SLIDE 1
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
SLIDE 2 V1a V1a V1b V1b V2 V2 Expression site Expression site action action Vessels Vessels vasoconstriction vasoconstriction Anterior pituitary Anterior pituitary ACTH secretion ACTH secretion Renal tubules Renal tubules antidiuresis antidiuresis Subtype Subtype
pituitary-
ACTH secretion
uterus-
Muscle contraction
liver-
Glycogenolysis
kidney-
Anti-
diuresis
platelet-
Aggregation
Cardiovascular-
Vasoconstriction
pancreas-
Insulin secretion secretion
adrenal-
Hormone secretion
Arginine Arginine-
Vasopressin (AVP) AVP) is involved in regulating diverse functions
These physiological effects of These physiological effects of AVP AVP are mediated via the AVP receptor subfamily.
SLIDE 3 SBP (mmHg) SBP (mmHg) MAP (mmHg) MAP (mmHg)
80 80 120 120 100 100 60 60 40 40 20 20 *
WT (18) WT (18) V1aR V1aR-
KO (15) V1bR V1bR-
KO (14)
* 100 100 120 120 80 80 60 60 40 40 20 20
Pressor Response in Perfused Mesenteric Arterial Beds
Reduced Blood Pressure (BP) in V1aR Reduced Blood Pressure (BP) in V1aR-
KO
Increase in Increase in pressure (mmHg) pressure (mmHg) 20 20 40 40 A VP (50 50 mM)
*
8 12 12 4 ng/ml ng/ml
Catecholamine Catecholamine AVP AVP
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.
8 12 12 4 pg/ml pg/ml
*
SLIDE 4 BP change in the hypertension model BP change in the hypertension model
80.0 80.0 90.0 90.0 100.0 100.0 110.0 110.0 120.0 120.0 130.0 130.0 140.0 140.0 150.0 150.0
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 Control (WT) (12) Control (WT) (12) V1aR V1aR-
KO (11) SBP (mmHg) SBP (mmHg)
Bladder Bladder
1% NaCl 1% NaCl
2 4 6 8 10 160 150 140 130 120 110 MAP (mmHg) ＊＊ ＊ ◆ control (WT) (6) ■ V1aR-KO (11) (min)
Effect of the V1aR antagonist (10μg/kg, i.v.)
- n BP in the hypertensive mice
Subtotal nephrectomy Subtotal nephrectomy
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.
SLIDE 5 2.
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 .
BP and HR response to the AVP stimulation BP and HR response to the AVP stimulation
SLIDE 6 BP and HR change after the AVP injection into the intra-cereberoventricle (icv) AVP 10 ng (icv) was injected into icv. V1aR-KO 20 40
∆MAP (mmHg)
Control (WT) MAP 50 100
∆HR (bpm)
5 10 15 20 25 30 (min) HR Catecolamines after the A VP injection (icv) ng/mL AVP (ng) 5 10 15 1 2 3 Vehicle 3 10 30 100 * * Epinephrine(EP) Norepinephrine(NE)
5000 10000
Epi NE
pg/mL * * AVP(100ng) 3.
- 3. Decreased sympathetic nerve activity in response to A
VP could cause the decreased BP in KO mice.
Control (WT) V1aR KO
Control (WT) Control (WT) Decreased EP Decreased EP and NE in KO and NE in KO
Oikawa et al., Eur J Pharmacol. 2007
Decreased sypatethic nerve activity in CNS of V1aR-KO
SLIDE 7 Reduced Reduced Blood Volume and Plasma aldosterone level in V1aR in V1aR-
KO Blood volume (ml) Blood volume (ml)
*
2.0 2.0 3.0 3.0 2.5 2.5 1.5 1.5 1.0 1.0 0.5 0.5 3.5 3.5 4.0 4.0 6.0 6.0 5.0 5.0 3.0 3.0 2.0 2.0 1.0 1.0
WT (5) WT (5) V1aR V1aR-
KO (5)
Aoy agi et al., Endocrinology. 2007
*
Aldosterone (ng/ml) Aldosterone (ng/ml)
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 .
Birumachi et al. Birumachi et al. Eur J Pharmacol. 2007 Eur J Pharmacol. 2007
Impaired aldosterone release Impaired aldosterone release in in V1aR V1aR-
KO adrenal cells A VP stimulates aldosterone release A VP stimulates aldosterone release from adrenal gland cells via V1aR. from adrenal gland cells via V1aR.
SLIDE 8
SLIDE 9 Decreased plasma renin activity and angiotension II in V1aR-KO
Aoyagi et al., Aoyagi et al., Amer J Physiol Amer J Physiol 2008 2008
SLIDE 10 Decreased renin expression in the kidney of V1aR-KO
Immunohistochemistry with renin antibody in V1aR-KO Number of renin positive cells in the kidney Renin RNA expressions in the kidney Renin expressions in the kidney
Aoyagi et al., Aoyagi et al., Amer J Physiol Amer J Physiol 2008 2008
SLIDE 11 Regulation of renin secretion by NO and PGE2 in MD cells
http://www.gik.gr.jp/~skj/ht/ht-kidney_p.php3より
NO, PGE2 ↑ renin↑ nNOS, COX2 ↑ Urinary Cl-concentration↓ （Macradensa cell）
Immunohistochemistry with COX2 and nNOS antibody
- No. of nNOS positive cells
in the kidney COX2 expressions in the kidney PGE2 levels in V1aR-KO
V1aR is involved in regulating NOS and COX2, and decreased expressions cause the reduced renin production
Decreased expression of nNOS and COX-2 in the kidney of V1aR-KO
Decreased expression of nNOS and COX-2 in the kidney of V1aR-KO
SLIDE 12
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 nNOS with V1aR in Macradensa (MD) cell
SLIDE 13
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 COX-2 with V1aR in MD cell Co-localization of COX-2 with V1aR in TAL
SLIDE 14
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.
Co-localization of renin with V1aR in granule cells Co-localization of renin with V1aR in renal tubles
SLIDE 15 ① V1aR mediates the renin production by regulating the nNOS and COX-2 in MD cells. ② ・A
VP-aldosterone system ・Renin-angiotensin-aldosterone system
Summary of AVP function on regulating RAS and blood volume
Which are impaired in V1aR-KO, leading to decreased aldosterone and blood volume A VP regulates RAS via V1aR in the kidney A VP V1aR Renin Angiotensinogen Angiotensin I Angiotensin II Aldosterone ACE Water retension nNOS COX2
Physiological role of AVP on regulating blood volume
Na reabsorption A VP nNOS/NO COX-2/PGE2 COX-2/PGE2
MD cell Distal tubule
renin
granule cell
renin
Proximal tubule
Renin-angiotensin-aldosterone system
V1aR V1aR
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.
SLIDE 16 Impaired glucose tolerance in V1aR Impaired glucose tolerance in V1aR-
KO mice
Glucose Tolerance Test (GTT) Glucose Tolerance Test (GTT)
Fasting 18 h, Glucose 1.5 g/kg i.p Fasting 18 h, Glucose 1.5 g/kg i.p 50 100 150 200 250 300 350 30 60 120 Blood glucose (mg/dl) Blood glucose (mg/dl) 90 Time (min) Time (min) 200 400 600 800 1000 1200 Insulin (pg/ml) Insulin (pg/ml) 30 60 120 90 WT, male (12) V1aR-KO, male (10) Time (min Time (min
Hiroyama et al., Hiroyama et al., J Physiol J Physiol 2007 2007
Hyperinsulinemic-euglycemic clamp test
Decreased phsophorylation of A kt in V1aR-KO
Glucose tolerance was impaired due to increased hepatic glucose production, and suppressed insulin signal in V1aR-KO.
20 40 60 80 100 Blood glucose (mg/dl) Blood glucose (mg/dl)
＊ ＊
SLIDE 17 BW, fat weight and glucose tolerance after feeding with the high fat diet
Histology of fat and liver after loading with HF diet GTT after HF diet
Aoyagi et al. Endocrinology 2007 WT on NC V1aR-KO on NC WT on HF V1aR-KO on HF
Body weight
Subcutaneous
Total-WAT Epididymal
Retroperitoneal
Visceral
adipose tissue w eight (% of body w eight)
5 10 15 20 25 30 ** * WT KO * WT KO WT KO ** WT KO *** WT KO
Normal chow HFD
Fat weight
Glucose intolerance was accelerated by the HF diet, leading to hyperglycemia, excessive obesity, and fatty liver in V1aR-KO.
SLIDE 18 Impaired insulin release from cultured islets in V1bR Impaired insulin release from cultured islets in V1bR-
KO
AVP mediates the insulin secretion via the V1b receptors and AVP- stimulated insulin secretion was impaired in V1bR-KO.
SLIDE 19 Increased insulin sensitivity in V1bR Increased insulin sensitivity in V1bR-
KO mice
20 40 60 80 100 120
V1bR-KO (25) WT (16)
Blood glucose (mg/dl) Blood glucose (mg/dl)
V1aR-KO (20)
＊ ＊
GTT under the HF diet condition GTT under the HF diet condition GTT GTT
○ V1bR+/+ V1bR+/+ ■ V1bR V1bR-/-
ITT
○ V1bR+/+ V1bR+/+ ■ V1bR V1bR-/-
Fujiwara et al., J Physiol. 2007 J Physiol. 2007
Insulin sensitivity was enhanced in V1bR-KO due to the enhanced insulin signal in adipocytes.
SLIDE 20
Decreased insulin sensitivity in V1abR-KO
GTT on NC diet
V1abR-KO WT
GTT on high fat diet
V1abR-KO WT WT WT V1abR-KO V1abR-KO
ITT on NC diet ITT on high fat diet Insulin sensitivity was decreased in V1abR-KO, similar to V1aR-KO.
SLIDE 21
Decreased phosphorylation of Akt by the insulin stimulation in V1abR KO
Hiroyama et al., 2007 Fujiwara et al., 2007
Insulin sensitivity was decreased in V1abR-KO due to the suppressed insulin signal similar to V1aR-KO.
SLIDE 22
V1aR-KO and V1abR-KO are pre-diabetic on NC diet and diabetic on HF diet
Classification of glucose intolerance in V1aR-KO or V1abR KO mice
SLIDE 23
AVP V1a receptor V1b receptor Water recruitment aldosteron release Insulin signal
(Akt Phosphorylation)
Glucose tolerance Hepatic glucose production insulin release
Possible mechanism for regulating the glucose tolerance by AVP
Aoyagi et al., Endocrinology 2007 modified
renin
A VP regulates the insulin secretion from the pancreas and insulin signal in fat via the V1b receptor, and A VP regulates hepatic glucose production, insulin signal and aldosteron secretion via the V1a receptor, which consequently affect the glucose tolerance in vivo.
SLIDE 24 Acknowledgement Acknowledgement
Kyoto University Kyoto University ・Prof. Tsujimoto
・Dr. Koshimizu
Tokyo University of Pharmacy and Life Science
・Prof. Takeo
・Dr.Nasa Dr.Nasa ・Dr. Oikawa
Kitasato University Kitasato University ・Prof. Kawahara
・Dr. Yasuoka
Kumamoto University Kumamoto University ・Dr. Nonoguchi
・Dr. Izumi
