Biochemical studies of olfaction: isolation, characterization, and odorant binding activity of cilia from rainbow trout olfactory rosettes.

Abstract

The role of cilia in recognition of olfactory stimuli has been controversial. Cilia from the intact olfactory rosettes of the rainbow trout Salmo gairdneri were isolated, characterized biochemically, and examined by electron microscopy. The markers studied are those associated with cilia in other organisms. Dynein arms contain Mg2+-ATPase; this enzyme was enriched in the isolated cilia preparation. Guanine nucleotides are associated with the outer microtubule doublets of cilia but adenine nucleotides are not; a substantial enrichment in guanine, relative to adenine, was found in the cilia preparation. Tubulin, the structural protein component of microtubules, occurs in large amounts in cilia. Disc gel electrophoresis indicated tubulin in the cilia preparation. Electron microscopy confirmed the presence of cilia in the isolated preparation. Rainbow trout have an acute sense of smell and many amino acids are odorants to this species. Functional activity of the cilia preparation relevant to odorant recognition was assessed by using binding of radioactively labeled odorant amino acids. L-Alanine, Lserine, L-threonine, L-lysine, and Dalanine bound to the cilia preparation. This study provides direct biochemical evidence that olfactory cilia bind odorant molecules and supports the hypothesis that odorant recognition sites are integral parts of the cilia. The biochemical basis of odorant recognition is beginning to be understood. The numerous hypotheses proposed to explain odor specificity (1) are generally based upon structure-activity correlations made either psychologically in human subjects or electrophysiologically in various animal species. The rainbow trout Salmo gairdneri, which has a functional olfactory system, provides a suitable biochemical model for studying the specificity of odorant interactions with receptor sites. Their olfactory receptors are sensitive to amino acids as stimuli (2, 3). Amino acids bind specifically to a sedimentable fraction isolated from rainbow trout olfactory tissue (4). The extent of binding corresponds to the stimulatory effectiveness recorded electrophysiologically (3) from the olfactory bulb of the brain.The hypothesis that olfactory cilia are the loci of olfactory receptor sites had been postulated for many years (5, 6) but has remained controversial. A widely cited preliminary report (7) described results suggesting that removal of cilia, by use of detergents, from turtle olfactory epithelium resulted in a preparation that continued to be electrophysiologically active.Earlier experiments by Shibuya (8) wete unclear regarding involvement of cilia, but recent results described by Bronshtein and Minor (9) support the hypothesis. They showed that, following removal of frog olfactory cilia with detergent, the electroolfactogram responses to chemical stimulation declined. Upon regeneration of cilia, observed by electron microscopy, the responses reappeared.tThe publication costs of this article were defrayed in part by page charge payment. This article must therfore be here...

A partially purified preparation of isolated chemosensory cilia from the olfactory epithelium of the bullfrog, Rana catesbeiana

“…Earlier protocols employed a 10% ethanol solution or other organic solvents during the calcium shock-induced detachment of the cilia (Chen and Lancet, 1984;Linck, 1973;Rhein and Cagan, 1980). We observed that the calcium shock alone, in the absence of organic solvent, is equally effective for detaching the cilia from the epithelium.…”

“…Electron-microscopic observations of freeze-fracture replicas reveal numerous particles in membranes of cilia from olfactory epithelium, while only few particles are detected in cilia from respiratory epithelium (Menco, 1980;Menco et al, 1976). In addition, Rhein and Cagan (1980) have described binding sites for odorant amino acids on cilia isolated from the olfactory rosette of the rainbow trout.…”

“…Based on these studies, Rhein and Cagan (1980) applied a calcium shock to detach and isolate cilia from the olfactory organ of the rainbow trout. Subsequently, Chen and Lancet (1984) reported the isolation of olfactory cilia from the frog, Rana ridibunda, using a similar method.…”

Odorants induce the phosphorylation of the cAMP response element binding protein in olfactory receptor neurons

“…Many aspects of the mechanism of odor detection in the sensory cilia of ORNs are well established from patch clamp recordings (1) and molecularcloning studies (2)(3)(4)(5). Signal transduction is initiated when odorants interact with specific receptors on cilia (6)(7)(8)(9). Receptors subsequently couple to a G protein, which activates adenylyl cyclase (10)(11)(12).…”

Role of nitric oxide during neurogenesis in the olfactory epithelium

“…Olfactory transduction begins when the odorants bind to their receptors, located in the chemosensory cilia (Rhein and Cagan, 1980;Menco et al, 1997). These receptors are proteins belonging to the G proteincoupled receptor family, with seven transmembrane domains (Buck and Axel, 1991).…”