Stress upregulates arterial matrix metalloproteinase expression and activity via endothelin A receptor activation.

Degradation of the extracellular matrix proteins by matrix metalloproteinases (MMP) is an important regulatory step in the vascular remodeling process. Recent studies demonstrated that ETA receptors regulate cardiac MMP activity and fibrosis in DOCA-salt hypertension. However, little is known about endothelin (ET)-1 regulation of vascular MMP activity in hypertension. Thus early changes in ET-1-mediated regulation of MMP activity were measured in borderline hypertensive rats that develop impaired vasorelaxation and hypertension with chronic exposure to stress. Experiments were performed after 10 days of exposure to the behavioral stressor, air-jet stress, but before the onset of stress-induced hypertension. Study groups were 1) control (n = 8); 2) air-jet stress for 10 days (n = 8); 3) control plus ETA antagonist ABT-627 (n = 4), and 4) air-jet stress plus ETA antagonist (n = 4). MMP activity in the thoracic aorta was assessed by gelatin zymography. MMP protein and tissue ET-1 levels were evaluated by immunohistochemistry, and ET receptor density was determined by immunoblotting. Exposure to stress caused a twofold increase in plasma ET-1 levels (P < 0.05), and there was increased ET-1 staining at the tissue level. Total MMP activity and expression of MMP-2 and MMP-9 were increased in the stress group. ETA receptor antagonism prevented the increase in MMP expression and activation in the stress group. These results provide evidence that the MMP system is activated before the development of hypertension and ET-1 mediates these early events in vascular remodeling.

Decreased endothelin binding and [Ca2+]i signaling in microvessels of DOCA-salt hypertensive rats.

OBJECTIVES AND DESIGN: The deoxycorticosterone acetate (DOCA)-salt model of hypertension is characterized by elevated vascular endothelin-1 (ET-1) and by reduced contraction to ET-1 in isolated mesenteric small arteries. The decreased contraction to ET-1 may be a compensatory mechanism caused by elevations in ET-1 and arterial pressure. The present study was designed to determine whether down-regulation of endothelin receptors or altered Ca2+ signaling contribute to the decreased contraction to ET-1. METHODS AND RESULTS: Contraction to ET-1 (10 to 10 mol/l) was significantly reduced in isolated mesenteric small arteries (87-286 microm intraluminal diameter) from DOCA-salt rats compared with placebo rats. Membrane protein was obtained for measurement of [125I]ET-1 receptor binding and ET receptor expression. Maximum binding was significantly reduced in vascular membranes from DOCA-salt rats (670 +/- 71 fmol/mg protein) compared with placebo rats (1165 +/- 75 fmol/mg protein), but binding affinity was unchanged. Conversely, ETA receptor protein was increased in DOCA-salt rat vessels. To assess Ca2+ signaling, freshly dissociated mesenteric small artery smooth muscle cells were loaded with fura-2 for measurement of the average myoplasmic free Ca2+ concentration ([Ca2+ ] ). The ET-1 (10 mol/l) induced increase in [Ca2+ ] was significantly less in cells from DOCA-salt rats compared with from placebo rats. This effect was not due to a loss of L-type Ca2+ channels since expression was increased in membrane protein from DOCA-salt rats compared with placebo rats, as measured by Western blot analysis. CONCLUSIONS: These findings indicate that decreases in receptor binding and Ca2+ signaling contribute to the impaired contraction to ET-1 in DOCA-salt hypertensive rats. However, these changes are not due to reduced expression of ETA receptors or L-type Ca2+ channels.

Role of ET-1 receptor binding and [Ca(2+)](i) in contraction of coronary arteries from DOCA-salt hypertensive rats.

Hypertension is associated with an increase in coronary artery disease, but little is known about the regulation of coronary vascular tone by endothelin-1 (ET-1) in hypertension. The present study evaluated the mechanisms mediating altered contraction to ET-1 in coronary small arteries from deoxycorticosterone acetate (DOCA)-salt hypertensive rats. DOCA-salt rats exhibited an increase in systolic blood pressure and plasma ET-1 levels compared with placebo rats. Contraction to ET-1 (1 x 10(-11) to 3 x 10(-8) M), measured in isolated coronary small arteries maintained at a constant intraluminal pressure of 40 mmHg, was largely reduced in vessels from DOCA-salt rats compared with placebo rats. To determine the role of endothelin receptor binding in the impaired contraction to ET-1, (125)I-labeled ET-1 receptor binding was measured in membranes isolated from coronary small arteries. Maximum binding (fmol/mg protein) and binding affinity were similar in coronary membranes from DOCA-salt rats compared with placebo rats. Changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) were measured in freshly dissociated coronary small artery smooth muscle cells loaded with fura 2. ET-1 (10(-9) M) produced a 30 +/- 9% increase in [Ca(2+)](i) in smooth muscle cells from placebo rats, but had no effect on cells from DOCA-salt rats (2 +/- 2%). In summary, the ET-1-induced coronary artery contraction and increase in [Ca(2+)](i) are impaired in DOCA-salt hypertensive rats, whereas endothelin receptor binding is not altered. These results suggest endothelin receptor uncoupling from signaling mechanisms and indicate that impaired [Ca(2+)](i) signaling contributes to the decrease in ET-1-induced contraction of coronary small arteries in DOCA-salt hypertensive rats.

Potassium (BK(Ca)) currents are reduced in microvascular smooth muscle cells from insulin-resistant rats.

Insulin resistance (IR) syndrome is associated with impaired vascular relaxation; however, the underlying pathophysiology is unknown. Potassium channel activation causes vascular smooth muscle hyperpolarization and relaxation. The present study determined whether a reduction in large conductance calcium- and voltage-activated potassium (BK(Ca)) channel activity contributes to impaired vascular relaxation in IR rats. BK(Ca) channels were characterized in mesenteric microvessels from IR and control rats. Macroscopic current density was reduced in myocytes from IR animals compared with controls. In addition, inhibition of BK(Ca) channels with tetraethylammonium (1 mM) or iberiotoxin (100 nM) was greater in myocytes from control (70%) compared with IR animals (approximately 20%). Furthermore, activation of BK(Ca) channels with NS-1619 was three times more effective at increasing outward current in cells from control versus IR animals. Single channel and Western blot analysis of BK(Ca) channels revealed similar conductance, amplitude, voltage sensitivity, Ca2+ sensitivity, and expression density between the two groups. These data provide the first direct evidence that microvascular potassium currents are reduced in IR and suggest a molecular mechanism that could account for impaired vascular relaxation in IR.