Mechanisms of cytosolic Ca2+ suppression by prostaglandin E2 receptors in rat melanotrophs.

We have previously reported that voltage-dependent Ca2+ (VDC) channels of rat melanotrophs are inhibited by prostaglandin E2 (PGE2). In this study, mechanisms involved in the inhibitory actions of PGE2 receptors of rat melanotrophs were analysed using reverse transcriptase-polymerase chain reaction (RT-PCR), Ca2+-imaging and whole-cell, patch-clamp techniques with recently developed EP agonists, each of which is selective for the known four subclasses of EP receptors (EP1-4). PGE2 reversibly suppressed the cytosolic Ca2+ concentration ([Ca2+]i). The maximum reduction in [Ca2+]i by PGE2 was comparable to that by dopamine or to that by extracellular Ca2+ removal. RT-PCR analysis of all four EP receptors revealed that EP3 and EP4 receptor mRNAs were expressed in the intermediate lobe. The effects of PGE2 to suppress [Ca2+]i were mimicked by the selective EP3 agonist, ONO-AE-248, whereas three other EP agonists, ONO-DI-004 (EP1), ONO-AE1-259 (EP2) and ONO-AE1-329 (EP4), had little or no effect on [Ca2+]i. All four G-protein activated inward rectifying K+ (GIRK) channel mRNAs were identified in intermediate lobe tissues by RT-PCR. Dopamine concentration-dependently activated GIRK currents, whereas PGE2 did not activate GIRK currents, even at the concentration causing maximal inhibition of VDC channels. These results suggest that PGE2 acts on EP3 receptors to suppress Ca2+ entry of rat melanotrophs by selectively inhibiting VDC channels of these cells. We have compared the possible cellular and molecular mechanisms of inhibition by dopamine and PGE2.

Production of cAMP by adrenomedullin in human oligodendroglial cell line KG1C: comparison with calcitonin gene-related peptide and amylin.

The actions and the presence of adrenomedullin (AM) were investigated in cultured human oligodendroglial cell line KG1C. AM and AM mRNA were detected in KG1C cells by immunohistochemistry and RT-PCR. mRNAs for calcitonin receptor-like receptor (CRLR) and receptor-activity-modifying proteins (RAMPs) 1, 2 and 3 but not for calcitonin receptors were detected in the cells, while mRNAs for CRLR, calcitonin receptors and all RAMPs were detected in the human cerebellum. Application of AM resulted in time- and concentration-dependent increases in the cAMP level of KG1C cells. Calcitonin gene-related peptide (CGRP) and amylin, peptides structurally related to AM, also increased cAMP. The potencies for the cAMP production of the three peptides were CGRP > or =AM >> amylin with EC(50) of 8, 18, 90 nM, respectively. The responses induced by AM were strongly inhibited by the CGRP(1) receptor antagonist human CGRP(8-37), and inhibited also by the AM receptor antagonist human AM(22-52). In contrast, the responses induced by CGRP or amylin were inhibited only by CGRP(8-37) and not by AM(22-52). The responses induced by all three peptides were unaffected by the amylin receptor antagonist human amylin(8-37). The CGRP(2) receptor agonist human [Cys(Acm)(2,7)]CGRP significantly increased the cAMP level but the increase was smaller than that caused by CGRP. This increase in cAMP was unaffected by CGRP(8-37), AM(22-52) or by amylin(8-37). These results suggest that in KG1C cells, AM increases cAMP through AM and CGRP(1) receptors, whereas CGRP does so through CGRP(1) and CGRP(2) receptors, and amylin exerts its effects through CGRP(1) receptors. Collectively, these findings imply that AM released from oligodendroglial cells may play a role in the regulation of oligodendrocytes via autocrine/paracrine through AM receptors and CGRP(1) receptors.

Dual phases of functional change in norepinephrine transporter in cultured bovine adrenal medullary cells by long-term treatment with clozapine.

The effects of long-term treatment with clozapine, a prototype of atypical antipsychotic drugs, on the functional activity, synthesis and mRNA of norepinephrine (NE) transporter were examined in bovine adrenal medullary cells in culture. Treatment of cells with clozapine at 0.1-3.0 microM concentrations produced dual phases of changes in [(3)H]NE uptake, i.e. the first phase showed a decrease in [(3)H]NE uptake at 2-48 h, and the following phase showed an increase in uptake at 72-168 h. Treatment with clozapine for 6 h decreased V(max) to 40% of the control without changing the K(m) value for [(3)H]NE uptake. However, treatment with clozapine for 96 h increased V(max) by 56% over the control without a change in K(m). Scatchard plot analysis of [(3)H]desipramine (DMI) binding to membranes isolated from cells treated with clozapine for 6 h revealed a decrease in B(max) without any change in K(d); in contrast, treatment with clozapine for 96 h caused an increase in B(max) without any change in K(d). Both actinomycin D and cycloheximide, which are inhibitors of protein synthesis, suppressed the clozapine (96 h)-induced increase in [(3)H]NE uptake. Treatment of cells with clozapine for 12-96 h increased the level of NE transporter mRNA in a concentration-dependent manner (0.3-3.0 microM). These findings suggest that treatment of cells with clozapine results in the down-regulation and subsequent up-regulation of NE transporter. The latter change may be caused by the synthesis of new proteins of NE transporter via an increase in its mRNA.

Heterogeneous increases of cytoplasmic calcium: distinct effects on down-regulation of cell surface sodium channels and sodium channel subunit mRNA levels.

1. Long-term (> or = 12 h) treatment of cultured bovine adrenal chromaffin cells with A23187 (a Ca(2+) ionophore) or thapsigargin (TG) [an inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)] caused a time- and concentration-dependent reduction of cell surface [(3)H]-saxitoxin (STX) binding capacity, but did not change the K:(D:) value. In A23187- or TG-treated cells, veratridine-induced (22)Na(+) influx was reduced (with no change in veratridine EC(50) value) while it was enhanced by alpha-scorpion venom, beta-scorpion venom, or Ptychodiscus brevis toxin-3, like in nontreated cells. 2. The A23187- or TG-induced decrease of [(3)H]-STX binding was diminished by BAPTA-AM. EGTA also inhibited the decreasing effect of A23187. A23187 caused a rapid, monophasic and persistent increase in intracellular concentration of Ca(2+) ([Ca(2+)](i)) to a greater extent than that observed with TG. 2,5-Di-(t-butyl)-1,4-benzohydroquinone (DBHQ) (an inhibitor of SERCA) produced only a rapid monophasic increase in [Ca(2+)](i), without any effect on [(3)H]-STX binding. 3. Reduction in [(3)H]-STX binding capacity induced by A23187 or TG was attenuated by Gö6976 (an inhibitor of conventional protein kinase C) or calpastatin peptide (an inhibitor of calpain). When the internalization rate of cell surface Na(+) channels was measured in the presence of brefeldin A (an inhibitor of vesicular exit from the trans-Golgi network), A23187 or TG accelerated the reduction of [(3)H]-STX binding capacity. 4. Six hours treatment with A23187 lowered Na(+) channel alpha- and beta(1)-subunit mRNA levels, whereas TG had no effect. 5. These results suggest that elevation of [Ca(2+)](i) caused by A23187, TG or DBHQ exerted differential effects on down-regulation of cell surface functional Na(+) channels and Na(+) channel subunit mRNA levels.

Stimulation of catecholamine synthesis in cultured bovine adrenal medullary cells by leptin.

Recently, we characterized leptin receptors in bovine adrenal medullary cells (Yanagihara et al. 2000). Here we report the stimulatory effect of leptin on catecholamine synthesis in the cells. Incubating cells with leptin (10 nM) for 20 min increased the synthesis of 14C-catecholamines from [14C]tyrosine, but not from L-3,4-dihydroxyphenyl [3-14C]alanine. The stimulation of catecholamine synthesis in the cells by leptin was associated with the phosphorylation and activation of tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosynthesis. The incubation of cells with leptin resulted in a rapid activation of the mitogen-activated protein kinases (MAPKs). An inhibitor of MAPK kinase, U0126, nullified the stimulatory effect of leptin on the synthesis of 14C-catecholamines. Leptin potentiated the stimulatory effect of acetylcholine on 14C-catecholamine synthesis, whereas leptin failed to enhance the phosphorylation and activation of tyrosine hydroxylase induced by acetylcholine. These findings suggest that leptin stimulates catecholamine synthesis via the activation of tyrosine hydroxylase by two different mechanisms, i.e., one is dependent on tyrosine hydroxylase phosphorylation mediated through the MAPK pathway and the second is independent of enzyme phosphorylation.

Inhibition by neuromuscular blocking drugs of norepinephrine transporter in cultured bovine adrenal medullary cells.

UNLABELLED: Pancuronium stimulates the cardiovascular system, whereas vecuronium, a derivative of pancuronium, has far fewer effects. The inhibition of norepinephrine transporter (NET) in the sympathetic nervous system may partly account for the stimulatory actions of pancuronium. To investigate the mechanism of action of pancuronium on NET, we examined the effects of pancuronium on NET activity by using cultured bovine adrenal medullary cells and compared pancuronium with other neuromuscular blocking drugs. Pancuronium (1-300 microM) inhibited desipramine-sensitive [(3)H]norepinephrine (NE) uptake in a concentration-dependent manner. Vecuronium (100-300 microM) and d-tubocurarine (300 microM) also decreased [(3)H]NE uptake but were less potent than pancuronium at clinical concentrations. Succinylcholine had little effect on [(3)H]NE uptake. Saturation analysis showed that pancuronium and vecuronium reduced an apparent maximum velocity (V(max)) of [(3)H]NE uptake without altering Michaelis-Menten constant, indicating noncompetitive inhibition. Pancuronium did not inhibit the specific binding of [(3)H]desipramine to plasma membranes isolated from bovine adrenal medulla. A protein kinase C inhibitor, GF109203X, did not affect the inhibition of [(3)H]NE uptake by pancuronium. Pancuronium enhanced the inhibition of NET induced by ketamine. These results suggest that pancuronium, with clinically relevant concentrations, inhibits NET activity by interacting with a site distinct from the recognition site for NE and the desipramine binding site on the transporter. IMPLICATIONS: In this study, pancuronium inhibited norepinephrine uptake and was the most potent of the neuromuscular blocking drugs we tested, including pancuronium, vecuronium, d-tubocurarine, and succinylcholine. Pancuronium may affect the sympathetic nervous system by inhibiting the activity of the presynaptic norepinephrine transporter at clinically relevant concentrations.

Inhibitory effects of clozapine and other antipsychotic drugs on noradrenaline transporter in cultured bovine adrenal medullary cells.

The effects of clozapine and other antipsychotic drugs on noradrenaline (NA) transport were examined in cultured bovine adrenal medullary cells and in transfected Xenopus laevis oocytes expressing the bovine NA transporter. Incubation of adrenal medullary cells with clozapine (30-1000 ng/ml) inhibited desipramine (DMI)-sensitive uptake of [3H]NA in a concentration-dependent manner (IC50=110 ng/ml or 336 nM). Other antipsychotic drugs such as haloperidol, chlorpromazine, and risperidone also decreased [3H]NA uptake (IC50= 144, 220, and 210 ng/ml or 383, 690, and 512 nM, respectively). Eadie-Hofstee analysis showed that clozapine reduced V(max) of uptake of [3H]NA and increased K(m). Furthermore, clozapine inhibited specific binding of [3H]DMI to plasma membranes isolated from bovine adrenal medulla (IC50=48 ng/ml or 146 nM). Scatchard plot analysis of [3H]DMI binding revealed that clozapine decreased both B(max) and K(d). Other antipsychotic drugs, including haloperidol, chlorpromazine, and risperidone, also reduced [3H]DMI binding to the membranes. In transfected Xenopus oocytes expressing the bovine NA transporter, clozapine inhibited [3H]NA uptake in a concentration-dependent manner similar to that observed in adrenal medullary cells. These results suggest that clozapine and haloperidol directly inhibit transport of NA by acting on the site of an NA transporter that influences both substrate transport and binding of tricyclic antidepressants.

Characterization and functional role of leptin receptor in bovine adrenal medullary cells.

We report here the characterization and functional roles of the leptin receptor (ObR) in bovine adrenal medullary cells. The plasma membranes isolated from bovine adrenal medulla showed a single class of specific binding sites of (125)I-leptin with an apparent K(d) of 6.6 nM and B(max) of 62 fmol/mg protein. ObRa but not ObRb mRNA was detected in bovine adrenal medulla by reverse transcriptase-polymerase chain reaction. Incubation of cultured adrenal medullary cells with leptin (3-30 nM) for 20 min resulted in a significant increase in [(14)C]catecholamine synthesis from [(14)C]tyrosine without any change in catecholamine secretion. These findings suggest that leptin stimulates catecholamine synthesis through its receptors in bovine adrenal medullary cells.

Prostaglandin E2 induces Ca2+ release from ryanodine/caffeine-sensitive stores in bovine adrenal medullary cells via EP1-like receptors.

Prostaglandin E2 (PGE2) causes Ca2+ release from intracellular Ca2+ stores and stimulates phosphoinositide metabolism in bovine adrenal medullary cells. These results have been interpreted as PGE2 induces Ca2+ release from inositol trisphosphate (IP3)-sensitive stores. However, we have recently shown that pituitary adenylate cyclase-activating polypeptide (PACAP), bradykinin, and angiotensin II release Ca2+ from caffeine/ryanodine-sensitive stores, although they cause a concomitant increase of intracellular IP3. In light of these results, the mechanism of PGE2-induced Ca2+ release was investigated in the present study. PGE2 dose-dependently caused a transient but consistent Ca2+ release from internal Ca2+ stores. The PGE2-induced Ca2+ release was unaffected by cinnarizine, a blocker of IP3-induced Ca2+ release. By contrast, it was potently inhibited by prior application of caffeine and ryanodine. Although IP3 production in response to PGE2 was abolished by the phospholipase C inhibitor U-73122, Ca2+ release in response to PGE2 was unaffected by U-73122. The PGE2-induced Ca2+ release was unaffected by Rp-adenosine 3',5'-cyclic monophosphothioate, an inhibitor of protein kinase A, and forskolin, a cyclic AMP (cAMP)-elevating agent, did not cause Ca2+ release. The EP1 agonist 17-phenyl-trinorPGE2 and the EP1/EP3 agonist sulprostone mimicked the Ca(2+)-releasing effects of PGE2, whereas the EP2 agonist butaprost or the EP2/EP3 agonist misoprostol caused little or no Ca2+ release. The EP1 antagonist SC-51322 significantly suppressed the Ca2+ release response induced by PGE2, whereas the EP4 antagonist AH-23828B had little effect. These results suggest that PGE2, acting on EP1-like receptors, induces Ca2+ release from ryanodine/caffeine-sensitive stores through a mechanism independent of IP3 and cAMP and that PGE2 may share the same mechanism with PACAP and the other peptide ligands in causing Ca2+ release in bovine adrenal medullary cells.

Modification of low density lipoprotein potentiates its inhibitory effect on catecholamine secretion in cultured bovine adrenal medullary cells.

Low density lipoprotein (LDL) and lipoprotein(a) suppress catecholamine secretion in cultured adrenal medullary cells. Modification of LDL by oxidation or acetylation potentiates various atherogenic actions of LDL. In the present study, we investigated whether the modification of LDL influences catecholamine secretion in cultured bovine adrenal medullary cells. The exposure of LDL to CuSO4 caused a time-dependent oxidation of LDL. Maximal oxidation of LDL was observed after exposure to CuSO4 for 24 h. Native LDL inhibited catecholamine secretion induced by carbachol to 68.5% of control. Oxidized LDL caused further inhibition of carbachol-evoked secretion to 37.6% of control. Acetylated LDL inhibited it to 41.0% of control. There was a good correlation between the extent of LDL oxidation and the inhibition of catecholamine secretion. These results suggest that oxidation or acetylation of LDL augments its inhibitory effect on the secretion of catecholamines. Since catecholamines are a risk factor of atherosclerosis, the inhibitory effect by such modified LDL may be a mechanism inhibiting atherosclerotic progression.

Carbamazepine-induced up-regulation of voltage-dependent Na+ channels in bovine adrenal medullary cells in culture.

Treatment of cultured bovine adrenal medullary cells with carbamazepine (CBZ) for 5 days caused an increase in catecholamine secretion induced by veratridine, an activator of voltage-dependent Na+ channels. However, no increase was stimulated by carbachol, an agonist of nicotinic receptors, or by 56 mM K+, a depolarizing agent that activates voltage-dependent Ca++ channels. CBZ (30 microg/ml) treatment enhanced veratridine-induced catecholamine secretion in a time-dependent manner (increases of 25%, 65% and 70% for 3, 5 and 7 days of treatment, respectively). CBZ treatment (5 days) increased veratridine-induced catecholamine secretion in a concentration-dependent manner (increases of 27%, 36%, 45% and 55% at 10, 15, 20 and 30 microgram/ml of CBZ, respectively). CBZ treatment also increased 22Na+ influx and 45Ca++ influx stimulated by veratridine. The stimulatory effect of CBZ treatment on catecholamine secretion was blocked by either actinomycin D or cycloheximide, an inhibitor of protein synthesis. Additive responses of catecholamine secretion and 22Na+ influx induced by veratridine were associated with combined exposure of the cells to CBZ and dibutyryl cyclic AMP. CBZ treatment (30 microg/ml, 5 days) significantly increased the specific binding of [3H]saxitoxin to cell membranes. A Scatchard analysis of [3H]saxitoxin binding revealed that CBZ increased the Bmax value without any change in the dissociation constant. These findings suggest that CBZ up-regulates the density and activity of voltage-dependent Na+ channels.

Ketamine interacts with the noradrenaline transporter at a site partly overlapping the desipramine binding site.

Effects of the intravenous anaesthetic ketamine on the desipramine-sensitive noradrenaline transporter (NAT) were examined in cultured bovine adrenal medullary cells and in transfected Xenopus laevis oocytes expressing the bovine NAT (bNAT). Incubation (1-3 h) of adrenal medullary cells with ketamine (10-300 microM) caused an increase in appearance of catecholamines in culture medium. Ketamine (10-1000 microM) inhibited desipramine-sensitive uptake of [3H]noradrenaline (NA) (IC50=97 microM). Saturation analysis showed that ketamine reduced Vmax of [3H]NA uptake without changing Km, indicating a non-competitive inhibition. Other inhibitors of NAT, namely cocaine and desipramine, showed a competitive inhibition of [3H]NA uptake while a derivative of ketamine, phencyclidine, showed a mixed type of inhibition. Ketamine (10-1000 microM) also inhibited the specific binding of [3H]desipramine to plasma membranes isolated from bovine adrenal medulla. Scatchard analysis of [3H]desipramine binding revealed that ketamine increased Kd without altering Bmax, indicating a competitive inhibition. In transfected Xenopus oocytes expressing the bNAT, ketamine attenuated [3H]NA uptake with a kinetic characteristic similar to that of cultured adrenal medullary cells. These findings are compatible with the idea that ketamine non-competitively inhibits the transport of NA by interacting with a site which partly overlaps the desipramine binding site on the NAT.

Adrenomedullin increases intracellular Ca2+ and inositol 1,4,5-trisphosphate in human oligodendroglial cell line KG-1C.

The effects of adrenomedullin (AM), a hypotensive peptide, were investigated in cultured human oligodendroglial cell line KG-1C. Human AM increased the intracellular Ca2+ concentration ([Ca2+]i) at concentrations greater than 10(-7) M. Human calcitonin gene-related peptide (CGRP), a peptide structurally related to AM, also increased [Ca2+]i with a potency similar to that of AM. AM increased [Ca2+]i in the absence of extracellular Ca2+. Further, AM increased inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) level in a concentration-dependent manner similar to that of AM-induced [Ca2+]i, suggesting that AM-induced elevation of [Ca2+]i is due to Ca2+ release from Ins(1,4,5)P3-sensitive stores. AM (10(-9) to 10(-6) M) increased cAMP in a concentration-dependent manner. Forskolin also increased cAMP, but did not mimic the [Ca2+]i-raising effect of AM. These findings suggest that functional AM receptors are present in oligodendroglial KG-1C cells and that AM increases [Ca2+]i through a mechanism independent of cAMP.

Activation of inwardly rectifying K+ channels by GABA-B receptors expressed in Xenopus oocytes.

In Xenopus oocytes coinjected with poly(A)+ RNA derived from the rat cerebellum and cRNAs for the cloned G protein-gated inwardly rectifying K+ channel (GIRK), GIRK1 and GIRK2, the GABA-B agonist baclofen elicited inwardly rectifying K+ currents. The inward K+ currents elicited by baclofen were inhibited by the selective GABA-B antagonists 2-OH saclofen and CGP 35348, and by the GIRK inhibitor Ba2+. In contrast, baclofen caused no currents in oocytes injected with the cerebellar poly(A)+ RNA alone, the poly(A)+ RNA and cRNA for GIRK1 or GIRK2, or only cRNAs for GIRK1 and GIRK2. These findings indicate that GABA-B receptors in the rat cerebellum were functionally expressed in Xenopus oocytes and activated the cloned GIRKs composed of GIRK1 and GIRK2 as heteromultimers.

An active metabolite of carbamazepine, carbamazepine-10,11-epoxide, inhibits ion channel-mediated catecholamine secretion in cultured bovine adrenal medullary cells.

We have recently reported inhibitory effects of carbamazepine (CBZ) on ion channel-mediated secretion of catecholamines in bovine adrenal medullary cells. Here, we report the effects of carbamazepine-10,11-epoxide (CBZ-E), an active metabolite of CBZ, and carbamazepine-10,11-diol (CBZ-D), a non-active metabolite, on 22Na+ influx, 45Ca2+ influx and catecholamine secretion in cultured adrenal medullary cells. CBZ-E, but not CBZ-D inhibited 22Na+ influx, 45Ca2+ influx and catecholamine secretion induced by carbachol or veratridine with a half-maximal inhibitory concentration (IC50) of 0.26 or 0.68 microg/ml, respectively. CBZ-E also inhibited high K+-evoked 45Ca2+ influx and catecholamine secretion (IC50 = 0.3 microg/ml), but CBZ-D did not. These findings suggest that CBZ-E, but not CBZ-D, attenuates catecholamine secretion by inhibiting nicotinic acetylcholine receptor-associated ion channels, voltage-dependent Na+ channels and voltage-dependent Ca2+ channels in the cells. This inhibition of CBZ-E as well as CBZ may be related to the clinical effects in neuropsychiatric disorders.

Down-regulation of the noradrenaline transporter by interferon-alpha in cultured bovine adrenal medullary cells.

The aim of this study was to investigate the effect of long-term treatment with interferon (IFN)-alpha on the noradrenaline transporter of bovine adrenal medullary cells. Treatment of cultured adrenal medullary cells with IFN-alpha caused a decrease in uptake of [3H]noradrenaline by the cells in time (4-48 h)- and concentration (300-1,000 U/ml)-dependent manners. IFN-beta also inhibited [3H]noradrenaline uptake to a lesser extent than did IFN-alpha, whereas IFN-gamma had little effect. An anti-IFN-alpha antibody reduced the effect of IFN-alpha on [3H]noradrenaline uptake. Saturation analysis of [3H]noradrenaline uptake showed that the inhibitory effect of IFN-alpha was due to a reduction in the maximal uptake velocity (Vmax) values without altering apparent Michaelis constant (Km) values. Incubation of cells with IFN-alpha caused a translocation of protein kinase C from the soluble to the particulate fraction in the cells. The effect of IFN-alpha on [3H]noradrenaline uptake was diminished in protein kinase C-down-regulated cells. Incubation of cells with IFN-alpha for 48 h significantly reduced the specific binding of [3H]desipramine to crude plasma membranes isolated from cells. Scatchard analysis of [3H]desipramine binding revealed that IFN-alpha decreased the maximal binding (Bmax) values without any change in the dissociation constant (K(D)) values. These findings suggest that IFN-alpha suppresses the function of noradrenaline transporter by reducing the density of the transporter in cell membranes through, at least in part, a protein kinase C pathway.

Pituitary adenylate cyclase-activating polypeptide causes Ca2+ release from ryanodine/caffeine stores through a novel pathway independent of both inositol trisphosphates and cyclic AMP in bovine adrenal medullary cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) causes both Ca2+ release and Ca2+ influx in bovine adrenal chromaffin cells. To elucidate the mechanisms of PACAP-induced Ca2+ release, we investigated expression of PACAP receptors and measured inositol trisphosphates (IP3), cyclic AMP, and the intracellular Ca2+ concentration in bovine adrenal medullary cells maintained in primary culture. RT-PCR analysis revealed that bovine adrenal medullary cells express the PACAP receptor hop, which is known to couple with both IP3 and cyclic AMP pathways. The two naturally occurring forms of PACAP, PACAP38 and PACAP27, both increased cyclic AMP and IP3, and PACAP38 was more potent than PACAP27 in both effects. Despite the effects of PACAP on IP3 production, the Ca2+ release induced by PA-CAP38 or by PACAP27 was unaffected by cinnarizine, a blocker of IP3 channels. The potencies of the peptides to cause Ca2+ release in the presence of cinnarizine were similar. The Ca2+ release induced by PACAP38 or by PACAP27 was strongly inhibited by ryanodine and caffeine. In the presence of ryanodine and caffeine, PACAP38 was more potent than PACAP27. PACAP-induced Ca2+ release was unaffected by Rp-adenosine 3',5'-cyclic monophosphothioate, an inhibitor of protein kinase A. Ca2+ release induced by bradykinin and angiotensin II was also inhibited by ryanodine and caffeine, but unaffected by cinnarizine. Although IP3 production stimulated by PACAP38 or bradykinin was abolished by the phospholipase C inhibitor, U-73122, Ca2+ release in response to the peptides was unaffected by U-73122. These results suggest that PACAP induces Ca2+ release from ryanodine/caffeine stores through a novel intracellular mechanism independent of both IP3 and cyclic AMP and that the mechanism may be the common pathway through which peptides release Ca2+ in adrenal chromaffin cells.

Stimulatory effect of lymphocyte-derived factor on catecholamine efflux from cultured bovine adrenal medullary cells.

The effects of lymphocytes and their conditioned medium on catecholamine efflux and uptake were examined in cultured bovine adrenal medullary cells. Co-culture of adrenal medullary cells with lymphocytes for 3 days caused an increase in appearance of catecholamines in the culture medium. Treatment of adrenal medullary cells with a conditioned medium prepared from lymphocytes also enhanced the appearance of catecholamines in culture medium in time- (8-48 h) and concentration-dependent manners. Heat treatment of the conditioned medium at 60 and 100 degrees C for 10 min reduced its stimulatory effect to 59 and 20% of control, respectively. After gel filtration on a Sephadex G-25 column or dialysis (<8 kDa molecular mass cutoff), the stimulatory activity of the conditioned medium was found in a high molecular fraction. The conditioned medium had little effect on the activity of lactate dehydrogenase in the medium of cultured adrenal medullary cells and on desipramine-sensitive [3H]norepinephrine uptake by the cells. These findings suggest that lymphocytes release a heat-sensitive factor(s) (molecular mass of more than 8 kDa) which increases efflux of catecholamines from cultured adrenal medullary cells.

Interferon-alpha reduces catecholamine secretion from bovine adrenal chromaffin cells stimulated by acetylcholine.

A long-term pretreatment (72 h) of bovine adrenal chromaffin cells with recombinant human interferon (IFN) -alpha-2b (1500 units/ml) produced a decrease in the secretion of catecholamines from the cells stimulated by acetylcholine (ACh) (25 micromol/l) but not that with human fibloblast IFN-beta (3000 units/ml) or recombinant human IFN-gamma (3000 units/ml). IFN-alpha-2b inhibited the ACh-induced secretion in a concentration- (30-1500 units/ml) and time-dependent manner (18-72 h). The content of catecholamines in the cells treated with IFN-alpha-2b for 72 h did not change. The inhibitory effect of IFN-alpha-2b on the secretion was abolished when the cells were simultaneously treated with anti-IFN-alpha antibody, and it was overcome by the increase in the external ACh concentration. IFN-alpha-2b also inhibited ACh-induced Ca2+ influx into the cells in a concentration-dependent manner similar to that of the IFN-alpha-2b inhibiting ACh-induced secretion. On the other hand, IFN-alpha-2b failed to reduce the secretion from the cells induced by high K+. These results strongly suggest that IFN-alpha-2b reduces the ACh-induced secretion of catecholamines from bovine adrenal chromaffin cells due to modulating the gene expression of the nicotinic ACh receptor-operated cation channels rather than due to directly affecting the channels. The results further indicate that the IFN-alpha-2b inhibition may be associated with the psychiatric side effects of IFN-alpha (depression, neurasthenica and somnolence, etc.), and that immune systems may regulate the function of (autonomic) nervous systems or adrenal medulla via IFN-alpha in vivo.