The charge of this invited review is to present a convincing case for the fact that cells release their ATP for physiological reasons. Many of our “purinergic” colleagues as well as ourselves have ...experienced resistance to this concept, because it is teleologically counter-intuitive. This review serves to integrate the three main tenets of extracellular ATP signaling: ATP release from cells, ATP receptors on cells, and ATP receptor-driven signaling within cells to affect cell or tissue physiology. First principles will be discussed in the Introduction concerning extracellular ATP signaling. All possible cellular mechanisms of ATP release will then be presented. Use of nucleotide and nucleoside scavengers as well as broad-specificity purinergic receptor antagonists will be presented as a method of detecting endogenous ATP release affecting a biological endpoint. Innovative methods of detecting released ATP by adapting luciferase detection reagents or by using “biosensors” will be presented.
Because our laboratory has been primarily interested in epithelial cell physiology and pathophysiology for several years, the role of extracellular ATP in regulation of epithelial cell function will be the focus of this review. For ATP release to be physiologically relevant, receptors for ATP are required at the cell surface. The families of P2Y G protein-coupled receptors and ATP-gated P2X receptor channels will be introduced. Particular attention will be paid to P2X receptor channels that mediate the fast actions of extracellular ATP signaling, much like neurotransmitter-gated channels versus metabotropic heptahelical neurotransmitter receptors that couple to G proteins. Finally, fascinating biological paradigms in which extracellular ATP signaling has been implicated will be highlighted. It is the goal of this review to convert and attract new scientists into the exploding field of extracellular nucleotide signaling and to convince the reader that extracellular ATP is indeed a signaling molecule.
Cilia are complex organelles involved in sensory perception and fluid or cell movement. They are constructed through a highly conserved process called intraflagellar transport (IFT). Mutations in IFT ...genes, such as Tg737 , result in severe developmental defects and disease. In the case of the Tg737 orpk mutants, these pathological alterations include cystic kidney disease, biliary and pancreatic duct abnormalities, skeletal patterning defects, and hydrocephalus. Here, we explore the connection between cilia dysfunction and the development of hydrocephalus by using the Tg737 orpk mutants. Our analysis indicates that cilia on cells of the brain ventricles of Tg737 orpk mutant mice are severely malformed. On the ependymal cells, these defects lead to disorganized beating and impaired cerebrospinal fluid (CSF) movement. However, the loss of the cilia beat and CSF flow is not the initiating factor, as the pathology is present prior to the development of motile cilia on these cells and CSF flow is not impaired at early stages of the disease. Rather, our results suggest that loss of cilia leads to altered function of the choroid plexus epithelum, as evidenced by elevated intracellular cAMP levels and increased chloride concentration in the CSF. These data suggest that cilia function is necessary for regulating ion transport and CSF production, as well as for CSF flow through the ventricles.
ERIK M. SCHWIEBERT ,
DALE J. BENOS ,
MARIE E. EGAN ,
M. JACKSON STUTTS , AND
WILLIAM B. GUGGINO
Department of Physiology and Biophysics, Gregory Fleming James CF Research Center, University of ...Alabama at Birmingham, Birmingham, Alabama; Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut; Department of Medicine, UNC CF Research Group, University of North Carolina, Chapel Hill, North Carolina; and Department of Physiology and Division of Pediatrics, Department of Medicine, Johns Hopkins CF Research Group, Johns Hopkins University School of Medicine, Baltimore, Maryland
Schwiebert, Erik M., Dale J. Benos, Marie E. Egan, M. Jackson Stutts, and William B. Guggino. CFTR Is a Conductance Regulator as well as a Chloride Channel. Physiol. Rev. 79, Suppl. : S145-S166, 1999. Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter gene family. Although CFTR has the structure of a transporter that transports substrates across the membrane in a nonconductive manner, CFTR also has the intrinsic ability to conduct Cl at much higher rates, a function unique to CFTR among this family of ABC transporters. Because Cl transport was shown to be lost in cystic fibrosis (CF) epithelia long before the cloning of the CF gene and CFTR, CFTR Cl channel function was considered to be paramount. Another equally valid perspective of CFTR, however, derives from its membership in a family of transporters that transports a multitude of different substances from chemotherapeutic drugs, to amino acids, to glutathione conjugates, to small peptides in a nonconductive manner. Moreover, at least two members of this ABC transporter family ( mdr-1 , SUR) can regulate other ion channels in the membrane. More simply, ABC transporters can regulate somehow the function of other cellular proteins or cellular functions. This review focuses on a plethora of studies showing that CFTR also regulates other ion channel proteins. It is the hope of the authors that the reader will take with him or her the message that CFTR is a conductance regulator as well as a Cl channel.
During the past two decades, several cell membrane receptors, which preferentially bind extracellular nucleotides, and their analogs have been identified. These receptors, collectively known as ...nucleotide receptors or "purinergic" receptors, have been characterized and classified on the basis of their biological actions, their pharmacology, their molecular biology, and their tissue and cell distribution. For these receptors to have biological and physiological relevance, nucleotides must be released from cells. The field of extracellular ATP release and signaling is exploding, as assays to detect this biological process increase in number and ingenuity. Studies of ATP release have revealed a myriad of roles in local regulatory (autocrine or paracrine) processes in almost every tissue in the body. The regulatory mechanisms that these receptors control or modulate have physiological and pathophysiological roles and potential therapeutic applications. Only recently, however, have ATP release and nucleotide receptors been identified along the renal epithelium of the nephron. This work has set the stage for the study of their physiological and pathophysiological roles in the kidney. This review provides a comprehensive presentation of these issues, with a focus on the renal epithelium.
Department of Physiology and Biophysics and Department of Cell
Biology, University of Alabama at Birmingham, Birmingham, Alabama
35294 - 0005
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.
purinergic receptors; cytosolic calcium; ectoadenosinetriphosphatases; exocytosis; nitric oxide
Department of Physiology and Biophysics, Department of Cell
Biology, Gregory Fleming James Cystic Fibrosis Research Center,
University of Alabama at Birmingham, Birmingham, Alabama 35294-0005
The ...concept that the cystic fibrosis (CF) transmembrane
conductance regulator, the protein product of the CF gene, can conduct larger multivalent anions such as ATP as well as
Cl is controversial. In
this review, I examine briefly past findings that resulted in
controversy. It is not the goal of this review to revisit these
disparate findings in detail. Rather, I focus intently on more recent
studies, current studies in progress, and possible future directions
that arose from the controversy and that may reconcile this issue.
Important questions and hypotheses are raised as to the physiological
roles that ATP-binding cassette (ABC) transporter-facilitated ATP
transport and signaling may play in the control of epithelial cell
function. Perhaps the identification of key biological paradigms for
ABC transporter-mediated extracellular nucleotide signaling may unify
and guide the CF research community and other research groups
interested in ABC transporters toward understanding why ABC
transporters facilitate ATP transport.
cystic fibrosis transmembrane conductance regulator; multidrug
resistance protein; P-glycoprotein; autocrine; paracrine; signaling; ion transport; cystic fibrosis; ATP-binding cassette
It is well established that ATP is co-secreted with insulin and zinc from pancreatic beta-cells (β-cells) in response to elevations in extracellular glucose concentration. Despite this knowledge, the ...physiological roles of extracellular secreted ATP and zinc are ill-defined. We hypothesized that secreted ATP and zinc are autocrine purinergic signaling molecules that activate P2X purinergic receptor (P2XR) channels expressed by β-cells to enhance glucose-stimulated insulin secretion (GSIS). To test this postulate, we performed ELISA assays for secreted insulin at fixed time points within a “real-time” assay and confirmed that the physiological insulin secretagogue glucose stimulates secretion of ATP and zinc into the extracellular milieu along with insulin from primary rat islets. Exogenous ATP and zinc alone or together also induced insulin secretion in this model system. Most importantly, the presence of an extracellular ATP scavenger, a zinc chelator, and P2 receptor antagonists attenuated GSIS. Furthermore, mRNA and protein were expressed in immortalized β-cells and primary islets for a unique subset of P2XR channel subtypes, P2X
2
, P2X
3
, P2X
4
, and P2X
6
, which are each gated by extracellular ATP and modulated positively by extracellular zinc. On the basis of these results, we propose that, within endocrine pancreatic islets, secreted ATP and zinc have profound autocrine regulatory influence on insulin secretion via ATP-gated and zinc-modulated P2XR channels.
PDF
