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Department of Cell Biology and Physiology, Vascular Physiology Group, University of New Mexico, Health Sciences Center, Albuquerque, New Mexico
Submitted 31 October 2007 ; accepted in final form 7 December 2007
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-selective inhibitor (rottlerin, 3 µM) significantly decreased ET-1-mediated constriction in E-IH but not in sham sMA. ET-1 increased PKC phosphorylation in E-IH but not sham sMA. In contrast, ET-1 constriction in thoracic aorta from both sham and E-IH rats was inhibited by Gö-6976 but not by rottlerin. These observations support our hypothesis that E-IH exposure significantly increases ET-1-mediated constriction of sMA through PKC activation and modestly augments ET-1 contraction in thoracic aorta through activation of one or more cPKC isoforms. Therefore, upregulation of a PKC pathway may contribute to elevated ET-1-dependent vascular resistance in this model of hypertension.
sleep apnea; intermittent hypoxia; hypercapnia; endothelin-1; protein kinase C ; vascular smooth muscle cell; mesenteric arteries
In a previous study, we observed that the augmented constriction to ET-1 in arteries from E-IH rats is not accompanied by greater increases in vascular wall Ca2+ concentration ([Ca2+]) (1). This suggests that E-IH exposure increases ET-1-dependent vasoconstriction by elevating Ca2+-sensitization rather than increased Ca2+ signaling.
Ca2+ sensitization increases vascular tone by elevating phosphorylation of the myosin regulatory light chain (MLC20) independent of changes in intracellular [Ca2+]. The degree of MLC20 phosphorylation is determined by the activities of both Ca2+-activated MLC kinase (MLCK) and Ca2+-independent MLC phosphatase (MLCP). Therefore, constriction can be initiated by phosphorylation of myosin by MLCK and/or by MLCP inhibition (38). Activation of both Rho-associated kinase (ROK) and protein kinase C (PKC) can lead to MLCP inhibition. Indeed some recent studies suggest elevated ROK activation contributes to augmented vasoconstriction in hypertension. However, we recently reported that ROK contributes to ET-1-induced vasoconstriction in the thoracic aorta but not in resistance-sized mesenteric arteries (2). Furthermore, most studies of ROK regulation of vasoconstriction have been conducted in large arteries or using agonists other than ET-1. In contrast, PKC may play a more important role than ROK in mediating ET-1-dependent Ca2+ sensitization and constriction in smaller arteries (5). Therefore, this study investigated the hypothesis that increased PKC-induced Ca2+ sensitization is responsible for augmented ET-1 vasoconstriction following exposure to E-IH.
PKC is a large family of serine/threonine kinases activated by many agonists, including ET-1 (10, 33). The 13 isoforms of PKC are grouped into three subclasses; classical PKC (cPKC) isoforms (
, βI, βII, and 
) requiring both Ca2+ and diacylglycerol (DAG) for activation; novel PKC (nPKC) isoforms (, 
, 
, and 
) requiring only DAG for activation; and atypical PKC isoforms (
, µ, and 
) that are activated by binding phosphatidylserine but not DAG or Ca2+ (17). Multiple PKC isoforms can inhibit MLCP indirectly by phosphorylating and activating PKC-potentiated inhibitory protein (CPI-17) (6). Based on our previous observation that augmented ET-1 vasoconstriction in E-IH arteries is not accompanied by increased intracellular [Ca2+] (1) or ROK activation (2), we hypothesized that one or more nPKC isoform mediate augmented ET-1 constriction in small mesenteric arteries from E-IH-exposed rats. To test this hypothesis, endothelium-disrupted mesenteric arteries or thoracic aorta segments were exposed to increasing concentrations of ET-1 (10–10 to 10–8 M) in the presence or absence of PKC inhibitors. The contribution of phospholipase C (PLC) to ET-1-mediated constriction was also evaluated because PLC liberates DAG, the activator of nPKC isoforms. Finally, we determined expression levels and ET-1-stimulated phosphorylation of PKC in small mesenteric arteries from E-IH and sham rats.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Male Sprague Dawley rats (weighing 250–300 g) were exposed to E-IH as described previously (1, 16). Briefly, animals were housed in Plexiglas boxes with free access to food and water and exposed 7 h/day for 14 days to either E-IH (20 brief exposures/h to 5% O2-5% CO2) or air-air cycling (sham, constant stream of room air). We have demonstrated that this protocol lowers PO2 to 
35 mmHg with no change in PCO2 (37), simulating moderate to severe sleep apnea where O2 saturation falls as low as 70% (22, 23). Body weight, systolic blood pressure (SBP), and heart rate were recorded on days 0 and 14 before the start of the daily E-IH or air-air exposure using a standard tail-cuff apparatus (IITC). Approximately 16 h after the final E-IH exposure, animals were deeply anesthetized with pentobarbital sodium (150 mg/kg), and the mesenteric arterial arcade or the thoracic aorta was collected for constrictor studies. All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the University of New Mexico Health Science Center and conform to National Institutes of Health guidelines for animal use.
Isolated Mesenteric Arteriole Preparation
Isolation. The intestinal arcade was removed and placed in a Silastic-coated petri dish containing chilled physiological salt solution [PSS (in mmol/l): 129.8 NaCl, 5.4 KCl, 0.83 MgSO4, 19 NaHCO3, 1.8 CaCl2, and 5.5 glucose]. Small mesenteric artery (sMA) segments (4th to 5th order, diameter <250 µm) were dissected from the 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 mmHg with PSS using a servocontrolled peristaltic pump (Living Systems) and superfused with oxygenated 37°C PSS at a rate of 5 ml/min.
Endothelium disruption. The endothelium was disabled in all mesenteric artery experiments by passing 1 ml of air through the lumen. Disruption of the endothelium was assessed by exposing phenylephrine (PE, 10 µmol/l)-constricted arterioles to ACh (1 µmol/l), and only arteries where ACh-mediated vasodilation was eradicated were used.
Vessel wall intracellular [Ca2+] detection. After endothelium disruption, pressurized mesenteric arteries (inner diameter: sham = 144.0 ± 3.4 µm; E-IH = 148.2 ± 3.4 µm) were loaded with the cell-permeable ratiometric Ca2+-sensitive fluorescent dye fura 2-AM (Molecular Probes). Fura 2-AM was dissolved in anhydrous dimethyl sulfoxide (DMSO; 1 mmol/l) with 20% pluronic acid and then added to PSS for a final concentration of 2 µmol/l fura 2-AM and 0.05% pluronic acid. Pressurized arteries were incubated 45 min in the dark at room temperature in fura 2-AM solution receiving normoxic gas. After incubation, arteries were washed with 37°C PSS for 15 min to remove excess dye and allow complete deesterification of the compound. Fura 2-loaded 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 were collected with a photomultiplier tube [ratio of fluoresecence at 340 nm (F340) to that at 380 nm (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 (28).
Constrictor studies. After determining baseline internal diameter and F340/F380, sMA were pretreated for 10 min with one of the following in the superfusion media; the nonselective PKC inhibitor GF-109203x (3 µM; Sigma), the cPKC inhibitor Gö-6976 (1 µM; Calbiochem), the nPKC inhibitor rottlerin (3 µM; Calbiochem), the PLC inhibitor U-73122 (1 µM; Calbiochem), or vehicle (DMSO, 2.9 mM; Sigma). Effective concentrations of inhibitors were determined from concentration-response curves with each inhibitor on sMA constricted 50% with either ET-1 or phorbol dibutyrate (3 µM). Concentrations used in studies were maximally effective at reversing constriction. After inhibitor incubation, arteries were exposed to increasing concentrations of ET-1 (10–10 to 10–8 mol/l) in the superfusion media. Arteries were exposed to each concentration of agonist for 5 min in the continued presence of the inhibitors, and each artery was used for only one concentration-response curve. After ET-1 exposure, sMA were superfused with Ca2+-free PSS to cause complete dilation and demonstrate that the constriction was caused by active tone. Only sMA demonstrating 100% or greater dilation were used. Constrictions are expressed as percent baseline diameter. Vessel wall [Ca2+] is expressed as the F340/F380 ratio because the ratio is linearly related to the true molar [Ca2+] when the dissociation constant of fura 2 does not differ between treatment groups (11).
Permeabilized studies. sMA were prepared as described above. After fura 2 loading, arteries were superfused with Ca2+-free PSS to achieve complete dilation. Once completely dilated, 3 µM ionomycin (Calbiochem) was added to the Ca2+-free superfusate to permeabilize sMA to Ca2+ as described previously (2). After permeabilization, arteries were equilibrated in PSS with a calculated free [Ca2+] of 300 nM to hold intracellular [Ca2+] ([Ca2+]i) constant at approximate basal levels throughout the study. This maintained diameter and fura 2 ratios near baseline [diameters: sham 98.1 ± 3.3%, E-IH 100.6 ± 2.4%; ratios: sham (basal vs. ionomycin + Ca2+) 1.18 ± 0.05 vs. 1.02 ± 0.06 F340/F380, E-IH 1.08 ± 0.08 vs. 0.86 ± 0.04 F340/F380].
Aortic Ring Contraction Studies
Isolation, denuding, and contractile studies. Thoracic aortas were collected from E-IH and sham rats and placed in chilled PSS. Arteries were cleaned of blood, connective tissue, and surrounding fat and cut into 3-mm segments. Endothelial cells were removed by gently rubbing the lumen with fine-tipped forceps. Aortic rings were suspended between two stainless steel hooks in a water-jacketed tissue bath containing PSS maintained at 37°C and bubbled with 21% O2-6% CO2-73% N2. The hooks were attached on one side to a rigid Plexiglas support and on the other side to a force transducer (Grass FT03) connected to an amplifier (Gould) that fed into a data acquisition program (version 3.01; Dataq). Rings were stretched with 2,500 mg of passive tension and equilibrated for 60 min. After equilibration, viability was confirmed by contraction to PE (10–6 mol/l), and endothelial integrity was assessed by relaxation to ACh (10–6 mol/l) in rings contracted with PE. Segments generating >1 g tension and relaxing <5% were used. Tissues were washed repeatedly over 60 min to remove PE and ACh and then exposed to increasing concentrations of ET-1 (10–10 to 10–7 mol/l) in the presence or absence of the inhibitors GF-109203X (3 µmol/l), Gö-6976 (1 µmol/l) or rottlerin (3 µmol/l). Each ring was used to generate only one concentration-response curve. Contractions are expressed as the percentage of PE (10–6 M) contraction (7).
Western analysis.
To determine whether E-IH alters ET-1 activation of PKC in mesenteric arteries, PKC translocation and levels of phosphorylated PKC (P-PKC) were evaluated as described previously (1). Briefly, mesenteric artery cascades (2nd to 5th order inclusive) from sham and E-IH rats were cleaned of connective and adipose tissue, incubated with either vehicle or ET-1 (10–8 mol/l) for 30 min at 37°C, frozen in liquid nitrogen, and homogenized on ice in Tris·HCl buffer (pH 7.4) containing protease inhibitors. Homogenates were centrifuged at 1,500 g at 4°C for 10 min to remove insoluble material. For the translocation studies, homogenates were then separated into cytosolic and particulate fractions by high-speed centrifugation (100,000 g at 4°C for 60 min). The cytosolic fraction was removed, and the particulate fraction was reconstituted in 100 µl of Laemmli buffer. For the phosphorylation study, total tissue homogenates were solubilized in Laemmli buffer. Proteins were separated in 4–20% gradient polyacrylamide gels (Bio-Rad) and transferred to polyvinylidene difluoride membranes. After being blocked, membranes were incubated 2 h at 25°C and then overnight at 4°C with a mouse monoclonal antibody specific for rat total PKC (1:1,000, translocation study; Santa Cruz) or for P-PKC (1:500; Santa Cruz). After being washed, blots were incubated with peroxidase-labeled goat anti-mouse IgG (1:5,000; Bio-Rad), followed by chemiluminescence labeling (Amersham). Band densities (SigmaGel; SPSS) were normalized to the total protein loaded per lane as determined by Coomassie blue staining of the membranes.
Statistical analysis. Constriction and [Ca2+]-response curves were analyzed using two-way repeated-measures ANOVA with Student-Newman Keul's post hoc analysis for differences between groups and treatments. Percent data were transformed to the square root of the arcsin before analysis to approximate a normal distribution (SigmaStat Software; SPSS). P < 0.05 was considered statistically significant for all analyses.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ET-1-mediated constriction was greater in sMA from vehicle-treated E-IH rats than in sMA from vehicle-treated sham rats and was not accompanied by an increase in vessel wall [Ca2+], which is consistent with our previous observations (1). Therefore, DMSO does not appear to affect ET-1-mediated constriction or vessel wall [Ca2+] (Fig. 1 and Table 2).
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The PKC inhibitor GF-109203x (3 µM) reduced ET-1 constriction in endothelium-disrupted sMA from E-IH rats (Fig. 2B). GF-109203x-treatment increased the calculated EC50 (effective concentration to constrict to 50% of the maximum) and decreased the calculated maximal response (Emax, Table 2). GF-109203x had no significant effect on ET-1-dependent constriction in sham sMA (Fig. 2A). Because GF-109203x (3 µM) inhibits both classical Ca2+-dependent (cPKC) and novel Ca2+-independent (nPKC) isoforms of PKC, specific cPKC and nPKC inhibitors were also used to determine the contribution of different PKC isoforms to ET-1-mediated vasoconstriction.
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Gö-6976 is a potent inhibitor of cPKC isoforms , β1, β2, and µ with an inhibitory constant of 0.1 µM (12). Gö-6976 (1 µM) did not affect ET-1-mediated constriction in either group of sMA (Fig. 2, C and D, and Table 2). To verify that the concentration used was effective, we administered increasing concentrations of Gö-6976 to reverse phorbol 12,13-dibutyrate (PDBu, 3 µM)-mediated constriction. PDBu-mediated constriction in endothelium-disabled mesenteric arteries was completely reversed with 1.0 µM Gö-6976 (data not shown). Therefore, cPKC isoforms do not appear to contribute to ET-1-mediated vasoconstriction in sMA.
Rottlerin (3 µM) greatly inhibited ET-1-mediated constriction in E-IH sMA (Fig. 3B) similar to the effect of the nonselective PKC inhibitor (Fig. 2B). This was seen as an increase in EC50 and a decrease in Emax (Table 2). At this concentration, rottlerin specifically inhibits PKC and only affects other PKC isoforms at 10-fold greater concentrations (13). This suggests the effects of GF-109023x (Fig. 2B) were primarily through PKC inhibition. In contrast, rottlerin had no effect on ET-1-mediated constriction in sham sMA (Fig. 3A), suggesting that ET-1 does not normally signal through PKC to constrict sMA.
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and suggests that activation of this nPKC isoform mediates augmented ET-1-mediated vasoconstriction independent of increases in [Ca2+]i. Effects of PLC Inhibition on ET-1-mediated sMA Constriction
Both cPKC and nPKC require DAG for activation. Therefore, we investigated the contribution of the DAG-liberating enzyme PLC to ET-1-mediated constriction. U-73122 (1 µM), a phosphatidylinositol-PLC inhibitor, significantly reduced ET-1-mediated constriction in E-IH sMA (Fig. 4B) but not in sham arteries (Fig. 4A). This suggests that ET-1 activates PLC upstream of PKC activation in the E-IH sMA.
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Western Blots
Western blots revealed that PKC is present in both E-IH and sham mesenteric arteries (Fig. 5A) and that a fraction of it is phosphorylated (Fig. 5B). Although ET-1 increased the amount of P-PKC in E-IH arteries but not in sham arteries, it did not cause translocation of PKC in any of the sMA (Fig. 5A). This suggests that ET-1 activates PKC in sMA from E-IH rats but not in those from sham rats.
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PKC inhibition with GF-109203x (3 µmol/l) diminished contraction in arteries from both sham (Fig. 6A) and E-IH (Fig. 6B) rats, but the inhibition appeared to be greater in the E-IH arteries (Table 2).
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In contrast to the observations in sMA, rottlerin (3 µM) did not affect ET-1-induced contraction in aortic rings from either sham (Fig. 6C) or E-IH (Fig. 6D) rats. However, cPKC inhibition with Gö-6976 (1 µM) significantly inhibited contraction in aorta from both sham (Fig. 6C) and E-IH (Fig. 6D) rats.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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, appears to be activated by and contribute to ET-1 vasoconstriction in E-IH sMA while only cPKC participates in contraction of aorta. 3) Inhibition of the PKC upstream activator PLC attenuates ET-1 vasoconstriction in E-IH but not in sham sMA. 4) When [Ca2+]i is held constant, PKC mediates constriction in E-IH sMA, but some other mechanism leads to Ca2+ sensitization-mediated constriction in sham sMA.
ET-1, like many other G protein-coupled receptor agonists, causes Ca2+-independent contraction of vascular smooth muscle cells by activating Ca2+-sensitizing pathways (20). PKC can increase Ca2+ sensitivity in smooth muscle by phosphorylating CPI-17, which inhibits MLCP thus maintaining MLC20 in a phosphorylated state (38). ET-1 can activate both Ca2+-dependent PKC isoforms (cPKC) and Ca2+-independent PKC isoforms (nPKC) in coronary smooth muscle cells (27), and PKC inhibition significantly reduced ET-1-stimulated constriction in the superior mesenteric artery of portal-hypertensive rats (40). Thus ET-1 can cause Ca2+ sensitization and constriction through the activation of PKC. Our previous studies showed that, in sMA from E-IH rats, the ET-1 constrictor responses were augmented without an apparent increase in vessel wall [Ca2+]i and mostly independent of ROK activation. These data suggested that ET-1 increases Ca2+-sensitivity in E-IH arteries more than in sham arteries (1). The current studies demonstrate that this augmented Ca2+ sensitization is at least in part through activation of PKC, a pathway that does not appear to contribute to ET-1 constriction in sham sMA. Therefore, E-IH exposure appears to enable ET-1 activation of PKC-dependent constriction in resistance-size arteries via a pathway that is completely inactive in the sham arteries. Interestingly, the same inhibitor had no effect in aorta from either sham or E-IH rats.
In contrast, the cPKC inhibitor Gö-6976, which potently inactivates PKC and PCKβ1 isoforms (26), did not reduce ET-1-mediated constriction in either E-IH or sham sMA but was the most effective inhibitor of ET-1 contraction in aortic rings. This supports our hypothesis that cPKC isoforms are not involved in ET-1 constriction in mesenteric arteries and demonstrates that ET-1-dependent vasoconstriction is mediated by very different signaling pathways in these two arteries. Additionally, this suggests that the inhibition of ET-1-mediated constriction in sMA by GF-109203x is through its effects on one or more nPKC isoforms. Other studies have shown that cPKC inhibition with Gö-6976 selectively reduces norepinephrine-mediated contraction of thoracic aorta from deoxycorticosterone acetate-salt hypertensive rats (21) and UK-14304 contraction in aorta from rats made hypertensive with a nitric oxide synthase inhibitor (7). Therefore, increased activity of cPKC isoforms appears to contribute to elevated constrictor responses in large arteries in many models of hypertension. However, these PKC isoforms do not appear to be a component of ET-1 signaling in sMA nor to contribute to the E-IH-induced augmented Ca2+ sensitization in this vascular bed.
In contrast to the results with Gö-6976, the PKC inhibitor rottlerin significantly reduced ET-1-mediated constriction in E-IH (Fig. 4B) but not in sham sMA (Fig. 4A), similar to the effects of GF-109203x (Fig. 2). This same concentration of rottlerin, which is quite selective for PKC (13), had no effect on ET-1 contraction of thoracic aorta from either group, suggesting E-IH exposure bed specifically facilitates ET-1 activation of PKC-dependent vasoconstriction. This observation was confirmed using ionomycin-permeabilized sMA to evaluate Ca2+ sensitization when [Ca2+]i was held constant. In these studies, rottlerin almost completely inhibited ET-1-mediated constriction in E-IH sMA (Fig. 5B), supporting the hypothesis that PKC mediates ET-1 vasoconstriction in small arteries only after exposure to simulated sleep apnea. We have previously shown that E-IH exposure results in an increase in oxidative stress (39), and others have shown that intermittent hypoxia-induced oxidative stress can activation PKC in cardiomyocytes (19). Thus elevated oxidative stress may underlie the observed PKC upregulation.
Activation of PKC requires binding of DAG to facilitate translocation from the cytosol to the particulate fraction (32). However, the duration of the translocation appears to be isoform, stimulus, and tissue specific (25). In ventricular myocytes, PKC and PKC
are both translocated to the particulate fraction for a prolonged period by hypoxia in cultured cells (8, 29) and by intermittent hypoxia in vivo (19). However, PKC was equally distributed between the particulate and cytosolic fractions under resting conditions for both sham and E-IH arteries, and neither E-IH nor ET-1 appeared to stimulate translocation (Fig. 5). This is similar to a previous report from Sirous and coworkers (36) that in cultured coronary artery smooth muscle cells most PKC is in the particulate fraction under basal conditions and ET-1 causes little translocation. Our constrictor studies, however, strongly indicate that ET-1 signals through PKC in sMA from E-IH-exposed rats. The apparent lack of ET-1-mediated PKC translocation may thus be because of high basal levels in the membrane or very rapid, reversible translocation as has previously been shown in cultured cardiac myocytes. Because PKC remains activated after it returns to the soluble fraction as long as it is phosphorylated (25), the ET-1-stimulated increase in Ser643-P-PKC in the E-IH sMA suggests that this is also the pattern in mesenteric arteries and supports the conclusion that ET-1-dependent contraction is dependent on PKC-mediated Ca2+ sensitization. Future studies to find the target(s) of the activated PKC will determine if this is through the well-described phosphorylation of CPI-17 (14, 18) or some other target.
Although we did not investigate targets downstream of PKC, we did determine that the inhibition of PLC greatly diminished ET-1-mediated constriction in E-IH but not sham sMA. These data suggest that E-IH augmentation of ET-1 activation of PKC is in part dependent on increased PLC activation. Because PLC inhibition did not reduce ET-1-mediated constriction as effectively as PKC inhibition, PKC may be activated in part through other pathways such as phosphatidylcholine-PLC or PLD (9, 24, 35).
Overall, this study has established that E-IH exposure increases ET-1 activation of Ca2+ sensitization to augment constriction in isolated endothelium-disabled sMA. It also demonstrates that ET-1-induced vasoconstriction is PKC dependent in both mesenteric arteries and thoracic aorta following E-IH but the PKC pathway is not activated in sMA from sham rats. This suggests that exposure to E-IH uniquely couples ET-1 receptors with PLC and PKC to augment sMA constriction. Furthermore, two different isoforms of PKC appear to mediate ET-1-dependent contraction in aorta and sMA. Therefore, these data suggest that ET-1 signaling and PKC activation are vascular targets of E-IH and these two pathways are potential sites of intervention for controlling hypertension and vascular pathologies in sleep apnea and other states with intermittent exposures to hypoxia.
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GRANTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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2-AR vasoreactivity in chronic NOS inhibition hypertension. Am J Physiol Heart Circ Physiol 284: H309–H316, 2003. upregulation and cardioprotection induced by chronic intermittent hypoxia. Am J Physiol Heart Circ Physiol 292: H224–H230, 2007. isoform translocation and enhancement of tonic contractions of gastrointestinal smooth muscle. Am J Physiol Gastrointest Liver Physiol 292: G887–G898, 2007.This article has been cited by other articles:
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  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]
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) or PKC inhibitors (
). The nonselective PKC inhibitor GF-103209x reduced constriction in E-IH arteries (B) but not in sham arteries (A). The classical PKC (cPKC) selective inhibitor Gö-6976 (1 µmol/l) did not affect constriction in either group (C and D). *Significant difference from vehicle for P < 0.05.
) does not alter ET-1-stimulated contraction in either group, but cPKC-selective inhibition reduces ET-1-induced contraction of aortic segments from both sham and E-IH rats (gray squares). *Significant difference from vehicle for P < 0.05.