Further study on the role of HSP70 on Ca2+ homeostasis in rat ventricular myocytes subjected to simulated ischemia

We hypothesized that activation of heat shock protein 70 (HSP70) by preconditioning, which is known to confer delayed cardioprotection, attenuates the impaired handling of Ca²⁺ at multiple sites. To test the hypothesis, we determined how the ryanodine receptor (RyR), sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA), and Na⁺/Ca²⁺ exchanger (NCX) handled Ca²⁺ in rat ventricular myocytes preconditioned with a -opioid receptor agonist, U50488H (UP), followed by blockade of HSP70 with a selective antisense oligonucleotide and subsequently subjected to simulated ischemia. We determined the following: 1) the Ca²⁺ transients induced by electrical stimulation and caffeine, which provide the overall picture of Ca²⁺ homeostasis; 2) expression of RyR, SERCA, and NCX; and 3) Ca²⁺ fluxes via NCX by the use of ⁴⁵Ca²⁺ in the rat ventricular myocyte. We found that UP increased the activity of RyR, SERCA, and NCX and the expression of RyR and SERCA. These effects led to increases in the release of Ca²⁺ from the sarcoplasmic reticulum via RyR and in the removal of Ca²⁺ from the cytoplasm by reuptake of Ca²⁺ to the SR via SERCA and by extrusion of Ca²⁺ out of the cell via NCX. UP also reduced mitochondrial Ca²⁺ accumulation. All of the effects of UP were either abolished or significantly attenuated by blockade of HSP70 synthesis with a selective antisense oligonucleotide. The results are evidence that activation of HSP70 by preconditioning improves the ischemia-impaired Ca²⁺ homeostasis at multiple sites in the heart, which may be responsible, at least partly, for attenuated Ca²⁺ overload, improved recovery in contractile function, and cardioprotection. intracellular Ca²⁺, -opioid receptor; Na⁺/Ca²⁺ exchanger; ryanodine receptor; sarco(endo)plasmic reticulum Ca²⁺-ATPase     

Chronic intermittent hypoxia alters Ca2+ handling in rat cardiomyocytes by augmented Na+/Ca2+ exchange and ryanodine receptor activities in ischemia-reperfusion

This study examined Ca²⁺ handling mechanisms involved in cardioprotection induced by chronic intermittent hypoxia (CIH) against ischemia-reperfusion (I/R) injury. Adult male Sprague-Dawley rats were exposed to 10% inspired O₂ continuously for 6 h daily from 3, 7, and 14 days. In isolated perfused hearts subjected to I/R, CIH-induced cardioprotection was most significant in the 7-day group with less infarct size and lactate dehydrogenase release, compared with the normoxic group. The I/R-induced alterations in diastolic Ca²⁺ level, amplitude, time-to-peak, and the decay time of both electrically and caffeine-induced Ca²⁺ transients measured by spectrofluorometry in isolated ventricular myocytes of the 7-day CIH group were less than that of the normoxic group, suggesting an involvement of altered Ca²⁺ handling of the sarcoplasmic reticulum (SR) and sarcolemma. We further determined the protein expression and activity of ⁴⁵Ca²⁺ flux of SR-Ca²⁺-ATPase, ryanodine receptor (RyR) and sarcolemmal Na⁺/Ca²⁺ exchange (NCX) in ventricular myocytes from the CIH and normoxic groups before and during I/R. There were no changes in expression levels of the Ca²⁺-handling proteins but significant increases in the RyR and NCX activities were remarkable during I/R in the CIH but not the normoxic group. The augmented RyR and NCX activities were abolished, respectively, by PKA inhibitor (0.5 µM KT5720 or 0.5 µM PKI_14-22) and PKC inhibitor (5 µM chelerythrine chloride or 0.2 µM calphostin C) but not by Ca²⁺/calmodulin-dependent protein kinase II inhibitor KN-93 (1 µM). Thus, CIH confers cardioprotection against I/R injury in rat cardiomyocytes by altered Ca²⁺ handling with augmented RyR and NCX activities via protein kinase activation. cardioprotection; intracellular calcium     

Adenylyl cyclase expression and modulation of cAMP in rat taste cells

The present study determined Ca²⁺ handling in the hearts of rats subjected to chronic hypoxia (CH). Spectrofluorometry was used to measure intracellular Ca²⁺ concentration ([Ca²⁺]_i) and its responses to electrical stimulation, caffeine, and isoproterenol in myocytes from the right ventricle of rats breathing 10% oxygen for 1, 3, 7, 14, 21, 28, and 56 days and age-matched controls. The protein expression of sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) and its ryanodine receptor (RyR) were measured. The uptake of ⁴⁵Ca²⁺ by SERCA, release by RyR, and extrusion by Na⁺/Ca²⁺ exchange (NCX) were determined. It was found that Ca²⁺ homeostasis and Ca²⁺ responses to -adrenoceptor stimulation reached a new equilibrium after 4 wk of CH. Ca²⁺ content in the sarcoplasmic reticulum (SR) was reduced, but cytosolic Ca²⁺ remained unchanged after CH. Expression of SERCA and its Ca²⁺ uptake, Ca²⁺ release via RyR, and NCX activity were suppressed by CH. The results indicate impaired Ca²⁺ handling, which may be responsible for the attenuated Ca²⁺ responses to -adrenoceptor stimulation in CH. intracellular calcium ion concentration; calcium-adenosinetriphosphatase; ryanodine receptor; sodium/calcium exchange; sarcoplasmic reticulum; -adrenoceptor; chronic hypoxia     

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     

Role of plasma membrane Ca2+-ATPase in contraction-relaxation processes of the bladder: evidence from PMCA gene-ablated mice

We investigated the roles and relationships of plasma membrane Ca²⁺-ATPase (PMCA), sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA)2, and Na⁺/Ca²⁺ exchanger (NCX) in bladder smooth muscle contractility in Pmca-ablated mice: Pmca4-null mutant (Pmca4^–/–) and heterozygous Pmca1 and homozygous Pmca4 double gene-targeted (Pmca1^+/–Pmca4^–/–) mice. Gene manipulation did not alter the amounts of PMCA1, SERCA2, and NCX. To study the role of each Ca²⁺ transport system, contraction of circular ring preparations was elicited with KCl (80 mM) plus atropine, and then the muscle was relaxed with Ca²⁺-free physiological salt solution containing EGTA. We measured the contributions of Ca²⁺ clearance components by inhibiting SERCA2 (with 10 µM cyclopiazonic acid) and/or NCX (by replacing NaCl with N-methyl-D-glucamine/HCl plus 10 µM KB-R7943). Contraction half-time (time to 50% of maximum tension) was prolonged in the gene-targeted muscles but marginally shortened when SERCA2 or NCX was inhibited. The inhibition of NCX significantly inhibited this prolongation, suggesting that NCX activity might be augmented to compensate for PMCA4 function in the gene-targeted muscles under nonstimulated conditions. Inhibition of SERCA2 and NCX as well as gene targeting all prolonged the relaxation half-time. The contribution of PMCA to relaxation was calculated to be 25–30%, with that of SERCA2 being 20% and that of NCX being 70%. PMCA and SERCA2 appeared to function additively, but the function of NCX might overlap with those of other components. In summary, gene manipulation of PMCA indicates that PMCA, in addition to SERCA2 and NCX, plays a significant role in both excitation-contraction coupling and the Ca²⁺ extrusion-relaxation relationship, i.e., Ca²⁺ homeostasis, of bladder smooth muscle. ATP2B; sarco(endo)plasmic reticulum Ca²⁺-ATPase 2; Na⁺/Ca²⁺ exchanger; homeostasis     

Determinants of cardiac Na(+)/Ca(2+) exchanger temperature dependence: NH(2)-terminal transmembrane segments

The cardiacNa⁺/Ca²⁺ exchanger (NCX) in troutexhibits profoundly lower temperature sensitivity in comparison to themammalian NCX. In this study, we attempt to characterize the regions of the NCX molecule that are responsible for its temperature sensitivity. Chimeric NCX molecules were constructed using wild-type trout andcanine NCX cDNA and expressed in Xenopus oocytes.NCX-mediated currents were measured at 7, 14, and 30°C using thegiant excised-patch technique. By using this approach, the differentialtemperature dependence of NCX was found to reside within theNH₂-terminal region of the molecule. Specifically, we foundthat ~75% of the Na⁺/Ca²⁺ exchangedifferential energy of activation is attributable to sequencedifferences in the region that include the first four transmembranesegments, and the remainder is attributable to transmembrane segmentfive and the exchanger inhibitory peptide site.

     

Mechanism of shortened action potential duration in Na+-Ca2+ exchanger knockout mice

In cardiac-specific Na⁺-Ca²⁺ exchanger (NCX) knockout (KO) mice, the ventricular action potential (AP) is shortened. The shortening of the AP, as well as a decrease of the L-type Ca²⁺ current (I_Ca), provides a critical mechanism for the maintenance of Ca²⁺ homeostasis and contractility in the absence of NCX (Pott C, Philipson KD, Goldhaber JI. Excitation-contraction coupling in Na⁺-Ca²⁺ exchanger knockout mice: reduced transsarcolemmal Ca²⁺ flux. Circ Res 97: 1288–1295, 2005). To investigate the mechanism that underlies the accelerated AP repolarization, we recorded the transient outward current (I_to) in patch-clamped myocytes isolated from wild-type (WT) and NCX KO mice. Peak I_to was increased by 78% and decay kinetics were slowed in KO vs. WT. Consistent with increased I_to, ECGs from KO mice exhibited shortened QT intervals. Expression of the I_to-generating K⁺ channel subunit Kv4.2 and the K⁺ channel interacting protein was increased in KO. We used a computer model of the murine AP (Bondarenko VE, Szigeti GP, Bett GC, Kim SJ, and Rasmusson RL. Computer model of action potential of mouse ventricular myocytes. Am J Physiol Heart Circ Physiol 287: 1378–1403, 2004) to determine the relative contributions of increased I_to, reduced I_Ca, and reduced NCX current (I_NCX) on the shape and kinetics of the AP. Reduction of I_Ca and elimination of I_NCX had relatively small effects on the duration of the AP in the computer model. In contrast, AP repolarization was substantially accelerated when I_to was increased in the computer model. Thus, the increase in I_to, and not the reduction of I_Ca or I_NCX, is likely to be the major mechanism of AP shortening in KO myocytes. The upregulation of I_to may comprise an important regulatory mechanism to limit Ca²⁺ influx via a reduction of AP duration, thus preventing Ca²⁺ overload in situations of reduced myocyte Ca²⁺ extrusion capacity. genetically altered mice; cardiac myocytes; short QT interval; transient outward current     

Differential regulation of Ca(2+) sparks and Ca(2+) waves by UTP in rat cerebral artery smooth muscle cells

Uridine 5'-triphosphate (UTP), a potent vasoconstrictor that activatesphospholipase C, shifted Ca²⁺ signaling from sparks towaves in the smooth muscle cells of rat cerebral arteries. UTPdecreased the frequency of Ca²⁺ sparks and transientCa²⁺-activated K⁺ (K_Ca) currentsand increased the frequency of Ca²⁺ waves. The UTP-inducedreduction in Ca²⁺ spark frequency did not reflect adecrease in global cytoplasmic Ca²⁺, Ca²⁺influx through voltage-dependent Ca²⁺ channels (VDCC), orCa²⁺ load of the sarcoplasmic reticulum (SR), since globalCa²⁺ was elevated, blocking VDCC did not prevent theeffect, and SR Ca²⁺ load did not decrease. However,blocking protein kinase C (PKC) with bisindolylmaleimide I did preventUTP reduction of Ca²⁺ sparks and transient K_Cacurrents. UTP decreased the effectiveness of caffeine, which increasesthe Ca²⁺ sensitivity of ryanodine-sensitiveCa²⁺ release (RyR) channels, to activate transientK_Ca currents. This work supports the concept thatvasoconstrictors shift Ca²⁺ signaling modalities fromCa²⁺ sparks to Ca²⁺ waves through the concertedactions of PKC on the Ca²⁺ sensitivity of RyR channels,which cause Ca²⁺ sparks, and of inositol trisphosphate(IP₃) on IP₃ receptors to generateCa²⁺ waves.

     

Expression levels of RyR1 and RyR3 control resting free Ca2+ in skeletal muscle

To better understand the role of the transient expression of ryanodine receptor (RyR) type 3 (RyR3) on Ca²⁺ homeostasis during the development of skeletal muscle, we have analyzed the effect of expression levels of RyR3 and RyR1 on the overall physiology of cultured myotubes and muscle fibers. Dyspedic myotubes were infected with RyR1 or RyR3 containing virions at 0.2, 0.4, 1.0, and 4.0 moieties of infection (MOI), and analysis of their pattern of expression, caffeine sensitivity, and resting free Ca²⁺ concentration ([Ca²⁺]_r) was performed. Although increased MOI resulted in increased expression of each receptor isoform, it did not significantly affect the immunopattern of RyRs or the expression levels of calsequestrin, triadin, or FKBP-12. Interestingly, myotubes expressing RyR3 always had significantly higher [Ca²⁺]_r and lower caffeine EC₅₀ than did cells expressing RyR1. Although some of the increased sensitivity of RyR3 to caffeine could be attributed to the higher [Ca²⁺]_r in RyR3-expressing cells, studies of [³H]ryanodine binding demonstrated intrinsic differences in caffeine sensitivity between RyR1 and RyR3. Tibialis anterior (TA) muscle fibers at different stages of postnatal development exhibited a transient increase in [Ca²⁺]_r coordinately with their level of RyR3 expression. Similarly, adult soleus fibers, which also express RyR3, had higher [Ca²⁺]_r than did adult TA fibers, which exclusively express RyR1. These data show that in skeletal muscle, RyR3 increases [Ca²⁺]_r more than RyR1 does at any expression level. These data suggest that the coexpression of RyR1 and RyR3 at different levels may constitute a novel mechanism by which to regulate [Ca²⁺]_r in skeletal muscle. ryanodine receptor; calcium release; ryanodine binding; muscle fibers     

Na+/Ca2+ exchange activity in neonatal rabbit ventricular myocytes

Much less is known about the contributions of the Na⁺/Ca²⁺ exchanger (NCX) and sarcoplasmic reticulum (SR) Ca²⁺ pump to cell relaxation in neonatal compared with adult mammalian ventricular myocytes. Based on both biochemical and molecular studies, there is evidence of a much higher density of NCX at birth that subsequently decreases during the next 2 wk of development. It has been hypothesized, therefore, that NCX plays a relatively more important role for cytosolic Ca²⁺ decline in neonates as well as, perhaps, a role in excitation-contraction coupling in reverse mode. We isolated neonatal ventricular myocytes from rabbits in four different age groups: 3, 6, 10, and 20 days of age. Using an amphotericin-perforated patch-clamp technique in fluo-3-loaded myocytes, we measured the caffeine-induced inward NCX current (I_NCX) and the Ca²⁺ transient. We found that the integral of I_NCX, an indicator of SR Ca²⁺ content, was greatest in myocytes from younger age groups when normalized by cell surface area and that it decreased with age. The velocity of Ca²⁺ extrusion by NCX (V_NCX) was linear with [Ca²⁺] and did not indicate saturation kinetics until [Ca²⁺] reached 1–3 µM for each age group. There was a significantly greater time delay between the peaks of I_NCX and the Ca²⁺ transient in myocytes from the youngest age groups. This observation could be related to structural differences in the subsarcolemmal microdomains as a function of age. ontogeny of cardiac excitation-contraction coupling; sodium/calcium exchanger; cytosolic calcium concentration; subsarcolemmal calcium concentration; sarcoplasmic reticulum calcium content     

Intermittent hypoxia protects cardiomyocytes against ischemia-reperfusion injury-induced alterations in Ca2+ homeostasis and contraction via the sarcoplasmic reticulum and Na+/Ca2+ exchange mechanisms

We have previously demonstrated that intermittent high-altitude (IHA) hypoxia significantly attenuates ischemia-reperfusion (I/R) injury-induced excessive increase in resting intracellular Ca²⁺ concentrations ([Ca²⁺]_i). Because the sarcoplasmic reticulum (SR) and Na⁺/Ca²⁺ exchanger (NCX) play crucial roles in regulating [Ca²⁺]_i and both are dysfunctional during I/R, we tested the hypothesis that IHA hypoxia may prevent I/R-induced Ca²⁺ overload by maintaining Ca²⁺ homeostasis via SR and NCX mechanisms. We thus determined the dynamics of Ca²⁺ transients and cell shortening during preischemia and I/R injury in ventricular cardiomyocytes from normoxic and IHA hypoxic rats. IHA hypoxia did not affect the preischemic dynamics of Ca²⁺ transients and cell shortening, but it significantly suppressed the I/R-induced increase in resting [Ca²⁺]_i levels and attenuated the depression of the Ca²⁺ transients and cell shortening during reperfusion. Moreover, IHA hypoxia significantly attenuated I/R-induced depression of the protein contents of SR Ca²⁺ release channels and/or ryanodine receptors (RyRs) and SR Ca²⁺ pump ATPase (SERCA2) and SR Ca²⁺ release and uptake. In addition, a delayed decay rate time constant of Ca²⁺ transients and cell shortening of Ca²⁺ transients observed during ischemia was accompanied by markedly inhibited NCX currents, which were prevented by IHA hypoxia. These findings indicate that IHA hypoxia may preserve Ca²⁺ homeostasis and contraction by preserving RyRs and SERCA2 proteins as well as NCX activity during I/R. intracellular Ca²⁺ concentration; Ca²⁺ transients; Ca²⁺ transporters; myofilament Ca²⁺ sensitivity     

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     

Temperature dependence of cloned mammalian and salmonid cardiac Na(+)/Ca(2+) exchanger isoforms

The cardiacNa⁺/Ca²⁺ exchanger (NCX), an importantregulator of cytosolic Ca²⁺ concentration in contractionand relaxation, has been shown in trout heart sarcolemmal vesicles tohave high activity at 7°C relative to its mammalian isoform. Thisunique property is likely due to differences in protein structure. Inthis study, outward NCX currents (I_NCX) of thewild-type trout (NCX-TR1.0) and canine (NCX 1.1) exchangers expressedin oocytes were measured to explore the potential contributions ofregulatory vs. transport mechanisms to this observation. cRNA wastranscribed in vitro from both wild-type cDNA and was injected intoXenopus oocytes. I_NCX of NCX-TR1.0 and NCX1.1 were measured after 3-4 days over a temperature range of 7-30°C using the giant excised patch technique. TheI_NCX for both isoforms exhibitedNa⁺-dependent inactivation and Ca²⁺-dependentpositive regulation. The I_NCX of NCX1.1exhibited typical mammalian temperature sensitivities withQ₁₀ values of 2.4 and 2.6 for peak and steady-statecurrents, respectively. However, the I_NCX ofNCX-TR1.0 was relatively temperature insensitive with Q₁₀values of 1.2 and 1.1 for peak and steady-state currents, respectively.I_NCX current decay was fit with a singleexponential, and the resultant rate constant of inactivation () wasdetermined as a function of temperature. As expected,  decreasedmonotonically with temperature for both isoforms. Although  wassignificantly greater in NCX1.1 compared with NCX-TR1.0 at alltemperatures, the effect of temperature on  was not differentbetween the two isoforms. These data suggest that thedisparities in I_NCX temperature dependencebetween these two exchanger isoforms are unlikely due to differences intheir inactivation kinetics. In addition, similar differences intemperature dependence were observed in both isoforms after-chymotrypsin treatment that renders the exchanger in a deregulatedstate. These data suggest that the differences in I_NCX temperature dependence between the twoisoforms are not due to potential disparities in either theI_NCX regulatory mechanisms or structuraldifferences in the cytoplasmic loop but are likely predicated ondifferences within the transmembrane segments.

     

In squid nerves intracellular Mg(2+) promotes deactivation of the ATP-upregulated Na(+)/Ca(2+) exchanger

We investigated the role of intracellular Mg²⁺(Mg_i²⁺) on the ATP regulation ofNa⁺/Ca²⁺ exchanger in squid axons and bovineheart. In squid axons and nerve vesicles, the ATP-upregulated exchangerremains activated after removal of cytoplasmic Mg²⁺, evenin the absence of ATP. Rapid and complete deactivation of theATP-stimulated exchange occurs upon readmission ofMg_i²⁺. At constant ATP concentration, the effectof intracellular Mg²⁺ concentration([Mg²⁺]_i) on the ATP regulation of exchangeris biphasic: activation at low [Mg²⁺]_i,followed by deactivation as [Mg²⁺]_i isincreased. No correlation was found between the above results and thelevels of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] measured innerve membrane vesicles. Incorporation ofPtdIns(4,5)P₂ into membrane vesicles activates Na⁺/Ca²⁺ exchange in mammalian heart but not insquid nerve. Moreover, an exogenous phosphatase prevents MgATPactivation in squid nerves but not in mammalian heart. It is concludedthat 1) Mg_i²⁺ is an essentialcofactor for the deactivation part of ATP regulation of the exchangerand 2) the metabolic pathway of ATP upregulation of theNa⁺/Ca²⁺ exchanger is different in mammalianheart and squid nerves.

     

Ca(2+) regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts

Osteoblasts subjected to fluid shearincrease the expression of the early response gene, c-fos, andthe inducible isoform of cyclooxygenase, COX-2, two proteins linked tothe anabolic response of bone to mechanical stimulation, in vivo. Theseincreases in gene expression are dependent on shear-induced actinstress fiber formation. Here, we demonstrate that MC3T3-E1osteoblast-like cells respond to shear with a rapid increase inintracellular Ca²⁺ concentration([Ca²⁺]_i) that wepostulate is important to subsequent cellular responses to shear. Totest this hypothesis, MC3T3-E1 cells were grown on glass slides coatedwith fibronectin and subjected to laminar fluid flow (12 dyn/cm²). Before application of shear, cells were treatedwith two Ca²⁺ channel inhibitors or various blockers ofintracellular Ca²⁺ release for 0.5-1 h. Althoughgadolinium, a mechanosensitive channel blocker, significantly reducedthe [Ca²⁺]_i response, neithergadolinium nor nifedipine, an L-type channel Ca²⁺ channelblocker, were able to block shear-induced stress fiber formation andincrease in c-fos and COX-2 in MC3T3-E1 cells. However, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceticacid-AM, an intracellular Ca²⁺ chelator, or thapsigargin,which empties intracellular Ca²⁺ stores, completelyinhibited stress fiber formation and c-fos/COX-2 production in shearedosteoblasts. Neomycin or U-73122 inhibition of phospholipase C, whichmediates D-myo-inositol 1,4,5-trisphosphate (IP₃)-induced intracellular Ca²⁺ release, alsocompletely suppressed actin reorganization and c-fos/COX-2 production.Pretreatment of MC3T3-E1 cells with U-73343, the inactive isoform ofU-73122, did not inhibit these shear-induced responses. These resultssuggest that IP₃-mediated intracellular Ca²⁺release is required for modulating flow-induced responses in MC3T3-E1 cells.

     

Ca(2+)-dependent activation of c-jun NH(2)-terminal kinase in primary rabbit proximal tubule epithelial cells

Previous work from this laboratorydemonstrated that arachidonic acid activates c-junNH₂-terminal kinase (JNK) through oxidative intermediatesin a Ca²⁺-independent manner (Cui X and Douglas JG.Arachidonic acid activates c-jun N-terminal kinase throughNADPH oxidase in rabbit proximal tubular epithelial cells. ProcNatl Acad Sci USA 94: 3771-3776, 1997.). We now report thatJNK can also be activated via a Ca²⁺-dependent mechanism byagents that increase the cytosolic Ca²⁺ concentration(Ca²⁺ ionophore A₂₃₁₈₇, Ca²⁺-ATPaseinhibitor thapsigargin) or deplete intracellular Ca²⁺stores [intracellular Ca²⁺ chelator1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid(BAPTA)-AM]. The activation of JNK by BAPTA-AM occurs despite adecrease in cytosolic Ca²⁺ concentration as detected by theindicator dye fura 2, but appears to be related to Ca²⁺metabolism, because modification of BAPTA with two methyl groups increases not only the chelation affinity for Ca²⁺, butalso the potency for JNK activation. BAPTA-AM stimulates Ca²⁺ influx across the plasma membrane, and the resultinglocal Ca²⁺ increases are probably involved in activation ofJNK because Ca²⁺ influx inhibitors (SKF-96365, nifedipine)and lowering of the free extracellular Ca²⁺ concentrationwith EGTA reduce the BAPTA-induced JNK activation.

     

Coupling strength between localized Ca(2+) transients and K(+) channels is regulated by protein kinase C

Localized Ca²⁺ transients resulting from inositoltrisphosphate (IP₃)-dependent Ca²⁺ releasecouple to spontaneous transient outward currents (STOCs) in murinecolonic myocytes. Confocal microscopy and whole cell patch-clamptechniques were used to investigate coupling between localizedCa²⁺ transients and STOCs. Colonic myocytes were loadedwith fluo 3. Reduction in external Ca²⁺([Ca²⁺]_o) reduced localized Ca²⁺transients but increased STOC amplitude and frequency. Simultaneous recordings of Ca²⁺ transients and STOCs showed increasedcoupling strength between Ca²⁺ transients and STOCs when[Ca²⁺]_o was reduced. Gd³⁺ (10 µM) did not affect Ca²⁺ transients but increased STOCamplitude and frequency. Similarly, an inhibitor of Ca²⁺influx,1-2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole (SKF-96365), increased STOC amplitude and frequency. A protein kinase C(PKC) inhibitor, GF-109203X, also increased the amplitude and frequencyof STOCs but had no effect on Ca²⁺ transients. Phorbol12-myristate 13-acetate (1 µM) reduced STOC amplitude and frequencybut did not affect Ca²⁺ transients. 4-Phorbol (1 µM)had no effect on STOCs or Ca²⁺ transients. Single channelstudies indicated that large-conductance Ca²⁺-activatedK⁺ (BK) channels were inhibited by aCa²⁺-dependent PKC. In summary 1)Ca²⁺ release from IP₃ receptor-operated storesactivates Ca²⁺-activated K⁺ channels;2) Ca²⁺ influx through nonselective cationchannels facilitates activation of PKC; and 3) PKC reducesthe Ca²⁺ sensitivity of BK channels, reducing the couplingstrength between localized Ca²⁺ transients and BK channels.

     

Role of aspartate residues in Ca(2+) affinity and permeation of the distal ECaC1

The Ca²⁺ affinity andpermeation of the epithelial Ca²⁺ channel (ECaC1) wereinvestigated after expression in Xenopus oocytes. ECaC1displayed anomalous mole-fraction effects. Extracellular Ca²⁺ and Mg²⁺ reversibly inhibited ECaC1 wholecell Li⁺ currents: IC₅₀ = 2.2 ± 0.4 µM (n = 9) and 235 ± 35 µM (n = 10), respectively. These values compare well with theCa²⁺ affinity of the L-type voltage-gated Ca²⁺(Ca_V1.2) channel measured under the same conditions,suggesting that high-affinity Ca²⁺ binding is awell-conserved feature of epithelial and voltage-gated Ca²⁺channels. Neutralization of D550 and E535 in the pore region had nosignificant effect on Ca²⁺ and Mg²⁺ affinities.In contrast, neutralization of D542 significantly decreasedCa²⁺ affinity (IC₅₀ = 1.1 ± 0.2 mM,n = 6) and Mg²⁺ affinity(IC₅₀ > 25 ± 3 mM, n = 4).Despite a 1,000-fold decrease in Ca²⁺ affinity in D542N,Ca²⁺ permeation properties and theCa²⁺-to-Ba²⁺ conductance ratio remainedcomparable to values for wild-type ECaC1. Together, our observationssuggest that D542 plays a critical role in Ca²⁺ affinitybut not in Ca²⁺ permeation in ECaC1.

     

Changes in intracellular Ca2+ and pH in response to thapsigargin in human glioblastoma cells and normal astrocytes

Despite extensive work in the field of glioblastoma research no significant increase in survival rates for this devastating disease has been achieved. It is known that disturbance of intracellular Ca²⁺ ([Ca²⁺]_i) and intracellular pH (pH_i) regulation could be involved in tumor formation. The sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) is a major regulator of [Ca²⁺]_i. We have investigated the effect of inhibition of SERCA by thapsigargin (TG) on [Ca²⁺]_i and pH_i in human primary glioblastoma multiforme (GBM) cells and GBM cell lines, compared with normal human astrocytes, using the fluorescent indicators fura-2 and BCECF, respectively. Basal [Ca²⁺]_i was higher in SK-MG-1 and U87 MG but not in human primary GBM cells compared with normal astrocytes. However, in tumor cells, TG evoked a much larger and faster [Ca²⁺]_i increase than in normal astrocytes. This increase was prevented in nominally Ca²⁺-free buffer and by 2-APB, an inhibitor of store-operated Ca²⁺ channels. In addition, TG-activated Ca²⁺ influx, which was sensitive to 2-APB, was higher in all tumor cell lines and primary GBM cells compared with normal astrocytes. The pH_i was also elevated in tumor cells compared with normal astrocytes. TG caused acidification of both normal and all GBM cells, but in the tumor cells, this acidification was followed by an amiloride- and 5-(N,N-hexamethylene)-amiloride-sensitive recovery, indicating involvement of a Na⁺/H⁺ exchanger. In summary, inhibition of SERCA function revealed a significant divergence in intracellular Ca²⁺ homeostasis and pH regulation in tumor cells compared with normal human astrocytes. fura-2; BCECF; store-operated calcium channels     

Changes in regulation of sodium/calcium exchanger of avian ventricular heart cells during embryonic development

It has been suggested that the sodium/calcium exchanger NCX1 may have a more important physiological role in embryonic and neonatal hearts than in adult hearts. However, in chick heart sarcolemmal vesicles, sodium-dependent calcium transport is reported to be small and, moreover, to be 3–12 times smaller in hearts at embryonic day (ED) 4–5 than at ED18, the opposite of what would be expected of a transporter that is more important in early development. To better assess the role of NCX1 in calcium regulation in the chick embryonic heart, we measured the activity of NCX1 in chick embryonic hearts as extracellular calcium-activated exchanger current (I_NCX) under controlled ionic conditions. With intracellular calcium concentration ([Ca²⁺]_i) = 47 nM, I_NCX density increased from 1.34 ± 0.28 pA/pF at ED2 to 3.22 ± 0.55 pA/pF at ED11 (P = 0.006); however, with [Ca²⁺]_i = 481 nM, the increase was small and statistically insignificant, from 4.54 ± 0.77 to 5.88 ± 0.73 pA/pF (P = 0.20, membrane potential = 0 mV, extracellular calcium concentration = 2 mM). Plots of I_NCX density against [Ca²⁺]_i were well fitted by the Michaelis-Menton equation and extrapolated to identical maximal currents for ED2 and ED11 cells (extracellular calcium concentration = 1, 2, or 4 mM). Thus the increase in I_NCX at low [Ca²⁺]_i appeared to reflect a developmental change in allosteric regulation of the exchanger by intracellular calcium rather than an increase in the membrane density of NCX1. Supporting this conclusion, RT-PCR demonstrated little change in the amount of mRNA encoding NCX1 expression from ED2 through ED18. NCX1; chick embryo; allosteric regulation; sodium/calcium exchange current