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Am J Physiol Heart Circ Physiol 293: H2281-H2288, 2007. First published July 27, 2007; doi:10.1152/ajpheart.00566.2007
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Altered role of smooth muscle endothelin receptors in coronary endothelin-1 and {alpha}1-adrenoceptor-mediated vasoconstriction in Type 2 diabetes

S. B. Bender and R. E. Klabunde

Department of Biomedical Sciences, Ohio University College of Osteopathic Medicine, Athens, Ohio

Submitted 15 May 2007 ; accepted in final form 23 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Regulation of vascular tone and blood flow involves interactions between numerous local and systemic vascular control signals, many of which are altered by Type 2 diabetes (T2D). Vascular responses to endothelin-1 (ET-1) are mediated by endothelin type A (ETA) and type B (ETB) receptors that have been implicated in cross talk with {alpha}1-adrenoceptors ({alpha}1-AR). ETA and ETB receptor expression and plasma ET-1 levels are elevated in T2D; however, whether this influences coronary {alpha}1-AR function has not been examined. Therefore, we examined the effect of ETA and ETB receptor inhibition on coronary vasoconstriction to ET-1 and {alpha}1-AR activation in a mouse model of T2D. Coronary vascular responses were examined in isolated mouse hearts from control and diet-induced T2D C57BL/6J mice. Responses to ET-1 and the selective {alpha}1-AR agonist phenylephrine (PE) were examined alone and in the presence of the nitric oxide synthase inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME) alone or in combination with selective ETA or ETB receptor inhibitors BQ-123 and BQ-788, respectively. Vasoconstriction to ET-1 was enhanced, whereas ETB, but not ETA, receptor blockade reduced basal coronary tone in T2D hearts. In the presence of L-NAME, ETA receptor inhibition attenuated ET-1 vasoconstriction in both groups, whereas ETB inhibition abolished this response only in control hearts. In addition, ETA inhibition enhanced {alpha}1-AR-mediated vasoconstriction in T2D, but not control, hearts following L-NAME treatment. Therefore, in this model, enhanced coronary ET-1 responsiveness is mediated primarily through smooth muscle ETB receptors, whereas the interaction with {alpha}1-ARs is mediated solely through the ETA receptor subtype.

isolated heart; C57BL/6J mouse; diet-induced diabetes

TYPE 2 DIABETES (T2D) is characterized by endothelial dysfunction and the abnormal release and/or action of endothelium-derived vasoactive substances (28). Experimental studies have demonstrated elevated plasma levels of the potent endothelium- derived vasoconstrictor endothelin-1 (ET-1) along with enhanced expression and/or coupling of vascular ET-1 receptors in T2D (15, 17, 43). Activation of endothelin type A (ETA) and type B (ETB) receptors on vascular smooth muscle by ET-1 induces vasoconstriction, whereas activation of ETB receptors on endothelial cells leads to ET-1-mediated vasodilation through stimulation of nitric oxide (NO) production by NO synthase (NOS), as well as prostacyclin release in some vessels (35, 37). Although changes in receptor gene and protein expression have been demonstrated, the functional consequence of the diabetes-related alterations in these receptors remains unclear due to reports of enhanced (15, 17), reduced (40), or unchanged (36) ET-1-mediated vasoconstriction in this state.

This disparity in ET-1-mediated vasoconstriction in T2D may be due to differences in species studied or disease severity but may also indicate alterations in signaling cross talk between vasoactive systems. Concomitant with diabetes-induced elevations in plasma ET-1, studies have reported the presence of sympathetic overdrive and enhanced adrenergic receptor expression in this state (20, 24, 45). Recently, cross talk between the ETA and {alpha}1-adrenergic receptors ({alpha}1-AR) has been reported. In rat-1 fibroblasts transfected with hamster {alpha}1b-ARs, ETA activation resulted in {alpha}1-AR phosphorylation and inhibition of {alpha}1-AR activation (38). This is supported in vivo by the attenuation of stress-induced, {alpha}1-AR-mediated vasoconstriction by endogenous ET-1 in Dahl salt-resistant and ET-1-dependent, salt-sensitive, hypertensive rats (6, 7). Little is currently known, however, concerning this interaction within the Type 2 diabetic milieu.

Coronary tone and blood flow are normally maintained by an appropriate balance of endogenous vasodilator and vasoconstrictor stimuli acting on coronary smooth muscle. This balance, however, is offset in T2D by a reduction in NO bioavailability, allowing for the enhanced release and/or action of vasoconstrictors, such as ET-1, the actions of which are normally blunted by the vasodilator influence of NO (18, 42). Therefore, enhanced activation of ET-1 receptors in diabetes, either singularly or through cross talk with other vasoconstrictor systems, may contribute to the elevated risk of myocardial ischemia and infarct in this patient population.

We hypothesized that 1) coronary vasoconstriction to ET-1 would be enhanced via both the ETA and ETB receptor subtypes located on vascular smooth muscle and 2) {alpha}1-AR-mediated vasoconstriction would be attenuated by basal ETA receptor activation in T2D. These hypotheses were tested using isolated hearts from control and diabetic mice that were perfused by the Langendorff method. The hearts were pretreated with a NOS inhibitor so that these interactions could be studied without the confounding influences of different endogenous NO levels.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Induction of obesity and T2D. All protocols were approved by the Institutional Animal Care and Use Committee of Ohio University. Male C57BL/6J mice were obtained from Jackson Laboratories (Bar Harbor, ME) at 3, 7, or 9 wk of age. Three-wk-old mice were given ad libitum access to water and a high-fat, high-simple carbohydrate, low-fiber diet containing 20.5% protein, 35.8% fat, 0.4% fiber, 3.6% ash, 3.1% moisture, and 36.8% carbohydrate (F1850; Bio-Serve, Frenchtown, NJ) to induce T2D for 15 wk as previously described (34). Control 7- and 9-wk-old mice were fed a standard rodent chow containing 22.0% protein, 5.0% fat, 5.0% fiber, 6.0% ash, 2.5% added minerals, and 59.5% carbohydrate (Prolab RMH 3000; LabDiet, Richmond, IN) for 11 or 9 wk, respectively. Animals were housed four per cage in a temperature-controlled room with a 12-h:12-h light-dark cycle.

Blood collection. Fasted mice (25–50 g) were weighed before blood glucose determination from tail vein blood using a calibrated OneTouch Ultra glucometer.

Isolated heart preparation. Mice were heparinized (300 IU ip) and anesthetized with pentothal sodium (100 mg/kg ip). Hearts were rapidly excised by bilateral thoracotomy and placed into ice-cold buffer, and the aorta were cannulated. Each heart was submerged, perfused, and beating spontaneously on a nonrecirculating constant-flow Langendorff perfusion apparatus at 37°C with filtered Krebs-Henseleit buffer (KHB) consisting of (in mmol/l): 118.5 NaCl, 25 NaHCO3, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, and 11.1 glucose, gassed with 95% O2-5% CO2. A small polyethylene vent tube was inserted through the mitral valve into the left ventricle. Coronary perfusion pressure (CPP) was monitored using a PowerLab workstation (ADInstruments, Colorado Springs, CO). Hearts were equilibrated for 20 min before beginning any experimental protocol, after which they were removed, blotted, and weighed.

Experimental protocols. Four protocols were utilized in this study in isolated hearts from control and T2D animals. Baseline CPP for all protocols was 60 mmHg.

The first protocol examined the role of endogenous ET-1 in basal coronary tone. Following equilibration, hearts were perfused for 30 min with the selective ETA receptor antagonist BQ-123 (10–6 mol/l) or the selective ETB receptor antagonist BQ-788 (10–6 mol/l). Coronary vascular resistance (CVR) changes were measured after 30 min.

The second protocol examined coronary reactivity to exogenous ET-1 and the role of ETA and ETB receptors in this response. Coronary responses to ET-1 (10–10 mol/l) were determined with or without pretreatment with the NOS inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME) (10–5 mol/l) alone or in combination with BQ-123 (10–6 M) or BQ-788 (10–6 M) for 30 min. In experiments where no pretreatment was used, hearts were perfused with KHB for 30 min before the addition of ET-1. L-NAME was included in this protocol and protocol 3 to eliminate the negative influence of both shear stress and endothelial ETB-mediated NO release on coronary vasoconstrictor responses. This allows for the selective examination of smooth muscle ETB receptors by blockade with BQ-788. Following pretreatment, CPP was readjusted to baseline and ET-1 was added to the perfusate reservoir for 1 h, at which time CPP was measured. ET-1-mediated vasoconstriction in the isolated mouse heart is a slow-onset phenomenon (45–60 min for maximum response); therefore, to keep total protocol length <3 h (reasonable preparation stability limit), only a single dose of ET-1 was utilized. The dose used was chosen based on reported EC50 values for ET-1 in numerous mouse vessels, including septal coronary arteries, which fall around 10–9 M (23, 25, 36, 47). The use of a single submaximal dose of ET-1 is necessary to study the potentiation of vasoconstriction following NOS inhibition.

The third protocol examined coronary {alpha}1-AR reactivity and the involvement of endogenous ET-1 via ETA and ETB receptors. Dose responses to the selective {alpha}1-AR agonist phenylephrine (PE, 10–9–10–5 mol/l) were performed following the same pretreatments used in protocol 2. Similarly, in experiments where no pretreatment was used, hearts were perfused with KHB for 30 min before PE dose-response measurements. Following pretreatment, CPP was readjusted to baseline and PE dose response was performed. PE was infused into the coronary perfusion cannula through a 0.22-µm syringe filter with a syringe infusion pump at 5% of coronary flow. Responses to PE were measured when a stable CPP was achieved at each dose (within 8 min).

The fourth protocol examined the role of ET-1 in the vasoconstrictor response to NOS inhibition by L-NAME. Following equilibration, hearts were perfused for 30 min with L-NAME (10–5 M) combined with either BQ-123 (10–6 M) or BQ-788 (10–6 M) to block ETA and ETB receptors, respectively. CVR changes were measured after 30 min.

Drugs and chemicals. ET-1, BQ-123, and BQ-788 were obtained from American Peptide (Sunnyvale, CA). All other drugs and chemicals were obtained from Sigma-Aldrich (St. Louis, MO). ET-1 was prepared in phosphate-buffered saline with 0.05% bovine serum albumin. BQ-123 and BQ-788 were prepared in deionized water. ET-1, BQ-123, and BQ-788 stock solutions were frozen and thawed on the day of the experiment. All other solutions, including KHB, were prepared on the day of the experiment.

Statistics. CVR was calculated by dividing CPP by coronary flow normalized per gram of heart. Descriptive statistics (means ± SE) were computed, and data were graphed using Excel. Data were expressed as percent change from baseline CVR and analyzed using InStat (GraphPad Software, San Diego, CA) and NCSS (Kaysville, UT) software. Dose-response curves for PE were compared by two-way ANOVA for repeated measures with Fisher least significant difference (LSD) post hoc test. ET-1 and L-NAME responses were compared by one-way ANOVA with Fisher LSD post hoc test. Other data were compared by Student's unpaired t-test. A P value <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Induction of obesity and T2D. Fasting blood glucose concentration increased from 6.9 ± 0.2 mmol/l (126.3 ± 3.1 mg/dl) in control animals (n = 96) to 9.5 ± 0.3 mmol/l (172.9 ± 5.8 mg/dl) in T2D animals (n = 104). Body weight increased in high-fat fed T2D mice compared with controls by 39% (28.9 ± 0.2 vs. 40.1 ± 0.6 g). Heart weight also increased in T2D mice by 16% (169.9 ± 2.8 vs. 146.7 ± 2.2 mg). Average mouse ages were 136.7 ± 2.4 and 130.8 ± 0.8 days for control and T2D groups, respectively. We previously reported a 316% increase in plasma insulin in this model (1).

Baseline coronary flow values normalized per gram of tissue for each treatment group before any drug pretreatment are presented in Table 1. While some instances of different flows before drug pretreatments between groups exist, baseline CVRs following pretreatments (reported below for each group) and before the administration of an agonist were not different between groups.


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  		Table 1. Baseline coronary flows per gram of heart weight in all treatment groups before inhibitor pretreatments

 
Protocol 1: effects of ETA and ETB blockade on CVR. The role of endogenous ET-1 in basal coronary tone was examined by individual blockade of ETA and ETB receptors. Coronary perfusion with BQ-123 did not alter CVR in control or T2D hearts from baselines of 6.2 ± 1.0 and 6.2 ± 1.1 mmHg·ml–1·min–1·g–1, respectively (P > 0.05, Fig. 1A). Perfusion with BQ-788, however, induced vasodilation in T2D hearts, but not control (Fig. 1B). CVR decreased 27% from a baseline of 5.9 ± 0.9 mmHg·ml–1·min–1·g–1 in T2D hearts. Baseline CVR in control hearts treated with BQ-788 was 9.2 ± 1.8 mmHg·ml–1·min–1·g–1(P > 0.05 vs. T2D baseline).


Figure 1
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  		Fig. 1. Coronary responses in control and Type 2 diabetic (T2D) hearts to selective endothelin type A (ETA) (A) or endothelin type B (ETB) (B) receptor inhibition with BQ-123 (10–6 mol/l) and BQ-788 (10–6 mol/l), respectively. Vertical bars are means ± SE; sample size in parentheses. *P < 0.05 vs. control (within group). CVR, coronary vascular resistance.

 
Protocol 2: coronary responses to exogenous ET-1. ET-1 (10–10 mol/L) produced significantly greater vasoconstriction in T2D hearts (97% CVR increase) than in control hearts (27% CVR increase) (Fig. 2, A and B). Baseline CVRs in these groups were 6.9 ± 0.5 and 6.4 ± 0.7 mmHg·ml–1·min–1·g–1, respectively (P > 0.05). Elimination of endogenous NO production by NOS inhibition enhanced this response in control (82% CVR increase), but not T2D, hearts (Fig. 2). Baseline CVRs following L-NAME pretreatment in these groups were 13.0 ± 2.6 and 12.1 ± 0.8 mmHg·ml–1·min–1·g–1, respectively (P > 0.05). Since ET-1 responses were similar in control and T2D hearts following NOS inhibition, examination of ETA and ETB receptor involvement in ET-1-mediated vasoconstriction was performed in the presence of L-NAME.


Figure 2
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  		Fig. 2. Coronary vasoconstrictor responses in control (A) and T2D (B) hearts to endothelin-1 (ET-1) (10–10 mol/l) alone or following pretreatment with the NO synthase (NOS) inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME) (10–5 mol/l) alone or in combination with the ETA receptor antagonist BQ-123 (10–6 mol/l) or the ETB receptor antagonist BQ-788 (10–6 mol/l). Vertical bars are means ± SE; sample size in parentheses. *P < 0.05 vs. all other treatments (within group); **P < 0.05 vs. response in the presence of L-NAME only (within group); {dagger}P < 0.05 vs. L-NAME + BQ-123 (within group); {ddagger}P < 0.05 vs. control ET-1 alone (between groups).

 
Evaluation of ETA and ETB receptor involvement in ET-1-mediated vasoconstriction revealed a significant role for each receptor. Pretreatment with L-NAME plus the ETA blocker BQ-123 attenuated coronary responses to exogenous ET-1 (10–10 mol/l) in both control and T2D hearts (Fig. 2). ET-1-mediated vasoconstriction was reduced by 52% and 60% in control and T2D hearts compared with responses without ETA receptor inhibition. Baseline CVRs were 9.8 ± 1.3 and 5.9 ± 1.4 mmHg·ml–1·min–1·g–1, respectively (P > 0.05). Pretreatment with L-NAME plus the ETB blocker BQ-788 completely inhibited coronary ET-1 vasoconstriction in control hearts but had no effect in T2D hearts (Fig. 2). Baseline CVRs were 7.8 ± 0.9 and 5.7 ± 1.4 mmHg·ml–1·min–1·g–1, respectively (P > 0.05).

Protocol 3: coronary responses to PE. PE induced similar dose-dependent coronary constriction in control versus T2D animals with and without NOS inhibition (Fig. 3). Under basal conditions, high-dose PE (10–5 mol/l) increased CVR 30% in control hearts and 37% in T2D hearts from baselines of 8.0 ± 0.7 and 6.6 ± 0.7 mmHg·ml–1·min–1·g–1, respectively (P > 0.05). Following NOS inhibition, vasoconstriction to PE was enhanced in control, but not T2D, hearts (Fig. 3). Following L-NAME pretreatment, PE (10–5 mol/l) increased CVR 49% in control hearts and 51% in T2D hearts from baselines of 16.0 ± 2.2 and 11.3 ± 2.0 mmHg·ml–1·min–1·g–1, respectively (P > 0.05). Since NOS inhibition affected PE-induced vasoconstriction in control hearts, an examination of ET-1 receptor involvement in this response was done in the presence of L-NAME to eliminate the negative influence of NO.


Figure 3
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  		Fig. 3. Coronary vasoconstrictor responses in control (A) and T2D (B) hearts to {alpha}1-adrenoceptor ({alpha}1-AR) activation with phenylephrine (PE) (10–9–10–5 mol/l) alone or following pretreatment with the NOS inhibitor L-NAME (10–5 mol/l) alone or in combination with the ETA receptor antagonist BQ-123 (10–6 mol/l) or the ETB receptor antagonist BQ-788 (10–6 mol/l). Vertical bars are means ± SE; sample size in parentheses. *P < 0.05 vs. PE alone (within group); {dagger}P < 0.05 vs. PE alone and plus L-NAME (within group); {ddagger}P < 0.05 vs. all other treatments (within group).

 
Evaluation of endogenous ET-1 acting via ETA and ETB receptors in PE-mediated vasoconstriction revealed that ETA receptor activation acts to blunt this response in T2D hearts. Following L-NAME plus BQ-123 pretreatment, PE responses were enhanced in T2D, but not control, hearts (Fig. 3). PE (10–5 mol/l) increased CVR 86% in T2D hearts compared with 44% in control hearts. Baseline CVRs following pretreatment were 7.9 ± 1.0 and 8.6 ± 1.9 mmHg·ml–1·min–1·g–1, respectively (P > 0.05). Inhibition of ETB receptors, however, did not affect PE-induced vasoconstriction in either group. Following L-NAME plus BQ-788 pretreatment, PE responses were unchanged from responses with L-NAME alone in each group (Fig. 3). Baseline CVRs were 12.5 ± 2.9 and 9.8 ± 1.6 mmHg·ml–1·min–1·g–1 in control and T2D hearts, respectively (P > 0.05).

Protocol 4: role of ET-1 in L-NAME-induced vasoconstriction. The use of combination pretreatments (L-NAME plus ETA or ETB antagonists) in this study also revealed that endogenous ET-1 contributes to the coronary constriction induced by NOS inhibition in control, but not T2D, hearts. We have previously shown that coronary vasoconstriction to NOS inhibition is reduced in T2D hearts compared with control (77% vs. 101% CVR increase) (1). In the current study, ETA and ETB receptor inhibition reduced L-NAME-mediated coronary constriction by 55% and 42% in control and T2D hearts, respectively (Fig. 4). Baseline CVRs in the combined pretreatment groups were 5.8 ± 0.6 and 7.5 ± 0.5 mmHg·ml–1·min–1·g–1 for L-NAME plus BQ-123 or BQ-788, respectively. These combined pretreatments did not affect coronary vasoconstriction to L-NAME in T2D hearts, as noted by similar CVR increases in the absence of the selective ETA and ETB receptor antagonists (Fig. 4). Baseline CVRs in the T2D-combined pretreatment groups were 4.9 ± 0.6 and 4.5 ± 0.4 mmHg·ml–1·min–1·g–1 for L-NAME plus BQ-123 or BQ-788, respectively.


Figure 4
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  		Fig. 4. Coronary vasoconstrictor responses in control (A) and T2D (B) hearts to NOS inhibition with L-NAME (10–5 mol/l) alone or in combination with the ETA receptor antagonist BQ-123 (10–6 mol/l) or the ETB receptor antagonist BQ-788 (10–6 mol/L). Vertical bars are means ± SE; sample size in parentheses. *P < 0.05 vs. L-NAME alone (within group); **P < 0.05 vs. L-NAME alone (between groups).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Current evidence remains unclear concerning the effect of T2D on the role of vascular ETA and ETB receptors in vascular regulation although a number of studies suggest an enhanced contribution of vasoconstrictor pathways mediated through these receptors. Recent data also demonstrate that significant functional cross talk may exist between the ETA and {alpha}1-adrenergic receptor subtypes (6, 7, 38). Therefore, this study evaluated the hypotheses that 1) coronary vasoconstriction to ET-1 would be enhanced via both the ETA and ETB receptor subtypes located on vascular smooth muscle and 2) {alpha}1-AR-mediated vasoconstriction would be attenuated by basal ETA receptor activation in T2D. These hypotheses were evaluated in the presence of NOS blockade to alleviate the problem of differences in background NO bioavailability that are known to interact with endothelin vasoconstrictor pathways. The principle findings of this study, as illustrated in Fig. 5, are that 1) endogenous ET-1 contributes to basal coronary tone in T2D mouse hearts via ETB receptor activation; 2) basal smooth muscle ETB receptor activation is necessary for ETA-mediated vasoconstriction to ET-1 in control, but not T2D, hearts; and 3) basal ETA activation attenuates {alpha}1-AR-mediated vasoconstriction in T2D, but not control, hearts.


Figure 5
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  		Fig. 5. T2D-mediated alterations in the role of ETA and ETB receptors in coronary vasoconstriction to ET-1 and {alpha}1-AR activation in isolated mouse hearts. Actions that are enhanced or reduced by T2D are signified by + and –, respectively. Solid arrows, known actions of receptors/agonist; dotted arrows, T2D-mediated alterations in these actions as demonstrated in the present study; L-arg, L-arginine; [Ca2+]i, intracellular Ca2+ concentration.

 
Both ETA and ETB receptors are members of the G protein-coupled superfamily and activate numerous downstream signaling pathways (22). The most common result of activation of these receptors is an increase in intracellular calcium. In vascular smooth muscle this occurs through phospholipase C activation and subsequent inositol triphosphate production, resulting in smooth muscle contraction and vasoconstriction (27). Elevations in endothelial cell cytosolic calcium due to ETB receptor activation lead to NOS activation and NO release through calcium/calmodulin- and tyrosine kinase-dependent pathways (37). Endothelial prostacyclin release has also been shown to contribute to endothelial ETB-mediated vasodilation (35).

Effect of diabetes on ETA and ETB involvement in vasoconstriction to ET-1. Activation of smooth muscle ETA and, to a lesser extent, ETB receptors accounts for vasoconstriction to the potent endothelium-derived peptide ET-1 (17, 26, 36, 47), a response that is altered in the Type 2 diabetic vasculature (15, 17, 30, 31, 36). Our findings demonstrate a generalized enhancement of ET-1-mediated coronary vasoconstriction in T2D animals indicated by the elevated role of endogenous ET-1 in basal tone and enhanced vasoconstriction to exogenous ET-1. A similar enhancement of coronary ET-1-mediated vasoconstriction at this dose (10–10 M) has previously been reported in isolated hearts from streptozotocin diabetic rats (39). It is important to note, however, that the necessary use of a single dose of ET-1 in this study precludes us from differentiating whether the enhanced ET-1-mediated vasoconstriction is due to alterations in coronary ET-1 sensitivity, responsiveness, or both.

We found enhanced ET-1-mediated vasoconstriction in T2D hearts before pretreatment with the NOS inhibitor L-NAME. This finding may be the consequence of reduced NO bioavailability since NO, in healthy vessels, attenuates ET-1 production and/or activity (5, 11, 33). We have previously reported reduced coronary NO bioavailability, which was returned to control levels with the superoxide scavenger Tempol in this model (1). In addition, although not examined in this study, it is possible that the enhanced ET-1 responses in T2D coronary vessels are partly due to increased stimulation of endogenous vasoconstrictor release by ET-1. ET-1-mediated contraction of normal C57BL/6J mouse aorta is attenuated by cyclooxygenase inhibition (36). Furthermore, it is reported that thromboxane receptor and synthase expression in obese, probably diabetic, mouse aorta is elevated, suggesting an enhanced contribution of these pathways in this state. Unfortunately, the effect of cyclooxygenase inhibition on ET-1-mediated vasoconstriction in obese animals was not examined (36).

Historically, the assessment of endothelin receptors has focused on the ETA receptor subtype due to the assumption that most of the pathological effects of ET-1 are mediated by this receptor. Evidence is slowly accumulating, however, that suggests that changes in the smooth muscle ETB receptor subtype may also play a role in vascular pathologies. Elevated ETB receptor protein and mRNA expression have been reported in human atherosclerotic aortas and mesenteric arteries from insulin-resistant Zucker rats, respectively (14, 43). Furthermore, dual ETA/ETB receptor inhibition enhanced forearm blood flow more than selective ETA receptor inhibition in patients with atherosclerosis (4). Recently, Shemyakin et al. (32) reported enhanced endothelium-dependent vasodilation following dual ETA/ETB, but not selective ETA, receptor inhibition in the forearm vasculature of insulin-resistance patients, indicating an enhanced ETB-mediated vasoconstriction in these patients. This is consistent with our current results in the diabetic mouse coronary vasculature that also demonstrate an enhanced ETB-mediated vasoconstrictor component in basal coronary tone.

Our examination of smooth muscle ETA and ETB receptor involvement in coronary ET-1-mediated vasoconstriction was done in the presence of NOS inhibition with L-NAME to 1) eliminate NO-dependent endothelial ETB-mediated vasodilation and 2) eliminate the differential effects of endogenous NO since coronary NO bioavailability is reduced in this model of T2D (1). In addition to NO, it has been demonstrated that endothelial ETB-mediated vasodilation may also involve prostacyclin release, although this is unclear in the coronary vasculature (8, 35, 46). Recent experiments in our laboratory using the cyclooxygenase inhibitor meclofenamate have shown that basal coronary cyclooxygenase-derived vasodilator influences are not different between control and T2D hearts (O. Y. Suer and R. E. Klabunde, unpublished observations); however, we do not know whether diabetes alters prostanoid release that may occur via endothelial ETB receptor activation. Therefore, further studies are necessary to fully delineate the effect of T2D on the mechanism of coronary endothelial ETB-mediated vasodilation.

Following NOS inhibition, the ETA receptor-dependent component of ET-1 vasoconstriction was similar in both groups, suggesting little effect of T2D on normal ET-1/ETA receptor interactions. Our results examining the role of the ETB receptor, however, suggest that T2D also impacts the intracellular coupling or downstream signaling of this receptor. Combined ETB and NOS inhibition abolished coronary ET-1-mediated vasoconstriction in control animals but had no effect on this response in T2D coronary vessels. These results suggest that activation of smooth muscle ETB receptors is necessary for ETA-mediated vasoconstriction. The mechanism through which this occurs is currently unknown. Berthiaume et al. (2, 3) have previously reported in mice that ETB receptor inhibition with BQ-788 eliminated systemic, renal, and mesenteric vasoconstrictor responses to ET-1. Inscho et al. (13) have also demonstrated a similar phenomena in rat renal afferent arterioles where selective ETA and ETB receptor antagonism each inhibited low-dose (10–12–10–10 M) ET-1-mediated vasoconstriction. Whether this involves any direct interaction between smooth muscle ETA and ETB receptors is currently unknown; however, Just et al. (16) have previously proposed that ETA-mediated responses in the renal circulation may vary as a function of ETB receptor activity and vice versa. A recent report of ETA/ETB receptor heterodimerization may account for the findings of the present study if this heterodimerization is necessary for normal coronary ET-1 vasoconstriction and is disrupted by T2D (12). Therefore, current evidence suggests that vascular responses to ET-1 are governed by a complex series of interactions that remain to be fully elucidated.

Effect of diabetes on ETA and ETB involvement in vasoconstriction to {alpha}1-AR activation. In vitro and in vivo evidence has demonstrated that significant interactions exist between the ET-1 and {alpha}1-AR systems. In transfected rat-1 fibroblasts, ETA receptor activation leads to {alpha}1B/D-AR phosphorylation and desensitization (9, 38). Additionally, the suppression of {alpha}1-AR-mediated elevations in blood pressure by ET-1 have been demonstrated (6, 7, 10). Our results demonstrate, for the first time in a model of T2D, that endogenous ET-1 acting via ETA, and not via ETB, receptors attenuates {alpha}1-AR-mediated vasoconstriction following NOS inhibition. The mechanism mediating this response remains unclear but may involve downstream signaling through PKC. PKC activation with subsequent {alpha}1-AR phosphorylation has been reported in cell culture and may be suggested in the T2D vasculature due to elevated PKCbeta activity in mesenteric vessels of the db/db mouse (9, 19, 38). Similarly, suppression of sympathetic vasoconstriction has been reported in the ETB receptor-deficient rat, a model of ET-1-dependent hypertension (6). Overall, these data suggest that the ETA receptor acts to protect against myocardial ischemia due to enhanced sympathetically mediated coronary vasoconstriction in T2D.

The elevated maximal {alpha}1-AR-mediated vasoconstriction following ETA inhibition also may suggest {alpha}1-AR upregulation in T2D coronary vessels that is not functionally manifest in the presence of ETA-mediated {alpha}1-AR suppression. In other words, any possible enhancement in {alpha}1-AR-mediated vasoconstriction due to upregulation of these receptors is offset by the ETA-mediated reduction in {alpha}1-AR responsiveness in T2D. Yoshida et al. (45) demonstrated aortic {alpha}1B-AR upregulation and enhanced {alpha}1-AR sensitivity in T2D rat cremaster arterioles. In transfected rat-1 fibroblasts, human {alpha}1-ARs are differentially influenced by overstimulation in a manner that may result in a net upregulation (44). Although {alpha}1-AR expression was not measured in our model, upregulation may occur due to coronary {alpha}1-AR overstimulation resulting from sympathetic overdrive as reported in this model (20).

Effect of diabetes on the ET-1-dependent component of L-NAME vasoconstriction. The current experimental design also allowed for an examination of the role of endogenous ET-1 in vasoconstriction to NOS inhibition with L-NAME. Coronary resistance changes to these pretreatments revealed, as others have shown, that endogenous ET-1-mediated vasoconstriction via ETA and ETB receptors partially accounts for the coronary vasoconstriction to NOS inhibition in control animals (21, 29, 41). This is not surprising given the negative influence of NO on ET-1 production and/or activity (5, 11, 33). Conversely, the ET-1-dependent component of this response was absent in T2D animals. When coupled with the results that NOS inhibition did not enhance ET-1- or {alpha}1-AR-mediated vasoconstriction in T2D animals, these data suggest that basal and shear stress-sensitive production and/or bioavailability of NO is reduced such that coronary vasoconstriction or the local release of ET-1 in T2D mouse coronary vessels is not limited (1). The possibility does exist, however, that maximal coronary vasoconstriction was achieved with the single dose of ET-1 utilized in this study, thereby masking any potentiation of this response with NOS inhibition. Regardless of this, however, our data support a marked role for endogenous NO in the maintenance of basal coronary tone in control animals that remains, albeit to a lesser degree, in T2D.

Conclusions. The present study demonstrates significant diabetes-related alterations in ETA and ETB receptor subtype function in response to activation by ET-1 and in cross talk with {alpha}1-ARs. In this model of T2D, it appears that coronary ET-1 responsiveness is altered through smooth muscle ETB receptors, including their interaction with ETA receptors, whereas the interaction with {alpha}1-ARs is mediated solely through the ETA receptor subtype. To our knowledge, our data are the first to directly suggest significant effects of T2D on ETB receptor function and underscore the need for better examination of this receptor in subsequent studies examining vascular responses in T2D.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was funded by the Ohio University College of Osteopathic Medicine and Office of Vice President for Research.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the technical assistance of Emily Drummond and Dr. Leon Wince and critical review of this manuscript by Dr. M. Harold Laughlin and Dr. Sean Newcomer. Current address for Dr. Bender: E102 Vet Med Bldg., Dept. of Biomedical Sciences, University of Missouri, Columbia, MO, 65211.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. E. Klabunde, Dept. of Biomedical Sciences, I-304, Ohio Univ. College of Osteopathic Medicine, Athens, OH 45701 (e-mail: klabunde{at}ohio.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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