Urinary trypsin inhibitor prevents uterine muscle contraction by inhibition of Ca++ influx

doi:10.1016/0002-9378(95)90189-2

American Journal of Obstetrics and Gynecology

Volume 173, Issue 1, July 1995, Pages 192-199

https://doi.org/10.1016/0002-9378(95)90189-2 Get rights and content

Abstract

OBJECTIVE: The aim of this research was to elucidate the mechanism of action of urinary trypsin inhibitor, a Kunitz-type protease inhibitor, in suppressing uterine muscle contraction.

STUDY DESIGN: An isometric uterine contraction test was used to study this inhibitory effect of urinary trypsin inhibitor on the myometrium. Oxytocin, prostaglandin F_2α, and lipopolysaccharide were used to stimulate myometrial contraction. Prostaglandins F_2α and E₂ were measured in the buffer solution. Influx of calcium into uterine smooth muscle cells was assessed by digital imaging microscopy.

RESULTS: After incubation with urinary trypsin inhibitor of fetal urine, myometrial contractions stimulated by oxytocin, prostaglandin F_2α or lipopolysaccharide were suppressed completely. The concentrations of prostaglandins F_2α and E₂ in the buffer solution during the isometric contraction test were significantly increased by lipopolysccharide stimulation, but when urinary trypsin inhibitor was present in the buffer solution the concentrations of prostaglandins F_2α or E₂ did not change significantly. Preincubation with urinary trypsin inhibitor also inhibited calcium influx, resulting in no detectable change in the intracellular free calcium concentration of smooth muscle cells.

CONCLUSION: We proposed that urinary trypsin inhibitor from fetal urine inhibits uterine muscel contraction by regulation of intracellular Ca⁺⁺.

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2017, Journal of Diabetes and its Complications
Citation Excerpt :
When bound to IαI, bikunin lacks some of its known activities, and there is evidence that its release, by partial proteolytic degradation, may function as a regulatory mechanism. Beside its proteinase inhibitory activities, for example, toward plasmin during tumor cell invasion and metastasis (Kobayashi H, Gotoh J, et al. 1995; Kobayashi, Shinohara H et al., 1996; Kobayashi H et al. 2003), bikunin plays additional roles, such as inhibition of interleukin (IL)-8 gene expression induced by lipopolysaccharide (Maehara et al., 1995), smooth muscle contraction (Kanayama et al., 1995, 1998), neutrophil release of elastase (Hiyama, Takeda, Kotake, Morisaki, & Fukushima, 1997), mast cell release of histamine (Kobayashi, Shibata, Fujie, & Terao, 1998), suppression of immune cells (Cowan et al., 1996; Kato, Nagao, & Kurosawa, 1995; Nakatani & Takeshita, 1999), and urolithiasis (Atmani, Glenton, & Khan, 1999, Khan & Kok, 2004), as well as stabilization of lysosomal membranes (Kato et al., 1998; Nakakuki et al., 1996). Free bikunin is rapidly cleared from circulation by both tissue uptake and renal excretion (Kaczmarczyk, Blom, Alston-Smith, Sjöquist, & Fries, 2005) and it is found in urine as the urinary trypsin inhibitor (UTI) also referred to as LSC-PG (low-sulfated chondroitin sulfate proteoglycan).
Because of the high incidence of kidney disease in diabetic patients, the early diagnosis of renal impairment is a key point for intervention and management. Although urinary albumin excretion currently represents the accepted standard to assess both diabetic nephropathy and cardiovascular risk, it has some limitations as structural changes in the glomerular basement membrane may occur before the onset of microalbuminuria. It is therefore important to identify urinary markers that may provide greater sensitivity, earlier detection, and greater predictive power for diabetes complications. In this respect, urinary glycosaminoglycans/proteoglycans (GAGs/PGs) have been long associated with several kidney diseases as well as diabetic nephropathies as their levels increase more readily than albuminuria. In particular, heparan sulfate, a key component of the glomerular basement membrane responsible for its charge-dependent permeability, is excreted into urine at higher concentrations during the early kidney remodeling events caused by the altered glucose metabolism in diabetes. Over the past few years, also urinary trypsin inhibitor has been linked to a chronic inflammatory condition in both type 1 and 2 diabetes. The underlying mechanisms of such increase are not completely known since either a systemic inflammatory condition or a more localized early renal impairment could play a role. Nevertheless, the association with other inflammatory markers and a detailed urinary trypsin inhibitor structural characterization in diabetes remain to be elucidated.
This review will discuss a great deal of information on the association between urinary GAGs/PGs and type 1 and 2 diabetes, with particular emphasis on renal involvement, and their potential as markers useful in screening, diagnosis and follow up to be associated with the current standard tests.
Glycosaminoglycan and transforming growth factor β1 changes in human plasma and urine during the menstrual cycle, in vitro fertilization treatment, and pregnancy
2009, Fertility and Sterility
To evaluate transforming growth factor β1 (TGF-β1) and glycosaminoglycans (GAG) changes in human plasma and urine during the menstrual cycle, IVF-ET, and pregnancy.
Prospective clinical study.
University hospital.
Thirteen women with apparently normal menstrual cycle (group 1); 18 women undergoing IVF-ET (group 2); and 14 low-risk pregnant women (group 3).
We assayed plasma and urine concentrations of TGF-β1, urine content, and distribution of GAG. Blood and urine samples were collected during days 2 to 3, 12 to 13, and 23 to 24 in group 1; in group 2, samples were obtained at menstrual phase, oocyte pick-up day, and 15 days after ET; in group 3, samples were obtained during gestational weeks 10–12, 22–24, and 30–32 and 1 month after delivery.
Changes in TGF-β1 and GAG content.
The mean value of total urinary trypsin inhibitor/chondroitin sulfate (UTI/CS) showed a distinct peak at day 12 of the menstrual cycle in the fertile women in whom we monitored the ovulatory period. In the IVF-ET group, GAG distribution and TGF-β1 levels showed significant differences during the cycle. We observed increased levels of plasma TGF-β1 15 days after ET. A significant increase of total UTI/CS value with increasing gestation was detected.
Transforming growth factor β1 and GAG levels could represent an additional tool to monitor reproductive events and could be useful, noninvasive markers of ovulation and ongoing pregnancy.
Molecular structure and function analysis of bikunin on down-regulation of tumor necrosis factor-α expression in activated neutrophils
2008, Cytokine
Objective: We performed a detailed molecular analysis of bikunin-mediated anti-inflammation (suppressive effect of cytokine release, MAP kinase activation, and nuclear translocation of NF-kB) using a truncated form of bikunin. Materials and methods: We obtained bikunin derivatives that contained O-glycoside-linked N-terminal glycopeptide (Bik-m1), N-glycoside-linked C-terminal tandem Kunitz domains (Bik-m2), bikunin lacking O-glycoside (Bik-c), asialo bikunin (Bik-a), bikunin lacking N-glycoside (Bik-n), and purified C-terminal Kunitz domain II (kII) of bikunin (HI-8). Enzyme-linked immunosorbent assay and Western blot were carried out to measure secreted TNF-α and MAP kinase activation. Results: We examined the TNF-α secretion in control and lipopolysaccharide (LPS)-treated neutrophils and did not see any changes of its protein levels in the cells pretreated with Bik-m1, Bik-m2, Bik-c, or HI-8. In all of the derivatives tested, only the derivatives that lacked N-glycoside side chain showed a significant suppression of TNF-α secretion by LPS. Only a small (21 amino acids) deletion of the N-terminal portion of bikunin (which corresponds to Bik-m2) abolished its suppressing activity of TNF-α secretion, thus suggesting that the N-terminal 21 amino acids play a critical role in anti-inflammation. Bik-m1 alone failed to show anti-inflammatory response. Bikunin failed to inhibit ionomycin-induced phosphorylation of MAP kinases. Conclusion: These data allow us to conclude that the cytokine expression was inhibited only by the O-glycoside-linked core protein without the N-glycoside side chain. Our results also suggest a possible role of bikunin for receptor-dependent MAP kinase activation.
PAR-2 activating peptide-induced stimulation of pregnant rat myometrium contractile activity partly involves the other membrane receptors
2007, European Journal of Obstetrics and Gynecology and Reproductive Biology
Citation Excerpt :
An inhibitor of kallikrein and trypsin, aprotinin was shown to attenuate contractility of the pregnant rat uterus [22] although, the oxytocic effect of kallikrein could be due to direct effect on the myometrium [23,24]. Urinary trypsin inhibitor, from neonatal or healthy adult urine, inhibited oxytocin and PGF2α-induced contraction and intracellular Ca2+-increase in human myometrium [25]. Recently the presence of thrombin receptors (PAR-1) in non-pregnant and pregnant rat myometrium was demonstrated [26–28].
To study if spontaneous contractions augmented by proteinase-activated receptor-2 (PAR-2)-activating peptide serine-leucine-isoleucine-glycine-arginine-leucine (SLIGRL) involve coactivation of membrane chemoceptors and are associated with expression of PAR-2 mRNA in non-pregnant and pregnant rat myometrium.
Non-pregnant, mid-pregnant, and late pregnant rat uterine horn and small intestine segments were snap-frozen in liquid nitrogen to determine PAR-2 mRNA levels by real time polymerase chain reaction (PCR). Uterine rings were used for isometric tension recording. Effect of SLIGRL (0.1 mM) on spontaneous contractions before and after exposure to ibuprofen (cyclooxygenase inhibitor, 1.0 μM), SQ-29548 (thromboxane A₂ receptor inhibitor, 1.0 μM), ketotifen (histamine 1 receptor inhibitor, 10 μM), WEB-2170BS (platelet-activating factor (PAF) receptor inhibitor, 10 μM), atropine (muscarinic receptor inhibitor, 0.1 μM), or ketanserin (serotonin receptor inhibitor, 10 μM) were compared. Paired t-test and one-way ANOVA followed by Dunnett's or Newman–Keuls post hoc tests were used for statistical analysis when appropriate.
P < 0.05.
The agents did not significantly affect time-associated decay in spontaneous contractile activity in any group of the tissues. Activation of spontaneous contractions induced by SLIGRL in non-pregnant rat myometrium did not involve coactivation of membrane chemoceptors, while in mid-pregnant rat myometrium coactivation of prostanoid, histamine, and serotonin receptors and in late pregnant rat myometrium coactivation of thromboxane receptors was noted. Expression of PAR-2 mRNA was similar in non-pregnant, mid-pregnant, and late pregnant rat myometrium.
Expression of PAR-2 in rat myometrium is not dependent on gestational age. Stimulation of PAR-2 is associated with production/release of cyclooxygenase pathway product(s) activating thromboxane/prostaglandin H2 receptors, partial involvement of histamine H1 receptors and serotonin receptors in midpregnancy and thromboxane A2/prostaglandin H2 receptors in late pregnancy.
Tocolytic effect of a Rho-kinase inhibitor in a mouse model of lipopolysaccharide-induced preterm delivery
2005, American Journal of Obstetrics and Gynecology
Citation Excerpt :
These results suggest that the up-regulation of the expression of RhoA/Rho-kinase cascade may also play a physiologic role in normal pregnancy and in inflammation-related premature labor. As for the mechanisms of lipopolysaccharide-induced uterine contraction, it has been shown that lipopolysaccharide stimulates Ca2+ influx into human26 and rat27 myometrial cells. It has been demonstrated that lipopolysaccharide activates RhoA in endothelial cells,28 which led us to speculate that lipopolysaccharide could induce RhoA activation in myometrial cells.
The small guanosine triphosphatase RhoA/Rho-kinase cascade has been implicated in uterine contraction. Our purpose was to evaluate the tocolytic effect of a Rho-kinase inhibitor, Y-27632, in lipopolysaccharide-induced preterm delivery in mice.
We used an animal model of lipopolysaccharide-induced preterm delivery in C3H/HeN × B6D2F1 pregnant mice. Y-27632 was delivered continuously through an osmotic pump that was implanted into the peritoneal cavity 6 hours before lipopolysaccharide treatment. The primary outcome was the preterm delivery rate. To further study the possible involvement of this cascade in lipopolysaccharide-induced preterm delivery, we determined the effect of lipopolysaccharide and prostaglandin F_2α on RhoA activation in mouse myometrial cells and uterine smooth muscle tissues.
The rate of preterm delivery for lipopolysaccharide-treated animals was 94.4%. The administration of Y-27632 (1 or 10 mg/kg/d) significantly reduced the preterm delivery rate to 61.1% or 15.8%, respectively. The level of guanosine triphosphate–bound RhoA was increased after the addition of lipopolysaccharide or prostaglandin F₂α.
The RhoA/Rho-kinase cascade is involved in lipopolysaccharide-induced preterm delivery, which suggests that Rho-kinase could be used as a new therapeutic target for the prevention of preterm labor.
Mechanisms of lipopolysaccharide-induced changes in effects of contractile agonists on pregnant rat myometrium
2004, American Journal of Obstetrics and Gynecology
Citation Excerpt :
Increased uterine L-type Ca++ channel activity is further evident from our studies, wherein it was observed that Ca++ channel agonist BAY K8644 was more potent in contracting uterine strips from LPS-treated rats compared with controls. Acute treatment with LPS has earlier been shown to stimulate Ca++ influx into human myometrial cells.16 Enhancement of L-type Ca++ channel current by LPS in isolated smooth muscle cells of rat tail artery has also been demonstrated.17
The study was undertaken to investigate the mechanisms underlying enhanced uterine contractility induced by lipopolysaccharide (LPS) in pregnant rats.
Wistar rats were administered intrauterine either LPS (50 μg) or normal saline solution (0.05 mL) on day 17 of gestation. On day 19, the animals were killed and uterus was isolated for isometric recording, ⁴⁵Ca⁺⁺ influx measurement, and determination of plasma membrane Na⁺-K⁺-ATPase.
Uterine strips, taken from LPS-treated rats, displayed a marked increase in amplitude of spontaneous rhythmic contractions compared with controls. Enhancement in the sensitivity of uterine strips to agonists such as oxytocin, 5-hydroxytryptamine (5-HT), and BAY K8644 was also observed in rats treated with LPS. Cyclo-oxygenase-2 inhibitor, nimesulide (10 μmol/L) had no significant effect on the LPS-induced increase in spontaneous rhythmic contractions. On the other hand, nimesulide attenuated the increased sensitivity of uterine strips to oxytocin induced by LPS. Nimesulide significantly inhibited 5-HT–induced uterine contractions in both control and LPS-treated rats. However, the enhanced sensitivity of uterine strips to 5-HT was evident even in the presence of nimesulide in rats treated with LPS. Nifedipine-sensitive ⁴⁵Ca⁺⁺-influx into uterine strips both in the basal state as well as those stimulated by high K⁺ (80 mmol/L) and 5-HT (1 μmol/L) was greater in LPS-treated group compared with the controls. LPS treatment caused a marked inhibition in the Na⁺-K⁺-ATPase activity of the uterine plasma membrane compared with controls. LPS had no effect on plasma 17β-estradiol levels.
LPS appears to increase uterine contractility of pregnant rats both through the release of endogenous prostaglandins and increased influx of Ca⁺⁺ through L-type Ca⁺⁺ channels. Inhibition of sodium pump by LPS may be an additional mechanism in augmentation of uterine excitability.

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^☆: Supported in part by Grants-in-Aid for Specific Research (No. 06454469) from the Japanese Ministry of Education, Science, and Culture and by an Adeza Biomedical grant (United States).

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General obstetrics and gynecologyUrinary trypsin inhibitor prevents uterine muscle contraction by inhibition of Ca++ influx☆

Abstract

Am J Obstet Gynecol

Am J Obstet Gynecol

Prostaglandins

Biochim Biophys Acta

J Biol Chem

J Biol Chem

Urinary trypsin inhibitor (mingin): transformation into a new trypsin inhibitor by acid hydrolysis or by sialidase

Nature

The purification of trypsin inhibitor from human pregnancy urine

J Lab Clin Med

Structure of inter-α-inhibitor (inter-α-trypsin inhibitor) and pre-α-inhibitor: current state and proposition of a new terminology

Biol Chem Hoppe-Seyler

General obstetrics and gynecology
Urinary trypsin inhibitor prevents uterine muscle contraction by inhibition of Ca⁺⁺ influx☆