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Effects of prostanoids on phenylephrine-induced contractions in the mesenteric vascular bed of rats with streptozotocin-induced diabetes mellitus
Life Sciences 76 (2004) 239 – 247 www.elsevier.com/locate/lifescie
Current topics
Effects of prostanoids on phenylephrine-induced contractions in the mesenteric vascular bed of rats with streptozotocin-induced diabetes mellitus Andreia Fernanda Carvalho Leone, Eduardo Barbosa Coelho* Department of Internal Medicine, Nephrology Division, Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, Brazil Received 9 December 2003; accepted 17 June 2004
Abstract The main aim of this study was to compare the vascular reactivity of the perfused (Krebs, 4 ml/min) mesenteric vascular bed (MVB) isolated from rats with streptozotocin (STZ)-induced diabetes of 8 weeks duration to that of the MVB from non-diabetic (ND) Wistar rats. There were no differences in basal perfusion pressure between the MVB isolated from STZ and ND rats. The addition of indomethacin to the perfusate increased the basal perfusion pressure in both ND (18.8 F 0.7 vs 29.4 F 3.7 mmHg, p b 0.05) and STZ rats (18.3 F 0.9 vs 27.2 F 2.6 mmHg, p b 0.05), suggesting the release of a vasodilator prostaglandin. Remotion of the endothelium did not affect this response, indicating that prostaglandin was released from vascular smooth muscle. The response to phenylephrine was reduced in STZ rats compared to ND rats (2.3 [1.6–3.8] vs 8.3 [3.5–19.4], ED50. [IC 95%]) and was not modified by removal of the endothelium or by perfusion of L-nitro-arginine (50 AM). In contrast, indomethacin was able to reduce the response to phenylephrine in ND but not in STZ rats (2.3 [1.6–3.8] vs 4.7 [3.2–6.0], ED50. [IC 95%], p=0.02), suggesting that the blunted response to phenylephrine observed in STZ was due to the abolition of the release of prostaglandin by vascular smooth muscle. In conclusion, experimental diabetes induction in the rat is followed by a reduction of the contractile effect of phenylephrine due to the lack of release of a vasoconstrictor prostaglandin from vascular smooth muscle. D 2004 Elsevier Inc. All rights reserved. Keywords: Diabetes mellitus; Vascular reactivity; Indomethacin; Prostaglandins; Adrenergic agents; Mesenteric artery
* Corresponding author. Departamento de Clı´nica Me´dica, FMRP-USP., Av Bandeirantes 3900, CEP 14049-900, Brazil. Tel.: +55 16 6022543; fax: +55 16 6336695. E-mail address: [email protected] (E.B. Coelho). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.06.018
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Introduction Diabetes mellitus type I (DM) causes profound metabolic and cardiovascular modifications. Rats treated with streptozotocin (STZ), an agent that destroys pancreatic beta cells, develop hyperglycemia and tissue lesions similar to those observed in human DM I (Taylor et al., 1992). Among the changes observed, the predominant one is an increase in vascular reactivity to a1-adrenergic agents in different vascular beds (Abebe et al., 1990; Inazu et al., 1991; Taylor and Poston, 1994; White and Carrier, 1990), although a reduction (Oyama et al., 1986; Ramanadham et al., 1984) and no change (Chang and Stevens, 1992; Heygate et al., 1995) in this response have also been reported. These discrepancies in the results are attributed to the time of exposure to hyperglycemia and to the animal species and vascular bed studied. The response to adrenergic agonists depends on the sum of the responses directly linked to the activation of the contractile machinery and of the release of vasoactive substances from the vascular endothelium and/or smooth muscle. In diabetic rats, an increase in the endothelium-dependent vasodilator response is observed in the early phase of the disease, followed by an important reduction during the later phases of the disease (Fitzgerad and Brands, 2000; Fortes et al., 1983; Pieper and Gross, 1988; Tesfamariam et al., 1989). This phenomenon is being attributed to a lower bioavailability of nitric oxide (NO) due to an increase in the vascular production of superoxide anions (Consentino et al., 1997). Prostanoids are substances produced and released by endothelium and vascular smooth muscle that make an important contribution to the maintenance of vascular homeostasis. Changes in arachidonic acid metabolism have been reported to occur in diabetic animals (Peredo et al., 1999). However it is not known if these changes occur in the endothelium or in the vascular smooth muscle in this model. The objective of the present study was to investigate if prostanoids participates in the vascular reactivity changes to the adrenergic agonist a1 phenylephrine in perfused mesenteric vascular bed (with or without endothelium) isolated from rats with STZ-induced DM.
Materials and methods Male Wistar rats weighing 180 F 10 g were used. DM was induced by the administration of streptozotocin (STZ-Calbiochem, Darmstadt, Germany) in 0.1 M citrate buffer, pH 4.5, at the dose of 45 mg/kg. The animals were evaluated for glycemia and body weight two weeks later. The non-diabetic only received an equivalent volume of the buffer solution. Only animals with glycemia above 200 mg/dL were included in the DM group. NPH insulin (1 IU) sc was administered to all DM animals to prevent glycemia from exceeding 400 mg/dL. The rat mesenteric vascular bed isolated and perfused in vitro was used as a model of resistance vascular territory according to the method of Macgregor, 1965, with minor modifications. Rats were anesthetized with 2.5% tribromoethanol (1 ml/100 g body weight, ip), the abdominal cavity was opened and the intestinal loops were exposed. The superior mesenteric artery was dissected close to its origin in the abdominal aorta and cannulated with a PE-50 polyethylene catheter. The vascular bed was perfused with 1 ml of Krebs solution (120.0 mM NaCl, 4.7 mM KCl, 25.0 mM NaHCO3, 2.4 mM CaCl2.2H2O, 1.4 mM MgCl2.6H2O, 1.17 mM KH2PO4, and 11.0 mM glucose) containing 500 IU of heparin. The intestinal loops were removed en bloc, the mesenteric bed was separated by cutting close to the intestinal loops, and the preparation was placed in a cuvette heated to 37 8C. The cannulated superior mesenteric artery was coupled to a perfusion pump (LKB 2215 Multiperpex pump, Broma, Sweden) and the mesenteric bed was perfused with Krebs solution bubbled
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with 95% O2 and 5% CO2, pH 7.4, at a constant flow of 4 ml /min. A pressure transducer (R 511A, Beckman Inst., Schiller Park, IL, USA) was coupled in a byQ arrangement to the system for perfusion pressure recording. The pre-amplified and filtered outlet signal was coupled to the data acquisition system DATAQ DI-150 (Akron, OH, USA) connected to the RS 232 parallel port of a Pentium II personal computer (Intel, USA), and stored for later analysis with the Windaq software, version 2.5 (DATAQ). Vascular reactivity to vasoconstrictive agents The dose-response curve for phenylephrine was first constructed for non-diabetic and diabetic animals. The mesenteric bed was perfused with Krebs solution and left to rest for 15 minutes for the stabilization of the basal perfusion pressure, whose mean values were continuously recorded. Increasing doses of phenylephrine (0.5 to 80 Ag) were then injected in bolus with a 50 Al Hamilton syringe. The interval between injections was 5 minutes or the time needed for perfusion pressure to return to initial values. After the control curve, indomethacin (10 AM) or L-nitroarginine (L-NNA) (50 AM) was added to the Krebs solution and the dose-response curve was repeated. A maximum of three dose-effect curves were constructed per preparation. In some preparations the endothelium was removed with a solution of sodium deoxycholate (1 mg/ml–2 ml, in bolus). After 10 min for equilibration, a dose-response curve was constructed using the parameters previously described. At the end of the experiment, the mesenteric vascular bed (MVB) was pre-contracted with a dose of phenylephrine capable of increasing the basal perfusion pressure by 60 mmHg. An injection of acetylcholine in bolus (10 nM) was applied and the absence of a dilating response indicated the efficiency of removal of the vascular endothelium. Drugs The drugs used in the present study were acetylcholine, purchased from Merck (San Diego, CA, USA), streptozotocin purchased from Calbiochem (Darmstadt, Germany), and phenylephrine, NG-nitroL-arginine, and indomethacin, all from Sigma (St. Louis, MO, USA). Statistical analysis The results are reported as mean F SEM. Data considered to be parametric were compared by the Student t-test for unpaired samples. The ED50 (effective dose needed to promote 50% of the maximum effect of the agonist) was calculated for each dose-effect curve based on the maximum effect (Emax) values obtained experimentally using the Instat program version 4.0 (Graph Pad Software, USA). The Emax and ED50 of the various pharmacological blockades were compared by the unpaired nonparametric Mann-Whitney test. For comparison of the perfusion pressure we used ANOVA followed by the nonparametric Kruskal-Wallis test and the Dunn post-test. Differences were considered to be significant when p b 0.05.
Results Fig. 1A illustrates the basal perfusion pressure of the isolated and perfused MVB from nondiabetic (ND) and diabetic (STZ) rats after treatment with indomethacin. No difference in basal perfusion
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Fig. 1. Basal perfusion pressure of the isolated and perfused AMB from non-diabetic (ND) and diabetic (STZ) rats after treatment with indomethacin (filled bars) and without (open bars) indomethacin (10 AM). Panel A represents experiments with endothelium and the panel B without endothelium. The bars represents the values of the means F S.E.M (* p b 0.05 compared with indomethacin vs. without indomethacin). Kruskal-Wallis and Dunn’s test post. (N = 7 for group).
pressure of the MVB with or without endothelium was observed between ND and STZ rats (with: 18.8 F 0.7, n=7 vs 18.3 F 0.9, n=7, panel A; without: 22.5 F 0.8 ND vs 19.0 F 0.7 STZ, panel B, respectively). Perfusion with indomethacin (10 AM) increased the basal perfusion pressure in both groups even in the absence of vascular endothelium (ND with endothelium (panel A) 18.8 F 0.7, vs 29.4 F 3.7, p b 0.05, and ND without endothelium (panel B) 22.5 F 0.8 vs 26.4 F 0.8, p b 0.05; STZ with endothelium (panel A) 18.3 F 0.9, vs 27.2 F 2.6, p b 0.05; STZ without endothelium (panel B) 19.0 F 0.7 vs 25.1 F 1.1, p b 0.05; means F SEM represents respectively control and indomethacin groups). Treatment with L-NNA did not change the basal perfusion pressure of the MVB of ND rats compared to STZ rats (16.0 F 0.4, n=7 vs 20.3 F 1.0. n=5; data not shown). Sensitivity to phenylephrine, determined on the basis of the ED50, was lower in STZ animals (2.3 mg [1.6–3.8] vs 8.3 mg [3.5–19,4], p b 0.01, median, [95%CI]) compared to ND animals (Fig. 2A). Perfusion with the nitric oxide synthase (NOS) inhibitor L-NNA increased the maximum vasoconstrictive effect of phenylephrine in ND animals (136 [110.8–158.1] vs 180.6 [150.3–211.8], p=0.03) and tended to increase the maximum effect, although not significantly, in STZ rats (104.5 [75.9–130.7] vs 119.8 [100.4–158.5]). L-NNA reduced the ED50 in both groups (1.4 [0.8–3.1] and 4.3 [0.05–10.6] for ND and STZ, respectively). However, perfusion with L-NNA did not eliminate the difference in reactivity to phenylephrine between ND and STZ (Fig. 2C), whereas the difference was abolished by perfusion with indomethacin (Fig. 2B) (ED50 4.7 [3.2–6.0] vs 6.0 [3,7–10.5], median, [95%CI], ND vs STZ). Fig. 3 illustrates the effect of endothelium removal on the vasoconstrictive effect of phenylephrine on the isolated and perfused MVB of ND and STZ animals. Endothelium removal potentiated the response to phenylephrine in both groups (2.3 [1.6–3.8] vs 0.9 [0.3–1.6], n=7, p=0.007, ND, and 8.3 [3.5–19.4] vs 3.4 [1.4–5.4], n=6, STZ, p=0.035). However, even in the absence of endothelium, phenylephrine had a lower vasoconstrictive effect on STZ animals (Fig. 3A). In contrast, perfusion with indomethacin of MVB in which the endothelium was removed abolished the difference in
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Fig. 2. Panel A-Dose effect curves for phenylephrine realized in AMB isolated (n control, 5 diabetic, n=7 for each) Panel BEffect of the perfusion of the indomethacin (10 AM). ( control, n=7 and o diabetic, n=6). Panel C-Effect of the perfusion of the L-NNA (50 AM) (E control, n=7 and D diabetic, n=6). The points represents the mean F S.E.M (*p = 0.01 vs. control (DE50); *p = 0.022 vs control (EMAX), test of Mann Whitney).
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reactivity to phenylephrine between groups (Fig. 3B) (3.7 [1.2–7.2] vs 2.0 [1.1–3.5], ED50 [95%CI], ND, n=6 vs STZ, n=6).
Discussion The results reported here show that the presence of DM does not modify the basal perfusion pressure. In addition, perfusion with L-NNA also did not modify this parameter, suggesting that no flow-mediated NO release occurred under our experimental conditions. On the other hand, in the presence of
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Fig. 3. Panel A and Panel B-Dose-effects curves for phenylephrine realized in AMB isolated without endothelium (z control, n=7; j diabetic, n=6). Panel B-Effect of the perfusion of indomethacin (10 AM) (x control; R diabetic, n=6 for group). The points represents the mean F S.E.M (*p = 0.004 vs. control (DE50), test of Mann Whitney).
indomethacin there was an increase in basal perfusion pressure of the MVB with and without endothelium in both experimental groups. These data suggest that under basal conditions there may be a continuous release from smooth muscle of a prostaglandin with a vasodilating effect. Papadaki et al., 1996 demonstrated the participation of vascular smooth muscle in the release of prostaglandins induced by shear stress. It should be pointed out that most investigators report the endothelial release of prostanoids mediated by shear stress, with emphasis on prostacyclin (Ando et al., 1988; Koller et al., 1994; Hanada et al., 2000). Only Alshihabi et al., 1996 reported the basal release of prostacyclin by vascular smooth muscle. The present protocol was not designed to answer the question about the nature of the released prostaglandin or about the exact mechanism of transduction between a mechanical stimulus and the formation of prostanoids. Thus, further experiments are needed to clarify this question. In the present study there was a reduction in the sensitivity to phenylephrine in the diabetic animals (eight weeks). Two hypotheses may be raised to explain this finding: an increase in the release or in the effect of a vasodilating substance, or a reduction of a vasoconstrictive component that may be formed together with the response induced by phenylephrine when it binds to the a adrenergic receptor. Perfusion with L-NNA, a non-selective NOS inhibitor, abolished the differences in the potency of phenylephrine, as observed by the ED50, between diabetic rats and controls. However, its maximum effect was increased only in non-diabetic animals. Thus, even in the presence of L-NNA, phenylephrine had a lower vasoconstrictive effect on diabetic animals compared to non-diabetic ones. This fact suggests that a greater NO production in diabetic rats may not be the mechanism responsible for the reduction in the response to phenylephrine. Indeed, the literature indicates an early endothelial dysfunction in experimental DM characterized by a reduction in the vasodilating response to vascular endothelium-dependent agents (Consentino and Lu¨scher, 1998; Hattori et al., 1991; Pieper, 1998; Tesfamariam, 1994). There was an increase in the vasoconstrictor response to phenilephrine after perfusion with L-NNA in both groups (ND and STZ rats). This result indicates that vascular endothelium NO is released when adrenergic a1-receptors are stimulated, as suggested by Ballejo et al., 2002. The reduction in the response to phenylephrine in diabetic animals was observed even in the absence of vascular endothelium (Fig. 3A). Thus, the increased formation or release of an endothelium-derived vasodilating agent, whether NO, endothelium-derived hyperpolarizing factor (EDHF) or prostacyclin, seems to be unlikely as a mechanism explaining the lower reactivity to phenylephrine in diabetic
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animals. In addition, this experiment rules out the hypothesis of a greater endothelial release of vasodilating agents as the cause of the reduced response to phenylephrine in diabetic animals. The inhibition of cyclooxygenase promoted a reduction of the response to phenylephrine, even in endothelium-free preparations. This fact suggests that phenylephrine may stimulate the production of vasoconstrictive prostanoids by smooth muscle in the MVB. Similar results were obtained by Manku and Horrobin, 1976, Peredo and Adler-Graschinsky, 2000, and Peredo, 2001. These investigators demonstrated that indomethacin reduced the vasoconstrictive effect of noradrenaline in the mesenteric arterial bed of rats and suggested that the constrictive effect of noradrenaline involves the participation of two different pathways of arachidonic acid metabolism, i.e., the cyclooxygenase pathway and the lipoxygenase pathway. In addition, the result reported by Peredo and Adler-Graschinsky, 2000 indicates the participation of the endothelium in the production of lipoxygenase-derived metabolites, but not of the prostanoids formed by cyclooxygenase. Indomethacin was unable to change the reactivity to phenylephrine in diabetic rats. In addition, in the presence of indomethacin the difference in reactivity between diabetic animals and controls was abolished. These data show that in diabetic animals phenylephrine is unable to release vasoconstrictive prostanoids from MVB smooth muscle and that this is the major mechanism explaining the difference in reactivity to this agent observed in diabetic rats. Using noradrenaline with an agonist, Peredo et al., 1999, Peredo, 2001 obtained similar data for rats with diabetes mellitus in two distinct models (STZ diabetic rats and rats with diabetes similar to the human type II). However, 8 weeks after STZ induction of diabetes, the vasoconstrictive effect of noradrenaline was again reduced by indomethacin perfusion. These authors quantified the production of prostaglandin-F2a and concluded that STZ-induced diabetes is associated with a lower production of this agent which is time dependent. Curiously, in rats with a DM type II model the prostanoid release by noradrenaline is abolished during later phases. In contrast to the cited study, our animals were treated with insulin to prevent hyperglycemia of more than 400 mg/dl, a fact that might have delayed the development of the vascular changes induced by DM in later phases. In conclusion, the interpretation of the present findings suggests that diabetes may selectively modify the release of prostaglandins from vascular smooth muscle, with preservation of basal vasodilating prostanoid production and abolition of the production of vasoconstrictive prostanoids stimulated by adrenergic agonists. The possibility is proposed that reduction of vasoconstrictive prostaglandin production by vascular smooth muscle may be a counter-regulating mechanism in response to the lower release of vasodilating substances such as nitric oxide observed in the vessels of diabetic rats. Acknowledgments Research supported by Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) and by Fundac¸a˜o de Apoio ao Ensino, Pesquisa e Assisteˆncia do Hospital das Clı´nicas da Faculdade de Medicina de Ribeira˜o Preto-USP (FAEPA). References Abebe, W., Harris, K.H., Macleod, K.M., 1990. Enhanced contractile responses of arteries from diabetic rats to a1-adrenoreceptor stimulation in the absence and presence of extracellular calcium. Journal of Cardiovascular Pharmacology 16, 239 – 248.
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Alshihabi, S., Chang, Y., Frangos, J., Tarbell, J., 1996. Shear stress-induced release of PGE2 and PGI2 by vascular smooth muscle cells. Biochemical Biophysical Research Communications 224, 808 – 814. Ando, J., Komatsuda, T., Kamiya, A., 1988. Cytoplasmic calcium response to fluid shear stress in cultured vascular endothelial cells. In Vitro Cellular and Developmental Biology 24 (9), 871 – 877. Ballejo, G., Barbosa, T.A., Coelho, E.B., Antoniali, C., Salgado, M.C., 2002. Pregnancy-associated increase in rat systemic arteries endothelial nitric oxide production diminishes vasoconstrictor but does not enhance vasodilator responses. Life Sciences 70 (26), 3131 – 3142. Chang, K.S.K., Stevens, W.C., 1992. Endothelium-dependent increase in vascular sensitivity to phenylephrine in long-term streptozotocin diabetic rat aorta. British Journal of Pharmacology 107, 983 – 990. Consentino, F., Hishikawa, K., Katusic, Z.S., Lqscher, T.F., 1997. High glucose increases nitric oxide synthase expression and superoxide anion generation in human aortic endothelial cells. Circulation 96, 25 – 28. Consentino, F., Lqscher, T.F., 1998. Endothelial dysfunction in diabetes mellitus. Journal of Cardiovascular Pharmacology 32 (suppl. 3), S54 – S61. Fitzgerad, S.M., Brands, M.W., 2000. Nitric oxide may be required to prevent hypertension at the onset of diabetes. American Journal of Physiology 279, E62 – E768. Fortes, Z.B., Garcia, J.L., Scivoletto, R., 1983. Vascular reactivity in diabetes mellitus: role of the endothelial cells. British Journal of Pharmacology 79, 771 – 781. Hanada, T., Hashimoto, M., Nosaka, S., Sasaki, T., Nakayama, K., Masumura, S., Yamauchi, M., Tamura, K., 2000. Shear stress enhances prostacyclin release from endocardial endothelial cells. Life Sciences 66 (3), 215 – 220. Hattori, Y., Kawasaki, H., Abe, K., Kanno, M., 1991. Superoxide dismutase recovers altered endothelium-dependent relaxation in diabetic rat aorta. American Journal of Physiology 261 (4 Pt 2), H1086 – 1094. Heygate, K.M., Lawrence, I.G., Bennett, M.A., Thurston, H., 1995. Impaired endothelium-dependent relaxation in isolated resistance arteries of spontaneously diabetic rats. British Journal of Pharmacology 116, 3251 – 3259. Inazu, M., Sakai, Y., Homma, I., 1991. Contractile responses and calcium mobilization in renal arteries of diabetic rats. European Journal of Pharmacology 203, 79 – 84. Koller, A., Sun, D., Huang, A., Kaley, G., 1994. Corelease of nitric oxide and prostaglandins mediates flow-dependent dilation of rat gracilis muscle arterioles. American Journal of Physiology 267 (1 Pt 2), H326 – 332. Macgregor, D.D., 1965. The effect of sympathetic nerve stimulation on vasoconstrictor responses in perfused mesenteric blood vessels of the rat. Journal of Physiology 177, 21. Manku, M.S., Horrobin, D.F., 1976. Indomethacin inhibits responses to all vasoconstrictors in the rat mesenteric vascular bed: restoration of responses by prostaglandin E2. Prostaglandins 12, 369 – 376. Oyama, Y., Kawasaki, H., Hattori, Y., Kanno, M., 1986. Attenuation of endothelium-dependent relaxation in aorta from diabetic rats. European Journal of Pharmacology 131, 75 – 78. Papadaki, M., Mcintire, L., Eskin, S.G., 1996. Effects of shear stress on the growth kinetics of human aortic smooth muscle cells in vitro. Biotechnology Bioengineering 50, 555 – 561. Peredo, H.A., Adler-Graschinsky, E., 2000. Effects of eicosanoids and nitric oxide on the noradrenaline-induced contractions in the rat mesenteric bed. Journal of Autonomic Pharmacology 20 (3), 163 – 170. Peredo, H.A., Feleder, E.C., Adler-Graschinsky, E., 1999. Time-course of the alterations in prostanoid production and in contractile responses of mesenteric beds isolated from streptozotocin diabetic rats. Prostaglandins, Leukotrienes and Essential Fatty Acids 60, 269 – 274. Peredo, H.A., 2001. Prostanoid release and constrictor responses to noradrenaline in the rat mesenteric vascular bed in noninsulin-dependent diabetes mellitus. Journal of Autonomic Pharmacology 21 (3), 131 – 137. Pieper, G.M., Gross, G.J.P., 1988. Oxygen-derived free radicals abolish endothelium-dependent relaxation in diabetic rat aorta. American Journal of Physiology 255, H825 – H833. Pieper, G.M., 1998. Review of alterations in endothelial nitric oxide production in diabetes: protective role of arginine on endothelial dysfunction. Hypertension 31 (5), 1047 – 1060. Ramanadham, S., Lyness, W.H., Tenner, T.E., 1984. Alteration in aortic and tail artery reactivity to agonists after streptozotocin treatment. Canadian Journal of Physiology and Pharmacology 62, 418 – 423. Taylor, P.D., Mccarthy, A.L., Thomas, C.R., Poston, L., 1992. Endothelium-dependent relaxation and noradrenaline sensitivity in mesenteric resistance arteries of streptozotocin-induced diabetic rats. British Journal of Pharmacology 107, 393. Taylor, P.D., Poston, L., 1994. The effect of hyperglycaemia on function of rat isolated mesenteric resistance artery. British Journal of Pharmacology 113 (3), 801 – 808.
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Current topics
Effects of prostanoids on phenylephrine-induced contractions in the mesenteric vascular bed of rats with streptozotocin-induced diabetes mellitus Andreia Fernanda Carvalho Leone, Eduardo Barbosa Coelho* Department of Internal Medicine, Nephrology Division, Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, Brazil Received 9 December 2003; accepted 17 June 2004
Abstract The main aim of this study was to compare the vascular reactivity of the perfused (Krebs, 4 ml/min) mesenteric vascular bed (MVB) isolated from rats with streptozotocin (STZ)-induced diabetes of 8 weeks duration to that of the MVB from non-diabetic (ND) Wistar rats. There were no differences in basal perfusion pressure between the MVB isolated from STZ and ND rats. The addition of indomethacin to the perfusate increased the basal perfusion pressure in both ND (18.8 F 0.7 vs 29.4 F 3.7 mmHg, p b 0.05) and STZ rats (18.3 F 0.9 vs 27.2 F 2.6 mmHg, p b 0.05), suggesting the release of a vasodilator prostaglandin. Remotion of the endothelium did not affect this response, indicating that prostaglandin was released from vascular smooth muscle. The response to phenylephrine was reduced in STZ rats compared to ND rats (2.3 [1.6–3.8] vs 8.3 [3.5–19.4], ED50. [IC 95%]) and was not modified by removal of the endothelium or by perfusion of L-nitro-arginine (50 AM). In contrast, indomethacin was able to reduce the response to phenylephrine in ND but not in STZ rats (2.3 [1.6–3.8] vs 4.7 [3.2–6.0], ED50. [IC 95%], p=0.02), suggesting that the blunted response to phenylephrine observed in STZ was due to the abolition of the release of prostaglandin by vascular smooth muscle. In conclusion, experimental diabetes induction in the rat is followed by a reduction of the contractile effect of phenylephrine due to the lack of release of a vasoconstrictor prostaglandin from vascular smooth muscle. D 2004 Elsevier Inc. All rights reserved. Keywords: Diabetes mellitus; Vascular reactivity; Indomethacin; Prostaglandins; Adrenergic agents; Mesenteric artery
* Corresponding author. Departamento de Clı´nica Me´dica, FMRP-USP., Av Bandeirantes 3900, CEP 14049-900, Brazil. Tel.: +55 16 6022543; fax: +55 16 6336695. E-mail address: [email protected] (E.B. Coelho). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.06.018
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Introduction Diabetes mellitus type I (DM) causes profound metabolic and cardiovascular modifications. Rats treated with streptozotocin (STZ), an agent that destroys pancreatic beta cells, develop hyperglycemia and tissue lesions similar to those observed in human DM I (Taylor et al., 1992). Among the changes observed, the predominant one is an increase in vascular reactivity to a1-adrenergic agents in different vascular beds (Abebe et al., 1990; Inazu et al., 1991; Taylor and Poston, 1994; White and Carrier, 1990), although a reduction (Oyama et al., 1986; Ramanadham et al., 1984) and no change (Chang and Stevens, 1992; Heygate et al., 1995) in this response have also been reported. These discrepancies in the results are attributed to the time of exposure to hyperglycemia and to the animal species and vascular bed studied. The response to adrenergic agonists depends on the sum of the responses directly linked to the activation of the contractile machinery and of the release of vasoactive substances from the vascular endothelium and/or smooth muscle. In diabetic rats, an increase in the endothelium-dependent vasodilator response is observed in the early phase of the disease, followed by an important reduction during the later phases of the disease (Fitzgerad and Brands, 2000; Fortes et al., 1983; Pieper and Gross, 1988; Tesfamariam et al., 1989). This phenomenon is being attributed to a lower bioavailability of nitric oxide (NO) due to an increase in the vascular production of superoxide anions (Consentino et al., 1997). Prostanoids are substances produced and released by endothelium and vascular smooth muscle that make an important contribution to the maintenance of vascular homeostasis. Changes in arachidonic acid metabolism have been reported to occur in diabetic animals (Peredo et al., 1999). However it is not known if these changes occur in the endothelium or in the vascular smooth muscle in this model. The objective of the present study was to investigate if prostanoids participates in the vascular reactivity changes to the adrenergic agonist a1 phenylephrine in perfused mesenteric vascular bed (with or without endothelium) isolated from rats with STZ-induced DM.
Materials and methods Male Wistar rats weighing 180 F 10 g were used. DM was induced by the administration of streptozotocin (STZ-Calbiochem, Darmstadt, Germany) in 0.1 M citrate buffer, pH 4.5, at the dose of 45 mg/kg. The animals were evaluated for glycemia and body weight two weeks later. The non-diabetic only received an equivalent volume of the buffer solution. Only animals with glycemia above 200 mg/dL were included in the DM group. NPH insulin (1 IU) sc was administered to all DM animals to prevent glycemia from exceeding 400 mg/dL. The rat mesenteric vascular bed isolated and perfused in vitro was used as a model of resistance vascular territory according to the method of Macgregor, 1965, with minor modifications. Rats were anesthetized with 2.5% tribromoethanol (1 ml/100 g body weight, ip), the abdominal cavity was opened and the intestinal loops were exposed. The superior mesenteric artery was dissected close to its origin in the abdominal aorta and cannulated with a PE-50 polyethylene catheter. The vascular bed was perfused with 1 ml of Krebs solution (120.0 mM NaCl, 4.7 mM KCl, 25.0 mM NaHCO3, 2.4 mM CaCl2.2H2O, 1.4 mM MgCl2.6H2O, 1.17 mM KH2PO4, and 11.0 mM glucose) containing 500 IU of heparin. The intestinal loops were removed en bloc, the mesenteric bed was separated by cutting close to the intestinal loops, and the preparation was placed in a cuvette heated to 37 8C. The cannulated superior mesenteric artery was coupled to a perfusion pump (LKB 2215 Multiperpex pump, Broma, Sweden) and the mesenteric bed was perfused with Krebs solution bubbled
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with 95% O2 and 5% CO2, pH 7.4, at a constant flow of 4 ml /min. A pressure transducer (R 511A, Beckman Inst., Schiller Park, IL, USA) was coupled in a byQ arrangement to the system for perfusion pressure recording. The pre-amplified and filtered outlet signal was coupled to the data acquisition system DATAQ DI-150 (Akron, OH, USA) connected to the RS 232 parallel port of a Pentium II personal computer (Intel, USA), and stored for later analysis with the Windaq software, version 2.5 (DATAQ). Vascular reactivity to vasoconstrictive agents The dose-response curve for phenylephrine was first constructed for non-diabetic and diabetic animals. The mesenteric bed was perfused with Krebs solution and left to rest for 15 minutes for the stabilization of the basal perfusion pressure, whose mean values were continuously recorded. Increasing doses of phenylephrine (0.5 to 80 Ag) were then injected in bolus with a 50 Al Hamilton syringe. The interval between injections was 5 minutes or the time needed for perfusion pressure to return to initial values. After the control curve, indomethacin (10 AM) or L-nitroarginine (L-NNA) (50 AM) was added to the Krebs solution and the dose-response curve was repeated. A maximum of three dose-effect curves were constructed per preparation. In some preparations the endothelium was removed with a solution of sodium deoxycholate (1 mg/ml–2 ml, in bolus). After 10 min for equilibration, a dose-response curve was constructed using the parameters previously described. At the end of the experiment, the mesenteric vascular bed (MVB) was pre-contracted with a dose of phenylephrine capable of increasing the basal perfusion pressure by 60 mmHg. An injection of acetylcholine in bolus (10 nM) was applied and the absence of a dilating response indicated the efficiency of removal of the vascular endothelium. Drugs The drugs used in the present study were acetylcholine, purchased from Merck (San Diego, CA, USA), streptozotocin purchased from Calbiochem (Darmstadt, Germany), and phenylephrine, NG-nitroL-arginine, and indomethacin, all from Sigma (St. Louis, MO, USA). Statistical analysis The results are reported as mean F SEM. Data considered to be parametric were compared by the Student t-test for unpaired samples. The ED50 (effective dose needed to promote 50% of the maximum effect of the agonist) was calculated for each dose-effect curve based on the maximum effect (Emax) values obtained experimentally using the Instat program version 4.0 (Graph Pad Software, USA). The Emax and ED50 of the various pharmacological blockades were compared by the unpaired nonparametric Mann-Whitney test. For comparison of the perfusion pressure we used ANOVA followed by the nonparametric Kruskal-Wallis test and the Dunn post-test. Differences were considered to be significant when p b 0.05.
Results Fig. 1A illustrates the basal perfusion pressure of the isolated and perfused MVB from nondiabetic (ND) and diabetic (STZ) rats after treatment with indomethacin. No difference in basal perfusion
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Fig. 1. Basal perfusion pressure of the isolated and perfused AMB from non-diabetic (ND) and diabetic (STZ) rats after treatment with indomethacin (filled bars) and without (open bars) indomethacin (10 AM). Panel A represents experiments with endothelium and the panel B without endothelium. The bars represents the values of the means F S.E.M (* p b 0.05 compared with indomethacin vs. without indomethacin). Kruskal-Wallis and Dunn’s test post. (N = 7 for group).
pressure of the MVB with or without endothelium was observed between ND and STZ rats (with: 18.8 F 0.7, n=7 vs 18.3 F 0.9, n=7, panel A; without: 22.5 F 0.8 ND vs 19.0 F 0.7 STZ, panel B, respectively). Perfusion with indomethacin (10 AM) increased the basal perfusion pressure in both groups even in the absence of vascular endothelium (ND with endothelium (panel A) 18.8 F 0.7, vs 29.4 F 3.7, p b 0.05, and ND without endothelium (panel B) 22.5 F 0.8 vs 26.4 F 0.8, p b 0.05; STZ with endothelium (panel A) 18.3 F 0.9, vs 27.2 F 2.6, p b 0.05; STZ without endothelium (panel B) 19.0 F 0.7 vs 25.1 F 1.1, p b 0.05; means F SEM represents respectively control and indomethacin groups). Treatment with L-NNA did not change the basal perfusion pressure of the MVB of ND rats compared to STZ rats (16.0 F 0.4, n=7 vs 20.3 F 1.0. n=5; data not shown). Sensitivity to phenylephrine, determined on the basis of the ED50, was lower in STZ animals (2.3 mg [1.6–3.8] vs 8.3 mg [3.5–19,4], p b 0.01, median, [95%CI]) compared to ND animals (Fig. 2A). Perfusion with the nitric oxide synthase (NOS) inhibitor L-NNA increased the maximum vasoconstrictive effect of phenylephrine in ND animals (136 [110.8–158.1] vs 180.6 [150.3–211.8], p=0.03) and tended to increase the maximum effect, although not significantly, in STZ rats (104.5 [75.9–130.7] vs 119.8 [100.4–158.5]). L-NNA reduced the ED50 in both groups (1.4 [0.8–3.1] and 4.3 [0.05–10.6] for ND and STZ, respectively). However, perfusion with L-NNA did not eliminate the difference in reactivity to phenylephrine between ND and STZ (Fig. 2C), whereas the difference was abolished by perfusion with indomethacin (Fig. 2B) (ED50 4.7 [3.2–6.0] vs 6.0 [3,7–10.5], median, [95%CI], ND vs STZ). Fig. 3 illustrates the effect of endothelium removal on the vasoconstrictive effect of phenylephrine on the isolated and perfused MVB of ND and STZ animals. Endothelium removal potentiated the response to phenylephrine in both groups (2.3 [1.6–3.8] vs 0.9 [0.3–1.6], n=7, p=0.007, ND, and 8.3 [3.5–19.4] vs 3.4 [1.4–5.4], n=6, STZ, p=0.035). However, even in the absence of endothelium, phenylephrine had a lower vasoconstrictive effect on STZ animals (Fig. 3A). In contrast, perfusion with indomethacin of MVB in which the endothelium was removed abolished the difference in
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Fig. 2. Panel A-Dose effect curves for phenylephrine realized in AMB isolated (n control, 5 diabetic, n=7 for each) Panel BEffect of the perfusion of the indomethacin (10 AM). ( control, n=7 and o diabetic, n=6). Panel C-Effect of the perfusion of the L-NNA (50 AM) (E control, n=7 and D diabetic, n=6). The points represents the mean F S.E.M (*p = 0.01 vs. control (DE50); *p = 0.022 vs control (EMAX), test of Mann Whitney).
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reactivity to phenylephrine between groups (Fig. 3B) (3.7 [1.2–7.2] vs 2.0 [1.1–3.5], ED50 [95%CI], ND, n=6 vs STZ, n=6).
Discussion The results reported here show that the presence of DM does not modify the basal perfusion pressure. In addition, perfusion with L-NNA also did not modify this parameter, suggesting that no flow-mediated NO release occurred under our experimental conditions. On the other hand, in the presence of
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Fig. 3. Panel A and Panel B-Dose-effects curves for phenylephrine realized in AMB isolated without endothelium (z control, n=7; j diabetic, n=6). Panel B-Effect of the perfusion of indomethacin (10 AM) (x control; R diabetic, n=6 for group). The points represents the mean F S.E.M (*p = 0.004 vs. control (DE50), test of Mann Whitney).
indomethacin there was an increase in basal perfusion pressure of the MVB with and without endothelium in both experimental groups. These data suggest that under basal conditions there may be a continuous release from smooth muscle of a prostaglandin with a vasodilating effect. Papadaki et al., 1996 demonstrated the participation of vascular smooth muscle in the release of prostaglandins induced by shear stress. It should be pointed out that most investigators report the endothelial release of prostanoids mediated by shear stress, with emphasis on prostacyclin (Ando et al., 1988; Koller et al., 1994; Hanada et al., 2000). Only Alshihabi et al., 1996 reported the basal release of prostacyclin by vascular smooth muscle. The present protocol was not designed to answer the question about the nature of the released prostaglandin or about the exact mechanism of transduction between a mechanical stimulus and the formation of prostanoids. Thus, further experiments are needed to clarify this question. In the present study there was a reduction in the sensitivity to phenylephrine in the diabetic animals (eight weeks). Two hypotheses may be raised to explain this finding: an increase in the release or in the effect of a vasodilating substance, or a reduction of a vasoconstrictive component that may be formed together with the response induced by phenylephrine when it binds to the a adrenergic receptor. Perfusion with L-NNA, a non-selective NOS inhibitor, abolished the differences in the potency of phenylephrine, as observed by the ED50, between diabetic rats and controls. However, its maximum effect was increased only in non-diabetic animals. Thus, even in the presence of L-NNA, phenylephrine had a lower vasoconstrictive effect on diabetic animals compared to non-diabetic ones. This fact suggests that a greater NO production in diabetic rats may not be the mechanism responsible for the reduction in the response to phenylephrine. Indeed, the literature indicates an early endothelial dysfunction in experimental DM characterized by a reduction in the vasodilating response to vascular endothelium-dependent agents (Consentino and Lu¨scher, 1998; Hattori et al., 1991; Pieper, 1998; Tesfamariam, 1994). There was an increase in the vasoconstrictor response to phenilephrine after perfusion with L-NNA in both groups (ND and STZ rats). This result indicates that vascular endothelium NO is released when adrenergic a1-receptors are stimulated, as suggested by Ballejo et al., 2002. The reduction in the response to phenylephrine in diabetic animals was observed even in the absence of vascular endothelium (Fig. 3A). Thus, the increased formation or release of an endothelium-derived vasodilating agent, whether NO, endothelium-derived hyperpolarizing factor (EDHF) or prostacyclin, seems to be unlikely as a mechanism explaining the lower reactivity to phenylephrine in diabetic
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animals. In addition, this experiment rules out the hypothesis of a greater endothelial release of vasodilating agents as the cause of the reduced response to phenylephrine in diabetic animals. The inhibition of cyclooxygenase promoted a reduction of the response to phenylephrine, even in endothelium-free preparations. This fact suggests that phenylephrine may stimulate the production of vasoconstrictive prostanoids by smooth muscle in the MVB. Similar results were obtained by Manku and Horrobin, 1976, Peredo and Adler-Graschinsky, 2000, and Peredo, 2001. These investigators demonstrated that indomethacin reduced the vasoconstrictive effect of noradrenaline in the mesenteric arterial bed of rats and suggested that the constrictive effect of noradrenaline involves the participation of two different pathways of arachidonic acid metabolism, i.e., the cyclooxygenase pathway and the lipoxygenase pathway. In addition, the result reported by Peredo and Adler-Graschinsky, 2000 indicates the participation of the endothelium in the production of lipoxygenase-derived metabolites, but not of the prostanoids formed by cyclooxygenase. Indomethacin was unable to change the reactivity to phenylephrine in diabetic rats. In addition, in the presence of indomethacin the difference in reactivity between diabetic animals and controls was abolished. These data show that in diabetic animals phenylephrine is unable to release vasoconstrictive prostanoids from MVB smooth muscle and that this is the major mechanism explaining the difference in reactivity to this agent observed in diabetic rats. Using noradrenaline with an agonist, Peredo et al., 1999, Peredo, 2001 obtained similar data for rats with diabetes mellitus in two distinct models (STZ diabetic rats and rats with diabetes similar to the human type II). However, 8 weeks after STZ induction of diabetes, the vasoconstrictive effect of noradrenaline was again reduced by indomethacin perfusion. These authors quantified the production of prostaglandin-F2a and concluded that STZ-induced diabetes is associated with a lower production of this agent which is time dependent. Curiously, in rats with a DM type II model the prostanoid release by noradrenaline is abolished during later phases. In contrast to the cited study, our animals were treated with insulin to prevent hyperglycemia of more than 400 mg/dl, a fact that might have delayed the development of the vascular changes induced by DM in later phases. In conclusion, the interpretation of the present findings suggests that diabetes may selectively modify the release of prostaglandins from vascular smooth muscle, with preservation of basal vasodilating prostanoid production and abolition of the production of vasoconstrictive prostanoids stimulated by adrenergic agonists. The possibility is proposed that reduction of vasoconstrictive prostaglandin production by vascular smooth muscle may be a counter-regulating mechanism in response to the lower release of vasodilating substances such as nitric oxide observed in the vessels of diabetic rats. Acknowledgments Research supported by Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) and by Fundac¸a˜o de Apoio ao Ensino, Pesquisa e Assisteˆncia do Hospital das Clı´nicas da Faculdade de Medicina de Ribeira˜o Preto-USP (FAEPA). References Abebe, W., Harris, K.H., Macleod, K.M., 1990. Enhanced contractile responses of arteries from diabetic rats to a1-adrenoreceptor stimulation in the absence and presence of extracellular calcium. Journal of Cardiovascular Pharmacology 16, 239 – 248.
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