INTRODUCTION

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CARDIOVASCULAR DISEASES SUCH as hypertension and atherosclerosis are often associated with an increase in vascular tone or an imbalance favoring the actions of vasoconstrictors. The cellular mechanisms that are altered to result in hypertension are still unknown and the subject of intense investigation. Although it has been suggested that alterations in cellular signaling favoring vasoconstriction are the cause of elevated blood pressure, others contend that the enhanced vascular reactivity observed in hypertension is the result of the rise in blood pressure. The separation of these two variables (increased vascular reactivity and increased blood pressure) is difficult. Epidermal growth factor (EGF), of which the enhanced mitogenic effect on vascular smooth muscle cells from hypertensive rats is well characterized (3, 5, 22), has proven to be a potent vasoconstrictor in DOCA salt-induced hypertension (10). EGF interacts with one of four receptors: ErbB1 (EGFR), ErbB2, ErbB3, or ErbB4. Of these receptors, the primary receptor for EGF is EGFR. EGF-induced contraction was reduced by tyrosine kinase inhibitors, both general and specific to the tyrosine kinase intrinsic to the EGF receptor, and appears to be enabled by an increased density of EGF receptors in the vascular smooth muscle of the DOCA-salt hypertensive rat. It was observed that the contraction to EGF appeared only after the systolic blood pressure (SBP) of the DOCA-salt rats was significantly elevated (10). These findings suggested that vascular changes necessary to enable the contractile response to EGF may be dependent on an increase in SBP.

Separating out the effects of elevated blood pressure and changes in vascular reactivity can be difficult, and thus one model we have chosen to test the above hypothesis is a rat strain that is resistant to mineralocorticoid-induced hypertension. The Wistar-Furth rat, a substrain of the Wistar rat, is relatively resistant to the hypertensive effects of excess mineralocorticoids (21). Others have demonstrated that contraction to norepinephrine and a membrane-depolarizing concentration of potassium chloride is enhanced in arteries from aldosterone-treated Wistar rats and unchanged in aldosterone-treated Wistar-Furth rats (1), suggesting that vascular signaling is altered in response to a sustained elevation in blood pressure. Our findings in the present studies have led to the focus of this paper investigating the possibility that mineralocorticoids themselves may function at the level of the vasculature to increase EGF-receptor expression and thereby enable EGF-induced contraction.

A secondary aspect of the present study was to determine whether an increase in blood pressure is necessary for increased contraction to EGF. It must be noted that a finding of a vascular alteration that proceeds rather than preceeds hypertension does not mean that the vascular alteration is not important to the hypertension. Although it is clear that such vascular change is not responsible for the initiation of the hypertension, changes can play a role in the maintanence of hypertension. For example, hyperreactivity to serotonin [5-hydroxytryptamine (5-HT)] occurs coincident with or just after an increase in blood pressure, and blockade of appropriate 5-HT receptors cannot decrease blood pressure in weeks 1 and 2 of DOCA-salt hypertension. This, however, changes in weeks 3 and 4 of DOCA-salt hypertension (29).

Goal. Thus the aim of this project was to examine contraction of isolated rat aorta to EGF in different forms of hypertension [the DOCA-salt, N-nitro-L-arginine (L-NNA) (6), and spontaneously hypertensive rat (SHR) models of hypertension]. In addition, we examined contraction to EGF in isolated aortas from Wistar DOCA-salt hypertensive rats and in aortas from Wistar-Furth rats on DOCA salt. In complementary in vitro experiments, aortas from Wistar and Wistar-Furth rats were exposed to aldosterone to determine whether mineralocorticoids, independent of blood pressure, could change EGFR mRNA expression. The effect of high salt and DOCA therapy alone on contraction to EGF was also examined. Throughout these studies, changes in contractile responses of EGF were compared with those of 5-HT, to which the vascular effects in hypertensive vessels have been well characterized (26). Collectively, these experiments investigated the dependence of contraction to EGF on mineralocorticoids and the association of an increased response to EGF with elevated blood pressure.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals. All animal procedures were followed in accordance with institutional guidelines established by Michigan State University. When the rats arrived at our facility, they were housed in clear plastic boxes with wood-chip bedding and allowed ad libitum access to standard rat chow (Teklab) and tap water.

DOCA-salt hypertension. Sprague-Dawley rats (225- 250 g) were purchased from Charles River (Portage, MI); Wistar and Wistar-Furth rats (200-225 g; age matched to Sprague-Dawley rats) were purchased from Harlan Laboratories (Indianapolis, IN). Under methoxyflurane (Metophane, Mallinckrodt Veterinary, Mundelin, IL) anesthesia, the area to be incised was shaved free of fur. The animals underwent uninephrectomy (flank incision, left side), and a Silastic (Dow Corning, Midland, MI) implant impregnated with DOCA (200 mg/kg) was implanted subcutaneously in the subscapular region. Sham rats were uninephrectomized but did not receive the DOCA implant.

L-NNA hypertension. Sprague-Dawley rats (250-300 g, Harlan Laboratories, Indianapolis, IN) received either normal tap water (sham) or tap water supplemented with L-NNA (0.5 g/l; Sigma Chemical, St. Louis, MO) for 14 days. The rats had ad libitum access to normal rat chow. On day 14, SBPs were measured using a tail-cuff method.

Wistar-Kyoto rats and SHR. Wistar-Kyoto (WKY) rats and SHR (12 wk old) were obtained from Charles River Laboratories. The rats had ad libitum access to normal rat chow. SBPs were measured using a tail-cuff method.

Isolated tissue bath protocol. Rats were euthanized (80 mg/kg ip pentobarbital sodium), and the thoracic aortas were removed. Arteries were dissected into helical strips (0.25 × 1 cm), and the endothelial cell layer was removed by rubbing the luminal side of the vessel with a moistened cotton swab. Tissues were placed in muscle baths filled with warmed (37^oC), aerated (95%O₂/5%CO₂) physiological salt solution containing (mmol/l): 130 NaCl, 4.7 KCl, 1.18 KH₂PO₄, 1.17 MgSO₄ · 7H₂O, 1.6 CaCl₂ · 2H₂O, 14.9 NaHCO₃, 5.5 dextrose, and 0.03 CaNa₂EDTA. One end of the preparation was attached to a glass rod, the other attached to a force transducer (FT03, Grass Instruments, Quincy, MA), and the strip was placed under optimum resting tension (1,500 mg for all tissues) and allowed to equilibrate for 1 h. Changes in isometric force were recorded on a Grass polygraph (Grass Instruments, Quincy, MA). After a 1-h equilibration, arteries were challenged with a maximal concentration of the ₁-adrenergic receptor agonist phenylephrine (PE; 10 umol/l). This response is listed in milligrams in the figure legend of each figure for each group. Tissues were washed, and the status of the endothelium was examined by observing arterial relaxation to the endothelium-dependent agonist acetylcholine (1 umol/l) in tissues contracted by a half-maximal concentration of PE (~10 nmol/l). Only strips that demonstrated minimal relaxation (15%) of the PE-induced contraction were used in this study. EGF (10 pmol/l to 300 nmol/l) was incubated with the tissue for ~10 min to allow the contraction to plateau before the addition of the next concentration. Cumulative concentration response curves to 5-HT (1 nmol/l to 300 umol/l) were conducted in separate aortic strips. In all experiments, the EGF vehicle (10 mmol/l acetic acid + 1 mg/ml bovine serum albumin) was without effect on arterial tone.

In vitro incubation of arteries with aldosterone. To assess whether the observed effects were a direct effect of aldosterone, aortas were removed from Wistar and Wistar Furth rats, cleaned, and incubated for 8 h under tissue culture conditions in PBS or PBS + aldosterone (10⁸ mol/l). RNA was extracted, and RT-PCR was carried out for EGFR (described in RT-PCR protocol).

RT-PCR protocol. RNA was extracted from aortas using a preparative kit (Qiagen), and 1 µg of RNA was used for first strand cDNA synthesis using oligo(dT) as a primer. Occasional RNA samples were subjected to the PCR procedure without prior reverse transcription to control for the presence of contaminating genomic DNA in the sample. PCR amplifications were carried out on a portion of the cDNA produced. Reactions were performed using a PE Applied Biosystems GeneAmp thermal cycler and Thermus aquaticus (Taq) DNA polymerase. The reaction contained 5 pmol/l of each oligonucleotide primer, 200 µmol/l dNTP, 0.2 units Taq, 1.5 µmol/l magnesium chloride, and 1 µCi of [³²P]dCTP in the manufacturer's buffer. Optimum annealing temperature, cycle number, and template dilution factor were determined for each amplicon before experimentation. The cDNA (487-bp product) was resolved on an 8% polyacrylamide gel, and the amount of DNA present was identified by phosphorimage analysis (Bio-Rad, Hercules, CA) and quantified using Multi-Analyst software. The results were normalized to the expression of the constitutively expressed gene cyclophilin. The specific oligonucleotide primers used were as follows: EGF-receptor upstream primer: GAC AGC AGA AGG GAT CAG TCA; downstream primer: CTG GAA GTT TGC AGA TGC CAA; cyclophilin upstream primer: TGT CTC TTT TCG CCG CTT GC; downstream primer: TGC TGG TCT TGC CAT TCC TG. All samples were produced in triplicate for eight separate pairs of aortas from Wistar and Wistar-Furth rats.

Statistics. Contractility data are presented as means ± SE as a percentage of the PE (10⁵ mol/l)-induced contraction for the number of animals indicated in parentheses. In the figure legends, the response (in mg) to PE is reported for each experimental group. Unpaired Student's t-tests were used where appropriate in comparing two groups' responses (P < 0.05 considered statistically significant). Agonist EC₅₀ values were calculated using a nonlinear regression analysis using the algorithm [effect = maximum response/1 + (EC₅₀/agonist concentraction)]. In many cases, a true maximum to EGF was not obtained in tissues from sham rats, and so the EC₅₀ values for shams are estimates of the response, and the true value must be considered equal to or greater than the values reported. One-way ANOVA followed by Student-Newman Keuls test was used when comparing three or more single point groups. When comparing cumulative concentration response curve data, we used a two-way repeated-measures ANOVA. To determine the association between maximal contraction to EGF and SBP, linear regression and correlation analysis were used. RT-PCR product was quantified using Multi-Analyst software. The results were normalized to the expression of the constitutively expressed gene cyclophilin.

Chemicals. Compounds were made in deionized water unless indicated otherwise in parentheses: acetylcholine chloride, aldosterone, 5-HT hydrochloride, PE hydrochloride, (Sigma, St. Louis, MO), EGF (10 mmol/l acetic acid + 1 mg/ml bovine serum albumin; GIBCO Life Technologies, Grand Island, NY).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Table 1 compiles the SBPs of the experimental groups and pharmacological parameters for EGF-induced contraction. We have also included blood pressure data on the graphs depicting EGF-induced contraction. Finally, Table 2 compiles pharmacological parameters for 5-HT, an agonist used in parallel for comparative purposes.

Contraction to EGF in aortas from sham and L-NNA hypertensive rats. The L-NNA-induced model of experimental hypertension was used to raise SBP by inhibiting the enzyme nitric oxide (NO) synthase (NOS) and thus the production of the vasodilator NO. The SBP of the L-NNA-treated rats (213 ± 9 mmHg) was significantly elevated compared with that of sham rats (130 ± 6 mmHg; Table 1, Fig. 1A). Aortic strips from the L-NNA hypertensive rats displayed an increased contraction to EGF compared with aortas from sham rats with respect to both potency and efficacy (Fig. 1A). The contractile agonist 5-HT contracted aortic strips from both sham and L-NNA rats; however, the maximum contraction to 5-HT was only slightly greater in strips from L-NNA rats (111.7 ± 5.3%) than in strips from sham rats (94.8 ± 3.0%; Table 2).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   A: concentration-dependent contraction of endothelium-denuded sham normotensive [phenylephrine (PE) contraction = 930 ± 114 mg] and N-nitro-L-arginine (L-NNA) hypertensive rat aortas (PE contraction 742 ± 65 mg) to epidermal growth factor (EGF). B: concentration-dependent contraction of endothelium-denuded Wistar-Kyoto normotensive (WKY; PE contraction = 779 ± 58) and spontaneously hypertensive rats (SHR; PE contraction = 669 ± 80) aortas to EGF. Points represent the mean, and vertical lines represent the SE of the mean for the number of animals indicated in parentheses; systolic blood pressures are also included in the parentheses. *Significant difference (P < 0.05) between responses of WKY and SHR aortas or sham and L-NNA aortas.

Contraction to EGF in aortas from WKY rats and SHR. We next determined whether EGF would cause contraction in aortas from genetically hypertensive rats. SHR and WKY rats were purchased at 12 wk of age. At this time point, the SBPs of the SHR rats (153 ± 2 mmHg) were significantly higher than those of the WKY rats (117 ± 1 mmHg; Table 1, Fig. 1B). As observed with the aortas of L-NNA hypertensive rats, a profound contraction to EGF was observed in aortas from SHR rats, whereas minimal contraction was demonstrated in aortas from WKY rats. The EC₅₀ values of EGF were similar in aortas of WKY and SHR (9.30 ± 0.38 and 9.86 ± 0.30, respectively), but the magnitude of contraction to EGF in aortic strips from SHR was over four times the contraction achieved in WKY strips (Fig. 1B). By comparison, 5-HT was only slightly more potent and efficacious in aortas of SHR vs. WKY rats (Table 2). Together, these data suggest that the enhanced contractions to EGF and 5-HT are not specific to the DOCA-salt model of hypertension but rather are common to several models of hypertension. However, the magnitude increase in contractile response between sham and hypertensive rats was much greater for EGF than 5-HT.

Contraction to EGF in aortas from Wistar and Wistar-Furth sham and DOCA-salt rats. Previous studies from our laboratory have demonstrated that contraction to EGF occurs in vessels from DOCA-salt hypertensive rats after a significant rise in blood pressure, suggesting elevated systolic pressure is required for contraction to EGF. The similar findings in aortas from L-NNA and SHR are consistent with this idea. Thus Wistar and Wistar-Furth sham and DOCA-salt rats were used because Wistar rats are sensitive to the hypertensive effects of DOCA salt, whereas Wistar-Furth rats are reported to not become hypertensive with the same treatment (1, 21). If an elevated blood pressure was necessary to observe an enhanced arterial response to EGF, then aortas from Wistar-Furth rats that do not get hypertensive when given DOCA salt should not respond to EGF. Table 3 reports the SBPs of these four groups of rats and the result of a statistical comparison of these blood pressures. Wistar DOCA-salt rats (176 ± 9 mmHg) had significantly higher blood pressures than Wistar shams (120 ± 4 mmHg; Table 3). The maximal contraction to EGF in aortas from Wistar DOCA-salt rats (35 ± 3% maximal PE-induced contraction) was nine times greater than the maximal contraction to EGF in aortas from Wistar sham rats (4 ± 3%; Fig. 2A). Interestingly, the Wistar-Furth DOCA-salt group, whose blood pressures (134 ± 6 mmHg) were only slightly but significantly elevated compared with the Wistar-Furth sham group (112 ± 3 mmHg) but statistically similar to the Wistar sham group (Table 3), achieved the same maximal contraction to EGF as observed in the Wistar DOCA-salt group (34 ± 9% maximal PE-induced contraction or 9 times greater than sham response; Fig. 2B). The concentration of EGF that caused a half-maximal contraction in aortas from Wistar and Wistar-Furth DOCA-salt rats was similar (9.62 ± 0.14 and 9.57 ± 0.33, respectively; Table 1). In addition, although maximal contraction to 5-HT was significantly greater in arteries from Wistar DOCA-salt rats and Wistar-Furth DOCA-salt rats compared with the corresponding sham rats, the absolute increase in contraction to 5-HT (Table 2) was not similar to the absolute increases in contraction observed with EGF.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   A: concentration-dependent contraction of endothelium-denuded Wistar sham normotensive (PE contraction = 903 ± 112 mg) and DOCA-salt hypertensive rat aortas (PE contraction = 988 ± 95 mg) to EGF. B: concentration-dependent contraction of endothelium-denuded Wistar-Furth sham normotensive (PE contraction = 1,056 ± 86 mg) and DOCA-salt hypertensive rat aortas (PE contraction = 1,164 ± 83 mg) to EGF. Points represent the mean, and vertical lines represent the SE of the mean for the number of animals indicated in parentheses; systolic blood pressures are also included in parentheses. *Significant difference (P < 0.05) between aortic responses from Wistar sham and Wistar DOCA-salt rats or between Wistar-Furth sham and Wistar-Furth DOCA-salt rats.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Comparison of aortic response to EGF in blood pressure-matched Wistar-Furth sham and Wistar-Furth DOCA-salt rats. All tissues responded to PE with a contraction between 900 and 1,100 mg tension.

Contraction to EGF in aortas from sham, salt-alone- and DOCA-alone-treated rats. To determine the influence of DOCA alone and high salt alone on contraction to EGF, Sprague-Dawley rats were placed on either DOCA (200 mg/kg DOCA, normal tap water) or high salt (no DOCA; 1.0% NaCl + 0.2% KCl) for 4 wk. Rats placed on high salt alone (151 ± 9 mmHg) had significantly higher SBPs than those of sham rats (113 ± 1 mmHg Table 1, Fig. 4A). Interestingly, rats on high salt, although they were hypertensive, did not demonstrate a contraction to EGF (9 ± 5%) that was of the same magnitude as that observed in aortas from DOCA-salt rats (67 ± 22%; Fig. 4A). To the contrary, the maximum response to 5-HT was significantly greater in strips from salt-alone-treated rats (133.1 ± 12.6%) than those observed in sham strips (93.0 ± 5.0%). DOCA alone also significantly raised the SBPs of DOCA rats (156 ± 11 mmHg) compared with sham rats (113 ± 1 mmHg). However, as seen in Fig. 4B, the contractile response to EGF was modestly increased in aortas from DOCA-alone-treated rats compared with sham rat aortas. DOCA alone did not affect contraction to 5-HT (Table 2).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   A: concentration-dependent contraction of endothelium-denuded sham normotensive (PE contraction = 1,320 ± 126), salt-alone (PE contraction = 918 ± 134) and DOCA-salt hypertensive rat aortas (PE contracton = 1,180 ± 135 mg) to EGF. B: Concentration-dependent contraction of endothelium-denuded sham normotensive (PE contraction = 1,320 ± 126), DOCA-alone (PE contraction = 1,280 ± 110 mg), and DOCA-salt hypertensive rat aortas (PE contraction = 1,180 ± 35 mg) to EGF. Points represent the mean, and vertical lines represent the SE of the mean for the number of animals indicated in parentheses; systolic blood pressures are also included in parentheses. *Significant difference (P < 0.05) between aortic responses from sham and DOCA-alone-treated rats.

Correlation of blood pressure with maximum arterial contraction to EGF. To determine whether enhanced contraction to EGF was associated with increasing SBP, a linear regression correlation was conducted using all of the sham and hypertensive rats in this study. There was a positive correlation (r = 0.733) between the maximal contraction to EGF and SBP (Fig. 5). However, it is clear that this correlation is not perfect, and there are groups that clearly deviate from a linear association with SBP, namely Wistar-Furth DOCA-salt, SHR, L-NNA, and even DOCA-salt groups. These data provide evidence that high blood pressure is associated with an enhanced contraction to EGF and may be one cause of an enhanced response to EGF, but the lack of a perfect correlation suggests that other factors, acting either independently or in concert with changes in blood pressure, ultimately determine arterial response to EGF.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Correlation of systolic blood pressure and maximum contraction to EGF [reported as %PE (10 uM) contraction] for aortas for all groups of rats.

Effect of aldosterone on EGF-receptor mRNA expression in aortas from Wistar and Wistar-Furth rats. The above experiments using Wistar-Furth rats suggest that mineralocorticoids may be an independent factor for increasing EGF-receptor expression. To examine this idea, we incubated aortas from the Wistar rats, an animal sensitive to the hypertensive effects of mineralocorticoids, and the Wistar-Furth rat, an animal relatively insensitive to the hypertensive effects of mineralocorticoids, with the mineralocorticoid aldosterone (10 nmol/l) for 8 h. RT-PCR was performed, and the results of these experiments were depicted in Fig. 6. EGF-receptor mRNA was detected in aortas from both Wistar and Wistar-Furth rats, and aldosterone significantly increased the density of EGF mRNA in aortas from both strains of rats (P < 0.05). These data confirm, as suggested above, that mineralocorticoids can directly increase EGF-receptor mRNA.

View larger version (64K): [in this window] [in a new window]   Fig. 6.   A: representative phosphorimage of EGF receptor (EGFR) and cyclophilin mRNA density in Wistar and Wistar-Furth aortas treated with vehicle or aldosterone (Aldo; 10 nmol/l). RT = no RT control. B: normalized results from RT-PCR for EGFR expression in aortas incubated with vehicle or Aldo. Bars represent means ± SE for triplicates performed in aortas from 8 Wistar and 8 Wistar-Furth rats. *Significant difference (P < 0.05) in relative EGFR density between vehicle- and Aldo-incubated aortas.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Since the discovery of EGF, several EGF-related ligands have been identified including transforming growth factor- (TGF-), heparin-binding EGF-like growth factor (Hb-EGF), betacellulin, amphiregulin, and epiregulin. Four different EGF-receptor subtypes have been discovered including EGF (ErbB1) receptor, ErbB2, ErbB3, and ErbB4 (28). Elucidating potential vascular effects, such as contraction, of EGF is crucial because several of these EGF-related growth factors are produced within the vascular wall. For instance, messenger RNA for Hb-EGF is not only found in arteries, but can be concentration and time dependently increased by hydrogen peroxide-generating compounds (4) and by angiotensin II (25) in rat aortic smooth muscle cells. Hb-EGF mRNA levels are elevated in the left ventricle of SHR compared with WKY rats (11). Just as important are reports in the literature that show the EGF receptor can be rapidly transactivated by several stimuli including agonists of G protein-coupled receptors, cytokine receptors, calcium, and cell stress (7, 8, 31). These findings support the concept that the EGF receptor can act as a central mediator for various physiological stimuli and thus can have a significant impact on vascular tone. Thus it is important to understand how the EGF receptor-dependent pathway is modulated in the vasculature.

EGF as a vasoactive substance. As classic growth factors, EGF, Hb-EGF, and epiregulin all stimulate proliferation of vascular smooth muscle (15, 22, 24). However, few studies have examined the vascular contractile or pressor responses of EGF. Medial thickening of pulmonary arteries (100-200 µm inner diameter) and a moderate elevation in mean pulmonary arterial pressure have been demonstrated in rats given intravenous infusion of human recombinant EGF for 1 wk (12). Our laboratory demonstrated that whereas EGF and TGF- stimulated contraction in arteries from DOCA-salt and Goldblatt one kidney-one clip hypertensive rats, minimal contraction was observed in sham normotensive arteries (10). Moreover, the contraction to EGF did not occur in arteries from DOCA-salt rats until after a significant rise in SBP, suggesting that contraction to EGF was dependent on an elevated blood pressure. Thus one purpose of the present experiments was to investigate the dependence of vascular contraction to EGF on blood pressure.

EGF in Wistar-Furth rats. With the observation that contraction to EGF occurs in arteries from multiple forms of experimentally hypertensive rats, the idea that contraction to EGF is dependent on an elevation in blood pressure or that elevated blood pressure could be a stimulus for the appearance of a contraction to EGF was addressed. Wistar and Wistar-Furth rats were placed on DOCA-salt therapy. Previous studies have determined that Wistar rats but not Wistar-Furth rats are sensitive to the hypertensive effects of mineralocorticoids (1, 21). As expected, contraction to EGF occurred in aortas from Wistar DOCA-salt rats but not from Wistar shams. Surprisingly, EGF stimulated a virtually identical contractile response in aortas from Wistar-Furth DOCA-salt rats as that observed from Wistar DOCA-salt rat. This was, however, in the presence of a small increase in SBP. In contrast, 5-HT stimulated contraction in vessels from all sets of rats, although the maximal contraction obtained was greater in aortic strips from Wistar and Wistar-Furth DOCA-salt rats. It must be mentioned that the Wistar and Wistar-Furth rats received the same dose of DOCA (200 mg/kg) and were treated for the exact same time period, and mineralocorticoids are absorbed similarly between Wistar and Wistar-Furth rats (21). In addition, water intake was also monitored, and both strains of rats drank equal amounts of water when corrected for body size (data not shown). Thus the difference in blood pressure between the two groups must be due to differences in mineralocorticoid sensitivity and not due to differences in DOCA and/or saltwater intake. In a different study, Ullian et al. (27) found that although contraction to angiotensin II and phenylephrine was potentiated in aortic rings from Wistar rats on DOCA-salt therapy, the responses were not potentiated in rings from Wistar-Furth DOCA-salt rats. Similar findings were also observed for norepinephrine and potassium chloride in aldosterone-treated Wistar rats (enhanced) and Wistar-Furth rats (unchanged) (1).

Association of blood pressure with response to EGF. It is fair to suggest that mineralocoritoids may have the potential to act independently of changes in blood pressure to enhance reactivity to EGF, but it is possible and likely that, in concert, elevations in blood pressure and mineralocorticoids have complex and interwoven effects on arterial function. A positive correlation was demonstrated between maximal contraction to EGF and SBP (Fig. 5). However, these variables are only partially associated, because contraction to EGF would have been observed in aortas from salt-treated (hypertensive) rats and not in aortas from some Wistar-Furth DOCA-salt rats (not hypertensive). Were the resonse to EGF purely blood pressure dependent, then none of the aortas from these animals should have responded to EGF. By comparison, contraction to 5-HT was enhanced in vessels from salt-treated rats but not in vessels from rats on DOCA therapy alone, and both of these groups had similar blood pressures. These findings suggest that the pathway that regulates contraction to EGF may be more sensitive to the effects of DOCA or mineralocorticoids, whereas the pathway that regulates contraction to 5-HT are more tightly regulated by changes in blood pressure. These findings also refute the idea that contraction to EGF is solely dependent on a sustained elevation in blood pressure and further suggest that the contractile response to EGF may be partially regulated by mineralocorticoids.

Perspectives

In summary, these studies determined that arterial contraction to EGF was common to several forms of experimental hypertension. However, contraction to EGF is not solely dependent on a significant elevation in blood pressure, because a dramatic contraction to EGF occurred in vessels from normotensive Wistar-Furth rats on DOCA salt. Thus it appears vascular contraction to EGF may be specifically regulated by mineralocorticoids. Additional experiments examining the influence of mineralocorticoids on the EGF receptor and other proteins associated with the EGF receptor are required to more accurately determine the effect of mineralocortoids on EGF signaling.