Modification of ATP-sensitive K+ channels by proteolysis in smooth muscle cells from pig urethra

Patch-clamp experiments have been performed to investigate the effects of endoproteases (such as trypsin, carboxypeptidase B) on both membrane currents and unitary currents in isolated smooth muscle cells from pig proximal urethra (conventional whole-cell configuration, cell-attached configuration, and inside-out patches). Application of either trypsin (1 mg/mL) or carboxypeptidase B (0.1 mg/mL) to the intracellular surface of the excised membrane patches stimulated the activity of a 2.1 pA K+ channel (in symmetrical 140 mM K+ conditions) at a holding potential of -50 mV. The trypsin-induced K+ channels in inside-out configuration exhibited the same amplitude and similar channel opening kinetics to the levcromakalim-induced ATP-sensitive K+ channel (i.e. K ATP channel) in cell-attached patches of the same membrane; however, the sensitivity of the channels to glibenclamide was greatly reduced after the trypsin-treatment. The activity of the trypsin-induced K+ channel was reversibly inhibited by cibenzoline in an inside-out configuration (Ki = 5 microM). It is concluded that trypsin and carboxypeptidase B reactivate the channel with an intact pore activity but the different pharmacological properties of the channels may reflect some change in the conformation in channel proteins after proteolysis.     

Localization and function of ATP-sensitive potassium channels in human skeletal muscle

The present study investigated the localization of ATP-sensitive K+ (KATP) channels in human skeletal muscle and the functional importance of these channels for human muscle K+ distribution at rest and during muscle activity. Membrane fractionation based on the giant vesicle technique or the sucrose-gradient technique in combination with Western blotting demonstrated that the KATP channels are mainly located in the sarcolemma. This localization was confirmed by immunohistochemical measurements. With the microdialysis technique, it was demonstrated that local application of the KATP channel inhibitor glibenclamide reduced (P < 0.05) interstitial K+ at rest from approximately 4.5 to 4.0 mM, whereas the concentration in the control leg remained constant. Glibenclamide had no effect on the interstitial K+ accumulation during knee-extensor exercise at a power output of 60 W. In contrast to in vitro conditions, the present study demonstrated that under in vivo conditions the KATP channels are active at rest and contribute to the accumulation of interstitial K+.     

Surface charge and properties of cardiac ATP-sensitive K+ channels

ATP-sensitive K+ (KATP) channels are present in a wide variety of tissues. The sensitivity of these channels to closure by cytosolic ATP (ATPi) varies significantly among different tissues and even within the same tissue. The purpose of this study was to test the hypothesis that negative surface charges modulate the sensitivity of the KATP channels to ATPi by influencing surface potential in the vicinity of the ATP- binding site(s) of the channel. Unitary currents through KATP channels were measured in inside-out membrane patches excised from rabbit ventricular myocytes using the patch-clamp technique. Agents known to be effective at screening negative surface charges were applied to the cytosolic surface of the patches, and their effects on ATP sensitivity were examined. These agents included Mg2+ (2-15 mM), Ba2+ (2-10 mM), and the polycations protamine (0.01-10 microM), poly-L-lysine (500 microM), and poly-L-arginine (0.5 microM). The divalent cations and the various polycations all dramatically reduced the concentration of ATPi required to half-maximally suppress current through KATP channels (Kd), from approximately 100 microM in the absence of these agents to 1.6-8 microM in their presence. The effects were dose dependent. Protamine also reduced the sensitivity of KATP channels to block by cytosolic ADP. The sensitivity of KATP channels to block by ATP was independent of membrane potential, suggesting that the ATP-binding site is not located within the transmembrane voltage field. The effects of the polycation poly-L-lysine on ATP sensitivity were also independent of membrane potential or the direction (inward or outward) of current through KATP channels. In addition to increasing ATP sensitivity, Mg2+, Ba2+, and the polycations all caused dose-dependent block of inward and outward currents through KATP channels over similar concentration ranges as their effects on ATP sensitivity. The block of inward current by polycations was not associated with reduction of single-channel conductance or evidence of fast open channel block. However, the polycations did cause a modest reduction in single-channel conductance of outward current. These results are consistent with the presence of negative surface charges that reduce the local ATP concentration at the ATP-binding site(s) on the channel, relative to the bulk cytosolic ATP concentration. Screening these negative surface charges with divalent cations or polycations decreases the local ATP gradient, resulting in a decrease in the apparent Kd for ATP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cl- channels in intact human T lymphocytes.

We recently described a large, multiple-conductance Cl- channel in excised patches from normal T lymphocytes. The properties of this channel in excised patches are similar to maxi-Cl- channels found in a number of cell types. The voltage dependence in excised patches permitted opening only at nonphysiological voltages, and channel activity was rarely seen in cell-attached patches. In the present study, we show that Cl- channels can be activated in intact cells at physiological temperatures and voltages and that channel properties change after patch excision. Maxi-Cl- channels were reversibly activated in 69% of cell-attached patches when the temperature was above 32 degrees C, whereas fewer than 2% of patches showed activity at room temperature. Upon excision, the same patches displayed large, multiple-conductance Cl- channels with characteristics like those we previously reported for excised patches. After patch excision, warm temperatures were not essential to allow channel activity; 37% (114/308) of inside-out patches had active channels at room temperature. The voltage dependence of the channels was markedly different in cell-attached recordings compared with excised patches. In cell-attached patches, Cl- channels could be open at cell resting potentials in the normal range. Channel activation was not related to changes in intracellular Ca2+ since neither ionomycin nor mitogens activated the channels in cell-attached patches, Ca2+ did not rise in response to warming and the Cl- channel was independent of Ca2+ in inside-out patches. Single-channel currents were blocked by internal or external Zn2+ (100-200 microM), 4-acetamido-4' isothiocyanostilbene-2,2'-disulfonate (SITS, 100-500 microM) and 4,4'-diisothiocyanostilbene 2,2'-disulfonate (DIDS, 100 microM). NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate) reversibly blocked the channels in inside-out patches.     

Calcium- and voltage-activated potassium channels in human macrophages.

Single calcium-activated potassium channel currents were recorded in intact and excised membrane patches from cultured human macrophages. Channel conductance was 240 pS in symmetrical 145 mM K+ and 130 pS in 5 mM external K+. Lower conductance current fluctuations (40% of the larger channels) with the same reversal potential as the higher conductance channels were noted in some patches. Ion substitution experiments indicated that the channel is permeable to potassium and relatively impermeable to sodium. The frequency of channel opening increased with depolarization and intracellular calcium concentration. At 10(-7) M (Ca++)i, channel activity was evident only at potentials of +40 mV or more depolarized, while at 10(-5) M, channels were open at all voltages tested (-40 to +60 mV). In intact patches, channels were seen at depolarized patch potentials of +50 mV or greater, indicating that the ionized calcium concentration in the macrophage is probably less than 10(-7) M.     

Effect of ATP-sensitive K+ channel regulators on cystic fibrosis transmembrane conductance regulator chloride currents.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that is regulated by cAMP-dependent phosphorylation and by intracellular ATP. Intracellular ATP also regulates a class of K+ channels that have a distinct pharmacology: they are inhibited by sulfonylureas and activated by a novel class of drugs called K+ channel openers. In search of modulators of CFTR Cl- channels, we examined the effect of sulfonylureas and K+ channel openers on CFTR Cl- currents in cells expressing recombinant CFTR. The sulfonylureas, tolbutamide and glibenclamide, inhibited whole-cell CFTR Cl- currents at half-maximal concentrations of approximately 150 and 20 microM, respectively. Inhibition by both agents showed little voltage dependence and developed slowly; > 90% inhibition occurred 3 min after adding 1 mM tolbutamide or 100 microM glibenclamide. The effect of tolbutamide was reversible, while that of glibenclamide was not. In contrast to their activating effect on K+ channels, the K+ channel openers, diazoxide, BRL 38227, and minoxidil sulfate inhibited CFTR Cl- currents. Half-maximal inhibition was observed at approximately 250 microM diazoxide, 50 microM BRL 38227, and 40 microM minoxidil sulfate. The rank order of potency for inhibition of CFTR Cl- currents was: glibenclamide < BRL 38227 approximately equal to minoxidil sulfate > tolbutamide > diazoxide. Site-directed mutations of CFTR in the first membrane-spanning domain and second nucleotide-binding domain did not affect glibenclamide inhibition of CFTR Cl- currents. However, when part of the R domain was deleted, glibenclamide inhibition showed significant voltage dependence. These agents, especially glibenclamide, which was the most potent, may be of value in identifying CFTR Cl- channels. They or related analogues might also prove to be of value in treating diseases such as diarrhea, which may involve increased activity of the CFTR Cl- channel.     

Endothelium-dependent and -independent vasorelaxation by a theophylline derivative MCPT: roles of cyclic nucleotides, potassium channel opening and phosphodiesterase inhibition

The vasorelaxation activities of MCPT, a newly synthesized xanthine derivative, were investigated in this study. In phenylephrine (PE)-precontracted rat aortic rings with intact endothelium, MCPT caused a concentration-dependent relaxation, which was inhibited by endothelium removed. This relaxation was also reduced by the presence of nitric oxide synthase inhibitor Nomega-nitro-L-arginine methylester (L-NAME, 100 microM), soluble guanylyl cyclase (sGC) inhibitors methylene blue (10 microM), 1 H-[1,2,4] oxidazolol [4,3-a] quinoxalin-1-one (ODQ, 1 microM), adenylyl cyclase (AC) blocker SQ 22536 (100 microM), ATP-sensitive K+ channel blocker (KATP) glibenclamide (1 microM), a Ca2+ activated K+ channels blocker tetraethylammonium (TEA, 10 mM) and a voltage-dependent potassium channels blocker 4-aminopyridine (4-AP, 100 microM). The vasorelaxant effects of MCPT together with IBMX (0.5 microM) had an additive action. In PE-preconstricted endothelium-denuded aortic rings, the vasorelaxant effects of MCPT were attenuated by pretreatments with glibenclamide (1 microM), SQ 22536 (100 microM) or ODQ (1 microM), respectively. MCPT enhanced cAMP-dependent vasodilator isoprenaline- and NO donor/cGMP-dependent vasodilator sodium nitroprusside-induced relaxation activities in endothelium-denuded aortic rings. In A-10 cell and washed human platelets, MCPT induced a concentration-dependent increase in intracellular cyclic GMP and cyclic AMP levels. In phosphodiesterase assay, MCPT displayed inhibition effects on PDE 3, PDE 4 and PDE 5. The inhibition % were 52 +/- 3.9, 32 +/- 2.6 and 8 +/- 1.1 respectively. The Western blot analysis on HUVEC indicated that MCPT increased the expression of eNOS. It is concluded that the vasorelaxation by MCPT may be mediated by the inhibition of phosphodiesterase, stimulation of NO/sGC/ cGMP and AC/cAMP pathways, and the opening of K+ channels.     

Galanin activates nucleotide-dependent K+ channels in insulin-secreting cells via a pertussis toxin-sensitive G-protein.

The effects of galanin (7-70 nM) on ATP-sensitive K+ channels (KATP channels), membrane potential and the release of insulin have been studied in the insulinoma cell line, RINm5F. Single-channel currents have been recorded from excised outside-out membrane patches as well as intact insulin-secreting cells and it is shown that galanin, added to the outside of the membrane, specifically activates KATP channels. Studies carried out using the fluorescent probe bisoxonol demonstrate that galanin hyperpolarizes RINm5F cells. Galanin was also found to abolish glyceraldehyde-stimulated immunoreactive insulin release from the insulinoma cells. Both the galanin-evoked hyperpolarization and inhibition of insulin release were abolished in cells pre-exposed to pertussis toxin. The possibility that the gating of KATP channels could be mediated by a G-protein was studied in patch-clamp experiments by adding F- to the solution bathing the inside of the cell membranes (open-cell), in order to generate the alumino-fluoride complex AlF4-. F- (1-10 mM) evoked dose-dependent activation of KATP channels and this effect was fully reversible. F- was also able to activate K+ channels inhibited by ATP. That the fluoride activation of KATP channels is mediated by the complex AlF4- was indicated by experiments in which AlCl3 (10 microM) was found to enhance further the activation of K+ channels evoked by 1 mM F- and by results showing that F(-)-stimulation of KATP channels was (i) abolished in the continued presence of F- by the Al3+ chelator deferoxamine (0.5 mM) and (ii) could be mimicked by VO4(3-) which has a structure similar to that of the AlF4- complex.     

N-bromoacetamide removes a calcium-dependent component of channel opening from calcium-activated potassium channels in rat skeletal muscle

Calcium-activated potassium channels from cultured rat skeletal muscle were treated with the protein-modifying reagent N-bromoacetamide (NBA) (0.3-1 mM) and studied in excised patches using patch-clamp techniques. After NBA treatment, channels opened only occasionally, and, in contrast to untreated channels, the open probability was no longer sensitive to intracellular surface calcium ions (1 nM to 100 microM). Channel activity did, however, exhibit a voltage dependence similar in direction and magnitude to that shown before NBA treatment (increasing e-fold with 19 mV depolarization). Distributions of open channel lifetimes revealed that NBA treatment virtually abolished openings of long duration, which suggests that this class of openings requires calcium sensitivity. These effects were not reversed by subsequent washing. Quantitatively similar open probability, voltage dependence, and open-interval distributions were observed in untreated channels in calcium-free medium. These results suggest that NBA removed a calcium-dependent component of channel opening, and that normal channels are able to open in the absence of significant intracellular calcium concentrations.     

Visual evidence and quantification of interaction of polymeric sulfonylurea with pancreatic islet

Using a polymeric sulfonylurea (PSU) designed from glibenclamide, we examined the interactions of sulfonylurea with pancreatic islets rather than genetically remodeled beta-cell lines to clarify the biological roles of ATP-sensitive K+ (KATP) channels to which sulfonylurea binds. PSU enhanced insulin secretion from the islets with 10 nM (SU equivalent) treatment, especially at low glucose concentration, but its activity was inhibited by 100 microM diazoxide. Confocal microscopy visualized PSU interactions with the islet and revealed that the modulation of intracellular Ca2+ occurred in the same region of an islet where PSU was also bound. In quantification method of the confocal microscopic images, competition of PSU with glibenclamide on its binding sites and glucose inhibition against PSU binding were confirmed. In this study, it was concluded that the PSU was a comparable drug with glibenclamide and offered a new standard method to study intact islets.     

Increased excitability of acidified skeletal muscle: role of chloride conductance

Generation of the action potentials (AP) necessary to activate skeletal muscle fibers requires that inward membrane currents exceed outward currents and thereby depolarize the fibers to the voltage threshold for AP generation. Excitability therefore depends on both excitatory Na+ currents and inhibitory K+ and Cl- currents. During intensive exercise, active muscle loses K+ and extracellular K+ ([K+]o) increases. Since high [K+]o leads to depolarization and ensuing inactivation of voltage-gated Na+ channels and loss of excitability in isolated muscles, exercise-induced loss of K+ is likely to reduce muscle excitability and thereby contribute to muscle fatigue in vivo. Intensive exercise, however, also leads to muscle acidification, which recently was shown to recover excitability in isolated K(+)-depressed muscles of the rat. Here we show that in rat soleus muscles at 11 mM K+, the almost complete recovery of compound action potentials and force with muscle acidification (CO2 changed from 5 to 24%) was associated with reduced chloride conductance (1731 +/- 151 to 938 +/- 64 microS/cm2, P < 0.01) but not with changes in potassium conductance (405 +/- 20 to 455 +/- 30 microS/cm2, P < 0.16). Furthermore, acidification reduced the rheobase current by 26% at 4 mM K+ and increased the number of excitable fibers at elevated [K+]o. At 11 mM K+ and normal pH, a recovery of excitability and force similar to the observations with muscle acidification could be induced by reducing extracellular Cl- or by blocking the major muscle Cl- channel, ClC-1, with 30 microM 9-AC. It is concluded that recovery of excitability in K(+)-depressed muscles induced by muscle acidification is related to reduction in the inhibitory Cl- currents, possibly through inhibition of ClC-1 channels, and acidosis thereby reduces the Na+ current needed to generate and propagate an AP. Thus short term regulation of Cl- channels is important for maintenance of excitability in working muscle.     

Decrease of the contraction force of skeletal muscle in mammals by the potassium channel activator, SR 44866

The ATP-sensitive K channel opener, SR 44866, was applied to intact extensor digitorum muscle of the mouse. Electrically stimulated maximal twitch contractions of the muscle were decreased by SR 44866 with a KD of 7.4 microM and a Hill coefficient of 1.2 at 34 degrees C. The effect of SR 44866 was antagonized by glibenclamide and reduced at 24 degrees C. We suggest that these results represent the consequence of activation of ATP-sensitive K channels in mammalian skeletal muscle.     

Hydrogen sulfide-induced relaxation of resistance mesenteric artery beds of rats

Hydrogen sulfide (H2S) has been shown recently to function as an important gasotransmitter. The present study investigated the vascular effects of H2S, both exogenously applied and endogenously generated, on resistance mesenteric arteries of rats and the underlying mechanisms. Both H2S and NaHS evoked concentration-dependent relaxation of in vitro perfused rat mesenteric artery beds (MAB). The sensitivity of MAB to H2S (EC50, 25.2 +/- 3.6 microM) was about fivefold higher than that of rat aortic tissues. Removal of endothelium or coapplication of charybdotoxin and apamin to endothelium-intact MAB significantly reduced the vasorelaxation effects of H2S. The H2S-induced relaxation of MAB was partially mediated by ATP-sensitive K+ (KATP) channel activity in vascular smooth muscle cells. Pinacidil (EC50, 1.7 +/- 0.1 microM, n=6) mimicked, but glibenclamide (10 microM, n=6) suppressed, the vasorelaxant effect of H2S. KATP channel currents in isolated mesenteric artery smooth muscle cells were significantly augmented by H2S. L-cysteine, a substrate of cystathionine-gamma-lyase (CSE), at 1 mM increased endogenous H2S production by sixfold in rat mesenteric artery tissues and decreased contractility of MAB. DL-propargylglycine (a blocker of CSE) at 10 microM abolished L-cysteine-dependent increase in H2S production and relaxation of MAB. Our results demonstrated a tissue-specific relaxant response of resistance arteries to H2S. The stimulation of KATP channels in vascular smooth muscle cells and charybdotoxin/apamin-sensitive K+ channels in vascular endothelium by H2S represents important cellular mechanisms for H2S effect on MAB. Our study also demonstrated that endogenous CSE can generate sufficient H2S from exogenous L-cysteine to cause vasodilation. Future studies are merited to investigate direct contribution of endogenous H2S to regulation of vascular tone.     

Properties of single voltage-gated proton channels in human eosinophils estimated by noise analysis and by direct measurement

Voltage-gated proton channels were studied under voltage clamp in excised, inside-out patches of human eosinophils, at various pHi with pHo 7.5 or 6.5 pipette solutions. H+ current fluctuations were observed consistently when the membrane was depolarized to voltages that activated H+ current. At pHi < or = 5.5 the variance increased nonmonotonically with depolarization to a maximum near the midpoint of the H+ conductance-voltage relationship, gH-V, and then decreased, supporting the idea that the noise is generated by H+ channel gating. Power spectral analysis indicated Lorentzian and 1/f components, both related to H+ currents. Unitary H+ current amplitude was estimated from stationary or quasi-stationary variance, sigmaH2. We analyze sigmaH2 data obtained at various voltages on a linearized plot that provides estimates of both unitary conductance and the number of channels in the patch, without requiring knowledge of open probability. The unitary conductance averaged 38 fS at pHi 6.5, and increased nearly fourfold to 140 fS at pHi 5.5, but was independent of pHo. In contrast, the macroscopic gH was only 1.8-fold larger at pHi 5.5 than at pHi 6.5. The maximum H+ channel open probability during large depolarizations was 0.75 at pHi 6.5 and 0.95 at pHi 5.5. Because the unitary conductance increases at lower pHi more than the macroscopic gH, the number of functional channels must decrease. Single H+ channel currents were too small to record directly at physiological pH, but at pHi < or = 5.5 near Vthreshold (the voltage at which gH turns on), single channel-like current events were observed with amplitudes 7-16 fA.     

Intracellular pH modulates the availability of vascular L-type Ca2+ channels

L-type Ca2+ channel currents were recorded from myocytes isolated from bovine pial and porcine coronary arteries to study the influence of changes in intracellular pH (pHi). Whole cell ICa fell when pHi was made more acidic by substituting HEPES/NaOH with CO2/bicarbonate buffer (pHo 7.4, 36 degrees C), and increased when pHi was made more alkaline by addition of 20 mM NH4Cl. Peak ICa was less pHi sensitive than late ICa (170 ms after depolarization to 0 mV). pHi-effects on single Ca2+ channel currents were studied with 110 mM BaCl2 as the charge carrier (22 degrees C, pHo 7.4). In cell-attached patches pHi was changed by extracellular NH4Cl or through the opened cell. In inside-out patches pHi was controlled through the bath. Independent of the method used the following results were obtained: (a) Single channel conductance (24 pS) and life time of the open state were not influenced by pHi (between pHi 6 and 8.4). (b) Alkaline pHi increased and acidic pHi reduced the channel availability (frequency of nonblank sweeps). (c) Alkaline pHi increased and acidic pHi reduced the frequency of late channel re- openings. The effects are discussed in terms of a deprotonation (protonation) of cytosolic binding sites that favor (prevent) the shift of the channels from a sleepy to an available state. Changes of bath pHo mimicked the pHi effects within 20 s, suggesting that protons can rapidly permeate through the surface membrane of vascular smooth muscle cells. The role of pHi in Ca2+ homeostases and vasotonus is discussed.     

Ischemic shortening of action potential duration as a result of KATP channel opening attenuates myocardial stunning by reducing calcium influx

Action potential duration (APD) shortening due to opening of sarcolemmal ATP-dependent potassium (KATP) channels has been postulated to protect the myocardium against postischemic damage by reducing Ca²⁺ influx. This hypothesis was assessed, assuming that increased postischemic stunning due to KATP channel inhibition with glibenclamide could be reverted by the addition of the Ca²⁺ channel blocker diltiazem. Percent wall thickening fraction (% WTh, conscious sheep) and APD (open-chest sheep) were obtained from the following groups: control: 12 min ischemia by anterior descending coronary artery occlusion followed by 2 h reperfusion; glibenclamide: same as control, with glibenclamide (0.4 mg/kg) infused 30 min before ischemia; diltiazem: same as control, with diltiazem (100 g/kg) administered prior to ischemia; glibenclamide+diltiazem: both drugs infused as in glibenclamide and diltiazem groups. APD was reduced in control ischemia. Conversely, KATP-channel blockade by glibenclamide lengthened APD and increased postischemic stunning (p < 0.01 vs. control); glibenclamide+diltiazem did not shorten APD but enhanced functional recovery (p < 0.01 vs. glibenclamide). Ca²⁺ channel blockade improvement of increased stunning provoked by KATP channel inhibition supports the hypothesis that APD shortening due to opening of KATP channels protects against postischemic stunning by limiting Ca²⁺ influx.     

Single chloride-selective channels active at resting membrane potentials in cultured rat skeletal muscle.

The patch-clamp technique was used to characterize channels that could contribute to the resting Cl-conductance in the surface membrane of cultured rat skeletal muscle. Two Cl- -selective channels, in addition to the Cl- -selective channel of large conductance described previously (Blatz and Magleby, 1983), were observed. One of these channels had fast kinetics and a conductance of 45 +/- 1.8 pS (SE) in symmetrical 100 mM KCl. The other had slow kinetics and a conductance of 61 +/- 2.4 pS. The channel with fast kinetics typically closed within 1 ms after opening and flickered between the open and shut states. The channel with slow kinetics typically closed within 10 ms after opening and displayed less flickering. Both channels were active in excised patches of membrane held at potentials similar to resting membrane potentials in intact cells, and both were open a greater percentage of time with depolarization. Under conditions of high ion concentrations, both channels exhibited nonideal selectivity for Cl- over K+ with the permeability ratio PK/PCl of 0.15-0.2. Additional experiments on the fast Cl- channel indicated that its activity decreased with lowered pHi and that SO2-4 and CH3SO-4 were ineffective charge carriers. These findings, plus the observation that the fast Cl- channel was also active in membrane patches on intact cells, suggest that the fast Cl- channel provides a molecular basis for at least some of the resting Cl- conductance. The extent to which the slow Cl- channel contributes is less clear as it was typically active only after excised patches of membrane had been exposed to high concentrations of KCl at the inner membrane surface.     

Evidence that Na+/H+ exchange regulates angiotensin II-stimulated diacylglycerol accumulation in vascular smooth muscle cells

Angiotensin II stimulation of vascular smooth muscle cells results in initial, rapid diacylglycerol (DG) formation from the polyphosphoinositides accompanied by intracellular acidification, as well as a more sustained DG accumulation which is accompanied by a prolonged intracellular alkalinization. To determine whether intracellular pH (pHi) modulates DG accumulation, NH4Cl and potassium acetate were used to alter pHi and DG formation was measured. NH4Cl (10 mM) increased pHi from 7.15 +/- 0.05 to 7.34 +/- 0.02 pH units and markedly enhanced the sustained (5 min), but not the initial (15 s), phase of DG formation in response to 100 nM angiotensin II (65 +/- 13% increase). Conversely, intracellular acidification with Na+-free buffer and potassium acetate (20 mM) decreased pHi to 6.93 +/- 0.08 and reduced subsequent angiotensin II-induced sustained DG formation by 82 +/- 9%. In intact cells, inhibition of angiotensin II-stimulated alkalinization by incubation in Na+-free buffer or by addition of the Na+/H+ exchange inhibitor dimethylamiloride (10 microM) decreased the ability of the cell to sustain DG formation, suggesting that active Na+/H+ exchange is necessary for continued DG formation. Thus, it seems that sustained, angiotensin II-induced diacylglycerol accumulation is regulated by intracellular alkalinization secondary to Na+/H+ exchange in cultured vascular smooth muscle cells.     

Voltage-dependent K+ currents and underlying single K+ channels in pheochromocytoma cells

Properties of the whole-cell K+ currents and voltage-dependent activation and inactivation properties of single K+ channels in clonal pheochromocytoma (PC-12) cells were studied using the patch-clamp recording technique. Depolarizing pulses elicited slowly inactivating whole-cell K+ currents, which were blocked by external application of tetraethylammonium+, 4-aminopyridine, and quinidine. The amplitudes and time courses of these K+ currents were largely independent of the prepulse voltage. Although pharmacological agents and manipulation of the voltage-clamp pulse protocol failed to reveal any additional separable whole-cell currents in a majority of the cells examined, single-channel recordings showed that, in addition to the large Ca++-dependent K+ channels described previously in many other preparations, PC-12 cells had at least four distinct types of K+ channels activated by depolarization. These four types of K+ channels differed in the open-channel current-voltage relation, time course of activation and inactivation, and voltage dependence of activation and inactivation. These K+ channels were designated the Kw, Kz, Ky, and Kx channels. The typical chord conductances of these channels were 18, 12, 7, and 7 pS in the excised configuration using Na+-free saline solutions. These four types of K+ channels opened in the presence of low concentrations of internal Ca++ (1 nM). Their voltage-dependent gating properties can account for the properties of the whole-cell K+ currents in PC-12 cells.     

Activation of KATP channels by Na/K pump in isolated cardiac myocytes and giant membrane patches.

Strophanthidin inhibits KATP channels in 2,4-dinitrophenol-poisoned heart cells (). The current study shows that the Na/K pump interacts with KATP current (IK-ATP) via submembrane ATP depletion in isolated giant membrane patches and in nonpoisoned guinea pig cardiac cells in whole-cell configuration. IK-ATP was inhibited by ATP, glibenclamide, or intracellular Cs+. Na/K pump inactivation by substitution of cytoplasmic Na+ for Li+ or N-methylglucamine decreased both IK-ATP by 1/3 (1 mM ATP, zero calcium), and IC50 of ATP for IK-ATP (0.3 +/- 0.1 mM) by 2/5. The Na+/Li+ replacement had no effect on IK-ATP at low pump activity ([ATP] </= 0.1 mM or 100 microM ouabain) or when IK-ATP was completely inhibited by 10 mM ATP. In whole-cell configuration, ouabain inhibited up to 60% of inwardly rectifying IK-ATP at 1 mM ATP in the pipette but not at 10 mM ATP and 10 mM phosphocreatine when IK-ATP was always blocked. However, mathematical simulation of giant-patch experiments revealed that only 20% of ATP depletion may be attributed to the ATP concentration gradient in the bulk solution, and the remaining 80% probably occurs in the submembrane space.