Simultaneous measurement of Ca2+ in muscle with Ca electrodes and aequorin. Diffusible cytoplasmic constituent reduces Ca(2+)-independent luminescence of aequorin

Estimates of cytoplasmic Ca2+ concentration ([Ca2+]i) were made essentially simultaneously in the same intact frog skeletal muscle fibers with aequorin and with Ca-selective microelectrodes. In healthy fibers under truly resting conditions [Ca2+]i was too low to be measured reliably with either technique. The calibration curves for both indicators were essentially flat in this range of [Ca2+], and the aequorin light signal was uniformly below the level to be expected in the total absence of Ca2+. When [Ca2+]i had been raised to a stable level below the threshold for contracture by increasing [K+]o to 12.5 mM, [Ca2+]i was 38 nM according to aequorin and 59 nM according to the Ca-selective microelectrodes. These values are not significantly different. Our estimates of [Ca2+]i are lower than most others obtained with microelectrodes, probably because the presence of aequorin in the cells allowed us to detect damaging microelectrode impalements that otherwise we would have had no reason to reject. The observation that the light emission from aequorin-injected fibers in normal Ringer solution was below the level expected from the Ca(2+)-independent luminescence of aequorin in vitro was investigated further, with the conclusion that the myoplasm contains a diffusible macromolecule (between 10 and 30 kD) that interacts with aequorin to reduce light emission in the absence of Ca2+.     

Measurements of cytoplasmic free Ca2+ concentration in human pancreatic islets and insulinoma cells

In human pancreatic islets an increase in the glucose concentration from 3 to 20 mM raised the free cytoplasmic Ca2+ concentration [( Ca2+]i), an effect being reversible upon withdrawal of the sugar. Depolarization with a high concentration of K+ or the sulphonylurea tolbutamide also raised [Ca2+]i. Addition of extracellular ATP produced a transient rapid rise in [Ca2+]i. Oscillations in [Ca2+]i were observed in the presence of 10 mM glucose. Insulinoma cells responded to glucose and tolbutamide with increases in [Ca2+]i, whereas the sulphonamide diazoxide caused a decrease in [Ca2+]i. These findings confirm previous results obtained in rodent beta-cells.     

Measurement and manipulation of cytoplasmic free calcium of ram and boar spermatozoa using quin 2

The highly selective fluorescent Ca2+ indicator 'quin 2' has been loaded into ram and boar spermatozoa as the acetoxymethyl ester, 'quin 2/AM', which is hydrolysed and trapped in the cytoplasm. Loadings of several mM were not toxic to spermatozoa as judged by motility. Fluorescence measurements (mean +/- S.E.M.) indicated a normal cytoplasmic free-calcium concentration, [Ca2+]i, of 193 nM +/- 0.2 (n = 10) for ejaculated ram sperm, 175 nM +/- 3.9 (n = 10) for cauda epididymal boar sperm and 105 nM +/- 10 (n = 10) for the caput sperm. After cold shock ejaculated ram and cauda epididymal boar sperm did not retain quin 2, due presumably to structural damage. However, cold shocked caput boar sperm could be readily loaded with quin 2 and had a [Ca2+]i similar to control sperm. Sodium azide, propranolol and caffeine did not affect the [Ca2+]i of ram and boar sperm, however theophylline, dibutyryl c-AMP and La3+ significantly reduced it. The inhibitors rotenone and antimycin A, and the uncouplers 2,4-DNP and CCCP caused a transient elevation of [Ca2+]i, most likely resulting from release of mitochondrial calcium. The increased [Ca2+]i following addition of the ionophore A23187, was highly pH dependent in ram spermatozoa and it was critical to increase the pH of the medium above 7.5; the increase in [Ca2+]i was apparently not dependent on the oxidative metabolism of the sperm as addition of the uncouplers 2,4-DNP and CCCP had no effect on [Ca2+ )i. Addition of filipin to ram and boar sperm resulted in a large increase in [Ca2+]i but addition of filipin to ionophore-treated sperm caused [Ca2+]i to fall well below control levels.     

Dual-fluorescence flow cytometric analysis of membrane potential and cytoplasmic free Ca2+ concentration in embryonic rat hippocampal cells

We have demonstrated simultaneous measurement of the membrane potential and cytoplasmic free Ca2+ concentration ([Ca2+]i) by utilizing a dual-laser flow cytometer in embryonic rat hippocampal cell suspensions. Veratrine, a Na+ channel activator, induced both membrane depolarization and elevation of [Ca2+]i. These actions of veratrine were all reversed by the presence of tetrodotoxin (TTX). These findings suggest that Na+ channels are functionally expressed in the cells and the activation of Na+ channels increases [Ca2+]i. The usefulness of the flow cytometric analysis in elucidating the expression of membrane functions in the embryonic central nervous systems (CNS) is discussed.     

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.     

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.     

Synchronous oscillation of the cytoplasmic Ca2+ concentration and membrane potential in cultured epithelial cells (Intestine 407)

Cultured epithelial Intestine 407 cells exhibit regular oscillations of the membrane potential with repeated hyperpolarizations. These hyperpolarizations were inhibited not only by K+ channel blockers (tetraethylammonium and nonyltriethylammonium) but also by inhibitors of the Ca2+-activated K+ channel (quinine and quinidine). Using Ca2+-selective microelectrodes, cyclic increases in the cytosolic free Ca2+ concentration of more than 1 X 10(-6) M were found to coincide with the cyclic membrane hyperpolarizations. Thus, it appears that the potential oscillation is brought about by the oscillation of the intracellular free Ca2+ level which induces periodic activation of the Ca2+-dependent K+ channels. Neither the deprivation of extracellular Ca2+ nor the application of Ca2+ channel blockers (Co2+ and Ni2+) abolished the potential oscillation. Mitochondrial inhibitors (KCN, NaN3, antimycin A, FCCP and dinitrophenol) inhibited the potential oscillation, whereas glycolytic inhibitors (iodoacetic acid and NaF) had no effects. Caffeine and oxalate, which affect the microsomal Ca2+ transport, failed to exert any effect upon the potential oscillation. It is concluded that the cytosolic Ca2+ oscillation results from cyclic releases of Ca2+ from the intracellular storage site, which depends upon mitochondrial activities.     

Measurement of intracellular Ca2+ in cultured arterial smooth muscle cells using Fura-2 and digital imaging microscopy

A rise in cytosolic free Ca2+ is the immediate trigger for contraction in vascular smooth muscle (VSM). We employed the fluorescent Ca2(+)-indicator, Fura-2, and digital imaging microscopy to study the spatial distribution of intracellular Ca2+ in cultured A7r5 cells and the changes evoked by activation with 5-HT. Several methodological considerations that affect the temporal and spatial resolution of Ca2+ images have been addressed. These include: cytoplasmic distribution of Fura-2, wavelength selection for ratio imaging, signal:noise ratio measurement and the effect of [Ca2+] on the limits of detectability under conditions in which [Ca2+] is changing. The distribution of apparent free Ca2+, [Ca2+]App, in A7r5 cells was heterogeneous. This reflects, in part, different pools of intracellular Ca2+. [Ca2+]App was lowest in the nucleus (113 +/- 14 nM; n = 20 cells) and highest in the organelle-rich perinuclear region (228 +/- 12; n = 20), while the surrounding cytoplasmic area (containing relatively few organelles) had intermediate [Ca2+]app levels (150 +/- 13; n = 20). 5-HT (1 microM) evoked transient increases in [Ca2+]App that began within 11 s as relatively modest elevations of [Ca2+]App in the periphery, near the sarcolemma, and subsequently spread to the entire cell, reaching a peak within 18-24 s. At the peak of the Ca2+ transients, [Ca2+]App was highest in the perinuclear region where it sometimes exceeded the maximal detectable levels of the system (1.9 microM). The average peak Ca2+ transient amplitude in the non-nuclear cytoplasm was 1083 +/- 208 nM (1 microM 5-HT; n = 20 cells). Despite the continued presence of 5-HT following the Ca2+ transients, [Ca2+]App then returned to pre-stimulation levels within 5 min. These observations indicate that digital imaging microscopy enables the study of subcellular regulation of intracellular Ca2+ in VSM. The results provide new insights into the role of localized changes in Ca2+ in the regulation of VSM contractility.     

Cytosolic free Ca2+ concentration and intracellular calcium distribution of Ca2+-tolerant isolated heart cells

The cytosolic free Ca2+ concentration of calcium-tolerant rat myocytes has been measured by the null point titration technique using arsenazo III as a Ca2+ indicator and digitonin to permeabilize the plasma membrane. The mean value obtained for 8 separate preparations was 270 +/- 35 nM. The distribution of releasable calcium between the mitochondrial and sarcoplasmic reticular compartments was measured by the successive additions of uncoupler and A23187 to cells pretreated with ruthenium red. The relative distribution of calcium in each pool was independent of the cell calcium content up to the maximum value of releasable calcium investigated (4.5 nmol/mg of cell dry weight) and was distributed in the approximate ratio of 2:1 in favor of the sarcoplasmic reticulum. The cells contained 1 nmol of calcium/mg of cell dry weight in a form nonreleasable by A23187, which was independent of the total cell calcium content as measured by atomic absorption spectroscopy. It is calculated that the calcium content of mitochondria in heart under physiological conditions is about 5 nmol/mg of mitochondrial protein. At this level, the mitochondria are likely to provide effective buffering of the cytosolic free Ca2+ concentration of quiescent heart cells. The corresponding intramitochondrial free Ca2+ is in a range above values needed to regulate the activity of Ca2+-dependent enzymes of the citric acid cycle in heart. The physiological calcium content of the sarcoplasmic reticulum in heart cells is estimated to be about 2.5 nmol/mg of cell dry weight, which is at least 5-fold greater than the amount of calcium release calculated to cause maximum tension development of cardiac muscle.     

The regulation of the cytoplasmic free Ca2+ concentration in aortic smooth muscle cells (A7r5 line) after stimulation by vasopressin and bombesin

The characteristics of intracellular Ca2+ transient induced by vasopressin and bombesin in aortic smooth muscle cells were studied using flow cytometric analysis of indo-1 loaded cells. The two hormones induced a rapid and transient rise in [Ca2+]i. This Ca2+ transient was independent of the presence of extracellular Ca2+. Addition of bombesin to cells that have already been stimulated by vasopressin (or conversely the addition of vasopressin to bombesin-stimulated cells) results in a second Ca2+ transient that has a smaller amplitude. This transient is the same when the external Ca2+ concentration is lowered from 1.8 mM to 50 nM, suggesting that the agonist-sensitive pool reloaded using the Ca2+ that has been previously released into the cytoplasm. Intracellular Ca2+ pools that have been depleted by a prolonged incubation of the cells in a low Ca2+ medium can be refilled by shifting cells to a high Ca2+ medium. The reloading was analyzed in detail and found to be a slow process. It is hardly affected by Ni2+ or by (-)D888, a potent inhibitor of the voltage-dependent Ca2+ channels. It is accelerated when Ca2+ uptake by the Na+/Ca2+ exchange system is stimulated. The results suggest that Ca2+ homeostasis in aortic smooth muscle cells is achieved using mechanisms that are distinct from those operating in various acini and in striated muscles.     

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.     

Critical intracellular Ca2+ concentration for all-or-none Ca2+ spiking in single smooth muscle cells.

Neurotransmitters induce contractions of smooth muscle cells initially by mobilizing Ca2+ from intracellular Ca2+ stores through inositol 1,4,5-trisphosphate (InsP3) receptors. Here we studied roles of the molecules involved in Ca2+ mobilization in single smooth muscle cells. A slow rise in cytoplasmic Ca2+ ([Ca2+]i) in agonist-stimulated smooth muscle cells was followed by a wave of rapid regenerative Ca2+ release as the local [Ca2+]i reached a critical concentration of approximately 160 nM. Neither feedback regulation of phospholipase C nor caffeine-sensitive Ca(2+)-induced Ca2+ release was found to be required in the regenerative Ca2+ release. These results indicate that Ca(2+)-dependent feedback control of InsP3-induced Ca2+ release plays a dominant role in the generation of the regenerative Ca2+ release. The resulting Ca2+ release in a whole cell was an all-or-none event, i.e. constant peak [Ca2+]i was attained with agonist concentrations above the threshold value. This finding suggests a possible digital mode involved in the neural control of smooth muscle contraction.     

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     

Radial spread of aequorin Ca2+ signal in single frog skeletal muscle fibers

The Ca²⁺-sensitive photoprotein aequorin was injected into single frog skeletal muscle fibers, and the intracellular aequorin light intensity during muscle activation with different maneuvers was mapped with digital imaging microscopy. During 50 Hz electrical activation (tetanus), the aequorin light intensity from different locations in the muscle fiber rose with very similar time course. Caffeine (10 mM) application, on the other hand, caused aequorin light signals to show significantly different time courses, with an earlier increase in Ca²⁺ concentration near the surface of the fiber than near the core. The non-uniform rise of intracellular Ca²⁺ concentration with caffeine treatment is consistent with the slow inward diffusion of caffeine and subsequent Ca²⁺ release from sarcoplasmic reticulum.