The effects of racemic cromakalim,BRL 38226 and levcromakalim on the membrane potential of the rat aorta and of BRL 38226 on the contractile activity of the rat aorta and portal vein

1 The effects of racemic cromakalim and the (+)-3R,4S and (-)-3S,4R isomers, BRL 38226 and levcromakalim respectively, on the membrane potential of the rat aorta have been determined. In addition, preliminary studies of the effects of BRL 38226 on the contractility of the rat aorta and portal vein have been made. 2 The rat aorta is hyperpolarized by racemic cromakalim at 10 μm and by levcromakalim at 1 μm , but is depolarized by BRL 38226 at 1 μm . 3 In the presence of depolarization with 20 μm KCl, racemic cromakalim and levcromakalim, but not BRL 38226, reversed the depolarization of the rat aorta. 4 In the presence of hyperpolarization with isoprenaline at 1 μm or in a CaCl₂-free Krebs solution, racemic cromakalim and BRL 38226, but not levcromakalim reversed the hyperpolarization. 5 Glibenclarnide at 0.3 and 1 μm depolarized the rat aorta. Pretreatment with glibenclamide at 1 μm prevented the hyperpolarizing action of racemic cromakalim but did not alter the depolarizing action of BRL 38226. Pretreatment with BRL 38226 at 10 μm also prevented the hyperpolarizing action of racemic cromakalim. 6 Contractility studies demonstrated that BRL 38226 and glibenclamide have no effect on the resting tone of the rat aorta in normal Krebs solution. However in a CaCl₂-free Krebs solution, BRL 38226 and glibenclamide, 0.1–100 μm , caused small contractions. BRL 38226 and glibenclamide increased the spontaneous contractile activity of the rat portal vein in normal Krebs solution. 7 The present study illustrates that levcromakalim opens the ATP-dependent potassium channels of the rat aorta to cause hyperpolarization. BRL 38226 and glibenclamide produce depolarization and contraction of the rat aorta and increase the contractile activity of the rat portal vein, possibly by blocking the ATP-dependent potassium channels.     

The potassium channel opening action of pinacidil; studies using biochemical,ion flux and microelectrode techniques

Summary In rat aorta and rat portal vein, (–)- and (+)-pinacidil each produced a concentration-dependent inhibition of tension development. Although the (–) isomer was the more potent, concentration effect curves for each isomer were steep with similar slopes. In rat portal vein, tetraethylammonium and procaine antagonised the relaxant effect of (±)-pinacidil, whereas 3,4-diamino-pyridine was without effect. Intracellular microelectrode recording in rat portal vein showed that low concentrations of (±)-pinacidil reduced the duration of multispike electrical complexes. In both rat aorta and rat portal vein, higher concentrations of (±)-pinacidil hyperpolarised the membrane towards the potassium equilibrium potential. (±)-Pinacidil increased ⁸⁶Rb efflux from rat aorta and rat portal vein in a concentration dependent manner. In a separate study, (±)-pinacidil increased ⁴²K efflux from rat portal vein. (±)-Pinacidil had no effect on cyclic GMP or cyclic AMP levels in rat aorta. It is concluded that pinacidil opens ⁸⁶Rb-permeable potassium channels in rat aorta and rat portal vein. This mechanism is independent of cyclic nucleotide changes and may be responsible for the antihypertensive effect of pinacidil. Send offprint requests to: A. H. Weston at the above address     

Evidence that acetylcholine-mediated hyperpolarization of the rat small mesenteric artery does not involve the K+ channel opened by cromakalim.

1. Acetylcholine causes a concentration-dependent hyperpolarization of the rat small mesenteric artery (diameter at 100 mmHg, 200-400 microns). In the absence of tone the average potential change was from approximately -60 to -75 mV. In the presence of tone induced by endothelin-1 (20 nM), acetylcholine caused vasorelaxation in association with a marked hyperpolarization; from approximately -32 to -71 mV. 2. A number of compounds known to antagonize the actions of cromakalim were tested for their ability to block responses to acetylcholine. Glibenclamide (0.1-3 microM), phentolamine (10-100 microM) and alinidine (1-30 microM) caused a concentration-dependent depolarization of the rat small mesenteric artery which was not dependent on an intact endothelium. Glibenclamide was approximately 10 times more potent than either phentolamine or alinidine, a similar ratio to their potency as antagonists of cromakalim. 3. In the presence of concentrations of the cromakalim antagonists which functionally inhibited responses to cromakalim, only phentolamine and alinidine had a significant effect on the hyperpolarization and functional responses to acetylcholine. Glibenclamide was without effect at the concentrations used. 4. Experiments on pig coronary artery, where acetylcholine causes vasoconstrictor responses, showed that phentolamine and alinidine have some anti-muscarinic activity which could account for their ability to affect vasorelaxant/hyperpolarization responses to acetylcholine in the rat small mesenteric artery. 5. The results suggest that the acetylcholine-mediated hyperpolarization observed in the rat small mesenteric artery does not involve K+ channels opened by cromakalim. This finding differs from other studies performed on the rabbit middle cerebral artery which show hyperpolarizing responses to acetycholine to be glibenclamide-sensitive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endogenous nitric oxide attenuates beta-adrenoceptor-mediated relaxation in rat aorta

1. Divergent evidence suggests that the intracellular signalling pathways for beta-adrenoceptor-mediated vascular relaxation involves either cAMP/protein kinase (PK) A or endothelial nitric oxide (NO) release and subsequent activation of cGMP/PKG. The present study identifies the relative roles of NO and cAMP, as well as dependence on the endothelium for beta-adrenoceptor-mediated relaxation of rat isolated aortas. 2. Cumulative concentration-response curves to isoprenaline (0.01-3 micromol/L) in phenylephrine (0.1 micromol/L)-preconstricted endothelium-intact and -denuded aortas were constructed. Isoprenaline-mediated relaxation was partially reduced by endothelium removal and the presence of the NO synthase inhibitor N(G)-monomethyl-L-arginine (0.1 mmol/L), but not by the cAMP antagonist (Rp)-cyclic adenosine-3',5'-monophosphorothioate (Rp-cAMPS; 0.5 mmol/L). 3. In contrast, in endothelium-denuded aortas, the isoprenaline-mediated relaxation was inhibited by Rp-cAMPS and this inhibition was lost in the presence of the NO donor sodium nitroprusside (1 nmol/L). This effect was not due to phosphodiesterase (PDE) activity because the non-selective PDE inhibitor 3-isobutyl-1-methylxanthine (1 micromol/L) failed to affect the isoprenaline vasorelaxant response. 4. The K(+) channel blocker tetraethylammonium (TEA; 1 mmol/L) attenuated isoprenaline-induced relaxation in endothelium-denuded aorta, but its effect was non-additive with Rp-cAMPS, suggesting that the K(+) channel component may involve cAMP. In endothelium-intact aortas, TEA but not Rp-cAMPS reduced isoprenaline relaxation, suggesting an additional non-cAMP component. 5. These findings suggest that beta-adrenoceptors induce vascular smooth muscle relaxation by acting through the NO-cGMP pathway and, when that is disrupted by endothelium removal or the presence of an NO synthase inhibitor, the cAMP pathway in smooth muscles is used. The lack of cAMP participation in endothelium-intact vessels may be because NO suppresses or overrides the cAMP effect.     

Negative inotropic effect of pinacidil on the rat left atria

Abstract— Cromakalim at 50 μm and pinacidil at 0·1–10 μm had no effect, but pinacidil at 0·1 Mm had a negative inotropic effect on the rat electrically-driven left atria without altering the positive inotropic responses to isoprenaline or phenylephrine alone. Glibenclamide had no effect but 4-aminopyridine, procaine (30 μm ) and tetraethylammonium (3 Mm ) augmented the cardiac stimulation response. The ability of pinacidil to attenuate the cardiac stimulation response was not altered by glibenclamide, 4-aminopyridine or procaine but was prevented by pretreatment with tetraethylammonium. Thus, on the rat left atria, pinacidil has a negative inotropic effect which is unrelated to the opening of ATP-sensitive potassium channels, but may be due to opening the inward rectifying potassium channels.     

Alpha-2 adrenoceptors are present in rat aorta smooth muscle cells, and their action is mediated by ATP-sensitive K(+) channels

The role of alpha(2)-adrenoceptors in the response of aorta smooth muscle rings to the alpha(2)-adrenoceptors agonists UK 14,304 and clonidine was studied. Stimulation by 1 - 10 nM UK 14,304 caused dose-dependent relaxant responses in BaCl(2)-contracted endothelium-denuded aorta rings, and hyperpolarization in rings with or without endothelium, which were inhibited by yohimbine and glibenclamide, but not affected by prazosin, propranolol, apamin or iberiotoxin. At higher concentrations (10 nM - 10 microM) UK 14,304 also induced a depolarizing effect which was potentiated by yohimbine and inhibited by prazosin. These results indicate that UK 14,304 acts on alpha(2)-adrenoceptors at lower concentrations and on both alpha(1)- and alpha(2)-adrenoceptors above 10 nM. In rings, with or without endothelium, noradrenaline had a depolarizing effect which was inhibited by prazosin. Adrenaline did not affect the membrane potential but in the presence of prazosin caused hyperpolarization, which was inhibited by yohimbine and glibenclamide. These results indicate that noradrenaline is more selective for alpha(1)-, whereas adrenaline has similar affinities for alpha(1)- and alpha(2)-adrenoceptors. In aortae with endothelium, L-NNA caused a small depolarization but did not affect the hyperpolarization induced by UK 14,304, indicating that NO is not involved in that response. Glibenclamide induced a small depolarization in aortae, with or without endothelium, indicating that ATP-sensitive K(+) channels may play a role in maintaining the smooth muscle's membrane potential. Our results indicate that, in rat aorta, alpha(2)-adrenoceptors are also present in the smooth muscle, and that these receptors act through small-conductance ATP-sensitive K(+) channels.     

Pharmacological differences between two muscarinic responses of the rat superior cervical ganglion in vitro.

1 Pharmacological differences have been observed between the muscarinic agonist-induced depolarizing and hyperpolarizing responses of the rat isolated superior cervical ganglion. 2 Pirenzepine (0.3 microM) selectively reduced the depolarizing response and unmasked the hyperpolarizing response. No such selectivity was observed with a concentration of N-methylatropine which was equipotent with pirenzepine in antagonizing the depolarizing response. 3 The neuromuscular blocking agents gallamine (10 microM) and pancuronium (3 microM) exhibited the oppositive selectivity to pirenzepine, both dramatically reduced the hyperpolarization but only slightly antagonized the depolarization. 4 The potencies of a range of agonists in evoking the depolarizing and hyperpolarizing responses, the latter in the presence of 0.3 microM pirenzepine, have been determined. Methylfurmethide failed to hyperpolarize the ganglion at concentrations which evoked maximal depolarizations. 5 The muscarinic hyperpolarization did not appear to be mediated by the secondary release of catecholamines. 6 It was concluded that the two muscarinic responses on the rat superior cervical ganglion, the slow depolarization and faster hyperpolarization, are mediated by different muscarinic receptor subtypes.     

Effects on cardiac muscle of the -adrenoceptor blocking drugs INPEA and LB46 in relation to their local anaesthetic action on nerve

1. As a local anaesthetic on frog nerve, LB46 had 0.4 times the activity of procaine and 2 times that of INPEA.2. In reducing the maximum rate of depolarization (MRD) of intracellularly recorded cardiac action potentials, LB46 was 20 times more potent than INPEA.3. As an antagonist of the chronotropic effect on atrial muscle and of the inotropic effects on ventricular muscle of isoprenaline the pA(2) values for LB46 were 9.05+/-0.15 and 9.30+/-0.06 respectively, and for INPEA 6.00+/-0.16 and 6.10+/-0.12.4. LB46 was 8 times more active than racemic propranolol in blocking the effects of isoprenaline on isolated cardiac muscle, and since its direct action on the cardiac membrane was only 0.1 times that of propranolol, there was a net gain of 80 times in the specificity of LB46 for class 2 (antisympathetic) over class 1 (depression of MRD) antidysrhythmic actions.5. INPEA was 2.5 times less active as a beta-adrenoceptor blocking drug on cardiac muscle than (+)-propranolol, and 1,300 times less active than LB46.6. INPEA, but not LB46, prolonged the duration of both atrial and ventricular intracellular action potentials.     

Effects of LP-805, a novel vasorelaxant agent, a potassium channel opener, on rat thoracic aorta.

1. In rat thoracic aorta, LP-805 (0.1-10 microM) caused the marked reduction of NE-induced maximum response and relaxed the low K+ (less than 35.9 mM)-induced contraction, in a concentration-dependent manner, but failed to relax the high K+ (65.9 mM)-induced contraction. 2. Glibenclamide (0.3-1 microM) caused a parallel shift of concentration-response curve produced by LP-805 for 25.9 mM K(+)-induced contraction and prevented the LP-805-induced reduction in maximum response evoked by NE in a concentration-dependent manner. 3. Glibenclamide (10 microM) prevented the LP-805 (10 microM)-induced decrease in cytosolic Ca2+ levels which was increased by 1 microM NE or 25.9 mM K+. 4. LP-805 (10 microM) increased basal 86Rb efflux, which was completely inhibited by 10 microM glibenclamide. 5. The results suggest that LP-805 causes a vasorelaxation as a consequence of the decrease in cytosolic Ca2+ levels due to the increase in K+ efflux via opening ATP-dependent K+ channels.     

The effects of (+/-)-, (+)- and (-)-celiprolol and bromoacetylalprenololmentane at the beta-adrenoceptors of rat isolated cardiovascular preparations

The effects of (+/-)-, (+)- and (-)-celiprolol and of bromoacetylalprenololmentane (BAAM, an irreversible beta-adrenoceptor antagonist) on the contractile responses of the electrically driven rat right ventricle strip to isoprenaline and on the relaxant responses of the rat aorta to procaterol, have been studied. Racemic and (-)-celiprolol or BAAM treatment of the ventricle produced non-parallel rightward shifts of the isoprenaline response curves with a reduction in the maximal response. Sotalol produced parallel rightward displacements of the procaterol response curves of the aorta with no effect on the maximal relaxations. Racemic and (+)- and (-)-celiprolol or BAAM treatment of the aorta produced non-parallel rightwards shifts of the procaterol relaxant curves with a reduction in the maximal relaxation. The BAAM data was used to demonstrate that the KA (dissociation constant) for isoprenaline at beta 1-adrenoceptors was 1.46 x 10(-7) M and for procaterol at beta 2-adrenoceptors was 2.34 x 10(-5) M. Calculation of receptor occupancy demonstrated that to produce a maximal response of the rat right ventricle, had to occupy 87% of the beta 1-adrenoceptors. Likewise, for a maximal response of the rat aorta, procaterol had to occupy 81% of the beta 2-adrenoceptors. It is suggested that the use of tissues with small beta-adrenoceptor reserves has shown that (+/-)- and (-)-celiprolol are slowly dissociating, rather than readily reversible, beta-adrenoceptor antagonists.     

Modulation of the isoprenaline-induced membrane hyperpolarization of mouse skeletal muscle cells.

1. The hyperpolarization of the resting membrane potential, Vm, induced by isoprenaline in the lumbrical muscle fibres of the mouse, was investigated by use of intracellular microelectrodes. 2. In normal Krebs-Henseleit solution (potassium concentration: K+o = 5.7 mM, 'control'), Vm was -7.40 +/- 0.2 mV; lowering K+o to 0.76 mM ('low K+o') resulted in either a hyperpolarization (Vm = -95.7 +/- 2.9 mV), or a depolarization (Vm = -52.0 +/- 0.3 mV). 3. Isoprenaline (> or = 200 nM) induced a hyperpolarization of Vm by delta Vm = -5.6 +/- 0.4 mV in control solution. 4. When Vm hyperpolarized after switching to low K+o, the addition of isoprenaline resulted in increased hyperpolarization Vm: delta Vm = -16.3 +/- 3.2 mV to a final Vm = -110.1 +/- 3.4 mV. Adding iso-prenaline when Vm depolarized in low K+o, leads to a hyperpolarization of either by -11.6 +/- 0.5 mV to -63.6 +/- 0.8 mV or by -51.7 +/- 2.7 mV to -106.9 +/- 3.9 mV. 5. Ouabain (0.1 to 1 mM) did not suppress the hyperpolarization by isoprenaline in 5.7 mM K+o (delta Vm = -6.7 +/- 0.4 mV) or the hyperpolarization of the depolarized cells in low K+- (delta Vm = -9.7 +/- 1.5 mV). 6. The hyperpolarization is a logarithmically decreasing function of K+o in the range between 2 and 20 mM (12 mV/decade). 7.IBMX and 8Br-cyclic AMP mimicked the response to isoprenaline whereas forskolin (FSK) induced in low K+o a hyperpolarization of -7.0 +/- 0.7 mV that could be augmented by addition of isoprenaline (delta Vm = -8.2 +/- 1.8 mV). 8. In control and low K+o, Ba2+ (0.6 mM) inhibited the hyperpolarization induced by isoprenaline, IBMX or 8Br-cyclic AMP. Other blockers of the potassium conductance such as TEA (5 mM) and apamin (0.4 microM) had no effect. 9. We conclude that in the lumbrical muscle of the mouse the isoprenaline-induced hyperpolarization is primarily due to an increase in potassium permeability.     

Na+-K+-ATPase is involved in the sustained ACh-induced hyperpolarization of endothelial cells from rat aorta

BACKGROUND AND PURPOSE: Inhibition of Na(+)-K(+)-ATPase is known to attenuate endothelium-dependent relaxation in many arteries. The purpose of this study was to evaluate the role of Na(+)-K(+)-ATPase in the regulation of endothelial membrane potential at rest and during stimulation by ACh. EXPERIMENTAL APPROACH: Membrane potential was recorded from the endothelium of rat aorta using the perforated patch-clamp technique. KEY RESULTS: Superfusion with K(+)-free solution produced a depolarization of about 11 mV from the resting value of -42.9+/-0.9 mV. Reintroduction of 4.7 mM K(+) transiently hyperpolarized endothelial cells to -52.4+/-1.8 mV and the membrane potential recovered within 10 min. Ouabain 500 microM depolarized endothelium by about 11 mV and inhibited the hyperpolarization induced by K(+) reintroduction into the K(+)-free solution. However, 500 nM ouabain did not affect the resting membrane potential or the hyperpolarization induced by K(+) reintroduction. Pre-exposure to ouabain 500 microM, but not 500 nM, attenuated the sustained component of hyperpolarization to ACh without affecting the amplitude of the transient peak hyperpolarization. In K(+)-free solution, the amplitude of peak hyperpolarization to ACh was increased, while the sustained component of hyperpolarization was attenuated. CONCLUSIONS AND IMPLICATIONS: These results indicate that electrogenic Na(+)-K(+)-ATPase partially contributes to the sustained hyperpolarization of endothelial cells from rat aorta in response to ACh. They also suggest that the alpha1, but not alpha2 or alpha3 isoforms, is involved in ACh-mediated hyperpolarization.     

The effects of (+)- and (-)-propranolol on 3H-transmitter efflux in guinea-pig atria and the presynaptic beta-adrenoceptor hypothesis.

The effects of isoprenaline and of the (+)- and (-)-isomers of propranolol on the stimulation-induced overflow of 3H-transmitter was assessed in guinea-pig atria to evaluate the hypothesis of presynaptic beta-adrenoceptors. 2 Isoprenaline (1.2 x 10(-8) M) enhanced the efflux of tritium at 2 and 5 Hz with 100 pulses and did so to a similar extent at both frequencies. 3 The (-)-isomer of propranolol (1.0 x 10(-7) M) blocked the enhancing effect of isoprenaline but did not by itself modify transmitter efflux. 4 The (+)-isomer of propranolol, almost devoid of beta-adrenoceptor blocking properties, was also effective at 1.0 x 10(-7) M in blocking the enhancement of tritium efflux by isoprenaline. 5 The (-)-isomer of propranolol (1.0 x 10(-7) M) blocked almost entirely the inotropic response to isoprenaline (3 x 10(-7) M) but even 3.0 x 10(-6) M (+)-propranolol was ineffective in antagonizing the beta-adrenoceptor-mediated contractile responses to the catecholamine. 6 It is concluded that the presynaptic site of isoprenaline action does not show the requisite stereo-specificity of beta-adrenoceptors and that a 'non-specific' action of the antagonist probably accounts for its reduction of the effect of isoprenaline.     

Reduced hyperpolarization of endothelial cells following high dietary Na+: effects of enalapril and tempol

1. High dietary Na(+) is associated with impaired vascular endothelial function. However, the underlying mechanisms are not completely understood. In the present study, we investigated whether the endothelial hyperpolarization response to acetylcholine (ACh) exhibited any abnormalities in Wistar rats fed a high-salt diet (HSD) for 1 month and, if so, whether chronic treatment with the angiotensin-converting enzyme inhibitor enalapril or the anti-oxidant tempol could normalize the response. Membrane potential was recorded using the perforated patch-clamp technique on the endothelium of rat aorta. 2. Acetylcholine (2 μmol/L) produced a hyperpolarization sensitive to TRAM-34, a blocker of intermediate-conductance Ca(2+) -sensitive K(+) channels (IK(Ca)), but not to apamin, a blocker of small-conductance Ca(2+)-sensitive K(+) channels (SK(Ca)). NS309 (3 μmol/L), an activator of SK(Ca) and IK(Ca) channels, produced a hyperpolarization of similar magnitude as ACh. 3. In the HSD group, the ACh-evoked hyperpolarization was significantly attenuated compared with that in the control group, which was fed normal chow rather than an HSD. Similarly, the hyperpolarization produced by NS309 was weaker in tissues from HSD-fed rats. 4. Combination of HSD with chronic enalapril treatment (20 mg/kg per day for 1 month) normalized endothelial hyperpolarizing responses to ACh. Chronic tempol treatment (1 mmol/L in tap water for 1 month) prevented the reduced hyperpolarization to ACh. 5. The results of the present study indicate that excess in dietary Na(+) results in a failure of endothelial cells to generate normal IK(Ca) channel-mediated hyperpolarizing responses. Our observations implicate oxidative stress mediated by increased angiotensin II signalling as a mechanism underlying altered endothelial hyperpolarization during dietary salt loading.     

The relaxing effect of BDF 9148 on the KCl-contracted aorta isolated from normo- and hyper-tensive rats

BDF 9148, a positive cardiac inotrope, relaxes the rat isolated portal vein and the KCl-contracted rat aorta. The aims of our study were to determine the mechanism of action of BDF 9148, and to ascertain whether the relaxing effect of BDF 9148 was maintained in the presence of the hypertrophy associated with hypertension, by investigating the effects of BDF 9148 on the contractility and electrophysiology of aortae of Wistar Kyoto normotensive rats (WKY) and Spontaneously Hypertensive Rats (SHRs). High concentrations of veratridine contracted the quiescent rat aorta. BDF 9148 had no effect on the quiescent, but relaxed the KCl-contracted WKY and SHR aorta by a tetrodotoxin insensitive mechanism, and these relaxations decreased with age but were not greatly altered by hypertrophy. The verapamil relaxations of the KCl-contracted aorta were not altered by age or hypertrophy. The ability of KCl to depolarise the aorta was reversed by verapamil, but not by BDF 9148. On the contracted rat aorta, the relaxant responses to acetylcholine were abolished by removal of the endothelium but potentiated by IBMX (10^–6 M), and the responses to isoprenaline were inhibited by propranolol (10^–6 M) but potentiated by forskolin (10^–7 M). The relaxation responses of the KCl-contracted aorta to BDF 9148 were not altered by removal of the endothelium, or by propranolol, forskolin and IBMX. In summary, the effects of verapamil and BDF 9148 on the aorta are different, and thus it is unlikely that the relaxant responses to BDF 9148 on the aorta are due to calcium channel blocking activity. The mechanism of the relaxant effect of BDF 9148 on the aorta remains unknown, but we have shown the response is endothelium-independent, and not mediated by sodium channel opening, hyperpolarization, β-adrenoceptors, or by stimulating adenylate cyclase or guanylate cyclase. Received: 12 March / Accepted: 8 October 1997     

Characterization of alpha2-adrenoceptors in smooth muscles of the spontaneously hypertensive rat aorta

Previous works have shown that the alpha(2)-adrenoceptor agonist UK 14,304 induced the relaxation and hyperpolarization of the rat aorta, mediated by alpha(2)-adrenoceptors present in the smooth muscles, through small-conductance, ATP-sensitive K(+) channels. We now report that in spontaneously hypertensive rat (SHR) aortic rings, UK 14,304 induced concentration-dependent hyperpolarizing responses, which were inhibited by yohimbine, an alpha(2)-adrenoceptor inhibitor, and by glibenclamide, a specific inhibitor of small-conductance, ATP-sensitive K(+) channels. The responses were also partially inhibited by iberiotoxin and by apamin. Treatment with N(omega)-nitro-L-arginine (L-NNA) did not affect the response to UK 14,304. These results indicate that alpha(2)-adrenoceptors are present in SHR aortic smooth muscle cell membranes, but differ from those of normotensive animals regarding the K(+) channels involved in their responses. Moreover, the resting membrane potential (RMP) was significantly more negative in SHR than in normotensive rats. This relative hyperpolarized state is probably due to Ca(2+)-dependent K(+) channels being constitutively open in SHR, since the addition of iberiotoxin caused a significant depolarization of the aortic smooth muscle membranes in this strain.     

Analysis of the hyperpolarizing effect of catecholamines on canine cardiac Purkinje fibres.

1. The hyperpolarization induced by catecholamines on barium-depolarized (0.2-0.8 mM BaCl) canine cardiac Purkinje fibres, in vitro, was studied by use of conventional microelectrode recordings of transmembrane electrical potentials. 2. Noradrenaline, adrenaline and isoprenaline hyperpolarized Purkinje fibres in a concentration-dependent manner from a threshold concentration around 5 nM. The three catecholamines were shown to be approximately equipotent. Tachyphylaxis was observed when the interval between catecholamine applications was less than 15 min. 3. Atenolol (10 microM) blocked the hyperpolarization reversibly and theophylline (0.5 mM) potentiated it. 4. Tetrodotoxin (5 microM) did not affect the hyperpolarization induced by isoprenaline. Acetylcholine and histamine, up to 10 microM, were not effective in hyperpolarizing Purkinje fibres. 5. Low extracellular potassium concentrations (zero and 1 mM) did not affect the hyperpolarization, but high extracellular potassium concentrations (10-20 mM), markedly reduced the effect of isoprenaline (100 nM). 6. Reduction of the extracellular sodium concentration produced a roughly proportional reduction in the isoprenaline-induced hyperpolarization. The hyperpolarization was reversibly blocked in 34 mM sodium Tris-Tyrode solution. 7. The hyperpolarization was not reduced in Tyrode solution containing 0.6 mM calcium, but was drastically reduced in zero-calcium Tyrode solution. This effect was reversible. 8. Addition of verapamil (5-10 microM) diminished the hyperpolarization, in a concentration-dependent manner. This effect was partially reversed after washing. 9. Ouabain (0.7-1 microM) significantly reduced the isoprenaline-induced hyperpolarization, but 2,4-dinitrophenol (0.2 mM) did not affect it. 10.Caesium chloride (20 mM) abolished the hyperpolarization. The blockade was only partially reversed upon washing.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thapsigargin- and cyclopiazonic acid-induced endothelium-dependent hyperpolarization in rat mesenteric artery.

1. The present study was designed to determine whether putative, selective inhibitors of the Ca(2+)-pump ATPase of endoplasmic reticulum, thapsigargin (TSG) and cyclopiazonic acid (CPA), induce endothelium-dependent hyperpolarization in the rat isolated mesenteric artery. The membrane potentials of smooth muscle cells of main superior mesenteric arteries were measured by the microelectrode technique. 2. In tissues with endothelium, TSG (10(-8)-10(-5) M) caused sustained hyperpolarization in a concentration-dependent manner. In tissues without endothelium, TSG did not cause any change in membrane potential. CPA (10(-5) M) also hyperpolarized the smooth muscle membrane, an effect that was endothelium-dependent and long-lasting. 3. The hyperpolarizing responses to these agents were not affected by indomethacin or NG-nitro-L-arginine (L-NOARG). 4. In Ca(2+)-free medium, neither TSG nor CPA elicited hyperpolarization, in contrast to acetylcholine which generated a transient hyperpolarizing response. 5. In rings of mesenteric artery precontracted with phenylephrine, TSG and CPA produced endothelium-dependent relaxations. L-NOARG significantly inhibited the relaxations to these agents, but about 40-60% of the total relaxation was resistant to L-NOARG. The L-NOARG-resistant relaxations were abolished by potassium depolarization. 6. These results indicate that TSG and CPA can cause endothelium-dependent hyperpolarization in rat mesenteric artery possibly by releasing endothelium-derived hyperpolarizing factor and that membrane hyperpolarization can contribute to the endothelium-dependent relaxations to these agents. The mechanism of hyperpolarization may be related to increased Ca2+ influx into endothelial cells triggered by depletion of intracellular Ca2+ stores due to inhibition of endoplasmic reticulum Ca(2+)-pump ATPase activity.     

Cromakalim and K+ channels in canine trachealis

The effects of cromakalim and glibenclamide on membrane properties and responses to acetylcholine of canine trachea were studied in the double sucrose gap to evaluate the presence and function of ATP-sensitive K⁺ channels. Cromakalim produced a concentration-dependent hyperpolarization of muscle membrane potential which at maximum brought the membrane potential near the potassium equilibrium potential. Current clamping by hyperpolarizing current to this equilibrium potential abolished the hyperpolarization but not the membrane resistance decrease to cromakalim. Glibenclamide had no effect on resting membrane properties but reduced or abolished effects of cromakalim. Another K⁺ channel antagonist, tetraethylammonium at 20 mM, also reduced the effects of cromakalim, but 4-aminopyridine (5 mM), Ba²⁺ (1 mM, and apamin (10⁻⁶M) had no antagonistic effect.The EJP produced on stimulation of cholinergic nerves sometimes increased just after cromakalim-induced hyperpolarization, but within 5–10 min as membrane resistance dramatically fell it was reduced, as was the depolarization to infused acetylcholine. Initially the reduction in EJP amplitude could be partially overcome by applying hyperpolarizing currents or by applying a second field stimulation; later the EJP was reduced further and was unaffected by these procedures. Even when depolarization to acetylcholine was markedly reduced, the contraction was not. Glibenclamide had no effects alone but antagonized all the effects of cromakalim.These results suggest that ATP-sensitive cromakalim activated K⁺ channels are present in canine trachea but are usually closed during resting conditions under our experimental conditions. When they are opened by cromakalim, they hyperpolarize to near E_K, markedly decrease membrane resistance and reduce the depolarization response to acetylcholine, probably by short circuiting the acetylcholine-induced current. There also appear to be ATP-sensitive K⁺ channels in cholinergic nerves which, when activated, reduced acetylcholine output.     

Actions of the GABAB agonist, (-)-baclofen, on neurones in deep dorsal horn of the rat spinal cord in vitro.

1. The electrophysiological actions of the GABAB agonist, (-)-baclofen, on deep dorsal horn neurones were studied using an in vitro preparation of the spinal cord of 9-16 day old rat. 2. On all neurones tested, (-)-baclofen (100 nM-30 microM) had a hyperpolarizing action which was associated with a reduction in apparent membrane input resistance. The increase in membrane conductance was dose-dependent and had a Hill coefficient of 1.0. 3. The (-)-baclofen-activated hyperpolarization persisted in the presence of bicuculline (50 microM) and Mg2+ (20 mM). 4. The reversal potential of the hyperpolarizing event was estimated at 102 mV and was made less negative by increasing the external concentration of potassium ions. 5. Over the same concentration range, (-)-baclofen also depressed the polysynaptic composite excitatory postsynaptic potentials (e.p.s.ps) evoked in these neurones by electrical stimulation of the dorsal root entry zone. 6. The potassium channel blockers caesium, applied intracellularly, and barium, applied extracellularly, depressed the postsynaptic response to baclofen but not its effect on e.p.s.ps. 7. We propose that (-)-baclofen has more than one mechanism of action in spinal dorsal horn: a postsynaptic action mediated via an increase in potassium conductance and a presynaptic action that is not associated with potassium channels and may be mediated via calcium channels. Since previous studies have demonstrated little effect of (-)-baclofen on transmitter release in spinal cord, it is possible that the postsynaptic hyperpolarizing action of (-)-baclofen may account for its clinical potency as an anti-spastic agent.