Effect of epinephrine on cyclic adenosine 3′,5′-phosphate and hexose phosphates in intestinal smooth muscle

BIOCHIMICA ET BIOPHYSICA ACTA

173

BBA 25 460 E F F E C T OF E P I N E P H R I N E

ON CYCLIC A D E N O S I N E 3 ' , 5 ' - P H O S P H A T E

AND H E X O S E P H O S P H A T E S IN I N T E S T I N A L SMOOTH MUSCLE E. BUEDING, R. W. BUTCHER, J. HAWKINS, A. R. TIMMS AND E. W. SUTHERLAND, JR. Department of Pathobiology, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Md., and Department of Physiology, School of Medicine, Vanderbilt University, Nashville, Tenn. (U.S.A.)

(Received July 7th, 1965)

SUMMARY Physiologically active concentrations of epinephrine produce an increase in the concentrations of cyclic adenosine 3',5'-phosphate in isolated intestinal smooth muscle preparations from the taenia coli of the guinea-pig. This effect is not associated with a change in the concentrations of hexose phosphate esters in this tissue.

INTRODUCTION I t has been observed that no activation of phosphorylase occurs during the physiological effect of epinephrine on intestinal smooth muscle (taenia coli of the guinea-pig) 1. Activation of phosphorylase in other tissues b y epinephrine is mediated through increased formation of adenosine 3',5'-monophosphate (cyclic 3',5'-AMP) 2-5. Therefore, the question arose whether epinephrine failed to increase phosphorylase a (EC 2.4.1.1 ) activity in taenia coli because it did not stimulate cyclic 3',5'-AMP formation or whether, in a given tissue, an increase in cyclic 3',5'-AMP is not necessarily associated with phosphorylase activation. Accordingly, an attempt was made to determine the concentrations of cyclic 3',5'-AMP in taenia coli in the presence and absence of physiologically active concentrations of epinephrine. EXPERIMENTAL Male guinea-pigs (350-55 ° g) were sacrificed b y a blow on the neck and exsanguinated from the jugular veins. The taenia coli was dissected in its entire length from the caecum, weighed (65-1oo mg), and placed in an isotonic salt medium 6. Both ends of the tissue sample were tied with nylon thread ("Ethicon") and suspended on a spring balance described b y GOODFORD AND HERMANSEN7 in such a manner that the doubled-up piece was attached at both ends to the lower part of the spring balance and, with its center, to the upper part. The sample was placed in a tissue b a t h containing 240 ml of a modified glucose-containing K r e b s - R i n g e r solution ~, maintained at 360 ~: o.5 °, and gassed with a mixture of O3 (97 %) and CO 2 (3 %)A tension of 2-3 g was applied. Tension could be determined within 0.2 g and the relaxation produced b y epinephrine was observed readily with this apparatus. Each Biochim. Biophys. Acta, 115 (1966) 173-178

174

E. BUEDINGel al.

guinea-pig supplied two samples of taenia coli--one from the ventral and one from the dorsal portion of the caecum. This permitted the use of one muscle strip for the control series and of the other strip of the same animal for the experimental series. After a period of 9o-12o min, the control sample was removed for the extraction of cyclic 3',5'-AMP. Immediately thereafter a freshly prepared solution of epinephrine was added to the tissue bath containing the experimental sample. Extraction of cyclic 3',5'-AMP from this sample was performed as soon as the maximal decrease in tension had occurred. This took place 7-15 sec after addition of the epinephrine solution.

Extraction of cyclic 3',5'-AMP The tissue was transferred immediately from the bath into either liquid N2 or isopentane cooled to --80 ° to --IOO °. After 3 ° see, the frozen sample was cut from the spring balance and transferred to a centrifuge tube (preheated in a boilingwater bath) containing 0. 5 ml of 0.05 N HC1. The mixture was stirred for 15 rain in a boiling-water bath. Subsequently the tube was placed in an ice bath for lO-2O min and centrifuged at 4000 × g for 20 rain at 0-2 °. The resulting supernatant was removed and the residue washed with 0.25 ml of 0.05 N HC1. After centrifugation, the second supernatant was combined with the first extract and this solution was neutralized to pH 7.0-7.4 (bromthymol blue was used as an external indicator). The resulting precipitate was separated by centrifugation at IOOOO ~'/. g for 20 min at 0-2 ° and the supernatant was frozen. Fractionation and analysis of cyclic 3',5'AMP were carried out according to BUTCHER el al. 8.

Analysis of hexose phosphate esters The tissue was homogenized in 3% HCI04 in an all-glass homogenizer (0.2°). The mixture was centrifuged at 6000 × g for io rain and a measured aliquot of the supernatant neutralized with 2 N KOH to pH 7.0. After recording the volume, followed by centrifugation at 40o0 × g for 15 min, the resulting supernatant was used for the analysis of GIc-6-P, Fru-6-P, and Fru-I,6-P~. Glc-6-P was determined by the enzymatic speetrophotometric procedure of •ARAHARA AND OZAND9 in a total volume of 0.8 ml. After the reaction had gone to completion, 0.02 ml of a glucosephosphate isomerase (EC 5.3.1.9) solution (0.05 mg/ml) was added to the mixture, and Fru-6-P was determined on the basis of the increase in absorbance at 340 m/~ as a result of the reduction of NADP+. Analysis of Fru-i,6-P 2 was carried out according to a procedure used previously 1°, except that triosephosphate isomerase (EC 5.3.I.I), in addition to glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) was used. In this manner the sensitivity of the method was doubled. The enzyme preparations used were products of Boehringer and Soehne, Mannheim, Germany. RESULTS Addition of epinephrine resulted in a decrease in tension varying between 40 and 9° % of its control level. The lower of the two concentrations of epinephrine used represents the physiological threshold level, i.e., the lowest concentration which consistently produces a decrease in both tension and spontaneous spike activity 11. In addition to these changes, hyperpolarization is produced by the higher concen-

Biochim. Biophys. Acta, 115 (1966) 173-178

3',5'-AMP

CYCLIC

IN S M O O T H M U S C L E

175

tration 11. While there was considerable variation in the control levels of cyclic 3',5'-AMP, both concentrations of epinephrine produced significant increases in the concentrations of the cyclic nucleotide (Table I). It has been reported that under certain conditions in vitro, cyclic 3',5'-AMP can activate phosphofructokinase (EC 2.7.1.11) of mammalian tissuesl~, TM. Although TABLE

I

E F F E C T OF E P I N E P H R I N E ON T H E C O N C E N T R A T I O N S OF CYCLIC

3t,5"-AMPIN

T A E N I A COLI

All figures represent m/~moles of cyclic 3',5'-AMP per g of tissue (wet weight).

Expt. No.

Control

Epinephrine

% change

{a) Final concentration of epinephrine 2. 5. IO -8 M I 2 3 4 5 6 7

o.18 o.18 0.47 0.48 o.18 0.09 °.34

0.24 0.22 0.59 0.70 o.21 0.23 0.47

+ 33 + 22 + 25 + 46 + 17 +156 + 38

Average

o.27

0.38

+

41

(b) Final concentration of epinephrine 5" zo-7 M 8 9 IO II 12 13 14

o.18 o.18 o.13 o.18 0.23 0.26 0.23

0.29 0.35 o.17 o.26 0.29 0.25 0.27

+ + + + + -+

61 94 31 44 26 4 17

Average

o.2o

0.27

+

38

TABLE

n

Fru-6-P

AND G I c - 6 - P C O N C E N T R A T I O N S ( I N ~ M O L E P E R g W E T %VEIGHT) OF T A E N I A COLI B E F O R E AND A F T E R E X P O S U R E TO E P I N E P H R I N E (2. 5" 1 0 - 8 M )

Expt. Before epinephrine No. Tension Fru-6-P (g)

After epinephrine Gle-6-P

Time after epinephrine

Tension (g)

Fru-6-P

Glc-6-P

1.2 1.2 0.8 i.i 1.2 1. 5 o.8 0.8

o.oo 7 o.oli O.Oli 0.007 o.oIo o.o12 o.oo 4 o.0o9

o,oi6 0.022 o.o21 o.o21 -o.o13 o.o22

i.I

0.009

0.020

(see) I

2.0

2 3 4 5 6 7 8

2. 7 2.0 2.0 2.0 2.o 2.0 2.0

IVIc a n 2 . I

0.005 0.009 O.Oli 0.006 o.olo O.Ol 3 0.003 o.oo6

O.Oli o.o18 0.025 0.025 o.o18 -o.o16 o.o2o

0.008

O.Ol 9

I2O 90 9° 60 60 9° 6o 60

o,o22

Biochim. Biophys. Acta, I 1 5 (1966) I 7 3 - I 7 8

E. BUEDINC et al.

17 6

the concentration of the cyclic nucleotide reported to produce this effect is much higher than that observed in the present study after exposure to physiologically active concentrations of epinephrine in taenia coli and in other tissues a4, the possibility was tested whether increased phosphofructokinase activity might be associated with epinephrine-induced relaxation of taenia coll. In this tissue the concentrations of the snbstrate (Fru-6-P) and of the product (Fru-I,6-P2) of the phosphofrnctokinase reaction remained unchanged following exposure of taenia coli to a concentration of epinephrine which consistently produced a marked reduction in tension and an increase in cyclic 3',5'-AMP (Tables I I and III). On the other hand, anaerobiosis produced an increase in the Fru-I,6-P 2 levels of the tissue (Table IV). A similar accumulation of this ester under anaerobic conditions has been reported by PARK et al. ~5, for heart muscle. TABLE

III

E r u - I , 6 - P 2 CONCENTRATIONS (IN /IMOLE PER g ~,VET WEIGHT) OF TAENIA COLI BEFORE AND AFTER EXPOSURE TO E P I N E P H R I N E (2. 5" IO 8 M)

Expt. No.

Before epinephrine

After epinephrine

Tension (g)

Fru-±,6-P 2

Tension (g)

Fru-z,6-P~

I 2 3 4 5

2,0 2,o 2,0 2,0 2.0

o.o31 o.o52 0.025 o.034 0.028

I. 3 1.4 I .o 0.6 1.3

0.029 o'o49 0.022 o.033 0.026

Mean

2,0

0.034

i.i

0.032

TABLE

IV

EFFECT OF ANAEROBIOSlS (5 m i n ) ON F r u - I , 6 - P a CONCENTRATION

(#MOLE PER g WET WI~.!GHT)

OF TAENIA COLI / 97 o/o/ 02-3 o/o C02

97 ~o/ N2-3 % C02

Tension (g)

Fru-z,6-P 2

Tension (g)

Fru-i,6-P 2

I 2 3 4 5 6

2.0 2.0 2.o 2.0 2.5 2.0

0.034 0.025 o.oI6 0.023 0.026 o.020

2.8 2.5 3.o 3.0 2. 5 3.0

0,063 o.o56 o.o48 0.045 o.o31 0.045

Mean

2.1

0.024

2.8

0.048

Expt. No.

DISCUSSION

The results reported in this paper suggest a positive correlation between the physiological effects of epinephrine on taenia coli and an increased concentration of cyclic 3',5'-AMP in this tissue. This correlation appears significant because the Biochirn. Biophys. Acla, 115 (1966) 1 7 3 - 1 7 8

CYCLIC 3',5'-AMP IN SMOOTH MUSCLE

177

increase in cyclic 3',5'-AMP was observable with the minimal concentration of epinephrine which produces a physiological effect. Therefore, an increase in cyclic 3',5'-AMP takes place during exposure of the intact tissue to epinephrine, while phosphorylase activation occurs only under certain conditions after removal from the tissue bath 1. Possibly this artifactual activation of phosphorylase after removal of the tissue from the epinephrine-containing medium is due to an activation of phosphorylase b kinase (EC 2,7.1.38 ) by extrusion of cyclic 3',5'-AMP into another cell compartment during manipulation of the muscle. It has been reported that relatively high concentrations of cyclic 3',5'-AMP can activate phosphofructokinase in vitro12,13. Since the steady-state levels of the substrate (Fru-6-P) and of the product (Fru-I,6-P2) of the phosphofructokinase reaction remain unchanged, there is no evidence that activation of this enzyme occurs in taenia coli during the physiological action of epinephrine, despite the increase in cyclic 3',5'-AMP observed under these conditions. Furthermore, phosphofructokinase activity of taenia coli is not affected by cyclic 3',5'-AMP when extracts of this tissue are incubated with concentrations of Fru-6-P, ATP, AMP, inorganic phosphate and Mg2+ equal to those prevailing in intact taenia eoli TM. Therefore, the hypothesis that the physiological effects of epinephrine on intestinal smooth muscle are brought about by an activation of phosphofructokinase receives no support. Activations of phosphorylase and of phosphofruetokinase are by no means the only known actions of cyclic 3',5'-AMP in vitro. For example, evidence has been reported indicating that this nucleotide increases lipolysisS, ~7, the incorporation of acetate into acetoacetate TM, the hydroxylation of steroids in the 13-11 position TM, and the permeability of the toad bladder to water 2~, that it decreases the active form of UDPG glycogen a-4-glucosyltransferase (EC 2.4.1.11) 21,22, and that it reactivates tryptophan pyrrolase (EC 1.13.1.12) 23. In addition to the known effects of cyclic 3',5'-AMP on a variety of biochemical reactions, there is a distinct possibility that this nucleotide may have hitherto unknown actions on many other systems. Some known or unknown effect of cyclic 3',5'-AMP might account for the recently observed increases in the tissue levels of energy-rich phosphate compounds following exposure of taenia coli to epinephrine24,25. The view that the electrophysiological and mechanical changes of intestinal smooth muscle produced by epinephrine are brought about by an increased supply of metabolic energy 26 might be related to such an effect, although it is recognized that other possibilities exist. For example, an effect of the increased level of ATP on the synthesis of cyclic 3',5'-AMP cannot be excluded. ACKNOWLEDGEMENTS

This work was supported by research grants from the National Institutes of Health (HE-o5268, 5TIGM 423, HE-o8332, and AM-o7642-oi AMP) and the American Heart Association (62-G-2I) REFERENCES I 2 3 4

E. T. E. E.

BUEDING, E. BOLBRING, H. KURIYAMA AND G. GERCKEN, Nature, 196 (1962) 944W. RALL AND E. W. SUTHERLAND, Jr., J. Biol. Chem., 323 (1958) lO6.5. W. SUTHERLAND, Jr. AND T. W. RALL, J. Am. Chem. Soc., 79 (1957) 36o8. I~REBS, D. J. GRAVES AND E. I-I. FISCHER, dr. Biol. Chem., 234 (1959) 2867.

Biochim. Biophys. Acta, 115 (I966) 173-178

178 5 6 7 8 9 IO ii 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

E. BUEDING et al.

E. W. SUTHERLAND, Jr. AND T. W. RALL, Pharmaeol. Rev., 12 (I96o) 265. J. AXELSSON AND E. ]BOLBRING, J. Physiol. London, 156 (1961) 344. P" S. GOODFORD AND K. HERMANSEN, dr. Physiol. London, 158 (1961) 426. R. W. BOTCHER, R. J. Ho, H. C. MEN~ AND E. W. SUXHERLAND, Jr., J. Biol. Chem., 240 (1965) 4515 • H. T. NARAHARA AND P. OZAND, Proe. Soc. Exptl. Biol. Med., lO 3 (196o) 529. E. BISEDING AND J. M. MANSOISR, Brit. J. Pharmacol., 12 (1957) 159. E. BULBRING, personal c o m m u n i c a t i o n . J. V. PASSONEAU AND O. L. LOWRY', Biochem. Biophys. Res. Commun., 7 (1962) io. T. E. MANS0OR, dr, Biol. Chem., 238 (1963) 2285. R. W. BUTCHER AND E. W. SUTHERLAND, Jr., J. Biol. Chem., 232 (1958) lO77. C. R. PARK, H. F. MORGAN, M. J. HENDERSON, O. M. REGEN, ]~. CADENAS AND R. L. PosT, Recent Progr. Hormone Res., 7 (I96I) 493. E. BUEDING AND M. J. FISHER, u n p u b l i s h e d o b s e r v a t i o n s . M. A. RIZACK, J. Biol. Chem., 239 (1964) 392. J. BERTHET, Proe. Intern. Congr. Biochem., 4th, Vienna, ±958, p. lO7. J. E. CREANGE AND S. ROBERTS, Biochem. Biophys. Res. Commun., 19 (1965) 73. J. ORLOFF AND J. S. HANDLER, J. Clin. Invest., 41 (1962) 702. E. BELICOmTOW, Arch. Biochem. Biophys., 93 (1961) 467 . M. ROSELL-PEREZ AND J. LARNER, Biochemistry, 3 (1964) 81. F. CHY'TIL AND J. SKRIVANOVA, Bioehim. Biophys. Acta, 67 (1963) 164. E. BUEDING, E. BOLBRING, G. GERCKEN AND H. KURIYAMA, dr. Physiol. London, 166 (1963) 8P. E. BUDDING AND BOLBRING, in E. Bt)LBRING, Pharmacology of Smooth Muscle, P e r g a m o n Press, 1964, P. 37. E. BOLBRIN6, Ciba Found. Syrup., Adrenergie Mechanisms, (196o) 275.

Biochim. Biophys. deta, 115 (1966) 173-178