Phorbol Ester and Calcium Regulation of Corticotrophin-Releasing Factor Receptor 1 Expression in a Neuronal Cell Line

Abstract: 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 4α-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.     

Regulation of the Release of Interleukin-6 from Human Astrocytoma Cells

Abstract: Recent evidence suggests that the level of interleukin-6 (IL-6) is elevated in Alzheimer's disease (AD) brains. IL-6 is produced by reactive glial cells and could potentially affect neuronal survival. Understanding the biochemical mechanism that regulates the production and release of IL-6 by astrocytic cells may help to identify potential targets for therapeutic intervention in AD. In the present study, glial fibrillary acidic protein-positive human U373MG astrocytoma cells were used as a model of reactive astrocytes. Production of IL-6 in response to drug treatment was monitored with an ELISA assay. Histamine (1–100 µ M ), substance P (SP; 1–100 n M ), and human interleukin-1β (IL-1β; 1–30 p M ) stimulated the release of IL-6 in a time- and concentration-dependent manner, with EC₅₀ values of 4.5 µ M , 8 n M , and 4.5 p M , respectively. The respective effects of histamine, SP, and IL-1β were effectively blocked by the histamine H₁, SP, and IL-1 receptor antagonists, supporting a receptor-mediated event for these agents. Both histamine and SP enhanced the formation of inositol phosphates and increase intracellular calcium levels, suggesting that the phosphatidyl-inositol bisphosphate/protein kinase C pathway may be involved in the IL-6 release process. Indeed, phorbol 12-myristate 13-acetate, a protein kinase C activator, also evoked IL-6 release from the U373MG cells. On the other hand, IL-1β, which produces a much more robust release of IL-6 than histamine or SP, has no effect on inositol phosphate formation or intracellular calcium levels. The biochemical mechanism of the release of IL-6 in response to IL-1β remains to be elucidated.     

Effects of Phorbol Esters and Forskolin on Basal and Histamine-induced Accumulation of Inositol Phosphates in Cultured Bovine Adrenal Chromaffin Cells

The effect of phorbol esters and forskolin pretreatment on basal and histamine-induced accumulation of inositol phosphates and catecholamine release was examined in cultures of bovine adrenal chromaffin cells. Histamine caused a dose-dependent, Ca2+-dependent accumulation of total inositol phosphates with an EC50 at approximately 1 microM and an eight- to 10-fold increase at 100 microM within 30 min of incubation. Histamine (10 microM) also caused the release of cellular catecholamines amounting to some 2.8% of cellular stores released over a 20-min period. Both the inositol phosphate and catecholamine responses were completely blocked by the H1-antagonist mepyramine and were insensitive to the H2-antagonist cimetidine. Examination of the time course of accumulation of the individual inositol phosphates stimulated by histamine revealed an early and sustained rise in inositol 1,4-bisphosphate content but not inositol 1,4,5-trisphosphate content at 1 min and the overall largest accumulation of inositol monophosphate after 30 min of stimulation. Pretreatment with the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) resulted in a dose-dependent, time-dependent inhibition of histamine-induced inositol phosphate formation and catecholamine secretion. In this inhibitory action, PMA exhibited high potency (IC50 of approximately 0.5 nM), an effect not shared by the inactive phorbol ester 4-alpha-phorbol 12,13-didecanoate. Pretreatment with forskolin, on the other hand, only marginally inhibited the histamine-induced inositol phospholipid metabolism and catecholamine secretion. These data suggest that protein kinase C activation in chromaffin cells may mediate a negative feedback control on inositol phospholipid metabolism.     

Nerve Growth Factor Potentiates Bradykinin-Induced Calcium Influx and Release in PC12 Cells

To investigate how the response to agonists changes during neuronal differentiation, we examined the effect of nerve growth factor (NGF) on bradykinin-induced calcium increases in PC12 cells. Short-term (1 h) treatment with NGF increased the potency of bradykinin to raise intracellular calcium by about 10-fold, whereas long-term (1 week) treatment, which was associated with the expression of the differentiated phenotype, increased the potency about 100-fold. Neither treatment affected the maximal response to bradykinin. NGF alone had no acute effect on calcium levels. Short-term potentiation appeared to be mainly a result of greater release of calcium from intracellular stores, whereas the effect of long-term treatment apparently was due to increases in both release from intracellular stores and calcium influx. [3H]Bradykinin binding to intact PC12 cells was unaltered by short-term NGF treatment, whereas differentiated cells displayed a 50% increase in receptor number and about a twofold increase in affinity as compared with cells not treated with NGF. The production of inositol phosphates in response to bradykinin correlated poorly with the calcium transients, in that large calcium responses were associated with small increases in inositol phosphates. Neither NGF treatment had a significant effect on the appearance of inositol phosphates in response to bradykinin. Experiments with permeabilized cells revealed that differentiated cells did not display a heightened response to exogenously added inositol 1,4,5-trisphosphate. Our results demonstrate that NGF modulates the bradykinin signaling pathway without acutely activating this pathway itself.     

Effects of Adrenocorticotropin Treatment on Estrogen,Luteinizing Hormone,and Progesterone Secretion in the Female Rhesus Monkey

Adrenocorticotropin (ACTH) was administered to female rhesus monkeys in order to determine the effects of adrenal axis activation on the endocrine events occurring during the menstrual cycle. ACTH injected twice daily during the follicular period and through the time of expected ovulation was found to prevent the rise of estrogens during the follicular phase. In addition, the ACTH administration also blocked the preovulatory surge of LH, prevented the luteal rise of progesterone, and extended the length of the menstrual cycle.     

Phorbol Ester-Induced Upregulation of Angiotensin II Receptors in Neuronal Cultures Is Potentiated by a Calcium Ionophore

Previous studies have suggested that protein kinase C is important in the regulation of angiotensin II receptors in neuronal cultures, because the C-kinase agonists, phorbol esters, are able to increase the number of these receptors. In the present study, we have further investigated the role of protein kinase C in angiotensin II receptor regulation. This enzyme is calcium dependent, and so we investigated the effects of A23187, a calcium ionophore, on phorbol ester-stimulated and basal angiotensin II receptor regulation. A23187, at concentrations that increased 45Ca2+ influx, caused a dose-dependent potentiation of phorbol-12-myristate-13-acetate (TPA)-stimulated upregulation of angiotensin II receptors. This potentiation by A23187 was a further increase in angiotensin II receptor number and was abolished in calcium-free medium. In the absence of TPA, A23187 caused a decrease in angiotensin II receptor number, an effect not observed in calcium-free medium. The results suggest at least two pathways for angiotensin II receptor regulation in neuronal cells: (a) by calcium-dependent protein kinase C and (b) via an influx of calcium into the cell.     

Inhibition of Bradykinin-Induced Phosphoinositide Hydrolysis and Ca Mobilisation by Phorbol Ester in Rat Cultured Vascular Smooth Muscle Cells

Regulation of the increase in inositol phosphate (IP) production and intracellular Ca²⁺ concentration ([Ca²⁺]_i by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 μM) for 30 min almost abolished the BK-induced IP formation and Ca²⁺ mobilisation. This inhibition was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The concentrations of PMA giving a half-maximal (pEC₅₀) and maximal inhibition of BK induced an increase in [Ca²⁺]_i, were 7.8 ± 0.3 M and 1 μM, n = 8, respectively. Prior treatment of VSMCs with staurosporine (1 μM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Paralleling the effect of PMA on the BK-induced IP formation and Ca²⁺ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-, βI, βII, δ, ε, and ζ isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 μM PMA for either 1 or 24 h did not significantly change the K_D and B_max of the BK receptor for binding (control: K_D = 1.7 ± 0.2 nM; B_max = 47.3 ± 4.4 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, these resuts demonstrate that translocation of PKC-, βI, βII, δ, ε, and ζ induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca²⁺ mobilisation in VSMCs.     

Phorbol Myristate Acetate Inhibition of Phospholipase C Activation by Carbachol in Slices of Rat Brain Cortex Is a Delayed and Indirect Effect

We examined the effect of phorbol esters on phospholipase C activation in rat brain cortical slices and membranes. There was little effect of concurrent addition of phorbol 12-myristate 13-acetate (PMA) with carbachol on phosphoinositide breakdown due to carbachol over a 1-h incubation of brain slices. However, if slices were preincubated for 3 h with 1 microM PMA or 200 microM sphingosine before addition of carbachol, there was a 35-50% inhibition of phosphoinositide breakdown. There was also a marked loss of protein kinase C (PKC) activity from both cytosol and membranes after a 3-h exposure to PMA. The loss in responsiveness to the muscarinic agonists in slices was not reflected in carbachol-stimulated phospholipase C activation using isolated membranes. However, the decrease in carbachol-induced phosphoinositide breakdown seen in slices after a 3-h exposure to PMA was abolished if the extracellular K+ concentration was elevated from 5.9 to 55mM. Because elevation of the K+ level induces depolarization and increases Ca2+ entry, we examined the effect of ionomycin, a Ca2+ ionophore. Ionomycin potentiated the effects of carbachol on phosphoinositide breakdown but was unable to reverse the effects of a 3-h incubation with PMA. Because apamin, an inhibitor of Ca2(+)-dependent K+ channels, mimicked the effects of exposure to PMA for 3 h, it is possible that these channels are involved in muscarinic cholinergic regulation of phosphoinositide breakdown in rat brain slices. These results support the hypothesis that prolonged PMA treatment in rat brain cortex has no direct effect on phospholipase C activation by muscarinic cholinergic stimulation.     

Activation of Protein Kinase C by Phorbol Esters and Arachidonic Acid Required for the Optimal Potentiation of Glutamate Exocytosis

Abstract: The effects of arachidonic acid and phorbol esters in the Ca²⁺-dependent release of glutamate evoked by 4-aminopyridine (4-AP) in rat cerebrocortical synaptosomes were studied. In the absence of arachidonic acid, high concentrations (500 n M ) of 4β-phorbol dibutyrate (4β-PDBu) were required to enhance the release of glutamate. However, in the presence of arachidonic acid, low concentrations of 4β-PDBu (1–50 n M ) were effective in potentiating glutamate exocytosis. This potentiation of glutamate release by phorbol esters was not observed with the methyl ester of arachidonic acid, which does not activate protein kinase C. Moreover, pretreatment of synaptosomes with the protein kinase inhibitor staurosporine also prevented the stimulatory effect by arachidonic acid and phorbol esters. These results suggest that the activation of protein kinase C by both arachidonic acid and phorbol esters may play a role in the potentiation of glutamate exocytosis.     

The Differential Role of Protein Kinase C Isozyme in the Rapid Induction of Neurofilament Phosphorylation by Nerve Growth Factor and Phorbol Esters in PC12 Cells

We examined the short-term regulation of the phosphorylation of the mid-sized neurofilament subunit (NF-M) by kinases which were activated in rat pheochromocytoma (PC12) cells by nerve growth factor (NGF) and/or 12-O-tetradecanoylphorbol 13-acetate (TPA). We found that NGF and TPA, alone or in combination, increased (a) the incorporation of [32P]Pi into NF-M and (b) the rate of conversion of NF-M from a poorly phosphorylated to a more highly phosphorylated form. This was not due to increased synthesis of NF-M, because NGF alone did not increase NF-M synthesis and TPA alone or TPA and NGF together inhibited the synthesis of NF-M. Further, an increase in calcium/phospholipid-dependent kinase (PKC) activity resulting from the treatment of PC12 cells with NGF and TPA was observed concomitant with the increased phosphorylation of NF-M. This PKC activity was determined to be derived from the PKC alpha and PKC beta isozymes. Finally, when PC12 cells were rendered PKC-deficient by treatment with 1 muM TPA for 24 h, NGF maintained the ability to induce an increase in NF-M phosphorylation, though not to the level attained in cells which were not PKC-deficient. These data suggest that NGF with or without TPA stimulates NF-M phosphorylation as a result of a complex series of events which include PKC-independent and PKC-dependent pathways.     

甲状旁腺高血压因子和甲状旁腺素对细胞钙通道的不同作用

1989年Lewanczuk等[1]报道在自发性高血压大鼠（spontaneouslyhvnertensiverat,SHR）的血浆中发现一种具有独特升压效应的高血压因子．通过激活平滑肌细胞膜钙通道,提高细胞内游离钙（[Ca2 ]）水平起作用．随之证明这种循环血中的高血压因子来源于甲状旁腺,故称“甲状旁腺高血压因子（Parathyroidhypertensivefactor,PHF）”[2]。我们经实验研究证明,当给SD（Sprague-Dawley）大鼠注射经透析的SHR血浆后30min其血压开始升高,45min达高峰,60min恢复到注射前水平．同时经透析的血浆可使SD大鼠尾动脉条的细胞45Ca2 摄取增加,其…     

Effects of Calcium Channel Antagonists on Calcium Entry and Glutamate Release from Cultured Rat Cerebellar Granule Cells

Abstract: Using a range of Ca²⁺ channel blockers we have investigated the Ca²⁺ channel subtypes that mediate the depolarisation-induced elevation of the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and glutamate release from cultured rat cerebellar granule cells. ω-Conotoxin-GVIA had little effect on either the transient or plateau phase of the depolarisation-induced [Ca²⁺]_i rise or on glutamate release, ruling out a significant role for N-type Ca²⁺ channels. Nifedipine substantially inhibited the initial transient rise in [Ca²⁺]_i and the plateau phase of the [Ca²⁺]_i rise and glutamate release, suggesting the involvement of L-type Ca²⁺ channels. Both ω-agatoxin and ω-conotoxin-MVIIC also inhibited the transient rise in [Ca²⁺]_i and glutamate release but not the plateau phase of the [Ca²⁺]_i rise. The inhibitions by nifedipine were not increased by coaddition of ω-conotoxin-MVIIC, suggesting overlapping sensitivity to these channel blockers. These data show that glutamate release from granule cells in response to depolarisation with a high KCI level involves Ca²⁺ currents that are sensitive to nifedipine, ω-agatoxin-IVA, and also ω-conotoxin-MVIIC. The overlapping sensitivity of the channels to these toxins prevents attribution of any of the phases of the [Ca²⁺]_i rise or glutamate release to distinct P-, Q-, or O-type Ca²⁺ currents.     

Effects of Protein Kinase C Down-Regulation on Secretory Events and Proopiomelanocortin Gene Expression in Anterior Pituitary Tumor (AtT-20) Cells

To elucidate the role of the diacylglycerol-protein kinase C (PKC) pathway in beta-endorphin synthesis and secretion in anterior pituitary corticotrope tumor cells (AtT-20), a procedure for down-regulating PKC activity in the cells was developed. Treatment of AtT-20 cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) led to an increase in [3H]phorbol 12,13-dibutyrate binding to PKC in the membrane fraction of these cells 30 s after its addition to the culture medium. Thereafter, a decrease in both [3H]phorbol 12,13-dibutyrate binding and PKC-specific phosphotransferase activity occurred in a time- and dose-dependent manner in both the cytosolic and membrane fractions. For example, treatment of the cells with 100 nM TPA for 24 h resulted in an almost complete depletion of PKC activity. Immunoreactive beta-endorphin secretion was found to be stimulated two- to fourfold in the control cells after incubation with corticotropin-releasing factor (10(-7) M), forskolin (10(-6) M), or TPA (10(-7) M) for 4 h. In cells rendered PKC deficient, TPA-stimulated immunoreactive beta-endorphin release was abolished, forskolin-stimulated release was unaffected, and corticotropin-releasing factor-stimulated release was depressed. Treatment of control cells with any one of the three stimulatory agents led to an increase in proopiomelanocortin mRNA levels, and these responses were also depressed after TPA pretreatment. The results suggest that physiological processes thought to be entirely cyclic AMP dependent, such as corticotropin-releasing factor-elicited secretion, may be partially dependent on PKC-mediated biochemical events.     

Synergistic Stimulation of Interleukin 6 Release and Gene Expression by Phorbol Esters and Interleukin 1β in Rat Cortical Astrocytes: Role of Protein Kinase C Activation and Blockade

Abstract: The involvement of protein kinase C and its interaction with interleukin 1β in the control of interleukin 6 release by cortical astrocytes was studied. The blockade of protein kinase C catalytic domain, by staurosporine, as well as the desensitization of protein kinase C by short-term phorbol 12-myristate 13-acetate pretreatment, increased the basal release of interleukin 6 by rat cortical astrocytes, whereas calphostin C, an antagonist of phorbol ester binding on protein kinase C regulatory domain, did not affect the basal release of the cytokine. The activation of protein kinase C by phorbol 12-myristate 13-acetate enhanced concentration- and time-dependently interleukin 6 release. This stimulatory action of phorbol 12-myristate 13-acetate was significantly reduced by staurosporine, by calphostin C, and by the desensitization of protein kinase C. Interleukin 1β increased interleukin 6 release in a concentration-related manner. Protein kinase C inhibition, by staurosporine or desensitization, potentiated severalfold, whereas calphostin C reduced interleukin 1β stimulation of interleukin 6 release. The treatment of cortical astrocytes with both interleukin 1β (3 ng/ml) and phorbol 12-myristate 13-acetate (10 nM) caused a synergistic stimulation of interleukin 6 release and its gene expression, an effect that was not relieved by either 20 nM staurosporine or by calphostin C but was slightly affected by protein kinase C desensitization. In conclusion, our data show that in rat cortical astrocytes the basal release of interleukin 6 is under a tonic inhibition exerted by a protein kinase C isoform or isoforms sensitive to blockade by staurosporine and desensitization but insensitive to calphostin C. Interleukin 1β stimulated interleukin 6 secretion via a mechanism that is also negatively modulated by a protein kinase C isoform or isoforms sensitive to staurosporine and desensitization. Finally, we showed that interleukin 1β and phorbol 12-myristate 13-acetate synergistically stimulated interleukin 6 release and its gene expression, operating in a manner insensitive to protein kinase C blockers and slightly reduced by protein kinase C desensitization.     

Stimulation of the P2Y Purinergic Receptor on Type 1 Astroglia Results in Inositol Phosphate Formation and Calcium Mobilization

Cultured astroglia express purinergic receptors that initiate phosphoinositide metabolism and calcium mobilization. Experiments were conducted to characterize the purinergic receptor subtype on type 1 astroglia responsible for stimulation these second-messenger systems. Inositol phosphate (IP) accumulation and calcium mobilization were measured after stimulation with ATP or purinergic receptor subtype-selective ATP analogues. ATP (10(-5) M) increased IP accumulation severalfold. Dose-effect assays monitoring astroglial IP accumulation revealed the order of potency that defines the P2Y receptor: 2-methylthioadenosine 5'-triphosphate greater than ATP greater than alpha beta-methyleneadenosine 5'-triphosphate greater than beta gamma-methyleneadenosine 5'-triphosphate. The influence of ATP on intracellular calcium levels in individual type 1 astroglia was examined using the calcium indicator dye, fura-2. Dose-effect experiments indicated that ATP was equally potent for generating inositol phosphates and increasing cellular calcium. The most prevalent response (87% of total responses) to ATP consisted of a rapid increase in calcium to a peak level that was approximately five times greater than the prestimulation level. This peak was followed by a decline to a plateau level that was significantly above baseline. This plateau phase of the calcium increase was maintained for at least 5 min in the presence of ATP and was dependent on external calcium. Many (23%) astroglia exhibited spontaneous calcium oscillations whose frequency and magnitude increased after the addition of 10(-5) M ATP. Immunocytochemical staining indicated that the responses occurred in glial fibrillary acidic protein positive cells. We conclude that type 1 astroglia express the P2Y purinergic receptor which regulates IP production and calcium mobilization.     

Mitochondria, Calcium Regulation, and Acute Glutamate Excitotoxicity in Cultured Cerebellar Granule Cells

Abstract: Exposure of cultured cerebellar granule cells to 100 µ M glutamate plus glycine in the absence of Mg²⁺ causes calcium loading of the in situ mitochondria and is excitotoxic, as demonstrated by a collapse of the cellular ATP/ADP ratio, cytoplasmic Ca²⁺ deregulation (the failure of the cell to maintain a stable cytoplasmic free Ca²⁺ concentration), and extensive cell death. Glutamate-evoked Ca²⁺ deregulation is exacerbated by the mitochondrial respiratory chain inhibitor rotenone. Cells maintained by glycolytic ATP, i.e., in the presence of the mitochondrial ATP synthase inhibitor oligomycin, remain viable for several hours but are still susceptible to glutamate; thus, disruption of mitochondrial ATP synthesis is not a necessary step in glutamate excitotoxicity. In contrast, the combination of rotenone (or antimycin A) plus oligomycin, which collapses the mitochondrial membrane potential, therefore preventing mitochondrial Ca²⁺ transport, allows glutamate-exposed cells to maintain a high ATP/ADP ratio while accumulating little ⁴⁵Ca²⁺ and maintaining a low bulk cytoplasmic free Ca²⁺ concentration determined by fura-2. It is concluded that mitochondrial Ca²⁺ accumulation is a necessary intermediate in glutamate excitotoxicity, whereas the decreased Ca²⁺ flux into cells with depolarized mitochondria may reflect a feedback inhibition of the NMDA receptor mediated by localized Ca²⁺ accumulation in a microdomain accessible to the mitochondria.     

Translocation and Activation of Protein Kinase C in Striatal Neurons in Primary Culture: Relationship to Phorbol Dibutyrate Actions on the Inositol Phosphate Generating System and Neurotransmitter Release

The actions of the tumor-promoting phorbol ester phorbol dibutyrate were examined, under identical physiological conditions, on three distinct cellular processes in striatal neurons: the distribution of protein kinase C, the carbachol-stimulated generation of [3H]inositol monophosphate, and the KCl-evoked release of gamma-[3H]aminobutyric acid ([3H]GABA). Phorbol dibutyrate induced a rapid (complete in 5 min), dose-dependent, entirely reversible (t0.5 = 15 min) translocation of protein kinase C from cytosol to membrane. On longer exposure to phorbol dibutyrate, membrane-associated protein kinase C returned toward the control level, and total cellular enzyme activity declined markedly. Phorbol dibutyrate also induced the dose-dependent attenuation of carbachol-stimulated [3H]inositol monophosphate production and potentiation of KCl-evoked release of [3H]GABA. The translocation of protein kinase C and the potentiation of KCl-evoked [3H]GABA release were both rapidly reversed following washout of phorbol dibutyrate. In addition, for both processes, the effect of a 1-h exposure to phorbol dibutyrate was markedly less than that observed following a 5-min exposure to the agent. In direct contrast, inhibition of carbachol-stimulated [3H]inositol monophosphate production was not rapidly reversed following washout of phorbol dibutyrate and was actually more pronounced following a 1-h exposure, compared with a 5-min exposure. These findings indicate that some, but not all, of the actions of phorbol dibutyrate are closely associated with the translocation of protein kinase C in striatal neurons in primary culture.     

Different Patterns of Agonist-Stimulated Increases of 3H-Inositol Phosphate Isomers and Cytosolic Ca2+ in Bovine Adrenal Chromaffin Cells: Comparison of the Effects of Histamine and Angiotensin II

Abstract: Bovine adrenal chromaffin cells (BCC) were used to compare histamine- and angiotensin II-induced changes of inositol mono-, bis-, and trisphosphate (InsP₁, InsP₂, and InsP₃, respectively) isomers, intracellular free Ca²⁺ ([Ca²⁺]_i), and the pathways of inositol phosphate metabolism. Both agonists elevated [Ca²⁺]_i by 200 nM 3–4 s after addition, but afterwards the histamine response was much more prolonged. Histamine and angiotensin II also produced similar four- to fivefold increases of Ins(1,4,5)P₃ that peaked within 5 s. Over the first minute of stimulation, however, Ins(1,4,5)P₃ formation was monophasic after angiotensin II, but biphasic after histamine, evidence supporting differential regulation of angiotensin II- and histamine-stimulated signal transduction. The metabolism of Ins(1,4,5)P₃ by BCC homogenates was found to proceed via (a) sequential dephosphorylation to Ins(1,4)P₂ and Ins(4)P, and (b) phosphorylation to inositol 1,3,4,5-tetrakisphosphate, followed by dephosphorylation to Ins(1,3,4)P₃, Ins(1,3)P₂, and Ins(3,4)P₂, and finally to Ins(1 or 3)P. In whole cells, Ins(1 or 3)P only increased after histamine treatment. Additionally, Ins(1,3)P₂ was the only other InsP₂ besides Ins(1,4)P₂ to accumulate within 1 min of agonist treatment [Ins(3,4)P₂ did not increase]. These results support a correlation between the time course of Ins(1,4,5)P₃ formation and the time course of [Ca²⁺]_i transients and illustrate that Ca²⁺-mobilizing agonists can produce distinguishable patterns of inositol phosphate formation and [Ca²⁺], changes in BCC. Different patterns of second-messenger formation are likely to be important in signal recognition and may encode agonist-specific information.     

Phorbol Ester- and Retinoic Acid-Induced Regulation of the Protein Kinase C Substrate MARCKS in Immortalized Hippocampal Cells

Abstract: The expression of MARCKS, a major protein kinase C (PKC) substrate, was examined in the immortalized hippocampal cell line HN33, following differentiation using phorbol esters or retinoic acid. In cells exposed to phorbol esters, MARCKS protein levels were reduced through an apparent PKC-dependent mechanism. Exposure to 1 µ M phorbol 12-myristate 13-acetate (PMA) for 10 min resulted in a rapid loss of PKC activity in the soluble fraction with a concurrent increase in membrane-associated PKC activity. PKC activity was reduced to <20% of control values in both soluble and membrane fractions following 1 h of PMA exposure. Significant reductions in MARCKS protein levels were initially observed in membrane and soluble fractions following PMA exposure for 4 and 8 h, respectively. The reduction in MARCKS protein levels was maximal following 24 h of PMA exposure. MARCKS protein expression was also down-regulated in a dose-dependent manner on exposure of HN33 cells to retinoic acid. In cells exposed to 10 µ M retinoic acid, the MARCKS protein level was reduced in the membrane fraction within 4 h. Reduction of MARCKS protein levels was maximal (>90%) by 12 h with no evidence for any alteration in PKC activity. Reduced levels of MARCKS protein were also observed in the soluble fraction of retinoic acid-exposed cells, but to a significantly lesser extent. Addition of the PKC inhibitor GF109203X blocked the down-regulation of MARCKS protein in PMA-treated cultures but not in retinoic acid-treated cells. These findings suggest that the down-regulation of MARCKS may play an important role in both phorbol ester- and retinoic acid-induced differentiation in cells of neuronal origin.     

Protein Kinase C Activation Enhances K+-Stimulated Endogenous Dopamine Release from Rat Striatal Synaptosomes in the Absence of an Increase in Cytosolic Ca2+

The possibility that protein kinase C modulates neurotransmitter release in brain was investigated by examining the effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on Ca2+ transport and endogenous dopamine release from rat striatal synaptosomes. TPA (0.16 and 1.6 microM) significantly increased dopamine release by 24 and 33%, respectively, after a 20-min preincubation with TPA followed by 60 s of depolarization with 30 mM KCl. Depolarization-induced 45Ca2+ uptake, measured simultaneously with dopamine release, was not significantly increased by TPA. Neither 45Ca2+ uptake nor dopamine release was altered under resting conditions. When the time course of K+-stimulated 45Ca2+ uptake and dopamine release was examined, TPA (1.6 microM) enhanced dopamine release after 15, 30, and 60 s, but not 1, 3, or 5 s, of depolarization. A slight increase in 45Ca2+ uptake after 60 s of depolarization was also seen. The addition of 30 mM KCl to synaptosomes which had been preloaded with the Ca2+-sensitive fluorophore fura-2 increased the cytosolic free Ca2+ concentration ([Ca2+]i) from 445 nM to 506 nM after 10 s of depolarization and remained elevated after 60 s. TPA had no effect on [Ca2+]i under depolarizing or resting conditions. Replacing extracellular Ca2+ with 100 microM EGTA reduced K+-stimulated (60 s) endogenous dopamine release by 53% and decreased [Ca2+]i to 120 nM. In Ca2+-free medium, 30 mM KCl did not produce an increase in the [Ca2+]i. TPA (1.6 microM) did not alter the [Ca2+]i under resting or depolarizing conditions, but did increase K+-stimulated dopamine release in Ca2+-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)