Diabetes-induced changes in endothelial mechanisms implicated in rabbit carotid arterial response to 5-hydroxytryptamine

The influence of diabetes on endothelial mechanisms implicated in the response of isolated rabbit carotid arteries to 5-hydroxytryptamine (5-HT) was studied. 5-HT induced a concentration-dependent contraction that was potentiated in arteries from diabetic rabbits with respect to that in arteries from control rabbits. Endothelium removal potentiated 5-HT contractions in arteries from both control and diabetic rabbits but increased the maximum effect only in arteries from diabetic rabbits. Incubation of arterial segments with N(G)-nitro-L-arginine (L-NA) enhanced the contractile response to 5-HT. This L-NA enhancement was greater in arteries from diabetic rabbits than in arteries from control rabbits. Aminoguanidine did not modify the 5-HT contraction in arteries from control and diabetic rabbits. Indomethacin inhibited the 5-HT-induced response, and this inhibition was higher in arteries from control rabbits than in arteries from diabetic rabbits. In summary, diabetes enhances the sensitivity of the rabbit carotid artery to 5-HT. In control animals, the endothelium modulated the arterial response to 5-HT by the release of both nitric oxide (NO) and a vasoconstrictor prostanoid. Diabetes enhances endothelial constitutive NO activity and impairs the production of the endothelial vasoconstrictor.     

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.     

Different role of endothelin ETA and ETB receptors and endothelial modulators in diabetes-induced hyperreactivity of the rabbit carotid artery to endothelin-1

The influence of diabetes on regulatory mechanisms and specific receptors implicated in the contractile response of isolated rabbit carotid arteries to endothelin-1 was examined. Endothelin-1 induced a concentration-dependent contraction that was greater in arteries from diabetic rabbits than in arteries from control rabbits. Endothelium removal or N(G)-nitro-L-arginine enhanced contractions in response to endothelin-1 only in control arteries, without modifying the endothelin-1 response in diabetic arteries. Indomethacin, furegrelate (thromboxane A(2) inhibitor), or cyclo-(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123; endothelin ET(A) receptor antagonist) inhibited the contractions in response to endothelin-1, the inhibition being greater in diabetic arteries than in control arteries. 2,6-Dimethylpiperidinecarbonyl-gamma-methyl-Leu-N(in)-(methoxycarbonyl)-D-Trp-D-Nle (BQ-788; endothelin ET(B) receptor antagonist) enhanced the contraction elicited by endothelin-1 in control arteries and displaced to the right the contractile curve for endothelin-1 in diabetic arteries. In summary, diabetes induces hyperreactivity of the rabbit carotid artery to endothelin-1 by a mechanism that at least includes: (1) enhanced activity of muscular endothelin ET(A) receptors; (2) impairment of endothelin ET(B) receptor-mediated nitric oxide (NO) release; and (3) enhancement of the production of thromboxane A(2).     

Mechanisms of the ergonovine-induced vasoconstriction in the rabbit main coronary artery

Summary The mean membrane potential of smooth muscle cells of the rabbit main coronary artery was-60.3 mV and an evoked action potential could be recorded in response to acetylcholine (ACh). Ergonovine or 5-hydroxytryptamine (5-HT) slightly depolarized the membrane and methysergide, a relatively selective antagonist for the 5-HT receptor, had a slight inhibitory action on these depolarizations. 5-HT produced larger contractions than ergonovine, and the concentration-effect relationships obtained for both agents shifted to higher concentrations following pre-equilibration with methysergide. ACh (10^–11 M) slightly hyperpolarized the membrane and relaxed the tissue, and high concentrations of ACh (>10^–8 M) depolarized the membrane, increased the membrane resistance and produced a contraction. ACh but not ergonovine or 5-HT, produced a contraction in Ca-free EGTA-containing solution. Following a 60 min pre-equilibration with indomethacin, the ergonovine-induced contraction was markedly enhanced but the 5-HT-or ACh-induced contractions were not. Removal of the endothelium by rubbing the vascular lumen enhanced the ergonovine-or ACh-induced contractions, but not those to 5-HT.The results obtained can be summarized as follows: ergonovine probably accelerates Ca influx and thereby produces contraction in the rabbit main coronary artery. This contraction is due to activation of the 5-HT receptor as an agonist, but the ergonovine-induced contraction is attenuated due to activation of the endothelium from which inhibitory prostanoid substances may be released. Ergonovine, therefore, may produce greater contractions in coronary arteries with damaged endothelium than in intact tissues.     

Mechanisms underlying diabetes enhancement of endothelin-1-induced contraction in rabbit basilar artery

The influence of alloxan-induced diabetes on the reactivity of rabbit basilar artery to endothelin-1 was examined. Endothelin-1 induced concentration-dependent contraction of basilar arteries that was higher in diabetic than in control rabbits. Endothelium removal produced a higher enhancement of the endothelin-1-induced contraction in control than in diabetic rabbits. N(G)-nitro-L-arginine (L-NOArg) enhanced the maximal contraction induced by endothelin-1 in control rabbits and potentiated this response in diabetic rabbits. Endothelin ETA receptor antagonist, cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123), inhibited endothelin-1-induced contraction in both rabbit groups. Endothelin ETB receptor antagonist, 2,6-Dimethylpiperidinecarbonyl-gamma-Methyl-Leu-Nin-(Methoxycarbonyl)-D-Trp-D-Nle (BQ-788), enhanced endothelin-1-induced contraction in control rabbits and decreased the potency of endothelin-1 in diabetic rabbits. Sodium nitroprusside-induced relaxation of basilar arteries was lower in diabetic than in control rabbits. These results suggest that mechanisms underlying rabbit basilar artery hyperreactivity to endothelin-1 include decreased endothelial modulation of endothelin-1-induced contraction, with impaired endothelial endothelin ETB receptor activity; decreased sensitivity to nitric oxide (NO) in vascular smooth muscle; and enhanced participation of muscular endothelin ETA and ETB receptors.     

Diabetes potentiates acetylcholine-induced relaxation in rabbit renal arteries

The response of rabbit renal arteries to acetylcholine and its endothelial modulation in diabetes were investigated. Acetylcholine induced concentration-related endothelium-dependent relaxation of renal arteries that was significantly more potent in diabetic rabbits than in control rabbits. Pretreatment with N(G)-nitro-L-arginine (L-NOArg), indomethacin, or L-NOArg plus indomethacin induced partial inhibition of acetylcholine-induced relaxation. Inhibition induced by L-NOArg plus indomethacin was significantly higher in arteries from diabetic rabbits than in arteries from control rabbits. In renal arteries depolarised with KCl 30 mM and incubated with L-NOArg plus indomethacin, acetylcholine-induced relaxation was almost abolished in both groups of rabbits and this response was not different from that obtained in arteries without endothelium. Sodium nitroprusside induced concentration-dependent relaxation of renal arteries from control and diabetic rabbits without significant differences between the two groups of animals. These results suggest that diabetes potentiates the acetylcholine-induced relaxation in rabbit renal arteries. Increased release of nitric oxide and prostacyclin could be responsible for the enhanced relaxant potency of acetylcholine in diabetes.     

Alterations of cyclo-oxygenase products and NO in responses to angiotensin II of resistance arteries from the spontaneously hypertensive rat.

1. The involvement of cyclo-oxygenase (COX) products and nitric oxide (NO) in contractile responses of resistance arteries to angiotensin II (AII) were investigated in small mesenteric arteries from spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats. 2. In endothelium intact vessels, AII induced concentration-dependent responses without any significant difference between the two strains. However, removal of functional endothelium resulted in enhanced sensitivity to AII, the pD2 value increasing from 8.4 +/- 0.2 to 8.9 +/- 0.2 (P < 0.05) in WKY and from 8.2 +/- 0.1 to 8.6 +/- 0.1 (P < 0.05) in SHR (not significantly different between strains, n = 9 - 12). In addition, endothelium removal enhanced maximal contractions elicited by AII in SHR (1.4 +/- 0.1 to 2.1 +/- 0.2 mN mm-1, n = 5; P < 0.05) but not in WKY (1.0 +/- 0.1 to 1.2 +/- 0.1 mN mm-1, n = 5) vessels. 3. In the absence of functional endothelium, the COX inhibitor indomethacin (10(-5) M) reduced contractile responses elicited by AII in SHR arteries, resulting in 33 +/- 5% (n = 5) decrease in maximal contraction. However, it produced minimal if any, effect on responses of WKY vessels. In both strains, the TP receptor antagonist GR32191 B (3 x 10(-6) M) did not modify contractions elicited by AII in these conditions. 4. In the presence of functional endothelium, indomethacin (10(-5) M) almost abolished the responses to AII in both strains. GR32191 B (3 x 10(-6) M) reduced the sensitivity of WKY arteries to AII (pD2 = 8.1 +/- 0.1, P < 0.01) without any effect on maximal contraction. In SHR arteries, it markedly reduced maximal contraction (47 +/- 3.5%). 5. In both strains, the NO synthase inhibitor NG-nitro-L-arginine methy lester (L-NAME; 10(-4) M) had no effect in the absence of functional endothelium but it markedly reduced the inhibitory influence of endothelium on contractile responses to AII. Furthermore, in arteries with endothelium, it reduced the effect of both indomethacin and GR32191 B to the same level as observed in vessels without functional endothelium. 6. The results suggest that enhanced contraction caused by COX products was counteracted by enhanced relaxation caused by endothelium-derived NO in resistance mesenteric arteries of the SHR exposed to AII, compared to WKY arteries. The COX products involved in alterations of SHR responses comprised an endothelium-derived prostaglandin activating TP receptors and another nonendothelial unidentified vasoconstrictor compound which did not activate these receptors.     

Role of endothelium in the response to prostaglandin H2 in isolated dog arteries

Prostaglandin (PG) H2 produced a transient contraction followed by a relaxation in helical strips of dog coronary, mesenteric and renal arteries contracted with PGF2 alpha. The contraction was in the order of mesenteric greater than renal greater than coronary artery. Removal of endothelium abolished the contraction in these arteries and significantly potentiated the relaxation only in mesenteric arteries. The relaxation was greater in mesenteric arteries than in renal and coronary arteries, denuded of endothelium. PGI2-induced relaxations were not influenced by endothelium denudation. In the arteries contracted with K+, PGH2-induced relaxations were attenuated, compared to those contracted with PGF2 alpha. Treatment with ONO3708, an antagonist of vasoconstrictor PGs, abolished the PGH2-induced contraction and potentiated the relaxation in the K+-contracted arteries. The relaxant response was suppressed by diphloretin phosphate, a PG receptor antagonist, as was the response to PGI2. PGH2-induced contractions in dog coronary, mesenteric and renal arteries would be due to vasoconstrictor PGs produced preferentially in the endothelium. However, production of PGI2 from PGH2 in endothelial and subendothelial tissues do not appear to differ.     

Mechanisms underlying the diabetes-induced hyporeactivity of the rabbit carotid artery to atrial natriuretic peptide

Atrial natriuretic peptide (ANP) plays an important role in the pathophysiology of the vascular complications in diabetes. The working hypothesis was that diabetes might modify the vascular actions of ANP in isolated rabbit carotid arteries and the mechanisms involved in these actions. ANP (10^?12–10^?7 M) induced a relaxation of precontracted carotid arteries, which was lower in diabetic than in control rabbits. In arteries from both groups of animals, endothelium removal increased the ANP-induced relaxation. Isatin inhibited the relaxation to ANP both in arteries with and without endothelium. Carotid arteries from diabetic rabbits showed a decreased natriuretic peptide receptor (NPR)-A expression and an enhanced NPR-C expression. Inhibition of NO-synthesis did not modify ANP-induced relaxation in control rabbits but inhibited it in diabetic rabbits. In arteries with endothelium indomethacin enhanced the relaxation to ANP in control rabbits but did not modify it in diabetic rabbits. In endothelium-denuded arteries indomethacin inhibited the relaxation to ANP in both groups of animals. In KCl-depolarised arteries, relaxation to ANP was almost abolished both in control and diabetic rabbits. Tetraethylammonium inhibited the relaxation to ANP, and this inhibition was higher in diabetic than in control rabbits. These results suggest that diabetes produces hyporeactivity of the rabbit carotid artery to ANP by a mechanism that at least includes a reduced expression of NPR-A, an enhanced expression of NPR-C and a reduced participation of K⁺-channels. Furthermore, diabetes enhances endothelial NO release and diminishes the ratio thromboxane A₂/prostacyclin. This increase of vasodilators could result from compensatory mechanisms counteracting the arterial hyporeactivity to ANP.     

Mechanisms involved in the relaxant action of testosterone in the renal artery from male normoglycemic and diabetic rabbits

Kidney disease is a frequent complication in diabetes, and significant differences have been reported between male and female patients. Our working hypothesis was that diabetes might modify the vascular actions of testosterone in isolated rabbit renal arteries and the mechanisms involved in these actions. Testosterone (10^?8 to 10^?4 M) induced relaxation of precontracted arteries, without significant differences between control and diabetic rabbits. Both in control and diabetic rabbits endothelium removal inhibited testosterone relaxant action. In arteries with endothelium, incubation with indomethacin (10^?5 M), N^G-nitro-l-arginine (10^?5 M) or tetraethylammonium (10^?5 M) did not modify relaxations to testosterone neither in control nor in diabetic rabbits. In endothelium-denuded arteries indomethacin enhanced the relaxant action of testosterone, both in control and diabetic rabbits. In arteries from diabetic rabbits, eNOS, iNOS and COX-1 expression and testosterone-induced release of thromboxane A₂ and prostacyclin were not significantly different from those observed in control rabbits. However, COX-2 expression was significantly lower in diabetic rabbits that in control rabbits. In nominally Ca²⁺-free medium, cumulative addition of CaCl₂ (10^?5 to 3 × 10^?2 M) contracted previously depolarized arteries. Testosterone (10^?4 M) inhibited CaCl₂ contractions of the renal artery both in control and diabetic rabbits. These results show that testosterone relaxes the renal artery both in control and diabetic rabbits. This relaxation is modulated by muscular thromboxane A₂, it is partially mediated by endothelial prostacyclin, and it involves the blocking of extracellular Ca²⁺ entry. Diabetes does not modify the mechanisms involved in the relaxant action of testosterone in the rabbit renal artery.     

Critical role of endothelial nitric oxide synthase and cyclooxygenase in response of rabbit basilar artery to serotonin

The modes of action of serotonin (5-HT) on the tone of the rabbit basilar artery were investigated in vitro with the aim of determining the exact role of the endothelium. After sacrificing the animal under pentobarbital anesthesia, 3-mm segments of the artery were removed and mounted in a 5-ml myograph for isometric tension recording. Vessels precontracted by histamine were relaxed by acetylcholine. Mean maximum relaxation at 10(-4) M was reduced from 79% to 22% (P < 0.001) by 10(-5) M N-nitro-L-arginine (L-NA), and from 73% to 63% (NS) by 3.12(-6) M indomethacin. Intact non-precontracted vessels were contracted by 5-HT (10(-9) M to 10(-5) M): 10(-5) M L-NA significantly increased the contractile force (approximately twofold), whereas 3.10(-6) M indomethacin significantly decreased it (to approximately 35%). In histamine-precontracted vessels, 5-HT induced at low concentrations (3.10(-9) M to 3.10(-8) M) a reduction in tone and induced an increase in tone at higher concentrations. At 10(-5) M, L-NA abolished the relaxant phase of the response, whereas 3.10(-6) M indomethacin potentiated it. In uridine triphosphate-precontracted segments, there was not a net reduction in tone under 5-HT at 3.10(-9) to 3.10(-8) M, but further contraction appeared at higher concentrations. The presence of 10(-5) M L-NA significantly increased the contraction to 5-HT, but 3.10(-6) M indomethacin did not significantly reduce it. Endothelial lesion reduced by about 50% the contractile response of L-NA-treated arteries to 5-HT; and conversely, endothelial lesion increased approximately twofold the contraction of indomethacin-treated arteries to 5-HT. We conclude that 5-HT causes the release from the endothelium of two vasoactive factors, one of which is probably the vasodilator nitric oxide, but the size of the relaxation may depend on the prevailing level of nitric oxide synthase activation. The second factor is a cyclooxygenase-dependent contractile agent. However, the contraction to 5-HT was not modified by the presence of the thromboxane synthase inhibitor CGS 13080 (10(-4) M), suggesting that thromboxane A2 is not the main contractile agent released.     

Different modulation by cyclooxygenase inhibitors of the response to angiotensin II in monkey arteries and veins

In coronary, renal and femoral arteries and mesenteric veins isolated from Japanese monkeys, tachyphylaxis to angiotensin (ANG) II (10(-7) M)-induced contraction rapidly developed. Contractions caused by ANG II in coronary arteries were attenuated by treatment with indomethacin and aspirin and also by endothelium denudation. Indomethacin inhibited the response of the arteries with and without endothelium to a similar extent. OKY 046, a thromboxane A2 synthesis inhibitor, failed to inhibit the response. In contrast, contractions of renal arteries were potentiated and prolonged by the cyclooxygenase inhibitors. ANG II-induced contractions of mesenteric veins were prolonged but those of femoral arteries were not altered by indomethacin. It is concluded that ANG II contracts monkey coronary arteries, possibly due to the release of vasoconstrictor prostanoids but not thromboxane A2 from endothelial and subendothelial tissues and also due to its direct action on smooth muscle, whereas contractions of renal arteries and mesenteric veins are blunted by vasodilator prostanoids, possibly PGI2. Cyclooxygenase products even if released do not appear to regulate femoral artery contractions produced by ANG II.     

Modulation of contractions to ergonovine and methylergonovine by nitric oxide and thromboxane A2 in the human coronary artery

This study explored the modulatory effects of nitric oxide and thromboxane A2 on contractions to ergonovine and methylergonovine in human coronary arteries. To elucidate the different role of nitric oxide synthase in the response to the ergot alkaloids, the serotonin (5-HT) receptors involved in nitric oxide synthase in the response to the ergot alkaloids, the 5-HT receptors involved in nitric oxide release and the contraction of the vascular smooth muscle were characterized with more selective 5-HT-receptor agonists and antagonists. Rings of human coronary arteries from explanted hearts were suspended in organ chambers for isometric tension recording. After testing for contractile (potassium chloride, 60 mM) and endothelial function (substance P, 10(-8) M), respectively, they were exposed to ergot alkaloids or other agonists in the absence or presence of U 46619 (10(-9) M), or nitro-L-arginine (10(-4) M), or both. Ergonovine and methylergonovine were comparable, weak vasoconstrictors in untreated preparations. Contractions to ergonovine were augmented by U 46619, but not by nitro-L-arginine. Contractions to methylergonovine were augmented only by combining U 46619 and nitro-L-arginine. Serotonin and methylergonovine, but not ergonovine, elicited endothelium-dependent, nitric oxide-mediated relaxations. Nonselective 5-HT(1B/1D)-receptor stimulation caused both contractions and relaxations; selective 5-HT1B stimulation caused relaxations only. In the human coronary artery, contractions to ergonovine are not dependent on NO release but are synergistically augmented by thromboxane. Methylergonovine causes similar effects on the vascular smooth muscle, but contractions are inhibited by the release of NO from the endothelium. The 5-HT receptor on the endothelium appears to be different from the receptor on the vascular smooth muscle, which mediates the contractile response to the ergot alkaloids.     

Endothelium-derived relaxing factor released by 5-HT: distinct from nitric oxide in basilar arteries of normotensive and hypertensive rats.

1. The role of the endothelium in cerebrovascular responses to 5-hydroxytryptamine (5-HT) was investigated in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) in vitro. 2. Cumulative addition of 5-HT caused concentration-dependent contractions in ring preparations of SHR basilar arteries; the contractile response was smaller in WKY basilar arteries. 3. Removal of the endothelium enhanced markedly the contractile responses to 5-HT in WKY arteries but had only a slight effect in SHR arteries. The responsiveness to 5-HT in WKY arteries after removal of endothelium was comparable to that in SHR arteries. 4. The endothelium-dependent relaxation induced by acetylcholine in WKY basilar arteries was almost abolished by treatment with 10 microM methylene blue or 10 microM NG-nitro-L-arginine (L-NOARG). However, the response to 5-HT was not affected by treatment with methylene blue, L-NOARG or indomethacin. 5. Application of 10-20 mM K+ or 3.2 mM tetraethylammonium (TEA) did not change significantly, or only increased slightly, the resting tension, but markedly enhanced the contractile response to 5-HT in WKY arteries with endothelium. In contrast, the submaximal response to 5-HT in SHR arteries with endothelium was significantly enhanced by 0.3 mM TEA. 6. In the presence of 1 mM TEA, the application of 10 microM L-NOARG further enhanced the responses of 5-HT in WKY arteries with endothelium. In SHR arteries with endothelium, 10 microM L-NOARG per se enhanced slightly but significantly the responses to 5-HT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biological evaluation of the nitric oxide-trapping agent, N-methyl-D-glucamine dithiocarbamate-Fe2+, as a probe of nitric oxide activity released from control and diabetic rat endothelium.

We utilized the nitric oxide (NO) scavenger N-methyl-D-glucamine dithiocarbamate-Fe2+ (MGD-Fe) to characterize the role of NO in basal and acetylcholine (ACh)-stimulated relaxation arising from the endothelium of control vs diabetic rat aortic rings. In phenylephrine-contracted rings, MGD-Fe produced an additional increment in tension that was indomethacin-insensitive (i.e., excluding a role of prostanoids in this action). This MGD-Fe-sensitive component was more pronounced in control rings than diabetic rings and of the same magnitude achieved in rings without MGD-Fe treatment after removal of endothelium or treatment with the NO synthase inhibitor L-nitroarginine (L-NA). This suggests complete scavenging of basal NO by MGD-Fe and supports reduced basal NO in diabetic rings. ACh fully relaxed both control and diabetic rings. This relaxation was abolished by removal of the endothelium and was inhibited by L-NA (by 100% and 90% in control and diabetic rings, respectively). In contrast, MGD-Fe only partially inhibited ACh-induced relaxation in control (65+/-5% inhibition) and diabetic (41+/-11% inhibition) rings. The MGD-Fe-resistant component was not further modified by indomethacin. Addition of L-arginine (L-ARG) (but not D-arginine (D-ARG) enhanced the ACh-induced relaxation of MGD-Fe-treated diabetic (but not control) rings. These data provide evidence about endothelium-dependent relaxation in control and diabetic rings which cannot be discerned by use of L-NA alone. This study suggests that ACh produces a NO synthase-dependent vasodilation, a portion of which is due to free NO radical (*NO) or due to NO in a form or location that is unavailable for scavenging by MGD-Fe.     

Oxygen-derived free radicals mediate endothelium-dependent contractions in femoral arteries of rats with streptozotocin-induced diabetes

BACKGROUND AND PURPOSE:The present experiments were designed to study the contribution of oxygen-derived free radicals to endothelium-dependent contractions in femoral arteries of rats with streptozotocin-induced diabetes. EXPERIMENTAL APPROACH: Rings with and without endothelium were suspended in organ chambers for isometric tension recording. The production of oxygen-derived free radicals in the endothelium was measured with 2',7'-dichlorodihydrofluorescein diacetate using confocal microscopy. The presence of protein was measured by western blotting. KEY RESULTS: In the presence of L-NAME, the calcium ionophore A23187 induced larger endothelium-dependent contractions in femoral arteries from diabetic rats. Tiron, catalase, deferoxamine and MnTMPyP, but not superoxide dismutase reduced the response, suggesting that oxygen-derived free radicals are involved in the endothelium-dependent contraction. In the presence of L-NAME, A23187 increased the fluorescence signal in femoral arteries from streptozotocin-treated, but not in those from control rats, confirming that the production of oxygen-derived free radicals contributes to the enhanced endothelium-dependent contractions in diabetes. Exogenous H2O2 caused contractions in femoral arterial rings without endothelium which were reduced by deferoxamine, indicating that hydroxyl radicals contract vascular smooth muscle and thus could be an endothelium-derived contracting factor in diabetes. The reduced presence of Mn-SOD and the decreased activity of catalase in femoral arteries from streptozotocin-treated rats demonstrated the presence of a redox abnormality in arteries from rats with diabetes. CONCLUSIONS AND IMPLICATIONS: These findings suggest that the redox abnormality resulting from diabetes increases oxidative stress which facilitates and/or causes endothelium-dependent contractions.     

Altered intra- and extraluminal effects of 5-hydroxytryptamine in hypertensive mesenteric resistance arteries: contribution of the endothelium and smooth muscle.

Vasoconstrictor responses to intraluminal and extraluminal 5-hydroxytryptamine (5-HT) were studied in isolated mesenteric resistance arteries of Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). Third-order branches of mesenteric arteries were dissected free and mounted on glass cannulae in organ chambers. Changes in intraluminal diameter of the perfused and pressurized vessels were measured. Extraluminal 5-HT (10(-8)-10(-4)M) evoked concentration-dependent contractions that were augmented after removal of the endothelium. The sensitivity of arteries without but not of those with endothelium to 5-HT was increased in SHRs compared to WKY rats. The pA2 value for ketanserin using 5-HT as an agonist was identical in WKY rats and SHRs. The slope of the Schild plots did not significantly differ from 1. Intraluminal 5-HT caused smaller contractions in arteries with endothelium than extraluminal 5-HT. After endothelial removal, the contractions to intraluminal 5-HT were increased. The contraction induced by intraluminal 5-HT in arteries with endothelium was greater in SHRs than WKY rats. Thus, contractile responses to 5-HT are mediated by homogeneous 5-HT2 receptors in rat mesenteric resistance arteries. In SHRs, the affinity of the receptor to 5-HT is not altered. The sensitivity of vascular smooth muscle to 5-HT is increased and the protective role of the endothelium against intraluminal 5-HT is decreased in SHRs.     

Influence of the endothelium on contractile effects of 5-hydroxytryptamine and selective 5-HT agonists in canine basilar artery

1. We have investigated the influence of endothelial damage on the cerebrovascular reactivity to 5-hydroxytryptamine (5-HT) and some selective 5-HT agonists in canine basilar artery. 2. 5-HT, alpha-methyl 5-HT, GR 43175 (3-[2-dimethyl amino] ethyl-N-methyl-1H-indole-5-methane sulphonamide) and 5-carboxamidotryptamine (5-CT) produced concentration-dependent contractions of untreated dog basilar artery with a functional endothelium. Following endothelial damage by perfusion with Triton X-100 (0.1%), which abolished the relaxant response to substance P, the maximum contractile effect of 5-HT, alpha-methyl 5-HT, GR 43175 and 5-CT was markedly enhanced although there was little change in the EC50 values. Endothelial damage did not modify the vasoconstrictor effect of the thromboxane agonist, U46619, or potassium chloride. 3. Neither 5-HT nor 5-CT caused relaxation of untreated canine basilar arteries contracted with prostaglandin F2 alpha, U46619, uridine triphosphate or potassium chloride. 4. These results suggest that canine basilar artery spontaneously releases endothelium-derived relaxing factor which can attenuate the vasoconstrictor effect of 5-HT and selective 5-HT agonists. This effect appeared to be specific since the vasoconstrictor response to U46619 was not modified. 5. These results demonstrate that the cerebrovascular endothelium can markedly influence the reactivity of the vascular smooth muscle of canine basilar artery to 5-HT and 5-HT1-like receptor agonists. However we could find no evidence that 5-HT receptor activation stimulates endothelial cell function as it does in some other blood vessels.     

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.     

Vascular reactivity to vasopressin during diabetes: gender and regional differences

The isometric response to vasopressin of 2-mm-long segments of basilar, coronary, renal and tail arteries from male and female, control (normoglycemic) and streptozotocin-induced diabetic rats was studied. Vasopressin (10(-12)-3 x 10(-8) M) produced arterial concentration-dependent contraction, with a lower potency in coronary arteries from female than from male rats, and was similar for both genders in basilar, renal and tail arteries. This contraction was reduced by diabetes in basilar and coronary arteries, increased in renal arteries, and not modified in tail arteries, in both genders. Inhibition of nitric oxide synthesis with N(W)-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) increased the contraction to vasopressin in coronary arteries from control female and diabetic female rats; as well as in renal arteries from control male and control female rats, but not in any other experimental group. Inhibition of cyclooxygenase with meclofenamate (10(-5) M) reduced the contraction to vasopressin in basilar arteries from diabetic female rats and in renal arteries from diabetic male rats, but not in any other experimental group. These results suggest that the response to vasopressin (a) has lower potency in female coronary arteries due to higher nitric oxide production; (b) is reduced by diabetes in basilar and coronary arteries from both genders, by mechanisms independent of nitric oxide and prostanoids; and (c) is increased by diabetes in renal arteries due to reduced production of nitric oxide in females, and to both reduced production of nitric oxide and increased production of prostanoids in males. Therefore, the effects of diabetes on vascular reactivity to vasopressin may differ between vascular beds, and the mechanisms underlay these effects may be distinct between genders.