Muscarinic receptor subtypes and calcium signaling in Fischer rat thyroid cells

A specific and saturable binding site for [3H]N-methyl-scopolamine ([3H]NMS) was observed in plasma membrane of Fischer rat thyroid (FRT) cells with an equilibrium dissociation constant (K(d)) of 0.11 +/- 0.02 nM and a concentration of receptor sites (B(max)) of 14.1 +/- 3.9 fmol/mg protein. Pharmacological characterization of this binding site using pirenzepine, himbacine, (11(2-diethyl-amino)methyl)-1-piperidinylacetyl-5-11-dihydro-6H-pyrido(14) benzodiazepine (AF-DX 116), dicyclomine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), and hexahydro-sila-difenidol (HHSD) showed clear differences, in terms of affinities, between these muscarinic receptor antagonists. The order of potency for inhibiting [3H]NMS binding was HHSD = dicyclomine > 4-DAMP > pirenzepine = himbacine > AF-DX 116. These findings suggest that the muscarinic receptors found in FRT cells belong to the M3 subtype. Stimulation of FRT cells with carbachol produced a biphasic and dose-dependent increase in the intracellular calcium concentration ([Ca2+]i), which was blocked in pretreated cells with atropine and almost abolished by a low concentration of 4-DAMP and HHSD. Removal of extracellular Ca2+ from the incubation medium reduced the initial transient peak and completely abolished the plateau phase, while the transient phase was markedly reduced by the phospholipase C inhibitor U73122. These data indicate that [Ca2+]i results from both Ca2+ influx across Ca2+ channels and mobilization of Ca2+ from intracellular Ca2+ stores. The present data showed the presence of the M3 muscarinic acetylcholine receptor subtype in plasma membrane of FRT cells, which may influence cellular function via modulation of [Ca2+]i.     

M1 muscarinic receptors mediate intracellular calcium release in NB-OK1 human neuroblastoma cells

Summary Muscarine acetylcholine receptors were characterized in NB-OK1 cells using radioligand (³H-NMS) binding experiments and second messenger (calcium and phosphatidylinositol (PI) turnover) studies. In radioligand binding experiments the displacement curves of pirenzepine (K_I = 1.3 × 10^–8 M), AF-Dx 116 (KI = 8.2 × 10^–7 M), methoctramine (KI = 8.4 × 10^–8 M) and parafluorohexahydrosiladifenidol (pF-HHSiD) (K1 = 1.8 × 10^–8 M) were monophasic and indicated the presence of M 1 muscarinic receptors. Schild analysis with the muscarinic antagonists pirenzepine, AF-Dx 116, methoctramine and pF-HHSiD yielded pA₂ values of 8.40 +- 0.13, 6.48 =- 0.09, 7.61 +- 0.12 and 7.22 +- 0.08 in the calcium experiments and pA₂ values of 8.13 +- 0.30 and 6.26 +- 0.26, 7.65 +- 0.16 and 7.46 +- 0.11, respectively, in the PI turnover experiments. These results indicate that both the carbachol-induced increase in intracellular calcium and the increase in PI turnover are mediated by M1 muscarinic receptors. In calcium free buffer, stimulation with carbachol induced similar responses to those seen under control conditions. From functional and radioligand binding experiments we conclude that the muscarinic receptor expressed in NB-OK1 cells is the M1 subtype. In addition, the M1 receptor-induced calcium response is related to PI turnover and is independent on extracellular calcium. Send offprint requests to H. W. G. M. Boddeke at the above address     

Role of intracellular calcium in histamine release from rat mast cells

An aqueous extract of Panax Ginseng C.A. Meyer (G.S.) was prepared by boiling crushed G.S. roots in water. The extract obtained was adjusted to 125 mg G.S. per ml and was administered orally to mice for 5 to 6 days at the daily dose of 10, 50 and 250 mg G.S. per kg or was added to cultures of mouse spleen cells at concentrations varying between 0.25 and 8 mg G.S. per ml. The average total ginsenoside content of the G.S. roots used was determined by HPLC analysis and found to be 0.58% (w/w). Treated mice responded with enhanced antibody formation to either a primary or a secondary challenge with sheep red cells. The effects were dose-dependent. At the highest dose regimen, the primary IgM response was increased by 50% and the secondary IgG and IgM responses were increased by 50 and 100%, respectively. An even more pronounced effect was obtained with natural killer cell activity which was enhanced between 44 and 150% depending on the effector-to-target cell ratios used in the assay. In vitro, G.S. showed two main effects, an inhibition of stimulated and spontaneous lymphocyte proliferation at high, but not cytotoxic concentrations and an enhancement of interferon production particularly in non-stimulated spleen cells. The immunostimulating effects obtained in vivo are in agreement with the stimulation of interferon production observed in vitro. The inhibition of lymphocyte proliferation, however, cannot be reconciled with the immunostimulatory action of G.S. observed in vivo.     

delta- and mu-opioid receptor mobilization of intracellular calcium in SH-SY5Y human neuroblastoma cells.

1. In this study we have investigated delta and mu opioid receptor-mediated elevation of intracellular Ca2+ concentration ([Ca2+]i) in the human neuroblastoma cell line, SH-SY5Y. 2. The Ca(2+)-sensitive dye, fura-2, was used to measure [Ca2+]i in confluent monolayers of SH-SY5Y cells. Neither the delta-opioid agonist, DPDPE ([D-Pen2,5]-enkephalin) nor the mu-opioid agonist, DAMGO (Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol enkephalin) elevated [Ca2+]i when applied alone. However, when either DPDPE or DAMGO was applied in the presence of the cholinoceptor agonist, carbachol (100 nM-1 mM) they evoked an elevation of [Ca2+]i above that caused by carbachol alone. 3. In the presence of 1 microM or 100 microM carbachol, DPDPE elevated [Ca2+]i with an EC50 of 10 nM. The elevation of [Ca2+]i was independent of the concentration of carbachol. The EC50 for DAMGO elevating [Ca2+]i in the presence of 1 microM and 100 microM carbachol was 270 nM and 145 nM respectively. 4. The delta-receptor antagonist, naltrindole (30 nM), blocked the elevations of [Ca2+]i by DPDPE (100 nM) without affecting those caused by DAMGO while the mu-receptor antagonist, CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Pen-Thr-NH2) (100 nM-1 microM) blocked the elevations of [Ca2+]i caused by DAMGO (1 microM) without affecting those caused by DPDPE. 5. Block of carbachol activation of muscarinic receptors with atropine (10 microM) abolished the elevation of [Ca2+]i by the opioids. The nicotinic receptor antagonist, mecamylamine (10 microM), did not affect the elevations of [Ca2+]i caused by opioids in the presence of carbachol. 6. Muscarinic receptor activation, not a rise in [Ca2+]i, was required to reveal the opioid response. The Ca2+ channel activator, maitotoxin (3 ng ml-1), also elevated [Ca2+]i but subsequent application of opioid in the presence of maitotoxin caused no further changes in [Ca2+]i. 7. The elevations of [Ca2+]i by DPDPE and DAMGO were abolished by pretreatment of the cells with pertussis toxin (200 ng ml-1, 16 h). This treatment did not significantly affect the response of the cells to carbachol. 8. The opioids appeared to elevate [Ca2+]i by mobilizing Ca2+ from intracellular stores. Both DPDPE and DAMGO continued to elevate [Ca2+]i when applied in nominally Ca(2+)-free external buffer or when applied in a buffer containing a cocktail of Ca2+ entry inhibitors. Thapsigargin (100 nM), an agent which discharges intracellular Ca2+ stores, also blocked the opioid elevations of [Ca2+]i. 9. delta and mu Opioids did not appear to mobilize intracellular Ca2+ by modulating the activity of protein kinases. The application of H-89 (10 microM), an inhibitor of protein kinase A, H-7 (100 microM), an inhibitor of protein kinase C, protein kinase A and cyclic GMP-dependent protein kinase, or Bis I, an inhibitor of protein kinase C, did not alter the opioid mobilization of [Ca2+]i. 10. Thus, in SH-SY5Y cells, opioids can mobilize Ca2+ from intracellular stores but they require ongoing muscarinic receptor activation. Opioids do not elevate [Ca2+]i when applied alone.     

Distinct mixtures of muscarinic receptor subtypes mediate inhibition of noradrenaline release in different mouse peripheral tissues, as studied with receptor knockout mice

The muscarinic heteroreceptors modulating noradrenaline release in atria, urinary bladder and vas deferens were previously studied in mice in which the M(2) or the M(4) muscarinic receptor genes had been disrupted. These experiments showed that these tissues possessed both M(2) and non-M(2) heteroreceptors. The analysis was now extended to mice in which either the M(3), both the M(2) and the M(3), or both the M(2) and the M(4) genes had been disrupted (M(3)-knockout, M(2/3)-knockout and M(2/4)-knockout). Tissues were preincubated with (3)H-noradrenaline and then stimulated electrically (20 pulses per 50 Hz). In wild-type atria, carbachol (0.01-100 microM) decreased the electrically evoked tritium overflow by maximally 60-78%. The maximum inhibition of carbachol was reduced to 57% in M(3)-knockout and to 23% in M(2/4)-knockout atria. Strikingly, the effect of carbachol was abolished in M(2/3)-knockout atria.In wild-type bladder, carbachol (0.01-100 microM) reduced the evoked tritium overflow by maximally 57-71%. This effect remained unchanged in the M(3)-knockout, but was abolished in the M(2/4)-knockout bladder. In wild-type vas deferens, carbachol (0.01-100 microM) reduced the evoked tritium overflow by maximally 34-48%. The maximum inhibition of carbachol was reduced to 40% in the M(3)-knockout and to 18% in the M(2/4)-knockout vas deferens. We conclude that the postganglionic sympathetic axons of mouse atria possess M(2) and M(3), those of the urinary bladder M(2) and M(4), and those of the vas deferens M(2), M(3) and M(4) release-inhibiting muscarinic receptors.     

Adrenoreceptor mobilization of calcium in rat submandibular cells

The stimulation of adrenoreceptors by epinephrine leads to an alteration in the Ca2+ homeostasis of rat submandibular cells. The relative contributions of alpha- and beta-adrenoreceptors were assessed with specific adrenergic agents. Stimulation of alpha 1-adrenoreceptors resulted in enhanced unidirectional 45Ca2+ fluxes, while such effects following beta-adrenoreceptor stimulation, though suggestive, were equivocal. When the cytosolic Ca2+ level was followed in cells pre-loaded with the Ca2+-sensitive fluorescent indicator Quin 2, clear results were observed. Both alpha 1- and beta-adrenoreceptor stimulation were capable of mobilizing intracellular and extracellular Ca2+ pools, as assessed by differential responses of cells incubated in media (+/-)-ethylene-glycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid. However, Ca2+ mobilization following alpha 1-adrenoreceptor stimulation was more rapid (initial rate approximately 4-5 X) and to a greater extent (approximately 2 X) than seen with beta-adrenoreceptor stimulation.     

NMDA and AMPA receptors mediate intracellular calcium increase in rat cortical astrocytes

INTRODUCTION Almost 50 % of cells in central nervous systemare astrocytes. They play an important role in normalphysiological activity andhave intimate relationship withneurons. There is a close bidirectional communicationexisting between neurons and astrocytes[1]. Glutamate,as the most important excitatory transmitter in centralnervous system, is proved to be a crucial bridge be-tween astrocytes and neurons. Astrocytes respondedto glutamate released from neurons by intracellular C…     

The ACh-induced contraction in rat aortas is mediated by the Cys Lt1 receptor via intracellular calcium mobilization in smooth muscle cells

1. Our previously published data indicate that an endogenously produced 5-lipoxygenase metabolite can strongly contract isolated endothelium-preserved rat aortic strips when cyclo-oxygenase isoenzymes are inhibited. Therefore, we decided to investigate if cysteinyl-containing leukotrienes (Cys Lts) are involved in this endothelium-dependent contraction. 2. The isometric contraction of endothelium-preserved rat aortic strips was recorded in preparations preincubated with 5 microM indomethacin and precontracted with phenylephrine, adjusting resting tension at 0.7 g. Acetylcholine (ACh) contracted control strips. Montelukast and MK-571, selective type 1 Cys Lts receptor (Cys Lt(1)) antagonists and the Cys Lt(1)/Cys Lt(2) (type 2 Cys Lts receptor) antagonist BAYu9773 dose-dependently prevented ACh-induced contraction, their IC(50)s being 2.2, 3.1 and 7.9 nM respectively. The leukotriene B4 receptor antagonist U75302 was far less potent (IC(50) 1.5 microM). 3. In rat aorta smooth muscle cells (RASMs), Western blot analysis showed the presence of Cys Lt(1) and Cys Lt(2) receptors, the Cys Lt(1) receptor being predominantly expressed. 4. In fura-2 loaded RASMs, LTD4 (0.01-100 nM) and LTC4 (200-800 nM) dose-dependently increased intracellular calcium concentration ([Ca(2+)](i)). Montelukast (1-100 nM) reduced LTD4-induced [Ca(2+)](i) increase, its IC(50) being approximately 10 nM. BAY u9773 exhibited significantly low effectiveness. 5. LTD4 (10 nM) induced a redistribution of smooth muscle actin fibres throughout the cytoplasm as visualized by confocal microscopy. 6. In conclusion, Cys Lt(1) activation by endogenously produced Cys Lts, can contract rat aortas, while Cys Lt(2) only marginally influences aortic tone. Intracellularly, this effect is mediated by an increase in [Ca(2+)](i). Therefore, Cys Lts, by inducing vascular contraction, can contribute to systemic hypertension.     

Oxidizing effects of vanadate on calcium mobilization and amylase release in rat pancreatic acinar cells.

The effects of vanadate were examined by monitoring intracellular free calcium concentration ([Ca2+]i) and amylase secretion in collagenase-dispersed rat pancreatic acinar cells. Vanadate increased [Ca2+]i by mobilizing calcium from agonist-releasable intracellular calcium stores, since this increase was observed in the absence of extracellular calcium and vanadate failed to increase [Ca2+]i after treatment with thapsigargin in calcium-free medium. Moreover, pretreatment of acinar cells with vanadate prevented the cholecystokinin octapeptide (CCK-8)-induced signal of [Ca2+]i, whereas co-incubation with CCK-8 potentiated the plateau phase of calcium response to CCK-8 without modifying the transient calcium spike. The effects of vanadate on calcium mobilization were reversed by the presence of the sulfhydryl reducing agent dithiothreitol. Vanadate also activated the calcium influx, since an additional enhancement of calcium influx induced by thapsigargin-evoked intracellular store depletion was observed and vanadate reversed the inhibitory effect of lanthanum (an inhibitor of calcium entry) into acinar cells. In addition, vanadate evoked a concentration-dependent release of amylase from pancreatic acinar cells and moreover, reduced the secretory response to CCK-8. We conclude that, in pancreatic acinar cells, vanadate releases calcium from the agonist-releasable intracellular calcium pool and consequently induces amylase secretion. These effects are likely due to the oxidizing effects of this compound.     

Serotonin-induced platelet intracellular calcium mobilization in depressed patients

Serotonin(5-HT)-stimulated intracellular calcium(Ca) mobilization was measured in the platelets of depressed patients to assess 5-HT₂ receptor function. The 5-HT-induced Ca response was significantly higher in unmedicated patients with bipolar depression and melancholic major depression than in those with non-melancholic major depression and normal controls. The enhanced Ca response to 5-HT failed to correlate with severity of depressive symptoms. In patients with bipolar disorder and melancholic major depression, there was no significant difference in 5-HT-stimulated Ca response between the unmedicated group and those in remission. These results suggest that 5-HT₂ receptor function is increased in some types of depression, and raise the possibility that the enhanced Ca response to 5-HT may be trait dependent rather than state dependent.     

Effects of ginsenosides on the glutamate release and intracellular calcium levels in cultured rat cerebellar neuronal cells

These studies were designed to examine the effects of ginsenosides on glutamate neurotransmission. In primary cultures of rat cerebellar granule cells, ginsenosides (Rb1, Rc and Rg1, 500 μg/ml) increased glutamate release which was measured by HPLC, but Re did not show an elevation of glutamate release. However, all of these ginsenosides down-regulated N-methyl-D-aspartate (NMDA)-induced glutamate release. Rc strongly increased glutamate release and elevated intracellular calcium concentrations (Ca²⁺ _i) which was measured by ratio fluorometry with FURA-2 AM. These, results indicate that ginsenosides have a homeostatic effect on glutamate neurotransmission, and there is a structure-function relationship among the ginsenosides tested.     

Desnitro-imidacloprid activates the extracellular signal-regulated kinase cascade via the nicotinic receptor and intracellular calcium mobilization in N1E-115 cells

Imidacloprid (IMI) is the principal neonicotinoid (the only major new class of synthetic insecticides of the past three decades). The excellent safety profile of IMI is not shared with a metabolite, desnitro-IMI (DNIMI), which displays high toxicity to mammals associated with agonist action at the alpha4beta2 nicotinic acetylcholine receptor (nAChR) in brain. This study examines the hypothesis that IMI, DNIMI, and (-)-nicotine activate the extracellular signal-regulated kinase (ERK) cascade via primary interaction with the alpha4beta2 nAChR in mouse neuroblastoma N1E-115 cells. These three nicotinic agonists induce phosphorylation of ERK (p44/p42) in a concentration-dependent manner with an optimal incubation period of 30 min. DNIMI (1 microM)-induced ERK activation is blocked by nicotinic antagonist mecamylamine but not by alpha-bungarotoxin and muscarinic antagonist atropine. This activation is prevented by intracellular Ca(2+) chelator BAPTA-AM but not by removal of external Ca(2+) using EGTA and Ca(2+)-free medium. 2-Aminoethoxy-diphenylborate, a blocker for inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release from intracellular stores, inhibits DNIMI-induced ERK activation but a high level of ryanodine (to block ryanodine receptor-mediated Ca(2+) release) does not. The inhibitor U-73122 for phospholipase C (to suppress IP(3) production) prevents ERK activation evoked by DNIMI. Inhibitors for protein kinase C (PKC) (GF109203X) and ERK kinase (PD98059) block this activation whereas an inhibitor (H-89) for cyclic AMP-dependent protein kinase does not. Thus, neonicotinoids activate the ERK cascade triggered by primary action at the alpha4beta2 nAChR with an involvement of intracellular Ca(2+) mobilization possibly mediated by IP(3). It is further suggested that intracellular Ca(2+) activates a sequential pathway from PKC to ERK.     

Somatostatin (SRIF) modulates distinct signaling pathways in rat pituitary tumor cells; negative coupling of SRIF receptor subtypes 1 and 2 to arachidonic acid release

The somatotropin release-inhibiting factor somatostatin-14 (SRIF) is known to activate distinct receptor subtypes (sst1-5). In rat pituitary tumor cells (GC cells), sst2 but not sst1 receptors mediate the SRIF-induced inhibition of intracellular concentration of Ca2+ ([Ca2+]i) and are negatively coupled to cAMP-dependent pathways. In the present study, transduction mechanisms coupling distinct SRIF receptors to their specific functional role were investigated with the use of both SRIF agonists with well-known affinity at individual SRIF receptors and the sst2 receptor antagonist L-Tyr(8) isomer of Cyanamid 154806 (CYN-154806). Our results demonstrate that sst1 and sst2 receptors are coupled to distinct signaling pathways in GC cells. In particular, sst2 receptors are negatively coupled to the cAMP-dependent pathway and this pathway is partially responsible for the sst2 receptor-mediated inhibition of [Ca2+]i. In addition, sst1 and sst2 receptors are both coupled to a decrease of arachidonic acid (AA) release with an efficacy similar to that of SRIF, suggesting that SRIF reduces AA release through either a partial activation of both receptors or the activation of one at a time. This finding is important given the well-accepted role for phospholipase A2 (PLA2) as a positive signaling component in transduction pathways of SRIF receptors. sst1 and sst2 receptor negative coupling to PLA2/AA pathways does not seem to be implicated in the SRIF-induced inhibition of [Ca2+]i. The possible role for the SRIF-mediated inhibition of AA release in GC cell function remains to be elucidated.     

M3-subtype muscarinic receptor that controls intracellular calcium release and inositol phosphate accumulation in gastric parietal cells.

The muscarinic receptor subtype which triggers acid secretion was investigated in isolated rabbit gastric parietal cells. Cytosolic free Ca2+ concentration ([Ca2+]i), measured with the fluorescent indicator FURA-2, increased rapidly after full agonist (carbachol) stimulation (6-8 sec), then returned to an intermediate sustained value. Other M2-agonists, oxotremorine and arecoline, produced a partial [Ca2+]i increase, whereas M1-agonists, pilocarpine and [4-m-chlorophenylcarbamoyloxyl]-2-butynyl-trimethylammonium, were without any significant effect. [Ca2+]i rise was inhibited by selective muscarinic antagonists: atropine greater than 4-diphenylacetoxy-N-methyl-piperidine methbromide greater than quinuclidinylbenzilate (QNB) greater than pirenzepine greater than 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one, this sequence being characteristic of the involvement of an M3-subtype. This inhibition was shown to be stereoselective; dexetimide and (-)QNB were more potent than levetimide and (+)QNB. The IC50 values for inhibition of [Ca2+]i increase by muscarinic antagonists were in good agreement with those obtained for inhibition of phospholipase C activation. In conclusion, the muscarinic receptor that controls acid secretion appears to be of the M3-subtype and the biochemical events coupled to the activation of this receptor system are also controlled through the same subtype.     

Different mu receptor subtypes mediate spinal and supraspinal analgesia in mice

To examine the relative roles of mu 1- and mu 2-receptors in spinal and supraspinal analgesia, we assessed the effects of naloxonazine, naloxone, beta-funaltrexamine (beta-FNA), and ICI-154,129 on tail-flick analgesia produced by intrathecal or intracerebroventricular injections of the highly mu-selective agonist, [D-Ala2,Me-Phe4,Gly(ol)5]enkephalin (DAGO; mu 1 and mu 2), [D-Ser2,Leu5]enkephalin-Thr6 (DSLET; mu 1 and delta), and the selective delta-receptor agonist [D-Pen2,D-Pen5]enkephalin (DPDPE) in mice. Both DAGO and DSLET supraspinal analgesia were mediated through mu 1-receptors. Naloxonazine shifted the supraspinal DAGO dose-response curve 4-fold to the right without changing the curve for spinal DAGO. Likewise, naloxonazine pretreatment shifted supraspinal DSLET analgesia 10-fold, whereas spinal DSLET analgesia was not affected. DPDPE analgesia was not antagonized spinally or supraspinally by naloxonazine pretreatment. These findings suggest that DAGO produces analgesia spinally and supraspinally through different sets of mu-receptors. Moreover, at least two distinct receptor subtypes mediated spinal analgesia. First, naloxone inhibited spinal DAGO analgesia more potently than DPDPE analgesia. Second, the irreversible mu-antagonist, beta-FNA, blocks spinal DAGO analgesia. Since spinal DAGO was insensitive to naloxonazine, ruling out a mu 1 mechanism, these results indicate a role for mu 2-receptors. Spinal DAGO analgesia also developed tolerance to morphine far more slowly than supraspinal DAGO analgesia even though mu-receptors mediate both, as indicated by their sensitivity towards beta-FNA. Finally, the delta-antagonist ICI-154,129 is a more potent inhibitor of spinal DPDPE analgesia than spinal DAGO analgesia. Thus, delta-receptors mediate spinal DPDPE analgesia.     

Methylmercury affects multiple subtypes of calcium channels in rat cerebellar granule cells

We tested the ability of methylmercury (MeHg) to block calcium channel current in cultures of neonatal cerebellar granule cells using whole-cell patch clamp techniques and Ba(2+) as charge carrier. Low micromolar concentrations of MeHg (0.25-1 microM) reduced the amplitude of whole cell Ba(2+) current in a concentration- and time-dependent fashion; however, this effect was not voltage-dependent and the current-voltage relationship was not altered. Increasing the stimulation frequency hastened the onset and increased the magnitude of block at both 0.25 and 0.5 microM MeHg but not at 1 microM. In the absence of stimulation, all concentrations of MeHg were able to decrease current amplitude. The ability of several Ca(2+) channel antagonists (omega-conotoxin GVIA, omega-conotoxin MVIIC, omega-agatoxin IVA, calcicludine, and nimodipine) to alter the MeHg-induced effect was tested in an effort to determine if MeHg targets a specific subtype of Ca(2+) channel. Each of the antagonists tested was able to decrease a portion of whole cell Ba(2+) current under control conditions. However, none were able to attenuate the MeHg-induced block of whole cell Ba(2+) current, suggesting either that the mechanism of MeHg-induced block involves sites other than those influenced specifically by Ca(2+) channel antagonists or that MeHg was able to "outcompete" these toxins for their binding sites. These results show that acute exposure to submicromolar concentrations of MeHg can block Ba(2+) currents carried through multiple Ca(2+) channel subtypes in primary cultures of cerebellar granule cells. However, it is unlikely that the presence of a specific Ca(2+) channel subtype is able to render granule cells more susceptible to the neurotoxicologic actions of MeHg.     

Oligopeptides interfering with calcium channels inhibit prolactin and growth hormone release by cultured anterior pituitary cells of the rat

A number of oligopeptides, protected at their N termini and possessing an aldehyde residue at their C terminal amino acids, are able to inhibit 45Ca2+ influx into anterior pituitary cells grown in monolayer culture and depolarized with high extracellular potassium concentration. In addition, the same oligopeptides interfere with hormone release, especially with that produced by lactotrophs. Our findings imply that oligopeptides may represent a new class of calcium channel ligands, and the pituitary cells are sensitive targets for them.     

Characterization of the neuropeptide Y-induced intracellular calcium release in human erythroleukemic cells.

Human erythroleukemic (HEL) cells, loaded with fura-2, respond to neuropeptide Y (NPY) with a fast and transient increase in intracellular calcium. The Y1 receptor-specific agonist (Leu-31,Pro-34)-NPY is 4-fold more potent and the carboxyl-terminal fragment NPY13-36 is 150-fold less potent than NPY. Thus, it is concluded that the response is mediated through the activation of a Y1 type of NPY receptor. HEL cells do not respond to a second addition of NPY but do respond to a further addition of alpha-thrombin (alpha-T). However, in a calcium-free medium, prior stimulation with NPY largely inhibits a subsequent response to alpha-T. Moreover, prior stimulation with alpha-T in the absence of external calcium completely prevents the response to the addition of NPY, indicating a common effector pathway. The latter is further reinforced by using thapsigargin (TG), which has been shown to deplete the Inositol 1,4,5-trisphosphate-dependent calcium pool in other systems. HEL cells preincubated with TG in calcium-free medium fail to respond to either NPY or alpha-T. Likewise, prior stimulation with NPY or alpha-T in calcium-free medium significantly inhibits the response to TG. Preincubation of cells with phorbol esters strongly inhibits the NPY-induced release of intracellular Ca2+ in HEL cells, an effect that is partially prevented by preincubation of the cells with H7, a protein kinase C inhibitor. However, neither the homologous nor the apparent heterologous desensitization of the NPY receptor can be prevented by H7. It is concluded that NPY releases intracellular Ca2+ from an inositol 1,4,5-trisphosphate-sensitive calcium pool, which is restored by external calcium, and that NPY receptor desensitization is protein kinase C independent.