The presence and effects of nociceptin (N/OFQ) and nocistatin (NST) in the central nervous system have been reasonably well described, but less data are available on their peripheral functions. Besides their presence in several peripheral organs (white blood cells, airway, liver, skin, vascular and intestinal smooth muscles, ovary, and testis), they have been found in the pregnant myometrium in both rat and human. The level of their precursor prepronociceptin is elevated in the preterm human myometrium as compared with full-term samples, whereas it gradually increases toward term in the pregnant rat uterus. Both N/OFQ and NST inhibit myometrial contractions, an effect which can be enhanced by naloxone and blocked by Ca2+-dependent K+ channel (BKCa) inhibitors. Both compounds increase the myometrial cAMP level which may be responsible for the activation of this channel and subsequent intracellular hyperpolarization. NST releases calcitonin gene-related peptide from the sensory nerve ends, which explains its cAMP-elevating effect. In contrast with the nervous system, where they behave as antagonists, N/OFQ and NST are able to potentiate the uterine-relaxing effect of each other in both rat and human tissues. Further studies are required to clarify the roles of N/OFQ and NST in the regulation of the myometrial contractions and the perception of pain during delivery.

Aims: Our aims were to examine the effects of a simultaneous stimulation of beta 2-adrenergic receptors and inhibition of uterine phosphodiesterases (PDE), in the pregnant rat uterus in vivo and on human uterine tissue in vitro. We also set out to measure cAMP levels and detect the expressions of the isoenzymes PDE4B and PDE4D in human uterine tissue samples. Material and Methods: Preterm birth was induced in Sprague-Dawley rats with bacterial lipopolysaccharide. The uterine effects of terbutaline alone or in combination with rolipram were tested in vivo. Human myometrial strips from cesarean sections at full-term pregnancy and at preterm labor were stimulated with oxytocin, and the inhibitory effects of theophylline, rolipram and terbutaline were studied. The myometrial accumulation of cAMP in the presence of rolipram and terbutaline was determined by enzyme immunoassay. The expressions of PDE4B and PDE4D proteins were detected by Western blotting. Results: The selective PDE4 inhibitor rolipram was more effective than the non-selective PDE inhibitor theophylline in inhibiting the oxytocin-induced contractions in the human uterus. The uterus-relaxing effects of low doses of terbutaline were markedly potentiated by rolipram, both in rats and in human tissues. The changes in uterine cAMP levels correlated with these results. At preterm labor, PDE4B was the predominant form of PDE4 expressed; at full term, PDE4D was expressed more strongly. Conclusions: A combination of selective PDE4 inhibitors and beta 2-agonists should be considered for the treatment of preterm contractions.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

The endogenous neuropeptide nociceptin/orphanin FQ, translated from the prepronociceptin gene, exerts a contraction-inhibitory effect on the rat uterus. As nocistatin has been reported to cause functional antagonism of the pro-nociceptive effects of nociceptin, we set out to investigate its effects on the pregnant rat uterus and to elucidate its signalling pathway. The expression of prepronociceptin mRNA in the uterus and nocistatin levels in the uterus and the plasma were confirmed by RT-PCR and radioimmunoassay. The uterine levels of prepronociceptin mRNA and nocistatin were significantly increased by the last day of pregnancy, while the plasma nocistatin levels remained unchanged. In the isolated organ bath studies nocistatin inhibited the prostaglandin- and the KCl-evoked contractions in the uterus dose-dependently. This latter effect was decreased by preincubation with capsaicin. Incubation with calcitonin gene-related peptide after capsaicin treatment caused an elevation in the contraction-inhibitory effect of nocistatin. The effect of nocistatin was also decreased by the Ca2+-dependent K+ channel inhibitor paxilline, against spontaneous uterine contractions. Nociceptin potentiated the action of nocistatin. Naloxone decreased the effect of nocistatin administered either alone or in combination with nociceptin. In Ca2+-poor environment, this effect of naloxone was suspended. Enzyme immunoassay for the uterine intracellular cAMP levels partially confirmed the results of in vitro contractility studies. We conclude that nocistatin, generated locally in the uterus, exerts an inhibitory effect, the mechanism being mediated in part by Ca2+-dependent K+ channels, the elevation of cAMP levels and sensory neuropeptides.

A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3'-diamino-4,4'-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating beta-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure.

A diabetes mellitus (DM) egyike a leggyakoribb terhessegi komplikacioknak, jelentős anyai es magzati kockazati tenyező. Ugyanakkor keves adat all rendelkezesre a DM uterus funkciokra gyakorolt hatasaval kapcsolatban. Jelen munkank celja a streptozotocinnal kivaltott DM mymetrialis kovetkezmenyeinek feltarasa volt patkanyban, a gesztacios kor fuggvenyeben. Szuperfuzios technikat alkalmazva azt talaltuk, hogy DM-ban jelentősen gyorsul az uterus adrenerg denervacioja, ami csokkent noradrenalin felvevő kapacitasban nyilvanul meg. A diabetes posztszinaptikus hatasainak leirasara kontraktilitasi mereseket vegeztunk izolalt uterusgyűrűkon. Nem terhes allatban a DM fokozza az ?-, es csokkenti a ?-adrenerg stimulaciora adott kontrakcios, ill. relaxacios valaszt. E valtozasokat reszben magyarazza az ?1B-receptorok DM hatasara fokozodo mRNS szintű expresszioja, amit RT-PCR technikaval mutattunk ki. A terhesseg alatt nem talaltunk jelentős DM-fuggő elterest az uterus motoraktivitasaban. Megallapitottuk tovabba, hogy DM eseten fokozodik az exogen oxytocin uterotonikus hatasa a gesztacio 15. es 21. napjan, mely kulonbseg a terminusra (22. nap) megszűnik. Igazoltuk, hogy DM hatasara fokozodik a myometrialis oxytocin receptorok expresszioja. Eredmenyeinkből valoszinűsithető, hogy ? hasonlo human viszonyok eseten ? a diabetessel szovődott terhessegek fokozott koraszulesi kockazatanak elharitasara az oxytocin-antagonistak lehetnek a legalkalmasabbak. | Diabetes mellitus (DM) is one of the most frequent complications during pregnancy being a significant fetal and maternal risk factor. However our knowledge concerning the effects of DM on uterine functions is limited. The aim of the present work was to elucidate the myometrial consequences of the streptozotocin-induced DM as a function of gestational age of the rat. The results of superfusion experiments revealed that DM increases the adrenergic denervation of the uterus evidenced by a decreased nordrenaline utake capacity . The postsynaptic effects of DM were characterized by recording the contractility of isolated uterine rings exposed to sympathomimetics. DM resulted in an increase in alpha-receptor-mediated contractions and a decrease in relaxation elicited by terbutaline on non-pregnant uterine rings. These changes could be partly explained by a higher myometrial level of PCR-product of alpha1B-receptor No DM-related change in uterine reactivity was described during pregnancy. Additionally, a more pronounced effect of oxytocin was evidenced in late pregnancy (days 15 and 21) which was diminished by term (day 22). Higher level of oxytocin receptors was detected from the uterine samples of diabetic animals. Presuming similar human regulatory mechanisms, it could be suggested that oxytocin antagonists are drugs of choice for the treatment of DM-elicited risk for premature delivery.

Aims: Previous results by our group showed that the in vitro uterus-relaxing potency of beta(2)-adrenergic receptor (beta(2)-AR) agonists and uterine cAMP accumulation are enhanced in case of visceral inflammation. Our aim was to study the effects of the non-selective phosphodiesterase (PDE) inhibitor theophylline and the selective PDE4 inhibitor rolipram on the uteri of intact late-pregnant female rats (on days 20 and 22 of pregnancy) and of pregnant rats treated with lipopolysaccharide (LPS) to evoke preterm labor (on day 20).Main methods: The effects of theophylline and rolipram alone and of rolipram with terbutaline were investigated in isolated organ system. Contractions were evoked with KCl. The forskolin- and terbutaline-stimulated cAMP accumulations were determined by enzyme immunoassay, with or without rolipram.Key findings: The maximum uterus-relaxing effects of theophylline and rolipram decreased significantly (p<0.05) with the progression of pregnancy in intact rats. The most pronounced effect of rolipram was detected in rats challenged with LPS on day 20. Rolipram increased the in vitro effect of terbutaline both in intact and in LPS-treated rats. In the presence of rolipram, the forskolin- and terbutaline-stimulated cAMP accumulations were higher in LPS-treated than in intact rats.Significance: The previous findings led us to conclude that the combined administration of PDE4 inhibitors with beta(2)-agonists is of therapeutic value for the inhibition for uterine contractions, especially in the case of genital inflammation, which often triggers preterm birth. Combination therapy in general is associated with lesser side-effects, as a consequence of lower effective doses of each drug. (C) 2010 Elsevier Inc. All rights reserved.

The premature labour is one of the major challenges in the clinical practice. Finding new agents and mechanisms in the control of uterine activity is the main objective of the last decade's experiments. One of the new targets is the α 2 -adrenoceptors (α 2 -AR). The purpose of this study was to determine the effect of the α 2B/C -adrenoceptor blocker ARC 239 on the myometrial contractions and the cervical resistance on pregnant rats, in vitro. We identified the α 2 -adrenoceptor subtypes proteins both in the myometrial and the cervical samples. In isolated organ studies, the ARC 239 exerted a strong inhibitory effect on noradrenaline-stimulated contractions. The effect of ARC 239 on labour-induced myometrial samples was also convincing. In the stretching test, the cervical resistance was increased and decreased in by ARC 239 on pregnancy days 18 and 20, respectively. ARC 239 did not have effect on the 22-day pregnant cervical samples. These results were supported by the cAMP studies. We can conclude that, the α 2B -adrenoceptors predominate and mediate contraction, while the α 2A - and α 2C -ARs decrease the contractile response to noradrenaline in 22-days-pregnant animals. In the pregnant cervix the α 2 -adrenoceptors can couple to both G i - and G s -proteins in the 18- and 20-day-pregnant samples, respectively, resulting in increase or decrease in the cervical resistance. Based on these facts we suggest that ARC 239 may open new perspective in the influence of premature labour.

The premature labour is one of the major challenges in the clinical practice. Finding new agents and mechanisms in the control of uterine activity is the main objective of the last decade's experiments. One of the new targets is the alpha2-adrenoceptors (alpha2-AR). The purpose of this study was to determine the effect of the alpha2B/C-adrenoceptor blocker ARC 239 on the myometrial contractions and the cervical resistance on pregnant rats, in vitro. We identified the alpha2-adrenoceptor subtypes proteins both in the myometrial and the cervical samples. In isolated organ studies, the ARC 239 exerted a strong inhibitory effect on noradrenaline-stimulated contractions. The effect of ARC 239 on labour-induced myometrial samples was also convincing. In the stretching test, the cervical resistance was increased and decreased in by ARC 239 on pregnancy days 18 and 20, respectively. ARC 239 did not have effect on the 22-day pregnant cervical samples. These results were supported by the cAMP studies. We can conclude that, the alpha2B-adrenoceptors predominate and mediate contraction, while the alpha2A- and alpha2C-ARs decrease the contractile response to noradrenaline in 22-days-pregnant animals. In the pregnant cervix the alpha2-adrenoceptors can couple to both G(i)- and G()-proteins in the 18- and 20-day-pregnant samples, respectively, resulting in increase or decrease in the cervical resistance. Based on these facts we suggest that ARC 239 may open new perspective in the influence of premature labour.

Although the published results regarding the function of the beta(3)-adrenergic receptors (beta(3)-ARs) in the regulation of smooth muscle activity are very promising, the question of the mechanism of beta(3)-ARs' action in the pregnant myometrium cannot be fully answered by human investigations. To assess whether it possesses an essential role in the regulation of uterine contractility in pregnant rats, as in humans, we performed functional, western blotting and molecular biology experiments on the late-pregnant rat myometrium. The influence of progesterone on the function of the beta(3)-ARs was also investigated. We demonstrated the presence and the functional activity of the beta(3)-ARs in the late-pregnant rat myometrium. The maximum dose-dependent uterus-relaxing effect of the selective beta(3)-agonist BRL 37344 was recorded at the end of pregnancy in rats, similarly as in humans. The extent of its relaxing action was regarded as moderate. The expression of beta(3)-AR protein and mRNA remained unchanged during the investigated period. The administration of progesterone had no effect on the beta(3)-AR mRNA and protein expression or the maximum relaxation effect of BRL 37344, but shifted the dose-response curve to the right and decreased the synthesis of the second messenger, cAMP. It can be concluded that the beta(3)-ARs play an additional role in the regulation of the contractile activity of the pregnant rat uterus. The inhibitory effect of progesterone on the functional activity of the beta(3)-ARs may have important consequences in the case of human application if this effect is also demonstrated in pregnant human myometrial tissue.

The roles of the α2-adrenoceptor subtypes in the regulation of cervical resistance have previously not been investigated. The aim of the present study was to identify these receptors in the late-pregnant cervix and determine their functions in vitro in the rat. The expressions of the α2-adrenoceptor subtypes were determined by means of RT-PCR and Western blotting techniques. The changes in cervical resistance due to subtype-selective antagonists were investigated in stretching tests. The cyclic AMP immunoassay technique was used to detect the level of cyclic AMP following stimulation of the α2-adrenoceptors with or without pertussis toxin. On pregnancy days 18, 20, 21 and 22, the RT-PCR and Western blotting studies revealed the expressions of all three α2-adrenoceptor subtype mRNAs and proteins. On days 18 and 20, noradrenaline increased and decreased the resistance, respectively. Its effect was blocked by each of the antagonists used, except ARC 239 on both days. On day 21, noradrenaline again increased the resistance, this effect being maintained only in the presence of spiroxatrine. Noradrenaline was ineffective on day 22. These results were supported by the changes in cyclic AMP levels. Pertussis toxin pretreatment eliminated the changes in the cyclic AMP level on days 18 and 21. We presume that the α2A- and α2C-adrenoceptors play predominant roles in the regulation of cervical resistance on days 18–21. Depending on the day of pregnancy, stimulation of these α2-adrenoceptors could even result in opposite effects. This fluctuation can be explained by the changes in the Gi/Gs-coupling of the α2A- and α2C-adrenoceptors.

A lezarult OTKA tamogatassal vegzett kutatasi periodus főbb eredmenyeinek a kovetkezőket tartom: 1) Elsőkent allapitottuk meg es tovabbi adatokat szolgaltattunk arra vonatkozoan, hogy a periferias idegek seruleset kovető un. gerincvelői sprouting jelenseg morfologiai szubsztratumat nem a vastag myelinhuvelyes spinalis afferens rostok ill. azok burjanzasa, hanem a velőtlen, nociceptiv capsaicin-erzekeny C-tipusu afferensek kepezik. Ezzel jelentősen hozzajarultunk a fajdalomkutatasban mintegy egy evtizedig uralkodo dogma megdontesehez; 2) Kvantitativ in situ hibridizacios es immunhisztokemiai vizsgalatainkban kimutattuk, hogy a capsaicin receptor feherje expressziojanak tartos, es a TRPV1 mRNS reszleges es tranziens down-regulacioja szerepet jatszhat a capsaicin es mas vanilloidok, mint a resiniferatoxin antinociceptiv hatasaban; 3) Elektrofiziologiai es immunhisztokemiai modszerekkel karakterizaltuk az inzulin receptor-pozitiv ganglionsejtek uj populacioit; 4) Felismertuk, hogy a glukozilceramide-szintaz gatlasa jelentősen befolyasolja a nociceptiv erző ganglionsejtek capsaicin-erzekenyseget. Megfigyelesunk utat nyithat egy uj kutatasi terulethez, a glykolipideknek fajdalomerzes modulaciojaban betoltott szerepenek tanulmanyozasahoz. | The main findings of the past research period may be summarized as follows: 1) We provided evidence for the first time for unmyelinated nociceptive C-fibre afferents being the anatomical substrate of nerve lesion induced increased transganglionic transport of choleratoxin B subunit, a highly specific marker and receptor of GM1 ganglioside and membrane lipid rafts. This observation disproved the decade old dogma of pain research which suggested that myelinated primary afferent sprouting may be responsible for this phenomenon which, in turn, may play a role in the development of chronic pain states; 2) Using quantitative immunohistochemistry and in situ hybridization techniques we demonstrated that permanent elimination of the TRPV1 (capsaicin) receptor protein and transient and partial down regulation of TRPV1 mRNA may significantly contribute to the antinociceptive, pain relieving effect of perineurally administered capsaicin and similar vanilloids such as resiniferatoxin; 3) By using electrophysiological and immunohistochemical techniques we characterized new populations of primary sensory neurons which express the insulin receptor; 4) We demonstrated that inhibition of neuronal glucosylceramide synthase markedly reduced the sensitivity of cultured dorsal root ganglion cells to capsaicin. This finding may lead to new observations concerning pain modulation and pain relief.

The objective of this study was to assess the in vitro uterus relaxing potency of β 2 -adrenergic receptor (β 2 -AR) agonists in pregnant rats after in utero administration of the bacterial lipopolysaccharide, Escherichia coli endotoxin (LPS). The LPS (100 μg/kg) was injected into the uterine lumen on day 16 of pregnancy. The effects of β 2 -AR agonist terbutaline was tested in vitro , in isolated uterine rings precontracted by electric field stimulation. Uterine β 2 -AR densities were detected by radioligand binding assay, the activated G-protein levels were investigated by a radiolabelled GTP binding assay. Uterine cAMP accumulation and the serum tumor necrosis factor-α (TNF-α) levels were measured by enzyme immunoassay. The endotoxin-evoked preterm delivery occurred on day 21. Higher pD 2 values of terbutaline ( p < 0.001) were detected in endotoxin-treated rats: 9.14 ± 0.36 vs. 7.71 ± 0.12 compared with sham-operated rats. The densities or the equilibrium dissociation constants of β 2 -ARs were not different ( p > 0.05) in LPS-treated vs. control animals. Serum TNF-α level rose threefold after LPS treatment, but this rise was abolished by thalidomide. In LPS + thalidomide-treated rats, the effect of terbutaline became similar to that in sham-operated controls. By the measurement of myometrial cAMP levels, we documented that the concentration–response curve of terbutaline on cAMP accumulation was shifted to the left in the LPS-treated rats, with a significant rise in the pD 2 . We concluded that in the case of uterine inflammation, the in vitro uterus-relaxing potency of β 2 -agonists enhances, which is possibly mediated by TNF-α and uterine cAMP levels and that may serve as a rationale for the use of β 2 -AR agonists in the attenuation of preterm uterine contractions on an inflammatory basis.

The roles of the alpha(2)-adrenoceptor (alpha(2)-AR) subtypes (alpha(2A)-, alpha(2B)- and alpha(2C)-AR) in uterine contractility have not been investigated. The aims of this study were to identify these receptors in the non-pregnant and the late-pregnant rat myometrium and to determine their roles in contractions. We found that the myometrial alpha(2)-AR subtypes are involved differently in the control of late-pregnant contractions, while they have no influence on the contractions of the non-pregnant myometrium.The myometrial expressions of the alpha(2)-AR subtypes were determined by RT-PCR and Western blotting techniques. In vitro contractions were stimulated with noradrenaline, and its effect was modified with the selective antagonists BRL 44408 (alpha(2A)), ARC 239 (alpha(2B/C)) and spiroxatrine (alpha(2C)). cAMP production was followed by noradrenaline stimulation in the presence of isobutylmethylxanthine and forskolin, and alterations induced in it by the antagonists were determined with an Enzyme Immunoassay Kit. The most effective antagonist was tested on labour-induced uteri in vitro.All the alpha(2)-AR subtypes were identified in both non-pregnant and pregnant uteri. Noradrenaline was not able to contract the non-pregnant tissue in the presence of propranolol and doxazosin, while its contracting effect in the pregnant uteri was enhanced by BRL 44408, spiroxatrine and the combination BRL 44408 + spiroxatrine. ARC 239 exerted a strong inhibitory effect on noradrenaline-stimulated contractions. The increasing and the decreasing effects of the compounds were confirmed by the changes in the intracellular cAMP levels. The effect of ARC 239 on the labour-induced myometrium was similar to that on the 22-day-pregnant myometrium.The stimulation of alpha(2)-ARs does not evoke contractions in the non-pregnant uterus. The alpha(2A)- and alpha(2C)-ARs mediate decreases, while the alpha(2B)-AR mediates an increase in the contractions in the 22-day-pregnant myometrium. These differences may offer new targets for drugs against premature contractions in pregnancy. (C) 2007 Published by Elsevier Ltd.

A kutatas celkitűzese az adrenerg hatoanyagok hatasanak vizsgalata a terhes patkany cervixen es corpuson. Irodalmi adatok segitsegevel beallitottunk es tovabbfejlesztettunk egy cervix rezisztencia merő modszert, melynek segitsegevel kimutattuk, hogy a terbutalin in vitro kepes fokozni a cervix nyujtassal szembeni rezisztenciajat a terhesseg vegen (18-22. nap). Igazoltuk, hogy a hatas hattereben az aktivalt G-protein mennyiseget csokkentő tulajdonsaga jatszik szerepet. A corpuson a terbutalin relaxalo hatast fejt ki, mely a terhesseg előrehaladtaval csokken, ennek oka a G-protein aktivalo hatasanak csokkeneseben mutatkozik. A terbutalin corpusban mutatott gyengulő hatasa progeszteron kezelessel visszafordithato, melyet a G-protein aktivacio erősodese is kiser. In vivo koraszules modellben is sikerult igazolnunk a betamimetikumok es a gesztagenek kozotti potencialo szinergizmust, mely terapias jelentőseggel birhat. Az altipusszelektiv α1-adrenerg receptor blokkoloknal kimutattuk, hogy az α1A antagonista WB4101 gatolja az uterusz kontrakciokat es jelentős mertekben fokozza a terhesseg vegen a cervix rezisztenciat. A tobbi altipusszelektiv antagonista (α1B, α1D) nem volt hatasos e tekintetben. A WB4101 cervix rezisztenciat fokozo hatasa magyarazatakent G-protein aktivalo hatasat talaltuk, melynek hattereben a vegyulet inverz agonista tulajdonsaga all. Mindezek alapjan az α1A antagonistak esetleges tokolitikus alkalmazasa tovabbi megerősitest nyert. | The principal goal of the study was to investigate the pharmacological effect of adrenergic compounds on the pregnant rat uterus and cervix. On the basis of literature data we have adjusted and further developed an in vitro model to assess cervical resistance, by which method we have demonstrated that terbutaline enhanced cervical resistance by the end of pregnancy (days 18-22). In the background of this effect we have justified that terbutaline decreased G-protein activation in the cervix. Terbutaline relaxes the pregnant uterus, which effect declines towards term, this phenomenon is due to decreased G-protein activation. The decrease in the effect of terbutaline on the uterus can be reversed by the administration of progesterone, which results in enhanced G-protein activation. We have also demonstrated a potentiating synergism between the alpha-adrenergic agonists and progesterone in an in vivo rat preterm birth model, which may have therapeutic benefit in obstetrical practice. The subtype-selective alpha1A-adrenergic receptor antagonist WB 4101 either relaxed the pregnant uterus and increased cervical resistance. Other (alpha1B-, 1D) antagonists were not proved to be effective in neither tissue. The enhanced cervical resistance by WB 4101 was confirmed by its increasing effect on G-protein activation, due to its partial agonist property. Upon these results we have provided further evidence on the possible tocolytic use of alpha1A-adrenergic receptor antagonists.