Catecholamine secretion from digitonin-treated PC12 cells. Effects of Ca2+, ATP, and protein kinase C activators

PC12 cells, a cloned rat pheochromocytoma cell line, were treated with digitonin to render the plasma membrane permeable to ions and proteins. At a cell density of 2-6 X 10(5) cells/cm2, incubation with 7.5 microM digitonin permitted a Ca2+-dependent release of 25-40% of the catecholamine within 18 min in the presence of 10 microM Ca2+. Half-maximal secretion occurred at 0.5-1 microM Ca2+. PC12 cultures at lower cell densities were more sensitive to digitonin and gave more variable results. Secretion in the presence of digitonin and Ca2+ began after a 2-min lag and continued for up to 30 min. When cells were treated for 3 min in digitonin and then stimulated with Ca2+ in the absence of digitonin, secretion occurred in the same manner but without the initial lag. Optimal secretion from PC12 cells was also dependent upon the presence of Mg2+ and ATP. Permeabilized PC12 cells exhibited a slow time-dependent loss of secretory responsiveness which was correlated with the release of a cytosolic marker, lactate dehydrogenase (134 kDa). This suggests that digitonin permeabilization allows soluble constituents necessary for secretion to leave the cell in addition to allowing Ca2+ and ATP access into the cell interior. Ca2+-dependent secretion was completely inhibited by exposure of digitonin-permeabilized cells to 100 micrograms/ml trypsin (27 kDa), whereas secretion was only slightly inhibited by trypsin exposure prior to digitonin treatment. Thus, an intracellular, trypsin-sensitive protein is probably involved in secretion. The data also indicate that the same population of digitonin-treated cells which responded to Ca2+ was permeable to a 27-kDa protein. 1,2-Dioctanoylglycerol and phorbol esters which activate protein kinase C enhanced the Ca2+-dependent and Ca2+-independent secretion in digitonin-permeabilized PC12 cells. Thus, protein kinase C appears to be involved in the regulation of catecholamine secretion from permeabilized PC12 cells.     

High voltage electron microscopy studies of axoplasmic transport in neurons: a possible regulatory role for divalent cations

Light and high voltage electron microscopy (HVEM) procedures have been employed to examine the processes regulating saltatory motion in neurons. Light microscope studies demonstrate that organelle transport occurs by rapid bidirectional saltations along linear pathways in cultured neuroblastoma cells. HVEM stereo images of axons reveal that microtubules (Mts) and organelles are suspended in a continuous latticework of fine microtrabecular filaments and that the Mts and lattice constitute a basic cytoskeletal structure mediating the motion of particles along axons. We propose that particle transport depends on dynamic properties of nonstatic microtrabecular lattice components. EXperiments were initiated to determine the effects of changes in divalent cation concentrations (Ca2+ and Mg2+) on: (a)the continuation of transport and (b) the corresponding structural properties of the microtrabecular lattice. We discovered that transport continues or is stimulated to a limited extent in cells exposed to small amounts of exogenously supplied Ca2+ and Mg2+ ions (less than 0.1 mM). Exposure of neurons to increased dosages of Ca2+ and Mg2+ (0.2-1.0 mM) stimulates transport for 2-4 min at 37 degrees C, but after a 5- to 20-min exposure the saltatory movements of organelles are observed gradually to become shorter in duration and rate particle motion ceases to occur. HVEM observations demonstrated that Ca2+ - and with the cessation of motion. Ca2+-containing solutions produced contractions of the microtrabecular filaments, whereas Mg2+-containing solutions had the opposing effect of stimulating an elongation and assembly (expansion) of microtrabeculae. On the basis of these observations we hypothesize that cycles of Ca2+/Mg2+-coupled contractions and expansions of the microtrabecular lattice probably regulate organelle motion in nerve cells.     

Loss and Ca2+-Dependent Retention of Scinderin in Digitonin-Permeabilized Chromaffin Cells: Correlation with Ca2+-Evoked Catecholamine Release

Exposure of chromaffin cells to digitonin causes the loss of many cytosolic proteins. Here we report that scinderin (a Ca(2+)-dependent actin-filament-severing protein), but not gelsolin, is among the proteins that leak out from digitonin-permeabilized cells. Chromaffin cells that were exposed to increasing concentrations (15-40 microM) of digitonin for 5 min released scinderin into the medium. One-minute treatment with 20 microM digitonin was enough to detect scinderin in the medium, and scinderin leakage levelled off after 10 min of permeabilization. Elevation of free Ca2+ concentration in the permeabilizing medium produced a dose-dependent retention of scinderin. Results were confirmed by immunofluorescence microscopy of digitonin-permeabilized cells. Subcellular fractionation of permeabilized cells showed that scinderin leakage was mainly from the cytoplasm (80%); the remaining scinderin (20%) was from the microsomal fraction. Other Ca(2+)-binding proteins released by digitonin and also retained by Ca2+ were calmodulin, protein kinase C, and calcineurins A and B. Scinderin leakage was parallel to the loss of the chromaffin cell secretory response. Permeabilization in the presence of increasing free Ca2+ concentrations produced a concomitant enhancement in the subsequent Ca(2+)-dependent catecholamine release. The experiments suggest that: (1) scinderin is an intracellular target for Ca2+, (2) permeabilization of chromaffin cells with digitonin in the presence of micromolar Ca2+ concentrations retained Ca(2+)-binding proteins including scinderin, and (3) the retention of these proteins may be related to the increase in the subsequent Ca(2+)-dependent catecholamine release observed in permeabilized chromaffin cells.     

The devapamil-binding site of the purified skeletal muscle receptor for organic-calcium channel blockers is modulated by micromolar and millimolar concentrations of Ca2+.

The interaction of 2,7-dimethyl-3-(3,4-dimethoxyphenyl)-3-cyan-7-aza-9-(3- methoxyphenyl) nonahydrochloride (devapamil), a stereospecific analog of (3-[2-(3,4-dimethoxyphenyl)ethyl]- methylaminopropyl-3,4-dimethoxy-(1-methylethyl)benzeneacetonitr ile (verapamil), with the purified skeletal muscle receptor for calcium channel blockers (CaCB) was studied at 4 degrees C and 30 degrees C in the absence and presence of calcium. The purified CaCB receptor bound 0.9 mol devapamil/mol calcium-channel alpha 1 subunit, with an apparent Kd of 13 +/- 2.6 nM at 4 degrees C in the presence of 0.4 microM Ca2+. The affinity, and not the density, of the devapamil-binding site was decreased by lowering the pH from 8.5-6.5, or by increasing the Ca2+ concentration from 0.4 microM to 100 mM. The same results were obtained at 30 degrees C, although the ligand-receptor complex was not stable at Ca2+ concentrations below 10 microM. These binding data were confirmed by kinetic experiments. The rate constants calculated for a pseudo-first-order and a second-order reactions were identical and yielded fourfold lower k-1/k+1 (KD) values than the equilibrium experiments performed using 1 nM and 0.4 microM Ca2+, but the same values using 1 mM Ca2+. 1 mM Ca2+ increased the k-1/k+1 (KD) by decreasing 10-fold the association rate at 4 degrees C. The dissociation rate was increased about 10-fold by 5 microM devapamil or 100 microM D-cis-diltiazem, suggesting that the high affinity site is negatively regulated allosterically by millimolar Ca2+ concentrations and by the occupation of a second low-affinity site. Incubation of the CaCB receptors in the absence of Ca2+ and devapamil at 30 degrees C, but not at 4 degrees C, resulted in an apparent loss of devapamil-binding sites. The decrease in binding sites was caused by a reduced affinity. This apparent loss of binding sites was prevented by the addition of Ca2+ with an apparent median effective concentration of 0.4 microM. The apparent half-maximal inactivation times of the devapamil-binding site were 90 s and 12 min in the presence of 1 nM and 0.4 microM Ca2+, respectively. These results show that micromolar Ca2+ concentrations stabilize the CaCB receptor in a conformation which allows high-affinity binding of phenylalkylamines. Millimolar Ca2+ concentrations induce a low-affinity state of the devapamil-binding site on a stable CaCB receptor.     

Temperature and Ca2+ dependence of [3H]ryanodine binding in the burbot (Lota lota L.) heart

Opening and closing of the cardiac ryanodine (Ry) receptor (RyR) are coordinated by the free intracellular Ca2+ concentration, thus making the Ca2+ binding properties of the RyR important for excitation-contraction coupling. Unlike mammalian cardiac RyRs, which lose their normal function at low temperatures, RyRs of ectothermic vertebrates remain operative at 2-4 degrees C, as indicated by Ry sensitivity of contractile force. To investigate the mechanisms of low temperature adaptation of ectothermic RyRs, we compared Ca2+-dependent kinetics of [3H]ryanodine binding in cardiac preparations of a fish (burbot, Lota lota) and a mammal (rat). The number of ventricular [3H]ryanodine binding sites determined at 20 degrees C was 1.54 times higher in rat than burbot heart (0.401 +/- 0.039 and 0.264 +/- 0.019 pmol/mg protein, respectively) (P < 0.02), while the binding affinity (Kd) for [3H]ryanodine was similar (3.38 +/- 0.63 and 4.38 +/- 1.14 nM for rat and burbot, respectively) (P = 0.47). The high-affinity [3H]ryanodine binding to burbot and rat cardiac preparations was tightly coordinated by the free Ca2+ concentration at both 20 degrees C and 2 degrees C and did not differ between the two species. Half-maximal [3H]ryanodine binding occurred at 0.191 +/- 0.027 microM and 0.164 +/- 0.034 microM Ca2+ for rat and at 0.212 +/- 0.035 microM and 0.188 +/- 0.039 microM Ca2+ for burbot (P = 0.65), at 2 degrees C and 20 degrees C, respectively. In two other fish species, rainbow trout (Oncorhynchus mykiss) and crucian carp (Carassius carassius), the Ca2+-binding affinity at 20 degrees C was 4.4 and 5.9 times lower, respectively, than in the burbot. At 20 degrees C, the rate of [3H]ryanodine binding to the high-affinity binding site was similar in rat and burbot but was drastically slowed in rat at 2 degrees C. At 2 degrees C, [3H]ryanodine failed to dissociate from rat cardiac RyRs, and at 10 degrees C and 20 degrees C, the rate of dissociation was two to three times slower in rat than burbot preparations. The latter finding is compatible with a channel gating mechanism, where the closing of the Ca2+ release channel is impaired or severely retarded by low temperature in rat but less so in burbot preparations. The stronger effect of low temperature on association and dissociation rate of [3H]ryanodine binding in rat compared with burbot suggests that RyRs of the ectothermic fish, unlike those of endothermic rat, are better able to open and close at low temperatures.     

Kinetic studies of calcium and cardiac troponin I peptide binding to human cardiac troponin C using NMR spectroscopy

Ca2+ and human cardiac troponin I (cTnI) peptide binding to human cardiac troponin C (cTnC) have been investigated with the use of 2D [1H,15N] HSQC NMR spectroscopy. The spectral intensity, chemical shift, and line-shape changes were analyzed to obtain the dissociation ( K(D)) and off-rate ( k(off)) constants at 30 degrees C. The results show that sites III and IV exhibit 100-fold higher Ca2+ affinity than site II ( K(D(III,IV)) approximately 0.2 microM, K(D(II)) approximately 20 microM), but site II is partially occupied before sites III and IV are saturated. The addition of the first two equivalents of Ca2+ saturates 90% of sites III and IV and 20% of site II. This suggests that the Ca2+ occupancy of all three sites may contribute to the Ca2+-dependent regulation in muscle contraction. We have determined a k(off) of 5000 s(-1) for site II Ca2+ dissociation at 30 degrees C. Such a rapid off-rate had not been previously measured. Three cTnI peptides, cTnI(34-71), cTnI(128-147), and cTnI(147-163), were titrated to Ca2+-saturated cTnC. In each case, the binding occurs with a 1:1 stoichiometry. The determined K(D) and k(off) values are 1 microM and 5 s(-1) for cTnI(34-71), 78+/-10 microM and 5000 s(-1) for cTnI(128-147), and 150+/-10 microM and 5000 s(-1) for cTnI(147-163), respectively. Thus, the dissociation of Ca2+ from site II and cTnI(128-147) and cTnI(147-163) from cTnC are rapid enough to be involved in the contraction/relaxation cycle of cardiac muscle, while that of cTnI(34-71) from cTnC may be too slow for this process.     

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.     

Inositol 1,4,5-trisphosphate mobilizes glucose-incorporated calcium from pancreatic islets

Mobilization of intracellular calcium from beta-cell-rich pancreatic islets of ob/ob-mice was studied by measuring unidirectional 45Ca efflux at 37 degrees and 18 degrees C during perifusion with a K+-rich medium deficient in Ca2+ and Na+. Addition of 100 microM carbachol induced a prominent peak of Ca2+ efflux from islets preexposed to glucose. After cell permeabilization with digitonin D-myo-inositol 1,4,5-trisphosphate (IP3) caused glucose-dependent mobilization of calcium. In demonstrating that not only carbachol but also IP3 can mobilize calcium incorporated in response to glucose, the present data suggests that the endoplasmic reticulum participates in glucose-induced lowering of cytoplasmic Ca2+ activity in the pancreatic beta-cells.     

Influence of temperature upon contractile activation and isometric force production in mechanically skinned muscle fibers of the frog

Increasing temperature (4-22 degrees C) increases the Ca2+ concentration required for activation of mechanically skinned frog muscle fibers. The pCa required for 50% maximal force (pCa50) was inversely proportional to absolute temperature. Assuming that relative force is directly related to fractional occupancy of the Ca2+-binding sites on troponin that regulate force, the shift was consistent with a Gibbs free energy change of binding (delta G) of about -7.8 kcal/mol. This is close to the delta G for Ca2+ binding to the calcium-specific sites on troponin C reported by others. Decreasing Mg2+ from 1 mM to 60 microM shifts the force-pCa curves at either 4 or 22 degrees C to higher pCa, but the shift of pCa50 with temperature over this range (0.4 log units) was the same at low and high Mg2+. Maximal force increased with temperature for the entire range 4-22 degrees C with a Q10 of 1.41, and over the restricted range 4-15 degrees C with a Q10 of 1.20. From the dual effects of temperature on Ca2+ activation and maximal force, one would expect that force would respond differently to temperature change at high or low Ca2+. At high Ca2+, a temperature increase will lead to an increased force. However, at low to intermediate Ca2+ levels (below the intersection of the force-pCa curves for the initial and final temperatures), steady state force should decrease with increasing temperature. The inverse responses should occur with a decrease in temperature. These responses are observed when temperature is changed by rapid solution exchange.     

Ca2+,Mg2+-ATPase of microsomal membranes from bovine aortic smooth muscle. Identification and characterization of an acid-stable phosphorylated intermediate of the Ca2+,Mg2+-ATPase

An acid-stable phosphoprotein was formed in a microsomal membrane fraction isolated from bovine aortic smooth muscle in the presence of Mg2+ + ATP and Ca2+. The microsomes also showed Ca2+ uptake activity. The Ca2+ dependence of phosphoprotein formation and of Ca2+ uptake occurred over the same range of Ca2+ concentration (1-10 microM), and resembled similar findings from rabbit skeletal microsomes. The molecular weight of the phosphorylated protein, estimated by SDS-gel electrophoresis, was approximately 105,000. The phosphoprotein was labile at alkaline pH, and its decomposition was accelerated by hydroxylamine. Half-maximum incorporation of 32P in the presence of 10 microM Ca2+ occurred at 60 nM ATP. The calcium-dependent phosphoprotein formation was not affected by 5 mM NaN3, but was inhibited in a dose-dependent fashion by ADP with a 50% inhibition occurring at 180 microM. Fifty mM MgCl2 was required for the maximal phosphorylation. The rate of phosphoprotein decomposition after adding 2 mM EGTA was accelerated by varying the Mg2+ concentration from 10 microM to 3 mM. Alkaline pH (9.0) slowed the rate of phosphoprotein decay. Optimal Ca2+-dependent phosphoprotein occurred at 15 degrees C over a broad pH range (6.4 to 9.0). The activation energy of EGTA-induced phosphoprotein decomposition was 25.6 kcal/mol between 0 and 16 degrees C and 14.6 kcal/mol between 16 and 30 degrees C. The phosphoprotein formed by aortic microsomes was thus quite similar to the acid-stable phosphorylated intermediate of the Ca2+-transport ATPase of sarcoplasmic reticulum from skeletal and cardiac muscle. These data suggest that the Ca2+-dependent phosphoprotein is a reaction intermediate of the Ca2+,Mg2+-ATPase of the aortic microsomes.     

Ca2+- and Mg-ATP-dependent shape change of human erythrocyte ghosts and triton shells

Human erythrocyte membranes (ghosts) prepared from fresh blood changed in shape from spherical to crenated, when suspended in 10(-7)-10(-6) M Ca2+-EGTA buffers. Although the ghosts from long-stored ACD blood (10 weeks) were less sensitive to 10(-7)-10(-6) M Ca2+, the ghosts obtained from this blood after it had been preincubated with adenine and inosine for 3 h at 37 degrees C were highly sensitive to Ca2+. When these highly sensitive ghosts were incubated in 10 mM Tris-Cl buffer (pH 7.4) or 1 mM MgCl2 (pH 7.4) at 0 degrees C, they gradually lost Ca2+ sensitivity within 60 min, but they recovered Ca2+ sensitivity again after re-incubation with 2 mM Mg-ATP for 20 min at 37 degrees C followed by washing with 1 mM MgCl2 (pH 7.4). The shape of these highly Ca2+-sensitive ghosts immediately changed from crenate to disc on addition of 1 mM Mg-ATP even at 6 degrees C in the presence of 10(-7)-10(-6) M Ca2+. A similar shape change was also observed when ghosts treated with 0.5% Triton X-100 (Triton shells) were used. Triton shells from fresh blood ghosts or from long-stored blood ghosts which had been preincubated with 2 mM Mg-ATP for 20 min at 37 degrees C shrank immediately in the presence of 10(-6) M Ca2+ and then swelled on addition of 1 mM Mg-ATP. The specificity to ATP and the dependency on ATP concentration are in agreement with those of the ghost shape change at step 2 (Jinbu, Y. et al., Biochem biophys res commun 112 (1983) 384-390) [18]. These results suggest that cytoskeletal protein phosphorylation enhances sensitivity to Ca2+ and induces erythrocyte shape change in the presence of physiological concentrations of ATP and Ca2+.     

Calcium uptake and release characteristics of the dense tubules of digitonin-permeabilized human platelets

The kinetics of ATP-driven Ca2+ uptake by the dense tubules were studied in digitonin-permeabilized human blood platelets. Digitonin at 3 micrograms/ml was shown capable of permeabilizing the plasma membrane to lactate dehydrogenase and the cytoplasmic Ca2+ indicator Quin2 without increasing the passive permeability of the dense tubular membrane for Ca2+. Experimentation was carried out with platelets treated with 3 micrograms/ml digitonin reisolated and resuspended in detergent-free medium ('digitonin-permeabilized' platelets). Active Ca2+ accumulation, which occurs over a period of minutes, was monitored by the increase in the fluorescence of chlorotetracycline after the addition of Mg-ATP (37 degrees C). The active uptake is inhibited by 15 microM trifluoperazine. The process is saturable with respect to external [Ca2+], with a Km of 180 +/- 5 nM and a Hill coefficient (n) of 1.40 +/- 0.05. Analysis of the maximal uptake in steady state gave similar results (Km = 160 +/- 5 nM, n = 1.50 +/- 0.05). The rate of uptake at [Ca2+] approximately Km is increased when the digitonin-permeabilized platelets are preincubated with 100 nM phorbol 12-myristate 13-acetate. Actively accumulated Ca2+ is rapidly released (less than 1 min) by addition of D-myo-inositol trisphosphate (IP3). The maximal extent of release is 50%; the EC50 for IP3 is approx. 12 microM. The data are compared with findings for fractionated dense tubular membrane vesicles and for the intact platelet.     

Ca2+ uptake in bovine adrenocortical microsomes: formation of phosphorylated intermediate of Ca2+ dependent ATPase

Bovine adrenocortical microsomes were prepared and partially purified by discontinuous sucrose density gradient. Light fractions of the microsomes at the interface between 15 and 30% sucrose solution, exhibited ATP dependent Ca2+ uptake. The Ca2+ uptake was dependent on temperature and stimulated by free Ca2+ (the concentration for half maximal activation = 1.0 microM) and Mg2+. The Ca2+ uptake was inhibited by ADP but not affected by 10 mM NaN3 or 0.5 mM ouabain. Calcium release from the microsomes was accelerated by a Ca2+ ionophore, A23187, but not by a Ca2+ antagonist, diltiazem. A microsomal protein with a molecular weight of 100-110 kDa was phosphorylated by [gamma-32P]ATP in the presence of Ca2+, and the Ca2+ dependency was over the same range as the Ca2+ uptake (the concentration for half maximal activation = 3.0 microM). The phosphorylated protein (EP) was stable at acidic pH but labile at alkaline pH and sensitive to hydroxylamine. The rate of EP formation at 0 degrees C in the presence of 1 microM ATP and 10 microM Ca2+ (half time = 0.2 s) was less than that in the sarcoplasmic reticulum (SR) of rabbit skeletal muscle (half time = 0.1 s). The rate of EP decomposition at 0 degrees C after adding EGTA was about 6.7 times slower (rate constant: kd = 4.3 X 10(-3) s-1) than that of SR. It was suggested that adrenocortical microsomes contain a Ca2+ dependent ATPase which function as a Ca2+ pump with similar properties to that of SR.     

A polymorphism in thrombospondin-1 associated with familial premature coronary heart disease causes a local change in conformation of the Ca2+-binding repeats

A single nucleotide polymorphism that substitutes a serine for an asparagine at residue 700 in the Ca2+-binding repeats of thrombospondin-1 is associated with familial premature coronary heart disease. We expressed the Ca2+-binding repeats alone (Ca) or with the third epidermal growth factor-like module (E3Ca), without (Asn-700) or with (Ser-700) the disease-associated polymorphism. The intrinsic fluorescence of a single tryptophan (Trp-698) adjacent to the polymorphic residue was quenched cooperatively by adding Ca2+. The third epidermal growth factor-like repeat dramatically altered the Ca2+-dependent fluorescence transition for the Asn-700 constructs; the half-effective concentration (EC50) of Ca Asn-700 was 390 microM, and the EC50 of E3Ca Asn-700 was 70 microM. The Ser-700 polymorphism shifted the EC50 to higher Ca2+ concentrations (Ca Ser-700 EC50 of 950 microM and E3Ca Ser-700 EC50 of 110 microM). This destabilizing effect is due to local conformational changes, as the Ser-700 polymorphism did not influence the secondary structure of E3Ca or Ca as assessed by far UV circular dichroism. At 200 microM Ca2+, in which both E3Ca Asn-700 and Ser-700 are in the Ca2+-replete conformation at 37 degrees C, the fluorescence of E3Ca Ser-700 reverted to the Ca2+-depleted spectrum at 50 degrees C compared with 65 degrees C for E3Ca Asn-700. These findings indicate that the Ser-700 polymorphism subtly but significantly sensitizes the calcium-binding repeats to removal of Ca2+ and thermal denaturation.     

The amplitude and time course of the myoplasmic free [Ca2+] transient in fast-twitch fibers of mouse muscle

Bundles of 10-100 fibers were dissected from the extensor digitorum longus muscle of mouse, mounted in an apparatus for optical recording, and stretched to long sarcomere length (> or = 3.6 microns). One fiber within the bundle was microinjected with furaptra, a fluorescent indicator that responds rapidly to changes in myoplasmic free [Ca2+] (delta [Ca2+]). Twitches and brief tetani were initiated by external stimulation. At myoplasmic furaptra concentrations of approximately 0.1 mM, the indicator's fluorescence signal during fiber activity (delta F/F) was well resolved. delta F/F was converted to delta [Ca2+] under the assumption that furaptra's myoplasmic dissociation constant for Ca2+ is 98 microM at 16 degrees C and 109 microM at 28 degrees C. At 16 degrees C, the peak amplitude of delta [Ca2+] during a twitch was 17.8 +/- 0.4 microM (+/-SEM; n = 8) and the half-width of delta [Ca2+] was 4.6 +/- 0.3 ms. At 28 degrees C, the peak and half-width values were 22.1 +/- 1.8 microM and 2.0 +/- 0.1 ms, respectively (n = 4). During a brief high-frequency tetanus, individual peaks of delta [Ca2+] were also well resolved and reached approximately the same amplitude that resulted from a single shock; the initial decays of delta [Ca2+] from peak slowed substantially during the tetanus. For a single twitch at 16 degrees C, the amplitude of delta [Ca2+] in fast-twitch fibers of mouse is not significantly different from that recently measured in fast- twitch fibers of frog (16.5 +/- 0.9 microM; Zhao, M., S. Hollingworth, and S.M. Baylor. 1996. Biophys. J. 70:896-916); in contrast, the half- width of delta [Ca2+] is surprisingly brief in mouse fibers, only about half that measured in frog (9.6 +/- 0.6 ms). The estimated peak rate at which Ca2+ is released from the sarcoplasmic reticulum in response to an action potential is also similar in mouse and frog, 140-150 microM/ms (16 degrees C).     

Calcium transport in brain synaptosomes during depolarization. The role of potential-dependent channels and Na+/Ca2+ metabolism

The contribution of Ca2+ channels and Na+/Ca2+ exchange to Ca2+ uptake in rat brain synaptosomes upon long- (t greater than or equal to 30 s) and short-term (t less than 30 s) depolarization by high K+ was studied by measuring the 45Ca content and free Ca2+ concentration (from Quin-2 fluorescence). At 37 degrees C, the system responsible for the K+-stimulated uptake of 45Ca (t greater than or equal to 30 s) and the Na+/Ca+ exchanger are characterized by a similar concentration dependence of external Ca2+ (Ca0(2+] and K0+ as well as by an equal sensitivity to verapamil (Ki = approximately 20-40 microM) and La2+ (Ki = approximately 50 microM). These data and the results from predepolarization suggest that the 45Ca entry into synaptosomes at t greater than or equal to 30 s is due to the activation of Na+/Ca+ exchange caused by its electrogenic component, while the insignificant contribution of Ca2+ channels can be accounted for by their inactivation. At low temperatures (2-4 degrees C) which decelerate the inactivation, the initial phase of 45Ca uptake is fully provided for by Ca2+ channels, showing a lower (as compared to the exchanger) affinity for Ca0(2+) (K0.5 greater than 1 mM)m a greater sensitivity to La3+ (Ki = approximately 0.2-0.3 microM) and verapamil (Ki = approximately 2-3 microM); these channels are fully inactivated by predepolarization with K0+, ouabain and batrachotoxin. The Ca2+ channels can be related to T-type channels, since they are not blocked by nicardipine and niphedipine.     

Ca2+ stimulates the Mg2(+)-ATPase activity of brush border myosin I with three or four calmodulin light chains but inhibits with less than two bound.

Brush border myosin I from chicken intestinal microvilli is a membrane-associated, single-headed myosin composed of a 119-kDa heavy chain and several calmodulin light chains. We first describe in detail a new procedure for the rapid purification of brush border myosin I in greater than 99% purity with a yield of 40%, significantly higher than for previous methods. The subunit stoichiometry was determined to be 4 calmodulin light chains/myosin I heavy chain by amino acid compositional analysis of the separated subunits. We have studied the effects of Ca2+ and temperature on dissociation of calmodulin from myosin I and on myosin I Mg2(+)-ATPase and contractile activities. At 30 degrees C the actin-activable ATPase activity is stimulated 2-fold at 10-700 microM Ca2+. Dissociation of 1 calmodulin occurs at 25-50 microM Ca2+, but this has no effect on actin activation. The contractile activity of myosin I, expressed as superprecipitation, is greatly enhanced by Ca2+ under conditions in which 1 calmodulin is dissociated. This calmodulin is thus not essential for actin activation or superprecipitation. Myosin I was found to be highly temperature-sensitive, with an increase to 37 degrees C resulting in dissociation of 1 calmodulin at below 10(-7) M Ca2+ and an additional 1.5 calmodulins at 1-10 microM Ca2+. A complete loss of actin activation accompanies the Ca2(+)-induced calmodulin dissociation at 37 degrees C. Our conclusion is that physiological levels of Ca2+ can either stimulate or inhibit the mechanoenzyme activities of brush border myosin I in vitro, with the mode of regulation determined by the number of associated calmodulin light chains.     

Fast kinetics of calcium release induced by myo-inositol trisphosphate in permeabilized rat hepatocytes

We used a stopped-flow method for determining the kinetic properties (between 10 ms and 10 s) of the Ca2+ release induced by inositol 1,4,5-trisphosphate (InsP3) in saponin-treated rat hepatocytes. Preliminary experiments ensured that the indicator was able to monitor rapid changes in free Ca2+ reliably. At 20 degrees C, a maximally efficient concentration of 10 microM InsP3 released Ca2+ with a half-time of 150-300 ms, the initial rate being about 1-2 nmol of Ca2+/mg of cell protein/s. The delay between the addition of 10 microM InsP3 and the onset of Ca2+ release was shorter than 20 ms, suggesting that the opening process of Ca2+ channels after binding of InsP3 to receptors is completed within a few milliseconds. Half-maximal initial rates for Ca2+ release occurred at about 1 microM InsP3 (Hill index was 1.6). The resulting Ca2+ efflux had a moderate temperature dependence. It could not be fitted to a single exponential. After low speed centrifugation of saponin-treated cells (1000 x g for 1 min), part of the InsP3-sensitive Ca2+ pool was recovered in the cell-free supernatant fraction, suggesting that the response to InsP3 arises from a vesicular fraction which may diffuse from the saponin-treated cells into the medium.     

Serratia marcescens Hemolysin (ShlA) Binds Artificial Membranes and Forms Pores in a Receptor-independent Manner

A non-discharge mutant of Paramecium tetraurelia (nd12-35 degrees C, lacking exocytotic response upon stimulation with the nonpermeable polycationic secretagogue aminoethyldextran, AED), in the pawnA genetic context (d4-500r, lacking ciliary voltage-dependent Ca2+ influx), was shown to lack (45)Ca2+ entry from outside upon AED stimulation. In contrast, cells grown at 25 degrees C behave like the wildtype. To check the functional properties in more detail, fluorochrome-loaded 35 degrees C cells were stimulated, not only with AED (EC(100) = 10(-6) M in wildtype cells), but also with 4-chloro-meta-cresol, (4CmC, 0.5 mM), a permeable activator of ryanodine receptor-type Ca2+ release channels, usually at extracellular [Ca2+] of 50 microM, and eventually with a Ca2+ chelator added. We confirm that pwA-nd12(35 degrees C) cells lack any Ca2+ influx and any exocytosis of trichocysts in response to any stimulus. As we determined by x-ray microanalysis, total calcium content in alveolar sacs (subplasmalemmal stores) known to be mobilized upon exocytosis stimulation in wild-type cells, contain about the same total calcium in 35 degrees C as in 25 degrees C cells, and Ca2+ mobilization from alveoli by AED or 4CmC is also nearly the same. Due to the absence of any AED-induced Ca2+ influx in 35 degrees C cells and normal Ca2+ release from stores found by x-ray microanalysis one can exclude a "CICR"-type mechanism (Ca2+-induced Ca2+ release) and imply that normally a store-operated Ca2+ ("SOC") influx would occur (as in 25 degrees C cells). Furthermore, 35 degrees C cells display a significantly lower basal intracellular [Ca2+], so that any increase upon stimulation may be less expressed or even remain undetected. Under these conditions, any mobilization of Ca2+ from stores cannot compensate for the lack of Ca2+ influx, particularly since normally both components have to cooperate to achieve full exocytotic response. Also striking is our finding that 35 degrees C cells are unable to perform membrane fusion, as analyzed with the Ca2+ ionophore, A23187. These findings were corroborated by cryofixation and freeze-fracture analysis of trichocyst docking sites after AED or 4CmC stimulation, which also revealed no membrane fusion. In sum, in nd12 cells increased culture temperature entails multiple defects, notably insensitivity to any Ca2+ signal, which, moreover, cannot develop properly due to a lower basal [Ca2+] level and the lack of Ca2+ influx, despite normal store activation.