Endothelium-derived relaxing factor and the effects of acetylcholine and histamine on resistance blood vessels.

1. The role of endothelium-derived relaxing factor (EDRF) in the action of vasodilator (acetylcholine, histamine, nitroprusside) and vasoconstrictor (noradrenaline, vasopressin) drugs on vascular resistance in the isolated perfused kidney and mesentery of the rat was studied. 2. Acetylcholine (EC50 = 0.18 +/- 0.05 nmol and 3.1 +/- 0.06 nmol, n = 8) and histamine (EC50 = 31.2 +/- 4.9 nmol and 46.2 +/- 3.9 nmol, n = 8) produced dose-related vasodilatation in noradrenaline-preconstricted (i.e. 'high tone') rat renal and mesenteric blood vessels. The response to both vasodilators (but not nitroprusside) was abolished by infusion of CHAPS (4.7 mg ml-1, 30 s). By use of an immunocytochemical staining procedure CHAPS was demonstrated to remove vascular endothelial cells lining intrarenal blood vessels. 3. Gossypol (3 microM), metyrapone (10 microM) and nordihydroguaiaretic acid, (NDGA, 30 microM), presumed inhibitors of EDRF biosynthesis, reduced or abolished the response to acetylcholine and histamine in perfused kidney and mesentery of the rat without affecting vasodilatation due to nitroprusside. Mepacrine (10 microM) similarly abolished the response to acetylcholine and histamine but in addition, reduced the response to nitroprusside in both preparations. 4. Methylene blue (100 microM), a presumed antagonist of the effect of EDRF, abolished vasodilatation due to acetylcholine and histamine and reduced the response to nitroprusside in perfused rat kidney and mesentery. Superoxide dismutase, SOD (15 u ml-1), was without effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic GMP release and vasodilatation induced by EDRF and atrial natriuretic factor in the isolated perfused kidney of the rat.

1. Guanosine 3':5'-cyclic monophosphate (cyclic GMP) release and vascular tone was measured in the isolated kidney of the rat perfused at constant flow with Krebs-Henseleit solution. The effects of 3 vasodilators, acetylcholine (ACh), atrial natriuretic factor (ANF) and sodium nitroprusside (SNP) on the renal release of cyclic GMP and vascular tone were examined. The ability of the endothelial-derived relaxing factor (EDRF) inhibitors, haemoglobin and gossypol, to modify vasodilatation and vasodilator-induced changes in cyclic GMP releases from the kidney was also investigated. 2. Renal cyclic GMP release was elevated 8 fold by ANF (0.01 microM), 5 fold by SNP (1 microM) and 3 fold by ACh (0.3 microM). 3. For ACh, both the increase in renal cyclic GMP release and the vasodilatation were reduced by the EDRF inhibitors, haemoglobin (1 microM) and gossypol (15 microM). For SNP, neither the increase in renal cyclic GMP release nor vasodilatation were inhibited by gossypol (15 microM). 4. For ANF, neither the increase in cyclic GMP release from the kidney nor its vasodilator activity were affected by haemoglobin (1 microM). 5. EDRF inhibitors reduced the basal release of cyclic GMP from 0.32 +/- 0.06 pmol min-1 to 0.18 +/- 0.03 pmol min-1, gossypol being more effective than haemoglobin. 6. The results are consistent with the ability of ACh to induce EDRF-mediated vasodilatation in the isolated perfused kidney of the rat. Basal EDRF release appears to contribute approximately 50% to the basal release of cyclic GMP from this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of arginine and nitric oxide on resistance blood vessels of the perfused rat kidney.

1. The vasodilator effects of arginine, nitric oxide (NO), acetylcholine (ACh) and sodium nitroprusside (NP) in the noradrenaline-preconstricted ('high tone') perfused rat kidney have been examined. 2. L-Arginine (0.6-23 mumol) caused a biphasic change in renal perfusion pressure. D-Arginine (0.6-23 mumol) was without effect. The second vasodilator component was abolished and the first vasoconstrictor effect augmented following CHAPS-induced removal of the vascular endothelium suggesting that vasodilatation was endothelium-dependent. 3. L-Arginine salts produced transient and dose-related vasodilatation. L-Arginine methylester was the most potent with an ED50 of 2.2 +/- 0.4 mumol (n = 6). The rank order of potency of the salts tested was: methylester greater than hydroxamate greater than chloride. L-Homoarginine chloride was also vasodilator (ED50, 12.0 +/- 1.3 mumol, n = 5). D-Arginine chloride was without effect at doses up to 170 mumol. Responses to L-arginine chloride were endothelium-derived relaxing factor (EDRF)-dependent being abolished by CHAPS (4.7 mg ml-1, 30 s) and significantly inhibited (greater than 70%) by gossypol (3 microM) and nordihydroguaiaretic acid (NDGA, 10 microM). 4. Vasodilatation due to NO was unaffected by CHAPS and gossypol treatment but inhibited by NDGA. NO was approximately 3 times less potent than ACh but 3000 times more potent than L-arginine methylester. 5. Kidneys perfused for 1 h with Krebs solution containing L-arginine chloride (100 microM) or L-canavanine (50 microM) showed no change in sensitivity towards ACh or NP. Higher concentrations of L-arginine chloride (500 microM) or L-canavanine (150 microM) significantly reduced the response to both vasodilators.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histamine-induced vasodilation in the perfused kidney of STZ-diabetic rats: role of EDNO and EDHF

In this study, we have examined the contribution of endothelium-derived nitric oxide (EDNO) and endothelium-derived hyperpolarizing factor (EDHF) to histamine-induced endothelium-dependent relaxation in the perfused kidney of rats treated with streptozotocin (STZ) to induce diabetes. Histamine-induced vasodilatation in the perfused kidney preparations of both control and diabetic animals, which was not significantly different. Sodium nitroprusside (SNP)-induced relaxation was also not affected in diabetic and control rats. In order to isolate the EDHF component of histamine-induced vasodilator response, L-NAME (10(-4)M) and indomethacin (10(-6)M) were added to the Krebs' solution throughout the experiment. TBA (0.5 mM) produced a significant reduction in histamine-induced maximal vasodilator response in both preparations from control and diabetic animals, indicating the involvement of K+ channels in mediating this response. Charybdotoxin (0.05 microM) but not glibenclamide (0.1 microM) produced significant reduction in histamine-induced vasodilator responses. To test the contribution of EDNO in mediating histamine-induced vasodilatation, the vascular preparations were perfused with 20 mM K+ -Krebs' solution to inhibit the EDHF component of the response. Under this condition, histamine-induced vasodilator response was not significantly different in both preparations from control and diabetic rats. Pre-treatment with L-NAME (10(-4)M) attenuated histamine-induced vasodilatation. There was a more significant attenuation in histamine-induced vasodilatation in the vascular preparations from diabetic rats. The vasodilator effect of calcium ionophore A23187 was investigated in preparations from control and diabetic rats to examine receptor dysfunction associated with diabetes. A23187 produced dose-dependent vasodilator response in the preparations from both control and diabetic rats. In conclusion, our results indicate that histamine-induced vasodilatation in the perfused kidney of the STZ-induced diabetic rats is mediated by the two vasodilator components, namely EDHF and EDNO. The EDHF component was not significantly affected by diabetes. However, histamine-induced vasodilatation mediated by the EDNO component was more significantly reduced in diabetic rats. Results have also indicated that the EDHF component of histamine-induced vasodilatation was mediated through Ca2+ -activated K+ channels in perfused kidney preparations from both control and diabetic rats.     

The role of endothelin in FK506-induced vascular reactivity changes in rat perfused kidney

Tacrolimus (FK506) is an immunosuppressant agent that is widely used in transplanted patients. The aim of this study was to investigate the role of endothelin in the acute effects of FK506 on the vascular reactivity in perfused isolated rat renal and mesenteric vasculature. Left kidney/mesentery of male Wistar rats (230-300 g) were perfused by a constant flow and perfusion pressure was recorded. The responses to noradrenaline and sodium nitroprusside were obtained both in the absence and presence of FK506 (10(-7) M) or polyoxyethylene hydrogenated castor oil 60 (HCO-60 and solvent of the drug at equivalent concentrations). FK506 significantly increased the noradrenaline-induced vasoconstrictor responses in renal, but not in mesenteric vascular beds. Bosentan (10(-5) M), a nonselective endothelin ET-1 receptor antagonist given by perfusion, reversed the increase in noradrenaline responses in the kidney. Sodium nitroprusside-induced vasodilator responses in both renal and mesenteric vascular beds were significantly decreased by FK506. However, in renal vasculature, there was no significant difference between the inhibitory effects of FK506 and HCO-60, although the effect of the solvent was not significantly different from that of the control. While in the mesenteric bed, the solvent significantly inhibited nitroprusside-induced vasodilation, similar to that of FK506. The effect of FK506 on vasodilation in both vascular beds was not reversed by bosentan. Our results indicated that FK506 increased the reactivity of the renal vascular bed to noradrenaline through endothelin ET-1 receptor activation. The mechanism of impaired vasodilation due to FK506 appears to be due to its solvent action and is independent of endothelin release.     

Possible involvement of endothelium in the responses of various vasoactive agents in rabbit isolated perfused kidney.

1. Perfusion of the kidney with methylene blue, a soluble guanylate cyclase inhibitor, significantly enhanced the vasoconstrictor effects of angiotensin II, noradrenaline and phenylephrine but significantly reduced the vasodilator effect of acetylcholine without altering that of iloprost. 2. In the kidneys, which were perfused with Triton X-100 to remove endothelium, acetylcholine-induced vasodilation was completely abolished and angiotensin II-, noradrenaline- and phenylephrine-induced vasoconstriction was greatly reduced. 3. The vasodilator effect of iloprost was unchanged after perfusion of kidney with Triton X-100. 4. Neither methylene blue nor Triton X-100 significantly altered urine volume form normal and angiotensin II induced increase of urine volume. 5. These results were taken as evidence for the involvement of renal vascular endothelium originated EDRF in the responses of various vasoactive agents in the rabbit isolated perfused kidney.     

Hydroxylamine dilates resistance blood vessels of the perfused rat kidney and mesentery

Hydroxylamine (ED50 values, 47 +/- 8.9 nmol and 320 +/- 39 nmol) dilates resistance arterioles of the perfused noradrenaline-preconstricted rat kidney and mesentery. In this respect hydroxylamine was approximately 63x and 320x less potent than acetylcholine (ACh) and 15x and 128x less potent than nitroprusside in the two perfused organs studied. The vasodilator effect of hydroxylamine (unlike that of ACh) was unaffected by CHAPS de-endothelialization suggesting that its effect is independent of endothelium-derived relaxing factor (EDRF).     

Epidermal growth factor receptor tyrosine kinase-mediated signalling contributes to diabetes-induced vascular dysfunction in the mesenteric bed

In order to characterize the roles of tyrosine kinases (TKs) and epidermal growth factor receptor (EGFR) in diabetes-induced vascular dysfunction, we investigated the ability of a chronic administration of genistein, a broad-spectrum inhibitor of TKs and AG1478, a specific inhibitor of EGFR TK activity to modulate the altered vasoreactivity of the perfused mesenteric bed to common vasoconstrictors and vasodilators in streptozotocin (STZ)-induced diabetes in rats. The vasoconstrictor responses induced by norepinephrine (NE), endothelin-1 (ET-1) and angiotensin II (Ang II), were significantly increased, whereas vasodilator responses to carbachol and histamine were significantly reduced in the perfused mesenteric bed of STZ-induced diabetic rats in comparison with healthy rats. Treatment of diabetic animals with genistein or AG1478 produced a significant normalization of the altered agonist-induced vasoconstrictor and vasodilator responses without affecting blood glucose levels. In contrast, neither inhibitor had any effect on the vascular responsiveness of control (nondiabetic) animals. Treatment of diabetic animals with diadzein, an inactive analogue of genistein, did not affect the vasoconstrictor and vasodilator responses in control or diabetic animals. Phosphorylated EGFR levels were markedly raised in the mesenteric bed from diabetic animals and were normalized upon treatment with AG1478 or genistein. These data suggest that activation of TK-mediated pathways, including EGFR TK signalling are involved in the development of diabetic vascular dysfunction.     

Effect of early stage of experimental diabetes on vascular functions in isolated perfused kidneys

1 The present study investigates the renal vascular responsiveness to vasoactive agents in diabetic rats which present an early stage of renal failure. 2 Adult male Wistar rats were administered alloxan (150 mg kg^–1, s.c.). Seven days later the right kidneys were isolated and perfused. Renal perfusion pressure was measured continuously. Concentration-response curves were plotted for noradrenaline (NA), sodium nitroprusside (SNP) and carbachol. 3 In basal conditions, kidneys from diabetic rats presented a decreased vascular resistance compared with those from control rats. 4 The vasoconstrictor response to NA showed decreased EC₅₀ values in preparations from diabetic rats compared with control ones (EC₅₀ nmols, control: 2.03 ± 0.44, n = 8; diabetic: 0.84 ± 0.18, n = 6, P < 0.05). This enhanced sensitivity to NA could be in line with the decreased glomerular filtration rate and cortical renal plasma flow previously described in vivo in our laboratory ( Garcia et al. 1998 ) . Vasoconstrictor responses to phenylephrine were not however, different between diabetic and control rat kidneys. This suggests that the increased sensitivity to NA was due to impaired neuronal uptake since phenylephrine is not a substrate for neuronal uptake. 5 After precontraction with phenylephrine, both endothelium-dependent (carbachol) and endothelium independent (SNP) vasodilator agents caused similar response in the preparations taken from the two groups of animals. So, the enhanced sensitivity to NA is not associated with a deficient dilator responsiveness of the renal vasculature. 6 The vasodilator response to carbachol was the same in absence or presence of l -arginine in the perfusate, suggesting no alteration in its availability at this stage of diabetes. 7 Diabetic animals showed increased plasma level of fructosamine and glycosylated haemoglobin (Hb A_1c), indicating the presence of early glycated products at this stage of diabetes, which could be involved in a possible structural alteration of the vessels.     

Histamine-induced vasodilatation in the perfused mesenteric arterial bed of diabetic rats

In this study, we have examined the contribution of endothelium-derived nitric oxide (EDNO) and endothelium-derived hyperpolarizing factor (EDHF) to histamine-induced endothelium-dependent relaxation in the perfused mesenteric arterial bed of rats treated with streptozotocin (STZ) to induce diabetes. Histamine (10(-10) to 5 x 10(-6) mol) produced dose-dependent vasodilator response in the perfused mesenteric arterial bed of both control and diabetic animals. In order to isolate the EDHF component of histamine-induced vasodilator response, NG-nitro-L-arginine-methyl ester hydrochloride (L-NAME) (10(-4) M) and indomethacin (10(-6) M) were added to the Krebs solution throughout the experiment. Histamine induced vasodilatation in the perfused mesenteric bed in preparations from both control and diabetic rats. The vasodilator response to histamine was slightly potentiated in the diabetic rat preparations. Sodium nitroprusside (SNP)-induced relaxation was similar in diabetic and control rats. The role of EDNO in histamine-induced vasodilatation was also examined. Vascular preparations were perfused with 20 mM K(+)-Krebs solution to inhibit the EDHF contribution to histamine-induced vasodilatation. Under this condition, histamine induced a vasodilator response in preparations from both control and diabetic rats. However, relative to nondiabetic control animals, histamine-induced maximal response was significantly reduced in preparations from diabetic animals. Pretreatment with L-NAME (10(-4) M) attenuated histamine-induced vasodilatation in both preparations, indicating an NO-mediated vasodilator response. There was a significant attenuation in histamine-induced vasodilatation in the vascular preparations from diabetic rats. The vasodilator effect of calcium ionophore A23187 was investigated in preparations from control and diabetic rats to investigate receptor dysfunction associated with diabetes. A23187 (10(-11) to 10(-7) mol)-induced vasodilator response was not significantly different in the preparations from control and diabetic animals. In conclusion, our results indicated that histamine-induced vasodilation in the perfused mesenteric arterial bed of the STZ-induced diabetic rats is mediated by two vasodilator components, namely EDHF and EDNO. Under diabetic conditions, the EDHF component was potentiated, while histamine-induced vasodilation mediated by the EDNO component was attenuated.     

Reduction of endothelial function with age in the mesenteric arterial bed of the normotensive rat.

1. Age-related changes in endothelial vasodilator function were studied in an in vitro preparation of the mesenteric arterial bed removed from male, normotensive, Wistar rats. 2. Animals were killed at 2, 12 or 22 months of age, the superior mesenteric artery was cannulated and the gut removed. The mesenteric arterial bed was perfused at a constant flow rate of 4 ml min-1 and perfusion pressure was taken as an index of arteriolar tone. 3. The muscarinic agonist, carbachol, antagonized noradrenaline-induced vasoconstriction in the presence, but not in the absence, of endothelium. This cholinoceptor agonist-induced release of endothelial-derived relaxing factor (EDRF) was impaired in 22 month old rats. 4. Noradrenaline-induced vasoconstriction increased following removal of endothelium suggesting that agonist-induced release of EDRF attenuates vasoconstrictor responses to noradrenaline measured in the presence of endothelium. 5. Removal of endothelium had less effect on noradrenaline-induced vasoconstriction in old rats suggesting once again that agonist-induced release of EDRF is impaired in old rats. 6. The noradrenaline dose-response curve established in the presence of endothelium was shifted to the left in 22 month old rats. 7. In conclusion, aging in the rat appears to lead to a reduction in endothelial vasodilator function in a resistance vessel.     

Chronic treatment with the angiotensin I converting enzyme inhibitor, perindopril, protects in vitro carbachol-induced vasorelaxation in a rat model of vascular calcium overload.

1. Treatment of young rats with vitamin D3 plus nicotine produced 31 and 4 fold increases in the calcium content of the aorta and the mesenteric arterial bed, respectively. 2. Aortic rings and perfused mesenteric arterial beds from vitamin D3/nicotine-treated animals showed a diminished contractile response to noradrenaline in vitro. 3. In vascular preparations from vitamin D3/nicotine-treated animals, precontracted with noradrenaline, relaxation by the endothelium-dependent vasodilator, carbachol, was attenuated but responses to sodium nitroprusside were not modified. 4. Prolonged treatment with the angiotensin I converting enzyme inhibitor, perindopril, at a dose (1 mg kg-1) which did not significantly modify blood pressure, failed to prevent vascular calcium overload. 5. Perindopril treatment diminished noradrenaline-evoked vasoconstrictor responses of aortic rings in both groups, but restored responses in mesenteric arterial beds of vitamin D3/nicotine-treated rats. 6. Perindopril treatment also restored the maximal responses to carbachol of both aortic rings and mesenteric arterial beds of vitamin D3/nicotine-treated rats. 7. In conclusion, in the vitamin D3 plus nicotine model of calcium overload, reduced endothelial-mediated relaxation can be prevented by perindopril treatment.     

Effect of phenobarbitone pretreatment upon endothelium-dependent relaxation to acetylcholine in rat superior mesenteric arterial bed.

1. Pretreatment of rats for 5 days with phenobarbitone (80 mg kg-1 day-1) enhanced the potency enhanced the potency of acetylcholine in opposing noradrenaline-induced vasoconstriction in the isolated perfused superior mesenteric arterial bed; in 10 saline-pretreated control animals the ED50 was 14.0 +/- 3.9 ng whereas it was 3.23 +/- 1.00 ng in 10 phenobarbitone-pretreated animals. 2. In both saline- and phenobarbitone-pretreated rats acetylcholine was ineffective at opposing noradrenaline vasoconstriction after the mesentery had been perfused for 90s with a 0.3% solution of the detergent CHAPS in distilled water (to remove the endothelium), but pressor responses to noradrenaline were unaffected. 3. Pretreatment with phenobarbitone had no effect on the opposition by sodium nitroprusside of noradrenaline pressor responses. Also, the effects of nitroprusside were not affected by perfusion with CHAPS in either control or barbiturate-pretreated groups. 4. Inclusion of indomethacin (10 microM) in the perfusion fluid had no effect on the enhancement by phenobarbitone pretreatment of the endothelium-dependent opposition by acetylcholine of noradrenaline pressor responses; the ED50 values in the absence and presence of indomethacin were, respectively, 2.40 +/- 0.31 ng and 1.87 +/- 0.27 ng (n = 6). 5. The concentration of cytochrome P450 in the microsomal fraction obtained from the mesenteric preparation was increased from 204 +/- 32 (saline-pretreated; n = 7) to 784 +/- 249 pmol g-1 wet wt (n = 7) by the phenobarbitone pretreatment. 6. It is concluded that the increase in potency of acetylcholine as an endothelium-dependent vasodilator by phenobarbitone pretreatment is most probably at the level of the endothelium rather than the vascular smooth muscle.     

Attenuation of vasoconstriction by endogenous nitric oxide in rat caudal artery.

1. The effects of NG-nitro-L-arginine (L-NNA), NG-nitro-L-arginine methyl ester (L-NAME), haemoglobin and methylene blue have been examined on vascular reactivity in the rat isolated caudal artery. The effects of L-NNA and sodium nitroprusside were also investigated on the stimulation-induced (S-I) efflux of noradrenaline in the rat caudal artery. 2. L-NNA (10 microM) and L-NAME (10 microM) significantly attenuated the vasodilator responses to acetylcholine (1 nM-1 microM), but had no effect on vasodilator responses to papaverine (1-100 microM). 3. Vasoconstrictor responses to sympathetic nerve stimulation (3 Hz, 10 s), noradrenaline (0.01-1 microM), methoxamine (1-10 microM), 5-hydroxytryptamine (0.01-0.3 microM), phenylephrine (0.1-10 microM), endothelin-1 (10 nM) and KCl (40 mM) were significantly enhanced by 10 microM L-NNA. L-NAME (10 microM) caused a significant enhancement of vasoconstrictor responses to noradrenaline and sympathetic nerve stimulation in endothelium-intact, but not in endothelium-denuded tissues. 4. Haemoglobin and methylene blue (both 10 microM) enhanced the vasoconstrictor responses to sympathetic nerve stimulation and noradrenaline. The enhancements were absent in endothelium-denuded arterial segments. 5. In endothelium-denuded arterial segments precontracted with phenylephrine, the vasodilator responses to the nitric oxide donor, sodium nitroprusside (0.1-300 nM) were decreased by increasing the level of precontraction. 6. L-NNA (10 microM) had no effect on the S-I efflux of radioactivity from arteries in which transmitter stores had been labelled with [3H]-noradrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vasodilator actions of acetylcholine, A23187 and bradykinin in the guinea-pig isolated perfused heart are independent of prostacyclin

1. The involvement of prostacyclin (PGI2) in the vasodilator responses to acetylcholine (ACh), A23187 and bradykinin (Bk) has been investigated in guinea-pig, isolated, Krebs-perfused hearts. 2. ACh (0.01-10 nmol), A23187 (0.1-1.0 nmol) and Bk (0.3-10 pmol) each elicited dose-related and shortlasting (approximately 2 min) reductions in perfusion pressure. Larger maximal responses were obtained in preparations with coronary vascular tone elevated by platelet-activating factor (100 pmol) than in preparations at basal perfusion pressure. 3. Bk and A23187 elicited dose-related increases in the generation of PGI2 as measured by its chemically-stable breakdown product, 6-oxo-PGF1 alpha. Indomethacin (2.8 microM) prevented both basal and the stimulated generation of 6-oxo-PGF1 alpha, whereas the magnitudes of the vasodilator responses were unaffected. 4. Attempts to identify the release of vasodilator materials by on-line superfusion bioassay of cardiac effluent were unsuccessful, indicating a possible role for a labile vasodilator such as endothelium-dependent relaxing factor (EDRF). In addition, the inhibitors of EDRF action/production, mepacrine (3 microM) or diethylcarbamazine (300 microM), attenuated vasodilator responses to ACh without altering those to the endothelium-independent vasodilator, verapamil (1 nmol). 5. Haemoglobin (10 microM) reduced vasodilator responses to ACh, Bk and verapamil and abolished those induced by A23187. Inhibition of the endothelium-independent vasodilator, verapamil, was significantly less than that for the other compounds. 6. The present data indicate the existence of an indomethacin-resistant vasodilator mechanism in the coronary microcirculation in response to ACh, A23187 and Bk. EDRF is a candidate for mediating these responses; however, a direct vasodilator action of these substances cannot be excluded.     

The effect of ischaemia on endothelium-dependent vasodilatation and adrenoceptor-mediated vasoconstriction in rat isolated hearts.

1. The aim of this study was to investigate whether global ischaemia and reperfusion in rat isolated hearts affects endothelium-dependent vasodilatation and adrenoceptor-mediated vasoconstriction. In addition, it was first determined whether inhibition of the actions of nitric oxide (NO) influenced the responses to alpha-adrenoceptor agonists in the rat coronary vasculature. 2. In rat isolated, Langendorff perfused hearts, inhibition of NO with haemoglobin (Hb, 6 microM) significantly inhibited the vasodilator responses to the endothelium-dependent vasodilators, acetylcholine (ACh, 3-100 pmol), carbachol (CCh, 10-300 pmol), bradykinin (Bk, 1-30 pmol) and histamine (0.3-10 nmol) but did not affect responses to the endothelium-independent vasodilator, sodium nitroprusside (SNP, 0.01-1 nmol). 3. Inhibition of the action of NO by Hb significantly enhanced the vasoconstrictor response to the non-selective alpha-adrenoceptor agonist, noradrenaline (NA, 0.1-10 nmol) and the alpha 2-adrenoceptor agonist, B-HT 920 (0.001-1 mumol) but had no effect on the vascular response to the alpha 1-adrenoceptor agonist, methoxamine (MTX, 10-300 nmol). 4. In the perfused hearts ischaemia, induced by 30 min perfusion at 5% of the normal rate of flow, followed by 15 min of reperfusion (ischaemia/reperfusion) selectively impaired the vasodilator responses to ACh and CCh which act by muscarinic receptor stimulation but did not affect responses to the other endothelium-dependent vasodilators Bk and histamine or to the endothelium-independent dilator SNP. 5. After ischaemia/reperfusion the coronary vasoconstrictor responses to B-HT 920 were slightly but significantly enhanced whereas the responses to NA and MTX were unaffected. 6. Thus, in the rat isolated heart, low flow induced-ischaemia and reperfusion causes a selective impairment of muscarinic receptor-mediated vasodilatation but does not impair responses to all endothelium-dependent vasodilators. Enhanced constrictor responses to noradrenaline and B-HT 920 in the presence of Hb indicates that endogenous NO modulates the constriction of coronary resistance vessels in response to stimulation of alpha 2-adrenoceptors. Ischaemia and reperfusion in this isolated vascular bed caused only a small increase in the coronary vasoconstrictor response to alpha 2-adrenoceptor stimulation. It appears that in the rat isolated heart the degree of endothelial dysfunction caused by ischaemia/reperfusion is insufficient to cause a functionally significant change in alpha-adrenoceptor-mediated constriction.     

Endothelium-dependent relaxation in isolated renal arteries of diabetic rabbits

1 In this study, we have investigated the vasodilator response to acetylcholine under diabetes conditions in isolated renal arteries of rabbits. We have also examined the contribution of endothelium-derived nitric oxide (EDNO) and endothelium-derived hyperpolarizing factor (EDHF) to the endothelium-dependent relaxation caused by acetylcholine in the renal arteries of alloxan-induced diabetic rabbits. 2 Acetylcholine (10(-10) - 10(-4) M) produced cumulative concentration-response curve in the renal arteries of both control and diabetic rabbits. The EC50 values and maximal responses to acetylcholine were not significantly different relative to diabetic conditions. In order to isolate the EDHF component of acetylcholine-induced vasodilator response, L-nitro-methyl arginine ester (L-NAME, 10(-4) M) and indomethacin (10(-6) M) were added to the Krebs' solution throughout the experiment. Under these conditions, acetylcholine induced vasodilatation in the isolated renal arteries from both control and diabetic rabbits. The vasodilator response to acetylcholine was not affected under diabetic conditions. 3 Sodium nitroprusside (SNP)-induced relaxation was increased in the diabetic rabbits compared with the control animals. 4 Tetrabutyl ammonium (TBA, 0.5 mM) produced a significant reduction in acetylcholine-induced vasodilatation in both preparations from control and diabetic animals, consistent with involvement of K+ channels in mediating this response. Glibenclamide (1 microM) attenuated acetylcholine-induced vasodilatation in preparations from control animals only, while iberiotoxin (0.05 microM) significantly reduced the vasodilator response to acetylcholine in preparations from both control and diabetic animals. 5 The role of EDNO in mediating acetylcholine-induced vasodilatation was examined. The vascular preparations were incubated with 20 mM K(+)-Krebs' solution to inhibit the EDHF contribution to acetylcholine-induced vasodilatation. Under this condition, acetylcholine induced a vasodilator response in both preparations from control and diabetic rats. Pretreatment with L-NAME (10(-4) M) attenuated acetylcholine-induced vasodilatation in both preparations, indicating an nitric oxide-mediated vasodilator response. 6 Our results indicated that acetylcholine-induced vasodilatation in the isolated renal arteries of alloxan-induced diabetic rabbits was not affected under diabetic conditions. Acetylcholine-induced vasodilatation is mediated by two vasodilator components; namely, EDHF and EDNO. The contribution of EDHF and EDNO to acetylcholine-induced vasodilatation was not affected under diabetic conditions and there was no indication of endothelial dysfunction associated with diabetes. EDHF component was found to act mainly through high conductance Ca(2+)-activated K+ channels under normal and diabetic conditions, while the adenosine triphosphate-dependent K+ channels were involved in mediating acetylcholine vasodilator response in the control preparations only.     

Vasodilator action of endothelin-1 in the perfused rat heart.

The effects of bolus injections of porcine endothelin (ET-1, 1-100 pmol) on the coronary microvasculature of isolated perfused rat heart were examined. Results show that ET-1 possesses dose-dependent vasodilator as well as vasoconstrictor properties. The vasodilator effect was transient and preceded its more pronounced and persistent vasoconstrictor action. ET-1-induced vasodilation in rat heart was not associated with release of prostacyclin (PGI2), as shown by radioimmunoassay (RIA) analysis of cardiac effluent and was not blocked by the cyclooxygenase inhibitors flurbiprofen (2 microM) or BW755c (7.5 microM). Neither was the dilatory response to ET-1 inhibited by haemoglobin (10 microM) or potentiated by superoxide dismutase (20 U/ml) but it was abolished by methylene blue (20 microM). However, methylene blue itself caused coronary dilation which could mask the vasodilator action of ET-1. These results show that in isolated perfused rat heart ET-1 possesses a vasodilator action that is not mediated by PGI2 and that may also be independent of release of endothelium-derived relaxing factor (EDRF).     

Enhancement of aorta tonus evoked by hypertonic solutions is endothelium-dependent.

1. The effect of NaCl hypertonic solutions on the contractions induced by noradrenaline or prostaglandin F2 alpha and on the relaxant effect of acetylcholine and sodium nitroprusside was investigated in aortae with or without endothelium isolated from rats. 2. Hypertonic solutions enhanced the contractions elicited by the vasoconstrictor agents in preparations with endothelium and this phenomenon was not altered by previous treatment of the animals with reserpine or the preparations with methylene blue or indomethacin. 3. In hypertonic medium acetylcholine but not sodium nitroprusside vasodilator effect was inhibited. 4. We suggest that NaCl hypertonic solutions modify vascular reactivity probably through: (i) the release of an endothelium-derived contracting factor not sensitive to cyclooxygenase inhibitors; (ii) a diminished release of an endothelium-derived relaxing factor not related to the cGMP system.     

Human and rat alpha-CGRP but not calcitonin cause mesenteric vasodilatation in rats

A single gene encodes both calcitonin and the calcitonin gene-related peptide (CGRP). Human and rat alpha-CGRP were compared with sodium nitroprusside in the rat and rabbit isolated mesenteric vascular preparation perfused at constant flow. In the presence of the vasoconstrictor noradrenaline (10(-5) M), rat alpha-CGRP was about 10 times as potent as either human alpha-CGRP or sodium nitroprusside as a vasodilator in the rat mesenteric vasculature. In the rabbit mesenteric vasculature the order of potency was rat alpha-CGRP greater than human alpha-CGRP greater than sodium nitroprusside. Human and salmon calcitonin showed no vasodilator activity at doses 100 times greater than human alpha-CGRP. These results show that human and rat alpha-CGRP are potent vasodilators in the mesenteric vasculature, an effect not mimicked by the alternative gene product, the plasma calcium lowering hormone calcitonin.