Chem. Senses 27: 635-642,
2002
© Oxford University Press 2002
Comparison of Mechanical Agitation and Calcium Shock Methods for Preparation of a Membrane Fraction Enriched in Olfactory Cilia
Departments of Cellular and Molecular Physiology and Neuroscience, Tufts University School of Medicine, Boston, MA 02111, USA
Correspondence to be sent to: Barbara R. Talamo, Department of Neuroscience, Tufts University, Boston MA, USA. e-mail: barbara.talamo{at}tufts.edu
Calcium plays an important regulatory role in olfactory signal
transduction. Many investigations into the regulation of the olfactory
signaling pathway have been performed using fractions enriched in ciliary
membranes from olfactory sensory neurons. The traditional method of preparing
ciliary fractions uses high calcium concentrations, thought to dislodge cilia
from the dendritic knobs of the olfactory neurons in the nasal epithelium.
However, calcium, an important second messenger in the odorant signaling
cascade, modulates the activity of many enzymatic reactions in this cascade.
Pre-exposure of cilia to high calcium concentrations may modify these
signaling events. Therefore, we sought to develop a method of isolating
cilia-enriched membranes that avoids exposing the cilia to high calcium
concentrations. Our method of isolation, referred to as the mechanical
agitation method, involves mechanical disruption and sonication of the
olfactory epithelium to dislodge the cilia. To evaluate this method of cilia
preparation, basal adenylyl cyclase activity, as well as forskolin- and
odorant-activated adenylyl cyclase, were analyzed. Specific activity of
adenylyl cyclase and protein yield were compared for the mechanical agitation
and the high calcium preparations. Immunoblots were analyzed for the presence
of transduction components enriched in olfactory cilia: adenylyl cyclase type
III (ACIII), heterotrimeric G-protein subunit G
olf and the 1 C2 isoform
of phosphodiesterase (PDE 1 C2). Based on these analyses, the ciliary fraction
prepared by the mechanical agitation method appears to be very similar to that
prepared by the high calcium method, with a higher yield.
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