Z Kardiol 90: Suppl 3, III/99 – III/105 (2001)© Steinkopff Verlag 2001

K. M. Kim Cellular mechanism of calcification and its prevention in glutaraldehyde treated vascular tissue

sis. [Ca2+]i increase in fibroblaststreated with various Ca2+-CBs wascompared with the untreated con-trol. To study the role of Ca2+ influxin calcification and to find out theportals of Ca2+ entry, porcine aorticvalve fibroblasts and freshlyremoved rat aorta were treated withverapamil + ryanodine, or vera-pamil + econazole, fixed with GAand incubated in Hank’s balancedsalt solution with 2.5 mmol/L cal-cium. The progress of calcificationwas monitored by the rate of Caand Pi depletions from the super-natant. Calcified cells and tissueswere identified by calcein fluores-cence. Results. Verapamil + ryan-odine or econazole inhibited theGA-induced Ca2+ influx and pre-vented calcification of the cells andrat aorta. The effect of verapamilwas additive to that of ryanodineand econazole. Conclusions. Find-ings further support the influx the-ory of calcification. Ca2+ enters GA-

treated cells mainly through thestore operated and the L-typeCa2+channels. Ca2+-CBs may beuseful for prevention of calcifica-tion in GA-treated vascular bio-prostheses. Cell culture serves as aconvenient model for screeningdrug effects on calcification.

M Key words Calcification – glu-taraldehyde – prevention

M Abbreviations GA: glutaralde-hyde; GFVPs: glutaraldehyde fixedvalvular prostheses; [Ca2+]i: intra-cellular Ca2+; [Pi]i: intracellularphosphate; AV: aortic valve; Ca2+-CBs: calcium channel blockers;CDPs: cellular degradation prod-ucts; ER: endoplasmic reticulum;SR: sarcoplasmic reticulum; SOCC:store operated calcium channel;CICR: Ca2+ induced Ca2+ releasechannel; HBSS: Hank’s balancedsalt solution; HBSS2.5: HBSS with2.5 mmol/L calcium.

Kookmin M. Kim, M.D. (Y)VA Medical Center510 Stoner Ave.Shreveport, Louisiana 71101, USAE-mail: kirkmkim@cs.com

M Summary Objectives. Preventionof calcification in glutaraldehyde(GA) treated porcine aortic valvefibroblasts and rat aorta withCa2+channel blockers (Ca2+-CBs).Background. GA causes a massiveincrease in [Ca2+]i and a many foldincrease in [Pi]i followed by calcifi-cation of porcine aortic valvefibroblasts. The influx of extracel-lular Ca2+ into [Pi]i rich cells appar-ently underlies the mechanism ofcalcification. Inhibition of Ca2+

influx is likely to prevent calcifica-tion in GA-treated cells. Methods.[Ca2+]i in GA-treated cells was mea-sured by fluorescence image analy-

Introduction

Because of the limited supply of human heart valves, dis-eased valves are commonly replaced with glutaraldehyde(GA) fixed valvular prostheses (GFVPs) prepared fromanimal tissues. Unfortunately, GFVPs frequently fail dueto calcification. Despite extensive studies, the mecha-nism of calcification in GFVPs has remained controver-sial.

In aging human aortic valves, calcification occurs in alayer of lipids deposited in the fibrosa (1). The lipids cor-respond to membranous cellular degradation products(CDPs) observed by electron microscopy. Calcificationoccurs mainly in association with CDPs (2). In a varietyof dystrophic calcifications, calcific deposits are observedin CDPs as well (3). In GFVPs grafted in rat subcutis,calcification has been shown to begin intracellularlyfollowed by calcification of the extracellular matrix (4). Infailed GFVPs removed from human hearts, calcific

deposits have been observed in membranous cell debris,similar to CDPs in aging vascular connective tissue (5).The mechanism of calcification in GFVPs may be akin tothat of other dystrophic calcifications.

Calcification has long been noted in necrotic tissues(6). Of tissue components, cells are most vulnerable toinjury. Cell injury to canine aortic valve (AV) fibroblasts,including GA treatment, has resulted in calcification (7).Since cell injury has been known to cause an influx ofextracellular Ca2+ and to increase intracellular phosphate([Pi]i) (8, 9), Ca2+ influx into [Pi]i rich cells is likely thecause of calcification (3). Indeed, GA causes a massiveincrease in intracellular Ca2+([Ca2+]i) and a many-foldincrease in [Pi]i within hours, followed by calcification ofporcine AV fibroblasts in a few days. Calcification beginsin blebs similar to CDPs (10). Hence, the blockade of Ca2+

influx is likely to prevent calcification in GA-treatedtissues.

The above study with cell cultures has not consideredthe role of matrix in calcification. To compare the capac-ity to nucleate apatite by cells vs. the matrix, lipid-extracted porcine AVs and fibroblasts cultured from thesame valves were placed in lipid-extracted rat intestinalpouches, fixed with GA and grafted in rat subcutis. Cal-cification occurred in the cells and spread to the wall ofthe pouch in 3 weeks, whereas the pouches with thematrix did not calcify for 9 weeks (unpublished data).Evidently, cells, rather than the matrix, nucleate apatitein GA-fixed tissue and an attempt to prevent calcificationshould take into account the cell’s role in calcification.

To test further the role of Ca2+ influx on calcificationand to find out the portals of Ca2+ entry, the blockade ofCa2+ influx into GA-treated porcine AV fibroblasts wasattempted using various Ca2+-channel blockers (Ca2+-CBs). A number of Ca2+-CBs inhibited Ca2+ influx andcalcification of the cells and rat aorta. Findings furthersupport the influx theory of calcification and raise thepossibility of preventing calcification in GFVPs withCa2+-CBs.

Materials and methods

Fetal bovine serum was purchased from Hyclone (Logan,UT). Reagent grade chemicals and culture media werefrom SIGMA (St. Louis, MO). Calcium green-1/AM(CaGr-1) and tetramethylrhodamine (TMRE) were pur-chased from Molecular Probes (Eugene, OR). Glu-taraldehyde (50 %) was from E. F. Fullam (Shenectady,NY).

M Cell culture

Cells were cultured as described previously (10). Briefly,porcine AVs were obtained from a local slaughterhouse

within 30 min of sacrifice. The valves were transported inice chilled RPMI 1640 with 20 % fetal bovine serum, 50 µg/ml gentamycin sulfate, and 1.25 µg/ml ampho-tericin B. After removal of endothelium, the valves werefinely minced, explanted, and the cells were cultured inminimal essential medium supplemented with 10 % fetalbovine serum, 100 IU penicillin, and 100 µg/ml strepto-mycin under 5 % CO2 at 37 °C.

M Measurement of [Ca2+]i

Because GA quenches ratiometric dyes, [Ca2+]i was meas-ured by a single wavelength fluorescence image analysisusing CaGr-1 as the indicator (10). A 25 mm round coverslip with semiconfluent cells was mounted on an Atto-fluor cell chamber (Molecular Probes). The cells wereloaded with 5 µmol/L CaGr-1 for 30 min in Hank’s bal-anced salt solution in which the total calcium wasadjusted to 2.5 mmol/L with CaCl2, additionally bufferedwith 25 mmol/L HEPES, and pH adjusted to 7.40(HBSS2.5). The cells were allowed to de-esterify for anadditional 30 min. GA-induced [Ca2+]i increase wasmeasured at an excitation wavelength of 488 nm and anemission wavelength of 531 nm using a BioRad MRC1000confocal microscope. To study the effect of Ca2+-CBs onCa2+ influx, cells were pretreated for 3 days with L-typeCa2+-CBs (verapamil, diltiazem, and nifedipine), inhi-bitors of Ca2+ release from the ER (ryanodine, neomycin,ruthenium red, Gd3+), and inhibitors of Ca2+entrythrough store operated Ca2+channels (econazole,SKF96365) and the [Ca2+]i measurement was repeated.Cells aliquoted from the same culture flask and loadedwith CaGr-1 aliquoted from the same vial served as thecontrol. Cells with signs of injury and uneven loading ofCaGr-1 were avoided. The measurements were repeatedmore than three times to confirm consistency of theresults.

Of Ca2+-CBs tested, verapamil, ryanodine, and econa-zole were selected for prevention of calcification. Thedose of each Ca2+-CB was determined by treating cellswith gradient concentrations of Ca2+-CBs for three daysfollowed by GA fixation and incubation in HBSS2.5. Thehighest dose of Ca2+-CB without cell injury, i.e., blebbingor detachment of cells from the culture surface, and with-out consumption of Ca2+ and Pi from the supernatant fora week, was chosen.

M Measurement of membrane potentials

Since GA preserves the cell structure, the commonly usedmorphological evaluation for GA-induced cell death isnot well suited. GA is also likely to devitalize cells rapidly,and the devitalization was monitored with changes in themembrane potential. Semiconfluent cells were treatedwith a fluorescent potentiometric indicator, TMRE

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(3 µmol/L), for 30 min and the effect of GA on the poten-tial was monitored at an excitation wavelength of 488 nmand emission wavelength of 522 nm (11).

M Calcification of cells

Confluent cells on 24 well plates in triplicates were pre-treated with 30 µmol/L verapamil + 100 nmol/L ryan-odine, or 10 µmol/L verapamil + 10 µmol/L econazole for3 days and fixed with 0.6 % GA in HBSS for 24 h. Afterwashing GA away, the cells in each well were incubatedin 1.0 ml of HBSS2.5 at 37 °C and 100 % humidity in a CO2incubator. Ca2+-CBs were added to the fixative but not tothe incubation medium. Cells without Ca2+-CB treatmentserved as the control. Samples were harvested weekly forup to 4 weeks. The total calcium in the supernatant wasmeasured by atomic absorption spectrophotometry. Piwas measured by an ammonium molybdate color reac-tion (12). The cells were stained with 0.03 % calcein for 1 min. Calcific deposits were identified by calcein fluo-rescence at an excitation wavelength of 488 nm and anemission wavelength of 520 nm (10).

M Calcification of rat aorta

The effect of Ca2+-CBs on calcification of the vasculartissue was studied using rat aorta. The entire aortae were

removed from 250 gm Sprague-Dawley rats under Iso-flurane anesthesia (Abbott Lab., Chicago, IL), placed in12 ml of RPMI 1640 with 15 % serum, 30 µM verapamiland 20 µM econazole in screw-capped plastic tubes, andincubated at 37 °C for 5 days. Cells in tissue apparentlytolerate higher doses of Ca2+-CBs than those in culture.Aortae without Ca2+-CBs treatment served as the control.Aortae were cut to 1 cm segments, fixed in 0.6 % GA inCa2+, Mg2+-free HBSS for 24 h. After washing, each seg-ment was placed in a plastic tube with 7 ml of HBSS2.5and incubated at 37 °C. Samples in triplicate were har-vested weekly. Ionic Ca2+ and Pi in the supernatant weremeasured with a Ca2+-selective electrode and NH4-molybdate color reaction, respectively. Five µm thickcryostat sections of aortae were stained with 0.03 % cal-cein for 1 min, rinsed, cover-slipped using Slow-Fade(Molecular Probes) and fluorescence images were cap-tured.

M Statistical analysis

was performed using the SigmaStat software (SPSS,Chicago, IL). Paired t test was applied to the comparisonof two sample populations.

Results

In control cells treated with 0.6 % GA, an immediate[Ca2+]i spark occurred. The spark occurred even whenthe cells were in Ca2+, Mg+-free HBSS. The amplitude ofthe spark was approximately 150–300 nmol/L and thespark lasted for 30 s. The spark was followed in a few sec-onds by a progressive rise of [Ca2+]i approaching theextracellular Ca2+ concentration within 1 h. Addition of1.6 mM 4-Br-A23187 after 1 h of GA treatment furtherincreased [Ca2+]i only slightly (data not shown). Vera-pamil, diltiazem, and nifedipine, the prototypes of threegroups of L-type Ca2+-CBs, warded off approximatelyone-third of the GA-induced Ca2+ influx. The effect ofeach Ca2+-CB was similar and the combination of allthree Ca2+-CBs exerted no additive effect. Inhibitors ofCa2+ release from the ER, e.g., ruthenium red, neomycin,and Gd3+, exhibited a milder inhibition of the influx thanverapamil (Fig. 1). Of the ER Ca2+-CBs, ryanodine inhib-ited more effectively the increase in [Ca2+]i than others.Econazole (20 µmol/L) tended to be more effective thanryanodine (100 nmol/L) or SKF96365 (50 µmol/L) forinhibition of the influx. The effect of verapamil was addi-tive to that of ryanodine and econazole (Fig. 1). Vera-pamil saturably binds to the channel; fluorescencelabeled verapamil stained the control cells intensely,whereas cells pretreated with verapamil hardly displayed

K. M. Kim III/101Prevention of calcification

Fig. 1 [Ca2+]i measurement by fluorescence image analysis in cells loaded withCaGr-1. GA caused a small [Ca2+]i spark followed by a progressive increase in [Ca2+]iin control cells. The spark occurred even when the cells were in Ca2+, Mg2+-free HBSS(inset). Ca2+-CB pretreated cells showed less increases in [Ca2+]i than the control. OfCa2+-CBs tested, ryanodine and econazole more effectively inhibited [Ca2+]i incre-ase than verapamil. Ruthenium red mildly inhibited [Ca2+]i increase. C: Control. RR:Ruthenium red, 3 (emol/L). V: Verapamil 30. R: Ryanodine, 0.1. E: Econazole, 20. RV:Ryanodine, 0.1 + verapamil, 30. EV: Econazole, 20 + verapamil, 30.

any fluorescence by the same stain (data not shown).Many animal toxins, e.g., phylotoxins and calciseptins,are efficient blockers of Ca2+ entry into GA-treated cells.However, they are prohibitively expensive and are diffi-cult to obtain in a sufficiently large quantity.

Addition of 0.6 % GA to TMRE stained cells induceda sharp increase in the plasmalemmal potential followedby a complete depolarization in 2 min (Fig. 2).

For prevention of calcification, verapamil, ryanodine,and econazole were chosen. Cells treated with 30 mM ver-

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Fig. 2 Plasma membrane potential measured with a potentiometric indicator. Cellswere treated with 3 emol/L TMRE for 30 min and fluorescence intensity was moni-tored after addition of 0.6 % GA. GA caused a sharp rise in the membrane potentialfollowed by a total collapse of the potential in 2 min.

Fig. 3 Ion depletions from HBSS2.5 by cells fixed with 0.6 % GA and incubated inHBSS2.5. Control cells progressively depleted Ca (A) and Pi (B), whereas cells pre-treated for 3 days with 100 nmol/L ryanodine + 30 emol/L verapamil showed nonotable ion depletions for 4 weeks.

Fig. 4 Calcein fluorescence micrographs of cells fixed with 0.6 % GA and incubatedin HBSS2.5 for 4 weeks. The control shows intense calcein fluorescence in blebs(arrowhead) and entire cells (A). Ca2+-CB treated cells show the fluorescence in occa-sional small blebs but no calcified cells (B). X600.

Fig. 5 Rat aorta treated with 30 mmol/L verapamil and 15 mmol/L econazole for 5days, fixed with 0.6% GA in HBSS for 24 h and incubated in HBSS2.5. The untreatedcontrol began to deplete Ca2+ and Pi from the supernatant in week 2, whereas Ca2+-CB treated aorta did not deplete the ions.

apamil or 10 µM econazole only inhibited GA-inducedcalcification for more than a week (data not shown).Control cells fixed with 0.6 % GA in HBSS and incubatedin HBSS2.5 showed a progressive decrease in Ca in thesupernatant. A notable Pi depletion began in the secondweek and accelerated after 3 weeks. Cells treated with 30 µmol/L verapamil + 100 nmol/L ryanodine did notdeplete the ions or calcify for 4 weeks (Figs. 3 and 4).Although live cells tolerated 20 µmol/L econazole, econa-zole combined with verapamil tended to be toxic to thecells, causing blebbing and cell detachment. The toxicityusually became apparent during GA fixation and incu-bation in HBSS2.5 but not during the pretreatment. Atreduced doses, verapamil (30 µmol/L) + econazole (10 µmol/L) inhibited calcification of GA-treated cellssimilar to verapamil + ryanodine (data not shown). Con-trol cells developed blebs with calcein fluorescence in aweek in a small number of cells. What appeared to bemitochondria also showed an intense fluorescence. Afterthe second week, the entire cells displayed calceinfluorescence, which subsequently grew more intense andextensive (Fig. 4). Cells pretreated with Ca2+-CBs showedonly occasional blebs but no calcified cells after 4 weeksof incubation.

Rat aortae treated with GA similarly depleted Ca2+

and Pi from HBSS2.5 and calcified in 3 weeks, whereasCa2+-CB treated aortae did not deplete ions for 4 weeks(Fig. 5). Calcification was seen in fusiform smooth mus-cle cells between eleastic fibers (Fig. 6).

Discussion

Moderation of atherosclerosis and vascular calcificationin patients treated with Ca2+-CBs has been known forsome time (13). The decrease in calcification has been

attributed to the regression of atherosclerosis, and therole of Ca2+-CBs on calcification has rarely beenaddressed. The L-type Ca2+-CBs have been shown toinhibit calcium uptake by rat aortic valve in vivo andcalcification of hypertrophic chondrocytes in vitro (14,15). Findings in this study demonstrate further that Ca2+-CBs have a capacity to prevent calcification by blockingCa2+ influx into GA-treated cells. Ca2+-CBs have an addedbenefit of the continuous medication to the recipients ofGFVPs, as required. As opposed to other cell injury, GAtreatment preserves the membrane structure and servesas a convenient model for the study of Ca2+ influx andcalcification. Calcification of smooth muscle cells in rataorta (Fig. 6) demonstrates that cells are primarilyresponsible for apatite nucleation in vascular tissue aswell.

Functions of cells in culture differ from that of thecells in tissue. Cells bind to the extracellular matrix viaintegrin receptors of the cell surface, and the matrixmodulates ion transports of the attached cells (16). Pre-vention of calcification in rat aorta by Ca2+-CBs and theoccurrence of calcification in smooth muscle cells indi-cate that the Ca2+ influx causes apatite nucleation in thetissue as well. Since Ca2+ influx begins within seconds ofGA treatment, Ca2+-CBs treatment was applied to freshlyremoved rat aorta. A delay of GA fixation of tissues fromthe time of harvest has been shown to intensify the sub-sequent calcification of GFVPs (17, 18). This intensifica-tion can be attributed to Ca2+ influx due to hypoxic cellinjury during the delay.

Phopholipid bilayer is highly impermeable to waterand water soluble ions. Ion transports across cell mem-brane occur through ion channels and pumps that areubiquitous in biological systems, ranging from microor-ganisms to mammals (19). The mechanism of Ca2+ entryis complex and varies from one cell type to the other.Four portals of Ca2+ entry into functioning cells areknown: (1) voltage or receptor dependent Ca2+ channels,(2) store operated Ca2+ channels (capacitative Ca2+ entrychannels), (3) non-specific leak through other ion chan-nels, and (4) Na+-Ca2+ exchange (20). Of the six knowntypes of voltage-gated Ca2+ channels (L, N, P, Q, R & T),the slow L-type channels are widespread in non-excitablecells including fibroblasts (21, 22). Voltage-gated ionchannels assume three states, open, closed, and resting(21). Depolarization of plasma membrane opens the rest-ing channels in milliseconds. The rate of Ca2+ flowthrough the open channels approximates that of free iondiffusion (21). Most Ca2+ channel blockers (Ca2+-CBs) inclinical use are for the L-type channels. Ca2+-CBs prefer-entially bind to the inactivated channels, inhibit confor-mational changes of the channels back to the open stateand, thus, inhibit Ca2+ entry. In addition to membranedepolarization, agonist binding to the receptors alsoopens voltage-gated Ca2+ channels (22). The receptoractivation also allows Ca2+ entry through non-selective

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Fig. 6 Calcein stained cryosections of GA-treated rat aorta after 4 weeks of incu-bation in HBSS2.5. Spindle shaped smooth muscle cells between elastic fibers (E)emit strong fluorescence (A), whereas Ca2+-CBs treated aorta shows faint back-ground noise only (B). X300.

cation channels that are insensitive to Ca2+-CBs (20). Itrecently became apparent that the release of intracellularstores of Ca2+, by, e.g., inositol trisphosphate, triggersCa2+ entry through the store-operated Ca2+ channels(SOCC) of plasma membrane (23, 24). Depolarization ofthe membrane has no effect on SOCC. Although therelease of Ca2+ from the ER by any means tends to openSOCC, the mechanism of signal transduction from the ERCa2+ release to the opening of plasma membrane SOCCis not yet clear.

The mechanism of Ca2+ entry in cell injury, includingGA treatment, is poorly understood. The chronology ofrapid succession of ionic and other cellular events in GA-treated cells remains to be determined. A sharp rise of themembrane potential followed by depolarization of GA-treated cells indicates the occurrence of a rapid redistri-bution of ions across the membrane. In view of the depo-larization of plasma membrane within minutes of GAtreatment (Fig. 2), the cells have sufficient time to openand close ion channels, which takes place in split sec-onds. The massive increase in [Ca2+]i in GA-treated cellsappears to be due in part to the loss of Ca2+-pump activ-ity. GA has been shown to bind specifically to the ATPbinding sites of Ca2+-pumps of the SR and abolish thepump activity (25). GA is likely to increase net [Ca2+]i bysimilarly paralyzing plasmalemmal Ca2+-pumps and, atthe same time, allowing Ca2+ entry into the cell throughthe channels.

The inhibition of GA-induced Ca2+ influx by vera-pamil indicates that the influx occurs in part through theL-type channels, presumably secondary to membranedepolarization and activation of agonist receptors by GA.In view of the occurrence of [Ca2+]i spark even when thecells are treated with GA in a Ca2+ free solution, the sparkis evidently due to the release of [Ca2+]i from the ER.Hence, it is likely that GA activates agonist receptors,releases Ca2+ from the ER, and opens SOCC. GA is alsolikely to open voltage-gated Ca2+ channels secondary tomembrane depolarization. The occurrence of Ca2+ influxinto GA-treated cells and its inhibition by verapamil sug-gests that some of the L-type channels are fixed in theopen status. Retardation of calcification in cells pre-treated with verapmil or econazole alone indicates that a partial blockade of Ca2+ entry may be sufficient toprevent calcification.

A greater inhibition of GA-induced Ca2+ influx byryanodine and econazole than verapamil indicates thatSOCC is the major portal Ca2+ influx into GA-treatedcells. The additive effect of verapamil and ryanodine oreconazole demonstrates that the Ca2+ influx occurs

through two distinctive channels. Ryanodine receptors ofthe sarcoplasmic reticulum coincide with the Ca2+-induced Ca2+ release channel (CICR) of myocardiocytes(26, 27). Ryanodine in nmol/L has been shown to lockCICR in the open state and to enhance Ca2+ release fromthe SR, whereas at higher concentrations it has beenshown to inhibit the release (26, 27). Inhibition of Ca2+

influx into GA-treated cells by other inhibitors of ryano-dine receptors (Fig. 1) ascertains the involvement ofryanodine receptors in GA-induced Ca2+ influx. Fibrob-lasts appear to have well developed ryanodine receptors.The inhibition of Ca2+ influx by inhibitors of ryanodinereceptors appears to be due to the restraining of openingSOCC by inhibiting the Ca2+ release from the ER. A sim-ilar inhibition of Ca2+ influx by econazole and SKF96365supports the occurrence of Ca2+ entry through SOCC ofGA-treated cells. Econazole and SKF96365 have beenused extensively as experimental blockers of SOCC.

The blockade of Ca2+ entry and prevention of calcifi-cation by verapamil + ryanodine or econazole indicatesthat Ca2+ influx through existing Ca2+ channels is prima-rily responsible for calcification in GA-treated cells. Theeffect of each Ca2+-CB is not absolutely specific. Forinstance, econazole has been shown to elicit variableeffects on calcium transports depending on the cell type(28). An L-type Ca2+-CB has been shown to inhibit Ca2+

current through SOCC of skeletal muscle in a study (29).In view of the incomplete blockade of GA-induced Ca2+

entry by a combination of verapamil, econazole, andryanodine, there apparently are additional portals ofCa2+ entry into GA-treated cells, perhaps including non-specific leakage of Ca2+ through other ion channels.Identification of these additional portals would aid a fur-ther inhibition of calcification of the cells. With the devel-opment of more efficient Ca2+-CBs, it may eventually bepossible to completely and irreversibly lock Ca2+ chan-nels in the inactive state. Na+-Ca2+ exchanger activelytransports Ca2+ in excitable cells. However, the exchangeris poorly expressed in non-excitable cells and such anenergy dependent ion transport is unlikely in GA-treatedcells.

Ca2+ influx into GA-treated cells occurs mainlythrough the store operated and the L-type Ca2+ channels.Inhibition of the Ca2+ influx and calcification by Ca2+-CBsin GA-treated cells and rat aorta further substantiates theinflux theory of calcification. Ca2+-CBs may be proved tobe useful for prevention of GFVP calcification.

Acknowledgment Supported in part by American Heart Association,Louisiana Affiliate, Inc., LA97GS08.

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