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Pericytes exert an important influence on the control of retinal blood flow; however, little is known regarding the molecular basis of retinal pericyte excitability. The purpose of this study was to elucidate the signaling pathway of how prostacyclin (PGI2), an important endogenous regulator of retinal blood flow, stimulates potassium channel activity in retinal pericytes. Retinal pericytes were isolated from porcine eyeballs and plated on glass coverslips. Immunocytochemistry was performed to verify expression of the pericyte-specific ganglioside marker, 3G5 and smooth muscle alpha-actin. Activity of the large-conductance, voltage- and calcium-activated potassium (BKCa) channel was measured in retinal pericytes via single-channel patch-clamp, and channel identification was confirmed via biophysical and pharmacological characterization. PGI2 (10 microM) or beraprost (30 microM; more stable prostacyclin analog) dramatically stimulated the activity of BKCa channels isolated in cell-attached patches. These experiments strongly suggested that PGI2 stimulated BKCa channel activity via a diffusible second messenger. Similarly, chlorophenylthio (CPT)-cAMP (100 microM; membrane permeable cAMP derivative) induced a significant increase in BKCa channel activity; however, inhibition of the cAMP-dependent protein kinase (PKA) with 300 nM KT5720 could not reverse the stimulatory effect of either PGI2 or CPT-cAMP. In contrast, activation of BK(Ca) channels with either CPT-cAMP or PGI2 was abolished by 300 nM KT5823 (n=5, p

SummaryThe acrosome reaction (AR) is a fundamental event for fertilization, which is induced in concert with acrosome reaction-inducing substance (ARIS) and asterosap, both of which are components of starfish egg jelly (EJ). During the AR, a spermatozoon undergoes a series of physiological changes, such as in intracellular cGMP concentration ([cGMP]i), pHiand intracellular Ca2+concentration ([Ca2+]i). Affinity purification of cGMP-binding protein resulted in the isolation of a regulatory subunit of the cAMP-dependent protein kinase A (PKA), suggesting the involvement of a cAMP-dependent pathway in the AR. By using a cAMP enzyme immunoassay, [cAMP]iwas found to increase in starfish spermatozoa when stimulated with ARIS and asterosap. ARIS could also increase the [cAMP]iin the presence of high pH seawater. Pretreatment of spermatozoa with two specific and cell-permeable PKA inhibitors, H89 and KT5720, prevented the induction of the AR in a concentration-dependent manner. These results suggest that PKA activity participates in the induction of the AR with ARIS and asterosap. To investigate this, we have cloned a gene that encodes a regulatory subunit of PKA that had been identified in starfish spermatozoa.

In medusae of the hydrozoan Cytaeis uchidae, oocyte meiotic maturation and spawning occur as a consequence of dark–light transition. In this study, we investigated the mechanism underlying the initiation of meiotic maturation using in vitro (isolated oocytes from ovaries) and in vivo (ovarian oocytes in medusae) systems. Injection of cAMP derivatives into isolated oocytes induced meiotic maturation in a dose-dependent manner. Meiotic maturation was also achieved in isolated oocytes preloaded with caged cAMP and exposed to UV irradiation. The caged cAMP/UV irradiation-induced meiotic maturation was completely inhibited by blockers of protein kinase A (PKA), H-89, KT5720, and Rp-cAMPS. The medusae from which most parts of the umbrella were removed (umbrella-free medusae) survived for at least 2 weeks, during which time oocyte meiotic maturation and spawning occurred. When H-89 and Rp-cAMPS were injected into ovarian oocytes of umbrella-free medusae within 3 min of dark–light stimulation, meiotic maturation was inhibited or delayed. An increase in intracellular cAMP was confirmed by FlCRhR, a fluorescent cAMP indicator, in ovarian oocytes exposed to dark–light transition as well as in isolated oocytes stimulated by caged cAMP/UV irradiation. These results indicate that the cAMP/PKA signaling pathway positively contributes to light-triggered physiological oocyte meiotic maturation in Cytaeis uchidae.

Our recent study showed that intravenous ethanol selectively inhibited the pressor effects elicited by the microinjection of N-methyl-D-aspartate (NMDA) into rostral ventrolateral medulla (RVLM) and acute tolerance to the inhibition was observed during prolonged application of ethanol in anesthetized Sprague-Dawley rats. In this study, we examined the role of the cAMP-dependent protein kinase (PKA) signaling pathway in acute tolerance to ethanol inhibition of NMDA-induced responses in rat RVLM. A significant increase in the level of PKA-regulated phosphoserine 897 on the NMDA NR1 subunit was found in the rostroventral medulla during acute ethanol tolerance. Reduction of NMDA-induced pressor effects was observed at 10 min but disappeared at 40 min after continuous ethanol infusion. This effect was dose-dependently blocked by microinjection of KT5720 (0.04-4 pmol, a selective PKA inhibitor) or cAMPS-Rp (0.02, 0.2 pmol, a cAMP antagonist) into the RVLM 10 min post-injection of ethanol; KT 5720 or cAMPS-Rp alone at doses tested had no significant effects on NMDA-induced responses. Post-treatment with cAMPS-Sp (10 pmol, a cAMP activator) did not affect acute ethanol tolerance. Interestingly, administration of KT 5720 (0.4, 4 pmol) or cAMPS-Rp (2,10 pmol) into the RVLM 20 min before the injection of ethanol also reduced the inhibitory effects of ethanol on NMDA-induced pressor effects in a dose-dependent manner. Our results provide the first in vivo evidence that PKA signaling pathways participate in acute tolerance to ethanol inhibition of NMDA receptor function. Furthermore, PKA-mediated signaling pathways may also be involved in the interaction between ethanol and NMDA receptors.

T. annulata, an intracellular pathogenic parasite of the Aplicomplexa protozoan family infects bovine B-lymphocytes and macrophages. Parasitized cells that become transformed survive and proliferate independently of exogenous growth factors. In the present study, we used the isogenic non parasitized BL3 and parasitized TBL3 B cell lines, as a model to evaluate the contribution of two-major PI3-K- and PKA-dependent anti-apoptotic pathways in the survival of T. annulata parasitized B lymphocytes. We found that T. annulata increases PKA activity, induces over-expression of the catalytic subunit and down-regulates the pro-survival phosphorylation state of Akt/PKB. Consistent with a role of PKA activation in survival, two pharmacological inhibitors H89 and KT5720 ablate PKA-dependent survival of parasitized cells. To specifically inhibit PKA pro-survival pathways we linked the DPTsh1 peptide shuttle sequence to PKI(5-24) and we generated DPT-PKI, a cell permeable PKI. DPT-PKI specifically inhibited PKA activity in bovine cell extracts and, as expected, also inhibited the PKA-dependent survival of T. annulata parasitized TBL3 cells. Thus, parasite-dependent constitutive activation of PKA in TBL3 cells generates an anti-apoptotic pathway that can protect T. annulata-infected B cells from apoptosis. These results also indicate that DPT-PKI could be a powerful tool to inhibit PKA pathways in other cell types.

Resveratrol (3,4′,5-trihydroxy-trans-stilbene), a naturally occurring polyphenolic compound found abundantly in grape skins and red wines, has been found to pharmacologically precondition the heart against ischemia reperfusion injury through the potentiation of a survival signal involving cAMP response element-binding protein-dependent phosphatidylinositol 3-kinase-Akt-BclII pathway. The present study was designed to determine whether, similar to ischemic preconditioning, resveratrol uses mitogen-activated protein kinases (MAPKs) as upstream signaling targets. The isolated rat hearts were preperfused for 15 min with Krebs-Henseleit bicarbonate buffer in the absence (control) or presence of extracellular signal-regulated kinase (ERK) 1/2 inhibitor 2′-amino-3′-methoxyflavone (PD98059), p38 MAPK inhibitor 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1 H -imidazole (SB-202190), mitogen- and stress-activated protein kinase 1 (MSK-1) inhibitor N -[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89), protein kinase A inhibitor (9 S ,10 S ,12 R )-2,3,9,10,11,12-hexahydro-10hydroxy-9-methyl-1-oxo-9,12-epoxy-1 H -diindolo[1,2,3 fg : 3′,2′,1′- kl ]-pyrrolo[3,4- i ][1,6]benzodiazocine-10-carboxylic acid hexyl ester (KT5720), resveratrol only, resveratrol plus PD98059, resveratrol plus SB-202190, resveratrol plus H89, or resveratrol plus KT5720. Consistent with previous reports, resveratrol provided cardioprotection as evidenced by its ability to improve postischemic ventricular function, reduction of myocardial infarct size, and cardiomyocyte apoptosis. The cardioprotection afforded by resveratrol was partially abolished with PD98059 or SB-202190, suggesting that ERK1/2 and p38 MAPK play roles in resveratrol-mediated preconditioning. An MSK-1 inhibitor, H89, abolished resveratrol-mediated preconditioning, indicating MSK-1 to be the downstream target molecule for both ERK1/2 and p38 MAPK. KT5720 had no effect on resveratrol-mediated cardioprotection. Corroborating these results, Western blot analysis revealed phosphorylation of ERK1/2, p38 MAPK, MAPK-activated protein (MAPKAP) kinase 2, and MSK-1 with resveratrol and inhibition of phosphorylation with corresponding inhibitors. These results showed for the first time that resveratrol triggers an MAPK signaling pathway involving ERK1/2 and p38 MAPK, the former using MSK-1 as the downstream target and the latter, using both MAPKAP kinase 2 and MSK-1 as downstream targets.

Summary Mycobacterium bovis bacillus Calmette–Guerin (BCG)-induced tumor necrosis factor (TNF)-α secretion via an extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase-dependent mechanism is an important host defence mechanism against Mycobacterium tuberculosis in human monocytes. We now define distinct signaling pathways that regulate induction of TNF-α and activation of ERK1/2 by intracellular signaling mechanisms during M. bovis infection. We determined that M. bovis BCG-induced ERK 1/2 activation occurs through a mechanism that requires intracellular calcium and likely involves a calmodulin-sensitive step. In contrast, M. bovis BCG can induce p38 mapk activation by a calcium (Ca 2+ )/calmodulin-independent mechanism. Interestingly, we present evidence that M. bovis BCG activates protein kinase A (PKA), since pretreatment of monocytes with H-89, a inhibitor of PKA activity, blocked the ability of M. bovis BCG to induce ERK1/2 activation. These results were further supported by the fact that treatment of cells with KT5720, another well-described inhibitor of PKA activity, significantly diminished the effect of M. bovis BCG on ERK1/2 activation. Furthermore, secretion of TNF-α in M. bovis -infected human monocytes was also dependent on Ca 2+ /calmodulin, and PKA pathways. Finally, addition of H-89 significantly diminished TNF-α mRNA expression in M. bovis -infected human monocytes. These results indicate that the Ca 2+ /calmodulin, and PKA pathways play important regulatory roles in monocyte signaling upon M. bovis infection.

The molecular events that regulate phagocytosis, an important innate immune response, in invertebrate defence cells (haemocytes) are poorly understood. Lymnaea stagnalis haemocytes were used as a model to elucidate the role of cell signalling pathways in phagocytosis by molluscan defence cells. The phosphatidylinositol 3-kinase (PI3-K) inhibitor, LY294002, significantly impaired haemocyte phagocytic activity in a dose-responsive manner with 10 microM LY294002 reducing internalization of fluorescent-conjugated Escherichia coli by 62% (P < or = 0.001). In contrast, the protein kinase A (PKA) inhibitor KT5720 was without effect. Therefore, PI3-K, but not PKA, appears to control phagocytosis by haemocytes in these gastropod molluscs.

Osteoblasts, normally derived from undifferentiated mesenchymal precursor cells, acquire their characteristic phenotypes when induced by various regulatory factors, one of which is bone morphogenetic protein-2 (BMP-2). Our recent studies suggest that expression of cAMP-dependent protein kinase (PKA) inhibitor G (PKIG) is down-regulated as human mesenchymal stromal cells (MSCs) undergo BMP-2-induced osteoblastic differentiation. This raises our hypothesis that the PKA pathway is involved in osteogenesis. In this report, we demonstrated that PKIG in human MSCs and its murine homologue PKA inhibitor gamma (PKIgamma) in murine pre-myoblast C2C12 cells were down-regulated when these cells were treated with BMP-2. On the contrary, the PKA activity of C2C12 cells was increased upon BMP-2 treatment. Overexpression of PKIgamma in C2C12 cells was shown to repress mRNA expression of early osteoblastic markers osterix and type I collagen while inhibiting the PKA activity. This correlated with decreased alkaline phosphatase (ALP) activities. Furthermore, inhibition of the PKA activity using its specific inhibitor KT5720 was found to have the similar effect, whereas 8-Br-cAMP, a specific PKA activator, accelerated BMP-2-induced ALP activities. Finally, this study showed that BMP-2 treatment promoted activities of transcription regulatory elements including cAMP response element (CRE) and activating protein-1 (AP1). This effect of BMP-2 was diminished in PKIgamma-overexpressed C2C12 cells. Taken together, our results indicate that the activation of the PKA pathway may be one of key BMP-2-activated signaling events that lead to osteogenesis and that downregulation of PKIgamma may be prerequisite for the PKA activation during the osteoblastic differentiation of precursor cells.

The highly dynamic nature of voltage-gated Na+ channel (VGSC) expression and its controlling mechanism(s) are not well understood. In this study, we investigated the possible involvement of nerve growth factor (NGF) in regulating VGSC activity in the strongly metastatic Mat-LyLu cell model of rat prostate cancer (PCa). NGF increased peak VGSC current density in a time- and dose-dependent manner. NGF also shifted voltage to peak and the half-activation voltage to more positive potentials, and produced currents with faster kinetics of activation; sensitivity to the VGSC blocker tetrodotoxin (TTX) was not affected. The NGF-induced increase in peak VGSC current density was suppressed by both the pan-trk antagonist K252a, and the protein kinase A (PKA) inhibitor KT5720. NGF did not affect the Nav1.7 mRNA level, but the total VGSC alpha-subunit protein level was upregulated. NGF potentiated the cells' migration in Transwell assays, and this was not affected by TTX. We concluded that NGF upregulated functional VGSC expression in Mat-LyLu cells, with PKA as a signaling intermediate, but enhancement of migration by NGF was independent of VGSC activity.

Disturbances in Galpha(s-L) levels and function have been implicated in the pathophysiology of bipolar disorder, but the role of these changes in the development of the illness is not clear. In view of the critical role of Galpha(s)-mediated cAMP signaling in regulating cell survival, we investigated the potential role of Galpha(s-L) in modulating susceptibility to cellular stressors in human SH-SY5Y neuroblastoma cells. Overexpression of Galpha(s-L) to a level twice that of the vector-transfected cells did not directly affect cell viability but significantly increased the sensitivity to induction of cell death by serum deprivation and other apoptotic stimuli, including staurosporine, H(2)O(2), and tunicamycin. This enhanced sensitivity was associated with increased caspase-3 activation and appearance of fragmented nuclei (Hoechst 33342 staining). The broad-spectrum caspase inhibitor z-VAD-fmk completely suppressed cell death evoked by these apoptotic insults in both vector-transfected and Galpha(s-L)-overexpressing cells. The increased vulnerability conferred by increased Galpha(s-L) expression was neither mimicked by cAMP analogs 8-Br-cAMP, 8-CPT-cAMP, and 8-CPT-2Me-cAMP nor attenuated by PKA inhibitors Rp-cAMPS and KT5720. These data indicate that Galpha(s-L) may modulate apoptotic processes in a caspase-dependent manner through a signaling cascade that is independent of the cAMP/PKA or cAMP/Epac pathway. These results suggest that enhanced Galpha(s-L) expression, as was observed in post-mortem brain of bipolar patients, may impair cellular resilience in response to intracellular stress signals resulting from mitochondrial and/or endoplasmic reticulum dysfunction implicated in this disorder.

The molecular mechanisms underlying long-term potentiation (LTP) in the CA1 region of the hippocampus are known to vary with developmental age. The physiological factors regulating this developmental change, however, have not yet been elucidated. Here we show that mild neonatal isolation accelerates the developmental switch in the signalling cascades for hippocampal CA1 LTP induction from a cyclic AMP-dependent protein kinase (PKA)- to a Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent pattern via the activation of the corticotrophin-releasing factor (CRF) system. Furthermore, this action appears to be mediated through an increased transcription of the α isoform of the CaMKII (CaMKIIα) gene. We also demonstrate that application of CRF to cultured hippocampal neurones significantly increases the expression of CaMKIIα, which is blocked by the non-specific CRF receptor antagonist astressin, the specific CRF receptor 1 antagonist NBI 27911, and the PKA inhibitor KT5720, but not by the CRF receptor 2 antagonist K 41498, or the protein kinase C inhibitor, bisindolylmaleimide I. CRF signalling also mediates the normal maturation of LTP. These results suggest a novel role for CRF in regulating early developmental events in the hippocampus, and indicate that, although maternal deprivation is stressful for the neonate, appropriate neonatal isolation can serve to promote an endocrine state that fosters the rate of maturation of the signalling cascades underlying the induction of LTP in the developing hippocampus.

There is an inverse relationship between renin and atrial natriuretic peptide (ANP) levels in the plasma. Since both the ANP and renin-angiotensin system (RAS) are upregulated in development and cardiac hypertrophy, we tested whether ANP differentially regulates RAS in cardiac cells. Cardiac fibroblasts isolated from neonatal rats were treated with ANP(1-28), a biologically active fragment of ANP. Renin and angiotensinogen (Ao) mRNA levels were measured by quantitative multiplex RT-PCR and protein levels determined by Western blot analysis. ANP(1-28) increased renin and Ao mRNA levels (737+/-131% and 178+/-51.3%) with EC50 values of 4.12+/-0.3 and 8.67+/-0.22 nmol/L, respectively. At the protein level, secretion of renin and Ao was significantly enhanced resulting in approximately 4-fold increase in ANG II level in the medium. The effect of ANP(1-28) on renin and Ao mRNA expression were reproduced by 8-bromo-cyclic GMP. Inhibition of protein kinase G (PKG) with KT5823 blunted ANP(1-28)-induced upregulation of renin, but not Ao mRNA, while inhibition of protein kinase A (PKA) with KT5720 attenuated the upregulation of both renin and Ao mRNA. These findings suggest that unlike in plasma, ANP positively regulates the RAS in cardiac fibroblasts, which may have a significant role in development of the fetal heart.

Exposure of fully grown fish and amphibian oocytes to a maturation-inducing steroid (MIS) activates numerous signal transduction pathways to initiate the final stage of oocyte maturation. These events culminate in the activation of maturation-promoting factor and germinal vesicle breakdown (GVBD). In most species, exposure to MIS causes a transient decrease in oocyte cAMP levels. Whether this reduction in oocyte cAMP concentration is sufficient to induce GVBD is unclear. The current study tested the hypothesis that activation of cAMP-independent signal transduction pathways by the naturally occurring MIS, 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S), is necessary for GVBD in Atlantic croaker (Micropogonias undulatus) oocytes. Results indicate that although 20beta-S treatment of oocyte membranes significantly reduced cAMP production, incubation of follicles with the cell-permeable cAMP-dependent protein kinase (Prka) inhibitors Rp-cAMP or KT5720 did not promote GVBD in the absence of 20beta-S. Additionally, treatment of follicles with the phosphodiesterase (Pde) inhibitors Cilostamide (Pde3) or Rolipram (Pde4) significantly reduced GVBD, but they were not able to completely block it. In contrast, pharmacologic inhibition of the cAMP-independent phosphatidylinositol 3-kinase (Pik3)/Akt signal transduction pathway using the Pik3 inhibitors Wortmannin or LY294002, or the Akt inhibitor ML-9, blocked 20beta-S-induced GVBD. Finally, mitogen-activated protein kinase (Mapk1/3) activity increased after treatment with 20beta-S; however, inhibition of Mapk1/3 activity using PD98059 or U0126 had no effect on GVBD. These results demonstrate that activation of cAMP-independent signaling pathways, especially the Pik3/Akt pathway, is necessary for 20beta-S-induced GVBD in Atlantic croaker oocytes.

Increased cAMP improves neuronal survival and axon regeneration in mammals. Here, we assess cAMP levels and identify activated pathways in a spontaneously regenerating central nervous system. Following optic nerve crush in goldfish, almost all retinal ganglion cells (RGC) survive and regenerate retinotectal topography. Goldfish received injections of a cAMP analogue (CPT-cAMP), a protein kinase A (PKA) inhibitor (KT5720), both compounds combined, or PBS (control). RGC survival in experimental groups was unaffected at any stage. The rate of axon regeneration was accelerated by the activator and decelerated both by the inhibitor and by combined injections, suggesting a PKA-dependent pathway. In addition, errors in regenerate retinotectal topography were observed when agents were applied in vivo and RGC response to the guidance cue ephrin-A5 in vitro was altered by the inhibitor. Our results highlight that therapeutic manipulation of cAMP levels to enhance axonal regeneration in mammals must ensure that topography, and consequently function, is not disrupted.

We previously showed that oestrogen confers cardioprotection by downregulating the cardiac beta1-adrenoceptor (beta1-AR). The present study examined the effect of oestrogen on the post beta1-AR signalling cascade, with particular emphasis on the activity of protein kinase A (PKA) and its influence on the L-type Ca2+ channel. Three groups of adult female Sprague-Dawley rats were used: sham-operated controls, bilaterally ovariectomized (Ovx) rats, and Ovx rats with oestrogen replacement (Ovx + E2), which restored the oestrogen concentration to normal. The electrically induced intracellular Ca2+ transient (E[Ca2+]i), 45Ca(2+)-uptake through cardiac L-type Ca2+ channels (Ca2+ channels), heart rate and force of contraction in response to beta-AR stimulation with 10 nM isoprenaline (Iso) in hearts from Ovx rats were significantly greater than those of control and Ovx + E2 rats. The basal and Iso-induced PKA activities were also higher in hearts from Ovx rats. KT5720, a selective PKA inhibitor, completely inhibited its potentiating effect on basal Ca2+ channel activity in the Ovx rat heart. On the other hand, expression of G proteins (G(alpha)s and G(alpha)i1-3)), basal and forskolin-stimulated cAMP accumulation, and responsiveness of PKA to cAMP, were not altered by Ovx. Interestingly, the PKA inhibitor at the same concentration significantly reduced the increases in PKA activity and Ca2+ channel activity upon beta-AR stimulation in all three groups of rats and the inhibitions were significantly greater in the Ovx rat than in the other two groups of rats. This study provides the first evidence that, in addition to downregulation of beta1-AR shown previously, suppression of PKA activity, which is partly responsible for the suppressed Ca2+ channel activity, also determines the E[Ca2+]i and cardiac contractility following beta-AR stimulation in the female rat.

Amphotropic murine leukemia virus (A-MuLV) utilizes the PiT2 sodium-dependent phosphate transporter as its cell surface receptor to infect mammalian cells. The process of A-MuLV infection requires cleavage of the R peptide from the envelope protein. This occurs within virions thereby rendering them competent to fuse with target cells. Envelope proteins lacking the inhibitory R peptide (e.g. envelope (R-) proteins) induce viral envelope-mediated cell-cell fusion (syncytium). Here we have performed studies to determine if cell signaling through protein kinases is involved in the regulation of PiT2-mediated A-MuLV envelope (R-)-induced syncytium formation. Truncated A-MuLV retroviral envelope protein lacking the inhibitory R peptide (R-) was used to induce viral envelope-mediated cell-cell fusion. Signaling through cyclic AMP to activate PKA was found to inhibit envelope-induced cell-cell fusion, whereas treatment of cells with PKA inhibitors H89, KT5720, and PKA Catalpha siRNA all enhanced this cell fusion process. It was noted that activation of PKC, as well as overexpression of PKCepsilon, up-regulated A-MuLV envelope protein-induced cell-cell fusion, whereas exposure to PKC inhibitors and expression of a kinase-inactive dominant-negative mutant of PKCepsilon (K437R) inhibited syncytium formation. v-ras transformed NIH3T3 cells were highly susceptible to A-MuLV envelope-induced cell-cell fusion, whereas expression of a dominant-negative mutant of Ras (N17Ras) inhibited this cell fusion process. Importantly, activation of Raf-1 protein kinase also is required for A-MuLV envelope-induced syncytium formation. Expression of constitutively active BXB Raf supported, whereas expression of a dominant-negative mutant of Raf-1 (Raf301) blocked, A-MuLV-induced cell-cell fusion. These results indicate that specific cell signaling components are involved in regulating PiT2-mediated A-MuLV-induced cell-cell fusion. Selective pharmacological modulation of these signaling components may be an effective means of altering cell susceptibility to viral-mediated cytopathic effects.

Objective— Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs). CYP2C9-derived EETs elicit endothelial cell proliferation and angiogenesis, but the signaling pathways involved are incompletely understood. Because cyclooxygenase-2 (COX-2) is involved in angiogenesis, we determined whether a link exists between CYP2C9 and COX-2 expression. Methods and Results— Human umbilical vein endothelial cells were infected with CYP2C9 sense or antisense adenoviral constructs. Overexpression of CYP2C9 increased COX-2 promoter activity, an effect accompanied by a significant increase in COX-2 protein expression and elevated prostacyclin production. The CYP2C9-induced expression of COX-2 was inhibited by the CYP2C9 inhibitor, sulfaphenazole, whereas 11,12-EET increased COX-2 expression. Overexpression of CYP2C9 and stimulation with 11,12-EET increased intracellular cAMP levels and stimulated DNA-binding of the cAMP-response element-binding protein. The protein kinase A inhibitor, KT5720, attenuated the CYP2C9-induced increase in COX-2 promoter activity and protein expression. Overexpression of CYP2C9 stimulated endothelial tube formation, an effect that was attenuated by the COX-2 inhibitor celecoxib. Identical responses were observed in cells preconditioned by cyclic strain to increase CYP2C expression. Conclusion— These data indicate that CYP2C9-derived EETs induce the expression of COX-2 in endothelial cells via a cAMP-dependent pathway and that this mechanism contributes to CYP2C9-induced angiogenesis.

Previously we have described the properties of store-operated channel currents (SOCs) in freshly dispersed rabbit portal vein smooth muscle cells. In addition to Ca2+ store depletion these SOCs could also be activated by α-adrenoceptor stimulation and diacylglycerol (DAG) via a protein kinase C (PKC)-dependent mechanism. In the present study we have investigated the effect of β-adrenoceptor stimulation on SOCs in rabbit portal vein myocytes. With whole-cell recording the selective β-adrenoceptor agonist isoprenaline reduced the current evoked by cyclopiazonic acid (CPA, sarcoplasmic/endoplasmic reticulum ATPase inhibitor) by over 85%. With cell-attached patch recording, bath application of isoprenaline produced a pronounced inhibition of SOC activity evoked by either CPA or the acetoxymethyl ester form of BAPTA (BAPTA-AM). SOC activity evoked by CPA, the DAG analogue, 1-oleoyl-acetyl-sn-glycerol (OAG) or the phorbol ester, phorbol-12,13-dibutyrate (PDBu) was also markedly inhibited by the adenylate cyclase activator, forskolin, and the cell-permeable non-hydrolysable analogue of cyclic adenosine monophosphate (cAMP), 8-Br-cAMP. With inside-out patches, bath application of PDBu evoked channel currents with similar properties to SOCs which were inhibited by over 90% by a catalytic subunit of protein kinase A (PKA) and by 8-Br-cAMP. Moreover bath application of PKA inhibitors, H-89, KT5720 and an inhibitory peptide to quiescent cell-attached or inside-out patches, activated channel currents with similar properties to SOCs. These data suggest that in rabbit portal vein myocytes, stimulation of β-adrenoceptors inhibits SOC activity via a cAMP-dependent protein kinase signal transduction cascade. In addition it is concluded that constitutive PKA activity has a profound inhibitory effect on SOC activity in this vascular preparation.

The present study was conducted to explore the influence of rolipram, a specific inhibitor of the phosphodiesterase type 4 (PDE4) isoform, on glutamate release in the rat prefrontal cortex, using isolated nerve terminal (synaptosome) preparation. In prefrontocortical nerve terminals, rolipram potentiated the Ca2+-dependent release of glutamate evoked by 4-aminopyridine (4AP) in a concentration-dependent manner. This potentiation of release was occluded by the activation of PKA by Sp-cAMP or β-adrenergic receptor agonist and prevented by the inhibition of PKA by Rp-cAMP or KT5720, indicating a PKA-mediated mechanism. The rolipram-mediated potentiation of glutamate release is associated with an increase both in the 4AP-evoked depolarization of the synaptosomal plasma membrane potential and in 4AP-evoked Ca2+ influx into synaptosomes. Moreover, Ca2+ ionophore ionomycin-induced glutamate release was also facilitated by rolipram. These results concluded that phosphodiesterase 4 inhibited by rolipram produces an increase in PKA activation, which subsequently enhances the voltage-dependent Ca2+ influx by increasing terminal excitability as well as the vesicular release machinery to cause an increase in evoked glutamate release from rat prefrontocortical nerve terminals. Synapse 59:41–50, 2006. © 2005 Wiley-Liss, Inc.

Brain-derived neurotrophic factor (BDNF) regulates neuronal survival, neurite outgrowth, and excitatory synaptic transmission. We reported recently that acute BDNF exposure decreased gamma-aminobutyric acid (GABA) responses in cultured mouse cerebellar granule cells through tyrosine receptor kinase B (TrkB) receptor-mediated signaling. In the present study, we extend this work to investigate BDNF-induced modulation of GABA responses and GABA(A) receptor-mediated synaptic events in cerebellar slices. Thin (200 microm) parasagittal slices of cerebellum were prepared from postnatal Day 7 and 14 mice. Purkinje cells and granule cells, both of which express TrkB-like immunoreactivity, were identified for whole-cell recording. BDNF promptly enhanced GABA responses in Purkinje cells but, consistent with our previous finding in culture, attenuated those recorded in granule cells. In Purkinje cells, BDNF exposure shifted rightward the cumulative peak amplitude distribution for miniature inhibitory postsynaptic currents (mIPSCs) without changing the mIPSC frequency. BDNF-induced potentiation of Purkinje cell responses to GABA was blocked by TrkB-Fc (receptor body that sequesters BDNF), K252a (inhibitor of TrkB receptor autophosphorylation), U73122 (inhibitor of phospholipase-Cgamma [PLCgamma]), KN62 (specific inhibitor of calcium/calmodulin-dependent kinase), KT5720 (specific cyclic AMP-dependent kinase inhibitor), and by intracellular dialysis of Rp-cyclic AMP or BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N, N',N'-tetraacetic acid). Overall, our results indicate that BDNF acutely potentiates GABA(A) receptor function in cerebellar Purkinje cells via the TrkB receptor-PLCgamma signal transduction cascade. In addition, we propose that cyclic AMP-mediated intracellular signaling mechanisms may facilitate manifestation of the BDNF-induced modulatory outcome.

Progesterone (P4) inhibits granulosa cell and spontaneously immortalized granulosa cell (SIGC) apoptosis by regulating membrane-initiated events. However, the nature of the signal transduction pathway that is induced by these membrane-initiated events has not been defined. To gain insights into the P4-regulated signal transduction pathway, mouse granulosa cells and SIGCs were cultured with 8-br-cGMP and P4. In culture, 8-br-cGMP mimicked P4's antiapoptotic actions. Because cGMP activates protein kinase G (PKG), the effect of PKG antagonists on P4-regulated SIGC viability was assessed. P4's antiapoptotic action was attenuated by the PKG inhibitors, Rp-8-pCPT-cGMP, KT5823, the PKG-1α-specific inhibitor, DT-3, and a dominant negative PKG-1α. Further, the type I isoform of PKG was shown to be expressed by SIGCs and activated by P4. P4's antiapoptotic action was not affected by the PKA inhibitor, KT5720. Collectively, these findings indicate that P4 maintains SIGC viability by activating PKG-1α. PKG-1...

In this study, we investigated adrenergic and photoneural regulation of p38MAPK phosphorylation in the rat pineal gland. Norepinephrine (NE), the endogenous neurotransmitter, dose-dependently increased the levels of phosphorylated MAPK kinase 3/6 (MKK3/6) and p38MAPK in rat pinealocytes. Time-course studies showed a gradual increase in MKK3/6 and p38MAPK phosphorylation that peaked between 1 and 2 h and persisted for 4 h post NE stimulation. In cells treated with NE for 2 and 4 h, the inclusion of prazosin or propranolol reduced NE-induced MKK3/6 and p38MAPK phosphorylation, indicating involvement of both α- and β-adrenergic receptors for the sustained response. Whereas treatment with dibutyryl cAMP or ionomycin mimicked the NE-induced MKK3/6 and p38MAPK phosphorylation, neither dibutyryl cGMP nor 4β-phorbol 12-myristate 13-acetate had an effect. The NE-induced increase in MKK3/6 and p38MAPK phosphorylation was blocked by KT5720 (a protein kinase A inhibitor) and KN93 (a Ca2+/calmodulin-dependent kinase inhibitor), but not by KT5823 (a protein kinase G inhibitor) or calphostin C (a protein kinase C inhibitor). In animals housed under a lighting regimen with 12 h of light, MKK3/6 and p38MAPK phosphorylation increased in the rat pineal gland at zeitgeber time 18. The nocturnal increase in p38MAPK phosphorylation was blocked by exposing the animal to constant light and reduced by treatment with propranolol, a β-adrenergic blocker. Together, our results indicate that activation of p38MAPK is under photoneural control in the rat pineal gland and that protein kinase A and intracellular Ca2+ signaling pathways are involved in NE regulation of p38MAPK.

Urocortin is a potent vasodilator, which plays physiological or pathophysiological roles in systemic circulation. However, little is known about its action on pulmonary circulation. The present study was aimed to characterize some cellular mechanisms underlying the relaxant effect of urocortin in isolated rat pulmonary arteries. Changes in isometric tension were measured on small vessel myographs. Urocortin inhibited U46619-induced contraction with reduction of the maximal response. Urocortin-induced relaxation was independent of the presence of endothelium. Inhibitors of nitric oxide (NO)-dependent dilator, NG-nitro- l -arginine methyl ester or 1H-[1,2,4]oxadizolo[4,3-a]quinoxalin-1-one, did not affect the relaxation. Astressin (100–500 nM), a corticotropin-releasing factor (CRF) receptor antagonist and KT5720, a protein kinase A (PKA) inhibitor reduced urocortin-induced relaxation. Urocortin produced less relaxant effect in 30 mM K+- than U46619-contracted arterial rings. Urocortin did not reduce CaCl2-induced contraction in 60 mM K+-containing solution. Ba2+ (100–500 μM) but not other K+ channel blockers reduced the relaxant responses to urocortin. Urocortin also relaxed the rings preconstricted by phorbol 12,13-diacetae in normal Krebs solution while this relaxation was less in a Ca2+-free solution. Our results show that urocortin relaxed rat pulmonary arteries via CRF receptor-mediated and PKA-dependent but endothelium/NO or voltage-gated Ca2+ channel-independent mechanisms. Stimulation of Ba2+-sensitive K+ channel may contribute to urocortin-induced relaxation. Finally, urocortin relaxed pulmonary arteries partly via inhibition of a PKC-dependent contractile mechanism.

cAMP significantly inhibits IL-1beta+IFNgamma-induced iNOS gene expression in hepatocytes, but the signaling pathways responsible for the effect are not known. PKA inhibitors, H89, PKI, and KT5720, had no effect on the recovery of the inhibitory effects of cAMP on cytokine-induced hepatocyte iNOS expression and activity. The JNK inhibitor, SP 600125, effectively reversed the inhibitory effects of cAMP on iNOS expression and significantly increased iNOS promoter activity. A cAMP analogue, dbcAMP, significantly induced JNK signaling and increased AP-1 binding activity in hepatocytes. The JNK activator, anisomycin, inhibited iNOS expression and transcription in hepatocytes as well as AP-1 binding activity; and SP600125 reversed this effect of anisomycin. Overexpression of c-Jun in hepatocytes inhibited IL-1beta+IFNgamma-induced nitrite accumulation and iNOS promoter activity while dominant negative c-Jun partially reversed the inhibitory effects of cAMP on nitrite accumulation. We conclude that JNK signaling plays an important role in the inhibitory effects of cAMP on IL-1beta+IFNgamma-induced iNOS gene expression in cultured hepatocytes.

Prostaglandin E2 (PGE2), an abundant eicosanoid in bone, has been implicated in a number of pathological states associated with bone loss and is also known to stimulate matrix metalloproteinase-1 synthesis and secretion in rat and human osteoblast cells, although the intracellular reactions responsible for this remain unclear. Interleukin-1beta (IL-1beta) is a cytokine that plays a critical role in bone remodeling and appears to act as a downstream effector of most bone-resorbing agents. However, the issue of whether PGE2 regulates the expression of IL-1beta in mouse osteoblasts has not been resolved. In this work, we demonstrate that PGE2 is a potent inducer of IL-1beta production by fetal osteoblasts and show that PGE2 stimulates the activity of the IL-1beta promoter in osteoblasts, suggesting that PGE2 controls IL-1beta gene expression at least at the transcriptional level. PGE2 was found to induce IL-1beta mRNA expression in the cells within 4 h and the level of expression was maintained for 36 h. A dose-related increase in IL-1beta production was found with 0.1-2.0 microM PGE2. The induction of IL-1beta protein in the medium paralleled the induction of IL-1beta mRNA levels. The role of cAMP activation in PGE2-mediated IL-1beta production was examined by the effects of forskolin, an adenylyl cyclase (AC) activator and dideoxyadenosine (DDA), an AC inhibitor. Forskolin enhanced and DDA blocked the production of IL-1beta by PGE2. In addition, PGE2-mediated IL-1beta induction was completely inhibited by the cAMP antagonist, Rp-cAMP, and protein kinase A (PKA) inhibitors of KT5720 and H89. The PGE2-induced production of IL-1beta was also blocked by the PKA inhibitor PKI14-22. However, a specific inhibitor of protein kinase C, calphostin C, had no affect on PGE2-induced IL-1beta gene expression. Among the potential agonists, forskolin was a potent inducer of IL-1beta expression, while phorbol myristate acetate and serum had little effect. These findings indicate that PGE2 involves the cAMP-PKA signaling pathway in regulating IL-1beta gene expression in osteoblasts.

Agents that elevate cellular cAMP are known to inhibit the activation of phospholipase D (PLD). We investigated whether PLD can be phosphorylated by cAMP-dependent protein kinase (PKA) and PKA-mediated phosphorylation affects the interaction between PLD and RhoA, a membrane regulator of PLD. PLD1, but not PLD2 was found to be phosphorylated in vivo by the treatment of dibutyryl cAMP (dbcAMP) and in vitro by PKA. PKA inhibitor (KT5720) abolished the dbcAMP-induced phosphorylation of PLD1, but dibutyryl cGMP (dbcGMP) failed to phosphorylate PLD1. The association between PLD1 and Val14RhoA in an immunoprecipitation assay was abolished by both dbcAMP and dbcGMP. Moreover, RhoA but not PLD1 was dissociated from the membrane to the cytosolic fraction in dbcAMP-treated cells. These results suggest that both PLD1 and RhoA are phosphorylated by PKA and the interaction between PLD1 and RhoA is inhibited by the phosphorylation of RhoA rather than by the phosphorylation of PLD1.

The firing activity of suprachiasmatic nuclei (SCN) neurones in vitro shows a circadian rhythm with the peak in average firing frequency during the middle of the projected day. Vasoactive intestinal polypeptide (VIP), which is abundant in the rat SCN, phase-advances the time of peak in SCN neuronal activity when applied in vitro during the late projected night; an effect most likely mediated via the VPAC(2) receptor. Employing extracellular single-unit electrophysiology, we investigated the influence of protein kinase A (PKA) inhibitor KT5720 or mitogen-activated protein kinase kinase (MAPKK) inhibitor U0126 on phase advances evoked during the late projected night by VIP or Ro 25-1553, a VPAC(2) receptor agonist. Both inhibitors blocked VIP or Ro 25-1553-induced phase advances, indicating that the late-night resetting actions of VIP via the VPAC(2) receptor require PKA and MEK.

Using pharmacological means we investigated the functional interactions between the estrogen receptor (ER)-, protein kinase A (PKA)-, and mitogen-activating protein kinase (MAPK)-mediated pathways in the regulation of lactotroph proliferation in primary culture. Treatment of lactotrophs for 28 h with the PKA inhibitor H89 or KT5720, an effective inhibitor of forskolin-induced proliferation, inhibited both insulin- and estradiol-induced proliferation. Inhibition of the MAPK activity by PD98059 or U0126 suppressed not only insulin-induced proliferation but also forskolin- and estradiol-induced proliferation. However, treatment for 28 h with the antiestrogens 4-hydroxy tamoxifen and ICI182780 failed to antagonize estradiol-induced lactotroph proliferation but instead enhanced it. Prolonging the antiestrogen treatment time from 28 to 88 h was effective in antagonizing estradiol-induced proliferation with this long-term treatment also inhibiting insulin- and forskolin-induced proliferation. There was no decrease in these mitogen-induced proliferations following treatment with a progesterone antagonist or protein kinase C inhibitor. These results suggest that cross-talk occurs between the ER-, PKA-, and MAPK-mediated signaling pathways in the regulation of lactotroph proliferation, and that antiestrogens stimulate and inhibit estradiol-induced proliferation in a time-dependent manner.

The roles of 3',5'-cyclic adenosine monophosphate (cAMP) and protein kinase A in 5-hydroxytryptamine (5-HT)7 receptor-mediated activation of extracellular-regulated kinase (ERK) were studied in cultured hippocampal neurons and transfected PC12 cells. Activation of ERK by neuronal Gs-coupled receptors has been thought to proceed through a protein kinase A-dependent pathway. In fact we identified coupling of 5-HT7 receptors to activation of adenylyl cyclase and protein kinase A. However, no inhibition of agonist-stimulated ERK activation was found when cells were treated with H-89 and KT5720 at concentrations sufficient to completely inhibit activation of protein kinase A. However, activation of ERK was found to be sensitive to the adenylyl cyclase inhibitor 9-(tetrahydrofuryl)-adenine, suggesting a possible role for a cAMP-guanine nucleotide exchange factor (cAMP-GEF). Co-treatment of cells with 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate, a direct activator of the cAMP-GEFs Epac1 and 2, reversed the inhibition of agonist-stimulated ERK activation induced by adenylyl cyclase inhibition. Additionally, over-expression of Epac1 enhanced 5-HT7 receptor-mediated activation of ERK. These results demonstrate that the activation of ERK mediated by neuronal Gs-coupled receptors can proceed through cAMP-dependent pathways that utilize cAMP-GEFs rather than protein kinase A.

Ammonia-induced apoptosis and its prevention by GABAC receptor stimulation were examined using primary cultured rat hippocampal neurons. Ammonia (0.5-5 mm NH4Cl) dose-dependently induced apoptosis in pyramidal cell-like neurons as assayed by double staining with Hoechst 33258 and anti-neurofilament antibody. A GABAC receptor agonist, cis-4-aminocrotonic acid (CACA, 200 microm), but not GABAA and GABAB receptor agonists, muscimol (10 micro m) and baclofen (50 microm), respectively, inhibited the ammonia (2 mm)-induced apoptosis, and this inhibition was abolished by a GABAC receptor antagonist (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA, 15 microm). Expression of all three GABAC receptor subunits was demonstrated in the cultured neurons by RT-PCR. The ammonia-treatment also activated caspases-3 and -9 as observed in immunocytochemistry for PARP p85 and western blot. Such activation of the caspases was again inhibited by CACA in a TPMPA-sensitive manner. The anti-apoptotic effect of CACA was blocked by inhibitors for MAP kinase kinase and cAMP-dependent protein kinase, PD98059 (20 microm) and KT5720 (1 microm), suggesting possible involvement of an upstream pro-apoptotic protein, BAD. Levels of phospho-BAD (Ser112 and Ser155) were decreased by the ammonia-treatment and restored by coadministration of CACA. These findings suggest that GABAC receptor stimulation protects hippocampal pyramidal neurons from ammonia-induced apoptosis by restoring Ser112- and Ser155-phospho-BAD levels.

The vasodilator-stimulated phosphoprotein (VASP) plays an important role in cGMP-induced platelet inhibition. Since VASP is an in vitro substrate for cGMP-dependent protein kinase (PKG), it has been presumed that VASP phosphorylation induced by cGMP is mediated by PKG. Here we show that, in human platelets, phosphorylation of VASP at Ser239 induced by either cGMP analogs or nitric oxide (NO) donor glyco-SNAP1 is inhibited by PKA inhibitors KT5720, PKI, Rp-Br-cAMPS, and H89, but not by PKG inhibitors KT5823 or Rp-pCPT-cGMPS. Unlike human platelets, cGMP analog–induced phosphorylation of VASP in mouse platelets is inhibited by both PKG and PKA inhibitors. Ineffectiveness of PKG inhibitors in inhibiting VASP phosphorylation in human platelets is not due to an inability to inhibit PKG, as these PKG inhibitors but not PKA inhibitors inhibit a different cGMP-induced intracellular signaling event: phosphorylation of extracellular signal–responsive kinase. Furthermore, PKA inhibitors reverse cGMP-induced inhibition of thrombin-induced platelet aggregation, whereas PKG inhibitors further enhance the inhibitory effect of cGMP analogs. Thus, PKA plays a predominant role in the cGMP-induced phosphorylation of VASP and platelet inhibition in human platelets.

It has been suggested that the cannabinoid receptor type 1 (CB1), a G protein-coupled receptor, is internalized after agonist binding and activation of the second messenger pathways. It is proposed that phosphorylation enhances the down-regulation of the CB1 receptor, thus contributing to tolerance. Alterations in phosphorylation of proteins in the signal transduction cascade after CB1receptor activation could also alter tolerance to cannabinoids. We addressed our hypothesis by evaluating the role of several kinases in antinociceptive tolerance to Delta(9)-tetrahydrocannabinol (THC). We evaluated cAMP-dependent protein kinase (PKA) using KT5720, a PKA inhibitor; protein kinase C (PKC) using bisindolylmaleimide I, HCl (bis), a PKC inhibitor; cGMP-dependent protein kinase (PKG) using KT5823, a PKG inhibitor; beta-adrenergic receptor kinase (beta-ARK) using low molecular weight heparin (LMWH), a beta-ARK inhibitor; and phosphatidylinositol-3 kinase (PI3-K) using 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), a PI3-K inhibitor and PP1, a Src family tyrosine kinase inhibitor. The cAMP analog used was dibutyryl-cAMP and the cGMP analog used was dibutyryl-cGMP. Our data indicate that selective kinases may be involved in cannabinoid tolerance. Mice and rats were rendered tolerant to Delta(9)-THC. The PKG inhibitor KT5823, the beta-ARK inhibitor LMWH, the PI3-K inhibitor LY294002, and inhibition of PKC by bis had no effect on tolerance. At a higher dose, bis attenuated the antinociceptive effect of delta(9)-THC in nontolerant mice. PP1, the Src family tyrosine kinase inhibitor, and KT5720, the PKA inhibitor, reversed THC-induced tolerance. In addition, inhibition of PKA reversed a decrease in dynorphin release shown to accompany THC tolerance in rats. These data support a role for PKA and Src tyrosine kinase in phosphorylation events in delta(9)-THC-tolerant mice.

Whereas the loss of ATP-sensitive K+ channel (KATP channel) activity in human pancreatic β-cells causes severe hypoglycemia in certain forms of hyperinsulinemic hypoglycemia, similar channel loss in sulfonylurea receptor-1 (SUR1) and Kir6.2 null mice yields a milder phenotype that is characterized by normoglycemia, unless the animals are stressed. While investigating potential compensatory mechanisms, we found that incretins, specifically glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), can increase the cAMP content of Sur1KO islets but do not potentiate glucose-stimulated insulin release. This impairment is secondary to a restriction in the ability of Sur1KO β-cells to sense cAMP correctly. Potentiation does not appear to require cAMP-activated protein kinase (PKA) because H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) and KT5720, inhibitors of PKA, do not affect stimulation by GLP-1, GIP, or exendin-4 in wild-type islets, although they block phosphorylation of cAMP-response element-binding protein. The impaired incretin response in Sur1KO islets is specific; the stimulation of insulin release by other modulators, including mastoparan and activators of protein kinase C, is conserved. The results suggest that the defect responsible for the loss of cAMP-induced potentiation of insulin secretion is PKA independent. We hypothesize that a reduced release of insulin in response to incretins may contribute to the unexpected normoglycemic phenotype of Sur1KO mice versus the pronounced hypoglycemia seen in neonates with loss of KATP channel activity.

1. In inflammatory kidney diseases procoagulatory activity (PCA) becomes evident. Glomerular fibrin deposits and capillary microthrombi are histopathological hallmarks in most forms of glomerulonephritis. 2. Therefore in this study the expression of tissue factor (TF) as the main inducer of thrombogenesis was examined in cultured human mesangial cells (MC) in response to proinflammatory stimuli such as interleukin-1 (IL-1 beta), tumour necrosis factor alpha (TNF-alpha) and lipopolysaccharide (LPS). Also main signalling pathways were investigated. 3. IL-1 beta, TNF-alpha and LPS induced TF in MC in a time and dose dependent manner on mRNA and protein levels. Highest activity was found after 12 h of stimulation. Induction of TF was completely blockable by BAPTA-AM, a chelator of intracellular [Ca(2+)](i) as well as calphostin, a protein kinase C (PKC) inhibitor. Activation of the protein kinase A (PKA) pathway had no influence on basal TF expression, but down-regulated cytokine-induced TF. The PKA blocker, KT5720, increased TF formation significantly. Since TF exerts its activity primarily on the surface of cells and after release of encrypted receptors we further tested TF activity in MC supernatants. IL-1 beta did not significantly increase TF activity in supernatants of intact cells. However, when MC were rendered apoptotic by oxidative metabolites, IL-1 beta treated MC released highly stimulated TF activity into the supernatants, suggesting that a paracrine activation of the coagulatory cascade can take place under such conditions. 4. Inflammatory mediators up-regulate TF expression in MC by a PKC dependent pathway whereas PKA can serve as a negative feed-back link. Apoptosis of inflammatory MC may trigger to spread PCA.

Embryonic astrocytes respond readily to serine/threonine kinase regulation in terms of cytoskeleton assembly, mitotic activity, and cell fate. We now present evidence that these responses include apoptosis. Staurosporine induced apoptosis in astrocyte cultures derived from chick embryo cerebral hemispheres, as assayed both by immunocytochemical detection of new 3-hydroxy DNA ends and production of 200-bp DNA fragment laddering. Staurosporine treatment also resulted in the prolonged (>24 h) activation of a 60-kDa serine/threonine protein kinase (PK60), increased ceramide formation (fourfold after 24 h), increased glutamine synthetase activity, and significant apoptosis (40%) after 24 h. PK60 was shown to be cytoskeleton associated and its activity, as measured by phosphorylation of myelin basic protein, was rapid, increased for up to 3 h, and was stable for at least 24 h. Other protein kinase C inhibitors, H8, sphingosine, calphostin C, or the protein kinase A inhibitor KT5720 did not induce either PK60 activation or apoptosis. The dose-dependent increase in [3H]palmitate labeling of ceramide and a specific decrease in labeling of its precursor sphingomyelin were not blocked by the biosynthetic inhibitor fumonisin beta1 but were increased (in a dose-dependent manner) by the coaddition of the ceramidase inhibitor oleoylethanolamine. Exogenous addition of C2-ceramide induced apoptosis but did not activate PK60. These results suggest that apoptosis in embryonic astrocytes involves pathways similar to those described in other cell types and that the activation of PK60 and formation of ceramide are early events in the pathway.

Urotensin-II (U-II), a peptide with multiple vascular effects, is detected in cholinergic neurons of the rat brainstem and spinal cord. Here, the effects of U-II on [Ca2+]i was examined in dissociated rat spinal cord neurons by fura 2 microfluorimetry. The neurons investigated were choline acetyltransferase-positive and had morphological features of motoneurons. U-II induced [Ca2+]i increases in these neurons with a threshold of 10−9 m, and a maximal effect at 10−6 m with an estimated EC50 of 6.2 × 10−9 m. The [Ca2+]i increase induced by U-II was mainly caused by Ca2+ influx from extracellular space, as the response was markedly attenuated in a Ca2+-free medium. Omega-conotoxin GVIA (10−7 m), a N-type Ca2+ channel blocker, largely inhibited these increases, whereas the P/Q Ca2+ channel blocker, omega-conotoxin GVIIC (10−7 m) and the l-type Ca2+ channel blocker, verapamil (10−5 m) had minimal effects. Down-regulation of protein kinase C by 4-α-phorbol 12-myristate 13-acetate (10−6 m) or enzyme inhibition using the specific inhibitor bisindolylmaleimide I (10−6 m) did not inhibit the observed effects. Similarly, inhibition of protein kinase G with KT5823 (10−6 m) or Rp-8-pCPT-cGMPS (3 × 10−5 m) did not modify U-II-induced [Ca2+]i increases. In contrast, protein kinase A inhibitors KT5720 (10−6 m) and Rp-cAMPS (3 × 10−5 m) reduced the response to 25 ± 3% and 42 ± 8%, respectively. Present results demonstrate that U-II modulates [Ca2+]i in rat spinal cord neurons via protein kinase A cascade.

Repair of tissues is a necessary step in restoring tissue function following injury consequent to inflammation. Many inflammatory mediators are capable of modulating not only the activity of "inflammatory cells" but also of modulating functions of parenchymal cells that may contribute to repair. Disordered repair is believed to contribute to tissue dysfunction in many inflammatory diseases, including bronchial asthma. The current study evaluated the ability of prostaglandin D(2) (PGD(2)) to modulate fibroblast repair using the in vitro contraction of three-dimensional native collagen gels as a model system. PGD(2) stimulated gel contraction in a concentration- and time-dependent manner. In contrast, the PGD(2) analog BW245C inhibited contraction. Both effects were blocked by a DP-receptor blocker (AH6809). Neither TP receptor blocker SQ29548 nor protein kinase (PK) A antagonist KT5720 hand an effect on PGD(2)-stimulated contraction, suggesting action through a novel prostaglandin D receptor. PKC inhibitor calphostin-C (10(-6) M) blocked the PGD(2) stimulation of gel contraction. A calcium-independent PKC-epsilon inhibitor (Ro31-8220), but not calcium-dependent PKC-alpha and -beta inhibitors, also blocked the PGD(2) effect on contraction, implying a role for a calcium-independent pathway. This study, therefore, supports a role for PGD(2) in tissue repair and remodeling. These effects of PGD(2) appear to be mediated through receptor-signal transduction pathways different from the cAMP-PKA pathways mediating the proinflammatory activity of PGD(2), creating the possibility for selective therapeutic manipulation.

MUC2 is a secretory mucin normally expressed by goblet cells of the intestinal epithelium. It is overexpressed in mucinous type colorectal cancers but down-regulated in colorectal adenocarcinoma. Phorbol 12-myristate 13-acetate (PMA) treatment of colon cancer cell lines increases MUC2 expression, so we have undertaken a detailed analysis of the effects of PMA on the promoter activity of the 5'-flanking region of the MUC2 gene using stably and transiently transfected promoter reporter vectors. Protein kinase C inhibitors (bisindolylmaleimide, calphostin C) and inhibitors of mitogen-activated protein/extracellular signal regulated kinase kinase (MEK) (PD98059 and U0126) suppressed up-regulation of MUC2. Src tyrosine kinase inhibitor PP2, a protein kinase A inhibitor (KT5720), and a p38 inhibitor (SB 203580) did not affect transcription. Western blotting and reverse transcription-PCR analysis confirmed these results. In addition, co-transfections with mutants of Ras, Raf, and MEK showed that the induction of MUC2 promoter activity by PMA required these three signaling proteins. Our results demonstrate that PMA activates protein kinase C, stimulating MAP kinase through a Ras- and Raf-dependent mechanism. An important role for nuclear factor kappaB (NF-kappaB) was also demonstrated using the inhibitor caffeic acid phenethyl ester and electrophoretic mobility shift assays. Such identification of pathways involved in MUC2 up-regulation by PMA in the HM3 colon cancer cell line may serve as a model for the effects of cytokines and growth factors, which regulate MUC2 expression during the progression of colorectal cancer.

Background: Tauroursodeoxycholate (TUDC) provides partial protection against taurolithocholate (TLC) induced cholestasis, possibly by inducing a signalling cascade activating protein kinase C (PKC). The potential protective effects of β muricholic acid (β-MC), another 7-β-hydroxylated bile salt, have not previously been studied in TLC cholestasis. Aims: To study the effect of β-MC on TLC induced cholestasis and also to investigate further the effects of agents affecting intracellular signalling, notably DBcAMP (a cell permeable cAMP analogue) and several protein kinase inhibitors. Methods: Functional studies were carried out analysing the proportion of hepatocyte couplets able to accumulate the fluorescent bile acid analogue cholyl-lysyl-fluorescein (CLF) into their sealed canalicular vacuole (cVA of CLF assay). Results: It was found that both β-MC and DBcAMP were as effective as TUDC in protecting against TLC induced cholestasis. The PKC inhibitors staurosporin and H7 but not the specific protein kinase A (PKA) inhibitor KT5720 abolished the protective effects of TUDC and β-MC. BAPTA/AM, a chelator of intracellular Ca2+, significantly decreased the protective effect of both bile salts, and that of DBcAMP. PKC and PKA inhibitors had no effect on protection with DBcAMP. Conclusions: β-MC was as effective as TUDC in protecting against TLC cholestasis. Mobilisation of Ca2+ and activation of PKC, but not of PKA, are involved in the anticholestatic effect of the two 7-β-hydroxylated bile salts. The hepatoprotective effects of DBcAMP involved Ca2+ mobilisation, but not PKC or PKA activation.

The effect of noradrenaline on the volume-sensitive chloride current (I(Cl(swell))) was studied with conventional whole-cell recording techniques in freshly dispersed isolated smooth muscle cells of the rabbit portal vein. In the absence of receptor antagonists, noradrenaline produced an increase in the amplitude of I(Cl(swell)) in some cells and a decrease in others. In the presence of the beta-adrenoceptor antagonist propranolol, noradrenaline increased I(Cl(swell)) and in the presence of the alpha(1)-adrenoceptor antagonist prazosin, noradrenaline reduced I(Cl(swell).) The phospholipase C (PLC) inhibitor U73122 reduced the amplitude of I(Cl(swell)) whereas the inactive analogue U73343 had no effect. The phorbol esters phorbol-12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu) increased the amplitude of I(Cl(swell)) by approximately 60 and 100 %, respectively, in a voltage-independent fashion. Inhibitors of protein kinase C (PKC) chelerythrine and calphostin-C decreased the amplitude of I(Cl(swell)) in a concentration-dependent but voltage-independent manner. Bath application of 8-Br-cAMP decreased I(Cl(swell)) by about 60 % whereas the inhibitor of protein kinase A (PKA) KT5720 increased the amplitude of I(Cl(swell)) by approximately 80-90 %. In the presence of propranolol, chelerythrine prevented the increase of I(Cl(swell)) by noradrenaline; in the presence of prazosin, KT5720 blocked the inhibitory action of noradrenaline. The results show that in rabbit portal vein myocytes noradrenaline enhances I(Cl(swell)) by acting on alpha(1)-adrenoceptors and reduces I(Cl(swell)) by stimulating beta-adrenoceptors. The data suggest that the potentiating and inhibitory effects of noradrenaline are mediated, respectively, by PKC and PKA.

It has been reported that theophylline induces growth inhibition and apoptosis in tumour cells. We report that theophylline induces growth inhibition and apoptosis of several human epithelial tumour cells with an IC:50 of 2.5 mM after 48 h of exposure. Moreover, 2.5 mM theophylline induces the accumulation of cancer cells in S-phase of the cell cycle with a concomitant reduction in the percentage of tumour cells in G(1)/G(0) phase. These effects are paralleled by cytoskeletal remodelling with a consequent redistribution of actin fibers and shape change as demonstrated by fluorescence microscopy. The apoptotic death of tumour cells occurs together with an increase in the expression and activity of the pro-apoptotic enzyme tissue transglutaminase (tTGase). All these effects are promptly antagonized by the specific PKA inhibitor KT5720, suggesting the involvement of cAMP intracellular elevation and, consequently, PKA activation. On the other hand, growth inhibition and tTGase expression and activity are potentiated by retinoic acid, a tTGase inducer. Therefore, a mechanistic model of theophylline action and anti-tumour strategies based on the concomitant use of theophylline and agents that potentiate tTGase activity can be hypothesized.

Functional gap junctional communication between vascular cells has been implicated in ascending dilatation and the cytochrome P-450 (CYP) inhibitor–sensitive and NO- and prostacyclin-independent dilatation of many vascular beds. Here, we assessed the mechanisms by which the epoxyeicosatrienoic acids (EETs) generated by a CYP 2C enzyme control interendothelial gap junctional communication. In CYP 2C–expressing porcine coronary endothelial cells, bradykinin, which enhances EET formation, elicited a biphasic effect on the electrical coupling and transfer of Lucifer yellow between endothelial cells, consisting of a transient increase in coupling followed by a sustained uncoupling. The initial phase was sensitive to the CYP 2C9 inhibitor sulfaphenazole and the protein kinase A (PKA) inhibitors Rp-cAMPS and KT5720 and could be mimicked by forskolin and caged cAMP as well as by the PKA activators 5,6-dichloro-1-β- d -ribofuranosylbenzimidazole 3′,5′-cyclic monophosphorothioate sodium salt and Sp-cAMPS. Gap junction uncoupling in bradykinin-stimulated porcine coronary endothelial cells was prevented by inhibiting the activation of extracellular signal–regulated kinase (ERK)1/2. In human endothelial cells, which express little CYP 2C, bradykinin elicited only an ERK1/2-mediated inhibition of intercellular communication. The CYP 2C9 product, 11,12-EET, also exerted a dual effect on the electrical and dye coupling of human endothelial cells, which was sensitive to PKA inhibition. These results demonstrate that an agonist-activated CYP-dependent pathway as well as 11,12-EET can positively regulate interendothelial gap junctional communication, most probably via the activation of PKA, an effect that is curtailed by the subsequent activation of ERK1/2.

1. Previous studies have reported discrepancies in the potencies of A(1) adenosine receptor agonists at mouse motor nerve terminals. In addition, conflicting results on the role of protein kinase A (PKA) in mediating the inhibitory effects of A(1) receptor agonists have been published. We thus decided to investigate the possibility of endogenous control of adenosine receptor sensitivity by protein kinases, using a variety of protein kinase inhibitors in conjunction with the adenosine receptor agonist 2-chloroadenosine (CADO). 2. CADO, at the concentration employed previously to study spontaneous ACh release in the mouse (1 microM), did not inhibit spontaneous ACh release in our experiments. However, a higher concentration of CADO (10 microM) produced highly statistically-significant reductions in spontaneous ACh release. 3. In the presence of the non-selective protein kinase inhibitor, H7 (50 microM), the potency of CADO was increased such that 1 microM CADO now reduced spontaneous quantal ACh release to approximately 63% of control. 4. Both H7, and the selective PKA inhibitor, KT5720 (500 nM) prevented increases in ACh release produced by CPT cyclic AMP (250 microM), suggesting these kinase inhibitors were blocking PKA. In contrast to H7, however, KT5720, did not reveal an inhibitory effect of 1 microM CADO. A number of other non-selective PKA inhibitors also failed to increase the potency of CADO. 5. The results suggest that an endogenous H7-sensitive process modulates the sensitivity of the mouse A(1) adenosine receptor and that the inhibitory effects of CADO are independent of cyclic AMP accumulation or PKA inhibition.

In a previous study, we showed that type 1 cannabinoid (CB(1)) receptor activation substantially depresses the corticostriatal glutamatergic transmission onto striatal neurons in the brain slice preparation. We now report that the adenylyl cyclase activator forskolin and cAMP analog (S)-p-8-(4-chlorophenythil) adenosine-3',5'-monophosphorothioate (Sp-8-CPT-cAMPS) strongly suppressed the synaptic depression induced by cannabimimetic aminoalkylindole, WIN 55,212-2. Application of the cAMP-dependent protein kinase (PKA) inhibitor KT5720 alone had no consistent effect on basal synaptic transmission but the synaptic enhancement elicited by forskolin was blocked. In addition, pretreatment of striatal slices with either KT5720 or another PKA inhibitor, H89, completely abolished the attenuation by forskolin on WIN 55,212-2-induced synaptic depression. The effect of forskolin on CB(1) receptor function was still observed in a low Ca(2+) bathing solution, suggesting that the forskolin's action is not attributable to its ability to saturate the presynaptic transmitter release processes. The possibility that forskolin acted by increasing CB(1) receptor phosphorylation was confirmed by demonstrating that the serine-phosphorylated component with CB(1) receptors was significantly increased after forskolin treatment. This forskolin effect was markedly attenuated in the presence of KT5720. Moreover, the activation of beta-adrenergic receptors by isoproterenol mimics forskolin to elicit a PKA-dependent inhibition of CB(1) receptor function. Together, these observations indicate that the presynaptic inhibitory action of CB(1) receptors at corticostriatal synapses could be negatively regulated by cAMP/PKA-mediated receptor phosphorylation. This effect of PKA may play a functional role in fine-tuning glutamatergic transmission at corticostriatal synapses.

Nociceptin/orphanin FQ (N/OFQ), an endogenous ligand for opioid receptor-like (ORL1) receptor, transduces signaling cascades implicated in MAPK, PKC, PLC, and calcium, etc. This study was designed to investigate the intracellular signaling mechanism of N/OFQ in human dopaminergic neuroblastoma SH-SY5Y cells. N/OFQ rapidly induced the phosphorylation of CREB, which was significantly suppressed by pretreatment of PKA inhibitor, but not by MAPK inhibitors. It also time-dependently increased the phosphorylation of MAPK, which was proven as ERKs, whereas it did not affect the PI3K activity. Interestingly, KT5720, a specific inhibitor of PKA, markedly suppressed the phosphorylation of MAPK by N/OFQ in SH-SY5Y cells. Furthermore, BAPTA-AM, an intracellular chelator of Ca(2+), completely abolished the phosphorylation of CREB as well as MAPK in N/OFQ-treated SH-SY5Y cells. Taken together, these results suggest that N/OFQ independently induces the activation of CREB prior to MAPK phosphorylation, which was also modulated by PKA. Furthermore, Ca(2+)-related signaling implicates in the phosphorylation processes of CREB and MAPK simultaneously.

The antiarrhythmic potential of betablockers contributes to their beneficial effects in the treatment of cardiac diseases, although the molecular basis of their class II antiarrhythmic action has not been clarified yet.To investigate a putative functional link between beta-adrenoreceptors and the fast component of cardiac delayed rectifier K(+) channels (I(Kr)), whole-cell patch-clamp experiments were performed with isolated guinea-pig ventricular myocytes. Tail currents of I(Kr) were measured at -40 mV after short (200 ms) test pulses to +40 mV.After application of the unspecific beta-receptor agonist isoproterenol (10 microM) for 12 min, the I(Kr) tail current was decreased by 72%, with an IC(50) of 1.4 microM. The specific beta(1)-blocker CGP207120A (10 microM) significantly attenuated the isoproterenol effect (net 24% decrease). The specific beta(1)-agonist xamoterol (10 microM), could mimic the isoproterenol effect (58% decrease). Modulators of beta(2)- or beta(3)-adrenoreceptors were far less effective. When isoproterenol or xamoterol were combined with KT5720 (2.5 microM), a specific inhibitor of protein kinase A (PKA), their effects were drastically reduced, indicating that PKA presumably mediates the beta(1)-adrenergic inhibition of I(Kr). Tail current reductions by cAMP, forskolin, PKA catalytic subunit and a combination of PKA holoenzyme and cAMP support an involvement of PKA in the regulation of I(Kr).The functional link between I(Kr) and the beta(1)-adrenergic receptor involving PKA may play an important role in arrhythmogenesis and contribute to the antiarrhythmic action of clinically used beta(1)-blockers.

This study was undertaken to assess the cellular localization of the inhibitory effect of a chemically synthetic isoquinoline compound 1-(3',4'-dimethoxybenzyl)-6,7-dichloroisoquinoline (DMDI) on the formyl-methionyl-leucyl-phenylalanine (fMLP)-induced respiratory burst in rat neutrophils. The DMDI concentration dependently inhibited the superoxide anion (O(2)(*-)) generation and O(2) consumption (IC(50) 12.2+/-4.9 and 15.2+/-8.4 microM, respectively) of neutrophils. DMDI did not scavenge the O(2)(*-) generated during the autoxidation of dihydroxyfumaric acid in a cell-free system. DMDI did not elevate cellular cyclic AMP levels. Inhibition of O(2)(*-) generation by DMDI in neutrophils was not reversed by a cyclic AMP-dependent protein kinase inhibitor, (8R,9S,11S)-(-)-9-hydroxy-9-hexoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde]trinden-1-one (KT5720). The DMDI concentration dependently inhibited the late plateau phase but not the initial spike of fMLP-induced [Ca(2+)](i) changes in the presence of extracellular Ca(2+). However, DMDI had no effect on the fMLP-induced [Ca(2+)](i) changes in the absence of extracellular Ca(2+). In addition, DMDI did not affect the fMLP-stimulated phosphatidylinositol 3-kinase (PI3-kinase) activation. DMDI produced a concentration-dependent reduction in the formation of phosphatidic acid and phosphatidylethanol in the presence of ethanol from fMLP-stimulated neutrophils (IC(50) 13.3+/-4.0 and 9.4+/-4.3 microM, respectively). On the basis of the immunoblot analysis of the phosphorylation of the mitogen-activated protein (MAP) kinase, DMDI attenuated the fMLP-stimulated MAP kinase phosphorylation in a similar concentration range. Collectively, these results indicate that the inhibition of the respiratory burst by DMDI in rat neutrophils is mediated through the blockade of phospholipase D and MAP kinase signaling pathways.

Introduction Hyperhomocysteinemia (HHcy) impairs nitric oxide endothelium-dependent vasodilation, consequently leading to atherosclerosis, a risk factor for cardiovascular disease. Novel treatments for HHcy are necessary. Aim We tested the hypothesis that alamandine, a vasoactive peptide of the renin-angiotensin system (RAS), could reverse HHcy-induced vascular dysfunction through the MrgD receptor, and that this is mediated by the protein kinase A (PKA) pathway. Furthermore, we sought to determine a putative binding model of alamandine to the MrgD receptor through docking and molecular dynamics simulations. Method The abdominal aorta was excised from New Zealand white rabbits (n=15) and incubated with 3 mM Hcy (to mimic HHcy) to induce vascular dysfunction in vitro. Vascular function was assessed by vasodilatory responses to cumulative doses of acetylcholine. Result Vasodilation was significantly impaired in HHcy-incubated aortic rings while alamandine reversed this effect (control, 74.2±5.0%; Hcy, 30.3±9.8%; alamandine+Hcy, 59.7±4.8%, p

Anabolic anti-osteoporotic agents are desirable for treatment and prevention of osteoporosis and fragility fractures. Osthole is a coumarin derivative extracted from the medicinal herbs Cnidium monnieri (L.) Cusson and Angelica pubescens Maxim.f. Osthole has been reported with osteogenic and anti-osteoporotic properties, whereas the underlying mechanism of its benefit still remains unclear. The objective of the present study was to investigate the osteopromotive action of osthole on mouse osteoblastic MC3T3-E1 cells and on mouse femoral fracture repair, and to explore the interaction between osthole-induced osteopromotive effect and cyclic adenosine monophosphate (cAMP) elevating effect. Osthole treatment promoted osteogenesis in osteoblasts by enhancing alkaline phosphatase (ALP) activity and mineralization. Oral gavage of osthole enhanced fracture repair and increased bone strength. Mechanistic study showed osthole triggered the cAMP/CREB pathway through the elevation of the intracellular cAMP level and activation of the phosphorylation of the cAMP response element-binding protein (CREB). Blockage of cAMP/CREB downstream signals with protein kinase A (PKA) inhibitor KT5720 partially suppressed osthole-mediated osteogenesis by inhibiting the elevation of transcription factor, osterix. In conclusion, osthole shows osteopromotive effect on osteoblasts in vitro and in vivo. Osthole-mediated osteogenesis is related to activation of the cAMP/CREB signaling pathway and downstream osterix expression.