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α1B-Adrenoceptor-mediated stimulation of Ca2+ mobilization and cAMP accumulation in isolated rat hepatocytes
European Journal of Pharmacology - Molecular Pharmacology Section, 246 (1993) 113-120
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© 1993 Elsevier Science Publishers B.V. All rights reserved 0922-4106/93/$06.00
EJPMOL 90460
ala-Adrenoceptor-mediated stimulation of C a 2 + mobilization and cAMP accumulation in isolated rat hepatocytes T a k a h i d e N o m u r a , H a r u h i t o K o n d o , Seiko H a s e g a w a , T o s h i k o W a t a n a b e , R i e Y o k o y a m a , K i k u k o Ukai, Masakatsu Tachibana, Chiho Sumi-Ichinose, Hiroko Nomura and Yasumichi Hagino Department of Pharmacology, Fujita Health University School of Medicine, Toyoake, Aichi, Japan
Received 16 December 1992; revised MS received 1 March 1993;accepted 23 March 1993
Noradrenaline stimulates not only Ca 2+ mobilization but also cAMP formation through activation of al-adrenoceptors in hepatocytes from mature male rats. We examined which subtype(s) of al-adrenoceptor mediate these signal transduction mechanisms. Treatment of hepatocytes with chloroethylclonidine produced a dose-dependent inhibition of noradrenaline-induced Ca 2+ mobilization, involving both transient and sustained components. Chloroethylclonidine also blocked noradrenaline-induced cAMP accumulation. It was observed that prazosin was much more potent than WB4101 (2-(2,6-dimethoxyphenoxyethyl)aminomethyl-l,4-benzodioxane) in antagonizing noradrenaline-induced Ca 2+ mobilization. The same potency order was found in cAMP formation studies. Pretreatment of rats with pertussis toxin did not affect al-adrenergic responsiveness. Incubations of hepatocytes with tumor-promoting phorbol esters eliminated both Ca 2+ mobilization and cAMP accumulation caused by noradrenaline. Our data suggest that in hepatocytes from mature mate rats, single alB-adrenoceptors are linked to cAMP formation as well as Ca 2+ mobilization. al-Adrenoceptors; ala-Adrenoceptors; Noradrenaline; Chloroethylclonidine; Ca2+; cAMP; Hepatocyte (Rat)
1. Introduction
It is well established that al-adrenergic agonists exert their effects through the hydrolysis of phosphatidylinositol 4,5-bisphosphate (reviewed by Exton, 1986). However, Morgan et al. (1983a,b) and Nomura et al. (1991) have documented that al-adrenoceptors are also coupled to a cAMP generation system in isolated hepatocytes from matured male rats. Similarly, activation of al-adrenoceptors in rat cerebral cortex slices has been reported to increase cAMP formation as well as phosphatidylinositol metabolism (Davis et al., 1978; Johnson and Minneman, 1986; Robinson and Kendall, 1990). At present, it is not clear by which mechanism ax-adrenoceptor activation leads to the accumulation of cAMP. Recent evidence suggests that a~-adrenoceptors are not homogeneous but are further divided into at least two pharmacologically distinct subtypes, i.e., alA and a m (reviewed by Minneman, 1988). al-Adrenoceptor subtypes have been defined using competitive antagonists and alkylating agents.
The OtlA subtype has a high affinity for the competitive antagonists WB4101 ( 2 - ( 2 , 6 - d i m e t h o x y p h e n o x y ethyl)aminomethyl-l,4-benzodioxane) (Morrow and Creese, 1986) and 5-methylurapidil (Gross et al., 1988) and is insensitive to alkylation by chloroethylclonidine (Han et al., 1987a; Minneman et al., 1988), whereas the alB subtype is potently inactivated by chloroethylclonidine and has a lower affinity for WB4101 and 5-methylurapidil. Therefore, these adrenergic antagonists appear to be useful probes for investigating the relationship between al-adrenoceptor subtypes and their signal transduction mechanisms. The aim of this study was to clarify whether noradrenaline-induced Ca 2+ mobilization and cAMP accumulation in isolated rat hepatocytes (Nomura et al., 1991) are mediated by a single class of al-adrenoceptor subtypes or by distinct subtypes.
2. Materials and methods 2.1. Materials
Correspondence to: Takahide Nomura, M.D., Department of Pharmacology, Fujita Health University School of Medicine, 1-98 Dengakugakubo, Kutsukake-cho, Toyoake, Aichi 470-11, Japan.
The sources of materials used in this work were as follows: collagenase (CLS 2) from Worthington Biochemical (Freehold, N J, USA), ( - ) - n o r a d r e n a l i n e lay-
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drochloride, (-)-phenylephrine hydrochloride, methoxamine hydrochloride and 4/3-phorbol 12-myristate 13-acetate (PMA) from Sigma (St. Louis, MO, USA), propranolol hydrochloride from Sumitomo Chemical (Osaka, Japan), prazosin hydrochloride from Pfeizer (Tokyo, Japan), chloroethylclonidine and WB4101 from Research Biochemicals (Natick, MA, USA), fura-2 acetoxymethyl ester (fura-2/AM) from Dojindo Laboratories (Kumamoto, Japan), [Arg-8]vasopressin from Peptide Institute (Minoh, Japan) and pertussis toxin (from Bordetella pertussis) from Kaken Pharmaceuticals (Shiga, Japan). 2.2. Animals Male Wistar rats (8-16 weeks of age, 300-500 g) were used. The rats were allowed free access to water and standard laboratory food. 2.3. Incubation of hepatocytes and analytical methods Isolated rat hepatocytes (Berry and Friend, 1969; Harris, 1975) were suspended at a concentration of 3 X 106 cells/ml in a HEPES buffer containing 120 mM NaCI, 5 mM KC1, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.3 mM CaCIz, 5 mM NaHCO 3 and 10 mM HEPES, pH 7.4. The incubation procedure in the absence of Ca 2+ has been described (Nomura et al., 1987). Intracellular Ca 2÷ concentration ([Ca2+] i) was measured with the fluorescent Ca 2÷ indicator dye, fura-2, essentially as described by Tohkin et al. (1988). Fura-2 loading was achieved by preincubating hepatocytes in the HEPES buffer medium supplemented with 5 ixM fura-2/AM for 20 min at 37°C. The cells were washed three times and kept on ice after being resuspended in the same HEPES buffer medium. Aliquots of cells were warmed immediately before use by incubating at 37°C for 1 min. Cellular fluorescence was determined in a Hitachi Model F-4010 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) equipped with a thermostat-controlled cuvette holder and magnetic stirring. Incubations were conducted for 105 s at 37°C. Agonists were added at 45 s and their effects were followed for 1 min. When the effects of prazosin, propranolol, WB4101 and PMA were being studied, these agents were added at 0 s. Increase in [Ca2+]i was calculated by subtracting [Ca2+]i value immediately before the addition of agonist from the value at the peak of response. To study the action of chloroethylclonidine, hepatocytes were preincubated for 20 min with this agent and then washed three times before being used. In the experiments for the determination of cAMP, hepatocytes were similarly treated, except that dimethylsulfoxide without fura-2/AM was present during the preincubation. Incubations were conducted for 2 min. Agonists were added at 1 min and samples
were removed at 2 min. When used, antagonists and PMA were added at 0 min. Hepatocytes were treated with chloroethylclonidine in the same way as for the measurement of [Ca2+]i . cAMP was determined by radioimmunoassay using a Y A M A S A cyclic AMP assay kit (Yamasa, Tokyo, Japan) in KOH-neutralized HC10 4 extracts of cell suspensions.
3. Results
3.1. Effect of pretreatment with chloroethylclonidine on noradrenaline-induced Ca e+ mobilization in hepatocytes in the presence and absence of extracellular Ca e ÷ In the presence of extracellular Ca 2+, noradrenaline (10 ~M) caused a rapid increase in [Ca2+]i, consisting of a quick transient peak followed by a more sustained component (fig. 1A). We found that pretreatment of hepatocytes with increasing concentrations of chloroethylclonidine produces a progressive inhibition of both components of Ca 2÷ mobilization induced by noradrenaline (fig. 1A). In the absence of extracellular Ca 2+, basal levels of [Ca 2+ ]i were significantly reduced (fig. 1B). Noradrenaline also increased [Ca2+] i in the absence of extracellular Ca z+, although this increase was transient and small, with no sustained component (fig. 1B). Pretreatment with chloroethylclonidine also abolished the response to noradrenaline under this condition (fig. 1B). Like noradrenaline, vasopressin (100 nM) produced increases in [Ca2+]i in both the presence and absence of extracellular Ca 2+ (fig. 1) However, the effect of vasopressin was not affected by chloroethylclonidine (fig. 1). Fig. 2 clearly illustrates that the increase in [Ca2+]i, which is calculated by subtracting the value of [Ca2+] i immediately before the addition of noradrenaline from that at the peak of the response, is dose-dependently blocked by chloroethylclonidine in both the presence and absence of extracellular Ca 2÷. Noradrenaline-induced increase in [Ca2+] i in the presence of extracellular Ca z+ was reduced by 18% and 66% by pretreatment with 10 /zM and 100 ~zM chloroethylclonidine, respectively. The effect of chloroethylclonidine was more marked in the absence of extracellular Ca 2÷, the inhibition being 50% and 93% by 10 /.~M and 100 tzM chloroethylclonidine, respectively. 3.2. Effect of pretreatment with chloroethylclonidine on noradrenaline-induced stimulation of cAMP accumulation in hepatocytes in the presence and absence of extracellular Ca 2 ÷ Fig. 3 indicates that preincubation with chloroethylclonidine dose-dependently inactivated cAMP accumulation induced by noradrenaline in both the presence
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Fig. 1. Effect of pretreatment with chloroethylclonidine (CEC) on Ca 2+ mobilization induced by noradrenaline (Nadr, 10/~M) and vasopressin (Vas, 100 nM) in the presence (A) and absence (B) of extracellular Ca 2+. Cells were pretreated with various concentrations of CEC as described in the text. Traces shown were representative of four to six experiments using different hepatocyte preparations.
and absence of extracellular C a 2+. In the presence of extracellular Ca 2+, noradrenaline-induced cAMP accumulation was reduced by 50% and 75% by pretreatment with 10 ~zM and 100 /zM chloroethylclonidine, respectively. Noradrenaline produced a more marked stimulation of cAMP accumulation in the absence of extracellular C a 2+, confirming the previous data (Nomura et al., 1991). The magnitude of the inhibition by chloroethylclonidine in the absence of extracellular Ca 2+ was similar to values observed in the presence of 200
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extracellular Ca 2+ (35% inhibition by 10 /zM chloroethylclonidine and 74% inhibition by 100 /xM chloroethylclonidine). The potent inhibition by chloroethylclonidine of noradrenaline-induced stimulation of cAMP accumulation observed in the present study is consistent with that observed in liver cells for other %-adrenergic responses including C a 2+ mobilization (figs. 1 and 2), phosphatidylinositol labeling (Torres-M~rquez et al., 1991) and lzsI-BE2254 binding (Tsujimoto et al., 1989).
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Fig. 2. Effect of pretreatment with chloroethylclonidine (CEC) on noradrenaline (10 /zM)-induced increase in [Ca 2+ ]i in the presence and absence of extracellular Ca 2÷. Cells were pretreated with various concentrations of CEC as described in the text. Increases in [Ca 2+ ]i were calculated by subtracting basal values of [Ca 2÷ ]i immediately before the addition of noradrenaline from those at the peaks of response. Basal cytosolic Ca 2 ÷ concentrations in the presence and absence of extracellular Ca 2÷ were 277 + 24 nM and 79 4-6 n M, respectively. The data shown are the m e a n s + S.E. of four to six hepatocyte preparations. * P < 0.05, compared with control incubations by Student's t-test.
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3.3. Comparison o f the antagonizing effects o f prazosin and WB4101 on noradrenaline-induced Ca 2 ÷ mobilization and cAMP accumulation
the response to phenylephrine (10 ~M) was also suppressed by prazosin (1 /xM) and by chloroethylclonidine (100/xM) (data not shown).
Fig. 4 illustrates the effects of prazosin and WB4101 on noradrenaline responsiveness in the presence (fig. 4, panels A and B) and absence (fig. 4, panels C and D) of extracellular Ca 2+. Both noradrenaline-induced Ca 2÷ mobilization (fig. 4, panels A and C) and cAMP accumulation (fig. 4, panels B and D) were blocked by these antagonists, although prazosin was approximately 100 times more potent than WB4101. The maximal effect of prazosin was seen at 10 to 100 nM while that of WB4101 was seen at 10 /zM. Since WB4101 is rapidly metabolized by hepatocytes (Han et al., 1990), the potency of WB4101 observed in the present study may be the one somewhat underestimated. Propranolol (10/xM) failed to affect these noradrenaline actions.
3.5. Pertussis toxin pretreatment o f rats does not affect noradrenaline-stimulated Ca 2 + mobilization and cAMP accumulation in hepatocytes
3.4. Effects of methoxamine and phenylephrine on Ca 2 + mobilization and cAMP accumulation in hepatocytes
Methoxamine (10 izM) exerted little effect on either Ca 2+ mobilization (fig. 5A) or cAMP accumulation (fig. 5B) in hepatocytes. In contrast, phenylephrine (10 ~M) significantly stimulated both, although the effect of this agent was less marked than that of noradrenaline (10/xM) (fig. 5, panels A and B). We observed that
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CEC (-10gM) Fig. 3. Effect of pretreatment with chloroethylclonidine (CEC) on noradrenaline (10 /zM)-induced increase in cAMP levels in the presence and absence of extracellular Ca 2+. Cells were pretreated with various concentrations of CEC as described in the text. Noradrenaline-induced increase in cAMP levels was calculated by subtracting basal cAMP levels at 1 min after saline addition from those at 1 min after noradrenaline addition. Basal cAMP levels in the presence and absence of extracellular Ca 2+ were 266+41 p m o l / g wet weight and 274_+45 p m o l / g wet weight, respectively. The data shown are the m e a n s _+S.E. of five to six hepatocyte preparations. * P < 0.05, compared with control incubations by Student's t-test.
It has been documented that a~-adrenoceptors of FRTL5 thyroid cells are coupled to two distinct signal transduction mechanisms, one of which is pertussis toxin-sensitive, and the other pertussis toxin-insensitive (Burch et al., 1986). Thus we decided to examine whether pertussis toxin discriminates between a~adrenoceptor-mediated second messenger responses in isolated rat hepatocytes. Male Wistar rats were injected intravenously with 10/xg toxin/kg body weight in a single dose, 3 days before the experiment was performed. Similar stimulations of cAMP accumulation and Ca 2+ mobilization were elicited by noradrenaline (10/xM) in hepatocytes isolated from toxin-treated rats as for non-treated rats. This is consistent with results reported by Pushpendran et al. (1983), who showed that stimulation of phosphatidylinositol labeling produced by adrenaline is not affected in liver cells from pertussis toxin-treated rats. The negative results of our experiments are not shown. 3.6. Effect of PMA on noradrenaline-induced Ca 2 + mobilization and cAMP accumulation
PMA has been shown to cause desensitization of aradrenoceptor responses in various tissues, including liver and DDTIMF-2 cells, through activation of protein kinase C (Leeb-Lundberg et al., 1985; Corvera et al., 1986). On the other hand, it has been reported that the a 1 response in rabbit aorta is not affected by PMA (Garcia-Sfiinz et al., 1985). To date, it is not clear what effect PMA has on a~-adrenoceptor-mediated stimulation of cAMP accumulation in rat hepatocytes. We, therefore, investigated the effect of PMA both on cAMP accumulation and Ca 2+ mobilization caused by noradrenaline. We found that PMA (1/~M) attenuated the stimulation of Ca 2+ mobilization caused by noradrenaline, while it did not alter the effect of vasopressin (fig. 6A). Similarly, PMA also abolished a noradrenaline-induced increase in cAMP accumulation.
4. Discussion
Differences between ata- and alB-adrenoceptor subtypes have been pharmacologically identified as follows: (1) The atB subtype is sensitive to inactivation by the alkylating agent, chloroethylclonidine, while the alA subtype is insensitive to it (Han et al., 1987a;
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Minneman et al., 1988; Minneman, 1988). (2) The potency orders of competitive antagonists, WB4101, 5-methylurapidil and prazosin, are prazosin >> 5methylurapidil > WB4101 in am-adrenoceptors and WB4101 >_ 5-methylurapidil = prazosin in axa-adrenoceptors (Torres-M~irquez et al., 1991). (3) Methoxamine potently stimulates a]a-adrenoceptors but has little effect on am-receptors (Garda-Sfiinz et al., 1985; Tsujimoto et al., 1989). Recently, the alc subtype has been identified by molecular biological approaches (Schwinn et al., 1990). This subtype has a high affinity for aaaselective antagonists but is also sensitive to inactivation by chloroethylclonidine (Schwinn et al., 1990; GardaSfiinz et al., 1992). Results of the present study clearly indicate that aFadrenoceptors mediating noradrenal i n e - i n d u c e d Ca 2+ mobilization and cAMP accumula-
tion are alB subtype, judging from the pharmacological criteria. This is the first report which documents that activation of OqB-adrenoceptors leads to cAMP formation as well as Ca 2+ mobilization in isolated rat hepatocytes. It has previously been demonstrated in rat cerebral cortex slices that the ai-adrenoceptors mediating two second messenger responses, namely, inositol phosphate accumulation and cAMP potentiation, are distinct from each other (Robinson and Kendall, 1990). Their findings are in contrast with those of the present study. It is well established that activation of avadrenoceptors causes elevation o f [Ca2+] i through formation of inositol 1,4,5-trisphosphate which promotes the release of stored intracellular Ca 2+ and through the influx of e x t r a c e l l u l a r Ca 2+ (Exton, 1986). It has been proposed
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Fig. 4. Effects of WB4101, prazosin and propranolol on noradrenaline-induced increase in [Ca 2+ ]i and cAMP. Panels A and B: effects of WB4101 ( • ) , prazosin ([]) and propranolol (zx) on noradrenaline (10/xM)-induced increase in [Ca 2+ ]i (A) and cAMP (B) in the presence of extracellular Ca 2+. Panels C and D: effects of WB4101 ( • ) , prazosin (rn) and propranolol (zx) on noradrenaline (10 ~M)-induced increase in [Ca2+ ]i (C) and cAMP (D) in the absence of extracellular Ca 2+. Noradrenaline-induced increases in [Ca 2+ ]i and cAMP were calculated as described for fig. 2 and fig. 3, respectively. Basal cytosolic Ca 2+ concentrations in the presence and absence of extracellular Ca 2+ were 133 ± 13 nM and 54 ± 3 nM, respectively. Basal cAMP levels in the presence and absence of extracellular Ca 2+ were 253 ± 25 p m o l / g wet weight and 267 ± 40 p m o l / g wet weight, respectively. Each point represents the mean ± S.E. of three to five hepatocyte preparations.
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(A)
that different a r a d r e n o c e p t o r subtypes are linked to different mechanisms for increasing intracellular Ca 2+ (Han et al., 1987b). Activation of o~tB subtype has recently been shown to lead to the stimulation of phosphoinositide turnover (Tsujimoto et al., 1989; Torres-Mfirquez et al., 1991). It is currently hypothesized that alB subtypes are associated with the formation of inositol 1,4,5-trisphosphate and mobilization of intracellular Ca 2+, while the alA subtypes are linked to gating Ca 2+ influx (Han et al., 1987b; Tsujimoto et al., 1989). However, we found that pretreatment with chloroethylclonidine abolished both components of noradrenaline-induced Ca 2+ mobilization, i.e., a quick transient component representing Ca 2+ release from intracellular stores (fig. 1, panels A and B) and a more sustained component representing Ca 2+ influx (fig. 1A). Thus, our data allows us to conclude that am-adrenoceptors of rat hepatocytes promote Ca 2+ influx as well as intracellular Ca 2+ mobilization. This is consistent with recent results in MDCK-D] cells reported by Klijn et al. (1991) and those in DDT~ MF-2 cells by Han et al. (1992). We emphasize that stimulation of cAMP accumulation in response to noradrenaline is not secondary to Ca 2+ mobilization for the following reasons: (1), vasopressin, which produces Ca 2+ mobilization in the same manner as noradrenaline (fig. 1), exerts no effect on cAMP levels (Nomura et al., 1991). (2), no parallel
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Fig. 6. Effect of P M A on noradrenaline (Nadr)-induced stimulation of Ca ~ + mobilization and cAMP accumulation. Panel A shows the effect of P M A (1 izM) on Ca 2÷ mobilization induced by Nadr (10 ~zM) and vasopressin (Vas, 100 nM) in the presence of extracellular Ca 2÷. Propranolol (Pro, 10/xM) was present when Nadr was added. Traces shown were representative of four or five experiments using different hepatocyte preparations. Panel B shows the effect of P M A (1 /~M) on Nadr (10 p~M)-induced stimulation of cAMP accumulation in the absence of extracellular Ca 2÷. Propranolol ( 1 0 / z M ) was added to all the incubations. The data shown are the m e a n s _+S.E. of four hepatocyte preparations. * P < 0.05, compared with saline addition by Student's t-test.
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Fig. 5. Comparison of the effects of noradrenaline (Nadr), methoxamine (Mtx) and phenylephrine (Phe) on Ca 2÷ mobilization and cAMP accumulation. Panel A shows the effects of Nadr (10 /~M), Mtx (10/zM) and Phe (10/xM) on [Ca 2+ ]i, which were studied in the presence of extracellular Ca 2+. Traces shown were representative of at least four experiments using different hepatocyte preparations. Panel B shows the effects of Nadr (10 /zM), Mtx ( 1 0 / z M ) and Phe (10 /~M) on cAMP levels which were studied in the absence of extracellular Ca z+. Results are expressed as percentages of basal cAMP levels at 1 min. Basal cAMP levels averaged 287 + 40 p m o l / g wet weight. The data shown are the m e a n s + S.E. of at least three hepatocyte preparations. * P < 0.05, compared with basal cAMP levels by Student's t-test.
relationship was found b e t w e e n [Ca2+]i and cAMP levels: namely, noradrenaline-induced increase in [Ca2+]i was larger in the presence of extracellular Ca 2÷ than in the absence of extracellular Ca z+ (fig. 2), while that in cAMP was less marked in the presence of extracellular Ca 2 ÷ than in the absence of extracellular Ca 2÷ (fig. 3). We observed that stimulation of cAMP accumulation by noradrenaline (10 ~M, plus 10 /zM propranolol) became more marked when it was added in the presence of isobutyl methylxanthine (IBMX, 100 /xM) (noradrenaline-induced increase in cAMP levels at 1 min in the absence of extracellular Ca2+: control; 97.6 _+ 14.1 p m o l / g wet weight, IBMX; 272.7 _+ 46.2 p m o l / g wet weight, n = 3). The data suggest that activation of adenylate cyclase is involved in am-adrenoceptor-mediated increases in cAMP levels. In the present study, neither pertussis toxin pretreatment nor PMA addition was found to discriminate
119
between aradrenoceptor-mediated second messenger responses in isolated hepatocytes. This indicates that G i is not involved in the receptor-mediated responsiveness and that both signaling pathways are equally desensitized by activation of protein kinase C. Is it possible that a single class of receptors simultaneously couples to multiple signals? Recently, it has been reported that activation of a single receptor, such as parathyroid hormone receptor (Abou-Samra et al., 1992) or a~adrenoceptor (Cotecchia et al., 1990), expressed in COS-7 cells stimulates intracellular accumulation of both cAMP and inositol phosphates. Furthermore, a2C10, a class of c~2-adrenoceptor subtypes has been demonstrated to couple simultaneously to both G i and G s proteins in CHO ceils (Eason et al., 1992). It remains to be investigated how activation of a madrenoceptors stimulates both cAMP formation and Ca 2÷ mobilization in isolated rat hepatocytes.
Acknowledgement This study was supported by a Grant-in-Aid from Fujita Health University.
References Abou-Samra, A.B., H. Jiippner, T. Force, M.W. Freeman, X.F. Kong, E. Schipani, P. Urena, J. Richards, J.V. Bonventre, J.T. Potts, Jr., H.M. Kronenberg and G.V. Segre, 1992, Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related peptide from rat osteoblast-like cells: a single receptor stimulates intracellular accumulation of both cAMP and inositol trisphosphates and increases intracellular free calcium, Proc. Natl. Acad. Sci. USA 89, 2732. Berry, M.N. and D.S. Friend, 1969, High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study, J. Cell Biol. 43, 506. Burch, R.M., A. Luini and J. Axelrod, 1986, Phospholipase A a and phospholipase C are activated by distinct GTP-binding proteins in response to al-adrenergic stimulation in FRTL5 thyroid cells, Proc. Natl. Acad. Sci. USA 83, 7201. Corvera, S., K.R. Schwarz, R.M. Graham and J.A. Garc~a-S~iinz, 1986, Phorbol esters inhibit as-adrenergic effects and decrease the affinity of liver cell al-adrenergic receptors for (-)-epinephrine, J. Biol. Chem. 261,520. Cotecchia, S., B.K. Kobilka, K.W. Daniel, R.D. Nolan, E.Y. Lapetina, M.G. Caron, R.J. Lefkowitz and J.W. Regan, 1990, Multiple second messenger pathways of a-adrenergic receptor subtypes expressed in eukaryotic cells, J. Biol. Chem. 265, 63. Davis, J.N., C.D. Arnett, E. Hoyler, L.P. Stalvey, J.W. Daly and P. Skolnick, 1978, Brain a-adrenergic receptors: comparison of [3H]WB4101 binding with norepinephrine-stimulated cyclic AMP accumulation in rat cerebral cortex, Brain Res. 159, 125. Eason, M.G., H. Kurose, B.D. Holt, J.R. Raymond and S.B. Liggett, 1992, Simultaneous coupling of aa-adrenergic receptors to two G-proteins with opposing effects: subtype-selective coupling of a2C10, a2C4 , and a2C2 adrenergic receptors to G i and Gs, J. Biol. Chem. 267, 15795. Exton, J.H., 1986, Mechanisms involved in calcium-mobilizing agonist responses, Adv. Cyclic Nucl. Protein Phosphorylation Res. 20, 211.
Garcla-S~iinz, J.A., M.T. Romero-Avila, R.A. Hern~indez, M. Macias-Silva, A. Olivares-Reyes and C. Gonz;ilez-Espinosa, 1992, Species heterogeneity of hepatic al-adrenoceptors: atn-, alnand alc-subtypes, Biochem. Biophys. Res. Comrnun. 186, 760. Garcla-S~inz, J.A., R. Villalobos-Molina, S. Corvera, J. HuertaBahena, G. Tsujimoto and B.B. Hoffman, 1985, Differential effects of adrenergic agonists and phorbol esters on the as-adrenoceptors of hepatocytes and aorta, Eur. J. Pharmacol. 112, 393. Gross, G., G. Hanft and C. Rugevics, 1988, 5-Methylurapidil discriminates between subtypes of the as-adrenoceptor, Eur. J. Pharmacol. 151,333. Han, C., K.M. Wilson and K.P. Minneman, 1990, as-Adrenergie receptor subtypes and formation of inositol phosphates in dispersed hepatocytes and renal cells, Mol. Pharmacol. 37, 903. Han, C., P.W. Abel and K.P. Minneman, 1987a, Heterogeneity of al-adrenergic receptors revealed by chlorethylclonidine, Mol. Pharmacol. 32, 505. Han, C., P.W. Abel and K.P. Minneman, 1987b, as-Adrenoceptor subtypes linked to different mechanisms for increasing intracellular Ca 2+ in smooth muscle, Nature 329, 333. Han, C., T.A. Esbenshade and K.P. Minneman, 1992, Subtypes of al-adrenoceptors in DDT s MF-2 and BC3H-1 clonal cell lines, Eur. J. Pharmacol. Mol. Pharmacol. 226, 141. Harris, R.A., 1975, Studies on the inhibition of hepatic lipogenesis by N 6,O2,_dibutyryl adenosine 3',5 '-monophosphate, Arch. Biochem. Biophys. 169, 168. Johnson, R.D. and K.P. Minneman, 1986, Characterization of a sadrenoceptors which increase cyclic AMP accumulation in rat cerebral cortex, Eur. J. Pharmacol. 129, 293. Klijn, K., S.R. Slivka, K. Bell and P.A. Insel, 1991, Renal as-adrenergic receptor subtypes: MDCK-D1 cells, but not rat cortical membranes possess a single population of receptors, Mol. Pharmacol. 39, 407. Leeb-Lundberg, L.M.F., S. Cotecchia, J.W. Lomasney, J.F. DeBernardis, R.J. Lefkowitz and M.G. Caron, 1985, Phorbol esters promote al-adrenergic receptor phosphorylation and receptor uncoupling from inositol phospholipid metabolism, Proc. Natl. Acad. Sci. USA 82, 5651. Minneman, K.P., 1988, al-Adrenergic receptor subtypes, inositol phosphates, and sources of cell Ca a÷, Pharmacol. Rev. 40, 87. Minneman, K.P., C. Han and P.W. Abel, 1988, Comparison of al-adrenergic receptor subtypes distinguished by chlorethylclonidine and WB4101, Mol. Pharmacol. 33, 509. Morgan, N.G., L.E. Waynick and J.H. Exton, 1983a, Characterisation of the al-adrenergic control of hepatic cAMP in male rats, Eur. J. Pharmacol. 96, 1. Morgan, N.G., P.F. Blackmore and J.H. Exton, 1983b, Age-related changes in the control of hepatic cyclic AMP levels by a s- and /32-adrenergic receptors in male rats, J. Biol. Chem. 258, 5103. Morrow, A.L. and I. Creese, 1986, Characterization of as-adrenergic receptor subtypes in rat brain: a reevaluation of [3H]WB4101 and [3H]prazosin binding, Mol. Pharmacol. 29, 321. Nomura, T., M. Tachibana, H. Nomura, M. Chihara and Y. Hagino, 1987, Effects of phorbol esters, A23187 and vasopressin on oleate metabolism in isolated rat hepatocytes, Lipids 22, 474. Nomura, T., Y. Nomura, M. Tachibana, H. Nomura, K. Ukai, R. Yokoyama and Y. Hagino, 1991, al-Adrenergic regulation of ketogenesis in isolated rat hepatocytes, Biochim. Biophys. Acta 1092, 94. Pushpendran, C.K., S. Corvera and J.A. Garcia-S~inz, 1983, Effect of pertussis toxin on hormonal responsiveness of rat hepatocytes, FEBS Lett. 160, 198. Robinson, J.P. and D.A. Kendall, 1990, Niguldipine discriminates between as-adrenoceptor-mediated second messenger responses in rat cerebral cortex slices, Br. J. Pharmacol. 100, 3. Schwinn, D.A., J.W. Lomasney, W. Lorenz, P.J. Szklut, R.T. Fremeau, Jr., T.L. Yang-Feng, M.G. Caron, R.J. Lefkowitz and S.
120 Cotecchia, 1990, Molecular cloning and expression of the cDNA for a novel al-adrenergic receptor subtype, J. Biol. Chem. 265, 8183. Tohkin, M., N. Yoshimatsu and T. Matsubara, 1988, Comparison of the action of epinephrine and a respiratory chain uncoupler, 2,4-dinitrophenol, on Ca2+-mobilization in isolated hepatocytes and perfused livers, Japan. J. Pharmacol. 46, 61. Torres-Mfirquez, M.E., R. Villalobos-Molina and J.A. Garcla-Sfiinz,
1991, al-Adrenoceptor subtypes in aorta ( a l A ) and liver (Otln) , Eur. J. Pharmacol. Mol. Pharmacol 206, 199. Tsujimoto, G., A. Tsujimoto, E. Suzuki and K. Hashimoto, 1989, Glycogen phosphorylase activation by two different al-adrenergic receptor subtypes: methoxamine selectively stimulates a putative t~l-adrenergic receptor subtype (Otla) that couples with Ca 2+ influx, Mol. Pharmacol. 36, 166.
113
© 1993 Elsevier Science Publishers B.V. All rights reserved 0922-4106/93/$06.00
EJPMOL 90460
ala-Adrenoceptor-mediated stimulation of C a 2 + mobilization and cAMP accumulation in isolated rat hepatocytes T a k a h i d e N o m u r a , H a r u h i t o K o n d o , Seiko H a s e g a w a , T o s h i k o W a t a n a b e , R i e Y o k o y a m a , K i k u k o Ukai, Masakatsu Tachibana, Chiho Sumi-Ichinose, Hiroko Nomura and Yasumichi Hagino Department of Pharmacology, Fujita Health University School of Medicine, Toyoake, Aichi, Japan
Received 16 December 1992; revised MS received 1 March 1993;accepted 23 March 1993
Noradrenaline stimulates not only Ca 2+ mobilization but also cAMP formation through activation of al-adrenoceptors in hepatocytes from mature male rats. We examined which subtype(s) of al-adrenoceptor mediate these signal transduction mechanisms. Treatment of hepatocytes with chloroethylclonidine produced a dose-dependent inhibition of noradrenaline-induced Ca 2+ mobilization, involving both transient and sustained components. Chloroethylclonidine also blocked noradrenaline-induced cAMP accumulation. It was observed that prazosin was much more potent than WB4101 (2-(2,6-dimethoxyphenoxyethyl)aminomethyl-l,4-benzodioxane) in antagonizing noradrenaline-induced Ca 2+ mobilization. The same potency order was found in cAMP formation studies. Pretreatment of rats with pertussis toxin did not affect al-adrenergic responsiveness. Incubations of hepatocytes with tumor-promoting phorbol esters eliminated both Ca 2+ mobilization and cAMP accumulation caused by noradrenaline. Our data suggest that in hepatocytes from mature mate rats, single alB-adrenoceptors are linked to cAMP formation as well as Ca 2+ mobilization. al-Adrenoceptors; ala-Adrenoceptors; Noradrenaline; Chloroethylclonidine; Ca2+; cAMP; Hepatocyte (Rat)
1. Introduction
It is well established that al-adrenergic agonists exert their effects through the hydrolysis of phosphatidylinositol 4,5-bisphosphate (reviewed by Exton, 1986). However, Morgan et al. (1983a,b) and Nomura et al. (1991) have documented that al-adrenoceptors are also coupled to a cAMP generation system in isolated hepatocytes from matured male rats. Similarly, activation of al-adrenoceptors in rat cerebral cortex slices has been reported to increase cAMP formation as well as phosphatidylinositol metabolism (Davis et al., 1978; Johnson and Minneman, 1986; Robinson and Kendall, 1990). At present, it is not clear by which mechanism ax-adrenoceptor activation leads to the accumulation of cAMP. Recent evidence suggests that a~-adrenoceptors are not homogeneous but are further divided into at least two pharmacologically distinct subtypes, i.e., alA and a m (reviewed by Minneman, 1988). al-Adrenoceptor subtypes have been defined using competitive antagonists and alkylating agents.
The OtlA subtype has a high affinity for the competitive antagonists WB4101 ( 2 - ( 2 , 6 - d i m e t h o x y p h e n o x y ethyl)aminomethyl-l,4-benzodioxane) (Morrow and Creese, 1986) and 5-methylurapidil (Gross et al., 1988) and is insensitive to alkylation by chloroethylclonidine (Han et al., 1987a; Minneman et al., 1988), whereas the alB subtype is potently inactivated by chloroethylclonidine and has a lower affinity for WB4101 and 5-methylurapidil. Therefore, these adrenergic antagonists appear to be useful probes for investigating the relationship between al-adrenoceptor subtypes and their signal transduction mechanisms. The aim of this study was to clarify whether noradrenaline-induced Ca 2+ mobilization and cAMP accumulation in isolated rat hepatocytes (Nomura et al., 1991) are mediated by a single class of al-adrenoceptor subtypes or by distinct subtypes.
2. Materials and methods 2.1. Materials
Correspondence to: Takahide Nomura, M.D., Department of Pharmacology, Fujita Health University School of Medicine, 1-98 Dengakugakubo, Kutsukake-cho, Toyoake, Aichi 470-11, Japan.
The sources of materials used in this work were as follows: collagenase (CLS 2) from Worthington Biochemical (Freehold, N J, USA), ( - ) - n o r a d r e n a l i n e lay-
114
drochloride, (-)-phenylephrine hydrochloride, methoxamine hydrochloride and 4/3-phorbol 12-myristate 13-acetate (PMA) from Sigma (St. Louis, MO, USA), propranolol hydrochloride from Sumitomo Chemical (Osaka, Japan), prazosin hydrochloride from Pfeizer (Tokyo, Japan), chloroethylclonidine and WB4101 from Research Biochemicals (Natick, MA, USA), fura-2 acetoxymethyl ester (fura-2/AM) from Dojindo Laboratories (Kumamoto, Japan), [Arg-8]vasopressin from Peptide Institute (Minoh, Japan) and pertussis toxin (from Bordetella pertussis) from Kaken Pharmaceuticals (Shiga, Japan). 2.2. Animals Male Wistar rats (8-16 weeks of age, 300-500 g) were used. The rats were allowed free access to water and standard laboratory food. 2.3. Incubation of hepatocytes and analytical methods Isolated rat hepatocytes (Berry and Friend, 1969; Harris, 1975) were suspended at a concentration of 3 X 106 cells/ml in a HEPES buffer containing 120 mM NaCI, 5 mM KC1, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.3 mM CaCIz, 5 mM NaHCO 3 and 10 mM HEPES, pH 7.4. The incubation procedure in the absence of Ca 2+ has been described (Nomura et al., 1987). Intracellular Ca 2÷ concentration ([Ca2+] i) was measured with the fluorescent Ca 2÷ indicator dye, fura-2, essentially as described by Tohkin et al. (1988). Fura-2 loading was achieved by preincubating hepatocytes in the HEPES buffer medium supplemented with 5 ixM fura-2/AM for 20 min at 37°C. The cells were washed three times and kept on ice after being resuspended in the same HEPES buffer medium. Aliquots of cells were warmed immediately before use by incubating at 37°C for 1 min. Cellular fluorescence was determined in a Hitachi Model F-4010 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) equipped with a thermostat-controlled cuvette holder and magnetic stirring. Incubations were conducted for 105 s at 37°C. Agonists were added at 45 s and their effects were followed for 1 min. When the effects of prazosin, propranolol, WB4101 and PMA were being studied, these agents were added at 0 s. Increase in [Ca2+]i was calculated by subtracting [Ca2+]i value immediately before the addition of agonist from the value at the peak of response. To study the action of chloroethylclonidine, hepatocytes were preincubated for 20 min with this agent and then washed three times before being used. In the experiments for the determination of cAMP, hepatocytes were similarly treated, except that dimethylsulfoxide without fura-2/AM was present during the preincubation. Incubations were conducted for 2 min. Agonists were added at 1 min and samples
were removed at 2 min. When used, antagonists and PMA were added at 0 min. Hepatocytes were treated with chloroethylclonidine in the same way as for the measurement of [Ca2+]i . cAMP was determined by radioimmunoassay using a Y A M A S A cyclic AMP assay kit (Yamasa, Tokyo, Japan) in KOH-neutralized HC10 4 extracts of cell suspensions.
3. Results
3.1. Effect of pretreatment with chloroethylclonidine on noradrenaline-induced Ca e+ mobilization in hepatocytes in the presence and absence of extracellular Ca e ÷ In the presence of extracellular Ca 2+, noradrenaline (10 ~M) caused a rapid increase in [Ca2+]i, consisting of a quick transient peak followed by a more sustained component (fig. 1A). We found that pretreatment of hepatocytes with increasing concentrations of chloroethylclonidine produces a progressive inhibition of both components of Ca 2÷ mobilization induced by noradrenaline (fig. 1A). In the absence of extracellular Ca 2+, basal levels of [Ca 2+ ]i were significantly reduced (fig. 1B). Noradrenaline also increased [Ca2+] i in the absence of extracellular Ca z+, although this increase was transient and small, with no sustained component (fig. 1B). Pretreatment with chloroethylclonidine also abolished the response to noradrenaline under this condition (fig. 1B). Like noradrenaline, vasopressin (100 nM) produced increases in [Ca2+]i in both the presence and absence of extracellular Ca 2+ (fig. 1) However, the effect of vasopressin was not affected by chloroethylclonidine (fig. 1). Fig. 2 clearly illustrates that the increase in [Ca2+]i, which is calculated by subtracting the value of [Ca2+] i immediately before the addition of noradrenaline from that at the peak of the response, is dose-dependently blocked by chloroethylclonidine in both the presence and absence of extracellular Ca 2÷. Noradrenaline-induced increase in [Ca2+] i in the presence of extracellular Ca z+ was reduced by 18% and 66% by pretreatment with 10 /zM and 100 ~zM chloroethylclonidine, respectively. The effect of chloroethylclonidine was more marked in the absence of extracellular Ca 2÷, the inhibition being 50% and 93% by 10 /.~M and 100 tzM chloroethylclonidine, respectively. 3.2. Effect of pretreatment with chloroethylclonidine on noradrenaline-induced stimulation of cAMP accumulation in hepatocytes in the presence and absence of extracellular Ca 2 ÷ Fig. 3 indicates that preincubation with chloroethylclonidine dose-dependently inactivated cAMP accumulation induced by noradrenaline in both the presence
115
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and absence of extracellular C a 2+. In the presence of extracellular Ca 2+, noradrenaline-induced cAMP accumulation was reduced by 50% and 75% by pretreatment with 10 ~zM and 100 /zM chloroethylclonidine, respectively. Noradrenaline produced a more marked stimulation of cAMP accumulation in the absence of extracellular C a 2+, confirming the previous data (Nomura et al., 1991). The magnitude of the inhibition by chloroethylclonidine in the absence of extracellular Ca 2+ was similar to values observed in the presence of 200
30
// +Ca 2+
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extracellular Ca 2+ (35% inhibition by 10 /zM chloroethylclonidine and 74% inhibition by 100 /xM chloroethylclonidine). The potent inhibition by chloroethylclonidine of noradrenaline-induced stimulation of cAMP accumulation observed in the present study is consistent with that observed in liver cells for other %-adrenergic responses including C a 2+ mobilization (figs. 1 and 2), phosphatidylinositol labeling (Torres-M~rquez et al., 1991) and lzsI-BE2254 binding (Tsujimoto et al., 1989).
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Fig. 2. Effect of pretreatment with chloroethylclonidine (CEC) on noradrenaline (10 /zM)-induced increase in [Ca 2+ ]i in the presence and absence of extracellular Ca 2÷. Cells were pretreated with various concentrations of CEC as described in the text. Increases in [Ca 2+ ]i were calculated by subtracting basal values of [Ca 2÷ ]i immediately before the addition of noradrenaline from those at the peaks of response. Basal cytosolic Ca 2 ÷ concentrations in the presence and absence of extracellular Ca 2÷ were 277 + 24 nM and 79 4-6 n M, respectively. The data shown are the m e a n s + S.E. of four to six hepatocyte preparations. * P < 0.05, compared with control incubations by Student's t-test.
116
3.3. Comparison o f the antagonizing effects o f prazosin and WB4101 on noradrenaline-induced Ca 2 ÷ mobilization and cAMP accumulation
the response to phenylephrine (10 ~M) was also suppressed by prazosin (1 /xM) and by chloroethylclonidine (100/xM) (data not shown).
Fig. 4 illustrates the effects of prazosin and WB4101 on noradrenaline responsiveness in the presence (fig. 4, panels A and B) and absence (fig. 4, panels C and D) of extracellular Ca 2+. Both noradrenaline-induced Ca 2÷ mobilization (fig. 4, panels A and C) and cAMP accumulation (fig. 4, panels B and D) were blocked by these antagonists, although prazosin was approximately 100 times more potent than WB4101. The maximal effect of prazosin was seen at 10 to 100 nM while that of WB4101 was seen at 10 /zM. Since WB4101 is rapidly metabolized by hepatocytes (Han et al., 1990), the potency of WB4101 observed in the present study may be the one somewhat underestimated. Propranolol (10/xM) failed to affect these noradrenaline actions.
3.5. Pertussis toxin pretreatment o f rats does not affect noradrenaline-stimulated Ca 2 + mobilization and cAMP accumulation in hepatocytes
3.4. Effects of methoxamine and phenylephrine on Ca 2 + mobilization and cAMP accumulation in hepatocytes
Methoxamine (10 izM) exerted little effect on either Ca 2+ mobilization (fig. 5A) or cAMP accumulation (fig. 5B) in hepatocytes. In contrast, phenylephrine (10 ~M) significantly stimulated both, although the effect of this agent was less marked than that of noradrenaline (10/xM) (fig. 5, panels A and B). We observed that
//
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CEC (-10gM) Fig. 3. Effect of pretreatment with chloroethylclonidine (CEC) on noradrenaline (10 /zM)-induced increase in cAMP levels in the presence and absence of extracellular Ca 2+. Cells were pretreated with various concentrations of CEC as described in the text. Noradrenaline-induced increase in cAMP levels was calculated by subtracting basal cAMP levels at 1 min after saline addition from those at 1 min after noradrenaline addition. Basal cAMP levels in the presence and absence of extracellular Ca 2+ were 266+41 p m o l / g wet weight and 274_+45 p m o l / g wet weight, respectively. The data shown are the m e a n s _+S.E. of five to six hepatocyte preparations. * P < 0.05, compared with control incubations by Student's t-test.
It has been documented that a~-adrenoceptors of FRTL5 thyroid cells are coupled to two distinct signal transduction mechanisms, one of which is pertussis toxin-sensitive, and the other pertussis toxin-insensitive (Burch et al., 1986). Thus we decided to examine whether pertussis toxin discriminates between a~adrenoceptor-mediated second messenger responses in isolated rat hepatocytes. Male Wistar rats were injected intravenously with 10/xg toxin/kg body weight in a single dose, 3 days before the experiment was performed. Similar stimulations of cAMP accumulation and Ca 2+ mobilization were elicited by noradrenaline (10/xM) in hepatocytes isolated from toxin-treated rats as for non-treated rats. This is consistent with results reported by Pushpendran et al. (1983), who showed that stimulation of phosphatidylinositol labeling produced by adrenaline is not affected in liver cells from pertussis toxin-treated rats. The negative results of our experiments are not shown. 3.6. Effect of PMA on noradrenaline-induced Ca 2 + mobilization and cAMP accumulation
PMA has been shown to cause desensitization of aradrenoceptor responses in various tissues, including liver and DDTIMF-2 cells, through activation of protein kinase C (Leeb-Lundberg et al., 1985; Corvera et al., 1986). On the other hand, it has been reported that the a 1 response in rabbit aorta is not affected by PMA (Garcia-Sfiinz et al., 1985). To date, it is not clear what effect PMA has on a~-adrenoceptor-mediated stimulation of cAMP accumulation in rat hepatocytes. We, therefore, investigated the effect of PMA both on cAMP accumulation and Ca 2+ mobilization caused by noradrenaline. We found that PMA (1/~M) attenuated the stimulation of Ca 2+ mobilization caused by noradrenaline, while it did not alter the effect of vasopressin (fig. 6A). Similarly, PMA also abolished a noradrenaline-induced increase in cAMP accumulation.
4. Discussion
Differences between ata- and alB-adrenoceptor subtypes have been pharmacologically identified as follows: (1) The atB subtype is sensitive to inactivation by the alkylating agent, chloroethylclonidine, while the alA subtype is insensitive to it (Han et al., 1987a;
117
Minneman et al., 1988; Minneman, 1988). (2) The potency orders of competitive antagonists, WB4101, 5-methylurapidil and prazosin, are prazosin >> 5methylurapidil > WB4101 in am-adrenoceptors and WB4101 >_ 5-methylurapidil = prazosin in axa-adrenoceptors (Torres-M~irquez et al., 1991). (3) Methoxamine potently stimulates a]a-adrenoceptors but has little effect on am-receptors (Garda-Sfiinz et al., 1985; Tsujimoto et al., 1989). Recently, the alc subtype has been identified by molecular biological approaches (Schwinn et al., 1990). This subtype has a high affinity for aaaselective antagonists but is also sensitive to inactivation by chloroethylclonidine (Schwinn et al., 1990; GardaSfiinz et al., 1992). Results of the present study clearly indicate that aFadrenoceptors mediating noradrenal i n e - i n d u c e d Ca 2+ mobilization and cAMP accumula-
tion are alB subtype, judging from the pharmacological criteria. This is the first report which documents that activation of OqB-adrenoceptors leads to cAMP formation as well as Ca 2+ mobilization in isolated rat hepatocytes. It has previously been demonstrated in rat cerebral cortex slices that the ai-adrenoceptors mediating two second messenger responses, namely, inositol phosphate accumulation and cAMP potentiation, are distinct from each other (Robinson and Kendall, 1990). Their findings are in contrast with those of the present study. It is well established that activation of avadrenoceptors causes elevation o f [Ca2+] i through formation of inositol 1,4,5-trisphosphate which promotes the release of stored intracellular Ca 2+ and through the influx of e x t r a c e l l u l a r Ca 2+ (Exton, 1986). It has been proposed
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Fig. 4. Effects of WB4101, prazosin and propranolol on noradrenaline-induced increase in [Ca 2+ ]i and cAMP. Panels A and B: effects of WB4101 ( • ) , prazosin ([]) and propranolol (zx) on noradrenaline (10/xM)-induced increase in [Ca 2+ ]i (A) and cAMP (B) in the presence of extracellular Ca 2+. Panels C and D: effects of WB4101 ( • ) , prazosin (rn) and propranolol (zx) on noradrenaline (10 ~M)-induced increase in [Ca2+ ]i (C) and cAMP (D) in the absence of extracellular Ca 2+. Noradrenaline-induced increases in [Ca 2+ ]i and cAMP were calculated as described for fig. 2 and fig. 3, respectively. Basal cytosolic Ca 2+ concentrations in the presence and absence of extracellular Ca 2+ were 133 ± 13 nM and 54 ± 3 nM, respectively. Basal cAMP levels in the presence and absence of extracellular Ca 2+ were 253 ± 25 p m o l / g wet weight and 267 ± 40 p m o l / g wet weight, respectively. Each point represents the mean ± S.E. of three to five hepatocyte preparations.
118
(A)
that different a r a d r e n o c e p t o r subtypes are linked to different mechanisms for increasing intracellular Ca 2+ (Han et al., 1987b). Activation of o~tB subtype has recently been shown to lead to the stimulation of phosphoinositide turnover (Tsujimoto et al., 1989; Torres-Mfirquez et al., 1991). It is currently hypothesized that alB subtypes are associated with the formation of inositol 1,4,5-trisphosphate and mobilization of intracellular Ca 2+, while the alA subtypes are linked to gating Ca 2+ influx (Han et al., 1987b; Tsujimoto et al., 1989). However, we found that pretreatment with chloroethylclonidine abolished both components of noradrenaline-induced Ca 2+ mobilization, i.e., a quick transient component representing Ca 2+ release from intracellular stores (fig. 1, panels A and B) and a more sustained component representing Ca 2+ influx (fig. 1A). Thus, our data allows us to conclude that am-adrenoceptors of rat hepatocytes promote Ca 2+ influx as well as intracellular Ca 2+ mobilization. This is consistent with recent results in MDCK-D] cells reported by Klijn et al. (1991) and those in DDT~ MF-2 cells by Han et al. (1992). We emphasize that stimulation of cAMP accumulation in response to noradrenaline is not secondary to Ca 2+ mobilization for the following reasons: (1), vasopressin, which produces Ca 2+ mobilization in the same manner as noradrenaline (fig. 1), exerts no effect on cAMP levels (Nomura et al., 1991). (2), no parallel
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200
13.
0 Saline
Nadr
Saline
Nadr
Fig. 6. Effect of P M A on noradrenaline (Nadr)-induced stimulation of Ca ~ + mobilization and cAMP accumulation. Panel A shows the effect of P M A (1 izM) on Ca 2÷ mobilization induced by Nadr (10 ~zM) and vasopressin (Vas, 100 nM) in the presence of extracellular Ca 2÷. Propranolol (Pro, 10/xM) was present when Nadr was added. Traces shown were representative of four or five experiments using different hepatocyte preparations. Panel B shows the effect of P M A (1 /~M) on Nadr (10 p~M)-induced stimulation of cAMP accumulation in the absence of extracellular Ca 2÷. Propranolol ( 1 0 / z M ) was added to all the incubations. The data shown are the m e a n s _+S.E. of four hepatocyte preparations. * P < 0.05, compared with saline addition by Student's t-test.
,~
~ 200
~
400
105
Nadr Mix Phe
Fig. 5. Comparison of the effects of noradrenaline (Nadr), methoxamine (Mtx) and phenylephrine (Phe) on Ca 2÷ mobilization and cAMP accumulation. Panel A shows the effects of Nadr (10 /~M), Mtx (10/zM) and Phe (10/xM) on [Ca 2+ ]i, which were studied in the presence of extracellular Ca 2+. Traces shown were representative of at least four experiments using different hepatocyte preparations. Panel B shows the effects of Nadr (10 /zM), Mtx ( 1 0 / z M ) and Phe (10 /~M) on cAMP levels which were studied in the absence of extracellular Ca z+. Results are expressed as percentages of basal cAMP levels at 1 min. Basal cAMP levels averaged 287 + 40 p m o l / g wet weight. The data shown are the m e a n s + S.E. of at least three hepatocyte preparations. * P < 0.05, compared with basal cAMP levels by Student's t-test.
relationship was found b e t w e e n [Ca2+]i and cAMP levels: namely, noradrenaline-induced increase in [Ca2+]i was larger in the presence of extracellular Ca 2÷ than in the absence of extracellular Ca z+ (fig. 2), while that in cAMP was less marked in the presence of extracellular Ca 2 ÷ than in the absence of extracellular Ca 2÷ (fig. 3). We observed that stimulation of cAMP accumulation by noradrenaline (10 ~M, plus 10 /zM propranolol) became more marked when it was added in the presence of isobutyl methylxanthine (IBMX, 100 /xM) (noradrenaline-induced increase in cAMP levels at 1 min in the absence of extracellular Ca2+: control; 97.6 _+ 14.1 p m o l / g wet weight, IBMX; 272.7 _+ 46.2 p m o l / g wet weight, n = 3). The data suggest that activation of adenylate cyclase is involved in am-adrenoceptor-mediated increases in cAMP levels. In the present study, neither pertussis toxin pretreatment nor PMA addition was found to discriminate
119
between aradrenoceptor-mediated second messenger responses in isolated hepatocytes. This indicates that G i is not involved in the receptor-mediated responsiveness and that both signaling pathways are equally desensitized by activation of protein kinase C. Is it possible that a single class of receptors simultaneously couples to multiple signals? Recently, it has been reported that activation of a single receptor, such as parathyroid hormone receptor (Abou-Samra et al., 1992) or a~adrenoceptor (Cotecchia et al., 1990), expressed in COS-7 cells stimulates intracellular accumulation of both cAMP and inositol phosphates. Furthermore, a2C10, a class of c~2-adrenoceptor subtypes has been demonstrated to couple simultaneously to both G i and G s proteins in CHO ceils (Eason et al., 1992). It remains to be investigated how activation of a madrenoceptors stimulates both cAMP formation and Ca 2÷ mobilization in isolated rat hepatocytes.
Acknowledgement This study was supported by a Grant-in-Aid from Fujita Health University.
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