Effect of calcium on rat intestinal alkaline phosphatase activity and molecular aggregation

Two fractions of rat intestinal alkaline phosphatase (IAP) were detected by Western blot: 168 ± 6 and 475 ± 45 kDa. The low molecular weight fraction constitutes 43% of the isolated proteins exhibiting 82% of the enzymatic activity, and a heavier fraction constitutes 57% of the isolated proteins and has 18% of the enzymatic activity. Calcium produced an increase of the 475-kDa form to the detriment of the 168-kDa form. This work also describes the kinetic and structural changes of IAP as a function of calcium concentration. With [Ca²⁺] < 10 mmole/L, the Ca²⁺-IAP interaction fitted a binding model with 7.8 ± 4.4 moles of Ca²⁺ /mole of protein, affinity constant = 19.1 ± 8.4 L/mmole, and enzymatic activity increased as a linear function of [Ca²⁺] (r = 0.946 p < 0.01). On the other hand, with [Ca²⁺] >10 mmole/L the data did not fit this model and, the enzymatic activity decreased as a function of [Ca²⁺] (r = ? 0.703 p < 0.05).     

Apelin decreases the SR Ca2+ content but enhances the amplitude of [Ca2+]i transient and contractions during twitches in isolated rat cardiac myocytes

Apelin has been reported to have a positive inotropic action in the isolated rat heart. However, the effect of apelin on sarcoplasmic reticulum (SR) Ca2+ content and its influence on intracellular Ca2+ transient during excitation-contraction coupling remains poorly understood. In the present study, we determined the effect of apelin on Ca2+ transient and contractions in isolated rat cardiomyocytes. When compared with control, treatment with apelin caused a 55.7 +/- 13.9% increase in sarcomere fraction shortening and a 43.6 +/- 4.56% increase in amplitude of electrical-stimulated intracellular Ca2+ concentration (E[Ca2+]i) transients (n = 14, P < 0.05). But SR Ca2+ content measured by caffeine-induced [Ca2+]i (C[Ca2+]i) transient was decreased 8.41 +/- 0.92% in response to apelin (n = 14, P < 0.05). Na+/Ca2+ exchanger (NCX) function was increased since half-decay time of C[Ca2+]i was decreased 16.22 +/- 1.36% in response to apelin. Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity was also increased by apelin. These responses can be partially or completely blocked by chelerythrine chloride, a PKC inhibitor. In addition, to confirm our data, we used indo-1 as another Ca2+ indicator and rapid cooling as another way to measure SR Ca2+ content, and we observed similar results. So we conclude that apelin has a positive inotropic effect on isolated myocytes, and increased amplitude of E[Ca2+]i is at least partially involved in the mechanism. NCX function and SERCA activity are increased by apelin, and the SR Ca2+ content is decreased by apelin during twitches. PKC played an important role in these signaling mechanisms.     

Chick heart cells with high intracellular calcium concentration have a higher affinity for cardiac glycosides than those with low intracellular calcium concentration, as revealed by affinity labelling with a digoxigenin derivative.

Digital-imaging fluorescence microscopy with fura-2 allows the determination of intracellular calcium concentration ([Ca2+]i) in single cells. At a cell density of 10(5) cells/petri dish 44% of the chick embryo heart cells had a high [Ca2+]i of 99.4 +/- 7.1 nM and 56% of the cells a low [Ca2+]i of 27.8 +/- 4.4 nM (mean +/- SE). This laboratory previously reported that high-[Ca2+]i and low-[Ca2+]i cells from chick embryo hearts differ in their sensitivity to cardiac glycosides, as shown by measuring the increase in [Ca2+]i to reach a new steady state [Ahlemeyer, B., Weintraut, H., Seibold, G. & Schoner, W. (1991) in The sodium pump: recent developments (Kaplan, J. H. & De Weer, P., eds) pp. 653-656, Rockefeller University Press, New York]. This time we used N-hydroxysuccinimidyl digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproate (HDMA) which binds irreversibly to amino groups of the Na+/K(+)-ATPase, and sheep anti-digoxigenin Fab fragments coupled with fluorescein isothiocyanate to identify different cardiac glycoside-binding sites. Half-maximal labelling of high-[Ca2+]i cells was obtained at 0.36 nM HDMA, and at 12.0 nM with the low-[Ca2+]i cells. Specific labelling of the cells by HDMA was 91% and 80% in high-[Ca2+]i and low-[Ca2+]i cells, respectively, as revealed by competition experiments with a 1000-fold excess of ouabain. HDMA half-maximally elevated the [Ca2+]i of high-[Ca2+]i cells at a concentration of 50 pM and that of low-[Ca2+]i cells at 8.0 nM. Concentrations higher than 0.1 microM produced signs of intoxication. When the labelled cells were subjected to a SDS/PAGE, a 100-kDa band was found to contain HDMA. The electrophoretic mobility of a protein labelled at 10 nM HDMA was slightly higher than that of a protein labelled at 1.0 microM. The data suggest that different isoforms of the alpha-subunit of Na+/K(+)-ATPase may exist in low-[Ca2+]i and high-[Ca2+]i cells of chick embryo heart.     

Cytosolic and stored calcium antagonistically control tyrosine phosphorylation of specific platelet proteins.

Depletion of intracellular calcium stores appears to increase plasma membrane permeability for calcium by an as yet obscure mechanism. We found that the Ca2+ ionophore, A23187, and thrombin elevate cytosolic calcium ([Ca2+]i) equally and cause tyrosine phosphorylation of a 130-kDa protein and to a lesser extent 80- and 60-kDa proteins. Chelation of [Ca2+]i by 1,2-bis(2-aminophenoxyethane)-N,N,N',N'-tetraacetic acid/acetomethoxy ester decreased thrombin-induced tyrosine phosphorylation responses. These results suggested that [Ca2+]i elevation promotes tyrosine phosphorylation. Tyrosine phosphorylation persisted in the presence or absence of extracellular calcium after thrombin stimulation but subsided rapidly after A23187 addition if extracellular calcium was present. When Ca2+/ATPase activity, which is apparently required to maintain calcium stores, is inhibited by low temperature, tyrosine phosphorylation of the 130-kDa protein occurs. Rewarming platelets reverses tyrosine phosphorylation only if extracellular calcium is present. Thapsigargin, a calcium ATPase inhibitor, also induces tyrosine phosphorylation of the 130-kDa protein and prevents dephosphorylation of this protein when added prior to rewarming. These observations suggest that homeostatic levels of calcium in storage compartments favor tyrosine dephosphorylation of specific proteins. Thus the levels of [Ca2+]i and stored calcium appear to control tyrosine phosphorylation antagonistically. Tyrosine phosphorylation may play a role in regulating calcium channel function.     

High levels of cytosolic free calcium inhibit dephosphorylation of insulin receptor and glycogen synthase.

Treatment of adipocytes with depolarizing concentrations of K+ (40 mM) for 60 min increased [Ca2+]i from 158 +/- 28 nM to 328 +/- 38 nM. This significantly reduced (up to 80% inhibition) dephosphorylation of insulin receptor (IR), EGF receptor (EGF-R) and glycogen synthase (GS). The calcium channel blocker, nitrendipine (30 microM), or Ca2+ free medium completely prevented K(+)-induced inhibition of phosphoprotein phosphatase (PPTase). This effect of high [Ca2+]i was completely reversible when the cells were returned into the non-depolarizing medium. Trypsin treatment (4 micrograms/ml) of the membrane fraction containing inhibited PPTase activity, restored dephosphorylation activity to normal suggesting that elevated [Ca2+]i may inhibit PPTase by promoting its association with the inhibitors. These observations indicate that dephosphorylation of IR and GS can be regulated by [Ca2+]i.     

Mouse mast cell secretory granules can function as intracellular ionic oscillators

Fluorescent Ca2+ probes and digital photo-sectioning techniques were used to directly study the dynamics of Ca2+ in isolated mast cell granules of normal (CB/J) and beige (Bg(j)/Bg(j)) mice. The resting intraluminal free Ca2+ concentration ([Ca2+]L) is 25 +/- 4.2 microM (mean +/- SD, n = 68). Exposure to 3 microM inositol 1,4,5-trisphosphate (InsP3) induced periodic oscillations of luminal Ca2+ ([Ca2+]L) of approximately 10 microM amplitude and a period around 8-10 s. The [Ca2+]L oscillations were accompanied by a corresponding oscillatory release of [Ca2+]L to the extraluminal space. Control experiments using ruthenium red (2 microM) and thapsigargin (100 nM) ruled out artifacts derived from the eventual presence of mitochondria or endoplasmic reticulum in the isolated granule preparation. Oscillations of [Ca2+]L and Ca2+ release result from a Ca2+/K+ exchange process whereby bound Ca is displaced from the heparin polyanionic matrix by inflow of K+ into the granular lumen via an apamin-sensitive Ca2+-sensitive K+ channel (ASK(Ca)), whereas Ca2+ release takes place via an InsP3-receptor-Ca2+ (InsP3-R) channel. These results are consistent with previous observations of [Ca2+]L oscillations and release in/from the endoplasmic reticulum and mucin granules, and suggest that a highly conserved common mechanism might be responsible for [Ca2+]L oscillations and quantal periodic Ca2+ release in/from intracellular Ca2+ storage compartments.     

Excitation-contraction coupling model to estimate the recirculating fraction of activator calcium in intact cardiac muscle.

Potentiated contractions were evoked with rapid pace pause maneuver in 14 length-clamped ferret papillary muscles paced 12 times/min at 25 degrees C. At 1.25 mM [Ca2+]o the average steady-state force was 2.94 +/- 1.08 g/mm2 and the potentiated contraction averaged 10.96 +/- 1.61 g/mm2. At 5.0 mM [Ca2+]o the steady-state force increased to 6.18 +/- 1.23 g/mm2 and the potentiated contraction averaged 12.08 +/- 1.15 g/mm2. Under the conditions of these experiments the potentiated contraction obtained at 5.0 mM [Ca2+]o is equal to the maximum twitch tension (Fmax) these muscles can generate. We have previously shown that Fmax is an equivalent of maximal calcium activated force. Since there is a beat to beat nearly exponential decay of the evoked potentiation, the fraction (= fraction x) of the potentiation that is not dissipated with each beat is nearly constant. Using an excitation-contraction coupling model we have previously found that x reflects a measure of the recirculating fraction of activator calcium. Because the tension-calcium relationship is better characterized by a sigmoidal curve, we have now incorporated the Hill equation in the model. To account for the inverse relationship between [Ca2+]i and the magnitude of the slow inward current, a term for negative feedback (h) was also included. We have determined the quantity (x-h) because x and h could not be determined separately. The quantity (x-h) was denoted as x'. The average values of x' at 1.25 and 5.0 mM [Ca2+]o were significantly different (p less than 0.0001), approximately 20% at the lower [Ca2+]o and about 50% at the higher [Ca2+]o.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alteration of cytosolic free calcium homeostasis by SIN-1: high sensitivity of L-type Ca2+ channels to extracellular oxidative/nitrosative stress in cerebellar granule cells

Exposure of cerebellar granule neurones in 25 mm KCl HEPES-containing Locke's buffer (pH 7.4) to 50-100 microm SIN-1 during 2 h decreased the steady-state free cytosolic Ca2+ concentration ([Ca2+]i) from 168 +/- 33 nm to 60 +/- 10 nm, whereas exposure to > or = 0.3 mm SIN-1 produced biphasic kinetics: (i) decrease of [Ca2+]i during the first 30 min, reaching a limiting value of 75 +/- 10 nm (due to inactivation of L-type Ca2+ channels) and (ii) a delayed increase of [Ca2+]i at longer exposures, which correlated with SIN-1-induced necrotic cell death. Both effects of SIN-1 on [Ca2+]i are blocked by superoxide dismutase plus catalase and by Mn(III)tetrakis(4-benzoic acid)porphyrin chloride. Supplementation of Locke's buffer with catalase before addition of 0.5-1 mm SIN-1 had no effect on the decrease of [Ca2+]i but further delayed and attenuated the increase of [Ca2+]i observed after 60-120 min exposure to SIN-1 and also protected against SIN-1-induced necrotic cell death. alpha-Tocopherol, the potent NMDA receptor antagonist (+)-MK-801 and the N- and P-type Ca2+ channels blocker omega-conotoxin MVIIC had no effect on the alterations of [Ca2+]i upon exposure to SIN-1. However, inhibition of the plasma membrane Ca2+ ATPase can account for the increase of [Ca2+]i observed after 60-120 min exposure to 0.5-1 mm SIN-1. It is concluded that L-type Ca2+ channels are a primary target of SIN-1-induced extracellular nitrosative/oxidative stress, being inactivated by chronic exposure to fluxes of peroxynitrite of 0.5-1 microm/min, while higher concentrations of peroxynitrite and hydrogen peroxide are required for the inhibition of the plasma membrane Ca2+ ATPase and induction of necrotic cell death, respectively.     

Steady-state [Ca2+]i-force relationship in intact twitching cardiac muscle: direct evidence for modulation by isoproterenol and EMD 53998.

We have developed a novel method for measuring steady-state force-[Ca2+]i relations in isolated, membrane-intact rat trabeculae that are microinjected with Fura-2 salt. Twitches are markedly slowed after inhibition of phasic Ca2+ release and uptake from the sarcoplasmic reticulum by addition of cyclopiazonic acid and ryanodine. During relaxation of slowed twitches, force and [Ca2+]i trace a common trajectory in plots of force versus [Ca2+]i, despite very different histories of contraction. The common trajectory thereby provides a high resolution determination of the steady-state relation between force and [Ca2+]i. Using this method, we show that 1 microM isoproterenol, a beta-adrenergic agonist, causes a rightward shift (Hill function K1/2 increased from 0.39 +/- 0.07 microM to 0.82 +/- 0.23 microM, p < 0.02, n = 6) and a decreased slope (nH decreased from 5.4 +/- 1.1 to 4.0 +/- 1.4, p < 0.02) of the steady-state force-[Ca2+]i curve, with no change in maximal force (Fmax = 99.2 +/- 2.2% of control). In contrast, 2 microM EMD 53998, a racemic thiadiazinone derivative, causes a leftward shift (K1/2 decreased from 0.42 +/- 0.02 microM to 0.30 +/- 0.06 microM, p < 0.02, n = 4) with no change in slope of the steady-state force-[Ca2+]i curve, accompanied by a modest increase in maximal force (Fmax = 107.1 +/- 4.6% of control, p < 0.02). To gain mechanistic insight into these modulatory events, we developed a simple model of cooperative thin filament activation that predicts steady-state force-[Ca2+]i relationships. Model analysis suggests that isoproterenol decreases cooperativity arising from nearest-neighbor interactions between regulatory units on the thin filament, without change in the equilibrium constant for Ca2+ binding. In contrast, the effects of EMD 53998 are consistent with an increase in the affinity of strong-binding cross-bridges, without change in either the affinity of troponin C for Ca2+ or cooperative interactions.     

Cytosolic and mitochondrial [Ca2+] in whole hearts using indo-1 acetoxymethyl ester: effects of high extracellular Ca2+.

Assessment of free cytosolic [Ca2+] ([Ca2+]c) using the acetoxymethyl ester (AM) form of indo-1 may be compromised by loading of indo-1 into noncytosolic compartments, primarily mitochondria. To determine the fraction of noncytosolic fluorescence in whole hearts loaded with indo-1 AM, Mn2+ was used to quench cytosolic fluorescence. Residual (i.e., noncytosolic) fluorescence was subtracted from the total fluorescence before calculating [Ca2+]c. Noncytosolic fluorescence was used to estimate mitochondrial [Ca2+]. In hearts paced at 5 Hz (N = 17), noncytosolic fluorescence was 0.61 +/- 0.06 and 0.56 +/- 0.07 of total fluorescence at lambda 385 and lambda 456, respectively. After taking into account noncytosolic fluorescence, systolic and diastolic [Ca2+]c was 673 +/- 72 and 132 +/- 9 nM, respectively, noncytosolic [Ca2+] was 183 +/- 36 nM and increased to 272 +/- 12 when extracellular Ca2+ was increased from 2 to 6 mM. This increase in noncytosolic [Ca2+] was inhibited by ruthenium red, a blocker of Ca2+ uptake by mitochondria. We conclude that cytosolic and mitochondrial [Ca2+] can be determined in whole hearts loaded with indo-1 AM by using Mn2+ to quench cytosolic fluorescence.     

Measurement of sarcoplasmic reticulum Ca2+ content in intact amphibian skeletal muscle fibres with 4-chloro-m-cresol.

Single skeletal muscle fibres were isolated from the toad (Bufo marinus) and isometric force and myoplasmic free calcium concentration ([Ca2+]i) were measured. Brief applications of 4-chloro- m-cresol (4-CmC, 0.2-5 mM) elevated [Ca2+]i reversibly in a dose-dependent manner. The lowest concentration of 4-CmC which reliably gave maximal [Ca2+]i was 2 mM and it was, therefore, used for measurement of sarcoplasmic reticulum (SR) Ca2+ content. Tetanic stimulations (100 Hz) increased [Ca2+]i from a resting level of 105 +/- 47 nM (n = 10) to 1370 +/- 220 nM (n = 6). Application of 2 mM 4-CmC produced a contracture that was 54 +/- 16% (n = 6) of the tetanic force and elevated [Ca2+]i to a peak of 3520 +/- 540 nM (n = 8). Both force and [Ca2+]i levels (resting and tetanic) were restored after 10 min of washout of 4-CmC. In skinned muscle fibres, the myofibrillar Ca(2+)-sensitivity was not changed by 4-CmC, but maximal force was reduced to 74 +/- 10% (n = 4). The magnitude of the peak of the 4-CmC-induced Ca2+ transient was not significantly changed by removal of extracellular Ca2+ nor by inhibiting the SR Ca2+ pump with 2,5-di-tert-butylhydroquinone. Treatment of intact fibres with 30 mM caffeine produced a peak Ca2+ level that was indistinguishable from 2 mM 4-CmC. These results indicate that it is possible to measure the SR Ca2+ content in the same fibre with 4-CmC without loss of normal muscle function.     

Extracellular Ca2+ mobilization in potential-dependent contraction of trachealis of maturing swine.

We studied the effect of maturation on potassium-induced parasympathetic activation and Ca2+ entry in tracheal smooth muscle (TSM) from fifteen 2-wk-old (2ws) and sixteen 10-wk-old (10ws) male domestic farm swine. Atropine (10(-7) M) caused inhibition of the maximal contraction elicited by potassium to 50.3 +/- 2.6% maximum of control response (P less than 0.001) in TSM from 2ws but had no significant effect in TSM from 10ws (94.6 +/- 4.2% maximum; P = NS vs. control). Verapamil (10(-7) M) plus 10(-7) M atropine reduced contraction elicited by potassium in both 2ws (23.7 +/- 5.8% maximum; P less than 0.001 vs. control) and 10ws (50.6 +/- 6.3% maximum; P less than 0.001 vs. control, P less than 0.05 vs. 2ws); 10(-6)M verapamil caused greater than 95% blockade of contraction caused by potassium in both 2ws and 10ws. In separate studies, atropine-treated strips were equilibrated with extracellular Ca2+ concentrations ([Ca2+]o) ranging from normal (1X [Ca2+]o) to four times normal (4x [Ca2+]o). Increasing [Ca2+]o increased maximal contractile response in atropine-treated TSM strips from 68.7 +/- 3.8% maximum for 1x [Ca2+]o to 100.8 +/- 4.8% maximum for 4x [Ca2+]o (P less than 0.001) in 2ws. Neither atropine nor [Ca2+]o affected maximal responses of TSM in 10ws (103.5 +/- 3.0% maximum for 1x [Ca2+]o; P = NS vs. control). However, in the presence of atropine and verapamil, 4x [Ca2+]o augmented KCl-elicited contraction of TSM from both 2ws (46.9 +/- 6.3% maximum; P less than 0.01 vs. control) and 10ws (78.6 +/- 2.3% maximum; P less than 0.005 vs. control).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of intracellular and extracellular concentrations of Ca2+, K+, and Cl- on the Na+-dependent Mg2+ efflux in rat ventricular myocytes

Intracellular Mg2+ concentration ([Mg2+]i) was measured in rat ventricular myocytes with the fluorescent indicator furaptra (25 degrees C). After the myocytes were loaded with Mg2+, the initial rate of decrease in [Mg2+]i (initial Delta[Mg2+]i/Deltat) was estimated upon introduction of extracellular Na+, as an index of the rate of Na+-dependent Mg2+ efflux. The initial Delta[Mg2+]i/Deltat values with 140 mM [Na+]o were essentially unchanged by the addition of extracellular Ca2+ up to 1 mM (107.3+/-8.7% of the control value measured at 0 mM [Ca2+]o in the presence of 0.1 mM EGTA, n=5). Intracellular loading of a Ca2+ chelator, either BAPTA or dimethyl BAPTA, by incubation with its acetoxymethyl ester form (5 microM for 3.5 h) did not significantly change the initial Delta[Mg2+]i/Deltat: 115.2+/-7.5% (seven BAPTA-loaded cells) and 109.5+/-10.9% (four dimethyl BAPTA loaded cells) of the control values measured in the absence of an intracellular chelator. Extracellular and/or intracellular concentrations of K+ and Cl- were modified under constant [Na+]o (70 mM), [Ca2+]o (0 mM with 0.1 mM EGTA), and membrane potential (-13 mV with the amphotericin-B-perforated patch-clamp technique). None of the following conditions significantly changed the initial Delta[Mg2+]i/Deltat: 1), changes in [K+]o between 0 mM and 75 mM (65.6+/-5.0% (n=11) and 79.0+/-6.0% (n=8), respectively, of the control values measured at 140 mM [Na+]o without any modification of extracellular and intracellular K+ and Cl-); 2), intracellular perfusion with K+-free (Cs+-substituted) solution from the patch pipette in combination with removal of extracellular K+ (77.7+/-8.2%, n=8); and 3), extracellular and intracellular perfusion with K+-free and Cl--free solutions (71.6+/-5.1%, n=5). These results suggest that Mg2+ is transported in exchange with Na+, but not with Ca2+, K+, or Cl-, in cardiac myocytes.     

Arteriolar smooth muscle Ca2+ dynamics during blood flow control in hamster cheek pouch.

Intracellular calcium concentration ([Ca2+]i) governs the contractile status of arteriolar smooth muscle cells (SMC). Although studied in vitro, little is known of SMC [Ca2+]i dynamics during the local control of blood flow. We tested the hypothesis that the rise and fall of SMC [Ca2+]i underlies arteriolar constriction and dilation in vivo. Aparenchymal segments of second-order arterioles (diameter 35 +/- 2 microm) were prepared in the superfused cheek pouch of anesthetized hamsters (n = 18) and perifused with the ratiometric dye fura PE-3 (AM) to load SMC (1 microM, 20 min). Resting SMC [Ca2+]i was 406 +/- 37 nM. Elevating superfusate O2 from 0 to 21% produced constriction (11 +/- 2 microm) that was unaffected by dye loading; [Ca2+]i increased by 108 +/- 53 nM (n = 6, P < 0.05). Cycling of [Ca2+]i during vasomotion (amplitude, 150 +/- 53 nM; n = 4) preceded corresponding diameter changes (7 +/- 1 microm) by approximately 2 s. Microiontophoresis (1 microm pipette tip; 1 microA, 1 s) of phenylephrine (PE) transiently increased [Ca2+]i by 479 +/- 64 nM (n = 8, P < 0.05) with constriction (26 +/- 3 microm). Flushing blood from the lumen with saline increased fluorescence at 510 nm by approximately 45% during excitation at both 340 and 380 nm with no difference in resting [Ca2+]i, diameter or respective responses to PE (n = 7). Acetylcholine microiontophoresis (1 microA, 1 s) transiently reduced resting SMC [Ca2+]i by 131 +/- 21 nM (n = 6, P < 0.05) with vasodilation (17 +/- 1 microm). Superfusion of sodium nitroprusside (10 microM) transiently reduced SMC [Ca2+]i by 124 +/- 18 nM (n = 6, P < 0.05), whereas dilation (23 +/- 5 microm) was sustained. Resolution of arteriolar SMC [Ca2+]i in vivo discriminates key signaling events that govern the local control of tissue blood flow.     

Control of cytosolic free calcium in rat and chicken osteoclasts. The role of extracellular calcium and calcitonin

Single cell [Ca2+], studies were performed in chicken and rat osteoclasts loaded with fura-2 and exposed to a variety of treatments. Under resting conditions, basal [Ca2+]i, was 79.2 +/- 47.3 and 84.3 +/- 65.7 nM (averages +/- S.D.; n = 141 and 126) in the osteoclasts of the two species, respectively. Basal [Ca2+]i was stable in all rat and in approximately 80% of chicken osteoclasts. In the remaining 20%, spontaneous, irregular [Ca2+], fluctuations were observed (amplitude range: 50-200 nm over basal values). Increase of [Ca2+]o over the concentration of the Krebs-Ringer incubation medium (2 mM) induced rises of [Ca2+] in almost all cells investigated. [Ca2+] rises were already appreciable with 0.5 mM [Ca2+]o additions and reached high values with 4 mM additions: 390 +/- 113 and 364 +/- 214 nM [Ca2+], in rat and chicken osteoclasts, respectively (n = 122 and 101). Qualitatively, the responses to [Ca2+]o additions consisted of discrete [Ca2+]i transients, biphasic (an initial spike followed by a plateau), or monophasic (either the spike or the plateau). In a few chicken osteoclasts, the [Ca2+]i increase occurring after [Ca2+]o addition consisted of multiple, irregular fluctuations, similar to those observed in 20% of these cells under resting conditions. In individual osteoclasts subsequently exposed to multiple [Ca2+]o increase pulses, the type of the [Ca2+]i transient (mono- or biphasic) was maintained, and the size was dependent on the magnitude of the [Ca2+]o additions. Effects similar to those of [Ca2+]o were induced by the addition of Cd2+ or Ba2+ (but not La3+ or Mg2+) into the medium. The Cd2+ effect was maintained in part even in a Ca2+-free medium. Of various hormones and factors, parathormone, 1,25-dihydroxyvitamin D3, and prostaglandin E2 were inactive. In contrast, calcitonin was active in rat osteoclasts (which express numerous receptors). [Ca2+]i increases were small (19 +/- 17.9 nM; n = 21) when the hormone was administered alone; they were synergistic (severalfold potentiation) when the hormone was administered before or after [Ca2+]o. The [Ca2+]i effects of calcitonin were mimicked by 8Br-cAMP (31 +/- 26 nM; n = 12) when the nucleotide was administered alone; marked synergism when it was administered in combination with [Ca2+]o. This paper demonstrates for the first time that changes of [Ca2+]i are induced in osteoclasts by treatments with [Ca2+]o and calcitonin and can therefore be involved in intracellular mediation of the physiological effects of these two extracellular signals.     

T-cell stimulation through the T-cell receptor/CD3 complex regulates CD2 lateral mobility by a calcium/calmodulin-dependent mechanism

T lymphocyte activation through the T cell receptor (TCR)/CD3 complex alters the avidity of the cell surface adhesion receptor CD2 for its ligand CD58. Based on the observations that activation-associated increases in intracellular [Ca2+] ([Ca2+]i) strengthen interactions between T cells and antigen-presenting cells, and that the lateral mobility of cell surface adhesion receptors is an important regulator of cellular adhesion strength, we postulated that [Ca2+]i controls CD2 lateral mobility at the T cell surface. Human Jurkat T leukemia cells were stimulated by antibody-mediated cross-linking of the TCR/CD3 complex. CD2 was labeled with a fluorescently conjugated monoclonal antibody. Quantitative fluorescence microscopy techniques were used to measure [Ca2+]i and CD2 lateral mobility. Cross-linking of the TCR/CD3 complex caused an immediate increase in [Ca2+]i and, 10-20 min later, a decrease in the fractional mobility of CD2 from the control value of 68 +/- 1% to 45 +/- 2% (mean +/- SEM). One to two hours after cell stimulation the fractional mobility spontaneously returned to the control level. Under these and other treatment conditions, the fraction of cells with significantly elevated [Ca2+]i was highly correlated with the fraction of cells manifesting significantly reduced CD2 mobility. Pretreatment of cells with a calmodulin inhibitor or a calmodulin-dependent kinase inhibitor prevented Ca2+-mediated CD2 immobilization, and pretreatment of cells with a calcineurin phosphatase inhibitor prevented the spontaneous reversal of CD2 immobilization. These data suggest that T cell activation through the TCR/CD3 complex controls CD2 lateral mobility by a Ca2+/calmodulin-dependent mechanism, and that this mechanism may involve regulated phosphorylation and dephosphorylation of CD2 or a closely associated protein.     

Thrombin activation of the Na+/H+ exchanger in vascular smooth muscle cells. Evidence for a kinase C-independent pathway which is Ca2+-dependent and pertussis toxin-sensitive

The mechanism by which human alpha-thrombin activates the Na+/H+ exchanger was studied in cultured neonatal rat aortic smooth muscle cells. Thrombin (0.4 unit/ml) caused a rapid cell acidification followed by a slow, amiloride-inhibitable alkalinization (0.10-0.14 delta pHi above base line). In protein kinase C down-regulated cells (exposed to phorbol 12-myristate 13-acetate for 24 or 72 h), the delta pHi induced by thrombin was only partially attenuated. This protein kinase C-independent activation of the Na+/H+ exchanger was blocked by pertussis toxin (islet activating protein (IAP)), reducing delta pHi by 50%. IAP did not directly inhibit Na+/H+ exchange activity as assessed by the response to intracellular acid loading. Thrombin also stimulated arachidonic acid release by 2.5 fold and inositol trisphosphate release by 6.2 fold. IAP inhibited both of these activities by 50-60%. Intracellular Ca2+ chelation with 120 microM quin2 prevented the thrombin-induced Ca2+ spike, inhibited thrombin-induced arachidonic acid release by 75%, and inhibited thrombin-induced activation of the Na+/H+ exchanger in protein kinase C-deficient cells by 65%. Increased intracellular [Ca2+] alone was not sufficient to activate the Na+/H+ exchanger, since ionomycin (0.3-1.5 microM) failed to elevate cell pH significantly. 10 microM indomethacin inhibited thrombin-induced delta pHi in both control and protein kinase C down-regulated cells by 30-50%. Thus, thrombin can activate the Na+/H+ exchanger in vascular smooth muscle cells by a Ca2+-dependent, pertussis toxin-sensitive pathway which does not involve protein kinase C.     

Calcium homeostasis in a clonal pituitary cell line of mouse corticotropes.

Calcium homeostasis was studied following a depolarization-induced transient increase in [Ca2+]i in single cells of the clonal pituitary cell line of corticotropes, AtT-20 cells. The KCl-induced increase in [Ca2+]i was blocked in (i) extracellular calcium-deficient solutions, (ii) external cobalt (2.0 mM), (iii) cadmium (200 microM), and (iv) nifedipine (2.0 microM). The mean increase in [Ca2+]i in single cells in the presence of an uncoupler of mitochondrial function [carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, FCCP, 1 microM] was 54 +/- 13 nM (n = 9). The increase in [Ca2+]i produced by FCCP was greater either during or following a KCl-induced [Ca2+]i load. However, FCCP did not significantly alter the clearance of calcium during a KCl-induced rise in [Ca2+]i. Fifty percent of the cells responded to caffeine (10 mM) with an increase in [Ca2+]i (191 +/- 24 nM; n = 21) above resting levels; this effect was blocked by ryanodine (10 microM). Thapsigargin (2 microM) and 2,5 di(-t-butyl)-1,4 hydroquinone (BuBHQ, 10 microM) produced increases in [Ca2+]i (47 +/- 11 nM, n = 6 and 22 +/- 4 nM, n = 8, respectively) that increased cell excitability. These results support a role for mitochondria and sarco-endoplasmic reticulum calcium stores in cytosolic [Ca2+]i regulation; however, none of these organelles are primarily responsible for the return of [Ca2+]i to resting levels following this KCl-induced [Ca2+]i load.     

Relationship between force and intracellular [Ca2+] in tetanized mammalian heart muscle

To determine features of the steady state [Ca2+]-tension relationship in intact heart, we measured steady force and intracellular [Ca2+] ([Ca2+]i) in tetanized ferret papillary muscles. [Ca2+]i was estimated from the luminescence emitted by muscles that had been microinjected with aequorin, a Ca2+-sensitive, bioluminescent protein. We found that by raising extracellular [Ca2+] and/or by exposing muscles to the Ca2+ channel agonist Bay K 8644, tension development could be varied from rest to an apparently saturating level, at which increases in [Ca2+]i produced no further rise in force. 95% of maximal Ca2+-activated force was reached at a [Ca2+]i of 0.85 +/- 0.06 microM (mean +/- SEM; n = 7), which suggests that the sensitivity of the myofilaments to [Ca2+]i is far greater than anticipated from studies of skinned heart preparations (or from previous studies using Ca2+-sensitive microelectrodes in intact heart). Our finding that maximal force was reached by approximately 1 microM also allowed us to calculate that the steady state [Ca2+]i-tension relationship, as it might be observed in intact muscle, should be steep (Hill coefficient of greater than 4), which is consistent with the Hill coefficient estimated from the entire [Ca2+]i-tension relationship derived from families of variably activated tetani (6.08 +/- 0.68; n = 7). Finally, with regard to whether steady state measurements can be applied directly toward understanding physiological contractions, we found that the relation between steady force and [Ca2+]i obtained during tetani was steeper than that between peak force and peak [Ca2+]i observed during physiological twitches.     

Thrombin-stimulated increases in cytosolic Ca2+ level and gonadotropin-releasing hormone release in GT1-7 neurons.

The effects of thrombin on cytosolic calcium levels ([Ca2+]cyt), and on gonadotropin-releasing hormone (GnRH) release, were characterized in cultured GT1-7 neurons. GnRH release from GT1-7 neurons was pulsatile with an average pulse amplitude of 14.3+/-5.8 pg x min x ml(-1) and an average pulse duration of 21.3+/-4.2 min. The [Ca2+]cyt response to 0.005 to 0.2 U/ml thrombin was saturable and concentration dependent (EC50 = 0.0268 U/ml). Ethyleneglycotetraacetic acid (EGTA) chelation of extracellular Ca2+ resulted in an approximately 70% attenuation of thrombin-stimulated increase in [Ca2+]cyt. By use of a special superfusion system, a 5-min exposure to 0.1 U/ml thrombin significantly increased the amplitude (193.2+/-67.8 pg x min x ml(-1); P = 0.001) but not the duration (22.5+/-2.4 min; P = 0.8) of GnRH release. These results suggest that thrombin increases [Ca2+]cyt and GnRH release from GT1-7 neurons via specific membrane-bound receptors.