Jun Sato

Accumulation of endogenous inhibitors for nitric oxide synthesis and decreased content of L-arginine in regenerated endothelial cells.

1 We examined regeneration of endothelial cells (ECs), neointima formation, decreased endothelium‐dependent relaxation (EDR) and changes in the contents of L‐arginine, NG–monomethyl‐L‐arginine (l‐NMMA), asymmetrical NG, NG–dimethylarginine (ADMA) and symmetrical NG, NG–dimethylarginine (SDMA) in the regnerated ECs, 6 weeks after balloon denudation of the rabbit carotid artery. 2 Regeneration of ECs was completed in 6 weeks and a significant neointima formation accompanied by the decreased EDR was observed. 3 L‐NMMA and ADMA contents in the regenerated ECs (23.5 ± 4.3 and 21.2 ± 2.0 pmol mg−1 DNA, respectively) were significantly (P<0.05 and P<0.01) higher than those in the control ECs (8.8 ± 3.0 and 7.4plusmn;1.9 pmol mg−1 DNA, respectively), whereas L‐arginine was significantly (P< 0.005) decreased in the regenerated ECs (31,470±1,050 pmol mg−1 DNA) as compared to that in the control ECs (47,870±1,890 pmol mg−1 DNA). SDMA content was below the assay limits. 4 L‐NMMA and ADMA, but not SDMA, inhibited the EDR induced by acetylcholine in a concentration‐dependent manner. The inhibition with L‐NMMA and ADMA was prevented by an addition of L‐arginine, but not by D‐arginine. 5 These results suggest that the accumulation of endogenous inhibitors for nitric oxide synthesis and decreased L‐arginine content are associated with decreased NO production/release from regenerated ECs and neointima formation.

Effect of nicotine on the intimal hyperplasia after endothelial removal of the rabbit carotid artery

1. The present experiments were designed to investigate the effect of long-term oral nicotine (10 mg/200 ml/kg/day for 7 weeks) on the intimal hyperplasia after endothelial removal of the rabbit carotid artery. 2. The plasma concentrations of nicotine were determined to be 11.7-12.5 ng/ml during the term of administration and corresponded to the plasma levels in human smokers. 3. Six weeks after the endothelial removal, light microscopy revealed a marked intimal hyperplasia. Administration of nicotine tended to accelerate the intimal hyperplasia, which was estimated by comparing the histological findings, DNA content and wet weight of the vessel wall. 4. Acetylcholine- and A23187-induced endothelium-dependent relaxations were greatly impaired in the hyperplastic artery strips. The impairment of relaxations tended to be accelerated in the nicotine group. Sodium nitroprusside-induced relaxation was not different between the control and the hyperplastic artery strips and remained unaffected in the nicotine group. 5. The concentrations of endogenous nitric oxide (NO) synthesis inhibitors, NG-monomethyl-L-arginine (L-NMMA) and asymmetrical NG,NG-dimethyl-L-arginine (ADMA) were significantly more increased in the regenerated endothelial cells compared with those in the control endothelial cells. The concentrations of L-NMMA and ADMA in the regenerated endothelial cells were significantly increased by as much as 1.3 x 10(-6) and 5.6 x 10(-7) M, respectively, in the nicotine group. 6. Immunoreactive endothelin-1 was significantly increased in the hyperplastic vessel wall (2.4 times that of the control) in 6 weeks. Administration of nicotine tended to increase the level. 7. It seems possible to assume from these results that, although, under the present experimental conditions, nicotine exhibited a tendency to accelerate the intimal hyperplasia after endothelial removal, the longer exposure to nicotine or a higher dose of the agent or both would significantly accelerate the intimal hyperplasia through the enhanced impairment of endothelium-derived relaxing factor/ NO production, which might be brought about by the enhanced increases in L-NMMA and ADMA concentrations, and the enhanced increase in endothelin-1 in the vessel wall.

Different localization of ETA and ETB receptors in the hyperplastic vascular wall.

We investigated which subtypes of endothelin-1 (ET-1) receptors are involved in the pathogenesis of angioplasty-induced lesion formation in the rabbit carotid artery. Four weeks after removing endothelial cells (EC), we noted a marked intimal hyperplasia. The Bmax values for [125I]ET-1 and [125I]IRL1620 (an agonist for the ETB receptors) bindings were greater in the hyperplastic artery, without changes in Kd values. [125I]ET-1 binding was completely inhibited by unlabeled ET-1 and Ro 462005, a mixed-type antagonist for the ETA and ETB receptors, but partially by BQ123, a selective antagonist for ETA receptors, and IRL1620. The [125I]ET-1 binding sites not inhibited with BQ123 were significantly increased in the hyperplastic artery. The binding study suggested the presence of non-ETA/non-ETB receptors. The rank order of the increasing ratio in the density of receptors was ETB > putative non-ETA/non-ETB > total ET-1 receptors > ETA. The histochemical experiments with biotinylated ET-1 at lysine-9 side chain alone or in combination with unlabeled ET-1, BQ123, Ro 46–2005, or IRL1620, showed the ETA receptors to be localized mainly in the media, whereas the ETB receptors localized mainly in the neointima. These results suggest that the increased ET-1 receptors, especially ETB and/or putative non-ETA/non-ETB, are closely related to the occurrence of the intimal hyperplasia after endothelial removal.

Endogenous asymmetrical dimethylarginine and hypertension associated with puromycin nephrosis in the rat

1 The present experiments were designed to investigate the role of asymmetrical NG,NG‐dimethyl‐L‐arginine (ADMA) in causing hypertension associated with the focal and segmental glomerulosclerosis (FSGS) produced by a single bolus of puromycin aminonucleoside (PAN) and successive injection of protamine for 7 days in rats which had undergone unilateral nephrectomy. 2 After the unilateral nephrectomy, and administering PAN and protamine, histological examinations of the kidney revealed a typical FSGS, that is, evident abnormalities including segmental mesangial proliferation, obliteration of glomerular capillary lumens and adhesions between the glomerulus and Bowmans capsule could be observed. Changes in the glomerular epithelial cells consisted of the swelling with bleb formation. 3 In the FSGS rats, urine volume and urinary protein were significantly (P<0.05 and P<0.005) increased throughout 4‐week experimental period, while the creatinine clearance was significantly (P<0.005) and transiently decreased, and recovered 4 weeks later. These changes were associated with the sustained elevation of the systolic blood pressure. 4 ADMA levels in aortic endothelial cells, plasma and urine were significantly (P<0.05 and P<0.005) increased in the FSGS rats, but the level in the kidney remained unchanged. 5 The basal level and net production of cyclic GMP in the aortic vessel wall with endothelium when stimulated by norepinephrine and acetylcholine were significantly (P<0.05 and P<0.01) attenuated in the FSGS rats. 6 There were significant and positive correlations between systolic blood pressure (y) and ADMA levels (x) in endothelial cells (y=4.43x+122.2, r=0.979, P<0.0001), plasma (y=0.10x+71.9, r=0.921, P<0.001) and urine (y=0.48x+126.9, r=0.699, P<0.005), but not significant in the kidney (y=0.06x+102.7, r=0.252, NS). 7 These findings suggest that ADMA as an endogenous inhibitor of NO synthesis may play an important role for the pathogenesis in the hypertension associated with the experimental FSGS in the rat.

Intimal hyperplasia in human uterine arteries accompanied by impaired synergism between prostaglandin I2 and nitric oxide.

1 The present experiments were designed to investigate the mechanisms causing intimal hyperplasia in connection with the impaired synergism between prostaglandin I2 (PGI2) and nitric oxide (NO) in human uterine arteries (UAs). 2 In order to assess the magnitude of intimal hyperplasia, the intimaimedia ratio (%) was estimated with the aid of an image analyser. Human UAs were classified into two groups, I and II on the basis of the ratio and the degree of elastin deposition of histologically normal specimens. The intimaimedia ratio in group II was determined to be 38.9 ± 7.7% (n = 6), which was significantly (P < 0.01) higher than that in group I (16.5 ± 1.5%, n = 7). Less deposition of elastin was found in group I than in group II. 3 The relaxation activities of iloprost (IP) as a stable analogue of PGI2 and sodium nitroprusside (SNP) as a NO donor were not different between the two groups. When the minimum concentrations (Cmin) of IP and SNP in producing relaxation were applied together to the UA strips, these compounds interacted synergistically in group I. The observed relaxation (48.7 ± 8.8%, n = 7) in this group was significantly (P < 0.01) greater than the predicted value of 18.8 ± 3.1% (n = 7) (the mathematical sum of the relaxations caused by IP and SNP alone). By contrast, these agents interacted in an additive manner in group II. The observed relaxation (20.8 ± 9.5%, n = 6) was not significantly different from the predicted value (18.6 ± 2.4%, n = 6) in this group. 4 During the relaxation produced by the addition of IP and SNP alone or in combination, the changes in cyclic nucleotides (cyclic AMP and cyclic GMP) contents (pmol mg−1 protein) were assayed. When IP and SNP at Cmin were applied together to the UA strips, these compounds interacted synergistically in increasing cyclic nucleotides in group I. The observed net increase in the content was determined to be 1.46 ± 0.30 (P < 0.05 vs. the predicted value of 0.67 ± 0.12) in this group (n = 7). By contrast, the observed net increase (0.40 ± 0.07, n = 6) did not exceed the predicted value (0.65 ± 0.07, n = 6) in group II. 5 These results suggest that the formation of intimal hyperplasia in group II may be closely related to the impaired synergism between PGI2 and NO in the human UAs.

Impaired endothelium-dependent relaxation in rabbit pulmonary artery after subarachnoid hemorrhage

We investigated the changes in endothelial function of pulmonary as well as basilar artery after experimental subarachnoid hemorrhage (SAH) induced by cisternal blood injection in rabbits. The animals were killed on day 2 and day 7, and the mechanical responses were determined on transverse strips of the main pulmonary artery and rings harvested from the basilar artery. To examine the role of the vagal nerve, we cut the left vagal nerve immediately before injecting cisternal blood. Relaxation response of pulmonary and basilar arteries to acetylcholine (ACh) and A23187 were abolished after endothelial removal and reality inhibited by either NG-nitro-L-arginine (NOARG) or methylene blue (MB). Indomethacin failed to modify these relaxation responses. Relaxation response of the main pulmonary artery strips to ACh was significantly (p < 0.05 and p < 0.01) attenuated on day 2 in 6 of 12 rabbits. In the remaining 6 rabbits, relaxation was not affected. No change in relaxation responses to A23187 and sodium nitroprusside (SNP) was observed in any of the 12 cases. In the vagotomized rabbits no decreased response to ACh was observed. On day 7, relaxation response to ACh returned to normal. ACh also produced decreased relaxation in basilar artery rings on day 2 with no change in A23187- and SNP-induced relaxation. Contractile responses of pulmonary and basilar arteries to norepinephrine (NE), endothelin-1 (ET-1), 5-hydroxytryptamine (5-HT), and U46619 were not affected after SAH. These results suggest that less functional muscarinic receptors that produce/release less endothelium-derived relaxation factor [EDRF/nitric oxide (NO)] are involved in causing the reduced relaxation of pulmonary and basilar arteries after SAH and the vagal nerve may play a role in regulating the receptor-mediated, endothelium-dependent relaxation in the main pulmonary artery after experimental SAH.