The relationship between mitogen-induced changes in the cytoplasmic pH and free Ca2+ concentration in rat thymocytes

The relationship between pHi and [Ca]i signals generated in rat thymocytes by the mitogen Con A has been investigated. It is shown that the mitogen-induced [Ca]i rise is dependent on Na+/H+ exchange or some other Na(+)-sensitive process. This conclusion is based on the following findings: (i) [Ca]i response to Con A weakens upon decreasing the concentration of extracellular Na+, or inhibiting Na+/H+ exchange; (ii) agents that alkalinize the cytoplasm (the phorbol ester TPA, the Na+/H+ ionophore monensin and NH4Cl) cause an increase in [Ca]i (Klip, A., Rothstein, A. and Mack, E. (1984) Biochem. Biophys. Res. Commun. 124, 14-22; Grinstein, S. and Goetz, J.D. (1985) Biochim. Biophys. Acta 819, 267-270); (iii) The effects of Con A, TPA and monensin on [Ca]i are not additive. The last observation suggests that all these agents activate the same Na+/H+ (Na+ and/or H+)-dependent system of Ca2+ transport. It is found that the pH i and [Ca]i responses in rat thymocytes are sensitive to changes in the intracellular levels of cyclic nucleotides, ATP and in temperature. These regulatory effects on the ionic signals are different for Con A, TPA and monensin. In particular, both the stimulation of Na+/H+ antiport and the [Ca]i rise brought about by Con A or TPA are inhibited upon elevating the cellular cAMP. In contrast, the monensin-induced [Ca]i signal is almost independent of cAMP but is highly sensitive to changes in cGMP and temperature. Reducing the ATP level eliminates both the pHi and [Ca]i responses to Con A but not to monensin. These different characteristics of [Ca]i signals elicited by the mitogen and the Na+/H+ ionophore indicate that these agents use different mechanisms to activate the Na+/H(+)-dependent Ca2+ transporting system. A [Ca]i response to monensin has been obtained in some other cell types, namely, in lymphoblastoid Raji cells, Ehrlich ascites tumor cells and also in platelets.     

The relationship between the free concentrations of Ca2+ and Ca2+-calmodulin in intact cells

Using stably expressed fluorescent indicator proteins, we have determined for the first time the relationship between the free Ca2+ and Ca2+-calmodulin concentrations in intact cells. A similar relationship is obtained when the free Ca2+ concentration is externally buffered or when it is transiently increased in response to a Ca2+-mobilizing agonist. Below a free Ca2+ concentration of 0.2 microM, no Ca2+-calmodulin is detectable. A global maximum free Ca2+-calmodulin concentration of approximately 45 nM is produced when the free Ca2+ concentration exceeds 3 microM, and a half-maximal concentration is produced at a free Ca2+ concentration of 1 microM. Data for fractional saturation of the indicators suggest that the total concentration of calmodulin-binding proteins is approximately 2-fold higher than the total calmodulin concentration. We conclude that high-affinity calmodulin targets (Kd /= 100 nM) occurs only where free Ca2+-calmodulin concentrations can be locally enhanced.     

Rapid kinetics of agonist-evoked changes in cytosolic free Ca2+ concentration in fura-2-loaded human neutrophils.

The initial kinetics of agonist-evoked rises in the cytosolic Ca2+ concentration [Ca2+]i were investigated in fura-2-loaded human neutrophils by stopped-flow fluorimetry. The rises in [Ca2+]i evoked by chemotactic peptide (fMet-Leu-Phe), platelet-activating factor and ADP all lagged behind agonist addition by 1-1.3 s. Lag times were not significantly different in the presence and in the absence of external Ca2+. Stimulation of the cells in the presence of extracellular Mn2+ resulted in a quench of fluorescence with a similar lag time to [Ca2+]i rise. The delay in onset of the rise in [Ca2+]i evoked by fMet-Leu-Phe was dependent on concentration, becoming longer at lower concentrations of agonist. These results indicate that both the agonist-evoked discharge of the intracellular Ca2+ stores and the generation of bivalent-cation influx lag behind agonist-receptor binding in neutrophils. Both pathways thus appear to be mediated by indirect mechanisms, rather than by a directly coupled process such as a receptor-operated channel. The temporal coincidence of the onset of store discharge with the commencement of bivalent-cation influx suggests that the two events may be causally linked.     

Resting cytoplasmic free Ca2+ concentration in frog skeletal muscle measured with fura-2 conjugated to high molecular weight dextran

Intact frog skeletal muscle fibers were injected with the Ca2+ indicator fura-2 conjugated to high molecular weight dextran (fura dextran, MW approximately 10,000; dissociation constant for Ca2+, 0.52 microM), and the fluorescence was measured from cytoplasm (17 degrees C). The fluorescence excitation spectrum of fura dextran measured in resting fibers was slightly red-shifted compared with the spectrum of the Ca(2+)-free indicator in buffer solutions. A simple comparison of the spectra in the cytoplasm and the in vitro solutions indicates an apparently "negative" cytoplasmic [Ca2+], which probably reflects an alteration of the indicator properties in the cytoplasm. To calibrate the indicator's fluorescence signal in terms of cytoplasmic [Ca2+], we applied beta-escin to permeabilize the cell membrane of the fibers injected with fura dextran. After treatment with 5 microM beta-escin for 30-35 min, the cell membrane was permeable to small molecules (e.g., Ca2+, ATP), whereas the 10-kD fura dextran only slowly leaked out of the fiber. It was thus possible to estimate calibration parameters in the indicator fluorescence in the fibers by changing the bathing solution [Ca2+] to various levels; the average values for the fraction of Ca(2+)-bound indicator in the resting fibers and the dissociation constant for Ca2+ (KD) were, respectively, 0.052 and 1.0 microM. For the comparison, the KD value was also estimated by a kinetic analysis of the indicator fluorescence change after an action potential stimulation in intact muscle fibers, and the average value was 2.5 microM. From these values estimated in the fibers, resting cytoplasmic [Ca2+] in frog skeletal muscle fibers was calculated to be 0.06-0.14 microM. The range lies between the high estimates from other tetracarboxylate indicators (0.1-0.3 microM; Kurebayashi, N., A. B. Harkins, and S. M. Baylor. 1993. Biophysical Journal. 64:1934-1960; Harkins, A. B., N. Kurebayashi, and S. M. Baylor. 1993. Biophysical Journal. 65:865-881) and the low estimate from the simultaneous use of aequorin and Ca(2+)-sensitive microelectrodes (< 0.04-0.06 microM; Blatter, L. A., and J. R. Blinks. 1991. Journal of General Physiology. 98:1141-1160) recently reported for resting cytoplasmic [Ca2+] in frog muscle fibers.     

Fluorescence measurements of free Ca2+ concentration in human erythrocytes using the Ca2+-indicator fura-2

We report here the use of the fluorescent Ca2+-chelator fura-2 to directly measure free Ca2+ concentration within intact human erythrocytes and the influence of viscosity on the fluorescence of this probe. The bright fluorescence of fura-2 has permitted the use of low concentrations of indicator and cells, thus minimizing the screening effect and the intrinsic fluorescence of haemoglobin. Erythrocytes (10(8) cells/ml) were loaded with 0.5 microM fura-2AM then diluted at 10(7) cells per ml for measurements. The extracellular signal was suppressed by addition of manganese ions just before recording spectra. Under these conditions, a blood sample of 100 microliter was sufficient for analysis. To study the influence of viscosity on fura-2 fluorescence, gelatin and polyvinylpyrrolidone at various concentrations were added to a physiological buffer to perform fura-2-Ca fluorescence standard curves. Fluorescence intensities and the apparent affinity constant for Ca2+ were modified by viscosity. When intra-erythrocytic viscosity was simulated with 21 g/l polyvinylpyrrolidone to obtain a mean viscosity of 14 mPa.s similar to that observed in human erythrocytes, the mean value of free Ca2+ concentration measured in erythrocytes from healthy subjects was 78 +/- 16 nM (mean +/- S.D., n = 29).     

Turgor regulation and cytoplasmic free Ca2+ in the algaLamprothamnium

Summary In internodal cells ofLamprothamnium succinctum, turgor regulation in response to hypotonie treatment is inhibited by lowering external Ca²⁺ concentration ([Ca²⁺]_e) from 3.9 (normal) to 0.01 (low) mM. In order to clarify whether a change in the cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_c) is involved in turgor regulation, the Ca²⁺ sensitive protein aequorin was injected into the cytoplasm of internodal cells. A large transient light emission was observed upon hypotonic treatment under normal [Ca²⁺]_e but not under low [Ca²⁺]_e. Thus hypotonic treatment induces a transient increase in [Ca²⁺]_c under normal [Ca²⁺]_e but not under low [Ca²⁺]_e.Abbreviations ASW artificial sea water - _i cellular osmotic pressure - [Ca²⁺]_c cytoplasmic free Ca²⁺ concentration - EDTA ethylenediamine-tetraacetic acid - EGTA ethylenglycol-bis(-aminoethyl ether(N,N-tetraacetic acid - [Ca²⁺]_e external Ca²⁺ concentration - _e external osmotic pressure - GM glass micropipette - GP glass plate - HEPES N-2-hydroxyethylpiperazine-N-2-ethansulfonic acid - MS microscope stage - OL objective lens - PIPES piperazine-N-N-bis(2-ethanesulfonic acid) - W Weight     

Studies on mitochondrial Ca2+-transport and matrix Ca2+ using fura-2-loaded rat heart mitochondria

Rat heart mitochondria were incubated for 5 min at 30 degrees C and at approx. 40 mg protein.ml-1 and in the presence of 10 microM fura-2/AM. This allowed the entrapment of free fura-2 within the mitochondrial matrix and its use as a probe for Ca2+, but without affecting the apparent viability of the mitochondria. Parallel measurements of the activities of the intramitochondrial Ca2+-sensitive enzymes, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase, allowed an assessment of their sensitivity to measured free Ca2+ within intact mitochondria incubated under different conditions; the enzymes responded to matrix Ca2+ over the approximate range 0.02-2 microM with half-maximal effects at about 0.3-0.6 microM Ca2+. Effectors of Ca2+-transport across the inner membrane (e.g., Na+, Mg2+, Ruthenium red, spermine) did not appear to affect these ranges, but did bring about expected changes in Ca2+ distribution across this membrane. Significantly, when mitochondria were incubated in the presence of physiological concentrations of both Na+ and Mg2+, and at low extramitochondrial Ca2+ (less than 400 nM), there was a gradient of Ca2+ (in:out) of less than unity; at higher extramitochondrial [Ca2+] (but still within the physiological range) the gradient was greater than unity indicating a highly cooperative nature of transmission of the Ca2+ signal into the matrix under such conditions.     

Effect of extracellular Ca2+ on plasma membrane Ca2+ inflow and cytoplasmic free Ca2+ in isolated hepatocytes

An initial rapid phase and a subsequent slow phase of 45Ca2+ uptake were observed following the addition of 45Ca2+ to Ca2+-deprived hepatocytes. The magnitude of the rapid phase increased 15-fold over the range 0.1-11 mM extracellular Ca2+ (Ca2+o) and was a linear function of [Ca2+]o. The increases in the rate of 45Ca2+ uptake were accompanied by only small increases in the intracellular free Ca2+ concentration. In cells made permeable to Ca2+ by treatment with saponin, the rate of 45Ca2+ uptake (measured at free Ca2+ concentrations equal to those in the cytoplasm of intact cells) increased as the concentration of saponin increased from 1.4 to 2.5 micrograms per mg wet weight cells. Rates of 45Ca2+ uptake by cells permeabilized with an optimal concentration of saponin were comparable with those of intact cells incubated at physiological [Ca2+o], but were substantially lower than those for intact cells incubated at high [Ca2+o]. It is concluded that Ca2+ which enters the hepatocyte across the plasma membrane is rapidly removed by binding and transport to intracellular sites and by the plasma membrane (Ca2+ + Mg2+)-ATPase and the plasma membrane Ca2+ inflow transporter is not readily saturated with Ca2+o.     

The relationship between depletion of intracellular Ca2+ stores and activation of Ca2+ current by muscarinic receptors in neuroblastoma cells

The relationship between the depletion of IP3-releasable intracellular Ca2+ stores and the activation of Ca(2+)-selective membrane current was determined during the stimulation of M1 muscarinic receptors in N1E-115 neuroblastoma cells. External Ca2+ is required for refilling Ca2+ stores and the voltage-independent, receptor-regulated Ca2+ current represents a significant Ca2+ source for refilling. The time course of Ca2+ store depletion was measured with fura-2 fluorescence imaging, and it was compared with the time course of Ca2+ current activation measured with nystatin patch voltage clamp. At the time of maximum current density (0.18 + .03 pA/pF; n = 48), the Ca2+ content of the IP3- releasable Ca2+ pool is reduced to 39 + 3% (n = 10) of its resting value. Calcium stores deplete rapidly, reaching a minimum Ca2+ content in 15-30 s. The activation of Ca2+ current is delayed by 10-15 s after the beginning of Ca2+ release and continues to gradually increase for nearly 60 s, long after Ca2+ release has peaked and subsided. The delay in the appearance of the current is consistent with the idea that the production and accumulation of a second messenger is the rate-limiting step in current activation. The time course of Ca2+ store depletion was also measured after adding thapsigargin to block intracellular Ca2+ ATPase. After 15 min in thapsigargin, IP3-releasable Ca2+ stores are depleted by > 90% and the Ca2+ current is maximal (0.19 + 0.05 pA/pF; n = 6). Intracellular loading with the Ca2+ buffer EGTA/AM (10 microM; 30 min) depletes IP3-releasable Ca2+ stores by between 25 and 50%, and it activates a voltage-independent inward current with properties similar to the current activated by agonist or thapsigargin. The current density after EGTA/AM loading (0.61 + 0.32 pA/pF; n = 4) is three times greater than the current density in response to agonist or thapsigargin. This could result from partial removal of Ca(2+)- dependent inactivation.     

Measurement of the matrix free Ca2+ concentration in heart mitochondria by entrapped fura-2 and quin2.

A method was developed to monitor continuously the matrix free Ca2+ concentration ([Ca2+]m) of heart mitochondria by use of the fluorescent Ca2+ indicators, fura-2 and quin2. The acetoxymethyl esters of fura-2 and quin2 were accumulated in and hydrolysed by isolated mitochondria. An increase of the mitochondrial Ca content from 0.3 nmol/mg of protein to 6 nmol/mg corresponded to a rise of [Ca2+]m from 30 to 1000 nM. The results indicate that physiological fluctuations of the mitochondrial Ca content elicit changes of [Ca2+]m in that range which regulates the matrix dehydrogenases.     

Parallel measurement of oxoglutarate dehydrogenase activity and matrix free Ca2+ in fura-2-loaded heart mitochondria

The entrapment of the Ca2+-sensitive fluorescence indicators fura-2 or quin2 in the matrix space of isolated heart mitochondria renders possible the direct monitoring of the matrix free Ca2+ [( Ca2+]m) [(1987) Biochem J. 248, 609-613]. In this paper the correlation between the [Ca2+]m and the in situ activity of oxoglutarate dehydrogenase (OGDH) in fura-2-loaded mitochondria is shown. At the initial value of [Ca2+]m, 64 nM, which corresponded to 0.36 nmol/mg mitochondrial Ca content, the OGDH activity was 12% of the maximal. Half-maximal and maximal activation were attained at 0.8 and 1.6 microM [Ca2+]m, respectively. The results indicate that an increase of the mitochondrial Ca content in the physiological range enhances the OGDH activity by means of elevation of [Ca2+]m.     

Unusual sensitivity of cytosolic free Ca2+ to changes in extracellular Ca2+ in rat C-cells

An essential function of C-cells is to monitor extracellular Ca2+ concentration ([Ca2+]e) and to respond to changes in [Ca2+]e by regulating hormone secretion. Using the calcitonin-secreting rat C-cell line rMTC 44-2, we have investigated a possible tight linkage between [Ca2+]e and cytosolic free Ca2+ ([Ca/+]i). We have demonstrated, using the Ca2+ indicator Quin 2, that the [Ca2+]i is particularly sensitive to changes in [Ca2+]e. Sequential increases in [Ca2+]e as small as 0.1 mM evoke clear elevations in [Ca2+]i. In contrast, other cell types tested did not alter their [Ca2+]i in response to increasing [Ca2+]e even to levels as high as 4.0 mM. Sequential 1.0 mM increments in [Ca2+]e caused the [Ca2+]i to rise from a base line of 357 +/- 20 nM Ca2+i at 1.0 mM Ca2+e to a maximum of 1066 +/- 149 nM Ca2+i at 5.0 mM Ca2+e. [Ca2+]e above 2.0 mM produced a biphasic response in [Ca2+]i consisting of an immediate (less than 5 s) spike followed by a decay to a new plateau. Treatment of rMTC 44-2 cells with either 50 mM K+ or 100 nM ionomycin at 1.0 mM Ca2+e caused an immediate spike in [Ca2+]i to micromolar levels. Pretreatment with EGTA or verapamil inhibited completely the increase in [Ca2+]i induced by 50 mM K+. However, pretreatment with EGTA only slightly attenuated the spike phase in [Ca2+]i produced by ionomycin, demonstrating that ionomycin released intracellular stores of calcium. We conclude that rMTC 44-2 cells regulate [Ca2+]i by monitoring small physiological changes in [Ca2+]e, the primary secretagogue for C-cells.     

The T-cell antigen receptor regulates sustained increases in cytoplasmic free Ca2+ through extracellular Ca2+ influx and ongoing intracellular Ca2+ mobilization.

Signal transduction by the T-cell antigen receptor involves the turnover of polyphosphoinositides and an increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). This increase in [Ca2+]i is due initially to the release of Ca2+ from intracellular stores, but is sustained by the influx of extracellular Ca2+. To examine the regulation of sustained antigen-receptor-mediated increases in [Ca2+]i, we studied the relationships between extracellular Ca2+ influx, the mobilization of Ca2+ from intracellular stores, and the contents of inositol polyphosphates after stimulation of the antigen receptor on a human T-cell line, Jurkat. We demonstrate that sustained antigen-receptor-mediated increases in [Ca2+]i are associated with ongoing depletion of intracellular Ca2+ stores. When antigen-receptor-ligand interactions are disrupted, [Ca2+]i and inositol 1,4,5-trisphosphate return to basal values over 3 min. Under these conditions, intracellular Ca2+ stores are repleted if extracellular Ca2+ is present. There is a tight temporal relationship between the fall in [Ca2+]i, the return of inositol 1,4,5-trisphosphate to basal values, and the repletion of intracellular Ca2+ stores. Reversal of the increase in [Ca2+]i preceeds any fall in inositol tetrakisphosphate by 2 min. These studies suggest that sustained antigen-receptor-induced increases in [Ca2+]i, although dependent on extracellular Ca2+ influx, are also regulated by ongoing inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ mobilization. In addition, an elevated concentration of inositol tetrakisphosphate in itself is insufficient to sustain an increase in [Ca2+]i within Jurkat cells.     

The relationship between extracellular K+ and Ca2+ on aminopyrine accumulation in rat parietal cells.

The effects of extracellular K+ in relation to extracellular Ca2+ on acid production were studied. Studies were performed in vitro using isolated cells from rat stomachs, and acid production was indirectly determined by 14C-aminopyrine (AP) accumulation. In the absence of K+ in the incubation medium histamine-stimulated AP accumulation ratios were significantly decreased independently in the presence or absence of extracellular Ca2+. Under basal conditions, in the absence of extracellular Ca2+, increasing concentrations of extracellular K+ enhanced AP accumulation ratios to significantly higher than those found in the presence of Ca2+. In histamine-, cAMP-, and carbachol-stimulated parietal cells, high K+ concentrations increased AP accumulation significantly less in Ca(2+)-free than in Ca(2+)-containing media. High K+ also induced significantly both an increase in cytosolic free Ca2+ concentration and 45Ca2+ uptake. The present results confirmed the importance of K+ in gastric acid production and suggested a role for Ca2+ as a modulator of mechanisms of parietal cell stimulation.     

The spatial relationship between Ca2+ channels and Ca2+-activated channels and the function of Ca2+-buffering in avian sensory neurons

In order to learn about the endogenous Ca2+-buffering in the cytoplasm of chick dorsal root ganglion (DRG) neurons and the distance separating the ryanodine receptor Ca2+ release channels (RyRs) from the plasma membrane, we monitored the amplitude and time course of Ca2+-activated Cl- currents (I(ClCa)) in protocols that manipulated Ca2+-buffering. I(ClCa)was activated by Ca2+ influx via voltage-gated Ca2+ channels or by Ca2+ release via RyRs activated by 10 mM caffeine. I(ClCa)was measured in neurons at 20 degrees C and 35 degrees C using the amphotericin perforated patch technique that preserves endogenous Ca2+-buffering, or at 20 degrees C in neurons dialyzed with pipette solutions designed to replace the endogenous Ca2+ buffers. The amplitude of I(ClCa)activated by Ca2+ influx or Ca2+ at 20 degrees C was similar in the amphotericin neurons and neurons dialyzed with an 'unbuffered' pipette solution containing 10 mM citrate and 3 mM ATP as the only Ca2+ binding molecules. Thus, endogenous mobile Ca2+ buffers are relatively unimportant in chick DRG neurons. Warming the neurons from 20 degrees C to 35 degrees C increased the amplitude and the rate of deactivation of I(ClCa)consistent with an increased rate of Ca2+ buffering by fixed endogenous Ca2+-buffers. Dialysis with 2 mM EGTA/0.1 microM free Ca2+ reduced the amplitude and increased the rate of deactivation of I(ClCa)activated by Ca2+ influx and abolished I(ClCa)activated by Ca2+ release. Dialysis with 2 mM BAPTA/0.1 microM free Ca2+ abolished I(ClCa)activated by Ca2+ influx or release. Dialysis with 42 mM HEEDTA/0.5 microM free Ca2+ caused the persistent activation of I(ClCa). Calculations using a Ca2+-diffusion model suggest that the voltage-gated Ca2+ channels and the Ca2+-activated Cl- channels are separated by 50-400 nm and that the RyRs are more than 600 nm from the plasma membrane.     

Coelenterate photoproteins as indicators of cytoplasmic free Ca2+ in small cells

The Ca2+-activated photoproteins aequorin and obelin are capable of detecting rapid changes in free Ca2+ over the range 10nM-100uM. Whilst they have been used to quantify free Ca transients in giant cells for some time, their use in small mammalian cells has been restricted because of the difficulty of incorporating them into live cells without impairment of cell function. We have developed three methods for incorporating photoproteins into small cells (a) reversible cell swelling (b) membrane fusion and (c) intracellular release from pinocytotic vesicles. Formation of the membrane attack complex of complement (C5b6789), via a specific cell surface antibody to activate complement, causes a rapid increase in cytoplasmic Ca2+ detectable within 5-10 s. It provides a specific method for quantifying cytoplasmic photoprotein. As a result new insights into the role of intracellular Ca2+ in cell physiology and pathology have been established.     

Association of cytoplasmic free Ca2+ gradients with subcellular organelles.

Previous investigations have identified gradients of intracellular free (Ca2+)i (Ca2+i) in the cytoplasm of human fibroblasts. In this study we have compared the spatial distribution of these gradients with the subcellular distribution of cytoplasmic organelles. Using the Ca(2+)-sensitive dye fura-2 and organelle-specific fluorescent dyes, we have found that the highest Ca2+ concentrations are found in the perinuclear cytoplasm and that these regions co-localize with the Golgi apparatus. The area occupied by the endoplasmic reticulum, which includes the Golgi region plus an adjacent area, is also significantly elevated above the average cellular (Ca2+)i. Most mitochondria are located in regions different from those with the highest (Ca2+)i. A variety of phenomena which could have given rise to artifactual (Ca2+)i gradients have been ruled out, including compartmentalization of fura-2 in subcellular organelles, incomplete hydrolysis of fura-2AM esters, and the presence of pH gradients which might change the Ca2+ binding characteristics of fura-2. The existence of gradients in (Ca2+)i between ER and Golgi containing regions of the cytoplasm supports the hypothesis (Sambrook: Cell 61:197-199, 1990) that the traffic of membrane bound vesicles from ER to Golgi is directed by local variations in (Ca2+)i.