Analysis of hystereses in force length and force calcium relations

Analysis of the hystereses in the force-length relationship at constant Ca(2+) concentration and in the force-calcium relationship at constant sarcomere length (SL) provides insight into the mechanisms that control cross-bridge (XB) recruitment. The hystereses are related here to two mechanisms that regulate the number of strong XBs: the cooperativity, whereby the number of strong XBs determines calcium affinity, and the mechanical feedback, whereby the shortening velocity determines the duration for which the XBs are in the strong state. The study simulates the phenomena and defines the role of these feedbacks. The model that couples calcium kinetics with XB cycling was built on Simulink software (Matlab). Counterclockwise (CCW) hysteresis, wherein the force response lags behind the SL oscillations, at a constant calcium level, is obtained in the force-length plane when neglecting the mechanical feedback and accounting only for the cooperativity mechanism. Conversely, the force response precedes the SL oscillations, yielding a clockwise (CW) hysteresis when only the mechanical feedback is allowed to exist. In agreement with experimental observations, either CW or CCW hysteresis is obtained when both feedbacks coexist: CCW hystereses are obtained at low frequencies (<3 Hz), and the direction is reversed to CW at higher frequencies (>3 Hz). The cooperativity dominates at low frequencies and allows the muscle to adapt XB recruitment to slow changes in the loading conditions. The changeover frequency from CCW to CW hysteresis defines the velocity limit above which the muscle absorbs rather than generates energy. The hysteresis in the force-calcium relation is conveniently explained by the same cooperativity mechanism. We propose that a single cooperativity mechanism that depends on the number of strong XBs can explain the hystereses in the force-length as well as in the force-calcium relationships.     

Filament compliance influences cooperative activation of thin filaments and the dynamics of force production in skeletal muscle

Striated muscle contraction is a highly cooperative process initiated by Ca²⁺ binding to the troponin complex, which leads to tropomyosin movement and myosin cross-bridge (XB) formation along thin filaments. Experimental and computational studies suggest skeletal muscle fiber activation is greatly augmented by cooperative interactions between neighboring thin filament regulatory units (RU-RU cooperativity; 1 RU = 7 actin monomers+1 troponin complex+1 tropomyosin molecule). XB binding can also amplify thin filament activation through interactions with RUs (XB-RU cooperativity). Because these interactions occur with a temporal order, they can be considered kinetic forms of cooperativity. Our previous spatially-explicit models illustrated that mechanical forms of cooperativity also exist, arising from XB-induced XB binding (XB-XB cooperativity). These mechanical and kinetic forms of cooperativity are likely coordinated during muscle contraction, but the relative contribution from each of these mechanisms is difficult to separate experimentally. To investigate these contributions we built a multi-filament model of the half sarcomere, allowing RU activation kinetics to vary with the state of neighboring RUs or XBs. Simulations suggest Ca²⁺ binding to troponin activates a thin filament distance spanning 9 to 11 actins and coupled RU-RU interactions dominate the cooperative force response in skeletal muscle, consistent with measurements from rabbit psoas fibers. XB binding was critical for stabilizing thin filament activation, particularly at submaximal Ca²⁺ levels, even though XB-RU cooperativity amplified force less than RU-RU cooperativity. Similar to previous studies, XB-XB cooperativity scaled inversely with lattice stiffness, leading to slower rates of force development as stiffness decreased. Including RU-RU and XB-RU cooperativity in this model resulted in the novel prediction that the force-[Ca²⁺] relationship can vary due to filament and XB compliance. Simulations also suggest kinetic forms of cooperativity occur rapidly and dominate early to get activation, while mechanical forms of cooperativity act more slowly, augmenting XB binding as force continues to develop.     

Sarcomere mechanics in uniform and non-uniform cardiac muscle: a link between pump function and arrhythmias

Starling's Law and the well-known end-systolic pressure-volume relationship (ESPVR) of the left ventricle reflect the effect of sarcomere length (SL) on stress (sigma) development and shortening by myocytes in the uniform ventricle. We show here that tetanic contractions of rat cardiac trabeculae exhibit a sigma-SL relationship at saturating [Ca2+] that depends on sarcomere geometry in a manner similar to skeletal sarcomeres and the existence of opposing forces in cardiac muscle shortened below slack length. The sigma-SL-[Ca2+]free relationships (sigma-SL-CaR) at submaximal [Ca2+] in intact and skinned trabeculae were similar, albeit that the sensitivity for Ca2+ of intact muscle was higher. We analyzed the mechanisms underlying the sigma-SL-CaR using a kinetic model where we assumed that the rates of Ca2+ binding by Troponin-C (Tn-C) and/or cross-bridge (XB) cycling are determined by SL, [Ca2+] or stress. We analyzed the correlation between the model results and steady state stress measurements at varied SL and [Ca2+] from skinned rat cardiac trabeculae to test the hypotheses that: (i) the dominant feedback mechanism is SL, stress or [Ca2+]-dependent; and (ii) the feedback mechanism regulates: Tn-C-Ca2+ affinity, XB kinetics or, unitary XB-force. The analysis strongly suggests that feedback of the number of strong XBs to cardiac Tn-C-Ca2+ affinity is the dominant mechanism that regulates XB recruitment. Application of this concept in a mathematical model of twitch-stress accurately reproduced the sigma-SL-CaR and the time course of twitch-stress as well as the time course of intracellular [Ca2+]i. Modeling of the response of the cardiac twitch to rapid stress changes using the above feedback model uniquely predicted the occurrence of [Ca2+]i transients as a result of accelerated Ca2+ dissociation from Tn-C. The above concept has important repercussions for the non-uniformly contracting heart in which arrhythmogenic Ca2+ waves arise from weakened areas in cardiac muscle. These Ca2+ waves can reversibly be induced in muscle with non-uniform excitation contraction coupling (ECC) by the cycle of stretch and release in the border zone between the damaged and intact regions. Stimulus trains induced propagating Ca2+ waves and reversibly induced arrhythmias. We hypothesize that rapid force loss by sarcomeres in the border zone during relaxation causes Ca2+ release from Tn-C and initiates Ca2+ waves propagated by the sarcoplasmic reticulum (SR). These observations suggest the unifying hypothesis that force feedback to Ca2+ binding by Tn-C is responsible for Starling's Law and the ESPVR in uniform myocardium and leads in non-uniform myocardium to a surge of Ca2+ released by the myofilaments during relaxation, which initiates arrhythmogenic propagating Ca2+ release by the SR.     

Characterization of positive inotropic effect of colforsin daropate [correction of dapropate] hydrochloride, a water-soluble forskolin derivative, in isolated adult rat cardiomyocytes.

The present study was undertaken to characterize the positive inotropic action of colforsin daropate [corrected] hydrochloride (NKH477), a novel water-soluble forskolin derivative, on isolated cardiomyocytes of adult rats. Simultaneous measurements of cellular contraction and intracellular calcium concentration ([Ca2+]i) were carried out. The effects of isoprenaline and ouabain on these parameters were also determined for comparison. The contraction and maximum [Ca2+]i of NKH477-, isoprenaline-, or ouabain-treated cells were increased concentration dependently. Peak shortening of NKH477-treated cells was positively correlated with the shortening velocity and inversely with the time to peak shortening. Maximum, but not minimum, [Ca2+]i in NKH477-treated cells was correlated with the rate of increase in [Ca2+]i and inversely with the time to maximum [Ca2+]i. Similar results were obtained with isoprenaline. In contrast, ouabain increased both maximum and minimum [Ca2+]i. Treatment with either NKH477 or isoprenaline increased cellular cAMP content, but treatment with ouabain did not. These results suggest that the positive inotropic action of NKH477 is associated with an increase in [Ca2+]i and acceleration of its kinetics.     

Calcium mobilization and influx during the biphasic cytosolic calcium and secretory responses in agonist-stimulated pituitary gonadotrophs

Stimulation of enriched pituitary gonadotrophs by gonadotropin-releasing hormone (GnRH) elicits dose-dependent biphasic elevations of cytosolic calcium ([Ca2+]i) and luteinizing hormone (LH) release, with rapid initial peaks followed by sustained plateaus during continued exposure to the agonist. A potent GnRH-antagonist, [N-acetyl-D-p-Cl-Phe1,2,D-Trp3,D-Lys6,D-Ala10]GnRH, prevented the biphasic [Ca2+]i and LH responses when added before GnRH, and rapidly abolished both responses to GnRH when added during the plateau phase. In low Ca2+ medium the LH peak responses to GnRH were reduced and the subsequent sustained responses were almost completely abolished; reduction of extracellular Ca2+ during exposure to GnRH caused a prompt decline of LH release. The initial [Ca2+]i peak is derived largely from intracellular calcium mobilization with a partial contribution from calcium influx, while the sustained phase is dependent on the entry of extracellular Ca2+ through both L-type and dihydropyridine-insensitive channels. The presence of L-type voltage-sensitive calcium channels (VSCC) in pituitary gonadotrophs was indicated by the ability of elevated extracellular [K+] to stimulate calcium influx and LH release, and the sensitivity of these responses to dihydropyridine agonist and antagonist analogs. In cells pretreated with high [K+], the peak [Ca2+]i response to GnRH was enhanced but the subsequent plateau phase was markedly attenuated. This divergent effect of sustained membrane depolarization on the biphasic [Ca2+]i response suggests that calcium entry through VSCC initially potentiates agonist-induced mobilization of Ca2+ from intracellular storage sites. However, established Ca2+ entry through depolarization-activated VSCC cannot be further increased by agonist stimulation because both processes operate through the same channels, probably by changes in their activation-inactivation kinetics. Finally, the reciprocal potentiation by the dihydropyridine agonist, BK 8644, and GnRH of [Ca2+]i and LH responses confirms that both compounds act on the same type of channels, i.e., L-type VSCC, that participate in agonist-mediated calcium influx and gonadotropin secretion.     

Thin filament cooperativity as a major determinant of shortening velocity in skeletal muscle fibers.

The mechanism underlying the calcium sensitivity of the velocity of shortening of skeletal muscle fibers was investigated using a multiple shortening protocol: within a single contraction, skinned rabbit psoas fibers were made to shorten repetitively under a light load by briefly stretching back to their initial length at regular intervals. At saturating [Ca2+], the initial fast shortening pattern was repeated reproducibly. At submaximal [Ca2+], the first shortening consisted of fast and slow phases, but only the slow phase was observed in later shortenings. When the fibers were held isometric after the first shortening, the velocity of the second shortening recovered with time. The recovery paralleled tension redevelopment, implying a close relationship between the velocity and the number of the preexisting force-producing cross-bridges. However, this parallelism was lost as [Ca2+] was increased. Thus, the velocity was modified in a manner consistent with the cooperative thin filament activation by strong binding cross-bridges and its modulation by calcium. The present results therefore provide evidence that the thin filament cooperativity is primarily responsible for the calcium sensitivity of velocity. The effect of inorganic phosphate to accelerate the slow phase of shortening is also explained in terms of the cooperative activation.     

A Spatially Detailed Model of Isometric Contraction Based on Competitive Binding of Troponin I Explains Cooperative Interactions between Tropomyosin and Crossbridges

Biophysical models of cardiac tension development provide a succinct representation of our understanding of force generation in the heart. The link between protein kinetics and interactions that gives rise to high cooperativity is not yet fully explained from experiments or previous biophysical models. We propose a biophysical ODE-based representation of cross-bridge (XB), tropomyosin and troponin within a contractile regulatory unit (RU) to investigate the mechanisms behind cooperative activation, as well as the role of cooperativity in dynamic tension generation across different species. The model includes cooperative interactions between regulatory units (RU-RU), between crossbridges (XB-XB), as well more complex interactions between crossbridges and regulatory units (XB-RU interactions). For the steady-state force-calcium relationship, our framework predicts that: (1) XB-RU effects are key in shifting the half-activation value of the force-calcium relationship towards lower [Ca²⁺], but have only small effects on cooperativity. (2) XB-XB effects approximately double the duty ratio of myosin, but do not significantly affect cooperativity. (3) RU-RU effects derived from the long-range action of tropomyosin are a major factor in cooperative activation, with each additional unblocked RU increasing the rate of additional RU’s unblocking. (4) Myosin affinity for short (1–4 RU) unblocked stretches of actin of is very low, and the resulting suppression of force at low [Ca²⁺] is a major contributor in the biphasic force-calcium relationship. We also reproduce isometric tension development across mouse, rat and human at physiological temperature and pacing rate, and conclude that species differences require only changes in myosin affinity and troponin I/troponin C affinity. Furthermore, we show that the calcium dependence of the rate of tension redevelopment k_tr is explained by transient blocking of RU’s by a temporary decrease in XB-RU effects.     

Contractile response of skeletal muscle to low peroxide concentrations: myofibrillar calcium sensitivity as a likely target for redox-modulation.

Endogenous peroxides and related reactive oxygen species may influence various steps in the contractile process. Single mouse skeletal muscle fibers were used to study the effects of hydrogen peroxide (H2O2) and t-butyl hydroperoxide (t-BOOH) on force and myoplasmic Ca2+ concentration ([Ca2+]i). Both peroxides (1010 to 105 M) decreased tetanic [Ca2+]i and increased force during submaximal tetani. Catalase (1 kU/ml) blocked the effect of H2O2, but not of t-BOOH. The decrease in tetanic [Ca2+]i was constant, while the effect on force was biphasic: A transitory increase was followed by a steady decline to the initial level. Myofibrillar Ca2+ sensitivity remained increased during incubation with either peroxide. Only the highest peroxide concentration (10 mM) increased resting [Ca2+]i and slowed the return of [Ca2+]i to its resting level after a contraction, evidence of impaired sarcoplasmic reticulum Ca2+ re-uptake. The peroxides increased maximal force production and the rate of force redevelopment, and decreased maximum shortening velocity. N-ethylmaleimide (25 mM, thiol-alkylating agent) prevented the response to 1 mM H2O2. These results show that myofibrillar Ca2+ sensitivity and cross-bridge kinetics are influenced by H2O2 and t-BOOH concentrations that approach those found physiologically, and these findings indicate a role for endogenous oxidants in the regulation of skeletal muscle function.     

Mild renal hypertension alters run training effects on the frequency response of rat cardiomyocyte mechanics.

We examined the effects of run training on the frequency dependence of cardiomyocyte mechanics and intracellular calcium concentration ([Ca2+]i) dynamics in rats with mild renal hypertension. Male Fischer 344 rats aged 2-3 mo underwent a sham operation or stenosis of the left renal artery, which increased systolic blood pressure 20-30 mmHg. Half of the rats in each group underwent treadmill run training for >16 wk. Isolated cardiomyocytes were paced at 1.0 and 0.2 Hz in 2 mM external Ca2+ concentration at 29 degrees C. Under these conditions, negative frequency responses, i.e., decreased value with increased frequency, were recorded for peak shortening, shortening velocity, and the integral of the [Ca2+]i transient in both groups. Run training amplified the negative frequency response for the integral of the [Ca2+]i transient in both groups, but it amplified the negative frequency response for the shortening dynamics only in the normotensive sham-operated and not in the hypertensive rats. These results, as well as others for relaxation parameters, suggest that renal hypertension altered the effects of run training on the frequency response for cardiomyocyte contractile apparatus and/or passive mechanical properties, which respond to [Ca2+]i.     

Spatial and temporal aspects of ACh-induced [Ca2+]i oscillations in porcine tracheal smooth muscle

This study evaluated the relationship between regional elevation in intracellular calcium concentration ([Ca2+]i) induced by acetylcholine (ACh) and the global cellular responses in porcine tracheal smooth muscle (TSM) cells. Regional (approximately 1.5 microm3) and global (whole cell) changes in [Ca2+]i were measured in fluo-3 loaded TSM cells using real-time confocal microscopy. Regional responses appeared as propagating [Ca2+]i oscillations whereas global responses reflected the spatiotemporal integration of these regional responses. Within a region, [Ca2+]i oscillations were 'biphasic' with initial higher frequencies, followed by slower steady-state oscillations. With increasing ACh concentration, the peak (maximum value relative to 0 nM) of regional [Ca2+]i oscillations remained relatively constant, whereas both frequency and propagation velocity increased. In contrast, the global spatiotemporal integration of the regional oscillatory responses appeared as a concentration-dependent increase in peak as well as mean cellular [Ca2+]i. We conclude that the significance of ACh-induced [Ca2+]i oscillations lies in the establishment of mean [Ca2+]i level for slower Ca2+-dependent physiological processes via modulation of oscillation frequency and propagation velocity.     

Thyrotropin-releasing hormone-induced spike and plateau in cytosolic free Ca2+ concentrations in pituitary cells. Relation to prolactin release

Using the acetoxymethyl ester of "Quin 2," a fluorescent Ca2+-indicator, we have loaded prolactin (PRL)-producing rat pituitary cells with non-toxic concentrations of Quin 2 and quantitated changes in cytosolic free calcium concentration ( [Ca2+]i) during stimulation of PRL release by thyrotropin-releasing hormone (TRH) and 40 mM K+. TRH induced a biphasic response, with an immediate (less than 1 s) spike in [Ca2+]i from basal levels (350 +/- 80 nM) to a peak of 1-3 microM, which decayed rapidly (t 1/2 = 8 s) to a near basal nadir, then rising to a plateau in [Ca2+]i of 500-800 nM. The TRH-induced spike phase was attenuated but not abolished by prior addition of EGTA, while the plateau phase was eliminated by EGTA. Addition of 40 mM K+ caused an immediate spike in [Ca2+]i to 1-3 microM which equilibrated slowly (t 1/2 = 1 min) directly to a plateau of 600-800 nM. The K+-induced spike and plateau phases were both abolished by prior addition of EGTA. The biphasic nature of TRH action on [Ca2+]i parallels the biphasic actions of TRH on 45Ca2+ fluxes and the biphasic release of PRL by GH cells in suspension. These findings provide evidence that Ca2+-dependent agonist-mediated increases in [Ca2+]i and hormone release are linked, and may generally have two modes: an acute "spike" mode, dependent primarily on redistribution of intracellular Ca2+ stores; and a sustained "plateau" mode, dependent on influx of extracellular Ca2+.     

Generation and amplification of the cytosolic calcium signal during secretory responses to gonadotropin-releasing hormone

Gonadotropin-releasing hormone (GnRH) stimulates characteristic biphasic increases in cytosolic calcium concentration ([Ca2+]i) and in luteinizing hormone (LH) release in cultured gonadotrophs, with an early peak followed by a prolonged plateau in both responses. Analysis of [Ca2+]i by dual-wavelength fluorimetric assay and of LH release at 5-sec intervals in perifused pituitary cells revealed increases in both responses within a few seconds of exposure to GnRH. The maximum elevation of [Ca2+]i occurred within 20 sec, and the peak gonadotropin release in 35 sec; the total duration of the spike phase for both [Ca2+]i and LH release was 2.5 min. Under extracellular Ca2(+)-deficient conditions, the GnRH-induced peak in [Ca2+]i was reduced by about 20% and the plateau phase was abolished. Concomitantly, the magnitude of the acute phase of LH release was reduced by 40% and that of the second phase by about 90%. Recovery of the plateau phase of LH release occurred within 25 sec after addition of 1.25 mM Ca2+ to Ca2(+)-deficient medium. In a dose-dependent manner, the non-selective Ca2+ channel blockers Co2+ and Cd2+ reduced the Ca2+ current measured by whole-cell recording in pituitary gonadotrophs and abolished the extracellular Ca2(+)-dependent component of LH release. The selective calcium channel blocker, nifedipine, decreased the magnitude of the Ca2+ current and reduced the plateau phase of LH release by 50%; conversely, the dihydropyridine agonist methyl, 1,4,dihydro-2,6-dimethyl 3-nitro-4-(2-trifluorome) (Bay K 8644) consistently enhanced the amplitudes of both Ca2+ current and GnRH-induced LH release. These data reveal a close temporal correlation between changes in [Ca2+]i and LH release during GnRH action, with Ca2+ mobilization during the spike phase and Ca2+ influx through dihydropyridine-sensitive and insensitive sets of receptor-operated calcium channels during the spike and plateau phases. In addition, analysis of the magnitudes of the [Ca2+]i and LH responses to a wide range of GnRH concentrations in the presence and absence of extracellular Ca2+ is consistent with amplification of the [Ca2+]i signal in agonist-stimulated gonadotrops.     

Computer-aided measurement of cell shortening and calcium transients in adult cardiac myocytes

The contractile cycle of the cardiac myocyte is essentially controlled by the concentration of intracellular calcium ([Ca2+]i). Measurement of [Ca2+]i using Ca2+-dependent fluorescence and simultaneous monitoring of cell dynamics enable characterization of a variety of substances interacting with ion channels and contractile proteins. In this report we describe a novel method featuring up to 480 frames/s for monitoring rapid changes in cellular calcium and cell length, in which every individual cycle allows effective evaluation of major cell parameters. Computers aid in determination of time to peak (in ms), time to 50% decrease (ms), diastolic Ca2+ (relative fluorescence units, rfu), systolic Ca2+ (rfu), Ca2+ transients (rfu), DeltaCa2+/Delta(t) rise (rfu/s), and DeltaCa2+/Delta(t) fall (rfu/s). Contractile parameters are as follows: maximum cell length (microm), minimum cell length (microm), absolute cell shortening (microm), peak DeltaL/Delta(t) shortening (microm/s), and peak DeltaL/Delta(t) relaxation (microm/s). In summary, we succeeded in demonstrating that this system is a unique and valuable tool that allows simultaneous and accurate assessment of contractile parameters and of calcium movements of isolated adult cardiac myocytes.     

Dual-phase response of bovine pulmonary artery endothelial cells to agonists which increase free cytoplasmic calcium concentration

The regulation of free cytoplasmic calcium concentration ([Ca2+]i) was studied in bovine pulmonary artery endothelial cells (BPAEC). The cells were seeded on the inner surface of glass cuvettes, grown to confluency and loaded with INDO-1. Using a multiwavelength method for estimation of [Ca2+]i it was shown that in Ca2+ containing medium a rapid rise of [Ca2+]i occurs in response to bradykinin, ATP or thrombin followed by a much slower decrease in free cytoplasmic calcium. Binding of extracellular Ca2+ by EGTA lowered basal [Ca2+]i but had no effect on the rate of agonist-induced [Ca2+]i increase or its absolute amount. In contrast, the kinetics of [Ca2+]i decrease were entirely different. A rapid (less than 0.5 min) decrease in [Ca2+]i to the basal level was observed immediately after the maximum had been achieved. If excess Ca2+ was added to the medium after EGTA, a second [Ca2+]i rise in response to the agonists occurred. The decrease in [Ca2+]i after the second peak was several times slower than the decrease in Ca2+ free medium. It is concluded that Ca2+ entry from the external medium had no effect on the maximal increase in [Ca2+]i but provides a severalfold increase in the duration the endothelial cell responses to the agonists.     

Spontaneous and agonist-induced calcium oscillations in pituitary gonadotrophs

Basal and receptor-regulated changes in cytoplasmic calcium concentration ([Ca2+]i) were monitored by fluorescence analysis in individual rat pituitary gonadotrophs loaded with the calcium-sensitive dye indo-1. Most gonadotrophs exhibited low amplitude spontaneous oscillations in basal [Ca2+]i that were interspersed by quiescent periods and abolished by removal of extracellular Ca2+ or addition of calcium channel blockers. Such random fluctuations in [Ca2+]i, which reflect the operation of a plasma membrane oscillator, were not coupled to basal gonadotropin secretion. The physiological agonist GnRH induced high amplitude [Ca2+]i oscillations; when a threshold [Ca2+]i level was reached, a cytoplasmic oscillator began to generate extremely regular Ca2+ transients. The time required to reach the threshold [Ca2+]i level was inversely correlated with agonist dose; the frequency, but not the amplitude, of agonist-induced Ca2+ spiking increased with agonist concentration. The duration of the latent period decreased and the frequency of Ca2+ spiking increased with the increase in ambient temperature. At high GnRH concentrations, the calcium transients merged into biphasic responses similar to those observed in cell suspensions at all GnRH concentrations. The presence of spontaneous fluctuations in basal [Ca2+]i did not significantly change the patterns of agonist-induced [Ca2+]i responses. Also, removal of extracellular Ca2+ did not interfere with the frequency or amplitude of Ca2+ spikes, but caused the loss of the plateau phase. Blockade of intracellular Ca(2+)-ATPase pumps by thapsigargin was usually accompanied by a subthreshold increase in [Ca2+]i. In such cells the agonist-induced oscillatory pattern was transformed into the biphasic response. In about 10% of the cells, however, high thapsigargin concentrations induced coarse [Ca2+]i oscillations; subsequent stimulation of such cells with GnRH was ineffective. The cytoplasmic oscillatory and biphasic responses may represent a mechanism for differential activation of Ca(2+)-dependent enzymes and their dependent cellular processes, including hormone secretion. The membrane oscillator is probably responsible for refilling of agonist-sensitive pools during and after agonist stimulation.     

Pulsatile Ca2+ extrusion from single pancreatic acinar cells during receptor-activated cytosolic Ca2+ spiking.

Ca2+ extrusion was measured simultaneously with the free intracellular Ca2+ concentration ([Ca2+]i) from single pancreatic acinar cells placed in microdroplets of extracellular solution (Tepikin, A. V., Voronina, S. G., Gallacher, D. V., and Petersen, O. H. (1992) J. Biol. Chem. 267, 3569-3572). Submaximal stimulation with cholecystokinin usually evoked discrete cytosolic Ca2+ spikes and each of these spikes was associated with a discrete and virtually synchronous pulse of Ca2+ extrusion into the extracellular microdroplet solution. When ACh evoked repetitive discrete [Ca2+]i spikes, each spike was also accompanied by a discrete pulse of Ca2+ extrusion. The velocity of Ca2+ extrusion oscillated with a time course similar to that of [Ca2+]i. The extracellular solution in our experiments had a low total calcium concentration (15-35 microM) and only a limited number of [Ca2+]i spikes (2-8) could be evoked. The magnitudes of the [Ca2+]i spikes and the amounts of Ca2+ extruded during each spike gradually decreased in each experiment. During the first cholecystokinin-evoked cytosolic Ca2+ spike the Ca2+ extrusion corresponded to a loss of 15-70% (mean value 39% +/- 12) of the mobilizable cellular calcium pool. The substantial pulsatile Ca2+ extrusion occurring synchronously with the receptor-activated cytosolic Ca2+ spikes is therefore an important element in repetitively bringing back [Ca2+]i to the resting level.     

Biphasic activation of cytosolic free calcium and LH responses by gonadotropin-releasing hormone

Gonadotropin-releasing hormone (GnRH) stimulates rapid peak increases in [Ca2+]i and LH release, followed by lower but sustained elevations of both [Ca2+]i and hormone secretion. Omission of extracellular Ca2+ only slightly decreased the peak of [Ca2+]i, but reduced the peak LH response by 40% and prevented the prolonged increases in [Ca2+]i and LH release. Dihydropyridine calcium antagonists did not affect the peak [Ca2+]i and LH responses, but reduced the sustained increases by up to 50%. Whereas GnRH-induced mobilization of intracellular calcium initiates the LH peak, and Ca2+ entry through dihydropyridine-insensitive channels contributes to the peak and plateau phases of LH release, dihydropyridine-sensitive L-type Ca2+ channels participate only in the sustained phase of gonadotropin secretion.     

Intracellular calcium 'signatures' evoked by different agonists in isolated bovine aortic endothelial cells.

Agonist induced increases in intracellular free calcium, [Ca2+]i, were measured in single Fura-2 loaded bovine aortic endothelial (BAE) cells by dual wavelength microspectrofluorimetry. Low doses of ATP (less than 10 microM) induced complex changes in [Ca2+]i. These changes usually consisted of a large initial transient peak with subsequent fluctuations superimposed upon a maintained rise in [Ca2+]i. Higher doses of ATP (greater than 50 microM) produced much simpler biphasic increases in [Ca2+]i in individual cells. Acetylcholine and bradykinin also elicited increases in [Ca2+]i in single cells in confluent monolayers of endothelial cells. However, only acetylcholine produced complex fluctuations. High doses of acetylcholine evoked simple rises in [Ca2+]i similar to those seen with high doses of ATP. In contrast, bradykinin evoked relatively simple rises in [Ca2+]i at all doses used. These results indicate that the mechanisms responsible for generating agonist induced increases in [Ca2+]i in BAE cells are not homogeneous. ATP and acetylcholine produced more complex Ca2+ changes or 'signatures' in single confluent bovine aortic endothelial cells than bradykinin. All three agonists appeared to release Ca2+ from intracellular stores as well as stimulating Ca2+ influx. The possible mechanisms underlying these phenomena are discussed.     

Intracellular calcium accumulation following eccentric contractions in rat skeletal muscle in vivo: role of stretch-activated channels

Although the accumulation of intracellular calcium ions ([Ca2+]i) is associated with muscle damage, little is known regarding the temporal profile of muscle [Ca2+]i under in vivo conditions, and, specifically, the effects of different contraction types [e.g., isometric (ISO); eccentric (ECC)] on [Ca2+]i remain to be determined. The following hypotheses were tested. 1) For 90 min at rest, an in vivo vs. in vitro preparation would better maintain initial [Ca2+]i. 2) Compared with ISO, ECC contractions (50 contractions, 10 sets, 5-min interval) would lead to a greater increase of [Ca2+]i. 3) Elevated [Ca2+]i during ECC would be reduced or prevented by the stretch-activated ion channel blockers streptomycin and gadolinium (Gd3+). Spinotrapezius muscles of Wistar rats were exteriorized (in vivo) or excised (in vitro). [Ca2+]i was evaluated by loading the muscle with fura 2-AM using fluorescence imaging. [Ca2+]i rose progressively beyond 40 min at rest under in vitro but not in vivo conditions during the 90-min protocol. In vivo [Ca2+]i increased more rapidly during ECC (first set) than ISO (fifth set) (P < 0.05 vs. precontraction values). The peak level of [Ca2+]i was increased by 21.5% (ISO) and 42.8% (ECC) after 10 sets (both P < 0.01). Streptomycin and Gd3+ abolished the majority of [Ca2+]i increase during ECC (69 and 86% reduction, respectively; P < 0.01 from peak [Ca2+]i of ECC). In conclusion, in vivo quantitative analyses demonstrated that ECC contractions elevate [Ca2+]i significantly more than ISO contractions and that stretch-activated channels may play a permissive role in this response.