1 The role of extracellular nucleotides in regulation of ion transport activities (short circuit current, Is¢) of human respiratory epithelia was studied. 2 Application of nucleotides to the apical or basolateral membrane of human nasal epithelium induced a concentration-dependent increase in IS.. 3 The rank order of potency of purine-or pyrimidine-induced changes in ISC of normal human nasal epithelium when applied to the apical membrane (UTP > ATP > ATPyS > 2MeSATP > ADPIIS > PyMeATP > aq#MeATP) or basolateral membrane (2MeSATP > UTP > ATP > ATPyS > afiMeATP > /JyMeATP) is consistent with involvement of a P2 purinoceptor. A similar rank order of potencies was observed for nucleotide effects on intracellular calcium measured by Fura-2 fluorescence using microspectrofluorimetry. 4 Similar nucleotide potency in the regulation of ion transport and intracellular calcium in cystic fibrosis (CF) airway epithelium (UTP > ATP) was observed, suggesting purinoceptors might be used to stimulate ion transport processes that would promote hydration of airway secretions and facilitate their clearance from CF lungs. 5 These data provide evidence for the regulation of ion transport by P2 purinoceptors in normal and cystic fibrosis human airway epithelium.

Epithelial cells exist in a complex setting in which responses to mucosal or serosal environments are mediated by receptors expressed on specialized cellular domains, such as apical versus basolateral cell membranes. We investigated whether airway epithelia can react selectively through G-protein-coupled receptors to stimuli in the mucosal or serosal environments by measuring inositol phosphate and intracellular Ca2+ responses in polarized human nasal epithelial monolayers. We report here that unilateral ATP (10(-4) M) administration stimulated P2 purinoceptors and tapped pools of intracellular Ca2+ associated with the plasma membrane ipsilateral but not contralateral to stimulated receptors. Similarly, activation of plasma membrane Ca2+ influx by ATP was confined to the membrane ipsilateral to receptor stimulation. These findings demonstrate that polarized epithelia restrict P2 receptor-mediated responses to a single domain of the cell, reflecting membrane-specific generation and catabolism of inositol phosphates and confinement of calcium influx regulation to the membrane ipsilateral to the stimulated receptors.

The blood–brain barrier (BBB), which forms the interface between the blood and the cerebral parenchyma, has been shown to be disrupted during retroviral-associated neuromyelopathies. Human T Lymphotropic Virus (HTLV-1) Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) is a slowly progressive neurodegenerative disease associated with BBB breakdown. The BBB is composed of three cell types: endothelial cells, pericytes and astrocytes. Although astrocytes have been shown to be infected by HTLV-1, until now, little was known about the susceptibility of BBB endothelial cells to HTLV-1 infection and the impact of such an infection on BBB function. We first demonstrated that human cerebral endothelial cells express the receptors for HTLV-1 (GLUT-1, Neuropilin-1 and heparan sulfate proteoglycans), both in vitro, in a human cerebral endothelial cell line, and ex vivo, on spinal cord autopsy sections from HAM/TSP and non-infected control cases. In situ hybridization revealed HTLV-1 transcripts associated with the vasculature in HAM/TSP. We were able to confirm that the endothelial cells could be productively infected in vitro by HTLV-1 and that blocking of either HSPGs, Neuropilin 1 or Glut1 inhibits this process. The expression of the tight-junction proteins within the HTLV-1 infected endothelial cells was altered. These cells were no longer able to form a functional barrier, since BBB permeability and lymphocyte passage through the monolayer of endothelial cells were increased. This work constitutes the first report of susceptibility of human cerebral endothelial cells to HTLV-1 infection, with implications for HTLV-1 passage through the BBB and subsequent deregulation of the central nervous system homeostasis. We propose that the susceptibility of cerebral endothelial cells to retroviral infection and subsequent BBB dysfunction is an important aspect of HAM/TSP pathogenesis and should be considered in the design of future therapeutics strategies.

The rate of Cl- secretion by human airway epithelium is determined, in part, by apical cell membrane Cl- conductance. In cystic fibrosis airway epithelia, defective regulation of Cl- conductance decreases the capability to secrete Cl-. Here we report that extracytosolic ATP in the luminal bath of cultured human airway epithelia increased transepithelial Cl- secretion and apical membrane Cl- permeability. Single-channel studies in excised membrane patches revealed that ATP increased the open probability of outward rectifying Cl- channels. The latter effect occurs through a receptor mechanism that requires no identified soluble second messengers and is insensitive to probes of G protein function. These results demonstrate a mode of regulation of anion channels by binding ATP at the extracellular surface. Regulation of Cl- conductance by external ATP is preserved in cystic fibrosis airway epithelia.

1 We have characterized an adenosine receptor subtype present in human airway epithelial cells by measuring the changes in the intracellular levels of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and the rate of transepithelial Cl-secretion. 2 Primary cultures of human nasal epithelium obtained from excised surgical airway epithelial tissues and the cell lines BEAS39 and CF/T43 derived from human airway epithelium were grown on plastic dishes and labelled with [3H]-adenine for measurement of intracellular cyclic AMP accumulation.Primary cultures were loaded with the calcium indicator fura-2 to measure [Ca2+]i and studied as polarized, ion transporting epithelia on collagen matrix supports for measurement of C1-secretion. 3 Adenosine analogues stimulated cyclic AMP accumulation with a rank order of potency characteristic of an A2-receptor: 5-N-ethyl-carboxamidoadenosine (NECA) > adenosine > R-phenylisopropyladenosine (R-PIA), 6-N-cyclopentyladenosine (CPA)> S-PIA. NECA