Endothelin-1-induced vasoconstriction does not require intracellular ...

Am J Physiol Heart Circ Physiol 306: H667–H673, 2014. First published January 10, 2014; doi:10.1152/ajpheart.00643.2013.

Endothelin-1-induced vasoconstriction does not require intracellular Ca2⫹ waves in arteries from rats exposed to intermittent hypoxia Jessica M. Osmond, Laura V. Gonzalez Bosc, Benjimen R. Walker, and Nancy L. Kanagy Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico Submitted 19 August 2013; accepted in final form 30 December 2013

Osmond JM, Gonzalez Bosc LV, Walker BR, Kanagy NL. Endothelin-1-induced vasoconstriction does not require intracellular Ca2⫹ waves in arteries from rats exposed to intermittent hypoxia. Am J Physiol Heart Circ Physiol 306: H667–H673, 2014. First published January 10, 2014; doi:10.1152/ajpheart.00643.2013.—Sleep apnea is associated with cardiovascular disease, and patients with sleep apnea have elevated plasma endothelin (ET)-1 concentrations. Rats exposed to intermittent hypoxia (IH), a model of sleep apnea, also have increased plasma ET-1 concentrations and heightened constriction to ET-1 in mesenteric arteries without an increase in global vascular smooth muscle cell Ca2⫹ concentration ([Ca2⫹]). Because ET-1 has been shown to increase the occurrence of propagating Ca2⫹ waves, we hypothesized that ET-1 increases Ca2⫹ wave activity in mesenteric arteries, rather than global [Ca2⫹], to mediate enhanced vasoconstriction after IH exposure. Male Sprague-Dawley rats were exposed to sham or IH conditions for 7 h/day for 2 wk. Mesenteric arteries from sham- and IH-exposed rats were isolated, cannulated, and pressurized to 75 mmHg to measure ET-1-induced constriction as well as changes in global [Ca2⫹] and Ca2⫹ wave activity. A low concentration of ET-1 (1 nM) elicited similar vasoconstriction and global Ca2⫹ responses in the two groups. Conversely, ET-1 had no effect on Ca2⫹ wave activity in arteries from sham rats but significantly increased wave frequency in arteries from IH-exposed rats. The ET-1-induced increase in Ca2⫹ wave frequency in arteries from IH rats was dependent on phospholipase C and inositol 1,4,5-trisphosphate receptor activation, yet inhibition of phospholipase C and the inositol 1,4,5-trisphosphate receptor did not prevent ET-1-mediated vasoconstriction. These results suggest that although ET-1 elevates Ca2⫹ wave activity after IH exposure, increases in wave activity are not associated with increased vasoconstriction. endothelin-1; intermittent hypoxia; Ca2⫹ oscillations

of vascular smooth muscle cell (VSMC) Ca2⫹ concentration ([Ca2⫹]) is complex and involves multiple ion channels (for a review, see Ref. 19), with the profile of the Ca2⫹ signal depending largely on the initiating molecular trigger. Generally, an increase in VSMC [Ca2⫹] occurs through Ca2⫹ entry into the cell or Ca2⫹ release from the sarcoplasmic reticulum (SR) (19). The SR expresses two Ca2⫹-release channels, the ryanodine-sensitive Ca2⫹-release channel (RyR) and the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R), that contribute to intracellular [Ca2⫹] (17, 29, 34). Confocal fluorescence microscopy allows imaging of dynamic Ca2⫹ events with the resolution to define the spatial and temporal characteristics of the signal in VSMCs of the arterial wall. The role of Ca2⫹ events to regulate VSMC function depends on the source of Ca2⫹ and the spatial and temporal THE REGULATION

Address for reprint requests and other correspondence: N. L. Kanagy, Dept. of Cell Biology and Physiology, MSC08 4750, 1 University of New Mexico, Albuquerque, NM 87131 (e-mail: [email protected]). http://www.ajpheart.org

profile of the events (for a review, see Ref. 14). For example, short-duration, discrete Ca2⫹ release from RyRs, known as Ca2⫹ sparks, activates large-conductance Ca2⫹-sensitive K⫹ channels and elicits VSMC hyperpolarization and relaxation (29). Conversely, longer-duration, propagating Ca2⫹ events, or Ca2⫹ waves, involve Ca2⫹ release from SR IP3Rs and have been associated with vasoconstriction (1, 2, 18, 32). Activation of VSMC G␣q protein-coupled receptors elicits phospholipase C (PLC) hydrolysis of inositol 4,5-bisphosphate into IP3 and diacylglycerol followed by IP3 activation of SR IP3Rs to release Ca2⫹ into the cytosol. The majority of studies evaluating Ca2⫹ wave activity downstream of agonists that elicit vasoconstriction have focused on ␣-adrenergic signaling. Iino and colleagues (15) visualized Ca2⫹ waves in individual VSMCs of rat tail arteries in response to norepinephrine and demonstrated that these Ca2⫹ oscillations require SR Ca2⫹ release. Adrenergic stimulation with phenylephrine (PE) also increases Ca2⫹ wave activity in rat mesenteric artery VSMCs (25, 27). A study (32) in the rabbit vena cava demonstrated a correlation between asynchronous Ca2⫹ waves and force generation in response to PE. Therefore, Ca2⫹ waves have been linked with constriction, especially in the presence of ␣-adrenergic receptor activation, but clear evidence showing that Ca2⫹ waves contribute to rather than associate with vasoconstriction has not been provided. Previous work from our laboratory (2) observed heightened constriction to the vasoactive peptide endothelin (ET)-1 in mesenteric arteries from rats made hypertensive by exposure to intermittent hypoxia (IH) to mimic the effects of sleep apnea. Humans with sleep apnea, as well as rats and mice exposed to IH, have elevated plasma levels of ET-1 (10, 18, 30), and inhibition of the ET type (ETA) receptor in rats exposed to IH prevents the IH-induced increased in blood pressure (19), suggesting that ET-1 is involved in blood pressure regulation during IH exposure. The reported increase in constriction to ET-1 in rats after IH exposure was not accompanied by an increase in global VSMC [Ca2⫹], as measured by the Ca2⫹ indicator fura-2 AM, but did require extracellular Ca2⫹. Because there is increasing evidence showing that local Ca2⫹ events are not detected as global Ca2⫹ changes (4, 27), we hypothesized that ET-1 increases Ca2⫹ wave activity in mesenteric arteries, rather than global [Ca2⫹], to mediate enhanced vasoconstriction after IH exposure. MATERIALS AND METHODS

Rodent model of sleep apnea. Male Sprague-Dawley rats (Harlan Laboratories, 250 –275 g) were used for all experiments. Rats were exposed to either sham or IH conditions for 2 wk as previously described (18). Briefly, rats were exposed to IH cycling between room air (21% O2-0% CO2) and hypoxia (5% O2-5% CO2), whereas sham

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Ca2⫹ WAVES AND VASOCONSTRICTION IN SLEEP APNEA

conditions consisted of exposure to room air only. All rats were housed in identical Plexiglas boxes that subjected control rats to air flow and sounds similar to those produced by the hypoxia cycler. Rats were exposed to 20 hypoxic episodes/h for 7 h/day during their sleep period and were maintained on a 12:12-h light-dark cycle. Rats were euthanized with a lethal concentration of pentobarbital sodium (200 mg/kg ip), and mesenteric arteries were collected for all experiments. The Institutional Animal Care and Use Committee of the University of New Mexico School of Medicine reviewed and approved all animal protocols. All protocols conformed with National Institutes of Health guidelines for animal use. Isolated mesenteric artery preparation. Mesenteric arteries were isolated in ice-cold HEPES buffer [containing (in mM) 134 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, 0.026 EDTA, and 10 glucose], cannulated, and then pressurized to 75 mmHg in a vessel chamber (Living Systems Instrumentation). Fourth- and fifth-order endotheliumintact arteries were allowed to equilibrate in physiological salt solution [PSS; containing (in mM) 129.8 NaCl, 5.4 KCl, 0.83 MgSO4, 0.43 NaH2PO4, 19 NaHCO3, 1.8 CaCl2, and 5.5 glucose], which was heated to 37°C, gassed (21% O2-6% CO2-balance N2), and superfused over the artery at a rate of 5 ml/min. Arteries generated spontaneous tone during equilibration; however, tone was similar between groups (sham: 15.3 ⫾ 3.3% vs. IH: 15.0 ⫾ 3.7%). Ca2⫹ measurements. The fast Ca2⫹ indicator fluo-4 AM (Life Technologies) was used to image VSMC Ca2⫹ waves. Arteries were incubated with fluo-4 AM (10 ␮M fluo-4 AM and 0.25% pluronic acid in HEPES buffer) in the dark at 28°C for 60 min. After cannulation and equilibration at 33°C, baseline Ca2⫹ wave activity was assessed by exciting arteries for 60 s with a solid state 488-nm laser at a rate of 10 Hz, and emissions ⬎ 525 nm were collected using an Olympus IX71 microscope with a ⫻60 water-immersion objective and a spinning-disk confocal scanning unit (Andor Technology). Wave activity in the presence of ET-1 (1 nM) was then recorded for another 60 s in the same arteries. Paired wave measurements were also conducted in arteries pretreated with 2-aminoethoxydiphenyl borate (2-APB; 50 ␮M, Enzo Life Sciences) or U-73122 (1 ␮M, Tocris Bioscience), inhibitors of IP3Rs and PLC, respectively. VSMC Ca2⫹ wave frequency was analyzed with SparkAn software (courtesy of Dr. Bonev and Dr. Nelson, University of Vermont), which allowed for the determination of the ratio of fluorescence to background fluorescence (F/Fo) over time. Ten cells per artery were analyzed individually for wave activity before and after ET-1 to calculate waves per cell per minute. Each cell was analyzed as a single region of interest. Events with an F/Fo value of at least 1.2 that propagated through the cell were considered Ca2⫹ waves. To measure global Ca2⫹ responses in VSMC, pressurized arteries were incubated with the ratiometric Ca2⫹ indicator fura-2 AM [2 ␮M fura-2 AM (Life Technologies) and 0.05% pluronic acid (Life Technologies) in HEPES buffer] in the dark at room temperature for 45 min. After incubation, arteries were washed with PSS and then allowed to equilibrate at 37°C. An IonOptix Hyperswitch dualexcitation light source was used to excite fura-2-loaded arteries at 340 and 380 nm, and the 510-nm emission was collected with a photomultiplier tube (Electron Tubes) throughout the experiment. Data are expressed as the change in the background-subtracted 340-nm-to380-nm fluorescence emission ratios in response to ET-1 (1 nM). Global Ca2⫹ changes were recorded in the absence or presence of 2-APB (50 ␮M), U-73122 (1 ␮M), the voltage-dependent Ca2⫹ channel (VDCC) inhibitor diltiazem (30 ␮M), the Rho kinase inhibitor Y-27632 (10 ␮M), and Ca2⫹-free PSS [containing (in mM) 129.8 NaCl, 5.4 KCl, 0.83 MgSO4, 0.43 NaH2PO4, 19 NaHCO3, 5.5 glucose, and 3.7 EGTA]. Constriction experiments. Constriction to a bolus of ET-1 was assessed in pressurized mesenteric arteries from sham and IH rats in the absence and presence of 2-APB (50 ␮M) and U-73122 (1 ␮M). After equilibration with one of these pharmacological agents or its respective vehicle, baseline lumen diameter was recorded with edge

detection software (IonOptix), and ET-1 (1 nM) was then added to the superfusion. A stable constriction was achieved within ⬃5 min. Data are expressed as the percentage of constriction compared with the baseline diameter. IP3R expression. Expression of the IP3R was assessed in mesenteric arterial cascades from sham and IH rats. After isolation, arteries were placed in ice-cold lysis buffer [containing 25 mM Tris·HCl (pH 7.6), 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% SDS, Thermo Scientific] containing 1⫻ Halt phosphatase inhibitor cocktail (Thermo Scientific), 2.5 mM PMSF (Sigma-Aldrich), and 1⫻ Complete protease inhibitor cocktail (Santa Cruz Biotechnology). Artery homogenates (25 ␮g protein) were separated on a 4 –15% gradient gel (Bio-Rad Laboratories) and then transferred to a polyvinylidene fluoride membrane, which was incubated overnight with an IP3R type 1(IP3R1)-specific antibody (Cell Signaling). IP3R protein (320 kDa) was analyzed using a LI-COR Odyssey Infrared Imaging System (LI-COR) and is expressed as a ratio of ␤-actin. Statistical analysis. Vasoconstriction and global Ca2⫹ measurements were compared in arteries from sham and IH rats by Student’s t-tests and in IH vehicle-, 2-APB-, and U-73122-treated groups by one-way ANOVA with Holm-Sidak post hoc analysis. Ca2⫹ wave frequency was compared by repeated-measures two-way ANOVA. Western blot densitometric levels were compared by Student’s t-test. Data are expressed as means ⫾ SE. The sample size for all experiments was n ⱖ 5, and P values of ⬍0.05 were considered statistically significant. RESULTS

Vasoconstriction to a single concentration of ET-1 (1 nM) was assessed in pressurized mesenteric arteries from sham and IH rats. At this concentration of ET-1, constriction was not different in the two groups (Fig. 1A). This finding was somewhat unexpected because of previous observations that this concentration produces greater constriction in arteries from IH rats when present as part of a cumulative concentration-response protocol, but it was useful for comparing Ca2⫹ responses in arteries with a similar degree of constriction. Global VSMC Ca2⫹ was also measured using the ratiometric Ca2⫹ indicator fura-2 AM under the same conditions. Representative traces of the global Ca2⫹ response over time are shown in Fig. 1B. The change in the fura-2 AM ratio in response to 1 nM ET-1 was also similar between arteries from sham and IH rats (Fig. 1C). Ca2⫹ wave frequency in VSMCs was evaluated under basal and ET-1-stimulated (1 nM) conditions in arteries from sham and IH rats. Representative traces of Ca2⫹ wave activity over time are shown in Fig. 2A. Two weeks of IH exposure did not affect Ca2⫹ wave frequency in VSMCs under baseline conditions but significantly increased wave frequency compared with baseline after the addition of 1 nM ET-1 (Fig. 2B). This concentration of ET-1 did not affect wave activity in arteries from sham rats despite eliciting vasoconstriction and global Ca2⫹ increases similar to those observed in arteries from IH rats, suggesting Ca2⫹ wave activity after the addition of ET-1 may not be linked to ET-1-induced vasoconstriction. There were no differences in Ca2⫹ wave kinetics between sham and IH under basal conditions, but the rise time, duration, and decay time of the waves were decreased in arteries from IH rats after the addition of ET-1 compared with arteries from sham rats (Fig. 2C), potentially contributing to or caused by the increased Ca2⫹ wave frequency. ET-1 stimulation also increased the percentage of cells displaying wave activity com-

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arteries from IH rats in the presence of the nonspecific IP3R inhibitor 2-APB (50 ␮M). Only arteries from IH rats were studied because ET-1 did not affect wave frequency in arteries from sham rats. Treatment with 2-APB decreased basal wave frequency and prevented the ET-1-induced increase in wave frequency (Fig. 4). Wave frequency in the presence of the PLC inhibitor U-73122 (1 ␮M) was also decreased, and U-73122 prevented the increase in wave frequency after ET-1 stimulation (Fig. 4), suggesting that PLC products regulate wave activity under basal conditions and downstream of ET-1 in arteries from IH-exposed rats. In contrast, neither IP3R inhibition with 2-APB nor PLC inhibition with U-73122 affected constriction to ET-1 or the change in the fura-2 AM ratio in response to ET-1 (Fig. 5). These findings underscore the separation of Ca2⫹ wave frequency and vasoconstriction to ET-1. Following the observation that 2-APB and U-73122 administration did not affect constriction to ET-1 in arteries from IH rats, other pathways were investigated. Specifically, the contribution of VDCCs and Rho kinase to this constriction was evaluated. Inhibition of VDCCs with diltiazem (30 ␮M) did not affect ET-1-mediated vasoconstriction; however, blockade of Rho kinase with Y-27632 (10 ␮M) greatly attenuated constriction (Table 1), suggesting that the ET-1-induced vasoconstriction is largely dependent on Ca2⫹ sensitization mechanisms. Also, as shown in Table 1, ET-1-mediated constriction requires extracellular Ca2⫹, since incubation with Ca2⫹-free PSS prevented vasoconstriction and global Ca2⫹ changes in response to ET-1. DISCUSSION

Fig. 1. Vasoconstriction (A) and vascular smooth muscle (VSM) Ca2⫹ responses (B and C) to 1 nM endothelin (ET)-1 in pressurized mesenteric arteries from sham and intermittently hypoxic (IH) rats. VSM Ca2⫹ was measured with fura-2 AM. ET-1 was added to the superfusion reservoir at time 0 and reached the vessel chamber after ⬃120 s (arrow). [Ca2⫹]i, intracellular Ca2⫹ concentration; F340/F380, 340-to-380-nm fluorescence ratio. Values are means ⫾ SE.

pared with sham conditions (sham: 54 ⫾ 14% vs. IH: 88 ⫾ 7%, P ⬍ 0.05). Ca2⫹ waves have been attributed to IP3R activation. The expression of IP3R1 was assessed in mesenteric artery homogenates from sham and IH rats by Western blot analysis to determine whether IH exposure increases IP3R expression. As shown in Fig. 3, IP3R1 expression was similar in sham and IH artery homogenates, indicating that increased ET-1-induced wave activation after IH exposure is not likely the result of increased IP3R expression. To confirm the relationship between IP3R activation and waves after ET-1 stimulation, wave frequency was measured in

The results from the present study demonstrate that exposure to IH, to mimic sleep apnea, enhanced ET-1 activation of Ca2⫹ wave activity. Furthermore, this heightened Ca2⫹ wave activity was elicited by a concentration of ET-1 that yielded a similar constrictor response in arteries from IH and sham rats. Although 2-APB and U-73122 treatment prevented the ET-1induced increase in wave activity, they had no effect on ET-1-mediated constriction. These findings suggest Ca2⫹ wave activity is not required for ET-1-induced vasoconstriction. An uncoupling of waves and constriction in rat mesenteric arteries has been previously reported by Miriel et al. (27). Although rat mesenteric arteries constricted when depolarized with KCl, KCl did not alter Ca2⫹ wave activity. Conversely, the addition of PE elicited vasoconstriction and enhanced Ca2⫹ wave generation. This study concluded that the IP3 generation occurring downstream of PE activation of ␣-adrenergic receptors but not after KCl-mediated depolarization of VSMC membrane potential caused both the Ca2⫹ waves and vasoconstriction. Although this does not clarify the reason for the uncoupling of waves and constriction downstream of ET-1, also a trigger for IP3 release, in the present study, these findings do support the concept that Ca2⫹ waves are not required for constriction under certain conditions. Additionally, Jaggar (16) demonstrated that increased Ca2⫹ wave frequency in response to elevated intraluminal pressure in small cerebral arteries actually opposes constriction. Thus, whether Ca2⫹ waves facilitate constriction under certain conditions, such as during ␣-adrenergic receptor stimulation, or are simply associated with it remains unclear.


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Fig. 2. Ca2⫹ wave activity under basal and ET-1-stimulated conditions measured with the fast Ca2⫹ indicator fluo-4 AM. A: representative traces of Ca2⫹ wave activity over time. B: wave frequency before and after the addition of ET-1 (1 nM) in arteries from sham and IH rats. C: kinetics of the Ca2⫹ wave measurements shown in B. Values are means ⫾ SE. *P ⬍ 0.05 vs. the respective sham condition; #P ⬍ 0.05 vs. baseline.

The differential coupling of Ca2⫹ waves and vasoconstriction observed between PE and ET-1 also does not appear to depend on differential stimuli of Ca2⫹ waves. Binding of either agonist to its receptor increases Ca2⫹ wave frequency (21, 27,

Fig. 3. Expression of inositol 1,4,5-trisphosphate receptor (IP3R)1 as determined by Western blot analysis and expressed as a ratio of ␤-actin. Values are means ⫾ SE.

28, 32, 33), and, as previously demonstrated (25, 28), inhibition of IP3R prevents agonist-induced wave activity. One key difference between PE- and ET-1-mediated effects may be receptor binding. ET-1 irreversibly binds its receptor (36), whereas PE binds its receptor reversibly (26). Additionally, in a comparison of Ca2⫹ sensitization in response to ET-1 and PE in pressurized rat mesenteric arteries, Shaw and colleagues (33) observed that ET-1 increases Ca2⫹ sensitivity more than

Fig. 4. Ca2⫹ wave frequency with and without ET-1 (1 nM) in arteries from IH rats in the absence and presence of the nonspecific IP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB; 50 ␮M) and the phospholipase C inhibitor U-73122 (1 ␮M). Values are means ⫾ SE. *P ⬍ 0.05 vs. baseline; #P ⬍ 0.05 vs. vehicle.


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Fig. 5. Vasoconstriction (A) and VSM Ca2⫹ responses (B) to 1 nM ET-1 in mesenteric arteries from IH rats in the absence and presence of the nonspecific IP3R inhibitor 2-APB (50 ␮M) and the phospholipase C inhibitor U-73122 (1 ␮M).

does PE. Therefore, even though both agonists trigger Ca2⫹ waves and elicit some degree of Ca2⫹ sensitization, it is possible that the balance between Ca2⫹ wave activation and Ca2⫹ sensitization may determine the driving factor for vasoconstriction. As shown in Table 1 and demonstrated by a previous study from our laboratory (3), ET-1 constriction in arteries from IH rats is greatly dependent on Rho kinase activity, highlighting the role of Ca2⫹ sensitization in the vasoconstriction response to ET-1 in these arteries. In the present study, Ca2⫹ wave activity was observed in mesenteric artery VSMCs from sham and IH rats under basal conditions (Fig. 2A), with no differences in basal wave frequency or kinetics between groups, suggesting that IH exposure does not alter Ca2⫹ wave activity in the absence of exogenous agonists. However, both the frequency (Fig. 2B) and kinetics (Fig. 2C) of ET-1-induced waves were altered in VSMCs in arteries from IH rats, as apparent in the representative trace shown in Fig. 2A. Also, ET-1 increased the percentage of cells exhibiting wave activity in arteries from IH rats. Both of these alterations appear to contribute to the heightened wave activity after ET-1 stimulation and may result from an increase in ETA receptor protein expression in the arteries from IH rats, as previously observed (2). The mechanism by which ET-1 alters Ca2⫹ wave kinetics after IH exposure is unclear but is an interesting avenue for future investigation. Previous investigations into the mechanism of Ca2⫹ waves indicate a dependency on IP3R activation and refilling of the

SR by sarco(endo)plasmic reticulum Ca2⫹-ATPase (21). Ca2⫹ released from IP3Rs has been proposed to activate other IP3Rs, as well as RyRs, to maintain wave activity (7). In the present study, experiments with the nonspecific IP3R inhibitor 2-APB not only prevented the ET-1-mediated increase in Ca2⫹ wave activity but also abolished basal wave activity (Fig. 4). This finding is consistent with previous studies indicating that IP3R activation is requisite for wave activity in VSMCs (21), including a study by Narayanan and colleagues (28) demonstrating that 10 nM ET-1 increases Ca2⫹ wave frequency in cerebral arteries in a manner that is completely prevented by IP3R inhibition. However, due to the nonspecific nature of 2-APB, the findings in the present study do not exclude the possibility that other 2-APB targets, such as transient receptor potential (TRP) channels, are involved. Although many studies are in agreement that VSMC Ca2⫹ waves are mediated by IP3R activation, the function of these Ca2⫹ events is not clear. There is evidence that 2-APB inhibits IP3R with an IC50 of 42 ␮M (24), but it is possible that the effects of 2-APB observed in the present study can be attributable to modulation of store-operated Ca2⫹ entry and/or TRP channel activity. It has been previously demonstrated that 2-APB facilitates storeoperated Ca2⫹ entry at lower concentrations (23, 31) and inhibits it at higher concentrations (20, 31). Furthermore, 2-APB inhibits multiple TRP channels (22, 35, 37), potential mediators of store-operated Ca2⫹ entry, as well as a variety of other signaling pathways (for reviews, see Refs. 5, 13, and 38). Although the exact mechanism by which 2-APB inhibits Ca2⫹ waves is not clear, there is a dissociation between ET-1induced Ca2⫹ wave frequency and constriction in the presence of 2-APB, suggesting that Ca2⫹ waves are not required for ET-1-mediated vasoconstriction. The present study was conducted using a single concentration of ET-1 (1 nM). Previous work from our laboratory compared effects of ET-1 on constriction and global [Ca2⫹] in arteries from sham and IH rats using cumulative concentrationresponse curves. With this approach, we detected concentration-dependent vasoconstriction to ET-1 from 1 to 30 nM in the absence of increases in global [Ca2⫹] (2). The single bolus exposure to ET-1 was necessary in the present study because the integrity of the fluo-4 signal decreases with prolonged laser exposure. The lowest concentration of ET-1 to elicit a greater constriction in arteries from IH rats compared with those from sham rats in previous studies was chosen to avoid the vasomotion elicited at higher concentrations of ET-1, compromising the ability to continuously track individual VSMCs over time while recording Ca2⫹ wave activity. This allowed us to Table 1. Effect of inhibition of voltage-dependent Ca2⫹ channels or Rho kinase on endothelin-1-induced constriction and global Ca2⫹ concentration changes in arteries from intermittently hypoxic rats Condition

Vehicle Diltiazem (30 ␮M) Y-27653 (10 ␮M) Ca2⫹-free physiological saline solution

Percent Constriction

Change in Vascular Smooth Muscle Ca2⫹ Concentration

55.4 ⫾ 6.0 50.5 ⫾ 7.5 24.6 ⫾ 9.3*

0.17 ⫾ 0.02 0.18 ⫾ 0.03 0.32 ⫾ 0.10

1.6 ⫾ 0.9*

0.00 ⫾ 0.06*

Values are means ⫾ SE. *P ⬍ 0.05 vs. vehicle.


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make the novel observation that at this low agonist concentration delivered as a bolus, there is no correlation between Ca2⫹ wave activity and VSMC contraction. Another novel observation of the present study was the degree of constriction obtained using a bolus exposure to ET-1. In previous cumulative-exposure studies (1–3), 1 nM ET-1 elicited 15–20% constriction. In contrast, a bolus of 1 nM ET-1 elicited a 50% constriction in the present study. Surprisingly, this robust constriction to a bolus of ET-1 was similar in sham and IH groups. The reasons for the heightened constriction elicited by a bolus of ET-1 and lack of differences in constriction between sham and IH are unclear but should be considered as a potential caveat of the study and a point for future investigations into the effect of repeated exposures to ET-1. Differential responses between a bolus and repeated exposure to ET-1 may be an effect of receptor-binding characteristics and ET-1 clearance. Interestingly, administration of a bolus of 3 nM ET-1 produced greater constriction than 1 nM ET-1 but did not further increase Ca2⫹ wave activity (data not shown), in contrast to a previous report (32) suggesting a concentrationdependent increase in Ca2⫹ wave activity elicited by PE. Although ET-1 elicited substantial constriction in arteries from sham rats (Fig. 1A), it did not affect wave activity in these arteries (Fig. 2B), highlighting the dissociation between vasoconstriction and Ca2⫹ wave activity in healthy and diseased arteries. A previous study (1) demonstrated that chronic or acute inhibition of the ETA receptor with BQ-123 did not affect blood pressure in sham rats but prevented or reversed the IH-induced increase in blood pressure. These findings are in agreement with the observation of higher plasma levels of ET-1 and increased expression of ETA receptor in mesenteric arteries from rats exposed to IH compared with rats exposed to sham conditions (18), suggesting that chronic exposure to ET-1 may sensitize VSMCs to ET-1-induced Ca2⫹ wave activity. In VSMCs, Ca2⫹ is involved in a variety of cellular processes, made possible by different spatial and temporal distributions of intracellular Ca2⫹ as well as multiple sources of Ca2⫹ (for a review, see Ref. 6). Among the processes regulated by intracellular Ca2⫹ are cell proliferation, metabolism, vesicle trafficking, apoptosis, and transcription. ET-1-induced increases in VSMC Ca2⫹ wave activity may therefore facilitate processes other than vasoconstriction that contribute to IHinduced increases in arterial pressure. For example, Ca2⫹ release from SR IP3Rs increases nuclear translocation of the transcription factor nuclear factor of activated T cells, which plays a role in VSMC differentiation and hypertrophy (9, 11, 12). Furthermore, de Frutos et al. (8) demonstrated that nuclear factor of activated T cells isoform c3 activity is upregulated by IH exposure and is required for IH-induced hypertension in mice. The higher circulating concentration of ET-1 could trigger VSMC Ca2⫹ release from the SR in the form of Ca2⫹ waves to regulate expression and activity of other transcription factors and proteins as well. Although our results demonstrate that Ca2⫹ waves are not required for constriction in response to a low concentration of ET-1 in mesenteric arteries, they also indicate that ET-1mediated Ca2⫹ wave activity is increased in this vascular bed in a rat model of sleep apnea. The significance of the increased Ca2⫹ wave activity after IH exposure is therefore an intriguing area for future investigations.

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