Dihydropyridine Receptor Regulation of Acetylcholinesterase Biosynthesis*

The dihydropyridine calcium channel antagonist ni- fedipine causes marked reductions in the amounts of acetylcholinesterase (AchE) molecular forms in pri- mary tissue cultures of avian pectoral muscle. These reductions are time-dependent, requiring passage of 3 h prior to any observable response, dose-dependent, with principal actions occurring in the l-100 nM range, are greater on the 7 S and 19 S forms than on the 11.4 S form, and, based on susceptibility of AchE to irreversible inhibition by a cationic inhibitor, occur almost exclusively with intracellular AchE coincident with a a-fold reduction in the rate of secretion. The effects are markedly mare pronounced in skeletal muscle than in neurons and differ from those observed for verapamil, diltiazem, and the calcium ionophore A23187. These reductions are incompatible with ac- celerated protein degradation, alterations in post- translational processing and assembly in the Golgi complex, or enhanced loss of enzyme to the medium, but instead indicate that nifedipine causes a reduction in AchE biosynthesis. AchE forms are to arise from a single gene, these findings imply a linkage in skeletal muscle between transcription and post-transcriptional processing of mRNA and ligand occu- pation of the dihydropyridine receptor.

The dihydropyridine calcium channel antagonist nifedipine causes marked reductions in the amounts of acetylcholinesterase (AchE) molecular forms in primary tissue cultures of avian pectoral muscle. These reductions are time-dependent, requiring passage of 3 h prior to any observable response, dose-dependent, with principal actions occurring in the l-100 nM range, are greater on the 7 S and 19 S forms than on the 11.4 S form, and, based on susceptibility of AchE to irreversible inhibition by a cationic inhibitor, occur almost exclusively with intracellular AchE coincident with a a-fold reduction in the rate of secretion. The effects are markedly mare pronounced in skeletal muscle than in neurons and differ from those observed for verapamil, diltiazem, and the calcium ionophore A23187.
These reductions are incompatible with accelerated protein degradation, alterations in posttranslational processing and assembly in the Golgi complex, or enhanced loss of enzyme to the medium, but instead indicate that nifedipine causes a reduction in AchE biosynthesis.
Since AchE forms are thought to arise from a single gene, these findings imply a linkage in skeletal muscle between transcription and posttranscriptional processing of mRNA and ligand occupation of the dihydropyridine receptor.
Synthesis of the different molecular forms of acetylcholinesterase (AchE)l in skeletal muscle depends on contractile activity as well as a number of putative, but as yet unidentified, neurotrophic factors (1)(2)(3)(4). AchE appears in muscle and neurons as a polymorphic family of globular catalytic species that are distinguishable from the larger and more asymmetric forms containing elongated, collagen-like tails (2,3). In contrast to the globular forms, encompassing multimeric, membrane-, and cell surface-associated intracellular species, the asymmetric forms are extracellular species noncovalently localized in the basal lamina of the neuromuscular junction (5, 6). The asymmetric forms attract interest because they appear coincident with innervation and disappear following denervation and blockade of contractile activity (4). Mobilization of Ca" and formation of second messengers appear to underlie at least part of these phenomena since, as seen in study of noncontracting cultures of rat and avian skeletal muscle, * This work was supported by Grant ES-03085 from the National EGTA, [ethylenebis(oxyethylenenitril)]tetraacetic acid. elevated concentrations of intracellular calcium mediated through the ionophore A23187, either alone (7) or in combination with phorbol ester activation of protein kinase C (8), promote the appearance of the asymmetric forms of AchE.
While membrane-active ionophores and membrane-permeable phorbol esters can be demonstrated to alter the presence of AchE in skeletal muscle, heterologous regulation of AchE biosynthesis through ligand-specific occupation of distinct receptors has not yet been demonstrated. Voltage-dependent calcium channels are implicated in a wide variety of cellular processes including calcium conductances, excitation-contraction coupling, and second messenger formation (9, lo), and therefore represent one plausible target for consideration. Skeletal muscle transverse tubules contain large numbers of calcium channels that are identified through their capacity to associate with a chemically heterogeneous family of ligand antagonists (9, 10). The ligands most extensively studied fall among the dihydropyridines, phenylalkylamines, and benzothiazepines, typified, respectively, by nifedipine, verapamil, and diltiazem. Dihydropyridines, in particular, associate in skeletal muscle at t-tubule sites with high affinity (KD = l-10 x 10s9 M) and cause inactivation of the slow L-type Ca'+ channel and a reduction in Ca2+ release from the sarcoplasmic reticulum (11). These actions lead to an overall reduction in intracellular Ca2+ and are to be distinguished from those of ionophores that elevate intracellular Ca*+ by promoting passive transfer of Ca2* into the cell.
This study examines the influence of the dihydropyridine antagonist nifedipine on biosynthesis of AchE molecular forms in skeletal muscle and in neurons. Results obtained with nifedipine are compared with those obtained for verapamil, diltiazem, and the calcium ionophore A23187. Particular attention focuses on the dose and time dependences of these actions in monolayers, resolution of intru-and extracellular forms of the enzyme, and accompanying alterations in secretion. AchE forms are examined in primary cultures of avian pectoral muscle because the kinetics of synthesis, degradation, and transport of AchE in this tissue are well documented (2,(12)(13)(14), providing a suitable frame of reference for interpreting changes in the amounts and distribution of AchE forms. Avian neural retina serves as the source of neurons; this tissue arises as an extension of the forebrain, follows an orderly program of growth and differentiation (15), and provides a rich source of glial-free neuronal cells (16). The influence of nifedipine on intra-uersus extracellular forms of AchE is resolved by employing a rapid, irreversible, cationic methylphosphonate inhibitor of AchE, @-(trimethylammonium)ethyl methylphosphonofluoridate (@(TMA)ethyl-MPF) (17). By virtue of its cationic charge, low concentrations of ,&(TMA)ethyl-MPF are expected to be cell-impermeant and therefore to react with cell surface forms of the enzyme, whereas higher concentrations might be expected to penetrate the cell and to react with intracellular forms of AchE. @-(TMA)ethyl-MPF is ad- forms of AchE (Fig. 1B). The 19 S form was conspicuously absent from neural retina. Compartmentalization of AchE in skeletal muscle and neural retina was assessed by measuring accessibility of the different molecular forms to irreversible inhibition by the cationic methylphosphonate inhibitor P-(TMA)ethyl-MPF present in the concentration range 10-l' to 10T5 M (Fig. 2). More than 90% of the initial 7 S AchE activity remained in skeletal muscle after treatment with P-(TMA)ethyl-MPF in the concentration range 10-l' to 10e7 M ( Fig. 2A). Activity of 7 S AchE fell sharply at P-(TMA)ethyl-MPF concentrations above 10T7 M. The 11.4 S form was not appreciably inhibited at P-(TMA)ethyl-MPF concentrations less than lo-' M. The extent of inhibition increased with increasing concentrations up to 10m7 M, at which it levelled at 40-50% of the initial enzyme activity. Complete inhibition of both the 7 S and 11.4 S forms was observed after treatment with 10e5~g-(TMA)ethyl-MPF.
The 19 S form displayed an exquisite sensitivity to the presence of inhibitor and fell sharply after treatment with concentrations of @-(TMA)ethyl-MPF in the range 10-l' to lo-' M; nearly complete inhibition of 19 S AchE occurred at low7 M, a concentration that was loo-fold lower than required for inhibition of the 7 S and 11.4 S forms. Because 19 S AchE was present in only small amounts, measurement of this form engendered a greater uncertainty than seen for the other more abundant forms. These data revealed that 7 S AchE was inaccessible to lower concentrations of inhibitor, suggesting that this AchE molecular form existed predominantly as an intracellular species. In contrast, 11.4 S AchE appeared to be partitioned equally between intra- 90% of the initial 7 S and 50% of the initial 11.4 S AchE remained after pulse inhibition by /3-(TMA)ethyl-MPF in the concentrations range lo-' to 10m7 M (Fig. 2B). At concentrations higher than 10e6 M, complete inhibition of both the 7 S and 11.4 S forms was observed. These results were virtually identical with those found for the corresponding forms in skeletal muscle. That is, the AchE molecular forms common to skeletal muscle and neural retina showed common cell compartmentalization.

Influence of Nifedipine on Appearance of AchE Molecular
Forms in Skeletal Muscle and Neural Retina-Nifedipine caused measurable reductions in the amounts of AchE in primary culture of pectoral muscle. The effects were timedependent, as measured over the duration O-48 h (Fig. 3A), and dose-dependent, over the range lo-' to low4 M (Fig. 4A). With regard to the time dependence, the presence of nifedipine (10T5 M) caused graded, pronounced reductions in the 7 S and 19 S forms of AchE. These reductions approached 50% loss after 10 h and remained essentially unchanged through the subsequent 48 h of observation.
The 11.4 S form underwent no measurable reduction during this time.
With respect to the dose dependence of these reductions, greater than 75% of the 7 S and 19 S forms remained after treatment with lo-' M nifedipine; more than 50% of these forms remained after treatment with higher nifedipine concentrations in the range lo-* to 10e6 M (Fig. 4A). In contrast to 7 S and 19 S AchE, the 11.4 S form of the enzyme was relatively unaffected by treatment with nifedipine over the concentration range lo-' to 10e6 M; measurable reductions of 11.4 S AchE were observed only at the highest concentrations employed, low5 to 10e4 M.
Several features of these data are noteworthy. Loss of 7 S and 19 S AchE required passage of 3 h prior to observations of any discernible reductions of AchE. range (l-100 x lo-' M) over which significant losses of AchE were observed was compatible with the dissociation constant for nifedipine binding at voltage-dependent calcium channels in skeletal muscle (10). After the longest times of treatment (24-48 h) with the highest concentration of nifedipine (10e6 to 10e4) the 7 S and 19 S forms were never abolished and remained essentially unchanged at 40-50% of their initial activities. As assessed by visual examination in the phasecontrast microscopic field, treatment with nifedipine caused neither loss of spontaneous contractile activity nor any visible morphological or cytochemical alterations. This latter observation contrasts with reports that all classes of calcium channel antagonists, including dihydropyridines, block the depolarization-activated contraction of cultured mouse muscle (23, 24).
In neural retina the time (Fig. 3B) and dose dependences (Fig. 4B) for reduction of 7 S and 11.4 S AchE forms in the presence of nifedipine were quite shallow and contrasted with the more striking time and dose dependences obtained for skeletal muscle. Nifedipine concentrations less than 10e6 M caused no discernible loss of the 7 S AchE and loss of only 20% of 11.4 S AchE. The time and dose dependence for these effects reveal a markedly lower sensitivity of neural retina to nifedipine than skeletal muscle. For comparison with nifedipine, the influence of the Ca2+ ionophore A23187 on AchE in skeletal muscle was examined over a duration of O-48 h (Fig. 5). In contrast with the behavior observed for nifedipine, treatment of skeletal muscle cultures with A23187 resulted in reductions of all forms of AchE. Within experimental uncertainty, the time course for reduction in 7 S and 11.4 S AchE was approximately linear (Fig. 5). Amounts of 19 S AchE increased during the 1st h of treatment with A23187, but sharply declined during the subsequent 9 h. The transient increase in 19 S AchE was similar to that reported for cultured rat (7) and avian (8) Fig, 6 as the percent activity of each molecular form remaining in cultures pulse-inhibited with /3-(TMA)ethyl-MPF after treatment with nifedipine. Results with nifedipine and /3-(TMA)ethyl-MPF alone are presented for comparison. Treatment of skeletal muscle cultures with nifedipine resulted in a 38% loss of 7 S AchE; treatment with /3-(TMA)ethyl-MPF alone resulted in a 16% loss (Fig. 6A). The 52% reduction of 7 S AchE after treatment with nifedipine followed by pulse-inhibition with &(TMA)ethyl-MPF was the approximate sum of effects seen after the individual treatments.
Similarly, for the 11.4 S and 19 S AchE forms, sequential treatment with nifedipine followed by pulse-inhibition with /3-(TMA)ethyl-MPF resulted in reductions in these forms that were the sums of the individual treatments. In neural retina, as shown in Fig. 6Z3, the effects of sequential treatment with nifedipine and @(TMA)ethyl-MPF on the 7 S and 11.4 S AchE molecular forms were approximately additive, and hence similar to those observed in skeletal muscle. Similar results were observed at nifedipine concentrations of lOma M. Hence, these effects on intra-and extracellular populations of AchE were independent of nifedipine concentration.
In all cases, the amount of enzyme available for inhibition by P-(TMA)ethyl-MPF after treatment with nifedipine was nearly identical with that available before nifedipine treatment. That is, the nifedipine-induced reductions of AchE occurred with a population of enzyme that was separate from the enzyme acted on by the cell-impermeant irreversible inhibitor. The reductions of AchE following treatment with @-(TMA)ethyl-MPF reflected direct inhibition of cell surface/ accessible AchE. The reductions of AchE following treatment with nifedipine were therefore concluded to occur exclusively with intracellular AchE. Cell surface or extracellular popula- Monolayer cultures of skeletal muscle (A) or neural retina (B) were treated with nifedipine (10m5 M) for 24 and 48 h, respectively. After this treatment, monolayers were pulse-inhibited at room temperature for 10 min with &(TMA)ethyl-MPF (10-r M), washed three times to remove any unreacted inhibitor and then harvested and examined on sucrose density gradients as described under "Experimental Procedures." Results are presented as the percent activity of each molecular form present in the untreated culture and represent the mean + SD. averaged over three to five separate sets of cultures. The percent activity present in untreated cultures (Cl) is compared with that found after treatment with nifedipine (El), after pulse-inhibition with @-(TMA)ethyl-MPF (lo-' M) (&l), and after treatment with nifedipine and subsequent inhibition with P-(TMA)ethyl-MPF (m). As seen in A and B, sequential treatment with nifedipine and /3-(TMA)ethyl-MPF caused reductions in AchE molecular forms that were the approximate sum of the individual treatments.
tions of the enzyme were relatively unaffected by treatment with nifedipine.
The Influence of Nifedipine on Secretion of Skeletal Muscle A&!&--The influence of nifedipine on secretion of AchE was examined by monitoring appearance of the enzyme in the culture medium. In one type of experiment AchE secretion from skeletal muscle cultures was monitored after long-term treatment (24 h) with nifedipine ( Fig. 7A), while in an alternative experiment secretion was monitored immediately after supplementation of the medium with nifedipine (Fig. 7B). In both cases secretion in the presence of lo-' M nifedipine was monitored over a time course of 8-10 h.
In the absence of nifedipine, AchE secretion into the medium was linear and was characterized by an average rate of 0.31 f 0.09 h-' (Fig. 7A). After treatment with nifedipine for 24 h, secretion remained linear but the rate was reduced to 0.21 + 0.05 h-'. When secretion was measured immediately after addition of nifedipine to the culture medium, appearance of AchE in the medium deviated from linearity, and was more adequately described as the sum of two kinetically distinct components (Fig. 7B). During the early phase of secretion encompassing O-3 h, the appearance of AchE in the medium was coincident with that of the untreated myotubes; the average rate for secretion under this condition was calculated to be 0.31 f 0.06 h-i, in excellent agreement with that determined in the absence of nifedipine. Secretion during the subsequent 3-10 h was linear and was characterized by an average rate of 0. A, secretion of AchE from primary cultures of chick skeletal muscle was monitored after 24 h treatment with nifedipine (lo-' M). Cultures were washed and replaced with fresh conditioned medium, as described under "Experimental Procedures." In untreated cultures secretion over 8 h occurred in a linear fashion at an apparent rate of 0.44 h-i. Averaged over 10 separate sets of cultures the rate was calculated to be 0.31 + 0.09 h-i. After treatment with nifedipine, the rate of secretion of AchE was determined to be 0.26 h-l. Averaged over five separate sets of cultures the rate was calculated to be 0.21 + 0.05 h-l. B, the time course for secretion of AchE into the medium was monitored immediately after replacing the complete culture medium with the conditioned medium either free of or containing nifedipine (10M5 M). In untreated cultures secretion of AchE over 10 h occurred in a linear fashion at an apparent rate of 0.27 h-l. After initiating treatment with nifedipine the secretion of AchE occurred coincident with that for the untreated cultures for a duration of 3-4 h; averaged over three separate determinations the rate was calculated to be 0.31+ 0.06 h-l. During the remaining 4-10 h, secretion of AchE deviated from the untreated cultures and occurred with an apparent rate of 0.15 h-l. Averaged over three separate sets of cultures, the rate constant for secretion during the 4-10 h duration was determined to be 0.22 f 0.07 h-i, in close agreement with that the value obtained after longterm treatment with nifedipine (A). 0, untreated cultures; 0, cultures treated with nifedipine.
with that determined for secretion after long-term treatment with nifedipine.

The Influence of Voltage-dependent
Calcium Channel Antagonists on AchE Expression in Skeletal Muscle-The effects of two other voltage-dependent calcium channel blockers, verapamil and diltiazem, were examined and compared with that seen for nifedipine and A23187 (Fig. 8). Treatment with verapamil and diltiazem caused no marked losses of 7 S AchE and measurable but variable reductions, 39 + 23 and 26 f 27%, respectively, in 11.4 S AchE. Most noticable were the losses in 19 S AchE of 78 r: 19 and 54 _+ 30% following treatment with verapamil and diltiazem, respectively (Fig. 8, B and C), and therefore were comparable with those seen for nifedipine. These results represent the mean f S.D. of seven to nine separate sets of cultures. Treatment with diltiazem and A23187, as noted above for nifedipine, caused neither reductions in spontaneous contraction nor any morphological alterations in the muscle fibers. Verapamil, in contrast, not only blocked spontaneous contraction but also caused marked alterations in appearance of the fibers. When examined with phase-contrast microscopy before and after Giemsa staining, verapamil-treated fibers displayed a grainy appearance, an increased number of cytoplasmic vacuoles, loss of striations, and a disrupted, irregular alignment of nuclei. These observations were more pronounced at higher verapamil concentrations in the range lo-' to 10e5 M, and were similar to those reported for rat skeletal muscle treated with D600, an analog of verapamil (25).

DISCUSSION
This paper reports that treatment of primary skeletal muscle cultures with the dihydropyridine calcium channel antagonist nifedipine causes marked reductions in the amounts of individual molecular forms of AchE. These reductions are time-and dose-dependent, are greater on the 7 S and 19 S forms than on the 11.4 S form, and are markedly more pronounced in skeletal muscle than in neurons. The primary effects occur in the l-100 nM dose range and, based on susceptibility of AchE to irreversible inhibition by a cationic inhibitor, occur exclusively on the intracellular rather than eztrucellular pools of the 7 S and 11.4 S forms. The effects in muscle require passage of 3 h prior to any observable reduction in amounts of AchE and prior to onset of a 2-fold reduction in the rate of secretion.
The graded dose-response relationship in skeletal muscle in the l-100 nM concentration range is compatible with the binding affinities for dihydropyridines at the calcium channel (lo), and contrasts with the shallow dependence in the l-10 pM range seen for neural retina. Skeletal muscle contains a large number of dihydropyridine receptors (80,000 fmol/mg protein) that are localized in t-tubules (10). Neural retina is known to contain a calcium channel that, compared to the Ltype channel in skeletal muscle, is present in much smaller amounts (98 fmol/mg protein), is less antagonized by dihydropyridines, and possesses a lower affinity for dihydropyridines (26). The effects seen with nifedipine differ from those seen with verapamil and diltiazem and, taken together, are consistent with a dihydropyridine receptor-mediated phenomenon. The reductions of all forms of AchE at the higher concentrations of nifedipine @PM), since they are observed in both skeletal muscle and neurons, are not suggestive of high affinity binding at a single site but more likely result from nonspecific actions of dihydropyridines on ATP-dependent Ca2+ transport (reviewed in Ref. 10).
As indicated through direct spectroscopic measurement in skinned muscle fibers, blockade of dihydropyridine sites reduces Ca2+ influx as well as Ca*+ dissociation from the sarcoplasmic reticulum leading to an overall reduction in intracellular free Ca*+ (11,27). AchE loss in the presence of nifedipine differs both quantitatively and qualitatively from the near linear relationships observed when intracellular Ca*+ is elevated by treatment with the calcium ionophore A23187. As such, these data are most compatible with a dihydropyridine receptor-mediated reduction rather than an increase in intracellular Ca*+.

Mechanism of Dihydropyridine
Receptor-mediated Reduction in AchE-AchE exists as a polymorphic family of glycoprotein molecular forms. Employing the nomenclature of Massoulie and Bon (28), AchE molecular forms can be described as monomeric, dimeric, and tetrameric globular catalytically active species, denoted as Gl, G2, and G4, respectively. In addition, there exists a unique family of asymmetric species containing the globular catalytic tetrameric units covalently linked to an elongated, fibrillar, collagen-like tail. The 19 S species in avian skeletal muscle, denoted Al2 AchE, represents 3 tetrameric units linked to individual strands of the triple-stranded collagen-like tail; 12 S and 15.5 S forms containing 1 and 2 tetrameric units are also known. AchE molecular forms in chick skeletal muscle and neural retina display similar if not identical sedimentation coefficients and compartmentalization; hence their physical and cellular properties are similar. The reduction in AchE following nifedipine treatment must therefore arise from differences in activity of dihydropyridine receptors in skeletal muscle and neural retina. The possible actions of nifedipine with respect to reductions in AchE are discussed with reference to alterations in glycosylation/assembly, secretion, degradation, and synthesis of the individual forms (2). Glycosylation/Assembly-As measured through their acquisition of high mannose asparagine-linked oligosaccharides, the 7 S and 11.4 S molecular forms of AchE are identifiable in the rough endoplasmic reticulum as unique catalytically active species within 5 min of synthesis. Assembly and glycosylation of these forms in the Golgi complex are complete within the subsequent 45-60 min (14,29). The 19 S form is assembled in the distal cisternae of the Golgi complex 90-120 min following appearance of the 11.4 S form in the rough endoplasmic reticulum (14). If nifedipine treatment of skeletal muscle were to lead to alterations in oligosaccharide processing of AchE in the Golgi complex, then it is predicted that reduction in 7 S AchE would be evident within 45-60 min of initiation of drug treatment, and the reduction of 19 S AchE would be evident within 1.5-2 h of treatment.
The 3-4 h lag preceding any measureable reduction in AchE exceeds these times. Indeed, from the known transit times for these individual forms, the 3-h delay encompasses the time during which synthesis of 7 S AchE is already completed and during which the 19 S form is still resident in the Golgi complex. In addition, while synthesis and assembly of 7 S and 19 S AchE follow different time courses, the temporal dependence of the nifedipine-induced reductions are indistinguishable. The time course for the nifedipine-induced reductions of AchE is therefore not compatible with alterations in post-translational processing and assembly in the Golgi complex.
Secretion-AchE secretion in the presence of nifedipine, measured over 10 h, is characterized by two kinetically distinct components (Fig. 7B). The early component (<3 h) is coincident with secretion in the absence of nifedipine, while the later component (>3 h) is 2-fold slower. The observation of a reduction rather than an increase in rate of AchE secretion is sufficient to rule out enhanced loss of the 7 S and 19 S to the extracellular medium. The resolution of two clear components is compatible with a conversion between two distinct states. The conversion point at 3 h coincides with the onset of reductions of intracellular AchE, indicating a linkage between amounts of enzyme in the monolayer and the amounts of enzyme secreted. Moreover, since AchE secretion occurs from newly synthesized enzyme (12, 13), the reduction in AchE secretion implies a reduction in the amounts of new rather than old enzyme available for secretion.
Degradation-In tissue-cultured muscle two principal rates of AchE degradation are known. Intracellular forms of AchE undergo turnover with half-lives of 1.5 h, whereas cell surface membrane-and basal lamina-associated forms undergo turnover with half-lives in the range 40-50 h (12, 30). The loss of AchE can not represent an accelerated degradation of the slower turnover cell surface forms since the primary action of nifedipine occurs almost exclusively on intracellular AchE. With respect to the fast turnover rates, nifedipine-induced loss of AchE occurs only after passage of 3 h, a time encompassing two turnover half-lives, and therefore deviates significantly from the first-order kinetic behavior characteristic of intracellular AchE degradation (12). The loss of AchE following treatment with nifedipine differs both quantitatively and qualitatively from the pattern of AchE loss when protein degradation prevails, as observed after treatment of cultured muscle with A23187 (31-33). Finally, even though approximately 50% of 11.4 S AchE is intracellular, this form undergoes only small loss following nifedipine treatment. Overall, loss of the different molecular forms of AchE following treatment with nifedipine is not compatible with accelerated protein degradation.
Synthesis-The nifedipine-induced reductions are not compatible with alterations in post-translational processing and assembly in the Golgi complex, increased rates of secretion, or intracellular protein degradation. By exclusion, the evidence points to dihydropyridine receptor-mediated reductions in biosynthesis of the individual molecular forms of AchE, an action that is selective to the 7 S and 19 S forms. This conclusion is supported by the requirement for a 3-h duration prior to onset of reduction of 5/7 S and 19 S AchE, and prior to reduction in the rate of AchE secretion. This time coincides with the 3-h interval separating de nouo synthesis and appearance of AchE and the nicotinic acetylcholine receptor in avian skeletal muscle (12, 13) and neurons (34). Since the 2fold reduction in AchE secretion (Fig. 7) parallels the 50% loss in amount of AchE after long-term treatment with nifedipine (Fig. 3A), and on the basis of unaltered protein degradation, the rate of synthesis is concluded to be reduced approximately 2-fold. AchE molecular forms in chick muscle and neurons appear to arise from allelic variants of a single gene (35,36). The appearance of AchE from a single gene requires that the multiple forms of AchE arise either through alternative splicing of a single primary transcript or through differential promotion, or both, leading to formation of multiple transcripts (37). While alternative splicing has been deduced as one mechanism underlying AchE polymorphism in Torpedo (3840), neither mechanism is yet known for regulation of AchE biosynthesis in skeletal muscle. If alternative splicing mechanisms prevail, then antagonist occupation of the dihydropyridine receptor can be concluded to alter AchE expression at post-transcriptional points such as mRNA processing and stability.
If differential promotor mechanisms prevail, then antagonist occupation of the dihydropyridine receptor can be concluded to alter AchE expression at the level of transcription.
Overall, any post-translational mechanisms must be subordinate to alterations occurring at transcriptional and post-transcriptional levels. While many examples of protein polymorphism through alternative splicing and differential promotion mechanisms are known (41-43), the cellular mechanisms that signal these processes have yet to be identified in molecular terms. The present results imply a linkage between transcription and post-transcriptional mRNA processing within the nucleus and ligand occupation of the dihydropyridine receptor at the t-tubule plasma membrane. Since dihydropyridines are known to cause a reduction in intracellular Ca", mobilization of Ca2+ subsequent to antagonist occupation of the dihydropyridine receptor represents one probable component in this mechanism. These results are specific to skeletal muscle since no such relationship is evident in neural retina and, by virtue of representing receptor-specific activity, differ from the more general examples of Ca2' ionophore-mediated alterations in gene expression (44-48). Moreover, since dihydropyridine blockers in chick myotubes also cause up-regulation of the nicotinic acetylcholine receptor (49), these findings are of general significance to regulation of choline@ proteins.
Precursor Relationships in AchE Synthesis-It is not known whether the asymmetric AchE forms undergo assembly from globular Gl or G4 units (14,34,50), and whether the G4 units within the Al2 species are identical with the catalytically detected 11.4 S species. In this regard it is of interest that the 7 S, and 19 S forms show behaviour common to one another but unique from that for 11.4 S AchE, analogous with observations characterizing reductions in AchE following denervation of rat anterior tibiulis (4).
The observed loss of 19 S AchE with neither reduction nor increase in 11.4 S AchE is incompatible with a sequence in which synthesis of Al2 AchE depends directly on availability of a common pool of tetrameric G4 AchE. Indeed, the parallel loss of 7 S and 19 S AchE (Figs. 3 and 4) is more compatible with assembly of Al2 AchE from globular species that are physically distinct from the catalytically active 11.4 S form. Hence, these results argue against assembly of the asymmetric AchE molecular forms from fully processed G4 species that are identical with 11.4 S AchE. This conclusion implies that the individual molecular forms of AchE are synthesized and assembled independent of one another, and finds indirect support in observations that at least two forms of AchE in avian muscle, the 7 S and 11.4 S species, undergo no interconversion and retain distinct identities throughout their intracellular residence (29). In light of these considerations, the final identity of the asymmetric AchE molecular forms must be determined at a stage no later than post-transcriptional processing of mRNA.
Functional Significance of Dihydropyridine Receptor Regulation of AchE-The dose-and time-dependent reductions in AchE following treatment with nifedipine are unique from those seen for verapamil and diltiazem, are not evident in neurons, and are thus compatible with a dihydropyridine receptor-mediated phenomenon specific for skeletal muscle. Dihydropyridines bind in skeletal muscle at receptor sites localized within the transverse tubules of the junctional triad. A small fraction of these receptor sites operate as voltagedependent slow calcium channels (51) while they, or a separate number, appear also to function as voltage sensors for excitation-contraction coupling (11,24,27,(52)(53)(54). Inositol 1,4,5-triphosphate is implicated in this process as a chemical second messenger linking t-tubule depolarization with calcium release from the sarcoplasmic reticulum (56-59).
As seen in noncontracting skeletal muscle treated with phorbol esters (8), calcium ionophores (7), and the Na' channel activator veratridine (25), evidence is accumulating that the dependence of AchE biosynthesis on innervation and contractile activity is attributable to formation of intracellular second messengers in response to membrane electrical activity rather than contraction per se (60). The present studies provide a strong indication that the dihydropyridine receptor, with an as yet incompletely defined role in excitation-contraction coupling, presents an abundant, localized, and highly coupled target for neuronal regulation of skeletal muscle. Evidence of an increasing number of endogenous ligands of neuronal origin that block both Ca'+ uptake and ligand association at voltage-dependent calcium channels serves to support such an outlook (61-65).