Isolated rat gastric parietal cells: Cholinergic response and pharmacology

Life Sciences, Vol. 28, pp. 609-621 Printed in the U.S.A.

Pergamon Press

ISOLATED RAT GASTRIC PARIETAL CELLS: CHOLINERGICRESPONSEAND PHARMACOLOGY Rainer Ecknauer, Elizabeth Dial, W. Joseph Thompson, Leonard R. Johnson and Gary C. Rosenfeld I Departments of Pharmacology and Physiology, The University of Texas Medical School at Houston, P. O. Box 20708, Houston, Texas 77025 (Received in final form November 18, 1980)

Summary Isolated, p a r t i a l l y purified or enriched rat gastric muscosal parietal cells were shown to respond to carbamycholine (EC50 : 2 uM) and other muscarinic cholinergic agonists as measured by an increased accumulation of 14C-aminopyrine, an indirect measure of acid secretion. The secretory response to carbamylcholine was shown to be inhibited stereoselectively and reversibly by nanomolar concentrations of muscarinic cholinergic antagonists. Non-muscarinic antagonists, including cimetidine, were either ineffective or very weak i n h i b i t o r s . The a f f i n i t y constants calculated for cholinergic antagonist i n h i b i t i o n of l¢C-aminopyrine accumulation induced by carbamylcholine were similar to those previously calculated from direct binding studies on purified parietal cell particulate fractions using 3H-QNB ( I ) . These studies support the existence of specific parietal c e l l muscarinic cholinergic receptors with which the natural secretagogue acetylcholine interacts to regulate gastric acid secretion. Methods recently developed in our laboratory and those of others to isolate mammalian parietal cells have allowed studies of the direct action of the gastric acid secretagogue acetylcholine (ACh), studies which were previously precluded because of the cellular heterogeneity of the gastric mucosa. Cholinergic agents have been shown to stimulate oxygen consumption, aminopyrine accumulation and potassium transport in cells isolated from various species (2-5), effects which were inhibited by atropine. Our previous studies on the binding of the t r i t i a t e d muscarinic cholinergic antagonist quinuclidinyl benzilate, (3H)-QNB, also demonstrated that the particulate fraction of purified rat parietal cells have high a f f i n i t y , saturable and stereoselective muscarinic cholinergic binding sites (1,6). The precise relationship between these putative receptors and the parietal cell response requires a more complete pharmacologic analysis than is currently available. This report, using isolated and enriched intact rat parietal cells, gives an analysis of the cholinergic pharmacology of hydrogen ion production. A cQmparison is made to the data previously obtained on the pharmacology of (JH)-QNB binding.

1 To whom reprint requests should be sent. 0024-3205/81/060609-13502.00/0 Copyright (c) 1981 Pergamon Press Ltd.

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Materials and Methods Materials The following drugs, chemicals and media were used in this study: scopolamine, N6,2'O-dibutyryl adenosine-3'5'-cyclic monophosphate (Aldrich Chem. Co., Inc., Milwaukee, WI); bovine serum albumin (Fraction V), physostigmine, neostigmine, acetyl ~-methylcholine, hexamethonium, ethyleneglycol-bis-(~-amino-ethyl ether) N, N ' - t e t r a - a c e t i c acid (Sigma Chemical Co., St. Louis, M O ) ; atropine, acetylcholine, pilocarpine, carbamylcholine, oxotremorine (Regis Chemical Co., Morton Grove, I L . ) ; cimetidine (gift from Smith, Kline and French, Philadelphia, PA); quinuclidinyl benzilate, dexetimide, levetimide ( g i f t s from Dr. Henry I. Yamamura, The University of Arizona, Tucson, AZ); pentagastrin (ICN Corporation, Irvine, CA); mepyramine ( g i f t from Dorsey Labs., Lincoln, NB); chlorpheniramine ( g i f t from USV Labs., Tuckahoe, NY); RPMI-1640 and Hanks' Balanced Salts without sodium bicarbonate (Flow Labs, Rockville, MD); Tris hydroxy-methyi)-amino-methane, pronase E (EM Labs, Darmstadt, Germany); 4C-dimethyl aminopyrine (N.E.N. Corp., Boston, MA; 82 mC/mmole.); Percoll (Pharmacia, Uppsala, Sweden).

l

Cell isolation P a r t i a l l y purified preparations of rat gastric parietal cells (20-25%) were isolated as previously described (7). In some exp#riments parietal cells were enriched (8), in which case approximately 2x10/ p a r t i a l l y pure cells were resuspended in 2 ml HBE media (Hanks' Balanced Salt media with added 0.1% BSA and 2 mM EGTA, aerated for 15 min before use with 95% 02 and 5% CO2 to pH 7.4) and layered over a discontinuous gradient consisting of 3 ml each of 10%, 20% and 30% Percoll in HBE contained in a 15 ml centrifuge tube (Corning #25300). The gradients were centrifuged at 20o for i0 min at 1100 x g in a Beckman Model TJ-6 centrifuge (Beckman I n s t r . , Inc., Palo Alto, CA). The enriched parietal cells banded at the 10%/20% Percoll interface and were collected with a Pasteur pipette. The cells were diluted 1:1 with HBE and centrifuged at 20o in 50 ml plastic centrifuge tubes (Corning #25330) for 7 min at 75 x g. After a second cell wash in HBE, the cell pellet was resuspended in RPMI-1640 medium (aerated as above and adjusted to pH 7.6 with Tris base)~ The recovery of parietal cells from the p a r t i a l l y pure preparation was 30-50% with an average cell purity of 70% (50-90%; Figure I) determined from 20 preparations by histological and morphological c r i t e r i a . Percoll prepared cell preparations with purity less than 50%, due primarily to interference in cell separation by contaminating mucous, were not used in these studies. Approximately 85-90% of the enriched parietal cells excluded trypan blue, an indication of v i a b i l i t y . In other studies, these cells were shown to respond to histamine (EC50 = 28 ~M) and dibutyryl cyclic AMP (EC50 = 29 ~M) with an increase in the accumulation of 14C-aminopyrine (5,9). 14C-Aminopyrine

accumulation

14C-aminopyrine ( A P ) accumulation, used as an indirect measure of hydrogen ion production, was determined using a modification of the procedure of Berglindh et al. (10). According to the pH-partition hypothesis (10) AP(PKa=5), which is unionized at physiological pH, w i l l accumulate in the acidified subcellular compartments of the parietal c e l l . Unless otherwise stated, 5 x 105 parietal cells were added to plastic 20 ml s c i n t i l l a t i o n vials containing 2 ml RPMI-1640 medium (pH 7.4), 14C-AP (0.2 ~C) and test

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FIGURE 1 Light microscopy of enriched rat gastric parietal cells. Parietal cells enriched by centrifugation through a discontinuous Percoll gradient (see Methods) were fixed in 3% glutaraldehyde and stained by the Papanicolau gynecological procedure with Gill hematoxylin, bluing agent, orange G-6 and eosin A-50 after fixation to glass slides with a Cytospin centrifuge (Shandon Southern Prods., Runcorn, England). Micrograph was photographed at 400X. drugs or drug vehicle. Incubation was for 30 min at 30o (unless otherwise indicated) with continuous gassing with 95% 02:5% CO2. Following incubation, two 0.9 ml aliquots were removed and centrifuged (Microfuge B; Beckman, Palo Alto, CA). The supernatant was aspirated and the pelleted c e l l s surface washed in 1.6 ml RPMI-1640 medium and then lysed in 0.5 ml d i s t i l l e d water. The lysed cells were transferred to s c i n t i l l a t i o n vials containing 6 ml Aquasol and r a d i o a c t i v i t y was determined by l i q u i d scintillation spectrometry with a counting e f f i c i e n c y of 72%. AP accumulation is expressed as % maximum accumulation or cpm per 106 parietal c e l l s per 30 min of incubation.

Results Basal 14C-Aminopyrine Accumulation The accumulation of AP in unstimulated cells was linear with increasing AP concentrations between 0.02 and 0.4 uC/assay (1-16 nmole; Figure 2A) and linearly related to parietal cell number between 0.1 and 1.0 x 106 cells per assay (Figure 2B). Equilibrium between cells and medium was reached by 30 min and half-maximum accumulation occurred at 8 min (Figure 2C).

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3,0

1.8 ?

'7 ':' 1,2 ×

2.0 x

1.0

0.6

0.1

0.2 0.3 14C-Aminopyrine (uC/assay)

0.5 Parietal Ceils ( x 106)

0.4

1.0

2.0 C

1.5

x

1,0

0,5

Minutes

Figure 2 Effect of aminopyrine concentration, parietal cell number and time on the accumulation of 14C-aminopyrine in isolated, p a r t i a l l y purified parietal cells. (A) 0.5 x i0 b parietal cells/ml; 30 min (B) 0 . i ~C 14C-aminopyrine/ml; 30 min (C) 0.5 x 106 parietal cells/ml, 0.1 uC z"C-aminopyrine/ml. Data are the average of three separate experiments. Carbamylcholine stimulated 14C-aminopyrine

accumulation

In the presence of 50 ~ M carbamylcholine parietal cell AP accumulation (Figure 3).

there

was an increase

in

The extent of AP accumulation was somewhat variable (Table I) and in some experiments there was no increase above unstimulated cells. However, in the presence of maximally stimulating concentrations of dibutyryl cyclic AMP (DbcAMP) carbamylcholine always resulted in a marked potentiation of AP accumulation (3-5, Table I ) . Because the presence of DbcAMP allowed a much greater cholinergic agonist stimulated accumulation of AP and therefore more accurate experimental determinations, the majority of studies describing the chol i nergic pharmacology of the parietal cells were carried out in the presence of i mM DbcAMP. In experiments which included i mM DbcAMP cholinergic stimulated 14C-AP accumulation was determined by subtracting that due to DbcAMP alone. None of the cholinergic antagonist drugs tested inhibited the response to dibutyryl cyclic AMP.

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-Yr

/ + Carbachol (5 x IO-SM) " ~ / /

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#

7

o,p,

4

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i/~/,6 I

|

I

I

10

20

30

40

I

50 Minutes

I

I

I

!

60

70

80

90

Figure 3 Effect of carbamylcholine (carbachol) on the rate of 14Caminopyrine accumulation in isolated p a r t i a l l y purified parietal cells. Data are the average of three separate experiments.

TABLE i Secretagogue stimulation of 14C-aminopyrine accumulation in isolated~ partiall~ purified parietal cells a Basal

9.96 ~ 1.16 (13)b

Carbamylcholine (50uM) DbcAMP (lmM) DbcAMP (ImM) + Carbamylcholine (50 ~M)

35.42 + 5.21 (11) 198.92 ~ 31.86(8) 411.06~ 47.14 (7)

a

Data is expressed as ~ol S.E.

14C-~inopyrine/106 parietal cells/30 min +

b

Numberof separate determinations

No significant difference was found in the sensitivity (EC50) of the parietal cell to carbamycholine in the presence (2.8 + 0.6 ~M; n=3) or absence (1.9 + 0.3 uM; n=3) of DbcAMP (Figure 4).

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f.~:,I

100 -

._-.----~/ /"

+DbcAMP

(10-3M)

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DbcAMP

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/ ~'"-

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-Log [Carbachol] Figure 4 Dose-related increase by carbamylcholine (carbachol) of 14Caminopyrine accumulation in isolated p a r t i a l l y purified parietal cells in the absence and presence of dibutyryl cyclic AMP(DbcAMP). Data is the average of three separate experiments.

Cholinergic a~onist and antagonist action on p a r t i a l l y purified parietal cell 14C-aminopyrine accumulation The effect on AP accumulation of several is shown in Figure 5. The concentrations response (EC50) were between 0.6 and 2.7 s t a t i s t i c a l l y s i g n i f i c a n t , in all comparisons were the most potent and carbamylcholine cholinergic agonists tested.

muscarinic cholinergic agonists necessary for a half-maximal ~M (Table I I ) . Although not acetylcholine and oxotremorine was the least potent of the

Carbamylcholine stimulated AP accumulation was concentrations (IC50 = lO-20nM; K i = l-2nM) by all cholinergic antagonists tested (Figure 6; Table I I ) .

inhibited at low of the muscarinic

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100 " • • • • o=

Parietal Cell Cholinergic Receptors

ACETYLCHOLINE OXOTREMORINE ACETYL-~-METHYLCHOLINE CARBAMYLCHOLINE

75

'~

50

C

E "F,

25

I 8

I 6

7

I 5

I 4

-Log [Agonist]

Figure 5 Dose-related increase by muscarinic cholinergic agonists of !4Caminopyrine accumulation in isolated partially purified parietal cells. DbcAMP (1 mM) was included in each assay. Data shown are representative of dose-response c u r v e s obtained from 2-8 experiments for each drug. 100 "0 0\ •~ .

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Figure 6 Inhibition by atropine of carbamylcholine stimulated 14Caminopyrine in isolated, partially purified parietal cells. Carbamylcholine (50 ~M) and DbcAMP (1 mM) were included in each assay. Data are the average of two separate experiments.

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TABLE II Comparison of EC50's and K i ' s of Muscarinic Cholinergic Agonists and Antagonists.

Agonists b

P a r t i a l l y Purified a EC50 (~M)

Enriched a - ~ M )

Pure b Ki (~M)

Acetylcholine

0.7 + .2(4)

0.8

18.9

Carbamylcholine

2.2 + .6(8)

3.9

74.6

Oxotremorine

0.6 + .1(5)

0.8

4.0

Acetyl ~-methylcholine

1.0 + .2(5)

1.4

26.3

Pilocarpine

2.7

-

18.9

Antagonists c

Ki (nM)a, d

Ki (nM)b

Atropine

1.3

2.0

QNB

3.1

0.8

Scopolamine

1.7

1.4

Dexetimide

1.4

i.i

Levetimide

600

1,900

Values without standard errors are the average of two separate experiments Ki is agonist or antagonist a f f i n i t y constant calculated from data obtained by displacement of (3H)-QNB binding from p u r i f i e d parietal cells as reported in reference #i. Agents inactive or i n h i b i t i n g less than 50 percent at concentrations to 100 ~M include: hexamethonium, phentolamine, cimetidine, metiamide Ki values were determined from equation of Cheng and Prusoff (12)

inhibition

curves

(IC50)

using

up the

Antagonist i n h i b i t i o n was also stereoselective as shown by the more than 400 f o l d difference in potency between the pharmacologically more active isomer, dexetimide, and the less active isomer, levetimide (Figure 7). In the presence of atropine the carbamylcholine dose response curve was displaced to the r i g h t (Figure 8) with no reduction in the maximal AP accumulation, indicating competitive interaction of the agonist and antagonist with the same receptor s i t e s .

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100

e=

Levetimide

75

~E


_ram 50 ~E

m

I

I

I

I

I

9

8

7

6

5

-Log [Antagonist] Figure 7 Stereospecific inhibit ion by the isomers of benzetimide of carbamyl choline stimulated 14C-aminopyrine accumulation in isolated, p a r t i a l l y purified parietal cells. Carbamylcholine (50 ~M) and DbcAMP (1 mM) were included in each assay. Data are the average of two separate experiments.

) - ~...'• , ~

100

/

® CONTROL • +ATROPINE (10"8M) • +ATROPINE (5xl0-'M)

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5

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3

-Log [Carbachol] Figure 8 Carbamyl choline stimulated 14C-aminopyri ne accumulation in isolated, p a r t i a l l y purified parietal cells in the absence and presence of atropine. DbcAMP (1 mM) was included in each assay. Data are the average of two separate experiments.

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Addition of atropine after maximal AP accumulation had been reached resulted in a rapid and complete reversal of AP accumulation (Figure 9). No e f f e c t of the antagonists on DbcAMP stimulated AP accumulation was seen at concentrations as high as I0 ~M.

15~k

12 Carbachol

\ I

\

m

0

>¢

\

E

O. tJ

Carb+achol Atropine, t=15" O

6

\ \

3-

0

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Carbachol +

\

Atropine, t=O"

15

I

I

I

30

45

60

Minutes Figure 9 Atropine reversal of carbamylcholine (carbachol) stimulated 14C-aminopyrine accumulation in isolated, partially purified parietal c e l l s . Atropine (1 ~M) was added e i t h e r at the s t a r t (t = 0 ' ) or 15 minutes (t = 15') a f t e r beginning incubation in the presence of carbamylcholine (50 uM). DbcAMP was not present in the assay. Data are the average of two separate experiments.

Cimetidine, a histamine H2-receptor antagonist, had only a weak i n h i b i tory e f f e c t on carbamylcholine stimulated AP accumulation (Figure 10); more than a 10,000 f o l d higher concentration of cimetidine than atropine was needed for inhibition. Mepyramine and chlorpheniramine, histamine Hi-receptor antagonists, both inhibited carbamylcholine stimulated AP accumulation with an IC50 of 7 ~M (Figure 10), a concentration much higher than needed f o r i n h i b i t i o n by muscarinic antagonists.

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Parietal Cell Chollnerglc Receptors

619

100

75

~'~

50

@ MEPYRAMINE

~

\

25

,\ 7

6

5

4

3

-Log [Antagonist] Figure 10 I n h i b i t i o n by histamine antagonists of carbamylcholine stimulated 14C-aminopyrine accumulation in isolated partially purified parietal cells. Carbamylcholine (50 ~M) and DcbAMP (I mM) were included in each assay, The curves shown for mepyramine and chlorpheniramine represent i n h i b i t i o n of the combined effect of carbamylcholine and DbcAMP. Similar results were obtained for each secretagogue alone (data pgt shown). Cimetidine was without effect on DbcAMP stimulated z~C-aminopyrine accumulati on (data not shown). Data are the average of two separate experiments.

Cholinergic pharmacology of enriched parietal cells No differences in the EC50 or Ki of several cholinergic agonists and antagonists on AP accumulation were found in a comparison of the s e n s i t i v i t y of partially purified and enriched cell preparations despite the approximately three fold increase in cell purity (Table I I ) Nor were differences observed in the extent of AP accumulation (cpm per 1"06 parietal cells) in p a r t i a l l y purified and enriched parietal cells from the same cell preparation or in enriched parietal cell responsiveness to DbcAMP (data not shown).

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Discussion The results of these studies demonstrate that preparations

of isolated

t parietal cells respond to direct cholinergic stimulation, as measured by ~~C-AP accumulation. These drugs act by interaction with specific muscarinic type cholinergic receptors. The receptors were shown to be stereoselective and to interact reversibly, competitively and specifically with micromolar concentrations of muscarinic cholinergic agonists and nanomolar concentrations of muscarinic cholinergic antagonists. No difference could be detected in the pharmacology of partially pure or enriched parietal cell preparations. Our studies would support a working hypothesis that muscarinic cholinergic agonists act directly on the elaboration of hydrogen ion production. Although limited in extent, pharmacological analyses of isolated parietal cells from dog (13; purity = 10-51%), guinea pig (2; purity = 70-80%) and isolated gastric glands of the rabbit (14) have also indicated a direct muscarinic cholinergic stimulation of the parietal cell. The observed inhibition of carbamylcholine stimulated AP accumulation by Hi-receptor antagonists may be due to their anticholinergic activity; however, the effect may also be nonspecific since these agents also inhibit at similar concentrations both DbcAMP and histamine stimulated AP accumulation (5,9). Other nonmuscarinic cholinergic agents had l i t t l e effect on carbamylcholine stimulated AP accumulation (Table I I ) . Previous results (1,6) obtained for cholinergic antagonist and agonist inhibition (Ki) of (3H)-QNB binding to the particulate fraction of nearly pure (~90%) parietal cells are shown in Table I I . The close correlation which exists between antagonist a f f i n i t y determined in these binding studies and their a f f i n i t y (Ki) determined in the present studies from inhibition of carbamylcholine stimulated AP accumulation in intact cells (Table I I ) , provides strong support for a direct effect and common binding site of the antagonists on parietal cells. There are, however, marked discrepancies between the potencies of agonists as inhibitors of (3H)-QNB binding and as stimulants of AP accumulation. The a f f i n i t y (Ki) of agonists determined from binding studies are 5-30 fold less than their a f f i n i t y (EC50's) determined in the present studies from stimulation of AP accumulation (Table I I ) . For example, acetylcholine stimulates AP accumulation half-maximally (EC50) at a concentration of 0.6 uM, but inhibits ( H)-QNB binding by 50% at a concentration of 18.9 uM. Such discrepencies have been noted in other systems (15,16). The possible explanation for such results have been considered (15-17) and include receptor desensitization, negatively cooperative interactions between agonist binding sites, and the presence of spare receptors. In a detailed analysis of these possibilities Birdsall et al. (17) have presented evidence of heterogeneity in the population of agonist binding sites, all of which have the same a f f i n i t y for antagonists. Two major sites and one minor site of differing a f f i n i t i e s for agonists were detected using the t r i t i a t e d muscarinic agonist oxotremorine-M. The use of such an agonist receptor probe in addition to the antagonist probe used in these studies, might be useful to define multiple receptor populations associated with parietal cells. The basis of the potentiation between cholinergic agonists and dibutyryl cyclic AMP as shown in this study, or between cholinergic agonists and histamine, dibutyryl cyclic AMP or phosphodiesterase inhibitors as found in our previous studies (3-5) as well as studies by Soll (I~), remains to be determined. Potentiation of acid secretion by combinations of secretagogues is known from in vivo investigations and has also been demonstrated in isolated gastric g l a - n ' d s ~ ) . The a b i l i t y to reproduce potentiation in isolated cell systems should lead to an understanding of the biochemical and molecular basis of secretagogue interaction.

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Acknowledgements A portion of this work was presented at the Meeting of the American Society for Pharmacology and Experimental Therapeutics in Houston, Texas, August 13-18, 1978 (4) and the 63rd Annual Meeting of the Federation' of American Societies for Experimental Biology in Dallas, Texas, April 1-10, 1979 (3). This work was supported by USPHS grants AM 20431 and AM 16505. Dr. Rosenfeld's work is supported by Research Career Developmental Award AM 00457 from the National Institutes of Health. The authors wish to acknowledge the contributions of Mr. David Jessup and Ms. Nancy Farrar with cell staining and photography and Mrs. Shirley Washington for her secretarial aid.

References 1. Ecknauer, R., W.J. Thompson, L.R. Johnson and G.C. Rosenfeld, Amer. J. Physiol. 239:G204-209 (1980). 2. Batzri, S~nd J.D. Gardner, Biochim. Biophys. Acta 508:328-338 (1978). 3. Ecknauer, R., W.J. Thompson, L.R. Johnson and G.C. Rosenfeld, Fed. Proc. 38:884 (1979). 4. ~senfeld, G.C., P. Gerba, S.J. Strada and W.J. Thompson. Pha6~acplogist 20:208 (1978). 5. ~senfeld, G.C., S.J. Strada, E.J. Dial, C.F. Bearer and W.J. Thompson. Adv. Cyclic Nucl. Res. 1__2:255-266, (1980). 6. Rosenfeld, G.C., R. Ecknauer, L.R. Johnson and W.J. Thompson, Proc. 7th Int. Congr. Pharmacol. p. 132 (1978). 7. Thompson, W.J., L.K. Chang and G.C. Rosenfeld, Amer. J. Physiol. (In Press). 8. Farrar, N., E. Dial, W.J. Thompson and G.C. Rosenfeld, Proc. Exp. Biol. Med. (In Press). 9. Dial, E.J., W.J. Thompson and G.C. Rosenfeld, Physiologist 22:29 (1979). 10. Berglindh, T., H.F. Helander and K.S. Obrink, Acta. Physiol. Scand. 97:401-414 (1976). 11. -S-hore, P.A., B.B. Brodie and C.A.M. Hogben, J. Pharmacol. Exp. Ther. 119:361-369 (1959). 12. Cheng, Y. and W.H. Prusoff, 8iochem. Pharmacol. 22:2099-3108 (1973). 13. Soil, A.H., J. Clin. Invest. 61:370-380 (1978). - 14. Berglindh, T., Biochim. Biophy~. Acta. 464:217-233 (1977). 15. Birdsall, N.J.M. and E.C. Hulme. J. Neurochem. 27:7-16 (1976). 16. Snyder, S.H., K.J. Chang, M.J. Kuhar and H.I. Yamamura, Fed. Proc. 34:1915-1921 (1975). 17. B~rdsall, N.J.M., A.S.V. Burgen and E.G. Hulme, M o l . Pharmacol. 14:723-736 (1978). 18. B~rglindh, T., Acta Physiol. Scand. 99:75-84 (1977).