Important differences in nitric oxide synthase activity and predominant isoform in reproductive tissues from human and rat

Abstract

For the extrapolation of data obtained from experimental animals to the human situation, it is important to know the similarities and differences between human and animal species. Some important characteristics of nitric oxide synthase (NOS) in myometrium and vagina from human and rat were compared. NOS-activity was measured by the formation of 14C-citrulline from 14C-arginine and the expression of NOS isoforms was examined by Western blotting. NOS activity in human uterus and vagina was significantly lower than in the tissues from rat. In contrast to the rat where NOS activity was predominantly found in the cytosolic fractions, NOS activity in particulate and cytosolic fractions from both human myometrium and vagina was similar. Data from Western blots confirmed that eNOS and nNOS isoforms were concentrated in the particulate and cytosolic fractions, respectively. Estrogen treatment of rats resulted in a down regulation of uterine cytosolic NOS activity. A down regulation of NOS in the cytosolic fraction was also seen in the human pregnant myometrium as compared with the nonpregnant myometrium. The vaginal NOS activity was considerably higher than the uterus in both species. In spite of some clear-cut qualitative and other differences between human and rat tissues, there are some interesting similarities. Downregulation in pregnancy of human uterine NOS is probably due to, at least in part, the influence of estrogen and progesterone.

Figure 1

NOS activity in cytosolic (hatched bars), particulate (solid bars) and Ca-independent (empty bars), fractions isolated from non-pregnant and pregnant human myometrium. Values are means ± SEM. Significance of differences between non-pregnant and pregnant myometrium is shown by *(p < 0.05) and ***(p < 0.005).

Figure 2

NOS activity in cytosolic (hatched bars) and particulate (solid bars) from non-pregnant and pregnant fractions isolated from human pregnant myometrium and from ovariectomised control (Ovx) and estrogen treated (Est) rat myometrium. Values are means ± SEM. Significance of differences between non-pregnant human and ovariectomized rats is shown by (p < 0.01), and between pregnant human and estrogenized rat is shown by (p < 0.005).

Figure 3

NOS activity in cytosolic (hatched bars) and particulate (solid bars) fractions isolated from non-pregnant human myometrium (Myo) and vagina (Vag), and rat ovariectomised rat myometrium and vagina. Values are means ± SEM. Significance of differences between human and rat tissues is shown by **(p < 0.01) and ***(p < 0.005).

Figure 4

Western blots of particulate (P) and cytosolic (C) fractions isolated from non-pregnant human myometrium and vagina (a) and ovariectomised rat myometrium and vagina (b) using an antibody directed against eNOS. The putative bands are seen at 133 kDa corresponding to eNOS protein, for which human placental villi (Pl villi) were used as a positive control.

Figure 5

Western blots of particulate (P) and cytosolic (C) fractions isolated from human (a) and rat (b) tissues using an antibody directed against nNOS. The bands are seen at 161 kDa corresponding to eNOS protein, for which rat cerebral cortex was used as a positive control.