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(Hypertension. 1995;26:1065-1069.)
© 1995 American Heart Association, Inc.

Articles

Effects of Dehydroepiandrosterone Sulfate on Cellular Calcium Responsiveness and Vascular Contractility

Mario Barbagallo; Jie Shan; Peter K.T. Pang; Lawrence M. Resnick

From the University of Palermo (Italy) (M.B.); Division of Endocrinology and Hypertension, Wayne State University, Detroit, Mich (M.B., L.M.R.); and Department of Physiology, University of Alberta, Edmonton, Alberta, Canada (J.S., P.K.T.P.).

	Abstract

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Abstract Dehydroepiandrosterone sulfate (DHEAS) is an endogenous steroid having a wide variety of biological effects, but its physiological role remains undefined. Since an age-related decline of DHEAS corresponds to the progressive onset of atherosclerosis, cardiovascular diseases, and overall mortality, we investigated a possible protective role of DHEAS in vascular disease by studying the effects of this hormone (10^-7 to 10^-5 mol/L) on cytosolic free calcium and contractility in different in vitro vascular tissue preparations. DHEAS produced a significant, dose-dependent relaxation of isolated helical strips of rat tail artery precontracted with KCl (60 mmol/L) (89.7±18.7%, P<.01), arginine vasopressin (3 nmol/L) (27.3±7.1%, P<.01), and norepinephrine (0.1 µmol/L) (49.2±18.2%, P<.01). In isolated vascular smooth muscle cells DHEAS reversibly inhibited KCl (30 mmol/L)–induced elevations of cytosolic free calcium to 69.8±8.4% and 43.8±7.4% of the control response at 5x10^-7 and 5x10^-6 mol/L, respectively (P<.05 at both doses). These results provide evidence of a direct vascular action of DHEAS, in doses reflecting circulating levels in vivo, and suggest the possibility that these effects are mediated by modulation of intracellular calcium metabolism. We hypothesize that physiologically, DHEAS may serve to buffer vascular responsiveness to a wide variety of depolarizing and constrictor hormonal stimuli.

Key Words: calcium • muscle, smooth, vascular • hormones • dehydroepiandrosterone sulfate (prasterone)

	Introduction

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Dehydroepiandrosterone is the most abundantly produced adrenal steroid, and serum concentrations of its sulfate ester, DHEAS, are approximately 20-fold higher than those of any other circulating steroid hormone.¹ Nevertheless, the physiological function of this endogenous steroid remains undefined.² A progressive decrease in circulating levels of DHEAS with age has long been recognized, peak levels occurring between the third and fourth decade of life and decreasing progressively thereafter by 90% to 95% after the age of 85.^{1 2 3 4} This decline in circulating DHEAS levels has been linked to the gradually increasing prevalence of atherosclerotic and cardiovascular disease with age,^{5 6} whereas higher plasma levels of DHEAS have been associated with fewer cardiovascular diseases and decreased mortality.⁷

Supporting these epidemiological studies are experimental data suggesting the vasculature as a target of DHEAS action. This hormone, in a dose-related fashion, prevents corticosteroid-induced hypertension⁸ and may inhibit the development of atherosclerosis.^{9 10 11} In clinical hypertension it has also been linked to the activity of the renin-angiotensin system and to dietary salt, calcium, and magnesium intake.¹²

Based on the above observations and our recent demonstration of direct calcium-related effects of other steroid hormones on vascular smooth muscle tissue,^{13 14 15} we undertook the present experiments to assess whether a direct effect of DHEAS could be demonstrated physiologically on the constrictor responsiveness in vitro of isolated rat tail artery strips and, at the cellular level, on [Ca²⁺]_i in isolated VSMCs.

	Methods

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Vascular Tension Studies
Vascular tension was measured in tail artery helical strips in which the endothelium had been removed. The assay was performed according to the method we previously described.¹⁶ Male Sprague-Dawley rats were anesthetized with pentobarbital, and the rat tail artery and aortic strips were suspended in a Sawyer-Bartlestone chamber containing aerated (95% O₂/5% CO₂) Krebs-Henseleit solution with the following composition (mmol/L): NaCl 115, KCl 5, CaCl₂ 2.1, MgSO₄ 1.2, NaH₂PO₄ 1.2, NaHCO₃ 25, and glucose 11. The free end of the artery was attached to a force displacement transducer (Grass Instrument Co) by a silk thread. Isometric contractions were recorded on a Grass model 7D polygraph. Before exposure to drug each preparation was equilibrated for 60 minutes under a resting tension of 0.7 g. During the equilibration period the artery strips were washed every 15 minutes. The solution in the tissue chambers was continuously oxygenated with a gas mixture of 95% O₂/5% CO₂ and maintained at 37°C. After the equilibration period tissues were tested two times with KCl (60 mmol/L) and then washed. Only tissues with reproducible contractions were used for studies. The rat tail helical strips were then contracted with KCl (60 mmol/L), AVP (3 nmol/L), or norepinephrine (0.1 µmol/L). When a steady tension was achieved a cumulative dose response for the vasodilator effect of DHEAS was obtained. DHEAS was added to the tissue bath at 5-minute intervals to reach the following concentrations: 5x10^-7, 1.5x10^-6, 5x10^-6, 1.5x10^-5, and 5x10^-5 mol/L. The entire dose-response curve was obtained within 45 minutes, a period during which a steady contractile effect of the vasoconstrictors was maintained. A dose-response curve with ethanol was also performed for each type of experiment.

VSMC Studies
Cell Preparation
All studies were performed on VSMCs isolated from male Sprague-Dawley rat tail artery as previously described.^{17 18} Cells were subcultured and used at passages 3 to 10. Male Sprague-Dawley rats (100 to 200 g) were anesthetized with sodium pentobarbital (65 mg/kg body wt IP), and the tail artery was dissected out, immersed in a Ca²⁺- and Mg²⁺-free solution (HBSS, GIBCO Laboratories) at 4°C, and cut into pieces of approximately 2 cm. The arterial strips were than processed as follows at 37°C: (1) Tissues were first rinsed in low-calcium enzyme solution I (0.2 mmol/L Ca²⁺, HBSS) composed of collagenase/dispase (1.5 mg/mL, Boehringer Mannheim Biochemicals), elastase (0.5 mg/mL, type II-a, Sigma Chemical Co), trypsin inhibitor (1 mg/mL, Sigma), and bovine serum albumin (2 mg/mL, fatty acid-free, Sigma) for 1.5 hours; (2) the tissues were rinsed twice in Ca²⁺-free HBSS; and (3) the medium was then changed to enzyme solution II, composed of Ca²⁺-free HBSS with collagenase (1 mg/mL, Sigma type II), trypsin inhibitor (0.3 mg/mL, Sigma type I-S), and bovine serum albumin (2 mg/mL, Sigma). Incubation in enzyme solution II lasted 1 hour. All incubations in enzyme solutions were carried out in a CO₂ incubator (95% O₂/5% CO₂ at 37°C). The dispersed cells were plated in 35-mm Petri dishes with DMEM, with 10% fetal bovine serum, and cultured in a humidified atmosphere of 5% CO₂/95% air at 37°C. More than 95% of cells were viable as shown by the trypan blue exclusion method.¹⁹ Furthermore, as previously described, the contractile responses of the cells to norepinephrine¹⁷ and the localization in these cells of fluorescent antibodies to -actin¹⁸ supported the identity of these cells as functional smooth muscle cells.

[Ca²⁺]_i Measurement
Cells were placed in 35-mm culture dishes in DMEM with 10% fetal bovine serum and maintained in a humidified atmosphere of 5% CO₂/95% O₂ at 37°C. Cells were harvested with 2 mL of 0.25% trypsin and subcultured weekly at a 1:10 dilution. Culture medium was changed every other day until the cells were confluent. Confluent cells were then plated onto glass coverslip (25-mm circle) at a density of approximately 1x10⁶/mL in DMEM and kept in culture until the cells became elongated and confluent (usually 24 to 48 hours). Cells were then incubated for 45 minutes in DMEM containing 5 µmol/L fura 2-AM (Molecular Probes, Inc) at 37°C in a dark compartment. Afterward, the cells were gently washed three times with the buffer ([mmol/L]: NaCl 145, KCl 5, MgCl₂ 1, glucose 10, CaCl₂ 1, NaH₂PO₄ 0.5, and HEPES 10, at pH 7.4) and kept in the same buffer. After about 5 minutes the coverslip with attached cells was placed in a Sykes-Moore chamber of 1 mL volume on the stage of a phase-contrast microscope (Phase Contrast-2, Nikon). Fluorometric data were obtained with a dual-wavelength excitation monochrometer spectrofluorometer (SPEX Industries Inc). Excitation wavelengths of 340 and 380 nm and an emission wavelength of 505 nm were used, and [Ca²⁺]_i was calculated according to the method described by Grynkiewicz et al²⁰ using the equation [Ca²⁺]_i (nmol/L)=K_dx(R-R_min/R_max-R)b, where R is the ratio of fluorescence in the sample at 340 and 380 nm, R_max is the fluorescence ratio obtained by addition of 2 µmol/L ionomycin, R_min is the fluorescence ratio subsequently obtained by addition of 5 mmol/L EGTA, and b is the ratio of the fluorescence of fura 2 at 380 nm at zero and saturating Ca²⁺ concentrations. K_d is the Ca²⁺–fura 2 dissociation constant, taken as 224 nmol/L.²⁰

Control [Ca²⁺]_i elevations were induced by KCl (30 mmol/L) before the addition of the hormone. Cells showing a lack of basal responsiveness to KCl (defined as an increase of [Ca²⁺]_i to 50% of basal) were excluded from further study. No differences in KCl responsiveness were noted in cells used from passages 3 to 4 (the majority used) compared with those of later passages. DHEAS (5x10^-7 and 5x10^-6 mol/L) was subsequently added to the Sykes-Moore chamber and incubated for 10 minutes. [Ca²⁺]_i was measured and compared with control. A second stimulus with KCl (30 mmol/L) was then performed and the response compared with control in the absence of the hormone. After washout of the hormone and a 10-minute recovery period, a third stimulus with KCl (30 mmol/L) was performed and the response compared with the previous two KCl stimuli.

Drugs
DHEAS (Sigma Chemical Co) was dissolved in 95% ethanol to make a stock solution of 5x10^-3 mol/L and was stored at 4°C. The same concentration of alcohol in a control solution had no effect in any of the in vivo or in vitro assays.

Statistics
Values are expressed as mean±SEM. The paired t test was used for comparison between mean values of control and those obtained after drug administration. In the case of multiple comparisons, ANOVA with the Newman-Keuls multiple range test was applied. A minimum of eight experiments was performed for each of the studies. A value of P<.05 was considered significant.

	Results

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In Vitro Short-term Effect on Isolated Tissues
Dose-related responses of precontracted rat tail artery strips to DHEAS are shown in Fig 1. Increasing DHEAS doses produced increasing vasorelaxant responses, starting at a dose of 1.5x10^-6 mol/L. The highest concentration tested (5x10^-5 mol/L) induced a relaxation of 49.2±18.2% (P<.01) in preparations precontracted with norepinephrine, 27.3±7.1% (P<.01) in those precontracted with AVP, and 89.7±18.7% in those precontracted with KCl (Fig 1). No effects of DHEAS on basal tone of the above isolated blood vessels were observed. In addition, a dose-response curve with the vehicle, ethanol alone, was performed for each experiment. Ethanol concentrations up to 2% (200 µL ethanol in a 10-mL bath solution) did not significantly change the vasoconstrictor responses of the artery strip preparation used (+2.04±1.1%, P=NS at the highest ethanol concentration).

View larger version (14K): [in this window] [in a new window]   Figure 1. Line graph shows effect of DHEAS on the developed force of helical strips of rat tail artery contracted with norepinephrine (NE, 0.1 µmol/L), AVP (3 nmol/L), and KCl (60 mmol/L) (n=8). *P<.05 vs control.

Effect of DHEAS on [Ca²⁺]_i of VSMCs
The effects of DHEAS on [Ca²⁺]_i in fura 2–loaded VSMCs are shown in Fig 2 (original recording in a group of monolayer VSMCs) and Fig 3. Stable resting signals of fura 2–loaded VSMCs were recorded for several minutes. Basal [Ca²⁺]_i averaged 110±5.4 nmol/L. KCl (30 mmol/L) increased [Ca²⁺]_i by 90.2±12.8% ([Ca²⁺]_i=+99.2±14.1 nmol/L) (Fig 2, trace A). The addition of DHEAS alone did not produce any significant alteration in resting [Ca²⁺]_i (Fig 2, trace B). However, when KCl (30 mmol/L) was again added after 10 minutes of incubation with DHEAS (5x10^-7 and 5x10^-6 mol/L), the [Ca²⁺]_i increase was significantly inhibited by 30.2±8.4% at the concentration of 5x10^-7 mol/L and by 56.2±7.4% at 5x10^-6 mol/L (P<.05 at both concentrations; Fig 2, trace C, and Fig 3). This effect of DHEAS was reversible, because after washout of hormone and a 5-minute recovery period, KCl responsiveness (30 mmol/L) was restored, [Ca²⁺]_i increasing to 82.3±5.8% of the control response (Fig 2, trace D, and Fig 3; P=NS, DHEAS washout versus control).

View larger version (13K): [in this window] [in a new window]   Figure 2. Actual recording of changes in [Ca2+]i in a monolayer of VSMCs.

View larger version (10K): [in this window] [in a new window]   Figure 3. Bar graph shows effect of DHEAS (5x10-7 and 5x10-6 mol/L) on the increment in intracellular free calcium induced by KCl (30 mmol/L) in VSMCs (n=8). *P<.05 vs control.

	Discussion

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The present study demonstrates that DHEAS, in doses reflecting circulating levels of the hormone in vivo,^{1 21} is a vasoactive hormone in both tissue and cellular in vitro preparations. Specifically, in helical rat tail artery strips DHEAS blunts the development of tension in response to depolarizing stimuli and the specific endogenous vasoconstrictor hormones norepinephrine and AVP. This suggests that the DHEAS effect is general rather than agonist specific. At the same time and in a parallel fashion at the cellular level, DHEAS blunts depolarization-induced cytosolic free calcium accumulation in VSMCs. We suggest that these direct actions of DHEAS on vascular smooth muscle tone derive from its ability to modulate cellular calcium homeostasis. Given these results, we hypothesize that DHEAS may serve physiologically to buffer the vasculature against excessive vasoconstrictor responses to a wide variety of depolarizing and hormonal stimuli.

Mechanistically, most well-accepted actions of steroid hormones are mediated by specific cytosolic receptor molecules, which, when bound to hormone, are translocated to the nucleus where they interact at specific DNA sites to promote or suppress the transcription of various genes. Whether DHEAS follows the same pattern is not known, although a specific high-affinity receptor for dehydroepiandrosterone has been reported.²² However, under our experimental conditions the vasorelaxant and cytosolic free calcium effects produced by DHEAS cannot easily be attributed to a genomic effect involving interaction of the hormone with DNA and subsequent RNA transcription, translation, and protein synthesis, because this mechanism presupposes a significant latency period, whereas our observations are of a more immediate type. Rather, since the constrictor and cytosolic calcium–elevating actions of KCl, norepinephrine, and AVP all depend on cellular calcium entry from the extracellular space, our observations support the view that DHEAS exerts its effects by a nongenomic, presumably plasma membrane–mediated mechanism. The possible contribution of the sulfate moiety of DHEAS as a calcium chelator to decreasing intracellular calcium responses must also be considered, although the fact that basal calcium levels were not affected makes this less likely.

Aside from the mechanism or mechanisms underlying our observations, the specificity of DHEAS action on the vasculature needs to be considered because other steroid hormones may affect calcium fluxes, and direct evidence for estrogen, progesterone, and vitamin D effects on vascular contractile responsiveness and cellular calcium handling have been recently reported.^{13 14 23} Thus, although glucocorticoids, at doses similar to those used for DHEAS in the present study, do not affect calcium transients,²⁴ effects similar to those reported here for DHEAS were previously reported for estradiol and progesterone.^{13 23} However, other steroid hormones, such as 1,25(OH)₂D₃, actually increase both L-type calcium channel currents and [Ca²⁺]_i in VSMCs.¹⁴ Last, despite DHEAS being a weak androgenic hormone, the more potent androgen, testosterone, also has vascular effects opposite to those of DHEAS, potentiating norepinephrine-mediated vascular contraction.²⁵ The above argues against the direct vascular actions of DHEAS reported here representing nonspecific steroid effects.

The clinical relevance of establishing a physiological role for DHEAS derives at least in part from increasing evidence suggesting a link between urine or serum levels of dehydroepiandrosterone and DHEAS and cardiovascular mortality,⁷ myocardial infarction,²⁶ hyperlipidemia,²⁷ and hypertension.^{12 28 29} DHEAS has been shown to prevent dexamethasone-induced hypertension in rats.⁸ Furthermore, DHEAS administration may also provide substantial protection against the development of aortic or coronary atherosclerosis. Thus, a reduction in aortic atherosclerosis in rabbits fed a high cholesterol diet was observed when the diet was supplemented with dehydroepiandrosterone,^{9 10} and long-term dehydroepiandrosterone administration also significantly retarded the progression of accelerated coronary atherosclerosis in transplanted hearts in a hypercholesterolemic rabbit model.¹¹ Although beneficial effects of dehydroepiandrosterone administration on serum lipids and a reduction of low-density lipoprotein cholesterol level have been reported,³⁰ in these above-mentioned animal experiments DHEAS exerted its antiatherogenic effects independently of its action on serum lipids.^{9 10 11} Other possible mechanisms for the antiatherogenic effects of dehydroepiandrosterone include inhibition of smooth muscle cell proliferation³¹ and inhibition of platelet aggregation.³² These above reports are all consistent with our current findings demonstrating blunting of cellular calcium uptake by DHEAS, because the role of cytosolic free calcium in mediating cellular responsiveness to a wide range of extracellular signals is well known,³³ and similar antihypertensive, antiatherosclerotic, antiaggregatory, and antiproliferative effects are also observed after administration of calcium channel antagonists.³⁴ It thus seems reasonable to suggest that DHEAS may function endogenously to physiologically buffer vascular responses to changing electrophysiological and hormonal stimuli. Other modulating effects may also be relevant to nonvascular tissues such as the adrenal glomerulosa, in which DHEAS has been shown to alter adrenal angiotensin receptor–mediated aldosterone production, stimulating responsiveness in that preparation.^{35 36} Overall, we hypothesize that the calcium-related vascular actions of DHEAS observed here may be at least one mechanism linking the age-related decline in DHEAS to the progression of atherosclerotic and cardiovascular disease in the elderly.

Further studies are needed to show whether these effects are also present in vivo. Furthermore, as the vascular preparations used here were taken from male animals, similar studies need to be carried out in female tissues, in view of the sex differences in the plasma levels of the hormone¹ and because no protective effect of DHEAS on cardiovascular mortality has been observed in postmenopausal women.³⁷

	Selected Abbreviations and Acronyms

 

AVP = arginine vasopressin DHEAS = dehydroepiandrosterone sulfate (Prasterone) DMEM = Dulbecco's modified Eagle's medium HBSS = Hanks' buffered saline solution VSMC = vascular smooth muscle cell

	Footnotes

 
Reprint requests to Lawrence M. Resnick, MD, Division of Endocrinology and Hypertension, Wayne State University Medical School, University Health Center, 4H, 4201, St Antoine, Detroit, MI 48201.

Received June 19, 1995; first decision August 18, 1995; accepted September 16, 1995.

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