Studies on adenyl cyclase in Necturus gastric mucosa

ARCHI\‘liH

OF

13IOCHF:~fIIHTRY

Studies

on

.\NlJ

Adenyl

STiM 10 IYAKASIMA, Dicision

of

IUOPIIYRI(!S

Guslroen/ero~ogy,

143,

12.7-1’36

Cyclase HASIT,

(1971)

in Necturus

I. HIRSCHOWITZ,

Depnrlnrenl of Afedicine, University Birmingham, A lobarna 35233

Received September

Gastric

Mucosa’

AND GEORGE

SACHS

nf .~~dmtna

of 1tfedicine,

fkhool

21, 1970; accepted December 23, 1970

Adenyl cyclasc activity of Secturlls gastric mucosa was det.ermined by measuring the amount of radioactive adenosine 3’,5’-cyclic monophosphate formed from SHlabeled adenosine triphosphat,e. Histamine, peut.agastrin, and fluoride added in vitro significantly increased fundic adenyl cyclase activity. The dose response curves show that the affinity of pentagastrin for adenyl cyclasc is great,er than that of histamine, whereas t,he peak response to pentagastrin is less thau that, to hist.amine. additive stimulation was not obtained when maximal doses of pcntagast rin and hist.amine were combined. These findings suggest. that gastric mucosa contains a single adenyl cyclase uuit which is coupled to dist,inctive selectivit,y sites for gast.rin and hist amiua. S-Benxyl-3-pyrrolidyl acrtat.e methobromide (AIIR-602), t,he mtlscarinic compound, caused a significant reduction in aden.yl cyclase activity, indicat,in, u a differcut mechanism of stimulation of gastric acid secretion for cholinergic muscariuir compounds.

Adenosine 3’) 5’-cyclic monophosphate (cyclic 3’) ?-AMP) provokes acid secretion in isolated amphibian gastric mucosa (1, 2). Cyclic S’,;“-AMP is formed from ATP in the reaction cat,alvaed by adenyl cyclase, and recent investigations (1, 2) indicate that, methylxanthine derivat,ivcs such as theoph.y!line stimulde gastric acid secretion by inhlblting phosphodiesterasc which catalyzes hydrolysis of the 3’- bond of cyclic 3’,5’-AJIP. The basic question arises as to whet.her the effects of other gastric secretnRoguesare mediated via cyclic 3’) 6’-AlIP. The objective of the present work was to determine the effect of ;I variety of gastric secretugoguesand fluoride on adenyl cyclnse activity in Necturus gastric mucosa. MA4TERIALS

AND METHODS

The gastric mucosae from A-eclurlts macdoszc.s f.common name: mudpuppy) were stripped of outer muscularis mucosa and the Rmdic mucosal membranes were minced aud homogenized wit,h a -._---.. -. ..1 This study was supported by the National Inst.itut.es of Health, Grants AM-08541, AM-09260 and TIAM-5286, and the ?iational Sciencse Foundation, Grant GB-8351.

motor-driven Teflon pestle (A. II. Thomas) in cold 0.2 M sucrose (10: 1, v/w). The crude homogenate was centrifuged at 3,600 g for 10 min. The supernatant was then cent,rifuged at 10,000 g for 20 min to obt,ain a crude mitochondrial fraction and the supernatant decanted; the particles were puspended in dist,illed water and protein concentration determined by the procedure of Lowry ef a.1. (3). The crude mitochondrial fractions of many tissues have been shown to contain significant adenyl cyclase act ivit.y (4-6). These results do not imply, however, that. adenyl cyclase is mitochondrial, since the mitochondrial frac+ons may well contain membrane fragments (7). The amount, of W-labeled cyclic 3’,5’-AMP formed was measured by a modification of the method of Krishna el al. (4). One-fifth milliliter aliquots of the particular fraction were incubated at. 37” for various times in a total volume of 0.6 ml with Tris-1ICl buffer, pH 7.4 (3 X lO+ M); MgSO( (3 X l(r3 M); theophylline (lW* M); ATP (1.6 X 10m3M) ; ‘VI-ATP (NW Englaud Suclear, 1.8 X 10-o MM).The fiual specific activit.y of 3H-ATP wae 16 &i/pmole. Additions of histamine, pentagastrin, N-benzyl-3-pyrrolidyl acetate methobromide (AHR-602), aud NaF were as indicated. The reactious were terminated by the addition of 0.1 ml (0.5 mg) of unlabeled cyclic 3’,5’-AMP, and by placing the tubes in boiling water for 3 min. After

123

124

NAKAJIMa4,

HIL
A?KL) SACIIS

I

2.000 ATP,ADP 2 % N

1.500

Histamine Pentagastrin

B 5 5 3 0

1.000

0.500

s 0 2

4

6

8 FRACTION

IO

12

14

16

I8

20

NUMBER

FIG. 1. Chromatographic separation of ATP, ADP, cyclic 3’,5’-AMP, and 5’-AMP on I)owex 50-H+ (5OW-S2, 200400 mesh) columns (1.6 X 2.0 cm). Each compound (0.5 mg) was added bo the incubation medium for adenyl cyclase assay which consisted of Tris-HCI, pff 7.4 (3 X 1O-8 M), MgSOl (3 X 1(r3 M), and theophylline (lO+ M). Columns were &ted with distilled water and 2-ml fractions were collected. The absorbance at 260 rnp wa8 determined.

centrifugation to remove t,he precipitate, the entire supernatant. from each tube was chromat,ographed on Dowex 50-H+ (J. T. Baker Chemical Co.; 5OW-X2, 200-400 mesh) columns (1.6 X 2.0 cm), prepared by pipetting 4 ml of a 50% (w/v) suspension of the resin into column8 and washing with water. The column was eluted with water and 2-ml fraction8 were collected. Approximately 45% of cyclic 3’,5’-AMP wa8 recovered in the sixth fraction (Fig. 1). ?u’ucleotides other than cyclic 3’,5’-AMP were precipitated twice with 0.2 ml each of 0.25 M ZnSOc and 0.25 M Ba(OH)p as described by Krishna et al. (4). The supernatant sol&ion (0.5 ml) was added t,o 15 ml of a 1,4-dioxane medium containg 70 g/l. naphthalene, 7 g/l. 2,5-diphenyloxazole (PPO), and 0.05 g/l. POPOP-1,4-bis2-(5-phenyloxazolyl)-benzene, and t,hen counted for 3H-labeled cyclic 3’,5’-AMP in a Beckman liquid scintillat,ion counter. The results were expressed a8 pmoles cyclic 3’,5’-AMP produced per milligram protein. The statistical significance of results was evaluated by application of t test for paired samples. RESULTS

Figure 2 illust.rat,es the efiect of dukion of incubat,ion on the formation of cyclic 3)) St-AMP by crude mitochondrial fraction from Necturus gastric mucosa with or without gastric secretagogues. The amount of cyclic 3’) 5’-AMP measured progressively

TIME

IminI

FIG. 2. The effect of l(ra 11 histamine, 5.0 X 10-G N pentagastrin, aud 1.6 X lO+ M AHR-602 on the formation of cyclic 3’,5’-AMP by adenyl cyclase in Neck-us gast.ric mucosa. Kach point represents the mean of five experiments. Vertical bars indicate SE of the mean.

SECRETAGOGUE

L

(M)

AHR

FIG. 3. Dose response curves for adenyl cyclase activity in response to pentagaatrin (PG), histamine (HA), and AHR-602. Incubation8 were carried out at 37” for 20 min. Each point represents t.he mean of three to five experiments.

increased with the time of incubation for 520 min. As can be seen from Fig. 2, adenyl cyclase activity was significant.ly increased by 1O-z11 hist.amine (p < 0.06 when compared with control at, 20 min), and to a lesserextent by 5.0 X lo+ hz pentagastrin (p < 0.05 compared with cont,rol at 15 and 20 min). The amounts of cyclic 3’,5’-AMP formed at. 15 and 20 min 1vit.h 1.(i X 10m411AHR-MI? were

ADENYL TABLE

CYCLASE

IN GASTRIC

I

THE COMBINED EFFECTS OF Maxrm~ DOSES OF PENTAGASTRIN A.I‘D HISTAWINE ON ADENSL CYCL~SE ACTIVITY IN Xecturus GASTRIC Mucosa Adenyl cyclase activitp (o/u increase from control)

secretagogue

Pentagastrin (lO+ XX) Histamine (10-a ?a) Pentagastrin (10-j M) -I- Histamin (1W8 M)

60.8 f 14.0 93.4 r.k 18.8 94.5 f 18.9

u Values are mean f SEM for five separate analyses. Incubations were carried out at 37” for 2OJnin. T

f:,/

1.52.0 /

//

/ P

Fluoride

/ 1.0 -

TIME

(min)

FIG. 4. The effect of 1W2 M sodium fluoride on the formation of cyclic 3’,5’-AMP by adenyl cyclase in Necturus gastric mucosa. Each point represents the mean of five experiments. Vertical bars indicate SE of the mean.

less t,han those wirithout, AHR602 (p < 0.05). Figure 3 shows dose response curves for pentagastrin, histamine, and AHR-602. The peak responseswere obt,ained with concentrations of about, 1.0 X lo+ M pentagastrin, 1.0 X lOwaN histamine, and 1.6 X 1O-4 11 AHR-602. The cyclase activation by combinat.ions of maximal dosesof pent.agastrin and histamine was not’ significantly different from that by maximal doses of histamine given singly (Table I, p > 0.05). As shown in Fig. 4, lo+ Y NaF had a marked st.imulatory effect on adenyl cyclase activit.y during incubat.ion periods varying

significant,ly

MUCOSA

1%

from 15 t.o 20 min (p < 0.05 when compared with control). I)ISCUSSION

Recently Perrier and Laster (8) have report,ed t.hut, l&amine and its analogs, bet,azole and aminoethyltriazole, and prostaglandins stimulate adenyl cyclase in guinea pig gastric mucosa t,hree- t.o four-fold, and that. choline cst,ers and gastrin have 110 effect. They concluded that t.heir observations might, support the hypothesis that. histamine is, or is related to, t.he final common stimulus for gastric acid secretion. The results of t,he present study, however, shorn that, in Necturus gastric mucosa, fundic adenyl cyclase is activated by histamine, pentagastrin and fluoride, and inhibited by AHR-602, the cholinergic muscarinic compound. Since many peptide hormones do not appear to affect, phosphodiesterase (9), the results presented suggest, that both histnmine and pentagastrin may increase the intracellular level of cyclic 3’) ,?‘-AMP. However, in previous studies ,in vitro, some differences were observed bet,ween the effects of pentagast.rin and t.heophyline (3). Furthermore, resting Nectuws gastric mucosa is relatively refractory to a large amount of histamine-requiring lo-” 11 for maximal acid responseof upproximat.ely 0.6 pEq/hr. cm!---a response which is significantly less than that to pentagastrin (10). Since combinat.ions of maximal doses of pent.agastrin and histamine do not. produce additive effcct.s on adenyl cyclase activity, it. is tentatively concluded that gastric mucosa contains a single adenT cyclase unit which is coupled to dist.inctwe selectivity sites for pcnt.agast.rin and histamine. While fluoride was t.he most,potent stimulant of adenyl cyclase activit,y in gastric mucosa, as also reported in other bissues(11)) it failed to evoke acid secretion in Necturus gastric mucosa in z&o, possibly because of its cyt.otoxic effect. Similarly, prostaglandin, which stimulates gastric adenyl cyclase (S), apparently inhibits gastric acid secretion in vitro (13). Fluoride has also been shown to inhibit, (Sa+ + Ii+)-ATPase activity from st.omach and heart, (13), brain (14), and erythrocytes (15). Schramm and Naim (16)

126

NAKAJIMA,

HIRSCHOWITZ,

postulate that, fluoride and Mg2+ form a complex wit,h the specific inhibit.ors of adenyl cyclase so that t.he inhibition is largely abolished. It should also be noted that, fluoride does not, increase cyclic 3’) 5’-AMP concentrations in thyroid slices despite the fact that it enhances thyroid adenyl cyclase activit.y (17). It is of special interest that AHR-602 caused a significant, inhibition of adenyl cyclase activity in Necturus gast,ric mucosa.. The muscarinic compounds, therefore, st.imulat,e acid secret.ion in vitro (10) by a mechanism which is unrelat,ed to activation of adenyl cyclase, and is highly susceptible to atropine. Similar effects of cholinergic compounds have been observed by hfurad et al. (18) who demonstrated bhat acetylcholine, carbachol and acetyl-/3-met,hylcholine inhibit cardiac adenyl cyclase and that) atropine prevents the inhibitory effect of carbachol. If acid secret.ion is mediated by cyclic 3’,5’-AMP, then the gastric secretagogues will either stimulate adenyl cyclase or inhibit phosphodiesterase. The effect of muscarinic compounds on phosphodiesterase act,ivity remains to be established. It has been shown, however, t.hat carbachol does not, increase t.he concentrations of cyclic 3’, 5’AMP in t.hvroid slices (17) and that, acet,ylcholine, which stimulates glucose oxidation, phospholipid synthesis, snd phosphorylaae activit,y (19, 20), does not activate adenyl cyclase in t.hyroid (21). Thus it seems probable that. the muscarinic compounds may exert similar met,abolic effects on gastric mucosa by a mechanism unrelated to co11centrations of cyclic 3’,5’-AMP. In the light of t.hese findings, it appears reasonable to quest.ion whether the effects of all gastric secretagogues are mediat,ed solely by t:he adenyl cyclase-cyclic 3’) 5’-AMP system. ACKNOWLEDGMENTS The aut.hors are grateful to Dr. T. Robitscher of Ayerst Research Laboratories, New York, New York, for pentagaatrin AY-6608 (I.C.I. 50,123) and

AND

SACHS

to Dr. J. W. Ward of Robins Research Laboratories, Richmond, Virginia, for AHR-602. REFERENCES HARI~IS, J. B., NIQON, K., AND ALONSO, D., Gustroenlero~ogy 67, 377 (1969). 2. NAKUIMA, S., SHOEM,\KE:R, R. L., HIRSCHOWITZ, B. I., AND SACHS, G., Amer. J. Physiol. 219, 1269 (1970). 3. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 266 (1961). 4. KRISHNA, G., WEISS, B., .~ND BRODIE, B. B., J. Pharmacol. Exp. Ther. 163,379 (1968). 6. SUTHERLAND, E. W., DYE, I., AND BUTCHER, IL W., Recent Progr. Hosm. Res. 21, 623 (1965). 6. HOLLINQXR, M. A., Life Sci. 9, 533 (1970). 7. BURKE, G., Endocrinology 86, 346 (1970) 8. PERRIER, C. V., AND LISTI~R, L., J. Clin. Znvest. 49, 73a (1970). 9. RO~ISON, G. A., BUTCHER, R. W., AND SUTRERLIND, R. W., Ann. Rev. Biochem. 87, 149 (1968). 10. N~KAJIMA, S., SHOEMAKER, It. L., HIRSCHOWITZ, B. I., AND SACHS, G., Amer. J. Physiol. 218, 990 (1970). 11. BRECKENRIDGE, B. M., Ann. Rev. Pharmacol. 10, 19 (1970). 12. WAY, L., :IND DURBIN, R. P., Nature London. 221, 874 (1969). 13. MOZSIK, G., Eur. J. Pharmacol. 7,319 (1969). 14. YOSHIDA, H., NAGAI, K., K.~MEI, M., AND NAKAGAWA, Y., Biochim. Biophys. Acta 156, 162 (1968). 15. FARIAS, It. N., GOLDEYBERG, A. L., AND TRUCCO, R. E., Arch. Biochem. Biophys. 139, 38 (1970). 16. SCHRAMM, M., AND NAIX, E., J. Biol. Chem. 246, 3226 (1970). 17. KANEKO, T., ZOR, U., END FIILD, J. B.. Science 163. 1062 (1969). 18. MURAD, F., CRI, Y. M., RALL, T. W., AND SUTHDRLAXD, E. W., J. Biol. Chem. 287,1233 (1962). 19. ALTMAN, M., OKA, H., AND FIELD, J. B., Biochim. Biophys. Acta 118, 586 (1966). 20. BOTCHER, F. R., AND SERIF, G. S., B&him. Biophys. Actu 166, 69 (1968). 21. PAST.~N, I., AND KATZEJI, R., Biochem. Biophys. Res. Commun. 29, 792 (1967). 1.