Intracellular Concentrations Of Na Research Articles

Sodium and Epithelial Sodium Channels Participate in the Regulation of the Capacitation-associated Hyperpolarization in Mouse Sperm

In a process called capacitation, mammalian sperm gain the ability to fertilize after residing in the female tract. During capacitation the mouse sperm plasma membrane potential (E(m)) hyperpolarizes. However, the mechanisms that regulate sperm E(m) are not well understood. Here we show that sperm hyperpolarize when external Na(+) is replaced by N-methyl-glucamine. Readdition of external Na(+) restores a more depolarized E(m) by a process that is inhibited by amiloride or by its more potent derivative 5-(N-ethyl-N-isopropyl)-amiloride hydrochloride. These findings indicate that under resting conditions an electrogenic Na(+) transporter, possibly involving an amiloride sensitive Na(+) channel, may contribute to the sperm resting E(m). Consistent with this proposal, patch clamp recordings from spermatogenic cells reveal an amiloride-sensitive inward Na(+) current whose characteristics match those of the epithelial Na(+) channel (ENaC) family of epithelial Na(+) channels. Indeed, ENaC-alpha and -delta mRNAs were detected by reverse transcription-PCR in extracts of isolated elongated spermatids, and ENaC-alpha and -delta proteins were found on immunoblots of sperm membrane preparations. Immunostaining indicated localization of ENaC-alpha to the flagellar midpiece and of ENaC-delta to the acrosome. Incubations known to produce capacitation in vitro or induction of capacitation by cell-permeant cAMP analogs decreased the depolarizing response to the addition of external Na(+). These results suggest that increases in cAMP content occurring during capacitation may inhibit ENaCs to produce a required hyperpolarization of the sperm membrane.

Na+ stimulates binding of dopamine to the dopamine transporter in cells but not in cell-free preparations.

Although Na+ is crucial for the function of the dopamine (DA) transporter (DAT), its role in the substrate binding step has been questioned. To address this issue, we investigated the effect of Na+ on DA binding by measuring the potency of DA in inhibiting the binding of the cocaine analogue [3H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT) in intact cells expressing DAT in their plasma membranes and in membranes isolated from these cells. In cells, Na+ substantially enhanced the potency of DA in inhibiting CFT binding. This effect of Na+ was independent of buffer compositions and substitutes (sucrose vs. NMDG), more pronounced at 4 degrees C than 25 degrees C, and correlated with its stimulatory effect on DA uptake Km. Removing extracellular Na+ had little effect on intracellular concentrations of Na+ and K+, or on membrane potential. These data suggest that extracellular Na+ most likely acts at the transporter level to enhance the binding of external DA during the transport cycle. In contrast, in cell-free membrane preparations the Na+ stimulation was abolished without impairment of the potency of DA in inhibiting CFT binding, regardless of whether sucrose was used to maintain the buffer osmolarity. The difference in Na+ dependence for DA to inhibit CFT binding between plasma membranes of intact cells and isolated membranes raises the possibility that intracellular ion environment, alone or in combination with other cellular factors, plays a critical role in determining DA-DAT interaction and the integration of Na+ modulation in this interaction.

Multi-barrelled ion-selective microelectrodes as tools for the investigation of volume regulation mechanisms in invertebrate nerve cells under hyperosmotic conditions

Changes in cell volume can be recorded by a potentiometric method based on ion-selective microelectrodes filled with the classical K + exchanger Corning 477317, which is very sensitive to large quaternary ammonium ions (PNAS 82 (1985) 6014; Electrochim. Acta 47 (2001) 309). We investigated the effect of hyperosmotic bathing solutions on cell volume, the intracellular concentrations of Na +, Cl −, and K + ([Na +] i, [Cl −] i, [K +] i) as well as on the membrane potential of leech Retzius neurones by using multi-barrelled ion-selective microelectrodes. We found that an increase in the extracellular NaCl concentration induced a shrinkage of the cells as well as an increase in [Na +] i, [Cl −] i and [K +] i. Provided that the change in osmolality was moderate (≤+40%), cell shrinkage and the changes in [Na +] i, [Cl −] i and [K +] i were proportional to the increase in the extracellular osmolality, i.e. the cells behaved like ideal osmometers. When the bath osmolality was increased more strongly, significant differences from the ideal behaviour were observed. In particular, the changes in [Na +] i and [Cl −] i were larger than expected from the changes in cell volume. The results show that leech neurones raise their intracellular osmolyte content in response to severe osmotic stress, thereby reducing changes in their cellular volume.

Differential expression of the Na+-Ca2+ exchanger transcripts and proteins in rat brain regions.

In the central nervous system (CNS), the Na(+)-Ca(2+) exchanger plays a fundamental role in controlling the changes in the intracellular concentrations of Na(+) and Ca(2+) ions. These cations are known to regulate neurotransmitter release, cell migration and differentiation, gene expression, and neurodegenerative processes. In the present study, nonradioactive in situ hybridization and light immunohistochemistry were carried out to map the regional and cellular distribution for both transcripts and proteins encoded by the three known Na(+)-Ca(2+) exchanger genes NCX1, NCX2, and NCX3. NCX1 transcripts were particularly expressed in layers III-V of the motor cortex, in the thalamus, in CA3 and the dentate gyrus of the hippocampus, in several hypothalamic nuclei, and in the cerebellum. NCX2 transcripts were strongly expressed in all hippocampal subregions, in the striatum, and in the paraventricular thalamic nucleus. NCX3 mRNAs were mainly detected in the hippocampus, in the thalamus, in the amygdala, and in the cerebellum. Immunohistochemical analysis revealed that NCX1 protein was mainly expressed in the supragranular layers of the cerebral cortex, in the hippocampus, in the hypothalamus, in the substantia nigra and ventral tegmental area, and in the granular layer of the cerebellum. The NCX2 protein was predominantly expressed in the hippocampus, in the striatum, in the thalamus, and in the hypothalamus. The NCX3 protein was particularly found in the CA3 subregion, and in the oriens, radiatum, and lacunoso-moleculare layers of the hippocampus, in the ventral striatum, and in the cerebellar molecular layer. Collectively, these results suggest that the different Na(+)-Ca(2+) exchanger isoforms appear to be selectively expressed in several CNS regions where they might underlie different functional roles.

Brain distribution of the Na+/Ca2+ exchanger-encoding genes NCX1, NCX2, and NCX3 and their related proteins in the central nervous system.

In the central nervous system, the Na(+)/Ca(2+) exchanger plays a fundamental role in controlling changes in the intracellular concentrations of Na(+) and Ca(2+) ions that occur in physiologic conditions such as neurotransmitter release, cell migration and differentiation, gene expression, as well as neuro-degenerative processes. Three genes, NCX1, NCX2, and NCX3, encoding for Na(+)/Ca(2+) exchanger isoforms have been cloned. In this review, by using non-radioactive in situ hybridization and light immunohistochemistry with NCX isoform-specific riboprobes and antibodies, respectively, a systematic brain mapping for both transcripts and proteins encoded by all three NCX genes is described. Intense expression of NCX transcripts and proteins was detected in the cerebral cortex, hippocampus, thalamus, metathalamus, hypothalamus, brainstem, spinal cord, and cerebellum. In these areas, NCX transcripts and proteins were often found with an overlapping distribution pattern, although specific brain areas displaying a peculiar expression of each exchanger isoform were also found. Furthermore, immunoelectron and confocal microscopy revealed the expression of the NCX1 isoform of the exchanger at both pre- and postsynaptic sites as well as in association with membranes of the endoplasmic reticulum. Collectively, these data suggest that the different isoforms of the Na(+)/Ca(2+) exchanger appear to be selectively expressed in several CNS regions where they might underlie different functional roles.

High salt‐treatment‐induced Na+ extrusion and low salt‐treatment‐induced Na+ accumulation in suspension‐cultured cells of the mangrove plant, Bruguiera sexangula

AbstractA suspension‐cultured cell strain of the mangrove plant (Bruguiera sexangula) was established from a callus culture and maintained in an amino acid medium in the absence of NaCl. NaCl non‐adapted cells were transferred to media containing 0–200 mm NaCl. The initial growth rate decreased gradually with increasing salt concentrations. However, at up to 150 mm NaCl, cell number growth at the highest point was almost the same as that at lower salt concentrations. Cells even continued to grow in the presence of 200 mm NaCl.Cells incubated in a medium containing 50 mm NaCl for 3 weeks accumulated Na+, while those incubated in 150 mm NaCl for 2 d showed only a transient increase in Na+ and Cl– concentrations. In the latter treatment, the intracellular concentration of Na+ returned to the original low level within 2 weeks. It took a longer time for Cl– to return to its original level. As a result, the Na+ and Cl– concentrations in cells cultured with 50 mm NaCl were much larger than those in cells cultured with 150 mm NaCl. The intracellular distribution of ions after transfer to the medium containing 150 mm NaCl was analysed by isolating the vacuoles.Treatment with amiloride, an inhibitor of the Na+/H+ antiporter, suppressed the recovery of Na+ to the original level in the cells. Treatment with 150 mm NaCl for 3 d stimulated the activities of both the vanadate‐dependent H+‐ATPase and the Na+/H+ antiporter in the plasma membrane fraction.

Alterations in the structure of phycobilisomes of the cyanobacterium, Spirulina platensis in response to enhanced Na+ level

Incubation of Spirulina platensis at enhanced Na+ concentrations resulted in 30% increased intracellular accumulation of Na+ ions. This accumulation of Na+ ions intracellularly altered the phycobilisome organization as revealed by transmission electron microscopic data and changes in the absorption spectrum. The room temperature emission peak at 638 nm in the control sample was blue-shifted by 7 nm in the treated samples suggesting phycobilisome disorganization. SDS-PAGE of phycobilisome polypeptides showed a significant increase in a 66.2 kDa polypeptide. This is the first report that the enhanced concentration of intracellular Na+ ion alters the structure of the phycobilisome in S. platensis.

Intracellular ion levels in erythrocytes and hepatocytes isolated from three teleost species

Intracellular concentrations of Na+ and K+ were similar (∼75 mmol l−1) in rainbow trout Oncorhynchus mykiss hepatocytes directly following isolation by collagenase digestion, but partial recovery occurred over 6 h with K+ levels increasing to 110 mmol l−1 and Na+ levels decreasing to 42 mmol l−1. Black bullhead Ameiurus melas hepatocytes exhibited higher intracellular concentrations of K+ (90 mmol l−1) than Na+ (55 mmol l−1) with no recovery occurring over 6 h following cell isolation. Concentrations of Na+, K+ and Cl− in eel Anguilla rostrata hepatocytes were similar (∼ 55 mmol l−1) following isolation, with no recovery occurring over time. Erythrocytes from all species apparently did not experience an intracellular ion imbalance following isolation as indicated by high K+ levels (<140 mmol l−1) and low Na+ levels (<40 mmol l−1) during the entire 24‐h monitoring period. Although hepatocytes from all species exhibited an ion imbalance post‐isolation, comparison of their in vitro intracellular Na+ and K+ concentrations with those in plasma demonstrated that directionally correct ion gradients still exist across the cell membrane, albeit differing from those that would be found in the tissue in vivo.

Hepatic Hemodynamics and Cell Functions in Human and Experimental Sepsis

Hepatic Hemodynamics and Cell Functions in Human and Experimental Sepsis

Hepatic hemodynamics and cell functions in human and experimental sepsis.

Hepatic hemodynamics and cell functions in human and experimental sepsis.

Both d- cis- and l- cis-diltiazem attenuate hydrogen peroxide-induced derangements in rat hearts

The effects of d- cis- and l- cis-diltiazem on the hydrogen peroxide (H 2O 2)-induced derangements of mechanical function and energy metabolism, and accumulation of intracellular Na + were studied in isolated rat hearts. The intracellular concentration of Na + ([Na +] i ) in the myocardium was measured using a nuclear magnetic resonance technique. H 2O 2 (600 μM) increased the left ventricular end-diastolic pressure, decreased the tissue level of ATP, and increased the release of lactate dehydrogenase from the myocardium. These alterations induced by H 2O 2 were significantly attenuated by d- cis-diltiazem (15 μM) or l- cis-diltiazem (15 μM). H 2O 2 (1 mM) produced a marked increase in the myocardial [Na +] i , which was effectively inhibited by tetrodotoxin (3 μM), d- cis-diltiazem (15 μM) or l- cis-diltiazem (15 μM). These results suggest that both d- cis- and l- cis-diltiazem protect the myocardium against the H 2O 2-induced derangements in the isolated, perfused rat heart. The protective action of d- cis- and l- cis-diltiazem may be due to their ability to inhibit the H 2O 2-induced increase in [Na +] i , at least in part.

Sodium pump evokes high density pump currents in rat midbrain dopamine neurons.

1. Patch pipettes contained various concentrations of Na+ ([Na+]pip) in order to record strophanthidin-sensitive currents under voltage clamp in dopamine neurons in slices of rat substantia nigra and ventral tegmental area. 2. When [Na+]pip was 40 mM and the external K+ concentration ([K+]o) was 2.5 mM, strophanthidin (10 microM) evoked 461 +/- 121 pA of inward current. This effect was concentration dependent, with an EC50 of 7.1 +/- 2.6 microM. At potentials of -60 to -120 mV, strophanthidin-induced currents were not associated with significant changes in chord conductance. 3. Strophanthidin (10 microM) evoked 234 +/- 43 pA of inward current when [Na+]pip was 0.6 mM, and 513 +/- 77 pA when [Na+]pip was 80 mM. Despite higher pump currents with greater [Na+]pip, the strophanthidin EC50 was not significantly different for any of six different [Na+]pip. 4. Sodium pump currents were half-maximal when the [Na+]pip was about 1.3 mM. Maximum pump current was estimated at 830 pA (29 microA cm-2) at concentrations of intracellular Na+ that were assumed to be saturating (50-100 mM). 5. Strophanthidin currents were smaller in a reduced [K+]o (EC50 = 0.2 mM). 6. These data show that intracellular Na+ loading evokes relatively large pump currents. Our results are consistent with the physiological role of the sodium pump in burst firing in midbrain dopamine neurons

Volume-induced chloride transport in HT29 cells studied by X-ray microanalysis.

Ion transport mediated by osmotically induced chloride channels in HT29 cells was investigated by X-ray microanalysis of cell cultures. The cells were cultured on cellulose nitrate filters, and exposed to isotonic, hypotonic, or hypertonic solutions. Then the NaCl-rich medium was removed by rinsing the cells with a washing fluid, the cells were frozen, freeze-dried, and analyzed. As washing solutions, distilled water, ammonium acetate, or mannitol (the latter two at different concentrations) were used. No major effects of the composition of the washing fluid was noted, except for mannitol, which left remnants that caused an apparent decrease in the intracellular elemental concentrations. In response to exposure to hypotonic solution (modified Krebs Ringer's buffer, KRB, with 90 mM NaCl), the cells lost Cl, K, and Na. On the other hand, exposure to hypertonic solution (modified KRB, 290 mM NaCl) caused an increase of the intracellular concentrations of Na and Cl and a decrease in K. In the presence of chloride channel blockers, chloride efflux induced by exposure to hypotonic medium was inhibited. This chloride efflux was also inhibited in the absence of extracellular calcium, indicating that the process is Ca2+ dependent.

Transient Na + stress in symbiotic dinoflagellates after isolation from coral-host cells and subsequent immersion in seawater

Dinoflagellates which live in intracellular symbiosis with corals (zooxanthellae), probably share the ionic conditions of their host cells, i.e. are subjected to lower sodium and calcium concentrations than ambient seawater. Although free-living zooxanthellae are not generally found in waters of reef ecosystems, they can be released in either a controlled diurnal regulation or an uncontrolled (coral bleaching) reaction by their animal hosts. Upon release, zooxanthellae experience new external ionic conditions. The aims of this study were to (1) examine the ionic conditions experienced by zooxanthellae in hospite, (2) determine changes in the intracellular Na+ concentration of dinoflagellates (Symbiodinium sp.) following isolation from the scleractinian coral Galaxea fascicularis (Linnaeus, 1767), and (3) characterize the mechanism of Na+ regulation and control. On the basis of equilibrium studies, it has been suggested that zooxanthellae in coral-host cells experience a Na+ concentration of ≃60 mM. The intracellular concentration of Na+ in zooxanthellae, as determined by flame photometry, was found to be ≃0.300 μequiv mg−1 protein, or 30 to 35 mM when the water content of cells was taken into account. Half the cell Na+ seems to be compartmentalized (i.e. non-exchangeable) in freshly isolated zooxanthellae (FIZ), while in cultured zooxanthellae (CZ) all the Na+ was exchangeable. Isolation of zooxanthellae into seawater from their intracellular environment caused a transient two-fold increase in Na+ concentration within the first 30 min. This increase was directly proportional to extracellular Na+ concentration, suggesting passive influx. After 30 min the Na+ concentration decreased, reaching its initial level within 1 to 3 h. Following isolation for up to 3 h, the Na+ influx rate measured during short incubations (5 min) was constant, suggesting that some Na+-regulation mechanism, probably a Na+ efflux system, was created or stimulated within the first 30 min. Since this mechanism is independent of DNA transcription (as proved by its insensitivity to 100 μM actinomycin D) and protein synthesis (insensitivity to 100 μM cycloheximide or emetin), we conclude that activation of an efflux system already present in the zooxanthella membrane occurred during the first 30 min after isolation in seawater. This mechanism was cyanide (CN)- and 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU)-sensitive and dependent on extracellular K+. However, while we found an ouabain-sensitive ATPase activity in zooxanthella homogenates, suggesting the involvement of a Na+/K+ ATPase, no correlation was found between Na+ efflux and K+ influx [measured using 86Rb (rubidium chloride) as a tracer]. The results presented show that isolation of symbiotic dinoflagellates into seawater causes major ionic stress, resulting in a two-fold increase in intracellular Na+ concentration. Within 30 min, a Na+-efflux mechanism (putatively involving Na+-ATPase), in the membrane of zooxanthellae in hospite is stimulated. The Na+ concentration of the cell consequently returns to its initial level 3 h after isolation.

The Na+-specific interaction between the LysR-type regulator, NhaR, and the nhaA gene encoding the Na+/H+ antiporter of Escherichia coli.

We used partially purified NhaR and a highly purified His-tagged NhaR derivative to identify the cis-regulatory sequences of nhaA recognized by NhaR and to study the specific effect of Na+ on this interaction. Gel retardation assay with DNase I footprinting analysis showed that NhaR binds a region of nhaA which spans 92 bp and contains three copies of the conserved LysR-binding motif. Na+, up to 100 mM, had no effect on the binding of NhaR to nhaA. The dimethylsulfate methylation protection assay in vivo and in vitro, showed that bases G-92, G-60, G-29 and A-24 form direct contacts with NhaR; in the absence of added Na+ in vivo, these bases were protected but became exposed to methylation in a DeltanhaR strain; accordingly, these bases were protected in vitro by the purified His-tagged NhaR. 100 mM Na+, but not K+, removed the protection of G-60 conferred by His-tagged NhaR in vitro. Exposure of intact cells to 100 mM Na+, but not K+, exposed G-60. The maximal effect of Na+ in vitro was observed at 20 mM and was pH dependent, vanishing below pH 7.5. In contrast to G-60, G-92 was exposed to methylation by the ion only in vivo, suggesting a requirement for another factor existing only in vivo for this interaction. We suggest that NhaR is both sensor and transducer of the Na+ signal and that it regulates nhaA expression by undergoing a conformational change upon Na+ binding which modifies the NhaR-nhaA contact points.

Differential effects of Ca 2+ channel blockers on Ca 2+ overload induced by lysophosphatidylcholine in cardiomyocytes

The effects of Ca 2+ channel blockers (verapamil, diltiazem, nicardipine, bepridil and flunarizine) on Ca 2+ overload induced by lysophosphatidylcholine were examined in rat isolated cardiomyocytes. Addition of lysophosphatidylcholine (15 μM) produced Ca 2+ overload as evidenced by a marked increase in the concentration of intracellular Ca 2+ and hypercontracture of cells. Verapamil, flunarizine and bepridil concentration dependently inhibited the lysophosphatidylcholine-induced Ca 2+ overload, whereas diltiazem and nicardipine did not. Lysophosphatidylcholine increased the release of creatine kinase, which was significantly attenuated by verapamil, flunarizine or bepridil (5 μM for each), but not by diltiazem or nicardipine (20 μM for each). Verapamil, flunarizine, bepridil (which attenuated the lysophosphatidylcholine-induced Ca 2+ overload) and nicardipine (which did not) inhibited the veratridine-induced increase in the concentration of intracellular Na + (indicated by the increase in fluorescence ratio of Na +-binding benzofuran isophthalate) and cell contracture, whereas diltiazem did not. These results suggest that verapamil, bepridil and flunarizine attenuate the Ca 2+ overload induced by lysophosphatidylcholine, and that the Ca 2+ channel blocking action of these drugs does not contribute substantially to this effect. The Na + channel inhibition together with high lipophilicity of these drugs may be important for the attenuation of the lysophosphatidylcholine-induced Ca 2+ overload.

X-ray microanalysis of intracellular ions in the anaerobic halophilic eubacterium Haloanaerobium praevalens

The intracellular concentrations of Na+, K+, and Cl− of the anaerobic halophilic eubacterium Haloanaerobium praevalens were assayed by means of X-ray microanalysis with the transmission electron microscope. Apparent intracellular cation concentrations between 1.22 and 1.91 M and chloride concentrations of 0.93–1.57 M were measured in cells growing exponentially in 2.6 M total salts. In exponentially growing cells, K+ was the major cation (70% of the cation sum). Stationary phase cells showed a high variability among individual cells, some of the cells containing higher Na+ than K+ concentrations.Key words: Haloanaerobium praevalens, intracellular ion concentrations, sodium, potassium, X-ray microanalysis.

Effects of ischemia on intracellular sodium and phosphates in the in vivo rat liver.

Metabolic factors that influence the transition form reversible to irreversible ischemic injury were studied in the rat liver in vivo with 31P-nuclear magnetic resonance (NMR) spectroscopy. Hepatic ischemia for 15, 35, or 65 min was produced by occlusion of the hepatic artery and portal vein in rats. Ischemia caused a rapid decrease in the ATP concentration ([ATP])-to-P(i) concentration ratio and pH within 5 min, but there was little change in these variables detectable by 31P-NMR with longer periods of ischemia. After reperfusion, the [ATP] and P(i) concentration returned toward normal values in livers exposed to 15 or 35 min of ischemia, but 65 min of ischemia were associated with only modest recovery in [ATP], and the [ATP] later decreased. Because the 31P-NMR spectrum was similar after brief compared with prolonged ischemia, it appears that neither ATP depletion, P(i) accumulation, nor acidosis predicts metabolic recovery. Hepatic intracellular NA+ was also measured in separate groups of animals by 23Na-NMR in the presence of a shift agent, thulium (III) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis (methylene-phosphonate) (TmDOTP5-), and by atomic absorption spectroscopy. Under baseline conditions, the concentration of intracellular Na+ was 15.2 mM by atomic absorption spectroscopy and 16.5 mM by 23Na-NMR. Although the 31P-NMR spectrum responded very rapidly to the onset of ischemia, intracellular Na+ concentration measured by 23Na-NMR increased gradually but steadily at approximately 1.0 mM/min during early (up to 15 min) ischemia. These observations demonstrate that a rise in intracellular Na+ does occur early ischemia, that TmDOTP5- can be applied in vivo for analysis of intracellular Na+ in the ischemic liver, and that 31P-NMR spectroscopy is very sensitive to early ischemic injury.

Evidence that reverse Na-Ca exchange can trigger SR calcium release.

Several results suggest that the Na-Ca exchange can function as a trigger promoting SR Ca release and ensuing contractions. First, if the Ca current was the sole trigger for contraction we would expect the relationship between triggered contractions and voltage to be similar to the relationship between Ca current and contraction. When Na is present in the pipette this is not observed. Between -40 and +10 mV the relationships between contractions and voltage and current and voltage are similar. At potentials positive to 10 mV the Ca current declines as expected but contractions either decline much more slowly or continue to increase depending upon the concentration of intracellular Na. In addition, we have observed that contractions can be activated when Ca current is largely or completely blocked. Since these contractions are sensitive to the presence of ryanodine and thapsigargin they appear to be triggered by Na-Ca exchange. Also, contractions that are activated in the presence of nifedipine are sensitive to the Na-Ca exchange inhibitor XIP. Finally, rapid removal of extracellular Na apparently stimulates enough reverse exchange triggering of SR Ca release without affecting the SR content. It is clear that the shape of the shortening voltage relationship depends upon the concentration of dialyzing Na. This is likely to occur for two reasons. Either the shape of the shortening voltage relationship depends upon the extent to which Na-Ca exchange contributes a trigger for SR Ca release or alternatively the shape of the shortening voltage relationship depends upon SR Ca content. The latter is known to depend upon the Na concentration. In addition it is now established that the gain of SR Ca release is influenced by SR content. However, we studied triggered contractions in the absence of a Na gradient when the only available trigger is the Ca current. We measured triggered contractions over a range of voltages between -30 and +60 mV. Between each measurement we reestablished the Na gradient and activated a series of conditioning pulses to standardize the SR Ca content. Just before a test pulse we removed extracellular Na and activated either 3 or 6 pulses to produce two different SR Ca loads (in the absence of a Na gradient entering Ca cannot be extruded and therefore changes the SR Ca content). Regardless of the number of prepulses in the absence of a Na gradient the shortening voltage relationship was similar and bell shaped. From this we conclude that the shape of the relationship between shortening and voltage does not depend upon SR Ca content. Therefore, we conclude that the asymmetry in the shortening voltage relationship that depends upon intracellular Na is due to a contribution of reverse Na-Ca exchange. It is too early to say what the physiological significance (if any) of triggering by reverse exchange actually is. However, it does seem likely that it might provide a powerful inotropic mechanism. For example intracellular Na might be expected to change with heart rate and to be elevated at higher heart rates. Presumably this increased intracellular Na would tend to favor triggering by reverse exchange and would therefore enhance contractility at a time when it would be most required.

Inhibition of Na+K+ATPase Activity in Membranes of Sindbis Virus-Infected Chick Cells

Alterations in intracellular concentrations of Na+and K+in Sindbis virus-infected cells result largely from inhibition of ouabain-sensitive Na+K+ATPase (Na+pump) activity. Here we report that membrane preparations derived from Sindbis virus-infected chick cells exhibit reduced Na+K+ATPase activity, indicating that limitation of cellular factors is not responsible for inhibition of ion transport.In vitrophosphorylation of the Na+K+ATPase by [32P]orthophosphate or [γ-32P]ATP is unaltered in membranes of Sindbis virus-infected cells, indicating that a loss of specific enzymatic functions unrelated to formation of Na+pump phosphoenzyme intermediates occurs during the course of viral infection. However, phosphoenzyme intermediates of the Na+K+ATPase prepared from membranes of Sindbis virus-infected cells are inherently less stable than those prepared with membranes of uninfected cells. The instability of these intermediatesin vitrois correlated with an altered capacity of the Na+pump to transport monovalent cations into virus-infected cells.22Na+transport studies reveal enhanced ouabain-sensitive Na+uptake by Sindbis virus-infected cells, suggesting that the Na+pump may catalyze enhanced Na+-Na+exchange in the infected cells. These results indicate that the capacity of the Na+K+ATPase to discriminate between binding of extracellular Na+and K+is specifically altered during the course of infection by Sindbis virus.