Chandrasekhar Yallampalli

Inhibition of nitric oxide synthesis in rats during pregnancy produces signs similar to those of preeclampsia

OBJECTIVES Preeclampsia is associated with hypertension, fetal growth retardation, and proteinuria. We hypothesized that impaired vascular nitric oxide synthesis during pregnancy may be an important causal factor in preeclampsia. STUDY DESIGN An inhibitor of nitric oxide synthase, L-nitro-arginine methyl ester, or a nitric oxide donor, nitroglycerin, was infused subcutaneously to rats at a constant rate from day 17 of gestation. Systolic blood pressure, day of spontaneous delivery, weight, and mortality rate of pups were recorded. RESULTS Systolic blood pressures in rats infused with L-nitro-arginine methyl ester at daily doses of both 25 and 50 mg were significantly elevated compared with controls. This treatment also caused a substantial decrease in the weight of pups, with an increase in mortality rate, without affecting the gestational length. These effects were dose dependent. Nitroglycerin infusion, on the other hand, affected neither the weight and mortality rate of the pups nor the length of gestation. CONCLUSIONS Infusion of an inhibitor of nitric oxide synthesis during pregnancy causes hypertension and fetal growth retardation, without affecting gestational length. These signs are similar to those of preeclampsia and indicate that an alteration in nitric oxide synthesis may be one of the factors responsible for this disorder. Treatment with nitric oxide inhibitors may be used in an animal model for preeclampsia, to test various therapeutic strategies.

Involvement of a nitric oxide-cyclic guanosine monophosphate pathway in control of human uterine contractility during pregnancy☆

OBJECTIVES The aims of the study were to investigate whether a nitric oxide-cyclic guanosine monophosphate relaxation pathway is present in the human uterus and whether it differentially inhibits contractility during pregnancy and labor. STUDY DESIGN Myometrial strips were obtained from pregnant women who were either in labor or not in labor and from nonpregnant women. Nitrites and cyclic guanosine monophosphate production by the tissues and contractile responses to nitric oxide modifiers were measured. RESULTS Biochemical assays revealed that nitric oxide (nitrites) and cyclic guanosine monophosphate are generated by the human uterus. Cyclic guanosine monophosphate production by the uterus was increased by L-arginine (the substrate for nitric oxide) and diethylamine/nitric oxide (a nitric oxide donor) and decreased by nitro-L-arginine methyl ester (an inhibitor of nitric oxide synthase). Spontaneous contractility in vitro was increased by nitro-L-arginine methyl ester and decreased by diethylamine/nitric oxide, which furthermore produced a dose-dependent inhibition of contractility, and the median effective dose of inhibition in tissues from nonlaboring pregnant patients (1.5 +/- 0.4 mumol/L) is substantially lower than in tissues from laboring pregnant (21.7 +/- 7.4 mumol/L or nonpregnant (20.8 +/- 4.4 mumol/L) women. These studies show that the nitric oxide-cyclic guanosine monophosphate system exists in the human uterus and that it inhibits contractility. Furthermore, the relaxation responsiveness to nitric oxide is elevated during pregnancy and decreased during labor. CONCLUSION A nitric oxide-cyclic guanosine monophosphate relaxation pathway is present in the human uterus and may be responsible for maintaining uterine quiescence during pregnancy. A decrease in uterine relaxation responsiveness to nitric oxide at term may play a role in the initiation of labor.

Gestational changes in L-arginine-induced relaxation of pregnant rat and human myometrial smooth muscle

OBJECTIVE We intended to demonstrate the presence of an L-arginine-nitric oxide system in human myometrium and to clarify the mechanisms of action of nitric oxide on rat myometrium during gestation. STUDY DESIGN By examining very small myometrial muscle strips (approximately 750 muscle cells), characteristic features of contraction of rat longitudinal muscle at the midstage of gestation (day 16) and during delivery at term were determined. RESULTS Spontaneous contractions were significantly different during delivery compared with the midstage of gestation of rat myometrium. L-Arginine relaxed spontaneous and carbachol-induced, but not potassium chloride-evoked, contractions at both stages. However, much higher concentrations of L-arginine were required during delivery, 8-Bromo-cyclic guanosine monophosphate inhibited spontaneous contractions from concentrations of 1 nmol/L in the midstage of gestation and from 0.1 mmol/L during delivery. In human myometrial tissues L-arginine also inhibited contractions during the late stages of gestation. CONCLUSION (1) The experimental model is sufficient to compare properties of longitudinal myometrial strips during gestation. (2) In rat and human myometrium an L-arginine-nitric oxide system has an important role in inhibiting uterine contractility and possibly maintaining pregnancy. (3) The relaxing effect of the nitric oxide system is largely because of the voltage-independent action of cyclic guanosine monophosphate systems.

Differential expression of cyclooxygenase-1 and -2 proteins in rat uterus and cervix during the estrous cycle, pregnancy, labor and in myometrial cells

The synthesis of prostaglandins in the uterus at term are modulated by two isoforms of the enzyme cyclooxygenase (COX): constitutive COX-1 and inducible COX-2. This study aims to characterize the expression of the protein for COX-1 and -2 in the rat uterus and cervix during the estrous cycle, pregnancy, and labor, and in cultured myometrial cells. Western immunoblotting of proteins was performed and quantitation of protein was obtained densitometrically. Results indicate: 1) the rat uteri, cervix, and isolated myometrial cells express both COX-1 and COX-2 proteins, 2) during pregnancy, both COX-1 and -2 increase, with a dramatic increase at parturition (250%-280%), 3) a 2-fold increase of cervical COX-2 is seen at spontaneous labor, 4) during proestrus and estrus, uterine expression of COX-2 is elevated, 5) both COX-1 and -2 were expressed by rat myometrial cells and treatment with IL-1 beta (10 ng/mL) produced a significant increase in COX-2, and 6) immunocytochemical studies show that both COX-1 and -2 were primarily localized to the epithelial cells of the endometrium and smooth muscle cells in the circular layers of the myometrium in the uterus and to the epithelial cells and smooth muscle cells in the cervix. Thus, we propose that increased expression of COX-2 may be involved at term in increased uterine contractility and cervical ripening.

Regulation of Calcitonin Gene-Related Peptide Expression in Dorsal Root Ganglia of Rats by Female Sex Steroid Hormones

Abstract Calcitonin gene-related peptide (CGRP), a potent vasodilator primarily synthesized in dorsal root ganglia (DRG) neurons, has been shown to decrease vascular resistance and thus regulate blood flow to a variety of organs in rats. Serum CGRP levels in the human have been reported to increase with pregnancy and decrease postpartum. It has been suggested that female sex steroid hormones play a role in cardiovascular function, but the mechanisms are unknown. In this study, we examined the effects of estradiol-17β (E2) and progesterone (P4) on the expression of CGRP in DRG in adult rats both in vivo and in vitro. Ovariectomized (ovx) animals were injected s.c. with 5 μg E2, 4 mg P4, or 5.0 μg E2 + 4 mg P4 in 0.5 ml sesame oil or with oil only, and groups of 4 rats were killed at 0, 24, or 48 h. DRGs were then removed and analyzed for CGRP mRNA and immunoreactive (i-)CGRP content by Northern blotting and RIA, respectively. Primary cultures of DRG neurons from adult female rats were used to assess the effects of varying doses of E2 (1, 10, 100 nM), P4 (10, 100, 1000 nM), or E2 (10 nM) + P4 (100 nM) in the absence or presence of nerve growth factor (NGF; 20 ng/ml); and CGRP mRNA content in the cells and i-CGRP in the medium were quantitated at 24 or 48 h after incubation. Results of in vivo studies showed that E2 caused a significant increase in CGRP mRNA at 24 h (1.8-fold) and in i-CGRP levels both at 24 h (2.8-fold) and at 48 h (3.4-fold) in DRG of ovx rats. P4 also stimulated expression of both CGRP mRNA and i-CGRP. In the in vitro studies, either E2 or P4 alone or the two in combination were without effect on CGRP expression in cultured DRG neurons at all the doses tested. However, in the presence of NGF, both CGRP mRNA and peptide levels were significantly enhanced by E2, P4, and E2+P4 in a time-dependent (2.0- to 2.8-fold at 24 h, 3.0- to 5.0-fold at 48 h) and dose-dependent manner, with maximal effects achieved at 1.0 nM (E2) and 100 nM (P4) at 24 h of incubation. In summary, both E2 and P4, either alone or in combination, stimulate CGRP peptide synthesis in DRG neurons through increasing CGRP mRNA. The effects of these steroid hormones are mediated through amplifying the NGF-induced synthesis of CGRP in these neurons. Thus, we propose that the cardiovascular functions of female sex steroid hormones may be mediated, at least in part, by the up-regulation of neuronal CGRP synthesis, via NGF-mediated mechanisms.

Immunocytochemical localization of nitric oxide synthase-III in reproductive organs of female rats during the oestrous cycle.

SummaryConstitutive endothelial nitric oxide synthase (NOS III) expression during the oestrous cycle was mapped immunocytochemically on 5 μm-thick paraffin sections of rat female reproductive organs. Ovarian NOS III immunoreactivity increased with follicular maturation (strongest in dioestrus corpora lutea), suggesting that nitric oxide may regulate folliculogenesis and luteal functions. Oviductal NOS III, localized in mucosal epithelium and muscular wall, was maximal during pro-oestrus and oestrus, suggesting that nitric oxide may impart periovulatory quiescence for reception, retention and fertilization of ovulated oocytes. Uterine NOS III, localized in endometrial and glandular epithelium, and in myometrial smooth muscle cells, was abundantly expressed during pro-oestrus and oestrus. The peri-implantation period in pregnant rats corresponds to the periovulatory period and the elevated NOS, and thus nitric oxide may provide uterine relaxation to facilitate embryo implantation following fertilization. Cervical NOS III, localized in the mucus-secreting epithelium and smooth muscle cells, exhibited enzyme abundance during pro-oestrus and oestrus, probably indicating cervical preparation to facilitate sperm entry following mating. Vaginal NOS III, found in the stratified squamous epithelial lining and in smooth muscle cells, was maximal during oestrus and pro-oestrus, suggesting that nitric oxide may stimulate vaginal secretions. Differential expression of NOS III by different reproductive organs during the oestrus cycle suggests a role for nitric oxide in modulating reproduction.

Female Steroid Hormones Modulate Receptors for Nerve Growth Factor in Rat Dorsal Root Ganglia

Abstract Calcitonin gene-related peptide (CGRP) is a vasodilatory peptide, and it is primarily synthesized in dorsal root ganglia (DRG). Plasma CGRP levels increase during pregnancy and with steroid hormones, and nerve growth factor (NGF) stimulates calcitonin/CGRP promoter and CGRP synthesis in DRG. We previously showed that CGRP levels in DRG were stimulated with steroid hormone treatments in vivo but not in vitro. Thus, the stimulation of CGRP by these hormones may be indirect through the upregulation of NGF effects. We hypothesized that the female sex steroid hormones upregulate NGF receptors, trkA and p75NTR, in DRG. We examined the effects of 17β-estradiol (E2) and progesterone (P4) on NGF receptors in DRG obtained from ovariectomized (ovx) rats. Groups of 4 ovx rats were injected s.c. with 5 μg E2, 4 mg P4, or 5 μg E2 + 4 mg P4 in 0.2 ml sesame oil or injected with oil only and were killed at 6, 24, and 48 h. In addition, ovx rats were also injected s.c. with varying doses (0.2, 1.0, 5.0, 25 μg) of E2 (0.5, 1.5, 4, 10 mg) P4, and (5 μg) E2 + (0.5, 1.5, 4.0, 10 mg) P4 in 0.2 ml sesame oil, or vehicle, and killed at 6 (for E2) or 24 (for P4 and E2 + P4) h. Furthermore, groups of ovx rats were also killed at 12 and 24 h; 3 and 7 days; 2, 4, and 6 wk after ovariectomy. The DRGs were collected from all groups and then processed for Western immunoblotting to examine both trkA and p75NTR levels. Estradiol increased trkA at 6 h but not p75NTR. Progesterone caused upregulation of trkA and p75NTR at 6 and 24 h. 17β-Estradiol + P4 increased trkA at 6 and 24 h and p75NTR at all time points examined. One microgram of E2 increased trkA but did not affect p75NTR levels. Progesterone at 4 and 10 mg upregulated trkA but only 10 mg P4 increased p75NTR. Five micrograms of E2 coinjected with P4 at 1.5 and 4 mg increased trkA, while p75NTR receptor was upregulated when coinjected with P4 at 1.5 to 10 mg. The ovariectomy caused a decrease in trkA receptors compared to proestrus rats, and these decreases were significant by 6 wk, but surprisingly p75NTR increased at 2 wk after ovariectomy. 17β-Estradiol increased trkA but not p75NTR receptors in DRG, whereas P4 caused increases in both trkA and p75NTR in DRG. In addition, the combination of these steroid hormones had more effect on both receptors than either hormone alone. Thus, we concluded that high levels of female steroid hormones such as those due to pregnancy or hormonal replacement therapy could increase NGF receptor expression in DRG that carry more NGF to elevate the CGRP synthesis in these groups. We suggested that the regulation of NGF receptors by ovarian steroids may underlie steroidal regulation of other factors such as CGRP.

Involvement of calcitonin gene–related peptide in the modulation of human myometrial contractility during pregnancy

Calcitonin gene-related peptide (CGRP) is a potent vasodilator and relaxes smooth muscle of a variety of tissues, but the effects of CGRP on human myometrial contractions and the changes in CGRP receptors (CGRP-Rs) in human myometrium have not been described. We report that CGRP induced dose-dependent relaxation in spontaneously contracting myometrium from pregnant women. This relaxation effect is diminished in myometrium obtained from patients during labor and in the nonpregnant state. CGRP-induced relaxations are inhibited by a CGRP-R antagonist (CGRP(8-37)), a soluble guanylate cyclase inhibitor (LY(83583)), and a nitric oxide synthase inhibitor (L-NAME). Both Western blotting and mRNA analysis showed that CGRP-Rs are present in human myometrium, and that the expression of these receptors is increased during pregnancy and decreased during term labor. Immunofluorescent staining revealed that CGRP-Rs are abundant in the myometrial cells of pregnant women who are not in labor, and are minimal in uterine specimens from women in labor and in the nonpregnant state. We conclude that increased CGRP-Rs in myometrium, and resulting enhanced myometrial sensitivity to CGRP, may play a role in maintaining human myometrium in a quiescent state during pregnancy, and that a decline in the CGRP-Rs at term could contribute to the initiation of labor.

Calcitonin Gene-Related Peptide Is a Depressor in NG-Nitro-l-Arginine Methyl Ester-Induced Hypertension During Pregnancy

Inhibition of nitric oxide production with NG-nitro-L-arginine methyl ester (L-NAME) increases blood pressure and fetal mortality in pregnant rats. We previously reported that administration of calcitonin gene-related peptide (CGRP) reduces the blood pressure and fetal death produced by L-NAME. To determine the hemodynamic role of endogenous CGRP in this setting, CGRP8-37, a CGRP receptor antagonist, was used. In addition, CGRP mRNA and peptide levels were determined in dorsal root ganglia. L-NAME or control rats had intravenous (for drug administration) and arterial (for continuous mean blood pressure monitoring) catheters surgically placed and were studied in the conscious unrestrained state. Baseline blood pressure was higher in the L-NAME than the control rats on days 19, 20, and 21 or pregnancy and postpartum day 1. Vehicle administration did not change blood pressure in any group, and CGRP8-37 (100 micrograms) did not change blood pressure in control groups. However, CGRP8-37 administration to the L-NAME rats further increased blood pressure (P < .05) on days 19 (8 +/- 1), 20 (12 +/- 2), and 21 (7 +/- 1) of gestation but was without effect on postpartum day 1. Furthermore, CGRP mRNA or peptide levels in dorsal root ganglia were not different between the L-NAME and control rats at any of the time points studied. These data indicate that in experimental preeclampsia, CGRP is playing a compensatory vasodilator role to attenuate the elevated blood pressure. The mechanism of this effect appears to be an enhanced vascular responsiveness to CGRP that is attenuated after the birth of pups.

Calcitonin gene-related peptide in pregnancy and its emerging receptor heterogeneity

Calcitonin gene-related peptide (CGRP) is the most potent vasodilator, and there is a growing body of evidence that this peptide might have multiple other functions. During pregnancy, circulating CGRP levels in rats increase up to the time of delivery, followed by a sharp decline at term and postpartum. In addition, the sensitivity of various vascular beds to CGRP in rats appears to increase with advancing pregnancy. This increased sensitivity might be involved in regulating uteroplacental blood flow, in addition to other vascular adaptations that occur during normal pregnancy. Furthermore, the uterine relaxation response to CGRP is elevated during pregnancy and decreased at term. Sex steroid hormones, estrogens and progesterone, regulate CGRP synthesis and its effects on both myometrial and uterine vascular tissues. These changes in smooth muscle relaxation sensitivity to CGRP appear to be a consequence of changes in CGRP-receptor levels in these tissues. There appear to be two receptors for CGRP: the CGRP-A receptor, a well-characterized receptor consisting of calcitonin receptor-like receptor and receptor activity modifying protein 1, and the CGRP-B receptor. The CGRP system might play a role in the maintenance of normal pregnancy, and a defect in this system might lead to complications.