The Effects of Extracellular Purines and Pyrimidines on Human Airway Smooth Muscle Cells

Abstract

Extracellular ATP and UTP modulate the function of many cell types through the stimulation of specific P2 receptors, and the inhalation of UTP has been proposed as a therapeutic means of increasing mucociliary clearance in cystic fibrosis patients. The aim of this study was to determine whether P2 receptors are present and functional in human airway smooth muscle (HASM) cells. Experiments were conducted on primary cultures of HASM cells. Reverse transcription-polymerase chain reaction and Western blot analysis showed that P2Y 1 , P2Y 2 , P2Y 4 , and P2Y 6 receptor subtypes are expressed. Exposure to extracellular ATP, UTP, ADP, and UDP at concentrations ranging from 10 Ϫ6 to 10 Ϫ4 M, produced significant increases in intracellular Ca 2ϩ that peaked to 491 Ϯ 51 nM (p Ͻ 0.001) with ATP 10 Ϫ5 M and to 321 Ϯ 30 nM with UTP 10 Ϫ4 M. ATP and UTP also induced HASM cell contraction, decreasing cell length by 9.9 Ϯ 4.3 and 5.6 Ϯ 2.0%, respectively. Pretreatment of the cells with UTP for short periods of time (10 and 30 min) enhanced the peak Ca 2ϩ release to UTP, whereas repeated and prolonged pretreatment with UTP decreased it. These results indicate that several subtypes of P2Y receptors are present and functional in HASM cells. They also show that the response of the receptors is increased after short periods of exposure to UTP and decreased after prolonged and repeated exposure. Considering that ATP and UTP are endogenous mediators and that analogs of UTP could be used as a therapeutic modality, the role of extracellular triphosphate nucleotides in physiological and pathophysiological processes in the airways warrants further investigation.Extracellular triphosphate nucleotides such as ATP, UTP, and related compounds have significant biological effects, including the modulation of synaptic transmission in the central and peripheral nervous systems, mast cell degranulation, and the modulation of visceral and vascular smooth muscle contractility (for review, see Ralevic and Burnstock, 1998). In the cardiovascular system, extracellular ATP has positive inotropic and chronotropic effects on the myocardium and induces contraction and relaxation of vascular smooth muscle cells (Ralevic and Burnstock, 1998). It also regulates vascular smooth muscle cell proliferation and apoptosis, and it is thought to be a significant mediator of vessel wall remodeling (Boarder and Hourani, 1998;Erlinge, 2004).Although the effects of extracellular purine nucleotides have been extensively investigated in the cardiovascular, urogenital, gastrointestinal, and nervous systems, research on the effects of these compounds in the respiratory system is more recent. In the respiratory system, ATP and UTP act on various cell types: ATP is one of the most powerful known agonists for surfactant secretion by alveolar type II cells, and it also increases Cl Ϫ secretion and arachidonic acid metabolism when applied to the apical surface of cultured airway epithelial cells (Knowles et al., 1991;Hwang et al., 1996). Mucin secretion by submucosal glan...

In airways ATP refills sarcoplasmic reticulum via P2X smooth muscle receptors and induces contraction through P2Y epithelial receptors

“…Thus, further research in this topic is required to elucidate the purinergic receptors involved in the suramin-and RB2-resistant response. ATP-induced [Ca 2+ ] i increment has been associated with activation of P2Y and P2X receptors in the airway smooth muscle from different species [4,17,27]. However, this is the first report in guinea pig airway smooth muscle showing that both purinergic receptors are responsible for the [Ca 2+ ] i increment induced by ATP.…”

“…Thus, the contraction induced by ATP in guinea pig trachea seems to be an indirect effect, i. e., does not involve a direct stimulation of purinergic receptors in the smooth muscle cells to produce contraction. Nevertheless, ATP-induced [Ca 2+ ] i increment has been associated with the stimulation of P2Y or P2X receptors in the airway smooth muscle [4,17,27].…”

“…P2 receptors are classified in P2X and P2Y subtypes. P2X receptors are ligand-gated nonselective cation channels [29], and P2Y receptors belong to G protein-coupled receptors linked to cytosolic Ca 2+ elevation through phosphoinositol hydrolysis [4,17].…”

Characterization of P2Y receptors mediating ATP induced relaxation in guinea pig airway smooth muscle: involvement of prostaglandins and K+ channels

“…These receptors are classified in P2X 1-7 and P2Y 1,2,4,6,11,12,13,14 subtypes [7]. P2X receptors are ligand-gated channels [20] and P2Y receptors are coupled to G proteins linked to IP 3 synthesis [4,11].…”

Purinergic Signaling in the Airways

“…In a later article, a more detailed analysis of the actions of ATP on human and rat bronchial smooth muscle led to the conclusion that ATP induces a transient contractile response mainly mediated by P2X4 receptors, but P2Y receptors were also present (Mounkaïla et al, 2005). RT-PCR and Western blot analyses carried out on primary cultures of human airway smooth muscle showed expression of P2Y 1 , P2Y 2 , P2Y 4 , and P2Y 6 receptors and ATP, UTP, ADP, and UDP all produced a significant increase in [Ca 2ϩ ] i and contraction (Govindaraju et al, 2005).…”