Modulation of the beta-adrenergic-response in cultured ... - Springer Link

Molecular and Cellular Biochemistry 102: 49-60, 1991. © 1991 Kluwer Academic Publishers. Printed in the Netherlands.

Modulation of the beta-adrenergic-response in cultured rat heart cells H. Mammary-derived growth inhibitor (MDGI) blocks induction o f beta-adrenergic supersensitivity. Dissociation from lipid-binding activity of M D G I Gerd Wallukat I, Frank-D. Boehmer2, Ulla Engstroem 3, Peter Langen 2, Morley Hollenberg4, Joachim Behlke 5, Hartmut Kuehn 6 and Richard Grosse 2

1CentralInstitutefor CardiovascularResearch and 2Departments of Cell Biology and 5Molecular Biophysics, Central Institute of Molecular Biology, Academy of Sciences, 1115 Berlin-Buch, Germany; Ludwig Institute for Cancer Research, Uppsala Branch, 75321 Uppsala, Sweden; 4Department of Pharmacology and Therapeutics, University of Calgary, Calgary, Canada T2N 4N1, 6Humboldt-Universitaet Berlin, Institutefor Biochemistry, Berlin, Germany Received 27 February 1990; accepted 9 August 1990

Key words: growth inhibitor, modulation of beta-adrenergic response, rat neonatal heart muscle cells, lipid binding, fatty acid-binding protein

Summary 'Mammary-derived growth inhibitor (MDGI)' is a 14.5 kDa polypeptide with growth-inhibitory activity for various mammary epithelial cells in vitro which is highly homologous to cardiac fatty acid-binding protein (H-FABP). Here we describe a new biological activity of MDGI: Inhibition of L(+)-lactate-, arachidonic acid- and 15-S-hydroxyeicosatetraenoic acid-induced supersensitivity of neonatal rat heart cells for betaadrenergic stimulation, concerning particularly a small population of beta2-receptors. Synthetic peptides corresponding to the MDGI-sequence, residue 121-131 mimic the effect of MDGI. Measurements of lipid-binding to MDGI and synthetic peptides excluded the binding of arachidonic acid, 15-S-hydroxyeicosatetraenoic acid or beta-adrenergic agonists to MDGI or the peptides as the mechanism for this effect. Also, no direct interference of MDGI and the synthetic peptides with the binding of the beta-adrenergic agent CGP 12177 to its receptor on A431 cells could be detected. We suggest that MDGI and the peptides act by interference with the function of the beta2-adrenergic receptor and that this mechanism might also be relevant for the growth-inhibitory activity of MDGI. Furthermore, the data point to a possible function of H-FABP for the modulation of beta-adrenergic sensitivity of cardiac myocytes.

Abbreviations: MDGI - mammary-derived growth inhibitor, HETE - hydroxyeicosatetraenoic acid, CGP 12177 - ((+/-)-4-(3-t-butylamino-2-hydroxypropoxy)-benzimidazole-2-one, PBS - phosphate buffered saline: 20 mM sodium phosphate, 150 mM sodium chloride, pH 7.4, HEPES - N - (2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid, SDS - sodium dodecylsulfate

50 Introduction

Materials and methods

MDGI is a 14.5 kDa polypeptide which has been isolated from lactating bovine mammary gland according to its growth inhibitory activity for Ehrlich ascites mammary carcinoma cells [1, 2]. The protein has been sequenced and found to belong to a superfamily of lipid-binding proteins including cellular retinoid-binding proteins, fatty acid-binding proteins and proteins of as yet unknown specificity of lipid-binding [3]. MDGI has been shown to inhibit the growth of a variety of mammary-epithelial cells in vitro [3, 4] and to be immunologically related to a 'flbroblast growth regulator (FGRs 13k)' [51. The expression of MDGI in the mammary gland has been found to be positively correlated with differentiation in terms of lactation [2, 6]. Large amounts of MDGI in lactating mammary tissue were localized intracellularly by immunological methods [6]. MDGI-related antigens have been characterized in milk fat globule membranes [7] and in a nuclear fraction of lactating mammary gland [6]. The mechanism of action of MDGI as well as its physiological function are unknown. MDGI has been demonstrated to possess lipid-binding activity and this activity has been suggested to be involved in the mechanism of growth inhibition [8]. As described in the accompanying paper [9] L(+)-lactate, arachidonic acid and 15-hydroxyeicosatetraenoic acid can induce a beta-adrenergic supersensitivity in neonatal rat heart muscle cells. The involvement of long-chain fatty acids in the induction mechanism on the one hand and the lipid-binding activity of MDGI as well as its close homology to H-FABP [3] on the other prompted us to test the influence of MDGI on the induction of beta-adrenergic supersensitivity. We found that MDGI can inhibit the induction of supersensitivity for beta-adrenergic stimulation. This inhibitory effect could, however, be dissociated from the lipidbinding activity of MDGI.

Lipids Arachidonic acid was from Serdary Research Laboratories (London, Ontario) thromboxane Be and the thromboxane A2 agonist U 46619 were a gift of Prof. C. Taube (Halle). 15-S-HETE was synthesized and purified as described in the accompanying paper [9]. All other lipids were from Sigma.

Radiochemicals [3H]-15-S-Hydroxyeicosatetraenoic acid (213 Ci/ mmol), [3H]-CGP 112177 (30--50 Ci/mmol), [3H]palmitic acid (50 Ci/mmol) and [3H]-prostaglandin E1 (40.7 Ci/mmol), were from Amersham. [3H]-prostaglandin E2 (185 Ci/mmol) was from New England Nuclear. [3H]-retinoic acid was a kind gift of Dr. Wuersch (Basel). Monoiodinated p25]I-tetraiodothyronine was kindly provided by L. Boehlke (Berlin).

MDGI MDGI was prepared from lactating bovine mammary gland as described [3]. Preparations used in this study were at least 95% pure, as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate.

Synthetic peptides Peptides were synthesized with an Applied Biosystems 430A Peptide Synthesizer (Forster City, CA, USA) using t-boc chemistry following the protocol recommended by the manufacturer. The products were cleaved from the polymeric support by hydrogen fluoride and purified by reversed phase HPLC on a 20 x 300 mm column of Nucleosil C18 with a gradient of 0--40% acetonitril in 0.1% trifluoroacetic acid. Identification of the purified peptides was accomplished by zszCf-plasma desorption mass spectrometry. The cysteine-containing

51 peptides were subjected to reduction by treatment with 5% (v/v) 2-mercaptoethanol at 100°C for 2 min and subsequent lyophilization before assaying.

Ultracentrifugation studies Ultracentrifugation experiments to determine the molecular mass of the peptides in solution were done as outlined in [10].

Induction of supersensitivity for beta-adrenergic stimulation and inhibition by MDGI and synthetic peptides Supersensitivity was induced by treatment of the neonatal rat heart muscle cells with L(+)-lactate (3 mM), arachidonic acid (10 nM) or 15-S-HETE (10 nM) for two hours at 37°C as described in the accompanying paper [9]. Beta-adrenergic stimulation was achieved by subsequent treatment of the cells with various concentrations of isoprenaline and measurement of the beating rate of the cells, as described in detail in the accompanying paper [9]. To test the effect of MDGI or synthetic peptides, the putative effectors were added to the cultures simultaneously with the inducing agent in the concentrations indicated in the figures and tables, and beta-adrenergic stimulation was measured as described above. In the figures mean values of measurements in 3-5 different cultures representing 30-50 cells or cell clusters are shown.

Lipid binding experiments Lipid binding to MDGI was measured using the Sephadex derivative Lipidex 1000 (Packard) for separation of free and protein-bound ligand [11], as described previously [8]. In brief, the radioactive ligand (about 3 x 10-TM) was incubated with 2-3nmol MDGI in a total volume of 0.4ml of HBSS (20 mM HEPES, 150 mM sodium chloride, pH7.4) or PBS (20mM sodium phosphate,

150mM sodium chloride, pH7.4) for 30min at room temperature. Then, the mixture was cooled to 4°C and chromatographed over a 2 ml Lipidex 1000 column, previously equilibrated with the same buffer. Protein-bound radioactive ligand was eluted in the void volume. After elution with 5 ml buffer, the column was subsequently eluted with 10 ml methanol. Aliquots of the 5 ml buffer eluate and the 10 ml methanol eluate were measured in a toluene-based liquid scintillation cocktail. For estimation of binding constants, various concentrations of unlabeled ligand were added in the incubation and separation of bound and free ligand was performed as described. Two corrections were made for the calculations: The 'real' ligand concentration was calculated based on the radioactivity of an aliquot of the incubation mixture to correct for ligand losses in the incubation. The amount of bound ligand was related to the sum of eluted ligand in the void volume and in the methanol fraction, to correct for losses in the chromatography. Data were then analysed according to Scatchard to determine the dissociation constants. To check putative ligands for interaction with MDGI in an indirect manner, a 1000-fold or 100fold excess of the respective lipid was incubated together with radioactive palmitic acid and the extent of displacement of [3H]-palmitic acid from binding was used as a measure for the strength of interaction of the lipids of unknown binding behaviour.

[~H]-CGP-12177 binding to A431 cells A431 cells were cultured in Minimal Essential Medium, buffered with 20 mM HEPES (pH 7.2) and supplemented with glutamine (2mM) 10% fetal calf serum and gentamycin (40 ~g/ml). Confluent cultures in 35 mm (ID)-dishes were used for the assays. The cells were washed three times with PBS. Incubation with [3H]-CGP 12177 was performed in a total volume of 2 ml HEPES-buffered Minimal Essential Medium without supplements but in the presence of 1% bovine serum albumin (Serva, Heidelberg) for 16 hours at 4° C under gentle shaking.

52 A

" ~ 100

o'~

O

t-- O

.c_ E

°o

,, " I / /

c

0

°~ -14

-13

-12 -11 -10 -g -8 -7 -6 -5 (-)lsoprenatine.log molar concentration

-4

B

100,4-."

O o

75

o

E c o

c c •~ o

÷

50

o

Fig. 1. Inhibition of L(+)-lactate induction of supersensitivity

0

25

o c

25

0

0.1

1

50

Concentration of MDGI [nglml.] The [3H]-CGP 12177-concentration was about 0.4 or 4nM, corresponding to about 0.5 × 105 and 0.5 × 106dpm/ml, respectively. Incubations were performed in the presence and absence of MDGI and the synthetic peptide D in the concentrations indicated under 'Results'. For determination of non-specific binding, incubations were performed in the presence of the beta-blocker (-)-propranolol (10-6 M).

for beta-adrenergic stimulation by MDGI. A. Concentrationresponse-curves for beta-adrenergic stimulation of cells without ((3) or with a 2h pretreatment with 3 mM L(+)-lactate (rT), with lactate and 50 ng/ml MDGI (0) or with lactate and 50 ng/ml heat-treated MDGI (5 min, 100° C) (11). The basal beating rate was in the range of 140-160 beats/min. B. Concentration-response histogram for inhibition of L(+)-lactate induction of supersensitivity by MDGI. Cells were treated with lactate as in A in the presence of MDGI at the indicated concentrations. Data are shown as % increase of the beating rate at a concentration of 10-9 M isoprenalin compared to cells not treated with lactate (0%).

The incubation was terminated by aspiration of the medium and the cells were washed three times with ice-cold PBS. The cells were then solubilized in 0.5ml of 0.1N NaOH containing 1% SDS and the radioactivity was measured in a liquid scintillation counter.

53 A

¢-

E I/3

80-

I:1 .13 '4,--

O I.-

~D

E

40

C c-

U~

~,o'

0 0

__c

0

t

-12 B

i

-10

J

!

i

i

i

i

-9

-8

-7

-6

-5

-4

(-)lsoprenatine, tog motar concentration

c-

E

i

-11

80

.-i-,

E~ G) ¢q O

..Q

E

40

C cco (3 c'-

0 -12

-11 -10 -9 -8 -7 -6 -5 -4 (-)lsoprenotine, tog motor concentrotion

Fig. 2. Inhibition of arachidonic acid and 15-S-HETE induction of supersensitivity for beta-adrenergic stimulation by MDGI. Concentration-response curves for beta-adrenergic stimulation in cells without pretreatment (O) or with 2 h pretreatment with 10- 8M arachidonic acid (A) or 15-S-HETE (B) in the absence (El) or presence (11) of 50ng/ml MDGI.

Results

1. M D G I inhibits the induction by various agents of supersensitivity to beta-adrenergic stimulation As described previously [12-14] and outlined in

detail in the accompanying paper [9], L(+)-lactate treatment of neonatal rat heart muscle cells leads to a highly increased sensitivity of these cells for betaadrenergic stimulation. Beta-adrenergic stimulation (measured as an increase of the beating-rate of

54 the cells) can be observed in lactate-treated cells upon exposure to as little as 10-12M isoprenaline whereas untreated cells require a threshold concentration of 10-9-10 -8 M isoprenaline. Simultaneous treatment of the cells with MDGI in addition to lactate prevents the characteristic induction of supersensitivity as shown in Fig. 1A, i.e. cells treated with both agents respond to isoprenaline as control cells, not treated with lactate. Heat treatment of MDGI destroys its respective inhibitory activity. The dose-response curve for this inhibitory activity of MDGI (as shown in Fig. 1B) is remarkably similar to the one obtained for growth inhibition in Ehrlich ascites mammary carcinoma cells [2] with clearly detectable activity at a concentration of 1 ng/ml and a saturation of the effect at 10 ng/ml. This property was proposed to form the basis for the development of a rapid bio-assay of MDGI [15]. As outlined and discussed in the accompanying paper [9], phospholipase A2-activators, arachidonic acid and 15-S-hydroxyeicosatetraenoic acid (15S-HETE) could likewise induce supersensitivity. Lipoxygenase inhibitors could interfere with the induction of supersensitivity by arachidonic acid but not with the induction by 15-S-HETE. As shown in Fig. 2, MDGI blocks both, arachidonic acid - as well as 15-S-HETE-induction of supersensitivity (Figs 2A and 2B, respectively). The inhibitory effect of MDGI on the induction of supersensitivity is thus distinct from the similar effect of lipoxygenase inhibitors.

2. Dissociation of inhibitory activity of MDGI from binding of supersensitivity-inducing lipids Since MDGI is known to bind long-chain fatty acids [8] it was reasonable to assume that the interference of MDGI with the induction of supersensitivity by arachidonic acid or 15-S-HETE could be due simply to a binding of the inducing fatty acids, thereby preventing them from acting on the target cells. We therefore studied the interaction of MDGI with a variety of lipids including arachidonic acid and various eicosanoids. The results are summa-

rized in Table 1. The binding was either measured directly using the appropriate radioactively labeled putative ligand or indirectly, checking the displacement of labeled palmitic acid by an excess of unlabeled putative ligand. Whereas arachidonic acid exhibited the expected interaction with MDGI with a similar affinity as other long chain fatty acids, 15-S-HETE did not show any binding when analysed by both methods. We therefore conclude that 15-S-HETE-binding cannot be the basis for the abrogation of 15-SHETE-induced supersensitivity by MDGI. Because of the relatively low affinity of arachidonic acid for MDGI (dissociation constant 1.5x Table 1. Binding of various lipids to MDGI

A. Direct binding measurements Lipid

Dissociation constant (10-6M)

palmitic acid oleic acid arachidonic acid

2.6 0.9 1.5

No specific binding detectable for: retinoic acid, prostaglandin E~, prostaglandin E2, tetraiodothyronine, 15-S-HETE. B. Indirect binding measurements by displacement of [aH]-palmitic acid Lipid

oleic acid arachidonic acid palmitic acid myristic acid caprylic acid retinoic acid retinol retinol acetate estradiol prostaglandin E~ prostaglandin E2 thromboxane B2 thromboxane A2 agonist 15-S-HETE (-)-isoprenaline

Displacement % of [3H]-palmitic acid displaced 1000-fold molar excess

100-fold molar excess

85 82 85 66 20 30 enhanced enhanced 8 0 5 58 64 1 0

88 88 58 32 6 0 12 8 4 n.d. n.d. 0 4 n.d. n.d.

55 10 -6 M) as compared with the very low concentration of arachidonic acid needed for the induction of supersensitivity (10-1°-10 -8 M) it is also extremely unlikely that arachidonic acid-binding would be the basis for inhibition of the arachidonic acid-induced supersensitivity by MDGI.

3. Synthetic peptides derived from MDGI structure mimic inhibitory activity of MDG1 Prompted by the finding that a synthetic peptide derived from the MDGI sequence, representing the 11 C-terminal amino acids, can mimic the growth inhibitory activity of MDGI for Ehrlich ascites mammary carcinoma cells [P. Langen et al., in preparation], various synthetic peptides were tested for their ability to block the arachidonic acid-induction of supersensitivity in neonatal rat heart muscle cells. As shown in Table 2, the peptide A, representing MDGI sequence 121-131, also effectively blocked arachidonic acid-induction of supersensitivity. Derivatives of this peptide differing in one amino-acid (peptide B-D, MDGI 121-131: R122, T127, S124, respectively) were indistinguishable from peptide A. Notably, peptide C is identical to sequence 121131 of bovine cardiac fatty acid-binding protein

[16]. The inhibitory activity required the peptides A - C to be in the reduced (i.e. monomeric) state. Dimeric forms (as judged by analytical ultracentrifugation, see 'Materials and methods') were inactive (not shown). A shorter sequence from the C-terminal part of MDGI (peptide E, MDGI 126-130) was insufficient to mimic MDGI activity. Also two peptides from distant regions of the MDGI sequence (F, G) were inactive, demonstrating the specificity of the observed inhibitory activities. Peptide D (selected for further studies because of the greater simplicity of handling compared with peptides A-C) can cause an inhibition of arachidonic acid-induced supersensitivity in remarkably low concentrations. Typical concentration-response curves for MDGI and peptide D, observed at three different concentrations of isoprenaline are shown in Fig. 3; peptide D (Fig. 3B) can be compared directly with MDGI (Fig. 3A). As expected from the structural requirements for lipid-binding [17, 18], peptide A and D did not show any significant fatty acid-binding, when analyzed by the described binding assay with Lipidex 1000 (not shown). Thus, lipid-binding cannot be the underlying mechanism for the inhibition of induction of supersensitivity by MDGI-derived peptides.

Table 2. Synthetic peptides mimic MDGI-Action Peptide sequence

Corresponding residues in M D G I

Increase in number of beats/min induced by 10 -8 M arachidonic acid in presence of the peptide at 10-1ZM

10-10M

10-SM

(-)-isoprenaline [m + SEM, for n = 25-35] no A B C D E F G

TAVCTRVYEKQ TRVCTVYEKQ TAVCTRTYEKQ TAVSTRVYEKQ RVYEK GQETSLVREMVD EFDETI'ADDR

121-131 121-131, R122 121-131, T127 121-131, S124 126-130 99-110 69- 78

10.8 1.2 3.2 0.4 0.4 9.0 8.4 12.0

+ + + + + + + +

0.4 0.8 1.2 2.0 1.2 1.2 0.8 1.2

19.2 3.2 0.8 1.6 0.8 20.4 14.0 22.8

+ 0.8 + 0.8 + 1.2 + 1.2 + 1.2 + 1.6 ___ 1.2 + 1.2

30.8 9.6 13.8 10.4 16.8 29.2 23.6 34.4

+ 2.4 + 1.2 ___ 1.5 + 1.2 + 1.6 + 2.4 + 1.6 + 2.0

Cells were treated with 10 -8 M arachidonic acid in the absence or presence of the indicated peptides at a concentration of 10 -7 M for 2 hours. Then, the beta-adrenergic stimulation was measured at the indicated concentrations of isoprenaline. The basal beating rate was in the range of 120-160.

56

A

B

c o n c e n t r a t i o n of

MD~I

tog motor concentration of

PEPTIDE D

[ng/mt]

¢-.

25

•

.

,

,

.

if}

E O

,

i

I

i

1

I

I

t']

/ / / / / /

E

10-

t-. i

24

'1

o c-

-12 -5

/I

/ / / / / /

-10

~l

-9

-12

,

,

/ / / / / / /

/1 .-1

_

/I T T / / / I / / / / / / f / /

l

15

.ID

c-

I

20

(A n

O

i

T ~

-10

-9

(-)lsoprenatine, tog motar concentration

Fig. 3. Concentration-response histogram for inhibition of arachidonic acid-induction of beta-adrenergic supersensitivity by synthetic peptide D in comparison with MDGI. Cells were pretreated with 10 -8 M arachidonic acid in the presence of the indicated concentrations of MDGI (A) or peptide D (B). Then, beta-adrenergic stimulation of either untreated (hatched bars) or pretreated cells (open bars) was measured at the indicated concentrations of (-)-isoprenaline. The basal beating rate was in the range of 140-160 beats/min.

4. MDGI does not directly interfere with the receptor binding of beta-adrenergic agonists As shown in Table 1, a large excess of isoprenaline had no effect on the binding of palmitic acid by MDGI. It can therefore be excluded that the inhibitory effect of MDGI on the induction of supersensitivity could be due to the binding of the betaadrenergic agonist at the lipid binding site of MDGI. We furthermore wanted to know whether MDGI can exert any direct effect on the binding of the beta-adrenergic agonist to its cellular receptor. This type of experiment is difficult to perform with the neonatal rat cardiac muscle cells since they have only a small number of beta2-adrenergic receptors compared to a large number of betaa-adrenergic receptors [19]. However, only beta2-adre-

nergic receptors seem to be involved in the induction of supersensitivity [12]. We therefore measured the binding of the hydrophilic beta-adrenergic agent [3H]-CGP 12177 to A431 cells, a cell type rich in beta2-adrenergic receptors [20]. The binding was performed in the cold in the presence and absence of MDGI and in the presence and absence of the MDGI-derived peptide D (Table 3). Non-specific binding, estimated by performing the experiment in the presence of 10 -6 M ( - ) propranolol, was substracted. Neither MDGI nor the peptide had an effect on specific binding of [3H]-CGP 12177 to A431 cells under the described conditions (Table 3). We therefore conclude that MDGI and MDGI-mimicking peptides do not inhibit induction of supersensitivity by a direct interference with the binding

57

of the beta-adrenergic agonists to the beta2-adrenergic receptor. The described binding experiments do not, however, allow us to exclude the possibility that the treatment of neonatal rat heart muscle cells with MDGI or MDGI-derived peptides might in turn result in a changed accessibility or function of the betaz-adrenergic receptor in these cells by an indirect mechanism. A431 cells might lack the respective metabolic pathways or putative MDGI-receptors required for such an interaction.

.......

~

• MDGI q I peptide D I ........

( beta-

1 GI

.

~ ~ adrenergic k........ agonist

.

.

1 I

cepto, I

" ~ .

I adrenergic I

f- L~ece~\

.

.

.

.

i L-l+)-toctate . . . . . . i. .

, '

i ?aCtjvQtjon9 I phosphotipase A2/

~ - ~

modutation [decrease)

rnodutation I (increase}

of sensitivity

[ of "\ k ; 15- [ipoxygenase / v sensitivity X~ " L~ 1__~

. . . .

~ . . . . .

increased beating rate

Discussion

In this paper we describe a new biological activity of MDGI, a polypeptide previously isolated on the basis of its growth inhibitory activity for different mammary epithelial cells in vitro. MDGI inhibits the induction of supersensitivity of neonatal rat cardiac muscle cells for beta-adrenergic stimulation by various agents. It is currently unknown whether the growth inhibition observed in some cell systems and the inhibition of a beta-adrenergic response described here are related phenomena. Growth stimulation can be elicited with beta-adrenergic agonists in a number of cell systems including mammary epithelial cells [21, 22] and similar signaling mechanisms might account for some growth stimulating factors on the one hand and beta-adrenergic agonists on the other [22]. Betazadrenergic receptors have been demonstrated in

Fig. 4. Schematic illustration of the possible mechanism of inhibition of the induction of beta-adrenergic supersensitivity by MDGI.

rat mammary epithelial cells with different levels depending on the different reproductive phases of the gland [23], suggesting a relationship to mammary gland growth and differentiation. It could be speculated, therefore, that the growth inhibitory effects of MDGI on mammary epithelial cells are relevant for mammary gland physiology and result from an interference with similar pathways as involved in the induction of beta-adrenergic supersensitivity. The new effect of MDGI should stimulate experiments to test this hypothesis. As discussed in the accompanying paper [9], the exact nature of the mechanism of induction of supersensitivity in the cardiac cells is not known. The

Table 3. Effect of MDGI and peptide D on the binding of the hydrophilic beta-adrenergic agent CGP 12177 to A431 cells Additions

[3H]-CGP 12177 bound (10 -2 dpm/106 cells) 4nM

no MDGI (50ng/ml) PeptideD (lO-6M)

0.4nM

total

non-specific

specific

total

non-specific

specific

108 102

62 61

46 41

59 53

13 14

46 40

104

67

37

54

12

42

(Non-specific binding was determined as binding in the presence of 10 -6 M (-)-propranolol). Values represent the averages of 3 replicates wherein individual measurements varied less than 10%.

58 data point to the involvement of a lipoxygenasemediated pathway. In particular, arachidonic acid metabolites generated by lipoxygenase catalysis can induce beta-adrenergic supersensitivity by an unknown mechanism. It is suggested that the release of lipoxygenase-products via a number of unknown steps ultimately leads to a facilitated access of hydrophilic beta-agonists to the beta2-adrenergic receptor or otherwise to a sensitization of beta2-receptors [9]. In this paper it is demonstrated that MDGI can block the inducing effect of various agents including the lipoxygenase product 15-SHETE. This finding points to an interference of MDGI with steps of the inducing pathway subsequent to the generation of lipoxygenase products. One possibility could be the interference with the interaction of 15-S-HETE or its metabolites with their putative receptors. Binding measurements for a hydrophilic betaadrenergic agent in the presence of MDGI argue against a direct interference of MDGI with receptor binding. However, the data do not exclude a modulation of the beta-adrenergic receptor by an indirect mechanism, for instance by a 'transmodulation' via a putative MDGI-receptor. Figure 4 summarizes schematically our current understanding of the mechanism of MDGI-action in the neonatal heart muscle cell system. Since it was previously found that MDGI has lipid-binding activity for long-chain fatty acids [8] it seemed reasonable to assume that the interference of MDGI with the induction of supersensitivity might result from simple binding of the inducing agents, in particular arachidonic acid and 15-SHETE. Two lines of evidence, however, disprove this hypothesis: First, it is shown in this paper that while MDGI can bind arachidonic acid albeit with a comparatively low affinity, MDGI, in contrast to findings of Raza et al. [24] for rat liver fatty acid-binding protein, does not bind 15-S-HETE. These findings make an involvement of lipid-binding activity in the observed effect of MDGI very unlikely. The results of lipid binding assays also rendered unlikely the possibility that MDGI could bind the beta-adrenergic agonist with high affinity. Second, relatively short synthetic peptides de-

rived from the MDGI primary structure do not bind lipid but mimic MDGI with respect to its inhibition of induction of supersensitivity. This effect is specific for a particular MDGI sequence (121-131); it seems reasonable to assume that MDGI and the synthetic peptides act via the same pathway. Taken together, these data strongly suggest that the described activity of MDGI is not related to its lipid-binding activity. It is tempting to speculate that the same might apply for the growth inhibitory activity of MDGI. Attention has been given to the possibility that supersensitivity to beta-adrenergic stimulation induced in cardiac muscle by raised lactate levels may aggravate disturbances of cardiac function occurring during an ischaemic insult [12]. The inhibitory activity of MDGI relating to the induction of supersensitivity for hydrophilic beta-adrenergic agonists might point to a physiological role for a close relative of MDGI - the cardiac fatty acid-binding protein (H-FABP) [16, 25]. H-FABP and MDGI have an almost identical structure or isoforms are even identical [26]. The synthetic peptide C corresponding to sequence 121-131 of bovine H-FABP [16] was indistinguishable from MDGI in its inhibition of the induction of beta-adrenergic supersensitivity. Thus, H-FABP might be a physiological modulator of beta-adrenergic responses in the cardiac muscle. The fact we describe in this manuscript that synthetic peptides identical to, or slightly differing from the MDGI sequence 121-131 can mimic the MDGI-activity at very low concentrations suggests that the C-terminal region of MDGI is crucial for biological activity. These MDGI-derived peptides are agents of potential pharmacological interest and should be investigated further for their biological activities in vivo.

Acknowledgements The expert technical assistance of Mrs. M. Wegener, Mrs. H. Schmidt, Mrs. K. Karczewski, and Mr. M. Grothe is gratefully acknowledged. F.-D.

59 Boehmer was recipient of a postdoctoral fellowship of the Alberta Heritage Foundation for Medical Research. We are indebted to Mrs. I. Wiznerowicz for preparation of the manuscript and thank Prof. Albert Wollenberger for critical reading and helpful discussions.

11.

12.

References 1. Boehmer FD, Lehmann W, Schmidt HE, Langen P, Grosse R: Purification of a growth inhibitor for Ehrlich ascites mammary carcinoma cells from bovine mammary gland. Exp Cell Res 150: 466~76, 1984 2. Boehmer FD, Lehmann W, Noll F, Samtleben R, Langen P, Grosse R: Specific neutralizing antiserum against a polypeptide growth inhibitor for mammary cells purified from bovine mammary gland. Biochim Biophys Acta 846: 145154, 1985 3. Boehmer FD, Kraft R, Otto A, Wernstedt C, Hellman U, Kurtz A, Mueller T, Rohde K, Etzold G, Lehmann W, Langen P, Heldin CH, Grosse R: Identification of a polypeptide growth inhibitor from bovine mammary gland. Sequence homology to fatty acid- and retinoid-binding proteins. J Biol Chem 262: 15137-15143, 1987 4. LehmannW, Widmair R, LangenP: Response of different mammary epithelial cell lines to a mammary-derived growth inhibitor (MDGI). Biomed Biochim Acta 48: 143151, 1989 5. Boehmer FD, Sun Q, Pepperle M, Mueller T, Eriksson U, Wang JL, Grosse R: Antibodies against mammary derived growth inhibitor (MDGI) react with a fibroblast growth inhibitor and with heart fatty acid binding protein. Biochem Biophys Res Comm 148: 1425-1431, 1987 6. Mueller T, Kurtz A, Vogel F, Breter H, Schneider F, Engstroem U, Mieth M, Boehmer FD, Grosse R: A mammary-derived growth inhibitor (MDGI) related 70 kDa antigen identified in nuclei of mammary epithelial cells. J Cell Physiol 138: 415-423, 1989 7. Brandt R, Pepperle M, Otto A, Kraft R, Boehmer FD, Grosse R: A 13-kilodalton protein purified from milk fat globule membranes is closely related to a mammary-derived growth inhibitor. Biochemistry 27: 1420-1425, 1988 8. Boehmer FD, Mieth M, Reichmann G, Taube C, Grosse R, Hollenberg MD: A polypeptide growth inhibitor isolated from lactating bovine mammary gland (MDGI) is a lipid-carrying protein. J Cell Biochem 38: 199-204, 1989 9. Wallukat G, Nemecz G, Farkas T, Kuehn H, Wollenberger A: Modulation of the beta-adrenergic response in cultured rat heart cells. I. Induction of beta-adrenergic supersensitivity via a phospholipase A2 and 15-1ipoxygenase involving pathway Mol Cell Biochem 102: 35-47, 1991 10. Behlke J, Mieth M, Boehmer FD, Grosse R: Hydrodynam-

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

ic and circular dichroic analysis of mammary-derived growth inhibitor (MDGI). Biochem Biophys Res Comm 161: 363--370, 1989 Dahlberg E, Snochowsk M, Gustafson JA: Removal of hydrophobic compounds from biological fluids by a simple method. Anal Biochem 106: 380-388, 1980 Wallukat G, Wollenberger A: Involvement of betaE-adrenergic receptors in the potentiation of the chronotropic action of isoprenaline evoked in rocker-cultured neonatal rat heart cells by pyruvate and L(+)-lactate. In: RE Beamish, V Panagia, NS Dhalla (eds.) Pharmacological Aspects of Heart Disease. Martinus Nijhoff Publishing, Boston, Dordrecht, Lancester, 1987, pp 217-231 Wallukat G, Wollenberger A: Differential alpha- and betaadrenergic responsiveness of beating rat heart myocytes after stationary and non-stationary cultivation. Acta Biol Med Germ 39: K7-K13, 1980 Wollenberger A, Wallukat G: Sensitization by lactate and pyruvate of rocker-cultured rat myocardial cells to isoproterenol. In: CM Caldarera and P Harris (eds.) Advances in Studies on Heart Metabolism. CLUEB, Bologna, 1982, pp 133-137 Grosse R, Boehmer FD, Langen P, Kurtz A, Lehmann W, Mieth M, Wallukat G: Purification, biological assay and immunoassay of mammary-derived growth inhibitor. Meth Enzymol, in press Billich S, Wissel T, Kratzin H, Hahn U, Hagenhoff B, Lezius AG, Spener F: Cloning of a full-length complementary DNA for fatty acid-binding protein from bovine heart. Eur J Biochem 175: 549-556, 1988 Jones TA, Bergfors T, Sedzik S, Unge T: The three-dimensional structure of P2 myelin protein. EMBO J 7: 15971604, 1988 Sacchettini JC, Gordon JI, Banaszak LJ: The structure of crystalline Escherichia coli-derived rat intestinal fatty-acid binding protein at 2.5 - A resolution. J Biol Chem 263: 5815-5819, 1988 Wollenberger A, WaUukat G: Adrenergic and antiadrenergic activity of cycloheximide in cultured rat heart ceils. Biomed Biochim Acta 42: 917-927, 1983 Hoebeke J, Durien O, Delavier C, Schmutz A, Strosberg AD: Biochemical and immunological studies of beta-adrenergic receptors on various cell types. Adv Cycl Nucleot Prot Phosphoryl Res 17: 73-80, 1984 Yang J, Guzman R, Richards J, Imagawa W, MoCormick K, Nandi S: Growth factor- and cyclic nucleotide-induced proliferation of normal and malignant mammary epithelial cells in primary culture. Endocrinology 107: 35-41, 1980 Dumont JE, Jauniaux JL, Roger PP: The cyclic AMPmediated stimulation of cell proliferation. Trends Biochem Sci 14: 67-71, 1989 Marchetti B, Fortier MA, Poyet P, Follea N, Pelletier G, Labrie F: Beta-adrenergic receptors in the rat mammary gland during pregnancy and lactation: characterization, distribution, and coupling to adenylate cyclase. Endocrinology 126: 565-574, 1990

60 24. Raza H, Pougubala JR, Sorof S: Specific high affinity binding of lipoxygenase metabolites of arachidonic acid by liver fatty acid binding protein. Biochem Biophys Res Comm 161: 448-455, 1989 25. Sacchettini JC, Said B, Sehulz H, Gordon JI: Rat heart fatty acid-binding protein is highly homologous to the murine adipocyte 422 protein and the P2 protein of peripheral nerve myelin. J Biol Chem 261: 8218-8223, 1986

26. Kurtz A, Vogel F, Funa K, Heldin CH, Grosse R: Developmental regulation of mammary-derived growth inhibitor expression in bovine mammary tissue. J Cell Biol 110: 17791789, 1990

Address for offprints: G. Wallukat, Central Institute of Cardiovascular Research, Academy of Sciences, Roessle-Strasse 10, Berlin-Buch, Germany