Autocrine and paracrine release of and extracellular signaling by ATP is a ubiquitous cell biological and physiological process. Despite this knowledge, the mechanisms and physiological roles of cellular ATP release are unknown. We tested the hypothesis that epithelia release ATP under basal and stimulated conditions by using a newly designed and highly sensitive assay for bioluminescence detection of ATP released from polarized epithelial monolayers. This bioluminescence assay measures ATP released from cystic fibrosis (CF) and non-CF human epithelial monolayers in a reduced serum medium through catalysis of the luciferase-luciferin reaction, yielding a photon of light collected by a luminometer. This novel assay measures ATP released into the apical or basolateral medium surrounding epithelia. Of relevance to CF, CF epithelia fail to release ATP across the apical membrane under basal conditions. Moreover, hypotonicity is an extracellular signal that stimulates ATP release into both compartments of non-CF epithelia in a reversible manner; the response to hypotonicity is also lost in CF epithelia. The bioluminescence detection assay for ATP released from epithelia and other cells will be useful in the study of extracellular nucleotide signaling in physiological and pathophysiological paradigms. Taken together, these results suggest that extracellular ATP may be a constant regulator of epithelial cell function under basal conditions and an autocrine regulator of cell volume under hypotonic conditions, two functions that may be lost in CF and contribute to CF pathophysiology.

These studies provide evidence that cystic fibrosis transmembrane conductance regulator (CFTR) potentiates and accelerates regulatory volume decrease (RVD) following hypotonic challenge by an autocrine mechanism involving ATP release and signaling. In wild-type CFTR-expressing cells, CFTR augments constitutive ATP release and enhances ATP release stimulated by hypotonic challenge. CFTR itself does not appear to conduct ATP. Instead, ATP is released by a separate channel, whose activity is potentiated by CFTR. Blockade of ATP release by ion channel blocking drugs, gadolinium chloride (Gd 3؉ ) and 4,4-diisothiocyanatostilbene-2,2di-sulfonic acid (DIDS), attenuated the effects of CFTR on acceleration and potentiation of RVD. These results support a key role for extracellular ATP and autocrine and paracrine purinergic signaling in the regulation of membrane ion permeability and suggest that CFTR potentiates ATP release by stimulating a separate ATP channel to strengthen autocrine control of cell volume regulation.ATP and its metabolites function as potent autocrine and paracrine agonists that act within tissues to control cell function through activation of P2 purinergic receptors (1-3) expressed by all cells and tissues. Purinergic agonists are essential for many specialized physiological functions (1-10). In cystic fibrosis (CF), 1 ATP and a related triphosphate nucleotide, UTP, stimulate epithelial chloride (Cl Ϫ ) channels alternative to CFTR via purinergic receptors (11-16). Supraphysiological concentrations of ATP also stimulate CFTR (17). Metabolites of ATP can also act as Cl Ϫ secretagogues (15,16,18). Despite the diverse roles of purinergic signaling, the cellular mechanisms that govern ATP release are not fully defined. CFTR and related ATP-binding cassette (ABC) transporters such as mdr-1 or P-glycoprotein have been implicated as facilitators of ATP release in some cell models (14, 19 -24), while other laboratories have failed to show evidence of CFTRfacilitated ATP conduction or release (25-30).Release of ATP via a conductive pathway has been implicated as an essential autocrine regulator of cell volume in rat hepatoma cells (5). Moreover, ABC transporters have been shown to modulate volume-sensitive Cl Ϫ channels and cell volume (31-34). As such, we tested the hypotheses that CFTR facilitates ATP release under constitutive and hypotonic conditions for autocrine control of cell volume regulation. These hypotheses were also based on the fact that airway surface liquid on CF epithelia is hypertonic with respect to NaCl (35) and/or reduced in volume (36) or both (37, 38) when compared with non-CF epithelia. These airway surface liquid composition abnormalities may reflect an inability of CF epithelial cells to sense changes in external mucosal environment and/or an inability of CF cells to regulate their own cell volume.To this end, complimentary observations using a variety of techniques suggest that expression of CFTR enhances ATP release and modulates the dynamic relationship between cell volume, puriner...

ATP and its metabolites are potent autocrine agonists that act extracellularly within tissues to affect epithelial function. In polycystic kidneys, renal tubules become dilated and/or encapsulated as cysts, creating abnormal microenvironments for autocrine signaling. Previously, our laboratory has shown that high-nanomolar to micromolar quantities of ATP are released from cell monolayers in vitro and detectable in cyst fluids from microdissected human autosomal dominant polycystic kidney (ADPKD) cysts. Here, we show enhanced ATP release from autosomal recessive polycystic kidney (ARPKD) and ADPKD epithelial cell models. RT-PCR and immunoblotting for P2Y G protein-coupled receptors and P2X purinergic receptor channels show expression of mRNA and/or protein for multiple subtypes from both families. Assays of cytosolic Ca2+concentration and secretory Cl− transport show P2Y and P2X purinergic receptor-mediated stimulation of Cl− secretion via cytosolic Ca2+-dependent signaling. Therefore, we hypothesize that autocrine purinergic signaling may augment detrimentally cyst volume expansion in ADPKD or tubule dilation in ARPKD, accelerating disease progression.

People with psychiatric disabilities experience disproportionately high rates of unemployment. As research evidence is mounting regarding effective vocational programs, interest is growing in identifying subgroup variations. Data from a multisite research and demonstration program were analyzed to identify demographic characteristics associated with employment outcomes, after adjusting for the effects of program, services, and study site. Longitudinal analyses found that people with more recent work history, younger age, and higher education were more likely to achieve competitive employment and to work more hours per month, while race and gender effects varied by employment outcome. Results provide strong evidence of demographic subgroup variation and need.

ATP and its metabolites regulate vascular tone; however, the sources of the ATP released in vascular beds are ill defined. As such, we tested the hypothesis that all limbs of an extracellular purinergic signaling system are present in vascular endothelial cells: ATP release, ATP receptors, and ATP receptor-triggered signal transduction. Primary cultures of human endothelial cells derived from multiple blood vessels were grown as monolayers and studied using a bioluminescence detection assay for ATP released into the medium. ATP is released constitutively and exclusively across the apical membrane under basal conditions. Hypotonic challenge or the calcium agonists ionomycin and thapsigargin stimulate ATP release in a reversible and regulated manner. To assess expression of P2X purinergic receptor channel subtypes (P2XRs), we performed degenerate RT-PCR, sequencing of the degenerate P2XR product, and immunoblotting with P2XR subtype-specific antibodies. Results revealed that P2X(4) and P2X(5) are expressed abundantly by endothelial cell primary cultures derived from multiple blood vessels. Together, these results suggest that components of an autocrine purinergic signaling loop exist in the endothelial cell microvasculature that may allow for "self-regulation" of endothelial cell function and modulation of vascular tone.

Extracellular nucleotides regulate NaCl transport in some epithelia. However, the effects of nucleotide agonists on NaCl transport in the renal inner medullary collecting duct (IMCD) are not known. The objective of this study was to determine whether ATP and related nucleotides regulate NaCl transport across mouse IMCD cell line (mIMCD-K2) epithelial monolayers and, if so, via what purinergic receptor subtypes. ATP and UTP inhibited Na(+) absorption [measured via Na(+) short-circuit current (I(Na)(sc))] and stimulated Cl(-) secretion [measured via Cl(-) short-circuit current (I(Cl)(sc))]. Using selective P2 agonists, we report that P2X and P2Y purinoceptors regulate I(Na)(sc) and I(Cl)(sc). By RT-PCR, two P2X receptor channels (P2X(3), P2X(4)) and two P2Y G protein-coupled receptors (P2Y(1), P2Y(2)) were identified. Functional localization of P2 purinoceptors suggest that I(Cl)(sc) is stimulated by apical membrane-resident P2Y purinoceptors and P2X receptor channels, whereas I(Na)(sc) is inhibited by apical membrane-resident P2Y purinoceptors and P2X receptor channels. Together, we conclude that nucleotide agonists inhibit I(Na)(sc) across mIMCD-K2 monolayers through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane, whereas extracellular nucleotides stimulate I(Cl)(sc) through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane.

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