Introduction

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nAChRs are found in a variety of tissues, including the neuromuscular junction, central nervous system, autonomic ganglia and adrenal medulla. This important class of ligand-gated ion channels mediates several physiological functions, such as synaptic transmission and modulation of neurotransmitter release, and may be involved in the regulation of neuronal development (for review, see Sargent, 1993; Galzi and Changeux, 1995). Neuronal nAChRs, like muscle nAChRs, are composed of multiple subunits. To date, eight  (2 to 9) and three  (2 to 4) neuronal nAChR subunits have been identified (Sargent, 1993; Elgoyhen et al., 1994). The diversity of nAChR subunits allows for the possibility of a daunting number of subtypes based upon subunit composition. Numerous combinations of the known neuronal nAChR subunits have been reported to form functional nAChRs in vitro (Luetje and Patrick, 1991; Gerzanich et al., 1994; Elgoyhen et al., 1994); however, little information is available regarding the in vivo subunit compositions of neuronal nAChRs. More importantly, though, contribution of multiple subunits/subtypes to the regulation of cellular function has not been established.

Oocyte expression studies have revealed that combinations of 2, 3 and 4 with either 2 or 4 subunits are required for the formation of functional nAChRs (Boulter et al., 1987; Luetje and Patrick, 1991). In contrast, the 7, 8 and 9 subunits can form functional homomeric receptors (Gerzanich et al., 1994; Elgoyhen et al., 1994). Furthermore, studies have shown that subunit composition influences the channel characteristics and pharmacology of nAChRs (for review, see McGehee and Role, 1995). This is demonstrated by the effects of the nAChR agonist cytisine and the antagonist -bgt. In the oocyte expression system, cytisine is a potent agonist on nAChRs containing 4 subunits but has little effect on nAChRs containing 2 subunits (Luetje and Patrick, 1991; Papke and Heinemann, 1993). -bgt has been shown to inhibit agonist-induced currents generated in 7, 8 and 9 homomeric nAChRs but has no effect on other neuronal nAChRs (Gerzanich et al., 1994; Elgoyhen et al., 1994).

It is becoming increasingly evident that nAChR populations within a given neuronal tissue may be heterogeneous. In chick ciliary ganglia at least three populations of nAChRs have been immunologically identified. These include a large population of 7-containing receptors that gate Ca⁺⁺ in response to nicotine and are inhibited by -bgt (Vijayaraghavan et al., 1992; Zhang et al., 1994; Pugh and Berg, 1994). These cells also contain a smaller population of 354 receptors that cross-react with the monoclonal antibody mAb35 (Conroy et al., 1992; Vernallis et al., 1993). Additionally, a small population of nAChRs that bind mAb35 contain the 2 subunit (Conroy and Berg, 1995). Patch-clamp techniques have also been used to identify multiple populations of nAChRs within given tissues. In cultured hippocampal neurons, four classes of nicotine-mediated currents (types IA, IB, II and III) can be demonstrated, based on channel characteristics and pharmacological profiles (Alkondon and Albuquerque, 1993; Lukas, 1995).

Activation of adrenal nAChRs leads to the release of a variety of secretory products, including epinephrine. Despite their physiological importance, very little information is available on these neuronal nAChRs. Patch-clamp and binding studies have indicated that the number of nAChRs on chromaffin cells is relatively low (Maconochie and Knight, 1992; Lee et al., 1992). Pharmacological studies support the presence of multiple populations of adrenal nAChRs. One population binds -bgt and probably is not involved in secretion (Wilson and Kirshner, 1977; Afar et al., 1994). Another population of nAChRs, which interact with the nAChR antibody mAb35, is also present. Our laboratory has demonstrated that adrenal chromaffin cells contain mAb35 binding sites (Lopez and McKay, in press) and that mAb35 potently and specifically reduces nAChR-stimulated catecholamine release (Gu et al., 1996). These effects of mAb35 develop slowly and are slowly reversible, suggesting that mAb35 induces nAChR down-regulation. The ability of mAb35 to modulate adrenal nAChRs has led to their identification as mAb35-nAChRs (Gu et al., 1996); a similar classification has been used for nAChRs found on chick ciliary ganglion neurons (Halvorsen and Berg, 1990; Conroy et al., 1992; Vernallis et al., 1993). The subunit composition of mAb35-nAChRs and other nAChR subtypes possibly involved in adrenal secretion is unknown. To date, only the 3 and 7 nAChR subunits have been cloned and sequenced in bovine adrenal chromaffin cells (Criado et al., 1992; García-Guzmán et al., 1995). Because -bgt has no effect on nicotine-stimulated release in these cells, it is unlikely that the 7 subunit plays an important role in secretory events. Furthermore, 3 subunits do not form functional homomeric channels in oocytes (Luetje and Patrick, 1991). Therefore, additional subunits are presumably present and contributing to populations of nAChRs that are involved in secretion.

Although it is recognized that subunit composition influences the pharmacology of nAChRs, the presence of receptor reserves (spare receptors) may also alter the pharmacological profiles of nAChR populations. Little or no data are available on the presence of spare nAChRs in neuronal tissues, and relatively little is known about the type or number of nAChRs present on adrenal chromaffin cells. The following studies were designed to investigate the presence and composition of adrenal nAChR reserves. In theses studies, the irreversible inhibitor brACh was used to inactivate nAChRs and the concentration-response profiles of the nAChR agonists nicotine, epibatidine and cytisine were determined. The monoclonal antibody mAb35 was used to provide information on the relative magnitude of populations of nAChRs involved in secretion. Finally, RT-PCR was used to identify nAChR subunits expressed in bovine adrenal chromaffin cells.

	Materials and Methods

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Materials. ()-Nicotine hydrogen tartrate, cytisine and components of N2+ medium were obtained from Sigma Chemical Co. (St. Louis, MO). DMEM and DMEM/F-12 medium were obtained from Life Technologies (Grand Island, NY). (±)-Epibatidine dihydrochloride and brACh bromide were purchased from Research Biochemicals Inc. (Natick, MA). DL-[³H]NE (specific activity, 12.0-15.0 Ci/mmol) was purchased from DuPont-New England Nuclear Corp. (Boston, MA).

Isolation and primary culture of bovine adrenal chromaffin cells. Adrenal chromaffin cells were dissociated from intact glands and plated in supplemented DMEM, as previously described (Maurer and McKay, 1994). Cells were plated at a density of 1 to 2 × 10⁵ cells/well on 24-well culture plates for secretion studies or 10⁷ cells/100-mm dish for RNA studies. Two days after plating, media were replaced with a modified serum-free N2+ medium previously described by our laboratory (Maurer and McKay, 1994). DMEM and N2+ media were supplemented with 250 ng/ml amphotericin B, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine and 10 µM 5-fluoro-2-deoxyuridine. One day before experimentation, the culture medium was removed and replaced with medium free of amphotericin B and 5-fluoro-2-deoxyuridine. Cells were used 4 to 7 days after isolation.

Catecholamine secretion studies. A [³H]NE assay was used to monitor catecholamine release from cultured cells (McKay and Schneider, 1984). Cells were incubated with 0.1 µM [³H]NE in a PSS containing 140 mM NaCl, 4.4 mM KCl, 1.2 mM MgSO₄, 3.6 mM NaHCO₃, 1.2 mM KH₂PO₄, 2 mM CaCl₂, 10 mM glucose, 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.2-7.4) and 0.5% bovine serum albumin and were extensively washed before all treatments. The amount of radioactivity released after a 5-min incubation with secretagogue (stimulated release) or without secretagogue (basal release) was determined using liquid scintillation counting. The radioactivity remaining in the cells was then extracted with 8% trichloroacetic acid and counted. The sum of the secreted and trichloroacetic acid-extractable radioactivity represented total incorporated [³H]NE. Results were expressed either as a percentage of total incorporated [³H]NE released under the treatment conditions (i.e., secreted [³H]NE divided by total incorporated [³H]NE × 100) or as a percentage of the net stimulated control response (percentage of control) where basal (nonstimulated) release was subtracted from all groups (i.e., treatment group release minus basal release divided by control nicotine-stimulated release minus basal release × 100).

mAb35 (anti-nAChR monoclonal antibody). A hybridoma cell line that secretes mAb35, a monoclonal antibody directed against the main immunogenic region of nAChRs, was obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured and the antibody was concentrated and purified using techniques described previously (Gu et al., 1996).

brACh treatment. Adrenal nAChRs were irreversibly inactivated by acetylation with brACh, using techniques modified from the work of Gardette et al. (1991). The cells were first treated with 1 mM dithiothreitol in PSS (pH 8) for 15 min at 37°C, to reduce nAChR disulfide bonds. After washing (1 ml PSS/well, 5 min), cells were treated with brACh for 6 min at room temperature and washed for 5 min. Finally, the disulfide bonds were reoxidized by incubation of the cells with 1 mM 5,5-dithio-bis(2-nitrobenzoic acid) in PSS for 15 min at 37°C. After a 5-min wash, cells were used immediately for acute studies or incubated in N2+ medium for chronic studies.

RT/PCR, cloning and sequencing. Total RNA was isolated from adrenal medulla using Trizol reagent (Life Technologies), according to the procedure of Chomczynski and Sacchi (1987). Before use as a template for RT, the RNA was treated with 1 U of DNase I (amplification grade; Life Technologies). The RT reaction was performed in a 20-µl volume containing 500 µM deoxynucleoside triphosphates, 10 mM dithiothreitol, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl₂, 200 U Superscript II (Life Technologies) and 100 ng random hexamers. RNA and random primers were heat-denatured and reverse transcribed for 10 min at 25°C and then for 50 min at 42°C, followed by treatment with 2 U of RNase H (Life Technologies) at 37°C for 20 min. Single-stranded cDNA was amplified by PCR in a MJ Research thermocycler, in a final volume of 100 µl containing 1.5 mM MgCl₂, 50 mM KCl, 20 mM Tris-HCl (pH 8.4), 500 nM levels of each primer, 500 µM deoxynucleoside triphosphates and 5 U of Taq polymerase (Life Technologies) overlaid with mineral oil. The design of degenerate PCR primers was based on homologous sequences of TM3 and TM4 of the bovine 3, rat 3, rat 2 and rat 4 subunit genes. The TM3 primer sequence was 5-GTMACCYTYTCCATYGTCA-3, and the TM4 primer sequence was 5-CGRTCTAYSACCATSGCMAC-3, where M is A or C, Y is C or T, S is G or C and R is A or G. Amplification was performed as follows. Samples were heated to 94°C for 5 min, after which the temperature was lowered to 80°C. Taq polymerase was then added and the reaction proceeded for 36 cycles (94°C for 1 min, 52°C for 1 min and 72°C for 1.5 min), followed by incubation for 10 min at 72°C to complete the extension. PCR products were cloned into the vector pCRII, using the TA cloning kit (Invitrogen, San Diego, CA), and sequenced. Sequencing was performed using the chain-termination method (Sanger et al., 1977), with Sequenase T7 polymerase (United States Biochemical, Cleveland, OH). A basic local alignment search tool (Altschul et al., 1990) search of nonredundant GenBank and EMBL sequences was used to identify similar sequences. Additional analysis of sequences was performed using the GeneWorks 2.1 program (IntelliGenetics, Mountain View, CA).

RNA analysis. RNA was purified from cultured bovine adrenal chromaffin cells using Trizol reagent (Life Technologies), according to the procedure of Chomczynski and Sacchi (1987). Northern blot analysis was performed using 1% (w/v) agarose gels containing 7.4% (v/v) formaldehyde in 20 mM 3-(N-morpholino)propanesulfonic acid, 1 mM EDTA disodium, 5 mM sodium acetate, pH 7. After electrophoresis, the RNA was transferred to Gene Screen Plus membranes (DuPont-New England Nuclear) in 10× standard saline citrate (1× standard saline citrate contains 0.15 M sodium chloride and 0.015 M sodium citrate) according to the manufacturer's instructions. The EcoRI inserts from the pCRII vector containing either bovine cDNA 4 or bovine 3 cDNA were ³²P-labeled with [-³²P]dCTP (3000 Ci/mmol; DuPont-New England Nuclear) by random priming (Feinberg and Vogelstein, 1983) and hybridized to Gene Screen Plus membranes in 5× SSPE (1× SSPE contains 0.15 M NaCl, 0.01 M NaH₂PO₄ and 1 mM EDTA, pH 7.4), 50% deionized formamide, 5× Denhardt's solution (1× Denhardt's solution contains 0.2 mg/ml polyvinylpyrrolidone, 0.2 mg/ml bovine serum albumin and 0.2 mg/ml Ficoll 400), 1% sodium dodecyl sulfate, 10% dextran sulfate, 100 µg/ml salmon sperm DNA, at 42°C. The filters were washed in 2× SSPE at room temperature, 2× SSPE/2% sodium dodecyl sulfate at 65°C and 0.1× SSPE at room temperature. Autoradiograms were developed after exposure to Kodak XAR-5 film at 70°C, with DuPont Cronex intensifying screens.

Calculations and statistics. Results were calculated from the number of observations performed in duplicate or triplicate. EC₅₀ and E_max values were obtained by averaging values generated from sigmoid nonlinear regression analyses (Inplot 3.1; GraphPad, San Diego, CA) of individual concentration-response curves. At high agonist concentrations, a reduction in secretory response was often seen; these data were omitted during nonlinear curve fitting. Results are expressed as arithmetic means ± S.E., except for EC₅₀ values, which are expressed as geometric means (with 95% confidence limits). Statistical analysis was performed using Dunnett's multiple-comparison procedure at a .05 level of significance.

	Results

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Previous studies have documented the utility of brACh treatment in the study of nAChRs (Leprince, 1983; Listerud et al., 1991; Gardette et al., 1991). We found that brACh was effective as an nAChR agonist (fig. 1A), with an EC₅₀ of approximately 4 µM. However, when brACh was used under acetylating conditions (see "Materials and Methods"), an immediate loss of nicotine-stimulated adrenal catecholamine release occurred. These effects were concentration-dependent (IC₅₀, ~0.3 µM). At concentrations greater than 10 µM, brACh eliminated approximately 90% of control nAChR-stimulated release (fig. 1B). brACh had no effect on 56 mM KCl-stimulated release under conditions that reduced nicotine-stimulated release (data not shown). The reduction in nAChR-stimulated release was not immediately reversible (fig. 2). nAChR-stimulated secretion returned slowly, with a functional recovery rate of approximately 2%/hr (fig. 2). Recovery of nAChR-stimulated function could be blocked by cycloheximide (1 µg/ml) treatment (data not shown), suggesting that the return of functional receptors involves protein synthesis and supporting the irreversible nature of brACh treatment.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   brACh-induced stimulation and inactivation of bovine adrenal nAChRs and concentration-dependent effects. brACh can be used as a nAChR agonist or, under acetylating conditions (see "Materials and Methods"), can be used to inactivate nAChRs. A, cultured bovine adrenal chromaffin cells were stimulated for 5 min with various concentrations of brACh, and catecholamine release during this stimulation period was determined. Results are expressed as a percentage of total catecholamine content. Values for nonstimulated (dotted line) and 10 µM nicotine-stimulated (dashed line) catecholamine release were 2.8 ± 0.8% and 24.2 ± 3.2%, respectively. Values represent means ± S.E. (n = 5). B, cultured bovine adrenal chromaffin cells were treated under acetylating conditions with various concentrations of brACh. The effects of brACh-induced nAChR inactivation on 10 µM nicotine-stimulated catecholamine release (5 min) were determined. Results are expressed as a percentage of total catecholamine content. Values for nonstimulated (dotted line) and 10 µM nicotine-stimulated (dashed line) catecholamine release were 1.5 ± 0.5 and 19.8 ± 1.9%, respectively. Values represent means ± S.E. (n = 6).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Time course for recovery from the inhibitory effects of brACh on adrenal catecholamine release. Cultured chromaffin cells were treated with 100 µM brACh under acetylating conditions. After this treatment, the cells were washed and placed in N2+ medium. Recovery of nAChR-mediated secretory function was then assessed; at the indicated times after nAChR inactivation, cells were stimulated for 5 min with 10 µM nicotine, and catecholamine release during this stimulation period was determined (). Results are expressed as a percentage of total catecholamine content. Nontreated/10 µM nicotine (NIC)-stimulated () and nonstimulated () groups were analyzed in parallel. Values represent means ± S.E. (n = 4-6).

Irreversible receptor antagonists are often used to investigate the presence of spare receptors. These antagonists produce increases in EC₅₀ values and decreases in E_max values, which are characteristics of systems containing receptor reserves. In the next series of experiments, brACh was used to irreversibly inactivate nAChRs and then the concentration-response profiles for the nAChR agonists nicotine, epibatidine and cytisine were determined. As seen in figures 3 and 4 and table 1, the EC₅₀ values for nicotine, epibatidine and cytisine were approximately 4.0 µM, 8.5 nM and 41.0 µM, respectively. With increasing concentrations of brACh, shifts to the right in the concentration-response curves of nicotine and epibatidine occurred (fig. 3; table 1). At a concentration of 100 nM, brACh caused a small increase in the EC₅₀ value of nicotine; at 1 µM brACh, an increase in the EC₅₀ value of approximately 3.7-fold was seen (fig. 3; table 1). Similarly, 1 µM brACh caused an increase in the EC₅₀ value of epibatidine of approximately 4.1-fold (fig. 3; table 1). At 10 µM brACh, there was an additional increase in EC₅₀ values for nicotine and epibatidine; however, the values were not significantly different than the EC₅₀ values generated after 1 µM brACh treatment (fig. 3; table 1). Additionally, curve fitting of the depressed values was difficult and introduced considerable variability. Unlike the observed shifts with nicotine and epibatidine, 1 µM brACh increased the EC₅₀ value of cytisine by approximately 2-fold (fig. 3; table 1). Finally, 100 nM brACh had no significant effect on the E_max values of the agonists. However, at 1 µM brACh, the E_max values for nicotine, epibatidine and cytisine were reduced by 44, 35 and 38%, respectively (fig. 3; table 1). Increasing the concentration of brACh to 10 µM caused an additional reduction in the E_max value for each agonist (fig. 3; table 1).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Concentration-response curves for nicotine, epibatidine and cytisine after brACh-induced nAChR inactivation. Cultured adrenal chromaffin cells either were not treated () or were treated under acetylating conditions with 100 nM (), 1 µM () or 10 µM () brACh. Cells were washed and then stimulated for 5 min with the indicated concentrations of nicotine (A), epibatidine (B) or cytisine (C), and catecholamine release during this stimulation period was determined. Results are expressed as a percentage of net stimulated control response. Control stimulated values (nontreated, 10 µM nicotine) were 18.5 ± 0.4% of total catecholamine content. Basal (nonstimulated) values were 0.9 ± 0.1% of total catecholamine content. Values represent means ± S.E. (n = 3-6).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Concentration-response curves for nicotine, epibatidine and cytisine after mAb35-induced nAChR modulation. Cultured adrenal chromaffin cells either were not treated () or were treated for 6 hr (), 24 hr () or 48 hr () with 50 nM mAb35. Cells were washed and then stimulated for 5 min with the indicated concentrations of nicotine (A), epibatidine (B) or cytisine (C), and catecholamine release during this stimulation period was determined. Results are expressed as a percentage of net stimulated control response. Control stimulated values (nontreated, 10 µM nicotine) were 14.4 ± 0.4% of total catecholamine content. Basal (nonstimulated) values were 1.2 ± 0.1% of total catecholamine content. Values represent means ± S.E. (n = 3-5).

The studies described above demonstrate that adrenal chromaffin cells contain a receptor reserve among the population of nAChRs involved in secretion. Previous studies from our laboratory indicate that more than one subtype of nAChR is involved in secretory function, and they suggest that mAb35-nAChRs are the principal receptors mediating secretory responses (Gu et al., 1996). The presence of mAb35-nAChRs is supported by the ability of mAb35 treatment to cause down-regulation of a population of adrenal nAChRs. The time course for the effects of mAb35 suggest a gradual disappearance of surface receptors (Gu et al., 1996). If this occurs, then mAb35 treatment should produce shifts in agonist concentration-response curves with increasing treatment times, as the antibody eliminates receptor reserves. To investigate these possibilities, we used an approach similar to the one described above for brACh. In these studies, the concentration-response relationships for nicotine, epibatidine and cytisine at various times after treatment with maximal inhibitory concentrations (50 nM) of mAb35 (Gu et al., 1996) were determined. mAb35 treatment produced a time-dependent shift in the agonist concentration-response curves for nicotine and epibatidine (fig. 4; table 1). After 6 hr, the EC₅₀ value for nicotine was increased by approximately 2-fold (table 1). After 24 and 48 hr, no additional increase in the EC₅₀ value of nicotine was seen; however, the E_max value was reduced by approximately 26% after 24 hr and by approximately 31% after 48 hr (fig. 4; table 1). As with nicotine, a 6-hr treatment with mAb35 increased the EC₅₀ value of epibatidine by approximately 2-fold (fig. 4; table 1). After 24 and 48 hr, no additional increases in the EC₅₀ value for epibatidine were seen; however, the E_max value was reduced by approximately 22% after 24 hr and by 26% after 48 hr (fig. 4; table 1). mAb35 pretreatment had no significant effect on the EC₅₀ value of cytisine, even after 48 hr of treatment (fig. 4; table 1). However, in as little as 6 hr, mAb35 pretreatment reduced the E_max value for cytisine by approximately 56% (fig. 4; table 1). After 24 and 48 hr, the E_max value for cytisine was reduced by approximately 63 and 64%, respectively (fig. 4; table 1). No additional reductions in E_max values for the agonists were seen after 72 hr of mAb35 treatment (data not shown). These results with mAb35 paralleled those observed with brACh treatment, providing evidence that mAb35 causes the down-regulation and loss of a population of nAChRs.

The subunit composition of mAb35-nAChRs in chromaffin cells is not known. In the chick ciliary ganglion (autonomic parasympathetic neurons), mAb35-nAChRs are composed of 3, 5, 4 and sometimes 2 subunits (Conroy et al., 1992; Vernallis et al., 1993; Conroy and Berg, 1995). Chromaffin cell nAChRs may be of similar composition. To complement studies of the pharmacology, turnover and expression of nAChRs in chromaffin cells, there is a need to clone and characterize additional nAChR subunits. We used knowledge of subunit sequences from other species to design a strategy to clone additional chromaffin nAChR subunits. Each nAChR subunit thus far identified has four putative TM regions. The sequences of these regions are conserved among nAChRs. The sequence between TM3 and TM4 forms an intracellular loop region that varies in length and sequence among nAChR subunits (for review, see Sargent, 1993) and is much less conserved than the TM regions. Oligonucleotide primers were designed to amplify sequences related to nAChR regions between TM3 and TM4 (TM3-TM4) after RT of bovine adrenal RNA. Using RT-PCR, we amplified bovine cDNA sequences with homology to nAChR sequences. When the bovine cDNAs were cloned and sequenced, two distinct classes of PCR products were identified. One was identical to a bovine 3 cDNA previously identified by Criado et al. (1992). Another cDNA was sequenced and was distinct from the bovine 3 sequence. This sequence, bovine cDNA 4, was used to search GenBank and EMBL databases for similar sequences. The sequences with the greatest similarity to bovine cDNA 4 were from the TM3-TM4 regions of nAChR genes. The GenBank search produced sequences with the highest homology to human and rat 4 subunits (fig. 5). The bovine cDNA 4 clone was also aligned pairwise with TM3-TM4 regions of human 4, rat 4 and rat 2 nAChR subunits. The bovine cDNA 4 sequence was identical to 81% of human 4 and 76% of rat 4 but only 56% of rat 2 TM3-TM4 nucleotide sequences. The bovine cDNA 4 was used to probe a Northern blot containing cultured bovine adrenal chromaffin RNA. One major and one minor band of about 3 kilobases were observed (fig. 6). This pattern was distinct from that observed using bovine 3 (fig. 6) or rat 3 (Gu et al., 1996) cDNAs as probes.

View larger version (105K): [in this window] [in a new window]   Fig. 5.   Alignment of bovine cDNA 4 with nAChR gene sequences. Single-stranded cDNA was synthesized by RT with random primers and amplified using degenerate PCR primers based upon TM sequences of nAChRs. The cDNA was cloned into pCRII and sequenced (see "Materials and Methods"). Bovine cDNA 4 was used for a basic local alignment search tool (Altschul et al., 1990) search of nonredundant GenBank and EMBL sequences. The two sequences with the highest degree of similarity are TM3-TM4 regions from nAChRs. The bovine cDNA 4 was aligned with the TM3-TM4 regions of the human and rat 4 nAChR subunit genes using the GeneWorks 2.1 program.

View larger version (37K): [in this window] [in a new window]   Fig. 6.   Northern blot analysis of bovine nAChR 3 subunit and bovine cDNA 4 RNAs. Ten micrograms of total bovine adrenal RNA from cultured cells were electrophoresed in a 1% agarose gel and transferred to Gene Screen Plus membranes (see "Materials and Methods"). The Northern blots were probed with the designated, random-primed, 32P-labeled probes. Autoradiograms were developed after exposure to Kodak XAR-5 film at 70°C with DuPont Cronex intensifying screens. The 18S (1.8 kilobases) and 28S (4 kilobases) rRNAs are indicated.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

In these studies, we have used the cholinergic agent brACh as a tool to investigate adrenal nAChR populations. We have shown that, under acetylating conditions, brACh reduces nicotine-stimulated catecholamine secretion in bovine adrenal chromaffin cells. This effect is receptor-specific (i.e., acetylation with brACh has no effects on catecholamine release induced by depolarizing concentrations of KCl). We have also found that the effects of brACh are slowly reversible, with full recovery of nAChR-mediated secretory function occurring within 24 to 48 hr. In the presence of the protein synthesis inhibitor cycloheximide, recovery of nAChR-mediated secretory function does not occur. These characteristics are consistent with the actions of brACh in a variety of muscle and neuronal nAChR preparations in which brACh covalently binds to nAChR subunits, producing irreversible nAChR inactivation (Moore and Raftery, 1979; Kao and Karlin, 1986).

In these studies, we have documented the presence of a nAChR reserve (i.e., spare receptors) using brACh to irreversibly inactivate adrenal nAChRs. According to spare receptor theory, as an irreversible antagonist eliminates a receptor reserve, the concentration-response curve of a full agonist shifts to the right. This produces an apparent increase in the EC₅₀ value for the agonist. When the irreversible antagonist has totally eliminated the receptor reserve, further elimination of functional receptors decreases the E_max value for the agonist (for review, see Ruffolo, 1982). In our studies, as nAChRs are inactivated with increasing concentrations of brACh, there are progressive increases in EC₅₀ values, followed by progressive decreases in E_max values for nicotine and epibatidine. These characteristics support the presence of a population of spare nAChRs. The magnitude of our nAChR reserve is difficult to estimate accurately because knowledge of K_d values is required. Theoretically, fractional receptor occupancy equals the concentration of the ligand divided by the sum of the K_d of the ligand plus the concentration of the ligand. However, K_d values obtained from nAChR binding studies may not be an accurate reflection of the relationship between nAChR affinity and functional responses. Contrary to the expected results, the K_d value of nicotine (nanomolar) is several orders of magnitude lower than the EC₅₀ value of nicotine (micromolar) for a variety of functional responses (Lee et al., 1992) (table 1). These lower K_d values are believed to represent binding to desensitized, higher affinity states of nAChRs (Sine and Taylor, 1979). K_d values can be calculated from functional data using Furchgott analysis (Furchgott, 1966). However, this type of analysis is difficult to perform, because of the relatively small changes in EC₅₀ values in our studies. For our determination of receptor occupancy, we have extrapolated apparent K_d values. Theoretically, when the receptor reserve has been eliminated, the E_max value should begin to decrease and the EC₅₀ value should no longer increase; at this point, the EC₅₀ value should equal the K_d. From table 1, we have estimated the K_d values of nicotine and epibatidine to be 13 µM and 35 nM, respectively. Using these K_d values in the receptor occupancy equation stated above, receptor occupancy at maximum functional response for nicotine (10 µM) and epibatidine (30 nM) is 43 and 46%, respectively. Accordingly, when considering nAChRs involved in secretion, bovine adrenal chromaffin cells possess >2 times the number of nAChRs than are required for maximum secretory responses.

To further investigate adrenal nAChR populations involved in secretory function, we have used the nAChR antibody mAb35. Compared with brACh treatment, mAb35 causes a more slowly developing and less complete down-regulation of nAChR-mediated secretory function (Gu et al., 1996). We show in these studies that the mAb35-induced loss of nAChR-mediated secretory function is characterized by a shift in the concentration-response curve to the right, as well as a decrease in E_max. The time course of this effect is consistent with a scenario where the antibody slowly down-regulates nAChRs (i.e., removes the receptor from the cell surface). The resulting loss of receptors can be seen as a shift in EC₅₀ followed by a reduction in E_max as the receptor reserve is eliminated. Furthermore, the maximum attainable reduction in E_max with mAb35 in our studies is 30%. Because full secretory responses are reached with only 45% receptor occupancy, this seemingly minor mAb35-induced reduction in E_max likely represents a >55% reduction in the total population of adrenal nAChRs involved in secretion. Although these calculations assume that nAChR populations are functionally similar, these studies provide further evidence that nAChR reserves exist and that mAb35-nAChRs are the principal receptors mediating adrenal catecholamine release.

In these studies, we have also used the nAChR agonist cytisine to study adrenal nAChR populations involved in secretion. Cytisine has been shown to have activity on 4-containing nAChRs and little or no activity on 2-containing nAChRs (Luetje and Patrick, 1991; Papke and Heinemann, 1993). We have shown that cytisine stimulates adrenal catecholamine release (EC₅₀, ~41 µM); however, cytisine is somewhat less efficacious than either nicotine or epibatidine (80-90% of the response attainable with either nicotine or epibatidine). Our data are consistent with cytisine acting as a partial agonist on adrenal nAChRs. In support of this, reductions in nAChR pools by either brACh or mAb35 produce progressive reductions in the E_max values of cytisine and have little effect on the EC₅₀ values of cytisine. These are characteristics associated with partial agonism. mAb35 treatment alters the concentration-response effects of cytisine to a much greater extent than that seen with nicotine and epibatidine. These findings are also consistent with the actions of cytisine as a partial agonist. However, we cannot rule out the possibility that cytisine may be acting as a full agonist on a subpopulation of nAChRs, the majority of which coincide with mAb35-nAChRs. Our data are unable to distinguish between these two possibilities.

As described above, cytisine, a nAChR agonist in bovine adrenal chromaffin cells, has been shown to have activity on 4-containing nAChRs (Luetje and Patrick, 1991). This suggests that 4 nAChR subunits may be present in these cells. In these studies, we present evidence that a 4 subunit is expressed in bovine adrenal chromaffin cells. Bovine cDNA 4 likely represents a segment of the bovine 4 nAChR subunit corresponding to the large cytoplasmic loop between TM3 and TM4. This clone has the highest degree of sequence similarity with analogous regions of 4 nAChR subunits from humans (81%) and rats (76%). Bovine cDNA 4 has a lesser degree of sequence similarity with the analogous region of rat 2 (56%), which is comparable to the degree of sequence identity between analogous regions of rat 4 and rat 2 nAChR subunits (54%). It is not surprising that 4 transcripts might be present in adrenal chromaffin cells, because 4 subunits are found in several tissues of similar embryonic origins. In chick ciliary ganglia, the 4 subunit is associated with a synaptic-type nAChR that also contains the 3, 5 and sometimes 2 subunits (Conroy et al., 1992; Vernallis et al., 1993; Conroy and Berg, 1995). In the rat PC12 cell line, 3, 5, 2 and 4 transcripts have been found (Boulter et al., 1990). Rat superior cervical ganglia also express the 4 subunit and contain nAChRs that can be activated by cytisine with an EC₅₀ of approximately 20 µM (Mandelzys et al., 1995), which is similar to the EC₅₀ for cytisine in our studies. Although comparing pharmacological/sequence data across species can be problematic, our studies suggest that bovine adrenal chromaffin cells contain a 4 subunit in nAChRs involved in secretion.

These studies demonstrate that a receptor reserve exists for nAChR-mediated adrenal catecholamine secretion. This finding represents the first time receptor reserves for nAChRs involved in secretion have been documented on neuronal tissues, and it has important functional and pharmacological implications. Spare receptors influence concentration-response relationships for agonists and antagonists (Zhu, 1993). Because of the presence of spare receptors, reductions in nAChRs may lead to alterations in the pharmacological profile of remaining nAChRs. In these studies, we also provide supporting evidence that the total population of nAChRs regulating secretory function may be heterogeneous, with mAb35-nAChRs representing no less than 55% of the total nAChR population involved in secretion. This observation may also have important functional and pharmacological implications. Finally, the presence of cytisine sensitivity and our observation of a 4-related subunit transcript in adrenal chromaffin cells support the presence of the 4 subunit in adrenal nAChRs involved in secretion.