Adenosine-enhanced ischemic preconditioning provides enhanced cardioprotection in the aged heart

BACKGROUND: Recently we have reported a novel myo-protective protocol "adenosine-enhanced ischemic preconditioning" (APC), which extends and amends the protection afforded by ischemic preconditioning (IPC) by both reducing myocardial infarct size and enhancing postischemic functional recovery in the mature rabbit heart. However, the efficacy of APC in the senescent myocardium was unknown. METHODS: The efficacy of APC was investigated in senescent rabbit hearts and compared with magnesium-supplemented potassium cardioplegia (K/Mg) and IPC. Global ischemia (GI) hearts were subjected to 30 minutes of global ischemia and 120 minutes of reperfusion. Ischemic preconditioning hearts received 5 minutes of global ischemia and 5 minutes of reperfusion before global ischemia. Magnesium-supplemented potassium cardioplegia hearts received cardioplegia just before global ischemia. Adenosine-enhanced ischemic preconditioning hearts received a bolus injection of adenosine in concert with IPC. To separate the effects of adenosine from that of APC, a control group (ADO) received a bolus injection of adenosine 10 minutes before global ischemia. RESULTS: Infarct size was significantly decreased to 18.9%+/-2.7% with IPC (p<0.05 versus GI); 17.0%+/-1.0% with ADO (p<0.05 versus GI); 7.7%+/-1.3% with K/Mg (p<0.05 versus GI, IPC, and ADO); and 2.1%+/-0.6% with APC (p<0.05 versus GI, IPC, ADO, and K/Mg; not significant versus control). Only APC and K/Mg significantly enhanced postischemic functional recovery (not significant versus control). CONCLUSIONS: Adenosine-enhanced ischemic preconditioning provides similar protection to K/Mg cardioplegia, significantly enhancing postischemic functional recovery and decreasing infarct size in the senescent myocardium.     

Effect of protein kinase C inhibitors and activators on corneal re-epithelialization in the rat

PURPOSE: To examine the ability of protein kinase C (PKC) inhibitors and activators to influence the rate of corneal re-epithelialization in the rat. METHOD: Rat corneas with 3 mm diameter central epithelial abrasions were organ-cultured in control medium or in medium with inhibitors or activators of PKC. RESULTS: In control corneas, the defect was completely re-epithelialized by 25 hr. In the presence of the PKC inhibitors staurosporine (100 nM), sphinganine (50 mumol/l), or H-7 (100 mumol/l) there were significantly larger epithelial defects than in controls after 5-25 hr of incubation. Re-epithelialization rates were similar to control corneas when the incubation medium contained HA1004 (100 mumol/l), an analogue of H-7 that is a potent inhibitor of cyclic adenosine monophosphate- and cyclic guanosine monophosphate-dependent protein kinases and a weak inhibitor of PKC. Two PKC activators, 1-oleoyl-2-acetyl-sn-glycerol (OAG) and phorbol 12-myristate 13-acetate (PMA), were unable to enhance the rate of epithelial wound healing. CONCLUSIONS: Our results suggest that PKC activity is an important factor in regulating corneal epithelial wound healing, presumably by influencing cell migration. Moreover, the results with OAG and PMA suggest that PKC is maximally activated during re-epithelialization in this organ-culture assay.     

Pharmacologic preconditioning induced by beta-adrenergic stimulation is mediated by activation of protein kinase C

Ischemic preconditioning (I-PC) occurs via activation of protein kinase C (PKC). This study was undertaken to determine whether pharmacologic preconditioning by beta-adrenergic stimulation (beta-PC) is mediated by PKC activation. Isolated rat hearts were subjected to 40-min ischemia and 30-min reperfusion. Beta-PC was induced by 0.25 microM isoproterenol pretreatment for 2 min followed by 10-min normoxic perfusion. Beta-PC enhanced the recovery of rate-pressure product of the ischemic/reperfused heart (79.1 +/- 8.4% vs. 12.4 +/- 1.6% of initial for Non-PC group, n = 6) and attenuated the release of creatine kinase during 30-min reperfusion (30.2 +/- 2.2 vs. 59.8 +/- 6.1 nmol/min/g wet wt for Non-PC group, n = 6), similar to an I-PC stimulus of 5-min ischemia and 5-min reperfusion. Treatment with 50 microM polymyxin B, a PKC inhibitor, abolished the cardioprotection of both beta-PC and I-PC. Furthermore, similar changes in subcellular distribution of PKC were induced by both beta-PC and I-PC. The changes in subcellular distribution of PKC-delta suggested its translocation from cytosol to membrane fraction, a marker of PKC activation. These results suggest that the cardioprotection induced by beta-PC, like I-PC, is mediated by PKC activation.     

No evidence for mediation of ischemic preconditioning by alpha 1-adrenergic signal transduction pathway or protein kinase C in the isolated rat heart

A model of vertical signal flow across a layered cortical structure is presented and analyzed. Neurons communicate through spikes, which evoke an excitatory or inhibitory postsynaptic potential (spike response model). The layers incorporate two anatomical features-dendritic and axonal arborization patterns and distance-dependent time delays. The vertical signal flow through the network is discussed for various stimulus conditions using two different, but typical, axonal arborization patterns. We find stationary as well as oscillatory response, but the oscillatory response may be restricted to a single layer. Confronted with conflicting stimuli the network separates the patterns through phase-shifted oscillations. We also discuss two hypothetical animals, to be called "cat" and "mouse." These have different axonal arborizations, which give rise to a different oscillatory response (if any) of the various layers.     

Time window for the contribution of the delta-opioid receptor to cardioprotection by ischemic preconditioning in the rat heart

The present study aimed to examine (1) whether the role of the opioid receptor in ischemic preconditioning (PC) is consistent regardless of the duration of ischemic insult and (2) which opioid receptor subtype contributes to PC. In the first series of experiments, the effects of PC, a nonselective opioid receptor antagonist (naloxone), and their combination on the infarct size after various durations of ischemia were assessed. In anesthetized, open-chest rats, the coronary artery was occluded for 20, 30, or 40 minutes to induce infarction and was reperfused for 3 hours, PC was performed with two cycles of 5-minute ischemia followed by 5-minute reperfusion before the sustained ischemia. At 25 minutes before the ischemia, naloxone was injected alone or in combination with subsequent PC. Infarct size was determined by tetrazolium staining and was expressed as a percentage of the risk area size (%IS/RA). In the second series of experiments, the effects of a delta-receptor-selective antagonist, naltrindole (NTI), and a kappa-receptor selective antagonist, nor-binaltrophimine (nor-BNI), on PC before 30-minute coronary occlusion were assessed. In untreated controls, %IS/RA was 53.1 +/- 3.2 after 20 minutes, 67.9 +/- 3.9 after 30 minutes, and 87.8 +/- 2.0 after 40 minutes of ischemia, respectively. PC significantly reduced %IS/RA after 20, 30, and 40 minutes of ischemia to 3.1 +/- 0.8, 12.8 +/- 1.1, and 42.1 +/- 4.3, respectively (P < 0.05 vs. each control). Naloxone (6 mg/kg) partially attenuated the protection afforded by PC when the sustained ischemia was 30 minutes (%IS/RA = 27.4 +/- 4.5; P < 0.05 vs. PC), but this inhibitory effect of naloxone was not detected when the duration of the ischemia was 20 or 40 minutes. NTI (10 mg/kg) also attenuated infarct size limitation by PC after 30 minutes of ischemia (%IS/RA = 25.6 +/- 3.7), but nor-BNI (10 mg/kg) failed to modify infarct size limitation by PC (%IS/RA = 13.3 +/- 3.2). The present results suggest that activation of the opioid delta-receptor partly contributes to preconditioning against infarction in the rat and that there may be a time window (at around 30 minutes after the onset of ischemia) for this opioid receptor-mediated protective mechanism.     

Adrenergic activation confers cardioprotection mediated by adenosine, but is not required for ischemic preconditioning

BACKGROUND: The aim of this study was to determine whether (1) adrenergic activation is cardioprotective, (2) adrenergic cardioprotection occurs via adenosine receptor activation, and (3) ischemic preconditioning requires alpha-adrenergic activation. METHODS: Anesthetised open chest rabbits underwent 30 min coronary occlusion and 3 h reperfusion. Ischemic preconditioning was elicited with 5 min coronary occlusion and 10 min reperfusion. Activation of adrenergic receptors with endogenous norepinephrine was achieved with tyramine (0.28 mg/kg/min intravenously for 5 min). Adenosine receptors were blocked with 8-p-sulfophenyl theophylline (10 mg/kg intravenously), alpha 1-adrenergic receptors were selectively blocked with prazosin (0.1 mg/kg intravenously), and alpha-adrenergic receptors were blocked with phentolamine (4 mg/kg intravenously). RESULTS: Ischemic preconditioning reduced risk-adjusted infarct volume by 79% (P < 0.0005). This protection was attenuated by adenosine receptor blockade. Tyramine infusion resulted in a 1305% change from baseline plasma norepinephrine concentration (P < or = 0.01), and reduced infarct volume by 55% (P = 0.01). Adenosine receptor blockade abolished this protection. Blockade of alpha 1-adrenergic receptors with prazosin failed to abolish ischemic preconditioning (79 versus 89% reduction in infarct volume, without and with prazosin, respectively). Similarly, non-selective blockade of alpha-adrenergic receptors also failed to abolish ischemic preconditioning (79 versus 57% reduction without and with phentolamine, respectively). CONCLUSIONS: We conclude that the cardioprotection of ischemic preconditioning and alpha-adrenergic activation both involve adenosine, but ischemic preconditioning does not require alpha-adrenergic activation.     

Inhibition of demecolcin-induced DNA synthesis by inhibitors of phospholipase C and protein kinase C

PURPOSE OF STUDY: Interleukin-2 (IL-2) is a potent activator of lymphocytes, but its effectiveness as an anti-cancer agent is compromised by several adverse side effects including pulmonary edema. One explanation for the pulmonary toxicity of IL-2 is that activated lymphocytes directly induce the pulmonary vascular endothelium to become more leaky. METHODS: To test this hypothesis the number of total lymphocytes, gamma delta T cells, and CD2-positive cells (alpha beta T cells and natural killer cells) in peripheral blood and lung lymph of sheep were compared before and after IL-2 infusion. Hemodynamic and lymph dynamic changes were also evaluated. RESULTS: IL-2 decreased mean aortic pressure, increased cardiac output, lowered systemic vascular resistance, and doubled lung lymph flow (P < or = 0.05), but had no effect on plasma or lymph oncotic pressure. The lymph protein concentration and the lymph-to-plasma protein concentration ratio were not different after IL-2 infusion. IL-2 had no effect on the number of total lymphocytes, gamma delta T cells, or CD2-positive cells in the peripheral blood. In contrast, the number of total lymphocytes, gamma delta T cells, and CD2-positive cells in lung lymph decreased significantly (P < or = 0.05). CONCLUSIONS: The lymphocyte populations decreased more than could be explained by the increase in lymph flow, demonstrating that lung lymphocytes were not reduced simply by dilution. These results imply that the pulmonary edema associated with IL-2 is not caused by activated lymphocytes.     

Terikalant, an inward-rectifier potassium channel blocker, does not abolish the cardioprotection induced by ischemic preconditioning in the rat

Recent results have shown that the sulfonylurea receptor couples to several types of inward-rectifier potassium (KIR) channels, which suggests that sensitivity to blockade of a pathophysiological phenomenon such as ischemic preconditioning (PC) by glibenclamide may not be the result of this compound selectively blocking the ATP-sensitive potassium (KATP) channel. Therefore, to address this possibility, a role for myocardial KIR v KATP channels in ischemic PC was evaluated in the rat. To test this hypothesis, anesthetized, open-chest, male Wistar rats were assigned to one of seven experimental protocols. Animals assigned to group I (control) received 30 min of occlusion and 2 h of reperfusion. Ischemic PC was produced by 3x5-min occlusion and 2-h reperfusion periods (group II). Terikalant (TK), an inward-rectifier potassium channel blocker, was used to test the role of other K+ channels, most notably the KIR, in the cardioprotective effect of ischemic PC in the rat. TK was given at a dose of 3 mg/kg, i.v., 15 min before the prolonged occlusion and reperfusion periods (group III). In groups IV, V, and VI terikalant (1, 3 and 6 mg/kg, i.v.) was given 15 min before ischemic PC (lowTK+PC, medTK+PC and hiTK+PC, respectively). Group VII consisted of glibenclamide (0.3 mg/kg, i.v.) given 30 min prior to ischemic PC (GLY+PC). Infarct size (IS) as a percent of the area at risk (AAR) was measured using the histochemical stain, 2,3, 5-triphenyltetrazolium chloride. The average IS/AAR for the control was 49.9+/-2.1%. Ischemic PC markedly reduced infarct size (8.6+/-1. 8%; * P<0.05 v control). Terikalant (TK; 1, 3 and 6 mg/kg, i.v.) did not abolish the cardioprotective effect of ischemic PC at any dose (15.5+/-6.4, 16.4+/-5.2 and 8.8+/-1.6%, respectively; * P<0.05 v control). TK itself had no effect on infarct size. GLY completely abolished the cardioprotective effect of ischemic PC (48.2+/-6.4%). In addition, the high dose of TK significantly (P<0.05) increased the action potential duration at 50% repolarization from 48+/-3 to 64+/-4 ms and 30 microM of TK, a concentration which produced a 39% decrease in the inward-rectifier potassium channel current in isolated guinea-pig ventricular myocytes in the whole-cell patch-clamp mode did not block the increase in K ATP current produced by the KATP opener bimakalim (3 microM). These results demonstrate that although the myocardial KATP channel belongs to the K IR superfamily, the endogenous myocardial KIR channel does not mediate ischemic PC in the rat heart; however, the K ATP channel does mediate its cardioprotective effect.     

The expression of constitutively active isotypes of protein kinase C to investigate preconditioning

The role of protein kinase C (PKC) in ischemic preconditioning remains controversial because of difficulties with both its measurement and pharmacological manipulation. We investigated preconditioning in isolated neonatal rat cardiocytes by expressing constitutively active isotypes of PKC. Observations at differing durations of simulated ischemia suggested beta-galactosidase (beta-gal) activity reflected viability within transfected myocytes. Preconditioning with 90 min of ischemia significantly increased beta-gal activity and myocyte survival after 6 h of ischemia; an effect abolished by PKC inhibitors. After co-transfection with plasmids encoding beta-gal and either constitutively active mutants of PKC-delta, PKC-alpha, wild type PKC-delta, or empty vector, cardiocytes were subjected to 6 h of ischemia. Only PKC-delta, rendered constitutively active by a limited deletion within the pseudosubstrate domain, consistently increased resistance to simulated ischemia (beta-gal activity was 85.6 +/- 11.9% versus 53.7 +/- 6.5% (p 相似文献   

Effect of protein kinase inhibitors on activity of mammalian small heat-shock protein (HSP25) kinase

The aim of this study was to investigate different protein kinase inhibitors (secondary metabolite-derived substances, synthetic compounds, and substrate-based peptides) for their potency to inhibit the mammalian small heat shock protein (HSP25) kinase (E.C. 2.7.1.37) isolated from Ehrlich ascites tumor cells. Among the secondary metabolite-derived inhibitors (staurosporine, K-252a, K-252b, KT5926, KT5720, erbstatin analog, and quercetin) and synthetic compounds (H-9, H-89, HA 1004, KN-62, ML-7, tyrphostin A25, and tyrphostin B42), KT5926, staurosporine, and K-252a inhibited HSP25 kinase most efficiently. Kinetic analysis revealed that inhibition by staurosporine (Ki = 32.4 nM) and K-252a (Ki = 13.7 nM) was competitive with ATP. Inhibition by KT5926 was competitive with the substrate peptide KKKALNRQLSVAA (Ki = 27.2 nM) and noncompetitive with respect to ATP (Ki = 38.8 nM). In comparison with other protein kinases, HSP25 kinase was relatively resistant to most of the inhibitors. KT5926 was the only tested inhibitor with certain preference for HSP25 kinase when compared with protein kinases A, C, and G. Among the tested substrate-based peptides, we identified one peptide (KKKALNRQLGVAA), which preferentially inhibited HSP25 kinase in comparison with protein kinases A and C and mitogen-activated protein kinase. This peptide inhibited HSP25 kinase competitively with the substrate peptide (Ki = 8.1 microM) and noncompetitively with ATP (Ki = 134 microM). A peptide (SRVLKEDKERWEDVK) derived from the putative autoinhibitory domain of the closely related human mitogen-activated protein kinase-activated protein kinase-2 did not inhibit HSP25 kinase activity, suggesting the existence of several species of HSP25 kinases. Furthermore, the data identified structural requirements for inhibitors of HSP25-kinase.     

The involvement of protein kinase C in proliferation and differentiation of human keratinocytes--an investigation using inhibitors of protein kinase C

Protein kinase C, the major cellular receptor for tumour-promoting phorbol esters, has been suggested as playing a key role in the regulation of proliferation and differentiation of epidermal cells. In the present study, we investigated the effects of various well-characterized inhibitors of protein kinase C on proliferation and differentiation of SV 40-transformed and normal human keratinocytes. The drugs were found to inhibit cell proliferation in a dose-dependent manner, displaying similar effects in both cell types and reflecting their potencies in inhibiting purified protein kinase C. In contrast, keratinocyte differentiation induced by treatment with a calcium ionophore or spontaneously, i.e. by exposure of cells grown in the presence of low calcium concentration (0.06 mM) to normal calcium concentration (1.6 mM), was not inhibited by the compounds tested. The potent protein kinase C inhibitor, staurosporine, was found even to enhance cell differentiation. Therefore, the present study provides evidence that the classical protein kinase C pathway plays a critical role in the regulation of keratinocyte proliferation rather than in calcium-induced differentiation.     

Effect of protein kinase C inhibitors and 2,3-butanedione monoxime on the long-term hypothermic preservation of isolated rat hearts

1. This study examines the protective effect of staurosporine, chelerythrine, Ro 31-8220 and 2,3-butanedione monoxime in rat hearts during hypothermic storage. 2. Hearts were microperfused at 4 degrees C for 24 or 48 h with a storage buffer that in some cases contained one of these protein kinase C inhibitors either alone or in combination with 2,3-butanedione monoxime. After hypothermic storage, hearts were rewarmed to 37 degrees C with Krebs-Henseleit buffer. Cardiac function was then assessed in either Langendorff mode or working heart mode. 3. Compared with values from fresh non-stored hearts, hypothermic stored hearts showed a significant decrease in both coronary flow and left ventricular developed pressure when the stored hearts were reperfused in Langendorff mode. The decrease in coronary flow and left ventricular developed pressure was more pronounced in hearts stored for 48 h than in those stored for 24 h. 4. Hearts stored for 24 or 48 h, with or without the protein kinase C inhibitors, and then perfused in working mode generated less aortic flow and less cardiac output than fresh unstored hearts. 5. Hearts preserved in solutions containing staurosporine, chelerythrine, Ro 31-8220 or 2,3-butanedione monoxime had significantly higher left ventricular developed pressure values on reperfusion than hearts stored without any such drug. 6. Addition of 2,3-butanedione monoxime to a storage buffer containing either staurosporine, chelerythrine or Ro 31-8220 further improved left ventricular developed pressure, aortic flow and cardiac output values in these stored hearts. The group of hearts stored in a buffer containing 2,3-butanedione monoxime and chelerythrine gave the highest left ventricular developed pressure value seen during reperfusion. 7. The ATP and creatine phosphate concentrations of hearts stored in buffer alone were significantly lower than those of fresh unstored hearts, irrespective of the duration of storage. ATP concentrations were better preserved in hearts stored in a buffer containing 2,3-butanedione monoxime or/and one of the protein kinase C antagonists than those stored without such antagonists. A positive correlation was found between peak cardiac output values and the concentrations of combined high-energy phosphates in various groups of stored and reperfused hearts. 8. The present study showed that inhibition of protein kinase C during long-term hypothermic storage significantly increased high-energy phosphate concentrations and also improved contractile function during reperfusion.     

Modulation of human CACO-2 intestinal epithelial cell phenotype by protein kinase C inhibitors

Protein kinase C (PKC) isoforms are altered in colon tumors and upon exposure of intestinal mucosa to nutrients. We evaluated the effects of the PKC inhibitors staurosporine and calphostin C on human Caco-2 intestinal epithelial proliferation, motility, and differentiation. Motility was quantitated by monolayer expansion and the brush border enzymes dipeptidyl dipeptidase (DPDD) and alkaline phosphatase (AP) by synthetic substrate digestion. Staurosporine (0.03-1.0 ng/ml) and calphostin C (10(-12) M-10(-4) M) dose-dependently inhibited monolayer expansion and AP but stimulated DPDD. Proliferation was also inhibited but the effects of each inhibitor on motility, AP, and DPDD were preserved after mitomycin C proliferative blockade, suggesting that these effects were proliferation-independent. PKC inhibitors independently inhibit motility, AP and proliferation in human intestinal Caco-2 epithelial cells, but selectively stimulate the small intestinal differentiation marker DPDD. PKC may regulate small intestinal epithelial differentiation.     

Modulation of protein kinase C and cAMP-dependent protein kinase by delta-opioid

Modulation of protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) activities by delta-opioid receptor specific agonist [D-Pen2, D-Pen5]-enkephalin (DPDPE) was investigated in neuroblastoma x glioma hybrid NG 108-15 cells. DPDPE activated PKC in a dose-dependent manner, with the maximal response at 5 min. The DPDPE-stimulated PKC activation could be blocked by naltrindole. The activation of PKC by DPDPE was dependent on Ca2+ and was inhibited by chelerythrine chloride (10 microM), but not by H89 (1 microM). Pretreatment of NG 108-15 cells with pertussis toxin (100 ng/ml for 24 h) completely abolished DPDPE-stimulated PKC activation. In contrast to the result from the acute treatment with DPDPE, which had no significant effect on PKA activity, chronic treatment of DPDPE (1 microM for 24 h) increased PKA activity, but reduced the basal activity of PKC. These results demonstrated that DPDPE differentially modulated PKC and PKA activities via a receptor-mediated, PTX sensitive pathway.     

Effect of protein kinase inhibitors on primary antibody induction in tissue cultures

The effects of protein-kinase-inhibitors (PKIs) on protein kinase C (PKCs) i.e., staurosporin, calphostin C, H-7, H-8, H-9, on phosphatidyl inositol 3-proteinkinase (PI3-K) i.e., wortmannin, and on protein tyrosine kinase (PTKs) i.e., genistein, herbimycin A, sanguinarin, lavendustin A and B were tested on the induction phase of the primary Ab-response in vitro. The inhibitory action of PKIs was the highest with herbimycin A, sanguinarin, H-9 and wortmannin. Although wortmannin inhibits the function of T-lymphocytes (Taub et al., 1997, Shi et al., 1997), we believe that this communication is the first report of PKIs immunosuppressive action on the inductive steps of Ab-formation.     

Effect of ischemic preconditioning on myocardial oxygen consumption during ischemia

Ischemic preconditioning (IPC) in the heart may reduce myocardial energy demand. The present study was undertaken to examine changes in myocardial oxygen consumption (MVO2) during ischemia by IPC in Langendorff perfused rat hearts. We assessed MVO2 during ischemia from the measurement of mitochondrial cyt. aa3 redox state by a two-wavelength reflectance spectrophotometry where T(1/2), the time from the onset of ischemia to the point for half reduction of cyt. aa3, was assumed to represent MVO2. The heart was preconditioned by three cycles of 5 min ischemia plus 5 min reperfusion and then subjected to 30 min global ischemia followed by reperfusion for 30 min. The T(1/2) was significantly longer in the preconditioned heart (30 +/- 6 s, n = 10) than the control heart (14 +/- 5 s, n = 9, P<0.001), indicating a reduction of MVO2 during ischemic period by IPC. The prolongation of T(1/2) was evident after only one IPC episode. When the heart was perfused with high K+ solution to abolish MVO2 for contractions, we still found the prolongation of T1(1/2) in the preconditioned heart (116 +/- 12 s, n = 6) compared to the control heart (86 +/- 10 s, n = 6, P<0.01), suggesting that decrease in contractile activity may be, in part but not completely, responsible for the reduction of MVO2. In contrast, the prolongation of T(1/2) was completely abolished by administration of a NO synthase inhibitor N omega-nitro-L-arginine in the high K+ arrested heart, demonstrating involvement of NO in the reduction of MVO2, presumably by suppression of mitochondrial respiratory chain. In conclusion, IPC reduces MVO2 during ischemia. The reduction of MVO2 in the preconditioned heart may be accounted for by decreased contractile activity and by depression of respiratory chain by NO.     

Translocation inhibitors define specificity of protein kinase C isoenzymes in pancreatic beta-cells

The protein kinase C (PKC) family consists of 11 isoenzymes. Following activation, each isoenzyme translocates and binds to a specific receptor for activated C kinase (RACK) (Mochly-Rosen, D. (1995) Science 268, 247-251) that provides an anchoring site in close proximity to the isoenzyme's specific substrate. Pancreatic islet cells contain at least six PKC isoenzymes (Knutson, K. L., and Hoenig, M. (1994) Endocrinology 135, 881-886). Although PKC activation enhances insulin release, the specific function of each isoenzyme is unknown. Here we show that following stimulation with glucose, alphaPKC and epsilonPKC translocate to the cell's periphery, while deltaPKC and zetaPKC translocate to perinuclear sites. betaC2-4, a peptide derived from the RACK1-binding site in the C2 domain of betaPKC, inhibits translocation of alphaPKC and reduces insulin response to glucose. Likewise, epsilonV1-2, an epsilonPKC-derived peptide containing the site for its specific RACK, inhibits translocation of epsilonPKC and reduces insulin response to glucose. Inhibition of islet-glucose metabolism with mannoheptulose blocks translocation of both alphaPKC and epsilonPKC and diminishes insulin response to glucose while calcium-free buffer inhibits translocation of alphaPKC but not epsilonPKC and lowers insulin response by 50%. These findings illustrate the unique ability of specific translocation inhibitors to elucidate the isoenzyme-specific functions of PKC in complex signal transduction pathways.     

Studies on protein kinase C inhibitors; structure-activity relationships in indolocarbazole and bis-indole series

To assess the role of the cyclic amide moiety in indolocarbazole and bis-indole protein kinase C inhibitors, five amides in these series were synthesized in which the amide group is acyclic. These new compounds have no inhibitory effect on protein kinase C, indicating that the rigid cyclic structure of the amide group is compulsory for biological activity.     

Seasonal fluctuations in the secretory response of neuroendocrine cells of Aplysia californica to inhibitors of protein kinase A and protein kinase C

The neuroendocrine bag cells of Aplysia provide an excellent model system for exploring the roles of second-messenger pathways regulating peptide hormone secretion. Both the cAMP and diacylglycerol second-messenger systems and their associated protein kinases (PKA and PKC) are involved in regulating membrane excitability in bag-cell neurons of Aplysia. The purpose of the present set of experiments was to determine if PKA and PKC also play roles in regulating egg laying hormone (ELH) secretion from bag-cell neurons. Abdominal ganglia with attached bag-cell clusters and connective nerves were dissected from reproductively mature Aplysia, and ELH secretion in response to electrically stimulated afterdischarges was measured by RIA. ELH secretion from bag cells treated with protein-kinase inhibitors (Rp-cAMPS to inhibit PKA; H-7 to inhibit PKC) was compared to that from untreated controls. Our experiments showed that 100 microM Rp-cAMPS significantly attenuated ELH secretion during the nonbreeding seasons (winter and spring) of 2 consecutive years. This suggested a role for PKA in regulating ELH secretion. However, Rp-cAMPS had no effect on ELH secretion during the breeding seasons (summer and fall) of 2 consecutive years, even when the dose of Rp-cAMPS was increased to 200 microM. These findings indicate that there is a seasonal fluctuation in responsiveness to PKA inhibition. We also investigated if there was a seasonal fluctuation in the ability of the PKC inhibitor H-7 to suppress ELH secretion. During the nonbreeding season, 10-100 microM H-7 significantly inhibited ELH secretion, but during the breeding season, only the highest dose (100 microM) of H-7 inhibited ELH release. These results confirm that PKC plays a role in regulating ELH secretion and indicate that there is a seasonal fluctuation in responsiveness to PKC inhibition. Overall, our findings suggest that both the cAMP and diacylglycerol second-messenger pathways are regulated on a seasonal basis.     

GDNF-induced neurite formation was stimulated by protein kinase inhibitors and suppressed by Ras inhibitors

Video-assisted endoscopic techniques have decreased the surgical aggression in abdominal and thoracic surgery. In our country, pediatric laparoscopy has been developing slowly, but this is not the case for thoracoscopy. The aim of this paper is to present the techniques and results of thoracoscopy pulmonary biopsy in our first patients. Pulmonary biopsies with this approach had been done in 9 patients (5 males, 3 females). Their age ranged between 30 months and 16 years. In all cases this was the last resort for the diagnosis of pulmonary condensation of unknown etiology. The biopsies were done with the pretied knot in 5 cases, stappler in 1 case and with biopsy forceps in 2 cases. Thoracotomy was necessary in one patient, due to intraoperative haemorrhage. Enough tissue for bacterial and pathological diagnosis was obtained. There was not mortality nor important morbidity related with the technique. Postoperative recovery is better when compared with conventional thoracotomy. Thoracoscopy is an adequate approach to perform pulmonary biopsies in children. The advantages if we compare with open thoracotomy are: 1. The possibility to choose the are to perform a minimally invasive biopsy. 2. To take samples of different pulmonary lobes. 3. Less postoperative pain and shorter hospital stay (36-48 hours).