Andrei Adrian Tica

Contractile effects by intracellular angiotensin II via receptors with a distinct pharmacological profile in rat aorta.

We studied the effect of intracellular angiotensin II (Ang II) and related peptides on rat aortic contraction, whether this effect is pharmacologically distinguishable from that induced by extracellular stimulation, and determined the Ca2+ source involved. Compounds were delivered into the cytoplasm of de‐endothelized aorta rings using multilamellar liposomes. Contractions were normalized to the maximum obtained with phenylephrine (10−5 M). Intracellular administration of Ang II (incorporation range: 0.01–300 nmol mg−1) resulted in a dose‐dependent contraction, insensitive to extracellular administration (10−6 M) of the AT1 receptor antagonist CV11947, the AT2 receptor antagonist PD 123319, or the non‐selective AT receptor antagonist and partial agonist saralasin ([Sar1,Val5,Ala8]‐Ang II (P<0.05). Intracellular administration of CV11947 or PD 123319 right shifted the dose‐response curve about 1000 fold or 20 fold, respectively. PD 123319 was only effective if less than 30 nmol mg−1 Ang II was incorporated. Contraction was partially desensitized to a second intracellular Ang II addition after 45 min (P<0.05). Intracellular administration of Ang I and saralasin also induced contraction (P<0.05). Both responses were sensitive to intracellular CV11947 (P<0.05), but insensitive to PD 123319. The response to Ang I was independent of intracellular captopril. Contraction induced by extracellular application of Ang II and of Ang I was abolished by extracellular pre‐treatment with saralasin or CV11947 (P<0.05), but not with PD 123319. Extracellular saralasin induced no contraction. Intracellular Ang II induced contraction was not affected by pre‐treatment with heparin filled liposomes, but completely abolished in Ca2+‐free external medium. These results support the existence of an intracellular binding site for Ang II in rat aorta. Intracellular stimulation induces contraction dependent on Ca2+‐influx but not on Ins(1,4,5)P3 mediated release from intracellular Ca2+‐stores. Intracellular Ang I and saralasin induce contraction, possibly via the same binding site. Pharmacological properties of this putative intracellular receptor are clearly different from extracellular stimulated AT1 receptors or intracellular angiotensin receptors postulated in other tissue.

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.

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.

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 Ca2+ signaling in hypothalamic neurons, we first assessed the effect of this peptide on cytosolic Ca2+ in cardiac pre‐ganglionic neurons of nucleus ambiguus. We provide evidence that nesfatin‐1 increases cytosolic Ca2+ concentration via a Gi/o‐coupled mechanism. The nesfatin‐1‐induced Ca2+ rise is critically dependent on Ca2+ influx via P/Q‐type voltage‐activated Ca2+ 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.

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 (InsP3). 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.

Urocortin 3 elevates cytosolic calcium in nucleus ambiguus neurons

J. Neurochem. (2012) 122, 1129–1136.

Recurrent partial hydatidiform mole, with a first twin pregnancy, after treatment with clomiphene citrate

We present a patient, treated for 3 months with clomiphen citrate after 5 years of infertility. This treatment resulted in a twin pregnancy, one degenerated into a partial hydatidiform mole and the other into a very early embryo death. The karyotype was a mosaic one: 63% of metaphases showed triploidy – 69 XXX and 37% diploidy – 46 XX. Despite all medical advice, she returned 8 months later with a new pregnancy, which proved to be a new partial hydatidiform mole, this time a single one. Karyotype was, also, a triploidy – 69 XXX. The genetic map of both genitors was performed, showing no aberrations. Unfortunately, the patient came back, once again, 5 months later, with a new positive pregnancy test. Ultrasonography revealed a new very early embryo death, the histopathological analysis establishing to be a single ‘pure’ stop in evolution of the pregnancy. As all the three pregnancies obtained after treatment with clomiphene were abnormal, two being partial hydatidiform moles and one being a premature miscarriage, without any genetic aberrations of the genitors, it seems very possible that clomiphene, apart from improving fertility, also increases the risk of abnormal ovum appearance.

Effects of VPAC1 activation in nucleus ambiguus neurons

The pituitary adenylyl cyclase-activating polypeptide (PACAP) and its G protein-coupled receptors, PAC1, VPAC1 and VPAC2 form a system involved in a variety of biological processes. Although some sympathetic stimulatory effects of this system have been reported, its central cardiovascular regulatory properties are poorly characterized. VPAC1 receptors are expressed in the nucleus ambiguus (nAmb), a key center controlling cardiac parasympathetic tone. In this study, we report that selective VPAC1 activation in rhodamine-labeled cardiac vagal preganglionic neurons of the rat nAmb produces inositol 1,4,5-trisphosphate receptor-mediated Ca2+ mobilization, membrane depolarization and activation of P/Q-type Ca2+ channels. In vivo, this pathway converges onto transient reduction in heart rate of conscious rats. Therefore we demonstrate a VPAC1-dependent mechanism in the central parasympathetic regulation of the heart rate, adding to the complexity of PACAP-mediated cardiovascular modulation.