INTRODUCTION

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SODIUM-potassium-activated ATPase (Na⁺-K⁺-ATPase or Na⁺ pump) is an integral membrane enzyme that pumps intracellular Na⁺ outward and extracellular K⁺ inward (10, 26). The enzyme is involved in several cellular functions, such as cell volume regulation, creation of transmembrane potential, regulation of intracellular ionic concentrations, contractility, growth, and differentiation (6, 10). In vascular smooth muscle cells, Na⁺-K⁺-ATPase contributes to the maintenance of an electrochemical gradient of Na⁺ and K⁺ across the cell membrane and plays a major role in the regulation of vascular tone (7, 17). An increase in Na⁺-K⁺-ATPase activity leads to hyperpolarization and relaxation of smooth muscle, whereas its inhibition by cardiac glycosides induces the opposite effects. Furthermore, digitalis-like substances that inhibit Na⁺-K⁺-ATPase activity in cardiovascular tissues have been found in patients and animal models with hypertension and diabetes (2, 4, 8, 14). Therefore, it has been suggested that inhibition of Na⁺-K⁺-ATPase may play a pathophysiological role in the genesis of essential hypertension (2).

Na⁺-K⁺-ATPase activity has been shown to be dynamically regulated in a number of tissues by hormones, neurotransmitters, and local factors (6). Vasodilators acting through the cyclic nucleotide pathways (cAMP and cGMP) increase Na⁺ pump activity by activating their specific protein kinases. Therefore, an activation of Na⁺-K⁺-ATPase has been suggested to contribute, at least partly, to the mechanism of nitric oxide- and -adrenoceptor agonist-induced vasodilation (20, 25, 28). Paradoxically, several vasoconstrictors may also stimulate Na⁺-K⁺-ATPase. Protein kinase C (PKC)-dependent activation of Na⁺-K⁺-ATPase has been reported with endothelin-1 (ET-1) in rabbit aorta (9), with the -adrenergic agonists phenylephrine and clonidine in canine femoral artery and saphenous vein (15), with ANG II in rat aorta (3), and with serotonin in cultured canine femoral artery cells (16). PKC phosphorylates Na⁺-K⁺-ATPase in several tissues, but conflicting results have been reported because PKC may increase, decrease, or not change the Na⁺-K⁺-ATPase activity (1, 6, 13). In vascular smooth muscle cells, it has been suggested (29) that PKC induces a direct stimulation of Na⁺-K⁺-ATPase, whereas it indirectly inhibits the enzyme by activation of phospholipase A₂ (PLA₂) and the subsequent release of arachidonic acid and its metabolites that are known to inhibit the enzyme (23, 24).

The activity of Na⁺-K⁺-ATPase is commonly measured by ouabain-sensitive ⁸⁶Rb⁺ influx, ²²Na⁺ efflux, or phosphate generation. However, little attention has been paid to the consequences of enzyme activity on cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) and vascular tone. In the present study, we analyzed the modulation by norepinephrine (NE), ET-1, and phorbol esters on Na⁺-K⁺-ATPase-induced changes in [Ca²⁺]_i and contractile force in isolated piglet mesenteric arteries. KCl-induced reduction in [Ca²⁺]_i and relaxation in arteries incubated in K⁺-free solution have been used as functional indicators of Na⁺-K⁺-ATPase activity (5, 27).

	METHODS

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Tissue preparation. Twenty-seven male piglets (2-3 wk old, 3-6 kg) were killed by exsanguination in the local abattoir, and the mesenteric vascular beds were rapidly immersed in cold (4°C) Krebs solution (composition in mM: 118 NaCl, 4.75 KCl, 25 NaHCO₃, 1.2 MgSO₄, 2.0 CaCl₂, 1.2 KH₂PO₄, and 11 glucose) and transported to the laboratory. The mesenteric arteries (internal diameter 1-2 mm) were carefully dissected free of surrounding tissue and cut into rings 2-3 mm in length (18). The endothelium was removed by gently rubbing the intimal surface of the rings with a metal rod. The endothelium removal was verified by the inability of acetylcholine (10⁶ M) to relax arteries precontracted with NE (10⁶ M). Two L-shaped stainless steel wires were inserted into the arterial lumen, and the rings were introduced into Allhin organ chambers filled with Krebs solution (gassed with 95% O₂-5% CO₂ at 37°C). Isometric tension was recorded as previously described (18). The preparations were stretched to their optimal resting tension (2 g) and allowed to equilibrate for 60-90 min.

Experimental protocols. After equilibration, rings were washed two times in a nominally K⁺-free solution (without KCl and replacing KH₂PO₄ with NaH₂PO₄) for 1 h. Rings were then stimulated with NE (10⁶ M) or ET-1 (3 × 10⁹ M). These concentrations induced similar submaximal contractions, representing 60-75% of the maximal response of the agonists. Unstimulated controls were further incubated for 20 min in K⁺-free solution. Thereafter, KCl (0.2-8.8 mM) was added in a cumulative fashion. The relaxant effect of each concentration of KCl was expressed as a percentage of relaxation of the contraction before the addition of KCl. Some arteries were treated with phorbol 12-myristate 13-acetate (PMA), staurosporine, ouabain, quinacrine, or propranolol just after the challenge to K⁺-free solution.

Simultaneous measurements of [Ca²⁺]_i and tension. Endothelium-denuded rings prepared as described in Tissue preparation were inverted and incubated for 4-6 h at room temperature in Krebs solution containing acetoxymethyl ester of fura 2 (fura 2-AM; 5 × 10⁶ M) and cremophor EL (0.05%). Thereafter, rings were mounted under 2 g of tension in a 5-ml organ bath filled with Krebs solution at 37°C gassed with a 95% O₂-5% CO₂ gas mixture in the absence of fura 2. The contraction was measured by an isometric force transducer. The bath was part of a fluorimeter (CAF 110; Jasco, Tokyo, Japan) that allowed us to estimate changes in the fluorescence intensity of fura 2 simultaneously with force development (21). The luminal face of the ring was alternatively illuminated (128 Hz) with two excitation wavelengths (340 and 380 nm) from a xenon lamp coupled with two monochromators. The emitted fluorescent light (filtered at 500 nm) at the two excitation wavelengths (F₃₄₀ and F₃₈₀) was recorded together with the force data by using data-acquisition hardware (model 8e, Mac Lab) and data-recording software (Chart v3.2; AD Instruments, Castle Hill, Australia). The ratio of emitted fluorescence (F₃₄₀/F₃₈₀) obtained at the two excitation wavelengths was used as an indicator of [Ca²⁺]_i. After equilibration for 30-45 min, vessels were initially stimulated with 80 mM KCl for 10-15 min, which induced a sustained increase in [Ca²⁺]_i and force. Because of the overall failure of the calibration techniques (11), all results of both [Ca²⁺]_i and force are expressed as a percentage, considering the values at rest in normal Krebs solution (4.75 mM KCl) and after 80 mM KCl-induced stimulation as 0 and 100%, respectively.

Drugs. The following drugs were used: l-NE bitartrate, acetylcholine chloride, ET-1, staurosporine, PMA, propranolol, quinacrine, cremophor EL, and ouabain (Sigma, Alcobendas, Madrid, Spain). Fura 2-AM (1 mM solution in dimethyl sulfoxide) was purchased from Calbiochem (La Jolla, CA). Stock solutions of all drugs were prepared in distilled deionized water (except for staurosporine, quinacrine, and PMA, which were dissolved in dimethyl sulfoxide), and further dilutions were made in Krebs solution. Ascorbic acid (10⁴ M) was added to the stock solution of NE to prevent oxidation.

Statistical analysis. Results are expressed as means ± SE, with n equal to the number of animals. Individual cumulative concentration-response curves were fitted to a logistic equation. The maximal effect (E_max) and the drug concentration producing 50% of E_max (pD₂, expressed as negative log molar) were obtained from the fitted concentration-response curve for each ring. Statistically significant differences between groups were calculated by an ANOVA followed by a Newman-Keuls test. P < 0.05 was considered statistically significant.

	RESULTS

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KCl-induced relaxation in K⁺-free solution. After equilibration, exposure of the rings to K⁺-free solution induced a slowly developing and sustained contraction with a time delay of ~20-30 min. Under these conditions, addition of KCl in a cumulative fashion (0.2-8.8 mM) induced a concentration-dependent relaxant response (Fig. 1). Table 1 shows the contractile responses induced by K⁺-free solution and calculated pD₂ and E_max values of the concentration-response curves to KCl. The relaxant response after each addition of KCl reached a steady state within 3-5 min. Addition of 10⁶ M NE or 3 × 10⁹ M ET-1 after 1 h of exposure to K⁺-free solution induced a further significant increase in tension. After this contractile response reached a steady state, addition of KCl fully relaxed precontracted rings in a concentration-dependent manner (Fig. 1). However, NE and ET-1 inhibited the relaxant effect of KCl, producing a significant rightward shift of the concentration-response curve without affecting the maximal relaxant response compared with unstimulated rings, this effect being significantly more marked for ET-1 than for NE (P < 0.05). Ouabain (10⁵ M), added just after the exposure to K⁺-free solution, almost completely inhibited the relaxant response to KCl in arteries treated with NE (E_max = 12 ± 5%, P < 0.01 vs. control with NE in absence of ouabain; Fig. 1).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Relaxant effects of KCl (0.2-8.8 mM) in endothelium-denuded piglet mesenteric arteries incubated in K+-free solution for 80 min (Ctrl), K+-free solution + norepinephrine (NE; 106 M), K+-free solution + endothelin-1 (ET-1; 3 × 109 M), or K+-free solution + ouabain (105 M) + NE (10 6 M). NE and ET-1 were present for the last 20-30 min and ouabain for the whole exposure to K+-free solution. Results are means ± SE. Brackets indicate concentration. Number of experiments, previous tone, and parameters of concentration-response curves are shown in Table 1.

Effects of phorbol esters. The simultaneous exposure to K⁺-free solution and 10⁷ M PMA induced a contractile response after 80 min equivalent to that induced by NE or ET-1 (Table 1). As shown in Fig. 2, in arteries treated with PMA, the maximal relaxant response to KCl was reduced but the pD₂ value was unaffected compared with arteries in the absence of PMA. In arteries exposed to K⁺-free solution and 10⁷ M PMA for 60 min, NE induced a further increase in tone, so that the final tone was higher than that in arteries treated with NE alone or PMA alone, but these differences did not reach statistical significance (Table 1). However, PMA inhibited the maximal relaxant response to KCl in arteries stimulated by NE (Fig. 2), but it had no effect on the pD₂ value.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Effects of phorbol 12-myristate 13-acetate (PMA; 107 M) on relaxant effects of KCl (0.2-8.8 mM) in endothelium-denuded piglet mesenteric arteries incubated in K+-free solution in absence and presence of NE. Ctrl indicates experiments in absence of NE and PMA. NE was present for the last 20-30 min and PMA for the whole exposure to K+-free solution. Results are means ± SE. Number of experiments, previous tone, and parameters of concentration-response curves are shown in Table 1.

Effects of staurosporine, quinacrine, and propranolol. To study the role of PKC and PLA₂ on the inhibitory actions of NE and ET-1, we pretreated mesenteric arteries with the protein kinase inhibitor staurosporine (10⁷ M) or the PLA₂ inhibitor quinacrine (2 × 10⁵ M; Fig. 3). Quinacrine decreased the contractile responses induced by K⁺-free solution after 60 min (235 ± 173 mg, n = 11), whereas staurosporine did not significantly modify these contractions (773 ± 244 mg, n = 13) compared with control values in parallel experiments (940 ± 200 mg, n = 21). Both drugs tended to inhibit the further contraction induced by addition of NE or ET-1 (Table 1). Staurosporine induced a leftward shift of the concentration-response curve to KCl in arteries stimulated by NE or ET-1, increasing the pD₂ values. Therefore, it partially prevented the rightward shift of the concentration-response curve induced by both NE and ET-1, this effect being more marked for ET-1 than for NE. In contrast, quinacrine had no effect on the relaxant response of KCl in arteries stimulated by NE, ET-1, or PMA + NE. Similarly, pretreatment with the -adrenoceptor antagonist propranolol (10⁶ M) had no effect on the relaxant effects of KCl in NE-contracted arteries (Table 1).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Effects of staurosporine (107 M) and quinacrine (2 × 105 M) on relaxant effects of KCl (0.2-8.8 mM) in endothelium-denuded piglet mesenteric arteries incubated in K+-free solution and stimulated by NE (106 M; A), ET-1 (3 × 109 M; B), and PMA (107 M) + NE (C). NE and ET-1 were present for the last 20-30 min and staurosporine, quinacrine, and PMA for the whole exposure to K+-free solution. Results are means ± SE. Number of experiments, previous tone, and parameters of concentration-response curves are shown in Table 1. Dashed line representing fitted curve of concentration-response curve of KCl in absence of stimulation (taken from Fig. 1) is given for reference.

Effects of KCl on [Ca²⁺]_i and contraction in K⁺-free solution. Figure 4 shows representative recordings of the changes in [Ca²⁺]_i and force in mesenteric arteries loaded with fura 2. When the bathing medium was changed from normal to 80 mM KCl Krebs solution, both [Ca²⁺]_i and force rapidly increased. The values at rest and after the steady-state increase induced by 80 mM KCl were considered to be 0 and 100%, respectively. Exposure to K⁺-free solution for 30 min produced no measurable change in [Ca²⁺]_i or force. However, cumulative addition of KCl (0.4-8.8 mM) to rings exposed to K⁺-free solution resulted in a concentration-dependent reduction in [Ca²⁺]_i below resting values without any change in force (Fig. 5 and Table 2).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Original recordings of effects of KCl (0.4-8.8 mM) on cytoplasmic free Ca2+ concentration ([Ca2+]i) and force in endothelium-denuded piglet mesenteric arteries incubated in K+-free solution. Arteries were initially exposed to 80 mM KCl, then to K+-free solution, stimulated by NE (106 M; A) and ET-1 (3 × 109 M; B), and finally KCl was added in a cumulative fashion. Results of [Ca2+]i (fluorescence ratio F340/F380) and force were expressed as a percentage of response induced by 80 mM KCl.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Effects of KCl (0.4-8.8 mM) on [Ca2+]i (A) and force (B) in endothelium-denuded piglet mesenteric arteries incubated in K+-free solution under control conditions (Ctrl) or stimulated by NE (106 M), ET-1 (3 × 109 M), or PMA (107 M) + NE (106 M). Experiments were similar to those shown in Fig. 4. Results of [Ca2+]i (ratio F340/F380) and force are expressed as a percentage of response induced by 80 mM KCl. Results are means ± SE of 3-5 experiments. Insets show normalized results.

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View larger version (21K): [in this window] [in a new window]   Fig. 6.   [Ca2+]i-force relationship in endothelium-denuded piglet mesenteric arteries incubated in K+-free solution, stimulated by NE (106 M), ET-1 (3 × 109 M), or PMA (107 M) + NE (10 6 M), and treated by increasing concentrations of KCl (0-8.8 mM). Data were obtained from results shown in Fig. 5. Dashed line indicates [Ca2+]i-force relationship of initial response to 80 mM KCl.

	DISCUSSION

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We analyzed the effects of NE, ET-1, and phorbol esters on the changes in [Ca²⁺]_i and force induced by activation of Na⁺-K⁺-ATPase after the addition of KCl in isolated piglet mesenteric arteries incubated in K⁺-free solution. To avoid interferences with endothelium-derived mediators, all the experiments were performed in endothelium-denuded arteries (22). The present results can be summarized as follows. KCl-induced relaxations in K⁺-free solution were associated with a reduction in [Ca²⁺]_i and inhibited by ouabain. However, KCl reduced [Ca²⁺]_i below resting values, whereas force was reduced to near-resting values. NE, ET-1, and PMA inhibited the relaxant effects of KCl, and this effect was attenuated by the PKC inhibitor staurosporine but not by the PLA₂ inhibitor quinacrine. However, ET-1 and PMA potentiated the [Ca²⁺]_i-reducing effect of KCl. Thus the [Ca²⁺]_i-force relationship for KCl was shifted leftward in PMA + NE-treated or ET-1-treated arteries compared with NE-treated arteries.

KCl-induced relaxation has been reported to be mediated by an activation of Na⁺-K⁺-ATPase when the previous K⁺ concentration is <5 mM, whereas inward rectifier K⁺ channels predominantly mediate K⁺-induced relaxations above the physiological K⁺ concentrations (5, 19, 27). In the present study, KCl induced a concentration-dependent relaxation in arteries exposed to K⁺-free solution that was strongly inhibited by ouabain, indicating that it was mainly mediated by an activation of the Na⁺-K⁺-ATPase. The link between activation of ATPase and relaxation is not well understood but must involve hyperpolarization and removal of Ca²⁺ from the area of the contractile proteins (e.g., by an activation of Na⁺/Ca²⁺ exchange). In experiments performed in conventional organ chambers, K⁺-free solution induced a slowly developing contractile response with a time delay of 20-30 min that was relaxed by readdition of KCl. This contraction was absent in the experiments performed in the fluorimeter mainly because of the shorter exposure (30 min) to K⁺-free solution (to avoid excessive fura 2 loss, the whole duration of experiments had to be drastically reduced). Nevertheless, KCl-induced relaxation in arteries treated with K⁺-free solution plus NE or ET-1 was similar in both types of experiments. Thus the presence of a contractile response to K⁺-free solution is not a prerequisite for K⁺-induced relaxation, suggesting that although both phenomena are initiated by an inhibition and activation of Na⁺-K⁺-ATPase, respectively, the mechanisms leading to contraction and relaxation, respectively, are different. This suggestion is also supported by the fact that KCl fully relaxes the contractile response induced by K⁺-free solution plus NE (not only the component of tone induced by K⁺-free solution).

Several physiological stimuli modulate Na⁺-K⁺-ATPase activity (6, 29). ET-1 stimulates the Na⁺-K⁺-ATPase activity in rabbit aorta (9) and capillary endothelium (12) by activating PKC. -Adrenergic agonists also increased Na⁺-K⁺-ATPase activity in canine femoral artery and saphenous vein (15) even when this effect was not observed with NE in cultures of smooth muscle from rat aorta (3). In the present study, NE, ET-1, and the phorbol ester PMA (a PKC activator) inhibited KCl-induced relaxations. Because NE and ET-1 can also activate PKC, this signaling pathway might be involved in the inhibitory effect of NE and ET-1 on KCl-induced relaxation. The nonselective PKC inhibitor staurosporine partially prevented, whereas quinacrine, an inhibitor of PLA₂, had no effect on, the inhibitory actions of NE and ET-1. These results suggested that the shifts in the concentration-response curves to KCl induced by NE and ET-1 are mediated at least partially by an activation of PKC, whereas PLA₂ does not appear to play any role. Furthermore, propranolol had no effect on the relaxant effects of KCl, indicating that a possible -adrenoceptor stimulation and an increase in cAMP is not implicated in NE-induced effects.

Although the main mediator for vascular smooth muscle contraction is an increase in [Ca²⁺]_i, receptor agonists and phorbol esters cause greater contraction than expected at a given [Ca²⁺]_i, i.e., they induce Ca²⁺ sensitization (11). In the present study, NE, ET-1, and PMA + NE induced a contractile response about two times as high as that induced by 80 mM KCl for an increase in [Ca²⁺]_i of about one-half of that induced by 80 mM KCl, indicating the Ca²⁺ sensitization induced by these drugs. To analyze the possible involvement of Ca²⁺ sensitization in the reduced relaxant effect of KCl in NE-, ET-1-, and PMA-treated arteries, we compared their effects on the changes induced by KCl on both [Ca²⁺]_i and contraction measured simultaneously. ET-1 and PMA potentiated the KCl-induced reduction of [Ca²⁺]_i, producing a leftward shift of the concentration-response curve to KCl, but did not affect the maximal [Ca²⁺]_i reduction. These results are consistent with a stimulatory effect of ET-1 and phorbol esters on Na⁺-K⁺-ATPase activity in vascular smooth muscle, as reported by direct measurements of ⁸⁶Rb⁺ uptake (9). However, it cannot be ruled out that these agents might interfere with the cascade of events from the activation of Na⁺-K⁺-ATPase to the [Ca²⁺]_i reduction (e.g., Na⁺/Ca²⁺ exchange). In contrast, NE induced a weak, nonsignificant, leftward shift of the concentration-response curve to KCl. Thus contradictory results were obtained, because ET-1 and PMA, but not NE, potentiated the [Ca²⁺]_i reduction, whereas all three stimulatory agents inhibited relaxation induced by KCl.

The plot of changes in [Ca²⁺]_i versus changes in force induced by KCl shows that the Ca²⁺-force relationship was shifted to the left in NE, ET-1, and PMA + NE (i.e., less reduction in force was observed for a given reduction in [Ca²⁺]_i) even when the effect was less marked with NE alone. Thus the apparent contradiction of inhibition of KCl-induced relaxation and potentiation of KCl-induced reduction in [Ca²⁺]_i by NE, ET-1, and PMA can be explained by the Ca²⁺ sensitization induced by these drugs. NE and ET-1 may induce Ca²⁺ sensitization through the stimulation of PKC so that the effect of staurosporine on KCl-induced relaxations in arteries stimulated by ET-1 and NE could also be interpreted in terms of an inhibitory action on PKC-induced Ca²⁺ sensitization rather than on PKC-induced changes on Na⁺-K⁺-ATPase.

Our results are in good agreement with the proposal that KCl-induced relaxation after incubation in K⁺-free solution is a functional indicator of Na⁺-K⁺-ATPase (27) because the initial event is an activation of the Na⁺-K⁺-ATPase. However, it does not accurately reflect Na⁺-K⁺-ATPase activity (5) because other factors may alter relaxation without an effect on the enzyme activity. Therefore, differences in KCl-induced relaxation can be found in different experimental conditions without or even with opposite changes in Na⁺-K⁺-ATPase activity (5). We provide evidence that changes in Ca²⁺ sensitization can explain these apparently contradictory results.

In conclusion, ET-1, PMA, and to a lesser extent, NE, are functional inhibitors of Na⁺-K⁺-ATPase-induced relaxant effects in endothelium-denuded piglet mesenteric arteries. This effect occurs even when the direct effect on the enzyme activity might be stimulatory rather than inhibitory. This apparent paradox can be explained because ET-1, PMA, and NE induce a Ca²⁺ sensitization for smooth muscle contraction, and therefore relaxations do not parallel the reductions in [Ca²⁺]_i after the activation of Na⁺-K⁺-ATPase.