The classically recognized functions of the renin-angiotensin system are mediated by type 1 (AT 1 ) angiotensin receptors. Whereas man possesses a single AT 1 receptor, there are two AT 1 receptor isoforms in rodents (AT 1A and AT 1B ) that are products of separate genes (Agtr1a and Agtr1b). We have generated mice lacking AT 1B (Agtr1b ؊͞؊) and both AT 1A and AT 1B receptors (Agtr1a ؊͞؊Agtr1b ؊͞؊). Agtr1b ؊͞؊ mice are healthy, without an abnormal phenotype. In contrast, Agtr1a ؊͞؊Agtr1b ؊͞؊ mice have diminished growth, vascular thickening within the kidney, and atrophy of the inner renal medulla. This phenotype is virtually identical to that seen in angiotensinogen-deficient (Agt؊͞؊) and angiotensin-converting enzyme-deficient (Ace ؊͞؊) mice that are unable to synthesize angiotensin II. Agtr1a ؊͞؊Agtr1b ؊͞؊ mice have no systemic pressor response to infusions of angiotensin II, but they respond normally to another vasoconstrictor, epinephrine. Blood pressure is reduced substantially in the Agtr1a ؊͞؊ Agtr1b ؊͞؊ mice and following administration of an angiotensin converting enzyme inhibitor, their blood pressure increases paradoxically. We suggest that this is a result of interruption of AT 2 -receptor signaling. In summary, our studies suggest that both AT 1 receptors promote somatic growth and maintenance of normal kidney structure. The absence of either of the AT 1 receptor isoforms alone can be compensated in varying degrees by the other isoform. These studies reaffirm and extend the importance of AT 1 receptors to mediate physiological functions of the renin-angiotensin system.The renin-angiotensin system (RAS) regulates blood pressure and body fluid balance and plays a role in growth and development (1-3). The biological functions of the RAS and its major effector peptide, angiotensin II, are mediated by specific receptors. Two angiotensin receptor subtypes, type 1 angiotensin receptor (AT 1 ) and AT 2 , can be distinguished pharmacologically. The classically recognized actions of the RAS are mediated by AT 1 receptors (1, 2). Whereas man possesses a single AT 1 receptor, rodents possess two AT 1 receptor isoforms, designated AT 1A and AT 1B . These receptors are the products of distinct but highly homologous genes (Agtr1a and Agtr1b) located on separate chromosomes (3, 4). Expression of the AT 1A receptor subtype predominates in nearly all tissues except the anterior pituitary gland and the adrenal cortex, where AT 1B receptors are more highly expressed (5-9). Because pharmacological AT 1 receptor antagonists block both AT 1A and AT 1B receptors, it has been difficult to separate their distinct functions (2).Experiments using gene targeting have provided insight into the roles of RAS genes in regulating blood pressure, body fluid homeostasis, and development. For example, mice that are unable to generate angiotensin II because of targeted mutations in the angiotensinogen (Agt Ϫ͞Ϫ) or angiotensinconverting enzyme (Ace Ϫ͞Ϫ) genes have virtually identical phenotypes characterized by reduced survival, l...

By inactivating the gene for L-gulono-␥-lactone oxidase, a key enzyme in ascorbic acid synthesis, we have generated mice that, like humans, depend on dietary vitamin C. Regular chow, containing about 110 mg͞kg of vitamin C, is unable to support the growth of the mutant mice, which require L-ascorbic acid supplemented in their drinking water (330 mg͞liter). Upon withdrawal of supplementation, plasma and tissue ascorbic acid levels decreased to 10 -15% of normal within 2 weeks, and after 5 weeks the mutants became anemic, began to lose weight, and die. Plasma total antioxidative capacities were approximately 37% normal in homozygotes after feeding the unsupplemented diet for 3-5 weeks. As plasma ascorbic acid decreased, small, but significant, increases in total cholesterol and decreases in high density lipoprotein cholesterol were observed. The most striking effects of the marginal dietary vitamin C were alterations in the wall of aorta, evidenced by the disruption of elastic laminae, smooth muscle cell proliferation, and focal endothelial desquamation of the luminal surface. Thus, marginal vitamin C deficiency affects the vascular integrity of mice unable to synthesize ascorbic acid, with potentially profound effects on the pathogenesis of vascular diseases. Breeding the vitamin C-dependent mice with mice carrying defined genetic mutations will provide numerous opportunities for systematic studies of the role of antioxidants in health and disease.

Microsomal and mitochondrial isoforms of glycerol-3-phosphate acyltransferase (GPAT; E.C. 2.3.1.15) catalyze the committed step in glycerolipid synthesis. The mitochondrial isoform, mtGPAT, was believed to control the positioning of saturated fatty acids at the sn-1 position of phospholipids, and nutritional, hormonal, and overexpression studies suggested that mtGPAT activity is important for the synthesis of triacylglycerol. To determine whether these purported functions were true, we constructed mice deficient in mtGPAT. mtGPAT ؊/؊ mice weighed less than controls and had reduced gonadal fat pad weights and lower hepatic triacylglycerol content, plasma triacylglycerol, and very low density lipoprotein triacylglycerol secretion. As predicted, in mtGPAT ؊/؊ liver, the palmitate content was lower in triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine. Positional analysis revealed that mtGPAT ؊/؊ liver phosphatidylethanolamine and phosphatidylcholine had about 21% less palmitate in the sn-1 position and 36 and 40%, respectively, more arachidonate in the sn-2 position. These data confirm the important role of mtGPAT in the synthesis of triacylglycerol, in the fatty acid content of triacylglycerol and cholesterol esters, and in the positioning of specific fatty acids, particularly palmitate and arachidonate, in phospholipids. The increase in arachidonate may be functionally significant in terms of eicosanoid production.

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A common polymorphrsm of the anglotenxm-convertmg enzyme (ACE) gene (ACE m humans, Ace m mice) 1s associated with differences m crrculatmg ACE levels that may confer a dlfferenttal rusk for cardiovascular diseases To study the effects of genetically deternnned changes m Ace gene functron wrthm a defined geneuc and environmental background, we have studied mice having one, two, or three functional copies of the Ace gene at its normal chromosomal location ACE actlvltles m the serum Increased progressively from 62% of normal In the one-copy ammals to 144% of normal m the three-copy animals (P< 10-'5, n= 132) The blood pressures of the mice havmg from one to three copies of the Ace gene did not differ slgmficantly, but the heart rates, heart weights, and renal tubulomterstltlal volumes decreased B lood pressure IS a complex trait determined by an array of mterlockmg homeostatrc systems with feedbacks that maintam homeostasis m the face of widely varying environmental factors In some mdlvrduals, however, a pathologrcal mteractron of genetrc and environmental factors results m hypertension, conferrmg increased risk for myocardial mfarctton, stroke, and renal failure A region of chromosome 10 was found to influence blood pressures m an F2 cross between stroke-prone spontaneously hypertensive rats and normotenstve Wlstar-Kyoto rats 1.2 Thts chromosomal region included the gene encoding ACE, a maJor enzyme of the renm-angtotensm and kalhkrem-kmm systems ACE catalyzes both the conversion of anglotensm I to the vasoconstnctmg pepttde angiotensm II and also the mactlvatton of the vasodllatmg pepttde bradykmm Inhtbttors of thts enzyme are commonly used to treat hypertension Thus, the ACE gene 1s a candidate gene for essential hypertension m humansTo study the genetics of hypertension, we have been mvestigatmg m mace the systemic effects of mduced mutations m candidate genes We have mserttonally mactivated the Ace gene and have observed that homozygous mutant mice that lack ACE have blood pressures reduced by -34 mm Hg compared with weld type 3 Key Words l genetlcs l heart rate l remn l miceThese data establish that the Ace gene 1s essenttal for maintenance of normal blood pressures A common polymorphtsm of the human ACE gene IS associated with quantitative differences m circulating ACE actrvrtres.4 To study the overall systemtc effects of quantitative genetic changes of thts type, we have described a strategy using gene targeting that mvolves the generation of one stram of mice canymg an macttvatton of the target gene and a second strain carrying a duphcatton of the target gene at its normal chromosomal location 5 Compared with normal mace that have two functtonal target genes, mace heterozygous for the inacttvatron have only one functional target gene and consequently a reduction u-r the overall level of target gene function, mice heterozygous for the duphcatron have three functional target genes and consequently an increase m the overall level of target gene function Thus, studies m race having one, two, and three...

CIB1 is a 22-kDa calcium binding, regulatory protein with ϳ50% homology to calmodulin and calcineurin B. CIB1 is widely expressed and binds to a number of effectors, such as integrin ␣IIb, PAK1, and polo-like kinases, in different tissues. However, the in vivo functions of CIB1 are not well understood. To elucidate the function of CIB1 in whole animals, we used homologous recombination in embryonic stem cells to generate Cib1 ؊/؊ mice. Although Cib1 ؊/؊ mice grow normally, the males are sterile due to disruption of the haploid phase of spermatogenesis. This is associated with reduced testis size and numbers of germ cells in seminiferous tubules, increased germ cell apoptosis, and the loss of elongated spermatids and sperm. Cib1؊/؊ testes also show increased mRNA and protein expression of the cell cycle regulator Cdc2/Cdk1. In addition, mouse embryonic fibroblasts (MEFs) derived from Cib1 ؊/؊ mice exhibit a much slower growth rate compared to Cib1 ؉/؉ MEFs, suggesting that CIB1 regulates the cell cycle, differentiation of spermatogenic germ cells, and/or differentiation of supporting Sertoli cells.

Polymorphisms in the human endothelial nitric oxide synthase (eNOS) gene ( NOS3 ) have been associated with advanced nephropathy in diabetic patients and with decreased expression in tissue culture. However, direct proof that modest genetic decreases in eNOS expression worsen diabetic nephropathy is lacking. To investigate this effect, we took advantage of the hybrid vigor and genetic uniformity of the F1 progeny (eNOS +/+ , eNOS +/− , or eNOS −/− with or without diabetes) of a cross between heterozygous 129S6/SvEvTac eNOS +/− inbred females and heterozygous C57BL/6J eNOS +/− inbred males carrying the dominant Akita diabetogenic mutation Ins2 C96Y/+ . Whereas all C57BL/6J inbred eNOS −/− and eNOS +/− diabetic mice died before 5 mo, almost half of the F1 hybrid eNOS −/− and eNOS +/− diabetic mice lived until killed at 7 mo. Heterozygous eNOS +/− diabetic mice expressed ∼35% eNOS mRNA in the kidney and ∼25% glomerular eNOS protein relative to their eNOS +/+ diabetic littermates. These decreases in eNOS elevated blood pressure (BP) but not blood glucose. Urinary albumin excretion, mesangial expansion, glomerulosclerosis, mesangiolysis, and glomerular filtration rate increased in the order: eNOS +/+ Akita < eNOS +/− Akita < eNOS −/− Akita, independently of BP. Glomerular basement membrane thickening depended on increased BP. Renal expression of tissue factor and other inflammatory factors increased with the nephropathy; Nos2 also increased. Surprisingly, however, decreased eNOS expression ameliorated the increases in oxidative stress and tubulointerstitial fibrosis caused by diabetes. Our data demonstrate that a modest decrease in eNOS, comparable to that associated with human NOS3 variants, is sufficient to enhance diabetic nephropathy independently of its effects on BP.

The Smith-Lemli-Opitz syndrome (SLOS) is a malformation/mental retardation syndrome resulting from an inborn error in 3beta-hydroxysteroid Delta7-reductase (DHCR7), the terminal enzyme required for cholesterol biosynthesis. Using a targeting strategy designed to virtually eliminate Dhcr7 activity, we have created a SLOS mouse model that exhibits commissural deficiencies, hippocampal abnormalities, and hypermorphic development of serotonin (5-HT) neurons. The latter is of particular interest with respect to current evidence that serotonin plays a significant role in autism spectrum disorders and the recent clinical observation that 50% of SLOS patients present with autistic behavior. Immunohistochemical analyses have revealed a 306% increase in the area of 5-HT immunoreactivity (5-HT IR) in the hindbrains of mutant (Dhcr7-/-) mice as compared to age-matched wild type animals. Amount of 5-HT IR was measured as total area of IR per histological section. Additionally, a regional increase as high as 15-fold was observed for the most lateral sagittal hindbrain sections. In Dhcr7-/- mice, an expansion of 5-HT IR into the ventricular zone and floor plate region was observed. In addition, the rostral and caudal raphe groups exhibited a radial expansion in Dhcr7-/- mice, with 5-HT IR cells present in locations not seen in wild type mice. This increase in 5-HT IR appears to represent an increase in total number of 5-HT neurons and fibers. These observations may help explain the behavioral phenotype seen in SLOS, and provide clues for future therapeutic interventions that utilize pharmacological modulation of the serotonergic system.