Heterologous regulation of muscarinic and beta-adrenergic receptors in rat cardiomyocytes in culture

PII SOO24-3205(98)00378-6

ELSEVIER

tile Sciences, Vol. 63, No. 13. pp. 1169-1182, 1998 Copyright 0 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0024-3205/98 $19.00 + .I0

HETEROLOGGUS REGULATION OF MUSCARINIC AND BETA-ADRENERGIC RECEPTORS IN RAT CARDIOMYOCYTES IN CULTURE Jaromir MysliveEek1~2, Vera Lisa’, Stanislav Trojan’ and Stanislav TuEek’* ‘Institute of Physiology, Academy of Sciences of the Czech Republic, Videiiska 1083, 14220 Prague, and *Department of Physiology, First Medical Faculty of Charles University, AIbertov 5, 12800 Prague, Czech Republic (Received

in final form July 14, 1998)

Summaw Previous work indicated that hyperstimulation of muscarinic receptors brings about profound changes not only in the density of the muscarinic receptors, but also of the l3-adrenoceptors in rat heart atria in vivo. We have now investigated whether a similar receptor cross-regulation occurs in cardiomyocytes in vitro. Cardiomyocytes from 3 - 4 day old rats were exposed to chemical agents on days 5 ic and P-adrenergic receptor were measured - 6 in culture. Densities of must ar!irH]methylscopolamine and [9H]CGP 12177, according to the binding of N-[ respectively, to cell surface membranes and cell homogenates. Exposure of cells to the muscarinic agonist carbachol (1 mmol/l) brought about a profound decrease in the number of muscarinic receptors. The number of P-adrenoceptors displayed biphasic changes, being augmented after 24 h (by 20 - 45 % on the cell surface and by 29 % in the homogenate) and diminished after 48 h and 72 h (after 48 h, decrease by 44 - 75% on the cell surface and by 36 % in the homogenate). These effects of carbachol were not prevented by dimethylaminopropyl-bisindolylmaleimide, the inhibitor of protein kinase C. Exposure of cells to the padrenoceptor agonist isoprenaline (0.1 mmol/I) strongly diminished the number of P-adrenoceptors on the cell surface and in the homogenate. The density of muscarinic receptors on the cell surface was diminished by 24 - 43 % after 24 h exposure to isoprenaline and unchanged after 48 h, whereas the concentration of muscarinic receptors in the homogenate was unchanged after 24 h and increased by 20 % after 48 h. The isoprenaline-induced decrease in the density of cell surface muscarinic receptors could not be simulated by forskolin and was not abolished by the protein kinase A inhibitors Rp-CAMPS and HA-1004. Dibutyryl cyclic AMP diminished the density of cell surface muscarinic receptors more than that of the padrenergic receptors. Our data reveal a novel phenomenon of a biphasic change (an increase followed by a loss) in the density of P-adrenoceptors during exposure of cardiocytes to carbachol. Activation of g-adrenoceptors brings about less conspicuous changes in the density of muscarinic receptors. The observed phenomena of receptor cross-regulation cannot be explained by simple activations of protein kinases A and C. Kq Words: muscarinic receptors, p-adrenoceptors, protein kinase C, protein kinase A

* Corresponding

receptor regulation, in vivo cardiomyocytes,

author; e-mail [email protected]

adenylate cyclase,

1170

Autonomic

Receptors

on Cardiomyocytes

Vol. 63, No. 13, 1998

Mechanisms regulating the expression of genes for neurotransmitter receptors, the density of these receptors and the transduction of signals from them remain far from clear in spite of numerous investigations. It has been recognized that changes in the activity of a certain receptor influence not only the density and life cycle of this particular receptor (homologous up- and downregulatron, (1)) but also the density and life cycle of other receptors and associated molecules (heterologous regulation; (2-4)). Recent investigations underlined the importance of the regulation of G protein-coupled receptors and of their interactions in cardiac pathophystology and pharmacology (S-9). We have found in our prevtous work (10) that chronic administration of an organophosphate inhibitor of cholinesterases to rats brought about biphasic changes (increases followed by decreases) in the denstty of muscarinic and p-adrenergic receptors in the heart atria The brphasrcity of the observed effects and the large extent to which the P-adrenoceptors were affected by cholinesterase inhibition (their 50% loss) were unexpected from the data m the literature and indicated that the interactions between muscarimc and P-adrenergrc receptors m the heart deserve more investrgation. Here we present informatron on selected aspects of these interactions, based on experiments on primary cultures of cardromyocytes isolated from 3 - 4 dayold rats and incubated 111vitro.

Materials and Methods I.soiaiIorl clf’cardi0my0cy1e.s

Wistar rats were bred and kept in the Animal House of the Institute of Physiology m accordance with the NIH Guide,for the Care and CJse ofLaboratov Animals. Pups of either sex and of 3 - 4 days of age were killed by decapitation. Their hearts were dissected, washed, minced and incubated (with stirring) at 37 “C in a physiological saline solution consisting of (mmolil) NaCl 137, KC1 5.4. Na2HP04 7.8, and KH2PO4 1.5, and containing 0.1 % trypsin. After 15 min. the solution was discarded and the minced tissue was incubated eight times for 10 min in fresh portions of the same solution. At the end of each 10 min incubation period, the solution was collected and the action of trypsin on the released cells was stopped by mixing the collected solution with 114 volume of the E 199 cell culture medium supplemented with 10% fetal calf serum.The released cells were sedimented by centrifugation at 250 g for 10 min, resuspended in fresh medium. filtered through medical gauze, recentrifuged and resuspended. Combined ce& from all eight incubation periods were used for preplatmg on untreated plastic dishes (28 cm-), with the aim to diminish the number of fibroblasts. After 45 min at 37 “C, unattached cells (mostly cardiomyocytes) wel;e collected, counted and plated on6collagenpretreated plastic plates (Corning) with 9.4 cm’ wells. at a density of close to 1 x 1P cells per dish Plates with 1.88 cm- wells were used m later experiments and close to 3 x 10 cells were seeded in each well Trcatnient of cultwes Cardiomyocytes were incubated m the E 199 medium supplemented with 10% fetal calf serum and 40 pgiml gentamicin, at 37 “C in an atmosphere of 5 % CO:! and 95 % humidified air. The incubation medium was exchanged on the 2nd, 3rd and 5th days of incubation; between the 2nd and 3rd days, it contained cytosine arabinoside (2 umolil) to prevent the proliferation of contaminating libroblasts. Agonists and other drugs were administered on the 5th day. In experiments with isoprenahne, the drug was dissolved m I mmol/l ascorbic acid and the same concentration of ascorbic acid was added to control cultures without isoprenaline; media containing isoprenalme were exchanged every 12 h Ethanol was used to dissolve forskolin and was then present in the medium at final concentrations of 0. I - 0.4 %; it was also added to control cultures. Dimethylaminopropyl-bis-indolylmaleimide (BIM) was dissolved in dimethylsulfoxide, which was then present in the medium at a final concentration of 1%.

Vol. 63, No. 13, 1998

The purity of cells. Two methods cardiomyocytes: (i) fluoresceine-labelled reagent.

Autonomic Receptors on Cardiomywytes

1171

cultures was systematically controlled by the microscopical appearance of the were applied to more strictly identify the prevailing cell population as immunostaining with mouse anti-pan-myosin antibodies, combined with swine anti-mouse IgG; (ii) staining of glycogen with periodic acid-Schiff s

Radioliganf binding experiments N-[ H]methylscopolampe ([3H]NMS) and (-)-4-(3-tert-butylamino-2_hydroxypropoxy)[5,7-3H]benzimidazol-2-one ([ H]CGP 12177) were applied as specific ligands to determine the numbers of the muscarinic and p-adrenergic binding sites, respectively. [3H]CGP I2177 is a hydrophilic compoyd which does not appreciably permeate the cell membranes (41). The nonspecific binding of [ H]NMS was measured in the presence of atropine (10 mol/l in experiments Y on intact cells and 5 pmol/l in experiments on homogenates) and that of [ H]CGP 12177 in the presence of propranolol (5 pmol/l on intact cells and 3.75 ymol/l in homogenates). [3H]Quinuclidinyl benzilate ( [3H]QNB), a cell-permeant highly specific muscarinic antagonist, was also applied in one group of experiments. The bulk of the data on changes in radioligand binding was obtained with the use of ‘single point’ measurements of the binding. During these measurements, the cells or homogenates were incubated with a single (high but not tilly saturating) concentration of the radioligand and the Bmax values were computed (if necessary) according to the equation Bmax = B x WI +K,) / [Ll (1) where L = radioligand and K, = Kd of the radioligand. The cofcentration of [3H]NMS applied for ‘single point’ measurements was 1.5 nmolA and that of [ H]CGP 12177 was 1.8 nmolA, whereas the respective Kd values were 397 + 74 pmolfl and 312 * 39 pmol/l in control cells and did not change significantly in the course of the treatments (Table 1). Saturation binding (Scatchard-type) experiments were performed to determine the & values, to check their stability during treatments, and to confirm the changes in Bmax values at certain key points of the treatments. In these experiments, cells or homogenates wfre incubated with six different concentration? of the radioligands (50 pmol/l - 1600 pmol/l for [ H]NMS and 62.5 pmol/l - 2000 pmol/l for [ H]CGP 12177), and the Kd and B,,, values were computed by non-linear regression (10,ll) To determine the numbers of the binding sites on the cell surface, the incubation medium was decanted, the attached cells were washed twice with 1 4 mmol/l NaCl, and then incubated at 3 room temperature with [3H]NMS ( + atropine) or [ H]CGP 12177 ( k propranolol) at appropriate concentrations; the radioligands were dissolved in phosphate buffered saline. After 60 min, the radioligand was sucked off, the dish was washed 5 times with 154 mmol/l NaCl, and the membranes were solubilized for 30 min with 1% Triton X-100. The radioactivity released into Triton X-100 was measured by liquid scintillation spectrometry in Bray’s solution. To determine the numbers of binding sites in cell homogenates (‘total’ number of binding sites), the medium was decanted and the cells washed twice with 154 mmol/l NaCl. Cells from several dishes were collected with a rubber policeman, pooled and homogenized with the UltraTurrax T25 homogenizer (Janke and Kunkel, Staufen, Germany) in the homogenization medium consisting of 20 mmol/l Na-HEPES CN-(2-hydroxyethyl)-piperazine-N’-(2-ethanesulfonic) acid] , pH 7.4, and 1 mmol/l MgC12. Aliquots of the homogenate were incubated at 25 “C in an incubation medium consisting of 20 mmgliI Na-HEPFS and 1 mmol/l MgC12 and supplemented with the appropriate concentrations of [ H]NMS, [ H]CGP 12177, atropine, or propranolol, as required for the determinations of the muscarinic or P-adrenergic binding sites. After 90 min, the incubation was stopped by filtration through Whatman GF/B glass fibre filters presoaked in 0.1% polyethylenimine, with the use of a Brandel cell harvester. The filters were dried overnight and the radloactivity retained on them was measured by liquid scintillation spectrometry.

1172

Autonomic Receptors on Cardiomyocytes

Vol. 63, No. 13, 1998

[3H]QM3 was used to determine the total number of muscarinic binding sites in intact cells in the experiments described in Fig. 4. Determination of proteins Peterson’s (12) method was used, with bovine serum albumin as standard Tieatment of data The number of cardiomyocytes that became attached and successfUlly developed was different in different experiments (different seedings) To facilitate comparisons between experiments, control cultures were grown in each experiment and data obtained after various treatments were expressed as multiples of control values obtained in the same experiment Student’s t-test was applied and differences with p < 0 05 were taken as significant. Soirrce 0 materials [ d H]NMS (84 Ci/mmol) was from DuPont-NEN (Dreielch, Germany), [3H]CGP 12177 (50 Ciimmol), [3H]QNB (42 Ci/mmol) and mouse pan-myosin antibody were from Amersham Int (Amersham, England), cytosme arabinoslde (Alexan) was from Mack Nachf. (Illertlssen. Germany), E 199 medium and fluorescein-labelled swine anti-mouse IgG were from USOL (Prague, Czechla), gentamicin was from Lek (Ljubljana, Slovenia), Rp-adenosine 3’,5’-cychc monophosphothioate triethylamine (Rp-CAMPS) and N-(2-guanidinoethyl)-5-isoquinolme sulfonamide (HA-1004) were from Research Biochemicals Int (Natick, MA, U.S.A.), and BIM, trypsin, carbachol (carbamoylcholine chloride), isoprenaline sulphate, forskolin, and dibutyryl cychc AMP (dbcAMP) were from Sigma (St. Louis. MO, U S.A.)

Results Itlcuhations with carhachol Data in Fig. 1 indicate that carbachol (1 mmolil) brought about a rapid loss of the [3H]NMS binding to3the cell surface (78% decrease after 6 h, 93% decrease after 74 h) and a less dramttic decline of [ H]NMS binding in the homogenates (68% decrease after 24 h). The binding of [ H]CGP 12177 became increased after 24 h exposure to carbachol (by 20% in the cell surface membranes and by 19% in the homogenate) but dlmnushed at later time intervals (decrease by 44% in the cell surface membranes and by 36% m the homogenates after 48 h incubatjon with carbachol). After 72 h exposure to carbachol (not shown in Fig. l), the binding of [ H]CGP 12177 on the cell surface was diminished by 36% (n = 17; p < 0.0001) and that in the homogenate by 11% (n = 4; p < 0.05). The biphasic change in [3H]CGP 12177 binding induced by carbfchol was confirmed in saturation binding experiments. As shown in Table I, the number of the [ H]CGP 12177 bmdmg sites was augmented b 45% after 24 h and diminished by 75% after 48 h of the exposure to Y carbachol The Kd for [ H]CGP 12.177 bmding did not change Iucubntions with isoprenalincj As shown in Fig. 2, [ H]CGP 12177 binding to the cell surface diminished rapidly during exposure to 100 ~moill isoprenaline (91’/, decline after 6 ) and the binding to the homogenates also declined (67% decrease after 24 h). The bmdmg of [ jlH]NMS to the cell surface binding sites underwent a substantial (43%) decrease after 24 h exposure to isoprenaline, but this change was transient and was not accompanied by a similar chang in the in the binding to the homogenates After 48 h exposure to isoprenaline, the binding of [5 H]NMS to the homogenate increased by 20%, without an accompanying elevation in the binding to the cell surface. The loss of the [3H]NMS binding sites in cell surface membranes occurring after 14 h exposure to isoprenaline was confirmed in saturation binding experiments (Table 1); it was not accompanied by a significant change of the & value.

1173

Autonomic Receptors on Cardiomyocytes

Vol. 63, No. 13, 1998

1.4-

Surface

receptors

0.6 0.6 -

\;*

0.4 z s

0.2 0.0

8

** ,, 01

I

6

Fi 1

.g

I

I

24

36

**

0 46

Total receptors 1.4 -

S z

1.2 t.0 0.6 Y

0.6 -

**

0.2 0.0

** ,,

1

I

1

1

01

6

incabat%

t3i

46

time 0

W

m-AcChR

] P-AR

Fig. 1 Effect of carbachol on the numbers of [3H]NMS (circles) and [3H]CGP 12177 (squares) binding sites measured on the surfac5 of cardiomyocytes (top) and in their homogenates (bottom). Cells were grown in 9.4 cm wells and ‘single point’ measurement of binding was applied. Abscissa: Hours of incubation of cultures in the presence of 1 mmol/l carbachol. Ordinate. Number of binding sites discovered, expressed as a multiple of the number of binding sites in parallel control cultures. Data are means + SE of 9 - 18 determinations for the surface binding sites and of 5 - 8 determinations (with incubation in duplicates) for the binding sites in homogenates. The number of binding sites (Bmax values, mean it SE, c mputed according to equation 1) in control samp s was 38.5 f 4.9 fmol/well for !? [‘H]NMS on the cell surface, 4 2 1 _ + 3 2 fmol/well for [ H]CGP 12177 on the cell surface, 7.6 50.9 fmol/mg protein for [ H]NMS m the homogenate, and 21 .O f 3.1 fmol/mg protein for [ H]CGP 12177 in the homogenate. Statistical significance of differences from control: *, p < 0.05; **, p < 0.001.

1174

Autonomic

Receptors

Vol. 63, No. 13, 1998

on Cardiomyocytes

cl, I-_-i:_., 0

z. ._ ” 3

6

Total receptors

1.4

E ;i.;;-

aJ

0.6-

\

,I *

0.4 0.2 1 0.0

b’

** ’ , 0

I

I

I

48

6

incudiion 0

m-AcChR

time [ h ] n

II-AR

Fig. 2 Effect of isoprenaline on the numbers of [3H]NMS (circles) and 13H]CGP 12177 (squares) binding sites measured on the surfac5 of cardiomyocytes (top) and in their homogenates (bottom). Cells were grown in 9.4 cm’ wells and ‘single point’ measurement of binding was applied. Abscissa: Hours of incubation of cultures in the presence of 100 umol/l isoprenaline and 1 mmol/l sodium ascorbate. Ordinate: Number of binding sites discovered, expressed as a multiple of the number of binding sites in parallel control cultures. Data are means k SE of 9 - 19 determinations for the surface binding sites and of 5 - 8 determinations (with incubation in duplicates) for the binding sites in homogenates. The number30f binding sites (Bmax values; mean + SE) in control samrjles was 42.7 f 8.7 fmoliwell for [ H]NMS on the cell surface, 37.2 k 739 finoliwell for [ H]CGP 12177 on the cell surface, 17.2 k 8.1 f$tol/mg protein for [ H]NMS in the homogenate, and 11 7 i 6 1 fmolimg protein for [ H]CGP 12177 in the homogenate. Statistical significance of differences from control *, p < 0.05; **, p < 0.001.

1175

Autonomic Receptors on Cardiomyocytes

Vol. 63, No. 13, 1998

TABLE I

Effects of Farbachol on the Binding of [3H]CGP 12177, and Effects of Isoprenaline on the Binding of [ H]NMS to Cell Surface Binding Sites: Results of Saturation Binding Experiments

Exposure

to carbachol

B,, for [3H]CGP 12177 n B,, as er cent of control K., for [ !?HjCGP 12177

n P Exposure

to isoprenaline

B, for [3H]NMS n B,, as er cent of control Kd for [PHlNMS n P

Control

24 hours

48 hours

16.1 k 1.6 4 100

23.4 + 2.4 5 145

4.5 k 1.0 3 25

312~~39 6

352 f 56 5 < 0.05

370 f 87 3 < 0.05

1 mmol/l

0.1 mmoUl

9.4 * 1.1 3 100 397 f 74 5

7.2 f 0.3 4 76 319f39 4 < 0.05

B values (fmol per incubation well) are means + SE of n saturatiqn binding e:Griments on cell cultures from two different seedings, grown in 1.88 cm’ wells. IQ (pmolflJ are means f SE of n saturation experiments on cell cultures grown in 1.88 or 9.4 cm wells, p = probability of no difference of B,, value from control.

Incubations with forskolin Forskolin is known for its ability to strongly activate adenylate cyclase and it was of interest to see whether it can siTlate the effects of isoprenaline. After 24 h incubation with 10 pmolil forskolin, the binding of [ H]CGP 12177 to the cell surface binding sites diminished by 2 l%, and forskolin at 20 umol/l and 40 pmol/l concentrations had comparable3effects (24% and 28% decreases, respectively, of [ H]CGP 12177 binding). The binding of [ H]NMS to cell surface sites was not altered significantly by 10 - 40 pmol/l forskolin during 24 h incubation. The effect of forskolin on [3H]CGP 12 177 binding was substantially smaller than the effect of isoprenaline but was similar to that of dbcAMP (Fig. 3). Incubations with dibutyryl cyclic AMP er 24 h incubation of cardiomyocytes with 1 mmol/l dbcAMP, the densi of cell Aft surface [ H]NMS binding sites was diminished by 47% and the total nymber of the ?[ H]NMS binding sites was reduced to a similar extent. The density of cell surface [ H]CGP 12177 binding sites was diminished by 27% Apparent difference between the effects of dbcAMP treatment on the total number of the [3H]CGP 12177 binding sites and on the density of these sites on the cell surface was not significant statistically (p > 0.05; Figs. 3 and 4).

1176

Autonomic

Receptors

on Cardiomyocytes

Vol. 63, No. 13, 1998

Fig. 3

ects of 24 h exposures to v rious agents alone or in combination on the numbers of ? [ H]NMS (shaded bars) and [ H]CGP 12177 (empty bars) binding sites in cell surface membranes of cardiomyocytes. Cells were incubated in 1.88 cm wells. Upper part: isoprenaline (100 umolil), forskolin (40 umol/l), isoprenaline (100 pmol/l) + RpCAMPS (100 umol/l), isoprenaline (100 umol/l) + HA- 1004 (10 umol/l), isoprenaline (100 pmol/l) + HA-1004 (20 pmol/l), HA-1004 (10 umol/l), HA-1004 (20 umol/l), and RpCAMPS (100 umolil). Lower part Isoprenaline (100 umol/l) + BIM (1 umol/l), dbcAMP (1 mmol/l), dbcAMP (1 mmol/l) + Rp-CAMPS (100 pmolil), dbcAMP (1 mmol/l) + HA- 1004 ( 10 umol/l), dbcAMP (1 mmol/l) + HA- 1004 (20 pmol/l), dbcAMP (1 mmol/l) + BIM (1 umol/l), and BIM (1 umol/l). Data are means f SE of 6 determinations for the effect of forskolin, of 8 determinations for the effect of the combination of dbcAMP + RpCAMPS, and of 93 45 determinations for the other agents. Control valu s were 13 - 15 fmol/dish for the [ H]NMS binding sites and 15 - 25 fmol/dish for the [ !?H]CGP 12177 binding sites. Statistical significance of differences from control: * , p < 0 05; ** , p < 0 001. Y

Vol. 63, No. 13, 1998

Incubations

Autonomic

with Rp-CAMPS* HA-1004

Receptors on Cardiomyocytes

1177

and BIM

BIM is an inhibitor of protein kinase C (13) and we yondered if it rngy block the actions of carbachol, isoprenaline and dbcAMP on the binding of [ H]NMS and [ H]CGP 12177. BIM alone (applied at a concentration of 1 pmol/l, i.e. 10 fold its IC503for the inhibition of protein P sites kinase C [39]) had no effect on the densities of the [ H]NMS and [ H]CGJ’ 12177 binpg (Fig. 3). As shown in Table 2, it did not alter the effects of carbachol on [ H]NMS or [ H]CGP I 71 77 binding t cell surface membranes. The effects of 24 h exposure to isoprenaline on 9 [3H]NMS and [ H]CGP 12177 binding were not affected by BJ’M either (Fig. 3). Rather surprisingly, the negative effect of dbcAMP on the density of the [ H]CGP 12177 binding sites was considerably enhanced in the presence of BIM (Fig. 3). The effects of isoprenaline on the numbers of the [3H]NMS and [3H]CGP 12 177 binding sites were not altered in the presence of two inhibitors of protein kinase A, i.e. of 100 umol/l RpCAMPS (14) or of 20 umolil HA- 1004 (15) (Fig. 3). The effect of dbcAIvIP on the number of the [‘HICGP 12 177 binding sites was not changed by Rp-CAMPS but was enhanced by HA-1004 (Fig. 3).

Discussion The main observations described in the present study may be summarized as follows: (a) Chronic exposure of cardiomyocytes to carbachol brought about not only the down-regulation of the muscarinic receptors, but also a biphasic change (an increase followed by a decrease) in the density and the total content of the P-adrenoceptors. (b) Chronic exposure of cardiomyocytes to isoprenaline brought about not only the downregulation of p-adrenoceptors, but also a transient down-regulation of muscarinic receptors in cell surface membranes, followed by an up-regulation of the total content of muscarinic receptors. (c) Exposure of cardiomyocytes to dbcAMP for 24 h brought about decreases in the densities of both muscarinic and P-adrenergic receptors, whereas forskolin diminished only the density of padrenergic receptors. In comparison with isoprenaline, dbcAMP had a higher effect on the muscarinic and a lower effect on the g-adrenergic receptors. (d) The effects of carbachol and of isoprenaline on the muscarinic and p-adrenergic receptors were not prevented by BIM, an inhibitor of protein kinase C. Losses of muscarinic and P-adrenergic receptors induced by isoprenaline or dbcAMP were not prevented by Rp-CAMPS and HA-1004, two inhibitors of protein kinase A. The loss of surface P-adrenoceptors induced by dbcAMP was enhanced by BIM. Many interactions between various receptor systems have been described in recent years, but it is apparent From the literature that the interactions between various receptors (plus their associated transduction pathways) vary depending on the types of cells used for investigations. Physiological relevance of data obtained on transformed or genetically engineered cells requires special evaluation because the stoichiometric relations between the components of cell singalling systems are frequently altered in these cells. The focus of our study was the specific situation in the cells of the mammalian heart. Interactions between cardiac muscarinic receptors (which are predominantly of the M2 subtype) and l3-adrenergic receptors are particularly interesting in the heart because these two types of receptors induce nearly exactIy opposite changes of cardiac function (16). The observed carbachol-induced decline in the density of muscarinic binding sites in rat cardiomyocytes corresponds to changes that had already been described in chick cardiac cells (1719), but the complex changes in the density of the P-adrenergic sites discovered during incubations with carbachol are new. They consist in an elevation followed by a decline in the density of padrenoceptors both in the homogenates and in the surface membranes. This is a novel observation which is in good agreement with what we had found in the heart atria of rats treated in vivo with

1178

Autonomic Receptors on Cardiomyocytes

1.0 **

Vol. 63, No. 13, 1998

P-AR

**

n-AcChR ** **

0.2

0.0

d

m 0

surface total

Fig. 4

Effect of dbcAMP on the numbers of cell surface and total muscarinic (mAChR) apd Badrenergic (P-AR) binding sites in cardiomyocytes. Cells were incubated in 1.88 cm- wells in the presen?e of 1 mM dbcAMP for 24 9. Cell surface binding sites were determined with 1.5 nmol/l [ H]NMS and 1.8 nmol/l [ 3H]CGP 12177 as described in Methods. Cell permeant specific muscarinic antagonist [ H]QNB (1 .I nmol/l) was used to measure the total number of muscarinic binding sites, whereas the total numbers of the B-adrenergic binding sites were determined in cell homogenates. Data are means ( f SE) of 20 - 26 determinations. Control values corresponded to 28.7. ? 2.2 fmol/well for total muscarimc sites, 13.1 I 0.4 fmol/well for cell surface muscarinic sites, 19.1 -t 1.5 fmoliwell for total l3adrenergic sites and 16.3 + 0.5 fmol/well for cell surface P-adrenergic sites. Statistical The difference between the significance of differences from control: **, p < 0.001. dbcAMP-induced loss of P-adrenergic sites on the cell surface and in the homogenates was not significant (p > 0.05).

an inhibitor of cholinesterases (10). We assume that the loss of p-adrenoceptors (observed after 48 - 72 h exposure to carbachol) occurs as a part of the process in which cells tend to establish a new equilibrium between the P-adrenoceptors and the down-regulated muscarinic receptors. A carbachol-induced loss of P-adrenoceptors has been noted in the smooth muscle cells of the trachea (20). The molecular mechanism of the observed heterologous down-regulation of padrenoceptors is not known. It could be hypothesized that phosphorylation of p- adrenoceptors by protein kinase C might play a role since cardiac muscarinic receptors are known to stimulate the hydrolysis of phosphoinositides (2 1) and hence the activity of protein kinase C, and protein kmase C is able to phosphorylate both p-adrenoceptors (22) and muscarinic receptors (23). In addition, the activation of protein kinase C by a phorbol ester diminished the transcription of the muscarinic m2 receptor gene in a human embryonic lung cell line (24). We have found, however, that BIM, a strong and selective inhibitor of protein kinye C (IC50 = 50 nmolil; (13)) did .not modify the effects of carbachol on the densities of the [ H]NMS and [ H]CGP 12177 bmdmg sites, which contradicts the above hypothesis, Similarly, Lai et al. (25) observed that the carbachol-induced

1179

Autonomic Receptors on Cardiomyocytes

Vol. 63, No. 13, 1998

TABLE II Carbachol (CCh) Effects on the Densities of the [-‘H]NMS and [‘H]CGP 12177 Binding Sites in the Cell Surface Membranes in the Presence of Dimethylaminopropyl-bis-indolylmaleimide (BIM) Control

CCh

CCh

CCh + BIM

CCh + BIM

24 h

48 h

24 h

48 .-..-_-.-~-..~--. h

13H]NMS Mean f SE

5.57 f 0.34

0.68 i 0.08

0.94 f 0.17

0.60 k 0.08

1.03 f 0.02

n

6

4

5

5

5

% of control

100

P

12

17

11

18

< 0.001

< 0.001

< 0.001

< 0.001

17.01 + 2.97

4.05 f 0.18

16.11 + 1.26

3.78 f 0.73

[3H]CGP 12177

Mean f SE n

4

% of control P

12.78 z!z0.45

100

6

5

6

4

133

31

125

30

< 0.05

< 0.001

< 0.001

< 0.001

Cardiomyocytes were grown in culture for 7 days in 1.88 cmL wells. Carbachol (1 mmolil) was added either 24 h or 48 h before the end of cultivation. Where indicated, BIM (1 pmol/l) was added to the wells 30 min before car$achol. Radioligand binding was determined with 1.5 nmol/l [3H]NMS or 1.8 nmol/l [ H]CGP 12177 as described in Methods for ‘single point’ measurements. Data in the Table indicate fmol of radioligand bound per well (mean + SE of n wells). p = probability of no difference from control.

desensitization of muscarinic receptors in neuroblastoma cells was not dependent on the activity of protein kinase C. Recent data indicate that nitric oxide acts as the mediator of some of the muscarinic effects in the heart (26) but, at the present state of knowledge, it would seem speculative to us to propose its participation in the phenomena we observed. Limas and Limas (27) described a small rapid decrease in the density of cell surface l3adrenoceptors in cardiomyocytes isolated from adult hearts, which occurred within 20 min of the action of carbachol but was not followed beyond a 30 min incubation period. That3decrease might have corresponded to the transient and statistically insignificant decrease of [ H]CGP 12177 binding sites in cell surface membranes seen in our work (Fig. 1) after 1 h exposure to carbachol. After 20 h of carbachol action, Paraschos and Karliner (28) found a 22% decrease in the density of P-adrenoceptors on the surface of rat cardiocytes, which was not accompanied by any change in their intracellular content. On the other hand, Reithman and Werdan (29) noted no change in the number of P-adrenoceptors on the surface of rat cardiomyocytes after 3 days of exposure to 1 mmol/l carbachol, although the response of adenylate cyclase to isoprenaline was enhanced by this treatment. Their data are at variance with our findings of small but significant increases in the number P-adrenoceptors after 24 h incubation with carbachol and of losses after 48 and 72 h, but

1180

Autonomic

Receptors

on Cardiomyocytes

Vol. 63, No. 13, I!998

the explanation of the differences is not obvious; it seems unlikely that the use of a serum-free medium by Reithmann and Werdan (29) might be responsible. Exposure to isoprenaline brought about not only the expected loss of p-adrenoceptors (I), but also a transient 43 % decrease in the density of cell surface muscarnnc receptors after 34 h, and a 20 % increase of total muscarinic receptors after 48 h exposure. Decreases in the number of muscarinic receptors were found in genetically engineered CHO cells (30) and transformed human lung cells (3 1) during prolonged P-adrenoceptor activation, whereas increases were induced by the Matthews et al. (34) found no P-agonist isoprenaline in cultured chick cardiomyocytes (32,33) effect of chronic infusions of isoprenaline on the density of cardiac muscarinic receptors measured in viva, although they did find changes in the concentrations of mRNAs coding for the muscarinic receptors in the right and left ventricles. Lee and Fraser (30) and Jackson and Nathanson (33) explained the isoprenahne-induced changes in the numbers of muscarinic receptors by an activation of protein kinase A, whereas Rouse11 et al. (3 1) discovered that the regulatory mechanism must be rather more complex In then experiments, the loss of muscarinic M2 receptors induced by the p-2 adrenergtc agonist procaterol was completely prevented not only by the inhibition of protein kinase A, but also by the inhrbmon of protein kinase C, which indicates that both protein kinases played unidentified but ptvotal roles in the P-receptor induced muscarinic receptor down-regulation. Several aspects of our findings strongly indicate that the tsoprenaline-induced decrease m the density of cell surface muscarinic receptors cannot be a simple consequence of an ac vatron of 9 adenylate cyclase and protein kinase A: (i) Forskolin had no effect on the binding of [ H]NMS, a&though it is known to induce maximum activation of adenylate cyclase (Fig. 3). (Ii) The loss of [ H]NMS binding was not prevented by Rp-CAMPS and HA-1004, two inhibitors of protein kinase A (Fig. 3). (iii) While isoprenaline affected only the cell surface muscarimc bmding sites (Fig. 3) both cell surface and intracellular binding sites were diminished by the exposure to dbcAMP (Fig. 4). Similarly, Reithmann et al. (32) concluded that the increase in the concentration of muscarmic receptors which they induced in chick cardiomyocytes by 72 h exposure to isoprenaline was cychc AMP-independent. It is not clear which transduction pathway is mvolved The effect of dbcAMP on cell surface p-adrenoceptors appeared much weaker than the effect of isoprenaline This agrees with the notion that agonists induce p-adrenoceptor sequestration by an activation of the P-adrenoceptor-specific kinase, rather than by an activation of the protein kinase A. The activation of protein kinase A might perhaps inhibit the synthesis of new receptors. We cannot offer a definite explanation, however, for the findings that the dbcAMPinduced loss of cell surface P-adrenoceptors was enhanced by the protein kinase C inhibitor BIM and by the protein kinase A inhibitor HA-1004 (Fig. 3). Since HA-1004 is also an inhibitor of the cychc GMP-dependent protein kinase (15) , it is a speculative possibility that its actron on this enzyme served to potentiate the down-regulation of p-adrenoceptors by dbcAMP. Recent investigations of the effects of agonists on the concentrations of mRNAs for their receptors yielded unexpected results in that, in several studies, agonists produced transient increases in the levels of mRNAs for then own receptors both in the case of P-adrenoceptors (35,36) and of muscarmic receptors (37-40). Although we have observed transient increases of muscarinic receptor numbers in the atria of rats treated wtth an inhibitor of cholinesterases in IQVO (i.e., homologous up-regulation (10)) , we drd not discover a simtlar pattern of changes in the cells ilr viiro. Collectively, our data clearly demonstrate that the mechanisms regulating the levels and life cycle of muscarinic and j3-adrenergic receptors are mutually interconnected in rat heart cells. Muscarinic activation induced an increase and subsquent substantial decrease of P-adrenoceptor numbers. The activation of p-adrenoceptors by isoprenaline produced a transient loss of muscarinic receptors from the surface membranes, followed by an increase in the total number of

Vol. 63, No. 13, 1998

Autonomic Receptors on Cardiomyocytes

1181

receptors in the cells. Data obtained with forskolin, dbcAMP and the itihibitors of protein kinases indicate that the mechanism of the observed interactions is complex and apparently cannot be simply reduced to receptor-mediated activations of either protem kinase A or protein kinase C.

AcknowledPements This work was supported by grant No. 30919611287 of the Grant Agency of the Czech Republic (to S. TuEek) and by NM FIRC Award No TWO0171 (to E. E. El-Fakahany). We thank Professor N. M. Nathanson for advice on culturing cardiomyocytes, Professor E. E. El-Fakahany for collaboration, and Dana Ungerova and Olga Hustbkovb for technical assistance

References 1. 7 ;: 4. 5. 6. 7.

8. 9. 10. 11. 12. 13.

14. 15. 16. 17. 18 19. 20. 21. 22. 23. 24. 25.

J.L. BENOVIC, M. BOUVIER, M.G. CARON and R.J. LEFKOWITZ, Annu. Rev. Cell. Biol. 4, 405-428 (1988). R.C. ARMSTRONG and M.R. MONTMINY, Annu. Rev. Neurosci. 16, 17-29 (1993). J.D. PORT and C.G. MALBON, Trends Cardiovasc. Med. 3, 85-92 (1993). T.T. CHUANG, L. IACOVELLI, M. SALLESE and A. DE BLASI, Trends Pharmacol. Sci. 17,416-421 (1996). O.-E. BRODDE, Pharmac. Ther. 60,405-430 (1993). O.-E. BRODDE, M.C. MICHEL and H.-R. ZERKOWSKI, Cardiovasc. Res. 30, 570-584 (1995). T. ESCHENHAGEN, U. MENDE, M. DIEDERICH, B. HERTLE, C. MEMMESHEIMER, A. POHL, W. SCHMITZ, H. SCHOLZ, M. STEINFATH, M. B&lM, M.C. MICHEL, O.E. BRODDE and A. RAAP, Circulation 93, 763-771 (1996). P. SCHNABEL and M. B&&I, Cell Signal. 8,413-423 (1996). M. CASTELLANO and M. B&IM, Hypertension 29,715-722 (1997). J. MYSLIVECEK, S. TROJAN and S. TUCEK, Life Sci. 58,2423-2430 (1996). J. JAKUBiK and S. TUCEK, Brit. J. Pharmacol. 113, 1529-1537 (1994). G.L. PETERSON, Analyt. Biochem. 83,346-356 (1997). D. TOULLEC, P. PIANETTI, H. COSTE, P. BELLEVERGUE, T. GRAND-PERRET, M. AJAKANE, V. BAUDET, P. BOISSIN, E. BOURSIER, F. LORIOLLE, L. DUHAMEL, D. CHARON and J. KIRILOVSKY, J. Biol. Chem. 266, 15771-15781 (1991). L.H.P. BOTELHO, J.D. ROTHERMEL, R.V. COOMBS and B. JASTORFF, Methods Enzymol. 159, 159-172 (1988). H. HIDAKA, M. INAGAKI, S. KAWAMOTO and Y. SASAKI, Biochemistry 23, 50365041 (1984). J.P. LINDEMANN and A.M. WATANABE, Physiology and Pathophysiology of the Heart, N. Sperelakis (ed) 423-452, Kluwer Academic Publishers, Boston (1989). J.B. GALPER and T.W. SMITH, J. Biol. Chem. 255, 9571-9579 (1980). J.B. GALPER, L.C. DZIEKAN, D.S. O’HARA and T.W. SMITH, J. Biol. Chem. 257, 10344-10356 (1982). E.M. SUBERS and N.M. NATHANSON, J. Mol. Cell. Cardiol. 20, 131-140 (1988). B.M. GRANDORDY, J.C.W. MAK and P.J. BARNES, Life Sci. 54, 185-191 (1994). S.L. BROWN and J. H. BROWN, Mol. Pharmacol. 24, 351-356 (1983). M. BOUVIER, L.M.F. LEEB-LUNDBERG, J.L. BENOVIC, M.G. CARON and R.J.LEFKOWITZ, J. Biol. Chem. 262, 3 106-3 113 (1987). W C. LILES, D.D. HUNTER, K.E. MEIER and N.M. NATHANSON, J. Biol. Chem. 261, 5307-53 13 (1986). J. ROUSELL, E.-B. HADDAD, J.C.W. MAK and P.J. BARNES, J. Biol. Chem. 270, 7213-7218 (1995). W.S. LAl, T.N. ROGERS and E.E. EL-FAKAHANY, Biochem. J. 267,23-29, (1990).

1182

26.

27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41.

Autonomic

Receptors

on Cardiomyocytes

Vol. 63, No. 13, 1998

J.M. HARE, J.F. KEANEY, J.-L. BALLIGAND, J. LOSCALZO and T.W. SMITH, J. Clin. Invest. 95, 360-366 (1995). C.J. LIMAS, and C. LIMAS, Biochem. Biophys. Res. Commun. 128, 699-704 (1985). A. PARASCHOS and J.S. KARLINER, Naunyn-Schmiedeberg’s Arch. Pharmacol. 350, 267-276 (1994). C. REITHMANN and K. WERDAN, Naunyn-Schmiedeberg’s Arch. Pharmacol. 351, 2734, (1995). N.H. LEE and C.M. FRASER J. Biol. Chem. 268, 7949-7957 (1993). J. ROUSELL, E.-B. HADDAD, J.C.W. MAK, B.L.J. WEBB, M.A. GIEMBYCZ and P.J. BARNES, Mol. Pharmacol. 49, 629-635 (1996). C. REITHMANN, B. PANZNER and K. WERDAN, Naunyn-Schmiedeberg’s Arch. Pharmacol. 345, 530-540 (1992). D.A. JACKSON and N.M. NATHANSON, J. Biol. Chem. 270, 22374-22377 (1995). J.M. MATTHEWS, P.H.J. FALCKH, P. MOLENAAR and R.J. SUMMERS, NaunynSchmiedeberg’s Arch. Pharmacol. 213, 2 13-225 (1996). S. COLLINS, M. BOUVIER, M.A. BOLANOWSKI, M.G. CARON and R.J. LEFKOWITZ, Proc. Natl. Acad. Sci. USA 86,4853-4857 (1989). S. COLLINS, J. ALTSCHMIED, 0. HERBSMAN, M.G. CARON, P.L. MELLON and R.J. LEFKOWITZ, J. Biol. Chem. 265, 19330-19335 (1990). S.-Z. ZHU, S.-Z. WANG, E.A.M. ABDALLAH and E.E. EL-FAKAHANY, Life Sci. 48, 2579-2584 (1991) H. CHOU, N. OGAWA, M. ASANUMA, H. HIRATA, Y. KONDO and A. MORI, Mol. Brain Res. 19, 211-214 (1993). M.C. STEEL and N.J. BUCKLEY, Mol. Pharmacol. 43,694-701 (1993). R. BRUSA, S.R. GAMALERO, E. GENAZZANI and C EVA, Eur. J. Pharmacol. 289, 9-16 (1995). M. STAEHELIN, P. SIMONS, K. JAEGGI and N. WIGGER, J. Biol Chem. 258, 34963502 (1983).