Introduction

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The renin-angiotensin system participates in the regulation of regional and systemic hemodynamics. In this regard, circulating angiotensin II increases peripheral vascular resistance by direct vasoconstriction (Timmermans et al., 1993), by augmenting noradrenergic neurotransmission (Moura et al., 1999), and by releasing catecholamines from the adrenal glands (Giacchetti et al., 1996). Moreover, blood-borne angiotensin II indirectly vasoconstricts the peripheral vasculature by stimulating central nervous system angiotensin receptors that lie outside the blood-brain barrier and that are linked to sympathetic activation (Hasser et al., 2000). Also, angiotensin II synthesized in local vascular beds may directly and indirectly affect peripheral vascular resistance (Zimmerman and Dunham, 1997).

An important issue is the regional vascular selectivity of angiotensin II. If angiotensin II is highly selective with regard to which vascular beds it vasoconstricts, this would have important implications concerning the physiological roles of the renin-angiotensin system. Another important issue is whether local and circulating angiotensin II produce similar or differential profiles of regional vascular resistance changes. If the profile of regional vascular effects of angiotensin II is different depending on whether the peptide is presented locally or produced systemically, this would imply fundamentally different hemodynamic roles for the renin-angiotensin system depending on the site of production and point of delivery of angiotensin II. On the other hand, if the regional vascular effects of the peptide are similar regardless of whether the peptide is produced locally or systemically, this would imply that the various mechanisms by which the renin-angiotensin system induces vasoconstriction evolved to support a common hemodynamic outcome.

Very few studies have compared the effects of angiotensin II on different vascular beds in the same animal. Furthermore, those few studies in which angiotensin II-induced changes in regional vascular resistances were determined in the same experimental animal were restricted in scope. Four decades ago, using electromagnetic flow probes, Assali and Westersten (1961) reported that bolus injections of angiotensin II decreased renal blood flow, increased femoral blood flow, and had no effect on carotid blood flow in anesthetized dogs and sheep. That same year, McGiff and Aviado (1961), using venous outflow measurements, reported that bolus injections of angiotensin II decreased renal blood flow and had biphasic effects on femoral blood flow in anesthetized dogs. Not until 23 years later did Lappe and Brody (1984) report that in conscious rats instrumented with miniaturized pulsed Doppler flow probes 10-min infusions of angiotensin II increased vascular resistances similarly in the mesenteric, renal, and hindquarter vascular beds. Two years later, Corder et al. (1986) published that bolus doses of angiotensin II increased renal vascular resistance but decreased femoral vascular resistance in cats instrumented with electromagnetic flow probes. Gardiner et al. (1988) reported similar increases in renal and mesenteric vascular resistances induced by bolus injections of angiotensin II in conscious Long Evans and Brattleboro rats instrumented with miniaturized pulsed Doppler flow probes. In this same study, angiotensin II had little effect on hindquarter vascular resistance. More recently, Clark et al. (1992) found that intra-arterial injections of angiotensin II were more effective in increasing mesenteric versus renal vascular resistances in anesthetized cats.

The above discussion indicates that 1) only a very limited database exists regarding the regional vascular effects of angiotensin II in the same experimental animal; 2) currently available studies are restricted in scope, examining only a few vascular beds with boluses or brief infusions of only one or two doses of angiotensin II; 3) published studies are inconsistent; and 4) there are no published within-study comparisons of the regional vascular effects of angiotensin II administered intravenously versus locally. Therefore, the purpose of this investigation was to determine the regional vascular selectivity of angiotensin II and to determine whether regional vascular selectivity differs depending on whether the peptide is infused systemically versus locally.

	Materials and Methods

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Animals. Studies were conducted in adult male Sprague-Dawley rats obtained from Charles River (Wilmington, MA) and weighing between 270 and 490 g. Rats were housed in the University of Pittsburgh Medical Center animal care facility for at least 1 week before experiments were conducted and were fed Prolab RMH 3000 rat chow (PMI Feeds, Inc., St. Louis, MO) containing 0.26% sodium and 0.82% potassium. All studies were approved by the Institutional Animal Care and Use Committee. Rats were anesthetized with pentobarbital (50 mg/kg intraperitoneal) and were placed on a Deltaphase Isothermal Pad (Braintree Scientific, Inc., Braintree, MA). Rats anesthetized with pentobarbital still have sympathetic tone and baroreceptor reflexes (Shimokawa et al., 1998). A heat lamp was also placed above the animal and its height above the animal was adjusted to maintain rectal temperature at 37°C.

Study A. In the first study, a polyethylene (PE) cannula (PE-240) was inserted into the trachea to facilitate respiration. Three PE-50 cannulas were inserted into the jugular vein for intravenous infusions of angiotensin II, intravenous boluses of saline for cardiac output determinations, and for supplementary doses of anesthetic. Also, another PE-50 cannula was inserted into the left femoral artery and connected to a digital blood pressure analyzer (MicroMed, Inc., Louisville, KY), which sampled arterial blood pressure at 1100 Hz with an 82% duty cycle. The blood pressure analyzer was set to time-average arterial blood pressure and heart rate every minute.

Study B. In the second study, animals were prepared exactly as described for study A, and after a 1-h rest period, baseline regional blood flows, arterial blood pressure, heart rate, and cardiac output were measured as described for study A. Arginine vasopressin in peptide buffer was infused at increasing doses (1, 3, and 10 pmol/min) for 10 min at each dose, and all measurements were repeated during the last minute of each 10-min infusion of vasopressin.

Study C. In the third study, a PE-240 cannula was inserted into the trachea to facilitate respiration, and a PE-50 cannula was inserted into the jugular vein for administration of supplementary anesthetic. Also, another PE-50 cannula was inserted into the left carotid artery and connected to a digital blood pressure analyzer for time-averaging arterial blood pressure each minute. Noncannulating, transit-time flow probes were placed on the left renal, mesenteric, and right carotid arteries and also on the lower abdominal aorta just proximal to the iliac bifurcation. Flow probes were connected to two 2-channel small animal transit-time flow meters and the synchronization features of the flow meters were adjusted to prevent cross talk among the four flow probes. In addition, a 32-gauge needle connected to an infusion pump via Silastic tubing was inserted into the mesenteric, left renal, and right carotid arteries and lower abdominal aorta. At each site, peptide buffer was infused at 20 µl/min. After the surgery was completed, all rats received an intravenous dose of captopril (30 mg/kg) to block the biosynthesis of endogenous angiotensin II.

Study D. In the fourth study, a PE-240 cannula was inserted into the trachea to facilitate respiration, and two PE-50 cannulas were inserted into the jugular vein for intravenous infusions of peptide buffer (60 µl/min) and for supplementary doses of anesthetic. Also, another PE-50 cannula was inserted into the left carotid artery and connected to a digital blood pressure analyzer for time-averaging arterial blood pressure each minute. Noncannulating, transit-time flow probes were placed on the left and right renal arteries and were connected to a two-channel small animal transit-time flowmeter. Next, a 32-gauge needle connected to an infusion pump via Silastic tubing was inserted into the right carotid artery, and an infusion of peptide buffer (20 µl/min) was begun. After the surgery was completed, all rats received an intravenous dose of captopril (30 mg/kg) to block endogenous angiotensin II synthesis.

Statistical Analysis. Statistical analyses were conducted with the Number Crunchers Statistical System (Kaysville, UT). Data were analyzed by one-factor analysis of variance (independent sampling or repeated measures as appropriate), and if significance was obtained by analysis of variance, differences among means were determined with a Fisher's least significant difference test. The criterion of significance was a p value less than 0.05.

	Results

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Study A. Table 1 lists baseline values for regional vascular blood flows and regional vascular resistances for study A. Angiotensin II increased arterial blood pressure and total peripheral resistance (Table 2), without affecting cardiac output (Table 3). As shown in Fig. 1, angiotensin II caused dose-dependent reductions in renal blood flows and increases in renal vascular resistances that were sustained for 2 h. Angiotensin II also decreased mesenteric blood flow and increased mesenteric vascular resistance (Fig. 2). However, in the mesentery, after approximately 30 min of infusion, the percentage increases in mesenteric vascular resistances induced by 100 and 300 pmol/min angiotensin II were similar (77 ± 14 versus 86 ± 19%, respectively, at 30 min; 85 ± 14 versus 90 ± 24%, respectively, at 60 min; 98 ± 16 versus 108 ± 32%, respectively, at 90 min; and 98 ± 15 versus 118 ± 35%, respectively, at 120 min). In contrast, the percentage increases in renal vascular resistances induced by 100 and 300 pmol/min angiotensin II were markedly different throughout the infusion of angiotensin II (115 ± 23 versus 304 ± 34%, respectively, at 30 min; 108 ± 24 versus 274 ± 30%, respectively, at 60 min; 93 ± 22 versus 260 ± 32%, respectively, at 90 min; and 83 ± 20 versus 252 ± 36%, respectively, at 120 min). Therefore, a dose of angiotensin II that was maximally effective in the mesenteric circulation (i.e., 300 pmol/min) had a much greater effect on renal vascular resistance compared with mesenteric vascular resistance.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Effects of intravenous angiotensin II on renal blood flow (top) and renal vascular resistance (bottom). *p < 0.05 compared with corresponding time period in 0 pmol/min group (i.e., vehicle/time control group).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Effects of intravenous angiotensin II on mesenteric blood flow (top) and mesenteric vascular resistance (bottom). *p < 0.05 compared with corresponding time period in 0 pmol/min group (i.e., vehicle/time control group).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Effects of intravenous angiotensin II on carotid blood flow (top) and carotid vascular resistance (bottom). *p < 0.05 compared with corresponding time period in 0 pmol/min group (i.e., vehicle/time control group).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Effects of intravenous angiotensin II on hindquarter blood flow (top) and hindquarter vascular resistance (bottom). *p < 0.05 compared with corresponding time period in 0 pmol/min group (i.e., vehicle/time control group).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Effects of intravenous angiotensin II on other tissue blood flow (top) and other tissue vascular resistance (bottom). *p < 0.05 compared with corresponding time period in 0 pmol/min group (i.e., vehicle/time control group).

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Effects of intravenous angiotensin II (300 pmol/min) on renal vascular resistance (RVR), mesenteric vascular resistance (MVR), carotid vascular resistance (CVR), other tissue vascular resistance (OTVR), and hindquarter vascular resistance (HQVR). The "line of complete autoregulation" refers to the percentage change in resistance that would have occurred if the given vascular bed fully autoregulated in response to the change in mean arterial blood pressure.

Study B. As shown in Table 4, intravenous infusions of vasopressin dose dependently increased mean arterial blood pressure and total peripheral resistance, and caused a reflex bradycardia and a reduction in cardiac output. Unlike angiotensin II, vasopressin increased similarly renal, mesenteric, carotid, hindquarter, and other tissue vascular resistances (Fig. 7).

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Effects of intravenous vasopressin on renal vascular resistance (RVR), mesenteric vascular resistance (MVR), carotid vascular resistance (CVR), hindquarter vascular resistance (HQVR), and other tissue vascular resistance (OTVR). Baseline values of RVR, MVR, CVR, HQVR, and OTVR were 11 ± 2, 21 ± 4, 15 ± 2, 14 ± 1, and 4 ± 1 mm Hg · min/ml, respectively. *Significant increase (p < 0.05) compared with basal period.

Study C. Table 5 lists baseline values for regional vascular blood flows and regional vascular resistances for study C. Intrarenal infusions of angiotensin II caused dose-related increases in renal vascular resistance, but did not affect vascular resistances of the mesenteric, carotid, or hindquarter vascular beds (Fig. 8). Infusions of angiotensin II into the mesenteric artery caused a dose-related increase in mesenteric vascular resistance, but did not affect vascular resistances of the renal, carotid, or hindquarter vascular beds (Fig. 8). The effects on mesenteric vascular resistance induced by intramesenteric artery infusions of angiotensin II were less than the effects on renal vascular resistance induced by intrarenal infusions of angiotensin II (Fig. 8). Infusions of angiotensin II into the carotid circulation only slightly increased carotid vascular resistance, did not change hindquarter vascular resistance, yet markedly increased both renal and mesenteric vascular resistances (Fig. 9). Infusions of angiotensin II into the hindquarter circulation only slightly increased hindquarter vascular resistance, did not change carotid vascular resistance, yet increased renal and mesenteric vascular resistances (Fig. 9). Intra-arterial infusions of angiotensin II into the renal, mesenteric, and hindquarter vascular beds only modestly increased mean arterial blood pressure; however, infusions of angiotensin II into the carotid circulation markedly increased arterial blood pressure (Table 6).

View larger version (22K): [in this window] [in a new window]   Fig. 8.   Changes in renal, mesenteric, carotid, and hindquarter vascular resistances induced by infusions of angiotensin II into the renal artery (top) and mesenteric artery (bottom). *Significant increase (p < 0.05) compared with basal period.

View larger version (23K): [in this window] [in a new window]   Fig. 9.   Changes in renal, mesenteric, carotid, and hindquarter vascular resistances induced by infusions of angiotensin II into the carotid artery (top) and lower abdominal artery (bottom). *Significant increase (p < 0.05) compared with basal period.

Study D. The increases in renal and mesenteric vascular resistances and arterial blood pressure induced by infusions of angiotensin II into the carotid artery could have been due to recirculation of angiotensin II and/or to a centrally mediated activation of the sympathetic nervous system by blood-borne angiotensin II acting on brain structures devoid of a blood-brain barrier. To determine the relative contribution of these factors to the vascular responses to intracarotid infusions of angiotensin II, we infused angiotensin II into the carotid artery of animals and measured vascular responses at baseline and following denervation of the left kidney. As illustrated in Fig. 10 and Table 7, infusions of angiotensin II into the carotid artery markedly increased vascular resistances of the left and right kidneys and increased arterial blood pressure. Denervation of the left kidney, either without or with adrenalectomy, did not alter the ability of intracarotid infusions of angiotensin II to increase renal vascular resistances or arterial blood pressure.

View larger version (20K): [in this window] [in a new window]   Fig. 10.   Effects of infusions into the carotid artery of angiotensin II on renal vascular resistance of the left and right kidneys during a control period, after denervation of the left kidney, and after denervation plus bilateral adrenalectomy. Baseline values of renal vascular resistance for the left kidney during the control period and after denervation of the left kidney were 12 ± 1 and 13 ± 2 mm Hg · min/ml, respectively. Baseline values of RVR for the right kidney during the control period and after denervation of the left kidney were 13 ± 2 and 15 ± 2 mm Hg · min/ml, respectively. *Significant increase (p < 0.05) compared with basal period.

	Discussion

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In study A, we continuously monitored blood flows to four vascular beds in the same animal using state-of-the-art transit-time flow probes. Others have validated this technology to be the most accurate for monitoring organ perfusion in small rodents (Welch et al., 1995). In addition, we measured total cardiac output and calculated by subtraction the blood flow to tissues other than the kidneys, mesentery, hindquarter, and carotid vascular beds. The study design used a 100-fold dose range of angiotensin II, monitored the effects of angiotensin II for 120 min, and included a vehicle/time control group.

The results of the current study strongly suggest that the kidneys and, to a lesser degree, the mesenteric vascular bed are the main physiological targets for the integrated vasoconstrictive response to circulating angiotensin II. In this regard, angiotensin II even at very high infusion rates did not increase vascular resistance in the hindquarter; and the resistance of the carotid vascular bed increased only slightly at the highest infusion rate of angiotensin II. Moreover, angiotensin II did not increase the calculated other tissue vascular resistance, which indicated little effect of angiotensin II on other well perfused vascular beds. An important caveat, however, is that the calculated other tissue vascular resistance would not have detected a marked vasoconstrictive effect of angiotensin II on vascular beds receiving a small percentage of other tissue flow, e.g., skin.

Intravenous infusions of angiotensin II caused large increases in arterial blood pressure, and these increases in arterial blood pressure may have triggered autoregulatory vasoconstriction in some tissues so as to maintain tissue perfusion relatively constant. In this regard, only the renal and mesenteric vascular beds demonstrated a vascular response to angiotensin II that exceeded that which could have been accounted for by autoregulation of tissue blood flow. Moreover, of the monitored vascular beds, only the renal and mesenteric vascular beds demonstrated a vigorous vascular response to angiotensin II. Comparing these two vascular beds, it appears that the kidney is even more affected by angiotensin II than is the mesenteric vascular bed. A dose of angiotensin II (300 pmol/min) that was maximal with regard to increasing mesenteric vascular resistance caused a far greater decrease in renal blood flow and increase in renal vascular resistance compared with the changes in blood flow and vascular resistance observed in the mesentery.

In contrast to angiotensin II, vasopressin caused widespread vasoconstriction. Doses of vasopressin in study B that increased arterial blood pressure and total peripheral resistance to a similar degree as the doses of angiotensin II used study A did not selectively increase renal and mesenteric vascular resistances. The vasopressin data indicate that the regional vascular selectivity observed with intravenous angiotensin II was not 1) an artifact of the methodology used in this study; 2) due to stronger or weaker autoregulatory responses in the various tissues; 3) due to reflex changes in sympathetic tone; or 4) due to maximal vasoconstriction in some vascular beds at baseline. If any of these factors were the explanation for the observed regional vascular selectivity of angiotensin II then vasopressin would have also display regional vascular selectivity.

In study A, we infused angiotensin II intravenously, thus delivering high concentrations of angiotensin II systemically. Therefore, responses to angiotensin II infusions in study A represented integrated responses mediated by the local and systemic actions of angiotensin II. In study C, we administered angiotensin II locally into specific vascular beds to determine the direct response of those vascular beds to angiotensin II. Importantly, the response to local infusions of angiotensin II matched the results with intravenously administered angiotensin II, i.e., angiotensin II strongly vasoconstricted the renal vasculature, moderately vasoconstricted the mesenteric vasculature, and had little effect on the carotid and hindquarter vascular beds.

Notably, infusion of angiotensin II into the carotid artery markedly increased renal and mesenteric vascular resistances. Blood-borne angiotensin II is well known to activate angiotensin receptors in circumventricular structures in the central nervous system lacking a blood-brain barrier. In this regard, blood-borne angiotensin II causes systemic vasopressor effects mediated by activation of the sympathetic nervous system (Fink et al., 1980; Andersson et al., 1995; Hasser et al., 2000). Thus, the result with intracarotid infusions could have been due to activation of the sympathetic nervous system and/or due to recirculation of angiotensin II. Study D clearly indicates that the ability of intracarotid infusions of angiotensin II to increase renal vascular resistance was not attenuated by renal denervation or renal denervation plus bilateral adrenalectomy. In the nondenervated right kidney, the first and second dose-response curves to angiotensin II were similar. This observation eliminates the possibility that the lack of effect of denervation in the left kidney was due to a time-related change in responsiveness of the kidneys to angiotensin II. It appears that vasoconstriction of the renal and mesenteric vascular beds induced by intracarotid infusions of angiotensin II was mediated by recirculation of angiotensin II. Recirculation of angiotensin II most likely also accounts for the increases in renal and mesenteric resistance following infusion of angiotensin II into the hindquarter.

When angiotensin II was infused into the kidney, mesenteric vascular resistance was not changed; and when angiotensin II was infused into the mesentery, renal vascular resistance was not altered. These results imply that the renal and mesenteric (plus hepatic) vascular beds extracted most of the locally infused angiotensin II. Indeed, Bauer et al. (1999) reported that 93% of angiotensin II that passes through the kidney is degraded. In contrast, as indicated in the preceding paragraph, infusions of angiotensin II into the carotid and hindquarter vascular beds resulted in pharmacologically active concentrations of recirculating angiotensin II. Clearly, the renal and mesentery (plus hepatic) vascular beds were more effective in degrading angiotensin II compared with the carotid and hindquarter vascular beds. Thus, it appears that those tissues that are more responsive to angiotensin II are also the tissues that more effectively degrade angiotensin II. This is biologically plausible since tissues that are highly responsive to angiotensin II would require mechanisms to protect against excessive angiotensin II-induced vasoconstriction.

The novel conclusion of this investigation is that regardless of whether angiotensin II is produced locally or produced systemically angiotensin II predominately vasoconstricts the renal and mesenteric circulations. Does this make evolutionary sense? One of the most important physiological roles for the renin-angiotensin system is to allow animals to adapt to extremes of salt intake while maintaining an adequate level of arterial blood pressure (Hall et al., 1980). Since the kidneys determine the long-term levels of arterial blood pressure (Cowley, 1992), angiotensin II can best accomplish its function to maintain constancy of arterial blood pressure in the face of changes in salt intake by having the kidney as its main vasoconstrictive target. In the setting of chronic low-salt intake, vasoconstriction of other organ beds would in fact be inappropriate.

Another important role for the renin-angiotensin system is to protect against hypotension during blood loss (Jakschik et al., 1974). By constricting the renal and mesenteric circulations, angiotensin II would increase total peripheral resistance without compromising blood flow to vital organs such as the heart and brain and without compromising blood flow to skeletal muscle. Thus, the renin-angiotensin system would serve as a homeostatic mechanism in times of injury with loss of vascular integrity. By temporarily reducing blood flow to the kidneys and gut, blood flow to the heart, brain, and skeletal muscle can be maintained to allow the organism to cope with the threat.

The cellular and biochemical mechanisms underlying the regional vascular selectivity of angiotensin II cannot be deduced from the current study. However, several nonmutually exclusive mechanisms may participate. First, it is possible that the regional selectivity to angiotensin II is mediated in part by differences in the regional distribution of AT₁ or AT₂ receptors or by differences in the ratio of AT₁ to AT₂ receptors in vascular smooth muscle cells from various vascular beds. Second, angiotensin II is well known to release a number of vasoactive agents that modulate vascular resistance. In this regard, angiotensin II increases the synthesis of arachidonic acid metabolites of cyclooxygenase (Nasjletti, 1998), lipoxygenase (Nasjletti, 1998), and cytochrome P450s (Chu et al., 2000; Croft et al., 2000), and stimulates the release of endothelin-1 (Moreau et al., 1997), nitric oxide (Millatt et al., 1999), and superoxide anion (Berry et al., 2000). It is possible, therefore, that angiotensin II induces differential release of vasoactive factors from various vascular beds. Finally, it is conceivable that the regional selectivity is mediated by differential expression of signal transduction mechanisms. Angiotensin II has the potential to activate, either directly or indirectly, most known signal transduction pathways (Berk and Corson, 1997; Sayeski et al., 1998). Thus, the overall vascular response to angiotensin II may be a function of which signal transduction pathways predominate in a given vascular bed.

In conclusion, the results of these studies indicate that angiotensin II is not a generalized vasoconstrictor that increases total peripheral resistance by nonselectively vasoconstricting all vascular beds. Instead, angiotensin II has evolved to constrict primarily the renal vascular bed, with somewhat lesser effects on the gut circulation. In this regard, the systemic and local effects of angiotensin II appear to work together, rather than as mechanisms serving to differentially regulate regional vascular tone. This remarkable selectivity and coordination of vasoconstrictive mechanisms assures that the renin-angiotensin system operates successfully to fulfill its function to protect animals from extremes of salt intake and from life-threatening situations involving blood loss.