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Although both cinacalcet and etelcalcetide are calcimimetics that directly inhibit parathyroid hormone (PTH) secretion by activating the calcium (Ca)-sensing receptor (CaSR), their binding sites are different. We report a first case of a hemodialysis (HD) patient with secondary hyperparathyroidism (SHPT), in whom cinacalcet, but not etelcalcetide, could reduce serum intact PTH (i-PTH) levels. A HD patient received total parathyroidectomy (PTx) with auto-transplantation 16 years earlier. Due to SHPT relapse, cinacalcet was started at 7 years after PTx. His i-PTH levels had been controlled with both 75-100 mg of cinacalcet and 4.5 μg/week of calcitriol for a year before switching from cinacalcet to etelcalcetide. At 1 month following the switch, his serum i-PTH level increased to 716 pg/mL. The dose of etelcalcetide was gradually increased and finally reached the maximal dose of 45 mg/week. Because even the maximal dose of etelcalcetide for > 4 months did not reduce his serum i-PTH levels to < 700 pg/mL, etelcalcetide was switched to 50 mg/day of cinacalcet, which reduced the levels to 208 pg/mL at 2 months after the switch. Genomic sequencing test using whole blood revealed no mutation in the portion including Cys 482 of CaSR gene. The patient was resistant to etelcalcetide treatment but not to cinacalcet, suggesting the possibility that the enlarged parathyroid gland has some change in the portion including Cys 482 in the CaSR gene. Therefore, considering the possibility of etelcalcetide resistance during SHPT treatment should be kept in mind., (© 2021. The Author(s).)

The Ca(2+) content in the sarcoplasmic reticulum (SR) determines the amount of Ca(2+) released, thereby regulating the magnitude of Ca(2+) transient and contraction in cardiac muscle. The Ca(2+) content in the SR is known to be regulated by two factors: the activity of the Ca(2+) pump (SERCA) and Ca(2+) leak through the ryanodine receptor (RyR). However, the direct relationship between the SERCA activity and Ca(2+) leak has not been fully investigated in the heart. In the present study, we evaluated the role of the SERCA activity in Ca(2+) leak from the SR using a novel saponin-skinned method combined with transgenic mouse models in which the SERCA activity was genetically modulated. In the SERCA overexpression mice, the Ca(2+) uptake in the SR was significantly increased and the Ca(2+) transient was markedly increased. However, Ca(2+) leak from the SR did not change significantly. In mice with overexpression of a negative regulator of SERCA, sarcolipin, the Ca(2+) uptake by the SR was significantly decreased and the Ca(2+) transient was markedly decreased. Again, Ca(2+) leak from the SR did not change significantly. In conclusion, the selective modulation of the SERCA activity modulates Ca(2+) uptake, although it does not change Ca(2+) leak from the SR., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Rapid lowering of the solution temperature (rapid cooling, RC) from 24 to 3°C within 3 s releases considerable amounts of Ca(2+) from the sarcoplasmic reticulum (SR) in mammalian cardiac muscles. In this study, we investigated the intracellular mechanism of RC-induced Ca(2+) release, especially the role of Ca(2+), in ferret ventricular muscle. Saponin-treated skinned trabeculae were placed in a glass capillary, and the amount of Ca(2+) released from the SR by RC and caffeine (50 mM) was measured with fluo-3. It was estimated that in the presence of ATP about 45% of the Ca(2+) content in the SR was released by RC. The amount of SR Ca(2+) released by RC was unchanged by the replacement of ATP by AMP-PCP (a non-hydrolysable ATP analogue and agonist for the ryanodine receptor but not for the Ca(2+) pump of SR), suggesting that the suppression of the Ca(2+) pump of SR at low temperature might not be a major mechanism in RC-induced Ca(2+) release. The free Ca(2+) concentration of the solution used for triggering RC-induced Ca(2+) release was estimated to be only about 20 nM with fluo-3 or aequorin. When this solution was applied to the preparation at 3°C, only a small amount of Ca(2+) was released from SR presumably by the Ca(2+)-induced Ca(2+) release (CICR) mechanism. Thus, in mammalian cardiac muscles, RC releases a part of the (<50%) stored Ca(2+) contained in the SR, and the mechanism of RC-induced Ca(2+) release may differ from that of CICR, which is thought to play a role in frog skeletal muscle fibres that express ryanodine receptors of different types.

In skeletal muscle, active force production varies as a function of sarcomere length (SL). It has been considered that this SL dependence results simply from a change in the overlap length between the thick and thin filaments. The purpose of this study was to provide a systematic understanding of the SL-dependent increase in Ca(2+) sensitivity in skeletal muscle, by investigating how thin filament "on-off" switching and passive force are involved in the regulation. Rabbit psoas muscles were skinned, and active force measurements were taken at various Ca(2+) concentrations with single fibers, in the short (2.0 and 2.4 μm) and long (2.4 and 2.8 μm) SL ranges. Despite the same magnitude of SL elongation, the SL-dependent increase in Ca(2+) sensitivity was more pronounced in the long SL range. MgADP (3 mM) increased the rate of rise of active force and attenuated SL-dependent Ca(2+) activation in both SL ranges. Conversely, inorganic phosphate (Pi, 20 mM) decreased the rate of rise of active force and enhanced SL-dependent Ca(2+) activation in both SL ranges. Our analyses revealed that, in the absence and presence of MgADP or Pi, the magnitude of SL-dependent Ca(2+) activation was (1) inversely correlated with the rate of rise of active force, and (2) in proportion to passive force. These findings suggest that the SL dependence of active force in skeletal muscle is regulated via thin filament "on-off" switching and titin (connectin)-based interfilament lattice spacing modulation in a coordinated fashion, in addition to the regulation via the filament overlap.

In heart failure, chronic catecholaminergic stimulation increases diastolic Ca(2+) leak from ryanodine receptors (RyRs) of sarcoplasmic reticulum (SR), possibly due to the phosphorylation of RyRs through the activation of protein kinase A (PKA) or Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In the present study, we attempted to identify which activated kinase is responsible for the enhanced Ca(2+) leak caused by beta-adrenergic stimulation. Trabeculae obtained from the hearts of adult male C57BL/6J mice were treated with isoproterenol and then permeabilized with saponin. To examine SR functions, Ca(2+) in SR was released with caffeine and measured with fluo-3. The Ca(2+) leak in isoproterenol-treated preparations was significantly increased when the PKA-dependent phosphorylation of RyR was increased without the involvement of CaMKII-dependent phosphorylation. Both the increase in Ca(2+) leak and the phosphorylation of RyR were blocked by a PKA inhibitor. Our results show that beta-adrenergic stimulation increases Ca(2+) leak from SR through PKA-dependent phosphorylation of RyR.

Protein kinase A (PKA)-dependent phosphorylation of troponin (Tn)I represents a major physiological mechanism during beta-adrenergic stimulation in myocardium for the reduction of myofibrillar Ca2+ sensitivity via weakening of the interaction with TnC. By taking advantage of thin filament reconstitution, we directly investigated whether or not PKA-dependent phosphorylation of cardiac TnI (cTnI) decreases Ca2+ sensitivity in different types of muscle: cardiac (porcine ventricular) and fast skeletal (rabbit psoas) muscles. PKA enhanced phosphorylation of cTnI at Ser23/24 in skinned cardiac muscle and decreased Ca2+ sensitivity, of which the effects were confirmed after reconstitution with the cardiac Tn complex (cTn) or the hybrid Tn complex (designated as PCRF; fast skeletal TnT with cTnI and cTnC). Reconstitution of cardiac muscle with the fast skeletal Tn complex (sTn) not only increased Ca2+ sensitivity, but also abolished the Ca2+-desensitizing effect of PKA, supporting the view that the phosphorylation of cTnI, but not that of other myofibrillar proteins, such as myosin-binding protein C, primarily underlies the PKA-induced Ca2+ desensitization in cardiac muscle. Reconstitution of fast skeletal muscle with cTn decreased Ca2+ sensitivity, and PKA further decreased Ca2+ sensitivity, which was almost completely restored to the original level upon subsequent reconstitution with sTn. The essentially same result was obtained when fast skeletal muscle was reconstituted with PCRF. It is therefore suggested that the PKA-dependent phosphorylation or dephosphorylation of cTnI universally modulates Ca2+ sensitivity associated with cTnC in the striated muscle sarcomere, independent of the TnT isoform.

As the left ventricular (LV) pressure curve and myocardial tension curve in heart are composed of contraction and relaxation processes, we have found that hybrid logistic (HL) function calculated as the difference between two logistic functions curve-fits better the isovolumic LV pressure curve and the isometric twitch tension curve than the conventional polynomial exponential and sinusoidal functions. Increase and decrease in intracellular Ca2+ concentration regulate myocardial contraction and relaxation. Recently, we reported that intracellular Ca2+ transient (CaT) curves measured using the calcium-sensitive bioluminescent protein, aequorin, were better curve-fitted by HL function compared to the polynomial exponential function in the isolated rabbit RV and mouse LV papillary muscles. We speculate that the first logistic component curve of HL fit represents the concentration of the Ca2+ inflow into the cytoplasmic space, the concentration of Ca2+ released from sarcoplasmic reticulum (SR), the concentration of Ca2+ binding to troponin C (TnC), and the attached number of cross-bridge (CB) and their time courses, and that the second logistic component curve of HL fit represents the concentration of Ca2+ sequestered into SR, the concentration of Ca2+ removal from the cytoplasmic space, the concentration of Ca2+ released from TnC, and the detached number of CB and their time courses. This HL approach for CaT curve may provide a more useful model for investigating Ca2+ handling, Ca(2+) -TnC interaction, and CB cycling.

A decrease in intracellular calcium (Ca2+) concentration in the cardiac muscle is one of the important factors to induce myocardial relaxation. A mono-exponential (m-E) function has been used for assessing myocardial relaxation curve of isometric tension and intracellular calcium transient (CaT) decay, and the m-E time constants for the relaxation curve of isometric tension (F tau E) and CaT decay (Ca tau E) have been recognised as lusitropic indices. However, we found that a half-logistic (h-L) function fits the relaxation curve of isometric tension much more precisely than the conventional m-E function in the ferret right ventricular (RV) papillary muscle. Moreover, we demonstrated that the goodness of the h-L fits for CaT decays was superior to the goodness of the m-E fits in the rabbit RV and murine left ventricular papillary muscles. The changes in the h-L time constants for the relaxation curves of isometric tension (F tau L) and CaT decays (Ca tau L) with the different onsets were significantly smaller than the changes in F tau E and Ca tau E, respectively. The differences in the h-L non-zero asymptotes for the relaxation curves of isometric tension and CaT decays with the different onsets were smaller than the changes in the m-E non-zero asymptotes. The h-L function model characterises the amplitudes and time courses of the relaxation curve of isometric tension and CaT decay more precisely than the m-E function model, and thus F tau L and Ca tau L serve as more novel and reliable lusitropic indices. Simultaneous analysis of myocardial relaxation curve of isometric tension and CaT decay using h-L functions can become a useful method for assessment of myocardial calcium handling.

Myocardial intracellular calcium (Ca2+) transients (CaTs) regulate tension generation and relaxation. Isometric tension curves are often analyzed using exponential equations; however, we previously demonstrated that hybrid logistic (HL) functions, which describe the difference between two S-shaped logistic functions, provide more accurate representations. In the present study, we investigated the potential application of HL functions for analyzing CaTs directly. CaTs were measured using the calcium-sensitive bioluminescent protein, aequorin, in 7 isolated rabbit right ventricular and 15 isolated mouse left ventricular papillary muscles. CaT data were fit by the least-squares method using HL and polynomial exponential (PE) function equations. The mean correlation coefficient (r) values of HL and PE fits were 0.9934 vs. 0.9523 in rabbit and 0.9980 vs. 0.9407 in mouse, respectively. The Z transformation of r value and the adjusted coefficient of determination (r squares) were higher, and the residual mean squares and Akaike information criterion values, which estimate goodness of fit between functions with different numbers of parameters, were lower for the HL curves than for the PE curves in both rabbit and mouse. There were significant correlations between the calculated values from the best-fit HL function curve and the primary CaT data. Thus the HL function curves more accurately described the amplitudes and time courses of CaTs in both rabbit and mouse papillary muscles. We speculate that the first logistic component curve reflects the concentration and time course of Ca2+ inflow into the cytoplasmic space, and that the second logistic component curve reflects the concentrations and time courses of Ca2+ removal from the cytoplasmic space as well as Ca2+ binding to troponin. This approach might provide a more robust model for studying CaTs and cardiac cycle regulation.

A decrease in myocardial intracellular calcium concentration ([Ca(2+)](i)) precedes relaxation, and a monoexponential function is typically used for fitting the decay of the Ca(2+) transient. However, a logistic function has been shown to be a better fit for the relaxation force curve, compared to the conventional monoexponential function. In the present study, we compared the logistic and monoexponential functions for fitting the [Ca(2+)](i) declines, which were measured using the aequorin method, and isometric relaxation force curves at 4 different onsets: the minimum time-derivative of [Ca(2+)](i) (d[Ca(2+)](i)/dt (min)) and force (dF/dt(min)), and the 10%, 20% and 30% lower [Ca(2+)](i) levels and forces over the data-sampling period in 7 isolated rabbit right ventricular and 15 isolated mouse left ventricular papillary muscles. Logistic functions were significantly superior for fitting the [Ca(2+)] (i) declines and relaxation force curves, compared to monoexponential functions. Changes in the normalized logistic [Ca(2+)] (i) decline and relaxation force time constants at the delayed onsets relative to their 100% values at d[Ca(2+)] (i)/dt(min) and dF/dt(min) were significantly smaller than the changes in the normalized monoexponential time constants. The ratio of the logistic relaxation force time constant relative to the logistic [Ca(2+)](i) decline time constant was significantly smaller in mouse than in rabbit. We conclude that the logistic function more reliably characterizes the [Ca(2+)](i) decline and relaxation force curve at any onset, irrespective of animal species. Simultaneous analyses using the logistic model for decay of the Ca(2+) transient and myocardial lusitropism might be a useful strategy for analysis of species-specific myocardial calcium handling.

Unlabelled: BACKGROUND The aim of the present study was to indicate the cross-bridge-dependent change in the Ca2+ affinity of troponin-C (TnC) during relaxation in an intact preparation, because the intracellular mechanism of relaxation is not fully understood, although several methods of evaluating global diastolic function have been reported., Methods and Results: The aequorin method was used with intact ferret papillary muscles and a tetanic contraction was induced by a repetitive electrical stimulation in the presence of ryanodine. The extra-Ca2+, the transient increase in the intracellular Ca2+ concentration in response to a rapid reduction in muscle length, which reflects the change in the Ca2+ affinity of TnC because of cross-bridge detachment, was measured, and the cross-bridge-dependent change in the Ca2+ affinity of TnC was estimated by observing the change in the slope of the extra-Ca2+ -tension relation. The extra-Ca2+ -tension relation measured during relaxation became steeper than that during contraction in all cases. The extra-Ca2+ -tension relation became steeper in the presence of 20 mmol/L caffeine during contraction in all cases., Conclusion: During relaxation, the downstream-dependent change in the Ca2+ affinity of TnC was enhanced, compared with that during contraction, because of a decrease in the number of attached cross-bridges.

We previously estimated the myofilament responsiveness to Ca(2+) in isolated intact ventricular myocytes, using the steady-state relationship between cytosolic Ca(2+) concentration ([Ca(2+)](i)) and cell-shortening during tetanus (Ca-L trajectory). This method was useful and easy; however, it could not be used for a high dose of Ca sensitizer because the instantaneous plots after the application of Ca sensitizer did not make a fixed point of shortening (we used 5% shortening). Therefore we must produce another method to investigate Ca(2+) responsiveness. For an estimation of a wider range of the Ca-L trajectory, we fitted the Ca-L trajectory data with the Hill equation to construct the Ca-shortening curve. To fit this curve, we measured the maximal shortening, which was on average 31.6%. The value of [Ca(2+)](i) to produce the half-maximal shortening (Ca(50)) was dose-dependently decreased by EMD57033 (sensitization). Either isoproterenol or 3-isobutyl-1-methylxanthine increased Ca(50) (desensitization) with a concomitant increase in intracellular c-AMP. EMD57439, a selective PDE-III inhibitor, did not significantly increase the c-AMP concentration and produced little change in Ca(50). These results are in agreement with previous reports with skinned or intact multicellular preparations. The Ca-shortening curve constructed in intact cardiac myocytes can be used to estimate the myofibrillar responsiveness to Ca(2+) in a wide range of [Ca(2+)](i).

Background: We hypothesized that the negative inotropic effect of nifedipine (Nif) on cardiac ventricular muscle is partly due to the cross-bridge-dependent decrease of Ca2+ sensitivity of the myofilaments as well as the decrease in Ca2+ influx., Method and Results: We used aequorin-injected ferret papillary muscles and measured the slope of the extra-Ca(2+)-tension relation which expresses the change in the Ca2+ sensitivity through the feedback from the cross-bridges. Twitch tension was decreased significantly by 0.5 micromol/L Nif accompanying a significant reduction of the Ca2+ transient peak. When Nif (0.2-0.5 micromol/L) was added to the solution with 8 mmol/L Ca2+, the slope of the extra-Ca2+-tension relation became steeper in a concentration-dependent manner, which was similar to the change in the slope when the concentration of Ca2+ was decreased from 8 to 1 mmol/L in the absence of Nif. BAY-K 8644 (0.3 micromol/L), a dihydropyridine receptor agonist, showed the opposite effect on the slope of the extra-Ca2+-tension relation to that observed in Nif. However, 2,3-butanedione monoxime (3 mmol/L), an inhibitor of the active cross-bridges, antagonized the effect of BAY-K 8644., Conclusion: Nif exerts its negative inotropic effect on cardiac muscle by suppressing Ca2+ binding to troponin C via the inhibition of the L-type Ca2+ channel, and by the cross-bridge-dependent decrease in the Ca2+ sensitivity, as in low extracellular Ca2+ concentration.

Genetic studies of families with familial Alzheimer's disease have implicated presenilin 2 (PS2) in the pathogenesis of this disease. PS2 is ubiquitously expressed in various tissues including hearts. In this study, we examined cardiac phenotypes of PS2 knockout (PS2KO) mice to elucidate a role of PS2 in hearts. PS2KO mice developed normally with no evidence of cardiac hypertrophy and fibrosis. Invasive hemodynamic analysis revealed that cardiac contractility in PS2KO mice increased compared with that in their littermate controls. A study of isolated papillary muscle showed that peak amplitudes of Ca2+ transients and peak tension were significantly higher in PS2KO mice than those in their littermate controls. PS2KO mouse hearts exhibited no change in expression of calcium regulatory proteins. Since it has been demonstrated that PS2 in brain interacts with sorcin, which serves as a modulator of cardiac ryanodine receptor (RyR2), we tested whether PS2 also interacts with RyR2. Immmunoprecipitation analysis showed that PS2, sorcin, and RyR2 interact with each other in HEK-293 cells overexpressing these proteins or in mouse hearts. Immunohistochemistry of heart muscle indicated that PS2 colocalizes with RyR2 and sorcin at the Z-lines. Elevated Ca2+ attenuated the association of RyR2 with PS2, whereas the association of sorcin with PS2 was enhanced. The enhanced Ca2+ transients and contractility in PS2KO mice were observed at low extracellular [Ca2+] but not at high levels of [Ca2+]. Taken together, our results suggest that PS2 plays an important role in cardiac excitation-contraction coupling by interacting with RyR2.

Ca2+ affinity of cardiac troponin C (TnC) is regulated by the active cross-bridges (downstream-dependent mechanism). In the present study, we showed one of the methods to evaluate the downstream-dependent change in the Ca2+ affinity of TnC during contraction using the aequorin-injected ferret papillary muscle. For this purpose, the tension-dependent change in the extra-Ca2+ (a transient increase in the intracellular Ca2+ concentration ([Ca2+]i) in response to a quick length reduction) was measured under various conditions. We examined whether the regression line between the magnitude of tension reduction and the magnitude of the normalized extra-Ca2+ (the extra-Ca2+ was divided by [Ca2+]i immediately before length change) (the normalized extra-Ca2+-tension relation) in twitch contraction can be used for the estimation of the downstream-dependent change in the Ca2+ affinity of TnC. The normalized extra-Ca2+-tension relation became shallow by EMD 57033 (EMD) (one of the Ca2+ sensitizers) and by an increase in Ca2+ concentration in the solution ([Ca2+]o) in a concentration-dependent manner. However, 2,3-butanedione monoxime (BDM) (one of the desensitizers) antagonized the effects of EMD and higher [Ca2+]o in a concentration-dependent manner. These effects of EMD and BDM were also observed in the normalized extra-Ca2+-tension relation in tetanic contraction. The normalized extra-Ca2+-tension relation became steep by shortening the initial muscle length before contraction in tetanic contraction. Length-tension relation in twitch contraction was significantly shifted upward by higher [Ca2+]o and EMD, but BDM showed the opposite effects on them in a concentration-dependent manner. Thus, the downstream-dependent change in the Ca2+ affinity of TnC which physiologically functions in intact cardiac muscle can be evaluated using the normalized extra-Ca2+-tension relation.

Background: Recent evidence suggests that ropivacaine exerts markedly less cardiotoxicity compared with bupivacaine; however, the mechanisms are not fully understood at the molecular level., Methods: Isolated ferret ventricular papillary muscles were microinjected with the Ca-binding photoprotein aequorin, and intracellular Ca transients and tension were simultaneously measured during twitch in the absence and presence of bupivacaine or ropivacaine., Results: Bupivacaine and ropivacaine (10, 30, and 100 microm) reduced peak systolic [Ca]i and tension in a concentration-dependent manner. The effects were significantly greater for bupivacaine, particularly on tension (approximately twofold). The percentage reduction of tension was linearly correlated with that of [Ca]i for both anesthetics, with the slope of the relationship being approximately equal to 1.0 for ropivacaine and approximately equal to 1.3 for bupivacaine (slope difference, P < 0.05), suggesting that the cardiodepressant effect of ropivacaine results predominantly from inhibition of Ca transients, whereas bupivacaine suppresses Ca transients and the reaction beyond Ca transients, i.e., myofibrillar activation, as well. BAY K 8644, a Ca channel opener, abolished the inhibitory effects of ropivacaine on Ca transients and tension, whereas BAY K 8644 only partially inhibited the effects of bupivacaine, particularly the effects on tension., Conclusion: The cardiodepressant effect of bupivacaine is approximately twofold greater than that of ropivacaine. Bupivacaine suppresses Ca transients more markedly than does ropivacaine and reduces myofibrillar activation, which may at least in part underlie the greater inhibitory effect of bupivacaine on cardiac contractions. These results suggest that ropivacaine has a more favorable profile as a local anesthetic in the clinical settings.

In cardiomyocytes, calcium plays important roles as a signal in cardiac hypertrophy and contraction-relaxation cycling. Elevation of Ca2+ concentration in myoplasm is associated with the onset and progression of hypertrophy as well as the enhancement of contractility. The cardiac Ca2+ ATPase (SERCA2a) of the sarcoplasmic reticulum plays a dominant role in lowering cytoplasmic calcium levels during relaxation and is regulated by phospholamban (PLN). To examine whether the modulation of SERCA2a activity results in the attenuation of cardiac hypertrophy and enhancement of contractility, we generated transgenic mice (TG) overexpressing a high calcium affinity SERCA2a mutant (K397/400E), lacking a functional association with PLN. In the TG hearts, the apparent affinity of SERCA2a for Ca2+ significantly increased compared with their nontransgenic littermate controls. The TG showed increased contraction and relaxation, with increases in the amplitude of Ca2+ transient and rapid Ca2+ decay. Upon induction of pressure overload by transverse aortic constriction, the TG developed less cardiac hypertrophy than littermate controls did. The activation of Ca2+-sensitive protein kinase C by pressure overload was significantly attenuated in the TG hearts. Our findings indicate an association of SERCA2a activity with cardiac hypertrophy and thus a new therapeutic target for the prevention and treatment of cardiac hypertrophy.

We investigated the mechanism of alpha(1)-adrenoceptor stimulation on the myofibrillar Ca(2+) responsiveness at steady-state in intact rat ventricular myocytes. We produced tetanus, and an instantaneous plot of [Ca(2+)](i) vs. cell length (Ca-L trajectory) was constructed to estimate the Ca(2+) responsiveness. An alpha(1)-agonist, phenylephrine, dose-dependently shifted the Ca-L trajectory to the left, corresponding to sensitization of the myofilaments. An alpha(1)-antagonist, prazosin, and inhibition of the Na/H exchange by ethylisopropylamiloride (EIPA) completely reversed the phenylephrine-induced shift. Phenylephrine increased pH(i) (DeltapH(i) = +0.1), which was reversed by prazosin and EIPA. Chelerythrine, an inhibitor of protein kinase C (PKC), completely blocked the effects of phenylephrine on Ca(2+) responsiveness and pH(i). When pH(i) was increased (DeltapH(i) = +0.1) without phenylephrine by changing pH(o), the Ca-L trajectory was shifted to the same extent as that observed with phenylephrine. We conclude that alpha(1)-adrenoceptor stimulation activates Na/H exchange through a PKC-mediated pathway and that an increase in pH(i) is mainly responsible for the increase in Ca(2+) responsiveness.

Background: Bupivacaine exhibits a cardiodepressant effect, the molecular mechanism(s) of which have yet to be fully understood. Bupivacaine may directly act on contractile proteins and thereby decrease myofibrillar Ca2+ sensitivity., Methods: Rat ventricular muscle was used. First, the effect of bupivacaine was examined on tetanic contractions in isolated intact myocytes. Next, Triton X-100-treated ventricular trabeculae were used to investigate the effect of bupivacaine on the pCa (= -log [Ca2+ ])-tension relation as well as on maximal Ca2+ -activated tension. Furthermore, to test whether bupivacaine inhibits the pathway downstream from Ca2+ binding to troponin C, tension was elicited in the skinned preparations by lowering the Mg-adenosine triphosphate (MgATP) concentration in the absence of Ca2+. The effect of bupivacaine on the pMgATP (= -log [MgATP])-tension relation was examined., Results: In myocytes, 3 microm bupivacaine significantly (P < 0.01) increased intracellular Ca2+ concentration required for 5% cell shortening from the resting cell length. In skinned preparations, bupivacaine shifted the pCa-tension relation to the lower pCa side; the midpoint of the pCa curve (pCa50) was significantly (P < 0.05) changed by 10 and 100 microm bupivacaine. A highly correlated linear relation (R = 0.81; P< 0.0005) was present between pCa50 and maximal Ca2+ -activated tension. Bupivacaine (10 and 100 microm) significantly (P < 0.05) shifted the midpoint of the pMgATP-tension relation to the higher pMgATP side., Conclusions: Bupivacaine decreases myofibrillar Ca2+ sensitivity in ventricular muscle, and this is coupled with the compound's inhibitory effect on the pathway beyond Ca2+ binding to troponin C, possibly on the actomyosin interaction. The current results may partly explain the overall cardiodepressant effect of bupivacaine in vivo.

We used the relation between intracellular Ca(2+) concentration ([Ca(2+)](i)) and cell shortening during tetanus to evaluate the endogenous characteristics of Ca(2+) responsiveness of myofibrils in mouse ventricular myocytes. Enzymatically isolated myocytes were loaded with fura-2 AM (4 microM for 10 min), and the fura-2 fluorescence ratio at 340 and 380 nm excitation wave length [F(340)/F(380)] and cell length were measured simultaneously. Following treatment with thapsigargin (0.2 microM) (an inhibitor of the Ca(2+) pump of sarcoplasmic reticulum), myocytes were stimulated at 10 Hz for 10 s to produce a tetanic contraction and an instantaneous plot of the fluorescence ratio signal versus cell length (R-L trajectory) was constructed. An increase in the extracellular Ca(2+) concentration ([Ca(2+)](o)) from 0.5 to 2 mM extended the R-L trajectory without a substantial shift of the relation. The R-L trajectory was shifted rightward by the nonselective phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xantine (IBMX, 200 microM) (desensitization of the myofibrils to Ca(2+)), and shifted leftward by the Ca(2+) sensitizing thiadiazinone derivative, EMD-57033 (0.5 microM) (sensitization of the myofibrils to Ca(2+)). Beta-adrenergic stimulant, isoproterenol (5 nM), also shifted the R-L trajectory to the right, suggesting that the membrane receptor could be preserved. These results suggest that the R-L trajectory is a useful method to estimate the myofibrillar responsiveness to Ca(2+) in isolated mouse myocytes and can be applied to various mouse models of heart disease.

X-ray diffraction studies were made using synchrotron radiation on ferret right ventricular papillary muscle under three different thyroid states: euthyroidism, hyperthyroidism, and hypothyroidism. The latter two states were induced by treatment with L-thyroxine and methimazole, respectively. The X-ray equatorial reflections were recorded at a time resolution of 10 ms to study the mass movement of myosin cross-bridges from thick to thin filaments. The myosin isomer content was measured by gel electrophoresis which showed that V3 isomer was predominant in euthyroid muscle and 27% of myosin was V1 isomer in hyperthyroid muscle. The intracellular free Ca concentration was measured by using the aequorin method. The peak Ca concentration was similar in all three states, but in the hypothyroid state the time-to-peak was longer and the decay was slower. The time-to-peak of twitch tension was shorter in hyperthyroidism and longer in hypothyroidism than in euthyroidism. The different time courses of twitch tension in different thyroid states accompanied a cross-bridge movement which closely followed the tension development. In hyperthyroidism, the cross-bridge movement significantly preceded tension development, suggesting that hyperthyroid myosin (V1) has a longer latency period between the shift to the vicinity of the thin filament and force development.

A multi-compartment model was used to estimate Ca2+ gradients in a sarcomere of a cardiac myocyte. The mathematical model assumed Ca2+ release from the sarcoplasmic reticulum as a driving function, and calculated Ca2+ binding to myoplasmic buffers, Ca2+ uptake by the sarcoplasmic reticulum, and diffusion of Ca2+ (and the buffers). During the fast Ca2+ transient similar to those observed during a twitch, the model predicted a large Ca2+ gradient in the sarcomere. A trajectory of the instantaneous relation between spatially averaged concentrations of Ca2+ and the Ca2+-troponin complex showed a counterclockwise loop, indicating non-equilibrium Ca2+ binding to troponin. During slow changes in [Ca2+] with time to peaks of approximately 500 ms or longer, the gradient of [Ca2+] was largely dissipated and the apparent equilibrium of the Ca2+-troponin binding reaction was suggested with little hysteresis of the trajectory. We conclude that a steady-state relation between [Ca2+] and mechanical activity can be achieved uniformly in the sarcomere by slowing the rate of Ca2+ release from the sarcoplasmic reticulum.

The mechanisms of the slower time courses of Ca2+ transients (CaT) and contraction in diabetic (diabetes mellitus, DM) myocardium were studied. The aequorin method was applied to papillary muscles of streptozotocin-induced DM and control rats. The time courses of CaT and tension of twitch in DM were slower than those in control, although the magnitudes of the CaT and contraction were identical. The dependence of CaT decay time and relaxation time on developed tension in DM and control rats differed. The length-tension relation in twitch and the pCa-tension relation in tetanus were identical in the two groups. The magnitude of extra Ca2+ (transient increase in intracellular Ca2+ concentration induced by a quick release in tetanus) was identical in both groups. pCa-tension relations of skinned trabeculae at different sarcomere lengths were nearly identical. The cross-bridge cycling rate (CCR) in DM was slower than that in control. These results indicate that the tension-dependent change in the Ca2+ affinity of troponin C in DM myocardium functions as in control myocardium. The slower time courses of CaT and tension in DM myocardium are caused by slower Ca2+ uptake by the sarcoplasmic reticulum and the slower CCR.

We investigated the contribution of sarcoplasmic reticulum (SR) and Na+/Ca2+ exchanger in the tension-dependent change in the decay of the Ca2+ transients (CaT) in euthyroid (Eu) and hyperthyroid (Hy) myocardium. Hy was induced by thyroxine treatment to enhance the rate of SR Ca2+ uptake. With the use of the aequorin method, CaT and tension in twitch contraction were simultaneously measured under various conditions (changing muscle length and Ca2+ concentration in solution). In both groups, the decay time of CaT (DT) showed a significant dependence on the developed tension, but the tension dependence of DT in Hy was significantly less than in Eu. In the presence of caffeine (3 mM), the tension dependence of DT in Hy became apparent as in Eu. Inhibition of Na+/Ca2+ exchanger by replacing Na+ with Li+ did not affect the dependence in Hy. The normalized extra Ca2+, which is the Ca2+ concentration change in response to a quick length change, in Hy was similar to that in Eu. pCa-tension relations of skinned trabeculae measured at different lengths (1.9 and 2.3 micrometer) were nearly identical in both groups. These results indicate that the tension-dependent change in the affinity of troponin C for Ca2+ works in both Eu and Hy myocardium and that the tension-dependent change in DT is influenced by the Ca2+ uptake rate of SR.

To study the effects of mechanical constraints on the Ca2+ affinity of cardiac troponin C, we analyzed the tension and aequorin light (AL) responses to sinusoidal length changes (5-10% of the initial muscle length) in aequorin-injected, tetanized cardiac muscles. The amplitude of the quasi-sinusoidal tension and AL responses decreased with increasing length-perturbation frequency from 0.5 to 1 Hz at 24 degreesC and from 1 to 3 Hz at 30 degreesC. The increase in AL corresponded well to the decrease in tension; likewise, the decrease in AL to the increase in tension and the tension response lagged behind the length change. A further increase in frequency (>1 Hz at 24 degreesC and >3 Hz at 30 degreesC) markedly increased the amplitude of the tension responses but decreased the amplitude of the AL responses. The increase in AL lagged behind the decrease in tension; likewise, the decrease in AL lagged behind the increase in tension, and the tension response led the length change. From previous mechanistic interpretations of the frequency dependence of the amplitude of tension response, we argue that the Ca2+ affinity of cardiac troponin C changes in parallel with the active tension (i.e., the number of active cross bridges) but not with the passive tension produced by the length perturbation-induced cross-bridge strain.

To estimate myofibrillar responsiveness to Ca2+, we used the relation between cell length and intracellular [Ca2+] ([Ca2+]i) during tetanic contractions of isolated ventricular myocytes. Enzymatically isolated rat ventricular myocytes were loaded with fura-2 AM (4 microM for 10 min) and excited alternately at 340 nm and 380 nm. The ratio (R) of fura-2 fluorescence at these wavelengths [F(340)/F(380), an index of [Ca2+]i] and cell length (L) were measured simultaneously. Following treatment with thapsigargin (0.2 microM), myocytes were stimulated at 10 Hz for 10 s to produce a tetanic contraction every min and an instantaneous plot of R vs L (R-L trajectory) was constructed. The R-L trajectory followed the same path during cell shortening and re-lengthening, suggesting that dynamic equilibrium between R and L was achieved during tetanus. Increasing the extracellular [Ca2+] from 1 to 8 mM extended the R-L trajectory without a substantial shift of the relation. The Ca2+-sensitizing thiadiazinone derivative, EMD57033 (1 microM), shifted the R-L trajectory to the left (sensitization of the myofibrils to Ca2+), whereas the non-selective phosphodiesterase inhibitor, 3-isobutyl-1-methylxantine (200 microM), shifted the R-L trajectory to the right (desensitization of the myofibrils to Ca2+), in agreement with previous results obtained using skinned preparations. We conclude that the R-L trajectory is useful for estimating the myofibrillar responsiveness to Ca2+ in isolated myocytes and may be beneficial for the evaluation of inotropic agents.

We investigated the effects of acidosis on the intracellular Ca2+ concentration ([Ca2+]i) and contractile properties of intact mammalian cardiac muscle during tetanic and twitch contractions. Aequorin was injected into ferret papillary muscles, and the [Ca2+]i and tension were simultaneously measured. Acidosis was attained by increasing the CO2 concentration in the bicarbonate (20 mM)-buffered Tyrode solution from 5% (pH 7.35, control) to 15% (pH 6.89, acidosis). Tetanic contraction was produced by repetitive stimulation of the preparation following treatment with 5 microM ryanodine. The relationship between [Ca2+]i and tension was measured 6 s after the onset of the stimulation and was fitted using the Hill equation. Acidosis decreased the maximal tension to 81 +/- 2% of the control and shifted the [Ca2+]i-tension relationship to the right by 0.18 +/- 0.01 pCa units. During twitch contraction, a quick shortening of muscle length from the length at which developed tension became maximal (Lmax) to 92% Lmax produced a transient change in the [Ca2+]i (extra Ca2+). The magnitude of the extra Ca2+ was dependent on the [Ca2+]i immediately before the length change, suggesting that the extra Ca2+ is related to the amount of troponin-Ca complex. Acidosis decreased the normalized extra Ca2+ to [Ca2+]i immediately before the length change, which indicates that the amount of Ca2+ bound to troponin C is less when [Ca2+]i is the same as in the control. The decrease in the Ca2+ binding to troponin C explains the decrease in tetanic and twitch contraction, and mechanical stress applied to the preparation induced less [Ca2+]i change in acidosis.

The possible contractile proteins, which are related to the length-dependent change in the relationship between intracellular Ca2+ concentration ([Ca2+]i) and tension, were investigated using aequorin-injected ferret papillary muscles. Tetanic contraction was produced by applying repetitive stimulation to the ryanodine-treated preparations, and the relationships between [Ca2+]i and tension were measured. When the muscle length was decreased from maximal length (Lmax), at which maximal tension is produced, to 95 and 90% Lmax, the maximal tension was significantly decreased. [Ca2+]i required for producing 50% of the maximal tension was significantly increased from 1.05 +/- 0.04 microM (Lmax) to 1.17 +/- 0.04 microM (95% Lmax) and to 1.22 +/- 0.04 microM (90% Lmax). Isoproterenol (Iso) accentuated the length-dependent change in the [Ca2+]i-tension relationship. The decrease in the Ca2+ sensitivity induced by Iso was larger at shorter muscle lengths compared with that at Lmax. It is, therefore, suggested that adenosine 3',5'-cyclic monophosphate-dependent phosphorylation of troponin I and/or C protein alters the length dependence of the [Ca2+]i-tension relationship and that troponin I and/or C protein might be involved in the length-tension-dependent change in the affinity of the contractile elements for Ca2+.

We investigated the role of mitochondria in the removal of intracellular Ca2+ which was increased by caffeine (15 mM, Caf), rapid lowering of the solution temperature from 30 to 4 degrees C (rapid cooling, RC), and electrical stimulation (0.07 Hz, ES). For this purpose, the intracellular Ca2+ concentration ([Ca2+]i) was measured using aequorin from the superficial cells of ferret papillary muscles. The three maneuvers induced transient changes in [Ca2+]i with different time courses. The decay time of the aequorin light signal (DT) in the Caf-induced Ca2+ release was significantly prolonged by the inhibitors for Na+-Ca2+ exchanger (Ni2+, Na+-free solution) at higher temperatures (> or = 12 degrees C). In the caffeine application at lower temperatures (< or = 12 degrees C), the inhibitors for mitochondria (ruthenium red, NaN3) significantly prolonged the DT but other inhibitors were ineffective. In the RC-induced Ca2+ release, DT was significantly prolonged by the mitochondrial inhibitors but other inhibitors were not effective. In the ES-induced Ca2+ release, each inhibitor for the sarcoplasmic reticulum (SR) (thapsigargin and 2,5-di(tert-butyl)-1,4-benzohydroquinone) prolonged the DT at all temperatures. The inhibitors for Na+-Ca2+ exchanger slightly prolonged the DT only at higher temperatures, and the mitochondrial inhibitors did not alter the DT at any temperature. These results suggest that mitochondria substantially transport Ca2+ when the Ca2+ uptake by the SR and Na+-Ca2+ exchanger are inhibited and [Ca2+]i is increased with a slower time course.

Objectives: The aim of the study is to test the hypothesis that tension-dependent change in the affinity of cardiac troponin-C influences the time courses of Ca2+ transients and tension in twitch contraction., Methods: The Ca(2+)-sensitive photoprotein, aequorin, was microinjected into superficial cells of ferret papillary muscles and the Ca2+ transients and tension were simultaneously measured. The peak of developed tension was altered by changing the extracellular Ca2+ concentration, initial muscle length, and the application of 2,3-butanedione monoxime., Results: In each maneuver, the decay time of Ca2+ transients was prolonged and the tension relaxation time was shortened when the peak of developed tension was decreased. In contrast, when the peak of developed tension was increased, the decay time of Ca2+ transients was shortened and the tension relaxation time was prolonged. The decay time of Ca2+ transients measured with different maneuvers was negatively correlated with the peak tension and the tension relaxation time was positively correlated with the tension peak., Conclusions: The changes in the decay time of Ca2+ transients and the tension relaxation time indicate that developed tension modulates the affinity of troponin-C for Ca2+ in normal twitch contraction.

Contraction of cardiac muscle is regulated by intracellular Ca2+ concentration ([Ca2+]i) change. The Ca2+ increased by the action potential is removed by Ca2+ removal mechanisms (sarcoplasmic reticulum, Na-Ca exchanger and Ca pump of the surface membrane) and is also bound to troponin. However, under some specific conditions which induced an overload of Ca2+, oscillatory changes in [Ca2+]i, which are considered to be due to regenerative Ca2+ release from the sarcoplasmic reticulum, appear. In Ca2+ overload, a transient increase in [Ca2+]i is observed after the falling phase of the Ca2+ transient which is induced by electrical stimulation. In accordance with the transient increase in [Ca2+¿i, the membrane transiently depolarizes (delayed after-depolarization). This membrane depolarization is considered to be due to the inward currents through the Na-Ca exchanger and non-specific cation channels. If the depolarization is large enough to reach the threshold, the action potentials are triggered (triggered activity). Thus, an increase in [Ca2+]i is one of possible factors in triggering of arrhythmia.

We investigated the direct effects of magnesium and hydrogen ions (Mg2+ and H+) on cardiac sarcoplasmic reticulum (SR) functions using saponin-treated ferret ventricular muscles. Ca2+ uptake function and the Ca(2+)-induced Ca2+ release (CICR) mechanism were examined. For the analysis of Ca2+ uptake, the SR was loaded with Ca2+ under various conditions and the Ca2+ content of SR was estimated by releasing Ca2+ with 50 mM caffeine. To determine CICR, following Ca2+ loading of the SR, solutions with various free Ca2+ concentrations ([Ca2+]) were applied to the preparation to release Ca2+, and the Ca2+ remaining in the SR was estimated. The amount of the released Ca2+ was measured with the fluorescent indicator fluo-3. Mg2+ (10mM) and H+ (pH 6.6) inhibited both Ca2+ uptake and CICR. The rate of Ca2+ accumulation was decreased but the steady-state Ca2+ content of the SR was not significantly altered in the loading solutions with a high [Mg2+] or low pH. CICR at around 1 microM [Ca2+] was clearly inhibited by a moderately low pH 6.6 but Mg2+ (0.6-4mM) did not significantly inhibit CICR except for at a very high [Mg2+] (10 mM). At the high [Ca2+] which shows maximal CICR, the Ca2+ release was not inhibited by either Mg2+ or H+. It is suggested that the elevation of intracellular H+ more profoundly influences the Ca2+ regulation mechanisms of the SR compared to the elevation of intracellular [Mg2+] under ischemic conditions.

A change in muscle length significantly alters the developed tension in mammalian cardiac muscle compared to that in skeletal muscle fibers. The intracellular mechanisms related to the length-dependent change in developed tension have been studied using Ca2+ indicators in intact preparations; a cross-bridge-dependent change in the affinity of troponin-C for Ca2+ is a possible mechanism. This hypothesis is further supported by the measurement of Ca2+ bound to troponin-C in skinned preparations. The molecular mechanism of the cross-bridge-dependent change in the affinity of troponin-C for Ca2+ is not fully understood although the studies which employ the substitution of troponin-C in skinned preparations, transgenic animals and in an animal model with heart disease have been performed. We reviewed the current studies by analyzing the intracellular mechanism responsible for the length-dependent change in tension development in mammalian cardiac muscle.

1. We measured the change in intracellular Ca2+ transients, using aequorin, in response to muscle length change during twitch contraction in ferret ventricular muscles. 2. Intracellular Ca2+ concentration ([Ca2+]i) was transiently increased when the muscle length was quickly shortened to 92% of maximum length (Lmax) at various times after stimulation (this increase in [Ca2+] is termed extra-Ca2+). The magnitude of extra-Ca2+, measured at different extracellular Ca2+ concentrations ([Ca2+]o), showed a dependence upon the magnitude of tension reduction and upon [Ca2+]i immediately before the length change. 3. In the presence of caffeine (5 mM), the difference between the Ca2+ transient at Lmax and at shorter lengths showed a time course similar to the difference between the developed tension at both lengths. A quick release in the caffeine-treated preparation produced the extra-Ca2+ with a slower time course compared with that observed in the absence of caffeine. Stretching the muscle from 96% Lmax to Lmax produced more active tension and decreased [Ca2+]i. 4. These results indicate that the affinity of troponin-C, a major Ca2+ binding protein, which controls contraction, is influenced by developed tension i.e. cross-bridge attachment and detachment.

We investigated the mechanisms of the effects of a newly synthesized cyclic AMP phosphodiesterase inhibitor (PDEI: 1,5-dihydro-7-(1-piperidinyl)imidazo[2,1-beta]quinazolin- 2(3H)-one dihydrochloride hydrate, DN-9693) on intracellular Ca2+ transients and tension in ferret papillary muscles. We used the aequorin method to measure Ca2+ transients in intact preparations. DN-9693 (1-100 microM) increased the peaks of both Ca2+ transients and tension. The potentiation of Ca2+ transients by DN-9693 was significant at lower concentrations (1-10 microM) but not at higher concentrations (50 and 100 microM). The peak of tension was significantly increased when the concentration of DN-9693 was increased (1-100 microM). In addition, the time to peak light was shortened at lower concentrations (1 and 5 microM). Other parameters of Ca2+ transients were not significantly altered. The time course of tension was not affected by DN-9693. In Triton X-100-treated skinned preparations, DN-9693 decreased the Ca2+ concentration required for half-maximal activation of the pCa-tension relation. These results suggest that the positive inotropic effect of DN-9693 is attributable to the alteration in Ca2+ handling of sarcoplasmic reticulum (SR) and Ca2+ sensitization of the myofilaments.

The effects of adenosine on Ca2+ transients and tension in ferret papillary muscles were investigated using the aequorin method. Adenosine (0.01-1 mM) reduced the peak of Ca2+ transients and caused a slight concentration-dependent decrease in tension. Adenosine prolonged the decay time of Ca2+ transients but did not alter the time course of tension. In isoproterenol (0.1 microM)-treated preparations, adenosine decreased the peak of Ca2+ transients but did not alter the peak of tension. Adenosine prolonged the isoproterenol-shortened decay time of Ca2+ transients. The effects of adenosine on Ca2+ transients were antagonized by the selective A1 receptor antagonist 8-cyclopentyl-1,3,-dipropylxanthine. In the presence of isoproterenol, adenosine (0.1 mM) shifted the intracellular [Ca2+]/tension relation to the left. These results can be explained by the hypothesis that adenosine inhibits the activity of adenylate cyclase via stimulation of the A1 receptor, other mechanisms however cannot be overlooked. The prolongation of the decay time of Ca2+ transients and the increase in the Ca2+ sensitivity of the contractile elements are the underlying mechanisms of adenosine which maintain developed tension in twitch response, although adenosine decreases the peak of Ca2+ transients.

We investigated the effects of a newly synthesized dihydropyridine (DHP) derivative (NZ-105) on intracellular Ca transients and contraction in ferret ventricular muscles, using the aequorin method. Low concentrations of NZ-105 (10(-9)-10(-7) M) showed no significant effects on the light signal of aequorin and tension in twitch. High concentrations of NZ-105 (10(-6)-10(-5) M) inhibited the peaks of the light signal and tension without altering their time courses. The relation between the peaks of the light signal and tension in twitch, measured in solutions with varying Ca2+ concentrations, was shifted to the left by NZ-105. The relation between [Ca2+]i and tension measured in tetanic contraction was also shifted to the left by NZ-105. These results suggest that the inhibitory effect of NZ-105 on contraction in mammalian cardiac muscle is curtailed by an increase in Ca sensitivity of the contractile elements.

In this short review, some of the factors which influence the intracellular Ca2+ transients and contraction in mammalian cardiac muscles which we have been examining are discussed. The processes concerning the change in [Ca2+]i and contraction are divided into three steps following the scheme proposed by Blinks [6]: Upstream, Central, and Downstream. However, these processes are interrelated. Although the Ca2+ transient is an important step for triggering contraction, it is influenced by the following Central and Downstream mechanisms. The evaluation of Ca2+ sensitivity of the contractile elements (Central mechanism) in intact preparations is discussed; measurement of the relation between [Ca2+]i and tension in tetanic contraction is one of the possible methods. A change in the Ca2+ binding to troponin-C is an important factor to determine the Ca2+ sensitivity but other mechanisms are also considered to be involved. In cardiac muscles, the feedback mechanism from the Downstream, in particular the cross-bridge attachment and detachment, to the Central processes is a physiologically important mechanism in the determination of contractile properties. This feedback mechanism alternatively influences the intracellular Ca2+ transient by changing the affinity of troponin-C for Ca2+. Thus, the interpretation of the measured Ca2+ transients is not simple and the numerous factors involved must be considered. Further study is necessary to elucidate the molecular mechanisms which are responsible for the feedback from the Downstream mechanism to the Central and Upstream mechanisms.

1. To study the effects of mechanical constraints on the calcium (Ca2+) affinity of cardiac troponin C, we analysed the tension and aequorin light (AL, intracellular Ca2+) transients in response to a step length change in aequorin-injected ferret right ventricular papillary muscles. The muscle preparations were continuously activated with ouabain (10(-4) M) (ouabain contracture) or with high frequency stimuli in the presence of ryanodine (5 microM) (tetanic contraction). 2. The tension transient in response to either the release or stretch was oscillatory: tension decreased rapidly during the release and then increased, after which it lapsed into a new steady level in a series of damped oscillations. The opposite was true for the stretch. The oscillatory responses were conspicuous and less damped in ouabain-activated preparations (oscillation frequency of 2.2-2.3 Hz at 22 degrees and 4.5-4.6 Hz at 30 degrees C) and much more damped in ryanodine-treated preparations. 3. The transient AL response was also oscillatory, the time course of which corresponded to that of the transient tension response. Regardless of the difference in the time course of the transients in two different preparations and at two different temperatures, the increase in AL corresponded to the decrease in tension, likewise the decrease in AL to the increase in tension. 4. The mean level of AL after release was lower than the control level present just prior to the release in ouabain-activated preparations, but the AL after release finally returned to the nearly control level in ryanodine-treated preparations. 5. When the ryanodine-treated muscle was further treated with 2,3-butanedione monoxime (BDM) (20 mM), the tetanic tension decreased remarkably without affecting the AL signal. The tension transient of this preparation was quite similar to that of the resting muscle, which changed in a nearly stepwise fashion; AL was hardly affected by step length changes, as in the resting muscle, in spite of the higher AL level. 6. These results suggest that the Ca2+ affinity of cardiac troponin C is increased with an increase in tension (i.e. the cross-bridge attachment) and decreased with a decrease in tension i.e. the cross-bridge detachment), and that the mean [Ca2+]i is lowered by release, at least in a Ca(2+)-overloaded condition, mainly through the sarcoplasmic reticulum.

The Ca(2+)-sensitive photoprotein aequorin was injected into single frog skeletal muscle fibers, and the intracellular aequorin light intensity during muscle activation with different maneuvers was mapped with digital imaging microscopy. During 50 Hz electrical activation (tetanus), the aequorin light intensity from different locations in the muscle fiber rose with very similar time course. Caffeine (10 mM) application, on the other hand, caused aequorin light signals to show significantly different time courses, with an earlier increase in Ca2+ concentration near the surface of the fiber than near the core. The non-uniform rise of intracellular Ca2+ concentration with caffeine treatment is consistent with the slow inward diffusion of caffeine and subsequent Ca2+ release from sarcoplasmic reticulum.

1. We investigated the mechanism of signal transduction during the effect of muscarinic receptor stimulation on Ca2+ transients, tension, Ca2+ sensitivity and the cross-bridge cycling rate (CCR). 2. Membrane-permeable derivatives of cyclic GMP (8-bromo-cyclic GMP and dibutyryl cyclic GMP) did not cause any significant changes in the peaks of Ca2+ transients and tension and the time courses of either signal modulated by isoprenaline (Iso) (0.1 microM). 3. Nitroprusside (0.1-1 mM) likewise did not change the peaks or the time courses of Ca2+ transients and tension in the Iso-treated preparations. 4. In papillary muscles excised from ferrets treated with pertussis toxin (islet-activating protein, IAP), which is known to abolish the function of GTP-binding proteins (Gi, Go and Gt), similar changes in Ca2+ transients and tension produced by treatment with Iso (0.1 microM) were noted as in non-IAP-treated preparations. However, no effects of acetylcholine (ACh; 1 microM) on either signal were observed. 5. The relation between [Ca2+]i and tension measured during the steady state of tetanic contraction was shifted to the right by Iso (0.1 microM), and cyclic GMP derivatives (1 mM) did not change the altered relation. In the IAP-treated preparations, ACh (1 microM) did not influence the relation altered by Iso (0.1 microM). 6. Cyclic GMP derivatives (1 mM) did not alter the Iso (0.1 microM)-increased CCR measured by perturbation analysis. ACh (1 microM) did not restore the Iso-increased CCR in the IAP-treated preparations. 7. These results suggest that signal transduction in muscarinic receptor stimulation is primarily mediated by inhibition of adenylate cyclase via IAP-sensitive GTP-binding proteins, and that cyclic GMP does not play an important role in the effect of muscarinic receptor stimulation on Ca2+ transients, tension, Ca2+ sensitivity or CCR.

1. To clarify the mechanism which regulates the time course of twitch tension when beta- and muscarinic receptors are stimulated, intracellular Ca2+ transients, Ca2+ sensitivity of the contractile element and the cross-bridge cycling rate (CCR) were measured in ferret ventricular muscles. 2. Isoprenaline (Iso; 0.1 microM) increased peaks of Ca2+ transients measured with aequorin and tension, and abbreviated the time courses of both signals. Addition of acetylcholine (ACh; 0.01-1 microM) to the Iso-treated preparation dose dependently decreased the peaks of both signals and restored the time course of Ca2+ transients. However, the time course of tension was not recovered by the addition of ACh, and the relaxation time in particular, was further shortened by ACh. Carbachol (1 microM) applied to the Iso-treated preparation yielded similar results. 3. [Ca2+]i and tension at a quasi-steady level of tetanic contraction, which was produced by ryanodine (5 microM) and repetitive stimulation, were measured and Ca2+ sensitivity of the contractile element was estimated. Iso (0.1 microM) decreased the Ca2+ sensitivity and the addition of ACh (1 microM) completely recovered it to the control level. 4. In order to measure CCR, the perturbation analysis method was applied to steady-state tension of tetanic contraction. The CCR was not altered even when the tetanic tension level was decreased to 50% by decreasing [Ca2+]o. Iso (0.1 microM) slightly decreased the tetanic tension level and increased the CCR from 2.73 to 3.25 Hz. The effect of Iso was observed when the Iso-decreased tension was recovered by an increase in [Ca2+]i. The addition of ACh (1 microM) recovered the CCR which was increased by Iso, to the control level. Atropine (10 microM) blocked the effect of ACh, and carbachol (1 microM) restored the CCR increased by Iso to the control level. 5. The time course of Ca2+ transients, Ca2+ sensitivity and CCR were antagonistically regulated by beta- and muscarinic receptor stimulation, but the time course of tension did not parallel the changes in these parameters. Therefore, these results suggest that the time course of tension, particularly the relaxation time, is not determined by the time course of Ca2+ transients, Ca2+ sensitivity and the CCR, and that other factors might be involved in the regulation of the time course of tension when beta- and muscarinic receptors are stimulated.

To elucidate the effects of mechanical constraints on the (Ca2+) affinity of cardiac troponin C, we studied the relationships among the myoplasmic Ca2+ concentration ([Ca2+]i), tension and length in steadily activated intact cardiac muscle. The Ca2+ sensitive photoprotein, aequorin, was micro-injected into cells of ferret right ventricular papillary muscles to monitor the [Ca2+]i. The muscle was then steadily activated with ouabain (10(-4) M)(ouabain contracture) or high frequency stimuli in the presence of ryanodine (5 microM)(tetanic contraction); the tension and aequorin light (AL) transients in response to a step length change were then analyzed. The tension transient response to either the stretch or release in length was oscillatory: tension decreased rapidly during the release and then increased, after which it lapsed into a new steady level in a series of damped oscillations. The opposite was true for the stretch. The oscillatory responses were conspicuous and less damped in the ouabain contracture. The transient AL response was also oscillatory, the time course of which corresponded exactly to that of the tension transient response, though no detectable changes in AL were observed at the initial phase of the stretch response. The increase in AL corresponded exactly to the decrease in tension, likewise the decrease in AL to the increase in tension. The steady level of AL after release was decreased in ouabain contracture, but was increased in tetanic contraction. These results suggest that the Ca2+ affinity of cardiac troponin C is increased with an increase in tension (i.e., the cross-bridge attachment) and decreased with a decrease in tension (i.e., the cross-bridge detachment), and that the myoplasmic calcium concentration is lowered by release, at least in a Ca(2+)-overloaded condition, mainly through the sarcoplasmic reticulum.

The fluorescent Mg2+/Ca2+ indicator, furaptra, was injected into single frog skeletal muscle fibers, and the indicator's fluorescence signals were measured and analyzed with particular interest in the free Mg2+ concentration ([Mg2+]) in resting muscle. Based on the fluorescence excitation spectrum of furaptra, the calibrated myoplasmic [Mg2+] level averaged 0.54 mM, if the value of dissociation constant (KD) for Mg2+ obtained in vitro (5.5 mM) was used. However, if the indicator reacts with Mg2+ with a two-fold larger KD in myoplasm, as previously suggested for the furaptra-Ca2+ reaction (M. Konishi, S. Hollingworth, A.B. Harkins, S.M. Baylor. 1991. J. Gen. Physiol. 97:271-301), the calculated [Mg2+] would average 1.1 mM. Thus, the value 1.1 mM probably represents the best estimate from furaptra of [Mg2+] in resting muscle fibers. Extracellular perfusion of muscle fibers with high Mg2+ concentration solution or low Na+ concentration solution did not cause any detectable changes in the [Mg2+]-related furaptra fluorescence within 4 min. The results suggest that the myoplasmic [Mg2+] is highly regulated near the resting level of 1 mM, and that changes only occur with a very slow time course.

Vitamin D metabolites exert both acute and chronic influences on proximal tubule function. To further evaluate vitamin D action on the kidney, we examined the immediate effects of vitamin D metabolites on cytoplasmic calcium ion concentration [( Ca2+]i), using fura-2 and patch-clamp method in cultured proximal straight tubule cells of rabbit kidney. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] and 25-hydroxyvitamin D3 [25(OH)D3] evoked a transient rise in [Ca2+]i, and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] caused a sustained rise in [Ca2+]i; all effects were dose dependent. [Ca2+]i transient, evoked by 1,25(OH)2D3 alone, was abolished in Ca(2+)-free media. Pretreatment of cells in Ca(2+)-free media with caffeine (4 mM) or ryanodine (1 microM) to deplete Ca2+ store of endoplasmic reticulum or with TMB-8 (5 mM) to block Ca2+ release from storage blunted the effect of 25(OH)D3 on [Ca2+]i but not of 24,25(OH)2D3. Data were also supported by activities of Ca-dependent K channel and show that these three vitamin D metabolites in pharmacological doses increase [Ca2+]i of proximal tubule cells from different sources.

Time-resolved X-ray equatorial diffraction studies on a single frog skeletal muscle fiber were performed with a 10 ms time resolution using synchrotron radiation in order to compare the time courses of the molecular changes of contractile proteins and the intracellular Ca2+ transient during an isometric twitch contraction at 2.7 degrees C. Measurements of the Ca2+ transient using aequorin as an intracellular Ca2+ indicator were conducted separately just before and after the X-ray experiments under very similar experimental conditions. The results, which showed a similar time course of tension to that observed in the X-ray experiment, were compared with the aequorin light signal, tension and the intensity changes of the 1,0 and 1,1 equatorial reflections. No appreciable change in both reflection spacings indicated that the effect of internal shortening of the muscle was minimized during contraction. The intensity change of the equatorial reflections generally occurred after the aequorin light signal. In the rising phase, the time course of increase in the 1,1 intensity paralleled that of the rise of the light signal and the intensity peak occurred 20-30 ms after the peak of the light signal. The decrease in the 1,0 intensity showed a time course similar to that of tension and the intensity minimum roughly coincided with the tension peak, coming at 80-90 ms and about 60 ms after the peaks of the light signal and the 1,1 intensity change, respectively. In the relaxation phase, the 1,1 intensity seemed to fall rapidly just before the tension peak and then returned to the original level in parallel with the decay of tension. The 1,0 intensity returned more slowly than the tension relaxation. Thus, the change of the 1,1 intensity was faster than that of the 1,0 intensity in both the rising and relaxation phases. When the measured aequorin light signal was corrected for the kinetic delay of the aequorin reaction with a first-order rate constant of either 50 or 17 s-1, the peak of the corrected light signal preceded that of the measured one by approx. 30 ms. Thus, the peak of the Ca2+ transient appeared earlier than the peaks of the 1,1 and 1,0 intensity changes by 50-60 and 110-120 ms, respectively. The time lag between the extent of structural change and the Ca2+ transient is discussed in relation to the double-headed attachment of a cross-bridge to actin.

Intracellular Ca(2+)-related optical signals during and after contraction (twitch and tetanus) were measured in single frog skeletal muscle fibers with fura-2 and aequorin. In twitch response, the peaks of [Ca2+]i estimated from the in vitro calibrations of fura-2 and aequorin were significantly different (0.5 microM for fura-2 and 5 microM for aequorin). Even 30s after twitch response, the fura-2 fluorescence ratio (F340/F380) signal did not recover to the resting level before stimulation. When the stimulation frequency was increased, an increase in the resting fura-2 ratio signal became obvious and after the cessation of stimulation this increase gradually recovered to the level before stimulation. After tetanus (50 Hz for 1 s), a higher fura-2 ratio signal than that before stimulation was sustained longer than 90 s. These results indicate that the dissociation of the released Ca2+ from the intracellular Ca2+ binding sites probably takes longer time than that previously reported. The present results, therefore, demonstrate that fura-2 is advantageous for qualitative monitoring of a slight change in the resting level of [Ca2+]i, which is not easily detectable with aequorin. In addition, the problems encountered in quantitative estimation of [Ca2+]i with fura-2 are also discussed.

1. The mechanism of modulation of Ca2+ transients and contraction by beta-adrenoceptor stimulation was studied in ferret ventricular muscles using aequorin to measure intracellular Ca2+. 2. Peaks of tension and light transients were increased by isoprenaline (10(-9) - 5 x 10(-7) M) which also abbreviated their time courses. 3. Time-to-peak tension was significantly shortened by 5 x 10(-9) M-isoprenaline and time-to-peak light was abbreviated by 10(-9) M-isoprenaline. 4. The time for the light to decay was shortened at 10(-9) M-isoprenaline. However, a higher concentration of isoprenaline (10(-8) M) was required for significant shortening of the half-relaxation time (TR50). 5. When isoprenaline was removed and beta-blocker (bupranolol, 1 microM) was applied, the time course of the light transients recovered but the time course of relaxation did not recover. 6. The relationship between [Ca2+]i and tension in tetanic contraction produced in the presence of ryanodine (5 microM) was shifted to the right by isoprenaline (10(-8) M). This was recovered by the replacement of isoprenaline with bupranolol (1 microM). 7. Isoprenaline (10(-7) M) added to the solution containing 20 mM [Ca2+]O and Bay K 8644 (1 microM), which produced maximal tension, caused a large light signal and enhancement of the initial phasic tension in tetanic contraction. However, the replacement of isoprenaline with bupranolol after immersing the preparation in 20 mM [Ca2+]O solution with Bay K 8644 and isoprenaline, did not significantly change the tension level, although the light signal decreased. Similar results were obtained in the ventricular muscle of young rats. 8. These results suggest that the dose dependence of modulation of the contractile element and sarcoplasmic reticulum (SR) by beta-adrenoceptor stimulation differs, and that additional factors, other than the faster Ca2+ uptake by SR and the decrease in Ca2+ sensitivity of the contractile element, might be involved in the shortening of the half-relaxation time by beta-adrenoceptor stimulation. In addition, beta-adrenoceptor stimulation does not produce a marked change in the maximal tension level.

Although animal cells swell in hypotonic medium, their volume is subsequently regulated by a net loss of KCl via Ca2(+)-dependent channels. A rise in intracellular free calcium ([Ca2+]i) thus appears to be an initial event in the adaptation of external tonicity, although details of this mechanism are not known. To investigate cell volume regulation, we measured [Ca2+]i (by use of fura-2) and cell diameters in single cells of cultured renal proximal convoluted tubule. We found that a rapid rise in [Ca2+]i occurred after cells were exposed to hypotonic solution (250 mosM) from 95.8 +/- 3.8 to 468.2 +/- 24 nM (n = 16). The rise in [Ca2+]i was not observed in cells exposed to Ca2(+)-free medium, and exposure to isotonic high-K or low-Na medium did not elicit a rise in [Ca2+]i, suggesting that this rise was a result of Ca2+ influx and not via voltage-dependent Ca2+ influx or decrease of Ca2+ efflux via Na(+)-Ca2+ pump. Pretreatment of cells with pertussis toxin dose dependently blocked the rise in [Ca2+]i. The hypotonic solution enhanced accumulations of inositol tris- and tetra-phosphate after a 1-min exposure. Studies that measured cell diameters suggest that recovery of cell volume may include the rise in [Ca2+]i. These data suggest that the regulatory volume decrease of proximal tubule cells involves a pertussis toxin-sensitive guanine nucleotide binding protein-operated Ca2+ influx.