INTRODUCTION

Top Abstract Introduction METHODS RESULTS DISCUSSION References

THE CALCIUM-ACTIVATED potassium (K_Ca) channel has been identified in all vascular smooth muscle cells investigated so far. The K current carried by K_Ca channels is one of the major outward currents of vascular smooth muscle cells. Recently, it has been shown that in arteries exposed to a physiological pressure level but not subjected to any vasoactive agonist this current belongs to the ion currents that establish the membrane potential, one of the main factors determining the contractile state of these arteries (5). Thus the contractile state of these arteries may be altered by either an increase of the K_Ca current, leading to membrane potential hyperpolarization and vasodilation, or by a decrease of the K_Ca current, leading to membrane potential depolarization and vasoconstriction. Indeed, an increase of the arterial K_Ca current has been shown to be important, for example, for the protein kinase G (PKG)-mediated vasodilation induced by NO (2) and estrogen (28) as well as for the protein kinase A (PKA)-mediated vasodilation induced by iloprost (23). Furthermore, a decrease of the arterial K_Ca current has been shown to be important for the vasoconstriction induced by 20-hydroxy-(5Z,8Z,11Z,14Z)-eicosatetraenoic acid (30). However, in contrast to numerous studies showing that vasodilations accompanied by an increase of the K_Ca current are mediated by intracellular messengers like PKA and PKG, the intracellular messengers mediating vasoconstrictions accompanied by a decrease of the K_Ca current are not well known. Yet, recent reports showing that another K current, the ATP-sensitive K current, is involved in the vasoconstriction induced by _2D receptor agonists (26) and that the decrease of this current produced by neuropeptide Y, phenylephrine, serotonin, and histamine (4) is mediated by protein kinase C (PKC) suggest that PKC may be an intracellular messenger mediating vasoconstrictions accompanied by a decrease of the K_Ca current. This hypothesis is supported by recent findings demonstrating that spontaneous transient outward currents, which represent K_Ca currents induced by local Ca release events, are inhibited by a PKC-mediated depletion of Ca stores in rabbit portal vein (13) or by a PKC-mediated inhibition of ryanodine receptor channel activity in rat cerebral arteries (3). In the latter study, it was additionally suggested that the PKC-mediated decrease of the arterial smooth muscle K_Ca current may be caused by some other mechanisms unrelated to local Ca release events. Thus an inhibition of K_Ca channels by PKC was proposed, although this question had not been studied in detail (3). This hypothesis is supported by the observation that activators of PKC can inhibit the activity of single K_Ca channels in cultured porcine coronary artery smooth muscle cells (18). However, considerable alterations can occur in cultured smooth muscle cells in comparison with native cells, including the appearance of K_Ca channels with atypical properties (25) and the disappearance of K_Ca channels (8). Thus the aim of this study was to explore in more detail the possibility that a PKC-mediated decrease of the arterial smooth muscle K_Ca current may be produced, at least partly, independently of effects of PKC on local Ca release events. To accomplish this, experiments were performed to investigate the effect of an activation of endogenous PKC by a diacylglycerol analog, 1,2-dioctanoyl-sn-glycerol (DOG), and of the application of PKC on the whole cell K_Ca current of freshly isolated arterial smooth muscle cells dialyzed with a strongly Ca-buffered solution to eliminate local Ca release events as well as the effect of the application of PKC on excised single K_Ca channels of these cells.

	METHODS

Top Abstract Introduction METHODS RESULTS DISCUSSION References

Cell isolation and solutions. Tail arteries were obtained from male Wistar-Kyoto rats, which were killed by stunning and subsequent decapitation. A 2- to 3-cm-long piece of an artery was placed into a microtube containing 1 ml of an enzyme solution and stored there overnight at 4°C. The enzyme solution contained (in mM) 110 NaCl, 5 KCl, 0.16 CaCl₂, 2 MgCl₂, 10 NaHEPES, 10 NaHCO₃, 0.5 KH₂PO₄, 0.5 NaH₂PO₄, 10 glucose, 0.49 EDTA, and 10 taurine at pH 7.0, and also 1.5 mg/ml papain (12,000 U/g), 1.6 mg/ml albumin, and 0.4 mg/ml DL-dithiothreitol. The next day, the microtube with the vessel was incubated for 5-10 min at 37°C. Single cells were released by trituration with a polyethylene pipette into the experimental bath solution. The experimental bath solution in the whole cell experiments contained (in mM) 135 NaCl, 6 KCl, 0.1 CaCl₂, 1 MgCl₂, 3 EGTA (purity 96%), and 10 HEPES at pH 7.4, with a calculated free Ca concentration of ~3 × 10⁹ M. The experimental bath solution in the inside-out experiments contained (in mM) 140 KCl, 3 EGTA (purity 96%), 3 HEDTA (purity 98%), an appropriate amount of MgCl₂ to get a free Mg concentration of 1 mM, an appropriate amount of CaCl₂ to get the desired free Ca concentration, and 10 HEPES at pH 7.4. For a first estimate of the free Ca concentrations, solution composition was calculated with the following apparent reaction constants (K) at pH 7.4: log K_CaEGTA = 7.17, log K_MgEGTA = 1.93, log K_CaHEDTA = 5.67, and log K_MgHEDTA = 4.37 (21). The exact free Ca concentration in the bath solution for the inside-out experiments was measured using fura 2, and, after calibration of the signal with K-based standard solutions (CALBUF-2; World Precision Instruments), a concentration of 2.5 µM was determined. The pipette solution contained (in mM) 102 KCl, 10 NaCl, 1 CaCl₂, 1 MgCl₂, 10 EGTA (purity 96%) or 10 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; purity 98%), 10 HEPES, and 0.1 MgATP at pH 7.4, with a calculated free Ca concentration of ~8 × 10⁹ M in the whole cell experiments and 135 NaCl, 6 KCl, 1 CaCl₂, 1 MgCl₂, and 10 HEPES at pH 7.4 in the inside-out experiments.

Patch-clamp recordings. All patch-clamp experiments were performed at room temperature (22-24°C). Patch pipettes were prepared from borosilicate glass (WP Instruments) and had a resistance in the range of 1-3 M when filled with the pipette solution. Recordings were made with an Axopatch 200 amplifier (Axon Instruments) with the conventional whole cell and inside-out configuration.

Whole cell experiments. In the whole cell experiments, stimulation of currents with pulse and ramp protocols, data sampling at a rate of 1 kHz for ion currents and of 50 kHz for cell capacitance and series resistance determination, and data analysis were all done with the software package ISO2 (MFK). Experiments were performed only on cells with negligible leaks. Series resistance compensation was not employed because often cells were lost even at slight overcompensation. Instead, series resistance was measured every 2 min during the course of the experiment. Data were used for analysis if the command voltage error due to series resistance changed <2 mV. In a few cases, a voltage error change of 5 mV was accepted, but these data were corrected using the current-voltage (I-V) relationship obtained during ramp pulses. Whole cell currents were elicited every 5 s by either 500-ms step pulses from a holding potential of 40 mV to a test potential of +70 mV or by 1,000-ms ramp pulses from 70 to +100 mV. Current amplitudes were either determined from step pulse stimulation and measured as the mean current of the last 100 ms of the average of three consecutive current traces or determined from ramp pulse stimulation at the desired potentials. Preliminary tests showed that the current amplitudes at a certain potential obtained from consecutive step pulse and ramp pulse stimulations do not differ significantly. The difference in command potential to obtain the same current amplitude with ramp pulse stimulation compared with step pulse stimulation at +70 mV was only 1.8 ± 0.3 mV (n = 34).

Isolation of the K_Ca current. The K_Ca current was defined as the difference of the net outward current amplitude under the desired condition, e.g., control or PKC application, and the outward current amplitude after application of 100 nM iberiotoxin, the specific inhibitor of K_Ca channels (9, 10), which was given at the end of each experiment. The correctness of this procedure was proven by the observation that the iberiotoxin-resistant currents after the application of DOG or PKC were not significantly different from the currents without application of any substance (data not shown). The threshold potential of the K_Ca current was determined as the potential of the intercept of the I-V relationships of the K_Ca current before and after iberiotoxin application. To be able to test the hypothesis that PKC may have an effect on the K_Ca current independently of effects of PKC on local Ca release events, freshly isolated arterial smooth muscle cells were dialyzed with a strongly Ca-buffered solution to eliminate local Ca release events. Two ligands were employed to buffer the intracellular Ca concentration, EGTA, a widely used Ca buffer, and BAPTA, a Ca buffer with fast binding kinetics. Under these conditions, it was observed that the control K_Ca current at +70 mV after 6 min was 87 ± 11% (n = 5) of the current at the beginning of the registration period when 10 mM EGTA was used in the intracellular solution and 93 ± 8% (n = 4) of the current at the beginning of the registration period when 10 mM BAPTA was used in the intracellular solution, which are not significantly different (P = 0.69). The active diacylglycerol analog DOG at 10 µM decreased the K_Ca current 6 min after the beginning of the application at +70 mV by 70 ± 4% (n = 8) compared with the current at the beginning of the application period when 10 mM EGTA was used in the intracellular solution and by 69 ± 8% (n = 6) compared with the current at the beginning of the application period when 10 mM BAPTA was used in the intracellular solution, which are not significantly different (P = 0.84). The catalytic subunit of PKC (PKC_C) at 0.1 U/ml decreased the K_Ca current 6 min after the beginning of the application at +70 mV by 47 ± 5% (n = 10) compared with the current at the beginning of the application period when 10 mM EGTA was used in the intracellular solution and by 40 ± 3% (n = 7) compared with the current at the beginning of the application period when 10 mM BAPTA was used in the intracellular solution, which are not significantly different (P = 0.28). Because there were no significant differences of the effect of DOG and PKC_C on the K_Ca current in the experiments using EGTA or BAPTA, respectively, in the intracellular solution, these data were pooled. In addition to the independence of the observed results from the Ca buffer used in the intracellular solution, further evidence for a successful elimination of local Ca release events under our experimental conditions are the inability to observe spontaneous transient outward currents and the absence of an effect of ryanodine, a well-known blocker of local Ca release events, and of cyclopiazonic acid, an inhibitor of the Ca pump in the sarcoplasmic reticulum, on the K_Ca current. Thus 10 µM ryanodine decreased the K_Ca current 6 min after the beginning of the application at +70 mV by 3 ± 6% (n = 7) compared with the current at the beginning of the application period. This is not significantly different from the 10 ± 7% (n = 9) decrease of the time-matched control current at +70 mV (P = 0.43). Furthermore, 10 µM cyclopiazonic acid increased the K_Ca current 6 min after the beginning of the application at +70 mV by 2 ± 7% (n = 9) compared with the current at the beginning of the application period. This is not significantly different from the 10 ± 7% (n = 9) decrease of the time-matched control current at +70 mV (P = 0.22).

Inside-out experiments. In the inside-out experiments, single-channel data were stored on a DTR-1800 data recorder (Biologic, France) and later replayed for analysis. They were filtered at 1 kHz with use of an eight-pole Bessel filter (model 902, Frequency Devices, USA) and digitized at 5 kHz. Thereafter, they were analyzed offline with the software package ASCD (G. Droogmans, Lab. Fysiologie, KU Leuven, Louvain, Belgium). The single-channel amplitudes were determined by fitting Gaussian distributions to the amplitude histograms of the closed and the open state, respectively. The activity of the channel in a patch was determined as NP_o, where P_o is the open probability of one channel and N is the number of channels in the patch, which could not be determined in most cases because the patch was lost before application of a high-Ca solution. NP_o was calculated as the sum of the times finding 1, 2, 3, · · · , N simultaneously open channels divided by the registration time. The registration time was 2-3 min in the control and 3-5 min during PKC and PKC inhibitor peptide application. All potentials are expressed as membrane potentials.

Drugs and chemicals. Albumin, DL-dithiothreitol, and the salts for the solutions were obtained from Sigma, except for BAPTA, which was purchased from Molecular Probes. Papain was from Ferak (Berlin, Germany). Iberiotoxin and ryanodine were from Research Biochemicals International. PKC_C, the PKC inhibitor peptide (1931), DOG, and calphostin C were obtained from Calbiochem, and 1,3-dioctanoyl-sn-glycerol was from Biomol.

Statistics. All data are presented as means ± SE; n is the number of cells. Statistical analysis was performed with use of paired and unpaired t-tests, one-way ANOVA followed by a Bonferroni test, or repeated measures ANOVA, as appropriate.

	RESULTS

Top Abstract Introduction METHODS RESULTS DISCUSSION References

In a recent publication, we characterized the properties of the outward current of rat tail artery smooth muscle cells in detail (22). Briefly, this current showed a marked outward rectification, a strong dependence on the intracellular Ca ion concentration, and a high sensitivity to iberiotoxin, the selective blocker of K_Ca channels (9, 10), which produced a considerable inhibition of this current. Neither glibenclamide, the selective blocker of ATP-sensitive K channels, nor 4-aminopyridine, a blocker of voltage-dependent K channels, affected this current. Additionally, it was shown in the cited publication that in excised patches from these cells a K channel with a high conductance, a steep voltage dependence of activation, and a prominent Ca sensitivity is the main channel, which was blocked by low concentrations of tetraethylammonium and by iberiotoxin. Thus, under our experimental conditions, the outward current of these cells consists largely of a K_Ca current, which is carried by K_Ca channels. The K_Ca current in the present study was defined as the difference of the net outward current amplitude under the desired experimental condition and the outward current amplitude after application of 100 nM iberiotoxin and was independent from local Ca release events (for details, see Isolation of the K_Ca current, above).

Effect of an activation of PKC on the K_Ca current. For the investigation of the effect of PKC on the K_Ca current of rat tail artery smooth muscle cells, DOG (7), a cell-permeable active analog of diacylglycerol, was used to activate PKC. DOG at a concentration of 10 µM was applied to the bath solution 2 min after the whole cell configuration was obtained. Control experiments had shown that this time was necessary but sufficient to get stable currents (data not shown). Therefore, the beginning of any application period was marked time 0. A typical example of the time course of the K_Ca current elicited with use of a voltage step from a holding potential of 40 mV to a test potential of +70 mV under control conditions and after DOG application is shown in Fig. 1A. A transient increase of the K_Ca current was observed ~1-2 min after the beginning of DOG application. This increase did not reach significance in comparison with the time-matched control current and was, therefore, not studied in detail. Subsequently, the K_Ca current started to decline, and 6-8 min after the beginning of DOG application, the K_Ca current reached a steady state at a considerably lower level compared with the time-matched control current. For example, in the control series, the K_Ca current elicited with use of a voltage step from a holding potential of 40 mV to a test potential of +70 mV was 131 pA at the beginning of the control application period and 99 pA 6 min after the beginning of the control application period (Fig. 1B, top). I-V relationships of the control K_Ca current were obtained with use of a voltage ramp from 70 mV to +100 mV based on a holding potential of 40 mV and showed that the changes of the control current with time were similar at all potentials tested (see Fig. 1B, bottom, for a typical example). In contrast, in the DOG series, the K_Ca current elicited with use of a voltage step from a holding potential of 40 mV to a test potential of +70 mV was, for example, 214 pA at the beginning of the DOG application period and only 33 pA 6 min after the beginning of the DOG application period (Fig. 1C, top). I-V relationships of the K_Ca current obtained with use of a voltage ramp from 70 mV to +100 mV based on a holding potential of 40 mV showed that DOG produced a marked decrease of the K_Ca current in the potential range tested. This is demonstrated in Fig. 1C, bottom, with a typical example and, in addition to the original traces, the I-V relationship of the pure K_Ca current, i.e., the difference of the net outward current and the iberiotoxin-insensitive current, is shown before and after DOG application (Fig. 1C, bottom right). In summary, the I-V relationship of the K_Ca current was altered significantly, seen as a reduction of this current (Fig. 2), and the threshold potential of the current changed significantly, from 23.6 ± 3.4 to 40.0 ± 2.9 mV or by 16.4 ± 2.8 mV (n = 7; P < 0.01). On average, 10 µM DOG decreased the K_Ca current 6 min after the beginning of the application at +70 mV by 70 ± 4% (n = 14) compared with the current at the beginning of the application period. This is a significant decrease in comparison with the 10 ± 7% (n = 9) decrease of the time-matched control current at +70 mV (see Fig. 4; P < 0.001).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Effect of 1,2-dioctanoyl-sn-glycerol (DOG) on Ca-activated K (KCa) current of rat tail artery smooth muscle cells. A: examples of time course of control (con) KCa current and of time course of effect of DOG and of inactive (inact) DOG on KCa current. KCa current (IKCa) was elicited with use of a voltage step from a holding potential of 40 mV to a test potential of +70 mV and was normalized to current at beginning of application period (time 0). B: examples of traces of KCa current under control conditions. Top: from left, stimulation protocol, control current at time 0 together with current after iberiotoxin (IBTX) application, and control current after 6 min together with current after IBTX application. Bottom: from left, stimulation protocol and control current-voltage (I-V) relationship at time 0 together with control I-V relationship after 6 min as well as I-V relationship after IBTX application. C: examples of traces of KCa current in DOG series. Top: from left, stimulation protocol, current at beginning (0) of DOG application together with current after IBTX application, and current after 6 min of DOG application together with current after IBTX application. Bottom: from left, stimulation protocol, I-V relationship at beginning of DOG application together with I-V relationship after 6 min of DOG application as well as I-V relationship after IBTX application, and I-V relationship of pure KCa current, i.e., difference of net outward I-V relationship and I-V relationship after IBTX application, at beginning of DOG application and after 6 min of DOG application. In A, data points of different experimental series do not coincide in time because of variable length of period for series resistance control. Also, capacitive artifacts shown in B and C are distorted due to filtering of current traces at low frequency and limited x-axis resolution of graphic presentation.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Dependence of KCa current density on membrane potential (MP). con, KCa current at beginning of application of 10 µM DOG. DOG, KCa current after 6 min of application of 10 µM DOG. A repeated measures ANOVA showed a significant difference between control and DOG (n = 7; P < 0.01).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Effect of protein kinase C (PKC) inhibitor calphostin C (cal) on DOG-induced decrease of KCa current of rat tail artery smooth muscle cells. A: examples of time course of effect of calphostin C on KCa current, of time course of effect of DOG on KCa current after calphostin C pretreatment, and of time course of effect of DOG on KCa current without any pretreatment (taken from Fig. 1 for comparison). KCa current was elicited with use of a voltage step from a holding potential of 40 mV to a test potential of +70 mV and was normalized to current at beginning of application period (time 0). B: examples of traces of KCa current in DOG series with calphostin C pretreatment. Top: from left, stimulation protocol, current at beginning of DOG application with calphostin C pretreatment together with current after IBTX application, and current after 6 min of DOG application with calphostin C pretreatment together with current after IBTX application. Bottom: from left, stimulation protocol and I-V relationship at beginning of DOG application with calphostin C pretreatment together with I-V relationship after 6 min of DOG application with calphostin C pretreatment as well as I-V relationship after IBTX application. In A, data points of different experimental series do not coincide in time because of variable length of period for series resistance control. Also, capacitive artifacts shown in B are distorted due to filtering of current traces at low frequency and limited x-axis resolution of graphic presentation.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Summary of effect of DOG (10 µM) on KCa current of rat tail artery smooth muscle cells 6 min after beginning of application. Data are expressed as ratio of KCa current 6 min after beginning of application under specified experimental conditions (exp) to KCa current at beginning of application period under same conditions (con). cal, 1 µM calphostin C; PKC-I, 1 µM PKC inhibitor peptide; inact DOG, 10 µM inactive DOG. * Significant difference compared with control determined by 1-way ANOVA (P < 0.001); no. of cells investigated appears on bar for each condition.

Effect of PKC on the K_Ca current. For the further investigation of the effect of PKC on the K_Ca current of rat tail artery smooth muscle cells, PKC_C (27), which is active in the absence of phospholipids and Ca, was employed. PKC_C was added to the pipette solution at 0.1 U/ml. A higher activity of PKC_C was not used so as to avoid possible artifacts caused by components of the PKC_C storage buffer. A typical example of the time course of the K_Ca current elicited with use of a voltage step from a holding potential of 40 mV to a test potential of +70 mV under control conditions and after PKC_C application is shown in Fig. 5A. The K_Ca current started to decline ~3-4 min after the beginning of PKC_C application, and 6-8 min after the beginning of PKC_C application the K_Ca current reached a steady state at a considerably lower level compared with the time-matched control current. For example, the K_Ca current elicited with use of a voltage step from a holding potential of 40 mV to a test potential of +70 mV was 209 pA at the beginning of the PKC_C application period and only 79 pA 6 min after the beginning of the PKC_C application period (Fig. 5B, top). I-V relationships of the K_Ca current obtained with use of a voltage ramp from 70 mV to +100 mV based on a holding potential of 40 mV showed that PKC_C produced a marked decrease of the K_Ca current in the potential range tested. This is demonstrated in Fig. 5B, bottom, with a typical example and, in addition to the original traces, the I-V relationship of the pure K_Ca current, i.e., the difference of the net outward current and the iberiotoxin-insensitive current, is shown before and after PKC_C application (Fig. 5B, bottom right). In summary, the I-V relationship of the K_Ca current was altered significantly, seen as a reduction of this current (Fig. 6), and the threshold potential of the current changed significantly, from 30.0 ± 2.2 to 41.1 ± 2.2 mV or by 11.1 ± 2.0 mV (n = 9; P < 0.01). On average, 0.1 U/ml PKC_C decreased the K_Ca current 6 min after the beginning of the application at +70 mV by 44 ± 3% (n = 17) compared with the current at the beginning of the application period. This is a significant decrease in comparison with the 10 ± 7% (n = 9) decrease of the time-matched control current at +70 mV (see Fig. 8; P < 0.01).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Effect of catalytic subunit of PKC (PKCC) on KCa current of rat tail artery smooth muscle cells. A: examples of time course of control KCa current (taken from Fig. 1 for comparison) and of time course of effect of PKCC on KCa current. KCa current was elicited with use of a voltage step from a holding potential of 40 mV to a test potential of +70 mV and was normalized to current at beginning of application period (time 0). B: examples of traces of KCa current in PKC series. Top: from left, stimulation protocol, current at beginning (0) of PKCC application together with current after IBTX application, and current after 6 min of PKC application together with current after IBTX application. Bottom: from left, stimulation protocol, I-V relationship at beginning of PKCC application together with I-V relationship after 6 min of PKCC application as well as I-V relationship after IBTX application, and I-V relationship of pure KCa current, i.e., difference of net outward I-V relationship and I-V relationship after IBTX application, at beginning of PKCC application and after 6 min of PKCC application. In A, data points of different experimental series do not coincide in time because of variable length of period for series resistance control. Also, capacitive artifacts shown in B are distorted due to filtering of current traces at low frequency and limited x-axis resolution of graphic presentation.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Dependence of KCa current density on membrane potential. con, KCa current at beginning of application of 0.1 U/ml PKCC. PKCC, KCa current after 6 min of application of 0.1 U/ml PKCC. A repeated measures ANOVA showed a significant difference between control and PKCC (n = 9; P < 0.01).

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Effect of heat-inactivated PKCC on KCa current of rat tail artery smooth muscle cells. A: examples of time course of effect of heat-inactivated PKCC on KCa current and of time course of effect of active PKCC on KCa current (taken from Fig. 5 for comparison). KCa current was elicited with use of a voltage step from a holding potential of 40 mV to a test potential of +70 mV and was normalized to current at beginning of application period (time 0). B: examples of traces of KCa current in series with heat-inactivated PKCC. Top: from left, stimulation protocol, current at beginning of application of heat-inactivated PKCC together with current after IBTX application, and current after 6 min of application of heat-inactivated PKCC together with current after IBTX application. Bottom: from left, stimulation protocol and I-V relationship at beginning of application of heat-inactivated PKCC together with I-V relationship after 6 min of application of heat-inactivated PKCC as well as I-V relationship after IBTX application. In A, data points of different experimental series do not coincide in time because of variable length of period for series resistance control. Also, capacitive artifacts shown in B are distorted due to filtering of current traces at low frequency and limited x-axis resolution of graphic presentation.

View larger version (16K): [in this window] [in a new window]   Fig. 8.   Summary of effect of PKCC on KCa current of rat tail artery smooth muscle cells 6 min after beginning of application. Data are expressed as ratio of KCa current 6 min after beginning of application under specified experimental conditions (0.1 U/ml active or heat-inactivated PKCC) and KCa current at beginning of application period under same conditions. * Significant difference compared with control determined by 1-way ANOVA (P < 0.01); no. of cells investigated appears on bar for each condition.

Effect of PKC on single K_Ca channels. For the investigation of the effect of PKC on the K_Ca current of rat tail artery smooth muscle cells, the direct effect of PKC_C on single K_Ca channels was tested. PKC_C was added to the bath solution at 0.1 U/ml together with 100 µM MgATP after the registration of channel control activity. In these single-channel experiments, a spontaneous decrease of the activity of the K_Ca channel (rundown) was often observed, the nature of which was not established in the present study. Therefore, all measurements showing a rundown in the registration of channel control activity were excluded from further analysis. Furthermore, to be able to differentiate the expected PKC-induced decrease of K_Ca channel activity from rundown, the experimental protocol included a test of the action of PKC inhibitor peptide after a stable level of channel activity was obtained after PKC application. Only experiments in which PKC inhibitor peptide was able to reverse the effect of PKC on the K_Ca current were included in the analysis. A typical example of the time course of the activity of single K_Ca channels at 0 mV under control conditions and after PKC_C and subsequent PKC inhibitor peptide application is shown in Fig. 9A. K_Ca channel activity started to decline ~2 min after the beginning of the application of PKC_C together with 100 µM MgATP, and K_Ca channel activity reached a steady state ~4 min after the beginning of PKC_C application. Subsequently, PKC inhibitor peptide application restored K_Ca channel activity, and, ~3 min after the beginning of PKC inhibitor peptide application, K_Ca channel activity reached a steady state. In this example (Fig. 9B), K_Ca channel activity (NP_o) at 0 mV was 0.56 in the control period, 0.07 during the steady state after PKC_C application, and 0.51 during the steady state after the subsequent addition of PKC inhibitor peptide. On average, 0.1 U/ml PKC_C together with 100 µM MgATP decreased K_Ca channel activity in the steady state at +0 mV significantly by 79 ± 9% (n = 6) compared with the activity in the control period (Fig. 9C; P < 0.001). PKC inhibitor peptide reversed the PKC_C-induced decrease of K_Ca channel activity, and in the steady state a nonsignificant decrease of channel activity by 5 ± 22% (n = 6) compared with the activity in the control period was observed (Fig. 9C; P = 0.82). The amplitude of single K_Ca channel openings was not changed after the addition of PKC_C or PKC inhibitor peptide (data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 9.   Effect of PKCC on single KCa channel activity of rat tail artery smooth muscle cells. A: example of a diary plot of channel number times open probability (NPo) of KCa channel at 0 mV with application of PKCC and PKC-I. B: examples of traces of KCa channel activity at 0 mV taken from same patch as in A during control (Ctrl), PKCC application and subsequent addition of PKC-I. C: summary of effect of PKCC on single KCa channel activity. Data are expressed as ratio of NPo of channel after reaching a steady state during application of 0.1 U/ml native PKCC, 0.1 U/ml heat-inactivated PKCC, or additional application of 1 µM PKC-I, respectively [NPo(exp)], and NPo of channel in control period [NPo(ctrl)]. * Significant difference compared with control (P < 0.001); no. of cells investigated appears on bar for each condition.

	DISCUSSION

Top Abstract Introduction METHODS RESULTS DISCUSSION References

Effect of an activation of PKC on the K_Ca current of rat tail artery smooth muscle cells. The aim of the first series of experiments was to investigate the effect of an activation of the endogenous PKC on the K_Ca current. To activate PKC, an active analog of the natural PKC activator diacylglycerol, DOG, was used (7). In the present experiments, the PKC activator DOG entered the cells from the extracellular space, in contrast to the natural conditions, in which the PKC activator is released from membrane phospholipids. Therefore, it was necessary to obtain evidence that the effect of DOG on the K_Ca current is mediated by PKC. In the present study the DOG-induced decrease of the K_Ca current was completely blocked by the specific PKC inhibitors calphostin C and PKC inhibitor peptide. Calphostin C acts by an interaction with the diacylglycerol binding site of the regulatory domain of PKC (14), and PKC inhibitor peptide acts as pseudo-substrate by binding to the active site of PKC. An unspecific action of DOG on the K_Ca current seems unlikely also because the inactive diacylglycerol analog 1,3-dioctanoyl-sn-glycerol was unable to reduce the K_Ca current. Therefore, the decrease of the K_Ca current induced by DOG is probably mediated by an activation of the PKC present in these cells.

Effect of PKC on the K_Ca current and single K_Ca channels of rat tail artery smooth muscle cells. The aim of the second series of experiments was to investigate the effect of exogenous PKC on the K_Ca current and on single K_Ca channels. To ensure a straightforward interpretation of these experiments, PKC_C was used, which does not require Ca or phosphatidylserine to be active (27). Thus the number of factors that could affect the K_Ca current or K_Ca channel on their own was limited. In the present study, it was shown for the first time that the application of PKC_C together with MgATP, which was permanently present in the intracellular solution in all experiments, considerably decreased the K_Ca current in freshly isolated arterial smooth muscle cells. Furthermore, the data on the direct inhibition of single-K_Ca channel activity by PKC_C together with MgATP in freshly isolated smooth muscle cells extend earlier findings showing an inhibition of single K_Ca channel activity in cultured smooth muscle cells from porcine coronary arteries after a direct application of PKC to this channel (18).

Mechanism of the PKC effect on the K_Ca current of rat tail artery smooth muscle cells. When discussing the mechanism of the PKC effect on the K_Ca current, it is important to note that in whole cell experiments the K_Ca current has been shown to be influenced markedly by changes of the intracellular Ca concentration induced by the inositol trisphosphate-mediated Ca release (13), the ryanodine-sensitive Ca release (19), or the Na/Ca exchange (6). The activity of these mechanisms regulating the intracellular Ca ion concentration is also affected by PKC (3, 15). Therefore, specific experimental conditions with a strongly buffered low intracellular Ca concentration and a low extracellular Ca concentration were selected, which have been shown to greatly reduce the influence of mechanisms regulating the intracellular Ca concentration on the K_Ca current (6). The absence of an effect of ryanodine, which blocks local Ca release events spontaneously occurring even in resting cells, on the K_Ca current demonstrated that under the selected experimental conditions it was possible to investigate the effect of PKC on the K_Ca current under conditions of a minimized influence of alterations of the intracellular Ca concentration.

Possible functional role of the PKC effect on the K_Ca current of rat tail artery smooth muscle cells. Because the K_Ca current in a variety of arterial smooth muscle cells in vessels exposed to a physiological pressure level but not subjected to any vasoactive agonist belongs to the ion currents establishing the membrane potential (5), a PKC-mediated decrease of this current may have important functional consequences. Thus a decrease of the K_Ca current, leading to membrane potential depolarization and vasoconstriction, may mediate the effect of transmural pressure or of different vasoconstrictors, because these stimuli have been shown to activate PKC (12, 15). However, the hypothesis of a functional importance of the PKC-mediated decrease of the vascular smooth muscle K_Ca current is highly speculative at the moment. Thus a phospholipase C-induced activation of PKC under physiological conditions is accompanied by an inositol trisphosphate-mediated release of Ca from intracellular stores. This Ca may increase the K_Ca current and, therefore, counteract its decrease by PKC. Additionally, the regulation of the K_Ca current by PKC-dependent local Ca release events has to be taken into account (3). The data available do not allow estimation of the net effect of the simultaneous action of Ca and PKC on the K_Ca current. Furthermore, PKC alters the activity of several other mechanisms, which also can produce vessel constriction. Limiting this consideration only to mechanisms regulating the membrane potential, PKC, for example, decreases the ATP-sensitive K current (4) and the voltage-dependent K current (1) and increases the voltage-dependent Ca current (16). Again, the data available do not allow estimation of whether the PKC-related response to the above-mentioned physiological stimuli is mediated mainly by the PKC-induced decrease of the K_Ca current or mainly by one of the other mechanisms. Additionally, although the membrane potential and the intracellular Ca concentration can be adjusted in patch-clamp experiments to simulate physiological conditions for the K_Ca channel, the functional state of a variety of other factors affecting its function is quite different in an intact artery compared with an isolated cell, e.g., PKA is activated by transmural pressure (11), PKG is activated by flow-induced release of NO (20), or G proteins are activated by epoxyeicosatrienoic acids (17). Moreover, their functional state in the intact artery is not known and, therefore, cannot be simulated in an isolated cell. Thus the functional role of the PKC effect on the K_Ca current can be established only in studies on intact vessel preparations. In conclusion, the complete mechanism and the functional role of the PKC-induced regulation of the K_Ca current have to be established in future studies.