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CARDIOVASCULAR
2-Adrenoceptor Interaction
Center for Clinical Pharmacology, Departments of Pharmacology (E.K.J.) and Medicine (E.K.J., L.G., C.Z.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
Received for publication
March 9, 2005
Accepted
May 17, 2005.
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Abstract |
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2-Adrenoceptors potentiate vascular responses to angiotensin II. The goal of this study was to test the hypothesis that the phospholipase C (PLC)/protein kinase C (PKC)/c-src/phosphatidylinositol 3-kinase (PI3K) pathway contributes to the vascular angiotensin II/2-adrenoceptor interaction. In rats in vivo, intrarenal infusions of angiotensin II (10 ng/kg/min) increased renal vascular resistance by 5.8 ± 0.5 units, and this response was enhanced (p < 0.05) to 9.1 ± 1.2 units by UK-14,304 [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine; 3 µg/kg/min; 2-adrenoceptor agonist]. Intrarenal infusions of U-73122 [1-[6-[[(17
)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]-hexyl]-1H-pyrrole-2,5-dione; 3 µg/min; PLC inhibitor], GF109203X [bisindolylmaleimide I; 10 µg/min; PKC inhibitor], CGP77675[1-(2-{4-[4-amino-5-(3-methoxyphenyl)pyrrolo[2,3-d]pyrimidin-7-yl]phenyl}ethyl)piperidin-4-ol; 5 µg/min; c-src inhibitor], and wortmannin (1 µg/min; PI3K inhibitor) abolished the angiotensin II/2-adrenoceptor interaction. In isolated perfused rat kidneys, angiotensin II (0.3, 1, and 3 nM) increased perfusion pressure (by 15 ± 8, 39 ± 4, and 93 ± 9 mm Hg, respectively), and UK-14,304 (1 µM) potentiated these responses (to 36 ± 4, 67 ± 7, and 135 ± 17 mm Hg, respectively). This angiotensin II/2-adrenoceptor interaction was abolished by U-73122 (10 µM), GF109203X (3 µM), CGP77675(5 µM), and wortmannin (0.2 µM). Preglomerular microvascular smooth muscle cells expressed phospholipase (PLC)-2, PLC-3, c-src, phospho(tyrosine 416)-c-src, and PI3K. In these cells, angiotensin II (0.1 µM) and UK-14,304 (1 µM) per se did not increase phospho-c-src; however, the combination of angiotensin II plus UK-14,304 doubled phospho-c-src, and this interaction was abolished by U-73122 (10 µM) and GF109203X (3 µM). In conclusion, the PLC/PKC/c-src/PI3K pathway may contribute importantly to the interaction between 2-adrenoceptors and angiotensin II on renal vascular resistance.
). For example, renal sympathetic nerves activate the renin-angiotensin system by stimulating renin release via 1-adrenoceptors, and angiotensin II (Ang II), via AT1 receptors, activates sympathetic nerves by increasing renal sympathetic tone (via a central action of blood-borne Ang II) and by augmenting norepinephrine release (via a peripheral action on renal sympathetic nerves). Thus, these two systems are physiologically intertwined with regard to the regulation of vascular function (DiBona, 2001).
There seems, however, to be another important mode of interaction between the sympathetic nervous system and the renin-angiotensin system on vascular function that has received very little attention. Our studies demonstrate that 2-adrenoceptors, which can be activated via norepinephrine released from sympathetic nerves or epinephrine and norepinephrine released from the adrenal medulla, enhance Ang II-induced increases in vascular resistance (Jackson et al., 2001; Gao et al., 2003). This interaction may be of particular biological significance because our studies demonstrate that the ability of 2-adrenoceptors to enhance Ang II-induced vasoconstriction both in vivo (Jackson et al., 2001) and in vitro (Gao et al., 2003) is much greater in animals with genetic hypertension compared with normotensive animals or animals with nongenetic hypertension.
Our previous study shows that inhibiting the Gi signal transduction pathway with pertussis toxin blocks the ability of 2-adrenoceptors to augment Ang II-induced vasoconstriction, indicating that 2-adrenoceptors modulate Ang II-induced vasoconstriction via the Gi signal transduction pathway (Gao et al., 2003). However, despite the potential physiological importance of 2-adrenoceptor-induced enhancement of Ang II-induced vasoconstriction, apart from our previous work with pertussis toxin there is no information regarding the mechanism of this interaction.
How does the Gi signal transduction pathway potentiate vasoconstrictor responses to Ang II? It is known that 
subunits released from Gi synergize with q subunits released from Gq with respect to activating phospholipase C (PLC)-2/3 (Selbie and Hill, 1998), and this is an example of a class of interactions called coincidence signaling. Because 2-adrenoceptors are coupled to Gi (Kurose et al., 1991) and AT1 receptors are coupled to Gq (Sano et al., 1997), this type of coincidence signaling may participate in the interaction between 2-adrenoceptors and Ang II on vascular resistance. Via increases in diacyglycerol and calcium, PLC stimulates protein kinase C (PKC) (Newton, 1995), and PKC activates c-src by phosphorylating c-src on tyrosine 416 (Moyers et al., 1993), and c-src mediates many of the actions of PKC (Raptis and Whitfield, 1986; Liebenhoff et al., 1994; Nagao et al., 1998; Lu et al., 1999; Matrougui et al., 2000; Brandt et al., 2002; Chang et al., 2002; Huang et al., 2003). Moreover, multiple studies indicate that c-src strongly stimulates phosphatidylinositol 3-kinase (PI3K) (Otsu et al., 1991; Conway et al., 1999; Daulhac et al., 1999; Versteeg et al., 2000; Encinas et al., 2001; Kubota et al., 2001; Gentili et al., 2002; Jimenez et al., 2002; Seshiah et al., 2002; Acosta et al., 2003; Nair and Sealfon, 2003). Therefore, we hypothesize that the PLC/PKC/c-src/PI3K pathway mediates the interaction between 2-adrenoceptors and Ang II on vascular resistance (Fig. 1), and the goal of the present study was to test this hypothesis.
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Materials and Methods |
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Experiments in SHR Kidney in Vivo. SHR (n = 79) were anesthetized (Inactin, 90 mg/kg i.p.), and body temperature was maintained at 37°C. The trachea was cannulated with polyethylene-240 tubing, and a cannula (polyethylene-50) was inserted in the left jugular vein and carotid artery. The arterial cannula was connected to a digital blood pressure analyzer for measurement of MABP.
The influence of endogenous catecholamines on 2-adrenoceptors was minimized by bilateral adrenalectomy and denervating the left kidney as described previously (Jackson et al., 2001). Adrenal steroids were replaced by infusing at 25 µl/min (via the jugular vein catheter) 0.9% saline containing aldosterone (20 ng/min) and hydrocortisone (20 µg/min).
A transit-time flow probe connected to a transit-time flowmeter was positioned around the left renal artery to monitor renal blood flow (RBF). A 32-gauge needle connected to a catheter was placed into the renal artery. This catheter was inserted into a four-way connector, and three lines linked the connector to three separate infusion pumps. Intrarenal infusion line 1 infused DMSO (5 µl/min), and intrarenal infusion lines 2 and 3 infused 0.9% saline (25 µl/min). Captopril (30 mg/kg) was given to minimize the influence of endogenous Ang II, and 0.9% saline (20 ml/kg) was administered to improve hemodynamic stability.
After a 1-h stabilization period, animals were randomly assigned to five groups according to the infusion via intrarenal infusion line 1: 1) nonpretreated group (DMSO), 2) U-73122-pretreated group (U-73122 at 3 µg/min; PLC inhibitor; Bleasdale et al., 1990) (Tocris Cookson Inc., Ellisville, MO), 3) GF109203X-pretreated group (GF109203X at 10 µg/min; PKC inhibitor; Ku et al., 1997 (Tocris Cookson Inc.), 4) CGP77675pretreated group (CGP77675at 5 µg/min; c-src inhibitor; Missbach et al., 1999) (Novartis, Basel, Switzerland), and 5) wortmannin-pretreated group (wortmannin at 1 µg/min; PI3K inhibitor; Vanhaesebroeck et al., 2001) (Sigma, St. Louis, MO). All inhibitors were dissolved in DMSO and infused at 5 µl/min. Ten minutes after starting the infusions of inhibitors, RBF and MABP were recorded just before and during the last minute of a 5-min intrarenal infusion of Ang II (10 ng/kg/min infused in 0.9% saline at 25 µl/min). The angiotensin II was administered via intrarenal infusion line 2. Renal vascular resistance (RVR) was calculated by dividing RBF (milliliters per minute per kilogram of body weight) into the MABP (mm Hg).
Within each of the five groups, while the infusions of inhibitors continued, an intrarenal infusion of UK-14,304 (3 µg/kg/min infused in 0.9% saline at 25 µl/min; 2-adrenoceptor agonist; Paris et al., 1989) was initiated via intrarenal infusion line 3 in some of the animals. Whether an animal continued to receive 0.9% saline or UK-14,304 via infusion line 3 was determined randomly. After 20 min, RVR responses to angiotensin II were redetermined.
Experiments in Isolated Perfused SHR Kidney. SHR (n = 29) were anesthetized, and the left kidney was isolated and perfused with Tyrode's solution as described previously (Gao et al., 2003). Kidneys were perfused (single pass mode) at a constant flow (5 ml/min). Perfusion pressure was monitored with a pressure transducer.
Kidneys were randomly assigned to five groups: 1) nonpretreated group, 2)U-73122-pretreated group (10 µM), 3) GF109203X-pretreated group (3 µM), 4) CGP77675pretreated group (5 µM), and 5) wortmannin-pretreated group (0.2 µM). Inhibitors were added directly to the Tyrode's solution. After a 30-min rest period, vasoconstrictor responses (changes in perfusion pressure) to Ang II (0.3, 1, and 3 nM; final concentration in perfusate) were obtained by infusing Ang II for 2 min, with a 10-min rest period between each concentration of Ang II. After a 30-min rest period, UK-14,304 (1 µM, final concentration in perfusate) was infused for 10 min, and vasoconstrictor responses to Ang II were repeated while the UK-14,304 infusion was maintained. Pilot studies demonstrated that concentration-response curves to Ang II were stable and reproducible for the duration of the experiment in the absence or presence of inhibitors.
Experiments in Preglomerular Microvascular Smooth Muscle Cells (PGSMCs). Primary preglomerular microvascular smooth muscle cells were cultured by explant from freshly isolated SHR microvessels using our previously described method (Mokkapatti et al., 1998). PGSMCs were maintained under standard cell culture conditions, and studies were conducted in PGSMCs with low passage number (<8). Protein extraction, SDS-polyacrylamide gel electrophoresis (20 µg of protein per sample), and Western blotting were performed as described previously (Jackson et al., 2002). We used one of five different primary antibodies for Western blotting: 1) anti-PLC-2 antibody (catalog no. sc-206; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 2) anti-PLC-3 antibody (catalog no. sc-403; Santa Cruz Biotechnology, Inc.), 3) anti-c-Src antibody (catalog no. sc-19; Santa Cruz Biotechnology, Inc.), 4) anti-phospho(tyrosine 416)-c-Src antibody (catalog no. 05-677; Upstate Biotechnology, Waltham, MA), 5) or anti-PI3K(p85 subunit) antibody (Chemicon International, Temecula, CA). To examine the interaction between Ang II and UK-14,304 on activation of c-src in PGSMCs, cells were grown to confluence in 12-well plates and then serum-starved for 24 h (0.4% fetal calf serum). The cells were then placed in phosphate-buffered saline without and with either Ang II (100 nM), UK-14,304 (1 µM), or both Ang II plus UK-14,304. Some PGSMCs were also cotreated with either U-73122 (10 µM) or GF109203X (3 µM). After 15 min of incubation with agonists and/or inhibitors, PGSMCs were harvested and proteins were extracted and subjected to Western blotting for c-src and phospho-c-src.
Statistics. In vivo data were analyzed using a Mann-Whitney U test or Wilcoxon signed rank test as appropriate. In vitro data were analyzed by repeated measures two-factor analysis of variance or one-factor analysis of variance as appropriate. The criterion of significance was p < 0.05. All values in text and tables are means ± S.E.M.
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Figure 2, A and B, illustrates RVR responses to Ang II in the nonpretreated group. In the time-control subgroup, RVR response to Ang II did not change from period 1 to period 2 (Fig. 2A). In the subgroup that received UK-14,304 during period 2, RVR responses to Ang II were significantly (p = 0.0122) increased compared with the response during period 1. This indicated that UK-14,302 potentiated RVR responses to Ang II.
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Figure 2, C and D, illustrates RVR responses to Ang II in the U-73122-pretreated group. RVR responses to Ang II did not change from period 1 to period 2 in this group regardless of whether vehicle (Fig. 2C) or UK-14,304 (Fig. 2D) was administered during period 2. This indicated that U-73122 completely blocked UK-14,304-induced potentiation of RVR responses to Ang II.
Figure 2, E and F, illustrates RVR responses to Ang II in the GF109203X-pretreated group. RVR responses to Ang II did not change from period 1 to period 2 in this group, regardless of whether vehicle (Fig. 2E) or UK-14,304 (Fig. 2F) was administered during period 2. This indicated that GF109203X completely blocked UK-14,304-induced potentiation of RVR responses to Ang II.
Figure 3, A and B, illustrates RVR responses to Ang II in the CGP77675pretreated group. In the time control subgroup, RVR responses to Ang II did not change from period 1 to period 2 (Fig. 3A). In the subgroup of the CGP77675pretreated group that received UK-14,304 during period 2, RVR responses to Ang II were not statistically different (p > 0.2) from responses during period 1 (Fig. 3B). This indicated that CGP77675completely blocked UK-14,304-induced potentiation of RVR responses to Ang II.
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Experiments in Isolated Perfused SHR Kidney. Table 2 lists the basal perfusion pressures in the five groups before and after administration of UK-14,304. As shown, UK-14,304 did not significantly alter basal perfusion pressure in any of the five groups.
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In Fig. 4A, Ang II concentration dependently (0.3, 1, and 3 nM) increased perfusion pressure, and this response was markedly enhanced by UK-14,304 (p = 0.0010). Figure 4B demonstrates a typical response to Ang II (3 nM) in the absence and presence of UK-14,304. These data show clearly that UK-14,304 greatly enhances the renovascular response to Ang II.
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Figure 4, C to F, illustrates the concentration-dependent effects of Ang II on perfusion pressure in the absence and presence of UK-14,304 and in the presence of U-73122 (Fig. 4C), GF109203X (Fig. 4D), CGP77675(Fig. 4E), and wortmannin (Fig. 4F). Although each of the inhibitors significantly attenuated Ang II-induced changes in perfusion pressure, responses to Ang II were nonetheless clearly observable. As in the in vivo experiments, all four inhibitors completely blocked the ability of UK-14,304 to potentiate Ang II-induced changes in perfusion pressure.
Experiments in PGSMCs. Figure 5 demonstrates that PGSMCs expressed PLC-2 (Fig. 5A), PLC-3 (Fig. 5B), c-src (Fig. 5C), phospho(tyrosine 416)-c-src (Fig. 5D), and the p85 subunit of PI3K (Fig. 5E). Thus, the signal transduction molecules necessary so support the proposed pathway exist in PGSMCs. Figure 6 shows that in the absence of inhibitors, neither Ang II nor UK-14,304 per se increased the expression of phospho-c-src; however, the combination of Ang II plus UK-14,304 increased phospho-c-src approximately 100% (p < 0.0001). This demonstrates that there indeed is an interaction between Ang II and UK-14,304 at the biochemical level that culminates in the synergistic phosphorylation of c-src on tyrosine 416. Importantly, when PGSMCs were coincubated with either U-73122 (Fig. 7) or GF109203X (Fig. 8), the interaction between Ang II and UK-14,304 with respect to phosphorylation of c-src was completely abrogated. These results suggest that the interaction between Ang II and UK-14,304 on c-src phosphorylation is mediated by PLC and PKC.
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). A potentially powerful interaction that has received very little attention is the interaction between 2-adrenoceptors and Ang II. Sympathetic nerve varicosities release norepinephrine, and the adrenal medulla releases epinephrine and norepinephrine. These catecholamines stimulate multiple adrenergic receptors, including 1-adrenoceptors and 2-adrenoceptors. 1-Adrenoceptors increase renin release, thereby augmenting circulating and local levels of Ang II. Although 2-adrenoceptors have little direct effect on the renal vasculature, our studies demonstrate that this class of adrenoceptors can dramatically augment Ang II-induced vasoconstriction in genetically predisposed animals (Jackson et al., 2001; Gao et al., 2003). Therefore, the interaction between 2-adrenoceptors and Ang II is yet another aspect of an elegant physiological interplay that intertwines the sympathetic nervous system with the renin-angiotensin system.
Our previous study using pertussis toxin demonstrates that the Gi signal transduction pathway mediates the ability of 2-adrenoceptors to potentiate Ang II-induced vasoconstriction (Gao et al., 2003). How the Gi pathway does this is unknown; however, in theory the Gi pathway could augment AT1-receptor-induced responses via coincidence signaling (Fig. 1). Activation of Gi releases subunits, and stimulation of AT1 receptors releases q from Gq (Sano et la., 1997). The availability of these G protein subunits provides for possible coincidence signaling mechanisms in which two or more G protein subunits converge on a signal transduction molecule. In this regard, one of the most well described coincidence signaling molecule is PLC-2/3 (Selbie and Hill, 1998). It is well accepted that the ability of q to stimulate PLC-2/3 is greatly enhanced by subunits because inhibits the GAP activity of PLC-2/3 (Chidiac and Ross, 1999). Moreover, a recent report demonstrates that subunits relieve competitive product inhibition of PLC-2 (Feng et al., 2005). Because PLC-2/3 stimulates PKC activity (Newton, 1995), the PLC-2/3/PKC pathway is an obvious candidate for mediating the interaction between 2-adrenoceptors and Ang II. Indeed, in the present study, the PLC inhibitor U-73122 and the PKC inhibitor GF108203X blocked the interaction between UK-14,304 and Ang II both in vivo and in vitro. These findings corroborate and extend our previously published preliminary results with U-73122 in vivo (Jackson et al., 2001).
An important finding of the present study is that blocking either PLC, PKC, c-src, or PI3K is sufficient to completely prevent 2-adrenoceptors from potentiating Ang II-induced vasoconstriction. These findings are consistent with the hypothesis outlined in Fig. 1. This hypothesis is also supported by a large body of published data showing that PKC phosphorylates and activates c-src (Raptis and Whitfield, 1986; Moyers et al., 1993; Liebenhoff et al., 1994; Nagao et al., 1998; Lu et al., 1999; Matrougui et al., 2000; Brandt et al., 2002; Chang et al., 2002; Huang et al., 2003) and that c-src activates PI3K (Otsu et al., 1991; Conway et al., 1999; Daulhac et al., 1999; Versteeg et al., 2000; Encinas et al., 2001; Kubota et al., 2001; Gentili et al., 2002; Jimenez et al., 2002; Seshiah et al., 2002; Acosta et al., 2003; Nair and Sealfon, 2003). Therefore, our data are consistent with the hypothesis that the PLC/PKC/c-src/PI3K pathway mediates the interaction between 2-adrenoceptors and Ang II on blood vessels (Fig. 1). Although published reports indicate that c-src activates PI3K, the present experiments do not rule out the possibility that PI3K activation participates in the interaction between 2-adrenoceptors and Ang II in parallel to the PLC/PKC/c-src pathway. Additional studies are required to confirm or refute the proposed position of PI3K in the signal transduction mechanism shown in Fig. 1.
Additional studies were performed in cultured PGSMCs to verify that the proposed signaling molecules are expressed in the renal microvasculature. Also, we investigated whether an interaction between 2-adrenoceptors and Ang II on phosphorylation of c-src at tyrosine 416, an event that activates c-src (Boggon and Eck, 2004), exists and whether this interaction could be blocked by inhibition of PLC and PKC. Our results in cultured PGSMCs also support the hypothesis that the PLC/PKC/c-src/PI3K pathway mediates the interaction between 2-adrenoceptors and Ang II on blood vessels. In this regard, PGSMCs express PLC-2, PLC-3, c-src, phospho-c-src, and the p85 subunit of PI3K. Thus, PGSMCs seem to express the cellular machinery necessary to support the mechanism illustrated in Fig. 1. Additional strong support for this mechanism is provided by the observations that angiotensin II and UK-14,304 per se do not increase phospho-c-src expression, yet the combination of angiotensin II plus UK-14,304 approximately doubles phospho-c-src expression. Moreover, this robust interaction is abolished by inhibition of either PLC or PKC. These findings are consistent with the hypothesis outlined in Fig. 1.
In the present study, all four inhibitors suppressed vascular responses to Ang II in the isolated, perfused kidney. This suggests that PI3K, c-src, and PLC/PKC participate in the vascular response to Ang II even in the absence of UK-14,304. It is possible that the reduction in basal responses to Ang II "non-specifically" prevented the potentiation of vascular responses to Ang II by UK-14,304. However, this seems unlikely for two reasons. First, in vivo the inhibitors blocked UK-14,304-induced potentiation of vascular responses to Ang II, yet the inhibitors, at the doses selected, did not alter basal vascular responses to Ang II. The reason for the lack of attenuation of basal vascular responses to Ang II by the inhibitors in vivo is most likely because higher concentrations of the inhibitors can be achieved in vitro. Such high levels cannot be achieved in vivo because of the systemic toxicity associated with recirculation of the inhibitors. Second, in the isolated perfused kidney, the inhibitors blocked the UK-14,304-induced enhancement of vascular responses to Ang II, even when the basal vascular responses were matched to equivalent vascular responses in the absence of the inhibitors. For example, in the absence of inhibitors, UK-14,304 potentiated vascular responses to 0.3 ng/kg/min Ang II. In the presence of inhibitors, 1 to 3 ng/kg/min Ang II was necessary to achieve the same vascular response elicited by 0.3 ng/kg/min. Yet, the inhibitors still completely blocked UK-14,304-induced enhancement of vascular responses to 1 and 3 ng/kg/min Ang II.
In the present investigation, we examined the role of the PLC/PKC/c-src/PI3K pathway in the interaction between 2-adrenoceptors and Ang II on renal vascular resistance changes in kidneys from spontaneously hypertensive rats. We selected this rat strain because our previous studies demonstrate that the interaction between 2-adrenoceptors and Ang II on vasoconstriction is particularly robust in the kidney of the spontaneously hypertensive rat (Jackson et al., 2001; Gao et al., 2003). This strategy maximized the magnitude of the phenomenon under study and therefore facilitated experimental manipulation.
Our previous studies demonstrate that the renovascular response to Ang II is enhanced in spontaneously hypertensive rats and that this may participate in the pathophysiology of genetic hypertension in this genetic model of hypertension (Li and Jackson, 1989; Kost and Jackson, 1993). Because the enhanced interaction between 2-adrenoceptors and Ang II on renovascular resistance may explain the greater renovascular response to Ang II in spontaneously hypertensive rats (Jackson et al., 2001; Gao et al., 2003), the fact that the PLC/PKC/c-src/PI3K pathway seems to be involved in the interaction between 2-adrenoceptors and Ang II suggests that defects, perhaps mutations, in this pathway may account for the greater renovascular response to Ang II and in part the hypertension in spontaneously hypertensive rats.
Studying the interaction between 2-adrenoceptors and Ang II in kidneys in vivo is challenging. To optimize conditions for observing the interaction, preactivation of 2-adrenoceptors and AT1 receptors by endogenous catecholamines and Ang II, respectively, must be inhibited so that the effects of an exogenous 2-adrenoceptor agonist and exogenous Ang II can be examined. This can be partially, but not completely, achieved. Furthermore, to study the role of the PLC/PKC/c-src/PI3K pathway in the interaction requires the intrarenal coadministration of three agents simultaneously, i.e., an 2-adrenoceptor agonist, Ang II, and inhibitors of the PLC/PKC/c-src/PI3K pathway. This is technically difficult because of the multiple intrarenal infusions that are required. Also, the systemic effects of the infused agents must be minimized; however, some recirculation of infused agents is unavoidable. Despite these difficulties, there is value in performing such experiments in vivo because the signal transduction systems are in their natural state. The aforementioned problems can be circumvented with in vitro experiments in the isolated perfused kidney. However, there is always concern regarding whether signal transduction systems remain in their natural state in vitro. In the present study, we compensated for the limitation of each method by conducting our experiments both in vivo and in vitro. Therefore, an important strength of the present study was that the inhibitors blocked the interaction between UK-14,304 and Ang II both in vivo and in vitro. The fact that similar conclusions were reached using both approaches provides some assurance that our conclusions were not biased by confounding variables in the experiments because each approach had a different set of confounding variables.
Because 2-adrenoceptors and AT1 receptors are located both postjunctionally and prejunctionally, it is important to address the site of the interaction between 2-adrenoceptors and Ang II in the kidney vasculature. Most likely the site of interaction is postjunctional. This conclusion is based on three considerations: 1) Prejunctional 2-adrenoceptors and AT1 receptors attenuate and augment, respectively, noradrenergic neurotransmission (Reid, 1992; Starke, 2001). Because prejunctional 2-adrenoceptors and AT1 receptors have opposing effects, a synergism at prejunctional sites is unlikely. 2) Although prejunctional 2-adrenoceptors and AT1 receptors can inhibit and augment, respectively, the neurotransmission process, these receptors have little or no effect on nonstimulated neurotransmitter release. In our in vivo experiments and in our experiments with isolated organs, the kidneys were denervated so that the renal sympathetic nerves were nonstimulated. 3) We observed a strong interaction between 2-adrenoceptors and Ang II on c-src activation in cultured PGSMCs, which do not have prejunctional receptors.
The sympathetic nervous system and renin-angiotensin system interact at multiple levels to regulate vascular tone and hence arterial blood pressure. Augmentation of any of these interactions could participate in the pathophysiology of high blood pressure. The present experiments suggest that the strong interaction between 2-adrenoceptors and Ang II on the vasculature involves coincidence signaling via the PLC/PKC/c-src/IP3K pathway. Therefore, alterations in the PLC/PKC/c-src/PI3K pathway may importantly influence the vascular effects of activation of the sympathetic nervous system and renin-angiotensin system and consequently may contribute to the pathophysiology of hypertension.
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Footnotes |
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ABBREVIATIONS: Ang II, angiotensin II; PLC, phospholipase C; PKC, protein kinase C; PI3K, phosphatidylinositol 3-kinase; MABP, mean arterial blood pressure; SHR, spontaneously hypertensive rat(s); RBF, renal blood flow; DMSO, dimethyl sulfoxide; RVR, renal vascular resistance; PGSMC, preglomerular microvascular smooth muscle cell; U-73122, 1-(6-((17)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl)-1H-pyrrole-2,5-dione; GF109203X, 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide; CGP77675 1-(2-{4-[4-amino-5-(3-methoxyphenyl)pyrrolo[2,3-d]pyrimidin-7-yl]phenyl}ethyl)piperidin-4-ol; UK-14,304, 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine.
Address correspondence to: Dr. Edwin K. Jackson, Center for Clinical Pharmacology, University of Pittsburgh School of Medicine, 100 Technology Dr., Suite 450, Pittsburgh, PA 15219-3130. E-mail: edj{at}pitt.edu
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