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The possible role of calmodulin in the inhibition of prolactin secretion by dopaminergic antagonists
Life Sciences, Vol. 33, pp. 889-893 Printed in the U.S.A.
Pergamon Press
THE POSSIBLE ROLE OF CALMODULIN IN THE INHIBITION OF PROLACTIN SECRETION BY DOPAMINERGIC ANTAGONISTS Janet E. Merritt, +Stephen TomlJnson and Barry L. Brown. Department of Human Metabolism and Clln~ca] Biochemistry, University of Sheffield Medical School, Beech Hill Road, Sheffield SIO 2RX and +Department of Medicine, Northern General Hospital, Sheffield $5, U.K. (Received in final form June 13, 1983) Summary Several previous reports have indicated that a number of dopamlnerg~c antagonists paradoxically inhibit prolactln secretion at mlcromolar concentrations. It is well known that some of these drugs, including plmozide and the phenothlazlnes, are inhibitors of calmodulln activity. Here we report that mlcromolar concentrations of several dopaminerglc antagonists inhibit prolactln secretion from isolated rat anterior pituitary cells and ealmodulin activity (calmodulln-actlvated cyclic GMP phosphodlesterase). Inhibition of calmodulln activity may thus,at least partially, explain the inhlb~tory effect of these drugs on prolactln secretion.
It is well known that administration of dopamlnerglc antagonists in vlvo leads to an elevation in the prolactln concentration in serum, and that these agents (at nanomolar concentrations) increase prolactln secretion in vitro in the presence of dopamlne. However, a number of dopamlnerglc antagonists have been reported to paradoxically inhibit basal prolactln release at high (mlcromolar) concentrations (I-4). This inhibitory effect has been reported for plmozlde (1-3), haloperldol (1,2,4), both d- and I- butaclamol (3), chlorpromazlne (4), metoclopramlde (4) and domperldone (4). It is now established that plmozlde, at mleromolar concentrations, inhibits calmodulln activity (5). It has been suggested that the inhibitory effect of plmozlde on LH secretion is evidence for a role for calmodulln in the secretory process (6). Similarly, phenothlazlnes have been shown to inhibit both calmodulin activity and prolactln secretion over a comparable concentration range (7,8).
We have examined whether the other dopaminerglc antagonists also inhibit prolactln secretion via an effect on calmodulin activity. Thus, the potencies of each drug on inhibition of prolactln secretion from isolated rat anterior pituitary cells and on inhibition of calmodulin-actlvated cyclic GMP phosphodlesterase have been compared.
Methods Materials. Pimozlde, domperldone and flusplriline were gifts from Janssen Pharmaceutlca, Beerse, Belgium. Metoelopramlde was a gift from Beecham Pharmaceuticals, Gt. Burgh, Epsom and haloperldol was a gift from G.D. Searle & Co. Ltd., Morpeth. [JH] cyclic GMP was from Amersham International plc., Amersham, Bucks. Cyclic GMP, snake venom (Ophlophagus hannah) and Dowex anion exchange resin (ix8, 200-400 mesh) were from Sigma (London) Chemical Co., 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
890
Calmoduiin and Prolactin Secretion
Vol. 33, No. 9, 1983
Poole, Dorset. Calmodulin-deficlent phosphodlesterase was prepared from pig brain essentially as described by Thompson et al (9) up to the DEAE cellulose step. Calmodulin was prepared from boiled (6 min) extracts of pig brain by DEAE cellulose chromatography. The materials for radioimmunoassay of prolactln were from the National Pituitary Agency, National Institutes of Health, USA. Assessment of drug effects on pro lactln secretion. Anterior pituitary cells were prepared from adult male Sprague Dawley rats as previously described (7,10), and maintained in monolayeg culture (in Ham FIO with 10% fetal calf serum) in multlwel] plates at 7.10 cells/well for 3 days before use in the incubations. The cells were incubated (in triplicate wells) with the drugs for 1 hr in Krebs-Ringer bicarbonate pH7.6 (I ml) as previously described (7). The drugs were first solubilized (at lOmM) in dlmethylsulphoxide (DMSO), which was also included in the control incubations at a final concentration of 1% throughout. Prolactin released into the medium in each well was measured in triplicate by radlolmmunoassay using the National Pituitary Agency's RP-I as the standard. Basal levels of prolactin secretion were in the range of 30-70 ng/ml/hr from each well. Assessment of drug effects on calmodulin activity in the calmodulln-actlvated cyellc GMP phosphodlesterase assay. Phosphodiesterase activity was measured using the method of Thompson et al (ll) with cyclic GMP as the substrate in a 15 minute incubation which was terminated by boiling. The product, 5'GMP, was then converted to guanosine by snake venom 5' nucleot~dase. The guanosine was separated from unreacted cyclic GMP using Dowex ~nion exchange resin. The cyclic GMP substrate (2.5~M) contained I00 nCi [ H]-cyclic GMP, and sufficient calmodulin was added to produce 60-80% of the maximal response. The drugs were first solubillsed in DMSO. All incubations were performed in duplicate within each experiment. Calculatlon of results. From each experiment, dose-response curves were obtained (see for example, Figure i) for the inhibition of either prolactln secretion or calmodulin activity from wblch the drug concentration required to produce half-maximal inhibition (IC50) was derived. Each drug was studied at 5 doses between 0.01~M-100~M in at least 2 separate experiments and the IC50 values are expressed as the mean and range.
Results Figure I shows the inhibitory effect of the dopamlnerglc antagonist, domperldone, on calmodulin activity assessed as the inhibition of calmodullnactivated cyclic GMP phosphodlesterase. A dose-dependent inhibitory effect is evident in the mlcromolar concentration range, which is similar to the previously observed effects on prolactln secretion (4). Table I shows the potencies of several dopamlnerglc antagonists in inhibiting both basal prolactln secretion from isolated rat anterior pituitary cells and calmodulln activity based on its activation of cyclic GMP phosphodlesterase. It is clear that a number of dopamlnerglc antagonists of several types inhibit both prolactln secretion and calmodulln activity at mlcromolar concentrations. It is of interest that metoclopramlde, which inhibits prolactln secretion, has no effect on calmodulln activity over the dose range studied. The effect of dopamlne on both systems is included for comparison and it is clear that it has no direct effect on calmodulln activity.
Discussion. The data demonstrate that several dopaminerglc antagonists, in addition
Vol. 33, No. 9, 1983
Calmodulin and Prolactin Secretion
891
125
100
75"
! .Is
| 26"
1
10
DOMPERIOONIE
100
O~M)
FIG. i The effect of domperidone on calmodulin-activated cyclic GMP phosphodiesterase activity, which is expressed as pmol cyclic GMP hydrolysed per 15 mln. Each point represents the mean and range of duplicate measurements. 'Basal' represents the enzyme activity in the absence of calmodulin.
to those known to inhibit calmodulin (e.g. pimozide, haloperidol and the phenothiazines) are Inhibitors of calmodulin activity at micromolar concentrations. These results could thus explain the previously reported "paradoxical" inhibitory effects of several other dopaminergic antagonists including domperidone on prolactin secretion (1-4). It is of interest that, of the drugs studied which inhibited prolactin secretion, only metoclopramide had no effect on calmodulin activity. This is in agreement with previously reported data that metoclopramide does not bind to calmodulin (5). The potency ranges for plmozide and haloperidol on calmodulin activity are also in agreement with those previously reported by Weiss et al (5). In addition, the potency of pimozide in inhibiting LH secretion (6) is similar to its effects on prolactin secretion reported here. Indeed, in the present study the maximal effect of these agents on prolactin secretion (to between 40% and 60% of control values) is similar to those previously reported (1-4).
892
Calmodulin and Prolactin Secretion
Vol. 33, No. 9, 1983
TABLE I
Comparison of Drug Potencies for Inhibition of Prolactin Secretion and Calmodulln Activity. The IC50 values are given as the mean and range.
IC50 (~M) Prolactln secretion
Drug
Pimozlde Domperldone Metoclopramlde Haloperldol Flusplrillne Dopamlne
0.25 4 6 9 0.2 0.001
(0.2-0.3) (3-5) (3-11) (5-16) (0.2-0.2)
IC50 (~M) Calmodulln activity
3 5 >I00 45 7 >100
(2-5) (5-6) (40-56) (4-13)
It has been recently reported that I0 nM domperidone inhibits prolactln, but not growth hormone, secretion with a slow onset and recovery on removal of the drug (12). Since this effect is apparently specific for prolactln secretion, is slow in onset and occurs at nanomolar concentrations, it is very unlikely to involve an effect on calmodulln activity. There is conslderable evidence that calcium is involved in prolactln secretion (13-15) and that calmodulin plays a role in stlmulus-secretlon coupling in the anterior pituitary gland (6-8,16). Our own studies, using the same cell incubation conditions as in the present study have shown that depolarizing doses of K + increase prolactln secretion, and that Inhlbltors of calcium flux and known calmodulln antagonists inhibit secretion (7,8,10,17). The inhibitory effects of mlcromolar concentrations of several dopamlnerglc antagonists may thus be, at least partially, mediated via an effect on ealmodulln activity. However, most of these drugs appear to have more potent inhibitory effects on prolactin secretion than calmodulln activity, which may suggest that they exert multiple inhibitory effects in the pituitary with a component of the inhibition acting through calmodulin. Another possible site of inhibition may be through an effect on phosphollplds since we have recently found that both domperidone and trlfluoperazlne inhibit both phosphollpld-dependent and calmodulln-dependent protein klnases in the pituitary (unpublished observations). Thus, while it appears that the paradoxical effects of dopamlnerglc antagonists may be mediated via calmodulln-dependent processes, care should be exercised in interpreting the effects of these drugs on prolactln secretion, since they may also act on other systems in the cell.
i. 2. 3. 4. 5. 6.
References. R.M.MacLEOD and S.W.J.LAMBERTS, Endocrinology, 103, 200-203 (1978). B.WEST and P.S.DANNIES, Endocrinology, 104, 877-880 (1979). C.DENEF and J.J.FOLLEBOUCKT, Life Sci., 23, 431-436 (1978). G.M.BESSER, G.DELITALA, A.GROSSMAN, W.A.STUBBS and T.YEO, Br. J. Pharmac., 71, 569-573 (1980). B.WEISS, W.PROZIALECK, M.CIMINO, M.SELLINGER-BARNETTE and T.L.WALLACE, Ann. NY Acad. Sci., 356, 319-345 (1980). P.M.CONN, D.C.ROGERS AND T.SHEFFIELD, Endocrinology, 109, 1122-1126 (1981).
Vol. 33, No. 9, 1983
7. 8.
9. i0. ii. 12. 13. 14. 15. 16. 17.
Calmodulin and Prolactin Secretion
J.E.MERRITT, S.TOMLINSON and B.L.BROWN, FEBS Lett. 135, 107-110 (1981). J.E.MERRITT, S.MAC NELL, S.TOMLINSON AND B.L.BROWN. J. Endocr. (In press). W.J.THOMPSON, P.M.EPSTEIN, S.J.STRADA, Biochemistry, 18, 5228-5237 (1979). M.P.SCHREY, B.L.BROWN and R.P.EKINS, Mol. Cell Endocr. 3, 271-282 (1977). W.J.THOMPSON, W.L.TERASKI, P.M.EPSTEIN and S.J.STRADA, Adv. Cycllc Nuc. Res., i0, 69-92 (1979). A.M.BENTLEY and M.WALLIS, J.Endocr., 94, 317-326 (1982). M.C.GERSHENGORN, Mol. Cell. Biochem., 45, 163-179(1982). M.O.THORNER, J.T.HACKETT, F.MURAD and R.M.MACLEOD, Neuroendocrinology, 31, 390-402 (1980). C.M.MORIARTY, Life Sei. 23,185-194 (1978). A.FLECKMAN, J.ERLICHMAN, U.K.SCHUBERT and N.FLEISCHER, Endocrlnology, 108, 2072-2077 (1981). B.L.BROWN, J.E.MERRITT AND S.TOMLINSON, Clin. Scl. 61, 23P (1981).
893
Pergamon Press
THE POSSIBLE ROLE OF CALMODULIN IN THE INHIBITION OF PROLACTIN SECRETION BY DOPAMINERGIC ANTAGONISTS Janet E. Merritt, +Stephen TomlJnson and Barry L. Brown. Department of Human Metabolism and Clln~ca] Biochemistry, University of Sheffield Medical School, Beech Hill Road, Sheffield SIO 2RX and +Department of Medicine, Northern General Hospital, Sheffield $5, U.K. (Received in final form June 13, 1983) Summary Several previous reports have indicated that a number of dopamlnerg~c antagonists paradoxically inhibit prolactln secretion at mlcromolar concentrations. It is well known that some of these drugs, including plmozide and the phenothlazlnes, are inhibitors of calmodulln activity. Here we report that mlcromolar concentrations of several dopaminerglc antagonists inhibit prolactln secretion from isolated rat anterior pituitary cells and ealmodulin activity (calmodulln-actlvated cyclic GMP phosphodlesterase). Inhibition of calmodulln activity may thus,at least partially, explain the inhlb~tory effect of these drugs on prolactln secretion.
It is well known that administration of dopamlnerglc antagonists in vlvo leads to an elevation in the prolactln concentration in serum, and that these agents (at nanomolar concentrations) increase prolactln secretion in vitro in the presence of dopamlne. However, a number of dopamlnerglc antagonists have been reported to paradoxically inhibit basal prolactln release at high (mlcromolar) concentrations (I-4). This inhibitory effect has been reported for plmozlde (1-3), haloperldol (1,2,4), both d- and I- butaclamol (3), chlorpromazlne (4), metoclopramlde (4) and domperldone (4). It is now established that plmozlde, at mleromolar concentrations, inhibits calmodulln activity (5). It has been suggested that the inhibitory effect of plmozlde on LH secretion is evidence for a role for calmodulln in the secretory process (6). Similarly, phenothlazlnes have been shown to inhibit both calmodulin activity and prolactln secretion over a comparable concentration range (7,8).
We have examined whether the other dopaminerglc antagonists also inhibit prolactln secretion via an effect on calmodulin activity. Thus, the potencies of each drug on inhibition of prolactln secretion from isolated rat anterior pituitary cells and on inhibition of calmodulin-actlvated cyclic GMP phosphodlesterase have been compared.
Methods Materials. Pimozlde, domperldone and flusplriline were gifts from Janssen Pharmaceutlca, Beerse, Belgium. Metoelopramlde was a gift from Beecham Pharmaceuticals, Gt. Burgh, Epsom and haloperldol was a gift from G.D. Searle & Co. Ltd., Morpeth. [JH] cyclic GMP was from Amersham International plc., Amersham, Bucks. Cyclic GMP, snake venom (Ophlophagus hannah) and Dowex anion exchange resin (ix8, 200-400 mesh) were from Sigma (London) Chemical Co., 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
890
Calmoduiin and Prolactin Secretion
Vol. 33, No. 9, 1983
Poole, Dorset. Calmodulin-deficlent phosphodlesterase was prepared from pig brain essentially as described by Thompson et al (9) up to the DEAE cellulose step. Calmodulin was prepared from boiled (6 min) extracts of pig brain by DEAE cellulose chromatography. The materials for radioimmunoassay of prolactln were from the National Pituitary Agency, National Institutes of Health, USA. Assessment of drug effects on pro lactln secretion. Anterior pituitary cells were prepared from adult male Sprague Dawley rats as previously described (7,10), and maintained in monolayeg culture (in Ham FIO with 10% fetal calf serum) in multlwel] plates at 7.10 cells/well for 3 days before use in the incubations. The cells were incubated (in triplicate wells) with the drugs for 1 hr in Krebs-Ringer bicarbonate pH7.6 (I ml) as previously described (7). The drugs were first solubilized (at lOmM) in dlmethylsulphoxide (DMSO), which was also included in the control incubations at a final concentration of 1% throughout. Prolactin released into the medium in each well was measured in triplicate by radlolmmunoassay using the National Pituitary Agency's RP-I as the standard. Basal levels of prolactin secretion were in the range of 30-70 ng/ml/hr from each well. Assessment of drug effects on calmodulin activity in the calmodulln-actlvated cyellc GMP phosphodlesterase assay. Phosphodiesterase activity was measured using the method of Thompson et al (ll) with cyclic GMP as the substrate in a 15 minute incubation which was terminated by boiling. The product, 5'GMP, was then converted to guanosine by snake venom 5' nucleot~dase. The guanosine was separated from unreacted cyclic GMP using Dowex ~nion exchange resin. The cyclic GMP substrate (2.5~M) contained I00 nCi [ H]-cyclic GMP, and sufficient calmodulin was added to produce 60-80% of the maximal response. The drugs were first solubillsed in DMSO. All incubations were performed in duplicate within each experiment. Calculatlon of results. From each experiment, dose-response curves were obtained (see for example, Figure i) for the inhibition of either prolactln secretion or calmodulin activity from wblch the drug concentration required to produce half-maximal inhibition (IC50) was derived. Each drug was studied at 5 doses between 0.01~M-100~M in at least 2 separate experiments and the IC50 values are expressed as the mean and range.
Results Figure I shows the inhibitory effect of the dopamlnerglc antagonist, domperldone, on calmodulin activity assessed as the inhibition of calmodullnactivated cyclic GMP phosphodlesterase. A dose-dependent inhibitory effect is evident in the mlcromolar concentration range, which is similar to the previously observed effects on prolactln secretion (4). Table I shows the potencies of several dopamlnerglc antagonists in inhibiting both basal prolactln secretion from isolated rat anterior pituitary cells and calmodulln activity based on its activation of cyclic GMP phosphodlesterase. It is clear that a number of dopamlnerglc antagonists of several types inhibit both prolactln secretion and calmodulln activity at mlcromolar concentrations. It is of interest that metoclopramlde, which inhibits prolactln secretion, has no effect on calmodulln activity over the dose range studied. The effect of dopamlne on both systems is included for comparison and it is clear that it has no direct effect on calmodulln activity.
Discussion. The data demonstrate that several dopaminerglc antagonists, in addition
Vol. 33, No. 9, 1983
Calmodulin and Prolactin Secretion
891
125
100
75"
! .Is
| 26"
1
10
DOMPERIOONIE
100
O~M)
FIG. i The effect of domperidone on calmodulin-activated cyclic GMP phosphodiesterase activity, which is expressed as pmol cyclic GMP hydrolysed per 15 mln. Each point represents the mean and range of duplicate measurements. 'Basal' represents the enzyme activity in the absence of calmodulin.
to those known to inhibit calmodulin (e.g. pimozide, haloperidol and the phenothiazines) are Inhibitors of calmodulin activity at micromolar concentrations. These results could thus explain the previously reported "paradoxical" inhibitory effects of several other dopaminergic antagonists including domperidone on prolactin secretion (1-4). It is of interest that, of the drugs studied which inhibited prolactin secretion, only metoclopramide had no effect on calmodulin activity. This is in agreement with previously reported data that metoclopramide does not bind to calmodulin (5). The potency ranges for plmozide and haloperidol on calmodulin activity are also in agreement with those previously reported by Weiss et al (5). In addition, the potency of pimozide in inhibiting LH secretion (6) is similar to its effects on prolactin secretion reported here. Indeed, in the present study the maximal effect of these agents on prolactin secretion (to between 40% and 60% of control values) is similar to those previously reported (1-4).
892
Calmodulin and Prolactin Secretion
Vol. 33, No. 9, 1983
TABLE I
Comparison of Drug Potencies for Inhibition of Prolactin Secretion and Calmodulln Activity. The IC50 values are given as the mean and range.
IC50 (~M) Prolactln secretion
Drug
Pimozlde Domperldone Metoclopramlde Haloperldol Flusplrillne Dopamlne
0.25 4 6 9 0.2 0.001
(0.2-0.3) (3-5) (3-11) (5-16) (0.2-0.2)
IC50 (~M) Calmodulln activity
3 5 >I00 45 7 >100
(2-5) (5-6) (40-56) (4-13)
It has been recently reported that I0 nM domperidone inhibits prolactln, but not growth hormone, secretion with a slow onset and recovery on removal of the drug (12). Since this effect is apparently specific for prolactln secretion, is slow in onset and occurs at nanomolar concentrations, it is very unlikely to involve an effect on calmodulln activity. There is conslderable evidence that calcium is involved in prolactln secretion (13-15) and that calmodulin plays a role in stlmulus-secretlon coupling in the anterior pituitary gland (6-8,16). Our own studies, using the same cell incubation conditions as in the present study have shown that depolarizing doses of K + increase prolactln secretion, and that Inhlbltors of calcium flux and known calmodulln antagonists inhibit secretion (7,8,10,17). The inhibitory effects of mlcromolar concentrations of several dopamlnerglc antagonists may thus be, at least partially, mediated via an effect on ealmodulln activity. However, most of these drugs appear to have more potent inhibitory effects on prolactin secretion than calmodulln activity, which may suggest that they exert multiple inhibitory effects in the pituitary with a component of the inhibition acting through calmodulin. Another possible site of inhibition may be through an effect on phosphollplds since we have recently found that both domperidone and trlfluoperazlne inhibit both phosphollpld-dependent and calmodulln-dependent protein klnases in the pituitary (unpublished observations). Thus, while it appears that the paradoxical effects of dopamlnerglc antagonists may be mediated via calmodulln-dependent processes, care should be exercised in interpreting the effects of these drugs on prolactln secretion, since they may also act on other systems in the cell.
i. 2. 3. 4. 5. 6.
References. R.M.MacLEOD and S.W.J.LAMBERTS, Endocrinology, 103, 200-203 (1978). B.WEST and P.S.DANNIES, Endocrinology, 104, 877-880 (1979). C.DENEF and J.J.FOLLEBOUCKT, Life Sci., 23, 431-436 (1978). G.M.BESSER, G.DELITALA, A.GROSSMAN, W.A.STUBBS and T.YEO, Br. J. Pharmac., 71, 569-573 (1980). B.WEISS, W.PROZIALECK, M.CIMINO, M.SELLINGER-BARNETTE and T.L.WALLACE, Ann. NY Acad. Sci., 356, 319-345 (1980). P.M.CONN, D.C.ROGERS AND T.SHEFFIELD, Endocrinology, 109, 1122-1126 (1981).
Vol. 33, No. 9, 1983
7. 8.
9. i0. ii. 12. 13. 14. 15. 16. 17.
Calmodulin and Prolactin Secretion
J.E.MERRITT, S.TOMLINSON and B.L.BROWN, FEBS Lett. 135, 107-110 (1981). J.E.MERRITT, S.MAC NELL, S.TOMLINSON AND B.L.BROWN. J. Endocr. (In press). W.J.THOMPSON, P.M.EPSTEIN, S.J.STRADA, Biochemistry, 18, 5228-5237 (1979). M.P.SCHREY, B.L.BROWN and R.P.EKINS, Mol. Cell Endocr. 3, 271-282 (1977). W.J.THOMPSON, W.L.TERASKI, P.M.EPSTEIN and S.J.STRADA, Adv. Cycllc Nuc. Res., i0, 69-92 (1979). A.M.BENTLEY and M.WALLIS, J.Endocr., 94, 317-326 (1982). M.C.GERSHENGORN, Mol. Cell. Biochem., 45, 163-179(1982). M.O.THORNER, J.T.HACKETT, F.MURAD and R.M.MACLEOD, Neuroendocrinology, 31, 390-402 (1980). C.M.MORIARTY, Life Sei. 23,185-194 (1978). A.FLECKMAN, J.ERLICHMAN, U.K.SCHUBERT and N.FLEISCHER, Endocrlnology, 108, 2072-2077 (1981). B.L.BROWN, J.E.MERRITT AND S.TOMLINSON, Clin. Scl. 61, 23P (1981).
893