Muscle Membrane Potential Research Articles

Different Effects of Estrogen and Progesterone on K + Currents Expressed in Xenopus Oocytes

Potassium channel plays an active role in the regulation of membrane potential in vascular smooth muscle and vascular tone. These channels not only participate in the physiological responses to endogenously occurring substances, but also become the therapeutic targets for many synthetic drugs. Information regarding the role of K+ channels in vascular effects of female sex steroid hormones is scarce. We previously showed that K+ channel activation contributed in part to the estrogen-mediated vasorelaxation. In order to examine further whether steroid hormones may have direct interaction with K+ channels, we have recently expressed two types of K+ channels, KCa and KV channels in Xenopus oocytes. It was found that 17ß-estradiol increased the large-conductance KCa currents in a concentration-related manner. Tetraethylammonium ions or iberiotoxin inhibited the effect of 17ß-estradiol. KCa current was increased by NS 1619 and inhibited by progesterone. BSA-conjugated estrogen also increased KCa currents. Progesterone reduced the estrogen-stimulated KCa currents. KV1.5 channels were also expressed in Xenopus oocytes and inhibited by 4-aminopyridine. Progesterone reduced the KV1.5 current, while estrogen had little effect. These results showed that estrogen could stimulate KCa channels without an effect on KV1.5 channels. Progesterone inhibited the activity of both KCa and KV1.5 channels expressed in Xenopus oocytes. Progesterone was described to antagonize the vascular action of estrogen. Inhibition of K+ channels may be involved in the reported antagonistic effect of progesterone against the estrogen-induced vasorelaxation. (supported by UPGC Direct Grant)

The Na-K-ATPase is a target for an EDHF displaying characteristics similar to potassium ions in the porcine renal interlobar artery.

The present study was performed to determine the characteristics of the endothelium-derived hyperpolarizing factor (EDHF) that mediates the nitric oxide (NO)- and prostacyclin (PGI2)-independent hyperpolarization and relaxation of porcine renal interlobar arteries. Bradykinin-induced changes in isometric force or smooth muscle membrane potential were assessed in rings of porcine renal interlobar artery preconstricted with the thromboxane analogue U46619 in the continuous presence of N(omega)-nitro-L-arginine and diclofenac to inhibit NO synthases and cyclo-oxygenases. 3 Inhibition of NO- and PGI2-production induced a rightward shift in the concentration-relaxation curve to bradykinin without affecting maximal relaxation. EDHF-mediated relaxation was abolished by a depolarizing concentration of KCl (40 mM) as well as by a combination of charybdotoxin and apamin (each 100 nM), two inhibitors of calcium-dependent K+ (K+(Ca)) channels. Charybdotoxin and apamin also reduced the bradykinin-induced, EDHF-mediated hyperpolarization of smooth muscle cells from 13.7+/-1.3 mV to 5.7+/-1.2 mV. 4 In addition to the ubiquitous alpha1 subunit of the Na-K-ATPase, the interlobar artery expressed the gamma subunit as well as the ouabain-sensitive alpha2, alpha3 subunits. A low concentration of ouabain (100 nM) abolished the EDHF-mediated relaxation and reduced the bradykinin-induced hyperpolarization of smooth muscle cells (13.6+/-2.8 mV versus 5.20+/-1.39 mV in the absence and presence of ouabain). Chelation of K+, using cryptate 2.2.2., inhibited EDHF-mediated relaxation, without affecting NO-mediated responses. Elevating extracellular KCl (from 4 to 14 mM) elicited a transient, ouabain-sensitive hyperpolarization and relaxation that was endothelium-independent and insensitive to charybdotoxin and apamin. 6 These results indicate that in the renal interlobar artery, EDHF-mediated responses display the pharmacological characteristics of K+ ions released from endothelial K+(Ca) channels. Smooth muscle cell hyperpolarization and relaxation appear to be dependent on the activation of highly ouabain-sensitive subunits of the Na-K-ATPase.

Expression of mRNA transcripts for ATP-sensitive potassium channels in human myometrium.

The molecular mechanisms regulating human uterine quiescence and parturition are poorly understood. Potassium channels are central to regulation of cell membrane potential and contractility of smooth muscle. The aim of this study was to examine the expression of ATP-sensitive potassium channel (K(ATP) channel) subunits in human myometrium, and to investigate for possible differential expression of these subunits in myometrium obtained from three different functional states: (i) non-pregnant (NP); (ii) late pregnant not in labour (PNL); and (iii) late pregnant in labour (PL). RT-PCR detected the presence of mRNA for four subunits of K(ATP) channels (Kir6.1, Kir6.2, SUR1 and SUR2B) in the three tissue types. Quantitative analysis of these subunits was achieved with real-time RT-PCR using Lightcycler(TM) technology. This analysis showed that there were significantly higher levels of Kir6.1 and SUR2B transcripts in NP myometrium compared with those measured in myometrium obtained during pregnancy (P < 0.001). Lower levels of Kir6.2 and SUR1 mRNA expression were found, although higher transcript levels in NP myometrium (P < 0.05) were still observed. Our results indicate that the major K(ATP) channel expressed in human myometrium is composed of Kir6.1 and SUR2B, and that down-regulation of this channel may facilitate myometrial function during late pregnancy.

Anthelmintic paraherquamides are cholinergic antagonists in gastrointestinal nematodes and mammals.

Oxindole alkaloids in the paraherquamide/marcfortine family exhibit broad-spectrum anthelmintic activity that includes drug-resistant strains of nematodes. Paraherquamide (PHQ), 2-deoxoparaherquamide (2DPHQ), and close structural analogs of these compounds rapidly induce flaccid paralysis in parasitic nematodes in vitro, without affecting adenosine triphosphate (ATP) levels. The mechanism of action of this anthelmintic class was investigated using muscle tension and microelectrode recording techniques in isolated body wall segments of Ascaris suum. None of the compounds altered A. suum muscle tension or membrane potential. However, PHQ blocked (when applied before) or reversed (when applied after) depolarizing contractions induced by acetylcholine (ACh) and the nicotinic agonists levamisole and morantel. These effects were mimicked by the nicotinic ganglionic blocker mecamylamine, suggesting that the anthelmintic activity of PHQ and marcfortines is due to blockade of cholinergic neuromuscular transmission. The effects of these compounds were also examined on subtypes of human nicotinic ACh receptors expressed in mammalian cells with a Ca2+ flux assay. 2DPHQ blocked nicotinic stimulation of cells expressing alpha3 ganglionic (IC50 approximately 9 microm) and muscle-type (IC50 approximately 3 microm) nicotinic cholinergic receptors, but was inactive at 100 microm vs. the alpha7 CNS subtype. PHQ anthelmintics are nicotinic cholinergic antagonists in both nematodes and mammals, and this mechanism appears to underlie both their efficacy and toxicity.

Effects of histamine on the contractile and electrical activity in isolated lymphatic vessels of the guinea-pig mesentery.

1 The effect of histamine on the rate of lymphatic vessel constrictions and lymphatic smooth muscle membrane potential was examined in the guinea-pig mesentery. 2 Histamine (0.01-5 micro M) increased the frequency and decreased the amplitude of constrictions in lymphatic vessels under intraluminal perfusion. This response was accompanied by a depolarization of the smooth muscle membrane potential, an increase in the activity of spontaneous transient depolarizations (STDs), the proposed pacemaker for constrictions in these vessels, and an increase in the occurrence of action potentials. 3 Responses to histamine were inhibited by the H(1) receptor antagonist pyrilamine (0.2 micro M), but unaffected by NO synthase inhibition with N(G)-nitro L-arginine (L-NOARG, 100 micro M) and lysis of the endothelium. 4 In about 50% of the vessels, a decrease in constriction frequency, STD activity and a smooth muscle hyperpolarization were observed in response to dimaprit (10 micro M), suggesting the presence of H(2) receptors. These vessels had also a significantly lower basal contractile rate. Lymphatic vessel pumping was not affected by R-alpha-methylhistamine (10-50 micro M), ruling out a role for H(3) receptor stimulation in the histamine response. 5 The present results suggest a direct action of histamine on the lymphatic smooth muscle via stimulation of H(1) (and in some vessels H(2)) receptors. H(1) receptors enhance and H(2) receptors slow down lymphatic pumping, the dominant effect being an increased contractile activity. Correlation of these effects with histamine-induced changes in membrane potential and STD activity suggests the involvement of these electrical changes in the initiation of the contractile response.

Effects of chloride transport on bistable behaviour of the membrane potential in mouse skeletal muscle.

The lumbrical skeletal muscle fibres of mice exhibited electrically bistable behaviour due to the nonlinear properties of the inwardly rectifying potassium conductance. When the membrane potential (V(m)) was measured continuously using intracellular microelectrodes, either a depolarization or a hyperpolarization was observed following reduction of the extracellular potassium concentration (K+o) from 5.7 mM to values in the range 0.76-3.8 mM, and V(m) showed hysteresis when K+o was slowly decreased and then increased within this range. Hypertonicity caused membrane depolarization by enhancing chloride import through the Na+-K+-2Cl- cotransporter and altered the bistable behaviour of the muscle fibres. Addition of bumetanide, a potent inhibitor of the Na+-K+-2Cl- cotransporter, and of anthracene-9-carboxylic acid, a blocker of chloride channels, caused membrane hyperpolarization particularly under hypertonic conditions, and also altered the bistable behaviour of the cells. Hysteresis loops shifted with hypertonicity to higher K+o values and with bumetanide to lower values. The addition of 80 microM BaCl2 or temperature reduction from 35 to 27 degrees C induced a depolarization of cells that were originally hyperpolarized. In the K+o range of 5.7-22.8 mM, cells in isotonic media (289 mmol x kg(-1)) responded nearly Nernstianly to K+o reduction, i.e. 50 mV per decade; in hypertonic media this dependence was reduced to 36 mV per decade (319 mmol x kg(-1)) or to 31 mV per decade (340 mmol x kg(-1)). Our data can explain apparent discrepancies in DeltaV(m) found in the literature. We conclude that chloride import through the Na+-K+-2Cl- cotransporter and export through Cl- channels influenced the V(m) and the bistable behaviour of mammalian skeletal muscle cells. The possible implication of this bistable behaviour in hypokalaemic periodic paralysis is discussed.

Potassium Channels and Human Corporeal Smooth Muscle Cell Tone: Diabetes and Relaxation of Human Corpus Cavernosum Smooth Muscle by Adenosine Triphosphate Sensitive Potassium Channel Openers

Sustained contraction of human corporeal smooth muscle depends on continuous transmembrane calcium flux through voltage gated calcium channels. K channels modulate corporeal smooth muscle membrane potential and, thus, ultimately affect transmembrane calcium flux. Therefore, we characterized relaxation responses elicited by the K channel modulators pinacidil and levcromakalim on isolated human corporeal tissue strips. We also evaluated the possibility that there may be alterations in adenosine triphosphate sensitive K channel pharmacology/function related to the presence of diabetes mellitus. A total of 215 isolated human corporeal tissue strips obtained from 57 male patients with organic erectile dysfunction were investigated. Cumulative concentration-response curves were constructed at half log increments for steady state relaxation responses elicited by pinacidil and levcromakalim on equivalently phenylephrine pre-contracted (to approximately 75% of maximum) isolated corporeal tissue strips. Potassium currents were measured using the cell attached whole cell patch clamp technique on freshly isolated corporeal smooth muscle cells. A concentration dependent, glibenclamide sensitive relaxation response of phenylephrine pre-contracted corporeal tissue strips was observed for pinacidil and levcromakalim. Consistent with such observations, electrophysiological recordings on freshly isolated myocytes revealed that pinacidil (10 microM.) and levcromakalim (10 microM.) induced whole cell potassium currents that were blocked by glibenclamide (10 microM.). In addition, statistical analysis revealed that phenylephrine pre-contracted corporeal tissue strips from patients without diabetes were more sensitive to relaxation by both compounds than corporeal tissue strips excised from those with diabetes. Furthermore, relaxation responses elicited by pinacidil and levcromakalim were not affected by charybdotoxin or 4-aminopyridine but were completely reversed by KCl or tetraethylammonium chloride. These data indicate that the adenosine triphosphate sensitive K channel subtype is likely to have an important role in the relaxation of isolated corporeal tissue strips and, moreover, they are the molecular target for the K channel modulators/openers levcromakalim and pinacidil. Such observations are consistent with the supposition that alterations in the structure/function/activity of these potassium channels may underlie at least some aspects of observed diabetes related differences in tissue sensitivity to K channel modulators.

Swimming and ammonia toxicity in salmonids: the effect of sub lethal ammonia exposure on the swimming performance of coho salmon and the acute toxicity of ammonia in swimming and resting rainbow trout

This study tested the hypothesis that swimming exacerbates ammonia toxicity in fish. Both sub-lethal and acute toxicity testing was conducted in a swim tunnel on swimming and resting coho salmon and rainbow trout, respectively. The sub lethal tests on coho salmon also considered the compartmentalization of ammonia within the fish. Coho salmon showed a significant linear decrease in U crit both with increasing water ammonia (0, 0.02, 0.04 and 0.08 mg per l NH 3) and increasing plasma ammonia. Data collected included plasma pH and ammonia, muscle pH and ammonia and muscle membrane potential. Based on results found in these experiments it was concluded that the reduction in swimming performance was due to both metabolic challenges as well as depolarization of white muscle. Acute toxicity testing on swimming and resting rainbow trout revealed that swimming at (60% U crit or approximately 2.2 body lengths/s) decreased the LC 50 level from 207±21.99 mg N per l in resting fish to 32.38±10.81. The LC 50 for resting fish was significantly higher than that for swimming fish. The acute value set forth by the US EPA at the same pH is 36.1 mg N per l and may not protect swimming fish. In addition the effect of water hardness on ammonia toxicity was considered. It was found that increased water calcium ameliorates ammonia toxicity in fish living in high pH water.

Inhibitory effects of protein kinase C on inwardly rectifying K+- and ATP-sensitive K+ channel-mediated responses of the basilar artery.

The structurally related, inwardly rectifying K+ (K(IR)) channel and the ATP-sensitive K+ (K(ATP)) channel are important modulators of cerebral artery tone. Although protein kinase C (PKC) activators have been shown to inhibit these channels with the use of patch-clamp electrophysiology, effects of PKC on K+ channel function in intact cerebral blood vessels are unknown. We therefore tested whether pharmacological alteration of PKC activity affects cerebral vasodilator responses to K(IR) and/or K(ATP) channel activators in vivo. We measured changes in basilar artery diameter using a cranial window preparation in anesthetized rats. In addition, intracellular recordings of smooth muscle membrane potential were made in isolated basilar arteries. K+ (5 to 15 mmol/L) and aprikalim (1 to 10 micromol/L) each elicited reproducible vasodilatation. The PKC activator phorbol-12,13-dibutyrate (PdBu) (50 nmol/L) inhibited responses to K+ (by 40% to 55%) and aprikalim (by 40% to 70%), whereas responses to papaverine were unaffected. The PKC inhibitor calphostin C (0.1 micromol/L) augmented responses to K+ (by 2- to 3-fold) and aprikalim (2-fold) but not papaverine. In addition, K+ (5 mmol/L) and aprikalim (3 micromol/L) each hyperpolarized the basilar artery. PdBu inhibited these responses to aprikalim by 45% but had no effect on K+-induced hyperpolarization. These data suggest that both basal and stimulated PKC activity inhibit K(IR) and K(ATP) channel-mediated cerebral vasodilatation in vivo. The inhibitory effect on K(ATP) channel-mediated vasodilatation occurs at least partly by inhibition of hyperpolarization mediated by K(ATP) channels. PKC inhibits K+-induced vasodilatation without affecting hyperpolarization, suggesting that the inhibitory effect of PKC on vasodilator responses to K+ does not involve altered K(IR) channel function.

Long-chain fatty acids increase basal metabolism and depolarize mitochondria in cardiac muscle cells.

The effects of long-chain (LC) fatty acids on rate of heat production (heat rate) and mitochondrial membrane potential (DeltaPsi) of intact guinea pig cardiac muscle were investigated at 37 degrees C. Heat rate of ventricular trabeculae was measured with microcalorimetry, and DeltaPsi was monitored in isolated ventricular myocytes with either JC-1 or tetramethylrhodamine ethyl ester (TMRE). Methyl-beta-cyclodextrin was used as fatty acid carrier. Application of 400 microM oleate or linoleate increased resting heat rate by approximately 30% and approximately 25%, respectively. When LC fatty acid was supplied as sole metabolic substrate, resting heat rate was decreased by 3-mercaptopropionic acid. In TMRE-loaded myocytes, neither 40-80 microM oleate nor 40 microM linoleate affected DeltaPsi. At a higher concentration (400 microM) both oleate and linoleate increased TMRE fluorescence by approximately 20% of maximum, obtained using 2,4-dinitrophenol (100 microM), indicating a depolarization of the inner mitochondrial membrane. We conclude that LC fatty acids, at sufficiently high concentration, increase heat rate and decrease DeltaPsi in intact cardiac muscle, consistent with a protonophoric uncoupling action. These effects may contribute to the high metabolic rate after reperfusion of postischemic myocardium.

Oxidative stress and potassium channel function.

1. Modulation of K+ channel activities by cellular oxidative stress has emerged as a significant determinant of vasomotor function in multiple disease states. 2. Evidence from in vitro and in vivo studies suggest that superoxide (O2-) and hydrogen peroxide (H2O2) enhance BKCa channel activity in rat and cat cerebral arterioles; however, activity is decreased by peroxynitrite (ONOO-) in rat cerebral arteries. The mechanisms of changes in BKCa channel properties are not fully understood and may involve oxidation of cysteine residues that are located in the cell membranes. 3. Studies further suggest that O2- increases KATP channel activity in guinea-pig cardiac myocytes, but decreases opening in cerebral vasculature. Both H2O2 and ONOO- enhance KATP channel activity in the myocardium and in coronary, renal, mesenteric and cerebral vascular beds. Alteration of KATP channels by free radicals may be due to oxidation of SH groups or changes in the cytosolic concentration of ATP. 4. It does appear that O2- produced by either reaction of xanthine and xanthine oxidase or elevated levels of glucose reduces Kv channel activity and the impairments can be partially restored by free radical scavengers, superoxide dismutase and catalase. 5. Thus, redox modulation of potassium channel activity is an important mechanism regulating cell vascular smooth muscle membrane potential.

THE IMPACT OF VITAMIN C ON DIABETES INDUCED ALTERATIONS AT MURINE NEUROMUSCULAR JUNCTION

Physiological functions of skeletal muscle are compromised in diabetes. This may involve free radical mechanisms and may be reversed by antioxidants. We have studied effects of vitamin C on twitch tension, resting membrane potential (RMP) and miniature endplate potentials (MEPPs) frequencies in dorsiflexor muscle of diabetic murine. Forty mice were divided randomly into 2 groups (n=20 each). One group served as control and the other was injected once with streptozotocin (STZ) solution (60 mg/kg, i.p) to induce diabetes. The animals were then divided further into two subgroups (n=10 each). Vitamin C (200 mg/kg, i.p) was administered daily to one control and one diabetic group for three weeks prior to recording day. Experiments were conducted four weeks following diabetes induction. Isometric twitch tension (evoked directly by muscle stimulation and indirectly by nerve stimulation) was measured in urethane anesthetized (2 mg/g, i.p) mice via a transducer connected to computer system. Utilizing intracellular recording method, resting membrane potential RMP and MEPPs frequencies were also measured. Compared to control, diabetic mice showed reduced twitch tension (4.2±0.5 g control versus 2.6±0.2 g diabetic) and demonstrated delayed half time of decay. Diabetic flexor muscle also displayed significant reduction in MEPPs frequencies with no changes in RMP. Vitamin C reversed tension reduction in diabetic mice (from 2.6±0.2 g to 3.9±0.3 g), impacted delayed half time of decay and increased MEPPs frequencies. Vitamin C improves diabetes-induced nerve and muscle dysfunction possibly via a free radical scavenging mechanism.

Toxic concentrations of selenite shortens repolarization phase of action potential in rat papillary muscle.

Selenium (Se) has long been recognized as both an essential mammalian nutrient and a hazardous element. Sodium selenite is commonly used as a dietary supplement for the treatment of Se deficiency. On the other hand, chronic Se toxicity has been demonstrated to affect the major organs, including the heart, in experimental animals. This study examines the effects of high concentrations of extracellular selenite (in the range of 0.001-1 mM) application into the recording bathon the electrical properties of rat papillary muscles. Conventional glass semifloating microelectrodes were used to record intracellular action potentials (APs) in isolated rat papillary muscles. The amplitude of APs and the resting membrane potential of papillary muscles were not changed following a 20-min selenite (1 mM) application compared to the first minute of its application. Freshly isolated ventricular myocytes by an enzymatic method were used to determine the selenite-induced alterations in Na+ currents. Na+ currents, measured at 22 degrees C, by the whole-cell patch-clamp technique, decreased by 38 +/- 8% in the presence of 1 mM selenite for 5 min. These selenite-induced effects were not reversed, but are restored by dithiothreitol (1 mM). These results demonstrated that toxic concentrations of selenite induced a significant shortening in AP duration as a result of an increase in the rate of repolarization. Our findings also suggest that a decrease in Na+ currents, in addition to Ca2+ currents, may play a role in the shortening of AP duration in rat papillary muscles.

β1‐Subunit of the Ca2+‐activated K+ channel regulates contractile activity of mouse urinary bladder smooth muscle

1. The large-conductance calcium-activated potassium (BK) channel plays an important role in controlling membrane potential and contractility of urinary bladder smooth muscle (UBSM). These channels are composed of a pore-forming alpha-subunit and an accessory, smooth muscle-specific, beta1-subunit. 2. Our aim was to determine the functional role of the beta1-subunit of the BK channel in controlling the contractions of UBSM by using BK channel beta1-subunit 'knock-out' (KO) mice. 3. The beta-galactosidase reporter (lacZ gene) was targeted to the beta1 locus, which provided the opportunity to examine the expression of the beta1-subunit in UBSM. Based on this approach, the beta1-subunit is highly expressed in UBSM. 4. BK channels lacking beta1-subunits have reduced activity, consistent with a shift in BK channel voltage/Ca2+ sensitivity. 5. Iberiotoxin, an inhibitor of BK channels, increased the amplitude and decreased the frequency of phasic contractions of UBSM strips from control mice. 6. The effects of the beta1-subunit deletion on contractions were similar to the effect of iberiotoxin on control mice. The UBSM strips from beta1-subunit KO mice had elevated phasic contraction amplitude and decreased frequency when compared to control UBSM strips. 7. Iberiotoxin increased the amplitude and frequency of phasic contractions, and UBSM tone of UBSM strips from beta1-subunit KO mice, suggesting that BK channels still regulate contractions in the absence of the beta1-subunit. 8. The results indicate that the beta1-subunit, by modulating BK channel activity, plays a significant role in the regulation of phasic contractions of the urinary bladder.

Osmolality influences bistability of membrane potential under hypokalemic conditions in mouse skeletal muscle: an experimental and theoretical study

The membrane potential in mouse skeletal muscle depends on both extracellular osmolality and potassium concentration. These dependencies have been related to two membrane transporters, Na +/K +/2Cl − co-transporter and the inward potassium rectifier channel. To investigate the relation of the Na +/K +/2Cl − co-transporter and the inward potassium rectifier channel in a qualitative way, a combined electrophysiological and modelling approach was used. The experimental results show that the bistability of the membrane potential, which is related to the conductive state of the inward potassium rectifier channel, is shifted to higher extracellular potassium values when medium osmolality is increased. These results are confirmed by the computer simulation calculations for increased co-transporter flux. The combined results indicate that the co-transporter is capable of modulating the conductive state of the inward potassium rectifier channel.

Axial stretching of extremity artery induces reversible hyperpolarization of smooth muscle cell membrane in vivo.

Circumferential stretch due to increases in pressure induces vascular smooth muscle cell depolarization and contraction known as the myogenic response. The aim of this study was to determine the in vivo effects of axial-longitudinal stretch of the rat saphenous artery (SA) on smooth muscle membrane potential (Em) and on external diameter. Consecutive elongations of the SA were carried out from resting length (L0) in 10% increments up to 140% L0 while changes in membrane potential and diameter were determined in intact and de-endothelized vessels. Axial stretching resulted in a small initial depolarization at 120% of L0 followed by a progressive 20 to 33% hyperpolarizaion of vascular smooth muscle between 130% and 140% of L0. At 140%, an average maximal 10.6 mV reversible hyperpolarization was measured compared to -41.2 +/- 0.49 mV Em at 100% L0. De-endothelialization completely eliminated the hyperpolarization to axial stretching and augmented the reduction of diameter beyond 120% L0. These results indicate that arteries have a mechanism to protect them from vasospasm that could otherwise occur with movements of the extremities.

Properties of smooth muscle hyperpolarization and relaxation to K+ in the rat isolated mesenteric artery.

Smooth muscle membrane potential and tension in rat isolated small mesenteric arteries (inner diameter 100-200 microm) were measured simultaneously to investigate whether the intensity of smooth muscle stimulation and the endothelium influence responses to exogenous K+. Variable smooth muscle depolarization and contraction were stimulated by titration with 0.1-10 microM phenylephrine. Raising external K+ to 10.8 mM evoked correlated, sustained hyperpolarization and relaxation, both of which were inhibited as the smooth muscle depolarized and contracted to around -38 mV and 10 mN, respectively. At these higher levels of stimulation, raising the K+ concentration to 13.8 mM still hyperpolarized and relaxed the smooth muscle. Relaxation to endothelium-derived hyperpolarizing factor, released by ACh, was not altered by the level of stimulation. In endothelium-denuded arteries, the concentration-relaxation curve to K+ was shifted to the right but was not depressed. In denuded arteries, relaxation to K+ was unaffected by the extent of prior stimulation and was blocked with 0.1 mM ouabain but not with 30 microM Ba2+. The ability of K+ to stimulate simultaneous hyperpolarization and relaxation in the mesenteric artery is consistent with a role as an endothelium-derived hyperpolarizing factor activating inwardly rectifying K+ channels on the endothelium and Na+-K+-ATPase on the smooth muscle cells.

Differential expression of voltage-gated K(+) channel genes in arteries from spontaneously hypertensive and Wistar-Kyoto rats.

Voltage-gated K(+) currents play an important role in determining membrane potential, intracellular Ca(2+), and contraction in arterial smooth muscle. In this study, the expression of genes encoding voltage-gated K(+) channels of the Kv1.X family was compared in arteries from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Expression of Kv1.X in thoracic aorta, mesenteric arteries, tail artery, and heart was determined, both qualitatively and quantitatively, by reverse transcription-polymerase chain reaction. Our results demonstrate distinct but overlapping patterns of expression in vascular tissues. In general, Kv1.2 and Kv1.5 were most highly represented, and the levels of Kv1.2 were significantly larger in all tissues from SHR. Levels of Kv1.5 in arteries did not differ significantly between strains but were greater in SHR heart. Moderate levels of Kv1.3 and Kvbeta1.1 expression were also found in all tissues and were larger in SHR. Kv1.1 expression was not different between the 2 strains, and no significant expression of Kv1.4 (except in heart and aorta), Kv1.6, or Kvbeta2.1 was observed in either strain. Kv1.2 and Kv1.5 transcripts represent approximately 1 to 2 parts/10(5) of total mesenteric arterial RNA with approximately 2- to 5-fold lower levels in aorta and tail artery. Whole-cell voltage-gated K(+) channel currents, recorded from mesenteric arterial myocytes, were larger in SHR than WKY (eg, at 0 mV: 7.3+/-0.8 versus 10.9+/-1.2 pA/pF). The voltage dependence of activation was more negative in SHR (V(0.5): -20+/-4 mV versus -32+/-3 mV) but that of availability was not different. These results indicate that Kv1.X genes are differentially expressed between WKY and SHR (especially Kv1.2 and Kvbeta1.1). These differences in gene expression are associated with a greater voltage-gated K(+) channel current density in SHR and shifted voltage-dependent activation compared with WKY. These differences may be a compensatory mechanism related to the membrane potential depolarization in SHR or some manifestation thereof.

Modulation of potassium current characteristics in human myometrial smooth muscle by 17beta-estradiol and progesterone.

The K(+) channel currents are important modulators of smooth muscle membrane potential and excitability. We assessed whether voltage-gated K(+) currents from human myometrium are regulated by placental steroid hormones during pregnancy and labor. Pregnant human myometrial cells were isolated from samples obtained at cesarean section. Primary cultured cells were treated with 100 nM 17beta-estradiol, 1 microM progesterone, or both hormones in combination for 24 h. Acute effects of the two hormones were also determined. The K(+) currents were recorded using the standard whole-cell, patch-clamp technique. Primary cultures possessed both delayed rectifier (I(KV)) and A-like (I(KA)) voltage-gated K(+) currents. The 24-h 17beta-estradiol treatment caused a hyperpolarizing shift in the steady-state inactivation of both I(KV) and I(KA). Progesterone treatment also shifted the inactivation of I(KA) and increased I(KV) amplitude by 60%-110%. Conversely, the combined treatment had no effect on these currents. Neither 17beta-estradiol (0.1-1 microM) nor progesterone (1-5 microM) had any effect on the K(+) current when applied acutely. These results show that 17beta-estradiol should inhibit myometrial K(+) channel activity, whereas progesterone is likely to have the opposite effect. These results are consistent with the respective procontractile and proquiescence roles for 17beta-estradiol and progesterone in human uterus during pregnancy.

Differences in electromechanical coupling between bradykinin and the nonpeptide kinin B2 receptor agonist, FR 190997, in the circular muscle of guinea-pig colon.

We have compared the effect of bradykinin (BK) and the nonpeptide kinin B2 receptor agonist, FR 190997, in producing changes in membrane potential and tension in the circular muscle of guinea-pig colon by the sucrose gap technique. In the presence of atropine (1 microM), S-ketoprofen (3 microM) and apamin (0.1 microM), BK (1 microM for 20 s) induced a transient depolarization of the membrane with superimposed action potentials (spikes) and transient contraction. Nifedipine (1 microM) eliminated the spikes and markedly inhibited the BK-induced contractions. FR 190997 (3-10 microM for 20 s) induced a slowly developing sustained small depolarization associated with a slowly developing and sustained contraction but, contrary to BK, FR 190997 was unable to trigger spikes. Nifedipine had no effect on depolarization and contraction induced by FR 190997. In the presence of 1 microM nifedipine, the combined application of a blocker of receptor-operated cation channels, SKF 96365 (50 microM for 30 min), and of an inhibitor of sarcoplasmic reticulum calcium pump, cyclopiazonic acid (CPA 10 microM for 30 min), reduced the BK-induced depolarization and contraction by about 45%-60%. The same treatment induced about 40% reduction of the sustained contraction induced by FR 190997, whereas the concomitant depolarization was not significantly affected. The tyrosine kinase inhibitor genistein (40 microM for 20 min) had no effect on the BK- and FR 190997-induced depolarization and contraction in the presence of nifedipine. In radioligand binding experiments performed in membranes of colonic smooth muscle cells, both agonists displaced the [3H]BK specific binding with a pIC50 of 9.6 and 8.5 for BK and FR 190997, respectively. These findings indicate a substantial qualitative difference in mechanisms of excitation contraction coupling activated by BK and FR 190997 via B2 receptors in guinea-pig colon.