European Journal of Pharmacology, 60 (1979) 41--46 © Elsevier/North-Holland Biomedical Press

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MK-771-INDUCED ELECTROMYOGRAPHIC (EMG) ACTIVITY IN THE RAT: COMPARISON WITH THYROTROPIN RELEASING HORMONE (TRH) AND ANTAGONISM BY NEUROTENSIN GEORGE G. YARBROUGH and JODIE C. MCGUFFIN-CLINESCHMIDT

Merck Institute for Therapeutic Research, West Point, Pennsylvania 19486, U.S.A. Received 13 March 1979, revised MS received 12 June 1979, accepted 30 August 1979

G.G. YARBROUGH and J.C. MCGUFFIN-CLINESCHMIDT, MK-771-induced electromyographic (EMG) activity in the rat: comparison with thyrotropin releasing hormone (TRH) and antagonism by neurotensin, European J. Pharmacol. 60 (1979) 41--46. Administered by either intravenous (i.v.) or intracisternal (i.cis.) injections, MK-771 and TRH induced a doserelated increase in EMG activity recorded from the flexor ulnaris muscle in pentobarbital-anesthetized rats. By the i.v. route, MK-771 was 6 times more potent than TRH and with i.cis, administration MK-771 was some 30 times more active than TRH. At equieffective doses of the two peptides, MK-771 exhibited a greater (approximately 3 fold) duration of action than TRH. In unanesthetized, spinally transected rats MK-771 was also more potent than TRH in eliciting EMG activity recorded from the biceps femoris muscle. Substance P, administered by the i.cis route failed to induce EMG activity. Intracisternally administered neurotensin, which did not affect EMG activity by itself, antagonized the actions of MK-771 while somatostatin was inactive in this regard. Neurotensin did not affect the EMG activity induced by physostigmine. While these studies do not delineate the mechanism whereby TRH and MK-771 induce EMG activity, it appears reasonable to suggest that TRH and related peptides, such as MK-771, may have some influence in functional disorders of human muscle.

MK-771

Thyrotropinreleasinghormone

Neurotensin

EMG

1. I n t r o d u c t i o n

2. Materials a n d m e t h o d s

T R H (pyroglutamyl-histidyl-prolineamide) is an endogenous neuropeptide which upon exogenous administration produces multiple biological actions in experimental animals and in m a n (cf. Iversen et al., 1978). It has recently been reported that T R H induces an elevation of muscle tonus in decerebrate cats via an action on the spinal cord (Cooper and Boyer, 1978). W e have taken advantage of this finding to examine and compare with T R H the effects of'a novel T R H analog, M K 771 (pyro -2~aminoadipyl-histidyl-thiazolidine4~:arboxamide) on the E M G activity of anesthetized and unanesthetized, spinally transected rats. Additionally, studies were undertaken to examine the possible effects of other neurally active peptides and their interaction with MK-771 on E M G activity.

2.1. Met hods F e m a l e , S p r a g u e - D a w l e y rats ( 1 8 0 - 2 1 0 g) w e r e used in t h e s e studies. M o s t o f t h e e x p e r i m e n t s w e r e c o n d u c t e d o n animals a n e s t h e tized w i t h p e n t o b a r b i t a l N a ( N e m b u t a l , 50 m g / k g i.p.). In studies designed t o e l i m i n a t e a possible a n t i a n e s t h e t i c c o m p o n e n t o f t h e peptides on EMG activation, complete midt h o r a c i c spinal t r a n s e c t i o n s w e r e p e r f o r m e d o n animals lightly a n e s t h e t i z e d w i t h ether. T h e animals w e r e a l l o w e d to r e c o v e r 2 - - 4 h p r i o r t o r e s t r a i n t (with t a p e a n d a small anim a l h o l d i n g cage) a n d r e c o r d i n g o f E M G a c t i v i t y . T h e p e p t i d e s w e r e dissolved in sterile saline (0.9%) a n d i n j e c t e d e i t h e r i n t r a v e n o u s l y ( t h r o u g h a jugular c a n n u l a e o r a tail vein: 0.1 m g / 1 0 0 g b o d y weight) or i n t r a c i s t e r n a l l y

42

(i.cis.; 5 /~l injection volume) with a Hamilton syringe. In studies examining the relative potencies of MK-771 and TRH, EMG activity was recorded for 30 rain following the injection. Each rat was used only once as tachyphylaxis to the musculotonic actions of T R H has been reported (Cooper and Boyer, 1978). EMG activity from either the flexor ulnaris muscle of the forelimb or the biceps femoris muscle of the hindlimb (Green, 1963) was recorded on a Grass polygraph and quantitated through the use of a Model 7P10B integrator. The differentially amplified (through a Textronix AM 502 amplifier) EMG analog signals from needle electrodes (approximately 6 mm apart) inserted into the muscles were fed into the intergration channel which automatically reset after a predetermined voltage amplitude was reached. The amplification of the EMG activity of each rat was adjusted so that the control (predrug) activity was constant (i.e. one deflection of the integrator pen approximately every 10 min). In the studies on the duration of drug action, EMG activity was recorded until it had subsided to a level 3 times the preinjection activity (i.e. one pen deflection within 3 min). In studies involving the administration of more than one c o m p o u n d , baseline EMG activity was amplified so that one deflection of the integrator pen occurred every 3-5 min. Drug-induced EMG activity was recorded for up to 30 min or if it occurred sooner until it had subsided to a level 3 times that of the control period. In studies employing physostigmine, a tracheal cannula was inserted after the induction of anesthesia, and the animals were artifically respirated with a Harvard small animal respirator.

2.2. Materials The following c o m p o u n d s were used: T R H and MK-771 (supplied b y the Department of Medicinal Chemistry, Merck Institute for Therapeutic Research, West Point, Pennsylvania), neurotensin, Substance P, somatostatin (Beckman) and physostigmine sulfate (K & K Laboratories).

G.G. YARBROUGH, J.C. MCGUFFIN-CLINESCHMIDT

3. Results

3.1. Comparison o f MK-771 and TRH An example of the EMG activity induced b y MK-771 and T R H is shown in fig. 1. The integrated EMG responses recorded for 30 min after intracisternal (fig. 2A) or intravenous (fig. 2B) administration of the peptides in anesthetized rats clearly revealed the greater p o t e n c y of MK-771 compared to T R H in evoking this activity. After both routes of administration, T R H and MK-771 elicited dose-related increases in EMG activity. Intracisternally administered MK-771 was some 30 times more potent than T R H (95% confidence limits 5.9--168.4). After intravenous administration, MK-771 was 6 times more potent than T R H (95% confidence limits 3.2--15.0). The limited range of doses studied and the large variability in the magnitude and onset of the EMG response (particularly after intracisternal injections of the peptides) render us reluctant to draw any firm conclusions as to the relative potencies of the two peptides with intracisternal as opposed to intravenous administration. However, it is clear that b y examining the threshold doses required for T R H or MK-771 to elicit an activation of muscle activity, both peptides are at least 1000 times more active when given intracisternally as opposed to intravenous administration. In separate studies the duration of the peptide evoked EMG activity was examined. Employing equal (on a mg basis) doses of the peptides in b o t h barbiturate anesthetized (fig. 3) and in unanesthetized spinalized (fig. 4) rats, MK-771 consistently evoked a longer lasting increase in EMG activity than did TRH. The possibility that this longer duration of action by MK-771 was simply a reflection of its greater p o t e n c y was n o t confirmed in a separate experiment comparing the duration of action of equiactive doses of the t w o peptides. Thus, a dose of MK-771 (0.4 mg/kg) and T R H (3.0 mg/kg), b o t h of which elicited approximately 60 pen deflections within 30

43

MK-771 AND TRH-INDUCED EMG ACTIVITY MK-771 (250 &kg

TRH

(1.5

mg/kg

i.v.)

i.v.)

I

IO uv

MIN.

Fig. 1. Examples of the EMG activity (in the flexor ulnaris muscle of the forearm) induced by intravenous administration of MK-771 and TRH to anesthetized rata.

min after i.v. injection (see fig. 2B) were examined for their duration of action. The results showed that 0.4 mg/kg of MK-7J elicited a response lasting 41.8 + 3.1 (X f S.E.M.; n = 6) min whereas the response-to 3.0 mg/kg of TRH lasted 13.0 f 0.5 (X r S.E.M.; n = 6) min. These results demon-

o.dol

0.000 I

0.125 0.25 0.5

cg/5pI

(i. cis.)

2.0 4.0 (i.v.)

mg/kg

0.5

Fig. 2. Dose response effects of intracisternally (A) administered MK-771 intravenously (B) and on EMG activity in (-) and TRH (o-----~) anesthetized rats. The points represent the mean *S.E.M. of the number of peaks (pen deflections of the integrated EMG activity) recorded’ from the flexor ulnaris muscle within the 30 min subsequent to the injection of the peptitle. The lines were fitted by linear regression analysis and the relative potencies determined by probit analysis and documented in the text determined to be statistically~ (P < 0.05) significant for both routes of administration. Number of animals tested at each dose of each pepkide.in parentheses. Ordinate: integrated EMG activity, within 30 min after injection.

44

G.G. YARBROUGH, J.C, MCGUFFIN-CLINESCHMIDT 50-

220200 180 160

140 120

m

tO0 80 60

40

I./ /i t1 lJ lJ lJ lJ 11 11 fJ lj 11 lJ 11

B.

40-

fJ fi lJ fJ fJ fJ fJ fJ fJ fJ fJ fJ fJ

30-

20-

II

20

A.

I0-

F'?L 0125

025

05

2.0

zJ fJ

4.0

2.0 mo/kg,i.v.

2.0 mg/kg,i v mg/kg ( i v )

Fig. 3. Duration of intravenously-administered MK771 (striped bars) and TRH (open bars) induced EMG activity in anesthetized rats (recorded from the flexor ulnaris muscle). The lowest dose of MK-771 examined (0.125 mg/kg) and that of TRH (0.25 rag/ kg) were ineffective and the bars shown at these doses represent control activity for 30 rain. With active dose of the peptides, EMG activity was recorded until it returned to three times that of the preinjection control activity, n = 4 animals with all treatments. The bars represent the mean ±S.E.M. of the duration of the integrated EMG activity induced by the peptides. The asterisks in this and fig. 4 indicate a significant (P < 0.05) difference between the effect of MK-771 and TRH (Student's t-test). Ordinate: duration of EMG activity (rain).

Fig. 4. Duration of EMG activity induced by 2.0 mg/ kg i.v. of MK-771 (striped bars) and TRH (open bars) in barbiturate-anesthetized (A) and unanesthetized, spinalized (B) rats. EMG activity was recorded from the biceps femoris muscle of the hindlimb in these experiments. The bars represent the mean ±S.E.M. of the duration of the integrated EMG activity until it had recovered to three times that of preinjection control activity (as in the experiments described above in fig. 3). n = 8 animals with all treatments. Ordinate: duration of EMG activity (min).

TABLE 1 Antagonism ofMK-771-induced EMG activity by neurotensin 1 Treatment 1

Treatment 2 2

EMG activity 3 + S.E.M.

(1) (2) (3) (4) (5) (6) (7) (8) (9)

Saline (i.cis.) Neurotensin (20 pg/i.cis.) Saline (i.cis.) Neurotensin (5 pg/i.cis.) Neurotensin (20 pg/i.cis.) Somatostatin (5 pg/i.cis.) Saline (i.cis.) Neurotensin (20 pg/i.cis.)

6.2 6.1 101.0 72.3 42.8 126.1 64.6 57.3 6.3

Saline (0.2 ml, i.v.) Saline (0.2 ml, i.v.) MK-771 (0.5 mg/kg i.v.) MK-771 (0.5 mg/kg i.v.) MK-771 (0.5 mg/kg i.v,) MK-771 (0.5 mg/kg i.v,) Physostigmine (2.5 mg/kg i.v.) Physostigraine (2.5 mg/kg i.v.) Substance P (2.5 pg/i.cis.)

+ 0.1 + 0.1 + 7.8 4 -+ 5.9 + 3.5 s -+ 7.4 + 15.5 + 10.6 + 0.2

I Rats were anesthetized 20 rain prior to Treatment 1 with pentobarbital Na (50 mg/kg i.p.), n = 6-8 per treatment. 2 5 rain after Treatment 1. 3 Number of integrated EMG activity pen deflections recorded within 30 rain after Treatment 1 or until activity had subsided to a level 3 times that of preinjection period. 4 p < 0.001 compared to Group 1 (t-test). s p < 0.01 compared to Group 3 (t-test).

MK-771 AND TRH-INDUCED EMG ACTIVITY strate that in this test MK-771 exhibits approximately a 3 fold (P < 0.01; Student's t-test) longer duration of action than TRH.

3.2. Drug interaction studies Neurotensin, administered i.cis, antagonized in a dose-related fashion the increase in EMG activity induced b y MK-771 (table 1). Neurotensin b y itself had no effect on EMG activity nor did it attenuate the EMG activity induced b y physostigmine. Somatostatin, in the highest sublethal dose tested, did not offset MK-771 induced EMG activity. Substance P, administered i.cis, at a dose of 2.5 ~g, failed to alter EMG activity b y itself and was not tested for an interaction with MK771.

4. Discussion These studies clearly compliment the previously reported stimulation b y TRH of muscle tonus in decerebrate cats (Cooper and Boyer, 1978). The greatly enhanced potency, as judged b y threshold doses required to elicit an effect on EMG activity, of T R H and MK-771 when given directly into the cisterna magna, is consistent with the notion that this action of these peptides is mediated via an action on the central nervous system (CNS). The demonstration of a greater potency and duration o f action o f MK-771 as compared to T R H in stimulating EMG may be of potential significance. The increased potency of systemically administered MK-771 over TRH has been a consistent finding in all other CNS test systems when the potencies of the two peptides have been compared (Veber et al., 1976; Porter et al., 1977; Yarbrough, 1978). This is, however, the first clear demonstration of a substantially longer duration of action of MK-771 as compared to TRH. In the initial studies in barbiturate~anesthetized rats, the longer duration of action of MK-771 might have been to some extent a reflection o f the greater activity of MK-771 against barbiturate

45

sedation as compared with T R H (Porter et al., 1977). Thus, E M G activation would clearly be a component of the reversal of barbiturate sedation and we did not conduct the experiments until E M G activity, induced by the peptides, had returned completely to preinjection control levels. However, the demonstration of a greater duration of E M G activation by MK-771 over T R H in unanesthetized, spinalized animals appears to obviate such concerns and suggests that this difference between these peptides is not strictly related to their relative potencies as barbiturate "antagonists". Furthermore, the enhanced duration of action of MK-771 is not a simple reflection of its greater potency since at approximately equipotent doses MK-771 still exhibited a longer duration of E M G activation than TRH. The basis for the enhanced activity of MK-771 over T R H in this experimental paradigm or other test procedures is not presently known. With regard to the possible mechanism mediating the peptide-induced increase in E M G activity, it has been reported that T R H has a predominantly direct depolarizing action on spinal motoneurons of the amphibian spinal cord (ASC) (Nicoll, 1976). W e have confirmed these findings with T R H and also demonstrated that MK-771 depolarizes the ventral roots of the A S C (Yarbrough and Singh, in press). Thus, the centrally mediated E M G activation by these peptides may be a consequence of their excitatory actions on motoneurons in the spinal cord. However, if this is the case the enhanced in vivo potency and duration of action of MK-771 as compared with T R H on E M G activity is not readily explainable on this basis since the two peptides appear equipotent on the in vitro A S C preparation. Moreover, Substance P is known to have a depolarizing action on spinal motoneurons (Otsuka et al., 1975) and relatively high doses of intracisternally administered Substance P did not stimulate E M G activity. The finding that neurotensin antagonized the stimulant actions of MK-771 but not

46

those of physostigmine is of interest since it indicates some degree o f specificity in the interaction of these t w o peptides in this test system. The significance of this observation is not apparent b u t T R H has been observed to antagonize neurotensin-induced hypothermia in rats (Bissette et al., 1978; Nemeroff et al., 1978), and we have found that T R H will offset the antinociceptive actions of neurotensin in mice (unpublished observations). It would appear that studies on the possible interactions of neurotensin with T R H and related peptides in other paradigms are warranted. Finally, despite the absence of definitive information pertaining to the mechanism whereby these peptides stimulate EMG activity in experimental animals, it does not seem unreasonable to suggest that t h e y might exhibit some influence in selected functional disorders o f human muscle.

Ackno wledgements The authors are grateful to Dr. N. Bohidar, Department of Biostatistics, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania for assistance with the statistical analysis of the data.

References Bissette, G., P. Manberg, C.B. Nemeroff and A.J. Prange, 1978, Neurotensin, a biologically active peptide, Life Sci. 23, 2173.

G.G. YARBROUGH, J.C. MCGUFFIN-CLINESCHMIDT Cooper, B.R. and C.E. Boyer, 1978, Stimulant action of thyrotropin-releasing hormone on cat spinal cord, Neuropharmacol. 17, 153. Greene, E.C., 1963, Anatomy of the Rat (Hafner Publishing Co. Inc., New York, NY). Iversen, L.L., R.A. Nicoll and W.W. Vale, 1978, Neurobiology of peptides, Neurosci. Res. Prog. Bull. 16 (2), 211. Nemeroff, C.B., G. Bissette, P.J. Manberg, A.J. Osbahr, G.R. Breese, P.T. Loosen, M.A. Lipton and A.J. Prange, Jr., 1978, Effects of pharmacological and endocrinological manipulation on neurotensininduced hypothermia, Society for Neuroscience, Abstr. No. 1302, 412. Nicoll, R.A., 1976, Excitatory action of TRH on spinal motoneurons, Nature 265,242. Otsuka, M., S. Konishi and T. Takahashi, 1975, Hypothalamic Substance P as a candidate for transmitter of primary afferent neurons, Federation Proc. 34 (10), 1922. Porter, C.C., V.J. Lotti and M.J. DeFelice, 1977, The effect of TRH and related tripeptide L-N~2~xopiperidin-6-yl-carbonyl)-histidyl-L-thiazolidine-4carboxyamide (MK-771, OHT) on the depressant action of barbiturates and alcohol in mice and rats, Life Sci. 21,811. Veber, D.F., F.W. Holly, S.L. Varga, R. Hirschmann, R.F. Nutt, V.J. Lotti and C.C. Porter, 1976, The dissociation of hormonal and CNS effects in analogs of TRH, Proc. 14th Europ. Peptide Syrup., ed. A. Loffet (Univ. Brussels Press, Belgium). Yarbrough, G.G., 1978, Studies on the neuropharmacology of thyrotropinreleasing hormone (TRH) and a new TRH analog, European J. Pharmacol. 48, 19. Yarbrough, G.G. and D.K. Singh, (1979), Effects of MK-771 on the isolated amphibian spinal cord: comparison with thyrotropin releasing hormone, Can. J. Physiol. Pharmacol. (in press).