[Translated article] Influence of lateralized reverse shoulder prosthesis design on tuberosity union in proximal humerus fractures.

INTRODUCTION: Proximal humerus fractures are the third most frequent type of fracture in elderly patients. Nowadays, surgical treatment is indicated one third of the time, being the reverse shoulder prosthesis an option especially in complex comminuted patterns. In this study we analyzed the effects of a lateralized reverse prosthesis in tuberosity union and its relationship with the functional results. MATERIAL AND METHODS: Retrospective case study of patients with proximal humerus fractures treated with a lateralized design reverse shoulder prosthesis with one-year minimum follow-up. Tuberosity nonunion was defined as a radiological concept: absence of tuberosity, distance >1cm from the tuberosity fragment to the humeral shaft or tuberosity above the humeral tray. Subgroup analysis was performed, group 1 (n=16) tuberosity union vs. group 2 (n=19) tuberosity nonunion. Groups were compared with the following functional scores: Constant, American Shoulder and Elbow Surgeons and Subjective Shoulder Value. RESULTS: A total of 35 patients were included in this study with a median age of 72.65 years. Postoperative radiographic analysis at one year after surgery revealed a tuberosity nonunion rate of 54%. Subgroup analysis revealed no statistically significant differences in terms of range of motion or functional scores. However, there were differences regarding the Patte sign (p=0.03) which was positive in a larger proportion of patients in the group with tuberosity nonunion. CONCLUSION: Even though there was a large percentage of tuberosity nonunion with the use of a lateralized prosthesis design, patients obtained good results in a similar manner to those found in the union group in terms of range of motion, scores, and patient satisfaction.

Análisis de la heterogeneidad en revisiones sistemáticas de cardiología publicadas entre 2000-2005 y 2011-2016 / Analysis of heterogeneity in cardiology systematic reviews published be-tween 2000-2005 and 2011-2016 / Análise da heterogeneidade em revisões sistemáticas de cardiologia publicadas entre 2000-2005 e 2011-2016

Introducción: la cardiología es una de las especialidades médicas que cuenta con más revisiones sistemáticas y metanálisis. Estudiar la metodología de las revisiones y anali­zar su heterogeneidad estadística es fundamental para garantizar su validez científica. Objetivo: describir la comparación de medidas de asociación, modelos estadísticos y grado de heterogeneidad en metanálisis de revisiones sistemáticas de intervenciones farmacológicas en cardiología, publicadas entre 2000-2005 y 2011-2016. Metodología: estudio analítico basado en la descripción y comparación de métodos estadísticos de revisiones sistemáticas de intervenciones farmacológicas en cardiología, publicadas en la biblioteca Cochrane. Para las variables cualitativas se estimaron frecuen­cias absolutas y relativas, mientras que para las cuantitativas se determinaron medias y desviaciones estándar, o medianas y rangos intercuartílicos, según su distribución. Para establecer la diferencia de medias se realizó la prueba t de Student y para la diferencia de proporciones el Chi cuadrado. Resultados: se incluyeron 54 revisiones sistemáticas, con un total de 1053 metanálisis, 6 revisiones con 240 metanálisis entre 2000-2005 y 48 revisiones con 813 metanálisis entre 2011-2016. La mayoría de metanálisis utilizaron el tratamiento estándar como grupo de comparación (56.6%), midieron desenlaces cualitativos nominales (86.3%), determinaron riesgos relativos (63.3%) y aplicaron modelos de efectos fijos (57.8%). En 2011-2016 se encontró una media del Índice de Higgins 17.5 menor que en 2000-2005 (p<0.05). Conclusión: se evidenció una disminución de la heterogeneidad estadística y un aumento en la implementación de modelos de efectos aleatorios, lo que da cuenta de una mayor rigurosidad a la hora de demostrar resultados verdaderamente significativos.

Introduction: cardiology is one of the medical specialties with the most systematic reviews and meta-analyses. Studying the methodology of the reviews and analyzing their statistical heterogeneity is essential to guarantee their scientific validity. Objective: to describe the comparison of association measures, statistical models and degree of heterogeneity in meta-analyses of systematic reviews of pharmacological interventions in cardiology, published between 2000-2005 and 2011-2016. Methodology: analytical study based on the description and comparison of statistical methods of systematic reviews of pharmacological interventions in cardiology, published in the Cochrane library. For the qualitative variables, absolute and relative frequencies were estimated, while for the quantitative ones, means and standard deviations, or medians and interquartile ranges, were determined, depending on their distribution. The Student's t test was used to establish the difference in means and the Chi square for the difference in proportions. Results: 54 systematic reviews were included, with a total of 1.053 meta-analyses, 6 reviews with 240 meta-analyses between 2000-2005, and 48 reviews with 813 meta-analyses between 2011-2016. Most meta-analyses used standard treatment as the comparison group (56.6%), measured nominal qualitative outcomes (86.3%), determined relative risks (63.3%), and applied fixed-effect models (57.8%). In the 2011-2016 period, an average of the Higgins Index was found to be 17.5 lower than in the 2000-2005 (p<0.05). Conclusion: there was evidence of a decrease in statistical heterogeneity and an increase in the implementation of random effects models, which accounts for greater rigor when it comes to demonstrating truly significant results.

Introdução: a cardiologia é uma das especialidades médicas com mais revisões sistemáticas e metanálises. Estudar a metodologia das revisões e analisar sua heterogeneidade estatística é essencial para garantir sua validade científica. Objetivo: descrever a comparação de medidas de associação, modelos estatísticos e grau de heterogeneidade em metanálises de revisões sistemáticas de intervenções farmacológicas em cardiologia, publicadas entre 2000-2005 e 2011-2016. Metodologia: estudo analítico baseado na descrição e comparação de métodos estatísticos de revisões sistemáticas de intervenções farmacológicas em cardiologia, publicadas na biblioteca Cochrane. Para as variáveis qualitativas foram estimadas frequências absolutas e relativas, enquanto para as quantitativas foram determinadas médias e desvios padrão, ou medianas e intervalos interquartis, dependendo de sua distribuição. O teste t de Student foi utilizado para estabelecer a diferença de médias e o qui-quadrado para a diferença de proporções. Resultados: foram incluídas 54 revisões sistemáticas, com um total de 1053 meta-análises, 6 revisões com 240 meta-análises entre 2000-2005 e 48 revisões com 813 meta-análises entre 2011-2016. A maioria das metanálises usou tratamento padrão como grupo de comparação (56.6%), mediu resultados qualitativos nominais (86.3%), determinou riscos relativos (63.3%) e aplicou modelos de efeito fixo (57.8%). Em 2011-2016, a média do Índice de Higgins foi 17.5 menor do que em 2000-2005 (p<0.05). Conclusão: evidenciou-se uma diminuição da heterogeneidade estatística e um aumento da implementação de modelos de efeitos aleatórios, o que confere maior rigor na demonstração de resultados verdadeiramente significativos.

Influence of lateralized reverse shoulder prosthesis design on tuberosity union in proximal humerus fractures. / Influencia de las prótesis inversas con diseño lateralizado en la consolidación de las tuberosidades en fracturas de húmero proximal.

INTRODUCTION: Proximal humerus fractures are the third most frequent type of fracture in elderly patients. Nowadays, surgical treatment is indicated one third of the time, being the reverse shoulder prosthesis an option especially in complex comminuted patterns. In this study we analyzed the effects of a lateralized reverse prosthesis in tuberosity union and its relationship with the functional results. MATERIAL AND METHODS: Retrospective case study of patients with proximal humerus fractures treated with a lateralized design reverse shoulder prosthesis with one-year minimum follow-up. Tuberosity nonunion was defined as a radiological concept: absence of tuberosity, distance>1cm from the tuberosity fragment to the humeral shaft or tuberosity above the humeral tray. Subgroup analysis was performed, group 1 (n=16) tuberosity union vs. group 2 (n=19) tuberosity nonunion. Groups were compared with the following functional scores: Constant, American Shoulder and Elbow Surgeons and Subjective Shoulder Value. RESULTS: A total of 35 patients were included in this study with a median age of 72.65 years. Postoperative radiographic analysis at one year after surgery revealed a tuberosity nonunion rate of 54%. Subgroup analysis revealed no statistically significant differences in terms of range of motion or functional scores. However, there were differences regarding the Patte sign (p=0.03) which was positive in a larger proportion of patients in the group with tuberosity nonunion. CONCLUSION: Even though there was a large percentage of tuberosity nonunion with the use of a lateralized prosthesis design, patients obtained good results in a similar manner to those found in the union group in terms of range of motion, scores, and patient satisfaction.

Proteostasis regulators as potential rescuers of PMM2 activity.

Phosphomannomutase 2 deficiency (PMM2-CDG) is the most common N-glycosylation disorder. To date there is no treatment. Following the identification of a number of destabilizing pathogenic variants, our group suggested PMM2-CDG to be a conformational disease. The aim of the present study was to evaluate the possible use of proteostasis network regulators to increase the stability, and subsequently the enzymatic activity, of misfolded PMM2 mutant proteins. Patient-derived fibroblasts transduced with their own PMM2 folding or oligomerization variants were treated with different concentrations of the proteostasis regulators celastrol or MG132. Celastrol treatment led to a significant increase in mutant PMM2 protein concentration and activity, while MG132 had a small effect on protein concentration only. The increase in enzymatic activity with celastrol correlated with an increase in the transcriptional and proteome levels of the heat shock proteins Hsp90 and Hsp70. The use of specific Hsp70 or Hsp90 inhibitors showed the positive effect of celastrol on PMM2 stability and activity to occur through Hsp90-driven modulation of the proteostasis network. The synergistic effect of celastrol and a previously described pharmacological chaperone was also examined, and a mutation-dependent synergistic effect on PMM2 activity was noted. These results provide proof-of-concept regarding the potential treatment of PMM2-CDG by proteostasis regulators, either alone or in combination with pharmacological chaperones.

Erratum: Optoacoustic response of gold nanorods in soft phantoms using high-power diode laser assemblies at 870 and 905 nm: erratum.

[This corrects the article on p. 1430 in vol. 8, PMID: 28663839.].

Optoacoustic response of gold nanorods in soft phantoms using high-power diode laser assemblies at 870 and 905 nm.

In the present paper we show the optoacoustic (OA) response of two solutions of gold nanorods dispersed in distilled water (0.8 mg/ml) and hosted in tissue-like phantoms by using small arrays of high-power diode lasers [corrected] at 870 and 905 nm as excitation sources. The high-power diode lasers [corrected] are coupled to a 7-to-1 optical fiber bundle with output diameter of 675 µm. Each solution of gold nanorods exhibits an absorption peak close to the operating wavelength, i.e. ~860 nm and ~900 nm, respectively, to optimize the generation of OA signals. The phantoms are made of agar, intralipid and hemoglobin to simulate a soft biological tissue with reduced properties of scattering. Three 3-mm diameter tubes done in the phantoms at different depths (0.9 cm, 1.8 cm, and 2.7 cm) have been filled with gold nanorods. In this way, OA signals with appreciable SNR are generated at different depths in the phantoms. The high OA response exhibited by gold nanorods suggests their application in OA spectroscopy as exogenous contrast agents to detect and monitor emerging diseases like metastasis and arteriosclerotic plaques.

Hydrogen sulphide as a signalling molecule regulating physiopathological processes in gastrointestinal motility.

The biology of H2 S is a still developing area of research and several biological functions have been recently attributed to this gaseous molecule in many physiological systems, including the cardiovascular, urogenital, respiratory, digestive and central nervous system (CNS). H2 S exerts anti-inflammatory effects and can be considered an endogenous mediator with potential effects on gastrointestinal motility. During the last few years, we have investigated the role of H2 S as a regulator of gastrointestinal motility using both animal and human tissues. The aim of the present work is to review published data regarding the potential role of H2 S as a signalling molecule regulating physiopathological processes in gastrointestinal motor function. H2 S is endogenously produced by defined enzymic pathways in different cell types of the intestinal wall including neurons and smooth muscle. Inhibition of H2 S biosynthesis increases motility and H2 S donors cause smooth muscle relaxation and inhibition of propulsive motor patterns. Impaired H2 S production has been described in animal models with gastrointestinal motor dysfunction. The mechanism(s) of action underlying these effects may include several ion channels, although no specific receptor has been identified. At this time, even though there is much experimental evidence for H2 S as a modulator of gastrointestinal motility, we still do not have conclusive experimental evidence to definitively propose H2 S as an inhibitory neurotransmitter in the gastrointestinal tract, causing nerve-mediated relaxation.

P2Y(1) receptors mediate purinergic relaxation in the equine pelvic flexure.

In the equine large intestine, the knowledge of the basic mechanisms underlying motility function is crucial to properly treat motility disorders. P2Y1 receptors are responsible for mediating purinergic colonic relaxation in several species. In vitro experimental studies of the circular muscle from the equine pelvic flexure (n = 6) were performed to characterize inhibitory and excitatory neuromuscular transmission. Electrophysiological studies showed that electrical field stimulation (EFS) evoked biphasic inhibitory junction potentials (IJPs) in smooth muscle cells: a fast IJP (IJPf) followed by a sustained IJP (IJPs). IJPs was sensitive to L-NNA 1 mM (a nitric oxide synthase inhibitor) (P <0.01), while IJPf was abolished by MRS2500 1 µM (a P2Y1 receptor antagonist) (P <0.001). EFS (5 Hz for 2 min) in the organ bath inhibited rhythmic contractions to 3.0 ± 2.5% of basal area under the curve (P <0.0001). EFS under MRS2500 1 µM or L-NNA 1 mM incubation inhibited contractions to 6.0 ± 2.8% (P <0.05) and 24.4 ± 11.3% respectively (P <0.05). Combination of MRS2500 1 µM and L-NNA 1 mM completely reversed the EFS-induced inhibition of colonic motility. Non-nitrergic, non-purinergic conditions were used to reveal voltage-dependent EFS-induced contractions sensitive to atropine 1 µM (P <0.001) and, therefore, cholinergic. In conclusion, nerve-mediated relaxation and contraction in the equine pelvic flexure involve the same mechanisms as those observed in the human colon. P2Y1 receptors mediate purinergic relaxations and are potential targets for the treatment of equine colonic motor disorders.

The effect of levosulpiride on in vitro motor patterns in the human gastric fundus, antrum, and jejunum.

BACKGROUND: Levosulpiride is a 5HT4 agonist/D2 antagonist prokinetic agent used to improve gastric emptying in patients with functional dyspepsia or gastroparesis. The aim of this study was to characterize its effect on the main in vitro motility patterns in the human fundus, antrum, and jejunum. METHODS: Circular muscle strips from human stomach (antrum and fundus) and jejunum, obtained from 46 patients undergoing bariatric surgery, were studied using organ baths. Enteric motor neurons (EMNs) were stimulated by electrical field stimulation (EFS). KEY RESULTS: Levosulpiride, caused an increase in the EFS-induced cholinergic contractions in the gastric antrum (+37 ± 15.18% at 100 µM, pEC50 = 4.46 ± 0.14; p < 0.05, n = 8) and jejunum (+45.4 ± 22.03% at 100 µM, pEC50 = 3.78 ± 6.81; p < 0.05, n = 5), but not in the gastric fundus. It also caused a slight decrease in tone and frequency of spontaneous contractions in the jejunum, but did not have any major effect on tone or spontaneous contractions in the stomach. It did not have any effect on EFS-induced relaxations mediated by nitric oxide (NO) in the stomach (antrum and fundus) and by NO and ATP in the jejunum. CONCLUSIONS & INFERENCES: Our results suggest that the prokinetic effects of levosulpiride in humans are mainly due to the facilitation of the release of acetylcholine by enteric motor neurons in the gastric antrum and the jejunum.

Inverse gradient of nitrergic and purinergic inhibitory cotransmission in the mouse colon.

AIM: Gastrointestinal smooth muscle relaxation is accomplished by the neural corelease of ATP or a related purine and nitric oxide. Contractions are triggered by acetylcholine and tachykinins. The aim of this work was to study whether regional differences in neurotransmission could partially explain the varied physiological roles of each colonic area. METHODS: We used electrophysiological and myography techniques to evaluate purinergic (L-NNA 1 mm incubated tissue), nitrergic (MRS2500 0.3 µm incubated tissue) and cholinergic neurotransmission (L-NNA 1 mm and MRS2500 0.3 µm incubated tissue) in the proximal, mid and distal colon of CD1 mice (n = 42). RESULTS: Purinergic electrophysiological responses elicited by single pulses (28 V) were greater in the distal (IJPfMAX = -35.3 ± 2.2 mV), followed by the mid (IJPfMAX = -30.6 ± 1.0 mV) and proximal (IJPfMAX = -11.7 ± 1.1 mV) colon. In contrast, nitrergic responses decreased from the proximal colon (IJPsMAX = -11.4 ± 1.1 mV) to the mid (IJPsMAX = -9.1 ± 0.4 mV), followed by the distal colon (IJPsMAX = -1.8 ± 0.3 mV). A similar rank of order was observed in neural mediated inhibitory mechanical responses including electrical field stimulation-mediated responses and neural tone. ADPßs concentration-response curve was shifted to the left in the distal colon. In contrast, NaNP responses did not differ between regions. Cholinergic neurotransmission elicited contractions of a similar amplitude throughout the colon. CONCLUSION: An inverse gradient of purinergic and nitrergic neurotransmission exists through the mouse colon. The proximal and mid colon have a predominant nitrergic neurotransmission probably due to the fact that their storage function requires sustained relaxations. The distal colon, in contrast, has mainly purinergic neurotransmission responsible for the phasic relaxations needed to propel dehydrated faeces.

Changes in the response to excitatory antagonists, agonists, and spasmolytic agents in circular colonic smooth muscle strips from patients with diverticulosis.

BACKGROUND: Colonic samples from asymptomatic diverticulosis (DS) patients presented enhanced electrical field stimulation (EFS)-contractions, in an earlier study of ours, suggesting increased endogenous responses. The aim of this study was to explore changes in excitatory neuromuscular transmission and to assess the pharmacodynamics of spasmolytic agents in DS. METHODS: Circular muscle strips from sigmoid colon of DS patients (n = 30; 69.5 ± 14.8 years) and controls (n = 32; 64.7 ± 16.2 years) were studied using organ baths to evaluate the direct effect of excitatory agonists (carbachol, neurokinin A [NKA] and substance P [SP]), and the effect of antagonists (atropine and NK2 antagonist GR94800) and spasmolytic drugs (otilonium bromide [OB] and N-butyl-hyoscine) on the contractions induced by EFS-stimulation of excitatory motorneurons. qRT-PCR was also performed to compare mRNA expression of M2 , M3 , NK2 receptors and L-type calcium channels. KEY RESULTS: Contractions to carbachol (Emax : 663.7 ± 305.6% control vs 2698.0 ± 439.5% DS; p < 0.0005) and NKA (Emax : 387.8 ± 35.6% vs 1102.0 ± 190.1%; p < 0.0005) were higher in DS group, without differences for SP. Higher potency for DS patients was observed in the concentration-response curves for atropine (pIC50  = 8.56 ± 0.15 control vs pIC50  = 9.95 ± 0.18 DS group; p < 0.005) and slightly higher for GR94800 (pIC50  = 7.21 ± 0.18 control vs pIC50  = 7.97 ± 0.32 group; p < 0.0001). Lower efficacy (Emax ) and potency (pIC50 ) was observed for spasmolytic drugs in DS, whereas no differences were found regarding the relative expression of the receptors evaluated between groups. CONCLUSIONS & INFERENCES: The greater response to cholinergic and tachykinergic agonists and greater potency for muscarinic and NK2 antagonists observed in DS might play a role in the spasticity found in diverticular disease.

Potential role of the gaseous mediator hydrogen sulphide (H2S) in inhibition of human colonic contractility.

BACKGROUND: Hydrogen sulphide (H2S) is an endogenous signalling molecule that might play a physiologically relevant role in gastrointestinal motility. Cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) are two enzymes responsible for H2S production. d,l-Propargylglycine (PAG) is a CSE inhibitor whereas both aminooxyacetic acid (AOAA) and hydroxylamine (HA) are CBS inhibitors. The characterization of H2S responses and its mechanism of action are crucial to define H2S function. METHODS: Human colonic strips were used to investigate the role of H2S on contractility (muscle bath) and smooth muscle electrophysiology (microelectrodes). NaHS was used as a H2S donor. RESULTS: Combination of PAG and AOAA depolarized the smooth muscle (5-6mV, n=4) and elicited a transient increase in tone (260.5±92.8mg, n=12). No effect was observed on neural mediated inhibitory junction potential or relaxation. In the presence of tetrodotoxin 1µM, NaHS concentration-dependently inhibited spontaneous contractions (EC50=329.2µM, n=18). This effect was partially reduced by the guanylyl cyclase inhibitor ODQ 10µM (EC50=2.6µM, n=12) and by l-NNA 1mM (EC50=1.4mM, n=8). NaHS reversibly blocked neural mediated cholinergic (EC50=2mM) and tachykinergic (EC50=5.7mM) contractions. NaHS concentration-dependently reduced the increase in spontaneous mechanical activity (AUC) induced by carbachol (EC50=1.9mM) and NKA (EC50=1.7mM AUC). CONCLUSIONS: H2S might be an endogenous gasomediator regulating human colonic contractility. Its inhibitory effect is observed at high concentrations and could be mediated by a direct effect on smooth muscle with a possible synergistic effect with NO, as well as by an interaction with the cholinergic and tachykinergic neural mediated pathways.

EP2 and EP4 receptors mediate PGE2 induced relaxation in murine colonic circular muscle: pharmacological characterization.

BACKGROUND: Prostaglandin E2 (PGE2) is a regulator of gastrointestinal motility that might be involved in impaired motor function associated to gut inflammation. The aim of the present work is to pharmacologically characterize responses to exogenous and endogenous PGE2 in the mouse colon targeting EP2 and EP4 receptors. METHODS: Wild type (WT) and EP2 receptor knockout (EP2-KO) mice were used to characterize PGE2 and butaprost (EP2 receptor agonist) effects on smooth muscle resting membrane potential and myogenic contractility in circularly oriented colonic preparations. RESULTS: In WT animals, PGE2 and butaprost concentration-dependently inhibited spontaneous contractions and hyperpolarized smooth muscle cells. Combination of both EP2 (PF-04418948 0.1µM) and EP4 receptor antagonists (L-161,982 10µM) was needed to block both electrical and mechanical PGE2 responses. Butaprost inhibitory responses (both electrical and mechanical) were totally abolished by PF-04418948 0.1µM. In EP2-KO mice, PGE2 (but not butaprost) concentration-dependently inhibited spontaneous contractions and hyperpolarized smooth muscle cells. In EP2-KO mice, PGE2 inhibition of spontaneous contractility and hyperpolarization was fully antagonized by L-161,982 10µM. In WT animals, EP2 and EP4 receptor antagonists caused a smooth muscle depolarization and an increase in spontaneous mechanical activity. CONCLUSIONS: PGE2 responses in murine circular colonic layer are mediated by post-junctional EP2 and EP4 receptors. PF-04418948 and L-161,982 are selective EP2 and EP4 receptor antagonists that inhibit PGE2 responses. These antagonists might be useful pharmacological tools to limit prostaglandin effects associated to dismotility in gut inflammatory processes.

Differential functional role of purinergic and nitrergic inhibitory cotransmitters in human colonic relaxation.

AIM: ATP and nitric oxide (NO) are released from enteric inhibitory motor neurones and are responsible for colonic smooth muscle relaxation. However, how frequency of neural stimulation affects this cotransmission process and the post-junctional responses has not been systematically characterized in the human colon. METHODS: The dynamics of inhibitory cotransmission were studied using different protocols of electrical field stimulation (EFS) to characterize the inhibitory junction potentials (IJP) and the corresponding relaxation in colonic strips obtained from 36 patients. RESULTS: Single pulses elicited a fast IJP (IJPf(MAX) = -27.6 ± 1.6 mV), sensitive to the P2Y1 antagonist MRS2500 1 µm, that ran down with frequency increase leaving a residual hyperpolarization at high frequencies (IJPf∞ = -3.7 ± 0.6 mV). Accordingly, low frequencies of EFS caused purinergic transient relaxations that cannot be maintained at high frequencies. Addition of the P2Y1 agonist MRS2365 10 µm during the purinergic rundown did not cause any hyperpolarization. Protein kinase C (PKC), a putative P2Y1 desensitizator, was able to reduce the amplitude of the IJPf when activated, but the rundown was not modified by PKC inhibitors. Frequencies higher than 0.60 ± 0.15 Hz were needed to evoke a sustained nitrergic hyperpolarization that progressively increased reaching IJPs∞ = -13 ± 0.4 mV at high frequencies and leading to a sustained inhibition of spontaneous motility. CONCLUSION: Changes in frequency of stimulation possibly mimicking neuronal firing will post-junctionally determine purinergic vs. nitrergic responses underlying different functional roles. NO will be responsible for sustained relaxations needed in physiological processes such as storage, while purinergic neurotransmission evoking sharp transient relaxations will be dominant in processes such as propulsion.

Nitrergic neuro-muscular transmission is up-regulated in patients with diverticulosis.

BACKGROUND: Neuro-transmission impairment could be associated to motility changes observed in patients with diverticular disease. Therefore, the objective was to characterize the inhibitory neuro-muscular transmission and gene expression changes of the enteric inhibitory pathways in patients with diverticulosis (DS). METHODS: Circular muscle strips from sigmoid colon of patients with DS and controls were studied using the organ bath technique to evaluate spontaneous contractility and enteric motor neurons stimulated by electrical field and qRT-PCR to assess the expression of nNOS, iNOS, P2Y1 R and PGP9.5. KEY RESULTS: Patients with DS presented decreased spontaneous rhythmic contractions (SRC) that were significantly enhanced after incubation with L-NNA (1 mM) and TTX (1 µM), and unaffected by the P2Y1 antagonist MRS2500 (1 µM). Stimulation on enteric motor neurons caused an increased duration of the latency of OFF-contractions in DS group (p < 0.001), antagonized by L-NNA and slightly affected by MRS2500 (1 µM). No differences in the IC50 between controls and DS patients were observed on inhibition of SRC for the NO-donor sodium nitroprusside (SNP) and the preferential P2Y agonist ADPßS. Moreover, nNOS relative expression was also up-regulated 2.3-fold in the DS group (p < 0.05) whereas there was no significant difference in relative expression of iNOS, P2Y1 R and the neuronal marker PGP9.5 between groups. CONCLUSIONS & INFERENCES: Patients with DS presented an over-expression of nNOS with increased endogenously NO-mediated responses suggesting enhanced NO-release. Up-regulation in the nitrergic pathway in early stages of the disease might play a role in colonic motor disorders associated to diverticular disease.

Nitrergic and purinergic mechanisms evoke inhibitory neuromuscular transmission in the human small intestine.

BACKGROUND: Inhibitory neuromuscular transmission in the human colon is due to nitrergic and purinergic (P2Y1 -mediated) inputs. The aim of this study was to determine the mechanisms of neuromuscular transmission in different regions of the human small intestine. METHODS: Ileal (n = 6) and jejunal (n = 6) samples underwent histological examination and were studied using sharp microelectrodes in smooth muscle cells and conventional muscle bath techniques. Electrical field stimulation (EFS) was used to stimulate inhibitory neurons. KEY RESULTS: No histological abnormalities were found. Resting membrane potential was -39.7 ± 1.5 and -45.5 ± 2.1 mV in the jejunum and ileum, respectively. Slow waves and spontaneous contractions were recorded at a frequency of about 8-9 and 6-7 cpm in the jejunum and ileum, respectively. In non-adrenergic, non-cholinergic conditions, EFS caused an inhibitory junction potential and mechanical relaxation. Both responses were blocked by tissue incubation with the nitric oxide synthase inhibitor (Nω-nitro-l-arginine 1 mM) and the P2Y1 receptor blocker 2'-deoxy-N(6) -methyladenosine 3',5'-bisphosphate tetrasodium salt (MRS2179; 10 µM). Both exogenous addition of sodium nitroprusside (1 µM) and the preferential P2Y1 receptor agonist ADPßS (1 µM) hyperpolarized and relaxed smooth muscle cells. MRS2179 (10 µM) blocked ADPßS-induced responses. CONCLUSIONS & INFERENCES: Similar to colon, inhibitory neurotransmission in the human small intestine is mainly mediated by purinergic (via P2Y1 receptors) and nitrergic inhibitory neurotransmission. Similar mechanisms of inhibitory neurotransmission are present in different regions of the human intestine.

Mechanisms of action of otilonium bromide (OB) in human cultured smooth muscle cells and rat colonic strips.

BACKGROUND: The pharmacological properties of otilonium bromide (OB) have been investigated using different experimental models, techniques, and conditions, and consequently, the results are not always easy to compare. The aim of the present work was to investigate the pharmacological properties of OB in human cultured colonic smooth muscle cells (HCSMCs), which is the main target of the drug 'in vivo'. Rat colonic strips were used to confirm the pharmacological properties. METHODS: Human cultured colonic smooth muscle cells were studied using the calcium imaging technique. Microelectrodes and muscle bath experiments were performed in rat colonic strips. KEY RESULTS: Otilonium bromide (OB) concentration dependently inhibited nifedipine-sensitive calcium transients induced by KCl (EC50  = 3.6 µM) and BayK8644 (EC50  = 4.0 µM). All the following experiments were performed in the presence of nifedipine. In HCSMC, carbachol-induced calcium transients were inhibited by OB (EC50  = 8.4 µM). Carbachol evoked 1-a smooth muscle depolarization (10 mV) that was antagonized by 100 µM OB; and 2-a contraction that was inhibited by OB (EC50  = 13.0 µM). 'Non-nitrergic (L-NNA 1 mM) non-purinergic (MRS2500 1 µM)' conditions were used to elicit endogenous excitatory responses. Electrical field stimulation caused 1-an atropine-sensitive excitatory junction potential that was inhibited by OB (EC50  = 8.9 µM) and 2-an atropine-sensitive contraction that was inhibited by OB (EC50  = 7.3 µM). In HCSMC, neurokinin A (NKA) and CaCl2 induced calcium transients that were inhibited by OB (NKA: EC50  = 11.7 µM; CaCl2 : EC50  = 17.5 µM). CONCLUSIONS & INFERENCES: Otilonium bromide causes inhibition of L-/T-type calcium channels, muscarinic, and tachykininergic responses that acting together explain the pharmacological properties of the compound.

Effects of hydrogen sulphide on motility patterns in the rat colon.

BACKGROUND AND PURPOSE: Hydrogen sulphide (H2 S) is an endogenous gaseous signalling molecule with putative functions in gastrointestinal motility regulation. Characterization of H2 S effects on colonic motility is crucial to establish its potential use as therapeutic agent in the treatment of colonic disorders. EXPERIMENTAL APPROACH: H2 S effects on colonic motility were characterized using video recordings and construction of spatio-temporal maps. Microelectrode and muscle bath studies were performed to investigate the mechanisms underlying H2 S effects. NaHS was used as the source of H2 S. KEY RESULTS: Rhythmic propulsive motor complexes (RPMCs) and ripples were observed in colonic spatio-temporal maps. Serosal addition of NaHS concentration-dependently inhibited RPMCs. In contrast, NaHS increased amplitude of the ripples without changing their frequency. Therefore, ripples became the predominant motor pattern. Neuronal blockade with lidocaine inhibited RPMCs, which were restored after administration of carbachol. Subsequent addition of NaHS inhibited RPMCs. Luminal addition of NaHS did not modify motility patterns. NaHS inhibited cholinergic excitatory junction potentials, carbachol-induced contractions and hyperpolarized smooth muscle cells, but did not modify slow wave activity. CONCLUSIONS AND IMPLICATIONS: H2 S modulated colonic motility inhibiting propulsive contractile activity and enhancing the amplitude of ripples, promoting mixing. Muscle hyperpolarization and inhibition of neurally mediated cholinergic responses contributed to the inhibitory effect on propulsive activity. H2 S effects were not related to changes in the frequency of slow wave activity originating in the network of interstitial cells of Cajal located near the submuscular plexus. Luminal H2 S did not modify colonic motility probably because of epithelial detoxification.

P2Y(1) knockout mice lack purinergic neuromuscular transmission in the antrum and cecum.

BACKGROUND: Pharmacological studies using selective P2Y(1) antagonists, such as MRS2500, and studies with P2Y(1)(-/-) knockout mice have demonstrated that purinergic neuromuscular transmission is mediated by P2Y(1) receptors in the colon. The aim of the present study was to test whether P2Y(1) receptors are involved in purinergic neurotransmission in the antrum and cecum. METHODS: Microelectrode recordings were performed on strips from the antrum and cecum of wild type animals (WT) and P2Y(1)(-/-) mice. KEY RESULTS: In the antrum, no differences in resting membrane potential and slow wave activity were observed between groups. In WT animals, electrical field stimulation elicited a MRS2500-sensitive inhibitory junction potential (IJP). In P2Y(1)(-/-) mice, a nitrergic IJP (N(ω) -nitro-l-arginine-sensitive), but not a purinergic IJP was recorded. This IJP was equivalent to the response obtained in strips from WT animals previously incubated with MRS2500. Similar results were obtained in the cecum: 1- the purinergic IJP (MRS2500-sensitive) recorded in WT animals was absent in P2Y(1)(-/-) mice 2- nitrergic neurotransmission was preserved in both groups. Moreover, 1- spontaneous IJP (MRS2500-sensitive) could be recorded in WT, but not in P2Y(1)(-/-) mice 2- MRS2365 a P2Y(1) agonist caused smooth muscle hyperpolarization in WT, but not in P2Y(1) (-/-) animals, and 3- ß-NAD caused smooth muscle hyperpolarization both in WT and P2Y(1)(-/-) animals. CONCLUSIONS & INFERENCES: 1- P2Y(1) receptor is the general mechanism of purinergic inhibition in the gastrointestinal tract, 2- P2Y(1)(-/-) mouse is a useful animal model to study selective impairment of purinergic neurotransmission and 3- P2Y(1)(-/-) mouse might help in the identification of purinergic neurotransmitter(s).