Methods

Materials.

5-[1,2-³H(N)]-Hydroxytryptamine, creatinine sulfate (sp. act. 27.6 Ci/mmol) was purchased from New England Nuclear Corp. (Boston, MA). Oxytocin was a commercial oxytocin distributed by Sandoz Laboratories in 5 UI/ml solution. 6-Nitroquipazine was purchased from RBI (Natick, MA). The other compounds were purchased from Sigma Chemical Co. (St. Louis, MO). DesGly⁹,d(CH₂)₅[Tyr(Me)²Thr⁴] (OVT₁₆) was a gift from Dr. M. Manning, Medical School of Toledo, OH). Rabbit polyclonal serum anti-5HT was purchased from Incstar Corp. (Stillwater, MN). The second antibody and the PAP complexes were obtained from Dako Corp. (Carpinterı́a, CA).

The RBS used in these experiments had the following composition (in mM): NaCl, 153; KCl, 5.3; MgCl₂, 1.8; NaHCO₃24.9; CaCl₂ 2; ascorbic acid 1.2 and glucose 2.7. It was bubbled with 95% O₂ and 5% CO₂. Na-free buffer was made by iso-osmotic substitution with LiCl. High K⁺ (100 mM) was made by iso-osmotic substitution of NaCl with KCl.

Animals.

Female albino mice, weighing 30 to 40 g, maintained under a dark/light cycle (12 hr dark/12 hr light) in a controlled temperature room (24–25°C) with free access to drinking water and laboratory food were used. The stage of the estrous cycle of the animal was determined through a cytochemical analysis of a vaginal smear and confirmed by both physical and anatomical aspects of the uterine horns once dissected. In addition ovariectomized mice were used, they were treated with progesterone as described (Montesinoet al., 1995).

Sympathectomy of mouse uterine horns with 6-OHDA.

A total of 50 μl of an l-ascorbic acid (10 mM) solution containing 5 mg of 6-OHDA was injected into the uterus through a dorsal incision made under anesthesia with pentobarbital (100 mg/kg). Control mice were injected with an equal volume of 10 mM of l-ascorbic acid. Animals were sacrificed 48 hr after 6-OHDA (Donoso et al., 1992).

Serotonin uptake measurements.

Uterine horns from mice in estrus, diestrus and ovariectomized-treated with progesterone were dissected, fragmented into two pieces, weighed and mounted on a support. The time course of the 5HT uptake was determined by incubating each uterine horn fragment (20–25 mg) at different periods in beakers containing 2 ml of RBS without Ca⁺⁺ at 30°C in which [³H]5HT (38 nM) was added. For estimating the kinetic parameters of 5HT uptake, seven concentrations of [³H]5HT were used, ranging from 25 to 240 nM, using a 30-min incubation period. Corresponding incubations were conducted in a Na⁺-free medium to correct for nonspecific uptake. Radioactivity not incorporated by the tissue was washed off by further incubation for 10 min in 100 ml of RBS. The tissue was homogenized in 2 ml of 10% perchloric acid at the end of the incubation. The resulting suspension was centrifuged at 600 × g for 5 min and 0.1 ml of the supernatant was analyzed for radioactivity.

Samples of tissue suspensions were lyophilized, redissolved in acetic acid and analyzed by paper chromatography to determine the proportion of radioactivity that remained as intact 5HT after incubations (Verbeuren et al., 1987).

Effect of oxytocin on 5HT uptake into uterine horns.

Horn fragments (30–40 mg) were incubated as indicated above to evaluate the inhibitory effect of oxytocin, in uterine horns from mice in estrus and in diestrus. To evaluate the magnitude of this inhibitory effect, different concentrations of oxytocin were used ranging from 0.1 fM to 30 nM, although [³H]5HT concentration was kept constant at 38 nM. Results were expressed as the ratio of the values of the [³H]5HT taken up in uterine fragments treated with and without oxytocin. A similar protocol was followed to evaluate the action of vasopressin. Concentration-response curves for inhibiting 5HT uptake into uterine horns were developed for imipramine, fluoxetine and 6-nitroquipazine to evaluate IC₅₀. A single concentration was tested for some other endogenous compounds, as indicated in the results. These experiments were done in triplicate by incubating tissue fragments in 2 ml of RBS without Ca⁺⁺ at 30°C for 40 min with [³H]5HT (38 nM) and a high concentration of the inhibitor as indicated. Results were corrected for nonspecific uptake as described.

Immunocytochemistry of 5HT.

Both native and 5HT (38 nM) incubated tissues were used for immunocytochemistry. The tissue was fixed in a 4% paraformaldehyde-phosphate buffer (pH 7.4) at 4°C. The PAP method was used with rabbit polyclonal serum anti-5HT, 1:500 dilution. The second antibody (goat anti-rabbit immunoglobulin, whole serum) was used at a 1:25 dilution in a Tris buffer, pH 7.8, containing 0.7% λ-carrageenan and 0.25% Triton X-100. The negative controls were performed by omitting the first antibody in the incubation and replacing it with normal serum at a dilution of 1:100. They did not show any positive reaction. Mast cells were stained with 0.1% toluidine blue in 0.7 mol/liter HCl for 15 min and counterstained with 0.01% fast green and picrosyrius 0.1%.

Serotonin release studies.

To study the basal and evoked³H outflow from [³H]5HT previously taken up by the tissue, the horns were incubated in a beaker containing [³H]5HT (38 nM) in 4 ml of RBS without Ca⁺⁺at 30°C for 40 min. Then the tissue was washed with 100 ml of the RBS and placed into beakers containing 2 ml of RBS and sequentially transferred after 1 min of incubation into a series of nine beakers. The stimulus to evoke ³H outflow (10 μg/ml Compound 48/80, 100 mM K⁺ or 20 μM veratridine) was applied when the tissue was in beaker no. 6. At the end of perfusion, uterine horns were weighed and homogenized in 2 ml of 10% perchloric acid. The resulting suspension was centrifuged at 600 × g for 5 min. Aliquots from each beaker and from the supernatant were analyzed for ³H. ³H outflow was calculated for every beaker as a percentage of fractional release, i.e., the percentage of the total amount of ³H remaining in the uterine horn, to reduce the error due to metabolization of [³H]5HT (Hughes and Roth, 1971). Basal outflow was monitored in beakers 4 and 5. The stimulus-evoked release was calculated as the net fractional³H outflow above basal levels from the application of the stimulus.

Contraction studies in mouse uterine horns.

The objective of this experiment was to evaluate the indirect effect of oxytocin on uterine contractility that could be masked by the direct one, due to the presence of oxytocin receptors in smooth muscle myometrial cells. Therefore, 6-nitroquipazine, a specific inhibitor of the 5HT transporter (Hashimoto and Goromaru, 1990) was used instead of oxytocin. Longitudinal pieces of uterine horns of mice in estrus were mounted vertically into an isolated organ bath containing oxygenated RBS at 35°C, as described (Rudolph et al., 1992). After a stabilization period, increasing concentrations of 5HT were added in a nonaccumulative protocol. The evoked contractions were recorded on a Grass model 7 polygraph. The effect of 6-nitroquipazine on the contractile action evoked by 5-HT, was studied by pretreating the uterine horns with the drug for 15 min before 5-HT was added.

Data analysis.

The Km_app value, which represents the concentration of the 5HT giving half-maximal uptake, and Vmax, which is the maximal entry rate, were determined on the basis of different experiments in which specific uptake was measured in function of different concentrations of [³H]5HT. The results were expressed by using a Lineweaver and Burk plot and linear regression analysis. Data are expressed as means ± S.E.M. of a number of different experiments (n). The difference was assessed by using a t test or analysis of variance. The level of significance was 0.05 in each comparison.