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Am J Physiol Heart Circ Physiol 289: H188-H195, 2005. First published March 18, 2005; doi:10.1152/ajpheart.00504.2004
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Restoration of normal vascular relaxation mechanisms in cerebral arteries by chromosomal substitution in consomic SS.13BN rats

Ines Drenjancevic-Peric,1 Shane A. Phillips,1 John R. Falck,2 and Julian H. Lombard1

1Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin; and 2Department of Biochemistry, University of Texas, Southwestern Medical Center, Dallas, Texas

Submitted 4 June 2004 ; accepted in final form 10 March 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study sought to identify the mechanisms of vascular relaxation that are rescued in middle cerebral arteries (MCA) of SS.13BN consomic rats by substituting chromosome 13 containing the renin gene from Brown Norway (BN) rats into the Dahl salt-sensitive (SS) genetic background. Isolated MCA from SS rats exhibited an indomethacin-sensitive constriction in response to acetylcholine (ACh) and hypoxia. ACh-induced dilation was NO dependent and hypoxic dilations were cyclooxygenase (COX) dependent in BN and SS.13BN rats. In SS rats, hypoxic dilation was restored by indomethacin and abolished by inhibiting cytochrome P-450 epoxygenases, suggesting a role for epoxyeicosatrienoic acids. MCA from SS and SS.13BN rats constricted and MCA from BN rats dilated in response to the stable prostacyclin analog iloprost. MCA from SS.13BN and BN rats (but not SS rats) dilated in response to the prostaglandin E2 receptor agonist butaprost. Hypoxia increased prostacyclin release in cerebral arteries from all the strains, whereas thromboxane A2 production was reduced in BN rat vessels only. These data suggest that SS rats may be less sensitive to vasodilator prostaglandins and that normalization of renin-angiotensin system regulation causes a switch from production of COX-derived vasoconstrictor metabolites (in SS rats) toward NO-dependent relaxation in response to ACh- and prostaglandin-dependent dilation in response to hypoxia in SS.13BN rats.

angiotensin II; vasodilation; hypoxia; endothelium; genetic models; physiological genomics; vascular relaxation

REGULATION OF THE ACTIVE TONE of resistance vessels is a complex and integrated process that involves multiple mechanisms. Alterations in the function of resistance arteries can have significant pathophysiological consequences. For example, an impaired relaxation in response to vasodilator stimuli has been documented in many experimental animal models of hypertension (9–11, 15, 32, 35, 39, 53) and in hypertensive humans (3, 13, 42–44, 49, 50). Other studies (31, 32, 34, 48) have shown that high-salt diet alone leads to impaired vasodilation in arterioles and resistance arteries of Sprague-Dawley rats, in the absence of a change in blood pressure.

Impairment of vascular relaxation in response to vasodilator stimuli during exposure to high-salt diet appears to be caused by the suppression of circulating levels of angiotensin II (ANG II) that occurs in response to elevated dietary salt intake (19, 21). The latter hypothesis is supported by recent findings that continuous intravenous infusion of a low dose of ANG II to prevent ANG II suppression with high-salt diet restores the dilation in response to acetylcholine (ACh) and hypoxia that is eliminated with high-salt diet in middle cerebral arteries (MCA) (34). Other studies have provided evidence that the protective effect of ANG II to maintain normal vasodilator responses in animals on high-salt diet can be prevented by blocking AT1 receptors with losartan (52).

Recent studies have demonstrated that restoration of the normal regulation of the renin-angiotensin system (RAS) in SS.13BN rats [a genetic rat model that has chromosome 13 including the renin gene of the Brown Norway (BN) rat introgressed into the salt-sensitive (SS) genetic background] restores the relaxation of MCA in response to ACh and hypoxia (11). In that study, the restored relaxations in SS.13BN consomic rats were similar to those occurring in BN rats. These vasodilator responses in SS.13BN rats were eliminated by feeding the rats a high-salt diet and by blocking angiotensin AT1 receptors with losartan (11). The latter observations provided further support for the hypothesis that the RAS plays a critical role in maintaining normal vascular relaxation mechanisms. However, the precise mechanisms leading to altered responses to ACh and hypoxia in SS rats are unknown. It is also unknown whether restoration of normal regulation of the RAS by introgression of BN chromosome 13 into the SS background restores the mechanisms that normally mediate vascular relaxation in response to vasodilator stimuli or whether alternate compensatory mechanisms of vascular relaxation emerge to restore vasodilator responses in the SS.13BN rats.

The goal of the present study was to elucidate the mechanisms of the vascular responses to ACh and hypoxia in 1) inbred SS rats, which exhibit low renin levels even when they are fed a low-salt diet (1, 8, 25); 2) BN rats, which show elevated plasma renin levels when fed a low-salt diet (8); and 3) consomic SS.13BN rats, which also show elevated plasma renin levels when fed a low-salt diet (8). Elucidation of the mechanisms controlling vascular responses to vasodilator stimuli in these novel rat strains should shed new light on the role of the RAS in maintaining normal vascular relaxation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
General procedures. Male SS/JrHsd/Mcwi (SS) rats, BN (BN/NHsdMcwi) rats, and SS.13BN/Mcwi (SS.13BN) consomic rats were maintained on a low-salt (0.4% NaCl) diet (Dyets, Bethlehem, PA) with tap water to drink ad libitum. The rats were 11–13 wk old at the time of the experiment, and each experimental group included 10–11 rats. The Medical College of Wisconsin Animal Care Committee approved all procedures used in this study.

Isolated cannulated vessel studies. On the day of the experiment, the rat was anesthetized with an injection of pentobarbital sodium (30–60 mg/kg ip; Abbott Laboratories, North Chicago, IL). The lower dose of anesthesia was employed in SS rats and consomic rats to compensate for the enhanced sensitivity of SS rats to anesthesia. Arterial pressure was measured by direct cannulation, after which the brain was quickly removed and immersed in physiological salt solution (PSS) having the following ionic composition (in mM): 119.0 NaCl, 4.7 KCl, 1.6 CaCl2, 1.18 NaH2PO4, 1.17 MgSO4, 24.0 NaHCO3, 5.5 dextrose, and 0.03 EDTA. MCA were isolated using a dissecting microscope (Leica, Buffalo, NY) and cannulated using previously described procedures (14).

After being mounted on micropipettes, the artery was stretched to its in situ length. Intravascular pressure was maintained at 80 mmHg, and the vessels were perfused and superfused with PSS equilibrated with a 21% O2-5% CO2-74% N2 gas mixture (14). Any vessel that did not show active tone at rest, as indicated by a large dilation in response to Ca2+-free PSS, was not used in the study.

After the control equilibration period, responses of the arteries to a variety of vasodilator stimuli were determined using television microscopy combined with an on-screen Video Microscaler (model IV-550; FOR.A, Tokyo, Japan). Vasodilator stimuli tested included addition of the endothelium-dependent dilator ACh (1 µM) to the tissue bath and a simultaneous reduction of the perfusate and superfusate PO2 to 40–45 mmHg, accomplished by equilibrating the PSS with a 0% O2-5% CO2-95% N2 gas mixture. Maximum diameter of the arteries also was determined by measuring the diameter increase that occurred during maximal dilation with a Ca2+-free relaxing solution containing the following constituents (in mM): 92.0 NaCl, 4.7 KCl, 1.17 MgSO4, 20.0 MgCl2, 1.18 NaH2PO4, 24.0 NaHCO3, 0.026 EDTA, 2.0 EGTA, and 5.5 dextrose. Active tone in the vessels was calculated as ({Delta}D/Dmax) x 100, where {Delta}D is the change from resting diameter in PSS to maximum diameter (Dmax) in the Ca2+-free relaxing solution.

To elucidate the mechanisms of the vascular responses to ACh and reduced PO2 in SS rats, SS.13BN rats, and BN rats on low-salt diet, we performed the following experiments: 1) the role of nitric oxide (NO) in mediating vessel responses to ACh and reduced PO2 was assessed by comparing the response to reduced PO2 and ACh before and during NO synthase (NOS) inhibition with 100 µM NG-monomethyl-L-arginine (L-NMMA); 2) the relative role of prostaglandins, e.g., PGI2 or PGE2, in mediating vascular responses to hypoxia and ACh was assessed by comparing vessel responses to these vasodilator stimuli in the presence and absence of the cyclooxygenase (COX) inhibitor indomethacin (1 µM); 3) the role of epoxyeicosatrienoic acids (EETs) in mediating the response to ACh and hypoxia was assessed by measuring vessel responses to these vasodilator stimuli in the presence or absence of the cytochrome P-450 (CYP450) epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide(MS-PPOH; 10 µM); 4) the role of changes in prostacyclin sensitivity in contributing to altered vascular response to reduced PO2 was evaluated by adding increasing concentrations of the stable prostacyclin analog iloprost (10–10–10–8 M) to the tissue bath; and 5) the role of changes in PGE2 sensitivity in contributing to altered vascular response to reduced PO2 was evaluated by measuring vessel responses to increasing concentrations (10–10–10–8 M) of the EP2 receptor agonist butaprost.

Release of prostacyclin and thromboxane A2 from MCA during hypoxia. In a separate set of experiments, cerebral arteries (MCA, basilar arteries, and similar sized vessels from the circle of Willis) from three rats of the same strain were pooled and equilibrated in 2 ml of PSS with a 21% O2-5% CO2-74% N2 gas mixture maintained at 37°C for 1 h. The PSS was discarded, and the vessels were equilibrated with the 21% O2 gas mixture for an additional hour, followed by a reduction in O2 concentration to 5% O2 for 1 h (to match the PO2 levels encountered in the perfused vessel studies). After each 1-h equilibration period, 2 ml of PSS were removed from the incubation chamber and immediately snap frozen in liquid N2.

The production of prostacyclin and thromboxane A2 (TXA2) during normoxia (21% O2) and hypoxia (5% O2) was evaluated in the Physiology Department Biochemical Assay Core facility at the Medical College of Wisconsin, utilizing commercially available enzyme immunoassay kits (Cayman Chemical, Ann Arbor, MI). Prostacyclin and TXA2 release were evaluated simultaneously by measuring the stable PGI2 metabolite 6-keto-prostaglandin F1{alpha} (6-keto-PGF1{alpha}) and the stable TXA2 metabolite TXB2 in the incubation medium.

Statistical analysis. In all experiments, data were summarized as means ± SE. Differences between group means in the response to a single reduction of perfusate/superfusate O2 concentration or to a single agonist were assessed using a paired Student's t-test. Differences in means of multiple experimental groups were assessed using one-way ANOVA with a post hoc Student-Newman-Keuls test. A probability of P < 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Blood pressure and vessel diameters. Mean arterial pressures (MAP) in different groups of rats on low-salt diet were as follows: SS rats, 125 ± 4 mmHg (n = 9); SS.13BN rats, 122 ± 7 mmHg (n = 7); and BN rats, 95 ± 4 mmHg (n = 6). MAP of BN rats on low-salt diet was significantly lower than those of SS and SS.13BN rats on low-salt diet. Diameters of BN vessels were significantly larger than those of SS and SS.13BN rats under all conditions of the study, whereas diameters of MCA from SS and SS.13BN rats were not significantly different under any of the conditions of the study (Table 1). There were no significant differences in the amount of active resting tone in MCA from any of the strains employed in this study (Table 1).


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  		Table 1. Resting diameters, maximum diameters, and active tone of MCA from SS rats, consomic SS.13BN rats, and BN rats on low-salt diet

 
Mechanisms of vascular relaxation in response to ACh. The response to ACh in MCA from the different strains of rat on low-salt diet in the presence and absence of the COX inhibitor indomethacin (1 µM), the NOS inhibitor L-NMMA (100 µM), or the CYP450 epoxygenase inhibitor MS-PPOH (10 µM) are presented in Fig. 1. MCA from SS rats exhibited a paradoxical constriction in response to ACh that was eliminated by COX inhibition but was unaffected by L-NMMA (Fig. 1A). Combined inhibition of NOS (with L-NMMA) and COX (with indomethacin) or combined inhibition of NOS, COX, and CYP450 epoxygenase (with MS-PPOH) had no additional effect on vessel responses to ACh compared with COX inhibition alone.



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  		Fig. 1. Response to acetylcholine (ACh) in middle cerebral arteries (MCA) from salt-sensitive (SS) rats (A), SS rats with chromosome 13 including the renin gene of the Brown Norway (BN) rat (SS.13BN rats; B), and BN rats (C) on low-salt diet in the presence and absence of the cyclooxygenase (COX) inhibitor indomethacin (INDO; 1 µM), the nitric oxide synthase (NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA; 100 µM), combined COX and NOS inhibition with INDO + L-NMMA, or combined inhibition of COX, NOS, and cytochrome P-450 (CYP450) epoxygenase with N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH; 10 µM). Because the response to ACh was eliminated by L-NMMA in MCA of SS.13BN and BN rats, the effects of MS-PPOH were not tested in those strains. Data are presented as means ± SE for 6–10 vessels per treatment. *P < 0.05 vs. control. See text for details.

 
The paradoxical constriction of the vessels in response to ACh in MCA from SS rats contrasts with the ACh-induced relaxation that occurred in MCA from SS.13BN rats (Fig. 1B) and BN rats (Fig. 1C). In those experiments, ACh-induced dilations of MCA from BN rats and SS.13BN rats were eliminated by L-NMMA and were unaffected by indomethacin, suggesting that ACh-induced dilation of MCA from those strains of rats is completely NO dependent.

Mechanisms of vascular relaxation in response to reduced PO2. Figure 2 summarizes the effects of COX inhibition, NOS inhibition, and CYP450 epoxygenase inhibition (to prevent EET formation) on the response of MCA to reduced PO2. In these experiments, reduction of PO2 to 40–45 mmHg induced a paradoxical constriction of MCA from SS rats on low-salt diet, and this was converted to dilation by the COX inhibitor indomethacin (Fig. 2A). Hypoxic dilation of indomethacin-treated MCA from SS rats on low-salt diet was unaffected by NOS inhibition with L-NMMA but was abolished in the presence of the CYP450 epoxygenase inhibitor MS-PPOH. The latter observation suggests that EETs mediate the restored dilation to hypoxia that emerges in the presence of COX inhibition in MCA from SS rats on low-salt diet.



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  		Fig. 2. Response to hypoxia in MCA from SS rats (A), SS.13BN rats (B), and BN rats (C) on low-salt diet in the presence and absence of the COX inhibitor INDO (1 µM), the NOS inhibitor L-NMMA (100 µM), combined inhibition of COX and NOS with INDO + L-NMMA, or combined inhibition of COX, NOS, and CYP450 epoxygenase with MS-PPOH (10 µM). Data are presented as means ± SE for 5–10 vessels per treatment. *P < 0.05 vs. control. See text for details.

 
The responses of MCA to hypoxia in the SS rats contrast with those obtained in MCA from SS.13BN rats (Fig. 2B) and BN rats (Fig. 2C). In those strains, the dilation of the MCA in response to reduced PO2 was eliminated by indomethacin but was not affected by L-NMMA. The latter findings indicate that hypoxic dilation of MCA from BN and SS.13BN consomic rats is mediated by the release of vasodilator COX metabolites (e.g., prostacyclin or PGE2) in response to reduced PO2. Because the vasodilator response to reduced PO2 was completely eliminated by indomethacin in the SS.13BN and BN rats, the effect of MS-PPOH on vessel responses to hypoxia was not tested in those strains.

The responses of MCA to increasing concentrations of the stable prostacyclin analog iloprost and the PGE2 EP2 receptor agonist butaprost are summarized in Figs. 3 and 4, respectively. MCA from BN rats dilated in response to iloprost, but MCA from SS rats and SS.13BN rats failed to dilate in response to the prostacyclin analog (Fig. 3). MCA from SS rats also failed to relax in response to butaprost, whereas MCA from SS.13BN rats and BN rats both exhibited a significant dilation in response to butaprost (Fig. 4).



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  		Fig. 3. Responses to the stable prostacyclin analog iloprost (10–10–10–8 M) in MCA from SS rats, SS.13BN rats, and BN rats. MCA from BN rats exhibited a significant dilation in response to iloprost (P < 0.05 vs. control), whereas arteries of SS and SS.13BN rats failed to dilate in response to the prostacyclin analog. Data are presented as means ± SE for 5–10 vessels per treatment.

 


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  		Fig. 4. Responses to PGE2 EP2 receptor agonist butaprost (10–10–10–8 M) in MCA from SS rats, SS.13BN rats, and BN rats. MCA from BN rats and SS.13BN rats exhibited a significant dilation in response to butaprost (P < 0.05 vs. control), whereas arteries of SS rats failed to dilate in response to the agonist. Data are presented as means ± SE for 4–8 vessels per treatment.

 
Changes in the production of prostacyclin and TXA2 by cerebral arteries during exposure to reduced PO2 are summarized in Fig. 5. Arteries of all three strains of rats exhibited an increased release of the stable prostacyclin breakdown product 6-keto-PGF1{alpha} during exposure to hypoxia. Cerebral vessels from BN rats also exhibited a significant reduction in the release of TXB2, the stable breakdown product of TXA2, in response to hypoxia. However, there were no significant changes in TXB2 release in response to reduced PO2 in arteries from SS.13BN rats and SS rats.



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  		Fig. 5. Effect of reduced PO2 on production of prostacyclin (top) and thromboxane A2 (TXA2; bottom) by cerebral arteries of SS rats, consomic SS.13BN rats, and BN rats. Data are expressed as means ± SE of production (in pg·ml–1·min–1·mg–1 vessel wt–1) of the stable breakdown products 6-keto-PGF1{alpha} and TXB2 during equilibration with 21% O2 and 5% O2. *P < 0.05 vs. normoxic control (21% PO2) value.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The goal of this study was to elucidate the mechanisms of the vascular responses to ACh and hypoxia in two parental strains and one consomic strain of rats that differ in their regulation of the RAS (1, 8, 25). Rats fed a high-salt diet exhibit a reduction of plasma renin activity (PRA) (22) and ANG II (19, 21) and impaired responses to vasodilator stimuli that are similar to those in SS rats in the present study. In a similar fashion, SS rats exhibit significantly lower PRA than SS.13BN rats (8) and congenic rats having the Dahl R renin gene introgressed into the SS genetic background (1, 25). SS rats have little or no ability to increase PRA in response to low-salt diet and sodium depletion, unlike SS.13BN rats or congenic rats having the Dahl R renin gene on the SS background (1, 8, 25). The specific goal of this study was to determine whether the vasodilator responses to ACh and hypoxia that are rescued in SS.13BN rats following restoration of normal regulation of the RAS by chromosomal substitution are mediated by the mechanisms that normally mediate vascular relaxation in response to these vasodilator stimuli or whether alternate compensatory mechanisms emerge to mediate vascular relaxation when normal regulation of the RAS is restored by chromosomal substitution.

Restoration of vascular relaxation mechanisms in response to ACh in SS.13BN rats. Numerous studies have demonstrated that ACh-induced dilation is endothelium dependent (18) and that it is generally mediated via NO release (5, 41). In some cases, other metabolites can mediate vascular relaxation responses to ACh, e.g., COX metabolites of arachidonic acid (24, 55) or an endothelium-derived hyperpolarizing factor (5, 28, 30), putatively identified as one or more EET (4, 23, 37, 51). Collectively, those studies suggest that multiple mechanisms could contribute to ACh-induced vascular relaxation in different rat strains or under different experimental conditions.

The present study provides exciting new evidence of a striking difference in vascular control mechanisms in various strains of rats that differ in their ability to regulate their RAS. These results demonstrate that the paradoxical constriction in response to ACh that occurs in SS rats on a low-salt diet is eliminated by the COX inhibitor indomethacin but is unaffected by the NOS inhibitor L-NMMA. In indomethacin-treated vessels, inhibition of NOS with L-NMMA or CYP450 epoxygenase with MS-PPOH had no further effect on vessel responses to ACh in SS rats (Fig. 1A). A novel observation in the present study is that COX-dependent vasoconstrictor metabolites can contribute to impaired dilation in response to ACh in cerebral arteries of normotensive SS rats, even when they are maintained on a low-salt diet and are normotensive. These findings contrast with the vasodilator responses to ACh in MCA from SS.13BN rats (Fig. 1B) and BN rats on low-salt diet (Fig. 1C), in which ACh caused a vasodilation that could be eliminated by inhibiting NOS with L-NMMA.

In addition to formation of vasoconstrictor metabolites of arachidonic acid, another consideration regarding the potential contribution of COX to altered vasodilator responses in cerebral vessels is its potential role in generating superoxide anions. The studies of Katusic and Vanhoutte (27) and Cosentino et al. (6) suggest that the latter alteration can lead to an impaired balance between vasodilator and vasoconstrictor factors, similar to that observed in the present study. The latter hypothesis is consistent with the results of a recent study (12) demonstrating that superoxide scavenging with Tempol restores vasodilator responses to ACh (and hypoxia) in MCA of SS rats fed a low-salt diet.

Restoration of vascular relaxation mechanisms in response to reduced PO2 in consomic SS.13BN rats. Another important stimulus contributing to local blood flow control is vascular relaxation in response to reduced PO2. Previous studies have demonstrated that hypoxic dilation of MCA from Sprague-Dawley rats on normal salt diet is mediated by prostacyclin-induced activation of glibenclamide-sensitive (ATP-sensitive) K+ channels, leading to vascular smooth muscle hyperpolarization (14, 33). However, prostacyclin is not the only vasoactive metabolite that can mediate hypoxic vasodilation, because a variety of other metabolites may regulate vascular responses to reduced PO2, including PGE2 (38, 40), NO (16, 20, 54), EETs (4, 17), adenosine (36, 45) from parenchymal cells, and reduced levels of 20-hydroxyeicosatetraenoic acid (16).

The wide variety of mechanisms that have been implicated in contributing to the vasodilator responses to reduced PO2 and the observation that different mechanisms may contribute to hypoxic dilation under different experimental conditions, in different species, or in different strains of rats underscore the importance of identifying the mechanisms that mediate the restored relaxation of MCA in response to reduced PO2 in the SS.13BN consomic rats. This is emphasized by studies providing striking examples of the emergence of alternate mechanisms of vascular relaxation under some conditions. For example, Kerkhof et al. (29) reported that inhibition of CYP450 4A {omega}-hydroxylase unmasks a large NO-dependent dilation of isolated cremasteric arterioles in response to reduced PO2, in contrast to studies demonstrating that hypoxic dilation of cremasteric arterioles (38) and skeletal muscle resistance arteries (32) is normally mediated by vasodilator metabolites of the COX pathway of arachidonic acid metabolism.

In the present study, inhibition of COX restored the dilation of the vessels in response to reduced PO2 in SS rats. This restored dilation was eliminated by inhibiting CYP450 epoxygenase with MS-PPOH, suggesting that EETs play an important role in hypoxic relaxation of the MCA in SS rats when other COX-dependent vasoconstrictor influences are eliminated. The apparent contribution of CYP450 epoxygenase products to hypoxic dilation under these conditions is highly novel, given the major role that COX metabolites usually play in hypoxia-induced relaxation (14, 16, 33, 38).

In contrast to the hypoxic vasoconstriction exhibited by MCA from SS rats, MCA from SS.13BN and BN rats both dilated in response to reduced PO2, and this hypoxic dilation could be eliminated by inhibiting COX with indomethacin. These results suggest that restoration of normal RAS regulation by chromosomal substitution in the consomic SS.13BN rats rescues the mechanisms that mediate hypoxic vasodilation in MCA of BN rats, namely, the release of vasodilator prostaglandins in response to reduced PO2. Those mechanisms are in stark contrast to the ones that mediate vascular responses to reduced PO2 in SS rats, in which there is either a switch toward the production of vasoconstrictor COX metabolites and/or an altered sensitivity of the vessels to COX metabolites to favor vasoconstrictor responses (rather than vasodilation).

Studies of spontaneously hypertensive rats (2) and spontaneously hypertensive hamsters (47) suggest that overproduction of COX-dependent vasoconstrictor metabolites such as PGH2 and TXA2 can contribute to an impaired relaxation in response to ACh. Similar changes in vascular control mechanisms likely occur during conditions characterized by RAS suppression. For example, the paradoxical constriction that occurs in response to reduced PO2 in cerebral arteries of Sprague-Dawley rats fed high-salt diet appears to be mediated through changes in the balance between prostacyclin and TXA2 released from the endothelium in response to the hypoxic stimulus (34). Together, the findings of the present study and those of previous studies in the literature (34) suggest that exposure to reduced levels of ANG II could lead to an increased production of vasoconstrictor prostaglandins or, alternatively, to a reduced sensitivity of the vessels to the relaxing effect of COX-derived vasodilator metabolites of arachidonic acid.

It is possible that changes in RAS control may be associated with alterations in the production of specific COX metabolites so as to shift the balance between vasodilator and vasoconstrictor metabolites of arachidonic acid. In the present study, cerebral vessels of all three strains of rats exhibited a significant increase in the production of 6-keto-PGF1{alpha} (the stable breakdown product of prostacyclin) in response to reduced PO2. Cerebral arteries from BN rats also exhibited a significant reduction in the release of TXB2 (the stable breakdown product of TXA2) during exposure to reduced PO2, whereas TXB2 release was not significantly affected by reduced PO2 in vessels from SS rats or SS.13BN rats. Together, these findings suggest that changes in vascular sensitivity to metabolites released in response to decreased PO2 (rather than alterations in the levels of specific metabolites themselves) are primarily responsible for the paradoxical constriction of MCA in response to reduced PO2 in SS rats on low-salt diet.

Initially, it was anticipated that MCA from the SS rats would be insensitive to the vasorelaxant effects of prostacyclin that normally mediate hypoxic dilation in MCA (33), whereas MCA from SS.13BN rats would relax in response to prostacyclin released during exposure of the vessels to reduced PO2. Surprisingly, MCA from SS rats and SS.13BN rats on low-salt diet both exhibited a paradoxical constriction in response to the stable prostacyclin analog iloprost, in contrast to the vasodilator effect of iloprost in MCA from BN rats (Fig. 3) and Sprague-Dawley rats (34). The paradoxical constriction of MCA in response to iloprost in SS rats on low-salt diet is a novel observation and is consistent with the lack of hypoxic dilation in these vessels despite the increase in prostacyclin release by SS vessels during exposure to reduced PO2 (Fig. 5). However, the impaired relaxation of MCA from SS.13BN rats in response to iloprost was unexpected and suggests that some other COX metabolite, most likely PGE2 (38, 40), mediates hypoxic dilation of MCA from SS.13BN rats. The latter hypothesis would require differences in the sensitivity of the vessels from SS rats and SS.13BN consomic rats to PGE2.

Our observation that MCA from SS rats failed to relax in response to the PGE2 EP2 receptor agonist butaprost whereas MCA from SS.13BN rats and BN rats both exhibited a significant dilation in response to butaprost (Fig. 4) support the conclusion that vascular sensitivity to the vasodilator effect of PGE2 is impaired in SS rats but not in SS.13BN rats and that the rescue of hypoxic dilation in MCA from SS.13BN rats is caused by normalization of the response of the vessels to PGE2. Such a contribution of PGE2 to hypoxic dilation in MCA of SS.13BN rats is consistent with the results of other studies indicating that this arachidonic acid metabolite plays a role in hypoxic relaxation of isolated arterioles of the cremaster muscle (38) and in the Langendorff-perfused rat heart (40). Because SS.13BN consomic rats are 98% genetically identical to SS rats and differ from the SS rats only at various alleles on chromosome 13 (7), the present findings also suggest that factors in the Dahl SS genetic background (other than the renin gene) contribute to an impaired vasodilator response to prostacyclin in SS rats.

In summary, the results of this study suggest that normalization of circulating ANG II levels by introgression of the BN renin gene into the SS genetic background rescues the mechanisms that normally mediate vascular relaxation in response to ACh and hypoxia. These observations provide further support for the hypothesis that the RAS plays an important role in maintaining normal vascular relaxation mechanisms in resistance arteries (11, 12, 34, 52).


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Grants HL-29587, HL-65289, and HL-72920 and by NHLBI Programs for Genomic Applications Grant U01 HL-66579. I. Drenjancevic-Peric was supported by a Croatian Ministry of Science and Technology fellowship grant.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. H. Lombard, Dept. of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: jlombard{at}mcw.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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