Microsomal Ca2 Research Articles

Muscarinic acetylcholine receptor activation induces Ca2+ mobilization and Na+/K+-ATPase activity inhibition in eel enterocytes.

The effect of carbachol (Cch) on intracellular calcium concentration ([Ca2+]i) in eel enterocytes was examined using the fluorescent Ca2+ indicator fura-2. Cch caused a biphasic increase in [Ca2+]i, with an initial spike followed by a progressively decreasing level (over 6 min) to the initial, pre-stimulated, level. The effect of Cch was dose-dependent with a 7.5-fold increase in [Ca2+]i over basal level induced by the maximal dose of Cch (100 microM). In Ca2+-free/EGTA buffer the effect of Cch was less pronounced and the [Ca2+]i returned rapidly to basal levels. The increment of [Ca2+]i was dose-dependently attenuated in cells pre-treated with U73122, a specific inhibitor of phospholipase C, suggesting that the Cch-stimulated increment of [Ca2+]i required inositol triphosphate formation. In the presence of extracellular Ca2+, thapsigargin (TG), a specific microsomal Ca2+-ATPase inhibitor, caused a sustained rise in [Ca2+]i whereas in Ca2+-free medium the increase in [Ca2+]i was transient; in both cases, subsequent addition of Cch was without effect. When 2 mM CaCl2 were added to the cells stimulated with TG or with Cch in Ca2+-free medium, a rapid increase in [Ca2+]i was detected, corresponding to the capacitative Ca2+ entry. Thus, both TG and Cch depleted intracellular Ca2+ stores and stimulated influx of extracellular Ca2+ consistent with capacitative Ca2+ entry. K+ depolarization obtained with increasing concentrations of KCl in the extracellular medium induced a dose-related increase in [Ca2+]i which was blocked by 2 microM nifedipine, a non-specific L-type Ca2+ channel blocker. Nifedipine also changed significantly the height of the Ca2+ transient, and the rate of decrement to the pre-stimulated [Ca2+]i level, indicating that Ca2+ entry into enterocytes also occurs through an L-type voltage-dependent calcium channel pathway. We also show that isolated enterocytes stimulated with increasing Cch concentrations (0.1-1000 microM) showed a dose-dependent inhibition of the Na+/K+-ATPase activity. The threshold decrease was at 1 microM Cch; it reached a maximum at 100 microM (50.5% inhibition) and did not decrease further with the use of higher dose. The effect of Cch on Na+/K+-ATPase activity was dependent on both protein kinase C (PKC) and protein phosphatase calcineurin activation since the PKC inhibitor calphostin C abolished Cch effects, while the calcineurin inhibitor FK506 augmented Cch effect. Collectively, these data establish a functional pathway by which Cch can modulate the activity of the Na+/K+-ATPase through a PKC-dependent (calphostin C-sensitive) pathway and a calcineurin-dependent (FK506-sensitive) pathway.

Role of regucalcin as an activator of sarcoplasmic reticulum Ca2+-ATPase activity in rat heart muscle.

The expression of regucalcin, a regulatory protein of Ca(2+) signaling, and its effect on Ca(2+) pump activity in the microsomes (sarcoplasmic reticulum) of rat heart muscle was investigated. The expression of regucalcin mRNA was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) analysis in heart muscle using rat regucalcin-specific primers. Results with Western blot analysis showed that regucalcin protein was present in the cytoplasm, although it was not detected in the microsomes. Microsomal Ca(2+)-ATPase activity was significantly increased in the presence of regucalcin (10(-10)-10(-8) M) in the enzyme reaction mixture. This increase was not seen in the presence of thapsigargin (TP) (10(-5) M), a specific inhibitor of the microsomal Ca(2+) pump enzyme. Regucalcin (10(-10)-10(-8) M) significantly stimulated ATP-dependent (45)Ca(2+) uptake by the microsomes. The effect of regucalcin (10(-8) M) in increasing microsomal Ca(2+)-ATPase activity was completely prevented in the presence of digitonin (10(-3) or 10(-2)%), which has a solubilizing effect on membranous lipid, or N-ethylmaleimide (NEM), a modifying reagent of sulfhydryl (SH) groups. Dithiothreitol (DTT; 5 mM), a protecting reagent of SH groups, increased markedly Ca(2+)-ATPase activity. In the presence of DTT (5 mM), regucalcin could not significantly enhance the enzyme activity. Also, the effect of regucalcin in increasing Ca(2+)-ATPase activity was completely inhibited by the addition of vanadate (1 mM), an inhibitor of phosphorylation of enzyme. In addition, the effect of regucalcin on Ca(2+)-ATPase activity was not significantly modulated in the presence of dibutyryl cyclic AMP (10(-4) M), inositol 1,4,5-trisphosphate (10(-3) M), or calmodulin (5 microg/ml) which is an intracellular signaling factor. The present study demonstrates that regucalcin can activate Ca(2+) pump activity in rat heart microsomes, and that the protein may act the SH groups of Ca(2+)-ATPase by binding to microsomal membranes.

Kolaviron Modulates cellular redox status and impairment of membrane protein activities induced by potassium bromate (KBrO3) in rats

In this study, we examined the modulatory effects of kolaviron, a biflavonoid from Garcinia kola seeds on the antioxidant defense mechanisms, cellular redox status and oxidative stress in the kidney and liver of rats pretreated with potassium bromate (KBrO3) intragastrically as a single dose of 300 mg kg−1weight for 4 weeks. Treatment of rats with KBrO3resulted in an insignificant difference (P> 0.05) in body weight compared to controls. However, a significant increase in kidney/body weight ratio (P< 0.001) was observed in rats treated with KBrO3while liver/body weight ratio was not affected. KBrO3depressed the activities of superoxide dismutase, glutathione peroxidase and catalase (P< 0.001) in the kidney but not in the liver. Kolaviron (200 mg kg−1body weight) administered three times a week for 4 weeks inhibited the decrease mediated by KBrO3of these enzymes in the kidney by 29, 88 and 45%, respectively. Similarly, kolaviron reduced the KBrO3-induced decrease in the activities of γ -glutamyltransferase and microsomal Ca2+ATPase by 73 and 63% in the kidney. In addition, the extract elicited a 27 and 25% decrease in the KBrO3-induced increase in malondialdehyde and lipid hydroperoxide formation in the kidney. Kolaviron also attenuated the KBrO3-decreased activities of glucose 6-phosphatase, 5 ′ nucleotidase and alkaline phosphatase (membrane enzymes) by 72, 57 and 25% respectively. The results of the present investigation indicate the antioxidative effect of kolaviron, a natural antioxidant, on drug-induced kidney toxicity. Kolaviron may therefore intervene in the cellular redox status and depression of membrane protein activities caused by KBrO3and other environmental carcinogens in the kidney.

Role of endogenous regucalcin in transgenic rats: suppression of kidney cortex cytosolic protein phosphatase activity and enhancement of heart muscle microsomal Ca2+-ATPase activity.

Rats were generated by pronuclear injection of the transgene with a cDNA construct encoding rat regucalcin that is a regulatory protein of Ca2+ signaling. Transgenic (TG) founders were fertile, transmitted the transgene at the expected frequency, and bred to homozygote. Western analysis of the cytosol prepared from the tissue of TG female rats (5-week-old) showed a remarkable expression of regucalcin (3.3 kDa) protein in the liver, kidney cortex, heart, lung, stomach, brain, spleen, muscle, colon, and duodenum. Regucalcin expression of TG male rats was seen in the liver, kidney cortex, heart, and lung. In wild-type (wt) male and female rats, regucalcin was mainly present in the liver and kidney cortex. Regucalcin inhibited protein phosphatase activity in rat kidney cortex cytosol and activated Ca2+-ATPase activity in rat heart muscle microsomes. The suppressive effect of regucalcin on protein phosphatase activity was significantly enhanced in the cytosol of kidney cortex of TG male and female rats as compared with those of wt rats. Likewise, heart muscle microsomal Ca2+-ATPase activity was significantly enhanced in TG rats. The changes in their enzyme's activities in TG rats were completely abolished in the presence of anti-regucalcin monoclonal antibody (100 ng/ml) in the enzyme reaction mixture. Moreover, the body weight of TG female rats was significantly lowered as compared with that of wt rats. Serum inorganic phosphorus concentration was significantly increased in TG male and female rats, while serum calcium, glucose, triglyceride, free cholesterol, albumin, and urea nitrogen concentrations were not significantly altered in TG rats. Regucalcin TG rats should be a useful model to define a regulatory role of endogenous regucalcin in the tissues in vivo.

Consequence of aflatoxin B1 ingestion on the membrane-bound liver endoplasmic reticulum Ca2+-ATPase of protein-undernourished Fischer F344 rats.

Ingestion of the mycotoxin Aflatoxin B1 (AFB1) by protein-undernourished Fischer F344 rats for twelve weeks resulted in significant (p < 0.05) increases in the proliferation of liver cells, reduced body weight and increased microsomal Ca2+-ATPase activity. Cyt. P450 content, gamma-Glutamyl Transferase (GGT) and Glutathione Transferase (GST) activities were unaffected. The ingestion of AFB, by normal rats had no effect on all the parameters investigated. It appears that the microsomal Ca2+-pumping ATPase of Protein-Undernourished (PU) Fischer F344 rats is more sensitive to the mycotoxin AFB1 than its counterpart in well-nourished rats. The use of PU rat as animal model for studies on AFB, toxicity, particularly the effect of AFB1 on the regulation of intracellular calcium ion concentration ([Ca2+]), is suggested.

Inhibition of microsomal ATPases by high concentration of Mg 2+ in tracheal epithelial cells

Previous work in our laboratory established the presence of two types of microsomal ATPases, a low-affinity vanadate-sensitive (LAVS) and a high-affinity vanadate-sensitive (HAVS) ATPases, in tracheal epithelial cells. These ATPases were identified as Ca 2+-ATPases by specific inhibitors and microsomal Ca 2+ uptakes. Since the regulatory roles of Mg 2+ on both cellular Ca 2+-signaling and epithelial transports were demonstrated, the effects of Mg 2+ on these ATPases were investigated. Mg 2+-dependence of ATPase activity appeared bell-shaped with a maximal activity at 1–2 mM Mg 2+ and Mg 2+ at higher than 2 mM inhibited these enzymes. In a kinetic analysis of the LAVS ATPase inhibition, high concentration of Mg 2+ appeared to inhibit the binding of ATP to a substrate-binding site. The microsomal 45Ca 2+ uptakes mediated by both ATPases were also inhibited by high concentration of Mg 2+. In order to test whether high concentration of Mg 2+ directly inhibits these enzymes, microsomes were made leaky by the treatment of Triton X-100 and the microsomal ATPases were solubilized with CHAPS. The leaky microsomal ATPases and CHAPS-solubilized ATPases were similarly inhibited by high concentration of Mg 2+, suggesting that Mg 2+ directly inhibit these enzymes. In conclusion, Mg 2+ has two types of modulatory effects on these enzymes, a catalytic effect at low concentration and an inhibitory effect at high concentration.

Ryanodine receptor acts as a sensor for redox stress.

Ryanoids have not attained importance as insecticides, but the increasing number of xenobiotic effectors known to influence Ca2+ signalling by interaction with ryanodine receptors (RyRs) may serve to identify new targets for insect control. A historical review of redox control of microsomal Ca2+ transport is given here, followed by recent evidence indicating that hyperactive Cys residues are an essential component of a transmembrane redox sensor. Focus is on the role of sulfhydryl chemistry in RyR regulation; metabolic quinonoid intermediates from pesticides and other environmental contaminants are of interest in this context.

Multiple pathways of σ 1 receptor ligand uptakes into primary cultured neuronal cells

Although many antipsychotics have affinities for σ receptors, the transportation pathway of exogenous σ 1 receptor ligands to intracellular type-1 σ receptors are not fully understood. In this study, σ 1 receptor ligand uptakes were studied using primary cultured neuronal cells. [ 3H](+)-pentazocine and [ 3H]( R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]methyl-2-pyrrolidinone l-tartrate (MS-377), used as a selective σ 1 receptor ligands, were taken up in a time-, energy- and temperature-dependent manner, suggesting that active transport mechanisms were involved in their uptakes. σ 1 receptor ligands taken up into primary cultured neuronal cells were not restricted to agonists, but also concerned antagonists. The uptakes of these ligands were mainly Na +-independent. Kinetic analysis of [ 3H](+)-pentazocine and [ 3H]MS-377 uptake showed K m values (μM) of 0.27 and 0.32, and V max values (pmol/mg protein/min) of 17.4 and 9.4, respectively. Although both ligands were incorporated, the pharmacological properties of these two ligands were different. Uptake of [ 3H](+)-pentazocine was inhibited in the range 0.4–7.1 μM by all the σ 1 receptor ligands used, including N, N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride (NE-100), a selective σ 1 receptor ligand. In contrast, the inhibition of [ 3H]MS-377 uptake was potently inhibited by haloperidol, characterized by supersensitivity (IC 50, approximately 2 nM) and was inhibited by NE-100 with low sensitivity (IC 50, 4.5 μM). Moreover, kinetic analysis revealed that NE-100 inhibited [ 3H]MS-377 uptake in a noncompetitive manner, suggesting that NE-100 acted at a site different from the uptake sites of [ 3H]MS-377. These findings suggest that there are at least two uptake pathways for σ 1 receptor ligands in primary cultured neuronal cells (i.e. a haloperidol-sensitive pathway and another, unclear, pathway). In addition, pretreatment of cells with a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), a myosin light chain kinase inhibitor, 1-(5-chloronaphthalene-1-sulfonyl)homopiperazine (ML-9), or microsomal Ca 2+-ATPase inhibitors resulted in a reduction of the amount of σ receptor ligand uptake. These findings suggest that the Ca 2+ pump on the endoplasmic reticulum and/or calmodulin-related events might be involved in the regulation of the uptake of σ receptor ligands into primary neuronal cells.

Activation of muscarinic acetylcholine receptors induces Ca 2+ mobilization in FRT cells

The effect of the muscarinic receptors agonist carbachol (Cch) on intracellular calcium concentration ([Ca 2+] i) and cAMP level was studied in polarized Fischer rat thyroid (FRT) epithelial cells. Cch provoked a transient increase in [Ca 2+] i, followed by a lower sustained phase. Thapsigargin, a specific microsomal Ca 2+-ATPase inhibitor, caused a rapid rise in [Ca 2+] i and subsequent addition of Cch was without effect. Removal of extracellular Ca 2+ reduced the initial transient response and completely abolished the plateau phase. Ryanodine, an agent that depletes intracellular Ca 2+ stores through stimulation of ryanodine receptors (RyRs), had no effect on [Ca 2+] i. However, the transitory activation of [Ca 2+] i was dose-dependently attenuated in cells pretreated with U73122, a specific inhibitor of phospholipase C (PLC). These data suggest that the Cch-stimulated increment of [Ca 2+] i required IP 3 formation and binding to its specific receptors in Ca 2+ stores. Further studies were performed to investigate whether the effect of Cch on Ca 2+ entry into FRT cells was via L-type voltage-dependent Ca 2+ channels (L-VDCCs). Nicardipine, a nonspecific L-type Ca 2+ channel blocker, decreased Cch-induced increase on [Ca 2+] i, while Bay K-8644, an L-type Ca 2+ channel agonist, slightly increased [Ca 2+] i in FRT cells. These data indicate that Ca 2+ entry into these nondifferentiated thyroid cells occurs through an L-VDCC, and probably through another mechanism such as a capacitative pathway. Cch did not affect the intracellular cAMP levels, but its effects on [Ca 2+] i were significantly reduced when cells were pretreated with forskolin, suggesting the existence of an intracellular cross-talk between PLC and cAMP mechanisms in the regulation of intracellular Ca 2+ mobilization in neoplastic FRT cells.

Evidence for a role of the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase in thapsigargin and Bcl-2 induced changes in Xenopus laevis oocyte maturation.

Thapsigargin (Tg), a selective inhibitor of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA), causes depletion of intracellular Ca(2+) stores, hence activation of capacitative Ca(2+) entry (CCE). Incubation of Xenopus laevis oocytes with Tg resulted in an increased rate of progesterone-induced meiotic maturation. Non-mitochondrial (45)Ca(2+) uptake by SERCA-containing microsomes prepared from control wild-type oocytes microinjected with sterile water was inhibited essentially 100% by Tg. However, overexpression of Bcl-2, an oncogene known to protect against Tg-induced apoptosis in certain cell types, resulted in only 40% inhibition of microsomal (45)Ca(2+) uptake by Tg while non-inhibited (45)Ca(2+) uptake remained unchanged. Moreover Bcl-2 overexpression also protected against inhibition of CCE. I(Cl(Ca)) was similar in Bcl-2-overexpressing and control oocytes when intracellular Ca(2+) store depletion was induced by microinjection of inositol 1,4,5-trisphosphate (InsP(3)) and other means and when CCE was induced by means independent of SERCA inhibition. Our data indicate that Bcl-2 affects neither the InsP(3) receptor nor Ca(2+) entry itself. At the end of a 24-h period after progesterone addition to the medium, only 25% of Bcl-2-overexpressing oocytes had matured compared to 85% of control oocytes. Our data suggest that SERCA participates in Xenopus oocyte maturation by controlling cytosolic Ca(2+) and/or intracellular Ca(2+) stores, hence CCE. An observed progesterone-dependent protein kinase-catalysed phosphorylation of SERCA is further indication of its role in oocyte maturation.

Nicotinic acid-adenine dinucleotide phosphate (NAADP) elicits specific microsomal Ca2+ release from mammalian cells.

Nicotinic acid-adenine dinucleotide phosphate (NAADP), a molecule derived from beta-NADP, has been shown to promote intracellular calcium release in sea urchin eggs. However, there is little information regarding the role of NAADP in the regulation of intracellular calcium fluxes in mammalian cells. We found recently that several mammalian tissues have a high capacity for NAADP synthesis, as assessed by sea urchin egg bioassay. To determine the functional significance of NAADP production by mammalian tissues, we sought to determine whether NAADP is capable of inducing calcium release from microsomes prepared from cultured cells. We found that NAADP, but not beta-NADP, activates a specific microsomal calcium release system in mesangial cells isolated from rat kidney; NAADP was without effect in renal tubular epithelial cells. NAADP-induced calcium release is not affected by inhibitors of the inositol 1,4,5-trisphosphate or ryanodine channels. However, NAADP-elicited calcium release was inhibited by L-type calcium channel blockers and by alkaline phosphatase treatment of NAADP. NAADP also promotes specific microsomal calcium release in rat vascular smooth muscle cells, cardiac myocytes, fibroblasts and a human leukaemia cell line, indicating that the capacity for NAADP-induced calcium release is widespread in mammalian cells. We propose that NAADP may be an important regulator of intracellular calcium in many mammalian tissues.

Nicotinic acid–adenine dinucleotide phosphate (NAADP) elicits specific microsomal Ca2+ release from mammalian cells

Nicotinic acid–adenine dinucleotide phosphate (NAADP), a molecule derived from β-NADP, has been shown to promote intracellular calcium release in sea urchin eggs. However, there is little information regarding the role of NAADP in the regulation of intracellular calcium fluxes in mammalian cells. We found recently that several mammalian tissues have a high capacity for NAADP synthesis, as assessed by sea urchin egg bioassay. To determine the functional significance of NAADP production by mammalian tissues, we sought to determine whether NAADP is capable of inducing calcium release from microsomes prepared from cultured cells. We found that NAADP, but not β-NADP, activates a specific microsomal calcium release system in mesangial cells isolated from rat kidney; NAADP was without effect in renal tubular epithelial cells. NAADP-induced calcium release is not affected by inhibitors of the inositol 1,4,5-trisphosphate or ryanodine channels. However, NAADP-elicited calcium release was inhibited by L-type calcium channel blockers and by alkaline phosphatase treatment of NAADP. NAADP also promotes specific microsomal calcium release in rat vascular smooth muscle cells, cardiac myocytes, fibroblasts and a human leukaemia cell line, indicating that the capacity for NAADP-induced calcium release is widespread in mammalian cells. We propose that NAADP may be an important regulator of intracellular calcium in many mammalian tissues.

Chlorpromazine inhibits store‐operated calcium entry and subsequent noradrenaline secretion in PC12 cells

1. The effect of chlorpromazine on the store-operated Ca2+ entry activated via the phospholipase C signalling pathway was investigated in PC12 cells. 2. Chlorpromazine inhibited the sustained increase after the initial peak in the intracellular Ca2+ concentration produced by bradykinin while having no effect on the initial transient response. The inhibition was lowered by the removal of extracellular free Ca2+. However, chlorpromazine did not inhibit bradykinin-induced inositol 1,4,5-trisphosphate production. 3. Chlorpromazine inhibited the bradykinin-induced noradrenaline secretion in a concentration-dependent manner (IC(50): 24+/-5 microM, n=3). 4. To test for a direct effect of chlorpromazine on store-operated Ca2+ entry, thapsigargin, an inhibitor of microsomal Ca(2+)-ATPase, was used to induce store-operated Ca2+ entry in PC12 cells. Chlorpromazine reduced the thapsigargin-induced sustained Ca2+ level (IC(50): 24+/-2 microM, n=3), and the inhibition also occluded the inhibitory action of 1-[-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenyl]-1H-imidazole hydrochloride (SK&F96365). 5. The results suggest that chlorpromazine negatively modulates the store-operated Ca2+ entry activated subsequent to PLC activation.

Neurochemical effects of environmental chemicals: in vitro and in vivo correlations on second messenger pathways.

Polychlorinated biphenyls (PCBs) are persistent, bioaccumulative, toxic, and widely distributed environmental chemicals. There is now both epidemiological and experimental evidence that PCBs cause cognitive deficits; however, the underlying cellular or molecular mechanism(s) is not known. We have hypothesized that altered signal transduction/second messenger homeostasis by PCBs may be associated with these effects since second messengers in signal transduction pathways, such as calcium, inositol phosphates (IP), and protein kinase C (PKC), play key roles in neuronal development and their function. In vitro studies using cerebellar granule neurons and isolated organelle preparations indicate that ortho-PCBs increase intracellular free Ca2+ levels by inhibiting microsomal and mitochondrial Ca2+ buffering and the Ca2+ extrusion process. Ortho-PCBs also increase agonist-stimulated IP accumulation and cause PKC translocation at low micromolar concentrations where no cytotoxicity is observed. On the other hand, non-ortho-PCBs are not effective in altering these events. Further SAR studies indicate that congeners with chlorine substitutions favoring non-coplanarity are active in vitro, while congeners favoring coplanarity are relatively inactive. Subsequent in vivo studies have shown that repeated exposure to a PCB mixture, Aroclor 1254, increases PKC translocation and decreases Ca2+ buffering in the brain, similar to in vitro studies. These changes in vivo are associated with elevated levels of non-coplanar ortho-PCB congeners at levels equivalent to 40-50 microM in brain, the concentrations that significantly inhibited second messenger systems in neuronal cultures in vitro. Current research is focusing on PCB-induced alterations in second messenger systems following developmental exposure.

Characterization of platelet-activating factor-induced cytosolic calcium mobilization in human eosinophils.

Activated eosinophils play an important role in the pathogenesis of bronchial asthma and other allergic diseases, and platelet-activating factor (PAF) is a potent activator of eosinophils. To characterize the cytosolic Ca2+ ([Ca2+]i) mobilization in human eosinophils in response to PAF. [Ca2+]i responses to PAF were examined in human eosinophils using a microscopic fura-2 fluorescence-ratio imaging system. PAF caused a significant and dose-dependent increase in (Ca2+)i, which consisted of an initial rapid rise followed by a sustained elevation. This PAF-induced (Ca2+)i rise was inhibited by WEB 2086, a specific PAF receptor antagonist. The addition of 5 mM EGTA or 1 mM Ni2+ to a nominally Ca2+-free solution did not appreciably reduce the initial rise but significantly inhibited the sustained rise. The application of a protein kinase C inhibitor, Ro31-8220, augmented the sustained increase by PAF. Thapsigargin, a microsomal Ca2+ ATPase inhibitor, induced no appreciable change in a nominally Ca2+-free solution but induced a marked increase in (Ca2+)i when changed to a Ca2+-containing solution. The initial rapid rise and the following sustained rise in (Ca2+)i by PAF depends on Ca2+ release from the intracellular Ca2+ stores and Ca2+ influx, respectively, which are regulated by protein kinase C in human eosinophils. Furthermore, the so called Ca2+-capacitative entry is possibly involved in the Ca2+ influx from the extracellular solution in human eosinophils.

Modulation of the formalin-induced nociceptive response by diabetes: possible involvement of intracellular calcium

We examined the involvement of cytosolic calcium in the modulation of the formalin-induced nociceptive response by diabetes. Injection of formalin into the hindpaw of mice produced a biphasic nociceptive response consisting of immediate (first phase) and tonic (second phase) components. Although the duration of the first-phase response was significantly longer in diabetic mice than in non-diabetic mice, the second phase was significantly shorter in diabetic mice. The first-phase response was dose-dependently and significantly reduced by pretreatment with ryanodine, which blocks Ca 2+ release from Ca 2+/caffeine-sensitive microsomal pools. The second-phase response was also significantly increased when diabetic mice were pretreated with ryanodine. However, ryanodine had no significant effect on either the first-phase or second-phase response in non-diabetic mice. On the other hand, pretreatment with thapsigargin, which inhibits Ca 2+ uptake into the inositol-1,4,5-trisphosphate-sensitive microsomal Ca 2+ pool, significantly enhanced the first-phase response in non-diabetic mice. Furthermore, thapsigargin significantly and dose-dependently reduced the second phase of the formalin-induced nociceptive response in non-diabetic mice. Thapsigargin administered i.t. did not significantly affect either the first- or the second-phase response in diabetic mice. These results suggest that the change in the formalin-induced nociceptive response in diabetic mice may be due, at least in part, to the modification of nociceptive transmission in the spinal cord by intracellular calcium.

The Ca2+-transporting activity of rat liver microsomes in response to protein undernutrition: implications for liver tumor promotion.

The effect of protein undernutrition on the activity of the smooth endoplasmic reticulum (microsomal) Ca2+-ATPase (SERCA) was investigated. After 12 weeks of ad libitum ingestion of low protein diet (5% protein), a significant depression (p <0.05) of liver ER Ca2+-ATPase activity (68.6% depression) was observed. However, no significant effects on cytochrome P450 activity and relative liver weight were found. It is proposed that low protein diet by inhibiting the rat liver SERCA activity, might increase the cytosolic free calcium ion concentration ([Ca2+]) and promote the development of liver tumor. The possible mechanisms of low protein diet induced inhibition of SERCA activity are highlighted.

Immediate cell signal induced by laminin in rat sertoli cells.

Rat Sertoli cells in primary culture have been studied for their ability to respond to extracellular matrix macromolecules by increases of [Ca(2+)](i). We observed that cells seeded on glass coverslips, loaded with the intracellular Ca(2+) indicator fura-2, responded to laminin, but not to fibronectin, with an immediate [Ca(2+)](i) raise, with a peak followed by a prolonged plateau. [Ca(2+)](i) increases were dependent upon Ca(2+) influx across the plasma membrane and Ca(2+) release from intracellular Ca(2+) pools. Ca(2+) influx was inhibited by extracellular Ca(2+) removal by EGTA, and by treatment with La(3+), or with the L-type voltage operated Ca(2+) channel blocker, nifedipine. Ca(2+) release from intracellular Ca(2+) storing organelles, was inhibited by the microsomal Ca(2+)-ATPase blocker thapsigargin. Responses were mimicked by synthetic peptides carrying the Arg-Gly-Asp adhesion sequence, but not by the control Arg-Gly-Glu-containing peptide, in which aspartic acid was replaced by glutamic acid. Laminin-dependent [Ca(2+)](i) increases were down-regulated by the follicle-stimulating hormone. However, this occurred only when cells were not subjected to homotypic cell-cell contact, and responded to the hormone with a significant [Ca(2+)](i) elevation. These results indicate that laminin may regulate Sertoli cells by intracellular signals that perturb Ca(2+) homeostasis. This role may be related to an effect exerted by the seminiferous epithelium basement membrane on the regulation of spermatogenesis.

Unmodified calcium concentrations in tumour necrosis factor receptor subtype-mediated apoptotic cell death.

Tumour necrosis factor-alpha (TNF) receptors mediate a variety of effects dependent on cell type. A role for Ca2+ in TNF-induced death remains uncertain. Here we investigated restricting intracellular/extracellular Ca2+ in HeLa epithelial carcinoma cells expressing low and high levels of p75TNFR receptor subtype and KYM-1 rhabdomyosarcoma cells, models of rapid TNF-induced apoptosis. Ca2+ -chelators EGTA and BAPTA-AM as well as microsomal Ca2+ -ATPase inhibitor thapsigargin, did not alter TNF-induced death. TNF was also unable to alter resting [Ca2+]i levels which remained < 200 nM even during times when these cells were undergoing apoptotic cell death. These findings indicate no role for modulated Ca2+ concentrations in TNF-induced apoptotic cell death.

Ischemia-induced inhibition of active calcium transport into gerbil brain microsomes: effect of anesthetics and models of ischemia.

The excessive increase in intracellular Ca2+ concentration is associated with events linking cerebral blood flow reduction to neuronal cell damage. We have investigated the possible effect of ischemia and ischemia-reperfusion injury on endoplasmic reticulum (ER) Ca2+ transport. Two different models of ischemia as well as two different anesthetics were used. 5 min and 15 min of global forebrain ischemia caused significant depression of the rate of microsomal Ca2+ accumulation in pentobarbital anesthetised gerbils. The Ca2+ uptake activity recovered partially after 1 hour of reperfusion. Unlike pentobarbital anesthetised gerbils, no significant changes were detected in the active microsomal Ca(2+)-transport after 10 min of global forebrain ischemia in gerbil forebrain and hippocampus under halothane anesthesia. In addition, using the model of decapitation ischemia, we observed significant changes of the Ca2+ uptake in both halothane and pentobarbital anesthetised gerbils. These findings indicate that ischemic insult alters the brain microsomal Ca2+ transport which is not due to inhibition of the Ca(2+)-ATPase activity. However, the effect of ischemia on this transport system is dependent on the model of ischemia and on the type of anesthetics.