Prolactin-induced cell proliferation in PC12 cells depends on JNK but not ERK activation.

The effects of pituitary and extrapituitary prolactin include cellular proliferation and differentiation. PC12 cells was used as a model to delineate respective signaling of prolactin. Prolactin acted as a mitogen for undifferentiated PC12 cells, as measured by significant increases in bromodeoxyuridine incorporation and in cell numbers, with an efficacy equal to epidermal growth factor. Both the long and short form of the prolactin receptor was expressed, yet only the long isoform was tyrosine-phosphorylated upon agonist binding. Functional prolactin receptor signaling was further demonstrated in the activation of JAK2 and phosphorylation activation of the transcription factors Stat1, -3, and -5a. Surprisingly, prolactin stimulated a sustained activation of Raf-B, without activation of the MAP kinases ERK1 or -2. Instead, in solid phase kinase assays using a glutathione S-transferase-c-Jun fusion protein (amino acids 1-79) as the substrate, a significant activation of the mitogen-activated protein Janus kinase (c-Jun N-terminal kinase; JNK) was observed. The prolactin-induced activation of JNK was prolonged and accompanied by a significant increase in c-Jun mRNA abundance and c-Jun protein synthesis. Moreover, analysis of bromodeoxyuridine incorporation at the single cell level revealed that epidermal growth factor-dependent incorporation was inhibited by PD98059 and independent of SB203580, whereas prolactin-induced incorporation was ERK and mitogen-activated protein kinase p38 independent but was abolished with JNK inhibition by 30 microm SB203580. Our studies suggest that prolactin may have a role in the growth of PC12 cells, where it stimulates concurrent mitogenic and differentiation-promoting signaling pathways.

Differential regulation of phenylethanolamine N-methyltransferase expression in two distinct subpopulations of bovine chromaffin cells.

Chromaffin cells were isolated from bovine adrenal glands and fractionated into two distinct subpopulations by density gradient centrifugation on Percoll. Cells in the more dense fraction stored epinephrine (E) as their predominant catecholamine (81% of total catecholamines), contained high levels of phenylethanolamine N-methyltransferase (PNMT) activity, and exhibited intense PNMT immunoreactivity. This population of chromaffin cells was termed the E-rich cell population. Cells in the less dense fraction, the norepinephrine (NE)-rich cell population, stored predominantly NE (75% of total catecholamines). Although the NE-rich cells had only 3% as much PNMT activity as did the E-rich cells, 20% of the NE-rich cells were PNMT immunoreactive. This suggested that the PNMT-positive cells in the NE-rich cell cultures contained less PNMT per cell than did E-rich cells and may not be typical adrenergic cells. The regulation of PNMT mRNA levels and PNMT activity in primary cultures of E-rich and NE-rich cells was compared. At the time the cells were isolated, PNMT mRNA levels in NE-rich cells were approximately 20% of those in E-rich cells; within 48 h in culture, PNMT mRNA in both populations declined to almost undetectable levels. Treatment with dexamethasone increased PNMT mRNA levels and PNMT activity in both populations. In E-rich cells, dexamethasone restored PNMT mRNA to the level seen in freshly isolated cells and increased PNMT activity twofold. In NE-rich cells, dexamethasone increased PNMT mRNA to levels twice those found in freshly isolated cells and increased PNMT activity sixfold. Cycloheximide blocked the effects of dexamethasone on PNMT mRNA expression in NE-rich cells but had little effect in E-rich cells. Angiotensin II, forskolin, and phorbol 12,13-dibutyrate elicited large increases in PNMT mRNA levels in E-rich cells but had no effect in NE-rich cells. Our data suggest that PNMT expression is regulated differently in the two chromaffin cell subpopulations.

Differences in the composition of chromaffin granules in adrenaline and noradrenaline containing cells of bovine adrenal medulla.

Several constituents of chromaffin granules were quantitatively determined in noradrenaline and adrenaline cells purified from bovine adrenal medulla. As far as secretory peptides are concerned noradrenaline granules contained slightly more secretogranin II, but much less chromogranin A than adrenaline granules. This can be explained by the dependence of the biosynthesis of chromogranin A on corticosteroids. Proteolytic processing of chromogranin A and secretogranin II was higher in noradrenaline cells which was paralleled by a higher content of the prohormone convertase PC2. Noradrenaline granules also contained a higher concentration of the vesicular monoamine transporter (vMAT2). No differences were found for dopamine beta-hydroxylase, prohormone convertase PC1, carboxypeptidase H and synaptophysin. These results indicate that the secretory cocktail of peptides released from these cells differs significantly between adrenaline and noradrenaline storing cells.

Inhibition of lymphocyte activation by catecholamines: evidence for a non-classical mechanism of catecholamine action.

The effects of noradrenaline and other adrenergic agonists on lymphocyte activation were studied. Spleen and thymus cells from BALB/c mice were stimulated by mitogens and lymphocyte activation was monitored by measuring the incorporation of [methyl-3H]thymidine into DNA. Noradrenaline, adrenaline, isoproterenol and dopamine all inhibited the activation of spleen and thymus cells by concanavalin A, a T-cell specific mitogen, and the activation of spleen cells by lipopolysaccharide, a T-independent B-cell mitogen. The various catecholamines were approximately equipotent, having IC50 of approximately 10 microM. alpha-adrenergic agonists (phenylephrine, clonidine) did not inhibit lymphocyte activation. Noradrenaline, adrenaline and isoproterenol also inhibited DNA synthesis in S49 T lymphoma cells. The effects of adrenergic receptor antagonists on lymphocyte function were also studied. The inhibition of lymphocyte activation by catecholamines could not be reversed by antagonists to beta-adrenergic receptors (propranolol), alpha-adrenergic receptors (phentolamine), or dopaminergic receptors (haloperidol). Experiments with human peripheral blood leucocytes revealed that, as with murine cells, the beta-adrenergic antagonists propranolol and nadalol did not affect the catecholamine-mediated inhibition of lymphocyte activation. Although lymphocytes contain beta-adrenergic receptors that are coupled to adenylyl cyclase activity, catecholamines appear to inhibit murine lymphocyte activation by a mechanism that is independent of these or other classical adrenergic receptors.

Neurotransmitter suppression of the in vitro generation of a cytotoxic T lymphocyte response against the syngeneic MOPC-315 plasmacytoma.

We have previously shown that, as a consequence of low-dose melphalan (L-phenylalanine mustard (L-PAM) therapy, the hitherto immunosuppressed spleen cells from BALB/c mice bearing a large MOPC-315 tumor (in contrast to spleen cells from normal mice) acquire the ability to generate a greatly enhanced anti-MOPC-315 cytotoxic T lymphocyte (CTL) response upon in vitro stimulation with MOPC-315 tumor cells. Here we show that the catecholamines norepinephrine, epinephrine, and isoproterenol suppressed the in vitro generation of anti-MOPC-315 cytotoxicity by spleen cells from mice that had just completed the eradication of a large MOPC-315 tumor following low-dose L-PAM therapy (L-PAM TuB spleen cells), as well as by spleen cells from normal mice. In contrast to the marked suppression obtained with catecholamines, the cholinergic agonist carbachol had no effect on the in vitro generation of splenic anti-MOPC-315 cytotoxicity. The inhibitory effect of the catecholamines was "mimicked" by the membrane penetrating analog of cAMP, dibutyryl-cAMP, and by cholera toxin at concentrations that stimulate the endogenous production of cAMP. The beta-adrenergic receptor antagonist propranolol did not block norepinephrine-induced inhibition of the generation of anti-MOPC-315 cytotoxicity by either normal or L-PAM TuB spleen cells. Since the curative effectiveness of low-dose L-PAM therapy for MOPC-315 tumor bearers requires the participation of CD8+ T cells that exploit a CTL response in tumor eradication, it is conceivable that norepinephrine may reduce the therapeutic outcome of low-dose chemotherapy by inhibiting the acquisition of CTL activity.

Glucocorticoids enhance histamine-evoked catecholamine secretion from bovine chromaffin cells.

The synthetic glucocorticoid dexamethasone enhanced histamine-evoked catecholamine secretion from cultured bovine chromaffin cells. Dexamethasone enhanced the effects of histamine on both adrenergic (epinephrine-rich) and noradrenergic (norepinephrine-rich) chromaffin cells but had a more dramatic effect on noradrenergic cells. Histamine-evoked secretion in noradrenergic cells appeared to become rapidly inactivated, whereas the rate of secretion in adrenergic cells was nearly constant for up to 2 h; dexamethasone treatment attenuated the inactivation seen in noradrenergic cells. The effect of dexamethasone appeared after a lag of several hours and was maximal by 24 h. The EC50 for dexamethasone was approximately 1 nM. The effect of dexamethasone was mimicked by the glucocorticoid agonist RU 28362 and was blocked by the antagonist RU 38486, indicating that the effects of these steroids were mediated by the glucocorticoid or type II corticosteroid receptor. Histamine-evoked catecholamine secretion in both dexamethasone-treated and untreated cells was blocked by the H1 histamine receptor antagonist mepyramine but was not affected by the H2 antagonist cimetidine; thus, dexamethasone appeared to enhance an H1 receptor-mediated process. In the absence of glucocorticoids, H1 receptor mRNA levels were higher in adrenergic than in noradrenergic cells. Dexamethasone increased H1 receptor mRNA levels in both cell types. The increased expression of H1 receptors presumably contributes to the enhancement of histamine-evoked catecholamine secretion by glucocorticoids. Glucocorticoids may play a physiological role in modulating the responsiveness of chromaffin cells to histamine and other stimuli.

1,25(OH)2 vitamin D3 activates PKC-alpha in Caco-2 cells: a mechanism to limit secosteroid-induced rise in [Ca2+]i.

Our laboratory recently reported that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly increases the breakdown of membrane phosphoinositides, raises intracellular calcium concentration ([Ca2+]i), and translocates protein kinase C (PKC) from the cytosolic to the particulate fraction of Caco-2 cells. In the present experiments, we found that Caco-2 cells contained predominantly the alpha- and zeta-isoforms of PKC, with minimally detectable amounts of PKC-beta and -epsilon by Western blotting. 1,25(OH)2D3 and the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) each caused time-dependent translocations of PKC-alpha, but not PKC-zeta. TPA treatment of these cells for 24 h induced a significant concentration-dependent downregulation of PKC-alpha, but not PKC-zeta. Since PKC inhibits phospholipase C-induced mobilization of Ca2+ in other cells, we examined the effects of staurosporine and H-7, PKC inhibitors, and TPA on 1,25(OH)2D3-stimulated increase in [Ca2+]i. As previously demonstrated by our laboratory, 1,25(OH)2D3 caused a biphasic increase in [Ca2+]i, with an initial elevation (transient phase) followed by a sustained increase (plateau phase). We previously demonstrated that the transient phase is mediated, at least in part, by an increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] stimulated by the secosteroid. Acute pretreatment with staurosporine or H-7 caused a significant stimulation, whereas acute TPA pretreatment caused a significant inhibition of the 1,25(OH)2D3-induced increase in the transient phase of [Ca2+]i. Preincubation of Caco-2 cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxy-methyl ester (BAPTA-AM) abolished both the rise in [Ca2+]i and the increase in particulate-associated PKC-alpha stimulated by 1,25(OH)2D3. Moreover, downregulation of PKC-alpha by chronic TPA treatment significantly augmented the transient phase of the 1,25(OH)2D3-stimulated rise in [Ca2+]i but had no effect on the 1,25(OH)2D3-induced change in Ins(1,4,5)P3 concentration. Furthermore, in these PKC-alpha downregulated cells staurosporine no longer increased the secosteroid-stimulated transient rise in [Ca2+]i. These results indicate that 1,25(OH)2D3, which increases [Ca2+]i and diacylglycerol, activates PKC-alpha, but not PKC-zeta. The alpha-isoform, in turn, limits the secosteroid-stimulated rise in [Ca2+]i, at a step distal to Ins(1,4,5)P3 accumulation in Caco-2 cells.

Tetraethylammonium selectively stimulates secretion from noradrenergic bovine chromaffin cells.

1. The effects of tetraethylammonium chloride (TEA) on catecholamine secretion from primary cultures of noradrenaline-rich (noradrenergic) and adrenaline-rich (adrenergic) bovine chromaffin cells were studied. TEA stimulated catecholamine secretion from both cell types but was a much more effective secretory stimulus for noradrenergic cells. 2. TEA-induced catecholamine secretion was dependent on extracellular Ca2+, was partially inhibited by nifedipine and by tetrodotoxin, and was potentiated by ouabain. Other K+ channel blocking agents including 4-aminopyridine, glibenclamide, and tolbutamide did not stimulate catecholamine secretion. 3. TEA had no effect on Ca(2+)-induced secretion from digitonin-permeabilized chromaffin cells. 4. TEA presumably evokes secretion by inhibiting K+ channels, depolarizing chromaffin cells, and activating voltage-gated Ca2+ channels in the cells. Noradrenergic cells appear to be more sensitive to K+ channel inhibition than are adrenergic cells. 5. The secretory response of the chromaffin cells to TEA increased with time in culture. 6. In addition to being a more effective secretagogue in noradrenergic cells, TEA was also more effective in stimulating catecholamine synthesis in these cells.

Histamine evokes greater increases in phosphatidylinositol metabolism and catecholamine secretion in epinephrine-containing than in norepinephrine-containing chromaffin cells.

Chromaffin cells have H1 histamine receptors. Histamine, acting at these receptors, increases the metabolism of inositol-containing phospholipids and stimulates catecholamine secretion from chromaffin cells. We have investigated the effects of histamine and other agents on the accumulation of inositol monophosphate (InsP1) and catecholamine secretion in purified cultures of norepinephrine-containing and epinephrine-containing bovine chromaffin cells. Histamine-stimulated InsP1 accumulation in epinephrine cells was three times greater than that in norepinephrine cells. In contrast, bradykinin caused roughly equivalent increases in InsP1 accumulation in the two chromaffin cell subtypes. Histamine-stimulated catecholamine secretion was also greater in epinephrine cells than in norepinephrine cells, whereas high K+, bradykinin, phorbol 12,13-dibutyrate, and angiotensin II all caused greater secretion from norepinephrine cells than from epinephrine cells. The density of H1 receptors in epinephrine cells was approximately three times greater than that in norepinephrine cells. The greater density of H1 receptors on epinephrine cells may account for the greater effects of histamine on InsP1 accumulation and catecholamine secretion in these cells.

Staurosporine activates a 60,000 M(r) protein kinase in bovine chromaffin cells that phosphorylates myelin basic protein in vitro.

Bovine chromaffin cells contain a family of renaturable protein kinases. One of these, a 60,000 M(r) kinase (PK60) that phosphorylated myelin basic protein in vitro, was activated fourfold when cells were treated with the protein kinase inhibitor staurosporine. Because staurosporine inhibits protein kinase C, the role of this kinase in the regulation of PK60 activity was investigated. Fifty nanomolar staurosporine produced half-maximal inhibition of protein kinase C activity in chromaffin cells, whereas approximately 225 nM staurosporine was required to induce half-maximal activation of PK60. Other protein kinase C inhibitors, H-7 and K-252a, did not mimic the effect of staurosporine on PK60 activity. Chromaffin cells have three protein kinase C isoforms: alpha, epsilon, and zeta. Prolonged treatment with phorbol esters depleted the cells of protein kinase C alpha and epsilon, but not zeta. Neither activation nor depletion of protein kinase C affected the basal activity of PK60. Moreover, staurosporine activated PK60 in cells depleted of protein kinase C alpha and epsilon; thus, staurosporine appeared to activate PK60 by a mechanism that does not require these protein kinase C isoforms. Incubation of cell extracts with staurosporine in vitro did not activate PK60. Incubation of these extracts with adenosine 5'-O-(3-thiotriphosphate), however, caused a twofold activation of PK60. Although this suggests that PK60 activity is regulated by phosphorylation, the mechanism by which staurosporine activates PK60 is not known. Staurosporine has been reported to promote neurite outgrowth from chromaffin cells. The role of PK60 in mediating the effects of staurosporine on chromaffin cell function remains to be determined.

Polyoma-induced neoplasms of the mouse adrenal medulla. Characterization of the tumors and establishment of cell lines.

BACKGROUND: Pheochromocytomas that are usually noradrenergic arise commonly in the adult rat adrenal medulla. The widely studied PC12 cell line, that is representative of these rat adrenal tumors, is also noradrenergic. The reasons for the absence of epinephrine production by most rat pheochromocytoma cells are unknown, and there are currently no adrenergic adrenal medullary cell lines. Pheochromocytomas are rare in mice. EXPERIMENTAL DESIGN: Tumors induced by polyoma virus in the adrenal medullas of postnatal mice were studied immunocytochemically for catecholamine biosynthetic enzymes in order to determine how their profiles of catecholamine production compared with those of rat pheochromocytomas. Clonal cell lines were established from a representative tumor and were evaluated for responsiveness to agents known to affect the development and function of normal and neoplastic rat chromaffin cells. RESULTS: Although adrenal medullary cells from normal rodents produce epinephrine before birth, polyoma-induced mouse adrenal tumor cells are immature or poorly differentiated. They synthesize norepinephrine, but not epinephrine, which during normal development is produced later than norepinephrine. They also produce relatively large quantities of dihydroxyphenylalanine, suggesting an abnormality of catecholamine biosynthesis such that tyrosine hydroxylase is not rate-limiting. Secretory granules are sparse, as demonstrated by electron microscopy or by staining for chromogranin A, and catecholamine stores are low. Further, the tumor cells appear to be phenotypically unstable, as judged from heterogeneous staining for tyrosine hydroxylase even in early passage, twice-cloned cell lines. Tumor cell morphology and catecholamine profiles appear to be unaffected or minimally affected by nerve growth factor, forskolin or dexamethasone, which are known to affect normal or neoplastic rat chromaffin cells. However, tumors formed after subcutaneous injection of cell lines into mice show up to a 10-fold increase in catecholamine stores, suggesting that the cells are subject to some forms of regulation. The cloned cell lines do not produce detectable polyoma virus, but express all three viral T antigens, including a characteristic, truncated form of large T. CONCLUSIONS: The findings suggest that the process of neoplastic transformation and/or the presence of polyoma virus T antigens results in suppression of the adrenergic phenotype in mouse adrenal chromaffin cells. T antigens might therefore be useful as tools for studying mechanisms that regulate the differentiation and maturation of chromaffin cells in normal and neoplastic states. Furthermore, although polyoma virus cannot be readily used to produce adrenergic cell lines from the mouse adrenal medulla, the lines that are produced might substitute for PC12 cells in some types of studies that require a mouse model.

Neuroimmune modulation: signal transduction and catecholamines.

In recent years, much interest has centered on the commonalities and bi-directional interactions between the nervous system and the immune system. This review focuses on mechanisms through which, catecholamines, a class of neuro-endocrine molecules, modulate immune functions. Catecholamines can be immune suppressive and inhibit lymphocyte activation of both T and B cells as well as the generation of immune-mediated anti-tumor responses. Some of these catecholamine-regulated activities appear to be modulated through the second messenger, cyclic AMP, whereas others appear to be catecholamine-dependent but cyclic AMP independent. Further delineation of the interacting ligand-receptor complexes, populations of responding cells and signal transduction mechanisms leading to the activation of specifically involved genes and gene products, will lead to enhanced understanding of the integratory functions of the nervous system in immune responses, the biology of stress, the role of stress-associated molecular mechanisms in perturbations of physiological homeostasis and the development of a new biological psychiatry with accompanying rational therapeutic modalities.

Nicotinic agonists, phorbol esters, and growth factors activate two extracellular signal-regulated kinases, ERK1 and ERK2, in bovine chromaffin cells.

Treatment of bovine chromaffin cells with nicotinic agonists, phorbol esters, and growth factors increases protein kinase activity toward microtubule-associated protein-2 and myelin basic protein (MBP) in vitro. To characterize the kinases that are activated by these agents, we separated chromaffin cell proteins by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels into which MBP had been incorporated, allowed the proteins to renature, and then assayed MBP kinase activity by incubating the gels with [gamma-32P]ATP. Chromaffin cells contain a family of kinases that phosphorylate MBP in vitro. Two of these kinases, of M(r) 46,000 and 42,000 (PK46 and PK42), were activated by treatment of the cells with dimethylphenylpiperazinium (DMPP), phorbol 12,13-dibutyrate (PDBu), or insulin-like growth factor I (IGF-I). Activation of PK46 and PK42 by DMPP was dependent on extracellular Ca2+, whereas the effects of PDBu and IGF-I were Ca2+ independent. Down-regulation of protein kinase C by incubation of the cells with PDBu abolished the activation of PK46 and PK42 by DMPP, PDBu, and IGF-I. Staurosporine, a protein kinase C inhibitor, prevented the activation of PK46 and PK42 by DMPP and PDBu but did not block the activation of these kinases by IGF-I. Immunoblotting experiments with antiphosphotyrosine (anti-PTyr) antibodies demonstrated that agents that increased the kinase activities of PK46 and PK42 also increased the apparent PTyr content of M(r) 46,000 and 42,000 proteins. PK46 and PK42 comigrated with proteins that reacted with antibodies against extracellular signal-regulated kinases (ERKs). Thus, PK46 and PK42 appear to be the bovine homologues of ERK1 and ERK2.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute and chronic cycle length dependent increase in ventricular pacing threshold.

Several factors have been shown to influence ventricular pacing threshold in humans, including pacing lead location (endocardial vs epicardial), lead maturation, and antiarrhythmic agents. To determine whether ventricular pacing rate has a significant influence on acute and chronic pacing thresholds, we measured pacing thresholds in 16 patients receiving an implantable antitachycardia pacemaker cardioverter defibrillator (Cadence). Ventricular pacing thresholds were determined using the device programmer at cycle lengths of 600, 400 and 300 msec at the time of implantation; prior to hospital discharge at 3-14 days; and during follow-up outpatient visits at 6-8 weeks, 3 months, and 6 months to 1 year. Eleven patients had an epicardial lead system and five an endocardial lead system. Eleven patients were being treated with antiarrhythmic drug therapy. Device output ranged from 1-10 V and was adjustable in 1-V increments (pulse width was held constant at 1 msec). A cycle length dependent increase in pacing threshold (defined as a > or = 1-V increase in threshold at 400 or 300 msec relative to 600 msec) was observed in 10/16 patients during 12/72 pacing trials at 400 msec, and in 15/16 patients during 31/67 trials at 300 msec. In trials in which an increase in pacing threshold occurred, the magnitude of the increase at 400 msec relative to 600 msec was only 1 V in all 12 trials, but at 300 msec the increase ranged from 4-9 V in 7/31 (23%) trials.(ABSTRACT TRUNCATED AT 250 WORDS)

Voltage-dependent phosphorylation may recruit Ca2+ current facilitation in chromaffin cells.

Bovine chromaffin cells have two components of whole-cell Ca2+ current: 'standard' Ca2+ currents that are activated by brief depolarizations, and 'facilitation' Ca2+ currents, which are normally quiescent but can be activated by large pre-depolarizations or by repetitive depolarizations to physiological potentials. The activation of protein kinase A can also stimulate Ca2+ current facilitation, indicating that phosphorylation can play a part in facilitation. Here we investigate the role of protein phosphorylation in the recruitment of facilitation Ca2+ currents by pre-pulses or repetitive depolarizations. We find that recruitment of facilitation by depolarization is a rapid first-order process which is suppressed by inhibitors of protein phosphorylation or by injection of phosphatase 2A into cells. Recruitment of facilitation Ca2+ current by voltage is normally reversible but phosphatase inhibitors render it irreversible. Our results indicate that recruitment of these Ca2+ currents by pre-pulses or repetitive depolarizations involves voltage-dependent phosphorylation of the facilitation Ca2+ channel or a closely associated regulatory protein. Voltage-dependent phosphorylation may therefore be a mechanism by which membrane potential can modulate ion channel activity.

Mouse hepatitis virus infection suppresses modulation of mouse spleen T-cell activation.

Natural infection by mouse hepatitis virus (MHV) can affect interpretation of immunological studies in mice. MHV, a collective term describing a group of corona viruses, is found in natural infections in over 70% of laboratory mouse populations in the U.S.A. and Canada. Natural outbreaks of MHV in our animal colony afforded us the opportunity to study MHV-induced immunosuppression as well as the effects of MHV infection on neurotransmitter immunomodulation. Concanavalin A (Con A)-stimulated DNA synthesis by spleen T lymphocytes from MHV-infected mice was 20-50% that of non-infected mice. The MHV infection also altered neurotransmitter modulation of spleen T-lymphocyte activation. In contrast to noradrenaline ablation of Con A-activated DNA synthesis by spleen lymphocytes from non-infected mice, DNA synthesis by the infected group was not inhibited by noradrenaline or dibutyryl-cAMP. These effects of MHV infection were specific for spleen T lymphocytes since MHV infection did not alter Con A stimulation of thymocytes, lipopolysaccharide stimulation of spleen B lymphocytes, or noradrenaline inhibition of thymocyte and B-cell DNA synthesis. MHV infection also did not alter spleen T-lymphocyte subset proportions. Thus, MHV infection inhibits spleen T-lymphocyte activation and blocks in vitro catecholamine and cAMP regulation of spleen T-cell activation but does not affect activation of thymic cells or spleen B cells.

Phorbol esters cause preferential secretion of norepinephrine from bovine chromaffin cells.

Differential secretion of norepinephrine and epinephrine was studied in cultured bovine chromaffin cells. Nicotinic agonists and 55 mM K+ evoked a slightly greater release of norepinephrine than of epinephrine: The percentage of norepinephrine secreted was 1.5 to two times greater than the percentage of epinephrine secreted. In contrast, when the cells were treated with phorbol 12,13-dibutyrate, the percentage of norepinephrine released was six to eight times greater than that of epinephrine released. Similar results were obtained in experiments with cultures highly enriched in either norepinephrine-containing cells or epinephrine-containing cells. In response to 55 mM K+, catecholamine release from norepinephrine-containing cells was two times greater than that from epinephrine-containing cells. In response to phorbol 12,13-dibutyrate, secretion from norepinephrine-containing cells was 13 times greater than that from epinephrine-containing cells. These results suggest that protein kinase C plays a specific role in the regulation of catecholamine secretion from norepinephrine-containing cells.

Omega-conotoxin GVIA blocks a Ca2+ current in bovine chromaffin cells that is not of the "classic" N type.

Previous studies have identified two components of whole-cell Ca2+ current in bovine chromaffin cells. The "standard" component was activated by single depolarizations, while "facilitation" could be activated by large prepulses or repetitive depolarizations. Neither current component was sensitive to changes in holding potential between -100 and -50 mV; thus neither appeared to be carried by N-type Ca2+ channels. We now report that the facilitation Ca2+ current is insensitive to omega-conotoxin GVIA (omega-CgTx), but that the toxin blocks approximately 50% of the standard Ca2+ current. In some cells the toxin blocks all of the standard Ca2+ current, in others about half of the current, while in others it has no effect. Kinetic differences in current activation are observed after toxin application. These results suggest that the standard component of chromaffin cell Ca2+ current is composed of two pharmacologically distinct channels-one is omega-CgTx sensitive and the other is not. Two kinetically distinct types of 14 pS Ca2+ channels that may correspond to the omega-CgTx-sensitive and -insensitive components were observed in single-channel experiments. Because omega-CgTx blocked Ca2+ channels that were not inactivated by a depolarized holding potential, the commonly used Ca2+ channel categorization scheme may be inadequate to describe the Ca2+ channels found in chromaffin cells.

Activation of a microtubule-associated protein-2 kinase by insulin-like growth factor-I in bovine chromaffin cells.

Treatment of bovine chromaffin cells with insulin-like growth factor-I (IGF-I) caused the activation of a protein kinase that phosphorylates microtubule-associated protein-2 (MAP-2) in vitro. Activation of MAP-2 kinase by IGF-I varied with the time of treatment (maximal at 10-15 min) and the concentration of IGF-I (maximal at 10 nM). The IGF-I-activated MAP-2 kinase was localized to the soluble fraction of chromaffin cell extracts and required Mg2+ for activity. The IGF-I-activated kinase also phosphorylated myelin basic protein, but had little or no activity toward histones or ribosomal S6 protein. To examine the role of protein tyrosine phosphorylation in the activation of the MAP-2 kinase, we isolated phosphotyrosine (PTyr)-containing proteins from chromaffin cells by immunoaffinity adsorption on anti-PTyr-Sepharose beads. Anti-PTyr-Sepharose eluates from IGF-I-treated cells showed increased MAP-2 kinase activity; thus, the MAP-2 kinase (or a closely associated protein) appears to be a PTyr-containing protein. Treatment of anti-PTyr-Sepharose eluates or crude chromaffin cell extracts with alkaline phosphatase significantly decreased kinase activity toward myelin basic protein, indicating that phosphorylation of the IGF-I-activated kinase is required for its activity.

Three types of bovine chromaffin cell Ca2+ channels: facilitation increases the opening probability of a 27 pS channel.

1. Cell-attached patch recordings from bovine chromaffin cells were performed with 90 mM-Ba2+ in the patch pipette and with isotonic potassium aspartate in the bathing solution to zero the membrane potential. Three different types of unitary Ca2+ channel activity could be distinguished in these recordings. 2. A 27 pS Ca2+ channel was distinguished by constructing amplitude histograms and measuring slope conductance. This channel activated over a broad range of potentials (depolarizations greater than -10 mV). 3. A second Ca2+ channel with a slope conductance of 14 pS could also be detected with amplitude histograms. This channel activated with depolarizations greater than -20 mV. 4. An 18 pS Ca2+ channel was observed infrequently indicating that this channel may carry only a small amount of the whole-cell current. This 18 pS channel was sensitive to changes in holding potential. Depolarizing the patch to +10 mV from a holding potential of -80 mV elicited robust unitary activity. Changing the patch holding potential to -40 mV while maintaining test depolarizations to +10 mV completely inactivated the 18 pS channel. Neither the 25 pS nor the 14 pS Ca2+ channels were affected by changes in holding potential in the range from -80 mV to -40 mV, indicating the 18 pS channel was a different type of channel. As the 18 pS channel was observed so infrequently, no detailed studies of it were possible. 5. Chromaffin cell Ca2+ currents exhibited facilitation. Large pre-depolarizations greatly augmented whole-cell currents observed in these cells. Whole-cell currents could double or triple after recruiting facilitation. The application of large pre-depolarizations altered the gating behaviour of the 27 pS Ca2+ channel manifested as dramatically increased channel opening probabilities measured during subsequent test pulses. Large pre-depolarizations induced unitary activity in the 27 pS Ca2+ channel similar to the long-lived openings exhibited by L-type Ca2+ channels in the presence of Bay K 8644. Large pre-depolarizations did not change the gating behaviour of the 14 pS Ca2+ channel. 6. Repetitive depolarizations in the physiological range could also induce facilitation. At the single-channel level facilitation was manifested as a striking increase in opening probability of the 27 pS Ca2+ channel. No effect of repetitive activity was observed on 14 pS channel gating. At the whole-cell level, repetitive depolarizations dramatically increased the current observed. 7. Facilitation of 27 pS Ca2+ channel activity could be induced by changing the holding potential to a depolarized level (greater than or equal to -10 mV).(ABSTRACT TRUNCATED AT 400 WORDS)