Pharmacological characterization of the effects of methylmercury and mercuric chloride on spontaneous noradrenaline release from rat hippocampal slices

The environmental contaminants methylmercury (MeHg) and mercuric chloride (HgCl2) stimulated the spontaneous release of [3H]noradrenaline ([3H]NA) from hippocampal slices in a time- and concentration-dependent manner. Both MeHg and HgCl2 were similarly potent, with an EC50 of 88.4 microM and 75.9 microM, respectively. The releasing effects of MeHg and HgCl2 increased in the presence of desipramine, showing that the mechanism does not involve reversal of the transmitter transporter, and were completely blocked by reserpine preincubation, indicating a vesicular origin of [3H]NA release. The voltage-gated Na+ channel blocker tetrodotoxin (TTX) did not affect the response to mercury compounds. [3H]NA release elicited by MeHg was partially dependent on extracellular Ca2+, since it decreased significantly in a Ca2+-free EGTA-containing medium whereas HgCl2 induced a release of [3H]NA independent of extracellular Ca2+. Neither Ca2+-channels blockers, cobalt chloride (CoCl2) and (omega-conotoxin-GVIA, nor the Na+/Ca2+-exchanger inhibitor benzamil reduced MeHg-evoked [3H]NA release. Moreover, thapsigargin or caffeine, endoplasmic reticulum Ca2+-depletors, did not modify metal-evoked [3H]NA release, whereas ruthenium red, which inhibits the mitochondrial Ca2+ transport, decreased the effect of both MeHg and HgCl2. All these data indicate that, in hippocampal slices, mercury compounds release [3H]NA from the vesicular pool by a mechanism involving Ca2+ mobilization from mitochondrial stores.     

Calcium dependence of beta-adrenoceptor mediated cyclic AMP accumulation in human lymphocytes

In intact human lymphocytes, cyclic AMP accumulation in response to isoproterenol was inhibited by 5 mM EDTA, by deletion of calcium ions from the medium and by 1 mM lanthanum chloride, but not by 1 microM verapamil or by 10 microM nifedipine. A23187 caused a modest increase in cyclic AMP content. Exposure of lymphocytes to 5 microM 1-isoproterenol desensitized the cells to subsequent beta-adrenergic stimulation, reducing cyclic AMP accumulation. With higher concentrations of 1-isoproterenol (50 microM), receptor density was reduced as well. None of the above agents attenuated losses in agonist-stimulated cyclic AMP accumulation induced by treatment with 5 microM isoproterenol for 90 min. These data suggest that calcium ions, both those present in the extracellular medium and those bound to the plasma membrane, are required for isoproterenol-stimulation of adenylate cyclase. In addition, it appears that neither the presence of extracellular calcium ions nor full activation of adenylate cyclase are required for desensitization.     

Effects of Angiotensin II on [3H]Noradrenaline Release and Phosphatidylinositol Hydrolysis in the Parietal Cortex and Locus Coeruleus of the Rat

Angiotensin II (ANGII) (3-100 nM) facilitated the potassium-evoked (22.5 mM) release of [3H]-noradrenaline ([3H]NA) from slices of parietal cortex in a concentration-dependent manner, but did not significantly alter the release of [3H]NA evoked in a similar manner from locus coeruleus slices. The facilitatory action of ANGII was blocked by saralasin (0.1-3 microM). Neither nimodipine (10-30 microM) nor phenylmethylsulphonyl fluoride (1 mM) altered either [3H]NA release or the facilitatory action of ANGII in the parietal cortex. Carbachol (0.01-3 mM) and raised potassium (22.5 mM), but not ANGII (3-100 nM), stimulated the production of inositol phosphates in parietal cortex slices. The potassium-evoked increase in inositol phosphate production was unaffected by ANGII (3-100 nM). In the locus coeruleus, ANGII (3-100 nM) did not stimulate inositol phosphate production. The mechanism underlying the ANGII facilitation of [3H]NA release from the parietal cortex does not appear to involve either nimodipine-sensitive calcium channels, or, as far as we have been able to determine, the release of calcium from intracellular stores following the breakdown of phosphoinositides.     

Evidence for a complex influence of nicotinic acetylcholine receptors on hippocampal serotonin release

The effects of nicotine on 5-hydroxytryptamine (5-HT) release from serotonergic nerve endings in rat dorsal hippocampal slices were studied. Nicotine (50-500 microM:) caused a concentration-dependent increase in 5-HT release. This effect was antagonised by mecamylamine (0.5 microM:), indicating an action at nicotinic receptors. Nicotine-evoked 5-HT release was not affected by tetrodotoxin (3 microM:), cadmium chloride (0.1 mM:), or the absence of Ca(2+) or Na(+) in the superfusion medium. Unexpectedly, higher concentrations of mecamylamine alone (1-50 microM:) increased 5-HT release. This suggested the presence of inhibitory input to 5-HT neurones and that these inhibitory neurones possess tonically active nicotinic receptors. The effect of mecamylamine (50 microM:) on 5-HT release was reduced by the muscarinic M(1) receptor agonist, McN-A-343 (100 microM:), but pirenzepine (0.005-1 microM:), which blocks M(1) receptors, alone increased 5-HT release. Hippocampal serotonergic neurones are known to possess both excitatory nicotinic receptors and inhibitory M(1) receptors. Although there may be several explanations for our results, one possible explanation is that nicotine stimulates 5-HT release by activating nicotinic heteroreceptors on 5-HT terminals. Mecamylamine (0.5 microM:) antagonises this effect, but higher concentrations increase 5-HT release indirectly by blocking the action of endogenous acetylcholine on nicotinic receptors situated on cholinergic neurones that provide muscarinic inhibitory input to 5-HT neurones.     

Na+,K+,2Cl(-)-cotransport and chloride permeability of the cell membrane in mezaton and histamine regulation of electrical and contractile activity in smooth muscle cells from the guinea pig ureter

Influence of Na+,K+,2Cl(-)-cotransport and chloride permeability of the cell membrane on electrically-induced action potential and contraction of smooth muscle cells from guinea pig ureter was examined with the methods of the double sucrose gap junction. Mesatone (10 microM) and histamine (10 microM) induced prolongation of the action potential and elevation of smooth muscle cell contraction, whereas hyperosmic medium (+150 mM sucrose), and recovery of solution osmolality in hyposmic condition (70 mM NaCl) after a single contraction. Inhibitor Na+,K+,2Cl(-)-cotransport bumetanide (10 microM) and chloride permeability blockers niflumic acid (10-100 microM) and SITS (10-500 microM) attenuated stimulating effects of mesatone, histamine and hyperosmic medium. In opposite to adenylate cyclase activation with forskolin (1 microM), guanylate cyclase activation with sodium nitroprusside (SN, 100 microM) decreased both inhibitory action of bumetanide, niflumic acid and activating effects of mesatone, histamine on action potential and elevation contraction of smooth muscle cells. Influence of forskolin rather and not SN on AP and SMC C was inhibited with tetraethylammonium (5 mM). These results suggest that influence of Na+,K+,2Cl(-)-cotransport on electrical and contractil properties of ureter smooth muscle cells is mediated by stimulation of Ca(2+)-activated chloride permeability of the cell membrane and modulated by intracellular cGMP, but not triggered by Ca2+ release from sarcoplasmic reticulum.     

Effect of miconazole on intracellular Ca2+ levels and proliferation in human osteosarcoma cells

The effect of miconazole, an anti-fungal drug, on cytoplasmic free Ca2+ concentrations ([Ca2+]i) in human osteosarcoma cells (MG63) was explored by using the Ca2+-sensitive dye fura-2. Miconazole acted in a concentration-dependent manner with an EC50 of 75 microM. The Ca2+ signal comprised a gradual rise and a sustained elevation. Removal of extracellular Ca2+ reduced 50% of the signal. In Ca2+-free medium, the [Ca2+]i rise induced by 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) was completely inhibited by pretreatment with 20 microM miconazole. Pretreatment with thapsigargin partly inhibited miconazole-induced Ca2+ release. The miconazole-induced Ca2+ release was not changed by inhibition of phospholipase C with 2 microM U73122. By using tetrazolium as a fluorescent probe, it was shown that 10-100 microM miconazole decreased cell proliferation rate in a concentration-dependent manner. Collectively, this study shows that miconazole induces [Ca2+]i rises in human osteosarcoma cells via releasing Ca2+ mainly from the endoplasmic reticulum in a manner independent of phospholipase C activity, and by causing Ca2+ influx. Furthermore, miconazole may be cytotoxic to the cells at higher concentrations.     

Modifications of Ca2+ mobilization and noradrenaline release by S-nitroso-cysteine in PC12 cells

The effects of nitrogen monoxide (NO)-related compounds on cytosolic free Ca2+ concentrations ([Ca2+]i) and noradrenaline (NA) release in neurosecretory PC12 cells were investigated. The addition of S-nitroso-cysteine (SNC) stimulated [Ca2+]i increases from an intracellular Ca2+ pool continuously in a concentration-dependent manner. Other NO donors, which stimulate cyclic GMP accumulation, did not cause [Ca2+]i increases. After treatment with 0.2 mM SNC, transient increases in [Ca2+]i from the Ca2+ pool induced by caffeine were completely abolished. The addition of N-ethylmaleimide (NEM) caused sustained [Ca2+]i increases from the intracellular Ca2+ pool. Furthermore, caffeine did not stimulate further [Ca2+]i increases in PC12 cells pretreated with NEM. These findings suggest that SNC and NEM predominantly interact with a caffeine-sensitive Ca2+ pool. The addition of dithiothreitol (DTT) to 0.4 mM SNC-stimulated cells reduced [Ca2+]i to basal levels, and the addition of DTT to NEM-stimulated cells locked [Ca2+]i at high levels. The stimulatory effects of SNC but not NEM were not abolished by pretreatment with DTT. These findings suggest that modification of the oxidation status of the sulfhydryl groups on the caffeine-sensitive receptors by SNC or NEM regulates Ca2+ channel activity in a reversible manner. SNC did not stimulate NA release by itself but did inhibit ionomycin-stimulated NA release. In contrast, NEM stimulated NA release in the absence of extracellular CaCl2 and further enhanced ionomycin-stimulated NA release. Ca2+ mobilization by SNC from the caffeine-sensitive pool was not a sufficient factor, and other factors stimulating NA release may be negatively regulated by SNC.     

Excessive release of [3H] noradrenaline by veratridine and ischemia in spinal cord

In this study, the properties of ischemic condition-induced and veratridine-evoked [3H]noradrenaline ([3H]NA) release from rat spinal cord slices were compared. It was expected that ischemia mimicked by oxygen and glucose deprivation results in the impairment of Na+/K+ -ATPase with a consequent elevation of the intracellular Na+ -level which reverses the NA carrier and promotes excessive NA release, and veratridine, by the activation of Na+ channels, releases NA both carrier-mediated and Ca2+ -dependent, i.e. vesicular manner. In our experiments, veratridine (1-100 microM) dose-dependently increased the resting [3H]NA release, and its effect was only partially blocked by low temperature or the lack of external calcium, whereas the sodium channel inhibitor tetrodotoxin (TTX, 1 microM) completely prevented it, indicating that veratridine induces NA release via axonal depolarization and reversing the transporters by eliciting Na+ -influx. In contrast to TTX, the local anesthetic lidocaine (100 microM) only partially blocked the veratridine-induced [3H]NA release due to its inhibitory action on K+ channels. The ischemia-induced [3H]NA release was abolished at 12 degrees C, a temperature known to block only the transporter-mediated release of transmitters. However, lidocaine was also partially effective to reverse the action of ischemia on the NA release, indicating that lidocaine is not a useful compound in the treatment of spinal cord-injured patients against the excessive excytotoxic NA release.     

Safrole-induced Ca2+ mobilization and cytotoxicity in human PC3 prostate cancer cells

The effect of the carcinogen safrole on intracellular Ca2+ mobilization and on viability of human PC3 prostate cancer cells was examined. Cytosolic free Ca2+ levels ([Ca2+]i) were measured by using fura-2 as a probe. Safrole at concentrations above 10 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 350 microM. The Ca2+ signal was reduced by more than half after removing extracellular Ca2+ but was unaffected by nifedipine, nicardipine, nimodipine, diltiazem, or verapamil. In Ca2+-free medium, after treatment with 650 microM safrole, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) failed to release Ca2+. Neither inhibition of phospholipase C with U73122 nor modulation of protein kinase C activity affected safrole-induced Ca2+ release. Overnight incubation with 0.65-65 microM safrole did not affect cell viability, but incubation with 325-625 microM safrole decreased viability. Collectively, the data suggest that in PC3 cells, safrole induced a [Ca2+]i increase by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C- and protein kinase C-independent fashion, and by inducing Ca2+ influx. Safrole can decrease cell viability in a concentration-dependent manner.     

Identification of 30 kDa protein for Ca(2+) releasing action of myotoxin a with a mechanism common to DIDS in skeletal muscle sarcoplasmic reticulum.

The molecular mechanism of Ca(2+) release by myotoxin a (MTYX), a polypeptide toxin isolated from the venom of prairie rattlesnakes (Crotalus viridis viridis), was investigated in the heavy fraction of sarcoplasmic reticulum (HSR) of rabbit skeletal muscles. [(125)I]MYTX bound to four HSR proteins (106, 74, 53 and 30 kDa) on polyvinylidene difluoride (PVDF) membrane. DIDS, 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, bound predominantly to 30 kDa protein on the PVDF membrane, the molecular weight of which was similar to one of the MYTX binding proteins. The maximum (45)Ca(2+) release induced by caffeine (30 mM) was further increased in the presence of MYTX (10 microM) or DIDS (30 microM), whereas that induced by DIDS (30 microM) was not affected by MYTX (10 microM). MYTX inhibited [(3)H]DIDS binding to HSR in a concentration-dependent manner. Furthermore, [(125)I]MYTX binding to 30 kDa protein was inhibited by DIDS in a concentration-dependent manner. These results suggest that MYTX and DIDS release Ca(2+) from HSR in a common mechanism. The 30 kDa protein may be a target protein for the Ca(2+) releasing action of MYTX and DIDS.     

Ketoconazole-evoked [Ca2+]i rises and non-Ca2+-triggered cell death in rabbit corneal epithelial cells (SIRC)

The effect of ketoconazole on cytosolic free Ca2+ concentrations ([Ca2+]i) and proliferation has not been explored in corneal cells. This study examined whether ketoconazole alters Ca2+ levels and causes cell death in SIRC rabbit corneal epithelial cells. [Ca2+]i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Ketoconazole at concentrations of 5 microM and above increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. The ketoconazole-induced Ca2+ influx was insensitive to L-type Ca2+ channel blockers and protein kinase C modulators. In Ca2+-free medium, after pretreatment with 50 microM ketoconazole, thapsigargin-(1 microM)-induced [Ca2+]i rises were abolished; conversely, thapsigargin pretreatment nearly abolished ketoconazole-induced [Ca2+]i rises. Inhibition of phospholipase C with 2 microM U73122 did not change ketoconazole-induced [Ca2+]i rises. At concentrations between 5 and 100 microM, ketoconazole killed cells in a concentration-dependent manner. The cytotoxic effect of 50 microM ketoconazole was not reversed by prechelating cytosolic Ca2+ with BAPTA. In summary, in corneal cells, ketoconazole-induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum and Ca2+ influx from unknown pathways. Furthermore, the cytotoxicity induced by ketoconazole was not caused via a preceding [Ca2+]i rise.     

Effect of dihydroergocryptine and dihydroergocristine on cyclic AMP accumulation and prolactin release in vitro: evidence for a dopaminomimetic action

Dihydroergocryptine and dihydroergocristine, two C-9, 10-hydrogenated ergot alkaloids, inhibited in a concentration-dependent manner prolactin release and cyclic AMP accumulation in cultured anterior pituitary cells. The inhibitory effect of dihydroergocryptine was more potent and started at lower concentrations than that of dihydroergocristine. Haloperidol and pimozide, two dopamine receptor antagonists, completely abolished the inhibitory activity of the ergot alkaloids. The involvement of the adenylate cyclase-cyclic AMP system in the inhibitory action of the two compounds was demonstrated by the antagonism by pertussis toxin of the reduction of both prolactin release and cyclic AMP accumulation produced by dihydroergocryptine and dihydroergocristine.     

The antidepressant mirtazapine-induced cytosolic Ca2+ elevation and cytotoxicity in human osteosarcoma cells

The effect of the antidepressant mirtazapine on cytosolic free Ca2+ concentration ([Ca2+]i) and viability has not been explored in any cell type. This study examined whether mirtazapine alters Ca2+ levels and causes cell death in osteoblast-like cells using MG63 human osteosarcoma cells as a model. [Ca2+]i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Mirtazapine at concentrations above 250 microM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 60% by removing extracellular Ca2+. The mirtazapine-induced Ca2+ influx was sensitive to blockade of nifedipine and verapamil. In Ca(2+)-free medium, after pretreatment with 1.5 mM mirtazapine, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 2 microM CCCP (a mitochondrial uncoupler), and 1 microM ionomycin failed to release more stored Ca2+; conversely, pretreatment with thapsigargin, CCCP and ionomycin abolished mirtazapine-induced Ca2+ release. Inhibition of phospholipase C with 2 microM U73122 did not change mirtazapine-induced [Ca2+]i, increase. Seal of Ca2+ movement across the plasma membrane with 50 microM extracellular La3+ enhanced 1 microM thapsigargin-induced [Ca2+]i increase, suggesting that Ca2+ efflux played a role in lowering thapsigargin-induced [Ca2+]i increase; however, the same La3+ treatment did not alter mirtazapine-induced [Ca2+]i increase. At concentrations of 500 microM and 1000 microM, mirtazapine killed 30% and 60% cells, respectively. The cytotoxicity was not reversed by chelating cytosolic Ca2+ with BAPTA. Collectively, in MG63 cells, mirtazapine induced a [Ca2+]i increase by causing Ca2+ release from stores and Ca2+ influx from extracellular space. Furthermore, mirtazapine caused cytotoxicity at higher concentrations in a Ca(2+)-dissociated manner.     

Alpha7 nicotinic receptor activation inhibits ethanol-induced mitochondrial dysfunction,cytochrome c release and neurotoxicity in primary rat hippocampal neuronal cultures

Primary hippocampal neuronal cultures exhibited a concentration- and time-dependent loss of cells when exposed to ethanol (EtOH). EtOH-induced neurotoxicity was attenuated by 2,4-dimethoxybenzilidene anabaseine (DMXB) which selectively activates alpha7 nicotinic receptors in a concentration-dependent manner. We further investigated the mechanisms of the protective effect of DMXB on EtOH- induced neurotoxicity. We found that EtOH decreased the mitochondrial membrane potential and released cytochrome c from mitochondria at neurotoxic concentrations. DMXB (3 microm) attenuated both of these actions in a manner that was in turn blocked with the nicotinic antagonist methyllyconitine (MLA) 100 nm. Neither DMXB nor MLA alone affected these parameters. These results suggest that the neuroprotection conferred by alpha7 nicotinic receptor activation may be mediated, at least in part, through preventing the decrease in the mitochondrial membrane potential and the increase in the release of cytochrome c caused by EtOH.     

Regulation of Neuronal Nitric Oxide and Cyclic GMP Formation by Ca2+

Nitric oxide (NO) acts as a messenger molecule in the CNS by activating soluble guanylyl cyclase. Rat brain synaptosomal NO synthase was stimulated by Ca2+ in a concentration-dependent manner with half-maximal effects observed at 0.3 microM and 0.2 microM when its activity was assayed as formation of NO and L-citrulline, respectively. Cyclic GMP formation was apparently inhibited, however, at Ca2+ concentrations required for the activation of NO synthase, indicating a down-regulation of the signal in NO-producing cells. Purified synaptosomal guanylyl cyclase was not inhibited directly by Ca2+, and the effect was not mediated by a protein binding to guanylyl cyclase at low or high Ca2+ concentrations. In cytosolic fractions, the breakdown of cyclic GMP, but not that of cyclic AMP, was highly stimulated by Ca2+, and 3-isobutyl-1-methylxanthine did not block this reaction effectively. The effects of Ca2+ on cyclic GMP hydrolysis and on apparent guanylyl cyclase activities were abolished almost completely in the presence of the calmodulin antagonist calmidazolium, whose effect was attenuated by added calmodulin. Thus, a Ca2+/calmodulin-dependent cyclic GMP phosphodiesterase is highly active in synaptic areas of the brain and may prevent elevations of intracellular cyclic GMP levels in activated, NO-producing neurons.     

2-Hydroxymethyl-1-naphthol diacetate (TAC) suppresses the superoxide anion generation in rat neutrophils

We have investigated the inhibitory effect of 2-hydroxymethyl-1-naphthol diacetate (TAC) on the respiratory burst of rat neutrophils and the underlying mechanism of action was also assessed in this study. TAC caused concentration-related inhibition of the formylmethionyl-leucyl-phenylalanine (fMLP) plus dihydrocytochalasin B (CB)- and phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O2*-) generation (IC50 10.2+/-2.3 and 14.1+/-2.4 microM, respectively) and O2 consumption (IC50 9.6+/-2.9 and 13.3+/-2.7 microM, respectively) of neutrophils. TAC did not scavenge the generated O2*- during dihydroxyfumaric acid autoxidation. TAC inhibited both the transient elevation of [Ca2+]i in the presence or absence of [Ca2+]o (IC50 75.9+/-8.9 and 84.7+/-7.9 microM, respectively) and the generation of inositol trisphosphate (IP3) (IC50 72.0+/-9.7 microM) in response to fMLP. Cytosolic phospholipase C (PLC) activity was also reduced by TAC at a same range of concentrations. The PMA-induced PKC-beta associated to membrane was attenuated by TAC (about 80% inhibition at 30 microM). Upon exposure to fMLP, the cellular cyclic AMP level was decreased in neutrophils pretreated with TAC. TAC attenuated fMLP-induced phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 (IC50 17.4+/-1.7 microM), but not p38. The cellular formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt) induced by fMLP was inhibited by TAC in a concentration-dependent manner (IC50 25.4+/-2.4 and 25.9+/-1.4 microM, respectively). TAC had no effect on the O2*- generation of PMA-stimulated and arachidonic acid (AA)-stimulated NADPH oxidase preparations. However, TAC caused concentration-related decrease of the membrane associated p47phoX in PMA-stimulated neutrophils (about 80% inhibition at 30 microM). We conclude that inhibition by TAC of the neutrophil respiratory burst is probably attributable to the blockade of the p42/44 MAPK and phospholipase D (PLD) pathways, the membrane translocation of PKC, and to the failure in assembly of a functional NADPH oxidase complex. Blockade of the PLC pathway by TAC probably plays a minor role.     

Differential presynaptic effects of hexachlorocyclohexane isomers on noradrenaline release in cerebral cortex.

To investigate presynaptic effects of hexachlorocyclohexane (HCH) isomers, the release of noradrenaline (NA) in brain tissue was analyzed using rat cerebral cortical slices preloaded with [3H]-NA. gamma-HCH (lindane) 50 microM significantly enhanced the [3H]-NA release evoked by 15-25 mM K+. alpha- and beta-HCH (50 microM) did not produce any significant effect on K(+)-evoked [3H]-NA release. delta-HCH (50 microM) induced a significant decrease of the 25 mM K(+)-evoked release of [3H]-NA. The effect of the gamma- and delta-HCH isomers on the presynaptic action of the alpha 2-agonist clonidine and the alpha 2-antagonist yohimbine was also studied. The presynaptic inhibitory effect of clonidine and the stimulatory effect of yohimbine on [3H]-NA release was attenuated by lindane and delta-HCH, respectively. These results are consistent with a presynaptic action of the HCH isomers on noradrenergic release processes.     

Enhancement of Stimulation-Evoked Catecholamine Release from Cultured Bovine Adrenal Chromaffin Cells by Forskolin

Acetylcholine (ACh) increased cyclic AMP levels in cultured bovine chromaffin cells with a peak effect at 1 min after the addition. Pretreatment with forskolin (0.3 microM) enhanced the ACh-evoked cyclic AMP increase. The catecholamine (CA) release induced by ACh was enhanced by forskolin, but forskolin alone did not enhance the CA release. The effect of forskolin increased dose-dependently up to 1 microM, but decreased at higher concentrations. Dibutyryl cyclic AMP (DBcAMP) also enhanced ACh-evoked CA release, but the effect was less potent than that of forskolin. Forskolin enhanced both [3H]norepinephrine ([3H]NE) and endogenous CA release evoked by 30 mM K+ from cells that were preloaded with [3H]NE. The effects of forskolin were substantial when CA release was evoked with low concentrations of ACh or excess K+, but decreased with higher concentrations of the stimulants. Forskolin also enhanced the CA release induced by ionomycin and veratrine, or by caffeine in Ca2+-free medium. The potentiation by forskolin of the ACh-evoked CA release was manifest in low Ca2+ concentrations in the medium, but decreased when Ca2+ concentration was increased. These results suggest that cyclic AMP may play a role in the modulation of CA release from chromaffin cells.     

Effects of Antidepressant Drug Treatments on α2-Adrenoceptor Control of [3H]Noradrenaline Release from Hypothalamic Synaptosomes

KCl (16 mM) stimulated the release of [3H]noradrenaline ([3H]NA) from rat hypothalamic synaptosomes in a Ca2+-dependent manner; this release was attenuated by clonidine (0.01-100 microM). Changes in the release of [3H]NA and the functional status of alpha 2-adrenoceptors in the medial hypothalamus of rats treated acutely and chronically with clorgyline (1 mg/kg/day) or desipramine (DMI, 10 mg/kg/day) were assessed using superfused synaptosomes in which the attenuating effects of clonidine (1 microM) or the potentiating effects of yohimbine (1 microM) on K+-evoked release of [3H]NA were measured. After acute administration of DMI, significantly less [3H]NA was accumulated into synaptosomes. Although total (spontaneous + K+-evoked) [3H]NA release from these synaptosomes was unchanged, a significant reduction was apparent in the K+-evoked release from the DMI-treated tissue. Attenuation of K+-evoked release by clonidine was abolished in both these acute treatment groups. Following the chronic antidepressant drug regimens, [3H]NA uptake into DMI-treated tissue remained significantly reduced although total percent and K+-evoked [3H]NA release were unchanged. The K+-evoked release of [3H]NA in S1 was significantly enhanced (by 22%) in the clorgyline treatment group. Attenuation of K+-evoked [3H]NA release by clonidine in both chronic antidepressant-treated tissues was not significantly changed. It is concluded that the functional sensitivity of alpha 2-adrenoceptors on nerve endings in the medial hypothalamus is unchanged by these chronic antidepressant drug regimens. In synaptosomes from untreated tissue, yohimbine significantly potentiated K+-evoked release of [3H]NA; this effect was unchanged after acute regimens and reduced after chronic administration of both the antidepressants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulatory release of lipoprotein lipase activity from rat fat pads by vanadate

Vanadate stimulated the release of lipoprotein lipase (LPL) activity from rat fat pads into the medium in a time- and dose-dependent manner. It exerted the synergetic effect with heparin. The stimulatory effects of vanadate and heparin were decreased by incubation in Na+- or Ca2+-free media but were well preserved in K+-free medium. Amiloride inhibited the vanadate-stimulated release of LPL activity in a dose-dependent manner, but did not inhibit the heparin-stimulated release of LPL activity. Colchicine, antimycin A, and carbonyl cyanide m-chlorophenylhydrazone suppressed the stimulatory effect of vanadate, but cycloheximide did not. Preincubation of the fat pads with the tetrakis (acetoxymethyl) ester of quin 2 (quin 2-AM) inhibited the vanadate-stimulatory release of LPL activity without affecting basal activity. The concentration required for half-maximal inhibition of the action of vanadate by quin 2-AM was calculated to be 39 microM, suggesting that the action of vandate was dependent on intracellular Ca2+ concentration. The heparin-stimulated release, on the other hand, was not inhibited even at higher concentrations of quin 2-AM (up to 200 microM). These findings suggest that vanadate stimulates the release of LPL activity through mechanisms of action involving amiloride-sensitive and calcium-dependent pathways with a requirement of metabolic energy.