Inhibitory effects of Gs on desensitization of beta-adrenergic receptors in tracheal smooth muscle.

We examined the reduction of mechanical responsiveness to beta-agonists after continuous or repeated exposure to agonists in guinea pig tracheal smooth muscle, using an isometric tension record. The inhibitory action of 0.1 microM isoproterenol (Iso) on contraction induced by 1 microM methacholine (MCh) was suppressed markedly after incubation with 0.0003-3 microM Iso for 1 h. The effects of Iso on MCh-induced contraction gradually decreased after repeated application of these agents at intervals of 1 h. When Iso was perfused cumulatively to tissues repeatedly precontracted by MCh, the effects of Iso were reduced at the second application. This reduced responsiveness to beta-agonists was not mimicked by forskolin and did not occur after preexposure of the strips to 2 micrograms/ml cholera toxin for 6 h. We concluded that this reduced responsiveness to beta-agonists is homologous desensitization and that the irreversible activation of the stimulatory G proteins, Gs, may play an important role in prevention of the reduced responsiveness to beta-agonists.

Asymmetric expression of Notch/Delta/Serrate is associated with the anterior-posterior axis of feather buds.

We studied the roles of Notch, Delta, and Serrate in vertebrate epithelial appendage morphogenesis using feather as a model and found the following. (1) C-Notch-1, C-Delta-1, and C-Serrate-1 are not expressed at the early placode stage and are therefore not involved in the determination of bud versus interbud compartments. (2) From symmetric short buds to asymmetric long buds, C-Delta-1 and C-Serrate-1 are expressed in the posterior bud mesenchyme in a nested fashion, while C-Notch-1 is expressed as a stripe perpendicular to the anterior-posterior (A-P) axis and positioned posterior to the midpoint. (3) Epithelial-mesenchymal recombination with rotation led to the disappearance of these genes followed by their reappearance with new positions appearing to predict their new morphological orientation. (4) Conditions leading to branched buds (e.g., recombination of later buds) show polarized staining patterns before branching occurs. (5) Conditions leading to symmetrical round buds (e.g., treated with the protein kinase A agonist forskolin) suppress expression of all three genes. These results lead us to hypothesize that Notch, Delta, and Serrate are involved in establishing the A-P asymmetry of feather buds.

Regulation of porcine granulosa cell steroidogenic acute regulatory protein (StAR) by insulin-like growth factor I: synergism with follicle-stimulating hormone or protein kinase A agonist.

The transfer of cholesterol from the outer to the inner mitochondrial membrane, where side-chain cleavage occurs to form pregnenolone, is a crucial event in the regulation of steroidogenesis and recently has been demonstrated to be mediated by steroidogenic acute regulatory protein (StAR). We generated a partial porcine StAR complementary DNA (280 bp) by RT-PCR and used the corresponding antisense riboprobe to quantify the control of StAR gene expression by FSH and insulin-like growth factor I (IGF-I) in hormonally responsive swine granulosa cells, which typically manifest synergistic steroidogenic stimulation by these two dominant intrafollicular regulators. RNase protection assays were implemented to investigate the time course of the actions of FSH (100 ng/ml), IGF-I (100 ng/ml), and FSH plus IGF-I on StAR messenger RNA accumulation in serum-free cultures granulosa cells. Treatment with FSH (1.6-fold) or IGF-I (2.7-fold) alone had a small but consistent stimulatory effect on StAR message accumulation (corrected for 18S ribosomal RNA in each lane) at 48 h, whereas only IGF-I stimulated StAR protein expression (at least 6-fold as assessed by Western blot). Notably, the combined effect of FSH plus IGF-I was strongly synergistic and already significant by 24 h and maximal at 48 h (P < 0.001). Protein kinase A agonist, 8-bromoadenosine 3',5'-cAMP (8-bromo-cAMP) (1 mM) alone elicited a 3.5-fold increase in StAR message and more than 3.7-fold increase in StAR protein expression by 48 h. The combination of IGF-I and FSH or 8-bromo-cAMP evoked a 26- to 40-fold (P < 0.001) synergistic rise in StAR message accumulation. StAR protein also showed a similar synergistic pattern of expression driven by IGF-I and FSH or 8-bromo-cAMP, namely a greater than 56- to 60-fold increase. In summary, two distinct first messenger regulatory molecules, FSH and IGF-I, interact synergistically to induce amplification of StAR messenger RNA and protein expression in serum-free monolayer cultures of immature (swine) granulosa cells.

Stimulation of Na(+)-K(+)-2Cl- cotransport in rat medullary thick ascending limb by dopamine.

Dopamine receptors are present in the medullary thick ascending limb (mTAL) of Henle, but their effect on ion transport in this nephron segment has not been tested. Therefore, we studied the short-term effects of dopamine on Na(+)-K(+)-2Cl- cotransport (assessed by 100 microM bumetanide-sensitive 86Rb uptake) in rat mTAL tubular suspensions. Dopamine (1 microM) stimulated bumetanide-sensitive 86Rb uptake (72.1 +/- 10.6% vs. control, n = 5) by increasing total 86Rb uptake and by decreasing bumetanide-insensitive 86Rb uptake; this effect was concentration dependent. The dopamine-induced stimulation of Na(+)-K(+)-2Cl- cotransport activity was mimicked by calyculin A, a protein phosphatase (PP) inhibitor, and Sp isomer of adenosine 3',5'-cyclic monophosphothioate (Sp-cAMP[S]), a protein kinase A (PKA) agonist, and blocked by Rp isomer of 8-(4-chlorophenylthio)-cAMP[S] (Rp-8-CPT-cAMP[S]), a PKA inhibitor (n = 5). Dopamine did not increase the stimulatory effect of the PP inhibitor. However, the stimulatory effect of the PP inhibitor and PKA agonist was additive and approached the stimulatory effect of dopamine. The stimulatory effects of dopamine, PP inhibitor, and PKA agonist persisted even when intracellular sodium was clamped by 5 microM monensin. When K+ channels were blocked by 1 mM BaCl2, the effects of dopamine and calyculin A on the cotransport were no longer apparent, although the stimulatory effect of the PKA agonist was attenuated. We conclude that dopamine stimulates Na(+)-K(+)-2Cl- cotransport activity. This action is mediated mainly by PKA-dependent phosphorylation/dephosphorylation processes and modulated by dopamine actions on K+ channels.

Cyclic AMP-dependent protein kinase enhances hippocampal dentate granule cell GABAA receptor currents.

1. The effects of activation of endogenous adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA), intracellular application of PKA and inhibition of endogenous PKA by protein kinase inhibitory peptide (PKIP) on hippocampal dentate granule cell gamma-aminobuturic acid A (GABAA) receptor (GABAR) currents were characterized. 2. GABAR currents evoked by repeated application of GABA (30 or 100 microM) were enhanced by application of 1 mM norepinephrine (52 +/- 26%; mean +/- SE; n = 11) and of 500 mM 8-bromo cAMP (15 +/- 2%, n = 7). 3. GABA concentration response curves were obtained from six dentate granule cells before and after application of 500 microM 8-bromo cAMP. The maximal current was increased significantly by 89 +/- 36%, but the mean EC50 was not significantly changed (68 +/- 42 microM vs. 25 +/- 10 microM). 4. The GABA concentration response relationship was studied in a group of 7 granule cells recorded with pipettes containing PKIP and 2 mM ATP and compared with another group of 12 cells recorded with 2 mM ATP in the pipette. When currents were recorded with intracellular PKIP, the mean EC50 for GABA was no different (43 +/- 9 microM vs. 45 +/- 16 microM); however, the maximal current obtained was smaller, (961 +/- 102 pA vs. 658 +/- 104 pA). 5. Concentration response data were obtained from four granule cells using recording pipettes containing the cPKA and an ATP regeneration system and compared with seven cells recorded with the ATP regeneration system. With cPKA, the maximal GABAR current was significantly larger (1,224 +/- 132 pA vs. 718 +/- 56 pA), but the EC50 for GABA was not significantly altered (21 +/- 2.0 microM vs. 79 +/- 25 microM).

Hepatocyte growth factor and its actions in growth plate chondrocytes.

Hepatocyte growth factor (HGF) has been implicated as a paracrine regulator of organogenesis and repair in many tissues. Here we have studied the expression and actions of HGF in intact rachitic rat growth plate and derived cultures of proliferative zone chondrocytes. In vivo and in vitro chondrocytes express HGF mRNA; 1,25(OH)2 has a three-fold maximal stimulatory effect, which can be blocked by H-7, an inhibitor of protein kinase C. Although HGF elaboration and action generally follow a paracrine model, chondrocytes appear capable of both expressing and responding to HGF. mRNA encoding the HGF receptor (c-met) was detected in both growth cartilage and derived chondrocyte cultures. HGF addition to chondrocyte cultures increased collagen II mRNA and alkaline phosphatase enzymatic activity to degrees comparable to that observed for active vitamin D metabolites. Combining HGF and 1,25-D evoked a synergistic response (ninefold) of alkaline phosphatase activity. To assess whether a similar stimulatory effect might be seen with bioactive peptides and HGF, we investigated the effect of HGF pretreatment on acute responses of chondrocytes to synthetic human calcitonin, an anabolic chondrocyte regulator whose skeletal action are mediated principally by cAMP elevation and subsequent protein kinase A activation. CT's maximal activation of protein kinase A was increased by prior HGF treatment from 56% to 78%. In concert, our findings indicate that in addition to HGF's classical paracrine role during skeletal growth, this growth factor may modulate hormonal sensitivity of the chondrocyte during proliferation, differentiation, and/or apoptosis.

cAMP promotes gap junctional coupling in T84 cells.

The effect of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) on dye and electrical coupling was studied in T84 cells, a cell line often used as a model for epithelial cell fluid secretion. Injections of lucifer yellow (LY) into single cells within a cluster of control cells resulted in LY localization to 1.3 +/- 0.2 (mean +/- SE) cells within a cluster. Twenty-six percent of control T84 cell pairs were electrically coupled as assayed by the dual patch-clamp technique. Treatment of cells with agents that either increase intracellular cAMP and/or activate protein kinase A (PKA) increased dye localization to 3.8 +/- 0.6 cells and the proportion of electrically coupled cell pairs to 65%. No electrical coupling was observed in the presence of the Rp diastereomer of adenosine-3',5'-cyclic monophosphothioate (Rp-cAMPS), a PKA antagonist. Excess of Rp-cAMPS prevented cell coupling elicited by 20 microM of the Sp diastereomer of adenosine-3',5'-cyclic monophosphothioate. Expression of connexin 32 mRNA, but not of connexins 26, 43, or 45, was detected by reverse transcription-polymerase chain reaction. These results suggest that communication between T84 cells is modulated by PKA, providing a mechanism for regulating multicellular activity, such as fluid secretion.

Modulation of the hyperpolarization-activated current (Ih) by cyclic nucleotides in guinea-pig primary afferent neurons.

1. Whole-cell patch-clamp recordings were made from dissociated guinea-pig nodose and trigeminal ganglion neurons in culture to study second messenger mechanisms of the hyperpolarization-activated current (Ih) modulation. 2. Prostaglandin E2 (PGE2) and forskolin modulate Ih in primary afferents by shifting the activation curve in the depolarizing direction and increasing the maximum amplitude. 3. The cAMP analogues, RP-cAMP-S (an inhibitor of protein kinase A (PKA)) and SP-cAMP-S (an activator of PKA), both shifted the activation curve of Ih to more depolarized potentials and occluded the effects of forskolin. These results suggest that Ih is modulated by a direct action of the cAMP analogues. 4. Superfusion of other cyclic nucleotide analogues (8-Br-cAMP, 8-(4-chlorophenylthio)-cAMP and 8-Br-cGMP) mimicked the actions of forskolin and PGE2, but dibutyryl cGMP, 5'-AMP and adenosine had no effect on Ih. 8-Br-cAMP and 8-Br-cGMP had similar concentration response profiles, suggesting that Ih has little nucleotide selectivity. 5. The inhibitor peptide (PKI), the catalytic subunit of PKA (C subunit) and phosphatase inhibitors (microcystin and okadaic acid) had no effect on forskolin modulation of Ih. 6. These results indicate that Ih is regulated by cyclic nucleotides in sensory neurons. Positive regulation of Ih by prostaglandins produced during inflammation may lead to depolarization and facilitation of repetitive activity, and thus contribute to sensitization to painful stimuli.

Adrenergic, dopaminergic, and muscarinic receptor stimulation leads to PKA phosphorylation of Na-K-ATPase.

Na-K-adenosinetriphosphatase (Na-K-ATPase) is a potential target for phosphorylation by protein kinase A (PKA) and C (PKC). We have investigated whether the Na-K-ATPase alpha-subunit becomes phosphorylated at its PKA or PKC phosphorylation sites upon stimulation of G protein-coupled receptors primarily linked either to the PKA or the PKC pathway. COS-7 cells, transiently or stably expressing Bufo marinus Na-K-ATPase wild-type alpha- or mutant alpha-subunits affected in its PKA or PKC phosphorylation site, were transfected with recombinant DNA encoding beta 2- or alpha 1-adrenergic (AR), dopaminergic (D1A-R), or muscarinic cholinergic (M1-AChR) receptor subspecies. Agonist stimulation of beta 2-AR or D1A-R led to phosphorylation of the wild-type alpha-subunit, as well as the PKC mutant, but not of the PKA mutant, indicating that these receptors can phosphorylate the Na-K-ATPase via PKA activation. Surprisingly, stimulation of the alpha 1B-AR, alpha 1C-AR, and M1-AChR also increased the phosphorylation of the wild-type alpha-subunit and its PKC mutant but not of its PKA mutant. Thus the phosphorylation induced by these primarily phospholipase C-linked receptors seems mainly mediated by PKA activation. These data indicate that the Na-K-ATPase alpha-subunit can act as an ultimate target for PKA phosphorylation in a cascade starting with agonist-receptor interaction and leading finally to a phosphorylation-mediated regulation of the enzyme.

The steroidogenic acute regulatory protein is induced by angiotensin II and K+ in H295R adrenocortical cells.

Adrenal steroid hormone biosynthesis can be activated by the protein kinase A pathway by ACTH, the protein kinase C pathway by angiotensin II (AII), or by increasing intracellular Ca2+ levels by AII or K+. Although their mechanisms of action are not known, each of these pathways is dependent upon the de novo synthesis of a protein that is required for the acute production of steroids. We have recently proposed the steroidogenic acute regulatory (StAR) protein as this required protein, therefore, we examined the effect of different agonists on StAR's expression in H295R human adrenocortical carcinoma cells. (Bu)2cAMP, AII, K+, BAYK8644 (a calcium channel agonist) and TPA are all shown to induce StAR. Aldosterone synthesis was stimulated by all the agonists with the exception of TPA, indicating that AII-stimulated steroid production is mediated by increases in intracellular calcium. Thus, these data suggest that regulation of StAR expression may represent a common mechanism for divergent pathways to acutely control adrenal steroidogenesis.

Activators of protein kinase A and of protein kinase C inhibit MDCK cell myo-inositol and betaine uptake.

Amino acid sequences of the myo-inositol and betaine cotransporters that are induced in MDCK cells by hypertonicity include consensus sequences for phosphorylation by protein kinase A and by protein kinase C. To test for the effect of activation of protein kinases A and C on the activity of those cotransporters, MDCK cells were exposed to activators of each kinase and the activity of both cotransporters was assayed. Incubation with 8-bromoadenosine 3':5'-cyclic monophosphate (8Br-cAMP) or 3-isobutyl-1-methylxanthine (IBMX), activators of protein kinase A, and incubation with an active phorbol ester or with an active diacylglycerol, activators of protein kinase C, inhibited the activity of both cotransporters by about 30%. The relative effect of the activation of protein kinase A and of protein kinase C was similar in hypertonic and isotonic cells. The effects of activators of protein kinase A and of protein kinase C were not additive. The two cotransporters behaved differently when protein kinase C activity was down-regulated by prolonged incubation with a higher concentration of phorbol 12-myristate 13-acetate. There was a doubling of activity of the myo-inositol cotransporter and no change in the activity of the betaine cotransporter in hypertonic and isotonic cells. Although the mechanisms of the effects of activation of the two kinases remain to be established, it is clear that the kinases can mediate post-translational regulation of the uptake of compatible osmolytes.

Regulation of porcine granulosa cell 3-hydroxy-3-methylglutaryl coenzyme A reductase by insulin and insulin-like growth factor I: synergism with follicle-stimulating hormone or protein kinase A agonist.

In species such as the pig and human, gonadal steroidogenesis is believed to be dependent upon the availability of low density lipoprotein (LDL) cholesterol. However, before ovulation, Graafian follicles are impermeant to lipoproteins in the LDL class. Thus, de novo cholesterol biosynthesis via the rate-determining enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is likely to provide a significant mechanism for generating sterol substrate for steroidogenesis by granulosa cells before follicular rupture. As serum-free monolayer culture of (swine) granulosa cells offers an in vitro model of hormonally responsive HMG-CoA reductase, we generated a (porcine) complementary DNA and homologous complementary RNA to investigate by sensitive and specific ribonuclease protection assay the hormonal regulation of HMG-CoA reductase gene expression in ovarian cells from immature Graafian follicles. Using reverse transcriptase-polymerase chain reaction, we cloned and sequenced a 238-base pair complementary DNA from porcine luteal tissue that encodes the catalytic region of HMG-CoA reductase. GenBank analysis of the DNA sequence homology between the pig and other species showed the greatest concordance with human (88%) and hamster (90%). Solution hybridization/ribonuclease protection analysis of total RNA isolated from serum-free monolayer cultures of porcine granulosa cells revealed that insulin (3 micrograms/ml) increased HMG-CoA messenger RNA (mRNA) concentrations corrected for constitutive 18S ribosomal RNA expression in a time-dependent fashion, with significant effects observed at 12 h and a 6-fold increase by 48 h. Recombinant human insulin-like growth factor I (IGF-I) peptide was able to mimic the action of insulin alone. Neither FSH (100 ng/ml) nor 8-bromo-cAMP (1 mM) had observable effects on HMG-CoA message accumulation at any time point studied. However, the combined action of either FSH and insulin or 8-bromo-cAMP and insulin resulted in synergistic increases in reductase mRNA by 31- and 17-fold, respectively. To assess the possible feedback effects of sterol on HMG-CoA gene expression, granulosa cells were treated with LDL. At physiological concentrations, LDL suppressed basal expression of HMG-CoA mRNA to levels below the control value. In addition, LDL inhibited insulin-stimulated HMG-CoA mRNA accumulation by 84% as well as the synergistic effects of insulin and FSH (by 94%) and of insulin and 8-bromo-cAMP (by 93%). We conclude that insulin alone or in combination with FSH or cAMP augments the accumulation of HMG-CoA reductase mRNA in ovarian (granulosa) cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction of midbrain dopaminergic neurons by Sonic hedgehog.

Midbrain dopaminergic neurons, whose loss in adults results in Parkinson's disease, can be specified during embryonic development by a contact-dependent signal from floor plate cells. Here we show that the amino-terminal product of Sonic hedgehog autoproteolysis (SHH-N), an inductive signal expressed by floor plate cells, can induce dopaminergic neurons in vitro. We show further that manipulations to increase the activity of cyclic AMP-dependent protein kinase A, which is known to antagonize hedgehog signaling, can block dopaminergic neuron induction by floor plate cells. Our results and those of other studies indicate that SHH-N can function in a dose-dependent manner to induce different cell types within the neural tube. Our results also provide the basis for a potential cell transplantation therapy for Parkinson's disease.

Induction of apoptosis in thymocytes: new evidence for an interaction of PKC and PKA pathways.

The second messenger cascades connected to PKC and PKA are involved in the induction of apoptosis. Here we study the interaction of those two second messenger pathways with respect to the induction of apoptosis by stimulation or inhibition. The stimulators used were phorbol dibutyrate for PKC and one of the cAMP agonists Sp-5,6 DCl-cBIMPS or Sp-cAMP for PKA. The inhibitors used were staurosporin for PKC and the cAMP antagonist Rp-cAMPS for PKA. We found a synergism between both second messenger systems with regard to the induction of apoptosis in thymus lymphocytes.

cAMP-associated inhibition of phenobarbital-inducible cytochrome P450 gene expression in primary rat hepatocyte cultures.

The effects of elevated intracellular cyclic adenosine monophosphate (cAMP) in regulating phenobarbital (PB)-inducible gene expression in primary rat hepatocyte cultures were investigated. Cells were exposed to various concentrations (0.1-100 microM) of cAMP analogs and/or activators of intracellular cAMP-dependent pathways. Effects of these treatments were assessed either using a 1-h pulse prior to PB (100 microM) exposure or in conjunction with PB during a 24-h exposure period. PB-inducible responses were measured in hepatocytes by hybridization to cytochrome P450 (CYP) CYP2B1, CYP2B2, and CYP3A1 mRNAs. The cAMP analogs, 8-bromo-cAMP, 8-(4-chlorophenylthio)-cAMP, dibutyryl cAMP, and (Sp)-5,6-DCl-cBiMPS ((Sp)-5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3', 5'-monophosphorothioate), and the activators of adenylate cyclase, forskolin and glucagon, dramatically inhibited PB-mediated induction of CYP2B1 and CYP2B2 in a concentration-dependent manner. A similar inhibition of PB-induced CYP3A1 mRNA levels was effected by the cAMP analogs and glucagon. The phosphodiesterase inhibitors isobutylmethylxanthine and RO 201724 potentiated the cAMP responses. Increasing the concentration of PB (0.05-1.00 mM) did not alleviate the cAMP-mediated repression. A requirement for protein kinase A (PKA) was demonstrated by the use of (Sp)-cAMPS, a highly specific activator of PKA, whereas the inactive diastereoisomer, (Rp)-cAMPS, was ineffective in modulating PB induction. The response to cAMP was specific since elevated intracellular cAMP levels did not perturb beta-naphtholflavone-mediated induction of CYP1A1, CYP1A2, microsomal epoxide hydrolase, or dexamethasone-mediated induction of CYP3A1 gene expression. Nor did elevated intracellular cAMP modulate the liver-selective albumin gene expression levels. The results of the present study demonstrated striking inhibition of PB-mediated CYP gene induction by cAMP and PKA activators, indicating a negative regulatory role for the cAMP signal transduction pathway on PB gene induction.

Luteinizing hormone induces prostaglandin endoperoxide synthase-2 and luteinization in vitro by A-kinase and C-kinase pathways.

The LH surge induces ovulation [prostaglandin synthase-2 (PGS-2)] and luteinization (progesterone synthesis) of preovulatory follicles by cAMP-dependent mechanisms. Peptides, such as GnRH and angiotensin-II, that activate other cellular signaling pathways have been shown to mimic some of the effects of LH. Therefore, the relative functional importance of different cellular signaling pathways in mediating the induction of PGS-2 and luteinization was analyzed using the agonists (LH, GnRH, and angiotensin-II) and selective inhibitors of A-kinase (H-89), C-kinase (calphostin-C), and calmodulin kinase-II (KN93). LH or GnRH, but not angiotensin-II, markedly induced PGS-2 protein in preovulatory follicles. H-89 and calphostin-C, but not KN93 inhibited LH induction of PGS-2, whereas calphostin-C selectively blocked induction by GnRH. In contrast, the A- and C-kinase inhibitors prevented both LH and GnRH induction of granulosa cell luteinization. Taken together, these results provide biological evidence that the response of granulosa cells to the LH surge appears to involve the activation of A- and C-kinase pathways.

Regulation of parathyroid hormone messenger RNA levels by protein kinase A and C in bovine parathyroid cells.

Secretion of parathyroid hormone (PTH) is regulated by Ca2+ as well as by protein kinases A and C. In this study we report that protein kinases A and C regulate PTH messenger RNA levels in vitro in dispersed bovine parathyroid cells. Incubation of bovine parathyroid cells with cholera toxin (10(-9) M), which activates adenylate cyclase and indirectly stimulates protein kinase A, increased PTH mRNA levels about 2-fold after 3 and 7 h incubation, but not at 24 h. Incubation with pertussis toxin (5 x 10(-9) M), which blocks the high-calcium-mediated inhibition of cyclic adenosine monophosphate accumulation in these cells, also reversed the inhibition of PTH mRNA levels at high Ca2+ (2.0 mM) with a marked increase in PTH mRNA levels. Pertussis toxin also increased PTH mRNA at a low extracellular Ca2+ concentration (0.7 mM) (4-fold increase) and a normal concentration (1.25 mM) (2-fold increase). Inhibition of protein kinase C both by staurosporine (1 x 10(-8) M) and by prolonged incubation with the phorbol ester phorbol 12-myristate 13-acetate (PMA) (1 x 10(-7) M), decreased PTH mRNA levels at 24 h, reaching approximately 40% and 5% of control, respectively. Staurosporine and PMA had no effect on PTH mRNA levels at 3 h. The inactive phorbol ester, phorbol 12-13-dibutyrate (PDBu), had no effect on PTH mRNA levels at 1 and 24 h. There were no changes in a control gene 18S RNA in these studies.(ABSTRACT TRUNCATED AT 250 WORDS)

The two faces of a steroid antagonist: when an antagonist isn't.

Activation of protein kinase A potentiates the transcriptional response mediated by the glucocorticoid receptor in responsive fibroblasts and in mammary carcinoma cells. This potentiation is ligand-dependent and occurs in responsive fibroblasts and in mammary carcinoma cells. This potentiation is ligand-dependent and occurs without detectable change in the phosphorylation of receptor. The transcriptional response to glucocorticoid or progestin agonists can be blocked by potent antagonists like RU 486. However, upon activation of protein kinase A, the antagonist action of RU 486 on both receptors is blunted. Indeed, RU 486 can itself activate transcription of a hormone-responsive promoter. The conditional agonist activity is observed with type II antagonists, those which recapitulate many of the early steps of ligand-dependent receptor activation, but not type I antagonists, which do not. These studies have now been extended to antimineralocorticoids. In COS-1 cells transfected with a mineralocorticoid receptor expression vector, treatment with 8-BromocAMP potentiates the response to the agonist aldosterone and elicits additional agonist activity in mineralocorticoid antagonists. A model is proposed wherein type II antagonist-receptor complexes occupy receptor binding sites on the genome. The antagonist, however, fails to promote a receptor conformation that can interact productively with a coactivator mediating the communication between receptor and the basal transcription apparatus. Activation of protein kinase A results in the recruitment or activation of a coactivator that permits recovery of receptor-mediated activation function.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular adenosine 3',5'-phosphate formation is essential for down-regulation of surface adenosine 3',5'-phosphate receptors in Dictyostelium.

Dictyostelium discoideum cells contain cell surface cyclic AMP (cAMP) receptors that bind cAMP as a first messenger and intracellular cAMP receptors that bind cAMP as a second messenger. Prolonged incubation of Dictyostelium cells with cAMP induces a sequential process of phosphorylation, sequestration and down-regulation of the surface receptors. The role of intracellular cAMP in down-regulation of surface receptors was investigated. Down-regulation of receptors does not occur under conditions that specifically inhibit the formation of intracellular cAMP (the drug caffeine or mutant cells lacking adenylate cyclase) or conditions that inhibit the function of intracellular cAMP (mutants lacking protein kinase A activity). Cell-permeable non-hydrolysable cAMP derivatives were used to investigate further the requirement of intracellular cAMP for down-regulation. The Sp isomer of 6-thioethylpurineriboside 3',5'-phosphorothioate (6SEth-cPuMPS) does not bind to the surface receptor, enters the cell and has relative high affinity for protein kinase A. 6SEth-cPuMPS alone has no effect on down-regulation. However, together with an agonist of the surface receptor, the analogue induces down-regulation in caffeine-treated wild-type cells and in mutant cells lacking adenylate cyclase, but not in mutant cells lacking protein kinase A. These results indicate that intracellular cAMP formation and activation of protein kinase A are essential for down-regulation of the surface cAMP receptor.