Interface induced diffusion.

Interface induced diffusion had been identified in a thin film system damaged by electron bombardment. This new phenomenon was observed in Al2O3 (some nm thick)/Si substrate system, which was subjected to low energy (5 keV) electron bombardment producing defects in the Al2O3 layer. The defects produced partially relaxed. The rate of relaxation is, however, was different in the vicinity of the interface and in the "bulk" parts of the Al2O3 layer. This difference creates an oxygen concentration gradient and consequently oxygen diffusion, resulting in an altered layer which grows from the Al2O3/Si substrate interface. The relative rate of the diffusion and relaxation is strongly temperature dependent, resulting in various altered layer compositions, SiO2 (at room temperature), Al2O3 + AlOx + Si (at 500 °C), Al2O3 + Si (at 700 °C), as the temperature during irradiation varies. Utilizing this finding it is possible to produce area selective interface patterning.

Electron irradiation induced amorphous SiO2 formation at metal oxide/Si interface at room temperature; electron beam writing on interfaces.

Al2O3 (5 nm)/Si (bulk) sample was subjected to irradiation of 5 keV electrons at room temperature, in a vacuum chamber (pressure 1 × 10-9 mbar) and formation of amorphous SiO2 around the interface was observed. The oxygen for the silicon dioxide growth was provided by the electron bombardment induced bond breaking in Al2O3 and the subsequent production of neutral and/or charged oxygen. The amorphous SiO2 rich layer has grown into the Al2O3 layer showing that oxygen as well as silicon transport occurred during irradiation at room temperature. We propose that both transports are mediated by local electric field and charged and/or uncharged defects created by the electron irradiation. The direct modification of metal oxide/silicon interface by electron-beam irradiation is a promising method of accomplishing direct write electron-beam lithography at buried interfaces.

Energetics of Na(+)-Ca(2+) exchange in resting cardiac muscle.

The energetic effect of extracellular Na(+) removal and readmission (in a nominally Ca(2+)-free perfusate) in Langendorff-perfused ventricles of transgenic mice (TM), which overexpress the sarcolemmal Na(+)-Ca(2+) exchanger; normal mice (NM); young (7-12 days old) rats (YR); and older (13-20 days old) rats (OR) was studied. In all heart muscles, extracellular Na(+) removal induced an increase in heat production (H(1)). Na(+) readmission further increased heat production to a peak value (H(2)) followed by a decrease toward initial values. These effects were more marked in the YR and TM as compared with the OR and NM groups, respectively. Caffeine (1 mM), ryanodine (0.2 microM), and verapamil (1 microM) decreased H(1) and H(2) in both rat groups. EGTA (1 mM) decreased H(1) and H(2) in the YR but not in the OR group. Thapsigargin (1 microM) decreased H(1) and H(2) in all four hearts preparations. A possible interpretation is that Na(+)-Ca(2+) exchange acts as an energy-saving mechanism to prevent Ca(2+) accumulation at the junctional sarcoplasmic reticulum zone (JSR) and thus prevents further release of Ca(2+). Extracellular Na(+) removal lead to Ca(2+) accumulation in the JSR inducing further SR-Ca(2+) release and increased energy release. Na(+) readmission removes the accumulated Ca(2+) at the JSR (cleft) zone by exchanging Ca(2+) with Na(+) producing a transitory increase in energy release due to Na(+)-K pump activation.

Subcellular Ca2+ distribution with varying Ca2+ load in neonatal cardiac cell culture.

Recent work in our laboratory has investigated and modeled subcellular calcium compartmentation and Ca2+ movement under steady-state control conditions. This experimental study is directed to the further description and quantitation of cellular calcium compartmentation patterns and movements as correlated with contraction in neonatal rat cardiac myocytes in culture under a variety of calcium loading conditions. Compartmental contents were assessed after incubations in various [Ca2+]o, 0 Na+/1 mM Ca2+, and 10 microM ouabain/1.0 mM Ca2+ test solutions. The cellular components investigated include sarcolemmal bound, sarcoplasmic reticulum (SR), and mitochondrial calcium. The results indicate that 1) sarcolemmal calcium binding is insensitive to changes in [Ca2+]o in the range tested (0.25-6.0 mM) while highly sensitive to changes in [Na+]i; 2) SR is sensitive to both changes in [Ca2+]o and [Na+]i and exhibits a maximum loading capacity of approximately 750 micromol Ca2+/kg dw; 3) in the [Ca2+]o range between 0.25 and 2.0 mM, contractile amplitude is proportional to SR content; 4) the mitochondria comprise a high-capacity calcium-containing compartment that is sensitive to changes in [Ca2+]o but does not reach saturation under the conditions tested (0.25-8.0 mM [Ca2+]o); 5) SR calcium is divided into at least two functionally discrete pools, one of which is available for release to the myofilaments during a normal ICa-triggered contraction and other of which is caffeine releasable but unavailable for release to the myofilaments during a normal triggered release; and 6) mitochondrial calcium serves as a reservoir of calcium capable of replenishing and/or augmenting SR stores with anywhere from 10% to 50% of mitochondrial calcium cycling through SR calcium compartments.

Dibucaine displaceable sarcolemmal Ca2+ fraction in guinea-pig cardiac myocytes.

In previous work we found that Ca2+ bound to the internal leaflet of sarcolemma of the neonatal cultured rat cardiomyocytes may be displaced by a local anesthetic, dibucaine (DBC). This resulted in inhibition of Na/Ca exchange. Now we found that the DBC displaceable Ca2+ fraction may be demonstrated also in single, enzymatically isolated cardiomyocytes of adult guinea pigs. Only cell length was used as an index of the free sarcoplasmic Ca2+ concentration since we found that DBC is fluorescent when irradiated with UV light, the wave length overlapping that of Indo 1 fluorescence. DBC (0.5 mM) induced a contracture whose amplitude ranged from 30%-100% of that of the electrically stimulated twitch. Blocking of Na/Ca exchange with 0.5 mM Ni2+, or 0Na+, 0Ca2+ solution did not affect the amplitude nor the time course of the contracture. Caffeine (15 mM) superfused during exposure of the cell to DBC initiated contraction which relaxed despite continued caffeine superfusion. DBC also initiated contracture in cells pretreated with thapsigargin, a blocker of sarcoplasmic reticulum Ca2+ uptake. DBC did not initiate contracture in skinned myocytes and decreased their sensitivity to Ca2+. These results suggest that DBC displaces Ca2+ from sarcolemma and that the displacement results in inhibition of Na/Ca exchange. Inhibition may however, be overcome by high subsarcolemmal Ca2+ concentration induced by its SR release with caffeine. DBC rapidly blocked sodium and calcium currents in voltage clamped cells. Calcium current recovered within about a minute upon washout of DBC, whereas recovery of sodium current started after about 8 min and was completed within about 12 min. Rapid depolarisation from a holding potential of -80 mV to 0 or +20 mV potentiated the response of the cell to DBC. This suggests that the Ca2+ fraction which is displaced by DBC is distinct from that released by depolarisation and therefore does not contribute to activation of a normal, electrically stimulated contraction.

Mitochondrial Ca2+ uptake, efflux, and sarcolemmal damage in Ca2+-overloaded cultured rat cardiomyocytes.

The purpose of this study was to determine mitochondrial Ca2+ accumulation and its possible role in initiation of mitochondrial permeability transition (MPT) and sarcolemmal damage in Ca2+-overloaded cardiomyocytes. Cellular Ca2+ overload, generated secondary to ouabain or p-chloromercuribenzoate-stimulated cell Na+ concentration increase, induced Ca2+ accumulation in mitochondria (approximately 3/4 of total net uptake) as identified by kinetic analysis and verified by use of mitochondrial inhibition. Mitochondrial Ca2+ uptake was followed by a rapid Ca2+ efflux ( approximately 1 mmol . kg dry wt-1 . min-1) that can be best explained by efflux via Ca2+-dependent nonspecific pores. Cell ATP concentration was stable during mitochondrial Ca2+ uptake and decreased in parallel with Ca2+ efflux. In addition, sarcolemmal damage was not related to the increase in mitochondrial Ca2+ concentration per se, but rather connected with the extent of Ca2+ efflux from the mitochondria. A decrease in the rate of this Ca2+ efflux, indicating also a decrease in a subpopulation of mitochondria with open pores, was followed by decreased sarcolemmal damage. Both dithiothreitol and cyclosporin A decreased rapid Ca2+ efflux and inhibited sarcolemmal damage, implicating MPT as an important component in the mechanism of sarcolemmal damage.

Dependence of cardiac cell Ca2+ permeability on sialic acid-containing sarcolemmal gangliosides.

The specific removal of negatively-charged sialic acid by neuraminidase produces a large increase in cardiac myocyte Ca uptake (17.3 +/- 1.1 mmol Ca/kg dry weight) and marked cell contracture. Importantly, the insertion of the negatively-charged amphiphile dodecyl sulfate in the sarcolemma eliminates the increased calcium uptake and preserves contractile function. In the present study, we further examine the role of sialic acid-Ca interaction and, specifically, the role of gangliosides (sialic acid-containing glycolipids) in cardiac cells' Ca permeability. Neonatal cell culture and adult ventricular myocytes were used. The major findings of this study are: (1) while dodecyl sulfate inhibits cellular calcium uptake and contracture development induced by sialic acid removal, cationic and neutral amphiphiles are without effect. (2) Ca channel blockers (nifedipine and protamine) and the Na/Ca exchange inhibitor Ni do not modify the effect of sialic acid removal. (3) A non-classical-channel related whole-cell current appears and increases after 21 +/- 2.2 min treatment with 0.02 U/ml neuraminidase (n = 4). Incubation with neuraminidase in the presence of dodecyl sulfate greatly delays the appearance of these currents to 44.4 +/- 6.1 min (n = 4). (4) The use of a specific probe for GM1 ganglioside, the cholera toxin B subunit (3 micrograms/ml), induces a moderate but clear increase in cellular Ca (1.63 +/- 0.3 mmol Ca kg dry weight; n = 8). However, this increase was not modified by treatment with dodecyl sulfate. (5) Finally, 50 mU/ml endoglycoceramidase, an enzyme which specifically cleaves the link between the sialic acid-containing oligosaccharide and ceramide of gangliosides, induced a significant increase in Ca uptake (4.4 +/- 0.9 mmol Ca kg dry weight, n = 4). These results indicate the importance of negatively charged sialic acid-containing gangliosides in the maintenance of cardiac cell physiological Ca permeability. The increase in Ca uptake induced by the removal of sialic acid seems to be mediated by development of a Ca "leak" via other than classical cation channels.

Calcium concentration and movement in the ventricular cardiac cell during an excitation-contraction cycle.

This paper extends the model for Ca movement in the cardiac ventricular cell from the diadic cleft space to the entire sarcomere. The model predicts the following: 1) Shortly after SR release there is a [Ca] gradient >3 orders of magnitude from cleft center to M-line which, 50 ms after release, is still >30. Outside the cleft, 40 ms after cessation of release, the axial gradient from Z to M-line is >3. 2) At the end of SR release, >50% of the total Ca released is bound to low-affinity inner sarcolemmal phospholipid binding sites within the cleft. 3) Halving the SR release almost doubles the fraction of release removed from the cell via Na/Ca exchange and reduces average sarcomeric free [Ca] by 70%. 4) Adding 100 microM fluo-3, which doubles the buffering capacity of the cytoplasm, reduces peak average sarcomeric [Ca] by >50% and increases the initial half-time for [Ca] decrease by approximately twofold. 5) A typical Ca "spark" can be generated by an SR release 20% of maximum (4 x 10(-20) moles) over 2 ms. Fluo-3 (100 microM) significantly "shrinks" the spark. 6) The "spark" is a consequence of elementary events within the diadic cleft space. For example, removal of cleft binding sites would cause average sarcomeric Ca to increase by >10 fold, fall 10 times more rapidly, decrease latency for appearance of the spark by >20 times, and reduce spark duration by 85%. 7) Dividing SR Ca release between cleft and corbular SR produces a secondary [Ca] peak and a "flattening" of the sarcomeric [Ca] transient. These changes probably could not be resolved with current confocal microscopic techniques.

pHe, [Ca2+]e, and cell death during metabolic inhibition: role of phospholipase A2 and sarcolemmal phospholipids.

This study measures cellular lactate dehydrogenase (LDH) release during metabolic inhibition as a monitor of sarcolemmal integrity as affected by variation of external pH (pHe) and Ca2+ concentration ([Ca2+]e). The sigmoidal relationship between pHe and LDH release and pHe and net Ca2+ uptake was essentially identical with the 50% maximal value occurring at pH 7.0 for both. This suggests that a process(es) sensitive to both pHe and [Ca2+]e plays a role in cell lysis during the course of metabolic inhibition. Variation of pHe during metabolic inhibition did not alter the decline in cellular ATP, nor did it affect changes in sarcolemmal phospholipid topology. Intracellular pH followed changes of pHe with a few minutes lag. Cell lysis increased in a graded manner as pHe and [Ca2+]e were increased, but pHe was the sole determinant of lysis, i.e., [Ca2+]e level had no effect, at the lowest (6.2) and the highest (8.0) pHe levels. pHe variation did not affect the release of radiolabeled arachidonic acid, nor did inhibitors of phospholipase A2 (PLA2) affect cell lysis at varying pHe. Therefore, cellular PLA2 activation could not be implicated for a role in cell lysis in the present model of metabolic inhibition. Alternatively, we propose that Ca2+ binding to the cytoplasmic leaflet, in combination with membrane alterations secondary to the metabolic insult, combine to destabilize the sarcolemma (20). This Ca2+ binding to the negatively charged phosphatidylserine results in the expression of the bilayer destabilizing effect of phosphatidylethanolamine. This Ca2+ binding is greatly diminished by lowered pH, resulting in an attenuation of cell lysis.

Matching Ca efflux and influx to maintain steady-state levels in cultured cardiac cells. Flux control in the subsarcolemmal cleft.

The study examines the feed-back mechanism by which Ca efflux via Na/Ca exchange adjusts to match Ca influx via Ca channels-the condition which must obtain if cellular steady-state Ca level is to be maintained. It is proposed that variation in Ca channel influx produces changes in sarcoplasmic reticulum content which, in turn, through interaction within the subsarcolemmal cleft space, sets the level of Na/Ca exchange-mediated efflux. We measured Ca channel influx with whole-cell voltage-clamp, Ca efflux specifically via Na/Ca exchange by high resolution, non-perfusion-limited 45Ca wash-out technique and sarcoplasmic reticulum (SR) Ca content by combination of the latter with measurement of sarcolemmal Ca-binding by use of instantaneous "gas-dissected" sarcolemmal membrane isolation. Ca channel influx was varied by Bay K 8644 or nifedipine. Ca influx via Ca channels was increased by 37.8% with Bay K and decreased by 68% with nifedipine. The SR contribution to Na/Ca exchange-mediated efflux was increased by 36.7% with Bay K and reduced by 66.8% by nifedipine-virtually identical changes. Application of the cleft space model to determine beat-to-beat efflux via Na/Ca exchange predicted values which closely matched beat-to-beat channel influx.

The effects of temperature upon calcium exchange in intact cultured cardiac myocytes.

Cardiac myocyte Ca transport systems, such as sarcoplasmic reticulum Ca-ATPase and sarcolemmal Na/Ca exchange (Na/Ca), are critically dependent on temperature. The purpose of this work was to study the effect of temperature on cellular Ca compartmentation and its exchange characteristics in intact functional neonatal cultured myocytes. The Na/Ca mediated Ca exchange (CaNa/Ca)--including its sarcoplasmic reticulum (SR) and sarcolemmal (SL) contributions, a slow exchange component related to mitochondrial Ca and the La displaceable Ca pool were studied combining isotopic and gas-dissection techniques for membrane isolation. The major findings of this study are: (i) The amount of Ca exchanged through CaNa/Ca is clearly dependent on temperature (Q10 approximately 1.6) in the range studied (17-37 degrees C); (ii) the addition of 1 microM nifedipine does not modify the temperature dependence of CaNa/Ca; (iii) the sarcolemmal bound fraction contributing to CaNa/Ca is not changed by temperature; (iv) the increase in CaNa/Ca with temperature is explained by an increment in the contribution of SR-Ca to CaNa/Ca; (v) a fraction of SR which does not exchange via CaNa/Ca at low temperatures can be released and mobilized by caffeine-this caffeine sensitive fraction is reduced as temperature is increased and is no longer measurable as a separate entity at 37 degrees C; (vi) if we consider (iv) and (v) together, SR content would be temperature dependent with a Q10 of approximately 1.5; (vii) a La displaceable pool, which represents over 66% of the total exchangeable Ca, increases in the range of 22-33 degrees C with a Q10 of 1.25 which is consistent with a pool distribution of 70% SL-bound and 30% SR-derived [Post J.A., Langer G.A. Cellular origin of the rapidly exchangeable calcium pool in the cultured neonatal rat heart cell. Cell Calcium 1992; 13: 627-634]; and (viii) the rate constant for the mitochondrial Ca component increases by 60% from 22 degrees C to 37 degrees C, but Ca content in this organelle is not modified over this temperature range.

Effect of sarcoplasmic reticulum Ca release into diadic region on Na/Ca exchange in cardiac myocytes.

The hypothesis that sarcoplasmic reticulum (SR) generates subsarcolemmal [Ca] higher than that in the bulk of sarcoplasm and that it affects the rate of Na+Ca exchange were tested. Voltage clamped cardiomyocytes of guinea-pigs and rats were stimulated by pre-pulses from a holding potential of -80 mV to -40 mV (20 ms) followed by 200 ms depolarizations to +5 mV. Single 5, 10, 20, 30, 50, 100 and 300 ms depolarizations were interposed between 200 ms pulses. The amplitude of the tail (Na/Ca exchange) currents recorded upon repolarization were compared with instantaneous fluorescence on Indo 1 loaded into cells. In both species amplitude of the tail currents were higher during the ascending phase of the Ca transient than during the descending phase, although the fluorescence was lower. The dissociation was abolished by thapsigargin (TG), the blocker of the SR Ca-ATPase. The results suggest that over the initial < 50 ms of the transient the Na/Ca exchangers are exposed to [Ca] higher than that in the bulk sarcoplasm and that it is generated by the SR.

Characterization of the calcium overload in cultured neonatal rat cardiomyocytes under metabolic inhibition.

We studied the calcium compartmentation of the Ca overload induced by 50 min of metabolic inhibition (MI) in cultured neonatal rat cardiomyocytes. MI was achieved by application of 1 mM iodoacetic acid (IAA) and 10 mM 2-deoxyglucose (2-DOG) with omission of glucose in the perfusate. MI per se abolished spontaneous beating, but 10 mM caffeine pulses at 15, 30 and 45 min were able to induce contractile responses. The contractile responses were abolished in the presence of Thg. After 20 min of MI, cells began to accumulate Ca. After 50 min of MI, the Ca exchanged via Na/Ca exchange (Na/Ca exchange-dependent Ca content) was increased by 3.21 +/- 0.15 mmol/kg dry weight (n = 5). The net increase remaining exchangeable via Na/Ca exchange represents 80% of the global net 45Ca uptake after 50 min in MI which was 3.98 mmol/kg dry weight, obtained in the "on-line" experiments. The release of this component of Ca is completed within 150s via Na/Ca exchange. There also was a 50% increase in the Ca content of the so-called "slow" compartment, 0.44 +/- 0.08 mmol/kg dry weight (n = 10) after 50 min under MI. The t1/2 of this compartment was 24.8 +/- 3.8 min typical of a mitochondrial origin in these cells. Thus 92% of the net Ca increase after 50 min MI remains exchangeable-80% of this is completed within 150s via Na/Ca exchange and 20% more slowly, t1/2 about 25 min. Cell ATP content, 27.35 +/- 2.07 nmoles/mg protein, was decreased after 10 min of MI to 2.25 +/- 0.29 nmoles/mg protein (n = 10) and remained close to this level after 20 min of MI (2.11 +/- 0.17 nmoles/mg protein; n = 6). It is to be noted that our results indicate no compromise of the Na/Ca exchange system after 50 min of MI although the global ATP is depleted in these cells.

Inner sarcolemmal leaflet Ca(2+) binding: its role in cardiac Na/Ca exchange.

A recently completed model of Ca concentration and movements in the cardiac cell diadic cleft space predicts that removal or neutralization of inner sarcolemmal (SL) leaflet anionic Ca-binding sites at the sarcolemmal border of this space will greatly diminish Na/Ca exchange-mediated Ca efflux. The present study tests this prediction using the local anesthetic dibucaine as a probe. It is shown, in isolated SL, that dibucaine competitively displaces Ca specifically from anionic phospholipid headgroups. Dibucaine also displaces Ca from the SL when applied to intact cells. It does not affect the content or release of Ca from sarcoplasmic reticulum (SR) in these cells. This eliminates a primary effect on SR Ca as a contributing factor to dibucaine's effect on Na/Ca exchange-mediated Ca efflux. Measurement of this efflux from whole cells shows a highly significant reduction of 58% (p < 0.001) by 0.5 mM dibucaine. The inhibiting effect of dibucaine on Na/Ca exchange-mediated Ca efflux can be significantly reversed by augmentation of Ca release from SR by caffeine at the time of activation of Na/Ca exchange. This supports the contention that the dibucaine-SL interaction is a competitive one vis-a-vis Ca. The results are supportive of the model in which inner SL leaflet Ca-binding sites account for the delay of Ca diffusion from the diadic cleft, thereby prolonging the time for which [Ca] remains elevated in the cleft. The prolonged increased [Ca] significantly enhances the ability of Na/Ca exchange to remove Ca from the cell during the excitation-contraction cycle.

Calcium concentration and movement in the diadic cleft space of the cardiac ventricular cell.

We model the space between the junctional sarcoplasmic reticulum (JSR) membrane and the inner leaflet of the transverse tubular ("T") sarcolemmal (SL) membrane, the diadic cleft, with respect to calcium (Ca) concentration and movement. The model predicts the following: 1) Ca influx via the "L" channel increases [Ca] to 1 microM within a distance of 50 nm from the channel mouth in < 500 microseconds. This is sufficient to trigger Ca release from a domain of 9 "feet." 2) By contrast, "reverse" Na/Ca exchange will increase [Ca] to approximately 0.5 microM throughout the cleft space in 10 ms, sufficient to trigger Ca release, but clearly to a lesser extent and more slowly than the channel. 3) After a 20-ms JSR release into the cleft via the "feet" [Ca] peaks at 600 microM (cleft center) to 100 microM (cleft periphery) and then declines to diastolic level (100 nM) within 150 ms throughout the cleft. 4) The ratio of flux out of the cleft via Na/Ca exchange to flux out of the cleft to the cytosol varies inversely as JSR Ca release. 5) Removal of SL anionic Ca-binding sites from the model will cause [Ca] to fall to 100 nM throughout the cleft in < 1 ms after JSR release ceases. This markedly reduces Na/Ca exchange. 6) Removal from or decreased concentration of Na/Ca exchangers in the cleft will cause [Ca] to fall too slowly after JSR release to permit triggered release upon subsequent excitation.

Functional coupling between sarcoplasmic reticulum and Na/Ca exchange in single myocytes of guinea-pig and rat heart.

It has been proposed that the cardiac Na/Ca exchanger is primed by high Ca2+ concentrations derived from the sarcoplasmic reticulum (SR) in a recently identified subsarcolemmal, "Na/Ca exchange-dependent Ca2+ compartment". We tested this hypothesis by investigating the effect on Na/Ca exchange of interventions affecting Ca2+ flux through SR. Experiments were performed in single, isolated myocytes of guinea-pig and rat hearts loaded with Indo 1-AM and free Ca2+ concentration was assessed by measuring the ratio of fluorescence at 405 and 495 nm wavelength. In guinea-pig 1.0 microM ryanodine (Ry), expected to increase Ca2+ flux through the SR, decreased the amplitude of electrically stimulated Ca2+ transients by 35% and inhibited their initial, rapid phase. Responses of these cells to brief superfusions with 15 mM caffeine were inhibited which suggests that 1.0 microM Ry depleted the SR Ca2+ stores. Diastolic Ca2+ concentration was slightly increased, but it dropped below control during prolonged rest. Decrease of the amplitude of the transients in these ryanodine-treated cells were reversed by 2 x 10(-7) M thapsigargin (TG), an inhibitor of the SR Ca(2+)-ATPase, by 1.0 mM Ry, a blocker of the SR Ca2+ release channels and by low Na+ (50.0 mM) superfusion. This suggests that the decreased transients in 1.0 microM Ry result from uptake of Ca2+ by the SR and its rapid release to the subsarcolemmal (cleft) space where a fraction is diverted out of the cell via Na/Ca exchange before it can diffuse to the myofilaments. Removal of the SR from the pathway (addition of TG on 1.0 mM Ry) or reversal of Na/Ca exchange diverts more transsarcolemmal Ca2+ influx to the myofilaments and increases the Ca2+ transient. Decrease of the resting Ca2+ concentration was blocked by 2 x 10(-7) M TG, 1.0 mM Ry, and by 5.0 mM Ni2+, a blocker of Na/Ca exchange. This result suggests that the effect of 1.0 microM Ry on resting Ca2+ concentration also resulted from increase of flux of Ca2+ through the SR and out of the cell by Na/Ca exchange. In rat 1.0 microM Ry decreased amplitude of the transients by approximately 75% but did not affect their kinetics. TG and 1.0 mM Ry decreased the rate of rise of the transients and greatly delayed their decay. This result suggests that normal kinetics of the transients in the cells treated with 1.0 microM Ry depended on the preserved Ca2+ flux through the SR. As SR was not able to retain Ca2+ in these cells, decay of the transients must have depended on stimulated Na/Ca exchange. The results in guinea-pig and rat taken together are compatible with the proposal that Ca2+ released from the SR interacts with the cell's Na/Ca exchanger most probably within the newly defined subsarcolemmal "Na/Ca exchange-dependent Ca2+ compartment".

Organization and function of sarcolemmal phospholipids in control and ischemic/reperfused cardiomyocytes.

The topic of this review is the lipidic part of the sarcolemma, the plasma membrane of the myocardial cell, and its role in (dis)function of the cardiomyocyte. First the isolation of the sarcolemma and its lipid composition are discussed. These phospholipids are not randomly distributed over the two monolayers of the lipid bilayer and negatively charged phospholipids are exclusively present in the cytoplasmic leaflet of the sarcolemma, which also contains the majority of phosphatidylethanolamine. This distribution is most likely caused by an active transport of these lipids and by an interaction of the headgroup of these lipids with the cytoskeleton. Subsequently the physicochemical properties of sarcolemmal phospholipids are discussed, where it is shown that certain phospholipids prefer non-bilayer phases, and the effects of sarcolemmal phospholipids on trans-sarcolemmal ion fluxes and calcium compartmentation are discussed. In the second part the effect of ischemia on sarcolemmal phospholipids is discussed with regard to: transbilayer distribution, hydrolysis, lateral distribution and sarcolemmal bilayer stability. In our view, onset of ischemia initiates a sequence of events leading to a loss of normal sarcolemmal phospholipid distribution with an outward migration of phosphatidylethanolamine. There follows, as ischemia progresses, loss of sarcolemmal bilayer stability due to the expression of the non-bilayer behavior of phosphatidylethanolamine, leading to irreversible disruption of the sarcolemma and cell death.

Increase in calcium leak channel activity by metabolic inhibition or hydrogen peroxide in rat ventricular myocytes and its inhibition by polycation.

Ca influx in cultured neonatal myocardium can be augmented by metabolic inhibition or free radical exposure. This increase cannot be prevented by blockade of L-type Ca channels or inhibition of Na-Ca exchange. It is speculated that a specific Ca leak may be involved in this process. In the present study patch clamp techniques were used to examine this hypothesis. Currents were measured in a recording configuration of cell attached or excised inside-out membrane patches in adult rat ventricular myocytes as affected by metabolic inhibition or free radical exposure. The metabolic inhibition was achieved by use of 1 mM iodoacetic acid and 10 mM 2-deoxyglucose with omission of glucose in the perfusate. Free radicals were generated by application of 100 microM hydrogen peroxide in the perfusate. Specific Ca leak channels were identified. The channels were not significantly permeable to monovalent cations. The activity of these channels was increased markedly over a period of minutes by metabolic inhibition or free radical exposure. In the presence of 100 microM hydrogen peroxide the open probability (NPo) of the channels increased from 0.0083 +/- 0.003 (mean +/- S.D.) in control to 0.09 +/- 0.024 (P < 0.01). During metabolic inhibition it was augmented from 0.0075 +/- 0.0035 to 0.062 +/- 0.018 (P < 0.01). The increase of the Ca leak channel activity under both conditions could be completely blocked by addition of polycationic protamine to the patch pipette solution. The results support the hypothesis that Ca leak channels are involved in the Ca overload induced by metabolic inhibition or free radical exposure. The inhibitory effect of polycation may have important therapeutic implications for the treatment of ischemic cardiac heart disease.