Glucocorticoid Modulates Vasodilator Response of Mesenteric Arterioles in Spontaneously Hypertensive Rats

The progressive development of hypertension in SHR is associated with a shift in hemodynamic resistance. During this period a greater than normal level of arteriolar tone in the skeletal muscle microcirculation of SHR can be demonstrated, to the smallest precapillary ramifications of the terminal arterioles (15 to 20 μm).¹ Among the factors that have been proposed to account for the increase in arteriolar resistance are smooth muscle medial hypertrophy,² rarefaction,³ and an elevation of tone in resistance arteries.⁴ The precise causal and temporal interrelation between the factors leading to this upward displacement of arteriolar tone and the hypertension per se remains largely speculative.

In light of the fact that circulating corticosteroid levels are markedly elevated in SHR⁵⁶⁷⁸ it was proposed that various vascular manifestations of the hypertensive condition may involve glucocorticoid pathways.⁹ Ancillary evidence is provided by the facts that progression of the SHR syndrome is associated with a hypertrophy of the cortical zones of the adrenal glands¹⁰ and that the hypertensive state can be prevented by adrenalectomy or by hypophysectomy. The demonstration that corticosterone supplementation in SHR was found to restore hypertension after adrenalectomy further strengthens the possibility that glucocorticoids per se may be a key factor in the development of hypertension.¹¹

We recently observed that recruitment of leukocytes in SHR is blunted when stimulated with proinflammatory stimuli.^12131415 At the same time we noted a less striking response of the arterioles to such proinflammatory substances.¹³ In this context we reported that the blunted response under steady-state conditions to histamine treatment appeared to be mediated by way of an endothelial cell–dependent pathway in mesenteric arterioles of SHR.¹⁶

Accordingly, the present intravital microscopic protocol was designed to clarify the contribution of adrenal glucocorticoids to the atypical arteriolar response to histamine in SHR. In particular, the analysis is designed to measure mesenteric arteriolar dilator response after topical application of histamine, an endothelium-dependent mediator, compared with sodium nitroprusside, an endothelium-independent dilator. The behavior of the small arterioles (20 to 35 μm) was used as a framework for the following reasons. (1) The arterioles in this range are physically aligned separately from the adjacent venules or lymphatics so that a vasodilation independent of adjacent endothelium could be assessed. (2) The arterioles in such a hierarchical subdivision offer a clear image, and there are small differences in the intravascular pressure levels between SHR and WKY compared with large arterioles.

Methods

Rat Preparation

All rat procedures were previously reviewed and approved by the University of California, San Diego, Animal Subject Committee. SHR (n=18) and WKY (n=18) (Charles River Breeding Laboratories, Wilmington, Mass) were used for the study. Twelve SHR and 12 WKY were subjected to bilateral adrenalectomy, and 6 SHR and 6 WKY were subjected to sham surgery at 12 weeks and studied at 13 weeks.¹⁵¹⁷ The cohort of adrenalectomized SHR and WKY was fed and watered ad libitum with standard rat chow and with saline in the drinking water and were maintained in a light-controlled holding facility for 1 week. The sham-operated SHR and WKY were fed and watered ad libitum standard rat chow and water.¹⁰¹⁵¹⁷ The animals appeared alert and in good health. All experiments were performed at the same time of day (11 am to 2 pm). Completeness of the adrenalectomy was verified by postmortem examination of the suprarenal region. To explore the relation of adrenocortical secretions to the observed vascular sequelae, in SHR we resorted to adrenalectomy with and without replacement therapy.

Blood Pressure Measurement

Each rat was maintained under general pentobarbital sodium anesthesia (30 mg/kg IM) as part of the routine for in vivo microscopy, arranged on a heating pad, and covered with a blanket heated to 37°C. A catheter (polyethylene PE-50 tubing, Clay Adams) was inserted into the femoral artery, and mean arterial pressure was measured.¹⁸ Blood pressure was continuously monitored during the entire period of microvascular observation from which average values were calculated. In a previous study¹⁵ we showed that the mean arterial blood pressure exhibited an only modestly lower value (10% to 15%) after general anesthesia with pentobarbital sodium compared with levels prevailing after a catheterization under local anesthesia (4% lidocaine hydrochloride SC, Astra Pharmaceutical Products, Inc). Comparable minor reductions in blood pressure were seen during general anesthesia in the four groups used here (sham WKY, sham SHR, adrenalectomized WKY, and adrenalectomized SHR).¹⁵¹⁷

Intravital Microscopic Observation of Mesentery

The abdomen was opened by a small midline incision. The ileocecal part of the mesentery was exteriorized carefully and draped over a plastic support for intravital microscopy as described previously.¹⁴ The preparation was kept at 37°C and continuously superfused (1.0 mL/min) with a Krebs-Henseleit bicarbonate-buffered solution saturated with 95% N₂/5% CO₂. Interruption of the suffusion over the tissue was avoided, since even short drying was found to result in rapid cell injury as assessed previously by intravital staining with propidium iodide.¹⁹

The mesenteric microcirculation was visualized through an intravital microscope (×55 water immersion objective lens, Leitz) using a digital color CCD camera (DEI-470, Optronics Engineering). Single, unbranched arterioles with a diameter between 20 and 35 μm and a length of ≈50 μm were selected for study. The images were stored in a videocassette recorder (model AG-127OP, Panasonic) for playback analysis. Vessel diameters were measured off-line with a video image–shearing monitor (model 907 IPM). All diameters reported in this study refer to inner lumen measurements at the magnification provided by a ×55 objective lens with final magnification ×2000 on the screen of the television monitor (CT-1383 VY, Panasonic).

After a 20-minute period was observed to allow hemodynamic parameters to reach steady state, concentration-response curves were determined by adding each vasodilator individually (histamine dihydrochloride and sodium nitroprusside, Sigma Chemical Co) in 10-fold increments (10⁻⁸ to 10⁻³ mol/L) to the Krebs-Henseleit bicarbonate superfusate solution.¹⁶ The arterioles were observed at each concentration for 20 minutes. Separate sets of rats were used for each dilator. After the diameter response at the highest concentration was recorded, maximal vasodilation was induced by topical application of papaverine 1.0 mg/mL. This dilator is used routinely for such measurements because it interacts directly with the smooth muscle cell and does not involve any specific receptor pathway. The initial measurements made for a particular concentration at steady state provided the values for d_ss. Measurements after application of papaverine provided d_max values. The 1.0-mg/mL dose of papaverine was sufficient to eliminate evidence of active tone in the arterioles because no further vessel dilation was observed at constant pressure with the addition of any other vasodilator.¹ Topical application of papaverine did not affect the arterial blood pressure, which was monitored throughout the experimental protocol.

The level of arteriolar tone (T) was computed as T=(d_max−d_ss)/d_max. As such, T is a nondimensional parameter that serves to reflect the degree of active smooth muscle constriction such that T=0% in dilated vessels and T=100% in fully constricted vessels with an occluded lumen.¹ d_ss was computed for vessels that exhibited vasomotion as a time average over at least 10 vasomotor cycles.

As a further precaution possible mast cell degranulation, which might have been caused by the surgical handling of the ileocecal mesentery, was evaluated at the end of each experiment by superfusion of 0.1% toluidine blue solution for 30 minutes. After staining is completed, mast cells that show degranulation can be identified by the presence of metachromatic granules in the contiguous extracellular space.²⁰ After toluidine blue staining was completed, the microscopic field under observation together with 3 to 4 surrounding fields was scanned to determine the percentage of mast cells that were degranulated. In tissues in which >10% of the mast cells showed degranulation the data were discarded to minimize the influence of an unregulated cofactor on endogenous histamine release from the mast cells.

Steroid Intervention

To assess the effects of glucocorticoids on blood pressure and arteriolar behavior in the adrenalectomized rats,¹⁵ dexamethasone 21-acetate (Sigma Chemical Co) was injected into a separate group of rats at a dose of 0.5 mg/kg body wt per day IM for 5 days, in line with a previous method.⁶¹⁵¹⁷

Statistical Analysis

Statistical differences between the two animal strains and treatment modalities were determined by one-way layout ANOVA and Scheffé’s-type multiple comparison test. All values are expressed as mean±SD. Statistical significance was set at P<.05.

Results

Sham-operated SHR under general anesthesia were used as a point of departure because mean arterial blood pressure remained significantly higher than normal, at a level of 166±9.3 mm Hg compared with 102.0±11.1 mm Hg for the sham-operated WKY (Table 1).

In the present experiment arterioles with the same range of d_max values were sampled in two strains. We emphasize that the average d_max value was not significantly different among the groups (Table 2), in line with previous measurements.¹¹⁷

A concentration-dependent reduction of arteriolar tone occurred in response to topical histamine in sham-operated as well as adrenalectomized SHR both with and without dexamethasone supplementation (Fig 1). The magnitude of the response in sham-operated SHR was significantly less than in sham-operated WKY for all concentrations between 10⁻⁸ and 10⁻⁴ mol/L (Fig 1). The EC₅₀ value of histamine with respect to the tone response in sham-operated SHR (2.8×10⁻⁴ mol/L, Fig 1, bottom, ○) was ≈40 times higher than in sham-operated WKY (6.8×10⁻⁶ mol/L, Fig 1, top, ○)(P<.05).

After adrenalectomy, the elevated EC₅₀ value of histamine for the tone response in SHR decreased significantly (4.4×10⁻⁶ mol/L, Fig 1, bottom, •)(P<.05), to the level characteristic of sham-operated WKY. Such a decrease in the EC₅₀ values in the adrenalectomized SHR could be prevented by supplementation with dexamethasone (1.0×10⁻³ mol/L, Fig 1, bottom, ▪) (P<.05). The EC₅₀ value of histamine for the tone response in the adrenalectomized WKY and dexamethasone-supplemented, adrenalectomized WKY groups was 3.3×10⁻⁶ mol/L and 2.8×10⁻⁶ mol/L. A comparable range of values was present in the sham-operated WKY (Fig 1, top).

When the endothelium-independent dilator sodium nitroprusside was used, the arteriolar tone response in SHR and WKY did not show the same trend as the response to histamine (Fig 2). There were no differences in the EC₅₀ value between the sham-operated SHR (1.0×10⁻⁶ mol/L, Fig 2, bottom, ○) and sham-operated WKY (2.0×10⁻⁶ mol/L, Fig 2, top, ○). In both adrenalectomized cohorts the EC₅₀ values for sodium nitroprusside fell into the same range (WKY, 2.0×10⁻⁶ mol/L; SHR, 1.7×10⁻⁶ mol/L, •). No significant differences were seen in the EC₅₀ values between adrenalectomized, dexamethasone-supplemented WKY (1.2×10⁻⁶ mol/L, Fig 2, top, ▪) and dexamethasone-supplemented, adrenalectomized SHR (1.0×10⁻⁶ mol/L, Fig 2, bottom, ▪).

Discussion

The present results indicate that the mesenteric arterioles of SHR are refractory to the dilator action of histamine but not to sodium nitroprusside. Although a refractory state was no longer evident after bilateral adrenalectomy, this state could be restored with glucocorticoid supplementation (Fig 1).

Several studies in the literature have reported that the vascular tone of arterioles is significantly elevated in hypertensive individuals.¹²¹ Although several specific modalities have been advanced to explain the upward displacement of steady-state arteriolar tone in SHR, this condition is likely to actually be the end result of several overlapping mechanisms acting in concert, including an increased myogenic response to elevated blood pressure,²² suppression of endothelial cell–derived relaxation,²³ and a shift in neurogenic stimulation,²⁴ among others. Previous investigations dealing with the microcirculation of hypertensive patients also reported morphological changes in the wall of arterioles²⁵ and changes in the development of the arteriolar network,³²⁶²⁷ which the authors of these reports thought could account for the elevation in peripheral resistance. In a recent report we showed that adrenalectomy served to prevent the development of the hypertension per se as well as enhanced arteriolar tone in SHR and that glucocorticoid but not mineralocorticoid supplementation served to restore increased blood pressure and arteriolar tone, emphasizing the importance of glucocorticoids in the development of hypertension (H.S., B.W.Z., G.W.S.-S. unpublished results, 1995).

Although intravascular pressure in the smaller mesenteric arterioles of SHR and WKY has not been reported, according to the report of Bohlen²⁸ of the arterioles of the wall of the intestine, the intravascular micropressures in the 20- to 35-μm mesenteric arterioles in SHR are moderately higher than those in WKY. Compared with the large difference in systemic blood pressure between SHR and WKY, however, the significance of this difference of intravascular pressures at this branching level between two strains is not as great. A myogenic effect due to the somewhat higher intravascular pressure is likely to be associated with the higher tone in SHR arterioles.

A series of studies has demonstrated that histamine-induced relaxations were endothelium dependent.²⁹³⁰³¹ We emphasize that there are differences in the endothelium-dependent vascular response among species and even between vessels in different tissues of the same species.³² For example the level of NO released by histamine in the guinea pig coronary circulation alone is believed to be sufficient to account for the increase in cGMP and the observed coronary vasodilation.³³ In general our observations,¹⁶ in line with those of previous workers,³³³⁴ support the idea that the histamine-induced vasodilator response in the rat mesenteric arterioles is an endothelium-dependent phenomenon.

In line with the proposal by Ignarro et al³⁵ that the modes of action of sodium nitroprusside and NO are similar, the present measurements suggest that the actual release of NO is decreased in hypertensive individuals. Endothelial cells and smooth muscle cells from SHR vessels apparently are modified to release less NO than cells from WKY.²³ Thus, the increase in flow associated with reduced arteriolar dilation in young SHR could be interpreted to be a consequence of an impairment of the NO-mediated part of the flow-dependent dilation.³⁶ Such a sequela is in line with the observation that the arterioles in the SHR mesentery are refractory to endothelium-dependent vasodilators (histamine and acetylcholine).¹⁶ Although an enhanced level of superoxide anion production in mesenteric arteriolar endothelial cells of SHR is no longer present after adrenalectomy, an elevated level of oxidative stress can be reinstated by dexamethasone supplementation.¹⁷ In line with such a scenario, if endogenously produced NO in SHR was in turn consumed by an overproduction of superoxide anion, reactions involving an endothelium-derived NO-dependent vasodilator effect would in turn be attenuated. The activity of xanthine oxidase, a key enzyme associated with microvascular oxidative stress in SHR,¹⁷ is increased in SHR myocardium compared with that of WKY.³⁷ Interestingly, epithelial cell gene expression of xanthine dehydrogenase and xanthine oxidase are enhanced by glucocorticoid application.³⁸ Glucocorticoids also blunt the induction of nitric oxide synthesis in smooth muscle.³⁹ Because in the absence of a source of adrenal glucocorticoids the suppressed vasodilator response of the SHR arterioles to histamine is no longer evident (Fig 1, bottom), the present results provide additional indirect evidence for a link between glucocorticoids and the altered production of endothelium-derived vasoactive agents.

In conclusion, the mechanism by which the dilator response of the arterioles to agents such as histamine is blunted in the SHR mesenteric microcirculation appears to be related to an elevation of adrenal glucocorticoid secretion in this genetic model of the hypertension syndrome.

Selected Abbreviations and Acronyms

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Footnotes