Mechanisms underlying the impairment of endothelium-dependent relaxation in the pulmonary artery of monocrotaline-induced pulmonary hypertensive rats

Abstract

1. It has been reported that endothelium-dependent relaxation is impaired in pulmonary hypertensive vessels. The underlying mechanisms for this phenomenon, however, have not yet been identified. In this study, the mechanisms responsible for decreased endothelium-dependent relaxation in the pulmonary artery isolated from monocrotaline (MCT)-induced pulmonary hypertensive rat (MCT rat) were examined. MCT (60 mg kg-1), or its vehicle was administered by a single subcutaneous injection to 6-week-old male Sprague Dawley rats. 2. Endothelium-dependent relaxation induced by carbachol or ionomycin in the MCT rat artery was significantly smaller than that in vehicle-treated rat (control rat) artery. Cyclic GMP levels, measured by enzyme-immunoassay, under resting or stimulation with carbachol or ionomycin were also smaller in the MCT rat artery. However, sodium nitroprusside-induced cyclic GMP accumulation in the endothelium-denuded artery was similar in control and MCT rats. These results suggest that MCT treatment decreases endothelial nitric oxide (NO) production. 3. Resting endothelial Ca2+ levels ([Ca2+]i) in the fura-PE3-loaded MCT rat artery, were not different from those in the control rat. However, the increase in endothelial [Ca2+]i elicited by carbachol was attenuated in the MCT rat. 4. In quantitative RT - PCR analysis, the expression of mRNA encoding endothelial NO synthase was rather increased in the MCT rat artery, suggesting an up-regulation of eNOS expression. 5. These results provide evidence that impaired NO-mediated arterial relaxation in the MCT rat is due to dissociation between eNOS expression and NO production. This dissociation may be derived from an inhibition of receptor-mediated Ca2+ metabolism and also from the apparent decrease in Ca2+ sensitivity of eNOS.

Figure 1

(A) Effects of carbachol on phenylephrine (1 μM)-induced contraction in the endothelium-intact pulmonary artery. (B) Effects of ionomycin on phenylephrine (1 μM)-induced contraction in the endothelium-intact pulmonary artery. Carbachol or ionomycin was added after the phenylephrine-induced contraction had reached a steady-state level. Results are expressed as mean±s.e.mean. (n=4–7). ^* and ^**; significantly different from the control rat with P<0.05 and P<0.01, respectively.

Figure 2

Changes in cyclic GMP content in the pulmonary artery of control and MCT rats. (A) shows the effect of SNP (1 μM) in the endothelium-denuded artery of control and MCT rats. (B) shows the effects of carbachol and ionomycin in the endothelium-intact pulmonary artery of control and MCT rats. Results are expressed as mean±s.e.mean. (n=6–21). ^* and ^**; significantly different between the control rat and MCT rat with P<0.05 and P<0.01, respectively. NS, not significant.

Figure 3

Effect of carbachol on endothelial [Ca²⁺]_i. (A) shows a typical recording of the change in [Ca²⁺]_i in endothelial cell measured in situ. After observation of [Ca²⁺]_i response to carbachol at 1.5 mM [Ca²⁺]_o, ionomycin (1 μM) was added at 6.5 mM [Ca²⁺]_o to obtain a maximum [Ca²⁺]_i level (R_max), and external Ca²⁺ was removed (with 4 mM EGTA) to obtain a minimum [Ca²⁺]_i level (R_min). (B) shows the effect of carbachol on endothelial [Ca²⁺]_i in control and MCT rats. Results are expressed as mean±s.e.mean. (n=6). ^**; significantly different between the control rat and MCT rat with P<0.01.

Figure 4

Quantitative RT–PCR for determination of mRNA of eNOS and iNOS in pulmonary artery with endothelium. Agarose-gel electrophoresis of RT–PCR products for eNOS, iNOS, and GAPDH after 28, 33, 38, and 43 cycles of amplification. Total RNA was isolated from the endothelium-intact artery of control and MCT rats. Agarose-gel electrophoresis demonstrated RT–PCR products of expected size corresponding to mRNA encoding the iNOS (442 base pairs), eNOS (459 base pairs), and GAPDH (308 base pairs).