Preparation of rat liver cells. I. Effect of Ca 2+ on enzymatic dispersion of isolated, perfused liver

Suspensions of rat liver cells were prepared by perfusion of the isolated liver with collagenase and hyaluronidase. By means of a novel dispersion assay, which measured swelling of the liver in a closed perfusion system, the time course of enzymatic dispersion could be followed. Ca²⁺ stimulated the enzymatic dispersion strongly, but a preliminary removal of Ca²⁺ with the chelator EGTA rendered the liver tissue more susceptible to the action of enzymes. The best result was thus obtained when the liver was first perfused 5 min with EGTA, then 5 min with enzymes and Ca²⁺. This sequential treatment converted the whole liver to a cellular suspension, in which about 95% of the cells were intact.     

Effects of alloxan and ninhydrin on mitochondrial Ca2+ transport

Alloxan at millimolar concentrations slightly inhibited the velocity of Ca²⁺ uptake by isolated rat liver mitochondria irrespective of the free Ca²⁺ concentration between 1 and 10 µM and was an effective concentration-dependent stimulator of mitochondrial Ca²⁺ efflux. Ninhydrin also slightly inhibited the velocity of mitochondrial Ca²⁺ uptake but only at free Ca²⁺ concentrations above 5 µM. However, ninhydrin was a strong stimulator of mitochondrial Ca²⁺ efflux even at micromolar concentrations, 10–50 times more potent than alloxan. The mitochondrial membrane potential was reduced 10–20% at most by alloxan and ninhydrin. Alloxan and ninhydrin also stimulated Ca²⁺ efflux from isolated permeabilized liver cells. When isolated intact liver cells had been pre-incubated with alloxan or ninhydrin before permeabilization of the cells the ability of spermine to induce mitochondrial Ca²⁺ uptake was abolished. Glucose provided the typical protection against the effects of alloxan on mitochondrial Ca²⁺ transport only in experiments with intact cells but not in experiments with permeabilized cells or isolated mitochondria. Therefore glucose protection is apparently due to inhibition of alloxan uptake into the cell. Glucose provided no protection against effects of ninhydrin under any of the experimental conditions. Thus both alloxan and ninhydrin are potent stimulators of Ca²⁺ efflux by isolated mitochondria but very weak inhibitors of the velocity of mitochondrial Ca²⁺ uptake. The direct effects of ninhydrin on mitochondrial Ca²⁺ efflux may contribute to the cytotoxic action of this agent whereas the direct effects of alloxan on mitochondrial Ca²⁺ transport require concentrations which are too high to be of relevance for the induction of the typical pancreatic B-cell toxic effects of alloxan. However, the effects on mitochondrial Ca²⁺ transport during incubation of intact cells which may result from the generation of cytotoxic intermediates during alloxan xenobiotic metabolism may well contribute to the pancreatic B-cell toxic effect of alloxan. Mol Cell Biochem 118: 141–151, 1992)     

Enzyme separation techniques for the study of growth of cells from layers of bovine dental pulp

Summary Effects of the enzymes trypsin, papain, bromelains and ficin in bovine dental pulp tissue were studied. Minced or whole pulps were subjected to each enzyme at 17°, 20° and 37°C for set time intervals, after which aliquots of supernatant fluid were removed for cell counts and viability tests. Pooled samples were subsequently cultured as monolayers in Eagle’s MEM plus 10% calf serum. The dissociation characteristics were quite distinct for each enzyme, although quite similar between minced and whole pulp. A parallel histological study was made of the residual pulp tissue. Ficin was found to be the most suitable enzyme for future studies on the growth of isolated pulp cells from various layers of the bovine pulp, due to its even rate of cell removal, and the good initial viability and subsequent growth of the separated cells in monolayer culture. Further studies on ficin may show that it is more suitable for enzymatic separation of tissues generally than the more commonly used trypsin, a major advantage being its use in media containing Ca²⁺ and Mg²⁺. Supported in part by NIH Grant No. DE 02908 and by United Health Foundation Summer Fellowships to J.T.F. and J.F.C. Supported in part by N.I.H. Grant 5 R01-DE 02908 to W.A.M. and by United Health Foundation Summer Fellowships to J.T.F. and J.F.C.     

Calcium-induced patterns of calcium-oxalate crystals in isolated leaflets of Gleditsia triacanthos L. and Albizia julibrissin Durazz

For experimental induction of crystal cells (=crystal idioblasts) containing calcium-oxalate crystals, the lower epidermis was peeled from seedling leaflets of Gleditsia triacanthos L., exposing the crystal-free mesophyll and minor veins to the experimental solutions on which leaflets were floated for up to 10 d under continous light. On 0.3–2.0 mM Ca-acetate, increasing numbers of crystals, appearing 96 h after peeling, were induced. The pattern of crystal distribution changed with Ca²⁺-concentration ([Ca²⁺]): at low [Ca²⁺], crystals formed only in the non-green bundlesheath cells surrounding the veins, believed to have a relatively low Ca²⁺-extrusion capacity; at higher [Ca²⁺], crystals developed in up to 90% of the mesophyll cells, and at supraoptimal [Ca²⁺], large extracellular crystals formed on the tissue surface. By sequential treatments with solutions of different [Ca²⁺], the following three phases were identified in the induction of crystal cells: (1) during the initial 24-h period (adaptive aging), Ca²⁺ is not required and crystal induction is not possible; (2) during the following 48 h (induction period), exposure to 1–2 mM Ca-acetate induces the differentiation of mesophyll cells into crystal cells; (3) crystal growth begins 72 h after the start of induction. In intact leaflets of Albizia julibrissin Durazz., calcium-oxalate crystals are found exclusively in the bundle-sheath cells of the veins, but crystals were induced in the mesophyll of peeled leaflets floating on 1 mM Ca-acetate. Exposure to inductive [Ca²⁺] will thus trigger the differentiation of mature leaf cells into crystal cells; the spatial distribution of crystals is determined by the external [Ca²⁺] and by the structural and functional properties of the cells in the tissue.     

Hysteretic activation of the Ca pump revealed by calcium transients in human red cells

The enzymatic basis for the Ca²⁺ pump in human red cells is an ATPase with hysteretic properties. The Ca²⁺-ATPase shifts slowly between a ground state deficient in calmodulin and an active state saturated with calmodulin, and rate constants for the reversible shifts of state were recently determined at different Ca²⁺ concentrations (Scharff, O. and Foder, B. (1982) Biochim. Biophys. Acta 691, 133–143). In order to study whether the Ca²⁺ pump in intact red cells also exhibits hysteretic properties we have analysed transient increases of intracellular calcium concentrations (Ca_i), induced by the divalent cation ionophore A23187. The time-dependent changes of Ca_i were measured by use of radioactive calcium (⁴⁵Ca²⁺) and analysed with the aid of a mathematical model, based partly on the Ca²⁺-dependent parameters obtained from Ca²⁺-ATPase experiments, partly on the A23187-induced Ca²⁺ fluxes determined in experiments with intact red cells. According to the model a delay in the activation of the Ca²⁺ pump is a prerequisite for the occurrence of A23187-induced calcium transients in the red cells, and we conclude that the Ca²⁺ pump in human red cells responds hysteretically. It is suggested that Ca²⁺ pumps in other types of cell also have hysteretic properties.     

Hormonal stimulation, mitochondrial Ca2+ accumulation, and the control of the mitochondrial permeability transition in intact hepatocytes

Ca²⁺ functions as an intracellular signal to transfer hormonal messages to different cellular compartments, including mitochondria, where it activates intramitochondrial Ca²⁺-dependent enzymes. However, excessive mitochondrial Ca²⁺ uptake can promote the mitochondrial permeability transition (MPT), a process known to be associated with cell injury. The factors controlling mitochondrial Ca²⁺ uptake and release in intact cells are poorly understood. In this paper, we investigate mitochondrial Ca²⁺ accumulation in intact hepatocytes in response to the elevation of cytosolic Ca²⁺ levels ([Ca²⁺]_c) induced either by a hormonal stimulus (vasopressin), or by thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺ pump. After stimulation, cells were rapidly permeabilized for the determination of the mitochondrial Ca²⁺ content (Ca^2+_m) and to analyze the susceptibility of the mitochondria to undergo the MPT. Despite very similar levels of [Ca²⁺]_c elevation, vasopressin and thapsigargin had markedly different effects on mitochondrial Ca²⁺ accumulation. Vasopressin caused a rapid (< 90 sec), but modest (< 2 fold) increase in Ca²⁺m that was not further increased during prolonged incubations, despite a sustained [Ca²⁺]_c elevation. By contrast, thapsigargin induced a net Ca²⁺ accumulation in mitochondria that continued for up to 30 min and reached Ca^2+_m levels 10–20 fold over basal. Accumulation of mitochondrial Ca²⁺ was accompanied by a markedly increased susceptibility to undergo the MPT. Both mitochondrial Ca²⁺ accumulation and MPT activation were modulated by treatment of the cells with inhibitors of protein kineses and phosphatases. The results indicate that net mitochondrial Ca²⁺ uptake in response to hormonal stimulation is regulated by processes that depend on protein kinase activation. These controls are inoperative when the cytosol is flooded by Ca²⁺ through artificial means, enabling mitochondria to function as a Ca²⁺ sink under these conditions. (Mol Cell Biochem 174: 173–179, 1997)     

Ultrastructural study of the Ca2+-dependent ATPase activity in rat adenohypophyseal cells

Summary Ca-dependent ATPase activity in the rat anterior pituitary was demonstrated in 50-m tissue slices of aldehyde-fixed tissue with the medium of Takano et al. (Cell Tissue Res. 243:91. 1986). — The outer surface of the plasma membrane of the parenchymal as well as the folliculo-stellate cells was lined with lead precipitate. The reaction deposit was particularly well localized in intercellular spaces both between two parenchymal cells, and between a parenchymal and a folliculo-stellate cell. A fine reaction deposit was also seen in the endoplasmic reticulum and Golgi apparatus of some parenchymal cells. Elimination of Ca²⁺ from the tissue and the substrate medium drastically reduced the amount of reaction product. If ATP was omitted or replaced by sodium -glycerophosphate, no reaction product was seen. Changing the Ca²⁺ concentration or addition of Mg²⁺ to the standard medium caused a decrease in reaction intensity. Substitution of Mg²⁺ for Ca²⁺ resulted again in well-localized lead deposition which we attribute to the activity of another enzyme. We suggest that the activity we described in the membrane of glandular cells may correspond to the enzyme involved in the long-term regulation of intracellular Ca²⁺ level.     

Effect of hydrocortisone and thyroxine on ATPase activities of neuronal and glial cell lines in culture

The effect of hydrocortisone and thyroxine, on the activities of Ca²⁺-and Mg²⁺-ATPase was studied in cultured neuronal (clone M1) and glial (clones NN and C6) cell lines. For M1 and NN cells an increase in Ca²⁺-and Mg²⁺-ecto-ATPase activity was found when the cells were cultured during 4–6 days in presence of hydrocortisone or together with thyroxine. In the same conditions, a decrease in Ca²⁺-and Mg²⁺-ecto-ATPase activity was found for the C6 cells. In C6 cells the effect of hormones was more pronounced for the Mg²⁺-than for the Ca²⁺-ecto-ATPase activity. The observed decrease may be related to the tumoral origin of the C6 cells. The activity of (Na⁺, K⁺)-ATPase in all three cell lines increased in presence of hydrocortisone or together with thyroxine when the cells were cultured during 4–6 days, in presence of the hormones, whereas the total Mg²⁺-ATPase activity increased only after 6 days of treatment. Thyroxine alone has very few effect either on Ca²⁺-and Mg²⁺-ecto-ATPase, or on (Na⁺, K⁺)-and total Mg²⁺-ATPase activity. These observations are interpreted to indicate that hormones may modulate or induce enzymatic activities involved in active transport phenomena in nervous tissue.     

Immunological detection of cell surface components related with aggregation of Chinese hamster and chick embryonic cells.

Previous studies suggested that Chinese hamster V79 cells possess two mechanisms for their mutual adhesion, Ca²⁺-dependent and Ca²⁺-independent ones. We could prepare cells with only the Ca²⁺-dependent mechanism intact by dispersing cell monolayers with trypsin (0.01%) containing Ca²⁺. In the present study, we found that cells dispersed with a very low concentration of trypsin (0.0001%) in the absence of Ca²⁺ retain only the Ca²⁺-independent mechanism intact. Fab fragments of antibodies directed against surface antigens of V79 cells inhibited the aggregation of V79 cells by the Ca²⁺-independent mechanism, but did not inhibit the aggregation of these cells by the Ca²⁺-dependent mechanism. These results suggest that the two mechanisms of cell adhesion are based on different cellular components. Molecules responsible for the Ca²⁺-independent adhesion mechanism are probably cell surface components, because they were released from cells by the treatment with 0.01% trypsin without losing their specific antigenicity. The presence of adhesion mechanisms similar to those in V79 cells was shown in neural retinal cells of chick embryos. It was assumed, therefore, that these mechanisms of cell adhesion are generally present among a variety of cell types.     

Effect of tetramethylpyrazine (TMP) on Ca2+ signal transduction and cell viability in a model of renal tubular cells

Tetramethylpyrazine (TMP) is a compound purified from herb. Its effect on Ca²⁺ concentrations ([Ca²⁺]_i) in renal cells is unclear. This study examined whether TMP altered Ca²⁺ signaling in Madin‐Darby canine kidney (MDCK) cells. TMP at 100–800 μM induced [Ca²⁺]_i rises, which were reduced by Ca²⁺ removal. TMP induced Mn²⁺ influx implicating Ca²⁺ entry. TMP‐induced Ca²⁺ entry was inhibited by 30% by modulators of protein kinase C (PKC) and store‐operated Ca²⁺ channels. Treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5‐di‐tert‐butylhydroquinone (BHQ) inhibited 93% of TMP‐evoked [Ca²⁺]_i rises. Treatment with TMP abolished BHQ‐evoked [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) abolished TMP‐induced responses. TMP at 200–1000 μM decreased viability, which was not reversed by pretreatment with the Ca²⁺ chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid‐acetoxymethyl ester. Together, in MDCK cells, TMP induced [Ca²⁺]_i rises by evoking PLC‐dependent Ca²⁺ release from endoplasmic reticulum and Ca²⁺ entry via PKC‐sensitive store‐operated Ca²⁺ entry. TMP also caused Ca²⁺‐independent cell death.     

Econazole-evoked [Ca2+]i Rise and Non-Ca2+-triggered Cell Death in Rabbit Corneal Epithelial Cells (SIRC)

The effects of econazole, an antifungal drug applied for treatment of keratitis and mycotic corneal ulcer, on cytosolic-free Ca²⁺ concentrations ([Ca²⁺]_i) and viability of corneal cells was examined by using SIRC rabbit corneal epithelial cells as model. [Ca²⁺]_i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Econazole at concentrations ≥ 1 µM increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. The econazole-induced Ca(2+) influx was insensitive to L-type Ca²⁺ channel blockers and protein kinase C modulators. In Ca²⁺-free medium, after pretreatment with 20 µM econazole, [Ca²⁺]_i rises induced by 1 µM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor) were abolished. Conversely, thapsigargin pretreatment also abolished econazole-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 µM U73122 did not change econazole-induced [Ca²⁺]_i rises. At concentrations between 10 and 80 µM, econazole killed cells in a concentration-dependent manner. The cytotoxic effect of 20 µM econazole was not reversed by prechelating cytosolic Ca²⁺ with BAPTA. This shows that in SIRC cells econazole induces [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx from unknown pathways. Econazole-caused cytotoxicity was independent from a preceding [Ca²⁺]_i rise.     

Ca 2+ -specific removal of Z lines from rabbit skeletal muscle

Removal of rabbit psoas strips immediately after death and incubation in a saline solution containing 1 mM Ca²⁺ and 5 nM Mg²⁺ for 9 hr at 37°C and pH 7.1 causes complete Z-line removal but has no ultrastructurally detectable effect on other parts of the myofibril. Z lines remain ultrastructurally intact if 1 mM 1,2-bis-(2-dicarboxymethylaminoethoxy)-ethane (EGTA) is substituted for 1 mM Ca²⁺ and the other conditions remain unchanged. Z lines are broadened and amorphous but are still present after incubation for 9 hr at 37°C if 1 mM ethylenediaminetetraacetate (EDTA) is substituted for 1 mM Ca²⁺ and 5 mM Mg²⁺ in the saline solution. A protein fraction that causes Z-line removal from myofibrils in the presence of Ca²⁺ at pH 7.0 can be isolated by extraction of ground muscle with 4 mM EDTA at pH 7.0–7.6 followed by isoelectric precipitation and fractionation between 0 and 40% ammonium sulfate saturation. Z-line removal by this protein fraction requires Ca²⁺ levels higher than 0.1 mM, but Z lines are removed without causing any other ultrastructurally detectable degradation of the myofibril. This is the first report of a protein endogenous to muscle that is able to catalyze degradation of the myofibril. The very low level of unbound Ca²⁺ in muscle cells in vivo may regulate activity of this protein fraction, or alternatively, this protein fraction may be localized in lysosomes.     

Change in Intracellular pH Causes the Toxic Ca<Superscript>2+</Superscript> Entry via NCX1 in Neuron- and Glia-Derived Cells

Brain hypoxia or ischemia causes acidosis and the intracellular accumulation of Ca²⁺ in neuron. The aims of the present study were to elucidate the interaction between intracellular pH and Ca²⁺ during transient acidosis and its effects on the viability of neuronal and glial cells. Intracellular Ca²⁺ and pH were measured using the fluorescence of fura-2 and 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester in neuroblastoma (IMR-32), glioblastoma (T98G), and astrocytoma (CCF-STTG1) cell lines. The administration of 5 mM propionate caused intracellular acidification in IMR-32 and T98G cells but not in CCF-STTG1 cells. After the removal of propionate, the intracellular pH recovered to the resting level. The intracellular Ca²⁺ transiently increased upon the removal of propionate in IMR-32 and T98G cells but not in CCF-STTG1 cells. The transient Ca²⁺ increase caused by the withdrawal of intracellular acidification was abolished by the removal of external Ca²⁺, diminished by a reduction of external Na⁺, and inhibited by benzamil. Transient acidosis caused cell death, whereas the cells were more viable in the absence of external Ca²⁺. Benzamil alleviated cell death caused by transient acidosis in IMR-32 and T98G cells but not in CCF-STTG1 cells. These results suggest that recovery from intracellular acidosis causes a transient increase in cytosolic Ca²⁺ due to reversal of Ca²⁺ transport via Na⁺/Ca²⁺ exchanger coactivated with Na⁺/H⁺ exchanger, which can cause cell death.     

The Effect of Combined Exposure of 900 MHz Radiofrequency Fields and Doxorubicin in HL-60 Cells

Human promyelocytic leukemia HL-60 cells were pre-exposed to non-ionizing 900 MHz radiofrequency fields (RF) at 12 µW/cm² power density for 1 hour/day for 3 days and then treated with a chemotherapeutic drug, doxorubicin (DOX, 0.125 mg/L). Several end-points related to toxicity, viz., viability, apoptosis, mitochondrial membrane potential (MMP), intracellular free calcium (Ca²⁺) and Ca²⁺-Mg²⁺ -ATPase activity were measured. The results obtained in un-exposed and sham-exposed control cells were compared with those exposed to RF alone, DOX alone and RF+DOX. The results indicated no significant differences between un-exposed, sham-exposed control cells and those exposed to RF alone while treatment with DOX alone showed a significant decrease in viability, increased apoptosis, decreased MMP, increased Ca²⁺ and decreased Ca²⁺-Mg^2+-ATPase activity. When the latter results were compared with cells exposed RF+DOX, the data showed increased cell proliferation, decreased apoptosis, increased MMP, decreased Ca²⁺ and increased Ca²⁺-Mg²⁺-ATPase activity. Thus, RF pre-exposure appear to protect the HL-60 cells from the toxic effects of subsequent treatment with DOX. These observations were similar to our earlier data which suggested that pre-exposure of mice to 900 MHz RF at 120 µW/cm² power density for 1 hours/day for 14 days had a protective effect in hematopoietic tissue damage induced by subsequent gamma-irradiation.     

Calcium acetate induces calcium uptake and formation of calcium-oxalate crystals in isolated leaflets of Gleditsia triacanthos L.

During treatment of isolated, peeled leaflets of Gleditsia triacanthos with 0.5–2 mM [⁴⁵Ca]acetate, saturation of the cell-wall free space with Ca²⁺ occurred within 10 min and was followed by a period of 6–10 h during which there was no significant Ca-uptake into the protoplast, but apoplastic Ca²⁺ was periodically released into the medium. Later, Ca²⁺ was absorbed for 3–4 d at rates of up to 2.2 mol Ca²⁺·h^-1·(g FW)^-1 to final concentrations of 350 mol Ca²⁺· (g FW)^-1. The distribution of absorbed Ca²⁺ between cell wall, vacuole and Ca-oxalate crystals was determined during Ca-uptake. Wheras intact, cut leaflets deposited absorbed Ca²⁺ as Ca-oxalate in the crystal cells, peeled leaflets lacking crystal cells accumulated at least 40–50 mol·(g FW)^-1 soluble Ca²⁺ before the absorbed Ca²⁺ was precipitated as Ca-oxalate. These observations indicate that the mechanisms for the continuous uptake of Ca²⁺, the synthesis of oxalate and the precipitation of Ca²⁺ as Ca-oxalate are operational in the crystal cells of intact leaflets, but not in the mesophyll cells of peeled leaflets where they must be induced by exposure to Ca²⁺. The precipitation of absorbed Ca²⁺ as Ca-oxalate by the crystal cells of isolated Gleditsia leaflets illustrates the role of these cells in the excretion of surplus Ca²⁺ which enters normal, attached leaves with the transpiration stream.In addition to acetate, only Ca-lactate and Ca-carbonate lead to Ca-uptake, but at rates well below those observed with Ca-acetate. Other small organic anions (citrate, glycolate, glyoxalate, malate) and inorganic anions (chloride, nitrate, sulfate) did not permit Ca-uptake. Acetate-¹⁴C was rapidly absorbed during Ca-uptake, but less than 20% was incorporated into Ca-oxalate; the rest remained mostly in the soluble fraction or was metabolized to CO₂. Acetate, as a permeable weak acid, may enable rapid Ca-uptake by stimulating proton extrusion at the plasmalemma and by serving as a counterion during Ca-accumulation in the vacuole, but is unlikely to function as the principal substrate for oxalate synthesis.     

Effects on N6, O2′-dibutyryladenosine-3′,5′ cyclic monophosphate on calcium accumulation by mouse mastocytoma cells

The accumulation of ⁴⁵Ca²⁺ by intact mouse mastocytoma cells was examined before and after treatment of the cells with N⁶,O^2′-dibutyryladenosine 3′,5′, cyclic monophosphate and theophylline to inhibit growth. In the presence of phosphate either glycolysis, respiration or ATP supported ⁴⁵Ca²⁺ uptake by the cells and in each case the accumulated ⁴⁵Ca²⁺ appeared to be retained by mitochondria. Inhibition of growth by drug treatment for 20h increased subsequent ⁴⁵Ca²⁺ accumulation when cells were incubated with ⁴⁵CaCl₂, succinate and phosphate. Since prior drug treatment did not increase ⁴⁵Ca²⁺ accumulation with glucose, ATP or malate the drugs appeared to increase ⁴⁵Ca²⁺ accumulation by affecting succinate metabolism.     

Ca2+ and cyclic nucleotide dependence of amylase release from isolated rat pancreatic acinar cells rendered permeable by intense electric fields

Enzyme digestion of rat pancreatic tissue yielded a preparation of isolated acinar cells, over 90% of which excluded trypan blue. These isolated cells responded to a variety of secretagogues, the responses being sensitive to the removal of extracellular calcium, increasing extracellular magnesium, and by trifluoperazine, an antagonist of Ca-dependent processes. When exposed to intense electric fields, isolated acinar cells became permeable to CaEGTA and MgATP, these markers gaining access to over 60% of the intracellular mileu within minutes. The accessability to these markers seemed independent of the ionised Ca²⁺ level. Less than 0.5% of the cellular amylase was released when cells were rendered leaky in a medium containing about 10^?9 M Ca²⁺, but typically 4% was released when the Ca²⁺ level was subsequently raised to 10^?5M levels, the EC₅₀ for Ca²⁺ being 2 μM. This amount of amylase released was comparable to the amounts secreted from intact cells in response to a variety of agonists. The cytosolic marker lactate dehydrogenase was also released from leaky cells, but the extent was independent of Ca²⁺ concentration. No amylase was released at 10^?7M Ca²⁺ when permeable cells were exposed to cyclic 3′,5′-AMP or cyclic 3′,5′-GMP. The calcium activation curve for amylase release seemed to be independent of cyclic nucleotides, but was markedly increased in both the extent of release and apparent affinity for Ca²⁺ in the presence of the phorbol ester 12-O-tetradecanoyl phorbol 13 acetate. These results suggest that when “functionally normal” isolated acinar cells are rendered permeable, Ca²⁺ — but not cyclic nucleotides — acts as a second messenger for amylase secretion, and furthermore that protein kinase C may be involved in the secretory process.     

Effect of Celecoxib on Ca2+ Fluxes and Proliferation in MDCK Renal Tubular Cells

The effect of celecoxib on renal tubular cells is largely unexplored. In Madin Darby canine kidney (MDCK) cells, the effect of celecoxib on intracellular Ca^{2 +} concentration ([Ca^{2 +}]_i) and proliferation was examined by using the Ca^{2 +}-sensitive fluorescent dye fura-2 and the viability detecting fluorescent dye tetrazolium, respectively. Celecoxib (≥1 μ M) caused an increase of [Ca^{2 +}]_i in a concentration-dependent manner. Celecoxib-induced [Ca^{2 +}]_i increase was partly reduced by removal of extracellular Ca^{2 +}. Celecoxib-induced Ca^{2 +} influx was independently suggested by Mn^{2 +} influx-induced fura-2 fluorescence quench. In Ca^{2 +}-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca^{2 +}-ATPase, caused a monophasic [Ca^{2 +}]_i increase, after which celecoxib only induced a tiny [Ca^{2 +}]_iincrease; conversely, pretreatment with celecoxib completely inhibited thapsigargin-induced [Ca^{2 +}]_i increases. U73122, an inhibitor of phospholipase C, abolished ATP (but not celecoxib)-induced [Ca^{2 +}]_i increases. Overnight incubation with 1 or 10 μ M celecoxib decreased cell viability by 80% and 100%, respectively. These data indicate that celecoxib evokes a [Ca^{2 +}]_i increase in renal tubular cells by stimulating both extracellular Ca^{2 +} influx and intracellular Ca^{2 +} release and is highly toxic to renal tubular cells in vitro.     

Augmentation of zinc ion stimulation of lymphoid cells by calcium and lithium

Stimulation of hamster lymph node cells by optimal concentrations of ZnCl₂ (10 μM) was found to be enhanced by addition of 1–25 mM LiCl to the serum-free cultures. Maximal enhancement occurred at 10 mM Li⁺. Similar concentrations of either KCl or NaCl did not potentiate stimulation. Addition of 1 mM CaCl₂, but not 1–25 mM MgCl₂, also potentiated Zn²⁺ stimulation of lymph node cells. When the cultures were supplemented with 1 mM Ca²⁺ + 10 mM Li⁺, a synergistic potentiation of Zn²⁺ stimulation occurred. In addition, the dose response curve for Zn²⁺ was shifted such that maximal stimulation occurred at 100–250 μM Zn²⁺, a concentration of Zn²⁺ which was toxic for the unsupplemented cultures. In Ca²⁺ + Li⁺ supplemented cultures, Zn²⁺ stimulated [³H]thymidine incorporation to levels comparable to those obtained when hamster lymphoid cells were stimulated with lectins. In addition to Zn²⁺ stimulation, Ca²⁺ + Li⁺ supplemented medium also enhanced Hg²⁺ stimulation of hamster lymph node cells but did not change the dose response curve for Hg²⁺. Therefore, the observed ionic effects on Zn²⁺ stimulation of lymphocytes were unique to this mitogen, when compared to either Hg²⁺ stimulation or previously reported lectin stimulation of hamster lymphoid cells.     

Intracellular Ca2+ Inhibits Smooth Muscle L-Type Ca2+ Channels by Activation of Protein Phosphatase Type 2B and by Direct Interaction with the Channel

Modulation of L-type Ca²⁺ channels by tonic elevation of cytoplasmic Ca²⁺ was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba²⁺ was used as charge carrier, and run down of Ca²⁺ channel activity in inside-out patches was prevented with calpastatin plus ATP. Increasing cytoplasmic Ca²⁺ in intact cells by elevation of extracellular Ca²⁺ in the presence of the ionophore A23187 inhibited the activity of L-type Ca²⁺ channels in cell-attached patches. Measurement of the actual level of intracellular free Ca²⁺ with fura-2 revealed a 50% inhibitory concentration (IC₅₀) of 260 nM and a Hill coefficient close to 4 for Ca²⁺- dependent inhibition. Ca²⁺-induced inhibition of Ca²⁺ channel activity in intact cells was due to a reduction of channel open probability and availability. Ca²⁺-induced inhibition was not affected by the protein kinase inhibitor H-7 (10 μM) or the cytoskeleton disruptive agent cytochalasin B (20 μM), but prevented by cyclosporin A (1 μg/ ml), an inhibitor of protein phosphatase 2B (calcineurin). Elevation of Ca²⁺ at the cytoplasmic side of inside-out patches inhibited Ca²⁺ channels with an IC₅₀ of 2 μM and a Hill coefficient close to unity. Direct Ca²⁺-dependent inhibition in cell-free patches was due to a reduction of open probability, whereas availability was barely affected. Application of purified protein phosphatase 2B (12 U/ml) to the cytoplasmic side of inside-out patches at a free Ca²⁺ concentration of 1 μM inhibited Ca²⁺ channel open probability and availability. Elevation of cytoplasmic Ca²⁺ in the presence of PP2B, suppressed channel activity in inside-out patches with an IC₅₀ of ∼380 nM and a Hill coefficient of ∼3; i.e., characteristics reminiscent of the Ca²⁺ sensitivity of Ca²⁺ channels in intact cells. Our results suggest that L-type Ca²⁺ channels of smooth muscle are controlled by two Ca²⁺-dependent negative feedback mechanisms. These mechanisms are based on (a) a protein phosphatase 2B-mediated dephosphorylation process, and (b) the interaction of intracellular Ca²⁺ with a single membrane-associated site that may reside on the channel protein itself.