Gene annotation of nuclear receptor superfamily genes in the kissing bug Rhodnius prolixus and the effects of 20-hydroxyecdysone on lipid metabolism.

The hormone 20-hydroxyecdysone is fundamental for regulating moulting and metamorphosis in immature insects, and it plays a role in physiological regulation in adult insects. This hormone acts by binding and activating a receptor, the ecdysone receptor, which is part of the nuclear receptor gene superfamily. Here, we analyse the genome of the kissing bug Rhodnius prolixus to annotate the nuclear receptor superfamily genes. The R. prolixus genome displays a possible duplication of the HNF4 gene. All the analysed insect organs express most nuclear receptor genes as shown by RT-PCR. The quantitative PCR analysis showed that the RpEcR and RpUSP genes are highly expressed in the testis, while the RpHNF4-1 and RpHNF4-2 genes are more active in the fat body and ovaries and in the anterior midgut, respectively. Feeding does not induce detectable changes in the expression of these genes in the fat body. However, the expression of the RpHNF4-2 gene is always higher than that of RpHNF4-1. Treating adult females with 20-hydroxyecdysone increased the amount of triacylglycerol stored in the fat bodies by increasing their lipogenic capacity. These results indicate that 20-hydroxyecdysone acts on the lipid metabolism of adult insects, although the underlying mechanism is not clear.

Cellular mechanisms involved in iso-osmotic high K+ solutions-induced contraction of the estrogen-primed rat myometrium.

The aim of the present study was to investigate the mechanisms involved in the contraction evoked by iso-osmotic high K+ solutions in the estrogen-primed rat uterus. In Ca2+-containing solution, iso-osmotic addition of KCl (30, 60 or 90 mM K+) induced a rapid, phasic contraction followed by a prolonged sustained plateau (tonic component) of smaller amplitude. The KCl (60 mM)-induced contraction was unaffected by tetrodotoxin (3 microM), omega-conotoxin MVIIC (1 microM), GF 109203X (1 microM) or calphostin C (3 microM) but was markedly reduced by tissue treatment with neomycin (1 mM), mepacrine (10 microM) or U-73122 (10 microM). Nifedipine (0.01-0.1 microM) was significantly more effective as an inhibitor of the tonic component than of the phasic component. After 60 min incubation in Ca2+-free solution containing 3 mM EGTA, iso-osmotic KCl did not cause any increase in tension but potentiated contractions evoked by oxytocin (1 microM), sodium orthovanadate (160 micrM) or okadaic acid (20 microM) in these experimental conditions. In freshly dispersed myometrial cells maintained in Ca2+-containing solution and loaded with indo 1, iso-osmotic KCl (60 mM) caused a biphasic increase in the intracellular Ca2+ concentration ([Ca2+]i). In cells superfused for 60 min in Ca2+-free solution containing EGTA (1 mM), KCl did not increase [Ca2+]i. In Ca2+-containing solution, KCl (60 mM) produced a 76.0 +/- 16.2% increase in total [3H]inositol phosphates above basal levels and increased the intracellular levels of free arachidonic acid. These results suggest that, in the estrogen-primed rat uterus, iso-osmotic high K+ solutions, in addition to their well known effect on Ca2+ influx, activate other cellular processes leading to an increase in the Ca2+ sensitivity of the contractile machinery by a mechanism independent of extracellular Ca2+.

Complexation of okadaic acid: a preliminary study.

A complexation study was carried out with okadaic acid (OA) and the univalent metal ions Li+, Na+ and K+, and the divalent metal ions Ca2+ and Mg2+. K+ binding was observed identical with a complex obtained from the natural source (OAC). The pharmacological trials demonstrated that this cation has a very important influence on the pharmacological activity of okadaic acid.

Effects of Mn2+ on the responses induced by different spasmogens in the oestrogen-primed rat uterus.

We investigated the effect of Mn2+ on the mechanical responses evoked by high K+ (60 mM) or low Na+ (25 mM) solutions, oxytocin and neurokinin A in the oestrogen-primed rat uterus. In a Ca2+-free, Mn2+ (0.54 mM)-containing solution, high K+ or low Na+ solutions produced contractions of smaller amplitude than those observed in a normal Ca2+ (0.54 mM) solution, which were abolished by nifedipine (1 microM). Oxytocin (1 microM) and neurokinin A (1 microM, in the presence of phosphoramidon 1 microM) evoked nifedipine-insensitive contractile responses similar to (oxytocin) or smaller (neurokinin A) in amplitude than those observed in Ca2+ (0.54 mM)-containing solution. In strips loaded with Ca2+ (2.16 mM) for 10 min and then exposed to a Ca2+- and Mn2+-free, EGTA (3 mM)-containing medium for 4 min, both oxytocin and neurokinin A induced transient contraction followed by a small sustained response. The transient component of the response was abolished by cyclopiazonic acid (10 microM). When preparations were loaded with Mn2+ (2.16 mM) for 10 min, only the small, tonic contraction was observed. In Ca2+-containing solution, Mn2+ (0.01-10 mM) inhibited in a concentration-dependent manner the rhythmic contractions developed either spontaneously or by electrical stimulation as well as high K+- and neurokinin A-induced contractions. Mn2+ also abolished the rhythmic, but not the tonic component of the response to oxytocin, and the preparation remained maximally contracted. These data suggest that in the oestrogen-primed rat uterus, Mn2+ acts as an antagonist of Ca2+ influx through L-type voltage-operated Ca2+ channels. In addition, Mn2+ enters the cell mainly through nifedipine-insensitive receptor-operated channels and, to a lesser degree, through L-type Ca2+ channels to produce contraction by directly activating the contractile machinery.

Regulation by oestrogens of tachykinin NK3 receptor expression in the rat uterus.

The expression of the tachykinin NK3 receptor and its regulation by ovarian steroids were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) in uteri from ovariectomized rats. A single transcript corresponding to the 325-bp product expected for the tachykinin NK3 receptor was detected in uteri from olive oil-treated (control) ovariectomized rats. The level of tachykinin NK3 receptor mRNA in progesterone-treated animals was similar to that observed in uteri from control ones. Tachykinin NK3 receptor mRNA levels were significantly smaller in uteri from oestrogen-treated ovariectomized rats, with approximately a 32-fold decrease. These findings suggest that oestrogen, but not progesterone, regulates the expression of tachykinin NK3 receptors in the rat uterus.

Inhibitory effects of okadaic acid on rat uterine contractile responses to different spasmogens.

In the present study, we examined the effects of okadaic acid, a selective inhibitor of type 1 and 2A protein phosphatases, on the mechanical responses evoked by oxytocin, K(+)- and Na(+)-modified solutions and ouabain in estrogen-primed rat myometrium. Oxytocin elicited a rapid, phasic contraction followed by rhythmic oscillations. The phasic response was partially resistant to the absence of external Ca2+. Okadaic acid (1 microM) and the L-type calcium channel blocker nifedipine (1 microM) abolished the oscillatory component and reduced the initial, phasic response to about 80% of the control response. High K+ (60 mM) solution, ouabain (1 mM), K(+)-free medium and low Na+ (25 mM) solution induced extracellular Ca(2+)-dependent biphasic responses composed by an early rapid (KCl, ouabain and K(+)-free solution) or slower developed (25 mM Na+ solution) phasic contraction followed by a sustained increase in tension. Okadaic acid and nifedipine, alone or in combination, abolished or decreased similarly the contractile response evoked by these stimulants. The okadaic acid- and nifedipine-insensitive responses to ouabain, K(+)-free and low Na+ solution were enhanced by increasing the extracellular concentration of Ca2+ in the medium and were inhibited in a dose-dependent manner by amiloride (0.05-0.5 mM). These data suggest that, in estrogen-primed rat uterus, dephosphorylating mechanisms by OA-sensitive protein phosphatases play an important role in regulating myometrial contractions elicited by Ca2+ entry through voltage-sensitive Ca2+ channels.

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).

Effect of inhibition of the electrogenic Na+/K+ pump on the mechanical activity in the rat uterus.

The effects of ouabain and K(+)-free solution were studied in estrogen-primed rat uterine strips under resting tone or repeatedly stimulated with KCl, acetylcholine or oxytocin applied for 20 minutes at 60 minute intervals. These effects were compared with those of the K+ channel opener cromakalim. In preparations under resting tone, ouabain (0.1 mM and 0.3 mM) induced rhythmic contractions which disappeared after 20-30 minutes whereas at a higher concentration (1 mM) it evoked a rapid, phasic response followed by a small tonic contraction. Exposure of the strip to a K(+)-free solution induced either rhythmic waves, which ceased after 8-10 minutes, or a single phasic contraction which was followed by a small and slow increase in the resting tone (54 +/- 10 mg after 180 min exposure). Nifedipine (0.3 microM) abolished the rhythmic or phasic component of these responses but failed to modify the late small tonic contraction induced by ouabain 1 mM or by K(+)-free solution. Ouabain (0.1-1 mM) or K(+)-free-evoked responses disappeared after short (4 min) or prolonged (60 min) exposure to a Ca(2+)-free, 3 mM EGTA-containing solution. Cromakalim (10 nM-0.1 mM) did not induce any variation in the resting tone either in the presence or in the absence of Ca2+ in the medium. In strips repeatedly stimulated with acetylcholine (0.1 mM) or oxytocin (1 microM), ouabain (0.3 mM), K(+)-free-solution and cromakalim (10 microM) reduced the amplitude of the initial, phasic response and progressively decreased the oscillatory component of the response to these agonists. Conversely, the successive responses evoked by KCl 60 mM in similar experimental conditions were not affected by ouabain or cromakalim. Ouabain (0.3 mM), K(+)-free solution and cromakalim (10 microM) decreased the Ca(2+)-independent, maintained contractions induced by acetylcholine or oxytocin after prolonged exposure to a Ca(2+)-free, EGTA-containing medium. These inhibitory effects were partially or completely reversed in the presence of the non-selective potassium channel blocker tetraethylammonium (10 mM) or in a Ca(2+)-free solution containing 60 mM K+. In conclusion, these results suggest that the response induced by ouabain or K(+)-free solution in estrogen-primed rat myometrium involves Ca2+ influx through potential-operated calcium channels but not Ca2+ release from intracellular stores. In addition, our results show that prolonged exposure to ouabain or K(+)-free medium decreases membrane receptor-mediated responses in rat uterus. This inhibitory effect seems to be the result, at least in part, of a decrease in the cytosolic level of K+, due to the inhibition of the electrogenic Na+ pump.