Loss of Endothelium and Receptor-Mediated Dilation in Pial Arterioles of Rats Fed a Short-Term High Salt Diet
Abstract—A high salt diet often is regarded as an accessory risk factor in hypertension, coincidental to the deleterious effect of high blood pressure on vasodilator function. The aim of this study was to determine whether short-term ingestion of a high salt diet per se impairs vasodilator function in the cerebral circulation independent of blood pressure changes. Adult Sprague-Dawley rats were fed a normal salt (0.8%) or high salt (4%) diet for 3 days. Mean arterial pressures were similar in the normal and high salt groups (123±2 and 125±2 mm Hg, respectively). Subsequently, the responses of the in situ pial arterioles to acetylcholine, iloprost, and sodium nitroprusside were determined in cranial windows using intravital videomicroscopy. Pial arterioles of rats fed normal and high salt diets showed similar resting diameters of 69±2 and 72±3 μm, respectively, but their reactivity patterns to vasodilator stimuli were markedly different. Arterioles of rats fed a normal salt diet dilated progressively up to 17±3% in response to the endothelium-dependent agent acetylcholine (10−9 to 10−6 mol/L) and dilated by 22±2% in response to the prostaglandin I2 receptor agonist iloprost (3×10−11 mol/L). In contrast, pial arterioles of rats fed a high salt diet constricted by 4±3% and 8±2% in response to acetylcholine and iloprost, respectively. Sodium nitroprusside (10−6 mol/L), a nitric oxide donor, dilated pial arterioles of rats fed low and high salt diets by a similar amount (19±3% and 16±2%, respectively), suggesting that signaling mechanisms for dilation distal to the vascular smooth muscle membrane were intact after high salt intake. These results provide the first evidence that the short-term ingestion of a high salt diet may severely impair the vasodilator function of the in situ cerebral microcirculation independent of blood pressure elevation.
- cerebral circulation
- pial arterioles
- sodium chloride, dietary
- muscle, smooth, vascular
Ahigh salt diet often is regarded as an accessory risk factor for vascular disease because of its association with high blood pressure in some individuals. However, many individuals ingest a high salt diet independent of hypertension, and little consideration has been given to the possibility that a high salt diet alone may increase their susceptibility to vascular disorders.1 2 In this regard, a recent report suggests that isolated skeletal muscle resistance arteries of rats with normal blood pressure show blunted endothelium- and receptor-mediated dilator responses after exposure to a long-term high salt diet.3 These findings imply that ingestion of high salt per se, distinct from blood pressure elevation, may represent an independent risk factor for vascular abnormalities.
Because the cerebral microcirculation relies on vigorous dilator responses to match local blood flow to cortical metabolism, the goal of the present study was to determine whether a high salt intake alters the dilator function of in situ cerebral arterioles in the absence of detectable blood pressure changes. The short-term ingestion of high salt is a frequent event in Western diets, and hence, in these initial experiments, we examined the effect of a 3-day high salt diet on endothelium- and receptor-induced vasodilator responses of the cerebral microcirculation.
Fourteen male Sprague-Dawley rats (Harlan Sprague Dawley, Inc, Indianapolis, Ind), 8 weeks of age, were fed a standard Purina rat chow containing either normal salt (0.8% NaCl) or high salt (4% NaCl) for 3 consecutive days with water ad libitum. On the fourth day, rats were anesthetized with sodium pentobarbital (60 mg/kg IP) and instrumented for chronic monitoring of cardiovascular parameters as described previously.4 The femoral artery was cannulated for measurement of blood pressure and blood gases, and the femoral vein was cannulated for administration of supplemental anesthetic as necessary. The trachea was cannulated with PE-240 tubing, and the rats were artificially ventilated with 30% O2 (balance N2) to maintain arterial Po2 between 100 and 140 mm Hg and arterial Pco2 between 35 and 40 mm Hg. Body temperature was maintained at 37°C with a water-circulated heating mat. All procedures were performed in accordance with institutional guidelines.
In Situ Monitoring of Pial Arteriolar Diameters in Cranial Windows
Using published methods,4 dura-open cranial windows were prepared for the observation of in situ rat pial arterioles. Briefly, a stainless steel frame containing a glass cranial window with 3 ports for perfusion inlet, perfusion outlet, and intracranial pressure measurement was implanted into a 5-mm opening in the parietal bone. The window area was superfused at a rate of 1 mL/min with artificial cerebrospinal fluid (ACSF) equilibrated with 21% O2 and 6% CO2 and balanced with nitrogen, and maintained at a pH of 7.33 and temperature of 37°C. Local intracranial pressure was maintained at 5 to 7 mm Hg by adjusting the height of the outflow tubing. Images of pial arterioles were monitored with a video camera (RCA-2000; field size, 930×690 μm) after the rat was positioned under an intravital microscope. Vessel diameters were measured on a video monitor (model 917, American Dynamics). Acetylcholine, iloprost, and sodium nitroprusside contained in ACSF were superfused onto the brain to examine their effect on the diameter of superficial pial arterioles on the cortical surface. Acetylcholine and sodium nitroprusside were purchased from Sigma Chemical Co, and iloprost was provided by Berlex Laboratories.
All data are expressed as mean±SEM. Statistical comparisons between groups were made with one-way repeated-measures ANOVA with a subsequent Newman-Keuls test. Significance was accepted at a value of P<0.05.
Comparison of Mean Arterial Pressures
Rats fed normal salt (n=7) and high salt (n=7) diets for 3 days showed average mean arterial pressures of 123±2 and 125±2 mm Hg, respectively. These pressures were not significantly different.
Responses of Pial Arterioles to Acetylcholine and Iloprost
The resting diameter of in situ pial arterioles of rats fed normal and high salt diets averaged 69±2 and 72±3 μm, respectively (both n=7). These arterioles were sequentially exposed for 5 minutes to acetylcholine (10−9 to 10−6 mol/L), iloprost (3×10−11 mol/L), and sodium nitroprusside (10−6 mol/L). Resting diameter values after washout of acetylcholine or iloprost did not differ significantly from initial diameters in drug-free ACSF, demonstrating the reversibility of drug-induced changes in arteriolar diameter.
Figure 1A⇓ compares the average diameter response to the endothelium-dependent dilator acetylcholine between pial arterioles of rats fed normal and high salt diets. Increasing concentrations of acetylcholine (10−9 to 10−6 mol/L) progressively dilated pial arterioles of rats fed a normal salt diet, reaching a value of 17±3% at 10−6 mol/L. In contrast, acetylcholine tended to constrict pial arterioles of rats fed a high salt diet, although this effect did not reach statistical significance. Similarly, Figure 1B⇓ shows that pial arterioles of rats fed a normal salt diet dilated by 22±2% in response to the prostaglandin I2 receptor agonist iloprost (3×10−11 mol/L). However, the diameter of pial arterioles of rats fed a high salt diet was significantly decreased by 8±2%. Figure 1C⇓ compares the average diameter change in response to 10−6 mol/L sodium nitroprusside between pial arterioles of rats fed normal and high salt diets. Sodium nitroprusside increased the diameter of arterioles of rats fed a normal salt diet by 19±3% and dilated arterioles of rats fed a high salt diet by a similar value of 16±2%.
The vasoactive effects of acetylcholine, iloprost, and sodium nitroprusside on pial arterioles of rats fed normal and high salt diets are illustrated in Figure 2⇓ (top and bottom panels, respectively). Selected images were captured in drug-free ACSF (control) or in ACSF containing 10−7 mol/L acetylcholine, 3×10−11 mol/L iloprost, or 10−6 mol/L sodium nitroprusside. In the top panel, a pial arteriole of a rat fed a normal salt diet was observed to dilate from 66 to 77 μm in response to acetylcholine. Iloprost dilated the same arteriole from 66 to 81 μm, and sodium nitroprusside changed diameter from 68 μm in drug-free ACSF to 88 μm. In the bottom panel, a pial arteriole of a rat fed a high salt diet was observed to constrict from 70 to 64 μm in response to acetylcholine. After washout of the acetylcholine, iloprost constricted the same arteriole from 70 to 60 μm. However, this arteriole dilated vigorously from 68 to 85 μm in response to sodium nitroprusside.
The present study provides the first evidence that a short-term elevation of dietary salt intake severely impairs the dilator function of the in situ cerebral microcirculation, in the absence of detectable blood pressure changes. Furthermore, these initial findings suggest that this impairment extends to both endothelium- and receptor-mediated dilator pathways because the dilator responses to acetylcholine and iloprost were both absent in the cerebral arterioles examined in this study. In this regard, acetylcholine is widely used as a detector of endothelium-dependent dilator function, whereas iloprost is a stable analogue of prostacyclin, which directly relaxes arterial smooth muscle by activating the adenylyl cyclase–cAMP signaling cascade in vascular myocytes.3 Prostacyclin production is stimulated by hypoxia,3 5 and its release may contribute to the dilation of the cerebral circulation in response to low Po2.5 Thus, the finding of the present study that a 3-day high salt diet results in a constrictor response rather than a dilator response of in situ cerebral arterioles to iloprost implies that ingestion of high salt may lead to a rapid impairment of this pathway for hypoxia-induced vasodilation.
Several earlier studies focusing on the skeletal muscle circulation have indicated that a high salt diet may have direct, deleterious effects on vasodilator function. For example, Liu et al3 recently reported that isolated resistance arteries from the skeletal muscle of normotensive Sprague-Dawley rats fed a high salt (4% NaCl) diet for 8 weeks showed blunted dilator responses to acetylcholine, iloprost, and hypoxia. Similarly, Boegehold6 7 observed an impaired dilator response to acetylcholine in in situ arterioles of the spinotrapezius muscle of normotensive, salt-resistant Dahl rats after ingestion of a high salt diet (7% NaCl) for 2 to 5 weeks, which he attributed to a suppression of the effect of nitric oxide on these small vessels. Interestingly, Fujita and Ito8 reported that the increases in forearm blood flow during arterial infusion of incremental potassium chloride were blunted in human subjects fed a high salt (180 mEq/d) diet for only 1 week, as compared with the blood flow response of subjects treated with diuretics to prevent volume expansion, a finding they attributed to a reduced activity of the vascular Na+-K+ pump. Our findings support these earlier observations indicating that a high salt diet may have profound effects on vascular relaxation mechanisms. Furthermore, they provide initial evidence that only a short-term ingestion of a high salt diet may abolish fundamental dilator responses in the cerebral microcirculation and may even transform these dilator responses into constrictor events.
This initial study did not resolve the issue of the precise mechanisms responsible for the rapid impairment of cerebral vasodilator function during a high salt diet. However, because the in situ pial arterioles of rats fed a normal or high salt diet in our study showed a similar and pronounced dilator response to the nitric oxide donor sodium nitroprusside, it appears that the impairment of smooth muscle dilation does not extend to this intracellular signaling pathway. Notably, normal dilator responses to sodium nitroprusside also persist in skeletal muscle vessels from normotensive rats maintained on long-term high salt diets.3 6 Thus, it would appear that a high salt diet may rapidly impair crucial signaling steps upstream from the intracellular action of nitric oxide on the guanylyl cyclase–cGMP cascade, perhaps altering initial events at the level of the vascular smooth muscle membrane.
Although further work will be required to pinpoint the dilator events that are altered by a high dietary salt intake, the present study strongly implicates the short-term ingestion of high salt as an unrecognized risk factor for impaired cerebral dilator function, independent of blood pressure status. Future efforts to assess the effect of dietary salt on vascular reactivity in animal and human forms of hypertension, and its effect as an independent risk factor for vascular disorders, will be needed to evaluate the final functional significance of this initial study.
This study was supported by National Heart, Lung, and Blood Institute grants P01 HL-29587 and R01 HL-59238 to Dr Rusch and grants P01 HL-29587, R01 HL-52211, and R01 HL-37374 to Dr Lombard. Dr Liu is a postdoctoral fellow of the Wisconsin Affiliate of the American Heart Association. The authors would like to thank Berlex Laboratories (Wayne, NJ) for their generous donation of iloprost for these studies.
- Received September 14, 1998.
- Revision received September 29, 1998.
- Accepted October 26, 1998.
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