Stimulation of the adenylyl cyclase activity in ... - Semantic Scholar

1 downloads 0 Views 668KB Size Report
Vasoactive intestinal peptide (VIP) has been shown to stimulate adenylyl cyclase activity in human endometrial membranes. The effect was dependent on the ...
Bioscience Reports, Vol. 13, No. 2, 1993

Stimulation of the Adenylyl Cyclase Activity in Human Endometrial Membranes by VIP and Related Peptides A . M. Bajo, 1 L. G. Guijarro, l M. G. Juarranz, 1 P. Valenzuela, 2 P. Martinez, 2 and J. C. Prieto ~'3 Received September 28, 1992; accepted December 3, 1992 Vasoactive intestinal peptide (VIP) has been shown to stimulate adenylyl cyclase activity in human endometrial membranes. The effect was dependent on the time and temperature of incubation as well as on the concentration of endometrial membrane proteins in the medium. In the presence of 1 ~tM GTP, half-maximal stimulation of adenylyl cyclase activity was observed at 25.0 + 7.0 nM VIP, whereas the maximal activity (at 1/~M VIP) corresponded to an increase of about 140% with respect to basal values (7.5 • 0.6 pmol cyclic AMP~rain/rag of protein). However, the maximal stimulation of adenylyl cyclase activity was obtained with helodermin (1 ~M) that increased the activity by 170% over the basal. The relative potency of VIP-related peptides upon the adenylyl cyclase activity was: helodermin (EDso = 1..8 • 1.4 nM) > VIP(EDso = 25.0 • 7.0 nM) > PHI (EDs0 = 725.0 • 127.2 nM). Secretin had a faint effect upon the adenylyl cyclase activity and glucagon was completely inefficient at this level. The presence of o~s and oq subunits of G proteins in human endometrium was detected by immunoblot. Preliminary results showed the presence of two classes of t25I-VIP receptors in human endometrial membranes with the following stoichoimetric parameters: high affinity receptor (Kd = 2.0 nM, binding capacity 0.1 pmol VIP/mg protein) and 10w affinity receptor (Kd = 0.43/~M, binding capacity 13.1 pmol VIP/mg protein). The present results together with the known presence of VIP in human uterus and the actions of this neuropeptide in the adjacent myometrial tissue support the idea that VIP and related peptides may have a role in human endometrium. KEY WORDS: VIP; adenylyI cyclase activity; uterus; endometrium; helodermin; PHI; secret• proteins.

G

INTRODUCTION Vasoactive intestinal peptide (VIP) is a basic 28-amino acid peptide, first isolated from porcine gut (1), with a widespread neuronal distribution in the body acting as a neurotransmitter or a neuromodulator (2). In the female genital tract of mammals, VIP-immunoreactive nerves have been localized innervating smooth 1Departamento de Bioquimica y Biologia Molecular, Universidad de Alcal~i, E-28871 Alcalfi de Henares, Spain. 2 Hospital Principe de Asturias, Universidad de Alcal~i, E-28871, Alcal~i de Henares, Spain. 3 To whom correspondence should be addressed. 69 0144-8463/93/0400-0069507.00/0Q 1993 Plenum Publishing Corporation

70

Bajo et aL

muscle cells, blood vessels and epithelial cells (3). In porcine myometrium, it has been shown that the presence of VIP receptors (4) is associated with an inhibitory effect on the contractile activity stimulated by oxytocin (5) or prostaglandin F2oL

(6). There are some reports in humans concerning the effect of VIP in the regulation of the vaginal flow (7) and uterine secretion (8), but not any paper has appeared regarding the characteristics of the VIP signal transduction pathway, i.e. VIP receptors and their coupling with the adenylyl cyclase system. Only a preliminary autoradiographic study showed VIP binding sites in human uterus mainly located on the endometrium (9). Since the enzyme adenylyl cyclase regulates several metabolic functions in uterus (10) and, indeed, cyclic AMP has been related to the human uterine growth (11), the purpose of this study was to investigate the effect of the VIP and of the related peptides helodermin, peptide histidine isoleucine (PHI), secretin and glucagon on the adenylyl cyclase activity from human endometrium, which paves the way to search the functions of VIP receptor at this level.

EXPERIMENTAL PROCEDURES

Materials

Highly purified natural porcine VIP and secretin were supplied by Prof. V. Mutt (Karolinska Institute, Stockholm, Sweden). Helodermin and PHI were obtained from Peninsula Laboratories. Glucagon, bacitracin, phenylmethylsulfonyl fluoride (PMSF), bovine serum albumin, GTP, and other nucleotides, were purchased from Sigma Chemical. Antibodies were supplied by New England Nuclear Dupont. All other reagents were of the highest purity commercially available. 125I-labelled VIP was prepared at a specific activity of 2250 Ci/g and possessed a biological activity similar to that of native VIP (12).

Preparation and Sources of Membranes

Menstruating women (30-50 years old) underwent total hysterectomy for fibromyomas, menorrhagia or premalignant dysplasia of the cervix. The endometrium was removed from the myometrium and finely minced. After homogenization, using a Polytron (setting 5, 3 min) (Brinkmann Instruments), in 0.01 M triethanolamine-HC1 buffer (pH 7.5) containing 0.25 M sucrose and 0.5 mM EDTA, the mixture was filtered ~rough two layers of medical gauze. The homogenate was centrifuged at 30,000 x g for 30 min at 4~ The final pellet was washed twice in 20mM Tris-HC1 buffer (pH 7.5) and then immediately frozen at -70~ until use. Protein concentration was determinated according to the method of Bradford (13) using bovine serum albumin as a standard.

VIP-stimulated adenylylcyclase

71

Adenylyl Cyclase Assay Cyclic AMP production by the enzymatic system was carried out as described previously (14) with some modifications. Briefly, endometrial membranes (0.010.15mg protein/ml) were incubated in 0.1ml of total volume with an ATPregenerating system (7.4 mg/ml creatine phosphate and 1 mg/ml creatine kinase) dissolved in 0.25 M triethanolamine-HC1 buffer (pH 7.4) in the presence of 1.5 mM ATP, 5 mM MgSOa, 1 mM 3-isobutyl-l-methylxanthine, 1 mM EDTA, 2 mg/ml bacitracin and test substances (VIP-related peptides and GTP). After 30 min incubation at 30~ under standard conditions, the reaction was stopped by heating the mixture for 3 min. After addition of 0.2 ml of an alumina slurry (0.75 g/ml in triethanolamine-HC1 buffer (pH 7.4) and centrifugation, the cyclic AMP in the supernatant was measured by the method of Gilman (15).

VIP Binding Assay Membranes (0.15mg of protein/ml) were incubated in 0.25 ml of 50mM Tris-HC1 buffer, pH 7.5, containing 1.4% bovine serum albumin, 1 mg/ml bacitracin, 0.05 mM PMSF and 50 pM 125I-VIP in the absence or presence of increasing concentrations of unlabelled peptide (0.01-1000 nM). After separation by filtration under vacuum using Whatman GF/C filters pretreated with 1% polyethylenimine followed by washing with 10 ml ice-cold Tris-HC1 buffer (pH 7.5), membrane-bound 12SI-VIP fixed to the filter was counted for radioactivity with a 70% of efficiency. The nonspecific 125I-VIP binding was determined in parallel incubations in the presence of an excess (1/zM) of unlabelled VIP; this component averaged about 5% of the total radioactivity and was subtracted from the total binding.

Immunodetection of a~ and a~ Subunits of G Proteins Membranes were solubilized in 60 mM Tris-HC1 buffer (pH 6.8) containing 10% glycerol, 0.001% bromophenol blue and 3% SDS and proteins were run on SDS-10% P A G E (16). After the transfer of proteins to a nitrocellulose sheet, the ol~ and oLi proteins were immunodetected with antisera for the w~ (RM/1) and o:i (AS/7) as described previously (16). Briefly, nitrocellulose sheets were cut in separated lanes and preincubated in 50raM Tris-HC1 buffer (pH 8.0) supplemented with 2 mM CaCI2, 80 mM NaC1, 0.2% (v/v) Nonidet P40 and 5% (w/v) non-fat dry milk. After washing with the same buffer, the different antisera (1/1000 dilution) were incubated for i h at room temperature. The immunoreactive proteins were revealed using 125I-labelled donkey antibodies against rabbit Ig G and the immunoblots exposed for 1-5 days at 80~ to a Trimax type XM film (3M) with a 3M-Trimax intensifying screen.

RESULTS VIP simulated adenylyl eyclase activity in human endometrial membranes in time- and temperature-dependent manner (Fig. 1). The initial rate of

72

Bajo

4oo

ooc

c~ 300

~

t

u

E

-6

o~ - ' ~ 7 ~

E

o_ 200

~I

U 100 ~7~0~ o

o

0

0

~ 0

30

15~ 60

90

Time, min Fig. 1. Time course of VIP-induced cyclic AMP formation by human endometrial membranes as a function of temperature. Membranes were incubated in the presence of 1 # M VIP plus 1 # M GTP at 15~ (O), 30~ ( 0 ) or 37~ ([]). The experiment is representative of two others.

100

E O 03 E O_

50

O_ ~E O

.J

O O

0 I

0

I

I

.05 ,10 [Protein], mg/ml

I

,15

Fig. 2. Effect of varying membrane concentrations on adenylyl cyclase activity stimulated by VIP (1 #M) plus GTP (1 #M). Endometrial membranes were incubated at 30~ for 30 min. Cyclic AMP values in the presence of the peptide are represented after subtraction of the basal unstimulated value, The results are the mean of triplicates.

et

VIP-stimulated adenylyl cyclase

73

stimulation increased with the temperature; however, cyclic AMP production decreased after 30 min at 37~ under that observed at 30~ whereas at this second temperature the response was linear with time up to 90 min. The cyclic AMP response at 15~ was lower in rate and extent than that obtained at the other temperatures with an apparent steady-state reached after 30min. All subsequent experiments were performed at 30~ for 30 min. Figure 2 represents the effect of increasing membrane protein concentrations on VIP stimulation of adenylyl cyclase activity. Under standard conditions the stimulation of the enzyme activity was linear at protein concentrations up to 0.15 mg protein/ml. Thus, membrane preparations at 0.15 mg of protein/ml were used in later experiments. Figure 3 shows results on the specificity of the adenylyl cyclase response. Adenylyl cyclase activity increased by about 140% with respect to basal values (7.5 + 0.6 pmol cyclic AMP/min/mg of protein) in membranes incubated under standard conditions in the presence of 1/~M of VIP plus 1 ~M GTP. The increase of adenylyl cyclase activity obtained with helodermin (1/,M) in presence of 1/~M GTP was 170% of the basal activity. VIP and some related peptides were able to stimulate adenylyl cyclase activity in a dose-dependent manner, with the following potency: helodermin (EDs0 = 1.8 • 1.4 nM) > VIP(EDs0 = 25.0 • 7.0 nM) > PHI (EDso = 725.0 • 127.2 nM). Secretin exerted a modest effect at very high doses, in the micromolar range, whereas glucagon was inactive on the stimulation of the enzyme activity from human endometrium. Adenylyl cyclase activity is bimodally modulated by the mobilization of a~s

~2o

e /

10

0

& 18 16 E

"~- 1 4 E 0

E 0..

-5 0

oT

L

~.

II

'

f

,

*

I

10

9

8

7

6

[Peptide], - t o g

, 5

M

Fig. 3. Ability of various VIP-related peptides to stimulate the adenylyl cyclase activity from endometrial membranes. Membranes (0.15mg protein/ml) were incubated in standard conditions (30 rain, 30~ with different concentrations of VIP (El), helodermin (0), PHI (A), secretin (O) and glucagon ( I ) . Values are the mean of four duplicate experiments.

74

Bajo et al.

Fig. 4. Immunodetection of the 0cs and txi subunits of G proteins. Endometrial, myometrial and prostate membranes were resolved on SDS/PAGE as described in the Experimental Procedures section. Protein were transferred to nitrocellulose and the inmunodetection was achieved using anti-o:s and anti-oli antisera. Left: ols subunits. Right: oli subunits. (stimulatory) and oq (inhibitory) subunits of G proteins. Figure 4 shows a comparison of the levels of trs and oli subunits in human endometrial and myometrial membranes as well as in rat prostate membranes. The 0~s level in human endometrium (20% of that in rat prostate) was comparable to that in the other two tissues studied (Figure 4, left). However, the tri band corresponding to human endometrium was much more faint (5.4% of the prostate) than those of human miometrium and rat prostate (Fig. 4, right). In order to explain the VIP effect upon the adenylyl cyclase activity, we tested the presence of 125I-VIP binding sites in human endometrial membranes. As shown by stoichiometric studies (Fig. 5), two classes of VIP receptors appear to exist in human endometrial membranes with the following characteristics: a high-affinity receptor (Kd = 2 . 0 n M ) with low capacity (0.1 pmol V I P / m g membrane protein) and a low affinity receptor (Kd = 0.43/~M) with high capacity (13.1 pmol V I P / m g m e m b r a n e protein).

DISCUSSION The results of the present study indicate that human uterine endometrium contains functional VIP receptors coupled in a positive manner to adenylyl cyclase through the trs subunit of a G-protein.

VIP-stimulated adenylyl cyclase

75

O4

0

o

4~ 4--

0

= ~~ o

3

2

~~-

~

-

-

-

-

~

-

[3 (pmol 0f VIP/m9 pr0t.)

"0 r" 3 0 33

EL >

2

"'--2.__

In Ol

[VlP], - l o g

M

Fig. 5. Competition binding of 125I-VIP to endometrial membranes. Membranes (0.15 mg of protein/ml) were incubated with 50pM 125I-VIP for 30min at 30~ in the presence of increasing concentrations of VIP. Nonspecific binding was determined in the presence of 1/zM VIP. The Scatchard analysis of the data is included at the top, where B/F represents the bound/free VIP and B represents the pmol VIP bound/rag protein. The results are the mean of three separate experiments performed in duplicate.

The dependence of the sensitivity of human endometrial adenylyl cyclase activity to VIP on time, temperature and membrane concentration is a general feature previously described in other systems from human or animal origin endowed with VIP receptors (17-19). However, the pharmacological characteristics assessed by the effect of VIP and related peptides in activating adenylyl cyclase activity were not similar to those described in other tissues including rat seminal vesicle (17) or prostatic epithelium (18) in the genitourinary tract, rat intestinal (19) or human gastric (20) epithelium in the digestive tract, rat and human lung (21) or human larynx (22) in the respiratory tract or proliferative tissues such as pancreatic adenocarcinoma (23) and melanoma (24). The most striking feature described in this study is that helodermin exhibited higher potency than VIP in stimulating the enzyme activity. This pharmacological pattern is similar to that reported by others (25, 26) who suggested the existence of "helodermin-preferring" VIP receptors in human SUP-T1 lymphoblasts (25) and in human small-cells lung carcinoma (26). At present, it is tempting to

76

Bajo et al.

speculate that human myometrium may be added to this incipient list. [t25I]helodermin has been shown to bind to VIP receptors in systems such as rat liver and brain membranes or human heart membranes (27). However, in human endometrium it is possible that helodermin possesses its own receptors because its effect upon the adenylyl cyclase system is more potent than that of VIP and follows a different pattern. VIP showed an adenylyl-cyclase stimulation curve with two slopes that may be interpreted in terms of two different classes of VIP receptors. From the Scatchard analysis (28), the first class ( K d = 2 . 0 n M ) represents only 1% of the total receptors and could be accounted for the first slope of the adenylyl cyclase stimulatory curve, whereas the second class (Kd = 0.43 #M) could explain the stimulatory effect of the VIP upon adenylyl cyclase activity in the micromolar range. In any case, this report joins new evidences to search for "helodermin-preferring" VIP receptors with specific probes in human endometrium. Interestingly, recent data from our laboratory show that the pituitary adenylyl cyclase activating peptide PACAP-27 was also less potent than helodermin in the stimulation of adenylyl cyclase activity in this tissue ( u n p u b l i s h e d results). Additional studies with this and other VIP-related peptides, including the truncated form of glucagon-like peptide-1 GLP-l(736)NH2 (which has been recently shown to behave as a mammalian analogue of VIP-related Helodermatidae peptides) (29), will be useful to gain knowledge on this subject. The physiological role of the VIP receptor family in human uterus remains to be clearly established. However, the presence of VIP receptors coupled to adenylyl cyclase stimulation in human endometrial membranes and VIP relaxatory effects upon the adjoining myometrial tissue (9) strongly support that possibility. Finally it is interesting to point out the involvement of the adenylyl cyclase system in cellular growth at this level, since it has been recently shown the suppression of growth-promoting activity in extracts from human uterine cancer by a cyclic AMP- mediated mechanism (11).

ACKNOWLEDGEMENTS This work was supported by the Direcci6n General de Investigaci6n Cientifica y T6cnica (Grant PM89-96) and the University of Alcal~.

REFERENCES 1. Said, S. I. and Mutt, V. (1969) Nature 224:11324-11330. 2. Fahrenkrug,J. (1979) Digestion 19:149-169. 3. Ottesen, B., Larsen,J. J., Fahrenkrug,J., Stjernquist, M. and Sundler, F. (1981) Am. J. Physiol. 240: E32-E36. 4. Ottesen, B., Staun-Olsen, P., Gammeltoft, S. and Fahrenkrug, J. (1982) Endocrinology 110:2037-2043. 5. Ottesen, B., Ulrichsen, H., Wagner, G. and Fahrenkrug, J. (1979) Act Physiol. Scand. 107.285-287.

VIP-stimulated adenylyl cyclase 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

77

Ottesen, B., Wagner, G. and Fahrenkrug, J. (1980) Prostaglandins 19:427-435. Palle, C., Bredkjaer, H. E., Ottesen, B. and Fahrenkrug, J. (1990) Peptides 11:401-404. Levin, R. J. (1991) Exp. Clin. EndocrinoL 98:61-69. Leroy, M. J., Tanguy, G., Vial, M., Rost6ne, W., Malassin6, A. and Ferr6, F. (1991) Clin. Exp. Pharmacol. Physiol. 18:205-215. Do Khac, L., Mokhtari, A. and Harbon, S. (1986) J. Pharrnacol. Exp. Ther. 239:236-242. Matsunami, K., Imai, A., Ohno, T. and Tamaya, T. (1991) Res. Commun. Chem. Pathol. Pharmacol. 73:371-374. Laburthe, M., Rousset, M., Boissard, C., Chevalier, G., Zweibaum, A. and Rosselin, G. (1978) Proc. Natl. Acad. Sci. USA 75:2772-2779. Bradford, M. (1976)Anal. Biochem. 72:248-254. Houslay, M. D., Metcalfe, J. C., Warren, G. B., Hesketh, T. R. and Smith, G. A. (1976) Biochim. Biophys. Acta 436:489-494. Gilman, A. G. (1970) Proc. NatL Acad. Sci. USA 67:305-312. Guijarro, L. G., Couvineau, A., Rodriguez-Pena, M. S., Juarranz, M. G., Rodriguez-Henche, N., Arilla, E., Laburthe, M. and Prieto, J. C. (1992) Biochem. J. 285:515-520. Rodriguez-Pena, M. S., Guijarro, L. G. and Prieto, J. C. (1991) Peptides 12:821-824. Carmena, M. J. and Prieto, J. C. (1983) Biochim. Biophys. Acta 763:414-418. Prieto, J. C., Laburthe, M., Hui Bon Hoa, D. and Rosselin, G. (1981) Acta Endocrinol. 96:100-106. Gespach, C., Bawab, W., Chastre, E., Emami, S., Yanaihara, N. and Rosselin, G. (1988) Biochem. Biophys. Res. Commun. 151:939-947. Robberecht, P., Tatemoto, K., Chatelain, P., Waelbroeck, M., Delhaye, M., Tatan, G., De Neef, P., Camus, J. C., Hense, D. and Christophe, J. (1982) ReguL Pept. 4:241-250. Prost, A., Emami, S., Rosselin, G. and Gespach, C. (1984) Biosci. Rep. 4: 1045-1050. Estival, A., Mounielou, P., Trocheris, V., Scemama, J. L., Clemente, F., Hollande, E. and Ribet, A. (1983)Biochem. Biophys. Res. Commun. 111:958-963 Martin, J. M., Luis, J., Marvaldi, J., Pichon, J. and Pic, P. (1989) Eur. J. Biochem. 180:435-439. Gourlet, P., De Neef, P., Woussen Colle, M. C., Vandermeers, A., Vandermeers-Piret, M. C., Robberecht, P. and Christophe, J. (1991) Biochim. Biophys. Acta 1066:245-251. Luis, J. and Said, S. I. (1990) Peptides 11:1239-1244. Robberecht, P., Waelbroeck, M., De Neef, P., Camus, J. C., Vandermeers, A., VandermeersPiret, M. C. and Christophe, J. (1984) FEBS Letters 172:55-58. Scatchard, G. (1949) Ann. N. Y. Acad. Sci. 51:660-675. Raufman, J. P., Singh, L., Sing, G. and Eng, J. (1992) J. Biol. Chem. 267:21432-21437.