INHIBITION OF a,-ADRENERGIC RECEPTOR-MEDIATED CYCLIC GMP FORMATION BY A PHORBOL ESTER, A PROTEIN KINASE C ACTIVATOR
Rama K. Jaiswal and Rameshwar K. Sharma
Section of Regulatory Biology Cleveland Clinic Research Institute 9500 Euclid Avenue Cleveland, Ohio 44195
Received September 15, 1988
@2-adrenergic receptor-mediated signal transduction in rat adrenocortical carcinoma cells occurs through the opposing regulation of two second messengers, cyclic GMP and cyclic AMP, in which guanylate cyclase is coupled positively and adenylate cyclase negatively to the receptor signal. We now show that in these cells phorbol 12- myristate 13-acetate (PMA), a known activator of protein kinase C, inhibits the x2- agonist (p-aminoclodine)-dependent production of cyclic GMP in a dose-dependent and time-dependent fashion. The half-maximal inhibitory concentration of PMA was 10-10 M. A protein kinase C inhibitor, 1-(5-isoquinolinyl-sulfonyl)-2-methyl piperazine (H- 7), caused the release of the PMA-dependent attenuation of p-aminoclodine-stimulated cyclic GMP formation. These results suggest that protein kinase C negatively regulates the a2-receptor coupled cyclic GMP system in these cells, a feature apparently shared with the other cyclic GMP-coupled receptors such as those of muscarine, histamine, and atrial natriuretic factor. · 1988 Academic Press, Inc.
Recent studies in a variety of mammalian systems show that cyclic GMP is one of the “second messengers” of atrial natriuretic factor (ANF) signal transductions (1; reviewed in 2). An intriguing aspect of this transmembrane signaling system is that in contrast to the corresponding cyclic AMP system where the receptor and adenylate cyclase components are distinct, one protein appears to contain both the receptor and guanylate cyclase (3-5). This raises an important question. Are all cyclic GMP- mediated transmembrane signaling mechanisms identical?
This does not appear to be the case, because rat adrenocortical carcinoma a- adrenergic receptors, which are exclusively of a2-subtype (6), demonstrate hormone- dependent positive coupling to guanylate cyclase only in an intact cell (7), no coupling is observed in cell-free membrane preparations. This suggests that the @2-agonist- dependent activation of guanylate cyclase, in contrast to ANF, is indirect. To precisely define the direct and indirect coupling mechanisms of guanylate cyclase with the receptor-mediated signal transductions, we now show that phorbol ester signal, probably through protein kinase C, negatively regulates the a2-receptor coupled guanylate cyclase, a feature apparently shared with the ANF receptor-coupled guanylate cyclase (8,9).
MATERIALS AND METHODS
The isolated adrenocortical carcinoma cells were prepared by the trypsin digestion method (10,11). These cells have been thoroughly characterized, morphologically and biochemically (11). They were suspended in Krebs-Ringer- bicarbonate buffer, pH 7.4, containing 4% albumin and 0.2% glucose. To study the influence of PMA on a2-agonist-dependent cyclic GMP formation, the cells (1 x 106) were incubated at 37 ℃ under 5% CO2/95% O2 with varying concentrations of PMA in the presence or absence of indicated concentrations of p-aminoclonidine in a total volume of 1.0 ml of Krebs-Ringer-bicarbonate buffer. Unless otherwise stated, the cells were preincubated for 10 min in the presence of PMA prior to initiation of the appropriate assay; PMA was present throughout the subsequent assay incubation period. The assay for cyclic GMP was conducted at 20 min. At the end of the incubation period, 1.0 ml of 1 M perchloric acid was added to terminate the reaction. The reaction mixture was neutralized by the addition of 10 M KOH and the tubes were centrifuged at 500 x g for 10 min. The cyclic GMP levels were determined by radioimmunoassay (12). The 35,000 x g post-mitochondrial cell pellet was used to determine the particulate guanylate cyclase activity as in (12).
Trypsin, soybean trypsin inhibitor, phorbol 12-myristate 13-acetate, 48-phorbol, 4a-phorbol 12,13-didecanoate, and H-7 were purchased from Sigma and p- aminoclonidine was from Research Biochemicals. All other reagents were of analytical grade from commercial sources.
RESULTS AND DISCUSSION
Rat adrenocortical carcinoma 494 tumor cells (11,13) contain a high density of @2-adrenergic receptors that are thoroughly characterized in their membrane-bound (6), solubilized (14) and pure forms (15). Incubation of these cells with a series of concentrations of p-aminoclonidine, an a - agonist, caused a concentration-dependent increase in the levels of cyclic GMP (Fig. 1). The concentration causing half-maximal response (EC50) was 1.5 uM. These cells do not contain a,-receptors (6), indicating that p-aminoclonidine stimulated cyclic GMP formation comes about exclusively through @2-
cyclic GMP (%above basal level)
400
300
200
100
0
0
1
10
100
PAC (UM)
CyCIIC GMVIP Tormea (pmoI / 20 min)
1.0
cyclic GMP formed (% of control)
100
0.8
80
0.6
60
0.4
40
PMA
0.2
PMA + H - 7
20
0
0
0
12
11
10
9
8
-7
-6
0
2
4
6
8
10
12
14
16
2
-Log [PMA] [M]
3
TIME (MIN)
receptors. The carcinoma cells used in these studies are devoid of cyclic GMP phosphodiesterase activity (16); therefore the @2-agonist-dependent elevations in cyclic GMP levels reflect guanylate cyclase activity. This interpretation is further supported by the fact that: a) in these cells p-aminoclondine stimulates membrane guanylate cyclase activity in a dose-dependent fashion (7); b) the hormone-dependent rise of cyclic GMP is selectively inhibited by the @2-antagonist yohimbine (7), and c) the nitrite generating agents, known to stimulate soluble guanylate cyclase (17) but ineffective toward particulate guanylate cyclase (18-20), do not stimulate cyclic GMP formation in the carcinoma cells (18).
To determine the possible interaction of a2-receptor coupled cyclic GMP formation with protein kinase C signal, the carcinoma cells were incubated with varying concentrations of protein kinase C activator phorbol 12-myristate 13-acetate (PMA) (21) in the presence of 10 uM p-aminoclonidine. PMA inhibited the p-aminoclonidine- dependent elevations of cyclic GMP levels in a dose-dependent fashion with a half- maximal concentration of 10-10 M (Fig. 2). The inhibition was time-dependent (Fig. 3), with an achievement of steady state in ~8 min after 10 min preincubation of cells with 1 UM PMA. At ~2.5 min (T1/2) a 50% of total inhibition was observed (Fig. 3). Introduction of H-7, a non-specific inhibitor of protein kinase C (22), in the incubation medium released the PMA-dependent inhibition in a dose-dependent fashion (Fig. 2). The two phorbol ester analogues, 48-phorbol and phorbol 12,13-didecanoate, which
| Drugs Guanylate cyclase activity (pmol/min/mg) (10 µM) | |
|---|---|
| Basal | 5.6 ± 0.10 |
| Para-aminoclonidine | 5.4 ± 0.12 |
| Clonidine | 4.9 ± 0.63 |
| (-)Epinephrine | 5.0 ± 0.46 |
Adrenocortical carcinoma membranes were prepared as described in Methods. The membrane suspensions (10-20 µg) protein were incubated with indicated drugs (10 M final) for 10 min in a total volume of 0.1 ml. The guanylate cyclase activity was determined as described in Methods. The experiments were conducted in triplicate and repeated at least three times for reproducibility. The results are typical of one experiment ± SE (n=3).
neither stimulate nor inhibit protein kinase C (23), did not interfere with the PMA- stimulated cyclic GMP formation (data not shown). Because the major phorbol ester receptor is protein kinase C, these results suggest that through the hypothetical @2- receptor-coupled guanylate cyclase, protein kinase C and @2-adrenergic receptor signals are linked. The a,-receptor signal positively regulates the cyclic GMP formation and the protein kinase C signal uncouples this positive regulation.
To determine the direct a2-agonist-dependent stimulation of the adrenocortical carcinoma particulate guanylate cyclase, the plasma membranes of these cells were incubated with p-aminoclonidine, clonidine, and epinephrine. None of these agents stimulated guanylate cyclase activity (Table 1), indicating hormonal independence of the @2-receptor coupled guanylate cyclase in the cell-free system. Since the enzyme is hormone-dependent in intact cells, the results indicate that the @2-receptor-guanylate cyclase coupling is indirect.
ANF-dependent guanylate cyclase is also negatively linked to the phorbol ester signal (8,9). In this case the direct inhibition of the 180-kDa hormone-dependent guanylate cyclase (23), and its direct phosphorylation with protein kinase C has been demonstrated (24). Similarly, type 1 vasopressin receptor-mediated signal negatively regulates the ANF-stimulated cyclic GMP formation (25). The linkage between the two receptor signals appears to be through protein kinase C, since in these cells also phorbol ester inhibits the ANF-dependent cyclic GMP accumulation. Similar to the situation in a2-receptor-mediated cyclic GMP formation, neurotransmitters (muscarinic and histaminic) receptor-mediated cyclic GMP responses occur only in intact neuroblastoma cells (6,7). In these cells also phorbol ester inhibits muscarinic- and histaminic- dependent cyclic GMP formation, suggesting that protein kinase C is the inhibitory mediator. These results suggest a pattern in that all hormonally positively coupled cyclic
GMP receptors, regardless of the manner of their coupling, i.e., direct or indirect, are negatively regulated by the protein kinase C receptor signal. Taking into consideration that the a,-receptors of the adrenocortical carcinoma cells negatively regulate hormone dependent adenylate cyclase (7), the present study indicates that a single &-receptor signal exhibits dual transmembrane regulation of cyclic GMP and cyclic AMP signal pathways and in turn is negatively regulated by the protein kinase C signal. This indicates an example of a coordinated interwoven nature of a receptor-mediated signal transduction.
ACKNOWLEDGEMENTS
We appreciate the support by the National Institutes of Health (NS-23744) and the National Science Foundation (DCB 83-00500).
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