Nesfatin-1 activates cardiac vagal neurons of nucleus ambiguus and elicits bradycardia in conscious rats.

Nesfatin-1, a peptide whose receptor is yet to be identified, has been involved in the modulation of feeding, stress, and metabolic responses. More recently, increasing evidence supports a modulatory role for nesfatin-1 in autonomic and cardiovascular activity. This study was undertaken to test if the expression of nesfatin-1 in the nucleus ambiguus, a key site for parasympathetic cardiac control, may be correlated with a functional role. As we have previously demonstrated that nesfatin-1 elicits Ca²âº signaling in hypothalamic neurons, we first assessed the effect of this peptide on cytosolic Ca²âº in cardiac pre-ganglionic neurons of nucleus ambiguus. We provide evidence that nesfatin-1 increases cytosolic Ca²âº concentration via a Gi/o-coupled mechanism. The nesfatin-1-induced Ca²âº rise is critically dependent on Ca²âº influx via P/Q-type voltage-activated Ca²âº channels. Repeated administration of nesfatin-1 leads to tachyphylaxis. Furthermore, nesfatin-1 produces a dose-dependent depolarization of cardiac vagal neurons via a Gi/o-coupled mechanism. In vivo studies, using telemetric and tail-cuff monitoring of heart rate and blood pressure, indicate that microinjection of nesfatin-1 into the nucleus ambiguus produces bradycardia not accompanied by a change in blood pressure in conscious rats. Taken together, our results identify for the first time that nesfatin-1 decreases heart rate by activating cardiac vagal neurons of nucleus ambiguus. Our results indicate that nesfatin-1, one of the most potent feeding peptides, increases cytosolic Ca²âº by promoting Ca²âº influx via P/Q channels and depolarizes nucleus ambiguus neurons; both effects are Gi/o-mediated. In vivo studies indicate that microinjection of nesfatin-1 into nucleus ambiguus produces bradycardia in conscious rats. This is the first report that nesfatin-1 increases the parasympathetic cardiac tone.

Urocortin 3 elevates cytosolic calcium in nucleus ambiguus neurons.

Urocortin 3 (also known as stresscopin) is an endogenous ligand for the corticotropin-releasing factor receptor 2 (CRF(2)). Despite predominant G(s) coupling of CRF(2), promiscuous coupling with other G proteins has been also associated with the activation of this receptor. As urocortin 3 has been involved in central cardiovascular regulation at hypothalamic and medullary sites, we examined its cellular effects on cardiac vagal neurons of nucleus ambiguus, a key area for the autonomic control of heart rate. Urocortin 3 (1 nM-1000 nM) induced a concentration-dependent increase in cytosolic Ca(2+) concentration that was blocked by the CRF(2) antagonist K41498. In the case of two consecutive treatments with urocortin 3, the second urocortin 3-induced Ca(2+) response was reduced, indicating receptor desensitization. The effect of urocortin 3 was abolished by pre-treatment with pertussis toxin and by inhibition of phospolipase C with U-73122. Urocortin 3 activated Ca(2+) influx via voltage-gated P/Q-type channels as well as Ca(2+) release from endoplasmic reticulum. Urocortin 3 promoted Ca(2+) release via inositol 1,4,5 trisphosphate receptors, but not ryanodine receptors. Our results indicate a novel Ca(2+) -mobilizing effect of urocortin 3 in vagal pre-ganglionic neurons of nucleus ambiguus, providing a cellular mechanism for a previously reported role for this peptide in parasympathetic cardiac regulation.

The Future in Standards of Care for Gynecologic Laparoscopic Surgery to Improve Training and Education.

Standards of care offer doctors and patients the confidence that an established quality, evidence-based, care is provided, and represent a tool for optimal responding to the population's needs. It is expected that they will increasingly express a multimodal relationship with gynecologic laparoscopy. Laparoscopy is, now, a standard procedure in operative gynecology, standards are embedded in many laparoscopic procedures, standardization of the skills/competency assessment has been progressively developed, and the proof of competency in laparoscopy may become a standard of care. A continuous development of surgical education includes standard equipment (that may bring value for future advance), standardized training, testing (and performance) assessment, educational process and outcome monitoring/evaluation, patients' care, and protection, etc. Standards of care and training have a reciprocally sustaining relationship, as training is an essential component of standards of care while care is provided at higher standards after a structured training and as credentialing/certification reunites the two. It is envisaged that through development and implementation, the European wide standards of care in laparoscopic surgery (in close harmonization with personalized medicine) would lead to effective delivery of better clinical services and provide excellent training and education.

Intracellular angiotensin II activates rat myometrium.

Angiotensin II is a modulator of myometrial activity; both AT(1) and AT(2) receptors are expressed in myometrium. Since in other tissues angiotensin II has been reported to activate intracellular receptors, we assessed the effects of intracellular administration of angiotensin II via microinjection on myometrium, using calcium imaging. Intracellular injection of angiotensin II increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) in myometrial cells in a dose-dependent manner. The effect was abolished by the AT(1) receptor antagonist losartan but not by the AT(2) receptor antagonist PD-123319. Disruption of the endo-lysosomal system, but not that of Golgi apparatus, prevented the angiotensin II-induced increase in [Ca(2+)](i). Blockade of AT(1) receptor internalization had no effect, whereas blockade of microautophagy abolished the increase in [Ca(2+)](i) produced by intracellular injection of angiotensin II; this indicates that microautophagy is a critical step in transporting the peptide into the endo-lysosomes lumenum. The response to angiotensin II was slightly reduced in Ca(2+)-free saline, indicating a major involvement of Ca(2+) release from internal stores. Blockade of inositol 1,4,5-trisphosphate (IP(3)) receptors with heparin and xestospongin C or inhibition of phospholipase C (PLC) with U-73122 abolished the response to angiotensin II, supporting the involvement of PLC-IP(3) pathway. Angiotensin II-induced increase in [Ca(2+)](i) was slightly reduced by antagonism of ryanodine receptors. Taken together, our results indicate for the first time that in myometrial cells, intracellular angiotensin II activates AT(1)-like receptors on lysosomes and activates PLC-IP(3)-dependent Ca(2+) release from endoplasmic reticulum; the response is further augmented by a Ca(2+)-induced Ca(2+) release mechanism via ryanodine receptors activation.

G protein-coupled estrogen receptor 1-mediated effects in the rat myometrium.

G protein-coupled estrogen receptor 1 (GPER), also named GPR30, has been previously identified in the female reproductive system. In this study, GPER expression was found in the female rat myometrium by reverse transcriptase-polymerase chain reaction and immunocytochemistry. Using GPER-selective ligands, we assessed the effects of the GPER activation on resting membrane potential and cytosolic Ca(2+) concentration ([Ca(2+)](i)) in rat myometrial cells, as well as on contractility of rat uterine strips. G-1, a specific GPER agonist, induced a concentration-dependent depolarization and increase in [Ca(2+)](i) in myometrial cells. The depolarization was abolished in Na(+)-free saline. G-1-induced [Ca(2+)](i) increase was markedly decreased by nifedipine, a L-type Ca(2+) channel blocker, by Ca(2+)-free or Na(+)-free saline. Intracellular administration of G-1 produced a faster and transitory increase in [Ca(2+)](i), with a higher amplitude than that induced by extracellular application, supporting an intracellular localization of the functional GPER in myometrial cells. Depletion of internal Ca(2+) stores with thapsigargin produced a robust store-activated Ca(2+) entry; the Ca(2+) response to G-1 was similar to the constitutive Ca(2+) entry and did not seem to involve store-operated Ca(2+) entry. In rat uterine strips, administration of G-1 increased the frequency and amplitude of contractions and the area under the contractility curve. The effects of G-1 on membrane potential, [Ca(2+)](i), and uterine contractility were prevented by pretreatment with G-15, a GPER antagonist, further supporting the involvement of GPER in these responses. Taken together, our results indicate that GPER is expressed and functional in rat myometrium. GPER activation produces depolarization, elevates [Ca(2+)](i) and increases contractility in myometrial cells.

A functional role for nicotinic acid adenine dinucleotide phosphate in oxytocin-mediated contraction of uterine smooth muscle from rat.

Conventionally, G protein-coupled receptors are thought to increase calcium via inositol 1,4,5-trisphosphate (InsP(3)). More recent evidence shows that an alternative second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), also has a role to play, causing researchers to question established calcium releasing pathways. With the recent development, by our group, of cell-permeant NAADP (NAADP-aceteoxymethyl ester) and a selective NAADP receptor antagonist (Ned-19; 1-(3-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid),the ability to investigate this signaling pathway has improved. Therefore, we investigated a role for NAADP in oxytocin-mediated responses in the rat uterus. Oxytocin- and NAADP-mediated effects were investigated by using contractile measurements of whole uterine strips from rat in organ baths. Responses were correlated to calcium release in cultured rat uterine smooth muscle cells measured by fluorescence microscopy. Inhibition of both oxytocin-induced contraction and calcium release by the traditional NAADP-signaling disrupter bafilomycin and the NAADP receptor antagonist Ned-19 clearly demonstrated a role for NAADP in oxytocin-induced signaling. A cell-permeant form of NAADP was able to produce both uterine contractions and calcium release. This response was unaffected by depletion of sarcoplasmic reticulum stores with thapsigargin, but was abolished by both bafilomycin and Ned-19. Crucially, oxytocin stimulated an increase in NAADP in rat uterine tissue. The present study demonstrates directly that NAADP signaling plays a role in rat uterine contractions. Moreover, investigation of this signaling pathway highlights yet another component of oxytocin-mediated signaling, stressing the need to consider the action of new components as they are discovered, even in signaling pathways that are thought to be well established.

Magnesium ion inhibits spontaneous and induced contractions of isolated uterine muscle.

AIM: Magnesium sulfate, mainly used in obstetrics to treat eclamptic convulsions, is currently questioned as to its clinical tocolytic effect. We aimed to study the relaxant action (if any) of magnesium sulfate on in vitro pregnant and non-pregnant myometrium. METHODS: Myometrial strips, harvested from five pregnant women (35-39 gestational weeks) during Cesarean procedures indicated for dystocia or scared uterus and five non-pregnant women during hysterectomy or myomectomy for benign conditions, were placed in a Krebs-Henseleit solution organ bath and the isometric force was registered. We assessed the effect of Mg2+ (magnesium sulfate) at different concentrations (0.50-10 mM) on spontaneous and oxytocin-induced (1 microM) myometrial contractility. RESULTS: Mg2+ temporarily reduced spontaneous myometrial contractions in a dose-dependent manner, with efficient regimens at 2.0-2.5 mM, and arrested contractility completely at 3 mM. Oxytocin-induced contractions were reduced by 30-40% at 8 mM and decreased further at 9-10 mM. Induced contractions were reduced, in a dose-dependent and time-dependent manner (maximum effect at 20 min), at higher Mg2+ concentrations and with non-significant proportional differences between pregnant and non-pregnant myometrium. CONCLUSIONS: The present in vitro study suggests a possible benefit of Mg2+ in the inhibition of spontaneous myometrial contractility, but not of uterine-induced hyperactivity.

Preemption dimensional study for obtaining statistically significant results for the variation of gamma-glutamyl-transferase during ovarian stimulation.

BACKGROUND: Ovarian stimulation with gonadotropins/ gonadotropin releasing factor agonists (a-GnRH), largely used currently for infertility treatment, can induce hepatic effects, demonstrated only in animals or in women with hyperstimulation syndrome. AIM: We wanted to estimate the number of included patients for which the variation of gamma-glutamyl-transferase (GGT) during and due to ovarian stimulation could be sustained by statistic validation. METHOD: In 23 consecutive patients, aged 23-45 years (mean 32.6 +/- 7.4 years) included in a fertility program, busereline, an a-GnRH was started the first day of the cycle and followed from the 14th ay with human menopausal gonadotropin. Ovulation was triggered with human chorionic gonadotropin. GGT was measured in the serum the first day (control), in the 14th, 19th, 24th day, the day before the triggering of the ovulation and two days after that. The statistic study used a distribution analysis (Student t test)--BMDP, SAS 6.0 and EpiInfo 5 software and calculated the necessary number of measurements in order to obtain significant (95%) variation for GGT (actual and = 5%). RESULTS AND CONCLUSIONS: The proper number of determinations which statistically support a possible significant difference is 29-38 and 46 for a 5% difference. The considered suppositions are able to support a correct estimation.