S-Nitrosylation in endothelial cells contributes to tumor cell adhesion and extravasation during breast cancer metastasis.

BACKGROUND: Nitric oxide is produced by different nitric oxide synthases isoforms. NO activates two signaling pathways, one dependent on soluble guanylate cyclase and protein kinase G, and other where NO post-translationally modifies proteins through S-nitrosylation, which is the modification induced by NO in free-thiol cysteines in proteins to form S-nitrosothiols. High levels of NO have been detected in blood of breast cancer patients and increased NOS activity has been detected in invasive breast tumors compared to benign or normal breast tissue, suggesting a positive correlation between NO biosynthesis, degree of malignancy and metastasis. During metastasis, the endothelium plays a key role allowing the adhesion of tumor cells, which is the first step in the extravasation process leading to metastasis. This step shares similarities with leukocyte adhesion to the endothelium, and it is plausible that it may also share some regulatory elements. The vascular cell adhesion molecule-1 (VCAM-1) expressed on the endothelial cell surface promotes interactions between the endothelium and tumor cells, as well as leukocytes. Data show that breast tumor cells adhere to areas in the vasculature where NO production is increased, however, the mechanisms involved are unknown. RESULTS: We report that the stimulation of endothelial cells with interleukin-8, and conditioned medium from breast tumor cells activates the S-nitrosylation pathway in the endothelium to induce leukocyte adhesion and tumor cell extravasation by a mechanism that involves an increased VCAM-1 cell surface expression in endothelial cells. We identified VCAM-1 as an S-nitrosylation target during this process. The inhibition of NO signaling and S-nitrosylation blocked the transmigration of tumor cells through endothelial monolayers. Using an in vivo model, the number of lung metastases was inhibited in the presence of the S-nitrosylation inhibitor N-acetylcysteine (NAC), which was correlated with lower levels of S-nitrosylated VCAM-1 in the metastases. CONCLUSIONS: S-Nitrosylation in the endothelium activates pathways that enhance VCAM-1 surface localization to promote binding of leukocytes and extravasation of tumor cells leading to metastasis. NAC is positioned as an important tool that might be tested as a co-therapy against breast cancer metastasis.

Differential expression of upstream stimulatory factor (USF) 2 variants in eutopic endometria from women with endometriosis: estradiol regulation.

BACKGROUND: Endometriosis, pro-inflammatory and invasive benign disease estrogen dependent, abnormally express in endometria the enzyme P450Arom, positively regulated by steroid factor-1 (SF-1). Our objective was to study the nuclear protein contents of upstream stimulating factor 2 (USF2a and USF2b), a positive regulator of SF-1, throughout the menstrual cycle in eutopic endometria from women with and without (control) endometriosis and the involvement of nuclear estrogen receptors (ER) and G-coupled protein estrogen receptor (GPER)-1. RESULTS: Upstream stimulating factor 2 protein contents were higher in mid (USF2b) and late (USF2a and USF2b) secretory phase in eutopic endometria from endometriosis than control (p < 0.05). In isolated control epithelial cells incubated with E2 and PGE2, to resemble the endometriosis condition, the data showed: (a) significant increase of USF2a and USF2b nuclear protein contents when treated with E2, PPT (specific agonist for ERα) or G1 (specific agonist for GPER1); (b) no increase in USF2 binding to SF-1 E-Box/DNA consensus sequence in E2-treated cells; (c) USF2 variants protein contents were not modified by PGE2; (d) SF-1 nuclear protein content was significantly higher than basal when treated with PGE2, E2 or G1, stimulation unaffected by ICI (nuclear ER antagonist); and (e) increased (p < 0.05) cytosolic protein contents of P450Arom when treated with PGE2, E2, PPT or G1 compared to basal, effect that was additive with E2 + PGE2 together. Nevertheless, in endometriosis cells, the high USF2, SF-1 and P450Arom protein contents in basal condition were unmodified. CONCLUSION: These data strongly suggest that USF2 variants and P450Arom are regulated by E2 through ERα and GPER1, whereas SF-1 through GPER1, visualized by the response of the cells obtained from control endometria, being unaffected the endogenously stimulated cells from endometriosis origin. The lack of E2 stimulation on USF2/SF-1 E-Box/DNA-sequence binding and the absence of PGE2 effect on USF2 variants opposite to the strong induction that they exert on SF1 and P450 proteins suggest different mechanisms and indirect regulations. The sustained USF2 variants protein expression during the secretory phase in eutopic endometria from women with endometriosis may participate in the pathophysiology of this disease strongly associated with infertility and its characteristic endometrial invasion to ectopic sites in the pelvic cavity.

Differential expression of upstream stimulatory factor (USF) 2 variants in eutopic endometria from women with endometriosis: estradiol regulation

BACKGROUND: Endometriosis, pro-inflammatory and invasive benign disease estrogen dependent, abnormally express in endometria the enzyme P450Arom, positively regulated by steroid factor-1 (SF-1). Our objective was to study the nuclear protein contents of upstream stimulating factor 2 (USF2a and USF2b), a positive regulator of SF-1, throughout the menstrual cycle in eutopic endometria from women with and without (control) endometriosis and the involvement of nuclear estrogen receptors (ER) and G-coupled protein estrogen receptor (GPER)-1. RESULTS: Upstream stimulating factor 2 protein contents were higher in mid (USF2b) and late (USF2a and USF2b) secretory phase in eutopic endometria from endometriosis than control (p < 0.05). In isolated control epithelial cells incubated with E2 and PGE2, to resemble the endometriosis condition, the data showed: (a) significant increase of USF2a and USF2b nuclear protein contents when treated with E2, PPT (specific agonist for ERa) or G1 (specific agonist for GPER1); (b) no increase in USF2 binding to SF-1 E-Box/DNA consensus sequence in E2-treated cells; (c) USF2 variants protein contents were not modified by PGE2; (d) SF-1 nuclear protein content was significantly higher than basal when treated with PGE2, E2 or G1, stimulation unaffected by ICI (nuclear ER antagonist); and (e) increased (p < 0.05) cytosolic protein contents of P450Arom when treated with PGE2, E2, PPT or G1 compared to basal, effect that was additive with E2 + PGE2 together. Nevertheless, in endometriosis cells, the high USF2, SF-1 and P450Arom protein contents in basal condition were unmodified. CONCLUSION: These data strongly suggest that USF2 variants and P450Arom are regulated by E2 through ERa and GPER1, whereas SF-1 through GPER1, visualized by the response of the cells obtained from control endometria, being unaffected the endogenously stimulated cells from endometriosis origin. The lack of E2 stimulation on USF2/SF-1 E-Box/DNA-sequence binding and the absence of PGE2 effect on USF2 variants opposite to the strong induction that they exert on SF1 and P450 proteins suggest different mechanisms and indirect regulations. The sustained USF2 variants protein expression during the secretory phase in eutopic endometria from women with endometriosis may participate in the pathophysiology of this disease strongly associated with infertility and its characteristic endometrial invasion to ectopic sites in the pelvic cavity.

The effects of intraganglionic injection of calcium/calmodulin-dependent protein kinase II inhibitors on pain-related behavior in diabetic neuropathy.

Calcium/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the transmission of nociceptive input in diabetic neuropathy. The aim of this study was to test whether intraganglionic (i.g.) injection of CaMKII inhibitors may alleviate pain-related behavior in diabetic rats. Diabetes was induced in Sprague-Dawley rats using 55 mg/kg streptozotocin intraperitoneally. Two weeks after diabetes induction, CaMKII inhibitors myristoil-AIP and KN93 were injected directly into the right L5 dorsal root ganglion (DRG). Behavioral testing with mechanical and thermal stimuli was performed before induction of diabetes, the day preceding the injection, as well as 2 and 24h after the i.g. injection. The expression of total CaMKII and its alpha isoform in DRG neurons was analyzed using immunohistochemistry. CaMKII inhibitors attenuated pain-related behavior in a modality-specific fashion. Attenuation of nociceptive behavior was accompanied with a corresponding decrease of CaMKII alpha expression in DRG neurons on the side of injection. A significant decrease of CaMKII alpha expression was seen in small- and medium-sized neurons. In conclusion, our study provides evidence that CaMKII inhibitors are potential pharmacological agents that should be further explored for treatment of diabetic neuropathy symptoms.

P450Arom induction in isolated control endometrial cells by peritoneal fluid from women with endometriosis.

OBJECTIVE: To study the effect of peritoneal fluid from women with (PF-E) and without (PF-C) endometriosis on P(450)Arom expression in endometrial cells. DESIGN: Experimental study. SETTING: University research unit. PATIENT(S): Forty women of reproductive age with (n = 22) or without (control; n = 18) endometriosis. INTERVENTION(S): Peritoneal fluid and eutopic endometrial samples were obtained during surgery from women with (n = 13 and 9, respectively) and without (n = 4 and 14, respectively) endometriosis. MAIN OUTCOME MEASURE(S): Expression study for P(450)Arom, steroid factor 1 (SF-1), chicken ovalbumin upstream transcription factor I (COUP-TFI), and COUP-TFII messenger RNA (reverse transcriptase-polymerase chain reacion) and/or protein (immunoblot) in isolated endometrial epithelial cells transfected or not with expression vector containing SF-1, COUP-TFI, or COUP-TFII complementary DNAs. RESULT(S): Basal messenger RNA and/or protein expression of P(450)Arom and SF-1 were augmented in endometriosis, and that of COUP-TF was diminished. In control cells, (Bu)(2)cAMP and PF-E increased P(450)Arom and SF-1 expression (but not COUP-TF expression) in a dose-dependent way, an effect not observed with PF-C, adsorbed PF-E, or 10(-5) M indomethacin. Transfected cells confirmed these results. Any treatments modified the studied molecules in endometriosis cells. CONCLUSION(S): These data indicate that molecules contained in PF-E favor an estrogenic microenvironment, suggesting a role in the etiopathogenesis of endometriosis enabling the survival, maintenance, and growth of endometrial implants in the ectopic locations.

Association between MMP1 and MMP9 activities and ICAM1 cleavage induced by tumor necrosis factor in stromal cell cultures from eutopic endometria of women with endometriosis.

Endometriosis is a benign gynecological pathology in which immune system deregulation may play a role in its initiation and progression. In endometriotic lesions, intercellular adhesion molecule-1 (ICAM1) is released from the cell membrane by proteolytic cleavage of its extracellular domain, a process that coincides with increased expression and proteolytic activity of metalloproteinases such as MMP1 and MMP9. The objective of our study was to investigate the association between MMP1 and MMP9 activities and ICAM1 cleavage mediated by tumor necrosis factor (TNF) in eutopic endometrial stromal cells from women with and without (control) endometriosis during culture. The RNA was evaluated by RT-PCR, and the protein was determined by western blot (ICAM1, MMP1), casein or gelatin zymographies (secreted active MMP1 or MMP9 respectively), ELISA (soluble ICAM1 (sICAM1)), and fluorescence assay (secreted active MMP1). Under basal conditions, proMMP9 dimer and MMP9 were higher in endometriosis cell cultures. In stromal cultures derived from control women and those with endometriosis, TNF augmented the intracellular proMMP1 (1.2-fold in control stromal cells) and ICAM1 (1.4- and 1.9-fold), greatly increased MMP1 and proMMP9 levels, and the sICAM1 concentration (2.3- and 4.3-fold) in their media compared with basal levels. The combination of TNF and MMP9 increased the sICAM1 concentration 14-fold in the endometriosis cell media, whereas GM6001 inhibited the stimulatory effect of TNF in both cell cultures. The deregulation of MMP9, and the TNF participation in the MMP1 and proMMP9 secretions, in the MMP9 expression and in the expression and cleavage of ICAM1 may contribute to the pathophysiology of this disease.

Nuclear factor kappaB pathway and interleukin-6 are affected in eutopic endometrium of women with endometriosis.

In order to investigate the role of the nuclear factor kappaB (NFKB) pathway on gene expression in the eutopic endometrium in endometriosis, and in particular of interleukin-6 (IL6), we evaluated RELA, IkappaB kinase (CHUK), NFKBIA and IL6 expressions and NFKB DNA binding in eutopic endometrium from women with endometriosis. Eutopic endometrium was obtained from 37 women with endometriosis and 42 fertile women during laparoscopy. We analysed RELA, CHUK, NFKBIA and IL6 mRNA levels (RT-PCR); RELA, CHUK and NFKBIA proteins and p-NFKBIA/NFKBIA ratio (western blot); and NFKB binding (DNA shift assay) and IL6 concentration (ELISA) in endometrial explants. Our results indicate that mRNA and cytoplasmic proteins of RELA and CHUK exhibit constant levels in normal endometrium during the menstrual cycle. A dramatic increase (P<0.05) in NFKBIA mRNA expression, RELA nuclear presence and the mRNA and the protein of IL6 during late secretory phase was also observed in this tissue. By contrast, in eutopic endometrium from endometriosis patients, a decrease (P<0.05) in IL6 mRNA and protein (61%), NFKBIA mRNA (46%), p-NFKBIA/NFKBIA ratio (42%), RELA nuclear stromal (68%) and CHUK (48%) proteins were found exclusively during the late secretory phase compared with normal endometrium. In conclusion, the canonical activation of NFKB pathway is deregulated and may have reduced transcriptional function affecting NFKBIA and IL6 expression, genes related local proinflammatory processes. These molecular alterations observed during the late secretory phase in eutopic endometrium from endometriosis patients constitute a NFKB system dysfunction, suggesting that NFKB could be an important factor in endometriosis aetiology.

Augmented cell survival in eutopic endometrium from women with endometriosis: expression of c-myc, TGF-beta1 and bax genes.

BACKGROUND: Endometriosis is a common gynaecological disorder characterized by the presence of endometrial tissue outside of the uterus. The fragments in normal menstruation are composed of necrotic and living cells, which do not survive in ectopic locations because of programmed cell death. The aim of this study was to evaluate if the balance between cell proliferation and apoptosis is changed in eutopic endometrium from women with endometriosis throughout the menstrual cycle by studying bax (pro-apoptotic), c-myc (regulator of cell cycle) and TGF-beta1 (involved in cell differentiation) genes. METHODS: Eutopic endometrium was obtained from: 30 women with endometriosis (32.8 +/- 5 years) and 34 fertile eumenorrheic women (36 +/- 5.3 years). We analyzed apoptosis (TUNEL: DNA fragmentation); cell proliferation (immunohistochemistry (IHC) for Ki67); c-myc, bax and TGF-beta1 mRNA abundance (RT-PCR) and TGF-beta1 protein (IHC) in endometrial explants. RESULTS: Cell proliferation strongly decreased from proliferative to late secretory phases in glands, but not in stroma, in both endometria. Positive staining in glands and stroma from proliferative endometrium with endometriosis was 1.9- and 2.2-fold higher than control endometrium, respectively (p < 0.05). Abundance of c-myc mRNA was 65% higher in proliferative endometrium from endometriosis than normal tissue (p < 0.05). TGF-beta1 (mRNA and protein) augmented during mid secretory phase in normal endometrium, effect not observed in endometrium with endometriosis. In normal endometrium, the percentage of apoptotic epithelial and stromal cells increased more than 30-fold during late secretory phase. In contrast, in endometrium from endometriosis, not only this increase was not observed, besides bax mRNA decreased 63% versus normal endometrium (p < 0.05). At once, in early secretory phase, apoptotic stromal cells increased 10-fold with a concomitant augment of bax mRNA abundance (42%) in endometria from endometriosis (p < 0.05). CONCLUSION: An altered expression of c-myc, TGF-beta1 and bax was observed in eutopic endometrium from endometriosis, suggesting its participation in the regulation of cell survival in this disease. The augmented cell viability in eutopic endometrium from these patients as a consequence of a reduction in cell death by apoptosis, and also an increase in cell proliferation indicates that this condition may facilitate the invasive feature of the endometrium.

Differential in vitro actions of nitric oxide on human endometrial cell survival.

OBJECTIVE: To investigate the presence of caspase-3 and Bcl-2 concentration in human endometrial tissue throughout the menstrual cycle, and study the effect of nitric oxide (NO) on cell proliferation and apoptosis during culture. DESIGN: Expression of caspase-3 and Bcl-2 concentration in endometrial explants, and examination of L-arginine (L-Arg) effect on epithelial and stromal cell proliferation and apoptosis in vitro. SETTING: Prospective study.Twenty-seven eumenorrheic women (37 +/- 1.2 years). INTERVENTION(S): Endometrial samples were obtained with Pipelle suction curette from the corpus of the uterus. MAIN OUTCOME MEASURE(S): Apoptosis (annexin V-FITC binding), Bcl-2 concentration (ELISA), caspase-3 (immunohistochemistry), cell proliferation (spectrophotometric assay), and gene expression (RT-PCR). RESULT(S): Caspase-3 was detected by immunoassay in epithelial tissue throughout the menstrual cycle and in stroma during secretory phase. The Bcl-2 concentration was similar in endometrial homogenates obtained throughout the menstrual cycle, but L-Arg decreased Bcl-2 only in endometrium from the proliferative phase. In epithelial cells, NO increased apoptosis by 2.1 +/- 0.2-fold, augmented mRNA expression of Bax, and reduced expression of Bcl-2 compared with basal cultures. In stromal cells, NO increased cell proliferation in a dose-dependent manner, an effect that was blocked by a NO synthase inhibitor. CONCLUSION(S): These data indicate that NO has a differential regulatory function on endometrial cell survival, as indicated by the results on stromal cell proliferation and epithelial cell apoptosis during culture, which suggests paracrine interactions between both cell types.

[P450Arom and estrogenic microenvironment of eutopic endometria in endometriosis]. / P450Arom y microambiente estrogénico en endometrios eutópicos de mujeres con endometriosis.

BACKGROUND: Endometriosis, a common gynecologic disorder characterized by endometrial glands and stroma outside the uterus, is diagnosed by direct visualization of peritoneal and ovarian implants during laparoscopy. AIM: To study the estrogenic microenvironment in eutopic endometria of women with and without endometriosis. PATIENTS AND METHODS: Eutopic endometria, obtained during laparoscopy from 23 women with endometriosis and 20 fertile cyclic women undergoing tubal sterilization, was studied. P450Arom mRNA expression (RT-PCR) was measured. Also, P450Arom activity was assessed measuring testosterone conversion to estradiol and the concentration of this last hormone in cultured endometrial explants. RESULTS: Age and body mass index was similar in both groups studied. Seventy nine percent of endometria from women with endometriosis and in 29.4% from control group expressed P450Arom mRNA (p <0.01). The intensity of the band was higher in secretory endometria from women with endometriosis when compared to controls (p <0.01), but it was similar during the proliferative phase. Estradiol secretion to the culture media by proliferative endometria explants from women with endometriosis was 3-fold higher than secretory endometria (p <0.01) and endometria from control women in both phases. P450Arom activity, in the presence of testosterone, was 7-fold higher in endometrial cultures from women with endometriosis, when compare with the basal culture (p <0.01). However, in endometrial explant cultures from control women, this activity was not statistical different. CONCLUSIONS: These results indicate that in women with endometriosis, the microenvironment in the endometria is estrogenic as a consequence of an increased expression and activity of the P450Arom.

Reduced natriuresis after oral sodium load in cholestatic rats: role of compartment volumes and ANP.

The purpose of this study was to assess the participation of the atrial natriuretic peptide (ANP)-cGMP system in electrolyte and volume handling of cholestatic rats submitted to an acute oral sodium load. Cholestasis was induced by ligation and section of the common bile duct (n = 51). Control rats were sham operated (n = 56). Three weeks after surgery, 24-hr urinary volume, sodium, potassium, cGMP and creatinine excretion were measured. Three days later, animals received 10 mmol/kg NaCl (1 M) by gavage, and urinary excretion was measured for 6 hr. In parallel groups of rats, plasma volume, electrolytes and ANP concentration, extracellular fluid volume (ECFV), and renal medullary ANP-induced cGMP production were determined in basal conditions or 1 hr after oral sodium overload. As compared with controls, cholestatic rats had a larger ECFV and higher plasma ANP (67.2 +/- 5.2 vs 39.7 +/- 3.5 pg/ml), but lower hematocrit and blood volume, and were hyponatremic. Cholestatic rats showed higher basal excretion of sodium, potassium, and volume than controls, but equal urinary cGMP. After the NaCl overload, cholestatic rats showed a reduced sodium excretion but equal urinary cGMP. One hr after sodium overload, both groups showed hypernatremia, but whereas in control rats ECFV and ANP increased (50.7 +/- 4.1 pg/ml), in cholestatic rats ECFV was unchanged, and plasma volume and ANP were reduced (37.5 +/- 5.8 pg/ml). ANP-induced cGMP production in renal medulla was similar in cholestatic and control nonloaded rats (14.2 +/- 5.2 vs 13.4 +/- 2.6 fmol/min/mg). One hr after the load, medullary cGMP production rose significantly in both groups, without difference between them (20.6 +/- 3.1 vs 22.7 +/- 1. 7 fmol/min/mg). We conclude that the blunted excretion of an acute oral sodium load in cholestatic rats is associated with lower plasma ANP due to differences in body fluid distribution and cannot be explained by renal refractoriness to ANP.

Croxatto's fifty-year pursuit: from pepsanurin to the discovery of a new kininogen-derived peptide (PU-D1).

This paper narrates Dr Héctor R Croxatto and collaborators' efforts over the past 50 years in search for peptidic hormones obtained by pepsin hydrolysis of blood plasma substrates. In the forties, Croxatto described three peptidic fractions characterized by their hypertensive, oxytocic and antidiuretic properties, designated as pepsitensin, pepsitocin and pepsanurin, respectively. While pepsitensin and pepsitocin were later identified as angiotensin I and metlys-bradykinin, pepsanurin was not identified and its research was halted for 35 years. During that time, Prof Croxatto and his group worked mostly on the renal kallikrein-kinin system, studying its physiological anti-hypertensive role, making significant contributions in the field of renovascular hypertension. After the discovery of atrial natriuretic peptide, Croxatto resumed his work with pepsanurin. In a series of papers from 1988 to 1998, it was shown that: 1) when injected intraperitoneally or in the intestinal lumen of anesthetized rats, or in the isolated perfused rat kidneys, pepsanurin is a potent inhibitor of the natriuretic effect of ANP; 2) plasma kininogens are identified as the substrates for pepsanurin formation; 3) bradykinin and prokinins exert the anti-ANP effect when injected either intravenously, intraperitoneally or intraduodenally, at small non-vasodilator doses; endogenous kinins also block ANP renal excretory effects; 4) a 20-amino acid peptide released by pepsin from domain 1 of purified LMW kininogen was isolated by Croxatto and collaborators, designed as PU-D1, and shown to exert similar anti-ANP effects as pepsanurin or kinins, but being more potent and longer lasting; 5) the anti-ANP effect of pepsanurin, kinins and PU-D1 is mediated by B2 kinin receptors, since it is blocked by a bradykinin receptor antagonist. Currently, Dr Croxatto is working on the hypothesis that intestinal-borne kinins and/or PU-D1 may reduce renal excretion during the prandial cycle.

[Glucose in conjunction with peptides derived from kininogens might act from digestive tract as blockers of atrial natriuretic peptide mediated diuresis-natriuresis]. / La glucosa en conjunto con péptidos derivados de cininógenos podrían actuar desde el aparato digestivo como bloqueadores de la acción diurética-natriurética del péptido natriurético auricular.

This paper describes long term research efforts which have lead: 1) to the identification of peptides present in pepsanurin, a peptidic fraction obtained by pepsin hydrolysis of plasma globulins that inhibits the renal excretory action of atrial natriuretic peptide (ANP) and 2) to the discovery of an unexpected role of glucose, as a requisite for these inhibitory effects. The active peptides identified in pepsanurin are derived from plasma kininogens, substrates of the kallikrein-kinin system. Pro-kinins of 15, 16 and 18 amino acids, and bradykinin itself, block ANP-induced diuresis and natriuresis when injected i.v., i.p. or into the duodenal lumen of anesthetized rats in picomol doses. Furthermore, a novel 20 amino acids fragment derived from kininogen dominium-1, named PU-D1, is the most potent and longer lasting blocker of ANP renal effects. The anti-ANP effects of those peptides are prevented by B2-kinin receptor antagonists. The inhibition of ANP by kinins and PU-D1 was evident only in rats infused with isotonic glucose; whereas the excretory effect of ANP was not affected in fasted rats not infused, or infused with saline. These findings provide evidence that glucose facilitates liquid retention through a kinin-mediated inhibition of ANP excretory action that may be related to the prandial cycle.

Kinins mediate the inhibition of atrial natriuretic peptide diuretic effect induced by pepsanurin.

Pepsanurin is a peptidic fraction resulting from pepsin digestion of plasma globulins, that inhibits ANP renal excretory actions. We studied whether kinin-like peptides mediate the anti-ANP effect by testing if pepsanurin: 1) was blocked by the kinin B2 receptor antagonist HOE-140, 2) was produced from kininogen, and 3) was mimicked by bradykinin. Anti-ANP activity was assessed in anesthetized female rats by comparing the excretory response to two ANP boluses (0.5 microgram i.v.) given before and after i.p. injection of test samples. Pepsanurin from human or rat plasma (1-5 mL/kg), and bradykinin (5-20 micrograms/kg), dose-relatedly inhibited ANP-induced water, sodium, potassium and cyclic GMP urinary excretion, without affecting arterial blood pressure. The same effect was exerted by pepsin hydrolysates of purified kininogen, whereas hydrolysates of kininogen-free plasma had no effect. HOE-140 (5 micrograms, i.v.) did not alter baseline, or ANP-induced excretion, but blocked the anti-ANP effects of pepsanurin. Histamine (15 micrograms/kg) plus seroalbumin hydrolysates did not affect ANP response, despite inducing larger peritoneal fluid accumulation as compared with pepsanurin or bradykinin. We concluded that kinins cleaved from kininogen mediate the anti-ANP effects of pepsanurin by activation of kinin B2 receptors, independently of changes in systemic arterial pressure or peritoneal fluid sequestration.

A peptide released by pepsin from kininogen domain 1 is a potent blocker of ANP-mediated diuresis-natriuresis in the rat.

A 20-amino acid peptide, KYEIKEGDCPVQSGKTWQDC (PU-D1), released by pepsin hydrolysis of LMW kininogen domain 1 was tested for its ability to antagonize the diuretic and natriuretic effect of ANP(103-125) in anesthetized rats. A single dose of 10.8 or 21.6 pmol (25 or 50 ng) PU-D1 given intravenously or into the duodenal lumen suppressed the diuresis-natriuresis induced by 209 pmol (500 ng) ANP by 43% to 59% and 69% to 96%, respectively. None of the doses tested (2.16 to 432 pmol, 5 ng to 1 microg) modified systemic blood pressure. Strikingly, a single IV dose of 10.8 pmol PU-D1 blocked the action of ANP for more than 3 hours. ANP blockade by PU-D1 was annulled completely by the bradykinin (BK) B2 receptor inhibitor Hoe 140. On a molar basis, PU-D1 is more effective than BK and kinins of 15, 16, and 18 amino acids for blocking the ANP-mediated diuresis-natriuresis. As with BK and other kinins, the inhibitory effect of Pu-D1 on ANP is obtained only within a small range of picomol doses. A single dose of 2.16 or 4.32 pmol PU-D1 or 47 pmol (50 ng) BK is ineffective against ANP if injected alone. However, when both substances are administered concomitantly at these subthreshold doses, they totally suppress ANP-induced diuresis-natriuresis. These results raise the question of whether PU-D1, released from kininogen domain 1, either alone or associated with BK, may interact with ANP in the regulation of urinary water and electrolyte excretion in physiological and pathological conditions.

A fragment of human kininogen containing bradykinin blunts the diuretic effect of atrial natriuretic peptide.

A synthetic 15 aminoacids kinin, named PU-15, is able to block the diuretic natriuretic action of Atrial Natriuretic Peptide (ANP). The structure of PU-15 is Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Iso, having the aminoacid sequence of a fragment of human kininogens. The increase in the urinary excretion of sodium, potassium, and water, elicited by a bolus of 0.5 microg of ANP in anesthetized rats, is blocked by PU-15 (100-150 ng) given either intravenously 3 min before ANP injection, or injected intraperitoneally or in the duodenal lumen, 40 min before ANP. This ANP blockade, which mimics the action of pepsanurin, is only obtained with doses of PU-15 in a narrow range around 100 picomol/rat, and do not modify blood pressure. Larger doses, 2- to 8-fold the effective dose, either do not change the response to ANP or raise the excretion of sodium and water. The administration of HOE-140, a bradykinin B2 receptor blocker, prior to PU-15, completely abolishes the anti-ANP action of PU-15. These findings lend support to the proposal that kinins released from the intestinal tract during prandial period can modulate renal excretory function.

Isolation of a bovine uterotropic placental factor and development of an enzyme immunoassay in peripheral serum.

Bovine uterotropic placental factor (bUTPF) was obtained from bovine term placentae that had been homogenized and extracted in acidic medium. The extracts were chromatographed on Sephadex G-75, a fraction named native bUTPF was obtained, and an antiserum was generated. The native bUTPF was chromatographed on Sephacryl S-300 HR, and a 230-kDa fraction was obtained. A competitive enzyme immunoassay (EIA) was developed to determine bUTPF levels during gestation. This EIA was linear between 0 and 80 micrograms/ml (with a detection limit of 0.2 microgram/ml) and showed intraassay and interassay coefficients of variation of 11.2% and 15.5%, respectively. Bovine UTPF serum levels during gestation were determined by means of a cross-sectional study between the first month of gestation and delivery. Maximal serum levels of bUTPF were found during the first month of gestation: 7.1 +/- 1.9 micrograms/ml (p < 0.05). In another cross-sectional study, blood samples were collected from 105 cows between 8 and 25 days postinsemination (day of insemination = Day 0). Levels of bUTPF were found to be significantly higher (p < 0.05) by Day 17 compared to Day 0 (5.8 +/- 2.2 micrograms/ml vs. 1.2 +/- 0.7 micrograms/ml). We conclude that bUTPF is detected by EIA in maternal peripheral serum early during gestation in the bovine species.

[Digestive process and regulation of renal excretory function: pepsanurin and prokinin inhibit the natriuretic peptide diuretic action]. / El proceso digestivo y la regulación de la función excretora renal: pepsanurina y procininas inhibidoras de la acción diurética del péptido natriurético auricular.

In order to clarify the blunting effect of peptides released by pepsin from blood plasma on ANP diuretic action, two prokinins designated PU-18 and PU-16 were tested. Both of them were able to inhibit in nanomolar doses the diuretic-saluretic action of 0.5 ug i.v. bolus of ANP given to anesthetized rats either by intravenous route or introduced in the duodenal lumen. PU-16 in doses of 0.5 and 1 ug were able to reduce in 72 and 96.5% respectively the natriuresis induced by 0.5 ug intravenous bolus of ANP. The data support the hypothesis that prokinins liberated in the digestive tract, could be physiological factors involved in hydrosaline homeostasis, moderating the ANP mediated increase of water, Na and K excretion during digestion.

Blunting effect of pepsanurin introduced in the duodenum on the atrial natriuretic peptide diuretic action in rats.

Pepsanurin (PU) is a peptide(s) obtained by pepsin hydrolysis of human plasma or its globulin fraction. We have reported that the accelerated renal excretory rate induced by atrial natriuretic peptide (ANP) can be considerably blunted by PU either in the intact rat or in the isolated perfused rat kidney. We explored whether or not PU can be part of a signaling mechanism originated in the digestive tract, involved in the regulation of water and electrolyte homeostasis. PU obtained either from human (0.5 ml) or rat plasma (0.25-0.5 ml) administered into the duodenal lumen of rats, counteracted significantly the diuretic-saluretic action of a 0.5- microgram bolus of ANP, reproducing qualitatively the effect of its intraperitoneal administration. Human PU reduced the ANP-stimulated renal excretion by 67-90% for Na (P < 0.001) and by 35-54% for water (P < 0.02-P < 0.001); the inhibition induced by rat PU was 45-96% for Na (P < 0.05-P < 0.01) and 35-65% for water (P < 0.05-P < 0.01). Rat PU (0.5 ml) abolished the rise of glomerular filtration rate induced by ANP without affecting fractional Na excretion. All the samples tested decreased K excretion, but in some experiments, the difference did not reach statistical significance. Contrary to the effect of PU, the introduction in the duodenum of (i) isotonic glucose solution, (ii) hydrolysate of bovine serum albumin, or (iii) hydrolysate of casein prepared after the same procedure yielding PU from plasma failed to produce an inhibition of the ANP stimulation of renal excretory rate. In addition, human plasma incubated at 37 degrees C for 24 to 48 hr, prior to pepsin digestion, did not yield PU, which indicates that PU is generated from a substrate sensitive to endogenous enzymes and/or that its stability is vulnerable to endogenous enzymes.