Caveolin-1/Endothelial Nitric Oxide Synthase Interaction Is Reduced in Arteries From Pregnant Spontaneously Hypertensive Rats.

Jéssica A. Troiano,Simone R. Potje,Cristina Antoniali,Rita C. Tostes,Murilo E. Graton,Emily T. Gonçalves

doi:10.3389/fphys.2021.760237

Abstract

We have investigated the role caveolae/caveolin-1 (Cav-1) plays in endothelial nitric oxide synthase (eNOS) activation and how it impacts pregnancy-induced decreased vascular reactivity in normotensive (Wistar rats) and spontaneously hypertensive rats (SHR). Wistar rats and SHR were divided into non-pregnant (NP) and pregnant (P). Nitrite levels were assessed by the Griess method in the aorta and mesenteric vascular bed. In functional studies, arteries were incubated with methyl-β-cyclodextrin (dextrin, 10mmol/L), which disrupts caveolae by depleting cholesterol, and concentration-response curves to phenylephrine (PE) and acetylcholine (ACh) were constructed. Electronic microscopy was used to determine endothelial caveolae density in the aorta and resistance mesenteric artery in the presence of vehicle or dextrin (10mmol/L). Western blot was performed to evaluate Cav-1, p-Cav-1, calmodulin (CaM), and heat shock protein 90 (Hsp90) expression. Cav-1/eNOS interaction in the aorta and mesenteric vascular bed was assessed by co-immunoprecipitation. Nitric oxide (NO) generation was greater in arteries from P groups compared to NP groups. Dextrin did not change vascular responses in the aorta from P groups or the number of caveolae in P groups compared to NP groups. Compared to NP Wistar rats, NP SHR showed smaller number of caveolae and reduced Cav-1 expression. Pregnancy did not alter Cav-1, CaM, or Hsp90 expression in the aorta or mesenteric vascular bed from Wistar rats or SHR. These results suggest that pregnancy does not alter expression of the main eNOS regulatory proteins, but it decreases Cav-1/eNOS interaction. Reduced Cav-1/eNOS interaction in the aorta and mesenteric vascular bed seems to be an important mechanism to increase eNOS activity and nitric oxide production in pregnant normotensive and hypertensive rats.

Highlights

MATERIALS AND METHODSCaveolae are invaginations of the plasma membrane of cells, including endothelial cells, and they consist mainly of cholesterol, sphingolipids, and proteins (Severs, 1981; Yao et al, 2009), such as caveolin (Cav) isoforms
We evaluated the effect of dextrin, a cholesterol-depleting agent that disrupts caveolae, on vascular reactivity to PE and ACh and compared these effects for the aorta from NP Wistar rats, P Wistar rats, NP spontaneously hypertensive rats (SHR), and P SHR
Caveolae are an important structure in vascular cells AND most endothelial nitric oxide synthase (eNOS) is in these invaginations of the plasma membrane

Summary

Introduction

Caveolae are invaginations of the plasma membrane of cells, including endothelial cells, and they consist mainly of cholesterol, sphingolipids, and proteins (Severs, 1981; Yao et al, 2009), such as caveolin (Cav) isoforms. Cav-1 is an essential protein in structural caveolae formation, and it is involved in different signal transduction pathways (Razani and Lisanti, 2001; Patel et al, 2008). Cav-1 interacts directly with endothelial nitric oxide synthase (eNOS), keeping eNOS inactive by preventing it from interacting with calmodulin (CaM). Under conditions where specific stimuli increase intracellular calcium, CaM recognizes calcium ions and is activated, binding to and activating eNOS (GarcíaCardeña et al, 1996; Ju et al, 1997). Due to the Cav-1 inhibitory function on eNOS activity, Cav-1 knockout mice show greater eNOS activity in endothelial cells (Razani et al, 2001). CaM (Forstermann et al, 1991) and heat shock protein 90 (Hsp90) (García-Cardeña et al, 1998) positively regulate eNOS activity

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