Suppression of LH-stimulated prostaglandin and progesterone accumulation in rat granulosa cells by isoquinolinesulfonamide protein kinase inhibitors

SUPPRESSION OF LH-STIMULATED PROSTAGLANDIN AND PROGESTERONE ACCUMULATION IN RAT GRANULOSA CELLS BY lSOQUlNOLlNESULFONAMIDE PROTEIN KINASE INHIBITORS Martin

R. Clark,a William

P. Hummel,a

and Kathleen

M. Eysterb

aDe artment of Obstetrics and Gynecology, The University of North Carolina at 8 hapel Hill, CB#7570 McNider, Chapel Hill, NC 27599; bDepartment of Physiology and Pharmacology, School of Medicine, University of South Dakota, Vermillion, SD 57069, USA Corresponding

author: Martin R. Clark, Ph.D.

ABSTRACT Rat granulosa cells were incubated with isoquinolinesulfonamide inhibitors of protein kinases A and C and/or LH, dibutyryl CAMP (dbcAMP), tetradecano lphorbol acetate (TPA), cholera toxin, or forskolin for 5 h. H7 (25 PM) was o 6 served to inhibit LH, cholera toxin or dbcAMP stimulation of prostaglandin (PGE), and progesterone accumulation. H7 produced inhibition when added as little as 2 min before and as long as 1 h after LH. HA1004 was ineffective against LH or cholera toxin stimulation of PGE or progesterone at up to 100 PM. H9 blocked some LH and forskolin responses at 25 pM, but required a 50 pM concentration to minimally affect TPA stimulation. Cytotoxicity was not observed at the concentrations and times of isoquinolinesulfonamides tested. H7 and H9, therefore, suppress LH stimulation of granulosa cell functions in a dose- and time-dependent manner consistent with inhibition of protein kinases A and/or C, and consonant with a requirement for such kinases in LH action. INTRODUCTION A variety of protein kinases have been implicated in the re ulation of cell function (1). Protein kinases activated by CAMP (protein 1. mases A) are believed to mediate the actions of substances acting on the adenyl cyclase second-messenger s stem. Similarly, Caz + and lipid-dependent kinases (protein kinases Cr are thought to mediate the responses to the phospholipase Uinositol polyphos hate/diacylgl cerol system. The involvement of CAMP in LH action on t 1 e ovary has b een well-established (2). Recently, LH has also been shown to stimulate the phospholipase C system (3,4). Activation of more than one second-messenger system in a tissue may also occur with other substances (5,6). Responses, proAa landin (PGE) and pro esterone accumulation, of ovarian cells to agonists o8 CAMP are similar to t il ose elicited by LH (2,7). On the other hand, agonists of diacylglycerol acutely elevate PGE markedly while increasing progesterone accumulation only modestly (8). To discern which actionsof LH are mediated by individual er pathways, it would be desirable to be able to selectively second-messen inhibit protein 1 inases A versus protein kinases C. Several isoquinolinesulfonamides (H7: l-(5-isoquinolinesulfonyl)-2-methylpiperizine; H8: N-[2(methylamino)ethyll-5-isoquinolinesulfonamide; H9: N-(2-aminoethyl)-5isoquinolinesulfonamide; HA1004: N-(2-guanidinoethyl)5isoquinolinesul-

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fonamide) have been suggested as candidates for such selective inhibition (9). Differential inhibitor constants for protein kinase A type I, cGMPdependent protein kinases, and protein kinases C were reported for these compounds using rabbit or pig tissues (9). One inhibitor, H8, was shown to penetrate cells (9), and another, HA1004, acted at submicromolar concentrations on smooth muscle trssue in vitro (10). Other studies have demonstrated cytotoxicity onl at relatively high concentrations ( > 32 PM H7 and > 320 pM HA1004 r in long-term cultures (11). The present investigations were performed to examine the efficacy and specificity of these inhibitors in blocking the PGE and progesterone responses to LH in rat granulosa cells. MATERIALS AND METHODS Materials Pregnant mare serum gonadotropin PMSG and LH (NIH 523) were provided by the National Hormone and Pituitary Program, NIADDK, NIH. Tetradecanoylphorbol acetate (TPA), dibutyryl CAMP (dbcAMP), cholera toxin, and forskolin were purchased from Sigma Chemical Co. (St. Louis, MO). Stock solutions of TPA were prepared in dimethylsulfoxide (I mg/ml) and stored in the dark at -70 C. Isoquinolinesulfonamides were obtained from Seikagaku America, Inc. (St. Petersburg, FL). Medium 199 (Grand Island Biologicals Co., Grand Island, NY) was provided by the Lineberger Cancer Research Center, Chapel Hill, NC. Phosphatidyl~rine was purchased from Su elco, Inc. (Houston, TX) and [y-32P]ATP (3000 Ci/mmol) from ICN 4-Pregnen-1 la-ol-3,20-dione Radioc 1 emicals (Irvine, CA). hemisuccinate/BSA was purchased from Steraloids, Inc. (Wilton, NH). Avidin-biotin-peroxidase reagents were supplied by Vector Laboratories, Inc. (Burlingame, CA). All other reagents have been identified in previous publications (7,8,12,13) or were obtained from common commercial sources. Preparation and incubation of aranulosa cells Granulosa cells were obtained from immature (27 day old) rats 48 h after an SC injection of 15 IU PMSG (12). Incubations were performed in triplicate at 37 C with approximately 106 viable cells each in a final volume of 1 ml of modified medium 199 under an atmosphere of 95%1 air-S% CO2. The modified medium 199 contained loh bovine serum albumin and 15 mM HEPES. After an initial IS-min incubation period, inhibitors were included for another 15 min (except where noted otherwise). Stimulatory substances were then added, this time designated “zero time,” and the incubations continued up to 5 additional hours. The incubatrons were sto ped by freezing cells and medium combined at -100 C (12). Animal stuBies were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals following protocols approved by the University Animal Care Committee.

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Protein kinase assavs Ovarian tissues were removed from immature rats treated as above and homogenized in buffer containing 20 mM Tris HCI, 2 mM EDTA, 5 mM EGTA, 0.25 M sucrose, and 50 mM 2-mercaptoethanol at pH 7.5 and 4 C (13). Ovarian cytosol was assayed in duplicate for protein kinase A activity in the presence of various concentrations of inhibitors, as described (13). Alternatively, cytosol was partially purified by chromatography on a Mono Q ion exchange column (14; without ATP) and assayed similarly for protein kinase C (13). Analvses of PGE and proqesterone PGE content was determined by extraction and RIA as described (8). The progesterone RIA previously employed (8) was replaced with a competitive enzyme-linked immunosorbent assay (ELISA). Briefly, a mouse IgG monoclonal antibody was produced in our laboratory to 4-pregnen-1 la-ol3,20-dione hemisuccinate BSA by published procedures, and with virtually identical cross-reactivity results (15). For the ELISA, the hemisuccinate conjugate was bound to microtiter plates, standard or samples added with monoclonal antibody for corn etition in the solution phase, and the bound antibody detected by the avi dpin-biotin-peroxidase complex technique (16). lntraassay and interassay coefficients of variation were 5.6 and lOoh, respectively. Net accumulation was calculated by subtracting the values for samples of cells plus medium priorto incubation (8). In one experiment, the control value indicated a small decrease over time, resulting in a net negative accumulation when the above blank wassubtracted (Fig. 6). Analvses of data Cell culture experiments were each performed on two or more separate occasions. Results are depicted as the means from triplicate cultures of individual experiments. The standard error bars have not been included since the coefficients of variation of less than 6-8Oh generally resulted in representations too small to be readil visualized. Unpaired two-tailed Student’s t-tests on a limited number or planned comparisons were carried out, and minor corrections to P values for multiple comparisons were made (17). Comparisons with P < 0.05 were considered significant. RESULTS The concentrations of inhibitors which blocked half the maximal activity of ovarian extracts in vitro were, for protein kinase A: H7 = 7 pM, HA1004 = 3 yn~:z = 0.3 pM; and for protein kinase C: H7 = 5 pM, HA1004 = 30 pM, = 7 pM. This examination of the inhibition of both types of kinases using ovarian tissue was consistent with prior data on the relative inhibitions of protein kinases A and C by these isoquinolinesufonamides obtained using extracts of rabbit tissues (9). Inhibition of stimulated (i.e., CAMP- or Ca2 +/lipid-dependent) kinase activity was observed in the ovarian extracts, whereas the basal protein phosphorylation was unchanged. H7 exhibited the lowest inhibitor constant for protein kinase C. HA1004 and H9 demonstrated preferential inhibition of protein kinase A.

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In ovarian granulosa cell cultures, H7 was an effective inhibitor of LHstimulated PGE and progesterone accumulation when added 15 min prior to LH (Fig. 1). Whereas the presence of 10 pM H7 produced partial and variable inhibition of both responses (not shown)., 25 and 50 M H7 markedly inhibited PGE and progesterone accumulatrons compare B to the values with LH only.

Figure 1. Su pression of Lli-stimulated ranulosa cell PGE and progesterone accumulation by H7. Cel Ps were incubated with 0, 5, or 50 M H7 for 15 min. followed by a 5-h incubation without (-) or with (+) the 9further a didition of LH (100 n ml). Mean values significantly different from the roup with no H7 or LH (H7: 0; LH: - 4’ are indicated by * above the bars. Means for 25 an 8 50 PM H7 plus LH were compared to LH alone (H7: 0; LH: + ) and statistical significance depicted by + .

An extended incubation with H7 prior to addition of LH to granulosa cells was not necessary (Fig. 2). Significant inhibition was observed with as little as 2 min or as great as 75 min of ex osure to H7 prior to LH, for both progesterone (Fig. 2A) and PGE (Fig. 2BP. If H7 inhibition were related to an early role of protein kinases in secondmessenger signaling, the efficacy of H7 might also be expected to be greatest at early times. H7 inhibited PGE and progesterone accumulation at 5 h when included 15 min before or 1 h after LH, but not if added 2 or 3 h post-LH (Fig. 3). Such data do not support a rapid cytotoxic action of H7 on ranulosa cells, since PGE accumulation does not increase until more than 3 8 after LH (12). Assessment of cell viability b vital dye exclusion after 5 h with H7 also demonstrated no overt toxicity (J ata not shown). To determine if H7 blocked the actions of s ecific protein kinase C and A agonists, granulosa cells were incubated wit R TPA or dbcAMP. As with LH, H7 markedly reduced PGE and progesterone accumulation in response to both agonists (Fig. 4).

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TIME OF ADDITION OF Ht (MINI

Figure 2. Dependence of Ii7 inhibition of granulosa cell PGE and progesterone accumulation on time of addition prior to LH. H7 (25 PM) was not added to tells (NONE) or added 2 or 75 min prior to incubation for 5 h in the absence fCTL) or presence of LH (tOO n@mt). Srgnificant dtfferences within the CTL groups from the group wtth no H7 or LH, and wrthin the LH group from the LH only values are indicated above the bars by * or + t re5pectivefy.

rogesterone Figure 3. Dependence of H7 inhibition of granulosa cell PGE and accumulation on time of addition after LH. H7 (25 PM) was not added to ccl Ps (CTL and LH) or added 15 min prior tot- 15 MIN) or at 1,2, or 3 h after addition of LH (100 q/ml) to cells for a 5-h incubation. Significant differences from the CTL or LH groups are indicated above the bars by * or + , respectively.

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Figure 4. Suppression of TPA- or dbcAMP- stimulated granulosa cell PGE and progesterone accumulation by H7. Cells were incubated as in Fig. 1 without additions (0). with 0, 25, or 50 pM H7 and TPA (25 ng/ml), or with 0, 25, or 50 pM H7 and dbcAMP (DBCAMP~ 5 mM). Significant differences from the group with no additions are indicated by *. Wrthin the TPA or dbcAMP groups, significant differences from the TPA only or dbcAMP only means are indicated by +

3 CTL

L”

&l

rio

LH*“t.1cm.(pM,

Fi ure 5. Lack of effect of HA1004 on LH-stimulated PGE or progesterone accumulation. Ce8 Is were incubated as in Fig. 1 without additions (CTL)! with LH only, or with 3,30, or 100 PM HA1004 added 15 min prior to LH. Significant drfferences from the CTL group are Indicated by *. No HA1004 means were different from LH only. H7 had not roven to be a selective inhibitor of protein kinase C in enzyme assays,or oP protein kinase C versus A a onists in whole cells, the in vitro analyses of kinase inhibition (above, an 3 (9)) suggested HA1004 would be specific for protein kinase A at some concentrations. HA1004, however, was not effective against LH-stimulated PGE or progesterone accumulation at the concentratrons tested (Fig. 5). Although

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Since protein kinase A inhibition might be less favored at high LH (and resultant intracellular CAMP) concentrations, lower levels of LH were also tested. No effects of HA1004 were observed at these reduced LH concentrations (Fig. 6). (40 130 120 110 a 5 100 Y @0 5; 110 *: 70 SP “0 e0

Fi ure 6. Lack of effect of HA1004 on PCE or progesterone accumulation stimulated by sui!I maximal LH concentrations. Cells were incubated as in Fig. 5 without additions (01, with LH only at 0.5, 5, or 15 nglml, or these concentrations of LH with 2.5 pM HA1 004 added 15 min prior to LH. Significant differences from the (0) group are mdlcated by *. No HA1004 means were different from respective LH only means.

HA1004 was next tested with cholera toxin, a substance believed to act by increasing CAMP (18). Cholera toxin stimulation of PGE and progesterone was not inhibited by the concentrations of HA1004 tested (Fig.7).

Figure 7. Lack of effect of HA1004 on cholera toxin stimulated PGE or progerterone accumulation. Cellswere incubated as in Fig. 5, substituting cholera toxin (1 of LH. Significant differences from the CTL group are indicated by *, an c%: :nhPol:t toxin only by +

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W9, which like HA1004 showed some selectivity for protein kinase A in enzyme assays,was effective in whole cells (Figs. 8,9). Forskofin stimulation of PGE and pro esterone accumulation were markedly inhibited by H9. On the other han 8 , inhibition of TPA stimulation was minimal, and occurred only at 50 M H9. The reduction of LH responses was intermediate, with no effect at 1 8 pM H9, and partial inhibition at 50 PM.

CTL

LH

F6M

WA

Figure 8. Suppression of LH-, TPA-, and forskoiin-stimulated PGE accumulation by H9. Cells were incubated without additions (CTL), or with 0, 10.25, or 50 PM H9 for 15 min. followed by a 5-h incubation with LH (100 nglml), TPA (25 ng/ml), or forskolin (FSK, 15 pM). Srgnificant differences from CTL are Indicated by l. Withrn the LH, TPA, or forskolin groups, si nificant differences from the LH only, TPA only, or forskolin only means are Indicated %y +.

6

ni

6 C?L

6

16

66 L”

66

6

16

66

66

6

36

66

66

-66

TPA

Fi ure 9. Suppression of LH-, TPA-, and forskolin-stimulated H !I . (See Fig. 8 legend.)

progesterone accumulation by

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DISCUSSION Isoquinolinesulfonamides H7 and H9 were potent inhibitors of LH stimulation of rat granulosa cell PGE and progesterone accumulation. This inhibition may have been caused by actions on protein kinases A and/or C, since these compounds were highly effective in blockin such ovarian kinases in cell-free assays. The relative inhibitions of protein ‘t;inasesA and C in ovarian extracts were consistent with the inhibition constants reported for other tissues (9). The concentrations of H7 and H9 required for inhibition of LH actions were approximately IO-fold greater than the concentrations necessary for half-maximal blockage of kinase activity. Previous reports have interpreted experiments with HA1004 as indicative of the involvement, or lack thereof, of protein kinase A (10,11,19). Our data do not support the appropriateness of this approach for rat granulosa cells. Although HA1004 did not inhibit LH stimulation, it also failed to affect cholera toxin actions, suggesting that HA1004 was generally ineffective against protein kinase A in whole rat granulosa cells under our experimental conditions at concentrations approaching toxic levels. The cause for the ineffe~iveness of HA1004 is unknown, since it had been shown to act on intacttissue in vitro (IO), and to be effective in our hands in protein kinase assays. Structurally, HA1004 is the only inhibitor tested with a guanidino function. H7 inhibition has been widely employed as an indicator of protein kinase C participation in cellular res onses to extracellular agents (9- 11,19-23). The relatively small difference Pabout 2-fold) in inhibition constants for protein kinase A compared to C, however, makes successful utilization of H7 for this distinction unlikely (9). Our results demonstrate that concentrations which inhibit dbcAMP stimulation also inhibit the rotein kinase C agonist, TPA. As stated by Hidaka and co-workers, H7 ma Re useful only when low levels of protein kinase A are present (9); or to ta z e this a bit further, when CAMP is not involved as shown by direct testing. H9 inhibited protein kinase A activity at approximately one order of magnitude lower concentration than that affecting protein kinase C in our rat ovarian extracts, and in rabbit tissues (9). In granulosa cells, marked inhibition of forskolin stimulation of PGE and pro esterone accumulation was demonstrated at concentrations 5-fold lower t i! an those which slightly reduced the responses to TPA. These findin s are interpreted as inhibition of the major mechanism of action of 3orskofin, that is, increased intracellular CAMP and activation of protein kinase A (24). lnte~erence with non-CAMP actions of forskolin, however, cannot be ruled out (25,26). The inhibition of protein kinases A by H9 may also depend on the magnitude of the CAMP response in the cell. Althou h the PGE and progesterone responses to LH and forskolin are comparab ?e, the elevations of intracellular CAMP may not be. Nonetheless, H9 appears to be a superior protein kinase A inhibitor in comparison with HA1004 in granulosa cells, and shows potential as a selective inhibitor of protein kinase A compared to C at concentrations lessthan 50 FM.

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The above observations were inconsistent with a simple cytotoxic action of the isoquinolinesulfonamides, since cell death was not produced during the 5-h incubation period. This result is in agreement with the reported toxicities of H7 and HA1004 in long-term culture only at concentrations above 32 and 320 PM, respectively (11). These inhibitors were effective only when included in mcubations during the first hour of exposure to LH. This is the principal period of second messenger generation and protein kinase activation (2,3). Failure to block LH actions by later addition also sug ests a limited scope of inhibition. Toxicity short of overt cell death, such asPass of protein or mRNA synthesis, would be expected to affect PGE and rogesterone accumulation at 3-5 h. These inhibitors have been shown to ge effective against a cGMP-dependent protein kinase at similar concentrations, so the possible involvement of this enzyme cannot be ruled out (9). These concentrations of the drugs would not be expected to inhibit all kinases or ATP-requiring enzymes, since effects on myosin light-chain kinase, casein kinases I and II, actomyosin ATPase, or (Ca2 + -Mgz + )-ATPase required 50-to lOOO-fold higher concentrations (9). The absolute specificity of the isoquinolinesulfonamides with respect to protein kinases in whole cells, however, will require considerable further testing. In conclusion, two isoquinolinesulfonamides were found to markedly inhibit LH stimulation of granulosa cell functions in a manner consistent with inhibition of protein kinase activities. Since endo enous protein kinase inhibitors also exist for both protein kinases A (27B and C (28,29), these drugs may provide insight into the possible effects of such endogenous inhibitors on LH action. ACKNOWLEDGMENTS We appreciate the excellent technical assistance of Marsha Clod and Dania Aguirre. Supported in part by grant HD-22299 from the NIH and a Departmental Research Grant. Portions of this work were presented at the 2lst Annual Meeting of the Society for the Study of Reproduction, Seattle, Washington, 1988 (Abstract 162). REFERENCES 1. 2.

3.

4.

Edelman AM, Blumenthal DK, and Krebs EC (1987). Protein serineAhreonine kinases. ANNU REV BIOCHEM 56:567-613. Williams MT, Clark MR, Ling WY., LeMaire WJ, Caron MC, and Marsh JM (1977). The role of cyclic AMP m the actions of luteinizing hormone on steroidogenesis in the corpus luteum. ADV CYCLIC NUCLEOTIDE RES9: 573-580. Davis JS, Weakland LL, West LA, and Farese RV (1986). Luteinizing Hormone Stimulates the formation of inositol trisphosphate and cyclic AMP in rat granulosa cells: Evidence for phospholipase C generated second messengers in the action of luteinizing hormone. BIOCHEM J 238: 597-604. Dimino MJ, Snitzer J, and Brown KM (1987). lnositol phosphate accumulation in ovarian granulosa after stimulation by luteinizing hormone. BIOL REPROD 37:1129-l 134.

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Yama uchi DT Hahn TJ Beeker TG Kleeman CR, and Muallem S (198851. Relationship of’cAMP and ialcium messenger systems in prostaglandin-stimulated UMR-106 cells. J BIOL CHEM 263:1074510753. Limbird LE (1988). Receptors linked to inhibition of adenylate cyclase: Additional signaling mechanisms. FASEBJ 2:2686-2695. Clark MR, Marsh JM, and LeMaire WJ (1978). Stimulation of prostaglandin accumulation in preovulatory rat follicles by adenosine 3’,5’-monophosphate. ENDOCRINOLOGY 102:39-44. Kawai Y and Clark MR (1985). Phorbol ester regulation of rat granulosa cell prostaglandin and progesterone accumulation. ENDOCRINOLOGY 116:2320-2326. Hidaka H, lnagaki M, Kawamoto 5, and Sasaki Y (1984). Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. BIOCHEMISTRY 23:5036-5041. Ishikawa T, lnagaki M, Watanabe M, and Hidaka H (1985). Relaxation of vascular smooth muscle by HA-1004, an inhibitor of cyclic nucleotide-dependent protein kinase. J PHARMACOL EXP THER 235:495-499. Martell RE, Sim son RU, and Hsu T (1988). Effects of protein kinase inhibitors 1 P5-isoquinolinesulfon I)-2-methylpiperazine uinolinedih drochloride (H7) and N-[2-guani J inoethyll-5-iso (HA1004) on calcitrio 9 -induced sul onamide h drochloride y differentiation o Y HL-60 cells. BIOCHEM PHARMACOL 37:635-640. Clark MR (1982). Stimulation of progesterone and prostaglandin E accumulation by luteinizing hormone-releasing hormone (LHRH) and LHRH analogs in rat granulosa cells. ENDOCRINOLOGY 110: 146-152. Davis JS and Clark MR (1983). Activation of protein kinase in the bovine corpus luteum by phospholipid and Ca2 + . BIOCHEM J 214:569574. Jeng AY, Sharkey NA, and Blumberg PM (1986). Purification of stable protein kinase C from mouse brain cytosol by specific ligand elution using fast protein liquid chromatography. CANCER RES46:1966-1971. White A, Anderson DC, and Daly JR (1982). Production of a highly specific monoclonal antibody to progesterone. J CLIN ENDOCRINOL M ETAB 54: 205-207. Hsu S-M, Raine L, and Fanger H (1981). Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures. J HISTOCHEM CYTOCHEM 29:577-580. Matthews DE and Farewell VT (1988). Using and Understanding Medical Statistics, Karger, New York, p 178. Finkelstein RA (1973). Cholera. CRC GRIT REV MICROBIOL 2:553-623. Struhar D and Harbeck RJ (1987). Inhibition of induced acute lung edema by a novel protein kinase C inhibitor. FASEBJ 1:116-l 18. lnagaki M, Kawamoto 5, and Hidaka H (1984). Serotonin secretion from human platelets may be modified by Ca2+ -activated, phospholipid-dependent myosin phosphorylation. J BIOL CHEM 259:14321-14323.

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Kawamoto S and Hidaka H (1984). I-(5-lsoquinolinesuIfonyl)-2methylpiperazine (H7) is a selective inhibitor of protein kinase C in rabbit platelets. BlOCHEM BIOPHYS RESCOMMUN 125:258-264. Berkow RL, Dodson RW, and Kraft AS (1987). The effect of a protein kinase inhibitor, H-7, on human neutrophil oxidative burst and degranulation. J LEUKOCYTE 8IOL41:441-446. Clark R8, Love JT Jr, Sgroi D, Lingenheld EG, and Sha’afi RI (1987). The protein kinase inhibitor, H-7, inhibits antigen and IL-2-induced proliferation of murine T cell lines. BIOCHEM BIOPHYS RES COMMUN 145:666-672. Daly JW (1984). Forskolin, adenylate cyclase, and cell physiolo y: An overview. ADV CYCLIC NUCLEOTIDE PROTEIN PHOSPHORYLATI 8 N RES 17:81-89. Hoshi T, Garber 55, and Aldrich RW (1988). Effect of forskolin on voltage-gated K+ channels is independent of adenylate cyclase activation. SCIENCE 240: 1652-1655. Wagoner PK and Pallotta 8s (1988). Modulation of acetylcholine receptor desensitization by forskolin is independent of CAMP. SCIENCE 240:1655-1657. McPherson JM, Whitehouse 5, and Walsh DA (1979). Possibilit of shape conformers of the protein inhibitor of the CAMP-depen J ent protein kinase. BIOCHEMISTRY 18:4835-4845. Huang C-K and Oshana SC (1986). Partial characterization of protein kinase C and inhibitor activity of protein kinase C in rabbit peritoneal neutrophils. J LEUKOCYTE BIOL 39:671-678. Schwantke N and Le Peuch CJ (1984). A protein kinase C inhibitory activity is present in rat brain homogenate. FEBSLETT 177:36-40.

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