In cardiac muscle, excitation-contraction (E-C) coupling is determined by the ability of the sarcoplasmic reticulum (SR) to store and release Ca(2+). It has been hypothesized that the Ca(2+) sequestration and release mechanisms might be functionally linked to optimize the E-C coupling process. To explore the relationships between the loading status of the SR and functional state of the Ca(2+) release mechanism, we examined the effects of changes in SR Ca(2+) content on spontaneous Ca(2+) sparks in saponin-permeabilized and patch-clamped rat ventricular myocytes. SR Ca(2+) content was manipulated by pharmacologically altering the capacities of either Ca(2+) uptake or leak. Ca(2+) sparks were recorded using a confocal microscope and Fluo-3 and were quantified considering missed events. SR Ca(2+) content was assessed by application of caffeine. Exposure of permeabilized cells to anti-phospholamban antibodies elevated the SR Ca(2+) content and increased the frequency of sparks. Suppression of the SR Ca(2+) pump by thapsigargin lowered [Ca(2+)](SR) and reduced the frequency of sparks. The ryanodine receptor (RyR) blockers tetracaine and Mg(2+) transiently suppressed the frequency of sparks. Upon washout of the drugs, sparking activity transiently overshot control levels. Low doses of caffeine transiently potentiated sparking activity upon application and transiently depressed the sparks upon removal. In patch-clamped cardiac myocytes, exposure to caffeine produced only a transient increase in the probability of sparks induced by depolarization. We interpret these results in terms of a novel dynamic control scheme for SR Ca(2+) cycling. A central element of this scheme is a luminal Ca(2+) sensor that links the functional activity of RyRs to the loading state of the SR, allowing cells to auto-regulate the size and functional state of their SR Ca(2+) pool. These results are important for understanding the regulation of intracellular Ca(2+) release and contractility in cardiac muscle.

Important among the responses of endothelial cells to f low stimuli are cytosolic-free calcium transients. These transients are mediated by several factors, including blood-borne agonists, extracellular calcium, and f luidimposed shear forces. A mathematical model has been developed describing the recognition and transduction of shear stress to the second messenger cytosolic calcium. Shear stress modulates the calcium response via at least two modalities. First, mass transfer of agonist to the cell surface is enhanced by perfusion and is thus related to shear stress. Second, the permeability of the cell membrane to extracellular calcium increases upon exposure to shear stress. A mass balance for agonist in the perfusate is coupled to a previously published calcium dynamics model. Computations indicate a f low region where the transient moves from transport limited to kinetically limited. Parametric studies indicate distinct contributions to the time course by each step in the process. These steps include the time to develop the concentration boundary layer of agonist, receptor activation, and the mobilization of calcium from intracellular stores. Exogenous calcium is presumed to enter the cell via shear stress-gated ion channels. The model predicts a sigmoidal dependence of calcium inf lux upon shear stress. The peak value of the transient is determined largely by the agonist pathway, whereas the plateau level is governed by calcium inf lux. The model predicts the modulation of the calcium transient in the physiologically relevant range of f low and the associated shear stress. This implies that hemodynamics is important in regulating endothelial biology. Intracellular transients in Ca2ϩ are a typical cellular response to many different stimuli, including shear stress (1). Flow enhances the delivery of blood-borne agonists to the cell surface (2). These agonists in turn mediate the release of calcium from intracellular stores into the cytosol. Shear stress increases the permeability of the cell membrane to extracellular calcium, thereby increasing the cytosolic concentration (3, 4). A third transduction mechanism is a direct shear stress effect upon the generation of inositol trisphosphate, which could mediate the release of calcium from intracellular stores (5). This last would operate via a putative mechanoreceptor behaving as a biological ''strain gauge.'' It is likely that all three pathways act in concert to elicit the calcium transient.The response of cytosolic calcium to a fluid shear stress has been illuminated to a large degree by experimental work. It is therefore natural to propose mathematical models that account for this behavior. Several models have been proposed that relate cytosolic-free calcium dynamics to stimulation by agonists (6, 7). The concentration profiles of agonists in flow chambers containing cell monolayers have been described (8).There has been at least one report of a mathematical model linking calcium transients to mechanical stimulation (9). There have been no reports of ...

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Important among the responses of endothelial cells are cytosolic free calcium transients. These transients are mediated by several factors, including blood-borne agonists, extracellular calcium, and fluid-imposed shear forces. The transients are characterized by a rapid rise followed by a plateau phase. A base mathematical model is presented that reasonably reproduces the measured calcium transient in cultured human umbilical vein endothelial cells responding to thrombin. Kinetic equations for receptor activation and calcium mobilization comprise the model. A graded response of intracellular free calcium to increasing concentrations of agonist is predicted. Also predicted is the elevation of the peak value and the plateau level by steady nonspecific leak of calcium across the plasma membrane. The influences of capacitative calcium entry, calcium-induced calcium release, and buffering by cytosolic proteins are investigated parametrically. The model predicts significant depletion of cellular calcium in response to agonist stimulation.