Influence of Bradykinin on Diacylglycerol and Phosphatidic Acid Accumulation in Cultured Bovine Adrenal Chromaffin Cells

Earlier studies have shown that bradykinin stimulated release of catecholamines from chromaffin cells by an influx of calcium through dihydropyridine-insensitive channels, and also that bradykinin stimulated (poly)phosphoinositide hydrolysis. To investigate membrane-bound second messengers in chromaffin cells, and to elucidate any role these may play in stimulus-secretion coupling, we have studied the influence of bradykinin on diacylglycerol and phosphatidic acid (PA). Using equilibrium labelling of primary cultures of chromaffin cells with [3H]arachidonic acid or [3H]glycerol, we found no influence of bradykinin (10 nM) on labelled diacylglycerol formation, either in the presence or absence of inhibitors of diacylglycerol lipase or kinase. However, when we used cells prelabelled with 32Pi for 2.5 h, we found that bradykinin produced a substantial stimulation of label found in PA, with an EC50 value of about 1 nM. This bradykinin stimulation of [32P]PA formation was only partially dependent on extracellular calcium, in contrast to the smaller response to nicotine, which was completely dependent on extracellular calcium. Short (10 min) pretreatment with tetradecanoylphorbol acetate (TPA) almost completely eliminated the bradykinin-stimulated formation of inositol phosphates, but failed to affect bradykinin stimulation of label in PA, suggesting that PA production in response to bradykinin is not downstream of phospholipase C activation. TPA alone failed to stimulate [32P]PA substantially, whereas long-term (24 or 48 h) treatment with TPA failed to attenuate the response to bradykinin. Diacylglycerol kinase inhibitors were also without effect on the bradykinin stimulation of [32P]PA. These results suggest that bradykinin stimulates PA production by a mechanism independent of the activation of protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synapsin II Phosphorylation and Catecholamine Release in Bovine Adrenal Chromaffin Cells: Additive Effects of Histamine and Nicotine

Primary cultures of bovine adrenal medullary chromaffin cells can be stimulated with nicotine, which mimics the cholinergic stimulus from the splanchnic nerve. Histamine also stimulates catecholamine release in a time- and dose-dependent manner. We have previously shown that nicotine stimulates incorporation of 32Pi into the vesicle-associated phosphoprotein synapsin II. We report here that histamine, too, stimulates an increase in 32Pi incorporation into synapsin II, which is blocked by the H1-histamine receptor-specific antagonist pyrilamine. The time course of histamine-stimulated synapsin II phosphorylation closely paralleled that of histamine-stimulated catecholamine release. Interestingly, histamine and nicotine produced an additive increase in both catecholamine release and synapsin II phosphorylation, suggesting that these two secretogogues stimulate the phenomena via independent mechanisms. When we investigated the dependence of these two agonists on extracellular calcium, we found that nicotine-stimulated release and synapsin II phosphorylation were reduced to basal levels at low calcium concentrations. However, the histamine-stimulated effects remained significantly elevated. This suggests that calcium arising from two separate pools can stimulate catecholamine release and synapsin II phosphorylation in bovine chromaffin cells. Taken together, these data support the hypothesis that synapsin II phosphorylation is a component of the secretory response from these cells.     

Synergistic Effect of Prostaglandin E2 and Ouabain on Catecholamine Release from Cultured Bovine Adrenal Chromaffin Cells

We recently reported that prostaglandin E2 (PGE2) stimulated phosphoinositide metabolism in cultured bovine adrenal chromaffin cells and that PGE2 and ouabain, an inhibitor of Na+,K+-ATPase, synergistically induced a gradual secretion of catecholamines from the cells. The effect on catecholamine release was specific for prostaglandin E1 (PGE1) and PGE2 among prostaglandins tested (E1 = E2 greater than F2 alpha greater than D2). The release evoked by PGE2 plus ouabain was greatly reduced in Na+-depleted medium and not observed in Ca2+-free medium. Here we examined the synergistic effect of PGE2 and ouabain on the release with specific reference to ion fluxes. Regardless of the presence of PGE2, ouabain stimulated the release in a dose-dependent manner with half-maximal stimulation at 1 microM, and omission of K+ from the medium, a condition which suppresses the Na+,K+-ATPase activity, also enhanced the release from chromaffin cells exposed to PGE2. Ouabain induced a continuous accumulation of 22Na+ and 45Ca2+, as well as secretion of catecholamines. Although PGE2 itself showed hardly any effects on these cellular responses, PGE2 potentiated all of them induced by ouabain. The time course of catecholamine release was correlated with that of accumulation of 45Ca2+ rather than with that of 22Na+. The release evoked by PGE2 and ouabain was inhibited in a dose-dependent manner by amiloride and the analogue ethylisopropylamiloride, inhibitors of the Na+,H+-antiport, but not by the Na+-channel inhibitor tetrodotoxin nor by the nicotinic receptor antagonist hexamethonium. Ethylisopropylamiloride at 1 microM inhibited PGE2-enhanced accumulation of 22Na+ and 45Ca2+ and release of catecholamine by 40, 83, and 71%, respectively. Activation of the Na+,H+-antiport by elevation of the extracellular pH from 6.6 to 8.0 increased the release of catecholamines linearly. Furthermore, PGE2 induced a sustained increase in intracellular pH by about 0.1 pH unit above the resting value, which was abolished by amiloride or in Na+-free medium. These results taken together indicate that PGE2 activates the Na+,H+-antiport by stimulating phosphoinositide metabolism and that the increase in intracellular Na+ by both inhibition of Na+,K+-ATPase and activation of Na+,H+-antiport may lead to the redistribution of Ca2+, which is the initial trigger of catecholamine release.     

Stimulation of Formation of Inositol Phosphates in Primary Cultures of Bovine Adrenal Chromaffin Cells by Angiotensin II, Histamine, Bradykinin, and Carbachol

Histamine, bradykinin, and angiotensin II stimulate release of catecholamines from adrenal medulla. Here we show, using bovine adrenal chromaffin cells in culture, that these agonists as well as carbachol (with hexamethonium) stimulate production of inositol phosphates. The histamine response was mepyramine sensitive, implicating an H1 receptor, whereas bradykinin had a lower EC50 than Met-Lys-bradykinin, and [Des-Arg9]-bradykinin was relatively inactive, implicating a BK-2 receptor. Total inositol phosphates formed in the presence of lithium were measured, with histamine giving the largest response. The relative contribution of chromaffin cells and nonchromaffin cells in the responses was assessed. In each case chromaffin cells were found to be responding to the agonists; in the case of histamine the response was solely on chromaffin cells. When the inositol phosphates accumulating over 2 or 5 min, with no lithium present, were separated on Dowex anion-exchange columns, bradykinin gave the greatest stimulation in the inositol trisphosphate fraction, whereas histamine gave a larger inositol monophosphate accumulation. On resolution of the isomers of stimulated inositol trisphosphate after 2 min of stimulation, the principal isomer present was inositol 1,3,4-trisphosphate in each case. Two hypotheses for the differential responses to histamine and bradykinin are discussed.     

Differential Regulation of Histamine- and Bradykinin-Stimulated Phospholipase C in Adrenal Chromaffin Cells: Evidence for Involvement of Different Protein Kinase C Isoforms

Abstract: In this report we investigate the isoforms of protein kinase C (PKC) present in cultured adrenal chromaffin cells with respect to their modulation by treatment with phorbol ester and their possible differential involvement in the regulation of responses to histamine and bradykinin. The presence of individual isoforms of PKC was investigated by using eight isoform specific antisera, as a result of which PKC-α, ε, and ζ were identified. To characterize down-regulation of these enzymes, cells were incubated for 6–48 h with 1 µM phorbol myristate acetate (PMA). PKC-ε down-regulated more rapidly than PKC-α. At 12 h, PMA pretreatment, for example, PKC-ε was maximally down-regulated (23 ± 4% of controls), whereas PKC-α was unchanged. PKC-α showed partial down-regulation by 24 h of PMA pretreatment. PKC-ζ did not down-regulate at any of the times tested. Translocation from cytosol to membrane in response to PMA was also more rapid for PKC-ε than for PKC-α. The accumulation of total ³H-inositol (poly)phosphates in response to bradykinin or histamine was essentially abolished by prior treatment with 10-min PMA treatment (1 µM). However, with 12-h exposure to PMA, the bradykinin response was restored to the level seen with no prior PMA exposure. The histamine response showed no recovery by 12 h of PMA, but showed partial recovery by 24 h of PMA pretreatment. These observations showed that the restoration of the response to bradykinin corresponds to the loss of PKC-ε, whereas the restoration of the histamine response corresponds to the loss of PKC-α. This picture was confirmed with further studies on cytosolic Ca²⁺. The results show that chromaffin cells exhibit an unusual pattern of down-regulation of PKC isoforms on prolonged exposure to PMA, and that there is a differential effect of exposure to PMA on the histamine and bradykinin responses, suggesting that different PLC-linked receptors in chromafin cells are differentially regulated by PKC isoforms.     

Arachidonic Acid Release and Catecholamine Secretion from Digitonin-Treated Chromaffin Cells: Effects of Micromolar Calcium, Phorbol Ester, and Protein Alkylating Agents

The relationship between catecholamine secretion and arachidonic acid release from digitonin-treated chromaffin cells was investigated. Digitonin renders permeable the plasma membranes of bovine adrenal chromaffin cells to Ca2+, ATP, and proteins. Digitonin-treated cells undergo exocytosis of catecholamine in response to micromolar Ca2+ in the medium. The addition of micromolar Ca2+ to digitonin-treated chromaffin cells that had been prelabeled with [3H]arachidonic acid caused a marked increase in the release of [3H]arachidonic acid. The time course of [3H]arachidonic acid release paralleled catecholamine secretion. Although [3H]arachidonic acid release and exocytosis were both activated by free Ca2+ in the micromolar range, the activation of [3H]arachidonic acid release occurred at Ca2+ concentrations slightly lower than those required to activate exocytosis. Pretreatment of the chromaffin cells with N-ethylmaleimide (NEM) or p-bromophenacyl bromide (BPB) resulted in dose-dependent inhibition of 10 microM Ca2+-stimulated [3H]arachidonic acid release and exocytosis. The IC50 of NEM for both [3H]arachidonic acid release and exocytosis was 40 microM. The IC50 of BPB for both events was 25 microM. High concentrations (5-20 mM) of Mg2+ caused inhibition of catecholamine secretion without altering [3H]arachidonic acid release. A phorbol ester that activates protein kinase C, 12-O-tetradecanoylphorbol-13-acetate (TPA), caused enhancement of both [3H]arachidonic acid release and exocytosis. The findings demonstrate that [3H]arachidonic acid release is stimulated during catecholamine secretion from digitonin-treated chromaffin cells and they are consistent with a role for phospholipase A2 in exocytosis from chromaffin cells. Furthermore the data suggest that protein kinase C can modulate both arachidonic acid release and exocytosis.     

Peptide E and Its Products, BAM 18 and Leu-Enkephalin, in Bovine Adrenal Medulla and Cultured Chromaffin Cells: Release in Response to Stimulation

Peptide E is a 25 amino acid opioid peptide which, if cleaved at the sole double basic (Lys-Arg) typical processing site, would generate two opioid fragments, the amino-terminal fragment BAM 18 and the carboxy-terminal fragment Leu-enkephalin. We have analysed extracts of bovine adrenal medulla in order to quantify these three opioid peptides (peptide E, BAM 18, and Leu-enkephalin). Here we present evidence that BAM 18 and Leu-enkephalin were present in similar amounts, whereas peptide E was present at a higher concentration. This is consistent with previous observations showing a preferential accumulation of larger peptides in the bovine adrenal, and also with the Lys-Arg bond being the principal site of cleavage of peptide E. However, when bovine adrenal chromaffin cells were maintained in culture for several days, Leu-enkephalin was found to be present in much greater amounts than was BAM 18-like immunoreactivity. The molar amounts of peptide E still exceeded the estimated levels of BAM 18 and Leu-enkephalin. We provide evidence that under conditions of basal release BAM 18 and peptide E were released, whereas Leu-enkephalin was released in much smaller amounts, if at all. On stimulation with nicotine results were consistent with an increased release of all three peptides with a preferential stimulation of Leu-enkephalin release. Under all conditions, the molar amounts of peptide E released apparently exceeded that of the other peptides. The results are discussed in terms of the regulation of partial proteolysis and the fate of peptide E.     

Introduction of Macromolecules into Bovine Adrenal Medullary Chromaffin Cells and Rat Pheochromocytoma Cells (PC12) by Permeabilization with Streptolysin O: Inhibitory Effect of Tetanus Toxin on Catecholamine Secretion

Conditions are described for controlled plasma membrane permeabilization of rat pheochromocytoma cells (PC12) and cultured bovine adrenal chromaffin cells by streptolysin O (SLO). The transmembrane pores created by SLO invoke rapid efflux of intracellular 86Rb+ and ATP, and also permit passive diffusion of proteins, including immunoglobulins, into the cells. SLO-permeabilized PC12 cells release [3H]dopamine in response to micromolar concentrations of free Ca2+. Permeabilized adrenal chromaffin cells present a similar exocytotic response to Ca2+ in the presence of Mg2+/ATP. Permeabilized PC12 cells accumulate antibodies against synaptophysin and calmodulin, but neither antibody reduces the Ca2+-dependent secretory response. Reduced tetanus toxin, although ineffective when applied to intact chromaffin cells, inhibits Ca2+-induced exocytosis by both types of permeabilized cells studied. Omission of dithiothreitol, toxin inactivation by boiling, or preincubation with neutralizing antibodies abolishes the inhibitory effect. The data indicate that plasma membrane permeabilization by streptolysin O is a useful tool to probe and define cellular components that are involved in the final steps of exocytosis.     

Differential Effect of Intrastriatal Kainic Acid on the Modulation of Dopamine Release by μ- and δ-Opioid Peptides: A Microdialysis Study

The present study investigated the effects of a striatal lesion induced by kainic acid on the striatal modulation of dopamine (DA) release by mu- and delta-opioid peptides. The effects of [D-Pen2,D-Pen5]-enkephalin (DPDPE) and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAGO), two highly selective delta- and mu-opioid agonists, respectively, were studied by microdialysis in anesthetized rats. In control animals both opioid peptides, administered locally, significantly increased extracellular DA levels. The effects of DPDPE were also observed in animals whose striatum had been previously lesioned with kainic acid. In contrast to the effects of the delta agonist, the significant increase induced by DAGO was no longer observed in lesioned animals. These results suggest that delta-opioid receptors modulating the striatal DA release, in contrast to mu receptors, are not located on neurons that may be lesioned by kainic acid.     

Glutamate as a Putative Transmitter in the Cerebellum: Stimulation by GABA of Glutamic Acid Release from Specific Pools

The aim of the present paper was to determine whether the release of glutamate from putative "glutamergic" terminals in the cerebellum is influenced by gamma-aminobutyric acid (GABA). In a group of preliminary experiments, we present biochemical evidence in favour of a neurotransmitter role of glutamate in the cerebellum: (1) endogenous glutamate was released from depolarized cerebellar synaptosomal preparations in a Ca2+-dependent away; (2) [14C]glutamate was synthesized from [14C]glutamine in cerebellar synaptosomes, and the newly synthesized [14C]glutamate was released released in a Ca2+-dependent way; (3) the elevation of cyclic GMP elicited by depolarization of cerebellar slices in the presence of Ca2+ was partly reversed by the glutamate antagonist glutamic acid diethyl ester, which probably prevented the interaction of endogenously released glutamate with postsynaptic receptors. GABA and muscimol at low concentrations (2--20 micrometers) potentiated the depolarization-induced release of D-[3H]aspartate (a glutamate analogue which labels the glutamate "reuptake pool") from cerebellar synaptosomes. The effect was concentration dependent and was largely prevented by two GABA antagonists, bicuculline and picrotoxin. The stimulation of D-[3H]aspartate release evoked by muscimol was linearly related to the logarithm of K+ concentration in the depolarizing medium. GABA did not affect the overall release of endogenous glutamate, but potentiated, in a picrotoxin-sensitive manner, the depolarization-evoked release of [14C]glutamate previously synthesized from [14C]glutamine. Since nerve endings are the major site of glutamate synthesis from glutamine, GABA and muscimol appear to exert their stimulatory effect at the level of "glutamergic" nerve terminals, probably after interacting with presynaptic GABA receptors. The possible functional significance of these findings is briefly discussed.     

Effects of Hypoxia on the Catecholamine Release, Ca2+ Uptake, and Cytosolic Free Ca2+ Concentration in Cultured Bovine Adrenal Chromaffin Cells

The purpose of the present study is to clarify the effects of hypoxia on catecholamine release and its mechanism of action. For this purpose, using cultured bovine adrenal chromaffin cells, we examined the effects of hypoxia on high (55 mM) K(+)-induced increases in catecholamine release, in cytosolic free Ca2+ concentration ([Ca2+]i), and in 45Ca2+ uptake. Experiments were carried out in media preequilibrated with a gas mixture of either 21% O2/79% N2 (control) or 100% N2 (hypoxia). High K(+)-induced catecholamine release was inhibited by hypoxia to approximately 40% of the control value, but on reoxygenation the release returned to control levels. Hypoxia had little effect on ATP concentrations in the cells. In the hypoxic medium, [Ca2+]i (measured using fura-2) gradually increased and reached a plateau of approximately 1.0 microM at 30 min, whereas the level was constant in the control medium (approximately 200 nM). High K(+)-induced increases in [Ca2+]i were inhibited by hypoxia to approximately 30% of the control value. In the cells permeabilized by digitonin, catecholamine release induced by Ca2+ was unaffected by hypoxia. Hypoxia had little effect on basal 45Ca2+ uptake into the cells, but high K(+)-induced 45Ca2+ uptake was inhibited by hypoxia. These results suggest that hypoxia inhibits high K(+)-induced catecholamine release and that this inhibition is mainly the result of the inhibition of high K(+)-induced increases in [Ca2+]i subsequent to the inhibition of Ca2+ influx through voltage-dependent Ca2+ channels.     

Inhibitory Effect of Fulvic Acid Extracted from Canadian Sphagnum Peat on Chemical Mediator Release by RBL-2H3 and KU812 Cells

Fulvic acid (FA) was extracted and purified from Canadian Sphagnum peat (CP-FA) and characterized by using an element analysis meter, Fourier transform infrared (FT-IR) spectroscopy, electron spin resonance (ESR) spectroscopy, and ¹³C-nuclear magnetic resonance (¹³C-NMR) spectroscopy. To investigate the antiallergic effect of CP-FA, we incubated rat basophilic leukemia (RBL-2H3) cells with 0.001–10.0 μg/ml of CP-FA and determined the β-hexosaminidase release inhibition at different response stages. The intracellular calcium [Ca²⁺] _i level was also determined by using Fluo 3-AM, a calcium-specific fluorescent probe, and the cytotoxicity of CP-FA was determined by the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. The results revealed that RBL-2H3 cells incubated for 48 h with 0.001–10.0 μg/ml of CP-FA did not show any decreased viability. CP-FA inhibited the β-hexosaminidase release by IgE-sensitized, antigen-stimulated RBL-2H3 cells at the antigen-antibody binding stage and the antibody-receptor binding stage. CP-FA also inhibited histamine release from A23187 plus PMA- or compound 48/80-stimulated KU812 cells. Furthermore, there was a decrease in the intracellular [Ca²⁺] _i level in IgE-sensitized cells incubated with CP-FA and stimulated with antigen. Our results show that CP-FA may be useful for the treatment or prevention of allergic diseases.     

Accumulation of Cyclic AMP Elicited by Vasoactive Intestinal Peptide Is Potentiated by Noradrenaline, Histamine, Adenosine, Baclofen, Phorbol Esters, and Ouabain in Mouse Cerebral Cortical Slices: Studies on the Role of Arachidonic Acid Metabolites and Protein Kinase C

In mouse cerebral cortical slices, noradrenaline (NA) potentiates cyclic AMP (cAMP) accumulation elicited by vasoactive intestinal peptide (VIP) through alpha 1-adrenergic receptors. This synergism is inhibited by indomethacin, and the prostaglandins E2 and F2 alpha mimic the effect of NA. In the present study, we observed that the synergism between VIP and NA is not inhibited by the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) or the diacylglycerol-lipase inhibitor RHC 80267, thus further stressing the role of phospholipase A2 activation. Various neuroactive agents that potentiate the stimulatory effect of VIP on cAMP formation were also examined. As with NA, the potentiation by histamine and adenosine is inhibited by indomethacin. In contrast to NA, histamine, and adenosine, the synergistic interaction between phorbol esters and VIP on cAMP formation is abolished by H-7 but not by indomethacin. The potentiation by baclofen, a gamma-aminobutyric acidB receptor agonist, is partially inhibited by the 5-lipoxygenase inhibitor nafazatrom. The synergism between ouabain and VIP is reduced by H-7 but not by indomethacin and nafazatrom. These data indicate that the stimulation of cAMP formation elicited by VIP is under the modulation of various neuroactive agents that trigger diverse intracellular mechanisms to potentiate the effect of the peptide.     

Inhibition of Histamine Synthesis in Brain by α-Fluoromethylhistidine, a New Irreversible Inhibitor: In Vitro and In Vivo Studies

a-Fluoromethylhistidine (α-FMH), a new potent inhibitor of histidine decarboxylase (HD), has been used for in vitro and in vivo studies of brain HD. Following a preincubation with (+)-α-FMH, brain HD activity was inhibited in a time-dependent and concentration-dependent manner. The enzyme activity was not restored by overnight dialysis against standard buffer. The (–) antimer of α-FMH was ineffective. When injected intraperitoneally in a single dose of 20 mg/kg, (±)-α-FMH induced a complete loss in HD activity in cerebral cortex and hypothalamus as well as in peripheral tissues, such as stomach. At a dosage of 100 mg/kg (±)-α-FMH did not alter histamine-N-methyltransferase, DOPA decarboxylase, and glutamate decarboxylase activities. The maximal decrease of HD activity occurred after 2 h in both cerebral cortex and hypothalamus, but the time course of the recovery of enzyme activity was slower in the cerebral cortex. The enzyme activity reached control value within 3 days in hypothalamus and was not fully restored after 4 days in cerebral cortex. Contrasting with the diminished HD activity, a substantial concentration of histamine remained present in five regions of mouse brain. Thus, α-FMH is a highly specific irreversible inhibitor of brain HD activity and its efficacy makes it useful to study the physiological role of brain histamine.     

Effect of Nipecotic Acid, a γ-Aminobutyric Acid Transport Inhibitor, on the Turnover and Release of γ-Aminobutyric Acid in Rat Cortical Slices

Abstract: To see the effect of a γ-aminobutyric acid GABA uptake inhibitor on the efflux and content of endogenous and labeled GABA, rat cortical slices were first labeled with [³H]GABA and then superfused in the absence or presence of 1 mM nipecotic acid. Endogenous GABA released or remaining in the slices was measured with high performance liquid chromatography, which was also used to separate [³H]GABA from its metabolites. In the presence of 3 mM K⁺, nipecotic acid released both endogenous and [³H]GABA, with a specific activity four to five times as high as that present in the slices. The release of labeled metabolite(s) of [³H]GABA was also increased by nipecotic acid. The release of endogenous GABA evoked by 50 mM K⁺ was enhanced fourfold by nipecotic acid but that of [³H]GABA was only doubled when expressed as fractional release. In a medium containing no Ca²⁺ and 10 mM Mg²⁺, the release evoked by 50 mMK⁺ was nearly suppressed in either the absence or the presence of nipecotic acid. In the absence of nipecotic acid electrical stimulation (bursts of 64 Hz) was ineffective in evoking release of either endogenous or [³H]GABA, but in the presence of nipecotic acid it increased the efflux of endogenous GABA threefold, while having much less effect on that of [³H]GABA. Tetrodotoxin (TTX) abolished the effect of electrical stimulation. Both high K⁺ and electrical stimulation increased the amount of endogenous GABA remaining in the slices, and this increase was reduced by omission of Ca²⁺ or by TTX. The results suggest that uptake of GABA released through depolarization is of major importance in removing GABA from extracellular spaces, but the enhancement of spontaneous release by nipecotic acid may involve intracellular heteroexchange. Depolarization in the presence of Ca²⁺ leads to an increased synthesis of GABA, in excess of its release, but the role of this excess GABA remains to be established.     

Expression of the Proenkephalin A Gene and [Met5]-Enkephalin Secretion Induced by Arachidonic Acid in Bovine Adrenal Medullary Chromaffin Cells: Involvement of Second Messengers

Abstract: We have previously reported that arachidonic acid (AA) increases the long-term secretion of [Met⁵]-enkephalin (ME) and the expression of proenkephalin A (proENK) mRNA in bovine adrenal medullary chromaffin (BAMC) cells. To characterize the underlying signal transductional mechanisms for the AA-induced responses, the interactions of AA with several second messenger systems were studied. Long-term (24-h) treatment with AA (100 µ M ) increased both the secretion of ME and the expression of proENK mRNA. Pretreatment of BAMC cells with nimodipine (1 µ M ), but not with ω-conotoxin GVIA (1 µ M ), inhibited the secretion of ME and the expression of proENK mRNA induced by AA. Calmidazolium (1 µ M ), a calmodulin antagonist, also significantly inhibited AA-induced responses. However, a protein kinase C (PKC) inhibitor, sphingosine (36 µ M ), was ineffective in blocking AA-induced responses. In addition, the down-regulation of PKC by phorbol 12-myristate 13-acetate (0.1 µ M ) for 48 h did not inhibit the AA-induced responses. Forskolin (5 µ M ), an adenyl cyclase activator, alone increased the secretion of ME as well as proENK mRNA levels and, when coincubated with AA, showed an additive effect on the secretion of ME and the levels of proENK mRNA. The results suggest that the Ca²⁺/calmodulin pathway, but not the protein kinase A or PKC pathway, is partially involved in mediating the AA-induced increases of the long-term secretion of ME and the levels of proENK mRNA.     

Brain Pharmacokinetics of Non‐Imidazole Biphenyl H3 Receptor Antagonists: a Liquid Chromatography/Electrospray‐Mass Spectrometry and ex vivo Binding Study in Rats

In the present article, we report on the kinetics of brain penetration in rats of the H3R antagonist 1,1′‐[1,1′‐biphenyl‐4,4′‐diylbis(methylene)]bis‐[piperidine] ( 1 ), which had shown a favorable in vitro pharmacological profile and in vivo potency in preventing scopolamine‐induced amnesia. Two different approaches were employed: high‐performance liquid chromatography/electrospray‐mass spectrometry (HPLC/ESI‐MS) and ex vivo binding against the labeled agonist [³H]‐(R)‐α‐methylhistamine ([³H]RAMHA). Starting from the structure of 1 , the rigid piperidine ring was replaced by a flexible dipropylamino group (see 2 ) or by a morpholino ring (see 3 ), endowed with lower basicity. The effect of replacement on rat plasma and brain disposition in the 24 h after administration was analyzed. High (μM ) and persistent concentrations of 1 were found in rat plasma, while plasma levels were significantly lower (range: 0–200 nM ) for the other two derivatives. This could be explained, among other factors, by the higher stability, observed for 1 , to liver metabolic cleavage. The applied chemical modulation had an important effect on in vivo brain disposition, as, despite the comparable physico‐chemical properties, 2 did not show the tendency to accumulate within the brain, as stated by its brain vs. plasma concentration ratios, if compared to 1 . These structure? property relationships should be taken into account in the pharmacokinetic optimization of new series of H3 receptor antagonists.     

Effects of Heavy Metal Cations and Other Sulfhydryl Reagents on Brain Dopamine D1 Receptors: Evidence for Involvement of a Thiol Group in the Conformation of the Active Site

To investigate aspects of the biochemical nature of membrane-bound dopamine D1 receptors, rat striatal homogenates were pretreated with heavy metal cations and some other chemical agents, and their effects on D1 receptors were subsequently determined using a standard [3H](R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1-N-3- benzazepine([3H]SCH 23390) binding assay. Incubation of striatal membranes with as little as 1 microM Hg2+, 10 microM Cu2+, and 10 microM Cd2+ completely prevented specific [3H]SCH 23390 binding. The effect of Cu2+, 1.5 microM, was noncompetitive in nature, whereas 3-5 microM Cu2+ afforded mixed-type inhibition. The inhibitory effect of Cu2+ was fully reversed by dithiothreitol (0.1-1 mM). Cu2+ (2 microM) did not affect the affinity of cis-flupenthixol or clozapine for remaining [3H]SCH 23390 sites. A second series of cations, Co2+ (30 microM), Ni2+ (30 microM), Mn2+ (1 mM), Ca2+ (25 mM), and Ba2+ (20 mM), inhibited specific [3H]SCH 23390 binding by 50% at the concentrations indicated. The thiol alkylating reagent N-ethylmaleimide (NEM) (0.2 mM) reduced specific binding by 70%. The effect of NEM was completely prevented by coincubation with a D1 receptor saturating concentration of SCH 23390 (20 nM) or dopamine (10 microM). The results indicated that the dopamine D1 receptor is a thiol protein and that a thiol group is essential for the ligand binding.     

Influence of Potassium Concentration in Microdialysis Perfusate on Basal and Stimulated Striatal Dopamine Release: Effect of Ceruletide, a Cholecystokinin-Related Peptide

Abstract: The in vivo microdialysis method was used to study the effect of the cholecystokinin-related peptide, ceruletide, on extracellular levels of dopamine (DA) in the striatum following perfusion with various K⁺ concentrations. Increasing the K⁺ concentration in the perfusate from 4 to 15 or 17.5 m M did not change basal DA release or release evoked by electrical stimulation of the medial forebrain bundle (MFB). However, when the perfusing solution contained 20 or 30 m M K⁺, dose-dependent reductions of both basal and MFB-stimulated DA release occurred. Subcutaneous administration of ceruletide at 160 μg/kg had no influence on the basal or MFB-stimulated DA release with 4 or 15 m M K⁺ in the perfusate. However, after perfusion with 17.5 m M K⁺, ceruletide significantly attenuated the basal and MFB-stimulated DA release. Carbachol (10 μ M ) locally applied via the dialysis probe also attenuated MFB-stimulated DA release after perfusion with 17.5 m M K⁺. From these results, we conclude that under appropriate depolarization of striatal DA terminals, ceruletide induces further depolarization and inactivation of nigrostriatal DA terminals. The present data suggest that this effect may be mediated via intrinsic cholinergic neurons in the striatum.