Clinical and Experimental Pharmacolog-vand Ph.vsioiogy (199 I ) 18, 205-2 15

THE EFFECT OF VASOACTIVE INTESTINAL PEPTIDE (VIP) ON THE CONTRACTILE ACTIVITY OF HUMAN UTERINE SMOOTH MUSCLE M. J. Leroy, G. Tanguy, M. Vial,* W. Rostene,* A. Malassine' and F. Ferre INSERM,' U.166, Maternitd Baudelocque, Paris, * I N S E R M , U.55, H6pital Saint-Antoine, Paris, 'Laboratoire de Ph-vsiologie Cellulaire, U F R Sciences, Poitiers, France

(Received 7 September 1990; revision received 28 November 1990)

SUMMARY 1. In the present study we examined the in vitro effect of vasoactive intestinal peptide (VIP) on spontaneous contractions in both inner and outer layers of non-pregnant human myometrium. A dose-dependent relaxation was observed, but with a marked difference in sensitivity to VIP between the two layers, with an ICSo value of 1 X 10-8 and 1 X 10-5 mol/L in the outer and inner layers, respectively. 2. We also established that VIP did not directly stimulate the adenylate cyclase activity. The only slight stimulations were observed in non-initial rate conditions. The maximal response of this indirect effect was obtained for VIP concentrations between 1 X and 1 X 10-8 mol/L and this occurred to the same extent (an approximately 1.4-fold increase) in both layers. However this response is specific, since structurally related peptides such as glucagon, gastric inhibitory polypeptide (GIP), secretin, or human growth hormone-releasing factor (hGRF) had no effect in our preparations. 3 . Autoradiographic studies revealed that specific VIP binding sites were located on the vascularization of the intermediate vascular layer and on arterioles and venules distributed in the inner and outer myometrial layers. They were also present in the endometrium, but not on smooth muscle cells of either layer. 4. Such observations could provide evidence for another signal transduction pathway to mediate the biological effect of VIP. An additional intermediate step on the vascularization distributed in all of the muscle cannot be excluded.

Key words: cyclic adenosine monophosphate formation process, human myometrium, relaxation, vasoactive intestinal peptide.

INTRODUCTION Much data has been accumulated showing that in the uterus, vasoactive intestinal peptide (VIP) is involved in the control of blood circulation of the vascular bed and in the relaxation of smooth muscle preparations from the female genital tract (Larsson et al. 1977; Helm et al. 1981; Ottesen et al, 1979, 1980; Hansen el

al. 1986, 1988). In the myometrium, VIP nerves innervate smooth muscles and blood vessels but considerable species differences have been noted in the concentration and localization of VIP immunoreactive elements (Polak et al. 1984; Heinrich er al. 1986). The presence of a high density of binding sites which

'INSERM: Institut National de la Santi: et de la Recherche MCdicale (France). Correspondence: Dr M. J. Leroy, U. 166, INSERM, Maternit6 Baudelocque, 123 Bld Port-Royal, 75014 Paris, France.

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display both high and low affinities for VIP has been described in the pig myometrium (Ottesen et af. 1982a); however only a small number of VIP immunoreactive nerve fibres were found in this tissue (Polak et af. 1984). The coupling of these receptors with the adenylate cyclase-cyclic adenosine monophosphate (CAMP) system has been proposed, similar to that observed in other smooth muscles (Suzuki et al. 1988), but the cellular events leading to the muscle relaxation following VIP receptor activation were not investigated in myometrium. In humans, in contrast to the observations for the cervix and vagina, only a few VIP nerve fibres were observed at the level of the uterine corpus. The data are in agreement with the low tissue concentration of V1P for the uterine corpus. (Goodnough et af. 1979; Helm et al. 1981). The inhibitory effect of VIP on uterine smooth muscle activity has previously been described both in vitro and in vivo (Ottesen et al. 1982b, 1983) in non-pregnant women. However it is now well established that the human myometrium is a heterogenous tissue, formed of several layers which differ in their embryological origin and which exhibit quite different histological and pharmacological properties (Sandberg et d. 1958; Schwalm et af. 1966; Deals et al. 1977; Breuiller et al. 1987). In the uterine corpus, we were able to distinguish the outer layer, where muscular bundles were oblique and longitudinal with regard to the peritoneum, and the inner layer, closer to the endometrium, where the direction of the fibres was parallel to the decidua. These two layers, composed mainly of smooth muscle cells, were separated by an intermediate layer containing a large amount of vascular and connective tissues. Much evidence has accumulated indicating that outer and inner layers have different physiological functions which might be important for regulation of the uterine contractile activity (Bengtsson 1982). In the present study we tested the in vitro relaxing effect of VIP on both the outer and inner myometrial layers of non-pregnant women and determined whether VIP action was mediated by stimulation of the cAMP formation process. Furthermore we demonstrated the presence of specific VIP binding sites in the non-pregnant human uterus, mainly located in the intermediate vascular layer and in the endometrium.

METHODS Materials 3-Iodotyrosylmono-iodo-[1251]-VIP (specific activity) 2000 Ci/ mmol), cAMP-['25I] assay system, 8-[3H]-

cAMP (sp. act. 26 Ci/mmol), and [I4C]-adenosine (sp. act. 500 Ci/mol) were supplied by the Radiochemical Centre (Amersham, UK). Natural porcine VIP was obtained from the Sigma Chemical Co. (St Louis, Missouri, USA) or from Professor Mutt (Stockholm, Sweden). Human growth hormone-releasing factor (hGRF) was a gift from Doctor Galland of SANOFI Recherche (Paris, France). Gastric inhibitory polypeptide (GIP), insulin, glucagon, secretin, bacitracin, forskolin, (-)isoproterenol, bovine serum albumin (BSA) (Fraction V, fatty acid free), adenosine triphosphate (ATP), creatine phosphate, creatine phosphokinase, ethylenglycoI-bis-/3-amino-ethyl ether N - N tetra-acetic acid (EGTA), 3-isobutyl-l-methylxanthine (IBMX), CAMP, 5'-AMP and snake venom (Croralux atrox) were obtained from Sigma Chemicals. Guanosine triphosphate (GTP) and guanyl-5'imidodi-phosphate (Gpp(NH)p) were purchased from Boehringer Mannheim (Germany) and anion exchange resin AGl-X2 (200-400 mesh) from Bio-Rad (Richmond, Virginia, USA). All other chemicals were reagent grades from various sources.

Subjects and tissue samples Menstruating women (34-46 years old) underwent total hysterectomy for fibromyomas, menorrhagia or premalignant dysplasia of the cervix. They were indifferently in the follicular or luteal phase of their cycle. All operations were performed with the patients under thiopental sodium-succinylcholine anaesthesia. No pre-operative analgesic medication except atropin was given. This study was approved by the Ethical Committee of the Institut National de la SantC et de la Recherche MCdicale (INSERM). Tissue samples were excised with a scalpel in the uterine corpus from normal muscle. For autoradiographic studies, blocks of I c m X 5 cm long were quickly frozen on dry ice, 20 pm serial sections were cut on a cryostat at - 15"C, in the entire thickness of the uterus. They were mounted onto gelatin-coated slices, stored overnight at -20°C and then kept at - 80" C until required. For tension measurements, tissue samples from the myometrial outer (longitudinal) and inner (circular) layers were immediately immersed in Krebs' solution (see composition below). The efficiency of these procedures, currently performed for other studies, has been confirmed by microscopic investigations as reported previously (Breuiller et al. 1987). For adenylate cyclase and cyclic nucleotide phosphodiesterase assays, biopsies excised in both myometrial layers were immediately used, or were quickly frozen at - 80°C. We confirmed

Ejject of' V I P in the human myometrium

that no change in the enzymatic activities was observed 3 months after storage at this temperature. Myometrium was homogenized in 50 vol (w/v) of ice-cold buffer 50 mmol/L Tris-HC1 (pH 7.4), as previously described (Litime et al. 1987, 1989).

Tension measurements Immediately after excision, myometrial tissue was placed in Krebs' solution previously gassed with 95% 02-5% C02 and maintained at 4°C until use. In general, six myometrial strips (8-12 mm long by 2-3 mm in cross-section) from each muscle biopsy were suspended in a 20 mL organ bath containing Krebs' solution pH 7.4, at 37OC, continuously aerated. The composition of the Krebs' solution was as follows (mrnol/L): NaCl 114.0, KCI 4.7, CaCL? 2.5, MgS04 1.2, KH2P04 1.2, NaHC03 25.0, glucose 11.7 and ascorbic acid 1.1. Isotonic constractions were measured using a Grass F T 0 3 transducer connected to a Grass 79B polygraph (Leroy et al. 1989). The preparations were allowed to equilibrate for 2 h, during which time the passive tension was maintained by repeated adjustments at 2 g. After this period, when spontaneous contractions became regular in frequency and intensity, inhibitory cumulative concentrationresponse curves were determined. VIP from 1 X 1O-Io to 3 X mol/ L was added mol/ L directly to the bath in the presence of 1 X bacitracin (Besson et al. 1987) and all concentrations given were final bath concentrations. Only one complete curve was obtained for each strip. The maximal initial spontaneous contraction (defined as the area under the graph during 10 min) was taken as 100% and all contractions were calculated subsequently as a percentage of this value. The inhibitory potency was determined at the level of ICSOvalues. Controls were made with distilled water which was the solvent used for the VIP dilutions.

Adenylate cyclase assay Adenylate cyclase activity was measured as previously reported (Litime et al. 1989) in a mixture (100 pL final volume) containing 50 mmol/ L Tris-HC1 (pH 7.4). 5 mmol/L Mg acetate, 0.05 mmol/L ATP, an ATP regenerating system (5 mmol/ L creatine phosphate and 50 UI/mL creatine phosphokinase) 0.1% BSA and 10-5 mol/ L bacitracin. We had previously checked that no inhibitory effect on adenylate cyclase activity was observed when 1 X 1 O - j mol/L bacitracin was added. The incubations were conducted in the presence of 0.2 mmol/L IBMX to inhibit cAMP degra-

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dation by phosphodiesterase. When indicated, the different agents to be tested (VIP, NaF, forskolin, etc.) were added to the incubation medium. The reaction was initiated by the addition of 20 p L of myometrial homogenate. The enzymatic assay was conducted at 30°C for various times with constant shaking. The reaction was stopped by transferring the tubes to a boiling water bath for 3 min. All assays were conducted in triplicate and carried out in conditions of linearity with respect to time and protein concentrations allowing measurement of the initial rates of the reaction. After centrifugation at 2500 g for 30 min at 4OC, the amount of cAMP formed was determined in the supernatants using a specific cAMP [125I]-radioimmunoassay kit with the acetylation method (Harper et al. 1975). We had previously verified that the cAMP isolated in the supernatants (90% of the total formed in all experimental conditions) could be interpreted as representing the cAMP formed by myometrial homogenates (Litime et al. 1987).

Phosphodiesterase assay Cyclic nucleotide phosphodiesterase (PDE) activities were measured by the two-step isotopic method (Thompson er al. 1971) as described previously (Leroy et al. 1985). The reaction mixture contained 5 mmol/ L MgS04, 80 mmol/L Tris-HCI (pH 8.0), 0.3 pCi of 8-[3H]-cAMP, unlabelled cAMP at concentrations previously defined as high (1 pmol/L CAMP)and low (0.5 mmol/ L CAMP) affinity conditions and enzymatic preparation (myometrial homogenate) in a final volume of 0.2 mL. 'Blank' values determined in the absence of PDE were subtracted from the experimental values. All assays, conducted in triplicate, were carried out in linearity conditions with respect to time and protein concentration allowing measurement of the initial rates of the reaction. The effect of VIP on cAMP P D E activity was determined in a concentration range of 1 X 10-10 to 1 X 10-5 mol/ L.

Protein determination Protein concentrations were determined by the Folin phenol reagent method (Lowry et al. 1951) using bovine serum albumin as the standard protein.

Autoradiographic studies Sections were brought to room temperature and incubated for 3 h with ['251]-VIP 60 pmol/L with or without unlabelled VIP ( 1 pmol/L) in 50 mmol/L Hepes/ KOH (pH 7.4) containing (mmol/ L): NaCl

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130.0, KC1 4.7, MgC125.0, MnCL2 5.0, EGTA 1.0, 1% BSA, and 1 mg/mL bacitracin. At the end of the incubation, slices were washed twice for 15 min at 4OC in the same buffer, dipped in cold distilled water to remove salts and dried with a cold stream of air (Besson et al. 1986). Then in some cases, film autoradiographs were produced by the apposition of [3H]-Ultrofilm (LKB) onto the radiolabelled slices in Kodak X-omatic film holders. Following 6-8 weeks of exposure at room temperature in the dark, the films were developed. In other cases, slices were post-fixed after incubation and washed with 4% glutaraldehyde in 50 mmol/L Sorensen buffer, then dehydrated and defatted in graded ethanol and xylene before dipping in Kodak NTB2 emulsion. Six weeks later, the film was developed in Kodak D- 19 developer. For histological localization, slices were post-fixed in Carnoy's solution and stained with Masson's trichrome. For the binding assay, tissues were wiped off the slices with Schleicher and Schuell filters and their radioactivity was measured in a gamma Packard Multiprias Counter.

RESULTS Contractile responses of my ometrial strips to VIP Spontaneous contractile activity was observed after a period of stabilization (at least 1 h) in approximately 90% of the myometrial strips studied. Figure 1 shows representative traces of the various types of spontaneous contractions which were obtained with samples from the outer myometrial layer of four different non-

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pregnant women. According to individuals, and/ or samples, contractions differ with respect to frequency, duration and amplitude. Similar variations in the recording of spontaneous contractile activities have been observed in the corresponding inner layers. Dose-dependent inhibition of spontaneous contractions was obtained when VIP ( I X 10-10 to 3 X 10-6 mol/ L) was introduced into the preparation. In the outer layer, the induction of this relaxant effect was observed at a VIP concentration as low as 5 X 10-10 mol/ L and the VIP concentration that produced 50% of the maximum response (ICN) was around 1.26 X 10-8 mol/ L. The maximum inhibition, which reached 75%. was obtained at 1 X mol/L VIP (Fig. 2). It should be noted that variation in the type of response elicited by VIP is as great between strips from the same individual as it is between individuals. Thus, depending on the strips, modifications in frequency and/or amplitude of the contractions could be observed. This could be attributed to differences in the total smooth muscle content of the myometrial samples obtained (Dyal et al. 1988). In any case, the inhibitory effect of VIP in the inner layer is less important than that in the outer layer, and the ~ C SisO over 3 X 10-6 mol/L (Fig. 2). In comparison, the padrenergic agonist, isoproterenol, exerts a preferential relaxant effect in the inner layer with an ICSOof about 2 X 10 -6 mol/ L, whereas the ICSOin the outer layer is only 4 X 10-5 mol/L (data not shown).

Effect of VIP on adenylate cyclase activity Basal adenylate cyclase activity was always higher in the inner (7.7 t 1.8 pmoles/mg protein/ min) than in

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Fig. 1. Representative tracings f r o m the various types of spontaneous in vitro contractions obtained f r o m four different non-pregnant women. Strips were prepared from the outer myometrial layer.

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the outer layer (4.8 t 1.9 pmoles/mg protein/min), as previously reported (Litime et al. 1989). In the homogenates of the two myometrial layers, the adenylate cyclase activity was not stimulated by VIP. Only a slight response to VIP was observed when adenylate cyclase activity was measured in non-initial rate conditions (Fig. 3). In that way, a stimulation over basal activity of 1.5- and 1.3-fold in the outer and inner layers, respectively, was obtained in four preparations. When ATP concentration (0.1, 0.5 and I mmol/L) was increased, the basal activities still remained linear for at least 20 min. VIP ( I X 10-9 mol/L) in such linear conditions never stimulated cAMP formation (data not shown). This pattern contrasts with that of other classical agents known to interfere with the different components of the cAMP enzyme formation system, such as forskolin, FNa, Gpp(NH)p or isoproterenol (Fig. 4). All these agents stimulate adenylate cyclase activity in initial rate conditions, that is, in the first minutes of incubation. For these four compounds, similar results were obtained for the inner layer. Furthermore mol/L we confirmed that the presence of 1 X Gpp(NH)p did not reveal a precocious effect of VIP on adenylate cyclase activity. Likewise, the presence of the phosphodiesterase inhibitor IBMX, tested from 1 X 10-4 to 1 X 10-3 mol/L did not reveal any rapid response to 1 X 10-9 mol/ L VIP (data not shown).

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Fig. 3. The time-course of VIP-stimulated adenylate cyclase activity in the inner layer of human myometrium. Results are the mean t s.e.m. of triplicate determinations carried out on three different myometria. Similar results were obtained for the outer layer.

In the same myometrial preparations we also tested whether VIP had a direct effect on the cAMP degradation process. This peptide has been tested in concentrations ranging from 1 X 10-10 to 1 X 10-5 mol/L. No modification in the specific activities of cAMP phosmol/ L CAMP) phodiesterase measured in low (5 X and high ( I X 10-6 mol/ L CAMP) affinity conditions was observed (data not shown). Increasing concentrations of VIP from 1 X 10-10 to 1 X 10-5 mol/L were tested for effects on cAMP formation in initial (3 min) and non-initial (15 min) rate conditions on homogenates of the two myometrial layers. The results presented in Fig. 5 confirm the lack of a VIP effect at 3 min, since no dosedependent response could be observed. At 15 min, however, dose-response effects were obtained. This result was more distinct in the outer layer; however the maximum stimulation obtained was generally beand 1 X mol/L, in both layers. tween 1 X In order to test the specificity of the VIP response, we determined the effect of various peptides structurally related to VIP, such as GIP, glucagon, secretin and hGRF, on cAMP production. For glucagon (2.5 X and 2.5X mol/L), secretin (1 X 10-9

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Fig. 4. The time-course of FNa-, forskolin-, Gpp(NH)p- and isoproterenol-stimulated adenylate cyclase activity in the outer layer of human myometrium. Results are the mean k s.e.m. of triplicate determinations carried out on three different

myometria. Similar results were obtained for the inner layer. and I X 10-5 mol/L), G I P (2.5X 10-9 and 2.5X 10-5 mol/L), and h G R F (1 X and 1 X mol/L), no effect was observed o n adenylate cyclase activities measured at 3 and 15 min (Fig. 6).

When sections (20 pm) were treated for high resolution autoradiography, all cellular components contained silver grains (Fig. 7C). A much higher density of labelling was still associated with the blood vessels of the vascular layer and the thin vascularization of the inner and outer layer (arterioles and venules).

Autoradiographic studies After development of the photographic emulsion, histological staining of t h e corresponding slices allowed the localization of V I P binding sites o n uterus sections. Although the thickness (20 pm) of the slices was not compatible with a fine structural definition, it was possible t o associate the high densities of labelling mainly with the blood vessels of the vascular layer (Fig. 7A), as well as in the thin vascularization distributed in the inner and outer layers (Fig. 7B) and in the endometrium (Fig. 7A). When excess unlabelled VIP (1 X 10-6 mol/L) was added to ['2sI]-VIP during incubation, we observed a decrease of about 40% in the labelling, indicating the specificity of the binding (Fig. 7).

DISCUSSION T h e present study clearly shows that VIP is a very potent relaxing agent mainly in the outer layer of the non-pregnant human myometrium. A previous in vitro study described the inhibitory effect of V I P o n uterine activity in non-pregnant women with a maxim u m effect obtained at 6 X 10-7 m o l / L concentration (Ottesen et al. 1982b). However no attention was paid t o the individual response of each muscular layer. Our results have shown that there is in fact a marked difference in V I P sensitivity between the outer and the inner layer, since the ICSOin the outer layer is around

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-log [VIP] mol/L Fig. 5. The dose-response curves for VIP stimulation of adenylate cyclase activity from the inner and outer layers of two different myometria ( X I 0 ) . Enzymatic activities were measured either under linearity ( 3 min of incubation, lower curves), or under non-linearity conditions (15 min of incubation, upper curves). Values represent the mean k s.e.m. of triplicate determinations for each myometrium.

1X moll L while it is over 3 X 10-6 mol/ L for the inner layer. In addition VIP in vitro appears to be a much more potent relaxant in the human myometrium than is isoproterenol. In other species, such as rabbit o r guinea pig, a similar inhibitory effect of V I P was observed in vitro o n the spontaneous activity of the whole myometrium, with a n ICSOin the 1 X 10-8 mol/ L range (Bolton et al. 1981; Bardrum et al. 1986). In non-pregnant women, we have little d a t a at this time about the specific properties and functions of the different myornetrial layers. We can only suspect their implication in the regulation of contractile activity during the menstrual cycle and at the time of menstruation. The distinct responses we observed in both layers have already been described in pregnant rat myometrium, the outer layer being (in vitro) much more sensitive t o oxytocin (El Alj et al. 1990). In women, both layers also react differently t o adrenergic stimulation during gestation (Breuiller et a/. 1987). Such results confirm that both inner and outer layers are physiologically and pharmacologically different, and partly support the concept of a regulatory heterogeneity of the human myometrium (Csapo et a/. 1977).

It has now been clearly established that c A M P and hormones or drugs which increase c A M P production, induce uterine relaxation. Since in other tissues the physiological responses t o V I P have generally been shown t o be mediated by the binding of V I P t o specific receptors coupled t o the c A M P formation process (Laburthe et al. 1978; Bardrum et al. 1986; Griffiths et al. 1987), we analysed this mechanism in homogenates of both myometrial layers. Our results show that there is no direct effect of VIP o n adenylate cyclase activity in either layer. The only slight increase we observed in non-initial rate conditions suggests that V I P acts by another transduction pathway which only secondarily modified c A M P production. Only the non-linearity conditions allowed us t o visualize this indirect response which is maximum between V I P concentrations of 1 X 10-9 and 1 X m o l / L in both layers. In the outer layer this optimal concentration was in good agreement with its in vitro relaxing activity, while this is not the case for the inner layer. Previous studies also suggested that c A M P may not be the only second messenger mediating in VIPinduced effects (Audigier et al. 1986). I n a similar way, VIP-induced relaxation in the lower oesophageal

M . J. LerqlJet al. sphincter is linked to decreases in inositol lipid breakdown (Szewczak et al. 1990). Intracellular calcium T levels are hereby reduced, causing the muscle to relax. Furthermore it has been suggested that other related peptides such as glucagon in hepatocytes (Irvine et al. 1988), or secretin in rat pancreatic acini (Trimble et al. 1987), mediate their effects through the stimulation of phospholipid metabolism. For the rat stomach, it has also been proposed that the relaxant action of VIP may be mediated at least in part by the production of CAMP and by the opening of Ca2+-dependent K’ channels (Kamata et al. 1988). Autoradiographic studies allowed us to localize specific VIP binding sites, mainly on blood vessels GIP Glucagon hGRF Secretin in the intermediate vascular myometrial layer, which contains large amounts of vascular and connective tissues. VIP binding sites were also found on arterioles and venules distributed in the inner and outer layers, which are composed mainly of smooth muscle fibres (Breuiller et al. 1987), and on the endometrium. In contrast, under our experimental conditions, we were not able to observe any specific labelling of smooth muscle cells of the myometrium. These observations are in good agreement with the immunohistochemistry studies of Heinrich et al. (1986) which have shown that VIP fibres are found around large uterine arteries and endometria1 arterioles. Nevertheless, we acknowledge that localization of VIP binding sites by autoradiography, requiring frozen slices of 20 pm thick, leads to an important non-specific binding which makes difficult the display of specific receptors. For Control GIP Glucagon hGRF Secretin the hypothesis of an absence of these VIP binding sites on smooth muscle myometrial cells, we might Fig. 6 . The effect of peptides with structural similarities to suspect that an indirect mechanism mediates in its VIP on adenylate cyclase activities from the outer layer. relaxant action, Indeed, VIP has been reported to Enzyme activities were measured at (a) 3 min, or (b) 15 min of produce independent and dependent endothelium incubation. The concentrations of G IP and glucagon used arterial relaxation (Furchgott et al. 1980; Sata et al. were 2.5X10-’ mol/L (0) and 2.5X10-5 mol/L (a), and 1986; Ignarro et al. 1987). The endothelium-dependent the concentrations of hGRF and secretin were 1 XIO-9 mechanism is associated with the production of difmol/L (0) and I x 10-5 mol/L (m). Values are the mean+ ferent relaxing factors by the endothelial cells, such as s.e.m. of triplicate determinations carried out on two different prostacyclin and endothelium-derived relaxing factor my ometria. (EDRF), and an accumulation of CAMP. These endo-

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Fig. 7.(A) The autoradiogram of a human uterus ( X 5). Frozen sections (20 p m ) were incubated with [1zsI]-VIP (60 pmol/ L). Note the presence of a high density of labelling on the endometrium (E) and in the vascular layer (VL) between the two other muscular layers (longitudinal or outer LL, and circular or inner CL). (B)The autoradiogram of a human uterus ( X 5 ) showing a high density of labelling on large vessels and also on the diffuse vascularization distributed throughout the myometrium (arrow heads). (C)A light-microscopic autoradiograph of small uterine arterioles ( X 900) obtained by incubating 20 pm slices with [{25I]-VIP (60 pmol/L). Many grains are located around the lumen of blood vessel and on the endothelium. This photomicrograph was made in the inner layer. [Note that coincubation of [1251]-VIP with an excess of unlabelled VIP resulted in a reduction of labelling (A’, B’) and of silver grains (C’).]

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thelial agents could be released either i n the s u r r o u n d ing vascular s m o o t h muscle cells, producing vasodilat a t i o n of t h e vessel o r in t h e b l o o d flow. Since in t h e myometrium we localized specific V I P binding sites mainly o n t h e vascular elements, it is still unclear h o w such mechanisms could have repercussions f o r myometrial s m o o t h muscle relaxation. We can only speculate t h a t modifications in t h e blood flow result in an increased availability of substances which act subsequently o n t h e myometrium. F u r t h e r m o r e , t h e presence of specific V I P binding sites in t h e endometrial region suggests t h a t this neuropeptide m a y also be involved in the c o n t r o l of o t h e r f u n c t i o n s , s u c h as uterine secretion (Heinrich et al. 1986) and ion transport. In conclusion, t h e sequence of cellular events leading t o myometrial relaxation induced by V I P is still n o t completely clarified a t present. The non-evident localization of V I P binding sites on t h e m y o m e t r i u m , a n d t h e absence of a direct linkage t o t h e C A M P messenger system, suggest t h a t a n o t h e r signal transduction p a t h w a y might be implicated. An additional, intermediate step in t h e vascularization becoming distributed throughout the muscle c a n n o t be excluded.

ACKNOWLEDGEMENTS We are grateful t o Drs G. Rosselin, D. Saltarelli a n d G. Butler-Browne f o r critical reading of t h e m a n u script, and we t h a n k M. Verger f o r h e r secretarial assistance.

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