Integration of rapid cytosolic Ca2+ signals by mitochondria in cat ventricular myocytes

Decoding of fast cytosolic Ca²⁺ concentration ([Ca²⁺]_i) transients by mitochondria was studied in permeabilized cat ventricular myocytes. Mitochondrial [Ca²⁺] ([Ca²⁺]_m) was measured with fluo-3 trapped inside mitochondria after removal of cytosolic indicator by plasma membrane permeabilization with digitonin. Elevation of extramitochondrial [Ca²⁺] ([Ca²⁺]_em) to >0.5 µM resulted in a [Ca²⁺]_em-dependent increase in the rate of mitochondrial Ca²⁺ accumulation ([Ca²⁺]_em resulting in half-maximal rate of Ca²⁺ accumulation = 4.4 µM) via Ca²⁺ uniporter. Ca²⁺ uptake was sensitive to the Ca²⁺ uniporter blocker ruthenium red and the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and depended on inorganic phosphate concentration. The rates of [Ca²⁺]_m increase and recovery were dependent on the extramitochondrial [Na⁺] ([Na⁺]_em) due to Ca²⁺ extrusion via mitochondrial Na⁺/Ca²⁺ exchanger. The maximal rate of Ca²⁺ extrusion was observed with [Na⁺]_em in the range of 20–40 mM. Rapid switching (0.25–1 Hz) of [Ca²⁺]_em between 0 and 100 µM simulated rapid beat-to-beat changes in [Ca²⁺]_i (with [Ca²⁺]_i transient duration of 100–500 ms). No [Ca²⁺]_m oscillations were observed, either under conditions of maximal rate of Ca²⁺ uptake (100 µM [Ca²⁺]_em, 0 [Na⁺]_em) or with maximal rate of Ca²⁺ removal (0 [Ca²⁺]_em, 40 mM [Na⁺]_em). The slow frequency-dependent increase of [Ca²⁺]_m argues against a rapid transmission of Ca²⁺ signals between cytosol and mitochondria on a beat-to-beat basis in the heart. [Ca²⁺]_m changes elicited by continuous or pulsatile exposure to elevated [Ca²⁺]_em showed no difference in mitochondrial Ca²⁺ uptake. Thus in cardiac myocytes fast [Ca²⁺]_i transients are integrated by mitochondrial Ca²⁺ transport systems, resulting in a frequency-dependent net mitochondrial Ca²⁺ accumulation. mitochondrial Ca²⁺; excitation-contraction coupling; cardiomyocytes     

L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis

We investigatedthe role of intracellular calcium concentration([Ca²⁺]_i) in endothelin-1 (ET-1) production,the effects of potential vasospastic agents on[Ca²⁺]_i, and the presence of L-typevoltage-dependent Ca²⁺ channels in cerebral microvascularendothelial cells. Primary cultures of endothelial cells isolated frompiglet cerebral microvessels were used. Confluent cells were exposed toeither the thromboxane receptor agonist U-46619 (1 µM),5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 µM) alone or after pretreatment with the Ca²⁺-chelatingagent EDTA (100 mM), the L-type Ca²⁺ channel blockerverapamil (10 µM), or the antagonist of receptor-operated Ca²⁺ channel SKF-96365 HCl (10 µM) for 15 min. ET-1production increased from 1.2 (control) to 8.2 (U-46619), 4.9 (5-HT),or 3.9 (LPA) fmol/µg protein, respectively. Such elevated ET-1biosynthesis was attenuated by verapamil, EDTA, or SKF-96365 HCl. Toinvestigate the presence of L-type voltage-dependent Ca²⁺channels in endothelial cells, the [Ca²⁺]_isignal was determined fluorometrically by using fura 2-AM. Superfusionof confluent endothelial cells with U-46619, 5-HT, or LPA significantlyincreased [Ca²⁺]_i. Pretreatment ofendothelial cells with high K⁺ (60 mM) or nifedipine (4 µM) diminished increases in [Ca²⁺]_i inducedby the vasoactive agents. These results indicate that 1)elevated [Ca²⁺]_i signals are involved in ET-1biosynthesis induced by specific spasmogenic agents, 2) theincreases in [Ca²⁺]_i induced by thevasoactive agents tested involve receptor as well as L-typevoltage-dependent Ca²⁺ channels, and 3) primarycultures of cerebral microvascular endothelial cells express L-typevoltage-dependent Ca²⁺ channels.

     

Nitric oxide induces [Ca2+]i oscillations in pituitary GH3 cells: involvement of IDR and ERG K+ currents

The role of nitric oxide (NO) in the occurrence of intracellular Ca²⁺ concentration ([Ca²⁺]_i) oscillations in pituitary GH₃ cells was evaluated by studying the effect of increasing or decreasing endogenous NO synthesis with L-arginine and nitro-L-arginine methyl ester (L-NAME), respectively. When NO synthesis was blocked with L-NAME (1 mM) [Ca²⁺]_i, oscillations disappeared in 68% of spontaneously active cells, whereas 41% of the quiescent cells showed [Ca²⁺]_i oscillations in response to the NO synthase (NOS) substrate L-arginine (10 mM). This effect was reproduced by the NO donors NOC-18 and S-nitroso-N-acetylpenicillamine (SNAP). NOC-18 was ineffective in the presence of the L-type voltage-dependent Ca²⁺ channels (VDCC) blocker nimodipine (1 µM) or in Ca²⁺-free medium. Conversely, its effect was preserved when Ca²⁺ release from intracellular Ca²⁺ stores was inhibited either with the ryanodine-receptor blocker ryanodine (500 µM) or with the inositol 1,4,5-trisphosphate receptor blocker xestospongin C (3 µM). These results suggest that NO induces the appearance of [Ca²⁺]_i oscillations by determining Ca²⁺ influx. Patch-clamp experiments excluded that NO acted directly on VDCC but suggested that NO determined membrane depolarization because of the inhibition of voltage-gated K⁺ channels. NOC-18 and SNAP caused a decrease in the amplitude of slow-inactivating (I_DR) and ether-à-go-go-related gene (ERG) hyperpolarization-evoked, deactivating K⁺ currents. Similar results were obtained when GH₃ cells were treated with L-arginine. The present study suggests that in GH₃ cells, endogenous NO plays a permissive role for the occurrence of spontaneous [Ca²⁺]_i oscillations through an inhibitory effect on I_DR and on I_ERG. voltage-gated potassium channels; ether-à-go-go-related gene potassium channels; slow-inactivating outward currents; fast-inactivating outward currents     

Cellular Responses to Mechanical Stress: Invited Review: Effects of flow on vascular endothelial intracellular calcium signaling of rat aortas ex vivo

To study the effects of flow on in situendothelial intracellular calcium concentration([Ca²⁺]_i) signaling, rat aortic rings wereloaded with fura 2, mounted on a tissue flow chamber, and divided intocontrol and flow-pretreated groups. The latter was perfused with bufferat a shear stress of 50 dyns/cm² for 1 h. Endothelial[Ca²⁺]_i responses to ACh or shear stresseswere determined by ratio image analysis. Moreover, ACh-induced[Ca²⁺]_i elevation responses were measured ina calcium-free buffer, or in the presence of SKF-96365, to elucidatethe role of calcium influx in the flow effects. Our results showed that1) ACh increased endothelial[Ca²⁺]_i in a dose-dependent manner, and theseresponses were incremented by flow-pretreatment; 2) thedifferences in ACh-induced [Ca²⁺]_i elevationbetween control and flow-pretreated groups were abolished by SKF-96365or by Ca²⁺-free buffer; and 3) in the presenceof 10⁵ M ATP, shear stress induced dose-dependent[Ca²⁺]_i elevation responses that were notaltered by flow-pretreatment. In conclusion, flow-pretreatment augmentsthe ACh-induced endothelial calcium influx in rat aortas ex vivo.

     

Sustained membrane depolarization and pulmonary artery smooth muscle cell proliferation

Pulmonary vasoconstriction and vascularmedial hypertrophy greatly contribute to the elevated pulmonaryvascular resistance in patients with pulmonary hypertension. A rise incytosolic free Ca²⁺ ([Ca²⁺]_cyt)in pulmonary artery smooth muscle cells (PASMC) triggers vasoconstriction and stimulates cell growth. Membrane potential (E_m) regulates[Ca²⁺]_cyt by governing Ca²⁺influx through voltage-dependent Ca²⁺ channels. Thusintracellular Ca²⁺ may serve as a shared signaltransduction element that leads to pulmonary vasoconstriction andvascular remodeling. In PASMC, activity of voltage-gated K⁺(Kv) channels regulates resting E_m. In thisstudy, we investigated whether changes of Kv currents[I_K(V)], E_m, and[Ca²⁺]_cyt affect cell growth by comparingthese parameters in proliferating and growth-arrested PASMC. Serumdeprivation induced growth arrest of PASMC, whereas chelation ofextracellular Ca²⁺ abolished PASMC growth. Resting[Ca²⁺]_cyt was significantly higher, andresting E_m was more depolarized, inproliferating PASMC than in growth-arrested cells. Consistently, wholecell I_K(V) was significantly attenuated in PASMCduring proliferation. Furthermore, E_mdepolarization significantly increased resting[Ca²⁺]_cyt and augmented agonist-mediatedrises in [Ca²⁺]_cyt in the absence ofextracellular Ca²⁺. These results demonstrate that reducedI_K(V), depolarized E_m, and elevated [Ca²⁺]_cyt may play a criticalrole in stimulating PASMC proliferation. Pulmonary vascular medialhypertrophy in patients with pulmonary hypertension may be partlycaused by a membrane depolarization-mediated increase in[Ca²⁺]_cyt in PASMC.

     

VCAM-1-induced inwardly rectifying K(+) current enhances Ca(2+) entry in human THP-1 monocytes

Hyperpolarization in human leukemia THP-1 monocytes adherent tovascular cell adhesion molecule (VCAM)-1 is due to an induction ofinwardly rectifying K⁺ currents(I_ir) (Colden-Stanfield M and Gallin EK,Am J Physiol Cell Physiol 275: C267-C277, 1998).We determined whether the VCAM-1-induced hyperpolarization issufficient to augment the increase in intracellular free calcium([Ca²⁺]_i) produced by Ca²⁺ storedepletion with thapsigargin (TG) and readdition of external CaCl₂ in fura 2-loaded THP-1 monocytes. Whereas there was a2.1-fold increase in [Ca²⁺]_i in monocytesbound to glass for 5 h in response to TG and CaCl₂ addition, adherence to VCAM-1 produced a 5-fold increase in[Ca²⁺]_i. Depolarization of monocytes adherentto VCAM-1 by I_ir blockade or exposure to high[K⁺] abolished the enhancement of the peak[Ca²⁺]_i response. In monocytes bound toglass, hyperpolarization of the membrane potential with valinomycin, aK⁺ ionophore, to the level of hyperpolarization seen incells adherent to VCAM-1 produced similar changes in peak[Ca²⁺]_i. Adherence of monocytes to E-selectinproduced a similar peak [Ca²⁺]_i to cellsbound to glass. Thus monocyte adherence to the physiological substrateVCAM-1 produces a hyperpolarization that is sufficient to enhanceCa²⁺ entry and may impact Ca²⁺-dependentmonocyte function.

     

Regulation of KCa current by store-operated Ca2+ influx depends on internal Ca2+ release in HSG cells

This study examines theCa²⁺ influx-dependent regulationof the Ca²⁺-activatedK⁺ channel(K_Ca) in human submandibulargland (HSG) cells. Carbachol (CCh) induced sustained increases in theK_Ca current and cytosolic Ca²⁺ concentration([Ca²⁺]_i),which were prevented by loading cells with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Removal of extracellularCa²⁺ and addition ofLa³⁺ orGd³⁺, but notZn²⁺, inhibited the increases inK_Ca current and[Ca²⁺]_i.Ca²⁺ influx during refill (i.e.,addition of Ca²⁺ to cells treatedwith CCh and then atropine inCa²⁺-free medium) failed to evokeincreases in the K_Ca current but achieved internal Ca²⁺ storerefill. When refill was prevented by thapsigargin,Ca²⁺ readdition induced rapidactivation of K_Ca. These dataprovide further evidence that intracellularCa²⁺ accumulation provides tightbuffering of[Ca²⁺]_iat the site of Ca²⁺ influx (H. Mogami, K. Nakano, A. V. Tepikin, and O. H. Petersen. Cell 88: 49-55, 1997). We suggestthat the Ca²⁺ influx-dependentregulation of the sustained K_Cacurrent in CCh-stimulated HSG cells is mediated by the uptake ofCa²⁺ into the internalCa²⁺ store and release via theinositol 1,4,5-trisphosphate-sensitive channel.

     

Ca2+ sensitivity of BK channels in GH3 cells involves cytosolic phospholipase A2

To test thehypothesis that intracellular Ca²⁺activation of large-conductanceCa²⁺-activatedK⁺ (BK) channels involves thecytosolic form of phospholipase A₂ (cPLA₂), we first inhibited theexpression of cPLA₂ by treating GH₃ cells with antisenseoligonucleotides directed at the two possible translation start siteson cPLA₂. Western blot analysis and a biochemical assay of cPLA₂activity showed marked inhibition of the expression ofcPLA₂ in antisense-treated cells.We then examined the effects of intracellularCa²⁺ concentration([Ca²⁺]_i)on single BK channels from these cells. Open channel probability (P_o) for thecells exposed to cPLA₂ antisenseoligonucleotides in 0.1 µM intracellularCa²⁺ was significantly lower thanin untreated or sense oligonucleotide-treated cells, but the voltagesensitivity did not change (measured as the slope of theP_o-voltagerelationship). In fact, a 1,000-fold increase in[Ca²⁺]_ifrom 0.1 to 100 µM did not significantly increaseP_oin these cells, whereas BK channels from cells in the other treatmentgroups showed a normalP_o-[Ca²⁺]_iresponse. Finally, we examined the effect of exogenous arachidonic acidon theP_oof BK channels from antisense-treated cells. Although arachidonic aciddid significantly increaseP_o,it did so without restoring the[Ca²⁺]_isensitivity observed in untreated cells. We conclude that although [Ca²⁺]_idoes impart some basal activity to BK channels inGH₃ cells, the steepP_o-[Ca²⁺]_irelationship that is characteristic of these channels involves cPLA₂.

     

Histamine-mediated increases in cytosolic [Ca2+] involve different mechanisms in human pulmonary artery smooth muscle and endothelial cells

Agonist stimulation of human pulmonary artery smooth muscle cells (PASMC) and endothelial cells (PAEC) with histamine showed similar spatiotemporal patterns of Ca²⁺ release. Both sustained elevation and oscillatory patterns of changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) were observed in the absence of extracellular Ca²⁺. Capacitative Ca²⁺ entry (CCE) was induced in PASMC and PAEC by passive depletion of intracellular Ca²⁺ stores with 10 µM cyclopiazonic acid (CPA; 15–30 min). The pyrazole derivative BTP2 inhibited CPA-activated Ca²⁺ influx, suggesting that depletion of CPA-sensitive internal stores is sufficient to induce CCE in both PASMC and PAEC. The recourse of histamine-mediated Ca²⁺ release was examined after exposure of cells to CPA, thapsigargin, caffeine, ryanodine, FCCP, or bafilomycin. In PASMC bathed in Ca²⁺-free solution, treatment with CPA almost abolished histamine-induced rises in [Ca²⁺]_cyt. In PAEC bathed in Ca²⁺-free solution, however, treatment with CPA eliminated histamine-induced sustained and oscillatory rises in [Ca²⁺]_cyt but did not affect initial transient increase in [Ca²⁺]_cyt. Furthermore, treatment of PAEC with a combination of CPA (or thapsigargin) and caffeine (and ryanodine), FCCP, or bafilomycin did not abolish histamine-induced transient [Ca²⁺]_cyt increases. These observations indicate that 1) depletion of CPA-sensitive stores is sufficient to cause CCE in both PASMC and PAEC; 2) induction of CCE in PAEC does not require depletion of all internal Ca²⁺ stores; 3) the histamine-releasable internal stores in PASMC are mainly CPA-sensitive stores; 4) PAEC, in addition to a CPA-sensitive functional pool, contain other stores insensitive to CPA, thapsigargin, caffeine, ryanodine, FCCP, and bafilomycin; and 5) although the CPA-insensitive stores in PAEC may not contribute to CCE, they contribute to histamine-mediated Ca²⁺ release. intracellular calcium stores; oscillations; pulmonary hypertension     

Regulation of intracellular calcium in N1E-115 neuroblastoma cells: the role of Na(+)/Ca(2+) exchange

In fura 2-loaded N1E-115 cells, regulationof intracellular Ca²⁺ concentration([Ca²⁺]_i) following a Ca²⁺ loadinduced by 1 µM thapsigargin and 10 µM carbonylcyanidep-trifluoromethyoxyphenylhydrazone (FCCP) wasNa⁺ dependent and inhibited by 5 mM Ni²⁺. Incells with normal intracellular Na⁺ concentration([Na⁺]_i), removal of bath Na⁺,which should result in reversal of Na⁺/Ca²⁺exchange, did not increase [Ca²⁺]_i unlesscell Ca²⁺ buffer capacity was reduced. When N1E-115 cellswere Na⁺ loaded using 100 µM veratridine and 4 µg/mlscorpion venom, the rate of the reverse mode of theNa⁺/Ca²⁺ exchanger was apparently enhanced,since an ~4- to 6-fold increase in [Ca²⁺]_ioccurred despite normal cell Ca²⁺ buffering. In SBFI-loadedcells, we were able to demonstrate forward operation of theNa⁺/Ca²⁺ exchanger (net efflux ofCa²⁺) by observing increases (~ 6 mM) in[Na⁺]_i. These Ni²⁺ (5 mM)-inhibited increases in [Na⁺]_i could onlybe observed when a continuous ionomycin-induced influx ofCa²⁺ occurred. The voltage-sensitive dyebis-(1,3-diethylthiobarbituric acid) trimethine oxonol was used tomeasure changes in membrane potential. Ionomycin (1 µM) depolarizedN1E-115 cells (~25 mV). This depolarization was Na⁺dependent and blocked by 5 mM Ni²⁺ and 250-500 µMbenzamil. These data provide evidence for the presence of anelectrogenic Na⁺/Ca²⁺ exchanger that is capableof regulating [Ca²⁺]_i after release ofCa²⁺ from cell stores.

     

Polyunsaturated fatty acids mobilize intracellular Ca2+ in NT2 human teratocarcinoma cells by causing release of Ca2+ from mitochondria

In a variety of disorders, overaccumulation of lipid in nonadipose tissues, including the heart, skeletal muscle, kidney, and liver, is associated with deterioration of normal organ function, and is accompanied by excessive plasma and cellular levels of free fatty acids (FA). Increased concentrations of FA may lead to defects in mitochondrial function found in diverse diseases. One of the most important regulators of mitochondrial function is mitochondrial Ca²⁺ ([Ca²⁺]_m), which fluctuates in coordination with intracellular Ca²⁺ ([Ca²⁺]_i). Polyunsaturated FA (PUFA) have been shown to cause [Ca²⁺]_i mobilization albeit by unknown mechanisms. We have found that PUFA but not monounsaturated or saturated FA cause [Ca²⁺]_i mobilization in NT2 human teratocarcinoma cells. Unlike the [Ca²⁺]_i response to the muscarinic G protein-coupled receptor agonist carbachol, PUFA-mediated [Ca²⁺]_i mobilization in NT2 cells is independent of phospholipase C and inositol-1,4,5-trisphospate (IP₃) receptor activation, as well as IP₃-sensitive internal Ca²⁺ stores. Furthermore, PUFA-mediated [Ca²⁺]_i mobilization is inhibited by the mitochondria uncoupler carboxyl cyanide m-chlorophenylhydrozone. Direct measurements of [Ca²⁺]_m with X-rhod-1 and ⁴⁵Ca²⁺ indicate that PUFA induce Ca²⁺ efflux from mitochondria. Further studies show that ruthenium red, an inhibitor of the mitochondrial Ca²⁺ uniporter, blocks PUFA-induced Ca²⁺ efflux from mitochondria, whereas inhibitors of the mitochondrial permeability transition pore cyclosporin A and bongkrekic acid have no effect. Thus PUFA-gated Ca²⁺ release from mitochondria, possibly via the Ca²⁺ uniporter, appears to be the underlying mechanism for PUFA-induced [Ca²⁺]_i mobilization in NT2 cells. arachidonic acid; mitochondrial Ca²⁺ uniporter; G protein-coupled receptor; IP₃ receptor     

Role of K(+) channel expression in polyamine-dependent intestinal epithelial cell migration

Polyamines are essential for cell migrationduring early mucosal restitution after wounding in the gastrointestinaltract. Activity of voltage-gated K⁺ channels (Kv) controlsmembrane potential (E_m) that regulates cytoplasmicfree Ca²⁺ concentration([Ca²⁺]_cyt) by governing thedriving force for Ca²⁺ influx. This study determinedwhether polyamines are required for the stimulation of cell migrationby altering K⁺ channel gene expression,E_m, and[Ca²⁺]_cyt in intestinal epithelialcells (IEC-6). The specific inhibitor of polyamine synthesis,-difluoromethylornithine (DFMO, 5 mM), depleted cellularpolyamines (putrescine, spermidine, and spermine), selectivelyinhibited Kv1.1 channel (a delayed-rectifier Kv channel) expression,and resulted in membrane depolarization. Because IEC-6 cells did notexpress voltage-gated Ca²⁺ channels, the depolarizedE_m in DFMO-treated cells decreased [Ca²⁺]_cyt as a result of reduceddriving force for Ca²⁺ influx through capacitativeCa²⁺ entry. Migration was reduced by 80% in thepolyamine-deficient cells. Exogenous spermidine not only reversed theeffects of DFMO on Kv1.1 channel expression, E_m,and [Ca²⁺]_cyt but also restoredcell migration to normal. Removal of extracellular Ca²⁺ orblockade of Kv channels (by 4-aminopyridine, 1-5 mM) significantly inhibited normal cell migration and prevented the restoration of cellmigration by exogenous spermidine in polyamine-deficient cells. Theseresults suggest that polyamine-dependent intestinal epithelial cellmigration may be due partially to an increase of Kv1.1 channelexpression. The subsequent membrane hyperpolarization raises[Ca²⁺]_cyt by increasing the drivingforce (the electrochemical gradient) for Ca²⁺ influx andthus stimulates cell migration.

     

Role of extracellular [Ca2+] in fatigue of isolated mammalian skeletal muscle

The possiblerole of altered extracellular Ca²⁺concentration([Ca²⁺]_o)in skeletal muscle fatigue was tested on isolated slow-twitch soleusand fast-twitch extensor digitorum longus muscles of the mouse. Thefollowing findings were made. 1) Achange from the control solution (1.3 mM[Ca²⁺]_o)to 10 mM[Ca²⁺]_o,or to nominally Ca²⁺-freesolutions, had little effect on tetanic force in nonfatigued muscle.2) Almost complete restoration oftetanic force was induced by 10 mM[Ca²⁺]_oin severely K⁺-depressed muscle(extracellular K⁺ concentration of10-12 mM). This effect was attributed to a 5-mV reversal of theK⁺-induced depolarization andsubsequent restoration of ability to generate action potentials(inferred by using the twitch force-stimulation strength relationship).3) Tetanic force depressed bylowered extracellular Na⁺concentration (40 mM) was further reduced with 10 mM[Ca²⁺]_o.4) Tetanic force loss at elevatedextracellular K⁺ concentration (8 mM) and lowered extracellular Na⁺concentration (100 mM) was partially reversed with 10 mM[Ca²⁺]_oor markedly exacerbated with low[Ca²⁺]_o.5) Fatigue induced by using repeatedtetani in soleus was attenuated at 10 mM[Ca²⁺]_o(due to increased resting and evoked forces) and exacerbated at low[Ca²⁺]_o.These combined results suggest, first, that raised[Ca²⁺]_oprotects against fatigue rather than inducing it and, second, that aconsiderable depletion of[Ca²⁺]_oin the transverse tubules may contribute to fatigue.

     

Elevated resting [Ca(2+)](i) in myotubes expressing malignant hyperthermia RyR1 cDNAs is partially restored by modulation of passive calcium leak from the SR

Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle triggered in susceptible individuals by inhalation anesthetics and depolarizing skeletal muscle relaxants. This syndrome has been linked to a missense mutation in the type 1 ryanodine receptor (RyR1) in more than 50% of cases studied to date. Using double-barreled Ca²⁺ microelectrodes in myotubes expressing wild-type RyR1 (_WTRyR1) or RyR1 with one of four common MH mutations (_MHRyR1), we measured resting intracellular Ca²⁺ concentration ([Ca²⁺]_i). Changes in resting [Ca²⁺]_i produced by several drugs known to modulate the RyR1 channel complex were investigated. We found that myotubes expressing any of the _MHRyR1s had a 2.0- to 3.7-fold higher resting [Ca²⁺]_i than those expressing _WTRyR1. Exposure of myotubes expressing _MHRyR1s to ryanodine (500 µM) or (2,6-dichloro-4-aminophenyl)isopropylamine (FLA 365; 20 µM) had no effects on their resting [Ca²⁺]_i. However, when myotubes were exposed to bastadin 5 alone or to a combination of ryanodine and bastadin 5, the resting [Ca²⁺]_i was significantly reduced (P < 0.01). Interestingly, the percent decrease in resting [Ca²⁺]_i in myotubes expressing _MHRyR1s was significantly greater than that for _WTRyR1. From these data, we propose that the high resting myoplasmic [Ca²⁺]_i in _MHRyR1 expressing myotubes is due in part to a related structural conformation of _MHRyR1s that favors "passive" calcium leak from the sarcoplasmic reticulum. ryanodine; FLA 365; bastadin 5; resting intracellular calcium concentration; sarcoplasmic reticulum     

Changes in intracellular Ca2+ concentration induced by L-type Ca2+ channel current in guinea pig gastric myocytes

We investigatedthe relationship between voltage-operatedCa²⁺ channel current and thecorresponding intracellular Ca²⁺concentration([Ca²⁺]_i)change (Ca²⁺ transient) in guineapig gastric myocytes. Fluorescence microspectroscopy was combined withconventional whole cell patch-clamp technique, and fura 2 (80 µM) wasadded to CsCl-rich pipette solution. Step depolarization to 0 mVinduced inward Ca²⁺ current(I_Ca) andconcomitantly raised[Ca²⁺]_i.Both responses were suppressed by nicardipine, an L-typeCa²⁺ channel blocker, and thevoltage dependence of Ca²⁺transient was similar to the current-voltage relation ofI_Ca. When pulseduration was increased by up to 900 ms, peakCa²⁺ transient increased andreached a steady state when stimulation was for longer. The calculatedfast Ca²⁺ buffering capacity(B value), determined as the ratio ofthe time integral ofI_Ca divided bythe amplitude of Ca²⁺ transient,was not significantly increased after depletion of Ca²⁺ stores by the cyclicapplication of caffeine (10 mM) in the presence of ryanodine (4 µM).The addition of cyclopiazonic acid (CPA, 10 µM), a sarco(endo)plasmicreticulum Ca²⁺-ATPase inhibitor,decreased B value by ~20% in areversible manner. When KCl pipette solution was used,Ca²⁺-activatedK⁺ current[I_K(Ca)]was also recorded during step depolarization. CPA sensitivelysuppressed the initial peak and oscillations of I_K(Ca) withirregular effects on Ca²⁺transients. The above results suggest that, in guinea pig gastric myocyte, Ca²⁺ transient is tightlycoupled to I_Caduring depolarization, and global[Ca²⁺]_iis not significantly affected byCa²⁺-inducedCa²⁺ release from sarcoplasmicreticulum during depolarization.

     

Effects of ATP, Mg2+, and redox agents on the Ca2+ dependence of RyR channels from rat brain cortex

Despite their relevance for neuronal Ca²⁺-induced Ca²⁺ release (CICR), activation by Ca²⁺ of ryanodine receptor (RyR) channels of brain endoplasmic reticulum at the [ATP], [Mg²⁺], and redox conditions present in neurons has not been reported. Here, we studied the effects of varying cis-(cytoplasmic) free ATP concentration ([ATP]), [Mg²⁺], and RyR redox state on the Ca²⁺ dependence of endoplasmic reticulum RyR channels from rat brain cortex. At pCa 4.9 and 0.5 mM adenylylimidodiphosphate (AMP-PNP), increasing free [Mg²⁺] up to 1 mM inhibited vesicular [³H]ryanodine binding; incubation with thimerosal or dithiothreitol decreased or enhanced Mg²⁺ inhibition, respectively. Single RyR channels incorporated into lipid bilayers displayed three different Ca²⁺ dependencies, defined by low, moderate, or high maximal fractional open time (P_o), that depend on RyR redox state, as we have previously reported. In all cases, cis-ATP addition (3 mM) decreased threshold [Ca²⁺] for activation, increased maximal P_o, and shifted channel inhibition to higher [Ca²⁺]. Conversely, at pCa 4.5 and 3 mM ATP, increasing cis-[Mg²⁺] up to 1 mM inhibited low activity channels more than moderate activity channels but barely modified high activity channels. Addition of 0.5 mM free [ATP] plus 0.8 mM free [Mg²⁺] induced a right shift in Ca²⁺ dependence for all channels so that [Ca²⁺] <30 µM activated only high activity channels. These results strongly suggest that channel redox state determines RyR activation by Ca²⁺ at physiological [ATP] and [Mg²⁺]. If RyR behave similarly in living neurons, cellular redox state should affect RyR-mediated CICR. Ca²⁺-induced Ca²⁺ release; Ca²⁺ release channels; endoplasmic reticulum; thimerosal; 2,4-dithiothreitol; ryanodine receptor     

Regulation of exocytosis by purinergic receptors in pancreatic duct epithelial cells

In epithelial cells, several intracellular signals regulate the secretion of large molecules such as mucin via exocytosis and the transport of ions through channels and transporters. Using carbon fiber amperometry, we previously reported that exocytosis of secretory granules in dog pancreatic duct epithelial cells (PDEC) can be stimulated by pharmacological activation of cAMP-dependent protein kinase (PKA) or protein kinase C (PKC), as well as by an increase of intracellular free Ca²⁺ concentration ([Ca²⁺]_i). In this study, we examined whether exocytosis in these cells is modulated by activation of endogenous P2Y receptors, which increase cAMP and [Ca²⁺]_i. Low concentrations of ATP (<10 µM) induced intracellular Ca²⁺ oscillation but no significant exocytosis. In contrast, 100 µM ATP induced a sustained [Ca²⁺]_i rise and increased the exocytosis rate sevenfold. The contribution of Ca²⁺ or cAMP pathways to exocytosis was tested by using the Ca²⁺ chelator BAPTA or the PKA inhibitors H-89 or Rp-8-bromoadenosine 3',5'-cyclic monophosphorothioate. Removal of [Ca²⁺]_i rise or inhibition of PKA each partially reduced exocytosis; when combined, they abolished exocytosis. In conclusion, ATP at concentrations >10 µM stimulates exocytosis from PDEC through both Ca²⁺ and cAMP pathways. secretion; amperometry; photometry; calcium, adenosine 3',5'-cyclic monophosphate     

Injection of a Ca2+-Chelating Agent into the Cytoplasm Retards the Progress of Turgor Regulation upon Hypotonic Treatment in the Alga, Lamprothamnium

The turgor regulation induced by hypotonic treatment (hypotonicturgor regulation) in the brackish-water alga Lamprothamniumsuccinctum is accompanied by a transient increase in the electricalconductance of the membrane, membrane depolarization and a transientincrease in the cytoplasmic concentration of free Ca²⁺ ([Ca²⁺([Ca²⁺]_c) (Okazaki and Tazawa 1990). In the present study, weloaded a Ca²⁺-chelating agent, EGTA, into the cytoplasm by mechanicalinjection in order to suppress the increase in [Ca²⁺]_c thatoccurs during the hypotonic turgor regulation. The rate of thecytoplasmic streaming was taken as an indirect indicator of[Ca²⁺]_c, since cytoplasmic streaming has been shown to be inhibitedby high [Ca²⁺]_c in Lamprothamnium cells. The lag time for theinhibition of the cytoplasmic streaming upon hypotonic treatmentwas significantly prolonged in EGTA-loaded cells as comparedto that in intact cells. This result indicates that the loadedcytoplasmic EGTA functioned as a buffer of Ca²⁺ to retard theincrease in [Ca²⁺]_c. It took a longer time for the membraneconductance to reach the peak value in EGTA-loaded cells thanin intact cells. Membrane depolarization was affected to aninsignificant extent by the cytoplasmic EGTA. The regulationof turgor pressure itself was partially inhibited. These resultsstrongly support the idea that the net efflux of ions that occursduring the recovery from hy-potonically induced changes in turgorpressure is controlled by [Ca²⁺]_c. (Received August 22, 1990; Accepted December 6, 1990)     

Increase in cytosolic Ca2+ induced by elevation of extracellular Ca2+ in skeletal myogenic cells

Cytoplasmic Ca²⁺concentration ([Ca²⁺]_i) variation is akey event in myoblast differentiation, but the mechanism by which itoccurs is still debated. Here we show that increases of extracellular Ca²⁺ concentration ([Ca²⁺]_o)produced membrane hyperpolarization and a concentration-dependent increase of [Ca²⁺]_i due to Ca²⁺influx across the plasma membrane. Responses were not related toinositol phosphate turnover and Ca²⁺-sensing receptor.[Ca²⁺]_o-induced[Ca²⁺]_i increase was inhibited byCa²⁺ channel inhibitors and appeared to be modulated byseveral kinase activities. [Ca²⁺]_i increasewas potentiated by depletion of intracellular Ca²⁺ storesand depressed by inactivation of the Na⁺/Ca²⁺exchanger. The response to arginine vasopressin (AVP), which inducesinositol 1,4,5-trisphosphate-dependent[Ca²⁺]_i increase in L6-C5 cells, was notmodified by high [Ca²⁺]_o. On the contrary,AVP potentiated the [Ca²⁺]_i increase in thepresence of elevated [Ca²⁺]_o. Other clones ofthe L6 line as well as the rhabdomyosarcoma RD cell line and thesatellite cell-derived C2-C12 line expressed similar responses to high[Ca²⁺]_o, and the amplitude of the responseswas correlated with the myogenic potential of the cells.

     

Activation of K(+) channels and increased migration of differentiated intestinal epithelial cells after wounding

Earlymucosal restitution occurs by epithelial cell migration to resealsuperficial wounds after injury. Differentiated intestinal epithelialcells induced by forced expression of the Cdx2 gene migrateover the wounded edge much faster than undifferentiated parental cellsin an in vitro model. This study determined whether thesedifferentiated intestinal epithelial cells exhibit increased migrationby altering voltage-gated K⁺ (Kv) channel expression andcytosolic free Ca²⁺ concentration([Ca²⁺]_cyt). StableCdx2-transfected IEC-6 cells (IEC-Cdx2L1) with highly differentiated phenotype expressed higher basal levels of Kv1.1 andKv1.5 mRNAs and proteins than parental IEC-6 cells. Neither IEC-Cdx2L1cells nor parental IEC-6 cells expressed voltage-dependent Ca²⁺ channels. The increased expression of Kv channels indifferentiated IEC-Cdx2L1 cells was associated with an increase inwhole cell K⁺ currents, membrane hyperpolarization, and arise in [Ca²⁺]_cyt. The migration rates indifferentiated IEC-Cdx2L1 cells were about four times those of parentalIEC-6 cells. Inhibition of Kv channel expression by polyamine depletiondecreased [Ca²⁺]_cyt, reduced myosin stressfibers, and inhibited cell migration. Elevation of[Ca²⁺]_cyt by ionomycin promoted myosin IIstress fiber formation and increased cell migration. These resultssuggest that increased migration of differentiated intestinalepithelial cells is mediated, at least partially, by increasing Kvchannel activity and Ca²⁺ influx during restitution.