activation of the purinoceptor component and, at 30 degrees C, to activation of both purinoceptor and alpha2-adrenoceptor components of the sympathetic response. (+info)
A large blood pressure-raising effect of nitric oxide synthase inhibition in humans.
In experimental animals, systemic administration of nitric oxide synthase (NOS) inhibitors causes large increases in blood pressure that are in part sympathetically mediated. The aim of this study was to determine the extent to which these conclusions can be extrapolated to humans. In healthy normotensive humans, we measured blood pressure in response to two NOS inhibitors, NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine methyl ester (L-NAME), the latter of which recently became available for use in humans. The major new findings are 3-fold. First, L-NAME produced robust increases in blood pressure that were more than 2 times larger than those previously reported in humans with L-NMMA and approximated those seen in experimental animals. L-NAME (4 mg/kg) raised mean arterial pressure by 24+/-2 mm Hg (n=27, P<0.001), whereas in subjects who received both inhibitors, a 12-fold higher dose of L-NMMA (50 mg/kg) raised mean arterial pressure by 15+/-2 mm Hg (n=4, P<0.05 vs L-NAME). Second, the L-NAME-induced increases in blood pressure were caused specifically by NOS inhibition because they were reversed by L-arginine (200 mg/kg, n=12) but not D-arginine (200 mg/kg, n=6) and because NG-nitro-D-arginine methyl ester (4 mg/kg, n=5) had no effect on blood pressure. Third, in humans, there is an important sympathetic component to the blood pressure-raising effect of NOS inhibition. alpha-Adrenergic blockade with phentolamine (0.2 mg/kg, n=9) attenuated the L-NAME-induced increase in blood pressure by 40% (P<0.05). From these data, we conclude that pharmacological inhibition of NOS causes large increases in blood pressure that are in part sympathetically mediated in humans as well as experimental animals. (+info)
Secretion of old versus new exportable protein in rat parotid slics. Control by neurotransmitters.
The possibility that old and new secretory granules do not mix and that older exportable protein can be secreted preferentially was tested on parotid gland in vitro. Slices from fasted animals were pulse labeled for 3 min with L-[3H]leucine. Subcellular fractionstion showed that after 1 90-min chase period, the formation of new labeled secretory granules was mostly completed. The ratio of label in secretory granules to label in microsomes increased 250-fold during the period 5--90 min postpulse. After the 90-min chase, a submaximal rate of secretion was initiated by adding a low concentration of isoproterenol to the slices. Preferential secretion of old unlabeled exportable protein was evident from the finding that the percent of total amylase secreted was 3.5-fold greater than the percent of labeled protein secreted. Preferential secretion of old unlabeled exportable amylase was undiminished even when the chase period before addition of isoproterenol was extended to 240 min. Such long chase incubations were still meaningful due to the fact that the spontaneous rat of amylase release and radioactive protein release from the slices was negligibly low. A high isoproterenol concentration added to the slices after a 90-min chase produced the following results. An initial phase of preferential secretion of old unlabeled protein was soon replaced by secretion of a random mixture of new and old exportable protein. Electron micrographs indicated that high rates of secretion involved sequential fusion of secretory granules so that the lumen extended deep into the cell where the new labeled granules were presumably located. At low rates of secretion, the lumen showed no such deep extensions. Experiments were also conducted on slices from glands which had been largely depleted of old granules by prior injection of isoproterenol into the animals. Secretion of labeled protein from such slices stopped with the export of 80% of the labeled protein. This finding indicates that about 20% of the radioactive protein is cellular nonexportable protein and that the slices are capable of exporting the entire amount of secretory protein which was symthesized in vitrol. In addition to the beta-adrenergic receptor which mediates protein secretion, the parotid acinar cell also possesses an alpha-adrenergic and a cholinergic receptor both of which cause K+ release, vacuole formation, and water secretion. Activation of either of the latter two receptors in conjunction with the beta-adrenergic receptor increased randomization of the protein secreted. It is concluded that in the rat parotid acinar cell there is little spontaneous mixing between old granules near the luminal cell membrane and new granules coming up behind from the Golgi complex. The neurotransmitters which induce secretion produce the observed randomization. (+info)
Alpha-adrenoceptor blockade prevents exercise-induced vasoconstriction of stenotic coronary arteries.
OBJECTIVES: The study aimed to evaluate the role of alpha-adrenergic mechanisms during dynamic exercise in both normal and stenotic coronary arteries. BACKGROUND: Paradoxical vasoconstriction of stenotic coronary arteries has been reported during dynamic exercise and may be due to several factors such as alpha-adrenergic drive, a decreased release of nitric oxide, platelet aggregation with release of serotonin, or a passive collapse of the vessel wall. METHODS: Twenty-six patients were studied at rest, during two levels of supine bicycle exercise and after 1.6 mg sublingual nitroglycerin. The alpha-blocker phentolamine was given to 16 patients before exercise, five of whom had also taken a beta-adrenergic-blocker the same morning. Ten patients served as controls. The cross-sectional areas of a normal and a stenotic coronary vessel were determined by biplane quantitative coronary arteriography. RESULTS: In the normal vessel segments, coronary cross-sectional area did not change after phentolamine injection, but increased in all patient groups similarly during exercise. Although coronary vasoconstriction existed in stenotic vessel segments in control patients, phentolamine-treated patients showed exercise-induced vasodilation without difference in patients with and without chronic beta-blockade. CONCLUSIONS: Exercise-induced vasoconstriction of stenotic coronary arteries is prevented by intracoronary administration of phentolamine. There was no difference in coronary vasomotion between patients receiving phentolamine alone and patients receiving phentolamine in addition to a beta-blocker. This finding suggests that exercise-induced vasoconstriction is mediated not only by endothelial dysfunction but also by alpha-adrenergic mechanisms. (+info)
In vivo measurement by [3H]Tamsulosin of alpha1 adrenoceptors in rat tissues in relation to the pharmacokinetics.
The present study was undertaken to simultaneously measure alpha1 adrenoceptors in rat tissues by [3H]tamsulosin in vivo. In vivo specific [3H]tamsulosin binding was observed in the prostate, vas deferens, aorta, submaxillary gland, spleen, heart, lung, and kidney after i.v. injection of the ligand but not in the cerebral cortex and liver. Specific [3H]tamsulosin binding in the kidney, lung, heart, and spleen was greatest at 3 min after i.v. injection and declined rapidly with the disappearance of [3H]tamsulosin from the plasma. On the other hand, [3H]tamsulosin binding in the prostate and aorta peaked at 10 to 60 min after i.v. injection, and a considerable level of specific binding in both tissues persisted up to 240 min. The most sustained binding of [3H]tamsulosin occurred in the submaxillary gland. In vivo specific [3H]tamsulosin binding in rat tissues was effectively inhibited by the coinjection of low doses of unlabeled tamsulosin, prazosin, and terazosin with the radioligand but not by relatively high doses of yohimbine and propranolol. Based on estimated ID50 values, in vivo inhibitory effect of tamsulosin compared with prazosin was 5 to 14 times greater in rat tissues except the spleen, which showed 1.6 times less potent than prazosin. From ratios of ID50 (spleen) to ID50 (submaxillary gland) or ID50 (prostate), tamsulosin was 9 and 19 times, respectively, greater than prazosin in selectivity of alpha1 adrenoceptors in the submaxillary gland and prostate versus the spleen, respectively, suggesting that tamsulosin binds to alpha1A subtype with higher affinity than alpha1B subtype in vivo. The present study suggests that [3H]tamsulosin is a useful radioligand for in vivo measurement of alpha1 adrenoceptors in rat tissues. (+info)