Abstract-To evaluate the role of thromboxane in hypertension and its complications, we studied mice with targeted disruption of the TXA2 receptor gene in an angiotensin-II-dependent model of hypertension. To determine whether genetic background might alter the physiological actions of the TP receptor, we studied two lines of TP knockout (Tp Ϫ/Ϫ ) mice with distinct genetic backgrounds (C57BL/6 and BALB/c). During chronic angiotensin II infusion (1000 ng/kg per minute ϫ 28 days by subcutaneous osmotic pump), TP deficiency prevented mortality in the C57BL/6 background but not in the BALB/c strain. Chronic angiotensin II infusion also caused a rapid and significant increase in blood pressure in wild-type (WT) C57BL/6 and BALB/c animals, which was significantly attenuated in Tp Ϫ/Ϫ mice on either background. After 28 days of infusion, cardiac hypertrophy only occurred in the C57BL/6 strain: heart/body weight ratio increased by 57%Ϯ8% in WT mice compared with 17%Ϯ6.5% for the Tp Ϫ/Ϫ mice (PϽ0.01). Chronic angiotensin II infusion caused albuminuria only in the C57BL/6 strain, and TP deficiency did not alter its development. Cyclooxygenase-1 knockout mice also had attenuated blood pressure increase during chronic angiotensin II infusion, suggesting that cyclooxygenase-1 metabolites are involved in angiotensin-II-dependent hypertension. Thus, on the C57BL/6 background, TP receptors contribute to cardiac hypertrophy but not proteinuria. However, irrespective of genetic background, the TP receptor makes a robust contribution to the pathogenesis of angiotensin II-dependent hypertension. (TxA2) is produced by the metabolism of arachidonic acid through the cyclooxygenase-1 (COX1) and cyclooxygenase-2 pathway. TxA2 is a potent vasoconstrictor and platelet aggregate. 1 In addition, TxA2 regulates renal hemodynamics and sodium handling. [2][3][4] Based on its biological actions, TxA2 has been implicated in the pathogenesis of cardiovascular diseases including ischemic heart disease, 5 atherosclerosis, 6 and eclampsia, 7 although the precise COX isoform mediating these actions is still unclear. Interactions between TxA2 and the renin-angiotensin system have also been established. For example, angiotensin II (Ang II) stimulates TxA2 synthesis in vascular and renal tissues. 8,9 Moreover, there is evidence for common actions of Ang II and TxA2 to promote systemic and renal vasoconstriction, sodium handling, 10 and vascular smooth muscle cell proliferation. 11 These interactions suggest a potential contribution of TxA2 acting through the thromboxane A2 (TP) receptor to the pathogenesis of hypertension and its complications. Here, using a genetic approach, we examine the role of TP receptors and the COX1 pathway in a model of Ang-II-dependent hypertension and its cardiac and renal complications. Methods Establishment of the TP Receptor and COX1 Knockout Mice LinesThe thromboxane A2 (TP) knockout (Tp Ϫ/Ϫ ) mice were generated as previously described. 12 The TP mutation was backcrossed onto two different inbred genetic backgrounds for...

Specific inhibitors of COX-2 have been associated with increased risk for cardiovascular complications. These agents reduce prostacyclin (PGI2) without affecting production of thromboxane (Tx) A2. While this abnormal pattern of eicosanoid generation has been implicated in the development of vascular disease associated with COX-2 inhibition, its role in the development of hypertension, the most common cardiovascular complication associated with COX-2 inhibition, is not known. We report here that mice lacking the receptor for PGI2 (IPKOs) develop salt-sensitive hypertension, cardiac hypertrophy, and severe cardiac fibrosis. Coincidental deletion of the TxA2 (TP) receptor does not prevent the development of hypertension, but cardiac hypertrophy is ameliorated and fibrosis is prevented in IPTP double knockouts (DKOs). Thus, deletion of the IP receptor removes a constraint revealing adverse cardiovascular consequences of TxA2. Our data suggest that adjuvant therapy that blocks unrestrained Tx actions might protect against end-organ damage without affecting blood pressure in patients taking COX-2 inhibitors.

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The cytochrome P450 (CYP) enzymes participate in a wide range of biochemical functions, including metabolism of arachidonic acid and steroid hormones. Mouse CYP2J5 is abundant in the kidney where its products, the cis-epoxyeicosatrienoic acids (EETs), modulate sodium transport and vascular tone. To define the physiological role of CYP2J5 in the kidney, knockout mice were generated using a conventional gene targeting approach. Cyp2j5 (-/-) mice develop normally and exhibit no overt renal pathology. While renal EET biosynthesis was apparently unaffected by the absence of CYP2J5, deficiency of this CYP in female mice was associated with increased blood pressure, enhanced proximal tubular transport rates, and exaggerated afferent arteriolar responses to angiotensin II and endothelin I. Interestingly, plasma 17beta-estradiol levels were reduced in female Cyp2j5 (-/-) mice and estrogen replacement restored blood pressure and vascular responsiveness to normal levels. There was no evidence of enhanced estrogen metabolism, or altered expression or activities of steroidogenic enzymes in female Cyp2j5 (-/-) mice, but their plasma levels of luteinizing hormone and follicle stimulating hormone were inappropriately low. Together, our findings illustrate a sex-specific role for CYP2J5 in regulation of blood pressure, proximal tubular transport, and afferent arteriolar responsiveness via an estrogen-dependent mechanism.

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A family of orphan transporters has been discovered that are structurally related to the Na ؉ -Cl ؊ -dependent neurotransmitter transporters, including the dopamine transporter. One member of this family, the mouse XT2 gene, is predominantly expressed in the kidney and has 95% homology to rat ROSIT (renal osmotic stressinduced Na ؉ -Cl ؊ organic solute cotransporter). To study the physiological functions of this transporter, we generated XT2-knockout mice by gene targeting. XT2؊/؊ mice develop and survive normally with no apparent abnormalities. To attempt to identify potential substrates for XT2, we screened urine from XT2-knockout mice by high-pressure liquid chromatography and mass spectrometry and found significantly elevated concentrations of glycine. To study glycine handling, XT2؉/؉ and XT2 ؊/؊ mice were injected with radiolabeled glycine, and urine samples were collected to monitor glycine excretion. After 2 h, XT2؊/؊ mice were found to excrete almost twice as much glycine as the XT2 ؉/؉ controls (P ‫؍‬ 0.03). To determine whether the absence of the XT2 transporter affected sodium and fluid homeostasis, we measured systolic blood pressure by computerized tail-cuff manometry. Systolic blood pressure was significantly higher in XT2 ؊/؊ mice (127 ؎ 3 mmHg) than in wild-type controls (114 ؎ 2 mmHg; P < 0.001). This difference in systolic blood pressure was maintained on high and low salt feeding. To examine whether the alteration in blood pressure and the defect in glycine handling were related, we measured systolic blood pressure in the XT2 ؊/؊ mice during dietary glycine supplementation. Glycine loading caused systolic blood pressure to fall in the XT2 ؊/؊ mice from 127 ؎ 3 to 115 ؎ 3 mmHg (P < 0.001), a level virtually identical to that of the wild-type controls. These data suggest that the XT2 orphan transporter is involved in glycine reabsorption and that the absence of this transporter is sufficient to cause hypertension.The transport of hydrophilic substances across cell membranes is mediated by substrate-specific transporter proteins that can be classified into several families of related genes. Within these families, there are sequence homology and conservation of transmembrane domain topology. For example, the superfamily of sodium-and chloride-dependent transporter is comprised of transporters for neurotransmitters, amino acids, and organic osmolytes (11,31,36). Based on the homology of transmembrane domains with those of the neurotransmitter family, a group of putative transporters (17) has been identified that includes XT2/ROSIT (16, 33), XT3/rB21a (16, 27), NTT4/XT1 (4, 12), NTT5 (6), and NTT7/v7-3 (24, 32). However, studies in transfected cells and oocyte expression systems failed to determine conclusive substrate specificity (4, 16, 27) for this family of orphan transporters. Although sharing relatively high amino acid similarity (28 to 41%) with classical neurotransmitter transporter family members, these orphan transporters have larger second and fourth extracellular loops. In addition, rel...

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.

G protein-coupled receptors (GPCRs) play a key role in regulating bone remodeling. Whether GPCRs exert anabolic or catabolic osseous effects may be determined by the rate of receptor desensitization in osteoblasts. Receptor desensitization is largely mediated by direct phosphorylation of GPCR proteins by a family of enzymes termed GPCR kinases (GRKs). We have selectively manipulated GRK activity in osteoblasts in vitro and in vivo by overexpressing a GRK inhibitor. We found that expression of a GRK inhibitor enhanced parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor-stimulated cAMP generation and inhibited agonist-induced phosphorylation of this receptor in cell culture systems, consistent with attenuation of receptor desensitization. To determine the effect of GRK inhibition on bone formation in vivo, we targeted the expression of a GRK inhibitor to mature osteoblasts using the mouse osteocalcin gene 2 (OG2) promoter. Transgenic mice demonstrated enhanced bone remodeling as well as enhanced urinary excretion of the osteoclastic activity marker dexoypyridinoline. Both osteoprotegrin and OPG ligand mRNA levels were altered in calvaria of transgenic mice in a pattern that would promote osteoclast activation. The predominant effect of the transgene, however, was anabolic, as evidenced by an increase in bone density and trabecular bone volume in the transgenic mice compared with nontransgenic littermate controls.