RESUMEN
BACKGROUND: Adenosine 5'-triphosphate (ATP) is released extracellularly as a neurotransmitter and an autocrine or paracrine mediator in numerous systems, including the gastrointestinal tract. It is rapidly degraded to active and inactive metabolites by membrane-bound enzymes. Investigators frequently use inhibitors of ATP hydrolysis such as ARL-67156 and POM-1 to suppress the catabolism of ATP and prolong its effects in pharmacological studies. Our aim was to investigate directly the effects of ARL-67156 and POM-1 on the degradation of ATP and adenosine 5'-diphosphate (ADP) in mouse colonic muscles. METHODS: The degradation of ATP and ADP was evaluated by superfusing tissues with 1,N(6) -etheno-ATP (eATP) and 1,N(6) -etheno-ADP (eADP) as substrates and monitoring the decrease in substrate and increase in products (i.e., eADP, eAMP, and e-adenosine) by high-performance liquid chromatography techniques with fluorescence detection. Relaxation responses to etheno-derivatized and non-derivatized ATP and ADP were examined in isometric tension experiments. KEY RESULTS: ARL-67156 inhibits the degradation of ADP but not of ATP, whereas POM-1 inhibits the degradation of ATP but not of ADP in murine colonic muscles. Consequently, ARL-67156 enhances relaxation responses to both ATP and ADP, whereas POM-1 reduces relaxation to ATP and does not affect relaxation to ADP. CONCLUSIONS & INFERENCES: Studies that use ARL-67156 to inhibit ATP degradation in smooth muscle likely evaluate responses to accumulated ADP rather than ATP. POM-1 appears to be a more selective inhibitor of ATP degradation in the mouse colon. The choice of pharmacological tools in studies on extracellular ATP signaling may affect the interpretation of experimental data in functional studies.
Asunto(s)
Adenosina Difosfato/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Colon/efectos de los fármacos , Adenosina Trifosfato/farmacología , Animales , Colon/metabolismo , RatonesRESUMEN
BACKGROUND: Purinergic signaling provides regulation of colonic motility. Smooth muscle cells (SMC), interstitial cells of Cajal (ICC), and platelet-derived growth factor receptor α-positive (PDGFRα(+) ) cells are electrically coupled and form a functional (SIP) syncytium that constitutes the receptive field for motor neurotransmitters in the tunica muscularis. Each cell type in the SIP syncytium has specialized functions in mediating motor neurotransmission. We compared gene transcripts for purinergic receptors and membrane-bound enzymes for purine degradation expressed by each cell type of the SIP syncytium. METHODS: Fluorescence-activated cell sorting (FACS) was used to purify SMC, ICC, and PDGFRα(+) cells from mixed cell populations of colonic muscles dispersed from reporter strains of mice with constitutive expression of green fluorescent proteins. Differential expression of functional groups of genes related to purinergic signaling was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). KEY RESULTS: We detected marked phenotypic differences among SMC, ICC, and PDGFRα(+) cells. Substantial numbers of genes of importance in purinergic neurotransmission were enriched in PDGFRα(+) cells in relation to SMC and ICC. Notably, genes related to mediating effects and extracellular biotransformation of enteric purinergic inhibitory neurotransmitters were strongly expressed by PDGFRα(+) cells. CONCLUSIONS & INFERENCES: Our results demonstrate differential expression of genes for proteins involved in purinergic signaling in the SIP syncytium. These results may further clarify the specific functions of each cell type, identify novel biomarkers for postjunctional cells, and provide hypotheses for further studies to understand the physiological roles of cells of the SIP syncytium.
Asunto(s)
Motilidad Gastrointestinal/genética , Células Intersticiales de Cajal/metabolismo , Miocitos del Músculo Liso/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transcriptoma , Animales , Colon/citología , Colon/metabolismo , Ratones , Ratones Transgénicos , Purinas , Receptores Purinérgicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: The purinergic component of enteric inhibitory neurotransmission is important for normal motility in the gastrointestinal (GI) tract. Controversies exist about the purine(s) responsible for inhibitory responses in GI muscles: ATP has been assumed to be the purinergic neurotransmitter released from enteric inhibitory motor neurons; however, recent studies demonstrate that ß-nicotinamide adenine dinucleotide (ß-NAD(+)) and ADP-ribose mimic the inhibitory neurotransmitter better than ATP in primate and murine colons. The study was designed to clarify the sources of purines in colons of Cynomolgus monkeys and C57BL/6 mice. METHODS: High-performance liquid chromatography with fluorescence detection was used to analyze purines released by stimulation of nicotinic acetylcholine receptors (nAChR) and serotonergic 5-HT(3) receptors (5-HT(3)R), known to be present on cell bodies and dendrites of neurons within the myenteric plexus. KEY RESULTS: Nicotinic acetylcholine receptor or 5-HT(3)R agonists increased overflow of ATP and ß-NAD(+) from tunica muscularis of monkey and murine colon. The agonists did not release purines from circular muscles of monkey colon lacking myenteric ganglia. Agonist-evoked overflow of ß-NAD(+), but not ATP, was inhibited by tetrodotoxin (0.5 µmol L(-1)) or ω-conotoxin GVIA (50 nmol L(-1)), suggesting that ß-NAD(+) release requires nerve action potentials and junctional mechanisms known to be critical for neurotransmission. ATP was likely released from nerve cell bodies in myenteric ganglia and not from nerve terminals of motor neurons. CONCLUSIONS & INFERENCES: These results support the conclusion that ATP is not a motor neurotransmitter in the colon and are consistent with the hypothesis that ß-NAD(+), or its metabolites, serve as the purinergic inhibitory neurotransmitter.
Asunto(s)
Adenosina Trifosfato/metabolismo , Neuronas Motoras/metabolismo , Plexo Mientérico/metabolismo , NAD/metabolismo , Adenosina Trifosfato/análisis , Animales , Cromatografía Líquida de Alta Presión , Colon/inervación , Colon/metabolismo , Macaca fascicularis , Ratones , Ratones Endogámicos C57BL , Neuronas Motoras/química , Músculo Liso/inervación , Músculo Liso/metabolismo , NAD/análisisRESUMEN
Using high performance liquid chromatography fraction analysis we have recently established that numerous smooth muscle preparations, including the canine mesenteric artery and vein, release beta-nicotinamide adenine dinucleotide upon short-pulse electrical field stimulation in tetrodotoxin- and omega-conotoxin GVIA-sensitive manners [ Release of beta-nicotinamide adenine dinucleotide upon stimulation of postganglionic nerve terminals in blood vessels and urinary bladder. J Biol Chem 279:48893-48903.]. The beta-nicotinamide adenine dinucleotide metabolites ADP-ribose and cyclic ADP-ribose are also present in the tissue superfusates. CD38 is a multifunctional enzyme involved in the degradation of beta-nicotinamide adenine dinucleotide to ADP-ribose and cyclic ADP-ribose. Western immunoblot analysis revealed that CD38 is expressed in both artery and vein. Confocal laser scanning microscopy established colocalization of CD38 with tyrosine hydroxylase, synaptotagmin and synaptic vesicle protein in both blood vessels. High performance liquid chromatography with fluorescence detection demonstrated that whole tissue segments metabolize 1,N(6)-etheno-nicotinamide adenine dinucleotide to 1,N(6)-etheno-ADP-ribose and nicotinamide-guanine dinucleotide to cyclic GDP-ribose, suggesting the presence of both nicotinamide adenine dinucleotide-glycohydrolase and ADP-ribosyl cyclase activities in these blood vessels. Both enzymes appear to be associated with the membrane fraction, and therefore might be attributed to CD38. These data demonstrate a previously uncharacterized localization of CD38 in perivascular autonomic nerve terminals. Therefore, the beta-nicotinamide adenine dinucleotide/CD38 system may provide new mechanisms in autonomic neurovascular control.
Asunto(s)
ADP-Ribosil Ciclasa 1/metabolismo , Vasos Sanguíneos/metabolismo , Ganglios Simpáticos/citología , Terminales Presinápticos/metabolismo , Animales , Vasos Sanguíneos/citología , Western Blotting/métodos , Cromatografía Líquida de Alta Presión/métodos , Perros , Femenino , Nucleótidos de Guanina/metabolismo , Inmunohistoquímica/métodos , Masculino , NAD/análogos & derivados , NAD/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fracciones Subcelulares/metabolismo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
The present study investigated the spatial organization of electrical activity in the canine rectoanal region and its relationship to motility patterns. Contraction and resting membrane potential (E(m)) were measured from strips of circular muscle isolated 0.5-8 cm from the anal verge. Rapid frequency [25 cycles/min (cpm)] E(m) oscillations (MPOs, 12 mV amplitude) were present across the thickness of the internal anal sphincter (IAS; 0.5 cm) and E(m) was constant (-52 mV). Between the IAS and the proximal rectum an 18 mV gradient in E(m) developed across the muscle thickness with the submucosal edge at -70 mV and MPOs were replaced with slow waves (20 mV amplitude, 6 cpm). Slow waves were of greatest amplitude at the submucosal edge. Nifedipine (1 micro M) abolished MPOs but not slow waves. Contractile frequency changes were commensurate with the changes in pacemaker frequency. Our results suggest that changing motility patterns in the rectoanal region are associated with differences in the characteristics of pacemaker potentials as well as differences in the sites from which these potentials emanate.
Asunto(s)
Canal Anal/inervación , Relojes Biológicos , Potenciales de la Membrana/fisiología , Recto/inervación , Canal Anal/anatomía & histología , Animales , Relojes Biológicos/efectos de los fármacos , Perros , Conductividad Eléctrica , Femenino , Masculino , Potenciales de la Membrana/efectos de los fármacos , Contracción Muscular , Músculo Liso/inervación , Nifedipino/farmacología , Recto/anatomía & histología , Vasodilatadores/farmacologíaRESUMEN
1. The modulatory effects of agonists and antagonists of prejunctional alpha2-adrenoceptors on the electrical field stimulation (EFS, 0.3 ms, 12 V)-induced release of endogenous noradrenaline (NA) and the cotransmitter adenosine 5' triphosphate (ATP) were measured in endothelium-denuded segments of canine inferior mesenteric artery and compared with effects in mesenteric vein. The overflow of NA and ATP was evoked by long-duration (2 min) EFS at low frequency (4 Hz) and high frequency (16 Hz) of stimulation and was analysed using HPLC techniques with electrochemical detection and fluorescence detection, respectively. 2. The EFS-evoked overflow of both NA and ATP was significantly reduced by tetrodotoxin (1 microM) and guanethidine (10 microM) in the artery and vein. Desipramine (10 microM), a blocker of neuronal uptake of NA, increased the EFS (4 and 16 Hz)-evoked overflow of NA in both artery and vein. EFS-evoked overflow of NA in vein exceeded the NA overflow in artery at both 4 and 16 Hz in control preparations as well as in the presence of desipramine. However, the EFS-evoked overflow of ATP was equal in the artery and vein. 3. Stimulation of alpha2-adrenoceptors with clonidine (0.1 microM) and oxymethazoline (0.3 microM) reduced the EFS evoked overflow of NA in both artery and vein at 4 Hz, whereas the NA overflow at 16 Hz remained unchanged in both blood vessels. The overflow of ATP as well as of ADP (and hence ATP:ADP ratio) was unaffected by the alpha2-adrenoceptor agonists in the artery and vein. 4. In artery, blockade of alpha2-adrenoceptors with yohimbine at a concentration of 0.1 microM caused no effect on the NA overflow neither at 4 Hz nor at 16 Hz of EFS. Yohimbine at a concentration of 1 microM increased the overflow of NA at 4 Hz but not 16 Hz of EFS. In vein, however, yohimbine (0.1 and 1 microM) increased NA overflow at both 4 and 16 Hz of stimulation. Idazoxan (1 microM) increased the NA overflow in artery only at 4 Hz, whereas in vein idazoxan increased the NA overflow at both 4 and 16 Hz. No changes of EFS-evoked ATP overflow were observed in the presence of 0.1 microM yohimbine in both artery and vein. Greater concentration of yohimbine (i.e. 1 microM) increased the overflow of ATP in both the artery and vein only at 4 Hz EFS. Idazoxan (1 microM) enhanced the ATP overflow only at 16 Hz in vein. The overflow of ADP was affected by both yohimbine and idazoxan in a similar manner to the ATP overflow so that the ATP:ADP ratios were not changed. 5. In conclusion, sympathetic nerves in both mesenteric arteries and veins appear to release ATP along with NA. Release of NA in veins exceeds release of NA in arteries, whereas both the canine artery and vein release equal amount of ATP. At long-duration nerve stimulation (as might occur during stress) the alpha2-adrenoceptors appear to rather modulate release of NA than release of the cotransmitter ATP. The prejunctional autoinhibition of NA release is more effective at lower frequencies of nerve stimulation. The alpha2-adrenoceptor-mediated neuromodulation plays a greater role in veins than arteries. Quantitative differences in alpha2-adrenoceptor-mediated neuromodulation in the arteries and veins may participate to differing contributions of mesenteric blood vessels to the control of blood flow and volume distribution in splanchnic circulation.
Asunto(s)
Adenosina Trifosfato/metabolismo , Arterias Mesentéricas/metabolismo , Venas Mesentéricas/metabolismo , Norepinefrina/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Adenosina Difosfato/metabolismo , Agonistas alfa-Adrenérgicos/farmacología , Antagonistas Adrenérgicos alfa/farmacología , Animales , Perros , Estimulación Eléctrica , Femenino , Masculino , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/inervación , Venas Mesentéricas/efectos de los fármacos , Venas Mesentéricas/inervación , Neurotransmisores/farmacología , Receptores Adrenérgicos alfa 2/efectos de los fármacos , Sistema Nervioso Simpático/fisiología , Transmisión Sináptica/efectos de los fármacosRESUMEN
1. The present study was designed to compare the overflow of sympathetic neurotransmitters of guinea-pig inferior mesenteric artery and mesenteric vein evoked by electrical field stimulation (EFS) with special emphasis on the simultaneous release of ATP and noradrenaline (NA). The stimulation-evoked overflow of ADP, AMP and adenosine was also evaluated. 2. Endothelium-denuded segments of inferior mesenteric arteries or veins were superfused in a small volume (200 microL)-chamber for EFS and subsequent detection of NA (HPLC- electrochemical detection) and adenine nucleotides and adenosine (HPLC-fluorescence detection) in samples of the superfusate. 3. Both arteries and veins responded to EFS (15 V, 4-16 Hz, 0.3 msec for 60 s) with overflow of ATP and NA in a tetrodotoxin (1 micromol/L)- and guanethidine (10 micromol/L)-sensitive manner. The EFS-evoked overflow of NA in veins exceeded the overflow of NA in arteries at all frequencies of stimulation, whereas the EFS-evoked overflow of ATP, ADP and AMP in veins exceeded the overflow of adenine nucleotides in arteries at 4 and 8 Hz but not at 16 Hz stimulation. The EFS-evoked overflow of adenosine was similar in arteries and veins. 4. Activation of alpha1-adrenoceptors with methoxamine (10 micromol/L) did not produce overflow of ATP. 5. Blockade of alpha1/alpha2-adrenoceptors with phentolamine (1 micromol/L) did not affect EFS-evoked overflow of ATP, ADP, AMP and adenosine. 6. It is concluded that overflow of ATP and NA from sympathetic nerves may constitute an effective mechanism in the complex balance between capacitance and resistance in splanchnic circulation.
Asunto(s)
Adenosina Trifosfato/metabolismo , Arterias Mesentéricas/metabolismo , Venas Mesentéricas/metabolismo , Norepinefrina/metabolismo , Adenosina/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Estimulación Eléctrica , Cobayas , Técnicas In Vitro , Masculino , Arterias Mesentéricas/fisiología , Venas Mesentéricas/fisiología , Neurotransmisores/metabolismo , Purinas/metabolismoRESUMEN
Excitatory junctional potentials (EJPs) elicited with brief duration (10 s) electrical field stimulation of guinea-pig mesenteric arteries were nearly abolished at all frequencies by pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 30 microM) but persisted following reserpinization. Suramin (100 microM) enhanced EJPs at 0.2-0.5 Hz responses and reduced them at 2-32 Hz. Phentolamine (1 microM) and yohimbine (0.1 microM) enhanced EJPs at 0.2-8 Hz but not at 16-32 Hz. Oxymetazoline (0.3 microM) reduced EJPs at 0.2-0.5 Hz but not at 1-32 Hz. Following reserpinization, EJPs were enhanced at 0.2-2 Hz but not at 4-32 Hz. Clonidine (0.1 microM) was without effect at all frequencies in control arteries but reduced EJPs at 0.2-2 Hz in reserpine-treated arteries. In conclusion, pre-junctional alpha(2)-adrenoceptors modulate ATP release during low frequency, brief duration sympathetic nerve stimulation.
Asunto(s)
Arterias Mesentéricas/fisiología , Unión Neuromuscular/fisiología , Fosfato de Piridoxal/análogos & derivados , Receptores Adrenérgicos alfa 2/fisiología , Agonistas de Receptores Adrenérgicos alfa 2 , Antagonistas de Receptores Adrenérgicos alfa 2 , Agonistas alfa-Adrenérgicos/farmacología , Antagonistas Adrenérgicos alfa/farmacología , Animales , Clonidina/farmacología , Estimulación Eléctrica , Potenciales Evocados/efectos de los fármacos , Cobayas , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/inervación , Unión Neuromuscular/efectos de los fármacos , Oximetazolina/farmacología , Fentolamina/farmacología , Fosfato de Piridoxal/farmacología , Reserpina/farmacología , Suramina/farmacología , Yohimbina/farmacologíaRESUMEN
Vasoconstrictor responses to exogenous adenine and pyrimidine nucleotides were measured in endothelium-denuded segments of guinea pig mesenteric vein and compared with responses in mesenteric artery. The rank order of potency for nucleotides in veins was: 2-MeSADP = 2-MeSATP > UTP > ATPgammaS = alpha,betaMeATP > UDP = ATP > ADP >> beta,gamma-D-MeATP = beta,gamma-L-MeATP. In contrast 2-MeSADP, UTP, and UDP were inactive in arteries, and the rank order of potency of other nucleotides differed; that is, alpha,betaMeATP > beta, gamma-D-MeATP > beta,gamma-L-MeATP = ATPgammaS = 2-MeSATP > ATP > ADP. In veins, UTP, ATP, and 2-MeSATP were more efficacious contractile agents than alpha,beta MeATP. In addition, the ability to desensitize responses to these nucleotides and inhibit them with various blockers differed. The response to alpha,betaMeATP in veins exhibited rapid desensitization and was inhibited by pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS) and suramin. The response to 2-MeSATP in veins did not desensitize; nor was it inhibited by prior alpha,betaMeATP desensitization, but it was inhibited by PPADS, suramin, and the selective P2Y(1) receptor antagonist adenosine 3',5'-bisphosphate (ABP, 10-100 microM). Responses to ATP and UTP in veins did not desensitize and were not inhibited by PPADS, suramin, ABP, or alpha, betaMeATP desensitization. In conclusion, our results suggest that venous contraction to a variety of nucleotides is mediated in large part by P2Y receptors including P2Y(1) receptors and an UTP-preferring P2Y receptor. A small component of contraction also appears to be mediated by P2X(1) receptors. This receptor profile differs markedly from that of mesenteric arteries in which P2X(1) receptors predominate.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Venas Mesentéricas/fisiología , Contracción Muscular/fisiología , Músculo Liso Vascular/fisiología , Fosfato de Piridoxal/análogos & derivados , Receptores Purinérgicos P2/fisiología , Nucleótidos de Adenina/farmacología , Adenosina Difosfato/farmacología , Adenosina Trifosfato/farmacología , Animales , Cobayas , Técnicas In Vitro , Masculino , Arteria Mesentérica Inferior/efectos de los fármacos , Arteria Mesentérica Inferior/fisiología , Venas Mesentéricas/efectos de los fármacos , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Cloruro de Potasio/farmacología , Antagonistas del Receptor Purinérgico P2 , Fosfato de Piridoxal/farmacología , Nucleótidos de Pirimidina/farmacología , Receptores Purinérgicos P2/clasificación , Receptores Purinérgicos P2X , Suramina/farmacología , Uridina Trifosfato/farmacologíaRESUMEN
Neuropeptide Y (NPY) is a cotransmitter with noradrenaline in guinea pig inferior mesenteric vein. Tyrosine hydroxylase-like immunoreactivity and NPY-like immunoreactivity were colocalized in a dense network of fibers within the adventitial layer of guinea-pig inferior mesenteric vein. Vasoconstrictor responses to electrical field stimulation (0.2-64 Hz, 0.1 ms, 12 V, for 10 s) appear to be mediated primarily by norepinephrine at 0.2 to 4 Hz and by NPY at 8 to 64 Hz. NPY Y1 receptors mediate the contractile responses to both endogenous and exogenous NPY. Norepinephrine and NPY are involved in neuromuscular transmission in guinea pig mesenteric vein suggesting that the sympathetic nervous system requires the coordinated action of norepinephrine and NPY to serve capacitance.
Asunto(s)
Venas Mesentéricas/efectos de los fármacos , Neuropéptido Y/farmacología , Neurotransmisores/farmacología , Norepinefrina/farmacología , Sistema Nervioso Simpático/fisiología , Antagonistas Adrenérgicos alfa/farmacología , Animales , Arginina/análogos & derivados , Arginina/farmacología , Estimulación Eléctrica , Cobayas , Técnicas In Vitro , Masculino , Venas Mesentéricas/inervación , Neuropéptido Y/análogos & derivados , Reserpina/farmacología , VasoconstricciónRESUMEN
Vasoconstrictor responses to electrical field stimulation (EFS, 0.2-32 Hz, 0.1 ms, 12 V, for 1 min) were measured in endothelium-denuded segments of guinea-pig mesenteric vein and compared to responses in mesenteric artery. The distribution of both tyrosine-hydroxylase-like immunoreactivity (TH-LI) and neuropeptide Y-like immunoreactivity (NPY-LI) was also studied using anti-TH and anti-NPY antibodies. The effect of exogenous NPY (10 nM) on EFS (8 Hz, 0.3 ms, 12 V, for 1 min)-evoked overflow of noradrenaline (NA) was also studied using an HPLC technique with electrochemical detection. Veins responded with contractions at lower frequencies of stimulation than arteries. Prazosin (0.1 microM) abolished the EFS-evoked contractions in artery at 0.5-32 Hz and in vein at 0.2-1 Hz of stimulation. However, in vein, the contractile responses to EFS at 2-32 Hz of stimulation were only reduced by prazosin. Phentolamine (1 microM) abolished the responses to 0.5-4 Hz and reduced the responses to 8-32 Hz of EFS in artery. In vein, phentolamine (1 microM) abolished the responses to 0.2-1 Hz and facilitated the contractions elicited by 16-32 Hz. The NPY-receptor antagonist BIBP3226 (1 microM), in combination with phentolamine, abolished contractions in vein. Yohimbine (0.1 microM) abolished the responses to lower frequencies of stimulation in both artery (0.5-2 Hz) and vein (0.2-1 Hz). The responses to greater frequency stimulation were not affected by yohimbine in artery, and were facilitated in vein. Pre-treatment of animals for 24 h with reserpine abolished contractile responses to EFS in artery, whereas in vein, responses to 0.2-2 Hz were abolished while responses to 4-32 Hz were unchanged. Suramin (100 microM) or alpha,beta-methylene ATP (alpha,beta MeATP; 10-100 microM) treatment did not affect the contractile responses to EFS in either artery or vein. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS; 30 microM), even potentiated the responses to 2-16 Hz in vein. However, following resperine-treatment, both PPADS and suramin reduced the nerve-evoked contractions of vein. Either BIBP3226 (1 microM) alone or BIBP3226 in combination with PPADS or suramin abolished the contractile response to EFS in reserpine-treated veins. NPY (100 nM) produced significantly more contraction in vein than in artery (i.e., 93 +/- 2.5 versus 7 +/- 4% of the response to 70 mM KCl, respectively). NPY (10 nM) significantly reduced the NA overflow evoked by EFS at 8 Hz. Flat mount preparations and cryostat sections of both mesenteric artery and vein revealed that TH-LI and NPY-LI were co-localized in a dense network of fibers within the adventitial layer. In conclusion, NA exclusively mediates the contractile response to sympathetic nerve stimulation in guinea-pig mesenteric artery, whereas at least three neurotransmitters [i.e., NA, adenosine 5'-triphosphate (ATP) and NPY] are involved in the neural response of mesenteric vein.
Asunto(s)
Arterias Mesentéricas/inervación , Venas Mesentéricas/inervación , Neuronas Eferentes/metabolismo , Circulación Esplácnica/fisiología , Fibras Simpáticas Posganglionares/metabolismo , Vasoconstricción/fisiología , Adenosina Trifosfato/metabolismo , Antagonistas Adrenérgicos alfa/farmacología , Animales , Cobayas , Inmunohistoquímica , Masculino , Arterias Mesentéricas/citología , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/fisiología , Venas Mesentéricas/citología , Venas Mesentéricas/efectos de los fármacos , Venas Mesentéricas/fisiología , Neuronas Eferentes/citología , Neuronas Eferentes/efectos de los fármacos , Neuropéptido Y/metabolismo , Norepinefrina/metabolismo , Antagonistas Purinérgicos , Receptores de Neuropéptido Y/antagonistas & inhibidores , Circulación Esplácnica/efectos de los fármacos , Fibras Simpáticas Posganglionares/citología , Fibras Simpáticas Posganglionares/efectos de los fármacos , Tirosina 3-Monooxigenasa/metabolismo , Vasoconstricción/efectos de los fármacosRESUMEN
1. The present study was undertaken to determine the modulatory effects of the endothelin peptides on the neurogenically-induced release of endogenous noradrenaline (NA) and the cotransmitter adenosine 5'-triphosphate (ATP) from the sympathetic nerves of endothelium-free segments of the rat isolated tail artery. The electrical field stimulation (EFS, 8 Hz, 0.5 ms, 3 min) evoked overflow of NA and ATP, in the absence of endothelins, was 0.035+/-0.002 pmol mg(-1) tissue and 0.026+/-0.002 pmol mg(-1) tissue, respectively. 2. Endothelin-1 (ET-1; 1-30 nM) significantly reduced the EFS evoked overflow of both NA and ATP. The maximum inhibitory effect was produced by a peptide concentration of 10 nM, the amount of NA overflow being 0.020+/-0.002 pmol mg(-1) and that of ATP overflow 0.015+/-0.001 pmol mg(-1). Higher peptide concentrations (100 and 300 nM) reversed the EFS-evoked overflow of NA to control levels and that of ATP to above control levels. The inhibitory effect of ET-1 (10 nM) was resistant to the selective ET(A) receptor antagonist cyclo-D-Trp-D-Asp(ONa)-Pro-D-Val-Leu (BQ-123) but was prevented by ET(B) receptor desensitization with sarafotoxin S6c (StxS6c) or by ET(B) receptor blockade with N, cis-2,6-dimethylpiperidinocarbonyl-L-gmethylleucyl-D-1-me thoxycarbonyltryptophanyl-D-norleucine (BQ-788). 3. StxS6c, upon acute application, exerted a dual effect on transmitter release. At concentrations of 0.001-0.3 nM the peptide significantly reduced the EFS-evoked NA overflow, whereas at concentrations of 1-10 nM it caused a significant increase in the evoked overflow of both ATP and NA. Both the maximum inhibitory effect of StxS6c at a concentration of 0.003 nM (approximately 85% reduction of NA overflow and 40% of ATP overflow) and the maximum facilitatory effect of the peptide at a concentration of 3 nM (approximately 400% increase of ATP overflow and 200% of NA overflow) were completely antagonized by either BQ-788 or by StxS6c-induced ET(B) receptor desensitization. 4. ET-3 (10-100 nM) did not affect the EFS evoked overflow of either ATP or NA, but at a concentration of 300 nM significantly potentiated the release of both transmitters (0.118+/-0.02 pmol mg(-1) tissue ATP overflow and 0.077+/-0.004 pmol mg(-1) NA overflow). This effect was prevented either by BQ-123 or by BQ-788. 5. In summary, the endothelin peptides exerted both facilitatory and inhibitory effects on the neurogenically-induced release of the sympathetic cotransmitters ATP and NA in the rat tail artery. Both transmitters were modulated in parallel indicating that the endothelins do not differentially modulate the release of NA and ATP in this tissue.
Asunto(s)
Endotelina-1/farmacología , Endotelina-2/farmacología , Músculo Liso Vascular/inervación , Receptores Presinapticos/efectos de los fármacos , Sistema Nervioso Simpático/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Arterias/efectos de los fármacos , Arterias/inervación , Estimulación Eléctrica , Antagonistas de los Receptores de Endotelina , Técnicas In Vitro , Masculino , Músculo Liso Vascular/efectos de los fármacos , Norepinefrina/metabolismo , Ratas , Ratas Wistar , Flujo Sanguíneo Regional/efectos de los fármacos , Cola (estructura animal)/irrigación sanguínea , Vasoconstrictores/farmacología , Venenos de Víboras/farmacologíaRESUMEN
1. Rat type-C natriuretic peptide (CNP) has been studied for its effects on the neurogenically induced overflow of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), adenosine (ADO) and noradrenaline (NA) in endothelium-free segments of rat isolated tail artery. The overflow of each was evoked by electrical field stimulation (EFS) of 0.5 ms pulses at 8 Hz for 3 min and the amount of ATP, ADP, AMP and ADO was quantified by high-performance liquid chromatography (HPLC)-fluorescent detection, while the amount of NA was quantified by HPLC-electrochemical detection. 2. Type-C natriuretic peptide (100 nmol/L) was found to cause a significant reduction of the overflow of all adenine purines and NA. However, at lower concentrations (1 and 10 nmol/L), CNP caused a significant reduction of the overflow of NA but did not change ATP overflow. 3. The overflow of ADP, AMP and ADO was significantly reduced by either concentration of CNP, so that the ratio ATP:ADP was diminished from 1:2 in controls to 1:1 after 1 nmol/L CNP and to 1:1.2 after 10 nmol/L CNP. 4. The production of inorganic phosphate (Pi) in response to the exogenous application of ATP was significantly reduced by 1, 10 or 100 nmol/L CNP. 5. Type-C natriuretic peptide exerts neuromodulatory effects on the neurogenically induced release of the cotransmitters ATP and NA in rat tail artery, consisting of an inhibition of the release of both ATP and NA. This effect is accompanied by inhibition of the breakdown of ATP by ecto-ATPases. Either effect results in apparent CNP-induced differential modulation of the overflow of the cotransmitters ATP and NA.
Asunto(s)
Nucleótidos de Adenina/metabolismo , Arterias/efectos de los fármacos , Péptido Natriurético Tipo-C/fisiología , Sistema Nervioso Simpático/fisiología , Transmisión Sináptica/fisiología , Adenosina/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Arterias/inervación , Estimulación Eléctrica , Masculino , Péptido Natriurético Tipo-C/farmacología , Norepinefrina/metabolismo , Ratas , Ratas Wistar , Flujo Sanguíneo Regional/efectos de los fármacos , Sistema Nervioso Simpático/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Cola (estructura animal)/irrigación sanguíneaRESUMEN
Two members of the natriuretic peptide family (rANF8-33 and pBNP1-32) and two members of the endothelin family (ET-1 and ET-2) have been studied for their effects on the neurogenically induced overflow of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), and adenosine (ADO) from the isolated guinea-pig vas deferens. rANF, pBNP, ET-1, and ET-2 each at 10 nM produced a significant increase in the evoked overflow of ATP, by 52, 85, 130, and 115%, respectively. None of the peptides altered the overflow of ADO. ET-1 and ET-2 each caused an increase in the overflow of ADP and AMP by an amount similar to their effects on ATP overflow, so that the ratio ATP:ADP remained 1:1 throughout. Natriuretic peptides, however, affected the overflow of ADP and AMP to a lesser extent than ATP, resulting in an ATP:ADP ratio of 2:1 after rANF and of 1.5:1 after pBNP. In addition, rANF or pBNP, but not ET-1 or ET-2, inhibited ecto-ATPase activity, suggesting that this mechanism may contribute to the facilitatory effect of the natriuretic peptides on the nerve-evoked overflow of ATP in this tissue.
Asunto(s)
Nucleótidos de Adenina/metabolismo , Adenosina Trifosfato/metabolismo , Adenosina/metabolismo , Factor Natriurético Atrial/farmacología , Endotelinas/farmacología , Péptido Natriurético Encefálico , Proteínas del Tejido Nervioso/farmacología , Fragmentos de Péptidos/farmacología , Conducto Deferente/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Estimulación Eléctrica , Endotelina-1/farmacología , Endotelina-2/farmacología , Cobayas , Técnicas In Vitro , Masculino , Músculo Liso/enzimología , Músculo Liso/inervación , Músculo Liso/metabolismo , Conducto Deferente/enzimología , Conducto Deferente/inervaciónRESUMEN
The role of P1 purinoceptor subtypes, the adenylyl cyclase (AC) pathway, and ATP-sensitive K+ (KATP) channels in adenosine (Ado)-induced membrane hyperpolarization was investigated in isolated segments of the guinea pig coronary artery using conventional microelectrode techniques. Ado (1-100 microM) elicited concentration-dependent hyperpolarization (half-maximal effective concentration 7.5 +/- 0.5 microM) that averaged 28.6 +/- 2.9 mV at 100 microM Ado. The A1 selective agonist N6-cyclopentyladenosine (CPA), the A1/A2 agonist 2-chloroadenosine, and the A2a/A2b agonist 5-(N-ethylcarboxamido)adenosine (NECA) each induced glibenclamide (3 microM)-sensitive hyperpolarization at 10 microM. However, the selective A2a-receptor agonists CGS-21680 and N6-[2-(3,5-dimethoxyphenyl)-2-(2-methoxyphenyl]ethyladenosine (10 microM each) were without effect. Responses to CPA and NECA were significantly reduced by the AC inhibitor SQ-22,536 (100 microM). Activation of the AC-adenosine 3',5'-cyclic monophosphate (cAMP)-protein kinase A (PKA) pathway by four additional methods, i.e., 1) forskolin (0.3-1 microM), 2) isoproterenol (0.1-1 microM), 3) combined milrinone (0.4 microM) and rolipram (30 microM), and 4) combined N6-phenyladenosine 3',5'-monophosphate, 8-(6-aminohexyl)aminoadenosine 3',5'-cyclic monophosphate, and the Sp-isomer of 5,6-dichloro-1-D-ribofuranosylbenzimidazole-3', 5'-cyclic monophosphothioate (100 microM each), also gave rise to glibenclamide-sensitive hyperpolarization. These results suggest that stimulation of A2b receptors coupled to AC represents the predominant mechanism by which Ado elicits hyperpolarization in this vessel. The ensuing increase in cAMP activates PKA, which then increases the activity of KATP channels. Our results further suggest that KATP channels are an important target for numerous pathways that raise cAMP levels in the coronary artery.
Asunto(s)
Adenosina/farmacología , Vasos Coronarios/fisiología , Canales de Potasio/fisiología , Receptores Purinérgicos P1/fisiología , 2-Cloroadenosina/farmacología , Adenosina/análogos & derivados , Adenosina-5'-(N-etilcarboxamida)/farmacología , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Vasos Coronarios/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Gliburida/farmacología , Cobayas , Técnicas In Vitro , Indometacina/farmacología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Nitroarginina/farmacología , Canales de Potasio/efectos de los fármacos , Agonistas del Receptor Purinérgico P1 , Receptor de Adenosina A2BRESUMEN
Endothelin-3 (10 nM) produced a significant facilitation of the release of ATP from the in vitro guinea-pig vas deferens. This effect was converted to an inhibition of release by pretreatment with BQ-123, cyclo-(D-Trp,D-Asp,L-Pro,D-Val,L-Leu), an endothelin ETA receptor antagonist. Desensitization of endothelin ETB receptors by sarafotoxin S6c antagonized, but did not reverse, the facilitatory effect of endothelin-3. The release of noradrenaline was not facilitated by endothelin-3; however, following pretreatment with BQ-123 the release of noradrenaline was reduced by the peptide. These results indicate that there may be both facilitatory and inhibitory prejunctional endothelin receptors and further suggest that the release of the sympathetic nerve cotransmitters ATP and noradrenaline may be differentially modulated.
Asunto(s)
Endotelinas/farmacología , Músculo Liso/metabolismo , Neurotransmisores/metabolismo , Conducto Deferente/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Estimulación Eléctrica , Antagonistas de los Receptores de Endotelina , Cobayas , Técnicas In Vitro , Masculino , Datos de Secuencia Molecular , Músculo Liso/efectos de los fármacos , Norepinefrina/metabolismo , Péptidos Cíclicos/farmacología , Conducto Deferente/efectos de los fármacos , Vasoconstrictores/farmacología , Venenos de Víboras/farmacologíaRESUMEN
1. The present study has been performed to evaluate the modulatory effects of endothelin-1 (ET-1) on the purinergic and adrenergic components of sympathetically-mediated contractile responses of endothelium-free rabbit saphenous artery preparations. 2. ET-1 increased the smooth muscle tone, the pD2 value being 7.77 +/- 0.05. 3. Postjunctionally, ET-1 enhanced the responses to exogenous adenosine 5'-triphosphate (ATP) and did not influence those to exogenous noradrenaline (NA). 4. ET-1 increased the contractile responses to short-lasting and to long-lasting electrical field stimulation at a frequency of 5 or 10 Hz, showing a tendency towards decreasing the prazosin-sensitive component and increasing the mATP-sensitive component of the contractile responses. 5. In prazosin-treated preparations ET-1 increased the residual mATP-sensitive responses and this effect was more pronounced after yohimbine. 6. In mATP-treated preparations ET-1 increased the residual electrically-induced contractions and this increase was abolished after yohimbine. 7. It is suggested that ET-1 modulates co-transmission in the rabbit saphenous artery by potentiating postjunctionally the purinergic component of the contractile responses to both exogenous ATP or electrical stimulation.
Asunto(s)
Adenosina Trifosfato/farmacología , Endotelinas/farmacología , Norepinefrina/farmacología , Sistema Nervioso Simpático/fisiología , Vasoconstricción/efectos de los fármacos , Animales , Estimulación Eléctrica , Técnicas In Vitro , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Prazosina/farmacología , ConejosRESUMEN
1. The effects of cumulatively applied galanthamine hydrobromide (0.1-300 microM) on the mechanical activity of isolated proximal jejunum and distal ileum were studied. 2. Galanthamine was found to increase the spontaneous mechanical activity and exert by itself an enhancement of the smooth muscle tone in the two segments, both effects being more pronounced in ileum than in jejunum. 3. The galanthamine-induced augmentation of the spontaneous mechanical activity was tetrodotoxin (TTX)-sensitive, whereas the contractile effect of the drug on the tone was TTX-insensitive in both segments. 4. The present results showed not only quantitative differences in the tonic effects of galanthamine in both intestinal segments but they suggest some qualitative differences in cholinergic neuronal control along the gastro-intestinal tract.
Asunto(s)
Inhibidores de la Colinesterasa/farmacología , Galantamina/farmacología , Músculo Liso/efectos de los fármacos , Animales , Íleon/efectos de los fármacos , Técnicas In Vitro , Yeyuno/efectos de los fármacos , Masculino , Contracción Muscular/efectos de los fármacos , Ratas , Ratas Wistar , Tetrodotoxina/farmacologíaRESUMEN
The effects of atrial natriuretic peptide (ANP) on the purinergic and adrenergic components of electrically induced contractile responses in the prostatic portion of the guinea pig vas deferens were studied. ANP (0.01-100 nM) was found to inhibit the two phases of the biphasic contractions induced by field electrical stimulation (300 pulses, 0.1 msec, 9 Hz). The effects of ANP at a concentration of 10 nM were examined further. ANP had no effect on l-phenylephrine-induced or exogenous ATP-induced contractions in the presence of tetrodotoxin, indicating a prejunctional rather than postjunctional modulatory effect of ANP. Neither yohimbine, nor indomethacin, nor naloxone influenced the inhibitory effect of ANP on either phase of the electrically induced contractions. However, the inhibitory effect of ANP on the first phase was verapamil-sensitive and nitrendipine-insensitive, whereas the effect on the second phase was sensitive to both verapamil and nitrendipine. Thus, at least two different calcium pools could be involved in the ANP inhibitory effect. The inhibitory effect of ANP on the purinergic component of the electrically induced contractile responses after prazosin remained the same, whereas after alpha,beta-methylene adenosine-5'-triphosphate there was no effect on ANP on the residual adrenergic component. Therefore, ANP exerted an inhibitory effect on the purinergic and not on the adrenergic component of the electrically induced mechanical responses in the guinea pig vas deferens via prejunctional mechanisms which do not depend on alpha-2 adrenoceptors, opiate receptors or the production of prostaglandins and depend on different calcium pools.