Veratridine-sensitive Na+ channels regulate human sperm fertilization capacity.

AIMS: The sperm plasma membrane contains specific ion channels and transporters that initiate changes in Ca2+, Na+, K+ and H+ ions in the sperm cytoplasm. Ion channels are key regulators of the sperm membrane potential, cytoplasmic Ca2+ and intracellular pH (pHi), which leads to regulate motility, capacitation, acrosome reaction and other physiological processes crucial for successful fertilization. Expression of epithelial sodium channels (ENaC) and voltage-gated sodium channels (Nav) in human spermatozoa has been reported, but the role of Na+ fluxes sodium channels in the regulation of sperm cell function remains poorly understood. In this context, we aimed to analyze the physiological role of Nav channels in human sperm. MAIN METHODS: Motility and hyperactivation analysis was conducted by CASA analysis. Flow cytometry and spectrophotometry approaches were carried out to measure Capacitation, Acrosome reaction, immunohistochemistry for Tyr-residues phosporylation, [Ca2+]i levels and membrane potential. KEY FINDINGS: Functional studies showed that veratridine, a voltage-gated sodium channel activator, increased sperm progressive motility without producing hyperactivation while the Nav antagonist lidocaine did induce hyperactivated motility. Veratridine increased protein tyrosine phosphorylation, an event occurring during capacitation, and its effects were inhibited in the presence of lidocaine and tetrodotoxin. Veratridine had no effect on the acrosome reaction by itself, but was able to block the progesterone-induced acrosome reaction. Moreover, veratridine caused a membrane depolarization and modified the effect of progesterone on [Ca2+]i and sperm membrane potential. SIGNIFICANCE: Our results suggest that veratridine-sensitive Nav channels are involved on human sperm fertility acquisition regulating motility, capacitation and the progesterone-induced acrosome reaction in human sperm.

The opioid peptide beta-endorphin stimulates acrosome reaction in human spermatozoa.

The acrosome reaction occurs in vivo following sperm capacitation and is essential for the acquisition of sperm fertilization ability. However, little is known about the molecular identity of the physiological acrosome reaction regulators. In addition to progesterone, which is produced by cumulus oophorus cells and known to regulate acrosome reaction by activating the specific calcium channel CatSper, endogenous opioid peptides such as beta-endorphin and met-enkephalin are present at high concentrations in the follicular fluid suggesting that the opioid system may be involved in the mechanisms regulating the acrosome reaction in humans. By using Reverse Transcription-PCR, western blot and immunofluorescence approaches, we described the presence and localization of the beta-endorphin precursor, pro-opiomelanocortinin the middle section and in flagellum of human spermatozoa, and inside the seminiferous tubules of human testis. Flow cytometry and intracellular calcium analyses showed that beta-endorphin causes an inversely dose-dependent increase in the percentage of acrosome-reacted sperm cells by a calcium-independent protein kinase C pathway. These findings are important for future studies of sperm physiology and provide new insight into the function of the opioid system as a target of fertility management.

Expression of neurokinin B/NK3 receptor and kisspeptin/KISS1 receptor in human granulosa cells.

STUDY QUESTION: Are neurokinin B (NKB), NK3 receptor (NK3R), kisspeptin (KISS1) and kisspeptin receptor (KISS1R) expressed in human ovarian granulosa cells? SUMMARY ANSWER: The NKB/NK3R and kisspeptin/KISS1R systems are co-expressed and functionally active in ovarian granulosa cells. WHAT IS KNOWN ALREADY: The NKB/NK3R and KISS1/KISS1R systems are essential for reproduction. In addition to their well-recognized role in hypothalamic neurons, these peptide systems may contribute to the control of fertility by acting directly on the gonads, but such a direct gonadal role remains largely unknown. STUDY DESIGN, SIZE, DURATION: This study analyzed matched mural granulosa cells (MGCs) and cumulus cells (CCs) collected from preovulatory follicles of oocyte donors at the time of oocyte retrieval. PARTICIPANTS/MATERIALS, SETTING, METHODS: The samples were provided by 56 oocyte donor women undergoing ovarian stimulation treatment. Follicular fluid samples containing MGCs and cumulus-oocyte complexes were collected after transvaginal ultrasound-guided oocyte retrieval. RT-PCR, quantitative real-time PCR, immunocytochemistry and western blot were used to investigate the pattern of expression of the NKB/NK3R and KISS/KISS1R systems in MGCs and CCs. Intracellular free Ca(2+) levels, [Ca(2+)]i, in MGCs after exposure to NKB or KISS1, in the presence or not of tachykinin receptor antagonists, were also measured. MAIN OUTCOME AND THE ROLE OF CHANCE: NKB/NK3R and KISS1/KISS1R systems were expressed, at the mRNA and protein levels, in MGCs and CCs, with significantly higher expression in CCs. Kisspeptin increased the [Ca(2+)]i in the cytosol of human MGCs while exposure to NKB failed to induce any change in [Ca(2+)]i. However, the [Ca(2+)]i response to kisspeptin was reduced in the presence of NKB. The inhibitory effect of NKB was only partially mimicked by the NK3R agonist, senktide and marginally suppressed by the NK3R-selective antagonist SB 222200. Yet, a cocktail of antagonists selective for the NK1, NK2 and NK3 receptors blocked the effect of NKB. LIMITATIONS, REASONS FOR CAUTION: The granulosa and cumulus cells were obtained from oocyte donors undergoing ovarian stimulation, which in comparison with natural cycles, may have affected gene and protein expression in granulosa cells. WIDER IMPLICATIONS OF THE FINDINGS: Our data demonstrate that, in addition to their indispensable effects at the central nervous system, the NKB/NK3R and kisspeptin/KISS1R systems are co-expressed and are functionally active in non-neuronal reproductive cells of the female gonads, the ovarian granulosa cells. STUDY FUNDING/ COMPETING INTERESTS: This work was supported by grants from Ministerio de Economía y Competitividad (CTQ2011-25564 and BFI2011-25021) and Junta de Andalucía (P08-CVI-04185), Spain. J.G.-O., F.M.P., M.F.-S., N.P., A.C.-R., T.A.A., M.H., M.R., M.T.-S. and L.C. have nothing to declare.

Effects of cysteinyl leukotrienes in small human bronchus and antagonist activity of montelukast and its metabolites.

BACKGROUND: Evidence suggests that small airways contribute to clinically significant processes in asthma. Cysteinyl leukotrienes (CysLTs) are considered to be pivotal mediators in the pathogenesis of asthma. Montelukast (MK), a specific CysLT1 receptor antagonist, is metabolized in two main hydroxylated metabolites (termed M5 and M6, respectively). OBJECTIVES: The aims of this study were to compare the responsiveness of small and large human bronchi to the three CysLTs, to evaluate the antagonist activity of MK, M5 and M6 in these preparations of human bronchi, and to characterize the CysLT receptors involved in the contractile response. METHODS AND RESULTS: In isolated small bronchus (i.d. 0.5-2 mm), the potencies (-log molar EC50) of LTC4, LTD4 and LTE4 were 9.3 (n=11), 9.1 (n=30) and 8.4 (n=14), respectively. The three CysLTs were about 30-fold more potent in small bronchi than in larger bronchi (i.d. 4-6 mm). In small bronchi, MK significantly shifted to the right the CysLT concentration-effect curves with pA2 values against LTC4, LTD4 and LTE4 of 9.1 (n=3), 9.0 (n=11) and 8.7 (n=5), respectively. The antagonist potencies of M6 and M5 were similar to MK and fivefold lower, respectively. A similar activity of MK against the three CysLTs suggested that CysLT1 receptors are involved in the contraction of human bronchus. Analysis by RT-PCR also indicated that human bronchus mainly expressed CysLT1 receptors. CONCLUSION: MK exerts a potent antagonist activity against the particularly potent constricting effects of CysLTs in isolated human small bronchi, which only expressed the CysLT1 receptor subtype. The metabolites of MK are also potent in vitro antagonists, but may not participate in the therapeutic activity of MK due to their low plasma concentrations in patients treated with the recommended dose of MK.

Hormonal regulation of the contractile response induced by okadaic acid in the rat uterus.

The contractile effect of okadaic acid (OA), a highly selective inhibitor of protein serine/threonine phosphatases, was analyzed in the rat uterus during the estrous cycle and during the course of pregnancy. Contractile effects were related to circulating levels of estrogen and progesterone and to mRNA levels of myosin light chain kinase (MLCK) and of myosin light chain protein phosphatase catalytic (PP1-delta) and larger regulatory subunit (MYPT). Both in nonpregnant and pregnant uteri, OA (20 microM) induced a transient contraction, which after plateauing, slowly decreased. In the nonpregnant uterus, the amplitude of this contraction varied at different stages of the estrous cycle, being higher at proestrus and lower at diestrus. In the pregnant uterus, the contraction to OA increased significantly during the course of pregnancy, reaching a maximum in day 21 pregnant rats, and declined after delivery. Whatever the day of pregnancy, the amplitude of the contraction to OA was not significantly modified when obtained in Ca(2+)-free solution. The magnitude of the OA-induced contraction in spontaneously cycling and pregnant rats was positively correlated to the ratio of estrogen/progesterone serum levels. Reverse transcription-polymerase chain reaction assays on myometrial tissue demonstrated that mRNA expression of PP1-delta and MYPT was higher at early (day 3) than at late (day 21) pregnancy. MLCK mRNA levels were similar in day 3 and day 21 pregnant rats. These data suggest that changes in the expression and activity of myosin phosphatase may contribute to modulating the level of uterine contractile force during the estrous cycle, pregnancy, and labor.

An analysis of the mechanisms involved in the okadaic acid-induced contraction of the estrogen-primed rat uterus.

The contractile effect of okadaic acid (OA) and its derivatives was investigated in the rat uterus. OA (20 microM) induced a transient contraction which, after plateauing, slowly decreased. The structurally related compound okadanol (20 microM) failed to induce any significant contraction. Conversely, the synthetic compound methyl okadaate (20 microM) and the naturally occurring ester 7'-hydroxy-4'-methyl-2'-methylen-hept-4'(E)-enyl okadaate (20 microM) were as active as the free acid. The OA-induced contraction was unaffected in the presence of neomycin (5 mM), mepacrine (30 microM), 1-[N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperaz ine (10 microM), calphostin C (3 microM) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (30 microM). The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (100 microM) did not modify the amplitude of the OA-induced contraction but significantly increased the rate of tension decay. The myosin light chain kinase inhibitor 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (1 mM) significantly reduced the peak amplitude of the contraction. Staurosporine (0.03-0.1 microM) did not modify the contractile component of the OA-induced response but inhibited the subsequent decrease in tension. In freshly dispersed myometral cells loaded with the fluorescent Ca++ indicator indo 1, OA did not produce any significant increase in [Ca++]i. OA (5- to 90-min contact) also failed to modify the intracellular levels of arachidonic acid, compared with basal values. These data suggest that in the rat uterus 1) the contractile effect of OA (20 microM) is specifically mediated by inhibition of protein phosphatases type 1 and/or 2A and is related to a direct interaction with the contractile machinery; 2) the decreasing phase of the OA-induced mechanical response could be mediated by a staurosporine-sensitive protein kinase different from protein kinase C.

Ca(2+)-independent contraction induced by hyperosmolar K(+)-rich solutions in rat uterus.

The present experiments were designed to investigate the mechanisms involved in the contractile responses evoked by KCl, added either isoosmotically or hyperosmotically, in the rat uterus. Exposure of uterine strips to a Ca(2+)-free, 3 mM EGTA-containing solution abolished the responses induced by isoosmotic KCl solutions. Conversely, addition of hyperosmolar KCl induced concentration-dependent tonic responses in a Ca(2+)-free, 3 mM EGTA-containing solution. The maximum increase in tension was reached with 210 mM K+. The response to hyperosmotic K+ was unaffected by previous depletion of intracellular Ca2+ stores with oxytocin (1 microM), by inhibition of refilling of the intracellular Ca2+ stores using cyclopiazonic acid (10 microM) or by increasing the concentration of EGTA in the medium to 10 mM. Sucrose and mannitol (60-420 mM) induced concentration-dependent sustained contractions which were not reproducible and were significantly smaller in size than those evoked by the maximally effective concentration of hyperosmotic K+ (210 mM). The contraction induced by hyperosmotic K+ in Ca(2+)-free solution was not altered by the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7, 100 microM), the Ca2+/calmodulin protein kinase II inhibitor 1-[N,O-bis(1,5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]-4-phenyl piperazine (KN-62, 10 microM) or the tyrosine kinase inhibitor genistein (10 microM). The protein kinase C inhibitor calphostin C (1-3 microM) failed to modify the K(+)-effect curve, which was however partially inhibited in the presence of the non-selective protein kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2 methylpiperazine dihydrochloride (H-7, 3-100 microM). The protein kinase inhibitor staurosporine (30-300 nM) depressed the contraction induced by hyperosmolar K+ in a concentration-dependent manner. The contraction induced by sucrose in Ca(2+)-free solution was unaffected by W-7 (100 microM) and KN-62 (10 microM) but was partially reduced by calphostin C (1 microM), H-7 (30 microM), staurosporine (100 nM) and genistein (10 microM). These results suggest that different mechanisms are involved in the responses evoked by isoosmotic and hyperosmotic KCl in the rat uterus. A component of the contraction induced by hypertonic KCl seems mainly independent of both external and internal Ca2+ and of hyperosmolar stress. This contraction is not mediated by protein kinase C, Ca2+/calmodulin-dependent kinases or protein tyrosine kinases but involves activation of other, at the present unknown, staurosporine-sensitive protein kinase(s).

Selective inhibition of calcium entry induced by benzylisoquinolines in rat smooth muscle.

The mechanism of relaxant activity of six benzylisoquinolines was examined in order to determine the minimal structural requirements that enable these compounds to have either a non-specific action like papaverine or an inhibitory activity on calcium entry via potential-operated channels. All the alkaloids tested totally or partially relaxed KCl-depolarized rat uterus and inhibited oxytocin-induced rhythmic contractions. Only glaucine and laudanosine inhibited K(+)-induced uterine contractions more than oxytocin-induced uterine contractions. In Ca(+)-free medium, sustained contractions induced by oxytocin or vanadate were relaxed by the alkaloids tested except for glaucine and laudanosine indicating no inhibitory effect on intracellular calcium release. Those alkaloids containing an unsaturated heterocyclic ring (papaverine, papaverinol, papaveraldine, N-methylpapaverine and dehydropapaverine) exhibited a more specific activity than those with a tetrahydroisoquinoline ring.

Inhibition of calcium entry induced by cularines and isocrasifoline in uterine smooth muscle.

The effects of nifedipine, papaverine and four benzylisoquinoline alkaloids (cularine, cularidine, celtisine and isocrasifoline) were studied in isolated rat uterus in order to clarify the mechanism of their relaxant action. All the compounds tested completely relaxed KCl-induced contractions and totally or partially inhibited oxytocin-induced rhythmic contractions. Only papaverine acted intracellularly, promoting relaxation of contractile responses induced by oxytocin or vanadate in a Ca(2+)-free medium. In spite of the structural relationship between papaverine and the other alkaloids, the mechanism of their relaxant action is not the same. The activities of cularine derivatives and of isocrasifoline were similar to that of nifedipine.