Olfactory-specific cytochrome P-450. cDNA cloning of a novel neuroepithelial enzyme possibly involved in chemoreception.

We isolated cDNA clones for cytochrome P-450 genes expressed in the olfactory neuroepithelium by screening a corresponding rat cDNA library. Sequence analysis and RNA blot hybridization revealed a new cytochrome P-450, designated cytochrome P-450olf1, which is the first reported cytochrome P-450 mRNA uniquely expressed in the chemosensory organ. Cytochrome P-450olf1 shows intermediate level of sequence similarity (38-53% identity) to several liver cytochrome P-450 enzymes, suggesting that it belongs to the cytochrome P-450II family, but defines a new subfamily (cytochrome P-450IIG) within it. Cytochrome P-450II enzymes are known to process diverse organic compounds, including odorants. This, together with the specificity of cytochrome P-450olf1 to the sensory neuroepithelium, may indicate a role for this protein in olfactory reception.

Cyclic AMP-dependent protein phosphorylation in chemosensory neurons: identification of cyclic nucleotide-regulated phosphoproteins in olfactory cilia.

Chemosensory dendritic membranes (olfactory cilia) contain protein kinase activity that is stimulated by cyclic AMP and more efficiently by the nonhydrolyzable GTP analog guanosine-5'-O-(3-thio)triphosphate (GTP gamma S). In control nonsensory (respiratory) cilia, the cyclic AMP-dependent protein kinase is practically GTP gamma S-insensitive. GTP gamma S activation of the olfactory enzyme appears to be mediated by a stimulatory GTP-binding protein (G-protein) and adenylate cyclase previously shown to be enriched in the sensory membranes. Protein kinase C activity cannot be detected in the chemosensory cilia preparation under the conditions tested. Incubation of olfactory cilia with [gamma-32P]ATP leads to the incorporation of [32P]phosphate into many polypeptides, four of which undergo covalent modification in a cyclic nucleotide-dependent manner. The phosphorylation of one polypeptide, pp24, is strongly and specifically enhanced by cyclic AMP at concentrations lower than 1 microM. This phosphoprotein is not present in respiratory cilia, but is seen also in membranes prepared from olfactory neuroepithelium after cilia removal. Cyclic AMP-dependent protein kinase and phosphoprotein pp24 may be candidate components of the molecular machinery that transduces odor signals.

Isolated frog olfactory cilia: a preparation of dendritic membranes from chemosensory neurons.

The recently introduced frog olfactory cilia preparation (Chen and Lancet, 1984; Pace et al., 1985) has been useful for studies of molecular chemosensory mechanisms. Here we describe in detail the properties of this cilia preparation. The "calcium shock" procedure leads to a complete removal of the cilia from the olfactory epithelial surface. Isolated cilia constitute segments of proximal regions with 9 X 2 + 2 microtubular arrangement and a large proportion of membrane vesicles, probably derived from the ciliary distal segments. Polypeptides unique to the olfactory cilia preparation, compared to a control preparation of palate respiratory cilia, are identified by Coomassie brilliant blue staining, silver staining, and radiolabeled lectin overlays, as well as by biosynthetic labeling with 35S-methionine in epithelial explants and protein phosphorylation in isolated cilia. The olfactory cilia preparation contains odorant-sensitive adenylate cyclase, which is absent in control membranes from deciliated epithelium. High activities of tyrosine and serine/threonine protein kinases are also present. The olfactory cilia preparation described should be instrumental in the further elucidation of the biochemistry and molecular biology of vertebrate olfaction.

Association of membrane and cytoplasmic proteins with the cytoskeleton in blood platelets.

The association of membrane and cytoplasmic proteins with the cytoskeleton of resting and activated platelets was studied. Glycoproteins were identified by labeling with 125I-labeled lectins (concanavalin A, wheat germ agglutinin, and Lens culinaris). Polypeptides, which are embedded in the lipid bilayer, have been identified by their photolabeling with the lipid-soluble reagent 5-[125I]iodonaphthyl 1-azide (125INA). Cytoplasmic proteins were identified by their photolabeling with the intracellular probe azidofluorescein diacetate. Results indicate that the Triton X-100 residue contains the membrane-associated glycoprotein Ia, a 95 000-dalton protein, and two other acidic proteins of molecular weights of 35 000-40 000, one labeled with 125INA and the other with azidofluorescein diacetate. The presence of part of these proteins in the Triton residue is dependent upon the mode of platelet activation. Glycoproteins IIb and III are embedded in the membrane lipid bilayer but sedimented with the Triton residue only after thrombin activation. Another protein with Mr 70 000, which is highly labeled by 125INA in resting platelets, is found only in the Triton-soluble fraction.

Measurement of the intracellular pH of blood platelets using a photolabel fluorescent probe.

The internal pH of blood platelets using the intracellular photolabel probe azidofluorescein diacetate was determined. No change of intracellular pH during thrombin activation of human platelets was observed. Platelets were found to adjust themselves very quickly to the external pH. Quantitative subcellular localization of the attachment sites of this probe reveals that most of it is bound to low molecular weight proteins or peptides.

Intracellular viscosity changes during activation of blood platelets: studies by fluorescence polarization.

The intracellular viscosity changes that occur in washed human platelets as a result of activation by thrombin or ADP were studied by the use of a fluorescent probe. Results obtained showed a sharp and quick decrease of the intracellular viscosity when platelets were activated by thrombin. This decreased preceded both the release and the aggregation. When platelets were activated by ADP, the decrease in the polarization of fluorescence (and the viscosity) was more moderate. The fluorescent probe is bound to small proteins and peptides in the cytoplasm and not in the granules. Therefore, these changes in the fluorescence polarization reflect changes in the cytoplasmic viscosity which might be due to reorganization of the contractile proteins' system.

Coronary hemodynamics and oxygen utilization after hematocrit variations in hemorrhage.

Twenty closed-chest dogs anesthetized with pentobarbital sodium were used for studying coronary hemodynamics and myocardial oxygen utilization during hemorrhagic hypotension, with the mean arterial pressure maintained constant at 50 mmHg. Variations of hematocrit (Hct) were achieved by exchange of blood with plasma or packed cells. Coronary blood flow (133Xe washout) varied inversely with Hct, whereas cardiac output (indicator dilution) showed a peak value at a Hct of approximately 25%. Coronary, systemic, and pulmonary flow resistances varied in the same direction with Hct, and the relationship was attributable to the change of blood viscosity with Hct. Analyses of vascular hindrance (= resistance/viscosity) suggested that during hemorrhagic hypotension, coronary vasodilation was maintained during variations of Hct. In systemic and pulmonary circulations, however, there were marked increases in vasoconstriction after hemodilution. The optimum Hct for maximum O2 transport was 25% for coronary circulatin and approximately 45% for systemic circulation. The O2 consumption (QO2) in the myocardium increased after hemodilution with a peak value at a Hct of approximately 25%. The QO2 in the total body was constant over a wide range of Hct between 25 and 45%, above and below which the QO2 decreased.