Increase In Cytosolic Free Ca2 Research Articles

Silica increases cytosolic free calcium ion concentration of alveolar macrophages in vitro

Rat alveolar macrophages were exposed to silica dust (quartz) suspended in culture medium (SiO 2, dry particle size less than 5 μm in diameter) and fluctuation in their cytosolic free calcium content ([Ca 2+]i) was detected in cell monolayers with a fluorescent calcium probe (Indo-1AM). Cytosolic free calcium content was correlated with lactate dehydrogenase (LDH) release, an index of cell damage. SiO 2 induced a concentration- and time-dependent increase of cytosolic free Ca 2+ ion concentration and LDH release. [Ca 2+]i was increased about fivefold when cells were exposed to 200 μg of SiO 2 per milliliter (3 ml per dish) for 2 hr. [Ca 2+]i changed within 15 min of SiO 2 treatment, whereas LDH release was measurably increased only after 30 min. Chelation of extracellular Ca 2+ by 2 m m ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetate did not prevent SiO 2-induced fluctuation of macrophage [Ca 2+]i, but did partially prevent the SiO 2-induced increase in LDH release ( p < 0.01). We conclude that a very early event in SiO 2-induced damage of alveolar macrophages involves mobilization of intracellular calcium pools to increase [Ca 2+]i. These results suggest that SiO 2-induced macrophage damage, a key event in the development of silicosis, may involve perturbation of intracellular calcium homeostasis.

Pertussis-toxin-sensitive inhibition by (-) baclofen of Ca signals in bovine chromaffin cells.

Ca signals in bovine adrenal chromaffin cells were studied both in Fura-2/AM-loaded intact cells, and in voltage-clamped cells under whole-cell patch-clamp conditions. The effects of gamma-aminobutyric acid b subtype (GABAb) receptor activation on K(+)-depolarization-induced signals and on voltage-activated Ca2+ currents were investigated. Both GABA (20 microM) plus bicuculline (20 microM) and (-)baclofen (20-100 microM), effectively inhibited the Ca signal in intact cells. The effects caused by baclofen continued to develop during the time interval between two successive stimuli. The restoration of the Ca signal during washout of baclofen was also delayed and continued in some experiments for 10-20 min. The inhibitory effect of baclofen on the Ca signal was eliminated by pre-treatment of the cells with pertussis toxin (PTX, 1 micrograms/ml, for 4-6 h at 37 degrees C). Baclofen (50 microM) inhibited Ca2+ current in whole-cell mode by at most 20%. The effect developed quickly and was reversible. Infusion into the cells of a non-hydrolyzable analogue of guanosine 5'-triphosphate GTP gamma S (100 microM), led to complete inhibition of the Ca2+ conductance and of voltage-evoked intracellular Ca ([CA]i) transients within 2 min. In paired cells intracellularly perfused with GTP gamma S-free solution, the Ca2+ current amplitude decreased by only about 30% for 5-6 min. It is concluded that bovine chromaffin cells have functional GABAb receptors the activation of which, mediated by a PTX-sensitive GTP-binding protein, inhibits the evoked increase in cytosolic free Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

The signal transduction pathway involved in the migration induced by a monocyte chemotactic cytokine.

Recombinant monocyte-chemotactic and activating factor (rMCAF; alternative acronyms MCP-1, TDCF, human JE) induced migration of human monocytes across polycarbonate or nitrocellulose filters. Maximal induction of migration was observed at a concentration of 10 ng/ml (10(-9) M). Checkerboard analysis revealed that rMCAF elicited true gradient-dependent chemotactic migration, although a gradient independent chemokinetic effect was observed at low concentrations (1-5 ng/ml). rMCAF caused a rapid (less than 5 s) and transient (approximately 1.5 min) increase of free cytosolic Ca2+ ions, as assessed by the fura-2 probe. No Ca2+ increase was detected in neutrophils or lymphocytes stimulated by rMCAF. Studies conducted in the absence of extracellular Ca2+ or in the presence of Ni2+ (an inhibitor of Ca2+ influx) suggested that the increase of intracellular Ca2+ induced by rMCAF is dependent on the influx of extracellular Ca2+ through plasma membrane channels. Bordetella pertussis toxin inhibited the intracellular Ca2+ elevation and chemotaxis caused by rMCAF. The possible involvement of Ca(2+)-dependent protein kinases in rMCAF signaling pathway(s) was explored using inhibitors. Inhibitors of GMP-dependent kinase and myosin L chain kinase had no effect on rMCAF-induced monocyte migration. In contrast, protein kinase C/cAMP-dependent kinase inhibitors (such as, C-I, H-7, HA-1004, KT5720, and Staurosporine) markedly decreased rMCAF induced chemotaxis suggesting the involvement of a serine/threonine protein kinase, possibly protein kinase C, in rMCAF signaling pathway.

Differential coupling of muscarinic receptors to Ca 2+ mobilization and cyclic AMP in SH-SY5Y and IMR 32 neuroblastoma cells

Muscarinic receptor-linked Ca 2+ mobilization and changes in cyclic AMP were studied in SH-SY5Y and IMR 32 human neuroblastoma cell lines. Muscarinic agonists acetylcholine, carbachol, methacholine and muscarine induced an increase in cytosolic free Ca 2+ in a pertussis toxin (100 ng/ml)-insensitive manner in both cell lines. The ED 50 values in IMR 32 cells (8–98 μM) were one order of magnitude higher than in SH-SY5Y cells (0.3 – 1.6 μM). Oxotremorine and pilocarpine failed to mobilize Ca 2+ in IMR 32 cells. Pirenzepine antagonized carbachol-induced Ca 2+ mobilization in SH-SY5Y cells with a K i value in the range of 150–189 nM whereas the corresponding values in IMR 32 cells were 24–28 nM. Atropine inhibited a carbachol-stimulated increase in cytosolic Ca 2+ with an equal potency in both cell lines (K i 2–3 nM). Carbachol stimulated cyclic AMP (cAMP) accumulation in SH-SY5Y cells in a pertussis toxin-insensitive manner. In IMR 32 cells carbachol inhibited prostaglandin E 1-stimulated cAMP accumulation. Treatment of IMR 32 cells with pertussis toxin abolished the inhibition of stimulated cAMP accumulation. These results suggest that in SH-SY5Y cells the M 3 muscarinic receptor couples to both Ca 2+ mobilization and stimulation of cAMP accumulation. In IMR 32 cells the M 1 receptor seems to couple to Ca 2+ mobilization whereas the inhibition of stimulated cAMP accumulation is coupled to a non-M 1 subtype by an inhibitory G-protein.

The inhibition of human platelet function by ganodermic acids.

Human gel-filtered platelets aggregate at greater than 20 microM-ganodermic acid S [lanosta-7,9(11),24-triene-3 beta, 15 alpha-diacetoxy-26-oic acid] [Wang, Chen, Shiao & Wang (1989) Biochim. Biophys. Acta 986, 151-160]. This study showed that platelets at less than 20 microM-ganodermic acid S displayed both concentration- and time-dependent inhibition of function, in which the agent potency in response to inducers was ADP-fibrinogen greater than collagen greater than thrombin. The agent caused a biphasic time-dependent effect on platelet phosphoinositide metabolism. The first phase involved the decrease in the pool size of phosphoinositide by 10-20%. The second phase, in which both the resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2) and the decrease of [32P]phosphatidic acid occurred, took place after 30 min. Scanning electron microscopy also revealed a time-dependent morphological change in platelets in the presence of the agent. The cells initially became spiculate discs, then swelled to a 'potato-like' morphology at 60 min. Further studies on the time-dependent inhibition of thrombin response revealed that: (1) the percentage inhibition of cell aggregation was comparable with that occurring with an increase of cytosolic free Ca2+ concentration [( Ca2+]i) or the phosphorylation of marker proteins; (2) [32P]Pi-labelled platelets showed the time-dependent inhibition of thrombin-stimulated PIP2 resynthesis as indicated by first-2-min time-course studies of phosphoinositide interconversion; (3) scanning electron microscopy revealed that the aged platelet population showed an increase in the percentage of non-responding cells on prolonged incubation. The results, taken together, enabled one to discuss a possible mechanism for the time-dependent inhibition by ganodermic acid S of platelet response to thrombin.

The role of intracellular Ca2+ in the regulation of the plasma membrane Ca2+ permeability of unstimulated rat lymphocytes

The mechanism responsible for the increase in cytosolic free Ca2+ concentration ([Ca2+]i) during mitogenic stimulation of lymphocytes has been widely investigated. By contrast, little is known about the processes underlying Ca2+i homeostasis in resting (unstimulated) cells. It has been suggested that [Ca2+]i is an important determinant of the rate of Ca2+ influx following mitogenic activation. Using rat thymic lymphocytes, we investigated whether the resting influx pathway is similarly controlled by [Ca2+]i. Otherwise untreated cells were Ca(2+)-depleted by loading with Ca2+ chelators while suspended in Ca(2+)-free solution. Ca2+ depletion induced an 8-fold increase in the rate of unidirectional Ca2+ uptake. The depletion-activated flux was voltage-sensitive and was blocked by La3+ and by compound SK&F 96365, a receptor-operated Ca2+ channel blocker. Upon reintroduction to Ca(2+)-containing solution, the increased influx brought about a rapid recovery of [Ca2+]i. Detailed analysis of the magnitude of the 45Ca2+ flux during this recovery indicated that [Ca2+]i is not the primary determinant of the plasmalemmal Ca2+ permeability. Instead, depletion of an internal thapsigargin-sensitive store correlates with and appears to be responsible for the increased permeability of the plasma membrane. Accordingly, the Ca2+ fluxes induced by intracellular Ca2+ depletion and by thapsigargin were pharmacologically indistinguishable. Mitogenic lectins also released Ca2+ from a thapsigargin-sensitive store and activated a plasmalemmal Ca2+ permeability displaying identical pharmacology. The data support the existence of a coupling process whereby the degree of filling of an internal Ca2+ store dictates the Ca2+ permeability of the plasma membrane. This coupling mechanism is important not only in mediating the effects of mitogens and other agonists, as suggested before, but seemingly also in the control of resting Ca2+i homeostasis in unstimulated cells.

Endothelin-1 is an autocrine/paracrine growth factor for human cancer cell lines.

We studied whether a novel vasoconstrictor peptide, endothelin-1 (ET-1), is synthesized by and released from human carcinoma cell lines, and whether ET-1 stimulates proliferation of these tumor cells. ET-1-like immunoreactivity was released from both HeLa and HEp-2 cells as a function of time. Reverse-phase HPLC of the conditioned media from HeLa cells revealed a major peak coeluting with standard ET-1. Northern blot analysis demonstrated the expression of mRNA for ET-1 precursor in both tumor cell lines. Both cell lines contained a single class of specific binding sites for ET-1. ET-1 dose-dependently induced increases in cytosolic free Ca2+ concentration in fura-2-loaded tumor cells, whose effect was completely abolished by chelating extracellular Ca2+ or by Ca(2+)-channel blocker. ET-1 stimulated proliferation of the quiescent cell lines in a dose-dependent manner, whose effect was inhibited by Ca(2+)-channel blocker. Polyclonal antibody for ET-1 inhibited proliferation of these cell lines, whereas nonimmune serum had no effect. These results demonstrate that ET-1 is synthesized by and released from human epithelial carcinoma cell lines, and that exogenous and endogenous ET-1 stimulates proliferation of the cells possibly through Ca2+ influx, suggesting its role as an autocrine/paracrine growth factor for certain tumor cells.

Dextromethorphan and phencyclidine receptor ligands: differential effects on K(+)- and NMDA-evoked increases in cytosolic free Ca2+ concentration.

The ability of dextromethorphan (DXM) and phencyclidine (PCP) receptor ligands to attenuate increases in cytosolic free Ca2+ concentration ([Ca2+]i) evoked by N-methyl-D-aspartate (NMDA) and high extracellular [K+] was examined using the fluorescent dye Fura 2 in cultured rat hippocampal pyramidal neurons. The DXM receptor ligand caramiphen (40 microM) reduced K(+)-evoked rises in [Ca2+]i to a greater extent than NMDA-evoked rises; the reverse was true for the PCP receptor ligands ketamine (10-40 microM) and dextrorphan (10 microM). DXM itself, which has affinity for both DXM and PCP receptors, reduced both K(+)- and NMDA-evoked increases in [Ca2+]i in a concentration-dependent manner. The results suggest that DXM receptor ligands may at least in part exert their known anticonvulsant and neuroprotective effects by reducing Ca2+ influx through voltage-activated Ca2+ channels.

Voltage dependence of desensitization to carbamylcholine in frog muscle fibers reverses in low-calcium solutions

1. The effect of reduction in external Ca concentration on the voltage dependence of desensitization to carbamylcholine was studied in frog muscle fibers to determine whether local increase in cytosolic free Ca2+ from inward movement through agonist-activated receptor channels is a factor producing increased desensitization rate, as has been proposed. A critical feature is whether or not the relation of desensitization rate with voltage decreases in a regular manner as potential is increased to levels above ECa. 2. A conventional two-electrode voltage clamp procedure was used for voltage control of the postjunctional membrane of K-depolarized muscle fibers, and a standard 1-mM solution of carbamylcholine was applied to voltage-clamped endplates by local superperfusion from a micropipette. Measurements of average sarcomere length were also made in separate experiments to detect persistent raised levels of internal Ca2+ in the high-K test media. 3. In high-K test solutions containing 1.8 mM Ca, desensitization rate decreased in a regular monotonic relation with increase in potential through the range from -40 to +30 mV, giving an exponential voltage sensitivity factor of 0.016 mV-1. 4. By contrast, desensitization onset rate in high-K test media containing Ca ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (CaEGTA) buffer (calculated free Ca2+ = 61 nM) showed a nonmonotonic, reversing relation with potential characterized by a decrease in rate between -40 and -20 mV and an increase of rate with increase in potential from -20 to +30 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of trimebutine on cytosolic Ca 2+ and force transitions in intestinal smooth muscle

The effects of trimebutine maleate on cytosolic free Ca 2+ and force transitions in the guinea-pig taenia cecum were studied by fura-2 fluorometry and tension recording. The addition of 80 mM K + induced a transient increase in cytosolic free Ca 2+ concentration ([Ca 2+] i) and tension, followed by a sustained increase. Trimebutine (10 μM) suppressed both [Ca 2+] i elevation and tension development. The tonic responses were more potently inhibited than the phasic responses. Phasic components gradually increased as the added K + increased (10–40 mM). The relationship between the peak increases in [Ca +] i and tension was not affected by trimebutine (10 μM). This means that trimebutine dose not affect the Ca 2+ sensitivity of contractile elements. In a high K + and Ca 2+-free medium, carbachol (10 μM) or caffeine (30 mM) caused transient [Ca 2+] i elevation and tension development in the smooth muscle. Trimebutine (10 μM) decreased the amplitude of both responses. Trimebutine (10 μM) inhibited the spontaneous fluctuations in [Ca 2+] i and motility of taenia cecum in the presence of tetrodotoxin (TTX; 0.3 μM). These results suggest that trimebutine has two types of inhibitory actions on intestinal smooth muscle; one, the inhibition of Ca 2+ influx through voltage-dependent calcium channels, and the other, the inhibition of Ca 2+ release from intracellular storage sites.

Endothelin- and oxytocin-induced calcium signaling in cultured human myometrial cells.

The demonstration that endothelin (ET) induces rat uterine contraction, coupled with the observation that ET is present in human amniotic fluid, suggests that the myometrium may be an important target organ for this hormone. We show that in quiescent human myometrial cells ET produced a dose-dependent increase in cytosolic free Ca2+ (Cai2+), which was markedly attenuated when the cells were studied in Ca2(+)-free media. Preincubation with nicardipine, diltiazem, or verapamil reduced the ET-evoked Cai2+ transient by 30, 40, and 65%, respectively. The presence of voltage sensitive Ca2+ channels was demonstrated by Mn2+ quenching of fura-2. Activation of the Na+/H+ antiport could not be demonstrated with ET stimulation. In nonquiescent cells, the ET-evoked Cai2+ transient was significantly reduced, while the response to oxytocin was retained. This is at least partially explained by a reduction in Bmax (maximal binding capacity) for ET (mean +/- SEM) from 3,506 +/- 268 binding sites/cell in quiescent cells to 2,411 +/- 300 binding sites/cell, as well as 72% increase in Kd (equilibrium dissociation constant), in the nonquiescent cells. We conclude that, in human myometrial cells, ET and oxytocin modulate Cai2+ through independent receptors and propose that ET, like oxytocin, is an important endogenous modulator of uterine contractility.

A possible role for eicosanoids in ATP-induced plasma membrane bleb formation in isolated hepatocytes

Extracellular ATP promotes Ca2+ uptake into isolated hepatocytes via the plasma membrane [l] but other nucleotides have no comparable effect 21. ATP is also associated with the inhibition of Gal+ efflux [3]. exterior of the cell by an ecto-ATPase but no stoichiometry is observed between these two processes [2]. It has been suggested that the underlying mechanism of ATPinduced Ca2+ uptake involves phosphorylation of a membrane component which increases the negative surface charge density and causes a relatively unspecific permeability change [ 21 . plasma membrane bleb formation [4], ATP-induced Ca2+ uptake may be linked to the observed formation of blebs on hepatocytes treated with ATP [4]. Associated with ATP-induced bleb formation is an increased rate of cellular proteolysis which is prevented by inhibitors of Ca2+-activated proteinases (e.g. leupeptin or antipain) [4]. Although these inhibitors prevent loss of viability of hepatocytes exposed to ATP they do not arrest the increase in cytosolic free Ca2+(41. coupled with the known effects of Ca2+ on the proteins associating the cytoskeletal network with the plasma membrane led to the suggestion that these proteins may be the target for Ca2+-activated proteinases and that their removal may lead to bleb formation [5]. Ca2+-induced cytotoxicity either by Ca2+ ionophore in vitro 161 or paracetamol in vivo [7] is prevented by inhibitors of phospholipase A2, cyclooxygenase or thromboxane synthase indicating a possible role for thromboxanes in the mechanism of During Ca2+ uptake, ATP is hydrolysed on the

ATP-induced activation of human B lymphocytes via P2-purinoceptors.

ATP-specific P2-purinoceptors expressed on various cell types have been shown to trigger cell activation via a phospholipase C pathway. In the present study, we provide evidence that P2-purinoceptors are expressed on B lymphocytes but not on T lymphocytes. ATP at concentrations of 10 to 100 microM triggered a dose-dependent increase in inositol 1,4,5-trisphosphate (IP3) levels as well as total inositol phosphate in human B lymphocytes. As expected from the changes in IP3, incubation of B cells with increasing concentrations of ATP lead to a dose-dependent increase in cytosolic free Ca+2 ([Ca+2]i). Extracellular ATP also induced increases in the levels of c-fos and c-myc mRNA. Because no responses were elicited by other nucleotides, the increase in IP3 production, the rise in [Ca+2]i levels, and the enhanced expression of c-fos and c-myc mRNA seem to be mediated by P2-purinoceptors. These responses were exclusive to B lymphocytes, in that ATP had no effect on IP3, [Ca+2]i, or oncogene expression in T cells. The results show that binding of extracellular ATP to P2-purinoceptors on quiescent B cells leads to the activation of genes associated with cell activation. This appears to be mediated via the phospholipase C signal transduction pathway.

Noncyclooxygenase metabolites of arachidonic acid amplify the vasopressin-induced Ca2+ signal in glomerular mesangial cells by releasing Ca2+ from intracellular stores

Noncyclooxygenase metabolites of arachidonic acid may be potent modulators of the mitogenic response of renal mesangial cells to the mitogenic vasoactive peptide arginine vasopressin (AVP). Since Ca2+ is a critical second messenger in the response of mesangial cells to AVP, and Ca2+ has been implicated in the regulation of growth, we determined whether noncyclooxygenase metabolites altered the phospholipase C-Ca2+ signalling cascade which is activated by AVP. Pretreatment of mesangial cells for 10 min with lipoxygenase and cytochrome P450 monooxygenase inhibitors, nordihydroguaiaretic acid (NDGA, 10(-5) M) or SKF-525A (2.5 x 10(-5) M), but not the cyclooxygenase inhibitor indomethacin (2 x 10(-5) M), reduced the magnitude of the AVP (10(-8) and 10(-7) M)-induced increase in cytosolic free Ca2+ concentration ([Ca2+]i) without affecting inositol trisphosphate production. With 10(-8) M AVP, [Ca2+]i increased to 250 +/- 47 nM in NDGA-treated cells versus 401 +/- 59 nM in control cells (p less than 0.01). [Ca2+]i, measured 2 min after exposure to AVP, was also lower with NDGA (152 +/- 21 nM) when compared with AVP alone (220 +/- 22 nM, p less than 0.01). 14,15-epoxyeicosatrienoic acid (EET) (10(-8) M), which had no effect on inositol trisphosphate production, completely reversed the NDGA-induced inhibition of the [Ca2+]i transient, whereas 5-hydroperoxyeicosatetraenoic acid (HPETE) (5 x 10(-7) M) did not. Pretreatment with higher concentrations of 14,15-EET (10(-7)-10(-6) M) markedly potentiated the AVP-induced increase in [Ca2+]i. NDGA-induced inhibition of the AVP-generated [Ca2+]i transient was also observed when cells were incubated in low Ca2+ media ([Ca2+] less than 5 x 10(-8) M), suggesting that NDGA pretreatment impaired intracellular release of Ca2+. Since NDGA had no direct effect on inositol 1,4,5-trisphosphate-induced Ca2+ release, we postulated that NDGA blocked production of a metabolite that releases Ca2+ from intracellular stores. 14,15-EET and 15-HPETE, but not 15-hydroxyeicosatetraenoic acid (each at 3 x 10(-7) M), raised [Ca2+]i when added directly to cells in low Ca2+ media. In permeabilized cells 14,15-EET and 15-HPETE (10(-7) M) potently released Ca2+ from intracellular stores. In summary, noncyclooxygenase metabolites of arachidonic acid, and in particular P450 metabolites, are potent endogenous amplifiers of the AVP-induced [Ca2+]i signal by mechanisms not directly involving phospholipase C activation. This effect is mediated, at least in part, by enhanced release of Ca2+ from intracellular storage sites by an inositol 1,4,5-trisphosphate-independent mechanism.

Ca2+ responses to interleukin 1 and tumor necrosis factor in cultured human skin fibroblasts. Possible implications for Reye syndrome.

Elevated concentrations of cytokines were found in the plasma of patients acutely ill with Reye syndrome (RS) but not in control subjects or recovered RS patients. To determine whether this disorder involves a genetically determined abnormal response to cytokines, the effects of tumor necrosis factor (TNF) and IL-1 on intracellular free Ca2+ were compared in cultured skin fibroblasts from control subjects and patients with RS. IL-1 and TNF caused rapid, transient, and concentration-dependent increases in cytosolic free Ca2+. The peak cytosolic free Ca2+ was greater and occurred at higher concentrations of IL-1 and TNF in patient cells than in cells from age-matched controls. In control cells, the Ca2+ transient diminished sharply with increasing amounts of IL-1 or TNF above the maximum stimulatory concentration. In contrast, in patient fibroblast this bell-shaped curve of concentration dependency was much less apparent. Bradykinin-stimulated Ca2+ transients were similar in the two groups and did not exhibit the bell-shaped concentration dependency. Thus, plasma cytokine levels are elevated in RS patients and the Ca2+ response to cytokines is increased in cells derived from these patients. We propose that the increased response reflects a genetic defect in cytokine receptor-modulated signal transduction.

Extracellular ATP-induced acidification leads to cytosolic calcium transient rise in single rat cardiac myocytes

The origin of the increase in cytosolic free Ca2+ concentration ([Ca2+]i) induced by extracellular ATP was investigated in single isolated cardiac myocytes loaded with indo-1. The nucleotide added at a concentration of 10 microM triggers a few Ca2+ spikes, followed by a cluster of Ca2+ oscillations, increasing [Ca2+]i to around 200 nM from a basal value of 70 nM. Neither caffeine nor ryanodine affects the magnitude of the Ca2+ transient, but both shorten it by preventing the Ca2+ oscillations. This indicates that the latter must be related to the release of Ca2+ from the sarcoplasmic reticulum. Since ATP also induces cell depolarization (as shown by experiments using the potential sensitive dye bis-oxonol), the initial Ca2+ spikes were attributed to the opening of voltage-dependent Ca2+ channels. A small Ca2+ transient still remains under experimental conditions designed to prevent Ca2+ influx from external medium (low-Ca2+ high-Mg2+ medium containing La3+) and after depletion of the sarcoplasmic-reticulum Ca2+ load with caffeine. Under these conditions, when this Ca2+ transient was buffered by 1,2-bis-(O-aminophenoxy)ethane-NNN'N'-tetra-acetic acid, ATP was unable to trigger the initial Ca2+ spikes. These results indicate that ATP mobilizes Ca2+ ions from an intracellular pool other than the sarcoplasmic reticulum and that this Ca2+ release is responsible for the depolarization. The effects of ATP on [Ca2+]i share the same characteristics as the acidification simultaneously induced by the nucleotide (as shown by experiments using the pH-sensitive probe snarf-1). These ionic variations are highly specific to ATP and its hydrolysis-resistant analogues. They both require the presence of Mg2+ and Cl- ions in the extracellular medium, and they are prevented by pretreatment of the cells with 4,4'-di-isothiocyanostilbene or probenecid. These results suggest that: (1) the ATP-induced acidification leads to displacement of Ca2+ ions from or close to the internal face of sarcolemma; (2) the Ca2+ ions activate a non-specific membrane conductance responsible for the depolarization of the cells; (3) the depolarization leads to a Ca2+ influx, owing to the opening of the voltage-dependent Ca2+ channels; (4) this increase in Ca2+ triggers the release of Ca2+ from the sarcoplasmic reticulum, which is facilitated by the increase in inositol trisphosphate following P2-purinergic stimulation.

In VitroModels for Biochemical Investigations on Programmed Cell Death

Isolated rat thymocytes treated with methylprednisolone (MPS), cultured human synovial (McCoy's) cells exposed to cold shock and human mammary adenocarcinoma (BT-20) cells treated with Tumour Necrosis Factor (TNF), all showed DNA fragmentation and the morphological and biochemical features of cell death characteristic of a process known as apoptosis.DNA fragmentation in MPS-treated thymocytes and in cold shock-exposed McCoy's cells was preceded by a marked increase in cytosolic free Ca2+concentration resulting either from a sustained influx of Ca2+from the extracellular medium or from the release of Ca2+from intracellular pools. On the other hand, in TNF-treated BT-20 cells, DNA fragmentation was not associated with any early, marked increase in the cytosolic free Ca2+level. In all three models, however, DNA fragmentation was efficiently prevented by intracellular Ca2+chelators, calmodulin inhibitors and activators of protein kinase C (PKC). These results suggest that the activation of common mechanisms involving Ca2+and PKC may be essential for the development of apoptosis.

Prostaglandin F2α-lnduced Calcium Transient in Ovine Large Luteal Cells: II. Modulation of the Transient and Resting Cytosolic Free Calcium Alters Progesterone Secretion*

A previous study demonstrated that prostaglandin F2 alpha (PGF2 alpha) stimulates a transient increase in cytosolic free Ca2+ levels [( Ca2+]i) in ovine large luteal cells. In the present study, the magnitude of the PGF2 alpha (0.5 microM)-induced calcium transient in Hanks' medium (87 +/- 2 nM increase above resting levels) was reduced (P less than 0.05) but not completely eliminated in fura-2 loaded large luteal cells incubated in Ca2(+)-free or phosphate- and carbonate-free medium (10 +/- 1 nM, 32 +/- 6 nM, above resting levels; respectively). Preincubation for 2 min with 1 mM LaCl3 (calcium antagonist) eliminated the PGF2 alpha-induced calcium transient. The inhibitory effect of PGF2 alpha on secretion of progesterone was reduced in Ca2(+)-free medium or medium plus LaCl3. Resting [Ca2+]i levels and basal secretion of progesterone were both reduced (P less than 0.05) in large cells incubated in Ca2(+)-free medium (27 +/- 4 nM; 70 +/- 6% control, respectively) or with 5 microM 5,5'-dimethyl bis-(O-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (40 +/- 2 nM; 49 +/- 1% control; respectively). In addition, secretion of progesterone was inhibited (P less than 0.05) by conditions that increased (P less than 0.05) [Ca2+]i; that is LaCl3 ([Ca2+]i, 120 +/- 17 nM; progesterone, 82 +/- 8% control) and PGF2 alpha ([Ca2+]i, 102 +/- 10 nM; progesterone, 82 +/- 3% control). In small luteal cells, resting [Ca2+]i levels and secretion of progesterone were reduced by incubation in Ca2(+)-free Hanks ([Ca2+]i, 28 +/- 2 nM; progesterone, 71 +/- 6% control), however, neither LaCl3 nor PGF2 alpha increased [Ca2+]i levels or inhibited secretion of progesterone. The findings presented here provide evidence that extracellular as well as intracellular calcium contribute to the PGF2 alpha-induced [Ca2+]i transient in large cells. Furthermore, whereas an adequate level of [Ca2+]i is required to support progesterone production in both small and large cells, optimal progesterone production in large cells depends upon an appropriate window of [Ca2+]i.

Ethanol causes desensitization of receptor-mediated phospholipase C activation in isolated hepatocytes

The effect of ethanol on receptor-mediated phospholipase C-linked signal transduction processes was investigated in isolated rat hepatocytes. Pretreatment of the cells with ethanol (6-300 mM) markedly inhibited a subsequent stimulation of phospholipase C by vasopressin, angiotensin II, or epidermal growth factor. By contrast, the effects of the alpha 1-adrenergic agonist phenylephrine and of glucagon were not affected by ethanol pretreatment. Ethanol inhibited the agonist-induced decrease in polyphosphoinositides, the formation of inositol phosphates, and the increase in cytosolic free Ca2+ levels, as detected with the intracellular Ca2+ indicator indo-1. The effects of ethanol were concentration dependent and were pronounced at low concentrations of agonists but were not significant at saturating levels. Pretreatment of the cells with the protein kinase C inhibitor H7 partly prevented the inhibition by ethanol of vasopressin-induced phospholipase C activation. By contrast, pretreatment of the cells with (Rp)-adenosine cyclic 3':5'-phosphorothioate [Rp)-cAMP-S), a competitive inhibitor of protein kinase A, potentiated the inhibitory effect of ethanol on the Ca2+ mobilization by vasopressin. (Rp)-cAMP-S similarly potentiated the inhibition of phospholipase C by the protein kinase C-activating phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). The kinase A inhibitor also made the Ca2+ mobilization by phenylephrine sensitive to ethanol, indicating that the formation of cAMP in the cells played a role in suppressing the sensitivity to ethanol. Pretreatment of the cells with ethanol enhanced the inhibitory effects of TPA on the vasopressin-induced phospholipase C activation at all concentrations of the hormone; however, these synergistic effects were prevented when TPA was added prior to ethanol, a condition that prevents the activation of phospholipase C by ethanol. The data indicate that ethanol causes desensitization of the receptor-mediated phospholipase C secondary to the ethanol-induced activation of phospholipase C and activation of protein kinase C. Ethanol treatment also affects the sensitivity of the phospholipase C system to control by protein kinases A and C. The data indicate that ethanol can affect the control of intracellular signal transduction processes in liver cells under physiologically relevant conditions.

Ca 2+ oscillations induced by histamine H 1 receptor stimulation in HeLa cells: Fura-2 and patch clamp analysis

The response of HeLa cells to histamine H 1 receptor stimulation is characterized by periodic increases in cytosolic free Ca 2+ concentration. The mechanisms underlying this oscillatory behaviour are not well understood. Fura-2 and patch clamp experiments carried out on HeLa cells have previously shown: (a) that Ca 2+ oscillations are not initially dependent on the presence of external Ca 2+, that external Ca 2+ is required to maintain the oscillatory activity; (b) that a depolarization of the cell membrane leads to an inhibition of Ca 2+ oscillations during the external Ca 2+ dependent phase of the process; and (c) that Ca 2+ oscillations can be abolished during this latter phase by the exogenous addition of Ca 2+ channel blocking agents, such as Co 2+ or La 3+. The contribution of the inositol phosphate pathway to Ca 2+ oscillations was more recently investigated in whole cell experiments performed with patch pipettes containing IP 3 or the non-hydrolysable GTP analogue GTP-γS. Clear periodic current fluctuations were recorded using both patch pipette solutions. Assuming that the intracellular IP 3 level remained constant under these conditions, these findings provide direct evidence that the Ca 2+ oscillations in HeLa cells do not arise from a periodic production of IP 3. The effect of the internal and external cell pri on the oscillatory process was also investigated in Fura-2 and patch clamp experiments. It was found that an increase in intracellular pH from 7.4 to 7.7 during the external Ca 2+ dependent phase of the histamine stimulation abolishes the appearance of Ca 2+ spikes whereas, a cellular acidification to pH 7.2 maintains or stimulates the Ca 2+ oscillatory activity. The former effect was observed in the absence of Ca 2+ in the bathing medium, indicating that the inhibitory action of alkaline pH was not related to a reduced Ca 2+ entry. An increase in extracellular pH from 7.3 to 9.0 in contrast elicited an intracellular Ca 2+ accumulation which resulted in most cases in an inhibition of the oscillatory process. This effect was dependent on external Ca 2+ and was observed in alkaline internal pH conditions (pH 7.7). These observations suggest: (a) that the net Ca 2+ influx in HeLa cells is strongly dependent on the cell internal and external pH; and (b) that the magnitude of this Ca 2+ influx controls to a large extent the oscillation frequency. Finally, an inhibition of the histamine induced Ca 2+ oscillatory activity was observed following the addition of the Ca 2+-induced Ca 2+-release (CICR) inhibitor adenine to the external medium. This finding indicates that there is a possible contribution of a CICR mechanism to the Ca 2+ oscillations in HeLa cells. A similar inhibitory action of adenine was also observed in the absence of Ca 2+ in the bathing solution. These observations tend therefore to support a model in which Ca 2+ oscillations are related to a CICR mechanism fueled by a pH dependent net Ca 2+ influx.