Increased MMPs expression and decreased contraction in the rat myometrium during pregnancy and in response to prolonged stretch and sex hormones

Normal pregnancy is associated with uterine relaxation to accommodate the stretch imposed by the growing fetus; however, the mechanisms underlying the relationship between pregnancy-associated uterine stretch and uterine relaxation are unclear. We hypothesized that increased uterine stretch during pregnancy is associated with upregulation of matrix metalloproteinases (MMPs), which in turn cause inhibition of myometrium contraction and promote uterine relaxation. Uteri from virgin, midpregnant (day 12), and late-pregnant rats (day 19) were isolated, and myometrium strips were prepared for measurement of isometric contraction and MMP expression and activity using RT-PCR, Western blot analysis, and gelatin zymography. Oxytocin caused concentration-dependent contraction of myometrium strips that was reduced in mid- and late-pregnant rats compared with virgin rats. Pretreatment with the MMP inhibitors SB-3CT (MMP-2/MMP-9 Inhibitor IV), BB-94 (batimastat), or Ro-28-2653 (cipemastat) enhanced contraction in myometrium of pregnant rats. RT-PCR, Western blot analysis, and gelatin zymography demonstrated increased mRNA expression, protein amount, and activity of MMP-2 and MMP-9 in myometrium of late-pregnant>midpregnant>virgin rats. Prolonged stretch of myometrium strips of virgin rats under 8 g basal tension for 18 h was associated with reduced contraction and enhanced expression and activity of MMP-2 and MMP-9, which were reversed by MMP inhibitors. Concomitant treatment of stretched myometrium of virgin rats with 17β-estradiol (E2), progesterone (P4), or E2+P4 was associated with further reduction in contraction and increased MMP expression and activity. MMP-2 and MMP-9 caused significant reduction of oxytocin-induced contraction of myometrium of virgin rat. Thus, normal pregnancy is associated with reduced myometrium contraction and increased MMPs expression and activity. The results are consistent with the possibility that myometrium stretch and concomitant increase in sex hormones during pregnancy are associated with increased expression/activity of specific MMPs, which in turn inhibit uterine contraction and promote uterine relaxation.     

Innate immunity is regulated by CD38, an ecto-enzyme with ADP-ribosyl cyclase activity

Through its production of cyclic adenosine diphosphate ribose, the ecto-enzyme CD38 regulates calcium mobilization in neutrophils responding to some, but not all, chemoattractants. This signaling defect results in reduced chemotaxis of CD38-deficient neutrophils to bacterial peptides and increased susceptibility of CD38-deficient mice to bacterial infections.     

Demonstration of adrenergic catecholamine receptors in rat myometrium and their regulation by sex steroid hormones.

The molecular basis of sex steroid hormone-modulation of catecholamine-regulated smooth muscle cell contraction in the uterus was investigated at the level of the catecholamine receptor in rat myometrium. Myometrial membrane binding sites for ³H)-dihydroergocryptine bound α-but not β-adrenergic antagonists and stereospecifically bound the α-agonists (?)-norepinephrine > (?)-epinephrine > phenylephrine. Binding sites for (?) (³H)-dihydroalprenolol were specific for β-adrenergic antagonists and stereospecifically bound (?)-isoproterenol > epinephrine ? norepinephrine. These results were consistent with the expected properties of the myometrial α- and β-adrenergic catecholamine receptors. Myometrial content of β- but not α-adrenergic catecholamine receptors was significantly elevated during proestrus and estrus, suggesting a role for sex steroid hormones in the regulation of these receptors. This posibility was substantiated in ovariectomized rats where castration resulted in a reduction in myometrial β-receptor content which was restored in a dose-dependent manner by estrodiol administration. We conclude: 1) rat uterus contains a substantial concentration of α- and β-adrenergic catecholamine receptors, 2) sex steroid hormones may modulated uterine contractility by regulation of these cell surface receptors; 3) modulation of cell responses to surface active hormones and agents by regulation of their cell surface receptors may be a major way in which sex steroids regulate target organ function.     

CD38/ADP-ribosyl cyclase in the rat sublingual gland: subcellular localization under resting and saliva-secreting conditions

CD38 is a 42–45 kDa transmembrane glycoprotein that exhibits ADP-ribosyl cyclase enzyme activity. In the rat, we have previously reported strong ADP-ribosyl cyclase activity in the sublingual salivary gland (Masuda W. and Noguchi T. Biochem. Biophys. Res. Commun. (2000) 270, 469–472). Here, we have examined the specific localization of CD38/ADP-ribosyl cyclase activity in this gland and whether that localization changes upon saliva-secretary stimulation. Under resting conditions, CD38/ADP-ribosyl cyclase activity in the post-nuclear fraction of SLG homogenates was separated into two major peaks by sucrose density gradient centrifugation. The first peak included the plasma membrane proteins Na⁺/K⁺ ATPase and aquaporin 5, while the second peak included mucous secretory protein mucin and vesicle-associated membrane protein 2. When rats were subjected to the muscarinic agonist pilocarpine, the CD38/ADP-ribosyl cyclase activity disappeared from the second peak, as did mucin and vesicle-associated membrane protein 2. Pre-treatment of rats with the muscarinic antagonist atropine before pilocarpine administration, or adrenergic stimulation with isoproterenol, the sucrose density gradient separation profiles were same as that seen under resting condition. Using an immunofluorescent strategy, we observed the preferential localization of CD38 in the basolateral plasma membrane and intracellular granule-like membrane in sublingual acinar cells under resting conditions.     

The influence of steroid hormones on the uterine cervix during pregnancy

This paper reviews the evidence concerning the actions of steroid hormones on the connective tissues of the pelvis. Most available data concern the effects of steroids on the cervix. The time course of cervical softening in rats, sheep and humans suggests the possibility that the changes in connective tissue biochemistry that underlie the physiological phenomenon of cervical softening are under hormonal control. Both oestrogens and progestogens have been implicated in the control of cervical softening. However, recent experiments using inhibitors of 3 beta-hydroxysteroid dehydrogenase suggest that cervical softening can be produced in both sheep and humans by progesterone withdrawal in the absence of high circulating concentrations of oestradiol-17 beta.     

The embryo influences adenylate cyclase activity and hormonal response in rabbit myometrium during early pregnancy

The effect of pseudopregnancy (PSPG; days: 0 (estrus), 1, 6.5, 9 and 15) and pregnancy (PG; days: 6.5, 9 and 15) on adenylate cyclase (AC) activity was verified in rabbit myometrium. During PSPG, there was a time related decline in basal activity from 71 +/- 16.2 (D 0) to 13.1 +/- 1.6 (PSPG-D9) pmoles cAMP formed/mg prot-min. Stimulation of the enzyme by GTP, Isoproterenol (ISO), Prostaglandin E2 (PGE2) or Sodium Fluoride (NaF) followed a similar pattern. AC activity was compared in myometrial tissues of pregnant animals (PG) separated into embryonic (ES) and interembryonic (IES) sites. On days 6.5 and 9, AC activity measured in tissues from PG rabbits (ES and IES) was always higher than that found in tissues from PSPG animals on corresponding days. On day 6.5, AC activity was slightly higher (p less than 0.01) in ES than in IES. This was confirmed on day 9 where basal as well as GTP, ISO and PGE2 stimulated activities were higher in ES than in IES (p less than 0.001). Dose response to ISO, expressed as % of GTP, were similar on D0, 1, 6.5 and 15 of PSPG. However, on day 9, there was a striking diminution in response reflected by a lower stimulation at suboptimal dose (0.1 microM; p less than 0.05) from 115 +/- 2 on day 0 to 104 +/- 4 on day 9. These results suggest that protein content which is increased during pseudopregnancy could be responsible for the decline of AC activity. However, results obtained on day 9 and 15 suggest that other factors are involved. Dose responses to ISO during PG showed an alteration in response on days 6.5 and 9 at ES. It was reflected by a higher stimulation at suboptimal (0.1 microM) and optimal doses (100 microM). These results suggest that myometrial AC activity could be regulated by the presence of the embryo.     

CD38/ADP-ribosyl cyclase: A new role in the regulation of osteoclastic bone resorption.

The multifunctional ADP-ribosyl cyclase, CD38, catalyzes the cyclization of NAD(+) to cyclic ADP-ribose (cADPr). The latter gates Ca(2+) release through microsomal membrane-resident ryanodine receptors (RyRs). We first cloned and sequenced full-length CD38 cDNA from a rabbit osteoclast cDNA library. The predicted amino acid sequence displayed 59, 59, and 50% similarity, respectively, to the mouse, rat, and human CD38. In situ RT-PCR revealed intense cytoplasmic staining of osteoclasts, confirming CD38 mRNA expression. Both confocal microscopy and Western blotting confirmed the plasma membrane localization of the CD38 protein. The ADP-ribosyl cyclase activity of osteoclastic CD38 was next demonstrated by its ability to cyclize the NAD(+) surrogate, NGD(+), to its fluorescent derivative cGDP-ribose. We then examined the effects of CD38 on osteoclast function. CD38 activation by an agonist antibody (A10) in the presence of substrate (NAD(+)) triggered a cytosolic Ca(2+) signal. Both ryanodine receptor modulators, ryanodine, and caffeine, markedly attenuated this cytosolic Ca(2+) change. Furthermore, the anti-CD38 agonist antibody expectedly inhibited bone resorption in the pit assay and elevated interleukin-6 (IL-6) secretion. IL-6, in turn, enhanced CD38 mRNA expression. Taken together, the results provide compelling evidence for a new role for CD38/ADP-ribosyl cyclase in the control of bone resorption, most likely exerted via cADPr.     

Gulonolactonase in the rat: influence of sex and sex hormones on development

Gulonolactonase (D(or L)-gulono-γ-lactone hydrolase, (EC 3.1.1.18) in the kidney and liver of the rat, which are known to be identical enzymes but exhibit different patterns of post-natal development. The hepatic enzyme is detectable one day after birth, increases sharply to the adult level after nine days, and shows no appreciable sex-related difference. The renal enzyme is not detectable until approximately four days after birth in the male and ten days after birth in the female. The level of renal enzyme increases slowly in both sexes until about day 27 at which time activity in the male begins to increase rapidly while it declines slowly in the female. At this time the adult male has about 20 times as much hepatic gulonolactonase as the female. Adult enzyme levels are reached at age 44 days.The normal development increase in male renal gulonolactonase is prevented by administration of 17β-estradiol but it can be restored by subsequent administration of testosterone. Testosterone alone, or in combination with glucose does not evoke precocious induction of gulonolactonase in the male, nor does it affect its level after development has begun.Of the androgens tested for their ability to induce gulonolactonase in the kidney of the adult female, the following potency was observed: 5-androstan-3α, 17β-diol > testosterone > androstandione = androstanolone. Androsterone was without effect.     

Changes in secretion of sex steroid hormones during stress in rats with various brain excitability

A stress procedure decreased the plasma progesterone faster in highly excitable rats than in the less excitable those. The decrease in gonadal and adrenal progesterone was still obvious in 24 hours in both groups of rats but more obvious in the former group in respect to the progesterone level. The data obtained may help to understand individual ability to respond to a stress in a different way depending on functional condition of the hypothalamo-pituitary-adrenal axis.     

Electrical coupling in rat myometrium during pregnancy.

Gap junctions were found in rat myometrium only during parturition of abortion. A comparison was made between the values of the length constants obtained from longitudinally cut strips of rat myometrium at the midterm stage of pregnancy and during parturition. No significant difference was found between these values. No significant difference was found between length constants measured in myometrium from rats near term (22 days) and those during parturition, or between midterm myometrium and myometrium taken from animals aborting at midterm following ovariectomy. It was concluded that the appearance of gap junctions in parturient myometrium does not alter the spread of passive current in the longitudinal axis of the cells in these tissues. It is suggested that gap junctions are not required for spread of passive current, and that other structures may provide an alternate path.     

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.     

The CD38-independent ADP-ribosyl cyclase from mouse brain synaptosomes: a comparative study of neonate and adult brain

cADPR (cADP-ribose), a metabolite of NAD+, is known to modulate intracellular calcium levels and to be involved in calcium-dependent processes, including synaptic transmission, plasticity and neuronal excitability. However, the enzyme that is responsible for producing cADPR in the cytoplasm of neural cells, and particularly at the synaptic terminals of neurons, remains unknown. In the present study, we show that endogenous concentrations of cADPR are much higher in embryonic and neonate mouse brain compared with the adult tissue. We also demonstrate, by comparing wild-type and Cd38-/- tissues, that brain cADPR content is independent of the presence of CD38 (the best characterized mammalian ADP-ribosyl cyclase) not only in adult but also in developing tissues. We show that Cd38-/- synaptosome preparations contain high ADP-ribosyl cyclase activities, which are more important in neonates than in adults, in line with the levels of endogenous cyclic nucleotide. By using an HPLC method and adapting the cycling assay developed initially to study endogenous cADPR, we accurately examined the properties of the synaptosomal ADP-ribosyl cyclase. This intracellular enzyme has an estimated K(m) for NAD+ of 21 microM, a broad optimal pH at 6.0-7.0, and the concentration of free calcium has no major effect on its cADPR production. It binds NGD+ (nicotinamide-guanine dinucleotide), which inhibits its NAD+-metabolizing activities (K(i)=24 microM), despite its incapacity to cyclize this analogue. Interestingly, it is fully inhibited by low (micromolar) concentrations of zinc. We propose that this novel mammalian ADP-ribosyl cyclase regulates the production of cADPR and therefore calcium levels within brain synaptic terminals. In addition, this enzyme might be a potential target of neurotoxic Zn2+.     

Evidence for an intracellular ADP-ribosyl cyclase/NAD+-glycohydrolase in brain from CD38-deficient mice

Cyclic ADP-ribose, a metabolite of NAD+, is known to modulate intracellular calcium levels and signaling in various cell types, including neural cells. The enzymes responsible for producing cyclic ADP-ribose in the cytoplasm of mammalian cells remain unknown; however, two mammalian enzymes that are capable of producing cyclic ADP-ribose extracellularly have been identified, CD38 and CD157. The present study investigated whether an ADP-ribosyl cyclase/NAD+-glycohydrolase independent of CD38 is present in brain tissue. To address this question, NAD+ metabolizing activities were accurately examined in developing and adult Cd38-/- mouse brain protein extracts and cells. Low ADP-ribosyl cyclase and NAD+-glycohydrolase activities (in the range of pmol of product formed/mg of protein/min) were detected in Cd38-/- brain at all developmental stages studied. Both activities were found to be associated with cell membranes. The activities were significantly higher in Triton X-100-treated neural cells compared with intact cells, suggesting an intracellular location of the novel cyclase. The cyclase and glycohydrolase activities were optimal at pH 6.0 and were inhibited by zinc, properties which are distinct from those of CD157. Both activities were enhanced by guanosine 5'-O-(3-thiotriphosphate), a result suggesting that the novel enzyme may be regulated by a G protein-dependent mechanism. Altogether our results indicate the presence of an intracellular membrane-bound ADP-ribosyl cyclase/NAD+-glycohydrolase distinct from CD38 and from CD157 in mouse brain. This novel enzyme, which is more active in the developing brain than in the adult tissue, may play an important role in cyclic ADP-ribose-mediated calcium signaling during brain development as well as in adult tissue.