Erik M. Schwiebert

Dysfunctional cilia lead to altered ependyma and choroid plexus function, and result in the formation of hydrocephalus

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 Tg737orpk 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 Tg737orpk mutants. Our analysis indicates that cilia on cells of the brain ventricles of Tg737orpk 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.

Bioluminescence detection of ATP release mechanisms in epithelia

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.

Membrane Trafficking of the Cystic Fibrosis Gene Product, Cystic Fibrosis Transmembrane Conductance Regulator, Tagged with Green Fluorescent Protein in Madin-Darby Canine Kidney Cells

The mechanism by which cAMP stimulates cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride (Cl−) secretion is cell type-specific. By using Madin-Darby canine kidney (MDCK) type I epithelial cells as a model, we tested the hypothesis that cAMP stimulates Cl− secretion by stimulating CFTR Cl− channel trafficking from an intracellular pool to the apical plasma membrane. To this end, we generated a green fluorescent protein (GFP)-CFTR expression vector in which GFP was linked to the N terminus of CFTR. GFP did not alter CFTR function in whole cell patch-clamp or planar lipid bilayer experiments. In stably transfected MDCK type I cells, GFP-CFTR localization was substratum-dependent. In cells grown on glass coverslips, GFP-CFTR was polarized to the basolateral membrane, whereas in cells grown on permeable supports, GFP-CFTR was polarized to the apical membrane. Quantitative confocal fluorescence microscopy and surface biotinylation experiments demonstrated that cAMP did not stimulate detectable GFP-CFTR translocation from an intracellular pool to the apical membrane or regulate GFP-CFTR endocytosis. Disruption of the microtubular cytoskeleton with colchicine did not affect cAMP-stimulated Cl− secretion or GFP-CFTR expression in the apical membrane. We conclude that cAMP stimulates CFTR-mediated Cl− secretion in MDCK type I cells by activating channels resident in the apical plasma membrane.

Extracellular ATP and cAMP as Paracrine and Interorgan Regulators of Renal Function ATP Release Mechanisms, ATP Receptors and Purinergic Signalling along the Nephron

1. Autocrine and paracrine signalling along the nephron of the kidney has been a widely held hypothesis for several decades. The lumen of the nephron is an ideal autocrine and paracrine signalling microenvironment. Any agonist, filtered at the glomerulus or released in the proximal tubule or other proximal segments, is subsequently trapped in the lumen of the nephron and present to interact with luminal receptors. Similar signalling in the renal interstitium is also possible and likely. Indeed, receptors for many autocrine and paracrine agonists have been characterized on the luminal membrane and serosal membrane of multiple nephron segments.

Epithelial P2X purinergic receptor channel expression and function

P2X purinergic receptor (P2XR) channels bind ATP and mediate Ca(2+) influx--2 signals that stimulate secretory Cl(-) transport across epithelia. We tested the hypotheses that P2XR channels are expressed by epithelia and that P2XRs transduce extracellular ATP signals into stimulation of Cl(-) transport across epithelia. Electrophysiological data and mRNA analysis of human and mouse pulmonary epithelia and other epithelial cells indicate that multiple P2XRs are broadly expressed in these tissues and that they are active on both apical and basolateral surfaces. Because P2X-selective agonists bind multiple P2XR subtypes, and because P2X agonists stimulate Cl(-) transport across nasal mucosa of cystic fibrosis (CF) patients as well as across non-CF nasal mucosa, P2XRs may provide novel targets for extracellular nucleotide therapy of CF.

ABC transporter-facilitated ATP conductive transport.

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.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.

Nucleotides regulate NaCl transport in mIMCD-K2 cells via P2X and P2Y purinergic receptors

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[Formula: see text])] and stimulated Cl- secretion [measured via Cl-short-circuit current ([Formula: see text])]. Using selective P2 agonists, we report that P2X and P2Y purinoceptors regulate [Formula: see text] and[Formula: see text]. By RT-PCR, two P2X receptor channels (P2X3, P2X4) and two P2Y G protein-coupled receptors (P2Y1, P2Y2) were identified. Functional localization of P2 purinoceptors suggest that [Formula: see text] is stimulated by apical membrane-resident P2Y purinoceptors and P2X receptor channels, whereas[Formula: see text] is inhibited by apical membrane-resident P2Y purinoceptors and P2X receptor channels. Together, we conclude that nucleotide agonists inhibit[Formula: see text] across mIMCD-K2 monolayers through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane, whereas extracellular nucleotides stimulate [Formula: see text]through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane.

Adenosine regulates a chloride channel via protein kinase C and a G protein in a rabbit cortical collecting duct cell line.

We examined the regulation by adenosine of a 305-pS chloride (Cl-) channel in the apical membrane of a continuous cell line derived from rabbit cortical collecting duct (RCCT-28A) using the patch clamp technique. Stimulation of A1 adenosine receptors by N6-cyclohexyladenosine (CHA) activated the channel in cell-attached patches. Phorbol 12,13-didecanoate and 1-oleoyl 2-acetylglycerol, activators of protein kinase C (PKC), mimicked the effect of CHA, whereas the PKC inhibitor H7 blocked the action of CHA. Stimulation of A1 adenosine receptors also increased the production of diacylglycerol, an activator of PKC. Exogenous PKC added to the cytoplasmic face of inside-out patches also stimulated the Cl- channel. Alkaline phosphatase reversed PKC activation. These results show that stimulation of A1 adenosine receptors activates a 305-pS Cl-channel in the apical membrane by a phosphorylation-dependent pathway involving PKC. In previous studies, we showed that the protein G alpha i-3 activated the 305-pS Cl- channel (Schwiebert et al. 1990. J. Biol. Chem. 265:7725-7728). We, therefore, tested the hypothesis that PKC activates the channel by a G protein-dependent pathway. In inside-out patches, pertussis toxin blocked PKC activation of the channel. In contrast, H7 did not prevent G protein activation of the channel. We conclude that adenosine activates a 305-pS Cl- channel in the apical membrane of RCCT-28A cells by a membrane-delimited pathway involving an A1 adenosine receptor, phospholipase C, diacylglycerol, PKC, and a G protein. Because we have shown, in previous studies, that this Cl- channel participates in the regulatory volume decrease subsequent to cell swelling, adenosine release during ischemic cell swelling may activate the Cl-channel and restore cell volume.

Loss of apical monocilia on collecting duct principal cells impairs ATP secretion across the apical cell surface and ATP-dependent and flow-induced calcium signals

Renal epithelial cells release ATP constitutively under basal conditions and release higher quantities of purine nucleotide in response to stimuli. ATP filtered at the glomerulus, secreted by epithelial cells along the nephron, and released serosally by macula densa cells for feedback signaling to afferent arterioles within the glomerulus has important physiological signaling roles within kidneys. In autosomal recessive polycystic kidney disease (ARPKD) mice and humans, collecting duct epithelial cells lack an apical central cilium or express dysfunctional proteins within that monocilium. Collecting duct principal cells derived from an Oak Ridge polycystic kidney (orpkTg737) mouse model of ARPKD lack a well-formed apical central cilium, thought to be a sensory organelle. We compared these cells grown as polarized cell monolayers on permeable supports to the same cells where the apical monocilium was genetically rescued with the wild-type Tg737 gene that encodes Polaris, a protein essential to cilia formation. Constitutive ATP release under basal conditions was low and not different in mutant versus rescued monolayers. However, genetically rescued principal cell monolayers released ATP three- to fivefold more robustly in response to ionomycin. Principal cell monolayers with fully formed apical monocilia responded three- to fivefold greater to hypotonicity than mutant monolayers lacking monocilia. In support of the idea that monocilia are sensory organelles, intentionally harsh pipetting of medium directly onto the center of the monolayer induced ATP release in genetically rescued monolayers that possessed apical monocilia. Mechanical stimulation was much less effective, however, on mutant orpk collecting duct principal cell monolayers that lacked apical central monocilia. Our data also show that an increase in cytosolic free Ca2+ primes the ATP pool that is released in response to mechanical stimuli. It also appears that hypotonic cell swelling and mechanical pipetting stimuli trigger release of a common ATP pool. Cilium-competent monolayers responded to flow with an increase in cell Ca2+ derived from both extracellular and intracellular stores. This flow-induced Ca2+ signal was less robust in cilium-deficient monolayers. Flow-induced Ca2+ signals in both preparations were attenuated by extracellular gadolinium and by extracellular apyrase, an ATPase/ADPase. Taken together, these data suggest that apical monocilia are sensory organelles and that their presence in the apical membrane facilitates the formation of a mature ATP secretion apparatus responsive to chemical, osmotic, and mechanical stimuli. The cilium and autocrine ATP signaling appear to work in concert to control cell Ca2+. Loss of a cilium-dedicated autocrine purinergic signaling system may be a critical underlying etiology for ARPKD and may lead to disinhibition and/or upregulation of multiple sodium (Na+) absorptive mechanisms and a resultant severe hypertensive phenotype in ARPKD and, possibly, other diseases.

The cytoskeleton and membrane transport.

This review summarizes recent studies demonstrating that the actin-based cytoskeleton regulates the activity of many ion channels and transport proteins. Stretch-activated ion channels; channels conductive to chloride, to sodium, and to potassium; electroneutral transport proteins, including the Na(+)-K(+)-2Cl-cotransporter, the Na+/H+ exchanger, and the organic osmolyte transporter; as well as water channels are all regulated by the actin cytoskeleton. Several potential mechanisms whereby the actin cytoskeleton and microtubules regulate transport protein activity are discussed.