Subclasses of muscarinic receptors in isolated gastric mucosal cells: Receptor characterization and parietal cell function

European Journal of Pharmacology, 94 (1983) 281-295

281

Elsevier

S U B C L A S S E S O F M U S C A R I N I C R E C E P T O R S IN I S O L A T E D G A S T R I C M U C O S A L CELLS: R E C E P T O R C H A R A C T E R I Z A T I O N AND P A R I E T A L CELL F U N C T I O N MARGITTA ALBINUS * and DIETRICH WINNE Department of Pharmacology, University of Tiibingen, Wilhelmstrasse 56, D- 7400 Tiibingen, F.R.G.

Received 22 November 1982, revised MS received 21 June 1983, accepted 25 July 1983

M. ALBINUS and D. WINNE, Subclasses of muscarinic receptors in isolated gastric mucosal cells: receptor characterization and parietal cell function, European J. Pharmacol. 94 (1983) 281-295. Muscarinic receptors were characterized in isolated intact chief and parietal cell enriched cell populations from canine and guinea-pig gastric mucosa by binding of tritiated N-methylscopolamine ([3H]NMS). Antagonist and agonist binding was studied by displacement of [3H]NMS with non-radioactive atropine, pirenzepine, pilocarpine and carbachol. Model analysis points to the existence of two binding sites in each of the two cell populations. The number of binding sites per cell was 1.7-1.8 times higher in parietal than in chief cell populations. Subclasses of muscarinic receptors as characterized by pirenzepine binding were compatible with the suggested A- and C- (high and low affinity) binding sites. The observation that in canine cells GMPPNP induced a conformational change of the high affinity binding site for pirenzepine could suggest that their proportion might depend on environmental factors. Binding parameters were related to specific parietal cell function as measured by aminopyrine accumulation as index for acid secretion. The carbachol effects depended on the calcium concentration and were competitively inhibited by pirenzepine. The physiological relevance of muscarinic receptor heterogeneity in gastric mucosal cells is unknown although the data support the hypothesis that involvement of muscarinic binding sites in calcium transport mechanisms connected with parietal cell function and possible conformational changes of the binding sites might be regulatory parameters in gastric secretory processes. Cholinergic muscarinic agonists and antagonists Isolated gastric mucosal cells

Parietal cell function

1. Introduction In 1980 H a m m e r et al. reported that pirenzepine, a new anticholinergic drug used as therapeutic agent for treatment of ulcer disease, was the first antagonist that could discriminate between subclasses of muscarinic receptors. Further studies with the drug revealed that there were different muscarinic receptors in gastric mucosa and gastric smooth muscle as demonstrated by binding and displacement studies performed in tissue homogenates. Furthermore the Hill coefficient for binding in the mucosa was less than 1.0

* To whom all correspondence should be addressed. 0014-2999/83/$03.00 © 1983 Elsevier Science Publishers B.V.

Heterogeneity of muscarinic receptors

suggesting more than one binding site to be present in the mucosa. Knowledge about the location of subclasses of muscarinic receptors in gastric mucosa is speculative, although from pharmacological studies with pirenzepine a high affinity binding site was claimed to account for the antisecretory effects (Hammer, 1982). Since the gastric mucosa consists of different cell types with different secretory functions, studies on isolated and enriched cell populations would help to clarify cellular receptor distribution in connection with specific cell responses. More recently it was reported that cholinergic stimulation by carbachol increased calcium uptake by canine parietal cells (Soll, 1981b). This process appeared to run parallel with H + secretion as measured by [14C]AP uptake.

282

The aim of this study was to characterize cholinergic muscarinic receptors in different cell populations from guinea-pig and canine gastric mucosa in order to determine whether subclasses of muscarinic receptors would be located on different cell types. [3H]NMS as the classical antimuscarinic ligand was used as marker because it is characterized by very high affinity to muscarinic receptors and low nonspecific binding (Hulme et al., 1978). It had been reported in a preliminary communication that muscarinic receptors in intact enriched parietal and chief cell populations from guinea-pig were identical (Albinus, 1981). This paper reports on further studies with guinea-pig and canine gastric cells where subclasses of muscarinic receptors were described by model analysis, the distribution quantitated and possible changes in receptor conformation investigated. Furthermore a possible correlation between receptor occupancy and cellular function was investigated in parietal cell fractions.

2. Materials and methods

2.1. Cell isolation Isolated intact cells from canine and guinea pig gastric mucosa were prepared by pronase and collagenase digestion in principle according to the method described by Soil (1978). Canine gastric mucosa was obtained from starved beagles which were killed by i.v. injection of T 61 either immediately, 2 or 4 weeks following urological operation. Fed male guinea-pigs (200-350 g) were sacrificed by a blow on the head, bled, the stomach excised and the mucosa scraped off. For separation of guinea-pig cells the EDTA step was omitted. The incubation was performed in a buffer containing: N a H 2 P O 4 0.5, N a 2 H P O 4 1.0, N a H C O 3 20.0, NaC1 70.0, KC1 5.0, CaC12 1.0, MgC12 1.5, glucose 11.0 mmol/1, bovine serum albumin 0.1% (medium I). After isolation the cells were washed three times to remove the enzymes. Subsequently ceils were enriched due to their different density with Percoll ®. Briefly, 6.5 ml cell suspension were mixed with 2.7 ml Percoll * and 0.8 ml Hepes buffer

(0.25 mol/1), final density 1.03 g/ml, and centrifuged for 40 min at 190 × g. The top layer containing 60-80% parietal cells was taken off. The pellet was also carefully sucked off and consisted mainly of mucous and other small not further identified cells. The same volume of buffer was added to the remaining cell suspension and this centrifuged again for 5 min at 1000 × g. Residual parietal cells were located in the top layer. In the pellet, chief cells were enriched up to 60% as compared to other nonparietal cells. The different cell fractions were washed three times. Cell viability was about 95% as determined by trypan blue exclusion.

2.2. Binding and displacement studies For binding studies, parietal and chief cell enriched fractions were incubated with [3H]N-methylscopolamine ([3H]NMS). Guinea-pig cells were incubated in a buffer containing: NaC1 132.4, KC1 5.4, N a 2 H P O 4 5.0, N a H 2 p O 4 1.0, MgSO 4 1.2, CaCI 2 1.0, glucose 11.0 mmol/1, bovine serum albumin 0.1% (medium II). Binding studies with canine cells were performed in medium I. Cell bound ligand was separated from free ligand by centrifugation through siliconoil. Samples, 250/~1, of each cell suspension were put into 500 F1 microtubes preloaded with 50/*1 K O H (3 mol/1) and 100 /~1 of a 1:1 mixture of two siliconoils and centrifuged for 60 s at 10000 x g. The cell pellet subsequently dissolved in the KOH. The tips were cut off 12 h later, 10 ml diotol scintillator was added and the radioactivity measured in a liquid scintillation counter (Mark II, Nuclear Chicago, U.S.A.). Assays were carried out in triplicate. Nonspecific binding was defined as binding of [3H]NMS in the presence of 10 -5 mol/1 atropine. Specific binding was calculated as the difference between total and nonspecific bound [3H]NMS. [3H]NMS binding was expressed as cpm/106 cells. Displacement studies were performed to determine the dissociation constants for the two antagonists atropine and pirenzepine and the agonists pilocarpine and carbachol as well. Agonists were included in the study since they have been shown to discriminate different muscarinic binding sites in rat brain (Birdsall and Hulme, 1976; Birdsall et al., 1978). For displacement studies both cell

283 populations were incubated for 30 min at 37°C with [3H]NMS (10-9 mol/1) in the presence of the competing ligands. 2.3. Parietal cell function

Functional studies were performed with parietal cells. Parietal cells were stimulated with carbachol and H ÷ secretion was measured indirectly as the [14C]aminopyrine ([14C]AP) uptake using in principle the method described by Berglindh et al. (1976). Cells were preincubated for 5 rain at 37°C in medium I containing isobutylmethylxanthine (IBMX; 10 -6 mol/1) and [a4C]AP (1.65 × 10 -6 or 0.66 x 10 -5 mol/1 resp.). Carbachol was added and the incubation continued for 60 min. The uptake process was stopped by centrifugation for 30 s at 8 000 x g, the supernatant discarded and the cell pellet washed by resuspension once with ice-cold buffer. Finally the cell sediment was dissolved in 200 /~1 KOH (3 mol/1). Solution was completed after 12 h, radioactivity was determined as described above. Secretion from different parietal cell preparations was estimated in quadruplicate. When the inhibitory effect of pirenzepine on parietal cell H ÷ secretion was investigated, preincubation was performed in the presence of the antagonist. Calcium dependency of [14C]AP uptake was investigated by adding adequate amounts of CaC12 to the incubation medium. Cell protein was estimated by the method of Lowry et al. (1951) with bovine serum albumin as standard. All substances were dissolved in distilled water and subsequently diluted with saline. 2.4. Materials

[dimethylamine-14C]Aminopyrine (spec. act. 30 mCi/mmol, 114 mCi/mmol, Amersham-Buchler, Braunschweig, FRG); atropine sulfate; carbachol chloride; collagenase Type I (Sigma, Mtinchen, FRG); 5-guanylimidodiphosphate (GMP-PNP, Boehringer, Mannheim, FRG); bovine serum albumin, N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (Hepes, Serva, Heidelberg, FRG); isobutylmethylxanthine (IBMX, Sigma, M0nchen, FRG); [N-methyl-3H]scopolamine methylchloride

(spec. act. 53.5 Ci/mmol, NEN, Dreieich, FRG); PercoU ® (Pharmacia Fine Chemicals, Uppsala, Sweden); pilocarpine hydrochloride (Boehringer, Ingelheim, FRG); pirenzepine hydrochloride (Thomae, Biberach, FRG); pronase E (Merck, Darmstadt, FRG); siliconoil AR 20 and AR 200 (Wacker Chemie, Miinchen, FRG); trypan blue (Serva, Heidelberg, FRG); T 61 (20% N-[2-(mmethoxy-phenyl)-2-ethylbutyl(1)]-3,-hydroxy-butylamide; 5% 4,4'-methylen-bis-(cyclohexyl-trimethylammoniumjodide; 0.5% 4'-butylaminobenzoyl2-dimethylamino-ethanol-hydrochloride) (Hoechst, FRG). All other substances used were of analytical quality. 2.5. Data analysis

The following equations were fitted to the experimental data by means of a nonlinear regression method (Hartley, 1961; Daniel and Wood, 1971). 2:5.1. Binding experiments One-site model

Btot - - Rtot • L , / ( K m + L) + k,," L,

(1)

Bu, = k,n. L1

(2)

Btot = total binding (cpm/106 cells), Bun = nonspecific binding (cpm/106 cells), R t o t = total number of binding sites (cpm/106 cells), L 1 = concentration of marker ligand (mol/l), K D 1 = dissociation constant of marker ligand (mol/1), k~n = proportionality coefficient (cpm/106 cells. 1/mol). Hill model Bto, = Rtot • L~/(Km + L~) + kun • L1

(3)

and Eq (2) (n = Hill coefficient). Two-site model B,ot = Rtot • L l [ f l / ( K m l + L1) +(1 - f l ) / ( K m 2 + L1)] + kunL1

(4)

and Eq (2). fl = fraction of low affinity binding sites, KDn , Krn 2 = dissociation constant of marker ligand relative to low and high affinity binding sites (tool/l), respectively.

284

2. 5. 2. Displacement experiments One-site model B t o t -~-

Rto t • L1/[KD1-(1 + L2/KD2 ) + L,] + k.r,L,

(5) Bun = k un" L1

(6)

L1, L 2 = concentration of marker and displacing ligand (mol/1), respectively; K o l , KD2 = dissociation constant of marker and displacing ligand (mol/1), respectively. Hill model Btot = Rio , - L'~/[Ko~ .(1 + L~/KD2 ) + L~] + k . . L ,

(7) and Eq (6). Two-site model Btot = R tot" LI ((fl - f 2 ) / [KDal(1 + L2/KD2,) + L,] + (1 - f,)/[KD12 -(1 + L2/KD22) + La] + f 2 / [ K o , , . (1 + L 2 / K D : 2) + L,]) + k unL1

(8) and Eq (6). KD21, KD22 = dissociation constant of displacing ligand relative to low and high affinity binding sites (mol/l), respectively; f~ = fraction of total receptors with low affinity to marker and displacing ligand; f2 = fraction of total receptors with low affinity to marker and high affinity to displacing ligand. The dependent variable was the total and nonspecific binding, the independent variable the concentration of the marker and of the displacing ligand. The unknown parameters w e r e R tot, k un for each preparation, KD1, n, fl, KD11, KD12 for the binding and KD2, n, f~, Ki~2a, K D 2 2 for the displacement experiments. The unit of R tot was transformed finally into fmol/106 cells by means of the specific activity of [3H]NMS. In the analysis of the displacement experiments the concentration of the marker ligand L~ amounted to 10-9 mol/1. In the one-site model the value 4.48.10 -1° m o l / l was taken for KD~ (guinea-pig), a weighted mean of the dissociation constants obtained from the separate analysis of

each preparation in the binding experiments with [3H]NMS. In the Hill model L~ amounted to (10-9) 0.759 = 1.4757.10 -7 (0.759 is the Hill coefficient of [3H]NMS, see table 1). In the two-site model KDa 1 = 2.131 • 10 - 9 , KD12 = 1.875 • 10 10 and f~ = 0.694 were taken from the corresponding analysis of the binding experiments (see table 1). The analysis of the displacement experiments with danine cells was performed with KD1 = KDll = KD12 = 1.7.10 -8 mol/1. In order to obtain homogeneous variances the regression analysis was performed after logarithmic transformation of the total and nonspecific binding values. The data obtained from all preparations for one ligand were analysed simultaneously using the separate parameters R tot and k u n for each preparation and common parameters for K D~, n, f~ and so on, since the separate analysis of the chief cell and parietal cell enriched preparations by means of the one-site model revealed no significant difference for KD1. The fitting of the Hill and two-site model, respectively, was compared with that of the one-site model by the appropriate F-test (reduction of residual sum of squares versus residual sum of squares).

2. 6. Analysis of functional studies [14C]AP uptake was calculated as pmol/106 parietal cells and basal uptake was taken as 100%. The response to stimulation was calculated as percentage of or change above basal. Data were analysed using Student's t-test for paired or unpaired data. Dose-response curves were established for carbachol from 2 × 10 - 7 to 2 × 10 - 3 tool/1. The higher concentrations that would actually have been necessary to overcome the inhibition of pirenzepine at 2 × 10- 5 and 2 × 10 - 4 mol/1 impaired cell integrity to some extent and have therefore not been included in the calculation. Although dose ratios in the presence of pirenzepine might be uncertain due to these facts they were calculated from ICs0(IC30 ) values and a plot of log (dose ratio - 1 ) on log (antagonist concentration) (Schild plot) was constructed to establish the nature of the antagonism (Arunlakshana and Schild, 1959).

285 500

3. R e s u l t s

400

Most of the binding and displacement experiments were performed with isolated cells from guinea-pig gastric mucosa. Results obtained with canine cells are cited separately, and any differences between the two species were evaluated.

300

3.1. Receptor characterization

F3

?e~

y, Q

_z

3.1.1. [3H]NMS binding [3H]NMS binding was correlated linearly with cell number and found to be less in chief cell enriched populations than in parietal cells prepared from guinea-pig (fig. 1). The time course ot association of [3H]NMS to muscarinic receptors in guinea-pig parietal cell enriched populations (76% p.c.) at 20 and 37°C is shown in fig. 2. The association of [3H]NMS at a concentration of 10 -9 mol/1 occurred with a half life of about 6 min. Specific binding of [3H]NMS showed saturation (fig. 3). The affinity of [3H]NMS to the muscarinic receptors was identical in both cell populations. The number of binding sites was about 1.7-1.8 times higher in parietal cell (p.c.) than in chief cell (c.c.) enriched fractions. The concentration of re-

Z

~200 ~J =

h

~

/

k

r = 0.989

8n 100

I

I

I

I

I

I

0.2

0.4

0.6

0.8

1.0

1.2

I

I

1.4 1.6 x 106 CELLS

Fig. 1. Relationship between specific [3H]NMS binding and cell number as measured in isolated gastric mucosai cells from guinea-pig. F 2 = chief cell enriched (56.4% c.c.), F3 = parietal cell enriched (62.9% p.c.) cell population.

800 700

37" C •f

600

% Eo. 500 u L., 400 (D Z

•

~

'

~

A

&

20"

C

•

300 z o

200

0

~E z -r 100 L~ 0

i 10

~0-o

i 20

o

o

i 30

~

_

O

z~

o

,, _ _ _ _ _ _ . _ _ _ -

i 40

i 50

37" C ZO'C

i

60 TIME CmirO

Fig. 2. Time course of associationof [3H]NMS (10 - 9 mol/1) to muscarinic receptorsof guinea-pigparietal cells (76% p.c.) at 20 and 37°C. Open symbols: nonspecificbinding in the presenceof 10-5 mol/l atropine; solid symbols: specificbinding.

286

ceptors was calculated to be 5.58 _+ 0.33 fmol/106 cells in c.c. fractions (57.2 _+ 1.7% chief cells, n = 24) and 9.93 + 0.59 fmol/106 cells in p.c. fractions (64.8 + 2.3% parietal cells, n = 24). Approximately 7000 binding sites per parietal cell were calculated from one preparation containing 86% p.c., assuming one binding site for each molecule of [3H]NMS. Receptor density expressed per mg protein was about 80 f m o l e s / m g guinea-pig p.c. enriched protein. The K D values for [3H]NMS as obtained by analysing the experimental data from guinea-pig gastric mucosal cells with the above described models are summarized in table 1. There was no significant difference between the c.c. and p.c.

1200 1ooo

% .q

E .o -~

3.1.2. Displacement The binding characteristics for the two antagonists atropine and pirenzepine as well as for the partial agonist pilocarpine and the pure agonist carbachol were derived from competition experiments against [3H]NMS ( 1 0 - 9 mol/1). The KD2 values as calculated by the one-site model revealed no significant differences between chief and parietal cells.

3.1.2.1. Guinea-pig, antagonists Atropine: The KD2 value calculated with the

500

z

I

I

i

0

i

i

I

i

i

5xlO -9

i

I

Ix10-a

[mo.t] [3H]NMS 3.5

,,-I 3.0

E

enriched fractions. It can be seen that the K o value as calculated by the one-site model was 4.6 + 0 . 5 . 1 0 -1° m o l / l which is in very good agreement with data obtained from a variety of rat organs (Hammer et al., 1980) and from canine fundic mucosa (Hammer, 1980). The two-site model analysis revealed two binding sites for [3H]NMS. However in canine cells only one binding site with rather low affinity was identified for [3H]NMS (table 2). The binding sites in the two cell populations were not different.

2.5

one-site model was 1.4 _ 0.1 • 1 0 - 9 mol/1 and thus did not deviate from data obtained for rat cerebral cortex (Hulme et al., 1978). The Hill coefficient of 0.88 + 0.06 was not significantly different from 1.0 (table 1). Pirenzepine: The binding characteristics as established for this antagonist (table 1) are in agreement with data from canine mucosal homogenates as reported by Hammer in 1980 and 1982, substantiating the suggested A- and C- (high and low affinity) binding sites.

u

3.1.2.2. Guinea-pig, agonists Pilocarpine: The K o values for the partial

,'-, 2.0

~

1.5

~ z

1.0

I

-10

/

-9.5

I

-9

I

-8.5

I

-8

log [rnol/I][3H] NMS

Fig. 3. [3H]NMS binding in a chief cell enriched cell population from guinea-pig gastric mucosa. The upper panel shows the binding in c p m / 1 0 6 ceils versus concentration of N-methylscopolamine and the lower panel shows the data on a log

agonist pilocarpine derived from competition experiments and analysed with the models, suggested two binding sites on both cell types, whereby the low affinity binding site for [3H]NMS appeared to be further differentiated into high and low affinity binding sites for pilocarpine (table 1). scale. Total binding (A A), nonspecific binding (zx Lx). The curves without symbols are the respective differences between the two other curves and represent specific binding.

287 TABLE 1 Parameters of the models for the binding of antagonists and agnnists to cholinergic muscarinic receptors in isolated gastric mucosal cells from guinea-pig. One-site model

Hill model

Dissociation constant (tool/l)

Dissociation constant (mol/1)

Hill coefficient

P1

Two-site model Dissociation constant (mol/l) High affinity Low affinity

Fraction fl f2

P1

Data points

[3H]NMS

4.65:0.5.10 -1°

1.64-3.4.10 -7

0.765:0.08

b

184

1.35:0.1.10 -9

2.25:2.5.10 -8

0.884-0.06

n~

0.69 +0.23 (0.69)

b

Atropine

181

2.25:0.2.10 -7

2.5+1.6.10 - s

0.705:0.05

c

0.62 +0.21 0

ns

Pirenzepine

1.95:1.7"10 -1° 2.15:2.8.10 -9 9.7 + 4.7.10-10 4.5+16.6"10 -8 2.8+0.5.10 - s 1.95:0.3.10 -6

c

186

Pilocarpine

8.35:1.1.10 -7

2.04-2.3.10 -5

0.795:0.09

a

(0.69)

83

3.2 5: 0.4-10- s

5.6 5: 4.4-10- 4

0.74 4- 0.08

b

0.46 5:0.10 0

a

Carbachol

5.45:1.4.10- 7 2.35:4.4.10 -4 4.85:1.4-10 -6 2.6+__0.7.10 -4

b

118

Antagonist

(0.69)

A gonist (0.69)

fl: fraction of total receptors with low affinity to the marker and displacing ligand; f2: fraction of total receptors with low affinity to the marker and high affinity to the displacing ligand. P1: significance levels of the comparison of the Hill and two-site model, respectively, with the one-site model: ns Non-significant, a 0.05 > P > 0.01, b 0.01 > P > 0.001, ~ P < 0.001.

Carbachol: B y m o d e l a n a l y s i s o f t h e d a t a f r o m c o m p e t i t i o n b e t w e e n [ 3 H ] N M S a n d c a r b a c h o l as p u r e a g o n i s t t w o b i n d i n g sites a l s o w e r e e v a l u a t e d , w i t h t h e l o w a f f i n i t y s i t e s r e p r e s e n t i n g a b o u t 70% ( t a b l e 1). T h e d i s p l a c e m e n t c u r v e s f o r a g o n i s t s a n d

3.1.2.3. Dog The analysed binding data obtained with canine ceils a r e s u m m a r i z e d i n t a b l e s 2 a n d 3. B i n d i n g a n d d i s p l a c e m e n t s t u d i e s w i t h c a n i n e cells w e r e p e r f o r m e d i n m e d i u m I, w h e r e N a + w a s 92.5 m m o l / 1 a n d M g 2÷ 1.5 m m o l / 1 i n s t e a d o f 1 4 3 . 4 m m o l / 1 f o r N a + a n d 1.2 m m o l / 1 f o r M g 2÷ i n

a n t a g o n i s t s as s h o w n i n fig. 4 a r e b e s t fit c u r v e s through experimental data points from guinea-pig p a r i e t a l cells. N o s i g n i f i c a n t c h a n g e s i n b i n d i n g p a r a m e t e r s o c c u r r e d w h e n C a 2+ w a s i n c r e a s e d

m e d i u m II, i n w h i c h g u i n e a - p i g cells h a d b e e n i n c u b a t e d . I n c a n i n e cells o n l y o n e b i n d i n g site w a s f o u n d f o r [ 3 H ] N M S w i t h r a t h e r l o w a f f i n i t y as

f r o m 1.0 t o 1.8 m m o l / 1 . TABLE 2

The effect of GMPPNP on parameters of the one-site model for the binding of antagonists and agonists to cholinergic muscarinic receptors in isolated canine gastric mucosal cells. Dissociation constant (mol/l) No addition

Data points

+ 10-4 mol/l GMPPNP

Data points

1.704- 2.05.10- s (1.88-10- 7 _ 1.54.10- 9) a 3.60 + 0.55.10- 9 5.86 4- 0.71.10- 7

24

Not measured

48 48

3.96 + 0.79.10 - 9 ns 1.28 + 0.19.10- 6 ¢

24 24

24

1.20 + 0.12.10- 4 ns

24

Antagonist [ 3H]NMS Atropine Pirenzepine

Agonist Carbachol

9.70 4-1.22.10- s

a Values in parentheses represent 95% confidence interval on the log scale. Significance levels; ns = non-significant, c p < 0.001.

288

% z

70

z m

60

2~ 40

,-

30

o

20

O. u~

\

UA

tlJ U

e,-

0 -10 t-~

0

I

-11

I

-10

I

I

I

I

I

I

I

I

l

-9

-8

-7

-6

-5

-4

-3

-2

-1

log [mol/l'] LIGAND Fig. 4. The influence of antagonists and agonists on specific [3H]NMS binding to isolated parietal cells from guinea-pig gastric mucosa. Solid lines: one-site model; broken lines: Hill model. Atropine © ©, pirenzepine [] rn, pilocarpine t, zx, carbachol ~ - ~ . The curves were calculated using the parameters listed in table 1. Each point is the mean of n = 7-9.

compared with the data obtained for guinea-pig cells. The reason for this has not been evaluated as yet. In contrast the binding characteristics for atropine, pirenzepine and carbachol, respectively, did not deviate much from those obtained with guinea-pig cells.

3.1.3. Influence of guanine nucleotides on muscarinic binding characteristics In order to test the influence of guanine nucleotides on muscarinic binding affinity, displacement studies with atropine, pirenzepine and carbachol against [3H]NMS were also done in the

TABLE 3 The effect of GMPPNP on parameters of the two-site model for the binding of antagonists and agonist to cholinergic muscarinic receptors in isolated canine gastric mucosal cells. + 10 -4 mol/1 GMPPNP

No addition Dissociation constant (mol/1) High affinity Low affinity

Fraction of low affinity

P~

Dissociation constant (mol/1) High affinity Low affinity

Fraction of low affinity

P1

P2

1.22+0.51.10 -9 4.99 _+3.62-10- 8 3.87 + 4.42.10- 8 1.20+0.52" 10 -6

0.24+0.09

b

0.34--+0.13

b

ns

0.70 -+0.14

~

8.75+5.35-10 -1° 2.80 + 2.03.10 - 8 1.57 5: 5.37.10- 8 1.54+0.42-10 -6

0.92 -+0.08

ns

b

8.82 + 4.94.10-6 2.91 +0.85-10 -4

0.53 + 0.09

~

2.33 _+1.10.10- 5 4.74+ 1.82-10 -4

0.44 + 0.11

Antagonist Atropine Pirenzepine

Agonist Carbachol

PI: Significance level of the comparison between one- and two-site model. P2: Significance level of the GMPPNP effect in the two-site model (see table 1).

289 presence of 10 -4 m o l / l GMPPNP, a non-hydrolysable GTP-analogue. In guinea-pig cells, no effect of G M P P N P was seen on the binding of atropine whereas the results obtained with pirenzepine in parietal cells were not as clearcut. From the pirenzepine displacement curve a slight increase in the low affinity for pirenzepine appeared to occur. The results for carbachol were very similar, also showing a small increase in the low affinity. These effects were however not significant. One-site model analysis of the pirenzepine data obtained from studies with canine cells in the presence of G M P P N P suggested a small but significant increase in the K D value for pirenzepine (table 2). Two-site model analysis however showed that the proportion of low affinity binding sites increased significantly whereas neither the high nor the low affinity for pirenzepine changed (table 3). The binding characteristics for atropine and carbachol were not altered in the presence of G M P P N P (table 3).

cells was measured indirectly and was shown to be stimulated by carbachol in a dose-dependent manner. The cellular response depended on the extracellular calcium concentration, when parietal cell acid secretion was stimulated via muscarinic receptors, whereas histamine H2-receptor-mediated acid secretion was not significantly influenced by changes in extracellular calcium. [14C]AP uptake was maximally stimulated by about 10 - 4 mol/1 carbachol when the calcium concentration was 1.8 mmol/1, whereas approximately 10-100 times higher carbachol concentrations were necessary to evoke a similar response when calcium was lowered to 1.0 mmol/1. The results of one representative experiment with canine cells are shown in fig. 5. Increasing Mg 2÷ from 1.5 to 2.7 mmol/1 in parallel with calcium from 1.0 to 1.8 mmol/1 resulted in a reduced acid secretion; this was comparable to the effect of 1.0 mmol/1 Ca 2÷ alone. The response to muscarinic receptor stimulation was easier to demonstrate in canine parietal cells than in guinea-pig cells, whereas no difference was found in the response to histamine. In both species the carbachol-stimulated []4C]AP uptake was dose dependently blocked by pirenzepine. Dose-response curves for carbachol at different pirenzepine concentrations are given in fig. 6. Data points are the means of 4-7 different experiments. The

3. 2. Parietal cell function In order to obtain a correlation between muscarinic receptor occupancy and cell response, acid secretion by canine and guinea-pig parietal

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Fig. 5. Dose-response relationship for carbachol-stimulated [14C]aminopyrineaccumulation by canine parietal cells at different calcium concentrations.100% refers to basal [14 C]aminopyrineaccumulation.

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Fig. 6. Dose-response curves of [14C]aminopyrine accumulation by isolated guinea-pig parietal cells stimulated by carbachol (© ©) in the presence of different concentrations of pirenzepine ( 2 x l O - 7 A ,X, 2 X 1 0 - 6 [] Iq, 2×10 -5 ~, 2 X 10-4 v v mol/l). Each point is the mean_+S.E.M., n = 4-7. The inset represents a plot of log (dose ratio -1) versus -log (pirenzepine concentration) (Schild plot) with a slope n = -0.874_+ 0.041, which is not different from unity. The arrow indicates the pA 2 value for pirenzepine. calculated pA 2 value was 7.25. The slope of the Schild plot was not different from unity suggesting that in this model also the antagonism of pirenzepine appeared to be of a competitive nature.

4. Discussion The data presented in this paper confirm the existence and functional responsiveness of muscarinic receptors in intact isolated chief and parietal cells from dog and guinea-pig gastric mucosa. Since the data analysis revealed a Hill coefficient significarltly less than unity, a twobinding site model was chosen instead of a onebinding site model which more accurately described the experimental data. But the standard errors of the parameters were partially rather large and therefore a certain degree of uncertainty for the absolute values given in tables 1-3 might be implied. Although the binding and displacement studies described were performed with parietal and chief cell enriched and not with pure cell populations, the model analysis of the data sug-

gested that two types of muscarinic receptors are present in each of the two gastric mucosal cell types. In highly enriched parietal cell populations, binding could be extrapolated to 100% parietal cells by subtraction of the amount of marker ligand bound to nonparietal cells. Chief cell enriched populations contained less than 5% parietal cells beside mainly mucous cells. Rather low [3H]NMS binding was found in mucous cell populations (Albinus, unpublished data). The number of total muscarinic binding sites per cell correlated with the surface area as has been calculated for parietal and chief cells, respectively, and was confirmed by the finding of 1.7-1.8 times more muscarinic receptors per parietal cell whereas binding was identical when calculated per mg membrane protein. In the present study the muscarinic receptor density in guinea-pig parietal cell enriched cell populations was calculated to be about 80 f m o l / m g protein (approximately 7000 binding sites per parietal cell). Ecknauer et al. (1980) found the muscarinic receptor concentration in membranes prepared from rat parietal cells to be about 54 f m o l / m g protein, referring to approximately 4900 binding sites per

291 cell. For canine gastric mucosal homogenates a low receptor density of 2 pmol/g (-- 2 fmol/mg) was reported (Hammer, 1980). In gastric smooth muscle however muscarinic receptor density appears to be higher as becomes evident from results obtained for cat fundus with 240 fmol/mg protein (Rimele et al., 1981) and for rat fundus with 630 fmol/mg protein (Morisset et al., 1981). Comparison of the present data with results from the literature shows that muscarinic receptor characterization performed either by direct binding studies or by competing experiments in intact cells produced respective apparent dissociation constants which agreed rather well with K D and IC~0 values obtained from either binding or pharmacological studies with muscarinic receptors from homogenates of a variety of other organs like brain, heart, smooth muscle and exocrine glands (Birdsall et al., 1978; Hulme et al., 1978; Berrie et al., 1979; Hammer et al., 1980). Until recently it was thought that antagonists like N-methylscopolamine indicate apparent homogeneity of muscarinic receptor populations. Even if the data have to be interpreted with care they suggested that N-methylscopolamine also could discriminate subclasses of muscarinic receptors. The two receptor subtypes as described by [3H]NMS binding in guinea-pig parietal cell as well as in chief cell enriched cell populations were characterized by K D values which are very similar to data recently published for rat myocardium (Hulme et al., 1981). In rat heart however, muscarinic receptor heterogeneity was induced by use of low ionic strength medium and could be discriminated by N-methylscopolamine, propylbenzilylcholine and pirenzepine, respectively. Sodium and other ions as well as GMPPNP have been shown to reveal differences in the nature of agonist and antagonist binding in subtypes of muscarinic receptors in various body regions (Birdsall et al., 1977, 1979b; Ehlert et al., 1980; Rosenberger et al., 1980), therefore the apparent existence of only one [3H]NMS binding site found in this study in canine cells might have been due to the difference in the ion concentration of the medium used. One binding site for atropine has been characterized in guinea-pig cells. The apparent ex-

istence of two binding sites for atropine found in canine gastric cells (table 3) needs further substantiation. However, one intriguing finding in rat cerebral cortex was reported where the Hill coefficient of the atropine binding curve was 0.87 (P < 0.05) (Hulme et al., 1978). The results obtained for pirenzepine support the assumption that muscarinic receptors in the gastric mucosa are heterogenous (Hammer, 1982) and suggest that high and low affinity subclasses of muscarinic receptors are present on both parietal and chief cells from canine and guinea-pig gastric mucosa. The K D values refer to the proposed Aand C-binding sites (Birdsall et al., 1980). The small proportion of approximately 20-30% of high affinity binding sites might be of the same nature as the high affinity receptor subtype demonstrated in peripheral sympathetic ganglia which is now classified as muscarinic M 1 receptor subtype (Hammer, 1980; Hammer and Giachetti, 1982). It has been speculated that the intramural plexus in the gastrointestinal tract could be affected selectively by pirenzepine (Hammer, 1982). From pharmacological and clinical studies (for ref. see Hammer, 1982) including measurements of pirenzepine plasma levels (Hammer, 1982) it has been suggested that high affinity binding sites are responsible for the antisecretory effects of pirenzepine but it was assumed that they are not located on the parietal cell itself (Hammer, 1982). Our findings make it probable that the inhibitory effect on acid secretion will originate at least in part from interference with cellular receptors on the parietal cell itself. This was shown by the dose-dependent inhibition of carbachol-stimulated acid secretion in isolated parietal cells. Corresponding studies on the inhibition of pepsinogen secretion of isolated chief cell preparations also point to direct cellular inhibition (Albinus, unpublished data). The inhibitory mechanism of pirenzepine appeared to be of a competitive nature as shown by the Schild plot with a slope not different from unity and this is in agreement with data on acid secretion from the isolated stomach of the mouse (Szelenyi, 1982). The heterogeneity of muscarinic receptors as confirmed by carbachol and pilocarpine binding in isolated gastric mucosal cells is consistent with the

292 known complexity of muscarinic agonist binding (Birdsall et al., 1976, 1978). Within the described low affinity binding site for [3H]methylscopolamine the partial agonist pilocarpine further differentiated between high and low affinity sites. The meaning of this finding is not yet clear. The role of guanine nucleotides in connection with gastric secretion is still not fully understood. In several types of cells, the cyclic GMP content is increased by muscarinic receptor stimulation (Lee et al., 1972; Kebabian et al., 1975), a process that might also be calcium-dependent (Schultz et al., 1973; Berridge, 1980). Guanine nucleotides modulate not only binding of agonists to adrenergic, dopamine and opiate receptors (Lefkowitz et al., 1972; Maguire et al., 1976; Blume, 1978; Glossman and Presek, 1979) but also agonist binding to muscarinic receptors with a reduction of the high affinity (Berrie et al., 1979; Rosenberger et al., 1979; Wei and Sulakhe, 1979) as was shown for heart tissue (Berrie et al., 1979; Rosenberger et al., 1979, 1980; Wei and Sulakhe, 1979; Hulme et al., 1981) and fundic smooth muscle (Hammer, 1980). In the present study, about 30% of the binding sites for carbachol showed high affinity. It therefore appeared strange that GMPPNP did not affect carbachol binding significantly although the data suggested a minor decrease in the high affinity for carbachol, more prominent in parietal than in chief cell fractions, a finding that probably reflects quantitatively more binding sites per parietal cell. It can probably be excluded that a possible failure of the nucleotide to gain access to intracellular binding sites in the whole cell preparation was the reason for the absence of, or weak effects, since in canine gastric mucosal homogenates GMPPNP also had no effect on carbachol binding (Hammer, 1980). The observation that the binding characteristics for the antagonist pirenzepine were changed to small but significant extent in the presence of the guanine nucleotide in canine gastric mucosal cells but not in the guinea-pig cells is still puzzling. As became evident from the one-site model analysis, the apparent increase in the K D2 was obviously due to an increase in the number of low affinity binding sites as shown by two-site model analysis whereas the K D values for the high and low affin-

ity binding sites appeared not to be changed. In the rat myocardium, where the appearance of induced heterogeneity of the antagonist binding sites was accompanied by the development of a substantial sensitivity of these sites to guanine nucleotides, the reversal from two binding sites to one site resulted in an increase in the number of the high affinity sites (Hulme et al., 1981). More recently the muscarinic receptor subtype in rat atria was classified as M e subtype where a dissociation constant of 6.2 × 10 - 7 mol/1 was evaluated for pirenzepine (Hammer and Giachetti, 1982). This value would actually indicate this subtype to represent the suggested B-binding site and seems therefore not to be identical with the C-binding site proposed to exist in the gastric mucosa (K t) = 1.2 × 10 - 6 mol/1). This differentiation could have been the reason for the opposite changes of the two receptor subtypes in the two tissues in the presence of GMPPNP. The question arises whether the low affinity binding site for pirenzepine in the gastric mucosa should then be classified as M 3 subtype provided that B- and C-binding sites are proved to be different. One could speculate from our data that high and low affinity binding sites for pirenzepine on gastric mucosal cells might be present in a dynamic state which could be changed under experimental conditions. One could further speculate that the high affinity binding sites, suggested for pirenzepine to be responsible for the inhibitory effects, (Hammer, 1982) might have undergone conformational variations or some degree of proteolysis during cell isolation and incubation procedures but might predominantly exist under physiological conditions. Biochemical studies by gel electrophoresis of the muscarinic receptor isolated from the brain of various species suggested that the receptor polypeptide itself might be homogenous (Birdsall et al., 1979a), whereas the heterogeneity in binding properties does obviously depend on further factors. It is not known conclusively whether the small changes in pirenzepine binding characteristics as observed in isolated canine gastric mucosal cells under in vitro conditions might be of physiological relevance. Therefore it remains to be elucidated whether endoge-

293 nous guanine nucleotides, eventually increased by muscarinic receptor stimulation will interfere with muscarinic receptor conformation. Stimulation of muscarinic receptors can promote a variety of physiological and biochemical responses (Birdsall and Hulme, 1976). In the gastric mucosa, acid and pepsinogen secretion are stimulated via muscarinic receptors. Preliminary studies with isolated chief cells have shown that pepsinogen secretion was dose dependently stimulated by carbachol in concentration ranges where carbachol binding occurred (Albinus, unpublished data). Measuring the accumulation of [14C]aminopyrine as an index for parietal cell H ÷ secretion (Berglindh et al., 1976; Soil, 1981a) stimulation by carbachol in the presence of the usual calcium concentration of 1.8 m m o l / l correlated with the binding of carbachol, and was consistent with earlier observations (Soil, 1980; Soil et al., 1980; Batzri and Dyer, 1981). Lesser responsiveness of parietal cells to carbachol stimulation at lower calcium concentrations reflects the calcium dependency of parietal cell function as was also shown by Berglindh et al. (1980) with isolated rabbit gastric glands and by Soil et al. (1980) with canine parietal cells. Studies by Soil (1981b) revealed further that cholinergic stimulation enhanced calcium uptake in parietal ceils only. Muscarinic receptors are calcium mobilizing receptors (Michell et al., 1975; Berridge, 1980), enhance breakdown a n d / o r turnover of inositol phospholipids in different tissues (Michell and Kirk, 1981; Jones et al., 1982) and promote calcium movements into or inside the cell. This might reflect the importance of muscarinic receptors in parietal cell function. The physiological relevance of muscarinic receptor heterogeneity in this connection is still not known. The fact that the impaired parietal cell response at lower calcium concentrations is overcome partly by increasing the carbachol concentration suggested possible relations between high and low affinity binding sites and calcium fluxes. The different sensitivity in guinea-pig and canine parietal cell response to carbachol stimulation could reflect species-dependent functional changes of the receptors. Soil (1981b) mentioned differences in calcium uptake even in the same cell preparation depending on experimental conditions, thus raising the question

of either impaired cell function or influences of some other unknown factors. The sequence of steps following muscarinic receptor stimulation on gastric mucosal cells is rather complex and still not completely understood. The chofinergic regulation of gastrin and somatostatinlike immunoradioactivity release from rat stomach is mediated via M 1 receptors (Sue et al., 1983). The data presented in this communication support the probable existence of two subtypes of muscarinic receptors for agonists and antagonists in isolated gastric mucosal cells, which appear to be identical in two different cell types. In contrast to studies on sympathetic ganglia, a functional explanation of the two muscarinic receptor subtypes on parietal and chief cells is still speculative. The presumed conformational change of the pirenzepine high affinity binding site to a low affinity site under the influence of a guanine nucleotide seen only in canine cells needs further investigation. The dependency of parietal cell function upon calcium fluxes possibly connected with different muscarinic agonist binding sites proved the isolated gastric mucosal cell to be a useful model to elucidate further mechanisms underlying muscarinic receptor-mediated effects.

Acknowledgement We thank Mrs. G. Frisch for very skilful technical assistance and for typing the manuscript.

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