- Email: [email protected]
80 induces endothelium-dependent and histamine release-independent relaxation in rabbit aorta
Available online at www.sciencedirect.com
Nitric Oxide 18 (2008) 87–92 www.elsevier.com/locate/yniox
Compound 48/80 induces endothelium-dependent and histamine release-independent relaxation in rabbit aorta Fernanda Viaro, Andre´a Carla Celotto, Verena Kise Capellini, Caroline Floreoto Baldo, Alfredo Jose´ Rodrigues, Walter V.A. Vicente, Paulo Roberto B. Evora * Laboratory of Endothelial Function, Department of Surgery and Anatomy, Ribeira˜o Preto Medical School, Sa˜o Paulo University, Av. Bandeirantes, 3900, 14049-900, Ribeira˜o Preto, SP, Brazil Received 30 May 2007; revised 16 November 2007 Available online 27 December 2007
Abstract Compound 48/80 (C48/80) is a synthetic condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde and is an experimental drug used since the 1950s to induce anaphylactic shock through histamine release. This study was carried out to further elucidate the mechanism by which this drug induces nitric oxide (NO) release. Our specific goals were: (a) to verify if C48/80’s relaxation occurs through the stimulation of histamine receptors; (b) to evaluate the endothelium-dependent relaxation induced by C48/80; (c) to identify NO as the endothelium-relaxing factor released by C48/80; (d) to identify the NO synthase (NOS) responsible for NO release; and (e) to verify if the relaxation induced by C48/80 is calcium and cyclic guanidine monophosphate (cGMP) dependent. Rabbit aorta segments, with and without endothelium, were suspended in organ chambers (25 ml) filled with Krebs solution maintained at 37 C, bubbled with 95% O2/5% CO2 (pH 7.4). Phenylephrine was used to contract the segments. Other protocol drugs included H1- and H2-receptor antagonists, cyclooxygenase, NOS, guanylyl cyclase and phospholipase C (PLC) inhibitors. Endothelium-dependent relaxation induced by C48/80 was also studied in calcium-free Krebs solution associated with a calcium chelator. In summary, our investigation demonstrated that the C48/80 vasodilating action: (a) does not depend on H1 and H2 histamine receptors; (b) is NO endothelium-dependent; (c) is dependent on the endothelial constitutive NOS (NOS-3) isoform activation; (d) is cGMP-dependent; and that NOS-3 activation by C48/80: (a) is independent of PLC up to 25 lg/ml and (b) is partially dependent of this lipase in higher doses. 2007 Elsevier Inc. All rights reserved. Keywords: Rabbit aorta; Compound 48/80; Endothelium; Vasorelaxation; Nitric oxide
Histamine is present in essentially all tissues. It is found in significant concentrations in the blood and also in the vessel walls [1]. Intravascular administration of histamine elicits a dose-dependent fall in blood pressure in most species [2], therefore it plays an important role on experimental hypotension and vasoplegic syndrome. Since 1950, compound 48/80 (C48/80) has been used as an in vivo histamine releaser to resemble anaphylactic shock. It is a synthetic condensation product of Nmethyl-p-methoxyphenethylamine with formaldehyde [3], that additionally to histamine release [4] also promotes *
Corresponding author. Address: Rua Rui Barbosa, 367, Apt. 15, 14015-120, Ribeira˜o Preto, SP, Brazil. Fax: +55 16 3602 2497. E-mail address: [email protected] (P.R.B. Evora). 1089-8603/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2007.11.004
G-protein activation [5,6], nitric oxide (NO) release [7], and phospholipase C (PLC) [8] and calmodulin inhibition [9]. G-protein-coupled receptors (GPCRs) are a group quite familiar to pharmacologists. It’s worthy to remember that NO was brought into light after stimulation of a GPCR existing in the endothelium: the acetylcholine muscarinic receptor [10]. They control a variety of fundamental cellular processes. G-protein-mediated signal transduction is a complex signaling network with converging and diverging transduction steps at each coupling interface [11]. The classic NO release pathway implicates in a sequence of events that starting through endothelial GPCRs activate PLC which transforms phosphatidylinositol-4,5-bisphosphate (PIP2) in inositol 1,4,5-trisphosphate (IP3), responsi-
88
F. Viaro et al. / Nitric Oxide 18 (2008) 87–92
ble for the release of calcium from the endoplasmatic reticulum, and diacylglycerol (DAG), responsible for the inward shift of calcium. This calcium in combination with calmodulin establishes the condition for the NO synthase (NOS) activation that is responsible for NO production from L-arginine [12]. To our concern, there are no in vitro studies in the literature that report the pharmacological effects of C48/80 on endothelial reactivity related to NO paracrine function. Thus, the purpose of this study was (a) to verify if C48/ 80’s relaxation occurs solely through the stimulation of histamine receptors; (b) to evaluate if the relaxation induced by C48/80 is endothelium-dependent; (c) to identify NO as the endothelium-relaxing factor released by C48/80; (d) to identify the NOS responsible for NO release; and (e) to verify if the relaxation induced by C48/80 is calcium and cyclic guanidine monophosphate (cGMP) dependent.
was determined by the response to Ach (10 4 M) in rings contracted with potassium ions (20 mM). The organ chambers were washed with Krebs solution and the arterial segments were allowed to equilibrate for 60 min in the presence of indomethacin (10 5 M, an unspecific cyclooxygenase (COX) inhibitor), pyrilamine (10 5 M, a histamine H1-receptor antagonist), cimetidine (10 5 M, a histamine H2-receptor antagonist), L-NAME (10 4 M, a non-specific NOS inhibitor), L-NIO (10 4 M, a specific endothelial constitutive NOS (NOS-3) inhibitor), L-NIL (10 5 M, a specific inducible NOS (NOS-2) inhibitor) methylene blue (10 5 M, a guanylyl cyclase inhibitor), ODQ (10 4 M, a guanylyl cyclase inhibitor), calciumfree Krebs solution plus EDTA (5 · 10 4 M, a calcium chelator) or U73122 (10 5 M, a PLC inhibitor). After this, Phe (10 5 M) was added to the organ bath permitting aorta segments to contract and when a stable contraction plateau was obtained, the relaxant response to cumulatively increasing concentrations of C48/80 (2.5, 5, 12.5, 25, 50 and 100 lg/ml) was assessed. In addition to C48/80 curves, concentration response curves were obtained for histamine (10 9 to 10 4 M) in aorta rings with endothelium pre-contracted or not with Phe to test H1- and H2-receptor antagonists efficacy.
Statistical analysis Experimental procedures Materials Acetylcholine (Ach), indomethacin, phenylephrine (Phe), compound 48/ 80 (C48/80), methylene blue, cimetidine, pyrilamine, histamine, U-73122, N5-(1-iminoethyl)-L-ornithine hydrochloride (L-NIO), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) were purchased from Sigma Chemical Company (St. Louis, MO, USA). NG-nitro-L-arginina metil ester (LNAME) was obtained from Calbiochem (San Diego, CA, USA). L-N6-(1iminoethyl) lysine dihydrochloride (L-NIL) was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). All drugs were prepared with distilled water, except for indomethacin that was dissolved in Na2CO3 (10 5 M).
Animals and vessel preparations Studies were performed with isolated abdominal aorta from 10 male adult New Zealand rabbits weighing 2 kg. The procedures and handling of the animals were reviewed and approved by the Institutional Animal Care Review Board. Rabbits were anesthetized and exsanguinated, the abdomen quickly opened, and the abdominal aorta carefully dissected free of connective tissue and immersed in a cooled and oxygenated Krebs solution of the following composition (mM): NaCl—118.0, KCl—4.7, CaCl2—2.5, KH2PO4—1.2, MgSO4—1.66, Glucose—11.1, NaHCO3—25.0, pH 7.4. Ring segments of abdominal aorta (4–5 mm in length) were prepared with great care taken not to touch the intimal surface. In some segments the endothelium was removed by gently rubbing the intimal surface of the blood vessel with a pair of watchmaker’s forceps. This procedure removes endothelium but does not affect the ability of vascular smooth muscle to contract or relax.
Statistical two-way repeated-measures ANOVA and Bonferroni posttest data evaluations were performed using the program SPSS 15.0 (Apache Software Foundation, 2000). Values were considered to be statistically significant when the p value was lower than 0.05.
Results The results are shown in the figures, in which the major endpoints of the investigation are displayed. In the figures, concentrations are expressed as final concentrations in the organ chambers. Changes in wall tension are expressed as percent of relaxation of the maximal contraction achieved following exposure to Phe, a convention that corrects inter-animal variability in the response of the tissue to the drug. Results are expressed as means ± SEM. Effect of the removal of the vascular endothelium on the relaxation induced by C48/80 in rabbit aorta segments Compound 48/80 caused dose-dependent relaxation of rabbit arteries that had been pre-contracted with Phe. In artery segments where the endothelium had been mechanically removed, C48/80 did not induce relaxation, indicating that this effect was endothelium-dependent. Aorta rings with endothelium showed statistical differences in relaxation from those without endothelium when stimulated with increased doses of C48/80 (Fig. 1).
Isometric tension recordings Abdominal aorta segments were mounted in organ chambers (25 ml) filled with Krebs solution maintained at 37 C and bubbled with 95% O2/5% CO2 (pH 7.4). Each arterial ring was suspended by two stainless steel clips placed through the lumen. One clip was anchored to the bottom of the organ chamber and the other was connected to a strain gauge for measurement of isometric force using Grass FT03 (Grass Instrument Company, Quincy, MA, USA). The rings were placed at optimal lengthtension of 2 g and allowed to equilibrate for 60 min. (The optimal length-tension was determined in a pilot study after maximal contraction obtained in response to progressive stretching in potassium chloride medium 20 mM). In all experiments, the presence or absence of endothelium
Effects of indomethacin on the relaxation the induced by C48/80 in rabbit aorta segments The relaxation induced by C48/80 on rabbit arterial rings that had been pre-contracted with Phe was not statistically affected by the use of indomethacin when compared to the relaxation of rings that were not pre-incubated with indomethacin (Fig. 2). Even though indomethacin lacked significant effects, pre-incubation will be assumed as control to ensure exclusion of COX-pathway participation.
F. Viaro et al. / Nitric Oxide 18 (2008) 87–92
89
of rings that were pre-incubated with indomethacin only. LNIO inhibited relaxation and this effect was not observed for L-NIL when compared to the relaxation of rings that were pre-incubated with indomethacin only (Fig. 4). Effects of methylene blue and ODQ on the relaxation induced by C48/80 in rabbit aorta segments The relaxation induced by C48/80 on rabbit arterial rings that had been pre-contracted with Phe was inhibited by methylene blue and ODQ at statistical level when compared to the relaxation of rings that were pre-incubated with indomethacin only (Fig. 5). Fig. 1. Vasodilator responses of rabbit aorta segments. Phe-preconstricted aorta rings with and without endothelium were relaxed with cumulative concentrations of C48/80. Results are reported as means ± SEM (two-way ANOVA). *Indicates statistical difference between groups (p < 0.01). # Within groups, indicates statistical difference with the preceding concentration (p < 0.001).
Effects of pyrilamine and cimetidine on the vascular reactivity induced by C48/80 and histamine in rabbit aorta segments The relaxation induced by C48/80 on rabbit arterial rings that had been pre-contracted with Phe was not statistically affected by the use of pyrilamine or cimetidine solely or associated when compared to the relaxation of rings that were pre-incubated with indomethacin only (Fig. 2). Positive controls with histamine were made to show effectiveness of these receptor antagonists (Fig. 3). Effects of L-NAME, L-NIO and L-NIL on the relaxations induced by C48/80 in rabbit aorta segments The relaxation induced by C48/80 on rabbit arterial rings that had been pre-contracted with Phe was inhibited by LNAME at a statistical level when compared to the relaxation
Effect of calcium-free Krebs solution associated with EDTA on the relaxation induced by C48/80 in rabbit aorta segments The relaxation induced by C48/80 on rabbit arterial rings that had been pre-contracted with Phe was not statistically affected by the removal of calcium from the organ bath when compared to the relaxation of rings that were pre-incubated with indomethacin only (Fig. 6). Effects of U-73122 on the relaxation induced by C48/80 in rabbit aorta segments The relaxation induced by C48/80 on rabbit arterial rings that had been pre-contracted with Phe was not significantly affected by the use of U-73122 up to 25 lg/ml. Above 25 lg/ ml, C48/80-mediated vasodilation was approximately reduced in 30% in the presence of U-73122 (Fig. 7). Discussion The aim of this study was to seek if the vasodilating effect of C48/80 occurred through histamine release. For this, rabbit aortas were chosen for their wide known in vitro responses to histamine. Histamine, in most prepara-
Fig. 2. Vasodilator responses of rabbit aorta segments. Phe-preconstricted aorta rings with endothelium were relaxed with cumulative concentrations of C48/80 after pre-incubated or not with indomethacin (10 5 M), indomethacin plus pyrilamine (10 5 M), indomethacin plus cimetidine (10 5 M) and indomethacin plus pyrilamine plus cimetidine. Results are reported as means ± SEM (two-way ANOVA). #Within all groups, indicates statistical difference with the preceding concentration (p < 0.01).
90
F. Viaro et al. / Nitric Oxide 18 (2008) 87–92
Fig. 3. Controls with histamine to show effectiveness of histamine receptor antagonists: cimetidine and pyrilamine. Phe-preconstricted aorta rings with endothelium were submitted to cumulative concentrations of histamine alone or in the presence of pyrilamine (10 5 M) or cimetidine (10 5 M) and pyrilamine (10 5 M) plus cimetidine (10 5 M) demonstrating effective blockage of H1 and H2 receptors. Results are reported as means ± SEM (two-way ANOVA). #Within all groups, indicates statistical difference with the preceding concentration (p < 0.01).
Fig. 4. Vasodilator responses of rabbit aorta segments. Phe-preconstricted aorta rings with endothelium were relaxed with cumulative concentrations of C48/80 and pre-incubation with indomethacin (10 5 M), indomethacin plus L-NAME (10 4 M), indomethacin plus L-NIO (10 4 M) and indomethacin plus L-NIL (10 5 M). Results are reported as means ± SEM (two-way ANOVA). *Indicates statistical difference between all groups compared with indomethacin (p < 0.007). #Within all groups except for indomethacin plus L-NAME, indicates statistical difference with the preceding concentration (p < 0.001).
tions reacts with vasoconstriction which may be blocked by H1-receptor antagonists. A vasodilating effect, which reflects normal in vivo observation, is only seen on pre-contracted preparations in the presence of H1 antagonist
Fig. 5. Vasodilator responses of rabbit aorta segments. Phe-preconstricted aorta rings with endothelium were relaxed with cumulative concentrations of C48/80 and pre-incubation with indomethacin (10 5 M), indomethacin plus methylene blue (10 5 M), and indomethacin plus ODQ (10 4 M). Results are reported as means ± SEM (two-way ANOVA). *Indicates statistical difference between all groups compared with indomethacin (p < 0.005). #Within groups indicates statistical difference with the preceding concentration (p < 0.01).
[13,14]. These same results were observed on the rabbit aortas used in this study. Since C48/80 is a histamine releaser [4] and histamine is a potent vasodilator via NO [7,15] and prostacyclin release [15], we tested the effect of histamine H1- and H2-receptor
F. Viaro et al. / Nitric Oxide 18 (2008) 87–92
Fig. 6. Vasodilator responses of rabbit aorta segments. Phe-preconstricted aorta rings with endothelium were relaxed with cumulative concentrations of C48/80 with pre-incubation with indomethacin (10 5 M) and indomethacin in calcium-free Krebs solution plus EDTA (5 · 10 4 M). Results are reported as means ± SEM (two-way ANOVA). There was no statistical difference between groups (p < 0.05). #Within both groups, indicates statistical difference with the preceding concentration (p < 0.01).
Fig. 7. Vasodilator responses of rabbit aorta segments. Phe-preconstricted aorta rings with endothelium were relaxed with cumulative concentrations of C48/80 with pre-incubation with indomethacin (10 5 M) and indomethacin plus U-73122 (10 5 M). Results are reported as means ± SEM (two-way ANOVA). *Indicates statistical difference between all groups compared with indomethacin (p < 0.01). #Within both groups indicates statistical difference with the preceding concentration (p < 0.01).
antagonists on vasodilation by C48/80. These antagonists had no effect on the endothelium-dependent relaxation induced by C48/80, showing that the action of C48/80 is independent of these receptors. The results observed after the use of the histamine antagonists allied to literature description of G-protein activation by C48/80 provides evidence to suggest NO release through direct activation of this protein, as described earlier. This agent is known to directly stimulate inhibitory G-protein (Gi) subfamily to promote guanidine diphosphate–guanidine triphosphate (GDP–GTP) exchange and dissociation into their constituent’s bc- and a-subunits [16,17]. The endothelium-dependent action of C48/80 was established by demonstration that relaxation did not take place
91
in aorta rings in which the endothelium had been removed. The use of indomethacin to block cyclic adenosine monophosphate (cAMP)-dependent vasodilatation secondary to prostacyclin demonstrated that the endothelium-dependent relaxation induced by C48/80 was not dependent on COX relaxing products. The proof that C48/80 endothelium-dependent relaxation is caused by NO release was established by the inhibition of relaxation with the L-arginine analog (L-NAME), which is a NOS-competitive inhibitor. Further on we confirmed the specific role of NOS-3 on C48/80 relaxation using L-NIO, a specific inhibitor of this type of enzyme. The same was not observed with the experimental use of L-NIL that is NOS-2 specific inhibitor. The experimental delineament resembling acute phenomenons may be the cause for the participation of NOS-3 rather than NOS-2, as time course is crucial for enzyme type expression and activation [7]. To further confirm the participation of NO, inhibition of relaxation was induced with methylene blue and ODQ, which are guanilyl cyclase blockers that inhibit the formation of cGMP, ultimately responsible for vascular smooth muscle cell relaxation. Considering that C48/80 inhibits calmodulin [9] and increases calcium influx [18,19] and that NOS-3 enzymatic activity is classically known to be calcium–calmodulin dependent, we studied the effect of extracellular calcium removal on C48/80 relaxation using a calcium-free Krebs solution and EDTA. None of these experimental strategies to remove calcium altered the endothelium-dependent relaxation induced by C48/80. From these results associated with literature reports, it is reasonable to assume that intracellular calcium pool of rabbit aorta may be sufficient for NOS-3 activity in a calmodulin-independent manner. The exclusion of the classic pathway for NO production opens possibilities of NOS-3 activation via parallel pathways which are performed by post-translational modifications through protein phosphorylation [20,21]. A variety of protein kinases can modulate NOS-3 activity depending on the type of the initial stimulus to the cell and on kinase sensitivity to calcium [21]. For example, agonist induced activation of the calmodulin-dependent protein kinase II is highly dependent on an increase in intracellular calcium concentration, whereas the shear stress induced activation of protein kinase B (PKB) is not affected by calcium removal [22,23]. The most widely post-translational NOS3 activation described is serine phosphorylation by PKB. This is activated by the 3-phosphorylated inositol lipids generated by phosphatidylinositol 3-kinase (PI-3K) [20]. The latter is a well characterized and necessary component of the machinery controlling PKB phosphorylation [24,25] that is activated by C48/80 either directly or indirectly [26]. Moreover, a number of other protein kinases have been implicated in NOS-3 activation, such as PKG, PKA, AMPK [20,27] and tyrosine kinase [28], although neither have yet been associated with C48/80 effects. Future studies comprise the use of kinase inhibitors to elucidate which pathway leads to NOS-3 activation after C48/80 stimulus.
92
F. Viaro et al. / Nitric Oxide 18 (2008) 87–92
Many studies mentioned C48/80 as a potent PLC inhibitor as observed in human platelets [8], but some experimental studies performed on rat mastocytes have shown that PLC activity is stimulated by C48/80, and that this activity occurs through regulatory proteins linked to the guanine nucleotide [5]. Considering these literature data we find important to investigate PLC as a messenger in the cascade of events of C48/80 NO/endothelium-dependent relaxation to verify if the NOS-3 activation is actually independent of the raise in intracellular calcium concentration responsible for calmodulin activation. Our results demonstrated that the use of U-73122 did not alter the endothelium-dependent relaxation induced by up to 25 lg/ml of C48/80 indicating that, until this point, NOS3 activation occurs through a pathway alternative to PLC. C48/80-mediated vasodilation in concentrations higher then 25 lg/ml was approximately reduced in 30% in the presence of U-73122, proposing that, at higher concentrations, PLC has a partial participation in relaxation. In this context, C48/80 seems to involve distinct mechanisms by which vasodilation is mediated, thus the pathways of NO release induced by C48/80 is still to be identified in future studies. In summary, our investigation demonstrated that the C48/80 vasodilating action: (a) does not depend on H1 and H2 histamine receptors; (b) is NO endothelium-dependent; (c) is dependent on the NOS-3 isoform activation; (d) is cGMP-dependent; and, that NOS-3 activation by C48/ 80: (a) is independent of PLC up to 25 lg/ml and (b) is partially dependent of this lipase in higher doses. Acknowledgments This study was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and Fundac¸a˜o de Apoio ao Ensino, Pesquisa e Assisteˆncia do Hospital das Clı´nicas da Faculdade de Medicina de Ribeira˜o Preto da Universidade de Sa˜o Paulo (FAEPAHCFMRP). References [1] M.J. Ryan, M.J. Brody, Neurogenic and vascular stores of histamine in the dog, J. Pharmacol. Exp. Ther. 181 (1972) 83–91. [2] D.A. Owen, Histamine receptors in the cardiovascular, Gen. Pharmacol. 8 (1977) 141–156. [3] W.D. Paton, Compound 48/80: a potent histamine liberator, Br. J. Pharmacol. Chemother. 6 (1951) 499–508. [4] T. Irman-Florjanc, F. Erjavec, Compound 48/80 and substance P induced release of histamine and serotonin from rat peritoneal mast cells, Agents Actions 13 (1983) 138–141. [5] M. Mousli, C. Bronner, J. Bockaert, B. Rouot, Y. Landry, Interaction of substance P, compound 48/80 and mastoparan with the alphasubunit C-terminus of G protein, Immunol. Lett. 25 (1990) 355–357. [6] M. Mousli, C. Bronner, Y. Landry, J. Bockaert, B. Rouot, Direct activation of GTP-binding regulatory proteins (G-proteins) by substance P and compound 48/80, FEBS Lett. 259 (1990) 260–262.
[7] S.M. Ko, M.K. Kim, J.C. Kim, The role of nitric oxide in experimental allergic conjunctivitis, Cornea 19 (2000) 84–91. [8] C. Bronner, C. Wiggins, D. Monte, F. Marki, A. Capron, Y. Landry, R.C. Franson, Compound 48/80 is a potent inhibitor of phospholipase C and a dual modulator of phospholipase A2 from human platelet, Biochim. Biophys. Acta 920 (1987) 301–305. [9] K. Gietzen, Comparison of the calmodulin antagonists compound 48/ 80 and calmidazolium, Biochem. J. 216 (1983) 611–616. [10] R.F. Furchgott, J.V. Zawadzki, The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature 288 (1980) 373–376. [11] T. Gudermann, T. Schoneberg, G. Schultz, Functional and structural complexity of signal transduction via G-protein-coupled receptors, Annu. Rev. Neurosci. 20 (1997) 399–427. [12] H.P. Rang, M.M. Dale, J.M. Ritter, P.K. Moore, Farmacologia, Elsevier, Rio de Janeiro, 2004. [13] R. Casteels, H. Suzuki, The effect of histamine on the smooth muscle cells of the ear artery of the rabbit, Pflugers Arch. 387 (1980) 17–25. [14] T.E. Tenner Jr., Comparison of H2 receptor agonists in rabbit atria and aorta, Pharmacology 22 (1981) 227–234. [15] S.J. Hill, Distribution, properties, and functional characteristics of three classes of histamine receptor, Pharmacol. Rev. 42 (1990) 45–83. [16] U. Tomita, A. Inanobe, I. Kobayashi, K. Takahashi, M. Ui, T. Katada, Direct interactions of mastoparan and compound 48/80 with GTP-binding proteins, J. Biochem. (Tokyo) 109 (1991) 184–189. [17] T. Tanaka, T. Kohno, S. Kinoshita, H. Mukai, H. Itoh, M. Ohya, T. Miyazawa, T. Higashijima, K. Wakamatsu, Alpha helix content of G protein alpha subunit is decreased upon activation by receptor mimetics, J. Biol. Chem. 273 (1998) 3247–3252. [18] M. Takei, M. Ueno, K. Endo, Role of intracellular Ca2+ on histamine release from rat peritoneal mast cells, J. Pharm. Sci. 81 (1992) 518–520. [19] D.E. McClain, M.A. Donlon, T.A. Hill, G.N. Catravas, Early kinetics of Ca2+ fluxes and histamine release in rat mast cells stimulated with compound 48/80, Agents Actions 15 (1984) 279–284. [20] D. Fulton, J.P. Gratton, W.C. Sessa, Post-translational control of endothelial nitric oxide synthase: why isn’t calcium/calmodulin enough? J. Pharmacol. Exp. Ther. 299 (2001) 818–824. [21] I. Fleming, R. Busse, Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase, Am. J. Physiol. Regul. Integr. Comp. Physiol. 284 (2003) R1–R12. [22] S. Dimmeler, I. Fleming, B. Fisslthaler, C. Hermann, R. Busse, A.M. Zeiher, Activation of nitric oxide synthase in endothelial cells by Aktdependent phosphorylation, Nature 399 (1999) 601–605. [23] I. Fleming, B. Fisslthaler, S. Dimmeler, B.E. Kemp, R. Busse, Phosphorylation of Thr(495) regulates Ca(2+)/calmodulin-dependent endothelial nitric oxide synthase activity, Circ. Res. 88 (2001) E68–E75. [24] D.R. Alessi, S.R. James, C.P. Downes, A.B. Holmes, P.R. Gaffney, C.B. Reese, P. Cohen, Characterization of a 3-phosphoinositidedependent protein kinase which phosphorylates and activates protein kinase Balpha, Curr. Biol. 7 (1997) 261–269. [25] D.R. Alessi, M. Deak, A. Casamayor, F.B. Caudwell, N. Morrice, D.G. Norman, P. Gaffney, C.B. Reese, C.N. MacDougall, D. Harbison, A. Ashworth, M. Bownes, 3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase, Curr. Biol. 7 (1997) 776–789. [26] R.D. Byrne, E. Rosivatz, M. Parsons, B. Larijani, P.J. Parker, T. Ng, R. Woscholski, Differential activation of the PI 3-kinase effectors AKT/PKB and p70 S6 kinase by compound 48/80 is mediated by PKCalpha, Cell. Signal. 19 (2007) 321–329. [27] X.P. Zhang, T.H. Hintze, cAMP signal transduction induces eNOS activation by promoting PKB phosphorylation, Am. J. Physiol. Heart Circ. Physiol. 290 (2006) H2376–H2384. [28] I. Fleming, R. Busse, Signal transduction of eNOS activation, Cardiovasc. Res. 43 (1999) 532–541.
Nitric Oxide 18 (2008) 87–92 www.elsevier.com/locate/yniox
Compound 48/80 induces endothelium-dependent and histamine release-independent relaxation in rabbit aorta Fernanda Viaro, Andre´a Carla Celotto, Verena Kise Capellini, Caroline Floreoto Baldo, Alfredo Jose´ Rodrigues, Walter V.A. Vicente, Paulo Roberto B. Evora * Laboratory of Endothelial Function, Department of Surgery and Anatomy, Ribeira˜o Preto Medical School, Sa˜o Paulo University, Av. Bandeirantes, 3900, 14049-900, Ribeira˜o Preto, SP, Brazil Received 30 May 2007; revised 16 November 2007 Available online 27 December 2007
Abstract Compound 48/80 (C48/80) is a synthetic condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde and is an experimental drug used since the 1950s to induce anaphylactic shock through histamine release. This study was carried out to further elucidate the mechanism by which this drug induces nitric oxide (NO) release. Our specific goals were: (a) to verify if C48/80’s relaxation occurs through the stimulation of histamine receptors; (b) to evaluate the endothelium-dependent relaxation induced by C48/80; (c) to identify NO as the endothelium-relaxing factor released by C48/80; (d) to identify the NO synthase (NOS) responsible for NO release; and (e) to verify if the relaxation induced by C48/80 is calcium and cyclic guanidine monophosphate (cGMP) dependent. Rabbit aorta segments, with and without endothelium, were suspended in organ chambers (25 ml) filled with Krebs solution maintained at 37 C, bubbled with 95% O2/5% CO2 (pH 7.4). Phenylephrine was used to contract the segments. Other protocol drugs included H1- and H2-receptor antagonists, cyclooxygenase, NOS, guanylyl cyclase and phospholipase C (PLC) inhibitors. Endothelium-dependent relaxation induced by C48/80 was also studied in calcium-free Krebs solution associated with a calcium chelator. In summary, our investigation demonstrated that the C48/80 vasodilating action: (a) does not depend on H1 and H2 histamine receptors; (b) is NO endothelium-dependent; (c) is dependent on the endothelial constitutive NOS (NOS-3) isoform activation; (d) is cGMP-dependent; and that NOS-3 activation by C48/80: (a) is independent of PLC up to 25 lg/ml and (b) is partially dependent of this lipase in higher doses. 2007 Elsevier Inc. All rights reserved. Keywords: Rabbit aorta; Compound 48/80; Endothelium; Vasorelaxation; Nitric oxide
Histamine is present in essentially all tissues. It is found in significant concentrations in the blood and also in the vessel walls [1]. Intravascular administration of histamine elicits a dose-dependent fall in blood pressure in most species [2], therefore it plays an important role on experimental hypotension and vasoplegic syndrome. Since 1950, compound 48/80 (C48/80) has been used as an in vivo histamine releaser to resemble anaphylactic shock. It is a synthetic condensation product of Nmethyl-p-methoxyphenethylamine with formaldehyde [3], that additionally to histamine release [4] also promotes *
Corresponding author. Address: Rua Rui Barbosa, 367, Apt. 15, 14015-120, Ribeira˜o Preto, SP, Brazil. Fax: +55 16 3602 2497. E-mail address: [email protected] (P.R.B. Evora). 1089-8603/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2007.11.004
G-protein activation [5,6], nitric oxide (NO) release [7], and phospholipase C (PLC) [8] and calmodulin inhibition [9]. G-protein-coupled receptors (GPCRs) are a group quite familiar to pharmacologists. It’s worthy to remember that NO was brought into light after stimulation of a GPCR existing in the endothelium: the acetylcholine muscarinic receptor [10]. They control a variety of fundamental cellular processes. G-protein-mediated signal transduction is a complex signaling network with converging and diverging transduction steps at each coupling interface [11]. The classic NO release pathway implicates in a sequence of events that starting through endothelial GPCRs activate PLC which transforms phosphatidylinositol-4,5-bisphosphate (PIP2) in inositol 1,4,5-trisphosphate (IP3), responsi-
88
F. Viaro et al. / Nitric Oxide 18 (2008) 87–92
ble for the release of calcium from the endoplasmatic reticulum, and diacylglycerol (DAG), responsible for the inward shift of calcium. This calcium in combination with calmodulin establishes the condition for the NO synthase (NOS) activation that is responsible for NO production from L-arginine [12]. To our concern, there are no in vitro studies in the literature that report the pharmacological effects of C48/80 on endothelial reactivity related to NO paracrine function. Thus, the purpose of this study was (a) to verify if C48/ 80’s relaxation occurs solely through the stimulation of histamine receptors; (b) to evaluate if the relaxation induced by C48/80 is endothelium-dependent; (c) to identify NO as the endothelium-relaxing factor released by C48/80; (d) to identify the NOS responsible for NO release; and (e) to verify if the relaxation induced by C48/80 is calcium and cyclic guanidine monophosphate (cGMP) dependent.
was determined by the response to Ach (10 4 M) in rings contracted with potassium ions (20 mM). The organ chambers were washed with Krebs solution and the arterial segments were allowed to equilibrate for 60 min in the presence of indomethacin (10 5 M, an unspecific cyclooxygenase (COX) inhibitor), pyrilamine (10 5 M, a histamine H1-receptor antagonist), cimetidine (10 5 M, a histamine H2-receptor antagonist), L-NAME (10 4 M, a non-specific NOS inhibitor), L-NIO (10 4 M, a specific endothelial constitutive NOS (NOS-3) inhibitor), L-NIL (10 5 M, a specific inducible NOS (NOS-2) inhibitor) methylene blue (10 5 M, a guanylyl cyclase inhibitor), ODQ (10 4 M, a guanylyl cyclase inhibitor), calciumfree Krebs solution plus EDTA (5 · 10 4 M, a calcium chelator) or U73122 (10 5 M, a PLC inhibitor). After this, Phe (10 5 M) was added to the organ bath permitting aorta segments to contract and when a stable contraction plateau was obtained, the relaxant response to cumulatively increasing concentrations of C48/80 (2.5, 5, 12.5, 25, 50 and 100 lg/ml) was assessed. In addition to C48/80 curves, concentration response curves were obtained for histamine (10 9 to 10 4 M) in aorta rings with endothelium pre-contracted or not with Phe to test H1- and H2-receptor antagonists efficacy.
Statistical analysis Experimental procedures Materials Acetylcholine (Ach), indomethacin, phenylephrine (Phe), compound 48/ 80 (C48/80), methylene blue, cimetidine, pyrilamine, histamine, U-73122, N5-(1-iminoethyl)-L-ornithine hydrochloride (L-NIO), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) were purchased from Sigma Chemical Company (St. Louis, MO, USA). NG-nitro-L-arginina metil ester (LNAME) was obtained from Calbiochem (San Diego, CA, USA). L-N6-(1iminoethyl) lysine dihydrochloride (L-NIL) was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). All drugs were prepared with distilled water, except for indomethacin that was dissolved in Na2CO3 (10 5 M).
Animals and vessel preparations Studies were performed with isolated abdominal aorta from 10 male adult New Zealand rabbits weighing 2 kg. The procedures and handling of the animals were reviewed and approved by the Institutional Animal Care Review Board. Rabbits were anesthetized and exsanguinated, the abdomen quickly opened, and the abdominal aorta carefully dissected free of connective tissue and immersed in a cooled and oxygenated Krebs solution of the following composition (mM): NaCl—118.0, KCl—4.7, CaCl2—2.5, KH2PO4—1.2, MgSO4—1.66, Glucose—11.1, NaHCO3—25.0, pH 7.4. Ring segments of abdominal aorta (4–5 mm in length) were prepared with great care taken not to touch the intimal surface. In some segments the endothelium was removed by gently rubbing the intimal surface of the blood vessel with a pair of watchmaker’s forceps. This procedure removes endothelium but does not affect the ability of vascular smooth muscle to contract or relax.
Statistical two-way repeated-measures ANOVA and Bonferroni posttest data evaluations were performed using the program SPSS 15.0 (Apache Software Foundation, 2000). Values were considered to be statistically significant when the p value was lower than 0.05.
Results The results are shown in the figures, in which the major endpoints of the investigation are displayed. In the figures, concentrations are expressed as final concentrations in the organ chambers. Changes in wall tension are expressed as percent of relaxation of the maximal contraction achieved following exposure to Phe, a convention that corrects inter-animal variability in the response of the tissue to the drug. Results are expressed as means ± SEM. Effect of the removal of the vascular endothelium on the relaxation induced by C48/80 in rabbit aorta segments Compound 48/80 caused dose-dependent relaxation of rabbit arteries that had been pre-contracted with Phe. In artery segments where the endothelium had been mechanically removed, C48/80 did not induce relaxation, indicating that this effect was endothelium-dependent. Aorta rings with endothelium showed statistical differences in relaxation from those without endothelium when stimulated with increased doses of C48/80 (Fig. 1).
Isometric tension recordings Abdominal aorta segments were mounted in organ chambers (25 ml) filled with Krebs solution maintained at 37 C and bubbled with 95% O2/5% CO2 (pH 7.4). Each arterial ring was suspended by two stainless steel clips placed through the lumen. One clip was anchored to the bottom of the organ chamber and the other was connected to a strain gauge for measurement of isometric force using Grass FT03 (Grass Instrument Company, Quincy, MA, USA). The rings were placed at optimal lengthtension of 2 g and allowed to equilibrate for 60 min. (The optimal length-tension was determined in a pilot study after maximal contraction obtained in response to progressive stretching in potassium chloride medium 20 mM). In all experiments, the presence or absence of endothelium
Effects of indomethacin on the relaxation the induced by C48/80 in rabbit aorta segments The relaxation induced by C48/80 on rabbit arterial rings that had been pre-contracted with Phe was not statistically affected by the use of indomethacin when compared to the relaxation of rings that were not pre-incubated with indomethacin (Fig. 2). Even though indomethacin lacked significant effects, pre-incubation will be assumed as control to ensure exclusion of COX-pathway participation.
F. Viaro et al. / Nitric Oxide 18 (2008) 87–92
89
of rings that were pre-incubated with indomethacin only. LNIO inhibited relaxation and this effect was not observed for L-NIL when compared to the relaxation of rings that were pre-incubated with indomethacin only (Fig. 4). Effects of methylene blue and ODQ on the relaxation induced by C48/80 in rabbit aorta segments The relaxation induced by C48/80 on rabbit arterial rings that had been pre-contracted with Phe was inhibited by methylene blue and ODQ at statistical level when compared to the relaxation of rings that were pre-incubated with indomethacin only (Fig. 5). Fig. 1. Vasodilator responses of rabbit aorta segments. Phe-preconstricted aorta rings with and without endothelium were relaxed with cumulative concentrations of C48/80. Results are reported as means ± SEM (two-way ANOVA). *Indicates statistical difference between groups (p < 0.01). # Within groups, indicates statistical difference with the preceding concentration (p < 0.001).
Effects of pyrilamine and cimetidine on the vascular reactivity induced by C48/80 and histamine in rabbit aorta segments The relaxation induced by C48/80 on rabbit arterial rings that had been pre-contracted with Phe was not statistically affected by the use of pyrilamine or cimetidine solely or associated when compared to the relaxation of rings that were pre-incubated with indomethacin only (Fig. 2). Positive controls with histamine were made to show effectiveness of these receptor antagonists (Fig. 3). Effects of L-NAME, L-NIO and L-NIL on the relaxations induced by C48/80 in rabbit aorta segments The relaxation induced by C48/80 on rabbit arterial rings that had been pre-contracted with Phe was inhibited by LNAME at a statistical level when compared to the relaxation
Effect of calcium-free Krebs solution associated with EDTA on the relaxation induced by C48/80 in rabbit aorta segments The relaxation induced by C48/80 on rabbit arterial rings that had been pre-contracted with Phe was not statistically affected by the removal of calcium from the organ bath when compared to the relaxation of rings that were pre-incubated with indomethacin only (Fig. 6). Effects of U-73122 on the relaxation induced by C48/80 in rabbit aorta segments The relaxation induced by C48/80 on rabbit arterial rings that had been pre-contracted with Phe was not significantly affected by the use of U-73122 up to 25 lg/ml. Above 25 lg/ ml, C48/80-mediated vasodilation was approximately reduced in 30% in the presence of U-73122 (Fig. 7). Discussion The aim of this study was to seek if the vasodilating effect of C48/80 occurred through histamine release. For this, rabbit aortas were chosen for their wide known in vitro responses to histamine. Histamine, in most prepara-
Fig. 2. Vasodilator responses of rabbit aorta segments. Phe-preconstricted aorta rings with endothelium were relaxed with cumulative concentrations of C48/80 after pre-incubated or not with indomethacin (10 5 M), indomethacin plus pyrilamine (10 5 M), indomethacin plus cimetidine (10 5 M) and indomethacin plus pyrilamine plus cimetidine. Results are reported as means ± SEM (two-way ANOVA). #Within all groups, indicates statistical difference with the preceding concentration (p < 0.01).
90
F. Viaro et al. / Nitric Oxide 18 (2008) 87–92
Fig. 3. Controls with histamine to show effectiveness of histamine receptor antagonists: cimetidine and pyrilamine. Phe-preconstricted aorta rings with endothelium were submitted to cumulative concentrations of histamine alone or in the presence of pyrilamine (10 5 M) or cimetidine (10 5 M) and pyrilamine (10 5 M) plus cimetidine (10 5 M) demonstrating effective blockage of H1 and H2 receptors. Results are reported as means ± SEM (two-way ANOVA). #Within all groups, indicates statistical difference with the preceding concentration (p < 0.01).
Fig. 4. Vasodilator responses of rabbit aorta segments. Phe-preconstricted aorta rings with endothelium were relaxed with cumulative concentrations of C48/80 and pre-incubation with indomethacin (10 5 M), indomethacin plus L-NAME (10 4 M), indomethacin plus L-NIO (10 4 M) and indomethacin plus L-NIL (10 5 M). Results are reported as means ± SEM (two-way ANOVA). *Indicates statistical difference between all groups compared with indomethacin (p < 0.007). #Within all groups except for indomethacin plus L-NAME, indicates statistical difference with the preceding concentration (p < 0.001).
tions reacts with vasoconstriction which may be blocked by H1-receptor antagonists. A vasodilating effect, which reflects normal in vivo observation, is only seen on pre-contracted preparations in the presence of H1 antagonist
Fig. 5. Vasodilator responses of rabbit aorta segments. Phe-preconstricted aorta rings with endothelium were relaxed with cumulative concentrations of C48/80 and pre-incubation with indomethacin (10 5 M), indomethacin plus methylene blue (10 5 M), and indomethacin plus ODQ (10 4 M). Results are reported as means ± SEM (two-way ANOVA). *Indicates statistical difference between all groups compared with indomethacin (p < 0.005). #Within groups indicates statistical difference with the preceding concentration (p < 0.01).
[13,14]. These same results were observed on the rabbit aortas used in this study. Since C48/80 is a histamine releaser [4] and histamine is a potent vasodilator via NO [7,15] and prostacyclin release [15], we tested the effect of histamine H1- and H2-receptor
F. Viaro et al. / Nitric Oxide 18 (2008) 87–92
Fig. 6. Vasodilator responses of rabbit aorta segments. Phe-preconstricted aorta rings with endothelium were relaxed with cumulative concentrations of C48/80 with pre-incubation with indomethacin (10 5 M) and indomethacin in calcium-free Krebs solution plus EDTA (5 · 10 4 M). Results are reported as means ± SEM (two-way ANOVA). There was no statistical difference between groups (p < 0.05). #Within both groups, indicates statistical difference with the preceding concentration (p < 0.01).
Fig. 7. Vasodilator responses of rabbit aorta segments. Phe-preconstricted aorta rings with endothelium were relaxed with cumulative concentrations of C48/80 with pre-incubation with indomethacin (10 5 M) and indomethacin plus U-73122 (10 5 M). Results are reported as means ± SEM (two-way ANOVA). *Indicates statistical difference between all groups compared with indomethacin (p < 0.01). #Within both groups indicates statistical difference with the preceding concentration (p < 0.01).
antagonists on vasodilation by C48/80. These antagonists had no effect on the endothelium-dependent relaxation induced by C48/80, showing that the action of C48/80 is independent of these receptors. The results observed after the use of the histamine antagonists allied to literature description of G-protein activation by C48/80 provides evidence to suggest NO release through direct activation of this protein, as described earlier. This agent is known to directly stimulate inhibitory G-protein (Gi) subfamily to promote guanidine diphosphate–guanidine triphosphate (GDP–GTP) exchange and dissociation into their constituent’s bc- and a-subunits [16,17]. The endothelium-dependent action of C48/80 was established by demonstration that relaxation did not take place
91
in aorta rings in which the endothelium had been removed. The use of indomethacin to block cyclic adenosine monophosphate (cAMP)-dependent vasodilatation secondary to prostacyclin demonstrated that the endothelium-dependent relaxation induced by C48/80 was not dependent on COX relaxing products. The proof that C48/80 endothelium-dependent relaxation is caused by NO release was established by the inhibition of relaxation with the L-arginine analog (L-NAME), which is a NOS-competitive inhibitor. Further on we confirmed the specific role of NOS-3 on C48/80 relaxation using L-NIO, a specific inhibitor of this type of enzyme. The same was not observed with the experimental use of L-NIL that is NOS-2 specific inhibitor. The experimental delineament resembling acute phenomenons may be the cause for the participation of NOS-3 rather than NOS-2, as time course is crucial for enzyme type expression and activation [7]. To further confirm the participation of NO, inhibition of relaxation was induced with methylene blue and ODQ, which are guanilyl cyclase blockers that inhibit the formation of cGMP, ultimately responsible for vascular smooth muscle cell relaxation. Considering that C48/80 inhibits calmodulin [9] and increases calcium influx [18,19] and that NOS-3 enzymatic activity is classically known to be calcium–calmodulin dependent, we studied the effect of extracellular calcium removal on C48/80 relaxation using a calcium-free Krebs solution and EDTA. None of these experimental strategies to remove calcium altered the endothelium-dependent relaxation induced by C48/80. From these results associated with literature reports, it is reasonable to assume that intracellular calcium pool of rabbit aorta may be sufficient for NOS-3 activity in a calmodulin-independent manner. The exclusion of the classic pathway for NO production opens possibilities of NOS-3 activation via parallel pathways which are performed by post-translational modifications through protein phosphorylation [20,21]. A variety of protein kinases can modulate NOS-3 activity depending on the type of the initial stimulus to the cell and on kinase sensitivity to calcium [21]. For example, agonist induced activation of the calmodulin-dependent protein kinase II is highly dependent on an increase in intracellular calcium concentration, whereas the shear stress induced activation of protein kinase B (PKB) is not affected by calcium removal [22,23]. The most widely post-translational NOS3 activation described is serine phosphorylation by PKB. This is activated by the 3-phosphorylated inositol lipids generated by phosphatidylinositol 3-kinase (PI-3K) [20]. The latter is a well characterized and necessary component of the machinery controlling PKB phosphorylation [24,25] that is activated by C48/80 either directly or indirectly [26]. Moreover, a number of other protein kinases have been implicated in NOS-3 activation, such as PKG, PKA, AMPK [20,27] and tyrosine kinase [28], although neither have yet been associated with C48/80 effects. Future studies comprise the use of kinase inhibitors to elucidate which pathway leads to NOS-3 activation after C48/80 stimulus.
92
F. Viaro et al. / Nitric Oxide 18 (2008) 87–92
Many studies mentioned C48/80 as a potent PLC inhibitor as observed in human platelets [8], but some experimental studies performed on rat mastocytes have shown that PLC activity is stimulated by C48/80, and that this activity occurs through regulatory proteins linked to the guanine nucleotide [5]. Considering these literature data we find important to investigate PLC as a messenger in the cascade of events of C48/80 NO/endothelium-dependent relaxation to verify if the NOS-3 activation is actually independent of the raise in intracellular calcium concentration responsible for calmodulin activation. Our results demonstrated that the use of U-73122 did not alter the endothelium-dependent relaxation induced by up to 25 lg/ml of C48/80 indicating that, until this point, NOS3 activation occurs through a pathway alternative to PLC. C48/80-mediated vasodilation in concentrations higher then 25 lg/ml was approximately reduced in 30% in the presence of U-73122, proposing that, at higher concentrations, PLC has a partial participation in relaxation. In this context, C48/80 seems to involve distinct mechanisms by which vasodilation is mediated, thus the pathways of NO release induced by C48/80 is still to be identified in future studies. In summary, our investigation demonstrated that the C48/80 vasodilating action: (a) does not depend on H1 and H2 histamine receptors; (b) is NO endothelium-dependent; (c) is dependent on the NOS-3 isoform activation; (d) is cGMP-dependent; and, that NOS-3 activation by C48/ 80: (a) is independent of PLC up to 25 lg/ml and (b) is partially dependent of this lipase in higher doses. Acknowledgments This study was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and Fundac¸a˜o de Apoio ao Ensino, Pesquisa e Assisteˆncia do Hospital das Clı´nicas da Faculdade de Medicina de Ribeira˜o Preto da Universidade de Sa˜o Paulo (FAEPAHCFMRP). References [1] M.J. Ryan, M.J. Brody, Neurogenic and vascular stores of histamine in the dog, J. Pharmacol. Exp. Ther. 181 (1972) 83–91. [2] D.A. Owen, Histamine receptors in the cardiovascular, Gen. Pharmacol. 8 (1977) 141–156. [3] W.D. Paton, Compound 48/80: a potent histamine liberator, Br. J. Pharmacol. Chemother. 6 (1951) 499–508. [4] T. Irman-Florjanc, F. Erjavec, Compound 48/80 and substance P induced release of histamine and serotonin from rat peritoneal mast cells, Agents Actions 13 (1983) 138–141. [5] M. Mousli, C. Bronner, J. Bockaert, B. Rouot, Y. Landry, Interaction of substance P, compound 48/80 and mastoparan with the alphasubunit C-terminus of G protein, Immunol. Lett. 25 (1990) 355–357. [6] M. Mousli, C. Bronner, Y. Landry, J. Bockaert, B. Rouot, Direct activation of GTP-binding regulatory proteins (G-proteins) by substance P and compound 48/80, FEBS Lett. 259 (1990) 260–262.
[7] S.M. Ko, M.K. Kim, J.C. Kim, The role of nitric oxide in experimental allergic conjunctivitis, Cornea 19 (2000) 84–91. [8] C. Bronner, C. Wiggins, D. Monte, F. Marki, A. Capron, Y. Landry, R.C. Franson, Compound 48/80 is a potent inhibitor of phospholipase C and a dual modulator of phospholipase A2 from human platelet, Biochim. Biophys. Acta 920 (1987) 301–305. [9] K. Gietzen, Comparison of the calmodulin antagonists compound 48/ 80 and calmidazolium, Biochem. J. 216 (1983) 611–616. [10] R.F. Furchgott, J.V. Zawadzki, The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature 288 (1980) 373–376. [11] T. Gudermann, T. Schoneberg, G. Schultz, Functional and structural complexity of signal transduction via G-protein-coupled receptors, Annu. Rev. Neurosci. 20 (1997) 399–427. [12] H.P. Rang, M.M. Dale, J.M. Ritter, P.K. Moore, Farmacologia, Elsevier, Rio de Janeiro, 2004. [13] R. Casteels, H. Suzuki, The effect of histamine on the smooth muscle cells of the ear artery of the rabbit, Pflugers Arch. 387 (1980) 17–25. [14] T.E. Tenner Jr., Comparison of H2 receptor agonists in rabbit atria and aorta, Pharmacology 22 (1981) 227–234. [15] S.J. Hill, Distribution, properties, and functional characteristics of three classes of histamine receptor, Pharmacol. Rev. 42 (1990) 45–83. [16] U. Tomita, A. Inanobe, I. Kobayashi, K. Takahashi, M. Ui, T. Katada, Direct interactions of mastoparan and compound 48/80 with GTP-binding proteins, J. Biochem. (Tokyo) 109 (1991) 184–189. [17] T. Tanaka, T. Kohno, S. Kinoshita, H. Mukai, H. Itoh, M. Ohya, T. Miyazawa, T. Higashijima, K. Wakamatsu, Alpha helix content of G protein alpha subunit is decreased upon activation by receptor mimetics, J. Biol. Chem. 273 (1998) 3247–3252. [18] M. Takei, M. Ueno, K. Endo, Role of intracellular Ca2+ on histamine release from rat peritoneal mast cells, J. Pharm. Sci. 81 (1992) 518–520. [19] D.E. McClain, M.A. Donlon, T.A. Hill, G.N. Catravas, Early kinetics of Ca2+ fluxes and histamine release in rat mast cells stimulated with compound 48/80, Agents Actions 15 (1984) 279–284. [20] D. Fulton, J.P. Gratton, W.C. Sessa, Post-translational control of endothelial nitric oxide synthase: why isn’t calcium/calmodulin enough? J. Pharmacol. Exp. Ther. 299 (2001) 818–824. [21] I. Fleming, R. Busse, Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase, Am. J. Physiol. Regul. Integr. Comp. Physiol. 284 (2003) R1–R12. [22] S. Dimmeler, I. Fleming, B. Fisslthaler, C. Hermann, R. Busse, A.M. Zeiher, Activation of nitric oxide synthase in endothelial cells by Aktdependent phosphorylation, Nature 399 (1999) 601–605. [23] I. Fleming, B. Fisslthaler, S. Dimmeler, B.E. Kemp, R. Busse, Phosphorylation of Thr(495) regulates Ca(2+)/calmodulin-dependent endothelial nitric oxide synthase activity, Circ. Res. 88 (2001) E68–E75. [24] D.R. Alessi, S.R. James, C.P. Downes, A.B. Holmes, P.R. Gaffney, C.B. Reese, P. Cohen, Characterization of a 3-phosphoinositidedependent protein kinase which phosphorylates and activates protein kinase Balpha, Curr. Biol. 7 (1997) 261–269. [25] D.R. Alessi, M. Deak, A. Casamayor, F.B. Caudwell, N. Morrice, D.G. Norman, P. Gaffney, C.B. Reese, C.N. MacDougall, D. Harbison, A. Ashworth, M. Bownes, 3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase, Curr. Biol. 7 (1997) 776–789. [26] R.D. Byrne, E. Rosivatz, M. Parsons, B. Larijani, P.J. Parker, T. Ng, R. Woscholski, Differential activation of the PI 3-kinase effectors AKT/PKB and p70 S6 kinase by compound 48/80 is mediated by PKCalpha, Cell. Signal. 19 (2007) 321–329. [27] X.P. Zhang, T.H. Hintze, cAMP signal transduction induces eNOS activation by promoting PKB phosphorylation, Am. J. Physiol. Heart Circ. Physiol. 290 (2006) H2376–H2384. [28] I. Fleming, R. Busse, Signal transduction of eNOS activation, Cardiovasc. Res. 43 (1999) 532–541.