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cAMP and IP3 act as secondary messengers in olfactory signal transduction and when activated, stimulate calcium levels in olfactory receptor cells. Little is known however, about the causal mechanism. We studied calcium kinetics in mouse olfactory receptor cells after odorant stimuli. Olfactory receptor cells were isolated from female BALB/c mice, treted with trypsin, and stained with Fura-2/AM. Changes in intracellular Ca2+ concentrations in stained cells were measured with a fluorescent microscopic image-processing device (ARGUS-50; Hamamatsu Photonix, Japan). We found that intracellular Ca2+ concentrations rose after exposure to a set of odorants, including 3-ethoxy-4-hydroxy-benzaldehyde, caprylic acid, heptanoic acid, nonanoic acid, eugenol, phenethyl alcohol, and n-amyl acetate. Adding 2', 5'-dideoxyadenosine, a cAMP inhibitor, beforehand suppressed olfactory receptor cell response to odorants. Intracellular Ca2+ concentrations increased substantially in response to stimulation by odorants in calcium-free Ringer's solution, but only a slight increase was seen in intracellular calcium concentration in response stimulation by a high concentration of K+ (145.6 mM) in calcium-free Ringer's solution. The increase in intracellular Ca2+ concentration after odorant stimuli was suppressed when olfactory receptor cells were pretreated with ryanodine, which releases Ca2+ from intracellular stores. These findings suggest that elevated Ca2+ concentrations may be involved in releasing Ca2+ from intracellular calcium stores in mouse olfactory receptor cells, in which cAMP functions as a secondary messenger in olfactory signal transduction.

Successive electro-acupuncture (EA) stimulation applied to bilateral anterior tibial muscles, where Zusanli (ST36) acupoints are located, once a day (30 min) for 3 successive days significantly enhanced splenic natural killer (NK) cell activity in BALB/c mice. The percentage of splenic NK cells, as measured by flow cytometry, was not affected in these mice. Interferon (IFN)-gamma level in splenic aqueous extract, prepared from the ST36 acupoint-stimulated mice, was significantly higher than that of the controls. In vivo treatment with neutralizing monoclonal antibody against mouse IFN-gamma completely abrogated the increase in splenic NK cell activity induced by ST36 acupoint stimulation. The same stimulation also significantly increased the concentration of splenic beta-endorphin, which coincided with the significant increase in splenic IFN-gamma production. Pre-administration of 10 mg/kg naloxone before initiation of EA stimulation every day reduced the enhancements of NK cell activity and IFN-gamma level. These observations strongly suggest that endogenous IFN-gamma mediates the up-regulation of NK cell activity by EA stimulation at the ST36 acupoints. Furthermore, endogenous beta-endorphin secreted by EA stimulation also plays an important role in the up-regulation of NK cell function, which may be realized through regulating IFN-gamma production.

Oral administration and nasal instillation of glucocorticoids are currently being conducted and are producing satisfactory results for the treatment of olfactory dysfunctions. However, there are still many unanswered questions about the actions of glucocorticoids on the olfactory cells. In the present study, the effects of glucocorticoids on the olfactory response in mouse olfactory cells were examined by measuring changes in the intracellular Ca2+ concentration. The olfactory cells isolated from the BALB/c female mouse mucosa were stained with a Fura-2/AM and the changes in the intracellular Ca2+ concentration were observed by using a fluorescent microscopic image processing device, ARGUS50. The cells were exposed to the following olfactory stimuli, 3-ethoxy-4-hydroxy-benzaldehyde, caprylic acid, nonanoic acid, phenethyl alcohol and n-amyl acetate. When 0.1 and 0.01% betamethasone was applied to the olfactory cells for short periods(1 or 3 minutes), no changes were observed. However, long-term(7 minutes) application of 0.1 and 0.01% (but not 0.001 and 0.0001%) betametathone significantly decreased intracellular Ca2+ concentration, which was increased by olfactory stimulation. The inhibitory effect of betamethasone on Ca2+ influx into olfactory cells was attenuated by pre-treatment olfactory cells with RU486, a glucocorticoid receptor antagonist.