Norbormide: a calcium entry blocker with selective vasoconstrictor activity in rat peripheral arteries

Norbormide is a unique vasoactive substance endowed with species- and tissue-specific, endothelium independent, vasoconstrictor activity that is restricted to the peripheral arteries of rat. In rat aorta and in all tested arteries of other species norbormide exhibits vasorelaxant property presumably due to the blockade of calcium channels. A calcium entry blocker effect of norbormide has also been described in isolated, perfused guinea pig hearts. In these preparations norbormide produced coronary vasodilator, as well as negative inotropic and dromotropic effects. In single ventricular myocytes of guinea pigs norbormide reduces L-type calcium current. The mechanism underlying the selective vasoconstrictor effect of norbormide is unknown. In rat caudal artery, a vessel contracted by norbormide, the drug activates phospholipase C (PLC) signal cascade which is the biochemical pathway involved in the contractile effect triggered by most receptor-activating vasoactive agents. Therefore, norbormide-induced contraction of rat peripheral vessels is likely to be due to the activation of a PLC-coupled receptor abundantly or selectively expressed in vascular smooth muscle cells. The identification of this putative receptor could facilitate the development of tissue-selective pharmacological agents.     

Ca(2+) entry blocking and contractility promoting actions of norbormide in single rat caudal artery myocytes

1 Aim of the present study was to investigate the effects of norbormide, a selective vasoconstrictor agent of the rat peripheral vessels, on the whole-cell voltage-dependent L-type Ca(2+) current (I(Ca(L))) of freshly isolated smooth muscle cells from the rat caudal artery, using either the conventional or the amphotericin B-perforated whole-cell patch-clamp method. 2 Norbormide decreased L-type Ca(2+) current in a concentration- and voltage-dependent manner, without modifying the threshold and the maximum of the current-voltage relationship. Norbormide-induced I(Ca(L)) inhibition was reversible upon wash-out. 3 Norbormide both shifted the voltage dependence of the steady-state inactivation curve to more negative potentials by about 16 mV, without affecting the activation curve, and decreased the slope of inactivation. Norbormide, however, did not modify both the activation and the inactivation kinetics of the I(Ca(L)). 4 Norbormide decreased I(Ca(L)) progressively during repetitive step depolarizations, with inhibition depending on the stimulation frequency (use-dependent block) as well as on the holding potential. 5 Addition of 50 micro M norbormide caused the contraction of all freshly isolated cells and also of those impaled with the perforated method, but not of those impaled with the conventional method (i.e. dialysed). 6 In conclusion, these results prove norbormide to be a vascular L-type Ca(2+) channel inhibitor, which preferentially acts on the inactivated and/or open state of the channel. In rat caudal artery smooth muscle, however, this mechanism does not result in a vasodilating effect since it is overwhelmed by the mechanism underlying norbormide-induced vasoconstriction.     

Role of gap junctions in the responses to EDHF in rat and guinea-pig small arteries

1. In guinea-pig internal carotid arteries with an intact endothelium, acetylcholine (10 microM) and levcromakalim (10 microM) each hyperpolarized the smooth muscle whereas a 5 mM elevation of extracellular K(+) was without effect. 2. Incubation of the carotid artery with the gap junction inhibitors carbenoxolone (100 microM) or gap 27 (500 microM) essentially abolished the hyperpolarization to acetylcholine but it was without effect on that to levcromakalim. Carbenoxolone had no effect on the acetylcholine-induced endothelial cell hyperpolarization but inhibited the smooth muscle hyperpolarization induced by the endothelial cell K(+) channel opener, 1-ethyl-2-benzimidazolinone (600 microM). 3. In rat hepatic and mesenteric arteries with endothelium, carbenoxolone (100 or 500 microM) depolarized the smooth muscle but did not modify hyperpolarizations induced by KCl or levcromakalim. In the mesenteric (but not the hepatic) artery, the acetylcholine-induced hyperpolarization was inhibited by carbenoxolone. 4. Phenylephrine (1 microM) depolarized the smooth muscle cells of intact hepatic and mesenteric arteries, an effect enhanced by carbenoxolone. Gap 27 did not have a depolarizing action. In the presence of phenylephrine, acetylcholine-induced hyperpolarization of both hepatic and mesenteric artery myocytes was partially inhibited by each of the gap junction inhibitors. 5. Collectively, the data suggest that gap junctions play some role in the EDHF (endothelium-derived hyperpolarizing factor) response in rat hepatic and mesenteric arteries. However, in the guinea-pig internal carotid artery, electrotonic propagation of endothelial cell hyperpolarizations via gap junctions may be the sole mechanism underlying the response previously attributed to EDHF.     

Effects of portal hypertension on responsiveness of rat mesenteric artery and aorta.

1. We have examined the effects of pre-hepatic portal hypertension on the responsiveness of rat small mesenteric arteries and aorta. Rats were made portal hypertensive by creating a calibrated portal vein stenosis, or sham-operated. 2. In rat mesenteric arteries, there was no significant difference between portal hypertensive and sham-operated animals in the contractile potency of noradrenaline (NA), but the maximum contractile responses to NA, U46619 and KCl were significantly increased in vessels from portal hypertensive animals. This altered maximum contractile response was not due to alterations in smooth muscle mass. 3. In rat mesenteric arteries, there were no significant differences between portal hypertensive and sham-operated animals in endothelium-dependent relaxations to acetylcholine (ACh). The difference between portal hypertensive and sham-operated rats in the maximum response to U46619 was maintained following a combination of methylene blue (1 microM) and NG-monomethyl-L-arginine (100 microM), suggesting that any differences in endothelial function do not explain differences in the response to vasoconstrictors. 4. In rat aorta, there were no significant differences between portal hypertensive and sham-operated animals in the contractile response to NA or KCl or in the endothelium-dependent relaxations to ACh. 5. In pithed rats, there was no difference between portal hypertensive and sham-operated animals in the pressor potency of NA. 6. It is concluded that portal hypertension produces an increase in the contractile response to the vasoconstrictors NA, U46619 and KCl in rat mesenteric arteries but not in the aorta. This suggests that the diminished responsiveness to vasoconstrictors reported in portal hypertensive rats in vivo is not due to a diminished responsiveness at the level of the vascular smooth muscle.     

Vascular actions of 17beta-oestradiol in rat aorta and mesenteric artery

1. It has been proposed that the cardiovascular protective actions of 17beta-oestradiol may involve calcium antagonistic actions. We have examined the effects of 17beta-oestradiol on contractions to noradrenaline and KCl in male rat small mesenteric artery and aorta. 2. In rat mesenteric artery, 17beta-oestradiol (10 microM) significantly reduced the maximum contraction to noradrenaline (67.7 +/- 5.8% of control) and KCl (38.8 +/- 3.1% of control) without affecting potency. 3. In rat aorta, 17beta-oestradiol (10 microM) also significantly reduced contractions to noradrenaline (77.5 +/- 4.8% of control), and the effects were mimicked by droloxifene (10 microM). The effect of oestrogen was not prevented by the protein synthesis inhibitor cycloheximide (10 microM). In experiments carried out in calcium-free solution in which calcium stores were depleted, 17beta-oestradiol (10 microM) significantly reduced the contraction to calcium restoration in rat aorta. 4. In aorta from female rats, 17beta-oestradiol (10 microM) significantly reduced contractions to noradrenaline (73.6 +/- 10.8% of control), but this effect of oestrogen was not prevented by cycloheximide (10 microM). 5. In summary, 17beta-oestradiol diminishes the maximum contractile response to noradrenaline in both rat small mesenteric artery and aorta, an effect which at least in the aorta is mimicked by the oestrogen receptor antagonist/partial agonist droloxifene, and may be due to restriction of calcium entry by a nongenomic action.     

Enhanced contractile responses of arteries from streptozotocin diabetic rats to sodium fluoride.

1. Previous studies from this laboratory have demonstrated that alpha 1-adrenoceptor-mediated increases in tension and phosphoinositide metabolism are enhanced in the aorta and mesenteric arteries from diabetic rats. The purpose of the present investigation was to determine whether contractile responses to sodium fluoride (NaF), which directly stimulates GTP-binding proteins (G-proteins), are also enhanced in diabetic arteries. 2. NaF (1-20 mM) in the presence of 10 microM aluminium chloride produced slowly developing, concentration-dependent contractions in mesenteric arteries from three month streptozotocin-diabetic (60 mg kg-1, i.v.) male Wistar rats and age-matched control rats. The maximum contractile response but not the sensitivity to NaF was significantly greater in mesenteric arteries from diabetic than from control rats, as was the response to noradrenaline (NA). Maximum contractile responses of aorta and caudal artery from diabetic rats to NaF were also significantly enhanced. 3. Removal of the endothelium and denervation with 6-hydroxydopamine did not significantly alter the maximum contractile response of mesenteric arteries from either control or diabetic rats to NaF. Similarly, NaF had no effect on cyclic AMP levels in aorta, and no difference in cyclic AMP levels, either basally or in the presence of NaF, was detected between control and diabetic rat aorta. 4. Contractile responses of mesenteric arteries from both control and diabetic rats to NaF were diminished in calcium-free Krebs solution, but the NaF response remained significantly elevated in mesenteric arteries from diabetic rats compared to control. 5. Ryanodine (30 microM) which depletes intracellular calcium stores, nifedipine (3 microM) which blocks dihydropyridine-sensitive calcium channels and calphostin C (0.5 microM) which selectively inhibits protein kinase C, all significantly inhibited maximum contractile responses of mesenteric arteries from control and diabetic rats to NaF. There were no significant differences between control and diabetic arteries in the relative magnitude of the inhibition produce by the three antagonist. 6. These data suggest that there may be increased activation of the same signalling processes that mediate NA-stimulated vasoconstriction, perhaps contraction-associated G-proteins or the effectors coupled to these G-proteins, in response to NaF in mesenteric arteries from diabetic rats. This may also be responsible for the enhanced contractile responses of these arteries to alpha 1-adrenoceptor stimulation.     

Human urotensin-II is an endothelium-dependent vasodilator in rat small arteries

The possible role of the endothelium in modulating responses to human urotensin-II (U-II) was investigated using isolated segments of rat thoracic aorta, small mesenteric artery, left anterior descending coronary artery and basilar artery. Human U-II was a potent vasoconstrictor of endothelium-intact isolated rat thoracic aorta (EC(50)=3.5+/-1.1 nM, R(max)=103+/-10% of control contraction induced by 60 mM KCl and 1 microM noradrenaline). However the contractile response was not significantly altered by removal of the endothelium or inhibition of nitric oxide synthesis with L-NAME (100 microM). Human U-II did not cause relaxation of noradrenaline-precontracted, endothelium-intact rat aortae. Human U-II contracted endothelium-intact rat isolated left anterior descending coronary arteries (EC(50)=1.3+/-0.8 nM, R(max)=20.1+/-4.9% of control contraction induced by 10 microM 5-HT). The contractile response was significantly enhanced by removal of the endothelium (R(max)=55.4+/-16.1%). Moreover, human U-II caused concentration-dependent relaxation of 5-HT-precontracted arteries, which was abolished by L-NAME or removal of the endothelium. No contractile effects of human U-II were found in rat small mesenteric arteries. However the peptide caused potent, concentration- and endothelium-dependent relaxations of methoxamine-precontracted vessels. The relaxant responses were potentiated by L-NAME (300 microM) but abolished in the additional presence of 25 mM KCl (which inhibits the actions of endothelium-derived hyperpolarizing factor). The present study is the first to show that human U-II is a potent endothelium-dependent vasodilator in some rat resistance vessels, and acts through release of EDHF as well as nitric oxide. Our findings have also highlighted clear anatomical differences in the responses of different vascular beds to human U-II which are likely to be important in determining the overall cardiovascular activity of this peptide.     

The resistance of some rat cerebral arteries to the vasorelaxant effect of cromakalim and other K+ channel openers.

1. Cromakalim (0.01-30 microM) and sodium nitroprusside (SNP, 0.01-100 microM) were tested for their ability to relax a number of pre-contracted small arteries (approximate diameter 200-700 microM at 100 mmHg) from the rat, rabbit and guinea-pig. 2. In the rat, SNP (0.01-100 microM) caused near maximal relaxation in all vessels studied including the middle cerebral, anterior cerebellar, basilar, mesenteric and renal arteries. Cromakalim (0.01-30 microM) relaxed pre-contracted mesenteric and renal arteries but was only a weak relaxant of all the rat cerebral arteries with the exception of the basilar artery. Similar experiments using mesenteric and cerebral vessels from the rabbit and guinea-pig showed cromakalim could relax pre-contracted vessels in a concentration-dependent manner. 3. Two other K+ channel openers, nicorandil and pinacidil, were also tested for their ability to relax rat cerebral arteries. Nicorandil (0.01-100 microM) was ineffective in the rat anterior cerebellar artery at concentrations up to 100 microM. Pinacidil (0.01-100 microM) caused significant vasorelaxation, although high concentrations were required (greater than 10 microM) and the response was insensitive to the effects of glibenclamide (3 microM). 4. Electrophysiological experiments with the rat anterior cerebellar artery showed that cromakalim (up to 30 microM) failed to influence the resting membrane potential of impaled single smooth muscle cells. 5. The results showed that some rat small cerebral arteries were resistant to the effects of K+ channel openers including cromakalim, pinacidil and nicorandil. This is peculiar to this vascular tree since the same vessels from other species do not exhibit the same behaviour.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity in the distribution of vascular gap junctions and connexins: implications for function

1. Gap junctions, which are comprised of members of a family of membrane proteins called connexins (Cx), permit the transfer of electrical and chemical information between adjacent cells in a wide variety of tissues. The aim of the present study was to compare the expression of Cx37, 40 and 43 in the smooth muscle and endothelium of a large elastic artery and two smaller muscular arteries of the rat. Serial section electron microscopy was also used to determine the presence of pentalaminar gap junctions in the smooth muscle and the incidence of myoendothelial gap junctions between the smooth muscle and endothelial cells in muscular arteries of different size. 2. Using immunohistochemistry, Cx37, 40 and 43 were found in the endothelium of the aorta, caudal and basilar arteries, with Cx43 being the least abundant. Connexin 43 was readily observed throughout the muscle layers of the aorta, but was not detected in the media of the caudal or basilar arteries. Connexin 40 was not detected in the media of any of the arteries, while very fine punctate staining was observed with Cx37 antibodies in the media of the caudal and basilar arteries, but not in the aorta. 3. Real-time polymerase chain reaction showed that the expression of mRNA for Cx43 was 15-fold greater in the aorta than in the caudal artery of the rat. 4. At the ultrastructural level, small pentalaminar gap junctions (< 100 nm) were found between the fine processes of adjacent smooth muscle cells and also between the smooth muscle and endothelial cells. The incidence of myoendothelial gap junctions in the mesenteric vascular bed and in the caudal artery increased as vessel size decreased. 5. In summary, heterogeneity exists within the vascular system with regard to the distribution of gap junctions and their constituent Cx. Such variation will have important consequences for the coordination and propagation of vascular responses. In muscular arteries, in comparison with elastic arteries, Cx37 may be more important than Cx43 for cell coupling within the smooth muscle layers. The correlation between the incidence of myoendothelial gap junctions and the role of endothelium-derived hyperpolarizing factor, relative to nitric oxide, in vasodilatory responses suggests that myoendothelial gap junctions play an important physiological role in the regulation of vascular tone.     

Mitochondrial uncoupler triclosan induces vasorelaxation of rat arteries

Our previous studies found that mitochondrial uncouplers induced vasodilation. Triclosan, the broad spectrum antibacterial agent, is the active ingredient in soaps and toothpastes. It was reported that triclosan induced mitochondrial uncoupling, so we aim to investigate the effects of triclosan on vascular function of rat mesenteric arteries and aorta. The isometric tension of rat mesenteric artery and thoracic aorta was recorded by multi-wire myograph system. The cytosolic [Ca²⁺]_i, mitochondrial reactive oxygen species (mitoROS), and mitochondrial membrane potential of smooth muscle cells (A10 cells) were measured using laser scanning confocal microscopy. Triclosan treatment relaxed phenylephrine (PE)- and high K⁺ (KPSS)-induced constriction, and pre-treatment with triclosan inhibited PE- and KPSS-induced constriction of rat mesenteric arteries. In rat thoracic aorta, triclosan also relaxed PE- and KPSS-induced constriction. Triclosan induces vasorelaxation without involving K_ATP channel activation in smooth muscle cells of arteries. Triclosan treatment increased cytosolic [Ca²⁺]_i, mitochondrial ROS production and depolarized mitochondrial membrane potential in A10 cells. In conclusion, triclosan induces mitochondrial uncoupling in vascular smooth muscle cells and relaxes the constricted rat mesenteric arteries and aorta of rats. The present results suggest that triclosan would indicate vasodilation effect if absorbed excessively in vivo.     

The pharmacological properties of the peptide, endothelin.

1. The effect of endothelin (ET-1) has been studied on isolated vascular and non-vascular preparations, using both functional and competition radioligand binding techniques. The effects of endothelin on blood pressure were studied in both anaesthetised, chemically denervated normotensive and spontaneously hypertensive rats (SHR). 2. Endothelin elicited contractile responses in the rat thoracic aorta, perfused mesenteric bed, rabbit mesenteric artery and portal vein. The maximal responses in the rat aorta were enhanced by removal of the endothelium, and were reduced in the presence of either a cyclo-oxygenase inhibitor (indomethacin) or a thromboxane receptor antagonist (SQ 29,548). In terms of potency, the most sensitive preparation was the rat endothelium-denuded aorta and rat perfused mesenteric bed (-log EC50 values = 8.2 +/- 0.07 and 8.2 +/- 0.12, mean +/- s.e.mean, n = 4, respectively). In the perfused mesenteric bed of the rat the maximum response to endothelin (219 +/- 12 mmHg, n = 4) was greater than that to either phenylephrine (maximal response = 67 +/- 9 mmHg; n = 4) or KCl (maximal response = 110 +/- 6 mmHg, n = 4). 3. Endothelin elicited contractile responses of the guinea-pig isolated ileum, oesophageal muscularis mucosae and uterus. Responses were also observed in the rat fundic strip and paced left atria. The guinea-pig urinary bladder, trachea, rat vas deferens and anococcygeus exhibited little or no response to endothelin at the concentrations studied (1 x 10(-12)-3.2 x 10(-8) M). Of the above preparations, the ileum and oesophageal muscularis mucosae were the most sensitive to endothelin (-log EC50 = 8.5 +/- 0.11 and 8.4 +/- 0.06, n = 6, respectively), exhibiting potencies similar to those observed in the endothelium-denuded aorta of the rat. 4. In competition-radioligand binding studies, endothelin did not displace either [3H]-PN 210-100 or [125I]-(-)-omega-conotoxin GVIA from binding sites in membranes from rat cerebral cortex and, skeletal muscle or from guinea-pig cerebral cortex and hippocampus, respectively. This indicates a lack of direct interaction of endothelin at the dihydropyridine binding site and the N-type calcium channel, respectively. However, in functional studies, contractile responses to endothelin (1 x 10(-8) M) in the endothelium-denuded aorta of the rat were potently reversed by nifedipine, verapamil, and prenylamine (-log IC50 values = 8.0 +/- 0.13, 7.2 +/- 0.09 and 6.6 +/- 0.08, n = 4-8, respectively).(ABSTRACT TRUNCATED AT 400 WORDS)     

Vascular actions of 17β-oestradiol in rat aorta and mesenteric artery

1 It has been proposed that the cardiovascular protective actions of 17β-oestradiol may involve calcium antagonistic actions. We have examined the effects of 17β-oestradiol on contractions to noradrenaline and KCl in male rat small mesenteric artery and aorta. 2 In rat mesenteric artery, 17β-oestradiol (10 μm ) significantly reduced the maximum contraction to noradrenaline (67.7 ± 5.8% of control) and KCl (38.8 ± 3.1% of control) without affecting potency. 3 In rat aorta, 17β-oestradiol (10 μm ) also significantly reduced contractions to noradrenaline (77.5 ± 4.8% of control), and the effects were mimicked by droloxifene (10 μm ). The effect of oestrogen was not prevented by the protein synthesis inhibitor cycloheximide (10 μm ). In experiments carried out in calcium-free solution in which calcium stores were depleted, 17β-oestradiol (10 μm ) significantly reduced the contraction to calcium restoration in rat aorta. 4 In aorta from female rats, 17β-oestradiol (10 μm ) significantly reduced contractions to noradrenaline (73.6 ± 10.8% of control), but this effect of oestrogen was not prevented by cycloheximide (10 μm ). 5 In summary, 17β-oestradiol diminishes the maximum contractile response to noradrenaline in both rat small mesenteric artery and aorta, an effect which at least in the aorta is mimicked by the oestrogen receptor antagonist/partial agonist droloxifene, and may be due to restriction of calcium entry by a nongenomic action.     

Relaxant and Ca2+ channel blocking properties of norbormide on rat non-vascular smooth muscles

We have investigated the effects of the rat-specific vasoconstrictor agent norbormide on the mechanical and electrophysiological properties of rat non-vascular smooth muscles. Norbormide (50 μM) did not affect the resting tone of urinary bladder, tracheal, and duodenal rings. In all tissues, KCl-induced concentration–response curves were shifted downward by norbormide (5 and 50 μM). In urinary bladder and tracheal rings, norbormide inhibited contractile responses to carbachol only at the higher concentration (50 μM). In single gastric fundus myocytes, 50 μM norbormide inhibited L-type Ca²⁺ current (I_Ca(L)) by about 60%, neither affecting both activation and inactivation rates of the current nor the current–voltage curve along the voltage axis. Our results indicate that rat non-vascular smooth muscles are relaxed by norbormide with a mechanism likely involving a reduction of Ca²⁺ entry through L-type Ca²⁺ channels.     

Activation of endothelial cell IK(Ca) with 1-ethyl-2-benzimidazolinone evokes smooth muscle hyperpolarization in rat isolated mesenteric artery.

1. In rat small mesenteric arteries contracted with phenylephrine, 1-ethyl-2-benzimidazolinone (1-EBIO; 3-300 microM) evoked concentration-dependent relaxation that, above 100 microM, was associated with smooth muscle hyperpolarization. 2. 1-EBIO-evoked hyperpolarization (maximum 22.1+/-3.6 mV with 300 microM, n=4) was endothelium-dependent and inhibited by charybdotoxin (ChTX 100 nM; n=4) but not iberiotoxin (IbTX 100 nM; n=4). 3. In endothelium-intact arteries, smooth muscle relaxation to 1-EBIO was not altered by either of the potassium channel blockers ChTX (100 nM; n=7), or IbTX (100 nM; n=4), or raised extracellular K(+) (25 mM). Removal of the endothelium shifted the relaxation curve to the right but did not reduce the maximum relaxation. 4. In freshly isolated mesenteric endothelial cells, 1-EBIO (600 microM) evoked a ChTX-sensitive outward K-current. In contrast, 1-EBIO had no effect on smooth muscle cell conductance whereas NS 1619 (33 microM) stimulated an outward current while having no effect on the endothelial cells. 5. These data show that with concentrations greater than 100 microM, 1-EBIO selectively activates outward current in endothelial cells, which presumably underlies the smooth muscle hyperpolarization and a component of the relaxation. Sensitivity to block with charybdotoxin but not iberiotoxin indicates this current is due to activation of IK(Ca). However, 1-EBIO can also relax the smooth muscle by an undefined mechanism, independent of any change in membrane potential.     

Resveratrol decreases calcium sensitivity of vascular smooth muscle and enhances cytosolic calcium increase in endothelium

Resveratrol causes endothelium dependent and independent relaxation of vascular smooth muscle. This study investigated the mechanisms behind the effect of resveratrol on vascular tone. Resveratrol (0.1 mM) inhibited KCl-stimulated contractions in endothelium-denuded rat aorta and this inhibition was not reversed by tetraethylammonium (TEA) (5 mM), glyburide (3 microM), ouabain (0.1 mM), thapsigargin (1 microM), or indomethacin (10 microM). KCl (90 mM) increased the intracellular free calcium concentration ([Ca2+]i) in the isolated smooth muscle cells from the rat aorta and resveratrol (0.1 mM) did not inhibit the KCl-stimulated [Ca2+]i increase. The CaCl2 (0.1-100 microM) stimulated contractions were inhibited by resveratrol (0.1 mM) in the Triton X-100 skinned smooth muscle of the aorta. In heart valve endothelium, resveratrol (0.1 mM) augmented the acetylcholine (10 microM) stimulated [Ca2+]i increase. Resveratrol-induced augmentation of the acetylcholine-stimulated [Ca2+]i elevation was reversed by glyburide (3 microM), but not by TEA (5 mM). The present study indicated that resveratrol affected vascular smooth muscle and endothelium in different ways. Resveratrol decreased the Ca2+ sensitivity but did not affect the KCl-stimulated [Ca2+]i increase in the vascular smooth muscle. In the endothelial cells, resveratrol enhanced the agonist-stimulated [Ca2+]i increase that might trigger nitric oxide synthesis from endothelial cells.     

Effects of the calcium channel facilitator, CGP 28,392, on different modes of contraction in smooth muscle of rabbit and rat aortae and guinea-pig taenia caeci.

Effects of a Ca2+ channel facilitator, CGP 28,392, on smooth muscle contractions were examined in order to delineate characteristics of Ca2+ channels in rabbit and rat aortae and guinea-pig taenia caeci. Application of increasing concentrations of KCl induced contractile responses in these smooth muscles and CGP 28,392 shifted the concentration-response curve for KCl to the left. The maximum response was also increased in rat aorta and guinea-pig taenia. CGP 28,392 also shifted the concentration-response curves for noradrenaline in rat aorta and for histamine in taenia to the left and increased the maximum response in rat aorta. However, the corresponding curve for noradrenaline in rabbit aorta was not affected by CGP 28,392. The sustained contractions induced by KCl were inhibited by cumulative application of verapamil in these smooth muscles. Pretreatment of the muscle with CGP 28,392 decreased the inhibitory effect of verapamil. The noradrenaline-induced contraction in rat aorta and the histamine-induced contraction in taenia were also inhibited by verapamil, and CGP 28,392 antagonized the effect of verapamil. The noradrenaline-induced contraction in rabbit aorta was only slightly inhibited by verapamil, and CGP 28,392 did not modify the effect of verapamil. In these smooth muscles, cumulative application of Ca2+ to the Ca2+-depleted, KCl-treated muscle induced contraction, and the concentration-response curve for Ca2+ was shifted to the left by CGP 28,392 and to the right by verapamil. The concentration-response curves for Ca2+ in Ca2+-depleted, noradrenaline-treated rabbit and rat aortae and in Ca2+-depleted, histamine-treated taenia were also shifted to the left by CGP 28,392 and to the right by verapamil.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pinacidil inhibits neuromuscular transmission indirectly in the guinea-pig and rabbit mesenteric arteries.

1. Effects of pinacidil were investigated on neuromuscular transmission in smooth muscle tissues of the rabbit and guinea-pig mesenteric arteries by both electrophysiological procedures and a bioassay of noradrenaline (NA) outflows. 2. Pinacidil (over 1 microM) hyperpolarized smooth muscle cell membranes in both tissues, in a concentration dependent manner. Pinacidil hyperpolarized and increased the ionic conductance of smooth muscle membrane more markedly in the rabbit mesenteric artery than in the guinea-pig. The hyperpolarization induced by pinacidil occurred in the presence or absence of endothelial cells and was blocked by glibenclamide. 3. Perivascular adrenergic nerve stimulation produced excitatory junction potentials (e.j.ps) and repetitive stimulation produced a facilitation of e.j.ps in both tissues. Pinacidil (over 1 microM) reduced the amplitude and the decay time of e.j.ps to a consistently greater extent in the rabbit mesenteric artery than in the guinea-pig. However, the facilitation process of e.j.ps was not modified following application of pinacidil (1 microM). The pinacidil-induced inhibition of e.j.ps was prevented by pretreatment with glibenclamide. 4. Pinacidil (30 microM) marginally increased the overflows of NA and its metabolite, 3,4-dihydroxyphenylglycol (DOPEG) released following repetitive perivascular nerve stimulations. 5. Pinacidil (10 microM) partly inhibited the voltage-dependent Ca channel, as estimated from the recovery process following removal of pinacidil, of action potentials evoked on e.j.ps. 6. It is concluded that pinacidil increases ionic conductance and hyperpolarizes smooth muscle cell membranes of the guinea-pig and rabbit mesenteric arteries and as a consequence, inhibits the neuromuscular transmission process occurring on adrenergic nerve stimulation with no reduction in the amount of released transmitter.     

Lack of a causal relationship between the vasodilator effect of papaverine and cyclic AMP production in the dog basilar artery.

The effects of papaverine and isoprenaline on smooth muscle cells of the dog basilar artery were investigated using radioimmunoassay, electrophysiological and isometric tension recording methods. For comparative purposes, the actions of these drugs on the guinea-pig basilar artery were also examined. Papaverine and isoprenaline (1 microM and 10 microM) increased the amount of cyclic AMP in both dog and guinea-pig basilar arteries. Papaverine (up to 100 microM) and isoprenaline (up to 1 microM) had no effect on the membrane potential and membrane resistance measured by recording the amplitudes of the electrotonic potentials in smooth muscle cells of the dog and guinea-pig basilar arteries. The action potential evoked by outward current pulses after pretreatment with tetraethylammonium chloride (5-10 mM) was inhibited by papaverine (greater than 1 microM) but not by isoprenaline (up to 10 microM) in smooth muscle cells of the dog and guinea-pig basilar arteries. In the dog basilar artery, papaverine (greater than 1 microM) consistently inhibited the contractions evoked by excess concentrations of [K]o (greater than 20.2 mM) or 5-hydroxytryptamine (10 nM-10 microM), dose-dependently. Isoprenaline (1 microM) had only slight effects on the contraction evoked by low concentrations of 5-hydroxytryptamine (10 nM). In the Ca2+-free solution containing EGTA (2 mM), the contraction evoked by 5-hydroxytryptamine (10 microM) or caffeine (10 mM) was dose-dependently inhibited by papaverine (greater than 1 microM). However, isoprenaline (1 microM) had no effect on these contractions. These results indicate that the vasodilator actions of papaverine on the dog basilar artery are mainly due to inhibition of the voltage-dependent influx of Ca2+ and also to inhibition of the receptor-activated release of Ca2+ stored in the cell. Since isoprenaline increased the cyclic AMP to the same extent as papaverine but had no effect on the electrical and mechanical responses, the inhibitory actions of papaverine on this tissue may not be causally related to the increased levels of cyclic AMP induced by inhibition of phosphodiesterase.     

Comparative cardiovascular actions of clentiazem, diltiazem, verapamil, nifedipine, and nimodipine in isolated rabbit tissues.

Clentiazem is an 8-chloro-substituted derivative of diltiazem. We compared the relative potency of clentiazem with that of diltiazem, verapamil, nifedipine, and nimodipine in isolated rabbit right atria and vascular smooth muscle removed from various arterial beds. In experiments with isolated right atria, calcium channel blockers were added cumulatively to study relative cardiodepressive potencies (as compared with vascular effects) with the following results: verapamil greater than or equal to diltiazem greater than clentiazem greater than or equal to nimodipine much greater than nifedipine. The aorta, pulmonary, renal, mesenteric, coronary, and basilar arteries were removed, cut helically in strips, and mounted in isolated tissue baths to measure isometric force. Vessels were contracted with either 40 mM KCl (opening voltage-operated calcium channels) or 1 x 10(-5) M norepinephrine (NE, opening receptor-operated calcium channels). Cumulative dose-response curves were generated for relaxation with each calcium channel blocker. All compounds were more potent at relaxing potassium-induced contractions than NE-induced contractions. In vessels precontracted with KCl, neither diltiazem, verapamil, or nifedipine showed selectivity for basilar artery as compared with the mesenteric artery. Both clentiazem and nimodipine were selective (6- and 30-fold, respectively) for basilar artery in blocking potassium-induced contractions. When NE was used to contract the arteries, clentiazem (12-fold), diltiazem (8-fold), verapamil (8-fold), and nifedipine (153-fold) were all more potent in relaxing the contraction in basilar artery than in mesenteric artery. Nimodipine failed to demonstrate selectivity for basilar artery as compared with mesenteric artery contracted with NE.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of S-(+)-boldine on the alpha 1-adrenoceptor of the guinea-pig aorta.

1. The cardiovascular activity of S-(+)-boldine, an aporphine alkaloid structurally related to papaverine, was determined. The work includes functional studies on guinea-pig isolated aorta contracted with noradrenaline, caffeine, KCl or Ca2+, and on guinea-pig trachea contracted with acetylcholine or histamine. 2. S-(+)-boldine inhibited in a concentration-dependent manner the contractile response evoked by noradrenaline (10 microM) in guinea-pig aorta (IC50 = 1.4 +/- 0.2 microM) while the KCl depolarizing solution (60 mM)- or the Ca2+ (1 mM)-induced contractions were only partially affected by boldine up to 300 microM. In contrast, papaverine relaxed noradrenaline (NA), KCl or Ca2+ induced contractions showing similar IC50 values in all cases. S-(+)-boldine had a greater potency on the contraction elicited by NA whereas papaverine acted in a non-selective manner. 3. S-(+)-boldine was found to be an alpha 1-adrenoceptor blocking agent in guinea-pig aorta as revealed by its competitive antagonism of noradrenaline-induced vasoconstriction (pA2 = 5.64 +/- 0.08), and its potency was compared with that of prazosin (pA2 = 8.56 +/- 0.24), a known potent alpha 1-adrenoceptor antagonist. In contrast, papaverine caused rightward shifts of the NA concentration-response curves with depression of maximal response indicating that it acts as a non-competitive antagonist. 4. Contraction of guinea-pig aorta induced by caffeine (60 mM) in a Ca(2+)-containing Krebs solution was not affected by a 60 min incubation period with different doses of S-(+)-boldine (1-300 microM). Papaverine inhibited partially this caffeine-induced contraction at the maximal dose used (100 microM). 5. Inositol phosphates formation induced by noradrenaline (10 microM) in guinea-pig thoracic aorta was inhibited by S-(+)-boldine (30 microM) but not by papaverine (10 microM). 6. Contractions of guinea-pig trachea caused by acetylcholine (100 microM) or histamine (10 microM) were not modified by S-(+)-boldine (0.1-100 microM). 7. These results provide evidence that S-(+)-boldine, an aporphine alkaloid, has interesting properties as an alpha 1-adrenoceptor blocker in vascular smooth muscle, and acts as a competitive antagonist of the alpha 1-adrenoceptor present in the guinea pig aorta.