Transduction Mechanisms Involved in Thrombin Receptor-Induced Nerve Growth Factor Secretion and Cell Division in Primary Cultures of Astrocytes

Abstract: In astrocytes, thrombin and thrombin receptor-activating activating peptide (TRAP-14), a 14-amino-acid agonist of the proteolytic activating receptor for thrombin (PART), significantly increased cell division as assessed by [³H]thymidine incorporation into DNA (EC₅₀ = 1 n M and +650% at 100 n M for thrombin; EC₅₀ = 3 µ M and +600% at 100 µ M for TRAP-14) and nerve growth factor (NGF) secretion (approximately twofold at 100 n M thrombin or 100 µ M TRAP-14). The [³H]thymidine incorporation was prevented by protein kinase C inhibitors (staurosporine and H7) or by down-regulation of this enzyme by chronic exposure of astrocytes to phorbol 12-myristate 13-acetate (PMA). Thrombin-induced NGF secretion was completely inhibited by protein kinase C inhibitors. Treatment with PMA stimulated NGF secretion 19-fold, and this effect was not further enhanced by thrombin. These data suggest an absolute requirement of protein kinase C activity for thrombin-induced NGF secretion and cell division. Pretreatment of astrocytes with pertussis toxin (PTX) reduced thrombin- and TRAP-14-induced DNA synthesis. PART activation caused a decrease in forskolin-stimulated cyclic AMP accumulation. PTX treatment prevented the inhibitory effect of PART activation on cyclic AMP accumulation, suggesting that a PTX-sensitive G protein, such as G_i or G_o, is involved in thrombin-induced cell division. In contrast, thrombin-induced NGF secretion was not inhibited by PTX. Finally, the protein tyrosine kinase inhibitor herbimycin A partially but significantly prevented thrombin- and TRAP-14-induced cell division but was without effect on NGF secretion. Taken together, these results demonstrate that, in astrocytes, PART(s)-triggered cell division or NGF secretion is mediated by distinct transduction mechanisms.     

Nerve Growth Factor Stimulates Phospholipid Methylation in Target Ganglionic Neurons Independently of the Cyclic AMP and Sodium Pump Responses

Suspensions of neurons prepared from embryonic day 12 (E12) chick sympathetic ganglia were incubated with [methyl-3H]methionine in the absence of nerve growth factor (NGF). Presentation of the factor for different periods of time resulted in an approximate three-fold stimulation of radioactivity incorporated into total phospholipid, followed by a rapid decline thereafter. Both the magnitude and the time of the response were dependent on the NGF concentration used. Also examined were possible relationships of phospholipid methylation to two other short-latency responses to NGF, i.e., control of the Na+,K+-pump and elevation of cyclic AMP content. Incubation of E12 sympathetic neurons with known transmethylase inhibitors (shown to be active in the present system) failed to prevent reactivation of the Na+,K+-pump in response to NGF administration. E16 sympathetic neurons and E15 sensory neurons, which do not depend on exogenous NGF for control of their Na+,K+-pump, still show a stimulation of phospholipid methylation when challenged with the factor. Blockage of the pump with ouabain also fails to prevent a methylation response. Thus, the pump and methylation responses to NGF occur independently of each other. Intact E8 chick dorsal root ganglia, but not E12 sympathetic ganglia, display a rapid and transient rise in their cyclic AMP content when presented with NGF. At a concentration of 10 biological units/ml, NGF elicits a peak of phospholipid methylation at 4 min, and a peak of cyclic AMP at 10 min. Methylation inhibitors prevent the methylation response, but not that of cyclic AMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of ζ-Protein Kinase C by Cyclic AMP in PC12 Cells Occurs Through Phosphorylation by Protein Kinase A

Abstract: Although cyclic AMP (cAMP) has been reported to cross talk with the protein kinase C (PKC) system, effects of elevated intracellular cAMP on the activities of specific PKC isoforms have not been studied. We report findings from a permeabilized cell assay that was used to examine changes in the activity of the atypical PKC isoforms brought about by exposure of PC12 cells to agents that elevate intracellular cAMP. We found that increases in intracellular cAMP led to rapid stimulation of atypical PKC activity, 40–70% above control, for a sustained period of time, a response that occurred independent of the phorbol 12-myristate 13-acetate (PMA)-sensitive PKC isoforms. Changes in intracellular cAMP levels resulted in a dose-dependent redistribution of ζ-PKC to the cytoplasm with a concomitant increase in the phosphorylation state of the enzyme. Incubation of purified ζ-PKC with increasing concentrations of PKA likewise caused a twofold increase in the phosphorylation state of ζ-PKC. In contrast to the positive effect that PKA-mediated phosphorylation had on the activity of ζ-PKC, the enzyme displayed reduced binding to ras when phosphorylated. Taken together, these findings are consistent with the hypothesis that protein phosphorylation of PKC acts as a positive effector of its enzyme activity and may serve as a negative modulator for interaction with other proteins.     

Increased Intracellular Cyclic AMP Differentially Modulates Nerve Growth Factor Induction of Three Neuronal Recognition Molecules Involved in Neurite Outgrowth

The relative expression of the immunoglobulin superfamily members Thy-1 and L1 and the neural cell adhesion molecule (N-CAM) in PC12 cells grown in the presence of nerve growth factor (NGF), cholera toxin, or both has been quantified. Whereas NGF treatment induced increases in the cell surface expression of all three glycoproteins, treatment with cholera toxin resulted in the specific induction of L1. During the first few days of culture, cholera toxin acted synergistically with NGF to promote increases in neuritic outgrowth and the synthesis and cell surface accumulation of the 140- and 180-kilodalton subunits of N-CAM. In contrast, over the same period of culture, cholera toxin inhibited the NGF induction of Thy-1 and L1. Over longer periods of culture (3-5 days), cholera toxin inhibited the NGF induction of N-CAM and neurite outgrowth. A similar pattern of synergistic and inhibitory responses was observed when differentiation was induced by fibroblast growth factor (FGF) rather than NGF or when cholera toxin was replaced with forskolin. These data suggest that intracellular cyclic AMP can differentially modulate cell surface glycoprotein expression induced by either NGF or FGF. Of the three cell surface glycoproteins we have studied, temporal changes in N-CAM expression correlate best with the morphological differentiation status of PC12 cells.     

Potentiation of Agonist-Stimulated Cyclic AMP Accumulation by Tyrosine Kinase Inhibitors in Rat Pinealocytes

Abstract: To study cross-talk mechanisms in rat pinealocytes, the role of tyrosine kinase or kinases in the regulation of adrenergic-stimulated cyclic AMP production was investigated. Both norepinephrine- and isoproterenol-stimulated cyclic AMP accumulation were increased by two distinct tyrosine kinase inhibitors, genistein or erbstatin, in a concentration-dependent manner. A similar increase was observed with two other inhibitors, tyrphostin B44 and herbimycin. In contrast, daidzein, an inactive analogue of genistein, was ineffective; whereas vanadate, a phosphotyrosine phosphatase inhibitor, reduced the adrenergic-stimulated cyclic AMP accumulation. The tyrosine kinase inhibitors were effective in potentiating the cholera toxin-or forskolin-stimulated cyclic AMP accumulation, indicating that their sites of action are at the postreceptor level. Neither an activator nor inhibitors of protein kinase C influenced the potentiation of the cyclic AMP responses by genistein, suggesting that the potentiation effect by tyrosine kinase inhibitors does not involve the phospholipase C/protein kinase C pathway. However, when the phosphodiesterase was inhibited by isobutylmethylxanthine, genistein failed to potentiate and vanadate did not inhibit the adrenergic-stimulated cyclic AMP accumulation, indicating that the phosphodiesterase is a probable site of action for these inhibitors. These results suggest that cyclic AMP metabolism in the pinealocytes is tonically inhibited by tyrosine kinase acting on the cyclic AMP phosphodiesterase.     

Role of Ca2+ in Differentiation Mediated by Nerve Growth Factor and Dibutyryl Cyclic AMP in PC12 Cells

Abstract: Nerve growth factor (NGF) and dibutyryl cyclic AMP (dbcAMP) have synergistic effects on the neurite outgrowth of rat pheochromocytoma PC12 cells. The sites of interaction between NGF and dbcAMP have been studied extensively; however, the role of Ca²⁺ in differentiation induced by the two agents remains unclear. To understand whether intracellular Ca²⁺ is involved in the differentiation induced by the two agents, PC12 cells were treated with NGF, dbcAMP, or NGF plus dbcAMP for 2 days, and then effects on neurite outgrowth, ATP-induced Ca²⁺ influx, and Ca²⁺ mobilization from intracellular Ca²⁺ pools were examined. NGF or dbcAMP alone enhanced neurite outgrowth and Ca²⁺ accumulation by nonmitochondrial Ca²⁺ pools or the thapsigargin (TG)-sensitive Ca²⁺ pool. The dbcAMP acted synergistically with NGF to increase neurite outgrowth and to enlarge the TG-sensitive Ca²⁺ pool. The synergistic effect occurred within the first hour of treatment with dbcAMP plus NGF. On the other hand, dbcAMP abolished NGF's ability to enhance ATP-induced influx of extracellular Ca²⁺. Therefore, NGF and dbcAMP induced different effects on Ca²⁺ signaling pathways through two different but interacting pathways. In PC12 cells pretreated with TG to deplete the TG-sensitive Ca²⁺ pool, the dbcAMP- or dbcAMP plus NGF-mediated neurite outgrowth was significantly inhibited, whereas NGF-mediated neurite outgrowth was not affected by TG pretreatment. Our results suggest that the intracellular nonmitochondrial Ca²⁺ pools were changed in the differentiation process and were necessary for the synergistic effect of NGF and dbcAMP.     

GM1 Ganglioside Activates the High-Affinity Nerve Growth Factor Receptor trkA

Abstract: The monosialoganglioside GM1 has been shown to possess neurotrophic activity in vitro and in vivo and is now used as an experimental treatment for a variety of neurological disorders and trauma. Little is known about the mechanism of action used by GM1. Because GM1 appears to enhance nerve growth factor (NGF) activity, we have used C6trk⁺ cells, a derivative of C6-2B glioma cells that express the high-affinity receptor for NGF trkA , to determine whether the neurotrophic effects of GM1 occurs through induction of trkA activity. Exposure of C6trk⁺ cells to NGF (10–50 ng/ml) resulted in a five- to 10-fold increase in trkA tyrosine phosphorylation within 5 min. Incubation of cells with GM1 resulted in a threefold increase in trkA phosphorylation beginning within 1 h and peaking between 3 and 6 h. Optimal responses to GM1 were obtained using 80–100 µ M concentrations. Moreover, tyrosine phosphorylation of known trkA target proteins, such as extracellular signal-regulated kinases, and suc -associated neurotrophic factor-induced tyrosine-phosphorylated target, were activated upon stimulation of C6trk⁺ cells with GM1. In addition, GM1 potentiated the NGF-mediated activation of tyrosine phosphorylation of trkA . GM1 failed to induce phosphorylation of trkA and target proteins in mock transfected cells. Thus, our data demonstrate that GM1 mimics some of the effects of NGF and suggest that the neurotrophic properties of GM1 may be attributed to its activation of trkA signal transduction.     

Intracellular pH on Protein Kinase C and Ionomycin Potentiation of Isoproterenol-Stimulated Cyclic AMP and Cyclic GMP Production in Rat Pinealocytes

In rat pinealocytes, alpha 1-adrenergic activation, which leads to cytoplasmic alkalinization, also potentiates the beta-adrenergic stimulated cyclic AMP (cAMP) and cyclic GMP (cGMP) responses. Both elevation of intracellular calcium ([Ca2+]i) and activation of protein kinase C are involved in the potentiation mechanism. Recently, intracellular pH has also been found to modulate the adrenergic-stimulated cyclic nucleotide responses, suggesting intracellular pH may also affect the potentiation mechanism. This possibility was examined in the present study. Cytoplasmic alkalinization by ammonium chloride had an enhancing effect on the isoproterenol and ionomycin-stimulated cAMP and cGMP accumulation. In comparison, cytoplasmic acidification by sodium propionate reduced the isoproterenol and ionomycin-stimulated cAMP and cGMP responses. Direct measurement of [Ca2+]i indicated that neither ammonium chloride nor sodium propionate had an effect on the ionomycin-stimulated elevation of [Ca2+]i, suggesting their effects on cyclic nucleotide responses may be independent of [Ca2+]i. In cells stimulated by isoproterenol and an activator of protein kinase C, ammonium chloride had an enhancing effect on both cAMP and cGMP responses, whereas sodium propionate had no effect. Taken together, these results suggest that a site distal to elevation of [Ca2+]i and activation of protein kinase C, of importance to the potentiation mechanism, is modulated by intracellular pH.     

Suramin Induces Phosphorylation of the High-Affinity Nerve Growth Factor Receptor in PC12 Cells and Dorsal Root Ganglion Neurons

Abstract: Suramin is a polysulfonated naphthylurea with demonstrated antineoplastic activity. Toxicity includes adrenal insufficiency and peripheral neuropathy. Although the mechanism of antitumor activity is unknown, inhibition of binding of growth factors to their receptors has been suggested. Growth factors inhibited by suramin include platelet-derived growth factor, fibroblast growth factor, transforming growth factor, epidermal growth factor, insulin-like growth factor, and nerve growth factor (NGF). In these studies, suramin was shown to be cytotoxic to PC12 cells in a dose-dependent manner. At lower doses and in surviving cells, we observed the induction of neurite outgrowth. To determine the mechanism of suramin-induced neurite outgrowth, PC12 cells were exposed to suramin and/or NGF for various time periods and treated cells were analyzed, by western blot analysis, for expression of tyrosine phosphoproteins. There was a similarity in the pattern of tyrosine-phosphorylated proteins in PC12 cells stimulated with suramin or NGF. Of particular interest was the rapid phosphorylation (by 1 min) of the high-affinity NGF (TrkA) receptor. Activation of other members of the signal-transduction cascade (Shc, p21 ^ras , Raf-1, ERK-1) revealed similar phosphorylation levels induced by suramin and NGF. Parallel studies were performed in rat dorsal root ganglion cultures; suramin potentiated neurite outgrowth and activated the NGF receptor on these cells. This finding of specific patterns of tyrosine phosphorylation of cellular proteins in response to suramin treatment demonstrated that suramin is a partial agonist for the NGF receptor in both PC12 cells and dorsal root ganglion neurons.     

Interferon Suppresses Sympathetic Neuronal Cell Death Caused by Nerve Growth Factor Deprivation

Cultured rat sympathetic neurons die within 48 h after being deprived of nerve growth factor. Addition of interferons (IFN-alpha/beta or IFN-gamma) prevented the cell death in a dose-dependent manner. Upon longer periods of nerve growth factor deprivation, IFNs failed to maintain survival. Thus, IFNs retarded neuronal death, but did not prevent it. Ligand binding, autoradiography, and cross-linking experiments demonstrated the presence of specific IFN-gamma receptors on sympathetic neurons similar to those seen on other cell types. The possible relationships of the death-suppressing actions of IFNs are compared to the mechanisms of the antiviral or antiproliferative actions of IFNs.     

Phorbol Esters Enhance Neurotransmitter-Stimulated Cyclic AMP Production in Rat Brain Slices

The effect of phorbol esters on cyclic AMP production in rat CNS tissue was examined. Using a prelabeling technique for measuring cyclic AMP accumulation in brain slices, it was found that phorbol 12-myristate, 13-acetate (PMA) enhanced the cyclic AMP response to forskolin and a variety of neurotransmitter receptor stimulants while having no effect on second messenger accumulation itself. A short (15-min) preincubation period with PMA was required to obtain maximal enhancement, whereas the augmentation was lessened by prolonged exposure (3 h) to the phorbol. The response to PMA was concentration dependent (EC50 = 1 microM) and regionally selective, being most apparent in forebrain, and was not influenced by removal of extracellular calcium or by inhibition of phosphodiesterase or phospholipase A2. Only those phorbols known to stimulate protein kinase C augmented the accumulation of cyclic AMP. Moreover, the membrane substrates phosphorylated by endogenous C kinase and by a partially purified preparation of this enzyme were similar. The results suggest that phorbol esters, by activating protein kinase C, modify the cyclic AMP response to brain neurotransmitter receptor stimulation in brain by influencing a component of the adenylate cyclase system beyond the transmitter recognition site.     

Ceramide Prevents Neuronal Programmed Cell Death Induced by Nerve Growth Factor Deprivation

Abstract: We examined the ability of ceramide and sphingomyelinase (SMase) to prevent neuronal programmed cell death (PCD). We found that a cell-permeable ceramide analogue prevented neuronal PCD when applied to established sympathetic neuron primary cultures at the time of nerve growth factor (NGF) deprivation. Other amphiphilic lipids such as oleic acid failed to prevent cell death. Exogenous SMase also showed the same effect, probably by raising the intracellular ceramide level by sphingomyelin (SM) breakdown. Phosphocholine, another hydrolytic product of SM by SMase, did not prevent cell death. Other phospholipases, such as phospholipase C and phospholipase A₂, could not prevent cell death. Given the recent findings that the SM cycle is activated to increase the intracellular ceramide level on NGF binding to the low-affinity NGF receptor (LNGFR) and that NGF binding to LNGFR suppresses apoptosis in neural cell lines, our results suggest the possibility of the SM cycle as a signaling mechanism transducing the PCD-preventing activity of NGF.     

Expression of Nerve Growth Factor and Nerve Growth Factor Receptor Genes in Human Tissues and in Prostatic Adenocarcinoma Cell Lines

Nerve growth factor (NGF) mRNAs were detected and quantified in a variety of normal and neoplastic human tissues by northern blot hybridization. Human heart contained the highest NGF mRNA levels, whereas lower but comparable levels were found in the placenta, prostate, and kidney. All tissues examined coexpressed the low-affinity NGF receptor (LNGFR), whereas none of these tissues expressed the high-affinity NGF receptor encoded by the trk protooncogene. The widespread distribution of the LNGFR suggests that it plays a role in the regulation of normal cell growth. No overexpression of NGF or LNGFR mRNA was detected in neoplastic tissues, whereas LNGFR-like immunoreactivity was localized outside of tumor cells. Transforming growth factor-alpha and protooncogene c-fos expression in these tissues did not show a systematic correlation with NGF/LNGFR expression. Furthermore, regulation of the human NGF gene was studied in DU145 cells, a prostatic adenocarcinoma cell line that synthesizes significant NGF mRNA levels. Serum induced, whereas dexamethasone inhibited, NGF mRNA synthesis in these cells. Serum induction was preceded by a rapid and transient activation of the c-fos protooncogene.     

K-252b Is a Selective and Nontoxic Inhibitor of Nerve Growth Factor Action on Cultured Brain Neurons

K-252b is a kinase inhibitor structurally related to K-252a, which is known to abolish selectively the effects of nerve growth factor (NGF) on PC12 cells and PNS neurons. We tested whether K-252b, K-252a, and staurosporine, another related compound, are effective and selective inhibitors of NGF actions on CNS neurons. All three compounds, at appropriate concentrations, completely and selectively prevented the NGF-mediated activity increase of the cholinergic marker enzyme choline acetyltransferase in cultures of rat basal forebrain cells. The stimulatory effects of basic fibroblast growth factor and insulin on choline acetyltransferase in these cultures and on dopamine uptake in cultures of dissociated ventral mesencephalon were not affected. No signs of toxicity were observed in cultures treated with K-252b. In contrast, K-252a and staurosporine, at concentrations required to block the NGF actions on cholinergic cells, were cytotoxic and produced cell loss. In addition, K-252a, at higher concentrations and in the absence of growth factors, increased cell numbers. Our study suggests that K-252b is a selective and nontoxic inhibitor of NGF actions in the brain and may become a useful tool to study these actions in vivo.     

Multiple Signalling Pathways Interact in the Regulation of Nerve Growth Factor Production in L929 Fibroblasts

Fibroblasts are one of several cell types producing nerve growth factor (NGF) in neuronal targets. In previous studies we found that NGF production is up-regulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) and serum, down-regulated by corticosterone, and unaffected by dibutyryl-cyclic AMP (db-cyclic AMP) in fibroblasts. As fibroblasts in vivo are likely to be exposed to regulatory effects by more than one of these agents at any given time, we examined the effects of combinations of them on NGF production using L929 fibroblasts as a model system. TPA and serum together stimulated NGF production 10-fold more than either agent alone. Corticosterone reduced NGF mRNA and NGF production to less than 10% of basal levels whether or not TPA or serum, or both, were present but not in the presence of the glucocorticoid antagonist RU486. Corticosterone did not increase the rate of NGF mRNA degradation. Forskolin and db-cyclic AMP prevented NGF mRNA induction by TPA and serum without changing basal levels. TPA induced c-fos and junB mRNAs transiently and preceding NGF mRNA induction but c-jun mRNA remained undetectable. Forskolin enhanced the induction of both junB and c-fos mRNA whereas corticosterone prolonged junB mRNA induction. Thus, TPA induction of NGF mRNA is modulated differentially by corticosterone and cyclic AMP. c-fos and junB may play a role in the underlying mechanisms.     

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)     

Acute Regulation of the Epidermal Growth Factor Receptor in Response to Nerve Growth Factor

PC12 cells possess specific receptors for both nerve growth factor and epidermal growth factor, and by an unknown mechanism, nerve growth factor is able to attenuate the propagation of a mitogenic response to epidermal growth factor. The differentiation response of PC12 cells to nerve growth factor, therefore, predominates over the proliferative response to epidermal growth factor. We have observed that the addition of nerve growth factor to PC12 cells rapidly produces a decrease in surface 125I-epidermal growth factor binding capacity. Unlike previously described nerve growth factor effects on 125I-epidermal growth factor binding capacity, which required several days of nerve growth factor exposure, the decreases we report occur within minutes of nerve growth factor addition: A 50% decrease in 125I-epidermal growth factor binding capacity is evident at 10 min. This rapid nerve growth factor response is concentration dependent; inhibition of 125I-epidermal growth factor binding is detectable at nerve growth factor levels as low as 0.2 ng/ml and is maximal at approximately 50 ng/ml, consistent with known ranges of biological activity. No demonstrable differences in the rate of epidermal growth factor receptor synthesis or degradation were observed in cells acutely exposed to nerve growth factor. Scatchard analysis revealed that acute nerve growth factor treatment decreased the number of both high- and low-affinity 125I-epidermal growth factor binding sites, while the receptor affinity remained unchanged. We have also investigated the involvement of various potential intracellular mediators of nerve growth factor action and of known intracellular modulatory systems of the epidermal growth factor receptor for their capacity to participate in this nerve growth factor activity.     

Pituitary Adenylate Cyclase-Activating Polypeptide: Control of Rat Pineal Cyclic AMP and Melatonin but Not Cyclic GMP

Abstract: In this study, the effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on cyclic nucleotide accumulation and melatonin (MT) production in dispersed rat pinealocytes were measured. Treatment with PACAP (10⁻⁷ M ) increased MT production 2.5-fold. PACAP (10⁻⁷ M ) also increased cyclic AMP accumulation four- to fivefold; this effect was potentiated two- to three-fold by α₁-adrenergic activation. This potentiation appears to involve protein kinase C (PKC) because α₁-adrenergic activation is known to translocate PKC and the PACAP-stimulated cyclic AMP accumulation was potentiated ninefold by a PKC activator, 4β-phorbol 12-myristate 13-acetate (PMA). Phenylephrine and PMA also potentiated the PACAP-stimulated MT accumulation. These results indicate that cyclic AMP is one second messenger of PACAP in the pineal gland and that the effects of PACAP on cyclic AMP and MT production can be potentiated by an α₁-adrenergic → PKC mechanism. In addition to these findings, it was observed that PACAP treatment with or without phenylephrine or PMA did not alter cyclic GMP accumulation. This indicates that PACAP is the first ligand identified that increases cyclic AMP accumulation in the pineal gland without increasing cyclic GMP accumulation. That PACAP fails to activate the vasoactive intestinal peptide/cyclic GMP pathway suggests that the vasoactive intestinal peptide receptors present in the pineal may be distinct from the type II PACAP receptors.     

Cyclic AMP stimulates dephosphorylation of specific proteins in intact S49 mouse lymphoma cells

Two-dimensional gel electrophoresis of proteins labeled with ³²P₁ in S49 mouse lymphoma cells revealed five phosphoproteins that were rapidly and reversibly dephosphorylated in response to elevation of cyclic AMP (cAMP). Under basal conditions, labeling of at least two of these proteins was limited by slow turnover of protein-bound phosphate. The rapid cAMP-mediated dephosphorylation of these species was attributable, therefore, to stimulation of a specific phosphoprotein phosphatase.