4euron.

Vol. 9, 671-678,

October,

1992, Copyright

0 1992 by Cell Press

Protein Kinase C Couples Membrane Excitation to Acetylcholine Receptor Gene Inactivation in Chick Skeletal Muscle (-hang-Fen

Huang,

Jian long,

and jakob

tlepartment of Biochemistry and Cell state University of New York at Stony itony Brook, New York 11794

Schmidt Biology Brook

Summary The signaling pathway connecting membrane depolarization and gene activity in skeletal muscle remains largely unknown. Using transcription elongation (runon) analysis we have found that electrical stimulation of denervated chick skeletal muscle in vivo rapidly and selectively results in inactivation of acetylcholine receptor (AChR) subunit genes. We have studied the possible involvement of protein kinase C (PKC) in this response and have observed that electrical stimulation increases the activity of PKC in the nucleus by over two orders of magnitude within 10 min; phorbol esters, within minutes after intramuscular application, block AChR subunit genes in the absence of electrical activity; and the activity-triggered gene inactivation is blocked by the protein kinase inhibitor staurosporine or by enzyme depletion resulting from chronic pretreatment of muscle with phorbol esters. We conclude that PKC is an integral component of the pathway coupling membrane excitation and AChR gene control. Introduction The mechanisms underlying the appearance of extrajunctional acetylcholine receptors (AChRs) in denervated skeletal muscle have been under investigation since 1959 when Axelsson and Thesleff rediscovered the phenomenon of denervation supersensitivity. A consideration of two early findings, namely, Fambrough’s observation that actinomycin D blocks supersensitivity (1970) and the demonstration by several investigators that the supersensitivity of denervated muscle can be reduced to near control levels by electrical stimulation (Drachman and Witzke, 1972; Lomo and Westgaard, 1975), suggests that a signaling pathway links the plasma membrane to the genes responsible for acetylcholine sensitivity. In 1975 Hall and Reiness showed that AChR synthesis can be blocked by electrical stimulation of organ-cultured rat diaphragm. When AChR-specific nucleic acid probes became available, it was observed that denervationtriggered up-regulation of receptor subunit messages can be prevented by chronic electrical stimulation in vivo (Goldman et al., 1988). In addition, it has been shown that acetylcholine supersensitivity is, at least in part, a consequence of AChR gene activation (Tsay and Schmidt, 1989) and that this activation can be blocked with inhibitors of protein synthesis (Tsay et al., 1990), suggesting the participation of a transcriptional activator.

We have recently returned to the original experimental paradigm of Lomo (1975) (i.e., electrical stimulation of muscle at the height of denervation supersensitivity) to investigate short-term effects of plasma membrane electrical activity. We observed that AChR a subunit (Neville et al., 1991) and myogenin (Neville et al., 1992) transcripts decline at a rate comparable to that seen upon actinomycin D administration; these findings were interpreted to mean that electrical activity rapidly shuts down these genes by posttranslational inactivation of a transcription factor. In this report we show directly by transcript elongation analysis that action potentials rapidly inactivate AChR genes. We also report evidence for the participation of protein kinase C (PKC) in the signaling pathway connecting electrical membrane activityto the inhibition of AChR subunit genes. We expect that the reduced time frame (significant effects can be seen as early as 10 min after onset of stimulation) will greatly facilitate further analysis of depolarization effects.

Results Electrical Stimulation of Denervated Differential Gene Inactivation

Muscle Causes

We have previously shown that denervation activates AChR subunit genes under conditions that leave several control genes unaffected (l-say and Schmidt, 1989). Our first aim therefore was to establish whether the reduction in receptor mRNAs that we have observed upon electrical stimulation of denervated muscle in vivo (Neville et al., 1991) also is a consequence of altered transcription rates. When run-on analysis was performed on innervated muscle, muscle denervated for 40 hr (so as to allow AChR gene activity to reach maximal levels), and denervated/stimulated muscle, it was observed that denervation increases a, y, and 6 subunit gene activity and that upon stimulation activity returns to control (innervated) levels; a number of non-AChR genes were analyzed in the same manner, but found to respond little to either cessation or resumption of plasma membrane activity (Figure 1). As reported previously (Tsay and Schmidt, 1989), there is limited but signific:ant antisense transcription of receptor genes whose activity echoes that of sense transcription in the experimental paradigms investigated (data not shown).

Stimulation Causes Rapid Inactivation Coding for AChR Subunits

of Genes

The mechanism of excitation-AChR control coupling comprises an unknown number of steps, perhaps even including a need for de novo protein synthesis, as was recently shown for receptor up-regulation induced by denervation in vivo (Neville et al., 1991) or by tetrodotoxin administration in vitro (Duclert et al., 1990). To gain further insight we determined the time

NeWOn 672

a

N

S

D

la.

&actin

1 b. 2b. 3b. 4b.

Pa. ras 3a. c-fos 4a. tropomyosin Figure

1. Specific

AChR

CI Subunit

Gene

Regulation

by Membrane

myosin a-tubulin aexonl GAPDH

IigM

chain

Activity

Transcript elongation was assayed in nuclei isolated from innervated (N), 40 hr denervated (D), and 40 hr denervated/30 min stimulated (S) muscle, as described in the Experimental Procedures section; IO’ nuclei were used in each run-on incubation, and 2 x IO6 cpm of JZP-labeled RNA for each hybridization. Results shown were obtained with probes specific for p-actin, ki-ras, c-fos, tropomyosin, myosin light chain, a-tubulin, AChR a subunit (a-exonl), and CAPDH.

required to affect gene activity after the onset of stimulation. As can be seen in Figure 2, significant reduction in transcriptional activity of AChR genes is evident within 10 min, and return to predenervation levels is seen within 60 min. This strongly suggests that the signaling pathway directly targets, for inactivation, a necessary transcription factor that is already present in the denervated muscle fiber. Recovery of

0 n-eronl . a-exon’l 06 .Y [7 tropomyoain . GAPDH

Figure Genes

2. Electrical

Activity

Rapidly

Inactivates

AChR

Subunit

AChR subunit transcript elongation was assayed in nuclei isolated from innervated muscle (N) and from muscle stimulated for the indicated periods of time, as described in Experimental Procedures. Each data point represents the average of 2-5 independent measurements. Results are normalized to the transcriptional activities in 40 hr denervated muscle; about 1 hr after the onset of stimulation, gene activities begin to fall below control (innervated) values. CAPDH and tropomyosin gene activities, shown for comparison, vary less than 10% between protocols, attestingtothespecificityofgenecontrolaswellasthereproducibility of the assay (see also Figure 4, Figure 5, and Figure 7).

transcription rates after short periods of stimulation proceeds slowly, requiring approximately a day for the return of AChR subunit gene activity to near control, unstimulated levels (data not shown). Acute Stimulation Causes an increase in the Activity of PKC in the Nucleus Of the posttranscriptional mechanisms possibly involved in excitation-AChR gene repression coupling, the inhibition of a transactivator by phosphorylation is a plausible one; activation of PKC has been observed in several depolarization-triggered cascades. We determined total enzyme levels by assaying individual fractions with added phorbol IZmyristate 13-acetate (PMA) and found that chick muscle, regardless of its state of innervation, contains PKC in the cytosol, nuclei, and nonnuclear membranes. Within innervated muscle, the enzyme is partially active in all subcellular fractions. The low activity in the nuclear compartment virtually ceases upon denervation; a less pronounced inactivation is observed in the nonnuclear particulate and in the cytosol. When PKC was analyzed in response to electrical stimulation, enzyme activity was observed to rise steadily in the cytosolic and nonnuclear membrane fractions, while in the nuclear compartment it increases abruptly more than IOO-fold between 3 and 10 min after onset of stimulation (Figure 3). This effect lingers: 90 min after ending the stimulation nuclear PKC still exhibits 85% and, 24 hr later, 10% of maximal activity. Enzyme activity in electrically stimulated muscle exceeds that in innervated muscle; this may reflect the difference between the tetanus caused by high frequency stimulation and the incomplete recruitment and intermittent activity of individual motor units under physiological conditions.

Depolarization,

PKC, and Receptor

Gene

Activity

673

.

N

0

10 Period

Figure

3. Electrical

Stimulation

of treatment

Activates

20

j tropomgaain GAPDH

30

(min)

Nuclear

PKC

PKC enzyme activity was assayed in the cytosol, nonnuclear particulatefraction, and nuclei from innervated muscle(N) from muscle denervated for 40 hr and stimulated for the indicated periods of time. Results are presented as the ratio of active to total enzyme in each subcellular preparation (for definition of “total enzyme” see Experimental Procedures). While PKC levels (total enzyme) were little affected by the treatments, enzyme activity in all cellular compartments decreased upon denervation and increased upon stimulation as shown.The broken linetraces the approximate increase in the activity of nonnuclear enzyme.

Short-Term Treatment with Phorbol Esters Inactivates Genes Coding for AChR Subunits To test whether the kinase activation in the nucleus has any consequences for AChR gene activity, denervated unstimulated muscle was treated with directly injected phorbol ester. This leads, within 30 min, to significant (30%-60%) block of receptor genes (Figure 4A). The incomplete inhibition of gene activity (compared with electrical stimulation) may result from incomplete penetration of the drug from the injection site. No suppression of gene activity was observed with either 4a-phorbol, a pharmacologically inactive compound, or the vehicle alone (Figure 4B). Staurosporine Enhances AChR Gene Activity in Normal and Denervated Muscle and Blocks the Effects of Electrical Stimulation Staurosporine blocks the activity of several kinases including PKC; it also inhibits theactivation of nuclear PKC caused by stimulation of denervated muscle (Figure SA). One test of the possible involvement of PKC in membrane-genome signaling therefore is to determine whether this inhibitor abolishes the receptor gene response toelectrical stimulation. Wefound that ;n the presence of staurosporine, AChR genes are no ionger inhibited byelectrical membraneactivity;etha‘101, which is used as a solvent, has no effect by itself ‘Figure SB). Long-Term Treatment with Phorbol Esters Down-Regulates PKC and Blocks the Signaling Pathway -:he staurosporine experiment suggests that a kinase, but not necessarily PKC, is involved in gene inhibition.

alpha1 alpha7

Figure

4. Phorbol

Ester

delta

Inactivates

gamma GAPDH

AChR

Subunit

tropomyosin

Genes

(A) Transcript elongation was assayed in nuclei isolated from innervated muscle (NJ and from denervated muscle, at the indicated times after in viva administration of PMA (20 pg in 20 ~1 of 20% dimethyl sulfoxide in ethanol [v/v], injected intramuscularly). (B) The bar graph shows nuclear run-on data obtained with probes specific for the indicated AChR subunit genes and for GAPDH and tropomyosin as internal controls. Within each set, results (from left to right) refer to the following: innervated; 40 hr denervated; 40 hr denervated/30 min PMA treated; 40 hr denervated/30 min dimethyl sulfoxide (20 ~1 of 20% in ethanol) treated; and 40 hr denervated/30 min 4a-phorbol (20 pg) treated muscle. Each data point represents the average of l-3 independent measurements; results are normalized to the transcriptional activities in 40 hr denervated muscle.

To block PKC specifically, a different approach was employed. Prolonged treatment with phorbol esters “depletes” PKC (Niedel and Blackshear, 1986); this is also true in skeletal muscle (Figure 6). Pretreatment with phorbol esters thus provides an opportunity to test the involvement of PKC in a signaling pathway, since depletion of the enzyme should result in the interruption of information flow. ‘This is indeed observed. When chronic treatment with phorbol esters precedes electrical stimulation of denervated muscle, the inhibition of the AChRgenes is markedly reduced and in the case of the a subunit hardly observed any longer (Figure 7). Discussion It has

long

been

known

that

stimulus

load

and

pattern

Neuron 674

ioogo80-

P

70.

.e .E 92

6o 50

B 40. 75 L+? 30.

/

20. lo07 t Denervation

DenervationiStimulation

0

Figure 6. Long-Term PKC in the Nuclear

1

0 Figure

alpha1 5. Effect

alpha7

of Staurosporine

delta

gamma

GAPDH

on PKC and Gene

,pomyosln

d

Activity

At 37 hr after denervation, staurosporine (1 pg in 20 PI of 50% phosphate-buffered saline in ethanol [v/v]), or ethanol alone, was injected into the denervated muscle. Three hours later, one group of animals received a 30 min stimulation, whilethecontrol group remained unstimulated before being sacrificed. In a parallel experiment animals received no drug. (A) PKC activity was assayed in the nuclear fraction as described in the Experimental Procedures. The average of two independent measurements is shown, normalized to the PKC activity in denervated/stimulated, drug-free control muscle. (B) The bar graph shows nuclear runon data obtained with probes specific for the indicated AChR subunit genes and for CAPDH and tropomyosin as an internal control. Within each set, results (from left to right) refer to the following: innervated;40 hr denervated;40 hrdenervated/30 min stimulated; 40 hr denervatedi3 hr staurosporine treated; 40 hr denervatedi3 hr staurosporine treated/30 min stimulated muscle; and 40 hr denervatedI3 hr ethanol treated/30 min stimulated muscle. Results of run-on assays are presented as the percentage of gene activity in denervated muscle.

over prolonged periods of time profoundly influence skeletal muscle phenotype (see, e.g., Pette et al., 1976; Heilmann and Pette, 1979; Jolesz and Sreter, 1981). Complete cessation of electromechanical activity as caused by denervation has a pronounced effect on many muscle properties, especially those related to innervation and excitability: sprouting of nearby axons is promoted; the myofiber becomes susceptible to reinnervation; the pharmacology of voltage-gated

0.17

0.5 1 6 Period of treatment

Treatment Fraction

with

16 (h)

Phorbol

40 Esters

72* Depletes

PMA (20 pg) was injected into denervated muscle. Atter the indicated times the animal was sacrificed, and leg muscle nuclei were isolated. Total PKC was assayed in the nuclear fraction as described in Experimental Procedures. Averagesof two Independent measurements are shown and presented as fraction of the PKC level in denervated, drug-free, control muscle. Asterisk indicates that in the 72 hr experiment, intramuscular injections of PMA were repeated at 24 hr intervals, with denervation taking place 32 hr after administration of the first dose ot PMA.

channels and the level of acetylcholinesterase change; and, most conspicuously, extrajunctional AChRs appear. The mechanisms underlying these changes are incompletely understood. Among the “neuronal” properties affected by membrane activity, AChR control has been most intensively studied. These studies established that chronic stimulation of denervated muscle reduces AChR expression (Lomo and Westgaard, 1975), presumably via down-regulation of subunit messages(Goldman et al., 1988). Our goal was to determine whether this downregulation has a transcriptional component and, if so, to identify elements of the signaling pathway linking membrane and genes. The experiments reported here confirm by direct run-on analysis what was previously deduced indirectly from mRNA analysis (Neville et al., 1991, 1992), namely, that AChR gene transcription is rapidly shut off in response to membrane depolarization. Electrical stimulation of denervated muscle, in which AChR subunit genes are actively transcribed, leads within minutes to a shutdown of receptor gene activity. Over the relatively short periods and within the limited ensemble of genes investigated here, the effect is specific for AChR subunit genes. A slight up-regulation of c-fos is apparent, which, in view of the impulse activity-driven stimulation of this gene within the CNS (Sonnenberg et al., 1989), is not entirely surprising. The signaling pathway that conveys information on membrane activity into the cell interior has previously been investigated with muscle cells in vitro. Electrical

Depolarization,

PKC, and Receptor

Gene

Activity

575

Figure 7. Long-Term bol Esters Inhibits Signaling

b

P/S

la a.exon1 2a. trcQonlyti 3& 7-Smmn

alpha1

alpha7

1b. adxon7 2b. ct.aubunll 3b. GAPDH

delta

gamma

GAPDH

stimulation of such cultures inhibits AChR synthesis (Shainberg and Burstein, 1976). Conversely, abolition of spontaneous electrical activity with the sodium channel blocker tetrodotoxin results in enhanced AChR expression. In vitro analysis has also suggested the participation of calcium ions (Pezzementi and Schmidt, 1981; Klarsfeld et al., 1989) and of PKC (Bursztajn et al., 1988; Klarsfeld et al., 1989) in the regulation of AChR in cultured myotubes. Short-term treatment with the PKC-activating phorbol esters reduces AChR expression, while prolonged exposure of cells to phorbol esters-a treatment known to result in loss of PKC-or the administration of the protein kinase inhibitor staurosporine leads to increases in a subunit mRNA and AChR protein (Klarsfeld et al., 1989). While in vitro systems have been of great value for the delineation of regulatory elements involved in myogenic cell differentiation and the accompanying up-regulation of AChR expression, it is less clear that it will be possible to study the phenomenon of denervation supersensitivitywith such preparations. In this paperwefocuson mechanisms underlyingthe regulation of AChR in intact chick muscle and identify PKC as a component in excitation-AChR repression cou-

Treatment with PhorMembrane-Cenome

Transcription elongation was assayed in nuclei isolated from innervated (NJ, 40 hr denervated (D), 40 hr denervatedI30 min stimulated (S), 72 hr PMA treated140 hr denervated (P), and and 72 hr PMA treated/40 hr denervated/30 min stimulated (P/S) muscle using the indicated driver DNAs. (Top panel) Autoradiograms of slot blots. (Battom panel) Quantitative evaluation by means of a beta scanner (see Experimental Procedures). Activities in nuclei isolated from (from left to right) innervated, denervated, denervatedktimulated, PMAtreated/denervated, and PMA-treated/denervatedistimulated muscle are presented as a percentage of gene activity in denervated muscle.

opomyosin

pling. This identification is based on three observations. First, electrical stimulation of skeletal muscle rapidly activates PKC; within the nuclear compartment, enzyme activity, which is barely detectable in unstimulated muscle, rises by two orders of magnitude. A rapid time course is necessary if PKC is to play a mediating role in a response that itself is fast. The time to half-maximal response in PKC activity is approximately6min,whilethe halftimeforAChRgeneinhibition is significantly longer (approximately 15 min), as would be expected if one preceded the other. PKC is activated in all cellular compartments investigated: cytosol, nucleus, and nonnuclear membranes. In contrast to the moderate increase in the nonnuclear fractions, activation of PKC in the nuclear compartment exceeds two orders of magnitude. Whether this is a consequence of activation of an enzyme that resides in the nucleus in an inactive form, or whether PKC is translocated from the sarcoplasm and then activated is not known at present. The finding that subcellular enzymedistribution remains largely unaffected bythe state of innervation or activity of the muscle favors a local activation over a translocation mechanism. PKC,

Nl?UVXl 676

once activated, remains active for extended periods; even 24 hr after a 30 min stimulation, enzyme activity in the nuclear fraction exceeds that of controls (nuclei from denervated, unstimulated muscle) by an order of magnitude. This time frame agrees with the slow return of AChR gene activities to control levels. Second, effects of electrical stimulation on gene activity can be mimicked with PKC activators. Phorbol esters are believed to activate PKC specifically, and administration of phorbol esters results in rapid and specific down-regulation of AChR genes. Since PKC activation is a result of membrane excitation and in turn can cause the same selective gene inhibition as that observed after electrical stimulation itself, we may conclude that it is a component of the signaling pathway that couples the sarcolemma to the genome. Intramuscularly injected phorbol ester does not completeley abolish gene activity, which may be a reflection of the inability of the drug to reach all muscle nuclei subsequently analyzed, or of the AChR genes’ partial independence of the PKC pathway. The latter explanation may also be invoked for the persistence of thelimitedAChRgeneactivityobserved in innervated muscle. Third, inhibition of PKC abolishes excitation-AChR repression coupling. It is important to establish that the effects of phorbol esters are actually mediated by PKC, since, like other drugs, phorbol esters lack perfect specificity. For example, in skeletal muscle they have been shown to beable,at below micromolar concentrations, to bind to and inhibit Mg*+-ATPase located in the transverse tubules (Kang et al., 1991). Two approaches were taken to confirm the role of PKC: blocking the stimulation response with staurosporine, a general protein kinase inhibitor that is unlikely to interfere with the action of some unknown or unrelated phorbol ester target, such as the Mg2’ATPase, and chronic pretreatment with phorbol esters, which leads to a down-regulation of PKC (Niedel and Blackshear, 1986). The link between plasma membrane activity and AChR gene activity has long been suspected, and the effects of either chronic paralysis (induced by drugs or nerve section) or chronic stimulation are amply documented. The present results reveal that electrical activity leads to rapid repression of receptor genes and furthermore indicate that PKC plays a crucial signaling role. What remains unknown now is the mechanisms whereby membrane depolarization activates PKC and how the kinase turns off gene transcription. Regarding the proximal limb of the pathway, it will be important to identify the phospholipase that supplies the diacylglycerol; it may be worth mentioning here that, in chick sympatheticganglion neurons, electrical stimulation results in the hydrolysis of phosphatidylcholine rather than phosphatidylinositol (Wakade et al., 1991). As for the distal segment, previous observations have suggested that an autocatalytic transactivating factor induces the chick muscle AChR a subunit gene (Neville et al., 1991) and possibly they and 8

subunit genes as well. Myogenic transcription factors such as MyoD and myogenin are thought to activate their own genes (Thayer et al., 1989), and it has recently been shown that denervation leads to an up-regulation of MyoD and myogenin (Duclert et al., 1991), which can be prevented by long-term electrical stimulation (Eftimie et al., 1991). An analysis of myogenic factors in the denervated chick muscle has pointed to a special role for myogenin (Neville et al., 1992), and it is therefore tempting to speculate that the phosphorylation of myogenin (or a factor similar to, or upstream of, it) may mediate suppression of extrasynaptic AChR in active muscle. Preliminary data indicate that both MyoD and myogenin gene activities also fall rapidly in response to electrical stimulation. That gene activity should decline so promptly suggests that this long-known effect of membrane activity on AChR expression can be studied in a time frame appropriate to intracellular signaling. The analysis of the effects of electrical activity can now focus on second messenger systems as well as on PKC targets; such studies are currently underway in the laboratory. Experimental

Procedures

Animal Experiments White Leghorn cockerels (Hall’s Brothers Hatchery, North Brookfield, MA), 3-4 days after hatching, were anesthetized with ketamineR intraperitoneally (SO-100 mg/kg), and sectioning of the sciatic nerve was performed as described previously (Shieh et al., 1988); anesthesia was maintained at a surgical level by supplementation as needed throughout the procedure. Unless stated otherwise, denervated muscle refers to the leg musculature between knee and ankle, 40 hr after nerve section, at which time AChRgenes are maximallyactive(Tsayand Schmidt, 1989). Drugs were administered intramuscularly; for the stimulation experiments, the protocol of Lomo and Westgaard (1975) was adopted. Briefly, the denervated leg musculature was stimulated for periods of up to 2 hr in 100 Hz trains, 2 s in duration and applied once every minute. At the desired time animals were killed by decapitation, and the leg musculature was processed immediately for subcellular fractionation and isolation of nuclei. All animal experimentation utilized protocols approved by the Institutional Animal Care and Use Committee. Subcellular Fractionation for PKC Analysis Muscles were homogenized using a Dounce homogenizer in 2 mM EDTA, 10 mM ECTA, 2 mM phenylmethylsulfonyl fluoride, 100 ug/ml leupeptine, and 20 mM Tris-HCI (pH 7.5) (buffer A) and centrifuged at 1000 x g for 10 min to yield a crude nuclear fraction and a supernatant that was subjected to another centrifugation for 1 hr at 100,000 x g; the second supernatant constitutes the cytosol fraction. The high speed pellet was treated for 1 hr at 4OC in buffer A containing 0.1% Triton X-100 and then centrifuged again at 180,000 x g for 1 hr; the resulting supernatant is designated the nonnuclear membrane fraction. The crude nuclear fraction was resuspended in buffer B (60 mM KCI, 15 mM NaCI, 15 mM HEPES [pH 7.51, 2 mM EDTA, 0.5 mM ECTA, 0.15 mM spermine, 0.5 mM spermidine, 14 mM 8mercaptoethanol) containing 10% sucrose, passed through adouble layer of cheese cloth (mesh width, 40 urn) twice, and purified by sedimentation through 30% sucrose in buffer 6 at4000 x g for IO min. The pellet was resuspended in buffer B, containing 0.1% of Nonidet P-40, and recentrifuged through 30% sucrose in buffer B. Nuclei were then processed as described by Fields et al. (1988). Briefly, they were treated with DNAase I and RNAase A, centrifuged, and resuspended in 50 mM Tris (pH 7.5), followed by extraction in 1.6 M NaCl and 140 mM P-mercaptoethanol. Finally, nuclear en-

;)7epolarization,

‘relopes $rt 5000

PKC, and Receptor

and high salt extract x g for 30 min.

were

Gene

Activity

separated

by sedimentation

PKC Assay ~~ytosol, solubilized nonnuclear membrane fraction, and nuclear high salt extract were chromatographed on DEAE-cellulose. Aliquots representing 10% of the samples eluted off DEAEcellulose between 100 and 400 mM NaCl were assayed for PKC, #ISdescribed by McArdle and Conn (1989), using 50 ug of histone ‘1s a substrate. Enzyme activity was expressed as the difference between the histone phosphorylation levels measured in the .Jresence and absence of factors specifically required for PKC 1 mM CaCh, 40 Rg/ml phosphatidylserine, and 4 uglml 1,2-diJlein). To determine the total amount of enzyme present in a :;iven subcellular fraction, PMA was added to the assay cocktail o a final concentration of 5 uM. ‘iynthesis of SingleStranded Antisense DNA C;ene-specific driver DNA, as required for the nuclear run-on experiments, was obtained as follows: A Pstl fragment of pC25.lbgl Wang et al., 1988), which contains 1.4 kb of the a subunit genonit sequence, including exon I and exon II, was inserted into ‘he polylinker of M13mplO in both orientations; it is designated r-exonl in this paper. The Hindlll-EcoRI fragment of pa7, which comprises exon VII of the a subunit gene, was cloned into M13mplO and M13mpll and is referred to as a-exon7; similarly, .he Hindlll-EcoRI fragment of pL3, a plasmid containing 4.8 kb lf the 5’ portion of the 8 subunit gene, including exons I-IV, and the Hindlll-Pstl fragment of pB5, a plasmid containing 0.5 kb of the 5’ region of they subunit gene, including exon I, each were ZIoned into M13mplO and M13mpll (Tsay and Schmidt, 1989; Tsay et al., 1990), giving rise to the single-stranded DNA sequences designated d and c, respectively. A full-length cDNA of chicken 8-actin (2.0 kb; Cleveland et al., 1980), a near full-length :DNA of chicken skeletal a-tropomyosin (the 1.07 kb clone T-15, which lacks 122 nt of the 5’ untranslated region; Gooding et al., 1987J, a BamHl fragment (1.2 kb)of chicken a-tubulin (Valenzuela et al., 1981), and a pSt1 fragment (1.2 kb) of human glyceraldehyde3-phosphate dehydrogenase (GAPDH; K. Marcu, unpublished data) were cloned into M13mp18 in both orientations. The Kpnl-BamHI fragment (565 bp) of the chicken c-fos plasmid pTZlgR/pch-fosdB (Molders et al., 1987), the Sstl-EcoRI fragment of pGFLC6A, which contains 780 bp of chick MLClf cDNAcloned into the EcoRl site of pCEM4 (Zhu et al., 1991), and the Pstl-Sau3A fragment of ki-ras cDNA, which contains 114 bpof exon I and 50 bp of exon o (Nakano et al., 1984), were cloned into M13mp18 and M13mp19. Single-stranded DNA was prepared as described in the Molecular Cloning Manual (Sambrook et al., 1989). Transcript Elongation (Run-on) Analysis Nuclei were isolated and assayed by established procedures (Tsay and Schmidt, 1989). As a rule, eight chicks were used for a nuclear preparation. Animals were sacrificed at the desired time; the shank musculature was dissected free of bone and connective tissue (wet weight, approximately 1 g per animal) and homogenized with a motor-driven tissue grinder (B pestle; Thomas Scientific, Philadelphia, PA) in buffer B (see”Subcellular Fractionation for PKC Analysis”) with 10% sucrose and 1 mM phenylmethylsulfonyl fluoride. The homogenate was filtered twice through a double layer of cheese cloth to remove residual connective tissue, layered over a cushion of 30% sucrose in buffer B, and spun for IO min at 4,000 x g at 4“C. The crude nuclear fraction was resuspended in 0.1% Nonidet P-40 in buffer B, left on ice for 5 min, layered over a cushion of 30% sucrose in buffer B, and recentrifuged. The resulting pellets were resuspended in 5 ml of buffer C (storage buffer: 50% glycerol, 20 mM Tris [pH 7.9],75 mM NaCI, 0.5 mM EDTA, 0.85 mM dithiothreitol, 0.125 mM phenylmethylsulfonyl fluoride), centrifuged for 30 sat 3000 x g at 4OC, and resuspended in 1 ml of buffer C containing 100 U/ml ribonuclease inhibitor. Nuclei were counted in hemocytometer and either used immediately or frozen in liquid nitrogen and stored at -70°C for up to 6 months without loss of activity. Fortranscriptelongation, IO’nuclei were incubated with

150 RCi of [SZP]UTP (3000 Cilmmol) at 26°C for 45 min in 100 ul of reaction buffer (300 mM (NH4),SO,, 100 mM Tris [pH 7.91, 4 mM MgCI,, 4 mM MnCI,, 200 mM NaCI, 0.4 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 1.2 PM dithiothreitol, 1 mM NTP [except UTP], 29% glycerol, and IO mM creatine phosphate). Nascent transcripts weretreated with RNAase-free DNAase(0.4 mg/ ml), followed by proteinase K (0.2 mglml), and purified using phenol-chloroform extraction, centrifugation through a SephadexG-50column, and trichloroacetic acid precipitation. Samples were then exposed to 0.2 M NaOH (10 min; ice bath), followed by quenching with 1 M HEPES (pH 5.5) and ethanol precipitation; without this brief base treatment hybridization signals are 5-10 times weaker. For hybridization, radioactively elongated transcripts were dissolved in small volumes of 50 mM HEPES (pH 7.0), 750 mM NaCI, 50% formamide, 0.5% SDS, 2 mM E.DTA, 10x Denhardt’s reagent, 200 Rglml salmon sperm DNA, and aliquots of 2 x IO6 cpm [32P]RNA were reacted for 15 hr al 42°C in small culture dishes with sections of nitrocellulose filters containing singlestranded DNA probes at 10 ug per slot. Results were visualized by autoradiography and quantified using a beta scanner (Ambis, San Diego, CA). To permit comparison of different experiments, genes little affected by the experimental manipulations (tropomyosin and GAPDH) were included as internal controls in the analysis. Drugs and Reagents Staurosporine, PMA, (Folch fraction III from histone type III-S were

4a-phorbol, crude phosphatidylserine bovine brain), diolein, and calf thymus purchased from Sigma (St. Louis, MO).

Acknowledgments We thank Marlies Schmidt for expert technical assistance, Marc Ballivet (Geneva, Switzerland) for the chick AChR plasmids, Patrick Hearing (Stony Brook, NY) for 8-actin cDNA, Steve Hughes (Frederick, MD) for the chicken skeletal muscle a-tropomyosin clone T-15, Michael Crow (Baltimore, MD) for chicken MLClf cDNA, Martin Zenke (Vienna, Austria) for the chicken c-fos plasmid pTZlgR/pch-fosdB, and Ken Marcu (Stony Brook, NY) for the human GAPDH clone. This research was supported in part by National Institutes of Health grant NS20233, grant BNS8819383 from the National Science Foundation. and a grant from the Muscular Dystrophy Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisemenP in accordance with 18 USC Section 1734 solely to indicate this fact. Received

April

30, 1992; revised

July 15, 1992.

References Axelsson, denervated 178-193.

J., and Thesleff, mammalian

S. (1959). A study of supersensitivity skeletal muscle. J. Physiol. (Land.)

Bursztajn, S., Schneider, L. W., Jong, Y. J., and (1988). Phorbol esters inhibit synthesisofacetylcholine in cultured muscle cells. Biol. Cell 63, 57-65.

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