Role of Na(+)/H(+) exchanger during O(2) deprivation in mouse CA1 neurons.
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To determine the role of membrane transporters in intracellular pH (pH(i)) regulation under conditions of low microenvironmental O(2), we monitored pH(i) in isolated single CA1 neurons using the fluorescent indicator carboxyseminaphthorhodafluor-1 and confocal microscopy. After total O(2) deprivation or anoxia (PO(2) approximately equal to 0 Torr), a large increase in pH(i) was seen in CA1 neurons in HEPES buffer, but a drop in pH(i), albeit small, was observed in the presence of HCO(3)(-). Ionic substitution and pharmacological experiments showed that the large anoxia-induced pH(i) increase in HEPES buffer was totally Na(+) dependent and was blocked by HOE-694, strongly suggesting the activation of the Na(+)/H(+) exchanger (NHE). Also, this pH(i) increase in HEPES buffer was significantly smaller in Na(+)/H(+) exchanger isoform 1 (NHE1) null mutant CA1 neurons than in wild-type neurons, demonstrating that NHE1 is responsible for part of the pH(i) increase following anoxia. Both chelerythrine and H-89 partly blocked, and H-7 totally eliminated, this anoxia-induced pH(i) increase in the absence of HCO. We conclude that 1) O(2) deprivation activates Na(+)/H(+) exchange by enhancing protein kinase activity and 2) membrane proteins, such as NHE, actively participate in regulating pH(i) during low-O(2) states in neurons. (+info)
High calcium and dobutamine positive inotropy in the perfused mouse heart: myofilament calcium responsiveness, energetic economy, and effects of protein kinase C inhibition.
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BACKGROUND: In perfused hearts, high calcium-induced inotropy results in less developed pressure relative to myocardial oxygen consumption compared to the beta-adrenergic agonist dobutamine. Calcium handling is an important determinant of myocardial oxygen consumption. Therefore, we hypothesized that this phenomenon was due to reduced myofilament responsiveness to calcium, related to protein kinase C activation. RESULTS: Developed pressure was significantly higher with dobutamine compared to high perfusate calcium of 3.5 mM (73 +/- 10 vs 63 +/- 10 mmHg, p < 0.05), though peak systolic intracellular calcium was not significantly different, suggesting reduced myofilament responsiveness to intracellular calcium with high perfusate calcium. The ratio of developed pressure to myocardial oxygen consumption, an index of economy of contraction, was significantly increased with dobutamine compared to high perfusate calcium (1.35 +/- 0.15 vs 1.15 +/- 0.15 mmHg/micromoles/min/g dry wt, p < 0.05), suggesting energetic inefficiency with high perfusate calcium. The specific protein kinase C inhibitor, chelerythrine, significantly attenuated the expected increase in developed pressure when increasing perfusate calcium from 2.5 to 3.5 mM (3.5 mM: 64 +/- 8 vs 3.5 mM + chelerythrine: 55 +/- 5 mmHg, p < 0.05), though had no effects on dobutamine, or lower levels of perfusate calcium (1.5 to 2.5 mM). CONCLUSIONS: By measuring intracellular calcium, developed pressures and myocardial oxygen consumption in perfused mouse hearts, these results demonstrate that high perfusate calcium positive inotropy compared to dobutamine results in reduced myofilament responsiveness to intracellular calcium, which is associated with energetic inefficiency and evidence of protein kinase C activation. (+info)
Early membrane estrogenic effects required for full expression of slower genomic actions in a nerve cell line.
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Interpretations of steroid hormone actions as slow, nuclear, transcriptional events have frequently been seen as competing against inferences of rapid membrane actions. We have discovered conditions where membrane-limited effects potentiate later transcriptional actions in a nerve cell line. Making use of a two-pulse hormonal schedule in a transfection system, early and brief administration of conjugated, membrane-limited estradiol was necessary but not sufficient for full transcriptional potency of the second estrogen pulse. Efficacy of the first pulse depended on intact signal transduction pathways. Surprisingly, the actions of both pulses were blocked by a classical estrogen receptor (ER) antagonist. Thus, two different modes of steroid hormone action can synergize. (+info)
Protein kinase C regulation of cell spreading in the molluscan Biomphalaria glabrata embryonic (Bge) cell line.
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Cellular adhesion and spreading are critical components involved in the processes of cell and tissue development, and immune responses in molluscs, but at present, little is known regarding the signaling pathways involved in these basic cellular functions. In the present study, the molluscan Biomphalaria glabrata embryonic (Bge) cell line was used as an in vitro model to study the signal transduction pathways regulating molluscan cell adhesion and spreading behavior. Western blot analysis using antibodies specific to mitogen-activated protein kinase (MAPK) revealed the presence of an MAPK-like immunoreactive protein in Bge cells, that was phosphorylated upon exposure to phorbol myristate acetate (PMA). Moreover, Bge cell treatment with inhibitors of protein kinase C (PKC), Ras and MAPK kinase (Mek) suppressed PMA-induced expression of activated MAPK, suggesting that PKC-, Ras- and Mek-like molecules may be acting upstream of MAPK. Similarly, in vitro Bge cell-spreading assays were performed in conjunction with the same panel of inhibitors to determine the potential involvement of PKC, Ras and Mek in cellular adhesion/spreading. Results revealed a similar pattern of inhibition of cell-spreading behavior strongly implying that the Bge cell spreading also may be regulated through a MAPK-associated signal transduction pathway(s) involving proteins similar to PKC, Ras and Mek. (+info)
Adenosine A(2A) and A(2B) receptor activation of erythropoietin production.
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We have examined the effects of adenosine receptors and protein kinases A and C in the regulation of erythropoietin (Epo) production using hepatocellular carcinoma (Hep3B) cells in culture and in vivo in normal mice under normoxic and hypoxic conditions. CGS-21680, a selective adenosine A(2A) agonist, significantly increased levels of Epo in normoxic Hep3B cell cultures and in serum of normal mice under both normoxic and hypoxic conditions. CGS-21680 also produced a significant increase in Epo mRNA levels in Hep3B cell cultures. SCH-58261, a selective adenosine A(2A) receptor antagonist, significantly inhibited the increase in medium levels of Epo in Hep3B cell cultures exposed to hypoxia (1% O(2)). Enprofylline, a selective adenosine A(2B) receptor antagonist, significantly inhibited the increase in plasma levels of Epo in normal mice exposed to hypoxia. Chelerythrine chloride, an antagonist of protein kinase C activation, significantly inhibited hypoxia-induced increases in serum levels of Epo in normal mice. A model is presented for adenosine in hypoxic regulation of Epo production that involves kinases A and C and phospholipase A(2) pathways. (+info)
Mechanisms of the apoptotic and necrotic actions of trimethyltin in cerebellar granule cells.
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In evaluating mechanisms of trimethyltin (TMT)-initiated neuronal damage, the present study focused on involvement of reactive oxygen species, protein kinase C (PKC), and glutamate receptors. Exposure of cerebellar granule cells to TMT (0.01-0.1 microM) produced primarily apoptosis, but higher concentrations were associated with cellular lactate dehydrogenase efflux and necrosis. TMT increased generation of cellular reactive oxygen species, which was inhibited by either L-NAME (inhibitor of nitric oxide synthase, NOS) or catalase, indicating that both NO and H(2)O(2) are formed on TMT exposure. Since chelerythrine (selective PKC inhibitor) also inhibited oxidative species generation, PKC appears to play a significant role in TMT-induced oxidative stress. The metabotropic glutamate receptor antagonist, MCPG, (but not MK-801) prevented oxidative species generation, indicating significant involvement of metabotropic receptors (but not NMDA receptors) in TMT-induced oxidative stress. NOS involvement in the action of TMT was confirmed through measurement of nitrite, which increased concentration dependently. Nitrite accumulation was blocked by L-NAME, chelerythrine, or MCPG, showing that NO is generated by TMT and that associated changes in NOS are regulated by a PKC-mediated mechanism. Oxidative damage by TMT was demonstrated by detection of elevated malondialdehyde levels. It was concluded that low concentrations of TMT (0.01-0.1 microM) cause apoptotic cell death in which oxidative signaling is an important event. Higher concentrations of TMT initiate necrotic death, which involves both an oxidative and a non-oxidative component. TMT-induced necrosis but not apoptosis in granule cells is mediated by glutamate receptors. (+info)
Activation of PKC decreases myocardial O2 consumption and increases contractile efficiency in rats.
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The effect of protein kinase C (PKC) activation on cardiac mechanoenergetics is not fully understood. To address this issue, we determined the effects of the PKC activator phorbol 12-myristate 13-acetate (PMA) on isolated rat hearts. Hearts were exposed to PMA with or without pretreatment with the PKC inhibitor chelerythrine. Contractile efficiency was assessed as the reciprocal of the slope of the linear myocardial O2 consumption (VO2) pressure-volume area (PVA) relation. PMA decreased contractility (Emax; -30 +/- 8%; P < 0.05) and increased coronary perfusion pressure (+58 +/- 11%; P < 0.01) without altering left ventricular end-diastolic pressure. Concomitantly, PMA decreased PVA-independent VO2 [nonmechanical energy expenditure for excitation-contraction (E-C) coupling and basal metabolism] by 28 +/- 8% (P < 0.05) and markedly increased contractile efficiency (+41 +/- 8%; P < 0.05) in a manner independent of the coronary vascular resistance. Basal metabolism was not affected by PMA. Chelerythrine abolished the PMA-induced vasoconstriction, negative inotropy, decreased PVA-independent VO2, and increased contractile efficiency. We conclude that PKC-mediated phosphorylation of regulatory proteins reduces VO2 via effects on both the contractile machinery and the E-C coupling. (+info)
Role of spinal NMDA receptors, protein kinase C and nitric oxide synthase in the hyperalgesia induced by magnesium deficiency in rats.
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1. Magnesium (Mg)-deficient rats develop a mechanical hyperalgesia which is reversed by a N-Methyl-D-Aspartate (NMDA) receptor antagonist. Given that functioning of this receptor-channel is modulated by Mg, we wondered whether facilitated activation of NMDA receptors in Mg deficiency state may in turn trigger a cascade of specific intracellular events present in persistent pain. Hence, we tested several antagonists of NMDA and non-NMDA receptors as well as compounds interfering with the functioning of intracellular second messengers for effects on hyperalgesia in Mg-deficient rats. 2. Hyperalgesic Mg-deficient rats were administered intrathecally (10 microl) or intraperitoneally with different antagonists. After drug injection, pain sensitivity was evaluated by assessing the vocalization threshold in response to a mechanical stimulus (paw pressure test) over 2 h. 3. Intrathecal administration of MgSO4 (1.6, 3.2, 4.8, 6.6 micromol) as well as NMDA receptor antagonists such as MK-801 (0.6, 6.0, 60 nmol), AP-5 (10.2, 40.6, 162.3 nmol) and DCKA (0.97, 9.7, 97 nmol) dose-dependently reversed the hyperalgesia. Chelerythrine chloride, a protein kinase C (PKC) inhibitor (1, 10.4, 104.2 nmol) and 7-NI, a specific nitric oxide (NO) synthase inhibitor (37.5, 75, 150 micromol x kg(-1), i.p.) induced an anti-hyperalgesic effect in a dose-dependent manner. SR-140333 (0.15, 1.5, 15 nmol) and SR-48968 (0.17, 1.7, 17 nmol), antagonists of neurokinin receptors, produced a significant, but moderate, increase in vocalization threshold. 4. These results demonstrate that Mg-deficiency induces a sensitization of nociceptive pathways in the spinal cord which involves NMDA and non-NMDA receptors. Furthermore, the data is consistent with an active role of PKC, NO and, to a lesser extent substance P in the intracellular mechanisms leading to hyperalgesia. (+info)