Impaired endothelium-dependent relaxation in isolated resistance arteries of spontaneously diabetic rats.

1. Previous studies have shown that endothelium-dependent relaxation in the aorta of spontaneously diabetic bio bred rats (BB) is impaired. 2. We have investigated noradrenaline (NA) contractility, endothelium-dependent acetylcholine (ACh) and bradykinin (BK) relaxation, and endothelium-independent sodium nitroprusside (SNP) relaxation in mesenteric resistance arteries of recent onset BB rats and established insulin treated BB rats, compared to their age-matched non diabetic controls. 3. There was no significant difference in the maximum contractile response or sensitivity to noradrenaline in either of the diabetic groups compared to their age-matched controls. 4. Incubation with the nitric oxide synthetase inhibitor NG-nitro-L-arginine (L-NOARG) resulted in a significant increase in maximum contractile response to noradrenaline in the recent onset age-matched control group (P < 0.05). Analysis of the whole dose-response curve (using ANOVA for repeated measures with paired t test) showed a significant left-ward shift following the addition of L-NOARG (P < 0.001). A similar but less marked shift (P < 0.01) was evident in vessels from recent onset diabetics. An overall shift in both sensitivity and maximum response was also evident in the age-matched non diabetic controls of the insulin-treated group (P < 0.05). However, by contrast, there was no significant change in sensitivity in the insulin-treated diabetic rats. 5. ACh-induced endothelium-dependent relaxation was significantly impaired in the recent onset diabetic rats compared to their age-matched controls (47 +/- 11% versus 92 +/- 2%, P < 0.05, n = 6), and in the insulin treated diabetic rats (34 +/- 5% versus 75 +/- 6%, P < 0.05, n = 6). The relaxation responses to BK also were significantly impaired in the diabetic rats compared to their age-matched controls (recent onset: 20 +/- 3% versus 72 +/- 7%, P < 0.05, n = 6; insulin treated: 12 +/- 9% versus 68 +/- 7%, P < 0.05, n = 7). 6. Incubation with either the nitric oxide synthetase substrate, U-arginine, or the free radical scavenging enzyme superoxide dismutase (150 mu ml-1) failed to improve the attenuated response of acetylcholine-induced relaxation in the diabetic vessels. 7. Endothelium-dependent relaxation mediated by ACh and BK was significantly attenuated in both the diabetic and control vessels after incubation with L-NOARG. 8. Pretreatment with a cyclo-oxygenase inhibitor, indomethacin, significantly enhanced the relaxation to ACh in both the recent onset and insulin treated diabetic rats (42 +/- 10%, n = 7 versus 64 +/- 7%, n = 7, P < 0.05, and 40 +/- 5%, n = 7 versus 65 +/- 9%, n = 6, P < 0.05). 9. Following endothelium removal, there was a marked impairment in endothelium-dependent relaxation responses to ACh and BK in both the diabetic and control vessels. 10. Incubation with the thromboxane A2 receptor antagonist SQ29548, did not significantly improve the ACh endothelium-dependent relaxation response in the diabetic vessels. 11. Endothelium-independent relaxation to sodium nitroprusside was significantly impaired in the first group of diabetic vessels studied; however, subsequent studies showed no impairment of the sodium nitroprusside response in the diabetic vessels. 12. In conclusion, the ability of the endothelium to regulate vascular contractility is reduced in recent onset diabetic vessels, and significantly impaired in established insulin treated diabetics. Relaxation to the endothelium-dependent vasodilators ACh and BK was impaired in both the recent onset and the established insulin treated diabetics, and the ACh response was significantly improved following pretreatment with indomethacin, suggesting a role for a cyclo-oxygenase-derived vasoconstrictor. Preliminary studies with a thromboxane A2, receptor antagonist, SQ29548 did not significantly improve the impaired relaxation to ACh, indicating that the vasoconstrictor prostanoid is not thromboxane A2.     

Inhibitory effect of streptozotocin-induced diabetes on non-cholinergic motor transmission in rat detrusor and its prevention by sorbinil.

1. Non-cholinergic motor transmission in the urinary bladder of streptozotocin (STZ)-diabetic rats was studied by recording contractile activity of strips of detrusor in vitro. 2. The neurogenic contractile responses to electrical field stimulation (EFS) of atropine-treated detrusor strips were decreased in 4, 8 and 12 week STZ-diabetic rats. The decrease was most marked in 12 week diabetic rats and least in 4 week ones. 3. Concentration-response curves showed no change in sensitivity of the detrusor to acetylcholine (ACh) in diabetic rats. The maximum tension generated by ACh was similar in diabetic and non-diabetic animals. 4. The contractile responses to EFS at frequencies greater than or equal to 1 Hz were not maintained during stimulation. The 'fade' was significantly greater in detrusor strips of diabetic rats. 5. The contractile response of detrusor to EFS was significantly greater in 12 week diabetic rats treated with the aldose reductase inhibitor sorbinil, than in untreated 12 week diabetic rats. The sensitivity to ACh was similar in the two groups. 6. It is concluded that the reduction of the neurogenic non-cholinergic responses of detrusor to EFS in STZ-diabetic rats is probably caused by a reduction in the release of the non-cholinergic motor transmitter. The results are discussed in relation to bladder dysfunction in human diabetes mellitus.     

Endothelium-dependent modulation of resistance vessel contraction: studies with NG-nitro-L-arginine methyl ester and NG-nitro-L-arginine.

1. The effect of NG-nitro-L-arginine methyl ester (L-NAME) and NG-nitro-L-arginine (L-NOARG) on noradrenaline (NA)-induced contractility and acetylcholine (ACh)-induced endothelium-dependent relaxation was studied in rat mesenteric resistance arteries. 2. Third order branches of mesenteric arteries were dissected and mounted on two forty micron wires in a Mulvany myograph. 3. Incubation with L-NAME and L-NOARG (10 microM) caused a time-dependent shift in the 50% response to NA (ED50) (0.01 microM-10 microM) but was not associated with an increase in the maximum contractile response. 4. L-NAME and L-NOARG (10 microM) caused a time-dependent inhibition of ACh (1 microM)-induced relaxation with a maximum effect after 120 min. 5. Following endothelium removal, incubation with either L-NAME or L-NOARG caused no significant shift in the ED50, although the residual relaxation response to ACh (1 microM) was further attenuated. 6. Incubation with the cyclo-oxygenase inhibitor, indomethacin, enhanced the relaxation to ACh and reduced the inhibitory effects of L-NAME and L-NOARG. 7. In conclusion, L-NAME and L-NOARG are potent inhibitors of acetylcholine-induced endothelium-dependent relaxation in mesenteric resistance arteries. The shift in ED50 associated with these inhibitors suggests a probable role for the endothelium in modulating the contractility of the resistance vasculature.     

Altered responsiveness of diabetic dog renal arteries to acetylcholine and phenylephrine: role of endothelium

The mechanical responses to acetylcholine (ACh), sodium nitroprusside and phenylephrine (PE) were determined in normal and diabetic dog renal arterial strips with and without endothelium. Experimental diabetes increased the sensitivity, IC50 = (2.9 +/- 0.4) X 10(-8) mol/l in normal and (9.6 +/- 1.5) X 10(-9) mol/l in diabetic (p less than 0.01, n = 6) to ACh of endothelium intact renal arterial strips without influencing the maximum relaxation induced by this agonist. In all intact vessels PE produced contractions of equal magnitude. Removal of the endothelium completely abolished the relaxant ability of ACh, and caused a slight increase in the contractile response of both diabetic and normal strips to PE. The maximum contractile force generated by the denuded diabetic vessels in response to PE was significantly (p less than 0.01) greater than the maximum tension produced by the denuded nondiabetic arteries. The sensitivity of the tissues to PE was, however, not modified by either diabetes or endothelium removal. The direct relaxant sodium nitroprusside elicited a similar degree of relaxation in the two groups of arteries. Cyclooxygenase blockade had no effect on either the relaxation or contractile responses of any of the preparations. These findings suggest that short-term diabetes makes dog renal arteries supersensitive to ACh and hyperreactive to PE.     

Enhanced role for the opening of potassium channels in relaxant responses to acetylcholine after myocardial ischaemia and reperfusion in dog coronary arteries

1. Anaesthetized dogs were subjected to 1 h occlusion of the left circumflex coronary artery followed by 2 h of reperfusion. Relaxant responses were examined in coronary artery rings removed proximal (nonischaemic) or distal (ischaemic) to the site of occlusion. 2. Relaxant responses to acetylcholine (ACh) were similar in nonischaemic and ischaemic artery rings. In addition ACh-induced relaxation of nonischaemic and ischaemic artery rings was equally susceptible to inhibition of nitric oxide (NO) synthase using L-N(G)-nitroarginine (L-NOARG, 10(-4) M), or to inhibition of soluble guanylate cyclase using 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ, 10(-5) M). 3. In nonischaemic arteries, the relaxation to ACh was unaffected by high K+ (67 mM) but in ischaemic arteries, the maximum relaxation to ACh was significantly reduced from 113+/-6 to 60+/-2% (ANOVA, P<0.05). Tetraethylammonium (TEA, 10(-3) M), an inhibitor of large conductance calcium activated potassium (BK(Ca)) channels did not inhibit the response to ACh in nonischaemic arteries but in ischaemic arteries TEA significantly shifted the concentration response curve to ACh to the right (pEC(50); nonischaemic, 7.07+/-0.25; ischaemic, 6.54+/-0.21, P<0.01, ANOVA) without decreasing the maximum relaxation. TEA did not affect the responses to sodium nitroprusside in either nonischaemic or ischaemic arteries. 4. In conclusion, ischaemia/reperfusion did not change the sensitivity of endothelium-dependent relaxation to L-NOARG or ODQ indicating that ischaemia did not affect the contribution of NO or cyclic GMP to ACh-induced relaxation. However, in ischaemic arteries the opening of the BK(Ca) channels contributed to relaxation caused by ACh whereas TEA had no effect in nonischaemic arteries. The factor responsible for the opening of this potassium channel was a factor other than NO and may be endothelium derived hyperpolarizing factor (EDHF).     

野黄芩甙元对糖尿病大鼠主动脉的影响

目的：研究野黄芩甙元对糖尿病大鼠血管合并症的预防作用。方法：利用平滑肌条离体研究装置。结果：在第六周的糖尿病大鼠主动脉：1）乙酰胆碱引起的内膜依赖性舒张作用较对照明显减弱（P＜0．01）;2）苯肾上腺素引起的收缩反应较对照明显增加,最大收缩增加约40％（P＜0．01）;3）糖尿病大鼠服用含0．5％的野黄芩甙元的饮水后,乙酰胆碱引起的内膜依赖性舒张作用较糖尿病组明显增加（P＜0．01）。但是,苯肾上腺素引起的收缩反应增加更显著,最大收缩较对照加约80％（P＜0．01）。结论：野黄芩甙元对糖尿病引起的血管内膜功能损害有防护作用,也可增强苯肾上腺素引起的收缩。     

Chronic administration of genistein improves aortic reactivity of streptozotocin-diabetic rats: mode of action

Diabetes mellitus is associated with major cardiovascular risk factors which are responsible for excess morbidity and mortality. Soy isoflavones like genistein are beneficial for correcting the hyperglycemia and preventing some diabetic complications. Thus, the effect of chronic administration of genistein was studied on aortic reactivity of streptozotocin (STZ)-diabetic rats. Male diabetic rats received genistein 1 mg/kg/day (i.p.) for 4 weeks 3 days after diabetes induction. Contractile responses to KCl and phenylephrine (PE) and relaxation responses to acetylcholine (ACh) and isosorbide dinitrate (ISD) were obtained from aortic rings. Maximum contractile response of endothelium-intact rings to KCL and PE was significantly lower in genistein-treated diabetic rats relative to untreated diabetic ones. Endothelium removal abolished the significant difference between genistein-treated and untreated diabetic groups regarding contractile response to KCl and PE. Meanwhile, endothelium-dependent relaxation to ACh was significantly higher in genistein-treated diabetic rats as compared to diabetic ones. Pretreatment of rings with N(omega)-L-arginine methyl ester (L-NAME) and indomethacin (INDO) significantly attenuated the observed responses. Meanwhile, one-month diabetes resulted in an elevation of malondialdehyde (MDA) and decreased superoxide dismutase (SOD) activity in aortic tissue and genistein treatment attenuated the increased MDA content and reduced activity of SOD. Therefore, chronic treatment of diabetic rats with genistein could prevent the abnormal functional changes in vascular reactivity in diabetic rats through nitric oxide- and prostaglandin-dependent pathways and via attenuating oxidative stress in the wall of aortic tissue.     

Nitric oxide and relaxation of pig lower urinary tract.

1. We studied the non-adrenergic, non-cholinergic (NANC) nerve-mediated relaxation induced by electrical stimulation in pig isolated lower urinary tract smooth muscle, and the possible involvement of the L-arginine (L-ARG)/nitric oxide (NO) pathway in this response. 2. Trigonal strips, precontracted by noradrenaline (NA), carbachol or endothelin-1 (ET-1), relaxed frequency-dependently in response to electrical stimulation. Maximum relaxation was obtained at 6-8 Hz, and amounted to 56 +/- 2%, 77 +/- 3% and 62 +/- 6% of the agonist-induced tension in preparations contracted by NA, carbachol, or ET-1, respectively. Exposure to NG-nitro-L-arginine (L-NOARG; 10(-7)-10(-5) M) concentration-dependently reduced the relaxant response in preparations contracted by NA. L-NOARG (10(-6) M) reduced the maximal response to 51 +/- 8% of control. L-NOARG (10(-5) M) abolished all relaxation, and unmasked a contractile component; D-NOARG had no effect. Also in trigonal preparations, where the tension had been raised by carbachol or ET-1, L-NOARG (10(-5) M) markedly reduced relaxations evoked by electrical stimulation. 3. In trigonal preparations contracted by NA, maximal relaxation was increased after pretreatment with L-ARG (10(-3) M), and the inhibitory effect of L-NOARG (10(-6) M) was prevented. Incubation of the trigonal strips with methylene blue had no effect on relaxations elicited at frequencies less than 6 Hz, but a small inhibition was observed at higher frequencies. 4. Administration of NO (present in acidified solution of NaNO2) induced concentration-dependent relaxations in trigonal preparations contracted by NA, carbachol, or ET-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of insulin treatment on smooth muscle contractility and endothelium-dependent relaxation in rat aortae from established STZ-induced diabetes.

1. This study involved the chronic administration of low or high insulin to rats with established streptozotocin (STZ)-induced diabetes. We studied the effect of such treatment on smooth muscle contractility and endothelium-dependent relaxation using aortic strips. 2. Aortae from diabetic rats, but not those from high-insulin-treated diabetic rats, showed an impaired endothelium-dependent in response to acetylcholine (ACh) by comparison with untreated controls. 3. Isotonic high K+-induced contractility was impaired in diabetic aortae. This impairment was prevented by high-insulin treatment. 4. Noradrenaline (NA)-induced contractility was enhanced in aortae from high-insulin-treated diabetic rats, but not in those from untreated diabetic or low-insulin treated diabetic rats. 5. In the combined presence of the nitric oxide inhibitor N(G)-nitro-L-arginine and the cyclo-oxygenase inhibitor indomethacin, NA-induced contractility was significantly greater in aortae from high-insulin-treated diabetic rats than in those from controls or untreated diabetic rats. 6. An increased expression of the mRNA for the alpha1D and alpha1B adrenergic receptors was found in aortae from high-insulin-treated diabetic rats. 7. These results demonstrate that in rats with established STZ-induced diabetes, high-insulin treatment prevents the development of an impaired endothelium-dependent relaxation in the aorta, and that such treatment enhances NA-induced contractility. This enhancement may be related to an upregulation in the expression of the mRNA for the alpha1B or alpha1D adrenergic receptor that is secondary to the hyperinsulinaemia.     

Chronic epigallocatechin-gallate improves aortic reactivity of diabetic rats: Underlying mechanisms

Diabetes mellitus is associated with major cardiovascular risk factors which are responsible for excess morbidity and mortality. Green tea catechins including epigallocatechin-3-gallate (EGCG) could exert beneficial health effects to ameliorate cardiovascular and metabolic diseases. Thus, the effect of chronic administration of EGCG was studied on aortic reactivity of streptozotocin (STZ)-diabetic rats. Male diabetic rats received EGCG 25 mg/kg/day for 8 weeks 1 week after diabetes induction. Contractile responses to KCl and phenylephrine (PE) and relaxation responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were obtained from aortic rings. Maximum contractile response of endothelium-intact rings to KCL and PE was significantly lower in EGCG-treated diabetic rats relative to untreated diabetic ones. Endothelium removal abolished the significant difference between EGCG-treated and untreated diabetic groups regarding contractile response to KCl and PE. Meanwhile, endothelium-dependent relaxation to ACh was significantly higher in EGCG-treated diabetic rats as compared to diabetic ones. Pretreatment of rings with N(omega)-L-arginine methyl ester (L-NAME) and indomethacin (INDO) significantly attenuated the observed responses. Meanwhile, two-month diabetes resulted in an elevation of malondialdehyde (MDA) and decreased superoxide dismutase (SOD) activity in aortic tissue and EGCG treatment attenuated the increased MDA content and reduced activity of SOD. Therefore, chronic treatment of diabetic rats with EGCG could prevent the abnormal functional changes in vascular reactivity in diabetic rats through nitric oxide- and prostaglandin-dependent pathways and via attenuation of aortic lipid peroxidation.     

Role of potassium channels in endothelium-dependent relaxation resistant to nitroarginine in the rat hepatic artery.

1. In the presence of indomethacin (IM, 10 microM) and N omega-nitro-L- arginine (L-NOARG, 0.3 mM), acetylcholine (ACh) induces an endothelium-dependent smooth muscle hyperpolarization and relaxation in the rat isolated hepatic artery. The potassium (K) channel inhibitors, tetrabutylammonium (TBA, 1 mM) and to a lesser extent 4-aminopyridine (4-AP, 1 mM) inhibited the L-NOARG/IM-resistant relaxation induced by ACh, whereas apamin (0.1-0.3 microM), charybdotoxin (0.1-0.3 microM), iberiotoxin (0.1 microM) and dendrotoxin (0.1 microM) each had no effect. TBA also inhibited the relaxation induced by the receptor-independent endothelial cell activator, A23187. 2. When combined, apamin (0.1 microM) + charybdotoxin (0.1 microM), but not apamin (0.1 microM) + iberiotoxin (0.1 microM) or a triple combination of 4-AP (1 mM) + apamin (0.1 microM) + iberiotoxin (0.1 microM), inhibited the L-NOARG/IM-resistant relaxation induced by ACh. At a concentration of 0.3 microM, apamin + charybdotoxin completely inhibited the relaxation. This toxin combination also abolished the L-NOARG/ IM-resistant relaxation induced by A23187. 3. In the absence of L-NOARG, TBA (1 mM) inhibited the ACh-induced relaxation, whereas charybdotoxin (0.3 microM) + apamin (0.3 microM) had no effect, indicating that the toxin combination did not interfere with the L-arginine/NO pathway. 4. The gap junction inhibitors halothane (2 mM) and 1-heptanol (2 mM), or replacement of NaCl with sodium propionate did not affect the L-NOARG/IM-resistant relaxation induced by ACh. 5. Inhibition of Na+/K(+)-ATPase by ouabain (1 mM) had no effect on the L-NOARG/IM-resistant relaxation induced by ACh. Exposure to a K(+)-free Krebs solution, however, reduced the maximal relaxation by 13% without affecting the sensitivity to ACh. 6. The results suggest that the L-NOARG/IM-resistant relaxation induced by ACh in the rat hepatic artery is mediated by activation of K-channels sensitive to TBA and a combination of apamin + charybdotoxin. Chloride channels, Na+/K(+)-ATPase and gap junctions are probably not involved in the response. It is proposed that endothelial cell activation induces secretion of an endothelium-derived hyperpolarizing factor(s) (EDHF), distinct from NO and cyclo-oxygenase products, which activates more than one type of K-channel on the smooth muscle cells. Alternatively, a single type of K-channel, to which both apamin and charybdotoxin must bind for inhibition to occur, may be the target for EDHF.     

AN EXAMINATION OF AUTONOMIC NERVOUS FUNCTION IN GENETICALLY DIABETIC MICE

1 This study was designed to determine whether the autonomic innervation of the heart and vas deferens in genetically diabetic mice exhibited dysfunction similar to those seen in chemically diabetic animals and diabetic patients. 2 Diabetic mutant mice (outcrossed from the C57 BL/KS db/db strain) were compared with their non-diabetic litter-mates at age 20 to 22 weeks. Right and left atria and vasa deferentia were removed from freshly killed animals and subjected to nerve stimulation and treatment with noradrenaline (NA) or acetyl-choline (ACh) in organ baths. 3 Right atria from diabetic animals were less responsive to noradrenergic nerve stimulation than control preparations but there was no such difference between the noradrenergic responses of left atria from the two groups of mice. Both atria were hypersensitive to exogenous NA. 4 Atria from diabetic mice responded to cholinergic nerve stimulation and exogenous ACh in a fashion similar to those of non-diabetic mice. Likewise the responses of vasa deferentia to nerve stimulation were similar in the two groups. These findings are indicative of some autonomic nervous dysfunction characteristic, to an extent, of diabetes mellitus.     

Endothelium-dependent relaxation to acetylcholine in bovine oviductal arteries: mediation by nitric oxide and changes in apamin-sensitive K+ conductance.

1. Mechanisms underlying the relaxant response to acetylcholine (ACh) were examined in bovine oviductal arteries (o.d. 300-500 microns and i.d. 150-300 microns) in vitro. Vascular rings were treated with indomethacin (10 microM) to prevent the effects of prostaglandins. 2. ACh elicited a concentration-related relaxation in ring segments precontracted with noradrenaline (NA), which was abolished by endothelium denudation. 3. The ACh-induced relaxation was attenuated but not abolished by NG-nitro-L-arginine (L-NOARG, 1 microM-1 mM), an inhibitor of nitric oxide (NO) formation. The inhibition caused by L-NOARG (10 microM) was reversed by addition of excess of L-arginine but not D-arginine (1 mM). 4. In high K+ (40-60 mM)-contracted rings, ACh was a much less effective vasodilator and its relaxant response was completely abolished by L-NOARG (100 microM). 5. In NA (10 microM)-contracted rings, ACh induced sustained and concentration-dependent increases in cyclic GMP, which were reduced below basal values by L-NOARG (100 microM), while potent relaxation persisted. Similar increases in cyclic GMP were evoked by ACh in high K+ (50 mM)-treated arteries and under these conditions, both cyclic GMP accumulation and relaxation were L-NOARG-sensitive. 6. S-nitroso-L-cysteine (NC), a proposed endogenous precursor of endothelial NO, also induced cyclic GMP accumulation in NA-contracted oviductal arteries. 7. Methylene blue (MB, 10 microM), a proposed inhibitor of soluble guanylate cyclase, inhibited both endothelium-dependent relaxation to ACh and endothelium-independent response to exogenous NO, whereas relaxation to NC remained unaffected. 8. The L-NOARG-resistant response to ACh was not affected by either ouabain (0.5 mM), glibenclamide (3 microM), tetraethylammonium (TEA, 1 mM) or charybdotoxin (50 nM), but was selectively blocked by apamin (0.1-1 microM). However, apamin did not inhibit either relaxation to ACh in high K(+)-contracted rings or endothelium-independent relaxation to either NO or NC. 9. Apamin and MB inhibited ACh-induced relaxation in an additive fashion, suggesting the involvement of two separate modulating mechanisms. 10. These results suggest that ACh relaxes bovine oviductal arteries by the release of two distinct endothelial factors: a NO-like substance derived from L-arginine, which induces cyclic GMP accumulation in smooth muscle, and another non-prostanoid factor acting by hyperpolarization mechanisms through alterations in apamin-sensitive K+ conductance.     

Endothelium-dependent relaxations mediated by inducible B1 and constitutive B2 kinin receptors in the bovine isolated coronary artery.

1. Rings of bovine left anterior descending coronary artery (LAD) were contracted with the thromboxane A2-mimetic, U46619 (1-30 nM), to approximately 40% of their maximum contraction to 125 mM KCl Krebs solution (KPSSmax) for comparison of responses to the B1 and B2 kinin receptor agonists, des-Arg9-bradykinin (des-Arg9-BK) and bradykinin (BK), respectively. Relaxation responses were normalized as percentages of the initial U46619-induced contraction level, while contractile responses were expressed as percentages of KPSSmax. 2. After 6 h of in vitro incubation in Krebs solution at 37 degrees C, des-Arg9-BK (pEC50, 8.00 +/- 0.08; maximum response (Rmax), 93.9 +/- 1.9%) and BK (pEC50, 9.75 +/- 0.07; Rmax, 100.1 +/- 0.7%) caused endothelium-dependent relaxations in precontracted rings of bovine LAD which were competitively and selectively antagonized by the B1 receptor antagonist, des-Arg9-[Leu8]-BK (pA2, 6.27 +/- 0.11) and the B2 receptor antagonist Hoc-140 (pA2, 9.63 +/- 0.14), respectively. 3. At 3 h of in vitro incubation, the sensitivity (pEC50, 7.45 +/- 0.10) and Rmax (84.6 +/- 3.3%) to des-Arg9-BK were significantly less than those obtained in the same tissues at 6 h (pEC50, 7.94 +/- 0.06; Rmax, 91.4 +/- 2.5%), whereas endothelium-dependent relaxations to BK and ACh were unaffected by incubation time. 4. Relaxation responses to des-ARg9-BK, but not BK, at both 3 h and 6 h were significantly attenuated by the protein synthesis inhibitors, cycloheximide (30 and 100 microM) and actinomycin D (2 microM). 5. At 6 h, the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine (L-NOARG, 100 microM), caused a significant 2 fold decrease in pEC50 (9.58 +/- 0.03) but had no effect on Rmax for BK. For des-Arg9-BK, L-NOARG (100 microM) caused a marked and significant decrease in both the pEC50 and Rmax and revealed contractions to low concentrations of des-Arg9-BK. In both cases, L-NOARG inhibition was reversed in the presence of L-arginine (10 mM). 6. At 6 h removal of the endothelium abolished relaxation responses to des-Arg9-BK and BK, and for des-Arg9-BK, but not BK, unmasked concentration-dependent contractions (pEC50, 7.57 +/- 0.09; Rmax, 83.4 +/- 9.1%). The sensitivity of contractions to des-Arg9-BK increased slightly from 3 h (pEC50, 7.37 +/- 0.08) to 6 h (pEC50, 7.62 +/- 0.12) of in vitro incubation; however, there was a small but significant depression in the maximum response over this time (Rmax, 126.8 +/- 8.5% and 103.3 +/- 8.6% for 3 h and 6 h of incubation respectively). 7. In conclusion, the bovine LAD contains inducible B1 and constitutive B2 endothelial cell kinin receptors, both of which mediate endothelium-dependent relaxation partly via the release of NO. B1 receptors were also present on the smooth muscle layer of the bovine LAD.     

Endothelium-derived hyperpolarizing factor and potassium use different mechanisms to induce relaxation of human subcutaneous resistance arteries.

This investigation examined the hypothesis that release of K(+) accounts for EDHF activity by comparing relaxant responses produced by ACh and KCl in human subcutaneous resistance arteries. Resistance arteries (internal diameter 244+/-12 microm, n=48) from human subcutaneous fat biopsies were suspended in a wire myograph. Cumulative concentration-response curves were obtained for ACh (10(-9) - 3x10(-5) M) and KCl (2.5 - 25 mM) following contraction with noradrenaline (NA; 0.1 - 3 microM). ACh (E(max) 99.07+/-9.61%; -LogIC(50) 7.03+/-0.22; n=9) and KCl (E(max) 74.14+/-5.61%; -LogIC(50) 2.12+/-0.07; n=10)-induced relaxations were attenuated (P<0.0001) by removal of the endothelium (E(max) 8.21+/-5.39% and 11.56+/-8.49%, respectively; n=6 - 7). Indomethacin (10 microM) did not alter ACh-induced relaxation whereas L-NOARG (100 microM) reduced this response (E(max) 61.7+/-3.4%, P<0.0001; n=6). The combination of ChTx (50 nM) and apamin (30 nM) attenuated the L-NOARG-insensitive component of ACh-induced relaxation (E(max): 15.2+/-10.5%, P<0.002, n=6) although these arteries retained the ability to relax in response to 100 microM SIN-1 (E(max) 127.6+/-13.0%, n=3). Exposure to BaCl(2) (30 microM) and Ouabain (1 mM) did not attenuate the L-NOARG resistant component of ACh-mediated relaxation (E(max), 76.09+/-8.92, P=0.16; n=5). KCl-mediated relaxation was unaffected by L-NOARG+indomethacin (E(max); 68.1+/-5.6%, P=0.33; n=5) or the combination of L-NOARG/indomethacin/ChTx/apamin (E(max); 86.61+/-14.02%, P=0.35; n=6). In contrast, the combination of L-NOARG, indomethacin, ouabain and BaCl(2) abolished this response (E(max), 5.67+/-2.59%, P<0.0001, n=6). The characteristics of KCl-mediated relaxation differed from those of the nitric oxide/prostaglandin-independent component of the response to ACh, and were endothelium-dependent, indicating that K(+) does not act as an EDHF in human subcutaneous resistance arteries.     

Impairment of endothelium- and nerve-mediated relaxation responses in the cavernosal smooth muscle of experimentally diabetic rabbits: role of weight loss and duration of diabetes

The effects of short- and long-term experimental diabetes on corporal nerve, endothelium and smooth-muscle responses were investigated, and the reasons for possible alterations in corporal smooth muscle responses such as hyperglycaemia, duration of experimental diabetes and/or altered tissue weight were evaluated. Rabbits were injected with alloxan (125 mg/kg) to induce diabetes. Age-matched non-diabetic and diabetic (3 and 9 weeks) and weight-matched non-diabetic groups (9 weeks) were used as control. In all groups, relaxation (carbachol, electrical field stimulation and sodiumnitroprusside) responses were examined. The relaxation responses were expressed as percentage of the precontraction to phenylephrine and as g response/g tissue weight. The effects of elevated glucose were also examined by incubating cavernosal strips in Krebs–Henseleit solution containing 44.4 mM glucose for 6 h. Cavernosal tissues of non-diabetic and 9-week diabetic rabbits were evaluated histologically. Sodiumnitroprusside (10⁻⁷−10⁻⁴ M) responses were similar in all groups. Relaxation responses to electrical field stimulation (10 s train; amplitude 50 V; frequency 0.5–32 Hz; width 0.8 ms) were only attenuated in the 9-week diabetic group compared to the non-diabetic group. Carbachol (10⁻⁸−3×10⁻⁵ M) responses were attenuated in both diabetic groups. When the relaxation responses expressed as g response/g tissue weight were evaluated, results were similar compared to those expressed as percentage of phenylephrine (10⁻⁵ M). Neither carbachol nor electrical field stimulation mediated responses were impaired with glucose incubation. No morphological degenerations were observed in the endothelium. Diabetes may interfere with the synthesis and/or release of nitric oxide from both nerves and endothelium in corpus cavernosum, and alterations in endothelium-derived responses occur earlier than neurological disturbances. The sensitivity of cavernosal smooth muscle to nitric oxide did not alter in diabetes. Attenuation of responses was not due to decreased tissue weight caused by diabetes.     

Contribution of endothelin receptors and cyclooxygenase-derivatives to the altered response of the rabbit renal artery to endothelin-1 in diabetes

The influence of diabetes on regulatory mechanisms and specific receptors implicated in the response of isolated rabbit renal artery to endothelin-1 was examined. Endothelin-1 induced a concentration-dependent contraction that was less potent in arteries from diabetic rabbits than in arteries from control rabbits. Endothelium removal or N(G)-nitro-L-arginine (L-NOARG) enhanced contractions to endothelin-1 either in control and diabetic arteries. Indomethacin inhibited endothelin-1-induced response in control arteries, but enhanced it in diabetic arteries. In contrast to that observed in rubbed and in L-NOARG treated arteries, in the presence of indomethacin the contractile action of endothelin-1 was higher in diabetic arteries than in control arteries. Nimesulide enhanced endothelin-1 contractions both in control and diabetic arteries. Cyclo-(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123, endothelin ET(A) receptor antagonist), attenuated endothelin-1 vasoconstriction in control rabbits, while vasoconstriction resulted increased in diabetic rabbits. 2,6-Dimethylpiperidinecarbonyl-gamma-Methyl-Leu-N(in)-(Methoxycarbonyl)-D-Trp-D-Nle (BQ-788, endothelin ET(B) receptor antagonist), enhanced the contractile response in control rabbit arteries without modifying this response in diabetic rabbits. In summary, diabetes decreases the sensitivity of the rabbit renal artery to endothelin-1 by decreasing the ratio between vasoconstrictor and vasodilator prostanoids released after activation of endothelin ET(A) receptors.     

Mechanisms responsible for the vascular effect of aqueous Trigonella foenum-graecum leaf extract in diabetic rats

Background and Objective:

Since a beneficial vascular effect of aqueous leaf extract of Trigonella foenum-graecum (TFG) has previously been reported, this study was conducted to evaluate the underlying mechanisms, including the role of nitric oxide (NO) and cyclooxygenase pathways, in diabetic rats.

Materials and Methods:

Male Wistar rats were divided into control, extract-treated control, diabetic, and extract-treated diabetic groups. Diabetes was induced by a single i.p. injection of streptozotocin (STZ; 60 mg/kg). Treatment groups received TFG extract (200 mg/kg; ip.) every other day for 1 month. Contractile reactivity of the thoracic aorta to KCl and noradrenaline (NA) and relaxation response to acetylcholine (ACh) were determined. For determination of the participation of NO and prostaglandins in the relaxation response to ACh, aortic rings were incubated for 30 min before the experiment with N-nitro-l-arginine methyl ester (L-NAME) and/or indomethacin (INDO).

Results:

The diabetic state significantly increased the maximum contractile response to KCl and NA (P < 0.01-0.005) and reduced the maximum relaxation due to ACh (P < 0.01) as compared to controls and treatment with TFG extract in the diabetic group significantly improved these changes relative to the untreated diabetic group (P < 0.05). With L-NAME pretreatment, no significant difference between diabetic and extract-treated diabetic groups was found out. On the other hand, there was a significant difference between these two groups following INDO pretreatment (P < 0.05).

Conclusion:

Intraperitoneal administration of aqueous leaf extract of TFG for one month could improve some functional indices of the vascular system in the diabetic state and endothelium-derived prostaglandins are essential in this respect.     

Evidence for mediation by endothelium-derived hyperpolarizing factor of relaxation to bradykinin in the bovine isolated coronary artery independently of voltage-operated Ca2+ channels.

1. The role of endothelium-derived hyperpolarizing factor and voltage-operated Ca2+ channels in mediating endothelium-dependent, NG-nitro-L-arginine (L-NOARG; 100 microM) -resistant relaxations to bradykinin (BK), was examined in isolated rings of endothelium-intact bovine left anterior descending coronary artery. 2. Rings of artery were contracted isometrically to approximately 40% or their respective maximum contraction to 125 mM KCl Krebs solution (KPSSmax) with the thromboxane A2-mimetic, U46619. Relaxations to BK and the endothelium-independent NO donor, S-nitroso-N-acetylpenicillamine (SNAP), were normalized as percentages of reversal of the initial contraction to U46619. All experiments were carried out in the presence of indomethacin (3 microM). 3. BK caused concentration-dependent relaxations [sensitivity (pEC50) 9.88 +/- 0.05; maximum relaxation (Rmax), 103.3 +/- 0.5%] in U46619-contracted rings of bovine coronary artery. L-NOARG (100 microM) caused a significant (P < 0.01) 3 fold reduction in the sensitivity to BK (pEC50, 9.27 +/- 0.11) without affecting the Rmax (101.8 +/- 2.3%). A similar, significant 3 fold reduction in sensitivity to BK with no change in Rmax was observed after treatment with oxyhaemoglobin (20 microM; pEC50, 9.18 +/- 0.13, P < 0.001) or a combination of oxyhaemoglobin (20 microM) and L-NOARG (100 microM; pEC50, 9.08 +/- 0.10, P < 0.001). Oxyhaemoglobin (20 microM) either alone or in combination with L-NOARG (100 microM) caused an approximate 600 fold decrease in the sensitivity to SNAP. 4. The L-type voltage-operated Ca2+ channel inhibitor, nifedipine (0.3 microM-3 microM), reduced the maximum contraction (Fmax) to isotonic 68 mM KCl Krebs solution (103.5 +/- 2.0% KPSSmax) by 85-90% (P < 0.001); yet, the highest concentration of nifedipine (3 microM) caused only a small but significant reduction in both the sensitivity and Fmax to U46619. By contrast, nifedipine (3 microM) had no effect on the relaxation response to BK. Furthermore, a combination of nifedipine (3 microM) and L-NOARG (100 microM) had no further inhibitory effects on relaxations to BK (pEC50, 8.79 +/- 0.10; Rmax, 101.7 +/- 2.4%) than did L-NOARG (100 microM) alone (pEC50, 9.05 +/- 0.12; Rmax, 99.62 +/- 1.19). Also, nifedipine (0.3 microM and 3 microM) had no effect on the maximum relaxation to the K+ channel opener, levcromakalim (0.3 microM). 5. In the presence of nifedipine (0.3 microM to control contractions induced by high KCl) and isotonic 68 mM KCl Krebs solution (to inhibit K+ channel activity), relaxations to BK (pEC50, 9.42 +/- 0.10; Rmax, 93.9 +/- 1.8%) were similar to those observed in normal Krebs solution (pEC50, 9.58 +/- 0.09; Rmax, 98.4 +/- 0.8%). However, in the presence of 68 mM KCl Krebs solution the inhibitory effect of L-NOARG (100 microM) on relaxations to BK (pEC50, 8.53 +/- 0.20; Rmax, 31.0 +/- 11.3%) was markedly greater than that in normal KCl Krebs solution (pEC50, 9.12 +/- 0.08; Rmax, 91.5 +/- 2.0%). Similar treatment with 68 mM KCl Krebs had no effect on relaxations to the NO donor, SNAP, yet abolished the response to the K+ channel opener, levcromakalim (0.3 microM). 6. In summary, this study has shown that (1) NO synthesis in response to BK in bovine coronary artery endothelial cells in situ is likely to be abolished by L-NOARG, (2) NO-independent relaxations to BK are markedly attenuated by 68 mM KCl-containing Krebs, which, in the absence of L-NOARG, had no effect, (3) nifedipine blocked contractions to a maximum-depolarizing stimulus (KCl) yet had no effect on NO-independent relaxations to BK, and (4) maximum relaxations to levcromakalim were abolished by 68 mM KCl Krebs but were not affected by nifedipine. Therefore, we hypothesize that if smooth muscle hyperpolarization is involved in non-NO-, endothelium-dependent relaxation in bovine coronary arteries contracted with U46619, then it can accomplish this via a mechanism which does not i     

Histamine relaxation of aortic rings from diabetic rats

The effect of histamine-induced relaxation on thoracic aortic rings from rats 5, 12, 24 and 52 weeks following streptozotocin-induced diabetes was determined. Preliminary studies confirmed the dependence of histamine-induced relaxation and the independence of nitroglycerin-relaxation (GTN) on the presence of endothelium (EDRF). Diabetes was confirmed by blood glucose levels exceeding 300 mg/dl. Rings with endothelium were depolarized several times with 50 mM KCL and then contracted with phenylephrine (10(-6)). Dose-response curves were plotted from data obtained following exposure to histamine (10(-7)-10(-3)) and GTN (10(-9)-10(-7)) and compared to responses from age-matched untreated controls, diabetic and diabetic rats treated with insulin (2 U/day). The relaxation produced by histamine on phenylephrine pre-contracted rings was similar in all three groups from 5-week age matched rats. However, histamine-induced relaxation from untreated diabetic rats was significantly depressed at 12, 24 and 52 weeks (p less than 0.001). Conversely there was no difference in the relaxation elicited by GTN on rings obtained from the three groups at any age. Pretreatment with diphenhydramine (5 x 10(-7)) on aortic rings from 12 and 52-week age matched rats resulted in qualitatively similar histamine dose-response curves that were displaced about two orders of magnitude to the right, indicative of H1 receptor competitive antagonism. These results demonstrate that the duration of diabetes alters the responsiveness of rat thoracic aortic rings to histamine but not to GTN and suggests that the responses elicited by certain agonists on target tissues may be significantly altered depending on the duration of diabetes.