Exploring type I angiotensin (AT1) receptor functions through gene targeting.

The renin-angiotensin system (RAS) modulates a diverse set of physiological processes including development, blood pressure, renal function and inflammation. The principal effector molecule of this system, angiotensin II, mediates most of these actions. The classically recognized functions of the RAS are triggered via the type 1 (AT(1)) class of angiotensin receptors. Pharmacological blockade of the AT(1) receptor lowers blood pressure and slows the progression of cardiovascular and renal diseases. Gene-targeting technology provides an experimental approach for precisely dissecting the physiological functions of the RAS. Here, we review how gene-targeting experiments have elucidated AT(1) receptor functions.

Expression of microsomal prostaglandin E synthase 1 in rheumatoid arthritis synovium.

OBJECTIVE: Microsomal prostaglandin E synthase 1 (mPGES-1) catalyzes the formation of PGE(2) from cyclooxygenase-derived PGH(2). Microsomal PGES-1 is induced by proinflammatory cytokines and is strongly linked to conditions that result in high PGE(2) biosynthesis. PGE(2) contributes to the pathogenesis of rheumatoid arthritis (RA), acting as a mediator of inflammation and promoting bone destruction. Induction of mPGES-1 in rheumatoid synoviocytes by proinflammatory cytokines has been demonstrated in vitro, indicating an important role in RA pathogenesis. Recent studies using mPGES-1-deficient mice demonstrated the importance of this gene in chronic inflammation. The aim of this study was to investigate the expression and localization of mPGES-1 in synovial biopsy specimens obtained from patients with RA. METHODS: Synovial tissue samples from 24 patients with RA were obtained, and immunohistologic analysis was performed using polyclonal antibodies against mPGES-1. Double immunofluorescence staining was performed with antibodies to CD3, CD19, CD20, CD68, CD163, and prolyl 4-hydroxylase. RESULTS: Intracellular mPGES-1 staining was observed in synovial membranes from all of the RA patients studied. Specifically, strong expression of mPGES-1 was detected in synovial lining cells. In sublining mononuclear and fibroblast-like cells, the extent of mPGES-1 staining was less than that in the synovial lining cells. In some patients, positive staining was observed in endothelial cells. With the double immunofluorescence technique, mPGES-1 production was detected in synovial macrophages and fibroblasts, while mPGES-1 expression was not observed in lymphocytes. CONCLUSION: The demonstration of mPGES-1 expression in synovial tissues from patients with RA suggests a role for mPGES-1 in the RA disease process. Microsomal PGES-1 might be a potential new target for treatment strategies to control PGE(2) synthesis in patients with RA, without the systemic side effects associated with cyclooxygenase inhibitors.

Deficiency of COX-1 causes natriuresis and enhanced sensitivity to ACE inhibition.

BACKGROUND: Prostanoid products of the cyclo-oxygenase (COX) pathway of arachidonic acid metabolism modulate blood pressure (BP) and sodium homeostasis. Conventional non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit both COX isoforms (COX-1 and -2), cause sodium retention, exacerbate hypertension, and interfere with the efficacy of certain anti-hypertensive agents such as angiotensin-converting enzyme (ACE) inhibitors. While a new class of NSAIDs that specifically inhibit COX-2 is now widely used, the relative contribution of the individual COX isoforms to these untoward effects is not clear. METHODS: To address this question, we studied mice with targeted disruption of the COX-1 (Ptgs1) gene. Blood pressure, renin mRNA expression, and aldosterone were measured while dietary sodium was varied. To study interactions with the renin-angiotensin system, ACE inhibitors were administered and mice with combined deficiency of COX-1 and the angiotensin II subtype 1A (AT1A) receptor were generated. RESULTS: On a regular diet, BP in COX-1-/- mice was near normal. However, during low salt feeding, BP values were reduced in COX-1-/- compared to +/+ animals, and this reduction in BP was associated with abnormal natriuresis despite appropriate stimulation of renin and aldosterone. Compared to COX-1+/+ mice, the actions of ACE inhibition were markedly accentuated in COX-1-/- mice. Sodium sensitivity and BP lowering also were enhanced in mice with combined deficiency of COX-1 and AT1A receptor. CONCLUSIONS: The absence of COX-1 is associated with sodium loss and enhanced sensitivity to ACE inhibition, suggesting that COX-1 inhibition does not cause hypertension and abnormal sodium handling associated with NSAID use.

The prostaglandin E2 EP1 receptor mediates pain perception and regulates blood pressure.

The lipid mediator prostaglandin E2 (PGE2) has diverse biological activity in a variety of tissues. Four different receptor subtypes (EP1-4) mediate these wide-ranging effects. The EP-receptor subtypes differ in tissue distribution, ligand-binding affinity, and coupling to intracellular signaling pathways. To identify the physiological roles for one of these receptors, the EP1 receptor, we generated EP1-deficient (EP1-/-) mice using homologous recombination in embryonic stem cells derived from the DBA/1lacJ strain of mice. The EP1-/- mice are healthy and fertile, without any overt physical defects. However, their pain-sensitivity responses, tested in two acute prostaglandin-dependent models, were reduced by approximately 50%. This reduction in the perception of pain was virtually identical to that achieved through pharmacological inhibition of prostaglandin synthesis in wild-type mice using a cyclooxygenase inhibitor. In addition, systolic blood pressure is significantly reduced in EP1 receptor-deficient mice and accompanied by increased renin-angiotensin activity, especially in males, suggesting a role for this receptor in cardiovascular homeostasis. Thus, the EP1 receptor for PGE2 plays a direct role in mediating algesia and in regulation of blood pressure.

Role of EP(2) and EP(3) PGE(2) receptors in control of murine renal hemodynamics.

The kidney plays a central role in long-term regulation of arterial blood pressure and salt and water homeostasis. This is achieved in part by the local actions of paracrine and autacoid mediators such as the arachidonic acid-prostanoid system. The present study tested the role of specific PGE(2) E-prostanoid (EP) receptors in the regulation of renal hemodynamics and vascular reactivity to PGE(2). Specifically, we determined the extent to which the EP(2) and EP(3) receptor subtypes mediate the actions of PGE(2) on renal vascular tone. Renal blood flow (RBF) was measured by ultrasonic flowmetry, whereas vasoactive agents were injected directly into the renal artery of male mice. Studies were performed on two independent mouse lines lacking either EP(2) or EP(3) (-/-) receptors and the results were compared with wild-type controls (+/+). Our results do not support a unique role of the EP(2) receptor in regulating overall renal hemodynamics. Baseline renal hemodynamics in EP(2)-/- mice [RBF EP(2)-/-: 5.3 +/- 0.8 ml. min(-1). 100 g kidney wt(-1); renal vascular resistance (RVR) 19.7 +/- 3.6 mmHg. ml(-1). min. g kidney wt] did not differ statistically from control mice (RBF +/+: 4.0 +/- 0.5 ml. min(-1). 100 g kidney wt(-1); RVR +/+: 25.4 +/- 4.9 mmHg. ml(-1). min. 100 g kidney wt(-1)). This was also the case for the peak RBF increase after local PGE(2) (500 ng) injection into the renal artery (EP(2)-/-: 116 +/- 4 vs. +/+: 112 +/- 2% baseline RBF). In contrast, we found that the absence of EP(3) receptors in EP(3)-/- mice caused a significant increase (43%) in basal RBF (7.9 +/- 0.8 ml. min(-1). g kidney wt(-1), P < 0.05 vs. +/+) and a significant decrease (41%) in resting RVR (11.6 +/- 1.4 mmHg. ml(-1). min. g kidney wt(-1), P < 0.05 vs. +/+). Local administration of 500 ng of PGE(2) into the renal artery caused more pronounced renal vasodilation in EP(3)-/- mice (128 +/- 2% of basal RBF, P < 0.05 vs. +/+). We conclude that EP(3 )receptors mediate vasoconstriction in the kidney of male mice and its actions are tonically active in the basal state. Furthermore, EP(3) receptors are capable of buffering PGE(2)-mediated renal vasodilation.

What can knockout mice contribute to an understanding of hypertension?

The generation of knockout mice using homologous recombination in embryonic stem cells is a powerful tool for physiologic investigations. This experimental approach has provided unique insights into the study of hypertension. Studies using knockout mice have shed new light on blood pressure regulatory mechanisms, molecular mechanisms of end-organ injury, and genetic mechanisms for hypertension. With the development of more accessible approaches for carrying out sophisticated manipulation of the mouse genome, there will be continuing utility of this technique for future studies of hypertension.

Insights into the functions of type 1 (AT1) angiotensin II receptors provided by gene targeting.

The renin-angiotensin system (RAS) has a wide range of actions in biological processes ranging from development and reproduction to cardiovascular and renal functions. Most of these actions are mediated by the octapeptide hormone angiotensin II. The identified family of angiotensin II receptors is divided into two pharmacological classes: type 1 (AT1) and type 2 (AT2). The classically recognized actions of the RAS are primarily mediated by the AT1 subtype of angiotensin receptors, and these receptors are the targets of a new class of anti-hypertensive agents. In recent years, our understanding of the physiological functions of AT1 receptors has been advanced through the use of gene-targeting technology. In this review, we will summarize the emerging picture of AT1 receptor functions that has been provided by gene-targeting experiments.

Cardiovascular responses to the isoprostanes iPF(2alpha)-III and iPE(2)-III are mediated via the thromboxane A(2) receptor in vivo.

BACKGROUND: Isoprostanes (iPs) are free radical-catalyzed products of arachidonic acid that reflect lipid peroxidation in vivo. Several iPs exert biological effects in vitro and may contribute to the functional consequences of oxidant stress. For example, iPF(2alpha)-III (8-iso PGF(2alpha)) and iPE(2)-III modulate platelet function and vascular tone. Although these effects are blocked by antagonists of the receptor (TP) for the cyclooxygenase product thromboxane A(2), it has been speculated that the iPs may activate a receptor related to, but distinct from, the TP. METHODS AND RESULTS: Transgenic mice (TPOEs) were generated in which the TP-beta isoform was under the control of the preproendothelin promoter. They overexpressed TP-beta in the vasculature but not in platelets and exhibited an exaggerated pressor response to infused iPF(2alpha)-III compared with wild-type mice. This was blocked by TP antagonism. The platelet response to the iP was unaltered in TPOEs compared with wild-type mice. By contrast, both the pressor response to iPF(2alpha)-III and its effects on platelet function were abolished in mice lacking the TP gene. This was also true of the effects of infused iPE(2)-III on mean arterial pressure and platelet aggregation. CONCLUSIONS: Both iPF(2alpha)-III and iPE(2)-III exert their effects on platelet function and vascular tone in vivo by acting as incidental ligands at membrane TPs rather than via a distinct iP receptor. Activation of TPs by iPs may be of importance in syndromes in which cyclooxygenase activation and oxidant stress coincide, such as in atherosclerosis and reperfusion after tissue ischemia.

Angiotensin II regulates cellular immune responses through a calcineurin-dependent pathway.

The renin-angiotensin system (RAS) is a key regulator of vascular tone and blood pressure. In addition, angiotensin II also has a number of cellular effects that may contribute to disease pathogenesis. Using Agtr1a(-/-) mice, which lack AT(1A) receptors for angiotensin II, we have identified a novel function of the RAS to modulate the immune system. We find that angiotensin II, acting through type 1 (AT(1)) receptors on immune cells, triggers the proliferation of splenic lymphocytes. These actions contribute to the vigor of cellular alloimmune responses. Within lymphoid organs, sufficient components of the RAS are present to activate AT(1) receptors during an immune response, promoting cell growth. These actions require activation of calcineurin phosphatase. In an in vivo model of cardiac transplantation, the absence of AT(1) signaling accentuates the immunosuppressive effects of the calcineurin inhibitor cyclosporine. We conclude that inhibition of AT(1) receptor signaling should be useful as an anti-inflammatory and immunosuppressive therapy. Furthermore, the actions of the RAS to promote lymphocyte activation may contribute to inflammation that characterizes a number of diseases of the heart and the vascular system.

Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice.

Platelet activation is characterized by shape change, induction of fibrinogen receptor expression and release of granular contents, leading to aggregation and plug formation. While this response is essential for hemostasis, it is also important in the pathogenesis of a broad spectrum of diseases, including myocardial infarction, stroke and unstable angina. Adenosine 5'-diphosphate (ADP) induces platelet aggregation, but the mechanism for this has not been established, and the relative contribution of ADP in hemostasis and the development of arterial thrombosis is poorly understood. We show here that the purinoceptor P2Y1 is required for platelet shape change in response to ADP and is also a principal receptor mediating ADP-induced platelet aggregation. Activation of P2Y1 resulted in increased intracellular calcium but no alteration in cyclic adenosine monophosphate (cAMP) levels. P2Y1-deficient platelets partially aggregated at higher ADP concentrations, and the lack of P2Y1 did not alter the ability of ADP to inhibit cAMP, indicating that platelets express at least one additional ADP receptor. In vivo, the lack of P2Y1 expression increased bleeding time and protected from collagen- and ADP-induced thromboembolism. These findings support the hypothesis that the ATP receptor P2Y1 is a principal receptor mediating both physiologic and pathological ADP-induced processes in platelets.

Identification of specific EP receptors responsible for the hemodynamic effects of PGE2.

To identify the E-prostanoid (EP) receptors that mediate the hemodynamic actions of PGE2, we studied acute vascular responses to infusions of PGE2 using lines of mice in which each of four EP receptors (EP1 through EP4) have been disrupted by gene targeting. In mixed groups of males and females, vasodepressor responses after infusions of PGE2 were significantly diminished in the EP2 -/- and EP4 -/- lines but not in the EP1 -/- or EP3 -/- lines. Because the actions of other hormonal systems that regulate blood pressure differ between sexes, we compared the roles of individual EP receptors in males and females. We found that the relative contribution of each EP-receptor subclass was strikingly different in males from that in females. In females, the EP2 and EP4 receptors, which signal by stimulating adenylate cyclase, mediate the major portion of the vasodepressor response to PGE2. In males, the EP2 receptor has a modest effect, but most of the vasodepressor effect is mediated by the phospholipase C-coupled EP1 receptor. Finally, in male mice, the EP3 receptor actively opposes the vasodepressor actions of PGE2. Thus the hemodynamic actions of PGE2 are mediated through complex interactions of several EP-receptor subtypes, and the role of individual EP receptors differs dramatically in males from that in females. These differences may contribute to sexual dimorphism of blood pressure regulation.

Reproductive failure and reduced blood pressure in mice lacking the EP2 prostaglandin E2 receptor.

Prostaglandins (PGs) are bioactive lipids that modulate a broad spectrum of biologic processes including reproduction and circulatory homeostasis. Although reproductive functions of mammals are influenced by PGs at numerous levels, including ovulation, fertilization, implantation, and decidualization, it is not clear which PGs are involved and whether a single mechanism affects all reproductive functions. Using mice deficient in 1 of 4 prostaglandin E2 (PGE2) receptors -- specifically, the EP2 receptor -- we show that Ep2(-/-) females are infertile secondary to failure of the released ovum to become fertilized in vivo. Ep2(-/-) ova could be fertilized in vitro, suggesting that in addition to previously defined roles, PGs may contribute to the microenvironment in which fertilization takes place. In addition to its effects on reproduction, PGE2 regulates regional blood flow in various vascular beds. However, its role in systemic blood pressure homeostasis is not clear. Mice deficient in the EP2 PGE2 receptor displayed resting systolic blood pressure that was significantly lower than in wild-type controls. Blood pressure increased in these animals when they were placed on a high-salt diet, suggesting that the EP2 receptor may be involved in sodium handling by the kidney. These studies demonstrate that PGE2, acting through the EP2 receptor, exerts potent regulatory effects on two major physiologic processes: blood pressure homeostasis and in vivo fertilization of the ovum.

Prostaglandin E-prostanoid-3 receptor activation of cyclic AMP response element-mediated gene transcription.

The prostaglandin E-prostanoid (EP)3 receptor signals primarily through the inhibitory G protein Gi, thereby decreasing intracellular cAMP levels. To study the signal transduction properties of the rabbit EP3 receptor, five splice variants were expressed in HEK293tsA201 cells: 72A, 74A, 77A, 80A and the novel splice variant NT, which lacks the C-terminal sequence. The ability of the EP3 receptor splice variants to modulate expression of a beta-galactosidase reporter gene under the control of a promoter containing cAMP response elements (CRE) was assessed. Each splice variant induced sulprostone-mediated increase in beta-galactosidase enzymatic activity with EC50 ranging from 0.8 nM for the NT splice variant to 3.1 nM for the 77A splice variant. Substitution of either Asp338 with Ala, or Arg329 with Ala or Glu in the 77A splice variant resulted in a loss of receptor-evoked increases in beta-galactosidase activity, whereas substitution of Lys300 with alanine had no effect on signal transduction. These phenotypes correlate with the inhibition of cAMP generation by direct cAMP measurement. Signal transduction was insensitive to pretreatment of cells with pertussis toxin, suggesting that a nonGi/Go pathway is activated by the EP3 receptor. Direct measurement of second messenger levels confirmed that there was no increase in cAMP levels mediated by the 77A splice variant, however, there was a modest increase in intracellular Ca2+. Partial blockade of the reporter activity with kinase inhibitors demonstrates that CRE activation is mediated in part by a Ca2+-dependent kinase pathway. These data suggest that the EP3 receptor signals through a novel cAMP response element binding protein/CRE pathway.

Regulation of glucose production by NEFA and gluconeogenic precursors during chronic glucagon infusion.

We previously reported that simulation of the chronic hyperglucagonemia seen during infection was unable to recreate the infection-induced increase in hepatic glucose production. However, chronic hyperglucagonemia was accompanied by a fall in the arterial levels of gluconeogenic precursors as opposed to a rise as is seen during infection. Thus our aim was to determine whether an infusion of gluconeogenic precursors could increase hepatic glucose production in a setting of hyperglucagonemia. Studies were done in 11 conscious chronically catheterized dogs in which sampling (artery and portal and hepatic veins) and infusion catheters (splenic vein) were implanted 17 days before study. Forty-eight hours before infusion of gluconeogenic (GNG) precursors, a sterile fibrinogen clot was placed into the peritoneal cavity. Glucagon was infused over the subsequent 48-h period to simulate the increased glucagon levels (approximately 500 pg/ml) seen during infection. On the day of the experiment, somatostatin was infused peripherally, and basal insulin and simulated glucagon were infused intraportally. After a basal period, a two-step increase in lactate and alanine was initiated (120 min/step; n = 5). Lactate (Delta479 +/- 25 and Delta1, 780 +/- 85 microM; expressed as change from basal in periods I and II, respectively) and alanine (Delta94 +/- 13 and Delta287 +/- 44 microM) levels were increased. Despite increases in net hepatic GNG precursor uptake (Delta0.7 +/- 0.3 and Delta1.1 +/- 0.4 mg glucose . kg-1 . min-1), net hepatic glucose output did not increase. Because nonesterified fatty acid (NEFA) levels fell, in a second series of studies, the fall in NEFA was eliminated. Intralipid and heparin were infused during the two-step substrate infusion to maintain the NEFA levels constant in period I and increase NEFA availability in period II (Delta -29 +/- 29 and Delta689 +/- 186 microM; n = 6). In the presence of similar increases in net hepatic GNG precursor uptake and despite increases in arterial glucose levels (Delta17 +/- 5 and Delta38 +/- 12 mg/dl), net hepatic glucose output increased (Delta0.6 +/- 0.1 and Delta0.7 +/- 0.2 mg . kg-1 . min-1). In summary, a chronic increase in glucagon, when combined with an acute increase in gluconeogenic precursor and maintenance of NEFA supply, increases hepatic glucose output as is seen during infection.