Cytotoxicity of Extracts from Petiveria alliacea Leaves on Yeast.

Petiveria alliacea L. is a plant used in traditional medicine harboring pharmacological properties with anti-inflammatory, antinociceptive, hypoglycemiant and anesthetic activities. This study assessed the potential cytotoxic, genotoxic and mutagenic effects of ethanolic extract of P. alliacea on Saccharomyces cerevisiae strains. S. cerevisiae FF18733 (wild type) and CD138 (ogg1) strains were exposed to fractioned ethanolic extracts of P. alliacea in different concentrations. Three experimental assays were performed: cellular inactivation, mutagenesis (canavanine resistance system) and loss of mitochondrial function (petites colonies). The chemical analyses revealed a rich extract with phenolic compounds such as protocatechuic acid, cinnamic and catechin epicatechin. A decreased cell viability in wild-type and ogg1 strains was demonstrated. All fractions of the extract exerted a mutagenic effect on the ogg1 strain. Only ethyl acetate and n-butanol fractions increased the rate of petites colonies in the ogg1 strain, but not in the wild-type strain. The results indicate that fractions of mid-polarity of the ethanolic extract, at the studied concentrations, can induce mutagenicity mediated by oxidative lesions in the mitochondrial and genomic genomes of the ogg1-deficient S. cerevisiae strain. These findings indicate that the lesions caused by the fractions of P. alliacea ethanolic extract can be mediated by reactive oxygen species and can reach multiple molecular targets to exert their toxicity.

Chitosan gels for buccal delivery of Schinus molle L. essential oil in dogs: characterization and antimicrobial activity in vitro.

Periodontal disease is considered the main oral cavity disorder in dogs. Essential oils have the potential for use in the prevention and treatment of oral diseases. The antimicrobial activity of Schinus molle L. essential oil (SMEO) has already been reported. Chitosan, a natural product with antimicrobial activity and good biocompatibility has potential in biodental applications. In this study, we evaluated the in vitro antimicrobial activity of SMEO against bacteria associated with periodontal disease in dogs, developed and evaluated the physicochemical properties of a novel chitosan-based buccal delivery system containing SMEO. SMEO showed antimicrobial activity against Gram positive and Gram negative bacteria associated with canine periodontitis, with MIC values of 750 µg.mL-1 for Staphylococcus spp. and Streptococcus spp, 1000 µg.mL-1 for Corynebacterium spp. and 1250 µg.mL-1 for Pseudomonas spp. All formulations evaluated presented adequate physicochemical properties, good stability, and pH values close to buccal pH (5.0-7.0). Chitosan gel loaded with SMEO showed potential as a SMEO delivery system, having the ideal physicochemical and rheological properties (pseudoplastic and apparent viscosities) required for application on buccal tissue. Thus, we can conclude that formulation has the potential to be used for buccal mucosa delivery in the prevention and treatment of periodontal disease in dogs.

Comparative bioinformatic and structural analyses of pepsin and renin.

Pepsin, the archetypal pepsin-like aspartic protease, is irreversibly denatured when exposed to neutral pH conditions whereas renin, a structural homologue of pepsin, is fully stable and optimally active in the same conditions despite sharing highly similar enzyme architecture. To gain insight into the structural determinants of differential aspartic protease pH stability, the present study used comparative bioinformatic and structural analyses. In pepsin, an abundance of polar and aspartic acid residues were identified, a common trait with other acid-stable enzymes. Conversely, renin was shown to have increased levels of basic amino acids. In both pepsin and renin, the solvent exposure of these charged groups was high. Having similar overall acidic residue content, the solvent-exposed basic residues may allow for extensive salt bridge formation in renin, whereas in pepsin, these residues are protonated and serve to form stabilizing hydrogen bonds at low pH. Relative differences in structure and sequence in the turn and joint regions of the ß-barrel and ψ-loop in both the N- and C-terminal lobes were identified as regions of interest in defining divergent pH stability. Compared to the structural rigidity of renin, pepsin has more instability associated with the N-terminus, specifically the B/C connector. By contrast, renin exhibits greater C-terminal instability in turn and connector regions. Overall, flexibility differences in connector regions, and amino acid composition, particularly in turn and joint regions of the ß-barrel and ψ-loops, likely play defining roles in determining pH stability for renin and pepsin.

In vitro efficacy of essential oils and extracts of Schinus molle L. against Ctenocephalides felis felis.

Extracts and essential oils from plants are important natural sources of pesticides. These compounds are considered an alternative to control ectoparasites of veterinary importance. Schinus molle, an endemic species of Brazil, produces a high level of essential oil and several other compounds. The aim of this work was to determinate the chemical composition of extracts and essential oils of S. molle and further to evaluate the activity against eggs and adults of Ctenocephalides felis felis, a predominant flea that infests dogs and cats in Brazil. In an in vitro assay, the non-polar (n-hexane) extract showed 100% efficacy (800 µg cm(-2); LD50 = 524·80 µg cm(-2)) at 24 and 48 h. Its major compound was lupenone (50·25%). Essential oils from fruits and leaves were evaluated, and had 100% efficacy against adult fleas at 800 µg cm(-2) (LD50 = 353·95 µg cm(-2)) and at 50 µg cm(-2) (LD50 = 12·02 µg cm(-2)), respectively. On the other hand, the essential oil from fruits and leaves was not active against flea eggs. This is the first study that reports the insecticidal effects of essential oils and extracts obtained from Schinus molle against Ctenocephalides felis felis.

Phenolic chemical composition of Petroselinum crispum extract and its effect on haemostasis.

From the aqueous extract (Pc) of Petroselinum crispum (Mill) flat leaves specimens were isolated and identified the flavonoids apigenin (1), apigenin-7-O-glucoside or cosmosiin (2), apigenin-7-O-apiosyl-(1 --> 2)-O-glucoside or apiin (3) and the coumarin 2",3"-dihydroxyfuranocoumarin or oxypeucedanin hydrate (4). The inhibitory activity toward clotting formation and platelet aggregation was assessed for Pc flavonoids (1) and (2), and the coumarin (4). Pc showed no inhibition on clotting activity when compared with the control. On the other hand, a strong antiplatelet aggregation activity was observed for Pc (IC50 = 1.81 mg/mL), apigenin (IC50 = 0.036 mg/mL) and cosmosiin (IC50 = 0.18 mg/mL). In all cases ADP was used as inductor of platelet aggregation. Our results showed that Pc, apigenin and cosmosiin interfere on haemostasis inhibiting platelet aggregation. To the best of our knowledge this is the first report for the cosmosiin antiplatelet aggregation in vitro activity.

Nicotinic stimulation of catecholamine synthesis and tyrosine hydroxylase phosphorylation in cervine adrenal medullary chromaffin cells.

The synthesis and secretion of catecholamines by the adrenal medulla is of major importance in the stress response. Tyrosine hydroxylase, the rate-limiting enzyme for catecholamine biosynthesis, has been extensively studied in adrenal medullary chromaffin cells from a number of species. Cervine chromaffin cells are of interest because the deer is known to be a relatively stress-prone reactive species. We report the first characterisation of tyrosine hydroxylase regulation in cervine chromaffin cells. Nicotinic receptor activation resulted in a time- and concentration-dependent increase in catecholamine synthesis, which was significantly reduced by the extracellular signal-regulated kinase (ERK)1/2 signalling pathway inhibitor PD98059 and the calcium/calmodulin protein kinase II inhibitor KN-93, but not by H89 or bisindolylmaleimide I, inhibitors of protein kinase A and C, respectively. Nicotinic stimulation also increased the phosphorylation of ERK1/2 and tyrosine hydroxylase. This latter response occurred on serine residues 19, 31 and 40 of the enzyme. The nicotinic-induced phosphorylation of ERK1/2 and serine 31 of tyrosine hydroxylase was suppressed by PD98059 but not bisindolylmaleimide I. These data indicate that nicotinic stimulation of tyrosine hydroxylase involves the phosphorylation of serine 31 via an ERK1/2-dependent, protein kinase C-independent pathway. Protein kinase C activation by phorbol 12-myristate 13-acetate also caused an ERK1/2-dependent increase in the serine 31 phosphorylation of tyrosine hydroxylase but, in contrast to the nicotinic response, was not accompanied by an increase in enzyme activity. Thus, ERK1/2-mediated serine 31 phosphorylation of tyrosine hydroxylase appears necessary but not sufficient for nicotinic activation of catecholamine synthesis in cervine chromaffin cells. These data present potentially important similarities and differences between the regulation of catecholamine synthesis in cervine and the more widely studied bovine adrenal medulla.

GSK1562590, a slowly dissociating urotensin-II receptor antagonist, exhibits prolonged pharmacodynamic activity ex vivo.

BACKGROUND AND PURPOSE: Recently identified antagonists of the urotensin-II (U-II) receptor (UT) are of limited utility for investigating the (patho)physiological role of U-II due to poor potency and limited selectivity and/or intrinsic activity. EXPERIMENTAL APPROACH: The pharmacological properties of two novel UT antagonists, GSK1440115 and GSK1562590, were compared using multiple bioassays. KEY RESULTS: GSK1440115 (pK(i)= 7.34-8.64 across species) and GSK1562590 (pK(i)= 9.14-9.66 across species) are high affinity ligands of mammalian recombinant (mouse, rat, cat, monkey, human) and native (SJRH30 cells) UT. Both compounds exhibited >100-fold selectivity for UT versus 87 distinct mammalian GPCR, enzyme, ion channel and neurotransmitter uptake targets. GSK1440115 showed competitive antagonism at UT in arteries from all species tested (pA(2)= 5.59-7.71). In contrast, GSK1562590 was an insurmountable UT antagonist in rat, cat and hUT transgenic mouse arteries (pK(b)= 8.93-10.12 across species), but a competitive antagonist in monkey arteries (pK(b)= 8.87-8.93). Likewise, GSK1562590 inhibited the hU-II-induced systemic pressor response in anaesthetized cats at a dose 10-fold lower than that of GSK1440115. The antagonistic effects of GSK1440115, but not GSK1562590, could be reversed by washout in rat isolated aorta. In ex vivo studies, GSK1562590 inhibited hU-II-induced contraction of rat aorta for at least 24 h following dosing. Dissociation of GSK1562590 binding was considerably slower at rat than monkey UT. CONCLUSIONS AND IMPLICATIONS: Whereas both GSK1440115 and GSK1562590 represent high-affinity/selective UT antagonists suitable for assessing the (patho)physiological role of U-II, only GSK1562590 exhibited sustained UT residence time and improved preclinical efficacy in vivo.

Interferon-alpha signalling in bovine adrenal chromaffin cells: involvement of signal-transducer and activator of transcription 1 and 2, extracellular signal-regulated protein kinases 1/2 and serine 31 phosphorylation of tyrosine hydroxylase.

Adrenal medullary chromaffin cells are an integral part of the neuroendocrine system, playing an important role in the physiological adaptation to stress. In response to a wide variety of stimuli, including acetylcholine released from the splanchnic nerve, hormones such as angiotensin II or paracrine signals such as prostaglandins, chromaffin cells synthesise and secrete catecholamines and a number of biologically active peptides. This adrenal medullary output mediates a complex and diverse stress response. We report that chromaffin cells also respond both acutely and chronically to interferon (IFN)-alpha, thus providing a mechanism of interaction between the immune system and the stress response. Incubation of isolated bovine chromaffin cells maintained in culture, with IFN-alpha resulted in a rapid, transient activation of the extracellular signal-regulated protein kinase (ERK)1/2, which was maximal after 5 min. IFN-alpha mediated activation of ERK1/2 appeared to be responsible for the increased phosphorylation of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis. This tyrosine hydroxylase phosphorylation was exclusively on serine 31, with no change in the phosphorylation of serine 19 or 40. This increase in the serine 31 phosphorylation of tyrosine hydroxylase was prevented by inhibition of protein kinase C or ERK1/2 activation. Incubation with IFN-alpha also resulted in a time- and concentration-dependent phosphorylation and nuclear translocation of signal transducer and activator of transcription proteins (STAT)1 and 2. This response was maximal after approximately 60 min. Prolonged treatment with IFN-alpha (12-48 h) resulted in increased expression of STAT1 and, to a lesser extent, STAT2. Thus, these findings demonstrate that adrenal medullary chromaffin cells are responsive to IFN-alpha and provide a possible cellular mechanism by which this immune-derived signal can potentially influence and integrate with the stress response.

Palosuran inhibits binding to primate UT receptors in cell membranes but demonstrates differential activity in intact cells and vascular tissues.

BACKGROUND AND PURPOSE: The recent development of the UT ligand palosuran (1-[2-(4-benzyl-4-hydroxy-piperidin-1-yl)-ethyl]-3-(2-methyl-quinolin-4-yl)-urea sulphate salt) has led to the proposition that urotensin-II (U-II) plays a significant pathological role in acute and chronic renal injury in the rat. EXPERIMENTAL APPROACH: In the present study, the pharmacological properties of palosuran were investigated further using a series of radioligand binding and functional bioassays. KEY RESULTS: Palosuran functioned as a 'primate-selective' UT ligand in recombinant cell membranes (monkey and human UT K(i) values of 4 +/- 1 and 5 +/- 1 nM), lacking appreciable affinity at other mammalian UT isoforms (rodent and feline K(i) values >1 microM). Paradoxically, however, palosuran lost significant (10- to 54-fold) affinity for native and recombinant human UT when radioligand binding was performed in intact cells (K(i) values of 50 +/- 3 and 276 +/- 67 nM). In accordance, palosuran also exhibited diminished activity in hUT (human urotensin-II receptor)-CHO (Chinese hamster ovary) cells (IC50 323 +/- 67 nM) and isolated arteries (K(b)>10 microM in rat aorta; K(b)>8.5 microM in cat arteries; K(b)>1.6 microM in monkey arteries; K(b) 2.2 +/- 0.6 microM in hUT transgenic mouse aorta). Relative to recombinant binding K(i) values, palosuran was subjected to a 392- to 690-fold reduction in functional activity in monkey isolated arteries. Such phenomena were peculiar to palosuran and were not apparent with an alternative chemotype, SB-657510 (2-bromo-N-[4-chloro-3-((R)-1-methyl-pyrrolidin-3-yloxy)-phenyl]-4,5-dimethoxybenzenesulphonamide HCl). CONCLUSIONS AND IMPLICATIONS: Collectively, such findings suggest that caution should be taken when interpreting data generated using palosuran. The loss of UT affinity/activity observed in intact cells and tissues cf. membranes offers a potential explanation for the disappointing clinical efficacy reported with palosuran in diabetic nephropathy patients. As such, the (patho)physiological significance of U-II in diabetic renal dysfunction remains uncertain.

The novel endocannabinoid receptor GPR55 is activated by atypical cannabinoids but does not mediate their vasodilator effects.

BACKGROUND AND PURPOSE: Atypical cannabinoids are thought to cause vasodilatation through an as-yet unidentified 'CBx' receptor. Recent reports suggest GPR55 is an atypical cannabinoid receptor, making it a candidate for the vasodilator 'CBx' receptor. The purpose of the present study was to test the hypothesis that human recombinant GPR55 is activated by atypical cannabinoids and mediates vasodilator responses to these agents. EXPERIMENTAL APPROACH: Human recombinant GPR55 was expressed in HEK293T cells and specific GTPgammaS activity was monitored as an index of receptor activation. In GPR55-deficient and wild-type littermate control mice, in vivo blood pressure measurement and isolated resistance artery myography were used to determine GPR55 dependence of atypical cannabinoid-induced haemodynamic and vasodilator responses. KEY RESULTS: Atypical cannabinoids O-1602 and abnormal cannabidiol both stimulated GPR55-dependent GTPgammaS activity (EC50 approximately 2 nM), whereas the CB1 and CB2-selective agonist WIN 55,212-2 showed no effect in GPR55-expressing HEK293T cell membranes. Baseline mean arterial pressure and heart rate were not different between WT and GPR55 KO mice. The blood pressure-lowering response to abnormal cannabidiol was not different between WT and KO mice (WT 20+/-2%, KO 26+/-5% change from baseline), nor was the vasodilator response to abnormal cannabidiol in isolated mesenteric arteries (IC50 approximately 3 micro M for WT and KO). The abnormal cannabidiol vasodilator response was antagonized equivalently by O-1918 in both strains. CONCLUSIONS: These results demonstrate that while GPR55 is activated by atypical cannabinoids, it does not appear to mediate the vasodilator effects of these agents.

P2X1 stimulation promotes thrombin receptor-mediated platelet aggregation.

P2X1 receptors are ATP-gated channel demonstrated to be involved in multiple platelet responses, although in vitro analysis has been complicated by the effects of rapid desensitization. To further investigate potential roles of P2X1 receptors in platelet activation, the current study employed methods which maximally preserved P2X1 functionality. In preliminary in vivo studies, P2X1-deficiency reduced thrombus formation following the laser-induced, but not FeCl3-induced injury. Given the multiple potential mechanisms involved in thrombus formation in vivo, including tissue-factor/thrombin generation pathways, subsequent studies were designed to investigate the effects of P2X1 inhibition or stimulation on platelet activation in vitro; specifically, the interaction of P2X1 with thrombin receptor stimulation. Aggregation initiated by low/threshold levels of a protease-activated receptor (PAR)4 agonist was reduced in P2X1-deficient murine platelets, and inhibition of P2X1 in wild-type platelets similarly reduced PAR4-mediated aggregation. In human platelets, aggregation to low/threshold stimulation of PAR1 was inhibited with the P2X1 antagonist MRS2159. In addition, P2X1 stimulation primed human platelet responses, such that subsequent sub-threshold PAR1 responses were converted into significant aggregation. Selective ADP receptor inhibitors attenuated P2X1-mediated priming, suggesting that the synergy between P2X1 and sub-threshold PAR1 stimulation was in part because of enhanced granular release of ADP. Overall, the present study defines a novel interaction between platelet P2X1 and thrombin receptors, with P2X1 functioning to amplify aggregation responses at low levels of thrombin receptor stimulation.

Urotensin II and renal function in the rat.

Urotensin II (UII) is a potent vasoactive hormone in mammals. However, despite its well-known effects on epithelial sodium transport in fish, little is known about its actions on the mammalian kidney. The aim of this study was to determine the effects of UII on renal function in the rat. Using standard clearance methods, the effects of rUII and the rat UII receptor (UT) antagonist, urantide, were studied. UII was measured in plasma and urine by radioimmunoassay. UII and UT were localized in the kidney by immunohistochemistry and mRNA expression quantified. Rat urinary [UII] was 1,650-fold higher than that in plasma. Immunoreactive-UII was localized to the proximal tubules, outer and inner medullary collecting ducts (IMCD); UT receptor was identified in glomerular arterioles, thin ascending limbs, and IMCD. UII and UT mRNA expression was greater in the medulla; expression was higher still in spontaneously hypertensive rats (SHRs) associated with raised plasma (UII). Injection of rUII induced reductions in glomerular filtration rate (GFR), urine flow, and sodium excretion. Urantide infusion resulted in increases in these variables. Endogenous UII appears to contribute to the regulation of GFR and renal sodium and water handling in the rat. While hemodynamic changes predominate, we cannot rule out the possibility of a direct tubular action of UII. Increased expression of UII and UT in the SHR suggests that UII plays a role in the pathophysiology of cardiovascular disease.

Is ultrasound useful for evaluating paediatric inflammatory neck masses?

OBJECTIVE: To determine the accuracy of ultrasound in detecting the presence of pus in a neck mass in children. DESIGN: Retrospective case sheet review. SETTING: A district general hospital. PARTICIPANTS: Children admitted from January 2000 to January 2004 with an acute cervical mass who were investigated by ultrasonography. MAIN OUTCOME MEASURES: The presence of a clinically relevant abscess that did not respond to clinical management was used to determine the accuracy of the ultrasound result. RESULTS: The sensitivity of ultrasound in the detection of an abscess was 65% and the specificity 88%. The predictive value of a positive ultrasound result was 81% and the predictive value of a negative test 77%. CONCLUSIONS: Ultrasound is a useful modality in the evaluation of acute cervical masses but is observer dependent and has a relatively low sensitivity in detecting whether or not pus is present. The clinical indicators for the presence of an abscess are discussed and clinicians should combine clinical findings with the ultrasound findings in order to determine treatment.

Endoscopic assisted external approach anterior ethmoidal artery ligation for the management of epistaxis.

Anterior ethmoidal artery ligation is a well-established surgical procedure in the management of epistaxis. We describe a procedure of anterior ethmoidal artery ligation via minimal access external surgery with the use of a rigid endoscope. This is, as far as we are aware, the first description of an external approach endoscopic anterior ethmoidal artery ligation.

Appreciation of music in adult patients with cochlear implants: a patient questionnaire.

Many cochlear implant candidates express hopes of enjoying music following implantation. Our aim was to assess the appreciation of music after cochlear implantation in adult patients. Thirty-five out of 45 cochlear implantees (78%) from the North East Programme responded to a questionnaire. Only 16 out of 35 patients (46%) listened to music after implantation. Enjoyment of music on a self-assessment scale was graded a mean of 8.7/10 before deafness but only 2.6/10 after implantation. Listening to music after implantation was more likely in younger patients, those with higher speech perception scores and those with a shorter length of deafness, but was not found to be related to gender, type of implant, processing strategy, time since implant or music enjoyment before becoming deaf. Appreciation of music after cochlear implantation is disappointingly low. Future developments in implant technology should strive to improve satisfaction with music listening.

A retrospective comparison of endonasal KTP laser dacryocystorhinostomy versus external dacryocystorhinostomy.

We retrospectively reviewed all primary external dacryocystorhinostomies (DCRs) and endonasal KTP laser DCRs performed for epiphora as a result of nasolacrimal duct obstruction in our unit between 1993 and 2000. Forty-nine patients underwent an external approach and 76 endonasal laser procedures were performed. The success rate of the external group was 94% with a mean follow-up of 9 months. In contrast, the endonasal group's success rate was 64% with a mean follow-up of 12 months. This difference reached statistical significance (P = 0.0002). However, when including revision procedures, the success rate in the endonasal group increased from 64% to 82%. The success rate in the endonasal group improved from 50% in the first 38 cases to 79% in the last 38 cases (P = 0.0084), thereby demonstrating a learning curve. Our study confirms external DCR as the 'gold standard' for a successful outcome. However, the endonasal technique has significant advantages, including being a quicker procedure with less morbidity, no cutaneous scar, and being more amenable to a bilateral procedure, daycase surgery and local anaesthetic. We are persisting with the endonasal technique because of its advantages but have moved towards more 'cold steel' techniques in an effort to improve results and emulate other series. In conclusion, for nasolacrimal duct obstruction, the endonasal technique is our approach of choice, with revision surgery if necessary, and the external technique is held in reserve.

The development of a General Nasal Patient Inventory.

Most available clinical outcome measures for rhinology patients relate to specific nasal disease or general quality of life. Fairley's validated 12-item questionnaire measures general nasal symptoms, but is a 'physician-derived' clinical tool and may not reflect all the problems that rhinology patients experience. Our aims were to develop a patient-orientated questionnaire, representing the concerns of a large number of rhinology patients, called the General Nasal Patient Inventory (GNPI) and compare this with the Fairley nasal questionnaire (FNQ). The GNPI was developed from the open-ended problem lists of 211 rhinology patients, from the 45 most frequent complaints. Both questionnaires were then administered to 153 general rhinology patients and the results compared. The highest-ranking items for each questionnaire were different, but the total scores were highly correlated (r = 0.79, P < 0.0001). Factor analysis showed six factors to account for 75% of FNQ variance and 18 factors for 78% of GNPI variance. The 45-item GNPI, the first patient-derived, comprehensive nasal questionnaire could be a time-saving tool in rhinology clinics and more sensitive to change after intervention than other available measures.

Central effects of urotensin-II following ICV administration in rats.

RATIONALE: Urotensin-II (U-II) has recently been identified as an agonist for the G-protein-coupled receptor, GPR14. Detection of both U-II and GPR14 mRNA in the brain and spinal cord is consistent with a role for U-II in the CNS. However, the effects of central administration of U-II in rodents have not been reported previously. OBJECTIVES: To determine the localisation of GPR14 mRNA in rat tissues and to investigate the behavioural and endocrine effects of human U-II (hU-II) following intracerebroventricular (ICV) administration in rats. METHODS: Experiments were carried out in male Sprague-Dawley rats. Expression of GPR14 mRNA in rat brain was determined by semi-quantitative RT-PCR. Effects of hU-II on general behaviours were assessed by an observer and the motor activity response was measured by an automated activity monitor. Plasma hormones and [DOPAC + HVA]/[DA] and [5-HIAA]/[5-HT] ratios in five brain areas were measured 20 min post-hU-II (ICV). RESULTS: GPR14 mRNA expression was found in whole brain tissue and in all CNS regions tested. GPR14 mRNA expression was also detected in the periphery; highest levels were found in the heart. Following ICV administration, hU-II (3-10 micrograms ICV) increased rearing and grooming, and increased motor activity in a familiar environment. Further, hU-II increased plasma prolactin and TSH but did not affect levels of corticosterone. hU-II had no effects on dopamine or 5-HT levels or their metabolites in the frontal cortex, hippocampus, hypothalamus, striatum and nucleus accumbens. CONCLUSIONS: These data provide further insight into the distribution of GPR14 mRNA within the CNS and show for the first time that hU-II causes marked behavioural and endocrine effects.

The role of endothelin in the pathogenesis of Chagas' disease.

Infection with Trypanosoma cruzi causes a generalised vasculitis of several vascular beds. This vasculopathy is manifested by vasospasm, reduced blood flow, focal ischaemia, platelet thrombi, increased platelet aggregation and elevated plasma levels of thromboxane A(2) and endothelin-1. In the myocardium of infected mice, myonecrosis and a vasculitis of the aorta, coronary artery, smaller myocardial vessels and the endocardial endothelium are observed. Immunohistochemistry studies employing anti-endothelin-1 antibody revealed increased expression of endothelin-1, most intense in the endocardial and vascular endothelium. Elevated levels of mRNA for prepro endothelin-1, endothelin converting enzyme and endothelin-1 were observed in the infected myocardium. When T. cruzi-infected mice were treated with phosphoramidon, an inhibitor of endothelin converting enzyme, there was a decrease in heart size and severity of pathology. Mitogen-activated protein kinases and the transcription factor activator-protein-1 regulate the expression of endothelin-1. Therefore, we examined the activation of mitogen-activated protein kinases in the myocardium by T. cruzi. Western blot demonstrated an extracellular signal regulated kinase. In addition, the activator-protein-1 DNA binding activity, as determined by electrophoretic mobility shift assay, was increased. Increased expression of cyclins A and cyclin D1 was observed in the myocardium, and immunohistochemistry studies revealed that interstitial cells and vascular and endocardial endothelial cells stained intensely with antibodies to these cyclins. These data demonstrate that T. cruzi infection of the myocardium activates extracellular signal regulated kinase, activator-protein-1, endothelin-1, and cyclins. The activation of these pathways is likely to contribute to the pathogenesis of chagasic heart disease. These experimental observations suggest that the vasculature plays a role in the pathogenesis of chagasic cardiomyopathy. Additionally, the identification of these pathways provides possible targets for therapeutic interventions to ameliorate or prevent the development of cardiomyopathy during T. cruzi infection.

Differential modulation of endothelin ligand-induced contraction in isolated tracheae from endothelin B (ET(B)) receptor knockout mice.

The role of endothelin B (ET(B)) receptors in mediating ET ligand-induced contractions in mouse trachea was examined in ET(B) receptor knockout animals. Autoradiographic binding studies, using [(125)I]-ET-1, confirmed the presence of ET(A) receptors in tracheal and bronchial airway smooth muscle from wild-type (+/+) and homozygous recessive (-/-) ET(B) receptor knockout mice. In contrast, ET(B) receptors were not detected in airway tissues from (-/-) mice. In tracheae from (+/+) mice, the rank order of potencies of the ET ligands was sarafotoxin (Stx) S6c>ET-1>ET-3; Stx S6c had a lower efficacy than ET-1 or ET-3. In tissues from (-/-) mice there was no response to Stx S6c (up to 0.1 microM), whereas the maximum responses and potencies of ET-1 and ET-3 were similar to those in (+/+) tracheae. ET-3 concentration-response curve was biphasic in (+/+) tissues (via ET(A) and ET(B) receptor activation), and monophasic in (-/-) preparations (via stimulation of only ET(A) receptors). In (+/+) preparations SB 234551 (1 nM), an ET(A) receptor-selective antagonist, inhibited the secondary phase, but not the first phase, of the ET-3 concentration-response curve, whereas A192621 (100 nM), an ET(B) receptor-selective antagonist, had the opposite effect. In (-/-) tissues SB 234551 (1 nM), but not A192621 (100 nM), produced a rightward shift in ET-3 concentration-response curves. The results confirm the significant influence of both ET(A) and ET(B) receptors in mediating ET-1-induced contractions in mouse trachea. Furthermore, the data do not support the hypothesis of atypical ET(B) receptors. In this preparation ET-3 is not an ET(B) receptor-selective ligand, producing contractions via activation of both ET(A) and ET(B) receptors.