Crosstalk between adenosine A1 and β1-adrenergic receptors regulates translocation of PKCε in isolated rat cardiomyocytes

Adenosine A(1) receptor (A(1)R)-induced translocation of PKCε to transverse (t) tubular membranes in isolated rat cardiomyocytes is associated with a reduction in β(1)-adrenergic-stimulated contractile function. The PKCε-mediated activation of protein kinase D (PKD) by endothelin-1 is inhibited by β(1)-adrenergic stimulated protein kinase A (PKA) suggesting a similar mechanism of A(1)R signal transduction modulation by adrenergic agonists may exist in the heart. We have investigated the influence of β(1)-adrenergic stimulation on PKCε translocation elicited by A(1)R. Immunofluorescence imaging and Western blotting with PKCε and β-COP antibodies were used to quantify the co-localization of PKCε and t-tubular structures in isolated rat cardiomyocytes. The A(1)R agonist CCPA increased the co-localization of PKCε and t-tubules as detected by imaging. The β(1)-adrenergic receptor agonist isoproterenol (ISO) inhibited this effect of CCPA. Forskolin, a potent activator of PKA, mimicked, and H89, a pharmacological PKA inhibitor, and PKI, a membrane-permeable PKA peptide PKA inhibitor, attenuated the negative effect of ISO on the A(1)R-mediated PKCε translocation. Western blotting with isolated intact hearts revealed an increase in PKCε/β-COP co-localization induced by A(1)R. This increase was attenuated by the A(1)R antagonist DPCPX and ISO. The ISO-induced attenuation was reversed by H89. It is concluded that adrenergic stimulation inhibits A(1)R-induced PKCε translocation to the PKCε anchor site RACK2 constituent of a coatomer containing β-COP and associated with the t-tubular structures of the heart. In that this translocation has been previously associated with the antiadrenergic property of A(1)R, it is apparent that the interactive effects of adenosine and β(1)-adrenergic agonists on function are complex in the heart.     

Extracellular signal-regulated kinases 1/2 control claudin-2 expression in Madin-Darby canine kidney strain I and II cells

The tight junction of the epithelial cell determines the characteristics of paracellular permeability across epithelium. Recent work points toward the claudin family of tight junction proteins as leading candidates for the molecular components that regulate paracellular permeability properties in epithelial tissues. Madin-Darby canine kidney (MDCK) strain I and II cells are models for the study of tight junctions and based on transepithelial electrical resistance (TER) contain "tight" and "leaky" tight junctions, respectively. Overexpression studies suggest that tight junction leakiness in these two strains of MDCK cells is conferred by expression of the tight junction protein claudin-2. Extracellular signal-regulated kinase (ERK) 1/2 activation by hepatocyte growth factor treatment of MDCK strain II cells inhibited claudin-2 expression and transiently increased TER. This process was blocked by the ERK 1/2 inhibitor U0126. Transfection of constitutively active mitogen-activated protein kinase/extracellular signal-regulated kinase kinase into MDCK strain II cells also inhibited claudin-2 expression and increased TER. MDCK strain I cells have higher levels of active ERK 1/2 than do MDCK strain II cells. U0126 treatment of MDCK strain I cells decreased active ERK 1/2 levels, induced expression of claudin-2 protein, and decreased TER by approximately 20-fold. U0126 treatment also induced claudin-2 expression and decreased TER in a high resistance mouse cortical collecting duct cell line (94D). These data show for the first time that the ERK 1/2 signaling pathway negatively controls claudin-2 expression in mammalian renal epithelial cells and provide evidence for regulation of tight junction paracellular transport by alterations in claudin composition within tight junction complexes.     

Involvement of protein kinase C and protein kinase A in the muscarinic receptor signalling pathways mediating phospholipase C activation, arachidonic acid release and calcium mobilisation.

The involvement of protein kinase C (PKC) and protein kinase A (PKA) in cholinergic signalling in CHO cells expressing the M3 subtype of the muscarinic acetylcholine receptor was examined. Muscarinic signalling was assessed by measuring carbachol-induced activation of phospholipase C (PLC), arachidonic acid release, and calcium mobilisation. Carbachol activation of PLC was not altered by inhibition of PKC with chelerythrine chloride, bisindolylmaleimide or chronic treatment with phorbol myristate acetate (PMA). Activation of PKC by acute treatment with PMA was similarly without effect. In contrast, inhibition of PKC blocked carbachol stimulation of arachidonic acid release. Likewise, PKC inhibition resulted in a decreased ability of carbachol to mobilise calcium, whereas PKC activation potentiated calcium mobilisation. Inhibition of PKA with H89 or Rp-cAMP did not alter the ability of carbachol to activate PLC. Similarly, PKA activation with Sp-cAMP or forskolin had no effect on PLC stimulation by carbachol. Carbachol-mediated release of arachidonic acid was decreased by H89 but only slightly increased by forskolin. Forskolin also increased calcium mobilisation by carbachol. These results suggest a function for PKC and PKA in M3 stimulation of arachidonic acid release and calcium mobilisation but not in PLC activation.     

Pre- and postsynaptic mechanisms in Hebbian activity-dependent synapse modification

We have used a three compartment tissue culture system that involved two separate populations of cholinergic neurons in the side compartments that converged on a common target population of myotubes in the center compartment. Activation of the axons from one population of neurons produced selective down-regulation of the synaptic inputs from the other neuronal population (when the two inputs innervated the same myotubes). The decrease in heterosynaptic inputs was mediated by protein kinase C (PKC). An activity-dependent action of protein kinase A (PKA) was associated with the stimulated input and this served to selectively stabilize this input. These changes associated with PKA and PKC activation were mediated by alterations in the number of acetylcholine receptors at the neuromuscular junction. These results suggest that neuromuscular electrical activity produces postsynaptic activation of both PKA and PKC, with the latter producing generalized synapse weakening and the former a selective synapse stabilization. Treatment of the neuronal cell body and axon to increase PKC activity by putting phorbal ester (PMA) in the side chamber did not affect synaptic transmission (with or without stimulation). By contrast, PKA blockade in the side compartment did produce an activity-dependent decrease in synaptic efficacy, which was due to a decrease in quantal release of neurotransmitter. Thus, when the synapse is activated, it appears that presynaptic PKA action is necessary to maintain transmitter output.     

Analysis of inhibition by H89 of UCP1 gene expression and thermogenesis indicates protein kinase A mediation of beta(3)-adrenergic signalling rather than beta(3)-adrenoceptor antagonism by H89

Although it has generally been assumed that protein kinase A (PKA) is essential for brown adipose tissue function, this has not as yet been clearly demonstrated. H89, an inhibitor of PKA, was used here to inhibit PKA activity. In cell extracts, it was confirmed that norepinephrine stimulated PKA activity, which was abolished by H89 treatment. In isolated brown adipocytes, H89 inhibited adrenergically induced thermogenesis (with an IC(50) of approx. 40 microM), and in cultured cells, adrenergically stimulated expression of the uncoupling protein-1 (UCP1) gene was abolished by H89 (full inhibition with 50 microM). However, H89 has been reported to be an adrenergic antagonist on beta(1)/beta(2)-adrenoceptors (AR). Although adrenergic stimulation of thermogenesis and UCP1 gene expression are mediated via beta(3)-ARs, it was deemed necessary to investigate whether H89 also had antagonistic potency on beta(3)-ARs. It was found that EC(50) values for beta(3)-AR-selective stimulation of cAMP production (with BRL-37344) in brown adipose tissue membrane fractions and in intact cells were not affected by H89. Similarly, the EC(50) of adrenergically stimulated oxygen consumption was not affected by H89. As H89 also abolished forskolin-induced UCP1 gene expression, and potentiated selective beta(3)-AR-induced cAMP production, H89 must be active downstream of cAMP. Thus, no antagonism of H89 on beta(3)-ARs could be detected. We conclude that H89 can be used as a pharmacological tool for elucidation of the involvement of PKA in cellular signalling processes regulated via beta(3)-ARs, and that the results are concordant with adrenergic stimulation of thermogenesis and UCP1 gene expression in brown adipocytes being mediated via a PKA-dependent pathway.     

Progesterone induces acrosome reaction in stallion spermatozoa via a protein tyrosine kinase dependent pathway

Progesterone (P(4)) is a physiological inducer of the acrosome reaction (AR) in stallion spermatozoa. However, the capacitation-dependent changes that enable progesterone binding, and the nature of the signaling cascade that is triggered by progesterone and results in induction of the AR, are poorly understood. The aim of the current study was, therefore, to investigate the protein kinase dependent signaling cascades involved in progesterone-mediated induction of the AR in stallion spermatozoa. In addition, we aimed to determine whether bicarbonate, an inducer of sperm capacitation, acted via the same pathway as P(4) or whether it otherwise synergized P(4)-mediated induction of the AR. We examined the effect on AR progression of specific inhibitors and stimulators of protein kinase A (PKA), protein kinase C (PKC), protein kinase G (PKG), and protein tyrosine kinase (PTK), in the presence or absence of 15 mM bicarbonate and/or 1 microg/ml progesterone. Progression of the AR was assessed using the Pisum sativum agglutinin conjugated to fluorescein iso thiocyanate (PSA-FITC) staining technique. Bicarbonate specifically activated a PKA-dependent signaling pathway, whereas the effect of P(4) was independent of PKA. Conversely, while P(4)-mediated AR induction was dependent on PTK, the effects of bicarbonate were PTK-independent. Finally, although the AR inducing effects of both P(4) and bicarbonate were sensitive to staurosporin, a potent blocker of PKC activity at moderate (50 nM) concentrations, the effect of P(4) was completely blocked at 50 nM staurosporin, whereas that of bicarbonate was only completely inhibited by much higher concentrations (2 microM) where staurosporin also inhibits PKA activity. In conclusion, P(4)-mediated activation of the AR is dependent on a pathway that includes both PTK and PKC. While the effects of bicarbonate on the AR are mediated via a separate PKA-dependent signaling pathway, P(4) and bicarbonate have synergistic effects on the AR.     

Stimulation of Cell Elongation in Teleost Rod Photoreceptors by Distinct Protein Kinase Inhibitors

Abstract: The rod photoreceptors of teleost retinas elongate in the light. To characterize the role of protein kinases in elongation, pharmacological studies were carried out with rod fragments consisting of the motile inner segment and photosensory outer segment (RIS-ROS). Isolated RIS-ROS were cultured in the presence of membrane-permeant inhibitors that exhibit selective activity toward specific serine/threonine protein kinases. We report that three distinct classes of protein kinase inhibitors stimulated elongation in darkness: (1) cyclic AMP-dependent protein kinase (PKA)-selective inhibitors (H-89 and KT5720), (2) a protein kinase C (PKC)-selective inhibitor (GF 109203X) that affects most PKC isoforms, and (3) a kinase inhibitor (H-85) that does not affect PKC and PKA in vitro. Other kinase inhibitors tested neither stimulated elongation in darkness nor inhibited light-induced elongation; these include the myosin light chain kinase inhibitors ML-7 and ML-9, the calcium-calmodulin kinase II inhibitor KN-62, and inhibitors or activators of diacylglycerol-dependent PKCs (sphingosine, calphostin C, chelerythrine, and phorbol esters). The myosin light chain kinase inhibitors as well as the PKA and PKC inhibitors H-89 and GF 109203X all enhanced light-induced elongation. These observations suggest that light-induced RIS-ROS elongation is inhibited by both PKA and an unidentified kinase or kinases, possibly a diacylglycerol-independent form of PKC.     

Pre‐ and postsynaptic mechanisms in Hebbian activity‐dependent synapse modification

We have used a three compartment tissue culture system that involved two separate populations of cholinergic neurons in the side compartments that converged on a common target population of myotubes in the center compartment. Activation of the axons from one population of neurons produced selective down‐regulation of the synaptic inputs from the other neuronal population (when the two inputs innervated the same myotubes). The decrease in heterosynaptic inputs was mediated by protein kinase C (PKC). An activity‐dependent action of protein kinase A (PKA) was associated with the stimulated input and this served to selectively stabilize this input. These changes associated with PKA and PKC activation were mediated by alterations in the number of acetylcholine receptors at the neuromuscular junction. These results suggest that neuromuscular electrical activity produces postsynaptic activation of both PKA and PKC, with the latter producing generalized synapse weakening and the former a selective synapse stabilization. Treatment of the neuronal cell body and axon to increase PKC activity by putting phorbal ester (PMA) in the side chamber did not affect synaptic transmission (with or without stimulation). By contrast, PKA blockade in the side compartment did produce an activity‐dependent decrease in synaptic efficacy, which was due to a decrease in quantal release of neurotransmitter. Thus, when the synapse is activated, it appears that presynaptic PKA action is necessary to maintain transmitter output. Published 2002 Wiley Periodicals, Inc. J Neurobiol 52: 241–250, 2002     

Contribution of protein kinase A and protein kinase C signalling pathways to the regulation of HSD11B2 expression and proliferation of MCF-7 cells

Contribution of the protein kinase A (PKA) and protein kinase C (PKC) signalling pathways to the regulation of 11beta-hydroxysteroid dehydrogenase type II (HSD11B2) gene expression was investigated in human breast cancer cell line MCF-7. Treatment of the cells with an adenylyl cyclase activator, forskolin, known to stimulate the PKA pathway, resulted in an increase in HSD11B2 mRNA content. Semi-quantitative RT-PCR revealed attenuation of the effect of forskolin by phorbol ester, tetradecanoyl phorbol acetate (TPA), an activator of the PKC pathway. It was also demonstrated that specific inhibitors significantly reduced the effect of activators of the two pathways. Stimulation of the PKA pathway did not affect, whereas stimulation of the PKC pathway significantly reduced MCF-7 cell proliferation in a time-dependent manner. A cell growth inhibitor, dexamethasone, at high concentrations, caused a 40% decrease in proliferation of MCF-7 cells and this effect was abolished under conditions of increased HSD11B2 expression. It was concluded that in MCF-7 cells, stimulation of the PKA signal transduction pathway results in the induction of HSD11B2 expression and that this effect is markedly reduced by activation of the PKC pathway. Activation of the PKC pathway also resulted in inhibition of cell proliferation, while activation of the PKA pathway abolished the antiproliferative effect of dexamethasone. These effects might be due to oxidation of dexamethasone by the PKA-inducible HSD11B2.     

Regulation of the MDCK Cell Tight Junction

The sodium flux across individual tight junctions (TJ) of low-resistance MDCK cell monolayers grown on glass coverslips was determined as a measure of paracellular permeability. Increases in perfusate glucose concentration from 5 to 25 mm decreased tight junction Na permeability. This permeability decrease was not specific as nonmetabolizable analogues of glucose caused similar diminutions in TJ Na permeability. Stimulation of protein kinase A increased TJ Na permeability, and inhibition of protein kinase A decreased TJ Na permeability. Transepithelial electrical resistance of monolayers grown on permeable supports did not change as predicted from the observed alterations in TJ Na permeability of monolayers grown on glass coverslips. Fluorescent labeling of cell F-actin showed that increased F-actin in the perijunctional ring correlated with higher TJ Na permeability. Although a low dose of cytochalasin D did not change TJ Na permeability, it disrupted the cytoskeleton and blocked the decrease in TJ Na permeability caused by glucose. Cytochalasin D failed to block the effects of protein kinase A stimulation or inhibition on TJ Na permeability. We conclude that tight junction sodium permeability is regulated both by protein kinase A activity and by other processes involving the actin cytoskeleton. Received: 17 June 1997/Revised: 28 August 1997     

The effect of sphingosine and phorbol ester on the signal transduction enzymes and fibronectin release in cell culture.

In testing the hypothesis that the stimulation of the release of fibronectin (FN) by 12-O-tetradecanoylphorbol 13-acetate (TPA) from human lung fibroblasts in culture is the result of activation of protein kinase C (PKC), we found that the PKC inhibitor sphingosine strongly inhibited FN release in presence and even in absence of TPA. However, a different PKC inhibitor, calphostin C, despite almost complete inhibition of PKC, had no effect on FN release. We concluded that sphingosine is a potent inhibitor of FN release from the cell surface, independent of its inhibition of PKC; and that TPA stimulates release of FN by a pathway other than activation of PKC. We found that the activation of PKC by TPA was accompanied by inhibition of the cAMP-dependent protein kinase (PKA). When PKA was inhibited by an antagonist (H8, a cAMP analogue) at a concentration specific for PKA inhibition, the release of FN was stimulated similar to the stimulation with TPA. Activation of PKA with forskolin resulted in decreased FN release. In conclusion, we have shown that: (1) sphingosine had a robust effect inhibiting the release of FN from fibroblasts, independent of its action on PKC; (2) TPA treatment of these cells resulted in inhibition of PKA; (3) inhibition of PKA stimulated FN release whereas its activation decreased this release. It is possible that PKA, by phosphorylating a protein, may function, directly or indirectly, in keeping FN attached to the cell surface of fibroblasts.     

Possible involvement of a cyclic AMP-dependent mechanism in PACAP-induced proliferation and ERK activation in astrocytes

In cultured astrocytes, PACAP activates extracellular signal-regulated kinase (ERK) and induces cell proliferation at picomolar concentrations. Here, we examined the role of cyclic AMP signaling underlying the effects of PACAP. PACAP38 induced accumulation of cyclic AMP in astrocytes at concentrations as low as 10(-12)M. PACAP38 (10(-12)-10(-9)M)-stimulated cell proliferation was completely abolished by the cyclic AMP antagonist Rp-cAMP, whereas the protein kinase A (PKA) inhibitor H89 had no effect. This PACAP38-mediated effect was also abolished by the ERK kinase inhibitor PD98059, suggesting the involvement of ERK in PACAP-induced proliferation. PACAP38 (10(-12)M)-stimulated phosphorylation of ERK lasted for at least 60 min. This effect was completely abolished by Rp-cAMP but not by H89. Dibutyryl cyclic AMP maximally stimulated the incorporation of thymidine and activation of ERK at 10(-10)M. These results suggest that PACAP-mediated stimulation of ERK activity and proliferation of astrocytes may involve a cyclic AMP-dependent, but PKA-independent, pathway.     

L-NAME-induced dispersion of melanosomes in melanophores activates PKC, MEK and ERK1.

Melanosome movement represents a good model of cytoskeleton-mediated transport of organelles in eukaryotic cells. We recently observed that inhibiting nitric oxide synthase (NOS) with Nomega-nitro-L-arginine methyl ester (L-NAME) induced dispersion in melanophores pre-aggregated with melatonin. Activation of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (PKA) or calcium-dependent protein kinase (PKC) is known to cause dispersion. Also, PKC and NO have been shown to regulate the mitogen/extracellular signal-regulated kinase (MEK)-ERK pathway. Accordingly, our objective was to further characterize the signaling pathway of L-NAME-induced dispersion. We found that the dispersion was decreased by staurosporine and PD98059, which respectively inhibit PKC and MEK, but not by the PKA inhibitor H89. Furthermore, Western blotting revealed that ERK1 kinase was phosphorylated in L-NAME-dispersed melanophores. L-NAME also caused dispersion in latrunculin-B-treated cells, suggesting that this effect is not due to inhibition of the melatonin signaling pathway. Summarizing, we observed that PKC and MEK inhibitors decreased the L-NAME-induced dispersion, which caused phosphorylation of ERK1. Our results also suggest that NO is a negative regulator of phosphorylations that leads to organelle transport.     

Selectivity of protein kinase inhibitors in human intact platelets

The specificity of commonly used protein kinase inhibitors has been evaluated in the intact human platelet. Protein kinase C (PKC) and cyclic AMP-dependent protein kinase (PKA) were activated selectively by treating platelets with phorbol dibutyrate (PDBu) or prostacyclin (PGl2). PKC activity was quantitated by measuring PDBu-specific phosphorylation of a 47,000 molecular weight protein, and PKA activity monitored by measuring prostacyclin-dependent phosphorylation of a 22,000 molecular weight protein. Staurosporine and 1-(5-isoquinolinylsulphonyl)-2-methyl-piperazine (H-7) were found to be non-specific inhibitors in the intact platelet, consistent with their effects on the isolated enzymes. Tamoxifen inhibited PKC activity (IC50 = 80 microM) but increased PKA-dependent protein phosphorylation. These results support the use of human platelets for measuring the specificity of protein kinase inhibitors and indicate that tamoxifen might have value for experimental purposes as a relatively selective PKC inhibitor.     

Growth hormone-releasing peptide-2 stimulates secretion and synthesis of adrenocorticotropic hormone in mouse pituitary

Growth hormone (GH)-releasing peptides (GHRPs) are synthetic peptides which induce strong GH release in both animals and humans. Among them, GHRP-2 is known to stimulate GH release by acting at both hypothalamic and pituitary sites, but also induces adrenocorticotropic hormone (ACTH) release in healthy subjects. GHRP-2 may stimulate ACTH release directly via GHRP receptor type 1a in ACTH-producing tumors. GHRP-2 increases ACTH secretion in rat in vivo, but not ACTH release from rat primary pituitary cells. In the present study, in order to elucidate the mechanism underlying ACTH secretion by GHRPs, mouse pituitary cells were stimulated by GHRP-2. GHRP receptor mRNA was expressed in the mouse pituitary, and GHRP-2 directly stimulated secretion and synthesis of ACTH in the mouse anterior pituitary cells. GHRP-2 increased intracellular cyclic AMP production. H89, a potent protein kinase A (PKA) inhibitor, and bisindolylmaleimide I, a selective protein kinase C (PKC) inhibitor, inhibited the GHRP-2-induced ACTH release, and that H89, but not bisindolylmaleimide I, inhibited the GHRP-2-induced proopiomelanocortin mRNA levels. Together, the GHRP-2-induced ACTH release was regulated via both PKA and PKC pathways in the mouse pituitary cells, while ACTH was synthesized by GHRP-2 only via the PKA pathway.     

Lysophosphatidic Acid Increases Tight Junction Permeability in Cultured Brain Endothelial Cells

Abstract: Brain capillary endothelial cells are coupled by a continuous belt of complex high-electrical-resistance tight junctions that are largely responsible for the blood-brain barrier. We have investigated mechanisms regulating tight junction permeability in brain endothelial cells cultured to maintain high-resistance junctions. The phospholipid lysophosphatidic acid (LPA) was found to cause a rapid, reversible, and dose-dependent decrease in transcellular electrical resistance in brain endothelial cells. LPA also increased the paracellular flux of sucrose, which, together with the resistance decrease, indicated increased tight junction permeability. Activation of protein kinase C attenuated the effect of LPA, suggesting that it was mediated by activation of a signalling pathway. LPA did not cause any obvious relocalization of adherens junction- or tight junction-associated proteins. However, it did stimulate the formation of stress fibres, the recruitment of focal adhesion components, and the appearance of tyrosine phosphorylated protein at focal contacts. Our study shows that LPA is a modulator of tight junction permeability in brain endothelial cells in culture and raises the possibility that it triggers blood-brain barrier permeability changes under (patho)physiological conditions.