Ca2+ and the regulation of neurotransmitter secretion

A new binary polymer matrix tablet for oral administration was developed. The system will deliver drug at variable rates according to zero-order kinetics for total drug content and is manufactured by direct compression technology. Highly methoxylated pectin and hydroxypropyl methylcellulose (HPMC) at different ratios were used as major formulation components, and prednisolone was used as the drug model. The results indicate that by increasing pectin:HPMC ratios, release rates are increased, but zero-order kinetics prevail throughout the dissolution period (e.g., 3-22 h). Different pectin:HPMC ratios provide a range of viscosities that modulates drug release and results in rapid hydration/gelation in both axial and radial directions, as evidenced by photomicrographic pictures. This hydration-gelation contributes to the development of swelling/erosion boundaries and consequently to constant drug release. Combination of these particular polymers facilitates rapid formation of necessary boundaries (i.e., gel layer and solid core boundaries) to control overall mass transfer processes. The drug fraction released (Mt/M infinity), release kinetics, and mechanism of release were analyzed by applying the simple power law expression Mt/M infinity = kt(n), where k is a kinetic constant and the exponent n is indicative of the release mechanism. The calculated n values for pectin:HPMC ratios of 4:5, 3:6, and 2:7 were >0.95, which is indicative of a Case II transport mechanism (polymer relaxation/dissolution). The achievement of total zero-order kinetics is due to the predictable swelling/erosion and final polymer chain deaggregation and dissolution that is regulated by the gelling characteristics of polymers in the formulation.     

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)     

Does a decrease in subplasmalemmal Ca2+ explain how store-operated Ca2+ channels are opened?

The activity of each of 99 intraparietal neurons was studied in three awake-behaving rhesus monkeys (Macaca mulatta) while subjects performed 100-900 delayed saccade trials. On each trial, a saccadic target was presented at one location selected randomly from a grid of 441 locations spanning 40 degrees of horizontal and vertical visual space. Individual neurons in our population were sensitive to both the direction and amplitude of saccades. Response fields, which plotted firing rate as a function of the horizontal and vertical amplitude of movements for each neuron, were characterized by a Cartesian two-dimensional gaussian model. The goodness-of-fit of these gaussian models was tested by: (1) comparing observed responses with predicted responses for each movement; and (2) by computing the percentage of variance explained by each model. Cartesian Gaussian models provided a good fit to the response fields of most neurons. Across our population, the Gaussian fit to the response field of each neuron accounted for more of the variance in neuronal activity when the data were plotted with regard to the horizontal and vertical amplitude of the saccade than when the same data were plotted with regard to the position of the saccadic target. The Gaussian functions were used to estimate the eccentricity and spatial tuning breadth of each neuronal response field. Modal response field radius was less than 5 degrees, whereas mean response field radius was about 10 degrees. Linear regression analysis demonstrated that response field eccentricity accounted for less than 30% of the variance in response field radius. Analysis of the horizontal distribution of response field centers showed an approximately normal distribution around central fixation. Most histologically recovered neurons were located on the lateral bank of the intraparietal sulcus, although a small number of saccade-related neurons were recorded from Brodmann's area 5 on the medial bank of the intraparietal sulcus.     

Ca2+-dependent structural changes in C-type mannose-binding proteins

C-type animal lectins are a diverse family of proteins which mediate cell-surface carbohydrate-recognition events through a conserved carbohydrate-recognition domain (CRD). Most members of this family possess a carbohydrate-binding activity that depends strictly on the binding of Ca2+ at two sites, designated 1 and 2, in the CRD. The structural transitions associated with Ca2+ binding in C-type lectins have been investigated by determining high-resolution crystal structures of rat serum mannose-binding protein (MBP) bound to one Ho3+ in place of Ca2+, and the apo form of rat liver MBP. The removal of Ca2+ does not affect the core structure of the CRD, but dramatic conformational changes occur in the loops. The most significant structural change in the absence of Ca2+ is the isomerization of a cis-peptide bond preceding a conserved proline residue in Ca2+ site 2. This bond adopts the cis conformation in all Ca2+-bound structures, whereas both cis and trans conformations are observed in the absence of Ca2+. The pattern of structural changes in the three loops that interact with Ca2+ is dictated in large part by the conformation of the prolyl peptide bond. The highly conserved nature of Ca2+ site 2 suggests that the transitions observed in MBPs are general features of Ca2+ binding in C-type lectins.     

Capacitative Ca2+ entry and the regulation of smooth muscle tone

In many non-excitable cells, activation of phospholipase C-linked receptors results in a biphasic increase in the cytosolic Ca2+ concentration; an initial transient increase, owing to the release of Ca2+ from the endoplasmic/sarcoplasmic reticulum (ER/SR), is followed by a much smaller but sustained elevation, which often involves capacitative Ca2+ entry, where depletion of Ca2+ within the ER signals the opening of store-operated Ca2+ channels in the plasma membrane. However, in excitable cells such as smooth muscle, the role of capacitative Ca2+ entry is less clear and the main Ca2+ entry mechanisms responsible for sustained cellular activation have been considered to be either voltage-operated or receptor-operated Ca2+ channels. Although store-regulated Ca2+ entry was known to occur following agonist activation of smooth muscle, it was believed to be important only for the re-filling of the depleted SR and not as a source of activator Ca2+ for the contractile mechanisms. Here, Alan Gibson, Ian McFadzean, Pat Wallace and Christopher Wayman review recent evidence that capacitative Ca2+ entry might indeed be important for the regulation of smooth muscle tone, and that it might provide an important for pharmacological intervention.     

Ca2+-dependent exocytosis in mast cells is stimulated by the Ca2+ sensor, synaptotagmin I

Mast cells secrete a variety of biologically active substances that mediate inflammatory responses. Synaptotagmin(s) (Syts) are a gene family of proteins that are implicated in the control of Ca2+-dependent exocytosis. In the present study, we investigated the possible occurrence and functional involvement of Syt in the control of mast cell exocytosis. Here, we demonstrate that both connective tissue type and mucosal-like mast cells express Syt-immunoreactive proteins, and that these proteins are localized almost exclusively to their secretory granules. Furthermore, expression of Syt I, the neuronal Ca2+ sensor, in rat basophilic leukemia cells (RBL-2H3), a tumor analogue of mucosal mast cells, resulted in prominent potentiation and acceleration of Ca2+-dependent exocytosis. Therefore, these findings implicate Syt as a Ca2+ sensor that mediates regulated secretion in mast cells to calcium ionophore.     

Binding of Ca2+ and Zn2+ to human nuclear S100A2 and mutant proteins

The Ca2+-binding protein S100A2 is an unusual member of the S100 family, characterized by its nuclear localization and down-regulated expression in tumorigenic cells. In this study, we investigated the properties of human recombinant S100A2 (wtS100A2) and of two mutants in which the amino-terminal Ca2+-binding site I (N mutant) and in addition the carboxyl-terminal site II (NC mutant) were replaced by the canonical loop (EF-site) of alpha-parvalbumin. Size exclusion chromatography and circular dichroism showed that, irrespective of the state of cation binding, wtS100A2 and mutants are dimers and rich in alpha-helical structure. Flow dialysis revealed that wtS100A2 binds four Ca2+ atoms per dimer with pronounced positive cooperativity. Both mutants also bind four Ca2+ atoms but with a higher affinity than wtS100A2 and with negative cooperativity. The binding of the first two Ca2+ ions to the N mutant occurred with 100-fold higher affinity than in wtS100A2 and a 2-fold increase for the last two Ca2+ ions. A further 2-3-fold increase of affinity was observed for respective binding steps of the NC mutant. The Hummel-Dryer method demonstrated that the wild type and mutants bind four Zn2+ atoms per dimer with similar affinity. Fluorescence and difference spectrophotometry showed that the binding of Ca2+ and Zn2+ induces considerable conformational changes, mostly attributable to changes in the microenvironment of Tyr76 located in site II. Fluorescence enhancement of 4,4'-dianilino-1, 1'-binaphthyl-5,5'-disulfonic acid clearly indicated that Ca2+ and Zn2+ binding induce a hydrophobic patch at the surface of wtS100A2, which, as in calmodulin, may be instrumental for the regulatory role of S100A2 in the nucleus.     

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.     

Calmodulin regulation of Ca2+ entry in Jurkat T cells

BACKGROUND: The authors have previously demonstrated abnormalities in glucose and insulin metabolism in nondiabetic black American (BA) adults versus white American (WA) adults. Whether similar glucoregulatory alterations extend to BA adolescents remain unknown. In addition, obesity, a known risk factor for insulin resistance and hyperinsulinemia, occurs in a greater proportion of BA adults and children when compared to WA. The objective of the present study was to examine the differential effects of obesity on glucose homeostasis in BA and WA adolescents. METHODS: We examined glucose homeostasis in BA and WA adolescents using oral glucose tolerance test (OGTT), intravenous glucose tolerance test (IVGTT), and [6,6-2H2]-glucose infusion. The study consisted of four age-, sex-, and pubertal stage-matched groups: 15 lean BA, 29 lean WA, 7 obese BA, and 9 obese WA. RESULTS: Both obese groups had significantly increased insulin and C-peptide area under the curve (AUC) during OGTT and IVGTT when compared to their same-race lean counterparts. During OGTT, obese BA demonstrated greater insulin and C-peptide when compared to obese WA. During IVGTT, first- and second-phase insulin were significantly greater in obese BA versus obese WA. CONCLUSION: In summary, BA adolescents demonstrated insulin resistance which is markedly exaggerated in the face of obesity when compared to WA adolescents, implying a differential impact for obesity on glucose homeostasis that is unique to the obese BA adolescent group. In conclusion, there is a need for early aggressive weight management in obese BA adolescents.     

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.     

Ca2+ regulation of gelsolin activity: binding and severing of F-actin

Regulation of the F-actin severing activity of gelsolin by Ca2+ has been investigated under physiologic ionic conditions. Tryptophan fluorescence intensity measurements indicate that gelsolin contains at least two Ca2+ binding sites with affinities of 2.5 x 10(7) M-1 and 1.5 x 10(5) M-1. At F-actin and gelsolin concentrations in the range of those found intracellularly, gelsolin is able to bind F-actin with half-maximum binding at 0.14 microM free Ca2+ concentration. Steady-state measurements of gelsolin-induced actin depolymerization suggest that half-maximum depolymerization occurs at approximately 0.4 microM free Ca2+ concentration. Dynamic light scattering measurements of the translational diffusion coefficient for actin filaments and nucleated polymerization assays for number concentration of actin filaments both indicate that severing of F-actin occurs slowly at micromolar free Ca2+ concentrations. The data suggest that binding of Ca2+ to the gelsolin-F-actin complex is the rate-limiting step for F-actin severing by gelsolin; this Ca2+ binding event is a committed step that results in a Ca2+ ion bound at a high-affinity, EGTA-resistant site. The very high affinity of gelsolin for the barbed end of an actin filament drives the binding reaction equilibrium toward completion under conditions where the reaction rate is slow.     

Taurine regulation of Ca2+ uptake and (Ca(2+)+Mg2+)-ATPase in developing chick B-cells

1. The objective of the present study was to determine the effect of age and taurine on chick B cell calcium uptake and membrane (Ca(2+)+Mg2+)-ATPase activity in 1-4-week-old chicks. 2. The calcium uptake rate decreased with age (P < 0.05) and was further decreased by taurine (P < 0.05). 3. (Ca(2+)+Mg2+)-ATPase activity increased with age (P < 0.05) and was stimulated by taurine (P < 0.05). 4. The data demonstrate that the flux of calcium across the B-cell membrane changes during early post-hatch development, and that taurine regulates both the influx and efflux of calcium in chick B-cells.     

Endoplasmic reticulum stress proteins block oxidant-induced Ca2+ increases and cell death

Oxidants are important human toxicants. Increased intracellular free Ca2+ may be critical for oxidant toxicity, but this mechanism remains controversial. Furthermore, oxidants damage the endoplasmic reticulum (ER) and release ER Ca2+, but the role of the ER in oxidant toxicity and Ca2+ regulation during toxicity is also unclear. tert-Butylhydroperoxide (TBHP), a prototypical organic oxidant, causes oxidative stress and an increase in intracellular free Ca2+. Therefore, we addressed the mechanism of oxidant-induced cell death and investigated the role of ER stress proteins in Ca2+ regulation and cytoprotection after treating renal epithelial cells with TBHP. Prior ER stress induces expression of the ER stress proteins Grp78, Grp94, and calreticulin and rendered cells resistant to cell death caused by a subsequent TBHP challenge. Expressing antisense RNA targeted to grp78 prevents grp78 induction sensitized cells to TBHP and disrupted their ability to develop cellular tolerance. In addition, overexpressing calreticulin, another ER chaperone and Ca2+-binding protein, also protected cells against TBHP. Interestingly, neither prior ER stress nor calreticulin expression prevented lipid peroxidation, but both blocked the rise in intracellular free Ca2+ after TBHP treatment. Loading cells with EGTA, even after peroxidation had already occurred, also prevented TBHP-induced cell death, indicating that buffering intracellular Ca2+ prevents cell killing. Thus, Ca2+ plays an important role in TBHP-induced cell death in these cells, and the ER is an important regulator of cellular Ca2+ homeostasis during oxidative stress. Given the importance of oxidants in human disease, it would appear that the role of ER stress proteins in protection from oxidant damage warrants further consideration.     

Dynamic regulation of intracellular Ca2+ concentration in aortic endothelial cells

In non-excitable cells, a Ca2+ entry pathway is opened after the depletion of intracellular Ca2+ store sites. We have tried to estimate the sensitivity of this pathway to Ca2+ release using bovine aortic endothelial cells. Single application of a high concentration (30 microM) of ATP released almost all stored Ca2+ in Ca(2+)-free extracellular solution, whereas a low concentration of ATP (30 nM) produced a partial (57.3 +/- 3.0%) release of Ca2+. By 10 min of Ca2+ re-perfusion, the Ca2+ store site was reloaded to 97.1% of its initial filling state. When thapsigargin was applied to this cell in Mn2+ solution, Mn(2+)-induced quenching of fura-2 dye started when 19.3 +/- 5.3% of Ca2+ release, produced by 30 nM ATP, had occurred. Therefore, Ca2+ release required for Mn2+ entry was estimated as 11.1 +/- 3.0% of stored Ca2+. These results indicate that intracellular Ca2+ concentration is controlled dynamically by simultaneously occurring Ca2+ release and entry in bovine aortic endothelial cells.     

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.     

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.     

Differences in Ca2+ channels governing generation of miniature and evoked excitatory synaptic currents in spinal laminae I and II

Many neurons of spinal laminae I and II, a region concerned with pain and other somatosensory mechanisms, display frequent miniature "spontaneous" EPSCs (mEPSCs). In a number of instances, mEPSCs occur often enough to influence neuronal excitability. To compare generation of mEPSCs to EPSCs evoked by dorsal root stimulation (DR-EPSCs), various agents affecting neuronal activity and Ca2+ channels were applied to in vitro slice preparations of rodent spinal cord during tight-seal, whole-cell, voltage-clamp recordings from laminae I and II neurons. The AMPA/kainate glutamate receptor antagonist CNQX (10-20 microM) regularly abolished DR-EPSCs. In many neurons CNQX also eliminated mEPSCs; however, in a number of cases a proportion of the mEPSCs were resistant to CNQX suggesting that in these instances different mediators or receptors were also involved. Cd2+ (10-50 microM) blocked evoked EPSCs without suppressing mEPSC occurrence. In contrast, Ni2+ (相似文献   

Differential regulation of N- and Q-type Ca2+ channels by cyclic nucleotides and G-proteins

Voltage-dependent Ca2+ channels play a central role in controlling neurotransmitter release at the synapse. They can be inhibited by certain G-protein-coupled receptors, acting by a pathway delimited to the membrane. In addition, modulation of Ca2+ channel activity by protein kinases also contributes to the dynamic regulation of neuronal physiology. Recently, differences in these modulations between Ca2+ channel subtypes have been shown in several neuronal preparations. Here we show that two types of presynaptic Ca2+ channel (N-type and Q-type) are differentially regulated by cAMP and G-proteins using a Xenopus oocyte expression system. Treatment to increase cytosolic cAMP concentration with forskolin and 3-isobutyl-1-methylxanthine (IBMX) markedly potentiated Q-type channel current, and the enhancement was reversed by protein kinase A inhibitors. Much smaller enhancement was observed in N-type channel current after the cAMP elevation. When large depolarizing prepulse was applied to the oocytes for evaluation of the tonic inhibition of Ca2+ channels by intrinsic G-protein activity, N-type channel current elicited a large prepulse facilitation but Q-type channels did not. The tonic inhibition of N-type channels was abolished by an intracellular perfusion with a 'cut-open' recording configuration, or by co-expression with G(alpha o). When kappa opioid receptors were co-expressed and stimulated with agonists, depolarization-resistant inhibition was more apparent in Q-type channels than in N-type channels. These results suggest that Q-type channels are more susceptible to the protein kinase A-mediated facilitation than N-type channels, and that activity of N-type channels can be more highly regulated in a voltage-dependent manner by G(betagamma) than that of Q-type channels. These differences may account for the selective regulation of neurotransmitter release by these Ca2+ channels.     

Force regulation by Ca2+ in skinned single cardiac myocytes of frog

Atrial and ventricular myocytes 200 to 300 microm long containing one to five myofibrils are isolated from frog hearts. After a cell is caught and held between two suction micropipettes the surface membrane is destroyed by briefly jetting relaxing solution containing 0.05% Triton X-100 on it from a third micropipette. Jetting buffered Ca2+ from other pipettes produces sustained contractions that relax completely on cessation. The pCa/force relationship is determined at 20 degrees C by perfusing a closely spaced sequence of pCa concentrations (pCa = -log[Ca2+]) past the skinned myocyte. At each step in the pCa series quick release of the myocyte length defines the tension baseline and quick restretch allows the kinetics of the return to steady tension to be observed. The pCa/force data fit to the Hill equation for atrial and ventricular myocytes yield, respectively, a pK (curve midpoint) of 5.86 +/- 0.03 (mean +/- SE.; n = 7) and 5.87 +/- 0.02 (n = 18) and an nH (slope) of 4.3 +/- 0.34 and 5.1 +/- 0.35. These slopes are about double those reported previously, suggesting that the cooperativity of Ca2+ activation in frog cardiac myofibrils is as strong as in fast skeletal muscle. The shape of the pCa/force relationship differs from that usually reported for skeletal muscle in that it closely follows the ideal fitted Hill plot with a single slope while that of skeletal muscle appears steeper in the lower than in the upper half. The rate of tension redevelopment following release restretch protocol increases with Ca2+ >10-fold and continues to rise after Ca2+ activated tension saturates. This finding provides support for a strong kinetic mechanism of force regulation by Ca2+ in frog cardiac muscle, at variance with previous reports on mammalian heart muscle. The maximum rate of tension redevelopment following restretch is approximately twofold faster for atrial than for ventricular myocytes, in accord with the idea that the intrinsic speed of the contractile proteins is faster in atrial than in ventricular myocardium.