Introduction

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The spontaneously hypertensive rat (SHR) is a widely employed model of human genetic hypertension; however, the pathophysiology of hypertension in SHRs has eluded four decades of intensive investigation. Nonetheless, progress has been made, and it is now clear that hypertension in SHRs requires the vasoactive peptide angiotensin II (Ang II) (Bunkenburg et al., 1991; Lee et al., 1991); yet SHRs do not have increased circulating (Shiono and Sokabe, 1976) or tissue concentrations of Ang II (Campbell et al., 1995). It is also apparent that the SHR kidney is critical to the hypertensive state in this genetic model. In this regard, transplantation studies indicate that hypertension tracks the SHR kidney (Rettig and Unger, 1991). A third requirement for full expression of hypertension in SHRs is an intact G_i signal transduction pathway. Blockade of the G_i signal transduction pathway with a single dose of pertussis toxin causes a prolonged antihypertensive response in adult SHRs (Kost et al., 1999) and delays the development of hypertension in young SHRs (Li and Anand-Srivastava, 2002). To understand the pathophysiology of hypertension in the SHR, the challenge is to propose and test hypotheses that reconcile these seemingly unrelated findings.

We hypothesize that hypertension in SHRs is caused in part by an augmented cross talk in the renal microcirculation between the G_i signal transduction pathway and the signal transduction pathway used by vasoactive peptides. This hypothesis predicts that activation of the G_i pathway in the renal vasculature should potentiate renovascular responses to vasoactive peptides in SHR kidneys, and more so than in kidneys from normotensive Wistar-Kyoto (WKY) rats. This hypothesis also predicts that inhibition of G_i with pertussis toxin should prevent potentiation of renovascular responses to vasoactive peptides by the G_i pathway in SHR kidneys. The purpose of this investigation was to test these predictions.

	Materials and Methods

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Animals. Studies utilized adult male SHRs and WKY rats from Taconic Farms (Germantown, NY). Some SHRs were anesthetized with halothane 3 to 4 days before the perfusion experiments and were administered an intravenous (via jugular) injection of 30 µg/kg pertussis toxin. We have shown previously that this technique blocks signaling via the adenosine A₁ receptor, a G_i-coupled receptor, in the kidney (Kost et al., 2000). The Institutional Animal Care and Use Committee approved all procedures.

Method of Isolated Perfused Rat Kidney. Rats were anesthetized with Inactin (100 mg/kg i.p.), the left ureter was cannulated with polyethylene-10 tubing, and the left inferior suprarenal artery was ligated and severed. The aorta was cleared above and below the left renal artery, and two silk ties were placed loosely above (ties A and B) and below (ties C and D) the left renal artery. Ties B and C were positioned just above and below the left renal artery, respectively; ties A and D were positioned approximately 1 cm above and below the left renal artery, respectively. Two silk ties (E and F) were also placed loosely around the left renal vein. In this regard, tie E was positioned near the inferior vena cava, and tie F was placed near the renal hilus.

Protocol with Isolated Perfused Rat Kidney. After at least a 30-min rest period, a vasoconstrictor response (change in perfusion pressure) to 10 nM Ang II was elicited by infusing Ang II for 2 min. After another rest period, during which baseline perfusion pressure had returned to normal, a vasoconstrictor response to 3 nM vasopressin was elicited by infusing vasopressin for 2 min. These experiments were performed in five groups of kidneys as follows: 1) control SHR, n = 6; 2) control WKY, n = 6; 3) UK-14,304-treated SHR, n = 6; 4) UK-14,304-treated WKY, n = 6; 5) pertussis toxin-pretreated and UK-14,304-treated SHR, n = 6. Treatment with UK-14,304 was begun 5 to 10 min before each response to Ang II or vasopressin and was continued for the 2-min infusion of Ang II or vasopressin.

Isolation of Preglomerular Microvessels. SHRs and WKY rats were anesthetized with Inactin (100 mg/kg i.p.), and the kidneys were flushed with room-temperature L-15 medium (Leibovitz medium; Sigma-Aldrich, St. Louis, MO). A 1% suspension of iron oxide (Aldrich Chemical Co., Milwaukee, WI) in L-15 medium (10 ml; 37°C) was flushed into the kidneys. The kidneys were placed in ice-cold L-15 medium, and the cortex was obtained, minced, suspended in ice-cold L-15 medium, and dispersed by pushing the cortical material through a series of needle hubs (three times through 16, 18, and 21 gauge and six times through 23 gauge). The cortical material was suspended in ice-cold L-15 medium, and a magnet was applied to the tube to retrieve the iron oxide-laden preglomerular microvessels (PGMVs) while the unwanted material was decanted. The glomeruli were removed from the microvessels by filtering the suspension of PGMVs through a 149-µm nylon mesh. The PGMVs were retrieved from the nylon mesh and placed in ice-cold phosphate-buffered saline (PBS).

Protein Extraction. PGMVs were frozen in liquid nitrogen and ground into a powder on liquid nitrogen. The ground tissues were placed in a tube with 0.5 ml of SDS buffer (50 mM Tris, pH 7.0, 2% SDS, 10% glycerol) containing protease inhibitors (2 µg/ml antipain, 1 µg/ml aprotinin, 2 µg/ml leupeptin, 1 mg/ml phenylmethylsulfonyl fluoride) and homogenized. The homogenate was centrifuged at 12,000 rpm at 4°C for 10 min, and the supernatant was recovered. Protein in the supernatant was determined by the copper bicinchoninic acid method, and samples were stored at -20°C.

Western Blotting. Laemmli buffer was added to samples, and samples were placed in boiling water for 5 min and then chilled on ice. Samples (30 µg of protein in each well) were loaded onto a 7.5% acrylamide gel and subjected to SDS-PAGE using the Bio-Rad mini-gel system (Bio-Rad, Hercules, CA). Proteins were then electroblotted onto a PVDF membrane (Millipore, Bedford, MA). The PVDF membrane was blocked with 5% milk for 1 h and incubated for 3 h at room temperature with the primary antibody. We used one of four different primary antibodies, either anti-AT₁ receptor antibody (Cat. No. sc-1173; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-AT₂ receptor antibody (Cat. No. RDI-ANG1O2R20br, Research Diagnostics, Flanders, NJ), anti-_2A-adrenoceptor antibody (Cat. No. A269; Sigma-Aldrich), or anti-V_1a receptor antibody (RDI-AVPRV1Axabr; Research Diagnostics). Primary antibodies were diluted 1:1000 in PBS containing 0.05% Tween 20. Membranes were washed in PBS containing 0.05% Tween 20 solution three times and then incubated at room temperature for 1 h with horseradish peroxidase-conjugated donkey anti-rabbit IgG secondary antibody (Amersham Biosciences Inc., Piscataway, NJ) at 1:5000 dilution. Membranes were exposed to films, and the signals were detected by a Supersignal Substrate kit (Pierce Chemical, Rockford, IL). Band densities were quantitatively measured using Scion Image software (Scion Corporation, Frederick, MD). Background signals were obtained in each lane and subtracted from the band densities to correct for the background signal.

Statistical Analysis. A global analysis of the data was performed by three-factor analysis of variance in which factor A was rat strain (WKY versus SHR), factor B was level of treatment with UK-14,304 (0 or 10 nM), and factor C was peptide agonist (Ang II or vasopressin). Additional comparisons were performed by one-factor analysis of variance. Post hoc comparisons were performed using Fisher's least significant difference test. The criterion of significance was P < 0.05, and all data are expressed as the mean ± S.E.M.

	Results

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As shown in Table 1, baseline perfusion pressures in the SHR control group were slightly, yet significantly (P < 0.05), greater compared with the WKY control group. However, baseline perfusion pressures were not affected significantly by UK-14,304 in either WKY or SHR, and baseline perfusion pressures were not significantly different in the SHR UK-14,304 group compared with the WKY UK-14,304 group. Pertussis toxin did not affect baseline perfusion pressure in SHRs (Table 1).

As illustrated in Fig. 1 (panel A), the renovascular response to Ang II (10 nM) was not significantly different in control WKY kidneys versus WKY kidneys pretreated with UK-14,304 (10 nM). In contrast, in SHR kidneys, UK-14,304 (10 nM) significantly increased vasoconstrictor responses to Ang II (10 nM) by approximately 2-fold (Fig. 1, panel B). However, in kidneys removed from SHRs pretreated 3 to 4 days before the experiment with pertussis toxin (30 µg/kg i.v.), UK-14,304 did not alter Ang II-induced renovascular responses (Fig. 1, panel B). Renovascular responses to Ang II in the absence of UK-14,304 were not significantly different in WKY versus SHR kidneys (Fig. 1, panel A versus panel B); however, in the presence of UK-14,304, renovascular responses to Ang II were significantly greater in SHRs compared with WKY kidneys (Fig. 1, panel A versus panel B).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Changes in perfusion pressure induced by angiotensin II (10 nM) in control WKY kidneys (panel A), UK-14,304 (10 nM)-treated WKY kidneys (panel A), control SHR kidneys (panel B), UK-14,304-treated SHR kidneys (panel B), and UK-14,304-treated kidneys removed from SHRs pretreated 3 to 4 days earlier with 30 µg/kg pertussis toxin (panel B). Values represent means ± S.E.M. from six animals.

As illustrated in Fig. 2 (panel A), the renovascular response to vasopressin (3 nM) was not significantly different in control WKY kidneys versus WKY kidneys pretreated with UK-14,304 (10 nM). In contrast, in SHR kidneys, UK-14,304 (10 nM) significantly increased vasoconstrictor responses to vasopressin (3 nM) by approximately 5-fold (Fig. 2, panel B). However, in kidneys removed from SHRs pretreated 3 to 4 days before the experiment with pertussis toxin (30 µg/kg i.v.), UK-14,304 did not alter vasopressin-induced renovascular responses (Fig. 2, panel B). Renovascular responses to vasopressin in the absence of UK-14,304 were not significantly different in WKY versus SHR kidneys (Fig. 2, panel A versus panel B); however, in the presence of UK-14,304, renovascular responses to vasopressin were nearly significantly greater (P < 0.1) in SHRs compared with WKY kidneys (Fig. 2, panel A versus panel B).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Changes in perfusion pressure induced by vasopressin (3 nM) in control WKY kidneys (panel A), UK-14,304 (10 nM)-treated WKY kidneys (panel A), control SHR kidneys (panel B), UK-14,304-treated SHR kidneys (panel B), and UK-14,304-treated kidneys removed from SHRs pretreated 3 to 4 days earlier with 30 µg/kg i.v. pertussis toxin (panel B). Values represent means ± S.E.M. from six animals.

A three-factor analysis of variance was performed to examine more completely the interaction between rat strain and the effects of UK-14,304 on renovascular responses to Ang II and vasopressin. In this analysis, the factors were rat strain (WKY versus SHR), level of UK-14,304 (0 versus 10 nM), and the vasoactive peptide (Ang II versus vasopressin). This analysis revealed a significant (P = 0.016) interaction between rat strain and UK-14,304 on renovascular responses to the vasoactive peptides; however, the interaction between rat strain and UK-14,304 was independent of the type of vasoactive peptide (P = 0.801 for three-way interaction between strain, UK-14,304, and vasoactive peptide).

Western blot analysis of PGMVs or whole kidney using the anti-AT₁ receptor antibody gave rise to a clear and prominent band at 41.7 kDa, which is approximately the nominal mass (41 kDa) of the AT₁ receptor. Expression of the AT₁ receptor was greater in PGMVs (Fig. 3, panel A) compared with whole kidney (Fig. 4, panel A); however, expression of the AT₁ receptor was not different in WKY versus SHR either in PGMVs (Fig. 3, panel A) or whole kidney (Fig. 4, panel A). AT₂ receptor expression could not be detected by Western blotting or by reverse transcription-polymerase chain reaction followed by ethidium bromide staining in either whole kidneys or freshly isolated PGMVs in either SHR or WKY (data not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Bar graphs illustrating the expression of AT1 receptors, V1a receptors, and 2A-adrenoceptors in freshly isolated PGMVs in SHRs and WKY rats. Samples (30 µg of protein in each well) were loaded onto a 7.5% acrylamide gel and subjected to SDS-PAGE using the Bio-Rad mini-gel system. Proteins were then electroblotted onto a PVDF membrane. The PVDF membrane was blocked with 5% milk for 1 h and incubated for 3 h at room temperature with the primary antibody (anti-AT1 receptor, anti-V1a receptors, or anti-2A-adrenoceptor). Membranes were washed in PBS containing 0.05% Tween 20 solution three times and then incubated at room temperature for 1 h with horseradish peroxidase-conjugated donkey anti-rabbit IgG secondary antibody. Membranes were exposed to films, and the signals were detected by Supersignal Substrate kit. Band densities were quantitatively measured using Scion Image software. Background signals were obtained in each lane and subtracted from the band densities to correct for the background signal. Values represent means ± S.E.M. for three observations.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Bar graphs illustrating the expression of AT1 receptors, V1a receptors, and 2A-adrenoceptors in whole kidneys from SHRs and WKY rats. Samples (30 µg of protein in each well) were loaded onto a 7.5% acrylamide gel and subjected to SDS-PAGE using the Bio-Rad mini-gel system. Proteins were then electroblotted onto a PVDF membrane. The PVDF membrane was blocked with 5% milk for 1 h and incubated for 3 h at room temperature with the primary antibody (anti-AT1 receptor, anti-V1a receptors, or anti-2A-adrenoceptor). Membranes were washed in PBS containing 0.05% Tween 20 solution three times and then incubated at room temperature for 1 h with horseradish peroxidase-conjugated donkey anti-rabbit IgG secondary antibody. Membranes were exposed to films, and the signals were detected by Supersignal Substrate kit. Band densities were quantitatively measured using Scion Image software. Background signals were obtained in each lane and subtracted from the band densities to correct for the background signal. Values represent means ± S.E.M. for three observations.

Western blot analysis of PGMVs or whole kidney using the anti-V_1a receptor antibody gave rise to a clear and prominent band at 46 kDa, which is approximately the nominal mass (44 kDa) of the V_1a receptor. Expression of the V_1a receptors was greater in PGMVs (Fig. 3, panel B) compared with whole kidney (Fig. 4, panel B); however, expression of the V_1a receptor was not different in WKY versus SHR either in PGMVs (Fig. 3, panel B) or whole kidney (Fig. 4, panel B).

Western blot analysis of PGMVs and whole kidney using the anti-_2A-adrenoceptor antibody gave rise to a clear and dominant band at 54 kDa, which is slightly larger than the nominal mass (49 kDa) of the _2A-adrenoceptor and may represent a glycosylated and/or palmitoylated form of the receptor. Expression of the _2A-adrenoceptor was greater in PGMVs (Fig. 3, panel C) compared with whole kidney (Fig. 4, panel C); however, expression of the _2A-adrenoceptor was not different in WKY versus SHR either in PGMVs (Fig. 3, panel C) or whole kidney (Fig. 4, panel C).

	Discussion

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In the SHR, hypertension tracts the kidney (Rettig and Unger, 1991) and requires an intact renin-angiotensin system (Bunkenburg et al., 1991; Lee et al., 1991); however, Ang II production is not excessive in SHRs (Shiono and Sokabe, 1976; Campbell et al., 1995). Previous studies indicate that these facts can be explained by an enhanced renovascular sensitivity to Ang II in vivo (Li and Jackson, 1989; Chatziantoniou et al., 1990; Chatziantoniou and Arendshorst, 1991; Kost and Jackson, 1993). The mechanism of this enhanced renal sensitivity is under intense investigation.

In a recent in vivo study (Jackson et al., 2001), we measured increases in renovascular resistance in response to intrarenal infusions of Ang II and vasopressin in the presence and absence of coactivation of ₂-adrenoceptors (i.e., receptors selectively coupled to G_i) with UK-14,304 in adrenalectomized, renal-denervated, captopril-pretreated SHRs and WKY rats. In that study, we observed that in SHRs, but not WKY rats, UK-14,304 markedly enhanced renovascular responses to Ang II and vasopressin. We also observed that UK-14,304 did not enhance renovascular responses to methoxamine (₁-adrenoceptor agonist) in either strain. Moreover, we observed that in rats with nongenetic hypertension, UK-14,304 had little effect on renovascular responses to Ang II. We concluded that activation of ₂-adrenoceptors selectively enhances renovascular responses to Ang II and vasopressin in vivo in animals with genetic hypertensive, but not in normotensive animals or animals with acquired hypertension.

Our previous results suggest that in SHRs there is a genetically mediated enhanced cross talk between the G_i signal transduction pathway and signal transduction pathways activated by angiotensin II and vasopressin, but not methoxamine. To further test our hypothesis, in subsequent experiments we attempted to examine the interaction between renal ₂-adrenoceptors and Ang II or vasopressin in vivo in SHRs pretreated with pertussis toxin to inhibit the G_i signal transduction pathway. However, we could not perform these experiments because pertussis toxin-pretreated rats could not tolerate the extensive manipulations (adrenalectomy, renal denervation, and captopril pretreatment) necessary to examine this interaction in vivo.

One goal of the present study was to overcome the limitations of our in vivo paradigm by developing an in vitro model system that would recapitulate the in vivo enhanced interaction between Ang II or vasopressin and ₂-adrenoceptors. Importantly, the results of the present study demonstrate that in the isolated, perfused SHR kidney, UK-14,304, a selective ₂-adrenoceptor agonist, markedly augments renovascular responses to both Ang II and vasopressin. Our results also establish that in contrast to SHR, UK-14,304 has little effect on renovascular responses to Ang II or vasopressin in the isolated, perfused WKY kidney. These results are important for two reasons. First, the present data confirm that our in vivo findings were not an artifact caused by uncontrolled variables in the in vivo experiments. Second, the present findings provide a useful in vitro model system in which to investigate the mechanism of the enhanced interaction between Ang II or vasopressin and ₂-adrenoceptors in the SHR kidney.

A second goal of the present study was to test the hypothesis that the interaction between Ang II or vasopressin and ₂-adrenoceptors in SHR kidneys is mediated by the G_i signal transduction pathway. In this regard, we utilized the technique of administering an intravenous bolus dose of pertussis toxin several days before the experiment (Kost et al., 2000). Pertussis toxin is well known to ADP-ribosylate G_i and thereby inactivate this key signal transduction pathway. Importantly, the present study demonstrates that the ability of UK-14,304 to potentiate Ang II-mediated or vasopressin-mediated renal vasoconstriction in SHR kidneys is blocked by inhibition of the G_i signal transduction pathway with pertussis toxin.

In vivo SHR kidneys are more responsive to Ang II (Li and Jackson, 1989; Chatziantoniou et al., 1990; Chatziantoniou and Arendshorst, 1991; Kost and Jackson, 1993) and vasopressin (Feng and Arendshorst, 1996, 1997) compared with WKY kidneys, and the results of the present study offer an explanation for this consistent finding. In this regard, the present findings strongly support the conclusion that in SHR kidneys there is an enhanced interaction between Ang II or vasopressin and the G_i signal transduction pathway. This conclusion is consistent with our previous reports that in vivo pertussis toxin normalizes augmented renovascular responses to Ang II in SHRs (Jackson, 1994; Jackson et al., 1999). This conclusion is also consistent with the observation in the present study that in isolated perfused SHR kidneys in the absence of UK-14,304, renovascular responses to Ang II and vasopressin are not augmented compared with WKY kidneys. In contrast, in the presence of UK-14,304 to stimulate the G_i signal transduction pathway, renovascular responses to both Ang II and vasopressin become greater compared with control or UK-14,304-treated WKY kidneys. Evidently, in vitro, in the absence of UK-14,304 there is insufficient activation of the G_i signal transduction pathway to render the SHR kidney hyper-responsive to Ang II and vasopressin, whereas in vivo, there is sufficient hormonal stimulation of the G_i pathway (for example, via norepinephrine, adenosine, and/or neuropeptide Y acting on ₂-adrenoceptors, A₁ receptors, and Y₁ receptors, respectively) to ensure hyper-responsiveness of SHR kidneys to Ang II and vasopressin. Importantly, the in vivo situation is reconstituted in vitro by the addition of an activator of the G_i signal transduction pathway.

The observations that blockade of the G_i signal transduction pathway with a single dose of pertussis toxin causes a prolonged antihypertensive response in adult SHRs (Kost et al., 1999) and delays the development of hypertension in young SHRs (Li and Anand-Srivastava, 2002) indicate that the enhanced interaction between the G_i signal transduction pathway and vasoactive peptides importantly contributes to the pathophysiology of hypertension in SHRs.

It is possible that the enhanced interaction between Ang II or vasopressin and ₂-adrenoceptors in the SHR kidney is merely due to increased expression of receptors for Ang II, vasopressin, or UK-14,304. Ang II, vasopressin, and UK-14,304 cause vasoconstriction via AT₁ receptors (Kost and Jackson, 1993), V_1a receptors (Feng and Arendshorst, 1996) and _2A-adrenoceptors (Gavin and Docherty, 1996), respectively. The present study demonstrates that neither AT₁ receptors nor V_1a receptors nor _2A-adrenoceptors are over-expressed in either the preglomerular microcirculation or in total kidney tissue of SHRs. Moreover, AT₂ receptor expression was undetectable in either whole kidneys or PGMVs from either SHR or WKY. These results suggest that in the SHR the enhanced cross talk between the G_i signal transduction pathway and signal transduction pathways activated by angiotensin II and vasopressin is not due to changes in receptor expression.

Vagnes et al. (2000) observed in SHR PGMVs a 2-fold increase in V_1a receptor mRNA expression by reverse transcription-polymerase chain reaction and a 2- to 3-times increase in V_1a receptor density by ligand binding. There are several key differences between our study and the study by Vagnes et al. (2000) that could explain the disparate results. These differences include age of animals (adult animals versus young animals, respectively), strain of animals (Taconic Farms versus Chapel Hill breeding colony, respectively) and technique of isolating PGMVs (no collagenase digestion versus collagenase digestion, respectively). The difference in results between the two studies invites further exploration of the factors that determine V_1a receptor expression.

What is the mechanism by which the G_i signal transduction pathway participates in the enhanced interaction between ₂-adrenoceptors and Ang II or vasopressin in SHRs? Both AT₁ receptors (Sano et al., 1997) and V₁ receptors (Thibonnier et al., 1993) are G_q-coupled receptors, albeit nonselectively, and Ang II and vasopressin cause renal vasoconstriction by activating AT₁ (Kost and Jackson, 1993) and V₁ receptors (Feng and Arendshorst, 1996), respectively. Therefore, one interpretation of the present findings is that there is an enhanced ability of the G_i signal transduction pathway to augment renovascular responses to G_q-coupled receptors in SHRs. In this regard, it is well established that even though subunits per se have no effect on phospholipase C-, the ability of _q to activate phospholipase C- is markedly enhanced by subunits released from G_i proteins (Selbie and Hill, 1998). These considerations suggest that the mechanism by which G_i-coupled receptors enhance the renovascular response to Ang II and vasopressin could involve enhanced coincident signaling between _q and at the level of phospholipase C-.

We have recently shown that phospholipase D2 is activated by Ang II in preglomerular microvascular smooth muscle cells, and this response is markedly enhanced in SHR versus WKY cells and is mediated by RhoA (Andresen et al., 2001). Moreover, recent studies show that ₂-adrenoceptors activate RhoA (Jinsi-Parimoo and Deth, 1997). Therefore, it is conceivable that the interaction between Ang II or vasopressin and ₂-adrenoceptors is mediated by coincident signaling between Ang II and vasopressin and the G_i signal transduction pathway at the level of a Rho GEF.

In conclusion, the present investigation demonstrates an augmented cross talk in the SHR renal microcirculation between the G_i signal transduction pathway and the signal transduction pathway used by vasoactive peptides. Consequently, drugs that interfere with the G_i signal transduction pathway may offer a novel approach for the treatment of high blood pressure in human beings.