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PubMed2002

Propofol increases pulmonary artery smooth muscle myofilament calcium sensitivity: role of protein kinase C.

BACKGROUND: Vascular smooth muscle tone is regulated by changes in intracellular free Ca2+ concentration ([Ca2+]i) and myofilament Ca2+ sensitivity. These cellular mechanisms could serve as targets for anesthetic agents that alter vasomotor tone. This study tested the hypothesis that propofol increases myofilament Ca2+ sensitivity in pulmonary artery smooth muscle (PASM) via the protein kinase C (PKC) signaling pathway. METHODS: Canine PASM strips were denuded of endothelium, loaded with fura-2/AM, and suspended in modified Krebs-Ringer's buffer at 37 degrees C for simultaneous measurement of isometric tension and [Ca2+]i. RESULTS: The KCl (30 mm) induced monotonic increases in [Ca2+]i and tension. Verapamil, an L-type Ca2+ channel blocker, attenuated KCl-induced increases in [Ca2+]i and tension to an equal extent. In contrast, propofol attenuated KCl-induced increases in [Ca2+]i to a greater extent than concomitant changes in tension and caused an upward shift in the peak tension-[Ca2+]i relation. Increasing extracellular Ca2+ in the presence of 30 mM KCl resulted in similar increases in [Ca2+]i in control and propofol-pretreated strips, whereas concomitant increases in tension were greater during propofol administration. The Ca2+ ionophore, ionomycin (0.1 microm), increased [Ca2+]i to approximately 50% of the value induced by 60 mm KCl. Under these conditions, propofol (10, 100 microm) caused increases in tension equivalent to 11 +/- 2 and 28 +/- 3% of the increases in tension in response to 60 mM KCl, whereas [Ca2+]i was slightly decreased. Similar effects were observed in response to the PKC activator, phorbol 12-myristate 13-acetate (PMA, 1 microm). Specific inhibition of PKC with bisindolylmaleimide I before ionomycin administration decreased the propofol- and PMA-induced increases in tension and abolished the propofol- and PMA-induced decreases in [Ca2+]i. Selective inhibition of Ca2+ -dependent PKC isoforms with Gö 6976 also attenuated propofol-induced increases in tension. CONCLUSION: These results suggest that propofol increases myofilament Ca2+ sensitivity in PASM, and this effect involves the PKC signaling pathway.

Actin Cytoskeleton↗
PubMed1993

Platelet calcium handling in spontaneously hypertensive rats and in three strains of normotensive rats.

OBJECTIVES: Abnormalities in platelet calcium handling have been reported in spontaneously hypertensive rats (SHR) compared with Wistar-Kyoto (WKY) rats. Furthermore, several reports have indicated that WKY rats differ from both SHR and Wistar rats. The objectives of the present study were to investigate platelet calcium handling in three normotensive stains and in SHR, and to confirm the abnormal calcium mobilization in SHR. DESIGN AND METHODS: We compared calcium handling in fura-2-loaded platelets of SHR, Wistar, Sprague-Dawley (SD) and WKY rats. RESULTS: The basal cytosolic free Ca2+ concentration in platelets was significantly higher in SHR and significantly lower in SD rats than in the other strains. The intracellular Ca2+ response to thrombin in the presence of extracellular Ca2+ was greater in SHR than in the three normotensive strains. The thrombin-induced intracellular Ca2+ rise in the absence of extracellular Ca2+ was also greater in SHR and lower in SD rats than in and Wistar rats at higher doses of thrombin. The intracellular ionomycin-released calcium fraction, which may indicate the size of intracellular calcium stores, was similar in SHR, WKY and Wistar rats, and was greater than in SD rats. No difference was detected between WKY and Wistar rats in resting and peak agonist-evoked intracellular Ca2+ concentrations. CONCLUSIONS: These results show that calcium handling in WKY rats is similar to that in Wistar rats with respect to platelet calcium metabolism and confirm the abnormality in SHR. Furthermore, the enhanced intracellular Ca2+ response to thrombin in SHR was not dependent on the size of ionomycin-released Ca2+ stores. In addition, substantial differences in platelet calcium handling may occur even among normotensive strains if the strains are not related.

Animals↗
PubMed1993

The antilymphocytic activity of brequinar sodium and its potentiation by cytidine. Effects on lymphocyte proliferation and cytokine production.

Based on its capacity to inhibit de novo pyrimidine biosynthesis by blocking dihydroorotate dehydrogenase activity, the antitumor agent brequinar sodium (BQR) has emerged as a new immunosuppressive agent. Since BQR is known to prevent the synthesis of nucleotides during cell proliferation, we hypothesized that it would be highly effective in controlling strong lymphocyte proliferative responses but might be less effective in controlling comparatively weak responses that do not necessarily involve new nucleotide synthesis. We addressed this question by culturing murine spleen cells with different types of stimuli, including Con A, phorbol myristate acetate +/- ionomycin, anti-CD3, and anti-Igs. Addition of BQR (0.001 microgram/ml to 10 micrograms/ml) at the start of a 72-hr culture period caused dose-dependent inhibition of strong proliferative responses, induced either by Con A (5 micrograms/ml) or PMA+ionomycin. A residual degree of proliferation persisted, however, even at the highest BQR concentrations. In contrast, no impairment of low-concentration Con A (0.5 or 0.1 microgram/ml), anti-CD3, or anti-Igs responses was observed. In order to ascertain its role in arresting nucleotide synthesis, we attempted to reverse the inhibitory effect of BQR by adding exogenous uridine or cytidine to lymphocyte cultures. BQR's inhibitory activity was reversed completely by adding uridine at 0.1 mM. In contrast, combination of BQR and cytidine (0.1 mM) potentiated BQR's activity and abrogated anti-CD3 or anti-Igs-induced lymphocyte proliferation in a dose-dependent manner. A synergistic inhibitory action between BQR and cytidine was observed when the BQR concentration was higher than 0.1 microgram/ml and with cytidine at 0.1 mM. Production of interleukin-2 and IL-4 was only slightly affected by BQR, but was significantly suppressed by coadministration of BQR and cytidine. Neither BQR (5 micrograms/ml) on its own, however, nor combination of BQR with cytidine affected production of mRNA for IL-2, IL-4, or interferon-gamma, as determined by reverse-transcription polymerase chain reaction. Our observations suggest that BQR may not only affect dihydroorotate dehydrogenase activity, but may also inhibit the enzyme cytidine deaminase, which converts cytidine to uridine. These antimetabolic effects of BQR complement the well-known cytokine synthesis inhibitory actions of FK506 or CsA. The combination of BQR and cytidine, however, offers a further possibility for inhibition of both cytokine production and T and B cell proliferation, and may have potential for the control of graft rejection.

Animals↗
PubMed2005

PP2 inhibits glutamate release from nerve endings by affecting vesicle mobilization.

Src kinase is widely expressed in the brain and its inhibition with PP2 has previously been shown to depress depolarization-evoked glutamate release from rat cerebrocortical synaptosomes by reducing voltage-dependent Ca2+ entry. In this study, we further showed that the inhibitory effect of PP2 on 4-aminopyridine-evoked glutamate release results from a reduction of vesicular exocytosis and not from an inhibition of non-vesicular release. In addition, PP2 significantly inhibited ionomycin-induced or hypertonic sucrose-induced glutamate release. Also, disruption of cytoskeleton organization with cytochalasin D occluded the inhibitory action of PP2 on 4-aminopyridine and ionomycin-evoked glutamate release. These results suggest that PP2-mediated inhibition of glutamate release involves the modulation of some exocytotic steps, possibly through a regulation of actin cytoskeleton dynamics.

Actin Cytoskeleton↗
PubMed1989

Interleukin 1 regulation of interleukin 2 and interferon-gamma gene activation in a human leukemic HSB.2 subclone.

The role of interleukin 1 (IL1) in causing IL2 and interferon-gamma (IFN-gamma) production and their associated gene activation has been studied in a human leukemic HSB.2 subclone. One of the subclones, HSB.2-C5B2 clone #28, which produced only low levels of IL2 and IFN-gamma when stimulated with phytohemagglutinin (PHA) or with a combination of phorbol-myristate-acetate (PMA) and Ca2+ ionophore, ionomycin, showed marked IL2 and IFN-gamma production in the presence of IL1. The augmentation by IL1 was observed in both dot and Northern blot analysis, indicating that IL1 regulates lymphokine genes at the pretranslational level. The kinetics of IL2 and IFN-gamma production were essentially similar for both lymphokines except that there was a faster accumulation of IFN-gamma mRNA than IL2 mRNA. In contrast to the IL2 and IFN-gamma gene activation, IL2 receptor (Tac/p55 antigen) expression was induced directly by IL1 itself as with PMA in this subclone. In these studies, IL1 action was not replaced by the drugs raising intracellular cyclic AMP (cAMP). Taken collectively, these experiments support the notion that IL1 does not trigger IL2 gene activation per se, but amplifies the preactivated lymphokine genes initiated by PMA and ionomycin, whereas IL1 can activate the IL2 receptor gene without any other known signal requirements.

Blotting, Northern↗
PubMed1991

A synthetic peptide homologous to retroviral transmembrane envelope proteins depresses protein kinase C mediated lymphocyte proliferation and directly inactivated protein kinase C: a potential mechanism for immunosuppression.

CKS-17, an immunosuppressive peptide homologous to certain retroviral transmembrane envelope protein, has been shown to inhibit lymphocyte proliferation in response to mitogens or alloantigens when covalently attached to bovine serum albumin (CKS-17-BSA). To define its site of action, we determined if CKS-17 conjugated to human serum albumin (CKS-17-HSA) could block the direct activation of lymphocytes by phorbol-12-myristate-13-acetate (PMA) or by a synthetic diacylglycerol, dioctanoylglycerol (DiC8). CKS-17-HSA inhibited lymphocyte proliferation in response to PMA and ionomycin in a dose-dependent manner with up to 88% inhibition occurring with 15 microM CKS-17-HSA. The conjugated peptide also inhibited the proliferation of lymphocytes in response to DiC8 and ionomycin by up to 57% at 15 microM CKS-17-HSA. Based on these findings we investigated the effect of CKS-17-HSA on the activity of protein kinase C (PKC), an enzyme directly activated by PMA and DiC8. PKC was isolated chromatographically from the cytosol of human neutrophils or the human lymphoblastoid cell line Jurkat. CKS-17-HSA caused a dose-dependent enzyme inhibition with a concentration giving half-maximal inhibition (IC50) of ca.3 microM and greater than 95% inhibition at 15 microM CKS-17-HSA. Inhibition of PKC by the conjugated peptide was not reversed by increasing concentrations of Ca2+, Mg2+, phosphatidylserine, diolein, or adenosine triphosphate (ATP), indicating that the conjugated peptide did not function as a chelator or competitive inhibitor. In contrast to its effects on PKC, CKS-17-HSA did not inhibit the activity of adenosine 3':5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase (PK-A) nor the calcium and phospholipid-independent form of PKC (PK-M). Moreover the peptide inhibited in vivo PKC activity in cytosol of intact cells and in membrane of PMA-stimulated cells. These results suggest that the inhibition of lymphocyte proliferation by CKS-17-HSA may be due to the direct inactivation of PKC.

Amino Acid Sequence↗
PubMed1995

Defective expression of early activation genes in cartilage-hair hypoplasia (CHH) with severe combined immunodeficiency (SCID).

Cartilage-hair hypoplasia (CHH) is an autosomal recessive disease of unknown etiology characterized by metaphyseal dysostosis, unpigmented hair, and defective cellular immunity. We studied peripheral blood mononuclear cells (PBMC) of a boy with CHH and combined immunodeficiency in an attempt to characterize further the immune defect in this disease. Stimulation of his PBMC with mitogens was associated with severely depressed IL-2 and interferon-gamma (IFN-gamma) synthesis and IL-2 receptor alpha-chain (IL-2R alpha) expression and resulted in poor lymphocyte proliferation that was only modestly upregulated by the addition of recombinant IL-2 (rIL-2). The defective proliferation and lymphokine synthesis were not corrected by the addition of phorbol myristate acetate (PMA) and ionomycin, agents that bypass receptor-mediated signalling, indicative of a distal abnormality. Importantly, the levels of mRNA encoding c-myc, IL-2R alpha, IL-2 and IFN-gamma were markedly decreased in patient lymphocytes stimulated with PMA+ionomycin as compared to control lymphocytes. The defect in the expression of these early activation genes was selective in that induction by mitogens of mRNA encoding other early activation gene products such as c-fos and c-jun was not impaired. These results suggest that the underlying defect in this patient and perhaps others with CHH may be an abnormality in a component of intracellular signalling pathways or in a trans-acting factor which regulates the expression of a selected number of early activation genes.

Child↗
PubMed1991

Mechanisms in neutrophil priming: characterization of the oxidative response induced by formylmethionyl-leucyl-phenylalanine in human exudated cells.

Exudate human polymorphonuclear neutrophils were isolated and investigated regarding oxidative responsiveness and priming ability. The exudate neutrophils were found to produce an increased amount of O2- and H2O2 when stimulated with formylmethionyl-leucyl-phenylalanine (fMLP), i.e. these cells were metabolically primed. Cytochalasin B (cyt B) pretreatment affected the production of O2- by exudate cells, although to a lesser extent than the production by peripheral blood cells, in which a substantial increase was induced. Addition of N-ethylmaleimide (NEM) to activated exudate and peripheral blood cells revealed no difference in oxidase inactivation rate. To induce further priming, the cells were incubated in vitro with a synthetic diacylglycerol (sn-1,2-didecanoylglycerol; diC10), or the Ca2+ ionophore ionomycin. Results of this procedure showed significant differences between exudate and peripheral blood neutrophils: the peripheral cells expressed a primed response, which was measured as increased fMLP-induced O2- production following incubation with both these substance; whereas the metabolic activity of exudated cells was not affected by diC10, but was significantly primed by ionomycin (P less than 0.01). The exact route for diacylglycerol priming is unknown. However, our results with human neutrophils primed during exudation indicate an exhausted diC10-priming pathway, with a retained sensitivity for priming [Ca2+]i rises.

Cytochalasin B↗
PubMed1995

Constitutive and inducible cytokine mRNA expression in the human mast cell line HMC-1.

The discovery that mast cells are a potential source of cytokines has suggested new ways in which mast cells can act in immunological and inflammatory responses. In this study we have used the HMC-1 cell line as a model for human mast cells to study the constitutive and inducible mRNA expression of interleukins, colony-stimulating factors, interferons, tumour necrosis factors alpha and beta, tumour growth factor beta and platelet-derived growth factor A and B. We found that HMC-1 cells constitutively expressed mRNA for TNF-alpha and TGF-beta, and a low level of M-CSF. After treatment with the phorbol ester TPA or the calcium ionophore ionomycin expression of several cytokines, i.e. IL-1 beta, IL-3, IL-6, GM-CSF, TNF-beta and PDGF-A, could be detected. Both TPA and ionomycin induced the same set of cytokines, but the effect of TPA was more prominent. The relative induction was calculated to be 70X for IL-1 beta and IL-3, 30X for GM-CSF and PDGF-A and 3 - 10X for IL-6, M-CSF and TNF-beta. This study shows that human mast cells have the capacity to express not only cytokines mediating an immune response but also cytokines affecting other cell types, e.g. fibroblasts and endothelial cells, involved in later steps of the inflammatory response.

Cell Line↗
PubMed1998

Interruption of T-cell signal transduction by lentivirus lytic peptides from HIV-1 transmembrane protein.

Two peptide segments designated LLP1 (residues 828-855) and LLP2 (residues 768-788) of the HIV-1 transmembrane (TM) envelope protein display structural and functional properties of calmodulin (CaM) binding. These LLP segments may contribute to cytopathogenesis by binding cellular CaM and inhibiting normal CaM-regulated signal transduction pathways. To determine whether these peptides could interrupt signal transduction in vivo, a cellular assay which uses a reporter gene linked to the nuclear factor of activated T cells (NF-AT) was used. Signal transduction perturbation was tested by exogenous addition of LLPs, W-7 or ionomycin; the LLPs inhibited NF-AT-mediated signal transduction as measured by reduced reporter activity. The LLP inhibition profile of NF-AT-driven luciferase activity was similar to the CaM inhibitor W-7. This was in direct contrast to ionomycin, a mobile calcium ion carrier which caused a significant increase in luciferase activity. These findings are consistent with the hypothesis that the CaM-binding properties of TM may contribute to defects in signal transduction leading to the T-cell anergy observed in patients infected with HIV-1.

HIV Envelope Protein gp41↗
PubMed1998

Interferon-gamma pretreatment of peripheral blood mononuclear cells partially restores defective cytokine production in children with atopic dermatitis.

Interferon-gamma (IFN-gamma) is considered an important determinant of the balance between T-helper type 1 and 2 cytokines and has been used experimentally for the treatment of atopic dermatitis. However, contrasting results have been reported relative to the Th-1/Th-2 cytokine profile in atopic patients. In this study, we examined cytokine production by polyclonally activated peripheral blood mononuclear cells (PBMC) from children with atopic dermatitis, and assessed the influence of in vitro IFN-gamma pretreatment on these cells. A fraction of PBMC isolated from children with severe atopic dermatitis, as well as from age-matched controls, was initially exposed to IFN-gamma. After washing, both treated and untreated cells were then put into culture either alone or with the addition of phytohemagglutinin (PHA) or phorbol myristate acetate (PMA) plus ionomycin. IL-4, IL-5, IL-10 and IFN-gamma production were measured in the supernatants using commercially available ELISAs. PBMC from atopic patients produced more IL-4 (P = 0.04) and IL-10 (P = 0.03) and less IFN-gamma (P = 0.01) than controls, when stimulated with PHA. Interestingly, in PMA + ionomycin stimulated cultures, the atopic cytokine profile was different with more IL-5 (P = 0.0068) and less IFN-gamma production (P = 0.00046) than the control group. When cells were pretreated with IFN-gamma, there were no significant differences between patients and controls. PBMC from children with atopic dermatitis show alterations in cytokine production, compatible in general terms with the Th-1/Th-2 model. Exposure of PBMC to IFN-gamma before activation results in a reduction of these differences, so that cytokine production becomes similar in the atopic and normal groups.

Adolescent↗
PubMed1991

The involvement of extracellular calcium in the formation of 5-lipoxygenase metabolites by human polymorphonuclear leukocytes.

We have addressed the question why in the presence of a Ca2+ ionophore human polymorphonuclear leukocytes generate leukotrienes in high yields, but in only low amounts after stimulation by receptor agonists like fMLF (fM, formylmethionine), leukotriene B4 or platelet-activating factor (PAF), although a significant release of intracellular calcium can be measured. Using ionomycin we can show that from the two enzymes involved, phospholipase A2 and 5-lipoxygenase, the first requires a threshold level of about 350-400 nM calcium whereas 5-lipoxygenase shows a linear dependence on calcium and saturates at this concentration. Our data indicate that the Ca2+ requirement of phospholipase A2 can only be met by an additional influx of extracellular calcium, whereas 5-lipoxygenase will operate already at levels provided by intracellular stores. Consequently, the complexing of extracellular calcium by EGTA stops phospholipase A2 activity immediately, whereas added arachidonate can be still adequately metabolized by intracellular Ca2+ release triggered by fMLF or PAF. Interestingly, PAF shows a stronger extracellular component in its Ca2+ transient than fMLF, and also generates more 5-lipoxygenase metabolites. However, a clear correlation between the amount of 5-lipoxygenase metabolites and the extracellular Ca2+ signal was lacking, since maximal activity was achieved before the bulk of the extracellular calcium was monitored. Ca2+ influx after PAF stimulation could be blocked after 2 min by EGTA, but a further increase in the formation of 5-lipoxygenase metabolites was observed. In contrast ionomycin-elicited 5-lipoxygenase activity could be stopped at any time shortly after EGTA addition.

Arachidonate 5-Lipoxygenase↗
PubMed1994

Serine 16 of oncoprotein 18 is a major cytosolic target for the Ca2+/calmodulin-dependent kinase-Gr.

Oncoprotein 18 (Op18) is a cytosolic protein that was initially identified due to its up-regulated expression in acute leukemia and its complex pattern of phosphorylation in response to diverse extracellular signals. We have previously identified in vivo phosphorylation sites and some of the protein kinase systems involved. Two distinct proline-directed kinase families phosphorylate Ser25 and Ser38 of Op18 with overlapping but distinct site preference. These two kinase families, mitogen-activated protein (MAP) kinases and cyclin-dependent cdc2 kinases, are involved in receptor-regulated and cell-cycle-regulated phosphorylation events, respectively. During analysis of Op18 phosphorylation in the Jurkat T-cell line, we also found that Ser16 of Op18 is phosphorylated in response to a Ca2+ signal generated by T-cell receptor stimulation or the Ca2+ ionophore ionomycin. As suggested by a previous study, T-cell-receptor-induced phosphorylation events may be mediated by the Ca2+/CaM-dependent protein kinase type Gr (CaM kinase-Gr). The present study shows that activation of this protein kinase correlates with phosphorylation of Ser16 of Op18, and in vitro experiments reveal efficient and selective phosphorylation of this residue. The CaM kinase-Gr is only expressed in certain lymphoid cell lines, and the present study shows that ionomycin-induced phosphorylation of Op18 Ser16 is restricted to cells expressing this protein kinase. Finally, CaM kinase-Gr-dependent in vitro phosphorylation of a crude cellular extract reveals a striking preference of this protein kinase for Op18 compared to other cellular substrates. In conclusion, the results suggest that Ser16 of Op18 is a major cytosolic target for activated CaM kinase-Gr.

Amino Acid Sequence↗
PubMed1992

Relationship Between Intracellular Free Calcium Concentration and NMDA-induced Cerebellar Granule Cell Survival In Vitro.

The survival of cerebellar granule cells in culture is stimulated by activation of the N-methyl-d-aspartate (NMDA) class of glutamate receptors. Activation of these receptors at the key period for cell survival in vitro (3 days; 3DIV) resulted in a sustained elevation of intracellular free calcium concentration [Ca2+]i over the same concentration range of NMDA that led to granule cell survival. Agents that release Ca2+ from intracellular stores led to only small, transient elevations of [Ca2+]i and were unable to stimulate granule cell survival. Addition of the Ca2+ ionophore ionomycin to granule cell cultures at 3DIV resulted in increased granule cell number at 7DIV. The ability of ionomycin to stimulate granule cell survival was related to the [Ca2+]i elicited, indicating that a rise in [Ca2+]i is sufficient to activate the processes leading to granule cell survival and that the extent of the elevation in [Ca2+]i is crucially important in determining granule cell fate.

Journal Article↗
PubMed1992

A Nitric Oxide Synthase Activity Selectively Stimulated by NMDA Receptors Depends on Protein Kinase C Activation in Mouse Striatal Neurons.

In mouse striatal neurons in primary culture, the maximal increase in intracellular cyclic guanosine monophosphate level evoked by N-methyl-d-aspartic acid (NMDA) receptor activation was twice that induced by kainate, KCl and ionomycin. Quisqualate was almost inactive. All responses were mediated by nitric oxide (NO) production since they were blocked by haemoglobin (a NO scavenger) and by l-NG-monomethylarginine and l-NG-nitroarginine, the effects of both arginine analogues being reversed by an excess of l-arginine. Several results indicate that NMDA receptors stimulate a specific NO synthase activity. This specifically NMDA-activated NO synthase was blocked by nanomolar concentrations of l-NG-nitroarginine, whereas the responses evoked by other agents, including kainate, KCl and ionomycin, were only blocked by micromolar concentrations of this NO synthase inhibitor. The NMDA response could not be totally reproduced by an increase in cytosolic calcium (Ca2+) alone. In contrast, in the presence of staurosporine, an inhibitor of protein kinases C (PKC), as well as after desensitization of PKC induced by long-term treatment with the phorbol ester, phorbol-12, 13-dibutyrate, NMDA-stimulated NO production was selectively reduced, reaching the level evoked by kainate or Ca2+ increase. In conclusion, our results suggest that in striatal neurons, NMDA selectively stimulates a NO synthase activity which is inhibited by low concentrations of l-NG-nitro-arginine, through a mechanism involving PKC.

Journal Article↗
PubMed1998

The effect of extracellularly applied divalent cations on cytosolic Ca2+ in murine leydig cells: evidence for a Ca2+-sensing receptor.

1. The effect of extracellularly applied divalent cations upon cytosolic Ca2+ levels ([Ca2+]) was investigated in fura-2-loaded mouse Leydig (TM3) cells. 2. The extracellular application of Ca2+ (2.5-15 mM) or Ni2+ (0.5-5 mM) elicited concentration-dependent elevations in cytosolic [Ca2+] that were followed by decays to baseline levels. Extracellular Mg2+ (0.8-15 mM) failed to influence cytosolic [Ca2+]. 3. Conditioning applications of Ca2+ (2.5-10 mM), Mg2+ (2.5-15 mM) or Ni2+ (0.5-5 mM) all attenuated the cytosolic Ca2+ response to a subsequent test application of 5 mM [Ni2+]. 4. The amplitude of Ni2+-induced cytosolic Ca2+ signals remained constant in low-Ca2+ solutions. Such findings suggest a participation of Ca2+ release from intracellular stores. In parallel, depletion of Ca2+ stores by either ionomycin (5 microM, in low-Ca2+ solutions) or thapsigargin (4 microM) abolished or attenuated Ni2+-induced Ca2+ transients. 5. Ionomycin (5 microM) elevated cytosolic [Ca2+] in Ca2+-free solutions even after prior Ni2+ application, indicating the presence of Ni2+-insensitive stores. 6. Caffeine (250 and 500 microM) elevated cytosolic [Ca2+] and attenuated Ni2+-induced Ca2+ release. Furthermore, TM3 cells stained intensely with a specific anti-ryanodine receptor antiserum, Ab34. These findings suggest that Ca2+ release is regulated by ryanodine receptors. 7. Both membrane depolarization and hyperpolarization, brought about by changes in extracellular [K+] ([K+]e) in the presence of valinomycin (5 microM), altered the waveform of the Ni2+-induced cytosolic Ca2+ signal. Hyperpolarization, in addition, diminished the response magnitude. Such voltage-induced response modulation localizes the regulatory events to the Leydig cell plasma membrane. 8. We propose the existence of a cell surface divalent cation (Ca2+) receptor in Leydig cells, the activation of which triggers Ca2+ fluxes through ryanodine receptors.

Animals↗
PubMed1999

Glucocorticoid modulation of Ca2+ homeostasis in human B lymphoblasts.

1. We determined the effect of cortisol (200 nM for 48 h) on the intracellular Ca2+ concentration ([Ca2+]i) and parameters of Ca2+i signalling in 19 lymphoblastoid cell lines (LCLs). 2. Using the fluorescent dye fura-2, the basal [Ca2+]i in Ca2+-containing medium was 63.5 +/- 2.4 nM in vehicle (ethanol)-treated LCLs and 55.7 +/- 2. 6 nM (mean +/- s.e.m.) in cortisol-treated LCLs. 3. Ca2+i signalling following platelet-activating factor (PAF, 100 nM) addition was enhanced by cortisol treatment, with LCLs having small PAF responses showing the largest percentage increase after cortisol treatment. Mean peak [Ca2+]i responses to PAF were enhanced 67.0% and 55.7% in Ca2+-free and Ca2+-containing medium, respectively. 4. The endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (100 nM) caused a transient increase in [Ca2+]i in Ca2+-free medium in which the peak change was increased in cortisol-treated cells (98.5 +/- 5.8 vs. 79.8 +/- 4.5 nM). Peak changes in the freely exchangeable Ca2+ in response to 5 microM ionomycin were also enhanced in cortisol-treated cells (923.7 +/- 113.9 vs. 652.2 +/- 64.5 nM) and correlated to the PAF-evoked [Ca2+]i response. 5. Cortisol-treated LCLs exposed to thapsigargin to empty intracellular Ca2+ stores (10 min treatment in Ca2+-free medium) and exposed to CaCl2 or MnCl2 had a greater rate of Ca2+ entry (18.6 +/- 1.8 vs. 13.8 +/- 1.5 nM s-1) and higher rate constant for Mn2+ entry (0.0345 +/- 0.0029 vs. 0. 0217 +/- 0.0020) than vehicle-treated cells. Peak [Ca2+]i in cells exposed to CaCl2 was also enhanced (869.4 +/- 114.7 vs. 562.6 +/- 61.7 nM). Parameters of divalent cation influx were highly correlated to the peak [Ca2+]i elicited by thapsigargin or ionomycin. 6. Inclusion of RU 486 (a glucocorticoid antagonist) with cortisol prevented the decrease in basal [Ca2+]i and stimulatory actions of cortisol on all Ca2+i parameters. RU 486 alone had no apparent effects on basal [Ca2+]i or Ca2+i signalling. 7. Based on data obtained over a wide range of responses (in the presence and/or absence of cortisol or RU 486), the results show that cortisol stimulation of glucocorticoid receptors decreases basal [Ca2+]i and enhances PAF-evoked [Ca2+]i signalling, most probably through its effects on intracellular Ca2+ stores. In turn, the extent of Ca2+ entry via store-operated plasma membrane Ca2+ channels is closely linked to the size of the Ca2+ stores.

B-Lymphocytes↗
PubMed2000

The role of Ca2+ stores in the muscarinic inhibition of the K+ current IK(SO) in neonatal rat cerebellar granule cells.

Cerebellar granule neurons (CGNs) possess a standing outward potassium current (IK(SO)) which shares many similarities with current through the two-pore domain potassium channel TASK-1 and which is inhibited following activation of muscarinic acetylcholine receptors. The action of muscarine on IK(SO) was unaffected by the M2 receptor antagonist methoctramine (100 nM) but was blocked by the M3 antagonist zamifenacin, which, at a concentration of 100 nM, shifted the muscarine concentration-response curve to the right by around 50-fold. Surprisingly, M3 receptor activation rarely produced a detectable increase in [Ca2+]i unless preceded by depolarization of the cells with 25 mM K+. Experiments with thapsigargin and ionomycin suggested that the endoplasmic reticulum Ca2+ stores in CGNs were depleted at rest. In contrast, cerebellar glial cells in the same fields of cells possessed substantial endoplasmic reticulum Ca2+ stores at rest. Pretreatment of the cells with BAPTA AM, thapsigargin or the phospholipase C (PLC) inhibitor U-73122 all blocked the muscarine-induced Ca2+ signal but had little or no effect on muscarinic inhibition of IK(SO). Raising [Ca2+]i directly with ionomycin caused a small but significant inhibition of IK(SO). It is concluded that muscarine acts on M3 muscarinic acetylcholine receptors both to inhibit IK(SO) and to mobilize Ca2+ from intracellular stores in CGNs. While the mobilization of Ca2+ occurs through activation of PLC, this does not seem to be the primary mechanism underlying muscarinic inhibition of IK(SO).

Animals↗