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Adenosine stimulates contraction of airway smooth muscle, but the mechanism is widely considered indirect, depending on release of contractile agonists from mast cells and nerves. The goal was to determine whether adenosine, by itself, directly regulates calcium signaling in human bronchial smooth muscle cells (HBSMC). Primary cultures of HBSMC from normal subjects were loaded with fura 2-AM, and cytosolic calcium concentrations ([Ca^sup 2+^]^sub i^) were determined ratiometrically by imaging single cells. The nonselective adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA), and the adenosine A^sub 1^ receptor agonist, N^sup 6^-cyclopentyladenosine (CPA), both stimulated rapid, transient increases in [Ca^sup 2+^]^sub i^. In contrast, there were no calcium responses to 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamido-adenosine (100 nM) or N^sup 6^-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (100 nM), selective agonists at adenosine A^sub 2A^ receptors and adenosine A^sub 3^ receptors, respectively. Calcium responses to NECA and CPA were inhibited by 8-cyclopentyl-1,3-dipropylxanthine, an adenosine A^sub 1^ receptor antagonist, and by pertussis toxin (PTX). In other experiments, NECA stimulated calcium transients in the absence of extracellular calcium, but not when cells were preincubated in cyclopiazonic acid or thapsigargin to empty intracellular calcium stores. Calcium responses were attenuated by xestospongin C and 2-aminoethoxydiphenylborane, inhibitors of inositol trisphosphate (IP^sub 3^) receptors, and by U73122, an inhibitor of phospholipase C. It was concluded that stimulation of adenosine A^sub 1^ receptors on HBSMC rapidly mobilizes intracellular calcium stores by a mechanism dependent on PTX-sensitive C proteins, and IP^sub 3^ signaling. These findings suggest that, in addition to its well-established indirect effects on HBSMC, adenosine also has direct effects on contractile signaling pathways.
Keywords: adenosine A^sub 1^ receptor; calcium; cAMP; human bronchial smooth muscle; insulin
Adenosinc stimulates airway smooth muscle contraction in normal tissues from humans and animals, and this response is upregulaled in tissues from individuals with asthma and in models of asthma (1-5). Most evidence suggests that adenosine causes airway smooth muscle contraction by stimulating mast cells and nerves to release mediators that secondarily contract the airway smooth muscle cell (1,3. 6-S). However, a lew studies have isolated human airway smooth muscle cells in culture and shown that adenosine has some direct effects on this cell type and that adenosine A^sub 1^, A^sub 2A^, and A^sub 2B^ receptors are all expressed on normal human airway smooth muscle cells. For example, A^sub 2B^...