RESUMO
STUDY DESIGN: Animal proof of principle study. OBJECTIVES: To determine whether capromorelin, a compound that causes defecation by stimulating ghrelin receptors within the lumbosacral defecation centers, is effective after spinal cord injury (SCI), and whether SCI significantly alters sensitivity to the compound. SETTING: University of Melbourne and Austin Hospital, Melbourne, Australia. METHODS: Rats were subjected to spinal cord contusion injury or were sham-operated. At 6 weeks after surgery, effects of capromorelin on blood pressure, heart rate and propulsive contractions of the colorectum were investigated. RESULTS: Capromorelin caused robust propulsive activity in the colorectum soon after its application. The compound was similarly effective in naïve, sham-operated and spinal cord-injured rats. Blood pressure increases caused by capromorelin were not exaggerated after SCI, and there was no evidence of phasic blood pressure increases when the colon was contracted by the compound. CONCLUSION: Capromorelin is a therapeutic compound that could potentially be used to relieve constipation by triggering defecation in spinal cord-injured patients.
Assuntos
Constipação Intestinal/tratamento farmacológico , Constipação Intestinal/fisiopatologia , Defecação/efeitos dos fármacos , Piperidinas/farmacologia , Pirazóis/farmacologia , Receptores de Grelina/agonistas , Traumatismos da Medula Espinal/fisiopatologia , Animais , Constipação Intestinal/etiologia , Defecação/fisiologia , Modelos Animais de Doenças , Hormônio do Crescimento/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de Grelina/fisiologia , Traumatismos da Medula Espinal/complicaçõesRESUMO
In haploid rad52 Saccharomyces cerevisiae strains unable to undergo homologous recombination, a chromosomal double-strand break (DSB) can be repaired by imprecise rejoining of the broken chromosome ends. We have used two different strategies to generate broken chromosomes: (i) a site-specific DSB generated at the MAT locus by HO endonuclease cutting or (ii) a random DSB generated by mechanical rupture during mitotic segregation of a conditionally dicentric chromosome. Broken chromosomes were repaired by deletions that were highly variable in size, all of which removed more sequences than was required either to prevent subsequent HO cleavage or to eliminate a functional centromere, respectively. The junction of the deletions frequently occurred where complementary strands from the flanking DNA could anneal to form 1 to 5 bp, although 12% (4 of 34) of the events appear to have occurred by blunt-end ligation. These types of deletions are very similar to the junctions observed in the repair of DSBs by mammalian cells (D. B. Roth and J. H. Wilson, Mol. Cell. Biol. 6:4295-4304, 1986). When a high level of HO endonuclease, expressed in all phases of the cell cycle, was used to create DSBs, we also recovered a large class of very small (2- or 3-bp) insertions in the HO cleavage site. These insertions appear to represent still another mechanism of DSB repair, apparently by annealing and filling in the overhanging 3' ends of the cleavage site. These types of events have also been well documented for vertebrate cells.
Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Recombinação Genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Fúngicos , Primers do DNA , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas Fúngicas/biossíntese , Fator de Acasalamento , Mitose , Dados de Sequência Molecular , Mutagênese Insercional , Peptídeos/genética , Feromônios/genética , Reação em Cadeia da Polimerase , Proteína Rad52 de Recombinação e Reparo de DNA , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae , Deleção de SequênciaRESUMO
Focal extracellular recording techniques were used to study nerve impulse propagation and the intermittent transmitter release mechanism in sympathetic nerve terminals of the guinea-pig vas deferens in vitro. In particular, the nature of impulse propagation in postganglionic nerve fibres was characterized following pre- or postganglionic stimulation. Conventional intracellular recording techniques were also used to study directly ganglionic transmission in cell bodies in the anterior pelvic ganglia. When brief electrical stimuli were applied to the hypogastric nerve trunk close to the prostatic end of the vas deferens, the nerve terminal impulses recorded extracellularly could be evoked either directly by stimulation of the parent axon (i.e. postganglionically) or indirectly by stimulation of the preganglionic nerve fibre. In 364 separate recordings, nerve terminal impulse conduction failure was not observed during trains of stimuli at 1 Hz. However, apparent "intermittent conduction" of nerve impulses was noted on 16 occasions. In these fibres, the degree of intermittent conduction decreased as the frequency of stimulation was increased. Conduction in these intermittent fibres was reversibly interrupted by removing calcium from the Krebs' solution or by the addition of the ganglion blocker, hexamethonium (30-100 microM). Thus, the cause of intermittent conduction is failure of the transmission of excitation in the sympathetic ganglia. Impulses evoked by postganglionic stimulation never failed to propagate into the nerve terminals, and changes in the shape or amplitude of the nerve terminal impulse during trains of stimuli were not detected. One effect of stimulation was a frequency-dependent increase in the latency of the nerve terminal impulse which developed during the train of stimuli. Thus, intermittence of transmitter release from individual varicosities cannot be attributed to failure of impulse propagation in sympathetic nerve terminals. Transmission in the anterior pelvic ganglia was investigated directly by making intracellular recordings from cell bodies whose terminals projected to the vas deferens. Many cell bodies received a strong synaptic input which generated an action potential in the postganglionic cell body on a one-to-one basis. However, in some cell bodies there was a low safety factor for the generation of the action potential by the excitatory postsynaptic potential. The safety factor for generating an action potential in the postganglionic cell body was raised by increasing the frequency of stimulation. These findings suggest that peripheral ganglia are not simple one-to-one relay stations, but may well play an important role in controlling the patterns of nerve impulse traffic in postganglionic sympathetic neurons.
Assuntos
Gânglios Simpáticos/fisiologia , Terminações Nervosas/fisiologia , Condução Nervosa/fisiologia , Sistema Nervoso Simpático/fisiologia , Ducto Deferente/fisiologia , Animais , Estimulação Elétrica , Cobaias , Técnicas In Vitro , Masculino , Ducto Deferente/inervaçãoRESUMO
1. Focal extracellular recording techniques were used to study the effects of clonidine, yohimbine and tyramine on the intermittent transmitter release mechanism in the guinea-pig vas deferens in vitro. Drugs were applied locally to the varicosities located within the recording electrode. Statistical methods were used to determine whether noradrenaline (NA) acts locally to inhibit secretion from the same or a closely related release site (local regulation) on an impulse-to-impulse basis. 2. The alpha-adrenoceptor agonist, clonidine, inhibited transmitter release, an effect reversed by the alpha-adrenoceptor antagonist, yohimbine. Yohimbine alone increased action potential-evoked transmitter release, findings consistent with the idea that transmitter release is regulated through prejunctional alpha-adrenoceptors. 3. The indirectly acting sympathomimetic, tyramine, powerfully inhibited evoked transmitter release, an effect reversed by both yohimbine and phentolamine. The inhibitory effects of tyramine were greatly reduced in tissues taken from animals pretreated with reserpine. Clonidine powerfully inhibited transmitter release in reserpinized tissues showing that prejunctional alpha-adrenoceptors were functionally intact. The inhibitory effects of tyramine on transmitter release are therefore mediated indirectly through the release of endogenous NA. 4. Paradoxically, when transmitter release from a small population of variscosities on a single nerve fibre was studied in the absence of alpha-adrenoceptor antagonists, no evidence was found for local regulation of transmitter release. 5. The intermittent character of the transmitter release process makes it difficult to envisage how impulse-to-impulse regulation could occur. Furthermore, it is unlikely that NA will accumulate to any appreciable extent in the vicinity of the secreting varicosity. 6. The pharmacological evidence clearly supports the view that NA released from sympathetic nerve terminals by nerve impulses modulates subsequent transmitter release. However, the evidence does not support the view that released NA acts locally to inhibit secretion from recently activated varicosities.
Assuntos
Receptores Adrenérgicos alfa/fisiologia , Sistema Nervoso Simpático/efeitos dos fármacos , Ducto Deferente/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Clonidina/farmacologia , Cobaias , Técnicas In Vitro , Masculino , Neurotransmissores/metabolismo , Sistema Nervoso Simpático/fisiologia , Tiramina/farmacologia , Ducto Deferente/inervação , Ioimbina/farmacologiaRESUMO
1. The effects of prostaglandin E1 (PGE1) and E2 (PGE2) on postjunctional electrical activity in the guinea-pig vas deferens evoked by sympathetic nerve stimulation were investigated using both intracellular and focal extracellular recording techniques in vitro. 2. Bath application of PGE1 (1-100 nM) or PGE2 (0.1-100 nM) concentration-dependently inhibited the amplitudes of all excitatory junction potentials (e.j.ps) evoked during short trains of stimuli (10 stimuli at 1 Hz). Increasing the duration of nerve stimulation (100 stimuli at 1 Hz) did not overcome this inhibitory effect. At these concentrations PGE1 and PGE2 were without any apparent inhibitory effect on the amplitudes of spontaneous e.j.ps. 3. Local application of PGE1 (10-100 nM) or PGE2 (10-30 nM) markedly reduced the frequency of occurrence of excitatory junction currents (e.j.cs) evoked by trains of 20-100 stimuli at 1 to 4 Hz without changing the amplitudes of spontaneous e.j.cs or the configuration of the nerve terminal impulse. 4. In the presence of PGE1 or PGE2, raising the frequency of stimulation (from 1 to 4 Hz), increased the likelihood of e.j.c. occurrence. 5. The postjunctional electrical activity recorded in the guinea-pig vas deferens is believed to be due to ATP released from the sympathetic nerve endings. Thus the present study demonstrates that both PGE1 and PGE2 powerfully inhibit quantal ATP release in the guinea-pig vas deferens.
Assuntos
Alprostadil/farmacologia , Dinoprostona/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Ducto Deferente/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Cobaias , Técnicas In Vitro , Cinética , MasculinoRESUMO
1. The effects of Ca2+ concentration and Ca2+ channel blockers on noradrenaline (NA) and adenosine 5'-triphosphate (ATP) release from postganglionic sympathetic nerves have been investigated in rat tail arteries in vitro. Intracellularly recorded excitatory junction potentials (e.j.ps) were used as a measure of ATP release and continuous amperometry was used to measure NA release. 2. Varying the extracellular Ca2+ concentration similarly affected the amplitudes of e.j.ps and NA-induced oxidation currents evoked by trains of ten stimuli at 1 Hz. 3. The N-type Ca2+ blocker, omega-conotoxin GVIA (omega-CTX GVIA, 0.1 microM) reduced the amplitudes of both e.j.ps (evoked by trains of ten stimuli at 1 Hz) and NA-induced oxidation currents (evoked by trains of ten stimuli at 1 Hz and 50 stimuli at 10 Hz) by about 90%. 4. The omega-CTX GVIA resistant e.j.ps and NA-induced oxidation currents evoked by trains of 50 stimuli at 10 Hz were abolished by the non-selective Ca2+ channel blocker, Cd2+ (0.1 mM), and were reduced by omega-conotoxin MVIIC (0.5 microM) and omega-agatoxin IVA (40 nM). 5. Nifedipine (10 microm) had no inhibitory effect on omega-CTX GVIA resistant e.j.ps and NA-induced oxidation currents. 6. Thus both varying Ca2+ concentration and applying Ca2+ channel blockers results in similar effects on NA and ATP release from postganglionic sympathetic nerves. These findings are consistent with the hypothesis that NA and ATP are co-released together from the sympathetic nerve terminals.
Assuntos
Trifosfato de Adenosina/metabolismo , Artérias/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Cálcio/farmacologia , Norepinefrina/metabolismo , Transmissão Sináptica/efeitos dos fármacos , ômega-Conotoxinas , Animais , Artérias/inervação , Artérias/metabolismo , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Eletrofisiologia , Potenciais Evocados/efeitos dos fármacos , Feminino , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Nifedipino/farmacologia , Peptídeos/farmacologia , Ratos , Ratos Wistar , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/metabolismo , Cauda , ômega-Conotoxina GVIARESUMO
1. Intracellularly recorded excitatory junction potentials (ej.ps) were used to study the effects of adenosine receptor antagonists on neurotransmitter release from postganglionic sympathetic nerve terminals in the guinea-pig vas deferens in vitro. 2. The A1 adenosine receptor antagonists, 8-phenyltheophylline (10 microM) and 8-cyclopentyl-1,3-dipropylxanthine (0.1 microM), increased the amplitude of e.j.ps evoked during trains of 20 stimuli at 1 Hz in the presence, but not in the absence, of the alpha2-adrenoceptor antagonist, yohimbine (1 microM) or the non-selective alpha-adrenoceptor antagonist, phentolamine (1 microM). 3. Adenosine (100 microM) reduced the amplitude of e.j.ps, both in the presence and in the absence of phentolamine (1 microM). This inhibitory effect of adenosine is most likely caused by a reduction in transmitter release as there was no detectable change in spontaneous ej.p. amplitudes. 4. In the presence of phentolamine, application of the adenosine uptake inhibitor, S-(p-nitrobenzyl)-6-thioinosine (0.1 microM), had no effect on ej.p. amplitudes. 5. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (100 microM), significantly increased the amplitudes of all e.j.ps evoked during trains of 20 stimuli at 1 Hz, both in the presence and in the absence of phentolamine (1 microM). 6. These results suggest that endogenous adenosine modulates neurotransmitter release by an action at prejunctional A1 adenosine receptors only when alpha2-adrenoceptors are blocked.
Assuntos
Trifosfato de Adenosina/metabolismo , Antagonistas de Receptores Purinérgicos P1 , Transmissão Sináptica/fisiologia , Ducto Deferente/efeitos dos fármacos , 1-Metil-3-Isobutilxantina/farmacologia , Adenosina/farmacologia , Adenosina/fisiologia , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Cobaias , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Junção Neuroefetora/efeitos dos fármacos , Junção Neuroefetora/fisiologia , Fentolamina/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Receptores Purinérgicos P1/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Teofilina/análogos & derivados , Teofilina/farmacologia , Tioinosina/análogos & derivados , Tioinosina/farmacologia , Ducto Deferente/inervação , Ducto Deferente/fisiologia , Xantinas/farmacologia , Ioimbina/farmacologiaRESUMO
Staphylococcus aureus 502A was grown in the presence of one-third of the minimal inhibitory concentration of clindamycin. Phagocytosis of the antibiotic-treated bacteria by human polymorphonuclear leukocytes was significantly enhanced, compared with that of the untreated control (P less than 0.001). Study of opsonization kinetics by a chemiluminescence assay demonstrated that clindamycin-treated staphylococci were opsonized more rapidly than control bacteria and that the serum concentration required for sufficient opsonization was lower. Complement was consumed much faster, and the opsonic fragment C3b was fixed more rapidly to the bacterial surface when the staphylococci were preincubated with clindamycin. Electron micrographs showed an alteration of the staphylococcal cell wall after clindamycin treatment.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Venenos de Moluscos/farmacologia , Terminações Nervosas/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Sistema Nervoso Simpático/efeitos dos fármacos , Animais , Cobaias , Técnicas In Vitro , Masculino , Músculo Liso/efeitos dos fármacos , Músculo Liso/inervação , Músculo Liso/metabolismo , Neurotransmissores/metabolismo , Sistema Nervoso Simpático/citologia , Ducto Deferente/inervação , Ducto Deferente/metabolismo , ômega-Conotoxina GVIARESUMO
1. The effects of prejunctional beta-adrenoceptor activation on electrically evoked noradrenaline (NA) and adenosine 5'-triphosphate (ATP) were studied by use of continuous amperometry and conventional intracellular recording techniques. Excitatory junction potentials (e.j.ps) were used as a measure of ATP release, and NA-induced slow depolarizations and oxidation currents as measures of NA release, from postganglionic sympathetic nerves innervating the rat tail artery in vitro. 2. Isoprenaline (0.1 microM) increased the amplitude of e.j.ps, slow depolarizations and oxidation currents evoked by short trains of stimuli at 1 to 4 Hz. The facilitatory effect of isoprenaline on e.j.ps and oxidation currents was most pronounced on responses evoked by the first stimulus in a train. 3. Isoprenaline (0.1 microM) did not detectably alter the amplitude-frequency distribution of spontaneous e.j.ps. 4. The facilitatory effect of isoprenaline on e.j.ps, slow depolarizations and oxidation currents was abolished by the beta-adrenoceptor antagonist, propranolol (0.1 microM). Propranolol alone had no effect on e.j.ps, slow depolarizations or oxidation currents. 5. Thus, activation of prejunctional beta-adrenoceptors increases the release of both NA and ATP from postganglionic sympathetic nerves. The findings are consistent with the hypothesis that NA and ATP are released from the same population of nerve terminals and presumably from the same vesicles.
Assuntos
Trifosfato de Adenosina/metabolismo , Artérias/inervação , Norepinefrina/metabolismo , Receptores Adrenérgicos beta/fisiologia , Sistema Nervoso Simpático/metabolismo , Cauda/irrigação sanguínea , Agonistas Adrenérgicos beta/farmacologia , Animais , Artérias/fisiologia , Eletroquímica , Isoproterenol/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Propranolol/farmacologia , Ratos , Sistema Nervoso Simpático/fisiologia , Simpatomiméticos/farmacologiaRESUMO
1. Intracellular recording was used to investigate the electrophysiological effects of activating peptidergic primary afferent axons with capsaicin in the smooth muscle of rat mesenteric arteries in vitro. In addition, continuous amperometry was used to monitor the effects of capsaicin on noradrenaline release from the sympathetic nerves. 2. Capsaicin (1 microm) produced a hyperpolarization (-11+/-2 mV) and a reduction in the time constant of decay of excitatory junction potentials (e.j.p.'s) evoked by electrical stimulation of the perivascular sympathetic nerves. These effects of capsaicin were mimicked by calcitonin gene-related peptide (CGRP; 1 and 10 nm) but not by substance P (50 nm), which produced a small hyperpolarization (maximum -3+/-1 mV) but did not change excitatory junction potential (e.j.p.) time course. 3. The hyperpolarization produced by capsaicin and CGRP was blocked by glibenclamide (10 microm) but was not changed by the CGRP antagonist, CGRP8-37 (0.5 microm). Mechanical denudation of the endothelium also did not reduce the effect of capsaicin on membrane potential. 4. Capsaicin (1 microm) increased the amplitude of e.j.p.'s. This effect was not mimicked by CGRP or substance P nor blocked by glibenclamide or CGRP8-37. 5. All effects of capsaicin desensitized. 6. Capsaicin (1 microm) had no effect on noradrenaline-induced oxidation currents evoked by electrical stimulation, indicating that noradrenaline release was unchanged. 7. These results suggest that CGRP released from primary afferent axons hyperpolarizes vascular smooth muscle by activating glibenclamide-sensitive K+ channels. The findings also indicate that an unknown factor released by the primary afferent axons increases e.j.p. amplitude.
Assuntos
Capsaicina/farmacologia , Artérias Mesentéricas/efeitos dos fármacos , Neurônios Aferentes/efeitos dos fármacos , Animais , Apamina/farmacologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Estimulação Elétrica , Eletrofisiologia , Endotélio Vascular/fisiologia , Feminino , Glibureto/farmacologia , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Artérias Mesentéricas/inervação , Artérias Mesentéricas/fisiologia , Neurônios Aferentes/metabolismo , Neurônios Aferentes/fisiologia , Norepinefrina/farmacologia , Fragmentos de Peptídeos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos Wistar , Substância P/farmacologia , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologiaRESUMO
1. The effects of continuous but asynchronous nerve activity induced by ciguatoxin (CTX-1) on the membrane potential and contraction of smooth muscle cells have been investigated in rat proximal tail arteries isolated in vitro. These effects have been compared with those produced by the continuous application of phenylephrine (PE). 2. CTX-1 (0.4 nM) and PE (10 microM) produced a maintained depolarization of the arterial smooth muscle that was almost completely blocked by alpha-adrenoceptor blockade. In both cases, the depolarization was more sensitive to the selective alpha-adrenoceptor antagonist, idazoxan (0.1 microM), than to the selective alpha 1-adrenoceptor antagonist, prazosin (0.01 microM). 3. In contrast, the maintained contraction of the tail artery induced by CTX-1 (0.2 nM) and PE (2 and 10 microM) was more sensitive to prazosin (0.01) microM, than to idazoxan (0.01 microM). In combination, these antagonists almost completely inhibited contraction to both agents. 4. Application of the calcium channel antagonist, nifedipine (1 microM), had no effect on the depolarization induced by either CTX-1 or PE but maximally reduced the force of the maintained contraction to both agents by about 50%. 5. We conclude that the constriction of the tail artery induced by CTX-1, which mimics the natural discharge of postganglionic perivascular axons, is due almost entirely to alpha-adrenoceptor activation. The results indicate that neuronally released noradrenaline activates more than one alpha-adrenoceptor subtype. The depolarization is dependent primarily on alpha 2-adrenoceptor activation whereas the contraction is dependent primarily on alpha 1-adrenoceptor activation. The links between alpha-adrenoceptor activation and the voltage-dependent and voltage-independent mechanisms that deliver Ca2+ to the contractile apparatus appear to be complex.
Assuntos
Artérias/fisiologia , Receptores Adrenérgicos alfa/fisiologia , Cauda/irrigação sanguínea , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Artérias/efeitos dos fármacos , Artérias/inervação , Bloqueadores dos Canais de Cálcio/farmacologia , Ciguatoxinas/farmacologia , Feminino , Idazoxano/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Nifedipino/farmacologia , Fenilefrina/farmacologia , Prazosina/farmacologia , Ratos , Receptores Adrenérgicos alfa/efeitos dos fármacosRESUMO
1. The effects of ciguatoxin-1 (CTX-1) on the membrane potential of smooth muscle cells have been examined in rat proximal tail arteries isolated in vitro. 2. CTX-1 (> or = 10 pM) increased the frequency of spontaneous excitatory junction potentials (s.e.j.ps). At 100-400 pM, there was also a marked and maintained depolarization (19.7 +/- 1.4 mV, n = 14, at 400 pM). 3. In 20-400 pM CTX-1, perivascular stimuli evoked excitatory junction potentials (e.j.ps) which were prolonged in time course relative to control. 4. Although threshold and latency of the e.j.p. were not affected by CTX-1 (< or = 400 pM), propagated impulses were blocked at > or = 100 pM. 5. The spontaneous activity and the depolarization produced by CTX-1 were reduced in the presence of Ca2+ (0.1 mM)/Mg2+ (25 mM), omega-conotoxin (0.1 microM) or Cd2+ (50-100 microM). 6. All effects of CTX-1 were abolished by tetrodotoxin (0.3 microM). 7. Raised Ca2+ (6 mM) reduced the depolarization and spontaneous activity produced by CTX-1. 8. In 400 pM CTX-1, the membrane repolarized (17 +/- 3.2 mV, n = 4) following the addition of phentolamine (1 microM). S.e.j.ps and e.j.ps were selectively abolished by suramin (1 mM), and the membrane repolarized by 1.3 +/- 1.6 mV (n = 4). 9. We conclude that CTX-1 releases noradrenaline and ATP by initiating asynchronous discharge of postganglionic perivascular axons. In 100-400 pM CTX-1, the smooth muscle was depolarized to levels resembling those recorded in this artery during ongoing vasoconstrictor discharge in vivo.
Assuntos
Artérias/fisiologia , Ciguatoxinas/farmacologia , Músculo Liso Vascular/fisiologia , Junção Neuroefetora/fisiologia , Fármacos Neuromusculares Despolarizantes/farmacologia , Animais , Artérias/efeitos dos fármacos , Artérias/inervação , Axônios/fisiologia , Cálcio/fisiologia , Estimulação Elétrica , Eletrofisiologia , Feminino , Técnicas In Vitro , Magnésio/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/inervação , Junção Neuroefetora/efeitos dos fármacos , Ratos , Receptores Adrenérgicos alfa/efeitos dos fármacos , Receptores Adrenérgicos alfa/fisiologia , Fluxo Sanguíneo Regional/efeitos dos fármacos , Suramina/farmacologia , Cauda/irrigação sanguínea , Tetrodotoxina/farmacologiaRESUMO
1. Characteristic features of noradrenaline (NA) and adenosine 5'-triphosphate (ATP) release from postganglionic sympathetic nerves in rat small mesenteric arteries in vitro have been investigated on an impulse-by-impulse basis. NA release was measured using continuous amperometry and ATP release was monitored by intracellular recording of excitatory junction potentials (e.j.ps). 2. Electrical stimuli evoked transient increases in oxidation current. During trains of ten stimuli at 0.5 - 4 Hz there was a depression in the amplitude of oxidation currents evoked following the first stimulus in the train. 3. The neuronal NA uptake inhibitor, desmethylimipramine (1 microM), increased the amplitude of the summed oxidation current evoked by ten stimuli at 1 Hz and slowed the decay of oxidation currents evoked by trains of ten stimuli at 1 and 10 Hz. 4. The alpha2-adrenoceptor antagonist, idazoxan (1 microM), increased the amplitudes of the oxidation currents evoked during trains of ten stimuli at 0.5 - 10 Hz but had no effect on the oxidation currents evoked by the first stimulus in the train. 5. Idazoxan (1 microM) increased the amplitude of all e.j.ps evoked during trains of stimuli at 0.5 and 1 Hz. In addition, the facilitatory effect of idazoxan on e.j.ps was significantly greater than that on oxidation currents. 6. The findings indicate that NA release from sympathetic nerves supplying small mesenteric arteries is regulated by activation of presynaptic alpha2-adrenoceptors and that clearance of released NA in this tissue depends, in part, upon neuronal uptake. The different effects of idazoxan on the oxidation currents and e.j.ps may indicate that the release of NA and ATP is differentially modulated.
Assuntos
Artérias Mesentéricas/inervação , Norepinefrina/metabolismo , Sistema Nervoso Simpático/metabolismo , Trifosfato de Adenosina/metabolismo , Inibidores da Captação Adrenérgica/farmacologia , Antagonistas de Receptores Adrenérgicos alfa 2 , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Cádmio/farmacologia , Corticosterona/farmacologia , Desipramina/farmacologia , Condutividade Elétrica , Estimulação Elétrica , Eletrofisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Idazoxano/farmacologia , Técnicas In Vitro , Cinética , Potenciais da Membrana/efeitos dos fármacos , Artérias Mesentéricas/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores Adrenérgicos alfa 2/metabolismo , Sistema Nervoso Simpático/efeitos dos fármacos , Tetrodotoxina/farmacologiaRESUMO
1. This study investigated the effects of BIIE0246, a novel neuropeptide Y (NPY) Y2 receptor antagonist, on the inhibition of cholinergic neuroeffector transmission in rat heart and guinea-pig trachea and purinergic neuroeffector transmission in guinea-pig vas deferens produced by the NPY Y2 receptor agonist, N-acetyl [Leu28,31] NPY 24-36. 2. In pentobarbitone anaesthetized rats, supramaximal stimulation every 30 s, of the vagus nerve innervating the heart, increased pulse interval by approximately 100 ms. This response was attenuated by intravenous administration of N-acetyl [Leu28,31] NPY 24-36 (10 nmol x kg(-1)). 3. Transmural stimulation of segments of guinea-pig trachea at 1 min intervals with 5 s trains of stimuli at 0.5, 5, 10, 20 and 40 Hz evoked contractions which were reduced in force by N-acetyl [Leu28,31] NPY 24-36 (2 microM). 4. In guinea-pig vasa deferentia, the amplitude of excitatory junction potentials evoked by trains of 20 stimuli at 1 Hz was reduced in the presence of N-acetyl [Leu28,31] NPY 24-36 (1 microM). 5. In all preparations BIIE0246 attenuated the inhibitory effect of N-acetyl [Leu28,31] NPY 24-36 but had no effect when applied alone. 6. The findings support the view that the nerve terminals of postganglionic parasympathetic and sympathetic neurones possess neuropeptide Y Y2 receptors which, when activated, reduce neurotransmitter release.
Assuntos
Arginina/análogos & derivados , Arginina/farmacologia , Benzazepinas/farmacologia , Junção Neuroefetora/efeitos dos fármacos , Receptores de Neuropeptídeo Y/antagonistas & inibidores , Acetilcolina/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Feminino , Cobaias , Frequência Cardíaca/efeitos dos fármacos , Técnicas In Vitro , Masculino , Junção Neuroefetora/fisiologia , Purinas/metabolismo , Ratos , Ratos Wistar , Traqueia/efeitos dos fármacos , Traqueia/fisiologia , Ducto Deferente/efeitos dos fármacosRESUMO
Continuous amperometry was used to monitor noradrenaline (NA) release from sympathetic nerves supplying rat mesenteric arteries in vitro. During electrical stimulation the amplitude of oxidation currents evoked by successive stimuli varied over a small range, with occasional events of larger amplitude. In the absence of stimulation, spontaneous oxidation currents (s.o.cs) were recorded. The frequency of s.o.cs was increased by alpha-latrotoxin (1 nM). This agent also increased the frequency of spontaneous excitatory junction potentials (s.e.j.ps), which monitor the packeted release of adenosine 5' triphosphate (ATP). The frequency of s.o.cs recorded 20-25 min after applying alpha-latrotoxin was about four times the control value, but that of s.e.j.ps was about 30 times the control value. The findings suggest that continuous amperometry can detect the spontaneous packeted release of NA, probably from large dense-cored vesicles. In contrast, s.e.j.ps monitor spontaneous release of neurotransmitter (ATP) from a different store, most likely the small dense-cored vesicles.
Assuntos
Artérias Mesentéricas/fisiologia , Terminações Nervosas/metabolismo , Norepinefrina/metabolismo , Sistema Nervoso Simpático/metabolismo , Animais , Estimulação Elétrica , Eletroquímica , Eletrofisiologia , Potenciais Evocados/efeitos dos fármacos , Feminino , Técnicas In Vitro , Artérias Mesentéricas/inervação , Artérias Mesentéricas/metabolismo , Músculo Liso Vascular/inervação , Músculo Liso Vascular/fisiologia , Oxirredução , Ratos , Ratos Wistar , Venenos de Aranha/farmacologia , Fatores de TempoRESUMO
An established fish cell line, epithelioma papillosum cyprini (EPC), originating from the carp (Cyprinus carpio) was found to be susceptible to the penaeid shrimp virus, infectious hypodermal and hematopoietic necrosis virus (IHHNV). This economically important pathogen of penaeid shrimp raised in mariculture has been previously limited to growth only in young post-larval shrimps. This is, to our knowledge, the first reported successful growth of the penaeid virus in a fish cell line; it promises to provide a sensitive and convenient cell culture system for the primary isolation and detection of IHHNV which will aid immensely the production of IHHNV-free penaeid shrimp stocks for mariculture. Of added significance is the replication of a crustacean virus in a fish cell line which has never before been reported. The use of this sensitive cell line will also greatly facilitate the characterization of the IHHN virus and other crustacean viruses.
Assuntos
Decápodes/microbiologia , Cultura de Vírus , Vírus/crescimento & desenvolvimento , Animais , Bromodesoxiuridina/farmacologia , Carpas , Linhagem Celular , Peixes , Replicação Viral/efeitos dos fármacos , Vírus/ultraestruturaRESUMO
A protocol was developed for the isolation and purification of the infectious hypodermal and hematopoietic necrosis virus (IHHNV) of penaeid shrimps. Cesium chloride-banded virus obtained from three sources of infected shrimps was found to have a buoyant density of 1.33 g/cm3. Also, electron microscopical studies employing negative stain revealed isometric particles having a size of 19 +/- 1 nm. Colorimetric analysis of its nucleic acid type indicated that these particles contain only RNA.
Assuntos
Decápodes/microbiologia , Vírus/isolamento & purificação , Animais , Centrifugação com Gradiente de Concentração , Microscopia Eletrônica , RNA Viral/análise , Vírus/ultraestruturaRESUMO
A streptavidin-biotin-enhanced nitrocellulose enzyme immunoassay was developed for the detection of the rhabdovirus of penaeid shrimps (RPS) in the tissues of infected animals. Initial tests indicate that the assay was capable of detecting as few as ten plaque-forming units of virus.
Assuntos
Decápodes/microbiologia , Técnicas Imunoenzimáticas , Rhabdoviridae/isolamento & purificação , Animais , Proteínas de Bactérias , Biotina , Colódio , Estudos de Avaliação como Assunto , Estreptavidina , Ensaio de Placa Viral/métodos , Virologia/métodosRESUMO
A new virus was isolated from infectious hypodermal and hematopoietic necrosis virus (IHHNV)-infected penaeid shrimps. The virus was isolated from two species of penaeid shrimps obtained from three different sources employing a previously developed cell-culture assay. Electron-microscopical studies of both purified virus and infected cells showed bullet-shaped particles identifying it as a rhabdovirus.
Assuntos
Decápodes/microbiologia , Rhabdoviridae/isolamento & purificação , Animais , Rhabdoviridae/ultraestrutura , Células Tumorais CultivadasRESUMO
A 50% tissue culture infectious dose assay (TCID50) using primary culture of shrimp lymphoid organ (Oka) cells was developed for the quantitative titration of yellow-head baculovirus (YBV), a newly isolated virus of penaeid shrimp. The assay protocol includes the use of Primaria-grade 96-well tissue culture plates to grow the primary lymphoid organ cells of penaeid shrimp. A 15% gill suspension from YBV-infected shrimp was determined to have an infectious virus titer of 5 x 10(5.75) TCID50/ml. This report represents the first convenient assay protocol using cell culture derived from penaeid shrimp to titer a shrimp virus.