Molecular Human Reproduction, Vol. 5, No. 11, 1040-1047,
November 1999
© 1999 European Society of Human Reproduction and Embryology
Molecular events in the uterus |
Localization of nitric oxide synthase and effects of nitric oxide donors on the human Fallopian tube
Department of Obstetrics and Gynaecology, Göteborg University, Sahlgrenska University Hospital, 413 45 Göteborg, Sweden
Abstract
The objective of the study was to assess the plausible existence of a nitric oxide (NO) system within the human Fallopian tube and to examine the effects of NO on tubal contractility. Tissue was obtained from fertile women at operations due to non-tubal diseases. Production of NO and sites of nitric oxide synthase (NOS) activity were assessed by the use of NADPH diaphorase staining and by immunoblots as well as immunohistochemistry for the isoforms of NOS. Effects of NO on tubal contractility in vitro were examined by adding either of two NO donors (nitroglycerin, spermine NONOate) or an analogue of its second messenger (8-bromo cyclic GMP). The production of NO was indicated by positive NADPH diaphorase staining. In immunoblots, endothelial and inducible NOS were demonstrated in all samples analysed. By immunohistochemistry, moderate staining for endothelial NOS was demonstrated in the luminal epithelial cells and in the endothelial cells of blood vessels. Moderate staining for inducible NO synthase was seen in smooth muscle cells and weak staining in epithelial cells. Nitroglycerin, spermine NONOate and 8-bromo cGMP all resulted in a concentration-dependent inhibition of contractility with significant contractility inhibition at 10–7 mol/l, 10–6 mol/l and 10–5 mol/l respectively. The study demonstrates the existence of an endogenous NO system, which may be of physiological importance in Fallopian tube function.
contractile activity/Fallopian tube/human/nitric oxide/nitric oxide synthase
Introduction
Nitric oxide (NO) is a multifunctional molecule involved in a variety of bioregulatory processes (Änggård, 1994
). It is a free radical gas, with a half-life of only a few seconds, generated from the metabolism of L-arginine by a family of enzymes known as the nitric oxide synthases (NOS) (Moncada and Higgs, 1993). Three isoforms of NOS have been characterized and cloned (Förstermann et al., 1994). Endothelial NOS (eNOS) and neuronal NOS (nNOS) are both constitutively expressed and are calcium-calmodulin dependent. Endothelial NOS and nNOS were first described in vascular endothelial cells and in neuronal cells respectively. One main pathway by which NO exerts its effects is by activating soluble guanylate cyclase. This enzyme catalyses the formation of cyclic GMP (cGMP), which in turn activates protein kinases and ultimately leads to the dephosphorylation of myosine light chains and muscle relaxation (Änggård, 1994). On the other hand, inducible NOS (iNOS), first described in macrophages, does not appear to be regulated by calcium, although iNOS does contain tightly bound calmodulin. Inducible NOS-derived NO is typically expressed at the site of an inflammatory process. Its main function is cytostatic and cytotoxic effects on target cells, i.e. tumour cells but also even on bacteria and viruses.
In the female reproductive tract, NO has been implicated as a modulator of a variety of physiological and pathophysiological processes. Thus, excessive NO production may be associated with menorrhagia, while shortage of NO may contribute to the development of pre-eclampsia and intrauterine growth retardation (Norman and Cameron, 1996). Nitric oxide inhibits myometrial contractions in vitro (Buhimschi et al., 1995; Ekerhovd et al., 1999) and NOS has been found in endometrium and myometrium as well as placenta (Tseng et al., 1996; Thomson et al., 1997a). Recently, it was shown that NO donors applied intravaginally induced cervical ripening (Thomson et al., 1997b) and that NO inhibits contractions of cervical smooth muscle in vitro (Ekerhovd et al., 1998). Furthermore, NO seems to be involved in the process of ovulation (Bonello et al., 1996), and a preliminary report from our laboratory has indicated an endogenous NO system in the human Fallopian tube (Ekerhovd et al., 1997). The aim of the present study was to follow-up that report by examining in more detail the presence and functions of the NO system within the Fallopian tube. The existence of NOS activity was studied by means of NADPH diaphorase staining, immunoblots and immunohistochemistry, while the in-vitro effects of NO on tubal contractile activity were examined by an extended number of pharmacological agents.
Materials and methods
Subjects and tissue collection
A total number of 36 regularly cycling women (aged 25–46 years) admitted for legal sterilization or operation for non-tubal diseases (myoma uteri or benign ovarian cysts) were included in the study. The phase of the menstrual cycle was determined on the basis of menstrual history, clinical examination prior to surgery and, in some cases, endometrial histology. The women had given their informed consent to participate in the study, which was approved by the Human Ethics Committee of Göteborg University. All women had regular menstrual periods. As the first intra-abdominal procedure at operation, tissue was obtained from the isthmic portion of the Fallopian tube at the level of the ampullary isthmic junction (AIJ). The tissue was either immediately frozen in liquid nitrogen (NADPH diaphorase staining or immunoblotting), fixed in 10% neutral buffered formalin (immunohistochemistry) or transferred into oxygenated ice-chilled buffer (contractility experiments).
NADPH diaphorase staining
To demonstrate NOS activity within the Fallopian tube, NADPH diaphorase staining, a non-specific marker for all the isoforms of NOS was used. Tissue specimens, stored at 70°C, were mounted in OCT Tissuetec® (Miles Inc, Elkhart, IN, USA). Sections (~5 µm thick) were cut on a cryostat and then fixed onto gelatine/chrome aluminium-coated slides. The slides were incubated with 1 mmol/l NADPH/0.2 mmol/l Nitro Blue Tetrazolium / 0.1 mol/l Tris–HCl, pH 7.2/0.2% Triton X-100 for 30 min at 37°C (Dawson et al., 1991). Negative controls were exposed to the staining solution but with the omission of NADPH. Using light microscopy, the localization and density of staining (no staining; weak staining; moderate staining; strong staining) in specimens from four women were evaluated independently by two observers. All substances used in these experiments were purchased from Sigma Chemical Co (St Louis, MO, USA).
Immunohistochemistry
Transverse sections of the Fallopian tube (~5 µm thick) were prepared from paraffin-embedded specimens and mounted on silane-coated slides. The tissues were then heated to 60°C for 35 min, deparaffinized in xylene and rehydrated in a graded alcohol series. Subsequently, the samples underwent enzymatic digestion in a 0.01% (w/v) solution of CaCl2 containing 0.01% (w/v) protease type XXIV (Sigma Chemical Co) for 10 min at 37°C. The specimens were pre-incubated with 3% (w/v) immunoglobulin-free bovine serum albumin (BSA; Sigma Chemical Co.) in phosphate-buffered saline (PBS; 10 mmol/l sodium phosphate, pH 7.5, 120 mmol/l NaCl) for 20 min at room temperature and then incubated for 1 h at room temperature with a monoclonal antibody raised against mouse eNOS (Transduction Laboratories, Lexington, KY, USA) diluted 1:100 in 3% BSA. Afterwards, the tissue samples were washed in 0.1% Triton-X (Sigma Chemical Co.), further washed twice in PBS and then incubated with biotinylated anti-mouse immunoglobulin, diluted in 3% BSA and 1.5% human serum, using a Vectastain Elite ABC kit® (Vector, Peterborough, UK). The sections were washed twice in PBS, incubated for 30 min with avidin DH/biotinylated horseradish peroxidase H reagent (Vectastain Elite ABC kit) in PBS, and then washed again. Diaminobenzidine tetrahydrochloride (1 mg/ml, 0.02% H2O2 in 50 mmol/l Tris–Cl, pH 7.6) was used to localize immunoreactive eNOS. Sections of human umbilical cord were used as positive controls.
For the demonstration of nNOS a polyclonal antibody raised against amino acids 724–739 of rat brain nNOS (Serotec, Oxford, UK) at 1:750 and a rabbit IgG Vectastain ABC kit were used. Paraffin-embedded rat cerebellum served as positive control. A polyclonal antibody (diluted 1:1000) raised against mouse iNOS (Affiniti, Nottingham, UK) was used to detect iNOS, with human lung tissue as the positive control. Negative controls for all isoforms examined were processed as described above but with the omission of the primary antibody.
Immunoblotting
Transverse sections of isthmic tubal tissue of 10 women were homogenized by a polytron in a PE-buffer (10 mmol/l potassium phosphate buffer, pH 6.8, and 1 mmol/l EDTA) containing 10 mmol/l 3-[(-3 cholamidopropyl)dimetylammonio] 1-propane sulphonate (CHAPS), aprotinin (200 kallikrein inhibitory units/ml), leupeptin (1 mg/ml), pepstatin (1 mg/ml) and Pefablock® (Boehringer Mannheim, Mannheim, Germany). The homogenate was then sonicated (2x15 s) and centrifugated for 10 min at 10 000 g at 4°C and supernatants were stored at –70°C until analysis. Immediately prior to electrophoresis, Nu PAGE reducing agent (NOVEX, San Diego, CA, USA) was added to the sample solutions to a final concentration of 10%. Samples were heated at 70°C for 10 min before loading on a NuPAGE 4–12% Bis–Tris gel with the MOPS SDS [3-(N-morpholino) propane sulphonic acid sodium dodecyl sulphate] running buffers. Protein concentration was measured with a BCA Protein Assay Reagent Kit (Pierce, Rockford, IL, USA). The total protein loaded in each lane was 50 µg and prestained standards (MultimarkTM, NOVEX) were used as markers. The proteins were transferred to a polyvinyldifluoride membrane (PVDF; Amersham, Buckinghamshire, UK) using a blotting system (Mighty SmallTM, Hoeffer, San Francisco, CA, USA). The membranes were then incubated with blocking buffers containing antibodies against eNOS (dilution 1:1000, mouse monoclonal), nNOS (dilution 1:1000, mouse polyclonal) and iNOS (dilution 1:1000, mouse polyclonal). The antibodies against eNOS and nNOS were purchased from Transduction Laboratories (Lexington, KY, USA), while the antibody against iNOS was purchased from Santa Cruz Biotechnology (San Diego, CA, USA). In each blot, one lane was loaded with protein from an appropriate positive control [rat aorta for eNOS, rat cerebellum for nNOS and interleukin-1ß (IL-1ß)-stimulated rat spleen for iNOS]. Immunoreactive proteins were visualized by chemiluminescence using alkaline-conjugated second goat antibody and CDP Star (Tropix, Bedford, MA, USA) as substrate. The filters were exposed to ECL film (Amersham, Buckinghamshire, UK) at room temperature and the films were subsequently developed.
For semiquantitative measurement of the proteins of the immunoblots, a software package in The Discovery Series Densitometric Systems (Pharmacia Biotech, Lund, Sweden) with Desk Top Plus Scanner, was used. The optical densityxmm2 from each band was measured.
Contractility experiments
Excised tissue from the isthmic portion of the Fallopian tube was immediately placed in ice-chilled buffer and taken to the laboratory. The composition of the buffer was as follows (mmol/l): NaCl 122, KCl 4.7, CaCl2 2.5, MgCl2·6H2O2 1.19, KH2PO4 1.19, glucose 11.5 and HEPES 5.0. The AIJ, identified under the microscope by introducing a probe (diameter 1 mm) into the distal end of the tubal segment until resistance was excised (Lindblom et al., 1978). Using microscissors, longitudinal strips measuring ~6x1x1 mm, comprising the tubal epithelium as well as the smooth muscle layers, were isolated under a stereomicroscope. The strips were mounted vertically in organ baths and incubated in buffer continuously bubbled with oxygen and maintained at 37°C. Each strip was suspended under a tension of 5 mN and was allowed to equilibrate for ~45 min when regular contractile activity had been established for at least 20 min. Contractile activity was recorded by a Grass FT 03D transducer (Grass Instruments, Quincy, MA, USA), connected to a RPS 7D Grass polygraph recorder.
Two different NO donors, nitroglycerin (10–7 to 10–5 mol/l) and spermine NONOate (10–6 to 10–5 mol/l) as well as 8-bromo cGMP (10–5 to 10–3 mol/l), a membrane permeable analogue of the second messenger cGMP, were added at increasing concentrations at intervals of 20 min without washout periods between each concentration. In separate experiments, the parent compound spermine (10–5 mol/l) was added to the organ baths, to confirm that the responses registered after the administration of spermine NONOate were due to NO release. All drugs were dissolved in buffer solution prior to the administration. Each strip was only exposed to one single drug.
Nitroglycerin was purchased from Tika AB (Lund, Sweden), while spermine and spermine NONOate were both obtained from Alexis Co (Läufelfingen, Switzerland). 8-bromo cGMP was a product from Sigma Chemical Co (St Louis, MO, USA).
Statistical analysis
Contractile activity was evaluated by estimating the area under the tension curve (mean ± SEM), using computerized planimetry (FlexiTrace 1.02, Tree Star; INC, Santa Barbara, CA, USA). Twelve strips obtained from 12 women were examined for each drug tested. After the establishment of regular contractile activity, a period of 15 min immediately prior to the administration of each drug tested, was chosen as a control period. Periods of 15 min, starting after the administration of each drug, were compared with control periods. Statistical comparisons were performed by means of analysis of variance (ANOVA) followed by unpaired Student t-test or the Boniferroni method. P < 0.05 was considered to be statistically significant.
Results
NADPH diaphorase staining
Positive NADPH diaphorase staining, histochemically visualized as a dark blue deposit, was observed in the tubal epithelium, all layers of smooth muscle and in the walls of blood vessels (Figure 1A). In all specimens (n = 4) staining intensity was strong in tubal epithelium and vascular endothelium, while staining of smooth muscle tissue was weak. Staining was absent in control sections.
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Immunohistochemistry
Endothelial NOS protein was seen in tubal epithelium and endothelium of blood vessels (Figure 1B
). In three of the four specimens examined, staining intensity was moderate in epithelial cells, while the fourth sample demonstrated weak intensity. The epithelium of the samples had a mature secretory appearance, and staining intensity varied between the different areas of epithelial cells. Staining for eNOS of the vascular endothelium was of moderate intensity in all four specimens. Although the rat cerebellum (positive control) clearly demonstrated staining for the other constitutive isoform nNOS, no staining was detectable in the tubal sections examined (data not shown).
Inducible NOS was localized in all four tissues samples, demonstrating a moderate intensity in the tubal smooth muscle cells and weak intensity in the tubal epithelium and in the walls of blood vessels (Figure 1C). Control slides, without the primary antibodies, were absent of staining (Figure 1D).
Immunoblotting
Endothelial NOS was clearly seen in all tissue samples (n = 10) examined, demonstrated as a protein band of 140 kDa (Figure 2). The amount of eNOS protein differed substantially between the various samples, and was lower than what was observed in the positive control sample.
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Neuronal NOS was not detectable in any of the tubal tissue specimens (n = 10) examined although the positive control showed the expression of nNOS proteins as a protein band of 155 kDa (data not shown).
Inducible NOS was clearly seen as a band of 130 kDa in all samples (n = 10) tested and the variation seemed less as compared to eNOS (Figure 3). The positive control, IL-1ß-stimulated rat spleen, showed a much higher iNOS content than the samples obtained from the Fallopian tube.
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Contractility experiments
Spontaneous contractions were registered within minutes after the montage of tubal strips. The strips exhibited regular contractile activity of high frequency and short duration, that persisted for at least 5–6 h. The frequency of contractions was higher in strips obtained at the peri-ovulatory interval (39 ± 2.3 (mean ± SEM) per 10 min; cycle days 10–16; n = 14) compared with early follicular (22 ± 1.4 per 10 min; cycle days 1–9; n = 8) and mid-luteal and late luteal (21 ± 1.6 per 10 min; cycle days 17–28; n = 14) phase.
The addition of nitroglycerin (10–7 – 10–5 mmol/l) caused a significant concentration-dependent inhibition of contractions within seconds (Figures 4 and 5). A decrease in frequency of contractions as well as amplitude of contractions were observed. At the highest concentration tested (10–5 mol/l) a decrease in basal tone was registered in six of the 12 strips studied. Persistent abolishment of contractions was not seen.
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The addition of spermine NONOate at two concentrations (10–6 and 10–5 mol/l) resulted in a significant inhibition at both concentrations, with an ~70% inhibition of contractile activity at 10–5 mol/l (Figures 6 and 7
). In four out of 12 strips (10–5 mol/l) contractile activity completely disappeared for 3–6 min followed by a gradual increase in contractile activity. In five of the 12 strips a decrease in basal tone was registered at 10–5 mol/l. Administration of spermine (10–5 mol/l), the parent compound of spermine NONOate, did not cause any change in contractile activity (Figures 6 and 7).
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The second messenger analogue, 8-bromo-cGMP (10–5 to 10–3 mol/l), inhibited contractile activity in a dose dependent manner (Figures 8 and 9
). A reduction in frequency as well as amplitude of contractions was registered. In contrast to the NO donors, this substance did not result in any observable change in basal tone or transient abolishment of contractile activity.
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Discussion
Peristalsis of the Fallopian tube, due to contractions of its smooth muscle cells, is physiologically related to ovum transport. Thus, functional disorders of tubal contractile activity, i.e. prolonged spasms of the isthmic region, may lead to the retention of the fertilized ovum, thereby causing infertility or tubal pregnancy (Uher et al., 1990).
The Fallopian tube is composed of distinct communicating regions of characteristic morphology, reflecting different functional roles of the various regions. In the intramural and isthmic segments, smooth muscle cells constitute the major tissue component, which is gradually diminished towards the ampulla and the infundibulum. The present study focuses on the muscle function of the isthmic segment, a part of the tube in which a sphincter-like contractile activity may be of special importance (Anand and Guha, 1978).
Several biological agents, such as catecholamines (Owman et al., 1976), prostaglandins (Lindblom et al., 1978), peptidergic transmittors (Alm et al., 1980, Sjöberg and Helm, 1991) and sex steriods (Forcedello et al. 1986, Nozaki and Ito, 1987) have previously been shown to be involved in regulation of contractility and motility of the Fallopian tube. The first evidence for the presence, as well as a physiological role for NO in regulating tubal contractility, was demonstrated in the bovine oviduct (Roselli et al., 1994). In a previous study, we have shown the effects of L-arginine (the substrate for NO synthesis) and L-NAME (N6-nitro-L-arginine methyl ester) (a competitive inhibitor of NOS) on contractile activity in tubal tissue obtained from fertile women (Ekerhovd et al. 1997). These experiments indicated that NO may regulate contractile activity in the human Fallopian tube. To further evaluate this phenomenon the present study examines the plausible existence of an endogenous NO system within the Fallopian tube and the effects of NO on tubal contractility by the use of well-established NO donors.
NADPH diaphorase staining has been used extensively as a histochemical marker for NOS activity. This histochemical reaction is specific for NOS only if there are no other active NADPH-requiring enzymes present. Although all known NOS isoforms are NADPH diaphorase positive, NOS-associated NADPH diaphorase may represent only a part of the total cellular NADPH diaphorase activity (Tracey et al., 1993). Therefore, positive NADPH staining can only be looked upon as a non-specific marker for NOS activity and other methods are necessary to verify the results by this method in relation to localization of NOS. Nevertheless, the positive NADPH diaphorase staining, as observed in the present study in all layers of the tubal wall, though with different intensities, indicates the presence of an endogenous NO system.
In the present study it was observed that eNOS staining was particularly prominent in luminal epithelial cells. The tubal epithelium consists of monostratified cylindrical cells in which secretory cells are the major cell type of the intramural and the isthmic segments, while ciliated cells dominate the ampulla and the infundibulum (Hunter, 1988). The intensity of the eNOS staining varied between luminal epithelial cells, the strongest intensity being observed within mature secretory cells. It has been known for several decades that tubal epithelial cells undergo cyclic changes (Novak and Everett, 1928). Further, the composition and rheological properties of the luminal fluid, formed by transudation from blood vessels and active secretion from the tubal mucosa, have been shown to vary with the stage of the menstrual cycle (Jansen, 1984). Nitric oxide produced by vascular endothelial cells is a well-known regulator of blood flow (Änggård, 1994). The luminal milieu of the Fallopian tube is regulated by the transport and permeability properties of the blood-tubal barrier, together with secretion from epithelial cells (García-Pascual et al, 1996). Based on these facts, it is tempting to speculate that NO produced by endothelial and tubal epithelial cells may play a regulatory role in the production of intra-tubal fluid.
Since the Fallopian tube is the site of fertilization and early embryonic development, it is feasible that NO may be involved in these processes (Rosselli et al, 1998). Nitric oxide may protect the ovum and the spermatozoa against oxygen free radical-induced damage. Likewise, NO regulates sperm motility. Studies have shown that low concentrations of NO enhance sperm motility, while moderate/high concentrations have an impeding effect (Hellstrom et al, 1994; Rosselli et al, 1995).
In contrast to eNOS which exhibited no staining in smooth muscle cells, iNOS was most evident in the smooth muscle layers and the immunoblots indicated less variation of this enzyme. However, the physiological importance of iNOS may however be considerable, since this isoform produces much higher quantities of NO than the constitutive enzymes.
The immunoblot analyses of the present study clearly show that human tubal tissue expresses eNOS as well as iNOS. Semiquantitative analyses demonstrated a considerable inter-individual variation in the amount of eNOS. This indicates that the expression of eNOS may vary during the menstrual cycle.
The occurrence of endogenous NO synthesis within the human Fallopian tube by measuring both the conversion of [3H]-L-arginine to [3H]-L-citrulline as well as the generation NO2/NO3 has previously been demonstrated (Rosselli et al., 1996). The present study confirms these results indicating that both endothelial and inducible NOS are present in human tubal tissue.
The fact that there was no detectable nNOS protein, neither in the immunoblots nor in the specimens used for immunohistochemistry, does not totally rule out that this isoenzyme still is involved in the regulation of tubal function. The Fallopian tube is innervated by parasympathetic and sympathetic nerves, as well as visceral nerves (Hodgson and Eddy, 1975) and neuronal mechanisms are involved in the regulation of contractile activity of the sphincter-like AIJ (Cheviakoff et al., 1976). Previous experiments have indicated that non-adrenergic, non-cholinergic nerves liberate a transmittor substance with relaxing effects on the AIJ (Lindblom et al., 1979). In fact, in a recently published study, immunohistochemistry of the ampullary segment of the tube demonstrated a weak staining for iNOS in epithelial cells (Tschugguel et al, 1998). In this study positive staining for eNOS was only seen in endothelial cells of blood vessels, while iNOS-like immunoreactivity was weak and only diffusely scattered in epithelial cells. The discrepancy of the results of the two studies may be due to the fact that different tubal segments (isthmus versus ampulla) were examined, differences in sensitivity between the immunohistochemical methods used, differences in specificity between the antibodies employed as well as cycle-dependent changes of NOS activity. In most organs, iNOS is only expressed in response to an immune response. On the other hand, positive staining for iNOS has been demonstrated in the pregnant uterus as well as the ovary (Cameron and Campbell, 1998; Rosselli et al., 1998), indicating that iNOS activity may be present under normal physiological conditions.
The findings of variations in tubal contractions, as registered in the present study, confirm previous observations of cycle dependent variations in spontaneous contractile activity of the circular and the longitudinal muscle layers of the Fallopian tube (Lindblom et al., 1980). In the present study the frequency of contractions was significantly higher in tissue taken from the periovulatory phase as compared to the early follicular and the mid-luteal and late luteal phase. The decreased frequency of contractions, as observed in the luteal phase of the cycle, may be of importance to facilitate the transport of the ovum to the uterus.
The inhibitory effects on contractile activity by the substances used in the present study give further support to the existence of an endogenous NO system within the human Fallopian tube. While nitroglycerin, a conventional organic vasodilator, needs metabolic activation for pharmacological action (Feelisch and Kelm, 1991; Stamler et al., 1992), spermine NONOate has the advantage of releasing NO spontaneously at a predictable rate once in aqueous solution (Maragos et al., 1991). The release of NO does not require cofactors, electron transfer or metabolic activation (Hrabie et al., 1993). At a temperature of 37°C and pH 7.4, as in the present study, the half-life of spermine NONOate is 39 min. The decomposition of one molecule of the complex results in two molecules of NO, but also one molecule of free spermine, a polyamine with several biochemical effects, including hypotensive activity. To exclude any relaxing effects of spermine, this substance was administered at 10–5 mmol/L. The addition of spermine did not result in any change in contractile activity, supporting the notion that the observed effects by spermine NONOate were due to NO.
Initially, the effects of NO were thought to be solely mediated via activation of soluble guanylate cyclase, thereby increasing the amount of cGMP. However, recent studies have demonstrated that NO also may exert its effects via non-cGMP-dependent pathways. Activation of calcium dependent potassium channels as well as direct effects on cyclo-oxygenase enzymes may both cause relaxation of smooth muscle (Bolotina et al., 1994; Salvemini, 1997). In the present study the concentrations of 8-bromo cGMP required to influence tubal contractility were higher than that of nitroglycerin and spermine NONOate. These results indicate that the NO mediated effects may be only partially cGMP-dependent. Similar observations were done when myometrial tissue specimens obtained from labouring and non-labouring women at term were examined in organ baths (Ekerhovd et al, 1999). Further studies are needed to elucidate the possible existence of second messengers of NO other than cGMP.
Acknowledgments
This study was supported by grants from the Göteborg Medical Society and Handlanden Hjalmar Svenssons Foundation. We are also grateful to Histocenter AB for helping us with the immunohistochemical work.
Notes
1 To whom correspondence should be addressed 
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Submitted on May 10, 1999; accepted on August 10, 1999.
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