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We examined the effect of ischaemia on the neurogenic and nitric oxide (NO)-mediated urethral relaxation. Rabbits were divided into control and urethral ischaemia (UI) groups, which was prepared by the partial occlusion of bilateral iliac arteries using blood vessel occluders. Neurogenic and NO-mediated proximal urethral relaxation induced by electrical field stimulation (EFS) was greatly impaired in the UI group, while relaxation by sodium nitroprusside (SNP) as a NO donor showed no difference between the two groups. Pretreatment with L-arginine significantly improved but did not normalize the impaired relaxation in the UI group. Not only basal level, but also stimulated production of cyclic GMP with EFS, were significantly decreased in the UI group. The tissue contents of NG-methyl-L-arginine (L-NMA) and asymmetric NG, NG-dimethyl-L-arginine (ADMA) in the proximal urethra were increased following ischaemia. While L-arginine and symmetric NG, N′G-dimethyl-L-arginine (SDMA) contents remained unchanged. Exogenously applied authentic L-NMA and ADMA (1 – 100 μM) concentration-dependently inhibited the EFS-induced urethral relaxation in the control group. The inhibition with L-NMA and ADMA was undetectable in the presence of 3 mM L-arginine. The Ca2+-dependent NOS activity in the urethra from the UI group was significantly lower than that from the control group and was not restored by an addition of 3 mM L-arginine. These results suggest that the impaired neurogenic and NO-mediated urethral relaxation with ischaemia is closely related to the increased accumulation of L-NMA and ADMA and decreased NOS activity, which would result in an accelerated reduction in NO production/release. British Journal of Pharmacology (2001) 133, 97–106; doi:10.1038/sj.bjp.0704050

We examined whether endogenous inhibitors of NO synthesis are involved in the augmentation of intimal hyperplasia in rabbits with hyperglycaemia induced by alloxan. Four weeks after the endothelial denudation of carotid artery which had been performed 12 weeks after alloxan, the intimal hyperplasia was greatly augmented with hyperglycaemia. The degree of hyperplasia was assessed using three different parameters of histopathological findings as well as changes in luminal area and intima : media ratio. There were positive and significant correlations between intima : media ratio, plasma glucose, and concentrations of NG-monomethyl-L-arginine (L-NMMA) and NG, NG-dimethyl-L-arginine (ADMA) in endothelial cells, that is, the intima : media ratio became greater as plasma glucose and endothelial L-NMMA and ADMA were increased. Furthermore, endothelial L-NMMA and ADMA were increased in proportion to the increase in plasma glucose. In contrast, there were inverse and significant correlations between cyclic GMP production by carotid artery strips with endothelium and plasma glucose, between cyclic GMP production and endothelial L-NMMA and ADMA, and between the intima : media ratio and cyclic GMP production. Exogenously applied L-NMMA and ADMA inhibited cyclic GMP production in a concentration-dependent manner. IC50 values were determined to be 12.1 μM for the former and 26.2 μM for the latter. The cyclic GMP production was abolished after the deliberate removal of endothelium from the artery strips. These results suggest that the augmentation of intimal hyperplasia with hyperglycaemia is closely related to increased accumulation of L-NMMA and ADMA with hyperglycaemia, which would result in an accelerated reduction in NO production/release by endothelial cells. British Journal of Pharmacology (1999) 126, 211–218; doi:10.1038/sj.bjp.0702298

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 Bowman's 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

1. The aim of the present study was to elucidate functional differences between embryologically different portions of the posterior urethra of male rabbits in response to muscarinic acetylcholine receptor (mAChR) stimulation using in vitro isometric tension experiments and radioligand binding studies. 2. In the in vitro isometric tension experiments, carbachol, produced a dose-dependent contraction of the proximal portion under the resting state, but did not change the basal tone of the distal portion. Contraction of the proximal portion by 10(-5) M noradrenaline (NA) was dose-dependently enhanced by carbachol either in the presence or absence of NG-nitro-L-arginine (NOARG). In contrast, carbachol induced relaxation of the distal portion contracted by 10(-5) M NA, which was reversed to dose-dependent contraction in the presence of NOARG. 3. Both portions of the urethra had a similar number of [3H]-quinuclidinyl benzilate ([3H]-QNB) binding sites (195.3+/-74.1 fmols mg(-1) protein for the proximal portion and 146.5+/-8.5 fmols mg(-1) protein for the distal portion) with similar affinities (115.0+/-45.4 pM for the proximal portion and 79.9+ 2.9 pM for the distal portion). 4. The concentration-response curves to carbachol in both portions were shifted to the right in a parallel manner in the presence of pirenzepine (an M1 antagonist), 11-[[2-[(diethylamino)methyl]-1-piperidinyl] acetyl]-5, 11-dihydro-6H-pyrido-2,3-b)-(1,4)-benzodiazepin-6-one (AFDX-116, an M2 antagonist) and 4-diphenyl-acetoxy-N-methyl-piperidine (4-DAMP, an M1/M3 antagonist). The pA2 values for pirenzepine, AFDX-116 and 4-DAMP were 7.5+/-0.1, 7.2+/-0.02 and 9.3+/-0.1 respectively for the contraction of the proximal portion, and 7.2+/-0.1, 7.1+/-0.2 and 9.1+/-0.2, respectively for the relaxation of the distal portion. 5. In conclusion mAChR subtypes distribute in a similar fashion throughout the length of the male rabbit posterior urethra with the discrepant responses to carbachol attributable to the differential involvement of the NO pathway in mAChR-generated reactions.

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 intima:media 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 intima:media 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.

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 regenerated 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 7.4 +/- 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.

1. The present experiments were designed to investigate the role of endothelium in the human uterine arteries during the normal menstrual cycle. 2. Acetylcholine (ACh) produced a concentration-dependent relaxation response during the higher level of plasma 17 beta-oestradiol (E2) (follicular and luteal phases, E2 = 131.9 +/- 15.9 pg ml-1, n = 13; group I). However, the agent did not produce a definite relaxation, but produced a slight contraction during the ovulatory and menstruation phases (E2 = 19.8 +/- 2.9 pg mg-1, n = 5; group II). During the follicular and luteal phases (E2 = 181.1 +/- 9.0 pg ml-1, n = 6), ACh produced a slight contraction, but not relaxation in 6 cases (group III). Relaxation in response to A23187 in group II was not different from that in group I, while it was significantly (P < 0.05 and P < 0.005) reduced in group III. Sodium nitroprusside (SNP)-induced relaxation was similar in the three groups. 3. Correlation between the maximum response to ACh and the plasma E2 was highly significant (gamma = 0.8142, P < 0.001) in 18 cases of groups I and II, but not in all 24 cases including group III (gamma = 0.1183, NS). 4. Relaxations in response to ACh in group I or A23187 in all groups were abolished after removal of the endothelium. In group I, ACh- and A23187-induced relaxations were greatly inhibited by methylene blue or NG-nitro-L-arginine (L-NOARG) and partially inhibited by indomethacin. None of these treatments except for methylene blue modified the SNP-induced relaxation, which was significantly inhibited by methylene blue.5. The A23187-induced relaxation was hardly affected by methylene blue or L-NOARG in group III,but was partially inhibited by these agents in group II. The effect of indomethacin in inhibiting the A23187 induced-relaxation was most potent (58.9%) in group III and least (16.9%) in group I.6. There were no histological changes in 14 cases out of 18 (groups I and II), but very slight intimal thickening was observed in 4 cases in group I. On the other hand, severe intimal thickening was observed in all 6 cases in group III.7. These results indicate that, in human uterine artery strips, ACh and A23187 cause endothelium dependent relaxations, which are mediated mainly through EDRF/NO in group I, mainly prostacyclin(PGI2) in group III, or both in group II. It is suggested that lack of the production/release of EDRF/NO and/or of interaction between EDRF/NO and PGI2 might play a role in the formation of intimal thickening in human uterine arteries.

1. To explore mechanisms of urinary obstruction in benign prostatic hyperplasia (BPH), the features of contraction and relaxation in human hyperplastic and non-hyperplastic (control) prostatic tissues were investigated for beta- and alpha 1-adrenoceptors by radioligand binding and in vitro isometric tension experiments. 2. Hyperplastic and control prostatic tissues had a similar number (per mg protein) of prazosin binding sites with similar affinities. Noradrenaline (NA) induced dose-dependent contraction in both tissues. Contraction induced by either exogenous NA or transmural stimulation was inhibited by prazosin in both tissues, indicating that the same contractile mechanisms mediated by alpha 1-adrenoceptors exist in hyperplastic and control tissues. 3. The number of dihydroalprenolol (DHA) binding sites (per mg protein) was less in hyperplastic tissues than in controls, whereas the affinity to the ligand was identical in both tissues. Isoprenaline caused a marked relaxation of the tonic contraction induced by KCl in control tissues, but not in hyperplastic tissues. Propranolol enhanced exogenous NA- or transmural stimulation-induced contraction more in control tissues than in hyperplastic tissues. Both tissues, however, similarly responded to forskolin by relaxation. 4. These results indicate that decreased beta-adrenoceptor-mediated relaxation in hyperplastic prostatic tissues, which is attributable at least in part to the decreased number of beta-adrenoceptors, may play a role in the urinary obstruction of BPH in addition to mechanical compression of the urethra by the enlarged prostate.

1. The present experiments were designed to investigate the role of local angiotensin II receptors in the myointimal proliferative response of the vascular wall after endothelial removal, by use of a novel, nonpeptide, angiotensin II receptor antagonist, losartan. 2. When administered 1 week before endothelial removal from the rabbit carotid artery and then continuously until animals were killed 6 weeks later, losartan in a dose of 10 mg kg-1 daily, p.o. had no significant effects on the carotid blood flow (CBF), mean arterial blood pressure (MBP) and heart rate (HR). 3. A full endothelial lining with increased density of regenerated endothelial cells was observed 6 weeks after the endothelial removal. These changes were unaffected by treatment with losartan. 4. Six weeks after endothelial removal, acetylcholine (ACh)- and adenosine diphosphate (ADP)-induced relaxations were greatly reduced though endothelial cells had regenerated. The reduction of the relaxations to these agonists were significantly restored by chronic treatment with losartan. The endothelial-independent, sodium nitroprusside (SNP)-induced relaxation remained unaffected in all groups. 5. There were no differences in the noradrenaline (NA)- and endothelin-1 (ET-1)-induced contractions of the carotid artery strips between vehicle and losartan-treated groups. In contrast, the contractile response of the strips to angiotensin II was significantly decreased in the losartan group, indicating the specific antagonism by chronic losartan against the angiotensin II receptor. 6. Six weeks after endothelial removal, marked myointimal proliferation resulting from new accumulation of proliferating smooth muscle cells and connective tissue was observed in the vehicle group. Losartan treatment greatly suppressed the myointimal proliferative response.7. These results suggest that the local angiotensin II receptors play a role in the myointimal proliferativeresponse of the vascular wall to removal of the endothelium.

1 N-(3'-4'-dimethoxycinnamoyl) anthranilic acid (N-5') exhibited a dose-dependent, potent inhibition of the passive cutaneous anaphylaxis (PCA) mediated by homocytotropic antibodies (HTA), which was hardly affected by anti-inflammatory agents such as phenylbutazone, indomethacin and prednisolone at any dose used. The HTA-induced PCA was significantly inhibited by combined treatment with diphenydramine and cyproheptadine. 2 Doses of N-5' which potently inhibited HTA-induced PCA inhibited only slightly the heterologous PCA produced by anti-bovine serum albumin (BSA) rabbit serum. This heterologous PCA was clearly inhibited by phenylbutazone, indomethacin and prednisolone. Diphenydramine and cyproheptadine, singly or combined inhibited the heterologous PCA only slightly. 3 The increased vascular permeability caused by histamine and 5-hydroxytryptamine was significantly inhibited by diphenyldramine or cyproheptadine, but not by N-5' and the anti-inflammatory agents used. 4 N-5' 150 mg/kg orally inhibited rat paw oedema induced by carrageenin by about 26% while phenylbutazone, indomethacin and prednisolone produced significant inhibition. 5 N-5' at concentrations of 100 and 1000 muM significantly inhibited (by about 52% and 95%, respectively) the histamine release from rat peritoneal cells induced by HTA; 10 muM N-5' had little effect. Histamine release was inhibited by phenylbutazone or indomethacin at 1000 muM but not at 100 muM. Prednisolone had no effect on histamine release at any of the concentrations used. 6 These findings suggest that the inhibition of the HTA-induced PCA by N-5' may be due to inhibition of histamine release and is clearly different from the actions of anti-inflammatory agents such as phenylbutazone, indomethacin and prednisolone.

1In cascade perfusion and superfusion experiments on rabbit tissues, when acetylcholine (ACh) was introduced into the circuit so as to perfuse the aorta under perfusion with noradrenaline (NA), the effluent relaxed the transverse aortic strip which had been denuded of endothelium. 2The effluent from the perfused aorta which was capable of relaxing the transverse aortic strip also significantly inhibited platelet aggregation induced by arachidonic acid (AA) in a volume-related manner. The inhibitory activity was decreased by the prolongation of transit time before addition of the effluent to platelet-rich plasma. 3Neither the inhibition of AA-induced aggregation nor the relaxation of the transverse strip by the effluent could be observed after the removal of endothelium from the aorta, or after pretreatment of aorta with mepacrine or nordihydroguaiaretic acid (NDGA). 4The AA-induced platelet aggregation was unaffected by pretreatment of platelets with mepacrine or NDGA at the concentration tested. 5Pretreatment of aorta with indomethacin failed to modify the relaxation of the transverse strip induced by the effluent. 6These results strongly suggest that endothelium-derived vascular relaxant factor (EDRF) possesses inhibitory activity on AA-induced aggregation in addition to its vasodilator activity.

1. In the rabbit isolated aorta and trigone of the bladder, noradrenaline, phenylephrine and clonidine elicited concentration-dependent contractions, which may be caused through activation of postsynaptic alpha 1-adrenoceptors. 2. SHI437, IK29, prazosin and yohimbine competitively antagonized the contractile responses induced by noradrenaline in the aorta and trigone. The pA2 values of SHI437, IK29, prazosin and yohimbine were 7.35 +/- 0.09, 7.47 +/- 0.10, 8.55 +/- 0.02 and 6.28 +/- 0.05 in the aorta, and 8.07 +/- 0.04, 8.30 +/- 0.03, 8.22 +/- 0.04 and 6.46 +/- 0.04 in the trigone, respectively. 3. SHI437, IK29, prazosin and yohimbine also possessed competitive alpha 2-adrenoceptor blocking properties, judging from their antagonism of the clonidine-induced inhibitory effect on the twitch responses in the electrically stimulated vas deferens of the rat. The pA2 values of SHI437, IK29, prazosin and yohimbine were determined to be 4.76 +/- 0.02, 4.74 +/- 0.02, 5.06 +/- 0.03 and 7.86 +/- 0.04, respectively. 4. SHI437, IK29 and prazosin inhibited the contractile responses elicited by transmural electrical stimulation without affecting the evoked 3H-overflow from the [3H]-noradrenaline-preloaded rabbit aorta. Yohimbine augmented the contractile responses and 3H-overflow. 5. SHI437 and IK29 at a concentration sufficient to inhibit noradrenaline-induced contraction failed to attenuate the contractile responses of aorta to KCl, 5-hydroxytryptamine and prostaglandin F2 alpha, and of the trigone to acetylcholine and histamine. 6. The present results suggest that SHI437 and IK29 are highly selective alpha 1-adrenoceptor antagonists, especially in the trigone of the bladder.