The ecto-enzymes CD73 and adenosine deaminase modulate 5′-AMP-derived adenosine in myofibroblasts of the rat small intestine

Abstract

Adenosine is a versatile signaling molecule recognized to physiologically influence gut motor functions. Both the duration and magnitude of adenosine signaling in enteric neuromuscular function depend on its availability, which is regulated by the ectoenzymes ecto-5′-nucleotidase (CD73), alkaline phosphatase (AP), and ecto-adenosine deaminase (ADA) and by dipyridamolesensitive equilibrative transporters (ENTs). Our purpose was to assess the involvement of CD73, APs, ecto-ADA in the formation of AMP-derived adenosine in primary cultures of ileal myofibroblasts (IMFs). IMFs were isolated from rat ileum longitudinal muscle segments by means of primary explant technique and identified by immunofluorescence staining for vimentin and αsmooth muscle actin. IMFs confluent monolayers were exposed to exogenous 5′-AMP in the presence or absence of CD73, APs, ecto-ADA, or ENTs inhibitors. The formation of adenosine and its metabolites in the IMFs medium was monitored by highperformance liquid chromatography. The distribution of CD73 and ADA in IMFs was detected by confocal immunocytochemistry and qRT-PCR. Exogenous 5′-AMP was rapidly cleared being almost undetectable after 60-min incubation, while adenosine levels significantly increased. Treatment of IMFs with CD73 inhibitors markedly reduced 5′-AMP clearance whereas ADA blockade or inhibition of both ADA and ENTs prevented adenosine catabolism. By contrast, inhibition of APs did not affect 5′-AMP metabolism. Immunofluorescence staining and qRT-PCR analysis confirmed the expression of CD73 and ADA in IMFs. Overall, our data show that in IMFs an extracellular AMP-adenosine pathway is functionally active and among the different enzymatic pathways regulating extracellular adenosine levels, CD73 and ecto-ADA represent the critical catabolic pathway.

Involvement of hyaluronan in the adaptive changes of the rat small intestine neuromuscular function after ischemia/reperfusion injury

“…Total RNA from small intestine LMMPs was extracted with TRIzol (Invitrogen) and treated with DNase I (DNase Free, Ambion), to remove possible traces of contaminating DNA. 2.5 μg of total RNA were then retrotranscribed using the High Capacity cDNA Synthesis Kit (Applied Biosystems, ThermoScientific, Massachusetts, USA) as previously described 64 . Quantitative RT-PCR (qRT-PCR) was performed with an Abi Prism 7,000 real-time thermocyclator (Applied Biosystems).…”

Glutamatergic Signaling Along The Microbiota-Gut-Brain Axis

“…Local intestinal reflexes are coordinated by several types of ascending and descending interneurons, which are characterized by different chemical coding, according to their projection and function [29]. A distinctive feature of the ENS is that enteric neurons communicate with different cell types, which constitute the enteric microenvironment including enteric glial cells, smooth muscle cells and the interstitial cells of Cajal, which are considered intestinal pacemaker cells, immunocytes of the gut-associated lymphoid tissue (which represent the most important immune cell reservoir of the human body), ECCs, which contain more than 20 identified hormones and microbes of the commensal flora [80,81,82,83]. In this latter context, there are several reports suggesting that the enteric microflora may influence the activity of both motor and sensory enteric neurons.…”

Therapeutic Perspectives of Adenosine Deaminase Inhibition in Cardiovascular Diseases

“…However, during inflammation or hypoxia, adenosine is produced from intracellular ATP under conditions of low energy charge. AMP, which originates from ATP degradation is rapidly transformed to adenosine that is not immediately deaminated to inosine due to high Km of ADA (25–100 µM) [ 17 ]. This results in temporary accumulation of adenosine, which is vigorously exported out the cell via equilibrative nucleoside transporters (ENTs) [ 18 ].…”