RESUMEN
The rat vas deferens has scattered sensory afferens plus a dense network of sympathetic motor efferens; these fibers are not known to interact functionally. We ascertained whether sensory fibers modulate the release of sympathetic transmitters through the release of calcitonin gene-related peptide (CGRP) and reciprocally assessed whether sympathetic transmitters modulate the overflow of ir-CGRP from sensory fibers. The tissue overflow of electrically evoked sympathetic co-transmitters (ATP/metabolites, noradrenaline (NA), and immunoreactive neuropeptide tyrosine (ir-NPY)) and the motor responses elicited were quantified following either exogenous CGRP or capsaicin application to elicit peptide release. Conversely, the outflow of ir-CGRP was examined in the presence of sympathetic transmitters. Exogenous CGRP reduced in a concentration-dependent manner the electrically evoked outflow of ATP/metabolites, NA, and ir-NPY with EC(50) values of 1.3, 0.18, and 1.9 nM, respectively. CGRP also reduced the basal NA overflow. The CGRP-evoked modulation was blocked by CGRP8-37 or H-89. Release of endogenous CGRP by capsaicin significantly reduced the basal overflow of NA, ir-NPY, and the electrically evoked sympathetic transmitter release. ADP, 2-methylthioadenosine-5'-O-diphosphate (2-MeSADP), or UTP decreased the electrically evoked ir-CGRP overflow, whereas clonidine, α,ß-methyleneadenosine 5'-triphosphate (α,ß-mATP), or adenosine (ADO) were inactive. CGRP acting postjunctionally also reduced the motor responses elicited by exogenous NA, ATP, or electrically evoked contractions. We conclude that CGRP exerts a presynaptic modulator role on sympathetic nerve endings and reciprocally ATP or related nucleotides influence the release of ir-CGRP from sensory fibers, highlighting a dynamic sympatho-sensory control between sensory fibers and sympathetic nerve ending. Postjunctional CGRP receptors further contribute to reduce the tissue sympathetic motor tone implying a pre and postjunctional role of CGRP as a sympathetic tone modulator.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina/metabolismo , Unión Neuroefectora/fisiología , Nucleótidos/metabolismo , Células Receptoras Sensoriales/fisiología , Sistema Nervioso Simpático/fisiología , Adenosina Trifosfato/metabolismo , Animales , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Workshops are an important part of the IFPA annual meeting. At IFPA Meeting 2010 there were twelve themed workshops, six of which are summarized in this report. 1. The immunology workshop focused on normal and pathological functions of the maternal immune system in pregnancy. 2. The transport workshop dealt with regulation of ion and water transport across the syncytiotrophoblast of human placenta. 3. The epigenetics workshop covered DNA methylation and its potential role in regulating gene expression in placental development and disease. 4. The vascular reactivity workshop concentrated on methodological approaches used to study placental vascular function. 5. The workshop on epitheliochorial placentation covered current advances from in vivo and in vitro studies of different domestic species. 6. The proteomics workshop focused on a variety of techniques and procedures necessary for proteomic analysis and how they may be implemented for placental research.
Asunto(s)
Feto/fisiología , Placenta/fisiología , Trofoblastos/fisiología , Animales , Educación , Epigénesis Genética/fisiología , Femenino , Feto/irrigación sanguínea , Feto/citología , Feto/inmunología , Humanos , Transporte Iónico/fisiología , Intercambio Materno-Fetal/fisiología , Placenta/irrigación sanguínea , Placenta/citología , Placenta/inmunología , Placentación/fisiología , Embarazo , Proteómica/métodos , Trofoblastos/citología , Trofoblastos/inmunologíaRESUMEN
Human cellular prion protein (PrP(C)) is involved in several neurodegenerative disorders; however, its normal function is unknown. We report here that a synthetic peptide corresponding to the four-octarepeat sequence of the PrP(C) (PrP(59-91)) protects hippocampal neurons against copper neurotoxic effects in vivo. Using a rat bilateral intrahippocampal injection model, we found that PrP(59-91) protects against copper-induced neurotoxicity, including a recovery in spatial learning performance and a reduced neuronal cell loss and astrogliosis. Previous studies from our laboratory indicated that a tryptophan (Trp) residue plays a key role in the reduction of copper(II) to copper(I); therefore several PrP(59-91) fragments lacking histidine (His) and Trp residues were tested for their capacity to protect from copper toxicity. A PrP(59-91) peptide lacking His residue shows as much neuroprotection as the native peptide; however, PrP(59-91) without Trp residues only partially protected against copper toxicity. The neuroprotective effect not only occurs with PrP(59-91), in fact a full neuroprotection was also observed using just one octamer of the N-terminal region of prion protein. We conclude that the N-terminal tandem octarepeat of the human PrP(C) protects neurons against copper toxicity by a differential contribution of the binding (His) and reducing (Trp) copper activities of PrP(59-91). Our results are consistent with the idea that PrP(C) function is related to copper homeostasis.
Asunto(s)
Cobre/toxicidad , Memoria/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Priones/farmacología , Percepción Espacial/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Astrocitos/efectos de los fármacos , Astrocitos/patología , Hipocampo/efectos de los fármacos , Hipocampo/patología , Hipocampo/fisiología , Inyecciones Intraventriculares , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Datos de Secuencia Molecular , Neurotoxinas/farmacología , Fragmentos de Péptidos/química , Priones/química , Ratas , Ratas Sprague-DawleyRESUMEN
To ascertain the role of extracellular adenosine 5'-triphosphate (ATP) receptors in human placenta circulation, we identified and pharmacologically characterized the P2X receptor population in its superficial vessels. Total RNA was extracted from segments of chorionic and umbilical arteries and veins of terminal placentae delivered by vaginal or Caesarian births. Polymerase chain reaction (PCR), followed by sequencing of the products, identified the presence of P2X 1, 4, 5, 6, and 7mRNAs in smooth muscle from chorionic and umbilical arteries and veins. Umbilical vessels proximal to the fetus expressed the same population of P2X subtypes, except for the P2X(5), but additionally expressed the P2X(2). Rings of chorionic vessels contracted upon addition of nucleotides and analogs with the following relative rank order of potencies in arteries and veins: alpha,beta-methyleneATP>beta,gamma-methyleneATP>PNP>ATP=diBzATP>2-MeSATP>ADP>AMP; in umbilical vessels alpha,beta-methyleneATP was at least 100-fold more potent than ATP. Nucleotide potency was less than that of PGF(2alpha) or endothelin-2, but had the same magnitude as serotonin. ATP-desensitized receptors evidenced cross desensitization to alpha,beta-methyleneATP, 2-MeSATP and diBzATP, effect not observed when desensitization was elicited by alpha,beta-methyleneATP, confirming the presence of various P2X receptor subtypes in the smooth muscles of these vessels. The vasocontractile efficacy of alpha,beta-methyleneATP was unaltered by endothelium removal, while that of ATP was significantly attenuated and those elicited by 2-MeSATP were blunted, indicating the presence of additional endothelial nucleotide receptors. These results suggest that P2X receptors participate in the humoral regulation of placental blood flow.
Asunto(s)
Corion/metabolismo , Músculo Liso Vascular/metabolismo , Receptores Purinérgicos P2/metabolismo , Arterias Umbilicales/metabolismo , Cordón Umbilical/metabolismo , Venas Umbilicales/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Adulto , Corion/irrigación sanguínea , Corion/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Técnicas In Vitro , Intercambio Materno-Fetal/fisiología , Músculo Liso Vascular/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Embarazo , ARN Mensajero/metabolismo , Receptores Purinérgicos P2/genética , Arterias Umbilicales/efectos de los fármacos , Cordón Umbilical/efectos de los fármacos , Venas Umbilicales/efectos de los fármacos , Vasoconstrictores/farmacologíaRESUMEN
Changes in responsiveness of the vas deferens and urinary bladder to bradykinin (BK) receptor agonists (Tyr8-BK and des-Arg9-BK), substance P (SP), and endothelin-1 (ET-1) were assessed 8 weeks after streptozotocin (STZ)-induced diabetes. Preparations from control or STZ-treated (60 mg/kg i.p.) male rats were tested for contractile and neurogenic twitch potentiating (TP, in VD only) effects of all four agonists (1 nM to 0.3 or 3 microM). In diabetic VD, contractile effects of Tyr8-BK, des-Arg9-BK, and SP were enhanced, but ET-1 effects were unchanged. In contrast, TP by des-Arg9-BK was unaffected, that by Tyr8-BK was decreased, and those by SP and ET-1 were increased. In diabetic UB, only contractions to des-Arg9-BK and SP were enhanced. Following insulin replacement (human, 1-3 U/day s.c.), starting 1 week after STZ, TP induced by Tyr8-BK and des-Arg9-BK in VD were further inhibited, but all other changes in both preparations were reversed at least partially. Insulin treatment of nondiabetic rats, however, also affected VD (but not UB) responsiveness, such that contractions to Tyr8-BK and TP by ET-1 were increased, but TP by Tyr8-BK was decreased. Thus, STZ-induced type I diabetes causes important alterations in responsiveness of non-vascular smooth muscle tissues of the rat to BK, SP, and ET-1. Long term insulin replacement, at doses normalising glycaemia, effectively reversed most changes in VD or UB responsiveness, but it is unclear if this is truly due to blocking of STZ-induced changes, since the treatment also affected responsiveness of nondiabetic tissues.
Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Insulina/farmacología , Péptidos/farmacología , Vejiga Urinaria/fisiología , Conducto Deferente/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Endotelina-1/farmacología , Endotelina-1/fisiología , Técnicas In Vitro , Masculino , Péptidos/fisiología , Ratas , Ratas Wistar , Receptores de Bradiquinina/agonistas , Receptores de Bradiquinina/fisiología , Sustancia P/farmacología , Sustancia P/fisiología , Vejiga Urinaria/efectos de los fármacos , Conducto Deferente/fisiologíaRESUMEN
1. To assess the involvement of endothelial alpha(2)-adrenoceptors in the clonidine-induced vasodilatation, the mesenteric artery of Sprague Dawley rats was cannulated and perfused with Tyrode solution (2 ml min(-1)). We measured perfusion pressure, nitric oxide (NO) in the perfusate using chemiluminescence, and tissue cyclic GMP by RIA. 2. In phenylephrine-precontracted mesenteries, clonidine elicited concentration-dependent vasodilatations associated to a rise in luminal NO. One hundred nM rauwolscine or 100 microM L(omega)-nitro-L-arginine antagonized the clonidine-induced vasodilatation. Guanabenz, guanfacine, and oxymetazoline mimicked the clonidine-induced vasorelaxation. 3. In non-contracted mesenteries, 100 nM clonidine elicited a maximal rise of NO (123+/-13 pmol); associated to a peak in tissue cyclic GMP. Endothelium removal, L(omega)-nitro-L-arginine, or rauwolscine ablated the rise in NO. One hundred nM aminoclonidine, guanfacine, guanabenz, UK14,304 and oxymetazoline mimicked the clonidine-induced surge of NO. Ten microM ODQ obliterated the clonidine-induced vasorelaxation and the associated tissue cyclic GMP accumulation; 10 - 100 nM sildenafil increased tissue cyclic GMP accumulation without altering the clonidine-induced NO release. 4. alpha(2)-Adrenergic blockers antagonized the clonidine-induced rise in NO. Consistent with a preferential alpha(2D)-adrenoceptor activation, the K(B)s for yohimbine, rauwolscine, phentolamine, WB-4101, and prazosin were: 6.8, 24, 19, 165, and 1489 nM, respectively. 5. Rat pretreatment with 100 mg kg(-1) 6-hydroxydopamine reduced 95% tissue noradrenaline and 60% neuropeptide Y. In these preparations, 100 nM clonidine elicited a rise of 91.9+/-15.5 pmol NO. Perfusion with 1 microM guanethidine or 1 microM guanethidine plus 1 microM atropine did not modify the NO surge evoked by 100 nM clonidine. 6. Clonidine and congeners activate endothelial alpha(2D)-adrenoceptors coupled to the L-arginine pathway, suggesting that the antihypertensive action of clonidine involves an endothelial vasorelaxation mediated by NO release, in addition to presynaptic mechanisms.
Asunto(s)
Agonistas alfa-Adrenérgicos/farmacología , Clonidina/farmacología , Óxido Nítrico/fisiología , Receptores Adrenérgicos alfa 2/efectos de los fármacos , Vasodilatación/efectos de los fármacos , 3',5'-GMP Cíclico Fosfodiesterasas , Acetilcolina/farmacología , Antagonistas Adrenérgicos alfa/farmacología , Animales , GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5 , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Inhibidores Enzimáticos/farmacología , Guanilato Ciclasa/antagonistas & inhibidores , Guanilato Ciclasa/metabolismo , Técnicas In Vitro , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/metabolismo , Arterias Mesentéricas/fisiología , Óxido Nítrico/metabolismo , Nitroarginina/farmacología , Oxadiazoles/farmacología , Oxidopamina/farmacología , Fenilefrina/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , Piperazinas/farmacología , Purinas , Quinoxalinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos alfa 2/metabolismo , Saponinas/farmacología , Citrato de Sildenafil , Solubilidad , Sulfonas , Simpaticolíticos/farmacología , Factores de Tiempo , Resistencia Vascular , Vasodilatadores/farmacología , Yohimbina/farmacologíaRESUMEN
To reveal a basal production of nitric oxide (NO) and guanosine 3',5' cyclic monophosphate (cGMP) in the rat arterial mesenteric bed, mesenteries were perfused in the absence and in the presence of selective blockers of the L-arginine cascade. Endothelium removal or inhibition of NO synthase significantly reduced the release of NO and tissue cGMP. A significant correlation between these messengers was shown. Blockade of soluble guanylyl cyclase with 0.3-10 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) only reduced basal cGMP production; 1-100 nM sildenafil (Sild), an inhibitor of phosphodiesterase V, increased basal tissue cGMP without modifying the release of NO. Acetylcholine (0.01-10 microM) caused a concentration-dependent rise in NO and cGMP evoking a proportional vasodilatation, demonstrating the interdependence between these messengers and vascular reactivity. Endothelium removal or NO synthase blockade reduced the acetylcholine-induced increase of messengers and the vasodilatation. ODQ attenuated only the increase in cGMP and the vasodilatation, while sildenafil increased cGMP without significantly altering luminal NO release. The present results highlight a tonic release of NO and its involvement in endothelial-smooth muscle signaling; NO and cGMP are determinants of vascular reactivity.
Asunto(s)
GMP Cíclico/biosíntesis , Arterias Mesentéricas/fisiología , Óxido Nítrico/metabolismo , Vasodilatación/fisiología , Acetilcolina/farmacología , Animales , Arginina/farmacología , Relación Dosis-Respuesta a Droga , Endotelio Vascular/fisiología , Inhibidores Enzimáticos/farmacología , Masculino , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/metabolismo , Nitroarginina/farmacología , Norepinefrina/farmacología , Oxadiazoles/farmacología , Piperazinas/farmacología , Purinas , Quinoxalinas/farmacología , Ratas , Ratas Sprague-Dawley , Citrato de Sildenafil , Sulfonas , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
BACKGROUND: Placental vessels are not innervated. Therefore the vasomotor activity and vascular tone is not regulated by the nervous system. AIM: To assess the existence of pacemaker mechanisms related to rhythmic motor activity of blood vessels. MATERIAL AND METHODS: Isometric contractions of rings from umbilical and chorionic vessels of term human placentas were monitored. RESULTS: Recordings of the circular layer of chorionic and umbilical vessels revealed rhythmic spontaneous contractions with a frequency of 1.4 +/- 0.05 cycles/min, the duration of each cycle was 42.8 +/- 0.24 s (n = 12). The amplitude of contractions was larger in veins than in arteries, predominating in umbilical vein biopsies, proximal to the fetus. Both the frequency and the amplitude of contractions were relatively constant during the first 30 min. However, after an hour, the frequency declined while the amplitude increased. The absence of the endothelium neither modified the frequency nor the amplitude of the rhythmic activity. Blockage of voltage dependent sodium channels or calcium channels did not alter the frequency of spontaneous contractions, although their magnitude was reduced. Glibenclamide, an ATP-dependent K+ channel blocker or the blockade of gap junctions ablated the frequency and amplitude of spontaneous contractions. CONCLUSIONS: We propose that rhythmic contractions are triggered by pacemaker cells located in the circular layer of the smooth muscle of blood vessels and spread via gap junctions; they likely contribute to the control of blood flow.
Asunto(s)
Relojes Biológicos , Vasos Sanguíneos/fisiología , Contracción Muscular/fisiología , Placenta/irrigación sanguínea , Venas Umbilicales/fisiología , Canales de Calcio/fisiología , Corion/irrigación sanguínea , Corion/fisiología , Electrofisiología , Endotelio Vascular/citología , Femenino , Uniones Comunicantes/fisiología , Humanos , Contracción Isométrica/fisiología , Músculo Liso Vascular/fisiología , Placenta/citología , Canales de Sodio/fisiologíaRESUMEN
BACKGROUND: It is known that the sympathetic varicosities co-store and co-release norepinephrine (NE) together with adenosine S-triphosphate (ATP) and neuropeptide Y (NPY). AIM: To describe the chemical characterization of stored and released NPY from the varicosities of sympathetic nerve terminals surrounding segments of the human saphenous vein, and the vasomotor activity of rings electrically depolarized or contracted by the exogenous application of the co-transmitters. MATERIAL AND METHODS: Saphenous vein tissues were obtained from patients undergoing elective cardiac revascularization surgery. RESULTS: The chromatographic profile of NPY extracted from biopsies is identical to a chemical standard of human NPY. Upon electrical depolarisation of the perivascular sympathetic nerve terminals, we demonstrated the release of NPY to the superfusion media, which did not exceed a 1% of its stored content. The release of the peptide is sensitive to guanethidine, and to extracellular calcium, suggesting that the mechanism of its release is exocytotic in nature. The electrically evoked release of NPY is dependent on the frequency and duration of the electrical pulses. Phenoxybenzamine reduces the electrically evoked release of NPY. Exogenous application of NE and ATP contract saphenous vein rings; the simultaneous application of NE plus ATP causes a synergic response, effect which is further potentiated by the joint co-application of 10 nM NPY. CONCLUSIONS: Present results highlight the role of NPY as a sympathetic co-transmitter in the regulation of human vascular tone.
Asunto(s)
Adenosina Trifosfato/farmacología , Neuropéptido Y/fisiología , Vena Safena/inervación , Transmisión Sináptica/fisiología , Estimulación Eléctrica , Humanos , Neuropéptido Y/análisis , Neuropéptido Y/farmacología , Neurotransmisores/farmacología , Norepinefrina/farmacología , Sistema Nervioso Periférico/efectos de los fármacos , Vena Safena/química , Vena Safena/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Resistencia VascularRESUMEN
The aim of the present study was to test whether the contractile responses elicited by KCl in the rat mesenteric bed are coupled to the release of nitric oxide (NO). Contractions induced by 70 mM KCl were coincident with the release of NO to the perfusate. The in vitro exposure to the nitric oxide synthase (NOS) inhibitor L-N(omega)-nitro-L-arginine methyl ester, L-NAME (1-100 microM) potentiated the vascular responses to 70 mM KCl and, unexpectedly, increased the KCl-stimulated release of NO. Moreover, even after the chronic treatment with L-NAME (70 mg/kg/day during 4 weeks), the KCl-induced release of NO was not reduced, whereas the potentiation of contractile responses was indeed achieved. The possibility that NOS had not been completely inhibited under our experimental conditions can be precluded because NOS activity was significantly inhibited after both L-NAME treatments. After the in vitro treatment with 1 to 100 microM L-NAME, the inhibition of NOS was concentration-dependent (from 50% to 90%). With regard to the basal release of NO, the inhibition caused by L-NAME was not concentration-dependent and reached a maximum of 40%, suggesting that basal NO outflow is only partially dependent on NOS activity. An eventual enhancement of NOS activity caused by KCl was disregarded because the activity of this enzyme measured in homogenates from mesenteric beds perfused with 70 mM KCl was significantly reduced. On the other hand, endothelium removal, employed as a negative control, almost abolished NOS activity, whereas the incubation with the Ca(2+) ionophore A23187, employed as a positive control, induced an increase in NOS activity. It is concluded that in the mesenteric arterial bed of the rat, the contractile responses elicited by depolarization through KCl are coincident with a NOS-independent release of NO. This observation, which differs from the results obtained with noradrenaline, do not support the use of KCl as an alternative contractile agent whenever the participation of NO is under study.
Asunto(s)
Mesenterio/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/metabolismo , Cloruro de Potasio/farmacología , Antagonistas Adrenérgicos alfa/farmacología , Animales , Inhibidores Enzimáticos/farmacología , Mediciones Luminiscentes , Masculino , Mesenterio/efectos de los fármacos , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Norepinefrina/metabolismo , Perfusión , Prazosina/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Contraction and relaxation of smooth muscle is a tightly regulated process involving numerous endogenous substances and their intracellular second messengers. We examine the key role of cyclic guanosine monophosphate (cGMP) in mediating smooth muscle relaxation. We briefly review the current art regarding cGMP generation and degradation, while focusing on the recent identification of the molecular mechanisms underlying cGMP-mediated smooth muscle relaxation. cGMP-induced SM relaxation is mediated mainly by cGMP-dependent protein kinase activation. It involves several molecular events culminating in a reduction in intracellular Ca(2+) concentration and a decrease in the sensitivity of the contractile system to Ca(2+). We propose that the cGMP-induced decrease in Ca(2+) sensitivity is a strategic way to achieve "active relaxation" of the smooth muscle. In summary, we present compelling evidence supporting a key role for cGMP as a mediator of smooth muscle relaxation in physiological and pharmacological settings.
Asunto(s)
GMP Cíclico/metabolismo , Relajación Muscular/fisiología , Músculo Liso/fisiología , Animales , Calcio/metabolismo , Monóxido de Carbono/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Guanilato Ciclasa/metabolismo , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Canales Iónicos/metabolismo , Músculo Liso/metabolismo , Óxido Nítrico/metabolismo , Péptidos/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Sistemas de Mensajero SecundarioRESUMEN
The rat ATP P2X4 receptor was expressed in Xenopus laevis oocytes to assess the effect of zinc and copper as possible regulators of purinergic mechanisms. ATP applied for 20 s evoked an inward cationic current with a median effective concentration (EC50) of 21.4+/-2.8 microM and a Hill coefficient (nH) of 1.5+/-0.1. Coapplication of ATP plus 10 microM zinc displaced leftward, in a parallel fashion, the ATP concentration-response curve, reducing the EC50 to 8.4+/-1.8 microM (p < 0.01) without altering the receptor nH. The zinc potentiation was fast in onset, easily reversible, and voltage-independent and did not require metal preexposure. The zinc EC50 was 2-5 microM, with a bell-shaped curve. At concentrations of 100-300 microM, zinc produced less potentiation, and at 1 mM, it inhibited 50% the ATP current. The effect of zinc was mimicked by cadmium. In contrast, copper inhibited the ATP-evoked currents in a time- and concentration-dependent fashion, reducing the maximal current (Imax) without altering the EC50. The copper-induced inhibition was slow in onset, slowly reversible, and voltage-independent. Whereas coapplication of 300 microM copper plus ATP reduced Imax to 36.2+/-5%, the coapplication of, or 60-s preexposure by, 10 microM copper reduced Imax to 79+/-9.2% (p < 0.05) and 39.6+/-8.7% (p < 0.01), respectively. The inhibition was noncompetitive in nature and mimicked by mercury. Cobalt, barium, and manganese did not modify significantly the ATP-evoked current, demonstrating metal specificity. The simultaneous 1-min preapplication of both metals revealed that the 10 microM zinc-induced potentiation was obliterated by 10 microM copper, whereas 30 microM copper not only reduced the potentiation, but inhibited the ATP response. Following coapplication of both metals for 20 s with ATP, at least 100 microM copper was required to counteract the 10 microM zinc-induced potentiation. The simultaneous preincubation with both metals provided evidence for a noncompetitive interaction. We hypothesize the existence of metal binding site(s), which are most likely localized in the extracellular domain of the P2X4 receptor structure. These sites are selective and accessible to extracellular metal applications and bind micromolar concentrations of metals. The present results are compatible with the working hypothesis that trace metals, such as copper and zinc, are physiological modulators of the P2X4 receptor. The modulation of brain purinergic transmission by physiologically and toxicologically relevant trace metal cations is highlighted.
Asunto(s)
Cobre/farmacología , Activación del Canal Iónico/efectos de los fármacos , Receptores Purinérgicos P2/fisiología , Zinc/farmacología , Adenosina Trifosfato/farmacología , Animales , Cadmio/farmacología , Cationes Bivalentes/farmacología , Interacciones Farmacológicas , Electrofisiología , Potenciales de la Membrana/efectos de los fármacos , Neurotoxinas/farmacología , Oocitos/fisiología , Ratas , Receptores Purinérgicos P2X4 , Estimulación Química , Xenopus laevisRESUMEN
To test whether migraine and subarachnoid hemorrhage (SAH) are associated with increased sympathetic tone, we compared the neuropeptide Y-like (NPY-LI) and chromogranin A-like immunoreactivities (LI) of cerebrospinal fluid (CSF) from migraneurs and SAH patients with those from control subjects. Increased sympathetic tone was expected to produce higher co-release of these co-stored peptides and concordant changes in their CSF levels. In addition, we investigated a possible disturbed nitric oxide homeostasis by measuring CSF nitrites (NO). More than 70% of CSF NPY-LI corresponded to the chromatographic peak (HPLC) for the intact molecule in all three groups. Migraneurs had 64% higher CSF NPY-LI, but no significant difference in CSF chromogranin A-LI, as compared to controls. In contrast, SAH patients had 74% less CSF chromogranin A-LI and a trend to lower NPY-LI, as compared to controls. No differences in CSF NO were detected among groups. These results argue against an increased sympathetic tone in patients with either migraine or SAH, and suggest that the higher CSF NPY-LI of migraneurs probably originates from central neurons. Furthermore, our findings in SAH patients argue in favor of a decreased sympathetic tone; this could be a homeostatic response to counterbalance vasoconstriction mediated by other mechanisms.
Asunto(s)
Proteínas del Líquido Cefalorraquídeo/análisis , Trastornos Migrañosos/líquido cefalorraquídeo , Proteínas del Tejido Nervioso/líquido cefalorraquídeo , Neuropéptido Y/líquido cefalorraquídeo , Hemorragia Subaracnoidea/líquido cefalorraquídeo , Sistema Vasomotor/fisiopatología , Adulto , Biomarcadores , Cromogranina A , Cromograninas/líquido cefalorraquídeo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Trastornos Migrañosos/fisiopatología , Neuronas/metabolismo , Óxido Nítrico/fisiología , Nitritos/líquido cefalorraquídeo , Estudios Prospectivos , Hemorragia Subaracnoidea/fisiopatología , Sistema Nervioso Simpático/fisiopatologíaRESUMEN
To assess the pharmacodynamic profile of ALEPH-2, a phenylisopropylamine derivative with alleged anxiolytic and hallucinogenic properties, Xenopus laevis oocytes were microinjected with either of the rat cRNA for the 5-HT2A or the 5-HT2C receptor. Concentration-response curves were obtained following the exposure of the oocytes to varying concentrations of either ALEPH-2 or 5-hydroxytryptamine (5-HT) for 10 s. ALEPH-2 is a partial agonist on the 5-HT2A receptor with a similar potency to 5-HT. In contrast, ALEPH-2 is a full 5-HT2C receptor agonist and is about 15-fold less potent than 5-HT. Pre-application of 1 microM ritanserin antagonized the responses induced by 5-HT and ALEPH-2 to the same extent; however, the 5-HT2A receptor is more sensitive to ritanserin blockade than the 5-HT2C receptor.
Asunto(s)
Ansiolíticos/farmacología , 2,5-Dimetoxi-4-Metilanfetamina/análogos & derivados , 2,5-Dimetoxi-4-Metilanfetamina/farmacología , Alucinógenos/farmacología , Propilaminas/farmacología , Receptores de Serotonina/metabolismo , Agonistas de Receptores de Serotonina/farmacología , 2,5-Dimetoxi-4-Metilanfetamina/química , Animales , Ansiolíticos/química , Alucinógenos/química , Oocitos , Propilaminas/química , Receptor de Serotonina 5-HT2A , Receptor de Serotonina 5-HT2C , Serotonina/farmacología , Xenopus laevisRESUMEN
To evaluate whether sympathetic activity induces nitric oxide (NO) production, we perfused the rat arterial mesenteric bed and measured luminally accessible norepinephrine (NE), NO, and cGMP before, during, and after stimulation of perivascular nerves. Electrical stimulation (1 min, 30 Hz) raised perfusion pressure by 97 +/- 7 mmHg, accompanied by peaks of 23 +/- 3 pmol NE, 445 +/- 48 pmol NO, and 1 pmol cGMP. Likewise, perfusion with 10 microM NE induced vasoconstriction coupled to increased NO and cGMP release. Electrically elicited NO release depended on stimulus frequency and duration. Endothelium denudation with saponin abolished the NO peak without changing NE release. Inhibition of NO synthase with 100 microM N(omega)-nitro-L-arginine reduced basal NO and cGMP release and blocked the electrically stimulated and exogenous NE-stimulated NO peak while enhancing vasoconstriction. Blocking either sympathetic exocytosis with 1 microM guanethidine or alpha1-adrenoceptors with 30 nM prazosin abolished the electrically evoked vasoconstriction and NO release. alpha2-Adrenoceptor blockade with 1 microM yohimbine reduced both vasoconstriction and NO peak while increasing NE release. In summary, sympathetically released NE induces vasoconstriction, which triggers a secondary release of endothelial NO coupled to cGMP production.
Asunto(s)
Endotelio Vascular/fisiología , Arterias Mesentéricas/fisiología , Mesenterio/irrigación sanguínea , Óxido Nítrico/fisiología , Sistema Nervioso Simpático/fisiología , Animales , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Arterias Mesentéricas/inervación , Mesenterio/inervación , Mesenterio/fisiología , Nitroarginina/farmacología , RatasRESUMEN
To ascertain the role of the neuropeptide Y Y1 receptors in the vascular manifestations of the sympathetic baroreflex, 10-s bilateral carotid occlusions were performed in anesthetized cats; systemic blood pressure was monitored continually. This maneuver rose systolic blood pressure in 23 +/- 2 mmHg. Following 100 microg/kg BIBP 3226 or BIBO 3304 i.v., the increase in blood pressure elicited by the occlusions was only 14 +/- 1 and 15 mmHg, respectively. Both BIBP 3226 and BIBO 3304 displaced significantly 5.5 fold rightward the pressor dose-response curve elicited by exogenous neuropeptide Y, without altering the norepinephrine curve. Prazosin (10 microg/kg) reduced the pressor response elicited by the carotid occlusion to 12 +/- 4 mmHg. The simultaneous administration of BIBP 3226 plus prazosin rose the systemic blood pressure following the occlusion only 9 +/- 2 mmHg, supporting the involvement of neuropeptide Y in vascular sympathetic reflexes.
Asunto(s)
Arginina/análogos & derivados , Barorreflejo/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Fármacos Cardiovasculares/farmacología , Neuropéptido Y/farmacología , Receptores de Neuropéptido Y/antagonistas & inhibidores , Animales , Arginina/farmacología , Barorreflejo/fisiología , Presión Sanguínea/fisiología , Arterias Carótidas/efectos de los fármacos , Gatos , Relación Dosis-Respuesta a Droga , Ligadura , Receptores de Neuropéptido Y/fisiologíaRESUMEN
Although abundant literature supports the notion that neuropeptide Y (NPY) synergizes in vivo and in vitro, the vasomotor activity elicited by norepinephrine (NE), the converse interaction (i.e., the adrenergic modulation of the NPY vasomotor response) has been less characterized. To assess whether NE synergizes the vasomotor effect of NPY, the rat arterial mesenteric bed was chosen as a model experimental system. Mesenteries were precontracted with NE and few minutes later were perfused with exogenous NPY. Under these conditions, NPY contracted the arterial mesenteric bed with an EC50 value of 0.72 +/- 0.06 nM. NPY was unable to contract this vascular territory without an agonist-induced precontraction. Other agonists, such as endothelin-1, a synthetic analog of prostaglandin F2alpha, or 5-hydroxytryptamine, also were effective primers because in their presence, NPY was a potent vasoconstrictor. In contrast, mesenteries precontracted with KCl failed to evidence the NPY-induced rise in perfusion pressure. Two structural analogs of NPY, PYY and [Leu31, Pro34]NPY, mimicked the activity of NPY. The NPY fragment 13-36 did not elicit such a response. All NPY analogs exhibited less efficacy and potency relative to NPY. The NPY- and related structural analog-induced vasoconstriction was competitively and reversibly antagonized by BIBP 3226; the pA2 of the NPY interaction was 7.0. The application of 0.1 to 1 microM BIBP 3226 or 0.1 to 10 nM prazosin at the peak of the NPY vasomotor response elicited a gradual blockade of the vasoconstriction. Although BIBP 3226 blocked the increase in perfusion pressure elicited by NPY, leaving unaffected the NE-induced tone, 10 nM prazosin blocked the full response, including the NE-induced component. Tissue preincubation with 200 nM nifedipine abolished the NPY-induced vasoconstriction; likewise, the acute application of 10 to 100 nM nifedipine blocked gradually the maximal NPY-induced contraction. Removal of the mesenteric endothelial layer increased the potency of NPY by 2-fold; it also slightly potentiated the antagonist activity of BIBP 3226. The synergism between NPY and NE backs the principle of sympathetic cotransmission.
Asunto(s)
Arginina/análogos & derivados , Arterias Mesentéricas/fisiología , Músculo Liso Vascular/fisiología , Neuropéptido Y/análogos & derivados , Neuropéptido Y/farmacología , Norepinefrina/farmacología , Vasoconstricción/efectos de los fármacos , Animales , Arginina/farmacología , Dinoprost/farmacología , Sinergismo Farmacológico , Endotelina-1/farmacología , Endotelio Vascular/fisiología , Masculino , Arterias Mesentéricas/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Nifedipino/farmacología , Fragmentos de Péptidos/farmacología , Cloruro de Potasio/farmacología , Prazosina/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Neuropéptido Y/antagonistas & inhibidores , Serotonina/farmacología , Vasoconstricción/fisiologíaRESUMEN
1. Human saphenous veins were used to assess the cooperative participation of adenosine 5-triphosphate (ATP), neuropeptide Y (NPY), and noradrenaline (NA) in the vasomotor responses elicited following electrical depolarization of the perivascular nerve terminals. Rings from recently dissected human biopsies were mounted to record isometric muscular contractions; the motor activity elicited in the circular muscle layer following electrical depolarization (2.5-20 Hz, 50 V, 0.5 msec) were recorded. 2. Incubation of the biopsies with either 100 nM tetrodotoxin (TTX) or 1 microM guanethidine abolished the vasomotor response elicited by electrical nerve depolarization. The independent application of either ATP or NA to vein rings induced concentration-dependent contractions. 3. Tissue incubation with 30 microM suramin or 10 nM prazosin produced 10 fold rightward displacements of the alpha,beta-methylene ATP and NA concentration-response curves respectively. NPY contracted a limited number of biopsies, the vasoconstriction elicited was completely blocked by 1 microM BIBP 3226. A 5 min incubation of the biopsies with 10-100 nM NPY synergized, in a concentration-dependent fashion, both the ATP and the ATP analogue-induced contractions. Likewise, tissue preincubation with 10 nM NPY potentiated the vasomotor responses evoked with 20-60 nM NA. 4. Neither suramin, BIBP 3226, nor prazosin was individually able to significantly modify the derived frequency-tension curves. In contrast, the co-application of 30 microM suramin and 10 nM prazosin or 30 microM suramin and 1 microM BIBP 3226, elicited a significant (P<0.01) downward displacement of the respective frequency-tension curves. 5. The simultaneous application of the three antagonists-30 microM suramin, 1 microM BIBP 3226 and 10 nM prazosin-caused a significantly greater displacement of the frequency-tension curve than that achieved in experiments using two of these antagonists. 6. Electrically-evoked vasomotor activity is blocked to a larger extent by tissue incubation with 2.5 microM chloroethylclonidine and 30 microM suramin rather than with 10 nM 5 methyl urapidil and 30 microM suramin. As a result, the alpha1-adrenoceptor involved in the vasomotor activity has tentatively been associated with the alpha1B adrenoceptor family subtype. 7. Results support the physiological role of ATP in sympathetic neurotransmission. The present results are consistent with the working hypothesis that human sympathetic vasomotor reflexes involve the coordinated motor action of ATP, NPY, and NA acting on vascular smooth muscle cells. The present results support the concept of sympathetic co-transmission in the human saphenous vein.
Asunto(s)
Adenosina Trifosfato/fisiología , Neuropéptido Y/fisiología , Neurotransmisores/fisiología , Norepinefrina/fisiología , Vena Safena/fisiología , Sistema Vasomotor/fisiología , Antagonistas Adrenérgicos alfa/farmacología , Ansiolíticos/farmacología , Arginina/análogos & derivados , Arginina/farmacología , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/fisiología , Humanos , Neurotransmisores/antagonistas & inhibidores , Prazosina/farmacología , Antagonistas Purinérgicos , Receptores de Neuropéptido Y/antagonistas & inhibidores , Receptores de Neuropéptido Y/fisiología , Receptores Purinérgicos/fisiología , Vena Safena/efectos de los fármacos , Suramina/farmacología , Sistema Nervioso Simpático/efectos de los fármacos , Sistema Nervioso Simpático/fisiología , Sistema Vasomotor/efectos de los fármacosRESUMEN
Nanomoles of neuropeptide Y (NPY) and noradrenaline (NA), administered i.v. to pentobarbital-anesthetized rats, caused nearly equipotent dose-dependent pressor responses in normotensive rats. However, in renovascular Goldblatt hypertensive rats, the dose-response curves for both NPY and NA were significantly displaced to the left, approximately threefold. Intravenous administration of BIBP 3226 (30-180 microg/kg) did not consistently lower blood pressure, per se, but did evoke competitive antagonism of the NPY pressor response in both rat populations. The magnitude of the NPY antagonism evoked by BIBP 3226 was comparable in normotensive and hypertensive rats. The absence of NA antagonism demonstrates the selectivity of the BIBP 3226 blockade.
Asunto(s)
Arginina/análogos & derivados , Presión Sanguínea/efectos de los fármacos , Hipertensión Renovascular/fisiopatología , Neuropéptido Y/antagonistas & inhibidores , Neuropéptido Y/farmacología , Animales , Arginina/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Riñón/efectos de los fármacos , Riñón/fisiología , Masculino , Norepinefrina/farmacología , Ratas , Ratas Sprague-Dawley , Arteria Renal/efectos de los fármacos , Arteria Renal/fisiología , Arteria Renal/cirugíaRESUMEN
Neuropeptide Y (NPY) appears to be involved in the sympathetic regulation of vascular tone. To assess the putative role of NPY in mesenteric circulation, the release and biological effect of NPY were examined after electrical stimulation of perimesenteric arterial nerves. Nerve stimulation with trains of 2-30 Hz increased the perfusion pressure of the arterially perfused rat mesenteric bed in a frequency- and time-dependent fashion. Trains of 15-30 Hz significantly displaced to the left, approximately threefold, the noradrenaline (NA)-induced pressor concentration-response curve, in addition to increasing significantly its efficacy. Perfusion with 10 nM exogenous NPY mimicked the electrical stimulation effect, causing a threefold leftward shift of the NA concentration-response curve and increasing the maximal NA response. These effects were antagonized by 100 nM BIBP 3226, indicating the activity of NPY-Y1 receptors. Electrical stimulation of the perimesenteric nerves released immunoreactive NPY (ir-NPY) in a frequency-dependent fashion; the ir-NPY coelutes with synthetic NPY as confirmed by HPLC. Both the electrically induced pressor response and the calcium-dependent release of NPY were obliterated in preparations perfused with 1 microM guanethidine or in rats pretreated intravenously for 48 h with 6-hydroxydopamine, thus revealing the sympathetic origin of these phenomena. Only a small proportion of the total NPY content in the perimesenteric arterial nerves is released after electrical stimulation. Chromatographic studies of the physiological sources of the ir-NPY support that NPY fragments are generated via peptidase degradation. The present findings demonstrate that NPY is released from the perimesenteric arterial sympathetic nerves and acts, via the activation of NPY-Y1 receptors, as the mediator responsible for the potentiation of NA's effect on perfusion pressure in the isolated rat mesenteric bed.