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.

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.