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.     

Epidermal growth factor potentiates cyclic AMP accumulation in A-431 cells.

Epidermal growth factor (EGF) treatment of A-431 cells potentiates up to 5-fold the intracellular cyclic AMP (cAMP) accumulation induced by isoproterenol, cholera toxin, forskolin, or 3-isobutyl-1-methylxanthine (IBMX). EGF potentiates cAMP accumulation in several epithelial cell lines which overexpress the EGF receptor including A-431 cells, HSC-1 cells, and MDA-468 cells, and in the A-431-29S clone which expresses a normal complement of EGF receptors. Although EGF potentiates cAMP accumulation, EGF by itself does not measurably alter the basal level of cAMP. EGF rapidly enhances cAMP accumulation (within 1 to 3 min) in A-431 cells treated with these cAMP-elevating agents. EGF potentiation of cAMP accumulation does not reflect enhancement of beta-adrenergic receptor activation and is not a consequence of intracellular cAMP elevation or the concomitant activation of cAMP-dependent protein kinase. Since EGF potentiates accumulation of both intracellular and extracellular cAMP in isoproterenol-treated A-431 cells, EGF does not potentiate intracellular cAMP accumulation by inhibition of cAMP export. EGF potentiation of cAMP accumulation is pertussis toxin-insensitive and does not result from EGF inhibition of cAMP degradation in A-431 cells. These results demonstrate that EGF transmembrane signaling includes an interaction with a component of the adenylate cyclase system and that this interaction stimulates cAMP synthesis resulting in enhancement of cAMP accumulation.     

Effect of adenylyl cyclase activation on intracellular and extracellular cAMP and cGMP in preimplantation cattle blastocysts

The effects of direct and indirect activation of adenylyl cyclase on the production of intracellular and extracellular cAMP and cGMP by 13- to 16-day-old cattle embryos were determined. Embryos were incubated for 2 h in a Krebs Ringer bicarbonate medium containing the phosphodiesterase inhibitor isobutyl-methylxanthine, to which stimulating agents forskolin (100 mumol l-1), cholera toxin (2 micrograms ml-1), or both were added. Total (intra- and extracellular) basal cAMP and cGMP concentrations ranged from 6.65 +/- 0.895 to 3.4 +/- 0.708 fmol microgram-1 protein in 13-day-old embryos and from 4.05 +/- 1.151 to 0.19 +/- 0.041 fmol microgram-1 protein in 16-day-old embryos. Forskolin induced an increase (P < 0.001) in cAMP that ranged from 5.4-fold on day 13 to 2.7-fold on day 16, whereas cholera toxin induced an increase (P < 0.001) that ranged from 30-fold at day 13 to 21-fold at day 16, similar to the effect of forskolin and cholera toxin combined. Individually, forskolin and cholera toxin had no effect on cGMP concentrations, but together they induced an increase (P < 0.05). cAMP (P < 0.01) and cGMP (P < 0.001) concentrations decreased with embryo age from day 13 to day 16 for all treatments; the decrease was greater for cGMP than cAMP (5-24-fold versus 1.6-3.3-fold, respectively). It is concluded that inducible adenylyl cyclase is present in 13- to 16-day-old cattle embryos and that the embryos secrete cAMP and cGMP into the incubation medium. In addition, basal and inducible concentrations of cAMP and cGMP decrease with embryo age from day 13 to day 16. These observations indicate that cAMP and cGMP may have a role in the rapid embryonic cell proliferation that occurs at this time or in signalling to the endometrium.     

Short and long term regulation of catecholamine biosynthetic enzymes by angiotensin in cultured adrenal medullary cells. Molecular mechanisms and nature of second messenger systems

The purpose of this study was to examine the effects of angiotensin on the enzyme activities and gene expression of two catecholamine synthesizing enzymes, tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT), in bovine adrenal medullary (AM) cells. Short term (15 min) incubation of cultured AM cells with 2 nM [Sar1]angiotensin II (s1-AII) did not increase basal secretion of catecholamines; however, longer incubations (3, 24, or 72 h) produced 4-10-fold increases. To determine whether angiotensin affects synthesis of catecholamines, the activities of TH and PNMT were examined. Incubation with s1-AII (15-30 min) decreased the Km of TH for its biopterine cofactor [6R)-5,6,7,8-tetrahydro-1-biopterin dihydrochloride (BH4] without affecting the Vmax, suggesting activation of TH. After long term incubation (72 h) the Km value was identical to that of control, while increases in the apparent Vmax were observed. PNMT activity was unaffected during a 30-min treatment with s1-AII; however, 2-fold increases occurred after a 48-72-h incubation. s1-AII (24 h) increased the relative abundance of TH and PNMT mRNAs, suggesting that the long term increase in enzyme activities reflected increased expression of TH and PNMT genes. Maximal increases were observed at 2 nM s1-AII and the changes were antagonized by saralasin. Induction of TH mRNA by s1-AII was additive to the effects of veratridine or forskolin indicating that effects of angiotensin were not due to membrane depolarization or increased cyclic AMP levels. Incubation with Ca2+ ionophore A23187 increased TH and PNMT mRNA levels in AM cells raising the possibility that the increase in cellular [Ca2+] could mediate effects of angiotensin. Angiotensin-induced increases in TH and PNMT mRNA were inhibited by nifedipine indicating involvement of voltage-dependent Ca2+ channels. In addition, the increases in TH, but not PNMT mRNA, were antagonized by dantrolene, which inhibits mobilization of Ca2+ from intracellular stores. Calmodulin involvement was suggested by the inhibition of s1-AII induced changes in mRNA with 1 microM calmidazolium.(ABSTRACT TRUNCATED AT 400 WORDS)     

cAMP-independent effects of cholera toxin on B cell activation. I. A possible role for cell surface ganglioside GM1 in B cell activation

Cholera toxin has been used as a tool to study the effects of cAMP on the activation of B cells but may have effects independent of its ability to elevate cAMP. We found five lines of evidence which suggested that cholera toxin suppressed mitogen-stimulated B cell activation through a cAMP-independent pathway. 1) Cholera toxin (1 microgram/ml) was consistently more suppressive than forskolin (100 microM) despite the induction of higher intracellular cAMP levels by forskolin. 2) Cholera toxin was more suppressive at 1 microgram/ml than at 0.1 microgram/ml despite equivalent elevations of cAMP. 3) Washing B cells following their incubation with cholera toxin reversed much of the inhibition without altering intracellular cAMP levels. 4) The A subunit of cholera toxin, which at high concentrations (10 micrograms/ml) induced levels of cAMP comparable to those induced by cholera toxin (1 and 0.1 microgram/ml), did not inhibit B cell activation. 5) cAMP derivatives at high concentrations were much less effective than was cholera toxin in suppressing B cell activation. Although the elevation of cAMP may cause a mild inhibition of B cell proliferation, we found that even a marked elevation of cAMP did not suppress B cell proliferation, unless the elevation was persistent. We did, however, observe that the degree of toxin inhibition more closely paralleled binding of the toxin to B cells than toxin stimulation of cAMP. This result raised the possibility that binding of cholera toxin to its ganglioside GM1 receptor mediated an inhibitory signal which suppressed B cell proliferation.     

Modulation of glucagon effects by changes in extracellular pH and calcium

We have examined the influence of extracellular pH and calcium concentration on the action of glucagon on isolated rat hepatocytes, perfused liver or plasma membrane preparations. Incubation of rat hepatocytes with 10 nM glucagon at pH 7.4 caused an immediate increase in cAMP concentrations (8-fold), and this rise was almost 50% lower at acidic extracellular pH (6.9). This effect of pH could not be explained by an alteration of the hormone binding to its receptor for glucagon concentrations higher than 1 nM. The effect of acidosis on cAMP production was still present with non-hormonal effectors, such as 10 microM Gpp[NH]p, 30 microM forskolin or 10 mM NaF. This suggests a direct action of acidosis on the regulatory component Ns and/or on the catalytic subunit of adenylate cyclase. Acidic pH also depressed mitochondrial processes responsive to glucagon (NAD(P)H fluorescence, glutamine breakdown). Whatever the experimental model, calcium appeared to be required for maximal stimulation of cAMP production by glucagon. On perfused rat liver, glycogenolysis was depressed in the absence of extracellular calcium in the perfusate. In isolated hepatocytes, the stimulation of phosphorylase alpha activity by glucagon was modulated by extracellular calcium concentrations lower than 0.2 mM. This suggests that, although glucagon action is chiefly cAMP-mediated, its effect on calcium mobilization (affecting various cellular process, including cAMP production itself) should also be taken into account. This work also confirmed the importance of calcium in the stimulation of mitochondrial metabolism of glutamine by glucagon.     

Calcitonin stimulates cAMP accumulation in chicken osteoclasts

Calcitonin causes an increase in the accumulation of cAMP in mammalian osteoclasts leading to an inhibition of bone resorption. Increases in cAMP subsequent to calcitonin stimulation have not been detected in previous studies of chicken osteoclasts as the only source of large numbers of highly purified cells. In this report, we studied the effects of salmon calcitonin (sCT), bovine parathyroid hormone -(1-34) [(bPTH -(1-34)] and forskolin (FSK) on cAMP accumulation in freshly isolated osteoclasts obtained non-enzymatically from the metaphysis of 5-7 week old rachitogenic chickens. Parathyroid hormone did not stimulate the accumulation of intracellular cAMP. Calcitonin and forskolin treatment caused a nearly 2.5 and 3.5-fold increase in cAMP respectively. This study demonstrates that chicken osteoclasts respond to calcitonin with an increase in cAMP accumulation and that the rachitogenic chicken may be a valuable source of hormonally sensitive cells for the study of osteoclast biology.     

Actions of cyclic adenosine monophosphate on the cytodifferentiation of ovarian cells: studies in cultured swine granulosa cells using a novel exogenous adenylate cyclase from Bordetella pertussis

The regulation by cAMP of cholesterol side-chain cleavage activity and the synthesis of immunoisolated cytochrome P-450scc and adrenodoxin proteins was investigated in primary cultures of swine ovarian (granulosa) cells. Administration of a novel adenylate cyclase toxin isolated from Bordetella pertussis increased granulosa-cell cAMP accumulation up to 200-fold over basal. These effects were additive with those of FSH, forskolin, and cholera toxin. In contrast, bacterial extracts BP 347 and BP 348 from mutant strains of B. pertussis that lack either all virulent factors or the adenylate cyclase toxin and hemolysin were devoid of effect. Granulosa-cell cAMP accumulation supported by active bacterial adenylate cyclase was accompanied by 2- to 11-fold, time-dependent increases in [35S]methionine incorporation into immunospecific cytochrome P-450scc and adrenodoxin. These increases in the synthesis of cholesterol side-chain cleavage proteins were associated with enhanced pregnenolone production in response to exogenous sterol substrate, 25-hydroxycholesterol, and augmented progesterone secretion both in the absence and presence of exogenous lipoprotein. Moreover, the effects of Bordetella adenylate cyclase toxin on granulosa cell steroidogenesis were functionally integrated with other regulatory responses, since the non-cAMP dependent effector, estradiol 17-beta, interacted synergistically with bacterial adenylate cyclase in stimulating progesterone production. We conclude that exogenous adenylate cyclase isolated from B. pertussis can be functionally integrated into the cAMP-dependent effector pathway of granulosa cells with a resulting increase in intracellular cAMP concentrations, augmented biosynthesis of progesterone and pregnenolone, enhanced synthesis of immunospecific cytochrome P-450scc and adrenodoxin, and synergistic interactions with a non-cAMP-dependent ovarian effector hormone (estradiol).(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic effects and cyclic AMP levels produced by glucagon, (1-N alpha-Trinitrophenylhistidine,12-homoarginine)glucagon and forskolin in isolated rat hepatocytes

[1-N alpha-Trinitrophenylhistidine,12-homoarginine]glucagon (THG) is a potent antagonist of the effects of glucagon on liver membrane adenylate cyclase. In isolated hepatocytes, this glucagon analogue was an extremely weak partial agonist for cAMP accumulation, and it blocked the stimulation of cAMP accumulation produced by glucagon. However, THG was a full agonist for the stimulation of glycogenolysis, gluconeogenesis and urea synthesis in rat hepatocytes, and did not antagonize the metabolic effects of glucagon under most of the conditions examined. Forskolin potentiated the stimulation of cAMP accumulation produced by glucagon or THG, but did not potentiate their metabolic actions. A much larger increase in cAMP levels seemed to be required for the stimulation of hepatocyte metabolism by forskolin than by glucagon or THG. This may suggest the existence of a functional compartmentation of cAMP in rat hepatocytes. The possible existence of compartments in cAMP-mediated hormone actions and the involvement of factors, besides cAMP, in mediating the effects of THG and glucagon is suggested.     

Glucagon stimulates exocytosis in mouse and rat pancreatic alpha-cells by binding to glucagon receptors

Glucagon, secreted by the pancreatic alpha-cells, stimulates insulin secretion from neighboring beta-cells by cAMP- and protein kinase A (PKA)-dependent mechanisms, but it is not known whether glucagon also modulates its own secretion. We have addressed this issue by combining recordings of membrane capacitance (to monitor exocytosis) in individual alpha-cells with biochemical assays of glucagon secretion and cAMP content in intact pancreatic islets, as well as analyses of glucagon receptor expression in pure alpha-cell fractions by RT-PCR. Glucagon stimulated cAMP generation and exocytosis dose dependently with an EC50 of 1.6-1.7 nm. The stimulation of both parameters plateaued at concentrations beyond 10 nm of glucagon where a more than 3-fold enhancement was observed. The actions of glucagon were unaffected by the GLP-1 receptor antagonist exendin-(9-39) but abolished by des-His1-[Glu9]-glucagon-amide, a specific blocker of the glucagon receptor. The effects of glucagon on alpha-cell exocytosis were mimicked by forskolin and the stimulatory actions of glucagon and forskolin on exocytosis were both reproduced by intracellular application of 0.1 mm cAMP. cAMP-potentiated exocytosis involved both PKA-dependent and -independent (resistant to Rp-cAMPS, an Rp-isomer of cAMP) mechanisms. The presence of the cAMP-binding protein cAMP-guanidine nucleotide exchange factor II in alpha-cells was documented by a combination of immunocytochemistry and RT-PCR and 8-(4-chloro-phenylthio)-2'-O-methyl-cAMP, a cAMP-guanidine nucleotide exchange factor II-selective agonist, mimicked the effect of cAMP and augmented rapid exocytosis in a PKA-independent manner. We conclude that glucagon released from the alpha-cells, in addition to its well-documented systemic effects and paracrine actions within the islet, also represents an autocrine regulator of alpha-cell function.     

Metabolic effects and cyclic AMP levels produced by glucagon, (1-Nα-trinitrophenylhistidine,12-homoarginine)glucagon and forskolin in isolated rat hepatocytes

[1-N^α-Trinitrophenylhistidine,12-homoarginine]glucagon (THG) is a potent antagonist of the effects of glucagon on liver membrane adenylate cyclase. In isolated hepatocytes, this glucagon analogue was an extremely weak partial agonist for cAMP accumulation, and it blocked the stimulation of cAMP accumulation produced by glucagon. However, THG was a full agonist for the stimulation of glycogenolysis, gluconeogenesis and urea synthesis in rat hepatocytes, and did not antagonize the metabolic effects of glucagon under most of the conditions examined. Forskolin potentiated the stimulation of cAMP accumulation produced by glucagon or THG, but did not potentiate their metabolic actions. A much larger increase in cAMP levels seemed to be required for the stimulation of hepatocyte metabolism by forskolin than by glucagon or THG. This may suggest the existence of a functional compartmentation of cAMP in rat hepatocytes. The possible existence of compartments in cAMP-mediated hormone actions and the involvement of factors, besides cAMP, in mediating the effects of THG and glucagon is suggested.     

Discordance in the effects of interleukin-1 on rat granulosa cell differentiation induced by follicle-stimulating hormone or activators of adenylate cyclase

Recombinant human interleukin-1 (IL-1) inhibits the follicle-stimulating hormone (FSH)-induced development of luteinizing hormone (LH) receptors and suppresses progesterone secretion in cultured rat granulosa cells. Since activation of adenylate cyclase by FSH is considered to be the primary second messenger system responsible for differentiation of granulosa cells, we examined whether IL-1 could alter the FSH, cholera toxin, or forskolin-induced accumulation of cyclic adenosine 3', 5'-monophosphate (cAMP) from these cells. In addition, we sought to determine if IL-1 could influence differentiation induced by the cAMP analog, 8-bromo cAMP. Cells collected from ovaries of immature, diethylstilbestrol-treated rats were stimulated to differentiate by addition of FSH, cholera toxin, forskolin, or 8-bromo cAMP to the cultures. IL-1 or interleukin-2 (IL-2) was added to some of the tubes, and the primary cultures were incubated for various periods of time. At the end of the culture, the tubes were centrifuged, the medium was saved for progesterone and cAMP radioimmunoassay, and the cells were assayed for specific 125I-human chorionic gonadotropin (hCG) binding to determine the number of LH receptors. In the presence of FSH, IL-1, at a dose as small as 5 ng/ml, but not IL-2, significantly inhibited LH receptor formation and suppressed progesterone secretion in a dose-related manner. IL-1 also significantly suppressed FSH-induced cAMP accumulation after 72 h of incubation but did not appear to do so in a dose-related fashion. In the presence of FSH, IL-1 did not significantly alter the protein content of granulosa cells at the end of culture. During stimulation of granulosa cells with cholera toxin, forskolin, or 8-bromo cAMP, IL-1 significantly reduced LH receptor formation compared to that observed in the absence of IL-1. However, in contrast to IL-1 in the presence of FSH, IL-1 significantly augmented the forskolin-induced secretion of progesterone and accumulation of cAMP after 72 h at subsaturating doses of forskolin. Thus, IL-1 appeared to inhibit forskolin-induced and cholera toxin-induced formation of LH receptors even when cAMP levels were elevated. Similar to forskolin, 8-bromo cAMP-stimulated progesterone secretion was significantly enhanced by IL-1, but LH receptor formation was inhibited. Over a 72-h time course at single doses of FSH or forskolin, IL-1 did not affect cAMP accumulation until 48 h of culture, at which time IL-1 significantly suppressed FSH-induced, but augmented forskolin-induced, accumulation of cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Actions of cholera toxin on dispersed Chief cells from guinea pig stomach

Although much is known about the actions of cholera toxin on intestinal and extra-gastrointestinal tissues, almost nothing is known about the interaction of this toxin with cells in the stomach. In the present study, we prepared 125I-labeled cholera toxin (1900 Ci/mmol) and examined the binding of this radioligand to dispersed Chief cells from guinea pig stomach. Moreover, we examined the actions of cholera toxin on cellular cAMP and pepsinogen secretion from Chief cells. Binding of 125I-labeled cholera toxin could be detected within 5 min, was maximal by 60 min, and was increased by increasing the radioligand or cell concentrations. Inhibition of binding by unlabeled toxin indicated a dissociation constant of 3 nM and 8.7 X 10(5) cholera toxin receptors per Chief cell. In contrast to the rapidity of binding, a cholera toxin-induced increase in cAMP and pepsinogen secretion was not detected until 30-45 min of incubation. A 3 to 6-fold increase in cAMP and pepsinogen secretion was observed with maximal concentrations of cholera toxin. Binding of 125I-labeled cholera toxin and the toxin's actions on cAMP and pepsinogen secretion were inhibited by the B subunit of the toxin. Binding was not altered by other agents that have been shown to stimulate pepsinogen secretion (carbachol, CCK-8, secretin, vasoactive intestinal peptide, prostaglandin E1, or forskolin). These data indicate that Chief cells from guinea pig stomach possess a specific class of cholera toxin receptors. Binding of cholera toxin to these receptors causes an increase in cellular cAMP that stimulates pepsinogen secretion.     

Bradykinin inhibition of cyclic AMP accumulation in D384 astrocytoma cells. Evidence against a role of cyclic GMP.

The present studies were performed in order to examine the possible role of cyclic GMP-stimulated phosphodiesterase (cGMP-PDE) activity in the inhibitory action of the inflammatory peptide bradykinin on cyclic AMP (cAMP) accumulation in D384 cells. Bradykinin decreased the forskolin-stimulated cAMP accumulation in the presence of the phosphodiesterase inhibitor rolipram, and caused a transient 50% rise in cellular cGMP in the presence of the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Both basal and bradykinin-stimulated cGMP accumulation were about 8 times higher in the presence of IBMX than in the presence of rolipram. Sodium nitroprusside, which caused a 20-70-fold increase in cGMP levels reduced forskolin stimulated cAMP accumulation, whereas hydroxylamine, which maximally caused a 16-fold increase in cGMP, did not. 8-bromo-cGMP or dibutyryl cGMP had no effect on cAMP accumulation induced by forskolin. The inhibitory effect of nitroprusside was totally reversed by blocking the soluble guanylate cyclase activity by methylene blue treatment; however, the inhibitory action of bradykinin on cAMP accumulation was not changed by this treatment. Additionally, inhibition of nitric oxide synthesis, which is known to be regulated by Ca2+ and in turn stimulates cGMP production, by N omega-nitro-L-arginine (L-NAME) treatment did not alter the inhibitory effect of bradykinin on forskolin-induced cAMP accumulation. These results indicate that large increases in cGMP may regulate cAMP via cGMP-PDE whereas the small increase induced by bradykinin is insufficient and that cGMP is not involved in the inhibitory action of bradykinin on cAMP levels in D384 cells.     

Pertussis toxin abolishes the inhibitory effects of prostaglandins E1, E2, I2 and F2 alpha on hormone-induced cAMP accumulation in cultured hepatocytes

Several prostaglandins inhibit the cAMP response to glucagon and beta-adrenergic stimulation in hepatocytes. To probe the mechanism of this inhibition, we have examined in primary hepatocyte cultures how pretreatment with pertussis toxin (islet-activating protein) influences the ability of the cells to respond to hormones and prostaglandins. Pertussis toxin augmented the effects of glucagon, epinephrine and isoproterenol, and also markedly enhanced the cAMP response to prostaglandin E1 (PGE1). Furthermore, whereas PGE1, PGE2, PGI2 and PGF2 alpha attenuated the cAMP responses to glucagon in control cultures, this inhibition was abolished in cells pretreated with pertussis toxin. A more detailed comparison was made of the effects of PGE1 and PGF2 alpha. In cells not treated with pertussis toxin, both these prostaglandins at high concentrations reduced the cAMP response to glucagon and isoproterenol by approximately 50%, but dose-effect curves showed that PGE1 was about 100-fold more potent as an inhibitor than PGF2 alpha. Pertussis toxin abolished the inhibitory effects of PGE1 and PGF2 alpha with almost identical time and dose requirements. The results obtained with PGE1, PGE2, PGI2 and PGF2 alpha suggest that prostaglandins of different series attenuate hormone-activable adenylate cyclase in hepatocytes through a common mechanism, dependent on the inhibitory GTP-binding protein.     

Long-Term Activation of Protein Kinase C by Angiotensin II in Cultured Bovine Adrenal Medullary Cells

Previous studies from our laboratory suggest that protein kinase C (PKC) is involved in the angiotensin II (AII)-induced increase in the expression of genes encoding proenkephalin and catecholamine biosynthesizing enzymes in primary cultured bovine adrenal medullary (BAM) cells. The purpose of this study was to examine the effects of [Sar1]-AII (S1-AII), an AII agonist, on PKC activity in BAM cells. Thirty-minute incubation with S1-AII produced a dose-dependent activation of PKC. The particulate PKC activity was significantly increased by 2 nM S1-AII after both 30 min and 12 h of incubation. A high concentration of S1-AII (200 nM) caused an increase in particulate PKC activity after 30 min of incubation and this increase was still observed after 18 h of continuous incubation. [Sar1, Thr8]-angiotensin II (S1, T8-AII) (100 microM), an AII antagonist, inhibited the effect of S1-AII (20 nM) on PKC activity, suggesting a specific AII receptor-mediated effect. An increase in BAM cell particulate PKC immunoreactivity after 18 h of S1-AII treatment was observed in Western blot analysis of PKC-immunoreactive protein (82 kDa). The persistent activation of PKC seen in this study is consistent with our hypothesis that PKC may mediate the S1-AII-induced increase in the expression of genes encoding proenkephalin and catecholamine synthesizing enzymes in BAM cells.     

Inhibitory action of forskolin on adenylate cyclase activity and cyclic AMP generation

In testicular Leydig cells, forskolin causes the expected stimulation of cAMP and testosterone production and potentiates gonadotropin-induced responses, when present in concentrations of 1-10 microM. In addition, when added at lower doses that did not affect cAMP generation and testosterone responses (100 nM), forskolin caused an increase in sensitivity to hormonal stimulation for all cAMP pools (extracellular, intracellular, and receptor-bound) and a 70% reduction in the ED50 for human chorionic gonadotropin (hCG) stimulation of testosterone production. Forskolin-induced increases in receptor-bound cAMP were less effective than those elicited by hCG in stimulating steroidogenesis. In contrast to the well-known stimulatory actions of forskolin, low doses of the diterpene (in the picomolar to nanomolar range) markedly inhibited the production of cAMP and testosterone. Such inhibitory actions of low-dose forskolin were prevented by preincubation of Leydig cells with pertussis toxin before addition of forskolin and/or hCG. Low concentrations of forskolin also inhibited adenylate cyclase activation by GTP and luteinizing hormone, and this effect was prevented by pretreatment of cell membranes with pertussis toxin. These studies have defined the stimulatory effects of forskolin on Leydig-cell cAMP pools, including potentiation of the hormonal increase in receptor-bound cyclic AMP by forskolin, and have provided additional evidence for the functional importance of cAMP compartmentalization during hormonal stimulation of steroidogenesis. We have also demonstrated a novel, high-affinity inhibitory action of forskolin upon adenylate cyclase activity and cyclic AMP generation, an effect that appears to be mediated by the Ni guanine nucleotide regulatory subunit of adenylate cyclase.     

Multiple changes induced by cholera toxin contribute to the stimulation of aerobic lactate production in rat-1 fibroblasts.

Exposure of rat-1 fibroblasts to cholera toxin increased aerobic lactate production 3- to 8-fold with maximal stimulation observed between 1 and 2 h at a concentration of 1-2 micrograms/ml. Concomitant with this change was a 10- to 40-fold elevation in the intracellular concentration of cAMP. The cell permeable cAMP analogue, N6,2'-O-dibutyryl cAMP and the cyclic nucleotide phosphodiesterase inhibitor RO-20-1724 also increased lactate production and intracellular cAMP levels, although less effectively. Cholera toxin and dibutyryl cAMP induced a 2- to 3-fold elevation of intracellular fructose 2,6-bisphosphate and 2- to 3-fold increases in both 3-O-methylglucose and inorganic phosphate transport. A survey of five additional cell lines revealed striking variabilities in their individual responses to cholera toxin and dibutyryl cAMP. All were observed to be considerably less sensitive to either agent than rat-1 cells. These data suggest that a cooperative effect involving multiple parameters may be responsible for the observed increases in aerobic lactate production in response to cAMP and that these parameters may vary significantly among cell lines.