Cyclic nucleotide regulation of teleost rod photoreceptor inner segment length

Retinal rod photoreceptors of teleost fish elongate in the light and shorten in the dark. Rod cell elongation and shortening are both mediated by actin-dependent mechanisms that occur in the inner segment myoid and ellipsoid. The intracellular signaling pathways by which light and dark regulate the actin cytoskeleton in the inner segment are unknown. To investigate the intracellular signals that regulate teleost rod motility, we have been using mechanically isolated rod inner/outer segments (RIS-ROS) obtained from the retinas of green sunfish, Lepomis cyanellus. In culture, RIS-ROS retain the ability to elongate in response to light; myoids elongate 15-20 microns in length during 45 min of light culture. A pharmacological approach was taken to investigate the role of cyclic nucleotides, cyclic nucleotide-dependent kinases, and protein phosphatases in the regulation of RIS-ROS motility. Millimolar concentrations of cAMP and cGMP analogues were both found to inhibit light-induced myoid elongation and two cyclic nucleotide analogues, SpCAMPS and 8BrcGMP, promoted myoid shortening after RIS-ROS had elongated in response to light. The cyclic nucleotide- dependent kinase inhibitor, H8, mimicked light by promoting myoid elongation in the dark. The effects of H8 were dose dependent, with maximal elongation occurring at concentrations of approximately 100 microM. Similar to the effects of cyclic nucleotide analogues, the phosphatase inhibitor, okadaic acid (0.1-10 microM), inhibited light- induced elongation and promoted shortening. The results presented here suggest that RIS-ROS motility is regulated by protein phosphorylation: phosphorylation in the dark by cyclic nucleotide-dependent protein kinases promotes rod shortening, while dephosphorylation in the light promotes rod elongation.     

Roles of protein kinase A and C in spontaneous maturation and in forskolin or 3-isobutyl-1-methylxanthine maintained meiotic arrest of bovine oocytes

Four hypotheses were tested using isolated bovine oocytes. (1) Cumulus oocyte complexes (COCs) or denuded oocytes (DOs) were cultured with the protein kinase A (PKA) inhibitor, H-89, to test if meiotic arrest induced by forskolin or IBMX was due to cAMP-stimulated PKA activity or nonspecific effects of these cAMP elevators. (2) COCs were cultured with a protein kinase C (PKC) stimulator (PDDβ) or inhibitor (GF109203x) to test if PKC modulation altered oocyte maturation. (3) COCs were prestimulated for 15 min with (a) PDDβ followed by cotreatment with forskolin, or (b) with H-89 or H-7 followed by cotreatment with GF109203x, to test for interaction between the PKA and PKC signal transduction pathways. (4) H-89 was added to spontaneously maturing COCs at intervals 0–18 hr to test if H-89 interfered with the transition between meiosis I and II. The results were as follows: H-89 interfered with forskolin or IBMX arrested oocytes in a dose-response manner (IBMX ED₅₀ = 41 μM for COCs; forskolin ED₅₀ = 9 μM for denuded oocytes). Prestimulation with PKC induced meiotic resumption in COCs in spite of the presence of forskolin [PDDβ followed by PDDβ + forskolin: 41–47% germinal vesicle (GV) oocytes; forskolin alone: 90–95% GV], while PKC inhibition induced meiotic arrest to a similar extent as forskolin (GF109230x, 85% GV; forskolin, 67–80% GV). Additionally, pretreatment of COCs with H-89 interfered with GF109203x induced arrest (41% vs. 90% GV, respectively). Finally, H-89 interfered with the timely progression of COCs from meiosis I and II. These results indicate that the PKA and PKC pathways can modulate the maturation of bovine oocytes in vitro. © 1996 Wiley-Liss, Inc.     

Cyclic AMP-dependent protein kinase A plays a role in the directed migration of human keratinocytes in a DC electric field.

Skin wound healing requires epithelial cell migration for re-epithelialization, wound closure, and re-establishment of normal function. We believe that one of the earliest signals to initiate wound healing is the lateral electric field generated by the wound current. Normal human epidermal keratinocytes migrate towards the negative pole, representing the center of the wound, in direct currents of a physiological strength, 100 mV/mm. Virtually nothing is known about the signal transduction mechanisms used by these cells to sense the endogenous electric field. To elucidate possible protein kinase (PK) involvement in the process, PK inhibitors were utilized. Two important findings have been described. Firstly, addition of 50 nM KT5720, an inhibitor of PKA, resulted in a 53% percent reduction in the directional response of keratinocytes in the electric field, while not significantly affecting general cell motility. The reduction was dose-dependent, there was a gradual decrease in the directional response from 5 to 50 nM. Secondly, addition of 1 microM ML-7, a myosin light chain kinase inhibitor, resulted in an approximate 31% decrease in the distance the cells migrated without affecting directional migration. The PKC inhibitors GF109203X at 4 microM and H-7 at 20 microM and W-7, a CaM kinase inhibitor, did not significantly alter either directed migration or cell migration, although they all resulted in a slight reduction in directional migration. D-erythro-sphingosine at 15 microM, a PKC inhibitor, had virtually no effect on either migration distance or directed migration. These findings demonstrate that divergent kinase signaling pathways regulate general cell motility and sustained directional migration and highlight the complexity of the signal transduction mechanisms involved. The inhibitor studies described in this paper implicate a role for PKA in the regulation of the directional migratory response to applied electric fields, galvanotaxis.     

Histamine-induced vasoconstriction involves phosphorylation of a specific inhibitor protein for myosin phosphatase by protein kinase C alpha and delta isoforms

Histamine stimulus triggers inhibition of myosin phosphatase-enhanced phosphorylation of myosin and contraction of vascular smooth muscle. In response to histamine stimulation of intact femoral artery, a smooth muscle-specific protein called CPI-17 (for protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa) is phosphorylated and converted to a potent inhibitor for myosin phosphatase. Phosphorylation of CPI-17 is diminished by pretreatment with either or GF109203x, suggesting involvement of multiple kinases (Kitazawa, T., Eto, M., Woodsome, T. P., and Brautigan, D. L. (2000) J. Biol. Chem. 275, 9897--9900). Here we purified and identified CPI-17 kinases endogenous to pig artery that phosphorylate CPI-17. DEAE-Toyopearl column chromatography of aorta extracts separated two CPI-17 kinases. One kinase was protein kinase C (PKC) alpha, and the second kinase was purified to homogeneity as a 45-kDa protein, and identified by sequencing as PKC delta. Purified PKC delta was 3-fold more reactive with CPI-17 compared with myelin basic protein, whereas purified PKC alpha and recombinant RhoA-activated kinases (Rho-associated coiled-coil forming protein Ser/Thr kinase and protein kinase N) showed equal activity with CPI-17 and myelin basic protein. inhibited CPI-17 phosphorylation by purified PKC delta with IC(50) of 0.6 microm (in the presence of 0.1 mm ATP) or 14 microm (2.0 mm ATP). significantly suppressed CPI-17 phosphorylation in smooth muscle cells, and the contraction of permeabilized rabbit femoral artery induced by stimulation with phorbol ester. GF109203x inhibited phorbol ester-induced contraction of rabbit femoral artery by 80%, whereas a PKC alpha/beta inhibitor, Go6976, reduced contraction by 47%. The results imply that histamine stimulation elicits contraction of vascular smooth muscle through activation of PKC alpha and especially PKC delta to phosphorylate CPI-17.     

A2B adenosine and P2Y2 receptors stimulate mitogen-activated protein kinase in human embryonic kidney-293 cells. cross-talk between cyclic AMP and protein kinase c pathways

Mitogen-activated protein kinase (MAPK) cascades underlie long-term mitogenic, morphogenic, and secretory activities of purinergic receptors. In HEK-293 cells, N-ethylcarboxamidoadenosine (NECA) activates endogenous A2BARs that signal through Gs and Gq/11. UTP activates P2Y2 receptors and signals only through Gq/11. The MAPK isoforms, extracellular-signal regulated kinase 1/2 (ERK), are activated by NECA and UTP. H-89 blocks ERK activation by forskolin, but weakly affects the response to NECA or UTP. ERK activation by NECA or UTP is unaffected by a tyrosine kinase inhibitor (genistein), attenuated by a phospholipase C inhibitor (U73122), and is abolished by a MEK inhibitor (PD098059) or dominant negative Ras. Inhibition of protein kinase C (PKC) by GF 109203X failed to block ERK activation by NECA or UTP, however, another PKC inhibitor, Ro 31-8220, which unlike GF 109203X, can block the zeta-isoform, and prevents UTP- but not NECA-induced ERK activation. In the presence of forskolin, Ro 31-8220 loses its ability to block UTP-stimulated ERK activation. PKA has opposing effects on B-Raf and c-Raf-1, both of which are found in HEK-293 cells. The data are explained by a model in which ERK activity is modulated by differential effects of PKC zeta and PKA on Raf isoforms.     

The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C

Staurosporine is the most potent inhibitor of protein kinase C (PKC) described in the literature with a half-maximal inhibitory concentration (IC50) of 10 nM. Nevertheless, this natural product is poorly selective when assayed against other protein kinases. In order to obtain specific PKC inhibitors, a series of bisindolylmaleimides has been synthesized. Structure-activity relationship studies allowed the determination of the substructure responsible for conferring high potency and lack of selectivity in the staurosporine molecule. Several aminoalkyl bisindolylmaleimides were found to be potent and selective PKC inhibitors (IC50 values from 5 to 70 nM). Among these compounds GF 109203X has been chosen for further studies aiming at the characterization of this chemical family. GF 109203X was a competitive inhibitor with respect to ATP (Ki = 14 +/- 3 NM) and displayed high selectivity for PKC as compared to five different protein kinases. We further determined the potency and specificity of GF 109203X in two cellular models: human platelets and Swiss 3T3 fibroblasts. GF 109203X efficiently prevented PKC-mediated phosphorylations of an Mr = 47,000 protein in platelets and of an Mr = 80,000 protein in Swiss 3T3 cells. In contrast, in the same models, the PKC inhibitor failed to prevent PKC-independent phosphorylations. GF 109203X inhibited collagen- and alpha-thrombin-induced platelet aggregation as well as collagen-triggered ATP secretion. However, ADP-dependent reversible aggregation was not modified. In Swiss 3T3 fibroblasts, GF 109203X reversed the inhibition of epidermal growth factor binding induced by phorbol 12,13-dibutyrate and prevented [3H] thymidine incorporation into DNA, only when this was elicited by growth promoting agents which activate PKC. Our results illustrate the potential of GF 109203X as a tool for studying the involvement of PKC in signal transduction pathways.     

Fluid flow induces Rankl expression in primary murine calvarial osteoblasts

Mechanical loading of bone generates fluid flow within the mineralized matrix that exerts fluid shear stress (FSS) on cells. We examined effects of FSS on receptor activator of nuclear factor kappa B ligand (RANKL), a critical factor for osteoclast formation. Primary murine osteoblasts were subjected to pulsatile FSS (5 Hz, 10 dynes/cm(2)) for 1 h and then returned to static culture for varying times (post-FSS). Protein levels were measured by Western analysis and mRNA by Northern analysis, RT-PCR and quantitative PCR. There were 20- to 40-fold increases in RANKL mRNA at 2-4 h post-FSS. RANKL protein was induced by 2 h post-FSS and remained elevated for at least 8 h. Effects were independent of cyclooxygenase-2 activity. Small increases (up to three-fold) in mRNA of the decoy receptor for RANKL, osteoprotegerin, were seen. Five min of FSS, followed by static culture, was as effective in stimulating RANKL mRNA as 4 h of continuous FSS. FSS induced cAMP activity, and H-89, a protein kinase A (PKA) inhibitor, blocked the FSS induction of RANKL. H-89 also inhibited the PKC pathway, but specific PKC inhibitors, GF109203X and Go6983, did not inhibit FSS-induced RANKL. FSS induced phosphorylation of ERK1/2, and PD98059, an inhibitor of the ERK pathway, inhibited the FSS induction of RANKL mRNA 60%-90%. Thus, brief exposure to FSS resulted in sustained induction of RANKL expression after stopping FSS, and this induction was dependent on PKA and ERK signaling pathways. Increased RANKL after mechanical loading may play a role in initiating bone remodeling.     

The kinase inhibitor iso-H7 stimulates rat satellite cell differentiation through a non-protein kinase C pathway by increasing myogenin expression level

We analysed the signaling pathways involved in myogenic differentiation of primary cultures of rat satellite cells using substances targeting the protein kinase C (PKC) and the cAMP protein kinase (PKA) pathways. We have previously shown that iso-H7, which putatively inhibits both PKC and PKA, strongly stimulates satellite cell differentiation, as well as the PKA inhibitor HA 1004. In the study reported here, the effects of iso-H7 on satellite cell differentation were compared to those observed in the presence of agents which reduce PKC activity. It was shown that treatments with the highly specific PKC inhibitor GF109203X or with 12-O-tetradecanoylphorbol 13-acetate (TPA) which induced a partial PKC downregulation, did not significantly alter myogenic differentiation. Northern blot analyses showed that iso-H7 activated the expression of myogenin but not that of MyoD mRNA. Concurrently, iso-H7 increased myosin light-chain mRNA expression. In contrast, TPA had no effect on these syntheses. Taken together, these results showed that iso-H7 did not act intracellularly as a PKC inhibitor but rather as a PKA inhibitor as previously suggested. Our results are compatible with the hypothesis that a reduction in PKA activity controls satellite cell myogenesis through an increased myogenin mRNA expression.Abbreviations PKC protein kinase C - PKA cAMP-dependent protein kinase - CK creatine kinase - iso-H7 1-(5-isoquinolinesulfonyl)-3-methylpiperazine - H7 1-(5-isoquinolinesulfonyl)-2-methylpiperazine - HA 1004 N-(3-hydroxyethyl)-1-piperazine ethanesulfonate - TPA 12-O-tetradecanoyl phorbol 13-acetate - MLC myosin light chain - GAPDH glyceraldehyde 3-phosphate deshydrogenase     

Heparin- and superoxide anion-dependent capacitation of cryopreserved bovine spermatozoa: requirement of dehydrogenases and protein kinases

Capacitation is part of an oxidative process necessary for bovine spermatozoa to acquire fertilizing capacity. This process includes the generation of reactive oxygen species (ROS) and the participation of protein kinases such as A (PKA), C (PKC) and tyrosine kinase (PTK). A redox status is required to support both sperm motility and capacitation. Our aim was to determine the requirement of lactate dehydrogenase C4 (LDH-C4) and isocitrate dehydrogenase (NADP-ICDH) and of protein kinases in cryopreserved bovine sperm capacitation. The presence of inhibitors of both LDH-C4 and NADP-ICDH prevented the heparin-induced capacitation. H89, GF109203X or genistein blocked capacitation triggered by heparin or the superoxide (O(-*)(2))generator system xanthine-xanthine oxidase-catalase (XXOC) suggesting the requirement of PKA, PKC and PTK in this process. Taken together these results suggest that LDH-C4 and NADP-ICDH contribute with the redox status to support bovine sperm capacitation and that PKA, PKC and PTK are involved in different mechanisms induced by different inducers that lead bovine spermatozoa to be capacitated.     

Regulation of matrix Gla protein by parathyroid hormone in MC3T3-E1 osteoblast-like cells involves protein kinase A and extracellular signal-regulated kinase pathways

Inhibition of osteoblast-mediated mineralization is one of the major catabolic effects of parathyroid hormone (PTH) on bone. Previously, we showed that PTH induces matrix gamma-carboxyglutamic acid (Gla) protein (MGP) expression and established that this induction is critical for PTH-mediated inhibition of osteoblast mineralization. In the present study, we focus on the mechanism through which PTH regulates MGP expression in osteoblastic MC3T3-E1 cells. Following transient transfection of these cells with a -748 bp murine MGP promoter-luciferase construct (pMGP-luc), PTH (10 (-7) M) induced promoter activity in a time-dependent manner with a maximal four- to six fold induction seen 6 h after PTH treatment. Both H-89 (PKA inhibitor) and U0126 (MEK inhibitor), suppressed PTH induction of MGP promoter activity as well as the MGP mRNA level. In addition, forskolin (PKA activator) stimulated MGP promoter activity and mRNA levels confirming that PKA is one of the signaling molecules required for regulation of MGP by PTH. Co-transfection of MC3T3-E1 cells with pMGP-luc and MEK(SP), a plasmid encoding the constitutively active form of MEK, led to a dose-dependent increase in MGP promoter activity. Both MGP promoter activity and MGP mRNA level were not affected by the protein kinase C (PKC) inhibitor, GF109203X. However, phorbol 12-myristate 13-acetate (PMA), a selective PKC activator induced MGP mRNA expression through activation of extracellular signal-regulated kinase (ERK). Taken together, these results indicate that PTH regulates MGP via both PKA- and ERK-dependent pathways.     

Translocation of protein kinase Cepsilon and protein kinase Cdelta to membrane is required for ultraviolet B-induced activation of mitogen-activated protein kinases and apoptosis.

UV-induced signal transduction may be involved in tumor promotion and induction of apoptosis. The role of protein kinase C (PKC) in UVB-induced signal transduction is not well understood. This study showed that UVB markedly induced translocation of membrane-associated PKCepsilon and PKCdelta, but not PKCalpha, from cytosol to membrane. Dominant negative mutant (DNM) PKCepsilon or PKCdelta inhibited UVB-induced translocation of PKCepsilon and PKCdelta, respectively. UVB-induced activation of extracellular signal-regulated protein kinases (Erks) and c-Jun NH2-terminal kinases (JNKs) was strongly inhibited by DNM PKCepsilon and PKCdelta, whereas the DNM of PKCalpha was less effective on the UVB-induced phosphorylation of Erks and JNKs. Among the PKC inhibitors used only rottlerin, a selective inhibitor of PKCdelta, markedly inhibited the UVB-induced activation of Erks and JNKs, but not p38 kinases. Safingol, a selective inhibitor for PKCalpha, did not show any inhibitory effect on UVB-induced mitogen-activated protein kinase activation. GF109203X is a stronger inhibitor of classical PKC than novel PKC. Lower concentrations of GF109203X (<10 microM) had no effect on UVB-induced activation of Erks or JNKs. However, at higher concentrations (over 20 microM), GF109203X inhibited UVB-induced activation of JNKs, Erks, and even p38 kinases. Meanwhile, rottlerin and GF109203X markedly inhibited UVB-induced apoptosis of JB6 cells, whereas safingol had little inhibitory effect. DNM-Erk2 cells and PD98059, a selective inhibitor for mitogen-activated protein kinase/extracellular signal-regulated kinase 1 that directly activates Erks, inhibited UVB-induced apoptosis. DNM-JNK1 cells also blocked UVB-induced apoptosis, whereas SB202190, a specific inhibitor for p38 kinases, did not produce the inhibitory effect. These data demonstrate that PKCdelta and PKCepsilon, but not PKCalpha, mediate UVB-induced signal transduction and apoptosis in JB6 cells through activation of Erks and JNKs.     

Control of Cl- transport in the operculum epithelium of Fundulus heteroclitus: long- and short-term salinity adaptation

The eurohaline fish, Fundulus heteroclitus, adapts rapidly to enhanced salinity by increasing the ion secretion by gill chloride cells. An increase of approximately 70 mOsm in plasma osmolarity was previously found during the transition. To mimic this in vitro, isolated opercular epithelia of seawater-adapted Fundulus mounted in a modified Ussing chamber were exposed to an increase in NaCl and/or osmolarity on the basolateral side, which immediately increased I(SC). Various Cl(-) channel blockers as well as the K(+) channel blocker Ba(2+) added to the basolateral side all inhibited the steady-state as well as the hypertonic stimulation of I(SC). The exists -agonist isoproterenol stimulates I(SC) in standard Ringer solutions. In contrast, when cell volume was kept at the larger value by simultaneous addition of water, the stimulation with isoproterenol was abolished, suggesting that the key process for activation of the Na(+), K(+), 2Cl(-) cotransporter is cell shrinkage. The protein kinase C (PKC) inhibitor chelerythrine and the myosin light chain kinase (MLCK) inhibitor ML-7 had strong inhibitory effects on the mannitol activation of I(SC), thus both MLCK and PKC are involved. The two specific protein kinase A (PKA) inhibitors H-89 and KT 5720 had no effect after mannitol addition whereas isoproterenol stimulation was completely blocked by H-89. This indicates that PKA is involved in the activation of the apical Cl(-) channel via c-AMP whereas the shrinkage activation of the Na(+), K(+), 2Cl(-) cotransporter is independent of PKA activation. The steady-state Cl(-) secretion was stimulated by an inhibitor of serine/threonine phosphatases of the PP-1 and PP-2A type and inhibited by a PKC inhibitor but not by a PKA inhibitor. Thus, it seems to be determined by continuous phosphorylation and dephosphorylation involving PKC but not PKA. The steady-state Cl(-) secretion and the maximal obtainable Cl(-) secretion were measured in freshwater-adapted fish and in fish retransferred to saltwater. No I(SC) could be measured in freshwater-adapted fish or in the fish within the first 18 h after transfer to saltwater. As evidenced from Western blot analysis using antiserine-antibodies, a heavily serine phosphorylated protein of about 190 kDa was consistently observed in the saltwater-acclimated fish, but was only weakly present in freshwater-acclimated fish. This observation indicates that acclimatization to saltwater stimulates the expression of this 190-kDa protein and/or a serine/threonine kinase, which subsequently phosphorylates the protein.     

H-89 potentiates adipogenesis in 3T3-L1 cells by activating insulin signaling independently of protein kinase A

Among four kinds of protein kinase A (PKA) inhibitors tested, H-89 exhibited a unique action to remarkably enhance adipocyte differentiation of 3T3-L1 cells, whereas the other three PKA inhibitors, PKA inhibitor Fragment 14-22 (PKI), Rp-cAMP, and KT 5720, did not enhance adipocyte differentiation. H-85, which is an inactive form of H-89, exhibited a similar enhancing effect on adipocyte differentiation. H-89 also potentiated the phosphorylation of Akt and extracellular signal-regulated kinase (ERK) 1/2 in 3T3-L1 cells, which function as downstream signaling of insulin. Phosphoinositide 3-kinase (PI3K) inhibitor wortmannin and mitogen-activated protein kinase kinase (MEK) inhibitor PD 98059 suppressed both the H-89-induced promotion of adipocyte differentiation and the H-89-induced potentiation of phosphorylation of Akt and ERK1/2. Rho kinase inhibitor Y-27632 also promoted the phosphorylation of both Akt and ERK1/2 and enhanced adipocyte differentiation, although its effect was somewhat less than that of H-89. Even when cells were treated with a mixture of Y-27632 and H-89, the additive enhancing effects on both the insulin signaling and adipocyte differentiation were not detected. Therefore, it is suggested that the major possible mechanism whereby H-89 potentiates adipocyte differentiation of 3T3-L1 cells is activation of insulin signaling that is elicited mostly by inhibiting Rho/Rho kinase pathway.     

Nicotine activates the extracellular signal-regulated kinase 1/2 via the alpha7 nicotinic acetylcholine receptor and protein kinase A, in SH-SY5Y cells and hippocampal neurones

Neuronal nicotinic acetylcholine receptors (nAChR) can modulate many cellular mechanisms, such as cell survival and memory processing, which are also influenced by the serine/threonine protein kinases ERK1/2. In SH-SY5Y cells and hippocampal neurones, nicotine (100 microM) increased the activity of ERK1/2. This effect was Ca2+ dependent, and prevented by the alpha7 nAChR antagonist alpha-bungarotoxin (alpha-Bgt) and an inhibitor (PD98059) of the upstream kinase MEK. To determine the intervening steps linking Ca2+ entry to MEK-ERK1/2 activation, inhibitors of Ca2+-dependent kinases were deployed. In SH-SY5Y cells, selective blockers for PKC (Ro 31-8220), CaM kinase II (KN-62) or PI3 kinase (LY 294002) failed to inhibit the nicotine-evoked increase in ERK1/2 activity. In contrast, two structurally different inhibitors of PKA (KT 5720 and H-89) completely prevented the nicotine-dependent increase in ERK1/2 activity. Inhibition of the nicotine-evoked increase in ERK1/2 activity by H-89 was also observed in hippocampal cultures. Down stream of PKA, the activity of B-Raf was significantly decreased by nicotine in SH-SY5Y cells, as determined by direct measurement of MEK1 phosphorylation or in vitro kinase assays, whereas the modulation of MEK1 phosphorylation by Raf-1 tended to increase. Thus, this study provides evidence for a novel signalling route coupling the stimulation of alpha7 nAChR to the activation of ERK1/2, in a Ca2+ and PKA dependent manner.     

Alpha(1)-AR-induced positive inotropic response in heart is dependent on myosin light chain phosphorylation

The possible involvement of different kinases in the alpha(1)-adrenoreceptor (AR)-mediated positive inotropic effect (PIE) was investigated in rat papillary muscle and compared with beta-AR-, endothelin receptor- and phorbol ester-induced changes in contractility. The alpha(1)-AR-induced PIE was not reduced by the inhibitors of protein kinase C (PKC), MAPK (ERK and p38), phosphatidyl inositol 3-kinase, or calmodulin kinase II. However, PKC inhibition attenuated the effect of phorbol 12-myristate 13-acetate (PMA) on contractility. alpha(1)-AR-induced PIE was reduced by approximately 90% during inhibition of myosin light chain kinase (MLCK) by 1-(5-chloronaphthalene-1-sulfonyl)1H-hexahydro-1,4-diazepine (ML-9). Endothelin-induced PIE was also reduced by ML-9, but ML-9 had no effect on beta-AR-induced PIE. The Rho kinase inhibitor Y-27632 also reduced the alpha(1)-AR-induced PIE. The alpha(1)-AR-induced PIE in muscle strips from explanted failing human hearts was also sensitive to MLCK inhibition. alpha(1)-AR induced a modest increase in (32)P incorporation into myosin light chain in isolated rat cardiomyocytes. This effect was eliminated by ML-9. The PIE of alpha(1)-AR stimulation seems to be dependent on MLCK phosphorylation.     

G(q)-dependent signalling by the lysophosphatidic acid receptor LPA(3) in gastric smooth muscle: reciprocal regulation of MYPT1 phosphorylation by Rho kinase and cAMP-independent PKA

The present study characterized the signalling pathways initiated by the bioactive lipid, LPA (lysophosphatidic acid) in smooth muscle. Expression of LPA(3) receptors, but not LPA(1) and LPA(2), receptors was demonstrated by Western blot analysis. LPA stimulated phosphoinositide hydrolysis, PKC (protein kinase C) and Rho kinase (Rho-associated kinase) activities: stimulation of all three enzymes was inhibited by expression of the G(alphaq), but not the G(alphai), minigene. Initial contraction and MLC(20) (20 kDa regulatory light chain of myosin II) phosphorylation induced by LPA were abolished by inhibitors of PLC (phospholipase C)-beta (U73122) or MLCK (myosin light-chain kinase; ML-9), but were not affected by inhibitors of PKC (bisindolylmaleimide) or Rho kinase (Y27632). In contrast, sustained contraction, and phosphorylation of MLC(20) and CPI-17 (PKC-potentiated inhibitor 17 kDa protein) induced by LPA were abolished selectively by bisindolylmaleimide. LPA-induced activation of IKK2 {IkappaB [inhibitor of NF-kappaB (nuclear factor kappaB)] kinase 2} and PKA (protein kinase A; cAMP-dependent protein kinase), and degradation of IkappaBalpha were blocked by the RhoA inhibitor (C3 exoenzyme) and in cells expressing dominant-negative mutants of IKK2(K44A) or RhoA(N19RhoA). Phosphorylation by Rho kinase of MYPT1 (myosin phosphatase targeting subunit 1) at Thr(696) was masked by phosphorylation of MYPT1 at Ser(695) by PKA derived from IkappaB degradation via RhoA, but unmasked in the presence of PKI (PKA inhibitor) or C3 exoenzyme and in cells expressing IKK2(K44A). We conclude that LPA induces initial contraction which involves activation of PLC-beta and MLCK and phosphorylation of MLC(20), and sustained contraction which involves activation of PKC and phosphorylation of CPI-17 and MLC(20). Although Rho kinase was activated, phosphorylation of MYPT1 at Thr(696) by Rho kinase was masked by phosphorylation of MYPT1 at Ser(695) via cAMP-independent PKA derived from the NF-kappaB pathway.     

N(omega)-nitro-L-arginine inhibits inducible HSP-70 via Ca(2+), PKC, and PKA in human intestinal epithelial T84 cells

The nitric oxide (NO) synthase inhibitor N(omega)-nitro-L-arginine (L-NNA) inhibits heat stress (HS)-induced NO production and the inducible 70-kDa heat shock protein (HSP-70i) in many rodent organs. We used human intestinal epithelial T84 cells to characterize the inhibitory effect of L-NNA on HS-induced HSP-70i expression. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured using fura-2, and protein kinase C (PKC), and PKA activities were determined. HS increased HSP-70i mRNA and protein in T84 cells exposed to 45 degrees C for 10 min and allowed to recover for 6 h. L-NNA treatment for 1 h before HS inhibited the induction of HSP-70i mRNA and protein, with an IC(50) of 0.0471 +/- 0.0007 microM. Because the HS-induced increase in HSP-70i mRNA and protein is Ca(2+) dependent, we measured [Ca(2+)](i) after treating cells with L-NNA. L-NNA at 100 microM significantly decreased resting [Ca(2+)](i). Likewise, treatment with 1 microM GF-109203X or H-89 (inhibitors of PKC and PKA, respectively) for 30 min also significantly decreased [Ca(2+)](i) and inhibited HS-induced increase in HSP-70i. GF-109203X- or H-89-treated cells failed to respond to L-NNA by further decreasing [Ca(2+)](i) and HSP-70i. L-NNA effectively blocked heat shock factor-1 (HSF1) translocation from the cytosol to the nucleus, a process requiring PKC phosphorylation. These results suggest that L-NNA inhibits HSP-70i by reducing [Ca(2+)](i) and decreasing PKC and PKA activity, thereby blocking HSF1 translocation from the cytosol to the nucleus.     

Activation of phospholipase D by 8-Br-cAMP occurs through novel pathway involving Src, Ras, and ERK in human endometrial stromal cells

We investigated the mechanism of 8-Br-cAMP-mediated phospholipase D (PLD) activation using a primary cell culture system of human endometrial stromal cells (ES cells). PLD activity was increased by the treatment of ES cells with 8-Br-cAMP, maximally at 5 min. To determine whether the effects of 8-Br-cAMP on PLD occurred as a consequence of PKC activation, ES cells were preincubated for 15 min with RO320432 (1 microM) and GF109203X (1 microM), the PKC inhibitors, or they were pretreated for 24h with phorbol myristate acetate (100 nM) to downregulate PKC. However, these treatments had no effects on PLD activation induced by 8-Br-cAMP. Furthermore, 8-Br-cAMP had no effects on the subcellular distribution of PKC alpha and PKC betaI, confirming no involvement of PKC. 8-Br-cAMP activated ERK1/2, maximally at 5 min, and PD98059 (MEK inhibitor: 50 microM) and transfection of ES cells with dominant negative (DN)-MEK completely inhibited 8-Br-cAMP-induced PLD activation, suggesting that ERK1/2 mediates the PLD activation. To investigate the involvement of protein kinase A (PKA), Src, and Ras in 8-Br-cAMP-induced PLD activation, we used PKA inhibitor, H89 and Rp-cAMPs, and transfections of DN-Src and DN-Ras. H-89 and Rp-cAMPs completely blocked 8-Br-cAMP-mediated PLD and ERK activation, implying the involvement of PKA in this PLD activation. In addition, transfection of ES cells with DN-Src, or DN-Ras partially inhibited 8-Br-cAMP-induced ERK1/2 and consequently PLD activation, whereas cotransfection of DN-Src and DN-Ras completely inhibited ERK1/2 and PLD activation, suggesting that Src and Ras independently regulate ERK/PLD activation. Taken together, these results demonstrate a novel pathway in ES cells that 8-Br-cAMP activate PLD through PKA and ERK1/2 and this ERK/PLD activation by 8-Br-cAMP is mediated by Src and Ras, separately.     

Examination of the involvement of protein kinase A in D2 dopamine receptor antagonist-induced immediate early gene expression

Immediate early genes (IEGs) are induced by different signaling pathways. It has been proposed that D2 dopamine receptor blockade induces IEG expression through activation of protein kinase A (PKA), although few studies have examined this issue in vivo. We infused the PKA inhibitor H-89 into the striatum of male rats, followed 30 min later by systemic administration of eticlopride. Eticlopride-induced c-fos and zif268 mRNA expression in striatum was not blocked by H-89. In addition, eticlopride did not produce measurable levels of PKA activity in striatum, whereas the cAMP activator Sp-8-Br-cAMPs increased levels of activated PKA. Neither the adenosine A2a receptor agonist CGS 21680 nor the phosphodiesterase-4 inhibitor rolipram, each of which should increase PKA activation, potentiated eticlopride-induced IEG expression. To test whether other signaling pathways are involved in eticlopride-mediated gene induction, we also infused inhibitors of the mitogen-activated and calcium/calmodulin-dependent protein kinases into animals and then treated them with eticlopride. The data suggest that eticlopride-induced IEG expression is not solely dependent on these kinases either. These data suggest that PKA activation may not be necessary for induction of IEGs by D2 dopamine receptor antagonists and that other intracellular signaling pathways may be involved.