(Received for publication, March 20, 1995; and in revised form, July 20, 1995)
From the
Incubation of cultured embryonic chicken heart cells with the
-adrenergic agonist isoproterenol resulted in a dose-dependent
increase in the number of mAChR on the surface of intact cells. The
isoproterenol-mediated increase in mAChR number was time dependent and
reached a maximum by 48 h. Chick heart cells treated with isoproterenol
exhibited a greater than 6-fold increase in the sensitivity for
carbachol-mediated inhibition of adenylyl cyclase activity as compared
to control. Stimulation of cultured heart cells for 24 h with
isoproterenol resulted in a 25-35% increase in cm2 mRNA levels as
compared to control cm2 mRNA levels. In contrast, the level of cm4 mRNA
was not significantly affected by isoproterenol treatment. cm2 mRNA
levels were maximally elevated by 15 h following isoproterenol
stimulation and remained elevated for up to 72 h. Incubation of cells
with isoproterenol in the presence of Rp-cAMP, an inhibitor of
cAMP-dependent protein kinase, blocked the increase in the level of cm2
mRNA. Thus, prolonged activation of
-adrenergic receptors results
in an increase in mAChR number and muscarinic responsiveness in chick
heart cells due to a cAMP-dependent protein kinase mediated increase in
cm2 mRNA levels.
Muscarinic acetylcholine receptors (mAChRs) ()are
members of a large family of transmembrane spanning receptors that
couple to guanine nucleotide-binding proteins (G-proteins) upon agonist
activation. Five different genes encoding the mammalian mAChRs subtypes
(m
-m
) have been identified (Kubo et
al., 1986a, 1986b; Peralta et al., 1987a, 1987b; Bonner et al., 1987, 1988; Braun et al., 1987; Shapiro et al., 1988). The chick heart expresses at least three mAChR
subtypes (cm2, cm3, and cm4) that share a high degree of homology to
the mammalian m
, m
, and m
mAChR
(Tietje et al., 1990; Tietje and Nathanson, 1991; Gadbut and
Galper, 1994). Agonist activation of mAChRs have been shown to result
in a variety of physiological and biochemical events that include
inhibition of adenylyl cyclase (Nathanson et al., 1978),
stimulation of guanylyl cyclase (Renaud et al., 1980),
stimulation of phospholipase C (Brown and Masters, 1984), and
alteration of ion channel conductances (Galper et al., 1982;
Hunter and Nathanson, 1986). Activation of phospholipase C results in
the generation of the second messengers diacylglycerol and inositol
trisphosphate (Orellana and Brown, 1985) and subsequent activation of
protein kinase C and increase in intracellular calcium levels (Hirasawa
and Nishisuka, 1985). In the continued presence of acetylcholine or
other agonists, mAChR in the heart and other tissues undergo
sequestration and a subsequent decrease in receptor number (Nathanson,
1987). Long term treatment of embryonic chick heart cells with
muscarinic agonists results not only in a down-regulation of muscarinic
receptor number, but also in a significant reduction in the levels of
mRNA encoding the cm2 and cm4 mAChRs (Habecker and Nathanson, 1992;
Habecker et al., 1993). In addition to the agonist-induced
down-regulation of mAChRs in the chick heart, activation of adenosine
(Ad1) and angiotensin II (AngII) receptors has also been shown to
affect chick heart mAChR number and mRNA levels (Habecker and
Nathanson, 1992). Chick cardiac cells also express
-adrenergic
receptors which mediate stimulation of adenylyl cyclase activity.
Stimulation of mAChRs results in inhibition of
-adrenergic
stimulation of cardiac contractility due at least in part through
inhibition of adenylyl cyclase (Hartzell, 1988).
We report here that
persistent stimulation with the -adrenergic receptor agonist
isoproterenol increases mAChR number and muscarinic responsiveness in
chick heart cells due to a selective increase in cm2 mRNA levels with
no effect on cm4 mRNA levels.
Figure 1:
Concentration-response curve for
isoproterenol-mediated increase in [H]NMS binding
to intact chick heart cell cultures. Embryonic chick hearts cells were
cultured in serum-free defined media. Isoproterenol, dissolved in 10
µM ascorbic acid, was added once daily for 72 h. The media
was changed on the third day, and assays were performed on the fourth
day of culture. The binding of [
H]NMS to cell
surface mAChR on intact chick heart cells in culture was performed as
described under ``Experimental Procedures.'' Data are
presented as the mean ± S.D. from three separate experiments
which each had from six to nine independent determinations. The
increase in [
H]NMS binding is expressed as the
percent increase compared to control (10 µM ascorbic
acid). The mean value of [
H]NMS binding to
control heart cells was 960 ± 40 fmol/100-mm
plate.
Figure 2:
Inhibition of isoproterenol stimulation of
increase in mAChR by the -adrenergic antagonist nadolol and time
course of isoproterenol- induced increase of mAChR number. A,
antagonism of isoproterenol effect by pretreatment with nadolol.
Primary heart cell cultures were pretreated with 2 µM nadolol (final concentration) 10 min prior to addition of
isoproterenol. Cells were treated once daily for 3 days with either
nadolol, 100 nM isoproterenol, vehicle (10 µM ascorbic acid), or nadolol with isoproterenol. Medium was changed
on the third day of culture, and the binding of
[
H]QNB to membrane homogenates was performed on
the fourth day. Average [
H]QNB binding in
membrane homogenates of control treated cells was 310 ± 20
fmol/mg protein. The isoproterenol values were significantly different
from nadolol values, p < 0.01, and nadolol plus
isoproterenol, p = 0.01. Data are presented as the
means ± S.D. from three separate experiments which each had from
six to eight independent determinations. There were no significant
differences between nadolol alone and nadolol plus isoproterenol, p > 0.2. Statistical significance was calculated according to the
Student's t test for unpaired observations, with p < 0.05 taken as significant. B, heart cells
were treated for 24, 48, or 72 h with 100 nM isoproterenol
prior to determination of [
H]QNB
binding.
To ensure that the increase in mAChR numbers by isoproterenol
stimulation was due to activation of -adrenergic receptors, we
pretreated the cells with the
-adrenergic antagonist nadolol.
Pretreatment of embryonic chick cell cultures with nadolol prior to the
addition of 100 nM isoproterenol significantly attenuated the
increase in mAChR number (Fig. 2A). Treatment of cells
with nadolol alone did not produce a significant effect on mAChR number
as compared to control (Fig. 2A). In addition, we also
observed a time-dependent increase in mAChR numbers in cells stimulated
by isoproterenol. Muscarinic receptor number began to increase by 24 h
and was maximally elevated by 48 h after isoproterenol stimulation (Fig. 2B).
Figure 3:
Isoproterenol-induced increase in mAChR
number results in an increased sensitivity to carbachol-mediated
inhibition of forskolin-stimulated cAMP accumulation compared to
vehicle-treated cells. Cultures were incubated with either vehicle (closed squares) or 10 µM isoproterenol (open
squares) for 72 h. Heart cells were incubated with 100 µM forskolin and the indicated concentrations of carbachol for 5 min
and cellular cAMP levels were then determined. Values are presented as
percent of control cAMP (± S.D.). The control values of cAMP,
measured in the absence of carbachol, were 4330 ± 590 pmol/mg
protein and 4370 ± 780 pmol/mg protein for vehicle and 10
µM isoproterenol-treated cells, respectively. Each
experiment was performed in triplicate, and the results shown are the
average of three separate experiments. At carbachol concentrations of
10 (*) and 10
M (**),
isoproterenol-treated cell were significantly different compared to
control treated cells, p = 0.02 and p <
0.01, respectively.
Figure 4: Isoproterenol stimulation of embryonic chick heart cells results in an increase in cm2 mRNA levels. The level of cm2 mRNA was determined by solution hybridization. A, incubation of chick heart cells with 100 nM isoproterenol maximally increased cm2 mRNA levels by 15 h (p = 0.01). B, pretreatment of cell culture with 100 µM Rp-cAMP, 20 min prior to the addition of 100 nM isoproterenol and incubation for 24 h. There was no significant difference between Rp-cAMP-treated cells and Rp-cAMP plus isoproterenol-treated cells, p > 0.1. However, there was a significant difference between isoproterenol-treated cells and cells that were treated with isoproterenol plus Rp-cAMP, p = 0.05. Each point represents the average of three or more independent experiments (± S.D.), each performed in duplicate or quadruplicate.
We used the competitive inhibitor of cAMP-dependent protein
kinase, Rp-cAMP (Botelho et al., 1988), to test whether the
increase in cm2 mRNA levels by isoproterenol was a result of activation
of cAMP-dependent protein kinase. Incubation of cells with
isoproterenol in the presence of Rp-cAMP did not lead to an increase in
the level of cm2 mRNA (Fig. 4B), demonstrating that the
-adrenergic receptor-mediated increase inmAChR expression is
mediated through cAMP-dependent protein kinase.
This work demonstrates that chronic stimulation of cultured
chick heart cells -adrenergic receptors results in an increased
mAChR number and muscarinic responsiveness in chick heart cells due to
a selective increase in cm2 mRNA levels with no effect on cm4 mRNA
levels. Chick
-adrenergic receptors appear to be pharmacologically
similar to the mammalian
-1 subtype (Port et al., 1992).
Isoproterenol-mediated activation of these
-adrenergic receptors
results in stimulation of adenylyl cyclase activity and a subsequent
increase in cAMP accumulation (Port et al., 1992).
Pretreatment of cultured heart cells with the cAMP-dependent protein
kinase inhibitor, Rp-cAMP, blocked the isoproterenol-mediated increase
in cm2 mRNA levels. Thus, the isoproterenol-mediated increase in cm2
mRNA levels involves activation of cAMP-dependent protein kinase.
Interestingly, the isoproterenol-mediated increase in mAChRs numbers
was due to a differential effect on cm2 and cm4 mRNA levels. Although
cm2 mRNA levels were maximally increased by 25-35%, cm4 mRNA did
not exhibit any changes following isoproterenol stimulation. Prolonged
agonist activation of chick heart mAChRs results in a subsequent
decrease in both mAChR number (Galper and Smith, 1980) in mRNA levels
encoding both the cm2 and cm4 receptors (Habecker and Nathanson, 1992).
Treatment of chick heart cells with muscarinic agonists causes both
inhibition of adenylyl cyclase and stimulation of phospholipase C
(Brown and Brown, 1984). Coupling of the mAChR to both second messenger
pathways is required for agonist-mediated regulation of cm2 and cm4
mRNA. Simultaneous activation of the Ad1 receptor, which results in
inhibition of adenylyl cyclase in the chick heart, and AngII receptors,
which activates phospholipase C in the chick heart, also resulted in a
reduction in mAChR numbers and cm2 and cm4 mRNA levels (Habecker and
Nathanson, 1992). These results indicate that there is a complex role
of second messenger pathways in the regulation of mAChR gene
expression. While the mRNA encoding the cm2 and cm4 receptors are
regulated in a similar fashion by activation of mAChR or Ad1 receptors
and AngII receptors, the expression of cm2 and cm4 mRNAs are
differentially regulated by
-adrenergic receptor activation.
Reithmann et al.(1992) previously reported that chick
cardiac cells which were treated with -adrenergic agonists did not
exhibit an increased magnitude of inhibition of adenylyl cyclase
activity in response to a single high concentration of carbachol.
However, because of the presence of spare receptors, an increase in
mAChR number should lead not to an increase in extent of inhibition but
to a decrease in the concentration of carbachol required for inhibition
of adenylyl cyclase activity (Halvorsen and Nathanson, 1981; Hunter and
Nathanson, 1986). Indeed, we show here that the isoproterenol-mediated
increase in mAChR numbers leads to a greater responsiveness to
carbachol-mediated inhibition of forskolin stimulation of adenylyl
cyclase (Fig. 3).
We have shown here that chronic stimulation
of -adrenergic receptors by isoproterenol causes an increase in
mAChR numbers in heart cells. In addition, these cells exhibit an
increased sensitivity for mAChR-mediated inhibition of forskolin
stimulation of adenylyl cyclase. Furthermore, we have shown that
-adrenergic activation results in a selective increase in mRNA
levels encoding the cm2 mAChR subtype. This increase in cm2 mRNA levels
is dependent upon activation of cAMP-dependent protein kinase.
Long
term treatment with -adrenergic antagonists has been shown to
induce an increase in
-adrenergic receptor number and a
concomitant decrease in muscarinic m2 receptor number and functional
responsiveness in mammalian heart (Motomura et al., 1990;
Marquetant et al., 1992). These changes in neurotransmitter
receptor levels are likely to contribute to the adverse clinical
effects of abrupt withdrawal of chronic
-adrenergic antagonist
administration (Prichard et al., 1983; Fishman, 1987). The
regulation of mAChR expression by
-adrenergic receptors thus is
not only an interesting example of the regulation of neurotransmitter
receptors by heterologous receptor activation but also has important
clinical implications.