Increased availability of angiotensin AT 1 receptors leads to sustained arterial constriction to angiotensin II in diabetes - role for Rho-kinase activation

Abstract

Background and purpose: Antagonists of angiotensin AT(1) receptors elicit beneficial vascular effects in diabetes mellitus. We hypothesized that diabetes induces sustained availability of AT(1) receptors, causing enhanced arterial constriction to angiotensin II.

Experimental approach: To assess functional availability of AT(1) receptors, constrictions to successive applications of angiotensin II were measured in isolated skeletal muscle resistance arteries (∼150 µm) of Zucker diabetic fatty (ZDF) rats and of their controls (+/Fa), exposed acutely to high glucose concentrations (HG, 25 mM, 1 h). AT(1) receptors on cell membrane surface were measured by immunofluorescence.

Key results: Angiotensin II-induced constrictions to first applications were greater in arteries of ZDF rats (maximum: 82 ± 3% original diameter) than in those from +/Fa rats (61 ± 5%). Constrictions to repeated angiotensin II administration were decreased in +/Fa arteries (20 ± 6%), but were maintained in ZDF arteries (67 ± 4%) and in +/Fa arteries vessels exposed to HG (65 ± 6%). In ZDF arteries and in HG-exposed +/Fa arteries, Rho-kinase activities were enhanced. The Rho-kinase inhibitor, Y27632 inhibited sustained constrictions to angiotensin II in ZDF arteries and in +/Fa arteries exposed to HG. Levels of surface AT(1) receptors on cultured vascular smooth muscle cells (VSMCs) were decreased by angiotensin II but were maintained in VSMCs exposed to HG. In VSMCs exposed to HG and treated with Y27632, angiotensin II decreased surface AT(1) receptors.

Conclusions and implications: In diabetes, elevated glucose concentrations activate Rho-kinase which inhibits internalization or facilitates recycling of AT(1) receptors, leading to increased functional availability of AT(1) receptors and sustained angiotensin II-induced arterial constriction.

Figure 1

Original records (A,B) and summary data (C,D) of constrictions (%) to repeated applications of angiotensin II (Ang II; 10 pM–10 nM, n = 9) in skeletal muscle arteries from control heterozygous (+/Fa, n = 7) or Zucker diabetic fatty (ZDF, n = 7) rats. Data are means ± SEM. *P < 0.05, significantly different from first application. # indicate differences from control (+/Fa).

Figure 2

Original records (A,B) and summary data (C,D) of constrictions (%) to repeated applications of angiotensin II (Ang II; 10 pM–10 nM, n = 9) in skeletal muscle arteries from control heterozygous (+/Fa) rats in the presence of normal (5.5 mM, n = 5) or high glucose (25 mM, n = 5) concentration. Data are means ± SEM. *P < 0.05, significantly different from first application. # indicates difference from control (+/Fa).

Figure 3

Rho-kinase activity in skeletal muscle arteries obtained from control heterozygous (+/Fa, n = 4) or Zucker diabetic fatty (ZDF, n = 4) rats, or in arteries obtained from +/Fa rats in the presence of normal (5.5 mM, n = 4) or high glucose (25 mM, n = 4) concentration. Data are means ± SEM. *P < 0.05, significantly different from (A) Fa/+ or from (B) 5.5 mM glucose.

Figure 4

Summary data of constrictions (%) to repeated applications of angiotensin II (Ang II; 10 pM–10 nM, n = 9) in skeletal muscle arteries from +/Fa (n = 5) or Zucker diabetic fatty (ZDF, n = 5) rats (A,B) or arteries from +/Fa rats (n = 5) exposed to normal (5.5 mM glu) and high glucose (25 mM glu) concentration (C,D) in the presence of the Rho-kinase inhibitor, Y27632 (1 µM). Data are means ± SEM. *P < 0.05, significantly different from first application. Asterisks indicate significant differences (P < 0.05).

Figure 5

Detection of surface AT₁ receptors in vascular smooth muscle cells (VSMCs). Representative images (A) and summary data (B) show changes in extracellular AT₁ receptor immunofluorescence of VSMCs exposed to normal (5 mM) or high glucose (25 mM) concentrations, before and after stimulation with angiotensin II (Ang II; 100 nM, for 10 min) and in the absence and presence of Rho-kinase inhibitor, Y27632 (Y27; 10 µM). Images and data represent three separate experiments. Data are means ± SEM. *P < 0.05, significantly different from control (100%).