Juan J. Ballesta

Neuronal Nicotinic Acetylcholine Receptors on Bovine Chromaffin Cells: Cloning, Expression, and Genomic Organization of Receptor Subunits

Abstract: Neuronal nicotinic acetylcholine receptors from bovine adrenomedullary chromaffin cells play a primary role in triggering catecholamine secretion. In the present study, their constituent subunits were characterized. In addition to the α3 subunit, which we have previously cloned, the presence of α5 and β4 but not of β2 subunits was detected by reverse transcription‐PCR analysis of mRNA from adrenal medulla. In situ hybridization indicated that α3, α5, and β4 subunits are coexpressed in all chromaffin cells. The primary structure of α5 and β4 subunits was determined and functional receptors were obtained upon coinjection of subunit cRNAs into Xenopus oocytes. In contrast to other β4‐containing nicotinic receptors, the ones formed by the bovine β4 subunit are insensitive to the agonist cytisine. Finally, we characterized the intergenic region of α3 and α5 subunits, which together with the β4 subunit, form a gene cluster in rats and chickens. RNase assays and the existence of overlapping cDNAs indicate that, in the bovine genome, the α3 and α5 genes overlap at their 3′ ends. This fact is probably due to inefficient transcription termination, as a result of weak polyadenylation signals.

Separate Binding and Functional Sites for ω co‐Conotoxin and Nitrendipine Suggest Two Types of Calcium Channels in Bovine Chromaffin Cells

Abstract: Purified adrenomedullary plasma membranes contain two high‐affinity binding sites for l25I‐ω‐conotoxin, with KD values of 7.4 and 364 pM and Bmax values of 237 and 1,222 fmol/mg of protein, respectively. Dissociation kinetics showed a biphasic component and a high stability of the toxin‐receptor complex, with a t1/2 of 81.6 h for the slow dissociation component. Unlabeled ω‐conotoxin inhibited the binding of the radioiodinated toxin, adjusting to a two‐site model with Ki1 of 6.8 and Ki2 of 653 pM. Specific binding was not affected by Ca2+ channel blockers or activators, cho‐linoceptor antagonists, adrenoceptor blockers, Na+ channel activators, dopaminoceptor blockers, or Na+/H+ antiport blockers, but divalent cations (Ca2+, Sr2+, and Ba2+) inhibited the toxin binding in a concentration‐dependent manner. The binding of the dihydropyridine [3H]nitrendipine defined a single specific binding site with a KD of 490 pM and a Bmaxof 129 fmol/mg of protein. At 0.25 μM, co‐conotoxin was notable to block depolarization‐evoked Ca2+ uptake into cultured bovine adrenal chromaffin cells depolarized with 59 mMK+for 30 s, whereas under the same conditions, 1 μM nitrendipine inhibited uptake by ∼60%. When cells were hyper‐polarized with 1.2 mM K+ for 5 min and then Ca2+ uptake was subsequently measured during additions of 59 mMK+, ω‐conotoxin partially inhibited Ca2+ uptake in a concentration‐dependent manner. These results suggest that two different types of Ca2+ channels might be present in chromaffin cells. However, the molecular identity of ω‐conotoxin binding sites remains to be determined.

Secretory and radioligand binding studies on muscarinic receptors in bovine and feline chromaffin cells

1. Muscarinic agonists enhanced catecholamine release from perfused cat adrenal glands with the following relative order of potencies: methacholine greater than oxotremorine greater than McN‐A‐343 greater than pilocarpine greater than bethanechol greater than muscarine. Because a continuous online electrochemical detection system was used to monitor catecholamine release, this sequence could be obtained at concentrations much lower (1‐10 microM) and during much shorter stimulation times (3‐30 s) than in previous reports. 2. All muscarinic agonists used secreted adrenaline preferentially over noradrenaline. Methacholine evoked a sustained, non‐desensitizing response in the cat adrenal, which declined to basal levels of secretion immediately after Ca2+ removal: upon Ca2+ restoration secretion was restored to the previous plateau. 3. In addition to evoking a direct secretory response, low concentrations of methacholine, pilocarpine, bethanechol or muscarine clearly potentiated cat adrenal secretory responses evoked by pulses of nicotine (2 microM for 30 s) or high K+ (17.7 mM for 30 s). 4. [3H]Quinuclydinyl benzylate (QNB) specifically bound to cat adrenomedullary membranes with a saturating monophasic curve, suggesting a single binding site with a KD of 23 pM and a Bmax of 67 fmol (mg protein)‐1. Preferential displacement by atropine over pirenzepine suggests that the binding site is associated to a M2‐type muscarinoceptor. 5. Methacholine (3‐300 microM) did not enhance the spontaneous catecholamine release from perfused bovine intact adrenal glands or superfused chromaffin cells. Neither did the drug affect secretion evoked by dimethylphenylpiperazinium (10 microM for 3 s) or K+ (35 mM for 3 s) from isolated superfused bovine adrenal chromaffin cells. 6. [3H]QNB bound to purified bovine adrenomedullary plasma membranes with a KD of 29 pM and a Bmax of 89 fmol (mg protein)‐1. Displacement by pirenzepine suggests the presence of two binding sites (Hill coefficient = 0.64) with Ki1 of 39 nM and Ki2 of 2734 nM. 7. Because the ionophore A23187 enhanced K(+)‐evoked secretion in both, bovine and cat adrenals, it seems that a similar cytosolic Ca2+ rise induced by muscarinic stimulation might constitute the underlying mechanism both to cause a secretory response per se as well as the potentiation of catecholamine release evoked by nicotinic or high K+ stimulation. However, it is unclear why the bovine behaves differently from the feline chromaffin cell as far as the muscarine‐evoked effects are concerned.(ABSTRACT TRUNCATED AT 400 WORDS)

Multiple Functional Sp1 Domains in the Minimal Promoter Region of the Neuronal Nicotinic Receptor α5 Subunit Gene

The α5 subunit is a component of the neuronal nicotinic acetylcholine receptors, which are probably involved in the activation step of the catecholamine secretion process in bovine adrenomedullary chromaffin cells. The promoter of the gene coding for this subunit was isolated, and its proximal region was characterized, revealing several GC boxes located close to the site of transcription initiation (from −111 to −40). Deletion analysis and transient transfections showed that a 266-base pair region (−111 to +155) gave rise to ∼77 and 100% of the maximal transcriptional activity observed in chromaffin and SHSY-5Y neuroblastoma cells, respectively. Site-directed mutagenesis of five different GC motifs indicated that all of them contribute to the activity of the α5 gene, but in a different way, depending on the type of transfected cell. Thus, in SHSY-5Y cells, alteration of the most promoter-proximal of the GC boxes decreased α5 promoter activity by ∼50%, whereas single mutations of the other GC boxes had no effect. In chromaffin cells, by contrast, modification of any of the GC boxes produced a similar decrease in promoter activity (50–69%). In both cell types, however, activity was almost abolished when four GC boxes were suppressed simultaneously. Electrophoretic mobility shift assays using nuclear extracts from either chromaffin or SHSY-5Y cells showed the specific binding of Sp1 protein to fragment −111 to −27. Binding of Sp1 to the GC boxes was also demonstrated by DNase I footprint analysis. This study suggests that the general transcription factor Sp1 plays a dominant role in α5 subunit expression, as has also been demonstrated previously for α3 and β4 subunits. Since these three subunits have their genes tightly clustered and are expressed in chromaffin cells, probably as components of the same receptor subtype, we propose that Sp1 constitutes the key factor of a regulatory mechanism common to the three subunits.

GC- and E-box Motifs as Regulatory Elements in the Proximal Promoter Region of the Neuronal Nicotinic Receptor α7 Subunit Gene

The α7 subunit is a component of α-bungarotoxin-sensitive nicotinic acetylcholine receptors expressed in bovine adrenomedullary chromaffin cells. The proximal promoter of the gene coding for this subunit contains several GC-boxes and one E-box. Deletion analysis and transient transfections showed that a 120-base pair region (−77 to +43) including all of these elements gave rise to ∼70 and 95% of the maximal transcriptional activity observed in chromaffin and SHSY-5Y neuroblastoma cells, respectively. Site-directed mutagenesis of the different elements indicated that both GC and E motifs contribute to the activity of the α7 gene in a very prominent way. Using electrophoretic mobility shift assays, the upstream stimulatory factor (USF) was shown to be a component of the complexes that interacted with the E-box when nuclear extracts from chromaffin and SHSY-5Y cells were used. Binding of the early growth response gene transcription factor (Egr-1) to three different GC-boxes was also demonstrated by shift assays and DNase I footprint analysis. Likewise, α7 promoter activity increased by up to 5-fold when α7 constructs and an Egr-1 expression vector were cotransfected into chromaffin cell cultures. Mutagenesis of individual GC-boxes had little effect on Egr-1 activation. By contrast, pairwise suppression of GC-boxes abolished activation, especially when the most promoter-proximal of the Egr-1 sites was removed. Taken together, these studies indicate that the α7 gene is likely to be a target for multiple signaling pathways, in which various regulatory elements are involved.

Immunohistochemical localization of the voltage-gated potassium channel subunit Kv1.4 in the central nervous system of the adult rat

A large set of voltage-gated potassium channels is involved in regulating essential aspects of neuronal function in the central nervous system, thus contributing to the ability of neurons to respond to a given input. In the present study, we used immunocytochemical methods to elucidate the regional, cellular and subcellular distribution of the voltage-gated potassium channel subunit Kv1.4, a member of the Shaker subfamily, in the brain. At the light microscopic level, the Kv1.4 subunit showed a unique distribution pattern, being localized in specific neuronal populations of the rat brain. The neuronal regions expressing the highest levels of Kv1.4 protein included the cerebral cortex, the hippocampus, the posterolateral and posteromedial ventral thalamic nuclei, the dorsolateral and medial geniculate nuclei, the substantia nigra and the dorsal cochlear nucleus. The Kv1.4 subunit was also present in other neuronal populations, with different levels of Kv1.4 immunoreactivity. In all immunolabeled regions, the Kv1.4 subunit was mostly diffusely distributed and, to a lesser extent, it stained cell bodies and proximal dendrites. Furthermore, Kv1.4 immunoreactivity was also detected in nerve terminals and axonal terminal fields. At the electron microscopic level, Kv1.4 was located postsynaptically in dendritic spines and shafts at extrasynaptic sites, as well as presynaptically in axon and active zone of axon terminals, in the neocortex and hippocampus. The findings indicate that Kv1.4 channels are widely distributed in the rat brain and suggest that activation of this channel would have different modulatory effects on neuronal excitability.

Phorbol ester activation of the neuronal nicotinic acetylcholine receptor α7 subunit gene : Involvement of transcription factor Egr-1

Abstract:α‐Bungarotoxin‐sensitive neuronal nicotinic acetylcholine receptors from bovine adrenomedullary chromaffin cells are up‐regulated by long‐term exposure to phorbol esters. The rise in receptor density is paralleled by an increase in transcripts corresponding to the α7 subunit, which is a component of this receptor subtype. Transcriptional activation of the α7 subunit gene is evidenced in reporter gene transfection experiments, in which phorbol esters increase α7 promoter activity by up to 14‐fold. About 80% of this activation is abolished when at least two of the three sites for the immediate‐early transcription factor Egr‐1, present in the proximal promoter region of the α7 subunit gene, are mutated simultaneously. In addition, phorbol esters elevate both Egr‐1 mRNA and Egr‐1 protein levels in chromaffin cells, whereas electrophoretic mobility shift assays show that the Egr‐1 component of the complexes that originate at the α7 promoter increases in cells treated with phorbol esters. These results suggest that the transcription factor Egr‐1 is involved in triggering expression of α‐bungarotoxin‐sensitive nicotinic receptors in response to external stimuli, such as the ones resulting from phorbol ester treatment, and support our previous hypothesis that the α7 subunit gene is one of the specific targets for Egr‐1.

A residue in the middle of the M2-M3 loop of the β4 subunit specifically affects gating of neuronal nicotinic receptors

An aspartate residue in the M2‐M3 loop of neuronal nicotinic receptor α7 subunits is a major determinant of the channel functional response. This residue is conserved in most β4 subunits, e.g. human and rat, but not in others, e.g. bovine. We have used these differences to examine the mechanism by which this residue alters the functional properties of α3β4 receptors. Having ruled out an effect on the macroscopic binding ability of the agonist, the level of receptor expression, or the single channel conductance, the results suggest that receptors lacking that residue have a deficient coupling between binding and gating.

A Two-Dimensional Electrophoresis Study of Phosphorylation and Dephosphorylation of Chromaffin Cell Proteins in Response to a Secretory Stimulus

Abstract: Phosphorylated proteins of bovine chromaffin cells, radioactively labeled with [32P]orthophosphate, have been analyzed by two‐dimensional polyacrylamide gel electrophoresis and autoradiography. Complex two‐dimensional electrophoretograms were studied with the aid of computer‐assisted image analysis (CAIA). A database map of 32P‐labeled proteins was constructed; ∼500 polypeptides have been detected, numbered, and characterized according to the intensity of labeling, molecular weight, and iso‐electric point. The database was constructed from cells kept in resting conditions or stimulated with 59 mM K+ in 2.5 mM Ca2+ or in 0 Ca2+ solution. These manipulations caused statistically significant changes in the degree of phosphorylation of 20 proteins; they were classified as Ca2+‐dependent substrates for the phosphorylation or dephosphorylation processes. These changes were also shown in cells stimulated in the presence of the Ca2+ channel activator Bay K 8644. New proteins that show as much as a fivefold increase in their phosphorylation state during cell stimulation have been located with this methodology, as well as many others that had not previously been detected with conventional methods. These experiments provide the first CAIA database of chromaffin cell phosphoproteins; the map constructed with these data will allow the location of specific phosphoproteins and serve as a reference for future ongoing studies. The database will continue to grow to identify more proteins and to facilitate the comparison of complex patterns obtained in different laboratories for normal and transformed pheochromocytoma PC 12 ceils.

Activity of the Nicotinic Acetylcholine Receptor α5 and α7 Subunit Promoters in Muscle Cells

The acetylcholine receptor α5 and α7 subunits are components of different nicotinic receptor subtypes expressed in the nervous system. However, they are also present in non-neuronal tissues. We have detected α5 and α7 transcripts in mouse C2C12 muscle cells. Moreover, on differentiation of myoblasts into myotubes, the amount of α7 transcripts increased significantly, whereas α5 remained unchanged. In order to explore how the expression of these neuronal genes is regulated in muscle, we have characterized their promoter activities. Deletion and mutagenesis analysis with transfected reporter genes showed that transcriptional activity was controlled by regulatory elements also operative in neuronal-like cells. Thus, the activity of the α5 subunit core promoter decreased to ~50% on alteration of one, two, or three of the five Sp1 binding sites present in this region and was almost abolished when four or five sites were mutated simultaneously. In the case of the α7 subunit promoter, the upstream stimulatory fa...