Periodontal Treatment Reduces Circulating Pro-Inflammatory Cytokine and Chemokine Levels in African American HIV+ Individuals with Virological Suppression.

Introduction: Periodontal Disease (PD), a chronic inflammatory disease, is highly prevalent among Persons Living With HIV (PLWH) and is characterized by microbial symbiosis and oxidative stress. Our hypothesis stipulates that periodontal therapy attenuates systemic inflammatory and bacterial burden while improving periodontal status in PLWH. Methods: Sixteen African Americans (AA) with suppressed HIV viremia on long-term Antiretroviral Therapy (ART) were recruited to this study. Participants were placed into two groups, based on their dental care status: group 1 (In-Care, IC) and group 2 (Out of Care, OC). Periodontal health was investigated at baseline, 3 months, 6 months, and 12 months. Cytokine/chemokines, microbial phyla, and Asymmetric Dimethylarginine (ADMA, a marker for endothelial cell dysfunction) levels were assessed in the serum. Statistical comparisons between groups and at different visits were performed using multiple comparison tests. Results: Across longitudinal visits, periodontal treatment significantly reduced the levels of several cytokines and chemokines. At baseline, the out of care group had significantly higher blood levels of ADMA and actinobacteria than the IC group. Periodontal treatment significantly altered the abundance of circulating genomic bacterial DNA for various phyla in out of care group. Conclusions: Periodontal treatment interventions effectively attenuated circulating pro-inflammatory cytokines and altered microbial translocation, both critical drivers of systemic inflammation in PLWH.

Impairment of Nrf2- and Nitrergic-Mediated Gastrointestinal Motility in an MPTP Mouse Model of Parkinson's Disease.

BACKGROUND: Gastrointestinal (GI) motility dysfunction is the most common non-motor symptom of Parkinson's disease (PD). Studies have indicated that GI motility functions are impaired before the onset of PD. AIMS: To investigate the underlying mechanism of PD-induced GI dysmotility in MPTP (1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine)-induced animal model. METHODS: C57BL/6 mice were administered with or without a selective dopamine neurotoxin, MPTP, to induce parkinsonian symptoms. In addition to in vivo studies, in vitro experiments were also conducted in colon specimens using l-methyl-4-phenylpyridinium (MPP+), a metabolic product of MPTP. Gastric emptying, colon motility, nitrergic relaxation, and western blot experiments were performed as reported. RESULTS: MPTP-induced PD mice showed decreased expression of nuclear factor erythroid 2-related factor (Nrf2) and its target phase II genes in gastric and colon neuromuscular tissues. Decreased levels of tetrahydrobiopterin (BH4, a critical cofactor for nNOS dimerization) associated with uncoupling of nNOS in gastric and colon tissues exposed to MPTP. Impaired enteric nitrergic system led to delayed gastric emptying and slower colonic motility compared to the control mice. In vitro results in colon specimens confirm that activation of Nrf2 restored MPP+-induced suppression of alpha-synuclein, tyrosine hydroxylase (TH), Nrf2, and heme oxygenase-1. In vitro exposure to L-NAME [N(w)-nitro-L-arginine methyl ester], a NOS synthase inhibitor, reduced protein expression of TH in colon tissue homogenates. CONCLUSIONS: Loss of Nrf2/BH4/nNOS expression in PD impairs antioxidant gene expression, which deregulates NO synthesis, thereby contributing to the development of GI dysmotility and constipation. Nitric oxide appears to be important to maintain dopamine synthesis in the colon.

Chronic estrogen deficiency causes gastroparesis by altering neuronal nitric oxide synthase function.

BACKGROUND: Gastroparesis affects predominantly females; however, the biological basis for this gender bias is completely unknown. Several lines of evidence suggest that nitrergic dependent stomach motility function is reduced in diabetic gastroparesis and that nNOS is estrogen-regulated. AIMS: The purpose of this study was to investigate whether reduced levels of estradiol-17ß (E2) down-regulates tetrahydrobiopterin (BH4, a cofactor for nNOS dimerization and enzyme activity) biosynthesis and therefore nNOS mediated gastric motility would be impaired in a mouse model of chronic estrogen deficiency, follicle stimulating hormone receptor knock-out female mice (FORKO). METHODS: In-bred 12-week-old female FORKO mice were obtained from our FORKO breeding colony. Gastric emptying was measured in overnight fasting mice. Nitrergic relaxation (AUC/mg tissue) was measured at 2 Hz through electric field stimulation using gastric antrum strips prepared from WT and FORKO mice. Protein expression for nNOSα, BH4 biosynthesis enzymes (GCH-1, DHFR) and estrogen receptors (α, ß) were measured in gastric antrum by western blotting. Levels of BH4 and oxidized BH2, B biopterin levels were determined by HPLC. RESULTS: In FORKO, compared to wild type (WT) stomachs we indentified (1) reduced (%) gastric emptying (64 ± 2.5 vs. 77.6 ± 0.88), (2) greater reduction in nitregic relaxation (-0.13 ± 0.012 vs. -0.28 ± 0.012), (3) increased nNOS dimerization (0.48 ± 0.02 vs. 0.34 ± 0.05), (4) decreased NO release whether measured at 24 h (0.6 ± 0.04 vs. 1.7 ± 0.22, p < 0.05) or at 48 h (3.4 ± 0.26 vs. 5.0 ± 0.15, p < 0.05) of incubation, (5) decreased GCH-1 (1.9 ± 0.06 vs. 2.3 ± 0.04), DHFR (1.8 ± 0.14 vs. 2.4 ± 0.07) and ERα (2.7 ± 0.4 vs. 3.9 ± 0.4) and (6) increased oxidized biopterin levels and decreased ratio of BH4 versus BH2 + B. CONCLUSION: We conclude that chronic estrogen deficiency negatively modifies the function of both BH4 and nNOS thereby contributing to the development of gastroparesis in a FORKO mouse model.

Gender bias in gastroparesis: is nitric oxide the answer?

Accumulating evidence suggests that gender-related differences are prominent in gastric motility functions in both health and disease. Women are more susceptible to gastroparesis than men. Though the mechanism(s) involved are not fully understood, impairment of the nitrergic system is one of the main factors responsible for the disease. Uncoupling of neuronal nitric oxide synthase (nNOS) causes a decreased synthesis of NO leading to a reduction in smooth muscle relaxation. Tetrahydrobiopterin (BH(4)) (an essential cofactor for nNOS) is a key regulator of nNOS activity for stomach dysfunction and gastroparesis. In addition, BH(4) has been shown to be a potent antioxidant and anti-inflammatory agent. Well established by results from our laboratory, a diminished intracellular (BH(4):total biopterin) ratio in diabetic female rats significantly impairs nNOS activity and function. Recent research has been focused on BH(4) biosynthesis and gastroparesis because reduced BH(4) cofactor levels can alter the production of NO by nNOS. Researchers are now paying more attention to the possibility of using BH(4) as a therapeutic strategy in gastroparesis. The purpose of this review is to provide an overview of the regulation and function of nNOS by sex hormones and BH(4) and its potential role in the treatment of gastroparesis.

Impairment of nitrergic system and delayed gastric emptying in low density lipoprotein receptor deficient female mice.

BACKGROUND: In the current study, we have investigated whether low density lipoprotein receptor knockout mice (LDLR-KO), moderate oxidative stress model and cholesteremia burden display gastroparesis and if so whether nitrergic system is involved in this setting. In addition, we have investigated if sepiapterin (SEP) supplementation attenuated impaired nitrergic system and delayed gastric emptying. METHODS: Gastric emptying and nitrergic relaxation were measured in overnight fasting mice. nNOSα dimerization, anti-oxidant markers such as Nrf2, GCLM, GCLC, HO-1, catalase (CAT), and superoxide dismutase (SOD1) were measured using standard methods. Biopterin levels and intestinal transit time were measured using HPLC and dye migration assay, respectively. Wild type (WT) and LDLR-KO were supplemented with SEP. KEY RESULTS: In LDLR null stomachs: (i) significant reduction in rate of gastric emptying, gastric pyloric and fundus nitrergic relaxation and nNOSα dimerization, (ii) elevated oxidized biopterins and reduced ratio of BH(4) /BH(2) + B, (iii) reduced Nrf2 and GCLC protein expression and no change in GCLM, HO-1, CAT, SOD1, and (iv) accelerated small intestinal motility were noticed. Supplementation of SEP restored delayed gastric emptying, impaired pyloric and fundus nitrergic relaxation with restoration of nNOS dimerization and nNOS expression. CONCLUSIONS & INFERENCES: This novel data suggests that hyperlipidemia and/or suppression of selective antioxidants may be a potential cause of developing gastroparesis in diabetic patients.

Sepiapterin reverses the changes in gastric nNOS dimerization and function in diabetic gastroparesis.

BACKGROUND: We have demonstrated previously that in vivo supplementation of tetrahydrobiopterin (BH4); a co-factor for neuronal nitric oxide synthase (nNOS) significantly restored delayed gastric emptying and attenuated nitrergic relaxation in diabetic rat. In this study, we have investigated whether supplementation of sepiapterin (SEP), a precursor for BH4 biosynthesis via salvage pathway restores gastric emptying and nitrergic system in female diabetic rats. METHODS: Diabetic rats (streptozotocin-induced) were supplemented with BH4 or SEP (20 mg kg⁻¹ body weight). Gastric nitrergic relaxation in the presence or absence of high glucose and SEP were measured by electric field stimulation. Gastric muscular strips from healthy or diabetic female rats were incubated in the presence or absence of high glucose, SEP and/or methotrexate (MTX). Nitric oxide release was measured colorimetrically by NO assay kit. The expression of nNOSα and dimerization was detected by Western blot. KEY RESULTS: In vitro studies on gastric muscular tissues showed that MTX, an inhibitor of BH4 synthesis via salvage pathway, significantly decreased NO release. In vivo treatment with MTX reduced both gastric nitrergic relaxation and nNOSα dimerization. Supplementation of SEP significantly attenuated delayed gastric emptying in diabetic rats. In addition, SEP supplementation restored impaired nitrergic relaxation, gastric nNOSα protein expression, and dimerization in diabetic rats. CONCLUSIONS & INFERENCES: The above data suggests that supplementation of SEP accelerated gastric emptying and attenuated reduced gastric nNOSα expression, and dimerization. Therefore, SEP supplementation is a potential therapeutic option for female patients of diabetic gastroparesis.

Inhibitors of advanced glycation end-products prevent loss of enteric neuronal nitric oxide synthase in diabetic rats.

Gastrointestinal dysfunction is common in diabetes, and several studies indicate that loss of neuronal nitrergic inhibition may play an important role in its pathogenesis. However, the mechanisms responsible for this effect remain largely unknown. We have previously shown that advanced glycation end-products (AGEs) formed by non-enzymatic glycation dependent processes, can inhibit the expression of intestinal neuronal nitric oxide synthase (nNOS) in vitro acting via their receptor, receptor for AGEs. We now hypothesized that this effect may also be important in experimental diabetes in vivo. We aimed to evaluate the role of AGEs on duodenal nNOS expression and the effects of aminoguanidine (a drug that prevents AGE formation) and ALT-711 (AGE cross-link breaker) in experimental diabetes. Streptozotocin induced diabetic rats were randomized to no treatment, treatment with aminoguanidine (1 g L(-1) daily through drinking water) at the induction of diabetes, or treatment with ALT-711 (3 mg kg(-1) intraperitoneally), beginning at week 6. A fourth group was used as healthy controls. We performed real time polymerase chain reaction, Western blotting and immunohistochemistry to detect nNOS expression. AGE levels were analysed using sandwich ELISA. Diabetes enhanced accumulation of AGEs in serum, an effect that was prevented by treatment with aminoguanidine and ALT-711. Further, diabetic rats showed a significant reduction in duodenal nNOS expression by mRNA, protein and immunocytochemistry, an effect that was prevented by aminoguanidine. ALT-711 had similar effects on nNOS protein and immunohistochemistry (but not on mRNA levels). The generation of AGEs in diabetes results in loss of intestinal nNOS expression and may be responsible for enteric dysfunction in this condition. This study suggests that treatment directed against AGEs may be useful for the treatment of gastrointestinal complications of diabetes.

Mesenteric arterial relaxation to calcitonin gene-related peptide is increased during pregnancy and by sex steroid hormones.

The present study investigated whether pregnancy and circulatory ovarian hormones increase the sensitivity of the mesenteric artery to calcitonin gene-related peptide (CGRP)-induced relaxation and possible mechanisms involved in this process. Mesenteric arteries from young adult male rats or female rats (during estrous cycle, after ovariectomy, at Day 20 of gestation, or Postpartum Day 2) were isolated, and the responsiveness of the vessels to CGRP was examined with a small vessel myograph. The CGRP (10(-10) to 10(-7) M) produced a concentration-dependent relaxation of norepinephrine-induced contractions in mesenteric arteries of all groups. Arterial relaxation sensitivity to CGRP was significantly (P < 0.05) greater in female rats compared with male rats. Pregnancy increased the sensitivity to CGRP significantly (P < 0.05) compared to ovariectomized and Postpartum Day 2 rats. In pregnant rats, CGRP-receptor antagonist, CGRP(8-37), inhibited the relaxation responses produced by CGRP. The CGRP-induced relaxation was not affected by N(G)-nitro-l-arginine methyl ester (nitric oxide inhibitor, 10(-4) M) but was significantly (P < 0.05) attenuated by an inhibitor of guanylate cyclase (1H-[1 , 2 , 4 ]oxadizaolo[4 , 3 -a]quinoxalin-1-one, 10(-5) M). Relaxation responses of CGRP on mesenteric arteries were blocked (P < 0.05) by a cAMP-dependent protein kinase A inhibitor, Rp-cAMPs (10(-5) M). The CGRP-induced vasorelaxation was significantly (P < 0.05) attenuated by calcium-dependent (tetraethylammonium, 10(-3) M), but not ATP-sensitive (glybenclamide, 10(-5) M), potassium channel blocker. Therefore, the results of the present study suggest that mesenteric vascular sensitivity to CGRP is higher during pregnancy and that cAMP, cGMP, and calcium-dependent potassium channels appear to be involved. Therefore, we propose that CGRP-mediated vasodilation may be important to maintain vascular adaptations during pregnancy.

Studies on the effects of the N-terminal domain antibodies of calcitonin receptor-like receptor and receptor activity-modifying protein 1 on calcitonin gene-related peptide-induced vasorelaxation in rat uterine artery.

The vascular relaxation sensitivity to calcitonin gene-related peptide (CGRP) is enhanced during pregnancy, compared with nonpregnant human and rat uterine arteries. In the rat uterine artery, two types of CGRP receptors have been shown to coexist, CGRP-A receptor, which is a complex of calcitonin receptor-like receptor (CRLR), and receptor activity-modifying protein (RAMP(1)) and CGRP-B receptor, which is different from CRLR. In the present study, we hypothesized that: 1) CGRP-induced vasorelaxation in rat uterine artery is mediated through CGRP-A receptor and 2) N-terminal (Nt) domain of CRLR (Nt-CRLR) has a major contribution in ligand binding and mediating CGRP- induced relaxation effects in rat uterine artery. Polyclonal antibodies against Nt-domain of CRLR and RAMP(1) (Nt-RAMP(1)) were raised in rabbits and characterized for their specificity and were used to inhibit CGRP-induced vasorelaxation in rat uterine artery. For vascular relaxation studies, uterine arteries from Day 18 pregnant rats were isolated, and responsiveness of the vessels to CGRP was examined with a small vessel myograph. CGRP (10(-10) to 10(-7) M) produced a concentration-dependent relaxation of norepinephrine-induced contractions in Day 18 pregnant rat uterine arteries. These effects were significantly (P < 0.05) inhibited when uterine arteries were incubated with the antibody raised against Nt-CRLR (PD(2) = 6.75 +/- 0.20) and were totally abolished in presence of antibodies for both Nt-CRLR and Nt-RAMP(1) (PD(2) = 6.14 +/- 0.35). In contrast, a monoclonal antibody for CGRP-B receptor had no effect on CGRP-induced rat uterine artery relaxation. These studies suggest that CGRP effects in rat uterine artery are mediated through CGRP-A receptor and that Nt-domain of CRLR may play a predominant role in CGRP binding and thus in causing CGRP-induced uterine artery relaxation.

Female sex steroid hormones and pregnancy regulate receptors for calcitonin gene-related peptide in rat mesenteric arteries, but not in aorta.

Calcitonin gene-related peptide (CGRP) is a potent vasodilator neuropeptide known to be involved in the regulation of vascular tone. Results of previous studies from our laboratory and others suggest that vascular sensitivity to CGRP is enhanced during pregnancy and that the female sex steroid hormones estradiol-17beta (E2) and progesterone (P4) may be involved in this process. We hypothesized that CGRP receptors in the mesenteric artery are increased during pregnancy and with sex steroid hormone treatments. In the present study, we investigated whether pregnancy and female sex steroid hormones modulate the CGRP-receptors CGRP-A and CGRP-B in the mesenteric artery in the rat. The CGRP-A receptor consists of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying protein 1 (RAMP1); however, the CGRP-B receptor needs to be further characterized. Messenger RNA levels for CRLR and RAMP1 were assessed by reverse transcription-polymerase chain reaction, and CGRP-B receptor proteins levels were determined by Western blot analysis. In addition, [125I]CGRP binding was measured by Scatchard analysis. Both mRNA for CGRP-A (CRLR and RAMP1) and the protein for CGRP-B receptors in mesenteric arteries were increased with pregnancy compared to nonpregnant, diestrous animals. A P4 antagonist, RU-486, downregulated and P4 upregulated these receptors in mesenteric arteries (P < 0.05) in pregnant rats. In adult ovariectomized rats, P4 upregulated CRLR and RAMP1 mRNA levels as well as [125I]CGRP-binding sites. The CGRP-B-receptor protein levels were significantly (P < 0.05) elevated by P4 and by combined E2 and P4 treatment. Together with earlier findings, these data suggest that increases in the expression of CGRP-A (CRLR and RAMP1) and CGRP-B receptors in mesenteric arteries may be important in reducing vascular resistance and in vascular adaptations that occur during pregnancy; in addition, P4 may be involved in this process.

Mechanisms involved in calcitonin gene-related Peptide-induced relaxation in pregnant rat uterine artery.

Human and rodent studies have demonstrated that calcitonin gene-related peptide (CGRP), a potent vasodilator, relaxes uterine tissue during pregnancy but not during labor. The vascular sensitivity to CGRP is enhanced during pregnancy, compared to nonpregnant human uterine arteries. In the present study, we hypothesized that uterine artery relaxation effects of CGRP are enhanced in pregnant rats compared to nonpregnant diestrus rats (NP-DE) and that several secondary messenger systems are involved in this process. We also hypothesized that the expression of CGRP-A receptor components, calcitonin receptor-like receptor (CRLR), receptor activity-modifying protein (RAMP1), and CGRP-B receptors are greater in pregnant rats. For vascular relaxation studies, uterine arteries from either NP-DE or Day 18 pregnant rats were isolated, and responsiveness of the vessels to CGRP was examined with a small vessel myograph. CGRP-A and CGRP-B receptor expressions were assessed by RT-PCR and Western immunoblotting, respectively. CGRP (10(-10)--10(-7) M) produced a concentration-dependent relaxation of norepinephrine-induced contractions in both NP-DE and Day 18 pregnant rat uterine arteries. Pregnancy increased the vasodilator sensitivity to CGRP significantly (P < 0.05) compared to NP-DE rats. CGRP receptor antagonist, CGRP8-37, inhibited CGRP-induced relaxation of pregnant uterine arteries. The CGRP-induced relaxation was not affected by NG-nitro-l-arginine methyl ester (L-NAME) (nitric oxide inhibitor, 10(-4) M) but was significantly (P < 0.05) attenuated by inhibitors of guanylate cyclase (ODQ, 10(-5) M) and adenylate cyclase (SQ 22536, 10(-5) M). CGRP-induced vasorelaxation was significantly (P < 0.05) attenuated by potassium channel blockers KATP (glybenclamide, 10(-5) M) and K(CA) (tetraethylammonium, 10(-3) M). The expression of CRLR and RAMP1 was significantly (P < 0.05) elevated during pregnancy compared to nonpregnant diestrus state (NP-DE). However, CGRP-B receptor proteins in uterine arteries were not altered with pregnancy compared to those of NP-DE. These studies suggest that CGRP-induced increases in uterine artery relaxation may play a role in regulating blood flow to the uterus during pregnancy and, therefore, in fetal growth and survival.

Changes in the expression of calcitonin receptor-like receptor, receptor activity-modifying protein (RAMP) 1, RAMP2, and RAMP3 in rat uterus during pregnancy, labor, and by steroid hormone treatments.

Calcitonin gene-related peptide (CGRP) and its related peptide, adrenomedullin (AM), are potent smooth muscle relaxants in a variety of tissues. The CGRP has been reported to play an important role in maintaining uterine relaxation during pregnancy. We have previously reported that CGRP-induced uterine relaxation was gestationally regulated. Calcitonin receptor-like receptor (CRLR), a seven-domain transmembrane protein functions as CGRP-A receptor, in association with receptor activity-modifying protein (RAMP) 1, a single-domain transmembrane protein, whereas CRLR and RAMP2 or RAMP3 constitute a receptor for AM. In the present investigation, we examined the mRNA expression of CRLR, RAMP1, RAMP2, and RAMP3 in rat uterus (n = 8) by reverse transcriptional analysis and polymerase chain reaction to assess the changes in the expression of CGRP-A- and AM-receptor components during pregnancy and labor and by steroid hormone treatments in adult ovariectomized rats. The changes in mRNA are expressed relative to the 18S mRNA in the uterus of rats at various stages: nonpregnant, pregnant on Day 18, spontaneous labor at term, Day 2 postpartum, and in pregnant rats on treatment with RU486. Ovariectomized rats treated for 3 days twice daily s.c. with estradiol-17beta (2.5 microg/injection), progesterone (2 mg/injection), and the combination of estradiol-17beta and progesterone (same doses as above) were also examined for the expression of various receptor components. Results showed that mRNA expression of the receptor components was significantly higher (P < 0.001 for CRLR, P < 0.01 for RAMP1, P < 0.05 for RAMP2, and P < 0.01 for RAMP3) in pregnant compared to nonpregnant rats. Except for RAMP3, expression of all the other three genes decreased significantly (P < 0.05) during labor. A progesterone antagonist, RU486 significantly decreased (P < 0.01 for CRLR, P < 0.05 for RAMP1, RAMP2, and RAMP3) all the receptor components during pregnancy. In adult ovariectomized rats, progesterone caused significant increases in CRLR (P < 0.001), RAMP1 (P < 0.05), and RAMP2 (P < 0.01). Levels of RAMP3 were unaffected by the progesterone treatment. Estradiol-17beta treatment decreased all of the four receptor components significantly (P < 0.01 for CRLR, P < 0.05 for RAMP1, RAMP2, and RAMP3). Our results demonstrate that both CGRP and AM may play a role in uterine quiescence during pregnancy and that their receptor components are regulated by the steroid hormones.

Sex steroid hormones enhance hypotensive effects of calcitonin gene-related Peptide in aged female rats.

The aim of the present study is to investigate whether vascular protective effects of steroid hormones in aged female rats are mediated through calcitonin gene-related peptide (CGRP), a known potent vasodilator. This rat model reflects the postmenopausal state in humans. We examined whether blood pressure lowering effects of CGRP are enhanced in aged female rats when steroid hormone treatments are administered. We observed that 1) continuous infusion of CGRP lowered blood pressures in rats treated with estradiol-17beta and progesterone (P < 0.05), 2) acute hypotensive effects of CGRP were significantly (P < 0.05) greater in the presence of steroid hormones than in vehicle-treated groups, 3) blood pressure decreases in response to CGRP are lower in aged female rats than they are in young adult ovariectomized rats, and 4) age-related differences in the hypotensive effects of CGRP were nullified when animals were treated with steroid hormones. These data suggest that female sex steroid hormones may modulate arterial blood pressure by regulating the CGRP effector system in female rats regardless of age.

Infusion of pregnant rats with calcitonin gene-related peptide (CGRP)(8-37), a CGRP receptor antagonist, increases blood pressure and fetal mortality and decreases fetal growth.

Calcitonin gene-related peptide (CGRP) is the most potent endogenous vasodilatory peptide, and is involved in the regulation of blood flow to vital organs. We have previously shown that CGRP may be involved in vascular adaptations that occur during pregnancy, and that steroid hormones may be involved in these mechanisms. We hypothesized that endogenous CGRP is required for maintaining blood pressure and fetoplacental growth in pregnant rats, and that progesterone will enhance CGRP effects. The vasodilatory effects of CGRP are known to be inhibited by a competitive CGRP receptor antagonist, the C-terminal fragment CGRP(8-37). In the present study, we investigated whether continuous s.c. infusion of CGRP(8-37) to pregnant rats will reduce fetoplacental growth and increase systolic blood pressure. We also assessed whether progesterone will alter the effects of CGRP(8-37) on blood pressure during postpartum. Groups of five pregnant rats were s.c. infused with varying doses of CGRP(8-37) from Day 17 of pregnancy. Daily systolic blood pressures, pup weight, mortality at term delivery, and fetoplacental weights on Day 20 of gestation were measured. CGRP(8-37) at a dose of 0.083 mg day(-1) kg(-1) body weight (BW) showed no effects; however, doses of 0.33 and 1.33 mg day(-1) kg(-1) BW increased (P < 0.05) blood pressure during pregnancy, and these elevated blood pressures persisted during postpartum with the highest dose used. Progesterone (2 mg per injection, twice a day; s.c.) treatment significantly elevated blood pressure in rats infused with CGRP(8-37) during postpartum, suggesting that progesterone regulates CGRP-induced vascular effects. CGRP(8-37) infusion caused significant reductions in pup weight with an increase in mortality rate, and these effects were dose-dependent. Placental and fetal weights were also decreased prior to term on Day 20 of gestation, 72 h after CGRP(8-37) infusion, indicating effects on uteroplacental tissues. Therefore, we suggest that endogenous CGRP plays an important role in maintaining normal fetoplacental development, fetal survival, and vascular adaptations during pregnancy.

Gestational changes in calcitonin gene-related peptide, nerve growth factor, and its receptors in rat dorsal root ganglia.

In dorsal root ganglia (DRG) cell cultures, levels of calcitonin gene-related peptide (CGRP) are increased in the presence of ovarian hormones and nerve growth factor (NGF). In addition, injection of ovariectomized rats with ovarian hormones led to an increase in levels of two NGF receptors, TrkA and p75(NTR), in DRG. Thus, we hypothesized that increased levels of ovarian hormones during pregnancy may elevate the synthesis of CGRP and NGF receptors in the DRG. DRG harvested from rats on specific days of pregnancy, on Day 2 postpartum, and after ovariectomy were subjected to radioimmunoassay, Western blot analysis, and NGF immunoassay to determine levels of CGRP, TrkA and p75(NTR), and NGF, respectively. CGRP levels in rat DRG were significantly higher during pregnancy than at Day 2 postpartum or in ovariectomized rats. Levels of both TrkA and p75(NTR) in DRG increased during pregnancy and remained elevated at Day 2 postpartum, but CGRP levels declined. Levels of NGF reached a statistically significant peak at Day 18 of gestation, and were not significantly reduced at Day 2 postpartum. Increased levels of ovarian steroid hormones during pregnancy may be involved in the synthesis of CGRP, however, the postpartum decreases in CGRP synthesis appear to be unrelated to NGF and its receptors.

Pregnancy and steroid hormones enhance the systemic and regional hemodynamic effects of calcitonin gene-related peptide in rats.

Calcitonin gene-related peptide (CGRP) is a potent vasodilator neuropeptide known to be involved in the regulation of vascular resistance. Several lines of evidence suggest that CGRP plays a role in the vascular adaptations that occur during normal pregnancy; however, the effects of exogenous CGRP on systemic and regional hemodynamics during pregnancy remain unknown. Therefore, the purpose of this study was to determine the hemodynamic effects of systemically administered CGRP in adult pregnant (Day 19) and ovariectomized (ovx) rats using the radioactive microsphere technique. In addition, we also used ovariectomized rats treated for 3 days with estradiol (E2), progesterone (P4), E2 + P4 in sesame oil, or oil only to assess if these hormones regulate the CGRP-induced hemodynamic changes. On the day of study, catheters were inserted into the left cardiac ventricle (through the right carotid artery), right jugular vein, and caudal tail artery. Hemodynamic studies using radioactive microspheres were then performed in conscious rats 3 h after recovery from anesthesia. Blood pressure and heart rate were continuously monitored, and left ventricular pressure was determined immediately prior to each microsphere injection. Microspheres labeled with either (141)Ce or (85)Sr were injected prior to and 2 min following the i.v. bolus injection of CGRP (270 pmol/kg body weight [BW]). Mean arterial pressure (MAP) and total vascular resistance in pregnant rats was lower than in ovx rats, and this was further decreased with an i.v. bolus injection of 270 pmol CGRP/kg BW. Cardiac output was elevated with further increases upon CGRP administration in pregnant but not in ovx rats. The CGRP-induced changes in MAP, total vascular resistance, and cardiac output in E2 + P4 -treated rats were similar to that observed in Day 19 pregnant rats, indicating that CGRP effects on these parameters during pregnancy may be modulated by steroid hormones. Both pregnancy and E2 + P4 treatment in ovx rats caused significant decreases in CGRP-induced resistance in mesenteric, coronary, and renal vasculature. Thus, the vasodilatory sensitivity to CGRP during pregnancy may be mediated through decreased total vascular resistance, particularly to coronary, mesenteric, and renal vascular beds. Thus, CGRP-induced vasodilatory effects may play a role in mediating vascular adaptations that occur during pregnancy and that steroid hormones may modulate these CGRP effects.

Female steroid hormones modulate receptors for nerve growth factor in rat dorsal root ganglia.

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 p75(NTR), in DRG. We examined the effects of 17 beta-estradiol (E(2)) and progesterone (P(4)) on NGF receptors in DRG obtained from ovariectomized (ovx) rats. Groups of 4 ovx rats were injected s.c. with 5 microg E(2), 4 mg P(4), or 5 microg E(2) + 4 mg P(4) 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 microg) of E(2) (0.5, 1.5, 4, 10 mg) P(4), and (5 microg) E(2) + (0.5, 1.5, 4.0, 10 mg) P(4) in 0.2 ml sesame oil, or vehicle, and killed at 6 (for E(2)) or 24 (for P(4) and E(2) + P(4)) 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 p75(NTR) levels. Estradiol increased trkA at 6 h but not p75(NTR). Progesterone caused upregulation of trkA and p75(NTR) at 6 and 24 h. 17 beta-Estradiol + P(4) increased trkA at 6 and 24 h and p75(NTR) at all time points examined. One microgram of E(2) increased trkA but did not affect p75(NTR) levels. Progesterone at 4 and 10 mg upregulated trkA but only 10 mg P(4) increased p75(NTR). Five micrograms of E(2) coinjected with P(4) at 1.5 and 4 mg increased trkA, while p75(NTR) receptor was upregulated when coinjected with P(4) 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 p75(NTR) increased at 2 wk after ovariectomy. 17 beta-Estradiol increased trkA but not p75(NTR) receptors in DRG, whereas P(4) caused increases in both trkA and p75(NTR) 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.

Regulation of calcitonin gene-related peptide expression in dorsal root ganglia of rats by female sex steroid hormones.

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-17beta (E(2)) and progesterone (P(4)) 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 microg E(2), 4 mg P(4), or 5.0 microg E(2) + 4 mg P(4) 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 E(2) (1, 10, 100 nM), P(4) (10, 100, 1000 nM), or E(2) (10 nM) + P(4) (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 E(2) 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. P(4) also stimulated expression of both CGRP mRNA and i-CGRP. In the in vitro studies, either E(2) or P(4) 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 E(2), P(4), and E(2)+P(4) 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 (E(2)) and 100 nM (P(4)) at 24 h of incubation. In summary, both E(2) and P(4), 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.

Pregnancy and sex steroid hormones enhance circulating calcitonin gene-related peptide concentrations in rats.

Calcitonin-gene-related peptide (CGRP) is a 37 amino acid neuropeptide synthesized primarily in dorsal root ganglia (DRG) and distributed widely in the perivascular nerves, suggesting that this peptide may play a role in the regulation of peripheral vascular tone. Since female sex steroid hormones have been implicated in the regulation of peripheral vascular tone during pregnancy, we postulated that they may alter the concentration of CGRP in the circulation and thus modulate the increased blood flow observed during pregnancy. In the present study, we measured changes in plasma concentrations of CGRP in non-pregnant, pregnant, and post-partum rats. Groups of ovariectomized rats were treated s.c. for 3 days either with 17beta-oestradiol (2.5 microg per injection twice daily), progesterone (2 mg per injection twice daily), or vehicle. Another group of adult, non-pregnant rats at dioestrus stage of the oestrous cycle was also used in this study. Plasma concentrations of CGRP were higher (P < 0.05) in rats on day 19 of pregnancy (22.0 +/- 3.0 pmol/l) compared to that during delivery (5. 0 +/- 2.0), post-partum day 2 (2.0 +/- 0.7) or in non-pregnant (4.9 +/- 1.6) state. Furthermore, in adult ovariectomized (6.0 +/- 0.6) rats, plasma CGRP concentrations were increased significantly (P < 0. 05) by oestradiol (10.0 +/- 1.0), progesterone (9.5 +/- 1.0) and oestradiol + progesterone (14.0 +/- 1.0). Thus, circulating concentrations of CGRP are elevated during pregnancy and by oestrogen and progesterone, suggesting that the elevated concentrations of CGRP may play an important role in vascular adaptations that occur during pregnancy.

Increased blood pressure in alpha-calcitonin gene-related peptide/calcitonin gene knockout mice.

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 alpha-calcitonin gene-related peptide (alpha-CGRP) gene. Although the beta-calcitonin gene-related peptide (beta-CGRP) gene is intact in these mice, alpha-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 alpha-CGRP on baseline BP and beta-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 alpha-CGRP mRNA in the knockout mice. Substance P mRNA content in DRG was unchanged between the 2 groups; however, beta-CGRP mRNA levels were reduced 2-fold in the knockout mice. These results indicate for the first time that alpha-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.