Identification of cardiac stem cells within mature cardiac myocytes

Cardiac stem cells are described in a number of mammalian species including humans. Cardiac stem cell clusters consisting of both lineage-negative and partially committed cells are generally identified between contracting cardiac myocytes. In the present study, c-kit⁺, Sca⁺, and Isl1⁺ stem cells were revealed to be located inside the sarcoplasm of cardiac myocytes in myocardial cell cultures derived from newborn, 20-, and 40-day-old rats. Intracellularly localized cardiac stem cells had a coating or capsule with a few pores that opened into the host cell sarcoplasm. The similar structures were also identified in the suspension of freshly isolated myocardial cells (ex vivo) of 20- and 40-day-old rats. The results from this study provide direct evidence for the replicative division of encapsulated stem cells, followed by their partial cardiomyogenic differentiation. The latter is substantiated by the release of multiple transient amplifying cells following the capsule rupture. In conclusion, functional cardiac stem cells can reside not only exterior to but also within cardiomyocytes.     

Characterization of cardiomyocyte excitation-contraction coupling in the FVB/N-C57BL/6 intercrossed "chocolate" brown mice

Mice are extensively used for gene modification research and isolated cardiomyocytes are essential for evaluation of cardiac function without interference from non-myocyte contribution. This study was designed to characterize cardiomyocyte excitation-contraction coupling in FVB/N-C57BL/6 intercrossed brown mice. Mechanical and intracellular Ca(2+) properties were evaluated using an IonOptix softedge system including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR(90)), maximal velocity of shortening and relengthening (+/- dL/dt), intracellular Ca(2+) rise and decay rate. Resting cell length was longer in age- and gender-matched C57BL/6 and brown mice compared to FVB strain. PS and +/- dL/dt were significantly lower in brown mice compared to FVB/N and C57BL/6 groups. TPS was shortened in C57BL/6 mice and TR(90) was prolonged in brown mice compared to other groups. Resting intracellular Ca(2+) level and single exponential intracellular Ca(2+) decay constant were comparable among all three mouse lines. Rise in intracellular Ca(2+) in response to electrical stimulus was higher in C57BL/6 mouse myocytes whereas bi-exponential intracellular Ca(2+) decay was faster in brown mice. Myocytes from all three groups exhibited similar fashion of reduction in PS in response to increased stimulus frequency. These data suggest that inherent differences in cardiomyocyte excitation-contraction coupling exist between strains, which may warrant caution when comparing data from these mouse lines.     

Expression of the sodium/calcium exchanger in mammalian skeletal muscle cells in primary culture

Previous investigations have demonstrated molecular and functional expression, at early phases of development of skeletal muscle cells in primary culture, of cardiac isoforms of proteins involved in calcium transport and regulation, like the L-type calcium channel. Here the expression of the cardiac isoform of the Na(+)/Ca(2+) exchanger (NCX1) was studied in skeletal muscle cells developing in vitro, by using biochemical, immunological, and electrophysiological techniques. Northern and Western blot experiments revealed the presence of this cardiac exchanger and its increasing expression during the early phases of development. Confocal imaging of myotubes showed an NCX1 distribution that was predominantly sarcolemmal. The whole-cell patch-clamp technique allowed us to record ionic currents, the direction and the amplitude of which depended on extracellular sodium and calcium concentrations. The developmental changes of this functional expression could be correlated with the molecular NCX1 expression changes. Taken together these data demonstrate the presence of the NCX1 isoform of the Na(+)/Ca(2+) exchanger during in vitro myogenesis and reinforce the theory that significant levels of cardiac-type proteins are transiently expressed during the early phases of the skeletal muscle cell development.     

Two ryanodine receptor isoforms in nonmammalian vertebrate skeletal muscle: possible roles in excitation-contraction coupling and other processes

The ryanodine receptor (RyR) is a Ca²⁺ release channel in the sarcoplasmic reticulum in vertebrate skeletal muscle and plays an important role in excitation–contraction (E–C) coupling. Whereas mammalian skeletal muscle predominantly expresses a single RyR isoform, RyR1, skeletal muscle of many nonmammalian vertebrates expresses equal amounts of two distinct isoforms, α-RyR and β-RyR, which are homologues of mammalian RyR1 and RyR3, respectively. In this review we describe our current understanding of the functions of these two RyR isoforms in nonmammalian vertebrate skeletal muscle. The Ca²⁺ release via the RyR channel can be gated by two distinct modes: depolarization-induced Ca²⁺ release (DICR) and Ca²⁺-induced Ca²⁺ release (CICR). In frog muscle, α-RyR acts as the DICR channel, whereas β-RyR as the CICR channel. However, several lines of evidence suggest that CICR by β-RyR may make only a minor contribution to Ca²⁺ release during E–C coupling. Comparison of frog and mammalian RyR isoforms highlights the marked differences in the patterns of Ca²⁺ release mediated by RyR1 and RyR3 homologues. Interestingly, common features in the Ca²⁺ release patterns are noticed between β-RyR and RyR1. We will discuss possible roles and significance of the two RyR isoforms in E–C coupling and other processes in nonmammalian vertebrate skeletal muscle.     

Processes Maintaining Calcium Homeostasis in Acinar Cells of the Rat Submandibular Salivary Gland

Using a Ca²⁺-sensitive fluorescent indicator, fura-2/AM, we recorded calcium transients in secretory cells of isolated acini of the rat submandibular salivary gland; these transients were induced by hyperpotassium-induced depolarization (after an increase in [K⁺] _e up to 50 mM) of the plasma membrane of the above cells. Calcium transients were significantly suppressed by 50 M nifedipine. Addition of 10 M carbonyl cyanide m-chlorophenylhydrazone to the normal extracellular solution was accompanied by a rise in [Ca²⁺] _i , whereas when hyperpotassium solution is used the effect was less expressed. Blockers of CA²⁺-ATPase in the cellular membrane and in the endoplasmic reticulum, eosin Y (5 M) and cyclopiazonic acid (CPA, 5 M), respectively, evoked a significant increase in [Ca²⁺] _i and a decrease in the K⁺-depolarization-induced calcium transient. Extracellular application of caffeine (2, 10, or 30 mM) was accompanied by a concentration-dependent rise in [Ca²⁺] _i . Therefore, potassium depolarization of the plasma membrane of acinar cells of the rat submandibular salivary gland activates both the voltage-dependent Ca²⁺ influx and Ca²⁺-induced Ca²⁺ release from the endoplasmic reticulum; the initial level of [Ca²⁺] _i was restored at the joint involvement of Ca²⁺-ATPases in the plasma membrane and the membranes of the endoplasmic reticulum and mitochondria.     

A new structure from cardiac cells cultured in vitro: Cardiomyocyte-annulation of neonatal rats

To explore the formation, morphological characteristics, cell composition, and differentiation potential of cardiomyocyte annulation (cardio-annulation) during in vitro culture of cardiac cells. Cardiac cells were isolated and cultured. A live-cell imaging system was used to observe cardio-annulation. Cardiac troponin-T (cTnT) and vimentin were labeled with double immunofluorescence staining, and coexpressions of cTnT and connexin43 (Cx43), cTnT and nanog, c-kit and nanog, and c-kit and stem cell antigen (sca-1) were detected. The location of various types of cells within the cardio-annulation structure was observed. Adipogenic- and osteogenic-inducing fluids were used separately for in situ induction to detect the multidirectional differentiation potential of cells during the annulation process. After 3 to 6 days, cardiac cells migrated and formed an open or closed annulus with a diameter of 800 to 3500 μm. The annulus wall comprised the medial, middle, and lateral regions. The cells in the medial region were small, abundant, and laminated, while those in the middle region were larger with fewer layers, and those in the lateral region were less abundant, and loosely arranged in a single layer. Cardiomyocytes were distributed mainly on the surface of the medial region; nanog⁺, c-kit⁺, and sca-1⁺ cells were located mainly at the bottom of the annulus wall and fibroblasts were located mainly between these layers. The annulus cavity contained a large number of small, round cells, and telocytes. Cx43 was expressed in all cell types, and nanog, c-kit, and sca-1 were coexpressed in the cardio-annulation cells, which possess adipogenic and osteogenic differentiation potential. Cardio-annulation was discovered during an in vitro culture of cardiac cells. The structure contains cardiomyocytes, fibroblasts, telocytes, and abundant stem cells. These results provide insight into the relationship among cardiac cells in vitro.     

Regulation of Ca2+ mobilization by prolactin in mammary gland cells: possible role of secretory pathway Ca2+-ATPase type 2

Regulatory role of prolactin (PRL) on Ca2+ mobilization in human mammary gland cell line MCF-7 was examined. Direct addition of PRL did not affect cytoplasmic Ca2+ concentration ([Ca2+]i); however, treatment with PRL for 24h significantly decreased the peak level and duration time of [Ca2+]i elevation evoked by ATP or thapsigargin (TG). Intracellular Ca2+ release by IP3 or TG in permeablized cells was not decreased after PRL-treatment, indicating that the Ca2+ release was not impaired by PRL treatment. Extracellular Ca2+ entry evoked by ATP or TG was likely to be intact, because entry of extracellular Ba2+ was not affected by PRL treatment. Among Ca2+-ATPases expressed in MCF-7 cells, we found significant increase of secretory pathway Ca2+-ATPase type 2 (SPCA2) mRNA in PRL-treated cells by RT-PCR experiments including quantitative RT-PCR. Knockdown of SPCA2 by siRNA in PRL-treated cells showed similar Ca2+ mobilization to that in PRL-untreated cells. The present results suggest that PRL facilitates Ca2+ transport into Golgi apparatus and may contribute the supply of Ca2+ to milk.     

Diversity of mitochondrial Ca signaling in rat neonatal cardiomyocytes: Evidence from a genetically directed Ca probe,mitycam-E31Q

I_Ca-gated Ca²⁺ release (CICR) from the cardiac SR is the main mechanism mediating the rise of cytosolic Ca²⁺, but the extent to which mitochondria contribute to the overall Ca²⁺ signaling remains controversial. To examine the possible role of mitochondria in Ca²⁺ signaling, we developed a low affinity mitochondrial Ca²⁺ probe, mitycam-E31Q (300–500 MOI, 48–72 h) and used it in conjunction with Fura-2AM to obtain simultaneous TIRF images of mitochondrial and cytosolic Ca²⁺ in cultured neonatal rat cardiomyocytes. Mitycam-E31Q staining of adult feline cardiomyocytes showed the typical mitochondrial longitudinal fluorescent bandings similar to that of TMRE staining, while neonatal rat cardiomyocytes had a disorganized tubular or punctuate appearance. Caffeine puffs produced rapid increases in cytosolic Ca²⁺ while simultaneously measured global mitycam-E31Q signals decreased more slowly (increased mitochondrial Ca²⁺) before decaying to baseline levels. Similar, but oscillating mitycam-E31Q signals were seen in spontaneously pacing cells. Withdrawal of Na⁺ increased global cytosolic and mitochondrial Ca²⁺ signals in one population of mitochondria, but unexpectedly decreased it (release of Ca²⁺) in another mitochondrial population. Such mitochondrial Ca²⁺ release signals were seen not only during long lasting Na⁺ withdrawal, but also when Ca²⁺ loaded cells were exposed to caffeine-puffs, and during spontaneous rhythmic beating. Thus, mitochondrial Ca²⁺ transients appear to activate with a delay following the cytosolic rise of Ca²⁺ and show diversity in subpopulations of mitochondria that could contribute to the plasticity of mitochondrial Ca²⁺ signaling.     

Role of Ca2+ signaling in initiation of stretch-induced apoptosis in neonatal heart cells

Abnormal mechanical load, as seen in hypertension, is found to induce heart cell apoptosis, yet the signaling link between cell stretch and apoptotic pathways is not known. Using an in vitro stretch model mimicking diastolic pressure stress, here we show that Ca(2+) signaling participates essentially in the early stage of stretch-induced apoptosis. In neonatal rat cardiomyocytes, the moderate 20% stretch resulted in tonic elevation of intracellular free Ca(2+) ([Ca(2+)](i)). Buffering [Ca(2+)](i) by EGTA-AM, suppressing ryanodine-sensitive Ca(2+) release, and blocking L-type Ca(2+) channels all prevented the stretch-induced apoptosis as assessed by phosphatidylserine exposure and nuclear fragmentation. Notably, Ca(2+) suppression also prevented known stretch-activated apoptotic events, including caspase-3/-9 activation, mitochondrial membrane potential corruption, and reactive oxygen species production, suggesting that Ca(2+) signaling is the upstream of these events. Since [Ca(2+)](i) did not change without activating mechanosensitive Ca(2+) entry, we conclude that stretch-induced Ca(2+) entry, via the Ca(2+)-induced Ca(2+) release mechanism, plays an important role in initiating apoptotic signaling during mechanical stress.     

Characterization of the 1,25-dihydroxycholecalciferol-stimulated calcium influx pathway in CaCo-2 cells

The present studies were conducted to investigate the mechanisms underlying the 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃)-induced increase in intracellular Ca²⁺ ([Ca²⁺] _i ) in individual CaCo-2 cells. In the presence of 2mm Ca²⁺, 1,25(OH)₂D₃-induced a rapid transient rise in [Ca²⁺] _i in Fura-2-loaded cells in a concentration-dependent manner, which decreased, but did not return to baseline levels. In Ca²⁺-free buffer, this hormone still induced a transient rise in [Ca²⁺] _i , although of lower magnitude, but [Ca²⁺] _i then subsequently fell to baseline. In addition, 1,25(OH)₂D₃ also rapidly induced⁴⁵Ca uptake by these cells, indicating that the sustained rise in [Ca²⁺] _i was due to Ca²⁺ entry. In Mn²⁺-containing solutions, 1,25(OH)₂D₃ increased the rate of Mn²⁺ influx which was temporally preceded by an increase in [Ca²⁺] _i . The sustained rise in [Ca²⁺] _i was inhibited in the presence of external La³⁺ (0.5mm). 1,25(OH)₂D₃ did not increase Ba²⁺ entry into the cells. Moreover, neither high external K⁺ (75mm), nor the addition of Bay K 8644 (1 μm), an L-type, voltage-dependent Ca²⁺ channel agonist, alone or in combination, were found to increase [Ca²⁺] _i , 1,25(OH)₂D₃ did, however, increase intracellular Na⁺ in the absence, but not in the presence of 2mm Ca²⁺, as assessed by the sodium-sensitive dye, sodium-binding benzofuran isophthalate. These data, therefore, indicate that CaCo-2 cells do not express L-type, voltage-dependent Ca²⁺ channels. 1,25(OH)₂D₃ does appear to activate a La³⁺-inhibitable, cation influx pathway in CaCo-2 cells.     

On the involvement of cyclic AMP and extracellular Ca2+ in the regulation of hormone release from rat pituitary tumour (GH3) cells in culture

Thyroliberin (TRH), dibutyryl cyclic AMP (db-cAMP), and 3-isobutyl-l-methylxanthine (MIX) had a stimulatory effect on prolactin (PRL) and growth hormone (GH) release from GH 3 cells. Half-maximal and maximal effects were observed for TRH at 2.5 nM and 10 nM; for db-cAMP at 0.6 mM and 5 mM, respectively. MIX (0.1 mM–1 mM) induced a dose-dependent accumulation of cellular cyclic AMP, while the hormone release was already maximally stimulated at 0.1 mM MIX. The maximal effects on hormone release of TRH and db-cAMP, but not of TRH and MIX, were additive.The Ca²⁺ channel blockers Co²⁺ (5 mM) and verapamil (100 M) and the Ca²⁺ chelator EGTA (4 mM) abolished the stimulatory effect of TRH (1 M) on hormone release. Co²⁺ and verapamil, but not EGTA, inhibited the stimulatory effect of db-cAMP (5 mM) on hormone release. The inhibitory effects of Co²⁺ and verapamil on GH release were counteracted by the combination of TRH and db-cAMP. For PRL release Co²⁺, but not verapamil, was able to inhibit the combined action of TRH and db-cAMP. Co²⁺, verapamil, and EGTA eliminated the stimulatory effect of MIX (1 mM) on PRL release while only Co²⁺ and EGTA affected the GH release. Hormone release in the presence of MIX plus verapamil or EGTA, but not Co²⁺, was increased by TRH.The calmodulin antagonist trifluoperazine (TFP) at 30 M inhibited basal hormone release and hormone release stimulated by TRH (1 M), db-cAMP (5 mM), and MIX (1 mM). The Ca²⁺ ionophore A23187 (5 M) had a stimulatory effect on basal hormone release which was abolished by 30 M TFP.     

Cardiomyocyte-like cells differentiated in vitro from embryonic carcinoma cells P19 are characterized by functional expression of adrenoceptors and Ca2+ channels

Summary P19 embryonal carcinoma cells were differentiated via embryolike aggregates (embryoid bodies) into spontaneously beating myocytes. During the whole process of differentiation the functional expression of cardiac-specific receptors and ionic channels was characterized by measuring the chronotropic reactivity, action potentials, and ionic currents in response to various cardioactive drugs. Positive chronotropic effects obtained at different maximal effective concentrations of adrenoceptor-mediated agonists indicated differential adrenoceptor expression during the in vitro development of cardiomyocyte-like cells. No cardiac-specific response was obtained with the muscarinic cholinoceptor agonist carbachol. Single beating cells were enzymatically isolated and investigated by the patch-clamp technique. Pacemaker action potentials similar to those of embryonal cardiomyocytes exhibited amplitudes ranging from 50 to 85 mV. The action potentials were synchronous to the mechanical contractions and, comparable to the chronotropic effects, were modulated by BayK 8644, isradipine, and adrenaline. The functional expression of L-type Ca²⁺ channels was demonstrated by the Ca²⁺ channel blockers isradipine, nisoldipine, gallopamil, and diltiazem causing negative chronotropic responses, as well as by the Ca²⁺ channel activator BayK 8644 causing positive chronotropic responses. These effects gradually increased with time of differentiation. The expression of L-type Ca²⁺ channels and of nicotinic acetylcholine receptors was confirmed in voltage-clamp experiments. The study demonstrates that P19 embryonal carcinoma cells can be induced to differentiate into cardiomyocyte-like cells comparable to embryonal and neonatal heart cells lacking the muscarinic cholinoceptor response only.     

Molecular basis of Ca(2)+ activation of the mouse cardiac Ca(2)+ release channel (ryanodine receptor).

Activation of the cardiac ryanodine receptor (RyR2) by Ca(2)+ is an essential step in excitation-contraction coupling in heart muscle. However, little is known about the molecular basis of activation of RyR2 by Ca(2)+. In this study, we investigated the role in Ca(2)+ sensing of the conserved glutamate 3987 located in the predicted transmembrane segment M2 of the mouse RyR2. Single point mutation of this conserved glutamate to alanine (E3987A) reduced markedly the sensitivity of the channel to activation by Ca(2)+, as measured by using single-channel recordings in planar lipid bilayers and by [(3)H]ryanodine binding assay. However, this mutation did not alter the affinity of [(3)H]ryanodine binding and the single-channel conductance. In addition, the E3987A mutant channel was activated by caffeine and ATP, was inhibited by Mg(2)+, and was modified by ryanodine in a fashion similar to that of the wild-type channel. Coexpression of the wild-type and mutant E3987A RyR2 proteins in HEK293 cells produced individual single channels with intermediate sensitivities to activating Ca(2)+. These results are consistent with the view that glutamate 3987 is a major determinant of Ca(2)+ sensitivity to activation of the mouse RyR2 channel, and that Ca(2)+ sensing by RyR2 involves the cooperative action between ryanodine receptor monomers. The results of this study also provide initial insights into the structural and functional properties of the mouse RyR2, which should be useful for studying RyR2 function and regulation in genetically modified mouse models.     

Differential roles of PKC-theta in the regulation of intracellular calcium concentration in primary T cells

Activation of T lymphocytes requires protein kinase C theta (PKC-theta) and an appropriately elevated free intracellular Ca2+ concentration ([Ca2+]i). Here, we show that phorbol 12 myristate 13-acetate (PMA) inhibited Ca2+ influx in wild-type but not PKC-theta-/- T cells, suggesting that PKC-theta plays a role in PMA-mediated inhibition of Ca2+ influx. In contrast, T cell receptor (TCR) crosslinking in the same PKC-theta-/- T cells did result in significantly decreased [Ca2+]i compared to wild-type T cells, suggesting a positive role for PKC-theta in TCR-mediated Ca2+ mobilization. In PKC-theta-/- mice, peripheral mature T cells, but not developing thymocytes, displayed significantly decreased TCR-induced Ca2+ influx and nuclear factor of activated T cells (NFAT) translocation upon sub-optimal TCR crosslinking. The decreased intracellular free Ca2+ was due to changes in Ca2+ influx but not efflux, as observed in extracellular and intracellular Ca2+ mobilization studies. However, these differences in Ca2+ influx and nuclear factor of activated T cells (NFAT) translocation disappeared with increasing intensity of TCR crosslinking. The enhancing effect of PKC-theta on Ca2+ influx is not only dependent on the strength of TCR crosslinking but also on the developmental stage of T cells. The underlying mechanism involved phospholipase Cgamma1 activation and inositol triphosphate production. Furthermore, knockdown of endogenous PKC-theta expression in Jurkat cells resulted in significant inhibition of TCR-induced activation of NFAT, as evidenced from NFAT reporter studies. Forced expression of a constitutively active form of calcineurin in PKC-theta-/- Jurkat cells could readily overcome the above inhibition. Thus, PKC-theta can both positively and negatively regulate the Ca2+ influx that is critical for NFAT activity.     

Role of protein kinase C in the regulation of cytosolic Ca2+ in A431 cells: Separation of growth factor and bradykinin pathways

Summary Calcium signaling systems in nonexcitable cells involve activation of Ca²⁺ entry across the plasma membrane and release from intracellular stores as well as activation of Ca²⁺ pumps and inhibition of passive Ca²⁺ pathways to ensure exact regulation of free cytosolic Ca²⁺ concentration ([Ca²⁺] _i ). A431 cells loaded with fura-2 cells were used as a model system to examine regulation of Ca²⁺ entry and intracellular release. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-) both stimulated Ca²⁺ entry and release while bradykinin appeared only to release Ca²⁺ from intracellular stores. The possible role of protein kinase C (PKC) in modulating the [Ca²⁺] _i response to these agonists was examined by four methods. Low concentrations of TPA (2×10^–10 m) had no effect on Ca²⁺ release due to EGF, TGR- or bradykinin but resulted in a rapid return of [Ca²⁺] _i to baseline levels for EGF or TGF-. Addition of the PKC inhibitor staurosporine (1 and 10nm)_completely inhibited the action of TPA on EGF-induced [Ca²⁺] _i changes. An inhibitor of diglyceride kinase (R59022) mimicked the action of TPA. Down-regulation of PKC by overnight incubation with 0.1 or 1 m TPA produced the converse effect, namely prolonged Ca²⁺ entry following stimulation with EGF or TGF-. To show that one effect of TPA was on Ca²⁺ entry, fura-2 loaded cells were suspended in Mn²⁺ rather than Ca²⁺ buffers. Addition of EGF or TGF- resulted in Ca²⁺ release and Mn²⁺ entry. TPA but not the inactive phorbol ester, 4--phorbol-12,13-didecanoate, inhibited the Mn²⁺ influx. Thus, PKC is able to regulate Ca²⁺ entry due to EGF or TGF- in this cell type. A431 cells treated with higher concentrations of TPA (5×10^–8 m) inhibited not only Ca²⁺ entry but also Ca²⁺ release due to EGF/TGF- but had no effect on bradykinin-mediated Ca²⁺ release, suggesting differences in the regulation of the intracellular stores responsive to these two classes of agonists. Furthermore, sequential addition of EGF or TGF- gave a single transient of [Ca²⁺] _i , showing a common pool of Ca²⁺ for these agonists. In contrast, sequential addition of EGF (or TGF-) and bradykinin resulted in two [Ca²⁺] _i transients equal in size to those obtained with a single agonist. Ionomycin alone was able to fully deplete intracellular Ca²⁺ stores, whereas ionomycin following either EGF (or TGF-) or bradykinin gave an elevation of the [Ca²⁺] _i signal equal to that of the second agonist. These data indicate that there are separate pools of intracellular Ca²⁺ for EGF-mediated Ca²⁺ release which also respond differently to TPA.     

Ca2+ Modulation of sarcoplasmic reticulum Ca2+ release in rat skeletal muscle fibers

Ca²⁺ transients and the rate of Ca²⁺ release (dCa_REL/dt) from the sarcoplasmic reticulum (SR) in voltage-clamped, fast-twitch skeletal muscle fibers from the rat were studied with the double Vaseline gap technique and using mag-fura-2 and fura-2 as Ca²⁺ indicators. Single pulse experiments with different returning potentials showed that Ca²⁺ removal from the myoplasm is voltage independent. Thus, the myoplasmic Ca²⁺ removal (dCa_REM/dt) was studied by fitting the decaying phase of the Ca²⁺ transient (Melzer, Ríos & Schneider, 1986) and dCa_REL/dt was calculated as the difference between dCa/dt and dCa_REM/dt. The fast Ca²⁺ release decayed as a consequence of Ca²⁺ inactivation of Ca²⁺ release. Double pulse experiments showed inactivation of the fast Ca²⁺ release depending on the prepulse duration. At constant interpulse interval, long prepulses (200 msec) induced greater inactivation of the fast Ca²⁺ release than shorter depolarizations (20 msec). The correlation (r) between the myoplasmic [Ca²⁺]_i and the inhibited amount of Ca²⁺ release was 0.98. The [Ca²⁺]_i for 50% inactivation of dCa_REL/dt was 0.25 m, and the minimum number of sites occupied by Ca²⁺ to inactivate the Ca²⁺ release channel was 3.0. These data support Ca²⁺ binding and inactivation of SR Ca²⁺ release.This work was supported by Grant-in-Aid from the American Heart Association (National) and Muscular Dystrophy Association (USA). Part of this work was developed in Dr. Stefani's laboratory at Baylor College of Medicine.     

Oscillations of cytosolic free calcium in bombesin-stimulated HIT-T15 cells

The mechanism underlying the generation of cytosolic free Ca²⁺ ([Ca²⁺_i) oscillations by bombesin, a receptor agonist activating phospholipase C, in insulin secreting HIT-T15 cells was investigated. At 25 μM, 61% of cells displayed [Ca²⁺]_i oscillations with variable patterns. The bombesin-induced [Ca²⁺]_i oscillations could last more than 1 h and glucose was required for maintaining these [Ca²⁺ fluctuations. Bombesin-evoked [Ca²⁺]_i oscillations were dependent on extracellular Ca²⁺ entry and were attenuated by membrane hype rpolarization or by L-type Ca²⁺ channel blockers. These [Ca²⁺]_i oscillations were apparently not associated with fluctuations in plasma membrane Ca²⁺ permeability as monitored by the Mn²⁺ quenching technique. 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) and 4-chloro-m-cresol, which interfere with intracellular Ca²⁺ stores, respectively, by inhibiting Ca²⁺-ATPase of endoplasmic reticulum and by affecting Ca²⁺-induced Ca²⁺ release, disrupted bombesin-induced [Ca²⁺]_i oscillations. 4-chloro-m-resol raised [Ca²⁺]_i by mobilizing an intracellular Ca²⁺ pool, an effect not altered by ryanodine. Caffeine exerted complex actions on [Ca²⁺]_i It raised [Ca²⁺]_i by promoting Ca²⁺ entry while inhibiting bombesin-elicited [Ca²⁺]_i oscillations. Our results suggest that in bombesin-elicited [Ca²⁺]_i oscillations in HIT-T15 cells: (i) the oscillations originate primarily from intracellular Ca²⁺ stores; and (ii) the Ca²⁺ influx required for maintaining the oscillations is in part membrane potential-sensitive and not coordinated with [Ca²⁺]_i oscillations. The interplay between intracellular Ca²⁺ stores and voltage-sensitive and voltage-insensitive extracellular Ca²⁺ entry determines the [Ca²⁺]_i oscillations evoked by bombesin.     

RYR2 proteins contribute to the formation of Ca(2+) sparks in smooth muscle

Calcium release through ryanodine receptors (RYR) activates calcium-dependent membrane conductances and plays an important role in excitation-contraction coupling in smooth muscle. The specific RYR isoforms associated with this release in smooth muscle, and the role of RYR-associated proteins such as FK506 binding proteins (FKBPs), has not been clearly established, however. FKBP12.6 proteins interact with RYR2 Ca(2+) release channels and the absence of these proteins predictably alters the amplitude and kinetics of RYR2 unitary Ca(2+) release events (Ca(2+) sparks). To evaluate the role of specific RYR2 and FBKP12.6 proteins in Ca(2+) release processes in smooth muscle, we compared spontaneous transient outward currents (STOCs), Ca(2+) sparks, Ca(2+)-induced Ca(2+) release, and Ca(2+) waves in smooth muscle cells freshly isolated from wild-type, FKBP12.6(-/-), and RYR3(-/-) mouse bladders. Consistent with a role of FKBP12.6 and RYR2 proteins in spontaneous Ca(2+) sparks, we show that the frequency, amplitude, and kinetics of spontaneous, transient outward currents (STOCs) and spontaneous Ca(2+) sparks are altered in FKBP12.6 deficient myocytes relative to wild-type and RYR3 null cells, which were not significantly different from each other. Ca(2+) -induced Ca(2+) release was similarly augmented in FKBP12.6(-/-), but not in RYR3 null cells relative to wild-type. Finally, Ca(2+) wave speed evoked by CICR was not different in RYR3 cells relative to control, indicating that these proteins are not necessary for normal Ca(2+) wave propagation. The effect of FKBP12.6 deletion on the frequency, amplitude, and kinetics of spontaneous and evoked Ca(2+) sparks in smooth muscle, and the finding of normal Ca(2+) sparks and CICR in RYR3 null mice, indicate that Ca(2+) release through RYR2 molecules contributes to the formation of spontaneous and evoked Ca(2+) sparks, and associated STOCs, in smooth muscle.