A comparison of the effects of SCA40, NS 004 and NS 1619 on large conductance Ca(2+)-activated K+ channels in bovine tracheal smooth muscle cells in culture

1. The effects of imidazopyrazine derivative, SCA40, on the activity of single large conductance, Ca(2+)-activated K+ (BKCa) channels in inside-out and outside-out patches from bovine tracheal smooth muscle (BTSM) cells in culture have been compared with those of two established BKCa channel openers, NS 004 and NS 1619. 2. The presence of BKCa channels on inside-out patches of BTSM membranes was confirmed by the single channel conductance (240 pS), selectivity for K+, dependence of channel activity on [Ca2+]i, and sensitivity to the selective BKCa channel blocker, iberiotoxin. 3. NS 004 and ND 1619 (3-30 microM) induced concentration-related increases in open state probability of BKCa channels when applied to either inside-out or outside-out BTSM patches, thus confirming that these compounds are activators of the BKCa channel in this preparation. 4. SCA40 (0.1-10 microM) had no effect on the activity of BKCa channels when applied to either inside-out or outside-out patches which subsequently responded to the application of NS 004 (10-20 microM). 5. It is concluded that SCA40 does not have a direct effect on BKCa channel activity in BTSM patches and that the previously reported relaxant action of SCA40 on tracheal smooth muscle is unlikely to be mediated by this mechanism.     

Nitric oxide (NO)-induced activation of large conductance Ca2+-dependent K+ channels (BK(Ca)) in smooth muscle cells isolated from the rat mesenteric artery

1. To assess the action of nitric oxide (NO) and NO-donors on K+ current evoked either by voltage ramps or steps, patch clamp recordings were made from smooth muscle cells freshly isolated from secondary and tertiary branches of the rat mesenteric artery. 2. Inside-out patches contained channels, the open probability of which increased with [Ca2+]i. The channels had a linear slope conductance of 212+/-5 pS (n = 12) in symmetrical (140 mM) K+ solutions which reversed in direction at 4.4 mV. In addition, the channels showed K+ selectivity, in that the reversal potential shifted in a manner similar to that predicted by the Nernst potential for K+. Barium (1 mM) applied to the intracellular face of the channel produced a voltage-dependent block and external tetraethylammonium (TEA; at 1 mM) caused a large reduction in the unitary current amplitude. Taken together, these observations indicate that the channel most closely resembled BK(Ca). 3. In five out of six inside-out patches, NO (45 or 67 microM) produced an increase in BK(Ca) activity. In inside-out patches, BK(Ca) activity was also enhanced in some patches with 100 or 200 microM 3-morpholino-sydnonimine (SIN-1) (4/11) and 100 microM sodium nitroprusside (SNP) (3/8). The variability in channel opening with the NO donors may reflect variability in the release of NO from these compounds. 4. In inside-out patches, 100 microM SIN-1 failed to increase BK(Ca) activity (in all 4 patches tested), while at a higher (500 microM) concentration SIN-1 had a direct blocking effect on the channels (n = 3). NO applied directly to inside-out patches increased (P < 0.05) BK(Ca) activity in two patches. 5. In the majority of cells (6 out of 7), application of NO (45 or 67 microM) evoked an increase in the amplitude of whole-cell currents in perforated patches. This action was not affected by the soluble guanylyl cyclase inhibitor, 1H-[1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one (ODQ). An increase in whole-cell current was also evoked with either of the NO donors, SIN-1 or SNP (each at 100 microM). With SIN-1, the increase in current was blocked with the BK(Ca) channel blocker, iberiotoxin (50 nM). 6. With conventional whole-cell voltage clamp, the increase in the outward K+ current evoked with SIN-1 (50-300 microM) showed considerable variability. Either no effect was obtained (11 out of 18 cells), or in the remaining cells, an average increase in current amplitude of 38.7+/-10.2% was recorded at 40 mV. 7. In cell-attached patches, large conductance voltage-dependent K+ channels were stimulated by SIN-1 (100 microM) applied to the cell (n = 5 patches). 8. These data indicate that NO and its donors can directly stimulate BK(Ca) activity in cells isolated from the rat mesenteric artery. The ability of NO directly to open BK(Ca) channels could play an important functional role in NO-induced relaxation of the vascular smooth muscle cells in this small resistance artery.     

Bioassay of an endothelium-derived hyperpolarizing factor from bovine coronary arteries: role of a cytochrome P450 metabolite

An endothelium-derived hyperpolarizing factor (EDHF) mediates a part of the vasodilatory action of bradykinin. A bioassay method was developed to investigate the properties of EDHF on bovine coronary arterial smooth muscle cells. Cannulated bovine coronary arteries with an intact endothelium that were treated with indomethacin and NG-nitro-L-arginine methyl ester served as the EDHF donor. The effect of the donor vessel perfusate was examined on a 240 pS single-channel calcium (Ca2+)-activated potassium (K+) current (KCa) and resting membrane potential in recipient coronary arterial smooth muscle cells. The open state probability (NPo) of the channel averaged 0.011 +/- 0.003 during basal perfusate flow. After stimulation of the donor vessels with bradykinin (10(-10)-10(-6) M), the perfusate induced a 1.2- to 5-fold increase in the NPo (n = 7, p < 0.001). This increase in channel activity was attenuated by either removing the endothelium of the donor arterial segment or upon inhibition of cytochrome P450 in the donor arterial segment with the combination of 17-octadecynoic acid and miconazole. The resting cell membrane averaged -60 +/- 2 mV, and hyperpolarized to -69 +/- 1.5 mV (n = 6, p < 0.05) in response to the perfusate following stimulation of the donor vessel with bradykinin. Addition of 14, 15-epoxyeicosatrienoic acid mimicked the effects of the perfusate and increased the NPo of the KCa channel from 0.01 +/- 0.001 to 0.05 +/- 0.001. These findings suggest that bradykinin stimulates the release of a transferable endothelial factor that activates KCa channels and hyperpolarizes coronary arterial smooth muscle cell membranes. These findings support the hypothesis that coronary arteries release an EDHF which is a cytochrome P450 metabolite of arachidonic acid.     

Protein kinase C inhibits the Ca(2+)-activated K+ channel of cultured porcine coronary artery smooth muscle cells

The effect of protein kinase C (C-kinase) on the Ca(2+)-activated K+ channel (KCa-channel) was studied in cultured smooth muscle cells from porcine coronary artery by the patch-clamp technique. In cell-attached patches, bath application of phorbol 12-myristate 13-acetate (PMA, 1 microM), a C-kinase activator, significantly decreased the open probability of the activated KCa-channel in the presence of the calcium ionophore A23187 (20 microM), which increases intracellular Ca2+. This decrease in the open probability was reversed by subsequent application of staurosporine (1 nM), a C-kinase inhibitor. Application of 1-oleoyl-2-acetylglycerol (OAG, 30 microM) or 1,2-dioctanoylglycerol (DG8; 30 microM), activators of C-kinase, also inhibited KCa-channel activation by A23187, and these inhibitions were also reversed by staurosporine. PMA (1 microM) also inhibited KCa-channel activation by dibutylyl cyclic AMP (db-cAMP, 2 mM) or caffeine (30 mM). In inside-out patches, bath application of the C-kinase fraction from rat brain in the presence of ATP (1 mM) and PMA (1 microM) markedly inhibited the KCa-channel. These results indicate that activation of C-kinase inhibits the KCa-channel and may cause membrane depolarization and vascular contraction.     

A single non-L-, non-N-type Ca2+ channel in rat insulin-secreting RINm5F cells

Single high-voltage-activated (HVA) Ca2+ channel activity was recorded in rat insulinoma RINm5F cells using cell-attached and outside-out configurations. Single-channel recordings revealed three distinct Ca2+ channel subtypes: one sensitive to dihydropyridines (DHPs)-(L-type), another sensitive to omega -conotoxin (CTx)-GVIA (N-type) and a third type insensitive to DHPs and omega -CTx-GVIA (non-L-, non-N-type). The L-type channel was recorded in most patches between -30 and +30 mV. The channel had pharmacological and biophysical features similar to the L-type channels described in other insulin-secreting cells (mean conductance 21 pS in control conditions and 24 pS in the presence of 5 microM Bay K 8644). The non-L-, non-N-type channel was recorded in cells chronically treated with omega -CTx-GVIA in the presence of nifedipine to avoid the contribution of N- and L-type channels. Channel activity was hardly detectable below -10 mV and was recruited by negative holding potentials (< -90 mV). The channel open probability increased steeply from -10 to + 40 mV. Different unitary current sublevels could be detected and the current voltage relationship was calculated from the higher amplitude level with a slope conductance of 21 pS. Channel activity lasted throughout depolarizations of 300-800ms with little sign of inactivation. Above 0 mV the channel showed a persistent flickering kinetics with brief openings (tau o 0.6 ms) and long bursts (tau burst 60 ms) interrupted by short interburst intervals. The third HVA Ca2+ channel subtype, the N-type, had biophysical properties similar to the non-L-, non-N-type and was best identified in outside-out patches by its sensitivity to omega -CTx-GVIA. The channel was detectable only above -10 mV from a -90 mV holding potential, exhibited a fast flickering behaviour, persisted during prolonged depolarizations and had a slope conductance of about 19 pS. The present data provide direct evidence for a slowly inactivating non-L-, non-N-type channel in insulin-secreting RINm5F cells that activates at more positive voltages than the L-type channel and indicate the possibility of identifying unequivocally single HVA Ca2+ channels in cell-attached and excised membrane patches under controlled pharmacological conditions.     

Characterization of the calcium-activated chloride channel in isolated guinea-pig hepatocytes

Macroscopic and unitary currents through Ca(2+)-activated Cl- channels were examined in enzymatically isolated guinea-pig hepatocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. When K+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+]i) was set at 1 microM (pCa = 6), membrane currents were observed under whole-cell voltage-clamp conditions. The reversal potential of the current shifted by approximately 60 mV per 10-fold change in the external Cl- concentration. In addition, the current did not appear when Cl- was omitted from the internal and external solutions, indicating that the current was Cl- selective. The current was activated by increasing [Ca2+]i and was inactivated in Ca(2+)-free, 5 mM EGTA internal solution (pCa > 9). The current was inhibited by bath application of 9-anthracenecarboxylic acid (9-AC) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in a voltage-dependent manner. In single channel recordings from outside-out patches, unitary current activity was observed, whose averaged slope conductance was 7.4 +/- 0.5 pS (n = 18). The single channel activity responded to extracellular Cl- changes as expected for a Cl- channel current. The open time distribution was best described by a single exponential function with mean open lifetime of 97.6 +/- 10.4 ms (n = 11), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 21.5 +/- 2.8 ms (n = 11) and that for the slow component of 411.9 +/- 52.0 ms (n = 11). In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The relationship between [Ca2+]i and channel activity was fitted by the Hill equation with a Hill coefficient of 3.4 and the half-maximal activation was 0.48 microM. These results suggest that guinea-pig hepatocytes possess Ca(2+)-activated Cl- channels.     

Effects of halothane and isoflurane on bradykinin-evoked Ca2+ influx inbovine aortic endothelial cells

BACKGROUND: Volatile anesthetics, such as halothane and isoflurane, have been reported to affect the endothelium mediated relaxation of vascular smooth muscle cells. Because the activity of the constitutive nitric oxide synthase in endothelial cells depends on the availability of intracellular Ca2+, there is a definite possibility that the observed inhibitory effect of volatile anesthetics involves an action on the agonist-evoked internal Ca2+ mobilization and/or Ca2+ influx in these cells. Therefore, a study was undertaken to determine how halothane and isoflurane affect the Ca2+ signalling process in vascular endothelial cells. METHODS: The effect of halothane and isoflurane on the Ca2+ response to bradykinin of bovine aortic endothelial (BAE) cells was investigated using the fluorescent Ca2+ indicator fura-2. Halothane or isoflurane was applied either to resting cells or after bradykinin stimulation. The agonist-evoked Ca2+ influx in BAE cells was estimated by measuring either the rate of fura-2 quenching induced by Mn2+ or the increase in cytosolic Ca2+ concentration initiated after readmission of external Ca2+ after a brief exposure of the cells to a Ca(2+)-free external medium. The effects of halothane on cell potential and intracellular Ca2+ concentration were measured in cell-attached patch-clamp experiments in which a calcium-activated K+ channel and an inward rectifying Ca(2+)-independent K+ channel were used as probes to simultaneously monitor the intracellular Ca2+ concentration and the cell transmembrane potential. In addition, combined fura-2 and patch-clamp cell-attached recordings were carried out, to correlate the variations in internal Ca2+ caused by halothane and the activity of the Ca(2+)-dependent K+ channels, which are known in BAE cells to regulate intracellular potential. Finally, a direct action of halothane and isoflurane on the gating properties of the Ca(2+)-activated K+ channel present in these cells was investigated in patch-excised inside-out experiments. RESULTS: The results of the current study indicate that the initial Ca2+ increase in response to bradykinin stimulation is not affected by halothane, but that pulse applications of halothane (0.4-2 mM) or isoflurane (0.5-1 mM) reversibly reduce the sustained cytosolic Ca2+ increase initiated either by bradykinin or by the Ca2+ pump inhibitor thapsigargin. In addition, halothane appeared to dose-dependently inhibit the Ca2+ influx evoked by bradykinin, and to cause, concomitant to a decrease in cytosolic Ca2+ concentration, a depolarization of the cell potential. Halothane failed, however, to affect internal Ca2+ concentration in thapsigargin-treated endothelial cells, which were depolarized using a high K+ external solution. Finally, halothane and isoflurane decreased the open probability of the Ca(2+)-dependent K+ channel present in these cells. CONCLUSIONS: These observations suggest that the effects of halothane and isoflurane on Ca2+ homeostasis in BAE cells reflect, for the most part, a reduction of the thapsigargin- or bradykinin-evoked Ca2+ influx, which would be consequent to a cellular depolarization caused by an inhibition of the Ca(2+)-dependent K+ channel activity initiated after cell stimulation.     

Multiple effects of nordihydroguaiaretic acid on ionic currents in rat isolated type I carotid body cells

1. The effects of the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) on the ionic currents of rat carotid body type I cells were investigated by use of whole-cell and outside-out patch clamp techniques. 2. NDGA (5-50 microM) produced a concentration-dependent inhibition of whole-cell K+ currents at all activating test potentials (holding potential -70 mV). The time-course of the inhibition was also concentration-dependent and the effects of NDGA were only reversible following brief periods of exposure (<2 min). Another lipoxygenase inhibitor, phenidone (5 microM), was without effect on whole-cell K+ currents in carotid body type I cells. 3. NDGA (5-50 microM) also inhibited whole-cell Ca2+ channel currents (recorded with Ba2+ as charge carrier) in a concentration-dependent manner. 4. Isolation of voltage-gated K+ channels by use of high [Mg2+] (6 mM), low [Ca2+] (0.1 mM) solutions revealed a direct inhibition of the voltage-sensitive component of the whole-cell K+ current by NDGA (50 microM). 5. In excised, outside-out patches NDGA (20-50 microM) increased large conductance, Ca2+ activated K+ channel activity approximately 10 fold, an effect which could be reversed by either tetraethylammonium (10 mM) or charybdotoxin (30 nM). 6. It is concluded that NDGA activates maxi-K+ channels in carotid body type I cells and over the same concentration range inhibits voltage-sensitive K+ and Ca2+ channels. The inhibition of whole cell K+ currents seen is most likely due to a combination of direct inhibition of the voltage-sensitive K+ current and indirect inhibition of maxi-K+ channel activity through blockade of Ca2+ channels.     

Immunohistochemical detection of vascular growth factors in angiomatous and atypical meningiomas, as well as hemangiopericytomas

Nonselective cation channels have been identified and linked to important cell functions in rat hepatocytes. In this study, we characterized inward rectifying nonselective cation channels in detail by the patch clamp technique in human HepG2 cells. Channel properties were studied with high resistance borosilicate pipettes in cell-attached and inside-out configurations. With Ringer's solution and KCl as pipette solutions, the conductances were 19.7 +/- 2.1 and 22.2 +/- 0.0 picosiemens (pS), and reversal potentials were 30.9 +/- 3.5 and 31.3 +/- 4.6 mV, respectively. The channel was permeable to Ba2+, and the sequence of permeability ratios was Na+ > K+ > Cs+ > Ba2+. In the cell-attached configuration, the channel had a higher opening probability at depolarizing potential than at hyperpolarizing. In the inside-out patches with symmetric Ringer's solution, the current voltage curve was linear with conductance of 19.8 +/- 0.9 pS. Reversal potential shifted from -0.2 +/- 1.0 mV to 23.2 +/- 1.0 mV when the bath solution was replaced by dilute Ringer's solution. In the inside-out configuration, the gating was Ca(2+)-dependent, and the opening probability increased with increasing intracellular calcium concentration ([Ca2+]i). An outward rectifying channel appeared when [Ca2+]i was less than 1 mumol/L. The nonselective channel was reversibly blocked by 10 mumol/L internal flufenamic acid. We conclude that Ca(2+)- and voltage-dependent nonselective cation channels are present in human HepG2 cells. The channels might be involved in the regulation of Ca2+ influx and are associated with activation of other ion channels.     

B-type Ca2+ channels activated by chlorpromazine and free radicals in membrane of human atrial myocytes

The present study demonstrates that background or B-type calcium channel activity can be recorded in excised inside-out and cell-attached membrane patches from human atrial myocytes. In control conditions, with Ba2+ or Ca2+ as charge carrier, single-channel activity spontaneously appeared in irregular bursts separated by quiescent periods of 2-17 min, in nearly 25% of tested patches. Channel activity was recorded at steady-state applied membrane potentials including the entire range of physiological values, and displayed no "rundown" in excised patches. During activity, a variety of kinetic behaviors could be observed with more or less complex gating patterns. This type of channel activity was triggered or markedly increased when chlorpromazine (CPZ 20 or 50 microM) was applied to internal face of inside-out patches, with a proportion of active patches of approximately 25%. CPZ-activated channels were potential-independent in the physiological range of membrane potential. In 96 mM Ba2+ solution, three conductance levels: 23, 42 and 85 pS were routinely observed in the same excised membrane patch, sometimes combining to give a larger level. As previously observed by Wang et al. (1995) in membrane of rat ventricular myocytes, increasing free-radicals level and metabolic poisoning readily enhanced B-type channel activity in human atrial myocytes. Application of H2O2 (from 0.1-10 mM) in cell-attached mode induced an activation of Ba2+ permeable channel activity in a dose-dependent manner, with an estimated EC50 of 9.7 mM. In the same type of experiments, 10 mM deoxyglucose also induced similar Ba2+ permeable channel activity. When 500 microM CPZ were applied to myocytes studied in the whole-cell configuration and maintained at a holding potential of -80 mV in the presence of 5 mM external Ca2+, a noticeable inward current could be observed. The mean CPZ-activated current density determined from seven myocytes was 0.63 pA/pF.     

Inhibitory effects of genistein on ATP-sensitive K+ channels in rabbit portal vein smooth muscle

1. Effects on the pinacidil-induced outward current of inhibitors of tyrosine kinases and phosphatases were investigated by use of a patch-clamp method in smooth muscle cells of the rabbit portal vein. 2. A specific tyrosine kinase inhibitor, genistein, inhibited the pinacidil-induced current in a concentration-dependent manner with an IC50 of 5.5 microM. Superfusion of Ca2+-free solution did not affect this inhibitory effect of genistein. At higher concentrations, genistein inhibited the voltage-dependent Ba2+ and K+ currents with IC50 values of > 100 microM and 75 microM respectively. Tyrphostin B46 (30 microM), a tyrosine kinase inhibitor, also inhibited the pinacidil-induced current by 70% of the control. 3. Sodium orthovanadate (100 microM), an inhibitor of tyrosine phosphatase, slightly but significantly enhanced both the pinacidil-induced and delayed rectifier K+ currents. Daidzein (100 microM), an inactive analogue of genistein, did not inhibit these currents. 4. Neither herbimycin A (1 microM), lavendustin A (30 microM), tyrphostin 23 (10 microM), which are also tyrosine kinase inhibitors, nor wortmannin (10 microM), a phosphatidylinositol 3-kinase inhibitor, had an effect on either the pinacidil-induced or delayed rectifier K+ currents. Epidermal growth factor (EGF; 1 microg ml(-1)) did not induce an outward current or enhance the pinacidil-induced current. 5. Pinacidil alone, in the cell-attached configuration, or pinacidil with GDP, in the inside-out configuration, activated a 42 pS channel in the smooth muscle cells of the rabbit portal vein. Genistein (30 microM) reduced the channel's open probability without inducing a change in unitary conductance at any holding potential (-30 to +20 mV). 6. In the inside-out configuration, genistein at 30 microM did not change the mean channel open time, but reduced the burst duration. At 100 microM genistein abolished channel opening. The inhibitory potencies with which 30 and 100 microM genistein acted on the unitary current of the ATP-sensitive K+ channel were similar to those seen in the whole-cell voltage-clamp configuration. 7. Although direct inhibitory actions of genistein on the ATP-sensitive K+ channels are not ruled out, our results suggest that a protein tyrosine kinase may play a role in the regulation of ATP-sensitive K+ channel activity in the rabbit portal vein.     

Divalent cation channels activated by phenothiazines in membrane of rat ventricular myocytes

Phenothiazines (PTZ) such as chlorpromazine (CPZ) or trifluoperazine (TPZ) induced a sustained divalent cation-permeable channel activity when applied on either side of inside-out patches or on external side of cell-attached patches of adult rat ventricular myocytes. The percentage of active patches was approximately 20%. In the case of CPZ, the Kd of the dose-response curve was 160 microM. CPZ-activated channels were potential-independent in the physiological range of membrane potential and were permeable to several divalent ions (Ba2+, Ca2+, Mg2+, Mn2+). At least three levels of currents were usually detected with conductances of 23, 50 and 80 pS in symmetrical 96 mM Ba2+ solution and 17, 36 and 61 pS in symmetrical 96 mM Ca2+ solution. Saturation curves corresponding to the three main conductances determined in Ba2+ symmetrical solutions (tonicity compensated with choline-Cl) gave maximum conductances of 36, 81 and 116 pS (with corresponding half-saturating concentration constants of 31.5, 38 and 34.5 mM). The corresponding conductance values were estimated to 1.7, 3.3 and 5.2 pS in symmetrical 1.8 mM Ba2+ and to 1.1, 2.4 and 3.7 pS in symmetrical 1.8 mM Ca2+ (the value in normal Tyrode solution). Channels were poorly permeable to monovalent cations, such as Na, with a PBa/PNa ratio of 10. A PTZ-induced channel activity similar to that described in cardiac cells was also observed in cultured rat aortic smooth muscle cells but not in cultured neuroblastoma cells. PTZ-activated channels described in cardiac cells appear very similar to the sporadically active divalent ion permeable channels described in a previous paper (Coulombe et al., 1989). Surprisingly, when 100 microM CPZ were applied to myocytes studied in the whole-cell configuration, and maintained at a holding potential of -80 mV in the presence of 24 mM external Ca2+ or Ba2+, no detectable macroscopic inward current could be observed, whereas the L-type Ca2+ current triggered by depolarizing pulses was markedly and reversibly reduced. The possible reasons are discussed.     

MSX-1 gene expression and regulation in embryonic palatal tissue

1. During cardiac surgery, the heart is arrested and protected by hyperkalaemic cardioplegia. The coronary endothelium may be damaged by ischaemia-reperfusion and cardioplegia. Subsequently, this may affect cardiac function immediately after cardiac surgery and cause mortality and morbidity. 2. We investigated coronary endothelium-smooth muscle interaction after exposure to depolarizing (hyperkalaemic; K+ 20 or 50 mmol/L) and hyperpolarizing (the K+ channel opener aprikalim) cardioplegia and organ preservation solution (University of Wisconsin (UW) solution). Endothelium-dependent relaxation and hyperpolarization of the coronary smooth muscle were studied in the porcine and human large conductance and micro-coronary arteries. Intracellular free calcium concentration in endothelial cells was also measured. 3. The endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation to A23187, bradykinin, and substance P in arteries contracted by either U46619 (10 nmol/L) or K+ (25 mmol/L) was reduced after exposure to either high K+ or UW solution, but was maximally preserved after exposure to aprikalim. The hyperpolarization of the membrane potential in response to the above endothelium-derived relaxing factor stimuli was also reduced by exposure to depolarizing cardioplegia. Studies in microcoronary arteries are in accordance with findings in large arteries. The intracellular free calcium concentration remained unchanged after exposure to hyperkalaemia. 4. We concluded that: (i) during cardiac surgery, the function of coronary circulation may be changed due to exposure to depolarizing cardioplegia or preservation solutions; (ii) the functional change in the coronary circulation is related to the altered interaction between the endothelium and smooth muscle; (iii) depolarizing (hyperkalaemia) cardioplegia or hyperkalaemic organ preservation solutions affect endothelium-smooth muscle interaction through the EDHF pathway; (iv) EDHF relaxes the porcine large and microcoronary arteries through multiple K+ channels; and (v) that hyperpolarizing vasodilators (K+ channel openers) may protect EDHF-mediated endothelial function when used as cardioplegia.     

Genotoxicity of selected metal compounds in the SOS chromotest

We have recently proposed that anandamide, or a related cannabinoid, is the endothelium-derived hyperpolarizing factor (EDHF) and have now compared EDHF-mediated responses (induced by carbachol in the presence of both nitric oxide and prostanoid synthesis inhibitors) with those induced by anandamide in the rat isolated superior mesenteric arterial bed. Both EDHF-mediated and anandamide-induced relaxations were inhibited in the presence of high K+ (60 mM) and opposed by blockade of K+ channels with 10 mM tetraethylammonium. The cytochrome P450 inhibitors, and putative EDHF inhibitors, clotrimazole (10 microM) and proadifen (SKF 525A) (10 microM), opposed both anandamide-induced and EDHF-mediated relaxations and also relaxant responses to the K+ channel activator levcromakalim. Therefore, EDHF-mediated and anandamide-induced vasorelaxations show very similar pharmacological characteristics, with both responses being mediated via K+ channel activation. Further, the actions of EDHF and anandamide are both sensitive to proadifen and clotrimazole, EDHF antagonists which appear to act through K+ channel inhibition. Accordingly, these results support our proposal that an endocannabinoid is an EDHF.     

K+ is an endothelium-derived hyperpolarizing factor in rat arteries

In arteries, muscarinic agonists such as acetylcholine release an unidentified, endothelium-derived hyperpolarizing factor (EDHF) which is neither prostacyclin nor nitric oxide. Here we show that EDHF-induced hyperpolarization of smooth muscle and relaxation of small resistance arteries are inhibited by ouabain plus Ba2+; ouabain is a blocker of Na+/K+ ATPase and Ba2+ blocks inwardly rectifying K+ channels. Small increases in the amount of extracellular K+ mimic these effects of EDHF in a ouabain- and Ba2+-sensitive, but endothelium-independent, manner. Acetylcholine hyperpolarizes endothelial cells and increases the K+ concentration in the myoendothelial space; these effects are abolished by charbdotoxin plus apamin. Hyperpolarization of smooth muscle by EDHF is also abolished by this toxin combination, but these toxins do not affect the hyperpolarizaiton of smooth muscle by added K+. These data show that EDHF is K+ that effluxes through charybdotoxin- and apamin-sensitive K+ channels on endothelial cells. The resulting increase in myoendothelial K+ concentration hyperpolarizes and relaxes adjacent smooth-muscle cells by activating Ba2+-sensitive K+ channels and Na+/K+ ATPase. These results show that fluctuations in K+ levels originating within the blood vessel itself are important in regulating mammalian blood pressure and flow.     

The large conductance, voltage-dependent, and calcium-sensitive K+ channel, Hslo, is a target of cGMP-dependent protein kinase phosphorylation in vivo

Native large conductance, voltage-dependent, and Ca2+-sensitive K+ channels are activated by cGMP-dependent protein kinase. Two possible mechanisms of kinase action have been proposed: 1) direct phosphorylation of the channel and 2) indirect via PKG-dependent activation of a phosphatase. To scrutinize the first possibility, at the molecular level, we used the human pore-forming alpha-subunit of the Ca2+-sensitive K+ channel, Hslo, and the alpha-isoform of cGMP-dependent protein kinase I. In cell-attached patches of oocytes co-expressing the Hslo channel and the kinase, 8-Br-cGMP significantly increased the macroscopic currents. This increase in current was due to an increase in the channel voltage sensitivity by approximately 20 mV and was reversed by alkaline phosphatase treatment after patch excision. In inside-out patches, however, the effect of purified kinase was negative in 12 of 13 patches. In contrast, and consistent with the intact cell experiments, purified kinase applied to the cytoplasmic side of reconstituted channels increased their open probability. This stimulatory effect was absent when heat-denatured kinase was used. Biochemical experiments show that the purified kinase incorporates gamma-33P into the immunopurified Hslo band of approximately 125 kDa. Furthermore, in vivo phosphorylation largely attenuates this labeling in back-phosphorylation experiments. These results demonstrate that the alpha-subunit of large conductance Ca2+-sensitive K+ channels is substrate for G-Ialpha kinase in vivo and support direct phosphorylation as a mechanism for PKG-Ialpha-induced activation of maxi-K channels.     

Exogenous heat shock protein hsp70 activates potassium channels in U937 cells

With the use of patch clamp technique, the effect of exogenous heat shock protein hsp70 on ion channel properties in the plasma membrane of human promonocyte U937 cells has been examined. Cell-attached experiments showed that the addition of 30-100 micrograms/ml hsp70 to the pipette solution resulted in an activation of outward currents through potassium-selective channels of 9 pS unitary conductance. The activity of K(+)-selective channels did not depend on membrane voltage and could be controlled by the intracellular free calcium concentration as revealed in inside-out recordings. K+ channels with similar conductance and kinetic behaviour were found in normal cell-attached patches very rarely. Outside-out experiments showed that the addition of hsp70 to the external solution induced a channel-like stepwise increase of inward current which may provide cation entry from the extracellular medium. The interaction of extracellular hsp70 with the membrane surface of the native cell and of the excised fragment was found to be different. The results suggest that hsp70-induced activation of Ca-dependent K channels in monocyte-macrophage cells may be due to a local increase of free Ca2+ concentration just near the inner membrane side.     

3,4 dichlorobenzamil-sensitive, monovalent cation channel induced by palytoxin in cultured aortic myocytes

1. Smooth muscle cells were dispersed from rat aorta and then cultured. The action of palytoxin on rat aortic myocytes was analysed by measurement of 22Na+ uptake and single channel recording techniques. 2. Palytoxin induced an increase in 22Na+ uptake, with a concentration of 50 nM producing half-maximal activation. The action of palytoxin was inhibited by amiloride derivatives and by ouabain. The concentrations of inhibitor producing half-maximal inhibition were 10 microM for 3,4 dichlorobenzamil, 30 microM for benzamil, 100 microM for phenamil and 1 mM for ouabain. 3. In outside-out patches, palytoxin induced single channel currents that reversed near 0 mV with NaCl or KCl in the extracellular solution, but were outward with N-methyl-D-glucamine chloride or CaCl2 (110 mM), indicating that palytoxin induced a cation channel permeable to Na+ and K+ (PK/PNa = 1.2) but not to Ca2+ (PK/PCa > 30) or to N-methyl-D-glucamine (NMDG) (PK/PNMDG > 11) The unit channel conductance was 11-14 pS. 4. A high (> 0.1 mM) extracellular concentration of Ca2+ was necessary to observe channel activation by palytoxin. A high (150 mM) extracellular concentration of K+ partially prevented and reversed channel activation by palytoxin. 5. The channel activity was fully blocked by 3,4 dichlorobenzamil (20 microM) and partially blocked by phenamil (50 microM). It was not reduced by ouabain (200 microM).     

Protective effects of mercaptoethylguanidine, a selective inhibitor of inducible nitric oxide synthase, in ligature-induced periodontitis in the rat

We have demonstrated previously that photomodification permeabilizes cardiac cells as evidenced by activation of a whole-cell leak current. In this paper we report that photomodification induces in cell-attached and inside-out cardiac membrane patches a chaotic current. Unlike current recordings from many protein ion channels that show stepwise amplitude changes associated with open and closed states of the channel, the chaotic current consists of variable amplitude spike-like transitions. The amplitudes of these spikes can vary from tenths to tens of picoamperes at a constant transmembrane potential. We provide evidence that the chaotic current is transmembrane rather than trans-seal and has a voltage dependency expected for current flow through nonspecific conductance pathways. Photomodification can also induce high conductance states (greater than 500 pS) in cell-attached and inside-out patches. We present evidence that the high conductance state is also not related to seal breakdown. Our results suggest that both the chaotic current activity in and high conductance state of photomodified cardiac membrane patches result from the opening of many small conductance, nonspecific pathways through the membrane.     

Identification of a novel Rac3-interacting protein C1D

The actin cytoskeleton is an important contributor to the integrity of cellular shape and responses in neurons. However, the molecular mechanisms associated with functional interactions between the actin cytoskeleton and neuronal ion channels are largely unknown. Whole-cell and single channel recording techniques were thus applied to identified retinal bipolar neurons of the tiger salamander (Ambystoma tigrinum) to assess the role of acute changes in actin-based cytoskeleton dynamics in the regulation of voltage-gated ion channels. Disruption of endogenous actin filaments after brief treatment (20-30 min) with cytochalasin D (CD) activated voltage-gated K+ currents in bipolar cells, which were largely prevented by intracellular perfusion with the actin filament-stabilizer agent, phalloidin. Either CD treatment under cell-attached conditions or direct addition of actin to excised, inside-out patches of bipolar cells activated and/or increased single K+ channels. Thus, acute changes in actin-based cytoskeleton dynamics regulate voltage-gated ion channel activity in bipolar cells.