Cardiac Cholinergic Muscarinic Receptors: Changes in Multiple Affin ...

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Dec 10, 1984 - to methacholine perfusion. We wished to determine whether the muscarinic receptor forms were static or subject to change. We found changes.
0022-3565/85/2323-0754$02.OO/O THE JOURNAL OF PHARMACOLOGY Copyright © 1985 by The American

AND

EXPERIMENTAL

Society

Vol. 232, No. 3 Printed in U.S.A.

THERAPEUTICS

for Pharmacology

and Experimental

Therapeutics

Cardiac Cholinergic Muscarinic Receptors: Affin ity Forms with Down-Regulation1 ROBERT

ROSKOSKI,

Departments Department

of Biochemistry (R.R., R.R.R., WE.) and Biometry of Chemistry (P.F.C.), North Texas State University,

Accepted

JR.,

for publication

RICK

December

A.

REINHARDT,

WOLF

ENSELEIT,

(W.D.J.), Denton,

Louisiana Texas

Changes

WILLIAM

D.

State UnWersity

JOHNSON Medical

Center,

in Multiple

and

PAUL

F. COOK2

New Orleans,

Louisiana

and

10,1984

ABSTRACT The isolated working rat heart exhibits dynamic changes in the cholinergic muscarinic receptor in response to perfusion with the acetylcholine congener methacholine. For example, perfusion with 4 zM methacholine for 2.5 hr mediates a statistically significant and reversible 1 0 to 1 5% decrease in receptor content in right atrium and left atrium and ventricle. The muscarinic receptor exhibits a single affinity state for antagonists but multiple affinity states for receptor agonists. When agonist 3H-antagonist competition experiments are performed, the concentration dependence of displacement by agonists is flattened and extends over more than 2 log units. From such curves, it is difficult to extract values for the relative proportions of the multiple receptors or their K0 values by inspection. We have developed a procedure

Although to control

the heart by the

exhibits

intrinsic

autonomic

rhythmicity,

nervous

system.

thetic innervation decreases heart rate whereas the sympathetic division mediates sponses. The postganglionic parasympathetic is acetylcholine

tissue

which

and the

muscarinic

interacts

myocardium

receptor

with

through

(Higgens

the the

it is subject

The and the

action

et at., 1973).

The

ofthe

July

3, 1984.

in part

by Grants

HL-24791

Recipient

of U.S.

Public

Health

Service

Research

Career

ABBREVIATIONS:

754

QNB, quinuclidinyl

benzilate;

1975; et at.,

Ward

and

Young,

1977),

as

well

as

rat

1979).

modified

(R. R.) and GM 31686 Development

GppNHp,

at.,

pacemaker

(P. F. C.). 2

et (Berrie

cholinergic

re-

characterization

AM 01155.

(Taylor

A variety of procedures have been used to calculate the KD and proportions of the multiple forms of receptor through the use of competition assays with radioactive and unlabeled agents (Hulme et aL, 1978). These procedures use

contractility opposite

and quantitation of this receptor was greatly facilitated by the development of [3H]QNB (a potent muscarinic receptor antagonist) as a radioligand by Yamamura and Snyder (1974). Hulme and co-workers (1978) reported that antagonists exhibit simple mass action binding to the rat brain muscarinic receptor. In contrast, the binding of agonists is more complex and is consistent with the existence oftwo or more affinity states (Birdsall et at., 1978). Similar results have been found with rabbit and porcine heart (Fields et at., 1978; Schimerlik and Searles, 1980), embryonic chick heart (Galper and Smith, 1980; Halvorsen and Nathanson, 1981; Galper et aL, 1982) and ileal smooth muscle Received for publication 1 This work was supported

formsof

heart

parasympa-

neurotransmitter

specialized

for plotting the competition curves so that the K1, values and proportion of multiple receptor forms can be estimated graphically. We determined these more accurately by computer using a nonlinear least-squares analysis. After perfusion of methacholine for 1 hr, there were increases in the KD of the low and high affinity the receptor in the right and left ama. After 2.5 hr of perfusion, the K1, of the high-affinity form increased further in the left atrium. In the right atrium, the two affinity states were converted into a single state of low affinity. Although there was a significant decrease in the amount of receptor in left ventricle, there were no changes in the K0 values or proportions of the two states. All changes in receptor reversed during an additional 2.5 hr of perfusion without methacholine.

Award

values

Eadie-Hofstee

analyses

(Molinoff

et

aL,

1981)

and

nonlinear least-squares curve fitting (Hancock et at., 1979). We have developed a general procedure for plotting the competition of one agent by another which requires the determination of bound and free radioligand concentration at each concentration of inhibitor and the KD of the radioligand in the absence of inhibitor (determined independently by a Scatchard analysis). Our procedure gives an intuitive grasp of the approximate KD values and proportions of the multiple states by inspection. We have also calculated the KD values and proportions of the multiple states using nonlinear least-squares fitting procedures. We reported previously that perfusion ofthe isolated working rat heart with 4 M methacholine for a 2.5-hr period brought about a 10 to 15% decrease in receptor in right and left atria and left ventricle, but not in right ventricle (Reinhardt and Roskoski, 1983). After an additional 2.5 hr in the absence of methacholine, the receptor content returned to control values. There was no detectable decrease in receptor content after 1 hr

5’-guanylylimidodiphosphate.

Cardiac

1985

of perfusion with methacholine. and proportions of the multiple in response

to methacholine

whether the muscarinic to change. We found proportions

in two

We

measured

affinity

perfusion.

receptor changes

heart

the

forms We wished

where

occurs

(right and left atria). We found no changes in KD values or in the proportions of the two states in right or left ventricle in response to methacholine perfusion even though down-regulation was observed in the latter chamber.

Materials Agonist Sprague

3H-antagonist Dawley

absence of 4 M

and Methods assays.

competition

rats

g) were

(200-250

perfused

Hearts in

the

from presence

male or

for the specified times as described previously (Reinhardt and Roskoski, 1983) using the procedure of Morgan et at. (1980). After perfusion, the hearts were dissected and stored frozen in liquid nitrogen. At the time of the assay, hearts were homogenized in Ringer’s solution (1:32 for atria and 1:16 for ventricle, w/v). Duplicate tubes containing a given concentration of [3H]QNB of about 25 pM and about 22 concentrations of unlabeled methacholine (from 1 M to 3 mM, final) in Ringer’s solution were prepared. Portions (25 d) of homogenate were added to initiate the reaction (1 ml final volume). After 30 mm at 37’C bound [3H]QNB was collected on G/FA glass fiber filters as noted previously (Reinhardt and Roskoski, 1983). Nonspecific binding was determined in the presence of 1 MM unlabeled QNB or atropine in duplicate tubes. [3H]QNB binding was also determined in the absence of added methacholine. Protein was determined by the procedure of Lowry et at. (1951) using bovine serum albumin as methacholine

standard. Theory. A number of methods for graphical analysis ofbinding data from competition assays are currently in the literature (Molinoff et at., 1981; Hancock et at., 1979). These analyses usually consist of plots based on the linear transform of the Michaelis-Menten equation developed by Eadie-Hofstee. Equation 1 describes the competitive binding of ligand I and ligand L with receptor

-

B

Kq(1

)

B[L] +

+

(1)

EL]

i)

+

or subject and their

down-regulation

a concave

K(K1 BK1+ [I]) B

=

B

-

[K(K1 I K1+ [J])]B

(K

+ q

Equation

3 can

be rearranged B.(i

In this case [U) is plotted

+

upward

curve will be obtained.

of [3H]QNB

=

(5) EL]

+

Extracting

estimates

of

B

methods.

considered four computational methods ofthe nonlinear model: 1) the grid-search or Taylor series method, 3) the gradient and 4) Marquardt’s method which is a

We

for estimating the parameters method, 2) the Gauss-Newton

or steepest-descent compromise

methods

method

between

Gauss-Newton

were extremely

sensitive

and

steepest

descent.

to the choice of initial

four

All

estimates

but

usually

resulted in final estimates which were in close agreement for test data. For convenience, the results reported here were calculated by the Gauss-Newton method using the BMDP program P3R (Dixon et at., 1981). The algorithm for obtaining estimates of parameters of the modeluses iterative techniques to minimize the residual sum of squares. Convergence was usually obtained in less than 15 iterations with initial estimates chosen from graphs of data plotted according to equation 4. A statistical decision regarding the number of receptor classes (i.e., i vs. i + 1) was aided by the use of partial F tests, inspection of plots of residuals of the fitted model and comparison of the asymptotic S.E.s of the estimated parameter to the estimates (i.e., the coefficient of variation of the estimates). Two parameters were estimated for each class of receptor in a given model. Let SS1 and SS1+1 denote the residual sums of squares for i and i + 1 classes, respectively, and r the degrees associated

with

SS1. Then

F_SiSS1)

gives a suitable

statistic

r

ssi

-

for testing

2

the hypothesis

that

the appropriate

number of classes is i rather than i + 1. This statistic is approximately distributed as F with 2 and r degrees of freedom. Student’s t test was used to determine the significance between means among

and analysis means (Zar,

of variance

was used

to determine

differences

The KD values were calculated apparent KD values by the procedure of Cheng and Prusoff correct for receptor sites occupied by [3H]QNB. 1984).

from

the

(1973)

to

Results FEll]

We examined (4)

is plotted on the ordinate and B.(K[I]/ on the absissa. B is obtained directly as the ordinate intercept with -1/K1 as the slope. Kq (66 pM) was obtained from more than 10 Seatchard analyses of the binding of the ligand (L) to receptor under our experimental conditions (present work and Reinhardt and Roskoski, 1983). In the case in which more than one class of receptor exists which has different affinities for a competing ligand (I), but the same affinity for ligand (L), the following equation obtains. + K/[L])

)

(3)

K11[L]

B,,.K[I] [L]

+

binding.

Statistical

as follows.

j#{231}\ B [L]J

\

(2)

Kq(1

and the apparent K1 which have known statistical properties requires an iterative fitting procedure as will be discussed in the following section. By visual inspection, the negativeof the reciprocal of the slope of each portion of the curve gives the apparent KD (as in a Scatchard analysis); the maximum ordinate intercept represents 100% of the multiple states and the extrapolated ordinate intercept of each affinity form yields an estimate of its proportion of the total binding sites. The total receptor (maximum binding) is not given by this competition procedure and can be determined independently by Scatehard analysis

where

B. and B represent the amount of L bound to receptor at any concentration below saturation and saturation, respectively, Kq is the dissociation constant for L from the R/L (receptor/ligand) complex and K1 is the dissociation constant of I from the Rh complex. If one rearranges the equation in Scatchard and Eadie-Hofstee form, respectively, equations 2 and 3 are obtained:

[L]

+

In equation 5, W and B” represent the amount ofL bound to receptor classes 1 and n, respectively (n represents the apparent number of classes of receptor), whereas K11 and K1,, represent the dissociation constant for R1/I and R5/I, respectively. All other terms have the same definition as above. When data are plotted in the form of equation 4,

of freedom

f_ [U

B5[L] +

B=

to determine

forms were static in the KD values

regions

receptor

755

Receptors

B’[L]

values

KD

of the

Muscarinic

regions 2.5 hr.

are required QNB from sion

the multiple

(fig.

to displace the

right

states

the

than

expected

that

subsaturating atrial

1). As reported

rat brain,

curve

such displacement tion or KD values rinic

affinity

in response to perfusion with 4 tM We found that higher concentrations

receptor

from

curves, of the for

after

by Hulme

from

the

multiple

control mass

affinity

If the

of [3H]

methacholine

et at. (1978)

a theoretical it is difficult

methacholine.

of methacholine

concentrations

receptor

obtained

of several rat heart methacholine for

hearts action

to estimate forms

data

perfu-

in the

of

is flatter

curve.

From

the proporof the

are

case

musca-

replotted

as

756

Roskoski

et al.

Vol. 232

accurate than that for the high-affinity state, which is the usual case for parameters obtained by graphical analysis of Scatchard-type plots. The KD values obtained after the procedure of Cheng and Prusoff (1973) were 2.9 ± 0.7 and 171 ± 17 tM,

a

a)

respectively.

After 2.5 hr of perfusion with 4 iM methacholine, higher concentrations of methacholine are required to inhibit [3H] QNB binding (fig. 1) from homogenates of right atria. When this is replotted, the results are shown in figure 2B. Computer fitting was best with a single affinity state with a KD of 166 ± 16 ,M (corrected KD = 120 ± 12 SM). There is a change from two states to a single state of intermediate affinity in the right atrium associated with receptor down-regulation. After an additional 2.5 hr of perfusion in the absence of methacholine, the amount of receptor returns to control levels and the two affinity states also return (table 1). These experiments indicate that the subtypes of the muscarinic receptor undergo reversible changes over relatively short time periods. We observed previously a decrease in receptor in left atrium

z a

io2

io-

[Methacholine], M Fig. 1. Competition of methacholine and [3H]QNB for right atrial receptor in heartS perfused with and without methacholine. Rat hearts were perfused 2.5 hr in the presence or absence of 4 M methacholine and the tissues were dissected and stored in liquid N2 as described previously (Reinhardt and Roskoski, 1 983). Right atrial homogenates were incubated with subsaturating concentrations (25 pM) of [3HJQNB and varying concentrations of methacholine as descilbed under “Materials and Methoris.” The points represent the experimental value and the curves were generated by computer fitting for 2 sites (0, control perfusion) and 1 site (#{149}, methacholine perfusion).

after

The

the

estimate

of the KD of the

low-affinity

state

was

of

perfusion

with

high-affinity

As in the

methacholine

(Reinhard

and

form

right

increased

from

4.4 to 17 M

and

the

low-

affinity form increased from 172 to 240 cM. The percentage of the high-affinity form, however, increases from 40 to 57%. Although comparable receptor decreases occurred in the right and left atrium, the response of the receptor affinity states differed. After an additional 2.5 hr of perfusion in the absence of methacholine, control state

1fM(47±2%);KD2=236±23M(53±2%).

visual

h

atrium, we find two affinity left atrium (fig. 3; table 1). We wanted to determine whether the two receptor subtypes were converted to a single affinity state after methacholine perfusion as we found in the right atrium. Instead, we found that the KD for

described in equation 4 (“Materials and Methods”), the results can be displayed in a more comprehensible fashion (fig. 2). The experimental data are plotted as open circles and the curved line gives the computer fit of the data assuming two affinity states. From the straight lines drawn by inspection, one can calculate the approximate KD values (from the negative reciprocal of the slopes) and the proportions (from the y-axis intercepts) thereby obtaining: K1,1 = 12 M (44%) and KD2 = 247 M (56%). By computer fitting using nonlinear least-squares analysis, the following uncorrected KD values were obtained: KD1=4±

2.5

Roskoski, 1983). states in control

the indicating

KD values that the

and proportions changes were

return reversible

to the (table

1).

more

5

4

z a z

a U

z a

z

0

a +

8

KQNB#{149} CMethacholineJ

I

[QNB] x 1O

(M2)

Fig. 2. MOdified Scatchard replot of the competition of methacholine and [3H]ONB (B) rat hearts. The data on the competition of methacholine and [3H]QNB 4. The points are the experimental values and the solid line is a computer fit lines were drawn by inspection. The negative reciprocal of the slope yields the !(. The ordinate intercept. used equation

Bobs#{149} KQNB#{149} [Methacholine]

/

[QNB]

x i#{252}15 (M2)

for the right atnal receptor in control (A) and methacholine from which figure 1 was derived were plOtted according to of the data from a 2-site (A) and 1-site (B) model. The broken percentages of each site are proportional to the length of the

Cardiac

1985

Muscarinic

757

Receptors

TABLE 1 Methacholine perfusion reversibly afters multiple affinfty states of the muscarinic receptor in selected heart regions The hearts were perfused in the absence of presence of 4 zM methacholine as described previously (Reinhardt and Roskoski. 1983). The K0 values and proportion the mulp$e

affinity

for methacholine

forms

Reon

were determined Perfuon

as decribed

Materials

under

and Methods.

The data represents

Total Receptor

Condeons

2.5 hr control 2.5 hr methacholine 2.5 hr methacholine +2.5 hr control 2.5hrcontrol

Leftatrium

Right ventricle

Right atrium Left atrium a C

252

2.5 hr methacholine 2.5 hr methacholine +2.5 hr control 2.5hrcontrol 2.5 hr methacholine 2.5 hr methacholine +2.5 hr control 2.5 hr control 2.5 hr methacholine 2.5 hr methacholine 2.5 hr control

Leftventricle

a

262 235

± ± ±

6#{176}

8.5

6.8 3.4

73.5±2.5 59 ± 2.5k 65.0 ± 8 3.5

84.5 ± 4 84.5 ± 4.5 258 ± 7

1 hr methacholine

279

±

pM

8

178 ± 15 (53 ± 2)

± 6.5b (1O0%)b ± 1.0(47 ± 3)

4.4±1.0(40±2)

± 7b ± 10

1 hr methacholine

Data from six hearts in each group (Reinhardt and ROSkOSki, different from control (P < .05). Sigr#{227}f’-’ntiy different from control and 2.5 hr of methacholine

120 3.3

285±7

±

of

determinations.

ofthree

isM

2.4 ± 0.9 (47 ± 2)

6

234 266

89.5

± S.E.M.

K,, (%)

md/mg

Right atrium

the mean

± ±

5.4±

174

1.0(30±2)

0.8 (24 0.9 (26 0.8 (28

8.8 8.4

± ±

0.8k (50 ± 2%) 09bc (40 ± 3%)

± 3)

202±15(70±2) 187 ± 15 (72

4.1 ± 1 .2 (28 ± 2) 5.4 ± 0.9 (32 ± 2) ± ± ±

14(53

201 ±15(60±2) 279 ± 17 (43 ± 3)#{176} 200 ± 14 (63 ± 2)

1 .6b (57 ± 3)b 0.9 (37 ± 2)

4.1 3.8 3.8

±

± 2) 195 ± 14 (68 ± 2)

± 3) ± 2) ± 2)

176

±

15 (76

±

3)

1 77 ± 14 (74 ± 2)

166

±

15 (72

±

2)

275 ± 20

273

±

(50 ± 2%) 16b (60 ± 3%)

1983). treatment (P