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
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Abstract |
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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. |
Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
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Signal cascade in olfactory sensory neurons
Our current understanding of olfactory signal pathways hypothesizes that
odorants traverse the mucus layer of the nasal epithelium and then bind to
G-protein-coupled seven-transmembrane receptors on the cilia of the olfactory
neurons. Activated receptors then stimulate the Golf subunit of a
heterotrimeric G-protein complex and thus activate adenylyl cyclase III
(ACIII), and formation of the second messenger molecule, cAMP. cAMP stimulates
the opening of a cyclic-nucleotide-gated channel on the plasma membrane,
admitting both Na+ and Ca2+ and depolarizing the cell.
Elevated intracellular Ca2+ then induces the opening of
calcium-activated chloride channels, further contributing to cell
depolarization and generation of an action potential
(Zufall and Leinders-Zufall,
2000
).
Desensitization of the signaling mechanism is thought to involve the second
messengers cAMP and calcium. cAMP-dependent protein kinase A (PKA), as well as
G-protein-coupled receptor kinase type 3 (GRK3) have been reported to
contribute to the desensitization of the receptor through phosphorylation
(Boekhoff et al.,
1997). Calcium also has been implicated in desensitization via
actions of CaMKII and calcium—calmodulin binding to the
cyclic-nucleotide-gated channel. Electrophysiological recordings suggest that
the rise in internal calcium levels leads to a decrease in the amplitude and
duration of the odor-induced response
(Kurahashi, 1989;
Kurahashi et al.,
1990). Additionally, injections of EGTA into chemosensory neurons
decrease the internal calcium levels, extending the duration of the
odor-stimulated response (Kurahashi and
Shibuya, 1990).
Biochemical analyses of the odor-stimulated response in enriched ciliary
membrane preparations have focused on modulation of the activity of the
adenylyl cyclase (AC) enzyme. Published reports with contradictory
interpretations indicate that regulation is complex. Several investigations
using cilia preparations presented data showing that increases in calcium
concentration reduce the activity of adenylyl cyclase, in agreement with
physiological desensitization studies
(Shirley et al.,
1986; Sklar et al.,
1986; Boekhoff et al.,
1996). However, data from another study
(Jaworsky et al.,
1995) suggested that the effects of calcium on adenylyl cyclase
activity might be more dynamic than previously shown. This study indicated
that intermediate calcium levels increase adenylyl cyclase activity, whereas
high calcium levels decrease adenylyl cyclase activity. On the other hand,
calcium—calmodulin has been reported both to enhance the activity of
adenylyl cyclase (Anholt and Rivers,
1990; Choi et al.,
1992) and to decrease the activity of adenylyl cyclase through
calcium—calmodulin-activated CamKII
(Wei et al., 1998).
It is possible that differing concentrations of calcium selectively affect
various components of signaling associated with ciliary membranes and thereby
modulate transduction in a complex manner.
All of the biochemical analyses discussed above were carried out with ciliary membrane fractions isolated by the calcium shock method, where treatment with a high calcium concentration under hypotonic buffer conditions is thought to free cilia from the olfactory neurons. In addition to the effects noted above, this treatment potentially could affect calcium-sensitive machinery such as calcium-activated proteases, calcium-mediated protein associations, and kinase and/or phosphatase activity. To obviate potential alterations by calcium of the basal state of the signaling system in ciliary membranes, we have developed a preparation that does not involve exposure to high calcium concentrations as a tool for further examining the role of calcium in the modulation of the signal cascade.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
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Initially, we planned to develop methods for isolation of sealed ciliary vesicles from olfactory sensory neurons. In the course of these studies, we observed that preparations enriched with ciliary membranes could be prepared using isotonic rather than hypotonic buffer conditions and that high calcium was not necessary. Previous calcium shock preparations enriched in ciliary membranes were prepared using hypotonic buffer with high calcium concentrations (Pace et al., 1985
; Sklar et al.,
1986). We, however, used isotonic buffer in this preparation. Isolation of a membrane fraction enriched in olfactory cilia from primary olfactory sensory neurons by the mechanical agitation method
Five CF-1 female mice, ranging from 3 to 10 weeks of age, were killed by
cervical dislocation followed by decapitation. The buffer for the membrane
preparations utilized HEPES instead of NaHCO3 for buffering. The
nasal epithelia were surgically removed and placed in 10 ml of solution A [145
mM NaCl, 5 mM KCl, 1.6 mM K2HPO4, 2.0 mM
MgSO4, 20 mM HEPES, 7.5 mM D-glucose, 6 µg/ml chymostatin, 1
µg/ml leupeptin, 1 µg/ml pepstatin, 0.1 mg/ml Pefabloc (Boehringer
Mannheim), 1 µg/ml aprotinin, pH 7.4] on ice. EDTA (final concentration 1
mM) was added to the pooled epithelia (solution B) and the solution was rocked
for 20 min at 4°C (setting number 4, intermediate speed on a 55S single
platform rocker; Reliable Scientific). All subsequent steps were carried out
at 4°C. The suspension was then centrifuged for 10 min at 1500 g
and the supernatant (S1) was transferred to a separate tube. The remaining
pellet was resuspended in 6 ml of solution B and sonicated at low power for 10
s using a microprobe (Micro Ultrasonic Cell Disrupter; Kontes). Tissue was
allowed to settle to the bottom of the tube and the supernatant (S2a) was
transferred to a new tube. The pellet was resuspended in 6 ml of solution B
and sonicated again at low power for 30 s. After tissue settled to the bottom
of the tube, the supernatant (S2b) was removed to a new tube. The final
settled tissue (`deciliated epithelium') was resuspended in 3 ml of solution B
and homogenized twice using a Brinkman Homogenizer (Polytron) for 5 s
separated by a period of 1 min on ice to ensure that the solution remained
cool. This homogenized solution and supernatants S2a and S2b were each
centrifuged for 10 min at 1500 g to remove debris. Each of the
supernatants from the homogenized tissue (S3) and supernatants S1, S2a and S2b
was centrifuged at 43 140 g for 25 min at 4°C to collect
membranes. Pellets were then resuspended in 200 (P1, P2A and P2B) or 400 µl
(pellet P3) of solution B with 5% glycerol using a Teflon pestle, apportioned
into small volumes and stored at -70°C. Protein was measured using the
Lowry method (Lowry et al.,
1951), with bovine serum albumin as standard. In six experiments,
the protein yield per mouse averaged 77 ± 17 µg for the P1 fraction,
164 ± 35 µg for fraction P2A, 133 ± 17 µg for the P2B
fraction and 351 ± 107 µg for the P3 fraction.
Isolation of a membrane fraction enriched in olfactory cilia from primary olfactory sensory neurons by the calcium shock method
Ciliary membranes were isolated using minor modifications of the high
calcium method previously described (Pace
et al., 1985; Sklar
et al., 1986). Cilia were dislodged from the epithelium
by gentle agitation in a high calcium buffer and the remaining tissue was
further disrupted by homogenization and centrifuged to generate a membrane
pellet. Ten CF-1 female mice, ranging from 3 to 10 weeks of age, were killed
by cervical dislocation followed by decapitation. The nasal epithelium from
each was surgically excised and pooled with the others in 9 ml of solution C
(120 mM NaCl, 5 mM KCl, 1.6 mM K2HPO4, 1.2 mM
MgSO4, 25 mM NaHCO3, 7.5 mM D-glucose, adjusted to pH
7.4). All steps were carried out at 4°C. The pooled tissue was allowed to
settle to the bottom of the tube and the supernatant was discarded. The tissue
was resuspended in 5 ml of solution C containing 10 mM CaCl2
(solution D). Solution D was prepared by slowly adding CaCl2 to a
final concentration of 10 mM while gassing with 5% CO2/95% air at
22°C to prevent Ca2PO4 from precipitating out of
solution. Tissue was rocked for 20 min at 4°C (as in the mechanical
agitation method) and then centrifuged at 7700 g for 5 min. This
supernatant was reserved in a new tube for subsequent pooling with membranes
dislodged in a second rocking step. The pellet was resuspended in 4 ml of
solution D and rocked for a second time for 20 min. Supernatant from this
step, collected by centrifugation at 7700 g for 5 min, was added to
the supernatant from the first calcium shock incubation. The combined
supernatant was referred to as S2. The remaining pellet was resuspended in 3
ml of solution C, homogenized twice (as in the mechanical agitation method)
for 5 s with an intervening 1 min cooling period on ice to prevent tissue from
increasing in temperature. Centrifugation for 5 min at 7700 g yielded
supernatant (S3). Both S2 and S3 supernatants were centrifuged for 15 min at
higher speed (27 000 g) to generate pellets P2 and P3. The
supernatants were discarded. P2 and P3 were resuspended in 200 and 400 µl,
respectively, of TEM buffer (10 mM Tris—HCl, 3 mM MgCl2, 2 mM
EDTA, pH 8.0) with 5% glycerol, homogenized with a Teflon pestle, apportioned
into small volumes and stored at -70°C. Protein was determined by the
Lowry method (Lowry et al.,
1951), with bovine serum albumin as standard. In four experiments,
the protein yield per mouse averaged 38 ± 14 µg for the P2 fraction
and 261 ± 22 µg for the P3 fraction.
Immunoblot analysis
For immunoblotting, membrane proteins were separated on 8% SDS—polyacrylamide gels and transferred to nitrocellulose membranes. Non-specific binding sites were blocked with 5% dry non-fat milk (Carnation) in TBST (10 mM Tris—HCl, pH 8.0, 150 mM NaCl and 0.4% Tween 20) for 1 h at room temperature. The blots were incubated with primary antibody for 1 h at room temperature or overnight at 4°C. Blots were washed three times with TBST and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. The secondary antibodies used were HRP-conjugated anti-rabbit antibody (1:1000; Santa Cruz), HRP-conjugated anti-mouse antibody (1:2000; Santa Cruz) and HRP-conjugated antibiotin antibody (1:2000; New England Biolabs). Blots were washed twice with TBST and once with TBS (10 mM Tris—HCl, pH 8.0, 150 mM NaCl). The NEN chemiluminescence system was used to monitor bound antibodies. Biotinylated mol. wt markers (New England BioLabs) were used for calibration.
Adenylyl cyclase assay
Adenylyl cyclase activity was assayed according to a modified version of
the method described previously (Salomon,
1979). The reaction tube (total volume 50 µl/tube) contained:
reaction buffer (20 µl), membranes (10 µl, 5 µg protein), stimulant
(10 µl) and dilution buffer (10 µl). The final reaction mix contained 20
mM HEPES, 5 mM MgCl2, 1 mM ATP-Na2, 10 µM
GTP-Na2, 5 mM phosphocreatine, 85 U/ml creatine phosphokinase, 1 mM
dithiothreitol, 1 µCi -[32P]ATP (30 Ci/mmol), pH 7.4,
with or without a stimulant (odor mixture or forskolin) and 50 µM cAMP to
inhibit breakdown of [32P]cAMP by phosphodiesterase. In some
experiments, 0.5 mM isobutyl-1-methylxanthine (IBMX), a non-selective
phosphodiesterase inhibitor, was added to confirm that [32P]cAMP
breakdown was completely blocked. Addition of IBMX yielded the same results as
excess cold cAMP, thereby indicating that cold cAMP is sufficient to block
breakdown. Stimulants were odorant mixture (citralva, eugenol and D-carvone;
final concentration of 0.1 mM each) or forskolin (final concentration of 5
µM), with a final ethanol concentration of 0.09 or 0.5%, respectively.
Corresponding controls contained the same amount of ethanol. Reactions were
incubated for 15 min at 37°C in a shaking water bath and terminated on ice
by the addition of 10 µl of ice-cold `stop' solution (5 mg/ml cAMP, 0.5 N
HCl, [3H]cAMP tracer, 1 x 105 d.p.m./sample to
calculate recovery). cAMP was separated from the reaction mix on SpinZyme
acidic alumina columns (Pierce Chemicals) according to manufacturer's
instructions. Radioactivity of the eluates was determined by liquid
scintillation spectrometry. The time course was linear for 15 min under these
conditions at protein concentrations ranging between 1 and 5 µg.
Statistical analysis
Data were analyzed by two way, repeated-measure ANOVA. The within-subject
factor was stimulus condition and the between-subject factor was preparation.
Significance levels were set at P 
0.05.
Materials
Two-week-old female CF-1 mice were purchased from Charles River
Laboratories. -[32P]ATP and 2,8-[3H]cAMP were
from Du Pont—New England Nuclear. Forskolin was purchased from
Calbiochem. The odorant mix was composed of three different chemicals:
D-carvone, eugenol (both from Fluka Chemicals) and citralva (International
Flavors and Fragrances Inc.). The protease inhibitors chymostatin and Pefabloc
were purchased from Boehringer-Mannheim. Aprotinin, leupeptin and pepstatin
all were purchased from Sigma Chemicals. Affinity-purified rabbit polyclonal
antibodies to adenylyl cyclase III and to Golf were purchased from
Santa Cruz Biotechnology. Rabbit polyclonal antibody to PDE1C2 was a generous
gift from Dr Joseph Beavo, Department of Pharmacology, University of
Washington, Seattle, WA. All other reagents were purchased from Sigma and were
ultrapure.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
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Two membrane preparations enriched in cilia from olfactory receptor neurons were evaluated: a newly developed `mechanical agitation' method and the traditional calcium shock method. Preliminary experiments evaluated effects of sonication strength and time on the specific activity of adenylyl cyclase and the yield. The optimal conditions are reported here. Both methods of cilia preparation are outlined in Figure 1. In the mechanical agitation method, nasal epithelial tissue is rocked gently in HEPES/saline buffer with EDTA, followed by a centrifugation step. The resulting pellet fraction is further disrupted by two sonication steps and fractions are collected by differential centrifugation. In the calcium shock method, the nasal epithelium is gently rocked in a high calcium buffer followed by differential centrifugation to collect fractions. Fractions from these two preparations were biochemically characterized by comparing average protein yields, basal adenylyl cyclase activity, forskolin-stimulated adenylyl cyclase activity, odorant-stimulated adenylyl cyclase activity and by detection of ciliary marker proteins using immunoblot analysis.
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Ciliary membrane enrichment in each fraction from both preparations was estimated by assay of odorant-stimulated adenylyl cyclase activity (an activity marker for ciliary membranes; Figure 2). Basal, forskolin-stimulated and odorant-stimulated adenylyl cyclase activity was observed in every fraction. Specific activity for odor-stimulated and forskolin-stimulated adenylyl cyclase activity was similar in the initial fraction of each preparation. Subsequent fractions had lower specific activity. There is no dramatic difference in fold-stimulation by odorant or forskolin across fractions, suggesting that components for stimulation are present in every fraction and that inhibitors are not concentrated in any particular fraction.
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In Table 1, basal activity has been subtracted from stimulated activity to show net specific activity. For mechanical agitation, the results show that P1 specific activity was approximately twice that of P2A for the odor-stimulated fraction, indicating that P1 was more enriched for ciliary membranes. For calcium shock, the specific activity of the odor-stimulated and forskolin-stimulated fraction P2 was comparable to that for mechanical agitation. Although the average value of P2 appeared somewhat higher than the comparable fraction, P1, from the mechanical agitation method, ANOVA showed no significant difference between P1 of the mechanical agitation preparation and P2 of the calcium shock preparation.
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The distribution of total cyclase activity from the mechanical agitation and the calcium shock procedure is outlined in Table 2. One-third of the total activity was recovered in the P1 fraction of the mechanical agitation procedure and the P2 fraction of the calcium shock procedure. Another 40% of the total activity was recovered in fraction P2A of the mechanical agitation procedure. However, the specific activity of the P2A fraction was only half that of the P1 fraction, thereby indicating that P2A was not as enriched for cilia as P1. Distribution of total activity across fractions indicated no evidence for separation of odor-stimulated and forskolin-stimulated components (Table 2). There did not appear to be any fraction in which odorant receptor-activated cyclase was enriched over another compartment containing cyclase uncoupled to receptor. The yield of odorant- and forskolin-stimulated adenylyl cyclase activity by the mechanical agitation method was significantly higher than the yield by the calcium shock method (ANOVA).
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Molecular components of both preparations were characterized by immunoblot
analysis of marker proteins of cilia. Immunohistochemical and immunoblot
analysis from previous reports showed that ACIII, Golf and PDE1C2 are
highly enriched in cilia structures (Yan
et al., 1995;
Belluscio et al.,
1998; Schandar et
al., 1998; Wei et
al., 1998; Wong et
al., 2000). Immunoblot characterization of the mechanical
agitation and the calcium shock fractions
(Figure 3) shows that ACIII,
Golf and PDE1C2 were enriched in the first fraction of membranes from
both procedures. The levels of these proteins diminished in subsequent
fractions. This result is consistent with the enzymatic analysis and indicates
that the P1 fraction from mechanical agitation and the P2 fraction isolated by
calcium shock were most enriched in ciliary membranes.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
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Mechanical agitation is an effective way of preparing enriched fractions of olfactory cilia without exposing the tissue to high concentrations of calcium. This new method of isolating membrane fractions enriched in cilia was compared to the calcium shock method, using isotonic buffer conditions for each preparation. We showed that membranes enriched with olfactory cilia can be isolated under isotonic conditions (a more physiological environment) without the use of high calcium concentrations. Maintaining a physiological environment may prevent perturbations of biochemical enzymes and pathways induced by hypotonic and high calcium buffer conditions.
The most active fractions from both preparations were equivalent in enrichment, as shown by several criteria: specific activity, fold stimulation and enrichment for specific ciliary marker proteins. In our hands, the mechanical agitation method produced a slightly better yield of odorant- and forskolin-stimulated adenylyl cyclase activity than the calcium shock method (Table 2).
The specific activity of adenylyl cyclase in fractions enriched in ciliary
membranes varies according to animal species and across laboratories. These
results are difficult to compare across studies because different odorants and
concentrations were employed. The relevant studies also assayed cAMP
production on a minute time scale and separated products by column
chromatography. In our laboratory, fractions enriched for ciliary membranes
that are isolated from mouse olfactory epithelial tissue by the mechanical
agitation and the calcium shock preparations show similar basal (10 µM GTP;
2.81 ± 0.22 and 2.26 ± 0.15, nmol/mg/min, respectively) and
forskolin-stimulated (18.73 ± 0.91 and 20.21 ± 2.71,
nmol/mg/min, respectively) specific activity responses. For both preparations,
odorant-stimulated activity exhibited a 1.7- to 2.3-fold increase over basal
conditions. In agreement with these values, a study that used membranes from
cilia dislodged by calcium shock from both male Sprague—Dawley rats and
frogs (Rana catesbeiana) reported comparable values. For both frog
and rat, the basal plus GTP activity ranged from 2 to 4 nmol/mg/min and 4
µM forskolin-stimulated activity ranged from 18 to 24 nmol/mg/min. At
maximum stimulation, the odorant (100 µM) activity was 
1.5- to
1.6-fold above basal plus GTP specific activity
(Sklar et al., 1986).
Other publications from Lancet's laboratory using ciliary membrane
preparations isolated by calcium shock from frog (Rana ridibunda) or
rat (BN/Mai from Weizmann Institute Animal Breeding Center) report a higher
basal and stimulated adenylyl cyclase specific activity. In frog, basal plus
GTP activity is 25 nmol/mg/min (Chen
et al., 1986) and maximal odorant stimulated activity at
50 nmol/mg/min (Pace et al.,
1985; Chen et al.,
1986). In a separate study from this laboratory, even higher
levels of basal and odorant-stimulated adenylyl cyclase activity have been
reported (Pace and Lancet,
1986).
Calcium has been thought to be essential for dislodging cilia from olfactory neurons in epithelial tissue, but it remains unclear how exposure of olfactory epithelial tissue to high calcium concentrations affects the signaling process. Reasons to be concerned are that signal transduction is sensitive to dynamic changes in calcium. Calcium concentrations affect calcium-sensitive machinery, such as calcium-activated proteases, calcium-mediated protein association and kinase and/or phosphatase activity. Changes in calcium concentrations may alter covalent modifications, signal complex associations and/or location of signal components in cilia of olfactory neurons that potentially could alter the state of the isolated cilia. Therefore, calcium might be expected to alter the basal or regulatory state of isolated cilia because it affects so many signaling proteins in the cell.
In the majority of olfactory neurons, odor-stimulated olfactory receptors
activate signal cascades that ultimately result in the increase of internal
calcium concentrations. Elevated cytosolic calcium levels lead to
calcium—calmodulin inhibition of the cyclic-nucleotide-gated channel and
also to activation of CaMKII. CaMKII phosphorylates and reduces the activity
of adenylyl cyclase, suggesting an involvement with signal termination
(Wei et al., 1998).
CaMKII activity also results in the phosphorylation of ERK I/II, leading to
the stimulation of Cre-mediated gene transcription
(Watt and Storm, 2001).
PDE1C2 also is calcium—calmodulin activated and breaks down the
second messenger molecule cAMP to 5'-AMP, contributing to the
termination of the signal event (Borisy
et al., 1992; Yan
et al., 1995). Thus, high calcium concentrations are
intimately involved in the dynamics of cAMP signal transduction in olfactory
cilia.
An additional reason for being concerned about the effect of high calcium
on cilia is that some isoforms of G-protein-coupled receptor kinases (GRKs)
are modulated by calcium. GRKs phosphorylate seven transmembrane receptors
resulting in the down regulation of receptor activity. GRK3 has been localized
to the cilia of rat olfactory neurons and appears to play a role in
desensitization of olfactory receptors
(Schleicher et al.,
1993). Calcium—calmodulin complexes inhibit GRK3, thereby
potentially modulating odor receptor activity
(Chuang et al.,
1996).
Protein kinase C (PKC) also has been implicated in odorant-mediated
signaling and desensitization (Boekhoff and
Breer, 1992; Boekhoff et
al., 1992; Schleicher
et al., 1993). In rat olfactory neurons, the 
,
and 
isoforms of PKC have been localized to the sensory cilia
by immuno-histochemistry (Muller et
al., 1998). The isoform of PKC, a member of the
`classic' group, depends upon calcium for its regulation
(Muller et al.,
1998). In frog olfactory epithelium, activators of PKC (PDBu and
the diterpene mezerein) potentiated forskolin-induced cAMP accumulation,
whereas inhibitors of PKC (Goe 6983, staurosporine and polymyxin) had no
effect (Frings, 1993).
Alterations of calcium concentrations also may alter PKC distribution
(Almholt et al., 1999)
or perturb odorant-signaling.
Neural proteinases (calpains) are calcium-dependent cysteine proteases that
should be considered in evaluating ciliary isolation protocols. Immunostaining
of calpain I in rat brain showed high levels of expression in primary
olfactory axons (Siman et al.,
1985). Calpains activate or alter the regulation of certain
enzymes, such as protein kinases and phosphatases, by limited proteolysis and
thus could potentially modify the state of proteins involved in odorant
signaling (Molinari and Carafoli,
1997), although no such effects have been reported as yet.
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Summary |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
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We have developed an alternative method of isolating membranes enriched with ciliary markers that does not depend upon high calcium. Since calcium participates in multiple regulatory steps in olfactory neuron signaling, the possible effects of high calcium treatment warrant careful consideration. The data from these experiments indicate that high calcium is not essential for releasing cilia from olfactory neurons in the epithelial tissue. One apparent advantage of the mechanical agitation procedure is that the yield of functional membranes is somewhat higher than that of the calcium shock method, with comparable specific activity of adenylyl cyclase. Future studies will evaluate whether differing methods of isolation alter the dynamics of signaling and regulation.
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Acknowledgments |
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This work was supported in part by a grant from DARPA (DAAK60-97-K-9502).
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Accepted June 6, 2002
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