RESULTS

CD38 Expression in Rat Uterine Smooth Muscle During Gestation

CD38 mRNA expression was determined in myometrium obtained from preterm and term rats. In uterine smooth muscle obtained from term rats, there was increased CD38 mRNA expression compared with smooth muscle from preterm rats (Fig. 1A). Radiometric analysis of the intensity of the bands showed that samples from term rats had approximately 1.5-fold higher CD38 mRNA expression compared with samples from preterm rats. The intensity of CD38 bands was normalized using corresponding p-actin bands. Real-time PCR of RNA extracted from myometrium of term rats confirmed increased CD38 mRNA expression. The mean Ct values were 25.8 ± 0.4 and 26.7 ± 0.1, respectively, for samples from term and preterm rats.

CD38 protein expression was determined in the micro-somes obtained from preterm and term rats. Densitometric analysis showed that there was approximately a 2.5-fold greater CD38 protein expression in microsomes from term rats than from preterm rats (Fig. 1B).

ADP-Ribosyl Cyclase and cADPR Hydrolase Activities in the Rat Myometrium During Gestation

CD38 is a bifunctional protein and has both ADP-ribosyl cyclase and cADPR hydrolase activities. ADP-ribosyl cyclase activity measured in microsomes obtained from term rats was significantly higher than in microsomes from preterm rats (Fig. 2A). This difference in activity was approximately 2-fold and consistent with increased CD38 expression. However, the cADPR hydrolase activity measured in microsomes from the two groups of rats showed no significant difference (Fig. 2B). No significant differences in the enzyme activities were observed between the samples from Days 14 and 17 of gestation.

cADPR Levels in the Rat Myometrium During Gestation

We measured cADPR levels in myometrial samples obtained from preterm and term rats using the cycling assay.

In myometrium samples obtained from term rats, cADPR levels were significantly (P < 0.05) higher than in myometrium from preterm rats. The levels of cADPR were 1056 and 448 fmoles per milligram protein in myometrium obtained from term and preterm rats, respectively (Fig. 2C).

Effect of Estrogen and Progesterone on CD38 Expression and Function

The differential regulation of ADP-ribosyl cyclase and cADPR hydrolase activities seen in myometrium from term rats was similar to estrogen effects reported in a previous study. We extended these studies in ovariectomized rats to examine the effect of different doses of progesterone on CD38 expression and the enzyme activities in uterine smooth muscle. Consistent with our previous study, estrogen resulted in increased CD38 protein expression. Estrogen-induced CD38 protein expression was unaffected by coadministration of 1 mg progesterone (Fig. 3). CD38 protein expression was lower in uterine microsomes obtained from rats treated with estrogen and either 10 or 20 mg progesterone (EP10 and EP20) than in microsomes from estrogen-treated rats (Fig. 3). Progesterone treatment alone had no significant effects on basal CD38 protein expression (data not shown).

ADP-ribosyl cyclase activity, measured as the conversion of NGD to cGDPR, in microsomes obtained from rats in E and EP1 groups was significantly higher than in mi-crosomes obtained from the other groups (C, EP10, EP20) (Fig. 4A). Progesterone treatment alone had no significant effects on basal ADP-ribosyl cyclase activity in rat uterine smooth muscle (data not shown). To further confirm the effects of progesterone, ADP-ribosyl cyclase activity was also measured by the calcium release assay using 3-deaza-NAD and uterine microsomes obtained from the five groups of rats (C, E, EP1, EP10, and EP20). The magnitude of calcium release from sea urchin egg homogenates confirmed the results obtained using the NGD assay (data not shown). The cADPR hydrolase activities in microsomes obtained from the five groups of rats were not statistically different from each other (P > 0.05; Fig. 4B).
Fig1Changes in-1
FIG. 1. Changes in CD38 mRNA and protein expression in rat uterine smooth muscle. A) CD38 mRNA expression was measured in both preterm and term-rat uterine smooth muscle. Reverse transcription-PCR was done as described in Materials and Methods. Expression of p-actin from the same samples was used as an internal control. A 100-bp DNA ladder (L) was used to detect size of the bands. Note an increase in the expression of CD38 mRNA in uterine smooth muscle at term compared with that at preterm. Three preterm and three term animals were used for studies of CD38 mRNA expression (n = 3). B) CD38 protein expression was measured in both preterm and term uterine smooth muscle samples by Western blot analysis using a goat-polyclonal anti-rat CD38 antibody. Microsomes obtained from rat heart were used as a positive control. Each sample represents the microsomes obtained from different animals and the data shown is a representative blot from five different animals from each group. The lower panel depicts the densitometric analysis of CD38 expression in uterine smooth muscle. Note an increased expression of CD38 protein in the microsomes obtained from term-rat uteri compared with that obtained from preterm-rat uteri (n = 5).

Fig2Changes in-2
FIG. 2. Changes in the activities of ADP-ribosyl cyclase and cADPR hydrolase and levels of cADPR in preterm- and term-rat uterine smooth muscle. The n represents an individual animal. Assays to measure specific enzyme activities and cADPR levels are described in Materials and Methods. A) Specific activity of ADP-ribosyl cyclase (n = 12-16). Note a significant (★ P < 0.05) increase in ADP-ribosyl cyclase activity in the uterine smooth muscle microsomes obtained from term rats compared with that from preterm rats. B) Specific activity of cADPR hydrolase (n = 13-18). Note no significant change in cADPR hydrolase activity in the uterine smooth muscle microsomes obtained from term rats compared with that from preterm rats. C) The cADPR levels. Note significantly (★ P < 0.05) higher cADPR levels in the uterine smooth muscle obtained from term rats compared with that from preterm rats (n = 5-6).

Fig3Changes in-3
FIG. 3. Effects of progesterone on estrogen-induced CD38 protein expression in uterine smooth muscle obtained from ovariectomized rats. Rats were injected with sesame oil (C), 200 ^g/kg estrogen (E), 200 ^g/kg estrogen and 1 mg progesterone (EP1), 200 ^g/kg estrogen and 10 mg progesterone (EP10), or 200 ^g/kg estrogen and 20 mg progesterone (EP20) for 5 days. Data shown are a representative Western blot, repeated using smooth muscle microsomes obtained from at least three different samples. For the control and progesterone-treated groups, myometrium from two rats were pooled for the preparation of microsomes. The n refers to one preparation (see Materials and Methods for details). The lower panel depicts the densitometric analysis of CD38 bands. Note an increase in the expression of CD38 in the rat uterine smooth muscle upon treatment with estrogen, which was attenuated by 10 or 20 mg, but not 1 mg, progesterone (n = 3-5).

Fig4Changes in-4
FIG. 4. ADP-ribosyl cyclase and cADPR hydrolase activities in uterine smooth muscle obtained from the five groups of ovariectomized rats, as described for Figure 3. See MaterialsandMethods section for explanation of n. A) ADP-ribosyl cyclase activity was measured by using NGD assay as described in Materials and Methods. Note a significant (★ P < 0.05) increase in the activity of ADP-ribosyl cyclase in the rat uterine smooth muscle upon treatment with estrogen, which was attenuated by 10 or 20 mg, but not 1 mg, progesterone (n = 3-6). B) The cADPR hydrolase assay was done as described in Materials and Methods. Note no significant changes (P > 0.05) in cADPR hydrolase activity in uterine smooth muscle among the groups (n = 5).