Journal of Physiology (1992), 451, pp. 307-328 With 12 figures Printed in Great Britain

307

MEMBRANE HYPERPOLARIZATION INHIBITS AGONIST-INDUCED SYNTHESIS OF INOSITOL 1,4,5-TRISPHOSPHATE IN RABBIT MESENTERIC ARTERY

BY TAKEG ITOH, NARIHITO SEKI, SATOSHI SUZUKI, SHINICHI ITO, JUNKO KAJIKURI AND HIROSI KURIYAMA From the Department of Pharmacology, Faculty of Medicine, Kyushu University 60, Fukuoka 812, Japan

(Received 11 October 1990) SUMMARY

1. Effects of membrane hyperpolarization induced by pinacidil on Ca2+ mobilization induced by noradrenaline (NA) were investigated by measuring intracellular Ca2+ concentration ([Ca2+]i), isometric tension, membrane potential and production of inositol 1,4,5-trisphosphate (1P3) in smooth muscle cells of the rabbit mesenteric artery. 2. Pinacidil (01-10 ,UM) concentration dependently hyperpolarized the smooth muscle membrane with a reduction in membrane resistance. Glibenclamide (1 ,JM) blocked the membrane hyperpolarization induced by 1 jM-pinacidil. NA (10 /SM) depolarized the smooth muscle membrane with associated oscillations. Pinacidil (1 /LM) inhibited this response and glibenclamide (1 ,tM) prevented the action of pinacidil on both the NA-induced events. 3. In thin smooth muscle strips, 10 /M-NA produced a large phasic and a subsequent small tonic increase in [Ca2+]i with associated oscillations. These changes in [Ca2+]i seemed to be coincident with phasic, tonic and oscillatory contractions, respectively. Pinacidil (0-1-1 (1M) inhibited the increases in [Ca2+]i and in tension induced by NA, but not by 128 mM-K+. Glibenclamide inhibited these actions of pinacidil. Pinacidil (1 UM) also inhibited the contraction induced by 10 tm-NA in strips treated with A23187 (which functionally removes cellular Ca2+ storage sites), suggesting that membrane hyperpolarization inhibits Ca2+ influxes activated by NA. 4. In Ca2+-free solution containing 2 mM-EGTA, NA (10 gm) transiently increased [Ca2+]i, tension and synthesis of IP3. Pinacidil (over 01 ,lM) inhibited the increases in [Ca2+]i, tension and synthesis of IP3 induced by 10 /tM-NA in Ca2+-free solution containing 5.9 mM-K+, but not in a similar solution containing 40 or 128 mm-K+. Glibenclamide (1 ,tM) inhibited these actions of pinacidil. These inhibitory actions of pinacidil were still observed in solutions containing low Na+ or low Cl-. These results suggest that pinacidil inhibits NA-induced Ca2' release from storage sites through an inhibition of IP3 synthesis resulting from its membrane hyperpolarizing action. 5. In ,-escin-treated skinned strips, NA (10 /tM) or IP3 (20 gM) increased Ca2+ in Ca2+-free solution containing 50 /%M-EGTA and 3 /tM-guanosine triphosphate (GTP) after brief application of 0 3 /tM-Ca2 , suggesting Ca2+ is released from intracellular MS 8862

308

T. ITOH AND OTHERS

storage sites. Heparin (500 jtg/ml, an inhibitor of the IP3 receptor), but not pinacidil (1 #M) or glibenclamide (1 tM), inhibited the Ca2l release from storage sites induced by NA or 'P3. These results suggest that membrane hyperpolarization is essential for the inhibitory action of pinacidil on the NA-induced Ca2+-releasing mechanism. Thus, the membrane hyperpolarization induced by pinacidil negatively controls the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) induced by NA and causes vasodilatation together with a blockade of spike generation in smooth muscle cells of the rabbit mesenteric artery. INTRODUCTION

In smooth muscle of the rabbit mesenteric artery, noradrenaline (NA) produces a phasic followed by a tonic contraction. The phasic contraction is preserved in Ca2+free solution containing EGTA (Itoh, Kuriyama & Suzuki, 1983). NA synthesizes IP3 in intact smooth muscle strips of the rabbit mesenteric artery and IP3 produces contraction as a result of Ca2+ release from storage sites in skinned strips (Hashimoto, Hirata, Itoh, Kanmura & Kuriyama, 1986). Thus, NA may generate its phasic contraction via the action of IP3 in smooth muscle of the rabbit mesenteric artery. By contrast, the tonic phase of the NA-induced contraction is partly inhibited by blockers of the voltage-dependent Ca2+ channel and completely abolished in Ca2+free solution (Kanmura, Itoh, Suzuki, Ito & Kuriyama, 1983; Makita, Kanmura, Itoh, Suzuki & Kuriyama, 1983). Pinacidil ((± )-N-cyano-4-pyridyl-N-1,2,2-trimethylpropylguanodine monohydrate) is a novel vascular relaxant which hyperpolarizes the membrane of vascular smooth muscle cells (Southerton, Weston, Bray, Newgreen & Taylor, 1988). Pinacidil hyperpolarizes the membrane of smooth muscle cells through an activation of the ATP-dependent and [Ca2+]i-insensitive K+ channel and inhibits the contraction induced by NA in the rabbit mesenteric artery (Standen, Quayle, Davies, Brayden, Huang & Nelson, 1989). These authors suggested that membrane hyperpolarization inhibits the NA-induced tonic contraction by closing the voltage-dependent Ca2+ channel. However, the effect of hyperpolarization on the phasic contraction induced by NA has not yet been examined in vascular smooth muscle. In oc-toxin- or fl-escin-treated skinned smooth muscle strips, NA with guanosine triphosphate (GTP) enhances Ca2+-induced contraction, and this action of NA is inhibited by neomycin (an inhibitor of phospholipase C) or guanosine-5'-O-(f,thiodiphosphate) (GDPflS) (Kitazawa, Kobayashi, Horiuti, Somlyo & Somlyo, 1989). These results suggest that NA enhances Ca2+-induced contraction through an activation of G-protein-mediated pathways. It was recently reported that in oc-toxintreated skinned smooth muscle strips of the rabbit mesenteric artery, a high concentration of pinacidil (100 ,tM) inhibited Ca2+-induced contraction in the presence but not in the absence of NA (Anabuki, Hori, Ozaki, Kato & Karaki, 1990). In ,-escin-treated skinned smooth muscle of the rabbit mesenteric artery, although pinacidil (10 /LM) inhibited the Ca2+-induced contraction in the presence or absence of NA (Itoh, Suzuki & Kuriyama, 1991), pinacidil (> 3 ,SM) inhibited Ca2+-induced contraction more in the presence of NA with GTP than in the absence of NA and GTP. These results suggest that a high concentration of pinacidil may directly

MEMBRANE HYPERPOLARIZATION AND IP0 309 inhibit the NA-activated hydrolysis of phosphatidylinositol-4,5-bisphosphate without its corresponding membrane hyperpolarizing action. To investigate the mechanisms of vasodilatation resulting from the membrane hyperpolarization induced by pinacidil in detail, we investigated the effects of pinacidil on changes in the [Ca2+]i, tension and 1P3 production induced by NA in intact and skinned smooth muscle of the rabbit mesenteric artery.

METHODS

Male albino rabbits, weighing 1-9-2 5 kg, were anaesthetized by injection of pentobarbitone sodium (40 mg/kg, i.v.), and were then exsanguinated. The third branch of the mesenteric artery was excised immediately and cleaned by removal of the connective tissue in Krebs solution at room temperature.

Electrophysiological experintents A glass microelectrode filled with 3 M-KCl was made from a borosilicate glass tube (o.d., 1-2 mm with a core inside, Higenberg, FRG). The resistance of the electrodes was 40-80 MQl. The electrode was inserted into smooth muscle cells from the serosal side. The membrane resistance in the presence or absence of drugs was estimated using the partition stimulating method (Abe & Tomita, 1968) as described previously (Kanmura et al. 1983). The responses were displayed on a cathode ray oscilloscope (VC-10, Nihon-Kohden) and also on a pen-writing recorder (Recticorder RJG-4024, Nihon-Kohden).

Ca2+ and ten8ion mea8urement To enable recording of isometric tension, fine circularly cut strips (03-05 mm length, 0 04-0 05 mm width, 0-02-003 mm thickness) were prepared as described previously (Itoh et al. 1983). Endothelial cells were removed by gentle rubbing of the internal surface of the vessels using small knives. The absence of the functions of endothelial cells was confirmed by the inability to cause relaxation with acetylcholine (1 /M) or A23187 (0-1 /M) during contractions induced by NA. The strips were transferred to a chamber of 0 3 ml volume and mounted horizontally on an inverted microscope (Diaphot TMD with special optics for epifluorescence, Nikon). To load Fura-2 into smooth muscle cells of the strip, 1 ,SM-acetoxy methyl ester of Fura-2 (Fura2 AM) dissolved in dry dimethyl sulphoxide (1 mm stock solution) was applied for 1 h in Krebs solution at room temperature (20-23 °C). After this period, the solution containing Fura-2 AM was washed with Krebs solution for 1 h to ensure sufficient esterification of Fura-2 AM in the cells. The strip was moved to the centre of the field and a mask (0 04 mm square) placed in an intermediate image plane of the microscope to reduce background fluorescence. The Fura-2 fluorescence emission at 510 nm (passed through an interference filter centred at 510 nm with a full width at halftransmission of 20 nm) was passed through the objective lens (20 x fluor, Nikon) and collected with a photomultiplier tube (R928, side-on type, Hamamatsu Photonics, Japan) via a dichroic mirror (DM-400, Nikon) which was substituted for the photochanger in a Nikon Diaphot-TMD microscope. Two alternative excitation wavelengths, 340 and 380 nm (each slit 5 nm), were applied by a spectrofluorimeter (Spex, NJ, USA) and the data were analysed using customized software provided by Spex (DM-3000CM). The concentration of Fura-2 in smooth muscle cells of the strip was not measured since appropriate methods are not available. Instead, we examined the maximum rate and the magnitude of contractions induced by 10 /tM-NA or 128 mM-K+ in the same strips before and after loading with Fura-2. As shown in Table 1, the maximum amplitude of contraction induced by either stimulant was not significantly different between Fura-2-loaded and unloaded strips. However, the time to half-decay after wash-out of the stimulant was slightly elongated and the amplitudes and frequency of the oscillatory contractions induced by NA were slightly lowered in Fura-2-loaded strips (not shown). The ratio of Fura-2 fluorescence intensities excited by 340 or 380 nm was calculated after

T. ITOH AND OTHERS

310

subtraction of the background fluorescence. Background fluorescence (including the autofluorescence of the strip) excited by 340 and 380 nm UV light was obtained by application of a solution containing 50 /SM-ionomycin, 20 mM-MnCl2, 110 mM-KCl and 10 mM-3-(N'-morpholino)propanesulphonic acid (MOPS; pH 4 8) after the experiment. Under these conditions, the background fluorescence intensity was 10-15 % of the Fura-2 signals in smooth muscle strips at

TABLE

1. Effects of Fura-2 on

the rate and magnitude of contraction induced by 128 mM-K+ 10 /M-NA in smooth muscle strips of the rabbit mesenteric artery 128 mM-K+ NA

Peak tension (,uN) Time to half-peak (s)

Unloaded 77-7 + 81

Fura-2 loaded

Unloaded

or

Fura-2 loaded

826 + 18-7 57-8 60 67-4 + 19-2 5-7+1-2 6-8+1-4 9.0+0.9* 5-7+±16 Time to half-decay (s) 9-8 24 15-5 4.4* 14-0 30 20-0 4.4* The values were compared using the same strips before and after application of /uM-Fura-2 for 60 min. Results are mean + S.D. of six observations. * represents significant difference from control (P < 0 05). Note that the time to half-decay of tension after wash-out of either stimulant was longer in Fura-2-loaded strips than that in unloaded strips. +

+

+

+

+

1

either excitation

wavelength. Cytosolic

Ca2+

concentrations

were

calculated using the formula

described by Grynkiewicz, Poenie & Tsien (1985) and in vitro calibration (Poenie, Alderton, Steinhart & Tsien, 1986; Becker, Singer, Walsh & Fay, 1989). The ratios of the maximum (Fmax) and of the minimum fluorescence (Fmin) were determined in the calibration solution after subtraction of background excitation by either 340 or 380 nm and the 380 nm signals of Fura-2 were assumed to decrease by 15 % in the cell due to the possible intracellular viscosity effects of the dye (Becker et al. 1989). The dissociation constant(Kd) for Fura-2 was estimated to be 200nM (Becker et al. 1989). This calculated [Ca2+]i might not be accurate if Fura-2 binds to some proteins in the cell (Konishi, Olson, Hollingworth & Baylor, 1988).

Experiment on chemically8kinned8mooth mu8cle Chemically skinned smooth muscle strips were made using f-escin (Kobayashi, Kitazawa, Somlyo & Somlyo, 1989). The methods used to skin the muscle strips and the compositions of the solutions used have been described elsewhere (Itoh, Kanmura & Kuriyama, 1986; Kobayashi etal. 1989). To measure release from the storage sites, 0'3 buffered with 4mM-EGTA was applied for 2 (to load Ca2+ into the storage sites) and was removed by application Ca2+of free solution containing 4 mm-EGTA for min. 05 Then, a solution containing 50/sM-EGTA, /M3 GTP and 2 was applied formin. 2 Finally, 10 Um-NA with / 3 tM-GTP or 20/tM-I1P was applied formin 2 in a solution containing 50 /tm-EGTA and 2 /Sm-Fura-2 (see also Fig. 12).

min

Ca2l

Ca2+4um-Ca2+

/tM-Fura-2

Measurement of IP3, cyclic AMP and cyclic GMP Endothelium-denuded strips (10 mm length, 2 2-2 5 mm width, 0 1 mm thickness) were equilibrated for over 2 h°C at 32 in Krebs solution. After this, the strips were transferredCa2+_ to free Krebs solution containing 2 mm-EGTA with 5.9 for m1in, solution containing 2 mm-EGTA with various concentration of high for m1 in and then 10 /m-NA was applied. Pinacidil or glibenclamide was given as pretreatment for 3 in Krebs solution, for 2 Ca2+_ in min free solution and during application of NA. The reaction was stopped by addition of a large amount of ice-cold trichloroacetic acid (final concentration 8 %) and the strips were homogenized. The homogenate was centrifuged and the supernatant fraction was treated with ether three times and assayed using a radioimmunoassay kit from Amersham International plc. Great care was taken to maintain the pH of the homogenate at 90-9 5 to optimize the binding of the binding protein to IP8. To this end, 50 mm-2-(cyclohexylamino)ethanesulphonic acid (CHES) was used instead of 0x1 mTris to adjust the pH of the homogenate in the presence of universal indicator. To minimize the loss of IP3, Teflon tubing was used instead of glassware after homogenization.

Ca2+-free K+mm-Kmin

MEMBRANLE HYPERPOLARIZATION AND IP3

311

The methods for the measurement of cyclic AMP and cyclic GMP have been reported previously (Kajikuri & Kuriyama, 1990). The assay was done using a radioimmunoassay kit for cyclic GMP or cyclic AMP from Amersham International plc. Pinacidil (0-1-10 ,tM) or NA (10 ,M) was applied for 10 min and for 2 min. Solutions The ionic composition of the Krebs solution was as follows (mM): Na+, 137-4; K+, 5-9; Mg2", 1P2; Ca2+, 2-6; HCO3-, 15-5; H2PO4-, 1-2; Cl-, 134; glucose, 11 -5. The concentration of K+ was modified by replacing NaCl with KCl, isosmotically. To prevent NA outflow from sympathetic nerve terminals and ,-adrenoreceptor stimulation by exogenously applied NA, 3 /tM-guanethidine and 0 3 /SM-propranolol were added to the Krebs solution throughout the experiment. Ca2+-free Krebs solution was made by adding an equimolar concentration of MgC12 instead of CaCl2 and adding 2 mM-EGTA. To make low-Na+-containing Krebs solution, NaCl was replaced with Tris-Cl. To make low-Cl--containing Krebs solution, Cl- was replaced with methanesulphonate. The solutions were bubbled with 95% 02 and 5% C02, and their pH maintained at 7 3-7 4. The calibration solution for the Ca2+ measurements in intact strips contained 11 mM-EGTA, 10 mM-KCI, 1 mM-MgCl2, 2 /SM-Fura-2 and 20 mM-N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES) (pH 7-1) with or without 11 mM-CaCl2. For experiments on skinned muscle, the composition of the relaxing solution was: 87 mmpotassium methanesulphonate (KMs), 20 mM-piperazine-N,N'-bis-(2-ethanesulphonic acid) (PIPES), 5-1 mM-Mg(Ms)2, 5-2 mM-ATP, 10 mM-phosphocreatine and 4 mM-ethyleneglycol-bis-(flaminoethyl)-N,N,N',N'-tetraacetic acid (EGTA). To enable measurement of Ca2+ release from skinned strips, the concentration of EGTA was reduced to 50,ClM and 2 ,SM-Fura-2 was added. Various Ca2+ concentrations were prepared by adding appropriate amounts of Ca(Ms)2 to 4 mMEGTA, based on the calculation reported previously (Itoh et al. 1986). The pH of the solution was adjusted to 7-1 at 25 °C with KOH and the ionic strength was standardized at 0-2 M by changing the amount of KMs added.

Drugs Drugs used were Fura-2, Fura-2 AM, EGTA, PIPES, CHES, HEPES and MOPS (Dojin, Japan), NA, IP3, GTP, heparin (4000-6000 molecular weight from porcine intestinal mucosa), fl-escin and glibenclamide (Sigma), guanethidine (Tokyo Kasei, Japan), ATP (sodium salt, Kojin, Japan), propranolol (Nacalai, Japan), A23187 and ionomycin (free acid, Calbiochem), universal indicator (BDH Co.) and pinacidil (Shionogi, Japan).

Statistics The values recorded were expressed as means+ S.D., and statistical significance determined using Student's t test. Probabilities less than 5% (P < 0-05) were considered significant.

RESULTS

Effects of pinacidil on membrane properties Smooth muscle cells of the rabbit mesenteric artery were electrically quiescent and the resting membrane potential was -73 to -70 mV (mean value, - 710 + 1 1 mV, n = 20). To prevent NA outflow from sympathetic nerves and to block activation of ,/-adrenoreceptors, 3 /,M-guanethidine and 0 3 ,tm-propranolol were added throughout the experiment (Mishima, Miyahara & Suzuki, 1984). Pinacidil in concentrations over 041 fM hyperpolarized the membrane in a concentration-dependent manner (Fig. 1B). Changes in the ionic conductance of the membrane were estimated from changes in the amplitude of the electrotonic potential evoked by application of constant inward and outward current pulses, alternately (Fig. IA). Following application of 1 /SM-pinacidil the amplitude of the electrotonic potential was reduced 11

PH Y 451

T. ITOH AND OTHERS

312

to about 07 times control (Fig. I A). When the current-voltage (I-V) relationships before and during application of 1 /uM-pinacidil were compared by alternative application of various intensities of inward and outward current pulses, the slope of the relationship in the presence of 1 tm-pinacidil was less steep than that under A

1

jM-pinacidil

jlo mV 1 min

B

5'> 70

co E E0. E o80-

i

-

0.

C

90o, . A......... ... ....

7 -log [pinacidill

6

5

(M)

Fig. 1. Effects of pinacidil on membrane potential in smooth muscle cells of rabbit mesenteric artery. A, 1 /SM-pinacidil was applied at arrow. Constant inward and outward current pulses (1 s duration and 0 2 V intensity) were alternately applied to the tissue by the partition stimulating method. Guanethidine (3/M) was present throughout the experiment to inhibit NA outflow from sympathetic nerves. The microelectrode was inserted into a cell 0-1 mm from the stimulating electrode. B, concentration-response relationship for effect of pinacidil on membrane potential. The membrane potential was measured 10 min after application of pinacidil. Guanethidine (3 /tM) was present throughout the experiment. Results shown are each the mean of five to eight observations with S.D. shown by vertical bars. C represents control.

control conditions. Since pinacidil hyperpolarized the membrane, we displaced the membrane potential in the presence of 1 guM-pinacidil to the resting level by application of an outward current and the I-V relation again observed. The slope of the relationship in the presence of pinacidil was the same whether or not the membrane potential was displaced to the control level. Glibenclamide (1 UM) depolarized the membrane to -64-5+1-7 mV (n = 5) from the resting level of - 71-0 + I-1 mV (n = 5) and inhibited the hyperpolarization induced by 1 gM-pinacidil (- 78-3 + 1-3 mV in the presence of 1 /tM-pinacidil alone and - 64-3 + 1-8 mV in the presence of / M-pinacidil with 1 ,SM-glibenclamide, n = 5). The hyperpolarization of the membrane induced by 1 /tM-pinacidil was preserved in low-Na+-containing Krebs solution (15 mM-Na'). In low-Na+-containing solution, the membrane was depolarized from -71 + 0-5 to - 64-3 + 1-9 mV (n = 5) and it was hyperpolarized to -74 + 2-1 mV by application of 1 /tM-pinacidil (n = 5). Pinacidil (1 JtM) also hyperpolarized the membrane in low-Cl- (14-8 mM-Cl--containing) Krebs solution (- 76-3 + 1 -0 and - 79-0 + 1-2 mV in the absence and presence of pinacidil,

MEMBRANE HYPERPOLARIZATION AND IP3

313

respectively, n = 5, P < 0 05). These results suggest that pinacidil activates a glibenclamide-sensitive K+ channel and causes membrane hyperpolarization.

Effects of pinacidil on changes in membrane potential [Caa2+]i and tension induced by NA NA (10 /LM) depolarized the membrane and caused associated oscillations in membrane potential in smooth muscle cells of the rabbit mesenteric artery. Pinacidil 0.1

A

pM-pinacidil

1 min

1 ,M-pinacidil

B

10jum-NA

C

10 mV

r

10 pM-NA 1 pM-glibenclamide

1

,UM-pinacidil

X

Fig. 2. Effects of pinacidil on membrane depolarization induced by 10 ,uM-NA in a smooth muscle cell of rabbit mesenteric artery. NA (10,M) or glibenclamide was applied at arrows. Pinacidil (0-1 or 1 ,UM) was applied in the presence of 10 Mm-NA. Guanethidine (3/SM) and propranolol (0 3 ,sM) were present throughout the experiment to prevent NA outflow from sympathetic nerves and fl-receptor activation by NA, respectively. These results (A-C) were obtained from a single smooth muscle cell of the rabbit mesenteric artery.

(0-1 or 1 ,UM) inhibited the membrane depolarization and the oscillation induced by 10 /M-NA in a concentration-dependent manner. Glibenclamide (1 /LM) completely abolished these effects of pinacidil (Fig. 2C). Since the membrane oscillations induced by NA are inhibited by nifedipine (Kanmura et al. 1983), these results suggest that pinacidil inhibits the voltage-dependent Ca2+ influx induced by NA, probably as a result of its membrane hyperpolarizing action. Figure 3 shows actual traces of effects of pinacidil on increases in the [Ca2+]i and tension induced by 10 guM-NA in a thin smooth muscle strip. Resting Ca2+ concentration in the strips was 125+ 15 nM (n = 10). NA was applied for 2 min at 30 min intervals to obtain reproducible responses and then pinacidil was applied for

5 min before and during the application of NA. As shown in Fig. 3, 10 #M-NA

produced a phasic followed by a small tonic increase in [Ca2+]i and tension. The changes in [Ca2+]i always preceded the tension development. In some strips, oscillatory changes in [Ca2+]i were observed superimposed on the tonic increase induced by 10 gtM-NA when NA was applied for over 3 min. Pinacidil (0 1 and 1 /,M) slightly lowered the resting [Ca2+]i and inhibited the evoked increases in [Ca2+]i and tension induced by 10 /tM-NA, in a concentration11-2

T. ITOH AND OTHERS

314

dependent manner (Figs 3 and 4). Glibenclamide (1 JtM) inhibited the effects of pinacidil on the resting [Ca2+]i, and also the changes in the [Ca2+]i (Fig. 3C) and tension (Fig. 3D) induced by 10 ,tM-NA (see also Fig. 4). The effects of glibenclamide in concentrations over 1 ,UM were not examined because the vehicle (dimethyl Pinacidil 10 nM

Control

100 nM

1

JiM [Ca2+]i (nM)

A

400 2 min b

aS < 10 tM-NA

b

X

c

-

d.

C -

300

~~~~~~~~~~200 1100

Tension

B a

b

d

2 min -

5mg

10 ,uM-NA

[Ca2+]J (nM) C

400 2 min a

b

c

d

_____-~~~~~~~~~~~100

10 giM-NA

D 2 min a

1-300 1200

b

c

d

Tension 5 mg

10 /M-NA

Fig. 3. Effects of pinacidil on changes in [Ca2+]1 and tension induced by 10 /tM-NA in a smooth muscle strip of the rabbit mesenteric artery. Changes in [Ca2+]i induced by 10 UMNA are shown in the presence (C) or absence (A) of 1 /SM-glibenclamide. Dashed lines indicate resting [Ca2+]i level in the control state. Changes in tension induced by 10 AM-NA are shown in the presence (D) or absence (B) of l/LM-glibenclamide. NA (10 M) was applied for 2 min at 30 min intervals in Krebs solution. Guanethidine (3/tM) and propranolol (0 3 /UM) were present throughout the experiment. Pinacidil was given as pretreatment for 10 min and was present during application of NA. Aa, Ba, Ca and Da are controls; Ab, Bb, Cb and Db, in the presence of 10 nM-pinacidil; Ac, Bc, Cc and Dc, +100 nM-pinacidil; Ad, Bd, Cd and Dd, +1 /uM-pinacidil. Glibenclamide (1 /tM) was applied for 10 min before, and throughout application of pinacidil. The elapsed time from Ad to Ca and from Bd to Da was 80 min which was sufficient for recovery of the control responses. The experiment was done using a single smooth muscle strip.

sulphoxide) used to dissolve glibenclamide itself inhibited the NA contraction (final concentration of vehicle was 0 001 %). Pinacidil (0O01-1 fSM) did not modify the maximum increase in [Ca2+]i and tension

MEMBRANE HYPERPOLARIZATION AND IP3

315

induced by 128 mM-K+ in the presence or absence of 1 /LM-glibenclamide (Fig. 5). The maximum increase in [Ca2+]i induced by 128 mM-K+ was 488 + 85 mm in control, and this was 493 + 82, 498 + 100 and 470 + 39 nm in the presence of 0 01, 0 1 and 1,UMpinacidil, respectively (n = 5). The maximum tension induced by 128 mM-K+ was A 400

E300

o200 100 . ..-.. .. C

0.01 Pinacidil

0.1

1.0

(gM)

B 1.2 -

cu: 0.8,x,0-4\ 0.60 .4.

0.0M.4. C

.

0.1 0.01 Pinacidil (pM)

1.0

Fig. 4. Concentration-response relationships of pinacidil on increase in [Ca2+] (A) and tension (B) induced by 10 /M-NA in the presence (0) or absence (0) of 1 ,UMglibenclamide in smooth muscle strips of the rabbit mesenteric artery. A, the maximum increase in [Ca2+]1 induced by 10 /sM-NA was shown in the presence or absence of pinacidil. In B, the maximum tension induced by 10/SM-NA in the absence of pinacidil and glibenclamide was normalized as relative tension of 1-0. Experimental protocols were the same as those shown in Fig. 3. Results represented are mean of five observations with S.D. shown by vertical bar.

not changed in the presence of pinacidil (0-98 + 0 03 times the control in the presence of 0 I ,SM-pinacidil and 0-98 + 0-04 times in the presence of 1 ,sM-pinacidil, n = 5). The tension induced by 128 mM-K+ was slightly inhibited by 10 /tM-pinacidil (0-85 + 0 03 times the control) without corresponding changes in [Ca2+]i (468+35 nM) in the presence or absence of 1 j/M-glibenclamide (n = 5). Nifedipine blocked the tonic contraction induced by 128 mM-K+ but only partly inhibited the tonic contraction induced by 10 gm-NA (Kanmura et al. 1983; Makita et al. 1983). In the presence of 1 ,SM-nifedipine, pinacidil (0 1 M) further inhibited the tonic contraction induced by 10 /Lm-NA. To eliminate the role of intracellular Ca21 storage sites during the NA contraction, 041 ItM-A23187 was applied to the strip for over 30 min (Itoh, Kanmura & Kuriyama,

T. ITOH AND OTHERS

316

1985). In such strips, 10/tM-NA produced a monophasic contraction (i.e. the phasic and oscillatory contractions were not evoked). Pinacidil inhibited the NA contraction in 0 1 ftM-A23187-treated strips more potently than in non-treated strips (1P4-2 times control, n = 5). Pinacidil 10 nM

Control

100 nM

1 ,UM

[Ca2+jj (nM)

A

600

F500 minF 400 ~ ~ ~ ~~~~~~~-300 b 2

200

100

128 mM-K+ B

Tension

b

a

c

dI

2 min 5 mg

128 mM-K+

[Ca2+Ji (nM) 600 -500 400 300

c

b

a 128

c

dj

2 mmin

- 200

mM-K+

D

2 min

Tension a

b

c

d

5 mg

128 mM-K+ Fig. 5. Effects of pinacidil on changes in [Ca2+]1 and tension induced by 128 mM-K+ in a smooth muscle strip of the rabbit mesenteric artery. Change in [Ca2+]i induced by 128 mMK+ in the presence (C) or absence (A) of 1 ,uM-glibenclamide. Changes in tension induced by 128 mM-K+ in the presence (D) or absence (B) of 1 #tM-glibenclamide. K+ (128 mM) was applied for 2 min at 30 min intervals in Krebs solution. Guanethidine (3 /M) and propranolol (0-3 /M) were present throughout the experiment. Pinacidil was given as pretreatment for 10 min and was present during application of 128 mM-K+. Aa, Ba, Ca and Da are controls; Ab, Bb, Cb and Db, in the presence of 10 nM-pinacidil; Ac, Bc, Cc and Dc, + 100 nM-pinacidil; Ad, Bd, Cd and Dd, +1,IM-pinacidil. Glibenclamide (1 M) was applied for 10 min before and throughout application of pinacidil. The elapsed time from Ad to Ca and from Bd to Da was 80 min which was sufficient for recovery of the control responses. The experiment was done using a single smooth muscle strip.

The phasic contraction induced by NA persists in Ca2+-free solution, and this has been presumed to be due to the NA-induced release of Ca2+ from the storage sites in smooth muscle cells of the rabbit mesenteric artery (Itoh et al. 1983). To study the

MEMBRANE HYPERPOLARIZATION AND IP3 317 effects of pinacidil on the NA-induced Ca2+ release, the effects of pinacidil on the Ca21 transient and contraction induced by 10 ,tM-NA were studied in Ca2+-free solution containing 2 mM-EGTA. At 30 min intervals, NA (10 ftM) was applied for 2 min in Ca2 -free solution containing 2 mm-EGTA after 2 min removal of Ca2 , the strips A

500 400

j 300 s CN

o 200

100 0

0

50

100 Time (s)

200

150

B

5 mg

10,uM-NA .

;0,

100 150 200 Time (s) Fig. 6. Effects of pinacidil on increases in [Ca2+]i (A) and tension (B) induced by 10 /MNA in Ca2+-free solution containing 2 mM-EGTA in a smooth muscle strip of rabbit mesenteric artery. NA (10 #M) was applied for 2 min after 2 min removal of Ca2 , then Ca2+-free solution was applied for 1 min to wash out NA, and finally Krebs solution (containing 2-6 mM-Ca2+) was applied for 25 min. This protocol was repeated at 30 min intervals. Pinacidil was applied for 5 min before and throughout application of NA. Guanethidine (3 SM) and propranolol (0 3 /sM) were present throughout the experiment. NA (10 #M) was applied from time 30 to 150 s. An expanded representation of the initial changes in [Ca2+]1 induced by NA is shown in the inset in A. a, control; b, in the presence of 001 4uM-pinacidil; c, +0I /LM-pinacidil; d, + 1 zM-pinacidil. Note that the resting [Ca2+]1 before application of NA was lower in the presence of pinacidil than in control. 0

50

being kept in Krebs solution for the 25 min between tests. Figure 6 shows the effects of pinacidil on the Ca2+ transient and contraction induced by 10 ,tM-NA in Ca2+-free solution on a thin strip of the rabbit mesenteric artery. Following application of Ca2+-

T. ITOH AND OTHERS

318

free solution containing 2 mM-EGTA, the resting [Ca2+]i was rapidly decreased from 125 + 15 to 88 + 15 nm within 1 s and then maintained at the new steady level (n = 5). NA (10 jam) transiently increased [Ca2+] and produced contraction in Ca2+-free solution. Pinacidil (over 01 gM) lowered the resting [Ca21]i, and inhibited both the A 500

3400 300

200 100

-

I

C0.01 Pinacidil

B

0.1

1|0

(gM)

12a 0.8 .706 -o.

-

{0

j0.4

02 0.2

C

/

001 01 Pinacidil (pM)

10

Fig. 7. Concentration-response relationships of pinacidil on increase in [Ca2+]i (A) and tension (B) induced by 10 #M-NA in Ca2+-free solution containing 2 mM-EGTA with (0) or without (@) 1 /sm-glibenclamide in smooth muscle strips of the rabbit mesenteric artery. In A, the maximum increases in [Ca2+]i induced by 10 JiM-NA in Ca2+-free solution were shown in the presence or absence of pinacidil. B, the maximum tension induced by 10 #M-NA in the absence of pinacidil and glibenclamide was normalized as relative tension of 1 0. Experimental protocols were the same as those shown in Fig. 7. Results represented are mean of five observations with S.D. shown by vertical bar.

rate of rise and the magnitude of the increase in [Ca2+]i and in tension development induced by NA in Ca2+-free solution, in a concentration-dependent manner (Figs 6 and 7). The effects of pinacidil on the resting and active [Ca2+]i and tension induced by 10 jam-NA were inhibited by 1 jam-glibenclamide (Figs 7 and 8). The concentrationdependent effects of pinacidil on the increase in [Ca2+]i and tension induced by 10 aZmNA in Ca2+-free solution containing 2 mM-EGTA with 5.9 mM-K+ were shown in the presence or absence of 1 jim-glibenclamide (Fig. 7). Effects of pinacidil on the increase in [Ca2+]i and tension induced by 10 gZm-NA were observed in Ca2+-free solution containing 2 mM-EGTA in -the presence of various concentrations of K+ (Fig. 9). The Ca2+-free solution with high K+ (20-128 mm) was applied for 1 min after 1 min removal of Ca2+ by Ca2+-free solution containing 5 9 mM-K+. NA (10 jam) was then applied for 2 min in Ca2+-free

MEMBRANE HYPERPOLARIZATION AND IP3

319

A 500

500

b

b

400

a

a

300 0

C

400

+

___

100

300

___

p-NA 10m

-

0 ~~~~~d 30

~~

C

NT

35

40

Time (s)

O 200

10 /pm-NA 50

0

150'

100 Time (s)

200

B

5 mg

10gM-I I

0

.

.

.

.

I

50

--.

.

.

I

.

100

.

.

I

150

.

200

Time (s)

Fig. 8. Effects of pinacidil on increases in [Ca2+]j (A) and tension (B) induced by 10 /tMNA in Ca2+-free solution in the presence of 1 4aM-glibenclamide in a smooth muscle strip of the rabbit mesenteric artery. NA (10 /UM) or pinacidil was applied using the same protocol as described in Fig. 7. Glibenclamide was applied for 5 min before and throughout application of NA or pinacidil. Guanethidine (3 ,UM) and propranolol (0-3 /sM) were present throughout the experiment. NA (10 ,M) was applied from time 30 s to 150 s. An expanded representation of the initial changes in [Ca2+]i induced by NA is shown in the inset in A. a, control; b, in the presence of 1 4uM-glibenclamide with 0-01 #uM-pinacidil; c, +1 tM-glibenclamide with 01 #uM-pinacidil; d, +1 /M-glibenclamide with 1 /,Mpinacidil.

solution containing high K+. Under these conditions, high K+ (20-128 mM) increased neither [Ca2+]i nor tension (not shown). As shown in Fig. 9, the inhibitory actions of 1 JtM-pinacidil on the increase in [Ca2+]i and tension induced by 10 /M-NA were greatly inhibited in Ca2+-free solution containing 40 or 128 mM-K+.

T. ITOH AND OTHERS

320

Pinacidil (1 /LM) had no effect on the [Ca2+]i increase and contraction induced by 2 mM-caffeine in Ca2+-free solution (0-98 + 0-02 times control for the peak [Ca2+]i and 0-96 + 0-05 times control for the peak tension, n = 5). Effects of pinacidil on IP3 production induced by NA IP3 releases Ca2+ from intracellular storage sites as a second messenger in smooth muscle cells of the rabbit mesenteric artery (Hashimoto et al. 1986). To investigate A 800 700. 600d 2 500 ;400 300 200100 Oo

7?

a 0 .0'''°

,

5.9

.

...

20 40 K+ (mM)

128

B 2.2 2.0. 0

, 1.50 .y

cDl.0 0.5

~

4

0 1

,. 5.9

1.1 20 40 K+ (mM)

, 128

Fig. 9. Effects of pinacidil on increases in [Ca2+]i (A) and tension (B) induced by 10 tMNA in Ca2+-free solution containing 2 mM-EGTA with various concentrations of K+ in smooth muscle strips of the rabbit mesenteric artery. The Ca2+-free solution containing high K+ (20-128 mM) was applied for 1 min after 1 min removal of Ca2+ by Ca2+-free solution containing 5-9 mM-K+. NA (10 /tM) was then applied for 2 min in Ca2+-free solution containing high K+, and finally Krebs solution (containing 2-6 mM-Ca2+) was applied for 25 min. This protocol was repeated at 30 min intervals. (-), control; (0), pinacidil was applied for 5 min before and throughout application of NA. Guanethidine (3 /tM) and propranolol (0 3 /SM) were present throughout the experiment. Results represented are means of five observations with S.D. shown by vertical bar.

further the mechanisms of the vasodilatation induced by pinacidil during the NA contraction, the effects of 1 ,tM-pinacidil on the 1P3 production induced by 10 /M-NA were observed in Ca2+-free solution containing 2 mM-EGTA. NA transiently increased IP3 within 10 s and the effect gradually decayed (Fig. IOA). Pinacidil (1 #M) did not significantly lower the concentration of 1P3 at the resting conditions (4-1 + 2-8 pmol/mg protein in control and 3-2 + 2 1 pmol/mg protein in the presence of 1 /tM-pinacidil, n = 14), and this agent inhibited the maximum increase in the

MEMBRANE HYPERPOLARIZATION AND IP3

321

synthesis of 1P3 induced by 10 J,m-NA measured at 10 s after application of NA, in a concentration-dependent manner (Fig. lOB). Glibenclamide (1 ,UM) itself did not significantly modify the 1P3 production induced by 10 /tm-NA but it blocked the inhibitory actions of 1 /tM-pinacidil on the NA-induced IP3 synthesis (Fig. lOB). A

-E

17 -T 15 -

CD 0

0) 10a E 0 E

a CL

0-

m

1

i

t

i t

2

3

i

0

1

j

+ 1

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I

4

4

Control

AL;| 1-

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i

-J

5

i I 10

Time (min) B gn-

15 0

E 10 0

E -9 5

Control

Control

1 PM 10 PM 1 pfM

Pinacidil M-Gli

1pm-Gli Fig. 10. Effects of pinacidil on 1P3 synthesis induced by 10 ,tM-NA in Ca2l-free solution containing 5-9 mM-K+ in smooth muscle strips of the rabbit mesenteric artery. A, NA (10 /M) was applied in Ca2+-free solution containing 2 mM-EGTA with 5-9 mM-K+ after 2 min removal of Ca2 . Pinacidil was applied for 5 min before and throughout application of NA. A, time-dependent changes in the synthesis of IP3 following application of 10 /tMNA in the presence (0) or absence (@) of 1 4uM-pinacidil. NA (10 /M) was applied at time zero. Results represented are means of fourteen observations with S.D. shown by vertical bar. B, effects of pinacidil on NA-induced IP3 synthesis in the presence of 1 ,UMglibenclamide in smooth muscle strips of rabbit mesenteric artery. NA (10/OM) was applied for 10 s after 2 min removal of Ca2+. Glibenclamide (Gli) was applied for 5 min before and throughout application of NA or pinacidil. Results represented are means of fourteen observations with S.D. shown by vertical bar. *indicates significant difference from response to NA only (P < 0-05). E, strips not stimulated by NA; U, NA-treated strips. 1

1 uM-Gli

The effects of 1 #m-pinacidil on the synthesis of IP3 induced by 10 /M-NA were studied in Ca2+-free solution containing various concentrations of high K+ (Fig. 11). The synthesis of IP3 was measured 10 s after the application of 10 /M-NA in Ca2+-free solution containing 2 mM-EGTA with various concentrations of high K+. The

T. ITOH AND OTHERS

322

experimental protocols were as follows: Ca2+-free solution containing 2 mM-EGTA with 5-9 mM-K+ was applied for 1 min, and Ca2+-free solution containing 2 mmEGTA with various concentrations of high K+ was applied for 1 min. Finally, 10 ,uMNA was applied for 10 s in Ca2+-free solution containing 2 mM-EGTA with high K+. 50 40 CL

3

c.

E E 20

_ t

10

Resting

5.9

20

40

128

K+ (mM) Fig. 11. Effects of 1 /M-pinacidil on IP3 synthesis induced by 10 JiM-NA in Ca2+-free solution containing 2 mM-EGTA with various concentrations of high K+ in smooth muscle strips of the rabbit mesenteric artery. The experimental protocols were as follows: Ca2+free solution containing 2 mM-EGTA with 5 9 mM-K+ was applied for 1 min, and Ca2+-free solution containing 2 mM-EGTA with various concentrations of high K+ was applied for 1 min. Finally, 10 ,sM-NA was applied for 10 s in Ca2+-free solution containing 2 mMEGTA with high K+. *, strips not stimulated with NA and pinacidil; O, NA-treated strips; M, NA-treated strips in the presence of 1 FuM-pinacidil. Results represented are means of five observations with S.D. shown by vertical bar. * indicates significant difference from the result obtained from the resting condition (P < 0'05). t, significant difference from the results obtained from the relevant control in the absence of 1 ,UMpinacidil (P< 0 05).

Under these conditions, high K+ (20-128 mM) itself did not change the resting concentration of IP3 in Ca2+-free solution (for example, the resting concentration of IP3 was 6-3 ± 1-3 pmol/mg protein in Ca2+-free solution containing 2 mM-EGTA with 5-9 mM-K+, and this was 6-3 + 1-2 pmol/mg protein in Ca2+-free solution containing 2 mM-EGTA with 128 mM-K+, n = 6). The absolute amount of IP3 synthesized by 10 /uM-NA was smaller in the presence of high K+ (20-128 mM) than that observed in the presence of 5-9 mM-K+ (Fig. 11). Pinacidil (1 ,M) inhibited the NA-induced synthesis of IP3 in Ca2+-free solution containing 5-9 or 20 mM-K+, but not in the solution containing 40 or 128 mM-K+ (Fig. 11). In ,-escin-treated skinned smooth muscle strips, NA can release Ca2+ from its storage sites through the action of 1P3 (Kobayashi et al. 1989). To investigate the site of action of pinacidil, the effects of 1 fM-pinacidil on NA-induced Ca2+ release were observed in smooth muscle strips skinned with fl-escin. After the strips had been skinned by application of 20 /tM-/3-escin for 25 min, 0 3 ,#m-Ca2+ buffered with 4 mMEGTA was applied for 2 min to load Ca2+ into the storage sites, and Ca2+-free solution

323 MEMBRANE HYPERPOLARIZATION AND IP3 containing 4 mM-EGTA was applied for 0 5 min to remove Ca2+ from the solution. Finally, 10 tM-NA was applied for 2 min in Ca2+-free solution containing 50,UMEGTA, 3 /LM-GTP and 2 /LM-Fura-2 following application of Ca2+-free solution containing 50 /M-EGTA, 3 ,m-GTP and 2 /IM-Fura-2 for 2 min (Fig. 12A). NA A l I l I 0.5 min 2 min 0.5 min 2 min

a

0.3 jiM

b

Ca2+ 0 4 mM EGTA Fura-2 0

I

2 min 0.5 min

3um-GTP

m-NA

50 jUM 2 ,iM

B

ic CCo4 S

C

160-

i a

140 120-

100 80 Fig. 12. Effects of 1 ,uM-pinacidil or 1 uM-glibenclamide on Ca2' release induced by 10 jMNA in f-escin-treated, skinned smooth muscle strips of the rabbit mesenteric artery. A, the protocol used in this experiment. Detailed description is given in 'Results'. Pinacidil or glibenclamide was applied for 2 min before and throughout application of NA. Heparin (500 jug/ml) was given as pretreatment for 10 min before and throughout application of NA.

increased Ca2l concentration under the above conditions, possibly due to release of Ca2+ from its storage sites (Fig. 12B) and this response was inhibited by 500 /tg/ml heparin (Fig. 12 C). Pinacidil (1 /tM) or glibenclamide (1 JtM) did not change the Ca2+ release induced by NA in fl-escin-treated skinned strips (Fig. 12B and C). Pinacidil (1 /tM) also had no inhibitory effect on Ca2+ release induced by 20 /tM-IP3 in the skinned strips (1I23 ± 0-13 times control, n = 6).

324

T. ITOH AND OTHERS

Effects of pinacidil on cyclic AMP and cyclic GMP production Cyclic AMP or cyclic GMP inhibits the synthesis of 1P3 induced by several agonists in vascular tissue (Rapoport, 1986; Kajikuri & Kuriyama, 1990). To investigate further the sites of action of pinacidil in inhibiting NA-induced lP3 production, the effects of NA or pinacidil (0 1-10 /M) on synthesis of cyclic AMP and cyclic GMP were observed in endothelium-denuded strips of the rabbit mesenteric artery in the presence of 3 JtM-guanethidine and 0 3 /tM-propranolol. Pinacidil (0 1-10 /tM) or NA (10 /tM) had no effects on the concentrations of cyclic AMP and cyclic GMP in smooth muscle strips (cyclic AMP concentration: 7-50 + 096 pmol/mg protein in control, 744 + 1P20 pmol/mg protein in the presence of 10/ uM-NA and 8-59 + 0-09 pmol/mg protein in the presence of 1 /tM-pinacidil; cyclic GMP concentration: 0-28 + 0 05 pmol/mg protein in control, 0-29 + 0 07 pmol/mg protein in the presence of 10 /tM-NA, 0-29 + 0 03 pmol/mg protein in the presence oft 1uM-pinacidil, n = 5-7). DISCUSSION

Pinacidil hyperpolarizes the membrane by activation of an ATP-sensitive and [Ca2+]i-insensitive K+ channel, and a sulphonylurea, glibenclamide, inhibits this channel in smooth muscle cells of the rabbit mesenteric artery (Southerton et al. 1988; Videbeek, Aalkjoerm & Mulvany, 1988; Quast & Cook, 1989; Standen et al. 1989), and also in cardiac and skeletal muscle cells and pancreatic f8-cells (see Ashcroft, 1988, for a review). In the present experiment, we confirmed these actions of pinacidil in that it hyperpolarized the membrane with a reduction of input resistance. The hyperpolarizing action of pinacidil persists in low-Na+ or low-ClKrebs solution and depends on the extracellular concentration of K+ in smooth muscle cells of the rabbit mesenteric artery (Nakashima, Li, Seki & Kuriyama, 1990). Moreover, glibenclamide inhibited the pinacidil-induced hyperpolarization. These results suggest that pinacidil activates the glibenclamide- and ATP-sensitive K+ channel and thus causes hyperpolarization of the smooth muscle cell membrane in the rabbit mesenteric artery.

General effects of membrane hyperpolarization on [Ca2+]i estimated from the actions of pinacidil In smooth muscle of the rabbit mesenteric artery, pinacidil lowered the resting [Ca2+]i in both Ca2+-containing and Ca2+-free solutions and these actions were prevented by glibenclamide. In Ca2+-free solution containing 2 mM-EGTA, the extracellular Ca2+ has been calculated to be below 10 nM (Itoh et al. 1986) and intracellular Ca2+ concentration estimated to be 50-90 nm. Under the above conditions, while influxes of Ca2+ may be minimized, pinacidil still reduced the [Ca2+]i. Thus, the membrane hyperpolarization negatively controls the resting

[Ca2+]i. In this tissue, NA (10 /lM) depolarized the membrane and induced oscillatory potential changes. NA produced a phasic increase in [Ca2+]i and tension, possibly induced by release of Ca2+ from its storage sites, while the subsequent tonic increase may be due to activation of voltage-dependent and independent Ca2+ influxes, as suggested from the tension measurements (Itoh et al. 1983; Kanmura et al. 1983;

MEMBRANE HYPERPOLARIZATION AND IP3

325

Makita et al. 1983). Pinacidil (1 /LM) inhibited the membrane depolarization and the oscillations induced by 10/tM-NA and also inhibited the increases in [Ca2+]i and tension. The tonic increases in [Ca2+]i and tension induced by NA were not evoked in Ca2+-free solution containing EGTA. Pinacidil (1 #M) almost abolished the tonic increases in [Ca2+]i and tension induced by 10 /tM-NA whereas nifedipine or nisoldipine only partly inhibits the tonic tension (Kanmura et al. 1983; Makita et al. 1983). Glibenclamide prevented the inhibitory actions of pinacidil on the membrane depolarization, increase in [Ca2+]i and tension induced by NA. Furthermore, the inhibitory action of pinacidil on the contraction induced by NA was preserved in A23187-treated strips. These results suggest that the membrane hyperpolarization induced by pinacidil inhibits increases in [Ca2+]i and tension induced by NA through inhibition of Ca2+ influxes. It is also plausible that the membrane hyperpolarization induced by pinacidil activates the Ca2+-extrusion mechanism mediated by Na+-Ca2+ exchange through activation of K+ efflux, and then causes vasodilatation. However, since pinacidil inhibited the NA-induced contraction in low-Na+ solution more potently than in Krebs solution, this mechanism cannot play a major role, at least in the smooth muscle of the rabbit mesenteric artery. Roles of membrane hyperpolarization in PIP2 hydrolysis by NA It has been supposed that NA binds to the ac-receptor and synthesizes 'P3 which releases Ca2+ from its storage sites in smooth muscle of the rabbit mesenteric artery (Hashimoto et al. 1986). Heparin is thought to be a blocker of the 1P3 receptor (Kobayashi et al. 1989) and, in the present experiment, heparin inhibited Ca2+ release induced by NA or 1P3 in /3-escin-treated skinned smooth muscles of the artery. Pinacidil inhibited the Ca2+ increase induced by NA, but not caffeine, in intact smooth muscle strips. The inhibitory action of pinacidil (< 1 ,CM) on the NA contraction was inhibited by glibenclamide or in the presence of high K+ (> 20 mM). Pinacidil (1 /LM) did not modify the resting concentrations of IP3 but inhibited the synthesis of IP3 induced by NA, and these actions were inhibited by 1 ,UMglibenclamide or in the presence of 40 or 128 mM-K+. Lemakalim, another ATPsensitive K+ channel activator, also inhibited the NA-induced synthesis of 1P3 in smooth muscle strips of the rabbit mesenteric artery (Ito, Kajikuri, Itoh & Kuriyama, 1991). Thus, the present results suggest that pinacidil selectively inhibits the NA-induced Ca2+ release, in a manner dependent on the membrane potential, through inhibition of NA-induced 'P3 synthesis. An increase in cellular cyclic AMP or cyclic GMP inhibits agonist-induced IP3 production in some vascular tissues (Rapoport, 1986; Lang & Lewis, 1989). The role of cyclic AMP or cyclic GMP in the inhibition of IP3 production by pinacidil, however, is negligible since pinacidil had no effects on the cellular concentrations of cyclic AMP or cyclic GMP in the present smooth muscle strips. In the present experiments, the synthesis of IP3 induced by 10 /uM-NA in Ca2+-free solution was smaller in the presence of high K+ (> 20 mM) than that observed in the presence of 5.9 mM-K+ (Krebs solution). The effects of high K+ seem not to depend on its concentration (20-128 mM). In contrast, in Ca2+-free solution containing 2 mmEGTA high K+ enhanced the increase in [Ca2+]i and tension induced by 10 /M-NA,

T. ITOH AND OTHERS 326 but this solution itself did not increase the [Ca2+]i and tension. These results indicate that a small membrane depolarization enhances the NA-induced Ca2+ release without a corresponding increase in the absolute amount of synthesized IP3, suggesting that membrane depolarization may enhance the sensitivity of the 'P3-induced Ca2+ release mechanism to IP3 in smooth muscle of the rabbit mesenteric artery. The underlying mechanisms of this remain to be clarified in future work. In a-toxin- or skinned smooth muscle strips, NA enhances the amplitude of Ca2+-induced contraction in the presence of GTP (Kitazawa et al. 1989; Kobayashi et al. 1989; Nishimura & van Breemen, 1989). Since this effect of NA was inhibited by neomycin (an inhibitor of phospholipase C) or GDPflS, the authors suggested thata-toxin- or,-escin-treated skinned smooth muscle strips retain the xreceptor-GTP binding protein-phospholipase C coupling mechanism. A high concentration of pinacidil (>3 /tM) inhibited the action of NA on the Ca2+-induced contraction ina-toxin- or,-escin-treated skinned smooth muscle strips of the rabbit mesenteric artery (Anabuki et al. 1990; Itoh et al. 1991). This inhibitory action of pinacidil on Ca2+-induced contraction in the presence of NA with GTP was not apparent in lower concentrations of this drug (< ,UM) and was not attenuated by glibenclamide (Itoh et al. 1991). In the present experiments, we found that ,tMpinacidil had no effect on Ca2+ release induced by NA orIP3 in smooth muscle strips skinned with ,3-escin. Thus, the lack of the inhibitory action of JuM-pinacidil on NAinduced Ca2+ release in fl-escin-skinned smooth muscle (which cannot generate a membrane potential) also supports the notion that membrane hyperpolarization itself may play an essential role in the inhibition of the NA-induced synthesis of 'P3 in smooth muscle. Since the membrane potential of skinned smooth muscle cell is negligible (due to the porous plasma membrane and the bath solution containing 140 mM-K+), but they are capable of synthesizingIP3, the action of the membrane hyperpolarization in inhibitingIP3 production may be exerted in the process oof receptor and GTP-binding protein interaction. 1,2-Diacylglycerol (DAG) is a co-product of IP3 in several types of cells (Nishizuka, 1984). Inhibition of NA-induced phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis by pinacidil may lead to an inhibition of the activation of protein kinase C through inhibition of DAG production. Since activators of protein kinase C, such as phorbol esters, increase the Ca2+ sensitivity of the contractile apparatus in skinned smooth muscles (Itoh, Kubota & Kuriyama, 1988), the inhibition ofPIP2 hydrolysis by membrane hyperpolarization may also play a role in this agent-induced inhibition of the tonic contraction induced by NA in smooth muscle. In conclusion, in smooth muscle cells of the rabbit mesenteric artery pinacidil hyperpolarizes the muscle membrane by increasing K+ permeability in the presence or absence of NA. Since glibenclamide or high K+ (>40mM) consistently inhibited the change in the membrane potential, [Ca2+]i,1P3 synthesis and tension induced by pinacidil, the hyperpolarization induced by pinacidil may in turn induce a reduction in the NA-induced synthesis of 'P3. We thank Dr R. J. Timms for the language editing. This work was partly supported by a Grant-

,-escin-treated

In-Aid from the Ministry of Education of Japan (02807010, 63440022 and 02404024).

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