cAMP-stimulated Protein Kinase Research Articles

PTH1R Mutants Found in Patients with Primary Failure of Tooth Eruption Disrupt G-Protein Signaling.

AimPrimary failure of tooth eruption (PFE) is causally linked to heterozygous mutations of the parathyroid hormone receptor (PTH1R) gene. The mutants described so far lead to exchange of amino acids or truncation of the protein that may result in structural changes of the expressed PTH1R. However, functional effects of these mutations have not been investigated yet.Materials and MethodsIn HEK293 cells, PTH1R wild type was co-transfected with selected PTH1R mutants identified in patients with PFE. The effects on activation of PTH-regulated intracellular signaling pathways were analyzed by ELISA and Western immunoblotting. Differential effects of wild type and mutated PTH1R on TRESK ion channel regulation were analyzed by electrophysiological recordings in Xenopus laevis oocytes.ResultsIn HEK293 cells, activation of PTH1R wild type increases cAMP and in response activates cAMP-stimulated protein kinase as detected by phosphorylation of the vasodilator stimulated phosphoprotein (VASP). In contrast, the PTH1R mutants are functionally inactive and mutant PTH1R/Gly452Glu has a dominant negative effect on the signaling of PTH1R wild type. Confocal imaging revealed that wild type PTH1R is expressed on the cell surface, whereas PTH1R/Gly452Glu mutant is mostly retained inside the cell. Furthermore, in contrast to wild type PTH1R which substantially augmented K+ currents of TRESK channels, coupling of mutated PTH1R to TRESK channels was completely abolished.ConclusionsPTH1R mutations affect intracellular PTH-regulated signaling in vitro. In patients with primary failure of tooth eruption defective signaling of PTH1R mutations is suggested to occur in dento-alveolar cells and thus may lead to impaired tooth movement.

Enhanced cAMP-stimulated protein kinase A activity in human fibrolamellar hepatocellular carcinoma.

Fibrolamellar hepatocellular carcinoma (FL-HCC) affects children without underlying liver disease. A consistent mutation in FL-HCCs leads to fusion of the genes encoding a heat shock protein (DNAJB1) and the catalytic subunit of protein kinase A (PRKACA). We sought to characterize the resultant chimeric protein and its effects in FL-HCC. The expression pattern and subcellular localization of protein kinase A (PKA) subunits in FL-HCCs were compared to paired normal livers by quantitative polymerase chain reaction (qPCR), immunoblotting, and immunofluorescence. PKA activity was measured by radioactive kinase assay, and we determined whether the FL-HCC mutation is present in other primary liver tumors. The fusion transcript and chimeric protein were detected exclusively in FL-HCCs. DNAJB1-PRKACA was expressed 10-fold higher than the wild-type PRKACA transcript, resulting in overexpression of the mutant protein in tumors. Consequently, FL-HCCs possess elevated cAMP-stimulated PKA activity compared to normal livers, despite similar Kms between the mutant and wild-type kinases. FL-HCCs in children and young adults uniquely overexpress DNAJB1-PRKACA, which results in elevated cAMP-dependent PKA activity. These data suggest that aberrant PKA signaling contributes to liver tumorigenesis.

Pyruvate restores β-adrenergic sensitivity of L-type Ca2+channels in failing rat heart: role of protein phosphatase

Oxidative stress plays a major role in the pathogenesis of heart failure, where the contractile response to β-adrenergic stimulation is profoundly depressed. This condition involves L-type Ca(2+) channels, but the mechanisms underlying their impaired adrenergic regulation are unclear. Thus the present study explored the basis for impaired adrenergic control of Ca(2+) channels in a rat infarction model of heart failure. Patch-clamp recordings of L-type Ca(2+) current (I(Ca,L)) from ventricular myocytes isolated from infarcted hearts showed a blunted response to intracellular cAMP that was reversed by treatment with exogenous pyruvate. Biochemical studies showed that basal and cAMP-stimulated protein kinase A activities were similar in infarcted and sham-operated hearts, whereas molecular analysis also found that binding of protein kinase A to the α(1C) subunit of voltage-gated Ca(2+) channel isoform 1.2 was not different between groups. By contrast, protein phosphatase 2A (PP2A) activity and binding to α(1C) were significantly less in infarcted hearts. The PP2A inhibitor okadaic acid markedly increased I(Ca,L) in sham-operated myocytes, but this response was significantly less in myocytes from infarcted hearts. However, pyruvate normalized I(Ca,L) stimulation by okadaic acid, and this effect was blocked by inhibitors of thioredoxin reductase, implicating a functional role for the redox-active thioredoxin system. Our data suggest that blunted β-adrenergic stimulation of I(CaL) in failing hearts results from hyperphosphorylation of Ca(2+) channels secondary to oxidation-induced impairment of PP2A function. We propose that the redox state of Ca(2+) channels or PP2A is controlled by the thioredoxin system which plays a key role in Ca(2+) channel remodeling of the failing heart.

Non-steroidal anti-inflammatory drugs activate NADPH oxidase in adipocytes and raise the H2O2 pool to prevent cAMP-stimulated protein kinase a activation and inhibit lipolysis

BackgroundNon-steroidal anti-inflammatory drugs (NSAIDs) —aspirin, naproxen, nimesulide, and piroxicam— lowered activation of type II cAMP-dependent protein kinase A (PKA-II) in isolated rat adipocytes, decreasing adrenaline- and dibutyryl cAMP (Bt2cAMP)-stimulated lipolysis. The molecular bases of insulin-like actions of NSAID were studied.ResultsBased on the reported inhibition of lipolysis by H2O2, catalase was successfully used to block NSAID inhibitory action on Bt2cAMP-stimulated lipolysis. NSAID, at (sub)micromolar range, induced an H2O2 burst in rat adipocyte plasma membranes and in whole adipocytes. NSAID-mediated rise of H2O2 was abrogated in adipocyte plasma membranes by: diphenyleneiodonium, an inhibitor of NADPH oxidase (NOX); the NOX4 antibody; and cytochrome c, trapping the NOX-formed superoxide. These three compounds prevented the inhibition of Bt2cAMP-stimulated lipolysis by NSAIDs. Inhibition of aquaporin-mediated H2O2 transport with AgNO3 in adipocytes allowed NOX activation but prevented the lipolysis inhibition promoted by NSAID: i.e., once synthesized, H2O2 must reach the lipolytic machinery. Since insulin inhibits adrenaline-stimulated lipolysis, the effect of aspirin on isoproterenol-stimulated lipolysis in rat adipocytes was studied. As expected, isoproterenol-mediated lipolysis was blunted by both insulin and aspirin.ConclusionsNSAIDs activate NOX4 in adipocytes to produce H2O2, which impairs cAMP-dependent PKA-II activation, thus preventing isoproterenol-activated lipolysis. H2O2 signaling in adipocytes is a novel and important cyclooxygenase-independent effect of NSAID.

Inhibition of the mitochondrial permeability transition by protein kinase A in rat liver mitochondria and hepatocytes

NO and cGMP administered at reperfusion after ischaemia prevent injury to hepatocytes mediated by the MPT (mitochondrial permeability transition). To characterize further the mechanism of protection, the ability of hepatic cytosol in combination with cyclic nucleotides to delay onset of the calcium-induced MPT was evaluated in isolated rat liver mitochondria. Liver cytosol plus cGMP or cAMP dose-dependently inhibited the MPT, required ATP hydrolysis for inhibition and did not inhibit mitochondrial calcium uptake. Specific peptide inhibitors for PKA (protein kinase A), but not PKG (protein kinase G), abolished cytosol-induced inhibition of MPT onset. Activity assays showed a cGMP- and cAMP-stimulated protein kinase activity in liver cytosol that was completely inhibited by PKI, a PKA peptide inhibitor. Size-exclusion chromatography of liver cytosol produced a single peak of cGMP/cAMP-stimulated kinase activity with an estimated protein size of 180-220kDa. This fraction was PKI-sensitive and delayed onset of the MPT. Incubation of active catalytic PKA subunit directly with mitochondria in the absence of cytosol and cyclic nucleotide also delayed MPT onset, and incubation with purified outer membranes led to phosphorylation of a major 31kDa band. After ischaemia, administration at reperfusion of membrane-permeant cAMPs and cAMP-mobilizing glucagon prevented reperfusion injury to hepatocytes. In conclusion, PKA in liver cytosol activated by cGMP or cAMP acts directly on mitochondria to delay onset of the MPT and protect hepatocytes from cell death after ischaemia/reperfusion.

PDE4 inhibitors roflumilast and rolipram augment PGE2 inhibition of TGF-β1-stimulated fibroblasts

Fibrotic diseases are characterized by the accumulation of extracellular matrix together with distortion and disruption of tissue architecture. Phosphodiesterase (PDE)4 inhibitors, by preventing the breakdown of cAMP, can inhibit fibroblast functions and may be able to mitigate tissue remodeling. Transforming growth factor (TGF)-beta1, a mediator of fibrosis, can potentially modulate cAMP by altering PGE(2) metabolism. The present study assessed whether PDE4 inhibitors functionally antagonize the profibrotic activity of fibroblasts stimulated by TGF-beta1. The PDE4 inhibitors roflumilast and rolipram both inhibited fibroblast-mediated contraction of three-dimensional collagen gels and fibroblast chemotaxis toward fibronectin in the widely studied human fetal lung fibroblast strain HFL-1 and several strains of fibroblasts from adult human lung. Roflumilast was approximately 10-fold more potent than rolipram. There was a trend for PDE4 inhibitors to inhibit more in the presence of TGF-beta1 (0.05 < P < 0.08). The effect of the PDE4 inhibitors was mediated through cAMP-stimulated protein kinase A (PKA), although a PKA-independent effect on gel contraction was also observed. The effect of PDE4 inhibitors depended on fibroblast production of PGE(2) and TGF-beta1-induced PGE(2) production. PDE4 inhibitors together with TGF-beta1 resulted in augmented PGE(2) production together with increased expression of COX mRNA and protein. The present study supports the concept that PDE4 inhibitors may attenuate fibroblast activities that can lead to fibrosis and that PDE4 inhibitors may be particularly effective in the presence of TGF-beta1-induced fibroblast stimulation.

Down-Regulation of Soluble Guanylyl Cyclase Expression by Cyclic AMP Is Mediated by mRNA-Stabilizing Protein HuR

We analyzed whether the cyclic AMP induced down-regulation of the nitric oxide (NO) receptor soluble guanylyl cyclase (sGC) is mediated by the mRNA-protecting protein HuR. Exposure (up to 24 h) of isolated rat aortic segments to the activator of adenylyl cyclase, forskolin (10 microM), and to both activators of cAMP-stimulated protein kinase (PKA), 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3',5'-cyclic monophosphorothioate, Spisomer (Sp-5,6-DCl-cBIMPS; 400 nM), and N6-phenyl-cAMP (10 microM), strongly reduced sGCalpha1beta1 and HuR protein and mRNA expression in a time-dependent and actinomycin D (10 microM)-sensitive fashion. In vitro degradation of sGCalpha1 and beta1 poly(A)+ mRNA by native rat aortic protein was markedly increased by pretreatment of intact aortas with forskolin. Native protein extract from rat aorta shifted the electrophoretic mobility of biotin-labeled riboprobes from the 3'-untranslated region of sGCalpha1 and beta1 mRNA, and these bands was supershifted by a monoclonal antibody directed against the mRNA-stabilizing protein HuR. Forskolin decreased the HuR-sGCalpha1 and beta1 mRNA interaction and HuR protein expression in rat aorta, and this was prevented by the PKA inhibitory cAMP analog 3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS). In cultured smooth muscle cells from rat aorta, forskolin induced a rapid increase in Fos/p-Fos protein levels and activator protein 1 (AP-1) binding activity. Inhibition of this transcription factor by an AP-1 decoy prevented the forskolin-induced down-regulation of HuR. We conclude that forskolin/cAMP decrease the expression of heterodimeric sGC in rat aortic smooth muscle cells via activation of Fos/AP-1, which decreases the expression of HuR and thus destabilizes the sGCalpha1 and beta1 mRNA.

Growth hormone can regulate functions of porcine ovarian granulosa cells through the cAMP/protein kinase A system

The action of growth hormone (GH) on the production of hormones, growth factors, growth factor binding protein and the occurrence of apoptosis in porcine ovarian granulosa cells, as well as the role of cAMP-stimulated protein kinase A (PKA) in the mediation of these effects, were studied. For this purpose, the effects of exogenous pGH (1–10,000 ng/ml), PKA blockers KT5720 (100 ng/ml) and Rp-cAMPS (1 μmol), alone and in combination, on insulin-like growth factor type I (IGF-I), insulin-like binding protein 3 (IGFBP-3), oxytocin (OT) and prostaglandin F alpha (PGF) secretion, PKA and cAMP response element binding transcription factor (CREB) content and the occurrence of apoptosis were investigated. It was found (using RIA/IRMA) that GH addition to culture medium significantly stimulated IGF-I and PGF release and inhibited IGFBP-3 and OT secretion. GH significantly decreased the incidence of apoptosis (TUNEL method) in cultured cells. Immunocytochemical study and Western immunoblotting showed, that addition of GH caused a dramatic increase in the accumulation of immunoreactive PKA within the cells, whilst Western blotting did not reveal marked influence of GH on content of CREB in cell lysates. PKA blockers, given alone, were able to decrease IGFBP-3 output (Rp-cAMPS, but not KT5720), reduce basal OT release (either Rp-cAMPS and KT5720) and increase PGF accumulation (KT5720, but not Rp-cAMPS). Furthermore, PKA blockers were able to prevent stimulatory effects of GH on IGF-I and PGF release, and inhibitory effect of GH on IGFBP-3, OT output and on apoptosis. These observations suggest the involvement of GH and a PKA-dependent intracellular mechanism in the control of IGF-I, IGFBP-3, OT, PGF, cAMP and apoptosis in porcine ovarian granulosa cells. Stimulation of PKA by GH and the prevention of GH-induced effects by PKA blockers suggest that both stimulatory and inhibitory effects of GH on porcine ovarian cells are probably mediated by the cAMP/PKA system.

Role of the PI3K/PKB signaling pathway in cAMP-mediated translocation of rat liver Ntcp.

cAMP stimulates Na(+)-taurocholate (TC) cotransport by translocating the Na(+)-TC-cotransporting peptide (Ntcp) to the plasma membrane. The present study was undertaken to determine whether the phosphatidylinositol-3-kinase (PI3K)-signaling pathway is involved in cAMP-mediated translocation of Ntcp. The ability of cAMP to stimulate TC uptake declined significantly when hepatocytes were pretreated with PI3K inhibitors wortmannin or LY-294002. Wortmannin inhibited cAMP-mediated translocation of Ntcp to the plasma membrane. cAMP stimulated protein kinase B (PKB) activity by twofold within 5 min, an effect inhibited by wortmannin. Neither basal mitogen-activated protein kinase (MAPK) activity nor cAMP-mediated inhibition of MAPK activity was affected by wortmannin. cAMP also stimulated p70(S6K) activity. However, rapamycin, an inhibitor of p70(S6K), failed to inhibit cAMP-mediated stimulation of TC uptake, indicating that the effect of cAMP is not mediated via p70(S6K). Cytochalasin D, an inhibitor of actin filament formation, inhibited the ability of cAMP to stimulate TC uptake and Ntcp translocation. Together, these results suggest that the stimulation of TC uptake and Ntcp translocation by cAMP may be mediated via the PI3K/PKB signaling pathway and requires intact actin filaments.

GH regulates secretory activity and apoptosis in cultured bovine granulosa cells through the activation of the cAMP/protein kinase A system.

We have studied the action of GH on the production of hormones, growth factors, growth factor-binding protein and the occurrence of apoptosis in bovine ovarian granulosa cells, as well as the role of cAMP-stimulated protein kinase A (PKA) in the mediation of these effects. For this purpose we investigated the effects of exogenous bovine GH (0.001-10 microgram/ml), PKA blockers KT5720 (100 ng/ml) and adenosine-3',5'-monophosphothiodate (Rp-cAMPS) (1 micromol), alone and in combination, on IGF-I, IGF-binding protein (IGFBP)-3, oxytocin, progesterone and estradiol secretion, cAMP and PKA content and the occurrence of apoptosis. The secretion of hormones, IGF-I and IGFBP-3 into the culture medium was measured using RIA/IRMA. The presence of PKA was detected using immunocytochemistry and Western immunoblotting. The presence of cAMP in cells was demonstrated using immunocytochemistry, whilst the proportion of apoptotic cells was determined by the TUNEL method. It was found that the addition of GH to the culture medium strongly (P<0.05) stimulated IGF-I (at a concentration of 0.001-10 microgram GH/ml medium), IGFBP-3 (0.001-1 microgram GH/ml) and oxytocin (0.01-10 microgram GH/ml) secretion. Low concentrations (1-100 ng/ml) of GH stimulated, whilst a higher concentration (10 microgram/ml) inhibited estradiol output. GH slightly (P<0.05) inhibited progesterone (1-100 ng GH/ml) secretion and significantly (P<0.05) decreased the incidence of apoptosis (0.01-1 microgram GH/ml) in cultured cells. The addition of GH (100 ng/ml) caused a dramatic (P<0.05) increase in the proportion of cells possessing the immunoreactive catalytic subunit of PKA and a slight decrease in the proportion of cells containing the regulatory PKA subunit.PKA blockers KT5720 and Rp-cAMPS significantly (P<0.05) reduced the proportion of granulosa cells containing cAMP, and the catalytic and (in the case of KT5720) regulatory subunits of PKA. KT5720 given alone significantly (P<0.05) inhibited the secretion of IGFBP-3, but not that of IGF-I or progesterone. Rp-cAMPS decreased (P<0.05) the secretion of oxytocin but not that of estradiol output or the occurrence of apoptosis. KT5720 and Rp-cAMPS fully or partially prevented the GH effect on IGF-I, IGFBP-3, oxytocin, progesterone, estradiol and apoptosis. These observations suggest the involvement of GH and a cAMP/PKA-dependent intracellular cascade in the control of IGF-I, IGFBP-3, oxytocin, progesterone, estradiol, cAMP and apoptosis in bovine ovarian granulosa cells. The stimulation of PKA by GH and the prevention of GH-induced effects by PKA blockers suggest that the observed GH effects on bovine ovarian cells are probably mediated by the cAMP/PKA system.

Involvement of the Cyclic AMP‐Responsive Element Binding Protein Gene Transcription Factor in Genetic Preference for Alcohol Drinking Behavior

Cyclic adenosine 3',5'-monophosphate (cAMP)-responsive element binding (CREB) protein is a gene transcription factor that can integrate the signals mediated via the cAMP second messenger cascade at the gene expression level, which then controls neuronal functions. To examine if the protein kinase A --> CREB signaling cascade is involved in genetic alcohol drinking preference, different measures of CREB were determined in various brain structures of alcohol-preferring (P) and alcohol-nonpreferring (NP) rats. We show here that CRE-DNA binding activity is significantly decreased in the amygdala but not in the cortex, hippocampus, or striatum of P rats compared with NP rats. The levels of total CREB and phosphorylated CREB protein in the amygdala are significantly lower in P rats compared with NP rats. On the other hand, levels of the alpha-isoform of the catalytic subunit of protein kinase A protein, and basal as well as cAMP-stimulated protein kinase A activity are similar in the amygdala of both P and NP rats. Because P and NP rats are genetically bred for high and low alcohol drinking behavior, respectively, these results suggest the possibility that decreased expression of CREB protein in the amygdala may be associated with the high alcohol drinking behavior of P rats.

CPG16, a Novel Protein Serine/Threonine Kinase Downstream of cAMP-dependent Protein Kinase

Gene expression is necessary for the formation and consolidation of long term memory in both invertebrates and vertebrates. Here, we describe the expression and characterization of candidate plasticity gene 16 (cpg16), a protein serine/threonine kinase that was previously isolated from rat hippocampus as a plasticity-related gene. CPG16, when expressed in and purified from bacteria and COS7 cells, was only capable of autophosphorylation and phosphorylation of myelin basic protein but failed to phosphorylate many other peptides and proteins in in vitro phosphorylation assays. Recombinant CPG16, when overexpressed and purified from COS7 cells, had a relatively low level of autophosphorylation activity. This activity was significantly stimulated when cAMP-elevating agents (forskolin, 8-bromo-cAMP) were added to the cells but not by any other extracellular stimuli tested, e.g. serum, phorbol esters, and a calcium ionophore. Although the stimulation of CPG16 activity was inhibited by the cAMP-dependent protein kinase inhibitor H-89, it did not serve as a direct substrate for this kinase. This suggests that CPG16 may be activated by a cAMP-stimulated protein kinase cascade. Immunolocalization studies in COS7 and NIH-3T3 cells showed mostly cytoplasmic localization of CPG16 that turned partially nuclear upon stimulation with 8-bromo-cAMP. Moreover, overexpression of CPG16 seems to partially inhibit cAMP-stimulated activity of the transcription factor CREB (cAMP response element-binding protein), suggesting its involvement in the down-regulation of cAMP-induced transcription. Thus, CPG16 is a protein serine/threonine kinase that may be involved in a novel signaling pathway downstream of cAMP-dependent protein kinase.

Developmental changes in phosphorylation of MAP-2 and synapsin I in cytosol and taxol polymerised microtubules from chicken brain

In cytosol, cyclic AMP stimulated phosphorylation of microtubule associated protein-2 (MAP-2) increased from 2 days to adult in proportion to the increase in the concentration of MAP-2. By contrast, the calmodulin stimulated phosphorylation of MAP-2 decreased in proportion to the decrease in the concentration of calmodulin stimulated protein kinase II (CMK II). Similarly, the cAMP stimulated phosphorylation of the site on synapsin I labeled by the cAMP stimulated protein kinase (PKA) changed little during development whereas the calcium/calmodulin stimulated phosphorylation of the CMK II site decreased dramatically in proportion to the decrease in the concentration of CMK II. The decrease in the concentration of CMK II which occurs in cytosol during synapse maturation was also observed in taxol polymerised microtubules and the effects of the change in the relative concentrations of CMK II and PKA on the phosphorylation of MAP-2 and synapsin I in this fraction were similar to that observed in the cytosol. These results are consistent with the hypothesis that the developmental changes in phosphorylation of endogenous substrates by PKA is controlled largely by changes in the concentration of those substrates, whereas the concentration of CMK II is limiting so that the developmental changes in the phosphorylation of endogenous substrates by CMK II are a function of the concentration of CMK II itself as well as the concentration of endogenous substrates. Some possible functional consequences of this during synapse maturation are discussed.

The Role of Protein Kinase in the Lens

Publisher Summary This chapter discusses the role of protein kinase in the lens. The major impetus for research on the lens has been to understand the mechanisms involved in the regulation of lens metabolism and function, and the processes leading to lens dysfunction and cataracts. One major mechanism of cellular regulation is the phosphorylation dephosphorylation of enzymes and proteins. The lens is composed only of epithelial cells and fiber cells that are derived from epithelial cells. The anterior surface of the lens is a single layer of epithelial cells. Near the edges of the lens, the cells undergo cell division. These cells move posterially and, in the presence of vitreous factors, undergo differentiation into mature fiber cells. There is a wide range of values reported for the protein kinase activity in the buffer-soluble fraction of bovine cortex. Using histones as substrates, the specific activity of the basal protein kinase in buffer extracts of calf cortex was 15 pmol/min-mg protein and the cAMP-stimulated activity was 290 pmol/min-mg protein. One factor controlling in vivo physiological activity of cAMP-dependent protein kinases is the intracellular concentration of cAMP. That protein kinases may regulate certain cellular processes in the lens is supported by several observations. cAMP-stimulated protein kinase activity is present throughout the lens. cAMP is found throughout the lens, although the levels in the nucleus are quite low. Specific endogenous proteins are phosphorylated by the protein kinases in each region.

Cyclic changes in rat uterine proliferation during the estrous cycle are preceded by changes in protein kinase activity.

A protein kinase activity was defined in cytosols and membranes of rat uterus by using two types of enzyme inhibitors. The heat stable protein kinase inhibitor inhibited the cAMP-stimulated protein kinase but not the basal activity measured in the absence of the cyclic nucleotide. However, this basal activity was inhibited by quercetin in a dose-dependent manner, whereas the cAMP-stimulated protein kinase was not inhibited by this bioflavonoid. Thus, quercetin can be used as a tool to define a specific fraction of cAMP-independent protein kinase activity in rat uterine cytosols and membranes. The mature rat uterus is characterized by short (4 days) cyclic changes in tissue growth. Cell proliferation expressed as total uterine DNA content is observed on proestrus, reaches its peak at estrus, and declines sharply during metestrus and diestrus. The quercetin-inhibited protein kinase activity in the membranes of this tissue also changes cyclically and precedes by one phase the cyclic change in tissue proliferation. The clearest effect was seen when the protein substrates for this protein kinase activity were endogenous membrane proteins partially solubilized by Triton X-100 treatment. In contrast no change was observed in cytosolic, quercetin-inhibited protein kinase activity. The cAMP-stimulated protein kinase activity decreased slightly on diestrus. These results support our working hypothesis that tissue proliferation in the uterus and other tissues such as rat mammary gland and mammary tumors, correlates with cAMP-independent, quercetin-inhibited, protein kinase activity in these tissues.

Modulation of soluble ovarian adenosine 3',5'-monophosphate-dependent protein kinase activity during prepubertal development of the rat.

Studies were conducted to evaluate the ontogeny of cAMP-dependent protein kinases in the soluble fraction of rat ovaries obtained throughout prepubertal ovarian maturation (days 5-37). Protein kinase activity stimulated by cAMP and inhibited by the heat-stable protein kinase inhibitor, designated cAMP-dependent protein kinase activity, was already present in ovaries of 5-day-old rats. This kinase activity subsequently increased to reach maximal values between days 16 and 18, then plunged 4-fold to a nadir between days 21 and 23. cAMP-dependent protein kinase activity began to rise again by day 24, increasing 5-fold to a maximum on day 27. Experiments were conducted to evaluate the basis for the 80% reduction of cAMP-dependent protein kinase activity in the soluble extract of ovaries of 21- to 23-day-old rats. To determine whether the decrease in kinase activity was accompanied by a concomitant reduction of cAMP-binding activity, the ability of ovarian cytosol to bind [3H]cAMP was evaluated. Results showed that at the time of decreased cAMP-dependent protein kinase activity, total soluble cAMP-binding activity was not significantly reduced. Additionally, the cAMP-binding activity of the regulatory subunits RI and RII (regulatory subunits of the type I and II isoenzyme forms of cAMP-dependent protein kinase), as detected by photoaffinity labeling with 8-N3-[32P] cAMP, was not changed. To determine whether the decline in kinase activity was due to an actual disappearance of the catalytic subunit from the soluble ovarian extracts of ovaries of 21- to 23-day-old rats, the relative amounts of catalytic subunit were quantified by an enzyme-linked immunosorbent assay using an antiserum directed against bovine heart catalytic subunit. Results showed that the decrease in protein kinase catalytic activity was not due to a reduction in the amount of catalytic subunits. Experiments were conducted to determine whether the reduction of protein kinase catalytic activity in unfractionated cytosol was expressed after anion exchange chromatography. Results showed that the estimated total cAMP-stimulated protein kinase activity measured after DEAE-cellulose chromatography of the soluble ovarian extracts of 21- to 23-day-old rats was no longer depressed. Since the 80% reduction of catalytic kinase activity in ovarian extracts of 21- to 23-day-old rats was detectable in unfractionated cytosol before but not after DEAE-cellulose chromatography, we tested for the presence of an endogenous inhibitor of catalytic kinase activity in the cytosol fraction.

Adenosine 3',5'-monophosphate-dependent protein kinase in supernatants from dispersed bovine parathyroid cells.

The presence and characteristics of protein kinase(s) were studied in supernatants of sonicates of dispersed bovine parathyroid cells. cAMP caused a 3- to 5-fold stimulation of protein kinase activity in such extracts, with half of the maximal activation at 4--5 x 10(-8) M cAMP. Protein kinase inhibitor nearly totally abolished both basal and cAMP-stimulated activity, suggesting that most of the activity was cAMP dependent. About 90% of the cAMP-stimulated protein kinase activity eluted from a DEAE-cellulose column at 0.15 m NaCl, consistent with a type II enzyme. The presence of a type II enzyme was also supported by the effects of histone and salt concentrations on enzyme activity; both the basal and cAMP-stimulated activity ratios (activity minus cAMP divided by activity plus 10(-6) M cAMP) were stable in 0.4 M NaCl. The basal activity ratio was not increased by concentrations of histone as high as 10 mg/ml in the protein kinase assay. The predominance of the type II enzyme in dispersed bovine parathyroid cells made it possible to develop conditions for extracting the enzyme from intact intact cells (0.4 m NaCl and 5 mg/ml charcoal), whereby the state of activation of the enzyme remained relatively constant. These studies demonstrate the presence of cAMP-dependent protein kinase activity in dispersed bovine parathyroid cells and define conditions which make it possible to assess the effects of various secretagogues on protein kinase activation in intact parathyroid cells.

The effect of theophylline on adenosine 3′, 5′-monophosphate-dependent protein kinase and ACTH 1–24 stimulated steroidogenesis in bovine adrenal cortical cells

Theophylline (theo), a known phosphodiesterase (PDE) inhibitor, was tested for its effects on ACTH 1–24 regulated steroidogenesis in isolated bovine adrenal cortical cells. Theo produced a dose related inhibition of ACTH 1–24 stimulated cortisol synthesis with half maximal inhibition occuring at 7 mM. Theo enhanced ACTH 1–24 stimulated cellular adenosine 3′, 5′-monophosphate (cAMP) levels above that produced by ACTH 1–24 alone confirming its inhibition of cAMP PDE. When tested on cAMP binding protein and cAMP-dependent protein kinase activities in cytosol prepared from bovine adrenal cortex, theo displaced 3H-cAMP binding to cAMP binding protein and inhibited cAMP-stimulated protein kinase activity. The half maximal inhibition of cAMP binding and protein kinase activity was observed at 10 and 5 mM, respectively. Inhibition of cAMP-dependent protein kinase by theo provides a possible explanation of its inhibitory effects on adrenal steroidogenesis and further implicates cAMP-dependent protein kinase in mediating ACTH stimulated steroidogenesis. Furthermore these studies suggest a novel mechanism of action for theo in addition to its known action on cAMP PDE.

Insulin Release during Fasting: Studies on Adenylate Cyclase, Phosphodiesterase, Protein Kinase, and Phosphoprotein Phosphatase in Isolated Islets of Langerhans of the Rat*

The role of enzymes of the adenylate cyclasecAMP- protein phosphorylation system in the decreased insulin release observed during fasting has been investigated. Islets of Langerhans were isolated by collagenase digestion from paired 48-h fasted male Wistar rats and fed controls. Islets of both groups were perifused to measure rates and patterns of insulin release when exposed to 16.7 mM D-glucose or 10 mM D-glyceraldehyde. Similarly, islets from both groups were sonicated and assayed for adenylate cyclase, phosphodiesterase, protein kinase, cAMP-stimulated protein kinase, and phosphoprotein phosphatase activities. Basal insulin secretion under perifusion conditions with 2.8 mM D-glucose was not different in the two groups of islets (13.7 ± 1.6 pg insulin released islet-1 min-1 from fasted rats compared with 16.7 ± 1.2 for controls; P < 0.2). In response to 16.7 mM Dglucose, insulin release was diminished in islets from fasted animals by 50%, whereas D-glyceraldehyde-stimulated insulin release was not affe...

Effects of Diamide on Basal and Thyrotropin-Stimulated Thyroid Metabolism

The effect of diamide on basal and TSH-stimulated thyroid metabolism was studied using bovine and dog thyroid slices. Diamide (10 mM) inhibited basal and TSH-stimulated net cAMP production, basal and cAMP-stimulated protein kinase activity, and stimulation by TSH of colloid droplet formation and organification of iodide and abolished basal and TSH-stimulated uptake of 32P into phospholipids. In thyroid slices incubated with 0.1 mM diamide, none of these activities, whether basal or TSH-stimulated, was affected. However, 0.1 mM diamide increased the net basal production of cAMP and potentiated the effect of TSH on this process. These results demonstrate that the previously reported inhibition of protein kinase by 2-20 mM diamide is not specific and that this compound cannot be used to determine which of the metabolic effects of TSH are dependent upon cAMP activation of protein kinase. While 0.1 mM diamide increased cAMP in thyroid slices, it did not reproduce any of the other effects of TSH.