CLIN. AND EXPER. HYPER.-THEORY

AND PRACTICE, A14(4), 667-683 (1992)

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CHLORTHALIDONE REDUCES VASCULAR HYPERRESPONSIVENESS IN DOCA-SALT HYPERTENSIVE RATS

A. M. Cabral, M. N. Musso, N.S. Bissoli, F.B. Carvalhinho and E. C. Vasquez Dept. Physiological Sciences Biomedical Center, UFES C. P. 780, 29001 Vitoria, ES, Brazil

Key words: DOCA-salt hypertension, chlorthalidone, vascular reactivity, serotonin, norepinephrine, vasopressin

ABSTRACT The mechanisms of anti-hypertensive effect of diuretics remain unknown. The purpose of this study was to test the hypothesis that long-term treatment with chlorthalidone decreases the responsiveness of resistance vessels to neurohormones. The study was performed in deoxycorticosteroneacetate @OCA)-salt hypertensive rats with and without treatment with chlorthalidone (Chlor. 8 mg/day, for 20 days). Resting mean arterial pressure in freely moving state was significantly reduced in DOCA-saltChlor rats when compared to DOCA-salt rats (116f3 vs 147+7 mmHg, respectively). Chlorthalidone treatment reduced the high plasma sodium content observed in DOCAsalt rats to the same levels observed in normotensive control groups. Results obtained in isolated perfused mesenteric arteries showed: a) the increase in perfusion pressure elicited by norepinephrine (NE), serotonin (SE) and vasopressin (VP) was significantly greater in DOCA-salt than in DOCA-salt+Chlor rats or control normotensive rats; b) the endothelium removal increased the pressor responses to NE, SE and VP in a similar way in all groups. These data provide evidence that long-term chlorthalidone treatment reduces vascular hyperresponsivenessto these neurohormones. In addition, these results indicate that this reduction in vascular hyperresponsiveness, associated with a decrease in extracellular sodium level, could be a possible mechanism by which the diuretics reduce the high blood pressure.

66 7 Copyright 0 1992 by Marcel Dekker, Inc.

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INTRODUCTION Although diuretics have been frequently used in the long-term treatment of hypertension, the precise mechanism by which these anti-hypertensive agents lower blood pressure is still unknown. The initial response of the treatment with these drugs

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is a small reduction in extracellular fluid volume and salt depletion due to the diuretic and natriuretic effects. This negative sodium and fluid balance could be the first mechanism of reduction in blood pressure (1-3). After long-term administration, fluid volume and cardiac output

return almost to normal values (4-7) whereas total

peripheral resistance falls (8). The exact mechanism involving reduction in vascular resistance and consequently falls in high blood pressure is still uncertain. Alterations in salt and water balance could be one of the most important mechanisms. Diuretics diminish the excess of sodium in blood vessels (9,10), followed by a reduction in intracellular calcium concentration and thereby causing a diminution in the response of smooth muscle to vasoconstrictor hormones (1 1). Therefore, endogenous agents such as norepinephrine, serotonin and vasopressin could have a decreased vasoactive effect under diuretic treatment. The DOCA-salt is a well appropriate model of experimental hypertension to understand possible diuretic-induced alterations in the responsiveness of resistance vessels. The present study was designed to investigate the influence of long-term treatment with chlorthalidone on noradrenergic, serotonergic, and vasopressinergic sensitivity of microvessels from DOCA-salt hypertensive rats.

MATERIAL AND METHODS Male 45day old Wistar rats were uninephrectomized under ether anesthesia. Four days later the animals were treated with either Deoxycorticosterone acetate (DOCA, Sigma Chemical Co., St. Louis, MO, USA, injected subcutaneously, 2 mg/animal) or

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vehicle (soybean oil, 0.25 ml/animal). This treatment was repeated twice a week for 20 days. Rats treated with vehicle were provided either with plain drinking water (normal control) or like all DOCA-treated animals, with drinking water containing 1% NaCl and 0.03% KCI (salt control). Simultaneously to DOCA treatment, half the rats were Clin Exp Hypertens Downloaded from informahealthcare.com by Nyu Medical Center on 02/12/15 For personal use only.

given chlorthalidone (Chlor, Ciba-Geigy, Basel, Switzerland, 8 mg/animal/day, added to food) until the 20th day. Fluid intake and urinary volume were measured over a 24 h period for 20 days. Samples of urine were collected and frozen for estimation of sodium and potassium contents which were measured in a flame photometer (CorningEEL-100/13681, Evans Electroselenium Ltd., Essex, England). At the end of the treatment period, the femoral artery and jugular vein were cannulated with a polyethylene catheter (PE-50 with tapered end, and filled with heparinized saline, 40 Ulml) under ether anesthesia. Six hours later, mean arterial pressure (MAP) and heart rate (HR) measurements were made in conscious unrestrained animals using a pressure transducer (1280C, Hewlett-Packard, Massachusetts, USA) coupled to a polygraph (7754B, Hewlett-Packard). After recording the hemodynamic parameters, the animals were anesthetized with ether, and portions of blood were collected for estimation of plasma sodium and potassium concentration, and the superior mesenteric artery, with its branches, was isolated and perfused with warmed (37 "C) gassed (5% C 0 2 in 95%

02 physiological salt solution of the following composition: 130 mM NaCl, 4.7 mM KCI, 1.6 mM CaCl2, 1.17 mM MgS04, 14.9 mM NaHC03, 1.18 mM KH2P04, 0.026 mM EDTA, and 11.1 mM glucose, pH 7.4. Perfusion was maintained at a constant rate of 4 ml/min with a roller pump (BP-100, Incibras, Sio Paulo, Brazil) and the perfusion pressure was recorded by means of a pressure transducer (1280C, Hewlett-Packard ) placed in a side part proximal to the preparation and coupled to a polygraph (7754B, Hewlett-Packard). After an equilibration period of 45 min basal perfusion pressure (approximately 40 m a g ) , dose-response curves to the pressor effects of

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norepinephrine HCL and serotonin creatinin sulfate (Sigma Chemical Co.) and the pressor response to vasopressin (Sigma Chemical Co., 1.5 pg, repeated at interval of

10 min) were obtained. Dose-response curve to vasopressin was not performed due to the tachyphylactic effect. The drugs were dissolved in 0.9% NaCl and injected as a 0.1

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ml bolus into the Tygon tubing close to the mesenteric artery. These procedures were

made in presence and absence of endothelium which was removed by perfusion of the mesenteric vascular bed (MVB) with the non-ionic, nondenaturing detergent 3-[(3-

(cholamidopropyl)dimethyl-ammonio]-l-propanesulfonate (CHAPS, Sigma Chemical Co.) at 0.3% (w/v) for 60 sec. followed by a 45 min. reequilibration period (12). The presence of functional endothelium was tested by injections in bolus of single combined doses of Acetylcholine chloride (100 pg) plus Norepinephrine bitartarate (Sigma Chemical Co., 16 pg) before and after CHAPS. Statistical analysis of data was performed by a two-way repeated measures analysis of variance (ANOVA) followed by a post-hoc protected T test (Tukey test).

RESULTS As shown in table 1, MAP was significantly higher (35%) in the DOCA-salt rats than in normal-control and salt-control animals. Treatment with chlorthalidone significantly reduced, but did not completely block, hypertension in the DOCA-salt rats. The basal HR of DOCA-salt rats was significantly higher (9%) than those measured in both control groups. Chlorthalidone treatment reduced the basal HR to the same control levels observed in both control groups. During the chlorthalidone treatment, liquid intake, urinary volume, urinary sodium/potassium concentration, and plasma sodium/potassium concentration were measured; liquid intake and urinary volume observed in normal-control rats increased from 34 ml and 7.5 ml to 55 ml and

23 ml in salt-control grpup, respectively. After chlorthalidonetreatment, the high liquid

CHLORTHALIDONE AND VASCULAR RESPONSIVENESS

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TABLE 1 Influence of Concomitant Treatment with Chlorthalidone (Chlor) on Cardiovascular Parameters and Body SodiumD'otassium Stores in Rats Rendered Hypertensive with DOCA.

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GROUPS NormalControl (14)

SaltContro1 (15)

DOCA-Salt (18)

DOCA-Salt + Chlor (18)

110f2

106f2

147f7**

116f3++

Heart Rate (beatdmin)

339+4

341 f6

371 +9**

345f6+

Sodium (mEq/l) Plasma Urine

141 f 1 150f6

142+ 1 2 14fqXx

145f la 208 f6##

142k 1 206 f3'#

MEASUREMENTS

Mean Arterial Pressure (mmW

Potassium (mEq/l) Plasma Urine

++ 4.9f0.1 37&4

4.5f0.1 22 f2"

4.8k0.2 22 f2##

4.0f0.1** 21 f2"

Each value is meanf SEM for the number of rats shown in parentheses. **P

Chlorthalidone reduces vascular hyperresponsiveness in DOCA-salt hypertensive rats.

The mechanisms of anti-hypertensive effect of diuretics remain unknown. The purpose of this study was to test the hypothesis that long-term treatment ...
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