Richard C. Bruch

Ultrastructural localization of olfactory transduction components: the G protein subunit Golfα and type III adenylyl cyclase

Electron microscopy and postembedding immunocytochemistry on rapidly frozen, freeze-substituted specimens of rat olfactory epithelia were used to study the subcellular localization of the transduction proteins Golf alpha and type III adenylyl cyclase. Antibody binding sites for both of these proteins occur in the same receptor cell compartments, the distal segments of the olfactory cilia. These segments line the boundary between organism and external environment inside the olfactory part of the nasal cavity. Therefore, they are the receptor cell regions that most likely first encounter odorous compounds. The results presented here provide direct evidence to support the conclusion that the distal segments of the cilia contain the sites of the early events of olfactory transduction.

Bromocriptine, a dopamine D2 receptor agonist, inhibits adenylyl cyclase activity in rat olfactory epithelium

The presence of large numbers of dopaminergic neurons in the olfactory bulb suggests that dopamine plays an important role in olfaction. Dopamine D2 receptors are produced in olfactory sensory neurons [Shipley et al. (1991) Chem. Senses 16, 5] and found in relatively high concentrations in their terminals in the nerve and glomerular layers of the olfactory bulb [Nickell et al. (1991) NeuroReport 2, 9-12]. In other systems D2 receptors are linked to adenylyl cyclase by an inhibitory G-protein, and activation of the receptors results in inhibition of the enzyme. We examined rat olfactory mucous membrane to determine whether the D2 receptors were linked functionally to adenylyl cyclase as they are in other tissues. Adenylyl cyclase is found in both the olfactory cilia of the sensory epithelium and olfactory nerve terminals in the bulb. Bromocriptine, a D2 receptor agonist, was added to olfactory epithelium membrane preparations from normal and unilaterally bulbectomized adult rats and the preparations were assayed for forskolin-stimulated adenylyl cyclase activity. In unoperated animals bromocriptine significantly inhibited adenylyl cyclase activity, and the inhibition was abolished following pertussis toxin treatment. In mucosa from unilaterally bulbectomized animals we saw significantly lower adenylyl cyclase activity on the operated side and a further decrease in response to bromocriptine. The data indicate that bromocriptine decreases adenylyl cyclase activity in olfactory tissue, specifically in the sensory neurons, and the reaction is dependent on a pertussis toxin-sensitive G-protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning of a phosphoinositide-specific phospholipase C from catfish olfactory rosettes

A 2.7 kb clone encoding the partial (about 66%) sequence of a phosphoinositide-specific phospholipase C (PLC) was isolated from a cDNA library constructed from channel catfish (Ictalurus punctatus) olfactory rosettes. The clone, designated 30c7, was completely sequenced by automated DNA sequencing and was found to share significant homology with rat and bovine PLCs of the delta 1 isotype. In situ hybridization showed that 30c7 transcripts were expressed in a small subpopulation of olfactory neurons, as well as in other cell types in the olfactory epithelium. Polymerase chain reaction (PCR) analysis indicated that the enzyme was also expressed in several additional tissues, including brain, gill, heart, liver and skeletal muscle. These results suggest that the PLC encoded by clone 30c7 is expressed in several tissues and therefore may have a role in mediating transduction events in diverse tissues as well as in a small group of olfactory neurons.

Identification of three phospholipase C isotypes expressed in rat olfactory epithelium

The expression of three phosphoinositide-specific phospholipase C (PLC) isotypes in rat olfactory epithelium was investigated using monoclonal antibodies. In intact animals, PLC beta 1 was not expressed in the olfactory epithelium but was found in glands below the epithelium. However, following unilateral olfactory bulbectomy (OBX), PLC beta 1 was expressed in the dentrites of some olfactory receptor neurons, primarily in the endoturbinates on the unoperated side. PLC gamma 1 immunoreactivity was found in the apices of sustentacular cells and in glands below the epithelium. PLC delta 1 immunoreactivity was found in the glands and in the perinuclear region and dendrites of some receptor neurons. Since none of the PLC isotypes studied were expressed in the cilia of receptor neurons, the results suggest that another PLC isotype is likely to be involved in mediating olfactory transduction.

Amitriptyline-mediated inhibition of neurite outgrowth from chick embryonic cerebral explants involves a reduction in adenylate cyclase activity.

We have previously shown that amitriptyline, a tricyclic antidepressant, inhibited neurite outgrowth from chick embryonic cerebral explants, and that dibutyryl cyclic AMP, 3‐isobutyl‐l‐methylxanthine, or theophylline can enhance neurite outgrowth from embryonic olfactory explants. In the present study, we examined the mechanism(s) underlying amitriptyline‐mediated inhibition of neurite outgrowth by studying the effects of amitriptyline on adenylate cyclase activity and cyclic AMP levels. In cultured chick embryonic cerebral explants, dibutyryl cyclic AMP or theophylline, but not dibutyryl cyclic GMP, enhanced neurite outgrowth and partially reduced the inhibitory effects of amitriptyline on neurite outgrowth. Explants treated with amitriptyline for 2 days showed decreased cyclic AMP levels that significantly correlated with the degree of neurite outgrowth. Amitriptyline inhibited both basal and forskolin‐stimulated adenylate cyclase activity in vitro, but only in the presence of GTP. Taken together, these data suggest that amitriptyline inhibits the activity of adenylate cyclase via a GTP‐dependent mechanism, and that the subsequent decrease in cyclic AMP level may be involved in amitriptyline‐mediated inhibition of neurite outgrowth.

Rat olfactory neurons express a 200 kDa neurofilament

Neurofilament expression in peripheral olfactory neurons of adult rats was investigated by immunoblotting and immunohistochemistry using monoclonal antibodies specific for each of the 3 neurofilament proteins. Immunoblotting analysis of olfactory epithelium extracts demonstrated the presence of only the 200 kDa (NFH) polypeptide; the 68 kDa (NFL) and 160 kDa (NFM) neurofilaments were not detected. Similarly, no immunoreactivity was observed in tissue sections using the NFL and NFM antibodies. In contrast, when sections were probed with the antibody to NFH, immunoreactivity was localized primarily in the dendritic knobs and near the cell bodies of the receptor cells.

Growth Factor Regulation of Olfactory Cell Proliferation

Vertebrate olfactory sensory cells are unique neurons in that they are continually replaced throughout the life of the animal [1–3]. Because neurogenesis is continuous, at any given time in an adult animal the olfactory sensory neuronal population is a mixture of cells of different ages. It was believed that the life span of the olfactory sensory cell was about 1 month [4, 5], but it has since been shown that under some conditions cells can either live considerably longer [6, 7] or die prematurely [8–10].

Signal transducing gtp-binding proteins in olfaction

1. Several members of the family of heterotrimeric signal transducing GTP-binding proteins have been identified in the olfactory epithelium of vertebrates by biochemical and molecular cloning techniques. 2. Biochemical and electrophysiological evidence indicates that one or more GTP-binding proteins mediate many olfactory responses by coupling stimulus receptors to second messenger systems. 3. Although GTP-binding proteins may function in additional transduction events, a novel GTP-binding protein, expressed only in olfactory neurons, may mediate stimulus activation of adenylate cyclase in olfactory cilia.