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PubMed2003

Various inotropic effects of angiotensin II in post-ischaemic rat hearts depending on ischaemic time with possible involvement of protein kinase C.

AIMS: The present study investigated if the inotropic effect of angiotensin II (AngII) is altered during post-ischaemic reperfusion in hearts subjected to mild and severe ischaemia. The possible involvement of protein kinase C (PKC) in the change in the inotropic effect was also investigated. METHODS: Isolated Langendorff-perfused rat hearts were perfused under constant flow with oxygenated Krebs-Henseleit buffer and paced at 360 beats min(-1). A saline-filled balloon catheter inserted into the left ventricle was used for measurement of contractile force. In the first series of experiments, hearts were subjected to continuous perfusion, 15- or 25-min global ischaemia followed by 45-min reperfusion. At the end of reperfusion, 0.1 micromol L(-1) AngII was infused for 5 min. In a second series of experiments, AngII was infused in hearts subjected to 25-min ischaemia followed by 45-min reperfusion in the absence or presence of the PKC inhibitor chelerythrine chloride (5 micromol L(-1)). RESULTS: The current study demonstrates that AngII exerts a positive inotropic effect in normoxic hearts with an increase of left ventricular developed pressure (LVDP) by 11% (P<0.05 vs. prior to AngII infusion). In post-ischaemic hearts subjected to 15-min ischaemia no effect of AngII was observed. In hearts subjected to 25 min of ischaemia, however, AngII evoked a negative inotropic response with a decrease of LVDP by 18% (P<0.05 vs. prior to AngII infusion). The negative inotropic effect of AngII was inhibited by the PKC inhibitor chelerythrine chloride. CONCLUSIONS: AngII exerts negative inotropic effect in severely injured post-ischaemic heart, possibly through the PKC pathway.

Alkaloids↗
PubMed2003

Chelerythrin activates caspase-8, downregulates FLIP long and short, and overcomes resistance to tumour necrosis factor-related apoptosis-inducing ligand in KG1a cells.

KG1a cells (CD34+/38-) express FAS and TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand) receptors but are resistant to FAS-ligand and TRAIL/APO2-L (apoptosis antigen-2 ligand)-induced apoptosis. KG1a cells are sensitized to FAS-induced apoptosis by chelerythrin, an inhibitor of protein kinase C (PKC). As cytoplasmatic adaptor molecules of FAS, e.g. FLIP [Fas-associated death domain protein (FADD)-like interleukin 1 beta-converting enzyme [FLICE (caspase-8)-inhibitory protein]], also modulate TRAIL signals, we determined whether chelerythrin affected TRAIL-mediated apoptosis. Chelerythrin by itself induced apoptosis in KG1a cells, and apoptosis was associated with activation of caspase-8. While TRAIL alone failed to activate caspase-8 or induce apoptosis, the addition of TRAIL to chelerythrin-treated cells significantly enhanced cleavage of caspase-8 and apoptosis. Chelerythrin-pretreated KG1a cells showed decreased phosphorylation of protein kinase C (PKC)-zeta and downregulation of both FLIP long and FLIP short proteins. Downregulation of FLIP and induction of apoptosis were partially abrogated by pretreatment with the specific caspase-8 inhibitor, Z-IETD-FMK. The decrease in FLIP protein expression induced by chelerythrin was accompanied by a progressive increase in mRNA levels of both FLIP long and FLIP short. CD34+ precursors from normal human marrow were also sensitive to chelerythrin but, in contrast to KG1a cells, were not sensitized to TRAIL-mediated apoptosis. Thus, resistance to TRAIL-induced apoptosis in leukaemic KG1a cells but not in normal CD34+ precursors was overcome in the presence of chelerythrin. The mechanism appeared to involve inhibition of PKC. Central targets were FLIP long and FLIP short, and their interactions with caspase-8. Whether such a pathway can be exploited to selectively target leukaemic progenitor cells remains to be determined.

Alkaloids↗
PubMed1998

Varying very low-density lipoprotein secretion of rat hepatocytes by altering cellular levels of calcium and the activity of protein kinase C.

BACKGROUND: Calcium antagonists lower plasma levels of lipoproteins and suppress hepatic very low-density lipoprotein (VLDL) secretion. Similar effects have been observed with the calcium ionophore A23187. We studied further the effect of calcium on VLDL metabolism. METHODS: Hepatocytes from male Wistar rats were isolated and cultured in the presence or absence of calcium-mobilizing hormones, or compounds that either stimulate or inhibit the activity of protein kinase C. Secreted VLDL (d < 1.006 g mL-1) was isolated by centrifugation (145,000 x g), and lipids and apolipoprotein B were analysed. RESULTS: VLDL secretion reached maximum in hepatocytes cultured in medium containing calcium 0.8-2.4 mmolL-1. Depleting the cells of calcium by incubating in calcium-free medium or by treating the cells with the Ca(2+)-ATPase inhibitor thapsigargin (5 x 10-7 molL-1) suppressed lipid secretion to less than 15% of control, and this was accompanied by an increase in cellular levels of triacylglycerol. Calcium loading (medium calcium > 2.4 mmolL-1) suppressed both lipoprotein secretion and cellular levels of lipids, suggesting a reduced overall rate of lipid synthesis. At an extracellular calcium concentration of 0.8 mmolL-1, angiotensin II, vasopressin, endothelin-1 (10(-7) molL-1) or phenylephrine (10(-4) molL-1) suppressed VLDL secretion (maximum to 37% of control), and elevated medium calcium attenuated this effect. The protein kinase C inhibitor chelerythrine (5 x 10(-5) molL-1) and the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) (10(-6) molL-1), suppressed VLDL secretion to 18% and 60% of control, respectively, whereas the protein kinase C-inactive 4 alpha-PMA was without an effect. No effect on ketogenesis was observed by these compounds, indicating that suppressed lipid secretion was not due to an enhanced oxidation of lipids. CONCLUSIONS: Hepatic VLDL secretion can be related to changes in hepatocyte levels of calcium and the activity of protein kinase C.

Alkaloids↗
PubMed1998

Lysis-sensitive targets stimulate an elevation of cAMP in human natural killer cells.

Natural killer (NK) cells are lymphocytes that are capable of destroying tumour cells and virally infected cells (cytolysis) without prior sensitization. When cAMP is artificially elevated in NK cells, it is a potent inhibitor of their cytolytic function. We investigated whether NK-cell cAMP levels are modulated in response to tumour target cells to determine the potential of cAMP as a physiological regulator of NK cytotoxic function. When NK cells are exposed to a range of lysis-sensitive (LS) tumour-target cells there is an increase in intracellular cAMP levels in the NK cells over a 60-min period. The peak increase in cAMP (200-400% above control) occurs at 30 min for all LS targets tested. There is no increase in NK-cell cAMP in response to lysis-resistant (LR) tumour-target cells. The cAMP elevation may be dependent on both LS-target-stimulated adenylyl cyclase (AC) activation and LS-target-stimulated phosphodiesterase (PDE) inhibition. When the NK cells are pretreated with the protein tyrosine kinase (PTK) inhibitor, genistein (30 micrograms/ml), the AC-activation component of the cAMP elevation is abolished. Thus, the AC-activation component appears to require PTK activation. When NK cells are pretreated with the protein kinase C (PKC) inhibitor, chelerythrine chloride (10 microM) the cAMP elevation in response to LS targets was not diminished. This indicates that neither the AC-activation component nor any PDE-inhibition component require PKC activation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2000

Diverse intracellular signalling systems used by growth hormone-releasing hormone in regulating voltage-gated Ca2+ or K channels in pituitary somatotropes.

Influx of Ca2+ via Ca2+ channels is the major step triggering exocytosis of pituitary somatotropes to release growth hormone (GH). Voltage-gated Ca2+ and K+ channels, the primary determinants of the influx of Ca2+, are regulated by GH-releasing hormone (GHRH) through G-protein-coupled intracellular signalling systems. Using whole-cell patch-clamp techniques, the changes of the Ca2+ and K+ currents in primary cultured ovine and human somatotropes were recorded. Growth hormone-releasing hormone (10 nmol/L) increased both L- and T-type voltage-gated Ca2+ currents. Inhibition of the cAMP/protein kinase A (PKA) pathway by either Rp-cAMP or H89 blocked this increase in both L- and T-type Ca2+ currents. Growth hormone-releasing hormone also decreased voltage-gated transient (IA) and delayed rectified (IK) K+ currents. Protein kinase C (PKC) inhibitors, such as calphostin C, chelerythrine or downregulation of PKC, blocked the effect of GHRH on K+ currents, whereas an acute activation of PKC by phorbol 12, 13-dibutyrate (1 micromol/L) mimicked the effect of GHRH. Intracellular dialysis of a specific PKC inhibitor (PKC19-36) also prevented the reduction in K+ currents by GHRH. It is therefore concluded that GHRH increases voltage-gated Ca2+ currents via cAMP/PKA, but decreases voltage-gated K+ currents via the PKC signalling system. The GHRH-induced alteration of Ca2+ and K+ currents augments the influx of Ca2+, leading to an increase in [Ca2+]i and the GH secretion.

Alkaloids↗
PubMed1999

A pre- and postsynaptic modulatory action of 5-HT and the 5-HT2A, 2C receptor agonist DOB on NMDA-evoked responses in the rat medial prefrontal cortex.

Intracellular recordings were made from pyramidal neurons in layers V and VI of the rat medial prefrontal cortex in slice preparations to investigate the effect of the serotonin 5-HT2A,2C receptor agonist (-)-1-2,5-dimethoxy-4-bromophenol-2-aminopropane (DOB) and 5-hydroxytryptamine (5-HT) on N-methyl-D-aspartate (NMDA)-induced responses. Bath application of either DOB or 5-HT [in the presence of antagonists to 5-HT1A, 5-HT3 and gamma-aminobutytric acid (GABA) receptors] produced a concentration-dependent biphasic modulation of the NMDA responses. They facilitated and inhibited NMDA responses at low (</= 1 microM DOB and </= 50 microM 5-HT) and higher concentrations, respectively. Both the facilitating and inhibitory action were blocked by the highly selective 5-HT2A receptor antagonist R-(+)-alpha-(2, 3-dimethoxyphenil)-1-[4-fluorophenylethyl]-4-piperidineme thanol (M100907) and the 5-HT2 receptor antagonist ketanserin, thus indicating that both facilitation and inhibition were mediated by the activation of the 5-HT2A receptor subtype. However, the facilitating, but not inhibitory, action of DOB showed a marked desensitization, suggesting that the facilitation and inhibition of NMDA responses resulted from activation of different 5-HT2A receptor subtypes and/or signal-transduction pathways. Indeed, the selective PKC inhibitor chelerythrine and the Ca2+/CaM-KII inhibitor KN-93 prevented the facilitating and inhibitory action of DOB, respectively. We have generated several lines of evidence to indicate the following scenario. Low concentrations of DOB, at presynaptic nerve terminals, markedly enhance NMDA-induced release of excitatory amino acids (EAAs), which then act upon both NMDA and non-NMDA receptors to elicit inward current. The massive inward current masks the postsynaptic inhibitory action of DOB. At higher concentrations, DOB inhibits the release of EAAs and discloses the postsynaptic inhibitory action.

Alkaloids↗
PubMed2000

A role for protein kinase C in a form of metaplasticity that regulates the induction of long-term potentiation at CA1 synapses of the adult rat hippocampus.

The possibility that protein kinase C (PKC) is involved in the induction of N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) at CA1 synapses in the hippocampus has been the subject of considerable investigation. However, many of the conclusions have been drawn from the use of relatively nonspecific PKC inhibitors. In the present study we have examined the role of PKC in tetanus-induced LTP of AMPA receptor-mediated synaptic transmission in hippocampal slices obtained from adult rats. In particular, we have investigated the possible role of PKC in a molecular switch process that is triggered by the synaptic activation of metabotropic glutamate receptors and regulates the induction of LTP. We find that the three PKC inhibitors examined, chelerythrine, Ro-31-8220 and Gö 6983, all block the setting of the molecular switch at concentrations consistent with inhibition of PKC. In contrast, these inhibitors are without affect on the induction of LTP, even when applied in very much higher concentrations. A PKA inhibitor, Rp-cAMPS, had no effect on either process. We suggest that neither PKC nor PKA is required to induce LTP at this synapse. However, PKC is involved in the regulation of LTP induction, via the molecular switch process.

Alkaloids↗
PubMed2002

Neurotensin modulates the amplitude and frequency of voltage-activated Ca2+ currents in frog pituitary melanotrophs: implication of the inositol triphosphate/protein kinase C pathway.

Many excitatory neurotransmitters and neuropeptides regulate the activity of neuronal and endocrine cells by modulating voltage-operated Ca2+ channels. Paradoxically, however, excitatory neuromediators that provoke mobilization of intracellular calcium from inositol trisphosphate (IP3)-sensitive stores usually inhibit voltage-gated Ca2+ currents. We have recently demonstrated that neurotensin (NT) stimulates the electrical and secretory activities of frog pituitary melanotrophs, and increases intracellular calcium concentration in these cells. In the present study, we have investigated the effects of NT on Ca2+ currents in cultured frog melanotrophs by using the perforated patch-clamp technique. Frog neurotensin (f NT) reduced the amplitude and facilitated the inactivation of both L- and N-type Ca2+ currents. Application of the membrane-permeant Ca2+ chelator BAPTA-AM, the sarcoendoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin, or the IP3 receptor antagonist 2-APB suppressed the reduction of Ca2+ currents induced by f NT. Incubation of melanotrophs with the diacylglycerol analogue PMA, which causes desensitization of protein kinase C (PKC), or with the PKC inhibitors chelerythrine and calphostin C, reduced the inhibitory effect of f NT. The NT-induced action potential waveforms, applied as voltage-clamp commands, decreased the amplitude of Ca2+ currents, and enhanced Ca2+ influx by increasing the Ca2+ spike frequency. Altogether, these data indicate that the inhibitory effect of f NT on Ca2+ currents results from activation of the IP3/PKC pathway. The observation that NT controls Ca2+ signalling through both amplitude and frequency modulations of Ca2+ currents suggests that NT might induce spacial and temporal changes of intracellular Ca2+ concentration leading to stimulation of exocytosis.

Alkaloids↗
PubMed2003

Potentiation of glycine responses by dideoxyforskolin and tamoxifen in rat spinal neurons.

Dideoxyforskolin, a forskolin analogue unable to stimulate adenylate cyclase, and tamoxifen, an antioestrogen widely used against breast cancer, are both known to block some Cl- channels. Their effects on Cl- responses to glycine or GABA have been tested here by using whole-cell recording from cultured spinal neurons. Dideoxyforskolin (4 or 16 microm) and tamoxifen (0.2-5 microm) both potentiate responses to low glycine concentrations. They also induce blocking effects, predominant at high glycine concentrations. At 5 microm, tamoxifen increased responses to 15 microm glycine by a factor >4.5, reaching 20 in some neurons. Potentiation by extracellular dideoxyforskolin or tamoxifen persisted after intracellular application of the modulator and was not due to Zn2+ contamination. Potentiation by tamoxifen also persisted in a Ca2+-free extracellular solution, after intracellular Ca2+ buffering and protein kinase C blockade. Thus, the critical sites of action are not intracellular. The EC50 for glycine was lowered 6.6-fold by 5 microm tamoxifen. The kinetics and voltage-dependence of the effects of tamoxifen on glycine responses support the idea that this hydrophobic drug may act from a site located within the membrane. Tamoxifen (5 micro m) also increased responses to 2 micro m GABA by a factor of 3.5, but barely affected peak responses to 20 microm GABA. The demonstration that tamoxifen affects some of the main inhibitory receptors should be useful for better evaluating its neurological effects. Furthermore, the results identify a new class of molecules that potentiate glycine receptor function.

Alkaloids↗
PubMed2003

Kinase-dependent loss of Na+ channel slow-inactivation in rat CA1 hippocampal pyramidal cell dendrites after brief exposure to convulsants.

Na+ channels in the dendrites of rat CA1 pyramidal neurons display a profound activity-dependent inactivation, termed slow inactivation, that limits excitability in the dendrites even at low physiological rates of firing. The magnitude of this slow inactivation is powerfully modulated by a protein kinase C-dependent process. Because activation of kinases is a rapid and common feature of a number of seizure models, we hypothesized that a loss of slow inactivation of Na+ channels might exacerbate other changes in excitability. Thus, we observed the effects of a brief (5 min) chemical convulsant treatment on Na+ currents and action potentials in hippocampal slices. We found that slow inactivation decreased significantly and remained decreased for at least 30 min after return to control conditions. Pretreatment with either chelerythrine, a protein kinase C inhibitor, or U0126, a mitogen-activated protein kinase/extracellular signal regulated kinase kinase (MEK) inhibitor, blocked this reduction of slow inactivation. These results demonstrate that a brief period of hyperexcitability leads to a rapid, protein kinase-dependent loss of slow inactivation of Na+ channels that would contribute to and perhaps prolong the hyperexcitable state.

Action Potentials↗
PubMed1997

Double-blind randomized placebo-controlled study of homoeopathic prophylaxis of migraine.

Homoeopathic remedies for migraine are widely available over the counter, statutorily offered by the national health service in the UK, and apparently popular with patients. Do they work? Sixty-three outpatients with migraine with or without aura by IHS criteria entered a 4-month randomized placebo-controlled, double-blind parallel-groups trial of individualized homoeopathic prophylaxis, the first month being baseline with all patients on placebo. Three patients (4.8%) dropped out, leaving 30 in each treatment group. There were chance differences in attack frequency and severity between the groups at baseline (attacks were more frequent but less severe in the placebo group). Both groups improved on therapy, but neither to a great extent on the primary outcome measure of attack frequency (verum: -19%; placebo: -16%). Reduction was mostly in mild attacks on placebo, more in moderate and severe attacks on homoeopathy. Few adverse events were reported. Overall, there was no significant benefit over placebo of homoeopathic treatment. The course of change differed between groups, and suggested that improvement reversed in the last month of treatment on placebo. On this evidence we cannot recommend homoeopathy for migraine prophylaxis, but cannot conclude that it is without effect.

Adult↗
PubMed1994

Phorbol ester administration transiently increases aromatic L-amino acid decarboxylase activity of the mouse striatum and midbrain.

Aromatic L-amino acid decarboxylase (AAAD) is required for the synthesis of catecholamines, serotonin, and the trace amines. We found that the protein kinase C activator phorbol 12-myristate 13-acetate administered intracerebroventricularly transiently increased AAAD activity by 30-50% over control values within approximately 30 min in the striatum and midbrain of the mouse. The enzyme increase was manifested as an apparent increase of Vmax with little change of Km for either L-3,4-dihydroxyphenylalanine or pyridoxal phosphate. Chelerythrine, a protein kinase C inhibitor, prevented the phorbol ester-induced increase of AAAD. Moreover, okadaic acid, a serine/threonine-selective protein phosphatase 1 and 2A inhibitor, also increased AAAD activity in the mouse striatum and midbrain. Taken together, these observations suggest that protein kinase C-mediated pathways modulate AAAD activity in vivo.

Alkaloids↗
PubMed1994

Evidence that protein kinase C activities involved in regulating neurite growth are localized to distal neurites.

Previously, we observed that long-term treatment of distal nerve fibers of rat sympathetic neurons in compartmented cultures with phorbol 12-myristate 13-acetate (PMA) caused a reduction in the rate of neurite elongation by > 50%. In the present report we show that protein kinase C (PKC) activity could be measured in extracts of distal neurites by an assay of the Ca(2+)-dependent phosphorylation of a PKC-specific octapeptide substrate. We found that local application of 1 microM PMA for 24 h to distal neurites caused nearly complete down-regulation of Ca(2+)-dependent PKC activity measured in this manner. We determined that the inhibition of neurite elongation by PMA was mediated by local mechanisms in the neurites because local application of PMA to center compartments containing cell bodies and proximal neurites did not inhibit the rate of elongation of distal neurites. We then investigated the effects of the recently available PKC inhibitors, calphostin C and chelerythrine, finding that, like PMA, these inhibited the growth of distal neurites when applied locally to them, and had no effect when applied to cell bodies and proximal neurites. However, the inhibition of neurite growth by calphostin C occurred at a concentration far below its IC50 value for protein kinase inhibition, and both calphostin C and chelerythrine inhibited distal neurite growth even in neurons pretreated with PMA. Thus, it appears that these agents do not all inhibit neurite growth through the same mechanisms. Although the PKC activities involved in neurite elongation in sympathetic neurons have not been precisely defined, these data presented in this study indicate that protein kinases localized to growth cones play a complex and important role in regulating axonal growth.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Activation of protein kinase C by trimethyltin: relevance to neurotoxicity.

The differentiated PC12 cell neuronal model was used to determine the effect of trimethyltin (TMT) on protein kinase C (PKC). Cells treated with 5-20 microM TMT showed a partial and sustained PKC translocation within 30 min and persisted over a 24-h period. TMT treatment was accompanied by a low level of PKC down-regulation over 24 h, which was small compared with that produced by phorbol esters. Confocal imaging of differentiated PC12 cells showed that PKC translocates to the plasma membrane and the translocation is blocked by the PKC inhibitor chelerythrine (1 microM). Phorbol myristate-induced PKC down-regulation or inhibition with chelerythrine provided protection against TMT-induced cytotoxicity. It was concluded that TMT-induced PKC translocation and activation contribute to the cytotoxicity of TMT in differentiated PC12 cells.

Alkaloids↗
PubMed1996

Effects of protein kinase inhibitors on morphology and function of cultured bovine adrenal chromaffin cells: KN-62 inhibits secretory function by blocking stimulated Ca2+ entry.

In cultured bovine adrenal chromaffin cells, a nonselective protein kinase inhibitor, staurosporine, inhibits secretory function and induces neurite outgrowth. In the present study, effects of other nonselective protein kinase inhibitors (K-252a, H-7, and H-8) and reportedly selective protein kinase inhibitors (KN-62 and chelerythrine chloride) were examined on bovine adrenal chromaffin cell morphology, secretory function, and 45Ca2+ uptake. Treatment of chromaffin cells with 10 microM K-252a, 50 microM H-7, or 50 microM H-8 induced changes in cell morphology within 3 h; these compounds also induced a time-dependent inhibition of stimulated catecholamine release. Chelerythrine chloride, a selective inhibitor of Ca2+/phospholipid-dependent protein kinase, did not induce outgrowth or inhibit secretory function under our treatment conditions. KN-62, a selective inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMK II), significantly inhibited stimulated catecholamine release (IC50 value of 0.32 microM), but had no effect on cell morphology. The reduction of secretory function induced by 1 microM KN-62 was significant within 5 min and rapidly reversible. Unlike H-7, H-8, and staurosporine, KN-62 significantly inhibited stimulated 45Ca2+ uptake. KN-04, a structural analogue of KN-62 that does not inhibit CaMK II, inhibited stimulated 45Ca2+ uptake and catecholamine release like KN-62. These studies indicate that KN-62 inhibits secretory function via the direct blockade of activated Ca2+ influx. The nonselective inhibitors, K-252a, H-7, H-8, and staurosporine, inhibit secretory function by another mechanism, perhaps one involving alterations in the cytoskeleton.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1996

Dual signalling by the adenosine A2a receptor involves activation of both N- and P-type calcium channels by different G proteins and protein kinases in the same striatal nerve terminals.

Many Gs-linked receptors have been reported to use multiple signalling pathways in transfected cels but few in their normal cell environment. We show that the adenosine A2a receptor uses two signalling pathways to increase the release of acetylcholine from striatal nerve terminals. One pathway involves activation of Gs, adenylyl acylase, protein kinase A, and P-type calcium channels; the other is mediated by a cholera toxin-insensitive G protein, protein kinase C, and N-type calcium channels. The effects of these two pathways are not additive, the second pathway being inhibited by the first; but they are equally sensitive to the A2a receptor antagonist KF17837. This demonstrates that the A2a receptor activates two signalling systems in striatal cholinergic neurons.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

Evidence that the early loss of membrane protein kinase C is a necessary step in the excitatory amino acid-induced death of primary cortical neurons.

A rapid loss of protein kinase C (PKC) activity is a prognostic feature of the lethal damage inflicted on neurons by cerebral ischemia in vivo and by hypoxic and excitotoxic insults in vitro. However, it is not known if this inactivation of PKC is incidental or is an essential part of the neurodegenerative process driven by such insults. To address this issue, the effects of glutamate on PKC activity and neurotoxicity were studied in immature [8 days in vitro (DIV)] and mature (15-20 DIV) embryonic day 18 rat cortical neuronal cultures. Exposing 16 DIV neurons to as little as 20-50 microM glutamate for 15 min was neurotoxic and induced a rapid (approximately 1-2 h) Ca(2+)-dependent inactivation of membrane PKC. By contrast, neurons 8 DIV were resistant to > 800 microM glutamate, and no evidence of PKC inactivation was observed. Reverse transcription-polymerase chain reaction analysis of NMDA and AMPA receptor subtypes and fluorometric intracellular Ca2- concentration measurements of the effects of NMDA, AMPA, kainate, and metabotropic glutamate receptor activation demonstrated that this striking difference in vulnerability was not due to an absence of functional glutamate receptors on neurons 8 DIV. However, 8 DIV neurons became highly vulnerable to low (< 20 microM) concentrations of glutamate when PKC activity was inhibited by 50 nM staurosporine, 1 microM calphostin C, 5 microM chelerythrine, or chronic exposure to 100 nM PMA. A 15-min coapplication of 50 nM staurosporine with glutamate, NMDA, AMPA, or kainate killed between 50 and 80% of 8 DIV cells within the ensuing 24 h. Moreover, cell death was observed in these cells even when PKC inactivation was delayed up to 4 h after glutamate removal. The evidence indicates that a loss of PKC activity is an essential element of the excitotoxic death of neurons 8 DIV and that cellular event(s) responsible for linking glutamate-mediated Ca2+ influx to PKC inactivation in vulnerable neurons 16 DIV are undeveloped in resistant cells 8 DIV. These results also suggest that the loss of neuronal PKC activity observed in cerebral ischemia may indeed be an important part of the neurodegenerative process. The 8 DIV/16 DIV cortical cell model may prove to be valuable in discerning those intracellular signaling events critical to glutamate-mediated neuronal death.

Alkaloids↗
PubMed1997

Phorbol ester and calcium regulation of corticotrophin-releasing factor receptor 1 expression in a neuronal cell line.

We have previously demonstrated that corticotrophin-releasing factor receptor 1 (CRF-R1) mRNA levels can be down-regulated via activation of the cyclic AMP pathway in CATH.a cells, a neuronal cell line. In this study, we show evidence for down-regulation of CRF-R1 mRNA levels via activation of the protein kinase C (PKC) and calcium second messenger pathways. Incubation of CATH.a cells with phorbol 12-myristate 13-acetate (PMA), an activator of PKC, resulted in a time- and concentration-dependent down-regulation of CRF-R1 mRNA levels. Pretreatment with the inactive phorbol ester 4alpha-phorbol failed to influence significantly CRF-R1 mRNA levels. Incubation with carbachol, a cholinergic agonist known to activate PKC and increase intracellular calcium levels via phosphatidylinositol breakdown, also down-regulated CRF-R1 mRNA levels. Intracellular calcium levels were directly increased using A23187, a calcium ionophore, and thapsigargin, a calcium-ATPase inhibitor. Elevation of intracellular calcium content using either A23187 or thapsigargin significantly down-regulated levels of CRF-R1 mRNA. Furthermore, chelation of calcium with EGTA or blockade of voltage-dependent calcium channels with nifedipine inhibited agonist-mediated down-regulation of CRF-R1 mRNA levels. These results indicate that activation of PKC or calcium signal transduction pathways is sufficient to cause down-regulation of CRF-R1 mRNA levels and that calcium is required for agonist-mediated down-regulation of this receptor.

Alkaloids↗