Pandu R. Gangula

Nitric oxide donor alleviates ovariectomy-induced bone loss

Nitric oxide (NO) has been reported to inhibit osteoclastic bone resorption. We examined the bone-sparing effect of NO after 6 weeks of administration into estrogen-deficient rats. 30 female Sprague-Dawley rats, 12 weeks of age, underwent ovariectomy (OVX), and 5 rats were sham-operated. OVX rats were assigned to six groups (n = 5/group) treated respectively with: vehicle; 17-beta-estradiol (E2); nitroglycerine (NG, NO donor); NG-nitro-L-arginine methyl ester (L-NAME, NO synthase inhibitor); combination of E2 + NG; and a combination of E2 + L-NAME. Prior to treatment and at the end of the treatment period, bone mineral density (BMD) of rats was measured by dual-energy X-ray absorptiometry (DXA) scanning. OVX animals had significantly lower BMD and femur weights in comparison to sham operated rats (p < 0.01), and this was completely prevented by the administration of E2 (p < 0.01). Administration of NG alone prevented OVX-induced bone loss (p < 0.05). The combination of E2 + NG did not further enhance the bone mass or femur weight, and the OVX-induced bone loss was not further aggravated by L-NAME. However, in the presence of L-NAME, E2 was totally ineffective in reversing the bone loss, suggesting that the protective effect of estrogens against bone loss may be mediated through NO. In summary, the results suggest that NO counteracts the bone loss associated with OVX.

Increased Blood Pressure in α-Calcitonin Gene–Related Peptide/Calcitonin Gene Knockout Mice

Abstract —Nerves that contain calcitonin gene–related peptide (CGRP) are components of the sensory nervous system. Although these afferent nerves have traditionally been thought to sense stimuli in the periphery and transmit the information centrally, they also have an efferent vasodilator function. Acute administration of a CGRP receptor antagonist increases the blood pressure (BP) in several models of hypertension, which indicates that this potent vasodilator plays a counterregulatory role to attenuate the BP increase in these settings. To determine the role of this peptide in the long-term regulation of cardiovascular function, including hypertension, we obtained mice that have a deletion of the α-calcitonin gene–related peptide (α- CGRP ) gene. Although the β-calcitonin gene–related peptide (β- CGRP ) gene is intact in these mice, α- CGRP is by far the predominant species of CGRP produced in dorsal root ganglia (DRG) sensory neurons. Initially, we examined the effect of deletion of the α- CGRP on baseline BP and β- CGRP and substance P mRNA expression. Systolic BP was significantly higher in the knockout mice (n=7) compared with wild-type in both male (160±6.1 vs 125±4.8 mm Hg) and female (163±4.8 vs 135±33 mm Hg) mice. Next, groups (n=7) of knockout and wild-type mice had catheters surgically placed in the right carotid artery for mean arterial pressure recording. With the animals fully awake and unrestrained, the knockout mice displayed an elevated mean arterial pressure compared with wild-type in both male (139±4.9 vs 118±4.9 mm Hg) and female (121±3.4 vs 107±3.1 mm Hg) mice. Northern blot analysis of DRG RNA samples confirmed the absence of α- CGRP mRNA in the knockout mice. Substance P mRNA content in DRG was unchanged between the 2 groups; however, β- CGRP mRNA levels were reduced 2-fold in the knockout mice. These results indicate for the first time that α- CGRP may be involved in the long-term regulation of resting BP and suggest that these mice are particularly sensitive to challenges to BP homeostasis because of the loss of a compensatory vasodilator mechanism.

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.

Role and Regulation of Nitric Oxide in the Uterus During Pregnancy and Parturition

Objective: To review the English-language literature as it relates to the role and regulation of uterine nitric oxide (NO) during pregnancy and parturition, special emphasis being placed on the interactions of NO with other uterotonic agents. Methods: A thorough literature review of the English-language literature using a Medline search was performed. Results: Current data support the view that NO is generated in the uterus and inhibits uterine contractility. Gestation, parturition, steroid hormones, and prostaglandins modulate both the generation and the effects of NO on the uterus. The changes in NO and its effects are consistent with the theory that NO plays a role in uterine quiescence during pregnancy. A change in this system at term or preterm could play a role in inhibition of labor and delivery. Conclusion: Uterine NO may play a role in maintaining uterine quiescence during pregnancy.

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.

Active Invasion of Oral and Aortic Tissues by Porphyromonas gingivalis in Mice Causally Links Periodontitis and Atherosclerosis

Atherosclerotic vascular disease is a leading cause of myocardial infarction and cerebrovascular accident, and independent associations with periodontal disease (PD) are reported. PD is caused by polymicrobial infections and aggressive immune responses. Genomic DNA of Porphyromonas gingivalis, the best-studied bacterial pathogen associated with severe PD, is detected within atherosclerotic plaque. We examined causal relationships between chronic P. gingivalis oral infection, PD, and atherosclerosis in hyperlipidemic ApoEnull mice. ApoEnull mice (n = 24) were orally infected with P. gingivalis for 12 and 24 weeks. PD was assessed by standard clinical measurements while the aorta was examined for atherosclerotic lesions and inflammatory markers by array. Systemic inflammatory markers serum amyloid A, nitric oxide, and oxidized low-density lipoprotein were analyzed. P. gingivalis infection elicited specific antibodies and alveolar bone loss. Fluorescent in situ hybridization detected viable P. gingivalis within oral epithelium and aorta, and genomic DNA was detected within systemic organs. Aortic plaque area was significantly increased in P. gingivalis-infected mice at 24 weeks (P<0.01). Aortic RNA and protein arrays indicated a strong Th2 response. Chronic oral infection with P. gingivalis results in a specific immune response, significant increases in oral bone resorption, aortic inflammation, viable bacteria in oral epithelium and aorta, and plaque development.