Cyclic AMP and the nicotinic response of bovine adrenal chromaffin cells

Brain Research, 586 (1992) 344-347 ,~'~1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00

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BRES 25292

Cyclic AMP and the nicotinic response of bovine adrenal chromaffin cells A d r i e n n e E. D u b i n i, M a r g a r e t M. R a t h o u z , K a r e n S. M a p p a n d Darwin K. B e r g Department of Biohlgy. Unirt,rsity of (.'aliJ'ontiu. San Diego. La Jolhi, CA 92093-0322 (USA) (Accepted 5 May 1992)

Key word~." Cyclic AMP: Ni,cotinic: Acctylchtdinc receptor: Adrenal chromaffin; Adrenal medullary: Bovine

I"11ceffects of cAMP analog~ on the nicotinic responses of bovine adrenal chronlaffin ceils were examined by monitoring nicotine-induced whole cell current.,,. [~ll]norepinephrinc release, and nlcmhr:mc conductance changes. None of the three methods revealed an increased nicotinic re~pon,~c after treatment with cAMP analogs. The compounds did increase ['~H]norcpinephrine release from the cells but the effect was not exerted at the level of nicotinic receptors. Bovine adrenal chromaffin cells differ in this rc.~pcct from chick ciliary ganglion neurons which do .~ilm~ incre:,.~cd nicotinic rc.~ponscs after treatment with cAMP analogs.

Second messenger regulation of ncurotransmittcr receptors may provide .'1 mechanism for moduhlting syn:lptic transmission. One of the most studied second messengers, cAMP. has been shown to decrc,'lse the response of muscle acetylcholine receptors (AChRs) by increasing agonist.induced desensitization ~,t. The efI'ect is likely to be mediated by protein kinase A-dependent phosphorylation of the receptor as occurs with electric organ AChRs ~. At least one chlss of neuronal AChRs is regulated differently: cAMP analogs increase the nicotinic response of chick ciliary ganglion neurons and do so without requiring protein synthesis tf,.ix. Bovine adrenal chromaffin cells have AChRs that share many physiological and pharmacological properties both with muscle and with neuronal AChRs .~.~,,~.l,.. cAMP analogs have variously bccn reported to enhance the nicotinic response of bovine adrenal chromaffin cells 13. to have no specific effect on the nicotinic response 122 or to decrease it ¢,.i,J We have re-examined this issue with bovine adrenal chromaffin cells, using three different methods to measure the effects of cAMP analogs. Some of our preliminary results have been cited in a 'note added in proot" :".

Whole cell voltage clamp recording techniques '~'~''t~''t7were used to record nicotinic responses from bovine adrenal chromaffin cells after exposure to cAMP an:dogs and compare the magnitude of the 1ncan response to that obtained from untreated control co!Is. The preparation of the cultures and the criteria fi~r retaining data were as previously described i,..i,~,1,.=~. Cells',were incubated with 2 mM 8-bromo-cAMP and I mM 3-~sobutyrl-i-methylxanthine (IBMX) tbr 6 h since these were the test conditions previously shown to enhance the ACh response of chick ciliary ganglion neurons " ~ . Normalizing the responses for cell size (i,c. dividing the whole cell current in pA by the capacitance in pF) failed to reveal a difference between treated and untreated cells. Thus 63 treated cells challenged with 30 p,M nicotine had a mean response that was Ill + 12% of that observed for 58 untreated control cells examined in parallel (Fig. IA). A monoclonal antibody, mAb 35, has previously been used to quantitatc AChRs on the surface of bovine adrenal chromaffin cells i., Using IZSl-mAb 35 under similar conditions here to measure the number of AChRs confirmed that the incubation with 8-bromo-

('orr('~pundencc: D,K. Berg, Department of Biolugv, 0322, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0322, LISA,

Previuusly, Adrienne E, McEachern, Present address: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Slanfiwd, ('A 94305, USA,

345

niques 20,24 were used to measure the effects of cAMP analogs since this procedure had also been used previously to obtain evidence for a specific increase in the nicotinic response ~3. Incubating the cells in 1-2 mM 8-bromo-cAMP for 6-18 h (+ 1 mM IBMX) appeared to produce a decrease in the nicotinic response of adrenal chromaffin cells measured by this method, rather than an increase. The reason for this apparent discrepancy between results obtained with intracellular recording and those obtained with the other two methods is unclear, but all three methods concurred in showing no increased nicotinic response as a consequence of treating cells with a cAMP analog. The cyclic nucleotide treatment did increase the ACh response of chick ciliary ganglion neurons in culture as previously reported ~6,18, using intracellular recording to measure the responses (Fig. 1C). Failure to find a cAMP-dependent enhancement of nicotinic responses in bovine adrenal chromaffin cells raised questions about a related conclusion in the original studies, namely that newly synthesized AChRs on the cells produced a greater aggregate nicotinic response than did old AChRs on the cells and that this resulted from a differential effect of cAMP on new and old receptors ~3,~4. To re-examine this, cells having predominantly old AChRs were produced by incubating them in tunicamycin to block insertion of new

cAMP and IBMX for 6 h produced little change in the number of receptors on the cell surface (Fig. 1A). Because previous experiments in this laboratory measuring [3H]norepinephrine release from the cells suggested a positive effect of cAMP analogs on the nicotinic response s3, similar experiments were carried out here. Cells loaded with [3H]norepinephrine were tested for release as previously described ~2.~3except that nicotine-induced and K+-induced release were measured in parallel on quadruplicate sets of cultures rather than in tandem on the same cultures, and basal release, i.e. release occurring in the absence of secretogogue, was also always measured in parallel on separate cultures. Basal release was usually less than a third of that induced by secretogogue and was subtracted to calculate specific release. Typically, [3H]norepinephrine release specifically induced by nicotine (5 /zM) represented about 5-10% of the total [3H]norepinephrine associated with the cells. Incubation with 2 mM 8-bromo-cAMP for 24 h did produce an increase of about 40% in [3H]norepinephrine release but the increase was not specific for the nicotinic response. Nicotine-induced release and K+-induced release were increased by approximately the same amount (Fig. 1B). Another cAMP analog, dibutyrl cAMP, had a similar non-specific effect (data not shown). As a third approach, intracellular recording tech-

• CONTROL I=1 ~oAMPTREATED A

C

B

(12)

E .o (-) I

120

0 ,~",ws-"p (CLAMP)

Ac~s (MABSS8n'E8)

.~'~is~ (NE REL)

~.e~ (ICEflEL)

.~'~i,, (INT llEC)

~-,iL,. (INT flEC)

Fig. I. Effects of cAMP analogs on t~icotinic responses. A: bovine adrenal chromaffin (Chrom) cells were incubated with or without 2 mM 8.bromo-cAMP and 1 mM IBMX for 6 h and then either were tested for nicotinic responses using whole cell voltage clamp techniques (Nic Resp; Clamp) or were measured for relative levels of AChRs using S2Sl-mAb35 (AChRs; mAb 35 Sites). B: alternatively, chromaffin cells were incubated with or without I mM 8-bromo-cAMP for 24 h and tested for nicotine-induced (Nic Resp; NE ReD and K+-induced (K Resp; NE Rel) [3H]norepinephrine release. C: Chromaffin cells and ciliary ganglion (CG) neurons, grown in culture as previously described z3, were also incubated with or without I-2 mM 8-bromo-cAMP ± I mM IBMX for 5-18 h and examined for nicotinic responses with intracellular recording techniques (Nic Resp; Int gee). For each technique, results have been compiled by expressing individual values from an experiment as a percent of the mean control value for that experiment, and then averaging the values to obtain a grand mean ± S.E.M. for several experiments. For physiological experiments individual values represent single cells, and geometric means were calculated since the values did not conform to a normal distribution to. For binding and release experiments individual values represent triplicate and quadruplicate cultures, respectively, and arithmetic means were calculated. The numbers in parentheses indicate the total number of cells (physiology) or cultures (binding, release) used. For control chromaffin cells, 30 ~M nicotine induced a mean peak current of 0.72 + 0.06 nA (58 cells) recorded with whole cell voltage clamp techniques; I-5 ~M nicotine induced a mean peak conductance change of 3.0±0.5 nS (36 cells) measured with intracellular recording. For ciliary ganglion neurons, 2 ,~M nicotine induced a mean peak conductance change of 0.8 + 0.2 nS (l I cells) measured with intracellular recording. Average t2Sl-mAb 35 binding for control chromaffin cells was 8.9 ± 0.2 fmol/culture well (n = 12 wells). None of the three techniques (whole cell voltage clamp, [3H]norepinephrine release, and intracellular recording) showed a specific increase in the AChR response of chromaffin cells after treatment with 8-bromo-cAMP though an increase in the response of ciliary ganglion neurons was observed as previously reported.

346 AChRs into the plasma membrane as previously described t4. Cells having predominantly new AChRs were produced by removing existing AChRs by antigenic modulation with mAb 35 and then allowing new AChRs to accumulate =4. When the cells were examined for nicotine-induced [3H]norepinephrine release, little difference was found between cells with old receptors and those with new ones. Normalizing the data for the number of AChRs detected on the cells with tZSl-mAb 35, yielded values of 1.0 + 0.1-fold (16 cultures from 4 separate experiments) and 1.2 + 0.1-fold (24 cultures from 6 experiments) for old and new receptors, respectively (setting untreated control cultures equal to 1.0). lntracellular recordings revealed similar results: normalizing the ACh response for the number of AChRs measured on sister cultures yielded values of 0.9 + O.l-fold (53 cells from 4 experiments) for cells with predominantly old receptors, and 0.7 + 0.2-fold (71 cells from 4 experiments) for cells with predominantly new receptors compared to untreated control cells. Previous reports that cAMP analogs increase nicotine-induced ['~H]norepinephrine release from bovine adrenal chromaffin cells J,;.t4 may have resulted from the nicotine-induced release and K+.induced release having been carried out in t;mdem on the same cullures. Though control experiments initially endorsed this economy (L. Higgins, unpublished results), later work indicated that tandem assays might be unreliable because cAMP did not always enhance release to the same extent in subsequent trials as it did at the outset (D, Berg, unpublished results), Intracellular recording was used in the original reports to confirm the cAMPdependent effects t.t, but re-examination of the original data indicates that some recordings were rctaitied that should have been excluded on the basis of inadequate resting potentials or electrode imbalance. When these records are excluded, a nominal cAMP-dependent enhancement of the nicotinic response remains as reported, but the effect is not statistically significant bccausc of the large variation among cells, Thc reported difference in the responsiveness of old vs. new AChRs (which depended in part on differences in cAMP-dependent regulation t.~.t4) was also not observed in the present experiments, It is possible that the cell cultures or conditions used in the original experiments ~-~.~ differed in some way from those reported here. Nonetheless, with the present cultures it can be concluded that no evidence was obtained for a cAMP-dependent increase in the nicotinic response of bovine adrenal chromaffin cells, Adrenal chromaffin AChRs would be expected to resemble neuronal rather than muscle AChRs since

chromaffin cells derive from neural crest and can express neuronal properties in cell culture. Northern blot analysis confirms that adrenal chromaffin tissue as well as the pheochromocytoma cell line PCI2 express neuronal AChR genes 2-4.8. AChRs on bovine adrenal chromaffin cells have some of the same epitopes and toxin binding properties as chick ciliary ganglion AChRs t2. The two classes of receptors appear to differ, however, with respect to regulation by cAMP. Several studies demonstrate that cAMP analogs increase the ACh response of chick ciliary ganglion neurons 10.,,.m.z.~ while the present studies found no evidence for cAMP analogs increasing the nicotinic response of bovine adrenal chromaffin cells, it will be of interest to examine the basis for differences in second messenger regulation for AChRs expressed in these tissues. Grant support was provided by the National Institutes of Health (Grant POi NS25916) and the American ttear~ Association with fimds contributed in part by the California Heart Association.

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