Cellular Ca2+ ATPase activity in diabetes mellitus

Basal and maximal Ca2+ ATPase activity was studied in erythrocytes of 29 healthy controls, 15 patients with insulin-dependent diabetes mellitus (IDDM) and 22 patients with non-insulin-dependent diabetes mellitus (NIDDM). Basal and maximal Ca2+ ATPase activity was significantly decreased in insulin-dependent diabetes mellitus (8.4 +/- 0.5 and 22.5 +/- 1.1 pmol/10(6) RBC/min) and non-insulin-dependent diabetes mellitus (7.3 +/- 1.0 and 18.6 +/- 1.8 pmol/10(6) RBC/min) compared to healthy controls (9.3 +/- 1.0 and 24.6 +/- 1.1 pmol/10(6) RBC/min). Maximal Ca2+ ATPase activity showed a significant correlation to systolic blood pressure in both insulin-dependent diabetes mellitus and non-insulin-dependent diabetes mellitus. There was no significant correlation of maximal Ca2+ ATPase activity to fasting serum glucose concentration and to HbA1 levels. Maximal Ca2+ ATPase activity was significantly correlated to creatinine clearance in non-insulin-dependent diabetes mellitus, but not in insulin-dependent diabetes mellitus. It is concluded that a decreased cellular Ca2+ ATPase activity may predispose to the development of hypertension in diabetes mellitus.     

Creatine supplementation improves intracellular Ca2+ handling and survival in mdx skeletal muscle cells

Rapid review, digital recording, on-line quantification, and three-dimensional reconstruction are all essential in the evaluation of intracoronary ultrasound images during coronary interventions. We describe a low-cost method that offers all these necessary features. The proposed method uses the QuickTime compatible video digitizers of standard multimedia Apple Macintosh or PowerPC desktop computers and the freeware software Object Image 1.60.     

Effects of Mg2+ on Ca2+ handling by the sarcoplasmic reticulum in skinned skeletal and cardiac muscle fibres

The influence of myoplasmic Mg2+ (0.05-10 mM) on Ca2+ accumulation (net Ca2+ flux) and Ca2+ uptake (pump-driven Ca2+ influx) by the intact sarcoplasmic reticulum (SR) was studied in skinned fibres from the toad iliofibularis muscle (twitch portion), rat extensor digitorum longus (EDL) muscle (fast twitch), rat soleus muscle (slow twitch) and rat cardiac trabeculae. Ca2+ accumulation was optimal between 1 and 3 mM Mg2+ in toad fibres and reached a plateau between 1 and 10 mM Mg2+ in the rat EDL fibres and between 3 and 10 mM Mg2+ in the rat cardiac fibres. In soleus fibres, optimal Ca2+ accumulation occurred at 10 mM Mg2+. The same trend was obtained with all preparations at 0.3 and 1 microM Ca2+. Experiments with 2,5-di-(tert-butyl)-1,4-benzohydroquinone, a specific inhibitor of the Ca2+ pump, revealed a marked Ca2+ efflux from the SR of toad iliofibularis fibres in the presence of 0.2 microM Ca2+ and 1 mM Mg2+. Further experiments indicated that the SR Ca2+ leak could be blocked by 10 microM ruthenium red without affecting the SR Ca2+ pump and this allowed separation between SR Ca2+ uptake and SR Ca2+ accumulation. At 0.3 microM Ca2+, Ca2+ uptake was optimal with 1 mM Mg2+ in the toad iliofibularis and rat EDL fibres and between 1 and 10 mM Mg2+ in the rat soleus and trabeculae preparations. At higher [Ca2+] (1 microM), Ca2+ uptake was optimal with 1 mM Mg2+ in the iliofibularis fibres and between 1 and 3 mM Mg2+ in the EDL fibres.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Ca2+ transport mechanisms of mitochondria and Ca2+ uptake from physiological-type Ca2+ transients

Mitochondria contain a sophisticated system for transporting Ca2+. The existence of a uniporter and of both Na+-dependent and -independent efflux mechanisms has been known for years. Recently, a new mechanism, called the RaM, which seems adapted for sequestering Ca2+ from physiological transients or pulses has been discovered. The RaM shows a conductivity at the beginning of a Ca2+ pulse that is much higher than the conductivity of the uniporter. This conductivity decreases very rapidly following the increase in [Ca2+] outside the mitochondria. This decrease in the Ca2+ conductivity of the RaM is associated with binding of Ca2+ to an external regulatory site. When liver mitochondria are exposed to a sequence of pulses, uptake of labeled Ca2+ via the RaM appears additive between pulses. Ruthenium red inhibits the RaM in liver mitochondria but much larger amounts are required than for inhibition of the mitochondrial Ca2+ uniporter. Spermine, ATP and GTP increase Ca2+ uptake via the RaM. Maximum uptake via the RaM from a single Ca2+ pulse in the physiological range has been observed to be approximately 7 nmole/mg protein, suggesting that Ca2+ uptake via the RaM and uniporter from physiological pulses may be sufficient to activate the Ca2+-sensitive metabolic reactions in the mitochondrial matrix which increase the rate of ATP production. RaM-mediated Ca2+ uptake has also been observed in heart mitochondria. Evidence for Ca2+ uptake into the mitochondria in a variety of tissues described in the literature is reviewed for evidence of participation of the RaM in this uptake. Possible ways in which the differences in transport via the RaM and the uniporter may be used to differentiate between metabolic and apoptotic signaling are discussed.     

Polymorphonuclear leukocyte membrane fluidity and cytosolic Ca2+ concentration in diabetes mellitus

OBJECTIVE: To establish a rapid and efficient technique of constructing human chromosomal band specific probe pools and their libraries. METHODS: A modified method of combining chromosome microdissection with degenerate oligonucleotide primed PCR(DOP-PCR) was used. 3p23-p26, 3q21-q22 and 4p12- p16 band from human chromosomes were microdissected and amplified as probe pools. The origins of the PCR products were determined by chromosome fluorescence in situ hybridization. The PCR products and pUC19 were digested by Xho I and Sal I respectively, and linke up. The DH5alpha were transformed by the recombinated vectors as the specific band libraries. The inserts were digested by EcoR I and Hind III, then measured by electrophoretic analysis. And the copies of inserts were identified by in situ bacterial colony hybridization with genomic DNA. RESULTS: All the three probe pools showed the special yellow-green signals in their microdissection responsible bands. The sizes of DOP-PCR products ranged from 300bp to 1800bp. 3q21-q22 probe pool generated about 1.2 x 10(4) clones. The average size of inserts was about 420bp by analysis of 30 positive clones. The rate of single-copy and low-repeated sequences was about 81%(178/220), while the rate of middle-repeated and high- repeated sequences was about 19%(42/220). CONCLUSION: The results proved that the modified microdissection combining DOP-PCR technique provided a simple and efficient method to construct the human chromosome band-specific probe pools and might contribute to gene cloning and complete sequencing of human genome.     

Saltatory propagation of Ca2+ waves by Ca2+ sparks

Punctate releases of Ca2+, called Ca2+ sparks, originate at the regular array of t-tubules in cardiac myocytes and skeletal muscle. During Ca2+ overload sparks serve as sites for the initiation and propagation of Ca2+ waves in myocytes. Computer simulations of spark-mediated waves are performed with model release sites that reproduce the adaptive Ca2+ release observed for the ryanodine receptor. The speed of these waves is proportional to the diffusion constant of Ca2+, D, rather than D, as is true for reaction-diffusion equations in a continuous excitable medium. A simplified "fire-diffuse-fire" model that mimics the properties of Ca2+-induced Ca2+ release (CICR) from isolated sites is used to explain this saltatory mode of wave propagation. Saltatory and continuous wave propagation can be differentiated by the temperature and Ca2+ buffer dependence of wave speed.     

Effects of angiotensin II on inotropy and intracellular Ca2+ handling in normal and hypertrophied rat myocardium

The direct inotropic effect of angiotensin II on the myocardium is still controversial and little information exists as to its potential modification by heart disorders. Therefore, this study performed simultaneous measurements of isometric force and intracellular Ca2+ concentrations ([Ca2+]i) in left ventricular papillary muscles from sham-operated and aortic-banded rats at 10 weeks post-surgery. Angiotensin II (10(-6) M) induced a reduction of peak systolic [Ca2+]i (0.56 +/- 0.03 to 0.48 +/- 0.04 microM; P<0.05) and a parallel but insignificant diminution of developed tension (10.5 +/- 1.3 to 9.6 +/- 0.8 mN/mm2) in normal papillary muscles from sham-operated animals. Hypertrophied papillary muscles from aortic-banded rats demonstrated a significant decline in both peak systolic [Ca2+]i (0.51 +/- 0.02 to 0.44 +/- 0.01 microM; P<0.05) and developed tension (8.4 +/- 1.1 to 6.8 +/- 1.7 mN/mm2; P<0.05) after addition of angiotensin II. The time courses of the mechanical contraction and the intracellular Ca2+ signal were prolonged by angiotension II in both groups. Isoproterenol dose-dependently increased developed tension and peak systolic [Ca2+]i in papillary muscles from sham-operated rats. In contrast, the positive inotropic response to isoproterenol was markedly reduced in hypertrophied muscles despite a seemingly unimpaired increase in peak systolic [Ca2+]i. Pretreatment with angiotensin II (10(-6) M) resulted in a significant attenuation of the systolic [Ca2+]i response to isoproterenol stimulation in both normal and hypertrophied papillary muscles. Neither the bradykinin B2 antagonist icatibent (10(-6) M) nor the nitric oxide (NO) inhibitor L-NMMA (10(-6) M) abolished the depressant effects of angiotension II. Thus, ANG II induces a parallel decline of the mechanical performance and Ca2+ availability in rat myocardium. These effects are more distinct in hypertrophied than in normal muscle and become accentuated during beta-adrenergic stimulation. The underlying mechanism is not associated with the NO pathway but might involve a negative functional coupling between the angiotensin and beta-adrenergic-receptor complex.     

Studies of Ca2+ binding in spinach photosystem II using 45Ca2+

The Ca(2+)-binding properties of photosystem II were investigated with radioactive 45Ca2+. PS II membranes, isolated from spinach grown on a medium containing 45Ca2+, contained 1.5 Ca2+ per PS II unit. Approximately half of the incorporated radioactivity was lost after incubation for 30 h in nonradioactive buffer. About 1 Ca2+/PS II bound slowly to Ca(2+)-depleted membranes in the presence of the extrinsic 16- and 23-kDa polypeptides in parallel with restoration of oxygen-evolving activity. The binding was heterogeneous with dissociation constants of 60 microM (0.7 Ca2+/PS II) and 1.7 mM (0.3 Ca2+/PS II), respectively, which could reflect different affinities of the dark-stable S-states for Ca2+. The reactivation of oxygen-evolving activity closely followed the binding of Ca2+, showing that a single exchangeable Ca2+ per PS II is sufficient for the water-splitting reaction to function. In PS II, depleted of the 16- and 23-kDa polypeptides, about 0.7 exchangeable Ca2+/PS II binds with a dissociation constant of 26 microM, while 0.3 Ca2+ binds with a much weaker affinity (Kd > 0.5 mM). The rate of binding of Ca2+ in the absence of the two extrinsic polypeptides was significantly higher than with the polypeptides bound. The rate of dissociation of bound Ca2+ in the dark, which had a half-time of about 80 h in intact PS II, increased in the absence of the 16- and 23-kDa polypeptides and showed a further increase after the additional removal of the 33-kDa protein and manganese. The rate of dissociation was also significantly faster in weak light than in the dark regardless of the presence or absence of the 16- and 23-kDa polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+ in the dense tubules: a model of platelet Ca2+ load

In this work, we explored the relationship between the freely exchangeable Ca2+ (FECa2+) in the dense tubules (DT) and the sarco(endo)plasmic reticulum (SER) Ca2+-ATPase (SERCA) in circulating human platelets and examined the relationship between blood pressure (BP) and these platelet parameters. Studying platelets from 32 healthy men, we showed that the maximal reaction velocity (Vmax) of the SERCA significantly correlated with FECa2+ in the DT and with the protein expressions of SERCA 2 and 3. BP positively correlated with both the Vmax of the SERCA (r=.462, P=.010) and the FECa2+ sequestered in the DT (r=.492, P=.005). The relationships between these platelet Ca2+ parameters and BP were in part confounded by increased levels of serum triglycerides and diminished HDL cholesterol with a higher BP. No correlation was observed between the resting cytosolic Ca2+ and BP. Collectively, these findings indicate that (1) an increase in the cellular Ca2+ load in platelets is expressed by a higher activity of the SERCA and an increase in the expressions of SERCA 2 and 3 proteins, coupled with an increase in the FECa2+ in the DT, and (2) a higher BP is associated with an increase in platelet Ca2+ load in human beings, expressed by a rise in the FECa2+ in the DT and the upregulation of SERCA activity.     

Renal potassium handling in aging rats

The use of Gore-Tex (W. L. Gore & Associates, Flagstaff, AZ, U.S.A.) spheres as orbital implants is investigated. The left eyes of six New Zealand white rabbits were enucleated and spherical implants made of modified Gore-Tex were implanted. After 6 weeks of follow-up, the implants were harvested. No rabbit developed a postoperative infection and no cases of exposure or extrusion were noted. Histopathologic study revealed varying degrees of acute and chronic inflammation surrounding each implant. There was also evidence of inflammatory infiltration and fibrovascular ingrowth into each implant to a maximum distance of 500 microns. This preliminary study demonstrates that the Gore-Tex implant is well tolerated in vivo, allows cellular ingrowth, and may have a role as a permanent implant.     

Inositol trisphosphate receptors: Ca2+-modulated intracellular Ca2+ channels

The three subtypes of inositol trisphosphate (InsP3) receptor expressed in mammalian cells are each capable of forming intracellular Ca2+ channels that are regulated by both InsP3 and cytosolic Ca2+. The InsP3 receptors of many, though perhaps not all, tissues are biphasically regulated by cytosolic Ca2+: a rapid stimulation of the receptors by modest increases in Ca2+ concentration is followed by a slower inhibition at higher Ca2+ concentrations. Despite the widespread occurrence of this form of regulation and the belief that it is an important element of the mechanisms responsible for the complex Ca2+ signals evoked by physiological stimuli, the underlying mechanisms are not understood. Both accessory proteins and Ca2+-binding sites on InsP3 receptors themselves have been proposed to mediate the effects of cytosolic Ca2+ on InsP3 receptor function, but the evidence is equivocal. The effects of cytosolic Ca2+ on InsP3 binding and channel opening, and the possible means whereby the effects are mediated are discussed in this review.     

Cyclopiazonic acid and thapsigargin induce platelet aggregation resulting from Ca2+ influx through Ca2+ store-activated Ca2+-channels

The effects of cyclopiazonic acid and thapsigargin, selective inhibitors of the endoplasmic reticulum Ca2+-ATPase pump, on the platelet aggregation were investigated using washed rat platelets prepared by chromatography on Sepharose 2B columns. In Ca2+-free medium, cyclopiazonic acid and thapsigargin did not induce aggregation, but in the presence of 1 mM Ca2+, platelet aggregation was induced in a concentration-dependent manner. Cyclopiazonic acid- and thapsigargin-induced platelet aggregation was blocked by 1 mM Ni2+ but not by 100 microM indomethacin or 1 microM nifedipine. In aequorin-loaded platelets, cyclopiazonic acid and thapsigargin caused sustained elevation of the cytosolic Ca2+ concentration, an effect which was blocked by Ni2+, a non-selective Ca2+ channel blocker and SK&F 96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenyl]-1H-imidazole hydrochloride), a putative receptor-operated Ca2+ channel antagonist. The above results indicated that both cyclopiazonic acid and thapsigargin induced platelet aggregation and elevation of cytosolic Ca2+ concentration, that extracellular Ca2+ was essential for cyclopiazonic acid- and thapsigargin-induced platelet aggregation, and that platelet aggregation may be associated with Ca2+ influx through Ca2+ store-activated Ca2+ channels.     

Ca2+ pool emptying stimulates Ca2+ entry activated by S-nitrosylation

The entry of Ca2+ following Ca2+ pool release is a major component of Ca2+ signals; yet despite intense study, how "store-operated" entry channels are activated is unresolved. Because S-nitrosylation has become recognized as an important regulatory modification of several key channel proteins, its role in Ca2+ entry was investigated. A novel class of lipophilic NO donors activated Ca2+ entry independent of the well defined NO target, guanylate cyclase. Strikingly similar entry of Ca2+ induced by cell permeant alkylators indicated that this Ca2+ entry process was activated through thiol modification. Significantly, Ca2+ entry activated by either NO donors or alkylators was highly stimulated by Ca2+ pool depletion, which increased both the rate of Ca2+ release and the sensitivity to thiol modifiers. The results indicate that S-nitrosylation underlies activation of an important store-operated Ca2+ entry mechanism.     

Selective activation of Ca2+-activated K+ channels by co-localized Ca2+ channels in hippocampal neurons

Calcium entry through voltage-gated calcium channels can activate either large- (BK) or small- (SK) conductance calcium-activated potassium channels. In hippocampal neurons, activation of BK channels underlies the falling phase of an action potential and generation of the fast afterhyperpolarization (AHP). In contrast, SK channel activation underlies generation of the slow AHP after a burst of action potentials. The source of calcium for BK channel activation is unknown, but the slow AHP is blocked by dihydropyridine antagonists, indicating that L-type calcium channels provide the calcium for activation of SK channels. It is not understood how this specialized coupling between calcium and potassium channels is achieved. Here we study channel activity in cell-attached patches from hippocampal neurons and report a unique specificity of coupling. L-type channels activate SK channels only, without activating BK channels present in the same patch. The delay between the opening of L-type channels and SK channels indicates that these channels are 50-150 nm apart. In contrast, N-type calcium channels activate BK channels only, with opening of the two channel types being nearly coincident. This temporal association indicates that N and BK channels are very close. Finally, P/Q-type calcium channels do not couple to either SK or BK channels. These data indicate an absolute segregation of coupling between channels, and illustrate the functional importance of submembrane calcium microdomains.     

Fluorescence probe study of the lumenal Ca2+ of the sarcoplasmic reticulum vesicles during Ca2+ uptake and Ca2+ release

A limited amount of information is available about the lumenal Ca2+ kinetics of the sarcoplasmic reticulum (SR). Incubation of mag-fura-2AM permitted to incorporate a sufficient amount of the probe into the SR vesicles, as determined by Mn2+ quenching. Rapid changes in the lumenal [Ca2+] ([Ca2+]lum) during Ca2+ uptake and release could be monitored by following the signal derived from the lumenal probe while clamping the extra-vesicular Ca2+ ([Ca2+]ex) at various desired levels with a BAPTA/Ca buffer. Changes in the [Ca2+]lum during uptake and release show the characteristics intrinsic to the SR Ca2+ pump (the [Ca2+]ex-dependence of the activation and inhibition by thapsigargin) and the Ca2+ release channel (blocking by ruthenium red), respectively. A new feature revealed by the [Ca2+]lum measurement is that during the uptake reaction the free [Ca2+]lum showed a significant oscillation. Several pieces of evidence suggest that this is due to some interactions between the Ca2+ pump and lumenal proteins.     

Ca2+ spike initiation from sensitized inositol 1,4,5-trisphosphate-sensitive Ca2+ stores in megakaryocytes

Ca(2+)-mediated Ca2+ spikes were analysed in fura-2-loaded megakaryocytes. Direct Ca2+ loading using whole-cell dialysis induced an all-or-none Ca2+ spike on top of a tonic increase in cellular Ca2+ concentration ([Ca2+]i) with a latency of 3-7 s. The latency decreased with increasingly higher concentrations of Ca2+ in the dialysing solution. Spike size and its initiation did not correlate with the tonic level of [Ca2+]i. Thapsigargin completely abolished the Ca(2+)-induced spike initiation, suggesting that Ca2+ spikes originate from thapsigargin-sensitive Ca2+ pools. An inhibitor of phosphatidylinositide-specific phospholipase C (PLC), 2-nitro-4-carboxyphenyl-N,N-diphenyl-carbamate prolonged the latency without changes of spike size in most cases (6/9 cells), but abolished the spike initiation in the other cells (3/9). The results suggest that an increase in [Ca2+]i charges up the inositol-1,4,5-trisphosphate-(InsP3)- and thapsigargin-sensitive Ca2+ pools which progressively sensitize to low or slightly elevated levels of InsP3 by the action of Ca(2+)-dependent PLC until a critical Ca2+ content is reached, and then the Ca2+ spike is triggered. Thus, the limiting step of Ca2+ spike triggering is the initial filling process and the level of InsP3 in megakaryocytes.     

Canine bronchial sustained contraction in Ca2+-free medium: role of intracellular Ca2+

We evaluated whether cartilage was a source of Ca2+ and the possible role of Ca2+ recycling in the sustained bronchial contraction (SBC) induced by carbachol (Cch) in Ca2+-free medium. Canine first-order bronchi were studied with cartilage and epithelium (+CAR + EPI) and without these structures individually (-CAR + EPI and +CAR - EPI) or together (-CAR - EPI). After cartilage removal (-CAR - EPI or -CAR + EPI) Cch produced a transient contraction in Ca2+-free medium. Removal of the epithelium alone had minor effects on the magnitude of the SBC but increased the effect of removal of cartilage to diminish the SBC. Bronchial strips with cartilage were able to respond to Cch with lower Ca2+ concentrations (10-100 microM) than could dissected preparations. Preincubation with BAY K 8644 (30-1000 nM) or 60 mM KCl or -CAR - EPI tissues converted the transient contractions to Cch in Ca2+-free medium to sustained contractions. In microelectrode studies, 50 nM Cch induced membrane oscillations in solutions with 2.5 mM Ca2+ in bronchial preparations, plus or minus cartilage, and in undissected tissues in Ca2+-free medium but not in -CAR - EPI tissues. Preincubation with 1 microM BAY K 8644 in Ca(2+)-free medium restored these oscillations in -CAR - EPI tissues. The release of 45Ca2+ from cartilage was too rapid to provide a reservoir of Ca2+ to support multiple SBCs in Ca2+-free medium. Moreover, in the Ca2+-free medium (with 10 nM Ca2+ after tissue +CAR + EPI incubation) excitatory junction potentials rapidly disappeared. Addition of 1 microM nifedipine or 1 mM EGTA during the SBC of +CAR + EPI tissues produced complete relaxation. A transient contraction to Cch occurred with prior addition of nifedipine. Inhibition of the sarcoplasmic reticulum Ca2+ pump by tissue incubation with cyclopiazonic acid (CPA; 10 microM), or briefly with 1 mM EGTA significantly diminished the SBC induced by Cch in Ca2+-free medium. CPA and EGTA together abolished the Cch-induced SBC. Thus, cartilage plays a more complex role than as a Ca2+ reservoir to support the SBC induced by Cch in Ca2+-free solution; its removal affects the process supporting SBCs involving intracellular Ca2+ storage and Ca2+ entrance through voltage-dependent channels.     

Role of Ca2+/K+ ion exchange in intracellular storage and release of Ca2+

Although fluctuations in cytosolic Ca2+ concentration have a crucial role in relaying intracellular messages in the cell, the dynamics of Ca2+ storage in and release from intracellular sequestering compartments remains poorly understood. The rapid release of stored Ca2+ requires large concentration gradients that had been thought to result from low-affinity buffering of Ca2+ by the polyanionic matrices within Ca2+-sequestering organelles. However, our results here show that resting luminal free Ca2+ concentration inside the endoplasmic reticulum and in the mucin granules remains at low levels (20-35 microM). But after stimulation, the free luminal [Ca2+] increases, undergoing large oscillations, leading to corresponding oscillations of Ca2+ release to the cytosol. These remarkable dynamics of luminal [Ca2+] result from a fast and highly cooperative Ca2+/K+ ion-exchange process rather than from Ca2+ transport into the lumen. This common paradigm for Ca2+ storage and release, found in two different Ca2+-sequestering organelles, requires the functional interaction of three molecular components: a polyanionic matrix that functions as a Ca2+/K+ ion exchanger, and two Ca2+-sensitive channels, one to import K+ into the Ca2+-sequestering compartments, the other to release Ca2+ to the cytosol.     

Ca2+ transients in cardiac myocytes measured with high and low affinity Ca2+ indicators

Intracellular calcium ion ([Ca2+]i) transients were measured in voltage-clamped rat cardiac myocytes with fura-2 or furaptra to quantitate rapid changes in [Ca2+]i. Patch electrode solutions contained the K+ salt of fura-2 (50 microM) or furaptra (300 microM). With identical experimental conditions, peak amplitude of stimulated [Ca2+]i transients in furaptra-loaded myocytes was 4- to 6-fold greater than that in fura-2-loaded cells. To determine the reason for this discrepancy, intracellular fura-2 Ca2+ buffering, kinetics of Ca2+ binding, and optical properties were examined. Decreasing cellular fura-2 concentration by lowering electrode fura-2 concentration 5-fold, decreased the difference between the amplitudes of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes by twofold. Thus, fura-2 buffers [Ca2+]i under these conditions; however, Ca2+ buffering is not the only factor that explains the different amplitudes of the [Ca2+]i transients measured with these indicators. From the temporal comparison of the [Ca2+]i transients measured with fura-2 and furaptra, the apparent reverse rate constant for Ca2+ binding of fura-2 was at least 65s-1, much faster than previously reported in skeletal muscle fibers. These binding kinetics do not explain the difference in the size of the [Ca2+]i transients reported by fura-2 and furaptra. Parameters for fura-2 calibration, Rmin, Rmax, and beta, were obtained in salt solutions (in vitro) and in myocytes exposed to the Ca2+ ionophore, 4-Br A23187, in EGTA-buffered solutions (in situ). Calibration of fura-2 fluorescence signals with these in situ parameters yielded [Ca2+]i transients whose peak amplitude was 50-100% larger than those calculated with in vitro parameters. Thus, in vitro calibration of fura-2 fluorescence significantly underestimates the amplitude of the [Ca2+]i transient. These data suggest that the difference in amplitude of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes is due, in part, to Ca2+ buffering by fura-2 and use of in vitro calibration parameters.     

Termination of cytosolic Ca2+ signals: Ca2+ reuptake into intracellular stores is regulated by the free Ca2+ concentration in the store lumen

The mechanism by which agonist-evoked cytosolic Ca2+ signals are terminated has been investigated. We measured the Ca2+ concentration inside the endoplasmic reticulum store of pancreatic acinar cells and monitored the cytoplasmic Ca2+ concentration by whole-cell patch-clamp recording of the Ca2+-sensitive currents. When the cytosolic Ca2+ concentration was clamped at the resting level by a high concentration of a selective Ca2+ buffer, acetylcholine evoked the usual depletion of intracellular Ca2+ stores, but without increasing the Ca2+-sensitive currents. Removal of acetylcholine allowed thapsigargin-sensitive Ca2+ reuptake into the stores, and this process stopped when the stores had been loaded to the pre-stimulation level. The apparent rate of Ca2+ reuptake decreased steeply with an increase in the Ca2+ concentration in the store lumen and it is this negative feedback on the Ca2+ pump that controls the Ca2+ store content. In the absence of a cytoplasmic Ca2+ clamp, acetylcholine removal resulted in a rapid return of the elevated cytoplasmic Ca2+ concentration to the pre-stimulation resting level, which was attained long before the endoplasmic reticulum Ca2+ store had been completely refilled. We conclude that control of Ca2+ reuptake by the Ca2+ concentration inside the intracellular store allows precise Ca2+ signal termination without interfering with store refilling.