It has been suggested that an endogenous inhibitor of the sodium pump, identified as ouabain in several mammals including humans (1), contributes to the regulation of blood pressure and the pathogenesis of at least certain forms of hypertension through both peripheral and central mechanisms (2,3). Ouabain induces myogenic and/or neurogenic contractile responses in vascular preparations isolated from both laboratory animals and humans (4-8) and acts synergistically with vasoconstrictor agents on isolated vessels. Actually, ouabain potentiates agonist-induced contractions (9-11), and the contractile response to ouabain is amplified by an agonist such as noradrenaline (12,13). Furthermore, ouabain impairs endothelium-mediated relaxation in preparations of resistance arteries from humans (14). All these effects have been observed with short-term, in vitro exposure of isolated blood vessels to the glycoside. Whether long-term, in vivo treatment with ouabain modifies either the vascular smooth muscle contractile responses to vasoconstrictor stimuli or the influence of endothelium on these responses is unclear. The matter is of interest, because the concept that endogenous ouabain might be involved in the development of hypertension is still a controversial issue (15). Furthermore, the central nervous system, rather than the vascular wall (2) has been recently proposed as the major target tissue for endogenous ouabain (3,16).
This study was undertaken to investigate whether prolonged treatment of rats with ouabain modifies (a) the systolic blood pressure; (b) the contractile responses to phenylephrine and endothelin-1 of aortae isolated from treated rats; and (c) the influence of endothelium on these responses. In other words, whether long-term administered ouabain alters the function of the smooth muscle and endothelial cells of aortae isolated from treated rats.
Male normotensive Harlan Sprague-Dawley (Hsd:Sprague-Dawley) rats weighting 350-400 g, obtained from Harlan-Nossan (Milano, Italia), were housed in temperature- and humidity-controlled conditions, exposed to a 12-h light/dark cycle, and given tap water ad libitum and standard rat chow (Standard G.P.L. diet, containing average sodium, from M. Mucedola s.r.l., Settimo Milanese, Italia). Characteristics of this rat line are slow growth and a rather high systolic blood pressure. The systolic blood pressure of conscious, warmed, and restrained rats was measured by the tail-cuff plethysmographic method. For each rat, five measurements were made at 10-min intervals, and the mean was calculated. Systolic blood pressure measured 7 days after the arrival of the animals to our animal house was taken as the control value. A second series of measurements of systolic blood pressure was made 4 weeks after the beginning of the treatment, on the day before the rats were killed.
Rats were randomly assigned to receive either 50 μg/kg/day of ouabain dissolved in 0.9% saline (20 rats) or 0.9% saline (20 rats) continuously infused through miniosmotic pumps (model 2002; Alzet, Charles River Italia SpA) for 4 weeks, according to Ferrari et al. (17), who showed that this ouabain dose and regimen induced an increase in systolic blood pressure of Hsd: Sprague-Dawley rats. The miniosmotic pumps were implanted in a subcutaneous pocket created by making a small incision in the skin between the scapulae of the rats under ether anesthesia. The incision was closed by sutures. Each rat underwent this surgical procedure twice, because the miniosmotic pumps deliver solution for only 2 weeks. Three ouabain-treated and four control rats died during the treatment period.
Isolated aortic rings
Thoracic aortae excised from SD rats were cleaned of connective tissue and cut to obtain rings of 1.5 mm in length. When required, rings were deprived of endothelium by gently rubbing the lumen with the tip of round-nose pliers. Two steel hooks were inserted into the lumen of each ring, which was then vertically placed in an organ bath filled with 15 ml of physiologic salt solution (PSS) aerated with 95% O2 and 5% CO2 and maintained at 35°C (pH 7.35-7.40). One hook was connected to an isometric transducer coupled to a pen recorder (Battaglia-Rangoni TRB/200/2, Bologna, Italia) for monitoring the developed tension. The composition of the PSS was as follows (mM): NaCl, 125; KCl, 5; CaCl2, 2.7; MgSO4, 1; KH2PO4, 1.2; NaHCO3, 25; and glucose, 11. High-K+ PSS was made by increasing the KCl concentration to 60 mM without substituting for NaCl. Rings were stretched passively to impose a resting tension of 1.5 g and allowed to equilibrate for ≥60 min before starting the experiment. Each ring was then repeatedly stimulated with 1 μM phenylephrine until a reproduceable response was obtained. The presence of endothelium was verified by a relaxation of ≥70% induced by 1 μM carbamylcholine in phenylephrine-contracted rings. Each ring was then exposed to the contractile stimuli in the following sequence: 60 mM K+, 10 mM caffeine, phenylephrine, and endothelin-1. A 60-min washout was applied between one stimulus and the following.
Ouabain, L-phenylephrine hydrochloride, and carbamylcholine chloride were purchased from Sigma Chemical Co., St. Louis, MO, U.S.A. Ouabain was dissolved in 0.9% saline, and L-phenylephrine hydrochloride and carbamylcholine chloride were solubilized in bidistilled water. Endothelin-1 (Sigma Chemical Co.) was solubilized in 0.1 M acetic acid to give a stock solution of 4 μM. Caffeine (Sigma Chemical Co.) was dissolved in the PSS to obtain a 10 mM solution.
Data analysis and statistics
Responses of vascular preparations to contractile stimuli are expressed in milligrams of isometric developed tension. Concentration-response curves were constructed on the basis of the equation of a sigmoidal curve with variable slope. Means of Emax (maximal responses) and EC50 (half-maximal responses) ± SEM values were calculated. Student's t test for unpaired observations, with Welch's correction when required, was used to assess the significance of the differences between the means of two groups. Differences were considered significant at p < 0.05.
Systolic blood pressure of long-term treated rats
Systolic blood pressure values and body weights of control and ouabain-treated rats, measured before and after 4-week treatment, are shown in Table 1. No significant differences were found between systolic blood pressure values of control and ouabain-treated rats. Baseline systolic blood pressure values of rats that died during the treatment are included in the table. The exclusion of these values did not change the result.
Effect of prolonged ouabain treatment on the responses of endothelium-intact and endothelium-free rat aortic rings to contractile stimuli
Endothelium-intact aortic rings from ouabain-treated as compared with control rats exhibited higher responsiveness to endothelin-1 (Fig. 1a and Table 2), lower responsiveness to phenylephrine (Fig. 1b and Table 2), and unchanged response to high K+(Table 3). The comparison of endothelium-free aortic rings from ouabain-treated and control rats shows that endothelin-1, phenylephrine, and high-K+ induce lower responses in aortic rings from ouabain-treated than from control rats (Fig. 2a and b; Tables 2 and 3). Responses to caffeine of endothelium-intact and endothelium-free aortic rings from control and ouabain-treated rats are superimposable (Table 3).
Effect of prolonged ouabain treatment on the influence of endothelium on the responses of rat aortic rings to contractile stimuli
Responsiveness to endothelin-1 of aortic rings from control rats was unaffected, whereas that of aortic rings from ouabain-treated rats was greatly decreased by the removal of endothelium (Table 2). The increase of responsiveness and sensitivity to phenylephrine induced by endothelium removal in aortic rings from control rats was maintained in aortic rings from ouabain-treated rats (Table 2). Contractions to high K+ were decreased by the removal of endothelium in aortic rings from control as well as from ouabain-treated rats (Table 3).
In this study, rats treated for 4 weeks with ouabain did not develop hypertension. This result is in accordance with studies reporting lack of hypertensinogenic effect of prolonged ouabain administration to rats (18-21) and sheep (22) or of short-term ouabain administration to normal humans (23) and saline-loaded sheep (24). Otherwise, an increase in blood pressure has been reported in rats given long-term systemic ouabain (17,25,26), whose pressor effect was augmented by partial nephrectomy (25) or after short- or long-term injection into the brain of ouabain (27-29). On the basis of the hypothesis that the hypertensinogenic activity of ouabain could depend mainly on an action on the central nervous system (3), the discrepancies among these results may be due to differences in length, dosage, and route of administration (central vs. systemic); ouabain is a polar glycoside that does not readily cross the blood-brain barrier.
In this study, responsiveness to endothelin-1 is greater, whereas that to phenylephrine is less, in endothelium-intact aortic rings from ouabain-treated than from control rats. Thus long-term treatment with ouabain induces effects on the ex vivo rat aorta responsiveness to an α-adrenergic agonist such as phenylephrine and to endothelin-1 that are opposed and could balance out, resulting in a lack of effect on systolic blood pressure if they occur also in vivo on the microvasculature. Although the mechanisms of the amplified response to endothelin-1 induced by ouabain treatment have not been identified, it could be hypothesized that prolonged ouabain treatment makes endothelial cells able to release some contracting factor(s) in response to endothelin-1, all the more so because (a) the in vivo long-term treatment with ouabain seems to impair the ability of rat aortic smooth muscle to respond to several vasoconstrictor stimuli in vitro, because endothelium-free aortic rings from treated rats show lower responses not only to endothelin-1 but also to phenylephrine and high K+ than do endothelium-free aortic rings obtained from control rats; and (b) the comparison of the endothelium contribution to the contractile responses to endothelin-1 between control and ouabain-treated rat aortic rings shows that endothelium does not affect contractile responses to endothelin-1 of aortic rings from control rats, whereas it increases those from ouabain-treated rats. Therefore the increased responsiveness to endothelin-1 of ouabain-treated rat aortic rings with intact endothelium is related to the presence of endothelium. Interestingly, an analogous difference has been found between normotensive and spontaneously hypertensive rats: endothelium-derived contracting factors, identified as the prostanoids prostaglandin H2 and thromboxane A2, contribute to the endothelin-1-induced contractile responses of aortae only from hypertensive rats (30). It should be mentioned that, different from our results, endothelium removal increases the contractile response to endothelin-1 of several isolated vascular preparations (31,32).
Prolonged ouabain treatment does not impair the ability of endothelium to reduce contractile responses to α-adrenergic stimulation, because the difference between endothelium-intact and endothelium-free aortic rings from control rats is maintained in ouabain-treated rats. The amplification of α-adrenergic-stimulated contractile responses by the removal of endothelium is a well-known phenomenon (33,34) that has been ascribed to the release of nitric oxide from endothelium, because it is mimicked by the exposure of vascular preparations to inhibitors of nitric oxide synthesis (35-39). Both the maintenance of the ability to release relaxing factor(s) by endothelium and the impairment of the aortic smooth muscle to respond to vasoconstrictor stimuli likely account for the decreased responsiveness to α-adrenoceptor stimulation of endothelium-intact aortic rings from ouabain-treated as compared with control rats.
Our results differ from those obtained in isolated rat vessels exposed for a short while to ouabain or incubated in a medium decreasing the plasmalemmal sodium gradient (i.e., low extracellular potassium, which inhibits the sodium pump like ouabain does, or low extracellular sodium), which respond with amplified contractions to noradrenaline (9,40), serotonin, arginine-vasopressin (41), and phenylephrine (11). The fact that these maneuvers augment also caffeine-induced contraction (9,11,41), an indirect measure of the sarcoplasmic reticulum calcium content, implies that an increase in intracellular stored calcium is responsible for the increased vascular responsiveness to agonists. Conversely, our results show that the responses to caffeine of aortic rings from ouabain-treated rats are superimposable on those of control rat aortic rings, indicating that the amount of stored calcium in the sarcoplasmic reticulum is unaffected by in vivo, prolonged exposure to ouabain. Kimura et al. (42) reported unchanged contractility to caffeine in renal arteries from rats killed after prolonged peripheral infusion with ouabain. The lack of differences in caffeine-induced contractions between control and treated rats also indicates that the decrease in responsiveness to either phenylephrine or endothelin-1 of endothelium-free aortae from ouabain-treated rats is not attributable to a decrease in the sarcoplasmic reticulum calcium content. Accordingly, the high-potassium-induced response, which is mediated solely by calcium influx, is blunted as well.
In conclusion, long-term treatment of rats with ouabain induces effects on responsiveness to an α-adrenergic agonist and endothelin-1 of ex vivo aortae that are opposed. If this occurs also in vivo on the microvasculature, the two effects could balance out, accounting for the lack of an effect of long-term ouabain treatment on systolic blood pressure. Because systolic blood pressure was measured only at the beginning and conclusion of the treatment, we cannot exclude that blood pressure changes might have occurred during the 4 weeks of treatment. Further experiments are in progress to identify either the potential endothelium-derived contracting factor(s) involved in the amplification of the responsiveness to endothelin-1 of endothelium-intact aortae or the mechanism(s) responsible for the impairment of the responses to agonists and high potassium of aortic smooth muscle cells induced by prolonged ouabain treatment.
Acknowledgment: We thank Mrs. D. Cinquemani for skillful technical assistance. This work was supported by grants from CNR (FATMA 91.00165 PF41; 91.00414 CT04; FATMA "8" 91.00218 PF41 115.06.654) and Ministero Università e Ricerca Scientifica, Cofinanziamento 9806197882-002 to S.B.
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