Subcellular Ca2+ distribution with varying Ca2+ load in neonatal cardiac cell culture.
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Recent work in our laboratory has investigated and modeled subcellular calcium compartmentation and Ca2+ movement under steady-state control conditions. This experimental study is directed to the further description and quantitation of cellular calcium compartmentation patterns and movements as correlated with contraction in neonatal rat cardiac myocytes in culture under a variety of calcium loading conditions. Compartmental contents were assessed after incubations in various [Ca2+]o, 0 Na+/1 mM Ca2+, and 10 microM ouabain/1.0 mM Ca2+ test solutions. The cellular components investigated include sarcolemmal bound, sarcoplasmic reticulum (SR), and mitochondrial calcium. The results indicate that 1) sarcolemmal calcium binding is insensitive to changes in [Ca2+]o in the range tested (0.25-6.0 mM) while highly sensitive to changes in [Na+]i; 2) SR is sensitive to both changes in [Ca2+]o and [Na+]i and exhibits a maximum loading capacity of approximately 750 micromol Ca2+/kg dw; 3) in the [Ca2+]o range between 0.25 and 2.0 mM, contractile amplitude is proportional to SR content; 4) the mitochondria comprise a high-capacity calcium-containing compartment that is sensitive to changes in [Ca2+]o but does not reach saturation under the conditions tested (0.25-8.0 mM [Ca2+]o); 5) SR calcium is divided into at least two functionally discrete pools, one of which is available for release to the myofilaments during a normal ICa-triggered contraction and other of which is caffeine releasable but unavailable for release to the myofilaments during a normal triggered release; and 6) mitochondrial calcium serves as a reservoir of calcium capable of replenishing and/or augmenting SR stores with anywhere from 10% to 50% of mitochondrial calcium cycling through SR calcium compartments. (+info)
Relaxation of mammalian single cardiac cells after pretreatment with the detergent Brij-58.
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1. The influence of load and activation on relaxation of heart muscle has been studied. 2. Cardiac cells devoid of functioning sarcolemma were isolated from rat and cat ventricular myocardium. Pretreatment with the detergent Brij-58 destroyed residual sarcoplasmic reticulum function. In order to analyse the mechanical properties of relaxation in these cells, a new miniature transducer was designed which could measure force by feedback sensing (resolution of 1 microgram). Contraction was induced by ionophoretically released calcium ions. Activation, sequestration of calcium and loading conditions could be controlled independently. 3. The time course of relaxation was shown to be governed by the amount of calcium released, and unlike intact preparations from rat or cat heart (but like those from frog), to be independent of load and of alterations in load. 4. We conclude that relaxation of the cardiac contractile system is determined basically by an activation-dependent mechanism, which is masked by load dependence in intact muscle preparations with a well developed calcium sequestering membraneous system. (+info)
Local Ca2+ entry through L-type Ca2+ channels activates Ca2+-dependent K+ channels in rabbit coronary myocytes.
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Large-conductance Ca2+-dependent K+ channels (KCa), which are abundant on the sarcolemma of vascular myocytes, provide negative feedback via membrane hyperpolarization that limits Ca2+ entry through L-type Ca2+ channels (ICaL). We hypothesize that local accumulation of subsarcolemmal Ca2+ during ICaL openings amplifies this feedback. Our goal was to demonstrate that Ca2+ entry through voltage-gated ICaL channels can stimulate adjacent KCa channels by a localized interaction in enzymatically isolated rabbit coronary arterial myocytes voltage clamped in whole-cell or in cell-attached patch clamp mode. During slow-voltage-ramp protocols, we identified an outward KCa current that is activated by a subsarcolemmal Ca2+ pool dissociated from bulk cytosolic Ca2+ pool (measured with indo 1) and is dependent on L-type Ca2+ channel activity. Transient activation of unitary KCa channels in cell-attached patches could be detected during long step depolarizations to +40 mV (holding potential, -40 mV; 219 pS in near-symmetrical K+). This local interaction between the channels required the presence of Ca2+ in the pipette solution, was enhanced by the ICaL agonist Bay K 8644, and persisted after impairment of the sarcoplasmic reticulum by incubation with 10 micromol/L ryanodine and 30 micromol/L cyclopiazonic acid for at least 60 minutes. Furthermore, we provide the first direct evidence of simultaneous openings of single KCa (67 pS) and ICaL (3.9 pS) channels in near-physiological conditions, near resting membrane potential. Our data imply a novel sensitive mechanism for regulating resting membrane potential and tone in vascular smooth muscle. (+info)
Enhanced phosphorylation of phospholamban and downregulation of sarco/endoplasmic reticulum Ca2+ ATPase type 2 (SERCA 2) in cardiac sarcoplasmic reticulum from rabbits with heart failure.
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OBJECTIVES: To assess the phosphorylation of myocardial phospholamban (PLB) and quantify protein levels of PLB and sarco/endoplasmic reticulum Ca2+ ATPase type 2 (SERCA 2) in a rabbit model of heart failure. Furthermore, to correlate these parameters with the rate of Ca2+ uptake into sarcoplasmic reticulum (SR) vesicles. METHODS: Heart failure in the rabbit was indicated by the pronounced ventricular contractile dysfunction accompanied by post-mortem evidence of lung and liver congestion 8 weeks after a coronary artery ligation procedure. Phosphorylation of PLB was measured by reduced mobility of the phosphorylated forms on Tris-glycine gels. Phosphoserine and phosphothreonine-specific antibodies against PLB were used to determine the phosphorylated residues. Immunoblotting combined with densitometry was used to assess PLB and SERCA 2 levels. Finally, oxalate-supported Ca2+ uptake into SR vesicles was studied using the fluorescent indicator Fura-2. RESULTS: The phosphorylation state of PLB was significantly higher in myocardium isolated from left ventricles of heart failure rabbits (8.3 +/- 0.42 P-PLB) when compared with sham-operated animals (4.0 +/- 1.7 P-PLB). The kinase activity associated with SR vesicles isolated from animals with heart failure was a factor of 1.58 +/- 0.21-times higher than sham hearts, as assessed by the initial rate of phosphorylation of PLB. This higher kinase activity observed in heart failure was not completely abolished by inhibitors of either A-kinase, C-kinase or Ca2+/calmodulin-dependent protein kinase (CaM-kinase). Abundance of SERCA in heart failure myocardial homogenates was significantly less than sham values (0.68 +/- 0.11 vs. 1.74 +/- 0.27) as was PLB (0.41 +/- 0.08 vs. 0.69 +/- 0.13), similar reductions were seen in vesicle preparations. The rate constant of Ca2+ uptake into the isolated SR vesicles was lower in preparations from heart failure myocardium than from sham myocardium (2.50 +/- 0.23 ms vs. 4.43 +/- 0.3 ms). CONCLUSIONS: The higher level of phosphorylation of PLB observed in the left ventricle of rabbits with heart failure is associated with a higher intrinsic kinase activity of the SR. However, the abundance of both of SERCA 2 and PLB proteins are lower in heart failure. The net effect of these changes appears to be a reduced rate of Ca2+ uptake by the SR in heart failure. (+info)
Unloading induces transcriptional activation of the sarco(endo)plasmic reticulum Ca2+-ATPase 1 gene in muscle.
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Previous work showed that protein and mRNA levels of the "fast" isoform of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1) are markedly increased in unloaded slow-twitch soleus muscles, suggesting pretranslational control of gene expression [L. M. Schulte, J. Navarro, and S. C. Kandarian. Am. J. Physiol. 264 (Cell Physiol. 33): C1308-C1315, 1993]. However, because of the difficulty of measuring transcription rates from whole muscle, transcriptional activation of the SERCA1 gene with unloading has not been confirmed. Because SERCA1 pre-mRNA levels can reflect transcriptional activity, in the present study SERCA1 introns were sequenced to allow intron-directed RT-PCR measurement of SERCA1 pre-mRNA. These data were then compared with changes in SERCA1 mRNA expression in control and unloaded soleus muscles. After 2, 4, and 10 days of unloading, SERCA1 pre-mRNA and mRNA transcript levels increased significantly by two-, three-, and sevenfold, respectively (P < 0.01). Parallel increases in SERCA1 pre-mRNA and mRNA suggest transcriptional activation of the endogenous SERCA1 gene by muscle unloading. SERCA2, the cardiac/slow-twitch skeletal muscle isoform, was not markedly increased by unloading, and RNase protection assays showed no change in alternative splicing of SERCA1 or SERCA2 primary transcripts. With use of in vivo plasmid injection, the activity of a reporter gene driven by 3.6 kb of the SERCA1 5'-flanking region increased fivefold in 7-day-unloaded soleus muscles. Comparison of the magnitude of transcriptional activation of endogenous and constructed SERCA1 genes by unloading confirms the fidelity of using intronic RT-PCR to examine muscle gene transcription rates and suggests that cis-acting elements sufficient for regulating unloading-induced transcriptional activation are contained in this promoter construct. (+info)
Superficial buffer barrier and preferentially directed release of Ca2+ in canine airway smooth muscle.
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We examined cytosolic concentration of Ca2+ ([Ca2+]i) in canine airway smooth muscle using fura 2 fluorimetry (global changes in [Ca2+]i), membrane currents (subsarcolemmal [Ca2+]i), and contractions (deep cytosolic [Ca2+]i). Acetylcholine (10(-4) M) elicited fluorimetric, electrophysiological, and mechanical responses. Caffeine (5 mM), ryanodine (0.1-30 microM), and 4-chloro-3-ethylphenol (0.1-0.3 mM), all of which trigger Ca2+-induced Ca2+ release, evoked Ca2+ transients and membrane currents but not contractions. The sarcoplasmic reticulum (SR) Ca2+-pump inhibitor cyclopiazonic acid (CPA; 10 microM) evoked Ca2+ transients and contractions but not membrane currents. Caffeine occluded the response to CPA, whereas CPA occluded the response to acetylcholine. Finally, KCl contractions were augmented by CPA, ryanodine, or saturation of the SR and reduced when SR filling state was decreased before exposure to KCl. We conclude that 1) the SR forms a superficial buffer barrier dividing the cytosol into functionally distinct compartments in which [Ca2+]i is regulated independently; 2) Ca2+-induced Ca2+ release is preferentially directed toward the sarcolemma; and 3) there is no evidence for multiple, pharmacologically distinct Ca2+ pools. (+info)
Calcium recruitment in renal vasculature: NE effects on blood flow and cytosolic calcium concentration.
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This study provides new information about the relative importance of Ca2+ mobilization and entry in the renal vascular response to adrenoceptor activation. We measured renal blood flow (RBF) in Sprague-Dawley rats in vivo using electromagnetic flowmetry. We measured intracellular free Ca2+ concentration ([Ca2+]i) in isolated afferent arterioles utilizing ratiometric photometry of fura-2 fluorescence. Renal arterial injection of NE produced a transient decrease in RBF. The response was attenuated, in a dose-dependent manner, up to approximately 50% by nifedipine, an antagonist of L-type Ca2+ entry channels. Inhibition of Ca2+ mobilization by 3,4, 5-trimethoxybenzoic acid-8-(diethylamino)octyl ester (TMB-8) inhibited the renal vascular effects of NE in a dose-dependent manner, with maximal blockade of approximately 80%. No additional attenuation was observed when nifedipine and TMB-8 were administered together. In microdissected afferent arterioles, norepinephrine (NE; 10(-6) M) elicited an immediate square-shaped increase in [Ca2+]i, from 110 to 240 nM. This in vitro response was blocked by nifedipine (10(-6) M) and TMB-8 (10(-5) M) to a degree similar to that of the in vivo experiments. A nominally calcium-free solution blocked 80-90% of the [Ca2+]i response to NE. The increased [Ca2+]i elicited by depolarization with medium containing 50 mM KCl was totally blocked by nifedipine. In contrast, TMB-8 had no effect. Our results indicate that both Ca2+ entry and mobilization play important roles in the renal vascular Ca2+ and contractile response to adrenoceptor activation. The entry and mobilization mechanisms activated by NE may interact. That a calcium-free solution caused a larger inhibition of the NE effects on afferent arterioles than nifedipine suggests more than one Ca2+ entry pathway. (+info)
Properties and expression of Ca2+-activated K+ channels in H9c2 cells derived from rat ventricle.
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H9c2 is a clonal myogenic cell line derived from embryonic rat ventricle that can serve as a surrogate for cardiac or skeletal muscle in vitro. Using whole cell clamp with H9c2 myotubes, we observed that depolarizing pulses activated slow outward K+ currents and then slow tail currents. The K+ currents were abolished in a Ca2+-free external solution, indicating that they were Ca2+-activated K+ currents. They were blocked by apamin, a small-conductance Ca2+-activated K+ (SK) channel antagonist (IC50 = 6.2 nM), and by d-tubocurarine (IC50 = 49.4 microM). Activation of SK channels exhibited a bell-shaped voltage dependence that paralleled the current-voltage relation for L-type Ca2+ currents (ICa,L). ICa,L exhibited a slow time course similar to skeletal ICa, L, were unaffected by apamin, and were only slightly depressed by d-tubocurarine. RT-PCR analysis of the mRNAs revealed that rSK3, but not rSK1 or rSK2, was expressed in H9c2 myotubes but not in myoblasts. These results suggest that rSK3 channels are expressed in H9c2 myotubes and are primarily activated by ICa,L directly or indirectly via Ca2+-induced Ca2+ release from sarcoplasmic reticulum. (+info)