Effect of calcitonin gene-related peptide on skeletal muscle via specific binding site and G protein

Journal of the Neurological Sciences, 1989, 90:99-109 Elsevier

99

JNS 03135

Effect of calcitonin gene-related peptide on skeletal muscle via specific binding site and G protein Masaharu Takamori and Hiroaki Yoshikawa Department of Neurology, Kanazawa University School of Medicine, Kanazawa 920 (Japan) (Received 26 July, 1988) (Revised, received 14 November, 1988) (Accepted 17 November, 1988)

SUMMARY

In curarized rat skeletal muscle, rat calcitonin gene-related peptide (CGRP), a peptide coexisted with acetylcholine in the motor nerve terminal, increased the isometric twitch force, accompanied by an increase in the active state intensity of shortening, prolonged duration of the active state and additive effect of a phosphodiesterase inhibitor; the results reflect a potentiation in the sarcoplasmic calcium transport system. This CGRP effect was enhanced by cholera toxin, suggesting the activation of guanine nucleotide binding regulatory protein (G protein) that stimulates adenylate cyclase (Gs). The pertussis toxin (IAP), a factor to prevent the cyclic AMP decrease by inactivating the G protein that inhibits adenylate cyclase (Gi), provided no effect on the action of CGRP. The existence of CGRP binding site in the sarcolemmal membrane was confirmed by Scatchard analysis of binding data; affinity of the binding site for CGRP was decreased in the presence of guanosine-5'-[ ~,-thio]triphosphate (GTP ~,S). The Gs protein is thus implicated in the CGRP binding site and intraceUular processes of signal transduction. CGRP did not modify the neuromuscular transmission and cable properties of the muscle membrane.

Key words: Calcitonin gene-related peptide; Muscle contraction; Sarcoplasmic calcium transport; Guanine nucleotide-binding regulatory protein; Cyclic AMP

Correspondence to: Masaharu Takamori, M.D., Department of Neurology, Kanazawa University School of Medicine, 13-1, Takara-machi, Kanazawa 920, Japan. Tel: (0762)62-8151. 0022-510X/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

100 INTRODUCTION Recent evidence indicates that polypeptides are present in motor neurons where classical neurotransmitters also exist. The significance of such coexistence of peptides and transmitters is unclear, but the concept of multiple synaptic messengers may have implications for our understanding of pathophysiology in neuromuscular diseases (H0kfelt et al. 1986). Calcitonin gene-related peptide (CGRP) is a 37-amino acid residue peptide which can be transcribed by the same gene that encodes calcitonin (Rosenfeld et al. 1983). CGRP colocalizes with acetylcholine in neurons of motor systems (Fontaine et al. 1986; New and Mudge 1986; Takami et al. 1985a,b), and may thus serve as a coexisting neuronal messenger. CGRP increases the surface nicotinic acetylcholine receptor (Fontaine et al. 1986; New and Mudge 1986) and its ct-subunit mRNA (Fontaine et al. 1987), and also enhances skeletal muscle contraction (Takami et al. 1985b). Effects may be mediated by activated adenylate cyclase (Laufer and Changeux 1987; Kobayashi etal. 1987) and increased cyclic AMP (Laufer and Changeux 1987; Takami et al. 1986). The present study is to identify the CGRP binding site and intracellular pathway of CGRP action in the mammalian skeletal muscle contraction.

MATERIALSAND METHODS

Chemicals Synthetic rat-CGRP was obtained from the Peptide Institute Inc., 4-1-2, Mino-shi, Osaka, Japan 562.

Muscle contraction study Segmented diaphragms (19-28 mg as dry weight) were obtained from young adult male rats of the Wistar strain. Muscles were bathed in mammalian Ringer solution which was composed of 115-125 mM NaC1, 4.7 mM KC1, 15.5 mM NaHCO 3, 1.2 mM MgC12, 1.2 mM KH2PO4, 2.6 mM CaC12 and 11.5 mM glucose, and aerated with 02 + CO2 (95 : 5) at 37 ° C. They were massively stimulated by platinum plate electrodes to avoid inhomogenous activation, and were connected to a straha gauge and carrier amplifier for isometric recording. Direct stimulation with a rectangular pulse of 0.5-msec duration was assured by the curarization of the muscle (( + )-tubocurarine chloride, 6 #g/ml). Resting tension was set to obtain a maximal twitch. Tetanus was elicited by 166 Hz repetitive stimulation. Contractile responses were not altered in the control Ringer solution as long as the incubation period of 60 rain. In analysis of the muscle contraction, the 1st and 2nd derivatives of tension were recorded by differentiation (Takamori et al. 1981). Based on the concept of the active state (Close 1972; Takamori et al. 1978, 1981), the time component, reflecting kinetics of cross-bridge turnover modulated by activator Ca 2 + and troponin, was expressed by measurements of the duration from onset to point of maximum velocity of twitch development (T~,t/clt) and the time from onset to the half-relaxation level of twitch (T~/2a) (Takamori et al. 1981)

101 (Fig. 1). The active state intensity of shortening was measured by the maximum acceleration of twitch development (d2pt/dt2) to express the amount of sarcoplasmic Ca 2 + released in the initiation of excitation-contraction coupling; the intensity of load-bearing to express number and strength of actin-myosin cross-bridges was determined by maximum tetanic force (Takamori et al. 1981) (Fig. 1). In the pharmacologic study, CGRP was added to the Ringer solution in a concentration of 10 - 7 M; twitch and tetanus were recorded before and 10 min after the addition of this peptide. Theophylline was dissolved in a concentration of 2 x 10- 3 M alone or in combination with CGRP (10 - 7 M); each effect on contractile properties was estimated after 10 min incubation. To estimate the modification by an activator of adenylate cyclase, the muscle was pretreated with the solution containing cholera toxin (Sahyoun and Cuatrecasas 1975) in a concentration of 10 - 7 M for 1 h, and then treated with CGRP (10- 7 M) for 10 min. The effect of CGRP (10 - 7 M) treatment for 10 min was also studied in the muscle obtained from rats which were intravenously injected with pertussis toxin (IAP) (5#g/kg) 3days before sacrifice (Yajima etal. 1978; Nogimori et al. 1984).

control

CGRP (lO-rM)

pt

I2g s., ~

]l~"~sec :

dPt/dt

i'

':i::-i-i i i

~!\~

,

: ik

i

i i [0"3g//msec ~

dai~/dt •

msec

05g/msec =

90 msec

Po

~lOg . . . . . . . . . . . . . . . . . . .

~msec

Fig. l. Isometric twitch (Pt) and tetanus (1'o) analyzed by differentiations in control and 10 -7 M CGRPtreated muscle. Measurements of each of the active state properties are depicted. Contractile responses were elicited by massive stimulation in the solution containing +-tubocurarine chloride (6 #g/mi). Twitch potentiation by 10 min immersion with CGRP was associated with increased d2Pt/dt 2, and prolongation of Trip,/d, and T1/2R.

102

Microelectrode study Conventional intracellular microelectrodes were used to record the resting membrane potential, input resistance, miniature endplate potential (MEPP) and endplate potential (EPP, recorded in the bathing fluid containing +-tubocurarine chloride). Amplitude of the latter two were corrected for nonlinear summation and calculated for a standard resting membrane potential of - 7 7 mV. Acetylcholine (ACh) quantum content at 1 Hz stimulation was determined from the coefficient of variation of the EPP amplitude distribution (Takarnori et al. 1983). Immediately releasable ACh store and ACh mobilization rate during prolonged stimulation at 100 Hz were estimated according to the method of Lambert et al. (1976). On condition that the muscle was immersed in the solution containing dantrolene sodium (20 #g/ml) to avoid muscle contraction (Takamori et al. 1983), action potential was directly elicited by the stimulating microelectrode that was in touch with the muscle fiber to which the recording microeleetrode was inserted; its 1st derivative (dV/dt) was recorded by differentiation. These electrophysiologic measurements were done in the control Ringer solution and in the solution containing CGRP (10- 7 M), respectively.

Biochemical study Isolation ofsarcolemmal membrane. A modification of the method of Mickelson and Louis (1985) was used to isolate skeletal muscle sareolemma from young adult male rats of the Wistar strain. All procedures were performed at 0-4 °C. One hundred grams of hind-leg muscles were minced and homogenized.in 500 ml of 250 mM Tris-HC1, pH 7.5, 1 mM EDTA by Ultra-Turrax. The homogenate was centrifuged at 2000 × g for 20 min and the resulting pellet was resuspended in 500 ml 0.4 M LiBr, 20 mM Tris-HCl, pH 8.5. The suspension was stirred slowly overnight; this was then centrifuged at 1000 × g for 15 min, followed by centrifugation of the resultant supernatant at 10000 × g for 30 min to yield a crude membrane pellet. The pellet was resuspended in 50 ml 0.6 M KC1, 20 mM Tris-HC1, pH 8.0, by 8 strokes in a loose-fitting Dounce homogenizer and centrifuged at 10000 × g for 10 min. This supernatant was retained while the pellet was extracted twice more with the KCI-Tris medium. Three resultant supernatants were combined and centrifuged at 100000 x g for 30 rain. The pellet was resuspended in 0.6 M KC1, 20 mM Tris-HC1, pH 8.0, and further purified by differential centrifugation between 10000 × g (10 rain) and 100000 x g (30 min). The final 100 000 × g pellet was resuspended in distilled water and centrifuged at 100 000 x g for 30 min. This pellet was resuspended in a small volume of water; the volume was measured; an equal volume of 57~ sucrose (w/v), 20 mM histidine, pH 7.0, was slowly added. This suspension, transferred to centrifuge tubes and owrlaid with 15 ~o sucrose, 10 mM histidine, pH 7.0, was centrifuged at 150000 x g for 90 rain in an anglehead rotor. The band which was formed at the 28.5-15~ sucrose interface was collected, diluted with 4 vol of water, and centrifuged at 100000 x g for 30 min. This pellet was resuspended in a minimal volume of 10~o sucrose, aliquoted into small vials, frozen and stored at - 80 °C until use. Binding study. The binding assay was carried out according to the method of Fisher et al. (1981) and Henke et al. (1985). An assay tube contained the membrane

103 preparation (50-210 #g of protein) and 50 mM Hepes/Tris buffer, pH 7.4, with 1~o bovine serum albumin in a total volume of 0.5 ml. The reaction mixture was further supplemented with various concentrations of non-radioactive CGRP (with constant specific radioactivities) for the displacement study. For the measurement of kinetic parameters of binding, [ 125I]CGRP was added in various concentrations to the assay tube in the absence (total binding) or presence of 3 #M non-radioactive CGRP (nonspecific binding). Guanosine 5'-[ ),-thio]triphosphate (GTP~S, 100/~M) was added to the assay tube in association with MgCI2 (25 mM) to minimize the dissociation of GTP 7S from the 0t-subunit of guanine nucleotide-binding regulatory protein (G protein) that stimulates adenylate cyclase (Gs) (Higashijima et al. 1987). Incubation was allowed to proceed at 4 °C for 4 h and stopped by dilution with a 10-fold volume of buffer, followed by F~tration through a glass t'flter (Whatman GF/C) pretreated with polyethylenimine (Bruns et al. 1983). The filter was washed with 5 ml ice-cold buffer twice and counted in a gamma-counter. Protein was quantified using bovine serum albumin according to the method of Lowry et al. (1951). RESULTS

Immersion of the curarized muscle with the CGRP-containing solution for 10 min resulted in the potentiation of twitch (Pt), accompanied by increased acceleration of twitch development (d2pt/dt2), and prolonged time course (Tdet/dt and T~/2R) (Fig. 1 and Table 1). No change occurred in the tetanic force (Po) (Fig. 1). The CGRP-induced Pt potentiation was further augmented when the muscle was treated in combination with theophylline; this effect was on the top of the potentiation caused by theophylline alone (Fig. 2 and Table 2). The preincubation of muscle with cholera toxin caused the CGRP effect to further enhance, while the pretreatment with pertussis toxin (IAP) provided no effect on CGRP-induced Pt potentiation (Fig. 2 and Table 2). None of these pharmacological manipulations modified the tetanic force (Figs. 1 and 2). Microelectrode studies showed no significant effect of CGRP on synaptic functions estimated by MEPP and EPP, and on passive and active membrane properties estimated by resting membrane potential, input resistance, and directly elicited muscle action potential and its 1st derivative (dV/dt) (Fig. 3). Specific binding of CGRP with the membrane was demonstrated by the displacement of [ ~25I]CGRP by nonradioactive CGRP applied to the sarcolemmal memTABLE 1 E F F E C T O F C G R P (10 - 7 M) O N ACTIVE STATE P R O P E R T I E S D E T E R M I N E D BY T W I T C H ANALYSIS (%) Mean + SD values are indicated; 7 muscles were tested. An example is shown in Fig. 1.

Pt

dPt/dt

d2pt/dt 2

Tean/d,

TIleR

105 + 2

110 + 2

105 + 3

109 + 5

113 + 7

104

CGRP (lO-rM)

and

Control (pretreated with cholera toxin IO-TM, 1 hr.) C G R P ( l O - r M )

Control (pretreated with

t~.t~RP ( l l 3 - r M )

Fig. 2. Alterations of twitch (Pt) by pharmacologic manipulations to modify CGRP effect. The CGRPinduced Pt potentiation was augmented by theophylline and cholera toxin, but was not modified by pertussis toxin (IAP). No change was seen in tetanus (Po).

105 brahe; non-specific binding was approx. 299"0 of the total binding (Fig. 4A). Scatchard analysis of binding data revealed single classes of binding sites; the maximal binding (Bmax) of [ ~25I]CGRP was 94 fmol/mg of protein, and the binding affinity (Kd) was 6.3 nM (Fig. 4B). Affinity of the binding site for CGRP on the sarcolemmal membrane was measured based on various concentrations of [125I]CGRP; this was decreased in the presence of GTPyS (with Mg2+) (Fig. 5). TABLE 2 EFFECTS OF PHARMACOLOGIC AGENTS ON TWITCH (Pt) (%) Mean ± SD values are indicated; 6 muscles were tested for each. An example is shown in Fig. 2. CGRP (10 - 7 M) and theophylline (2 x 10 -3 M) Theophylline (2 x 10-3 M) alone CGRP (10 - 7 M) and cholera toxin (10 -7 M) CGRP (10- 7 M) and pertussis toxin (IAP) (5 #g/kg)

125 ± 112 ± 130 ± 105 ±

2* 3** 1'** 2****

* More potentiated than CGRP (Table 1) or theophylline alone (P < 0.001); ** less potentiated than CGRP plus theophylline (P < 0.001); *** more potentiated than CGRP alone (Table 1) (P < 0.001); **** not significantly different from CGRP alone (Table 1).

Control

CGRP (10-7M)

N(purornuscular transmission EPP, ACh quantum content at 1 Hz

233~:59 (r,:~5)

2 4 7 ± 6 1 (~181

Immediately releasable ACh store (quanta)

1340:1:445 (n:151

1 4 1 8 ~ 3 9 9 (~8~

5 5 3 4 ~ 7 5 0 (n:15)

6672~342

ACh-mobilization rate at 100 Hz (quanta per second) MEPP amplitude (mY) MEPP frequency (per second) Passive and active membrane properties Resting membrane potential (mY) Input resistance (MI3.)

0.75~0.15 (n:20)

(n:18)

0.74~0.17 (.:20)

4.3¢ 1.7 (n:20)

4.8¢ 1.0 (n:2O)

77=: 2.3 (n:2o)

77~ 2.3 (r=201

O. 18~0.06 <~ao)

O. 18±0.07 (~2o~ ( mean = S.D.)

Directly elicited action potential and first dedvative (dV/dt)

Fig. 3. Pre- and post-synaptic functions of neuromuscular transmission, and passive and active membrane properties in control and CGRP-treated muscle. Directly elicited muscle action potential and its 1st derivative (dV/dt) are shown as examples of results from 15 fibers in each muscle. Synaptic and cable properties were all unaffected by CGRP.

106

A

100

== ~.~_

80

._~ ~

40

20 0

"I~ 0

I 12

I 11

I1 10

-LOG (CGRP)

I 9

I 8

, 7

(M)

B 0.03 KO= 6.3 nM

LU 0.02 I.U

a z °m 0.Ol

o0

I

50 BOUND (fmol/mg protein)

1O0

Fig. 4. (A) Specificity of ['2sI]CGRP binding. Displacement of [12~I]CGRP binding by nonradioactive CGRP in the sarcolemmal membrane. Values represent means of duplicate determinations. (B) Scatchard plots of [~25I]CGRP binding in the sareolemmal membrane. Each point is the mean of duplicate determinations.

DISCUSSION

In the neuromuscular preparation from mammalian skeletal muscle, synaptic functions and cable properties of muscle membrane were all unaffected by CGRP (Fig. 3), so that the twitch potentiation induced by CGRP can be referred primarily to the contractile machineries modified by this peptide. Differential analysis of potentiated twitch showed an increase in the active state intensity of shortening and prolongation of the active state duration (Fig. 1 and Table 1). In view of the concept ofthe active state (Close 1972; Takamori et al. 1978, 1981), tla~e results suggest that CGRP stimulates the sarcoplasmie Ca2 ÷ pump, increatfin8 the-by the concentration of Ca 2 ÷ within the sarcoplasmic retieutum; the extra Ca2+ when released during subsequent activation may enhance the twitch tension (Gonzalez-Serratos et al. 1981). This effect may be mediated by cyclic AMP as assumed from the epinephrine effect on twitch (Gonzalez-Serratos et al. 1981; Takamori et al. 1978, 1981; Bowman et al. 1974), and as supported by evidences that CGRP increased cyclic AMP level in the muscle

107 2O

"-o~

irl the absence of G'TTJ[S

-S E v

10

7 en ~

5

O I

~'~

in the presence of GTP~S

0

0.,

°'.2 ('=l]-C

°'.3 GRP

0'.,

°'.5

(nM)

Fig. 5. Affinityspecificfor [125I]CGRPbinding in the presence or absence of GTP),S (100#M). 25 mM MgCI~ was added to the assay tube to minimize the dissociation of GTPTS from the e-subunit of Gs protein.

(Takami et al. 1986), and the CGRP effect on twitch was additive to the effect of a phosphodiesterase inhibitor (Fig. 2 and Table 2). Further details ofintracellular pathway in the action of CGRP were demonstrated by the experiment using cholera toxin and pertussis toxin (IAP). Enhancement of the CGRP effect on twitch by cholera toxin (Fig. 2 and Table 2) suggests that the CGRP increases cyclic AMP mediating through guanine nucleotide-binding regulatory protein (Gs)-mediated activation of adenylate cyclase (Moss and Vaughan 1981). Pretreatment of the muscle with pertussis toxin (IAP) did not modify the CGRP effect (Fig. 2 and Table 2), suggesting that the CGRP effect is not mediated through the prevention of a decrease in cyclic AMP by inactivating the inhibitory guanine nucleotide-binding regulatory protein (Gi) (Kurose and Ui 1985). The existence of specific CGRP binding site in the sarcolemmal membrane was confirmed by the significant displacement of [z25I]CGRP by nonradioactive CGRP (Fig. 4A) and also by Scatchard analysis of the binding data (Fig. 4B). Such data were similar to those of cerebellum, liver and spleen (Nakamuta et al. 1986). Decreased affinity of the binding site for CGRP in the presence of GTP~S (Fig. 5) suggests that the occupancy of the guanine nucleotide-binding site on G protein by GTP ?S lowers the affinity of coupled receptor for CGRP (Lefkowitz et al. 1976; Maguire et al. 1976).

ACKNOWLEDGEMENTS This study was supported by grants from the National Center for Nervous, Mental and Muscular Disorders and the Neuroimmunological Disorders Committee of the Ministry of Health and Welfare, and from the Ministry of Education (63480215).

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