Activation of protein kinase C potentiates cyclic AMP production and stimulates steroidogenesis in differentiated ovarian granulosa cells

Gonadotropin-stimulated steroidogenesis in the differentiating ovarian granulosa cell is mediated through the activation of cAMP-dependent protein kinase, and is also modulated by calcium-dependent mechanisms. Granulosa cells contain calcium-activated, phospholipid-dependent protein kinase (C kinase), and show an increase in phosphatidylinositol turnover in response to GnRH agonist analogs. To evaluate the role of C kinase in ovarian steroidogenesis, the potent phorbol ester, TPA, and the permeant diacylglycerol, OAG, were used to activate C kinase in granulosa cells from PMSG-treated immature rats. Both TPA and OAG caused dose-dependent stimulation of progesterone production without affecting intra- or extracellular cAMP levels. However, the maximum steroid responses to these compounds were less than those stimulated by cAMP. The ED50 for TPA-stimulated progesterone production was 3 nM, which is close to the known Km for activation of C kinase. Stimulation of steroidogenesis was only observed with biologically-active phorbol esters and permeant diacylglycerols such as OAG and DOG. Exposure of granulosa cells to phospholipase C also increased progesterone production in a dose-dependent manner without changing the cAMP content. Although TPA and OAG did not increase basal cAMP production, both agents enhanced the cAMP responses stimulated by hCG and forskolin; likewise, phospholipase C alone did not change cAMP production but caused a dose-dependent increase in the cAMP responses to hCG and forskolin. These results demonstrate that activation of C kinase promotes steroidogenesis in ovarian granulosa cells, and potentiates the activation of adenylate cyclase by hCG and forskolin. Such findings support the possibility that the calcium, phospholipid-dependent enzyme could be involved in the regulation of progesterone production by hormonal ligands such as gonadotropins and GnRH.     

Two different intrachain cAMP sites in the cAMP-dependent protein kinase of the dimorphic fungus Mucor rouxii

cAMP sites of the cAMP-dependent protein kinase from the fungus Mucor rouxii have been characterized through the study of the effects of cAMP and of cAMP analogs on the phosphotransferase activity and through binding kinetics. The tetrameric holoenzyme, which contains two regulatory (R) and two catalytic (C) subunits, exhibited positive cooperativity in activation by cAMP, suggesting multiple cAMP-binding sites. Several other results indicated that the Mucor kinase contained two different cooperative cAMP-binding sites on each R subunit, with properties similar to those of the mammalian cAMP-dependent protein kinase. Under optimum binding conditions, the [3H]cAMP dissociation behavior indicated equal amounts of two components which had dissociation rate constants of 0.09 min-1 (site 1) and 0.90 min-1 (site 2) at 30 degrees C. Two cAMP-binding sites could also be distinguished by C-8 cAMP analogs (site-1-selective) and C-6 cAMP analogs (site-2-selective); combinations of site-1- and site-2-selective analogs were synergistic in protein kinase activation. The two different cooperative binding sites were probably located on the same R subunit, since the proteolytically derived dimeric form of the enzyme, which contained one R and one C component, retained the salient properties of the untreated tetrameric enzyme. Unlike any of the mammalian cyclic-nucleotide-dependent isozymes described thus far, the Mucor kinase was much more potently activated by C-6 cAMP analogs than by C-8 cAMP analogs. In the ternary complex formed by the native Mucor tetramer and cAMP, only the two sites 1 contained bound cAMP, a feature which has also not yet been demonstrated for the mammalian cAMP-dependent protein kinase.     

cAMP-dependent protein kinase activation lowers hepatocyte cAMP

Rat hepatocyte protein kinase was activated by incubating the cells with various cAMP analogs. Boiled extracts were then prepared and Sephadex G-25 chromatography was carried out. The G-25 procedure separated the analogs from cAMP since the resin had the unexpected property of binding cyclic nucleotides with differing affinities. Separation was necessary because the analogs would otherwise interfere with the sensitive protein kinase activation method developed for assay of cAMP. The cAMP analogs, but not 5'-AMP, lowered basal cAMP by 50-70%. The effect was rapid, analog concentration-dependent, and occurred parallel with phosphorylase activation, suggesting that the cAMP analogs act through cAMP-dependent protein kinase activation. A cAMP analog completely blocked the cAMP elevation produced by relatively low concentrations of glucagon, but did not block the phosphorylase response, indicating that the cAMP analog substitutes for cAMP as the intracellular activator of protein kinase. One implication of the results is that elevation of cAMP and protein kinase activity by hormones has a negative feedback effect on the cellular cAMP level.     

Hormonal regulation of cyclic AMP-dependent protein kinase in cultured ovarian granulosa cells. Effects of follicle-stimulating hormone and gonadotropin-releasing hormone

The hormonal regulation of cAMP-dependent protein kinase was examined in granulosa cells from diethylstilbestrol-implanted immature rats. Follicle-stimulating hormone (FSH) increased the number of available cAMP-binding sites in a dose- and time-dependent manner, with a maximum 4-6-fold increase at 50-100 ng/ml between 6 and 48 h of culture after a transient decrease in available sites during the first 6 h. The potent gonadotropin-releasing hormone (GnRH) agonist [D - Ala6]des - Gly10 - GnRH - N - ethylamide (GnRHa) reduced the FSH-induced increase in cAMP-binding sites by approximately 50% at 24 and 48 h of culture. Photoaffinity labeling with 8-azido-[32P] cAMP revealed the existence of one major cAMP-binding protein (Mr = 55,000 +/- 400) which appeared to be the regulatory (R) subunit of type II cAMP-dependent protein kinase. While FSH induced a 5-10-fold increase in the labeling of R II both in vivo and in vitro, GnRHa reduced the amount of R II induced by FSH in granulosa cells cultured for 48 h. The large increase in R II subunit was not accompanied by a corresponding increase in protein kinase activity, which was only enhanced by 50% after 48 h of culture with FSH. Fractionation of granulosa cell cytosol from FSH-treated ovaries on DEAE-cellulose showed a single peak of cAMP-dependent phosphokinase activity with the elution properties of a type II protein kinase. However, the peak of cAMP binding activity (eluted at 0.20 M KCl) was not coincident with the protein kinase activity. FSH transiently stimulated cAMP-dependent protein kinase activity during the first 10-30 min of culture. GnRHa impaired the FSH-induced early increase in protein kinase activity, causing a delay in activation until 60 min. These findings suggest that a large dose- and time-dependent increase in the content of cAMP-binding sites may be a major factor in cAMP-mediated differentiation of granulosa cells. The inhibitory effect of GnRHa on both FSH-induced protein kinase activation during the first minutes of culture and on FSH-induced R II synthesis during the subsequent 48 h of culture could be crucial events in the prevention of granulosa cell maturation by GnRH agonists.     

Effect of some new cAMP analogs on cAMP-dependent protein kinase isoenzymes.

1. Ten new cAMP analogs were synthesized by replacing the purine ring with with indazole, benzimidazole or benztriazole and/or their nitro and amino derivatives. 2. Each analog proved effective in activating cAMP-dependent protein kinase I (PK-I) purified from rabbit skeletal muscle and cAMP-dependent protein kinase II (PK-II) from bovine heart and chasing 8-[3H]cAMP bound to regulatory subunits in the half-maximal effective concentrations of 2 x 10(-8)-8 x 10(-6) M. 3. The N-1-beta-D-ribofuranosyl-indazole-3'5'-cyclophosphate(I) proved a very poor chaser and activator of both isoenzymes, but when indazole was attached at its N-2 to ribose (IV) or when its H at C-4 (equivalent to the position of amino-group in adenine) was substituted by an amino-(III) or especially nitro-group (II) its efficiency was dramatically increased. 4. Analogs containing benztriazole ring proved as powerful as cAMP irrespective of the presence of substituents (VII-X). 5. Benzimidazole derivatives with amino-(VI) or nitro-group (V) activated PK-II 3 and 20 times better than PK-I. 6. Attaching of ribose to N-2 of indazole or benztriazole increased the affinity to PK-II 10 and 4 times, respectively. 7. Chasing efficiency of cAMP analogs at half-saturating [3H]cAMP tended to correlate with activating potency only for PK-I but at saturating [3H]cAMP concentration for both isoenzymes. 8. On the basis of synergistic activation with 8-Br-cAMP a site 2-selective binding of nitro-benzimidazole (V) and unsubstituted benztriazole (VII) derivatives to PK-II is suggested.     

Site-selective cAMP analogs induce nuclear translocation of the RII cAMP receptor protein in Ha-MuSV-transformed NIH/3T3 cells

Site-selective cAMP analogs, depending on the position of their substituents on the adenine ring, selectively bind to either site 1 or site 2 of the known cAMP binding sites of protein kinase. Treatment of Harvey murine sarcoma virus-transformed NIH/3T3 cells with such site-selective analogs results in growth inhibition and phenotypic reversion, and the combination of a C-8 thio or halogen analog (site 1 selective) with an N6 analog (site 2 selective) produces a synergistic effect. We report here that the growth inhibitory effect of the analogs correlates with the nuclear translocation of the RII cAMP receptor protein, the regulatory subunit of protein kinase type II. The transformed NIH/3T3 cells contained no detectable level of RII in the nucleus, whereas nontransformed NIH/3T3 cells exhibited a high level of nuclear RII. Within 30 min after treatment of the transformed cells with the site-selective analogs, immunofluorescence against the RII protein markedly increased in the cell nucleus. The nuclear translocation of the RII cAMP receptor protein is an early event in the reverse transformation of the fibroblasts treated with site-selective cAMP analogs.     

Effect of cyclic nucleotide analogs on intrachain site I of protein kinase isozymes

The effects of numerous cAMP analogs present in the [3H]cAMP binding reaction on subsequent dissociation of [3H]cAMP from the regulatory subunit of cAMP-dependent protein kinase I and II were analyzed. Certain analogs with modification at either C-8 or C-2 showed relative selectivity for one (site 1) of two intrachain cAMP binding sites of both isozymes. Modification at C-6 caused selectivity for the second site (site 2). The combination of a site-1-directed and site-2-directed analog inhibited [3H]cAMP binding much more than did either analog alone. In general, there was a correlation between the site 1 selectivity and the ability of the analog to stimulate the binding of [3H]cIMP, which selects site 2. The site-1-directed analogs stimulated the initial rate of [3H]cIMP binding. The stimulatory effect was enhanced in the presence of a polycationic protein such as histone and was inhibited by high ionic strength. The type I and II isozymes exhibited large differences in analog specificity for this effect. For type I, of the analogs tested the most efficacious for stimulating [3H]cIMP binding were those containing a nitrogen atom attached to C-8, 8-aminobutylamino-cAMP being the most effective. Type II responded best to analogs containing a sulfur atom attached to C-8, 8-SH-cAMP being the most effective of those tested. The stimulatory effect was accentuated in the presence of MgATP when using type I, but this nucleotide had no effect when using type II. It is proposed that in intact tissues cAMP binding to site 1 of either isozyme stimulates the binding to site 2.     

ATP analog specificity of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and phosphorylase kinase

The ATP analog specificities of the homogeneous cGMP-dependent protein kinase and the catalytic subunit of cAMP-dependent protein kinase have been compared by the ability of 27 analogs to compete with ATP in the protein kinase reaction. Although the data suggest general similarities between the ATP sites of the two homologous cyclic-nucleotide-dependent protein kinases, specific differences especially in the adenine binding pocket are indicated. These differences in affinity suggest potentially useful ATP analog inhibitors of each kinase. For example, apparent autophosphorylation of the purified regulatory subunit of the cAMP-dependent protein kinase is blocked by nebularin triphosphate, suggesting that the phosphorylation is catalyzed by trace contamination of cGMP-dependent protein kinase. Some of the ATP analogs have also been tested using phosphorylase b kinase in order to compare this enzyme with the cyclic-nucleotide-dependent enzymes. All three protein kinases have high specificity for the purine moiety of ATP, and lower specificity for the ribose or triphosphate. The similarity between the ATP site of phosphorylase b kinase to that of the cyclic-nucleotide-dependent protein kinases suggests that it is related to them. The ATP analog specificities of enzymes examined in this study are different from those reported for several unrelated ATP-utilizing enzymes.     

Evidence for involvement of protein kinase in the activation by adenosine 3':5'-monophosphate of brain tyrosine 3-monooxygenase.

Addition of adenosine 3':5'-monophosphate (cAMP) to high speed supernatant preparations obtained from rat brain caused a 3- to 4-fold increase in tyrosine 3-monooxygenase (tyrosine hydroxylase) activity. The tyrosine 3-monooxygenase remained in an activated state upon removal of the cAMP by passing the enzyme through a Sephadex G-25 column. Substances which inhibit cAMP-dependent protein kinase, namely, EDTA, ADP, and adenosine, and protein kinase modulator, each antagonized the activation of tyrosine 3-monooxygenase produced by cAMP. Furthermore, addition of partially purified brain cAMP-dependent protein kinase caused a several-fold increase in tyrosin 3-monooxygenase activity. The activation of tyrosine 3-monooxygenase by added cAMP and protein kinase required the presence of ATP and Mg-2+. These data suggests that the cAMP activation of tyrosine 3-monooxygenase may be mediated by a cAMP-dependent protein kinase.     

Activation of protein kinase isozymes by cyclic nucleotide analogs used singly or in combination. Principles for optimizing the isozyme specificity of analog combinations

104 cAMP analogs, most of them modified in the adenine moiety, were tested as activators of cAMP-dependent protein kinase I (from rabbit or rat skeletal muscle) and kinase II (from bovine heart or rat skeletal muscle). When tested singly, only 2-phenyl-1,N6-etheno-cAMP showed a considerably (sevenfold) higher potency as an activator of kinase II than of kinase I. Analogs containing an 8-amino modification preferentially activated kinase I, some being more than 10-fold more potent as activators of kinase I than kinase II. When two analogs were combined, the concentration of one (complementary) analog required to half-maximally activate each isozyme was determined in the presence of a fixed concentration of another (priming) analog. Analogs tested in combination had been analyzed for their affinity for the intrasubunit binding sites (A, B) of isozyme I and II. The degree to which complementary analogs preferentially activated one isozyme was plotted against the mean site selectivity, i.e. (affinity A/B isozyme I X affinity A/B isozyme II) 1/2. This plot produced a straight line, the slope of which reflected the ability of the priming analog to discriminate homologous sites on the isozymes. This means that the isozyme discriminating power of an analog pair can be quantitatively predicted from the affinity of the analogs for site A and B of the two enzymes. It also means that a systematic analysis of those features of analogs imparting a high mean site selectivity or the ability to discriminate between homologous isozyme sites will facilitate the synthesis of new even more isozyme-selective analogs.     

Luteinizing hormone receptor activation in ovarian granulosa cells promotes protein kinase A-dependent dephosphorylation of microtubule-associated protein 2D

The actions of LH to induce ovulation and luteinization of preovulatory follicles are mediated principally by activation of cAMP-dependent protein kinase (PKA) in granulosa cells. PKA activity is targeted to specific locations in many cells by A kinase-anchoring proteins (AKAPs). We previously showed that FSH induces expression of microtubule-associated protein (MAP) 2D, an 80-kDa AKAP, in rat granulosa cells, and that MAP2D coimmunoprecipitates with PKA-regulatory subunits in these cells. Here we report a rapid and targeted dephosphorylation of MAP2D at Thr256/Thr259 after treatment with human chorionic gonadotropin, an LH receptor agonist. This event is mimicked by treatment with forskolin or a cAMP analog and is blocked by the PKA inhibitor myristoylated-PKI, indicating a role for cAMP and PKA signaling in phosphoregulation of granulosa cell MAP2D. Furthermore, we show that Thr256/Thr259 dephosphorylation is blocked by the protein phosphatase 2A (PP2A) inhibitor, okadaic acid, and demonstrate interactions between MAP2D and PP2A by coimmunoprecipitation and microcystin-agarose pull-down. We also show that MAP2D interacts with glycogen synthase kinase (GSK) 3beta and is phosphorylated at Thr256/Thr259 by this kinase in the basal state. Increased phosphorylation of GSK3beta at Ser9 and the PP2A B56delta subunit at Ser566 is observed after treatment with human chorionic gonadotropin and appears to result in LH receptor-mediated inhibition of GSK3beta and activation of PP2A, respectively. Taken together, these results show that the phosphorylation status of the AKAP MAP2D is acutely regulated by LH receptor-mediated modulation of kinase and phosphatase activities via PKA.     

Phosphorylation of regulatory subunit of type I cyclic AMP-dependent protein kinase: biphasic effects of cyclic AMP in intact S49 mouse lymphoma cells

Intact S49 mouse lymphoma cells were used as a model system to study the effects of cyclic AMP (cAMP) and its analogs on the phosphorylation of regulatory (R) subunit of type I cAMP-dependent protein kinase. Phosphorylation of R subunit was negligible in mutants deficient in adenylate cyclase; low levels of cAMP analogs, however, stimulated R subunit phosphorylation in these cells to rates comparable to those in wild-type cells. In both wild-type and adenylate cyclase-deficient cells, R subunit phosphorylation was inhibited by a variety of N6-substituted derivatives of cAMP; C-8-substituted derivatives were generally poor inhibitors. Two derivatives that were inactive as kinase activators (N6-carbamoylmethyl-5'-AMP and 2'-deoxy-N6-monobutyryl-cAMP) were also ineffective as inhibitors of R subunit phosphorylation. Preferential inhibition by N6-modified cAMP analogs could not be ascribed simply to selectivity for the more aminoterminal (site I) of the two cAMP-binding sites in R subunit: Analog concentrations required for inhibition of R subunit phosphorylation were always higher than those required for activation of endogenous kinase; 8-piperidino-cAMP, a C-8-substituted derivative that is selective for cAMP-binding site I, was relatively ineffective as in inhibitor; and, although thresholds for activation of endogenous kinase by site I-selective analogs could be reduced markedly by coincubation with low levels of site II-selective analogs, no such synergism was observed for the inhibitory effect. The uncoupling of cyclic nucleotide effects on R subunit phosphorylation from activation of endogenous protein kinase suggests that, in intact cells, activation of cAMP-dependent protein kinase requires more than one and fewer than four molecules of cyclic nucleotide.     

Cyclic 3',5'-adenosine monophosphate-dependent kinase and phosphoproteins in human amnion

3',5'-Cyclic adenosine monophosphate (cAMP) modulates prostaglandin production in human amnion membranes. The major effects of cAMP are presumably mediated through the phosphorylation of specific regulatory phosphoproteins following cAMP activation of cAMP-dependent protein kinase. Cyclic AMP-dependent protein kinase and phosphoproteins have not previously been characterized in human amnion. Total homogenates, cytosol, and membrane fractions from human amnion were examined for [3H]cAMP binding activity and cAMP-dependent kinase activity. cAMP-dependent kinase activity was barely detectable in crude amnion fractions. Cytosol was therefore partially purified by DEAE column chromatography for further examination. Two peaks of coincident [3H]cAMP binding and cAMP-dependent kinase activity were demonstrated at 70 and 140 mM NaCl, characteristic of the Type I and Type II cAMP-dependent protein kinase isozymes. [3H]cAMP binding to the material from both peak fractions was saturable and reversible. Scatchard analysis of [3H]cAMP binding to the peak fractions was linear for peak I and curvilinear for peak II. Assuming a one-site model, [3H]cAMP binding to the Type I isozyme showed a KD = 4.17 x 10(-8) M and Bmax = 73 pmole/mg protein; using a two-site model, [3H]cAMP binding to the high-affinity site for the Type II isozyme had a KD = 3.94 x 10(-8) M and Bmax = 6.3 pmole/mg protein. Other cyclic nucleotides competed for these [3H]cAMP binding sites with a potency order of cAMP much greater than cGMP greater than (BU)2cAMP.cAMP caused a dose-dependent increase in cAMP-dependent kinase activity in the peak fractions; half-maximal activation was observed with 5.0 x 10(-8) M cAMP. The ability of cAMP to increase phosphorylation of endogenous proteins in both crude amnion cytosol and cytosol from cultures of amnion epithelial cells was assessed using [32P]ATP, SDS-polyacrylamide gel electrophoresis and autoradiography. cAMP stimulated 32P incorporation into three proteins having Mr = 80,000, 54,000, and 43,000 (P less than .01). Half-maximal 32P incorporation into these proteins occurred at 1.0 x 10(-7) M cAMP. cAMP-dependent kinase is present in human amnion; specific cAMP-enhanced phosphoproteins are also present. Hormones elevating cAMP levels in amnion may exert their effects by activating cAMP-dependent kinase and phosphorylating these phosphoproteins.     

Inhibition of cAMP-dependent protein kinase by adenosine cyclic 3'-, 5'-phosphorodithioate, a second cAMP antagonist

A single sulfur substitution for either the axial or the equatorial exocyclic oxygen of adenosine cyclic 3', 5'-phosphate (cAMP) results in diastereometric phosphorothioate analogs of cAMP with agonist versus antagonist properties towards activation of cAMP-dependent protein kinase. Sulfur substitutions for both of the exocyclic oxygens of cAMP results in a dithioate analog of cAMP, adenosine cyclic 3', 5'-phosphorodithioate (cAMPS2), which has antagonist properties. cAMPS2 displaced [3H]cAMP from the binding sites on bovine heart Type II cAMP-dependent protein kinase as demonstrated by equilibrium dialysis experiments with an apparent Kd of 6.3 microM. The addition of 10, 30, or 100 microM cAMPS2 when measuring cAMP-induced activation of pure porcine heart Type II cAMP-dependent protein kinase resulted in a concentration-dependent increase in the amount of cAMP required to produce half-maximal activation (EC50). A plot of the EC50 values as a function of the cAMPS2 concentration resulted in a straight line from which a KI value of 4 microM was derived. cAMPS2 had no significant effect on the degree of cooperativity (n) of cAMP activation of the holoenzyme. These data suggest that the most important structural requirement for the dissociation of the holoenzyme is an equatorial exocyclic oxygen.     

Regulation of cyclic adenosine 3',5'-monophosphate-dependent protein kinase activity and regulatory subunit RII beta content by basic fibroblast growth factor (bFGF) during granulosa cell differentiation: possible implication of protein kinase C in bFGF action.

We have previously shown that basic fibroblast growth factor (bFGF) inhibits the FSH-induced differentiation of cultured rat granulosa cells, as manifested by prominent reduction of the LH receptor expression. We now investigate the possible sites and mechanism of action of bFGF. Whereas bFGF decreased the cAMP formation induced by FSH, it enhanced the cAMP production caused by cholera toxin and forskolin, suggesting that bFGF exerted its inhibitory action on cell differentiation at a step to cAMP production. Photoaffinity labeling with 8-azido-[32P]cAMP revealed that bFGF markedly reduced the FSH-induced increase in the level of regulatory subunit RII beta of the cAMP-dependent protein kinase (PKA) type II. In contrast to its striking effect on RII beta expression (70-80% inhibition), bFGF decreased PKA enzymatic activity by only 30%. On the other hand, transforming growth factor-beta (TGF beta) slightly amplified the stimulatory action of FSH and antagonized the bFGF inhibitory effect on both LH receptor expression and RII beta synthesis. We report that the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA), which impaired granulosa cell differentiation, also abolished the RII beta synthesis induced by FSH. The activation of PKC by bFGF in granulosa cells was supported by the following findings: (i) bFGF markedly enhanced the production of diacylglycerol (2.3-fold stimulation at 5 min), the intracellular activator of PKC; (ii) bFGF promoted tight association of PKC to cellular membranes, a process that is believed to correlate with the enzyme activation; (iii) bFGF induced the phosphorylation of an endogenous M(r) 78,000/pI 4.7 protein that appears as a specific PKC substrate; (iv) bFGF mimicked the TPA-induced transmodulation of the epidermal growth factor (EGF) receptor, reducing by 36% the 125I-EGF binding on granulosa cells. We conclude that bFGF may exert its repressive action on RII beta synthesis, PKA activity, and granulosa cell differentiation by primarily targeting PKC activation.     

Cyclic AMP-dependent protein kinase and Epac mediate cyclic AMP responses in pancreatic acini

The pancreatic acinar cell has several phenotypic responses to cAMP agonists. At physiological concentrations of the muscarinic agonist carbachol (1 microM) or the CCK analog caerulein (100 pM), ligands that increase cytosolic Ca(2+), cAMP acts synergistically to enhance secretion. Supraphysiological concentrations of carbachol (1 mM) or caerulein (100 nM) suppress secretion and cause intracellular zymogen activation; cAMP enhances both zymogen activation and reverses the suppression of secretion. In addition to stimulating cAMP-dependent protein kinase (PKA), recent studies using cAMP analogs that lack a PKA response have shown that cAMP can also act through the cAMP-binding protein, Epac (exchange protein directly activated by cyclic AMP). The roles of PKA and Epac in cAMP responses were examined in isolated pancreatic acini. The activation of both cAMP-dependent pathways or the selective activation of Epac was found to enhance amylase secretion induced by physiological and supraphysiological concentrations of the muscarinic agonist carbachol. Similarly, activation of both PKA or the specific activation of Epac enhanced carbachol-induced activation of trypsinogen and chymotrypsinogen. Disorganization of the apical actin cytoskeleton has been linked to the decreased secretion observed with supraphysiological concentrations of carbachol and caerulein. Although stimulation of PKA and Epac or Epac alone could largely overcome the decreased secretion observed with either supraphysiological carbachol or caerulein, stimulation of cAMP pathways did not reduce the disorganization of the apical cytoskeleton. These studies demonstrate that PKA and Epac pathways are coupled to both secretion and zymogen activation in the pancreatic acinar cell.     

Regulation of mRNA expression of 3 beta-hydroxy-5-ene steroid dehydrogenase in porcine granulosa cells in culture: a role for the protein kinase-C pathway

We studied the effect of the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA), which activates protein kinase-C, on porcine granulosa cells in culture. PMA as well as cholera toxin, forskolin, and hCG increased cAMP accumulation. PMA further augmented the elevation in cAMP accumulation induced by cholera toxin, forskolin, and hCG. In the same cell culture model, hCG induced a time-dependent increase in the 3 beta-hydroxy-5-ene steroid dehydrogenase (3 beta HSD) mRNA levels with a maximal 3-fold stimulation obtained at 8-16 h of incubation with 1 IU hCG/ml. PMA inhibited the increase in 3 beta HSD mRNA levels induced by hCG in a dose-dependent manner. The phorbol ester also inhibited the increase in 3 beta HSD mRNA levels stimulated by LH as well as cholera toxin and forskolin and the cAMP analogs (Bu)2cAMP and 8-bromo-cAMP. Activation of protein kinase-C by mezerein similarly inhibited hCG stimulation of 3 beta HSD mRNA levels. The present data indicate that activation of the protein kinase-C pathway induces generation of cAMP, but causes a near-complete inhibition of the stimulatory effects of hCG, LH, forskolin, cholera toxin, and cAMP analogs on 3 beta HSD mRNA levels in porcine granulosa cells in culture.     

Catecholamine activation of the membrane transport of long chain fatty acids in adipocytes is mediated by cyclic AMP and protein kinase

Membrane transport of long chain fatty acids in the isolated adipocyte can be stimulated 5-10-fold by epinephrine (Abumrad, N. A., Perry, P. R., and Whitesell, R. R. (1985) J. Biol. Chem. 260, 9969-9971). This study shows that isoproterenol and norepinephrine are more potent than epinephrine in activating the transport process. The stimulatory effect on transport is mediated by beta-receptor interaction and cAMP. This was shown by the following. alpha-Receptor agonists and antagonists were ineffective; methylisobutylxanthine at low concentration (3 microM) potentiated the effect of a suboptimal dose (0.01 microgram/ml) of epinephrine and was stimulatory at high concentration (100 microM) in the absence of epinephrine; and cAMP analogs were very effective activators. Involvement of the cAMP-dependent protein kinase was indicated by two lines of evidence. 1) Combinations of cAMP analogs which are specific for sites 1 and 2 of the protein kinase, respectively, had synergistic effects on fatty acid transport. Combinations of analogs specific for the same site were only additive in their effects. This is similar to the pattern of protein kinase activation in vitro and to that of lipolysis activation in the intact adipocyte (Beebe, S. J., Holloway, R., Rannels, S. R., and Corbin, J. D. (1984) J. Biol. Chem. 259, 3539-3547). 2) Treatment of cells with various metabolic poisons abolished the stimulatory effect of norepinephrine. The response of fatty acid transport to catecholamines showed multiple parallels with that documented for lipolysis except that it was much more rapid. This suggested that the transport process was a regulatory step in fatty acid mobilization. This interpretation is supported by the observation that basal Vmax for transport is much too slow to accommodate the rate of fatty acid release which is observed following stimulation of intact cells with adrenergic hormones.