Intermediate Ca2 Research Articles

Pharmacological calcium sensitivity modulation of cardiac myofilaments.

As is well known, force generation and contractility of the heart may be controlled by calcium by way of regulating crossbridge attachment and the transition of attaching crossbridges into a force-generating state. These calcium activation processes do not occur in an all-or-none manner, but may be graded e.g. by altering the degree of calcium occupancy of troponin C (e.g. Pan and Solaro, 1987). Calcium occupancy depends on the free calcium concentration in the myoplasm in the range of 0.1 to 10 μM, but also, of course, on the calcium affinity of troponin C. Thus, at an intermediate cytosolic Ca2+ concentration allowing force to reach half maximum activation, any increase in calcium affinity would increase calcium occupancy and hence force and contractility. Thus, a lower Ca2+ concentration (or for that matter a higher pCa50 value) would then be required to reach half maximum activation under these conditions. This we refer to as Ca2+ sensitization of the myofilaments. The term calcium sensitivity modulation then is taken to mean a change in the pCa50 value, i.e. in the level of ionised calcium required for 50% activation of the myofilament activity normalized to the maximum contractile activity at saturating levels of calcium.KeywordsChronic Heart FailureCardiac TroponinCalcium SensitivityFree Calcium ConcentrationHalf Maximum ActivationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Changes in flip/flop splicing of astroglial AMPA receptors in human temporal lobe epilepsy.

Recent data suggested a role for glial cells in epilepsy. This study sought to identify and functionally characterize AMPA receptors expressed by astrocytes in human hippocampal tissue resected from patients with intractable temporal lobe epilepsy. Patch-clamp and fast application methods were combined to investigate astrocytes in situ and after fresh isolation from the stratum radiatum of the hippocampal CA1 subfield. Relying on presurgical and histopathologic analysis, we divided human specimens into two groups, Ammon's horn sclerosis (AHS) and lesion-associated epilepsy. Fast application of glutamate and kainate evoked receptor currents in all cells studied. Reversal-potential analysis revealed an intermediate Ca2+ permeability of the receptor channels that did not vary between the two groups of patients. However, preapplication of the AMPA receptor-specific modulator, cyclothiazide, disclosed differences in flip-flop splicing. This treatment considerably enhanced the receptor conductance, with potentiation being significantly stronger in cells from AHS specimens compared with lesion-associated cells, suggesting upregulation of AMPA receptor flip splice variants in astrocytes of the sclerotic tissue. Compelling evidence has been accumulated showing direct and rapid signaling between neurons and glial cells. Our data suggest that in AHS patients, neuronally released glutamate will lead to an enhanced and prolonged depolarization of astrocytes, which might be involved in seizure generation and spread in this particular condition of human temporal lobe epilepsy.

Three Ca2+ levels affect plasticity differently: the LTP zone, the LTD zone and no man's land

Three Ca²⁺ levels affect plasticity differently: the LTP zone, the LTD zone and no man's land

AMPA Receptor Calcium Permeability, GluR2 Expression, and Selective Motoneuron Vulnerability

AMPA receptor-mediated excitotoxicity is proposed to play a major pathogenic role in the selective motoneuron death of amyotrophic lateral sclerosis. Motoneurons have been shown in various models to be more susceptible to AMPA receptor-mediated injury than other spinal neurons. It has been hypothesized that this selective vulnerability of motoneurons is caused by the expression of highly Ca(2+)-permeable AMPA receptors and a complete or relative lack of the AMPA receptor subunit Glu receptor 2 (GluR2). The aim of this study was to quantify the relative Ca(2+) permeability of AMPA receptors and the fractional expression of GluR2 in motoneurons by combining whole-cell patch-clamp electrophysiology and single-cell RT-PCR and to compare these properties with those of dorsal horn neurons. Spinal motoneurons and dorsal horn neurons were isolated from embryonic rats and cultured on spinal astrocytes. As in previous studies, motoneurons were significantly more vulnerable to AMPA and kainate than dorsal horn neurons. However, all motoneurons expressed GluR2 mRNA ( approximately 40% of total AMPA receptor subunit mRNA), and their AMPA receptors had intermediate whole-cell relative Ca(2+) permeability (P(Ca(2+))/P(Cs(+)) approximately 0. 4). AMPA receptor P(Ca(2+))/P(Cs(+)) and the relative abundance of GluR2 varied more widely in dorsal horn neurons than in motoneurons, but the mean values did not differ significantly between the two cell populations. GluR2 was virtually completely edited at the Q/R site both in motoneurons and dorsal horn neurons. These results indicate that the selective vulnerability of motoneurons to AMPA receptor agonists is not determined solely by whole-cell relative Ca(2+) permeability of AMPA receptors.

Molecular Identification of the Ryanodine Receptor Ca2+ Sensor

We have investigated the molecular basis for ryanodine receptor (RyR) activation by Ca2+ by using site-directed mutagenesis together with functional assays consisting of Ca2+ release measurements and single channel recordings in planar lipid bilayers. We report here that a single substitution of alanine for glutamate at position 3885 (located in the putative transmembrane sequence M2 of the type 3 RyR) reduces the Ca2+ sensitivity, as measured by single channel activation, by more than 10,000-fold, without apparent changes in channel conductance and in modulation by other ligands (e.g. ATP and ryanodine). Co-expression of the wild type and mutant RyR proteins results in the synthesis of single channels that have intermediate Ca2+ sensitivities. These results suggest that the glutamates at position 3885 of each monomer may act in a coordinated way to form the Ca2+ sensor in the tetrameric structure corresponding to RyR.

Fundamental calcium release events revealed by two-photon excitation photolysis of caged calcium in Guinea-pig cardiac myocytes.

1. In cardiac muscle, 'Ca2+ sparks' have been proposed to underlie Ca2+-induced Ca2+ release (CICR), and to result from openings of clusters of Ca2+ channels (ryanodine receptors; RyRs) located in the sarcoplasmic reticulum membrane. 2. To investigate the elementary nature of these Ca2+ signals directly, a diffraction-limited point source of Ca2+ was created in single cardiac myocytes by two-photon excitation photolysis of caged Ca2+. Simultaneously, concentration profiles of released Ca2+ were imaged at high temporal and spatial resolution with a laser-scanning confocal microscope. 3. This approach enabled us to generate and detect photolytic Ca2+ signals that closely resembled the Ca2+ sparks occurring naturally, not only in amplitude and size, but also in their ability to trigger additional Ca2+ sparks or Ca2+ waves. 4. Surprisingly, at low photolytic power minuscule events with estimated Ca2+ release fluxes 20-40 times smaller than those calculated for a typical Ca2+ spark were directly resolved. These events appeared to arise from the opening of a more limited number of RyRs (possibly one) or from RyRs exhibiting a different gating mode and may correspond to the elusive 'Ca2+ quark'. 5. The Ca2+ quark represents the fundamental Ca2+ release event of excitable cells implementing hierarchical Ca2+ signalling systems with Ca2+ release events of various but distinct amplitude levels (i.e. Ca2+ quarks, Ca2+ sparks and full cellular Ca2+ transients). 6. A graded recruitment of nanoscopic Ca2+ release domains (i.e. Ca2+ quarks) exhibiting variable degrees of spatial coherence and coupling may then build up intermediate Ca2+ signalling events (i.e. Ca2+ sparks). This mechanism suggests the existence of Ca2+ sparks caused by gating of a variable fraction of RyRs from within an individual cluster. Additional mobilization of a variable number of these Ca2+ sparks enables cardiac cells to show graded cellular Ca2+ transients. Similar recruitment processes may underlie regulation of Ca2+ signalling on the cellular level in general.

Cytoplasmic calcium buffers in intact human red cells.

1. Precise knowledge of the cytoplasmic Ca2+ buffering behaviour in intact human red cells is essential for the characterization of their [Ca2+]i-dependent functions. This was investigated by using a refined method and experimental protocols which allowed continuity in the estimates of [Ca2+]i, from nanomolar to millimolar concentrations, in the presence and absence of external Ca2+ chelators. 2. The study was carried out in human red cells whose plasma membrane Ca2+ pump was inhibited either by depleting the cells of ATP or by adding vanadate to the cell suspension. Cytoplasmic Ca2+ buffering was analysed from plots of total cell calcium content vs. ionized cytoplasmic Ca2+ concentration ([CaT]i vs. [Ca2+]i) obtained from measurements of the equilibrium distribution of 45Ca2+ at different external Ca2+ concentrations ([Ca2+]o), in conditions known to clamp cell volume and pH. The equilibrium distribution of 45Ca2+ was induced by the divalent cation ionophore A23187. 3. The results showed the following. (i) The known red cell Ca2+ buffer represented by alpha, with a large capacity and low Ca2+ affinity, was the main cytoplasmic Ca2+ binding agent. (ii) The value of alpha was remarkably constant; the means for each of four donors ranged from 0.33 to 0.35, with a combined value of all independent measurements of 0.34 +/- 0.01 (mean +/- S.E.M., n = 16). This contrasts with the variability previously reported. (iii) There was an additional Ca2+ buffering complex with a low capacity (approximately 80 micromol (340 g Hb)(-1)) and intermediate Ca2+ affinity (apparent dissociation constant, K(D,app) approximately 4-50 microM) whose possible identity is discussed. (iv) The cell content of putative Ca2+ buffers with submicromolar Ca2+ dissociation constants was below the detection limit of the methods used here (less than 2 micromol (340 g Hb)(-1)). 4. Vanadate (1 mM) inhibited the Vmax of the Ca2+ pump in inosine-fed cells by 99.7%. The cytoplasmic Ca2+ buffering behaviour in these cells was similar to that found in ATP-depleted cells.

NMR studies of the E140Q mutant of the carboxy-terminal domain of calmodulin reveal global conformational exchange in the Ca2+-saturated state.

In the present investigation, the Ca2+ activation of the C-terminal domain of bovine calmodulin and the effects of replacing the bidentate Ca2+-coordinating glutamic acid residue in the 12th and last position of loop IV with a glutamine are studied by NMR spectroscopy. The mutation E140Q results in sequential Ca2+ binding in this domain and has far-reaching effects on the structure of (Ca2+)2 TR2C, thereby providing further evidence for the critical role of this glutamic acid residue for the Ca2+-induced conformational change of regulatory EF-hand proteins. Analyses of the NOESY spectra of the mutant under Ca2+-saturated conditions, such that 97% of the protein is in the (Ca2+)2 form, revealed two sets of mutually exclusive NOEs. One set of NOEs is found to be consistent with the closed structure observed in the apo state of the C-terminal domain of the wild-type protein, while the other set supports the open structure observed in the Ca2+-saturated state. In addition, several residues in the hydrophobic core exhibit broadened resonances. We conclude that the (Ca2+)2 form of the mutant experiences a global conformational exchange between states similar to the closed and open conformations of the C-terminal domain of wild-type calmodulin. A population of 65 +/- 15% of the open conformation and an exchange rate of (1-7) x 10(4) s(-1) were estimated from the NMR data and the chemical shifts of the wild-type protein. From a Ca2+ titration of the 15N-labeled mutant, the macroscopic binding constants [log(K1) = 4.9 +/- 0.3 and log(K2) = 3.15 +/- 0.10] and the inherent chemical shifts of the intermediate (Ca2+)1 form of the mutant were determined using NMR. Valuable information was also provided on the mechanism of the Ca2+ activation and the roles of the structural elements in the two Ca2+-binding events. Comparison with the wild-type protein indicates that the (Ca2+)1 conformation of the mutant is essentially closed but that some rearrangement of the empty loop IV toward the Ca2+-bound form has occurred.

Quantal release, incremental detection, and long-period Ca2+ oscillations in a model based on regulatory Ca2+-binding sites along the permeation pathway

Quantal release, incremental detection, and oscillations are three types of Ca2+ responses that can be obtained in different conditions, after stimulation of the intracellular Ca2+ stores by submaximum concentrations of inositol 1,4,5-triphosphate (InsP3). All three phenomena are thought to occur through the regulatory properties of the InsP3 receptor/Ca2+ channel. In the present study, we perform further analysis of the model (Swillens et al., 1994, Proc. Natl. Acad. Sci. USA. 91:10074-10078) previously proposed for transient InsP3-induced Ca2+ release, based on the bell-shaped dependence of the InsP3 receptor activity on the Ca2+ level and on the existence of an intermediate Ca2+ domain located around the mouth of the channel. We show that Ca2+ oscillations also arise in the latter model. Conditions for the occurrence of the various behaviors are investigated. Numerical simulations also show that the existence of an intermediate Ca2+ domain can markedly increase the period of oscillations. Periods on the order of 1 min can indeed be accounted for by the model when one assigns realistic values to the kinetic constants of the InsP3 receptor, which, in the absence of a domain, lead to oscillations with periods of a few seconds. Finally, theoretical support in favor of a positive cooperativity in the regulation of the InsP3 receptor by Ca2+ is presented.

Ca2+ Release channels of pigs heterozygous for malignant hyperthermia

Porcine malignant hyperthermia (MH) is an autosomal recessive disorder resulting from a mutation in the skeletal muscle sarcoplasmic reticulum (SR) Ca2+ release channel. The Ca2+ release properties of SR vesicles isolated from pigs heterozygous for the MH gene have been demonstrated previously to be intermediate to those of vesicles isolated from MH-susceptible (MHS) and normal pigs. The Ca2+ release channel is tetrameric, so the intermediate Ca2+ release properties of heterozygous pig SR preparations could result either from populations of MHS and normal homotetramers, or populations of heterotetrameric Ca2+ release channels with properties unique from those of the two types of homozygous channels. To discriminate between these possibilities, the single channel percent open time (Po) and channel dwell time distributions of SR Ca2+ release channels were analyzed. These data suggest that the heterozygous porcine Ca2+ release channel population must contain heterotetramers with properties distinct from those of either MHS or normal channels. The data also imply that the Ca2+ release channel population in MHS humans who are heterozygous for a dominant mutation in this protein also contains heterotetrameric channels.

Photophysics of the fluorescent Ca2+ indicator Fura-2

The photophysics of the complex forming reaction of Ca2+ and Fura-2 are investigated using steady-state and time-resolved fluorescence measurements. The fluorescence decay traces were analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution with BAPTA as Ca2+ buffer: k01 = 1.2 x 10(9)s-1, k21 = 1.0 x 10(11) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 2.2 x 10(7) s-1, and with EGTA as Ca2+ buffer: k01 = 1.4 x 10(9) s-1, k21 = 5.0 x 10(10) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 3.2 x 10(7) s-1. k01 and k02 denote the respective deactivation rate constants of the Ca2+ free and bound forms of Fura-2 in the excited state. k21 represents the second-order rate constant of binding of Ca2+ and Fura-2 in the excited state, whereas k12 is the first-order rate constant of dissociation of the excited Ca2+:Fura-2 complex. The ionic strength of the solution was shown not to influence the recovered values of the rate constants. From the estimated values of k12 and k21, the dissociation constant K*d in the excited state was calculated. It was found that in EGTA Ca2+ buffer pK*d (3.2) is smaller than pKd (6.9) and that there is negligible interference of the excited-state reaction with the determination of Kd and [Ca2+] from fluorimetric titration curves. Hence, Fura-2 can be safely used as an Ca2+ indicator. From the obtained fluorescence decay parameters and the steady-state excitation spectra, the species-associated excitation spectra of the Ca2+ free and bound forms of Fura-2 were calculated at intermediate Ca2+ concentrations.

Ca2+ load of guinea‐pig ventricular myocytes determines efficacy of brief Ca2+ currents as trigger for Ca2+ release.

1. In guinea-pig ventricular cells, the concentration of ionized cytosolic calcium ([Ca2+]o) was estimated from the fluorescence of 100 microM K5-indo-1. At 36 degrees C and 2 mM [Ca2+]o, the Ca2+ load of the cells was varied by 1 Hz trains of conditioning clamp pulses to -30 mV (low Ca2+ load), 0 mV (intermediate Ca2+ load) and paired pulses (high Ca2+ load). After seven pulses potentiation was steady and short test pulses to 0 mV were tested for their efficacy in triggering [Ca2+]c transients. The influx of trigger Ca2+ was graded by varying the test-pulse duration between 1 and 180 ms. 2. After a 3 min rest period, [Ca2+]c was 100 +/- 20 nM (mean +/- S.E.M.) and 2 ms test pulses were unable to induce [Ca2+]c transients. Test pulses of 2 ms duration, however, induced [Ca2+]c transients after potentiation with single or paired pulses. 3. At high cellular Ca2+ load, the amplitude of the [Ca2+]c transients (delta[Ca2+]c) gradually increased with pulse durations up to 8 ms. Pulse durations between 8 and 160 ms, however, did not further increase delta[Ca2+]c as if the largest part of the [Ca2+]c transient was due to regenerative contribution of Ca(2+)-induced Ca2+ release. 4. Pulses of 160 ms duration induced 'saturating' responses whose amplitudes delta[Ca2+]c, t = infinity decreased from 938 +/- 120 nM at high Ca2+ load, to 610 +/- 90 and 350 +/- 120 nM at intermediate and low Ca2+ loads, respectively. 5. Delta[Ca2+]c was more sensitive to the duration of Ca2+ influx at low or intermediate Ca2+ loads than at high Ca2+ load. When delta[Ca2+]c was plotted against the test-pulse duration, 50% of delta[Ca2+]c, t = infinity was found to be at 9 +/- 2 ms (low), 4.6 +/- 1 ms (intermediate) or 1.8 +/- 0.5 ms pulses (high Ca2+ load). Correspondingly, the efficacy of 2 ms test pulses in triggering [Ca2+]c transients increased with the Ca2+ load. 6. At high Ca2+ load, [Ca2+]c peaked nearly independently of pulse duration at 19 +/- 3 ms. At intermediate or low Ca2+ load, time to peak increased with pulse duration. 7. The results confirm the theory that sarcoplasmic reticulum (SR) Ca2+ release contributes an amount to the [Ca2+]c transient that increases with the cellular Ca2+ load. The results are compatible with the hypothesis that SR Ca2+ release can be activated by both Ca2+ influx and by SR Ca2+ release and that the latter mechanism constitutes a positive feedback, the amplification of which increases with the amount of releasable Ca2+.

Dynamics of Ca2+ and guanosine 5′-[γ-thio]triphosphate action on insulin secretion from α-toxin-permeabilized HIT-T15 cells

The time course of Ca2+ and GTP-analogue effects on insulin secretion was investigated in HIT-T15 cells permeabilized with Staphylococcus alpha-toxin. These cells responded to Ca2+ in the range 0.1-10 microM and could be used in a dynamic perifusion system because of the minimal run-down of the secretory response. High Ca2+ (10 microM) elicited a monophasic ATP-dependent stimulation of insulin secretion that reached a peak within 5 min (approximately 20-fold increase) and rapidly decreased during the subsequent 15 min to a plateau remaining above basal rates (0.1 microM Ca2+). The decrease in Ca(2+)-induced insulin secretion with time could not be attributed to decreased capacity to respond to Ca2+ after prolonged perfusion at low Ca2+ (run-down), nor to depletion of a particular secretory-granule pool. It was rather due to desensitization of the secretory machinery to Ca2+ that was not reversed by selective inhibition of the Ca2+/calmodulin-dependent kinase II with KN-62. However, an intermediate Ca2+ concentration (2 microM) increased insulin secretion to stable level without causing any desensitization. Imposed oscillations of Ca2+ (0.1-10 microM) produced phasic oscillations of insulin secretion, but did not prevent desensitization to Ca2+. Poorly hydrolysable GTP analogues increased insulin secretion at low Ca2+, whereas they strongly inhibited Ca(2+)-induced insulin secretion. By contrast, GTP did not affect basal secretion, and slightly increased Ca(2+)-evoked secretion. These results indicate the following. (1) Oscillations of insulin secretion are tightly coupled to cytosolic Ca2+ oscillations. (2) Oscillations of Ca2+ do not prevent high-Ca(2+)-induced desensitization to Ca2+; this result does not support the idea of a greater efficiency of oscillations compared with sustained Ca2+ rises in triggering exocytosis. (3) Activation of G-proteins modulates exocytosis in a bimodal manner.

Activation and relaxation mechanisms in single muscle fibres.

The effect of Ca2+ on the time course of force generation in frog skinned muscle fibres has been investigated using laser flash photolysis of the caged-calcium, either nitr-5 or DM-Nitrophen. Gradations in the rate and extent of contraction could be achieved by changing the energy of the laser pulse, which varied the amount of caged Ca2+ photolysed and hence the amount of calcium released. The half-time for force development at 12 degrees C was noticeably calcium-sensitive when small amounts of calcium were released (low energy pulses) but did not change appreciably for calcium releases which produced a final tension of more than 50% of the maximal tension at pCa 4.5. This result is unlikely to be due to calcium binding to the regulatory sites of troponin C when on the thin filament, as this process is considered rapid (kon 10(8) M-1 s-1, koff 100 s-1). Our experimental results show that force develops relatively rapidly at intermediate Ca2+ which produce only partial activation (i.e. 50% Pmax or greater). This would not be the case if the affinity of the regulatory sites changes slowly with crossbridge attachment. The kinetics of calcium exchange with the regulatory sites may be much more rapid than crossbridge cycling, so that if calcium binding to a particular functional unit induces crossbridge attachment and force production, the force producing state may be maintained long after calcium has dissociated from that particular functional unit. The relaxation of skinned muscle fibres has also been successfully studied following the rapid uptake of Ca2+ by a photolabile chelator Diazo-2, a photolabile derivative of BAPTA, which is rapidly (> 2000 s-1) converted from a chelator of low Ca2+ affinity (Kd 2.2 microM) to a high affinity chelator (Kd 0.073 microM). We have used single skinned muscle fibres from both frog (actin regulated) and scallop striated muscle (myosin regulated), to study the time course of muscle relaxation. This procedure has enabled us to examine the effects of the intracellular metabolites, ADP, Pi and H+ upon the rate of relaxation.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium-induced sensitization of the central helix of calmodulin to proteolysis

The rate of proteolysis of trypsin-sensitive bonds was used to examine the nature of the structural changes accompanying Ca2+ and Mg2+ binding to calmodulin. In the Ca(2+)-free form, the rates of proteolysis at Arg-106 and Arg-37 are rapid (greater than 300 and 28 nmol min-1 mL-1, respectively), the bonds at Arg-74, Lys-75, and Lys-77, in the central helix, are cleaved more slowly (10 nmol min-1 mL-1), and a lag in the cleavage at the remaining bonds (Lys-13, Lys-30, Arg-86, Arg-90, and Arg-126) suggests that they are not cleaved in the native protein. High concentrations of Ca2+, but not Mg2+, almost completely abolish proteolysis at Arg-106 and drastically reduce the rate of cleavage at Arg-37. Both Ca2+ and Mg2+ exert a moderate protective effect on the proteolysis of the central helix. These results suggest that the F-helix of domains III and, to a lesser extent, the F-helix of domain I are somewhat flexible in the Ca(2+)-free form and are stabilized by Ca2+. Whereas full occupancy of the four Ca(2+)-binding sites produces little change in the susceptibility of the central helix to proteolytic attack, binding of two Ca2+ produces a 10-fold enhancement of the rate of proteolysis in this part of the molecule. We propose that at intermediate Ca2+ levels the flexibility of the central helix of calmodulin is greatly increased, resulting in the transient formation of intermediates which have not been detected by spectroscopic techniques but are trapped by the irreversible action of trypsin.

Alterations in mitochondrial Ca 2+ flux by the antibiotic X-537A (lasalocid-A)

A previous communication (Pereira da Silva, L., Bernardes, C.F. and Vercesi, A.E. (1984) Biochem. Biophys. Res. Commun. 124, 80-86) presented evidence that lasalocid-A, at concentrations far below those required to act as a Ca2+ ionophore, significantly inhibits Ca2+ efflux from liver mitochondria. In the present work we have studied the mechanism of this inhibition in liver and heart mitochondria. It was observed that lasalocid-A (25-250 nM), like nigericin, promotes the electroneutral exchange of K+ for H+ across the inner mitochondrial membrane and as a consequence can cause significant alterations in delta pH and delta psi. An indirect effect of these changes that might lead to inhibition of mitochondrial Ca2+ release was ruled out by experiments showing that the three observed patterns of lasalocid-A effect depend on the size of the mitochondrial Ca2+ load. At low Ca2+ loads (5-70 nmol Ca2+/mg protein), under experimental conditions in which Ca2+ release is supposed to be mediated by a Ca2+/2H+ antiporter, the kinetic data indicate that lasalocid-A inhibits the efflux of the cation by a competitive mechanism. The Ca2+/2Na+ antiporter, the dominant pathway for Ca2+ efflux from heart mitochondria, is not affected by lasalocid-A. At intermediate Ca2+ loads (70-110 nmol Ca2+/mg protein), lasalocid-A slightly stimulates Ca2+ release. This effect appears to be due to an increase in membrane permeability caused by the displacement of a pool of membrane bound Mg2+ possibly involved in the maintenance of membrane structure. Finally, at high Ca2+ loads (110-140 nmol Ca2+/mg protein) lasalocid-A enhances Ca2+ retention by liver mitochondria even in the presence of Ca2(+)-releasing agents such as phosphate and oxidants of the mitochondrial pyridine nucleotides. The maintenance of a high membrane potential under these conditions may indicate that lasalocid-A is a potent inhibitor of the Ca2(+)-induced membrane permeabilization. Nigericin, whose chemical structure resembles that of lasalocid-A, caused similar results.

Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro.

Epidermal differentiation is characterized by a series of coordinated morphological and biochemical changes which result in a highly specialized, highly organized, stratified squamous epithelium. Among the specific markers expressed in differentiating epidermis are (a) two early spinous cell proteins, keratins 1 and 10 (K1 and K10); and (b) two later granular cell proteins, filaggrin and a cornified envelope precursor (CE). In vitro, epidermal basal cells are selectively cultured in 0.05 mM Ca2+ medium, and terminal differentiation is induced when the Ca2+ concentration is increased to 1 mM. However, only a small fraction of the cells express the markers K1, K10, CE, or filaggrin in the higher Ca2+ medium. To explore the factors required for marker expression, cultured epidermal cells were exposed to intermediate Ca2+ concentrations and extracts were analyzed using specific antibody and nucleic acid probes for the four markers of interest. These studies revealed that marker expression was enhanced at a restricted concentration of Ca2+ in the medium of 0.10-0.16 mM. At this Ca2+ concentration, both protein and mRNA levels for each marker were substantially increased, whereas at higher or lower Ca2+ concentrations they were diminished or undetected. The percentage of cells expressing each marker was increased two- to threefold in the permissive Ca2+ medium as determined by immunofluorescence analysis. This optimal level of Ca2+ was required both to initiate and sustain marker expression. At the permissive Ca2+ concentration, expression of the markers was sequential and similar to the order of appearance in vivo. K1 was expressed within 8-12 h and K10 was expressed in the ensuing 12-24-h period. CE and filaggrin were expressed in the subsequent 24 h. Inhibition of K1 expression by cycloheximide suggested that an inducible protein was involved. Other investigators have determined that a shallow Ca2+ gradient exists in epidermis, where the basal cells and spinous cells are in a Ca2+ environment substantially below serum Ca2+ levels. These in vitro results suggest that the Ca2+ environment is a fundamental regulator of expression of epidermal differentiation markers and provide an explanation for the existence of the Ca2+ gradient in vivo.

Guanine nucleotides induce Ca2+-independent insulin secretion from permeabilized RINm5F cells.

The role of guanine nucleotides in insulin secretion was investigated in electrically permeabilized RINm5F cells. Ca2+ stimulated insulin release (EC50 approximately 2 microM Ca2+). The GTP stable analog, GTP gamma S, elicited insulin secretion at vanishingly low Ca2+ concentrations (less than 10(-11) M), slightly potentiated the response to intermediate Ca2+ levels, but exerted less than additive effects at maximal Ca2+ concentrations. The GDP analog, GDP beta S, inhibited both GTP gamma S- and Ca2+-stimulated secretion. The action of GTP gamma S was not mediated by cAMP, as the latter only enhanced Ca2+-induced secretion. In contrast, 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, promoted insulin release at nonstimulatory Ca2+ levels as well as potentiating the Ca2+ response. GTP analogs stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2), as assessed by inositol phosphate generation. However, this could not fully explain guanine nucleotide-induced secretion because: GTP gamma S-stimulated PtdInsP2 breakdown was totally dependent on Ca2+ and abolished at Ca2+ below 10(-11) M; at these Ca2+ levels, activators of protein kinase C were weak or ineffective secretagogues; the GTP analog Gpp(NH)p was much less effective than GTP gamma S in activating PtdInsP2 hydrolysis, while fully mimicking the effect on Ca2+-independent secretion. Both GTP gamma S-induced PtdInsP2 hydrolysis and insulin release were insensitive to pertussis toxin and cholera toxin. The findings point to a guanine nucleotide-regulated site in the activation of insulin secretion different from the known transmembrane signalling systems.

Activation of actin-cardiac myosin subfragment 1 MgATPase rate by Ca2+ shows cooperativity intrinsic to the thin filament.

The magnesium adenosinetriphosphatase (MgATPase) rate of cardiac myosin subfragment 1 (S-1) was studied in the presence of regulated actin in order to investigate the mechanism by which Ca2+ cooperatively induces cardiac muscle contraction. The MgATPase rate increased cooperatively with Ca2+, exhibiting a Hill coefficient of 1.8 and 50% activation at pCa 5.75. This cooperative response occurred despite an experimental design excluding several potential sources of cooperativity. First, to exclude spurious cooperativity due to erroneous calculation of pCa at low ionic strength, the affinities of Ca2+ and Mg2+ for [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) were measured by a novel method using Quin 2. At pH 7.06, 25 degrees C, and mu = 30 mM, the KD was 140 nM for CaEGTA and 2.7 mM for MgEGTA. Second, the cooperativity was not produced by actin-myosin S-1 binding; myosin S-1 was bound to only 1 of every 300 actin promoters, and earlier work [Tobacman, L. S., & Adelstein, R. S. (1986) Biochemistry 25, 798-802] had shown that cardiac myosin S-1 binds with equal affinity to the thin filament at very low Ca2+ and at saturating Ca2+ concentrations. Furthermore, the adenosine 5'-triphosphate turnover rate of the myosin S-1 was independent of enzyme concentration at low, intermediate, and saturating Ca2+ concentrations. Finally, since cardiac troponin has only one regulatory Ca2+-specific site, cooperative interactions between such sites could not occur. These data suggest that part of the cooperativity conferred by interaction between adjacent troponin-tropomyosin complexes is intrinsic to the thin filament and independent of myosin.

Skinned smooth muscle: calcium-calmodulin activation independent of myosin phosphorylation.

In chemically skinned chicken gizzard smooth muscle fibers investigated shortly after preparation, a contraction may be induced by calcium and calmodulin which is independent of myosin phosphorylation at intermediate Ca2+-concentrations. However, fibers stored for a prolonged period also contract in the absence of exogenous calmodulin and exhibit a close relationship between force development and myosin phosphorylation.