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R Bolli

Publications and source records attributed to R Bolli.

At least 37 records · Page 2Linked to original sources
PubMed2000

The late phase of preconditioning.

Unlike the early phase of preconditioning (PC), which lasts 2 to 3 hours and protects against infarction but not against stunning, the late phase of PC lasts 3 to 4 days and protects against both infarction and stunning, suggesting that it may have greater clinical relevance. It is now clear that late PC is a polygenic phenomenon that requires the simultaneous activation of multiple stress-responsive genes. Chemical signals released by a sublethal ischemic stress (such as NO, reactive oxygen species, and adenosine) trigger a complex cascade of signaling events that includes the activation of protein kinase C, Src protein tyrosine kinases, and nuclear factor kappaB and culminates in increased synthesis of inducible NO synthase, cyclooxygenase-2, aldose reductase, Mn superoxide dismutase, and probably other cardioprotective proteins. An analogous sequence of events can be triggered by a variety of stimuli, such as heat stress, exercise, and cytokines. Thus, late PC appears to be a universal response of the heart to stress in general. Importantly, the cardioprotective effects of late PC can be reproduced pharmacologically with clinically relevant agents (eg, NO donors, adenosine receptor agonists, endotoxin derivatives, or opioid receptor agonists), suggesting that this phenomenon might be exploited for therapeutic purposes. The purpose of this review is to summarize current information regarding the pathophysiology and mechanism of late PC.

Adenosine↗
PubMed2000

Cyclooxygenase-2 mediates the cardioprotective effects of the late phase of ischemic preconditioning in conscious rabbits.

We examined the role of cyclooxygenase-2 (COX-2) in the late phase of ischemic preconditioning (PC). A total of 176 conscious rabbits were used. Ischemic PC (six cycles of 4-min coronary occlusions/4-min reperfusions) resulted in a rapid increase in myocardial COX-2 mRNA levels (+231 +/- 64% at 1 h; RNase protection assay) followed 24 h later by an increase in COX-2 protein expression (+216 +/- 79%; Western blotting) and in the myocardial content of prostaglandin (PG)E(2) and 6-keto-PGF(1alpha) (+250 +/- 85% and +259 +/- 107%, respectively; enzyme immunoassay). Administration of two unrelated COX-2 selective inhibitors (NS-398 and celecoxib) 24 h after ischemic PC abolished the ischemic PC-induced increase in tissue levels of PGE(2) and 6-keto-PGF(1alpha). The same doses of NS-398 and celecoxib, given 24 h after ischemic PC, completely blocked the cardioprotective effects of late PC against both myocardial stunning and myocardial infarction, indicating that COX-2 activity is necessary for this phenomenon to occur. Neither NS-398 nor celecoxib lowered PGE(2) or 6-keto-PGF(1alpha) levels in the nonischemic region of preconditioned rabbits, indicating that constitutive COX-1 activity was unaffected. Taken together, these results demonstrate that, in conscious rabbits, up-regulation of COX-2 plays an essential role in the cardioprotection afforded by the late phase of ischemic PC. Therefore, this study identifies COX-2 as a cardioprotective protein. The analysis of arachidonic acid metabolites strongly points to PGE(2) and/or PGI(2) as the likely effectors of COX-2-dependent protection. The recognition that COX-2 mediates the antistunning and antiinfarct effects of late PC impels a reassessment of current views regarding this enzyme, which is generally regarded as detrimental.

6-Ketoprostaglandin F1 alpha↗
PubMed2000

Transgenic overexpression of constitutively active protein kinase C epsilon causes concentric cardiac hypertrophy.

To test the hypothesis that activation of the protein kinase C (PKC) epsilon isoform leads to cardiac hypertrophy without failure, we studied transgenic mice with cardiac-specific overexpression of a constitutively active mutant of the PKCepsilon isoform driven by an alpha-myosin heavy chain promoter. In transgenic mice, the protein level of PKCepsilon in heart tissue was increased 9-fold. There was a 6-fold increase of the membrane/cytosol ratio, and PKC activity in the membrane fraction was 4.2-fold compared with wild-type mice. The heart weight was increased by 28%, and upregulation of the mRNA for beta-myosin heavy chain and alpha-skeletal actin was observed in transgenic mouse hearts. Echocardiography demonstrated increased anterior and posterior wall thickness with normal left ventricular function and dimensions, indicating concentric cardiac hypertrophy. Isolated cardiomyocyte mechanical function was slightly decreased, and Ca(2+) signals were markedly depressed in transgenic mice, suggesting that myofilament sensitivity to Ca(2+) was increased. No differences were observed in either the levels of cardiac Ca(2+)-handling proteins or the degree of cardiac regulatory protein phosphorylation between wild-type and transgenic mice. Unlike mice with PKCbeta(2) overexpression, transgenic mice with cardiac-specific overexpression of the active PKCepsilon mutant demonstrated concentric hypertrophy with normal in vivo cardiac function. Thus, PKC isoforms may play differential functional roles in cardiac hypertrophy and failure.

Animals↗
PubMed2000

Evidence for an essential role of cyclooxygenase-2 as a mediator of the late phase of ischemic preconditioning in mice.

Recent studies have demonstrated that cyclooxygenase-2 (COX-2) is an essential mediator of the cardioprotective effects of the late phase of ischemic preconditioning (PC) in rabbits. The goal of this study was to determine whether COX-2 also plays an essential role in late PC in the mouse. B6129F2/J mice underwent a 30-min coronary occlusion followed by 24 h of reperfusion. Administration of the COX-2 selective inhibitor, NS-398, 30 min prior to the 30-min occlusion (5 mg/kg i.p.) had no appreciable effect on infarct size compared with untreated controls (58.8 +/- 2.1%, vs. 58.8 +/- 4.3% of the risk region, respectively). When mice were preconditioned with six cycles of 4-min coronary occlusion/4-min reperfusion 24 h prior to the 30-min occlusion, infarct size was markedly reduced (19.3 +/- 3.4%), indicating a late PC effect. The protective effect of late PC was completely abrogated by administration of NS-398 30 min before the 30-min coronary occlusion (67.7 +/- 3.0%), but not by administration of vehicle alone (23.6 +/- 3.7%). These results indicate that COX-2 mediates the late phase of ischemic PC in the mouse and imply that the role of this enzyme in cardioprotection is not species-specific.

Animals↗
PubMed2000

PKCepsilon modulates NF-kappaB and AP-1 via mitogen-activated protein kinases in adult rabbit cardiomyocytes.

We have previously shown that protein kinase C (PKC)-epsilon, nuclear factor (NF)-kappaB, and mitogen-activated protein kinases (MAPKs) are essential signaling elements in ischemic preconditioning. In the present study, we examined whether activation of PKCepsilon affects the activation of NF-kappaB in cardiac myocytes and whether MAPKs are mediators of this signaling event. Activation of PKCepsilon (+108% above control) in adult rabbit cardiomyocytes to a degree that has been previously shown to protect myocytes against hypoxic injury increased the DNA-binding activity of NF-kappaB (+164%) and activator protein (AP)-1 (+127%) but not that of Elk-1. Activation of PKCeta did not have an effect on these transcription factors. Activation of PKCepsilon also enhanced the phosphorylation activities of the p44/p42 MAPKs and the p54/p46 c-Jun NH(2)-terminal kinases (JNKs). PKCepsilon-induced activation of NF-kappaB and AP-1 was completely abolished by inhibition of the p44/p42 MAPK pathway with PD98059 and by inhibition of the p54/p46 JNK pathway with a dominant negative mutant of MAPK kinase-4, indicating that both signaling pathways are necessary. Taken together, these data identify NF-kappaB and AP-1 as downstream targets of PKCepsilon, thereby establishing a molecular link between activation of PKCepsilon and activation of NF-kappaB and AP-1 in cardiomyocytes. The results further demonstrate that both the p44/p42 MAPK and the p54/p46 JNK signaling pathways are essential mediators of this event.

Animals↗
PubMed2000

Differential role of K(ATP) channels in late preconditioning against myocardial stunning and infarction in rabbits.

The role of ATP-sensitive potassium (K(ATP)) channels in the late phase of ischemic preconditioning (PC) remains unclear. Furthermore, it is unknown whether K(ATP) channels serve as end effectors both for late PC against infarction and against stunning. Thus, in phase I of this study, conscious rabbits underwent a 30-min coronary occlusion (O) followed by 72 h of reperfusion (R) with or without ischemic PC (6 4-min O/4-min R cycles) 24 h earlier. Late PC reduced infarct size approximately 46% versus controls. The K(ATP) channel blocker 5-hydroxydecanoic acid (5-HD), given 5 min before the 30-min O, abrogated the infarct-sparing effect of late PC but did not alter infarct size in non-PC rabbits. In phase II, rabbits underwent six 4-min O/4-min R cycles for 3 consecutive days (days 1, 2, and 3). In controls, the total deficit of systolic wall thickening (WTh) after the sixth reperfusion was reduced by 46% on day 2 and 54% on day 3 compared with day 1, indicating a late PC effect against myocardial stunning. Neither 5-HD nor glibenclamide, given on day 2, abrogated late PC. The K(ATP) channel opener diazoxide, given on day 1, attenuated stunning, and this effect was completely blocked by 5-HD. Thus the same dose of 5-HD that blocked the antistunning effect of diazoxide failed to block the antistunning effects of late PC. Furthermore, when diazoxide was administered in PC rabbits on day 2, myocardial stunning was further attenuated, indicating that diazoxide and late PC have additive anti-stunning effects. We conclude that K(ATP) channels play an essential role in late PC against infarction but not in late PC against stunning, revealing an important pathogenetic difference between these two forms of cardioprotection.

Adenosine Triphosphate↗
PubMed2000

Biphasic response of cardiac NO synthase isoforms to ischemic preconditioning in conscious rabbits.

In conscious rabbits, a sequence of six 4-min coronary occlusion/4-min reperfusion cycles, which elicits late preconditioning (PC), caused rapid activation of calcium-dependent nitric oxide (NO) synthase (NOS) [cNOS; endothelial NOS (eNOS) and/or neuronal NOS (nNOS)], whereas calcium-independent NOS [inducible NOS (iNOS)] activity remained unchanged. The enhanced cNOS activity was associated with increased myocardial levels of NO(2) and/or NO(3) (NO(x)). Twenty-four hours after ischemic PC was induced, the opposite pattern was observed, i.e., there was a pronounced increase in cytosolic iNOS activity but no change in cNOS activity. The initial burst of ischemia-induced cNOS activity was not affected by pretreatment with the antioxidant N-2-mercaptopropionyl glycine (MPG), the protein kinase C (PKC) inhibitor chelerythrine, or the tyrosine kinase inhibitor lavendustin A, indicating that it is independent of the generation of oxidant species and the activation of PKC and tyrosine kinases. In contrast, the delayed upregulation of iNOS 24 h after PC was prevented by pretreatment with N(omega)-nitro-L-arginine, MPG, or chelerythrine before the PC ischemia, indicating that it is triggered by a signaling mechanism that involves the generation of NO, the formation of oxidant species, and the activation of PKC. Taken together, these results demonstrate that, in conscious animals, ischemic PC elicits a biphasic response in cardiac NOS activity, i. e., an immediate activation of cNOS (most likely eNOS) followed 24 h later by a delayed upregulation of iNOS. To our knowledge, this is the first study to directly measure NOS activity after brief myocardial ischemia in vivo. In conjunction with previous functional studies, the data support a distinctive role of NOS isoforms in late PC, with eNOS serving as the trigger on day 1 and iNOS as the mediator on day 2.

Animals↗
PubMed2000

Late preconditioning enhances recovery of myocardial function after infarction in conscious rabbits.

It is unknown whether late preconditioning (PC) enhances the recovery of left ventricular (LV) function after a myocardial infarction. Thus 25 conscious rabbits were subjected to a 30-min coronary occlusion followed by 28 days of reperfusion after PC 24 h earlier with either ischemia or nitric oxide donor administration [S-nitroso-N-acetylpenicillamine (SNAP)]. The recovery of wall thickening (WTh) after reperfusion was significantly improved in the ischemic PC and SNAP PC groups compared with controls, both at rest and during dobutamine stress. Interestingly, neither ischemia- nor SNAP-induced late PC attenuated myocardial stunning from day 1 through day 14. Infarct size was smaller in the ischemic PC and SNAP PC groups compared with controls. In all groups, WTh at 28 days was positively and linearly related to the percentage of viable tissue in the region underlying the ultrasonic crystal (r = 0.90), indicating that the improvement in LV function after both ischemia-induced and NO donor-induced late PC can be fully explained by the reduction in infarct size; a separate effect of late PC on LV remodeling or LV contractility need not be invoked. In conclusion, in conscious rabbits late PC, induced either by ischemia or pharmacologically, not only limits infarct size but also enhances the recovery of LV function after myocardial infarction. This finding has important clinical implications and provides triphenyltetrazolium chloride-independent evidence that late PC limits myocellular death after sustained ischemia.

Animals↗
PubMed2000

Product equivalence study comparing the tolerability, pharmacokinetics, and pharmacodynamics of various human immunoglobulin-G formulations.

In this randomized, double-blind, parallel-group study, a commercially available human immunoglobulin-G product, IVIG, was compared with two test formulations: (1) IVIG-N, which is a nanofiltered formulation of IVIG, and (2) IVIG-L, which is a nanofiltered, liquid, ready-for-use IgG formulation containing nicotinamide, L-proline, and L-isoleucine as stabilizers. Three groups of 10 healthy subjects each received a single 0.6 g/kg dose of one of the formulations infused over 3.5 to 6.8 hours, depending on the total volume to be infused. Blood samples were obtained over a 6-week period to assess pharmacokinetics, immunogenicity, and the pharmacodynamic effects on leukocytes and TNF-alpha. A blood sample was taken at 6 months for a viral safety check. Administrations were generally well tolerated with only one reference IVIG infusion stopped prematurely due to headache. The IgG Cmax and AUC over the 6-week blood sampling period from both test formulations satisfied equivalence criteria compared with the reference formulation. In subjects receiving IVIG-L, peak concentrations of the stabilizer nicotinamide ranged from 0.34 to 0.47 mmol/L and of nicotinamide-N-oxide from 0.03 to 0.04 mmol/L, which are below those reported to cause adverse events. During the infusion of IVIG, leukocyte counts initially declined from a baseline of 5.7 +/- 1.1 x 10(9)/L to 3.7 +/- 0.8 x 10(9)/L at 2 to 4 hours and returned to baseline by 24 hours. TNF-alpha levels, reflecting activation of the monocyte-macrophage system by the infused IVIG, rose from a baseline of 13 +/- 4 pg/mL to a peak of 272 +/- 324 pg/mL at 2 to 4 hours and returned to baseline by 24 hours. These patterns were generally similar for the test formulations, with the exception that the increase in TNF-alpha levels was dampened for IVIG-N, although this was not statistically significant. There was no evidence of immunogenicity or viral transmission from any of the formulations. Hence, these three formulations were generally well tolerated, yielded similar systemic exposure to IgG over a 6-week period after administration, and did not give rise to immunogenicity or viral safety concerns.

Adolescent↗
PubMed1999

Adenosine as an adjunct to thrombolytic therapy for acute myocardial infarction: results of a multicenter, randomized, placebo-controlled trial: the Acute Myocardial Infarction STudy of ADenosine (AMISTAD) trial.

OBJECTIVES: The Acute Myocardial Infarction STudy of ADenosine (AMISTAD) trial was designed to test the hypothesis that adenosine as an adjunct to thrombolysis would reduce myocardial infarct size. BACKGROUND: Reperfusion therapy for acute myocardial infarction (MI) has been shown to reduce mortality, but reperfusion itself also may have deleterious effects. METHODS: The AMISTAD trial was a prospective, open-label trial of thrombolysis with randomization to adenosine or placebo in 236 patients within 6 h of infarction onset. The primary end point was infarct size as determined by Tc-99 m sestamibi single-photon emission computed tomography (SPECT) imaging 6+/-1 days after enrollment based on multivariable regression modeling to adjust for covariates. Secondary end points were myocardial salvage index and a composite of in-hospital clinical outcomes (death, reinfarction, shock, congestive heart failure or stroke). RESULTS: In all, 236 patients were enrolled. Final infarct size was assessed in 197 (83%) patients. There was a 33% relative reduction in infarct size (p = 0.03) with adenosine. There was a 67% relative reduction in infarct size in patients with anterior infarction (15% in the adenosine group vs. 45.5% in the placebo group) but no reduction in patients with infarcts located elsewhere (11.5% for both groups). Patients randomized to adenosine tended to reach the composite clinical end point more often than those assigned to placebo (22% vs. 16%; odds ratio, 1.43; 95% confidence interval, 0.71 to 2.89). CONCLUSIONS: Many agents thought to attenuate reperfusion injury have been unsuccessful in clinical investigation. In this study, adenosine resulted in a significant reduction in infarct size. These data support the need for a large clinical outcome trial.

Adenosine↗
PubMed1999

The late phase of ischemic preconditioning is abrogated by targeted disruption of the inducible NO synthase gene.

The goal of this study was to interrogate the role of inducible NO synthase (iNOS) in the late phase of ischemic preconditioning (PC) in vivo. A total of 321 mice were used. Wild-type mice preconditioned 24 h earlier with six cycles of 4-min coronary occlusion/4-min reperfusion exhibited a significant (P < 0.05) increase in myocardial iNOS protein content, iNOS activity (assessed as calcium-independent L-citrulline formation), and nitrite + nitrate tissue levels. In contrast, endothelial NOS protein content and calcium-dependent NOS activity remained unchanged. No immunoreactive neuronal NOS was detected. When wild-type mice were preconditioned 24 h earlier with six 4-min occlusion/4-min reperfusion cycles, the size of the infarcts produced by a 30-min coronary occlusion followed by 24 h of reperfusion was reduced markedly (by 67%; P < 0.05) compared with sham-preconditioned controls, indicating a late PC effect. In contrast, when mice homozygous for a null iNOS allele were preconditioned 24 h earlier with the same protocol, infarct size was not reduced. Disruption of the iNOS gene had no effect on early PC or on infarct size in the absence of PC. These results demonstrate that (i) the late phase of ischemic PC is associated with selective up-regulation of iNOS, and (ii) targeted disruption of the iNOS gene completely abrogates the infarct-sparing effect of late PC (but not of early PC), providing unequivocal molecular genetic evidence for an obligatory role of iNOS in the cardioprotection afforded by the late phase of ischemic PC. Thus, this study identifies a specific protein that mediates late PC in vivo.

Animals↗
PubMed1999

Demonstration of selective protein kinase C-dependent activation of Src and Lck tyrosine kinases during ischemic preconditioning in conscious rabbits.

Src tyrosine kinases have been shown to mediate cellular responses to stress in noncardiac cells. However, the effect of myocardial ischemia on Src tyrosine kinases is unknown. Furthermore, the identity of the tyrosine kinase(s) involved in the genesis of ischemic preconditioning (PC) remains obscure. Here, we present the first evidence that ischemic PC (6 cycles of 4-minute coronary occlusion and 4-minute reperfusion) induces selective activation of 2 members of the Src family of tyrosine kinases, Src and Lck, in the heart of conscious rabbits. The activation of Src in the particulate fraction was not evident at 5 minutes after ischemic PC but became apparent at 30 minutes (+119% versus control), whereas the activation of Lck in the particulate fraction was apparent both at 5 minutes (+103% versus control) and at 30 minutes (+89%) after ischemic PC. The activity of the other 5 members of the Src tyrosine kinases expressed in the rabbit heart (Fyn, Fgr, Yes, Lyn, and Blk) was not affected by ischemic PC. Ischemic PC had no effect on the activity of epidermal growth factor receptor kinases, either at 5 or at 30 minutes. The activation of Src and Lck was completely abrogated by the tyrosine kinase inhibitor lavendustin A, given at doses that have previously been shown to block the protective effect of ischemic PC in this same conscious rabbit model, suggesting that Src and Lck kinases are essential for the development of ischemic PC. The activity of the epsilon isoform of protein kinase C (PKC) in the particulate fraction increased at 5 minutes (+72%) and at 30 minutes (+67%) after ischemic PC. Pretreatment with lavendustin A had no effect on the activation of PKCepsilon, whereas pretreatment with the PKC inhibitor chelerythrine (given at doses that have previously been shown to block ischemic PC) blocked not only the activation of PKCepsilon but also that of Src and Lck, indicating that Src and Lck are downstream of PKCepsilon in the signaling cascade of ischemic PC. This study identifies a new component of the signaling mechanism of ischemic PC. The results support the concept that, in conscious rabbits, 2 specific members of the Src family of tyrosine kinases, Src and Lck, play an important role in the genesis of late PC by serving as downstream elements of PKC-mediated signal transduction.

Animals↗
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↗
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

Ischemic preconditioning increases iNOS transcript levels in conscious rabbits via a nitric oxide-dependent mechanism.

Recent studies implicate iNOS as the mediator of the late phase of ischemic preconditioning (PC). However, it is unknown whether induction of iNOS activity is mediated by transcriptional, post-transcriptional, translational, or post-translational mechanisms. To address this issue, we isolated and sequenced a partial iNOS cDNA expressed in preconditioned rabbit myocardium. Using a rabbit-specific probe generated from this sequence, we measured the steady state levels of the iNOS transcript after ischemic PC [six cycles of 4-min occlusion/4-min reperfusion (O/R)]. Three hours after ischemic PC, the iNOS mRNA levels in the ischemic/reperfused region were increased approximately three-fold relative to samples from the non-ischemic region and from control rabbits. This increase in mRNA levels was completely abolished by pretreatment with the NOS inhibitor Nomega -nitro- L-arginine. Conversely, administration of the NO donor nitroglycerin induced an increase in iNOS mRNA levels similar to that induced by ischemic PC. We conclude that in the conscious rabbit, ischemic PC induces an increase in iNOS mRNA levels, and that this induction is triggered by increased generation of NO during the PC stimulus. These results provide direct evidence that upregulation of iNOS is a natural response of the heart to a brief ischemic stress and that NO itself, in the absence of ischemia, upregulates myocardial iNOS transcript levels, a finding that may have implications for nitrate therapy. This previously unrecognized NO-dependent upregulation of iNOS mRNA is likely to play an important role in the development of late PC as well as in many other pathophysiological conditions in which NO is implicated.

Amino Acid Sequence↗
PubMed1999

Bradykinin-induced preconditioning in patients undergoing coronary angioplasty.

OBJECTIVES: The purpose of this study was to determine whether administration of bradykinin reproduces the cardioprotective effects of ischemic preconditioning (PC) in patients undergoing percutaneous transluminal coronary angioplasty (PTCA). BACKGROUND: Experimental studies suggest that activation of the bradykinin B2 receptor is an important trigger of ischemic PC. However, it is unknown whether bradykinin can precondition human myocardium against ischemia in vivo. Multicenter clinical trials have demonstrated an anti-ischemic effect of angiotensin-converting enzyme inhibitors, which has been postulated to result from potentiation of bradykinin; however, direct evidence for an anti-ischemic action of bradykinin in patients is lacking. METHODS: Thirty patients were randomized to receive a 10-min intracoronary infusion of bradykinin (2.5 microg/min) or normal saline. Ten minutes later they underwent PTCA (three 2-min balloon inflations 5 min apart). RESULTS: In control patients, the ST-segment shift on the intracoronary and surface electrocardiogram was significantly greater during the first inflation than during the second and third inflations, consistent with ischemic PC. In bradykinin-treated patients, the ST-segment shift during the first inflation was significantly smaller than in the control group, and there were no appreciable differences in ST-segment shift during the three inflations. Measurements of chest pain score and regional wall motion during inflation (quantitative two-dimensional echocardiography) paralleled those of ST-segment shift. Infusion of bradykinin had no hemodynamic effects and no significant adverse effects. Thus, intracoronary infusion of bradykinin before PTCA rendered the myocardium relatively resistant to subsequent ischemia, and the degree of this cardioprotective effect was comparable to that afforded by the ischemia associated with the first balloon inflation in control subjects. In a separate cohort of seven patients given the same dose of bradykinin, coronary hyperemia resolved completely within 10 min after the end of the infusion, indicating that bradykinin-induced vasodilation cannot account for the protective effects observed during the first balloon inflation. CONCLUSIONS: Bradykinin preconditions human myocardium against ischemia in vivo in the absence of systemic hemodynamic changes. Pretreatment with bradykinin appears to be just as effective as ischemic PC and could be used prophylactically to attenuate ischemia in selected patients undergoing PTCA.

Adult↗