INTRODUCTION

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PROSTAGLANDINS ARE critical modulators of vascular tone in both physiological and pathophysiological conditions. In particular, PGF₂ exerts a complex effect on vascular smooth muscles including coronary artery, cerebral artery, aorta, renal artery, and jugular vein (8, 19, 43, 46, 53).

Under various experimental conditions, PGF₂ was reported to have contractile effects on vascular smooth muscles through the following mechanisms. First, PGF₂ contracted rat aorta through phosphoinositide (PI) hydrolysis, and this PI hydrolysis did not appear to depend on extracellular Ca²⁺ flux (43). Second, PGF₂ contracted bovine middle cerebral artery by promoting Ca²⁺ uptake from low-affinity binding sites through receptor-operated channels sensitive to Ca²⁺ antagonists (50). This constriction by PGF₂ was near maximally inhibited in Ca²⁺-deficient solutions but only partially inhibited by Ca²⁺ antagonists. Third, PGF₂-induced contractions in human pial artery and feline basilar artery were relatively independent of free extracellular Ca²⁺. Incubation of these arteries in a Ca²⁺-free medium did not abolish contraction induced by PGF₂ (46, 47). Fourth, PGF₂ contracted rat aorta and ferret aorta partly by sensitization of the contractile filaments to Ca²⁺ (18, 44).

In contrast to the above observations, Chen et al. (8) showed that PGF₂ potently relaxed the rabbit jugular vein. This effect of relaxations elicited by PGF₂ appears to be mediated by nitric oxide and K⁺ channels.

Thus the mechanism underlying action of PGF₂ in vascular smooth muscles has not yet been investigated fully. Furthermore, whether PGF₂ affects vascular smooth muscle cells (SMCs) per se (especially via membrane potentials, K⁺ channels, and L-type Ca²⁺ channels) remains to be resolved. In vascular SMCs, K⁺ channels are implicated in the genesis and regulation of membrane potential and in this way are critically involved in the mechanism of vascular SMC contraction. In particular, Ca²⁺-activated K⁺ (K_Ca) channels, at high density in artery, participate in the maintenance or regulation of arterial tone (2, 34, 51) and delayed rectifier K⁺ (K_V) channels, also one of the dominant K⁺ channels, act mainly to limit membrane depolarization in arterial SMCs (20, 28). The activation or opening of these K⁺ channels in vascular SMCs results in an efflux of K⁺, membrane hyperpolarization and, ultimately, vascular relaxation due to the closure of L-type Ca²⁺ channels and a reduced concentration of intracellular Ca²⁺, and vice versa (5, 34). Therefore, it is important to know whether PGF₂ affects membrane potentials and membrane currents. However, no studies to date have addressed the effects of PGF₂ on electrophysiological properties of cerebral arterial SMCs.

In this study, we examined the direct effects of PGF₂ on membrane potentials, K_Ca channels, K_V channels, and L-type Ca²⁺ channels with the patch-clamp technique in single rabbit middle cerebral arterial SMCs.

	METHODS

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Cell isolation. Single vascular SMCs were isolated as previously reported (23). New Zealand White rabbits (n = 50) of either sex, weighing 0.8-1.2 kg, were anesthetized with an injection of pentobarbital sodium (1 mg/kg) and euthanized by exsanguination. The middle cerebral arteries were removed and cleaned of extraneous connective tissues in a dissecting solution containing (in mM) 137 NaCl, 5.6 KCl, 0.42 Na₂HPO₄, 0.44 NaH₂PO₄, 4.17 NaHCO₃, 1 MgCl₂, 2.6 CaCl₂, and 10 HEPES, pH 7.3 with NaOH. The arteries were then transferred to an isolation solution (in mM: 55 NaCl, 6 KCl, 88 L-glutamic acid, 10 HEPES, 10 glucose, pH 7.3 with NaOH) for 20 min. Cerebral arteries were digested for 10 min in an isolation solution containing (in mg/ml) 1 albumin, 1 papain, and 1 dithioerythritol and then for 10 min in a isolation solution containing (in mg/ml) 1 albumin, 1 collagenase F, and 1 hyaluronidase type I-S. Single arterial SMCs were obtained by gentle trituration with a wide-bore pipette in fresh isolation solution with albumin (1 mg/ml), stored at 4°C, and used within 12 h.

Electrical measurement and data analysis. The whole cell and excised-patch configurations of the patch-clamp technique were utilized in this study. The data were recorded with a patch-clamp amplifier (Axopatch-1D; Axon Instruments, Foster City, CA). Pipettes of 5- to 10-M resistance were pulled from borosilicate glass capillaries (Clark Electrochemical, Pangbourne, UK) with a vertical puller (Narishige PP-83). Their tips were coated with Sylgard and fire polished. Membrane potentials and whole cell currents were filtered at 5 kHz and stored in digitized format on digital audiotapes with a DTR-1200 recorder (Biologic, Grenoble, France). Single-channel currents were digitized at a sampling rate of 48 kHz and stored in digitized format on digital audiotapes with a DTR-1200 recorder. For analysis, the data were transferred to a computer (IBM-PC, Pentium III 450; Busan, Korea) with pCLAMP software (version 6.0; Axon Instruments, Union City, CA) through an analog-to-digital converter interface (Digidata-1200, Axon Instruments).

Solutions and drugs. The solutions used in whole cell experiments for membrane potentials and K⁺ currents contained (in mM) 133 K-aspartic acid, 7 KCl, 2.5 Mg-ATP, 2.5 Na-ATP, 2.5 Tris-creatine phosphate, 2.5 Na-creatine phosphate, 5 HEPES, and 0.05 or 10 EGTA, pH 7.3, for the pipette and 143 NaCl, 5.4 KCl, 5 HEPES, 0.33 NaH₂PO₄, 1 MgCl₂, 16.6 glucose, 0 or 1.8 CaCl₂, pH 7.4, for the bath. The solutions used in outside-out patch experiments contained (in mM) 140 KCl, 1.2 KH₂PO₄, 2.5 CaCl₂, and 5 HEPES, pH 7.3, for the bath; the composition of the pipette solution was similar, except that the Ca²⁺-to-EGTA ratio was adjusted to give a pCa of 6 × 10⁷, at which simultaneous opening of one to three channels could typically be observed. Stock solutions of PGF₂ were dissolved in 100% ethanol and diluted 1,000-fold for final solutions. At this concentration, ethanol did not affect channel activity. All chemicals and drugs in this study were obtained from Sigma (St. Louis, MO).

	RESULTS

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Effects of PGF₂ on membrane potentials. We first tested the effects of PGF₂ on membrane potentials with the current clamp mode. The membrane potentials were not quiescent but showed spontaneous hyperpolarizations as shown in Fig. 1A. In the following discussion, we refer to the minimum negative level of potential as the membrane potential. The mean value of membrane potentials was 42 ± 3.07 mV (n = 7; Fig. 1A, inset) with a low concentration (0.05 mM) of EGTA in the pipette. The bath application of 100 nM PGF₂ induced significant hyperpolarization (51 ± 4.94 mV, n = 7; Fig. 1A, inset). PGF₂ also increased spontaneous hyperpolarization activity. The superfusion of 100 nM iberiotoxin (IBTX) eliminated the hyperpolarization by 100 nM PGF₂ (39 ± 5.03 mV, n = 7; Fig. 1A, inset).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Effects of PGF2 on membrane potentials in rabbit middle cerebral arterial smooth muscle cells (SMCs). A: cerebral vascular SMCs were intracellularly perfused with a solution containing 0.05 mM EGTA. On extracellular application of 100 nM PGF2, the membrane hyperpolarized. This was eliminated by application of 100 nM iberiotoxin (IBTX). Right: effects of PGF2 and IBTX on membrane potential (Em). B: cerebral arterial SMCs were intracellularly perfused with a solution containing 10 mM EGTA and extracellularly perfused with a solution containing 1 mM tetraethylammonium (TEA) and 1 µM nicardipine. Application of 100 nM PGF2 hyperpolarized the membrane potential. This was eliminated by application of 1 mM 4-aminopyridine (4-AP). Right: effects of PGF2 and 4-AP on membrane potential. *P < 0.05 vs. control; **P < 0.05 vs. PGF2 treated.

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Effects of PGF₂ on whole cell K+ currents. With whole cell voltage-clamp recordings, a step of voltage from 60 mV to +50 mV in 10-mV increments was applied from a holding potential of 60 mV. Control currents showed time- and voltage-dependent outward currents. Stepping to positive potentials evoked much noisier outward currents on which spontaneous transient outward currents (STOCs; Ref. 2) were often superimposed. The application of 100 nM IBTX inhibited outward currents by ~50% (Fig. 2A). The application of PGF₂ (100 nM) activated outward currents that exceeded the amplitudes of the control currents and markedly increased STOCs, which were K_Ca currents. The further application of IBTX (100 nM) eliminated many of the outward K⁺ currents (Fig. 2B). The current-voltage relationship is shown in Fig. 2C. The peak current amplitudes in each condition were normalized relative to the maximum in control current amplitude.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Effects of PGF2 on whole cell K+ currents in rabbit middle cerebral arterial SMCs. A: representative families of currents were recorded in the absence and presence of 100 nM IBTX with step protocols. IBTX decreased outward currents. B: representative families of currents were recorded under control conditions, with application of 100 nM PGF2, and with the further application of 100 nM IBTX with step protocols. PGF2 increased outward currents, which was inhibited by IBTX. C: current-voltage (I-V) relationships in each condition. PGF2 significantly increased currents, and IBTX blocked the current below control levels. *P < 0.05 vs. control; **P < 0.05 vs. PGF2 treated.

Effects of PGF₂ on K_Ca currents. To address the contribution of K_Ca currents to the action of PGF₂, the effect of PGF₂ on K_Ca channels was investigated in the outside-out patch configuration. The application of 100 nM IBTX inhibited outward currents by ~50% (Fig. 2A). The application of PGF₂ (100 nM) activated outward currents. In the experiment shown in Fig. 3A, 100 nM PGF₂ increased P_o from 0.25 to 0.41, (1.6-fold) at +50 mV (Fig. 3, A and B). The mean increase in P_o to PGF₂ was 1.4 ± 0.8-fold (n = 5, P < 0.05; Fig. 3C). PGF₂ had no effect on unitary currents (11.1 ± 0.1 pA before PGF₂ and 11.2 ± 0.2 pA after PGF₂; n = 4, +50 mV; Fig. 4A).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Effect of PGF2 on single-channel Ca2+-activated K+ (KCa) current in rabbit middle cerebral arterial SMCs. A: representative current trace was recorded at membrane potential of +50 mV. Dotted line denotes the current level when the channel is closed. B: open probability (Po) from the same patch as in A, corresponding to the top record, calculated over 30-s intervals. C: summarized data (n = 6) before (open bar), during (filled bar), and after (gray bar) application of 100 nM PGF2. *P < 0.05 vs. control.

View larger version (39K): [in this window] [in a new window]   Fig. 4.   Effect of PGF2 on KCa channel kinetics in rabbit middle cerebral arterial SMCs. A: unitary current recordings and all-points amplitude histograms obtained from an outside-out membrane patch are shown before (a) and during (b) application of 100 nM PGF2. Patch membrane potential was held at +50 mV. Insets, examples of current records in each condition. Dotted lines denote the current level when the channel is closed. Note that the amplitude of the channel was not affected by 100 nM PGF2. B: effect of 100 nM PGF2 on single-channel kinetics. In the histograms of the open times, the closed times (insets) <50 ms were obtained before (a) and after (b) addition of 100 nM PGF2 from an outside-out patch at +50 mV. Each histogram was best fitted to 2 exponential distributions. Insets, examples of current records in each condition. Dotted line indicates the closed level.

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Effects of PGF₂ on K_V currents. The whole cell K⁺ currents consisted of K_Ca currents and K_V currents when the external solution contained 1.8 mM Ca²⁺ and the pipette solution contained a low concentration (0.05 mM) of EGTA (Fig. 2). Removal of extracellular Ca²⁺, addition of extracellular 1 mM TEA and 1 µM nicardipine, and increase of the intracellular EGTA concentration (from 0.05 to 10 mM) completely eliminated the K_Ca current and Ca²⁺ current components (40, 48). Figure 5 shows representative traces of the K_V currents evoked by step depolarizations from 60 mV to +50 mV from a holding potential of 60 mV. The application of 1 mM 4-AP inhibited outward currents (Fig. 5A). The application of PGF₂ (100 nM) increased the amplitude of the K_V currents, and the further application of 4-AP (1 mM) reduced this current (Fig. 5B). Figure 5C summarizes the effect of PGF₂ on K_V currents at 0 and +50 mV.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Effects of PGF2 on delayed rectifier K+ (KV) currents in rabbit middle cerebral arterial SMCs. A: representative families of KV currents were recorded in the absence and presence of 1 mM 4-AP with step protocols. 4-AP decreased KV currents. B: representative families of KV currents were recorded under control conditions, with application of 100 nM PGF2, and with the further application of 1 mM 4-AP. PGF2 increased KV currents, which was inhibited by 4-AP. C: mean ± SE currents (n = 6) at the peak evoked by voltage steps to 0 and +50 mV from a holding potential of 60 mV were plotted for control conditions, the application of PGF2 and the further application of 4-AP. Current amplitude was normalized to the peak current in control. D: mean ± SE activation points (n = 4) were determined by fitting the deactivation tail at 35 mV after voltage steps to various potentials. The voltage steps that preceded deactivation varied in length to return to 35 mV at the peak of the current. The amplitude of each tail was normalized using the largest tail. These points were fitted with the Boltzmann distribution equation derived by least-squares fitting method. The activation curve was shifted to the left by PGF2. E: mean ± SE inactivation points (n = 4) represent normalized peak outward current at +40 mV after holding at various potentials until this current stabilized. The smooth curve through these points is the best fit to the Boltzmann distribution equation derived by least-squares fitting method. The inactivation curve was shifted to the left by PGF2. F: activation time constants plotted on a natural logarithmic scale against the test potential. The time constants were decreased by PGF2 (n = 5). The fitted straight lines have correlation coefficients of 0.987 for controls and 0.979 for PGF2-treated SMCs. *P < 0.05 vs. control; **P < 0.05 vs. PGF2 treated.

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Effects of PGF₂ on L-type Ca²+ currents. The compositions of the internal and external solutions were chosen so as to minimize all ionic currents except those through Ca²⁺ channels where appropriate. Ca²⁺ channel currents in middle cerebral artery SMCs are relatively small. We recorded Ca²⁺ channel currents with an external solution that contained 10 mM Ba²⁺ instead of 1.8 mM Ca²⁺, similar to that reported by other investigators (27). We applied depolarizing step pulses from 40 mV to +50 mV in 10-mV increments from the holding potential of 60 mV. Various Ca²⁺ channel blockers (1 µM nicardipine or verapamil) abolished the current activated by this protocol, and any remaining time-dependent current was negligible (data not shown). This result indicated that our protocol activated the Ca²⁺ current with little contamination from other membrane currents and could therefore be used to investigate the effect of PGF₂ on Ca²⁺ currents.

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View larger version (21K): [in this window] [in a new window]   Fig. 6.   Effects of PGF2 on Ca2+ currents in rabbit middle cerebral arterial SMCs. A: I-V relationships of normalized peak Ca2+ currents to membrane capacitance. PGF2 (100 nM) decreased the amplitude of Ca2+ currents without any significant change in threshold or apparent reversal potential. *P < 0.05 compared with control. B: time constants of Ca2+ current inactivation. The inactivation phase of representative original current records elicited by a test potential to +10 mV is shown, to which theoretical curves have been fitted with single-exponential functions. In these examples time constants () are 275.36 ms in the control and 173.60 ms in PGF2-treated SMC. C: steady-state voltage dependence of the activation and inactivation of Ca2+ currents in control conditions. Depolarizing and hyperpolarizing prepulse potentials ranging from 80 to +40 mV were applied for a duration of 2 s. After a 5-ms interpulse interval at a potential of 80 mV, the membrane was displaced to test potential of +10 mV for 200 ms. Steady-state inactivation was measured as the ratio I/Imax. Characteristics of the steady-state activation variable were determined from the positive limb of the I-V relationship. The points were fitted to a Boltzmann distribution equation. D: steady-state voltage dependence of the activation and inactivation of Ca2+ currents in the PGF2-treated condition. E: time course of the effect on Ca2+ current amplitude. Membrane potential was depolarized at 10-s intervals from 60 mV to +10 mV to elicit Ca2+ currents while the cell was superfused with PGF2. Ca2+ currents are plotted against the time of experiment.

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	DISCUSSION

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PGF₂ is an extremely potent and fast-acting vasoactive substance. In particular, PGF₂ has been also implicated in the pathogenesis of delayed vasospasm after subarachnoid hemorrhage (7, 10, 49). Most studies have focused on the association of PGF₂ with constriction of cerebral arteries in vivo and in vitro (4, 47, 52). On the basis of these findings, however, it is not possible to determine the precise mechanisms of the change of vascular SMC contractility by PGF₂. Electrophysiological changes of vascular SMCs are critically involved in the mechanism of vascular smooth muscle contractility. Because there had been no study on the effects of PGF₂ on electrophysiological properties of cerebral arterial SMCs, we tested the direct effects of PGF₂ on membrane potentials and membrane currents.

Membrane potential hyperpolarization by PGF₂. The values of membrane potentials that we report are similar to those measured in other types of isolated vascular SMCs with the patch-clamp technique and an intracellular solution containing 0.1 mM EGTA, i.e., rabbit coronary artery cells (32.2 mV; Ref. 28), rabbit portal vein myocytes (48.7 mV; Ref. 31), human coronary artery cells in culture (32 mV; Ref. 42), and human saphenous vein (41 mV; Ref. 30). However, with intracellular microelectrodes, a membrane potential of 62 ± 5 mV was measured in rabbit cerebral arterial SMCs of intact vessels (25). It must be taken into account that in intact vessels, many factors may modify steady-state membrane potentials, such as the release of endothelial factors or endogenous neurotransmitters, which may hyperpolarize vascular SMCs.

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Whole K+ current activation by PGF₂. Recently, it was reported that arterial diameter and activity of K⁺ channels, as a major regulator of membrane potential in vascular SMCs, are closely correlated, i.e., opening of K⁺ channels leads to artery dilatation and vice versa. We demonstrated the activation of whole cell K⁺ current as well as membrane potential hyperpolarization by PGF₂, which could attenuate PGF₂-induced contractions.

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K_Ca channel activation by PGF₂. K_Ca channels have large unitary conductance. Therefore, opening a few K_Ca channels has a significant impact on membrane potentials (34, 36). In fact, Gokina et al. (15) demonstrated that blockade of K_Ca channels by charybdotoxin and TEA resulted in membrane depolarization, an increase of the amplitude and duration of action potentials, and marked contraction of SMCs in human pial arteries. Asano et al. (1) suggested that the resting tone of cerebral arteries is determined by at least two opposite components: contraction due to increased basal Ca²⁺ influx (probably through activation of myosin light chain kinase) and relaxation due to activation of K_Ca channels and other Ca²⁺ extraction systems. The net balance of these two components results in contraction or relaxation. These studies suggested that an important function of K_Ca channels in vascular smooth muscle might be to act as a buffering system to limit contraction. The present study, using isolated rabbit middle cerebral arterial SMCs, demonstrated that PGF₂ activates K_Ca channel activities without affecting channel amplitude and kinetic properties in outside-out patches. These results suggest that PGF₂ increases K_Ca channel activities in a membrane-delimited manner rather than through an intracellular second messenger and that the activation of K_Ca channels by PGF₂ could counteract the PGF₂-induced vasoconstriction. Our results have something in common with other studies.

K_V channel activation by PGF₂. K_V channel is also one of the dominant K⁺ channels, and its importance in determining membrane potential has been well characterized in cerebral artery (25, 34). K_V current represents the dominant repolarizing conductance within the physiological range of membrane potentials and is critical in determining membrane potential at low or normal intracellular Ca²⁺ levels (39). Thus membrane potential is directly related to the whole cell K_V currents. In the present study, PGF₂ increased K_V currents with a leftward shift of activation and inactivation curves and a decrease of activation time constant. Although not directly addressed in this study, the expected physiological consequence of this increase in K_V currents could counteract the PGF₂-induced vasoconstriction.

Ca²+ current inhibition by PGF₂. Ca²⁺ released from internal stores not only initiates smooth muscle contraction but also modulates the ion conductances of the plasma membrane. The latter modifies the membrane potential of SMCs, thereby regulating the influx of Ca²⁺ through L-type Ca²⁺ channels (6, 21). In the present study, PGF₂ reduced the amplitude of the L-type Ca²⁺ channel current without any significant change in threshold or apparent reversal potential. On the other hand, erythrocyte lysates increase intracellular Ca²⁺ and contract cerebral arteries in vitro. Hemolysate, however, does not increase intracellular Ca²⁺ by activation of L-type Ca²⁺ channels (22). Together, our results therefore suggest that PGF₂-induced vasoconstriction is not caused via activation of L-type Ca²⁺ channels. This finding is supported by the fact that PGF₂-induced contractions are not completely relaxed by addition of Ca²⁺ channel antagonists (46, 50).

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