Role of cyclic AMP in steroid action in rat intestine

287

Biochimicaet BiophysicaActa, 970 (1988) 287-291 Elsevier BBA12280

Role of cyclic A M P in steroid action in rat intestine Faustino Franco and Ronald S. Shaft Departmento/ Zooio~, The University.Sheffield(U.K.) (Received 4 December 1987)

Key words: cycfic AMP: Steroid: Sodium ion transport: (Rat intestine)

We have investigated the effect of mineraloeortieolds and glucoenrtieoids on intestinal fluid and eleetroge~ glucose-linked Na + transport across isolated sections of the rat small intestine. A rapid m vitro response is observed that contrasts with the delay normally associated with stemkl hormone responses. Cyclic AMP is known to affect intestinal glucose, water and Na ÷ transport and the effects of the steroids may be understoed in terms d an inhibitory effect on intestinal ¢ydic AMP production. An inin'bitery effect d stereids on membrane.bound adenylate cydase has been demonstrated and dose-response effects suggest me presence of specific membrane.bound glucocorficoid receptors, Introduction The rat smaU intestine plays a vital role in helping to conserve the large amounts of fluid secreted into the intestine during digestion. Different sections of the adult rat intestine display different levels of activity and if the intestine is divided into five equal parts it is the mid section that shows the highest fluid uptake activity [1]. The higher levels in the proximal and mid sections are mainly determined by a glucose-dependent transfer, known to be Na+-dependent and involving two distinct pathways, one of which is electro-neutral the other electrogenic [2]. Both glucocorticoids and mineralocorticoids affect fluid and Na + transport [3] and receptors for both steroids are present throughout the intestine [4,5]. Aldosterone is known to play a~aimport,:nt role as

Abbreviations: raft, mucosal fluid transfer; PD, electrical potential difference. Corr~pondence (present address): R.S. Shaft, INSERM 33, Hopital De Bicetre, Bicetre 94270, France.

a mineralocorticoid in the colon [6], but its role in the small intestine is less well dcf;ned. There is good evidence that aldosterone affects ionic movements in ",he small intestine, but it is generally accepted that glucoc~rt~coids play a more important role [7-10]. We have previously investigated the in vivo effects of aldosterone and corticosterone on a sodium-linked ammonia release in the intestine [11]. Aldosterone has an effect at physiological concentrations, but corticosterone is able to stimulate even higher levels of exchange. Our present work on the effects of aldosterone and corticosterone on fluid and glucose.linked electrogenic sodium ion transport was undertaken to investigate further their mechanism of action. Methods Male Wistar rats, 21 days old, were killed by a sharp blow to the head and the small intestine was excised from stomach to caecum, washed through with 0.9% saline and everted using a thin metal rod. The everted gut was held (without stretching) in a bath containing phosphate Ringer solution

0167-4889/88/$03.50 © 1988 FJsevier Science Publishers B.V. (Biomedical Division)

288

(133 mM NaCI, 10 mM KCI, 0.7 mM CaCI2, 0.6 mM Nail,PC4, 3.1 mM Na2HPO,, and 0.01 mM sodium acetate) at a room temperature of 20 ° C. Ligatures were tied around the everted intestine to produce five sections of equal length, designated I-V in the direction of stomach to caecum. The intestine was then cut at each of the ligatures to produce five open ended sacs. Each sac was blot dried, weighed then filled with a volume of phosphate Ringer solution, roughly equivalent to tl?o. weight of the empty sac, and then reweighed. The sacs took approx. 2 rain to prepare, therefore the fifth sac remained in the trough almost 8 rain longer than the first. The experiment was repeated several times and proximal and distal sections were alternatively set up first. Each everted sac was incubated, at 37 o C, for 1 h in 80 ml phosphate buffer containing 30 mM glucose. The mucosal solution was bubbled with oxygen throughout the incubation period after which the sacs were removed, blot dried and reweighed. In vitro steroid treatment involved use of a luminal mucosal bathing medium containing either 10 ag aldosterone (0.35 aM) or 10 ag dexamethasone (0.32 aM). Restdts for the mucosal fluid transfer are expressed as means :t: S.E. g/h per g wet weight tissue and the number of experiments are indicated. An unpaired t-test was used to establish the level of significance for the difference between the means. The difference being taken as significant when P < 0.05. Several animals were weaned at the age of 21 days and placed on a Labsure high salt diet with free access to tap water. At various ages, small groups were killed and the values for intestinal mucosal fluid transfer was measured in several sections of the intestine. The results were averaged to give a mucosal fluid transfer value for the intestine as a whole. In electrophysiologieal measurements, the everted gut was divided into eight 6-em sections (A-H) in the direction stomach to caecum (proximal to distal) after discarding about 6 cm at each end. Four sections of gut was attached to perfusion chambers (Fig. 1) that were filled with phosphate Ringer solution (10 ml) and oxygenated using an oxygen lift pump. Each chamber was then placed in a beaker contains 80 ~ phosphate

i

""-plas|k:tiOs

Fig. 1. Shows the perfusion chamber used to study membrane potentials across isolated section of the rat small intestine. Each intestinal section (thick lines) was held between plastic cones, connected by rubber tubing (hatched lines) to the glass chamber, that was then Idled with 10 ~ phosphate Ringer solution. This Ringer solution was circulated by an oxygen lift pump and the outside bathing Ringer solution (80 ml) was oxygenated by bubbling.

Ringer solution, maintained at 37°C, and efficiently oxygenated by bubbling. The electrical potential difference (PD) across each membrane was measured using KC! salt bridge,s connected via Calomel electrodes to an amplifier and the input terminals of a BBC computer. After 2 mill equilibration, the membrane potentials were recorded and glucose was added to both sides of each of the four membranes sufficient to bring its concentration up to 4 raM. Membrane potentials were then recorded every 20 s and sufficient glucose was added every 80 s to increase its concentration each time by 4 raM, up to a final glucose concentration of 32 mM. The PD readings were recorded onto a disc and the experiment was repeated several times until at least six determinations on each section had been obtained. A similar procedure was then carried out in the presence of dexamethasone (100 pg/1). In a final series of experiments a number of mid sections (C-F) were studied in the presence of varying concentrations of dexamethasone or aldosterone. For measurement of adenylate cyclase activities in crude membrane fractions, the small intestine

was taken from four young animals and rapidly washed with 0.9% saline. Mid sections were isolated and the epithelialceUs removed by scraping the mucosal surface with a ~croscope slide. These calls were then homogenised in phosphate Ringer and centrifuged at 100000 x g for 1 h after which the pellet was taken for adenylate cyclase assays using the technique described by Salomon et at. [12]. The measurements were repeated, using the same enzyme preparation, in the presence of several concentrations of dexamethasone, aldosterone or corticosterone and the results are expressed in pmol/mg protein per rain. Results

In these young animals, intestinal mucosal fluid transfer is significantly lower in proximal sections of the small intestine (Fig. 2) and the effect of in vitro treatment with steroids is to significantly increase the mucosal fluid transfer in proximal and mid sections. At these concentrations, both steroids had a stimulatory effect but no attempt was made to determine the most potent steroid as III

r"

~ ~.6, 1.4,

n

i"

7

I

,4] o

1.2

289

~x L

Mucoscl fluid tronfer

1.0

\

~'; Q6[ ~Eo2| " /

~-° UL

50

100

150

2O0

250

Age t 'lays) Fig. 3. Shows the intestinal macosal fluid transfer as an

average measured in five sectionsof the small int~fin¢ of eight rats taken at various ages after weaning onto a high salt diet, at 21 days old. 'I'n,,.results represent the mcan:l:S.F., the fast group of animals being taken 1 week after weaning.

the sensitivity of Ae response is limited and a detailed dose.response study would have proved difficult. After weaning onto the high salt d~et, intestinal mucosal fluid transfer gradually decreases and is low in adult animals (Fig. 3). It was impossible to fred any in vitro effects of steroids on the adult intestine mu~sal fluid transfer or an in rive effect within 4 h following interpefitonead injection with aldostcrone (19 pg). However, aldosterone (10/~g) injected iatcraperitoneally into 234-day-old rats increased the intestinal mucosal fluid transfer to 1.11 :t: 0.12 (n = 10) g/h per g tissue, when measured after a 27-h delay. The membrane potential results for particular sections of the intestine were analysed by computer to obtain average PIE)4- S.E. values at each recorded

~ 11.8,

'! OA" 0.2Control

Aldosterone

n:14

n:6

(i)

(ii)

Oexomethosone

n:8 (iii)

Fig. 2. Shows(i) the pattern of mucosal fluid transfer (increase in sac weight/h per g wet weight tissue) measured in proximal to distal sections (I-V) of the rat small intestine, taken from H-day-old rats. Similar measurements in the pres~ac¢ of (ii) aldosterone (0.35/LM) or (iii)dexamethasone ~" 32 t~M). (i),

(ii) and (iii) represent the mean:t:S.E. for 1/ ments, respectively.

'

sd 8 experi-

~ 0.1 ,.. I

l

l

INCREASEDPOTENTIAL0|FFERENCE(QFOmV) Fig. 4. A typical computer plot of APD/glucos¢ concentration against APD from which an 'apparent Kin' and APDmL, have been calculated.

290

E

20

jdr ~',

//" /I °

~ ._c

n u~ ~.s s o

E 10

¢=

5

<

t

~

A

B

i

|

i

C D E section

i

i

G

H

i

F

Fig. 5. Shows how the 'apparent Kin' values vary along the length of the rat small intestine in the absence (e) or presence (o) of dexamethasone (3.2 /~M). Each point represents the mean of at least six determinations and the bar defines the standard deviation as obtained by regression analysis of the Eadie Hofstee plots.

intervaland the cumulative increasein PD (APD) was determined following each glucose addition. These values were used to construct an Eadie Hofstcc plot (Fig. 4) from which two 'apparent Km' and ZiPDma x values have been calculated. Regression analysisof the resultswas carried out in order to determine the standard deviation of the coefficients.The basis for this treatment is that the A PD value is determined by the rate of electrogenic glucose-l~nked Na + transport and, when analysed by NfichaelisMenten kinetics,the

,~ 15 E

)examethason~ E 1o ~ i,~~ , T/Aldostepone

v

:}

X cl

<

0 0.0010.01 0.1 1 Steroid concentrotion ( # 9

10 °1o)

Fig. 6. Dose-response curves obtained for the 'appar¢nt K m' values determined in the presence of various concentrationsof dexarnethasoneor aldosterone.The results represent the means ± S.D. of at least six determinations using isolated ~ d sections of H-day-old rat small intestine,

i

jlc

Aid°//

,~[

e.. -- -eJ / e ~

Dexa

5 6 ,

L

7

B •

;i

i

Steroid concentration (-(og M )

Fig. 7. Shows the dose-response curves obtained for aldosterone (o) corticosterone (e) and dexamethasone (&) in. hibition of intestinal adenylate cyclase activity. The results repre~nt the mean of duplicate assays at each steroid con-

centration. All assays were carried out using the method of Salomonet al. [12]with the sameenzymepreparation.

APDm= may be considered equivalent to a V ~ and the 'apparent Km' equivalent to the ratio of the rate constants determining glucose uptake. The 'apparent Km' values for glucose-stimulated membrane potentials in various sections of

the small intestine are shown (Fig. 5). Proximal sections of the intestine have characteristically higher 'apparent Km' values and in the presence of dexamethasone these values are increased. The APDmax values tend to increase in parallel with the 'apparent Kin' values, but not consistently and not to the same extent. The dose-response curve (Fig. 6) obtained for dexamethasone-increased 'apparent Kin' values in mid sections is characteristic of the saturation of corticosteroid receptors. In contrast, aldosteroneincreased 'apparent Km' values occur when using concentrations well above physiological levels. Adenylate cyclase activity measured in pellet fractions obtained from rat intestinal mucosa demonstrates an inhibitory effect in the presence of steroids. The dose-response effects (Fig. 7) show that corticosterone is the more potent steroid and that it is active at physiological concentrations. The response to aldosteronc occurs at conccntra-

291

tions above its physiological concentration range and would appear to reflect its saturation of specific gluco~orticoid receptors. Discussion

Many hormone receptors exert their effect at the cell surface interacting with guanine nucleotide-binding regulatory proteins (G proteins). These proteins directly regulate the production of a second messenger, which in the case of many hormones is cyclic AMP. The activity of adenylate eyclase, the cyclic AMP producing enzyme, is controlled by two G proteins one of which (Gs) stimulates the enzyme, the other (Gi) inhibits the enzyme. Cyclic AMP is known to have a fluid secretory role in the intestine [131, so an increased mueosal fluid transfer could well reflect a decreased cyclic AMP concentration. The in vitro effects of steroids on mucosal fluid transfer, evident within 15 rain, could, therefore, be understood in terms of an inhibibition of eyelie AMP production. Intestinal glucose absorption, that may be characterized by analysis of associated electrical events [15,16] is also affected by cyclic AMP [14]. The increased 'apparent Kin' values following steroid treatment suggests a decreased affinity for glucose absorption that might also be expected to accompany a decreased cyclic AMP concentration. The dose-response characteristics for these steroid effects on glucose-stimulated membrane potentials and on adenylate cyclase activity in membrane fractions is therefore taken to suggest the presence of glucocorticoid-specifi¢ membrane receptors coupled to a G i protein at the cell surface. Preliminary [3H]dexamethasone-binding studies (results not shown) suggest that the effect is indeed due to the presence of steroid receptors located within the membrane. This is not the first report of a steroid inhibitory effect on membrane-bound adenylate cyclase. Finidori-Lepicard et al. [17] have reported a progesterone inhibition of membrane-bound adenylate cyelase in Xenopus laevis oocytes. It therefore appears that steroid hormones may ezereise three levels of developmental and functional control: they induce permanent changes in tissues such as the intestine [18], probably by a mechanism in.

volving chromatin activation; they induce gene activation by well-charaeterised mechanisms in. volving steroid receptor interaction with responsive DNA elements upstream of the promoter [19], and the present work suggests that they may also exercise a post transcriptional control involving membrane receptors coupled to G proteins. Although the receptors are gluccr.~rticoidspecific, they appear to be dependent on aldosterone stimulation. Intestines taken from adult animals on a high salt diet no longer demonstrate in vitro responses to the steroid, and only after 27 h following a single aldosterone injection is the intestinal response evident. The time lag suggests either an aldosterone-dependent synthesis of membrane rec~'vtors that take some time to be come incorporated into the membrane or, as suggested by Crocker and Munday [9], may reflect the activation of crypts cells that take some time to move into the villus region. References 1 Barry, B.A., Matthews, J. and Smyth, D.H. (1961) J. Physiol. 157, 279-288. 2 Lyon, L and Crane, R.K. (1986) Biochim. Biophys. Acta 112, 278-291. 3 LeAn, RJ. 0969) J. Endocrinol. 45, 315-348. 4 Lentze, M.T., Morey, P.C. and Trier, J.S. (1978) Gastroenterology 74,1055-1065. 5 Pressley, L. and Funder, J.W. (1975) Endocrinology,97, 588-596. 6 Edmonds, CJ. (1972) J. Ster. Biochent 3,150-154. 7 Levin, RJ. (1965) J. Physiol. 177, 58-73. 8 Levin, R.J,, Newey, H. and Smyth, D.H. (1965) J, Physiol. 177, 58-73. 9 Crocker, A. and Munday, K.A. (1969) J. Physiol. 202, 329-364. 10 Debnam, E.S. and Levin, R.J. (1975) J, Physiol. 252, 681-700. 11 Coates, D.R. and Shaft. R.S. (1984) J. Physiol. 354, 1-IO. 12 Salomon, Y., Londos, C. and Rodbell, M. (1974) Anal. Biochem. 58, 541-548. 13 Binder, H,L (1979) Mechanism of intestinal secretions. Kroe Foundation Series, Vol. 12, Allan R. Liss, New York. 14 Luche, H., Kinne, R. and Muter, H. (1979) in Mefhanisms of Intestinal Secretions (Binder, HJ. ed.) pp. 111-116, Allan R. Liss, New York. 15 Levin, R.J. and Syme, G. (1971) J. Physiol. 213, 46-48P. 16 Fromter, E. (1982) Pfluser Arch. 393, 179-189. 17 Finidori-Lepieard, J., Sehorderet-Slatkine,,S., Hanotme, J. and Baulieu, E.E. (1981) Nature 292, 255-257. 18 Henning, EJ. and Jefferson, M.S. (1979) Endoerinolo~ 104,1158-1163. 19 Buetti, E. and Kuhnel, B. (1986) J. Mol. Biol. 190, 379-389.