Cytosolic Ca Levels Research Articles

Protein synthesis in presynaptic endings from squid brain: Modulation by calcium ions

Previous biochemical, autoradiographic, and ultrastructural data have shown that, in the synaptosomal fraction of the squid optic lobe, protein synthesis is largely due to the presynaptic terminals of the retinal photoreceptor neurons (Crispino et al. [1993a] Mol. Cell. Neurosci. 4:366–374; Crispino et al. [1993b] J. Neurochem. 61:1144–1146; Crispino et al. [1997] J. Neurosci. 17:7694–7702). We now report that this process is close to its maximum at the basal concentration of cytosolic Ca++, and is markedly inhibited when the concentration of this ion is either decreased or increased. This conclusion is supported by the results of experiments with: 1) compounds known to increase the level of cytosolic Ca++, such as A23187, ionomycin, thapsigargin, and caffeine; 2) compounds sequestering cytosolic calcium ions such as BAPTA-AM; and 3) agents that block the role of Ca++ as second messenger, such as TFP and W7, which inhibit calmodulin, and calphostin, which inhibits protein kinase C. We conclude that variations in the level of cytosolic Ca++ induced in presynaptic terminals by neuronal activity may contribute to the modulation of the local synthesis of protein. J. Neurosci. Res. 55:776–781, 1999. © 1999 Wiley-Liss, Inc.

Protein synthesis in presynaptic endings from squid brain: modulation by calcium ions.

Previous biochemical, autoradiographic, and ultrastructural data have shown that, in the synaptosomal fraction of the squid optic lobe, protein synthesis is largely due to the presynaptic terminals of the retinal photoreceptor neurons (Crispino et al. [1993a] Mol. Cell. Neurosci. 4:366-374; Crispino et al. [1993b] J. Neurochem. 61:1144-1146; Crispino et al. [1997] J. Neurosci. 17:7694-7702). We now report that this process is close to its maximum at the basal concentration of cytosolic Ca++, and is markedly inhibited when the concentration of this ion is either decreased or increased. This conclusion is supported by the results of experiments with: 1) compounds known to increase the level of cytosolic Ca++, such as A23187, ionomycin, thapsigargin, and caffeine; 2) compounds sequestering cytosolic calcium ions such as BAPTA-AM; and 3) agents that block the role of Ca++ as second messenger, such as TFP and W7, which inhibit calmodulin, and calphostin, which inhibits protein kinase C. We conclude that variations in the level of cytosolic Ca++ induced in presynaptic terminals by neuronal activity may contribute to the modulation of the local synthesis of protein.

Cold acclimation enhances the activity of plasma membrane Ca 2+ ATPase in winter rye leaves

Exposure of plant cells and tissues to low or freezing temperatures often lead to uncontrolled and detrimental ion leakage. Therefore, when plants acclimate to low temperatures, processes that control ionic homeostasis are important. Here we characterized H + ATPase and ATP-dependent Ca 2+ transport activities in isolated plasma membranes of cold-acclimated and non-acclimated winter rye leaves ( Secale cereale L. cv. Voima). Cold acclimation resulted in a two-fold higher Ca 2+ transport activity, significantly different ( P = 0.021) from that of non-acclimated rye, whereas only a small increase in H + ATPase activity, measured as ATP hydrolysis, was observed in cold-acclimated compared to non-acclimated preparations. In plasma membranes, extensively washed with EDTA and Brij 58 to remove endogenous calmodulin, Ca 2+ transport activity increased to about double by calmodulin addition, with both non-acclimated and cold-acclimated material. Uptake of Ca 2+ was seen within the pHrange analyzed (pH 6–8), with an optimum at pH 7.2 with both materials, and both in the absence and in the presence of calmodulin. The increase in activity of ATP-dependent Ca 2+ transport in cold-acclimated rye plasma membranes probably reflects the capacity needed to sustain the resting level of cytosolic Ca 2+ concentration that is characteristic to the cold-acclimated situation.

Effects of HMG-CoA reductase inhibitors on excitation–contraction coupling of rat skeletal muscle

3-Hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors currently used as cholesterol-lowering drugs produce side effects in patients, one of which is myopathy. In the present study we compared the effect of a 3-month chronic treatment with two different compounds, simvastatin and pravastatin, on the excitation–contraction coupling of rat skeletal muscle fibers, the mechanism which links membrane depolarization to the movements of cytosolic Ca 2+ from intracellular stores. The voltage threshold for mechanical activation of extensor digitorum longus muscle fibers in response to depolarizing pulses of various durations was studied in vitro by the two intracellular microelectrode method in `point' voltage clamp mode. Simvastatin (5–50 mg/kg) modified the mechanical threshold of striated fibers in a dose-dependent manner. The muscle fibers of rats treated with 10 mg/kg and 50 mg/kg of simvastatin needed significantly less depolarization to contract than did untreated fibers at each pulse duration, suggesting that levels of cytosolic Ca 2+ were higher. Consequently, the rheobase voltage for fiber contraction was significantly shifted toward more negative potentials with respect to controls by 2.4 mV and 7.1 mV in the 10 mg/kg and 50 mg/kg simvastatin-treated animals, respectively. Pravastatin treatment at 100 mg/kg did not produce any alteration of excitation–contraction coupling since the rheobase voltage was similar to that of controls. The different physicochemical properties of the two drugs may underlie the different effect observed because lipophilic agents, such as simvastatin, have been shown to affect sterol biosynthesis in many tissues, whereas the hydrophilic pravastatin is hepato-selective.

Human SERCA2a levels correlate inversely with age in senescent human myocardium

Objectives. This study sought to characterize functional impairment after simulated ischemia-reperfusion (I/R) or Ca2+bolus in senescent human myocardium and to determine if age-related alterations in myocardial concentrations of SERCA2a, phospholamban, or calsequestrin participate in senescent myocardial dysfunction.Background. Candidates for elective cardiac interventions are aging, and an association between age and impairment of relaxation has been reported in experimental animals. Function of the sarcoplasmic reticulum resulting in diastolic dysfunction could be dysregulated at the level of cytosolic Ca2+uptake by SERCA2a, its inhibitory subunit (phospholamban), or at the level of Ca2+binding by calsequestrin.Methods. Human atrial trabeculae from 17 patients (45–75 years old) were suspended in organ baths, field simulated at 1 Hz, and force development was recorded during I/R (45/120 min). Trabeculae from an additional 12 patients (53–73 years old) were exposed to Ca2+bolus (2–3 mmol/L bath concentration). Maximum ± dF/dt and the time constant of force decay (tau) were measured before and after I/R or Ca2+bolus and related to age. SERCA2a, phospholamban, and calsequestrin from 12 patients (39–77 years old) were assessed by immunoblot.Results. Functional results indicated that maximum ±dF/dt and tau were prolonged in senescent (>60 years) human myocardium after I/R (p < 0.05). Calcium bolus increased the maximum ±dF/dt and decreased tauin younger, but not older patients (p < 0.05). SERCA2a and the ratio of SERCA2a to either phospholamban or calsequestrin were decreased in senescent human myocardium (p < 0.05).Conclusions. Senescent human myocardium exhibits decreased myocardial SERCA2a content with age, which may, in part, explain impaired myocardial function after either I/R or Ca2+exposure.

An all-or-nothing rise in cytosolic

The peritrich ciliate Vorticella sp. exhibits cellular contraction of an all-or-nothing type in response to a mechanical stimulus. Many authors have suggested that the contraction may be controlled by the cytosolic level of Ca2+, since glycerol-extracted Vorticella contracts when Ca2+ is added to the external solution. However, no direct evidence for the increase in cytosolic [Ca2+] has yet been obtained in living Vorticella. In the present study, by injecting a fluorescent Ca2+ indicator into living Vorticella and monitoring the cytosolic [Ca2+] with a confocal microscope, we have demonstrated that a mechanical stimulus evoked an all-or-nothing rise in cytosolic [Ca2+] (Ca2+ 'spike'). The onset of the Ca2+ spike was similar in its time course to that of cellular contraction. Since the Ca2+ spike was recorded in a Ca2+-deprived solution containing 1 mmol l-1 EGTA, we concluded that release of Ca2+ from intracellular Ca2+ storage site(s) is responsible for the Ca2+ spike.

Fluid shear stress induces actin polymerization in human neutrophils

We have previously reported that a physiological range of shear stress induces neutrophil homotypic aggregation mediated by lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-3 (ICAM-3) interactions. To further characterize the homotypic aggregation, actin polymerization was investigated in neutrophils stimulated by shear stress in comparison with formyl-methionyl-leucyl-phenylalanine (fMLP). In fMLP-stimulated neutrophils, actin polymerization was localized in the pseudopods, and this reaction was not mediated by a cytosolic level of Ca2+. In contrast to fMLP stimulation, the actin polymerization induced by shear stress in a cone-plate viscometer was localized in cell-cell contact regions, and this polymerization required the increase of intracellular Ca2+. This shear stress-induced actin polymerization was not observed when neutrophils were pretreated with anti-LFA-1 or anti-ICAM-3 antibody. In conclusion, LFA-1 and ICAM-3 interaction mediated by the increase of [Ca2+]i generated the intercellular signal in order to accumulate F-actin in the cell-cell contact regions. © 1996 Wiley-Liss, Inc.

Desensitization of P2U receptor in neuronal cell line. Different control by the agonists ATP and UTP, as demonstrated by single-cell Ca2+ responses.

The neuronal cell line NG108-15 (180CC15) responds to extracellular stimuli of ATP or UTP with a transient increase in the level of cytosolic Ca2+. Desensitization was investigated by recording single-cell Ca2+ responses induced by consecutive, regularly spaced (100 s intervals), brief pulses of the nucleotides. The two natural ligands of the P2U receptor, ATP and UTP, were applied at a concentration that evoked responses of a comparable size. With two pulses of UTP (10 microM), a substantial decrease (of 43%) was observed in the size of the second response. The magnitude of response was determined by measuring either the maximal amplitude or the total response, represented by the area of the Ca2+ transient. The analogous studies with ATP pulses showed a much smaller decrease (of 12%). Comparable experiments performed to investigate the mutual interaction between ATP and UTP revealed that after stimulation with ATP the response to UTP was slightly (12%) diminished, whereas the response to ATP after UTP was greatly (52%) decreased. The different degree of desensitization by either UTP or ATP of P2U receptors could be due to (1) a difference in the mode of activation of the receptor by the two ligands or (2) recruitment of another effector mechanism besides elevated Ca2+. Our results indicate the existence of a novel mechanism of receptor control, involving different modes of the receptor, that are induced by two different, activating ligands. We also investigated the crosstalk between the bradykinin B2 receptor and the nucleotide receptor. ATP and UTP, even when eliciting responses of comparable size in the neuronal cell line, affect the desensitization of the bradykinin receptor differently. This suggests regulatory binding sites for the nucleotides on either the nucleotide receptor or the peptide receptor.

A Possible Mechanism for the Stimulation of Metalloproteinase Production in Human Aortic Intimal Smooth Muscle Cells by Linoleic Acid Hydroperoxide

To approach the mechanism of the stimulating effect of linoleic acid hydroperoxide on the production of matrix metalloproteinases (MMPs) in human aortic intimal smooth muscle cells (ISMC), we investigated the effect of the hydroperoxide on the cytosolic level of Ca2+. Linoleic acid hydroperoxide provoked an increase in the cytosolic Ca2+level, but it had no effect on the level of inositol phosphates (IPs) in these cells, in contrast with the effect of platelet-derived growth factor (PDGF), which elevated the level of both Ca2+and IPs in these cells. A23187, a calcium ionophore, stimulated ISMC to produce matrix prometalloproteinase 1. These results indicate that linoleic acid hydroperoxide stimulates the production of MMPs in ISMC by elevation of the cytosolic Ca2+level without the intervention of IPs. In addition, we found that the hydroperoxide has no effect on the binding of PDGF to its specific receptor on ISMC.

The cytosolic free Ca2+ in ectromelia (mousepox) virus stimulated cytotoxic T lymphocytes.

The objective of these studies was to assess the intracellular calcium (Ca2+) signal involved in the activation of ectromelia virus (EV)-specific cytotoxic T lymphocytes (CTL) upon stimulation with EV-sensitized (A20) target cells or concanavalin A (Con A). The CTL originated from mice previously infected with EV. The level of cytosolic Ca2+ in EV-specific CTL was measured using Fluo-3 AM. In both cases transient [Ca2+]i rise and a sustained plateau (1723 nM) were observed in buffer with 1 mM extracellular calcium. The [Ca2+]i response of EV-specific CTL to EV-sensitized target cells or Con A in extracellular calcium free buffer consisted of only one peak at 852 nm. The cytotoxic activity EV-specific CTL assessed in normal medium was 53.0%, but significantly reduced to 15% when verapamil was added to the buffer. No enhancement of [Ca2+]i response in EV-specific CTL was observed upon costimulation with PMA. Association of the level of intracellular [Ca2+]i in EV-specific CTL and the percentage of their cytotoxicity was found. These results show partial dependence of EV-specific CTL killing on extracellular calcium.

Free and membrane-bound calcium in microgravity and microgravity effects at the membrane level

The changes of [Ca 2+] i controlled is known to play a key regulatory role in numerous cellular processes especially associated with membranes. Previous studies from our laboratory have demonstrated an increase in calcium level in root cells of pea seedlings grown aboard orbital station “Salyut 6” /1/. These results: 1) indicate that observed Ca 2+-binding sites of membranes also consist in proteins and phospholipids; 2) suggest that such effects of space flight in membrane Ca-binding might be due to the enhancement of Ca 2+ influx through membranes. In model presented, I propose that Ca 2+-activated channels in plasma membrane in response to microgravity allow the movement of Ca 2+ into the root cells, causing a rise in cytoplasmic free Ca 2+ levels. The latter, in its turn, may induce the inhibition of a Ca 2+ efflux by Ca 2+-activated ATPases and through a Ca 2+/H + antiport. It is possible that increased cytosolic levels of Ca 2+ ions have stimulated hydrolysis and turnover of phosphatidylinositols, with a consequent elevation of cytosolic [Ca 2+] i. Plant cell can response to such a Ca 2+ rise by an enhancement of membranous Ca 2+-binding activities to rescue thus a cell from an abundance of a cytotoxin. A Ca 2+-induced phase separation of membranous lipids assists to appear the structure nonstable zones with high energy level at the boundary of microdomains which are rich by some phospholipid components; there is mixing of molecules of the membranes contacted in these zones, the first stage of membranous fusion, which was found in plants exposed to microgravity. These results support the hypothesis that a target for microgravity effect is the flux mechanism of Ca 2+ to plant cell.

Cellular injury induced by oxidative stress is mediated through lysosomal damage

Cultured primary hepatocytes pretreated (protected) with the iron chelator deferoxamine or the antioxidant N,N′-diphenyl-p-phenylenediamine (DPPD) were resistant to the toxicity of 5 μM naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) during a 180-min exposure. Hepatocytes exposed to naphthazarin without any protection were abruptly depleted of intracellular reduced glutathione, and the level of cytosolic Ca 2+ was rapidly increased. This was followed by lipid peroxidation, measured as accumulation of malondialdehyde (MDA) and 4-hydroxyalkenals (4-HNA) intra- and extracellularly; decrease in ATP levels; destabilization of lysosomes; and finally cell death. The stability of the lysosomal membranes was evaluated by determining retention of the lysosomotropic weak base acridine orange (AO). Naphthazarin exposure caused leakage of protons from the acidic compartment, as indicated by relocalization of AO to the cytosol. Protection of the cell cultures with deferoxamine or DPPD prevented destabilization of lysosomes and cell killing. It also reduced the loss of ATP but did not prevent the depletion of glutathione or the increase in Ca 2+. In cells subjected to naphthazarin exposure, DPPD protection also completely inhibited lipid peroxidation, whereas deferoxamine pretreatment only slightly reduced the intracellular accumulation of MDA and 4-HNA but completely prevented cell rupture and the leakage of these lipid peroxidation products to the medium that took place in large amounts from unprotected cells exposed to naphthazarin. Deferoxamine is taken up by endocytosis and is thus transported to the acidic vacuolar apparatus, whereas the lipophilic DPPD is rapidly distributed throughout the cells. Inhibiting endocytosis during deferoxamine pretreatment, by incubating at +4°C or by preexposure to a mixture of the endocytosis-inhibitors cytochalasin B and monensin, abolished the protective effect of deferoxamine. The findings suggest that naphthazarin-induced cell killing is not caused directly by either thiol oxidation or an increase in cytosolic free Ca 2+, but rather is preceded by lysosomal destabilization, which may be prevented either by inhibition of cellular peroxidation in general or by prevention of iron-catalyzed oxidative reactions, and involves peroxidation of cellular membranes, energy depletion, and leakage of lysosomal content. DPPD would protect against cell killing by preventing lipid peroxidation of cellular membranes in general, whereas deferoxamine seems to allow a limited general cellular peroxidation but specifically prevents peroxidation and fragmentation of lysosomal membranes by chelating intralysosomal iron and, consequently, leakage of destructive lysosomal contents with ensuing cell rupture and death. Thus, a certain degree of cellular peroxidation does not appear to be lethal as long as lysosomal membranes are protected, placing lysosomes into a category of cellular loci minora resistentia.

Modification of gap junction conductance by divalent cations and protons in neonatal rat heart cells

Myocytes were isolated from neonatal rat hearts and grown in culture dishes. Pairs of cells were selected to study the effect of divalent cations and protons on the conductance of gap junctions, gj. The experimental approach involved the dual voltage-clamp method and cell dialysis via patch pipette, i.e. gj was monitored while the cytosolic level of Ca2+, Mg2+, Sr2+, Ba2+ or H+ was modified in one of the cells. A dose-dependent decrease in gj developed when pCa of the pipette solution was lowered (range: pCa = 7.7-2.42, equivalent to a [Ca2+] of 20 nM-3.8 mM). The gj/pCa-relationship revealed a Hill coefficient n of 0.87 and a half-maximal concentration pKCa of 3.5. Pretreatment with 3 mM NiCl2 and 1 micron ryanodine to minimize the removal of cytosolic Ca2+ did not significantly affect the response to gj. Similarly, gj was decreased in a dose-dependent fashion when pHi in the pipette solution was lowered (range: pH = 7.2-5.0, corresponding to a [H+] of 63 nM-10 microns). The gj/pH-relationship yielded an n of 0.92 and a pKH of 5.85. Pretreatment with 1 mM amiloride to minimize the extrusion of protons enhanced the effects of pH on gj. Simultaneous alterations in pCa and pH demonstrated an additive type of action of Ca2+ and H+ on gj. This is consistent with the existence of two types of sensors which contribute separately to the functional state of gj. No significant decrease in gj was detectable when the pipette solution contained Mg2+ or Ba2+ (up to 5 mM). Partial uncoupling was observed with pipette solution containing 5 mM Sr2+. We conclude that gj of neonatal and adult cardiomyocytes exhibit different ionic sensitivities. This discrepancy may reflect differences in connexin expression and/or molecular intermediates involved in regulating gj.

Cyclic ADP-ribose and related compounds activate sheep skeletal sarcoplasmic reticulum Ca2+ release channel

It has been suggested that adenosine 5'-cyclic-diphosphoribose (cADPR) can activate only nonskeletal isoforms of the ryanodine-sensitive Ca2+ release channel. We now demonstrate that cADPR is an effective activator of sheep skeletal sarcoplasmic reticulum (SR) Ca2+ release channels incorporated into planar phospholipid bilayers in the presence of activating levels of cytosolic Ca2+. In addition, the precursor of cADPR, beta-NAD+, and the metabolite, adenosine diphosphoribose (ADP-ribose), also increase the open probability (Po) of skeletal SR Ca2+ release channels in micromolar concentrations. At low concentrations of cADPR (1 microM), the mechanism for the increase in Po is an increase in the frequency of channel openings with no increase in the duration of the open events. We also show that the effect of cADPR is dependent on luminal [Ca2+]. cADPR has no effect on Po when the luminal [Ca2+] is < 40 microM. However, at millimolar concentrations of luminal Ca2+, cADPR 1 and 10 microM) increases Po in the presence of activating cytosolic Ca2+.

Effects of Hg2+ and Cu2+ on the cytosolic Ca2+ level in molluscan blood cells evaluated by confocal microscopy and spectrofluorimetry

In the present work the effect of Hg2+ and Cu2+ on the level of cytosolic Ca2+ in mussel (Mytilus edulis L.) haemolymph cells were investigated by confocal microscopy and spectrofluorimetry utilizing the fluorescent dye Fluo3. In the blood cells of marine molluscs, exposure to Cu2+ and Hg2+ in the nanomolar and micromolar range causes a time-and concentration-dependent increase in the cytosolic Ca2+ level. Both the presence of a low-calcium containing medium and pretreatment of the cells with the channel blocker Verapamil greatly reduced the effects of higher (50 μM) Hg2+ concentrations, this indicating that Hg2+ enhances the influx of extracellular Ca2+ partly through activation of voltage-dependent Ca2+ channels. Low concentrations of Hg2+ (1 μM) and also of Cu2+ (0.5 μM), an “essential” element, were able to induce a sustained increase in cytosolic Ca2+, which was not affected either by Verapamil pretreatment or by lowering the extracellular calcium concentration. These data indicate that in mussel haemocytes heavy metal cations impair Ca2+ homeostasis not only by affecting Ca2+ channels, but also by interfering with other mechanisms of calcium transport across cellular membranes, such as the Ca2+-ATPases. The resulting increase in cytosolic Ca2+ could activate Ca-dependent processes which may be involved in many of the biochemical and physiological alterations observed in the cells of metal-exposed mussels. Specimens used in these experiments were collected from the river Linker near Plymouth, U.K. in June 1991.

Effect of glucocorticosteroid treatment on intracellular calcium homeostasis in mouse lymphoma cells.

There is growing evidence for the involvement of Ca2+ in the programmed cell death (apoptosis) of lymphocytes, but the nature of glucocorticoid-induced Ca2+ fluxes and their role in the cell death pathway are poorly understood. In the study reported here, we assessed the effect of glucocorticoid treatment on intracellular Ca2+ homeostasis in W7MG1 mouse lymphoma cells. Levels of cytosolic Ca2+ were measured using the intracellular Ca2+ indicator fura2 AM, and total cellular Ca2+ was measured by atomic absorbance spectroscopy. The level of Ca2+ within internal stores, including the endoplasmic reticulum (ER), was estimated by measuring the increase in cytosolic Ca2+ induced by either ionomycin, an ionophore that mobilizes Ca2+ from a variety of internal stores, and by thapsigargin, a specific inhibitor of the ER-associated Ca(2+)-ATPase that mobilizes Ca2+ from the ER. Glucocorticoid treatment induced a significant decrease in ionomycin- and thapsigargin-mobilizable Ca2+ stores that was accompanied by an initial decrease in total cellular Ca2+, followed by a modest increase in both total cellular Ca2+ and cytosolic Ca2+. The glucocorticoid-induced depletion of internal Ca2+ stores was receptor mediated and occurred after a delay corresponding to the time required for glucocorticoid receptor complexes to regulate gene transcription. Mobilization of ER-associated Ca2+ stores by thapsigargin treatment induced DNA fragmentation and cell death similar to that observed after glucocorticoid treatment. These findings suggest that a mobilization of Ca2+ from internal stores may be a critical step in the apoptotic pathway of mouse lymphoma cells.

Effects of phorbol ester on gap junctions of neonatal rat heart cells.

Myocytes were isolated from the ventricles of neonatal rat hearts and cultured for 1-3 days. Newly formed cell pairs were used to examine the conductance of gap junctions, gj. Measurements were performed using a dual voltage-clamp method in conjunction with a whole-cell, tight-seal recording. Exposure to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, 100-160 nM) led to a decrease in gj. Single-channel events recorded immediately before complete uncoupling yielded a single-channel conductance, gamma j, of 40.5 pS, implying that TPA affects the channel kinetics rather than gamma j. TPA-induced uncoupling was observed at subphysiological levels of cytosolic Ca2+ (pipette solution = 18 nM), not at physiological levels (pipette solution = 170 nM). The effects of TPA could not be mimicked by 250 microM 1-oleoyl-2-acetyl-glycerol (OAG). Preincubation with TPA (up to 24 h) revealed no changes in gj attributable to down-regulation of protein kinase C, PKC. Pretreatment with PKC inhibitors, staurosporine or PKCI, prevented the TPA-dependent decrease in gj. TPA-dependent uncoupling was not impaired by 4-bromophenacyl bromide, an inhibitor of phospholipase A2, PLA2; conversely, an arachidonic acid-dependent decrease in gj was not prevented by PKCI. This suggests that gj regulation does not involve an interaction between PLA2 and PKC.

Protein prenylcysteine analog inhibits agonist-receptor-mediated signal transduction in human platelets.

Signal transduction components, including the Ras superfamily of low molecular weight GTP-binding proteins and the gamma subunits of heterotrimeric G proteins, are reversibly carboxyl methylated at C-terminal prenylcysteine residues. We have previously shown that the prenylcysteine analog N-acetyl-S-trans,trans-farnesyl-L-cysteine (AFC) inhibits carboxyl methylation of these proteins in human platelets. Here we show that concentrations of AFC that inhibit Ras carboxyl methylation (10-50 microM) also block responses to agonists such as ADP, collagen, arachidonic acid, U46619 (a stable analog of prostaglandin H2), thrombin, and guanosine 5'-[gamma-thio]triphosphate. AFC does not inhibit aggregation induced by effectors such as ionomycin, phorbol 12,13-dibutyrate, and bacterial phospholipase C that bypass G proteins to activate platelets at the level of cytosolic Ca2+ concentration and protein kinase C. These findings indicate that AFC inhibits agonist-receptor-mediated signal transduction in human platelets.

Effects of secretagogues on intracellular free calcium and magnesium concentrations in rat pancreatic acinar cells

1. 1. Rat pancreatic acinar cells were loaded with Fura 2 AM or Magfura AM and levels of cytosolic Ca 2+ ([Ca 2+] i) and Mg 2+ ([Mg 2+] i were observed. 2. 2. Addition of acetylcholine (ACh) evoked a transient rise in [Ca 2+] i. The component of the rise dependent on extracellular Ca 2+ sources, but not intracellular sources, was seen to be enhanced when both ACh and 5 mM Ca 2+ were present in the medium. In the presence of elevated extracellular Mg 2+ (10 mM) and ACh both components of the Ca 2+ transient were inhibited. 3. 3. Both GTPγS and fluoroaluminate, which can directly stimulate G-proteins, evoked a transient rise in [Ca 2+] i in acinar cells. These responses were inhibited in the presence of elevated Mg 2+. 4. 4. Resting [Mg 2+] i was seen to be 1.36 mM ± 0.08 ( n = 29) for cells in normal medium, 1.8 mM ± 0.08 ( n = 6) in elevated Mg 2+ medium and 0.93 mM ± 0.02 ( n = 5) in cells bathed and Mg 2+-free medium. Addition of ACh led to reductions in [Mg 2+] i in cells bathed in normal medium and Mg 2+-free medium but not elevated Mg 2+ medium. 5. 5. It is concluded that levels of extracellular Mg 2+ strongly influence [Mg 2+] i and [Ca 2+] i mobilization during ACh-evoked responses. Mg 2+ does not appear to be exerting its effects by influencing receptor-agonist interactions or by competing with Ca 2+ at extracellular sites of Ca 2+ uptake.

Chronic treatment with D600 enhances development of sodium channels in cultured chick skeletal muscle cells

We have studied the long-term effects of D600, a blocker of L-type voltage-dependent Ca channels (VDCC), on the development of voltage-dependent Na channels (VDNC) that are sensitive to tetrodotoxin (TTX), by electrophysiological measurements of the maximum rate of rise of the TTX-sensitive Na spike in cultured chick skeletal muscle cells. Chronic treatment with D600 (2–20 μM) caused a dose-dependent increase in the density of VDNC. The density of VDNC was increased by 150–250% when D600 was added to the cultures at 20 μM from the second day of culture onward. Co-treatment with an inhibitor of the transcription of RNA from DNA, α-amanitin, or with cycloheximide, an inhibitor of protein synthesis, prevented the up-regulation by D600. Nifedipine, a different type of blocker of L-type VDCC, was also effective in increasing the density of VDNC, and BAY K 8644, an agonist of L-type VDCC, had the opposite effect. It is suggested that the effect of D600 was mediated via a mechanism specific for L-type VDCC that involves regulation of cytosolic levels of Ca 2+ and protein synthesis de novo.