Myocardial calcium compartmentation and contractile control.

Under the condition of rapid perfusion, the time course of contractile response of single ventricular cells to extracellular calcium (Ca) depletion and repletion identifies "fast" and "slow" cellular Ca pools. 45Ca exchange was studied in these cells under the same conditions of on-line rapid perfusion. Four kinetically-defined compartments were distinguished: (1) A "rapid" compartment containing 2.6 mmoles Ca/kg dry wt of lanthanum (La) displaceable Ca, t1/2 less than 1 sec.; (2) An "intermediate" compartment(s) containing 2.1 mmoles, t1/2 = 3 and 19 sec. Caffeine displaced significant amounts of Ca from this compartment whereas La displaced none; (3) A "slow" compartment containing 1.6 mmoles, t1/2 = 3.6 min. Addition of inorganic phosphate to the perfusate adds significant amounts of Ca to this compartment; (4) An "inexchangeable" compartment, containing 1.2 mmoles. The "rapid" compartment's flux is greater than or equal to 300 mumoles Ca/kg wet wt/sec. Its exchange rate indicates that it is the kinetic counterpart of the functionally-defined "fast" pool. Its subcellular locus is undefined. The "intermediate" compartment is best correlated with the "slow" pool and represents Ca in the sarcoplasmic reticulum. The "slow" compartment contains a significant fraction from the mitochondria. The results indicate that greater than or equal to 40% of cellular Ca can turn over within the period of one contraction cycle. These results are consistent with the following sequence: (1) Upon sarcolemmal depolarization, Ca moves through the Ca channel to arrive at the SR and at the myofilaments. (2) Ca induced Ca release occurs via the "feet" at the SR-inner SL region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations in cellular calcium handling as a result of systemic calcium deficiency in the developing chick embryo: I. Erythrocytes.

Chick embryos rendered calcium (Ca) deficient by shell-less (SL) culture develop hypertension and tachycardia. Since hypocalcemia is accompanied by hypernatremia systemically but not by lower cellular Ca (Koide and Tuan, 1989), we speculate that cellular Ca handling may be altered in the SL embryo, perhaps involving Na transport. Using erythrocytes (RBC) from day-14 SL and normal (NL) embryos as the experimental cell, cellular Ca handling was studied under varying extracellular osmotic and ionic conditions by analyzing 45Ca uptake and cell volume regulation. Two agents, p-chloromercuriphenylsulfonate (PCM), and inosine/iodoacetamide (INI) were used to treat the RBCs to modify plasma membrane ion permeability and to deplete cellular ATP, respectively. Other cellular functions and activities related to Ca homeostasis, including ATP content and Ca(2+)-ATPase activity, were also analyzed. These analyses showed: (1) in NaCl, Ca uptake was similar in NL and SL cells, except after INI treatment, which resulted in slower Ca uptake by the SL cells, (2) in choline and sucrose, Ca uptake by SL RBCs was higher, (3) Ca uptake by RBCs of both embryos changed depending on the osmotic agent (Na < K < or = choline < sucrose), (4) Ca(2+)-ATPase activity was higher in SL RBC, although there was no change in the size or charge of the enzyme, and (5) in any osmotic agent, cellular Na was significantly lower, whereas cellular K was higher, in SL RBC. Based on these results, three features of RBC Ca handling were apparent: (1) Na-Ca exchange was functional and was more active in SL RBCs, (2) Ca uptake was dependent on the total ionic electrochemical gradient but not on bulk H2O movement, and (3) Ca pumping out capacity was directly correlated with Ca(2+)-ATPase activity. Elevated Ca uptake in sucrose-treated SL RBC is therefore indicative of its greater ion permeability. Taken together, these findings indicate that cellular Ca handling of the RBCs of SL chick embryos is characterized by a more active Na-Ca exchange system, greater ion permeability, and higher Ca pumping out capacity, thereby suggesting an up-regulated Ca handling function in the SL RBCs. The abnormal cellular Ca handling may be a direct result of the systemic Ca deficiency of the SL chick embryo and may be functionally related to its hypertension and tachycardia.     

Na-Ca exchange and the trigger for sarcoplasmic reticulum Ca release: studies in adult rabbit ventricular myocytes.

The importance of Na-Ca exchange as a trigger for sarcoplasmic reticulum (SR) Ca release remains controversial. Therefore, we measured whole-cell Ca currents (ICa), Na-Ca exchange currents (INaCa), cellular contractions, and intracellular Ca transients in adult rabbit cardiac myocytes. We found that changing pipette Na concentration markedly affected the relationship between cell shortening (or Ca transients) and voltage, but did not affect the Ca current-voltage relationship. We then inhibited Na-Ca exchange and varied SR content (by changing the number of conditioning pulses before each test pulse). Regardless of SR Ca content, the relationship between contraction and voltage was bell-shaped in the absence of Na-Ca exchange. Next, we rapidly and completely blocked ICa by applying nifedipine to cells. Cellular shortening was variably reduced in the presence of nifedipine. The component of shortening blocked by nifedipine had a bell-shaped relationship with voltage, whereas the "nifedipine-insensitive" component of contraction increased with voltage. With the SR disabled (ryanodine and thapsigargin pretreatment), ICa could initiate late-peaking contractions that were approximately 70% of control amplitude. In contrast, nifedipine-insensitive contractions could not be elicited in the presence of ryanodine and thapsigargin. Finally, we recorded reverse Na-Ca exchange currents that were activated by membrane depolarization. The estimated sarcolemmal Ca flux occurring by Na-Ca exchange (during voltage clamp steps to +30 mV) was approximately 10-fold less than that occurring by ICa. Therefore, Na-Ca exchange alone is unlikely to raise cytosolic Ca concentration enough to directly activate the myofilaments. We conclude that reverse Na-Ca exchange can trigger SR Ca release. Because of the sigmoidal relationship between the open probability of the SR Ca release channel and pCa, the effects of ICa and INaCa may not sum in a linear fashion. Rather, the two triggers may act synergistically in the modulation of SR release.     

A mathematical treatment of integrated Ca dynamics within the ventricular myocyte

We have developed a detailed mathematical model for Ca2+ handling and ionic currents in the rabbit ventricular myocyte. The objective was to develop a model that: 1), accurately reflects Ca-dependent Ca release; 2), uses realistic parameters, particularly those that concern Ca transport from the cytosol; 3), comes to steady state; 4), simulates basic excitation-contraction coupling phenomena; and 5), runs on a normal desktop computer. The model includes the following novel features: 1), the addition of a subsarcolemmal compartment to the other two commonly formulated cytosolic compartments (junctional and bulk) because ion channels in the membrane sense ion concentrations that differ from bulk; 2), the use of realistic cytosolic Ca buffering parameters; 3), a reversible sarcoplasmic reticulum (SR) Ca pump; 4), a scheme for Na-Ca exchange transport that is [Na]i dependent and allosterically regulated by [Ca]i; and 5), a practical model of SR Ca release including both inactivation/adaptation and SR Ca load dependence. The data describe normal electrical activity and Ca handling characteristics of the cardiac myocyte and the SR Ca load dependence of these processes. The model includes a realistic balance of Ca removal mechanisms (e.g., SR Ca pump versus Na-Ca exchange), and the phenomena of rest decay and frequency-dependent inotropy. A particular emphasis is placed upon reproducing the nonlinear dependence of gain and fractional SR Ca release upon SR Ca load. We conclude that this model is more robust than many previously existing models and reproduces many experimental results using parameters based largely on experimental measurements in myocytes.     

Interactions of the exchange inhibitory peptide with Na-Ca exchange in bovine cardiac sarcolemmal vesicles and ferret red cells.

The Na-Ca exchange inhibitory peptide (XIP), which corresponds to residues 251-270 of the Na-Ca exchange protein, specifically inhibits exchange activity (Li, Z., Nicoll, D. A, Collins, A., Hilgemann, D. W., Filoteo, A. G., Penniston, J. T., Weiss, J. N., Tomich, J. M., and Philipson, K. D. (1991) J. Biol. Chem. 266, 1014-1020). We have found that XIP decreased Na+i-dependent Ca2+ uptake to 46 and 20% of control in mixed and inside-out bovine sarcolemmal (SL) vesicles, respectively, and to 22% of control in ferret red cell vesicles. XIP inhibited uptake in bovine SL vesicles after proteolytic digestion. XIP also inhibited Na+o-dependent Ca2+ efflux in bovine SL vesicles but did not inhibit Ca2+ uptake in reconstituted proteoliposomes. Extracellular XIP did not inhibit Ca2+ uptake into intact ferret red cells. Inhibition of uptake in bovine SL vesicles was reduced as the ionic strength was increased. 125I-labeled XIP (1 microM) was cross-linked to proteins of bovine SL vesicles, ferret red cell vesicles, and intact ferret red cells. Labeling of bands at approximately 75, 120, and 220 kDa (in bovine SL vesicles) and bands at 55 and 85 kDa (in ferret red cell vesicles) was detected. No cross-linking was detected in intact ferret red cells. We conclude that XIP inhibition is insensitive to proteolytic digestion and is partially dependent on charge association and conformation of the exchanger. XIP binds to and interacts with the intracellular side of the Na-Ca exchanger.     

Activation of Na-Ca exchange current by photolysis of "caged calcium".

Intracellular photorelease of Ca2+ from "caged calcium" (DM-nitrophen) was used to investigate the Ca(2+)-activated currents in ventricular myocytes isolated from guinea pig hearts. The patch-clamp technique was applied in the whole-cell configuration to measure membrane current and to dialyze the cytosol with a pipette solution containing the caged compound. In the presence of inhibitors for Ca2+, K+, and Na+ channels, concentration jumps of [Ca2+]i induced a rapidly activating inward Na-Ca exchange current which then decayed slowly (tau approximately 500 ms). The initial peak of the inward current and the time-course of current decay were voltage-dependent, and no reversal of the current direction was found between -100 and +100 mV. The observed shallow voltage dependence can be described in terms of the movement of an apparently fractional elementary charge (+0.44e-) across an energy barrier located symmetrically in the electrical field of the membrane. The currents were dependent on extracellular Na+ with a half-maximal activation at 73 mM and a Hill coefficient of 2.8. No change of membrane conductance was activated by the Ca2+ concentration jump when extracellular Na+ was completely replaced by Li+ or N-methyl-D-glucamine (NMG) or when the Na-Ca exchange was inhibited by extracellular Ni2+, La3+, or dichlorobenzamil (DCB). The velocity of relengthening after a twitch induced by photorelease of Ca2+ was only reduced drastically when both the sarcoplasmic reticulum and the Na-Ca exchange were inhibited suggesting that all other Ca2+ removing mechanisms have a low transport capacity under these conditions. In conclusion, we have used a novel approach to study Na-Ca exchange activity with photolysis of "caged" calcium. We found that in guinea pig heart muscle cells the Na-Ca exchange is a potent mechanism for Ca2+ extrusion, is weakly voltage-dependent (118 mV for e-fold change) and can be studied without contamination with other Ca(2+)-activated currents.     

Modification of intracellular calcium concentration in cardiomyocytes by inhibition of sarcolemmal Na+/H+ exchanger

Although the Na(+)/H(+) exchanger (NHE) is considered to be involved in regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) through the Na(+)/Ca(2+) exchanger, the exact mechanisms of its participation in Ca(2+) handling by cardiomyocytes are not fully understood. Isolated rat cardiomyocytes were treated with or without agents that are known to modify Ca(2+) movements in cardiomyocytes and exposed to an NHE inhibitor, 5-(N-methyl-N-isobutyl)amiloride (MIA). [Ca(2+)](i) in cardiomyocytes was measured spectrofluorometrically with fura 2-AM in the absence or presence of KCl, a depolarizing agent. MIA increased basal [Ca(2+)](i) and augmented the KCl-induced increase in [Ca(2+)](i) in a concentration-dependent manner. The MIA-induced increase in basal [Ca(2+)](i) was unaffected by extracellular Ca(2+), antagonists of the sarcolemmal (SL) L-type Ca(2+) channel, and inhibitors of the SL Na(+)/Ca(2+) exchanger, SL Ca(2+) pump ATPase and mitochondrial Ca(2+) uptake. However, the MIA-induced increase in basal [Ca(2+)](i) was attenuated by inhibitors of SL Na(+)-K(+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+) transport. On the other hand, the MIA-mediated augmentation of the KCl response was dependent on extracellular Ca(2+) concentration and attenuated by agents that inhibit SL L-type Ca(2+) channels, the SL Na(+)/Ca(2+) exchanger, SL Na(+)-K(+)-ATPase, and SR Ca(2+) release channels and the SR Ca(2+) pump. However, the effect of MIA on the KCl-induced increase in [Ca(2+)](i) remained unaffected by treatment with inhibitors of SL Ca(2+) pump ATPase and mitochondrial Ca(2+) uptake. MIA and a decrease in extracellular pH lowered intracellular pH and increased basal [Ca(2+)](i), whereas a decrease in extracellular pH, in contrast to MIA, depressed the KCl-induced increase in [Ca(2+)](i) in cardiomyocytes. These results suggest that NHE may be involved in regulation of [Ca(2+)](i) and that MIA-induced increases in basal [Ca(2+)](i), as well as augmentation of the KCl-induced increase in [Ca(2+)](i), in cardiomyocytes are regulated differentially.     

Na-Ca exchanger as a calcium influx pathway in adrenal glomerulosa cells

When aequorin-loaded glomerulosa cells were incubated in isotonic Na2+-free medium containing N-methyl-D-glucamine instead of NaCl, there was an increase in cytoplasmic free calcium concentration, [Ca2+] c, which was not observed when extracellular calcium concentration was reduced to 1 microM. Upon removal of extracellular sodium, there was nearly five-fold increase in fractional efflux ratio of calcium. The reduction of extracellular sodium resulted in a stimulation of calcium influx rate, the magnitude of which was dependent on extracellular sodium concentration. Similar stimulation of calcium influx was observed when extracellular sodium was replaced with lithium. Nitrendipine did not affect the calcium influx induced by the reduction of extracellular sodium while a derivative of amiloride 3',4'-dichlorobenzamil, which inhibits Na-Ca exchange, attenuated calcium influx observed in sodium-free medium. These results indicate that removal of extracellular sodium leads to an increase in [Ca2+] c by stimulating calcium influx and that calcium enters the cell via Na-Ca exchanger.     

Measurements of Ca2+ entry and sarcoplasmic reticulum Ca2+ content during the cardiac cycle in guinea pig and rat ventricular myocytes.

This study investigates the contribution of Ca2+ entry via sarcolemmal (SL) Ca2+ channels to the Ca2+ transient and its relationship with sarcoplasmic reticulum (SR) Ca2+ content during steady-state contraction in guinea pig and rat ventricular myocytes. The action potential clamp technique was used to obtain physiologically relevant changes in membrane potential. A method is shown that allows calculation of Ca2+ entry through the SL Ca2+ channels by measuring Cd(2+)-sensitive current during the whole cardiac cycle. SR Ca2+ content was calculated from caffeine-induced transient inward current. In guinea pig cardiac myocytes stimulated at 0.5 Hz and 0.2 Hz, Ca2+ entry through SL Ca2+ channels during a cardiac cycle was approximately 30% and approximately 50%, respectively, of the SR Ca2+ content. In rat myocytes Ca2+ entry via SL Ca2+ channels at 0.5 Hz was approximately 3.5% of the SR Ca2+ content. In the presence of 500 nM thapsigargin Ca2+ entry via SL Ca2+ channels in guinea pig cardiac cells was 39% greater than in controls, suggesting a larger contribution of this mechanism to the Ca2+ transient when the SR is depleted of Ca2+. These results provide quantitative support to the understanding of the relationship between Ca2+ entry and the SR Ca2+ content and may help to explain differences in the Ca2+ handling observed in different species.     

In situ SR function in postinfarction myocytes.

Previous studies have shown lower systolic intracellular Ca(2+) concentrations ([Ca(2+)](i)) and reduced sarcoplasmic reticulum (SR)-releasable Ca(2+) contents in myocytes isolated from rat hearts 3 wk after moderate myocardial infarction (MI). Ca(2+) entry via L-type Ca(2+) channels was normal, but that via reverse Na(+)/Ca(2+) exchange was depressed in 3-wk MI myocytes. To elucidate mechanisms of reduced SR Ca(2+) contents in MI myocytes, we measured SR Ca(2+) uptake and SR Ca(2+) leak in situ, i.e., in intact cardiac myocytes. For sham and MI myocytes, we first demonstrated that caffeine application to release SR Ca(2+) and inhibit SR Ca(2+) uptake resulted in a 10-fold prolongation of half-time (t(1/2)) of [Ca(2+)](i) transient decline compared with that measured during a normal twitch. These observations indicate that early decline of the [Ca(2+)](i) transient during a twitch in rat myocytes was primarily mediated by SR Ca(2+)-ATPase and that the t(1/2) of [Ca(2+)](i) decline is a measure of SR Ca(2+) uptake in situ. At 5.0 mM extracellular Ca(2+), systolic [Ca(2+)](i) was significantly (P 相似文献   

Mechanisms of lysophosphatidic acid-induced increase in intracellular calcium in vascular smooth muscle cells

Although lysophosphatidic acid (LPA) is known to cause an increase in intracellular Ca2+ concentration ([Ca2+]i) in vascular smooth muscle cells (VSMCs), the mechanisms of [Ca2+]i mobilization by LPA are not fully understood. In the present study, the effect of LPA on [Ca2+]i mobilization in cultured A10 VSMCs was examined by Fura-2 fluorescence technique. The expression of LPA receptors was studied by immunostaining. LPA was observed to increase [Ca2+]i in a concentration-dependent manner; this increase was dependent on the concentration of extracellular Ca2+. Both sarcolemmal (SL) Na(+)-Ca2+ exchange inhibitors (amiloride, Ni2+ and KB-R7943) and Na(+)-H+ exchange inhibitor (MIA) as well as SL store-operated Ca2+ channel (SOC) antagonists (SK&F 96365, tyrphostin A9 and gadolinium), unlike SL Ca2+ channel antagonists (verapamil and diltiazem), inhibited the LPA-induced increase in [Ca2+]i. In addition, sarcoplasmic reticulum (SR) Ca2+ channel blocker (ryanodine), SR Ca2+ channel opener (caffeine), SR Ca2+ pump ATPase inhibitor (thapsigargin) and inositol 1,4,5-trisphosphate (InsP3) receptor antagonists (xestospongin and 2-aminoethoxydiphenyl borate) were found to inhibit the LPA-induced Ca2+ mobilization. Furthermore, phospholipase C (PLC) inhibitor (U 73122) and protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate) attenuated the LPA-induced increase in [Ca2+]i. These results indicate that Ca2+ mobilization by LPA involves extracellular Ca2+ entry through SL Na(+)-Ca2+ exchanger, Na(+)-H+ exchanger and SL SOCs. In addition, ryanodine-sensitive and InsP(3)-sensitive intracellular Ca2+ pools may be associated with the LPA-induced increase in [Ca2+]i. Furthermore, the LPA-induced [Ca2+]i mobilization in VSMCs seems to be due to the activation of both PLC and PKC.     

Unidirectional Na+, Ca2+, and K+ fluxes through the bovine rod outer segment Na-Ca-K exchanger

The properties of the Na-Ca exchanger in the plasma membrane of rod outer segments isolated from bovine retinas (ROS) were studied. Unidirectional Ca2+, Na+, and K+ fluxes were measured with radioisotopes and atomic absorption spectroscopy. We measured K+ fluxes associated with the Ca-Ca self-exchange mode of the Na-Ca exchanger to corroborate our previous conclusion that the ROS Na-Ca exchanger differs from Na-Ca exchangers in other tissues by its ability to transport K+ (Schnetkamp, P. P. M., Basu, D. K. & Szerencsei, R. T. (1989) Am. J. Physiol. 257, C153-C157). The Na-Ca-K exchanger was the only functional cation transporter in the plasma membrane of bovine ROS with an upper limit of a flux of 10(5) cations/ROS/s or a current of 0.01 pA contributed by other cation channels, pumps, or carriers; cation fluxes via the Na-Ca-K exchanger amounted to 5 x 10(6) cations/ROS/s or a current of 1 pA. Ca2+ efflux via the forward mode of the Na-Ca-K exchanger did not operate with a fixed single stoichiometry. 1) The Na/Ca coupling ratio was increased from three to four when ionophores were added that could provide electrical compensation for the inward Na-Ca exchange current. 2) The K/Ca coupling ratio could vary by at least 2-fold as a function of the external Na+ and K+ concentration. The results are interpreted in terms of a model that can account for the variable Ca/K coupling ratio: we conclude that the Ca2+ site of the exchanger can translocate independent of translocation of the K+ site, whereas translocation of the K+ site requires occupation of the Ca2+ site, but not its translocation. The results are discussed with respect to the physiological role of Na-Ca-K exchange in rod photoreceptors.     

Influence of long-term treatment of imidapril on mortality, cardiac function, and gene expression in congestive heart failure due to myocardial infarction

Although it is generally accepted that the efficacy of imidapril, an angiotensin-converting enzyme inhibitor, in congestive heart failure (CHF) is due to improvement of hemodynamic parameters, the significance of its effect on gene expression for sarcolemma (SL) and sarcoplasmic reticulum (SR) proteins has not been fully understood. In this study, we examined the effects of long-term treatment of imidapril on mortality, cardiac function, and gene expression for SL Na+/K+ ATPase and Na+ -Ca2+ exchanger as well as SR Ca2+ pump ATPase, Ca2+ release channel (ryanodine receptor), phospholamban, and calsequestrin in CHF due to myocardial infarction. Heart failure subsequent to myocardial infarction was induced by occluding the left coronary artery in rats, and treatment with imidapril (1 mg.kg(-1).day(-1)) was started orally at the end of 3 weeks after surgery and continued for 37 weeks. The animals were assessed hemodynamically and the heart and lung were examined morphologically. Some hearts were immediately frozen at -70 degrees C for the isolation of RNA as well as SL and SR membranes. The mortality of imidapril-treated animals due to heart failure was 31% whereas that of the untreated heart failure group was 64%. Imidapril treatment improved cardiac performance, attenuated cardiac remodeling, and reduced morphological changes in the heart and lung. The depressed SL Na+/K+ ATPase and increased SL Na+-Ca2+ exchange activities as well as reduced SR Ca2+ pump and SR Ca2+ release activities in the failing hearts were partially prevented by imidapril. Although changes in gene expression for SL Na+/K+ ATPase isoforms as well as Na+-Ca2+ exchanger and SR phospholamban were attenuated by treatments with imidapril, no alterations in mRNA levels for SR Ca2+ pump proteins and Ca2+ release channels were seen in the untreated or treated rats with heart failure. These results suggest that the beneficial effects of imidapril in CHF may be due to improvements in cardiac performance and changes in SL gene expression.     

The effect of monovalent cations upon 45Ca fluxes in frog sciatic nerve

Investigation of Ca fluxes in desheathed bundles of myelinated nerve of frog indicates an intracellular Ca concentration of 5 x 10(-4) mol . kg-1 (axoplasm) and an average transmembrane flux of 6 x 10(-8) mol. kg-1 . s-1 at an extracellular Ca concentration of 1 mM. Replacement of extracellular Na by isosmotic sucrose increases Ca influx threefold and decreases efflux by 50%. Similar, but significantly smaller, effects are observed when Tris or choline are substituted for Na. Li replaces Na without significant changes in Ca fluxes. The data demonstrate that Ca transmembrane fluxes in this preparation are sensitive to changes in the Na gradient. The observed flux changes, however, are too small to establish a Na-Ca exchange as the sole homeostatic mechanism for intracellular Ca. Moreover, as Li appears to serve as a good Na substitute and even Tris and choline interact with Ca flux, the exchange does not show the specificity described for squid axon.     

Expression of active p21-activated kinase-1 induces Ca2+ flux modification with altered regulatory protein phosphorylation in cardiac myocytes

p21-Activated kinase-1 (Pak1) is a serine-threonine kinase that associates with and activates protein phosphatase 2A in adult ventricular myocytes and, thereby, induces increased Ca2+ sensitivity of skinned-fiber tension development mediated by dephosphorylation of myofilament proteins (Ke Y, Wang L, Pyle WG, de Tombe PP, Solaro RJ. Circ Res 94: 194-200, 2004). We test the hypothesis that activation of Pak1 also moderates cardiac contractility through regulation of intracellular Ca2+ fluxes. We found no difference in field-stimulated intracellular Ca2+ concentration ([Ca2+]i) transient amplitude and extent of cell shortening between myocytes expressing constitutively active Pak1 (CA-Pak1) and controls expressing LacZ; however, time to peak shortening was significantly faster and rate of [Ca2+]i decay and time of relengthening were slower. Neither caffeine-releasable sarcoplasmic reticulum (SR) Ca2+ content nor fractional release was different in CA-Pak1 myocytes compared with controls. Isoproterenol application revealed a significantly blunted increase in [Ca2+]i transient amplitude, as well as a slowed rate of [Ca2+]i decay, increased SR Ca2+ content, and increased cell shortening, in CA-Pak1 myocytes. We found no significant change in phospholamban phosphorylation at Ser16 or Thr17 in CA-Pak1 myocytes. Analysis of cardiac troponin I revealed a significant reduction in phosphorylated species that are primarily attributable to Ser(23/24) in CA-Pak1 myocytes. Nonstimulated, spontaneous SR Ca2+ release sparks were significantly smaller in amplitude in CA-Pak1 than LacZ myocytes. Propagation of spontaneous Ca2+ waves resulting from SR Ca2+ overload was significantly slower in CA-Pak1 myocytes. Our data indicate that CA-Pak1 expression has significant effects on ventricular myocyte contractility through altered myofilament Ca2+ sensitivity and modification of the [Ca2+]i transient.     

Perpetuation of misinterpretations due to lack of methodical insight. A critical re-evaluation of the determination of 45Ca release from intact guinea-pig atria

1. The evaluation of still more pretentious and complicated methods is accompanied by a decline of methodical knowledge outside of the own technical field. Interpretations or extrapolations are taken as granted without critical examination of the methodical steps applied. An example is given by re-evaluating the 45Ca release from isolated cardiac tissue and the possible interpretations. 2. 45Ca release and tissue Ca content were measured in isolated guinea-pig left atria during Ca equilibrium and under conditions known to induce net Ca movements. 3. At equilibrium condition (1.8 mM Na2+0) 3 exponential phase of 45Ca release from the atria were observed. The compartments contained 61%, 29% and 10% of total 45Ca; the t1/2 were 2, 12 and 90 min, respectively. 4. The release of 45Ca from the slowly exchanging compartment (t1/2 90 min) decreased during incubation in nominal Ca-free solution, although a net loss of tissue Ca occurred. Addition of EGTA (5 x 10(-5) M) to the washout medium abolished this retardation of 45Ca release. 5. At external Na+ concentrations below 40 mM (substituted by sucrose), the 45Ca release from the slowly exchanging compartment decreased. Simultaneously, the tissue Ca content increased massively. The 45Ca release was further reduced in Na-poor, nominal Ca-free solution. Under both conditions, the presence of EGTA in the washout medium normalized the rate of 45Ca release. 6. The results suggest that the apparent decline of 45Ca release from intact atria upon reduction of the external Ca and Na concentration does not reflect a decrease of the cellular efflux rate, but is the consequence of an enhanced re-uptake of 45Ca from the extracellular space into the myocardial cells. The probability for the released 45Ca either to escape into the organ bath or to become reabsorbed depends on the specific radioactivity of 45Ca in the extracellular space during the washout phase. Thus, this experimental procedure is not suited to demonstrate a Na-Ca exchange at the cardiac sarcolemma.     

External Na-independent Ca extrusion in cultured ventricular cells. Magnitude and functional significance

The relative magnitudes and functional significance of Ca extrusion by Na-Ca exchange and by an Nao-independent mechanism were investigated in monolayer cultures of chick embryo ventricular cells. Abrupt exposure of cells in 0-Nao, nominally 0-Cao solution to 20 mM caffeine produced a large contracture (3.94 +/- 0.90 micron of cell shortening) that relaxed with a t1/2 of 8.60 +/- 1.22 s. An abrupt exposure to caffeine plus 140 mM Na resulted in a contracture that was smaller in amplitude (1.53 +/- 0.50 micron) and relaxed much more rapidly (t1/2 = 0.77 +/- 0.09 s). An abrupt exposure to caffeine in 0-Nao solutions produced an increase in 45Ca efflux that persisted for 20 s, and a net loss of Ca content, determined by atomic absorption spectroscopy (AAS), of approximately 4 nmol/mg protein, within 35 s. A comparable net loss of Ca was demonstrated in the presence of 100 microM [Ca]o. The abrupt exposure of cultured cells to 0 Nao in 1.8 mM Ca produced a Ca uptake, estimated with 45Ca, of 3.2 nmol/mg protein X 15 s, but produced no increase in cell Ca content (AAS). In cells in which a 30% increase in Nai was produced by 5 min exposure to 10(-6) M ouabain, the abrupt exposure to 0 Nao produced a Ca uptake of 6 nmol/mg protein X 15 s and an increase in Ca content (AAS) of 4 nmol/mg protein. We conclude that there is an Nao-independent mechanism for Ca extrusion in these cells, presumably a Ca-ATPase Ca pump, with a limited Ca transport capacity of no more than 2 nmol/mg protein X 15 s. This is five times smaller than the demonstrated maximum capacity of the Na-Ca exchanger in these cells. The relaxation of twitch tension in these cells seems to be dependent primarily on sarcoplasmic reticulum uptake of Ca, with a secondary role provided by the Na-Ca exchanger. The Ca pump appears to contribute little to beat-to-beat relaxation.     

Positive inotropic effects of ouabain in isolated cat ventricular myocytes in sodium-free conditions

The inotropic and toxic effects of cardiac steroids are thought to result from Na(+)-K(+)-ATPase inhibition, with elevated intracellular Na(+)(Na)causing increased intracellular Ca(2+)(Ca) via Na-Ca exchange. We studied the effects of ouabain on cat ventricular myocytes in Na(+)-free conditions where the exchanger is inhibited. Cell shortening and Ca transients (with fluo 4-AM fluorescence) were measured under voltage clamp during exposure to Na(+)-free solutions [LiCl or N-methyl-D-glucamine (NMDG) replacement]. Ouabain enhanced contractility by 121 +/- 55% at 1 micromol/l (n = 11) and 476 +/- 159% at 3 micromol/l (n = 8) (means +/- SE). Ca transient amplitude was also increased. The inotropic effects of ouabain were retained even after pretreatment with saxitoxin (5 micromol/l) or changing the holding potential to -40 mV (to inactivate Na(+) current). Similar results were obtained with both Li(+) and NMDG replacement and in the absence of external K(+), indicating that ouabain produced positive inotropy in the absence of functional Na-Ca exchange and Na(+)-K(+)-ATPase activity. In contrast, ouabain had no inotropic response in rat ventricular myocytes (10-100 micromol/l). Finally, ouabain reversibly increased Ca(2+) overload toxicity by accelerating the rate of spontaneous aftercontractions (n = 13). These results suggest that the cellular effects of ouabain on the heart may include actions independent of Na(+)-K(+)-ATPase inhibition, Na-Ca exchange, and changes in Na.     

Inner sarcolemmal leaflet Ca(2+) binding: its role in cardiac Na/Ca exchange.

A recently completed model of Ca concentration and movements in the cardiac cell diadic cleft space predicts that removal or neutralization of inner sarcolemmal (SL) leaflet anionic Ca-binding sites at the sarcolemmal border of this space will greatly diminish Na/Ca exchange-mediated Ca efflux. The present study tests this prediction using the local anesthetic dibucaine as a probe. It is shown, in isolated SL, that dibucaine competitively displaces Ca specifically from anionic phospholipid headgroups. Dibucaine also displaces Ca from the SL when applied to intact cells. It does not affect the content or release of Ca from sarcoplasmic reticulum (SR) in these cells. This eliminates a primary effect on SR Ca as a contributing factor to dibucaine's effect on Na/Ca exchange-mediated Ca efflux. Measurement of this efflux from whole cells shows a highly significant reduction of 58% (p < 0.001) by 0.5 mM dibucaine. The inhibiting effect of dibucaine on Na/Ca exchange-mediated Ca efflux can be significantly reversed by augmentation of Ca release from SR by caffeine at the time of activation of Na/Ca exchange. This supports the contention that the dibucaine-SL interaction is a competitive one vis-a-vis Ca. The results are supportive of the model in which inner SL leaflet Ca-binding sites account for the delay of Ca diffusion from the diadic cleft, thereby prolonging the time for which [Ca] remains elevated in the cleft. The prolonged increased [Ca] significantly enhances the ability of Na/Ca exchange to remove Ca from the cell during the excitation-contraction cycle.     

Does decreasing extracellular Na inhibit in vitro renin secretion by affecting NaCa exchange?

It has been shown previously that in vitro renin secretion is inhibited by partial replacement of extracellular NaCl with either mannitol or choline chloride; the inhibitory effect is attributed to an increase in intracellular Ca, resulting from a decreased rate of Ca efflux via Na-Ca exchange. In the present experiments, we confirmed that partially replacing NaCl with choline chloride inhibited renin secretion from rat renal cortical slices, but we found that atropine completely blocked the effect, suggesting cholinergic mediation. Partially replacing NaCl with mannitol also inhibited renin secretion, but the effect could not be attributed specifically to a reduction in extracellular Na. Moreover, the stimulatory effect of Ca chelation on renin secretion was antagonized by either mannitol- or choline chloride -containing incubation media. These results do not support the hypothesis that lowering extracellular Na inhibits renin secretion by a mechanism involving decreased Ca efflux via Na-Ca exchange.