Directionality in drug action on sodium-calcium exchange.

In pathological conditions, the exchanger may generate deleterious calcium entry. A drug that inhibited calcium entry, while still allowing transport of calcium out of the cell would then seem attractive. In fact, this is impossible for thermodynamic reasons. Inhibitors may appear to be more effective when the exchanger is operating in net calcium entry mode than in calcium exit mode. This is, however, always attributable to differences in conditions because there is strong internal sodium dependence of drug action on the exchanger. When the exchanger is operating near equilibrium, drug action is found to be equally effective in both directions.

Na/Ca exchanger and PMCA localization in neurons and astrocytes: functional implications.

Immunocytochemistry reveals that the Na/Ca exchanger (NCX) in neuronal somata and astrocytes is confined to plasma membrane (PM) microdomains that overlie sub-PM (junctional) endoplasmic reticulum (jER). By contrast, the PM Ca(2+) pump (PMCA) is more uniformly distributed in the PM. At presynaptic nerve terminals, the NCX distribution is consistent with that observed in the neuronal somata, but the PMCA is clustered at the active zones. Thus, the PMCA, with high affinity for Ca(2+) (K(d) congruent with 100 nM), may keep active zone Ca(2+) very low and thereby "reprime" the vesicular release mechanism following activity. NCX, with lower affinity for Ca(2+) (K(d) congruent with 1,000 nM), on the other hand, may extrude Ca(2+) that has diffused away from the active zones and been temporarily sequestered in the endoplasmic reticulum. The PL microdomains that contain the NCX also contain Na(+) pump high ouabain affinity alpha2 (astrocytes) or alpha 3 (neurons) subunit isoforms (IC(50) congruent with 5-50 nM ouabain). In contrast, the alpha1 isoform (low ouabain affinity in rodents; IC(50) >10,000 nM), like the PMCA, is more uniformly distributed in these cells. The sub-PM endoplasmic reticulum in neurons (and probably glia and other cell types as well) and the adjacent PM form junctions that resemble cardiac muscle dyads. We suggest that the PM microdomains containing NCX and alpha 2/alpha 3 Na(+) pumps, the underlying jER, and the intervening tiny volume of cytosol (<10(-18) l) form functional units (PLasmERosomes); diffusion of Na(+) and Ca(2+) between these cytosolic compartments and "bulk" cytosol may be markedly restricted. The activity of the Na(+) pumps with alpha 2/alpha 3 subunits may thus regulate NCX activity and jER Ca(2+) content. This view is supported by studies in mice with genetically reduced (by congruent with 50%) alpha 2 Na(+) pumps: evoked Ca(2+) transients were augmented in these cells despite normal cytosolic Na(+) and resting Ca(2+) concentrations ([Na(+)](CYT) and [Ca(2+)](CYT)). We conclude that alpha 2/alpha 3 Na(+) pumps control PLasmERosome (local) [Na(+)](CYT). This, in turn, via NCX, modulates local [Ca(2+)](CYT), jER Ca(2+) storage, Ca(2+) signaling, and cell responses.

Structural complexity and functional diversity of endoplasmic reticulum Ca(2+) stores.

Considerable evidence, including recent direct observations, suggest that endoplasmic reticulum (ER) Ca(2+) stores in neurons, glia, and other cell types, consists of spatially-distinct compartments that can be individually loaded and unloaded. In addition, sub-plasmalemmal ('junctional') components of the ER (jER) are functionally coupled to the overlying plasmalemmal (PL) microdomains in PL-jER units named 'PLasmERosomes'. The PL microdomains and the jER contain clusters of specific transport proteins that regulate Na(+) and Ca(2+) concentrations in the tiny cytosolic space between the PL and jER. This organization helps the ER to produce the many types of complex local and global Ca(2+) signals that are responsible for the simultaneous control of numerous neuronal and glial functions.

Interaction of a toxin from the scorpion Tityus serrulatus with a cloned K+ channel from squid (sqKv1A).

A toxin from the scorpion Tityus serrulatus (TsTX-Kalpha) blocks native squid K(+) channels and their cloned counterpart, sqKv1A, at pH 8 ((native)K(d) approximately 20 nM; (sqKv1A)K(d) approximately 10 nM). In both cases, decreasing the pH below 7.0 significantly diminishes the TsTX-Kalpha effect (pK = 6.6). In the cloned squid channel, the pH dependence of the block is abolished by a single point mutation (H351G), and no change in toxin affinity was observed at higher pH values (pH > or =8.0). To further investigate the TsTX-Kalpha-sqKv1A interaction, the three-dimensional structure of TsTX-Kalpha was determined in solution by NMR spectroscopy, and a model of the TsTX-Kalpha-sqKv1A complex was generated. As found for other alpha-K toxins such as charybdotoxin (CTX), site-directed mutagenesis at toxin residue K27 (K27A, K27R, and K27E) significantly reduced the toxin's affinity for sqKv1A channels. This is consistent with the TsTX-Kalpha-sqKv1A model reported here, which has K27 of the toxin inserted into the ion conduction pathway of the K(+) channel. This toxin-channel model also illustrates a possible mechanism for the pH-dependent block whereby lysine residues from TsTX-Kalpha (K6 and K23) are repelled by protonated H351 on sqKv1A at low pH.

Ouabain augments Ca(2+) transients in arterial smooth muscle without raising cytosolic Na(+).

Ouabain and other cardiotonic steroids (CTS) inhibit Na(+) pumps and are widely believed to exert their cardiovascular effects by raising the cytosolic Na(+) concentration ([Na(+)](cyt)) and Ca(2+). This view has not been rigorously reexamined despite evidence that low-dose CTS may act without elevating [Na(+)](cyt); also, it does not explain the presence of multiple, functionally distinct isoforms of the Na(+) pump in many cells. We investigated the effects of Na(+) pump inhibition on [Na(+)](cyt) (with Na(+) binding benzofuran isophthalate) and Ca(2+) transients (with fura 2) in primary cultured arterial myocytes. Low concentrations of ouabain (3-100 nM) or human ouabain-like compound or reduced extracellular K(+) augmented hormone-evoked mobilization of stored Ca(2+) but did not increase bulk [Na(+)](cyt). Augmentation depended directly on external Na(+), but not external Ca(2+), and was inhibited by 10 mM Mg(2+) or 10 microM La(3+). Evoked Ca(2+) transients in pressurized small resistance arteries were also augmented by nanomolar ouabain and inhibited by Mg(2+). These results suggest that Na(+) enters a tiny cytosolic space between the plasmalemma (PL) and the adjacent sarcoplasmic reticulum (SR) via an Mg(2+)- and La(3+)-blockable mechanism that is activated by SR store depletion. The Na(+) and Ca(2+) concentrations within this space may be controlled by clusters of high ouabain affinity (alpha3) Na(+) pumps and Na/Ca exchangers located in PL microdomains overlying the SR. Inhibition of the alpha3 pumps by low-dose ouabain should raise the local concentrations of Na(+) and Ca(2+) and augment hormone-evoked release of Ca(2+) from SR stores. Thus the clustering of small numbers of specific PL ion transporters adjacent to the SR can regulate global Ca(2+) signaling. This mechanism may affect vascular tone and blood flow and may also influence Ca(2+) signaling in many other types of cells.

Unloading and refilling of two classes of spatially resolved endoplasmic reticulum Ca(2+) stores in astrocytes.

Signaling by two classes of endoplasmic reticulum (ER) Ca(2+) stores was studied in primary cultured rat astrocytes. Cytosolic and intra-ER Ca(2+) concentrations ([Ca(2+)](CYT) and [Ca(2+)](ER)) were measured with, respectively, Fura-2 and Furaptra, in separate experiments. The agonists, glutamate and ATP, released Ca(2+) primarily from cyclopiazonic acid (CPA)-sensitive ER Ca(2+) stores (CPA inhibits ER Ca(2+) pumps). Agonist-evoked release was abolished by prior treatment with CPA but was unaffected by prior depletion of caffeine/ryanodine (CAF/RY)-sensitive ER Ca(2+) stores. Conversely, prior depletion of the CPA-sensitive stores did not interfere with Ca(2+) release or reuptake in the CAF/RY-sensitive stores. Unloading of the CPA-sensitive stores, but not the CAF/RY-sensitive stores, promoted Ca(2+) entry through "store-operated channels." Resting [Ca(2+)](ER) averaged 153 microM (based on in situ calibration of Furaptra: K(D) = 76 microM, vs 53 microM in solution). The releasable Ca(2+) in both types of ER Ca(2+) stores was increased by Na(+) pump inhibition with 1 mM ouabain or K(+)-free medium. Using high spatial resolution imaging and image subtraction methods, we observed that some regions of the ER (45-58% of the total ER) unloaded and refilled when CPA was added and removed. Other regions of the ER (24-38%) unloaded and refilled when CAF was added and removed. The overlap between these two classes of ER was only 10-18%. These data indicate that there are two structurally separate, independent components of the ER and that they are responsible for the functional independence of the CPA-sensitive and CAF/RY-sensitive ER Ca(2+) stores.

Regulatory processes on the cytoplasmic surface of the Na(+)/Ca(2+) exchanger from lobster exoskeletal muscle.

A partially purified preparation of the lobster muscle Na(+)/Ca(2+) exchanger was reconstituted with, presumably, random orientation in liposomes. Ca(2+) efflux from (45)Ca-loaded vesicles was studied in exchanger molecules in which the transporter cytoplasmic surface was exposed to the extravesicular (ev) medium. Extravesicular Na(+) (Na(ev))-dependent Ca(2+) efflux depended directly upon the extravesicular Ca(2+) concentration ([Ca(2+)](ev)) with a half-maximal activation at [Ca(2+)](ev) = 0.6 microm. This suggests that the lobster muscle exchanger is catalytically upregulated by cytoplasmic Ca(2+), as in most other species. In contrast, at low [Na(+)](ev), the Ca(ev)-binding site (i.e., on the cytoplasmic surface) for Ca(2+) transported via Ca(2+)/Ca(2+) exchange was half-maximally activated by about 7.5 microm Ca(2+). Mild proteolysis of the Na(+)/Ca(2+) exchanger by alpha-chymotrypsin also upregulated the Na(ev)-dependent Ca(2+) efflux. Following proteolytic digestion in Ca-free medium, the exchanger was no longer regulated by nontransported ev Ca(2+). Proteolytic digestion in the presence of 1.9 microm free ev Ca(2+), however, induced only a 1. 6-fold augmentation of Ca(2+) efflux, whereas, after digestion in nominally Ca-free medium, a 2.3-fold augmentation was observed; Ca(2+) also inhibited proteolytic degradation of the Na(+)/Ca(2+) exchanger measured by immunoblotting. These data suggest that Ca(2+), bound to a high affinity binding site, protects against the activation of the Na(+)/Ca(2+) exchanger by alpha-chymotrypsin. Additionally, we observed a 6-fold increase in the Na(+)/Ca(2+) exchange rate, on average, when the intra- and extravesicular salt concentrations were increased from 160 to 450 mm, suggesting that the lobster muscle exchanger is optimized for transport at the high salt concentration present in lobster body fluids.

Location of calcium transporters at presynaptic terminals.

The plasma membrane ATP-driven Ca2+ pump (PMCA) and the Na+/Ca2+ exchanger (NCX) are the major means of Ca2+ extrusion at presynaptic nerve terminals, but little is know about the location of these transporters relative to the major sites of Ca2+ influx, the transmitter release sites. We used immunocytochemistry to identify these transport proteins in a calyx-type presynaptic nerve terminal from the ciliary ganglion of the chick. The PMCA clusters were localized to the transmitter release sites, as identified by staining for the secretory vesicle-specific protein synaptotagmin I. This colocalization was not due to the presence of the pump on the secretory vesicle itself because membrane fractionation of chick brain synaptosomes demonstrated comigration of the pump with surface membrane and not vesicle markers. In contrast, the NCX did not colocalize with synaptotagmin but tended to be located at nonsynaptic regions of the terminal. The PMCA location, near the transmitter release sites, suggests that it plays a role in priming the release site by maintaining a low free Ca2+ level, facilitating the dissociation of the ion from its binding sites. The PMCA may also replenish external Ca2+ in the synaptic cleft following periods of synaptic activity. In contrast, the NCX location suggests a role in the rapid emptying of cytoplasmic Ca2+ uptake organelles which serve as the main line of defence against high free Ca2+.

Structural and functional differences of two toxins from the scorpion Pandinus imperator.

The Pandinotoxins, PiTX-K alpha and PiTX-K beta, are members of the Charybdotoxin family of scorpion toxins that can be used to characterize K+ channels. PiTX-K alpha differs from PiTX-K beta, another peptide from Pandinus imperator, by one residue (P10E). When the two toxins are compared in a physiological assay, the affinity of PiTX-K beta for voltage-gated, rapidly inactivating K+ channels in dorsal root ganglia (DRG) neurons is 800-fold lower than that of PiTX-K alpha (K alpha-IC50 = 8.0 nM versus K beta-IC50 = 6,500 nM). To understand this difference, the three-dimensional structure of PiTX-K beta was determined by nuclear magnetic resonance (NMR) spectroscopy and compared to that of PiTX-K alpha. This comparison shows that structural differences between the two toxins occur at a residue that is critical for blocking K+ channels (K27) as well as at the site of the natural mutation (P10E). In PiTX-K beta, the negatively charged carboxylate oxygen of E10 can approach the positive charge of K27 and presumably reduces the net positive charge in this region of the toxin. This is likely the reason why PiTX-K beta binds K+ channels from DRG neurons with a much lower affinity than does PiTX-K alpha.

Na(+) entry via store-operated channels modulates Ca(2+) signaling in arterial myocytes.

In many nonexcitable cells, hormones and neurotransmitters activate Na(+) influx and mobilize Ca(2+) from intracellular stores. The stores are replenished by Ca(2+) influx via "store-operated" Ca(2+) channels (SOC). The main routes of Na(+) entry in these cells are unresolved, and no role for Na(+) in signaling has been recognized. We demonstrate that the SOC are a major Na(+) entry route in arterial myocytes. Unloading of the Ca(2+) stores with cyclopiazonic acid (a sarcoplasmic reticulum Ca(2+) pump inhibitor) and caffeine induces a large external Na(+)-dependent rise in the cytosolic Na(+) concentration. One component of this rise in cytosolic Na(+) concentration is likely due to Na(+)/Ca(2+) exchange; it depends on elevation of cytosolic Ca(2+) and is insensitive to 10 mM Mg(2+) and 10 microM La(3+). Another component is inhibited by Mg(2+) and La(3+), blockers of SOC; this component persists in cells preloaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid to buffer Ca(2+) transients and prevent Na(+)/Ca(2+) exchange-mediated Na(+) entry. This Na(+) entry apparently is mediated by SOC. The Na(+) entry influences Na(+) pump activity and Na(+)/Ca(2+) exchange and has unexpectedly large effects on cell-wide Ca(2+) signaling. The SOC pathway may be a general mechanism by which Na(+) participates in signaling in many types of cells.

Local and cellular Ca2+ transients in smooth muscle of pressurized rat resistance arteries during myogenic and agonist stimulation.

1. Confocal laser scanning microscopy was used to visualize Ca2+ transients in the vascular smooth muscle cells (VSMC) of intact, pressurized rat mesenteric resistance arteries loaded with fluorescent calcium indicators. Vasoconstriction was assessed by measuring inner arterial diameter. All arteries were studied at 70 mmHg intralumenal pressure and 37 C. 2. In the control condition of myogenic tone the arteries were constricted to 62 % (n = 10) of their passive diameter (p.d.). The [Ca2+]i in most VSMC of these arteries was constant over time. In a small percentage (< 10 %) of cells in each artery, [Ca2+]i oscillated regularly. Local calcium transients (Ca2+ sparks) were observed in five arteries studied with confocal linescan imaging. 3. Activation of alpha-adrenoceptors by phenylephrine (PE, 1.0 microM) induced further vasoconstriction of pressurized arteries (to 27 % of p.d.). In this condition, [Ca2+]i oscillations were prominent in a large percentage (83 %) of the VSMC. The Ca2+ oscillations ranged in frequency from 4 to 22 min-1, and were usually asynchronous between cells. 4. High [KCl]o (65 mM) induced nearly comparable vasoconstriction to PE (37 % of p.d.) but [Ca2+]i oscillated in only about 13 % of cells in each artery. 5. Block of L-type Ca2+ channels (with nifedipine) in arteries activated by PE caused nearly full vasodilatation, but did not abolish the Ca2+ oscillations. Subsequent block of the sarcoplasmic reticulum Ca2+ pump (with cyclopiazonic acid) abolished Ca2+ oscillations in all cells. 6. We conclude that Ca2+ entering VSMC via L-type Ca2+ channels has an obligatory role in force development, both in myogenic tone and during alpha1-adrenoceptor activation. The oscillatory pattern of [Ca2+]i that persists in the absence of Ca2+ entry via L-type Ca2+ channels is ineffective in activating contraction.

Sodium/calcium exchange: its physiological implications.

The Na+/Ca2+ exchanger, an ion transport protein, is expressed in the plasma membrane (PM) of virtually all animal cells. It extrudes Ca2+ in parallel with the PM ATP-driven Ca2+ pump. As a reversible transporter, it also mediates Ca2+ entry in parallel with various ion channels. The energy for net Ca2+ transport by the Na+/Ca2+ exchanger and its direction depend on the Na+, Ca2+, and K+ gradients across the PM, the membrane potential, and the transport stoichiometry. In most cells, three Na+ are exchanged for one Ca2+. In vertebrate photoreceptors, some neurons, and certain other cells, K+ is transported in the same direction as Ca2+, with a coupling ratio of four Na+ to one Ca2+ plus one K+. The exchanger kinetics are affected by nontransported Ca2+, Na+, protons, ATP, and diverse other modulators. Five genes that code for the exchangers have been identified in mammals: three in the Na+/Ca2+ exchanger family (NCX1, NCX2, and NCX3) and two in the Na+/Ca2+ plus K+ family (NCKX1 and NCKX2). Genes homologous to NCX1 have been identified in frog, squid, lobster, and Drosophila. In mammals, alternatively spliced variants of NCX1 have been identified; dominant expression of these variants is cell type specific, which suggests that the variations are involved in targeting and/or functional differences. In cardiac myocytes, and probably other cell types, the exchanger serves a housekeeping role by maintaining a low intracellular Ca2+ concentration; its possible role in cardiac excitation-contraction coupling is controversial. Cellular increases in Na+ concentration lead to increases in Ca2+ concentration mediated by the Na+/Ca2+ exchanger; this is important in the therapeutic action of cardiotonic steroids like digitalis. Similarly, alterations of Na+ and Ca2+ apparently modulate basolateral K+ conductance in some epithelia, signaling in some special sense organs (e.g., photoreceptors and olfactory receptors) and Ca2+-dependent secretion in neurons and in many secretory cells. The juxtaposition of PM and sarco(endo)plasmic reticulum membranes may permit the PM Na+/Ca2+ exchanger to regulate sarco(endo)plasmic reticulum Ca2+ stores and influence cellular Ca2+ signaling.

Heterogeneity of mitochondrial matrix free ca2+: resolution of Ca2+ dynamics in individual mitochondria in situ.

The role of mitochondria in Ca2+ homeostasis is controversial. We employed the Ca2+-sensitive dye rhod 2 with novel, high temporal and spatial resolution imaging to evaluate changes in the matrix free Ca2+ concentration of individual mitochondria ([Ca2+]m) in agonist-stimulated, primary cultured aortic myocytes. Stimulation with 10 microM serotonin (5-HT) evoked modest cytosolic Ca2+ transients [cytosolic free Ca2+ concentration ([Ca2+]cyt) <500 nM; measured with fura 2] and triggered contractions in short-term cultured myocytes. However, 5-HT triggered a large mitochondrial rhod 2 signal (indicating pronounced elevation of [Ca2+]m) in only 4% of cells. This revealed heterogeneity in the responses of individual mitochondria, all of which stained with MitoTracker Green FM. In contrast, stimulation with 100 microM ATP evoked large cytosolic Ca2+ transients (>1,000 nM) and induced pronounced, reversible elevation of [Ca2+]m (measured as rhod 2 fluorescence) in 60% of cells. This mitochondrial Ca2+ uptake usually lagged behind the cytosolic Ca2+ transient peak by 3-5 s, and [Ca2+]m declined more slowly than did bulk [Ca2+]cyt. The uptake delay may prevent mitochondria from interfering with rapid signaling events while enhancing the mitochondrial response to large, long-duration elevations of [Ca2+]cyt. The responses of arterial myocytes to modest physiological stimulation do not, however, depend on such marked changes in [Ca2+]m.

Induction of seizures by the potent K+ channel-blocking scorpion venom peptide toxins tityustoxin-K(alpha) and pandinustoxin-K(alpha).

The scorpion venom peptide toxins tityustoxin-K(alpha) (TsTx-K(alpha)) and pandinustoxin-K(alpha) (PiTx-K(alpha)) are novel, highly potent and selective blockers of voltage-activated K+ channels. PiTx-K(alpha) preferentially blocks rapidly inactivating (A-type) K+ channels whereas TsTx-K(alpha) is selective for slowly inactivating (delayed rectifier-type) channels. K+ channel blockers are known to induce seizures, but the specific K channel types that can serve as convulsant targets are not well defined. To address this issue, we examined for convulsant activity the K+ channel type-specific scorpion toxins and the selective K+ channel antagonists 4-aminopyridine (4-AP), an inhibitor of A-type voltage-activated K+ channels, and paxilline, a selective blocker of large conductance (maxi K) Ca(2+)-activated K+ channels. Intracerebroventricular injection of recombinant TsTx-K(alpha) and PiTx-K(alpha) in mice produced limbic and clonic-tonic seizures. The severity of the seizures increased during the 60-min period following injection, culminating in continuous clonic seizure activity (status epilepticus), tonic hindlimb extension, and eventually in death. The estimated doses producing limbic and clonic seizures in 50% of animals (CD50) for TsTx-K(alpha) and PiTx-K(alpha) were 9 and 33 ng, respectively. 4-AP produced seizure activity similar to the toxins (CD50, 76 ng) whereas paxilline failed to induce seizures at doses up to 13.5 microg. Carbamazepine protected fully against the toxin- and 4-AP-induced seizures whereas phenytoin had variable activity against the clonic component although it was protective against tonic hindlimb extension. The AMPA receptor antagonist GYKI 52466 also conferred full protection against toxin-induced seizures, but the NMDA receptor antagonists (R)-CPP and dizocilpine failed to affect limbic and clonic seizures, although they protected against hindlimb extension. We conclude that selective blockade of delayed rectifier- or A-type voltage-activated K+ channels can produce limbic, clonic and tonic seizures, whereas blockade of maxi K-type Ca(2+)-activated K+ channels does not. The convulsant effects may be related to enhanced glutamate release and, in the case of the limbic and clonic convulsions, activation of AMPA receptors.

Physiological effects of Na+/Ca2+ exchanger knockdown by antisense oligodeoxynucleotides in arterial myocytes.

Antisense oligodeoxynucleotides (AS-oligos) targeted to the Na+/Ca2+ exchanger (NCX) inhibit NCX-mediated Ca2+ influx in mesenteric artery (MA) myocytes [Am. J. Physiol. 269 (Cell Physiol. 38): C1340-C1345, 1995]. Here, we show AS-oligo knockdown of NCX-mediated Ca2+ efflux. In initial experiments, the cytosolic free Ca2+ concentration ([Ca2+]cyt) was raised, and sarcoplasmic reticulum (SR) Ca2+ sequestration was blocked with caffeine and cyclopiazonic acid; the extracellular Na+-dependent (NCX) component of Ca2+ efflux was then selectively inhibited in AS-oligo-treated cells but not in controls (no oligos or nonsense oligos). In contrast, the La3+-sensitive (plasmalemma Ca2+ pump) component of Ca2+ efflux was unaffected in AS-oligo-treated cells. Knockdown of NCX activity was reversed by incubating AS-oligo-treated cells in normal media for 5 days. Transient [Ca2+]cyt elevations evoked by serotonin (5-HT) at 15-min intervals in AS-oligo-treated cells were indistinguishable from those in controls. When cells were stimulated every 3 min, however, the peak amplitudes of the second and third responses were larger, and [Ca2+]cyt returned to baseline more slowly, in AS-oligo-treated cells than in controls. Peak 5-HT-evoked responses in the controls, but not AS-oligo-treated cells, were augmented more than twofold in Na+-free media. This implies that NCX is involved in Na+ gradient modulation of SR Ca2+ stores and cell responsiveness. The repetitive stimulation data suggest that the NCX may be important during tonic activation of arterial myocytes.

Na+/Ca2+ exchange in neonatal rat heart cells: antisense inhibition and protein half-life.

Cardiac Na+/Ca2+ exchanger (NCX) protein half-life (t1/2) and antisense knockdown were studied in primary cultured neonatal rat cardiomyocytes. Protein t1/2 was determined using [35S]methionine with a pulse-chase protocol. The 35S signal in NCX was identified by immunoprecipitation and Western blotting. The t1/2 of NCX protein was 33 h. Low concentrations (0.5 microM) of chimeric, phosphorothioated antisense oligodeoxynucleotides (AS-oligos) targeted to the region around the start codon of NCX1 transcript were used to knock down NCX protein and activity. Control myocytes (no oligos or scrambled oligos for at least 4 days) exhibited spontaneous Ca2+ transients (measured with fura 2). The sustained ("diastolic") Ca2+ concentration in the cytosol ([Ca2+]cyt) of control cells was unaffected by cyclopiazonic acid (CPA) plus caffeine (Caf), which promote depletion of sarcoplasmic reticular Ca2+ stores, but [Ca2+]cyt rose in control cells when external Na+ was removed. In contrast, approximately 60% of cells treated with AS-oligos for at least 4 days did not exhibit spontaneous Ca2+ transients or respond to Na+-free medium; however, CPA + Caf did induce a prolonged elevation in [Ca2+]cyt in these cells. In all cells, 50 mM K+ increased [Ca2+]cyt. NCX protein was reduced by approximately 50% in cells treated with AS-oligos for 7 days but was not reduced after only 2 days. These biochemical data are consistent with the physiological evidence of NCX knockdown in approximately 60% of cells.

The cellular mechanism of action of cardiotonic steroids: a new hypothesis.

Arterial smooth muscle (ASM) contraction is triggered by agonist-evoked Ca2+ mobilization from sarcoplasmic reticulum (SR). The amount of Ca2+ released, and thus, the magnitude of the contractions, depends directly on SR Ca2+ content. Na+ pump inhibition by cardiotonic steroids (CTS) indirectly increases the Ca2+ content of the SR and, thus, contractility. This sequence of events does not, however, account for the multiple Na+ pump alpha subunit isoforms with different affinities for Na+ and for CTS, nor does it explain the cardiotonic and vasotonic effects of low doses of CTS that do not elevate cytosolic Na+ or Ca2+. We show that the Na+ pump high ouabain affinity (alpha3) isoform and the plasmalemmal (PM) Na/Ca exchanger are confined to PM domains that overlie junctional SR in ASM, while low ouabain affinity alpha1 and the PM Ca2+ pump are uniformly distributed in the PM. Thus, low doses of CTS, including an endogenous ouabain-like compound, influence cytosolic Na+ and (indirectly) Ca2+ concentrations only in the cytoplasmic clefts between the PM and junctional SR (a functional unit we call the "plasmerosome"). In turn, this modulates the Ca2+ content of the junctional SR and cell responsiveness.

Different effects of low and high dose cardiotonic steroids on cytosolic calcium in spontaneously active hippocampal neurons and in co-cultured glia.

The Na+ pump is crucial for the regulation of [Na+]i (the intracellular Na+ concentration) in all cells. Three Na+ pump alpha subunit isoforms, alpha1, alpha2 and alpha3, are expressed in rat hippocampal neurons, and alpha1 and alpha2 are expressed in glia, but the significance of these isoforms is not understood. We exploited the different ouabain affinities of the Na+ pump alpha subunit isoforms in rat (alpha1, low ouabain affinity; alpha2 and alpha3, high ouabain affinity) to probe their possible physiological roles. Low and intermediate doses (1-10 microM) of ouabain and its readily reversible analog, dihydroouabain, altered the spontaneous elevations of [Ca2+]i (the intracellular Ca2+ concentration) in neurons and induced [Ca2+]i transients in glia. Complete inhibition of all Na+ pump isoforms (>/=100 microM ouabain) caused sustained increases in global neuronal [Ca2+]i in rat neuronal/glial hippocampal co-cultures and transient [Ca2+]i increases in surrounding glia. High dose ouabain was also associated with increased [Na+]i and [H+]i in neurons and glia. In contrast, 1 microM ouabain (a concentration that completely inhibits only alpha2 and alpha3) was not associated with sustained increases in global neuronal [Ca2+]i or the sustained derangements in [Na+]i and [H+]i observed with high dose ouabain. Reduction of [K+]o to 1 mM suppressed the spontaneous [Ca2+]i oscillations in neurons and induced Ca2+ transients in some glia; removal of external K+ induced sustained elevation of neuronal [Ca2+]i. These studies indicate that the alpha1 isoform is the 'housekeeper' required for maintenance of the global Na+ gradient. As suggested by their restricted plasmalemmal distribution, the high ouabain-affinity Na+ pump isoforms may have more specific roles in neurons and glia.