Alpha-stat calibration of indo-1 fluorescence and measurement of intracellular free calcium in rat ventricular cells at different temperatures.

To explore how to manage pH when calibrating Ca2+ probes at different temperatures, the dissociation constant (Kd) of indo-1 was determined both in pH-stat (pH is fixed despite the temperature) and in alpha-stat (pH changes with temperature as in cells). The results showed that the Kd was much more sensitive to temperature in pH-stat than in alpha-stat, demonstrating that alpha-stat calibration should be preferred when using a Ca2+ probe to measure intracellular free calcium ([Ca2+]i) at different temperatures. Based on the calibration in situ and in alpha-stat, we showed a striking increase of [Ca2+]i from 141+/-8 nM at 30 degrees C to 218+/-22 nM at 10 degrees C in indo-1 loaded rat ventricular cells, which supports that intracellular calcium overload takes place in cardiac myocytes of non-hibernating mammals during hypothermia.     

Glucose induces temperature-dependent oscillations of cytoplasmic Ca2+ in single pancreatic beta-cells related to their electrical activity

Glucose induces large amplitude oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i) in pancreatic beta-cells. The effects of temperature on these oscillations were examined by monitoring [Ca2+]i continuously in single beta-cells from ob/ob-mice using dual wavelength microfluorometry. The oscillations of [Ca2+]i disappeared when the temperature was increased above 42 degrees C and were reversibly inhibited below 30 degrees C. However, cooling did not prevent a glucose response in terms of the average rise of [Ca2+]i. Since patch clamp studies of single beta-cells have indicated a random occurrence of glucose-induced action potentials at room temperature, it was important to explore how the sugar affected the electrical activity at 37 degrees C. Using the cell-attached configuration of the patch clamp technique for such analyses, the action potentials were found to occur in bursts with durations similar to the large amplitude oscillations of [Ca2+]i.     

Role of Ca(2+)-ATPase in spontaneous oscillations of cytosolic free Ca2+ in GH3 rat pituitary cells.

The relative contribution of voltage-sensitive Ca2+ channels, Ca(2+)-ATPases, and Ca2+ release from intracellular stores to spontaneous oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) observed in secretory cells is not well characterized owing to a lack of specific inhibitors for a novel thapsigargin (Tg)-insensitive Ca(2+)-ATPase expressed in these cells. We show that spontaneous [Ca2+]i oscillations in GH3 cells were unaffected by Ca2+ depletion in inositol-1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores by the treatment of Tg, but could be initiated by application of caffeine. Moreover, we demonstrate for the first time that these spontaneous [Ca2+]i oscillations were highly temperature dependent. Decreasing the temperature from 22 to 17 degrees C resulted in an increase in the frequency, a reduction in the amplitude, and large inhibition of [Ca2+]i oscillations. Furthermore, the rate of ATP-dependent 45Ca2+ uptake into GH3-derived microsomes was greatly reduced at 17 degrees C. The effect of decreased temperatures on extracellular Ca2+ influx was minor because the frequency and amplitude of spontaneous action potentials, which activate L-type Ca2+ channels, was relatively unchanged at 17 degrees C. These results suggest that in GH3 secretory cells, Ca2+ influx via L-type Ca2+ channels initiates spontaneous [Ca2+]i oscillations, which are then maintained by the combined activity of Ca(2+)-ATPase and Ca(2+)-induced Ca2+ release from Tg/IP3-insensitive intracellular stores.     

Protein kinase C-activated calcium channel in the osteoblast-like clonal osteosarcoma cell line UMR-106

The effects of protein kinase C stimulation on free cytosolic Ca2+ [( Ca2+]i) were studied in Fura 2-loaded UMR-106 cells. Stimulation of the protein kinase C with the tumor-promoting phorbol esters 12-O-tetradecanoylphorbol 13-acetate (TPA) and phorbol 12,13-diacetate or 1-oleoyl-2-acetylglycerol was followed by an increase in [Ca2+]i. The protein kinase C-induced increase in [Ca2+]i has a lag period, the duration of which was dependent on the stimulant and medium Ca2+ concentrations. With 2 microM TPA, the rise in [Ca2+]i peaked within 1.5 min, after which [Ca2+]i returned partially toward base line. The increase in [Ca2+]i was absolutely dependent on the presence of medium Ca2+ and was inhibited by the Ca2+ channel blockers nicardipine and verapamil. Cell stimulation also results in Ca2+ release from intracellular pool(s) which appears to be mediated by a Ca2+-dependent Ca2+ release mechanism. The reduction in [Ca2+]i was due to channel inactivation. Pretreatment of the cells with 1 nM TPA, 2 units/ml parathyroid hormone (PTH), or 15 microM forskolin blocked the effect of 2 microM TPA on [Ca2+]i. TPA and PTH were more potent inhibitors than was forskolin. The properties of this channel are compared to the cAMP-independent PTH-stimulated Ca2+ channel present in these cells.     

Temperature dependence and thermodynamic properties of Ca2+ sparks in rat cardiomyocytes

To elucidate the temperature dependence and underlying thermodynamic determinants of the elementary Ca2+ release from the sarcoplasmic reticulum, we characterized Ca2+ sparks originating from ryanodine receptors (RyRs) in rat cardiomyocytes over a wide range of temperature. From 35 degrees C to 10 degrees C, the normalized fluo-3 fluorescence of Ca2+ sparks decreased monotonically, but the Delta[Ca2+]i were relatively unchanged due to increased resting [Ca2+]i. The time-to-peak of Ca2+ sparks, which represents the RyR Ca2+ release duration, was prolonged by 37% from 35 degrees C to 10 degrees C. An Arrhenius plot of the data identified a jump of apparent activation energy from 5.2 to 14.6 kJ/mol at 24.8 degrees C, which presumably reflects a transition of sarcoplasmic reticulum lipids. Thermodynamic analysis of the decay kinetics showed that active transport plays little role in early recovery but a significant role in late recovery of local Ca2+ concentration. These results provided a basis for quantitative interpretation of intracellular Ca2+ signaling under various thermal conditions. The relative temperature insensitivity above the transitional 25 degrees C led to the notion that Ca2+ sparks measured at a "warm room" temperature are basically acceptable in elucidating mammalian heart function.     

Oscillations of intracellular calcium induced by vasopressin in individual fura-2-loaded mesangial cells. Frequency dependence on basal calcium concentration, agonist concentration, and temperature

Intracellular free calcium concentration ([Ca2+]i) was measured in fura-2-loaded single rat mesangial cells by dual wavelength spectrofluorometry. Stimulation with arginine vasopressin (AVP) caused an initial sharp rise of [Ca2+]i followed by repetitive spikes. The frequency of the oscillations was dependent on the concentration of AVP. At 0.1, 1.0, 10.0, and 100.0 nM AVP, the frequencies of oscillations were 0.17 +/- 0.05 (n = 6), 0.32 +/- 0.05 (n = 6), 0.49 +/- 0.05 (n = 6), and 0.48 +/- 0.05 min-1 (n = 5), respectively. Reduction in extracellular [Ca2+] reduced the frequency of AVP-induced oscillations but did not abolish the oscillations. The frequency of calcium oscillations, upon stimulation with 1.0 nM AVP, was directly correlated with the basal [Ca2+]i prior to stimulation. Oscillation frequency increased with increasing temperature. An Arrhenius plot between 24 and 37 degrees C indicated a strong temperature dependency of the oscillations with a Q10 of 3.0. Protein kinase C stimulation by active phorbol esters inhibited AVP-induced calcium oscillations but not the initial [Ca2+] response to AVP. These observations are consistent with a model incorporating a feedback loop linking [Ca2+]i to the mechanism of [Ca2+]i increase. Ca(2+)-induced Ca2+ release may be involved, whereby inositol 1,4,5-trisphosphate (inositol 1,4,5-P3) formation releases Ca2+ from an inositol 1,4,5-P3-sensitive pool, with subsequent Ca2+ uptake and release from an inositol 1,4,5-P3-insensitive pool.     

Temperature sensitivity of catecholamine secretion and ion fluxes in bovine adrenal chromaffin cells

The effects of temperature on ion fluxes and catecholamine secretion that are mediated by nicotinic acetylcholine receptors (nAChRs), voltage-sensitive calcium channels (VSCCs), and voltage-sensitive sodium channels (VSSCs) were investigated using bovine adrenal chromaffin cells. When the chromaffin cells were stimulated with DMPP, a nicotinic cholinergic agonist, or 50 mM K+, the intracellular calcium ([Ca2+]i) elevation reached a peak and decreased more slowly at lower temperatures. The DMPP-induced responses were more sensitive to temperature changes compared to high K+-induced ones. In the measurement of intracellular sodium concentrations ([Na+]i), it was found that nicotinic stimulation required a longer time to attain the maximal level of [Na+]i at lower temperatures. In addition, the VSSCs-mediated [Na+]i increase evoked by veratridine was also reduced as the temperature decreased. The measurement of [3H]norepinephrine (NE) secretion showed that the secretion within the first 3 min evoked by DMPP or high K+ was greatest at 37 degrees C. However, at 25 degrees C, the secretion evoked by DMPP, but not that by the 50 mM K+, was greater after 10 min of stimulation. This data suggest that temperature differentially affects the activity of nAChRs, VSCCs, and VSSCs, resulting in differential [Na+]i and [Ca2+]i elevation, and in the [3H]NE secretion by adrenal chromaffin cells.     

The correlation between Ca2+ influx and inositol 1, 4, 5-trisphosphate (IP3) formation in platelets stimulated by various agonists

The relationship between Ca2+ influx (delta [Ca2+]i) and the formation of inositol 1,4,5-trisphosphate (IP3) was investigated in human platelets stimulated by various agonists. Both delta [Ca2+]i and IP3 were increased in proportion to the amount of the agonists (thrombin, ADP, PAF, STA2), the receptors of which were demonstrated in platelets, and were correlated with each other. However, the ratio of delta [Ca2+]i to IP3 was significantly varied among agonists. Furthermore, in thrombin stimulated platelets, IP3 was small at low temperature (20 degrees C) compared with that at high temperature (37 degrees C) in spite of the similar delta [Ca2+]i. Thus, Ca2+ influx in human platelets seems to be regulated directly through the receptor operated mechanism and IP3 may not be involved in it.     

Regulation of intracellular Ca2+ oscillation in AR42J cells.

Recordings of [Ca2+]i in single AR42J cells loaded with Fura 2 were used to study regulation of [Ca2+]i oscillation. Continuous stimulation with the cholecystokinin analogue, (t-butyloxycarbonyl-Tyr-(SO3)-norleucine-Gly-Trp-Nle-Asp-2-phenylethyl ester) or carbachol evoked long lasting oscillation in [Ca2+]i. Removal of CCK-JMV-180 after brief stimulation did not abruptly stop the oscillation. Rather, removal of CCK-JMV-180 resulted in time-dependent reduction in amplitude with little change in frequency of oscillation. The patterns of [Ca2+]i oscillation were affected by activation of protein kinase C and protein kinase A. However, down-regulation of protein kinase C activity did not prevent stimulation of [Ca2+]i oscillation. Hence, we conclude that an active protein kinase C pathway is not crucial for [Ca2+]i oscillation in this cell line. Variation in extracellular Ca2+ concentration (Ca2+out) was used to further characterize the oscillation. Reducing Ca2+out to approximately 10 microM resulted in a time dependent inhibition of [Ca2+]i oscillation. Subsequent step increases in Ca2+out up to 2-3 mM resulted in increased amplitude and frequency of oscillation. Further increase in Ca2+out or an increase in plasma membrane permeability to Ca2+, brought about by an increase in pHo, resulted in increased amplitude, decreased frequency, and modified shape of the [Ca2+]i spikes. These observations point to the existence of regulatory mechanisms controlling the duration of Ca2+ release and entry during [Ca2+]i oscillation.     

Ca2+ mobilization can occur independent of acceleration of Na+/H+ exchange in thrombin-stimulated human platelets

Intracellular free Ca2+ [( Ca2+]i) and pH (pHi) were measured simultaneously by dual wavelength excitation in thrombin-stimulated human platelets double-labeled with the fluorescent probes fura2 and 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein to determine the relationship between changes in [Ca2+]i and pHi, respectively. At 37 degrees C, thrombin (0.5 or 0.1 units/ml) increased [Ca2+]i with no detectable lag period to maximum levels within 13 s followed by a slow return to resting levels. There was a transient decrease in pHi within 9 s that was immediately followed by an alkalinization response, attributable to activation of Na+/H+ exchange, that raised pHi above resting levels within 22 s. At 10-15 degrees C, thrombin-induced changes in [Ca2+]i and pHi were delayed and therefore better resolved, although no differences in the magnitude of changes in [Ca2+]i and pHi were observed. However, the increase in [Ca2+]i had peaked or was declining before the alkalinization response was detected, suggesting that Ca2+ mobilization occurs before activation of Na+/H+ exchange. In platelets preincubated with 5-(N-ethyl-N-isopropyl)amiloride or gel-filtered in Na+-free buffer (Na+ replaced with N-methyl-D-glutamine) to inhibit Na+/H+ exchange, thrombin stimulation caused a rapid, sustained decrease in pHi. Under these conditions there was complete inhibition of the alkalinization response, whereas Ca2+ mobilization was only partially inhibited. Nigericin (a K+/H+ ionophore) caused a rapid acidification of more than 0.3 pH unit that was sustained in the presence of 5-(N-ethyl-N-isopropyl)amiloride. Subsequent stimulation with thrombin resulted in slight inhibition of Ca2+ mobilization. These data show that, in human platelets stimulated with high or low concentrations of thrombin, Ca2+ mobilization can occur without a functional Na+/H+ exchanger and in an acidified cytoplasm. We conclude that Ca2+ mobilization does not require activation of Na+/H+ exchange or preliminary cytoplasmic alkalinization.     

Two components of hormone-evoked calcium release from intracellular stores of pancreatic acinar cells.

Dispersed pancreatic acini loaded with Fura 2 were used to study the effect of hormonal stimulation on [Ca2+]i (free cytosolic Ca2+ concentration). Stimulation of acini with cholecystokinin octapeptide or carbachol resulted in two components of increase in [Ca2+]i. The maximal increase in [Ca2+]i and the time to maximum for both components was dependent on hormone concentration. The first component reached a maximum after 2-10 s of stimulation, whereas the second component required 30-60 s of stimulation for maximal effect. Both components of the [Ca2+]i increase can be observed in the presence or absence of Ca2+ in the incubation medium. The two components of Ca2+ release from intracellular stores showed similar dependency on agonist concentration. Termination of cell stimulation with specific antagonist revealed two, kinetically separated, rates of decrease in [Ca2+]i. The initial decrease in [Ca2+]i, was completed within 2.5-7 s, whereas the secondary decrease in [Ca2+]i, back to resting values, required approx. 40 s. The magnitude of the antagonist-induced initial (rapid) and secondary (slow) decrease in [Ca2+]i was dependent on the duration of cell stimulation. Hence it appears that stimulation of pancreatic acinar cells with Ca2+-mobilizing hormones results in two, kinetically separated, components of Ca2+ release from intracellular stores.     

Muscarinic receptor-mediated increase in cytoplasmic free Ca2+ in isolated bovine adrenal medullary cells. Effects of TMB-8 and phorbol ester TPA

The change in cytoplasmic free calcium, [Ca2+]i in isolated bovine adrenal medullary cells during stimulation by acetylcholine (ACh) in Ca2+-free incubation medium was measured using the fluorescent Ca2+ indicator quin2. ACh (1-100 microM) caused an increase in [Ca2+]i by mobilization of Ca2+ from the intracellular pool. Nicotine (10 microM) did not increase [Ca2+]i in the absence of extracellular Ca2+. Pretreatment of the cells with atropine (10 microM) completely inhibited ACh-induced increase in [Ca2+]i, whereas pretreatment with hexamethonium (100 microM) did not. The intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), inhibited ACh-induced increase in [Ca2+]i. The activator of protein kinase C 12-O-tetradecanoylphorbol-13-acetate (TPA), but not its 'inactive' analog 4 alpha-phorbol-12,13-didecanoate (PDD), also inhibited ACh-induced increase in [Ca2+]i. These findings suggest that in bovine adrenal medullary cells, stimulation of muscarinic ACh receptor causes an increase in [Ca2+]i by mobilizing Ca2+ from the intracellular pool and that protein kinase C is involved in 'termination' or 'down regulation' of this response.     

Modulation of Ca2+ mobilization by protein kinase C in rat submandibular acinar cells

The effects of protein kinase C (PKC) activation and inhibition on the inositol 1,4,5-trisphosphate (IP3) and cytosolic Ca2+ ([Ca2+]i) responses of rat submandibular acinar cells were investigated. IP3 formation in response to acetylcholine (ACh) was not affected by the PKC activator phorbol 12-myristate 13-acetate (PMA), nor by the PKC inhibitor calphostin C (CaC). The ACh-elicited initial increase in [Ca2+]i in the absence of extracellular Ca2+ was not changed by short-term (0.5 min) exposure to PMA, but significantly reduced by long-term (30 min) exposure to PMA, and also by pre-exposure to the PKC inhibitors CaC and chelerythrine chloride (ChC). After ACh stimulation, subsequent exposure to ionomycin caused a significantly (258%) larger [Ca2+]i increase in CaC-treated cells than in control cells. However, pre-exposure to CaC for 30 min did not alter the Ca2+ release induced by ionomycin alone. These results suggest that the reduction of the initial [Ca2+]i increase is due to an inhibition of the Ca2+ release mechanism and not to store shrinkage. The thapsigargin (TG)-induced increase in [Ca2+]i was significantly reduced by short-term (0.5 min), but not by long-term (30 min) exposure to PMA, nor by pre-exposure to ChC or CaC. Subsequent exposure to ionomycin after TG resulted in a significantly (70%) larger [Ca2+]i increase in PMA-treated cells than in control cells, suggesting that activation of PKC slows down the Ca2+ efflux or passive leak seen in the presence of TG. Taken together, these results indicate that inhibition of PKC reduces the IP3-induced Ca2+ release and activation of PKC reduces the Ca2+ efflux seen after inhibition of the endoplasmic Ca2+-ATPase in submandibular acinar cells.     

Activation of the plasma membrane Ca2+ pump during agonist stimulation of pancreatic acini.

The role of internal stores and plasma membrane Ca2+ pumps in controlling [Ca2+]i during agonist stimulation and their regulation by agonists are not well understood. We report here measurements of intracellular ([Ca2+]i) and extracellular ([Ca2+]o) Ca2+ concentrations in agonist-stimulated pancreatic acini in an effort to directly address these questions. Stimulation of acini suspended in Ca(2+)-free or Ca(2+)-containing medium with Ca2+ mobilizing agonists resulted in a typical transient increase in [Ca2+]i. Thapsigargin, a specific inhibitor of internal Ca2+ pumps, inhibited the rate of [Ca2+]i reduction after agonist stimulation by approximately 40%. Under the same conditions, thapsigargin had no effect on the rate of the unidirectional Ca2+ efflux across the plasma membrane as revealed by measurements of [Ca2+]o. These findings suggest that internal Ca2+ pumps actively remove Ca2+ from the cytosol during continued agonist stimulation. The correlation between the reduction in [Ca2+]i and the increase in [Ca2+]o showed that Ca2+ efflux from cells stimulated with agonist and thapsigargin represent Ca2+ efflux across the plasma membrane. Inhibition of cells exposed to agonist and thapsigargin with a specific antagonist sharply reduced the rates of the [Ca2+]i decrease and the accompanied [Ca2+]o increase. Hence, at comparable [Ca2+]i, Ca2+ efflux from stimulated cells was about 3-fold faster than that from resting cells, indicating that agonists directly activate the plasma membrane Ca2+ pump. To study the role of [Ca2+]i increase in plasma membrane Ca2+ pump activation the acini were loaded with 1,2-bis-(2-aminophenoxyethane-N,N,N',N')-tetraacetic acid (BAPTA), and [Ca2+]o was measured during agonist stimulation. Surprisingly, although BAPTA completely prevented the increase in [Ca2+]i, Ca2+ efflux rate was reduced by only 34%. These findings provide the first evidence for Ca(2+)-independent activation of the plasma membrane Ca2+ pump by Ca2+ mobilizing agonists.     

Determination of Fura-2 dissociation constants following adjustment of the apparent Ca-EGTA association constant for temperature and ionic strength

The accurate calibration of Fura-2 fluorescence in living cells is dependent upon the apparent dissociation constant (Kd) of Fura-2 for Ca2+. If Ca-EGTA calibration buffers are used to construct an in vitro calibration curve, then the calculated value of the apparent Ca-EGTA association constant (K'CaEGTA) will have an important influence on the Kd of Fura-2 and thus the calculated free [Ca2+] in cells. In order to simulate experimental conditions, the individual proton and Ca2+ association constants for EGTA in these experiments were adjusted for both ionic strength and temperature using a semi-empirical form of the Debye-Huckel limiting law and the Van't Hoff isochore, respectively, as described by Harrison and Bers. The modified individual binding constants were then employed in the calculation of K'CaEGTA using the SPECS computer program of Fabatio. At pH = 7.05, ionic strength = 0.15 M, temp = 20 degrees C, K'CaEGTA = 3.232 x 10(6) M-1; at pH = 6.84, temp = 36 degrees C, K'CaEGTA = 1.652 x 10(6) M-1. These values differed substantially from those obtained with unadjusted individual association constants. Calibration buffers of varying [Ca2+] were prepared using the corrected values of K'CaEGTA, and Fura-2 fluorescence ratios were measured during superfusion of these buffers in the experimental chamber at both 20 degrees C and 37 degrees C. The Kd of Fura-2 for Ca2+ was determined to be 236 nM at 20 degrees C and 285 nM at 37 degrees C, utilizing the value of K'CaEGTA adjusted by the method of Harrison and Bers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synergistic signals in the mechanism of antigen-induced exocytosis in 2H3 cells: evidence for an unidentified signal required for histamine release

The aim of this study was to determine whether the increase in cytosolic free Ca2+ concentration ([Ca2+]i) in response to antigen (aggregated ovalbumin) on IgE-primed 2H3 cells was sufficient to account for exocytosis. When the [Ca2+]i responses to antigen and the Ca2+ ionophore A23187 were compared, A23187 was much less effective at releasing histamine at equivalent [Ca2+]i increases, and little or no stimulated histamine release occurred with A23187 concentrations that matched the [Ca2+]i response to antigen concentrations that stimulated maximal histamine release. The [Ca2+]i response to antigen is not, therefore, sufficient to account for exocytosis, although extracellular Ca2+ is necessary to initiate both the [Ca2+]i response and histamine release: the antigen must generate an additional, unidentified, signal that is required for exocytosis. To determine whether this signal was the activation of protein kinase C, the effects of the phorbol ester 12-0-tetradecanoyl phorbol 13-acetate (TPA) on the responses to antigen were examined. TPA blocked the antigen-induced [Ca2+]i response and the release of inositol phosphates but had little effect on histamine release and did not stimulate exocytosis by itself. The unidentified signal from the antigen is therefore distinct from the activation of protein kinase C and is generated independently of the [Ca2+]i response or the release of inositol phosphates. Taken together with other data that imply that there is very little activation of protein kinase C by antigen when the rate of histamine release is maximal, it is concluded that the normal exocytotic response to antigen requires the synergistic action of the [Ca2+]i signal together with an unidentified signal that is not mediated by protein kinase C.     

Studies on the changes in calcium fluxes in ts-RSV LA 90 cells transformation

To study the role of Ca2+ fluxes and [Ca2+]i in cell transformation by the v-src gene, ts-RSV LA 90 cells was used in this experiment. Ca2+ fluxes across the plasma membrane was measured with radioisotopes. The relative [Ca2+]i in LA 90 cells loaded Indo-1AM was measured by computer-based Optical Multichannel Analyzer connected with fluorescence microscopy. It was observed that changes in rate of Ca2+ fluxes across the plasma membrane are one of the earliest detectable changes of LA 90 cells transformation. Rates of Ca2+ fluxes in transformed LA 90 cells (40 degrees C) is higher than that in normal LA 90 cells (33 degrees C) and rates of Ca2+ fluxes increased in 25 minutes when LA 90 cells shifted from nonpermissive (40 degrees C) to permissive (33 degrees C) temperature. TMB-8 inhibited increases in rate of Ca2+ efflux induced by pp 60 v-src, and increase in rate of Ca2+ efflux in normal LA 90 cells was stimulated by calf serum. The rate of Ca2+ efflux was related to the changes in temperature. The increase in rate of Ca2+ influx induced by pp 60 v-src could be blocked by verapamil. The rate of Ca2+ influx was not affected by the changes in temperature. The increase in relative [Ca2+]i induced by pp 60 v-src is one of the early events in the transformation process. The level of [Ca2+]i in transformed LA 90 cells was about 2-3 times as much as that in normal LA 90 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of hypothyroidism on the cytosolic free Ca2+ concentration in rat hepatocytes during rest and following stimulation by noradrenaline or vasopressin

The mean resting concentration of cytosolic free Ca2+ [( Ca2+]i) in parenchymal liver cells, as determined with the intracellular Ca2+ indicator quin2, was lowered by about 30% in hypothyroidism (0.17 microM vs. 0.27 microM in normal cells). The [Ca2+]i level in hypothyroid cells at 10 s following stimulation by noradrenaline (1 microM) was about 64% lower than in normal cells (0.33 microM vs. 1.0 microM). The response to noradrenaline in hypothyroid cells was slower in onset (significant at 5 s vs. 3 s in euthyroid cells), and the maximum of the initial [Ca2+]i increase was reached later (14 s vs. 8 s in normal cells). In hypothyroid hepatocytes the initial increase was followed by a slow but prolonged secondary increase in [Ca2+]i. With vasopressin similar results were found. Chelation of extracellular Ca2+ with EGTA immediately prior to stimulation had no effect on the initial [Ca2+]i increase. Treatment with T3 in vivo (0.5 micrograms/100 g body weight daily during 3 days) completely restored the basal and stimulated [Ca2+]i in hypothyroid cells. The half-maximally effective dose of noradrenaline was the same in euthyroid and hypothyroid liver cells (1.8 X 10(-7) M). Hypothyroidism had no significant effect on the number of alpha 1-receptors determined by [3H]prazosin labeling in crude homogenate fractions, while the Kd for [3H]prazosin was 21% lower than in the euthyroid group. These results show that thyroid hormone has a general stimulating effect on intracellular Ca2+ mobilization by Ca2+-mobilizing hormones, probably at a site distal to the binding of the agonist to its receptor. The results also support our idea that thyroid hormone may control metabolism during rest and activation, at least partially, by altering Ca2+ homeostasis.     

Uptake of Ca2+ and refilling of intracellular Ca2+ stores in Ehrlich-ascites-tumour cells and in rat thymocytes.

We have studied the uptake of Ca2+ and its redistribution between the cytoplasm and the intracellular stores in Ehrlich-ascites-tumour cells and rat thymocytes previously depleted of Ca2+ by incubation in Ca2(+)-free medium. Measurements included changes of the cytoplasmic Ca2+ concentration ([Ca2+]i), uptake of 45Ca2+ and uptake of Mn2+, a Ca2+ surrogate for Ca2+ channels. Refilling of the Ca2+ stores in thymocytes was very fast (half-filling time: 4 s at 37 degrees C) and very sensitive to temperature (10 times slower at 20 degrees C). It was always preceded by increase of [Ca2+]i. In the Ehrlich cell, both refilling and increase of [Ca2+]i were about one order of magnitude slower. The increase of [Ca2+]i and the refilling of the intracellular stores were both almost completely blocked by Ni2+ in thymocytes, but only partially in the Ehrlich cell. The rates of 45Ca2+ and Mn2+ uptake varied consistently with temperature and the kind of cell. These results suggest that the intracellular stores are refilled by Ca2+ taken up from the cytoplasm. We also find that filling of the Ca2+ stores decreases by about 90% the rate of Mn2+ uptake in thymocytes. This is direct evidence of modulation of the plasma-membrane Ca2+ entry by the degree of filling of the intracellular stores. This modulation occurs in the absence of agonists, suggesting some kind of signalling between the intracellular stores and the Ca2+ entry pathways of the plasma membrane.     

Calcium and proton activities in rat cardiac mitochondria. Effect of matrix environment on behaviour of fluorescent probes.

The ionic composition of the mitochondrial matrix, under both physiological and pathophysiological conditions, remains controversial. Although fura-2 and 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), fluorescent probes for [Ca2+] and [H+] respectively, have successfully been loaded into mitochondria [Lukács & Kapus (1987) Biochem. J. 248, 609-613; Davis, Altschuld, Jung & Brierley (1987) Biochem. Biophys. Res. Commun. 49, 40-45], the adaptation of fluorescence-ratio spectroscopy to the study of the matrix ion content poses unique problems. In this report, we describe a method for successfully attaching viable rat cardiac mitochondria to glass coverslips, allowing continuous superfusion of isolated organelles during fluorescence microscopy. This technique obviated the need to correct for the accumulation of ion-sensitive and -insensitive fluorescent species of dye both within the matrix and outside of mitochondria in suspension in a cuvette, a particular problem with fura-2. By using this technique for superfusion of immobilized mitochondria, we found the pKa of BCECF for H+ at 25 degrees C shifted from 6.8 in buffer to 7.2 in rat cardiac mitochondria, with a marked hysteresis effect noted for intramitochondrial BCECF calibration curves. At higher pH, photobleaching of BCECF was enhanced. The dissociation constant (Kd) of fura-2 for Ca2+ was found to be 315 nM at 25 degrees C, pH 8.0, but only at [Ca2+] below 1 microM. At matrix [Ca2+] greater than 1 microM, the Kd shifted into the micromolar range, an effect that appeared to be pH-dependent. Importantly, the matrix [Ca2+] was determined to be between 10 and 100 nM at perfusion buffer [Ca2+] below 500 nM, but rose rapidly at the higher extramitochondrial [Ca2+] reported to occur in ischaemic cardiac myocytes. Importantly, mitochondrial transmembrane H+ and Ca2+ gradients both appeared to be maximal at perfusion buffer [H+] and [Ca2+] that approximate those of the cytosol of many resting cells.