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To investigate the physiological role of the α1D-adrenergic receptor (α1D-AR) subtype, we created mice lacking the α1D-AR (α1D–/–) by gene targeting and characterized their cardiovascular function. In α1D–/– mice, the RT-PCR did not detect any transcript of the α1D-AR in any tissue examined, and there was no apparent upregulation of other α1-AR subtypes. Radioligand binding studies showed that α1-AR binding capacity in the aorta was lost, while that in the heart was unaltered in α1D–/– mice. Non-anesthetized α1D–/– mice maintained significantly lower basal systolic and mean arterial blood pressure conditions, relative to wild-type mice, and they showed no significant change in heart rate or in cardiac function, as assessed by echocardiogram. Besides hypotension, the pressor responses to phenylephrine and norepinephrine were decreased by 30–40% in α1D–/– mice. Furthermore, the contractile response of the aorta and the pressor response of isolated perfused mesenteric arterial beds to α1-AR stimulation were markedly reduced in α1D–/– mice. We conclude that the α1D-AR participates directly in sympathetic regulation of systemic blood pressure by vasoconstriction

Vasopressin receptor subtype(s) responsible for stimulation of insulin release from pancreatic ␤ cells were investigated by using subtype-selective antagonists and mice that were genetically lacking either V1a or V1b receptors. Arginine vasopressin (AVP) increased insulin release from isolated mouse islet cells in a concentration-dependent manner, with a submaximal response at 100 nM. Reverse transcription-polymerase chain reaction (RT-PCR) analysis detected V1b and oxytocin, but not V1a or V2, receptor transcripts in mouse islet cells. We characterized the recently synthesized vasopressin receptor subtype antagonists

The neurohypophyseal peptide [Arg8]-vasopressin (AVP) exerts major physiological actions through three distinct receptor isoforms designated V1a, V1b, and V2. Among these three subtypes, the vasopressin V1b receptor is specifically expressed in pituitary corticotrophs and mediates the stimulatory effect of vasopressin on ACTH release. To investigate the functional roles of V1b receptor subtypes in vivo, gene targeting was used to create a mouse model lacking the V1b receptor gene (V1bR–/–). Under resting conditions, circulating concentrations of ACTH and corticosterone were lower in V1bR–/– mice compared with WT mice (V1bR+/+). The normal increase in circulating ACTH levels in response to exogenous administration of AVP was impaired in V1bR–/– mice, while corticotropin-releasing hormone–stimulated ACTH release in the V1bR–/– mice was not significantly different from that in the V1bR+/+ mice. AVP-induced ACTH release from primary cultured pituitary cells in V1bR–/– mice was also blunted. Furthermore, the increase in ACTH after a forced swim stress was significantly suppressed in V1bR–/– mice. Our results clearly demonstrate that the V1b receptor plays a crucial role in regulating hypothalamic-pituitary-adrenal axis activity. It does this by maintaining ACTH and corticosterone levels, not only under stress but also under basal conditions

To study the functional role of individual ␣ 1 -adrenergic (AR) subtypes in blood pressure (BP) regulation, we used mice lacking the ␣ 1B -AR and/or ␣ 1D -AR with the same genetic background and further studied their hemodynamic and vasoconstrictive responses. Both the ␣ 1D -AR knockout and ␣ 1B -/ ␣ 1D -AR double knockout mice, but not the ␣ 1B -AR knockout mice, had significantly (p Ͻ 0.05) lower levels of basal systolic and mean arterial BP than wild-type mice in nonanesthetized condition, and they showed no significant change in heart rate or in cardiac function, as assessed by echocardiogram. All mutants showed a significantly (p Ͻ 0.05) reduced catecholamine-induced pressor and vasoconstriction responses. It is noteworthy that the infusion of norepinephrine did not elicit any pressor response at all in ␣ 1B -/␣ 1D -AR double knockout mice. In an attempt to further examine ␣ 1 -AR subtype, which is involved in the genesis or maintenance of hypertension, BP after salt loading was monitored by tail-cuff readings and confirmed at the endpoint by direct intra-arterial recording. After salt loading, ␣ 1B -AR knockout mice developed a comparable level of hypertension to wild-type mice, whereas mice lacking ␣ 1D -AR had significantly (p Ͻ 0.05) attenuated BP and lower levels of circulating catecholamines. Our data indicated that ␣ 1B -and ␣ 1D -AR subtypes participate cooperatively in BP regulation; however, the deletion of the functional ␣ 1D -AR, not ␣ 1B -AR, leads to an antihypertensive effect. The study shows differential contributions of ␣ 1B -and ␣ 1D -ARs in BP regulation.Catecholamines released from sympathetic nerve terminals cause vascular smooth muscle contraction primarily by activating ␣ 1 -adrenergic receptors (␣ 1 -ARs) in arteries (Hoffman, 2001). Thus, blockade of ␣ 1 -AR leads to a fall in peripheral vascular resistance. Because of their consistent effect in lowering systemic blood pressure (BP), ␣ 1 -AR blockers have been widely used as an antihypertensive drug. However, a large clinical trial unexpectedly disclosed that doxazosin, a nonselective ␣ 1 -AR antagonist, was associated with an increased incidence of heart failure (ALLHAT Collaborative Research Group, 2000). This raised a serious concern about the long-term use of ␣ 1 -AR antagonists in the treatment of hypertension (HT) (ALLHAT Collaborative Research Group, 2000). On the other hand, clinical efficacy of a subtypeselective inhibition of ␣ 1 -AR has not been fully determined.This work was supported in part by research grants from the Scientific Fund of the Ministry of Education, Science, and Culture of Japan, the Japan Health Science Foundation and Ministry of Human Health and Welfare.C.H. and T.K. contributed equally to this work. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.104.007500. ABBREVIATIONS: ␣

Abstract-In an attempt to elucidate whether there is a specific ␣ 1 -adrenergic receptor (␣ 1 -AR) subtype involved in the genesis or maintenance of hypertension, the ␣ 1D -AR subtype was evaluated in a model of salt-induced hypertension. The ␣ 1D -AR-deficient (␣ 1D Ϫ/Ϫ ) and control (␣ 1D ϩ/ϩ ) mice (nϭ8 to 14 in each group) were submitted to subtotal nephrectomy and given 1% saline as drinking water for 35 days. Blood pressure (BP) was monitored by tail-cuff readings and confirmed at the end point by direct intraarterial BP recording. The ␣ 1D Ϫ/Ϫ mice had a significantly (Pϭ0.0004) attenuated increase in BP response in this protocol (baseline 94.6Ϯ2.8 versus end point 107.4Ϯ4.5 mm Hg) compared with that of their wild-type counterparts (␣ 1D ϩ/ϩ ), from a baseline 97.4Ϯ2.9 to an end point 139.4Ϯ4.5 mm Hg. Seven of 15 ␣ 1D ϩ/ϩ mice died with edema, probably owing to renal failure, whereas 14 of 15 ␣ 1D Ϫ/Ϫ mice were maintained for 35 days. Body weight, renal remnant weight, and residual renal function were similar in the 2 groups, whereas the values of plasma catecholamines (epinephrine, norepinephrine, and dopamine) were higher in ␣ 1D ϩ/ϩ than in the ␣ 1D Ϫ/Ϫ mice. These data suggest that ␣ 1D -AR plays an important role in developing a high BP in response to dietary salt-loading, and that agents having selective ␣ 1D -AR antagonism could have significant therapeutic potential in the treatment of hypertension. (Hypertension. 2002;40:101-106.)

An analysis of arginine-vasopressin (AVP) V1a receptor-deficient (V1aR−/−) mice revealed that glucose homeostasis and lipid metabolism were altered in the mutant mice. Here, we used V1aR−/− mice to investigate whether the deficiency of the V1a receptor, which led to altered insulin sensitivity, affected protein metabolism. The serum 3-methylhistidine levels were increased in V1aR−/− mice under feeding conditions, indicating that proteolysis was enhanced in muscle tissue from V1aR−/− mice. Furthermore, serum amino acid profiling revealed that the amino acid levels, including glycogenic and branched-chain amino acids, were reduced in V1aR−/− mice. In addition, an alanine-loading test showed that gluconeogenesis was enhanced in V1aR−/− mice. Blood ammonia, which is a by-product of amino acid catabolism, was two times higher in V1aR−/− mice without hepatopathy under the feeding and fasting conditions than in wild-type mice. Amino acid profiling also revealed that the amino acid pattern was not typical of a urea-cycle enzymatic disorder. An ammonia tolerance test and an indocyanine green elimination test showed that V1aR−/− mice had lower ammonia clearance due to a decreased intrahepatic circulating blood volume. Metabolic acidosis, including lactic-and keto-acidosis, was not observed in V1aR−/− mice. These results provide evidence that proteolysis promotes the production of glucose in the muscles of V1aR−/− mice and that hyperammonaemia is caused by promoted protein catabolism and reduced intrahepatic blood volume. Thus, our study with V1aR−/− mice indicates that AVP plays a physiological role via the V1a receptor in regulating both protein catabolism and glucose homeostasis.