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PubMed1995

Does ischemic preconditioning in the human involve protein kinase C and the ATP-dependent K+ channel? Studies of contractile function after simulated ischemia in an atrial in vitro model.

Protein kinase C (PKC) and the ATP-dependent K+ channel (KATP channel) have been implicated in the mechanism of ischemic preconditioning in animal models. This study investigated the role of KATP channels and PKC in preconditioning in human myocardium and whether KATP channels are activated via a PKC-dependent pathway. Right atrial trabeculae were superfused with Tyrode's solution and paced at 1 Hz. After stabilization, muscles underwent one of nine different protocols, followed by simulated ischemia (SI) consisting of 90 minutes of hypoxic substrate-free superfusion paced at 3 Hz and then by 120 minutes of reperfusion. Preconditioning consisted of 3 minutes of SI and 7 minutes of reperfusion. The experimental end point was recovery of contractile function after SI, presented here as percentage recovery (%Rec) of baseline function. %Rec was significantly improved by preconditioning by the KATP channel opener cromakalim (CK), and by the PKC activator 1,2-dioctanoyl-sn-glycerol (DOG) compared with nonpreconditioned controls when these treatments were given before the SI insult (control group, 29.5 +/- 3.6%; preconditioned group, 63.5 +/- 5.4%, CK-treated group, 52.9 +/- 3.1%; and DOG-treated group, 48.0 +/- 3.5%; P < .01). The effects of CK could be blocked by the KATP channel blocker glibenclamide (%Rec, 17.8 +/- 3.5%). Preconditioning could be blocked by the PKC antagonist chelerythrine (%Rec, 24.1 +/- 5.0%) and the KATP blocker glibenclamide (%Rec, 24.8 +/- 3.1%). The effects of DOG could also be blocked by glibenclamide (%Rec, 23.1 +/- 2.3%).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1997

Functional effects of protein kinase C activation on the human cardiac Na+ channel.

The cardiac Na+ current plays an important role in determining normal and abnormal impulse propagation in the heart. We have investigated the effects of protein kinase C (PKC) activation on the recombinant human cardiac Na+ channel (hH1) following heterologous expression in Xenopus laevis oocytes. Phorbol 12-myristate 13-acetate (PMA), which directly activates PKC, reduced current amplitude at all test potentials (43 +/- 12% at -10 mV). In contrast to the rat brain IIA (rBIIA) channel, there was no apparent change in either macroscopic Na+ current decay or the voltage dependence of channel gating. Further experiments indicate that the effects of PMA were mediated by PKC activation: (1) an inactive stereoisomer, 4 alpha-PMA, had no effect; (2) preincubation with the protein kinase inhibitor chelerythrine prevented the PMA effects; and (3) a hydrolyzable diacylglycerol analogue, 1-oleoyl-2-acetyl-glycerol, also reduced current (22 +/- 5%). In addition, when the alpha 1B-adrenergic receptor was coexpressed with hH1, the alpha-receptor agonist methoxamine reduced hH1 current (45 +/- 10%), an effect that could be eliminated by chelerythrine preincubation. When a conserved consensus PKC site (serine 1503) in the III-IV interdomain linker thought to be responsible for the PKC effects on rBIIA was mutated, PMA still reduced Na+ current, but the magnitude of the effect was smaller compared with that for the wild-type channel. Similar findings were obtained with alpha 1-receptor stimulation following receptor coexpression with the mutant channel. We conclude that activation of PKC modulates the human cardiac Na+ channel by at least two mechanisms, one similar to that seen with rat brain channels, involving a conserved putative PKC site, and a second more specific to the cardiac isoform.

Alkaloids↗
PubMed1997

Positive inotropy mediated by diacylglycerol in rat ventricular myocytes.

Many neurohormones stimulate phospholipid hydrolysis and elevate diacylglycerol in the mammalian heart, but the physiological consequences of these intracellular events are unclear. Regulation of myocardial contraction by diacylglycerol was investigated in the present study by releasing the diacylglycerol analogue dioctanoylglycerol (diC8) within adult rat ventricular myocytes by using a light-sensitive caged compound. This approach permitted us to avoid exposure of myocytes to extracellular diC8 and yet to control the amount of diC8 released into the cells. Photorelease of diC8 produced a slowly developing (half-time, 1.9 +/- 0.1 minute; n = 26) but robust (406 +/- 42%) enhancement of twitch amplitude in electrically paced myocytes (0.5 Hz, 1 mmol/L Ca2+, Ringer's solution [pH 7.4], 22 degrees C). This positive inotropic effect was dose dependent, stereospecific for the S-enantiomer of diC8, synergistically enhanced by arachidonic acid, and blocked by the protein kinase C inhibitor chelerythrine. The data provide evidence that diacylglycerol can induce a strong positive inotropic effect in mammalian ventricular muscle, possibly by activating protein kinase C. By contrast, perfusion of diC8 extracellularly onto myocytes caused a 42 +/- 2% decline in twitch amplitude, in accordance with previous reports. To account for this dependence on how diC8 is applied, we postulate that diC8 has distinct physiological actions at intracellular and extracellular sites. The peptide neurohormone endothelin-1, which elevates diacylglycerol in cardiac tissues, produced a positive inotropic effect that was similar to the response to photoreleased diC8. The diacylglycerol/protein kinase C pathway has now become a good candidate for mediator of at least a component of the positive inotropy associated with agents that stimulate phospholipid turnover in adult mammalian myocardium.

Alkaloids↗
PubMed1997

Modulation of Na+ current inactivation by stimulation of protein kinase C in cardiac cells.

Modulation of the inward Na+ current (I(Na)) by protein kinase C (PKC) was investigated by intracellular perfusion of a peptide corresponding to the catalytic subunit of PKC (PKCP). The effects of PKC activation independent of membrane-receptor pathways were studied in neonatal rat ventricular myocytes using whole-cell patch-clamp techniques. Perfusion with 2 nmol/L PKCP caused a depolarizing shift in steady state half-inactivation relative to control (-83.2+/-1.3 versus -74.9+/-1.6 mV for control versus PKCP, respectively) without a change in current-voltage relationships or peak I(Na). The development of resting inactivation was slowed by PKCP (tau, 69.1+/-7.6 [control] versus 100.4+/-5.1 ms). Open-channel inactivation, estimated by measuring I(Na) decay from peak current at test voltages between -10 and +30 mV was significantly slowed by PKCP. Recovery from inactivation was more rapid during PKCP perfusion, with a shortening of both the fast (tau(f)) and slow (tau(s)) components of tau (tau(f), 38.5+/-7.0 [control] versus 14.2+/-4.7 ms; tau(s), 163.4+/-47.9 [control] versus 51.3+/-9.2 ms). All of the effects of PKCP on I(Na) were antagonized by the PKC inhibitors chelerythrine chloride or staurosporine or by down-regulation of PKC using phorbol ester preincubation. We conclude that the actions of PKC on the Na+ channel result in slowing the development of inactivation and accelerating reactivation, resulting in less resting inactivation.

Alkaloids↗
PubMed1998

Angiotensin II activates Na+-independent Cl--HCO3- exchange in ventricular myocardium.

The effect of angiotensin II (Ang II) on the activity of the cardiac Na+-independent Cl--HCO3- exchanger (anionic exchanger [AE]) was explored in cat papillary muscles. pHi was measured by epifluorescence with BCECF-AM. Ang II (500 nmol/L) induced a 5-(N-ethyl-N-isopropyl)amiloride-sensitive increase in pHi in the absence of external HCO3- (HEPES buffer), consistent with its stimulatory action on Na+-H+ exchange (NHE). This alkalinizing effect was not detected in the presence of a CO2-HCO3- buffer (pHi 7.07+/-0.02 and 7.08+/-0.02 before and after Ang II, respectively; n=17). Moreover, in Na+-free HCO3--buffered medium, in which neither NHE nor Na+-HCO3- cotransport are acting, Ang II decreased pHi, and this effect was canceled by previous treatment with SITS. These findings suggested that the Ang II-induced activation of NHE was masked, in the presence of the physiological buffer, by a HCO3--dependent acidifying mechanism, probably the AE. This hypothesis was confirmed on papillary muscles bathed with HCO3- buffer that were first exposed to 1 micromol/L S20787, a specific inhibitor of AE activity in cardiac tissue, and then to 500 nmol/L Ang II (n=4). Under this condition, Ang II increased pHi from 7.05+/-0.05 to 7.22+/-0.05 (P<.05). The effect of Ang II on AE activity was further explored by measuring the velocity of myocardial pHi recovery after the imposition of an intracellular alkali load in a HCO3--containing solution either with or without Ang II. The rate of myocardial pHi recovery was doubled in the presence of Ang II, suggesting a stimulatory effect on AE. The enhancement of the activity of this exchanger by Ang II was also detected when the AE activity was reversed by the removal of extracellular Cl- in a Na+-free solution. Under this condition, the rate of intracellular alkalinization increased from 0.053+/-0.016 to 0.108+/-0.026 pH unit/min (n=6, P<.05) in the presence of Ang II. This effect was canceled either by the presence of the AT1 receptor antagonist, losartan, or by the previous inhibition of protein kinase C with chelerythrine or calphostin C. The above results allow us to conclude that Ang II, in addition to its stimulatory effect on alkaline loading mechanisms, activates the AE in ventricular myocardium and that the latter effect is mediated by a protein kinase C-dependent regulatory pathway linked to the AT1 receptors.

Alkaloids↗
PubMed1998

Stretch-induced alkalinization of feline papillary muscle: an autocrine-paracrine system.

Myocardial stretch is a well-known stimulus that leads to hypertrophy. Little is known, however, about the intracellular pathways involved in the transmission of myocardial stretch to the cytoplasm and nucleus. Studies in neonatal cardiomyocytes demonstrated stretch-induced release of angiotensin II (Ang II). Because intracellular alkalinization is a signal to cell growth and Ang II stimulates the Na+/H+ exchanger (NHE), we studied the relationship between myocardial stretch and intracellular pH (pHi). Experiments were performed in cat papillary muscles fixed by the ventricular end to a force transducer. Muscles were paced at 0.2 Hz and superfused with HEPES-buffered solution. pHi was measured by epifluorescence with the acetoxymethyl ester form of the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF-AM). Each muscle was progressively stretched to reach maximal developed force (Lmax) and maintained in a length that was approximately 92% Lmax (Li). During the "stretch protocol," muscles were quickly stretched to Lmax for 10 minutes and then released to Li; pHi significantly increased during stretch and came back to the previous value when the muscle was released to Li. The increase in pHi was eliminated by (1) specific inhibition of the NHE (EIPA, 5 micromol/L), (2) AT1-receptor blockade (losartan, 10 micromol/L), (3) inhibition of protein kinase C (PKC) (chelerythrine, 5 micromol/L), (4) blockade of endothelin (ET) receptors with a nonselective (PD 142,893, 50 nmol/L) or a selective ETA antagonist (BQ-123, 300 nmol/L). The increase in pHi by exogenous Ang II (500 nmol/L) was also reduced by both ET-receptor antagonists. Our results indicate that after myocardial stretch, pHi increases because of stimulation of NHE activity. This involves an autocrine-paracrine mechanism in which protein kinase C, Ang II, and ET play crucial roles.

Alkalies↗
PubMed1999

Role of protein kinase C in mitochondrial KATP channel-mediated protection against Ca2+ overload injury in rat myocardium.

Growing evidence exists that ATP-sensitive mitochondrial potassium channels (MitoKATP channel) are a major contributor to the cardiac protection against ischemia. Given the importance of mitochondria in the cardiac cell, we tested whether the potent and specific opener of the MitoKATP channel diazoxide attenuates the lethal injury associated with Ca2+overload. The specific aims of this study were to test whether protection by diazoxide is mediated by MitoKATP channels; whether diazoxide mimics the effects of Ca2+ preconditioning; and whether diazoxide reduces Ca2+ paradox (PD) injury via protein kinase C (PKC) signaling pathways. Langendorff-perfused rat hearts were subjected to the Ca2+ PD (10 minutes of Ca2+ depletion followed by 10 minutes of Ca2+ repletion). The effects of the MitoKATP channel and other interventions on functional, biochemical, and pathological changes in hearts subjected to Ca2+ PD were assessed. In hearts treated with 80 micromol/L diazoxide, left ventricular end-diastolic pressure and coronary flow were significantly preserved after Ca2+ PD; peak lactate dehydrogenase release was also significantly decreased, although ATP content was less depleted. The cellular structures were well preserved, including mitochondria and intercalated disks in diazoxide-treated hearts compared with nontreated Ca2+ PD hearts. The salutary effects of diazoxide on the Ca2+ PD injury were similar to those in hearts that underwent Ca2+ preconditioning or pretreatment with phorbol 12-myristate 13-acetate before Ca2+ PD. The addition of sodium 5-hydroxydecanoate, a specific MitoKATP channel inhibitor, or chelerythrine chloride, a PKC inhibitor, during diazoxide pretreatment completely abolished the beneficial effects of diazoxide on the Ca2+ PD. Blockade of Ca2+ entry during diazoxide treatment by inhibiting L-type Ca2+ channel with verapamil or nifedipine also completely reversed the beneficial effects of diazoxide on the Ca2+ PD. PKC-delta was translocated to the mitochondria, intercalated disks, and nuclei of myocytes in diazoxide-pretreated hearts, and PKC-alpha and PKC-epsilon were translocated to sarcolemma and intercalated disks, respectively. This study suggests that the effect of the MitoKATP channel is mediated by PKC-mediated signaling pathway.

Adenosine Triphosphate↗
PubMed1999

Isoform-selective activation of protein kinase C by nitric oxide in the heart of conscious rabbits: a signaling mechanism for both nitric oxide-induced and ischemia-induced preconditioning.

Although isoform-selective translocation of protein kinase C (PKC) epsilon appears to play an important role in the late phase of ischemic preconditioning (PC), the mechanism(s) responsible for such translocation remains unclear. Furthermore, the signaling pathway that leads to the development of late PC after exogenous administration of NO in the absence of ischemia (NO donor-induced late PC) is unknown. In the present study we tested the hypothesis that NO activates PKC and that this is the mechanism for the development of both ischemia-induced and NO donor-induced late PC. A total of 95 chronically instrumented, conscious rabbits were used. In rabbits subjected to ischemic PC (six 4-minute occlusion/4-minute reperfusion cycles), administration of the NO synthase inhibitor Nomega-nitro-L-arginine (group III), at doses previously shown to block the development of late PC, completely blocked the ischemic PC-induced translocation of PKCepsilon but not of PKCeta, indicating that increased formation of NO is an essential mechanism whereby brief ischemia activates the epsilon isoform of PKC. Conversely, a translocation of PKCepsilon and -eta quantitatively similar to that induced by ischemic PC could be reproduced pharmacologically with the administration of 2 structurally unrelated NO donors, diethylenetriamine/NO (DETA/NO) and S-nitroso-N-acetylpenicillamine (SNAP), at doses previously shown to elicit a late PC effect. The particulate fraction of PKCepsilon increased from 35+/-2% of total in the control group (group I) to 60+/-1% after ischemic PC (group II) (P<0.05), to 54+/-2% after SNAP (group IV) (P<0.05) and to 52+/-2% after DETA/NO (group V) (P<0.05). The particulate fraction of PKCeta rose from 66+/-5% in the control group to 86+/-3% after ischemic PC (P<0.05), to 88+/-2% after SNAP (P<0.05) and to 85+/-1% after DETA/NO (P<0.05). Neither ischemic PC nor NO donors had any appreciable effect on the subcellular distribution of PKCalpha, -beta1, -beta2, -gamma, -delta, - micro, or -iota/lambda; on total PKC activity; or on the subcellular distribution of total PKC activity. Thus, the effects of SNAP and DETA/NO on PKC closely resembled those of ischemic PC. The DETA/NO-induced translocation of PKCepsilon (but not that of PKCeta) was completely prevented by the administration of the PKC inhibitor chelerythrine at a dose of 5 mg/kg (group VI) (particulate fraction of PKCepsilon, 38+/-4% of total, P<0.05 versus group V; particulate fraction of PKCeta, 79+/-2% of total). The same dose of chelerythrine completely prevented the DETA/NO-induced late PC effect against both myocardial stunning (groups VII through X) and myocardial infarction (groups XI through XV), indicating that NO donors induce late PC by activating PKC and that among the 10 isozymes of PKC expressed in the rabbit heart, the epsilon isotype is specifically involved in the development of this form of pharmacological PC. In all groups examined (groups I through VI), the changes in the subcellular distribution of PKCepsilon protein were associated with parallel changes in PKCepsilon isoform-selective activity, whereas total PKC activity was not significantly altered. Taken together, the results provide direct evidence that isoform-selective activation of PKCepsilon is a critical step in the signaling pathway whereby NO initiates the development of a late PC effect both after an ischemic stimulus (endogenous NO) and after treatment with NO-releasing agents (exogenous NO). To our knowledge, this is also the first report that NO can activate PKC in the heart. The finding that NO can promote isoform-specific activation of PKC identifies a new biological function of this radical and a new mechanism in the signaling cascade of ischemic PC and may also have important implications for other pathophysiological conditions in which NO is involved and for nitrate therapy.

Alkaloids↗
PubMed1999

Chloride channel inhibition blocks the protection of ischemic preconditioning and hypo-osmotic stress in rabbit ventricular myocardium.

The objective of this study was to examine the role of chloride (Cl-) channels in the myocardial protection of ischemic preconditioning (IP). Isolated rabbit ventricular myocytes were preconditioned with 10-minute simulated ischemia (SI) and 20-minute simulated reperfusion (SR) or not preconditioned (control). The myocytes then received 180-minute SI or 45-minute SI/120-minute SR. Indanyloxyacetic acid 94 (IAA-94, 10 micromol/L) or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 1 micromol/L) was administered before IP or before SI or SI/SR to inhibit Cl- channels. Electrophysiological studies indicate that these drugs, at the concentrations used, selectively abolished Cl- currents activated under hypo-osmotic conditions (215 versus 290 mOsm). IP significantly (P<0.001) reduced the percentage of dead myocytes after 60-minute (30.8+/-1.3%, mean+/-SEM), 90-minute (35.3+/-1.3%), and 120-minute (39.2+/-1.7%) SI compared with controls (44.7+/-1.6%, 54.5+/-1.3%, and 58.9+/-1.8%, respectively) and after 45-minute SI/120-minute SR (36.3+/-0.6%) compared with control (56.6+/-2.2%). Hypo-osmotic stress also produced protection similar to IP. IAA-94 or NPPB abolished the protection of both IP and hypo-osmotic stress. In buffer-perfused rabbit hearts preconditioned with three 5-minute ischemia/10-minute reperfusion cycles given before the 40-minute long ischemia and 60-minute reperfusion, IP significantly (P<0.0001) reduced infarct size (IP+vehicle, 4.7+/-0.9%, versus control+vehicle, 26.6+/-3.3%; mean+/-SEM). Again, IAA-94 or NPPB abolished the protection of IP. Our results implicate Cl- channels in the IP protection of the myocardium against ischemic/reperfusion injury and demonstrate that hypo-osmotic stress is capable of preconditioning cardiomyocytes.

Alkaloids↗
PubMed1999

Nuclear factor-kappaB plays an essential role in the late phase of ischemic preconditioning in conscious rabbits.

Although it is recognized that late preconditioning (PC) results from upregulation of cardioprotective genes, the specific transcription factor(s) that govern this genetic adaptation remains unknown. The aim of this study was to test the hypothesis that the development of late PC is mediated by nuclear factor-kappaB (NF-kappaB) and to elucidate the mechanisms that control the activation of NF-kappaB after an ischemic stimulus in vivo. A total of 152 chronically instrumented, conscious rabbits were used. A sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles, which elicits late PC, induced rapid activation of NF-kappaB, as evidenced by a marked increase in p65 content (+164%; Western immunoblotting) and NF-kappaB DNA binding activity (+306%; electrophoretic mobility shift assay) in nuclear extracts isolated 30 minutes after the last reperfusion. These changes were attenuated 2 hours after ischemic PC and resolved by 4 hours. Competition and supershift assays confirmed the specificity of the NF-kappaB DNA complex signals. The mobility of the NF-kappaB DNA complex was shifted by anti-p65 and anti-p50 antibodies but not by anti-c-Rel antibodies, indicating that the subunits of NF-kappaB involved in gene activation after ischemic PC consist of p65-p50 heterodimers. Pretreatment with the NF-kappaB inhibitor diethyldithiocarbamate (DDTC; 150 mg/kg IP 15 minutes before ischemic PC) completely blocked the nuclear translocation and increased DNA binding activity of NF-kappaB. The same dose of DDTC completely blocked the cardioprotective effects of late PC against both myocardial stunning and myocardial infarction, indicating that NF-kappaB activation is essential for the development of this phenomenon in vivo. The ischemic PC-induced activation of NF-kappaB was also blocked by pretreatment with Nomega-nitro-L-arginine (L-NA), a nitric oxide synthase (NOS) inhibitor, N-2-mercaptopropionyl glycine (MPG), a reactive oxygen species (ROS) scavenger, chelerythrine, a protein kinase C (PKC) inhibitor, and lavendustin A, a tyrosine kinase inhibitor (all given at doses previously shown to block late PC), indicating that ischemic PC activates NF-kappaB via formation of NO and ROS and activation of PKC- and tyrosine kinase-dependent signaling pathways. A subcellular redistribution and increased DNA binding activity of NF-kappaB quantitatively similar to those induced by ischemic PC could be reproduced pharmacologically by giving the NO donor diethylenetriamine/NO (DETA/NO) (at a dose previously shown to elicit late PC), demonstrating that NO in itself can activate NF-kappaB in the heart. Taken together, these results provide direct evidence that activation of NF-kappaB is a critical step in the signal transduction pathway that underlies the development of the late phase of ischemic PC in conscious rabbits. The finding that four different pharmacological manipulations (L-NA, MPG, chelerythrine, and lavendustin A) produced similar inhibition of NF-kappaB suggests that this transcription factor is a common downstream pathway through which multiple signals elicited by ischemic stress (NO, ROS, PKC, tyrosine kinases) act to induce gene expression. To our knowledge, this is the first demonstration that NO can promote NF-kappaB activation in the heart, a finding that identifies a new biological function of NO and may have important implications for various pathophysiological conditions in which NO is involved and for nitrate therapy.

Alkaloids↗
PubMed2000

Effects of long-term nitroglycerin treatment on endothelial nitric oxide synthase (NOS III) gene expression, NOS III-mediated superoxide production, and vascular NO bioavailability.

Long-term nitroglycerin (NTG) treatment has been shown to be associated with cross-tolerance to endothelium-dependent vasodilators. It may involve increased production of reactive oxygen species (such as superoxide, O(2)(.-)) that rapidly inactivate the nitric oxide (NO) released from the endothelial cells. It remains to be elucidated, however, whether long-term treatment with NTG alters the activity and expression of the endothelial NO synthase (NOS III) and whether this enzyme can contribute to O(2)(.-) formation. We studied the influence of long-term NTG treatment on the expression of NOS III as assessed by RNase protection assay and Western blot. Tolerance was measured ex vivo in organ chamber experiments with rat aortic rings. O(2)(.-) and NO formation were quantified using lucigenin- and Cypridina luciferin analog-enhanced chemiluminescence as well as electron spin resonance (ESR) spectroscopy. Treatment of Wistar rats with NTG (Alzet osmotic minipumps, NTG concentration 10 microg x kg(-1) x min(-1)) for 3 days caused marked tolerance, cross-tolerance to the endothelium-dependent vasodilator acetylcholine, and a significant increase in O(2)(.-)-induced chemiluminescence. Tolerance was associated with a significant increase in NOS III mRNA to 236+/-28% and NOS III protein to 239+/-17%. In control vessels, the NOS inhibitor N(G)-nitro-L-arginine (L-NNA) increased the O(2)(.-)-mediated chemiluminescence, indicating that basal production of endothelium-derived NO depresses the baseline chemiluminescence signal. In the setting of tolerance, however, L-NNA decreased steady-state O(2)(.-) levels, indicating the involvement of NOS III in O(2)(.-) formation. Likewise, A23187-induced, NOS III-mediated O(2)(.-) production was more pronounced in tolerant than in control vessels. Vascular NO bioavailability as assessed with ESR spectroscopy using iron-thiocarbamate as a trap for NO was significantly reduced in tolerant vessels. Pretreatment of tolerant tissue in vitro with the protein kinase C (PKC) inhibitors reduced basal and stimulated NOS III-mediated O(2)(.-) production and partially reversed vascular tolerance. These findings suggest that NTG treatment increases the expression of a dysfunctional NOS III gene, leading to increased formation of O(2)(.-) and decreased vascular NO bioavailability. Normalization of NOS III-mediated O(2)(. -) production and improvement of tolerance with PKC inhibition suggests an important role for PKC isoforms in mediating vascular dysfunction caused by long-term NTG treatment.

Acetylcholine↗
PubMed2000

The epsilon subtype of protein kinase C is required for cardiomyocyte connexin-43 phosphorylation.

Gap junctions (GJs), composed of connexins, are intercellular channels ensuring electric and metabolic coupling between cardiomyocytes. We have shown previously that an endogenous mitogenic and cardioprotective protein, fibroblast growth factor-2 (FGF-2), decreases cardiomyocyte GJ permeability by stimulating phosphorylation of connexin-43 (Cx43). Identifying the kinase(s) phosphorylating cardiac Cx43 may thus provide a way of modulating cardiac intercellular communication. Because FGF-2 activates receptors linked to protein kinase C (PKC) and mitogen-activated protein kinase, we first investigated participation of these enzymatic systems in Cx43 phosphorylation. The inhibitor PD98059 blocked activation of mitogen-activated protein kinase, but it did not prevent the FGF-2 effects on GJs. In contrast, the PKC inhibitor chelerythrine blocked the effects of FGF-2 on Cx43 phosphorylation and permeability. Because the epsilon-isoform of PKC localizes to plasma membrane sites, we examined whether it is directly involved in the FGF-2-induced Cx43 phosphorylation. In nonstimulated myocytes, PKCepsilon displayed a discontinuous pattern of localization at intercellular contact sites and partial colocalization with Cx43. Treatment with FGF-2 or phorbol 12-myristate 13-acetate induced a more continuous pattern of PKCepsilon distribution, whereas the anti-Cx43 staining appeared to overlap extensively with that of PKCepsilon. In immunoprecipitation experiments using specific anti-Cx43 antibodies, PKCepsilon but not PKCalpha coprecipitated with Cx43. FGF-2 increased levels of coprecipitated PKCepsilon, suggesting increased association between PKCepsilon and Cx43 on stimulation. Transient gene transfer and overexpression of cDNAs coding for truncated or mutated dominant-negative forms of PKCepsilon decreased cardiomyocyte Cx43 phosphorylation significantly. We conclude that PKC mediates the FGF-2-induced effects on cardiac GJs and that PKCepsilon likely interacts with and phosphorylates cardiac Cx43 at sites of intercellular contact.

Alkaloids↗
PubMed2000

Opening of mitochondrial K(ATP) channels triggers the preconditioned state by generating free radicals.

The critical time for opening mitochondrial (mito) K(ATP) channels, putative end effectors of ischemic preconditioning (PC), was examined. In isolated rabbit hearts 29+/-3% of risk zone infarcted after 30 minutes of regional ischemia. Ischemic PC or 5-minute exposure to 10 micromol/L diazoxide, a mito K(ATP) channel opener, reduced infarction to 3+/-1% and 8+/-1%, respectively. The mito K(ATP) channel closer 5-hydroxydecanoate (200 micromol/L), bracketing either 5-minute PC ischemia or diazoxide infusion, blocked protection (24+/-3 and 28+/-6% infarction, respectively). However, 5-hydroxydecanoate starting 5 minutes before long ischemia did not affect protection. Glibenclamide (5 micromol/L), another K(ATP) channel closer, blocked the protection by PC only when administered early. These data suggest that K(ATP) channel opening triggers protection but is not the final step. Five minutes of diazoxide followed by a 30-minute washout still reduced infarct size (8+/-3%), implying memory as seen with other PC triggers. The protection by diazoxide was not blocked by 5 micromol/L chelerythrine, a protein kinase C antagonist, given either to bracket diazoxide infusion or just before the index ischemia. Bracketing preischemic exposure to diazoxide with 50 micromol/L genistein, a tyrosine kinase antagonist, did not affect infarction, but genistein blocked the protection by diazoxide when administered shortly before the index ischemia. Thus, although it is not protein kinase C-dependent, the protection by diazoxide involves tyrosine kinase. Bracketing diazoxide perfusion with N:-(2-mercaptopropionyl) glycine (300 micromol/L) or Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (7 micromol/L), each of which is a free radical scavenger, blocked protection, indicating that diazoxide triggers protection through free radicals. Therefore, mito K(ATP) channels are not the end effectors of protection, but rather their opening before ischemia generates free radicals that trigger entrance into a preconditioned state and activation of kinases.

Adenosine Triphosphate↗
PubMed1999

Oxidized low-density lipoprotein enhances myogenic tone in the rabbit posterior cerebral artery through the release of endothelin-1.

BACKGROUND AND PURPOSE: Cerebral arteries develop stretch-induced myogenic tone, which plays an important role in the regulation of blood flow to the brain. Although the effect of oxidized LDL (Ox-LDL) on many aspects of the vascular endothelial and smooth muscle cell function have been extensively investigated, its influence on myogenic activity has not been studied. METHODS: The effect of Ox-LDL on the myogenic tone that develops in the perfused rabbit posterior cerebral artery at intramural pressures between 40 and 90 mm Hg was examined. RESULTS: Ox-LDL (10 microg/mL) significantly enhanced myogenic tone by 21.4+/-6.1% to 28.5+/-1.8% at 60 to 90 mm Hg pressure (P<0.05) but had no influence on norepinephrine- (0.5 to 1 micromol/L) and KCl (20 mmol/L)-induced constriction. Ox-LDL was effective whether the artery was exposed to it from the intraluminal or the extraluminal surface. Lysophosphatidylcholine (10 micromol/L), a lipid component of Ox-LDL, had an equivalent potentiating effect. Native LDL (100 microg/mL) was inactive. The myogenic tone-potentiating effect of Ox-LDL was abolished by endothelium removal but was not influenced by the NO synthase inhibitor N(G)-nitro-L-nitro-arginine methyl ester (50 micromol/L). This effect was reversed by the endothelin-1 (ET-1) antagonist BQ-123 (1 micromol/L). This concentration blocked 1 to 3 nmol/L ET-1-induced constriction without altering constriction induced by 40 mmol/L KCl. The potentiating effect was suppressed by the specific protein kinase C inhibitor chelerythrine (1 micromol/L). CONCLUSIONS: Ox-LDL enhances myogenic tone through the release of ET-1 from the endothelium of the rabbit posterior cerebral artery.

Alkaloids↗
PubMed2006

Elevated endothelin-1 levels impair nitric oxide homeostasis through a PKC-dependent pathway.

BACKGROUND: Endothelin-1 (ET-1) plays an important role in the maintenance of vascular tone and pathological states such as ischemia/reperfusion (I/R) injury, coronary vasospasm, and cardiac allograft vasculopathy. We assessed the effects of elevated ET-1 levels as seen after I/R to determine if ET-1 modulates nitric oxide (NO) production via the translocation of specific protein kinase C (PKC) isoforms. METHODS AND RESULTS: Human saphenous vein endothelial cells (HSVECs) (n=8) were incubated with ET-1 or phosphate-buffered saline (PBS) for 24 hours. NO production was determined in the supernatant by measuring nitrate/nitrite levels. Protein expression of endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), caveolin-1 and PKC were determined. Lastly, PKC translocation and activity were assessed after exposure to the drug of interest. HSVECs exposed to ET-1 displayed decreased NO production. PKC inhibition reduced NO production, whereas PKC activation increased production. NO production was maintained when HSVECs exposed to ET-1 were treated with the PKC agonist, PMA. eNOS protein expression was reduced after ET-1 treatment. PKC inhibition also downregulated eNOS protein expression, whereas PMA upregulated expression. ET-1 exposure led to a significant increase in PKCdelta and PKCalpha translocation compared with control, whereas translocation of PKClambda was inhibited. ET-1 exposure significantly reduced overall PKC activity compared with control. CONCLUSIONS: Our study demonstrates that high levels of ET-1 impair endothelial NO production via an isoform-specific PKC-mediated inhibition of eNOS expression. ET-1 antagonism with bosentan stimulates translocation of PKClambda and leads to increased PKC activity and NO production. ET-1 antagonism may provide a novel therapeutic strategy to improve vascular homeostasis.

Alkaloids↗
PubMed2005

Role of the protein kinase C-epsilon-Raf-1-MEK-1/2-p44/42 MAPK signaling cascade in the activation of signal transducers and activators of transcription 1 and 3 and induction of cyclooxygenase-2 after ischemic preconditioning.

BACKGROUND: Although Janus kinase (JAK)-mediated Tyr phosphorylation of signal transducers and activators of transcription (STAT) 1 and 3 is essential for the upregulation of cyclooxygenase-2 (COX-2) and the cardioprotection of late preconditioning (PC), the role of Ser phosphorylation of STAT1 and STAT3 in late PC and the upstream signaling mechanisms responsible for mediating Ser phosphorylation of STAT1 and STAT3 remain unknown. METHODS AND RESULTS: In mice preconditioned with six 4-minute coronary occlusion/4-minute reperfusion cycles, we found that (1) ischemic PC activates the Raf1-mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase (MEK) 1/2-p44/42 MAPK signaling pathway, induces phosphorylation of STAT1 and STAT3 on the Ser-727 residue, and upregulates COX-2 expression; (2) pSer-STAT1 and pSer-STAT3 form complexes with pTyr-p44/42 MAPKs in preconditioned myocardium, supporting the concept that Ser phosphorylation of these 2 factors is mediated by activated p44/42 MAPKs; and (3) activation of the Raf-1-MEK-1/2-p44/42 MAPK-pSer-STAT1/3 pathway and induction of COX-2 during ischemic PC are dependent on protein kinase C (PKC)-epsilon activity, as determined by both pharmacological and genetic inhibition of PKCepsilon. CONCLUSIONS: To our knowledge, this is the first study to demonstrate that ischemic PC causes Ser phosphorylation of STAT1 and STAT3 and that this event is governed by PKCepsilon via a PKCepsilon-Raf1-MEK1/2-p44/42 MAPK pathway. Furthermore, this is the first report that COX-2 expression in the heart is controlled by PKCepsilon. Together with our previous findings, the present study implies that STAT-dependent transcription of the genes responsible for ischemic PC is modulated by a dual signaling mechanism that involves both JAK1/2 (Tyr phosphorylation) and PKCepsilon (Ser phosphorylation).

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PubMed1997

Expression of vascular endothelial growth factor by human eosinophils: upregulation by granulocyte macrophage colony-stimulating factor and interleukin-5.

Vascular endothelial growth factor (VEGF) is a pleiotropic polypeptide that mediates endothelial-cell-specific responses such as induction of proliferation and vascular leakage. We examined the expression of VEGF messenger RNA (mRNA) and protein by human eosinophils in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-5 (IL-5). Immunoreactive VEGF protein was detected in freshly isolated eosinophils by immunocytochemistry. Eosinophils spontaneously released VEGF protein in culture medium, and this release was upregulated by GM-CSF or IL-5. Freshly isolated eosinophils constitutively expressed VEGF mRNA. Although incubation of eosinophils in culture medium reduced steady-state VEGF mRNA levels, eosinophil VEGF mRNA levels were enhanced by GM-CSF and IL-5, and this enhancement was blocked by the transcription inhibitor actinomycin D. Analysis of alternatively spliced mRNA species revealed that eosinophils contained transcripts mainly encoding for the 121- and 165-amino-acid forms of VEGF. VEGF mRNA expression and VEGF release in cytokine-stimulated eosinophils were significantly reduced by treatment with a glucocorticosteroid, a protein-tyrosine kinase inhibitor, or a protein kinase C inhibitor. Cytokine-activated eosinophils may be an important source of a vascular permeability factor, namely VEGF, thus contributing to tissue edema formation at sites of allergic inflammation.

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PubMed1998

Effect of cigarette smoke extract on nitric oxide synthase in pulmonary artery endothelial cells.

Cigarette smoking is associated with impaired endothelium-dependent vasodilation and reduced nitric oxide (NO) in the exhaled air of smokers. To explore the mechanism for the impairment of NO-mediated vasodilation, we studied the effect of cigarette smoke extract (CSE) on NO synthase (eNOS) activity and content in pulmonary artery endothelial cells (PAEC). Incubation of PAEC with CSE resulted in a time- and dose-dependent decrease in eNOS activity. The inhibitory effect of CSE on eNOS activity was not reversible. Both gas-phase and particulate-phase extracts of CSE contributed to the inhibition of eNOS activity. The protein kinase c (PKC) inhibitors staurosporine and chelerythrine did not affect the CSE-induced inhibition of eNOS activity. Catalase, superoxide dismutase (SOD), vitamin C, vitamin E, glutathione, and dithiothreitol (DTT) also did not prevent the CSE-induced inhibition of eNOS activity, and incubation of PAEC with 3 mM nicotine did not change the activity of eNOS. Treatment of PAEC with CSE also caused a nonreversible, time-dependent decrease in eNOS protein content detected by Western blot analysis, and in eNOS messenger RNA (mRNA) detected by Northern blot analysis. Treatment of PAEC with CSE had no effect on cell protein or glutathione contents or on lactate dehydrogenase (LDH) release. These results indicate that exposure to CSE causes an irreversible inhibition of eNOS activity in PAEC, and suggest that the decreased activity is secondary to reduced eNOS protein mass and mRNA. The decrease in eNOS activity may contribute to the high risk of pulmonary and cardiovascular disease in cigarette smokers.

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