Biomedical subjects

Amal K Dutta

Publications and source records attributed to Amal K Dutta.

4 recordsLinked to original sources

PubMed2004

Detecting ATP release by a biosensor method.

Cells release adenosine 5'-triphosphate (ATP) into the extracellular space in response to various stimuli. This released ATP plays an important physiological role in cell-to-cell signal transduction. The bulk ATP concentration can be detected using a conventional luciferin-luciferase assay. However, the ATP concentration in the vicinity of the cell surface is often different from the bulk concentration because of its rapid degradation by ecto-ATPases and because of delayed diffusion due to unstirred layer effects. Here, we describe a simple biosensor method to measure the local ATP concentration on the cell surface in real time. The method is based on the ATP-dependent opening of ligand-gated cation channels of purinergic P2X receptors expressed in undifferentiated pheochromocytoma (PC12) cells or in human embryonic kidney 293 (HEK293) cells stably transfected with recombinant P2X2 purinergic receptors. Under the whole-cell configuration of patch-clamp, a sensor PC12 cell or HEK293 is positioned within the proximity of a target cell, and the P2X-mediated currents induced by ATP released from a given site on the target cell surface is measured. The ATP release is quantified by a calibration procedure utilizing local puff applications of ATP at preset concentrations.

Adenosine Triphosphate↗

PubMed2004

Role of ATP-conductive anion channel in ATP release from neonatal rat cardiomyocytes in ischaemic or hypoxic conditions.

It is known that the level of ATP in the interstitial spaces within the heart during ischaemia or hypoxia is elevated due to its release from a number of cell types, including cardiomyocytes. However, the mechanism by which ATP is released from these myocytes is not known. In this study, we examined a possible involvement of the ATP-conductive maxi-anion channel in ATP release from neonatal rat cardiomyocytes in primary culture upon ischaemic, hypoxic or hypotonic stimulation. Using a luciferin-luciferase assay, it was found that ATP was released into the bulk solution when the cells were subjected to chemical ischaemia, hypoxia or hypotonic stress. The swelling-induced ATP release was inhibited by the carboxylate- and stilbene-derivative anion channel blockers, arachidonic acid and Gd3+, but not by glibenclamide. The local concentration of ATP released near the cell surface of a single cardiomyocyte, measured by a biosensor technique, was found to exceed the micromolar level. Patch-clamp studies showed that ischaemia, hypoxia or hypotonic stimulation induced the activation of single-channel events with a large unitary conductance (approximately 390 pS). The channel was selective to anions and showed significant permeability to ATP4- (PATP/PCl approximately 0.1) and MgATP2- (PATP/PCl approximately 0.16). The channel activity exhibited pharmacological properties essentially identical to those of ATP release. These results indicate that neonatal rat cardiomyocytes respond to ischaemia, hypoxia or hypotonic stimulation with ATP release via maxi-anion channels.

Adenosine Triphosphate↗

PubMed2003

Ischemia-induced enhancement of CFTR expression on the plasma membrane in neonatal rat ventricular myocytes.

Pathophysiological functions of cardiac cystic fibrosis transmembrane conductance regulator (cCFTR) in ischemia are not well known. Using neonatal rat ventricular cardiomyocytes in primary culture in this study, we thus examined whether the CFTR protein is expressed and is functioning as a cAMP-activated anion channel on the plasma membrane under ischemic conditions. After the cells were subjected to simulated ischemia (O(2) and glucose deprivation), an up-regulation of the CFTR expression was transiently observed in the membrane fraction by Western blot. A peak expression of mature CFTR protein was found at 3 h of ischemia, and thereafter the signal diminished gradually. In contrast, the results of Northern blot indicated that the expression level of CFTR mRNA changed little until 3 h of ischemia, whereas the level slightly decreased after 8 h of ischemia. An immunohistochemical examination showed, in agreement with the results of Western blot analysis, that the expression of CFTR protein on the plasma membrane became most prominent at 3 h of ischemia, whereas the plasmalemmal CFTR signal was markedly reduced after 8 h of ischemia. Whole-cell recordings showed that the cardiomyocytes responded to cAMP with an activation of time- and voltage-independent currents that contained an anion-selective component sensitive to CFTR Cl(-) channel blockers (NPPB and glibenclamide) but not to a stilbene-derivative conventional Cl(-) channel blocker (SITS). This cAMP-activated Cl(-) channel current was found to be enhanced after an application of ischemic stress for 3 to 4 h. These findings indicate that a plasmalemmal expression of CFTR is transiently enhanced under glucose-free hypoxic conditions presumably because of a posttranslational control.

Animals↗

PubMed2002

Regulation of an ATP-conductive large-conductance anion channel and swelling-induced ATP release by arachidonic acid.

Mouse mammary C127 cells responded to hypotonic stimulation with activation of the volume-dependent ATP-conductive large conductance (VDACL) anion channel and massive release of ATP. Arachidonic acid downregulated both VDACL currents and swelling-induced ATP release in the physiological concentration range with K(d) of 4- 6 microM. The former effect observed in the whole-cell or excised patch mode was more prominent than the latter effect observed in intact cells. The arachidonate effects were direct and not mediated by downstream metabolic products, as evidenced by their insensitivity to inhibitors of arachidonate-metabolizing oxygenases, and by the observation that they were mimicked by cis-unsaturated fatty acids, which are not substrates for oxygenases. A membrane-impermeable analogue, arachidonyl coenzyme A was effective only from the cytosolic side of membrane patches suggesting that the binding site is localized intracellularly. Non-charged arachidonate analogues as well as trans-unsaturated and saturated fatty acids had no effect on VDACL currents and ATP release, indicating the importance of arachidonate's negative charge and specific hydrocarbon chain conformation in the inhibitory effect. VDACL anion channels were inhibited by arachidonic acid in two different ways: channel shutdown (K(d) of 4- 5 microM) and reduced unitary conductance (K(d) of 13-14 microM) without affecting voltage dependence of open probability. ATP(4-)-conducting inward currents measured in the presence of 100 mM ATP in the bath were reversibly inhibited by arachidonic acid. Thus, we conclude that swelling-induced ATP release and its putative pathway, the VDACL anion channel, are under a negative control by intracellular arachidonic acid signalling in mammary C127 cells.

Adenosine Triphosphate↗