Direct Activation Of Protein Kinase C Research Articles

Implication of endogenous β-endorphin in the inhibition of the morphine-induced rewarding effect by the direct activation of spinal protein kinase C in mice

It has often been proposed that opioid addiction does not arise as a consequence of opioid treatment for pain. Recently, we demonstrated that activated protein kinase C (PKC) in the spinal cord associated with chronic pain-like hyperalgesia suppressed the morphine-induced rewarding effect in mice. In the present study, we investigated whether a gene deletion for an endogenous mu-opioid peptide beta-endorphin could affect pain-like behavior and the suppression of the morphine-induced rewarding effect by the direct activation of PKC in the spinal cord. We found that activation of spinal PKC by intrathecal (i.t.) treatment with phorbol 12,13-dibutyrate (PDBu), a specific PKC activator, caused thermal hyperalgesia, pain-like behaviors and suppression of the morphine-induced rewarding effect. This suppression of morphine reward was eliminated in mice that lacked beta-endorphin. In contrast, thermal hyperalgesia and pain-like behaviors were not affected in beta-endorphin knockout mice. These results suggest that the activation of PKC in the spinal cord may play an essential role in the suppression of the morphine-induced rewarding effect in mice with neuropathic pain through the constant release of beta-endorphin.

206. Differential regulation of activin and inhibin production by interleukin 1 (IL1), transforming growth factor β1 (TGFβ1) and protein kinase C (PKC) in the Sertoli cell and granulosa cell

Activin and inhibin are gonadal regulatory proteins comprising an α-subunit and either a βA-subunit or βB-subunit (inhibin A or B), or two βA-subunits (activin A). Synthesis of the α-subunit, and the inhibins, is regulated by FSH via cAMP/protein kinase A. Regulation of the β-subunits in the gonads is less well defined, but the IL1/MAP kinase, TGFβ /Smad and PKC pathways have been implicated. Sertoli cells and granulosa cells were isolated from 18–22 day-old Sprague-Dawley rats under standard conditions and cultured with IL1, TGFβ1 and the PKC agonists, gonadotrophin releasing hormone (GnRH) or phorbol myristate acetate (PMA). Activin A, inhibin A and inhibin B were measured in culture medium (at 48h) by ELISA. Subunit mRNA expression was measured in cell extracts (at 4 h and 8h) using quantitative RT–PCR. IL1 stimulated βA-subunit and activin A production and inhibited α-subunit and βB-subunit expression and inhibin B production in Sertoli cells, but had no effect in granulosa cells. TGFβ1 stimulated activin A in both cell types, as well as the inhibins in granulosa cells. Surprisingly, TGFβ1 had no effect on Sertoli cell α-subunit or βA-subunit mRNA expression, but did cause a slight reduction of βB-subunit expression. GnRH increased activin A and inhibin A, but not inhibin B, production by granulosa cells and had no effect on Sertoli cells, which lack the GnRH receptor. However, direct activation of PKC by PMA stimulated βA-subunit mRNA expression and activin A production and decreased βB-subunit and inhibin B production by Sertoli cells, with marginal effects on inhibin A. These results indicate that activation of the TGFβ or PKC signalling pathways preferentially stimulates βA-subunit expression and/or translation, leading to increased activin A secretion by Sertoli cells and both activin A and inhibin A secretion by granulosa cells. The ability of IL1 to stimulate activin A is confined to the Sertoli cell.

(−)-Epigallocatechin gallate suppresses endothelin-1-induced interleukin-6 synthesis in osteoblasts: Inhibition of p44/p42 MAP kinase activation

We previously showed that endothelin-1 (ET-1) stimulates the synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells, and that protein kinase C (PKC)-dependent p44/p42 mitogen-activated protein (MAP) kinase plays a part in the IL-6 synthesis. In the present study, we investigated the effect of (−)-epigallocatechin gallate (EGCG), one of the major flavonoids containing in green tea, on ET-1-induced IL-6 synthesis in osteoblasts and the underlying mechanism. EGCG significantly reduced the synthesis of IL-6 stimulated by ET-1 in MC3T3-E1 cells as well primary cultured mouse osteoblasts. SB203580, a specific inhibitor of p38 MAP kinase, but not SP600125, a specific SAPK/JNK inhibitor, suppressed ET-1-stimulated IL-6 synthesis. ET-1-induced phosphorylation of p38 MAP kinase was not affected by EGCG. On the other hand, EGCG suppressed the phosphorylation of p44/p42 MAP kinase induced by ET-1. Both the IL-6 synthesis and the phosphorylation of p44/p42 MAP kinase stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA), a direct activator of PKC, were markedly suppressed by EGCG. The phosphorylation of MEK1/2 and Raf-1 induced by ET-1 or TPA were also inhibited by EGCG. These results strongly suggest that EGCG inhibits ET-1-stimulated synthesis of IL-6 via suppression of p44/p42 MAP kinase pathway in osteoblasts, and the inhibitory effect is exerted at a point between PKC and Raf-1 in the ET-1 signaling cascade.

Protein kinase signalling requirements for metabotropic action of kainate receptors in rat CA1 pyramidal neurones

Hippocampal pyramidal neurones display a Ca(2+)-dependent K(+) current responsible for the slow afterhyperpolarization (I(sAHP)), a prominent regulator of excitability. There is considerable transmitter convergence onto I(sAHP) but little information about the interplay between the kinase-based transduction mechanisms underlying transmitter action. We have added to existing information about the role of protein kinase C (PKC) in kainate receptor actions by demonstrating that direct postsynaptic activation of PKC with either 1-oleoyl-2-acethylsn-glycerol (OAG) or indolactam is sufficient to inhibit I(sAHP). The physiological correlate of this action - activation of PKC by kainate receptors - requires G alpha(i/o) proteins. The cAMP/PKA system is well documented to subserve the actions of monoamine transmitters. We have found an additional role for the cAMP/PKA system as a requirement for kainate receptor-mediated inhibition of I(sAHP). Inhibition of adenylyl cyclase with dideoxyadenosine or PKA with either H89 or RpcAMPs blocked kainate receptor-mediated actions but did not prevent the actions of direct PKC activation with either OAG or indolactam. We therefore propose that the PKA requirement is upstream from the actions of PKC. We additionally report a downstream link in the form of increased mitogen-activated protein (MAP) kinase activity, which may explain the long duration of metabotropic actions of kainate receptors on I(sAHP).

Activation of Mitogen-Activated Protein Kinase by Hepatocyte Growth Factor Is Stimulated by Both α1- and β2-Adrenergic Agonists in Primary Cultures of Adult Rat Hepatocytes

We investigated the effects of α1- and β2-adrenergic agonists on hepatocyte growth factor (HGF)-stimulated mitogen-activated protein kinase (MAPK) isoforms in primary cultures of adult rat hepatocytes. Hepatocytes were isolated and cultured with HGF (5 ng/ml) and/or α- and β-adrenergic agonists. Phosphorylated MAPK isoforms (p42 and p44 MAPK) were detected by Western blotting analysis using anti-phospho-MAPK antibody. The results show that HGF increased phosphorylation of p42 MAPK by 2.2-fold within 3 min. The HGF-induced MAPK activation was abolished by AG1478 treatment (10−7 M). The MEK (MAPK kinase) inhibitor 10−M) completely inhibited the HGF-dependent increase in MAPK activity. Phenylephrine (10−6 M alone had no effect in the absence of HGF, but significantly increased p42 MAPK induction by HGF. Moreover, the cell-permeable cAMP analog, 8-bromo cAMP (10−7 M), and phorbol 12-myristate 13 acetate (10−7 M) potentiated HGFinduced MAPK phosphorylation. The effects of these analogs were antagonized by the protein kinase A (PKA) inhibitor H-89 (10−7 M) and the protein kinase C (PKC) inhibitor sphingosine (10−6 M), respectively. These results suggest that direct or indirect activation of both PKA and PKC represent a positive regulatory mechanism for stimulating MAPK induction by HGF.

Cross-regulation of VPAC2 receptor desensitization by M3 receptors via PKC-mediated phosphorylation of RKIP and inhibition of GRK2

In gastrointestinal smooth muscle cells, VPAC(2) receptor desensitization is exclusively mediated by G protein-coupled receptor kinase 2 (GRK2). The present study examined the mechanisms by which acetylcholine (ACh) acting via M(3) receptors regulates GRK2-mediated VPAC(2) receptor desensitization in gastric smooth muscle cells. Vasoactive intestinal peptide induced VPAC(2) receptor phosphorylation, internalization, and desensitization in both freshly dispersed and cultured smooth muscle cells. Costimulation with ACh in the presence of M(2) receptor antagonist (i.e., activation of M(3) receptors) inhibited VPAC(2) receptor phosphorylation, internalization, and desensitization. Inhibition was blocked by the selective protein kinase C (PKC) inhibitor bisindolylmaleimide, suggesting that the inhibition was mediated by PKC, derived from M(3) receptor activation. Similar results were obtained by direct activation of PKC with phorbol myristate acetate. In the presence of the M(2) receptor antagonist, ACh induced phosphorylation of Raf kinase inhibitory protein (RKIP), increased RKIP-GRK2 association, decreased RKIP-Raf-1 association, and stimulated ERK1/2 activity, suggesting that, upon phosphorylation by PKC, RKIP dissociates from its known target Raf to associate with, and block the activity of, GRK2. In muscle cells expressing RKIP(S153A), which lacks the PKC phosphorylation site, RKIP phosphorylation was blocked and the inhibitory effect of ACh on VPAC(2) receptor phosphorylation, internalization, and desensitization and the stimulatory effect on ERK1/2 activation were abolished. This study identified a novel mechanism of cross-regulation of G(s)-coupled receptor phosphorylation and internalization by G(q)-coupled receptors. The mechanism involved phosphorylation of RKIP by PKC, switching RKIP from association with Raf-1 to association with, and inhibition of, GRK2.

Metformin reduces angiotensin-mediated intracellular production of reactive oxygen species in endothelial cells through the inhibition of protein kinase C

Oxidative stress plays a major role in the pathogenesis and in the onset of macrovascular complications of diabetes. We previously reported that the antihyperglycaemic drug metformin was able to decrease significantly intracellular reactive oxygen species (ROS) production of bovine aortic endothelial cells (BAEC) activated by high levels of glucose and angiotensin II (ANG). The aim of the present study was to investigate whether the antioxidant effect of metformin on BAEC could be mediated through a modulation of protein kinase C (PKC) activity, which plays a key role in the pathophysiology of diabetes. The effects of metformin on intracellular ROS production, PKC translocation and activity were studied on endothelial cells stimulated by PMA (a direct PKC activator), ANG or high levels of glucose as pathophysiological stimuli of endothelial dysfunction in diabetes. We showed that metformin decreased ROS production on PMA-, ANG- and glucose-stimulated BAEC in a similar manner to that obtained by PKC specific inhibitors (calphostin C, chelerythrine) alone. On the other hand, metformin reduced both PKC membrane translocation and kinase activity in ANG-stimulated cells. In PMA-activated cells, metformin reduced membrane PKC activity but we did not observe any alteration of PKC membrane translocation. Finally, in vitro incubation with purified PKC indicated that metformin had no direct effect on PKC activity. Taken together, our results suggest that metformin exerted intracellular antioxidant properties by decreasing ROS production through the inhibition of PKC activity.

Activation of PKC‐ε occurs upstream of mitoK ATP channel activation in the mechanism of diazoxide‐induced cardioprotection

In this study, we examined which isoforms of protein kinase C (PKC) family are modulated by diazoxide (DZX) and evaluated their roles in hypoxia-induced apoptotic cell death mediated via mitochondrial death pathway. We further investigated the interrelationship between PKC isoforms and the mitochondrial ATP-sensitive potassium (mitoKATP) channel in the mechanism of cardioprotection by DZX in rat heart-derived H9c2 cell lines and neonatal rat cardiomyocytes. Treatment with 100 μM DZX induced isoform-specific translocation of PKC-ε from the cytosolic to the mitochondrial fraction in both H9c2 and neonatal cardiomyocytes. A specific blockade of PKC-ε translocation by εV1-2 peptide abrogated the anti-apoptotic effect of DZX, suggesting an essential role of PKC-ε in cardioprotection. DZX-induced flavoprotein oxidation was completely inhibited by 10 μM εV1-2, and DZX-induced translocation of PKC-ε remained unaltered after treatment with 100 μM 5-hydroxydecanoate (5-HD). A direct PKC activation by phorbol ester mimicked flavoprotein-oxidizing effect of DZX, and this effect was completely blocked by εV1-2 or 5-HD. During hypoxia, the treatment with 100 μM DZX prevented the increase in mitochondrial Ca2+, mitochondrial depolarization and cytochrome c release induced by hypoxia, and all these effects of DZX were almost completely blocked by εV1-2 as well as 5-HD. In summary, these results suggest that DZX induces translocation of PKC-ε as an upstream signaling molecule for the mitoKATP channel, leading to cardioprotection through inhibition of mitochondrial death pathway.

Loss of metabotropic glutamate receptor‐mediated regulation of glutamate transport in chemically activated astrocytes in a rat model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motor neurones accompanied by intense gliosis in lesioned areas of the brain and spinal cord. Glutamate-mediated excitotoxicity resulting from impaired astroglial uptake constitutes one of the current pathophysiological hypotheses explaining the progression of the disease. In this study, we examined the regulation of glutamate transporters by type 5 metabotropic glutamate receptor (mGluR5) in activated astrocytes derived from transgenic rats carrying an ALS-related mutated human superoxide dismutase 1 (hSOD1(G93A)) transgene. Cells from transgenic animals and wild-type littermates showed similar expression of glutamate-aspartate transporter and glutamate transporter 1 (GLT-1) after in vitro activation, whereas cells carrying the hSOD1 mutation showed a three-fold higher expression of functional mGluR5, as observed in the spinal cord of end-stage animals. In cells from wild-type animals, (S)-3,5-dihydroxyphenylglycine (DHPG) caused an immediate protein kinase C (PKC)-dependent up-regulation of aspartate uptake that reflected the activation of GLT-1. Although this effect was mimicked in both cultures by direct activation of PKC using phorbol myristate acetate, DHPG failed to up-regulate aspartate uptake in cells derived from the transgenic rats. The failure of activated mGluR5 to increase glutamate uptake in astrocytes derived from this animal model of ALS supports the theory of glutamate excitotoxicity in the pathogenesis of the disease.

Hydrogen peroxide causes uncoupling of dopamine D1-like receptors from G proteins via a mechanism involving protein kinase C and G-protein-coupled receptor kinase 2

Dopamine, via activation of D1-like receptors, inhibits Na,K-ATPase and Na,H-exchanger in renal proximal tubules and promotes sodium excretion. This effect of dopamine is not seen in conditions associated with oxidative stress such as hypertension, diabetes, and aging due to uncoupling of D1-like receptors from G proteins. To identify the role of oxidative stress in uncoupling of the D1-like receptors, we utilized primary cultures from rat renal proximal tubules. Hydrogen peroxide (H 2O 2), an oxidant, treatment to the cell cultures increased the level of malondialdehyde, a marker of oxidative damage. Further, H 2O 2 decreased membranous D1-like receptor numbers and proteins, D1-like agonist (SKF 38393)-mediated [ 35S]GTPγS binding and SKF 38393-mediated inhibition of Na,K-ATPase. Moreover, H 2O 2 treatment to the cultures caused membranous translocation of G-protein-coupled receptor kinase 2 (GRK 2) and increased serine phosphorylation of D1A receptors accompanied by an increase in protein kinase C (PKC) activity. Interestingly, PKC inhibitors blocked the H 2O 2-mediated stimulation of GRK 2 and serine phosphorylation of D1A receptors. Further, GRK 2 antisense but not scrambled oligonucleotides attenuated the effect of H 2O 2 on membranous expression of GRK 2. Moreover, direct activation of PKC with phorbol ester (PMA) resulted in reduction of SKF 38393-mediated [ 35S]GTPγS binding. We conclude that H 2O 2 stimulates PKC leading to the activation of GRK 2, which causes serine phopshorylation of D1A receptors and receptor G-protein uncoupling in these cells, resulting in impairment in D1-like receptor function.

Protein kinase C-dependent NF-kappaB activation is altered in T cells by chronic stress.

Chronic stress has been associated with impaired immune function. In this work we studied the effect of chronic mild stress (CMS) exposure on the early intracellular pathways involved in T cells after stimulation with mitogen. We found that mitogen stimulation of T lymphocytes from CMS-exposed mice resulted in a reduction of the intracellular [Ca2+] rise, an impairment of growth-promoting protein kinase C (PKC) activation, a lower NF-kappaB activation and an increase in the inhibitory cAMP-protein kinase A (PKA) pathway activity with respect to those found in control lymphocytes. However, T cell activation with the direct PKC activator phorbol 12-myristate 13-acetate plus calcium ionophore led to a similar proliferative response in both CMS and control lymphocytes, indicating that signals downstream of PKC would not be affected by stress. In summary, our results show that chronic stress induced an alteration in T cell early transduction signals that result in an impairment of the proliferative response.

Protein Kinase C Activity is Necessary for Estrogen-Induced Erk Phosphorylation in Neocortical Explants

Our laboratory showed previously that estrogen activates ERK in neocortical cultures. To further elucidate the precise signaling sequelae that lead to estrogen-induced ERK activity, we evaluated the involvement of protein kinase C (PKC). We found that neocortical explants expressed primarily PKC gamma and PKC epsilon. Consistent with the involvement of PKC in mediating estrogen-induced ERK phosphorylation, we found that estrogen treatment induced translocation of these PKC isoforms to the plasma membrane. Importantly, inhibition of these isoforms abolished the ability of estrogen to phosphorylate ERK. While direct activation of PKC mimicked the effect of estrogen on ERK, both in pattern of activation and resulting intraneuronal distribution of ERK, PKC-induced ERK phosphorylation required the activity of MEK but not B-Raf. Collectively, these data suggest a critical role for PKC in mediating estrogen induction of ERK activation in the developing brain via a MEK-dependent but B-Raf-independent pathway.

Tiludronate inhibits prostaglandin F 2α-induced vascular endothelial growth factor synthesis in osteoblasts

We have previously reported that prostaglandin F 2α (PGF 2α) activates p44/p42 mitogen-activated protein (MAP) kinase through protein kinase C (PKC), resulting in the synthesis of vascular endothelial growth factor (VEGF) in osteoblast-like MC3T3-E1 cells, and that incadronate, a bisphosphonate, amplifies the VEGF synthesis. In the present study, we investigated the effects of tiludronate and etidronate, other bisphosphonates, on the PGF 2α-stimulated VEGF synthesis in these cells. Tiludronate reduced the synthesis of VEGF induced by PGF 2α. The PGF 2α-stimulated phosphorylation of p44/p42 MAP kinase was suppressed by tiludronate. On the other hand, etidronate affected neither the VEGF synthesis nor the phosphorylation of p44/p42 MAP kinase elicited by PGF 2α. Tiludronate attenuated the phosphorylation of both Raf-1 and MEK1/2 induced by PGF 2α. The VEGF synthesis stimulated by 12- O-tetradecanoylphorbol-13-acetate (TPA), a direct activator of PKC, was suppressed by tiludronate. The TPA-induced phosphorylations of Raf-1, MEK1/2 and p44/p42 MAP kinase were inhibited by tiludronate. These results strongly suggest that tiludronate but not etidronate suppresses the PGF 2α-stimulated VEGF synthesis in osteoblasts, and that the effect of tiludronate is exerted at the point between PKC and Raf-1.

Nicotine Exposure, Mimicked Smoking, Directly and Indirectly Enhanced Protein Kinase C Activity in Isolated Canine Basilar Artery, Resulting in Enhancement of Arterial Contraction

Cigarette smoking is a significant risk factor in the incidence of cerebrovascular disorders. Among the many compounds in cigarette smoke, nicotine is considered to most significantly affect cerebral arterial tone. The purpose of this study is to investigate precise pharmacological effects of nicotine on the regulation of cerebral arterial tone. To mimic smoking, a low concentration of nicotine (10(-6) mol/L), which is equivalent to the serum level of habitual smokers, was treated for 1 hour in an isometric tension study and for 24 hours in a study using cultured vascular endothelial cells (VECs). Using the canine basilar artery, the effect of nicotine on uridine 5'-triphosphate (UTP)-induced vasoconstriction was examined in the isometric tension study. Protein kinase C (PKC) activity in the canine basilar artery was measured by enzyme immunoassay. Endothelial function was assessed by endothelium-dependent vasodilatation and endogenous nitric oxide (NO) synthesis in VECs using a fluorescent indicator, diaminofluorescein-FM diacetate (DAF-FM/DA). Nicotine significantly enhanced UTP-induced contraction and PKC activity in the artery, and attenuated endothelium-dependent vasodilatation and NO synthesis in VECs. Because PKC activity was increased by de-endothelialization itself, endothelial dysfunction by nicotine enhances PKC activity. Because PKC was further activated by nicotine even in the de-endothelialized artery, nicotine directly affects PKC activities in smooth muscle. These results indicate that nicotine potentiates contractile response through direct and indirect PKC activation in the canine basilar artery.

Protein Kinase C Modulates Agonist-sensitive Release of Ca2+ from Internal Stores in HEK293 Cells Overexpressing the Calcium Sensing Receptor

This study examined the mechanism of Ca2+ entry and the role of protein kinase C (PKC) in Ca2+ signaling induced by activation of the calcium sensing receptor (CaR) in HEK293 cells stably expressing the CaR. We demonstrate that influx of Ca2+ following CaR activation exhibits store-operated characteristics in being associated with Ca2+ store depletion and inhibited by 2-aminoethoxydiphenyl borate. Inhibition of PKC with GF109203X, Go6983, or Go6976 and down-regulation of PKC activity enhanced the release of Ca2+ from internal stores in response to the polyvalent cationic CaR agonist neomycin, whereas activation of PKC with acute 12-O-tetradecanoylphorbol-13-acetate treatment decreased the release. In contrast, overexpression of wild type PKC-alpha or -epsilon augmented the neomycin-induced release of Ca2+ from internal stores, whereas dominant negative PKC-epsilon strongly decreased the release, but dominant negative PKC-alpha had little effect. Prolonged treatment of cells with 12-O-tetradecanoylphorbol-13-acetate effectively down-regulated immunoreactive PKC-alpha but had little effect on the expression of PKC-epsilon. Together these results indicate that diacylglycerol-responsive PKC isoforms differentially influence CaR agonist-induced release of Ca2+ from internal stores. The fundamentally different results obtained when overexpressing or functionally down-regulating specific PKC isoforms as compared with pharmacological manipulation of PKC activity indicate the need for caution when interpreting data obtained with the latter approach.

Acute Hyperglycaemia Enhances Platelet Activation: Involvement of Multiple Mechanisms.

Diabetes mellitus is associated with platelet dysfunction, and hypoglycemic treatments may counteract this diabetic alteration. We thus studied how acute hyperglycemia influences platelet function. Fast blood (n=20) was incubated with different levels of glucose (5, 15, and 30 mM) for 5 min, and without or with in vitro stimuli. Platelet activation was monitored by whole blood flow cytometry, while platelet aggregation was also measured using impedance aggregometry. Blood glucose levels had little influence on unstimulated platelets. Hyperglycemia enhanced ADP- and TRAP-induced platelet P-selectin expression. Hyperglycemia also increased TRAP-induced platelet fibrinogen binding. High glucose levels mildly increased ADP- and TRAP-induced platelet-leukocyte aggregation. The blockade of protein kinase C (PKC) slightly attenuated ADP-induced, and markedly inhibited TRAP-induced platelet activation. PKC blockade had, however, little effect on hyperglycemia effect. Platelet P-selectin expression induced by the direct PKC activator phorbol 12-myristate 13-acetate (PMA) was not augmented by hyperglycemia. Superoxide anion scavenging by superoxide dismutase (SOD) reduced, whistle cyclo-oxygenase (COX) inhibition by naproxen did not reduced the enhancing effect of platelet activation by hyperglycemia. In conclusion, acute hyperglycemia enhances platelet activation in whole blood. Superoxide anions contribute importantly to hyperglycemia-enhanced platelet activation, while other mechanisms not identified presently also contribute to hyperglycemic effects.

Differential participation of protein kinase C and Rho kinase in α1-adrenoceptor mediated contraction in rat arteries

The major functional alpha1-adrenoceptor in the rat aorta is of the alpha1Dsubtype and that in the caudal artery is of the alpha1A subtype. In the present study, the participation of protein kinase C (PKC) and Rho kinase (RhoK) in contractile responses to stimulation of the alpha1-adrenoceptors in these two arteries was investigated. Both the PKC inhibitor Ro-318220 and the RhoK inhibitor Y-27632 significantly blocked contractile responses of the aorta to phenylephrine (PE) and the selective alpha1A-adrenoceptor agonist A61603. When used in combination, the inhibitors had an additive blocking effect. In the caudal artery, Y-27632 but not Ro-318220 inhibited contractile responses to PE and A61603, and, in combination, the antagonism produced was no greater than that by Y-27632 alone. Contractile responses to direct activation of PKC with phorbol 12,13-dibutyrate were much smaller and levels of CPI-17 (PKC-activated protein phosphatase inhibitor of 17 kDa) were much lower in the caudal artery than the aorta. The results suggest that both PKC and RhoK contribute independently to contractile responses to stimulation of alpha1D-adrenoceptors in the aorta. However, RhoK, but not PKC, participates in contractile responses to stimulation of alpha1A-adrenoceptors in the caudal artery. This difference may largely be due to differences between the two arteries in the extent to which PKC participates in contraction.

Corticotropin-releasing factor and acute stress prolongs serotonergic regulation of GABA transmission in prefrontal cortical pyramidal neurons.

The stress-related neuropeptide corticotropin-releasing factor (CRF) and the serotonin system are both critically involved in the pathophysiology of mental disorders, including anxiety and depression. To understand the potential link between them, we investigated the impact of CRF on 5-HT functions in pyramidal neurons of the prefrontal cortex (PFC), a brain region that is crucial for the control of emotion and cognition. One prominent function of serotonin in PFC is to regulate GABAergic inhibitory transmission, as indicated by a 5-HT-induced large, desensitizing (approximately 4 min) enhancement of the amplitude and frequency of spontaneous IPSCs (sIPSCs). In PFC slices exposed to CRF treatment, the regulation of sIPSCs by 5-HT was significantly prolonged (8-10 min), and this effect of CRF was blocked by treatment with the competitive CRF receptor antagonist alpha-helical CRF9-41 and with the CRF-R1-specific antagonist astressin. Inhibiting phospholipase C or protein kinase C (PKC) abolished the prolongation by CRF of the effects of 5-HT on sIPSCs. In PFC slices prepared from animals previously exposed to acute stress (forced swim or elevated platform), the regulation of sIPSCs by 5-HT was significantly prolonged, mimicking the effect of CRF treatment. The stress-induced prolongation of the effects of 5-HT on sIPSCs was diminished by alpha-helical CRF9-41 treatment, mimicked by direct activation of PKC, and reversed by short-term treatment with drugs that have anxiolytic efficacy. These results show that in response to stressful stimuli, CRF alters the serotonergic regulation of GABA transmission through a mechanism that is dependent on PKC. The interaction between CRF and 5-HT may play an important role in psychiatric disorders, in which both are highly implicated.

Preconditioning: evolution of basic mechanisms to potential therapeutic strategies.

Preconditioning describes the phenomenon by which a traumatic or stressful stimulus confers protection against subsequent injury. Originally recognized in dog heart subjected to ischemic challenges, preconditioning has been demonstrated in multiple species, can be induced by various stimuli, and is applicable in different organ systems. Tremendous progress has been made elucidating the signal transduction cascade of preconditioning. Preconditioning represents a potent tissue-protective condition, and mechanistic understanding may allow safe clinical application. This review recalls the history of preconditioning and how it relates to the history of the investigation of endogenous adaptation; summarizes the current mechanistic understanding of acute preconditioning; outlines the signal transduction cascade leading to the development of delayed preconditioning; discusses preconditioning in noncardiac tissue; and explores the potential of using preconditioning clinically.

Endothelin-1 promotes Ca2+ antagonist-insensitive coronary smooth muscle contraction via activation of epsilon-protein kinase C.

Certain forms of coronary artery disease do not respond to treatment with Ca2+ channel blockers, and a role for endothelin-1 (ET-1) in Ca2+ antagonist-insensitive forms of coronary vasospasm has been suggested; however, the signaling mechanisms involved are unclear. We tested the hypothesis that a component of ET-1-induced coronary smooth muscle contraction is Ca2+ antagonist-insensitive and involves activation of protein kinase C (PKC). Cell contraction was measured in smooth muscle cells isolated from porcine coronary artery, [Ca2+]i was measured in fura-2 loaded cells, and the cytosolic and particulate fractions were examined for PKC activity and reactivity with isoform-specific PKC antibodies using Western blot analysis. In Hank's solution (1 mmol/L Ca2+), ET-1 (10(-7) mol/L) caused a transient increase in [Ca2+]i (236+/-14 nmol/L) followed by a maintained increase in [Ca2+]i (184+/-8 nmol/L) and 35% cell contraction. The Ca2+ channel blockers verapamil and diltiazem (10(-6) mol/L) abolished the maintained ET-1-induced [Ca2+]i, but only partially inhibited ET-1-induced cell contraction to 18%. The verapamil-insensitive component of ET-1 contraction was inhibited by the PKC inhibitors calphostin C and epsilon-PKCV1-2. ET-1 caused translocation of Ca2+-dependent alpha-PKC and Ca2+-independent epsilon-PKC from the cytosolic to the particulate fraction that was inhibited by calphostin C. Verapamil abolished ET-1-induced translocation of alpha-PKC, but not that of epsilon-PKC. Phorbol 12-myristate 13-acetate (10(-6) mol/L), a direct activator of PKC, caused 22% cell contraction, with no increase in [Ca2+]i, and translocation of epsilon-PKC that was inhibited by calphostin C, but not by verapamil. KCl (51 mmol/L), which stimulates Ca2+ influx, caused 35% cell contraction and increase in [Ca2+]i (291+/-11 nmol/L) that were inhibited by verapamil, but not by calphostin C, and did not cause translocation of alpha- or epsilon-PKC. In Ca2+-free (2 mmol/L EGTA) Hank's solution, ET-1 caused 15% cell contraction, with no increase in [Ca2+]i, and translocation of epsilon-PKC that were inhibited by epsilon-PKC V1-2 inhibitory peptide. Thus, a significant component of ET-1-induced contraction of coronary smooth muscle is Ca2+ antagonist-insensitive and involves activation and translocation of Ca2+-independent epsilon-PKC, and may represent a signaling mechanism of Ca2+ antagonist-resistant forms of coronary vasospasm.