Temporal Relationship between Action Potential and Ca2+ Transient in Characean Cells

Temporal relationship between the action potential and the changein cytosolic Ca²⁺ concentration was investigated in cells offour species of Characeae, Chara corallina, Nitellopsis obtusa,Nitella flexilis and Nitella axilliformis. The Ca²⁺ transientwas detected by light emission from Ca²⁺-sensitive photoproteinaequorin injected into the cytoplasm. Action potential was triggeredby an outward or sometimes inward electric current pulse of20–50 ms in most cases. In all species the action potentialstarted at almost the same time as the time at which the lightemission from aequorin began to increase. Also the peak of actionpotential almost coincided with that of light emission, whichis in contrast with the slower Ca²⁺ transient in Chara reportedby Thiel et al. [(1997) J. Exp. Bot. 48: 609]. A discussionwas made on the origin of Ca²⁺ transient and the ionic processesduring membrane excitation. (Received July 2, 1998; Accepted October 5, 1998)     

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.

     

Studies on Mechano-Perception in Characeae: Effects of External Ca2+ and Cl-

The effects of modification of extracellular concentrationsof Ca²⁺ and C^– on mechano-perception were studied in internodalcells of Chara corallina. Cells were stimulated by droppinga piece of glass tubing on them, and the resulting receptorpotentials and action potentials were analyzed. When the Ca²⁺concentration was extremely lowered by adding EGTA, the amplitudesof both receptor potentials and action potentials were attenuated,suggesting the involvement of Ca²⁺ channels. However, the possibilityremained that attenuation of the amplitude of the receptor potentialwas caused by modification of membrane characteristics by extremelowering of [Ca²⁺]_o. When the plasma membrane was depolarizedto about 0 mV by adding 100 mM KC1, responses in the negativedirection were induced upon mechanical stimulation. When theplasma membrane was depolarized by adding 50 mM K²SO⁴, responsesin the positive direction were induced. Thus, Cl^– channelsmay be involved in responses induced by mechanical stimulationunder K+-induced depolarization. (Received January 16, 1996; Accepted March 25, 1997)     

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)     

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.

     

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.

     

Palytoxin-induced cell death cascade in bovine aortic endothelial cells

The plasmalemmal Na⁺-K⁺-ATPase (NKA) pump is the receptor for the potent marine toxin palytoxin (PTX). PTX binds to the NKA and converts the pump into a monovalent cation channel that exhibits a slight permeability to Ca²⁺. However, the ability of PTX to directly increase cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) via Na⁺ pump channels and to initiate Ca²⁺ overload-induced oncotic cell death has not been examined. Thus the purpose of this study was to determine the effect of PTX on [Ca²⁺]_i and the downstream events associated with cell death in bovine aortic endothelial cells. PTX (3–100 nM) produced a graded increase in [Ca²⁺]_i that was dependent on extracellular Ca²⁺. The increase in [Ca²⁺]_i initiated by 100 nM PTX was blocked by pretreatment with ouabain with an IC₅₀ < 1 µM. The elevation in [Ca²⁺]_i could be reversed by addition of ouabain at various times after PTX, but this required much higher concentrations of ouabain (0.5 mM). These results suggest that the PTX-induced rise in [Ca²⁺]_i occurs via the Na⁺ pump. Subsequent to the rise in [Ca²⁺]_i, PTX also caused a concentration-dependent increase in uptake of the vital dye ethidium bromide (EB) but not YO-PRO-1. EB uptake was also blocked by ouabain added either before or after PTX. Time-lapse video microscopy showed that PTX ultimately caused cell lysis as indicated by release of transiently expressed green fluorescent protein (molecular mass 27 kDa) and rapid uptake of propidium iodide. Cell lysis was 1) greatly delayed by removing extracellular Ca²⁺ or by adding ouabain after PTX, 2) blocked by the cytoprotective amino acid glycine, and 3) accompanied by dramatic membrane blebbing. These results demonstrate that PTX initiates a cell death cascade characteristic of Ca²⁺ overload. necrosis; vital dyes; membrane blebs; time-lapse video microscopy; fura-2     

Differing temporal roles of Ca2+ and cAMP in nicotine-elicited elevation of tyrosine hydroxylase mRNA

The involvement of cAMP- andCa²⁺-mediated pathways in theactivation of tyrosine hydroxylase (TH) gene expression by nicotine wasexamined in PC-12 cells. ExtracellularCa²⁺ and elevations inintracellular Ca²⁺ concentration([Ca²⁺]_i)were required for nicotine to increase TH mRNA. The nicotine-elicited rapid rise in[Ca²⁺]_iwas inhibited by blockers of either L-type or N-type, and to a lesserextent P/Q-, but not T-type, voltage-gatedCa²⁺ channels. With continualnicotine treatment,[Ca²⁺]_ireturned to basal levels within 3-4 min. After a lag of~5-10 min, there was a smaller elevation in[Ca²⁺]_ithat persisted for 6 h and displayed different responsiveness toCa²⁺ channel blockers. This secondphase of elevated[Ca²⁺]_iwas blocked by an inhibitor of store-operatedCa²⁺ channels, consistent with theobserved generation of inositol trisphosphate.1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM), when added before or 2 h after nicotine,prevented elevation of TH mRNA. Nicotine treatment significantly raised cAMP levels. Addition of the adenylyl cyclase inhibitor2',5'-dideoxyadenosine (DDA) prevented thenicotine-elicited phosphorylation of cAMP response element bindingprotein. DDA also blocked the elevation of TH mRNA only when addedafter the initial transient rise in [Ca²⁺]_iand not after 1 h. This study reveals that several temporal phases areinvolved in the induction of TH gene expression by nicotine, each ofthem with differing requirements forCa²⁺ and cAMP.

     

Maternal Effects of mto1 Mutation, That Causes Overaccumulation of Soluble Methionine, on the Expression of a Soybean {beta}Conglycinin Gene Promoter-GUS Fusion in Transgenic Arabidopsis thaliana

ß-Conglycinin, the 7S seed storage protein of soybean(Glycine max [L.] Merr.), is comprised mainly of three subunits,designated , ' and ß. Expression of the gene encodingthe ß subunit is unique because its expression hasbeen shown to be down-regulated by exogenously applied L-methioninein immature soybean cotyledon cultures in vitro. Arabidopsisthaliana strain carrying a mto1-1 mutation overaccumulates solublemethionine. By using this mutant, we analyzed the effects ofmethionine on expression of the ß subunit gene invivo. Reciprocal crosses were made between the mto1-1 mutantand a transgenic A. thaliana strain, designated SNTß3,which carries a ß-glucuronidase (GUS) reporter geneunder the control of the promoter region of the ßsubunit gene. Analysis of GUS activity in F₁ seeds indicatedthat the GUS activity was dramatically repressed when the mto1-1mutant plants were used as female parents. We constructed astrain which carries both the transgene and mto1-1 mutationin the homozygous state. Analyses of the GUS activity in seedsof this double homozygous strain indicated that the GUS activitywas repressed to 2.5% of control by introduction of the mto1-1mutation. These results indicate that the ß subunitgene promoter activity in seeds is down-regulated by maternalgenotype and suggest that soluble methionine, or its mobilemetabolite, is translocated from mother plants to repress ßsubunit gene expression in seeds. ⁵Present address: Division of Biological Sciences, GraduateSchool of Science, Hokkaido University, Kita-ku, Sapporo, 060Japan ⁶Present address: Department of Biotechnology, Faculty of Agriculture,The University of Tokyo, Bunkyo-ku, Tokyo, 113 Japan     

Two types of voltage-dependent potassium channels in outer hair cells from the guinea pig cochlea

Cell-attached and cell-free configurations of the patch-clamptechnique were used to investigate the conductive properties andregulation of the major K⁺channels in the basolateral membrane of outer hair cells freshly isolated from the guinea pig cochlea. There were two majorvoltage-dependent K⁺ channels. ACa²⁺-activatedK⁺ channel with a high conductance(220 pS,P_K/P_Na = 8) was found in almost 20% of the patches. The inside-out activityof the channel was increased by depolarizations above 0 mV andincreasing the intracellular Ca²⁺concentration. External ATP or adenosine did not alter thecell-attached activity of the channel. The open probability of theexcised channel remained stable for several minutes without rundown andwas not altered by the catalytic subunit of protein kinase A (PKA)applied internally. The most frequentK⁺ channel had a low conductanceand a small outward rectification in symmetricalK⁺ conditions (10 pS for inwardcurrents and 20 pS for outward currents, P_K/P_Na = 28). It was found significantly more frequently in cell-attached andinside-out patches when the pipette contained 100 µM acetylcholine. It was not sensitive to internalCa²⁺, was inhibited by4-aminopyridine, was activated by depolarization above 30 mV,and exhibited a rundown after excision. It also had a slow inactivationon ensemble-averaged sweeps in response to depolarizing pulses. Thecell-attached activity of the channel was increased when adenosine wassuperfused outside the pipette. This effect also occurred with permeantanalogs of cAMP and internally applied catalytic subunit of PKA. Bothchannels could control the cell membrane voltage of outer hair cells.

     

Maitotoxin activates a nonselective cation channel and a P2Z/P2X7-like cytolytic pore in human skin fibroblasts

Maitotoxin (MTX),a potent cytolytic agent, activatesCa²⁺ entry via nonselective cationchannels in virtually all types of cells. The identity of the channelsinvolved and the biochemical events leading to cell lysis remainunknown. In the present study, the effect of MTX on plasmalemmalpermeability of human skin fibroblasts was examined. MTX produced atime- and concentration-dependent increase in cytosolic freeCa²⁺ concentration that dependedon extracellular Ca²⁺ and wasrelatively insensitive to blockade by extracellular lanthanides. MTXalso produced a time- and concentration-dependent increase inplasmalemma permeability to larger molecules as indicated by 1) uptake of ethidium (314 Da),2) uptake of YO-PRO-1 (375 Da), 3) release of intracellular fura 2 (636 Da), 4) uptake of POPO-3 (715 Da), and, ultimately,5) release of lactate dehydrogenase (relative molecular weight of 140,000). At the single cell level, uptake of YO-PRO-1 correlated in time with the appearance of large MTX-induced membrane currents carried by the organic cation,N-methyl-D-glucamine (167 Da). Thus MTX initially activatesCa²⁺-permeable cation channels andlater induces the formation of large pores. These effects of MTX onplasmalemmal permeability are similar to those seen on activation ofP2Z/P2X₇ receptors ina variety of cell types, raising the intriguing possibility that MTXand P2Z/P2X₇ receptor stimulationactivate a common cytolytic pore.

     

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.

     

Phospholipase D from Soybean (Glycine max L.) Suspension-Cultured Cells: Purification, Structural and Enzymatic Properties

Phospholipase D (phosphatidylcholine phosphatidohydrolase EC3.1.4.4 [EC] ) from soybean (Glycine max L.) suspension-cultured cellwas purified around 1,200-fold to homogeneity by acetone precipitation,Macro-Prep High Q anion exchange, and octyl-Sepharose CL-4Baffinity chromatography. The purified enzyme released 1,600µmol of choline per min per mg of protein. The enzymeis monomeric with a molecular mass of 92 kDa, as estimated bySDS-PAGE. One of the most interesting characteristics of thepurified soybean phospholipase D was the dependence of the pHoptimum on the Ca²⁺ ion concentration in the assay. With 10mM, 20 mM and 40 mM Ca²⁺ ions, the optima were at pH 7.5, 6and 5.5, respectively. The specific adsorption of phospholipaseD onto octyl-Sepharose gel suggests that the molecule becomesmore hydrophobic in the presence of Ca²⁺ ions. The amino acidsequence of the first 18 N-terminal residues of soybean phospholipaseD revealed a high degree of homology with those previously publishedfor cabbage leaf and castor bean endosperm enzymes. Westernblots of the soybean phospholipase D showed an immunoreactivitywith antibodies raised against a synthetic peptide correspondingto the 15 N-terminal aminoacid residues of phospholipase D fromcabbage leaves. (Received March 13, 1995; Accepted May 29, 1995)     

Pregnant rat myometrial cells show heterogeneous ryanodine- and caffeine-sensitive calcium stores

IntracellularCa²⁺ release channels such asryanodine receptors play crucial roles in theCa²⁺-mediated signaling thattriggers excitation-contraction coupling in muscles. Although theexistence and the role of these channels are well characterized inskeletal and cardiac muscles, their existence in smooth muscles, andmore particularly in the myometrium, is very controversial. We have nowclearly demonstrated the expression of ryanodine receptorCa²⁺ release channels in ratmyometrial smooth muscle, and for the first time, intracellularCa²⁺ concentration experimentswith indo 1 on single myometrial cells have revealed the existence of afunctional ryanodine- and caffeine-sensitive Ca²⁺ release mechanism in 30% ofrat myometrial cells. RT-PCR and RNase protection assay on wholemyometrial smooth muscle demonstrate the existence of all threeryr mRNAs in the myometrium:ryr3 mRNA is the predominant subtype,with much lower levels of expression forryr1 andryr2 mRNAs, suggesting that theryanodine Ca²⁺ release mechanismin rat myometrium is largely encoded byryr3. Moreover, using intracellularCa²⁺ concentration measurementsand RNase protection assays, we have demonstrated that the expression,the percentage of cells responding to ryanodine, and the function ofthese channels are not modified during pregnancy.

     

Auxin and heat shock activation of a novel member of the calmodulin like domain protein kinase gene family in cultured alfalfa cells

A calmodulin like domain protein kinase (CPK) homologue wasidentified in alfalfa and termed MsCPK3. The full-length sequenceof cDNA encoded a 535 amino acid polypeptide with a molecularweight of 60.2 kDa. The deduced amino acid sequence showed allthe conserved motifs that define other members of this kinasefamily, such as serine-threonine kinase domain, a junction regionand four potential Ca²⁺-binding EF sites. The recombinant MsCPK3protein purified from E. coli was activated by Ca²⁺and inhibitedby calmodulin antagonist (W-7) in in vitro phosphorylation assays.The expression of MsCPK3 gene increased in the early phase ofthe 2,4-D induced alfalfa somatic embryogenesis. Heat shockalso activated this gene while kinetin, ABA and NaCl treatmentdid not result in MsCPK3 mRNA accumulation. The data presentedsuggest that the new alfalfa CPK differs in stress responsesfrom the previously described homologues and in its potentialinvolvement in hormone and stress-activated reprogramming ofdevelopmental pathways during somatic embryogenesis. Key words: Medicago sativa, CPK, stress, 2,4-D, phosphorylation, somatic embryogenesis.     

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.

     

Altered Ca(2+) handling by ryanodine receptor and Na(+)-Ca(2+) exchange in the heart from ovariectomized rats: role of protein kinase A

Our previous study has demonstrated that ovariectomy (Ovx) significantly increased the left ventricular developed pressure (LVDP) and the maximal rate of developed pressure over time (±dP/dt_max) in the isolated perfused rat heart and the effects were reversed by female sex hormone replacement. In the present investigation, we studied the effects of Ovx for 6 wk on Ca²⁺ homeostasis that determines the contractile function. Particular emphasis was given to Ca²⁺ handling by ryanodine receptor (RyR) and Na⁺-Ca²⁺ exchange (NCX). ⁴⁵Ca²⁺ fluxes via the RyR, NCX, and Ca²⁺-ATPase (SERCA) were compared with their expression in myocytes from Ovx rats with and without estrogen replacement. Furthermore, we correlated the handling of Ca²⁺ by these Ca²⁺ handling proteins with the overall Ca²⁺ homeostasis by determining the Ca²⁺ transients induced by electrical stimulation and caffeine, which reveals the dynamic changes of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in the heart. In addition, we determined the expression and contribution of protein kinase A (PKA) to the regulation of the aforementioned Ca²⁺ handling proteins in Ovx rats. It was found that after Ovx there were 1) increased Ca²⁺ fluxes via RyR and NCX, which were reversed not only by estrogen replacement, but more importantly by blockade of PKA; 2) an increased expression of PKA; and 3) no increase in expression of NCX and SERCA. We suggest that hyperactivities of RyR and NCX are a result of upregulation of PKA. The increased release of Ca²⁺ through RyR and removal of Ca²⁺ by NCX are believed to be responsible for the greater contractility and faster relaxation after Ovx. ovariectomy