- Email: [email protected]
Modulation of Ca2+-activated K+ current by isoprenaline, carbachol, and phorbol ester in cultured (and fresh) rat aortic vascular smooth muscle cells
ISSN 0306-3623/96 15.00 + .00 0306-3623(95)02005-X All rights reserved
Gen. Pharmac. Vol. 27, No. 2, pp. 319-324, 1996 Copyright © 1996 Elsevier Science Inc. Printed in the USA. ELSEVIER
Modulation of Ca2+-Activated K ÷ Current by Isoprenaline, Carbachol, and Phorbol Ester in Cultured (and Fresh) Rat Aortic Vascular Smooth Muscle Cells Hiroyasu Satoh DEPARTMENTOF PHARMACOLOGY,N^gA MEDICALUNIWRS~, KASmHAR*,NAP.A634, JAPAN ABSTRACT~ 1. Effects ofisoprenaline (ISO), carbachol, and phorbol ester on the outward K + currents in single cultured (or fresh) rat aortic vascular smooth muscle (A7r5 and A. 10) cells were examined using a whole-ceU voltage-clamp (at room temperature 22°C). 2. W i t h 10 m M E G T A in the pipette solution, the delayed rectifier K + current (It,) was activated by Ca 2+ at pCa 7 more than at pCa 10, a n d was T E A (10 mM) and apamin (200 nM) sensitive, which represents a CaZ+-activated K + current (IKc,). 3. I n cultured A7r5 cells, isoprenaline (1 and 5 LaM) and carbachol (0.1 and 1 lab0 inhibited IKc,. Phorbol ester, 4-~-phorbol-12, 13-dibutyrate (PDB), at 0.1 a n d 1 laM also inhibited IKc~, a n d increased the inhibitory effects induced by isoprenaline (1 laM). 4. In fresh aortic cells, these drugs, at the same concentrations, also produced the similar effects. 5. In A-10 cells, PDB (1 ~M) enhanced the transient outward current (4-AP.sensitive), but ISO (1 laM) inhibited the current. 6. These results suggest that the IKe., current would be inhibited by cyclic nucleotides (cAMP a n d cGMP) and also by PK.C stimulation, and thereby be directly contributed to excitation-contraction coupling of the vascular smooth muscle cells. GEN PHA~AC 27;2:319-324, 1996. I
KEY WORDS. Isoprenaline, carbachol, phorbol esters, Ca2+-activated K + current, aortic vascular smooth muscle ceils INTRODUCTION The physiological functions of vascular smooth muscle (VSM) are affected by phosphorylation with cAMP-dependent and cGMP-dependent protein kinases (PK-A and PK-G), and with protein kinase C (PK-C). The effects of these kinases on cytosolic free Ca 2+ concentration ([Cali) may be one key regulator of vascular tone. The cAMP accumulation is associated with vascular relaxation through ~-adrenoceptor stimulation by catecholamines, due mainly to an inhibition of Ca 2+ current (Satoh 1992, S atoh and Sperelakis 1991, 1995) and a depolarization of the membrane (Ousterhout and Sperelakis, 1987). Also, PK-A and PK-G enhance the delayed rectifier K + current (IK)(Bkaily et at., 1988). Both cAMP and cGMP elevations have been implicated in the relaxation of VSM cells in response to some vasodilators, such as endothelium-derived releasing factor (EDRF), atrial natriuretic factor (ANF), and nitroprusside. ACh, through muscarinic receptors, can stimulate phosphatidyl inositol (PI) turnover and produce inositol 1,4,5triphosphate (IP3) and diacylglycerol (DAG) for stimulation of PK-C (Rasmussen and Barrett, 1984). PK-C stimulates the contraction of VSM cells. Therefore, A C h might contract the VSM cells. Thus, the contraction of VSM cells is regulated by cyclic nucleotides and PK-C stimulation. However, the effects of the protein kinases on the ionic currents of VSM cells are still controversial. Therefore, this article focuses on the Ca2+-activated current (Ikca)in cultured (A7r5 and A-10) and fresh rat aortic VSM cells, and the effects of isoprenaline (ISO), carbachol, and phorbol ester (for stimulation of PK-C) on the whole-cell IKcacurrent were examined. Since the IK current is reported to be Ca2+-activated, [Ca]~ level in this study was buffered at pCa 7 to amplify the current amplitude, according to calculations by Fabiato and Fabiato (1979) and Received 10 April 1995.
Tsien and Rink (1980). Effects on the transient outward current (Ito) were also examined. A preliminary report of a part of this work was given recently (Satoh and Sperelakis, 1991). MATERIALS AND METHODS
Cell culture a n d preparation Stable VSM cell lines (A7r5 and A10), derived from rat aorta, were purchased from the American Tissue Type Culture Collection (ATCC, Bethesda, MD). These cell lines present many characteristics of adult aortic VSM cells, including a well-developedrough endoplasmic reticulum, dispersed contractile filaments, and muscle-type creatine phosphokinase isozyme, and were prepared as described previously (Satoh and Sperelakis, 1995). Fresh aortic VSM cells were obtained from adult rats. Dissected aortas were incubated by collagenase and/or tripsin for 15-20 min. The ceils were transferred to glass coverslips (placed in 35-mm Petri dishes) and bathed in tissue culture medium (Gibco, Grand Island, NY) containing 10% fetal calf serum. The cells were incubated at 37°C in a humidified atmosphere under 5% CO2 and 95% air. The cells were used for experiments after maintaining for 1 or 2 days.
Electrical measurements The membrane currents were measured by a whole-ceU voltage-clamp technique. The currents were amplified with an Axopatch patch-clamp amplifier (Axon Instruments, Burlingame, CA), and the data were stored and analyzed on an IBM-AT microcomputer using the PCLAMP analysis program. Test pulses were applied once every 20 s. Current traces were filtered at a cutoff frequency of 2 kHz for plotting. The amplitude of the inward current was measured at the peak of each current record. All experiments were carried out at room temperature (22°C).
320
H. Satoh
D
+70 mV -40 L
pAIpF
]
30
ls
A pCa 10 20
B pCa 7
A.A.A-A" ~
--
"
" I
III I
qllH
I•
i
F
l/zx"
10
C
.----
B-A
II ~ . A
~.~)
-20
0
_..] 100 pA 0 100 ms
.,r I
Experimental solutions The composition of modified Tyrode solution used was (in raM): NaC1 110, KC15.4, CaCI2 20, MgC12 1, NaH2PO4 0.3, glucose 5, and HEPES 5. The pH was adjusted to pH 7.4. Nifedipine I IxM (Sigma Chemical Co.) was also added to block Ic, current. The pipette solution was composed of (mM): KOH 110, KCI 20, MgC12 1, Mg-ATP 5, EGTA 10, creatine phosphate 5, aspartic acid 100, and HEPES 5 (adjusted to pH 7.2). Drugs (Sigma) used were: L-isoprenaline, carbachol, and 4-[~-phorbol12,13-dibutyrate (PDB). PDB was dissolved with dimethyl sulphoxide (DMSO) at 10 mM and was stocked at - 10°C. Tetraethylammonium (TEA) and apamin (Sigma) were also used.
Statistical analysis The values represent mean_+ SEM. The differences of the mean values were analyzed by the Student t test for paired data, and a P value of less than 0.05 was considered significant. RESULTS
Dependence on [Call f o r IK current The delayed rectifier K + current (IK)was activated with an increase in cellular Ca z+ concentration ([Cal0 in A7r5 cells. As shown in Figure 1, the IK current was activated at pCa 7 more than at pCa 10. Thus, the difference is a part of CaZ+-activated K + current (IKc~) (Fig. 1C). The measured capacitance was 12.2-+ 1.3 pF (n=25), and the values were given as a current density at + 70 mV; 10.5 + 2.2 p A / p F (n = 8) at pCa I0; and 27.5 + 2.4 p A / p F (n-- 8) at pCa 7. The current was inhibited by I0 mM tetraethylammonium (TEA) and 200 nM apamin. The current difference (IKc~)in the A7r5 cells was ca. 16.3 pA/pF. The characteristics are consistent with previous reports (Romey and Lazdunski, 1984; Blatz and Magleby, 1986; Lang and Ritchie, 1990; Van Renterghem
,°=., I
+40
I
I
+80 mV
F I G U R E 1. Modulation of the delayed rectifier K + current by change in intracellular Ca z+ concentration in rat aortic vascular smooth muscle (ATrS) c e l l Holding potential was - 40 mV. Teat pulses were applied, with increments of 10 mV, from - 2 0 to + 70 m V for 1-s duration. Nifedipine (1 ldVl) and 4-aminopyridine (4-AP, 3 mM) were added to avoid the influence of other currents. (A) Delayed rectifier K + current (/K) in p e a 10 solution. (13)IK current traces in p e a 7 solution. (C) Subtracted current traces from (A) and 03). The short line at the left of the current records represents the zero current level. (D) Current-voltage relationships in pCa 10 (open circles), pCa 7 (solid circles) solutions, and the differce (IKC,) current (triangles). Cur. rent density values represent mean_+ SEM.
and Lazdunski, 1992). Thus, in the present experiments, [Ca]i buffered by 10 mM EGTA was at pCa 7 to facilitate the current.
Isoprenaline effects on Irm Test pulses for 1 s were applied from - 20 to + 70 mV from a holding potential of - 4 0 mV. In the A7r5 cell line, 1 I~M ISO inhibited the IKca as shown in Figure 2A and 2B. Figure 2D shows the currentvoltage relationships in the absence and presence of 1 I~M ISO. ISO at 1 I/M inhibited IKca(at +70 mV) only by 28.8+3.4% (n--8, P<0.01) and at 5 laM by 46.7-+3.2% (n=8, P<0.001). Addition of 1 I~M 4-1~phorbol-12,13-dibutyrate (PDB) to ISO-containing (1 p.M) solution inhibited IKcaby 18.9-+2.3% (n=6, P<0.01) (Fig. 1C). After 10-rain washout, the current amplitude was recovered to approximately 87% of control.
Carbachol effects on Irc, Carbachol (0.1 I~M) decreased Irca(at + 70 mV) by approximately 20% (Fig. 3A and B). The current-voltage curves are given in Figure 3C. Carbachol at 0.1 IlM inhibited IKC~(at + 70 mV) by 7.4_+2.3% (n=7, P>0.05), and at 1 I~M by 21.0_+3.1% (n = 7, p<0.01). The effects were reversible by about 80-90%.
P K . C stimulation with phorbol eater PDB is a potent stimulator of PK-C among phorbol esters (Satoh and Hashimoto, 1988; Satoh et al., 1992). PDB at 1 I~M decreased IKca(Fig. 4A and B). Current-voltage relationships in both control and PDB (1 ~tM) are shown in Figure 4C. The inhibition was 10.2-+2.7% (n=5, P<0.05) at 0.1 I~M, and 34.4-+2.0% (n=6, P<0.01) at 1 pM PDB. A phorbol ester, 4-et-phorbol-12,13-didecanoate,PDD (an inactive analog of phorbol esters), did not affect IKca. In a fresh aorta VSM cell, PDB at 0.1-1 ~tM inhibited IKC~(at +70
Effects on Ca2+-Activated K + Current
321
pAlpF F I G U R E 2. Effect of isoprena. line o n the Ca z+-activated K + c u r . rent in a cultured A7r5 cell. Test pulses were applied between - 20 a n d + 70 mV from a holding potential of - 40 mV. (A, 13)Current traces in the absence a n d presence of 1 pM isoprenaline (ISO). (C) Current traces in the!presence of ISO (1 ItM) plus PDB {1 pM). T h e short line at the left of the current records represents the Zero current level. (D) Current-Voltage relattonshtp forlK m control (open circles, n = 8), in the presence of 1 jIM ISO (solid circles, n =8), and in 1 pM ISO plus 1 pM PD• (triangles, n =6). T h e values plotted as a current density are mean_+SEM. •
•
•
,
D 30
i?
0 /ISO 1 ,uM
A Control
_J
Control /
20
(n=8~/
6/
(n=8) ~.~•
,e
i
^/
_,~F
/-, _,ll,
B ISO 1 ,tiM
_J
/ #'jIll I~,/+PDB 1 .tiM ,:~/~a .i~l" (n=6)
10
.
C +PDB 1 jiM
.,s',l"
1
/f--~
O
_3g
I
100 ms
mV) by 46.2-65.7% in a concentration-dependent manner (Fig. SAC). The pCa was also 7. In juSt three cells, the amplitude (at +70 mV) was decreased by 39.5+3.8% (P<0.01) at 0.1 I~M, and by 60.4_+3.7% (P<0.001) at I I~M PDB. Addition of ISO (1 ttM) to PDB-containing (1 I~M) solution potentiated to inhibit IKc, by 24.1 _+2.2% (n = 3) (Fig. 5D). After a washout, the current was recovered by approximately 65-75%• A small transient outward current (I~o)in an A-10 cell was observed at + 70 mV, which was 4-AP sensitive (Fig. 6A). Test pulses were applied between - 2 0 and +70 mV of a holding potential of - 4 0 mV. The pCa was 7. The amplitudes were plotted as a current density, and the current-voltage curves are given in Figure 6B. The threshold of I,o was 0 to +10 mV. PDB (1 gM) enhanced I,o at +70 mV by 64.5_+3.9% (n=3, P<0.01). Addition o f i S O (1 ttM) to PDB-containing (1 tiM) solution decreased the enhanced I,o by 68.2_+5.1% (n = 3, P<0.001).
I
I
-20
I
I
I
0
I
i
i
i
+40
i
+80 mV
DISCUSSION Voltage-dependent outward K ÷ current, activated by depolarization, has been described in many excitable cells including VSM cells. The current exhibits at least three types: a delayed rectifier current (IK) (slowly activating and noninactivating) (Van Renterghem eta/., 1988; McFadzean and England, 1992); a transient outward current (I,o)(rapidly activating and inactivating) (Cassel and McLachlan, 1986); and a Ca2+-dependent K + current (IKc,)(Romey and Lazdunski, 1984; Lynch, 1985; Blatz and Magleby, 1986; Lang and Ritchie, 1990; Van Renterghem et al., 1992). The contraction of smooth muscles is associated with calmodulin, a multifunctional Ca2+-binding protein, different from that of cardiac and skeletal muscles (Cheung, 1980; Means and Chafouleas, 1982). Voltage-dependent Ca 2+ channels are a major pathway of C a 2+ entry
C
pA/pF 30
F I G U R E 3 . Depression of the CaZ+-activated K + current in the A7r5 cell line by carbachoL (A, B) Current traces in coatrol a n d in 1 ItM carbachol. Test pulse was applied between - 20 and ÷ 70 mV from a holding p o t e n t i a l of - 40 mV. T h e short line at the left of the current records represents the zero current level. (C) Current-voltage relationship in controi (open circles, n = ?),in 0.1 ~M carbachol (solid circles, n = 7), a n d 1 pM carbachol (triangles, n = 7). Current demity values are represented as mean_+SEM.
A Control ,,
_
B Carbachol 0.1 ,uM
~r
F 'I
20
:
lY#I
1 / 6 ; ~)/~'~Ca r bachol / 0 , 0 x~ 0.1 ~JM(n:7) 10
,ml~-I
I T.._j 100 ms
, I
-20
I
I
0
I
I
'
i
+40
i
i
i
s
+80 mV
322
H. Satoh
across the cell membrane. Also, the outward K + currents could regulate contractility due to alteration of their conductances; that is, repolarization and membrane hyperpolarization (or depolarization). The changes in the K + current can indirectly modulate activation of the Ca 2÷ channels. In the present experiments, the threshold potential for IK was - 10 to --20 mV. The IK was a CaZ+-activated, TEA- and apamin-sensitive current which can coexist in the same cell. These properties are in accord with those reported previously (Romey and Lazdunski, 1984; Blatz and Magleby, 1986; Lang and Ritchie, 1990; Van Renterghem et al., 1992). Therefore, this is confirmed to be a IKca current.
Modulation on Igc, b y i s o p r e n a l i n e I]-Adrenoceptor stimulation accelerates the activation of the voltagedependent channels. The accelerating action is thought to be due to phosphorylation of channel proteins with PK-A (via cAMP synthesized through activation of adenylate cydase) (Seamon et al., 1981), and also to association with the related GTP-binding proteins (Sperelakis, 1990). ISO and forskolin produce smooth muscle relaxation by increasing the intracellular level of cAMP (Volicer and Hynie, 1971; Muller and Baer, 1983). In the present experiments, the application oflSO depressed IKca significantly. This depression may be due to elevation of cAMP. However, Sperelakis and colleagues (Ousterhout and Sperelakis, 1987; Bkaily et al., 1988) have showed that 8-Br-cAMP enhanced IK (but which was not IKc,). The lack of uniformity was also observed in the effects on Ic, (Meisheri and Van Breemen, 1982; Satoh and Sperelakis, 1995; Fukumitsu et al., 1990). The discrepancy might arise from difference of species (including the aging) and experimental environments. In the A7r5 cells, ISO inhibits IK,presumably due to the cAMP production, and may do so by a direct action. PK-A phosphorylates the myosin light-chain kinase (MLCK) which diminishes its Ca 2+ sensitivity, resulting somewhat in contribution to the vasodilation (Sperelakis et al., 1994).
Modulation through m u s c a r / n i c receptors In smooth muscle preparations with endothelium, A C h increases the amount of cGMP in VSM mainly through release of EDRF (Cherry et al., 1982; Furchgott, 1983). And the contraction induced by ACh is due to endothelin released from the endothelium (Furchgott and Zawadzki, 1980). Endothelin would be an endogenous modulator of Ca 2+channels. However, because single A7r5 cultured cells were used in the present experiments, the effects via endothelium would be neglected. Therefore, these results suggest that carbachol alone inhibits IKca current. Similarly, there are no consistent reports for effects of ACh on the IKcurrent in smooth muscle cells. ACh and dibutyryl analogs ofcGMP enhanced IK (Bkaily et al., 1988), and inhibited the action potentials (Ousterhout and Sperelakis, 1987). In frog visceral muscle cells, high ACh concentration increased IK, due to involvement with PK-C activation (Vivaudou et al., 1988). The responses to ACh are closely related to PI turnover. Therefore, the effect induced by carbachol might be related to PK-C stimulation (via DAG) and IP3 accumulation.
Regulation by PK.C stimulation Activation of phospholipase C by appropriate membrane receptors and G-coupling proteins stimulates PI turnover with IP3 and DAG production. DAG activates PK-C to phosphorylate ionic channels. I/)3 acts on the Ca2+-release channels of the SR to release Ca 2+ and elevate the [Ca]i level. Raising {Call produces vasoconstriction. PDB inhibited IKCawhich is consistent with the membrane depolarization by phorbol esters in cultured rat aortic cells (Bkaily et al., 1988; Sperelakis et al., 1994), suggesting IK inhibition. Inhibition was also produced in cardiac muscle cells (Satoh and Hashimoto, 1988; Satoh, 1995). Additional application of PDB in the presence of ISO increased the depressant effect induced by ISO on the IKCacurrent, which is consistent with our recent reports (Satoh and Hashimoto, 1988; Satoh
pAIpF C lS +70 mV
A Control
10
Control (~) / (n=8)//
O/
B PDB 1 ,uM
1100 100 ms
pA
-20
0
+40
+80 mV
F I G U R E 4. Inhibition by phorbol ester on the Ca 2+-actlvated K + current in the A7r5 cell. (A, ]3) Current traces in the absence and presence of I pM PDB (4-[I-phor. bob12,13-dibutyrate). T h e holding potential was - 40 mV. Test pulse was applied to + 20 to + 70 mV. T h e short line at the left of the current records represents the zero current level. (C) Currentvoltage curve s in control (open circles, n = 8) a n d in 1 ltM PDB (solid circles, n = 6). T h e values are represented as mean_+ SEM.
Effects on Ca2÷-Activated K + Current
Control
323
I KCa Fresh Aorta cell
A
+70 mV
:401 le !
Control
....
_J 1oo pA 100 ms
PDB 0.1 ~JM
B pAIpF 40
B
ITO
PDB 1 ,tiM/~ (n=3) /V Control (n=7
].
1 juM
30
C +ISO 1 ).iM
20
A-IO cells pCa7
:
D m
1°1"
et al., 1992; Satoh, 1994; Satoh and Sperelakis, 1995). Sugden et al. (1985) reported that addition of phorbol esters to ISO potentiated the cAMP accumulation. The presence of both drugs might modulate membrane phospholipids and Ca 2~ transiently to activate and stabilize the enzyme in association with the membrane. PK-C activation may result in the rapid phosphorylation of a component of the system synthesizing cAMP (B-adrenoceptor, GTP-binding proteins, and adenylate cyclase catalytic subunit) or degrading cAMP (phosphodiesterase). PK-A has been reported to inhibit PL-C, and thereby decrease the production of IP3 and D A G and thus diminish its Ca z+ sensitivity (Sperelakis et al., 1994). The Itois one of the currents to play an important role in repolarization in a wide variety of excitable cells. The Ito current found in aortic cells has presented some similaritie# to the IAcurrent reported in other tissues such as the cardiomyocyte (Satoh and Sperelakis, 1994), Drosophila flight muscle (Salkoff and W~man, 1981), and neurons (Bader et al., 1985). In this study, PDB markedly enhanced/to. However, activation of PK-C suppresses I~o,leadir~g to increased action potential duration in cardiac myocytes (Apkon and Nerbonne, 1988; Fedida et al., 1990). The discrepancy might also :come from different environments and species, like other currents (Di-oogmans et al., 1987; Pacaud et al., 1987; Benham and Tsien, 1988).
-20
Z~/(,.)
I
I
/ /
F I G U R E 5. Effects of phorbol ester on the CaZ+-activated K + current in a fresh aortic cell 0frat. Test pulses were applied between - 20 and + 70 m V from a holding potential of - 40 mV. The pCa was 7. T h e short line at the left of the current records represents the zero current level. (A) Ctlrrent traces in control. (B, C) Current traces in 4-1~-phorbol-12,13-dibutyrate (PDB). PDB (0.1 and 1 pM) was cumulatively administered to the bath solution. (D) Effects of addition of 1 pM isoprenali/ae (ISO) in the presence of I pM PDB.
]
A'~.w"PDB 1 .ul~l --'-O + ISO 1 ,uMI 0
+40
+80 mV
FIGURE 6. Enhancement of the transient outward current by phorbol ester in the A-10 cell. T h e p e a was 7. (A) Current traces in control and in 1 pM 4-1~-phorbol-12,I3-dibutyrate (PDB). Test pulses were applied to + 70 m V from a holding potential of - 40 mV. T h e short line at the left of the current records represents the zero current level. (B) Current-voltage relationship for the transient outward current (/to) in the absence (open circles, n = 7) and presence of I ~ 4 PDB (triangles, n = 3), and in PDB (1 plus ISO-containing ( 1 ItM) solution (solid circles, n = 3). The values plotted as current density are mean_+SEM.
Phorbol esters have already been described to have a biphasic effect: an initial stimulation and subsequent reduction, which may generate the nonuniform actions on the ionic currents. Other possibilitiesinclude: ( I ) the possibility that these actions may result from a negative feedback control (Nishizuka, 1988; Mond et al., 1991); (2) the basal PK-C activity in the cultured cells may have already been at its maximum before application ofphorbol ester, resulting in no effect of added phorbol ester; (3) because our experiments were carried out only at room temperature (22°C), the effect of phorbol ester may have been masked, due to the fact that regulation of ionic channels by protein kinase is temperaturedependent (Walsh and Kass, 1988); and (4) PK-C effects may be indirect, and may not correspond to a direct phosphorytation of the ionic channel protein itself (Marks et al., 1990). CONCLUSIONS The modulation of IKc, current was examined in the A7r5 (or A-10) cell line. All drugs used in this study caused an inhibitory effect on Irca current. There are differences between the effects on ionic currents,
324 but the explanation of this variability is still not clear. T h e inhibition, by PK-A and PK-G mediated t h r o u g h 15-adrenoceptor a n d muscarinic receptors and by PK-C, would modulate t h e relaxation or contraction of V S M cells. However, an understanding of the factors which influence these changes may provide important clues as to how mechanisms which regulate excitability work in vivo. To elucidate the very complex mechanism for relaxation (contraction) of V S M cells, further experiments are required.
References Apkon M., and Nerbonne J. M. (1988) Ctl-Adrenergic agonists selectively suppress voltage-dependent K + currents in rat ventricular myocytes. Proc. Natl. Acad. Sci. USA 85, 8756-8760. Bader C. R., Bertrand D., and Dupin E. (1985) Voltage-dependent potassium currents in developing neurones from quail mesencephalic neural crest. ]. Physiol. 366, 129-151. Benham C. D., and Tsien R. W. (1988) Noradrenaline modulation of calcium channels in single smooth muscle ceils from rabbit ear artery. J. Physiol. 404, 767-784. Bkaily G., Preyrow M., Yamamoto T., Sculptoreanu A., Jacques D., and Sperelakis N. (1988) Macroscopic Ca 2+, Na + and K + currents in single heart and aortic cells. Mol. Cell. Biochem. 80, 59-72. Blatz A. L. and Marleby K. L. (1986) Single apamin-blocked Ca-activated K + channels of small conductance in cukured rat skeletal muscle. Nature 323, 718-720. Cassel J. F. and McLachlan E. M. (1986) The effect of a transient outward current (IA) on synaptic potentials in sympathetic ganglion cells of guinea-pig. J. Physiol. 474, 273-288. Cherry P. D., Furchgott R. F., Zawadzki J. V., and Jothianandan D. (1982) Role of endothelial cells in relaxation of isolated arteries by bradykinin. Proc. Natl. Acad. Sci. USA 79, 2106-2110. Cheung, W. Y. (1980) Calmodulin plays a pivotal role in cellular regulation. Science 207, 19-27. Droogmans G., Declerck I., and Casteels R. J. (1987) Effect of adrenergic agonists on Ca++-channel currents in single vascular smooth muscle cells. Pflagers Arch. 409, 7-12. Fabiato A. and Fabiato F. (1979) Calcium programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J. Physiol. 75,463-505. Fedida D., Shimoni Y., and Giles W. R. (1990) Qt-Adrenergic modulation of the transient outward current in rabbit atrial myocytes. J. Physiol. 423, 257277. Fukumitsu T., Hayashi H., Tokuno H., and Tomita T. (1990) Increase in calcium channel current by 13-adrenoceptor agonists in single smooth muscle cells isolated from porcine coronary artery. Br. J. Pharmacol. 100, 593-599. Furchgott R. F. (1983) Role of endothelium in responses of vascular smooth muscle. Circ. Res. 53, 557-573. Furchgott R. F. and Zawadzki J. V. (1980) The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288, 373-376. Galizzi J. P., Qar J., Fosset M., Van Renterghen C., and Lazdunski M. (1987) Regulation of calcium channels in aortic muscle cells by protein kinase C activators (diacylglycerine and phorbol esters) and by peptides (vasopressin and bombesin) that stimulate phosphoinositide breakdown. J. Biol. Chem. 262, 6947-6950. Lang D. G. and Ritchie A. K. (1990) Tetraethylammonium blockade of apaminsensitive and insensitive Cag+-activated K + channels in a pituitary cell line. J. Physiol. 425, 117-132. Lynch C. (1985) Ionic conductances in frog short skeletal muscle fibers with slow delayed rectifier currents. J. Physiol. 368, 358-378. Marks T. N., Dubyak G. R., and Jones S. W. (1990) Calcium currents in the A7r5 smooth muscle-derived cell line. Pflagers Arch. 417, 433-439. McFadzean I. and England S. (1992) Properties of the inactivating outward current in single smooth muscle ceils isolated from the rat anococcygeus. Pflfigers Arch. 421, 117-124. Means A. R. and Chafouleas J. G. (1982) Calmodulin in endocrine cells. Annu. Rev. Physiol. 44, 667-682. Meisheri K. D. and Van Breemen C. (1982) Effects of 13-adrenergic stimulation on calcium movements in rabbit aortic smooth muscle: relationship with cyclic AMP. 1. Physiol. 331, 429-441. Mond J. J., Feuerstein N., June C. H., Balapure A. K., Glazer R. I., Witherspoon
H. S a t o h K., and Brunswick M. (1991) Bimodal effect of phorbol ester on 13cell activation. J. Biol. Chem. 266, 4458--4463. Muller M. J. and Baer H. P. (1983) Relaxant effects of forskolin in smooth muscle. Role of cyclic AMP. Naunyn-Schmiedeberg's Arch. Pharmac. 322, 78-82. Nishizuka Y. (1988) The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature 334, 661-665. Ousterhout J. M. and Sperelakis N. (1987) Cyclic nucleotides depress action potentials in cultured aortic smooth muscle cells. Eur. ]. Pharmac. 144, 714. Pacaud P., Loirand G., Mironneau C., and Mironneau J. (1987) Opposing effects of noradrenaline on the two classes of voltage-dependent calcium channels of single vascular smooth muscle cells in short-term primary culture. P~higers Arch. 410, 507-509. Rasmussen H. and Barrett P. Q. (1984) Calcium messenger system: an integrated view. Physiol. Rev. 64, 938-984. Romey G. and Lazdunski M. (1984) The coexistence in rat muscle cells of two distinct classes of CaZ+-dependent K + channels with different pharmacological properties and different physiological functions. Biochem. Biophys. Res. Commun. 118, 669-674. Salkoff L. and Wyman R. (1981) Outward currents in developing Drosophila flight muscle. Sc/ence 212, 461-463. Satoh H. (1992) Inhibition in L-type Ca 2+ channel by stimulation of protein kinase C in isolated guinea-pig ventricular cardiomyocytes. Gen. Pharmac. 23, 1097-1102. Satoh H. (1994) Elevation of intracdlular Ca z+ concentration by protein kinase C stimulation in isolated single rabbit sino-atrial node. cells. Gen. Pharmac. 25, 325-332. Satoh H. (1995) Depression of the spontaneous activity by phorbol esters in young embryonic chick cardiomyocytes. ]pn. ]. Pharmac. 67,297-304. Satoh H. and Hashimoto K. (1988) On electrophysiological responses to phorbol esters which stimulate protein kinase C in rabbit sino-atrial node cells. Naunyn-Schmiedeberg's Arch. Pharmacol. 337, 308-315. Satoh H., and Sperelakis N. (1991) Calcium and potassium currents in cultured rat aortic vascular smooth cell lines. In. Ionic Channels of Vascular Smooth Musde Cells and Endothelial Cells (edited by Sperelakis N. and Kuriyama H.), pp. 55-64. Elsevier, New York. Satoh H. and Sperelakis N. (1994) Identification of and developmental changes in transient outward current in embryonic chick cardiomyocytes. J. Devel. Physiol 20, 149-154. Satoh H. and Sperelakis N. (1995) Modulation of L-type Ca z+ cur rent by isoprenaline, carbachol and phorbol ester in cultured rat aortic vascular smooth muscle (A7r5) cells. Gen. Pharmacol. 26, 369-379. Satoh H., Nakashima K., and Tsuchida K. (1992) The responses to phorbol esters which stimulated protein kinase C in canine Purkinje fibers. Gen. Pharmac. 23, 847-852. Seamon K., Padgett W., and Daly J. W. (1981) Forskolin: unique diterpene activator of adenylate cyclase in membranes and in intact cells. Proc. Natl. Acad. Sci. USA 78, 3363-3367. Sperelakis N. (1990) Properties of calcium channels in cardiac muscle and vascular smooth muscle. Proceedings of a conference on membranes. Mol. Cell. BIOchem. 99, 97-109. Sperelakis N., Tohse N., Ohya Y., and Masuda H. (1994) Cyclic GMP regulation of calcium slow channels in cardiac muscle and vascular smooth muscle calls. Adv. Pharmac. 26, 217-252. Sugden D., Vanecek J., Klein D. C., Thomas T. P., and Anderson W. B. (1985) Activation of protein kinase C potentiates isoprenaline-induced cyclic AMP accumulation in rat pinealocytes. Nature 314, 359-361. Tsien R. Y., and Rink T. J. (1980) Neutral carrier ion-selective microelectrodes for measurements of intracellular free calcium. Biochem. Biophys. Acta 599, 623-638. Van Renterghem C., and Lazdunski M. (1992) A small-conductance charybdotoxin-sensitive, apamin-resistant Ca2+-activated K + channel in aortic smooth muscle cells (line A7r5 line and primary culture). Pflagers Arch. 420, 417-423. Van Renterghem C., Romey G., and Lazdunski M. (1988)Vasopressin modulates the spontaneous electrical activity in aortic cells (line A7r5) by acting on three different types of ionic channels. Pr0c. Natl. Acad. Sci. USA 85, 93659369. Vivaudou M. B., Clapp L. H., Walsh J. V., and Singer J. J. (1988) Regulation of one type of Ca z+ current in smooth muscle cells by diacylglycerol and acetylcholine. FASEB J. 2, 2497-2504. Volicer L. and Hynie S. (1971) Effect of catecholamines and angiotensin on cyclic AMP in rat aorta and tail artery. Fur. J. Immunol. 15, 214-220. Walsh K. B. and Kass R. S. (1988) Regulation of a heart potassium channel by protein kinase A and C. Science 242, 67-69.
Gen. Pharmac. Vol. 27, No. 2, pp. 319-324, 1996 Copyright © 1996 Elsevier Science Inc. Printed in the USA. ELSEVIER
Modulation of Ca2+-Activated K ÷ Current by Isoprenaline, Carbachol, and Phorbol Ester in Cultured (and Fresh) Rat Aortic Vascular Smooth Muscle Cells Hiroyasu Satoh DEPARTMENTOF PHARMACOLOGY,N^gA MEDICALUNIWRS~, KASmHAR*,NAP.A634, JAPAN ABSTRACT~ 1. Effects ofisoprenaline (ISO), carbachol, and phorbol ester on the outward K + currents in single cultured (or fresh) rat aortic vascular smooth muscle (A7r5 and A. 10) cells were examined using a whole-ceU voltage-clamp (at room temperature 22°C). 2. W i t h 10 m M E G T A in the pipette solution, the delayed rectifier K + current (It,) was activated by Ca 2+ at pCa 7 more than at pCa 10, a n d was T E A (10 mM) and apamin (200 nM) sensitive, which represents a CaZ+-activated K + current (IKc,). 3. I n cultured A7r5 cells, isoprenaline (1 and 5 LaM) and carbachol (0.1 and 1 lab0 inhibited IKc,. Phorbol ester, 4-~-phorbol-12, 13-dibutyrate (PDB), at 0.1 a n d 1 laM also inhibited IKc~, a n d increased the inhibitory effects induced by isoprenaline (1 laM). 4. In fresh aortic cells, these drugs, at the same concentrations, also produced the similar effects. 5. In A-10 cells, PDB (1 ~M) enhanced the transient outward current (4-AP.sensitive), but ISO (1 laM) inhibited the current. 6. These results suggest that the IKe., current would be inhibited by cyclic nucleotides (cAMP a n d cGMP) and also by PK.C stimulation, and thereby be directly contributed to excitation-contraction coupling of the vascular smooth muscle cells. GEN PHA~AC 27;2:319-324, 1996. I
KEY WORDS. Isoprenaline, carbachol, phorbol esters, Ca2+-activated K + current, aortic vascular smooth muscle ceils INTRODUCTION The physiological functions of vascular smooth muscle (VSM) are affected by phosphorylation with cAMP-dependent and cGMP-dependent protein kinases (PK-A and PK-G), and with protein kinase C (PK-C). The effects of these kinases on cytosolic free Ca 2+ concentration ([Cali) may be one key regulator of vascular tone. The cAMP accumulation is associated with vascular relaxation through ~-adrenoceptor stimulation by catecholamines, due mainly to an inhibition of Ca 2+ current (Satoh 1992, S atoh and Sperelakis 1991, 1995) and a depolarization of the membrane (Ousterhout and Sperelakis, 1987). Also, PK-A and PK-G enhance the delayed rectifier K + current (IK)(Bkaily et at., 1988). Both cAMP and cGMP elevations have been implicated in the relaxation of VSM cells in response to some vasodilators, such as endothelium-derived releasing factor (EDRF), atrial natriuretic factor (ANF), and nitroprusside. ACh, through muscarinic receptors, can stimulate phosphatidyl inositol (PI) turnover and produce inositol 1,4,5triphosphate (IP3) and diacylglycerol (DAG) for stimulation of PK-C (Rasmussen and Barrett, 1984). PK-C stimulates the contraction of VSM cells. Therefore, A C h might contract the VSM cells. Thus, the contraction of VSM cells is regulated by cyclic nucleotides and PK-C stimulation. However, the effects of the protein kinases on the ionic currents of VSM cells are still controversial. Therefore, this article focuses on the Ca2+-activated current (Ikca)in cultured (A7r5 and A-10) and fresh rat aortic VSM cells, and the effects of isoprenaline (ISO), carbachol, and phorbol ester (for stimulation of PK-C) on the whole-cell IKcacurrent were examined. Since the IK current is reported to be Ca2+-activated, [Ca]~ level in this study was buffered at pCa 7 to amplify the current amplitude, according to calculations by Fabiato and Fabiato (1979) and Received 10 April 1995.
Tsien and Rink (1980). Effects on the transient outward current (Ito) were also examined. A preliminary report of a part of this work was given recently (Satoh and Sperelakis, 1991). MATERIALS AND METHODS
Cell culture a n d preparation Stable VSM cell lines (A7r5 and A10), derived from rat aorta, were purchased from the American Tissue Type Culture Collection (ATCC, Bethesda, MD). These cell lines present many characteristics of adult aortic VSM cells, including a well-developedrough endoplasmic reticulum, dispersed contractile filaments, and muscle-type creatine phosphokinase isozyme, and were prepared as described previously (Satoh and Sperelakis, 1995). Fresh aortic VSM cells were obtained from adult rats. Dissected aortas were incubated by collagenase and/or tripsin for 15-20 min. The ceils were transferred to glass coverslips (placed in 35-mm Petri dishes) and bathed in tissue culture medium (Gibco, Grand Island, NY) containing 10% fetal calf serum. The cells were incubated at 37°C in a humidified atmosphere under 5% CO2 and 95% air. The cells were used for experiments after maintaining for 1 or 2 days.
Electrical measurements The membrane currents were measured by a whole-ceU voltage-clamp technique. The currents were amplified with an Axopatch patch-clamp amplifier (Axon Instruments, Burlingame, CA), and the data were stored and analyzed on an IBM-AT microcomputer using the PCLAMP analysis program. Test pulses were applied once every 20 s. Current traces were filtered at a cutoff frequency of 2 kHz for plotting. The amplitude of the inward current was measured at the peak of each current record. All experiments were carried out at room temperature (22°C).
320
H. Satoh
D
+70 mV -40 L
pAIpF
]
30
ls
A pCa 10 20
B pCa 7
A.A.A-A" ~
--
"
" I
III I
qllH
I•
i
F
l/zx"
10
C
.----
B-A
II ~ . A
~.~)
-20
0
_..] 100 pA 0 100 ms
.,r I
Experimental solutions The composition of modified Tyrode solution used was (in raM): NaC1 110, KC15.4, CaCI2 20, MgC12 1, NaH2PO4 0.3, glucose 5, and HEPES 5. The pH was adjusted to pH 7.4. Nifedipine I IxM (Sigma Chemical Co.) was also added to block Ic, current. The pipette solution was composed of (mM): KOH 110, KCI 20, MgC12 1, Mg-ATP 5, EGTA 10, creatine phosphate 5, aspartic acid 100, and HEPES 5 (adjusted to pH 7.2). Drugs (Sigma) used were: L-isoprenaline, carbachol, and 4-[~-phorbol12,13-dibutyrate (PDB). PDB was dissolved with dimethyl sulphoxide (DMSO) at 10 mM and was stocked at - 10°C. Tetraethylammonium (TEA) and apamin (Sigma) were also used.
Statistical analysis The values represent mean_+ SEM. The differences of the mean values were analyzed by the Student t test for paired data, and a P value of less than 0.05 was considered significant. RESULTS
Dependence on [Call f o r IK current The delayed rectifier K + current (IK)was activated with an increase in cellular Ca z+ concentration ([Cal0 in A7r5 cells. As shown in Figure 1, the IK current was activated at pCa 7 more than at pCa 10. Thus, the difference is a part of CaZ+-activated K + current (IKc~) (Fig. 1C). The measured capacitance was 12.2-+ 1.3 pF (n=25), and the values were given as a current density at + 70 mV; 10.5 + 2.2 p A / p F (n = 8) at pCa I0; and 27.5 + 2.4 p A / p F (n-- 8) at pCa 7. The current was inhibited by I0 mM tetraethylammonium (TEA) and 200 nM apamin. The current difference (IKc~)in the A7r5 cells was ca. 16.3 pA/pF. The characteristics are consistent with previous reports (Romey and Lazdunski, 1984; Blatz and Magleby, 1986; Lang and Ritchie, 1990; Van Renterghem
,°=., I
+40
I
I
+80 mV
F I G U R E 1. Modulation of the delayed rectifier K + current by change in intracellular Ca z+ concentration in rat aortic vascular smooth muscle (ATrS) c e l l Holding potential was - 40 mV. Teat pulses were applied, with increments of 10 mV, from - 2 0 to + 70 m V for 1-s duration. Nifedipine (1 ldVl) and 4-aminopyridine (4-AP, 3 mM) were added to avoid the influence of other currents. (A) Delayed rectifier K + current (/K) in p e a 10 solution. (13)IK current traces in p e a 7 solution. (C) Subtracted current traces from (A) and 03). The short line at the left of the current records represents the zero current level. (D) Current-voltage relationships in pCa 10 (open circles), pCa 7 (solid circles) solutions, and the differce (IKC,) current (triangles). Cur. rent density values represent mean_+ SEM.
and Lazdunski, 1992). Thus, in the present experiments, [Ca]i buffered by 10 mM EGTA was at pCa 7 to facilitate the current.
Isoprenaline effects on Irm Test pulses for 1 s were applied from - 20 to + 70 mV from a holding potential of - 4 0 mV. In the A7r5 cell line, 1 I~M ISO inhibited the IKca as shown in Figure 2A and 2B. Figure 2D shows the currentvoltage relationships in the absence and presence of 1 I~M ISO. ISO at 1 I/M inhibited IKca(at +70 mV) only by 28.8+3.4% (n--8, P<0.01) and at 5 laM by 46.7-+3.2% (n=8, P<0.001). Addition of 1 I~M 4-1~phorbol-12,13-dibutyrate (PDB) to ISO-containing (1 p.M) solution inhibited IKcaby 18.9-+2.3% (n=6, P<0.01) (Fig. 1C). After 10-rain washout, the current amplitude was recovered to approximately 87% of control.
Carbachol effects on Irc, Carbachol (0.1 I~M) decreased Irca(at + 70 mV) by approximately 20% (Fig. 3A and B). The current-voltage curves are given in Figure 3C. Carbachol at 0.1 IlM inhibited IKC~(at + 70 mV) by 7.4_+2.3% (n=7, P>0.05), and at 1 I~M by 21.0_+3.1% (n = 7, p<0.01). The effects were reversible by about 80-90%.
P K . C stimulation with phorbol eater PDB is a potent stimulator of PK-C among phorbol esters (Satoh and Hashimoto, 1988; Satoh et al., 1992). PDB at 1 I~M decreased IKca(Fig. 4A and B). Current-voltage relationships in both control and PDB (1 ~tM) are shown in Figure 4C. The inhibition was 10.2-+2.7% (n=5, P<0.05) at 0.1 I~M, and 34.4-+2.0% (n=6, P<0.01) at 1 pM PDB. A phorbol ester, 4-et-phorbol-12,13-didecanoate,PDD (an inactive analog of phorbol esters), did not affect IKca. In a fresh aorta VSM cell, PDB at 0.1-1 ~tM inhibited IKC~(at +70
Effects on Ca2+-Activated K + Current
321
pAlpF F I G U R E 2. Effect of isoprena. line o n the Ca z+-activated K + c u r . rent in a cultured A7r5 cell. Test pulses were applied between - 20 a n d + 70 mV from a holding potential of - 40 mV. (A, 13)Current traces in the absence a n d presence of 1 pM isoprenaline (ISO). (C) Current traces in the!presence of ISO (1 ItM) plus PDB {1 pM). T h e short line at the left of the current records represents the Zero current level. (D) Current-Voltage relattonshtp forlK m control (open circles, n = 8), in the presence of 1 jIM ISO (solid circles, n =8), and in 1 pM ISO plus 1 pM PD• (triangles, n =6). T h e values plotted as a current density are mean_+SEM. •
•
•
,
D 30
i?
0 /ISO 1 ,uM
A Control
_J
Control /
20
(n=8~/
6/
(n=8) ~.~•
,e
i
^/
_,~F
/-, _,ll,
B ISO 1 ,tiM
_J
/ #'jIll I~,/+PDB 1 .tiM ,:~/~a .i~l" (n=6)
10
.
C +PDB 1 jiM
.,s',l"
1
/f--~
O
_3g
I
100 ms
mV) by 46.2-65.7% in a concentration-dependent manner (Fig. SAC). The pCa was also 7. In juSt three cells, the amplitude (at +70 mV) was decreased by 39.5+3.8% (P<0.01) at 0.1 I~M, and by 60.4_+3.7% (P<0.001) at I I~M PDB. Addition of ISO (1 ttM) to PDB-containing (1 I~M) solution potentiated to inhibit IKc, by 24.1 _+2.2% (n = 3) (Fig. 5D). After a washout, the current was recovered by approximately 65-75%• A small transient outward current (I~o)in an A-10 cell was observed at + 70 mV, which was 4-AP sensitive (Fig. 6A). Test pulses were applied between - 2 0 and +70 mV of a holding potential of - 4 0 mV. The pCa was 7. The amplitudes were plotted as a current density, and the current-voltage curves are given in Figure 6B. The threshold of I,o was 0 to +10 mV. PDB (1 gM) enhanced I,o at +70 mV by 64.5_+3.9% (n=3, P<0.01). Addition o f i S O (1 ttM) to PDB-containing (1 tiM) solution decreased the enhanced I,o by 68.2_+5.1% (n = 3, P<0.001).
I
I
-20
I
I
I
0
I
i
i
i
+40
i
+80 mV
DISCUSSION Voltage-dependent outward K ÷ current, activated by depolarization, has been described in many excitable cells including VSM cells. The current exhibits at least three types: a delayed rectifier current (IK) (slowly activating and noninactivating) (Van Renterghem eta/., 1988; McFadzean and England, 1992); a transient outward current (I,o)(rapidly activating and inactivating) (Cassel and McLachlan, 1986); and a Ca2+-dependent K + current (IKc,)(Romey and Lazdunski, 1984; Lynch, 1985; Blatz and Magleby, 1986; Lang and Ritchie, 1990; Van Renterghem et al., 1992). The contraction of smooth muscles is associated with calmodulin, a multifunctional Ca2+-binding protein, different from that of cardiac and skeletal muscles (Cheung, 1980; Means and Chafouleas, 1982). Voltage-dependent Ca 2+ channels are a major pathway of C a 2+ entry
C
pA/pF 30
F I G U R E 3 . Depression of the CaZ+-activated K + current in the A7r5 cell line by carbachoL (A, B) Current traces in coatrol a n d in 1 ItM carbachol. Test pulse was applied between - 20 and ÷ 70 mV from a holding p o t e n t i a l of - 40 mV. T h e short line at the left of the current records represents the zero current level. (C) Current-voltage relationship in controi (open circles, n = ?),in 0.1 ~M carbachol (solid circles, n = 7), a n d 1 pM carbachol (triangles, n = 7). Current demity values are represented as mean_+SEM.
A Control ,,
_
B Carbachol 0.1 ,uM
~r
F 'I
20
:
lY#I
1 / 6 ; ~)/~'~Ca r bachol / 0 , 0 x~ 0.1 ~JM(n:7) 10
,ml~-I
I T.._j 100 ms
, I
-20
I
I
0
I
I
'
i
+40
i
i
i
s
+80 mV
322
H. Satoh
across the cell membrane. Also, the outward K + currents could regulate contractility due to alteration of their conductances; that is, repolarization and membrane hyperpolarization (or depolarization). The changes in the K + current can indirectly modulate activation of the Ca 2÷ channels. In the present experiments, the threshold potential for IK was - 10 to --20 mV. The IK was a CaZ+-activated, TEA- and apamin-sensitive current which can coexist in the same cell. These properties are in accord with those reported previously (Romey and Lazdunski, 1984; Blatz and Magleby, 1986; Lang and Ritchie, 1990; Van Renterghem et al., 1992). Therefore, this is confirmed to be a IKca current.
Modulation on Igc, b y i s o p r e n a l i n e I]-Adrenoceptor stimulation accelerates the activation of the voltagedependent channels. The accelerating action is thought to be due to phosphorylation of channel proteins with PK-A (via cAMP synthesized through activation of adenylate cydase) (Seamon et al., 1981), and also to association with the related GTP-binding proteins (Sperelakis, 1990). ISO and forskolin produce smooth muscle relaxation by increasing the intracellular level of cAMP (Volicer and Hynie, 1971; Muller and Baer, 1983). In the present experiments, the application oflSO depressed IKca significantly. This depression may be due to elevation of cAMP. However, Sperelakis and colleagues (Ousterhout and Sperelakis, 1987; Bkaily et al., 1988) have showed that 8-Br-cAMP enhanced IK (but which was not IKc,). The lack of uniformity was also observed in the effects on Ic, (Meisheri and Van Breemen, 1982; Satoh and Sperelakis, 1995; Fukumitsu et al., 1990). The discrepancy might arise from difference of species (including the aging) and experimental environments. In the A7r5 cells, ISO inhibits IK,presumably due to the cAMP production, and may do so by a direct action. PK-A phosphorylates the myosin light-chain kinase (MLCK) which diminishes its Ca 2+ sensitivity, resulting somewhat in contribution to the vasodilation (Sperelakis et al., 1994).
Modulation through m u s c a r / n i c receptors In smooth muscle preparations with endothelium, A C h increases the amount of cGMP in VSM mainly through release of EDRF (Cherry et al., 1982; Furchgott, 1983). And the contraction induced by ACh is due to endothelin released from the endothelium (Furchgott and Zawadzki, 1980). Endothelin would be an endogenous modulator of Ca 2+channels. However, because single A7r5 cultured cells were used in the present experiments, the effects via endothelium would be neglected. Therefore, these results suggest that carbachol alone inhibits IKca current. Similarly, there are no consistent reports for effects of ACh on the IKcurrent in smooth muscle cells. ACh and dibutyryl analogs ofcGMP enhanced IK (Bkaily et al., 1988), and inhibited the action potentials (Ousterhout and Sperelakis, 1987). In frog visceral muscle cells, high ACh concentration increased IK, due to involvement with PK-C activation (Vivaudou et al., 1988). The responses to ACh are closely related to PI turnover. Therefore, the effect induced by carbachol might be related to PK-C stimulation (via DAG) and IP3 accumulation.
Regulation by PK.C stimulation Activation of phospholipase C by appropriate membrane receptors and G-coupling proteins stimulates PI turnover with IP3 and DAG production. DAG activates PK-C to phosphorylate ionic channels. I/)3 acts on the Ca2+-release channels of the SR to release Ca 2+ and elevate the [Ca]i level. Raising {Call produces vasoconstriction. PDB inhibited IKCawhich is consistent with the membrane depolarization by phorbol esters in cultured rat aortic cells (Bkaily et al., 1988; Sperelakis et al., 1994), suggesting IK inhibition. Inhibition was also produced in cardiac muscle cells (Satoh and Hashimoto, 1988; Satoh, 1995). Additional application of PDB in the presence of ISO increased the depressant effect induced by ISO on the IKCacurrent, which is consistent with our recent reports (Satoh and Hashimoto, 1988; Satoh
pAIpF C lS +70 mV
A Control
10
Control (~) / (n=8)//
O/
B PDB 1 ,uM
1100 100 ms
pA
-20
0
+40
+80 mV
F I G U R E 4. Inhibition by phorbol ester on the Ca 2+-actlvated K + current in the A7r5 cell. (A, ]3) Current traces in the absence and presence of I pM PDB (4-[I-phor. bob12,13-dibutyrate). T h e holding potential was - 40 mV. Test pulse was applied to + 20 to + 70 mV. T h e short line at the left of the current records represents the zero current level. (C) Currentvoltage curve s in control (open circles, n = 8) a n d in 1 ltM PDB (solid circles, n = 6). T h e values are represented as mean_+ SEM.
Effects on Ca2÷-Activated K + Current
Control
323
I KCa Fresh Aorta cell
A
+70 mV
:401 le !
Control
....
_J 1oo pA 100 ms
PDB 0.1 ~JM
B pAIpF 40
B
ITO
PDB 1 ,tiM/~ (n=3) /V Control (n=7
].
1 juM
30
C +ISO 1 ).iM
20
A-IO cells pCa7
:
D m
1°1"
et al., 1992; Satoh, 1994; Satoh and Sperelakis, 1995). Sugden et al. (1985) reported that addition of phorbol esters to ISO potentiated the cAMP accumulation. The presence of both drugs might modulate membrane phospholipids and Ca 2~ transiently to activate and stabilize the enzyme in association with the membrane. PK-C activation may result in the rapid phosphorylation of a component of the system synthesizing cAMP (B-adrenoceptor, GTP-binding proteins, and adenylate cyclase catalytic subunit) or degrading cAMP (phosphodiesterase). PK-A has been reported to inhibit PL-C, and thereby decrease the production of IP3 and D A G and thus diminish its Ca z+ sensitivity (Sperelakis et al., 1994). The Itois one of the currents to play an important role in repolarization in a wide variety of excitable cells. The Ito current found in aortic cells has presented some similaritie# to the IAcurrent reported in other tissues such as the cardiomyocyte (Satoh and Sperelakis, 1994), Drosophila flight muscle (Salkoff and W~man, 1981), and neurons (Bader et al., 1985). In this study, PDB markedly enhanced/to. However, activation of PK-C suppresses I~o,leadir~g to increased action potential duration in cardiac myocytes (Apkon and Nerbonne, 1988; Fedida et al., 1990). The discrepancy might also :come from different environments and species, like other currents (Di-oogmans et al., 1987; Pacaud et al., 1987; Benham and Tsien, 1988).
-20
Z~/(,.)
I
I
/ /
F I G U R E 5. Effects of phorbol ester on the CaZ+-activated K + current in a fresh aortic cell 0frat. Test pulses were applied between - 20 and + 70 m V from a holding potential of - 40 mV. The pCa was 7. T h e short line at the left of the current records represents the zero current level. (A) Ctlrrent traces in control. (B, C) Current traces in 4-1~-phorbol-12,13-dibutyrate (PDB). PDB (0.1 and 1 pM) was cumulatively administered to the bath solution. (D) Effects of addition of 1 pM isoprenali/ae (ISO) in the presence of I pM PDB.
]
A'~.w"PDB 1 .ul~l --'-O + ISO 1 ,uMI 0
+40
+80 mV
FIGURE 6. Enhancement of the transient outward current by phorbol ester in the A-10 cell. T h e p e a was 7. (A) Current traces in control and in 1 pM 4-1~-phorbol-12,I3-dibutyrate (PDB). Test pulses were applied to + 70 m V from a holding potential of - 40 mV. T h e short line at the left of the current records represents the zero current level. (B) Current-voltage relationship for the transient outward current (/to) in the absence (open circles, n = 7) and presence of I ~ 4 PDB (triangles, n = 3), and in PDB (1 plus ISO-containing ( 1 ItM) solution (solid circles, n = 3). The values plotted as current density are mean_+SEM.
Phorbol esters have already been described to have a biphasic effect: an initial stimulation and subsequent reduction, which may generate the nonuniform actions on the ionic currents. Other possibilitiesinclude: ( I ) the possibility that these actions may result from a negative feedback control (Nishizuka, 1988; Mond et al., 1991); (2) the basal PK-C activity in the cultured cells may have already been at its maximum before application ofphorbol ester, resulting in no effect of added phorbol ester; (3) because our experiments were carried out only at room temperature (22°C), the effect of phorbol ester may have been masked, due to the fact that regulation of ionic channels by protein kinase is temperaturedependent (Walsh and Kass, 1988); and (4) PK-C effects may be indirect, and may not correspond to a direct phosphorytation of the ionic channel protein itself (Marks et al., 1990). CONCLUSIONS The modulation of IKc, current was examined in the A7r5 (or A-10) cell line. All drugs used in this study caused an inhibitory effect on Irca current. There are differences between the effects on ionic currents,
324 but the explanation of this variability is still not clear. T h e inhibition, by PK-A and PK-G mediated t h r o u g h 15-adrenoceptor a n d muscarinic receptors and by PK-C, would modulate t h e relaxation or contraction of V S M cells. However, an understanding of the factors which influence these changes may provide important clues as to how mechanisms which regulate excitability work in vivo. To elucidate the very complex mechanism for relaxation (contraction) of V S M cells, further experiments are required.
References Apkon M., and Nerbonne J. M. (1988) Ctl-Adrenergic agonists selectively suppress voltage-dependent K + currents in rat ventricular myocytes. Proc. Natl. Acad. Sci. USA 85, 8756-8760. Bader C. R., Bertrand D., and Dupin E. (1985) Voltage-dependent potassium currents in developing neurones from quail mesencephalic neural crest. ]. Physiol. 366, 129-151. Benham C. D., and Tsien R. W. (1988) Noradrenaline modulation of calcium channels in single smooth muscle ceils from rabbit ear artery. J. Physiol. 404, 767-784. Bkaily G., Preyrow M., Yamamoto T., Sculptoreanu A., Jacques D., and Sperelakis N. (1988) Macroscopic Ca 2+, Na + and K + currents in single heart and aortic cells. Mol. Cell. Biochem. 80, 59-72. Blatz A. L. and Marleby K. L. (1986) Single apamin-blocked Ca-activated K + channels of small conductance in cukured rat skeletal muscle. Nature 323, 718-720. Cassel J. F. and McLachlan E. M. (1986) The effect of a transient outward current (IA) on synaptic potentials in sympathetic ganglion cells of guinea-pig. J. Physiol. 474, 273-288. Cherry P. D., Furchgott R. F., Zawadzki J. V., and Jothianandan D. (1982) Role of endothelial cells in relaxation of isolated arteries by bradykinin. Proc. Natl. Acad. Sci. USA 79, 2106-2110. Cheung, W. Y. (1980) Calmodulin plays a pivotal role in cellular regulation. Science 207, 19-27. Droogmans G., Declerck I., and Casteels R. J. (1987) Effect of adrenergic agonists on Ca++-channel currents in single vascular smooth muscle cells. Pflagers Arch. 409, 7-12. Fabiato A. and Fabiato F. (1979) Calcium programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J. Physiol. 75,463-505. Fedida D., Shimoni Y., and Giles W. R. (1990) Qt-Adrenergic modulation of the transient outward current in rabbit atrial myocytes. J. Physiol. 423, 257277. Fukumitsu T., Hayashi H., Tokuno H., and Tomita T. (1990) Increase in calcium channel current by 13-adrenoceptor agonists in single smooth muscle cells isolated from porcine coronary artery. Br. J. Pharmacol. 100, 593-599. Furchgott R. F. (1983) Role of endothelium in responses of vascular smooth muscle. Circ. Res. 53, 557-573. Furchgott R. F. and Zawadzki J. V. (1980) The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288, 373-376. Galizzi J. P., Qar J., Fosset M., Van Renterghen C., and Lazdunski M. (1987) Regulation of calcium channels in aortic muscle cells by protein kinase C activators (diacylglycerine and phorbol esters) and by peptides (vasopressin and bombesin) that stimulate phosphoinositide breakdown. J. Biol. Chem. 262, 6947-6950. Lang D. G. and Ritchie A. K. (1990) Tetraethylammonium blockade of apaminsensitive and insensitive Cag+-activated K + channels in a pituitary cell line. J. Physiol. 425, 117-132. Lynch C. (1985) Ionic conductances in frog short skeletal muscle fibers with slow delayed rectifier currents. J. Physiol. 368, 358-378. Marks T. N., Dubyak G. R., and Jones S. W. (1990) Calcium currents in the A7r5 smooth muscle-derived cell line. Pflagers Arch. 417, 433-439. McFadzean I. and England S. (1992) Properties of the inactivating outward current in single smooth muscle ceils isolated from the rat anococcygeus. Pflfigers Arch. 421, 117-124. Means A. R. and Chafouleas J. G. (1982) Calmodulin in endocrine cells. Annu. Rev. Physiol. 44, 667-682. Meisheri K. D. and Van Breemen C. (1982) Effects of 13-adrenergic stimulation on calcium movements in rabbit aortic smooth muscle: relationship with cyclic AMP. 1. Physiol. 331, 429-441. Mond J. J., Feuerstein N., June C. H., Balapure A. K., Glazer R. I., Witherspoon
H. S a t o h K., and Brunswick M. (1991) Bimodal effect of phorbol ester on 13cell activation. J. Biol. Chem. 266, 4458--4463. Muller M. J. and Baer H. P. (1983) Relaxant effects of forskolin in smooth muscle. Role of cyclic AMP. Naunyn-Schmiedeberg's Arch. Pharmac. 322, 78-82. Nishizuka Y. (1988) The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature 334, 661-665. Ousterhout J. M. and Sperelakis N. (1987) Cyclic nucleotides depress action potentials in cultured aortic smooth muscle cells. Eur. ]. Pharmac. 144, 714. Pacaud P., Loirand G., Mironneau C., and Mironneau J. (1987) Opposing effects of noradrenaline on the two classes of voltage-dependent calcium channels of single vascular smooth muscle cells in short-term primary culture. P~higers Arch. 410, 507-509. Rasmussen H. and Barrett P. Q. (1984) Calcium messenger system: an integrated view. Physiol. Rev. 64, 938-984. Romey G. and Lazdunski M. (1984) The coexistence in rat muscle cells of two distinct classes of CaZ+-dependent K + channels with different pharmacological properties and different physiological functions. Biochem. Biophys. Res. Commun. 118, 669-674. Salkoff L. and Wyman R. (1981) Outward currents in developing Drosophila flight muscle. Sc/ence 212, 461-463. Satoh H. (1992) Inhibition in L-type Ca 2+ channel by stimulation of protein kinase C in isolated guinea-pig ventricular cardiomyocytes. Gen. Pharmac. 23, 1097-1102. Satoh H. (1994) Elevation of intracdlular Ca z+ concentration by protein kinase C stimulation in isolated single rabbit sino-atrial node. cells. Gen. Pharmac. 25, 325-332. Satoh H. (1995) Depression of the spontaneous activity by phorbol esters in young embryonic chick cardiomyocytes. ]pn. ]. Pharmac. 67,297-304. Satoh H. and Hashimoto K. (1988) On electrophysiological responses to phorbol esters which stimulate protein kinase C in rabbit sino-atrial node cells. Naunyn-Schmiedeberg's Arch. Pharmacol. 337, 308-315. Satoh H., and Sperelakis N. (1991) Calcium and potassium currents in cultured rat aortic vascular smooth cell lines. In. Ionic Channels of Vascular Smooth Musde Cells and Endothelial Cells (edited by Sperelakis N. and Kuriyama H.), pp. 55-64. Elsevier, New York. Satoh H. and Sperelakis N. (1994) Identification of and developmental changes in transient outward current in embryonic chick cardiomyocytes. J. Devel. Physiol 20, 149-154. Satoh H. and Sperelakis N. (1995) Modulation of L-type Ca z+ cur rent by isoprenaline, carbachol and phorbol ester in cultured rat aortic vascular smooth muscle (A7r5) cells. Gen. Pharmacol. 26, 369-379. Satoh H., Nakashima K., and Tsuchida K. (1992) The responses to phorbol esters which stimulated protein kinase C in canine Purkinje fibers. Gen. Pharmac. 23, 847-852. Seamon K., Padgett W., and Daly J. W. (1981) Forskolin: unique diterpene activator of adenylate cyclase in membranes and in intact cells. Proc. Natl. Acad. Sci. USA 78, 3363-3367. Sperelakis N. (1990) Properties of calcium channels in cardiac muscle and vascular smooth muscle. Proceedings of a conference on membranes. Mol. Cell. BIOchem. 99, 97-109. Sperelakis N., Tohse N., Ohya Y., and Masuda H. (1994) Cyclic GMP regulation of calcium slow channels in cardiac muscle and vascular smooth muscle calls. Adv. Pharmac. 26, 217-252. Sugden D., Vanecek J., Klein D. C., Thomas T. P., and Anderson W. B. (1985) Activation of protein kinase C potentiates isoprenaline-induced cyclic AMP accumulation in rat pinealocytes. Nature 314, 359-361. Tsien R. Y., and Rink T. J. (1980) Neutral carrier ion-selective microelectrodes for measurements of intracellular free calcium. Biochem. Biophys. Acta 599, 623-638. Van Renterghem C., and Lazdunski M. (1992) A small-conductance charybdotoxin-sensitive, apamin-resistant Ca2+-activated K + channel in aortic smooth muscle cells (line A7r5 line and primary culture). Pflagers Arch. 420, 417-423. Van Renterghem C., Romey G., and Lazdunski M. (1988)Vasopressin modulates the spontaneous electrical activity in aortic cells (line A7r5) by acting on three different types of ionic channels. Pr0c. Natl. Acad. Sci. USA 85, 93659369. Vivaudou M. B., Clapp L. H., Walsh J. V., and Singer J. J. (1988) Regulation of one type of Ca z+ current in smooth muscle cells by diacylglycerol and acetylcholine. FASEB J. 2, 2497-2504. Volicer L. and Hynie S. (1971) Effect of catecholamines and angiotensin on cyclic AMP in rat aorta and tail artery. Fur. J. Immunol. 15, 214-220. Walsh K. B. and Kass R. S. (1988) Regulation of a heart potassium channel by protein kinase A and C. Science 242, 67-69.