Effects of adenosine and related compounds on adenylate cyclase and cyclic AMP levels in smooth muscle

European Journal of Pharmacology, 41 (1977) 193--203 © Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands

193

EFFECTS OF ADENOSINE AND RELATED COMPOUNDS ON ADENYLATE CYCLASE AND CYCLIC AMP LEVELS IN SMOOTH MUSCLE SHEILA G. McKENZIE *, ROBERT FREW AND tIANS-PETER BAR **

Department of Pharmacology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada Received 20 May 1976, revised MS received 20 July 1976, accepted 7 October 1976

S.G. McKENZIE, R. FREW and H.-P. B~,R, Effects of adenosine and related compounds on adenylate cyclase and cyclic AMP levels in smooth muscle, European J. Pharmacol. 41 (1977) 193--203. The hypotheses were tested that the relaxant effect of adenosine and related compounds in the longitudinal muscle of the rabbit small intestine involves interaction with adenylate cyclase and]or the elevation of tissue cAMP levels. Adenylate cyclase was prepared by gentle homogenization of an isolated smooth muscle cell fraction obtained after collagenase digestion of longitudinal muscle strips. A number of analogs and derivatives of adenosine possessing a primary or secondary 6-amino group were found to inhibit the enzyme similarly to adenosine; however, there was no correlation between compounds known to relax the intact tissue and the existence, or the degree of, cyclase inhibition. Isolated muscle strips were exposed to adrenaline, isoprenaline, adenosine or ATP, at doses causing 30--60% relaxation, for 60 sec prior to sampling and analysis of cAMP content. While small increments in cAMP levels were found after administering adrenaline or isoprenaline, no change was found with adenosine in the absence or presence of theophylline or 1-methyl-3-isobutylxanthine. Neither adenylate cyclase inhibition nor changes in cAMP levels appear to be part of the mechanism of the smooth muscle relaxant action of adenosine or ATP. Smooth muscle relaxation Intersubject variability

Rabbit intestine Adenylate cyclase

1. Introduction

Adenine compounds and adenosine have been known as effective smooth muscle relaxants for a long time. Physiological roles for this class of compounds have been proposed, including functions in the regulation of coronary blood flow (Berne, 1963) and as chemical transmitters in non-cholinergic, nonadrenergic inhibitory nerves in various intestinal and other smooth muscles {Burnstock et al., 1970) as well as in the central nervous system (McIlwain, 1974). Regarding the mechanism of action of adenine compounds, no definitive evidence has been produced, although the literature contains a few data and specula* Present address: Neurologische Universit~tsklinik, 78 Freiburg, Hansastr. 9a, W. Germany. ** To whom reprint requests should be sent.

Cyclic AMP

Adenosine

ATP

tions. Suggested mechanisms of adenosineinduced relaxation include chelation or precipitation of calcium (Feldberg and Hebb, 1948; Falk, 1956), interference with the conductance of calcium through the smooth muscle plasma membrane (Axelson and Holmberg, 1969), induction of a specific increase in potassium conductance (Tomita and Watanabe, 1973), inhibition of adenylate cyclase (Schaumann et al., 1970) and, in contrast, enhancement of adenosine-3',5'~yclic monophosphate (cAMP) accumulation in smooth muscle or its supporting tissue (Raberger et al., 1970). While the ultimate parameter of functional importance in contractility is undoubtedly calcium ion, it is likely that the regulation of its intracellular distribution and concentration involves preceding biochemical reactions. We

194 have considered the possibility that the cAMP/adenylate cyclase system may play an intermediary role in this respect. This hypothesis was based on the analogous, albeit controversial, hypothesis for the mechanism of ~adrenergic drug action on smooth muscle (Andersson, 1972; cf. B ~ , 1974) and it was further stimulated by observations of the profound stimulatory action of adenosine on cAMP levels in guinea-pig cerebral cortex slices (Sattin and Rall, 1970; Shimizu and Daly, 1970), neuroblastoma cells (Schulz and Hamprecht, 1973; Blume et al., 1973), astroc y t o m a cells (Clark et al., 1974), bone cells (Peck et al., 1974), t h r o m b o c y t e s (Mills and Smith, 1971; Haslam and Rossom, 1975) and rat lung (Palmer, 1971). These latter findings suggested that certain cells, nerve cells in particular, contain adenosine sensitive cyclase systems. Although adenosine action on smooth muscle does not seem to involve nerve elements (McKenzie et al., 1977), the possibility that smooth muscle cells may also show a direct response to adenosine or ATP in terms of an increase in cAMP levels was a hypothesis to be tested. The stimulation of cAMP levels by adenosine seems to involve an extracellular receptor site, similar to receptors of many peptide hormones and catecholamines; however, it generally has n o t been possible to demonstrate stimulatory effects of adenosine on adenylate cyclase in broken cells preparations. Instead, inhibition of enzyme activity has been observed (Moriwaki and Foa, 1970; Fain et al., 1972; McKenzie and B~r, 1973; Weinryb and Michel, 1974) with two exceptions. These include cyclase preparations from human platelets (Haslam and Lynham, 1972) and from a mouse neuroblastoma cell line (Blume and Foster, 1975). In the case of platelets, gentle cell breakage was reported to be essential for preservation of adenosine sensitivity of the cyclase, suggesting that the adenosine receptor is a rather labile entity or that its coupling to the cyclase system is readily disturbed by mechanical or other manipulation. This might explain the unsuccessful attempts

S.G. MCKENZIE ET AL. of demonstrating adenosine sensitive adenylate cyclase in tissues such as brain. In the case of neuroblastoma cells, there appears to be no need for 'gentle' methods of cell breakage and the adenylate cyclase from these cells was found to be quite sensitive to adenosine and 2-chloroadenosine following homogenization. In order to investigate the possibility of cAMP involvement in adenosine-induced smooth muscle relaxation we have studied both the effect of adenosine on adenylate cyclase from the longitudinal muscle of rabbit ileum and the effect of adenosine or ATP on cAMP levels in the same tissue. General features of the action of adenosine and ATP on this muscle are described in a preceding paper (McKenzie et al., 1977).

2. Materials and methods 2.1. Materials and drugs

New Zealand White rabbits were used in all studies, and preparations of the longitudinal muscle of the intestine were dissected as described earlier (McKenzie et al., 1977). Tyrode solution contained (mM): 133.2 NaC1, 4.7 KCI, 1.9 CaCI~, 0.78 MgC12, 1.2 NaH~PO4, 18.6 NaHCO3 and 11.1 glucose and was aerated with 95% O~--5% CO2; the pH was 7.3-7.4. Adenosine, theophylline (aminophylline), 2'-deoxyadenosine, D,L-isoprenaline • HC1, Ladrenaline bitartrate and collagenase (Type I) were purchased from Sigma Chemical Co. Sources of other nucleosides and drugs were as follows: Nucleoside derivatives: Pharma Waldhof, Germany; 6-thioxanthine derivatives: May & Baker Ltd., England; CS'-alkyl derivatives of adenosine: Dr. H.P.C. Hogenkamp, University of Iowa, U.S.A; adenosine5'-nitrate: Dr. F.W. Lichtenthaler, Technische Hochschule Darmstadt, Germany; 1-methyl3-isobutylxanthine: Aldrich Chemical Co., U.S.A.

ADENOSINE EFFECTS IN SMOOTH MUSCLE

195

2.2. Preparation and assay of adenylate cyclase

bations were carried o u t at 37°C and usually lasted 15 min.

Longitudinal muscle was dissected from the entire length of the small intestine. After mincing finely, the tissue was incubated with 3 volumes of collagenase medium (containing 140 mM NaC1, 9.1 mM sodium phosphate, 5 mg/ml collagenase, 1 mg/ml glucose and 5 mg/ml purified bovine serum albumin, pH 7.4) at 37°C for 3 h and shaking at 140/min. Following digestion the contents of the vials were poured through nylon netting and the residues were washed with medium not containing collagenase. The filtrate was centrifuged at about 500 X g for 2 min and the sedimented cells (0.1--0.2 ml packed cell volume per g tissue) were washed twice with 5 volumes of phosphate buffer. Individual spindle-shaped cells could be observed microscopically under phase contrast. The cells were susceptible to mechanical damage unless resuspended gently and the yield o f cells was considerably reduced when the collagenase incubation exceeded 3 h. After resuspension in 5 volumes of 10 mM Tris-HC1, pH 7.5, and 1 mM MgC12 at 4°C, homogenization was carried o u t with a glass homogenizer and a hand-driven teflon pestle. This procedure disrupted all cells and the homogenate was centrifuged at 1000 X g for 15 min. The resulting pellet was washed and resuspended in a small volume of the same buffer. Using this procedure, the membrane preparation neither sedimented nor coagulated during incubation or after freezing and thawing, and basal and sodium fluoridestimulated activities were approximately 15--30 and 50--60 pmoles/mg/min, respectively. The assay of adenylate cyclase was carried out as described previously (B~ir, 1975). Triplicate assays contained in a 0.05 ml volume 25 mM sodium N-2-hydroxymethylpiperazine-N'-2-ethanesulphonate (pH 8), 5 mM MgC12, 0.5 mM sodium cAMP, 0.1 mg/ml creatine kinase, 10 mM sodium creatine phosphate, 0.1 mM ATP-a-32P (500,000 cpm), enzyme and other additions as indicated. Incu-

2.3. Measurement o f cAMP in longitudinal muscle strips Strips of the longitudinal muscle of the rabbit small intestine were mounted in organ baths as described previously (McKenzie et al., 1977) and contractile activity was measured via isometric force transducers (Grass FT3, Beckman dynograph recorder). The baths were m o u n t e d in such a way that they could be dropped rapidly by pressing a lever, thus allowing quick freeze-clamping of the tissue with liquid nitrogen-precooled Wollenberger clamps. The total time required for this procedure including the time of freezing was less than 2 sec. Sampling was carried o u t as closely as possible to the peak of the isometric contractions. Individual frozen strips were enclosed in a Teflon capsule containing 0.3 ml 5% trichloroacetic acid and a tungsten carbide ball, all at the temperature of liquid nitrogen. The capsule was fitted to a microdismembrator (Braun Melsungen) and vibrated for 30 sec (5 mm amplitude). The resultant 'homogenate' powder was collected in plastic centrifuge vials (1.5 ml), allowed to thaw and a b o u t 2000 cpm 3H-cAMP were added to allow monitoring of the recovery of tissue cAMP through the purification steps. After centrifugation at about 10,000 X g (Eppendorf Microcentrifuge) and removal of the supernatant, the protein pellet was washed with 0.1 ml 5% trichloroacetic acid and the pooled supernatants were applied to 0.5 X 7 cm columns containing Dowex AG50W-X4 ion exchanger (H ÷, 100--200 mesh) previously acid-washed and equilibrated with distilled water. Successive elutions with 0.5 ml volumes of distilled water were carried out. The neutral cAMP fraction was collected in 3--6 ml, depending on the resin batch, and after freeze drying was redissolved in 0.1 ml H20 and stored frozen until assay. The cAMP was assayed by a protein-binding method (Gilman, 1970} utilizing a cAMP-binding protein pre-

] 96 pared f r o m b e e f heart (Bfir, 1 9 7 5 ) . Aliquots o f the fractions were c o u n t e d for r e c o v e r y o f 3H-cAMP. F r o m time t o t i m e cyclic nucleotide p h o s p h o d i e s t e r a s e - t r e a t e d c o n t r o l s were p e r f o r m e d , using an e n z y m e p r e p a r e d f r o m pig cerebral c o r t e x (Ebadi, 1 9 7 2 ) . Over the c o n c e n t r a t i o n range o f cAMP c o n t e n t usually e n c o u n t e r e d , the n u c l e o t i d e was h y d r o l i z e d over 90% b y the e n z y m e , as c h e c k e d b y using 3H-cAMP as substrate and p a p e r c h r o m a t o g r a p h y (1 M a m m o n i u m a c e t a t e : 95% e t h a n o l , 30 : 75). T h e p r o t e i n pellets o b t a i n e d a f t e r extraction and c e n t r i f u g a t i o n were dissolved in 0.2 ml o f 0.5 M N a O H and t h e n the p r o t e i n conc e n t r a t i o n was d e t e r m i n e d b y the m e t h o d o f L o w r y et al. (1951). T h e cAMP c o n t e n t was expressed as p m o l e s / m g acid-precipitable and NaOH-solubilized p r o t e i n .

3. Results

3.1. Preparation of adenylate cyclase from rabbit intestine Initial studies with a d e n y l a t e cyclase f r o m m i n c e d longitudinal muscle o f the r a b b i t small intestine ( 1 0 0 0 ) , ' g washed pellet) revealed m a j o r p r o b l e m s . T h e activity was highly c o n t a m i n a t e d with ATP h y d r o l i z i n g enz y m e s leading t o rapid substrate d e p l e t i o n even in the presence of an ATP regenerating system and the m e m b r a n e material sedimented during assay and s h o w e d a m a r k e d t e n d e n cy t o coagulate, especially u p o n freezing and thawing. This p r o b l e m was p r o b a b l y caused b y collagen fibres. Basal activity m e a s u r e d u n d e r these c o n d i t i o n s was below 10 p m o l e s / m g / m i n and n o sensitivity t o adrenaline was noted. When the tissue was digested with collagenase and cells were isolated and h o m o g e n i z e d as described u n d e r Materials and m e t h o d s , basal activity was m u c h i m p r o v e d . Fig. 1 shows details of the time course o f the cAMP f o r m a t i o n and ATP levels u n d e r the conditions o f o u r assay in the absence and presence

s.G. MCKENZIE ET AL. i

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Fig. 1. Effect of enzyme concentration on the time course of cyclic AMP formation and ATP levels. Assays were carried out in a total volume of 0.2 ml and at time intervals single 3--5 pl aliquots were chromatographed on PEI plates as described by B~ir(1975). Protein concentrations were 0.19 mg/ml (o . . . . . . o) and 0.47 mg/ml (c). . . . . . o). Dotted lines are in the presence of 10 mM NaF. Upper graph: cyclic AMP formation. Lower graph: ATP levels as % of initial theoretical value (0.1 mM).

o f 10 mM NaF. Based on these results we have a d o p t e d a standard i n c u b a t i o n time o f 15 min at a p r o t e i n c o n c e n t r a t i o n o f a b o u t 0 . 3 - - 0 . 5 mg/ml. A l t h o u g h the e n z y m e c o u l d be p r e p a r e d b y a relatively mild h o m o g e n i z a tion p r o c e d u r e and the resulting specific activity was unusually high, the e n z y m e still did n o t show a n y sensitivity t o adrenaline (lO-S--lO -4 M).

3.2. Effect of adenosine and nucleosides on adenylate cyclase T h e e f f e c t o f a n u m b e r o f nucleosides (1 mM) on the e n z y m e activity ( b o t h freshly p r e p a r e d and a f t e r freezing and thawing) assayed u n d e r s t a n d a r d c o n d i t i o n s was determ i n e d . All c o m p o u n d s had been t e s t e d previously for t h e i r s m o o t h muscle r e l a x a n t

ADENOSINE EFFECTS IN SMOOTH MUSCLE

197

TABLE1 Effect of analogs and derivatives of adenosine on smooth muscle adenylate cyc]ase. " F r e s h " enzyme was assayed within 20 min of preparation without prior freezing. " F r o z e n " enzyme is from a different preparation and was stored under liquid nitrogen prior to the assay. Basal activities in the absence of any nucleoside were 25 and 15 pmoles/mg/min for fresh and frozen enzyme respectively. Numbers in parentheses 5 I preceding C -alkyl-substltuted nucleosides refer to those given in the original reference (Walker et al., 1973). Compounds marked by an asterisk show marked relaxant activity (McKenzie et al., 1977). Analog

% Activity in presence of 1 mM analog Frozen enzyme

Fresh enzyme

Substiluted purine ribonucleosides (PRN's) PRN ~ 6-Amino-PRN (adenosine)* 6-Hydroxy-PRN (inosine) 6-Chloro-PRN 6-Methyl-PRN 6-Methylamino-PRN* 6-Dimethylamino-PRN 6-Hydroxyamino-PRN* 6-Isopentenylamino-PRN* 6-Methoxy-PRN 6-Mercapto-PRN 6-Methylmercapto-PRN 2-Chloro-6-amino-PRN* 2 -Amino -6-chloro-PRN 2 -Ami no -6 -mercapto-PRN Adenosine-N 1-oxide*

87 56 104 98 89 85 96 64 102 110 68 62 66 97 69 107

86 55 82 92 93 90 85 68 91 87 95 -72 92 82 75

8-Substituted nucleosides 8-Bromoinosine 8-Bromoadenosine 8-Bromoguanosine

102 87 82

102 99 86

47 88 89 81 81

55 94 89 81 80

Nucleosides with alterations at the sugar 2'-Deoxyadenosine 2',3'-Isopropylidene-inosine 2',3'-Isopropylidene-adenosine Adenosine-5'-nitrate* 2',3'-Diacetyl-adenosine

Analog

a c t i v i t y ( M c K e n z i e e t al., 1 9 7 7 ) . A d e n o s i n e caused about 50% inhibition of enzyme activity. The results from one series of assays carried out with single enzyme preparations are s h o w n in t a b l e 1; g r o u p s o f n u c l e o s i d e s w e r e assayed previously with different preparations yielding essentially the same effects.

% Activity in presence of 1 mM analog Frozen enzyme

CS'-Alkyl-substituted adenosine derivatives C s' HOH--CH3* (3) C s' HOH--CH2NO2* (4) C s', HOH--CH2NH 2 (8) C s H=CH-CO(OCH2---CH3 )* (9) C s' H--CH--COOH* (10) C s' H2--CH2-CO(OCH2--CH3 )* (11) C s' H2---CH2--COOH* (12) C s' H2--CH2--CH2OH* 6-Thioxanthines (TX) 1,3-Dipropyl-TX 1,3-Dibutyl-TX 1-Methyl-3(3P-methoxypropyl)-TX 1-Methyl-3-(~3-phenethyl )-TX 1 -Methyl -3 -furfu ryl-TX 1,3-Diethyl-TX 1-Methyl-3-isobutyl-TX 1-n-Butyl-3-methyl-TX 1 -Methyl -3 n -propyl-TX 1-Methyl-3-benzyl-TX

Fresh enzyme

64 66 66

84 62 --

53 62

66 55

77 96 71

74 61 64

90 89

115 105

55 62 55 76 61 66 66 82

95 105 87 85 97 112 77 102

Compounds which showed pronounced relaxant activity on the same smooth muscle p r e p a r a t i o n ( M c K e n z i e e t al., 1 9 7 7 ) a r e m a r k e d b y a n a s t e r i s k . I t is e v i d e n t t h a t w i t h i n t h i s group two compounds which effectively relax the muscle do not cause any cyclase inhibit i o n a t all, n a m e l y a d e n o s i n e - N l - o x i d e a n d

198 6-isopentenylaminopurine ribonucleoside. On the other hand, three compounds caused better than 30% inhibition of the enzyme without possessing any relaxant effect, including 6-mercaptopurine, 2-amino-6-mercaptopurine ribonucleoside and 2'-deoxyadenosine. A number of other compounds show weaker enzyme inhibition while also possessing no relaxant activity. None of the compounds used caused any stimulation of the cyclase activity.

3.3. Variability of cAMP measurements In spite of the fact that the spontaneous contractile behaviour of strips from the longitudinal muscle of rabbit ileum obtained from different animals was very similar and reproducible, it soon became evident that the cAMP content of this muscle was very varialbe. In particular, the variability between animals was considerable and was greater than that between strips from control and treatment groups from a single animal. This finding does not appear to be caused by an artifact of the tissue processing or the methods used, since no correlation existed between cAMP levels and parameters such as the size of samples or the recovery of labelled nucleotide during the purification procedure; there was no statistically significant difference in cAMP c o n t e n t of fresh strips and those stored for 3 h at 4°C and then equilibrated again for 1 h at 37°C; there also was no difference between strips sampled by freeze-clamping as closely as possible to the relaxed and contracted state; repeated assays of a group of samples invariably confirmed any seemingly high or low values of cAMP content in such a group. Measurement of cAMP content in two strips each out of 18 animals yielded a range of 1.77--17.75 pmoles/mg (mean +S.E. = 6.1 -+ 0.54). This variability required that statistical analysis of data be performed in a paired or block design, comparing drug-treated tissues only with controls obtained from the same animal under closely identical circumstances

S.G. MCKENZIE ET AL. including timing and m e t h o d of handling.

3.4. Effect of drugs on cAMP levels Since the hypothesis of a possible involvement of cAMP in adenosine-induced relaxation was largely based on the analogous pathway proposed for ~-adrenergic drug action, isoprenaline and adrenaline were chosen as control drugs to verify the applicability of our m e t h o d to detecting changes in cAMP content. Although adrenaline relaxes the muscle predominantly via a-adrenergic receptors, a/3-adrenergic c o m p o n e n t is also present. The pattern of adenosine-induced relaxation closely resembled that to adrenaline in exhibiting an initial fast c o m p o n e n t followed by a sustained phase of lesser magnitude. With isoprenaline the initial fast c o m p o n e n t was absent. To determine roughly the time dependence of the cAMP response with the drugs to be tested, strips were treated with 1 pM isoprenaline, adrenaline and adenosine, and sampling was performed 10 and 60 sec following drug addition. As shown in table 2, adenosine at this concentration did n o t induce any change in cAMP levels at either time of exposure, while causing about 44 and 23% relaxation at 10 and 60 sec, respectively. Isoprenaline and adrenaline, on the other hand, appreciably elevated cAMP levels at both time intervals, the effect being significant at the 60 sec time point with adrenaline and at both 10 and 60 sec with isoprenaline. In view of these results all further studies were confined to the 60 sec interval following drug applications which corresponds to the stable, secondary phase of the relaxant response to adenosine and adrenaline. During investigation of the effects of other doses of adenosine and the catecholamines on contractility and cAMP levels, the influence of theophylline on these parameters was evaluated simultaneously. This was of interest because 0.1 mM theophylline was found to potentiate relaxant responses to isoprenaline and adrenaline while antagonizing those to

A D E N O S I N E E F F E C T S IN S M O O T H M U S C L E

199

TABLE 2 E f f e c t o f drugs o n c o n t r a c t i l i t y a n d tissue c A M P c o n t e n t at d i f f e r e n t times. C o n t r a c t i l i t y was r e c o r d e d a n d s a m p l i n g p e r f o r m e d at 10 a n d 60 sec f o l l o w i n g drug a d d i t i o n as d e t a i l e d in t h e text. Results are f r o m 6 p r e p a r a t i o n s a n d are related to u n t r e a t e d c o n t r o l tissues b y paired t-test. Mean values a n d s t a n d a r d errors are listed, c A M P : p m o l e s / m g , p values refer to t h e significance o f increases in c A M P c o n t e n t . Drug 1 pM

Results

Sampling time after drug addition 10 sec

60 sec

Isoprenaline

% Relaxation cAMP p

52.3 ± 6.2 +1.93 ± 0.82 < 0.05

85.0 ± 3.7 + 6 . 3 0 ± 2.52 < 0.05

Adrenaline

% Relaxation cAMP p

86.2 ± 3.9 +0.83 ± 0.40 0.1 > p > 0.05

87.8 ± 2.1 +1.91 ± 0.26 < 0.05

Adenosine

% Relaxation cAMP p

43.8 ± 7.1 +0.86 ± 1.16 NS

22.8 ± 2.9 + 0 . 3 0 ± 0.36 NS

TABLE 3 Effect of drugs o n c A M P levels a n d c o n t r a c t i l i t y . In a given analysis, 4 strips f r o m each o f 6 animals were p r o c e s s e d s i m u l t a n e o u s l y in o r g a n b a t h s (see t e x t for details) a n d were e x p o s e d t o (a) n o t r e a t m e n t , (b) to a drug ( i s o p r e n a l i n e , a d r e n a l i n e or a d e n o s i n e ) , (c) t o t h e o p h y l l i n e or MIX, a n d (d) t o b o t h a drug plus t h e o p h y l l i n e or M I X at t h e c o n c e n t r a t i o n s i n d i c a t e d . A: T h e range o f cAMP values over t h e six r e p l i c a t i o n s o f m e a s u r e m e n t s in each t r e a t m e n t g r o u p is listed t o g e t h e r w i t h t h e m e a n (5 S.E.M.) m a g n i t u d e o f t h e r e l a x a n t r e s p o n s e (negative values = increase in c o n t r a c t i l i t y ) . B: T h e d a t a o n cAMP c o n t e n t were processed b y a 2-way analysis o f variance ( A N O V A ) , a n d t h e r e s u l t a n t significance statem e n t s are given for each t r e a t m e n t individually a n d for t h e i n t e r a c t i o n b e t w e e n drugs a n d t h e o p h y l l i n e or MIX. Analysis

A: s u m m a r y o f effects

B: A N O V A results

Addition

% Relaxation

pmoles cAMP/mg (range)

Source of variation

Significance

None 0.05 p M I s o p r e n a l i n e 0.1 m M T h e o p h y l l i n e Both

-43 ± 6 (--) 5 ± 6 72 ± 4

2 . 5 0 - - 8.66 2 . 9 1 - - 8.66 2.39--11.13 3 . 3 3 - - 9.98

Animals Isoprenaline Theophylline Interaction

p < 0.001 0.1>p> 0.05 0.05 > p > 0 . 0 2 5 NS

None 0.05 p M A d r e n a l i n e 0.1 m M T h e o p h y l l i n e Both

-43 ± 7 (--)11 ± 5 55 ± 6

1.77--10.18 2 . 2 7 - - 7.55 2 . 7 4 - - 9.21 2.75--10.13

Animals Adrenaline Theophylline Interaction

p < 0.001 NS NS NS

None 10 g M A d e n o s i n e 0.1 m M T h e o p h y l l i n e Both

-31 ± 6 (--) 8 ± 5 11 ± 2

3.71--17.75 4 . 2 2 - - 8.84 4 . 1 0 - - 8.13 4 . 0 1 - - 9.41

Animals Adenosine Theophylline Interaction

0.25>p> NS NS 0.25>p>

-66 ± 7 45 ± 6 73 ± 4

2 . 2 6 - - 6.9 2 . 5 2 - - 5.65 2 . 8 2 - - 7.24 3.03--11.50

Animals Adenosine MIX Interaction

p < 0.001 0.25>p> 0.1 0 . 0 2 5 > p > 0.01 NS

None 0.1 m M A d e n o s i n e 10/~M M I X Both

0.1

0.1

200

S.G. MCKENZIE ET AL.

adenosine (data not shown). At a dose of 0.1 m M , t h e o p h y l l i n e e n h a n c e d t h e c o n t r a c t i l e a c t i v i t y s l i g h t l y (+8 +- 3%, n = 18, s i g n i f i c a n t ) . In a d d i t i o n , t h e e f f e c t o f a n o t h e r xanthine derivative and cyclic nucleotide phosphodiesterase inhibitor, 1-methyl-3-isob u t y l - x a n t h i n e ( M I X ) , w h i c h was r e p o r t e d t o antagonize adenosine-induced accumulation o f c A M P levels in b r a i n slices ( H u a n g e t al., 1 9 7 2 ) , was e v a l u a t e d on a d e n o s i n e - i n d u c e d r e s p o n s e s in t h e r a b b i t i n t e s t i n a l m u s c l e . Tissues f r o m o n e a n i m a l w e r e d i s s e c t e d , p o o l e d a n d a l l o c a t e d at r a n d o m t o a given t r e a t m e n t group. 4 strips were processed s i m u l t a n e o u s l y as a b l o c k of d i f f e r e n t l y t r e a t ed s a m p l e s . A f t e r t h e e q u i l i b r a t i o n p e r i o d in the organ bath, any relaxant response to a given t r e a t m e n t was r e c o r d e d in d u p l i c a t e p r i o r t o s a m p l i n g . T h e 4 s t r i p s in a b l o c k , e a c h u n d e r its o w n s p e c i f i e d c o n d i t i o n s o f time and drug treatment, were then sampled w i t h i n a t i m e p e r i o d o f 10 m i n b y r a p i d l y d r o p p i n g the bath and i m m e d i a t e l y freeze-

c l a m p i n g . 1 s t r i p s e r v e d as a c o n t r o l , 2 s t r i p s were treated with 1 drug each and the fourth was t r e a t e d w i t h b o t h drugs in c o m b i n a t i o n . T h i s e x p e r i m e n t a l design p e r m i t t e d e v a l u a t i o n o f t h e a b s e n c e or p r e s e n c e o f i n d i v i d u a l d r u g e f f e c t s as well as o f t h e i r i n t e r a c t i o n in t e r m s o f p o t e n t i a t i o n or a n t a g o n i s m b y a 2 - w a y a n a l y s i s o f v a r i a n c e . 6 r e p l i c a t i o n s f r o m different animals comprised the complete block design. A s u m m a r y of t h e r e s u l t s o b t a i n e d is c o n t a i n e d in t a b l e 3. I s o p r e n a l i n e at 0 . 0 5 pM c a u s e d a b o u t 43% r e l a x a t i o n a n d i n d u c e d a b a r e l y s i g n i f i c a n t i n c r e a s e in c A M P c o n t e n t (0.1 > p > 0 . 0 5 ) . T h e o p h y l l i n e a t 0.1 mM p o t e n t i a t e d t h e r e s p o n s e b u t t h e e f f e c t of b o t h drugs on c A M P levels was n o t significantly different from additive. A d o s e o f 0 . 0 5 pM a d r e n a l i n e f a i l e d t o e l e v a t e c A M P levels a l t h o u g h c a u s i n g a b o u t t h e s a m e e x t e n t of r e l a x a t i o n as i s o p r e n a l i n e . Theophylline potentiated the adrenalinei n d u c e d r e l a x a t i o n b y a b o u t 30% b u t d i d n o t

TABLE 4 Effect of high adenosine or ATP concentration on cAMP levels. 6 tissues from 1 preparation were used in each of the 3 treatment groups (+/-- adenosine-ATP) in an experiment and the effect of drug treatraent on tissue cAMP content was evaluated by a 1-way analysis of variance. The least significant increases (L.S.I.) were calculated by substituting the error mean squares (obtained from the analysis of variance) as the closest estimates of population variance in the equation for t (unpaired Student's t-test, 1-tailed), prechoosing a l value of 5%. Experiments 1 and 2 were performed with tissues from one animal (as were 3 and 4), those using ATP being done 30--60 min after those using adenosine. AR = adenosine. Experiment

Treatment

cAMP (pmoles/mg) mean ± S.E.M.

Difference from Control

L.S.I.

11.19 -+ 1.40 8.01 -+ 0.32 8.92 ± 0.81

---3.18 --2.27

+2.32

1

Control 0.1 mM AR 1 mM AR

2

Control 0.1 mM ATP 1 mM ATP

1.91 + 0.35 2.20 ! 0.30 2.29 ± 0.25

-+0.29 +0.33

+0.78

3

Control 0.1 mM AR 1 mM AR

5.04 ± 0.37 5.27 -+ 0.38 5.20 -+ 0.53

-+0.23 +0.16

+1.04

4

Control 0.1 mM ATP 1 mM ATP

1.85 ± 0.20 2.03 ± 0.15 1.95 +- 0.23

-+0.18 +0.10

+0.48

ADENOSINE EF F EC T S IN SMOOTH MUSCLE

show any interaction with adrenaline on cAMP content. At 10 pM adenosine did not influence cAMP content but relaxed the muscle by a b o u t 31%. This effect corresponded to a b o u t 50% of the maximal obtainable response seen usually with 0.1--0.4 mM adenosine. Theophylline (0.1 mM) reduced the relaxant response and showed no effect on cAMP levels alone or in combination with adenosine. Adenosine was further tested at a concentration of 0.1 mM in combination with 10 pM MIX. At this concentration MIX relaxed the muscle by a b o u t 45% and 0.1 mM adenosine alone relaxed it by a b o u t 66%. MIX significantly elevated cAMP levels by itself; however, adenosine, which relaxed the muscle to a larger extent than MIX, did n o t show a significant increase in this parameter. Also, no interaction between the two drugs was detected. In a final series of experiments we investigated the possibility that high doses (0.1 and 1 mM) of adenosine or ATP may increase cAMP levels in the muscle. Since at these concentrations maximal responsiveness of the tissue was invariably obtained, muscle relaxant activity was not measured in parallel and muscle strips were incubated without the imposition of tension. Results of the study are summarized in table 4 and show that in 4 experiments, each consisting of three treatment groups with 6 replicate observations from one animal, no statistically significant effect on cAMP content in the muscle was observed using a one-way analysis of variance.

4. Discussion The findings of the present study do not support the involvement of the cAMP/ adenylate cyclase system in the relaxant action of adenosine and ATP in the rabbit intestine. This conclusion is based on our negative findings of a relationship between adenosine responses and cAMP levels in the tissue. The absence of a stimulatory effect of adenosine on isolated adenylate cyclase, however, does

201

not allow any definite conclusions since cell breakage conceivably may cause the loss of sensitivity of the enzyme to hormones or humoral agents. Although our method of cell breakage was rather gentle it is interesting to note that despite this procedure no adrenaline sensitivity of the enzyme could be observed. This finding contrasts with our observation that 1 pM adrenaline elevates cAMP in muscle strips and with the conclusion of others that the intact muscle contains a catecholamine sensitive cyclase system. The possibility must be considered that this enzyme is actually not located in the muscle cell itself; we have not ascertained whether our preparation of isolated muscle cells still retained catecholamine sensitivity in terms of a cAMP response. The method of isolation of intestinal muscle cells described here may be of general interest and it is conveivable that refined methodology along these lines may lead to the isolation of clean muscle fibres also suitable for other biological studies. The procedure may also be applicable to other smooth muscles. The lack of any correlation between the relaxant activity of nucleosides and their inhibitory effect on isolated adenylate cyclase clearly rules out any functional connection between these parameters. The fact that inhibition of adenylate cyclase from many sources has been observed previously suggests that this effect is not likely to be related to any specific physiological function. It is noteworthy in this context that 1 mM adenosine had no effects on endogenous cAMP levels while inhibiting in vitro cyclase activity by about 50% The effects of catecholamines on tissue cAMP levels were not very pronounced although they were statistically significant at the higher concentration (1 pM). We would find it difficult if not impossible to derive unambiguous data on the causal relationship between ~-adrenergic drug action on relaxation and on tissue cAMP levels. Both the variability of the cAMP content, in particular between animals, and the magnitude of the effect

202

would complicate this task. Nevertheless, under conditions where increases of cAMP levels were seen with drugs such as adrenaline, isoprenaline and MIX, adenosine did not show any effect on this parameter although causing degrees of relaxation similar to catecholamines. Even at high concentrations neither ATP nor adenosine showed any effect on cAMP levels and no interaction with inhibitors of phosphodiesterase was evident. Since theophyUine is known to antagonize adenosine effects in other systems, one would expect this drug to block possible effects of adenosine on cAMP levels a s well. MIX, on the other hand, should lead to a potentiation of the effect on endogenous cAMP levels due to inhibition of intracellular phosphodiesterase. The absence of any type of interaction between these drugs and adenosine with respect to cAMP levels strongly speaks against a role of the cyclic nucleotide in adenosine- or ATPmediated relaxation. We would not extrapolate our conclusions to other smooth muscle systems in the absence of experimental data, although the possibility of entirely different results in other muscle preparations appears unlikely to us. The longitudinal muscle preparation has a relatively high content of muscle cells. Some smooth muscle preparations also contain a high proportion of other cell types including nerve cells. In view of the marked effects of adenosine on cAMP levels in the systems referred to in the Introduction, any effects of the nucleoside on cAMP levels in a given tissue would be difficult to interpret in relation to its physiological function.

Acknowledgement This study was supported by the Alberta Heart Foundation. S.M. was the recipient of a studentship by the Medical Research Council of Canada.

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