To study effects of mitochondrial complex I (CI, NADH:ubiquinone oxidoreductase) deficiency, we inactivated the Ndufs4 gene, which encodes an 18 kDa subunit of the 45-protein CI complex. Although small, Ndufs4 knockout (KO) mice appeared healthy until approximately 5 weeks of age, when ataxic signs began, progressing to death at approximately 7 weeks. KO mice manifested encephalomyopathy including a retarded growth rate, lethargy, loss of motor skill, blindness, and elevated serum lactate. CI activity in submitochondrial particles from KO mice was undetectable by spectrophotometric assays. However, CI-driven oxygen consumption by intact tissue was about half that of controls. Native gel electrophoresis revealed reduced levels of intact CI. These data suggest that CI fails to assemble properly or is unstable without NDUFS4. KO muscle has normal morphology but low NADH dehydrogenase activity and subsarcolemmal aggregates of mitochondria. Nonetheless, total oxygen consumption and muscle ATP and phosphocreatine concentrations measured in vivo were within normal parameters.

1The human P2u-purinoceptor was stably expressed in 1321N1 human astrocytoma cells and the pharmacological selectivity of the expressed receptor was studied by measurement of inositol lipid hydrolysis.2 High basal levels of inositol phosphates occurred in P2U-purinoceptor-expressing cells. This phenomenon was shown to be due to release of large amounts of ATP from 1321N1 cells, and could be circumvented by adoption of an assay protocol that did not involve medium changes. 3 UTP, ATP and ATPyS were full and potent agonists for activation of phospholipase C with EC50 values of 140 nM, 230 nM, and 1.72 pM, respectively. 5BrUTP, 2ClATP and 8BrATP were also full agonists although less potent than their natural congeners. Little or no effect was observed with the selective P2y-, P2X-, and P2T-purinoceptor agonists, 2MeSATP, a,fi-MeATP, and 2MeSADP, respectively.4 Diadenosine tetraphosphate, Ap4A, was a surprisingly potent agonist at the expressed P2u-purinoceptor with an EC50 (720 nM) in the range of the most potent P2U-purinoceptor agonists. Ap4A may be a physiologically important activator of P2u-purinoceptors.

1. Four different phospholipase C (PLC)-activating P2Y receptors have been cloned and stably expressed in 1321N1 human astrocytoma cells. These include the human homologues of the P2Y1, P2Y2 and P2Y4 receptors and the rat homologue of the P2Y6 receptor. 2. The nucleotide selectivities of these four receptors have been compared directly by measuring inositol phosphate accumulation in response to nucleotides under conditions in which the initial purity and stability of agonist was rigidly assured and quantitatively assessed. 3. The P2Y1 receptor is specific for adenine nucleotides and slightly more sensitive to disphosphates than triphosphates. When expressed in 1321N1 astrocytoma cells, it couples selectively to the stimulation of PLC and not to the inhibition of adenylyl cyclase. 4. The P2Y2 receptor is activated by UTP and ATP with similar potency and is not activated by nucleoside diphosphates. Diadenosine terraphosphate is a potent agonist at this receptor. 5. The P2Y4 receptor is highly selective for UTP over ATP and is not activated by nucleoside disphosphates. 6. The P2Y6 receptor is activated most potently by UDP, but weakly or not at all by UTP, ADP and ATP. The P2Y6 receptor appears to be identical to the uridine nucleotide-specific receptor previously characterized in C6-2B rat glioma cells. 7. We have identified a P2Y receptor on C6 glioma cells that inhibits adenylyl cyclase but has no effect on PLC. This receptor exhibits a pharmacological selectivity similar but not identical to that of the P2Y1 receptor. When the P2Y1 receptor was expressed in these C6 cells, it conferred an inositol lipid signalling response to adenine nucleotides that was pharmacologically identical to that of the P2Y1 receptor. Thus, the P2Y receptor of C6 glioma cells represents an additional receptor that exhibits the classical pharmacological selectivity of a P2Y1-R, but which couples to adenylyl cyclase rather than to PLC.

Olfactory sensory neurons (OSNs) can be sensitized to odorants by repeated exposure, suggesting that an animal's responsiveness to olfactory cues can be enhanced at the initial stage of detection. However, because OSNs undergo a regular cycle of apoptosis and replacement by ostensibly naive, precursor-derived neurons, the advantage of sensitization would be lost in the absence of a mechanism for odorant-enhanced survival of OSNs. Using recombinant adenoviruses in conjunction with surgical and electrophysiological techniques, we monitored OSN survival and function in vivo and find that odorant exposure selectively rescues populations of OSNs from apoptosis. We further demonstrate that odorant stimuli rescue OSNs in a cAMP-dependent manner by activating the MAPK/CREB-dependent transcriptional pathway, possibly as a result of expression of Bcl-2.

To test for the role of the P2Y 2 receptor (P2Y 2 -R) in the regulation of nucleotide-promoted Ca 2؉ signaling in the lung, we generated P2Y 2 -R-deficient (P2Y 2 -R(؊/؊)) mice and measured intracellular Ca Extracellular ATP induces a wide variety of responses in many cell types, including muscle contraction and relaxation, vasodilation, neurotransmission, platelet aggregation, ion transport regulation, and cell growth (1-3). The cell surface receptors mediating these diverse effects of ATP were originally termed P2 purinoceptors to distinguish them from the adenosine-activated P1 purinoceptors (4). Subsequently, pyrimidine nucleotides were also shown to regulate a broad range of cell functions, leading to speculation about the existence of separate pyrimidoceptors (5, 6). It is likely, however, that a common receptor for uridine and adenine nucleotides is present in many cell types, including neutrophils, pituitary cells, skin fibroblasts, smooth muscle cells, and specific endothelial and epithelial cell types (2). This receptor was originally named the P2U purinoceptor but has been subsequently reclassified as the P2Y 2 receptor (P2Y 2 -R).1 The cloning of the murine P2Y 2 -R gene (7) and its human counterpart (8) made possible the definitive identification of this signaling protein as a G-protein and phospholipase C-coupled receptor that is equipotently activated by ATP and UTP but not by diphosphate nucleotides (9 -11).The lack of specific agonists or antagonists for the growing number of nucleotide receptor subtypes (e.g. seven P2X and five P2Y receptors have been identified to date (12, 13)) constitutes a major obstacle in identifying the specific nucleotide receptor mediating a given cellular function. One example of the difficulty in assigning receptor subtypes to cellular responses is illustrated in studies of fibroblasts. Following original studies by Okada et al. (14), who observed that ATP induced change in the membrane potential of mouse L cells and human fibroblasts, a variety of adenosine-and ATP-induced responses in fibroblasts were reported. These actions of adenosine and ATP, which include regulation of cell growth, cytoskeletal contraction, Ca 2ϩ efflux, and LDH and nucleotide release (15-19), were proposed to be mediated by A 1 , A 2 , P2X, P2Z (in current terminology P2X 7 ), and P2Y 1 receptors (15-23). In one study with human skin fibroblasts, actions of ATP on Ca 2ϩ mobilization and phospholipase C activity were mimicked by UTP (24), although no further characterization of the receptor(s) mediating UTP responses in fibroblasts was provided.The effects of extracellular nucleotides have also been extensively studied on airway epithelia, and attempts have been made to link the cellular responses to specific nucleotide receptors. Both ATP and UTP equipotently regulate epithelial electrolyte and water transport (3, 25), trigger mucin secretion (26,27), and increase ciliary beat frequency (28 -30). ATP and UTP equipotently stimulate inositol phosphate formation (29) and Ca 2ϩ i mobilization and...

The presence of the P2Y 2 (P 2U -purinergic) receptor on the apical surface of airway tissue raises the possibility that aerosolized UTP might be used therapeutically to induce Cl ؊ secretion in individuals with cystic fibrosis. However, the duration of the effects of UTP may be limited by enzymatic degradation. We therefore have analyzed the metabolism of UTP and its metabolite UDP on polarized human nasal epithelium (HNE), and have compared the pharmacological activities of these two uridine nucleotides. Extracellular ATP regulates physiological responses in many tissues and cell types through a multi-gene family of P2 receptors (1-4). The existence of receptors that selectively recognize pyrimidines was proposed more than a decade ago (see ref. 5). However, support for the idea of specific pyrimidinergic receptors was lessened by the fact that the stimulatory activity of UTP in many tissues was mirrored by similar effects of ATP and by the eventual cloning of the P2Y 2 (P 2U -purinergic) receptor, which is equipotently activated by the purine ATP and the pyrimidine UTP. Nonetheless, a novel receptor was identified on C6-2B rat glioma cells that is activated by UDP but not by adenine nucleotides (6), and two recently cloned G protein-coupled P2Y receptors, the P2Y 4 and P2Y 6 receptors, show high selectivity for uridine nucleotides and are activated only weakly or not at all by adenine nucleotides (7-10). The P2Y 6 receptor, which is potently and selectively activated by UDP, subsequently was shown to be expressed endogeneously by C6-2B cells (11). Although mRNA for the P2Y 6 receptor was detected in several tissues, including aortic smooth muscle cells, spleen, kidney, stomach, lung, intestine, and heart (7, 8), functional evidence for uridine nucleotide selective receptors in native tissues is limited.Uridine nucleotide analogues that might discriminate between uridine nucleotide-selective receptors and the widely expressed P2Y 2 receptor have not been available. The instability of most nucleotides in aqueous solution and metabolic interconversion of UTP and UDP during incubation with cells contribute additional problems. For example, we (12) and others (13) initially reported that UDP was a full agonist at the P2Y 2 receptor, and Communi et al. (8) reported that UDP was a full and potent agonist at the P2Y 4 receptor. However, we recently have demonstrated that UTP-free UDP is essentially inactive at both receptor types (11). Conversely, while Chang et al. (7) originally reported that UTP was the most potent agonist at the P2Y 6 receptor, we have observed that UDP is at least 100-fold more potent than UTP (11), and the initially reported effects of UTP were likely due to contamination with UDP or formation of UDP during incubation with cells.In this study we have adopted conditions that allow for examination of the biological effects of UDP on primary cultures of polarized human nasal epithelial cells. We report that UDP potently stimulates formation of inositol phosphates, calcium mobilization, an...

1 The defective Cl-secretion characteristic of cystic fibrosis airway epithelial cells can be bypassed by an alternative Ca2+ dependent Cl-secretory pathway that is activated by extracellular nucleotides, e.g. uridine-5'triphosphate (UTP), acting on P2U purinoceptors. Since UTP is susceptible to hydrolysis by nucleotidases and phosphatases present in the airways, the identification of stable P2U-purinoceptor agonists would be of therapeutic relevance. 2 Uridine-5'-O-(3-thiotriphosphate) (UTPyS) was synthesized by nucleoside diphosphate kinasecatalyzed transfer of the y-phosphorothioate from guanosine-5'-O-(3-thiotriphosphate) (GTPyS) or adenosine-5' = 0-(3-thiotriphosphate) (ATPyS)

The cystic fibrosis (CF) transmembrane regulator (CFTR) is a cyclic AMP-dependent Cl