Local calcium release activation by DHPR calcium channel openings in rat cardiac myocytes

The principal role of calcium current in the triggering of calcium release in cardiac myocytes is well recognized. The mechanism of how calcium current ( I _Ca) controls the intensity of calcium release is not clear because of the stochastic nature of voltage-dependent gating of calcium channels (DHPRs) and of calcium-dependent gating of ryanodine receptors (RyRs). To disclose the relation between DHPR openings and the probability of calcium release, local calcium release activation by I _Ca was investigated in rat ventricular myocytes using patch-clamp and confocal microscopy. Calcium spikes were activated by temporally synchronized DHPR calcium current triggers, generated by instantaneous 'tail' I _Ca and modulated by prepulse duration, by tail potential, and by the DHPR agonist BayK 8644. The DHPR–RyR coupling fidelity was determined from the temporal distribution of calcium spike latencies using a model based on exponentially distributed DHPR open times. The analysis provided a DHPR mean open time of ∼0.5 ms, RyR activation time constant of ∼0.6 ms, and RyR activation kinetics of the 4th order. The coupling fidelity was low due to the inherent prevalence of very short DHPR openings but was increased when DHPR openings were prolonged by BayK 8644. The probability of calcium release activation was high, despite low coupling fidelity, due to the activation of many DHPRs at individual release sites. We conclude that the control of calcium release intensity by physiological stimuli can be achieved by modulating the number and duration of DHPR openings at low coupling fidelity, thus avoiding the danger of inadvertently triggering calcium release events.     

Alterations of ultrastructure and elemental composition in cultured neonatal rat cardiac myocytes after metabolic inhibition with iodoacetic acid

The purpose of this study was to document changes in cellular fine structure and elemental composition, and their relationship to progression of cell injury, in cultured neonatal rat cardiac myocytes in which impaired energy metabolism was produced by the metabolic inhibitor, iodoacetic acid (IAA). In order to quantitate changes in the concentrations of elements and their subcellular distribution in individual myocytes, electron probe x-ray microanalysis was performed on freeze-dried cryosections of rapidly frozen cells. After 1 hour of exposure to IAA, ATP level was not significantly reduced. Most cells exhibited minimal ultrastructural alterations and had normal elemental profiles, whereas some cells (10 to 25%) had increased sodium and calcium in mitochondria and cytoplasm. After exposure to IAA for 1.5, 2, or 4 hours, the ATP level was reduced to below one third of control, and remained decreased 24 hours after removal of IAA, indicating irreversible depression of this variable. After exposure to IAA for 1.5 hours no longer, many cells showed severe ultrastructural alterations, including contraction or swelling of mitochondria and distortion of the cristae, myofibrillar hypercontraction, and formation of fluid-filled blebs. At 1.5 and 2 hours, approximately 75% or more of the myocytes had increased sodium and calcium and decreased potassium and magnesium in mitochondria, nuclei, and cytoplasm. Thus, the development of an increased calcium concentration in cytoplasm as well as mitochondria of most myocytes was a feature of this transitional period. These data indicate that progressive alterations in the levels and distribution of elements accompany the development of severe ultrastructural changes and irreversible injury in response to impaired energy metabolism in cultured myocytes. These elemental alterations include accumulation of calcium in cytoplasm and mitochondria of myocytes in this model.     

Effects of calcium loading and impaired energy production on metabolic and ultrastructural features of cell injury in cultured neonatal rat cardiac myocytes

We evaluated the contributions of calcium loading and impaired energy production to metabolic and ultrastructural manifestations of cell injury in a cultured neonatal rat ventriculocyte model. Direct calcium loading was produced by incubation in K(+)-free medium to inhibit the Na+,K(+)-ATPase and promote Na(+)-Ca2+ exchange, and inhibition of energy metabolism was produced by incubation with 30 microM iodoacetic acid (IAA). Measurements were made of total cell calcium, [3H] arachidonic acid (AA) release (an index of membrane phospholipid degradation), ATP, and ultrastructural features of cell damage. Inhibition of the Na(+),K(+) pump resulted in the rapid onset of cellular calcium loading, increased [3H]AA release, and moderate ATP reduction. After return to control medium for 24 hours, myocytes previously exposed to K(+)-free medium for 1 hour showed recovery of ATP level and little additional [3H]AA release. However, after 2 to 3 hours of calcium loading, the ATP level remained moderately depressed, residual [3H]AA release was greater, and a mixed population of relatively normal and severely damaged myocytes was observed by electron microscopy. IAA treatment for 1 hour resulted in moderate ATP reduction without calcium accumulation or [3H]AA release, whereas IAA treatment for 3 hours resulted in marked ATP reduction associated with calcium accumulation and [3H]AA release. Reversal experiments showed substantial recovery of ATP level after 1 hour of IAA exposure, and marked ATP depression and [3H]AA release associated with widespread irreversible injury after 3 hours. Thus, the data indicate that increased calcium accumulation itself can initiate accelerated membrane phospholipid degradation, but that progression to irreversible injury is influenced by other factors, including the magnitude of ATP depression associated with calcium loading.     

Kinetics of calcium spikes in rat cardiac myocytes

The local calcium release flux signals (calcium spikes) evoked by membrane depolarization were recorded at high temporal resolution (2000 lines s⁻¹) in isolated ventricular myocytes of male rats, using combination of scanning confocal microscopy and the patch-clamp technique. The kinetic properties of calcium spikes were investigated. The time course of calcium spike activation could be described reliably by a model with higher-order ( n = 3) kinetics, but not by a first-order exponential process. A model of calcium spike with calcium release termination coupled to its activation was preferential to a model with the release termination independent of its activation. Three fluorescent calcium dyes (OG-5N, fluo-3, and fluo-4) were compared for calcium spike measurements. Experimental measurements as well as simulations showed that the occurrence and latency of calcium spikes could be measured faithfully with all indicators, while the kinetics of calcium spikes was reliably traced only with OG-5N. Calcium spikes evoked by a step depolarization from −50 to 0 mV commenced with a mean latency of 4.1 ± 0.2 ms and peaked 6.7 ± 0.2 ms later. Their full amplitudes were normally distributed. The activation time constant of calcium spikes was 3.1 ± 0.1 ms, and the time constant of termination was 5.5 ± 0.2 ms. A negative correlation was observed between the observed amplitude of calcium spikes and their time constant of activation, but there was no correlation between their observed amplitude and time constant of termination, in agreement with the concept of steep calcium-dependent activation and fateful inactivation of calcium release flux.     

Digital-imaging microscopy analysis of calcium release from sarcoplasmic reticulum in single rat cardiac myocytes

Digital imaging microscopy of fura-2 fluorescence has allowed us to assess the dynamic patterns of local Ca increase in newly isolated rat myocardial cells. Of the myocytes bathed in a saline solution (1.8 mM Ca²⁺, 37°C, pH 7.4), 10%–20% exhibited local spontaneous contractions. The resting intracellular free calcium concentration ([Ca²⁺]_i) of these cells was 106±4nM versus 77±3 nM for non-contracting cells. The spontaneous contractile activity appeared to be closely related to internal spontaneous Ca waves that spread across the myoplasm (velocity 50 m/s, maximal Ca amplitude=195±11nM) along the major axis of the cells. Precise topographical examination of Ca wave propagation indicated a refractory period for internal Ca release. The occurrence of both the generation and propagation of spontaneous Ca increases appeared to be closely dependent on the extent of Ca loading of the cells. Most of our observations were in accordance with the assumption that local Ca overload of the sarcoplasmic reticulum (SR) is the main parameter involved in the spontaneous Ca-release phenomena. Using the same approach, the increase in internal Ca evoked by KCl (50 mM) addition was investigated, and compared with that seen during spontaneous activity. Total [Ca²⁺]_i increase induced by K⁺ depolarization involved three consecutive local Ca-release patterns: (a) a peripheral Ca enhancement that remained during the total [Ca²⁺]_i increase, (b) subsequent transversal local Ca increases occurring in Z-line regions, (c) longitudinal local Ca increases. In addition, a weak heterogeneous Ca distribution was detected in both peripheral and central parts of resting cardiac cells. Thus, the total Ca increase seemed to result consecutively from a peripheral Ca pool, from junctional SR and from longitudinal structures (possibly longitudinal SR).     

Stimulation of murine cultured mast cells under anaerobic conditions: inhibition of arachidonic acid release

The exocytosis of beta-hexosaminidase from either IgE-antigen- or calcium ionophore A23187-stimulated murine bone-marrow-derived mast cells was not affected by oxygen-depleted conditions regardless of the absence of glucose from the medium. No detectable changes in the content of ATP were observed when the cells were triggered immunologically under anaerobic conditions in the absence of glucose in the medium. Depletion of oxygen from mast cells activated by both stimuli almost completely inhibited the specific release of arachidonic acid, which indicates that arachidonate does not play a significant role in the secretion of preformed mediators.     

Troglitazone-induced inhibition of l-type calcium currents is augmented in streptozotocin-induced diabetic rat cardiac ventricular myocytes

Troglitazone-induced inhibition of l-type calcium currents is augmented in streptozotocin-induced diabetic rat cardiac ventricular myocytes     

Reversible inhibition of gap junctional communication by tamoxifen in cultured cardiac myocytes

 Gap junction channels provide a cell-to-cell conduction pathway for direct exchange of ions and small molecules. The intercellular diffusion of a fluorescent dye, quantified in cardiac myocytes from neonatal rats by monitoring the fluorescence recovery after photobleaching, was found to be interrupted after short-term exposure (15 min) to tamoxifen, an anti-oestrogen drug often used in the treatment of human breast cancer. This diffusional uncoupling was dose dependent, occurred in the concentration range 3–25 μM and reversed after tamoxifen withdrawal. Some possible mechanisms of junctional channel closure have been examined. The cytosolic calcium concentration, examined using the fluorescent indicator Indo-1, did not vary during the short-term action of tamoxifen. A second anti-oestrogen agent (clomiphene) was able to impair gap junctional communication, whereas a third (nafoxidine) had no effect. Protein-kinase-C-inhibitor properties of tamoxifen did not seem to be involved in its uncoupling action. The characteristics of tamoxifen’s action (i.e. channel inhibition delay, active concentration range, reversibility, etc.) were very similar to the previously observed effects of several other lipophilic compounds (e.g. 17β-oestradiol, etc.) on junctional channels, and to recently reported effects of tamoxifen on voltage-gated calcium currents. Received: 23 July 1996 / Received after revision: 24 October 1996 / Accepted: 1 November 1996     

Troglitazone -induced inhibition of L -type calcium currents is augmented in streptozotocin -induced diabetic rat cardiac ventricular myocytes

Troglitazone -induced inhibition of L -type calcium currents is augmented in streptozotocin -induced diabetic rat cardiac ventricular myocytes     

Regulation of an ATP-conductive large-conductance anion channel and swelling-induced ATP release by arachidonic acid

Mouse mammary C127 cells responded to hypotonic stimulation with activation of the volume-dependent ATP-conductive large conductance (VDACL) anion channel and massive release of ATP. Arachidonic acid downregulated both VDACL currents and swelling-induced ATP release in the physiological concentration range with K _d of 4– 6 μ m . The former effect observed in the whole-cell or excised patch mode was more prominent than the latter effect observed in intact cells. The arachidonate effects were direct and not mediated by downstream metabolic products, as evidenced by their insensitivity to inhibitors of arachidonate-metabolizing oxygenases, and by the observation that they were mimicked by cis -unsaturated fatty acids, which are not substrates for oxygenases. A membrane-impermeable analogue, arachidonyl coenzyme A was effective only from the cytosolic side of membrane patches suggesting that the binding site is localized intracellularly. Non-charged arachidonate analogues as well as trans -unsaturated and saturated fatty acids had no effect on VDACL currents and ATP release, indicating the importance of arachidonate's negative charge and specific hydrocarbon chain conformation in the inhibitory effect. VDACL anion channels were inhibited by arachidonic acid in two different ways: channel shutdown ( K _d of 4– 5 μ m ) and reduced unitary conductance ( K _d of 13–14 μ m ) without affecting voltage dependence of open probability. ATP^4--conducting inward currents measured in the presence of 100 m m ATP in the bath were reversibly inhibited by arachidonic acid. Thus, we conclude that swelling-induced ATP release and its putative pathway, the VDACL anion channel, are under a negative control by intracellular arachidonic acid signalling in mammary C127 cells.     

枸橼酸铁铵对原代培养的大鼠心肌细胞铜蓝蛋白、膜铁转运辅助蛋白和膜铁转运蛋白表达的影响

目的 观察枸橼酸铁铵(FAC)对原代培养的大鼠心肌细胞铁释放相关蛋白表达的影响,探讨枸橼酸铁铵对心肌细胞铁代谢的影响机制. 方法 以原代培养的乳鼠心肌细胞为材料,分为对照组、20mg/L枸橼酸铁铵组、40mg/L枸橼酸铁铵组和80mg/L枸橼酸铁铵组,每组6个重复.然后检测心肌细胞存活率、搏动频率,免疫组织化学检测铜蓝蛋白(CP)、膜铁转运辅助蛋白(HP)和膜铁转运蛋白(FP1)表达的变化. 结果 各剂量枸橼酸铁铵对大鼠心肌细胞存活率无明显影响;心肌细胞搏动频率减慢,停止跳动的细胞数量明显增加,收缩幅度逐渐降低;随着枸橼酸铁铵浓度的增加,心肌细胞铜蓝蛋白、膜铁转运辅助蛋白和膜铁转运蛋白的表达均增加. 结论 枸橼酸铁铵影响大鼠心肌细胞的生理功能,IRP-IRE可能参与膜铁转运蛋白表达的调控,铜蓝蛋白、膜铁转运辅助蛋白表达的升高可能与铁处理增加细胞的氧化紧张性有关.     

FMLP-induced arachidonic acid release,phospholipid metabolism,and calcium mobilization in human monocytes

Radiolabeled human peripheral blood monocytes released [³H]arachidonic acid upon challenge with the calcium ionophore A23187 (10M), or f-Met-LeuPhe (FMLP, 1M). Chromatographic analysis of [³H]arachidonic acid labeled phospholipids showed that stimulation by FMLP reduced the amount of labeled phosphatidylcholine exclusively. Treatment of the monocytes with 10^–3 M dibutyryl cyclic AMP (d-cAMP) or 5×10^–4 M isobutylmethylxanthine (IBMX) substantially inhibited [³H]arachidonic acid release (30%) and depletion from labeled phosphatidylcholine (PC) in FMLP—but not calcium ionophore—stimulated cells. Using the fluorescent probe Indo-1, the FMLP-induced cytosolic calcium increase was unaffected by 10^–3 M dibutyryl cyclic AMP. The results suggest that FMLP-stimulated phospholipase activity is regulated by cyclic AMP, but not by depressing receptor-medicated increases in cytoplasmic free calcium.     

Altered inhibition of the rat skeletal ryanodine receptor/calcium release channel by magnesium in the presence of ATP

Magnesium-induced inhibition of the skeletal ryanodine receptor/calcium-release channel (RyR) was studied in the presence and absence of ATP under isolated conditions and in situ, by examining the RyR incorporated into a planar lipid bilayer and the calcium release flux (Rrel) in isolated single fibres mounted in the double Vaseline gap system. When the incorporated RyR had been activated by calcium (50 microM) in the absence of ATP, the magnesium-induced inhibition showed co-operativity with a Hill coefficient (N) of 1.83 and a half-inhibitory concentration (IC50) of 635 microM. When the open probability was measured in the presence of 5 mM ATP and at a low calcium concentration, the magnesium-induced inhibition was non-cooperative (N=1.1, IC50= 860 microM). In isolated muscle fibres, in the presence of ATP, lowering the intracellular magnesium concentration ([Mg2+]i) increased the maximal Rrel and shifted its voltage dependence to more negative membrane potentials. Increasing [Mg2+]i had the opposite effect. The concentration dependence was described with an IC50 of 174 microM, N=1, under depolarized conditions and showed a tenfold increase in affinity in polarized fibres. At the concentration required for the measurements from isolated fibres, ATP had a full activatory effect on the isolated channel. At a low calcium concentration, the RyR had two ATP-binding sites with half-activatory concentrations of 19 and 350 microM, respectively.     

Irreversible injury of isolated adult rat myocytes. Osmotic fragility during metabolic inhibition.

Isolated myocytes can be established as a valid model for studying changes in cytoskeletal proteins during the development of irreversible injury only if isolated cells develop lesions similar to those that occur during irreversible injury to intact hearts, specifically osmotic fragility and subsarcolemmal blebs. In the first experiment, isolated cells were irreversibly injured by metabolic inhibition with 5 mM Iodoacetic acid (IAA) and 6 mM amobarbital (Amy). Osmotic fragility of control and injured cells was determined by comparing the rates of development of trypan blue permeability during 60 minutes of isotonic or hypotonic (50% reduction in osmolality) incubations. Cell morphology was monitored by light and electron microscopy. Control cells remained elongated and excluded trypan blue. Metabolically inhibited cells rapidly contracted to a nearly square shape. The inhibited squared cells initially excluded trypan blue, but during 60 minutes of incubation became permeable to trypan blue. Cells in hypotonic buffer developed blue staining at a more rapid rate than cells in isotonic buffer, indicating increased osmotic fragility. In a second experiment, control and inhibited cells were first incubated for 25 minutes in isotonic buffer and then in either isotonic or hypotonic buffer. In this experiment, inhibited cells also developed more extensive and rapid permeability increases when transferred to the hypotonic buffer than cells maintained in the isotonic buffer. In both experiments, increased permeability of cells to trypan blue was accompanied by formation of subsarcolemmal blebs along the lateral cell border and at the intercalated disks. The results show that metabolically inhibited, isolated myocytes do exhibit morphologic lesions and increased osmotic fragility properties similar to those reported during anoxic or ischemic injury to intact hearts. Therefore, isolated myocytes may be a useful model with which to study cytoskeletal-sarcolemmal membrane changes during development of irreversible injury.