| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Hypertension. 2005;45:705.)
© 2005 American Heart Association, Inc.
Original Articles |
From the Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico, Health Sciences Center, Albuquerque.
Correspondence to Nancy L. Kanagy, PhD, Vascular Physiology Group, Department of Cell Biology and Physiology, MSC 08-4750, 1 University of New Mexico, Albuquerque, NM 87131. E-mail nkanagy{at}salud.unm.edu
| Abstract |
|---|
|
|
|---|
Key Words: rats arteries sleep apnea syndromes
| Introduction |
|---|
|
|
|---|
Chronic elevations in circulating ET-1 have been demonstrated in other models of hypertension.6,7 However, in studies in which vascular sensitivity to ET-1 has been evaluated, increases in circulating ET-1 are accompanied by desensitization and internalization of ET receptors.8,9 We demonstrated previously that depressor responses to ET-1 antagonists in IH-exposed rats causes a profound depressor response that is not seen in Sham rats.5 Thus, it is possible that unlike other models of ET-1dependent hypertension, IH exposure leads to increases in ET-1 and ET-1vascular sensitivity. Furthermore, the sympathetic nervous system has been suggested to play a major role in IH-induced hypertension as well,10 so that increases in vascular sensitivity to adrenergic constriction might also be augmented. This study was therefore designed to examine vascular responses to the adrenergic agonist phenylephrine (PE), depolarization-induced vasoconstriction by KCl, and constriction to ET-1. We hypothesized that small mesenteric arteries from IH rats would have a selective increase in constrictor sensitivity to ET-1 caused by alterations in signaling unique from that mediated by PE and KCl.
| Methods |
|---|
|
|
|---|
70% are seen in patients with moderate to severe sleep apnea.11 In the current protocol, rats were exposed to PO2 <10% for approximately 1 minute of every 3-minute cycle. This level of hypoxia is similar to that used by other investigators to simulate IH in sleep apnea.12,13 Systolic blood pressure (SBP) and heart rate were recorded on days 0 and 14 to confirm the effect of the IH protocol on blood pressure. SBP was recorded before the start of the daily IH or airair exposure using a standard tailcuff apparatus (IITC). Body weight was also recorded to determine whether the exposure protocol altered weight gain. Approximately 16 hours after the final IH exposure, animals were deeply anesthetized with sodium pentobarbital (50 mg/kg) and mesenteric arteries collected for constrictor studies. Blood was also collected to measure hematocrit and hearts to measure left ventricle plus septum mass (left ventricle/body weight [LV/BWT]) as an index of systemic hypertension-induced hypertrophy. All animal protocols were reviewed and approved by the institutional animal care and use committee of the University of New Mexico School of Medicine and conform to National Institutes of Health guidelines for animal use.
Isolated Vessel Preparation
The intestinal arcade was removed and placed in a Silastic-coated Petri dish containing chilled physiological saline solution (PSS; [in mmol/L] 129.8 NaCl, 5.4 KCl, 0.83 MgSO4, 19 NaHCO3, 1.8 CaCl2, and 5.5 glucose). Fourth-order artery segments were dissected from the mesenteric vascular arcade and placed in fresh PSS oxygenated with normoxic gas (21% O2-6% CO2-73% N2). Cleaned arterioles were transferred to a vessel chamber (Living Systems), cannulated with glass micropipettes, and secured with silk ligatures. The vessels were slowly pressurized to 60 mm Hg with PSS using a servo-controlled peristaltic pump (Living Systems) and superfused with oxygenated 37°C PSS at a rate of 5 mL per minute.
Endothelium Removal
The endothelium was disabled in all experiments by passing 1 mL of air through the lumen. The integrity of the endothelium was assessed before and after denuding by exposing PE (10 µmol/L)constricted arterioles to acetylcholine (1 µmol/L). Acetylcholine-mediated vasodilation was eradicated in vessels successfully disabled.
Fura 2-Acetoxymethyl Ester Loading
After denuding, pressurized mesenteric arteries were loaded with the cell-permeable ratiometric Ca2+-sensitive fluorescent dye fura 2-acetoxymethyl ester (fura 2-AM; Molecular Probes). Fura 2-AM was dissolved in anhydrous dimethyl sulfoxide (DMSO; 1 mmol/L). Directly before loading, fura 2-AM was mixed with a 20% solution of pluronic acid in DMSO, then added to PSS yielding an end concentration of 2 µmol/L fura 2-AM and 0.05% pluronic acid. Pressurized arteries were incubated 45 minutes in the dark at room temperature in oxygenated fura 2-AM solution. After incubation, arteries were washed with 37°C PSS for 15 minutes to remove excess dye and allow complete esterification of the compound. Fura 2loaded vessels were alternately excited at 340 and 380 nm at a frequency of 10 Hz with an IonOptix Hyperswitch dual-excitation light source and the respective 510-nm emissions collected with a photomultiplier tube (F340/F380). Background-subtracted F340/F380 emission ratios were calculated with Ion Wizard software (IonOptix) and recorded continuously throughout the experiment with simultaneous measurement of inner diameter from bright-field images as described previously.14
Constrictor Studies
After determining baseline internal diameter and F340/F380, constrictions were produced by exposing arteries to increasing concentrations of ET-1 (1010 to 108 mol/L), PE (108 to 105 mol/L), or KCl (15 to 75 mmol/L) in the superfusion media. Vessels were exposed to each concentration of agonist for 5 minutes, and each artery was exposed to only 1 agonist. After exposure, arteries were superfused with Ca2+- free PSS to cause complete relaxation and to demonstrate that the constriction was caused by reversible active tone. Only vessels demonstrating complete dilation were used. Constrictions are expressed as percent baseline. Vessel wall [Ca2+] is expressed as F340/F380 ratio because the ratio is linearly related to true molar [Ca2+] when the dissociation constant of fura-2 does not differ between treatment groups.15
Western Blot Analysis
To determine whether IH alters expression of vascular ETA and ETB receptors, protein levels were evaluated as described previously.4 Briefly, mesenteric artery cascades from Sham and IH rats were cleaned of connective and adipose tissue, frozen in liquid nitrogen, and homogenized on ice in Tris-HCl buffer (pH 7.4) containing protease inhibitors. Homogenates were centrifuged 1500g at 4°C for 10 minutes to remove insoluble material. After determining supernatant protein concentration by the Bradford method (Pierce protein assay), 25 µg protein was separated in 4% to 20% gradient polyacrylamide gels (BioRad) and transferred to polyvinyldifluoride membranes. After blocking, membranes were incubated for 2 hours at 25°C, then overnight at 4°C with a rabbit monoclonal antibody specific for rat ETA (1:2500) or ETB (1:5000; Biodesigns, Inc.) in TBS with 0.05% Tween-20. After washing, blots were incubated for 1 hour with peroxidase-labeled goat anti-rabbit IgG (1:5000; Bio-Rad), followed by chemiluminescence labeling (enhanced chemiluminescence assay; Amersham). ETA and ETB band densities (SigmaGel; SPSS) were normalized to the total protein loaded per lane as determined by Coomassie staining of the membranes.
Statistical Analysis
Constriction and Ca2+ concentration response curves were analyzed using 2-way repeated-measures ANOVA with Student-NewmanKeuls post hoc analysis for differences between groups, concentrations, and interactions. Percent data were transformed to the square root of the arcsin before analysis to assure normalized data (SigmaStat Software; SPSS). Covariate analysis of fura-2 ratios and percentage vasoconstriction were further analyzed to determine the dependence of constriction on vessel wall [Ca2+]. Calculated slope and y-intercept values were analyzed using SASS statistical analysis software. P<0.05 was considered statistically significant for all analyses.
| Results |
|---|
|
|
|---|
|
Contractile Studies
Concentration-dependent responses to ET-1, PE, and KCl in IH and Sham arteries are expressed as percentage vasoconstriction from baseline, with 100% being a completely closed lumen. IH arteries had augmented constrictor sensitivity to ET-1 compared with Sham arteries demonstrated as a leftward shift in the curve (Figure 1A). PE and KCl constrictions were not different between IH and Sham arteries (Figure 1B and 1C).
|
Vessel Wall [Ca2+]
Changes in vessel wall [Ca2+] were determined as the F340/F380 measured simultaneously with inner diameter. In contrast to the difference in constriction for ET-1, F340/F380 were not different between groups (Figure 2A), although they did approach significance as indicated by the P values in Figure 2. Similarly, PE and KCl F340/F380 responses were not different between groups (Figure 2B and 2C). Interestingly, in the Sham arteries, all agents increased vessel wall [Ca2+] simultaneous with and parallel to vasoconstriction. However, in the IH arteries, only PE and KCl caused concentration-dependent increases in [Ca2+]. Thus, ET-1 constriction in IH arteries was not accompanied by changes in vessel wall [Ca2+], and the ET-1 constriction in the IH arteries appears to be caused by mechanisms independent of increases in [Ca2+].
|
Calcium Sensitivity
When vasoconstriction was plotted as a function of vessel wall [Ca2+], constriction at any given intracellular [Ca2+] was greater in IH arteries than in Sham arteries in the presence of ET-1 (Figure 3A). In contrast, constrictions to PE and KCl occurred in the presence of similar increases in [Ca2+] in the IH and Sham arteries. Thus, ET-1 constriction appears to rely more on increased calcium sensitivity in IH arteries than in Sham arteries, whereas PE and KCl produce constrictions with similar increases in [Ca2+] and calcium sensitivity in the 2 groups. This suggests IH exposure causes a specific alteration in the ET-1 signaling pathway.
|
Receptor Expression
To determine whether the increased constrictor response to ET-1 in IH mesenteric arteries was attributable to ET receptor upregulation, ETAR and ETBR receptor protein expression was evaluated. Western analysis using commercially distributed antibodies produced blots with a single band migrating at 63 kDa for ETAR and a doublet at 48 kDa for ETBR, as has been reported previously in vascular tissue.9 Densitometric analysis of blots normalized to the density of Coomassie blue staining indicated that expression of ETAR was increased, but expression of ETBR was not different between groups. These data suggest there is increased expression of ETAR protein but no change in ETBR protein in IH arteries (Figure 4). This increased receptor expression may explain in part the augmented ET-1 constriction.
|
| Discussion |
|---|
|
|
|---|
Key findings in this study are: (1) arteries from hypertensive IH rats have augmented vasoconstrictor responses to ET-1 compared with arteries from Sham rats; (2) when vasoconstriction is plotted as a function of vessel wall [Ca2+], IH arteries constrict more for any given [Ca2+] in the presence of ET-1; (3) IH arteries do not have augmented calcium sensitivity compared with Sham arteries in the presence of PE and KCl; and (4) IH arteries express more ETA receptors than Sham arteries. These data indicate that IH exposure selectively augments ET-1 vasoconstriction independent of increased calcium signaling and suggest IH causes a selective alteration in ET-1 signal transduction in vascular smooth muscle cells independent of calcium influx pathways and unique from signaling pathways used by PE and KCl.
Our observation that ETAR expression is increased in IH arteries compared with Sham although ETBR expression is unchanged indicates that IH exposure upregulates rather than downregulates ETAR expression. Glucocorticoids transcriptionally upregulate preproendothelin and ETAR.9 Therefore, stress-induced increases in glucocorticoid production during IH exposure may contribute to the elevated ET-1 and ETAR expression.3 However, ET-1 constriction in IH arteries is independent of increases in vessel wall [Ca2+], whereas that in Sham arteries is accompanied by increased [Ca2+]. Thus, the augmented response in the IH arteries does not appear to be caused by activation of more receptors. Rather, ETAR activates a different pathway in the IH arteries that is linked exclusively to elevated calcium sensitivity. These data suggest that the mechanism of ET-1mediated constriction is altered at a postreceptor site in the IH arteries.
ET-1 vasoconstrictor responses were compared with KCl and PE to clarify potential points of upregulation in the ET-1 signaling pathway. KCl, an electro-mechanocoupling agent, causes vasoconstriction by decreasing the driving force for K+, ultimately depolarizing vascular smooth muscle cells. This depolarization activates voltage-gated calcium channels leading to Ca2+ influx. Conversely, PE is a pharmaco-mechanocoupling agent that causes vasoconstriction by activating the G-proteincoupled
1-adrenergic receptor. Thus, a nonspecific increase in calcium sensitivity or in calcium channel function should augment all 3 responses, whereas an increase in receptor-activated second messenger responses common to ETA and
1 adrenergic receptors would augment PE and ET-1 constrictions. The increased sensitivity to only ET-1 indicates that the alteration in IH arteries is unique to the ET-1 pathway.
ETA receptors act primarily through Gq proteins and cause constriction by increasing intracellular [Ca2+] and vasoconstrictor sensitivity to calcium.1619 In the present study, ET-1 caused a concentration-dependent increase in [Ca2+]i that paralleled constriction in Sham arteries. However, in IH arteries, constriction occurred in the absence of an increase in [Ca2+]i. This suggests calcium handling is altered in the IH arteries. ET-1 increases [Ca2+]i by efflux from the sarcoplasmic reticulum and by influx through membrane channels.16,17,20,21 Furthermore, ET-1 can cause constriction by increasing calcium sensitivity via Rho kinase and protein kinase C activation, with subsequent inhibition of myosin light chain phosphatase.18 This study indicates that ET-1 constriction of arteries from IH rats is independent of [Ca2+]i changes. Thus, 14 days of exposure to IH alters ETAR signaling in small mesenteric arteries, increasing activation of calcium-sensitizing pathways and decreasing activation of Ca2+ influx. Furthermore, although similar intracellular signaling pathways can be activated by
1-adrenergic and ETA receptors,18,22 there is clearly an ET-1exclusive pathway that is altered by IH exposure. Although only 1 vascular bed was examined, small mesenteric arteries are good models of resistance arteries with dense innervation and significant contributions to systemic vascular resistance.23,24 Thus, selective vascular reactivity to ET-1 in resistance arteries may explain a portion of sleep apneainduced hypertension
Perspectives
IH-exposed rats have increased circulating ET-1 similar to that reported in sleep apnea patients,4 suggesting hypertension in IH rats simulates at least some aspects of this medical condition. Patients with sleep apnea also have elevated sympathetic activation, increased daytime blood pressure, and increased risk of cardiovascular morbidity. Thus, the novel observation in this study that increases in vascular sensitivity to ET-1 are present in an animal model of sleep apneainduced, ET-1dependent hypertension5 highlights the potential benefit of blocking this pathway to prevent the cardiovascular morbidity common in sleep apnea patients.2,25,26
| Acknowledgments |
|---|
Received October 12, 2004; first decision November 1, 2004; accepted December 9, 2004.
| References |
|---|
|
|
|---|
This article has been cited by other articles:
![]() |
P. Levy, J-L. Pepin, C. Arnaud, R. Tamisier, J-C. Borel, M. Dematteis, D. Godin-Ribuot, and C. Ribuot Intermittent hypoxia and sleep-disordered breathing: current concepts and perspectives Eur. Respir. J., October 1, 2008; 32(4): 1082 - 1095. [Abstract] [Full Text] [PDF] |
||||
![]() |
K. J. Allahdadi, T. W. Cherng, H. Pai, A. Q. Silva, B. R. Walker, L. D. Nelin, and N. L. Kanagy Endothelin type A receptor antagonist normalizes blood pressure in rats exposed to eucapnic intermittent hypoxia Am J Physiol Heart Circ Physiol, July 1, 2008; 295(1): H434 - H440. [Abstract] [Full Text] [PDF] |
||||
![]() |
T. V. Serebrovskaya, E. B. Manukhina, M. L. Smith, H. F. Downey, and R. T. Mallet Intermittent Hypoxia: Cause of or Therapy for Systemic Hypertension? Experimental Biology and Medicine, June 1, 2008; 233(6): 627 - 650. [Abstract] [Full Text] [PDF] |
||||
![]() |
S. de Frutos, L. Duling, D. Alo, T. Berry, O. Jackson-Weaver, M. Walker, N. Kanagy, and L. Gonzalez Bosc NFATc3 is required for intermittent hypoxia-induced hypertension Am J Physiol Heart Circ Physiol, May 1, 2008; 294(5): H2382 - H2390. [Abstract] [Full Text] [PDF] |
||||
![]() |
F. Lopez-Jimenez, F. H. Sert Kuniyoshi, A. Gami, and V. K. Somers Obstructive Sleep Apnea: Implications for Cardiac and Vascular Disease Chest, March 1, 2008; 133(3): 793 - 804. [Full Text] [PDF] |
||||
![]() |
K. J. Allahdadi, L. C. Duling, B. R. Walker, and N. L. Kanagy Eucapnic intermittent hypoxia augments endothelin-1 vasoconstriction in rats: role of PKC{delta} Am J Physiol Heart Circ Physiol, February 1, 2008; 294(2): H920 - H927. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. B. Snow, V. Kitzis, C. E. Norton, S. N. Torres, K. D. Johnson, N. L. Kanagy, B. R. Walker, and T. C. Resta Differential effects of chronic hypoxia and intermittent hypocapnic and eucapnic hypoxia on pulmonary vasoreactivity J Appl Physiol, January 1, 2008; 104(1): 110 - 118. [Abstract] [Full Text] [PDF] |
||||
![]() |
K. J. Allahdadi, B. R. Walker, and N. L. Kanagy ROK contribution to endothelin-mediated contraction in aorta and mesenteric arteries following intermittent hypoxia/hypercapnia in rats Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H2911 - H2918. [Abstract] [Full Text] [PDF] |
||||
![]() |
D. B. Zoccal, L. G. H. Bonagamba, F. R. T. Oliveira, J. Antunes-Rodrigues, and B. H. Machado Increased sympathetic activity in rats submitted to chronic intermittent hypoxia Exp Physiol, January 1, 2007; 92(1): 79 - 85. [Abstract] [Full Text] [PDF] |
||||
![]() |
G. E. Foster, M. J. Poulin, and P. J. Hanly Sleep Apnoea & Hypertension: Physiological bases for a causal relation: Intermittent hypoxia and vascular function: implications for obstructive sleep apnoea Exp Physiol, January 1, 2007; 92(1): 51 - 65. [Abstract] [Full Text] [PDF] |
||||
![]() |
C. J. Lai, C. C. H. Yang, Y. Y. Hsu, Y. N. Lin, and T. B. J. Kuo Enhanced sympathetic outflow and decreased baroreflex sensitivity are associated with intermittent hypoxia-induced systemic hypertension in conscious rats J Appl Physiol, June 1, 2006; 100(6): 1974 - 1982. [Abstract] [Full Text] [PDF] |
||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
Hypertension Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search Copyright © 2005 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited. |