Tyrosine Kinases Are Required for Catecholamine Secretion and Mitogen-Activated Protein Kinase Activation in Bovine Adrenal Chromaffin Cells

Abstract: Nicotine-induced catecholamine secretion in bovine adrenomedullary chromaffin cells is accompanied by rapid tyrosine phosphorylation of multiple cellular proteins, most notably the mitogen-activated protein kinases (MAPKs). The requirement for activation of tyrosine kinases and MAPKs in chromaffin cell exocytosis was investigated using a panel of tyrosine kinase inhibitors. Genistein and tyrphostin 23, two compounds that inhibit tyrosine kinases by distinct mechanisms, were found to inhibit secretion by >90% in cells stimulated by nicotine, 55 m M KCI, or the Ca²⁺ ionophore A23187. Inhibition of secretion induced by all three secretagogues correlated with a block in both protein tyrosine phosphorylation and activation of the MAPKs and their activators (MEKs) in situ. However, neither genistein nor tyrphostin 23 inhibited the activities of the MAPKs or MEKs in vitro. These results indicate that the target(s) of inhibition lie down-stream of Ca²⁺ influx and upstream of MEK activation. This Ca²⁺-activated tyrosine kinase activity could not be accounted for entirely by c-Src or Fyn (two nonreceptor tyrosine kinases that are expressed abundantly in chromaffin cells), because their in vitro kinase activities were not inhibited by tyrphostin 23 and only partially inhibited by genistein. These results demonstrate that an unidentified Ca²⁺-activated tyrosine kinase(s) is required for MAPK activation and exocytosis in chromaffin cells and suggest that MAPK participates in the regulation of secretion.     

Fibroblast Growth Factors Stimulate Protein Tyrosine Phosphorylation and Mitogen-Activated Protein Kinase Activity in Primary Cultures of Hippocampal Neurons

Abstract: Fibroblast growth factors (FGFs) are not only mitogens, but they also promote the differentiation of various cell types. For instance, basic FGF (bFGF) provides a critical trophic support for hippocampal neurons in culture. To elicit their biological effects, FGFs interact with high-affinity receptors that are transmembrane proteins with a cytoplasmic portion containing a tyrosine kinase activity. The tyrosine phosphorylation pattern was examined in primary cultures of hippocampal neurons derived from rat embryos. In these cultures grown for 3 days in the absence of serum, the addition of bFGF causes a rapid increase of tyrosine phosphorylation for various proteins with an optimal level after 5 min of bFGF exposure. Concomitantly, bFGF activates mitogen-activated protein kinase (MAP kinase) activity measured with a selective MAP kinase peptide. The activity increased rapidly after the addition of bFGF and remained elevated even when cultures were treated for 1 h with bFGF. Both acidic and basic FGF were able to enhance protein tyrosine phosphorylation and MAP kinase activity, whereas nerve growth factor and epidermal growth factor did not elicit any of these responses. These data indicate that some of the transduction signals (i.e., tyrosine phosphorylation and activation of MAP kinase) that have been described for the proliferative effect of FGFs are also involved when FGFs act as trophic factors for postmitotic neurons in culture.     

Modulation of Dopamine Transporter Activity by Nicotinic Acetylcholine Receptors and Membrane Depolarization in Rat Pheochromocytoma PC12 Cells

To elucidate the regulation of the rat dopamine transporter (rDAT), we established several PC12 variants overexpressing the rDAT. Treating these cells with a nicotinic agonist (1,1-dimethyl-4-phenylpiperazinium iodide, 30 microM) depolarized the plasma membrane potential from -31 +/- 2 to 43 +/- 5 mV and inhibited rDAT activity significantly in a calcium- and protein kinase C-independent manner. Membrane depolarization by a high external K+ concentration or two K+ channel blockers (tetraethylammonium hydroxide and BaCl2) also resulted in a marked inhibition of rDAT activity. Such inhibition of dopamine uptake is due to a reduction in Vmax, with no marked effect on the Km for dopamine. The potency of cocaine in inhibiting dopamine uptake was not significantly altered, whereas that of amphetamine was slightly enhanced by membrane depolarization. Removing extracellular Ca2+ or blocking the voltage-sensitive L-type calcium channels using nifedipine did not exert any significant effect on the inhibition of rDAT activity by depolarization. These data confirm that calcium influx on depolarization is not required for inhibition of the rDAT. Collectively, our data suggest that rDAT activity can be altered by a neurotransmitter that modulates the membrane potential, thus suggesting an exquisite mechanism for the fine-tuning of dopamine levels in the synapse.     

Hydrogen Peroxide-Induced Phospholipase D Activation in Rat Pheochromocytoma PC12 Cells: Possible Involvement of Ca2+-Dependent Protein Tyrosine Kinase

Abstract: The mechanism for hydrogen peroxide (H₂O₂)-induced phospholipase D (PLD) activation was investigated in [³H]palmitic acid-labeled PC12 cells. In the presence of butanol, H₂O₂ caused a great accumulation of [³H]phosphatidylbutanol in a concentration- or time-dependent manner. However, treatment with H₂O₂ of cell lysates exerted no effect on PLD activity. Treatment with H₂O₂ had only a marginal effect on phospholipase C (PLC) activation. A protein kinase C (PKC) inhibitor, Ro 31-8220, did not inhibit but rather slightly enhanced H₂O₂-induced PLD activity. Thus, H₂O₂-induced PLD activation is considered to be independent of the PLC-PKC pathway in PC12 cells. In contrast, pretreatment with tyrosine kinase inhibitor herbimycin A, genistein, or ST638 resulted in a concentration-dependent inhibition of H₂O₂-induced PLD activation. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands after the H₂O₂ treatment and tyrosine phosphorylation of these proteins was inhibited by these tyrosine kinase inhibitors. Moreover, depletion of extracellular Ca²⁺ abolished H₂O₂-induced PLD activation and protein tyrosine phosphorylation. Extracellular Ca²⁺ potentiated H₂O₂-induced PLD activation in a concentration-dependent manner. Taken together, these results suggest that a certain Ca²⁺-dependent protein tyrosine kinase(s) somehow participates in H₂O₂-induced PLD activation in PC12 cells.     

Calmodulin Inhibitor-induced Apoptosis was Prevented by Glycogen Synthase Kinase-3 Inhibitors in PC12 Cells

Calmodulin is known to transduce Ca²⁺ signals by interacting with specific target proteins. In order to determine the role of calmodulin in regulating neuronal survival and death, we examined, whether calmodulin inhibitors induce caspase-dependent apoptotic cell death, and whether glycogen synthase kinase-3 is involved in calmodulin inhibitor-induced cell death in PC12 cells. W13, a calmodulin specific inhibitor increased apoptotic cell death with morphological changes characterized by cell shrinkage and nuclear condensation of fragmentation. Glycogen synthase kinase-3 inhibitors prevented calmodulin inhibitor-induced apoptosis. In addition, nerve growth factor and cycloheximide, a protein synthesis inhibitor, completely blocked cell death. Moreover, caspase-3 activation was accompanied by calmodulin inhibitor-induced cell death and inhibited by nerve growth factor. These results suggest that calmodulin inhibitors induce caspase-dependent apoptosis, and the activation of glycogen synthase kinase-3 is involved in the death of PC12 cells.     

Differential Activation of Mitogen-Activated Protein Kinase Pathways in PC12 Cells by Closely Related α1-Adrenergic Receptor Subtypes

Coupling of the three known alpha1-adrenergic receptor (alpha1-AR) subtypes to mitogen-activated protein kinase (MAPK) pathways were studied in stably transfected PC12 cells. Subclones stably expressing alpha1A-, alpha1B-, and alpha1D-ARs under control of an inducible promoter, or at high and low receptor density, were isolated and characterized. Radioligand binding showed similar ranges of expression of each subtype. Norepinephrine (NE) increased inositol phosphate formation and intracellular Ca2+ level in these cells in a manner dependent on receptor density. However, alpha1A-ARs activated these second messenger responses more effectively than alpha1B-ARs, whereas alpha1D-ARs were least effective. NE stimulated activation of extracellular signal-regulated kinases (ERKs) in cells expressing all three alpha1-AR subtypes, although alpha1A- and alpha1B-ARs caused larger ERK activation than did alpha1D-ARs. Nerve growth factor (NGF) caused similar levels of ERK activation in all subclones. NE also activated p38 MAPK in alpha1A- and alpha1B- but not alpha1D-transfected cells and activated c-Jun NH2-terminal kinase (JNK) only in alpha1A-transfected cells. NE, but not NGF, strongly stimulated tyrosine phosphorylation of a 70-kDa protein only in alpha1A-transfected PC12 cells. NE caused neurite outgrowth only in alpha1A-expressing PC12 cells, but not in alpha1B- or alpha1D-transfected cells, whereas NGF caused neurite outgrowth in all cells. These studies show that alpha1A-ARs activate all three MAPK pathways, alpha1B-ARs activate ERKs and p38 but not JNKs, and alpha1D-ARs only activate ERKs. Only the alpha1A-AR-expressing cells differentiated in response to NE. The relationship of these responses to second messenger pathways activated by these subtypes is discussed.     

Mitogen-Activated Protein Kinase-Dependent and Protein Kinase C-Dependent Pathways Link the m1 Muscarinic Receptor to β-Amyloid Precursor Protein Secretion

Abstract: Full and functionally selective M1 muscarinic agonists (carbachol and AF102B, respectively) activate secretion of the soluble form of amyloid precursor protein (APPs) in PC12 cells expressing the m1 muscarinic receptor (PC12M1 cells). This activation is further augmented by neurotrophins such as nerve growth factor and basic fibroblast growth factor. Muscarinic stimulation activates two transduction pathways that lead to APPs secretion: protein kinase C (PKC)-dependent and mitogen-activated protein kinase (MAPK)-dependent pathways. These pathways operate in parallel and converge with transduction pathways of neurotrophins, resulting in enhancement of APPs secretion when both muscarinic agonist and neurotrophins stimulate PC12M1 cells. These conclusions are supported by the following findings: (a) Only partial blockade of APPs secretion is observed when PKC, p21^ras, or MAPK is fully inhibited by their respective specific inhibitors, GF109203X, S-trans,trans -farnesylthiosalicylic acid, and PD98059. (b) K252a, which blocks PKC and phorbol 12-myristate 13-acetate-induced APPs secretion, enhances both muscarinic-stimulated MAPK activation and APPs secretion. (c) Activation of MAPK in PC12M1 cells by muscarinic agonists is Ras-dependent but PKC-independent and is enhanced synergistically by neurotrophins. These results suggest that muscarinic stimulation of APPs secretion is mediated by at least two independent pathways that converge and enhance the signal for APPs secretion at the convergence point.     

Neurotrophic Factors Prevent Ceramide-Induced Apoptosis Downstream of c-Jun N-Terminal Kinase Activation in PC12 Cells

Abstract: Neurotrophic factors prevent apoptosis of PC12 cells in serum-free medium. The present study determines whether neurotrophic factors can prevent ceramide-induced apoptosis in PC12 cells and investigates the role that c-Jun N-terminal kinase (JNK) activation may play in this system. Ceramide-induced apoptosis was inhibited by nerve growth factor, basic fibroblast growth factor, pituitary adenylyl cyclase-activating peptide, 4-(8-chlorophenylthio)cyclic AMP, and the caspase inhibitor benzyloxycarbonyl-Val-Ala- dl -Asp fluoromethyl ketone (zVAD-FMK). It was surprising that inhibition of extracellular signal-regulated kinase and/or phosphatidylinositol 3-kinase did not markedly block the protective effects exerted by neurotrophic factors against ceramide-induced apoptosis, suggesting that neurotrophic factors can promote survival independently of these signaling pathways. Treatment of PC12 cells with ceramide resulted in a time-dependent increase in JNK activity. However, neither neurotrophic factors nor zVAD-FMK attenuated ceramide-stimulated JNK activation. Further experiments indicated that ceramide-induced apoptosis in PC12 cells requires new protein synthesis, and that nerve growth factor and zVAD-FMK can prevent apoptosis after JNK activity has been detected. These results indicate that ceramide-induced JNK activation is an early event and may be required for the expression of essential components of the apoptotic machinery. It is anticipated that neurotrophic factors inhibit ceramide-induced apoptosis by affecting signaling events downstream of JNK activation.     

Activation and Tyrosine Phosphorylation of 44-kDa Mitogen-Activated Protein Kinase (MAPK) Induced by Electroconvulsive Shock in Rat Hippocampus

Abstract: Electroconvulsive shock (ECS) has been reported to induce the phosphorylation and activation of 42-kDa, but not 44-kDa, mitogen-activated protein kinase (MAPK) in rat hippocampus. We studied the activation and tyrosine phosphorylation of MAPKs in rat brain after ECS. We observed the increase of the activities of both 42- and 44-kDa MAPKs in rat hippocampus after ECS. The activities reached peak at 2 min and returned to basal levels by 15 min after ECS. We also observed the increased phsophorylation on the tyrosine residue of 42-kDa MAPK in rat hippocampus after ECS, but not on that of 44-kDa MAPK. However, when we examined the immunoprecipitated 44-kDa MAPK, we could demonstrate that the tyrosine phosphorylation of 44-kDa MAPK at 2 min after ECS was markedly increased, in accordance with the increase of kinase activity. These results indicate that ECS induces the transient activation and tyrosine phosphorylation of 44-kDa MAPK, as well as 42-kDa MAPK, in rat hippocampus, although the amount of tyrosine phosphorylation is far less and the kinase activity is lower in 44-kDa MAPK than in 42-kDa MAPK.     

Nerve Growth Factor-Induced Down-Regulation of Calmodulin-Dependent Protein Kinase III in PC12 Cells Involves Cyclic AMP-Dependent Protein Kinase

Treatment of PC12 cells with nerve growth factor (NGF), epidermal growth factor (EGF), or agents that raise intracellular cyclic AMP (cAMP) levels (e.g., forskolin) reduces the activity of calmodulin-dependent protein kinase III (CaM-PK III) over a period of 8 h. The mechanism of this effect of NGF has now been examined in more detail, making use of a mutant PC12 cell line (A126-1B2) that is deficient in cAMP-dependent protein kinase activity. Control experiments showed that A126-1B2 cells retain other NGF-mediated responses (e.g., the induction of ornithine decarboxylase, a cAMP-independent event) and contain a complement of CaM-PK III and its substrate, elongation factor-2, comparable to that of wild-type cells. The ability of NGF or forskolin, but not of EGF, to down-regulate CaM-PK III was markedly attenuated in A126-1B2 compared to wild-type cells. Treatment of wild-type cells with the cAMP phosphodiesterase inhibitor, isobutylmethylxanthine, enhanced the effects of NGF, but not of EGF. The possibility that NGF led to a stimulation of cAMP-dependent protein kinase activity in wild-type cells was assessed by measurement of the "activation ratio" (-cAMP/+cAMP) of this enzyme before and at various times after NGF addition. A small, but significant, increase in the activation ratio from 0.3 to 0.48 was observed, reaching a peak 5 min after NGF treatment. EGF had no effect on the activation ratio in wild-type cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trimethyltin Stimulates Protein Kinase C Translocation Through Receptor-Mediated Phospholipase C Activation in PC12 Cells

Abstract: Trimethyltin (TMT) is a potent neurotoxic compound that initiates a delayed neuronal cell death. Previously we have shown that TMT-induced cytotoxicity is associated with protein kinase C (PKC) translocation and activation. The present study investigates the mechanism underlying TMT-stimulated PKC translocation in PC12 cells. TMT exposure led to a rapid increase in intracellular levels of inositol 1,4,5-trisphosphate (IP₃), a product of phospholipase C (PLC). This was significantly decreased by pretreating cells with antagonists to either the cholinergic muscarinic receptor (atropine) or the glutamatergic metabotropic receptor [(+)-α-methyl-4-carboxyphenylglycine; (+)-MCPG]. Furthermore, the rise in IP₃ level was blocked by pretreating cells with a PLC inhibitor (U-73122) or by a combination of atropine and (+)-MCPG. This pretreatment also significantly decreased TMT-stimulated PKC translocation, indicating that TMT-mediated PKC translocation was related to PLC activation, presumably through formation of diacylglycerol, an endogenous activator of PKC and product of PLC. It is interesting that atropine and (+)-MCPG did not provide protection against TMT-induced cytotoxicity in these cells. However, these data suggest that TMT causes the release of cellular constituents that activate G protein-coupled receptors, ultimately leading to PKC translocation.     

Activation of p42 Mitogen-Activated Protein Kinase by Glutamate Receptor Stimulation in Rat Primary Cortical Cultures

Abstract— Recent studies have identified at least two homologous mitogen-activated protein (MAP) kinases that are activated by phosphorylation of both tyrosine and threonine residues by an activator kinase. To help define the role of these MAP kinases in neuronal signalling, we have used primary cultures derived from fetal rat cortex to assess the regulation of their activity by agonist stimulation of glutamate receptors and by synaptic activity. Regulation was assayed by monitoring changes in both tyrosine phosphorylation on western blots and in vitro kinase activity toward a selective MAP kinase substrate peptide. In initial studies, we found that phorbol ester treatment increased tyrosine phosphorylation of p42 MAP kinase and stimulated MAP kinase activity. A similar response was elicited by three agonists of metabotropic glutamate receptors, i.e., trans -(±)-1-amino-1,3-cyclopentane dicarboxylic acid, quisqualate, and (2S,3S,4S)-α-(carboxycyclopropyl)glycine. MAP kinase activity and p42 MAP kinase tyrosine phosphorylation were also stimulated by the ionotropic glutamate receptor agonist, kainate, but not by N -methyl- d -aspartate. To examine regulation of MAP kinase by synaptic activity, cultures were treated with picrotoxin, an inhibitor of GABA_A receptor-mediated inhibition that enhances spontaneous excitatory synaptic activity. Treatment of cultures with picrotoxin elicited activation of MAP kinase. This response was blocked by tetrodotoxin, which suppresses synaptic activity. These results demonstrate that p42 MAP kinase is activated by glutamate receptor agonist stimulation and by endogenous synaptic activity.     

Roles for Protein Kinase C and Mitogen-Activated Protein Kinase in Nicotine-Induced Secretion from Bovine Adrenal Chromaffin Cells

Abstract: Both the Ca²⁺/phospholipid-dependent protein kinases (protein kinases C, PKCs) and mitogen-activated protein kinases (MAPKs) have been implicated as participants in the secretory response of bovine adrenomedullary chromaffin cells. To investigate a possible role for these kinases in exocytosis and the relationship of these kinases to one another, intact chromaffin cells were treated with agents that inhibited each of the kinases and analyzed for catecholamine release and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)/MAPK activation after stimulation with secretagogues of differential efficacy. Of the three secretagogues tested, inactivation of PKCs by long-term phorbol 12-myristate 13-acetate (PMA) treatment or incubation with GF109203X had the greatest inhibitory effect on nicotine-induced catecholamine release and MEK/MAPK activation, a moderate effect on KCl-induced events, and little, if any, effect on Ca²⁺ ionophore-elicited exocytosis and MEK/MAPK activation. These results indicate that PKC plays a significant role in events induced by the optimal secretagogue nicotine and a lesser role in exocytosis elicited by the suboptimal secretagogues KCl and Ca²⁺ ionophore. Treatment of cells with the MEK-activation inhibitor PD098059 completely inhibited MEK/MAPK activation (IC₅₀ 1–5 µM) and partially inhibited catecholamine release induced by all secretagogues. However, PD098059 was more effective at inhibiting exocytosis induced by suboptimal secretagogues (IC₅₀～10 µM) than that induced by nicotine (IC₅₀～30 µM). These results suggest a more prominent role for MEK/MAPK in basic secretory events activated by suboptimal secretagogues than in those activated by the optimal secretagogue nicotine. However, PD098059 also partially blocked secretion potentiated by short-term PMA treatment, suggesting that PKC can function in part by signaling through MEK/MAPK to enhance secretion. Taken together, these results provide evidence for the preferential involvement of MEK/MAPK in basic secretory events activated by the suboptimal secretagogues KCl and Ca²⁺ ionophore and the participation of both PKC and MEK/MAPK in optimal secretion induced by nicotine.     

Neurite Outgrowth Stimulated by the Tyrosine Kinase Inhibitor Herbimycin A Requires Activation of Tyrosine Kinases and Protein Kinase C

Abstract: Activation of tyrosine kinases is established as an important mechanism for controlling growth cone motility and neurite outgrowth. We have tested the effects of a range of tyrosine kinase inhibitors on neurite outgrowth from postnatal day 4 cerebellar granule cells cultured over confluent monolayers of 3T3 fibroblasts. The only agent that had any effect was herbimycin A, which stimulated neurite outgrowth. The response is shown to be attributable to a direct effect of this tyrosine kinase inhibitor on neurones. The neurite outgrowth response to herbimycin A was inhibited by two other tyrosine kinase inhibitors, which on their own did not affect neurite outgrowth. The data suggest that the response to herbimycin A reflects either a direct or indirect activation of one or more protein tyrosine kinases. Independent signalling events downstream from tyrosine kinase activation underlying the neurite outgrowth response to herbimycin A include increased activity of protein kinase C and calcium influx into neurones through both N-and L-type calcium channels.     

玉米根尖质膜的受钙激活蛋白激酶的特性

以生长3－4d的玉米（ZeamaysL．）根尖为材料,用两相法得到高纯度的质膜,鉴定了质膜上存在一受钙激活的蛋白激酶。该类激酶主要位于质膜内侧;钙的半激活浓度为50μmol／L;外源钙调素对激酶没有明显的活化作用;钙调素拮抗剂三氟拉嗪（TFP）可明显抑制激酶活性,半抑制浓度为75μmol／L;该类激酶对外源底物组蛋白ⅢS型有较高的特异性。结果显示此激酶属于钙依赖钙调素不依赖蛋白激酶。质膜上33kD和58kD两种蛋白可能是这种钙依赖蛋白激酶的内源底物。     

Protein Kinase C Modulates Calcium Sensitivity of Nitric Oxide Synthase in Cerebellar Slices

Abstract: The possible modulation of nitric oxide (NO) synthase (NOS) activity by protein kinase C (PKC) was investigated. Incubation of rat cerebellar slices with the specific metabotropic glutamate receptor agonist, (±)-1-aminocyclopentane- trans -1,3-dicarboxylate ( trans -ACPD) increased cyclic GMP concentration two-fold. The increase was dose-dependently blocked by the protein kinase inhibitors staurosporine and calphostin C. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased cyclic GMP concentration without glutamate receptor activation. The cyclic GMP increases induced by PMA and trans -ACPD were independent of extracellular calcium blocked by N ^ω-nitro- l -arginine, a specific NOS inhibitor, and were not additive. Measurement of citrulline formation in cerebellar slices confirmed that NOS was activated by trans -ACPD and the activation was blocked by calphostin C. These results suggest that metabotropic glutamate receptor activates NOS through PKC. The calcium dependency of NOS activation was assessed in slices incubated with PMA and okadaic acid. NOS in both PMA-treated and untreated slices had similar activities at 100 n M free calcium, whereas at 25–70 n M free calcium, NOS in PMA-treated slices was more active than that in untreated slices. These results suggest that PKC regulates NO release in resting neurons by modulating the sensitivity of NOS at low calcium concentrations.     

Activation of Protein Kinase C in Permeabilized Human Neuroblastoma SH-SY5Y Cells

The activation of protein kinase C was investigated in digitonin-permeabilized human neuroblastoma SH-SY5Y cells by measuring the phosphorylation of the specific protein kinase C substrate myelin basic protein4-14. The phosphorylation was inhibited by the protein kinase C inhibitory peptide PKC19-36 and was associated to a translocation of the enzyme to the membrane fractions of the SH-SY5Y cells. 1,2-Dioctanoyl-sn-glycerol had no effect on protein kinase C activity unless the calcium concentration was raised to concentrations found in stimulated cells (above 100 nM). Calcium in the absence of other activators did not stimulate protein kinase C. Phorbol 12-myristate 13-acetate was not dependent on calcium for the activation or the translocation of protein kinase C. The induced activation was sustained for 10 min, and thereafter only a small net phosphorylation of the substrate could be detected. Calcium or dioctanoylglycerol, when applied alone, only caused a minor translocation, whereas in combination a marked translocation was observed. Arachidonic acid (10 microM) enhanced protein kinase C activity in the presence of submaximal concentrations of calcium and dioctanoylglycerol. Quinacrine and p-bromophenacyl bromide did not inhibit calcium- and dioctanoylglycerol-induced protein kinase C activity at concentrations which are considered to be sufficient for phospholipase A2 inhibition.     

Pertussis Toxin-Insensitive G Protein Mediates Carbachol Activation of Phospholipase D in Rat Pheochromocytoma PC12 Cells

In the present study, an activation mechanism for phospholipase D (PLD) in [3H]palmitic acid-labeled pheochromocytoma PC12 cells in response to carbachol (CCh) was investigated. PLD activity was assessed by measuring the formation of [3H]phosphatidylethanol ([3H]PEt), the specific marker of PLD activity, in the presence of 0.5% (vol/vol) ethanol. CCh caused a rapid accumulation of [3H]-PEt, which reached a plateau within 1 min, in a concentration-dependent manner. The [3H]PEt formation by CCh was completely antagonized by atropine, demonstrating that the CCh effect was mediated by the muscarinic acetylcholine receptor (mAChR). A tumor promoter, phorbol 12-myristate 13-acetate (PMA), also caused an increase in [3H]-PEt content, which reached a plateau at 30-60 min after exposure, but an inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, did not. Although a protein kinase C (PKC) inhibitor, staurosporine (5 microM), blocked PMA-induced [3H]PEt formation by 77%, it had no effect on the CCh-induced formation. These results suggest that mAChR-induced PLD activation is independent of PKC, whereas PLD activation by PMA is mediated by PKC. NaF, a common GTP-binding protein (G protein) activator, and a stable analogue of GTP, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), also stimulated [3H]PEt formation in intact and digitonin-permeabilized cells, respectively. GTP, UTP, and CTP were without effect. Furthermore, guanosine 5'-O-(2-thiodiphosphate) significantly inhibited CCh- and GTP gamma S-induced [3H]PEt formation in permeabilized cells but did not inhibit the formation by PMA, and staurosporine (5 microM) had no effect on [3H]PEt formation by GTP gamma S.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of Calcium activated Protein Kinase in the Plasma Membrane of Corn Root Tip Cells

Plasma membrane (PM) of high purity was prepared through a two-phase aqueous system from roots of 3 ～ 4 d old com ( Zea mays L. ) seedling, and within which calcium-activated protein kinase was identified. The kinase was mainly located in the inner side of the PM with a semi-activation concentration to free calcium ion of 50 μmol/L. Exogenous CaM had no agonistic effect on the protein kinase but CaM antagonist TFP could strongly inhibit its activity with a semi-inhibition concentration of 75 μmol/L. The protein kinase showed high specificity for the exogenous substrate, Histone Ⅲ S. These results indicated that this protein kinase could be a calcium-dependent and calmodulin-independent protein kinase (CDPK). Moreover, two proteins (33 kD and 58 kD) in PM might be the endogenous substrates of the CDPK.