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Tropical Journal of Pharmaceutical Research, Vol. 8, No. 3, June 2009, pp. 209-214 Research Article Altered Endothelium-Dependent Vasoreactivity of Aortic Rings Follows L-Arginine and Potassium Co-Supplementation in Rats Raymond I Ozolua* and Eric KI Omogbai Department of Pharmacology & Toxicology, University of Benin, PMB 1154, Benin City 300001, Nigeria Received: 13 Sept 2008 Revised accepted: 14 Jan 2009 Code Number: pr09028 Abstract Purpose: The use of L-arginine and potassium chloride separately as supplements has been reported to result in altered vascular reactivity. The concentration of either agent used has varied widely and there has been no report on the outcome of combined supplementation with both agents on vascular reactivity. We therefore designed this study to measure thoracic aortic ring reactivity after five-week co-supplementation with 1% L-arginine and 0.75% potassium chloride. Keywords: Potassium chloride, L-arginine, co-supplementation, aortic rings INTRODUCTION Prevention of hypertensive disease has focused, in recent times, on the use of dietary supplements, several of which are rich in potassium1. This has arisen following several studies in humans and animals, which show that potassium supplementation and ingestion of potassium-rich diets reduce blood pressure and the tendency to develop the disease1-3. In our previous study, we showed that 0.75% potassium supplementation in drinking water reduces blood pressure in normotensive animals and blunts responses to some vasoactive drugs4. L-arginine as a precursor of nitric oxide has also been widely reported to reduce blood pressure in normotensive humans and animals although the concentrations used vary with the investigators5-7. Several mechanisms have been proposed for the anti-hypertensive effect of potassium. These include increased endotheliummediated dilatation of blood vessels8,9 , increased activity of vascular superoxide dismutase and Na+, K+-ATPase enzymes thereby leading to hyperpolarization of blood vessels10,11 , and reduced response to vasoconstrictors such as angiotensin12. While the possible mechanisms of action of potassium seem to be poorly understood and have generated some controversy, the use of L-arginine seems more defined and better understood. Supplementation with L-arginine (the precursor of the vasodilatory autacoid nitric oxide) has been widely reported to increase the tendency of blood vessels to dilate13,14 . Inhibition of the synthesis of nitric oxide by administration of L-nitroarginine methyl ester has been known to result in hypertension in several animal studies15,16 . There has been no report on the vascular effects of co-supplementation with both potassium chloride (KCl) and L-arginine. We therefore designed experiments in order to study the effects of co-supplementation on the reactivity of aortic rings obtained from Sprague-Dawley rats and the dependence of these effects on the endothelium. MATERIALS AND METHODS Animals Sprague-Dawley rats weighing between 200 and 300 g were obtained from the animal house of the Department of Physiology, Ambrose Alli University, Ekpoma, Edo State, Nigeria. The animals were allowed two weeks of acclimatization before they were divided into two experimental groups (six animals per group). One group (the control) was given tap water while the second group (treated) received 0.75 %w/v KCl + 1 %w/v L-arginine in the tap water. Supplementation was done for a period of 5 weeks since our previous reports on potassium supplementation was for that length of time4,10 . All animals were housed in standard cages and had free access to pellets (Bendel Feeds and Flour Mills Ltd, Ewu, Nigeria) and the particular drinking fluid. The animals were exposed to natural lighting conditions and were handled according to international protocols for use of animals in experiments (National Institute of Health USA: Public Health Service Policy on Humane Care and Use of Laboratory Animals, 2002; and the 1986 European Convention for the protection of Vertebrate Animals Used for Experimental and other Scientific Purposes) in the absence of a functional local ethical committee on the use of animals. At the end of supplementation, animals were sacrificed under thiopentone sodium (15 mg/kg) anaesthesia. Measurement of fluid intake In order to ascertain that the animals actually drank the fluid which contained the supplements, average daily fluid intake in the groups was measured for the 5 weeks of supplementation. Animals drank from a graduated water bottle and at the same time each day, the decrement in the amount of fluid consumed over 24 h was measured by subtracting the day’s reading from that of the previous day. Isolated aortic ring experiments The thoracic aortae were quickly dissected out of the sacrificed animals and placed in physiological salt solution (PSS). The tissues were cleaned of adherent connective tissues and cut into rings of 2 mm length. In endothelium-denuded rings, denudation was achieved by gently rubbing the intimal layer of the vessel with a wire. The rings were suspended in L-shaped wire loops in 10 ml organ baths containing PSS. The upper loop was attached to a Grass Model FT03 force transducer connected to a Grass Model 7D polygraph (Grass Instruments Co., Quincy, MA, USA). The PSS was made of the following composition (mM/L): NaCl 119, KCl 4.7, NaHCO3 24.9, KH2PO4 1.2, MgSO4 1.2, CaCl2 1.6, glucose 11.5. The PSS was bubbled throughout with 95% O2 -5% CO2 gas mixture (BOC Gases, Nig. Plc) with the pH and temperature maintained at 7.4 and 37oC respectively. The rings were given a resting force of 1 g10. An equilibration period of 60 min was allowed after which rings were stimulated twice at 15 min intervals with 100 mM K+. Aortic tissue viability was closely monitored within 6-hour period of experiments. This was ascertained by the observation that force produced by 100 mM K+ for each ring was reproducible within this period. Rings from both groups were exposed to cumulative concentrations of noradrenaline ranging from 1x10-10 M to 1 x 10-4 M. Vasorelaxant effects of carbachol were studied by precontracting the rings with noradrenaline at a concentration producing 75% of maximal noradrenaline-induced contraction obtained from pilot study (1 x 10-6 M for control and 1 x 10-7 M for the treated). Contractile responses from the pilot studies were 75.2 ± 4.7% and 74.3 ± 5.5% for 1 x 10-6 M NA (control) and 1 x 10-7 M NA (treated) respectively. This was followed by the addition of the relaxant; after steady contraction had been established (timedependent control experiments showed that the precontraction induced by noradrenaline remained stable for the duration of the concentration-response tests). Drugs Noradrenaline and carbachol were obtained from Sigma (UK) and were prepared fresh each day by dissolving in distilled water. Larginine and KCl for supplementation were obtained from BDH chemicals (UK) and were prepared once a week. All other chemicals and reagents were of analytical grade and were obtained from either of Sigma, May & Baker or BDH chemicals. Statistics Aside from daily fluid intake in the two groups which is presented as means, all other data are presented as mean ± SEM (standard error of the mean) and n represents the number of rats from which aortic rings were taken. Emax (the maximum response by aortic rings) are presented either as contractile force (mg) produced by rings exposed to noradrenaline, or as percentage of maximum relaxation response to carbachol following noradrenaline precontraction. Comparison of Emax and pD2 (negative log of the concentration producing 50% contractile response to noradrenaline) values were made by use of Student’s t-test. In all cases, P < 0.05 was taken as indicating statistically significant difference between compared data. RESULTS The daily fluid intake by the animals as measured for 35 days shows fluctuating but insignificantly different patterns of consumption between the two groups (Fig. 1). Mean daily volume of the fluid consumed by the control group was 133.0 ± 2.2 ml and 130.0 ± 2.0 ml for the L-arginine + KCl cosupplementation group. Co-supplementation with L-arginine and KCl significantly enhanced the relaxation of endothelium-denuded aortic rings to effective concentrations of carbachol (Fig. 2). There was increase in the Emax compared to values in control (22.3 ± 3.6 versus 12.1 ± 2.7 %). Treatment also significantly altered the maximum relaxation to carbachol in endothelium-intact aortic rings (63.4 ± 2.8 versus 49.2 ± 4.3%). Endothelium-intact aortic rings obtained from rats which received the supplements showed significantly attenuated responses to noradrenaline (Fig. 3). Emax values were significantly lower in endothelium-intact rings from the treated group compared to the control: 1315.5 ± 106.2 versus 1690.1 ± 68.1 mg (P < 0.05). Similarly, the pD2 values were significantly lower in the endothelium-intact rings from the treated group than the control (7.0 ± 0.1 versus 7.4 ± 0.1, P < 0.05). Although endothelium-denuded rings from the treated group showed lower Emax and higher pD2 values than rings obtained from the controls, these values were not significantly different. The respective values are 2069.5 ± 80.1 versus 2356.8 ± 228.0 mg (Emax ) and 8.0 ± 0.2 versus 7.8 ± 0.1 (pD2 ). DISCUSSION The combination of 1% L-arginine and 0.75% KCl as supplements in drinking fluid of rats has been shown in the present study to alter vascular reactivity to noradrenaline and carbachol. In rats which received the supplements, responses of endotheliumintact aortic rings to noradrenaline were attenuated while there was enhanced relaxation by aortic rings to carbachol irrespective of the status of the endothelium. In our previous study we showed that while supplementation with 0.75% KCl for five weeks did not affect acetylcholine-induced relaxation in endothelium-intact aortic rings, it significantly enhanced acetylcholine-induced relaxation in endothelium-denuded rings17 . Similarly, responses to vasoconstrictors such as noradrenaline and 5-HT were attenuated in aortic rings from potassium-supplemented rats17. Decreased vasoconstriction to noradrenaline and enhanced relaxation to acetylcholine have also been reported in DOCA-salt hypertensive rats which were placed on 0.8% L-arginine supplementation18. Consequently, the present data suggest that the combined supplements further enhanced carbachol-induced relaxation in an endothelium-independent manner. This enhanced carbachol-induced relaxation is possibly due to the fact that basal nitric oxide production was increased by the increased provision of the precursor Larginine although serum and urinary Larginine levels were not measured. Several workers have found that supplementation with L-arginine enhances relaxant action of acetylcholine while improving haemodynamics in human and animal experiments7,18,19 . Nitric oxide itself causes relaxation through the cyclic GMP pathway while also facilitating the opening of potassium channels with the resultant vascular smooth muscle cell hyperpolarisation20. Similarly, several reports have suggested that potassium supplementation enhances endotheliumdependent relaxations, increased vascular activity of superoxide dismutase and Na+,K+-ATPase enzymes and reduced vascular reactivity to pressor agents8-12. Although membrane potentials were not measured in the present study, it seems that vascular smooth muscle cell hyperpolarisation may only be a part mechanism since in our previous report potassium channel blockade with tetraethylammonium only partially reversed the enhanced relaxant effect of levcromakalim and sodium nitroprusside in aortic rings obtained from potassium supplemented rats17. Although several protocols and concentrations have been used each for Larginine and KCl, previous workers have indicated that the concentrations used in the present study are well tolerated by the animals. For example the animals become adapted to the increased potassium load after weeks of supplementation21. In the case of L-arginine, concentrations far in excess of what has been used in the present study have been reportedly well tolerated22. The ability of the animals to tolerate the supplements is accentuated by the facts that fluid consumption was not significantly different in the two groups and that there were no obvious signs of toxicity. CONCLUSION Combining potassium chloride and L-arginine as supplements enhances carbachol-induced relaxation in an endothelium-independent manner while blunting vasoconstriction by noradrenaline in endothelium-intact aortic rings. These are beneficial vascular effects in the non-pharmacologic prophylaxis of hypertension and its sequelae. ACKNOWLEDGEMENTS The authors are grateful to Miss Miriam Ezeanuna who assisted with the work and to BOC Gases Nigeria Plc for offering a discount on the cost of O2-CO2 gas mixture. REFERENCES
© Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. The following images related to this document are available:Photo images[pr09028f2.jpg] [pr09028f1.jpg] [pr09028f3.jpg] |
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