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Dopamine receptors mediating the stimulation and the inhibition of adenylate cyclase in rat prostate gland
66
Neuroscience Letters, 77 (1987) 66 70 Elsevier Scientific Publishcrs Ireland Ltd
NSL 04586
Dopamine receptors mediating the stimulation and the inhibition of adenylate cyclase in rat prostate gland Daniela A m e n t a 1. Carlo Cavallotti I and Francesco A m e n t a 1,2 IDipartimento di Scienze Neurologiche, Universith 'La Sapienza' and 2Dipartimento di Sanitgt Pubblica e Biologia Cellulare, UniversittJ 'Tor Vergata', Rome (Italy) (Received 8 September 1986; Revised version received 19 December 1986; Accepted 20 January 1987) Key words." Prostate gland; Adenylate cyclase; Dopamine; SCH 23390; Bromocriptine; Rat The effect of dopamine on the 3',5'-cyclic adenosine monophosphate (cAMP) generating system of membrane particles of rat prostate gland was studied. Dopamine increased the concentration of cAMP in a dose-dependent manner. The selective D~ receptor inhibitor SCH 23390 caused a decrease in dopamine-elicited cAMP levels. Any effect of dopamine on prostatic cAMP concentration was abolished by the simultaneous addition to the incubation medium of SCH 23390 and of the D2 receptor blocking agent ( - )-sulpiride. Also the D2 receptor agonist bromocriptine decreased cAMP levels. The present data indicate the existence, in the rat prostate gland, of two types of dopamine receptors mediating, respectively, the activation (D~ effect) and the inhibition (D~ effect) of adenylate cyclase activity.
Ever since the identification of peripheral dopamine (DA) receptors a great effort has been made to characterize physiologically, biochemically and pharmacologically this receptor system and to study the similarities and the dissimilarities between central and peripheral dopamine receptors (for a review see refs. 1 and 2). Although the subject is still under debate, the current opinion is that peripheral DA1 and DA2 receptors are similar to, but not identical to, the central D1 and D2 receptors [1, 2, 4]. The central actions of DA have been demonstrated to be widely mediated by the activation (Di effect) or by the inhibition (D2 effect) of a DA-sensitive adenylate cyclase activity [4, 7, 8]. Since a growing body of evidence indicates the existence of a dopaminergic system in the prostate gland [10] as well as the existence of a DA-dependent cAMP generating system [9, 12, 13], we analyzed, in the present study, the existence of DA receptors coupled to the inhibition of adenylate cyclase in the rat prostate gland.
Correspondence: F. Amenta, Dipartimento di Scienze Neurologiche, Via A. Borelli 50, 1-00161 Roma, Italy. 0304-3940/87,'$ 03 50 © 1987 Elsevier Scientific Publishers Ireland Ltd.
67
Membrane particles of the prostate gland were obtained according to the procedure described by Shima et al. [12]. Ten rats of the Wistar strain (250 g body wt.) were sacrificed by decapitation. The prostate gland was removed, minced and homogenized in ice-cold 10 mM Tris-HCl buffer (pH 7.4) containing EGTA (1.2 mM) by the use of an Ultra-Turrax. The homogenates were centrifuged at 1000 g × 15 rain, the pellets were discarded and the supernatant was centrifuged at 50,000 g x 25 min at 4'~C. The resulting pellets were washed with incubation buffer, resuspended and centrifuged. The final pellets were used for adenylate cyclase assay. Adenylate cyclase was assayed in a 500-i~1 reaction mixture containing Tris-HC1 (80 raM), EGTA (0.6 mM), IBMX (0.5 mM), ATP (2 mM), MgSO4 (16 mM), phosphocreatine (5 mM), creatine phosphokinase (50 U/ml) and ascorbic acid (0.02%). The reaction was started by the addition of 0.5 mg of prostate gland membrane particles. After incubation at 30°C for 20 min, the samples were placed in a boiling water bath for 3 min, and cAMP was isolated by chromatography on Dowex 50W-X4 followed by neutral alumina [11]. Cyclic A M P levels were then measured by radioimmunoassay using the Amersham Radiochemical Center (U.K.) kit. Increasing concentrations on DA (from 10 6 to 10 '~ M) induced a significant increase in cAMP levels (Fig. 1). The stimulatory effect of DA on prostatic cAMP content was slightly potentiated by the simultaneous addition to the incubation medium of the specific D2 receptor antagonist (-)-sulpiride (see ref. 8; Fig. 1). On the contrary the concomitant presence in the incubation medium of the selective D1 receptor
mm(-)-sulp/ride •
dOlummino
•
E H 23390 + (-}-sulpirlde
/ram--"--"-'-
/=.
/
/,~-,
•
gl
-o-
i |
&_
~0 - - - " - - - ~
I 6
O
~
0 ~
I $
0 ~
0 ~
I i
Cy.....__.__.~ 0
I 3
DA (-~M)
Fig. 1. Effect of increasing concentrations of D A alone or in the presence of SCH 23390 (1 /zM), ( - ) sulpiride (l /~M) and both 1 ,uM SCH 23390 and ( - ) - s u l p i r i d e on c A M P content of m e m b r a n e particles of rat prostate gland. Values are the means_+ S.E.M. of 5 7 separate experiments carried out in triplicate. Basal c A M P values were 2.28 _+0.31 pmol/mg tissue/min.
68 "FABLE t EFFECT OF DOPAMINE AND OF ADRENERGIC AND DOPAMINERGIC RECEPTOR BLOCKERS ON ADENYLATE CYCLASE ACTIVITY OF MEMBRANE PARTICLES OF RAT PROSTATE GLAND A quantity of 0.5 mg of prostate gland membrane particles were incubated with the various agents indicated at 30°C for I0 rain. DA was then added and the incubation was continued for a further 20 m m Each result is the mean _+S.E.M. of 6 7 separate incubations carried out in triplicate. *P < 0.001 compared with the value in the presence of DA alone (two-tailed Student's t-test). Drugs ~ M )
cAMP concentration (pmol/mg tissue/min)
None DA (100) DA (100) + Phentolamine (10) DA (100)+(-)-Propranolol (1) DA (100) + Haloperidol (10) DA (I 00) + Fluphenazine (10)
2.28___0.31 6.95__+0.51 6.08_+0.63 6.21 +0.44 3.29_+0.12" 2.07 -+0.09*
antagonist SCH 23390 [3] caused a decrease in cAMP levels below basal values (Fig. 1). It is also interesting that any effect of DA on prostatic adenylate cyclase was abolished by the simultaneous addition of (-)-sulpiride and SCH 23390 (Fig. 1). The stimulatory action of DA on the prostatic cAMP generating system, very likely, was due to an interaction of the catecholamine with specific DA receptors. In fact the above-mentioned effect was unaltered by adrenoceptor blocking drugs, but it was selectively inhibited by the D receptor blockers haloperidol and fluphenazine (Table I).
c 2.1
E
%
i
"X
~, 1.8
\
,. 1.5
1.2 I
0
Ii
1
1
I
I
7
6
5
4
bromocriptlne
....
( - Io 9 M )
Fig. 2. Concentration-dependent inhibition of prostatic adenylate cyclase by bromocriptine. Values are the means _+S.E.M. of 3 5 separate experiments carried out in triplicate.
69
The DA D2 receptor agonist bromocriptine [14] decreased cAMP levels in a dosedependent manner (Fig. 2), suggesting the hypothesis of the existence, in rat prostate gland, of two kinds of DA receptors mediating, respectively, the stimulation (Di effect) and the inhibition (D2 effect) of adenylate cyclase. The identification of the selective Di receptor antagonist SCH 23390 allowed an easier characterization of the effects of DA on the cAMP generating system, and showed that, in the central nervous system, D2 receptors were linked in an inhibitory manner to adenylate cyclase activity [7, 8]. The same mechanism seems to occur in the peripheral organs, where DA receptors coupled in a stimulatory as well as in tin inhibitory manner to adenylate cyclase activity have been described in the rat adrenal cortex as well as in the rabbit renal and mesenteric arteries [5, 6]. Our data which show that DA has both stimulatory and inhibitory effects on adenylate cyclase activity of membrane particles of the rat prostate gland suggest the existence, in the gland, of dopamine Di and D2 receptors. Moreover, these findings seem to indicate that rat central and prostatic DA receptors are identical, at least with regard to Da-sensitive adenylate cyclase activity. The present study was supported in part by grants from the University 'La Sapienza" and from the Italian C.N.R. The kind help of Drs. M. De Rossi and W.L. Collier in the preparation of the manuscript as well as the careful secretarial work done by C. Collier are gratefully acknowledged. SCH 23390 was a gift of Dr. Ongini (Essex Italia): bromocriptine was a gift of Sandoz Prodotti Farmaceutici (Milan, Italy). I Clark, B.J., The role of dopamine in the periphery. In E. Flfickiger, E.E. Mfiller and M.O. Thorner (Eds.), The Dopaminergic System, Springer, Berlin, 1985, pp. 27 39. 2 Goldberg, L.I. and Kohli, J.D., Peripheral pre- and post-synaptic dopamine receptors: are they differcnt from dopamine receptors in the central nervous system'?, Commun. Psychopharmacol., 3 (1979) 447 456. 3 Hyttcl, J., SCH 23390: the first selective dopamine D~ antagonist, Eur. J Pharmacol., 91 (1983) 153 154. 4 Kebabian, J.W. and Calne, D.E., Multiple receptors for dopamine, Nature (London), 277 (1979) 93 96. 5 Missale, C., Memo, M., Liberini, P., Carrubba, M.O. and Spano, P.F., Evidence for the presence of D~ and D: dopamine receptors in the rat adrenal cortex. Eur. J. Pharmacol., 109 (1985) 315 316. 6 Missale, C., Pizzi, M., Memo, M., Picotti, G.B., Carrubba, M.O. and Spano, P.F.. Postsynaptic Dk and D, dopamine receptors are present in rabbit renal and mesenteric arteries, Neurosci. Lcn., 61 (1985) 207 211. 7 ()halt, P., Olianas, M.C. and Gessa, G . L , Selective blockade ofdopamine D~ receptors by SCIt 2339(1 discloses striatal dopamine D, receptors mediating the inhibition of adenylate cyclase in rats, Eur..l. Pharmacol., 9 (1984) 127 128. 8 0 n a l i , P., Olianas, M.C. and Gessa, G.L., Characterization of dopamine receptors mediating inhibition of adenylaie cyclase activity in rat striatum, Mol. Pharmacol., 28 (1985) 138 145. 9 Purvis, K., Rut, H., Gordeladze, J.O. and Attramadal, H., Hormonal activation of the adenylyl cyclases of the rat and human prostate gland, Prostate, 8 (1986) 11 24. I0 Relja, M., Dopaminergic receptors in genitourinary system: basic and clinical aspects. In E.S. Vizi and K. Magyar (Eds.), Regulation of Transmitter Function (Proc. 5th Meeting Eur. Soc. Neurochem.), Akademiai Kiado, Budapest, [984, pp. 39 47.
70 11 Salomon, Y.D., Londos, D. and Rodbell, M., A highy sensitive adenylate cyclase assay, Anal. Biochem., 58 (1974) 541-548. 12 Shima, S., Kawashima, Y., Hirai, M., Asakura, M. and Kouyama, H., Effect ofadrenergic stimulation on adenylate cyclase activity in the prostate, Biochim. Biophys. Acta, 628 (1980) 255 262, 13 Shima, S., Hirai, M., Asakura, M. and Kouyama, H., Regulation of adenylate cyclase system by catc cholamines in the prostate gland, Naunyn-Schmiedeberg's Arch. Pharmacol., 312 (1980) 19 22. 14 Trabucchi, M., Spano, P.F., Tonon, G.C. and Frattola, L., Effects of bromocriptine on central dopaminergic receptors, Life Sci., 19 (1976) 225 232.
Neuroscience Letters, 77 (1987) 66 70 Elsevier Scientific Publishcrs Ireland Ltd
NSL 04586
Dopamine receptors mediating the stimulation and the inhibition of adenylate cyclase in rat prostate gland Daniela A m e n t a 1. Carlo Cavallotti I and Francesco A m e n t a 1,2 IDipartimento di Scienze Neurologiche, Universith 'La Sapienza' and 2Dipartimento di Sanitgt Pubblica e Biologia Cellulare, UniversittJ 'Tor Vergata', Rome (Italy) (Received 8 September 1986; Revised version received 19 December 1986; Accepted 20 January 1987) Key words." Prostate gland; Adenylate cyclase; Dopamine; SCH 23390; Bromocriptine; Rat The effect of dopamine on the 3',5'-cyclic adenosine monophosphate (cAMP) generating system of membrane particles of rat prostate gland was studied. Dopamine increased the concentration of cAMP in a dose-dependent manner. The selective D~ receptor inhibitor SCH 23390 caused a decrease in dopamine-elicited cAMP levels. Any effect of dopamine on prostatic cAMP concentration was abolished by the simultaneous addition to the incubation medium of SCH 23390 and of the D2 receptor blocking agent ( - )-sulpiride. Also the D2 receptor agonist bromocriptine decreased cAMP levels. The present data indicate the existence, in the rat prostate gland, of two types of dopamine receptors mediating, respectively, the activation (D~ effect) and the inhibition (D~ effect) of adenylate cyclase activity.
Ever since the identification of peripheral dopamine (DA) receptors a great effort has been made to characterize physiologically, biochemically and pharmacologically this receptor system and to study the similarities and the dissimilarities between central and peripheral dopamine receptors (for a review see refs. 1 and 2). Although the subject is still under debate, the current opinion is that peripheral DA1 and DA2 receptors are similar to, but not identical to, the central D1 and D2 receptors [1, 2, 4]. The central actions of DA have been demonstrated to be widely mediated by the activation (Di effect) or by the inhibition (D2 effect) of a DA-sensitive adenylate cyclase activity [4, 7, 8]. Since a growing body of evidence indicates the existence of a dopaminergic system in the prostate gland [10] as well as the existence of a DA-dependent cAMP generating system [9, 12, 13], we analyzed, in the present study, the existence of DA receptors coupled to the inhibition of adenylate cyclase in the rat prostate gland.
Correspondence: F. Amenta, Dipartimento di Scienze Neurologiche, Via A. Borelli 50, 1-00161 Roma, Italy. 0304-3940/87,'$ 03 50 © 1987 Elsevier Scientific Publishers Ireland Ltd.
67
Membrane particles of the prostate gland were obtained according to the procedure described by Shima et al. [12]. Ten rats of the Wistar strain (250 g body wt.) were sacrificed by decapitation. The prostate gland was removed, minced and homogenized in ice-cold 10 mM Tris-HCl buffer (pH 7.4) containing EGTA (1.2 mM) by the use of an Ultra-Turrax. The homogenates were centrifuged at 1000 g × 15 rain, the pellets were discarded and the supernatant was centrifuged at 50,000 g x 25 min at 4'~C. The resulting pellets were washed with incubation buffer, resuspended and centrifuged. The final pellets were used for adenylate cyclase assay. Adenylate cyclase was assayed in a 500-i~1 reaction mixture containing Tris-HC1 (80 raM), EGTA (0.6 mM), IBMX (0.5 mM), ATP (2 mM), MgSO4 (16 mM), phosphocreatine (5 mM), creatine phosphokinase (50 U/ml) and ascorbic acid (0.02%). The reaction was started by the addition of 0.5 mg of prostate gland membrane particles. After incubation at 30°C for 20 min, the samples were placed in a boiling water bath for 3 min, and cAMP was isolated by chromatography on Dowex 50W-X4 followed by neutral alumina [11]. Cyclic A M P levels were then measured by radioimmunoassay using the Amersham Radiochemical Center (U.K.) kit. Increasing concentrations on DA (from 10 6 to 10 '~ M) induced a significant increase in cAMP levels (Fig. 1). The stimulatory effect of DA on prostatic cAMP content was slightly potentiated by the simultaneous addition to the incubation medium of the specific D2 receptor antagonist (-)-sulpiride (see ref. 8; Fig. 1). On the contrary the concomitant presence in the incubation medium of the selective D1 receptor
mm(-)-sulp/ride •
dOlummino
•
E H 23390 + (-}-sulpirlde
/ram--"--"-'-
/=.
/
/,~-,
•
gl
-o-
i |
&_
~0 - - - " - - - ~
I 6
O
~
0 ~
I $
0 ~
0 ~
I i
Cy.....__.__.~ 0
I 3
DA (-~M)
Fig. 1. Effect of increasing concentrations of D A alone or in the presence of SCH 23390 (1 /zM), ( - ) sulpiride (l /~M) and both 1 ,uM SCH 23390 and ( - ) - s u l p i r i d e on c A M P content of m e m b r a n e particles of rat prostate gland. Values are the means_+ S.E.M. of 5 7 separate experiments carried out in triplicate. Basal c A M P values were 2.28 _+0.31 pmol/mg tissue/min.
68 "FABLE t EFFECT OF DOPAMINE AND OF ADRENERGIC AND DOPAMINERGIC RECEPTOR BLOCKERS ON ADENYLATE CYCLASE ACTIVITY OF MEMBRANE PARTICLES OF RAT PROSTATE GLAND A quantity of 0.5 mg of prostate gland membrane particles were incubated with the various agents indicated at 30°C for I0 rain. DA was then added and the incubation was continued for a further 20 m m Each result is the mean _+S.E.M. of 6 7 separate incubations carried out in triplicate. *P < 0.001 compared with the value in the presence of DA alone (two-tailed Student's t-test). Drugs ~ M )
cAMP concentration (pmol/mg tissue/min)
None DA (100) DA (100) + Phentolamine (10) DA (100)+(-)-Propranolol (1) DA (100) + Haloperidol (10) DA (I 00) + Fluphenazine (10)
2.28___0.31 6.95__+0.51 6.08_+0.63 6.21 +0.44 3.29_+0.12" 2.07 -+0.09*
antagonist SCH 23390 [3] caused a decrease in cAMP levels below basal values (Fig. 1). It is also interesting that any effect of DA on prostatic adenylate cyclase was abolished by the simultaneous addition of (-)-sulpiride and SCH 23390 (Fig. 1). The stimulatory action of DA on the prostatic cAMP generating system, very likely, was due to an interaction of the catecholamine with specific DA receptors. In fact the above-mentioned effect was unaltered by adrenoceptor blocking drugs, but it was selectively inhibited by the D receptor blockers haloperidol and fluphenazine (Table I).
c 2.1
E
%
i
"X
~, 1.8
\
,. 1.5
1.2 I
0
Ii
1
1
I
I
7
6
5
4
bromocriptlne
....
( - Io 9 M )
Fig. 2. Concentration-dependent inhibition of prostatic adenylate cyclase by bromocriptine. Values are the means _+S.E.M. of 3 5 separate experiments carried out in triplicate.
69
The DA D2 receptor agonist bromocriptine [14] decreased cAMP levels in a dosedependent manner (Fig. 2), suggesting the hypothesis of the existence, in rat prostate gland, of two kinds of DA receptors mediating, respectively, the stimulation (Di effect) and the inhibition (D2 effect) of adenylate cyclase. The identification of the selective Di receptor antagonist SCH 23390 allowed an easier characterization of the effects of DA on the cAMP generating system, and showed that, in the central nervous system, D2 receptors were linked in an inhibitory manner to adenylate cyclase activity [7, 8]. The same mechanism seems to occur in the peripheral organs, where DA receptors coupled in a stimulatory as well as in tin inhibitory manner to adenylate cyclase activity have been described in the rat adrenal cortex as well as in the rabbit renal and mesenteric arteries [5, 6]. Our data which show that DA has both stimulatory and inhibitory effects on adenylate cyclase activity of membrane particles of the rat prostate gland suggest the existence, in the gland, of dopamine Di and D2 receptors. Moreover, these findings seem to indicate that rat central and prostatic DA receptors are identical, at least with regard to Da-sensitive adenylate cyclase activity. The present study was supported in part by grants from the University 'La Sapienza" and from the Italian C.N.R. The kind help of Drs. M. De Rossi and W.L. Collier in the preparation of the manuscript as well as the careful secretarial work done by C. Collier are gratefully acknowledged. SCH 23390 was a gift of Dr. Ongini (Essex Italia): bromocriptine was a gift of Sandoz Prodotti Farmaceutici (Milan, Italy). I Clark, B.J., The role of dopamine in the periphery. In E. Flfickiger, E.E. Mfiller and M.O. Thorner (Eds.), The Dopaminergic System, Springer, Berlin, 1985, pp. 27 39. 2 Goldberg, L.I. and Kohli, J.D., Peripheral pre- and post-synaptic dopamine receptors: are they differcnt from dopamine receptors in the central nervous system'?, Commun. Psychopharmacol., 3 (1979) 447 456. 3 Hyttcl, J., SCH 23390: the first selective dopamine D~ antagonist, Eur. J Pharmacol., 91 (1983) 153 154. 4 Kebabian, J.W. and Calne, D.E., Multiple receptors for dopamine, Nature (London), 277 (1979) 93 96. 5 Missale, C., Memo, M., Liberini, P., Carrubba, M.O. and Spano, P.F., Evidence for the presence of D~ and D: dopamine receptors in the rat adrenal cortex. Eur. J. Pharmacol., 109 (1985) 315 316. 6 Missale, C., Pizzi, M., Memo, M., Picotti, G.B., Carrubba, M.O. and Spano, P.F.. Postsynaptic Dk and D, dopamine receptors are present in rabbit renal and mesenteric arteries, Neurosci. Lcn., 61 (1985) 207 211. 7 ()halt, P., Olianas, M.C. and Gessa, G . L , Selective blockade ofdopamine D~ receptors by SCIt 2339(1 discloses striatal dopamine D, receptors mediating the inhibition of adenylate cyclase in rats, Eur..l. Pharmacol., 9 (1984) 127 128. 8 0 n a l i , P., Olianas, M.C. and Gessa, G.L., Characterization of dopamine receptors mediating inhibition of adenylaie cyclase activity in rat striatum, Mol. Pharmacol., 28 (1985) 138 145. 9 Purvis, K., Rut, H., Gordeladze, J.O. and Attramadal, H., Hormonal activation of the adenylyl cyclases of the rat and human prostate gland, Prostate, 8 (1986) 11 24. I0 Relja, M., Dopaminergic receptors in genitourinary system: basic and clinical aspects. In E.S. Vizi and K. Magyar (Eds.), Regulation of Transmitter Function (Proc. 5th Meeting Eur. Soc. Neurochem.), Akademiai Kiado, Budapest, [984, pp. 39 47.
70 11 Salomon, Y.D., Londos, D. and Rodbell, M., A highy sensitive adenylate cyclase assay, Anal. Biochem., 58 (1974) 541-548. 12 Shima, S., Kawashima, Y., Hirai, M., Asakura, M. and Kouyama, H., Effect ofadrenergic stimulation on adenylate cyclase activity in the prostate, Biochim. Biophys. Acta, 628 (1980) 255 262, 13 Shima, S., Hirai, M., Asakura, M. and Kouyama, H., Regulation of adenylate cyclase system by catc cholamines in the prostate gland, Naunyn-Schmiedeberg's Arch. Pharmacol., 312 (1980) 19 22. 14 Trabucchi, M., Spano, P.F., Tonon, G.C. and Frattola, L., Effects of bromocriptine on central dopaminergic receptors, Life Sci., 19 (1976) 225 232.