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PubMed1996

Role of protein kinase C in angiotensin II-induced renal vasoconstriction in genetically hypertensive rats.

The renal vasculature of young spontaneously hypertensive rats (SHR) responds to angiotensin II (ANG II) with exaggerated vasoconstriction, due in part to defective buffering by the adenosine 3',5'-cyclic monophosphate (cAMP) pathway. In vitro studies suggest greater activation of phospholipase C and protein kinase C (PKC) in cultured mesangial cells and vascular smooth muscle cells. The present studies evaluated the role of PKC activation in renal vascular responses to ANG II receptor activation and the relative contributions in SHR vs. Wistar-Kyoto control rats (WKY). Renal blood flow was measured in 8-wk-old anesthetized SHR and WKY pretreated with indomethacin. ANG II (2 ng) injection into the renal artery produced a transient 45-50% maximum reduction of renal blood flow in both rat strains. Intrarenal infusion of either staurosporine or chelerythrine into the renal artery effectively attenuated the vasoconstriction elicited by ANG II in a dose-dependent manner, with maximum inhibition of 60-70%. The PKC inhibitory effects were significant and independent of strain. Coadministration of the PKC inhibitors produced maximal inhibition similar to that observed with one agent, suggesting action via a common pathway. In other studies, the linkage of the PKC pathway to the AT1 receptor was evaluated using sub and maximal doses of losartan to antagonize 50-80% of ANG II-induced vasoconstriction. The same degree of inhibition was observed when a PKC inhibitor was coadministered with losartan. These findings support the views that the PKC system is a major intracellular signaling pathway coupled to the AT1 receptor in renal resistance vessels and that PKC activation is involved to similar degrees in the renal vasoconstriction elicited by ANG II in young WKY and SHR. Exaggerated vascular reactivity to vasoconstrictor agents in genetically hypertensive animals is probably due to a defect in cAMP generation in the presence of a normally operating PKC pathway.

Alkaloids↗
PubMed1998

Dopamine inhibits vasopressin-dependent cAMP production in the rat cortical collecting duct.

Dopamine inhibits Na+ and water reabsorption in the rat cortical collecting duct (CCD) in the presence of arginine vasopressin (AVP). This inhibition appears to involve the D4 dopamine receptor isoform, which inhibits cAMP production; however, the D1A receptor, which stimulates cAMP production, is also expressed in the CCD. To discriminate between these opposing effects, we measured cAMP production in intact CCD segments. The basal rate of cAMP production ranged from 6.5 to 10 fmol/mm of tubule length over a 7-min incubation period, and it was unaffected by either dopamine or the D1A-specific agonist fenoldopam. AVP increased cAMP production to the range of 85-153 fmol . mm-1 . 7 min-1. Whereas neither 0.1 nor 1.0 microM fenoldopam affected AVP-dependent cAMP production, dopamine reduced it in a dose-dependent manner, achieving a maximum inhibition of 50% at 10 microM. This effect was reversed by the D4 receptor antagonist clozapine but not by pimozide or spiperone (antagonists of D2 and D3 receptors) or by calphostin C or chelerythrine (inhibitors of protein kinase C). We conclude that dopamine inhibits transepithelial Na+ transport and osmotic water permeability in the presence of AVP by inhibition of cAMP production, which is mediated by the D4 receptor isoform linked via the inhibitory G protein Gi.

Alkaloids↗
PubMed1999

Hormone-stimulated Ca2+ transport in rabbit kidney: multiple sites of inhibition by exogenous ATP.

Exogenous ATP markedly reduced 1-desamino-8-D-arginine vasopressin (dDAVP)-stimulated Ca2+ transport and cAMP accumulation in primary cultures of rabbit connecting tubule and cortical collecting duct cells. Similarly, ATP inhibited the stimulatory effect of 8-bromo-cAMP. At first sight, this is in agreement with the "classic" concept that dDAVP exerts its stimulatory effect via cAMP. However, dDAVP-stimulated Ca2+ transport was markedly reduced by the protein kinase C (PKC) inhibitor chelerythrine, reported previously to inhibit the cAMP-independent pathway responsible for parathyroid hormone-, [Arg8]vasopressin-, PGE2-, and adenosine-stimulated Ca2+ transport. Chelerythrine also inhibited the increase in Ca2+ transport evoked by the cAMP-independent A1 receptor agonist N6-cyclopentyladenosine (CPA). Downregulation of phorbol ester-sensitive PKC isoforms by chronic phorbol ester treatment has been shown before to be without effect on hormone-stimulated Ca2+ transport, indicating that the chelerythrine-inhibitable pathway consists of a phorbol ester-insensitive PKC isoform. Here, this maneuver did not affect ATP inhibition of dDAVP-stimulated Ca2+ transport and cAMP formation, while abolishing ATP inhibition of CPA-stimulated Ca2+ transport. These findings show that ATP acts via 1) a phorbol ester-sensitive PKC isoform to inhibit hormonal stimulation of Ca2+ transport at the level of the chelerythrine-inhibitable pathway involving a phorbol ester-insensitive PKC isoform and 2) a phorbol ester-insensitive mechanism to inhibit V2 receptor-mediated concomitant activation of this pathway and adenylyl cyclase.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2001

Adenosine A(2A) and A(2B) receptor activation of erythropoietin production.

We have examined the effects of adenosine receptors and protein kinases A and C in the regulation of erythropoietin (Epo) production using hepatocellular carcinoma (Hep3B) cells in culture and in vivo in normal mice under normoxic and hypoxic conditions. CGS-21680, a selective adenosine A(2A) agonist, significantly increased levels of Epo in normoxic Hep3B cell cultures and in serum of normal mice under both normoxic and hypoxic conditions. CGS-21680 also produced a significant increase in Epo mRNA levels in Hep3B cell cultures. SCH-58261, a selective adenosine A(2A) receptor antagonist, significantly inhibited the increase in medium levels of Epo in Hep3B cell cultures exposed to hypoxia (1% O(2)). Enprofylline, a selective adenosine A(2B) receptor antagonist, significantly inhibited the increase in plasma levels of Epo in normal mice exposed to hypoxia. Chelerythrine chloride, an antagonist of protein kinase C activation, significantly inhibited hypoxia-induced increases in serum levels of Epo in normal mice. A model is presented for adenosine in hypoxic regulation of Epo production that involves kinases A and C and phospholipase A(2) pathways.

Adenosine↗
PubMed2003

Mechanisms of myocardial ischemic preconditioning are age related: PKC-epsilon does not play a requisite role in old rabbits.

Data obtained from adult cohorts have implicated activation/translocation of protein kinase C (PKC)-epsilon as an important cellular mediator of myocardial infarct size reduction with ischemic preconditioning (PC). Age-related alterations in cellular signaling may, however, confound the extrapolation of mechanistic insight derived from adults to the aging population, the specific subset in which cardioprotection is undoubtedly most relevant. Accordingly, our aim was to investigate the role of PKC-epsilon as a mediator of infarct size reduction with PC in old vs. adult rabbits. In protocol 1, we assessed the effect of PKC-epsilon translocation inhibitor peptide (PKC-epsilon-TIP) and the pan-PKC inhibitor chelerythrine on infarct size reduction with PC in adult and approximately 4-yr-old rabbits, a population previously shown to exhibit definitive hallmarks of cardiovascular aging. Rabbits received 5 min of PC ischemia or a matched control period followed by 30 min of coronary artery occlusion and 3 h of reperfusion, with infarct size (delineated by tetrazolium staining) serving as the primary endpoint. In protocol 2, we obtained insight (by Western immunoblotting) into the subcellular redistribution of PKC-epsilon in response to the 5-min PC stimulus in adult and old rabbits. In adults, infarct size reduction with PC was abrogated by both PKC-epsilon-TIP and chelerythrine. However, in old rabbits, 1). PC-induced cardioprotection was maintained despite inhibitor treatment and 2). brief PC ischemia was not associated with activation/translocation of PKC-epsilon. Thus the mechanisms responsible for PC are age related in the rabbit heart, with no apparent, requisite role of PKC-epsilon in aging animals.

Aging↗
PubMed1998

Protein kinase C is required for long-lasting synaptic enhancement by the neuropeptide DRNFLRFamide in crayfish.

The FMRFamide-related neuropeptide AspArgAsnPheLeuArgPhe-NH2 (DRNFLRFamide, DF2) induces a long-lasting enhancement of synaptic transmission at neuromuscular junctions on the crayfish deep abdominal extensor muscles. Here we investigated the function of protein kinase C (PKC) in this effect because PKC has been implied in the control of long-term synaptic modulation in other systems. The general kinase antagonist staurosporine reduced both the initial increase in excitatory postsynaptic potential (EPSP) amplitude and the duration of synaptic enhancement. Unlike staurosporine, the selective PKC inhibitors, chelerythrine and bisindolylmaleimide, augmented the initial EPSP increase. However, like staurosporine, they also reduced the duration of synaptic enhancement. The PKC activator, phorbol-12-myristate 13-acetate, induced a long-lasting synaptic enhancement that was blocked by chelerythrine. These results show that synaptic enhancement by DF2 is mediated by different intracellular signaling systems that act in temporal sequence. The initial increase in EPSP amplitudes is negatively regulated by PKC and involves another, staurosporine-sensitive, kinase; whereas, the maintenance of synaptic enhancement requires PKC.

Alkaloids↗
PubMed1998

Activation of protein kinase C increases neuronal excitability by regulating persistent Na+ current in mouse neocortical slices.

Effects of the protein kinase C activating phorbol ester, phorbol 12-myristate 13-acetate (PMA), were studied in whole cell recordings from layer V neurons in slices of mouse somatosensory neocortex. PMA was applied intracellularly (100 nM to 1 microM) to restrict its action to the cell under study. In current-clamp recordings, it enhanced neuronal excitability by inducing a 10- to 20-mV decrease in voltage threshold for action-potential generation. Because spike threshold in neocortical neurons critically depends on the properties of persistent Na+ current (INaP), effects of PMA on this current were studied in voltage clamp. After blocking K+ and Ca2+ currents, INaP was revealed by applying slow depolarizing voltage ramps from -70 to 0 mV. Intracellular PMA induced a decrease in INaP at very depolarized membrane potentials. It also shifted activation of INaP in the hyperpolarizing direction, however, such that there was a significant increase in persistent inward current at potentials more negative than -45 mV. When tetrodotoxin (TTX) was added to the bath, blocking INaP and leaving only an outward nonspecific cationic current (Icat), PMA had no apparent effect on responses to voltage ramps. Thus PMA did not affect Icat, and it did not induce any additional current. Intracellular application of the inactive PMA analogue, 4 alpha-PMA, did not affect INaP. The specific protein kinase C inhibitors, chelerythrine (20 microM) and calphostin C (10 microM), blocked the effect of PMA on INaP. The data suggest that PMA enhances neuronal excitability via a protein kinase C-mediated increase in INaP at functionally critical subthreshold voltages. This novel effect would modulate all neuronal functions that are influenced by INaP, including synaptic integration and active backpropagation of action potential from the soma into the dendrites.

Alkaloids↗
PubMed2000

Dopamine D1/D5 receptor activation modulates a persistent sodium current in rat prefrontal cortical neurons in vitro.

The effects of dopamine (DA) on a persistent Na(+) current (I(NaP)) in layer V-VI prefrontal cortical (PFC) pyramidal cells were studied using whole cell voltage-clamp recordings in rat PFC slices. After blocking K(+) and Ca (2+) currents, a tetrodotoxin-sensitive I(NaP) was activated by slow depolarizing voltage ramps or voltage steps. DA modulated the I(NaP) in a voltage-dependent manner: increased amplitude of I(NaP) at potentials more negative than -40 mV, but decreased at more positive potentials. DA also slowed the inactivation process of I(NaP). The D1/D5 dopamine receptor agonists SKF 38393, SKF 81297, and dihydrexidine (3-10 microM), but not the dopamine D2/D3 receptor agonist qiunpirole (1-20 microM), mimicked the effects of DA on I(NaP). Modulation of I(NaP) by D1/D5 agonists was blocked by the D1/D5 antagonist SCH23390. Bath application of specific protein kinase C inhibitor, chelerhythrine, or inclusion of the specific protein kinase C inhibiting peptide([19-36]) in the recording pipette, but not protein kinase A inhibiting peptide([5-24]), blocked the effect of D1/D5 agonists on I(NaP). In current-clamp recordings, D1/D5 receptors activation enhanced the excitability of cortical pyramidal cells. Application of the D1/D5 agonist SKF 81297 induced a long-lasting decrease in the first spike latency in response to depolarizing current ramp. This was associated with a shift in the start of nonlinearity in the slope resistance to more negative membrane potentials. We proposed that this effect is due to a D1/D5 agonist-induced leftward shift in the activation of I(NaP). This enables DA to facilitate the firing of PFC neurons in response to depolarizing inputs.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed2002

ATP-inhibition of M current in frog sympathetic neurons involves phospholipase C but not Ins P(3), Ca(2+), PKC, or Ras.

Suppression of the voltage-activated, noninactivating K(+) conductance (M conductance; g(M)) by muscarinic agonists, P(2Y) agonists or bradykinin increases neuronal excitability. All agonist effects are mediated, at least in part, via the Gq/(11) class of G protein. We found, using whole cell or perforated patch recording from bullfrog sympathetic B neurons that ATP-induced suppression of g(M) was attenuated by the phospholipase C (PLC) inhibitor, U73122 (IC(50) approximately 0.14 microM) but not by the inactive isomer, U73343. The ability of extracellularly applied U73122 to inhibit PLC was confirmed by its antagonism of ATP-induced elevation of intracellular Ca(2+) as measured by fura-2 photometry. ATP-induced g(M) suppression was not antagonized by the protein kinase C (PKC) inhibitor, chelerythrine (5 microM extracellular +10 microM intracellular), by the Ca(2+)-ATPase inhibitor, thapsigargin (5 microM), or by inositol trisphosphate (InsP(3)) receptor antagonists, heparin (approximaterly 300 microM) or xestospongin C (1.8 microM). The effect of ATP on g(M) was thus dependent on PLC yet independent of PKC and of InsP(3)-induced release of intracellular Ca(2+). We therefore tested the involvement of a PKC-independent action of diacylglycerol (DAG) that could occur via activation of Ras. This low-molecular-weight G protein is activated following DAG binding to Ras-GRP, a neuronal Ras-GTP exchange factor. However, impairment of Ras function by culturing neurons with isoprenylation inhibitors (perillic acid, 0.1 mM, or alpha-hydroxyfarnesyl-phosphonic acid, 10 microM) failed to affect ATP-induced g(M) suppression. Inhibition of MEK (mitogen-activated protein kinase), a downstream target of Ras, by using PD 98059 (10 microM) was also ineffective. The transduction mechanism used by ATP to suppress g(M) in frog sympathetic neurons therefore differs from the PLC-independent mechanism used by muscarine and from the PLC and Ca(2+)-dependent mechanism used by bradykinin and UTP in mammalian ganglia. The possibility remains that "lipid-signaling" mechanisms, perhaps involving PLC-induced depletion of phosphatidylinositol bisphosphate, are involved in PLC-mediated inhibition of g(M) by ATP in amphibian sympathetic neurons.

Adenosine Triphosphate↗
PubMed2005

Protein kinase C zeta is required for epidermal growth factor-induced chemotaxis of human breast cancer cells.

Chemotaxis plays an important role in cancer cell metastasis. In this study, we showed that epidermal growth factor (EGF) was a more potent chemoattractant than chemokine SDF-1alpha/CXCL12 for human breast cancer cell MDA-MB-231. Different inhibitors were used to evaluate the involvement of 12 protein kinase C (PKC) isotypes in the chemotactic signaling pathway. Chelerythrine chloride, an inhibitor of all PKC isotypes, blocked chemotaxis, whereas inhibitors of classic and novel PKC, such as Gö6976, Gö6850, or calphostin C, only impaired EGF-induced chemotaxis to a minor extent by not greater-than32% inhibition. These data suggested that atypical PKC were involved. The ligand-induced actin polymerization and cell adhesion were also similarly dependent on atypical PKC. Immunofluorescent staining showed an EGF-induced, LY294002-sensitive translocation of PKCzeta from the cytosol to the plasma membrane, indicating that EGF was capable of activating PKCzeta, probably via phosphoinositide 3 kinases. A myristoylated PKCzeta pseudosubstrate blocked the chemotaxis with an IC(50) of 20 mumol/L. To expand our investigation, we further showed that in MCF-7 and T47D, two additional human breast cancer cell lines, EGF-activated PKCzeta and the PKCzeta pseudosubstrate, inhibited chemotaxis. Taken together, our data suggest that PKCzeta is an essential component of the EGF-stimulated chemotactic signaling pathway in human breast cancer cells.

Alkaloids↗
PubMed2006

Protein kinase C zeta mediates epidermal growth factor-induced growth of head and neck tumor cells by regulating mitogen-activated protein kinase.

Protein kinase C (PKC) zeta has been implicated as a mediator of epidermal growth factor (EGF) receptor (EGFR) signaling in certain cell types. Because EGFR is ubiquitously expressed in squamous cell carcinomas of the head and neck (SCCHN) and plays a key role in tumor progression, we determined whether PKCzeta is required for tumor cell proliferation and viability. Examination of total and phosphorylated PKCzeta expression in normal oral mucosa, dysplasia, and carcinoma as well as SCCHN tumor cell lines revealed a significant increase in activated PKCzeta expression from normal to malignant tissue. PKCzeta activity is required for EGF-induced extracellular signal-regulated kinase (ERK) activation in both normal human adult epidermal keratinocytes and five of seven SCCHN cell lines. SCCHN cells express constitutively activated EGFR family receptors, and inhibition of either EGFR or mitogen-activated protein kinase (MAPK) activity suppressed DNA synthesis. Consistent with this observation, inhibition of PKCzeta using either kinase-dead PKCzeta mutant or peptide inhibitor suppressed autocrine and EGF-induced DNA synthesis. Finally, PKCzeta inhibition enhanced the effects of both MAPK/ERK kinase (U0126) and broad spectrum PKC inhibitor (chelerythrine chloride) and decreased cell proliferation in SCCHN cell lines. The results indicate that (a) PKCzeta is associated with SCCHN progression, (b) PKCzeta mediates EGF-stimulated MAPK activation in keratinocytes and SCCHN cell lines, (c) PKCzeta mediates EGFR and MAPK-dependent proliferation in SCCHN cell lines; and (d) PKCzeta inhibitors function additively with other inhibitors that target similar or complementary signaling pathways.

Alkaloids↗
PubMed2006

Enhancement of UVB radiation-mediated apoptosis by sanguinarine in HaCaT human immortalized keratinocytes.

In this article, we studied the chemopreventive effects of sanguinarine on UVB-mediated responses in human HaCaT immortalized keratinocytes. For our studies, HaCaT cells were treated with a low dose (50 nmol/L) of sanguinarine for 24 hours followed by irradiation with UVB (15 or 30 mJ/cm2). Our data showed that UVB exposure, at both doses, resulted in decreased cell viability and increased apoptosis. Interestingly, pretreatment of the cells with sanguinarine caused a significant enhancement in the antiproliferative response of UVB. These responses on UVB and/or sanguinarine treatments were associated with (a) decrease in Bcl-2 and Bcl-X(L) and (b) increase in Bax, Bid, and Bak protein levels. Bax knockdown and Bcl-2 overexpression resulted in a rescue of HaCaT cells from sanguinarine-mediated apoptosis. DNA cell cycle analysis revealed that UVB treatment resulted in an accumulation of cells in the G2-M phase of the cell cycle, whereas pretreatment of sanguinarine resulted in a significant shift of cells in the S phase at a low UVB dose and a further accumulation of cells in the G2-M phase at a higher UVB dose. These effects on cell cycle were accompanied with modulations in the protein levels of cyclin (B1, E, and A) and cdc2 and cyclin-dependent kinase 1. Furthermore, sanguinarine treatment was found to result in significant modulations in p53, p66Shc, MsrA, and superoxide dismutase levels. Based on our data, we suggest the sanguinarine may protect skin cells from UVB-mediated damages via apoptotic elimination of damaged cells that escape programmed cell death and therefore possess a potential of clonal expansion.

Adaptor Proteins, Signal Transducing↗
PubMedDate not supplied

Evidence for the involvement of protein kinase C in depression of endothelium-dependent vascular responses in spontaneously hypertensive rats.

The goal of the present study was to evaluate the role of protein kinase C (PKC) in the depression of endothelium-dependent vacular response in spontaneously hypertensive Okamoto rats (SHR). Aortae from SHR demonstrated a decreased relaxant response to acetylcholine (Ach) as compared to aortae from normotensive Wistar-Kyoto (WKY) rats, while papaverine lowered the force of aorta to a similar degree in both strains of rats. PKC inhibitors, H-7 (5 x 10(-6) M) and chelerythrine chloride (10(-6) M), produced a greater decrease in the force developed by the aortae from SHR vs. WKY rats both in intact and chemically permeabilized tissues. In SHR aortae PKC inhibitors enhanced relaxation to Ach to a greater extent as compared to WKY aortae. Furthermore, in the presence of PKC inhibitors, the constrictor responses of SHR aortae to Ach were transformed into relaxant responses, and the concentration-response curve to Ach was shifted to the left. The sensitivity of aortae from SHR to authentic nitric oxide (NO) was lowere compared to WKY rats. EC50s for authentic NO in SHR and WKY rat aortae were different: -2.9 +/- 0.15 x 10(-6) M and 4.58 +/- 0.1 x 10(-7) M (n = 15, p < 0. 001), respectively. Bioassay experiments using SHR aortae showed that the addition of chelerythrine (10(-6) M) to the detector superfusate caused relaxation during treatment of the donor segment with Ach, indicating that the sensitivity of the aortae to NO had been restored. When SHR detector ring was substituted for denuded aortae from WKY rats and PKC inhibitors were not added to the detector superfusate, the relaxation of the detector aortae was also close to the normal Ach-induced relaxation. WKY aortae demonstrated a positive relationship between Ach-stimulated NO release and relaxant response amplitudes (correlation coefficient r = 0.905, p < 0.001, n = 10). In contrast, there was a significant negative correlation in SHR aortae (r = -0.712, p < 0.05, n = 10). Detection of NO release by chemiluminescence showed no significant difference in NO release in SHR and WKY aortae. Taken together, these data suggest that the blunted endothelium-dependent relaxations seen in SHR aortae are mainly due to a decreased sensitivity of vascular smooth muscle to EDRF/NO resulting from an increased PKC activity.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2002

Roles of the cytoskeleton and of protein phosphorylation events in the osmotic stress response in eel intestinal epithelium.

The eel intestinal epithelium responds to an acute hypertonic challenge by a biphasic increase of the rate of Cl(-) absorption (measured as short circuit current, Isc, and creating a negative transepithelial potential, V(te), at the basolateral side of the epithelium). While the first, transient phase is bumetanide-insensitive, the second, sustained phase is bumetanide-sensitive, reflecting activation of the apically located Na(+)-K(+)-2Cl(-) (NKCC) cotransporter, which correlates with the cellular RVI response. Here, we investigated the involvement of the cytoskeleton and of serine/threonine phosphorylation events in the osmotic stress-induced ion transport in the eel intestinal epithelium, focusing on the sustained RVI phase, as well as on the previously uncharacterized response to hypotonic stress. The study was carried out using confocal laser scanning microscopy, a quantitative F-actin assay, and transepithelial electrophysiological measurements (V(te) and Isc) in Ussing chambers. Hypertonic stress did not detectably alter either net F-actin content or F-actin organization. In contrast, a brief exposure to hypotonic stress decreased the total cellular F-actin content in eel intestinal epithelium by about 15%, detectable morphologically mainly as a decrease in the intensity of the apical brush border F-actin labeling.The bumetanide-sensitive response of V(te) and Isc to hypertonicity was potently inhibited by treatment with either cytochalasin, latrunculin A, colchicine, the protein kinase C (PKC) inhibitor chelerythrine, the myosin light chain kinase (MLCK) inhibitor ML-7, or the serine/threonine protein phosphatase inhibitor Calyculin A, but was unaffected by the PKA inhibitor H-89. The electrophysiological response of the epithelium to hypotonic stress was characterized by a sustained decrease of V(te) and Isc, which was smaller and recovered faster in the presence of either cytochalasin, latrunculin A, or colchicine. It is concluded that in eel intestinal epithelium, the changes in ion transport in response to both hyper- and hypotonic stress require the integrity of both F-actin and microtubules. In addition, the shrinkage-induced activation of NKCC appears to require the activity of both PKC and MLCK. It is suggested that NKCC regulation by hypertonic stress involves an interaction between the cytoskeleton and protein phosphorylation events.

Actins↗
PubMedDate not supplied

Attenuation of canine cerebral vasospasm after subarachnoid hemorrhage by protein kinase C inhibitors despite augmented phosphorylation of myosin light chain.

The purpose of the present study is to assess the roles of protein kinase C (PKC) isoforms, especially PKC delta and alpha, and 20-kD myosin light chain (MLC(20)) phosphorylation in the mechanism of cerebral vasospasm following subarachnoid hemorrhage (SAH). We had shown that those PKC isoforms are involved in the development of cerebral vasospasm. Using PKC isoform-specific inhibitors in a 'two- hemorrhage' canine model, we examined changes in the development of cerebral vasospasm, translocation of PKC isoforms and MLC(20) phosphorylation level in canine basilar arteries. A PKC inhibitor (5 microM rottlerin for PKC delta or chelerythrine for PKC alpha) was injected into the cisterna magna on day 4 before the second hemorrhage. The treatment was continued daily until day 7. Rottlerin inhibited the initial phase of vasospasm and PKC delta translocation, but did not significantly inhibit PKC alpha translocation. Chelerythrine inhibited cerebral vasospasm, and the translocation of both PKC delta and alpha throughout the entire course of the study. Although cerebral vasospasm after SAH was inhibited by each PKC inhibitor, the MLC(20) phosphorylation level remained elevated as in the untreated hemorrhage-control study. We conclude that cerebral vasospasm following SAH depends on PKC delta and alpha, while the enhancement of MLC(20) phosphorylation contributes little to this form of vasospasm.

Acetophenones↗
PubMed2005

Protective effects of insulin during ischemia-reperfusion injury in hamster cheek pouch microcirculation.

OBJECTIVE: The effects of insulin (0.18 nM-0.18 microM) on reduced capillary perfusion, microvascular permeability increase and leukocyte adhesion induced by ischemia-reperfusion injury were investigated in the hamster cheek pouch microcirculation. To gain insight into the insulin's mechanism of action, the effects of its higher concentration (0.18 microM) were investigated after inhibition of tyrosine kinase (TK), nitric oxide synthase (NOS), protein kinase C (PKC), phosphatidylinositol 3-kinase and K+(ATP) channels, alone or in combination. Two concentrations for each inhibitor were used. METHODS: Microcirculation was visualized by fluorescence microscopy. Perfused capillary length, microvascular permeability, leukocyte adhesion to venular walls, vessel diameter and capillary red blood cell velocity were assessed by computer-assisted methods. Measurements were made at baseline (B), after 30 min of ischemia (I), and after 30 min of reperfusion (R). RESULTS: In control animals, perfused capillary length decreased by 63 +/- 5% of baseline at R. Microvascular permeability increased at I and R, while leukocyte adhesion was most pronounced in V1 postcapillary venules at R. Insulin dose-dependently preserved capillary perfusion at R (-28 +/- 6 and -15 +/- 6% of baseline), but was unable to prevent the increase in permeability at I (0.25 +/- 0.05 and 0.29 +/- 0.06 Normalized Grey Levels, NGL) and R (0.49 +/- 0.10 and 0.53 +/- 0.09 NGL), according to the concentrations. Adhesion of leukocytes was observed mostly in V3 venules at R (9 +/- 2 and 10 +/- 2/100 microm venular length, with the lower and higher concentration, respectively). Nitric oxide synthase inhibition by N(G)-nitro-L-arginine-methyl ester prior to insulin did not affect capillary perfusion at R (-18 +/- 3% of baseline with higher concentration), but prevented permeability increase (0.20 +/- 0.04 NGL, according to higher concentration) and reduced leukocyte adhesion in V3 venules at R (1.5 +/- 1.0/100 microm of venular length, with higher concentration). Blockade of K+(ATP) channels by glibenclamide prior to insulin decreased perfused capillary length at R (-58 +/- 6% of baseline with higher concentration), attenuated leakage at R (0.30 +/- 0.04 NGL, according to higher concentration) and caused leukocyte adhesion mainly in V1 venules at R (9.0 +/- 1.5/100 microm of venular length, with higher concentration). Inhibition of either TK, PKC or phosphatidylinositol 3-kinase did not affect microvascular responses to insulin. Simultaneous inhibition of TK and NOS did not increase protection. CONCLUSIONS: Insulin prevents ischemia-reperfusion injury by promoting capillary perfusion through an apparent activation of K+(ATP) channels and increase in nitric oxide release.

Alkaloids↗
PubMed2006

Inhibition of experimental proliferative vitreoretinopathy with protein kinase C inhibitor (chelerythrine chloride) and melatonin.

PURPOSE: To investigate whether a protein kinase C (PKC) inhibitor and melatonin prevent proliferative vitreoretinopathy (PVR). METHODS: Twenty pigmented rabbits were used in this study. All rabbits except controls received an intravitreal injection of 0.15 ml (75,000 units) of platelet-rich plasma into their left eye. The animals were divided into four groups: group I was treated with intravitreal injection of 0.1 ml (100 micromol/ml) of PKC inhibitor (chelerythrine chloride), group II received 1 ml (4 mg/kg) of intraperitoneal melatonin for 3 days, group III received nothing (blank group), and group IV (control group) received only 0.5 ml of 1% ethanol intraperitoneally for 3 days. Proliferative changes were graded in a masked fashion by indirect ophthalmoscopy for a 15-day follow-up period. The malondialdehyde (MDA), reduced glutathione (GSH) and total nitrite levels were measured in the vitreous humor. RESULTS: The grades of PVR were A and B in group I and II, treated with PKC inhibitor and melatonin, respectively. The PVR grade in the blank group was C-D. The mean MDA level in group I (4.2 +/- 0.9 micromol/l) was significantly lower than in the blank group (6.0 +/- 1.0 micromol/l; p < 0.05). The mean GSH level in group I (66.3 +/- 8.8 micromol/l) was not significantly different from that in the blank group (p > 0.05). The MDA and GSH levels in group II were 3.2 +/- 0.7 and 70.1 +/- 13.3 micromol/l, respectively. Both these levels were significantly different from those of the blank group (p < 0.05). The NO levels in both treatment groups were significantly lower than in the blank group (p < 0.001). CONCLUSION: These findings suggest an inhibitory effect of PKC inhibitor and melatonin on PVR. The inhibition of PVR development was associated with lower MDA and NO levels with higher GSH levels in the treatment groups.

Alkaloids↗
PubMed2006

Lysophosphatidylcholine augments Ca(v)3.2 but not Ca(v)3.1 T-type Ca(2+) channel current expressed in HEK-293 cells.

Lysophosphatidylcholine (LPC) has been shown to induce electrophysiological disturbances to arrhythmogenesis. However, the effects of LPC on the low-voltage-activated T-type Ca(2+) channels in the heart are not understood yet. We found that LPC increases the T-type Ca(2+) channel current (I(Ca.T)) in neonatal rat cardiomyocytes. To further investigate the underlying modulatory mechanism of LPC on T-type Ca(2+) channels, we utilized HEK-293 cells stably expressing alpha1G and alpha1H subunits (HEK-293/alpha1G and HEK-293/alpha1H), by use of patch-clamp techniques. A low concentration of LPC (10 micromol/l) significantly increased Ca(v)3.2 I(Ca.T) (alpha1H) that were similar to those observed in neonatal rat cardiomyocytes. Activation and steady-state inactivation curves were shifted in the hyperpolarized direction by 5.1 +/- 0.2 and 4.6 +/- 0.4 mV, respectively, by application of 10 micromol/l LPC. The pretreatment of cells with a protein kinase C inhibitor (chelerythrine) attenuated the effects of LPC on I(Ca.T) (alpha1H). However, the application of LPC failed to modify Ca(v)3.1 (alpha1G) I(Ca.T) at concentrations of 10-50 micromol/l. In conclusion, these data demonstrate that extracellularly applied LPC augments Ca(v)3.2 I(Ca.T) (alpha1H) but not Ca(v)3.1 I(Ca.T) (alpha1G) in a heterologous expression system, possibly by modulating protein kinase C signaling.

Alkaloids↗