Evidence that depletion of internal calcium stores sensitive to noradrenaline elicits a contractile response dependent on extracellular calcium in rat aorta.

1. Noradrenaline 1 microM induced a contractile response in rat isolated aorta in the presence or in the absence of extracellular Ca2+ with depletion of intracellular Ca2+ stores. Thereafter, during incubation in the presence of Ca2+, an increase in the resting tone was observed. Such a contractile response did not occur after exposure to caffeine or 5-hydroxytryptamine. 2. This increase in tension was inhibited in a concentration-dependent manner by alpha-adrenoceptor antagonists (prazosin, phentolamine and yohimbine), the non-specific relaxing compound, papaverine and by the Ca(2+)-entry blocker, nifedipine. Therefore, this contractile process is related to depletion of Ca2+ stores sensitive to noradrenaline and is linked to Ca2+ entry through voltage-operated Ca2+ channels and alpha-adrenoceptors. 3. Phentolamine and yohimbine did not block the Ca2+ refill pathway; prazosin and nifedipine inhibited the reuptake of Ca2+ by an internal store sensitive only to noradrenaline; papaverine inhibited the refilling of caffeine- and noradrenaline-sensitive Ca(2+)-stores.     

Role of the endothelium in the vasodilator response of rat thoracic aorta to histamine

Despite their potent vasodilating action in vivo, acetylcholine and histamine often show a vasoconstricting action in vitro. As the endothelium has an important role in the vasodilating effect of acetylcholine, we investigated the possible role of the endothelium in the vasodilating effect of histamine in comparison to acetylcholine. Experiments were done on ring segments of rat thoracic aorta mounted for isometric tension measurements. We demonstrated that relaxation by histamine and acetylcholamine of pre-contracted rat aorta segments required the presence of endothelial cells. Acetylcholine acting on muscarinic receptors, and histamine acting on H₁-receptors seemed to initiate the production of mediator(s) from the endothelial cells, which leads to relaxation of the vascular smooth muscle cells. This production appeared to be depressed by ETYA and hydroquinone, and under hypoxic conditions.     

Influence of the vascular endothelium on agonist-induced contractions and relaxations in rat aorta

The influence of the vascular endothelium on agonist-induced contractions and relaxations has been measured using intact segments of rat aorta. Contiguous rubbed segments were used as controls. Angiotensin II, histamine, noradrenaline, U46619 and UK14304 contracted both rubbed and intact tissues. The threshold spasmogenic concentrations of these agonists were lower in rubbed tissues than in intact preparations. The sensitivity and responsiveness of tissues to angiotensin II, histamine, noradrenaline and UK14304 were greater in rubbed than in intact tissues. Acetylcholine and histamine relaxed the established spasms of intact tissues but not those of rubbed preparations, These relaxant effects of acetylcholine were abolished by pre-incubation with haemoglobin. In the presence of prazosin, noradrenaline or UK14304 relaxed established contractions in intact tissues. These effects were antagonized by idazoxan or by pre-incubation with haemoglobin. In intact preparations, idazoxan had no effect on the spasmogenic sensitivity and responsiveness to UK14304. Pre-incubation with haemoglobin augmented the spasmogenic actions of noradrenaline, U46619 or UK14304 in intact tissues, but had no effect on these responses in rubbed preparations. Tissue concentrations of cyclic GMP were greater in intact than in rubbed tissues. A concentration of acetylcholine (10 microM) evoking just maximal mechanical inhibition produced a significant increase in cyclic GMP concentration in intact preparations. However, no detectable changes in cyclic GMP concentration were produced by UK14304 (10 microM) or by acetylcholine (30 nM), concentrations which were equi-effective in inhibiting mechanical activity. In the presence of threshold spasmogenic concentrations of noradrenaline, the contractile effects of angiotensin II were augmented and became comparable to those observed in rubbed preparations. In the presence of greater concentrations of noradrenaline, angiotensin II always produced an additional contraction. It is concluded that the presence of the vascular endothelium limits the spasmogenic action of a variety of agonists. Although spasmogens like noradrenaline and UK14304 can stimulate the release of endothelium-derived relaxing factor (EDRF) via alpha 2-adrenoceptors, the inhibitory effects of EDRF largely result from the spontaneous release of this substance.     

Effects of extracellular potassium on the pressure response to noradrenaline in the perfused hindquarters of the rat

1. The hindquarters of the rat were perfused with oxygenated Tyrode-Ficoll solution at constant flow and the blood pressure in the caudal artery was measured. All experiments were started with the resistance vessels maximally dilated (arterial pressure 20 mmHg). 2. Variations in the extracellular potassium concentration (from 0.5 mmol/1 to 33 mmol/1) had no direct effect on the arterial pressure. 3. Noradrenaline in the range 1.6–17 μmol/1 increased the arterial pressure in a dose-dependent manner at all potassium concentrations. 4. Variations in the potassium concentration within the physiological range did not affect the dose-dependent responses to noradrenaline.     

Effect of L-methionine on contractile response, calcium influx and calcium channel blocking agents in the rat aorta

L-Methionine incubated with aorta strips and S-adenosyl-L-methionine incubated with aorta membranes methylate membrane phospholipids. L-Methionine enhances the contractile response of helical strips of rat aorta to KCl. L-Methionine also enhances the slow component of the contractile response of rat aorta to norepinephrine associated with influx of exogenous calcium. L-Homocysteinethiolactone inhibits methylation of membrane phospholipids and depresses the contractile response to KCl and to norepinephrine. L-Methionine enhances and L-homocysteinethiolactone depresses KCl-stimulated influx of calcium into rat aorta strips. L-Methionine has no effect on calcium efflux. Tested against calcium channel blocking agents, L-methionine reduces the inhibition caused by diltiazem and chlorpromazine but not that caused by TMB 8 or verapamil. It is postulated that methylated intermediates of phospholipid methylation enhance the function of membrane calcium channels.     

Effect of calcium antagonists on the response to noradrenaline in the whole and bisected rat vas deferens

1 The present study was carried out to look at the effect of different calcium antagonists on the response to noradrenaline in the whole and bisected rat vas deferens considering that the response consisted of three components (I) the phasic response (II) the tonic response and (III) the spikes (rhythmic contractions). 2 Nifedipine (3 × 10^??9–1 × 10^??7 m ) inhibited all the components at the same concentration range, verapamil (1 × 10^??7–1 × 10^??5 m ) inhibited the phasic and tonic response but not the rhythmic activity. This latter component, at a certain concentration range and especially in the prostatic portion was markedly potentiated. Diltiazem and flunarizine lay in an intermediate position. 3 Papaverine, a Ca²⁺ antagonist that acts mainly intracellularly, inhibited preferentially the tonic component; ryanodine was practically inactive. 4 Cromakalim inhibited only partially the phasic and tonic components but totally inhibited the rhythmic contractions. 5 These results can be explained by postulating two types of calcium channels opened by α-adrenoceptor stimulation. The first one is verapamil- and nifedipine-sensitive and allows the entry of Ca²⁺ directly available for the contraction and responsible for the phasic and partially responsible for the tonic component. The second channel is merely nifedipine-sensitive and allows the entry of Ca²⁺ trigger which can release Ca²⁺ from intracellular sites: the mobilized Ca²⁺ is able to sustain the tonic component and is the main one responsible for the rhythmic activity. There is the possibility that this second channel is associated with ATP-sensitive K⁺ channels.     

Role of efficacy in the assessment of the actions of alpha-adrenoceptor agonists in rat aorta with endothelium

Concentration-effect curves to phenylephrine are shifted to the right in the presence of endothelium in rat aorta while responses to clonidine are practically abolished. Analysis of the concentration-effect curves showed that the effect of endothelium could possibly be explained by a reduction in relative intrinsic efficacy of the two agonists by between 3.5- and 6-fold. Published observations on the modulatory effects of agonist-induced contractions by endothelium in the rat aorta tend to support this explanation. It is further concluded that this reduction in efficacy could be related to the basal release of an endothelium-derived substance and that changes in tissue contractility in the presence of endothelium cannot necessarily be taken as evidence for a stimulated liberation of endothelium-derived products by agonists.     

Age influences responses of rat isolated aorta and pulmonary artery to the calcium channel agonist, Bay K 8664, and to potassium and calcium

Responses of isolated ring preparations of aorta and pulmonary artery from young (2 months) and aged (19-21 months) rats have been obtained to five contractile agents with differing mechanisms of action. The potencies (negative log EC50 values), but not the absolute maximum contractions (mN/mm2), for potassium, calcium (in K+-depolarized preparations), and the calcium channel activator, Bay K 8644, were all reduced in preparations from aged, compared with young, rats, and this reduction was more pronounced in aorta than in pulmonary artery. In contrast, the age of the rats had no influence on responses (potency or maximum contractions) to caffeine or the ionophore, A23187, indicating that the contractile ability of the preparations per se was retained in the aged rats. Aortic preparations from young rats contracted to Bay K 8644 without prior depolarization, whereas those from aged rats required partial depolarization (with 6 mM KCl) before consistent contractile responses to Bay K 8644 could be obtained. It is concluded that the influence of age on vascular contractions varies (a) between contractile agents, depending on their mechanisms of action and (b) between blood vessel types. To explain the reduced potencies of Bay K 8644, potassium, and calcium in aorta from aged rats, it is postulated that the resting membrane potential may be more negative in the preparations from the aged rats.     

Luteolin induces vasorelaxion in rat thoracic aorta via calcium and potassium channels

The aims of the present study were to investigate the vasoactive effects of luteolin and its mechanisms of action on the rat thoracic aorta. Luteolin (4.5-36 micromol/L) caused a concentration-dependent relaxation of endothelium-intact or endothelium-denuded aortic rings precontracted with phenylephrine (PE, 10(-6) mol/L) or a high level of K+ (6 x 10(-2) mol/L). Luteolin induced a shift of the PE concentration-response curve to the right and downward. L-NAME and propranolol did not influence the vascular effect of luteolin. However, 5-hydroxydecanoate, tetraethylammonium, BaCl2 and 4-aminopyridine significantly attenuated the vasorelaxant effect of luteolin. In Ca2+ -free medium, medium with graded concentrations of Ca2+, or K+ -free solution, luteolin reduced PE-induced contraction. It is concluded that luteolin induces endothelium-independent relaxation in rat thoracic aorta. The mechanism involves the inhibition of sarcolemmal Ca2+ channels, release from intracellular Ca2+ stores and activation of K+ channels.     

Influence of the endothelium on ex vivo tolerance and metabolism of glyceryl trinitrate in rat aorta

The influence of the endothelium on glyceryl trinitrate metabolism and relaxation and the relationship to tolerance induced by transdermal glyceryl trinitrate was explored in rat aorta. Metabolism was assessed in artery segments incubated with glyceryl trinitrate (1.0 microM) for 2 min and the contents of 1,2- and 1,3-glyceryl dinitrate measured by gas chromatography. In non-tolerant arteries mean contents of glyceryl trinitrate, 1,2-glyceryl dinitrate and 1,3-glyceryl dinitrate were 3.2, 0.23 and 0.10 nmol/g, respectively; in tolerant arteries the content of 1,2-glyceryl dinitrate was reduced by approximately 60%. Endothelium removal or nitric oxide synthase (NOS) inhibition did not affect metabolite contents but increased the relaxant response to glyceryl trinitrate in the tolerant artery to an extent that tolerance was significantly attenuated. It is concluded that (i) tolerance is associated with depression of glyceryl trinitrate metabolism by an endothelium-independent mechanism and (ii) the endothelium contributes to tolerance by a mechanism which is independent of metabolism and may be linked with endothelial NOS.     

Effect of felodipine, a new dihydropyridine vasodilator, on contractile responses to potassium, noradrenaline, and calcium in mesenteric resistance vessels of the rat

The effect of felodipine [4-(2,3-dichloro-phenyl)-1,4-dihydro-2,6-dimethyl-3-ethoxycarbonyl-5- methoxycarbonylpyridine on the contractile responses of mesenteric resistance vessels denervated in vitro was investigated. Sustained contractions of this vessel type were totally dependent on extracellular calcium. Felodipine (10-(15)-10-(9)M) had a concentration-dependent inhibitory action on contraction induced by maximal potassium (125 mM) and noradrenaline (10-(5)M) stimulation. Felodipine was more potent and more selective than D600 and nifedipine. Potassium and noradrenaline concentration-response characteristics were insurmountably antagonized by felodipine, the potassium sensitivity of vessels being unaffected while noradrenaline sensitivity was decreased. In calcium-depleted vessels, felodipine in all concentrations tested produced insurmountable antagonism of the potassium-activated calcium concentration-response characteristics, whereas the antagonism in low concentrations (10-(15)-10-(11)M) was surmountable in noradrenaline-activated vessels. Felodipine 10-(9) M blocked the response of potassium-activated vessels almost completely, whereas approximately 50% of the response of noradrenaline-activated vessels persisted. The results of this investigation indicate that the effect of felodipine may be caused primarily by selective inhibiton of responses evoked by membrane depolarization.     

The modulatory role of vascular endothelium in the interaction of agonists and antagonists with alpha-adrenoceptors in the rat aorta.

1. We have examined the effect of endothelium on the antagonistic action of prazosin, doxazosin, yohimbine and phentolamine against phenylephrine, clonidine and noradrenaline. 2. The action of prazosin against phenylephrine was similar to that earlier reported against noradrenaline, acting as a non-competitive antagonist in the presence of endothelium and as a competitive antagonist in the absence of endothelium. Prazosin also acted as a non-competitive antagonist against clonidine in the absence of endothelium. 3. Doxazosin behaved in a similar way to prazosin against noradrenaline, phenylephrine and clonidine acting as a non-competitive antagonist in the presence of endothelium and as competitive antagonist after removal of endothelium. In contrast, yohimbine and phentolamine acted as competitive antagonists both in the presence and in the absence of endothelium. 4. Analysis of the concentration-response curves for noradrenaline, phenylephrine and clonidine in the presence and in the absence of endothelium showed that the affinity for all three agonists was the same but not the efficacy and the receptor reserve, both of which were lower in the presence than in the absence of endothelium. 5. The rank order of agonist potency in the absence of endothelium was noradrenaline greater than phenylephrine greater than clonidine. The rank order of antagonist potency was prazosin greater than or equal to doxazosin greater than phentolamine greater than yohimbine. 6. The results show that vascular endothelium modulates the contractile response to alpha-adrenoceptor agonists and also modifies the action of the antagonists prazosin and doxazosin but not that of yohimbine and phentolamine. This effect of endothelium was related to a change in agonist efficacy and receptor reserve. These results also suggest that the alpha-adrenoceptors of the isolated aorta of the rat are predominantly, if not exclusively of the alpha 1-subtype.     

Characterization of an alpha 1D-adrenoceptor mediating the contractile response of rat aorta to noradrenaline.

1. The affinities of a number of alpha 1-adrenoceptor antagonists were determined by displacement of [3H]-prazosin binding from cloned human alpha 1A-adrenoceptors (previously designated cloned alpha 1c subtype), alpha 1B alpha 1D and rat alpha 1D-adrenoceptors, stably expressed in rat-1 fibroblasts. Functional affinity estimates for these compounds were also determined from noradrenaline-mediated contractions of rat aorta. 2. BMY 7378 displayed high affinity for cloned human alpha 1D-adrenoceptors (pKi = 8.2 +/- 0.10) and was selective over alpha 1A (pKi = 6.2 +/- 0.10) and alpha 1B subtypes (6.7 +/- 0.11). WB 4101, benoxathian and phentolamine displayed high affinity for alpha 1A and alpha 1D adrenoceptors compared to the alpha 1B subtype. Spiperone displayed high affinity and selectivity for alpha 1B adrenoceptors (pKi 8.8 +/- 0.16). 5-Methyl-urapidil was selective for cloned alpha 1A adrenoceptors. 3. Comparative binding affinities (pKi) for compounds at cloned human and rat1D adrenoceptors were almost identical (r = 0.99, slope = 1.08). 4. Prazosin, doxazosin and 5-methyl-urapidil were potent, competitive antagonists of noradrenaline-mediated contractions of rat aorta (pA2 values of 9.8, 8.8 and 7.8 respectively). The selective alpha 1D antagonist BMY 7378 was also a potent antagonist on rat aorta (pKB = 8.3 +/- 0.1) but the interaction of this compound was not consistent with competitive antagonism at a single population of receptors. 5. Functional affinities for compounds determined against noradrenaline-mediated contractions of rat aorta correlated well with binding affinities at cloned alpha 1D-adrenoceptors (r = 0.96), but not with alpha 1A (r = 0.61) or alpha 1B (r = 0.46) subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of mechanical endothelium removal techniques and conservation conditions on rat aorta responses.

Endothelial denudation of aortas induces a lack of relaxation to acetylcholine and enhancement of response to noradrenaline. These modified responses have been studied in rat aorta by using different techniques for mechanical removal of the endothelial layer and maintenance conditions. Rubbing the artery with cotton inside the organ bath preserves the enhancement of response to noradrenaline, and this effect is not present by rubbing the aortas with filter paper outside the organ bath. Both techniques abolished the relaxant response to acetylcholine. The endothelium dependent effects are not observed by conservation of the artery at room temperature during 1 h. The relaxant response to acetylcholine after this period is approximately 29% of the control. These effects were preserved when the conservation temperature was maintained at 4 degrees C and/or the solution was being bubbled with oxygen.     

Influence of calcium on noradrenaline release evoked by 5-hydroxytryptamine,tyramine and potassium from goat pial arteries

The release of tritium (³ H) evoked by tyramine, potassium (K⁺) and 5-hydroxytryptamine (5-HT) from goat pial arteries preloaded with [³ H]noradrenaline (³ H-NA) was studied. In normal Krebs-bicarbonate solution (KBS) all these agents caused a transient increase in radioactivity release over the basal spontaneous outflow. The pattern of release evoked by 5-HT was similar to that induced by tyramine with a slow onset and decline, but different from that induced by K⁺ which produced a rapid peak of ³ H release followed by a quick fall. The removal of Ca²⁺ from the medium did not modify the efflux of radioactivity caused by tyramine, but the ³ H efflux produced by K⁺ was markedly reduced. Nevertheless, in this Ca²⁺ -free medium the ³ H release evoked by 5-HT was partially, but significantly, decreased. These results indicate that K⁺ evokes NA release by a Ca²⁺ -dependent process, probably of an exocytotic nature, while tyramine mediates NA release by means of a Ca²⁺ -independent mechanism. However, 5-HT possesses a Ca²⁺ -dependent and a tyramine-like component.     

Influence of aging on the contractile response to endothelin of rat thoracic aorta.

Age-related changes in the contractile response to endothelin-1 and ACh were assessed in thoracic aortas isolated from 2-, 6- and 24-month-old male Fischer 344 rats. In aortic strips with an intact endothelium, the maximal contractile response to endothelin-1 decreased with development to maturity. Removal of the endothelium did not affect the contractile response to endothelin-1. Endothelin-1 did not elicit a relaxant response in phenylephrine-precontracted strips. The ACh-induced relaxation decreased in senescent rats. These results indicate that the contractile response of aortic smooth muscle to endothelin-1 decreases with age, and that the endothelial vasorelaxant factors do not contribute to this age-induced modulation.     

Influence of papaverine,D 600, and nifedipine on the effects of noradrenaline and calcium on the isolated aorta and mesenteric artery of the rabbit

Summary The effects of papaverine and of the organic calcium-antagonistic agents D 600 and nifedipine on the contraction induced by noradrenaline and calcium were studied on the isolated aorta and mesenteric artery. The affinities of both agonists, given as pD₂-values, were significantly higher on the aorta than on the mesenteric artery. Under our experimental conditions D 600, nifedipine and papaverine were found to act as antagonists against calcium and noradrenaline in a non-competitive fashion. In either vessel, the calcium-antagonistic activity of D 600 and nifedipine was about 1000-fold greater than that of papaverine, whereas their antagonistic activity against noradrenaline was about 1000-times weaker, i.e. D 600 as well as nifedipine were about equipotent with papaverine. The comparison between the calcium-and the noradrenaline-antagonistic activity offers the possibility to evaluate the specificity of calcium antagonistic agents.This work was supported by the Deutsche Forschungsgemeinschaft     

Influence of calcium on noradrenaline release evoked by 5-hydroxytrypamine and potassium from goat pial arteries

The release of tritium (3H) evoked by tyramine, potassium (K+) and 5-hydroxytryptamine (5-HT) from goat pial arteries preloaded with [3H]noradrenaline (3H-NA) was studied. In normal Krebs-bicarbonate solution (KBS) all these agents caused a transient increase in radioactivity release over the basal spontaneous outflow. The pattern of release evoked by 5-HT was similar to that induced by tyramine with a slow onset and decline, but different from that induced by K+ which produced a rapid peak of 3H release followed by a quick fall. The removal of Ca2+ from the medium did not modify the efflux of radioactivity caused by tyramine, but the 3H efflux produced by K+ was markedly reduced. Nevertheless, in this Ca2+-free medium the 3H release evoked by 5-HT was partially, but significantly, decreased. These results indicate that K+ evokes NA release by a Ca/+-dependent process, probably of an exocytotic nature, while tyramine mediates NA release by means of a Ca2+-independent mechanism. However, 5-HT possesses a Ca2+-dependent and a tyramine-like component.