Muscarinic modulation of endogenous noradrenaline release from adrenergic terminals in the guinea-pig colon

1 The present study examined the role of muscarinic receptors in the modulation of noradrenaline (NA) release in the guinea-pig isolated distal colon. The spontaneous endogenous NA overflow assayed by HPLC-ED was taken as an index of NA release from enteric noradrenergic nerve terminals. 2 Physostigmine (10 μm ) significantly enhanced spontaneous endogenous NA overflow. Hyoscine (muscarinic antagonist), (R)-(-)-trihexyphenidyl and telenzepine (M₁-selective antagonists), and 11[[2-[(diethylamino)methyl]-1-piperydil]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116, M₂-selective antagonist) inhibited NA overflow in a concentration dependent manner, with the following EC₅₀ values: 131.74 (18.19–953.96), 101.62 (58.83–175.60), 150 (60–330), 30 (5–170) nm , respectively. 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, M₁- and M₃- selective antagonist) had no significant effect up to 100 μm . 3 The muscarinic agonist oxotremorine inhibited NA overflow in a concentration dependent manner, with an EC₅₀ value of 0.67 (0.30–1.51) μm . The response to oxotremorine was inhibited by muscarinic antagonists with the following order of potency: hyoscine = (R)-(-)-trihexyphenidyl = telenzepine > 4-DAMP >> AF-DX 116. 4 In the presence of 3 μm tetrodotoxin (TTX), the effect of oxotremorine and 4-DAMP was unchanged, while hyoscine, (R)-(-)-trihexyphenidyl, telenzepine and AF-DX 116, instead of inhibiting, significantly enhanced NA overflow. 5 The present results indicate that, in the guinea-pig colon, endogenous acetylcholine sustains spontaneous NA release by activating muscarinic receptors possibly located on interneurones. In addition, inhibitory muscarinic receptors may exist on adrenergic terminals.     

Role of neuronal and inducible nitric oxide synthases in the guinea pig ileum myenteric plexus during in vitro ischemia and reperfusion

Background Intestinal ischemia and reperfusion (I/R) injury leads to abnormalities in motility, namely delay of transit, caused by damage to myenteric neurons. Alterations of the nitrergic transmission may occur in these conditions. This study investigated whether an in vitro I/R injury may affect nitric oxide (NO) production from the myenteric plexus of the guinea pig ileum and which NO synthase (NOS) isoform is involved. Methods The distribution of the neuronal (n) and inducible (i) NOS was determined by immunohistochemistry during 60 min of glucose/oxygen deprivation (in vitro ischemia) followed by 60 min of reperfusion. The protein and mRNA levels of nNOS and iNOS were investigated by Western‐immunoblotting and real time RT‐PCR, respectively. NO levels were quantified as nitrite/nitrate. Key Results After in vitro I/R the proportion of nNOS‐expressing neurons and protein levels remained unchanged. nNOS mRNA levels increased 60 min after inducing ischemia and in the following 5 min of reperfusion. iNOS‐immunoreactive neurons, protein and mRNA levels were up‐regulated during the whole I/R period. A significant increase of nitrite/nitrate levels was observed in the first 5 min after inducing I/R and was significantly reduced by N^ω‐propyl‐l ‐arginine and 1400 W, selective inhibitors of nNOS and iNOS, respectively. Conclusions & Inferences Our data demonstrate that both iNOS and nNOS represent sources for NO overproduction in ileal myenteric plexus during I/R, although iNOS undergoes more consistent changes suggesting a more relevant role for this isoform in the alterations occurring in myenteric neurons following I/R.     

Effect of a new cognition enhancer, alpha-glycerylphosphorylcholine, on scopolamine-induced amnesia and brain acetylcholine.

The present study investigates the effect of the administration of alpha-glycerylphosphorylcholine (alpha-GPC) on scopolamine-induced amnesia and on brain acetylcholine (ACh) levels and release in rats. The results indicate that alpha-GPC, when administered orally, reverses the amnesia caused by scopolamine in passive avoidance. The peak effect is observed using 600 mg/kg IG, 5 h before training. The effect of the drug is long lasting (up 30 h) in accordance with its pharmacokinetic characteristics. Since, alpha-GPC administered IG is cleaved within the gut mucosal cells to glycerophosphate and free choline, it is tempting to speculate that this drug acts by increasing the ACh precursor pool. This view is supported also by the observation that alpha-GPC partially counteracts the decrease of brain ACh levels elicited by scopolamine administration. The effect is observed in the hippocampus and cortex, but not in the striatum. Moreover, in ex vivo experiments, alpha-GPC is able to increase the amount of ACh released by rat hippocampus slices following potassium stimulation.     

Soy diet induces intestinal inflammation in adult Zebrafish: Role of OTX and P53 family

Inflammatory bowel diseases (IBDs) are a group of inflammatory conditions of the colon and small intestine, including Crohn''s disease and ulcerative colitis. Since Danio rerio is a promising animal model to study gut function, we developed a soy‐dependent model of intestinal inflammation in adult zebrafish. The soya bean meal diet was given for 4 weeks and induced an inflammatory process, as demonstrated by morphological changes together with an increased percentage of neutrophils infiltrating the intestinal wall, which developed between the second and fourth week of treatment. Pro‐inflammatory genes such as interleukin‐1beta, interleukin‐8 and tumour necrosis factor alpha were upregulated in the second week and anti‐inflammatory genes such as transforming growth factor beta and interleukin‐10. Interestingly, an additional expression peak was found for interleukin‐8 at the fourth week. Neuronal genes, OTX1 and OTX2, were significantly upregulated in the first two  weeks, compatible with the development of the changes in the gut wall. As for the genes of the p53 family such as p53, DNp63 and p73, a statistically significant increase was observed after two weeks of treatment compared with controls. Interestingly, DNp63 and p73 were shown an additional peak after four weeks. Our data demonstrate that soya bean meal diet negatively influences intestinal morphology and immunological function in adult zebrafish showing the features of acute inflammation. Data observed at the fourth week of treatment may suggest initiation of chronic inflammation. Adult zebrafish may represent a promising model to better understand the mechanisms of food‐dependent intestinal inflammation.     

Postnatal development of P2 receptors in the murine gastrointestinal tract

The actions of purine and pyrimidine compounds on isolated segments of the mouse intestine were investigated during postnatal development. The localization of P2Y(1), P2Y(2), P2Y(4), P2X(1,) P2X(2) and P2X(3) receptors were examined immunohistochemically, and levels of expression of P2Y(1), P2X(1) and P2X(2) were studied by Western immunoblot. From day 12 onwards, the order of potency for relaxation of longitudinal muscle of all regions was 2-MeSADP>or=alpha,beta-meATP>or=ATP=UTP=adenosine, suggesting P2Y(1) receptors. This was supported by the sensitivity of responses to 2-MeSADP to the selective antagonist MRS 2179 and P2Y(1) receptor immunoreactivity on longitudinal muscle and a subpopulation of myenteric neurons. A further alpha,beta-meATP-sensitive P2Y receptor subtype was also indicated. ATP and UTP were equipotent suggesting a P2Y(2) and/or P2Y(4) receptor. Adenosine relaxed the longitudinal muscle in all regions via P1 receptors. The efficacy of all agonists to induce relaxation of raised tone preparations increased with age, being comparable to adult by day 20, the weaning age. During postnatal development the contractile response of the ileum and colon was via P2Y(1) receptors, while the relaxant response mediated by P2Y(1) receptors gradually appeared along the mouse gastrointestinal tract, being detectable in the stomach from day 3 and in the duodenum from day 6. In the ileum and colon relaxant responses to 2-MeSADP were not detected until days 8 and 12, respectively. 2-MeSADP induced contractions on basal tone preparations from day 3, but decreased significantly at day 12 and disappeared by day 20. At day 8, contractions of colonic longitudinal muscle to ATP showed no desensitisation suggesting the involvement of P2X(2) receptors. Immunoreactivity to P2X(2) receptors only was observed on the longitudinal muscle of the colon and ileum from day 1 and on a subpopulation of myenteric neurons from day 3. These data suggest that P2Y(1) receptors undergo postnatal developmental changes in the mouse gut, with a shift from contraction to relaxation. Such changes occur 1 week before weaning and may contribute to the changes that take place in the gut when the food composition changes from maternal milk to solid food.     

Acid base disorders in patients with COVID-19

Purpose

Acid–base derangement has been poorly described in patients with coronavirus disease 2019 (COVID-19). Considering the high prevalence of pneumonia and kidneys injury in COVID-19, frequent acid–base alterations are expected in patients admitted with SARS-Cov-2 infection. The study aimed to assess the prevalence of acid–base disorders in symptomatic patients with a diagnosis of COVID-19.

Methods

The retrospective study enrolled COVID-19 patients hospitalized at the University Hospital of Modena from 4 March to 20 June 2020. Baseline arterial blood gas (ABG) analysis was collected in 211 patients. In subjects with multiple ABG analysis, we selected only the first measurement. A pH of less than 7.37 was categorized as acidemia and a pH of more than 7.43 was categorized as alkalemia.

Results

ABG analyses revealed a low arterial partial pressure of oxygen (PO₂, 70.2?±?25.1 mmHg), oxygen saturation (SO₂, 92%) and a mild reduction of PO₂/FiO₂ ratio (231?±?129). Acid–base alterations were found in 79.7% of the patient. Metabolic alkalosis (33.6%) was the main alteration followed by respiratory alkalosis (30.3%), combined alkalosis (9.4%), respiratory acidosis (3.3%), metabolic acidosis (2.8%) and other compensated acid–base disturbances (3.6%). All six patients with metabolic acidosis died at the end of the follow-up.

Conclusion

Variations of pH occurred in the majority (79.7%) of patients admitted with COVID-19. The patients experienced all the type of acid–base disorders, notably metabolic and respiratory alkalosis were the most common alterations in this group of patients.

     

Involvement of Ca-dependent PKCs in the adaptive changes of μ-opioid pathways to sympathetic denervation in the guinea pig colon

In the guinea pig colon, chronic sympathetic denervation entails supersensitivity to inhibitory μ-opioid agents modulating cholinergic neurons. The mechanism underlying such adaptive change has not yet been unravelled, although protein kinase C (PKC) may be involved. A previous study indirectly demonstrated that activation of μ-opioid receptors on myenteric neurons facilitates PKC activity. Such coupling may counteract the inhibitory action of μ-opioid agents on acetylcholine overflow, since PKC, per se, increases this parameter. After chronic sympathetic denervation such restraint abates, representing a possible mechanism for development of supersensitivity to μ-opioid agents. In the present study, this hypothesis was further investigated. After chronic sympathetic denervation, Ca²⁺-dependent PKC activity was reduced in colonic myenteric plexus synaptosomes. The μ-opioid agent, DAMGO, increased Ca²⁺-dependent PKC activity in synaptosomes obtained from normal, but not from denervated animals. In myenteric synaptosomes obtained from this experimental group, protein levels of Ca²⁺-dependent PKC isoforms βI, βII and γ decreased, whereas α levels increased. In whole-mount preparations, the four Ca²⁺-dependent PKC isoforms co-localized with μ-opioid receptors on subpopulations of colonic myenteric neurons. The percentage of neurons staining for PKCβII, as well as the number of μ-opioid receptor-positive neurons staining for PKCβII, decreased in denervated preparations. The same parameters related to PKCα, βI or γ remained unchanged. Overall, the present data strengthen the concept that μ-opioid receptors located on myenteric neurons are coupled to Ca²⁺-dependent PKCs. After chronic sympathetic denervation, a reduced efficiency of this coupling may predominantly involve PKCβII, although also PKCβI and γ, but not PKCα, may be implicated.