Neurotensin-induced antinociception and hypothermia in mice: antagonism by TRH and structural analogs of TRH

Regulatory Peptides, 8 (1984) 41-49

41

Elsevier RPT00257

Neurotensin-induced antinociception and hypothermia in mice: antagonism by TRH and structural analogs of TRH Daniel E. Hernandez 1, Charles B. Nemeroff 1,2, Miguel H. Valderrama i and Arthur J. Prange, Jr. 1,2 I Biological Sciences Research Center, 2 Department of Psychiatry, and the Neurobiology Program, University of North Carolina School of Medicine, Chapel Hill, NC 27514, U.S.A.

(Received 14 April 1983; revisedmanuscript received31 October 1983; acceptedfor publication 31 October 1983)

Summary Intracisternal (IC) administration of neurotensin (NT) in a dose of 10 #g produced a significant hypothermia and antinociception in the hot-plate test in mice. Both of these effects of IC N T were completely antagonii~ed by concomitant administration of equimolar doses of thyrotropin-releasing hormone (TRH) and several T R H congeners including 3-methyl-His-TRH (pGlu-3-methyl-His-Pro-NH 2), MK-771 (pyro-2-aminoadipyl-histidyl-thiazolidine-4-carboxamide), fl-ala-TRH (pGlu-His-Pro-fl-ala-NH2), and RX-77368 (pGlu-His-dimethyl-Pro-NH2). The antagonism by T R H and T R H analogs on NT-induced hypothermia and antinociception was dose-dependent. Of particular interest was the finding that RX-77368 not only blocked the effects of N T but also produced hyperalgesia. It appears that T R H analogs that are more resistant to biologic degradation are, like TRH, capable of blocking NT-induced behaviors. thyrotropin-releasing hormone; neurotensin; antinociception; hypothermia

Introduction Neurotensin (NT), an endogenous tridecapeptide (pGlu-Leu-Tyr-Glu-Asn-LysPro-Arg-Arg-Pro-Tyr-ILeu-Leu-OH) originally isolated from bovine hypothalami [8] Address all correspondence to: Daniel E. Hernandez, D.V.M., BiologicalSciencesResearch Center 220-H,

University of North Carolina School of Medicine, Chapel Hill, NC 27514, U.S.A. Telephone: 919 966-1480/1489. 0167-0115/84/$03.00 © 1984 ElsevierSciencePublishers B.V.

42

is heterogenously distributed in the central nervous system (CNS) of several vertebrate species [9]. When administered directly into the CNS, neurotensin produces a variety of effects [22]. After intracisternal (IC) administration neurotensin elicits a marked reduction in body temperature in several mammals [25]. This effect, which is exaggerated in a cold ambient [20], is also observed in neonatal rats [15]. Centrally administered NT also produces antinociception [10,11,20], muscle relaxation [23] and potentiation of the sedative effects of barbiturates and ethanol [19,3,16]. Thyrotropin-releasing hormone (TRH), a tripeptide (pGlu-His-Pro-NH2) produces a variety of CNS effects after peripheral or central administration [26]. These include reversal of pentobarbital- and ethanol-induced sedation and hypothermia [12,26]. Central or peripheral administration of TRH antagonizes many of the effects of neurotensin including hypothermia and antinociception [22,24]. Thus, it appears that TRH and neurotensin are antagonistic in their effects on the CNS. In addition, we have previously studied the structure-activity relationships of TRH, and TRH congeners, as regards reversal of barbiturate-induced narcosis and hypothermia [3]. This study was designed to evaluate the effects of TRH and several biologically active structural analogs of TRH on neurotensin-induced hypothermia and antinociception in the hot-plate test in mice.

Material and Methods

Adult, male Swiss-Webster mice (25-35 g) were purchased from Flow Laboratories (Dublin, VA) and were group housed (6/cage) in a controlled ~nvironment animal facility (12 h light, 12 h dark) with laboratory chow and water available ad libitum. The mice were housed for at least one week prior to experimentation. All experiments;were conducted between 0800 and 1200 at ambient temperatures of 24-26°C. Groups of mice (n = 8/group) were selected for each experiment, no animal being used in more than one experiment. Each mouse was injected IC under light ether anesthesia'as previously described [19] with either vehicle (10/~1 0.9% NaC1), neurotensin (NT), one of the following peptides: thyrotropin-releasing hormone (pGlu-His-Pro-NH2), pGlu-His-dimethyl-Pro-NH2 (RX-77368), pyro-2-aminoadipyl-histidyl-thiazolidine-4-carboxamide (MK-771), pGlu-His-Pro-fl-ala-NH 2 (fl-alaTRH) or pGlu-3-methyl-His-Pro-NH2 (3-methyl-His-TRH), or a combination of NT + TRH or a TRH congener. This basic experimental design included four groups of mice (n = 8/group) each one receiving one of the following IC treatments: (1) vehicle, (2) NT, (3) TRH or TRH analog and (4) NT + TRH or TRH analog. In the first series of experiments TRH and the different analogs of TRH were administered IC in a dose equimolar to 10 #g of NT. This dose of NT (10 ~tg) was chosen because it has been previously demonstrated to reliably produce hypothermia and antinociception in mice [20,21]. In the second series of experiments various doses of IC TRH and each of the other TRH congeners were evaluated for their effect in the antinociception and hypothermia induced by NT (10/xg IC). The IC

43 doses of TRH and TRH analogs tested were as follows: (1) TRH (2.17, 21.7, 217, and 2170 ng); (2) MK-771 (2.35, 23.5, 235, and 2350 ng); (3) 3-methyl-His-TRH (2.25, 22.5, 225, and 2250 ng); (4) RX-77368 (2.3, 23.0, 230, and 2300 ng), and (5) fl-ala-TRH (2.5, 25.0, 250, and 2500 ng). Body temperature was measured immediately after injection, and at 60 and 120 min post-injection, by insertion of a lubricated thermocouple probe (Bailey Instruments, Saddle Brook, NY) approximately 3 cm into the mouse rectum. Antinociception was assessed by using the hot-plate test [1]. In this test, mice are placed with all four paws on a heated copper plate and the time to the nearest tenth second for the mice to either lick their paws or jump was considered as a response to the noxious stimulus. The temperature of the hot-plate was set at 50-52°C. This hot-plate temperature permits repeated testing of the response of an individual mouse to the noxious stimulus without inflicting injury to the animals. An arbitrary cutoff was used to score animals not responding to the noxious stimulus within 30 s. Each animal was tested every 20 min for 2 h. Neurotensin was purchased from Bachem (Torrance, CA). Thyrotropin-releasing hormone was kindly provided by Abbott Laboratories (North Chicago, IL), fl-alaTRH was a gift from Dr. R.D. Studer (Hoffman LaRoche, Basel, Switzerland). MK-771 and 3-methyl-His-TRH were a gift from Merck Sharp Dohme Research Laboratories (West Point, PA) and RX-77368 was generously provided by Dr. G. Metcalf (Reckitt and Colman, Hull, England). Comparisons of the mean area (s x min) under the 2-h curve between control and experimental groups allows for evaluation of whether the¢ treatment produced a significant alteration in the response of the mice to the noxious stimulus. Furthermore, the difference between the area under the curve calculated by planimetry for the experimental mouse and that for the saline-treated mouse produced a measure representing both the magnitude and the duration of the effect of the substance under study. A significant increase in the response time for experimental compared to control animals was defined as antinociception. One-way analysis of variance followed by Dunnett's test for multiple comparison was used for this analysis [13].

Results

Figs. 1 and 2 illustrate the results of the first series of experiments in which a single equimolar IC dose of TRH (Figs. 1A, 2A), fl-ala-TRH (Figs. 1B, 2B), MK-771 (Figs. 1C, 2C) and 3-methyl-His-TRH (Figs. 1D, 2D) on NT-induced antinociception and hypothermia was evaluated. The results indicate that NT (10 #g) given IC produces significant antinociception and hypothermia thereby confirming previous reports [20,24]. In doses equimolar to 10 #g NT, TRH as well as the TRH analogs tested, significantly antagonized both NT-induced antinociception and hypothermia. In addition, Fig. 3, which illustrates the temporal effect of peptide treatment on antinociception or temperature shows that RX-77368 when given simultaneously with NT in a dose equimolar to 10 #g of NT produced a marked inhibition of

44 3O

50

A 25

25 20

15

15

I0

I0

=E i,i

4.1 5

u,l 0 I-w 25

i

C

i

i

i

i

I

I

I

L

I

I

25 /.

in

0

*

I

"'/"~ 2C

w

I1:15

15

I0

IO 5

0

I

I

I

I

I

20

40

60

80

I00

I

TIME

120 0

I

I

I

I

I

I

20

40

60

80

I00

120

(Minutes)

Fig. 1. Effect of IC administration of TRH (A), ~-ala-TRH (B), MK-771 (C) and 3-methyl-His-TRH (D) on NT-induce4i antinociception in .the hot-plate test in mice. Treatment regimens were as follows: (O) saline (10 lal ff.9% NaCl), (e) NT (10 ~g), (n) TRH or TRH analog (equimolar to 10 #g of NT) and ( i ) NT (10 # g ) + T R H or TRH analog (extuimolar to 10 Vg of NT). Asterisks indicate significant differences from saline-treated controls (* P < 0.05, ** P < 0.01, Dunnett's test for multiple comparisons).

NT-induced antinociception. Moreover when given alone RX-77368 provoked significant hyperalgesia (Fig. 3A). This structural analog of TRH also blocked NT-induced hypothermia, but did not itself produce hyperthermia (Fig. 3B). Finally, dose-response studies revealed that TRH (217 and 2170 ng IC) antagonized the antinociccption and hypothermia produced by the fixed dose of NT (10/~g IC) (Fig. 4A); the results with MK-771 indicate that only the equimolar dose to 10 #g of NT was effective in blocking the effects of NT (Fig. 4B); 3-methyl-His-TRH only antagonized NT-induced antinoeiception, but not hypothermia, in a dose-dependent manner (Fig. 4C); similar results were obtained with fl-ala-TRH (Fig. 4D). All the doses of RX-77368 employed (2.3-2300 ng IC) significantly antagonized NT-induced antinociception, but only the higher doses of this analog (230 and 2300 ng) were effective in reversing NT-induced hypothermia (Fig. 4E).

45

38 , . .

. ~ 38

36~

~

~E 34ILl O0

36

~

34

+I

** 32

32

A

ILl ,~ n~ i,,

0

60

381g_

120 0

_

_.il

60

120

38

ILl

()'1-/~-3634 rrL° .'~ f

~

-~3634

32~- C 01

I

32

**

1200

60

I

60

I

I

120

TIME (Minutes) Fig. 2. Effects of IC administration of T R H (A), fl-ala-TRH (B), MK-771 (C) and 3-methyl-His-TRH (D) on NT-induced hypothermia in mice. Treatment regimens and symbols identical to those described for Fig. 1. I

l

A.

3O

l

*

l

*$

!

i

4o

i

*

!

LIJ Or)

t.iJ o3 4-1 t.) Lo 20 o3 L~

!

8.

LIJ

OC ~36

rL~ o3 I0

34

bJ l-..J

Z

o3 n,-

32 ,Y,

0 20 40 60 80 I00 120 TIME(MIN) I

I

I

I

I

l

,

I

60 120 TIME ( MIN ) Fig. 3. The effect of RX-77368 (pGlu-His-dimethyl-Pro-NH2) on NT-induced antinociception in the hot-plate test (A) and hypothermia (B). Treatment regimens are as follows: ( O ) 0.9% NaCI 10 #1; (e) N T 10 #g, (D) RX-77368 (2.3 #g) and ( I ) NT 10 #g + RX-77368 2.3 ~tg. All substances were administered IC. RX-77368 was given in a dose equimolar to 10 #g of NT, n = 8 mice in each treatment group. * P < 0.05, • * P < 0.01 when compared with saline IC (Dunnett's test for multiple comparisons). 0

46

Analgesia

O|VE.H.r_~. ~

TRH

NTIlOjUg]..~ ~ ~+TRH ' 2.17 2L7 217 217Ong

Temperature

0

VEH

NTIlO.,ug) - ' - - J ~-'--+TRH --'-'~ 2.17 21,7 217 217Ong

o T I--] F [ VEH

I-'I' I'--I I--1 T--I NT (IO.,ug) ' ' ~ J L-.,-+ MK- 771 ,,,--J 2,35 25.5 2 3 5 2350ng

MK-771

O" VEH ~ " - - NT ( IO/,Jg) " - ' ' - j +MK-771-~J 2.35 25.5 235 2350n~

IJJ

(/) .H

3Methyl

- His- TRH

o °fooflill •i ool °:IiEo li w_

r-1 I-I r-1 r-~ r-1 r-j, VEH ~ N T (IO.ug)----" ~ ~+ 3 MethyI-TRH~ 2.25 225 225 2250 n9

0

IJ.I n<[

VEH ~ N T

(lO,u~l) ~+ 3 MethyI-TRH "~ 2,2~, 22.5 225 225Ong

B-ala-TRH 5000

o"

VEH L - - - - N T (IO/Jg) ~ L- +/9- OiO-TRH "-= 2..5 25 250 25OOng

oT r--i V7 V'-[ r - i i----i i--1 VEH ~ N T (tO#l) ~ ~+B.OIo-TRH

.,-.~

2.5 25 250 2500rig

RX-77368

O t v E H L~.--NT (IOA~l-----J-L-+RX-77386 - J 2.3 23 250 23(X)n0

OT_['~ L - - ~ - [ I-1 l - 3 1--1 VEH I , ,NT ( I O , l ~ l ) - - - - - j L-+ RX- T r 3 8 6 - - J 2 3

2 3 2 3 0 23OOn~11

47

Discussion

This study confirms previous findings that indicate that IC NT produces significant hypothermia and antinociception in mice [20]. All the TRH analogs tested, when administered in a dose equimolar to 10 #g of NT, shared with TRH the ability to significantly antagonize both NT-induced hypothermia and antinociception in a dose-dependent manner. An interesting observation was provided by our results with RX-77368. This analog of TRH produced hyperalgesia, but not hyperthermia in the previously untreated mice, indicating that certain of its efficacy in blocking the antinociceptive effect of NT may in fact be due to summation of the opposite sign of these two peptides. It is intriguing to speculate that the hyperalgesia induced by RX-77368 might well be due to blockade of the action of endogenous NT. These data confirm our previous findings of the antagonistic effect of TRH on many of the central actions of NT [7,24]. This antagonism by TRH, which is not only confined to the inhibition of NT-induced hypothermia and antinociception, but also demonstrated for NT-induced muscle relaxation [23], catalepsy [27], NT-induced potentiation of ethanol-induced narcosis [16], NT-induced suppression of food consumption [18] the effects of NT in a discrete trial conditioned avoidance paradigm [17], NT-induced gastric cytoprotection [14] is clearly not a nonspecific attribute of small peptides since another tripeptide, Pro-Leu-Gly-NH 2 (MIF-I) does not block the hypothermic and antinociceptive effects of NT*[24]. One possible mechanism that has been suggested to block the action of NT is that since TRH and NT are administered together, the two peptides might bind to each other, thereby reducing the NT available to act at receptor sites in vivo. This appears to be unlikely because the addition of TRH to a solution of NT does not alter NT immunoreactivity when measured by radioimmunoassay (Dr. Paul Manberg, personal communication, 1983). These results do not, of course, rule out this possibility. Whether this possibility might also apply to the TRH analogs tested in this study is not presently known. Our results pertaining to the characterization of the structural specificity for the antagonism by TRH on the effects of NT examined in this study (hypothermia and antinociception), suggest that a 3,3-dimethyl substituted proline amide residue makes the peptide particularly more resistant to enzymatic degradation, therefore increasing its biological stability, and producing a substantial enhancement of its neuropharmacological properties. This view is supported by the following. TRH is probably primarily inactivated by deamidation to TRH free acid (Pyr-His-Pro-OH), though other authors have also demonstrated the cleavage of pyroglutamate and proline [2,4,28]. In addition, it has been demonstrated that

Fig. 4. Effect of different doses of TRH (A), MK-771 (B), 3-methyl-His-TRH (C), fl-ala-TRH (D) and RX-77368 (E) on NT-induced antinociception and hypothermia in the hot-plate test. Asterisks represent significant antinociceptive and hypothermic response to 10 #g of IC NT in mice when compared to saline treated controls (* P < 0.05, ** P < 0.01), and the crosses indicate significant antagonism by TRH or the different TRH analogs of the NT-induced antinociception and hypothermia ( * P < 0.05, ** P < 0.01) when compared to NT-tested mice (Dunnett's test for multiple comparisons).

48 L-3,3-dimethylproline ( p G l u - H i s - D m p - N H 2, RX-77368) is m o r e p o t e n t t h a n L-3m e t h y l p r o l i n e ( p G l u - H i s - M e p - N H 2 ) in reversing the h y p o t h e r m i a a n d behavioral d e p r e s s i o n p r o v o k e d b y reserpine in mice [6]. F u r t h e r m o r e , the rate of m e t a b o l i c d e g r a d a t i o n of p G l u - H i s - D m p - N H 2 b y m o u s e b r a i n has been d e m o n s t r a t e d to be c o n s i d e r a b l y slower t h a n T R H a n d p G l u - H i s - M e p - N H 2 [5]. In conclusion, stabilised structural analogs of T R H such as RX-77368 a p p e a r to b e of p a r t i c u l a r interest b e c a u s e of their e n h a n c e d n e u r o p h a r m a c o l o g i c a l p o t e n c y , t h e r e b y suggesting that they m a y find clinical utility.

Acknowledgements This research was s u p p o r t e d b y N I M H g r a n t s MH-32316, MH-34121, MH-33127, M H - 2 2 5 3 6 , a n d N I C H H D g r a n t HD-03110. Miguel H. V a l d e r r a m a , M . D . was s u p p o r t e d b y a g r a n t f r o m the M e x i c a n P e p s i - C o l a C o m p a n y . W e are grateful to J u d y M. B a r n e t t for p r e p a r a t i o n of this m a n u s c r i p t . This r e p o r t will b e p r e s e n t e d in p a r t at the 7th E u r o p e a n N e u r o s c i e n c e Congress, H a m b u r g , F e d e r a l R e p u b l i c of G e r m a n y .

References 1 Ankier, S.I., New hot plate test to quantify antinociceptive and narcotic antagonists activities, Eur. J. Pharmacol., 27 (1974) 1-4. 2 Bauer, K., Degradation of hypothalamic hormones, Naunyn-Schmiedebergs Arch. Pharmacol., 297 (1977) $55-$56. 3 Bissette, G., Nemeroff, C.B., Loosen, P.T., Breese, G.R., Burnett, G.B., Lipton, M.A. and Prange, A.J., Jr., Modification of pentobarbital-induced sedation by natural and synthetic peptides, Neuropharmacology, 1i (1978) 229-237. 4 Brewster, D. and Waltham, K., TRH degradation rate varies widely between different animal species, Biochem. Pharmacol., 30 (1981) 619-622. 5 Brewster, D., Humphrey, M.J. and Wareing, M.V., Metabolism pharmacokinetics of TRH and an analogue with enhanced neuropharmacological potency, Neuropeptides, 1 (1981) 153-165. 6 Brewster, D., Dettmar, P.W. and Metcalf, G., Biologically stable analogues of TRH with increased neuropharmaeological potency, Neuropharmacology, 20 (1981) 497-503. 7 Burgess, S.K., Luttinger, D., Hernandez, D.E., Kalivas, P.W., Nemeroff, C.B. and Prange, A.J., Jr., Antagonism of the central nervous system effects of neurotensin by thyrotropin-releasing hormone. In E.C. Griffiths and G.W. Bennett (Eds.), Thyrotropin Releasing Hormone, Raven Press, New York, 1983, pp. 291-302. 8 Carraway, R. and Leeman, S.E., The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami, J. Biol. Chem., 248 (1973) 6854-6861. 9 Carraway, R. and Leeman, S.E., Characterization of radioimmunoassayable neurotensin in the rat, J. Biol. Chem., 251 (1976) 7045-7052. 10 Clineschmidt, B.V. and McGuffin, J.C., Neurotensin administered intracisternally inhibits responsiveness of mice to noxious stimuli, Eur. J. Pharmacol., 46 (1977) 395-396. 11 Clineschmidt, B.V., McGuffin, J.C. and Bunting, P.B., Neurotensin: Antinoeisponsive action in rodents, Eur. J. Pharmacol., 54 (1979) 129-139. 12 Cott, J.M., Breese, G.R., Cooper, B.R., Badow, T.S. and Prange, A.J., Jr., Investigations into the mechanism of reduction of ethanol sleep by thyrotropin-releasing hormone, J. Pharmacot. Exp. Ther., 196 (1976) 594-604.

49 13 Dunnett, C.W., New tables for multiple comparisons with a control, Biometrics, 20 (1964) 483-491. 14 Hernandez, D.E., Nemeroff, C.B., Orlando, R.C. and Prange, A.J., Jr., The effect of centrally administered neuropeptides on the development of stress-induced gastric ulcers in rats, J. Neurosci. Res., 9(2) (1983) 145-157. 15 Hernandez, D.E., Nemeroff, C.B. and Prange, A.J., Jr., Ontogeny of the hypothermic response to centrally administered neurotensin in rats, Dev. Brain Res., 3 (1982) 497-501. 16 Luttinger, D., Mason, G.A., Frye, G.D., Osbahr, A.J., Nemeroff, C.B. and Prange, A.J., Jr., Enhancement of ethanol-induced sedation and hypothermia by centrally administered, neurotensin, fl-endorphin and bombesin, Neuropharmacology, 20 (1981) 305-309. 17 Luttinger, D., Nemeroff, C.B. and Prange, A.J., Jr., The effects of neuropeptides on discrete-trial conditioned avoidance responding, Brain Res., 233 (1982) 183-192. 18 Luttinger, D., King, R.A., Sheppard, D., Strupp, J., Nemeroff, C.B. and Prange, A.J., Jr., The effect of neurotensin on food consumption in the rat, Eur. J. Pharmacol., 81 (1982) 499-503. 19 Nemeroff, C.B,, Bissette, G., Prange, A.J., Jr., Loosen, P.T. and Lipton, M.A., Neurotensin: central nervous system effec'is of a hypothalamic peptide, Brain Res., 128 (1977) 485-496. 20 Nemeroff, C.B., Osbahr, A.J., Manberg, P.J., Ervin, G.N. and Prange, A.J., Jr., Alterations in nociception and body temperature after intracisternal administration of neurotensin, fl-endorphin, other endogenous peptides, and morphine, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 5368-5371. 21 Nemeroff, C.B., Bissette, G., Manberg, P.J., Osbahr, A.J. III, Breese, G.R. and Prange, A.J., Jr., Neurotensin-induced hypothermia: evidence for an interaction with dopaminergic systems and the hypothalamic pituitary-thyroid axis, Brain Res., 195 (1980) 69-84. 22 Nemeroff, C.B., Luttinger, D. and Prange, A.J., Jr., Neurotensin: central nervous system effects of a neuropeptide, Trends Neurosci., 3 (1980) 212-215. 23 Osbahr, A.J., Nemeroff, C.B., Manberg, P.J. and Prange, A.J., Jr., Centrally administered neurotensin: activity in the Julou-Courvoisier muscle relaxation test in mice, Eur. J. Pharmacol., 54 (1979) 299-302. 24 Osbahr, A.J., Nemeroff, C.B., Luttinger, D., Mason, G.A. and Prange, A.J., Jr., Neurotensin-induced antinociception in mice: antagonism by thyrotropin-releasing hormone, J. Pharmacol. Exp. Ther., 217 (1981) 645-651. 25 Prange, A.J., Jr., Nemeroff, C.B., Bissette, G., Manberg, P.J., Osbahr, A.J., III, Burnett, G.B., Loosen, P.T. and Kraemer, G.W., Neurotensin: distribution of hypothermic response in mammalian and submammalian vertebrates, Pharmacoi. Biochem. Behav. 11 (1979) 473-477. 26 Prange, A.J., Jr., Nemeroff, C.B., Loosen, P.T., Bissette, G., Osbahr, A.J., III, Wilson, I.C. and Lipton, M.A., Behavioral effects of thyrotropin-releasing hormone in animals and man; a review. In Collu et al. (Eds.), Central Nervous System Effects of Hypothalamic Hormones and other Peptides, Raven Press, New York, 1979, pp. 75-96. 27 Snijders, R., Kramarcy, N.R., Hurd, R.W., Nemeroff, C.B. and Dunn, A.J., Neurotensin induces catalepsy in mice, Neuropharmacology, 21 (1982) 465-468. 28 'Taylor, W.L. and Dixon, J.E., Characterization of a pyroglutamate aminopeptidase from rat serum that degrades thyrotropin-releasing hormone, J. Biol. Chem., 253 (1978) 6943-6940.