H. Kuriyama

Two Ca-dependent K-channels classified by the application of tetraethylammonium distribute to smooth muscle membranes of the rabbit portal vein

Dispersed single smooth muscle cells of rabbit portal vein were prepared by treatment with collagenase and trypsin. The muscle cells were 100–300 μm in length, 5–10 μm in maximum width and cylindrical in shape. In insideout membrane patches, two different amplitudes of ionic currents were recorded, and these single channel conductances were 273 pS (Kl-channel) and 92 pS (Ks-channel), when both sides of the membrane were exposed to 142 mM K+ solution. The channel conductances depended on concentrations of K+ on both sides of the membrane. When K+ were replaced with Na+ or Tris+, these single-channel currents were abolished. When the concentration of Ca2+ inside the membrane was greater than 10−7 M, the channel activity was enhanced but there was enhancement when Ca2+ was applied to the extracellular membrane surface, in concentrations ranging between 10−9 and 10−3 M. During application of tetraethylammonium (TEA+; 1–10 mM) to the intracellular membrane surface, amplitudes of the single-channel current of both types of the K-channel were not modified. By contrast application of TEA+ (0.1–1 mM) to the extracellular membrane surface, reduced the amplitudes of the current and increased noise levels during the open-state of the Kl-channels, but did not have such an effect on the Ks-channel. We conclude that there are at least two different Ca-dependent K-channels distributed on the smooth muscle membrane of the rabbit portal vein. TEA+ applied to the extracellular membrane surface blocks activation of the Kl-channel, but not that of the Ks-channel. These two Ca-dependent K-channels do not seem to be important for maintenance of the resting membrane potential, but do play an important role in the repolarizing stage of the Ca spikes, in the rabbit portal vein.

Effects of acetylcholine and catecholamines on the smooth muscle cell of the porcine coronary artery.

The effects of acetylcholine (ACh), noradrenaline (NA) and isoprenaline (Isop) on the membrane and mechanical properties of smooth muscle cells of the pig coronary artery were investigated by micro‐electrode, double sucrose gap and isometric tension recording methods. (1) The mean membrane potential was ‐51.4 mV and the membrane was electrically quiescent. Application of outward current pulse generated a graded response. The current‐voltage relationship was linear for application of inward current pulses. The length constant of the tissue was 0.67 mm and time constant of the membrane was 290 msec. The tissue possessed cable‐like properties. (2) ACh (10(‐10) to 10(‐5) g/ml.) did not change the membrane potential and membrane resistance, but NA and Isop hyperpolarized the membrane and reduced the membrane resistance. These actions of catecholamines, presumably mediated by beta‐adrenergic receptors, were suppressed by propranolol but not phentolamine. (3) The minimum depolarization required to produce the contraction was 4 mV by excess extracellular K concentration and 6 mV by electrical displacement of the membrane potential. The amplitude of the contraction evoked by depolarization in excess K was consistently larger than that by the electrical displacement. The minimum concentration required to produce the contraction induced by ACh was 5 x 10(‐9) g/ml. NA and Isop consistently suppressed the contraction evoked by excess extracellular K or by ACh. (4) When the tissue was immersed in Ca‐free EGTA solution containing excess K or Ca‐free EGTA Krebs solution, the time taken for Ca depletion from the intracellular store site was longer with depolarized membranes than that with polarized membranes. ACh evoked contraction, even after the tissue had lost the ability to produce contraction by excess extracellular K in Ca‐free solution. (5) After the tissue had been immersed in Ca‐free solution containing excess K for more than 4 hr, or Ca‐free Krebs solution for more than 2 hr, application of Ca evoked the contraction. These effects of Ca were suppressed by application of catecholamines. The amplitudes of subsequent ACh‐induced contraction in Ca‐free excess K were not suppressed, while there was a suppression of the K‐induced contraction in Ca‐free Krebs solution. (6) It is concluded that the smooth muscle cell of the pig coronary artery possesses muscarinic and beta‐adrenergic receptors. The former mainly activates the mechanical response without affecting the surface membrane while the latter modifies both membrane and mechanical properties.

Dual regulation of cation‐selective channels by muscarinic and alpha 1‐adrenergic receptors in the rabbit portal vein.

1. The excitatory actions of phenylephrine (Phe) and acetylcholine (ACh) on the smooth muscle of rabbit portal vein were investigated and compared by using whole‐cell and single channel configurations of the patch clamp technique, in combination with a modified concentration jump method. 2. At negative holding potentials with KCl (0.1 mM EGTA) electrodes, rapid applications of Phe (> 1 microM) and ACh (> 10 microM) both resulted in biphasic responses consisting of fast outward or inward currents and a long‐lasting inward current with an increased noise level. 3. The slow inward current was still recorded when the cell was dialysed with caesium aspartate solution complemented with 10 mM EGTA, in order to eliminate contributions of calcium‐dependent conductances (K+ and Cl‐ currents). Phe was more potent at activating the current than ACh. Ion replacement experiments revealed that both the Phe‐ and ACh‐induced slow inward currents are cation‐selective conductances (CS currents). 4. The I‐V relationships of the Phe‐ or ACh‐induced CS currents were similar both for the instantaneous peak and the steady state. The tail current analysis over a wide range of membrane potentials (‐150 to +100 mV) showed that depolarizations to very positive potentials (> +50 mV) from near the resting membrane potential (‐40 mV) can produce a several‐fold increase in the steady‐state activation of the CS currents, but the maximal activations were in most cases not observed even at +100 mV. 5. Externally applied Cd2+ produced a quick and reversible inhibition of both the Phe‐ and ACh‐induced CS currents. This inhibition seemed almost voltage independent and the concentrations of half‐inhibition were 100 and 129 microM for Phe and ACh, respectively. 6. Single channel activities were recorded in the presence of Phe or ACh using the outside‐out membrane patches. The unitary conductances and reversal potentials of the Phe‐ and ACh‐activated channels were 23 pS, +5.2 mV and 25 pS, +4.1 mV, respectively, and the open lifetimes evaluated for 50 microM Phe and 500 microM ACh were of similar order (the longer open times at ‐60 mV for Phe and ACh were 4.3 and 4.4 ms, respectively). In addition, the relative open probability (Po,rel) was no more than 0.2 in the voltage range of ‐100 to ‐30 mV for both 50 microM Phe and 500 microM ACh, suggesting low open probabilities of the Phe‐ and ACh‐activated CS channels near the resting membrane potential.(ABSTRACT TRUNCATED AT 400 WORDS)

A newly identified Ca2+ dependent K+ channel in the smooth muscle membrane of single cells dispersed from the rabbit portal vein.

We found a new type of Ca2+-dependent K+ channel in smooth muscle cell membranes of single cells of the rabbit portal vein. A slope conductance of the current was 180 pS when 142 mM K+ solution was exposed to both sides of the membrane (this channel was named the KM channel, in comparison to the known KL and KS channels from the same membrane patch; Inoue et al. 1985). This KM channel was less sensitive to the cytoplasmic Ca2+ concentration, [Ca2+]i, but was sensitive to the extracellular Ca2+, [Ca2+]o, e.g. in the outside-out membrane patch, lowering the [Ca2+]o in the bath markedly reduced the open probability of this channel, and also in cell-attached configuration, lowering of the [Ca2+]o using the internally perfused patch clamp electrode device reduced the opening of KM channel. TEA+ (1–10 mM) reduced the amplitude of the elementary current through the KM channel applied from each side of the membrane, but this agent inhibited the KM channel to a greater extent when applied to the inner than to the outer surface of the membrane. Furthermore, this KM channel had a weak voltage dependency, and the open probability of the channel remained much the same within a wide range of potential (from −60 mV to +60 mV). Whereas most Ca2+-dependent K+ channels are regulated mainly by [Ca2+]i and possess a voltage dependency, these properties of the KM channel differed from other Ca2+-dependent K+ channels. The elucidation of this KM channel should facilitate explanations of the actions of external Ca2+ or TEA+ on the membrane potential, in the smooth muscles of the rabbit portal vein.

Characteristics of cromakalim-induced relaxations in the smooth muscle cells of guinea-pig mesenteric artery and vein.

1 The effects of cromakalim (BRL 34915) on the smooth muscle cells of guinea‐pig mesenteric artery and vein were investigated with microelectrode and tension recording methods. 2 Cromakalim (> 10 μm) produced membrane hyperpolarization with an increase in ionic conductance. The hyperpolarization occurred to a greater extent and lasted longer in the vein than in the artery. 3 The hyperpolarization induced by cromakalim in mesenteric vein comprised two components, one of which was Mn sensitive. In mesenteric artery, the hyperpolarization was relatively insensitive to Mn. 4 From the current‐voltage relationship measured from arterial smooth muscle membranes, the reversal potential of cromakalim was estimated to be −80 mV. The cromakalim‐induced hyperpolarization was not modified in Na‐ or Cl‐deficient solution. 5 In both mesenteric artery and vein, cromakalim relaxed tissues precontracted with high K with (below 40 mm) or without (above 40 mm) hyperpolarization of the membrane. 6 In the mesenteric artery, action potentials evoked by electrical stimulation ceased before the generation of hyperpolarization. 7 Cromakalim produced a cross‐desensitization with nicorandil on the evoked membrane hyperpolarization in mesenteric artery. 8 It is concluded that the relaxing actions of cromakalim result from the hyperpolarization which follows the opening of Ca‐dependent K channels. The inhibition of a voltage‐dependent Ca current may also be involved in this inhibitory effect.

Mechanisms of vasodilation induced by NKH477, a water-soluble forskolin derivative, in smooth muscle of the porcine coronary artery.

To study the mechanism of vasodilation induced by 6-(3-dimethylaminopropionyl) forskolin (NKH477), a water-soluble forskolin derivative, its effects on the acetylcholine (ACh)-induced contraction of muscle strips of porcine coronary artery were examined. [Ca2+]i, isometric force, and cellular concentrations of cAMP and inositol 1,4,5-trisphosphate were measured. NKH477 (0.1-1.0 microM), isoproterenol (0.01-0.1 microM), or forskolin (0.1-1.0 microM) increased cAMP and attenuated the contraction induced by 128 mM K+ or 10 microM ACh in a concentration-dependent manner. These agents, at concentrations up to 0.3 microM, did not change the amount of cGMP. NKH477 (0.1 microM) attenuated the contraction induced by 128 mM K+ without corresponding changes in the evoked [Ca2+]i responses. ACh (10 microM) produced a large phasic increase followed by a small tonic increase in [Ca2+]i and produced a sustained contraction. The ACh-induced phasic increase in [Ca2+]i, but not the tonic increase, disappeared after application of 0.1 microM ionomycin. NKH477 (0.1 microM) attenuated both the increase in [Ca2+]i and the force induced by 10 microM ACh in muscle strips that were not treated with ionomycin and inhibited the ACh-induced contraction without corresponding changes in [Ca2+]i in ionomycin-treated muscle strips. These results suggest that NKH477 inhibits ACh-induced Ca2+ mobilization through its action on ionomycin-sensitive storage sites. In ionomycin-treated and 128 mM K(+)-treated muscle strips, 0.1 microM NKH477 shifted the [Ca2+]i-force relation to the right in the presence or absence of 10 microM ACh. In beta-escin-skinned smooth muscle strips, 0.1 microM NKH477 shifted the pCa-force relation to the right but had no effects on Ca(2+)-independent contraction. We conclude that in smooth muscle of porcine coronary artery, NKH477 inhibits ACh-induced contraction by both attenuating ACh-induced Ca2+ mobilization and reducing the sensitivity of the contractile machinery to Ca2+, possibly by activating cAMP-dependent mechanisms.

Dual action of FRC8653, a novel dihydropyridine derivative, on the Ba2+ current recorded from the rabbit basilar artery.

Actions of FRC8653 on the macroscopic and unitary Ba2+ currents were studied using the rabbit basilar artery. Application of (+/-)-FRC8653 (less than 1 microM) increased the amplitude of the inward current when depolarization pulses more negative than -10 mV were applied but inhibited it when depolarization was more positive than 0 mV (in each case from a holding potential of -80 mV). At a holding potential of -40 mV, (+/-)-FRC8653 (greater than 0.1 nM) consistently inhibited the inward current. (-)-FRC8653 (greater than 1 nM) inhibited the amplitude of the inward current evoked by a depolarizing pulse more positive than -10 mV (the holding potential being -80 mV). At the holding potential of -80 mV, but not at -40 mV, (+)-FRC8653 (1 microM) enhanced the current amplitude evoked by a depolarizing pulse more negative than -10 mV but inhibited the current evoked by a pulse more positive than 0 mV. (+/-)-FRC8653 shifted the voltage-dependent inhibition curves to the left, and the slope of the curve became steeper (test pulse of +10 mV). Two types of single Ca2+ channel currents (12 and 23 pS) were recorded from the basilar artery by the cell-attached patch-clamp method. Opening of the 12-pS channel occurred with a depolarizing pulse (-20 mV) from a holding potential of -80 mV, but not from one of -60 mV. (+)-FRC8653 activated, and (-)-FRC8653 inhibited, the 23-pS channel.(ABSTRACT TRUNCATED AT 250 WORDS)

Factors inducing endothelium‐dependent relaxation in the guinea‐pig basilar artery as estimated from the actions of haemoglobin

1 Factors inducing dilatation of guinea‐pig basilar artery were investigated in intact and endothelium‐denuded tissues by measurement of isometric tension and by electrophysiological methods. 2 The amplitudes of contractions induced by 9,11,epithio‐11,12‐methanothromboxane A2 (STA2) and by high K+ were enhanced by haemoglobin (oxyhaemoglobin, Hb) in a concentration‐dependent fashion (above 1 μm). For the high K+‐induced contraction, the initial tonic component was enhanced to a greater extent than the secondary phasic component. Mechanical responses evoked by STA2 and by high K+ were greater in endothelium‐denuded tissues, but Hb (below 10 μm) had no effect on them. 3 Hb (10 μm) had no effect on the contractile proteins as estimated from the actions of Hb on Ca2+‐induced contractions in skinned muscle tissues. Further, Hb had no effect on the release of Ca2+ from intracellular stores but it accelerated the Ca2+ accumulation into the sarcoplasmic reticulum as judged from the caffeine‐ or STA2‐induced contraction generated in intact tissues. 4 Acetylcholine (ACh) relaxed tissues that were precontracted by STA2 but Hb prevented this relaxation, in a concentration‐dependent fashion. The ACh‐induced relaxation was sustained for over 10 min in the absence of Hb, but following application of Hb, ACh caused only a transient relaxation. 5 STA2 (up to 100 nm) did not modify the resting membrane potential of smooth muscle cells of the basilar artery. ACh (10 μm) caused transient hyperpolarization which was only slightly inhibited by Hb (10 μm) whether or not STA2 was present. The hyperpolarization induced by ACh required the presence of endothelial cells. 6 A23187 (0.01–1 μm) relaxed tissues which were precontracted by STA2, in a concentration‐dependent fashion but had no effect on the membrane potential. 7 These results suggest that in guinea‐pig basilar artery, ACh induces relaxation of tissues that were precontracted by STA2 by causing release of both endothelium‐derived relaxing (EDRF) and endothelial dependent hyperpolarizing factor (EDHF) (sustained and initial transient relaxation, respectively), but via different mechanisms. Hb inhibits the former and to a lesser extent, the latter. Since A23187 produced relaxation of pre‐contracted tissue but caused no detectable change in the membrane potential, this agent may release EDRF but not EDHF.

Effects of tetraethylammonium chloride on the membrane activity of guinea‐pig stomach smooth muscle

1. The effects of tetraethylammonium (TEA) on the membrane activity of the antral circular muscle of the guinea‐pig stomach were investigated with micro‐electrode and double sucrose gap methods.

Characteristic features of noradrenaline-induced Ca2+ mobilization and tension in arterial smooth muscle of the rabbit.

1. Effects of noradrenaline (NAd) on changes in cellular Ca2+ concentration ([Ca2+]i) and tension were investigated, and these effects were compared with those evoked by 128 mM K+ or caffeine in intact smooth muscle strips or by inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3) or caffeine in beta‐escin‐treated chemically skinned smooth muscle strips of the rabbit mesenteric artery. 2. In physiological solution containing 2.6 mM Ca2+, application of 128 mM K+ or 10 microM NAd produced a phasic, followed by a tonic increase in [Ca2+]i and tension. NAd (10 microM) produced a larger tonic tension than did 128 mM K+ but a smaller increase in [Ca2+]i. When the [Ca2+]i‐tension relationship was observed in ionomycin‐ and 128 mM K(+)‐treated muscle strips, 10 microM NAs shifted the relationship to the left and enhanced the maximum amplitude of contraction. These results suggest that NAd increases the sensitivity of contractile proteins to Ca2+ in smooth muscle of the rabbit mesenteric artery. 3. Noradrenaline (10 microM) or caffeine (10 mM), but not 128 mM K+, produced a phasic increase in both [Ca2+]i and tension in Ca(2+)‐free solution containing 2 mM EGTA. When 10 mM caffeine had been applied in Ca(2+)‐free solution, subsequent application of 10 microM NAd did not increase [Ca2+]i. By contrast, when 10 microM NAd had been applied in Ca(2+)‐free solution, subsequent application of 10 mM caffeine still increased [Ca2+]i. Ryanodine (50 microM) abolished the increase in [Ca2+]i induced by 10 mM caffeine or 10 microM NAd in intact and in skinned smooth muscle strips. These results suggest that NAd releases Ca2+ from the ryanodine‐sensitive Ca2+ storage sites. 4. Noradrenaline (10 microM) synthesized Ins(1,4,5)P3 in Ca(2+)‐free solution in intact smooth muscle strips. Following application of 10 microM NAd, a relatively long time lag (around 1 s) was always observed before the initiation of the increase in [Ca2+]i whether in the presence or absence of Ca2+. The maximum rate of rise of [Ca2+]i induced by 10 mM caffeine was much larger than that induced by 10 microM NAd in Ca(2+)‐containing or Ca(2+)‐free solution (containing 2 mM EGTA). Both [Ca2+]i and tension reached their peak in a shorter time with caffeine (10 mM) than with 10 microM NAd. In Beta‐escin‐treated skinned smooth muscle strips, 20 microM Ins(1,4,5)P3 10 mM caffeine or 10 microM NAd increased Ca2+ in Ca(2+)‐free solution following brief application of 0.3 microM Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)