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The vasotocin/vasopressin and isotocin/mesotocin/oxytocin family of nonapeptides regulate social behaviors and physiological functions associated with reproductive physiology and osmotic balance. While experimental and correlative studies provide evidence for these nonapeptides as modulators of behavior across all classes of vertebrates, mechanisms for nonapeptide inactivation in regulating these functions have been largely overlooked. Leucyl-cystinyl aminopeptidase (LNPEP) - also known as vasopressinase, oxytocinase, placental leucine aminopeptidase (P-LAP), and insulin-regulated aminopeptidase (IRAP) - is a membrane-bound zinc-dependent metalloexopeptidase enzyme that inactivates vasopressin, oxytocin, and select other cyclic polypeptides. In humans, LNPEP plays a key role in the clearance of oxytocin during pregnancy. However, the evolutionary diversity, expression distribution, and functional roles of LNPEP remain unresolved for other vertebrates. Here, we isolated and sequenced a full-length cDNA encoding a LNPEP-like polypeptide of 1033 amino acids from the ovarian tissue of Amargosa pupfish, Cyprinodon nevadensis. This deduced polypeptide exhibited high amino acid identity to human LNPEP both in the protein's active domain that includes the peptide binding site and zinc cofactor binding motif (53.1% identity), and in an intracellular region that distinguishes LNPEP from other aminopeptidases (70.3% identity). Transcripts encoding this LNPEP enzyme (lnpep) were detected at highest relative abundance in the gonads, hypothalamus, forebrain, optic tectum, gill and skeletal muscle of adult pupfish. Further evaluation of lnpep transcript abundance in the brain of sexually-mature pupfish revealed that lnpep mRNAs were elevated in the hypothalamus of socially subordinate females and males, and at lower abundance in the telencephalon of socially dominant males compared to dominant females. These findings provide evidence of an association between behavioral social status and hypothalamic lnpep transcript abundance and suggest that variation in the rate of VT/IT peptide inactivation by LNPEP may be a contributing component in the mechanism whereby nonapeptides regulate social behavior.
Insulin-regulated aminopeptidase (IRAP, cystinyl aminopeptidase, CysAP) and aminopeptidase M (alanyl aminopeptidase, AlaAP) are closely related enzymes involved in cognitive, metabolic, and cardiovascular functions. These functions may be modulated by the type of fat used in the diet. In order to analyze a possible coordinated response of both enzymes we determined simultaneously their activities in frontal cortex, liver, and plasma of adult male rats fed diets enriched with fats differing in their percentages of saturated, mono or polyunsaturated fatty acids such as sesame, sunflower, fish, olive, Iberian lard, and coconut. The systolic blood pressure, food intake, body and liver weight as well as glucose and total cholesterol levels in plasma were measured. The type of fat in the diet influences the enzymatic activities depending on the enzyme and its location. These results suggest cognitive improvement properties for diets with predominance of polyunsaturated fatty acids. Physiological parameters such as systolic blood pressure, food intake, and biochemical factors such as cholesterol and glucose in plasma were also modified depending on the type of diet, supporting beneficial properties for diets rich in mono and polyunsaturated fatty acids. Inter-tissue correlations between the analyzed parameters were also modified depending on the type of diet. If the type of fat used in the diet modifies the behavior and relationship between CysAP and AlaAP in and between frontal cortex, liver and plasma, the functions in which they are involved could also be modified.
This study analyzes the fluorimetric determination of alanyl- (Ala), glutamyl- (Glu), leucyl-cystinyl- (Cys) and aspartyl-aminopeptidase (AspAp) urinary enzymatic activities as early and predictive biomarkers of renal dysfunction in cisplatin-treated rats. Male Wistar rats (n = 8 each group) received a single subcutaneous injection of either saline or cisplatin 3.5 or 7 mg/kg, and urine samples were taken at 0, 1, 2, 3 and 14 days after treatment. In urine samples we determined Ala, Glu, Cys and AspAp activities, proteinuria, N-acetyl-β-D-glucosaminidase (NAG), albumin, and neutrophil gelatinase-associated lipocalin (NGAL). Plasma creatinine, creatinine clearance and renal morphological variables were measured at the end of the experiment. CysAp, NAG and albumin were increased 48 hours after treatment in the cisplatin 3.5 mg/kg treated group. At 24 hours, all urinary aminopeptidase activities and albuminuria were significantly increased in the cisplatin 7 mg/kg treated group. Aminopeptidase urinary activities correlated (p<0.011; r(2)>0.259) with plasma creatinine, creatinine clearance and/or kidney weight/body weight ratio at the end of the experiment and they could be considered as predictive biomarkers of renal injury severity. ROC-AUC analysis was made to study their sensitivity and specificity to distinguish between treated and untreated rats at day 1. All aminopeptidase activities showed an AUC>0.633. We conclude that Ala, Cys, Glu and AspAp enzymatic activities are early and predictive urinary biomarkers of the renal dysfunction induced by cisplatin. These determinations can be very useful in the prognostic and diagnostic of renal dysfunction in preclinical research and clinical practice.
In the present study, we analyzed the activity of several aminopeptidases (angiotensinases) involved in the metabolism of various angiotensin peptides, in pituitary and adrenal glands of untreated Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) or treated with the antihypertensive drugs captopril and propranolol or with the L-Arginine hypertensive analogue L-NG-Nitroarginine Methyl Ester (L-NAME). Intra- and inter-gland correlations between angiotensinase activities were also calculated. Membrane-bound alanyl-, cystinyl-, and glutamyl-aminopeptidase activities were determined fluorometrically using aminoacyl-β-naphthylamide as substrates. Depending on the type of angiotensinase analyzed, the results reflect a complex picture showing substantial differences between glands, strains, and treatments. Alanyl-aminopeptidase responsible for the metabolism of Ang III to Ang IV appears to be the most active angiotensinase in both pituitary and adrenals of WKY and particularly in SHR. Independently of treatment, most positive correlations are observed in the pituitary gland of WKY whereas such positive correlations are predominant in adrenals of SHR. Negative inter-gland correlations were observed in control SHR and L-NAME treated WKY. Positive inter-gland correlations were observed in captopril-treated SHR and propranolol-treated WKY. These results may reflect additional mechanisms for increasing or decreasing systolic blood pressure in WKY or SHR.
(1) Background: Prolonged feeding with a high-fat diet (HFD) acts as a stressor by activating the functions of the hypothalamic-pituitary-adrenal gland (HPA) stress axis, accompanied of hypertension by inducing the renin-angiotensin-aldosterone system. Angiotensinases enzymes are regulatory aminopeptidases of angiotensin metabolism, which together with the dipeptidyl peptidase IV (DPP-IV), pyroglutamyl- and tyrosyl-aminopeptidase (pGluAP, TyrAP), participate in cognitive, stress, metabolic and cardiovascular functions. These functions appear to be modulated by the type of fat used in the diet. (2) Methods: To analyze a possible coordinated response of aminopeptidases, their activities were simultaneously determined in the hypothalamus, adenohypophysis and adrenal gland of adult male rats fed diets enriched with monounsaturated (standard diet (S diet) supplemented with 20% virgin olive oil; VOO diet) or saturated fatty acids (diet S supplemented with 20% butter and 0.1% cholesterol; Bch diet). Aminopeptidase activities were measured by fluorimetry using 2-Naphthylamine as substrates. (3) Results: the hypothalamus did not show differences in any of the experimental diets. In the pituitary, the Bch diet stimulated the renin-angiotensin system (RAS) by increasing certain angiotensinase activities (alanyl-, arginyl- and cystinyl-aminopeptidase) with respect to the S and VOO diets. DPP-IV activity was increased with the Bch diet, and TyrAP activity decrease with the VOO diet, having both a crucial role on stress and eating behavior. In the adrenal gland, both HFDs showed an increase in angiotensinase aspartyl-aminopeptidase. The interrelation of angiotensinases activities in the tissues were depending on the type of diet. In addition, correlations were shown between angiotensinases and aminopeptidases that regulate stress and eating behavior. (4) Conclusions: Taken together, these results support that the source of fat in the diet affects several peptidases activities in the HPA axis, which could be related to alterations in RAS, stress and feeding behavior.
Oxytocin is a hormone with functions in: reproduction, maternal bonding, milk ejection, and feeding/social behavior, and is reported to be present in a variety of tissues. Our goal is to characterize oxytocin and leucyl and cystinyl aminopeptidase (LNPEP/oxytocinase), a key regulator of oxytocin in mares. We measured serum and tissue LNPEP by ELISA from ovulation (D0) until D21-22 in non-pregnant (n = 5) and pregnant mares (n = 6); and in periparturient and postpartum mares (n = 18). Placenta (n = 7) and homogenized tissue of diestrus mares (n = 6) were evaluated using protein determinations and LNPEP ELISAs. Identification of LNPEP and OXT protein in tissues was also performed via western blot, immunohistochemistry and liquid chromatography-mass spectrometry (LC-MS/MS). Furthermore, in situ hybridization was performed for LNPEP and OXT on endometrium, myometrium, pituitary and corpus luteum (CL). Serum LNPEP concentration were similar. Placental LNPEP U/mg protein was highest in the body and pregnant horn. The highest to lowest LNPEP U/mg protein by tissue were: myometrium > follicle wall > endometrium > kidney > CL > liver. Oxytocin was identified in the equine pituitary, CL and placenta and is likely to act in autocrine or paracrine manner, while LNPEP may act systemically and locally to regulate the availability of OXT.
High impulsivity characterizes a myriad of neuropsychiatric diseases, and identifying targets for neuropharmacological intervention to reduce impulsivity could reveal transdiagnostic treatment strategies. Motor impulsivity (impulsive action) reflects in part the failure of "top-down" executive control by the medial prefrontal cortex (mPFC). The present study profiled the complete set of mRNA molecules expressed from genes (transcriptome) in the mPFC of male, outbred rats stably expressing high (HI) or low (LI) motor impulsivity based upon premature responses in the 1-choice serial reaction time (1-CSRT) task. RNA-sequencing identified expression of 18 genes that was higher in the mPFC of HI vs. LI rats. Functional gene enrichment revealed that biological processes related to calcium homeostasis and G protein-coupled receptor (GPCR) signaling pathways, particularly glutamatergic, were overrepresented in the mPFC of HI vs. LI rats. Transcription factor enrichment identified mothers against decapentaplegic homolog 4 (SMAD4) and RE1 silencing transcription factor (REST) as overrepresented in the mPFC of HI rats relative to LI rats, while in silico analysis predicted a conserved SMAD binding site within the voltage-gated calcium channel subunit alpha1 E (CACNA1E) promoter region. qRT-PCR analyses confirmed that mRNA expression of CACNA1E, as well as expression of leucyl and cystinyl aminopeptidase (LNPEP), were higher in the mPFC of HI vs. LI rats. These outcomes establish a transcriptomic landscape in the mPFC that is related to individual differences in motor impulsivity and propose novel gene targets for future impulsivity research.
Thyroid disorders affect the hypothalamic-pituitary-adrenal axis with important consequences on the cardiovascular function in which the renin-angiotensin system plays a major role. Hypo and hyperthyroidism influence the classic main components of the renin-angiotensin system. However, the behavior of other elements of the renin-angiotensin system such as Ang III, Ang 2-10, Ang IV, or AT4, regulated by angiotensinase enzymes such as alanyl- (AlaAP), cystinyl- (CysAP), glutamyl- (GluAP), or aspartyl-aminopeptidase (AspAP), has not yet been described. In order to obtain a comprehensive view on the response of the renin-angiotensin system in the hypothalamic-pituitary-adrenal axis of animals with thyroid disorders, these enzyme activities were simultaneously analyzed fluorometrically, using arylamide derivatives as substrates in hypothalamus, anterior and posterior pituitary, adrenals and plasma of euthyroid, hypothyroid, and hyperthyroid rats, and their intra- and inter-tissue correlations were evaluated. The response is depending on the type of enzyme studied, its location and the thyroid status. Anterior pituitary, adrenals and plasma were mainly affected by the thyroid disorders. In the anterior pituitary, GluAP and AspAP increased in hypothyroid rats. In adrenals, AlaAP and CysAP decreased in hypothyroid whereas GluAP and AspAP decreased in hyperthyroid rats. In plasma, while AlaAP increased in hypo- and hyperthyroid rats, CysAP and GluAP decreased only in hyperthyroid. In comparison with euthyroid, intra-tissue correlations decreased in hypothyroid but inter-tissue correlations decreased mainly in hyperthyroid rats. Thyroid disorders also produced a disruption in the pattern of inter-tissue correlations observed in euthyroid. These results suggest that thyroid hormone levels hit components of the renin-angiotensin system and may influence the paracrine and endocrine cross talk between cells.
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