ATP-sensitive K+ channels in rat ventricular myocytes are blocked and inactivated by internal divalent cations

K⁺ currents were recorded from ATP-sensitive channels in inside-out patches from isolated rat ventricular myocytes. In the absence of internal divalent cations the current voltage relationship could be described by constant-field assumptions with a permeability of 1.25×10^–13 cm²/s; outward currents saturated under a high driving force for K⁺ movement. Internal 0.1–5.0 mM Mg²⁺, 0.1 M Ca²⁺ and 10 mM Na⁺ each depressed the flux of K⁺ ions moving outwards through open channels. Internal 0.1–5.0 mM Mg²⁺, 0.1–1.0 M Ca²⁺ and 1–10 M Ba²⁺ and Sr²⁺ blocked K⁺ channel activity in a dose-and voltage-dependent manner. Run-down channels could be reactivated by Mg-ATP, but not by AMP-PNP, ATPS or Mg-free ATP which suggested that phosphorylation of the channels was involved in their activity. Ca²⁺ (>=1 M) and Sr²⁺ (1 mM) markedly inactivated K⁺ ATP channels, millimolar Ba²⁺ or Mg²⁺ were less effective. This suggested that the run down of the channels was a Ca²⁺-dependent dephosphorylation of the K⁺ channel protein.     

Potassium channel openers act through an activation of ATP-sensitive K+ channels in guinea-pig cardiac myocytes

In a previous article (Escande et al. 1988a), we have shown that cromakalim (BRL 34915), a potassium channel opener (PCO), is a potent activator of ATP-sensitive K⁺ channels in cardiac cells. In the present article, the influence on K⁺ channels of two other potassium channel openers chemically unrelated to cromakalim, RP 49356 and pinacidil, has been investigated in patch-clamped isolated cardiac myocytes. In the whole-cell configuration, K⁺ currents were recorded in the presence of 50 M TTX and 3 M nitrendipine or 3 mM cobalt. Like cromakalim, RP 49356 or pinacidil activated a time-independent outward current at 33–35°C but not at 19–21°C, which showed little voltage-dependency in the potential range –60 to +60 mV. Its amplitude was a function of the agonist concentration, e.g. it was 2.1±0.4 nA at +60 mV with 30 M RP 49356 and 4.3±0.8 nA with 300 M. In control conditions, glibenclamide, a blocker of K⁺-ATP channels in pancreatic and heart cells, affected neither the inward rectifier,i _K1, nor the delayed K⁺ current,i _K. At 3 M, glibenclamide fully prevented the effects of 300 M RP 49356 or pinacidil. At lower concentrations, glibenclamide partially counteracted the activation by PCOs of a K⁺ current. In the cell-attached contiguration, externally applied RP 49356 or pinacidil caused opening of large channels which reversed around 0 mV in a high K⁺ external medium. In inside-out patches, both RP 49356 or pinacidil activated K⁺-ATP channels by increasing the time period for which the channels remained in the open state. It is concluded that, like cromakalim, RP 49356 and pinacidil are potent activators of K⁺-ATP channels in cardiac myocytes.     

Forskolin-induced block of delayed rectifying K+ channels in pancreaticβ-cells is not mediated by cAMP

K⁺ channels in the membrane of murine pancreatic -cells were studied using the patch-clamp technique. The delayed outward current was activated in whole-cell experiments by depolarizing voltage pulses to potentials between –30 mV and 0 mV. Forskolin blocked the current rapidly (<5 s) and reversibly with 50% inhibition at 13 M. The inhibition did not depend on a stimulation of the adenylate cyclase since it occurred even in presence of 1 mM cAMP in the pipette solution which replaced the cytoplasm. Membrane permeant cAMP analogues and phosphodiesterase inhibitors did not influence the delayed outward current. In experiments on outside-out patches forskolin (100 M) shortened the openings of a channel of about 10 pS conductance at 0 mV and a time course of activation and inactivation similar to the whole-cell current. Another smaller, slowly activating channel and the Ca²⁺- and ATP-dependent K⁺ channels were influenced only weakly or not at all. It is therefore concluded that the 10-pS channel generates most of the delayed outward K⁺ current in murine pancreatic -cells. The Ca²⁺-independent part of the delayed outward current in bovine adrenal chromaffin cells was also blocked by forskolin (100 M).     

Dual effects of ATP on K+ currents of mouse pancreatic β-cells

K⁺ currents through ATP-dependent channels were recorded from inside-out patches of -cell membrane as previously described (Rorsman and Trube 1985). Channels were opened by removing ATP from the intracellular side of the membrane. The open probability and/or the number of active channels declined spontaneously (run-down) when ATP was absent for periods longer than about 30 s. Channels subject to the run-down could be activated again after applying a blocking concentration (>0.1 mM) of ATP in presence of 1 mM MgCl₂ for at least 2 min. ATP in absence of Mg and the ATP-analogues AMP-PNP, AMP-PCP and ATPS were ineffective in reactivating the channels. This suggests that phosphorylation of the channels or associated proteins of hydrolysis of ATP may be necessary for keeping the channels available. In contrast to the differential effects on the run-down, ATP in presence and absence of Mg and the ATP analogues were similarly effective in blocking the channels at concentrations above 0.1 mM. Using an experimental protocol avoiding the run-down ghe dose-inhibition curve for ATP was found to reach 50% at 18 M.     

ATP-Dependent inhibition of Ca2+-activated K+ channels in vascular smooth muscle cells by neuropeptide Y

Neuropeptide Y(NPY) inhibits Ca²⁺-activated K⁺ channels reversibly in vascular smooth muscle cells from the rat tail artery. NPY (200 M) had no effect in the absence of intracellular adenosine 5triphosphate (ATP) and when the metabolic poison cyanide-M-chlorophenyl hydrozone (10 M) was included in the intracellular pipette solution. NPY was also not effective when ATP was substituted by the non-hydrolysable ATP analogue adenosine 5-[, -methylene]-triphosphate (AMP-PCP). NPY inhibited Ca²⁺-activated K⁺ channel activity when ATP was replaced by adenosine 5-O-(3-thiotriphosphate) (ATP [-S]) and the inhibition was not readily reversed upon washing. Protein kinase inhibitor (1 M), a specific inhibitor of adenosine 3, 5-cyclic monophosphatedependent protein kinase, had no significant effect on the inhibitory action of NPY. The effect of NPY on single-channel activity was inhibited by the tyrosine kinase inhibitor genistein (10 M) but not by daidzein, an inactive analogue of genistein. These observations suggest that the inhibition by NPY of Ca²⁺-activated K⁺ channels is mediated by ATP-dependent phosphorylation. The inhibitory effect of NPY was antagonized by the tyrosine kinase inhibitor genistein.     

Positive cooperativity in activation of the cardiac muscarinic K+ channel by intracellular GTP

Concentration-dependent effects of intracellular GTP on activation of the muscarinic K⁺ channel were examined in inside-out patches of cardiac atrial myocytes. The pipette solution contained 0.1 M ACh. GTP (0.01–30 M) and 0.5 mM MgCl₂ were applied to the inside side of the patch membrane. K⁺ channels were activated with GTP concentration above 0.1 M. Channel activation reached a maximal value with 1–3 M GTP. It decreased at GTP concentrations larger than 3 M, probably due to desensitization. The dependence of the open probability of the channel on intracellular GTP showed a sigmoidal relationship with a Hill coefficient of around 3. A positive cooperative effect of intracellular GTP on the K⁺ channel may play an important role in amplifying the signal from the membrane receptor to the K⁺ channel.     

A potassium current activated by lemakalim and metabolic inhibition in rabbit mesenteric artery

K⁺ channels which are inhibited by intracellular ATP (ATP_i) (K_ATP channels) are thought to be the physiological target site of the K⁺ channel opening drugs (2) and to underlie a variety of physiological phenomena including hypoxia induced vasodilation (3). However, electrophysiological evidence for ATP_i-regulated K⁺ currents in smooth muscle is scarce. We, therefore, investigated the effects of one K⁺ channel opener, lemakalim, and metabolic inhibition on the membrane conductance of freshly dissociated rabbit mesenteric artery smooth muscle cells, using the perforated-patch whole cell recording technique (6). The cells were metabolically inhibited with 1 mM iodoacetic acid and 50 M dinitrophenol. Both lemakalim (0.1–3 M) and metabolic inhibition activated a time-independent and glyburide sensitive K⁺ current at physiological membrane potentials. The similarities between the lemakalim and metabolic inhibition activated currents suggest that a single class of channels underlies both currents. These results are the first whole-cell current recordings to demonstrate the activation of a smooth muscle membrane conductance by metabolic inhibition, lending support to the view that hypoxia induced vasodilation arises from the activation of K_ATP channels.     

ATP-dependent activation of atrial muscarinic K+ channels in the absence of agonist and G-nucleotides

While opening of cardiac muscarinic K⁺ channels is mediated by a guanine nucleotide-binding protein, G_K, and normally requires both ACh and cytosolic GTP, we demonstrate that in the absence of agonist and G-nucleotides guinea-pig atrial muscarinic K⁺ channels are activated by cytosolic ATP (K₅₀ = 224 M). This activation involves a phosphorylating reaction and is most probably related to a transphosphorylation by a nucleoside diphosphate kinase (NDPK) from ATP to G_K-bound GDP. This ATP-induced response is completely inhibited by nanomolar concentrations of GDP or GTP, suggesting a G-nucleotide specific binding site on NDPK.This wort was supported by a grant of the National Fund for Scientific Research Belgium.     

Effect of Na+,K+,2Cl- Cotransport Inhibitor Bumetanide on Electrical and Contractile Activity of Smooth Muscle Cells in Guinea Pig Ureter

The effect of Na⁺,K⁺,2Cl^- cotransport inhibitor bumetanide on action potentials and contractions of smooth muscle cells in the ureter of guinea pigs evoked by electrical stimulation was studied by the method of double sucrose bridge. Bumetanide (10-100 M) dose-dependently suppressed action potential and contractions of smooth muscle cells induced by 1-10 M histamine, 10 M mesatone, 5 mM tetraethylammonium, and 100 M sodium nitroprusside. Our findings suggest that test substances modulate Na⁺,K⁺,2Cl^- cotransport in smooth muscle cells.     

Opposite effects of tolbutamide and diazoxide on the ATP-dependent K+ channel in mouse pancreatic β-cells

The influence of the antidiabetic sulphonylurea tolbutamide on K⁺ channels of mouse pancreatic -cells was investigated using different configurations of the patch clamp technique. The dominant channel in resting cells is a K⁺ channel with a single-channel conductance of 60 pS that is inhibited by intracellular ATP or, in intact cells, by stimulation with glucose. In isolated patches of -cells membrane, this channel was blocked by tolbutamide (0.1 mM) when applied to either the intracellular or extracellular side of the membrane. The dose-dependence of the tolbutamide-induced block was obtained from whole-cell experiments and revealed that 50% inhibition was attained at approximately 7 M. In cell-attached patches low concentrations of glucose augmented the action of tolbutamide. Thus, the simultaneous presence of 5 mM glucose and 0.1 mM tolbutamide abolished channel activity and induced action potentials. These were not produced when either of these substances was added alone at these concentrations. The inhibitory action of tolbutamide or glucose on the K⁺ channel was counteracted by the hyperglycaemic sulphonamide diazoxide (0.4 mM). Tolbutamide (1 mM) did not affect Ca²⁺-dependent K⁺ channels. It is concluded that the hypo- and hyperglycaemic properties of tolbutamide and diazoxide reflect their ability to induce the closure or opening, respectively, of ATP-regulated K⁺ channels.     

Fenamates and diltiazem modulate lipid-sensitive mechano-gated 2P domain K+ channels

A swelling-activated, background K⁺ current in the corneal epithelium is characteristically activated by fenamates and inhibited by diltiazem. Fatty acids also stimulate this current, indicating that its origin is a lipid-sensitive mechano-gated 2P domain K⁺ channel. In the present study, modulation of TREK-1, TREK-2, and TRAAK channels by fenamates and diltiazem was examined. TREK-1, TREK-2, and TRAAK currents transiently expressed in COS-7 cells were recorded by the perforated-patch configuration. As previously reported, arachidonic acid (20 M) stimulated all of these channels, and a volatile anesthetic, halothane (1 mM) augmented TREK-1 and TREK-2 but not TRAAK. Flufenamic acid (FA, 100 M), niflumic acid (NA, 100 M), and mefenamic acid (MA, 100 M) markedly stimulated TREK-1, TREK-2, and TRAAK. The potency sequence for the activation of TREK-1 and TREK-2 was FA > NA = MA, and the potency sequence for the activation of TRAAK was FA = NA > MA. Diltiazem (1 mM) inhibited TREK-1 and TREK-2, but not TRAAK. In conclusion, fenamates are openers of the lipid-sensitive mechano-gated 2P domain K⁺ channels, and diltiazem may be a specific blocker for TREK. These novel findings could help to further understand channel functions of the mechano-gated 2P domain K⁺ channels.     

A long lasting Ca2+ -activated outward current in guinea-pig atrial myocytes

Among other characteristics, the steady-state current-voltage relationship of patch-clamped single atrial myocytes from guinea-pig hearts is defined by an outward current hump in the potential region –15 to +40mV. This hump was reversibly suppressed by Co²⁺ (3 mM) or nitrendipine (5 M) and enhanced by Bay K 8644 (5 M). The maintained outward current component suppressed by Co²⁺ extended between –15.2±1.9 mV and +39.5 ±1.7 mV (mean±SEM of 14 cells) and has an amplitude of 95.7±9.4 pA at +10 mV. In isochronal I-V curves, the hump was already visible at 400 ms with essentially the same amplitude as at 1500 ms. The Co²⁺ -sensitive outward current underlying the hump was poorly time-dependent during 1.5 s voltage pulses but slowly relaxed upon repolarization. Tail currents reversed near the K⁺ equilibrium potential under our experimental conditions. The current hump of the steady-state I-V curve was also abolished by caffeine (10 mM) or ryanodine (3 M), both drugs that interfere with sarcoplasmic reticulum function. Apamin (1 M) or quinine (100 M) but not TEA (5–50 mM) markedly reduced its amplitude. However, at similar concentrations as required to inhibit the hump, both apamin and quinine appeared to be poorly specific for Ca²⁺ -activated K⁺ currents in heart cells since they also inhibited the L-Type Ca²⁺ current. It is concluded that a long lasting Ca²⁺ -activated outward current, probably mainly carried by K⁺ ions but not sensitive to TEA, exists in atrial myocytes which is responsible for the current hump of the background I-V curve.     

Metabolic inhibition and low internal ATP activate K-ATP channels in rat dopaminergic substantia nigra neurones

The patch-clamp technique was used to study whole-cell currents of acutely dissociated rat substantia nigra (SN) neurones. In perforated-patch current-clamp recordings, inhibition of mitochondrial metabolism by rotenone (5 M) produced a hyperpolarisation and inhibited electrical activity. These effects were reversed by the sulphonylureas tolbutamide (0.5 mM) or glibenclamide (0.5 M). Under voltageclamp conditions, rotenone induced a timeand voltage-independent K⁺ current which was selectively blocked by sulphonylureas. The glibenclamide-sensitive current reversed at –81.7±2.7 mV (n=5) and showed marked inward rectification. Intracellular dialysis with 0.3 mM adenosine 5-triphosphate (ATP), but not 2 mM or 5 mM ATP, in standard whole-cell recordings also resulted in activation of a sulphonylurea-sensitive K⁺ current with similar properties (reversal potential, –81.9±2.5 mV, n=5). The close similarity in the properties of the ATP-sensitive K⁺ current observed in whole-cell recordings and the K⁺ current activated by metabolic inhibition in perforated-patch recordings suggest that they both result from activation of the same type of ATP-sensitive K⁺ channel. Sulphonylureas had no effect on electrical activity or membrane currents in the absence of rotenone in perforated-patch recordings, or in cells dialysed with 5 mM ATP, indicating that in SN neurones these drugs are selective for the ATP-sensitive K⁺ current.     

ATP4− and ATP·Mg inhibit the ATP-sensitive K+ channel of rat ventricular myocytes

K⁺ currents were recorded from ATP-sensitive channels in inside-out membrane patches excised from isolated rat ventricular myocytes. ATP-sensitive K⁺ channel inhibition could be evoked by ATP in the absence of magnesium where most ATP would be present as the free acid ATP^4–. Channel inhibition was enhanced when the same total concentration of ATP was applied in the presence of magnesium, where most ATP would be bound as ATP·Mg. Dose-response relationships for ATP-sensitive K⁺ channel inhibition evoked by ATP had a Hill coefficient of 2 andK _i of 17 and 30 M for ATP in the presence and absence of magnesium respectively. This was the obverse of the expected results if ATP^4– were to be the sole form of ATP to effect channel closure. ATP-sensitive K⁺ channel inhibition evoked by ATPS, AMP-PNP and AMP-PCP was also enhanced in the presence of magnesium. It is concluded that the ATP-sensitive K⁺ channel of rat ventricular myocytes binds and is closed by both the free-acid and divalent-cationbound forms of ATP.     

Effect of depolarizing and hyperpolarizing agents on the membrane potential difference of primary cultures of rabbit aorta vascular smooth muscle cells

Vascular smooth muscle cells of rabbit aorta were enzymatically dispersed, kept in primary culture, and studied between days 1 and 7 in a bath rinsed with Ringer-like solution at 37°C. The electrical membrane potential difference (PD) was measured with microelectrodes. The mean value of PD was –50±0.4 mV (n=53). Cromakalim (BRL 34915), 1 mol/l and 10 mol/l, hyperpolarized the membrane potential by 9±1 mV (n=11) and 15±1 mV (n=53) respectively. Glibenclamide (10 mol/l) abolished the hyperpolarizing effect of cromakalim (n=6). Simultaneous addition of cromakalim and glibenclamide (both 10 mol/l, n=11) and glibenclamide itself (10 mol/l, n=7) had no effect on PD. In patch-clamp experiments in outside-out-oriented Ca²⁺-sensitive K⁺ channels, cromakalim increased the open probability (P _o) only slightly and only with a cytosolic Ca²⁺ activity of 1 mol/l. In all other series cromakalim had no effect on the P _o of these channels. Forskolin (10 mol/l) hyperpolarized PD by 6±1 mV (n=13). The nucleotides UTP, ATP and ITP (10 mol/l) depolarized PD by 12±1 mV (n=7), 8±1 mV (n=65) and 5±1 mV (n=6) respectively. GTP, [,-methylene]ATP and adenosine had no significant effect. Mn²⁺ (1 mmol/l, n=18), Ni²⁺ (1 mmol/l, n=13), Co²⁺ (1 mmol/l, n=11), Zn²⁺ (1 mmol/l, n=6) and the Ca²⁺-channel blockers verapamil and nifedipine (both 0.1 mmol/l, n=6) did not attenuate the depolarization induced by 10 mol/l ATP. Fetal calf serum (100 ml/l, n=7) depolarized PD by 11±2 mV. This effect was not abolished by nifedipine or by replacing NaCl by choline chloride. The data indicate that PD of vascular smooth muscle cells is depolarized by P₂ agonists and hyperpolarized by the K⁺-channel opener cromakalim. The effect of cromakalim is antagonized by glibenclamide. The effect of cromakalim is probably not mediated by the K⁺ channel identified in excised patches.Supported by DFG Gr 480/10     

Maxi K+ channels are stimulated by cyclic guanosine monophosphate-dependent protein kinase in canine coronary artery smooth muscle cells

By using a patch clamp technique, we examined the effect of cyclic guanosine monophosphate (cGMP)-dependent protein kinase (G kinase) on Ca²⁺-activated maxi K⁺ channels in canine coronary artery smooth muscle cells. Maxi K⁺ channels (274±4 pS in symmetrical 140 mM KCl at 24–26°C) were activated by cytoplasmic Ca²⁺ and were completely blocked by 100 nM charybdotoxin (CTX). G kinase (300 U/ml) added to the cytoplasmic face of the membrane patch shifted the voltage dependence of these channels by about 25 mV in the negative direction in the presence of 1 M Ca²⁺, 50 M cGMP and 1 mM magnesium adenosine triphosphate. At –50 mV and 1 M Ca²⁺, G kinase treatment increased the mean number of open channels 4.5-fold compared with the control. -Human atrial natriuretic peptide (ANP, 100 nM) reduced the isometric tension of coronary arterial rings elicited by 14 or 24 mM KCl, but failed to relax the artery contracted by 34 mM KCl. Addition of 100 nM CTX augmented tension development elicited by 24 mM KCl and totally prevented ANP from relaxing the arterial rings. These results indicate that G kinase-dependent protein phosphorylation activates maxi K⁺ channels in canine coronary smooth muscle, and further suggest that the G kinase-induced activation of maxi K⁺ channels may cause hyperpolarization and relaxation of coronary artery.     

Modulation of ionic currents in smooth muscle balls of the rabbit intestine by intracellularly perfused ATP and cyclic AMP

The effects of intracellularly perfused ATP and cyclic-AMP (c-AMP) on ionic currents recorded from fragmented smooth muscle cells (smooth muscle ball; SMB) were investigated, using the single electrode whole cell voltage clamp method. The Ca²⁺ current was distinguished from K⁺ currents, using pipette solution containing Cs⁺, TEA⁺ and 4 mM EGTA. ATP enhanced the Ca²⁺ current, dose-dependently between 0.3 and 10. mM, and slightly slowed the slow component of the decay of the Ca²⁺ current, while the steady-state inactivation curve remained unaffected. Intracellular application of 5-adenylyl-imidodiphosphate (AMP-PNP; 1mM) inhibited the Ca²⁺ current by competitionwith ATP, but c-AMP (up to 300 M) had no effect. With a high-K⁺ solution containing 0.3 mM EGTA and ATP in the pipette and physiological salt solution in the bath, a net inward current with transient (Ca²⁺ dependent) and delayed (Ca²⁺ independent) K⁺ outwart currents were evoked. Increased concentrations of ATP (above 1 mM) but not c-AMP (up to 100 M) in the pipette enhanced the transient K⁺ outward current Neither agent had any effect on the delayed outward current. When repetitive stimulations of intervals shorter than 5 s were applied, the amplitude of the transient outward current was markedly reduced, and 100 M c-AMP partially prevented this attenuation. ATP may act on the Ca²⁺ channel either by phosphorylating the channel protein or by other ATP requiring mechanisms, independently from those induced by the action of c-AMP. Thus, the different responses of cardiac and visceral smooth muscles induced by -adrenoceptor stimulation may be explained in part by the different natures of the Ca²⁺ channel in response to c-AMP.     

Actin-dependent activation of ion conductances in bronchial epithelial cells

Activation of Cl^– and K⁺ channels is necessary to drive ion secretion in epithelia. There is substantial evidence from previous reports that vesicular transport and exocytosis are involved in the regulation of ion channels. In the present study we examined the role of cytoskeletal elements and components of intracellular vesicle transport on ion channel activation in bronchial epithelial cells. To this end, cells were incubated with a number of different compounds which interact with either microtubules or actin microfilaments, or which interfere with vesicle transport in the Golgi apparatus. The effectiveness of these agents was verified by fluorescence staining of cellular microtubules and actin. The function was examined in ³⁶Cl^– efflux studies as well as in whole-cell (WC) patch-clamp and cell-attached studies. The cells were studied under control conditions and after exposure to (in mmol/l) ATP (0.1), forskolin (0.01), histamine (0.01) and hypotonic bath solution (HBS, NaCl 72.5). In untreated control cells, ATP primarily activated a K⁺ conductance whilst histamine and forskolin induced mainly a Cl^– conductance. HBS activated both K⁺ and Cl^– conductances. Incubation of the cells with brefeldin A (up to 100 mol/l) did not inhibit WC current activation and ³⁶Cl^– efflux. Nocodazole (up to 170 mol/l) reduced the ATP-induced WC current, and mevastatin (up to 100 mol/l) the cell-swelling-induced WC current. Neither had any effect on the WC current induced by forskolin and histamine. Also ³⁶Cl^– efflux induced by HBS, ATP, forskolin and histamine was unaltered by these compounds. Similarly, colchicine (10 mol/l) and taxol (6 mol/l) affected neither ³⁶Cl^– efflux nor WC current induced by ATP, forskolin, histamine or HBS. In contrast, depolymerisation of actin by cytochalasin D (10 mol/l) significantly attenuated ³⁶Cl^– effluxes and WC current activation by the above-mentioned agonists. Incubation with a C2 clostridial toxin (5 nmol/l) showed similar effects on WC currents. Moreover, when cytochalasin D (10 mol/l), C2 clostridial toxins (5 nmol/l), or phalloidin (10 mol/l) were added to the pipette filling solution current activation was markedly reduced. However, in excised inside-out membrane patches, cytochalasin D (10 mol/l), G-actin (10 mol/l) and phalloidin (10 mol/l) had no effect. These data suggest that actin participates in the activation of ion channels in 16HBE14o- epithelial cells and support the concept that exocytosis is a crucial step in the regulation of Cl^– and K⁺ channels in these cells.     

Modulation of single slow (L-type) calcium channels by intracellular ATP in vascular smooth muscle cells

Involvement of ATP in the regulation of slow (L-type) Ca²⁺ channels of vascular smooth muscle cells was investigated by recording single Ca²⁺ channel currents (single-channel conductance of 18 pS) using a patch clamp technique. In the cell-attached configuration, intracellular composition was modified by permeabilizing the cell membrane with mechanical disruption at one end of the cell. Single cells were freshly isolated from guinea-pig portal vein by collagenase treatment. For the channel recordings, the pipette solution contained 100 mM Ba²⁺ and the bath contained K⁺-rich solution (with 5 mM EGTA) to depolarize the membrane to near 0 mV. The channel activity decreased usually within 3 min after permeabilizing the cell end and exposure to ATP-free bath solution. If ATP (1–5 mM) was applied to the bath (access to cell interior) before complete disappearance of channel activity, channel activity was partially recovered. ATP did not change the current amplitude (i) or the mean open time of the channels, whereas the number of channels available for opening and/or the probability of their being open (NP _o) were increased by ATP. A non-hydrolyzable analogue of ATP, AMP-PNP, did not exert an ATP-like effect; ATP--S had a weak effect. With 1 M Bay-K-8644 (Ca²⁺ channel agonist) in the pipette, the activity of the Ca²⁺ channel was high; such activity persisted for more than 10 min after permeabilizing the cell and exposting to ATP-free solution containing KCN (1 mM) and 2-deoxy-d-glucose (10 mM). These results indicate that activation of slow Ca²⁺ channels requires ATP. The effect of ATP may be exerted by phosphorylation and/or an energy-requiring step. Bay-K-8644 may change the nature of the slow Ca²⁺ channel, making it resistant to rundown.     

Evidence for Na+/Ca2+ exchange in isolated smooth muscle cells: a fura-2 study

Isolated smooth muscle cells (SMC) from guinea pig taenia coli were employed. Suspension of cells were externally loaded in saline with the fluorescent calcium indicators quin-2/AM or fura-2/AM at 20–40 M or 4 M respectively, resulting in an estimated intracellular concentration of 100–200 M for quin-2 or 10–20 M fura-2 (free acid). On addition of 100 M carbachol or high K _o ⁺ (80 mM) depolarization, fura-2 loaded cells contracted (104±47 m,n=121 rest: 39±13 m,n=59 contracted) identically to control (103±35 m,n=232 rest: 39±16 m,n=89 contracted) cells, whereas quin-2 loaded cells were unresponsive to these protocols and there was no significant length change. The Ca _i ²⁺ of fura-2 loaded cells was 100±18 nM (mean±SD,n=15) and was not significantly different from quin-2 loaded cells 107±26 nM (n=13). Treatment of fura-2 loaded cells with 100 M ouabain saline for 10–60 min progressively elevated the Ca _i ²⁺ to a mean of 266±83 nM (n=15). Reduction of Na _p ⁺ (96% Li⁺ replaced) significantly increased Ca _i ²⁺ to 317±77 nM (n=8). After pretreatment with ouabain (100 M), Na _o ⁺ replacement (Li⁺) increased Ca _i ²⁺ at a significantly faster rate [3.6 nM min^–1 (control) cf. 19.8 nM min^–1 (ouabain)].