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Calcium and olfactory transduction
0300-9629jSS$3.00+ 0.00 Q 1988Pergamon Press pie
Camp. Biochem. Physiol. Vol. 91A, No. 2, pp. 309-315, 1988
Printed in Great Britain
CALCIUM
AND OLFACTORY
TRANSDUCTION
BRUCE D. WINEGAR,* EDWARD R. ROSICK~ and ROLLIE SCHAI=ER$ Department of Biological Sciences, University of North Texas, Denton, Texas 76203-5218, USA. Telephone: (512) 471-6971 (Received 26 January I988)
Abstract-1, Inorganic cations, organic calcium antagonists, and ~lrn~ulin antagonists were applied to olfactory epithelia of frogs (Runa @ens) while recording electroolfactogram (EOG) responses. 2. Inorganic cations inhibited EOGs in a rank order, reflecting their calcium channel blocking potency: La3+ > Zn2+ > Cd2+ > Al”+ > Ca*+ v Srr+ v Co2+ > Ba2+ > Mg 2+. Barium ion si~ificantiy enhanced EOGs immediateiy following application. 3. Dihiazem and verapamil produced dose-dependent EGG inhibition. 4. Calmodulin antagonists inhibited EOGs without correlation to their anti-calmodulin potency.
Olfactory transduction most probably begins when odorant molecules bind to receptor sites which exert
functional changes at ion channels, leading to the development of receptor potentials. These odorant receptor sites appear to be localized to olfactory cilia (Adamek et al., 1984; Anholt, 1987) and to the apical dendrites of olfactory receptor neurons. Odorantreceptor binding presumably affects the functional state of memb~ne ion channels by mechanisms similar to those which mediate the actions of chemical messengers such as neurotransmitters or hormones (Lance& 1986). These mechanisms may include: (1) conformational changes induced by messenger binding to a recognition site on a receptor/ ionophore (Karlin, 1983), including receptoroperated ion channels (Hurwitz, 1986); (2) modulation of ion channel conductance via protein phosphorylation by second messengers such as adenosine 3’:5’-cyclic monophosphate (CAMP) (DePeyer et al., 1982; Takai et al., 1982; Levitan ei al., 1983; Lancet and Pace, 1987) or ~almodulin (Cohen, 1982); (3) mobilization of Ca’+ and activation of protein kinase C by the signal-de~ndent breakdown of inositol phospholipids (Fisher et al., 1984; Nishizuka, 1984). of olfactory receptor potentials Sodium ions are generally regarded as the primary current source for olfactory receptor potentials (Lancet, 1986). Additionally, Vodyanoy and Murphy (1983) and Dionne (1986) have obtained evidence for chemosensitive potassium channels in rat and mouse olfactory receptor neurons. While Ca*+ is necessary for olfactory transduction (Suzuki, 1978; Kleene, 1986; Lance& 1986) its precise role is unknown. Ionic bases
*Present address: Department of Zoology, The University of Texas at Austin, Austin, TX 78712-1064, USA. 7Present address: Kellogg Eye Center, 1000 Wall Street, Ann Arbor, MI 48105, USA. $Gffprint requests to: R. Schafer at the above address.
Calcium ions may function as current carriers during olfactory receptor potential generation. Suzuki (1978) reported that La3+ or Co2+ (calcium channel blockers) or EGTA (a calcium chelator) blocked spike responses to L-a&nine in olfactory receptor cells of the lamprey (~nro~~~en~~ japonicus), while changes in Na+ and K+ concentrations had no effect. Takagi et al. (1969) reported decreases but not elimination of frog electroolfactogram (EOG) amplitudes after replacing Na+ with equimolar concentrations of Ca2+ or M$+. When they replaced Na+ with St?+, which is highly permeant in calcium channels, EOGs were recorded at slightly reduced amplitudes for 20 min but later decreased substantially. Replacement of Na+ with Zn2+, a calcium channel blocker, eliminated EOGs. Inward calcium currents can be blocked by certain inorganic cations, such as the following, listed in order of decreasing potency: La3+, Cd*+, Co’+, Ni2+, Mn*+ and Mg2+ (Hagiwara and Byerly, 1983). These cations appear to penetrate the calcium channel at different rates. Those which move relatively slowly through the channel act as competitive inhibitors of Ca2+ (Hurwitz, 1986). Lanthanum ion, a potent calcium channel blocker, may bind to a site (the “calcium coordination site”) on the outer mouth of the channel (Glossmann et al., 1982). If Ca*” influx is necessary for olfactory transduction, multivalent cations with greater calcium channel blocking potency should block the generation of olfactory receptor potentials. In contrast to the inhibitory action of many inorganic cations, Ba2+ conducts larger inward currents through calcium channels than Ca2+ itself (Hagiwara and Ohmori, 1982; Hille, 1984), and may have different effects from calcium channel blockers on olfactory transduction. However, low concentrations of extracellular Ca*+ can block Ba2+ currents, presumably because Caz+ binds more strongly to the outer site (Hess and Tsien, 1984). In this paper, we report on effects of inorganic cations, as well as organic ~l~iurn channel antagonists to depress amplitudes of the electroolfactogram (EOG), a measure of olfactory transduction. Calmodulin antagonists were also tested but did not
309
310
BRUCE
depress EGGS in a manner anticalmodulin potency.
consistent
with
D. WINEGARef al. their
MATERIAJ_S AND METHODS
Northern grass frogs (Ram pipiens) were anesthetized and immobilized with subcutaneous injections into the dorsal lymph sac of 250mg/kg urethane and 15 m&kg d-tubocurarine chloride. The ventral olfactory mucosa was surgically exposed by removal of the external nares and the roof of the olfacory sac. A standard odorant stimulating and recording system was used (Schafer et al., 1984). Clean air supplied by an air compressor was passed through activated charcoal, humidified, then delivered to the olfactory mucosa at a delivery rate of 850ml/min through 0.75 cm i.d. orifices situated 4.3 cm from the mucosa. The odorant reservoirs in the stimulating system contained sufficient odorant in liquid form (0.3-1.5 ml dispersed on a 5 cm diameter disk of filter paper) to saturate a 1000 ml air volume for many hours of constant stimulation. The standard odorant, isoamyl acetate @AA), was delivered as a saturated vapor in air to the surgically-exposed ventral olfactory mucosae every 100 set in 0.3 set pulses. Other odorants, including cyclohexanol, cyclopentanone, cyclopentanol, and dimethyl disulfide, were used periodically to verify that the effects observed with IAA were representative of the effects on responses to other odorant classes. Extracellular stimulating and recording methodology, described by Schafer et al. (1984), was used to record the slow, surface-negative potential (EOG) elicited by odorant delivery to the ventral olfactory epithelium. The EOG is primarily a spatial summation of receptor potentials from many olfactory receptor cells. A Ringer/agar-filled capillary electrode with a tip diameter of approximately 100 pm was placed onto the crest of the olfactory eminence, a moundlike landmark at the approximate center of the mucosa. El~tr~olfactogram responses were amplified by a Tektronix 5A22N differential amplifier. Waveforms were displayed and photographed in DC mode on a Tektronix Dl 1 analog storage oscilloscope or a 5223 digitizing oscilloscope, and the EOG amplitude recorded continuously with a Soltec 1242 chart recorder. Only preparations which gave an initial baseline EOG magnitude greater than 1 mV were used. The pre-treatment baseline was taken as the mean amplitude of five EOGs preceding the application of an agent. The course and extent of changes in the mV amplitude of EOGs following agent delivery were monitored over 33min, post-treatment (20 EOGs). The 20 post-tr~tment EOGs were expressed as a percentage of the baseline and in~vidu~ly averaged from four or more experiments to obtain the percent inhibition produced by a given agent at a given concentration. Electroolfactogram data were analysed by: (1) statistical testing {i.e. one-way analysis of variance for fixed effects); (2) construction of dose-response curves from dilution series; (3) rank ordering, as in the case of inorganic ions. Aerosol applicationof agents A quantitative aerosol delivery system developed in this laboratory was used to apply aqueous solutions to the epithehum. Aerosol application of reagents is preferable to irrigation techniques such as lavage, because the addition of fluids to the mucosa disrupts el~trophys~olo~~al recording (Schafer et al., 1987). The aerosol delivery system consisted of a DeVilbiss no. 40 glass nebulizer connected through ground glass fittings to a 0.75 cm i.d. orifice situated 4.3 cm from the mucosa. The nebulizer was loaded with solutions at or below the millimolar range and a uniform aerosol spray was generated by N, at a flowrate of 850ml/min (Hinds, 1982). The aerosol delivery system was calibrated by high performance liquid chromatography and acid-base titration
to deliver 2.7 n1/mm2. The solute concentration delivered to the olfactory mucosa was calculated from the area of the ventral olfa&ory mucosa (mean = 5.9 mm2) and assuming a mucous layer thickness of 0.03 mm (Getchell. 1980). However, the delivered concentration was probably attenuated by the mucous continually secreted from the olfactory epithelium. Qualitative observations of the rate of dye movement in the mucous suggested a copious secretion. The mucosa constantly cleanses itself through this secretion which is driven by cilia to a rapid drainage through the internal naris. chemical agents We tested chloride salts of inorganic cations which block or enhance inward calcium con&tctance (Hagiwara and Bverlv. 1983: Hille. 19841.These include Al3+. Cd* +. Co2 + . &+;'Mg2+'andin2f ' , which block calcium channels, and Ba2+, Ca’+ and Sr*+, which permeate calcium channels. Agents representing three classes of organic calcium channel antagonists (Garcia et al., 1986) were also tested, i.e. verapamil (an aralkylamine), diltiazem (a benzothiampine), and both nicardipine and nifedipine (1,4-dihydropyridines or DHPs). The anti-calmoduhn agents trifluoperazine (TFP), chlorpromazine (CPZ) and N-(6-aminohexyl)-5chloro-1-naphthalenesulfona~de (W-7) were used as probes for potential calmodulin involvement in olfactory transduction. Both TFP and CPZ ad~tionally affect other chemical messenger systems. Reagents were obtained from Aldrich, Calbiochem, Pfalz and Bauer, and Sigma Chemical companies. Hydrophilic agents were dissolved in double-distilled water. Hydrophobic agents were first dissolved in either dimethyl sulfoxide (DMSO) or polyethylene glycol400 (PEG 400) and then diluted to a 1% solution. Controls consisted of separate experiments with aerosol application of the appropriate solvent, but without the reagent. All organic solutions were freshly prepared each day and stored in darkness, because many are known to be lift-~nsitive (e.g. DHPs). RESULTS Solvents for drug delivery Aerosol experiments with delivery solvents alone were used to generate control recovery curves. In separate sets of experiments, aerosol application of distilled water, frog Ringer, 1% DMSO and 1% PEG 400, each produced slight to moderate decrements in averaged amplitudes of 20 post-treatment EOGs. Water inhibited by 5% (N = lo), frog Ringer by 9% (N = S), 1% DMSO (143 mM) by 19% (lv = 7) and 1% PEG 400 (28.15mM) by 18% (N =4). Since water was slightly less supressive to EOGs than frog Ringer (Fig. 1) it was chosen as the main delivery solvent. The first few EOGs following delivery of 1% PEG 400 were enhanced by as much as 20%, fol-
lowed by EGG supression that gives a net inhibitory effect. Since 1% PEG 400 could mask inhibitory effects of an agent when it enhances EOGs, DMSO was used as a solvent for hydrophobic agents, the only exception being the calmodulin antagonist, W-7. Ethanol, sometimes used to dissolve hydrophobic
~m~unds, is inapprop~ate for use with calcium antagonists because it blocks voltage-dependent calcium channels at physiologically relevant concentrations (Leslie et al., 1983, 1986). Ethanol, DMSO, and PEG 400 were each tested for enhancement or inhibition of EGGS following a 2 set aerosol spray of 100 mM solutions, which delivers about 15 mM to the olfactory mucosa (Fig. 1). All three solvents
311
Calcium and olfactory transduction E :: 1
(4)
AEROSOL
150
DELIVERY
.!, 1251
y
WAfER
RINGER’S
D&O
SOLVENTS
PEG400 (15
ETHANOL -
mM)
Fig. 1. Bar graph of percent inhibition averaged over 33 min post-treatment. Dimethyl sulfoxide, ethanol and PEG 400 were applied to give a concentration of 15 mM. Frog Ringer and distilled water are included as controls. Results are means f SD for the number of trials given in parentheses.
-10
-5
0
5
10
15
TIME
(MIN)
20
25
30
35
Fig. 3. Averaged recovery curves following 2 set aerosol sprays of the chloride salts of Ba2+ (closed circles, N = 7), A13+ (open circles, N = 5), Zn*+ (closed triangles, N = S), Cd*+ (open triangles, N = 6) and La’+ (closed squares, N = 5). All were applied to give a concentration of 7.5 mM. Bars represent SD.
EOGs: ethanol by 25% (N = 4), DMSO by 16% (N = 5) and PEG 400 by 18% (N = 4).
inhibited
Inorganic responses
calcium
channel
blockers
modijy
EOG
Inorganic di- and trivalent cations which block calcium channels in other tissues inhibited EOG responses by varying degrees, ranging from no effect to complete inhibition. They were applied by 2 set
aerosol spray to achieve a concentration of 7.5 mM on the olfactory mucosa. Averaged records of posttreatment EOGs in Fig. 2 illustrate inhibition of 13% by Co*+ (N = 6), 23% by Ca*+ (N = 4) and 25% by Srr+ (N = 5). Magnesium ions produced no measurable inhibition or enhancement (N = 5). Figure 3 shows averaged EOG records illustrating inhibition of 33% by Al’+ (N = 5), 61% by Cd*+ (N = 6) and 61% by Zn*+ (N = 5). The inhibition from each of these cations is partial and reversible at 7.5 mM, except for La’+, which virtually eliminates EOGs with no recovery over the 33 min post-treatment period (Fig. 3). Slight recovery was observed from La’+ inhibition in extended experiments over 1 hr, post-treatment. Barium ion significantly enhanced the first two post-treatment EOGs by as much as
s- 150
AEROSOL
8
30% (F = 43.04; P < 0.001, df = 19) then produced moderate inhibition with recovery to the baseline (Fig. 3). Averaging the 20 post-treatment EOGs after Ba*+ application gives 6% inhibition (N = 5). The above experiments are summarized in Fig. 4, illustrating the rank order of potency of the inorganic cations in inhibiting EOGs: La’+ > Zn*+ > Cd*+ > Al’+ > Ca*+ > St+*+> Co*+ > Ba*+ > Mg*+. Except for Al’+, which has not been tested extensively for calcium channel blocking action, the rank order of EOG inhibition by cations was compared with several rankings of calcium channel antagonism in other tissues: (1) rank order of Ca*+-activated potassium channel blocking effectiveness in human T lymphocytes (DeCoursey et al., 1984a); (2) rank order of inhibitory effectiveness against [‘Hlnitrendipine binding to homogenized rat cortex (Ehlert et al., 1982); (3) rank order of calcium channel blocking effectiveness in barnacle muscle fiber (Hagiwara and Takahashi, 1967); (4) general rank order of calcium channel blocking effectiveness in neuronal tissue (Hagiwara and Byerly, 1983). Since each of these rankings is very similar, a composite rank order of calcium channel blocking ability was derived by summing weighted values assigned to a given cation
DELIVERY
L
1 (4)
lOO--
b zm p: rns ~a zz ca: 50
(7)
80.-
(5)
CO-40.-
gs
aE
0 -10
-5
0
5
10
15
TIME
(MN)
20
25
30
J
35
Fig. 2. Averaged recovery curves following 2 set aerosol sprays of the chloride salts of Mg2+ (closed circles, N = S), Co*+ (open circles, N = 6), Sr*+ (closed triangles, N = 5) and Ca2+ (open triangles, N = 5). All were applied to give a concentration of 7.5 mM. Bars represent SD.
if
20 -0,
(5) ! MC=
BA=
CO*
SR”
MULTIVALENT
CA* CATIONS
AL%
CD”
(7.5
ZN”
LA”
mM)
Fig. 4. Bar graph of percent inhibition averaged over 33 min post-treatment. The chloride salts of multivalent cations were applied to give a concentration of 7.5 mM. Results are means f SD for the number of trials given in parentheses.
BRUCED.
312
WATER (CONTROL) CALCIUM
CHANNEL
DILTIAZEM
WINEGAR et al.
VERAPAMIL
I
(0.15
mM)
LOG
Fig. 5. Bar graph of percent inhibition averaged over 33 min post-treatment. Diltiazem and verapamil were applied to give a calculated final concentration of 0.15 mM. Distilled water is included as a control. Results are means f SD for the number of trials given in parentheses.
according to its position in each rank order (i.e. first position is given a value of 1, second position a value of 2, etc.): La3+ > Cd2+ > Zn2+ > Co2+ > Ba2+ > Ca2+ > Sr2+ > M$+. The Spearman rank correlation coefficient was computed to test for an association between the ranking of EGG inhibitory potency and the composite ranking. The correlation coefficient (R, = 0.79) is significant (P < 0.025), which supports the hypothesis that EGG inhibition by these cations involves their blockage of calcium channels. Certain organic inhibitory
calcium
-4
-5 ANTAGONISTS
channel
antagonists
are
The organic calcium channel blockers d-cisdiltiazem (hereafter referred to as diltiazem), (+)-verapamil (hereafter referred to as verapamil), nicardipine, and nifedipine were tested. Diltiazem and verapamil both inhibited EGGS following delivery of 0.15 mM. Verapamil was more inhibitory (Fig. 5), supressing EOGs by 36% (N = 5) while diltiazem produced 17% inhibition (N = 4). Dose-response curves for diltiazem and verapamil indicate that they both inhibited EOGs in a dosedependent manner. Verapamil produced significant inhibition compared with water control experiments at a concentration as low as 0.015 mM (F = 26.03; P = 0.0003; df = 12). Diltiazem had less intrinsic activity than verapamil, producing 34% inhibition (N = 5) at the highest concentration tested (1.5 mM) while verapamil inhibited by 54% (N = 5) at this concentration (Fig. 6). Neither of the DHPs tested (nicardipine and nifedipine) inhibited any more than 1% DMSO controls at 0.015 mM. Their extremely hydrophobic nature prevented experiments at higher drug concentrations in 1% DMSO solutions. Actions of calmodulin antagonists
The calmodulin antagonists trifluoperazine (TFP), chloropromazine (CPZ) and N-(daminohexyl)5-chloro-1-napthalenesulfonamide (W-7) inhibited EGG responses following aerosol delivery of 0.15 mM (Fig. 7). W-7, the most potent calmodulin antagonist of those we tested (Hidaka and Tanaka, 1982) inhibited the first post-treatment EOG up to 80% in
-3
CONCENTRATION
Fig. 6. Dose-response curves for verapamil (closed circles, N = 6) and diltiazem (open circles, N = 6). Each point represents the mean + SD.
comparison with the 1% PEG 400 control curve. However, the W-7 curve and the control curve merged rapidly. Both CPZ and TFP were less inhibitory than W-7 immediately following aerosol application, but they exerted longer-lasting inhibition. When averaged over 20 post-treatment EGGS, W-7 inhibited by 27% (N = 7), CPZ by 31% (N = 4) and TFP by 23% (N = 4) (Fig. 7). DISCUSSION
This study examined the role of Ca2+ in olfactory transduction by aerosol application of inorganic cations, organic calcium channel antagonists and calmodulin antagonists to the olfactory mucosae of anesthetized frogs during electrophysiological recording. Results of their effects on EOG amplitudes support a role for Ca2+ as a current carrier for olfactory receptor potentials, while a second messenger role of Ca2+, in conjunction with camodulin, appears unrelated to olfactory transduction. Tests for artifacts
Tests for correlations between pre- and posttreatment effects are useful to avoid errors of data
WATER (CONTROL)
CPZ
CALMODULIN
TFP ANTAGONISTS
PEG400 (CONTROL) (0
15
W-7 mM)
Fig. 7. Bar graph of percent inhibition, averaged over 33 mm post-treatment. Chlorpromazine (CPZ), trifluoperazine (TFP) and W-7 were applied to give a concentration of 0.15 mM. One percent PEG 400 (used to dissolve W-7) and water are included as controls. Results are means + SD for the number of trials given in parentheses.
Calcium and olfactory transduction anaylsis when using percentages alone. For instance, a positive correlation between baseline EOG amplitudes and their subsequent inhibition might give false indications of sensitivity to a given agent if many of the experiments had high pre-treatment baselines. A test of the null hypothesis (i.e. the true correlation is zero) was made with Student’s t-test to determine whether post-treatment EOG amplitudes were linearly correlated either negatively or positively with pre-treatment baselines. The values of t were determined from ten sets of experiments with N of not less than four for each set. No significant differences were observed, thus indicating that pretreatment EOG amplitudes did not bias the effects of these agents. Eflects of inorganic calcium channel blockers
The results reported here provide evidence that an inward CaZ+ current accompanies the EOG and appears to be necessary for olfactory transduction. Di- and trivalent inorganic cations inhibited EOG responses in the frog in a rank order which is highly correlated with their calcium channel blocking effectiveness in other tissues. The sole exception to this correlation is Co2+ which is less inhibitory than would be expected, compared to its relative effectiveness at blocking calcium channels. The EOGenhancing effect of Ba*+ suggests a current-carrying role for Ca2+, because substitution of Ba*+ for Ca*+ often produces greater currents in other tissues than Ca*+ itself (Hagiwara and Ohmori, 1982; Hille, 1984). If Ca*+ is a current carrier, it seems paradoxical that Ca*+ application results in inhibition of subsequent EOGs. However, in some tissues, elevated intracellular Caz+ inactivates voltage-dependent calcium channels (Reuter, 1983). Barium ions substitute well for Ca*+ as current carriers, but do not inactivate calcium channels (Hille, 1984). This phenomenon could explain the EOG enhancement observed with Ba*+, because Ba2+ entry might not inactivate calcium channels in olfactory receptor cells. An alternative explanation for EOG inhibition by Ca*+ is that high extracellular Ca*+ can reduce membrane permeability through a membranestabilizing action (Webb, 1982). Calcium ions in high concentration could bind to acidic phospholipids in the membrane, increasing the phase transition temperature and decreasing membrane fluidity (Housley and Stanley, 1982). Calcium ions could also inhibit through their effect on potassium channels. A common regulatory function of elevated intracellular Ca2+ is activation of Ca*+-dependent potassium channels which act to repolarize the membrane (Fishman and Spector, 1981). That Ba2+ may block potassium channels (Armstrong and Taylor, 1980) suggests the possibility that EOG enhancement by Ba2+ is the result of potassium channel blockage. However, in preliminary experiments (data not shown) we found no EOG enhancement in the frog after aerosol application of 4-aminopyridine and quinine, drugs which block potassium channels (DeCoursey et al., 1984b). These agents inhibited EOGs only slightly at concentrations in the millimolar range, indicating that potassium channel blockage is not associated with EOG enhancement.
313
Calcium channels can be made permeable to monovalent cations such as Na+ and Li+ by removing divalent cations (Lansman et al., 1985), an effect that may have significance for experiments where the ionic environment of the preparation is altered. For example, Yoshii and Kurihara (1983) recorded odorant-evoked activity in the olfactory bulbs of carp (Cyprinus carpio), rainbow trout (Salmo gairdneri), and bullfrog (Rana catesbeiana) while altering the ionic environment of the olfactory epithelium. Olfactory responses were eliminated by irrigating mucosae with EDTA, followed by deionized water. They found a number of organic and inorganic cations supported olfactory responses, including Ca*+, Cd*+, Co*+, Li+, Mn*+, choline+ and Tris+, which could have been due to an altered calcium channel selectivity in the absence of Ca*+ (Hess and Tsein, 1984). Effects of organic calcium channel antagonists
Many of the organic calcium channel antagonists tested blocked EOGs to some extent. The doseresponse curve for diltiazem had a lower asymptotic limit than the dose-response curve for verapamil (Fig. 6), indicating that these compounds differed in their intrinsic activity to inhibit EOGs. Diltiazem and verapamil were both partial inhibitors of EOGs (Fig. 7) which suggests that more types of channels contribute to EOGs than the calcium channels associated with their binding sites. The DHPs nicardipine and nifedipine, had little or no effect on EOG magnitude. Because DHPs typically exert physiological actions at a much lower concentration than we tested (e.g. 10m9M; Miller, 1985), it seems likely that DHP binding sites, if present on olfactory receptor cells, do not affect olfactory transduction. Inhibition of EOGs by inorganic cations but not by DHPs follows the pharmacological profile of N-type calcium channels characterized by Nowycky et al. (1985). Of the various known types of calcium channels, N-type channels are found on sensory neurons and are characterized by insensitivity to DHPs (Reuter, 1985). Additional evidence that suggests an involvement of N-type or other similar calcium channels in olfactory transduction is that the N-type channels are blocked by Cd*+ but enhanced by Ba2+ (Nowycky et al., 1985), the same effect seen with EOG responses in this study. Additionally, Cd*+ eliminates olfactory functioning in humans (Arvidson, 1981). Calmodulin
Calmodulin antagonists inhibited EOGs, but not in a rank order which corresponds to their binding affinities for calmodulin in other tissues. TFP has greater affinity for calmodulin than CPZ (Levin and Weiss, 1979), but CPZ inhibited EOGs somewhat more than TFP (Fig. 7). Inhibition of EOGs by CPZ and TFP may result from non-specific membrane effects rather than anti-calmodulin activity. Both CPZ and TFP are cationic, amphipathic agents. Because each possesses a hydrophobic region plus a positively charged nitrogen atom, these compounds can partition into and disrupt hydrophobic regions of protein and lipid in the cell membrane (Landry et al., 1981; Lieber et al., 1984). While EOG inhibition by
BRUCE D. WINEGAR et al.
314
the calmodulin antagonist W-7 (Fig. 7) suggests a role for camodulin in olfactory transduction, W-7 may have nonspecific effects as well. The CPZ and TFP results argue against a role of calmodulin in olfactory transduction as a modulator of EOGs. SUMMARY
The possible roles of Ca2+ in olfactory transduction were explored by pharmacological and electrophysiological methods, using agents which inhibit or promote membrane calcium conductance or modify calmodulin activity. These agents were applied by quantitative aeroseol spray onto the olfactory epithelia of urethane-anesthetized frogs (Rana pipiens) during extracellular recording and periodic odorant stimulation by isoamyl acetate. Inorganic cations known to block inward calcium currents in other tissues inhibited electoolfactogram (EOG) responses when applied by a 2 set aerosol spray, producing a concentration of 7.5 mM in the mucous layer overlying the epithelium. The rank order of inhibitory potency of the chloride salts is: La3+ > Zn2+ > Cd2+ > A13+ > Ca2+ > Sr2+ > Co’+ > Ba2+ > M$+. Aerosol exposure to La3+, virtually eradicates EGG responses, while Ba*+ initially produces significant enhancement (F = 43.04, P < 0.001, df = 19) followed by a slight inhibition. Barium ions can substitute for Ca2+ to carry greater currents in other tissues and may enhance olfactory responses by a similar mechanism. Magnesium ions have no inhibitory action at the same concentration. Calcium ions are significantly inhibitory (F = 5.74; P = 0.0355; df = 12) at a concentration of 0.15 mM. The organic calcium channel antagonists, diltiazem and verapamil, were applied at 0.15 mM by a 2 set aerosol spray. They reversibly inhibited EOG amplitudes by 17 and 36%, respectively. Verapamil produced significant inhibition (F = 17.17; P = 0.002; df = 11) after application at a concentration of 0.015 mM. By contrast, the 1,Cdihydropyridine calcium channel antagonists, nicardipine and nifedipine, did not inhibit significantly beyond 1% DMSO controls when tested at 0.015 mM. The calmodulin antagonists, trifluoperazine (TFP), chlorpromazine CPZ) and N-(6-aminohexyl)-5chloro- 1naphthalenesulfonamide (W-7) depressed EOG amplitudes without correlation to their order of potency as calmodulin antagonists. Following a 2 set aerosol delivery of 0.15 mM, CPZ and W-7 inhibited by 31 and 27%, respectively, while TFP inhibited by 23%. These results support the hypothesis that Ca2+ participates in olfactory transduction as a charge carrier. The pattern of electrophysiological enhancement and inhibition suggests that calcium channels with properties similar to N-type calcium channels participate in olfactory transduction. Acknowledgements-This work was supported by grants from the National Science Foundation (BNS-81-08842 and CHE-8509557) and the University of North Texas Faculty Research Fund to R. Schafer. We thank Drs M. Donahue and T. Yorio for the W-7, and Mr J. Gomez for technical assistance.
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Fishman M. C. and Spector I. (1981) Potassium current supression by quinidine reveals additional calcium currents in neuroblastoma cells. Proc. Natn. Acad. Sci., USA 18, 52455249.
Garcia M. L., King V. F., Siegl P. K. S., Reuben J. P. and Kaczorowski G. J. (1986) Binding of Ca2+ entry blockers to cardiac sarcolemmal membrane vesicles. J. biol. Chem. 26, 8146-8157. Getchell T. V., Heck G. L., DeSimone J. A. and Price S. (1980) The location of olfactory receptor sites: inferences from latency measurements Eiophys. J. 29, 397-411. Glossman H., Ferry D. R., Lubbecke F., Mewes R. and Hofman F. (1982) Calcium channels: direct identification with radioligand binding studies. Trena!s Pharmacol. Sci. 3, 431-437.
Hagiwara S. and Takahashi K. (1967) Surface density of calcium ions and calcium spikes in the barnacle muscle fiber membrane. J. gen. Physiol. 50, 583601. Hagiwara S. and Ohmori H. (1982) Studies of calcium channels in rat clonal pituitary cells with patch electrode voltage clamp. J. Physiol., Lond. 331, 231-252. Hagiwara S. and Byerly L. (1983) The calcium channel. Tremis Neurosci. 6, 189-193. Hess P. and Tsein R. W. (1984) Mechanism of ion permeation through calcium channels. Nature 309, 453-456. Hidaka H. and Tanaka T. (1982) Biopharmacological assessment of calmodulin function: utility of calmodulin antagonists. In Calmodulin and Inrracelhdar Ca2+ Receptors (edited by Kakiuchi S., Hidaka H. and Means A. R.), pp. 19-33. Plenum Press, New York. Hille B. (1984) Ionic Channels of Excitable Membranes, pp. 7698, Sinauer Associates Inc., Sunderland, Massachusetts. Hinds W. C. (1982) Aerosol Technology, pp. 40-48. John Wiley and Sons, New York. Houslay M. D. and Stanley K. K. (1982) Dynamics of Biological Membranes: InpUences on Synthesis, Structure and Function, 1st edn., p. 133. John Wiley and Sons,
New York.
Calcium
and olfactory
Hurwitz L. (1986) Pharmacology of calcium channels and smooth muscle. Ann Rev. Pharmacol. Toxicol. 26, 225-258. Karlin A. (1983) The anatomy of a recptor. Neurosci. Comm. 1, 111-123. Kleene S. J. (1986) Bacterial chemotaxis and vertebrate olfaction. Experienria 42, 241-250. Lancet D. (1986) Vertebrate olfactory reception. Ann Rev. Neurosci 9, 329-355. Lancet D. and Pace U. (1987) The molecular basis of odor recognition. Trends Biochem. Sci. 12, 63-66. Landry Y., Ruckstuhl A. and Ruckstuhl M. (1981) Can calmodulin inhibitors be used to probe calmodulin effects? Biochem. Pharmacol. 30, 203 I-2032. Lansman J. B., Hess P. and Tsien R. W. (1985) Direct measurement of entry and exit rates for calcium ions in single calcium channels. Biophys. J. 47, 67a. Leslie S. W., Barr E., Chandler J. and Farrar R. P. (1983) Inhibition of fast- and slow-phase depolarization dependent synaptosomal calcium uptake by ethanol. J. Pharmacol. exp. Therap. 225, 571-575. Leslie S. W., Woodward J. J., Wilcox R. E. and Farrar R. P. (1986) Chronic ethanol treatment uncouples striatal calcium entry and endogenous dopamine release. Brain Res. 368, 174-177. Levin R. M. and Weiss B. (1976) Mechanism by which psychotropic drugs inhibit adenosine cyclic 3’,5’-monophosphatephosphodiesterase of brain. Golec. Pharmacol. 12. 581-589. Le.& R. M. and Weiss B. (1979) Selective binding of antipsychotics and other psychoactive agents to the calcium-dependent activator of cyclic nucleotide phosphodiesterase. J. Pharamcol. exp. Therap. 208, 454-459. Levitan, I. B., Lemos J. R. and Novak-Hofer I. (1983) Protein phosphorylation and the regulation of ion channels. Trends Neurosci. 6, 496-499. Lieber M. R. Lange Y., Weinstein R. S. and Steck T. L. (1984) Interaction of chlorpromazine with the human erythrocyte membrane. J. biol. Chem. 259, 9225-9234. Miller R. J. (1985) How many types of calcium channels exist in neurones? Trends Neurosci. 8, 45-47.
transduction
315
Nishizuka Y. (1984) Turnover of inositol phospholipids and signal transduction. Science 225, 1365-1370. Nowycky M. C., Fox A. P. and Tsein R. W. (1985). Three types of neuronal calcium channels with different calcium agonist sensitivity. Nature 316, 440-443. Reuter H. (1983) Calcium channel modulation by neurotransmitters, enzymes, and drugs. Nature Ml, 569-574. Reuter H. (1985) A variety of calcium channels. Nature 316, 391. Schafer R., C&well D. W. Fracek S. P. Jr and Brower K. R. (1984) Olfactory studies using ethyl bromoacetate and other chemically active odorants. Chem. Senses 9, 3 l-52. Schafer R. Dickens J. C. and Fracek S. P. Jr (1987) Inhibition of protein synthesis does not block physiological responses to odors over the short term. Comp. Physiol. Biochem 86B, 5 13-5 18. Suzuki N. (1978) Effects of different ionic environments on the responses of single olfactory receptors in the lamprey. Comp. Biochem. Physiol. 61A, 461-467. Takagi S. F., Kitamura H., Imai K. and Takeuchi H. (1969) Further studies on the roles of sodium and potassium in the generation of the electro-olfactogram. J. gen. Physiol. 53, 115-130. Takai Y., Kaibuchi K., Matsubara T., Sana K., Nishizuka Yu and Nishizuka Y. (1982) Two transmembrane control mechanisms for protein phosnhorvlation in bidirectional _ _ regulation of cell functions. In Calmodulin and Inwacellular Ca++ Receptors (Edited bv Kakiuchi S., Hidaka H. and Means A. k.), pp. 3333347. Plenum Press, New York. Vodyanoy V. and Murphy R. B. (1983) Single-channel fluctuations in bimolecular lipid membranes induced by rat olfactory epithelial homogenates. Science 220, 717-719. Webb R. C. (1982) D-600 and the membrane stabilizing effect of calcium in vascular smooth muscle. Pharmacology 25, 250-26 1. Yoshii K. and Kurihara K. (1983) Role of cations in olfactory reception. Brain Res. 274, 239-248.
Camp. Biochem. Physiol. Vol. 91A, No. 2, pp. 309-315, 1988
Printed in Great Britain
CALCIUM
AND OLFACTORY
TRANSDUCTION
BRUCE D. WINEGAR,* EDWARD R. ROSICK~ and ROLLIE SCHAI=ER$ Department of Biological Sciences, University of North Texas, Denton, Texas 76203-5218, USA. Telephone: (512) 471-6971 (Received 26 January I988)
Abstract-1, Inorganic cations, organic calcium antagonists, and ~lrn~ulin antagonists were applied to olfactory epithelia of frogs (Runa @ens) while recording electroolfactogram (EOG) responses. 2. Inorganic cations inhibited EOGs in a rank order, reflecting their calcium channel blocking potency: La3+ > Zn2+ > Cd2+ > Al”+ > Ca*+ v Srr+ v Co2+ > Ba2+ > Mg 2+. Barium ion si~ificantiy enhanced EOGs immediateiy following application. 3. Dihiazem and verapamil produced dose-dependent EGG inhibition. 4. Calmodulin antagonists inhibited EOGs without correlation to their anti-calmodulin potency.
Olfactory transduction most probably begins when odorant molecules bind to receptor sites which exert
functional changes at ion channels, leading to the development of receptor potentials. These odorant receptor sites appear to be localized to olfactory cilia (Adamek et al., 1984; Anholt, 1987) and to the apical dendrites of olfactory receptor neurons. Odorantreceptor binding presumably affects the functional state of memb~ne ion channels by mechanisms similar to those which mediate the actions of chemical messengers such as neurotransmitters or hormones (Lance& 1986). These mechanisms may include: (1) conformational changes induced by messenger binding to a recognition site on a receptor/ ionophore (Karlin, 1983), including receptoroperated ion channels (Hurwitz, 1986); (2) modulation of ion channel conductance via protein phosphorylation by second messengers such as adenosine 3’:5’-cyclic monophosphate (CAMP) (DePeyer et al., 1982; Takai et al., 1982; Levitan ei al., 1983; Lancet and Pace, 1987) or ~almodulin (Cohen, 1982); (3) mobilization of Ca’+ and activation of protein kinase C by the signal-de~ndent breakdown of inositol phospholipids (Fisher et al., 1984; Nishizuka, 1984). of olfactory receptor potentials Sodium ions are generally regarded as the primary current source for olfactory receptor potentials (Lancet, 1986). Additionally, Vodyanoy and Murphy (1983) and Dionne (1986) have obtained evidence for chemosensitive potassium channels in rat and mouse olfactory receptor neurons. While Ca*+ is necessary for olfactory transduction (Suzuki, 1978; Kleene, 1986; Lance& 1986) its precise role is unknown. Ionic bases
*Present address: Department of Zoology, The University of Texas at Austin, Austin, TX 78712-1064, USA. 7Present address: Kellogg Eye Center, 1000 Wall Street, Ann Arbor, MI 48105, USA. $Gffprint requests to: R. Schafer at the above address.
Calcium ions may function as current carriers during olfactory receptor potential generation. Suzuki (1978) reported that La3+ or Co2+ (calcium channel blockers) or EGTA (a calcium chelator) blocked spike responses to L-a&nine in olfactory receptor cells of the lamprey (~nro~~~en~~ japonicus), while changes in Na+ and K+ concentrations had no effect. Takagi et al. (1969) reported decreases but not elimination of frog electroolfactogram (EOG) amplitudes after replacing Na+ with equimolar concentrations of Ca2+ or M$+. When they replaced Na+ with St?+, which is highly permeant in calcium channels, EOGs were recorded at slightly reduced amplitudes for 20 min but later decreased substantially. Replacement of Na+ with Zn2+, a calcium channel blocker, eliminated EOGs. Inward calcium currents can be blocked by certain inorganic cations, such as the following, listed in order of decreasing potency: La3+, Cd*+, Co’+, Ni2+, Mn*+ and Mg2+ (Hagiwara and Byerly, 1983). These cations appear to penetrate the calcium channel at different rates. Those which move relatively slowly through the channel act as competitive inhibitors of Ca2+ (Hurwitz, 1986). Lanthanum ion, a potent calcium channel blocker, may bind to a site (the “calcium coordination site”) on the outer mouth of the channel (Glossmann et al., 1982). If Ca*” influx is necessary for olfactory transduction, multivalent cations with greater calcium channel blocking potency should block the generation of olfactory receptor potentials. In contrast to the inhibitory action of many inorganic cations, Ba2+ conducts larger inward currents through calcium channels than Ca2+ itself (Hagiwara and Ohmori, 1982; Hille, 1984), and may have different effects from calcium channel blockers on olfactory transduction. However, low concentrations of extracellular Ca*+ can block Ba2+ currents, presumably because Caz+ binds more strongly to the outer site (Hess and Tsien, 1984). In this paper, we report on effects of inorganic cations, as well as organic ~l~iurn channel antagonists to depress amplitudes of the electroolfactogram (EOG), a measure of olfactory transduction. Calmodulin antagonists were also tested but did not
309
310
BRUCE
depress EGGS in a manner anticalmodulin potency.
consistent
with
D. WINEGARef al. their
MATERIAJ_S AND METHODS
Northern grass frogs (Ram pipiens) were anesthetized and immobilized with subcutaneous injections into the dorsal lymph sac of 250mg/kg urethane and 15 m&kg d-tubocurarine chloride. The ventral olfactory mucosa was surgically exposed by removal of the external nares and the roof of the olfacory sac. A standard odorant stimulating and recording system was used (Schafer et al., 1984). Clean air supplied by an air compressor was passed through activated charcoal, humidified, then delivered to the olfactory mucosa at a delivery rate of 850ml/min through 0.75 cm i.d. orifices situated 4.3 cm from the mucosa. The odorant reservoirs in the stimulating system contained sufficient odorant in liquid form (0.3-1.5 ml dispersed on a 5 cm diameter disk of filter paper) to saturate a 1000 ml air volume for many hours of constant stimulation. The standard odorant, isoamyl acetate @AA), was delivered as a saturated vapor in air to the surgically-exposed ventral olfactory mucosae every 100 set in 0.3 set pulses. Other odorants, including cyclohexanol, cyclopentanone, cyclopentanol, and dimethyl disulfide, were used periodically to verify that the effects observed with IAA were representative of the effects on responses to other odorant classes. Extracellular stimulating and recording methodology, described by Schafer et al. (1984), was used to record the slow, surface-negative potential (EOG) elicited by odorant delivery to the ventral olfactory epithelium. The EOG is primarily a spatial summation of receptor potentials from many olfactory receptor cells. A Ringer/agar-filled capillary electrode with a tip diameter of approximately 100 pm was placed onto the crest of the olfactory eminence, a moundlike landmark at the approximate center of the mucosa. El~tr~olfactogram responses were amplified by a Tektronix 5A22N differential amplifier. Waveforms were displayed and photographed in DC mode on a Tektronix Dl 1 analog storage oscilloscope or a 5223 digitizing oscilloscope, and the EOG amplitude recorded continuously with a Soltec 1242 chart recorder. Only preparations which gave an initial baseline EOG magnitude greater than 1 mV were used. The pre-treatment baseline was taken as the mean amplitude of five EOGs preceding the application of an agent. The course and extent of changes in the mV amplitude of EOGs following agent delivery were monitored over 33min, post-treatment (20 EOGs). The 20 post-tr~tment EOGs were expressed as a percentage of the baseline and in~vidu~ly averaged from four or more experiments to obtain the percent inhibition produced by a given agent at a given concentration. Electroolfactogram data were analysed by: (1) statistical testing {i.e. one-way analysis of variance for fixed effects); (2) construction of dose-response curves from dilution series; (3) rank ordering, as in the case of inorganic ions. Aerosol applicationof agents A quantitative aerosol delivery system developed in this laboratory was used to apply aqueous solutions to the epithehum. Aerosol application of reagents is preferable to irrigation techniques such as lavage, because the addition of fluids to the mucosa disrupts el~trophys~olo~~al recording (Schafer et al., 1987). The aerosol delivery system consisted of a DeVilbiss no. 40 glass nebulizer connected through ground glass fittings to a 0.75 cm i.d. orifice situated 4.3 cm from the mucosa. The nebulizer was loaded with solutions at or below the millimolar range and a uniform aerosol spray was generated by N, at a flowrate of 850ml/min (Hinds, 1982). The aerosol delivery system was calibrated by high performance liquid chromatography and acid-base titration
to deliver 2.7 n1/mm2. The solute concentration delivered to the olfactory mucosa was calculated from the area of the ventral olfa&ory mucosa (mean = 5.9 mm2) and assuming a mucous layer thickness of 0.03 mm (Getchell. 1980). However, the delivered concentration was probably attenuated by the mucous continually secreted from the olfactory epithelium. Qualitative observations of the rate of dye movement in the mucous suggested a copious secretion. The mucosa constantly cleanses itself through this secretion which is driven by cilia to a rapid drainage through the internal naris. chemical agents We tested chloride salts of inorganic cations which block or enhance inward calcium con&tctance (Hagiwara and Bverlv. 1983: Hille. 19841.These include Al3+. Cd* +. Co2 + . &+;'Mg2+'andin2f ' , which block calcium channels, and Ba2+, Ca’+ and Sr*+, which permeate calcium channels. Agents representing three classes of organic calcium channel antagonists (Garcia et al., 1986) were also tested, i.e. verapamil (an aralkylamine), diltiazem (a benzothiampine), and both nicardipine and nifedipine (1,4-dihydropyridines or DHPs). The anti-calmoduhn agents trifluoperazine (TFP), chlorpromazine (CPZ) and N-(6-aminohexyl)-5chloro-1-naphthalenesulfona~de (W-7) were used as probes for potential calmodulin involvement in olfactory transduction. Both TFP and CPZ ad~tionally affect other chemical messenger systems. Reagents were obtained from Aldrich, Calbiochem, Pfalz and Bauer, and Sigma Chemical companies. Hydrophilic agents were dissolved in double-distilled water. Hydrophobic agents were first dissolved in either dimethyl sulfoxide (DMSO) or polyethylene glycol400 (PEG 400) and then diluted to a 1% solution. Controls consisted of separate experiments with aerosol application of the appropriate solvent, but without the reagent. All organic solutions were freshly prepared each day and stored in darkness, because many are known to be lift-~nsitive (e.g. DHPs). RESULTS Solvents for drug delivery Aerosol experiments with delivery solvents alone were used to generate control recovery curves. In separate sets of experiments, aerosol application of distilled water, frog Ringer, 1% DMSO and 1% PEG 400, each produced slight to moderate decrements in averaged amplitudes of 20 post-treatment EOGs. Water inhibited by 5% (N = lo), frog Ringer by 9% (N = S), 1% DMSO (143 mM) by 19% (lv = 7) and 1% PEG 400 (28.15mM) by 18% (N =4). Since water was slightly less supressive to EOGs than frog Ringer (Fig. 1) it was chosen as the main delivery solvent. The first few EOGs following delivery of 1% PEG 400 were enhanced by as much as 20%, fol-
lowed by EGG supression that gives a net inhibitory effect. Since 1% PEG 400 could mask inhibitory effects of an agent when it enhances EOGs, DMSO was used as a solvent for hydrophobic agents, the only exception being the calmodulin antagonist, W-7. Ethanol, sometimes used to dissolve hydrophobic
~m~unds, is inapprop~ate for use with calcium antagonists because it blocks voltage-dependent calcium channels at physiologically relevant concentrations (Leslie et al., 1983, 1986). Ethanol, DMSO, and PEG 400 were each tested for enhancement or inhibition of EGGS following a 2 set aerosol spray of 100 mM solutions, which delivers about 15 mM to the olfactory mucosa (Fig. 1). All three solvents
311
Calcium and olfactory transduction E :: 1
(4)
AEROSOL
150
DELIVERY
.!, 1251
y
WAfER
RINGER’S
D&O
SOLVENTS
PEG400 (15
ETHANOL -
mM)
Fig. 1. Bar graph of percent inhibition averaged over 33 min post-treatment. Dimethyl sulfoxide, ethanol and PEG 400 were applied to give a concentration of 15 mM. Frog Ringer and distilled water are included as controls. Results are means f SD for the number of trials given in parentheses.
-10
-5
0
5
10
15
TIME
(MIN)
20
25
30
35
Fig. 3. Averaged recovery curves following 2 set aerosol sprays of the chloride salts of Ba2+ (closed circles, N = 7), A13+ (open circles, N = 5), Zn*+ (closed triangles, N = S), Cd*+ (open triangles, N = 6) and La’+ (closed squares, N = 5). All were applied to give a concentration of 7.5 mM. Bars represent SD.
EOGs: ethanol by 25% (N = 4), DMSO by 16% (N = 5) and PEG 400 by 18% (N = 4).
inhibited
Inorganic responses
calcium
channel
blockers
modijy
EOG
Inorganic di- and trivalent cations which block calcium channels in other tissues inhibited EOG responses by varying degrees, ranging from no effect to complete inhibition. They were applied by 2 set
aerosol spray to achieve a concentration of 7.5 mM on the olfactory mucosa. Averaged records of posttreatment EOGs in Fig. 2 illustrate inhibition of 13% by Co*+ (N = 6), 23% by Ca*+ (N = 4) and 25% by Srr+ (N = 5). Magnesium ions produced no measurable inhibition or enhancement (N = 5). Figure 3 shows averaged EOG records illustrating inhibition of 33% by Al’+ (N = 5), 61% by Cd*+ (N = 6) and 61% by Zn*+ (N = 5). The inhibition from each of these cations is partial and reversible at 7.5 mM, except for La’+, which virtually eliminates EOGs with no recovery over the 33 min post-treatment period (Fig. 3). Slight recovery was observed from La’+ inhibition in extended experiments over 1 hr, post-treatment. Barium ion significantly enhanced the first two post-treatment EOGs by as much as
s- 150
AEROSOL
8
30% (F = 43.04; P < 0.001, df = 19) then produced moderate inhibition with recovery to the baseline (Fig. 3). Averaging the 20 post-treatment EOGs after Ba*+ application gives 6% inhibition (N = 5). The above experiments are summarized in Fig. 4, illustrating the rank order of potency of the inorganic cations in inhibiting EOGs: La’+ > Zn*+ > Cd*+ > Al’+ > Ca*+ > St+*+> Co*+ > Ba*+ > Mg*+. Except for Al’+, which has not been tested extensively for calcium channel blocking action, the rank order of EOG inhibition by cations was compared with several rankings of calcium channel antagonism in other tissues: (1) rank order of Ca*+-activated potassium channel blocking effectiveness in human T lymphocytes (DeCoursey et al., 1984a); (2) rank order of inhibitory effectiveness against [‘Hlnitrendipine binding to homogenized rat cortex (Ehlert et al., 1982); (3) rank order of calcium channel blocking effectiveness in barnacle muscle fiber (Hagiwara and Takahashi, 1967); (4) general rank order of calcium channel blocking effectiveness in neuronal tissue (Hagiwara and Byerly, 1983). Since each of these rankings is very similar, a composite rank order of calcium channel blocking ability was derived by summing weighted values assigned to a given cation
DELIVERY
L
1 (4)
lOO--
b zm p: rns ~a zz ca: 50
(7)
80.-
(5)
CO-40.-
gs
aE
0 -10
-5
0
5
10
15
TIME
(MN)
20
25
30
J
35
Fig. 2. Averaged recovery curves following 2 set aerosol sprays of the chloride salts of Mg2+ (closed circles, N = S), Co*+ (open circles, N = 6), Sr*+ (closed triangles, N = 5) and Ca2+ (open triangles, N = 5). All were applied to give a concentration of 7.5 mM. Bars represent SD.
if
20 -0,
(5) ! MC=
BA=
CO*
SR”
MULTIVALENT
CA* CATIONS
AL%
CD”
(7.5
ZN”
LA”
mM)
Fig. 4. Bar graph of percent inhibition averaged over 33 min post-treatment. The chloride salts of multivalent cations were applied to give a concentration of 7.5 mM. Results are means f SD for the number of trials given in parentheses.
BRUCED.
312
WATER (CONTROL) CALCIUM
CHANNEL
DILTIAZEM
WINEGAR et al.
VERAPAMIL
I
(0.15
mM)
LOG
Fig. 5. Bar graph of percent inhibition averaged over 33 min post-treatment. Diltiazem and verapamil were applied to give a calculated final concentration of 0.15 mM. Distilled water is included as a control. Results are means f SD for the number of trials given in parentheses.
according to its position in each rank order (i.e. first position is given a value of 1, second position a value of 2, etc.): La3+ > Cd2+ > Zn2+ > Co2+ > Ba2+ > Ca2+ > Sr2+ > M$+. The Spearman rank correlation coefficient was computed to test for an association between the ranking of EGG inhibitory potency and the composite ranking. The correlation coefficient (R, = 0.79) is significant (P < 0.025), which supports the hypothesis that EGG inhibition by these cations involves their blockage of calcium channels. Certain organic inhibitory
calcium
-4
-5 ANTAGONISTS
channel
antagonists
are
The organic calcium channel blockers d-cisdiltiazem (hereafter referred to as diltiazem), (+)-verapamil (hereafter referred to as verapamil), nicardipine, and nifedipine were tested. Diltiazem and verapamil both inhibited EGGS following delivery of 0.15 mM. Verapamil was more inhibitory (Fig. 5), supressing EOGs by 36% (N = 5) while diltiazem produced 17% inhibition (N = 4). Dose-response curves for diltiazem and verapamil indicate that they both inhibited EOGs in a dosedependent manner. Verapamil produced significant inhibition compared with water control experiments at a concentration as low as 0.015 mM (F = 26.03; P = 0.0003; df = 12). Diltiazem had less intrinsic activity than verapamil, producing 34% inhibition (N = 5) at the highest concentration tested (1.5 mM) while verapamil inhibited by 54% (N = 5) at this concentration (Fig. 6). Neither of the DHPs tested (nicardipine and nifedipine) inhibited any more than 1% DMSO controls at 0.015 mM. Their extremely hydrophobic nature prevented experiments at higher drug concentrations in 1% DMSO solutions. Actions of calmodulin antagonists
The calmodulin antagonists trifluoperazine (TFP), chloropromazine (CPZ) and N-(daminohexyl)5-chloro-1-napthalenesulfonamide (W-7) inhibited EGG responses following aerosol delivery of 0.15 mM (Fig. 7). W-7, the most potent calmodulin antagonist of those we tested (Hidaka and Tanaka, 1982) inhibited the first post-treatment EOG up to 80% in
-3
CONCENTRATION
Fig. 6. Dose-response curves for verapamil (closed circles, N = 6) and diltiazem (open circles, N = 6). Each point represents the mean + SD.
comparison with the 1% PEG 400 control curve. However, the W-7 curve and the control curve merged rapidly. Both CPZ and TFP were less inhibitory than W-7 immediately following aerosol application, but they exerted longer-lasting inhibition. When averaged over 20 post-treatment EGGS, W-7 inhibited by 27% (N = 7), CPZ by 31% (N = 4) and TFP by 23% (N = 4) (Fig. 7). DISCUSSION
This study examined the role of Ca2+ in olfactory transduction by aerosol application of inorganic cations, organic calcium channel antagonists and calmodulin antagonists to the olfactory mucosae of anesthetized frogs during electrophysiological recording. Results of their effects on EOG amplitudes support a role for Ca2+ as a current carrier for olfactory receptor potentials, while a second messenger role of Ca2+, in conjunction with camodulin, appears unrelated to olfactory transduction. Tests for artifacts
Tests for correlations between pre- and posttreatment effects are useful to avoid errors of data
WATER (CONTROL)
CPZ
CALMODULIN
TFP ANTAGONISTS
PEG400 (CONTROL) (0
15
W-7 mM)
Fig. 7. Bar graph of percent inhibition, averaged over 33 mm post-treatment. Chlorpromazine (CPZ), trifluoperazine (TFP) and W-7 were applied to give a concentration of 0.15 mM. One percent PEG 400 (used to dissolve W-7) and water are included as controls. Results are means + SD for the number of trials given in parentheses.
Calcium and olfactory transduction anaylsis when using percentages alone. For instance, a positive correlation between baseline EOG amplitudes and their subsequent inhibition might give false indications of sensitivity to a given agent if many of the experiments had high pre-treatment baselines. A test of the null hypothesis (i.e. the true correlation is zero) was made with Student’s t-test to determine whether post-treatment EOG amplitudes were linearly correlated either negatively or positively with pre-treatment baselines. The values of t were determined from ten sets of experiments with N of not less than four for each set. No significant differences were observed, thus indicating that pretreatment EOG amplitudes did not bias the effects of these agents. Eflects of inorganic calcium channel blockers
The results reported here provide evidence that an inward CaZ+ current accompanies the EOG and appears to be necessary for olfactory transduction. Di- and trivalent inorganic cations inhibited EOG responses in the frog in a rank order which is highly correlated with their calcium channel blocking effectiveness in other tissues. The sole exception to this correlation is Co2+ which is less inhibitory than would be expected, compared to its relative effectiveness at blocking calcium channels. The EOGenhancing effect of Ba*+ suggests a current-carrying role for Ca2+, because substitution of Ba*+ for Ca*+ often produces greater currents in other tissues than Ca*+ itself (Hagiwara and Ohmori, 1982; Hille, 1984). If Ca*+ is a current carrier, it seems paradoxical that Ca*+ application results in inhibition of subsequent EOGs. However, in some tissues, elevated intracellular Caz+ inactivates voltage-dependent calcium channels (Reuter, 1983). Barium ions substitute well for Ca*+ as current carriers, but do not inactivate calcium channels (Hille, 1984). This phenomenon could explain the EOG enhancement observed with Ba*+, because Ba2+ entry might not inactivate calcium channels in olfactory receptor cells. An alternative explanation for EOG inhibition by Ca*+ is that high extracellular Ca*+ can reduce membrane permeability through a membranestabilizing action (Webb, 1982). Calcium ions in high concentration could bind to acidic phospholipids in the membrane, increasing the phase transition temperature and decreasing membrane fluidity (Housley and Stanley, 1982). Calcium ions could also inhibit through their effect on potassium channels. A common regulatory function of elevated intracellular Ca2+ is activation of Ca*+-dependent potassium channels which act to repolarize the membrane (Fishman and Spector, 1981). That Ba2+ may block potassium channels (Armstrong and Taylor, 1980) suggests the possibility that EOG enhancement by Ba2+ is the result of potassium channel blockage. However, in preliminary experiments (data not shown) we found no EOG enhancement in the frog after aerosol application of 4-aminopyridine and quinine, drugs which block potassium channels (DeCoursey et al., 1984b). These agents inhibited EOGs only slightly at concentrations in the millimolar range, indicating that potassium channel blockage is not associated with EOG enhancement.
313
Calcium channels can be made permeable to monovalent cations such as Na+ and Li+ by removing divalent cations (Lansman et al., 1985), an effect that may have significance for experiments where the ionic environment of the preparation is altered. For example, Yoshii and Kurihara (1983) recorded odorant-evoked activity in the olfactory bulbs of carp (Cyprinus carpio), rainbow trout (Salmo gairdneri), and bullfrog (Rana catesbeiana) while altering the ionic environment of the olfactory epithelium. Olfactory responses were eliminated by irrigating mucosae with EDTA, followed by deionized water. They found a number of organic and inorganic cations supported olfactory responses, including Ca*+, Cd*+, Co*+, Li+, Mn*+, choline+ and Tris+, which could have been due to an altered calcium channel selectivity in the absence of Ca*+ (Hess and Tsein, 1984). Effects of organic calcium channel antagonists
Many of the organic calcium channel antagonists tested blocked EOGs to some extent. The doseresponse curve for diltiazem had a lower asymptotic limit than the dose-response curve for verapamil (Fig. 6), indicating that these compounds differed in their intrinsic activity to inhibit EOGs. Diltiazem and verapamil were both partial inhibitors of EOGs (Fig. 7) which suggests that more types of channels contribute to EOGs than the calcium channels associated with their binding sites. The DHPs nicardipine and nifedipine, had little or no effect on EOG magnitude. Because DHPs typically exert physiological actions at a much lower concentration than we tested (e.g. 10m9M; Miller, 1985), it seems likely that DHP binding sites, if present on olfactory receptor cells, do not affect olfactory transduction. Inhibition of EOGs by inorganic cations but not by DHPs follows the pharmacological profile of N-type calcium channels characterized by Nowycky et al. (1985). Of the various known types of calcium channels, N-type channels are found on sensory neurons and are characterized by insensitivity to DHPs (Reuter, 1985). Additional evidence that suggests an involvement of N-type or other similar calcium channels in olfactory transduction is that the N-type channels are blocked by Cd*+ but enhanced by Ba2+ (Nowycky et al., 1985), the same effect seen with EOG responses in this study. Additionally, Cd*+ eliminates olfactory functioning in humans (Arvidson, 1981). Calmodulin
Calmodulin antagonists inhibited EOGs, but not in a rank order which corresponds to their binding affinities for calmodulin in other tissues. TFP has greater affinity for calmodulin than CPZ (Levin and Weiss, 1979), but CPZ inhibited EOGs somewhat more than TFP (Fig. 7). Inhibition of EOGs by CPZ and TFP may result from non-specific membrane effects rather than anti-calmodulin activity. Both CPZ and TFP are cationic, amphipathic agents. Because each possesses a hydrophobic region plus a positively charged nitrogen atom, these compounds can partition into and disrupt hydrophobic regions of protein and lipid in the cell membrane (Landry et al., 1981; Lieber et al., 1984). While EOG inhibition by
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the calmodulin antagonist W-7 (Fig. 7) suggests a role for camodulin in olfactory transduction, W-7 may have nonspecific effects as well. The CPZ and TFP results argue against a role of calmodulin in olfactory transduction as a modulator of EOGs. SUMMARY
The possible roles of Ca2+ in olfactory transduction were explored by pharmacological and electrophysiological methods, using agents which inhibit or promote membrane calcium conductance or modify calmodulin activity. These agents were applied by quantitative aeroseol spray onto the olfactory epithelia of urethane-anesthetized frogs (Rana pipiens) during extracellular recording and periodic odorant stimulation by isoamyl acetate. Inorganic cations known to block inward calcium currents in other tissues inhibited electoolfactogram (EOG) responses when applied by a 2 set aerosol spray, producing a concentration of 7.5 mM in the mucous layer overlying the epithelium. The rank order of inhibitory potency of the chloride salts is: La3+ > Zn2+ > Cd2+ > A13+ > Ca2+ > Sr2+ > Co’+ > Ba2+ > M$+. Aerosol exposure to La3+, virtually eradicates EGG responses, while Ba*+ initially produces significant enhancement (F = 43.04, P < 0.001, df = 19) followed by a slight inhibition. Barium ions can substitute for Ca2+ to carry greater currents in other tissues and may enhance olfactory responses by a similar mechanism. Magnesium ions have no inhibitory action at the same concentration. Calcium ions are significantly inhibitory (F = 5.74; P = 0.0355; df = 12) at a concentration of 0.15 mM. The organic calcium channel antagonists, diltiazem and verapamil, were applied at 0.15 mM by a 2 set aerosol spray. They reversibly inhibited EOG amplitudes by 17 and 36%, respectively. Verapamil produced significant inhibition (F = 17.17; P = 0.002; df = 11) after application at a concentration of 0.015 mM. By contrast, the 1,Cdihydropyridine calcium channel antagonists, nicardipine and nifedipine, did not inhibit significantly beyond 1% DMSO controls when tested at 0.015 mM. The calmodulin antagonists, trifluoperazine (TFP), chlorpromazine CPZ) and N-(6-aminohexyl)-5chloro- 1naphthalenesulfonamide (W-7) depressed EOG amplitudes without correlation to their order of potency as calmodulin antagonists. Following a 2 set aerosol delivery of 0.15 mM, CPZ and W-7 inhibited by 31 and 27%, respectively, while TFP inhibited by 23%. These results support the hypothesis that Ca2+ participates in olfactory transduction as a charge carrier. The pattern of electrophysiological enhancement and inhibition suggests that calcium channels with properties similar to N-type calcium channels participate in olfactory transduction. Acknowledgements-This work was supported by grants from the National Science Foundation (BNS-81-08842 and CHE-8509557) and the University of North Texas Faculty Research Fund to R. Schafer. We thank Drs M. Donahue and T. Yorio for the W-7, and Mr J. Gomez for technical assistance.
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