Role of the A(2B) receptor-adenosine deaminase complex in colonic dysmotility associated with bowel inflammation in rats.

BACKGROUND AND PURPOSE: Adenosine A(2B) receptors regulate several physiological enteric functions. However, their role in the pathophysiology of intestinal dysmotility associated with inflammation has not been elucidated. Hence, we investigated the expression of A2B receptors in rat colon and their role in the control of cholinergic motility in the presence of bowel inflammation. EXPERIMENTAL APPROACH: Colitis was induced by 2,4-dinitrobenzenesulfonic acid (DNBS). Colonic A(2B) receptor expression and localization were examined by RT-PCR and immunofluorescence. The interaction between A(2B) receptors and adenosine deaminase was assayed by immunoprecipitation. The role of A(2B) receptors in the control of colonic motility was examined in functional experiments on longitudinal muscle preparations (LMPs). KEY RESULTS: A(2B) receptor mRNA was present in colon from both normal and DNBS-treated rats but levels were increased in the latter. A(2B) receptors were predominantly located in the neuromuscular layer, but, in the presence of colitis, were increased mainly in longitudinal muscle. Functionally, the A(2B) receptor antagonist MRS 1754 enhanced both electrically-evoked and carbachol-induced cholinergic contractions in normal LMPs, but was less effective in inflamed tissues. The A(2B) receptor agonist NECA decreased colonic cholinergic motility, with increased efficacy in inflamed LMP. Immunoprecipitation and functional tests revealed a link between A(2B) receptors and adenosine deaminase, which colocalize in the neuromuscular compartment. CONCLUSIONS AND IMPLICATIONS: Under normal conditions, endogenous adenosine modulates colonic motility via A2B receptors located in the neuromuscular compartment. In the presence of colitis, this inhibitory control is impaired due to a link between A2B receptors and adenosine deaminase, which catabolizes adenosine, thus preventing A(2B) receptor activation.

Control of enteric neuromuscular functions by purinergic A(3) receptors in normal rat distal colon and experimental bowel inflammation.

BACKGROUND AND PURPOSE: Adenosine A(3) receptors mediate beneficial effects in experimental colitis, but their involvement in enteric neuromuscular functions during bowel inflammation is undetermined. This study investigated the regulatory role of A(3) receptors on colonic motility in the presence of experimental colitis. EXPERIMENTAL APPROACH: Colitis was induced in rats by 2,4-dinitrobenzenesulfonic acid. A(3) receptors and adenosine deaminase (ADA, adenosine catabolic enzyme) mRNA were examined by RT-PCR. Tissue distribution of A(3) receptors was detected by confocal immunofluorescence. The effects of 2,3-ethyl-4,5-dipropyl-6-phenylpyridine-3-thiocarboxylate-5-carboxylate (MRS1523) (MRS, A(3) receptor antagonist), 2-chloro-N(6) -(3-iodobenzyl)-adenosine-5'-N-methyluronamide (2Cl-IB-MECA) (CIB, A(3) receptor agonist), dipyridamole (DIP, adenosine transport inhibitor) and ADA were assayed on contractile responses evoked by electrical stimulation (ES) or carbachol in colonic longitudinal muscle preparations (LMP). KEY RESULTS: RT-PCR showed A(3) receptors and ADA mRNA in normal colon and their increased level in inflamed tissues. Immunofluorescence showed a predominant distribution of A(3) receptors in normal myenteric ganglia and an increased density during colitis. MRS enhanced ES-induced cholinergic contractions in normal LMP, but was less effective in inflamed tissues. After pretreatment with dipyridamole plus ADA, to reduce extracellular adenosine, CIB decreased cholinergic motor responses of normal LMP to ES, with enhanced efficacy in inflamed LMP. A(3) receptor ligands did not affect carbachol-induced contractions in LMP from normal or inflamed colon. CONCLUSIONS AND IMPLICATIONS: Normally, adenosine modulated colonic cholinergic motility via activation of A(3) receptors in the myenteric plexus. A(3) receptor-mediated tonic inhibitory control by adenosine was impaired in inflamed bowel, despite increased density of functioning and pharmacologically recruitable A(3) receptors.

Radiopharmaceutical pharmacokinetics in animals: critical considerations.

Since the advent of single photon emission computerized tomography (SPECT) and positron emission tomography (PET) various chemical ligands have been labeled with radionuclides and evaluated as tracer compounds in animal models to ascertain their suitability as potential radiopharmaceuticals for humans. In the absence of a defined algorithm to predict the diagnostic efficacy of a radiopharmaceutical, any new radioligand has to undergo preclinical evaluation even if it has excellent in vitro properties. Until now few studies have produced pharmacokinetic data that could be translated from animal models directly to humans. The purpose of this review is to highlight some critical aspects to consider during the development and validation phase of a new radiopharmaceutical. Interspecies differences and the absence of knowledge of physiological mechanism can become challenging drawbacks for obtaining a successful radiopharmaceutical. In this context, the influence of ABC transporters in neuroimaging, the effect of plasma protein binding and the consequence of anesthesia with reference to interspecies differences will be discussed with illustrative examples.

Cytochrome P450 and radiopharmaceutical metabolism.

Positron emission tomography (PET) is a powerful non-invasive probe to investigate human physiology. A large number of radiotracers have been studied as imaging agents, but only a few have found clinical applications in pharmacology. A potential radiopharmaceutical is designed with very specific physiochemical characteristics, but, generally, less attention is paid to its adsorption, distribution, metabolism, and excretion properties, especially metabolism. Understanding the metabolic fate of radiopharmaceutical probes is essential for an accurate analysis and interpretation of PET measurements. The inherent inability of PET to differentiate between a parent compound and its metabolites confounds the interpretation of images and may impact the identification of the pathologically induced biochemical changes under investigation. Cytochrome P450 plays a major role in mammalian xenobiotic biotransformation and many in vitro methods are available to study and predict drug metabolism. The purpose of this review is to highlight the existing in vitro techniques available to investigate the biotransformation of xenobiotics in a fashion analogous to small molecule drug discovery. The aim is to facilitate the development and validation phases of PET tracers during preclinical evaluation. Emphasis is placed also on describing how cross species comparisons are essential in establishing appropriate translational pharmacology. Procedures of analysis (tandem liquid chromatography-mass spectrometry), typically used for studying the metabolism of drugs, are proposed as quick and accurate tools for the determination of a radiopharmaceutical's metabolic stability at the tracer level.

A preliminary study of haemolymph from four Venezuelan populations of Panstrongylus geniculatus Latreille, 1811 (Hemiptera: Reduviidae) and its epidemiological significance.

SDS-PAGE profiles of both sexes of the haemolymphs of Panstrongylus geniculatus from different Venezuelan regions (savannas, piedmont, tropical forest and urban areas) were compared. It was determined that the haemolymphs showed a different electrophoretic profile, with proteins that ranged from 14 to 164 kDa. The most representative protein band in the profile of females was observed in two sectors: between 164 and 46 kDa and between 33 and 30 kDa. The main illustrative protein band in males was observed in one region: from 46 to 35 kDa. The Haemolymph composition of P. geniculatus from populations evaluated in this work expressed high homogeneity of this species with a clear difference between males and females. This similarity may be useful for control of these insects, taking into account that the genetic stability may be very important, since the use of an insecticide in a population with these characteristics is always more successful. According to the bibliographic review, this is the first study of haemolymph from Panstrongylus geniculatus.

99mTc-labeling experiments on CCK(4) by a direct method.

99mTc-labeling studies have been performed on CCK(4) fragment of cholecystokinin, starting from 99mTc-pertechnetate, by using tin(II)pyrophosphate or tin(II)gluconate as reducing agents, together with NaBH(4) acting as a stabilizing agent of tin(II). Gluconate has been used as exchange ligand in the carrier added experiments and in the syntheses of 99Tc-CCK(4) and Re-CCK(4) complexes to be able to reproduce at macroscopic level the same chemical reactions occurring at non carrier added conditions. 99mTc-labeling yields higher than 95% have been achieved depending on Sn(II) concentration, CCK(4)/gluconate ratio, reaction time and applied temperature. The species produced with 99mTc, 99Tc, and cold rhenium nuclides have been compared by means of HPLC measurements, which showed similar retention times and thus probably the same species in the three situations.

Evaluation of gallium-68 tris(2-mercaptobenzyl)amine: a complex with brain and myocardial uptake.

Previous research into development of a gallium-radiolabeled agent that crosses the blood-brain barrier has met with limited success. In this study, we focused our attention on a Ga(III) complex of a 4-coordinate amine trithiolate tripod ligand, tris(2-mercaptobenzyl) amine (S3N). The Ga(III) S3N complex is small, neutral, and lipophilic, meeting the requirements for a potential brain imaging agent. The Ga-68 complex was easily formed with a radiochemical purity of >95%. In vitro stability of the Ga-S3N complex, determined in rat serum incubated at 37 degrees C, was greater than 95% intact at 2 h by silica gel and reversed-phase radio-thin layer chromatography. Biodistribution studies conducted in female Sprague-Dawley rats showed the complex cleared rapidly from the blood with initial high liver uptake followed by rapid washout. Significant uptake was observed in the brain, with brain:blood ratios increasing from 0.11 at 2 min postinjection to 3.8 at 60 min postinjection. Uptake was also observed in the heart going from a heart:blood ratio of 2.3 at 2 min postinjection to 11 at 60 min postinjection. Molecular mechanics were used to determine the coordination number, and demonstrated that the Ga(III) complex prefers to be 4-coordinate. Imaging studies with 68Ga-S3N in a Nemestrina macaque showed significant brain uptake, similar to other lipophilic agents. The extraction of 68Ga-S3N into the brains of both rodents and primates, higher than any 68Ga agent reported in the literature, suggests that this compound may have potential as a brain imaging agent for positron emission tomography.