INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

SODIUM PUMPS IN THE PLASMALEMMA (PL) of animal cells maintain a low cytosolic Na⁺ concentration ([Na⁺]_cyt) (16).¹ These pumps also are receptors for exogenous cardiotonic steroids (CTS) such as ouabain (16, 44) as well as for an endogenous ouabain-like compound (OLC) (19). Four different Na⁺ pump catalytic () subunit isoforms (1-4) have been recognized. Each is the product of a different gene and, in the rat, can be distinguished by its affinity for ouabain and for [Na⁺]_cyt (5, 46).

Most cells in the rat express the high Na⁺/low ouabain affinity isoform (1) as well as one or more of the independently regulated low Na⁺/high ouabain affinity isoforms (2, 3, or 4) (5, 26, 46). Moreover, the different isoforms are differently distributed in the cells. For example, the 1-isoform is distributed uniformly in the PL of neurons, astrocytes, and mesenteric arterial myocytes (26). In contrast, the 3- and 2-isoforms, which are also present in neurons and arterial myocytes and in astrocytes, respectively, appear to be confined to PL microdomains adjacent to peripheral or "junctional" (j) elements of the sarco/endoplasmic reticulum (S/ER; jS/ER) (26). Despite the differences in their properties and localization, the functional significance of these isoforms has been unclear.

It is generally believed that CTS exert their effects by inhibiting Na⁺ pumps, elevating bulk [Na⁺]_cyt, and, in turn, raising cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) via Na/Ca exchange (3, 6, 28). Most of the entering Ca²⁺ is rapidly sequestered in the S/ER (6, 28), and this permits additional Ca²⁺ to be mobilized when the cells are activated (50). These steps have all been verified with relatively high ouabain concentrations, usually 1-1,000 µM (6, 31). A longstanding enigma, however, is that nanomolar (therapeutic) concentrations of CTS augment cardiac (31) and arterial (48) contractions, induce hypertension in rats (34), and have other effects, despite little or no change in whole cell ("bulk") [Na⁺]_cyt (31) or resting [Ca²⁺]_cyt (51). Such findings are incompatible with the generally accepted view of CTS action mentioned above. Furthermore, this view of CTS action does not account for the coexpression of multiple Na⁺ pump -subunit isoforms with different kinetic properties including different affinities for ouabain.

Recently, isoforms of the Na⁺ pump with high affinity for ouabain were shown to cluster with Na/Ca exchangers in PL microdomains of rat arterial smooth muscle cells (27) and some other cell types (21, 27, 30, 36). This clustering implies that these Na⁺ pumps have a specific function. We investigated the effects of low-dose (1-100 nM) ouabain and OLC on [Na⁺]_cyt and evoked Ca²⁺ transients in rat arterial myocytes, which coexpress 1 and 3 (26). The results show that selective block of 3-pumps amplifies Ca²⁺ transients when Ca²⁺ is released from intracellular stores in cultured cells and in small resistance arteries. In the cultured cells, these effects occurred under conditions in which there was no measurable rise in bulk [Na⁺]_cyt. These results indicate that the 3-isoform of the Na⁺ pump has a specific role in Ca²⁺ signaling and contraction in arterial smooth muscle.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cells and solutions. Rat mesenteric arterial myocytes were grown in primary culture (40) for 4-6 days. The cells, on 25-mm coverslips, were loaded with fura 2 or sodium-binding benzofuran isophthalate (SBFI) (for [Ca²⁺]_cyt or [Na⁺]_cyt determination, respectively) by incubation with 3.3 µM fura 2-AM for 30 min or 10 µM SBFI-AM (the respective acetoxymethyl esters; TEFLABS, Austin, TX) for 1 h at 22-25°C.

Imaging methods for cultured cells. [Ca²⁺]_cyt and [Na⁺]_cyt were determined with digital ratiometric imaging (9, 17): fura 2 excitation = 360 and 380 nm, emission = 510 nm; SBFI excitation = 340 and 380 nm, emission = 510 nm. A ×40 1.3-NA Nikon UV Fluor oil immersion objective was used for imaging. Fields containing 5-8 cells were imaged, background-subtracted, and transformed to [Ca²⁺] or [Na⁺] images (MetaFluor; Universal Imaging, West Chester, PA). Fluorescence ratio data for each cell were obtained from a 5 × 5-pixel (1.5 × 1.5-µm) nonnuclear region (1/cell). To enhance the signal-to-noise ratio, 32 frames were averaged at video frame rate, except when agonists and/or ouabain were added; then only four frames were averaged to improve temporal resolution. Images were acquired at a rate of two per minute, except during agonist application, when the rate was increased to two per second.

Isolated small mesenteric arteries. Sprague-Dawley male rats, 200-400 g, were anesthetized and decapitated, and small resistance arteries (length = 3-4 mm, maximum passive diameter = 200-220 µm; see Experimental protocol for blood vessels) were isolated (35). The arteries were dissected at 5-6°C in a "dissection solution" of the following composition (in mM): 145 NaCl, 5 KCl, 2.5 CaCl₂, 1 MgSO₄, 1 KH₂PO₄, 2 pyruvate, and 5 glucose, with 1% bovine serum albumin (pH = 7.4, osmolarity = 310-320 mosM). The vessels were denuded of endothelium by perfusion of the lumen (through a small pipette) with tiny air bubbles for 30 s. Removal of the endothelium was verified by the abolition of a 2 µM acetylcholine-evoked Ca²⁺ transient or vasodilation.

Experimental protocol for blood vessels. Resistance arteries were superfused (2.5 ml/min, 37°C) with modified Krebs solution of the following composition (in mM): 115 NaCl, 4.7 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 25 NaHCO₃, 10 glucose, and 1.8 HEPES (pH = 7.4, osmolarity = 310-320 mosM, bubbled with 95% air-5% CO₂); the solution also contained 5 µM guanethidine to deplete catecholamines from the nerve terminals. Before the experiments were started, the vessels were incubated for 1 h; maximum passive diameter was then determined by a 20-min wash in Ca²⁺-free Krebs containing 2 mM EGTA. For each experimental protocol, the Krebs solution also contained 50 µM tetrodotoxin during one entire experiment to verify that the responses were unaffected by the presence of this agent.

Statistical analysis. Data (fluorescence ratios and calibrated values) were analyzed and plotted with Sigma Plot software (Jandel, San Rafael, CA). The significance of the differences between means (±SE) for the same groups of cultured cells and for small arteries under various conditions was calculated by Student's paired t-test.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Nanomolar ouabain amplifies evoked Ca²+ transients in cultured arterial myocytes. Figure 1A illustrates the protocol and a key result. The responses to four consecutive 30-s applications of serotonin (5-HT) in PSS are shown. The first 5-HT application was a control. During the second and third exposures, ouabain (10 and 100 nM, respectively) and 5-HT were applied simultaneously. Ouabain was omitted during the fourth 5-HT application ("recovery"). The 5-HT-evoked Ca²⁺ transients were negligibly affected by 10 nM ouabain, whereas 100 nM ouabain reversibly augmented transients 1.8-fold (Fig. 1, A and B; Table 1 contains summary data). With a 60-s preincubation to permit more complete solution exchange and equilibration (see MATERIALS AND METHODS), 100 nM ouabain-augmented 5-HT evoked Ca²⁺ transients 2.6-fold (Fig. 1B); the apparent EC₅₀ was 8 nM, comparable with the IC₅₀ for block of the rat Na⁺ pump 3 isoform (5).

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Effects of nanomolar ouabain, Ca-free ("0" Ca) physiological salt solution (PSS), and reduced extracellular Na+ concentration ([Na+]O) on serotonin (5-HT)-evoked Ca2+ transients in primary cultured rat mesenteric arterial myocytes. A and D-F: representative data from single myocytes (Table 1 presents a statistical summary). A: representative Ca2+ transients evoked (10 min apart) by 500 nM 5-HT applied for 30 s under control conditions during simultaneous application of 10 nM and then 100 nM ouabain and after ouabain washout. B: ouabain dose-response data from multiple myocytes. Cells superfused with PSS were stimulated with 500 nM 5-HT (at 10-min intervals), and Ca2+-transient amplitudes were measured {equals peak cytosolic Ca2+ concentration ([Ca2+]cyt) amplitude  resting [Ca2+]cyt}. Ouabain (abscissa) was added either 60 s before and during 30 s of 5-HT application () or simultaneously with 5-HT (for 30 s; ). For each cell, test transient amplitudes were normalized to control transient amplitude (no ouabain = 1.0); data points correspond to means ± SE (cell nos. in parentheses). The 60-s preincubation data were fit by Hill equation: relative amplitude (RA) = 1.0 + {(RAMax  1.0) × ([ouabain]n/{[ouabain]n + (EC50)n})}, where RAMax (maximum RA) = 2.65, [ouabain] = ouabain concentration, EC50 = 8 nM, and n = 2. C: time course of recovery of Ca2+ transients evoked by 500 nM 5-HT after a 30-s exposure to 100 nM ouabain. The "0-time" transient is relative amplitude (compared with pre-ouabain control = 1.0) of transient evoked when 5-HT was applied as ouabain washout was started; abscissa indicates times at which subsequent transients were elicited. Data are means ± SE for 16 cells. Exponential decay curve fits equation: RA = 1.0 + {(RAMax  1.0) × exp(t/)}, where RAMax = 2.8, t = time minus 30 s after start of ouabain washout (to account for chamber washout t1/2  30 s; see MATERIALS AND METHODS), and time constant () = 47 s. D: Ca2+ transients in Ca-free PSS evoked (10 min apart) by 500 nM 5-HT before ouabain, during simultaneous application of 100 nM ouabain, and after ouabain washout. E: effects of external Ca2+ removal, external Na+ concentration ([Na+]O) reduction to 5 mM, and subsequent addition of 100 nM ouabain on 500 nM 5-HT-evoked Ca2+ transients (10 min apart). On average, Ca2+-transient amplitudes for 3 conditions did not differ significantly (372 ± 11, 390 ± 9, and 391 ± 9 nM Ca2+, respectively, in 22 cells). F: Ca2+ transients in Ca-free PSS evoked (10 min apart) by 500 nM 5-HT 2 min after removal of Ca2+ (first transient), 2 min after introduction of 0 Ca2+-5 mM Na+ PSS (second transient), and again during treatment with Ca-free PSS (third transient). The Ca2+-transient amplitude increased 1.85 ± 0.13-fold (32 cells) when evoked 2 min after introduction of 0 Ca2+-5 Na+ mM PSS; Na+ reduction did not affect transients evoked at same time Na+ was reduced in Ca-free PSS (not shown). Horizontal bars below x-axes (A and D-F) indicate times of application of solutions and agents (normal PSS was present unless otherwise indicated).

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Effects of low-dose human ouabain-like compound (OLC) on 5-HT-evoked Ca2+ transients in arterial myocytes. To conserve available OLC (19) in these experiments, superfusion flow was stopped when ouabain or OLC was present in bath (2 min); in controls, flow was stopped (without OLC or ouabain) for 2 min before 5-HT was added. A: representative single myocyte data; Ca2+ transients evoked by 30-s exposure to 500 nM 5-HT every 10 min. Cells were exposed to 30 nM ouabain for 2 min before second transient and to 30 nM human OLC (19) for 2 min before third transient; fourth transient was evoked 10 min after washing out OLC. B-a: original fura 2 fluorescent image (360-nm excitation and 510-nm emission). Small box at top right of a shows area used to collect data for graph in A. Bar in a = 5 µm. B, b-g: original Ca2+ images captured at times indicated on graph in A. C-a: effects of 10 and 30 nM human OLC and (plant) ouabain on relative amplitude of response to 500 nM 5-HT. C-b: effects of 10 mM Mg2+ and 10 µM La3+ on Ca2+-transient augmentation by 30 nM OLC (see Fig. 4, A and B, for protocol); nos. of cells observed for each condition are in parentheses. * P < 0.01 vs. control,  P < 0.01 vs. 30 nM OLC,  P < 0.01 vs. 30 nM OLC + 10 mM Mg2+, and § P < 0.05 vs. 30 nM OLC + 10 µM La3+.

Amplification by ouabain requires external Na+ but does not depend on a rise in bulk [Na+]_cyt. When external Ca²⁺ was removed 30 s before 5-HT application, ouabain amplified the peak Ca²⁺ transients evoked by 5-HT without increasing the duration of the transients (Fig. 1D; Table 1A). The augmentation of the peak transients was also unaffected by 10 µM nifedipine (Table 1C). This indicates that the amplified response was due to enhanced mobilization of stored Ca²⁺, and that the simultaneous entry of Ca²⁺ via L-type Ca²⁺ channels was not required.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effects of ouabain, veratridine, and reduced extracellular K+ concentration ([K+]O) on [Na+]cyt in sodium-binding benzofuran isophthalate (SBFI)-loaded rat mesenteric arterial myocytes. A: effects of 100 nM, 1 µM, and 1 mM ouabain on [Na+]cyt measured with SBFI. B: effects of reduced [K+]O (1 mM), 10 µM veratridine, and 1 mM ouabain on [Na+]cyt measured with SBFI. Insets: [Na+]cyt data on expanded time and concentration scales for periods (see arrows) when 100 nM ouabain was added (A) or [K+]O was reduced (B) and image capture rate was increased (and data scatter increased). Mean resting [Na+]cyt in normal PSS was 8.7 ± 0.5 mM (29 cells); after 10 min in PSS with 100 nM ouabain, [Na+]cyt was 8.3 ± 0.8 mM (16 cells), and after 10 min in 1 mM K+ PSS, [Na+]cyt was 8.5 ± 0.7 mM (13 cells). Horizontal bars indicate times of application of solutions and agents (normal PSS was present unless otherwise indicated).

Role of the Na/Ca exchanger in ouabain-augmented responses. Despite the finding that low-dose ouabain did not elevate whole cell [Na⁺]_cyt, the Na/Ca exchanger apparently contributed to the augmentation by ouabain. Ca²⁺ efflux via Na/Ca exchange is a steep, sigmoid function of external Na⁺ concentration ([Na⁺]_O) (Hill coefficient 3), with half-maximal activation at [Na⁺]_O 20-40 mM (7). As the chamber washout t_1/2 was 30 s (see MATERIALS AND METHODS), a 30-s washout of Na⁺ (Fig. 1E) would have lowered [Na⁺]_O only to 75 mM, which would still support Ca²⁺ extrusion via Na/Ca exchange. Yet, under these conditions, the ability of ouabain to augment evoked Ca²⁺ transients was prevented, presumably because of a marked reduction of Na⁺ entry (which may be a more linear function of [Na⁺]_O in this concentration range; see Ref. 22). With more prolonged (2 min) washout of Na⁺ to lower [Na⁺]_O to 10 mM, Ca²⁺ exit via Na/Ca exchange was inhibited, and the response to 5-HT was amplified in the absence of ouabain (Fig. 1F). Thus the inhibition of exchanger-mediated Ca²⁺ extrusion by a large reduction of [Na⁺]_O (Fig. 1F) or by inhibition of Na⁺ pump-mediated Na⁺ extrusion, with either nanomolar concentrations of ouabain (Fig. 1, A, B, and D, and Table 1, A and C) or reduced [K⁺]_O, augmented evoked Ca²⁺ transients to a similar extent. Collectively, this series of observations demonstrating the dependence of the ouabain augmentation on Na⁺ is most paradoxical, because in no instance was there any increase in bulk [Na⁺]_cyt (Fig. 3).

La³+ and elevated Mg²⁺ selectively block ouabain-induced augmentation. To address the question of whether this amplification depended on Na⁺ entry or was due to an action of Na⁺ at the external surface of the cells, we considered possible Na⁺ entry pathways. Most vascular smooth muscle cells, including mesenteric arterial myocytes, do not possess voltage-gated Na⁺ channels (53). Consistent with the electrophysiological data, neither veratridine (an Na⁺ channel opener; Fig. 3B) nor tetrodotoxin (50 µM, not shown) affected [Na⁺]_cyt. These myocytes do, however, possess store-operated channels (SOC) that are permeable not only to Ca²⁺ but also to Na⁺ (2) and are blocked by 10 µM La³⁺ and 10 mM Mg²⁺ (2, 23, 52). In unstimulated cells, La³⁺ and Mg²⁺ lowered resting [Na⁺]_cyt, possibly by blocking Na⁺ entry through a small number of SOC that may have been open at rest (2). La³⁺ and Mg²⁺ blocked both the ouabain- and the 1 mM K⁺-induced augmentation of 5-HT-evoked Ca²⁺ transients (Fig. 4 and Table 1, C and D), as well as the augmentation induced by human OLC (Fig. 2C-b). The data suggest that ouabain- and low [K⁺]_O-induced amplification of the Ca²⁺ transients depended on Na⁺ entry via an La³⁺- and Mg²⁺-inhibitable pathway, possibly mediated by SOC.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effects of Mg2+ and La3+ on augmentation of Ca2+ transients by ouabain and reduced external K+ concentration ([K+]O). A: effect of 10 mM Mg2+ on 500 nM 5-HT-evoked Ca2+ transients in absence and presence of 100 nM ouabain. B: effect of 10 µM La3+ on 500 nM 5-HT-evoked Ca2+ transients in 5 and 1 mM [K+]O. A and B: representative single myocyte data (for statistical summaries, see Table 1, C and D). All Ca2+ transients were evoked (10 min apart) by 5-HT. First Ca2+ transient in each panel is control; second, third, and fourth Ca2+ transients were evoked during simultaneous application of 5-HT and 100 nM ouabain (A) or low (1 mM)-K+ PSS (B). Both 10 mM Mg2+ (A) and 10 µM La3+ (B) were added to bath 1 min before ouabain and 5-HT (A) or 1 min before low [K+]O and 5-HT (B).

Effects of ouabain on PE-evoked Ca²+ transients in pressurized small arteries. The aforementioned experiments were performed on cultured myocytes from large, muscular mesenteric arteries. In small arteries and arterioles, myogenic tone plays a role in the regulation of vessel diameter and blood flow in response to changes in perfusion pressure. To determine the possible influence of ouabain on the function of these vessels, which are responsible for much of the peripheral vascular resistance, we studied evoked Ca²⁺ transients in pressurized small mesenteric arteries. In control experiments (not shown), a 60-min exposure to nanomolar ouabain had no detectable effect on [Na⁺]_cyt (measured with SBFI) or [Ca²⁺]_cyt in pressurized preparations. Treatment with 1 mM ouabain, however, markedly increased both [Na⁺]_cyt (i.e., the SBFI F_340/380 ratio) and [Ca²⁺]_cyt (i.e., the fura 2 F_360/380 ratio).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effect of nanomolar ouabain on [Ca2+]cyt transients [measured as changes in fluorescence (F360/F380) ratio] evoked by phenylephrine (PE) in pressurized, endothelium-denuded rat small mesenteric arteries. A: representative data from a single fura 2-loaded artery showing Ca2+ transient evoked by 5 µM PE before and during treatment with 3.3 nM ouabain and after washout of ouabain. Artery was pressurized to 70 mmHg. Horizontal bars indicate times of exposure to PE and to ouabain. B: dose-response curves showing effects of ouabain concentration on relative PE-evoked peak and plateau F360/F380 ratios; for each artery, amplitudes are normalized to respective control amplitudes (no ouabain = 1.0; see PE-evoked Ca2+ transient at left in A). Data points correspond to means ± SE for 7 arteries. The data fit Hill equation (see dose-response curve in Fig. 1B), with EC50 values of 4 nM (peak) and 8 nM (steady-state), and Hill coefficient n = 2. All solutions contained 10 µM nifedipine and 5 µM guanethidine.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Effect of 10 mM Mg2+ on Ca2+ transients evoked by PE in pressurized (70 mmHg), endothelium-denuded rat small mesenteric arteries. A: representative data from a single fura 2-loaded artery showing Ca2+ transient evoked by 5 µM PE before and during elevation of extracellular Mg2+ concentration ([Mg2+]O) to 10 mM and after return to Krebs solution containing 1.2 mM Mg2+. B: summary of data from A and 6 other arteries that were subjected to same protocol. Bars indicate absolute F360/F380 ratios (±SE) for resting [Ca2+]cyt and peak and plateau evoked by 5 µM PE. Resting [Ca2+]cyt and peak PE-evoked [Ca2+]cyt did not change significantly when [Mg2+]O was elevated (* P > 0.05 and  P > 0.05, respectively). PE-evoked plateau [Ca2+]cyt did, however, decline significantly ( P < 0.05) to a level that was not significantly different from resting [Ca2+]cyt (P > 0.05). All solutions contained 10 µM nifedipine and 5 µM guanethidine.

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Interactions between ouabain and Mg2+ on PE-evoked Ca2+ transients in pressurized (70 mmHg), endothelium-denuded rat small mesenteric arteries. A: representative data from an artery showing effect of 3.3 nM ouabain on Ca2+ transients evoked by 5 µM PE in media containing 1.2 mM and 10 mM [Mg2+]O. B: summary of data from experiment in A and 7 other arteries that were subjected to same protocol; recover in 1.2 mM Mg2+ was measured in 6 arteries. Bars indicate absolute F360/F380 ratios (±SE) for resting [Ca2+]cyt and peak and plateau evoked by 5 µM PE. Resting [Ca2+]cyt did not change significantly when ouabain was added or when [Mg2+]O was elevated (* P > 0.05). Ouabain (3.3 nM) significantly increased peaks and plateaus of PE-evoked transients ( P and § P < 0.01, respectively). Elevated [Mg2+]O significantly reduced PE-evoked peak ( P < 0.05) to a level that was not significantly different from peak F360/F380 ratio in presence of 1.2 mM Mg2+ and absence of ouabain (P > 0.05). Elevated [Mg2+]O also significantly reduced PE-evoked plateau (# P < 0.05) to a level that was not significantly different from resting F360/F380 ratio (P > 0.05). All solutions contained 10 µM nifedipine and 5 µM guanethidine.

View larger version (59K): [in this window] [in a new window]   Fig. 8.   Model of PLasmERosome with key transport proteins involved in augmentation of Ca2+ transients by low-dose ouabain and OLC and low [K+]O. Model shows junctional sarco/endoplasmic reticulum (S/ER) [with sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps], an adjacent plasmalemma (PL) microdomain [containing store-operated channels (SOC), 2/3 Na+ pumps, and Na/Ca exchanger], and tiny, diffusion-restricted cytosolic space between PL and junctional S/ER (PL-reticular space; PRS). 1 Na+ pumps are widely distributed in PL but may be excluded from these microdomains. We anticipate that PRS [Na+] is higher than bulk [Na+]cyt; shaded region (PRS) corresponds to [Na+] and [Ca2+] standing gradients, with higher concentrations of both ions at center of PRS than at periphery. A: situation under normal conditions. B: anticipated changes in local [Na+] and [Ca2+] after administration of low-dose ouabain (sufficient to inhibit only 3 or 2). ECF, extracellular fluid; IP3, inositol trisphosphate; IP3R, IP3 receptor; RYR, ryanodine receptor.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Rationale for a reexamination of the mechanism of action of ouabain. The idea that the cardiotonic (and other pharmacological) action(s) of low-dose CTS such as ouabain is initiated by Na⁺ pump inhibition and a global rise in [Na⁺]_cyt has long been accepted dogma (e.g., Ref. 28). The discovery of the Na/Ca exchanger established a critical link between Na⁺ pump activity and Ca²⁺ metabolism and provided the basis (3) for this widely held view. Nevertheless, the very low doses (<100 nM) of CTS that induce cardiotonic (11) and vasotonic effects (48) in the rat inhibit only a small fraction of the Na⁺ pumps of a cell, so that there should be negligible rise in whole cell [Na⁺]_cyt. Indeed, several investigators have pointed out that the cardiotonic effects of CTS can be observed without any detectable rise in [Na⁺]_cyt (9, 31), but their reports have generally been ignored. Our observations are not only consistent with these reports and support the view that the cardiotonic and vasotonic actions of low-dose CTS can occur in the absence of a rise in whole cell [Na⁺]_cyt but now suggest a plausible mechanism for these effects. Moreover, our results indicate specific and distinct functional roles for the two isoforms of the Na⁺ pump -subunit (1 and either 2 or 3) that are coexpressed but differently distributed in the PL of myocytes from arteries and skeletal muscle (26, 30) and perhaps cardiac muscle (25). Here, we discuss our findings from this perspective, and we propose a new theory of CTS action that embraces these previously unexplained observations.

Low-dose ouabain augments Ca²+ signaling in arterial myocytes with no elevation of whole cell [Na⁺]_cyt. In pressurized small mesenteric arteries as well as in cultured myocytes, we found that low concentrations of ouabain (1-30 nM) augmented Ca²⁺ transients. These same low doses of ouabain also augmented vasoconstrictor responses in small arteries (unpublished data). The augmentation of Ca²⁺ transients by ouabain depended on extracellular Na⁺. Similar augmentation was also observed with low-dose digoxin, OLC, and reduced [K⁺]_O in the cultured cells. Thus it appears that this effect was the result of Na⁺ pump inhibition and the reduction of active Na⁺ extrusion rather than some other effect of the CTS. For example, the effect cannot be attributed to "slip-mode conductance" of Ca²⁺ through voltage-gated Na⁺ channels (38) because mesenteric arterial myocytes do not express such channels (53) and because augmentation occurred in the absence of extracellular Ca²⁺ (Fig. 1D).

Na+ pump catalytic subunit () isoforms: their functional significance. Although Na⁺ pump inhibition may augment Ca²⁺ signaling by inhibiting, indirectly, Ca²⁺ extrusion via Na/Ca exchangers, it was necessary to find an explanation for the effect in the absence of elevation of whole cell [Na⁺]_cyt. Recent observations of differential Na⁺ pump isoform distribution and the localization of Na/Ca exchangers provided important clues that may help to resolve this dilemma. In mesenteric arterial myocytes, both the 3 Na⁺ pumps and the Na/Ca exchangers (27) are confined to discrete domains of PL that overlie the jSR (Fig. 8). In contrast, the low ouabain-affinity 1 Na⁺ pumps and the PL (ATP-driven) Ca²⁺ pumps are distributed uniformly over the remainder of the cell surface (27). Restricted localization of the 2- or 3-isoforms of the Na⁺ pump along with the Na/Ca exchanger in PL microdomains adjacent to jS/ER, have also been observed in other cell types (21, 27, 30, 36). Furthermore, transgenic studies reveal that, in mouse cardiac muscle, 1 and 2 are coexpressed, but the latter selectively mediates the cardiotonic effect of low-dose ouabain (25). In addition, several groups have reported that the activity of the cardiac Na/Ca exchanger is tightly coupled to the Na⁺ pump through a small compartment of cytosol (presumably sub-PL) that has restricted diffusional access to the bulk cytosol (14, 33, 45). Indeed, Wendt-Gallitelli et al. (49) directly demonstrated, in cardiac myocytes, the presence of tiny sub-PL regions of cytosol in which the local [Na⁺]_cyt was as high as 40 mM, whereas bulk [Na⁺]_cyt remained low. Also, microheterogeneity of [Ca²⁺]_cyt in sub-PL regions has been inferred from functional studies in arterial smooth muscle (10, 15, 43, 47) as well as in cardiac myocytes (29, 41).

19

Mechanism by which Mg²+ and La³⁺ antagonize the effect of the low-dose ouabain. The experiments with Mg²⁺ and La³⁺ support the aforementioned hypothesis. In mesenteric arterial myocytes, the SOC opened by SR Ca²⁺ depletion and through which Ca²⁺ stores are refilled are about one-tenth as permeable to Na⁺ as to Ca²⁺ (2). Because extracellular fluid contains 100 times more Na⁺ than Ca²⁺, however, paradoxically these channels may admit more Na⁺ than Ca²⁺ under physiological conditions. Block of SOC by La³⁺ or Mg²⁺ should reduce Na⁺ entry via this route. Neither La³⁺ nor Mg²⁺ is thought of as a specific SOC blocker. Nevertheless, the similarity of their effects at the concentrations used and the fact that they did not affect the 5-HT- or PE-evoked Ca²⁺ transient peaks in the absence of ouabain (Table 1, A-D; Ref. 39 and unpublished data) suggest that they may be relatively selective for SOC under the conditions of our experiments. Moreover, the localization of SOC in the PL microdomains at the PLasmERosome (24) might enhance the sensing of SR Ca²⁺ store depletion. Thus the effects of La³⁺ and elevated Mg²⁺ (Fig. 2C-b and Figs. 4 and 7; Table 1, C and D) provide further support for the participation of SOC as an entry pathway for Na⁺ that is critical to the ability of ouabain to amplify Ca²⁺ transients in the arterial myocytes.

Implications of the local modulation of Ca²+ signaling. The local regulation of [Na⁺]_cyt and [Ca²⁺]_cyt might affect numerous cell functions that are influenced by Ca²⁺, including many in neurons and in all types of muscle where PL-jS/ER complexes (13, 20, 42) with a similar complement of PL transport proteins are present. Our results in resistance arteries raise the possibility that local regulation of Na⁺ and Ca²⁺ may play an important role in the control of vascular tone and blood flow.