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Dibutyryl Cyclic AMP and Forskolin Inhibit Phosphatidylinositol Hydrolysis, Ca2+ Influx and Contraction in Vascular Smooth Muscle
Dibutyryl
Cyclic
Phosphatidylinositol
Hee Yul Ahni,
AMP
and Forskolin
Hydrolysis,
Cat + Influx
in Vascular
Smooth
Seung
Ki Churl
Eok
Kang2,
Inhibit and
Contraction
Muscle Chang3
and Hideaki
Karaki4, *
'Department of Pharmacology, College of Medicine, Chungbuk National University, Cheongju 360-763, Korea 2Department of Biochemistry, College of Pharmacy, Joongang University, Seoul 156-756, Korea 3Department of Pharmacology , College of Medicine, Gyeongsang National University, Chinju 660-280, Korea 4Department of Veterinary Pharmacology , Faculty of Agriculture, The University of Tokyo, Tokyo 113, Japan Received
March
25, 1992
Accepted
May
6, 1992
ABSTRACT-Dibutyryl cyclic AMP and forskolin inhibited the contraction induced by norepinephrine (NE) more strongly than the high K=induced contraction in isolated rat aorta. These inhibitors inhib ited the 45Ca2+ influx stimulated by NE but not that by high K+, and they inhibited NE-induced inosi tol monophosphate accumulation. These results suggest that cAMP inhibits NE-induced contraction, at least partly, by inhibiting the a-adrenoceptor-mediated signal transduction and high K=induced con traction by decreasing Ca 2+ sensitivity but not Ca 2+ influx. Keywords:
cyclic AMP,
Phosphatidylinositol
turnover,
In vascular smooth muscle, cyclic AMP (cAMP) has been shown to inhibit contraction by decreasing cytosolic Ca2+ levels ([Ca2+]i) (1) and the Ca2+ sensi tivity of contractile elements (1, 2). One of the charac teristics of the inhibitory effect of cAMP is to inhibit the contraction induced by norepinephrine (NE) more strongly than that induced by high K+ (1). To deter mine the reason for this difference, we examined the effects of dibutyryl cyclic AMP (db-cAMP) and fors kolin on muscle tension, Ca2+ influx and phosphatidyl inositol (PI) hydrolysis in vascular smooth muscle. Thoracic aorta was isolated from male Sprague Daw ley rats weighing about 200 g and cut into spiral strips (2-3-mm-wide and 10-15-mm-long). Endothelium was removed by gently rubbing the intimal surface with a cotton swab. The normal physiological salt solution (PSS) contained: 136.9 mM NaC1, 5.4 mM KCI, 5.5 mM glucose, 23.8 mM NaHCO3, 1.5 mM CaC12, 1.0 MM MgC12 and 0.01 mM ethylenediamine tetraacetic acid. High K+ solution was made by substituting 60 mM NaCI in the normal PSS with equimolar KC1. These solutions were saturated with 95%02 and 5% CO2 at 37°C. Muscle tension was recorded isometrically under
Vasodilation
a resting tension of 1 g. Since the contractions induced by 1,u M NE or 65.4 mM K+ reached a steady level approximately 5 min after the addition of a stimulant, muscle tension measured 20 min after the addition of NE or high K+ was taken as a sustained contraction. Inhibitor was added 30 min before the addition of a stim ulant. Ca2+ influx was measured as described previously (3). Muscle strips were incubated with 45Ca2+ (1.6 ,uCi/ml) for 5 min with or without stimulant. The in hibitor was added 30 min before the 45Ca2+ exposure. Tissues were then washed for 30 min with a solution containing 73.8 mM LaC13, 5.5 mM glucose and 24.0 mM tris, pH 6.8-6.9 at 0.5°C. After the wash, 45Ca2+ in the tissue was measured with a liquid scintillation counter (Beckman,. U.S.A.). For the determination of inositol monophosphate content, tissues were exposed to 20 u Ci/ml myo-2 [3H]inositol (19,uCi/mmol) for 5 hr followed by expo sure to NE for 60 min in the presence of 10 mM LiCI. Inhibitor was added 30 min before the addition of NE. Tissues were then rapidly frozen and inositol mono phosphate was assayed according to Berridge et al. (4). Db-cAMP (Yamasa, Japan), forskolin, NE bitartrate (Sigma, U.S.A.), "Ca 2+ and myo-2-[3H]inositol
(Amersham, Japan) were used. Results are expressed as the mean ± S.E.M. A difference was considered to be statistically significant when the P value was less than 0.05 using two-way analysis of variance in com bination with Dunnett's test. Results of the experiments are summarized in Fig. 1. As shown in this figure, 1 uM NE and 65.4 mM K+ in creased 45Ca2+influx and muscle tension. Although the NE-induced contraction was greater than that induced
by high K; 45Ca2+influx stimulatedby NE was small er than that due to high K This result is consistent with the finding that NE induces greater contraction than high K+ at a given [Ca2+]i in both intact and per meabilized smooth muscle preparations possibly be cause NE increases the Ca2+ sensitivity of the contrac tile elements (2, 5, 6). Db-cAMP (300,uM) and forskolin (1 ,aM) almost
completely inhibited the NE-induced increase in 45Ca2+ influx and muscle tension. It has been shown that the Ca2+ influx stimulated by NE or high K+ is mediated mainly by voltage-dependent Ca2+ channels in smooth muscle (6). However, cAMP does not seem to directly inhibit the Ca 2+ channels because neither db-cAMP (1 mM) nor forskolin (10 uM) inhibited the 45Ca2+ influx stimulated by high K+ even at concentrations 3 to 10 times higher than those that almost completely inhibit the NE-induced changes. In addition, it has been shown that intracellularly perfused cAMP had no effect on the voltage-dependent Ca2+ current in smooth muscle cells (7). These results suggest that cAMP does not directly inhibit voltage-dependent Ca2+ channels in rat aorta and that the cAMP-induced inhibition of NE-stimulated Ca2+ influx is indirect. Concerning
the mechanism
of cAMP-induced
Fig. 1. Effects of db-cAMP (A) and forskolin (B) on inositol monophosphate content, Ca2+ influx and muscle tension in isolated rat aorta. Muscle was stimulated with 1 uM NE or 65.4mM KCI. Inositol monophosphate accumulation (closed column): Difference between the levels in the absence (resting) and presence of NE is shown. The resting level was 145.6 ± 20.6 cpm/mg wet weight (n = 3) in A and 195.3 ± 21.5 cpm/mg wet weight (n = 3) in B. Values in the presence of high K+ was not determined (nd). Ca2+ influx (shaded column): Difference between the levels in the absence (resting) and presence of stimulant is shown. The resting level was 86.9 ± 5.5 nmol/g wet weight/5 min (n = 6) in A and 95.2 ± 8.5 nmol/mg wet weight/5 min (n = 6) in B. Muscle tension (open column): 100% represents the high K=induced contraction (n = 6). The actual contractile tension induced by NE or high K+ was 0.92 ± 0.04 g (n = 6) or 0.58 ± 0.08 g (n = 6), re spectively. The concentration of db-cAMP was 300,uM for NE and 1 mM for high K The concentration of forskolin was 1 uM for NE and 10,uM for high K *: Significantlydifferent from the value in the absence of an inhibitor (P < 0.05).
inhibi
tion of high K± induced contraction, it has been shown that forskolin only slightly decreases high K± stimulated [Ca 2±]; (1) and that cAMP inhibits Ca2=induced con traction in permeabilized smooth muscle (5). These re sults, together with the fact that cAMP did not inhibit the high K-stimulated Ca2+ influx, suggest that cAMP decreases the Ca2+ sensitivity of contractile elements. Thus, NE and cAMP seem to have opposite effects on Ca2+ sensitivity in smooth muscle. It has been shown that activation of a-adrenoceptor and several other receptors, but not high K+ depolar ization, increases PI turnover to produce inositol-1,4,5 trisphosphate (IP3) and diacylglycerol (8). It has also been shown that cAMP decreases inositol monophos phate accumulation stimulated by carbachol in iris smooth muscle and that by histamine in tracheal smooth muscle (9). In the present experiments, we found that db-cAMP and forskolin inhibited the NE induced inositol monophosphate accumulation in the vascular smooth muscle of rat aorta, suggesting that cAMP inhibits PI turnover mediated by a-adreno ceptors. We also found that db-cAMP (300 ,uM) and forskolin (1 ,uM) completely inhibited the NE-induced transient contraction in Ca2+ free solution (n = 4, data not shown). Since cAMP inhibits NE-induced Ca 2+ re lease (1) which is mediated by IP3 (8, 9), it is possible that cAMP-induced inhibition of IP3 formation is re sponsible for the inhibition of Ca 2+ release. Although the mechanism of receptor-mediated open ing of voltage-dependent Ca2+ channels is not clarified yet, either GTP binding protein coupled to a -adreno ceptors or products of P1 turnover such as inositol 1,3,4,5-tetrakisphosphate may activate the Ca2+ chan nels (10). cAMP may inhibit GTP-binding protein or phospholipase C (9), resulting in an inhibition of a adrenoceptor-mediated opening of the Ca 2+ channels. From these results, it is concluded that the inhibitory effect of cAMP on NE-induced contraction may be due, at least partly, to the inhibition of receptor-medi ated signal transduction. In contrast, inhibition of high K=induced contraction is not due to inhibition of Ca2+
influx,
but possibly
Ca 2-1 sensitivity
attributable
of the contractile
to the
decrease
in the
elements.
Acknowledgments Supported by a Grant-in-Aid (No. 883-0402-007-2) for Scientific Research from the Korea Science and Engineering Foundation and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. REFERENCES 1
2
3
4
5 6
Abe, A. and Karaki, H.: Effect of forskolin on cytosolic Ca2+ level and contraction in vascular smooth muscle. J. Pharmacol. Exp. Ther. 249, 895-900 (1989) Nishimura, J. and van Breemen, C.: Direct regulation of smooth muscle contractile elements by second messengers. Biochem. Biophys. Res. Commun. 163, 929-935 (1989) Karaki, H. and Weiss, G.B.: Alterations in high and low affinity binding of 45Ca in rabbit aortic smooth muscle by norepinephrine and potassium after exposure to lanthanum and low temperature. J. Pharmacol. Exp. Ther. 211, 86-92 (1979) Berridge, M.J., Downes, C.P. and Hanley, M.R.: Lithium amplifies agonist-dependent phosphoinositol responses in brain salivary glands. Biochem. J. 206, 587-595 (1982) Karaki, H.: Ca 2+ localization and sensitivity in vascular smooth muscle. Trends Pharmacol. Sci. 10, 320-325 (1989) Karaki, H., Sato, K. and Ozaki, H.: Different effects of vera
pamil on cytosolic Ca2+ and contraction in norepinephrine stimulated vascular smooth muscle. Japan. J. Pharmacol. 55, 35-42 (1991) 7 Ohya, Y., Kitamura, K. and Kuriyama, H.: Modulation of ionic currents in smooth muscle balls of the rabbit intestine by intracellularly perfused ATP and cyclic AMP. Pflugers Arch. 408, 465-473 (1987) 8 Michell, R.H., Drummond, A.H. and Downes, C.P. (Eds): Inositol Lipids in Cell Signalling, p. 1-519, Academic Press, Tokyo (1989) 9 Abdel-Latif, A.A.: Biochemical and functional interactions between the inositol 1,4,5-trisphosphate-Ca 2+ and cyclic AMP signalling systems in smooth muscle. Cell. Signal. 5, 371 385 (1991) 10 Meldolsi, J., Clementi, E., Fasolato, C., Zachetti, D. and Pozzan, T.: Ca 2+ influx following receptor activation. Trends Pharmacol. Sci. 12, 289-292 (1991)
Cyclic
Phosphatidylinositol
Hee Yul Ahni,
AMP
and Forskolin
Hydrolysis,
Cat + Influx
in Vascular
Smooth
Seung
Ki Churl
Eok
Kang2,
Inhibit and
Contraction
Muscle Chang3
and Hideaki
Karaki4, *
'Department of Pharmacology, College of Medicine, Chungbuk National University, Cheongju 360-763, Korea 2Department of Biochemistry, College of Pharmacy, Joongang University, Seoul 156-756, Korea 3Department of Pharmacology , College of Medicine, Gyeongsang National University, Chinju 660-280, Korea 4Department of Veterinary Pharmacology , Faculty of Agriculture, The University of Tokyo, Tokyo 113, Japan Received
March
25, 1992
Accepted
May
6, 1992
ABSTRACT-Dibutyryl cyclic AMP and forskolin inhibited the contraction induced by norepinephrine (NE) more strongly than the high K=induced contraction in isolated rat aorta. These inhibitors inhib ited the 45Ca2+ influx stimulated by NE but not that by high K+, and they inhibited NE-induced inosi tol monophosphate accumulation. These results suggest that cAMP inhibits NE-induced contraction, at least partly, by inhibiting the a-adrenoceptor-mediated signal transduction and high K=induced con traction by decreasing Ca 2+ sensitivity but not Ca 2+ influx. Keywords:
cyclic AMP,
Phosphatidylinositol
turnover,
In vascular smooth muscle, cyclic AMP (cAMP) has been shown to inhibit contraction by decreasing cytosolic Ca2+ levels ([Ca2+]i) (1) and the Ca2+ sensi tivity of contractile elements (1, 2). One of the charac teristics of the inhibitory effect of cAMP is to inhibit the contraction induced by norepinephrine (NE) more strongly than that induced by high K+ (1). To deter mine the reason for this difference, we examined the effects of dibutyryl cyclic AMP (db-cAMP) and fors kolin on muscle tension, Ca2+ influx and phosphatidyl inositol (PI) hydrolysis in vascular smooth muscle. Thoracic aorta was isolated from male Sprague Daw ley rats weighing about 200 g and cut into spiral strips (2-3-mm-wide and 10-15-mm-long). Endothelium was removed by gently rubbing the intimal surface with a cotton swab. The normal physiological salt solution (PSS) contained: 136.9 mM NaC1, 5.4 mM KCI, 5.5 mM glucose, 23.8 mM NaHCO3, 1.5 mM CaC12, 1.0 MM MgC12 and 0.01 mM ethylenediamine tetraacetic acid. High K+ solution was made by substituting 60 mM NaCI in the normal PSS with equimolar KC1. These solutions were saturated with 95%02 and 5% CO2 at 37°C. Muscle tension was recorded isometrically under
Vasodilation
a resting tension of 1 g. Since the contractions induced by 1,u M NE or 65.4 mM K+ reached a steady level approximately 5 min after the addition of a stimulant, muscle tension measured 20 min after the addition of NE or high K+ was taken as a sustained contraction. Inhibitor was added 30 min before the addition of a stim ulant. Ca2+ influx was measured as described previously (3). Muscle strips were incubated with 45Ca2+ (1.6 ,uCi/ml) for 5 min with or without stimulant. The in hibitor was added 30 min before the 45Ca2+ exposure. Tissues were then washed for 30 min with a solution containing 73.8 mM LaC13, 5.5 mM glucose and 24.0 mM tris, pH 6.8-6.9 at 0.5°C. After the wash, 45Ca2+ in the tissue was measured with a liquid scintillation counter (Beckman,. U.S.A.). For the determination of inositol monophosphate content, tissues were exposed to 20 u Ci/ml myo-2 [3H]inositol (19,uCi/mmol) for 5 hr followed by expo sure to NE for 60 min in the presence of 10 mM LiCI. Inhibitor was added 30 min before the addition of NE. Tissues were then rapidly frozen and inositol mono phosphate was assayed according to Berridge et al. (4). Db-cAMP (Yamasa, Japan), forskolin, NE bitartrate (Sigma, U.S.A.), "Ca 2+ and myo-2-[3H]inositol
(Amersham, Japan) were used. Results are expressed as the mean ± S.E.M. A difference was considered to be statistically significant when the P value was less than 0.05 using two-way analysis of variance in com bination with Dunnett's test. Results of the experiments are summarized in Fig. 1. As shown in this figure, 1 uM NE and 65.4 mM K+ in creased 45Ca2+influx and muscle tension. Although the NE-induced contraction was greater than that induced
by high K; 45Ca2+influx stimulatedby NE was small er than that due to high K This result is consistent with the finding that NE induces greater contraction than high K+ at a given [Ca2+]i in both intact and per meabilized smooth muscle preparations possibly be cause NE increases the Ca2+ sensitivity of the contrac tile elements (2, 5, 6). Db-cAMP (300,uM) and forskolin (1 ,aM) almost
completely inhibited the NE-induced increase in 45Ca2+ influx and muscle tension. It has been shown that the Ca2+ influx stimulated by NE or high K+ is mediated mainly by voltage-dependent Ca2+ channels in smooth muscle (6). However, cAMP does not seem to directly inhibit the Ca 2+ channels because neither db-cAMP (1 mM) nor forskolin (10 uM) inhibited the 45Ca2+ influx stimulated by high K+ even at concentrations 3 to 10 times higher than those that almost completely inhibit the NE-induced changes. In addition, it has been shown that intracellularly perfused cAMP had no effect on the voltage-dependent Ca2+ current in smooth muscle cells (7). These results suggest that cAMP does not directly inhibit voltage-dependent Ca2+ channels in rat aorta and that the cAMP-induced inhibition of NE-stimulated Ca2+ influx is indirect. Concerning
the mechanism
of cAMP-induced
Fig. 1. Effects of db-cAMP (A) and forskolin (B) on inositol monophosphate content, Ca2+ influx and muscle tension in isolated rat aorta. Muscle was stimulated with 1 uM NE or 65.4mM KCI. Inositol monophosphate accumulation (closed column): Difference between the levels in the absence (resting) and presence of NE is shown. The resting level was 145.6 ± 20.6 cpm/mg wet weight (n = 3) in A and 195.3 ± 21.5 cpm/mg wet weight (n = 3) in B. Values in the presence of high K+ was not determined (nd). Ca2+ influx (shaded column): Difference between the levels in the absence (resting) and presence of stimulant is shown. The resting level was 86.9 ± 5.5 nmol/g wet weight/5 min (n = 6) in A and 95.2 ± 8.5 nmol/mg wet weight/5 min (n = 6) in B. Muscle tension (open column): 100% represents the high K=induced contraction (n = 6). The actual contractile tension induced by NE or high K+ was 0.92 ± 0.04 g (n = 6) or 0.58 ± 0.08 g (n = 6), re spectively. The concentration of db-cAMP was 300,uM for NE and 1 mM for high K The concentration of forskolin was 1 uM for NE and 10,uM for high K *: Significantlydifferent from the value in the absence of an inhibitor (P < 0.05).
inhibi
tion of high K± induced contraction, it has been shown that forskolin only slightly decreases high K± stimulated [Ca 2±]; (1) and that cAMP inhibits Ca2=induced con traction in permeabilized smooth muscle (5). These re sults, together with the fact that cAMP did not inhibit the high K-stimulated Ca2+ influx, suggest that cAMP decreases the Ca2+ sensitivity of contractile elements. Thus, NE and cAMP seem to have opposite effects on Ca2+ sensitivity in smooth muscle. It has been shown that activation of a-adrenoceptor and several other receptors, but not high K+ depolar ization, increases PI turnover to produce inositol-1,4,5 trisphosphate (IP3) and diacylglycerol (8). It has also been shown that cAMP decreases inositol monophos phate accumulation stimulated by carbachol in iris smooth muscle and that by histamine in tracheal smooth muscle (9). In the present experiments, we found that db-cAMP and forskolin inhibited the NE induced inositol monophosphate accumulation in the vascular smooth muscle of rat aorta, suggesting that cAMP inhibits PI turnover mediated by a-adreno ceptors. We also found that db-cAMP (300 ,uM) and forskolin (1 ,uM) completely inhibited the NE-induced transient contraction in Ca2+ free solution (n = 4, data not shown). Since cAMP inhibits NE-induced Ca 2+ re lease (1) which is mediated by IP3 (8, 9), it is possible that cAMP-induced inhibition of IP3 formation is re sponsible for the inhibition of Ca 2+ release. Although the mechanism of receptor-mediated open ing of voltage-dependent Ca2+ channels is not clarified yet, either GTP binding protein coupled to a -adreno ceptors or products of P1 turnover such as inositol 1,3,4,5-tetrakisphosphate may activate the Ca2+ chan nels (10). cAMP may inhibit GTP-binding protein or phospholipase C (9), resulting in an inhibition of a adrenoceptor-mediated opening of the Ca 2+ channels. From these results, it is concluded that the inhibitory effect of cAMP on NE-induced contraction may be due, at least partly, to the inhibition of receptor-medi ated signal transduction. In contrast, inhibition of high K=induced contraction is not due to inhibition of Ca2+
influx,
but possibly
Ca 2-1 sensitivity
attributable
of the contractile
to the
decrease
in the
elements.
Acknowledgments Supported by a Grant-in-Aid (No. 883-0402-007-2) for Scientific Research from the Korea Science and Engineering Foundation and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. REFERENCES 1
2
3
4
5 6
Abe, A. and Karaki, H.: Effect of forskolin on cytosolic Ca2+ level and contraction in vascular smooth muscle. J. Pharmacol. Exp. Ther. 249, 895-900 (1989) Nishimura, J. and van Breemen, C.: Direct regulation of smooth muscle contractile elements by second messengers. Biochem. Biophys. Res. Commun. 163, 929-935 (1989) Karaki, H. and Weiss, G.B.: Alterations in high and low affinity binding of 45Ca in rabbit aortic smooth muscle by norepinephrine and potassium after exposure to lanthanum and low temperature. J. Pharmacol. Exp. Ther. 211, 86-92 (1979) Berridge, M.J., Downes, C.P. and Hanley, M.R.: Lithium amplifies agonist-dependent phosphoinositol responses in brain salivary glands. Biochem. J. 206, 587-595 (1982) Karaki, H.: Ca 2+ localization and sensitivity in vascular smooth muscle. Trends Pharmacol. Sci. 10, 320-325 (1989) Karaki, H., Sato, K. and Ozaki, H.: Different effects of vera
pamil on cytosolic Ca2+ and contraction in norepinephrine stimulated vascular smooth muscle. Japan. J. Pharmacol. 55, 35-42 (1991) 7 Ohya, Y., Kitamura, K. and Kuriyama, H.: Modulation of ionic currents in smooth muscle balls of the rabbit intestine by intracellularly perfused ATP and cyclic AMP. Pflugers Arch. 408, 465-473 (1987) 8 Michell, R.H., Drummond, A.H. and Downes, C.P. (Eds): Inositol Lipids in Cell Signalling, p. 1-519, Academic Press, Tokyo (1989) 9 Abdel-Latif, A.A.: Biochemical and functional interactions between the inositol 1,4,5-trisphosphate-Ca 2+ and cyclic AMP signalling systems in smooth muscle. Cell. Signal. 5, 371 385 (1991) 10 Meldolsi, J., Clementi, E., Fasolato, C., Zachetti, D. and Pozzan, T.: Ca 2+ influx following receptor activation. Trends Pharmacol. Sci. 12, 289-292 (1991)