Analysis of AVP functions via V1a and V1b receptors with knockout - PowerPoint PPT Presentation

Analysis of AVP functions via V1a and V1b receptors with knockout mice Akito Tanoue Department of Pharmacology, National Research Institute for Child Health and Development

Arginine Arginine- -Vasopressin ( Vasopressin (AVP) AVP) is involved in regulating diverse functions -kidney kidney- - -pituitary pituitary- - Anti- Anti -diuresis diuresis ACTH secretion ACTH secretion -liver liver- - Glycogenolysis Glycogenolysis -Cardiovascular Cardiovascular- - Vasoconstriction Vasoconstriction -pancreas pancreas- - Insulin Insulin -platelet platelet- - secretion secretion Aggregation Aggregation -uterus uterus- - -adrenal adrenal- - Muscle contraction Muscle contraction Hormone secretion Hormone secretion These physiological effects of These physiological effects of AVP AVP are mediated via the AVP receptor subfamily. V1a V1a V1b V1b V2 V2 Subtype Subtype Expression site Expression site Vessels Vessels Anterior pituitary Anterior pituitary Renal tubules Renal tubules action action vasoconstriction vasoconstriction ACTH secretion ACTH secretion antidiuresis antidiuresis

Reduced Blood Pressure (BP) in V1aR- Reduced Blood Pressure (BP) in V1aR -KO KO SBP (mmHg) SBP (mmHg) MAP (mmHg) MAP (mmHg) Catecholamine Catecholamine AVP AVP * WT (18) WT (18) 12 12 12 12 V1aR V1aR- -KO (15) KO (15) 120 120 120 120 V1bR- V1bR -KO (14) KO (14) 8 8 ng/ml ng/ml pg/ml pg/ml * * 100 100 100 100 4 4 80 80 80 80 0 0 Pressor Response in Perfused Mesenteric Arterial Beds 60 60 60 60 40 40 pressure (mmHg) pressure (mmHg) Increase in Increase in 40 40 40 40 20 20 20 20 20 20 * 0 A VP (50 50 mM) 0 0 Koshimizu et al., Proc Natl Acad Sci U S A. 2006 1. The 1. The decreased BP in V1aR-KO mice could result from the could result from the decreased vascular tonus.

BP change in the hypertension model BP change in the hypertension model 150.0 150.0 Control (WT) (12) Control (WT) (12) 140.0 140.0 SBP (mmHg) SBP (mmHg) 130.0 130.0 V1aR V1aR- -KO (11) KO (11) 120.0 120.0 110.0 110.0 100.0 100.0 90.0 90.0 1% NaCl 1% NaCl 80.0 80.0 -8 -4 0 0 4 4 8 8 12 12 16 16 20 20 24 24 28 28 32 32 36 36 days after subtotal nephrectomy days after subtotal nephrectomy Rt. K Lt. K Lt. K Effect of the V1aR antagonist (10 μ g/kg, i.v.) Bladder Bladder on BP in the hypertensive mice Subtotal nephrectomy Subtotal nephrectomy ＊＊ 160 ◆ control (WT) (6) MAP (mmHg) 150 ■ V1aR-KO (11) 140 130 120 ＊ 110 (min) 0 2 4 6 8 10 The V1a receptor is involved in developing and/or maintaining hypertension, and blockade of the V1a receptor results in decreasing BP in the hypertensive mice.

BP and HR response to the AVP stimulation BP and HR response to the AVP stimulation 2. 2. A VP stimulates vasoconstriction via V1aR and also stimulates vasodilatation via V2R, and the decreased pressor response to the A VP stimulation in KO mice could result in the decreased BP .

Decreased sypatethic nerve activity in CNS of V1aR-KO BP and HR change after the AVP injection into Catecolamines after the A VP injection (icv) the intra-cereberoventricle (icv) Control (WT) Control (WT) 40 ng/mL MAP 15 Epinephrine(EP) Decreased EP Decreased EP (mmHg) * ∆ MAP and NE in KO and NE in KO * 20 Control (WT) 10 pg/mL V1aR-KO Control (WT) 5 0 10000 V1aR KO 0 100 HR Norepinephrine(NE) 3 5000 (bpm) ∆ HR * 50 2 * 1 0 0 Epi NE 0 AVP(100ng) 0 5 10 15 20 25 30 Vehicle 3 10 30 100 (min) AVP 10 ng (icv) AVP (ng) was injected into icv. Oikawa et al., Eur J Pharmacol. 2007 3. Decreased sympathetic nerve activity in response to A 3. VP could cause the decreased BP in KO mice.

Reduced Reduced Blood Volume and Plasma aldosterone level in V1aR in V1aR- -KO KO Blood volume (ml) Blood volume (ml) Aldosterone (ng/ml) Aldosterone (ng/ml) A A VP stimulates aldosterone release VP stimulates aldosterone release from adrenal gland cells via V1aR. from adrenal gland cells via V1aR. 3.5 3.5 * WT (5) WT (5) 6.0 6.0 V1aR V1aR- -KO (5) KO (5) 3.0 3.0 5.0 5.0 2.5 2.5 * 4.0 4.0 2.0 2.0 3.0 3.0 1.5 1.5 2.0 2.0 1.0 1.0 1.0 1.0 0.5 0.5 Impaired aldosterone release in Impaired aldosterone release in 0 V1aR- V1aR -KO adrenal cells KO adrenal cells Aoy agi et al., Endocrinology. 2007 0 Birumachi et al. Eur J Pharmacol. 2007 Birumachi et al. Eur J Pharmacol. 2007 4. A 4. VP-stimulated aldosterone release was impaired in V1aR-KO mice, and impaired aldosterone release could result in the lower plasma aldosterone level and consequent lower blood volume and BP .

Decreased plasma renin activity and angiotension II in V1aR-KO Aoyagi et al., Aoyagi et al., Amer J Physiol Amer J Physiol 2008 2008

Decreased renin expression in the kidney of V1aR-KO Number of renin positive cells in the kidney Renin RNA expressions in the kidney Immunohistochemistry with renin antibody in V1aR-KO Renin expressions in the kidney Aoyagi et al., Aoyagi et al., Amer J Physiol Amer J Physiol 2008 2008

Decreased expression of nNOS and COX-2 in the kidney of V1aR-KO Decreased expression of nNOS and COX-2 Regulation of renin secretion by NO and PGE2 in MD cells in the kidney of V1aR-KO Urinary Cl - concentration ↓ （ Macradensa cell ） nNOS, COX2 ↑ PGE2 levels in V1aR-KO Immunohistochemistry with COX2 and nNOS antibody NO, PGE 2 ↑ renin ↑ No. of nNOS positive cells COX2 expressions in the kidney in the kidney http://www.gik.gr.jp/~skj/ht/ht-kidney_p.php3 より V1aR is involved in regulating NOS and COX2, and decreased expressions cause the reduced renin production

Co-localization of nNOS with V1aR in Macradensa (MD) cell Expression of V1a receptor in the MD cell. The co-localization of the V1aR mRNA and nNOS were determined by in situ hybridization and immunostaining in kidney mirror sections. Arrowheads indicate MD cells, where the V1aR mRNA was co-localized with nNOS.

Co-localization of COX-2 with V1aR in MD cell Co-localization of COX-2 with V1aR in TAL Expression of V1a receptor in the MD cell. The co-localization of the V1aR mRNA and COX-2 were determined by in situ hybridization and immunostaining in kidney mirror sections. Arrowheads indicate MD cells, or renal tubule cells where the V1aR mRNA was co- localized with COX-2.

Co-localization of renin with V1aR in granule cells Co-localization of renin with V1aR in renal tubles Expression of V1a receptor in the MD cell. The co-localization of the V1aR mRNA and renin were determined by in situ hybridization and immunostaining in kidney mirror sections. Arrowheads indicate MD cells, or renal tubule cells where the V1aR mRNA was co- localized with renin.

Summary of AVP function on regulating RAS and blood volume ① V1aR mediates the renin production by regulating the nNOS and COX-2 in MD cells. ② ・ A VP-aldosterone system Which are impaired in V1aR-KO, leading to ・ Renin-angiotensin-aldosterone system decreased aldosterone and blood volume A VP regulates RAS via V1aR in the kidney Physiological role of AVP on regulating blood volume A VP A VP Angiotensinogen V1aR V1aR MD cell Distal tubule nNOS nNOS/NO V1aR Renin COX2 COX-2/PGE2 COX-2/PGE2 Angiotensin I granule cell Proximal tubule ACE renin renin Angiotensin II Aldosterone Na reabsorption Renin-angiotensin-aldosterone system Water retension A VP not only stimulates aldosterone release directly from adrenal cortex via the V1a receptor, but also regulates nNOS, COX2 and renin via the V1a receptor in the kidney.

Impaired glucose tolerance in V1aR Impaired glucose tolerance in V1aR- -KO mice KO mice Glucose Tolerance Test (GTT) Glucose Tolerance Test (GTT) 100 ＊ Fasting 18 h, Glucose 1.5 g/kg i.p Fasting 18 h, Glucose 1.5 g/kg i.p ＊ 350 1200 Blood glucose (mg/dl) Blood glucose (mg/dl) 80 WT, male (12) 300 Blood glucose (mg/dl) Blood glucose (mg/dl) 1000 V1aR-KO, male (10) 250 Insulin (pg/ml) Insulin (pg/ml) 60 800 200 600 40 150 400 100 20 200 50 0 0 0 0 30 60 90 120 0 30 60 90 120 Time (min) Time (min) Time (min Time (min Hyperinsulinemic-euglycemic clamp test Decreased phsophorylation of A kt in V1aR-KO Hiroyama et al., J Physiol Hiroyama et al., J Physiol 2007 2007 Glucose tolerance was impaired due to increased hepatic glucose production, and suppressed insulin signal in V1aR-KO.

BW, fat weight and glucose tolerance after feeding with the high fat diet Fat weight Total-WAT Subcutaneous Epididymal 30 Retroperitoneal Visceral *** -WAT & BAT -WAT -WAT -WAT eight eight) 25 adipose tissue w Normal chow w 20 ** HFD (% of body Body weight 15 ** 10 * * 5 0 WT KO WT KO WT KO WT KO WT KO Histology of fat and liver after loading with HF diet GTT after HF diet WT on HF V1aR-KO on HF WT on NC V1aR-KO on NC Aoyagi et al. Endocrinology 2007 Glucose intolerance was accelerated by the HF diet, leading to hyperglycemia, excessive obesity, and fatty liver in V1aR-KO.

Impaired insulin release from cultured islets in V1bR Impaired insulin release from cultured islets in V1bR- -KO KO AVP mediates the insulin secretion via the V1b receptors and AVP- stimulated insulin secretion was impaired in V1bR-KO.