... The University of Texas Health Science Center at Dallas, 5323 Harry Hines Boulevard, Dallas, ..... Baraban, J. M., Gould, R. J.,Peroutka, S.J. & Snyder, S. H..
Proc. Nati. Acad. Sci. USA Vol. 83, pp. 4932-4936, July 1986 Medical Sciences
Activation of two different but complementary biochemical pathways stimulates release of hypothalamic luteinizing hormone-releasing hormone (protein kinase C/median eminence/prostaglandin E2/cyclic AMP/female development)
S. R. OJEDA*, H. F. URBANSKI*, K. H. KATZ*, M. E. COSTA*, AND P. M. CONNt *Department of Physiology, The University of Texas Health Science Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75235; and tDepartment of Pharmacology, University of Iowa College of Medicine, Iowa City, IA 52242
Communicated by S. M. McCann, March 18, 1986
ence of diacylglycerol becomes attached to membranes and acquires enhanced enzymatic activity (4, 5). Brain tissue is particularly rich in protein kinase C, which is, to a large extent, localized in the synaptosomal fraction (6, 7). These observations raise the possibility that LHRH secretion from median eminence nerve terminals may be regulated by mechanisms involving activation of protein kinase C, in addition to those dependent on PGE2 and cAMP production. The stimulation of LHRH release by NE (1, 2, 8) appears to be effected exclusively by an increase in PGE2 and cAMP formation (2, 3, 8). However, NE is not the only transmitter controlling LHRH secretion (9), thus raising the possibility of an additional transducer system regulating LHRH secretion. In the present study we have examined the response of the LHRH releasing system to each of the following compounds: a synthetic diacylglycerol that activates protein kinase C in intact cells (10), a phorbol ester known to directly activate the kinase (11), and phospholipase C, which activates protein kinase C through release of diacylglycerol from membrane inositol phospholipids (4, 5).
Evidence exists that a norepinephrine/ ABSTRACT prostaglandin E2 (PGE2)/cAMP pathway is involved in the regulation of luteinizing hormone-releasing hormone (LHRH) secretion. The aim of the present experiments was to determine if release of LHRH from the immature rat hypothalamus could also be stimulated by activation of protein kinase C. Median eminences from 28-day-old female rats were incubated in vitro with either dioctanoylglycerol (a synthetic diacylglycerol that selectively activates protein kinase C in intact cells) or 4fiphorbol 12(-myristate 13a-acetate (another protein kinase C activator). Both agents increased LHRH release, the response to dioctanoylglycerol being more pronounced than that to the phorbol ester. This direct activation of protein kinase C was not accompanied by changes in PGE2 formation. Activation of the PGE2/cAMP pathway by either norepinephrine, PGE2, or forskolin (a stimulator of adenylate cyclase) increased LHRH release. Dioctanoylglycerol or phorbol ester in conjunction with either norepinephrine, PGE2, or forskolin resulted in an additive effect on LHRH release suggesting coexistence of both pathways. Phospholipase C, which activates protein kinase C via formation of diacylglycerol, increased the release of both LHRH and PGE2. This suggests that an increase in endogenous phospholipase C activity caused by neurotransmitter inputs may lead to both activation of protein kinase C and PGE2 formation. Blockade of cyclooxygenase activity by indomethacin obliterated phospholipase C-induced PGE2 release. The same treatment reduced the LHRH response by only 50% indicating that protein kinase C activation can cause LHRH release in the absence of PGE2 synthesis. It is suggested that the median eminence of the rat possesses a protein kinase Cdependent pathway that is coupled positively to LHRH release and complements PGE2/cAMP-dependent mechanisms. Norepinephrine, however, does not appear to be the neurotransmitter responsible for activating the protein kinase C pathway. Simultaneous activation of both pathways may provide a mechanism by which a large increase in LHRH secretion occurs, such as in the afternoon of first proestrus.
MATERIALS AND METHODS Animals. Immature 28-day-old juvenile rats of the Sprague-
Dawley stock were purchased from Holtzman (Madison, WI) and were housed under controlled environmental conditions
(3).
Materials. PGE2 was purchased from Upjohn, and LHRH was from Peninsula Laboratories (Belmont, CA). Forskolin, a diterpene activator of adenylate cyclase (12), was obtained from Calbiochem. Phospholipase C (from Clostridium perfringens), 4,3phorbol 12,B-myristate 13a-acetate (PMA), the protease inhibitors phenylmethylsulfonyl fluoride, N-a-tosyllysine chloromethyl ketone (TLCK), and L-1-tosylamido2-phenylethyl chloromethyl ketone (TPCK) were all purchased from Sigma. Dioctanoylglycerol and its analog, 3thio-1,2-dioctanoylglycerol, were synthesized as described (10). Indomethacin was the generous gift of Merck, Sharp, and Dohme (Rahway, NJ). Dimethyl sulfoxide (Me2SO), used to dissolve PMA, was purchased from Baker Chemical Co. (Phillipsburg, NJ). Procedures. Drug solutions. PGE2, the diacylglycerols, and forskolin were initially dissolved in absolute ethanol and then further diluted in incubation medium (final ethanol concentration: 0.1% for PGE2 and forskolin, 1% for the diacylglycerols). The latter were purified (10), dissolved in chloroform, lyophilized, and stored at -20°C until resolubilization in ethanol at the time of the experiment. PMA was dissolved in
The intracellular mechanism by which norepinephrine (NE) elicits luteinizing hormone-releasing hormone (LHRH) release involves the activation of prostaglandin E2 (PGE2) and cAMP formation (1-3). Many neurotransmitters, however, are known to provoke phosphatidylinositol degradation upon binding to their specific membrane receptors and evoke a cascade of events (4) that, independently of cAMP, leads to regulation of cellular function. A key product of this cascade is diacylglycerol, which appears to control phosphorylation processes through activation of protein kinase C, a Ca2' activated, phospholipid-dependent kinase, which in the pres-
Abbreviations: PGE2, prostaglandin E2; LHRH, luteinizing hormone-releasing hormone; NE, norepinephrine; Me2SO, dimethyl sulfoxide; PMA, 43-phorbol 12,B-myristate 13a-acetate; TLCK, N-a-tosyllysine chloromethyl ketone; TPCK, L-1-tosylamido-2phenylethyl chloromethyl ketone.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
4932
Medical Sciences: 0 eda et al.
Proc. Natl. Acad. Sci. USA 83 (1986)
Me2SO, lyophilized, and stored at -70'C in darkness. At the time of the experiment it was redissolved in Me2SO and diluted in incubation medium (final Me2SO concentration, 0.01%). Phospholipase C was dissolved in 0.9% NaCl. Phenylmethylsulfonyl fluoride and TPCK were initially dissolved in absolute ethanol (at 50 mM) and TLCK in distilled H20. Indomethacin was dissolved in 0.1 M sodium phosphate (pH 7.5) at 1 mg/ml before final dilution in incubation medium. Incubations. The median eminences were dissected as described (13). Each median eminence was incubated (1, 3) in a polypropylene vial precoated with 0.1% gelatin. The incubation medium (250 ,u1) consisted of Krebs-Ringer bicarbonate buffer (pH 7.4) containing D-dextrose at 1 mg/ml (KRBG), and in an atmosphere of 95% 02/5% CO2. The tissues were preincubated for 15 min and then for 30 min with a change in medium. At the end of this period the medium was replaced by fresh KRBG containing the test substances. After a second 30-min incubation period the medium was centrifuged (1, 3), and the supernatant was assayed for LHRH and PGE2. Neither the medium nor any of the substances tested interfered with the RIAs for either LHRH or PGE2. Only dioctanoylglycerol altered the binding of [3H]PGE2 to its antibody. Bacitracin, an inhibitor of peptide degradation (14), was not included in the incubation medium
4933
32
24 _ E 0
16 _ 0'
8
U, n
E
al
a) a) -0
0
a,
50
100
200
0'
300
laM
0.
PMA
24 I
-J
16
1oI
(13).
8
Control flasks received KRBG alone or KRBG containing ethanol (0.1% or 1%) or Me2SO (0.01%)). When control values were similar, they were pooled for data analysis. The diacylglycerols were tested in the presence of 0.1% bovine serum albumin (10, 15). A control group was included in each experiment and with each experimental group that was retested. Radioimmunoassays. LHRH and PGE2 released into the incubation medium were assayed as described (1, 13) using specific antisera (16, 17). The LHRH assay was initiated immediately after each incubation to avoid having to acidify the medium (18). Similar LHRH values were obtained from untreated samples and from those acidified with HC1. Statistics. The results were analyzed with a one-way analysis of variance followed by the Student-NewmanKeuls multiple range comparison test for unequal replications. Effective dose, 50% (ED50) was obtained by linear
regression analysis. RESULTS Effect of Protein Kinase C Activators on LHRH Release. The synthetic diacylglycerol, dioctanoylglycerol, increased LHRH release (Fig. 1, Upper) with an apparent ED50 of 55 ,4M. Maximal effect was observed at 100 ,tM. The 3'sulfhydryl analog of dioctanoylglycerol, which does not activate protein kinase C, was ineffective even at 300 AM. Like dioctanoylglycerol, the phorbol ester PMA also enhanced LHRH release (Fig. 1, Lower) but the magnitude of the effect was smaller (P < 0.01) than that of dioctanoylglycerol. Maximal effect was attained at 10 ng/ml. Effect of Protein Kinase C Activators on PGE2 Release. The effect of dioctanoylglycerol on PGE2 release could not be assessed because dioctanoylglycerol inhibited [3H]PGE2 binding in the RIA for PGE2. PMA, however, did not affect the PGE2 assay. PMA was ineffective in increasing PGE2 release from the median eminence even at concentrations as high as 100 ng/ml (Fig. 2). All concentrations tended to decrease PGE2 levels, an inconsistent effect that was significant (P < 0.05 to P < 0.01) only at concentrations of 1 and 100 ng/ml. Effect of Protein Kinase C Activators in Conjunction with PGE2 or Forskolin on LHRH Release. As before, dioctanoylglycerol (100 4M) or PMA (25 or 100 ng/ml)
_0
10
20
J4o
30 ng /ml
100
FIG. 1. Stimulatory effect of two activators of protein kinase C on in vitro LHRH release from median eminences (MEs) of juvenile female rats. Dioctanoylglycerol (DiC8) (Upper) and PMA (Lower) stimulated LHRH release. A DiC8 analog, 3-thio-1,2-dioctanoylglycerol (DiC8-SH) in which the 3'-hydroxyl group was replaced by a sulfhydryl moiety (Upper) was ineffective. In this and subsequent figures vertical lines represent the SEM. Each point represents the mean of 5 (DiC8-SH) or 10 (DiC8) individual MEs. Different control groups were employed for the diacylyglycerols and the PMA.
stimulated (P < 0.01) LHRH release from isolated median eminences (Fig. 3). Maximally effective doses (1, 3) of PGE2 (2.8 uM) or forskolin (50 ,M) also enhanced LHRH release. Dioctanoylglycerol or PMA tested in conjunction with forskolin produced an effect similar to the sum of the individual effects (Fig. 3, Left). Dioctanoylglycerol or PMA plus PGE2 also resulted in an additive effect (Fig. 3, Right). c
I'rE 0
N
U
PMA 701-
501-
(9.a-
-0
a) (n
a, IV
301-
Ta) CD
0L
10 L ,
0
10
30
20 ng
100
/ ml
FIG. 2. Failure of PMA to stimulate in vitro PGE2 release from the median eminence (ME) of juvenile female rats. Each point represents the mean of 7-12 individual MEs.
4934
Proc. Natl. Acad. Sci. USA 83 (1986)
Medical Sciences: Ojeda et al. Ic
(37.7) (32.1) (33.1)
(39.7) (34.1) 50
-I
6,
I
5
0
6
._
w -o
EQi U,
l
40
30
9
97
-
10
8
0
a)
20-
0' 7
7
I
-J C
D IC8PMA25
F
F
F
+
+
PGE2
C
PGE2
PGE2
PGE2
+
+
+
DC8
DiC8 PMA-25
PMA- PMA100 25
FIG. 3. Additive effect of PGE2 or forskolin (F) with each of two activators of protein kinase C on in vitro LHRH release from median eminences (MEs) of juvenile female rats. Dioctanoylglycerol (DiC8), 100 AM; PMA, 25 or 100 ng/ml; F, 50 ,uM; PGE2, 2.8 AM. The numbers in parentheses represent the predicted additivity (i.e., the sum of the increments in LHRH release from basal release induced by each secretagogue individually). Numbers above bars indicate the number of MEs per group. C: control, basal release. The control group depicted in both panels is the same.
The effect of PMA plus forskolin or PGE2, or that of dioctanoylglycerol plus PGE2, was greater (P < 0.05 to P < 0.01) than that of forskolin or PGE2 alone. The effect of diQctanoylglycerol plus forskolin, however, was not significantly greater than that of forskolin alone. Effect of a Protein Kinase C Activator in Conjunction with NE on LHRH Release.'Joth NE (60 puM) and dioctanoylglycerol (100 AM), when tested separately, enhanced (P < 0.01) LHRH release from the median eminence (Fig. 4). As in the case of PGE2, concomitant administration of NE 'and dioctanoylglycerol'rqsulted in an additive effect on LHRH release. Effect of Phospholipase C on LHRH and PGE2 Release. Phospholipase C evoked a dose-relateA increase in both LHRH apd PGE2 release from the median eminence (Fig. 5). The apparent ED50s were 0.05 uinit/ml and 0.1 unit/nil for LHRH and PGE2, respectively. Maximal responses were observed at
0.12-0.25 unit/ml. Increasing the phospholipase C concentration to 1 unit/ml further enhanced LHRH. and PGE2 release (not shown), suggesting damage of the nerve terminals.
c
E
40
0
II 2:
w
30
E
J
T0
20
0
(a 0
10
0.
E
(25.8)
40
8
0
c
ti1 w
320
30 _
Uo
E
-o
0
0
20
240
10
0
10
160
0
.C
10
-
0
0' 0.
80
I
C
NE
DiC8
60MiM
1OO1LLM
NE +
DjC8
FIG. 4. Additive effect of NE (60 uM) with dioctanoylglycerol (DiC8, 100 ,aM) on in vitro LHRH release from median eminences (MEs) ofjuvenile female rats. The number in parentheses represents the predicted additivity. Numbers above bars indicate the number of MEs per group. C: control, basal release.
0
0.1
0.4 0.2 0.3 PLC (units/ ml )
0.5
FIG. 5. Stimulatory effect of phospholipase C (PLC) (from Clostridium perfringens) in vitro on LHRH and PGE2 release from median eminences (MEs) of juvenile female rats. Each point represents the mean of 5-11 individual MEs.
Proc. Natl. Acad. Sci. USA 83 (1986)
Medical Sciences: 0 eda et al. The possible contribution of protease contaminants in the phospholipase C preparation was tested by incubating median eminences with phospholipase C (0.1 unit/ml) in the presence of the protease inhibitors phenylmethylsulfonyl fluoride, TLCK, and TPCK at 100 ,AM each. The effect of phospholipase C was not diminished. On the contrary, both basal and phospholipase C-stimulated LHRH release were increased (data not shown). Effect of Blockade of Cyclooxygenase Activity on Phospholipase C and Dioctanoylglycerol-Induced LHRH and PGE2 Release. Indomethacin (50 ,uM) eliminated both basal and phospholipase C-induced PGE2 release from the median eminence (Fig. 6). In spite of this, phospholipase C was still effective (P < 0.01) in stimulating LHRH release. Indomethacin, however, reduced (P < 0.01) the LHRH response to phospholipase C indicating that part of the phospholipase C effect on LHRH release is prostaglandin dependent. In contrast, indomethacin failed to alter dioctanoylglycerolinduced LHRH release (data not shown).
DISCUSSION The results demonstrate the ability of three activators of protein kinase C to stimulate LHRH release from the median eminence of juvenile female rats. The probes, phospholipase C, dioctanoylglycerol, and PMA were selected because they simulate the sequence of events that lead to extracellular messenger-induced activation of protein kinase C. Phospholipase C from Clostridium perfringens is a potent secretagogue in other systems (19, 20). Its effectiveness presumably results from an ability to mimic the action of the endogenous, membrane-bound phospholipase C, which catalyzes the hydrolysis of membrane inositol phospholipids and, thereby, leads to the formation of inositol 1,4,5-tris(phosphate) and diacylglycerol (21). Diacylglycerol interacts with inactive, presumably cytosol-located protein kinase C, inducing its binding to the plasma membrane and its concomitant activation (22). To reproduce this action, we have used dioctanoylglycerol, a synthetic diacylglycerol whose fatty acid chain is sufficiently long to permit its passage to the inner cell membrane and, thereby, enhance protein kinase C activity (10). Dioctanoylglycerol releases leuteinizing hormone from pituitary cells in culture (15) and activates protein kinase C in
brain extracts (10) and platelets (23). Additionally, dioctanoylglycerol behaves like a phorbol ester (22) in that it amplifies the stimulatory effect of increasing the cytosolic concentration of free Ca2l on hormone secretion (15). Since phorbol esters can effectively substitute (5, 11) for diacylglycerol, PMA was used to directly increase the protein kinase C activity of median eminence nerve terminals. Dioctanoylglycerol and PMA, which appear to penetrate the membrane phospholipid bilayer without major alterations in lipid turnover, increased LHRH release independently of PGE2 synthesis. Thus PMA did not alter PGE2 release at doses that enhanced LHRH release, and indomethacin failed to block dioctanoylglycerol-induced LHRH release. Didecanoylglycerol, which does not cross the cell membrane as easily as dioctanoylglycerol (10), has been reported (24) to enhance PGE2 release from the median eminence without stimulating LHRH release unless lipooxygenase activity is first blocked; this suggests that didecanoylglycerol stimulates the formation of an inhibitory lipooxygenase-derived metabolite of arachidonic acid. It is now firmly established that the neurotransmitter NE elicits LHRH release through activation of PGE2 formation (1,2, 8). LHRH release can be evoked either with dibutyrylcAMP or by stimulation of endogenous cAMP formation (2, 3, 16). In addition, evidence has been presented that part of the mechanism by which PGE2 induces LHRH secretion may involve cAMP (2). These observations have led to the conclusion that activation of PGE2 and cAMP synthesis underlies the stimulatory, NE-directed, transmembrane control of LHRH secretion (2, 3). The inability of simultaneous in vitro administration of PGE2 and forskolin to induce more LHRH release than either substance individually (3) suggests that release is occurring from the same pool in both cases. In contrast, concomitant exposute of the median eminence to protein kinase C activators and either NE, PGE2, or forskolin resulted in an additive effect on LHRH release. This further indicates that activation of protein kinase C leads to LHRH release without involvement of PGE2 or cAMP and strongly suggests that both pathways operate in a complementary manner. Such a complementary operation may be maximal in situations during which LHRH secretion is enhanced. The proestrous surge of LHRH release may be one of these situations.
PGE2
LHRH
I
500 P-
4935
-A
75 .c 0
E 400
[1I
F
A 60
Qf) °
w
._
0 0 I
c.w
A E5E
300 F-
0'
0
U)
200 F-
30
104
0
0)
C31
100
15
a.
6 C
PLC
6
6
PLC
Id
C
PLC
PLC
Id
+
Id
Id
FIG. 6. Inhibitory effect of in vitro blockade of cyclooxygenase activity with indomethacin (Id) (50 uM) on phospholipase C (PLC) (0.25 unit/ml)-induced PGE2 and LHRH release from median eminences (MEs) of juvenile female rats. Numbers above bars indicate the number of individual MEs per group. C: control, basal release. Incomplete bars indicate undetectable PGE2 levels.
4936
Medical Sciences: Ojeda et al.
There is indeed substantial evidence that a complete cellular response to extracellular messengers fails to occur if only one intracellular pathway is operative. Thus the combination of A23187, a Ca2' ionophore, and a phorbol ester elicits a maximal release reaction from platelets, whereas each substance alone produces only a submaximal response (25). Similarly, cultured pituitary cells release more luteinizing hormone in response to A23187 plus PMA than in response to either agent alone (15). In pituitary somatotrophs PMA enhances the effect of both growth hormone-releasing factor and dibutyryl-cAMP on growth hormone release (20). Also, forskolin synergizes the effect of A23187 and a phorbol ester on insulin secretion (26). Directly relevant to the present results is the demonstration that phorbol esters facilitate the contractile response of the vas deferens to NE
(27).
The results obtained with exogenous phospholipase C are in harmony with the view that receptor-induced activation of the endogenous enzyme leads to protein kinase C activation and to increased availability of arachidonic acid (4, 28). While the latter can be inferred from the striking elevation in PGE2 levels induced by phospholipase C, the former is suggested by the finding that suppression of prostaglandin synthesis failed to block phospholipase C-induced LHRH release. The attenuation of the response, however, is in all probability the result of elimination of the PGE2 component. These effects of phospholipase C were not due to membrane lysis caused by protease contamination of the phospholipase C preparation. Inhibition of protease activity with a combination of phenylmethylsulfonyl fluoride, TLCK, and TPCK actually enhanced rather than diminished the LHRH response. Results indicate that part of this effect is due to the capability of TPCK to inhibit Tyr5-Gly6 endopeptidase activity (unpublished data). If NE induces LHRH release only through activation of PGE2 and cAMP synthesis, the question arises as to the identity of the transmitter that activates phospholipase C. NE itself can evoke inositol phospholipid hydrolysis via binding to a1-adrenergic receptors (29). However, NE and dioctanoylglycerol produced an additive effect on LHRH suggesting that an extracellular messenger, other than NE, is the primary signal for phospholipase C activation. Depending on the intensity of this stimulus a sequential activation of phospholipase C and phospholipase A2 would occur as described in other systems (30). Alternatively, simultaneous activation of both pathways by different agonists may be the predominant mechanism operating under normal conditions. Amplification of the effect of diacylglycerol and PMA on LHRH release by activation of the PGE2/cAMP pathway may be related to the capacity of PGE2 (31) and cAMP (32) to alter intracellular Ca2+ levels. Since NE also increases intracellular Ca2+ levels (27), the possibility emerges that NE-dependent changes in Ca2+ levels contribute to the activation of protein kinase C (4, 5). In conclusion, the foregoing results permit the suggestion that extracellular messenger (neurotransmitter?)-induced secretion of LHRH from the hypothalamus involves a protein kinase C- and a PGE2/cAMP-dependent pathway and that they operate in a complementary manner. The interactions between both transducing systems and the nature of the extracellular messenger primarily involved in protein kinase C activation remain to be defined. We are grateful to Dr. V. D. Ramirez (Department of Physiology, University of Illinois at Urbana-Champaign) for providing us with anti-LHRH serum and to Dr. W. B. Campbell (Department of
Proc. Natl. Acad. Sci. USA 83
(1986)
Pharmacology, UTHSCD) for supplying anti-PGE2 serum. We also thank Ms. Judy Scott for typing the manuscript. This research was supported by Grants HD-09988, project IV, and HD-19899 from the National Institutes of Health. 1. Ojeda, S. R., Negro-Vilar, A. & McCann, S. M. (1979) Endocrinology 104, 617-624. 2. Ramirez, V. D., Kim, K. & Dluzen, D. (1985) Recent Prog. Horm. Res. 41, 421-472. 3. Ojeda, S. R., Urbanski, H. F., Katz, K. H. & Costa, M. E. (1985) Endocrinology 117, 1175-1178. 4. Rasmussen, H. & Barrett, P. Q. (1984) Physiol. Rev. 64, 938-984. 5. Nishizuka, Y. (1983) Trends Biochem. Sci. 8, 13-16. 6. Takai, Y., Kishimoto, A., Isawa, Y., Kawahara, Y., Mori, T. & Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695. 7. Blumberg, P. M., Jaken, S., Konig, B., Sharkey, N. A., Leach, K. L., Jeng, A. Y. & Yeh, E. (1984) Biochem. Pharmacol. 33, 933-940. 8. Ojeda, S. R., Negro-Vilar, A. & McCann, S. M. (1982) Endocrinology 110, 409-412. 9. Ramirez, V. D., Feder, H. H. & Sawyer, C. H. (1984) in Frontiers in Neuroendocrinology, eds. Martini, L. & Ganong, W. F. (Raven, New York), Vol. 8, pp. 27-84. 10. Conn, P. M., Ganong, B., Ebeling, J., Staley, D., Neidel, J. E. & Bell, R. M. (1985) Biochem. Biophys. Res. Commun. 126, 532-539. 11. Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U. & Nishizuka, Y. (1982) J. Biol. Chem. 257, 7847-7851. 12. Seamon, K. B. & Daly, J. W. (1981) J. Cyclic Nucleotide Res. 7, 201-224. 13. Negro-Vilar, A., Ojeda, S. R. & McCann, S. M. (1979) Endocrinology 104, 1749-1751. 14. McKelvy, J. P., LeBlanc, P., Laudes, C., Perrie, S., GrimmJorgensen, Y. & Kordon, C. (1976) Biochem. Biophys. Res. Commun. 73, 507-515. 15. Harris, C. E., Staley, D. & Conn, P. M. (1985) Mol. Pharmacol. 27, 532-536. 16. Hartter, D. E. & Ramirez, V. D. (1985) Neuroendocrinology 40, 476-482. 17. Campbell, W. B., Gomez-Sanchez, C. & Adams, B. V. (1980) Hypertension 2, 471-476. 18. Gallardo, E. & Ramirez, V. D. (1977) Proc. Soc. Exp. Biol. Med. 155, 78-84. 19. Martin, T. F. J. & Kowalchyk, J. A. (1984) Endocrinology 115, 1517-1526. 20. Ohmura, E. & Friesen, H. G. (1985) Endocrinology 116, 728-733. 21. Berridge, M. J. (1983) Biochem. J. 212, 849-858. 22. Takai, Y., Kishimoto, A., Kawahara, Y., Minakuchi, R., Sano, K., Kikkawa, U., Mori, T., Yu, B., Kaibuchi, K. & Nishizuka, Y. (1981) Adv. Cyclic Nucleotide Res. 14, 301-313. 23. Lapetina, E. G., Reep, B., Ganong, B. R. & Bell, R. M. (1985) J. Biol. Chem. 260, 1358-1361. 24. Negro-Vilar, A., Conte, D. & Valenca, M. (1985) Program of the 67th Meeting of the Endocrine Society (Endocrine Society, Bethesda, MD), p. 184 (abstr.). 25. Kaibuchi, K., Takai, Y., Sawamura, M., Hoshijima, M., Fujikira, T. & Nishizuka, Y. (1983) J. Biol. Chem. 258, 6701-6704. 26. Zawalich, W., Brown, C. & Rasmussen, H. (1983) Biochem. Biophys. Res. Commun. 117, 448-455. 27. Baraban, J. M., Gould, R. J., Peroutka, S. J. & Snyder, S. H. (1985) Proc. Natl. Acad. Sci. USA 82, 604-607. 28. Lapetina, E. G. (1982) Trends Pharmacol. Sci. 3, 115-116. 29. Exton, J. H. (1985) Am. J. Physiol. Endocrinol. Metab. 11, E633-E647. 30. Siess, W., Weber, P. C. & Lapetina, E. G. (1984) J. Biol. Chem. 259, 8286-8292. 31. Ojeda, S. R. & Negro-Vilar, A. (1985) Endocrinology 116, 1763-1770. 32. Cachelin, A. B., DePeyer, J. E., Kokubrina, S. & Reuter, H. (1983) Nature (London) 304, 462-464.