Alteration of the transmembrane K+ gradient during development of delayed rectifier in isolated rat pulmonary arterial cells.

The properties of the tail current associated with the delayed rectifier K+ current (IK) in isolated rat pulmonary artery smooth muscle cells were examined using the whole cell patch clamp technique. The tail currents observed upon repolarization to -60 mV after brief (e.g., 20 ms) or small (i.e. to potentials negative of 0 mV) depolarizations were outwardly directed, as expected given the calculated K+ reversal potential of -83 mV. The tail currents seen upon repolarization after longer (e.g., 500 ms) and larger (e.g., to +60 mV) depolarizations tended to be inwardly directed. Depolarizations of intermediate strength and/or duration were followed by biphasic tail currents, which were inwardly directed immediately upon repolarization, but changed direction and became outwardly directed before deactivation was complete. When cells were depolarized to +60 mV for 500 ms both IK and the subsequent inward tail current at -60 mV were similarly blocked by phencyclidine. Both IK and the inward tail current were also blocked by 4-aminopyridine. Application of progressively more depolarized 30 s preconditioning potentials inactivated IK, and reduced the inward tail current amplitude with a similar potential dependency. These results indicated that the inward tail current was mediated by IK. The reversal potential of the tail current became progressively more positive with longer depolarizations to +60 mV, shifting from -76.1 +/- 2.2 mV (n = 10) after a 20-ms step to -57.7 +/- 3.5 mV (n = 9) after a 500-ms step. Similar effects occurred when extracellular K+ and Na+ were replaced by choline. When extracellular K+ was raised to 50 mM, the tail current was always inwardly directed at -60 mV, but showed little change in amplitude as the duration of depolarization was increased. These observations are best explained if the dependencies of tail current direction and kinetics upon the duration of the preceding depolarization result from an accumulation of K+ at the external face of the membrane, possibly in membrane invaginations. A mathematical model which simulates the reversal potential shift and the biphasic kinetics of the tail current on this basis is presented.

Intracellular Ca2+ release in cerebral arteries.

Vasoconstricting agonists elevate the intracellular Ca2+ concentration and induce tension development in vascular smooth muscle cells by inducing both Ca2+ influx from the extracellular space and Ca2+ release from cellular stores. The relative importance of Ca2+ release has been found to vary between different sites in the vasculature. This review examines the role of Ca2+ release in the activation of cerebral arteries produced by several vasoconstricting stimuli. Although the activation of cerebral arteries by agonists such as 5-hydroxytryptamine and noradrenaline has typically been found to have little dependence on Ca2+ release, other vasoconstrictors such as thromboxane A2, which may be released from the endothelium by other agonists, appear to induce a substantial intracellular Ca2+ release in cerebral arteries. The limited efficacy of Ca2+ influx blockers in the treatment of delayed cerebrovascular constriction occurring as a result of subarachnoid haemorrhage suggests that intracellular mechanisms such as Ca2+ release and/or the activation of protein kinase C may be important determinants of vasoconstriction under pathological conditions.

A role for a glibenclamide-sensitive, relatively ATP-insensitive K+ current in regulating membrane potential and current in rat aorta.

OBJECTIVE: ATP-sensitive K+ channels have been classified based on their inhibition by cytoplasmic ATP. Recent evidence in vascular smooth muscle has suggested that these channels show weak sensitivity to intracellular ATP. However, it is not known whether these channels regulate the resting K+ conductance in vascular smooth muscles. Therefore, the aim of the present investigation was to characterize this current in rat aorta myocytes and to examine whether it contributes to setting the membrane potential. METHODS: The conventional and nystatin-permeablised whole cell patch clamp techniques were used to characterize the effect of glibenclamide on membrane potential and K+ current in enzymatically dispersed rat aorta myocytes. RESULTS: The mean resting potential measured in current clamp mode using the permeabilized patch approach was -54 +/- 5 mV (n = 8). Glibenclamide (10 microM) caused a reversible 24-mV depolarization in these cells. In symmetrical K+ (135 mM) solution an inward glibenclamide-sensitive (10 microM) current (-4.1 +/- 0.7 pA/pF; n = 5), hereafter termed Iglib, was observed at a membrane potential of -80 mV when cells held at -60 mV were ramped from -80 to +80 mV. In the absence of any nucleotide in the pipette solution, Iglib measured by the conventional whole-cell method was -23.69 +/- 4.65 pA/pF (n = 9). With 1 and 3 mM ATP in the pipette, the average current density was -25 +/- 6.3 pA/pF (n = 8), and -9.4 +/- 2.7 pA/pF (n = 9), respectively. In the absence of ATP, 1 mM GDP significantly (P < 0.01) increased Iglib (-44.8 +/- 8.4 pA/pF; n = 13). Inclusion of 1 mM ATP in the GDP-containing pipette solution had no significant effect on the current amplitude (-56.4 +/- 10.7 pA/pF; n = 7). Iglib fell to -11.0 +/- 2.9 pA/pF (n = 10) if 1 mM GDP and 3 mM ATP were present. In symmetrical K+, the Iglib observed in the presence of 1 mM ATP in the pipette was increased by more than two-fold in the presence of 10 microM levcromakalim. In PSS containing 5 mM K+, a significant glibenclamide-sensitive current was observed at -45 mV membrane potential when cells dialyzed with 1 mM ATP were ramped between -80 to 30 mV. CONCLUSION: These results demonstrate that Iglib channels in rat aorta myocytes differ from classical KATP channels, being relatively insensitive to intracellular ATP. Iglib therefore appears to have an important role in contributing to the maintenance of the resting potential in rat aortic smooth muscle.

Hypoxia induces the release of a pulmonary-selective, Ca(2+)-sensitising, vasoconstrictor from the perfused rat lung.

OBJECTIVE: Sustained hypoxic pulmonary vasoconstriction is dependent upon the presence of an intact endothelium, strongly suggesting that an endothelium-derived constrictor factor is involved in this response. In the present study we have attempted to determine whether hypoxia induces the release of a vasoconstrictor(s) from the lung, and whether this vasoconstrictor shares mechanistic features with the hypoxic constrictor response. METHODS: The salt-perfused rat lung, coupled with a simple solid-phase extraction process, and a rat intrapulmonary artery functional bioassay were utilised in this study. RESULTS: Hypoxic, but not normoxic, perfusion of the isolated lung of the rat induced the release of a vasoconstrictor(s) which appeared to be selective for pulmonary over mesenteric arteries of the rat. The vasoconstriction observed was unaffected by inhibition of voltage-gated Ca(2+) channels, and was not associated with a rise in intracellular [Ca(2+)], suggesting Ca(2+)-sensitisation of the contractile apparatus. The vasoconstriction was also unaffected by the protein kinase C (PKC) inhibitor Ro-31-8220, or the endothelin-1 antagonists BQ123/BQ788 but was markedly potentiated in the presence of prostaglandin F(2alpha). CONCLUSION: We conclude that hypoxic perfusion of the rat lung results in the release of a vasoconstrictor(s) which shares some of the facets of the sustained hypoxic constriction of isolated intrapulmonary arteries of the rat, since it involves PKC-independent Ca(2+) sensitisation, is independent of voltage-gated Ca(2+) entry, and is potentiated by the presence of preconstriction.

Endothelium-dependent relaxation of rat aorta and main pulmonary artery by the phytoestrogens genistein and daidzein.

OBJECTIVE: The dietary phytoestrogens genistein and daidzein have been shown to relax agonist-preconstricted arteries in vitro; the mechanisms of relaxation remain incompletely understood. This study aimed to determine whether the relaxation of phenylephrine (PE)-constricted rat aorta and main pulmonary artery by genistein and daidzein was endothelium-dependent. METHODS: Effects of endothelial-denudation, and pretreatment with with 100 microM L-N(G)-nitroarginine methyl ester (L-NAME) and/or 10 microM indomethacin on relaxation of PE (1 microM)-preconstricted contractures by genistein (1-100 microM) and daidzein (3-100 microM) were assessed by measuring isometric force development by rat arterial rings. The effect of L-NAME on relaxation to 17beta-estradiol (10 microM) was also measured in aorta. RESULTS AND CONCLUSIONS: Genistein and daidzein caused concentration-dependent relaxation of aorta rings preconstricted with PE (1 microM). The IC50 values were 5.7 microM (n=8, 95% confidence limits 4.3-7.7 microM) and 36.7 microM (n=12, 95% confidence limits 25.7-44.1 microM), respectively. Removal of the endothelium and pretreatment with L-NAME (100 microM) significantly inhibited relaxation at 3, 10 and 30 microM genistein and 10 and 30 microM daidzein. The contracture evoked in rat aorta by depolarization with 75 mM K+ solution was similarly relaxed by genistein in a partially endothelium-dependent manner. 17Beta-estradiol (10 microM) caused a 48.7+/-5.0% (n=11) relaxation of the PE contracture, which was significantly reduced to 25.1+/-5.3% (n=7) by L-NAME. Relaxations brought about by 17beta-estradiol, genistein, and daidzein were not significantly affected by the genomic estrogen receptor antagonist ICI 182,780 (10 microM). Similar endothelium-dependent effects of genistein were observed in the main pulmonary artery. The results show that the relaxation of these rat arteries by concentrations of genistein and daidzein which overlap those present in human plasma after ingestion of soybean-containing meals is largely endothelium dependent.

Calcium antagonistic properties of the cyclooxygenase-2 inhibitor nimesulide in human myometrial myocytes.

The non-steroidal anti-inflammatory drug nimesulide is a selective inhibitor of cyclooxygenase-2 which relaxes spontaneously contracting human myometrium in vivo and is potentially a useful tocolytic drug. Part of the relaxant action of nimesulide may be via block of myometrial Ca2+ channels. Here, we describe the Ca2+ channel blocking properties of nimesulide in freshly dispersed human term-pregnant myometrial smooth muscle cells (HMSMCs). Both L- and T-components of the whole cell Ca2+ channel current were inhibited by 100 microM nimesulide (38+/-3 and 35+/-1% block, respectively). At physiological pH inside and outside the cell (pHo/pHi = 7.4/7.2), this block did not depend on the holding or test potential, although a degree of use-dependence was observed during high frequency stimulation at a higher concentration of drug (300 microM). At pHo/pHi = 6.8, under which condition the concentration of the non-ionized form of the drug is increased 3 fold compared to pH 7.4, nimesulide blocked the L-type current more potently (58+/-3% inhibition at 100 microM, P<0.01) compared to physiological pH. Nimesulide caused a 7 mV leftward shift in the availability curve of the current at pH 6.8, suggesting that the affinity of the drug for the inactivated channel is approximately 4 fold higher than its affinity for the closed channel. We speculate that acidification and depolarization of the myometrium during the intense and prolonged contractions of labour might increase the potency of nimesulide as a Ca2+ channel antagonist, promoting its action as a tocolytic agent.

Mechanism of butyrate-induced vasorelaxation of rat mesenteric resistance artery.

1. The vasorelaxant effect of the sodium salt of the short chain fatty acid, butyrate, on preconstricted rat small mesenteric arteries (mean inner diameter approximately 300 microns) was characterized. Isometric force development was measured with a myograph, and intracellular pH (pHi) was simultaneously monitored, in arteries loaded with the fluorescent dye BCECF in its acetomethoxy form. Sodium butyrate (substituted isosmotically for NaCl) was applied to arteries after noradrenaline (NA) or high K+ contractures were established. 2. Arteries preconstricted with a concentration of NA inducing an approximately half maximal contraction were relaxed by 91.5 +/- 6.3% by 50 mmol l-1 butyrate. This concentration of butyrate did not, however, cause a significant relaxation of contractures to a maximal (5 mumol l-1) NA concentration, and also failed to relax significantly contractures stimulated by high (45 and 90 mmol l-1) K+ solutions. Contractures elicited with a combination of NA (at a submaximal concentration) and 45 mmol l-1 K+ were, however, markedly relaxed by butyrate. 3. Investigation of the concentration-dependency of the butyrate-induced relaxation of the half maximal NA response revealed an EC50 for butyrate of approximately 22 mmol l-1. 4. Sodium butyrate (50 mmol l-1) caused pHi to decrease from 7.25 +/- 0.02 to 6.89 +/- 0.08 (n = 4, P < 0.001). However, the vasorelaxant effect of butyrate on the submaximal NA contracture was not significantly modified when this fall in intracellular pH was prevented by the simultaneous application of NH4Cl. 5. Butyrate-induced relaxation was also unaffected by endothelial denudation and inhibition of NO synthase with N omega-nitro-L-arginine methyl ester (100 mumol l-1). 6. The relaxation of the NA contracture by 50 mmol l-1 sodium butyrate was abolished in arteries pretreated with the cyclic AMP antagonist Rp-cAMPS (25 mumol l-1). 7. We conclude that the butyrate-induced relaxation of the NA contracture is independent of intracellular acidification. The ability of Rp-cAMPS to abolish the butyrate relaxation indicates that stimulation of the cyclic AMP second messenger system may play an important role in mediating this effect.

Effects of BRL 38227 on potassium currents in smooth muscle cells isolated from rabbit portal vein and human mesenteric artery.

1. Single smooth muscle cells were isolated from the rabbit portal vein and the human mesenteric artery and whole cell currents recorded at room temperature from either cell type by the whole cell voltage clamp technique. 2. In the rabbit portal vein cells addition of 10 microM BRL 38227 induced a quasi-instantaneous, voltage-insensitive and time-independent current which had a reversal potential of -75 mV under experimental conditions where the calculated EK was -83 mV. 3. Cells were held at 0 mV and BRL 38227 was added cumulatively to construct a dose-response relationship. BRL 38227 (0.03-10 microM) caused a dose-dependent outward shift in the holding current with an EC50 of 1.3 microM. 4. BRL 38227 (10 microM) had no effect on the delayed rectifier K+ current measured in the presence of 5 mM tetraethylammonium and no effect on the Ca(2+)-activated K+ current measured in the presence of 5 mM 4-aminopyridine. Similarly BRL 38227 had no effect on the Ca2+ current. 5. The BRL 38227-induced current was blocked by glibenclamide (10 microM) and phentolamine (100 microM), specific blockers of the ATP-sensitive K+ current in single cells. 6. In human isolated mesenteric artery cells, BRL 38227 (10 microM) induced a glibenclamide-sensitive current similar to, but smaller than, that observed in the rabbit portal vein. 7. We conclude that in these cells, BRL 38227 activates a potassium conductance which has the electrophysiological and pharmacological characteristics of ATP-sensitive K+ channels.

Effects of the 5-lipoxygenase activating protein inhibitor MK886 on voltage-gated and Ca2+-activated K+ currents in rat arterial myocytes.

1. The effects on the voltage-gated (IK) and Ca2+ activated (I(K,Ca)) K+ currents in rat arterial myocytes of the 5-lipoxygenase activating protein (FLAP) inhibitor MK886, and its inactive analogue L583,916 were evaluated. 2. In rat pulmonary arterial myocytes (RPAMs), MK886 caused a concentration-dependent reduction of the IK, with little obvious change in the kinetics of the current. Half maximal current block was observed at 75 nM MK886. 3. MK886 application led to a concentration-dependent increase in the amplitude of the TEA-sensitive I(K,Ca) current and single channel activity in RPAMs in whole cell and inside-out configurations, respectively. The threshold concentration for this effect was approximately 300 nM and a maximal 4-5 fold increase was observed at 10 microM MK886. MK886 also increased I(K,Ca) in rat mesenteric arterial myocytes (RMAMs). 4. L538,916, an analogue of MK886 which does not block FLAP, had no effect on either IK or I(K,Ca) at a concentration of 10 microM. 5. Leukotriene C4 (100 nM) had no effect on either IK or I(K,Ca) in RPAMs. MK886 produced its usual increase in I(K,Ca) and also blocked IK, in the presence of leukotriene C4. Similarly, leukotriene E4 (100 nM) did not alter the amplitude of IK. Also, the nonselective leukotriene receptor antagonist ICI 198,615 (3 microM) did not affect IK in RPAMs, and did not affect the response to MK886. 6. Arachidonic acid (10 microM) enhanced I(K,Ca) in both RPAMs and RMAMs. 7. The results show that MK886 markedly affects both IK and I(K,Ca) in a manner similar to that of arachidonic acid and independent of the endogenous production of leukotrienes. It is therefore possible that MK886, which is thought to compete with arachidonic acid for its binding to FLAP, may similarly occupy arachidonic acid binding sites on these K+ channels, and mimic its effects. Alternatively, MK886 might act via non-selective effects on other arachidonic acid metabolites which could modify K+ channel function.

The pharmacological properties of K+ currents from rabbit isolated aortic smooth muscle cells.

1. Using the whole-cell patch-clamp technique, the effects of several K+ channel blocking drugs on K+ current recorded from rabbit isolated aortic smooth muscle cells were investigated. 2. Upon depolarization from -80 mV, outward K+ current composed of several distinct components were observed: a transient, 4-aminopyridine (4-AP)-sensitive component (I1) and a sustained component (Isus), comprising a 4-AP-sensitive delayed rectifier current (IK(V)), and a noisy current which was sensitive to tetraethylammonium (TEA), and probably due to Ca(2+)-activated K+ current (IK(Ca)). 3. Several drugs in clinical or experimental use have as part of their action an inhibitory effect on specific K+ channels. Because of their differential K+ channel blocking effects, these drugs were used in an attempt to characterize further the K+ channels in rabbit aortic smooth muscle cells. Imipramine, phencyclidine, sotalol and amitriptyline failed to block selectively any of the components of K+ current, and were thus of little value in isolating individual channel contributions. Clofilium showed selective block of IK(V) in the presence of TEA, but only at low stimulation frequencies (0.07 Hz). At higher frequencies (1 Hz) of depolarization, both I1 and IK(V) were suppressed to a similar extent. Thus, the blocking action of clofilium was use-dependent. 4. The voltage-dependent inactivation of I1 and the delayed rectifier were very similar although a brief (100 ms) pre-pulse to -30 mV could preferentially inactivate I1. Together with the non-selective blocking effects of the K+ channel blockers, similarities in the activation and inactivation of these two components suggest that they may not exist as separate ionic channels, but as distinct kinetic states within the same K+ channel population. 5. The effects of all of these drugs on tension were examined in strips of rabbit aorta. The non-specific K+ channel blockers caused only minor increases in basal tension. TEA and 4-AP by themselves caused significant increases in tension and were even more effective when applied together. There appeared to be no correlation between the effects of the drugs tested on tension and their actions on currents recorded from isolated myocytes. Thus tension studies are an inappropriate means of investigating the mechanism of action of these drugs, and studies on ionic currents in isolated myocytes cannot easily predict drug actions on intact tissues.

Inhibition of sustained hypoxic vasoconstriction by Y-27632 in isolated intrapulmonary arteries and perfused lung of the rat.

We have examined the effects of Y-27632, a specific inhibitor of Rho-activated kinases (ROCK I and ROCK II) upon sustained hypoxic pulmonary vasoconstriction (HPV) in both rat isolated small intrapulmonary arteries (IPA) and perfused rat lungs in situ. Y-27632 (100 nM - 3 microM) was found to cause a concentration-dependent inhibition of acute sustained HPV in rat IPA. Application of Y-27632 (10-600 nM) in perfused rat lungs caused no change in basal perfusion pressure, but was found to inhibit HPV in a concentration-dependent manner, resulting in complete ablation of the pressor response to hypoxia at a concentration of 600 nM. Furthermore, addition of Y-27632 at any point during hypoxia caused a reversal of HPV in perfused rat lungs. These results suggest that activation of Rho-associated kinase may be a pivotal step in the generation of sustained HPV.

Effect of nimesulide and indomethacin on contractility and the Ca2+ channel current in myometrial smooth muscle from pregnant women.

The non-steroidal anti-inflammatory drug (NSAID) indomethacin inhibits both constitutive and inducible forms of cyclo-oxygenase (COX-1 and COX-2, respectively), while nimesulide is a selective COX-2 inhibitor. Uterine COX-2 is upregulated before and during term and pre-term labour, and prostaglandins play a crucial role in parturition. We therefore evaluated the effects of these drugs on myometrial contractility and the voltage-gated Ca2+ channel current in tissue strips and isolated human myometrial smooth muscle cells (HMSMC) from myometrial biopsies taken with informed consent from women undergoing caesarean section at term (not in labour). Nimesulide and indomethacin caused almost complete inhibition of spontaneous myometrial contractions at concentrations of 100 and 300 microM, respectively. The Ca2+ channel current was inhibited in a concentration-dependent manner by both drugs, with a 40% reduction of the current at 100 microM nimesulide and 300 microM indomethacin. Nimesulide also accelerated the decay of the Ca2+ channel current. The inhibition of the Ca2+ channel current by 100 microM nimesulide and 300 microM indomethacin was unaffected by the presence of either PGF2alpha or PGE2 (30 microM), and was of similar magnitude whether 10 mM Ba2+ or 1.5 mM Ca2+ was used as the charge carrier. The concentrations of indomethacin and nimesulide required to suppress spontaneous contractility in human pregnant myometrium were much higher than those necessary to inhibit prostaglandin production. The results suggest that both nimesulide and indomethacin inhibit myometrial contractility via mechanisms independent of cyclo-oxygenase inhibition. Blockade of the Ca2+ current may contribute to this effect.

pH-dependent block of the L-type Ca2+ channel current by diltiazem in human mesenteric arterial myocytes.

Inhibition of the L-type Ca2+ channel current (IBa) by diltiazem was characterised in human mesenteric arterial myocytes. External (pHo) and internal (pHi) pH was varied to alter the proportion of drug in charged and neutral forms. Diltiazem (20 microM) reduced IBa amplitude by approximately half at pHo 7.2 and 9.2 at holding potential -60 mV. The IBa decay was increased by diltiazem at pHo = 9.2 (97% uncharged), but not at 7.2. The IC50 for inhibition of IBa by diltiazem at holding potential -60 mV was decreased from 51 to 20 microM at pHo 7.2 and 9.2, respectively. At holding potential of -90 mV, but not -60 mV, tonic block increased and use-dependent block decreased as pHo was raised from 6.2 to 9.2. Diltiazem also caused a hyperpolarizing shift in IBa availability at alkaline pHo. The results suggest that raising pH promotes Ca2+ channel blockade by increasing the proportion of uncharged diltiazem.

Differential block by troglitazone and rosiglitazone of glibenclamide-sensitive K(+) current in rat aorta myocytes.

Thiazolidinediones are insulin-sensitising agents effective in controlling type II diabetes. These compounds also cause vasodilation. We evaluated the effects of the thiazolidinediones troglitazone and rosiglitazone on the glibenclamide-sensitive K(+) current in freshly isolated rat aorta myocytes. Troglitazone inhibited this current in a concentration-dependent manner (IC(50) approximately 1 microM). Rosiglitazone had a similar, but much less potent (IC(50) approximately 20 microM) action. Block of the glibenclamide-sensitive K(+) channels, in particular by troglitazone, may potentially affect the response of arteries to hypoxia and to certain endogenous and exogenous vasodilators.

Cromakalim does not act as a calcium antagonist in isolated human mesenteric artery cells.

The effects of cromakalim and its active enantiomer BRL 38226 on voltage-gated Ca2+ channels in smooth muscle cells isolated from human mesenteric arteries were studied using the whole cell patch-clamp technique. Neither of these drugs affected the Ca2+ channel current in these cells. These results suggest that the efficacy of cromakalim in lowering blood pressure in human beings does not involve a Ca2+ channel antagonistic effect.

Differential effects of insulin-sensitizers troglitazone and rosiglitazone on ion currents in rat vascular myocytes.

Insulin-sensitizing thiazolidinediones such as troglitazone and pioglitazone have been shown to lower blood pressure in vivo and cause vasorelaxation in vitro. Rosiglitazone (BRL 49653) is a novel thiazolidinedione which has been reported not to cause vasoleraxation. We therefore compared the effects of troglitazone and rosiglitazone on Ca2+ and K+ currents in rat aorta and pulmonary artery smooth muscle cells. Currents were recorded with the conventional whole cell patch clamp technique. Both drugs reduced the voltage-gated (L-type) Ca2+ current in rat aorta cells, with half-maximal current inhibition by troglitazone and rosiglitazone at 2 and 10 microM, respectively. Troglitazone, 2 microM and rosiglitazone, 20 microM caused a similar hyperpolarizing shift of 12 mV in the potential-dependence of Ca2+ current availability. Troglitazone (20 microM) produced a marked block of the tetraethylammonium- and paxilline-sensitive Ca2+ activated K+ current, while rosiglitazone (20 microM and 60 microM) slightly enhanced this current. Rat pulmonary artery smooth muscle cells have a prominent delayed rectifier K+ current. Troglitazone produced a potent block of this current (half-maximal inhibition at <1 microM), while rosiglitazone caused a smaller inhibition at 10 and 60 microM. These results show that troglitazone has relatively potent blocking effects on a wide variety of ion currents in vascular smooth muscle cells. Rosiglitazone exerts less potent, but similar effects on the Ca2+ current and delayed rectifier K+ current, but it enhances the Ca2+ activated K+ current. reserved.

Estimation of high K- and noradrenaline-induced 45Ca uptakes in isolated rat aorta: effects of washing in icecold solutions.

Isolated rat aorta rings, labelled for 10 min with 45Ca in control physiological saline solution (PSS), PSS with 10 microM noradrenaline (NA), or 80 mM potassium-PSS (80 K), were washed out into non-labelled, icecold PSS. NA and 80 K increased tissue 45Ca. Retention of stimulated 45Ca uptake during washout was studied by subtracting the control washout curve from the washout curves of the stimulated tissues. The measured stimulated uptake fell rapidly during the initial 15 min of washout, thereafter decreasing more slowly. These results suggested that a lengthy wash in icecold PSS would lead to an underestimate of stimulated cellular 45Ca uptake. The cellular location of the stimulated uptake was confirmed by using diltiazem (10 microM) and forskolin (10 microM) to block specifically the 45Ca influxes caused by 80 K and NA, respectively. A similar biphasic loss of stimulated 45Ca uptake was seen in icecold lanthanum (50 mM) solution. These data suggest that established methods for measuring cellular Ca in isolated blood vessels may have markedly underestimated the amount of Ca entering cells during stimulation.

Alterations in [Ca2+]i mediated by sodium-calcium exchange in smooth muscle cells isolated from the guinea-pig ureter.

1. Sodium-calcium exchange was studied in single enzymatically isolated cells of the guinea-pig ureter using the Ca2(+)-sensitive fluorescent dye Indo-1 to monitor the intracellular Ca2+ concentration ([Ca2+]i). Patch pipettes containing Indo-1 were used to introduce the dye into cells, to set the intracellular Na+ concentration ([Na+]i) and control the membrane potential during experiments. 2. With [Na+]i set at 11-12 mM and a membrane potential of -60 or -70 mV, brief depolarization of ureter cells elicited typical voltage-gated inward currents associated with rapid increases in [Ca2+]i which showed a bell-shaped potential dependence. If Ca2+ currents were blocked with nifedipine, depolarization led to slower rises in [Ca2+]i. The rates and amplitudes of these increased monotonically with progressively larger depolarizations up to +120 mV. 3. The nifedipine-resistant rises in [Ca2+]i elicited by depolarization were potentiated when the extracellular sodium concentration ([Na+]o) was reduced. Basal levels of [Ca2+]i also increased as [Na+]o was reduced, although the dependence of this effect on [Na+]o was smaller than would be predicted if [Ca2+]i was set only by a Na(+)-Ca2+ exchange process. 4. The nifedipine-insensitive rises in [Ca2+]i elicited by depolarization were potentiated at higher basal levels of [Ca2+]i. 5. The ability of cells to reduce [Ca2+]i rapidly following Ca2+ loading during voltage-gated transients was markedly inhibited if the Na+ concentration gradient was reversed, but was little affected if the Na+ gradient was decreased by 25 or 50%. Recovery from a Ca2+ load caused by reversal of the Na+ gradient could be induced by removal of Cao2+ in the continuing absence of Nao+, indicating the importance of a Na(+)-independent [Ca2+]i-lowering system. 6. The results demonstrate that Na(+)-Ca2+ exchange can modulate [Ca2+]i when [Na+]i and the membrane potential are set at or near their physiological levels in these smooth muscle cells. [Ca2+]i does not, however, appear to be markedly sensitive to the Na+ concentration gradient under the conditions employed for these experiments, suggesting that a Na(+)-independent Ca2+ extrusion system is mainly responsible for regulating [Ca2+]i under normal conditions.