Peptides (v.26, #8)

Contents (v-vi).

Preface by Réjean Couture (1285).

Combination cancer chemotherapy with one compound: Pluripotent bradykinin antagonists by John M. Stewart; Lajos Gera; Daniel C. Chan; Eunice J. York; Vitalija Simkeviciene; Paul A. Bunn; Laimute Taraseviciene-Stewart (1288-1291).
Lung and prostate cancers are major health problems worldwide. Treatments with standard chemotherapy agents are relatively ineffective. Combination chemotherapy gives better treatment than a single agent because the drugs can inhibit the cancer in different pathways, but new therapeutic agents are needed for the treatment of both tumor types. Bradykinin (BK) antagonists offer advantages of combination therapy in one compound. These promising multitargeted anti-cancer compounds selectively stimulate apoptosis in cancers and also inhibit both angiogenesis and matrix metalloprotease (MMP) action in treated lung and prostate tumors in nude mice. The highly potent, metabolism-resistant bradykinin antagonist peptide dimer, B-9870 [SUIM-(DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-Arg)2] (SUIM = suberimidyl; Hyp = 4-hydroxyproline; Igl = α-(2-indanyl)glycine; Oic = octahydroindole-2-carboxylic acid) and its non-peptide mimetic, BKM-570 [2,3,4,5,6-pentafluorocinnamoyl-(o-2,6-dichlorobenzyl)-l-tyrosine-N-(4-amino-2,2,6,6-tetramethylpiperidyl)amide] are superior to the widely used but toxic chemotherapeutic drugs cisplatin and taxotere. In certain combinations, they act synergistically with standard anti-cancer drugs. Due to its structure and biological activity, BKM-570 is an attractive lead compound for derivatization and evaluation for lung and prostate cancer drugs.
Keywords: Angiogenesis; Apoptosis; Bradykinin antagonist; Cancer; MMP inhibitor;

Treatment of severe pulmonary hypertension: A bradykinin receptor 2 agonist B9972 causes reduction of pulmonary artery pressure and right ventricular hypertrophy by Laimute Taraseviciene-Stewart; Robertas Scerbavicius; John M. Stewart; Lajos Gera; Yoshiki Demura; Carlyne Cool; Michael Kasper; Norbert F. Voelkel (1292-1300).
Bradykinin is an important modulator of endothelial cell function and has also a powerful cardioprotective effect. Here we report that treatment of severely pulmonary hypertensive rats (that recapitulate several of the physiological and pathological characteristics of the human pulmonary vascular disease, including dramatic right ventricular hypertrophy, pericardial effusion and death) with a newly synthesized long-acting bradykinin B2 receptor agonist B9972 caused reduction of the pulmonary artery pressure (PAP = 51 ± 2.0 versus 68 ± 2.8 of untreated animals) and of right ventricular hypertrophy (R v/L v  +  S  = 0.55 ± 0.02 versus 0.73 ± 0.03 of untreated rats) and activation of Akt. Long-term stimulation with B9972 in our animal model of SPH resulted in decreased expression of the B2 receptor in lung vasculature. Treatment with B9972 decreased the number of plexiform lesions in the lungs by inducing cell apoptosis in the obliterated vessels and by restoring caveolin-1 expression. B9972 also promoted eNOS activation. In vitro B9972 caused activation of caspase-3 as well as Erk and induction of prostacyclin production in rat pulmonary microvascular EC. Taken together our data suggest that a stable bradykinin B2 agonist B9972 demonstrates the capacity to reduce severe pulmonary hypertension, right ventricular hypertrophy and induce apoptosis of hyperproliferative cells in pre-capillary pulmonary arterioles.
Keywords: Severe pulmonary hypertension; Bradykinin agonist; Apoptosis; Caveolin;

Kallikrein–kinin system in hepatic experimental models by Maria Kouyoumdjian; Márcia Regina Nagaoka; Durval Rosa Borges (1301-1307).
The purpose of this brief review is to describe some characteristics of the kallikrein–kinin system (KKS) in the liver. The liver synthesizes kininogens and prekallikrein and the synthesis of both proteins is increased in rats during the acute phase reaction. It is also the main organ to clear tissue as well as plasma kallikrein from the circulation in normal and pathological conditions. Bradykinin (BK), yielded by the kallikrein–kinin system, is a potent arterial hypotensive peptide, but in the liver it induces a portal hypertensive response. The portal hypertensive action of bradykinin is mediated by B2 receptors located on sinusoidal cells of the periportal region and is followed by its hydrolysis by angiotensin-converting enzyme, which is primarily present in the perivenous (centrolobular) region.
Keywords: Kallikrein; Galectin; Bradykinin; Bradykinin-receptor; Portal hypertension; ACE;

Bradykinin B2 type receptor activation regulates fluid and electrolyte transport in the rabbit kidney by Richard L. Hébert; Domenico Regoli; Huaqi Xiong; Matthew D. Breyer; Gérard E. Plante (1308-1316).
Bradykinin is an important autacoid produced in the kidney, regulating both renal function and blood pressure. In vivo studies in anesthetized rabbits, revealed that BK induced diuresis (UV), natriuresis (UNaV) and was not associated with renal hemodynamic changes. These diuretic and natriuretic effects were blocked by the BK-B2 antagonist HOE-140. BK also inhibits vasopressin (AVP)-stimulated water flow (L p) in microperfused rabbit cortical collecting ducts (rCCD), in a concentration-dependent fashion, consistent with its in vivo diuretic effects. BK-B1 antagonist Leu8-des-Arg9-BK did not alter the effect of BK on L p, but HOE-140 completely blocked the inhibitory effects of BK on L p. While BK did not increase [Ca2+]i in fura-2 loaded freshly microdissected rCCD, BK increased [Ca2+]i in immortalized cultured rCCD cells demonstrating different signaling mechanisms are activated by BK in microdissected versus cultured rCCD. In microperfused rCCD, neither the protein kinase C inhibitor staurosporine nor the phospholipase C (PLC) inhibitor U-73,122 attenuated the BK response arguing against activation of PLC/PKC by BK in rCCD. We conclude: (1) BK induces UV and UNaV by a BK-B2 receptor; (2) BK inhibits AVP-stimulated L p by a BK-B2 receptor suggesting that its effects on L p are not via a PLC/PKC; (3) finally, BK raises [Ca2+]i in rCCD cells by a BK-B2 receptor mechanism.
Keywords: Bradykinin; Diuresis; Natriuresis;

Hypotensive effects of hemopressin and bradykinin in rabbits, rats and mice by Paul-André Blais; Jérôme Côté; Josée Morin; Annie Larouche; Gabrielle Gendron; Audrey Fortier; Domenico Regoli; Witold Neugebauer; Fernand Gobeil (1317-1322).
Hemopressin is a novel vasoactive nonapeptide derived from hemoglobin's α-chain as recently reported by Rioli et al. [Rioli V, Gozzo FC, Heimann AS, Linardi A, Krieger JE, Shida CS, et al. Novel natural peptide substrates for endopeptidase 24.15, neurolysin, and angiotensin-converting enzyme. J Biol Chem 2003;278(10):8547–55]. In anesthetized male Wistar rats, this peptide exhibited hypotensive actions similar to those of bradykinin (BK) when administered intravenously (i.v.), and was found to be metabolized both in vitro and in vivo by several peptidases, including the angiotensin-converting enzyme (ACE). In this study, these findings were expanded upon by examining: (i) the degradation kinetics following incubation with ACE purified from rabbit lung and (ii) the blood pressure lowering effects of HP and BK injected i.v. or intra-arterially (i.a.) in male rabbits, rats, and mice. Our findings demonstrate that, in vitro, HP and BK are both degraded by ACE, but at different velocity rates. Furthermore, both HP and BK induced transient hypotension in all animals tested, although the responses to HP relative to the administration sites were significantly lower (by 10–100-fold) on an equimolar basis compared to those of BK. In rabbits, the decrease of blood pressure induced by HP (10–100 nmol/kg) did not differ whether it was administered i.v. or i.a., suggesting an absence of pulmonary/cardiac inactivation in contrast to BK (0.1–1 nmol/kg). The in vivo effect of HP was significantly potentiated in rabbits immunostimulated with bacterial lipopolysaccharide (LPS), but was unaffected by both the B2 receptor antagonist HOE 140 (0.1 μmol/kg) and captopril (100 μg/kg), contrary to BK. Therefore, HP acts as a weak hypotensive mediator, which does not activate kinin B2 receptors, but uses a functional site and/or signaling paths appearing to be up-regulated by LPS.
Keywords: Hypotensive effect; Hemopressin; Bradykinin;

Increases of spinal kinin receptor binding sites in two rat models of insulin resistance by Adil El Midaoui; Brice Ongali; Mihai Petcu; Donata Rodi; Jacques de Champlain; Witold Neugebauer; Réjean Couture (1323-1330).
An autoradiographic study was conducted to determine whether kinin receptors are altered in the rat spinal cord in two experimental models of chronic hyperglycemia and insulin resistance. Sprague-Dawley rats were given 10% d-glucose in their drinking water alone or with insulin (9 mU/kg/min with osmotic pumps) for 4 weeks. Both groups and control rats were treated either with a normal chow diet or with an alpha-lipoic acid-supplemented diet as antioxidant therapy. After 4 weeks of treatment, glycemia, insulinemia, blood pressure, insulin resistance index, the production of superoxide anion in the aorta and the density of B1 receptor binding sites in the dorsal horn were significantly increased in the two models. These effects were prevented or attenuated by alpha-lipoic acid. In contrast, B2 receptor binding sites of most spinal cord laminae were increased in the glucose group only and were not affected by alpha-lipoic acid. Results show that chronic hyperglycemia associated with insulin resistance increases B1 and B2 receptor binding sites in the rat spinal cord through distinct mechanisms, including the oxidative stress for the B1 receptor.
Keywords: Bradykinin; B1 receptor; B2 receptor; Spinal cord; Diabetes mellitus; Hyperglycemia; Insulin resistance; Hypertension; Oxidative stress;

Effects of alpha-lipoic acid on kinin B1 and B2 receptor binding sites in the spinal cord of chronically angiotensin-treated rats by Mihai Petcu; Brice Ongali; Adil El Midaoui; Jacques de Champlain; Réjean Couture (1331-1338).
A quantitative autoradiographic study was performed to determine whether kinin receptors are altered in the rat spinal cord in an experimental model of arterial hypertension under antioxidant therapy with alpha-lipoic acid. Sprague–Dawley rats were fed for 4 weeks with a normal chow diet or with an alpha-lipoic acid supplemented diet (1000 mg/kg feed), and treated for the last 2 weeks with angiotensin II (AT II) (200 ng/kg/min with an osmotic pump implanted s.c.). Control rats received either diet but not AT II. A 2-week administration of AT II increased significantly systolic blood pressure, the production of superoxide anion in the aorta and B1 receptor binding sites in the thoracic spinal dorsal horn. This treatment did not affect spinal B2 receptor binding sites, glycemia and insulinemia. The diet supplemented with alpha-lipoic acid reduced significantly the increase in systolic blood pressure, the production of aortic superoxide anion and prevented the increases of B1 receptor binding sites. Results show an association between the oxidative stress and the increases of B1 receptors and arterial blood pressure induced by AT II. Data also exclude the possibility that arterial hypertension is a primary mechanism leading to an increase of B2 receptor binding sites in the rat spinal cord.
Keywords: Bradykinin; B1 receptor; B2 receptor; Spinal cord; Arterial hypertension; Oxidative stress;

Kinin and opioid receptors in the paratrigeminal nucleus modulate the somatosensory reflex to rat sciatic nerve stimulation by Janice Koepp; Cristofer André Caous; Giles Alexander Rae; Antonio Claudir Balan; Charles Julian Lindsey (1339-1345).
The influence of kinin and opioid receptor blockade in the paratrigeminal nucleus (Pa5) on the somatosensory reflex (SSR) to sciatic nerve stimulation (SNS) was assessed in anaesthetized–paralyzed rats. SNS (square 1 ms pulses at 0.6 mA and 20 Hz for 10 s) increased mean arterial pressure from 87 ± 3 to 106 ± 3 mmHg. Pressor responses to SNS were reduced 40–60% by HOE-140 and LF 16-0687 (B2 receptor antagonists; 20 and 100 pmol respectively), CTOP or nor-binaltorphimine (mu and kappa opioid receptor antagonists, respectively; 1 μg) but potentiated by naltrindole (delta opioid receptor antagonist) receptor antagonist microinjections into the contralateral (but not ipsilateral) Pa5. The SSR to sciatic nerve stimulation was not changed by B1 kinin receptor or NK1, NK2 and NK3 tachykinin receptor antagonists administered to the Pa5. Capsaicin pretreatment (40 mg/kg/day, 3 days) abolished the effects of the opioid receptor antagonists, but did not change the effect of kinin B2 receptor blockade on the SSR. Thus, the activity of B2 and opioid receptor-operated mechanisms in the Pa5 contribute to the SSR in the rat, suggesting a role for these endogenous peptides in the cardiovascular responses to SNS.
Keywords: Bradykinin; Blood pressure; Capsaicin-sensitive fibers; Cardiovascular reflex; Sciatic nerve; Nociception;

Chronic opioid use in the management of pain is limited by development of analgesic tolerance and physical dependence. The mechanisms underlying tolerance-dependence are not entirely clear, however, recent evidence suggests that spinal adaptations leading to increased activity of sensory neuropeptides (calcitonin gene-related peptide (CGRP), substance P) and their downstream signaling messengers derived from metabolism of arachidonic acid: prostaglandins (PG), lipoxygenase (LOX) metabolites, and endocannabinoids, plays an important role in this phenomenon. In this communication we review the evidence implicating these factors in the induction and expression of opioid tolerance and physical dependence at the spinal level.
Keywords: Opioid tolerance; Opioid physical dependence; Calcitonin gene-related peptide; Substance P; Cyclooxygenase; Lipoxygenase; Endocannabinoids;

There is an expanding repertoire of mammalian tachykinins produced by a variety of tachykinin genes, gene splicing events and peptide processing. Novel tachykinin-binding molecules/receptors are proposed, but only, three tachykinin receptors are identified with certainty. The question remains – do more tachykinin receptors exist or is there just the need to reappraise our understanding of the known receptors? The tachykinin NK1 receptor, the preferred receptor for both substance P and the peripheral SP-like endokinins, exists in several tissue-specific conformations and isoforms and may provide some clues. This review addresses recent advances in this exciting field and raises challenging new concepts.
Keywords: Endokinin; Hemokinin; Tachykinin; Neurokinin; Peptide; Receptor; Long; Short; Isoform; Substance P; Tachykinin NK1 receptor; Virokinin;

The tachykinins form one of the largest peptide families in nature. In this review, we describe the comparative features of the tachykinin peptides and their receptors, focusing particularly on amphibians. We also summarize our systematic studies of the localization, characteristics, and actions of bufokinin, a toad substance P-related peptide, in its species of origin. In addition, we discuss the establishment of multiple isoforms of the NK1-like receptor in the toad, and their structure, pharmacology and tissue distributions. We conclude that tachykinin peptides and receptors are well conserved in terms of their structures, physiological functions and coupling mechanisms during tetrapod evolution.
Keywords: Tachykinins; Bufokinin; Tachykinin receptors; Amphibian; Intestine; Cardiovascular system;

Neurokinin-1 (NK1) receptor antagonists have been reported to possess antidepressant and anxiolytic properties in controlled trials. Since antidepressant and anxiolytic drugs act mainly by enhancing serotonin (5-HT) and norepinephrine (NE) neurotransmission in forebrain areas, the main focus of the present review is to critically examine the electrophysiological effects of NK1 receptor antagonists on serotoninergic and noradrenergic neurons, and then hippocampal neurons. It is concluded that NK1 antagonists increase the firing and burst activity of 5-HT neurons, increase burst activity of NE neurons, and modulate postsynaptic transmission at the hippocampus level. Further research is needed in order to develop more selective ligands for the human NK1 receptor and to gain better knowledge of required brain penetration and optimal pharmacodynamic conditions for their use in patients.
Keywords: Antidepressant; Anxiolytic; Substance P; Antagonist of SP; NK1 receptor; Depression; Anxiety;

Role of serotonin (5-HT) in the antidepressant-like properties of neuropeptide Y (NPY) in the mouse forced swim test by John P. Redrobe; Yvan Dumont; Alain Fournier; Glen B. Baker; Rémi Quirion (1394-1400).
Neuropeptide Y (NPY) is thought to be implicated in depressive disorders. The mouse forced swim test (FST) is an animal model widely used as a predictor of the efficacy of antidepressant drugs. The present study was undertaken to explore the possible contribution of endogenous serotonin (5-HT) systems in the behavioral effects elicited by NPY in this model. The selective serotonin re-uptake inhibitor (SSRI), fluoxetine, was also tested for comparison. 5-HT was depleted prior to testing by the administration of the tryptophan hydroxylase inhibitor p-chlorophenylalanine (PCPA; 300 mg/kg, i.p., each day for 3 days; control mice received saline-vehicle over the same period). On the fourth day, mice received NPY (3 nmol, I.C.V.), fluoxetine (16 mg/kg, i.p.) or saline injections before testing in the FST. Both NPY and fluoxetine significantly reduced immobility time in saline-treated control animals. Pre-treatment with PCPA significantly blocked the effects of fluoxetine in the FST, confirming the role of endogenous 5-HT. Similarly, pre-treatment with PCPA also significantly attenuated the anti-immobility effects of NPY, thus suggesting a role for 5-HT in the effects of NPY in the FST. Quantitative receptor autoradiography revealed increases in specific [125I][Leu31, Pro34]PYY sites that were sensitive to BIBP3226 (Y1-like sites) in various brain regions. Specific [125I]GR231118 and [125I]PYY(3–36) binding levels were not changed following PCPA treatment, suggesting that depletion of endogenous 5-HT resulted in an apparent increase in the level of Y1 sites in their high-affinity state. Taken together, these results suggest a role for 5-HT-related systems in the antidepressant-like properties of NPY.
Keywords: NPY receptor subtypes; Forced swim test; Serotonin depletion; Receptor autoradiography; Mouse brain;

The AT2 receptor—A matter of love and hate by U.M. Steckelings; E. Kaschina; Th. Unger (1401-1409).
In 1989, the development of specific angiotensin receptor antagonists which distinguish between two angiotensin receptor subtypes (AT1 and AT2) led to a breakthrough in angiotensin research. It turned out, that the AT1 receptor was almost entirely responsible for the “classical” actions of angiotensin II related to the regulation of blood pressure as well as volume and electrolyte balance. However, actions and signal transduction mechanisms coupled to the AT2 receptor remained enigmatic for a long time. The present review summarizes the current knowledge of AT2 receptor distribution, signaling and function with an emphasis on growth/anti-growth, differentiation and the regeneration of neuronal tissue.
Keywords: Angiotensin; AT2 receptor; Renin–angiotensin-system;

Angiotensin II-induced increase of T-type Ca2+ current and decrease of L-type Ca2+ current in heart cells by Ghassan Bkaily; Adrian Sculptoreanu; Shimin Wang; Moni Nader; Khaled M. Hazzouri; Danielle Jacques; Domenico Regoli; Pedro D’Orleans-Juste; Levon Avedanian (1410-1417).
The effect of angiotensin II (Ang II) on the T- and L-type calcium currents (I Ca) in single ventricular heart cells of 18-week-old fetal human and 10-day-old chick embryos was studied using the whole-cell voltage clamp technique. Our results showed that in both, human and chick cardiomyocytes, Ang II (10−7  M) increased the T-type calcium current and decreased the L-type I Ca. The effect of Ang II on both types of currents was blocked by the AT1 peptidic antagonist, [Sar1, Ala8] Ang II (2 × 10−7  M). Protein kinase C activator, phorbol 12,13-dibutyrate, mimicked the effect of Ang II on the T- and L-type calcium currents. These results demonstrate that in fetal human and chick embryo cardiomyocytes Ang II affects the T- and L-type Ca2+ currents differently, and this effect seems to be mediated by the PKC pathway.
Keywords: Angiotensin II; Calcium currents; Heart cells; AT1 receptor;

Angiotensin II induced increase in frequency of cytosolic and nuclear calcium waves of heart cells via activation of AT1 and AT2 receptors by Ghassan Bkaily; Nesrine El-Bizri; Moni Nader; Khaled M. Hazzouri; Julie Riopel; Danielle Jacques; Domenico Regoli; Pedro D’Orleans-Juste; Fernand Gobeil; Levon Avedanian (1418-1426).
The aim of this work is to verify if Angiotensin II (Ang II) affects the frequency of spontaneous cytosolic and nuclear Ca2+ waves in chick embryonic cardiomyocytes and if this effect is mediated via the activation of AT1 and/or AT2 receptors. Using the rapid scan technique of confocal microscopy, we observed that Ang II (10−8  M) increases the frequency of cytosolic and nuclear Ca2+ waves. This effect was accompanied by a decrease in the amplitude of nuclear Ca2+ waves and an absence of effect on the amplitude of cytosolic Ca2+ waves. The effect of the octapeptide on both frequency and amplitude of the nuclear waves was prevented by the AT1 receptor antagonist L158809. However, blockade of the AT2 receptor using the antagonist PD123319 (10−7  M) only prevented the effect of Ang II on the frequency of Ca2+ waves. Furthermore, the effect was prevented by both a PKC inhibitor (bisindolylmaleimide) and a PKC activator (phorbol 12,13-dibutyrate). In addition, the Ang II effect was not prevented by the blocker of the pacemaker current I f. These results demonstrate that Ang II, via the activation of its receptors AT1 and AT2, affects the frequency of spontaneous Ca2+ waves and this effect seems to be mediated by the PKC pathway.
Keywords: Calcium waves; Heart; Angiotensin II; Angiotensin II receptors;

The distribution and density of ET-1 and its receptors are different in human right and left ventricular endocardial endothelial cells by Danielle Jacques; Magda Descorbeth; Dima Abdel-Samad; Chantale Provost; Claudine Perreault; Farah Jules (1427-1435).
Evidence suggests that endocardial endothelial cells (EECs) may play a role in the regulation of cardiac function by releasing ET-1. Furthermore, reports in the literature suggested that differences may exist in peptide receptor distribution between the left and right EECs. In this study, we verified if the distribution and density of ET-1 and its receptors could be different in right as compared to left ventricular EECs, and whether this difference may affect ET-1-induced increase of intracellular calcium. Using immunofluorescence and 3D confocal microscopy, our results showed that in both cell types, the ETA receptor is present and is homogeneously distributed throughout the two cell types. The relative density of the ETA receptor is similar in both right and left ventricular EECs. The ETB receptor is also present in right and left ventricular EECs, however, the relative density of the ETB receptor is higher in the nucleus as compared to the cytosol. In addition, the ETB receptor density was found to be higher in left EECs as compared to right EECs. In addition, our results showed that ET-1 is present in the cytosol and the nucleus of both types of cells and that the relative density of ET-1 is higher in right as compared to left ventricular EECs. Moreover, using the Fura-2 calcium measurement technique, our results showed that in left ventricular EECs, both ETA and ETB receptor activation mediated the effect of ET-1 on intracellular calcium, whereas in right ventricular EECs, this effect was solely mediated by the ETA receptor. In conclusion, our results showed that ET-1 and its receptors are present in both right and left ventricular EECs. However, the distribution and relative density of ET-1 and its receptors seem to be different in right EECs as compared to left EECs.
Keywords: Endocardial endothelium; Endothelin-1; ETA receptor; ETB receptor; Intracellular calcium;

PTHrP fragments 1-16 and 1-23 do not bind to either the ETA or the ETB endothelin receptors by Chantal Langlois; Myriam Létourneau; Kathy Turcotte; Michel Detheux; Alain Fournier (1436-1440).
Because of some isofunctional similarities with endothelin-1 (ET-1), it has been suggested that PTHrP(1-16) and PTHrP(1-23) could interact with osteoblast cells via ETA receptors. To document this interaction, we used the thoracic rat aorta and the guinea-pig lung parenchyma paradigms as ETA and ETB models, respectively. In addition, we also performed a series of competition experiments against [125I]ET-1, using transfected cells expressing the ETA or ETB receptor. So far, no agonistic nor antagonistic activities were observed in the ETA and ETB bioassays with the PTHrP fragments. Furthermore, both fragments were unable to displace [125I]ET-1 bound to cells expressing the ETA or ETB receptor.
Keywords: Endothelin; Parathyroid hormone-related protein (PTHrP); ETA; ETB; Breast cancer; Prostate cancer;

Development of an efficient strategy for the synthesis of the ETB receptor antagonist BQ-788 and some related analogues by Jean-Philippe Brosseau; Pedro D’Orléans-Juste; Witold A. Neugebauer (1441-1453).
BQ-788 [N-cis-2,6-dimethylpiperidine-1-carbonyl-l-γ-methylleucyl-d-1-methoxycarbonyltryptophanyl-d-norleucine sodium salt] is a very potent and selective ETB receptor antagonist. The formation of the highly hindered trisubstituted urea functionality in the peptide chain and the carbamination on the indole nitrogen of the tryptophan side chain are major challenges in the synthesis of this particular antagonist. Furthermore, the high cost of the unnatural amino acids in the sequence of BQ-788 and its reported synthesis render this pseudopeptide very expensive to produce. In order to improve the yield and to reduce the number of steps compared to previous reported syntheses, we developed an efficient strategy involving a novel one-pot procedure for the synthesis of a highly hindered trisubstituted urea. Under very mild conditions, the urea was obtained by using triphosgene and sodium iodide. This strategy allowed us to synthesize BQ-788 in seven steps with an overall yield of 53%. We also generalized the use of this powerful methodology by creating some new structural analogues of the cis-2,6-dimethylpiperidine moiety by replacing it with other bulky secondary amines. We evaluated the antagonist properties of those three new analogues of BQ-788 in two bioassays in vitro. These new antagonists were less potent than BQ-788 in an ETB rich preparation and inactive in an ETA rich preparation.
Keywords: BQ-788; ETB antagonist; Trisubstituted urea; Carbamoyl chloride; Triphosgene; IIDQ;

Endothelin-1 contributes to the sexual differences in renal damage in DOCA-salt rats by A.C.I. Montezano; G.E. Callera; A.L. Mota; Z.B. Fortes; D. Nigro; M.H.C. Carvalho; T.M.T. Zorn; R.C. Tostes (1454-1462).
We investigated whether gender differences in renal damage in DOCA-salt hypertension are associated with effects of ovarian hormones and/or endothelin-1 (ET-1). Renal injuries and renal pre-pro-ET-1 mRNA expression were enhanced in male and female ovariectomized (OVX) DOCA rats versus female DOCA rats. Treatment with estrogen plus progesterone or progesterone, but not estrogen alone, attenuated renal damage and pre-pro-ET-1 mRNA expression in OVX DOCA rats. The ETA antagonist BMS182874 greatly ameliorated renal damage in male and OVX DOCA rats. In conclusion, the ovarian hormones have a protective role on the renal structural alterations in female DOCA rats by modulating effects of ET-1, via ETA receptors.
Keywords: DOCA-salt hypertension; Endothelin; ETA receptor antagonist; Structural changes; Renal injury; Ovarian hormones; Estrogen; Progesterone;

Low-affinity state beta1-adrenoceptor-induced vasodilation in SHR by Mohamed Yassine Mallem; Olivier Reculeau; Olivier Le Coz; Marc Gogny; Jean-Claude Desfontis (1463-1467).
Low-affinity state beta1-adrenoceptor (β1-AR) was functionally expressed in some blood vessels and was different from β1, β2 and β3-AR. In rat aorta, low-affinity state β1-AR activation produced an endothelium-independent relaxation which was impaired in spontaneously hypertensive rats (SHRs). In the present work, we investigated whether renin–angiotensin system was involved in this alteration by evaluating the effects of enalapril, an angiotensin converting enzyme (ACE) inhibitor or losartan, an AT1 angiotensin receptor antagonist. Cumulative concentration–response curves to low-affinity state β1-AR agonists (CGP 12177, cyanopindolol or alprenolol) and to NS 1619, a large conductance Ca2+-activated K+ channels (BK) agonist were performed in denuded aortic rings isolated from control or treated Wistar Kyoto (WKY) rats or SHRs in different experimental conditions. The low-affinity state β1-AR-mediated aortic vasodilation was impaired in 5 and 12 weeks old SHRs when compared to age-matched WKY. Twelve days enalapril (5 mg/kg/day) or losartan (15 mg/kg/day) treatments reduced systolic blood pressure (SBP) only in 12 weeks old SHRs whereas no significant change was observed in other groups. These treatments improved low-affinity state β1-AR effect only in SHRs groups. In 12 weeks old WKY rats, CGP 12177-induced relaxation was insensitive to glibenclamide, a K ATP + channel blocker, but was reduced by TEA or iberiotoxin, two large conductance Ca2+-activated K+ channel (BK) blockers. The impairment of NS 1619-induced vasodilation in both 5 and 12 weeks old SHRs was restored by enalapril or losartan. These results suggested that improvement of the low-affinity state β1-AR-mediated vasodilation in 5 and 12 weeks old SHRs could be attributed to enhanced BK channels-induced hyperpolarization in SHRs independently of lowering of SBP.
Keywords: Low-affinity state; Beta-adrenoceptor; SHR; Potassium channel; Vasodilation; Enalapril; Losartan;

Urotensin II-induced hypotensive responses in Wistar–Kyoto (Wky) and spontaneously hypertensive (Shr) rats by Gabrielle Gendron; Fernand Gobeil; Simon Bélanger; Sandra Gagnon; Domenico Regoli; Pedro D’Orléans-Juste (1468-1474).
Human urotensin II (hU-II) is a potent vasoactive peptide which modulates some of the functions of the cardiovascular and other systems. The in vivo mechanism of action by which hU-II may influence blood pressure in developmental and pathological conditions, is poorly understood. Herein, the blood pressure effects of hU-II (0.1–10 nmol/kg) injected intravenously (i.v.) were studied on ketamine/xylazine anesthetized male WKY and SHR rats aged 4 and 8 weeks. hU-II elicited dose-dependent decreases in mean arterial pressure in both strains of animals. The hypotensive responses to hU-II were, however, significantly higher in SHR rats, independently of age. Four-week-old SHR rats (which are normotensive) were, however, less responsive than their hypertensive 8-week-old counterparts. A series of pharmacological inhibitors were used to identify putative endogenous (endothelial) factors that might account for the hU-II-mediated hypotension in 8-week-old SHR. These include the non-selective nitric oxide synthase inhibitor l-NAME (5 μmol/kg), the non-selective cyclooxygenase inhibitor meclofenamate (16 μmol/kg), the voltage-sensitive and ATP-sensitive K+-channel inhibitors, 4-aminopyridine (5 μmol/kg) and glybenclamide (10 μmol/kg), the cytochrome P450 CYP2C9 inhibitor sulfaphenazole (15 μmol/kg), the cytoskeletal fixation agent phalloidin (15 μmol/kg), the endothelin ETB receptor antagonist BQ-788 (35 μmol/kg), the bradykinin B2 receptor antagonist HOE 140 (0.5 μmol/kg), the angiotensin AT2 antagonist PD 123319 (10 μmol/kg) and the UT receptor antagonist urantide (10 μmol/kg). These agents were administered i.v. either at 2.5, 10 or 40 min prior hU-II injection (10 nmol/kg). Among these inhibitors, sulfaphenazole and phalloidin were able to reduce hU-II-induced hypotension. This suggests that the vasodepressor effect of hU-II is mediated by UT receptors and relies in part on the release of epoxide related products; increased microvascular permeability may also contribute to the blood pressure lowering effect of hU-II. Since urantide blocks the constrictor effects of hU-II on isolated aorta, but is inactive against the hypotensive action of hU-II in vivo, the results presented in this paper provide, for the first time, evidence for the existence of two different functional sites for hU-II.
Keywords: Blood pressure; Spontaneously hypertensive rat; Human urotensin; Vasodilatation inhibitors and antagonists;

Development and pharmacological characterization of “caged” urotensin II analogs by Steve Bourgault; Myriam Létourneau; Alain Fournier (1475-1480).
Urotensin-II (U-II) is a cyclic 11-amino acid peptide known as a potent mammalian vasoconstrictor. To study some purported intracellular actions of U-II, masked analogs of this peptide, becoming biologically active only upon UV exposure, were developed. Those analogs described as “caged” were derivatized with a photolabile 4,5-dimethoxynitrobenzyl group on the side chain of Lys-8 or Tyr-9. Both caged analogs of U-II showed a major decrease in their affinity towards the UT receptor. Nevertheless, upon UV irradiation, the native and biologically active U-II peptide was recovered. Thus, this work describes the development of new “caged” U-II derivatives and demonstrates that vasoactivity of U-II can be controlled by masking and unmasking two key residues.
Keywords: Urotensin-II; Caged; Photolabile; Dimethoxynitrobenzyl;

Design of novel melanotropin agonists and antagonists with high potency and selectivity for human melanocortin receptors by Minying Cai; Alexander V. Mayorov; Jinfa Ying; Magda Stankova; Dev Trivedi; Chris Cabello; Victor J. Hruby (1481-1485).
α-MSH and γ-MSH are the natural endogenous hormones for the human melanocortin-1, 3, 4 and 5 receptors (hMC1R, hMC3R, hMC4R and hMC5R). These and more potent, stable and prolonged acting analogues such as NDP-α-MSH, MT-II and SHU-9119 are not very receptor selective. To develop potent and selective agonist and antagonist ligands for the melanocortin receptors we have used state-of-the-art biophysical studies, computational chemistry, and design of conformational and topographical constraints with novel templates.
Keywords: Melanotropins; Melanocortin receptors; Structure-biological activity relationships; Conformation constraints; Cyclic melanotropins; Receptor selective melanotropin agonists; Receptor selective melanotropin antagonists; Melanocortin receptor pharmacophores;

Under congenital thyroid hypofunction, the oro-facial large and small calcitonin gene-related peptide (CGRP) immunoreactive motoneurons were classified into strong, moderate, weak and negative intensity in offspring weaned rats. While 50% of neurons in the trigeminal motor nucleus (Mo5) were of the large type, this value dropped to 30% in hypothyroid pups. Hypothyroid trigeminal accessory nucleus (Mo5-AC) contained 10% large motoneurons versus about 45% in normal pups. Normal facial nucleus (Mo7) had 20% large motoneurons in contrast with 10% in hypothyroid pups. These values are significant in comparison with the normal pattern of oro-facial CGRP positive immunoreactive motoneurons as well as those devoid of immunostaining.
Keywords: CGRP immunoreactivity; Jaw closing; Jaw opening; Facial; Motoneurons; Hypothyroidism; Weaned rats;

Nociceptin/orphanin FQ inhibits electrically induced contractions of the human bronchus via NOP receptor activation by Marco Basso; Paul André Risse; Emmanuel Naline; Girolamo Calo; Remo Guerrini; Domenico Regoli; Charles Advenier (1492-1496).
Nociceptin/orphanin FQ (N/OFQ) has been reported to inhibit neurogenic contractions in various tissues, including guinea pig airways. In the present study, we investigated the ability of N/OFQ to affect cholinergic contractions of human bronchi elicited by electrical field stimulation (EFS). Tissues were obtained from 23 patients undergoing surgery for lung cancer. EFS (20 Hz, 320 mA, 1.5 ms, 10 s) was applied five times every 20 min. Contractions induced by EFS were abolished by either TTX (1 μM) or atropine (1 μM) and concentration-dependently (10 nM–1 μM) inhibited by N/OFQ (E max, 11.5 ± 1.8% inhibition). The inhibitory effects of N/OFQ were mimicked by the N/OFQ receptor (NOP) ligand [Arg14, Lys15]N/OFQ which displayed however, higher significant maximal effects (17.7 ± 2.9% inhibition, P  < 0.05). The actions of N/OFQ and [Arg14, Lys15]N/OFQ were not affected by naloxone (1 μM) while prevented by the selective NOP receptor antagonist UFP-101 (10 μM). Moreover, the inhibitory effects of NOP agonists were no longer evident in tissues treated with tertiapin (10 μM), an inhibitor of inward-rectifier potassium channels. In conclusion, the present data demonstrate that N/OFQ inhibited acetylcholine (ACh) release in the human bronchi via NOP receptor activation. This effect may involve stimulation of potassium currents.
Keywords: Nociceptin/orphanin FQ; NOP receptor; Human bronchus; Cholinergic neurotransmission; Electrical field stimulation; Opioids; Airways;

Nociceptin/orphanin FQ stimulates human monocyte chemotaxis via NOP receptor activation by S. Trombella; R. Vergura; S. Falzarano; R. Guerrini; G. Calo; S. Spisani (1497-1502).
Nociceptin/orphanin FQ (N/OFQ) produces several biological actions by activating the N/OFQ peptide receptor (NOP). It has been previously shown that N/OFQ stimulates leukocyte chemotaxis both in vitro and in vivo. In the present study we investigated the ability of N/OFQ, in comparison with the proinflammatory peptide formyl-Met-Leu-Phe (fMLP), to stimulate human neutrophil and monocyte chemotaxis and the release of lysozyme and superoxide anion (O2 ) production from neutrophils. fMLP stimulated all the leukocyte functions examined. N/OFQ stimulated monocyte (pEC50 12.15) but not neutrophil chemotaxis. The production of O2 from neutrophils was not affected by N/OFQ while the release of lysozyme was increased in a concentration dependent manner (pEC50 11.00) although the maximal effects evoked by N/OFQ were about half of those of fMLP. The NOP ligands [Arg14, Lys15]N/OFQ, N/OFQ(1-13)NH2, Ro 64-6198, UFP-101 and the opioid antagonist naloxone were used for pharmacologically characterizing the receptor involved in the monocyte chemoattractant action of N/OFQ. [Arg14, Lys15]N/OFQ, N/OFQ(1-13)NH2, and Ro 64-6198 mimicked the action of N/OFQ showing similar maximal effects and the following order of potency: [Arg14, Lys15]N/OFQ (pEC50 13.22) > Ro 64-6198 (pEC50 12.96) > N/OFQ(1-13)NH2 (pEC50 12.67) > N/OFQ (pEC50 12.15). Moreover, the monocyte chemoattractant action of N/OFQ was not modified by naloxone 1 μM while antagonized by UFP-101 10 μM (pA2 7.00). Thus, the order of potency of agonists and the antagonist selectivity demonstrated that N/OFQ stimulates human monocyte chemotaxis via NOP receptor activation.
Keywords: Nociceptin/orphanin FQ; fMLP; Human NOP receptor; Monocytes; Neutrophils; Chemotaxis;

We studied the expression of the osteogenic and antinociceptive C-terminal histone H4-related peptide fragments, H4-(89–102) (OGP) and H4-(86–100), respectively, within various rat tissues and isolated alveolar macrophages (AM) by radioimmunoassay (RIA). OGP was located mainly within the bone marrow, spleen, thymus, and lungs whereas H4-(86–100) was more concentrated within the bone marrow, lymph nodes, spinal cord, pituitaries and thymus. The expression pattern of the two peptides showed similarities with the tissue expression pattern of the histone H4 mRNA variant H4-v.1. In rat AM, OGP and H4-(86–100) levels were significantly stimulated (2.6- and 1.9-fold, respectively) by LPS (1 μg/ml), along with H4-v.1 mRNA (4.1-fold), but not whole histone H4 (1.1-fold) nor total histone H4 mRNA (1.1-fold). The results suggest that H4-v.1 mRNA may play a role in the synthesis of the naturally occurring peptides H4-(86–100) and OGP via the alternative translation product H4-(84–102), but not whole histone H4.
Keywords: Histone H4 fragments; Osteogenic growth peptide; Histogranin; Peptide processing; H4-v.1 mRNA variant; Radioimmunoassay;

Suppression of fat deposition for the life time with gene therapy by Stéphane Boghossian; Anne Lecklin; Rita Torto; Pushpa S. Kalra; Satya P. Kalra (1512-1519).
Unexpended energy is stored as fat in the body and increased rate of fat accretion culminates in obesity. Obesity increases the risks of many diseases several folds and shortens life span. A progressive deficit in the central feedback effects of leptin, a peptide produced by fat cells and hypothalamus, results in increased weight gain and obesity. This article summarizes our experimental findings to show that a stable increase in leptin availability in the hypothalamus alone with the aid of leptin gene therapy suppresses fat accretion and metabolic hormones for nearly the lifetime of laboratory rodents. Consequently, central leptin gene therapy is a novel modality that offers a viable therapeutic option to reduce fat depots and attendant metabolic sequelae implicated in obesity-related illnesses.
Keywords: Leptin; Gene therapy; Obesity; Lifetime;

The influence of the peptide NAP on Mac-1-deficient mice following closed head injury by Roy Zaltzman; Alexander Alexandrovich; Victoria Trembovler; Esther Shohami; Illana Gozes (1520-1527).
A single administration of the neuroprotective peptide NAP was previously shown to protect against death associated with closed head injury (CHI) and enhance recovery of the surviving mice. The protective effect was accompanied by down-regulation of the relative mRNA content of the complement receptor 3 (Mac-1, a marker for inflammation) as measured about a month after the injury. In contrast, the mRNA transcripts for activity-dependent neuroprotective protein (ADNP, the NAP containing protein) were shown to increase 29 days post CHI in the injured hemisphere of Mac-1 expressing mice. The present study was set out to investigate: (1) are Mac-1-deficient mice less susceptible to the adverse outcome of traumatic head injury; (2) does NAP treatment affect Mac-1-deficient mice subjected to head injury; and (3) is Mac-1 expression associated with ADNP expression. Results showed that (1) Mac-1-deficient mice were partially protected against death associated with severe head injury as compared to Mac-1 expressing mice. (2) Significant protection against death was observed in NAP-treated mice and an increase in recovery was observed in the NAP-treated Mac-1 mice 4 weeks after injury. (3) ADNP expression did not change in the Mac-1-deficient mice following head injury. Our working hypothesis is that a month following injury, gene expression in the injured brain is altered and competing proteins are expressed such as Mac-1 that is associated with inflammation and ADNP that is associated with neuroprotection. Obviously, this plasticity in gene expression is intimately interwoven with the genetic background of the animal. NAP treatment tilts the balance toward neuroprotection.
Keywords: Activity-dependent neuroprotective protein (ADNP); NAP; Mac-1; Closed head injury (CHI); Neurological severity score (NSS);

Peptide and non-peptide G-protein coupled receptors (GPCRs) in skeletal muscle by Gaël Jean-Baptiste; Zhao Yang; Chamel Khoury; Sabrina Gaudio; Michael T. Greenwood (1528-1536).
G-protein coupled receptors (GPCRs) represent a large class of cell surface receptors that mediate a multitude of functions. Over the years, a number of GPCRs and ancillary proteins have been shown to be expressed in skeletal muscle. Unlike the case with other muscle tissues like cardiac and vascular smooth muscle cells, there has been little attempt at systematically analyzing GPCRs in skeletal muscle. Here we have compiled all the GPCRs that are expressed in skeletal muscle. In addition, we review the known function of these receptors in both skeletal muscle tissue and in cultured skeletal muscle cells.
Keywords: Skeletal muscle; G-protein coupled receptors; C2C12 cells; L6 cells;