M. Luz Candenas

Tachykinin Receptor and Neutral Endopeptidase Gene Expression in the Rat Uterus: Characterization and Regulation in Response to Ovarian Steroid Treatment1

Tachykinin neuropeptides, such as substance P, are localized to a population of sensory fibers that innervate the mammalian female reproductive tract. In the present study, we have characterized tachykinin NK1 receptor (NK1R), NK2 receptor (NK2R), and NK3 receptor (NK3R) gene expression by semiquantitative RT-PCR in uteri from ovariectomized rats and studied their regulation in response to 17β-estradiol (E2), progesterone (P4), or a combination of both. In addition, we analyzed the expression and regulation of the neutral endopeptidase 24.11 (NEP), the most important enzyme involved in tachykinin degradation in the rat uterus. In uteri from control (olive oil-treated) rats, RT-PCR assays revealed single bands corresponding to the expected product sizes encoding complementary DNA for NK1R (232 bp), NK2R (491 bp), NK3R (325 bp), and NEP (221 bp). The identity of the amplified fragments was confirmed by DNA sequence analysis. Compared with control rats, NK1R messenger RNA (mRNA) was increased by 2-fold in ut...

A Role for Tachykinins in Female Mouse and Rat Reproductive Function

Abstract Tachykinins may be involved in reproduction. A reverse transcription-polymerase chain reaction assay was used to analyze the expression of tachykinins and tachykinin receptors in different types of reproductive cells from mice. The preprotachykinin (PPT) genes, PPT-A, PPT-B and PPT-C, that encode substance P/neurokinin A, neurokinin B, and hemokinin-1, respectively, and the genes that encode the tachykinin NK1, NK2, and NK3 receptors were all expressed, at different levels, in the uterus of superovulated, unfertilized mice. The mRNA of neprilysin (NEP), the main enzyme involved in tachykinin metabolism, was also expressed in the uterus. Isolated cumulus granulosa cells expressed PPT-A, PPT-B, PPT-C, and NEP and low levels of the tachykinin NK1 and NK2 receptors. Mouse oocytes expressed PPT-A and -B mRNA transcripts. A low expression of the three tachykinin receptors was observed but PPT-C and NEP were undetectable. Two- and 8- to 16-cell mouse embryos expressed only a low-abundance transcript corresponding to the NK1 receptor. However, the mRNAs of PPT-B, PPT-C and NEP appeared in blastocyst-stage embryos. A low-abundance transcript corresponding to the NK2 receptor was the only target gene detected in mice sperm. Female mice or rats treated neonatally with capsaicin showed a reduced fertility. A reduction in litter size was observed in female rats treated in vivo with the tachykinin NK3 receptor antagonist SR 142801. These data show that tachykinins of both neuronal and nonneuronal origin are differentially expressed in various types of reproductive cells and may play a role in female reproductive function.

Tachykinins and tachykinin receptors in human uterus

Studies were undertaken to determine the nature of the receptors mediating contractile effects of tachykinins in the uteri of nonpregnant women, and to analyse the expression of preprotachykinins (PPT), tachykinin receptors and the cell‐surface peptidase, neprilysin (NEP), in the myometrium from pregnant and nonpregnant women. The neurokinin B (NKB) precursor PPT‐B was expressed in higher levels in the myometrium from nonpregnant than from pregnant women. Faint expression of PPT‐A mRNA was detectable in the myometrium from nonpregnant but not pregnant women. PPT‐C, the gene encoding the novel tachykinin peptide hemokinin‐1 (HK‐1), was present in trace amounts in the uteri from both pregnant and nonpregnant women. Tachykinin NK2 receptors were more strongly expressed in tissues from nonpregnant than from pregnant women. NK1 receptor mRNA was present in low levels in tissues from both pregnant and nonpregnant women. A low abundance transcript corresponding to the NK3 receptor was present only in tissues from nonpregnant women. The mRNA expression of the tachykinin‐degrading enzyme NEP was lower in tissues from nonpregnant than from pregnant women. Substance P (SP), neurokinin A (NKA) and NKB, in the presence of the peptidase inhibitors thiorphan, captopril and bestatin, produced contractions of myometrium from nonpregnant women. The order of potency was NKA≫SP≥NKB. The potency of NKA was unchanged in the absence of peptidase inhibitors. The tachykinin NK2 receptor‐selective agonist [Lys5MeLeu9Nle10]NKA(4–l0) was approximately equipotent with NKA, but the tachykinin NK1 and NK3 receptor‐selective agonists [Sar9Met(O2)11]SP and [MePhe7]NKB were ineffective in the myometrium from nonpregnant women. The uterotonic effects of [Lys5MeLeu9Nle10]NKA(4–10) were antagonized by the tachykinin NK2 receptor‐selective antagonist SR48968. Neither atropine, nor phentolamine nor tetrodotoxin affected responses to [Lys5MeLeu9Nle10]NKA(4–10). These data are consistent with a role of tachykinins in the regulation of human uterine function, and reinforce the importance of NK2 receptors in the regulation of myometrial contraction.

Changes in the Expression of Tachykinin Receptors in the Rat Uterus During the Course of Pregnancy

Abstract In the mammalian female reproductive tract, tachykinin neuropeptides, such as substance P (SP), are localized to a population of sensory fibers and their precise physiological role is still unknown. The aim of the present study was to characterize the population of tachykinin receptors in the pregnant rat uterus and to assess their regulation during the course of pregnancy and after delivery. The expression of the tachykinin NK1 receptor (NK1R), the tachykinin NK2 receptor (NK2R), and the tachykinin NK3 receptor (NK3R) in uteri from rats at different stages of pregnancy and on Day 1 postpartum was investigated by using a semiquantitative reverse transcription-polymerase chain reaction. The contractile effect of tachykinin receptor agonists acting selectively on the NK1R, the NK2R, or the NK3R was investigated by conventional organ bath techniques. Serum levels of estrogen and progesterone were measured by RIA. Our data show that the expression and function of NK1R and NK3R varied along the course of pregnancy and at postpartum. Uterine NK2R mRNA levels remain stable during the course of pregnancy and at Day 1 postpartum; and the contractions elicited by activating selectively the NK2 receptor in the presence of the neutral endopeptidase inhibitor phosphoramidon (1 μM) were similar in early, mid, or late pregnancy. These results show that the expression and function of tachykinin receptors within the uterus vary with reproductive state and length of gestation, supporting a role for tachykinins in pregnancy and/or parturition in the rat.

Identification of a tachykinin NK2 receptor splice variant and its expression in human and rat tissues

The tachykinins substance P, neurokinin A and neurokinin B are implicated in different diseases and play an important role in neuroimmunomodulation. These peptides interact with three distinct types of tachykinin receptors termed NK(1), NK(2) and NK(3). While most mammalian genes encoding G protein-coupling cell membrane receptors are intron-less, the three tachykinin receptors contain introns in their genomic structures. In the present study, we have identified a splice variant of the tachykinin NK(2) receptor that results from skipping of exon 2 in the processing of the tachykinin NK(2) receptor mRNA. By using reverse transcription-polymerase chain reaction analysis, we observed that the tachykinin NK(2) receptor splice variant, that we named NK(2)beta, appeared in different human and rat tissues that also express the wild type, tachykinin NK(2)alpha isoform. Compared to tachykinin receptor NK(2)alpha isoform mRNA levels, the NK(2)beta isoform was strongly expressed in human and rat uteri, expressed in a moderate degree in the rat urinary bladder, colon, duodenum and stomach and unexpressed in the rat cerebral cortex, kidney, thoracic aorta, skeletal muscle and heart. These data describe the first known tachykinin receptor splice variant and suggest that the variety of tachykinin receptors may be further expanded through the generation of splicing isoforms. The presence of the truncated isoform may have a physiological significance in the regulation of tachykinin NK(2) receptor protein levels.

Characterization of tachykinin receptors in the uterus of the oestrogen-primed rat

The aim of our study was to characterize the tachykinin receptor population in the oestrogen‐primed rat uterus. For this purpose, we investigated the receptor type(s) responsible for tachykinin‐induced contraction of longitudinally‐arranged smooth muscle layer. The effects of substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and several of their analogues with well‐defined selectivities for tachykinin NK1, NK2 and NK3 receptors were studied and their inhibition by the selective nonpeptide tachykinin receptor antagonists (S)1‐(2‐[3‐(3,4‐dichlorophenyl)‐1‐(3‐isopropoxyphenylacetyl)piperidin‐3‐yl]ethyl)‐4‐phenyl‐1‐azoniabicyclo[2.2.2]octane chloride (SR 140333, NK1‐selective), (S)‐N‐methyl‐N[4‐(4‐acetylamino‐4‐phenylpiperidino)‐2‐(3,4‐dichlorophenyl)butyl]benzamide (SR 48968, NK2‐selective) and (R)‐(N)‐(1‐(3‐(1‐benzoyl‐3‐(3,4‐dichlorophenyl)piperidin‐3‐yl)propyl)‐ 4‐phenylpiperidin‐ 4‐yl)‐N‐ methylacetamide (SR 142801, NK3‐selective) was evaluated. Additionally, expression of tachykinin receptor mRNA was examined by using the reverse transcription‐polymerase chain reaction (RT‐PCR). SP, NKA, [Nle10]‐NKA(4‐10), the analogue with selectivity at the tachykinin NK2 receptor type, and NKB elicited concentration‐dependent contractions of the rat uterus. The pD2 values were 5.95±0.19; 6.73±0.21; 7.53±0.12 and 5.76±0.21, respectively. The selective agonist for the tachykinin NK1 receptor [Sar9Met(O2)11]‐SP produced a small phasic response in the nanomolar concentration range. The selective tachykinin NK3 receptor agonist [MePhe7]‐NKB failed to induce any significant contraction. In the presence of the neutral endopeptidase inhibitor phosphoramidon (1 μM), the log concentration‐response curves to exogenous tachykinins and their analogues were shifted significantly leftwards. The pD2 values were 6.12±0.10, 8.04±0.07, 7.89±0.03 and 6.59±0.07 for SP, NKA, [Nle10]‐NKA(4‐10) and NKB, respectively. In the presence of phosphoramidon (1 μM), [Sar9Met(O2)11]‐SP (1 nM–0.3 μM) induced concentration‐dependent contractions of increasing amplitude when only one concentration of drug was applied to each uterine strip and the pD2 value was 7.61±0.89. [MePhe7]‐NKB induced small, inconsistent contractions and, therefore, a pD2 value could not be calculated. In experiments performed in the presence of phosphoramidon (1 μM), SR 48968 (3 nM–0.1 μM) caused parallel and rightward shifts in the log concentration‐response curves of NKA. The calculated pKB value was 9.16±0.08 and the slope of the Schild regression was 1.28±0.24. SR 48968 (0.1 μM) also antagonized responses to SP with an apparent pKB value of 7.63±0.13. SR 48968 (0.1 μM) inhibited contractions elicited by NKB (1 nM–3 μM) and [Nle10]‐NKA(4–10) (0.1 nM–3 μM) but had no effect on the response evoked by [Sar9Met(O2)11]‐SP (0.1 μM). SR 140333 (0.1 μM) inhibited responses to SP with an apparent pKB value of 7.19±0.22. This compound did not significantly affect responses to NKA, [Nle10]‐NKA(4‐10) and NKB, but suppressed [Sar9Met(O2)11]‐SP (0.1 μM)‐induced contraction. SR 142801 (0.1 μM) had no effect on responses to natural tachykinins or their analogues. Total RNA was extracted from some of the uteri used in functional studies. RT‐PCR assays revealed single bands corresponding to the expected product sizes encoding cDNA for tachykinin NK1 (587 base pairs) and NK2 receptors (491 base pairs) (n=6 different animals). A very low abundance transcript corresponding to the 325 base pairs product expected for the tachykinin NK3 receptor was detected. The present data show that functionally active tachykinin NK1 and NK2 receptors are expressed in the oestrogen‐primed rat uterus. The NK2 receptor type seems to be the most important one involved in the contractile responses elicited by tachykinins. NK3 receptors are present in trace amounts and seem not to be involved in tachykinin‐induced contractions.

Functional and Molecular Characterization of Tachykinins and Tachykinin Receptors in the Mouse Uterus

Abstract The aim of this study was to analyze the function and expression of tachykinins, tachykinin receptors, and neprilysin (NEP) in the mouse uterus. A previous study showed that the uterotonic effects of substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) in estrogen-treated mice were mainly mediated by the tachykinin NK1 receptor. In the present work, further contractility studies were undertaken to determine the nature of the receptors mediating responses to tachykinins in uteri of late pregnant mice. Endpoint and real-time quantitative RT-PCR were used to analyze the expression of the genes that encode the tachykinins SP/NKA, NKB, and hemokinin-1 (HK-1) (Tac1, Tac2, and Tac4); and the genes that encode tachykinin NK1 (Tacr1), NK2 (Tacr2), and NK3 (Tacr3) receptors in uteri from pregnant and nonpregnant mice. The data show that the mRNAs of tachykinins (particularly NKB and HK-1), tachykinin receptors, and NEP are locally expressed in the mouse uterus, and their expression changes during the estrous cycle and during pregnancy. The tachykinin NK1 receptor is the predominant tachykinin receptor in the nonpregnant and early pregnant mouse and may mediate tachykinin-induced uterine contractions in the nonpregnant mouse. The tachykinin NK2 receptor is predominant in the late pregnant mouse and is the main receptor mediating uterotonic responses to tachykinins at late pregnancy. The tachykinin NK3 receptor is expressed in considerable amounts only in uteri from nonpregnant diestrous animals, and its physiological significance remains to be clarified.

Expression and activity profiles of DPP IV/CD26 and NEP/CD10 glycoproteins in the human renal cancer are tumor-type dependent

BackgroundCell-surface glycoproteins play critical roles in cell-to-cell recognition, signal transduction and regulation, thus being crucial in cell proliferation and cancer etiogenesis and development. DPP IV and NEP are ubiquitous glycopeptidases closely linked to tumor pathogenesis and development, and they are used as markers in some cancers. In the present study, the activity and protein and mRNA expression of these glycoproteins were analysed in a subset of clear-cell (CCRCC) and chromophobe (ChRCC) renal cell carcinomas, and in renal oncocytomas (RO).MethodsPeptidase activities were measured by conventional enzymatic assays with fluorogen-derived substrates. Gene expression was quantitatively determined by qRT-PCR and membrane-bound protein expression and distribution analysis was performed by specific immunostaining.ResultsThe activity of both glycoproteins was sharply decreased in the three histological types of renal tumors. Protein and mRNA expression was strongly downregulated in tumors from distal nephron (ChRCC and RO). Moreover, soluble DPP IV activity positively correlated with the aggressiveness of CCRCCs (higher activities in high grade tumors).ConclusionsThese results support the pivotal role for DPP IV and NEP in the malignant transformation pathways and point to these peptidases as potential diagnostic markers.

Differential expression of neurokinin B and hemokinin-1 in human immune cells

The tachykinins are a group of related peptides that are mainly synthesized in the central and peripheral nervous system, but are also present in peripheral non-neuronal cells. In humans, substance P (SP) is the most extensively studied tachykinin and is present, along with the NK-1 receptor, in several inflammatory and immune cells. The release of SP under the appropriate stimulus may act as a paracrine or autocrine signal that may help to initiate and/or propagate inflammation. In the present study we have determined the expression pattern of NKB and HK-1 mRNA in human lymphocytes, monocytes, neutrophils and eosinophils. In addition, we have detected for the first time the presence of NKB protein in these cellular types. These findings reinforce the suggestion that tachykinins play a central role in the pathophysiology of the inflammatory process.

Expression of preprotachykinin-B, the gene that encodes neurokinin B, in the rat uterus

Neurokinin B, a peptide belonging to the tachykinin family, is undetectable in peripheral tissues from nonpregnant animals. In the present study, we analysed the expression of the preprotachykinin-B (PPT-B) gene, which encodes neurokinin B, in the rat uterus. Preprotachykinin-B mRNA was expressed in the uterus and its levels varied greatly depending upon the hormonal conditions. This is consistent with a role of this tachykinin in the regulation of uterine functions.