Camp. Biochem. Physiol. Vol 89C, No. 1, pp. 45-52, 1988 0306~4492/88 $3.00 + 0.00 Printed in Great Britain 0 1988 Pergamon Journals Ltd

EFFECTS OF NICORANDIL ON ISOLATED SMOOTH MUSCLE CELLS FROM GUINEA-PIG

TAENIA CAECI

YASLJSHI ITO and KAZUO OBARA

Department of Physiology, Sapporo Medical College, Sapporo, Hokkaido 060, Japan

(Received 3 February 1987)

Abstract-l. The effects of nicorandil on guinea-pig taenia caeci were investigated with the use of isolated smooth muscle cells and glycerin-treated muscle fiber bundles.

2. Nicorandil inhibited high K-, Ca2+- and carbachol-induced contractions in a dose-dependent manner without affecting 45Ca fluxes in isolated cells.

3. Nicorandil had no effect on ATP-induced contraction of glycerin-treated muscle fiber bundles. 4. The present results suggest that nicorandil may inhibit the contraction by action on the contractile

proteins m an indirect manner in guinea-pig taenia caeci.

INTRODUCTION

The action of nicorandil, a newly synthesized anti- angina1 agent which has a significant relaxing action in vascular smooth muscles, has heen investigated from the standpoints of pharmacology, physiology and clinical science by many workers. Some in- vestigators have shown that nicorandil inhibits mus- cle contractions by hyperpolarizing the membrane through an increase in K conductance in guinea-pig coronary artery (Furukawa et al., 1981) dog mesen- teric artery and trachea (Inoue et al., 1983).

There have also been suggestions that the vaso- dilative action of nicorandil is due to the inhibition of intracellular Ca2+ mobilization in miniature pig coronary artery (Nabata and Sakai, 1983), and that nicorandil might reduce the force development by an interaction with the activation process of contractile proteins (Kajiwara et al., 1984). These observations imply that the effects of nicorandil may differ accord- ing to animal species and smooth muscle region.

Yamanaka et al. (1985) have provided electro- physiological evidence that nicorandil hyperpolarizes the membrane in guinea-pig small intestine. How- ever, the effects of nicorandil on the contractile properties and the Ca’+ movements in gastro- intestinal smooth muscles still remain to be in- vestigated.

It should be noted that a study using whole smooth muscle tissue holds some technical difficulties owing to the large matrix of extracellular space (ECS) and the relatively minute total cell volume. The reason for this is that the matrix of ECS forms diffusional barriers for drugs and Ca’+, and makes it difficult to analyse the Ca2+ movements with the use of 4sCa owing to the enormous number of Ca2+-binding sites in the matrix. Nor can the intervention of neural elements in the drug effects be excluded in as far as muscle strips containing such elements are used for the investigation of smooth muscles. To overcome these problems, isolated smooth muscle cells devoid of ECS and neural elements are available for in- vestigating Ca2+ movements and the direct actions of various drugs in the cells.

In this report, the effects of nicorandil on the contractile responses and Ca2+ movements of smooth muscle cells induced by high K depolarization and by a cholinergic drug, carbachol (CCh), are investigated with the use of isolated cells from guinea-pig taenia caeci, and the inhibitory mechanism of nicorandil in the cells is discussed.

MATERIALS AND METHODS

Cell preparation

A suspension of isolated smooth muscle cells, hereafter referred to as isolated cells, from guinea-pig taenia caeci was prepared by the method of Obara (1984). Male guinea-pigs weighing 30@-600 g were used as tissue donors. Strips of taenia caeci, &5 cm in length, were carefully isolated from freshly killed guinea-pigs. They were incubated in 0.18 mM CaZ+ physiological salt solution (PSS) for 30 min and then in Ca2+-free PSS for 15 min bubbled with air at 35°C. Each strip was minced and then suspended in 2 ml of Ca2+-free PSS containing 0.3% collagenase, 0.6% trypsin inhibitor and 1.0% bovine serum albumin for 30min, with gentle stirring 2-3 times/set. The suspension was diluted with 8 ml of Ca2+-free PSS containing albumin and was centrifuged at 1,000 rpm for 3 min. The precipitate was suspended in 3 ml of Ca2+-free PSS for 15min with gentle stirring and then dispersed by pipetting with a wide-bored pipette (diameter 2 mm). The suspension was allowed to stand for 2-3 min and, thereafter, the dispersed cells in the supematant were subjected to the experiments.

Measurement of contractile response

Changes in the cell length were measured by the method of Obara (1984). Aliquots consisting of 104cells in 1 ml of Ca2+-free PSS were prepared by adding CaCl, to a final concentration of 1.8 mM for IO min or more prior to the addition of stimulants except in the case of Ca*+-induced contraction. For Ca2+-induced contraction, the cell sus- pension was resuspended in high K (71 mM) Ca2+-free solution. At the end of the reaction time,‘acrolein was added to make a final concentration of 1%. An aliquot of the cell suspension, fixed with acrolein, was placed on a glass slide under a cover slip and was randomly scanned on a screen bv using a microoroiector (XM 500-H. Tivoda). The lengths _ - __ of the first 20&3OOcells counted randomly ‘in successive fields were measured along the curved long axes of the

45

46 YASUSHI ITO and KAZUO OBARA

elongated cells. The contractile response was defined as the decrease in the average length of a population of muscle cells exposed to stimulants.

Resting 45Ca uptake

Isolated cells in Ca2+-free PSS were suspended in 0.18 mM Car+ PSS containing 45Ca (2 nCi/ml) and incu- bated for various durations at 35°C. After the desired incubation time, a sample of the cell suspension was trans- ferred to ice-cold Ca2+-free PSS containing 5 mM ethyl- eneglycol- bis- (1 -aminoethylether)-N,N’- tetraacetic acid (EGTA). After 10 min, this suspension was passed through a Millipore filter (LCWP 02500) and the filter was washed 5 times with 2m1 of ice-cold Ca*+-free EGTA PSS to remove 45Ca adhering to the membrane filter. Samples were prepared with a scintillation cocktail (Scintisol EX-H, Dojin) and counted by a liquid scintillation counter (LS- 9000, Beckman). The cellular content of Ca2+ (nmol/mg protein) was calculated as follows:

cpm/mg protein

cpm/ml mcubation solution x total Ca2+ concentration

in incubation solution (nmol/ml).

Protein concentration was determined by the method of Lowry et al. (1951).

45Ca uptake in high K or CCh-containing solution

Isolated cells were suspended in 0.18 mM Car+ PSS without 45Ca for 10 min or more. High K (71 mM) or CCh (10e4 M) containing 45Ca (2 pCi/ml) was added to the sus- pension and the cells were incubated for various durations. After the desired incubation time, a sample of the cell suspension was transferred to ice-cold Ca2+-free EGTA PSS, filtered, washed and counted as described above in the section on resting 45Ca uptake.

45Ca eflux

The 45Ca efllux from isolated cells was estimated from the amount of 45Ca remaining in the cells. The cells were incubated in 0.18 mM Ca2+ PSS containing 45Ca (2 pCi/ml) for 30 min and then transferred to Ca2+-free 5 mM EGTA PSS. After the desired time, a sample of the cell suspension was treated with ice-cold Ca2+-free EGTA PSS, filtered, washed and counted as described above. The cellular 45Ca-content was expressed as a fraction of 45Ca-content of the cells incubated in 0.18 mM Ca*+ PSS containing 45Ca for 30 min.

The composition of the physiological salt solution (PSS) was 137 mM NaCl, 2.7mM KCl, 1.8mM CaCl,, l.OmM M&l,, 5.6 mM glucose and 4.2 mM N-2-hydroxyethyl- piperazine-N’-2-ethanesulfonic acid (HEPES). High K (71 mM) solution was prepared by replacing NaCl with KC1 isosmotically. These solutions were adjusted to pH 7.4 and kept at a temperature of 35°C.

Glycerin -treated muscle fiber bundles A strtp of taema caeci was fixed on a thin bamboo stick

at the in situ length without stretching and then stored in 50% glycerin solution (50% glycerin, 2mM EGTA, 20 mM Tris-maleate, pH 6.8) at - 18°C for 1 month. Before the experiment, the strip fixed on the stick was transferred from 50% glycerin solution to 20% glycerin solution (20% glycerin. 2 mM EGTA, 20 mM Tri-maleate, pH 6.8) for 2 hr at room temperature (approximately 20°C). Then, the glycerin-treated muscle fiber bundle was prepared by cutting strips into thin fiber bundles with a diameter of 20&300 pm and a length of 20 mm.

After the glycerin-treated muscle fiber bundle was washed in relaxing solution (130 mM KCI, 4 mM MgCl,, 2 mM EGTA, 20 mM Tris-maleate, pH 6.8) for 15 min, it was incubated in pCa 6 solution containing ATP ( 130 mM KCl, 4 mM MgCI,, 4 mM ATP, 1 mM CaCl,, 2 mM EGTA,

20 mM Tris-maleate, pH 6.8) and ATP-induced free short- ening of the glycerin-treated muscle fiber bundle was ob- served at room temperature. The length of the bundle was measured by slide caliper under a binocular microscope. The contractile response was expressed as a ner cent decrease of the length in the relaxing-solution. The-binding constant for Ca2+-EGTA was considered to be lo6 M-t at DH 6.8 (Harafuji and Ogawa, 1980).

Chemicals

Sources of reagents are as follows: collagenase (Type I), soybean trypsin inhibitor and bovine serum albumin (Frac- tion V) were obtained from Sigma Chemical Co. Carbachol came from Aldrich Chemical Co. HEPES and glycerin were purchased from Wako Pure Chemical Industries, Ltd. EGTA was obtained from Doiin Chemicals Co. ATP di- sodium salt came from Boehringer Mannheim GmbH. 45CaC12 was purchased from New England Nuclear. Nico- randil was kindly provided by Mitsubishi-yuka Pharma- ceutical Co. Ltd.

Statistical analysis

Values were expressed as the mean f SE and statistical analysts was performed with Student’s r-test.

RESULTS

Time course of contractile response to various stimulants

As shown in Figs 1A and C, high K (71 mM)- and CCh ( 10m4 M)-induced contractions attained peaks at 15 set and then isolated cells relaxed spontaneously. The maximal responses obtained by high K and by CCh expressed as the per cent decrease in mean cell length from the control were 18.9 f 0.9% (N = 7) and 41.1 + 1.6% (N = 14) respectively. Ca2+ (1.8 mM)-induced contraction was obtained by the addition of Ca2+ to the cell suspension in high K Ca2+-free solution. As shown in Fig. lB, Ca2+- induced contraction slowly reached a peak at 60 set and then the cells relaxed slightly. The maximal response was a 21.1 f 1.6% (N = 10) decrease in cell length.

In the following experiments, acrolein was added to the cell suspension at the time when the maximal responses to respective stimulants were obtained (high K and CCh, 15 set; Ca*+, 60 set).

Dose-response curve to CCh

As shown in Fig. 2, the contractile responses of isolated cells to CCh increased in a dose-dependent manner in 1.8 mM Ca2+ PSS. The cells responded to CCh at concentrations of lo-‘M or more and the maximal response was obtained at a concentration of 10m4 M. The EDGE, estimated graphically, was about 3.3 x 10-6M.

Effects of nicorandil on high K-, Ca2+- and CCh-induced contractions

Figure 3 shows the effects of nicorandil at various concentrations on high K (71 mM)-, Ca2+ (1.8 mM)- and CCh ( 10m4 M)-induced contractions. Nicorandil inhibited high K-, Ca2+- and CCh-induced con- tractions in a dose-dependent manner. The half max- imal concentrations of nicorandil in the inhibition of high K-, Ca2+- and CCh-induced contractions, esti- mated graphically, were 2.8 x 10e3 M, 2.4 x 10d3 M and 6.3 x 10m3M, respectively. In 0.18mM Ca2+

Effects of nicorandil 41

Fig. 1. Time courses of contractile responses of Isolated cells to various stimulants m 1.8 mM Car+ solution. Contractile responses are expressed as the per cent decrease in cell length. A: high K (71 mM)-induced contraction (IV = 47). B: Ca?+ (1.8 mM)-induced contraction (N = 336). C: CCh (10m4 M)-induced contraction (N = 7-14). Each point repre-

sents mean & SE.

solution, 5 x 1O-3 M nicorandil completely sup- pressed high K-induced contraction and inhibited CCh-induced contraction to 26% of the contraction in the absence of nicorandil (unpublished data).

Effects of nicorandil on CCh-induced contraction in high K Ca2+-free EGTA solution

To investigate the effects of nicorandil on Ca2+ storage sites in the cell, the experiment was performed in high K Ca*+-free 7mM EGTA solution (Fig. 4). Isolated cells were incubated in 1.8 mM Ca2+ PSS for 10 min or more. As shown in column A in Fig. 4, the

Fig. 3. Effects of nicorandil on high K-, Ca2+-, and CCh- induced contractions. Nicorandil was applied 10 min before the addition of each stimulant and then exposed to high K, Ca2+, and CCh for 15, 60, and 15 set, respectively. Con- tractile responses are expressed as the per cent of response to each stimulant in the absence of nicorandil. 0: high K (71 mM)-induced contraction. 0: Ca*+ (I .8 mM)-induced contraction. A: CCh (lo-’ M)-induced contraction. Each _- . . _

Fig. 2. Dose-response curve of isolated cells to CCh in 1.8mM Ca*+ PSS. The cells were exposed to various concentrations of CCh for 15 sec. Contractile responses are expressed as a per cent of response to CCh (lo-’ M). Each

point represents mean f SE (N = 3-6).

cells did not contract in spite of high K depolar- ization which was induced by the addition of the high K Ca2+-free EGTA solution to the cell suspension. This means that the extracellular Ca*+ necessary for the initiation of high K-induced contraction was obviated at the moment of the addition of EGTA. These results suggest that with the use of isolated cells the extracellular environment can be exactly manipu- lated by the specific composition of solution.

As shown in column B in Fig. 4, there was a 27.0 k 2.5% (N = 8) decrease in cell length when 10e4 M CCh and high K Ca2+-free EGTA solution were added to the cell suspension simultaneously. This CCh-induced response was about 66% of the response in 1.8 mM Ca*+ PSS. Columns C and D in Fig. 4 show the effects of lo-’ M (C) and 5 x 10e3 M (D) nicorandil on this CCh-induced contraction. The decreases in cell length in the presence of 10m3 and 5 x lo-‘M nicorandil were 17.3 f 4.6% (N = 4) and 10.6 f 5.1% (N = 4), respectively. Nicorandil

potnt represents mean f SE (N = 3-14).

YASUSHI ITO and KAZUO OBARA

30 i

f r S

20 = Y I

-10 J A 0 C 0

Fig. 4. Effects of nicorandil on CCh-induced contraction in high K Ca2+-free EGTA solution. The cells were suspended in 1.8 mM Ca*+ PSS and then exposed to each stimulant for 15 sec. Nicorandil was applied 10 min before the addition of each stimulant. Contractile responses are expressed as the per cent decrease in cell leigth. A: high K (71 mM) Ca*+-free EGTA (7mM) solution. B: CCh (10m4M)- induced contraction in high K Caz+-free EGTA solution. C: CCh-induced contraction in high K Ca’+-free EGTA solu- tion in the oresence of nicorandil (10m3 M). D: CCH- induced co&action in high K Ca*+-free EGTk solution in the presence of nicorandil (5 x 10e3 M). Each value repre- sents mean k SE (N = 48). *Significantly different from carbachol-induced contraction in high K solution contain-

ing EGTA (P < 0.01).

(5 x lo-) M) inhibited this response, which was significantly different from the response in the ab- sence of nicorandil (P < 0.01).

Effects of nicorandil on resting 45Ca uptake

Figure 5 shows the time course of resting 45Ca uptake when isolated cells in Ca2+-free PSS were suspended in 0.18 mM Ca2+ PSS containing 45Ca (2 pCi/ml). 45Ca uptake reached a maximal value at

Fig. 5. Resting 45Ca uptake of isolated cells. After the desired incubation time in 0.18 mM Ca2+ PSS containing 45Ca (2 pCi/ml), the amount of 45Ca in the cells was measured. Nicorandil was annlied 10 min before the addi- tion of 4’Ca. Resting 45Ca-;ptake was observed in the presence of 10-l M (A), 5 x lo-’ M (A) nicorandil or in the absence of nicorandil (0). 45Ca uptake is expressed as nmol/mg protein. Each point represents mean f SE

(N = 3-12).

5 min and maintained a steady value thereafter. The steady value at 30 min was 0.163 + 0.017 nmol/mg protein (N = 8). The time course of 45Ca uptake in the presence of nicorandil was not different from that in the absence of nicorandil. The amounts of 45Ca uptake after 30min incubation in the presence of 10m3 M and 5 x lOA M nicorandil were 0.178 + 0.023 nmol/mg protein (N = 3) and 0.143 f 0.019 nmol/mg protein (N = 5), respectively.

Effects of nicorandil on 45Ca uptake in high K or CCh -containing solution

Isolated cells were incubated in 0.18 mM Ca2+ PSS without 45Ca for 10min or more and then high K (71 mM) or CCh ( 10M4 M) containing 45Ca (2 pCi/ml) was added to the cell suspension. Figure 6A shows 45Ca uptake in the high K solution. In the case of the control 45Ca uptake, only 45Ca without stimulant was added to the cell suspension. It is believed that control 45Ca uptake depended on the Ca2+-Ca2+ exchange mechanism. The control value at 30min was 0.140 k 0.010 nmol/mg protein (N = 8). 45Ca uptake in high K solution increased significantly compared with the control after 5min incubation (P < O.OOl), and the value at 30 min was 0.345 & 0.044 nmol/mg protein (N = 6).

High K-induced 45Ca uptake in the presence of 5 x 10m3 M nicorandil increased significantly com- pared with the control level after 5 min incubation (P < 0.005) and the value at 30 min in the presence of nicorandil was 0.304 k 0.035 nmol/mg protein (N = 4, P < 0.001 compared with control), but nico- randil did not affect high K-induced 45Ca uptake.

Figure 6B shows 45Ca uptake in CCh-containing solution. 45Ca uptake in CCh-containing solution was not different from the control 45Ca uptake and the value in this solution at 30min was 0.151 k 0.008 nmol/mg protein (N = 9). 45Ca uptake in CCh-containing solution with prior exposure to 5 x 10e3 M nicorandil was not different from either control 45Ca uptake or 45Ca uptake in CCh- containing solution. The value at 30min in the presence of nicorandil was 0.155 f 0.0 15 nmol/mg protein (N = 4).

EfSects of nicorandil on 45Ca efJlux

The experiment on 45Ca efflux was performed in Ca*+-free 5 mM EGTA PSS in order to eliminate the effects of Ca2+ back flux and Ca2+-Caz+ exchange.

As shown in Fig. 7, the cellular content of 4SCa decreased depending on exposure time to Ca*+-free EGTA PSS. The values of control 45Ca efflux were 0.578 f 0.026 (N = 9) at 4 min and 0.403 k 0.002 (N = 9) at 9 min. Control 45Ca efflux was suppressed significantly at low temperature (2°C) and the values were 0.92 at 4 min and 0.871 at 9 min (unpublished data). These results show that 45Ca efflux depends to a large degree on the temperature and suggest that an active Ca2+ extrusion mechanism is involved in 45Ca efflux.

When 10m4M CCh was added to the suspension after 3 min exposure to EGTA, 45Ca efflux was accelerated significantly compared with control 45Ca efflux (P < 0.05), and the values were 0.516 + 0.012 (N = 9) at 4 min and 0.355 f 0.009 (N = 9) at 9 min.

Effects of nicorandil 49

OY 0 IO 20 30

lncubal~an lime I mln 1

Fig. 6. Time course of 45Ca uptake in high K and CCh-containing solutions. After incubation in 0.18 mM Ca2+ PSS, high K (A) or CCh (B) containing 45Ca (2 &i/ml) was added to the suspension. After the desired incubation time, the amount of 45Ca in the cells was measured. Nicorandil was applied 10min before the addition of each stimulant. %a uptake is expressed as nmol/mg protein. 0: control. 0: high K (71 mM) in A or CCh (10m4M) in B. A: high K (A) or CCh (B) in the presence of nicorandil (5 x 10-j M). Each point represents mean + SE (A: N = 48. B: N = 4-9). Significantly different from

control (*, P < 0.005; *, P < 0.001).

When CCh and nicorandil (5 x 10m3 M) were added to the cell suspension simultaneously after 3 min exposure to EGTA, 4’Ca efflux was also accelerated significantly compared with the control (P < 0.05); the values were 0.524 f 0.016 (N = 6) at 4 min and 0.361 & 0.016 (N = 6) at 9 min. However, nicorandil did not affect CCh-stimulated 45Ca efflux.

Eflects of nicorandil on the free shortening of glycerin-treated muscle fiber bundle

To investigate the effects of nicorandil on con- tractile proteins, an experiment was performed with the use of a glycerin-treated muscle fiber bundle. Since glycerin-treated bundle is devoid of a sarco- lemma1 diffusion barrier, it is useful for studying direct effects of drugs on contractile proteins.

Figure 8 shows the time course of free shortening of glycerin-treated muscle bundle in pCa 6 solution containing ATP. ATP-induced contraction of glycerin-treated muscle fiber bundle was slow and reached its maximal response at 60min, with a 64.0 f 1.6% (N = 6) decrease in the bundle length. When the bundle was exposed to 5 x lo-‘M nico- randil 10 min before being transferred to pCa 6 solu- tion, ATP-induced contraction also reached the max- imal response (61 .O f 2.4%, N = 6) at 60 min. This was not significantly different from the contraction in the absence of nicorandil.

DISCUSSION

The present study shows that nicorandil inhibited both contractions induced by high K depolarization and by CCh in isolated cells from guinea-pig taenia caeci. We discuss the mechanisms of both con- tractions, and the inhibitory action of nicorandil below.

The contraction induced by high K depolarization of the membrane

Isolated cells from guinea-pig taenia caeci were contracted by the addition of high K solution (Fig. lA), and 45Ca uptake in high K solution increased significantly (Fig. 6A). However, high K-induced contraction was abolished when extracellular Ca*+ was removed by EGTA (Fig. 4). These results suggest that high K-induced contraction depends on the influx of extracellular Ca2+.

Sekiyama (1970) reported that Ca2+-induced con- traction depended on the influx of extracellular Ca*+, not on Ca*+ stored in the cell. Obara (1984) reported that Ca2+-induced contraction of isolated cells was extracellular Ca*+ concentration-dependent. There- fore, Ca*+-induced contraction in isolated cells seems to depend on Ca*+ influx. As shown in Figs 1A and B, high K-induced contraction was transient and reached a peak rapidly (Fig. 1 A), while Ca*+-induced contraction was sustained and reached a peak slowly

YASUSHI ITO and KAZUO OBARA

0 IO 20

Time ( mln I

Fig. 7. 45Ca efflux from isolated cells into Ca*+-free EGTA PSS. After 30 min incubation in 0.18 mM Ca*+ PSS contain- ing 45Ca (2 pCi/ml), the cells were transferred to Ca*+-free 5mM EGTA PSS. 45Ca efflux was estimated from the amount of 45Ca remaining in the cells. The cellular content of 45Ca is expressed as a fraction of the content incubated in solution containing 45Ca for 30min. CCh (10m4M) or CCh containing nicorandil (5 x 10m3 M) was added after 3 min incubation in Ca*+-free EGTA PSS (at arrow). 0: control efflux. 0: CCh-stimulated efflux. A: effect of nico- randil on CCh-stimulated efflux. Each point represents mean k SE (N = 3-12). *Significantly different from control

(P < 0.05).

(Fig. 1 B). The difference suggests that the mechanism of high K-induced contraction differs from that of Ca2+-induced contraction. Urakawa and Holland (1964) reported that high K-induced contraction in strips of guinea-pig taenia coli consisted of an initial rapid (phasic) contraction and a sustained (tonic) contraction, and suggested that in the phasic con- traction sufficient Ca2+ was released from a cellular site to initiate contraction, whereas in the tonic contraction enough Ca*+ crossed the membrane from outside of the cells to initiate it. The kinetics of high K- and Ca*+-induced contractions of isolated cells resemble the phasic and tonic contractions of the strips, respectively. These results suggest that, though Ca2+ influx is indispensable to both high K- and Ca2+-induced contractions, in the high K-induced contraction, influx Ca*+ may act as trigger Ca*+ and release Ca2+ from intracellular storage sites to initiate contraction (Ca*+-induced Ca2+ release mechanism), while in the Ca2+-induced contraction, influx Ca*+ may activate contractile proteins directly.

The contraction induced by CCh

The ED~ of CCh in isolated cells from guinea-pig

taenia caeci was 3.3 x 10e6 M (Fig. 2). Obara (1984) reported that the EDGE of acetylcholine (ACh) was 2.0 x lo-* M in isolated cells from guinea-pig taenia caeci. This means that isolated cells showed higher sensitivity to ACh than to CCh. Fay and Singer (1977) reported a difference in sensitivities to ACh and CCh in isolated smooth muscle cells from the stomach of Bufo marinus. They suggested that the difference may have been due to the absence of effective cholinesterases upon isolation of single cells. In our study this difference may have occurred for the same reason.

In spite of the elimination of extracellular Ca2+ by EGTA, isolated cells were contracted by CCh in the depolarized state in high K solution (Fig. 4). 45Ca efflux from isolated cells was stimulated by CCh (Fig. 7). These results suggest that intracellular Ca2+ is released by CCh in isolated cells. The response in- duced by CCh in high K Ca*+-free EGTA solution was 66% of that in 1.8 mM Ca2+ PSS. This fact indicates that the greater part of Ca2+ necessary for CCh-induced contraction of isolated cells may be released from intracellular storage sites.

45Ca uptake of isolated cells was not increased by CCh (Fig. 6). This result is very different from that of Nasu and Urakawa (1973), in which 45Ca uptake of the strips of guinea-pig taenia coli was increased by CCh in the early phase. Our experiment shows that 45Ca efflux was increased by CCh (Fig. 7). Therefore 45Ca efflux may be increased by CCh in the 45Ca uptake study. At low temperature (2°C) control 45Ca efflux was inhibited significantly and control 45Ca uptake increased to about one-and-a-half times that of 45Ca uptake at 35°C (unpublished data). This

OY, -,I-

0 IO 20 30 40 50 60 70

Time IIll”,

Fig. 8. Effects of nicorandil on the time course of ATP- induced contraction in glycerin-treated muscle fiber bundle. Glycerin-treated muscle fiber bundle was suspended in relaxing solution and then transferred to pCa 6 solution containing ATP. Nicorandil was applied 10 min before the transfer to pCa6 solution. Contractile responses are ex- pressed as the per cent decrease in the bundle length in relaxing solution. ATP-induced contraction was observed in the presence (0) or absence (0) of nicorandil(5 x lo-’ M).

Each point represents mean + SE (N = 6).

Effects of nicorandil 51

increase of 45Ca uptake at low temperature may be due to the inhibition of 45Ca efflux. The temperature dependency of 45Ca efflux and 45Ca uptake suggests that the degree of 45Ca efflux also affects the amount of 45Ca uptake.

The reasons why the 45Ca uptake of isolated cells in the early phase was not increased by CCh are that Ca2+ influx and Ca2+ efflux in isolated cells occurred very quickly and efficiently, and that the increase of Ca2+ influx was equal to the increase of Ca2+ efflux.

The effects of nicorandil

In isolated cells from guinea-pig taenia caeci, the half maximal concentration of nicorandil in the in- hibition of high K-induced contraction was 2.8 x 10m3 M. On the other hand, in strips of dog coronary artery, the half maximal concentration of nicorandil in the relaxation of high K-induced con- traction was between 10m4 M and 10m3 M (Imai et al., 1983). Vascular smooth muscle showed higher sensi- tivity to nicorandil than isolated cells from guinea-pig taenia caeci. These results make possible some de- ductions concerning the inhibitory effects of nicor- andil on the contraction of isolated cells.

(1) The hyperpolarization of the membrane. It has been shown that nicorandil hyperpolarizes the mem- brane of various smooth muscles (Furukawa et al., 1981; Inoue et al., 1983; Yamanaka et al., 1985). However, in dog mesenteric artery nicorandil did not alter the membrane potential in the presence of [K+], over 20.2mM (Inoue et al., 1983). Therefore, it may be possible that nicorandil does not hyper- polarize the membrane of isolated cells from guinea- pig taenia caeci in high K (71 mM) solution, and that the inhibition of contractile responses in high K solution is not due to hyperpolarization of the mem- brane but rather to some other action of nicorandil.

In the present study, though the effect of nicorandil on the membrane potential was not studied, the hyperpolarizing action of nicorandil, if any, may not be the main action, because nicorandil inhibited the contraction under conditions where the hyper- polarizing action of nicorandil did not occur.

(2) The inhibition of Ca2+ influx induced by high K depolarization of the membrane. The inhibition of high K- and Ca-induced contractions by nicorandil may be due to the inhibition of Ca2+ influx induced by high K depolarization. Imai et al. (1983) con- cluded that in dog coronary artery, the inhibitory action of nicorandil differed from that of the Ca2+ channel blocker, nifedipine, because nifedipine in- hibited 45Ca uptake in high K solution but nicorandil did not inhibit this 45Ca uptake. In our experiment, nicorandil did not affect 45Ca uptake in high K solution (Fig. 4A). Therefore, the inhibition of con- tractile responses by nicorandil in isolated cells may not be due to the inhibition of Ca2+ influx by depolarization.

(3) The inhibition of Ca2+ influx induced by CCh. Nicorandil inhibited CCh-induced contraction (Fig. 3). However, nicorandil did not affect 45Ca uptake and 45Ca efflux induced by CCh (Figs 6B and 7). These results suggest that nicorandil had no effect on the suspected increase of Ca2+ influx and Ca2+ efflux induced by CCh. Therefore, it would appear that the inhibition of CCh-induced contraction by nicorandil

is not due to the inhibition of Ca2+ influx induced by CCh.

(4) The inhibition of intracellularly stored Ca2+. Nicorandil inhibited CCh-induced contraction in high K Ca’+-free EGTA solution (Fig. 4). This result suggests that the inhibition of contractile responses by nicorandil may be due to a reduction of intra- cellularly stored Ca2 + or an inhibition of the release of intracellularly stored Ca2+. However, nicorandil did not affect resting 45Ca uptake (Fig. S), or CCh- stimulated 45Ca efflux which reflected the release of Ca2+ from intracellular storage sites (Fig. 7). These results suggest that there is little possibility that nicorandil may inhibit the contractions by reducing intracellularly stored Ca2+ or by inhibiting the release of intracellularly stored Ca’+. The fact that nicoran- dil inhibited Ca2+-induced contraction also supports this hypothesis.

(5) The acceleration of Ca2+ extrusion. Itoh et al. (1983) reported that in isolated cells from porcine coronary artery, nitroglycerin, a nitro-compound similar to nicorandil, inhibited the contraction through the acceleration of Ca2+ extrusion, since nitroglycerin increased 45Ca efflux stimulated by ACh from the cell. But in our experiment, nicorandil did not affect CCh-stimulated 45Ca efflux (Fig. 7). There- fore, the possibility that nicorandil may inhibit the contraction through the acceleration of Ca2+ ex- trusion seems to be denied.

(6) The inhibition of contractile proteins. Kajiwara et al. (1984) reported that in rabbit ear artery nicorandil might reduce the force development by an interaction with the activation process of contractile proteins, because nicorandil did not affect 45Ca fluxes. Our experiment also shows that nicorandil inhibited the contractions without affecting 45Ca fluxes, suggesting that in isolated cells from guinea- pig taenia caeci, the inhibitory action of nicorandil may be mediated at the level of the activation process of contractile proteins.

Nicorandil did not affect ATP-induced contraction of glycerin-treated muscle bundles (Fig. 8). This fact could indicate that the inhibitory action of nicorandil on contractile responses is not due to the direct action of nicorandil on contractile proteins.

The relaxation caused by nicorandil was accom- panied by a concomitant elevation of the cGMP level in bovine coronary artery (Holzmann, 1983) and in dog coronary artery (Endoh and Taira, 1983), and both reports suggested that nicorandil-induced relax- ation was mediated by the elevation of cGMP. Ptitzer et al. (1984) reported that the inhibitory effect of cGMP on the contraction was mediated at the level of contractile proteins. These observations im- ply that nicorandil does not act directly on con- tractile proteins, but acts indirectly through cGMP as a second messenger. In vascular smooth muscle, cGMP acts as a second messenger through the activation of cGMP-dependent protein kinase and the phosphorylation of specific proteins. Yamada and Obara (1985) showed that cGMP was related to smooth muscle relaxation in isolated cells from guinea-pig taenia caeci, and Pfitzer et al. (1986) showed that cGMP-dependent protein kinase relaxed skinned fibers from guinea-pig taenia coli. However, the precise action of cGMP in gastrointestinal

52 YASUSHI ITO and KAZIJO OBARA

smooth muscles has not yet been clarified, and studies comparing intracellular cGMP with Ca2+ movements and the activity of contractile proteins will be necessary in the future.

Acknowledgements-We wish to express our thanks to Professor Hideyo Yabu, Department of Physiology, Sap- poro Medical College, for his valuable suggestions and discussion.

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