In the Papaverine-Induced Relaxation of Medial Strips of Pig Coronary Artery Hiroki Aoki, Junji Nishimura, Sei Kobayashi & 'Hideo Kanaide

Home , Papaverine

Br. J. Pharmacol. (1994), 111, 489-496 '." Macmillan Press Ltd, 1994 Relationship between cytosolic calcium concentration and force in the papaverine-induced relaxation of medial strips of pig coronary artery Hiroki Aoki, Junji Nishimura, Sei Kobayashi & 'Hideo Kanaide

Division of Molecular Cardiology, Research Institute of Angiocardiology, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan

1 The mechanisms of vasorelaxation induced by papaverine were investigated using front-surface fluorometry and fura-2-loaded medial strips of the pig coronary artery. 2 In the presence of extracellular Ca2+ (1.25 x 10-3 M), histamine (10-4 M) induced abrupt elevations of cytosolic calcium concentration, [Ca2+Ji reaching a peak within 12 s (the first phase); after making a slight shoulder, [Ca2+Ji declined gradually to reach sustained levels (the second phase). Force rapidly rose to reach maximum levels in 3 min, then gradually declined. Papaverine (10-7- 10- M) inhibited both the first and the second phases of [Ca2+Ji elevation and the development of force induced by histamine, in a concentration-dependent manner. 3 In the absence of extracellular Ca2+, histamine (10-4 M) induced a transient increase in [Ca2+]i and force, both of which were inhibited in a concentration-dependent manner by papaverine (10-7- l0- M). When papaverine was washed out, a second application of 10-4M histamine also induced transient increases in [Ca2+]i and force. The smaller the first response, the greater was the second response. The total amount of [Ca2]I. released from intracellular stores by the first and second application of histamine in the presence of papaverine was smaller than in its absence, thereby indicating a reduction of Ca2+ in the histamine-sensitive store. However, while papaverine (10-5 M) did not affect the transient increase in [Ca2+], induced by 2 X 10-2 M caffeine, contractions were inhibited. 4 For a given level of [Ca2+1], the force developed with the cumulative application of histamine (10-7 M-10-1 M) was greater than that observed with the cumulative application of extracellular Ca2+ (0-7.5 x 10-3 M) during high K+ depolarization. Papaverine (10-7M-10-5M) suppressed, in a concentration-dependent manner, the increase in [Ca2+]i and the force induced by cumulative applications of both histamine and extracellular Ca2' during high K+ depolarization. The [Ca2+],-force curve obtained by depolarization with K+, but not that obtained during histamine application, was shifted to the right by papaverine. Diltiazem, 10-7 M, a concentration causing a similar degree of relaxation to 10-5 M papaverine, did not shift the [Ca2+]j-force curve obtained with high K+. Nitroglycerin (10-6 M) and isoprenaline (10-6 M) shifted the [Ca2J]i-force curve to the right to a greater extent than did 10-5 M papaverine. 5 These findings suggest that papaverine relaxes medial strips of the porcine coronary artery by two mechanisms. The first is mainly due to a decrease in [Ca2+1i, not only through inhibiting Ca2+ influx through either voltage-dependent or receptor-operated Ca2+ channels, but also by inhibiting agonist- induced intracellular Ca2+ release. This occurs presumably by interference with the signal transduction pathway for histamine and by a depletion of Ca2' in histamine-sensitive stores. Secondly, the [Ca2+],- sensitivity of certain contractile mechanisms may be minimally decreased. Keywords: Cytosolic calcium; papaverine; fura-2; coronary artery; vasorelaxation; histamine; vascular smooth muscle

Introduction Changes in cytosolic Ca2" concentration ([Ca2"]j) play a 1985). The Ca2l sensitivity of the contractile regulatory central role in regulating excitation-contraction coupling in apparatus (i.e. the [Ca2J]i-force relationship) is modulated by smooth muscle cells (Sommerville & Hartshorne, 1986). agonists (Morgan & Morgan, 1984; Hirano et al., 1990), [Ca2+]i can be elevated in smooth muscle cells, as in other coupled to G-proteins (Nishimura et al., 1988). cells, by the influx of extracellular Ca2" through voltage- Papaverine is a potent vasorelaxant and reduces the con- dependent (Putney et al., 1989) or ligand-gated (Benham & tractile responses to excitatory agonists (Tashiro & Tomita, Tsien, 1987) Ca2" channels of the surface membrane or by 1970; Ferrari, 1974). It has been well demonstrated that the release of intracellular Ca2" due to the activation of the papaverine inhibits the activity of cyclic nucleotide phos- phospholipase C/inositol trisphosphate (InsP3) cascade, phodiesterases (Ferrari, 1974) and, therefore, that it induces coupled to receptors by guanosine 5'-triphosphate (GTP)- the accumulation of cytosolic cyclic nucleotides (Miyamoto binding proteins, or both. It is generally accepted that phos- et al., 1976; Kramer & Wells, 1979). Adenosine 3':5'-cyclic phorylation of the 20 kDa myosin light chain (MLC) by a monophosphate (cyclic AMP) and guanosine 3':5'-cyclic Ca2+- and calmodulin-dependent enzyme, MLC kinase monophosphate (cyclic GMP) are identified as intracellular (MLCK), leads to activation of myosin ATPase by actin and, messengers for smooth muscle relaxation and are produced consequently, smooth muscle contraction (Kamm & Stull, by the activation of adenylate and guanylate cyclases, respectively (Adelstein et al., 1978; Ignarro & Kadowitz, 1987). It has been reported the isoprenaline, and hence, cyclic AMP might decrease [Ca2j]i of vascular smooth muscle in I Author for correspondence. porcine coronary arterial strips, regardless of whether they 490 H. AOKI et al. were at rest, under high-K+ or histamine stimulation (Ushio- 10-3 M K+) and 1.18 x 10-' M K+ PSS being 0% and 100%, Fukai et al., 1993). It was also reported that cyclic AMP respectively. The developed force induced by 1.18 x 10- M hyperpolarizes the cell membrane (Somlyo et al., 1970), prob- K+ was 0.91 ± 0.06 g (n = 20). Where indicated, papaverine, ably by opening Ca2"-activated K+-channels (Sadoshima et diltiazem, isoprenaline or nitroglycerin was applied for al., 1988), inhibits Ca2" influx into cells (Bfilbring & Tomita, 10 min prior to stimulation by high K+, histamine or 1987), and stimulates both Ca2" extrusion from cells caffeine. This incubation was for the purpose of equilibration (Bulbring & den Hertog, 1980) and Ca2" uptake into intra- (Hirano et al., 1990; Abe et al., 1990; Ushio-Fukai et al., cellular storage sites (Casteels & Raeymaekers, 1979). The 1993). findings that cyclic AMP-dependent protein kinase (A-kinase) [Ca2]i was assessed by changes in the fluorescence inten- phosphorylates purified MLCK to reduce its activity (Adel- sity of the fura-2-Ca2+ complex, which were monitored with stein et al., 1978) and relaxes skinned smooth muscle con- a specifically designed front-surface fura-2 fluorometer tracted with a constant Ca2+ concentration (Kerrick & Hoar, (model CAM-OF-1) developed by us in collaboration with 1981), suggest that there might be modulation of the Ca2+ the Japanese Spectroscopic Co. (Tokyo, Japan), as previously sensitivity of the contractile apparatus by A-kinase. Nitro- described (Abe et al., 1990; Hirano et al., 1990). In brief, glycerin (Kobayashi et al., 1985), which elevates cytosolic excitation light (340 nm and 380 nm) was obtained spectro- cyclic GMP, and 8-bromo-cyclic GMP (Kai et al., 1987), a scopically from a xenon light source. Strips were illuminated membrane-permeable cycle GMP analogue, reduce the by guiding the alternating (400 Hz) excitation light through [Ca2+], of vascular smooth muscle cells. Nitroglycerin reduces quartz optic fibres arranged in a circle (diameter = 3 mm). the Ca2+ sensitivity of the contractile apparatus and inhibits Surface fluorescence of strips was collected by optic glass agonist-induced release of intracellular Ca2+ (Abe et al., fibres arranged around those conducting the excitation light 1990). (diameter = 7 mm) and introduced through a 500 nm band- In the present study, using front-surface fluorometry (Abe pass filter into a photomultiplier. The ratio of the et al., 1990; Hirano et al., 1990) and fura-2-loaded porcine fluorescence intensities (collected at 500 nm) at 340 nm coronary arterial strips, we monitored simultaneously the excitation to those at 380 nm excitation was monitored and [Ca2+]i and force in intact coronary arterial strips, the objec- expressed as a percentage; the maintained values in normal tive being to characterize the multiple effects of papaverine (5.9 x 1O-3M K+) and 1.18 x 10-1 M K+ PSS were desig- on [Ca2+]i and force, at rest and as stimulated either by high nated 0% and 100%, respectively (Figure 1). The apparent K+ or histamine in the presence and absence of extracellular dissociation constant (Kd) of the fura-2:Ca2+ complex was Ca2+. The relative importance of these effects in the considered to be 2.24 x IO- M and the absolute value of mechanism of relaxation induced by papaverine was [Ca2+]i was calculated according to the method of Gryn- examined.

Methods a Tissue preparation -1000 Hearts from adult pigs of either sex were obtained from a 100l -700_ local abattoir immediately after the animals had been killed. -500 2 These hearts were placed in ice-cold physiological salt solu- m 1 --4 0 tion (PSS) and brought to the laboratory. The left circumflex 0 50 6 --a arteries were and 2-3 cm from C- x coronary isolated, segments X the origin were excised. The segments were cut open longi- Ir ._i tudinally and the luminal surface was rubbed with a cotton 0 -100 NC swab to remove the endothelium. After removal of the - adventitia, medial preparations were cut into approximately -30 1 mm x 5 mm (0.1 mm thick) strips of circular muscle. -50J 0 b10 15 Fura-2 loading Time (min) The strips were loaded with the Ca2" indicator dye, fura-2 by incubating in oxygenated (a mixture of 95% 02 and 5% C02) 15051b Dulbecco's modified Eagle's medium containing 2.5 x 10-5M 1.5 fura-2/AM (an acetoxymethyl ester form of fura-2) and 5% 100- foetal bovine serum for 3-4 h at 37°C. The fura-2-loaded -1.0 -C to remove the extracel- strips were washed with normal PSS 4) 0 0 lular dye, and further incubated in normal PSS for 1 h before 0 LL. 50. the start of measurements. Loading the strips with fura-2 in -0.5 U. itself did not affect contractility, as has been described previously (Abe et al., 1990; Hirano et al., 1990). o0 -0 Simultaneous measurement offorce and [Ca2+]i 6 5 1*0l15 Time (min) Isometric force of the strips, mounted vertically in a quartz organ bath, was measured by a force transducer (TB-612T, with Figure 1 Effects of various concentrations of papaverine on changes Nihon Kohden, Japan), simultaneously [Ca2+]i. During in fluorescence ratio (a) and force (b) induced by 10-4 M histamine in the 1 h fura-2 equilibration period, the strips were stimulated normal PSS. Strips were pretreated with 0 (0; control), 10-7 (0), with 1.18 x 10-' M K+ every 15 min, and the resting force 10-6 (A), 10-5 (0) M papaverine. Papaverine was applied for 10 min was adjusted to 250 mg. Consistent responses to 1.18 x before and was present during the application of histamine. The 10' M K+-depolarization were obtained before the experi- abscissa scale in (a) and (b) shows the time (in min) after the mental protocol was started. The developed force was ex- application of histamine. Mean values with s.e.mean are shown pressed as a percentage with the values in normal (5.9 x (n = 4). PAPAVERINE RELAXATIONS OF THE CORONARY ARTERY 491

kiewicz et al. (1985). The mean values of 10 different -1000 preparations of [Ca2"]i at rest (0%) and during 1.18 X 10-1 M 100.lOa -700 K+-depolarization (100%) were 1.08 ± 0.27 ( x 10-7 M) and - 500 2 7.15 ± 1.03 ( x IO- M), respectively. 0 - s BG50. x 0 _ Drugs and solutions cc 0' 100 0( Normal PSS was of the following composition (mM): NaCl 123, KCl 4.7, NaHCO3 15.5, KH2PO4 1.2, MgCl2 L2, CaCl2 -50C Ol - 30 1.25, and D-glucose 11.5. The Ca2+-free version of PSS COntrOI -7 -6 -5 -4 (Ca2+-free PSS) contained 2 x 10-3M ethyleneglycol-bis(p- Histamine aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) instead (log M) of 1.25 x 10-3 M CaC12. High potassium PSS was made by an equimolar substitution of KCl for NaCl. All solutions were gassed with a mixture of 5% CO2 and 95% 02 (pH 7.4 2.0 at 370C). Papaverine hydrochloride, histamine dihydro- chloride, and fura-2/AM were purchased from Katayama 15011b (Osaka, Japan), Wako (Osaka, Japan), and Dojindo 1.5 (Kumamoto, Japan), respectively. Nitroglycerin was obtained from Nihon-Kayaku (Tokyo, Japan). (-)-Isoprenaline hyd- 100' rochloride was purchased from Nikken-Kagaku (Tokyo, 11.0 0 I0- 0 Japan). Diltiazem hydrochloride was kindly donated by LL Tanabe Seiyaku Co. (Osaka, Japan). Fura-2/AM was dis- 0 solved in 100% dimethyl sulphoxide (DMSO) and diluted in LAL 50- 0.5 the medium just before loading the dye. The final concentra- was 5%. tion of DMSO 0* 0 Control -7 -6 -5 -4 Statistical analysis Histamine (log M) The measured values were expressed as mean ± standard error with the number of observations in different tissues from at least 3 different animals. t test Figure 2 Effects of papaverine on contractions induced by (= n) Student's was cumulative application of histamine (10-7M-10-4M). Changes in used to determine the statistical significance. Analysis of fluorescence ratio (a) and force (b) induced by cumulative applica- variance was used to determine the concentration-dependency tion of histamine to strips treated with 0 (0; Control), 10-7 (0), of effects of drugs. Analysis of covariance was used to deter- 10-6 (A) or 10-1 (O)M papaverine. Papaverine was applied for mine the non-overlapping (or shift) of the [Ca2+]i-force rela- 10 min before, and was present during, the cumulative applications tionship. P values <0.05 were considered to be significant. of histamine. Values are mean with s.e.mean (n = 4).

Results Effects ofpapaverine on increases in [Ca2+], andforce induced by cumulative application ofhistamine in the Effect ofpapaverine on the increase in [Ca2J]i andforce presence ofextracellular Ca2+ induced by histamine in the presence of extracellular Ca2+ In normal PSS, cumulative application of histamine (10-7-10-4 M) led to step-wise, concentration-dependent When histamine (10-4M) was applied in normal PSS, the (P<0.001 by analysis of variance, n = 4), increases in [Ca2+]J [Ca2+]i abruptly increased and reached a first peak at 12 s and force. Treatment with papaverine (10`-10- M) for (the first phase). After a slight shoulder, the [Ca2+]i declined 10 min before and during the cumulative application of his- gradually, but remained at higher than the prestimulation tamine (10-'-10-4 M) led to a concentration-dependent levels (the second phase) (Figure la). Force also developed inhibition of the increases in [Ca2+k (P<0.001 for rapidly and reached a maximum about 3 min after exposure papaverine, by two way analysis of variance, n =4; Figure to histamine, then declined gradually (Figure lb). The forces 2a). The elevation of [Ca2+]i by 10-4 M histamine was developed at the maximum and after 15 min were 123% reduced by 10-S M papaverine from 94.4 ± 2.8% (6.17 x (1.12 g) and 44% (0.40 g) of the contraction induced by high 10-7 M) to 74.1 ± 3.0% (4.09 x 10-7 M). Papaverine also K+, respectively (n = 4). inhibited the development of force in response to histamine, When IO-5M papaverine was applied for 10 min before in a concentration-dependent manner (P<0.001 for and during the applications of 10-4 M histamine, it reduced papaverine, by two way analysis of variance, n = 4; Figure both the first and the second phases of the increase in [Ca2+]i. 2b). The force developed in the presence of 10-4 M histamine Papaverine also inhibited the development of force induced was reduced by 10-5M papaverine from 138.8 ± 6.5% by 10-4 M histamine. Figure la and b shows that inhibition (1.26 g) to 82.4 ± 6.3% (0.75 g) (n = 4). of the increases in [Ca2J]i and force was concentration- dependent (10-7-10-5 M). Higher concentrations of Effects ofpapaverine on histamine- or caffeine-induced papaverine interfered with the fluorimetry and were not release of intracellular Ca-+ examined. These results, together with our previous findings that the first and the second phases of the increase in [Ca2+]i When vascular strips were exposed to Ca2+-free PSS contain- are mainly due to the release of intracellular Ca2+ and the ing 2 x 10-3 M EGTA, [Ca2+]i gradually declined to reach a influx of extracellular Ca2" into the cells, respectively steady state while the force remained unchanged (Figure 3a (Hirano et al., 1990), suggest that papaverine inhibits both and b). As shown in Figure 3a, a first application of 10-4 M the Ca2" release and Ca2" influx stimulated by histamine. histamine after 15 min incubation in Ca2"-free PSS caused 492 H. AOKI et al.

lob 1000 100- 700 - 500 2 a 1 1000 _ 1001n 700 0 _ 500 ol o co x

I:i O r.. x 100 N 0] ._: 100 z1 40 100 _ 40 1.0 100 10 min

LL ~~~~~~0 0 _ ~ ~ ~ *[ U 0 ~~~~~ L0 U. 0~~~~~~ U-L- L,-mw K+ 1.18x101 M Histamine 10-4 M Histamine 10-4 M K+ 1.18 x 10-1 M Histamine 10- M Histamine 10- M Papaverine 10-5 M EGTA2 x 10-3M EGTA 2 x 10-1 M

Figure 3 Effects of papaverine on histamine-induced contraction in Ca2+-free PSS containing EGTA, 2 x 10-3 M. Representative recordings of changes in fluorescence ratio and force in the absence (a) and presence (b) of 10-M papaverine. Strips were incubated in Ca2+-free PSS containing 2 x 1O-1 M EGTA for 15 min before and during the first application of histamine, with or without papaverine. Two min after the first application of 10-4 M histamine, strips were washed with Ca2"-free PSS and after a further 15 min, a second application of 10-M histamine was given.

rapid and transient elevations in [Ca2J+] and force, which incubating strips in Ca2l-free PSS containing 2 x 10-3 M began to decline within 1.5 min. After 2 min, histamine was EGTA for 15 min, 2 x 102 M caffeine induced transient in- washed out with Ca2"-free PSS. A second application of creases in [Ca2+]i and force (controls in Figure Sa and b). 10-4 M histamine caused only a slight elevation of [Ca2+]i and Treatment with I0-5 M papaverine 10 min before and during force. As shown in Figure 3b, preincubation with 10-5M the application of 2 x 10-2 M caffeine had no effect on the papaverine inhibited the increases in [Ca2+]i and force caused increases in [Ca2+]i (Figure 5a), hence papaverine probably by the first application of 10-4 M histamine. However, after did not affect the Ca2"-release induced by caffeine nor did it washing out papaverine and histamine with Ca2+-free PSS deplete the Ca2' present in the caffeine-sensitive storage sites. after 2min exposure to histamine, a second application of On the other hand, contractions induced by 2 x 10-2 M 10-4M histamine induced transient increases in [Ca2+]i and caffeine were inhibited by about 50% (P<0.01 by Student's force, and these responses were greater than those observed t test, n = 4) by 10-5 M papaverine (Figure Sb), thereby during the second application of histamine without suggesting that papaverine slightly inhibited the caffeine- papaverine treatment (compare traces in Figure 3a, b; Figure induced contraction, without affecting [Ca2+],. 4a, b, solid columns, control vs. 10-5 M papaverine). Figure 4 shows the mean effects of preincubation with various concen- Effects ofpapaverine on increases in [Ca2-]J andforce trations of papaverine only during a first exposure to his- induced by cumulative applications of extracellular Ca2- tamine on changes in [Ca2+]i and the force induced by the during high K+-depolarization first and second (no exposure to papaverine) applications of 10-4 M histamine in the absence of extracellular Ca2+. In the Effects of papaverine on K+-depolarization-induced contrac- presence of papaverine, there was a concentration-dependent tion were examined, using the following protocol. After inhibition of the increases in [Ca2+]i and force induced by the incubation in Ca2+-free PSS containing 2 x 10-3 M EGTA first application of histamine (P <0.05 by analysis of for 10 min and subsequently in Ca2"-free PSS without EGTA variance, n = 6). When responses to the first application of for 5 min, the vascular strips were depolarized with histamine were inhibited more markedly by a higher concen- 1.18 x 10-1 M K+- and Ca2+-free PSS. Cumulative applica- tration of papaverine, the responses to the second application tions of extracellular Ca2" (0-7.5 x 10-3 M) during high K+- of histamine became larger (P <0.05 by analysis of variance, depolarization induced step-wise increases in [Ca2'+l and n = 6). In addition, the total amount of released Ca2 , force, both of which were inhibited by papaverine estimated from the elevation of [Ca2+]i and its duration (10-7- l0-1 M), in a concentration-dependent manner (Matsumoto et al., 1990), by two applications of histamine (P< 0.001 for both [Ca2+J0 and the concentrations of combined with exposure to 10-5M papaverine during one papaverine, by two way analysis of variance, n = 4; Figure 6a application of histamine was smaller than that without and b). papaverine (P <0.05 by Student's t test, n = 6). Therefore, it is likely that inhibition of the histamine-induced Ca2+ release Effects ofpapaverine on the [Ca2+],-force relationship by papaverine may be due to interference with the histamine- stimulated signal transduction pathway, to the depletion of Effects of papaverine on the [Ca2+]j-force relationship of the Ca2+ present in intracellular stores sites, or to a combination contractions induced by histamine and by 1.18 ± 10-1 M K+- of these effects. depolarization are shown in Figure 7. This figure was made Effects of papaverine on caffeine-induced release of intra- by plotting the mean values of %ratio and force of Figures 2 cellular Ca2+ were determined with an experimental protocol and 6. The [Ca2+]j-force relationship determined by similar to that used for histamine-induced release. After cumulative applications of extracellular Ca2+ during PAPAVERINE RELAXATIONS OF THE CORONARY ARTERY 493

a I NS -700 5O.' -250

-500 - 0 50 m0 OR o * x 0 O -100 x m 0 ._ ._z cul + N 0 X 0 -100 u -50- -30 I I -40 Control Papaverine -50J i0-5 M Control -7 -6 -5 Papaverine (log M) b 20-1 b -0.2 r1 .0

cm 0 0 50 0 10 0 *0.5 Z1 '0.1 0 0 o 0 ULLo LL ULoL Lo Control -7 -6 -5 0* Papaverine Control Papaverine (log M) 1M-5M

Figure 4 Effects of various concentrations of papaverine on the Figure 5 Effects of lO-I M papaverine on the 2 x 10-2 M caffeine- increases in fluorescence ratio (a) and force (b) induced by 10- M induced increase in fluorescence ratio (a) and force (b) in the absence histamine in the absence of extracellular Ca2". Open and solid of extracellular Ca2". The bottom and top of columns indicate levels columns show the responses induced by the first and second applica- before, and peak response after, the stimulation by caffeine, respec- tions of 10- M histamine, respectively. The bottom and the top of tively. Data are means of 4 different measurements. Values are mean the column indicate levels before the stimulation and peak response with s.e.mean. (NS: not significant). after the stimulation by histamine, respectively. Data are means of 6 different measurements obtained from experimental procedures described in the legend for Figure 3. Values are mean with s.e.mean. determined by plotting the ratio and the force during cumulative applications of extracellular Ca2" (0-7.5 x 10-3 M) during high K+-depolarization in the presence and 1.18 X 10-1 M K+-depolarization in the presence of 10-5 M absence of 10-7 M diltiazem, 10-5 M papaverine, 10-6 M papaverine shifted slightly, but significantly (P < 0.05 by nitroglyercin or 10-6M isoprenaline. Each vasodilator was analysis of covariance, n = 4), to the right from that deter- applied 10 min before starting cumulative applications of mined in the absence of papaverine. On the other hand, the extracellular Ca2". As shown in Figure 8, in the presence of [Ca2+]J-force relationship obtained during cumulative applica- diltiazem, the [Ca2+]i-force relationship did not change, while tions of histamine in normal PSS in the presence of 10-5 M in the presence of papaverine, nitroglycerin or isoprenaline, papaverine did not differ from that determined in the absence the [Ca2J]i-force relationship was shifted to the right of papaverine. In addition, in the absence of papaverine, the (P<0.05 for papaverine and P<0.01 for nitroglycerin and developed force for given levels of [Ca2+]i in histamine- isoprenaline, by analysis of covariance, n = 4). In addition, in induced contractions was greater (P < 0.05 by analysis of the presence of nitroglycerin and isoprenaline, the [Ca2+]i- covariance, n =4) than that in 1.18 x 10-' M K+-depolar- force relationship shifted to the right more prominently than ization-induced contractions (that is, the control [Ca2J]i-force seen in the presence of papaverine (P<0.05 by analysis of relationship), thereby suggesting that histamine increases the covariance, n = 4). Thus, at a given [Ca2]i level, develop- Ca2` sensitivity of the myofilaments. ment of force in the presence of papaverine was higher than in the presence of nitroglycerin or isoprenaline but lower than in the presence of diltiazem or in the control. Effects of various vasodilators on [Ca2+],-force relationship during 1.18 x 10-' M K+-depolarization Discussion The effects of vasodilators (diltiazem 10-7 M, papaverine lo0- M, nitroglycerin 10-6 M and isoprenaline 10-6 M) on the Using front-surface fluorometry and fura-2 we have [Ca2+]i-force relationship during 1.18 x 10-1 M K+-depolar- examined the various effects of papaverine on [Ca2+]i and ization were investigated. In the presence of these vaso- force in pig coronary arterial strips. Both the first and second dilators, the force developed by 1.18 x 10-' M K+-depolari- phases of the elevation of [Ca2+]i induced by histamine were zation in the presence of 1.25 x 10-3 M extracellular Ca2` inhibited by 10-7 to 10-5 M papaverine (Figure 1) a was reduced by about 30%. [Ca2`]i-force relationships were concentration-range over which papaverine is known to 494 H. AOKI et al.

a 150-. 150 1.5 -1000 100* -700 -500 2 100 1.0 50- - x 0 0 CD U- + so ;e LL 0 50 LL -) 0.5 -50J -30 I 0 10-4 10o3 10-2 [Ca2+]O (M) O- LO

-50 60 5 100 150 Ratio (%) b I 1 150' 30 100 500 1000 '1.5 [Ca2+1, (x 10-9 M)

Figure 8 Effects of various vasodilators on the [Ca2+]j-force rela- 100 tionship determined by cumulative applications of Ca2+ during *1.0 1.18 x 10-1 M K+-induced depolarization in the strips pretreated 0 with I0- M papaverine (A), IO- M diltiazem (0), 10 6M nitro- Irl glycerin (A), 10-6 M isoprenaline (-) or the control (0). Data are ._co o means of 4 different measurements. The abscissa scale indicates 50- *0.5 U- fluorescence ratio andpcalculated [Ca2+1i, and the ordinate scales the force. The s.e. is omitted for purposes of clarity.

LO O0 inhibit cyclic nucleotide phosphodiesterases 10-5 M; -2 (ICm 0 10-4 10-3 102 Kramer & Wells, 1979). This, together with our previous finding that the first and the second phases of histamine- (M) [Ca"+]. induced responses are mainly due to Ca2" released from store sites and Ca2" influx through the cell membrane, respectively Figure 6 Effects of papaverine on increases in [Ca2+Ji and force induced by cumulative applications of extracellular Ca2+ during (Hirano et al., 1990), suggests that papaverine inhibits, with a not 1.18 x 10-1 M K+-induced depolarization: (a) and (b) show effects of similar potency, only Ca2" influx but also the Ca2" various concentrations of papaverine on the changes in fluoresence release stimulated by histamine. Inhibition of intracellular ratio and force, respectively, in the strips pretreated with 0 (0, Ca2" release was also observed in experiments carried out in control), lo-7 (0), 10-6 (A) or 10-5 (0) M papaverine. Values are the absence of extracellular Ca2" (Figure 3). Over the same mean ± s.e.mean (n = 4). range of concentrations, papaverine also inhibited the Ca2" changes induced by high K+-depolarization (Figure 6). Com- pared with these main effects of papaverine on [Ca2J]i tran- 150 sients, the slight shift of the [Ca2+]i-force curve during high K+-depolarization to the right in the presence of papaverine "1.5 appears to indicate a slight reduction in the sensitivity of the contractile apparatus to Ca2" (Figures 7 and 8). These results suggest that the main mechanism for the relaxation induced 100- '1.0 by papaverine is to decrease [Ca2J]i, through various cm mechanisms. The processes affected include inhibition of the

0 release of intracellularly stored Ca2" and inhibition of Ca2" L- 0

so 0 influx stimulated by either high K+-depolarization or by UL 50s -0.5 histamine.

Inhibition of the release of stored Ca2+

o0 .0 We found that papaverine inhibits intracellular Ca2` release induced by histamine (Figures 3 and 4), but not by caffeine -50 50 1i0 10o (Figure 5). Moreover the inhibition by papaverine of the Ratio (%) histamine-induced elevation of [Ca2+]i in Ca2"-free PSS was reversed after washing out papaverine, thereby indicating 30 100 500 1000 that papaverine interferes with histamine-activated Ca2+- [Ca2+1J (x 10-9 M) release mechanisms without affecting those related to caffeine-activated release. Another interpretation is that Figure 7 Effects of papaverine on the [Ca2+]i-force relationship, papaverine might deplete intracellular Ca2+ storage sites determined by cumulative applications of Ca2" during 1.18 x 10-1 M (Figure 4a). We reported that histamine-sensitive Ca2+- K+-induced and cumulative of depolarization (0, *) applications storage may histamine (A, A) in normal PSS. Open and closed symbols show the sites and caffeine-sensitive Ca2+-storage sites differ (Matsumoto et al., 1990) in rat aortic smooth muscle responses in the absence and presence of 10-1 M papaverine, respectively. Data are means of 4 different measurements. The abscissa cells in primary culture. Papaverine may deplete histamine- scales indicate fluorescence ratio and calculated [Ca2+]i and the sensitive storage sites, without affecting caffeine-sensitive ordinate scales indicate force. storage sites. Wang & Large (1991) reported that papaverine PAPAVERINE RELAXATIONS OF THE CORONARY ARTERY 495

inhibited the Ca2l-activated chloride and potassium currents, cause relaxation of the dog basilar artery. In other electro- and thus might induce relaxation in rabbit vascular smooth physiological studies (Itoh et al., 1981; Brading et al., 1983) muscle cells by depleting intracellular Ca2" stores; however it and 45Ca2l studies (Huddart et al., 1984), papaverine seemed was caffeine-sensitive. The difference in the tissues studied to inhibit Ca2l influx as a means of causing relaxation of (rabbit ear artery and portal vein vs. porcine coronary artery) smooth muscle. or concentration of papaverine ( > 10-5 M vs.

References ABE, S., KANAIDE, H. & NAKAMURA, M. (1990). Front-surface BRADING, A.F., BURDYGA, T.V. & SCRIPNYUK, Z.D. (1983). The fluorometry with fura-2 and effects of nitroglycerin on cytosolic effects of papaverine on the electrical and mechanical activity of calcium concentrations and on tension in the coronary artery of the guinea-pig ureter. J. Physiol., 334, 79-89. the pig. Br. J. Pharmacol., 101, 545-552. BOLBRING, G.E. & DEN HERTOG, A. (1980). The action of ADELSTEIN, R.S., CONTI, M.A. & HATHAWAY, D.R. (1978). Phos- isoprenaline on the smooth muscle of the guinea-pig taenia coli. phorylation of smooth muscle myosin light chain kinase by the J. Physiol., 304, 277-296. catalytic subunit of adenosine 3':5'-monophosphate-dependent BOLBRING, G.E. & TOMITA, T. (1987). Catecholamine action in protein kinase. J. Biol. Chem., 253, 8347-8350. smooth muscle. Pharmacol. Rev., 39, 49-96. BENHAM, C.D. & TSIEN, R.W. (1987). A novel receptor-operated Ca2+-permeable channel activated by ATP in smooth muscle. Nature, 328, 275-278. 496 H. AOKI et al.

CASTEELS, R. & RAEYMAEKERS, L. (1979). The action of acetyl- MADISON, J.M. & BROWN, J.K. (1988). Differential inhibitory effects choline and catecholamines on an intracellular calcium store in of forskolin, isoproterenol, and dibutyryl cyclic adenosine the smooth muscle cells of the guinea-pig taenia coli. J. Physiol., monophosphate on phosphoinositide hydrolysis in canine 294, 51-68. tracheal smooth muscle. J. Clin. Invest., 82, 1462-1465. CASTEELS, R. & SUZUKI, H. (1980). The effects of histamine on the MATSUMOTO, T., KANAIDE, H., SHOGAKIUCHI, Y. & NAKAMURA, smooth muscle cells of the ear artery of the rabbit. Pflugers Arch., M. (1990). Characteristics of the histamine-sensitive calcium store 387, 17-25. in vascular smooth muscle. Comparison with norepinephrine- or FERRARI, M. (1974). Effects of papaverine on smooth muscle and caffeine-sensitive stores. J. Biol. Chem., 265, 5610-5616. their mechanisms. Pharmacol. Res. Commun., 6, 97-115. MIYAMOTO, M., TAKAYANAGI, I., OHKUBO, H. & TAKAGI, K. FUJIOKA, M. (1984). Lack of causal relationship between the (1976). Actions of papaverine on intestinal smooth muscle and its vasodilator effect of papaverine and cyclic AMP production in inhibition of cyclic AMP and cyclic GMP phosphodiesterases. the dog basilar artery. Br. J. Pharmacol., 83, 113-124. Jpn. J. Pharmacol., 26, 114-117. GRYNKIEWICZ, G., POENIE, M. & TSIEN, R.Y. (1985). A new genera- MORGAN, J.P. & MORGAN, K.G. (1984). Stimulation-specific patterns tion of Ca2l indicators with greatly improved fluorescence pro- of intracellular calcium levels in smooth muscle of ferret portal perties. J. Biol. Chem., 260, 3440-3450. vein. J. Physiol., 351, 155-167. HALL, I.P., DONALDSON, J. & HILL, S.J. (1989). Inhibition of NISHIMURA, J., KOLBER, M. & VAN BREEMEN, C. (1988). histamine-stimulated inositol phospholipid hydrolysis by agents Norepinephrine and GTP-y-S increase myofilament Ca2+ sen- which increase cyclic AMP levels in bovine tracheal smooth sitivity in a-toxin permeabilized arterial smooth muscle. Biochem. muscle. Br. J. Pharmacol., 97, 603-613. Biophys. Res. Commun., 157, 677-683. HIRANO, K., KANAIDE, H., ABE, S. & NAKAMURA, M. (1990). NISHIZUKA, Y. (1983). Calcium, phospholipid turnover and trans- Effects of diltiazem on calcium concentrations in the cytosol and membrane signaling. Phil. Trans. R. Soc., B302, 101-112. on force of contractions in porcine coronary arterial strips. Br. J. PUTNEY, J.W., TAKEMURA, H., HUGHERS, A.R., HORSTMAN, D.A. Pharmacol., 101, 273-280. & THASTRUP, 0. (1989). How does inositol phosphate regulate HUDDART, H., LANGTON, P.D. & SAAD, K.H.M. (1984). Inhibition calcium signaling? FASEB J., 3, 1899-1905. by papaverine of calcium movements and tension in the smooth SADOSHIMA, J., AKAIKE, N., KANAIDE, H. & NAKAMURA, M. muscles of rat vas deferens and urinary bladder. J. Physiol., 349, (1988). Cyclic AMP modulates Ca2+-activated K' channel in 183-194. cultured smooth muscle cells of rat aortas. Am. J. Physiol., 255, IGNARRO, L.J. & KADOWITZ, P.J. (1987). The pharmacological and H754-H759. physiological role of cyclic GMP in vascular smooth muscle SOMLYO, A.V., HAEUSLER, G. & SOMLYO, A.P. (1970). Cyclic relaxation. Annu. Rev. Pharmacol. Toxicol., 25, 171-191. adenosine monophosphate: potassium-dependent action on vas- IGUCHI, M., NAKAJIMA, T., HISADA, T., SUGIMOTO, T. & cular smooth muscle membrane potential. Science, 169, 490-491. KURACHI, Y. (1992). On the mechanism of papaverine inhibition SOMMERVILLE, L.E. & HARTSHORNE, D.J. (1986). Intracellular cal- of the voltage-dependent Ca2" current in isolated smooth muscle cium and smooth muscle contraction. Cell Calcium, 7, 353-364. cells from the guinea pig trachea. J. Pharmacol. Exp. Ther., 26, SUEMATSU, E., HIRATA, M. & KURIYAMA, H. (1984). Effects of 194-200. cAMP and cGMP-dependent protein kinases, and calmodulin on ITOH, T., KAJIWARA, M., KITAMURA, K. & KURIYAMA, H. (1981). Ca21 uptake by highly purified sarcolemmal vesicles of vascular Effects of vasodilator agents on smooth muscle cells of the cor- smooth muscle. Biochem. Biophys. Acta, 773, 83-90. onary artery of the pig. Br. J. Pharmacol., 74, 455-468. SUPATrAPONE, S., DANOFF, S.K., THEIBERT, A., JOSEPH, S.K., KAI, H., KANAIDE, H., MATSUMOTO, T. & NAKAMURA, M. (1987). STEINER, J. & SNYDER, S.H. (1988). Cyclic AMP-dependent 8-Bromoguanosine 3':5'-cyclic monophosphate decreases intracel- phosphorylation of a brain inositol trisphosphate receptor lular free calcium concentrations in cultured vascular smooth decreases its release of calcium. Proc. Natl. Acad. Sci. U.S.A., 85, muscle cells from rat aorta. FEBS Lett., 221, 248-288. 8747-8750. KAMM, K.E. & STULL, J.T. (1985). The function of myosin and TASHIRO, N. & TOMITA, A. (1970). The effects of papaverine on the myosin light chain phosphorylation in smooth muscle. Annu. Rev. electrical and mechanical activity of the guinea-pig taenia coli. Pharmacol. Toxicol., 25, 593-620. Br. J. Pharmacol., 39, 608-618. KERRICK, W.G.L. & HOAR, P.E. (1981). Inhibition of smooth muscle USHIO-FUKAI, M., ABE, S., KOBAYASHI, S., NISHIMURA, J. & tension by cyclic AMP-dependent protein kinase. Nature, 292, KANAIDE, H. (1993). Effect of isoprenaline on cytosolic calcium 253-255. concentrations and on tension in the porcine coronary artery. J. KOBAYASHI, S., KANAIDE, H. & NAKAMURA, M. (1985). Cytosolic- Physiol., 462, 676-696. free calcium transients in cultured vascular smooth muscle cells: WANG, Q. & LARGE, W.A. (1991). Modulation of noradrenaline- microfluorometric measurements. Science, 229, 553-556. induced membrane currents by papaverine in rabbit vascular KRAMER, G.L. & WELLS, J.N. (1979). Effects of phosphodiesterase smooth muscle cells. J. Physiol., 439, 501-512. inhibitors on cyclic nucleotide levels and relaxation of pig coronary arteries. Mol. Pharmacol., 16, 813-822. (Received July 7, 1992 Revised Azlgust 9, 1993 Accepted October 14, 1993)