The Influence of Ca2+ Buffers on Free [Ca2+] Fluctuations and the Effective Volume of Ca2+ Microdomains

Intracellular calcium (Ca²⁺) plays a significant role in many cell signaling pathways, some of which are localized to spatially restricted microdomains. Ca²⁺ binding proteins (Ca²⁺ buffers) play an important role in regulating Ca²⁺ concentration ([Ca²⁺]). Buffers typically slow [Ca²⁺] temporal dynamics and increase the effective volume of Ca²⁺ domains. Because fluctuations in [Ca²⁺] decrease in proportion to the square-root of a domain’s physical volume, one might conjecture that buffers decrease [Ca²⁺] fluctuations and, consequently, mitigate the significance of small domain volume concerning Ca²⁺ signaling. We test this hypothesis through mathematical and computational analysis of idealized buffer-containing domains and their stochastic dynamics during free Ca²⁺ influx with passive exchange of both Ca²⁺ and buffer with bulk concentrations. We derive Langevin equations for the fluctuating dynamics of Ca²⁺ and buffer and use these stochastic differential equations to determine the magnitude of [Ca²⁺] fluctuations for different buffer parameters (e.g., dissociation constant and concentration). In marked contrast to expectations based on a naive application of the principle of effective volume as employed in deterministic models of Ca²⁺ signaling, we find that mobile and rapid buffers typically increase the magnitude of domain [Ca²⁺] fluctuations during periods of Ca²⁺ influx, whereas stationary (immobile) Ca²⁺ buffers do not. Also contrary to expectations, we find that in the absence of Ca²⁺ influx, buffers influence the temporal characteristics, but not the magnitude, of [Ca²⁺] fluctuations. We derive an analytical formula describing the influence of rapid Ca²⁺ buffers on [Ca²⁺] fluctuations and, importantly, identify the stochastic analog of (deterministic) effective domain volume. Our results demonstrate that Ca²⁺ buffers alter the dynamics of [Ca²⁺] fluctuations in a nonintuitive manner. The finding that Ca²⁺ buffers do not suppress intrinsic domain [Ca²⁺] fluctuations raises the intriguing question of whether or not [Ca²⁺] fluctuations are a physiologically significant aspect of local Ca²⁺ signaling.     

Mitochondrial Free [Ca] Increases during ATP/ADP Antiport and ADP Phosphorylation: Exploration of Mechanisms

ADP influx and ADP phosphorylation may alter mitochondrial free [Ca²⁺] ([Ca²⁺]_m) and consequently mitochondrial bioenergetics by several postulated mechanisms. We tested how [Ca²⁺]_m is affected by H₂PO₄⁻ (P_i), Mg²⁺, calcium uniporter activity, matrix volume changes, and the bioenergetic state. We measured [Ca²⁺]_m, membrane potential, redox state, matrix volume, pH_m, and O₂ consumption in guinea pig heart mitochondria with or without ruthenium red, carboxyatractyloside, or oligomycin, and at several levels of Mg²⁺ and P_i. Energized mitochondria showed a dose-dependent increase in [Ca²⁺]_m after adding CaCl₂ equivalent to 20, 114, and 485 nM extramatrix free [Ca²⁺] ([Ca²⁺]_e); this uptake was attenuated at higher buffer Mg²⁺. Adding ADP transiently increased [Ca²⁺]_m up to twofold. The ADP effect on increasing [Ca²⁺]_m could be partially attributed to matrix contraction, but was little affected by ruthenium red or changes in Mg²⁺ or P_i. Oligomycin largely reduced the increase in [Ca²⁺]_m by ADP compared to control, and [Ca²⁺]_m did not return to baseline. Carboxyatractyloside prevented the ADP-induced [Ca²⁺]_m increase. Adding CaCl₂ had no effect on bioenergetics, except for a small increase in state 2 and state 4 respiration at 485 nM [Ca²⁺]_e. These data suggest that matrix ADP influx and subsequent phosphorylation increase [Ca²⁺]_m largely due to the interaction of matrix Ca²⁺ with ATP, ADP, P_i, and cation buffering proteins in the matrix.     

Glucose and lactate regulate maitotoxin-activated Ca entry in spermatogenic cells: The role of intracellular [Ca]

Maitotoxin (MTX), a potent polyether marine biotoxin, induces Ca²⁺ entry in different mammalian cells by activation of Ca²⁺ channels. The identity and modulation of the MTX-activated Ca²⁺ entry pathway is not known. In this work, we show, for the first time, that glucose and lactate can modulate the excitability of spermatogenic cell MTX-activated Ca²⁺ channels. Physiological and pharmacological evidences indicate that glucose and lactate differentially affect MTX-activated Ca²⁺ entry mainly through changes that these substrates induce on intracellular Ca²⁺ stores and the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) in spermatogenic cells. Our findings strongly suggest that MTX-activated Ca²⁺ channels in spermatogenic cells can be regulated by a Ca²⁺-CaM-dependent protein kinase.     

The effect of [Ca]o on the resting potential in newborn and adult rat ventricular muscles

Microelectrode studies were carried out to examine the effect of varied [Ca]o on resting potentials of newborn and adult rat ventricular muscles. The ratio of membrane permeabilities to sodium and potassium (PNa/PK) estimated from the constant-field equation was about 0.03 in 2 mM [Ca]o in both newborn and adult preparations. Membrane hyperpolarization was observed in adult preparations when [Ca]o was increased from 1 mM to 10 mM, whereas the resting potential of newborn preparations was little hyperpolarized. In 10 mM [Ca]o, PNa/PK was 0.01 in adult and was still 0.03 in newborn. The hyperpolarization observed in adult tissue was reduced in low [Na]o, which is inconsistent with the constant-field equation. A possible mechanism of this hyperpolarization is discussed.     

Influence of polyhydroxysteroids on [Ca(2+)](i)

Recently, we have shown that two biologically active, disulfated polyhydroxysteroids from the Pacific brittle star Ophiopholis aculeata stimulate Ca(2+) influx into different cell types. In the present study, 45Ca(2+) and two fluorescent calcium probes, quin-2/AM and fura-2/AM, were employed to investigate the course and amplitude of calcium signals induced in different mouse cells using an radio-isotope, spectrofluorimetry, and microcytofluorimetry techniques. The cytotoxic and hemolytic effects were not observed for both steroids at the wide range of concentrations. Steroids did not influence [3H]-uridine incorporation in a variety of cells. The investigated steroids stimulated a rapid increase in cytosolic Ca(2+) content in Ehrlich mouse carcinoma cells, mouse spleen lymphocytes, and mouse peritoneal macrophages in the concentration range 1-100 microg/ml on a dose-dependent basis. Blockers of L-type calcium channels, such as verapamil, diltiazem, nifedipine (1 x 10(-7)M), and 1mM EGTA, inhibited this process and reduced the response of cells to steroid application. The stimulatory effect of steroids on human fibroblast proliferation and mouse macrophage lysosome activity was observed also. It is suggested that the investigated compounds may act as Ca(2+)-agonists and increase Ca(2+)-transport across cell membranes.     

Influence of Ca2+ channel modulators on [3H]purine release from rat cultured glial cells

[³H]Purine release from rat striatum astrocyte cultures was studied at 14 days in vitro (DIV). Superfusion of cultures with a Ca²⁺-free medium +0.5 mM ethylene glycol-bis(-aminoethylether)N,N,N,N-tetracetic acid (EGTA) reduced the electrically evoked [³H]purine release. Nimodipine only at the concentration of 10 M modified [³H]purine outflow whereas 0.1 M -conotoxin and 0.03–0.1 M nitrendipine reduced the evoked one. Superfusion of cultures with 0.1 M -conotoxin +0.1 M nitrendipine antagonized the evoked [³H]purine release similarly to each drug given alone. Neither nitrendipine nor -conotoxin influenced the uptake of⁴⁵Ca²⁺ by the cultures. The treatment of cells with the Ca²⁺ agonist Bay K 8644 did not affect [³H]purine release or the⁴⁵Ca²⁺ uptake. The drug did not either alter [Ca²⁺]_i, evaluated by loading the cells with 3 M Fura-2/AM. 10–30 M 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), a blocker of intracellular Ca²⁺ discharge, significantly reduced the evoked [³H]purine release. On the other hand, 2 M thapsigargin, an inhibitor of the ion store Ca²⁺ ATPase, was able to increase either the culture [³H]purine release or the [Ca²⁺]_i. Together, the findings indicate that voltage-sensitive calcium channels (VSCC_s) of the neuronal N and L-types are not involved in the modulation of [³H]purine release from rat cultured astrocytes whereas Ca²⁺ coming from intracytoplasmic stores seems to play a prevailing role. Moreover, agents which block VSCC_s seem to be able to affect [³H]purine outflow with mechanisms other than VSCC gating.     

The effect of insulin on the washout of [45Ca]calcium from adipocytes and soleus muscle of the rat.

Insulin stimulates the washout of 45Ca from preloaded isolated fat-cells, whole epididymal fat-pads and isolated soleus muscles of the rat. This effect occurs within 10 min after the addition of the hormone, and it can be detected at concentrations down to those measured in rat plasma. When K+ is omitted from the washout medium, the effect on soleus muscles is more pronounced and increases with the time of exposure.     

Effective reconstitution of sarcoplasmic reticulum Ca2+-ATPase using lubrol PX]

Ca2(+)-ATPase of sarcoplasmic reticulum was reconstituted in the proteoliposomes by the salting out procedure. Triton X-100, C12E8 and Lubrol PX were used for the solubilization of the Ca2(+)-ATPase. Using fluorescent probes (diS-C3-(5), chlortetracycline) as well pH-measuring method, the functional of the reconstituted Ca2(+)-ATPase was comparatively studied in three types of proteoliposomes. The efficiency of Ca2(+)-ATPase grew in the following detergent order: Triton X-100, C12E8, Lubrol PX.     

Assessment of intra-SR free [Ca] and buffering in rat heart.

To measure the free intrasarcoplasmic reticulum [Ca] ([Ca]SR) in isolated rat cardiac microsomes, ventricular tissue was homogenized in the presence of the low-affinity Ca indicator furaptra. Stepwise increases in cuvette [Ca] ([Ca]c) in the presence of ATP caused progressive increases in steady-state intravesicular fluorescence ratio to a maximum (Rmax). Steady-state [Ca]SR/[Ca]c was approximately 7000. Therefore the resting [Ca]SR may approach 700 microM in the rat cardiac myocyte at [Ca]c = 100 nM. The sarcoplasmic reticulum (SR) Ca pump requires a free energy of deltaG approximately 44 kJ x mol(-1) to generate this [Ca] gradient (e.g., approximately 74% of deltaG(ATP)). Total SR 45Ca uptake was also measured in digitonin-permeabilized myocytes as a function of [Ca]c in the absence of precipitating ions. The steady-state SR Ca content at 100 nM [Ca]c was approximately 400 micromol/liter cytosolic volume. Used together, these data allowed evaluation of the in situ SR Ca-buffering properties. The SR Ca-binding site concentration was approximately 14 mM, and Kd(Ca) approximately 0.638 mM [Ca]SR.     

A [Ca + Mg]-ATPase and active Ca transport in the plasma membranes isolated from ram sperm flagella

Plasma membranes isolated from the flagella of ram ejaculated sperm were found to contain a [Ca²⁺ + Mg²⁺]-ATPase. Freeze-fracture electron microscopy showed the membranes occur as vesicles. The membrane vesicles actively accumulate Ca²⁺, uptake was reversed by the ionophore A23187 and inhibited by either ruthenium red or La³⁺. The plasma membranes contain two major proteins, designated proteins A and B, with molecular weights of 109,000 and 18,300 daltons, respectively. Protein B is not detected in plasma membranes isolated from ram epididymal sperm. The plasma membrane Ca²⁺ pump may be modulated by protein factors present in seminal plasma.     

Influence of Seiche-Generated Light Field Fluctuations on Phytoplankton Growth

Phytoplankton, owing to the periodic ascent and descent caused by internal seiches, is exposed to experiential fluctuations in light intensity. The increase in light intensity associated with its ascent towards the surface is greater than the decrease caused by its descent. A simple mathematical model is used to derive relationships showing that 1) the mean light intensity in an epilimnion influenced by internal waves is not more than 6.1% lower than in a stationary system and 2) in the meta- and hypolimnion of lakes of low transparency (i.e. with a high extinction coefficient) internal seiches of large amplitude increase the mean light intensity by several orders of magnitude. This can produce a many-fold increase in the depth of the euphotic zone. The mean rates of photosynthesis of phytoplankton in the meta- and hypolimnion increase particularly at the antinodes of the waves. Internal seiches evidently may play an important role in causing plankton patchiness and in the presence of photosynthetically active phytoplankton at depths far below the 1% light limit.     

A decrease of both [Ca]e and [H]e produces cell damage in the perfused rat heart

Cell damage of the Langendorff-perfused rat heart in response to a decrease of both [Ca²⁺]_e and [H⁺]_e is described. At pH_e = 7.7, lactate dehydrogenase (LDH) release could be induced during perfusion with media of reduced [Ca²⁺]_e (0.1–0.4 mmol/I). Decreasing pH_e to normal abolished LDH release. The gap junction channel blocker heptanol (2 mmol/I) also reduced enzyme release, and polyethylene glycol (9% PEG₆₀₀₀) totally prevented cell damage. Elevation of buffer capacity of perfusion media or perfusion flow both increased LDH release. Cell damage could also be aggravated by substituting 10 mmol/I of [Na⁺]_e by foreign cations. At [Ca²⁺]_e = 0.1 mmol/I and pH_e = 7.7, [Ca²⁺]_i and [Na⁺]_i of non-lysed cells were markedly increased (in HCO₃/CO₂ buffered media to about 7.0 μmol/I and 36 mmol/I, respectively; in HEPES-buffered media, to about 5.0 μmol/I and 55 mmol/l; physiological values of [Ca²⁺]_i and [Na⁺]_i are around 0.1 μmol/I and 10 mmol/I, respectively), whereas pH_i was not appreciably elevated. In contrast to myocytes in the intact heart, [Ca²⁺]_i of isolated cardiomyocytes under similar conditions was decreased to about 75 nmol/I and LDH release was negligible; pH_i of isolated cardiomyocytes, as in intact myocardium, did not change appreciably. The results indicate that Ca²⁺ overload is produced at lowered [Ca²⁺]_e and [H⁺]_e by an influx of Ca²⁺ through gap junctional leaks.     

Effect of Na/Ca exchange on plateau fraction and [Ca]i in models for bursting in pancreatic beta-cells.

In the presence of an insulinotropic glucose concentration, beta-cells, in intact pancreatic islets, exhibit periodic bursting electrical activity consisting of an alternation of active and silent phases. The fraction of time spent in the active phase over a period is called the plateau fraction and is correlated with the rate of insulin release. However, the mechanisms that regulate the plateau fraction remain unclear. In this paper we investigate the possible role of the plasma membrane Na+/Ca2+ exchange of the beta-cell in controlling the plateau fraction. We have extended different single-cell models to incorporate this Ca2+-activated electrogenic Ca2+ transporter. We find that the Na+/Ca2+ exchange can provide a physiological mechanism to increase the plateau fraction as the glucose concentration is raised. In addition, we show theoretically that the Na+/Ca2+ exchanger is a key regulator of the cytoplasmic calcium concentration in clusters of heterogeneous cells with gap-junctional electrical coupling.