Inhibition Of PKA Activity Research Articles

Bile acids regulate SF-1 to alter cholesterol balance in adrenocortical cells via S1PR2

Glucocorticoid synthesis typically occurs in adrenal cortex and is influenced by cholesterol balance, since cholesterol is the sole precursor of steroids. Bile acids as the signaling molecules, have been shown to promote steroidogenesis in steroidogenic cells. However, whether bile acids directly regulate cholesterol balance remains elusive. In this study, we prepared cholestatic mouse models and cultured human adrenocortical cells (H295R) treated with taurochenodeoxycholic acid (TCDCA) to determine transcription levels of cholesterol metabolism associated genes and cholesterol concentrations in adrenocortical cells. Results showed that common bile duct ligation (CBDL) and chenodeoxycholic acid (CDCA) feeding elevated the mRNA levels of Abca1, Cyp51, Hmgcr, Srb1, and Mc2r in adrenals of mice. Meanwhile, the concentrations of total cholesterol and cholesteryl ester in adrenals of CBDL and CDCA-fed mice were dramatically lowered. The total and phosphorylation levels of HSL in adrenal glands of CBDL mice were also enhanced. Similarly, TCDCA treatment in H295R cells decreased intracellular concentrations of total cholesterol and cholesteryl ester and increased transcription levels of SRB1, MC2R, and HSL as well. Inhibition of bile acids’ receptor sphingosine 1‐phosphate receptor 2 (S1PR2), extracellular signal‐regulated kinase (ERK) phosphorylation, and steroidogenic factor 1 (SF-1) respectively successfully abolished effect of TCDCA on H295R cells. SF-1s was found to be phosphorylated at Thr75 in TCDCA-treated H295R cells. While a mild increase of intracellular cAMP concentration was detected upon TCDCA treatment, inhibition of PKA activity with Rp-Isomer in H295R cells failed to decrease the expression of SF-1 and its target genes. Our findings suggest that conjugated bile acids affect cholesterol balance through regulation of SF-1 in adrenocortical cells so as to provide an adequate cholesterol supply for glucocorticoid synthesis, which improves and enriches our understanding of the mechanism whereby bile acids regulate cholesterol balance to affect adrenal function.

P53-mediated regulation of mitochondrial dynamics plays a pivotal role in the senescence of various normal cells as well as cancer cells.

The tumor suppressor p53 is known as a critical mediator of many cellular processes, including cellular senescence, but its role in mitochondrial dynamics is not fully understood. We have previously shown that p53 regulates mitochondrial dynamics via the PKA-Drp1 pathway to induce cellular senescence. In this study, to further understand the role of p53-dependent regulation of mitochondrial dynamics, the effect of p53 expression on mitochondrial morphology was examined in various cancer cell lines and normal human cells. We found that p53 induced remarkable mitochondrial elongation and cellular senescence in various cancer cells regardless of their p53 status. p53 also induced mitochondrial elongation in various human primary normal cells, suggesting that p53-mediated mitochondrial elongation is a general phenomenon. Moreover, we found that p53 plays an essential role in mitochondrial elongation in H-Ras-induced cellular senescence and in the replicative senescence of normal human cells. Treatment with the MDM-2 antagonist Nutlin-3a also induced mitochondrial elongation through the PKA-Drp1 pathway in IMR90 normal human cells. Furthermore, the inhibition of PKA activity in late-passage normal cells significantly reduced both mitochondrial elongation and cellular senescence, suggesting that the p53-PKA pathway is essential for maintaining the senescence phenotype in normal cells. Together, these results further confirm the direct regulation of mitochondrial dynamics by p53 and the important role of p53-mediated mitochondrial elongation in cellular senescence.

Abstract 147: Metformin suppresses triple-negative breast cancer stem cells by targeting KLF5 for degradation

Abstract Out of the breast cancer subtypes, triple-negative breast cancer (TNBC) has the poorest prognosis without effective targeted therapies. A stem cell transcription factor KLF5 is over-expressed in basal type TNBC and promoting cell proliferation, survival and stemness. Previously, we demonstrated that Mifepristone suppresses basal TNBC stem cells by down-regulating KLF5 expression through inducing the expression of miR-153. In this study, Metformin, a first-line drug for type 2 diabetes mellitus, was demonstrated to target breast cancer stem cells selectively. However, the efficiency and the mechanism of action of metformin in TNBC are unclear. We demonstrated that metformin decreased the percentage of TNBC stem cells partially through the downregulation of the expression of KLF5 and its downstream target genes, such as Nanog and FGF-BP1, in TNBC cell lines. Metformin induced GSK3β-mediated KLF5 protein phosphorylation and degradation through the inhibition of PKA activity in TNBC cells. Consistently, PKA activators increased the expression levels of KLF5. We observed a positive correlation between p-CREB and KLF5 protein levels in human TNBC samples. These findings suggest that metformin suppresses TNBC stem cells partially through the PKA-GSK3β-KLF5 signaling pathway. Citation Format: Peiguo Shi, Zhongmei Zhou, Rong Liu, Ceshi Chen. Metformin suppresses triple-negative breast cancer stem cells by targeting KLF5 for degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 147. doi:10.1158/1538-7445.AM2017-147

Nesfatin-1 Suppresses Cardiac L-type Ca2+ Channels Through Melanocortin Type 4 Receptor and the Novel Protein Kinase C Theta Isoform Pathway

Background/Aims: Nesfatin-1 (NF-1), an anorexic nucleobindin-2 (NUCB2)-derived hypothalamic peptide, acts as a peripheral cardiac modulator and it can induce negative inotropic effects. However, the mechanisms underlying these effects in cardiomyocytes remain unclear. Methods: Using patch clamp, protein kinase assays, and western blot analysis, we studied the effect of NF-1 on L-type Ca²⁺ currents (I_Ca,L) and to explore the regulatory mechanisms of this effect in adult ventricular myocytes. Results: NF-1 reversibly decreased I_Ca,L in a dose-dependent manner. This effect was mediated by melanocortin 4 receptor (MC4-R) and was associated with a hyperpolarizing shift in the voltage-dependence of inactivation. Dialysis of cells with GDP-β-S or anti-G_β antibody as well as pertussis toxin pretreatment abolished the inhibitory effects of NF-1 on I_Ca,L. Protein kinase C (PKC) antagonists abolished NF-1-induced responses, whereas inhibition of PKA activity or intracellular application of the fast Ca²⁺-chelator BAPTA elicited no such effects. Application of NF-1 increased membrane abundance of PKC theta isoform (PKC_θ), and PKC_θ inhibition abolished the decrease in I_Ca,L induced by NF-1. Conclusion: These data suggest that NF-1 suppresses L-type Ca²⁺ channels via the MC4-R that couples sequentially to the βγ subunits of G_i/o-protein and the novel PKC_θ isoform in adult ventricular myocytes.

Exendin-4 promotes endothelial barrier enhancement via PKA- and Epac1-dependent Rac1 activation.

Among emerging antidiabetic agents, glucagon-like peptide-1 (GLP-1)-based therapies carry special cardiovascular implications, exerting both direct and indirect effects. The control of vascular permeability is of pivotal importance in vascular pathologies. The objective of the present study was to determine the effect of GLP-1 on endothelial barrier function and assess the underlying mechanism(s). Here we show for the first time that the stable GLP-1 analog exendin-4 attenuated the leakage of subcutaneous blood vessels in mice indexed by dye extravasation caused by injections of thrombin. Moreover, in cultured endothelial cells, exendin-4 significantly prevented the thrombin-induced FITC-dextran permeability of endothelial monolayers via GLP-1 receptor. Immunofluorescence microscopy reveals that exendin-4 abrogates detrimental effects of thrombin on VE-cadherin and the F-actin cytoskeleton, with decreased stress fiber and gap formation. Importantly, exendin-4 reduced thrombin-induced tyrosine phosphorylation of VE-cadherin at Y731 and Y658. Moreover, small GTPase Rac1 was significantly activated as a result of exendin-4 treatment. The efficacy of exendin-4 to counteract the barrier-compromising effect of thrombin was blunted when Rac1 was inactivated by Rac1 inhibitor NSC-23766. Inhibition of PKA activity or small-interfering RNA for exchange protein directly activated by cAMP 1 (Epac1) decreased exendin-4-induced Rac1 activation and barrier enhancement, indicating the participation of both PKA and Epac1 in the barrier-stabilizing effect of exendin-4 elicited on thrombin-impaired barrier function. Thus, our findings have uncovered an unpredicted role for exendin-4 in the coordination of vascular permeability and clarified the molecular underpinnings that contribute to barrier restoration initiated by exendin-4.

β-Agonist-mediated Relaxation of Airway Smooth Muscle Is Protein Kinase A-dependent

Inhaled β-agonists are effective at reversing bronchoconstriction in asthma, but the mechanism by which they exert this effect is unclear and controversial. PKA is the historically accepted effector, although this assumption is made on the basis of associative and not direct evidence. Recent studies have asserted that exchange protein activated by cAMP (Epac), not PKA, mediates the relaxation of airway smooth muscle (ASM) observed with β-agonist treatment. This study aims to clarify the role of PKA in the prorelaxant effects of β-agonists on ASM. Inhibition of PKA activity via expression of the PKI and RevAB peptides results in increased β-agonist-mediated cAMP release, abolishes the inhibitory effect of isoproterenol on histamine-induced intracellular calcium flux, and significantly attenuates histamine-stimulated MLC-20 phosphorylation. Analyses of ASM cell and tissue contraction demonstrate that PKA inhibition eliminates most, if not all, β-agonist-mediated relaxation of contracted smooth muscle. Conversely, Epac knockdown had no effect on the regulation of contraction or procontractile signaling by isoproterenol. These findings suggest that PKA, not Epac, is the predominant and physiologically relevant effector through which β-agonists exert their relaxant effects.

Cytokine-induced iNOS and ERK1/2 inhibit adenylyl cyclase type 5/6 activity and stimulate phosphodiesterase 4D5 activity in intestinal longitudinal smooth muscle.

This study identified a distinctive pattern of expression and activity of adenylyl cyclase (AC) and phosphodiesterase (PDE) isoforms in mouse colonic longitudinal smooth muscle cells and determined the changes in their expression and/or activity in response to proinflammatory cytokines (IL-1β and TNF-α) in vitro and 2,4,6 trinitrobenzene sulphonic acid (TNBS)-induced colonic inflammation in vivo. AC5/6 and PDE4D5, expressed in circular muscle cells, were also expressed in longitudinal smooth muscle. cAMP formation was tightly regulated via feedback phosphorylation of AC5/6 and PDE4D5 by PKA. Inhibition of PKA activity by myristoylated PKI blocked phosphorylation of AC5/6 and PDE4D5 and enhanced cAMP formation. TNBS treatment in vivo and IL-1β and TNF-α in vitro induced inducible nitric oxide synthase (iNOS) expression, stimulated ERK1/2 activity, caused iNOS-mediated S-nitrosylation and inhibition of AC5/6, and induced phosphorylation of PDE4D5 and stimulated its activity. The resultant decrease in AC5/6 activity and increase in PDE4D5 activity decreased cAMP formation and smooth muscle relaxation. S-nitrosylation and inhibition of AC5/6 activity were reversed by the iNOS inhibitor 1400W, whereas phosphorylation and activation of PDE4D5 were reversed by the phosphatidylinositol 3-kinase inhibitor LY294002 and the ERK1/2 inhibitor PD98059. The effects of IL-1β or TNF-α on forskolin-stimulated cAMP formation and smooth muscle relaxation reflected inhibition of AC5/6 activity and activation of PDE4D5 and were partly reversed by 1400W or PD98059 and completely reversed by a combination of the two inhibitors. The changes in the cAMP/PKA signaling and smooth muscle relaxation contribute to colonic dysmotility during inflammation.

Ca2+/Calcineurin-Dependent Inactivation of Neuronal L-Type Ca2+ Channels Requires Priming by AKAP-Anchored Protein Kinase A

Within neurons, Ca2+-dependent inactivation (CDI) of voltage-gated L-type Ca2+ channels shapes cytoplasmic Ca2+ signals. CDI is initiated by Ca2+ binding to channel-associated calmodulin and subsequent Ca2+/calmodulin activation of the Ca2+-dependent phosphatase, calcineurin (CaN), which is targeted to L channels by the A-kinase-anchoring protein AKAP79/150. Here, we report that CDI of neuronal L channels was abolished by inhibition of PKA activity or PKA anchoring to AKAP79/150 and that CDI was also suppressed by stimulation of PKA activity. Although CDI was reduced by positive or negative manipulation of PKA, interference with PKA anchoring or activity lowered Ca2+ current density whereas stimulation of PKA activity elevated it. In contrast, inhibition of CaN reduced CDI but had no effect on current density. These results suggest a model wherein PKA-dependent phosphorylation enhances neuronal L current, thereby priming channels to undergo CDI, and Ca2+/calmodulin-activated CaN actuates CDI by reversing PKA-mediated enhancement of channel activity.

Identification of a protein kinase A regulatory subunit from Leishmania having importance in metacyclogenesis through induction of autophagy

cAMP-mediated responses act as modulators of environmental sensing and cellular differentiation of many kinetoplastidae parasites including Leishmania. Although cAMP synthesizing (adenylate cyclase) and degrading (phosphodiesterase) enzymes have been cloned and characterized from Leishmania, no cAMP-binding effector molecule has yet been identified from this parasite. In this study, a regulatory subunit of cAMP-dependent protein kinase (Ldpkar1), homologous to mammalian class I cAMP-dependent protein kinase regulatory subunit, has been identified from L. donovani. Further characterization suggested possible interaction of LdPKAR1 with PKA catalytic subunits and inhibition of PKA activity. This PKA regulatory subunit is expressed in all life cycle stages and its expression attained maximum level in stationary phase promastigotes, which are biochemically similar to the infective metacyclic promastigotes. Starvation condition, the trigger for metacyclogenesis in the parasite, elevates LdPKAR1 expression and under starvation condition promastigotes overexpressing Ldpkar1 attained metacyclic features earlier than normal cells. Furthermore, Ldpkar1 overexpression accelerates autophagy, a starvation-induced cytological event necessary for metacyclogenesis and amastigote formation. Conditional silencing of Ldpkar1 delays the induction of autophagy in the parasite. The study, for the first time, reports the identification of a functional cAMP-binding effector molecule from Leishmania that may modulate important cytological events affecting metacyclogenesis.

Specific PKG inhibitors: do they really exist?

Background Cellular cGMP effects can be mediated by a number of effectors including cGMP-dependent protein kinases (PKGs), cGMP-stimulated phosphodiesterase (PDE2), cGMP-inhibited phosphodiesterase (PDE3), and cGMPgated channels. Pharmacological inhibitors of PKG are often used to discriminate between these diverse cGMP effects. Currently used PKG inhibitors can be divided into three classes: cyclic nucleotide binding site inhibitors like Rp-phosphorothioate analogs, ATP binding site inhibitors like KT5823, and substrate binding site inhibitors like the recently described DT-oligopeptides. However, several studies have observed no PKG inhibition by KT5823 in intact cells [1] or by Rp-cGMPS analogs in smooth muscle cells [2], as well as unspecific (PKG-independent) effects of Rp-cGMPS analogs in platelets [3]. Results We tested the inhibitory effects of (D)-DT2 and DT3 on PKG and its effects on intact cells. Our data show that (D)-DT2 selectively inhibited 2 nM purified PKG Ia and Ib with an IC50 of 8n M, and that up to 1µ M (D)-DT2 did not inhibit PKG II or PKA. In broken platelet cell experiments, PKG activity was inhibited by (D)-DT2 starting at 5 µM, with complete inhibition at 20 µM, but we also observed inhibition of PKA activity at these concentrations. However, concentrations of up to 200 µM of compounds failed to inhibit PKG activity (assessed by phosphorylation of the established PKG substrates VASP, PDE5 and GRP2) in intact human platelets, rat mesangial cells and neonatal mouse cardiac myocytes. It should be noted that the measured PKG concentration is about 7 µM in platelets and 0.1 – 0.5 µM in all other tested cells. (D)-DT2 effects on platelet function did not correlate with PKG activity. Preincubation of platelets with 10 nM (D)-DT2 strongly inhibited thrombin-induced platelet aggregation and calcium mobilization, whereas it potentiated these effects in collagen-stimulated platelets. Conclusion Interpretations of results based on PKG inhibitors require caution. None of the commercially available PKG inhibitors should be used without control experiments in intact cells since they may have unpredictable functional effects not mediated by PKG activity.

Abstract 5298: Human enhancer of filamentation 1 induction by Prostaglandin E2 enhances the cell motility of colorectal cancer cells

Abstract Prostaglandin (PG) E2 promotes cell motility during colorectal carcinogenesis. The mechanism by which PGE2 signaling regulates cell motility is not completely understood. Here, we demonstrated that PGE2 treatment induces HEF1 expression that links with cell motility in colorectal cancer cells. PGE2 rapidly stimulated HEF1 expression in colorectal cancer cells. Both PGE2 treatment and HEF1 overexpression activated Rac 1 and resulted in similar effects on cell spreading. Moreover, knockdown of HEF1 using shRNA suppressed PGE2-driven cell spreading. PKA activator forskolin induced HEF1 expression and cell spreading in a similar manner as PGE2. Inhibition of PKA activity by H-89 attenuated HEF1 expression induced by PGE2. Furthermore, PKA knockdown by siRNA blocked the HEF1 expression and cell spreading induced by PGE2. These data suggest that PGE2 induces HEF1 expression to promote cell motility through a PKA-involved pathway. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5298.

Abstract B39: Prostaglandin E2 induces human enhancer of filamentation 1 to promote the cell motility of colorectal carcinoma cells

Abstract PGE2 promotes cell motility during colorectal carcinogenesis. The mechanism by which PGE2 signaling regulates cell motility is not completely understood. Here, we demonstrated that PGE2 treatment induces HEF1 expression that links with cell motility in colorectal cancer cells. PGE2 rapidly stimulated HEF1 expression in colorectal cancer cells. Both PGE2 treatment and HEF1 overexpression activated Rac 1 and resulted in similar effects on cell spreading. Moreover, knockdown of HEF1 using shRNA suppressed PGE2-driven cell spreading. PKA activator forskolin induced HEF1 expression and cell spreading in a similar manner as PGE2. Inhibition of PKA activity by H-89 attenuated HEF1 expression induced by PGE2. Furthermore, PKA knockdown by siRNA blocked the HEF1 expression and cell spreading induced by PGE2. These data suggest that PGE2 induces HEF1 expression to promote cell motility through a PKA-involved pathway. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B39.

Genistein restricts leptin secretion from rat adipocytes

The isoflavones – genistein and daidzein – compounds found in high concentrations in soy play an important role in prevention of many diseases and affect some metabolic pathways. In the performed experiment it was demonstrated that genistein (5 mg/kg b.w.) administered intragastrically for three days to male Wistar rats substantially diminished blood leptin level. Studies with isolated rat adipocytes revealed that this phytoestrogen strongly restricted leptin secretion from these cells. These effects were not accompanied by any changes in leptin gene expression in adipocytes. Daidzein – an analogue of genistein – used at similar concentrations did not affect blood leptin concentration, leptin secretion and expression of its gene. To determine the influence of genistein and daidzein on leptin release, adipocytes isolated from the epididymal fat tissue were incubated for 2 h in Krebs–Ringer buffer. Leptin secretion stimulated by glucose with insulin was significantly diminished by genistein (0.25–1 mM). This effect of genistein may arise from several aspects of its action in adipocytes documented in the literature such as the inhibition of glucose transport and metabolism, the attenuation of insulin signalling, the inhibition of cAMP phosphodiesterase and the stimulation of lipolysis. However, the bypassing of the restrictive action of genistein on glucose transport and glycolysis (by the use of alanine instead of glucose) and on insulin action (by the use of nicotinic acid) was not sufficient to restore leptin secretion from isolated adipocytes. It was also demonstrated that the restriction of the stimulatory influence of genistein on cAMP/protein kinase A (PKA) pathway (by the inhibition of PKA activity) did not improve leptin release. Results obtained in our experiments point at the restriction of glucose metabolism following formation of pyruvate as the pivotal reason of the inhibitory action of genistein on leptin release.

Dopaminergic inhibition of secretin-stimulated choleresis by increased PKC-gamma expression and decrease of PKA activity.

To determine the role and mechanisms of action by which dopaminergic innervation modulates ductal secretion in bile duct-ligated rats, we determined the expression of D1, D2, and D3 dopaminergic receptors in cholangiocytes. We evaluated whether D1, D2 (quinelorane), or D3 dopaminergic receptor agonists influence basal and secretin-stimulated choleresis and lumen expansion in intrahepatic bile duct units (IBDU) and cAMP levels in cholangiocytes in the absence or presence of BAPTA-AM, chelerythrine, 1-(5-isoquinolinylsulfonyl)-2-methyl piperazine (H7), or rottlerin. We evaluated whether 1) quinelorane effects on ductal secretion were associated with increased expression of Ca(2+)-dependent PKC isoforms and 2) increased expression of PKC causes inhibition of PKA activity. Quinelorane inhibited secretin-stimulated choleresis in vivo and IBDU lumen space, cAMP levels, and PKA activity in cholangiocytes. The inhibitory effects of quinelorane on secretin-stimulated ductal secretion and PKA activity were blocked by BAPTA-AM, chelerythrine, and H7. Quinelorane effects on ductal secretion were associated with activation of the Ca(2+)-dependent PKC-gamma but not other PKC isoforms. The dopaminergic nervous system counterregulates secretin-stimulated ductal secretion in experimental cholestasis.

Insulin inhibits secretin-induced ductal secretion by activation of PKC alpha and inhibition of PKA activity

Insulin stimulates canalicular bile flow by interaction with hepatocytes. Insulin regulates the function of a number of epithelia through activation and membrane translocation of Ca 2+-dependent PKC isoforms. No information exists regarding insulin regulation of ductal bile secretion. The aim of the study was to determine the role and mechanisms of action of insulin in the regulation of cholangiocyte secretion in BDL rats. We determined the subcellular localization of insulin receptor in cholangiocytes. We measured the effect of insulin on (1) secretin-stimulated cAMP levels in cholangiocytes and duct expansion in intrahepatic bile duct units (IBDUs) in the absence or presence of BAPTA/AM, H7 or rottlerin and (2) bile flow. We evaluated (1) if insulin effects are associated with activation of PKC alpha and (2) if activation of PKC causes inhibition of secretin-stimulated cAMP levels and PKA activity. We found insulin receptors only in the apical domain of cholangiocytes. Insulin inhibited secretin-induced choleresis and secretin-stimulated cholangiocyte cAMP levels. Insulin inhibited secretin-induced secretion in IBDUs when applied at the basolateral membrane or microinjected into IBDU lumen. Insulin inhibitory effects on cholangiocyte secretion were blocked by BAPTA/AM and H7. Insulin induced activation of PKC alpha, which decreased secretin-stimulated cAMP and PKA activity. In conclusion, insulin inhibited secretin-induced ductal secretion of BDL rats through activation of PKC and inhibition of secretin-stimulated cAMP and PKA activity. In conclusion, insulin counter-regulates cholangiocyte secretory processes in the BDL model, which is characterized by cholangiocyte proliferation. (H EPATOLOGY 2002;36:641-651.)

Protein kinase A regulates β2 integrin avidity in neutrophils

The adhesive phenotype of neutrophils (PMN) depends largely on activating and deactivating intracellular signals regulating beta2 integrin avidity for ligand. Our hypothesis is that PKA is a negative regulator of beta2 integrin avidity. In this work, we examined the role of PKA in PMN alphaMbeta2 integrin activation. Elevation of cAMP inhibited alphaMbeta2 integrin-dependent adhesion of PMN to immune complexes (IC), but not PMA-induced adhesion. The PKA inhibitor KT5720 reversed the ability of cAMP to suppress adhesion to IC. Moreover, inhibition of PKA activity was sufficient to activate alphaMbeta2 integrin-dependent adhesion and increase beta2 integrin expression and binding of the monoclonal antibody CBRM1/5, which recognizes activated alphaMbeta2 specifically. However, PKA activity was necessary for sustained adhesion. Disruption of A kinase-anchoring, protein-PKA binding with a cell-permeant peptide derived from the AKAP Ht31 also activated adhesion. Unlike pharmacologic inhibition of PKA, AKAP peptide-induced adhesion was PKC dependent and did not affect beta2 integrin expression or CBRM1/5 binding. These data demonstrate that PKA appears to have a dual role in the mechanism regulating alphaMbeta2 integrin avidity and adhesion.

Regulation of endothelial barrier function by the cAMP-dependent protein kinase.

Elevation of cAMP promotes the endothelial cell (EC) barrier and protects the lung from edema development. Thus, we tested the hypothesis that both increases and decreases in PKA modulate EC function and coordinate distribution of regulatory, adherence, and cytoskeletal proteins. Inhibition of PKA activity by RpcAMPS and activation by cholera toxin was verified by assay of kemptide phosphorylation in digitonin permeabilized EC. Inhibition of PKA by RpcAMPS or overexpression of the endogenous inhibitor, PKI, decreased monolayer electrical impedance and exacerbated the decreases produced by agonists (thrombin and PMA). RpcAMPS directly increased F-actin content and organization into stress fibers, increased co-staining of actin with both phosphatase 2B and myosin light chain kinase (MLCK), caused reorganization of focal adhesions, and decreased catenin at cell borders. These findings are similar to those evoked by thrombin. In contrast, cholera toxin prevented the agonist-induced resistance decrease and protein redistribution. Although PKA activation attenuated thrombin-induced myosin light chain (MLC) phosphorylation, PKA inhibition per se did not cause MLC phosphorylation or affect [Ca2+]i. These studies indicate that a decrease in PKA activity alone can produce disruption of barrier function via mechanisms not involving MLCK and support a central role for cAMP/PKA in regulation of cytoskeletal and adhesive protein function in EC which correlates with altered barrier function.

Cyclic-AMP-Dependent Protein Kinase Activity Is Not Required by Parathyroid Hormone to Stimulate Phosphoinositide Signaling in Chondrocytes but Is Required to Transduce the Hormone′s Proliferative Effect

Parathyroid hormone (PTH), an activator of both cAMP and phosphoinositide (PI) signaling in growth plate chondrocytes (GPCs), is generally believed to trigger each of these pathways through interactions with separate G proteins. Recently, however, activation of cAMP-dependent protein kinase (pkA) has been found to cause a stimulation of the PI cascade in hepatocytes. This finding raises the possibility that PTH stimulation of PI metabolism in GPCs may really be a secondary event, mediated through a primary stimulation of pkA. Experiments discussed in the present report indicate that the PTH stimulation of PI metabolism in GPCs is independent of pkA activity. The data show that (1) unlike the Ca 2+ response evoked by PTH, the responses evoked by dibutyryl-cAMP or S p diastereomer of cyclic adenosine-3′,5′-monophosphothioate, two activators of pkA, require an extracellular Ca 2+ source; (2) also unlike PTH, activation of pkA by these same cAMP analogs does not cause an increase in cellular inositol-1,4,5-trisphosphate; and (3) specific inhibition of pkA with N-[2-( p-bromocinnamylamino)ethyl]-5-isoquinolinsulfanomide (H-89) or R p diastereomer of cylic adenosine-3′,5′-monophosphothioate (R p cAMPS) has no effect on the ability of PTH to evoke its normal Ca 2+ response. Furthermore, data presented indicate that the PTH stimulation of GPC proliferation does not require Ca 2+ signals, but rather is at least partially dependent on pkA. The data show that either loading the cells with the Ca 2+ buffer bis-( o-ami-nophenoxy)ethane- N,N,N′,N′-tetracetic acid or depleting the cells of intracellularly stored Ca 2+ is without effect on the stimulation of DNA synthesis by the hormone. Inhibition of pkA activity with H-89 or R p-cAMPS, in contrast, leads to a significant reduction in the ability of PTH to stimulate its proliferative effect.