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1.
Anal Chem ; 91(18): 12014-12020, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31453687

RESUMO

Serotonin (5-HT) is a key signaling molecule within the mucosal epithelium of the intestinal wall and has been shown to be an important modulator of motility. At present, no single approach has been established for simultaneous dual measurement of 5-HT overflow and circular muscle contraction. We developed a 3D-printed carbon black/polylactic acid (PLA) electrochemical sensor, which had a geometry suitable for ex vivo measurement in the anorectum. The device was characterized for sensitivity and stability for 5-HT measurements as well as suitability for accurate tracking of anorectal contractions. The 3D-printed electrochemical sensor had a linear range in physiological concentrations of 5-HT (1-10 µM) present within the intestinal tract and a limit of detection of 540 nM. The sensor was stable for 5-HT measurement following ex vivo tissue measurements. There was a signficant correlation in the amplitude and duration of individual contractions when comparing the measurements using an isometric force transducer and 3D-printed electrochemical sensor. Finally, in the presence of 1 µM fluoxetine, the sensor was able to monitor a reduction in contractility as well as an increase in 5-HT overflow as predicted. Overall, the 3D-printed sensor has the ability to conduct dual simultaneous measurements of 5-HT overflow and contractility. This single device will have significant potential for clinical measurements of anorectum function and signaling that can direct therapeutic management of patients with bowel disorders.


Assuntos
Técnicas Eletroquímicas , Músculo Liso/química , Impressão Tridimensional , Serotonina/análise , Animais , Cobaias , Masculino , Contração Muscular
2.
Neurogastroenterol Motil ; 30(10): e13426, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30062757

RESUMO

BACKGROUND: Increasing age is associated with an increase in the incidence of chronic constipation and fecal impaction. The contribution of the natural aging process to these conditions is not fully understood. This study examined the effects of increasing age on the function of the murine anorectum. METHODS: The effects of increasing age on cholinergic, nitrergic, and purinergic signaling pathways in the murine anorectum were examined using classical organ bath assays to examine tissue function and electrochemical sensing to determine age-related changes in nitric oxide and acetylcholine release. KEY RESULTS: Nitrergic relaxation increased between 3 and 6 months, peaked at 12 months and declined in the 18 and 24 months groups. These changes were in part explained by an age-related decrease in nitric oxide (NO) release. Cholinergic signaling was maintained with age by an increase in acetylcholine (ACh) release and a compensatory decrease in cholinesterase activity. Age-related changes in purinergic relaxation were qualitatively similar to nitrergic relaxation although the relaxations were much smaller. Increasing age did not alter the response of the anorectum smooth muscle to exogenously applied ACh, ATP, sodium nitroprusside or KCl. Similarly, there was no change in basal tension developed by the anorectum. CONCLUSIONS AND INFERENCES: The decrease in nitrergic signaling with increasing age may contribute to the age-related fecal impaction and constipation previously described in this model by partially obstructing defecation.


Assuntos
Envelhecimento/metabolismo , Canal Anal/metabolismo , Músculo Liso/metabolismo , Reto/metabolismo , Transdução de Sinais/fisiologia , Acetilcolina/análise , Acetilcolina/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Motores/metabolismo , Óxido Nítrico/análise , Óxido Nítrico/metabolismo
3.
Rev Sci Instrum ; 87(11): 11D421, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27910608

RESUMO

Our team has developed an experimental platform to evaluate the x-ray-generated stress and impulse in materials. Experimental activities include x-ray source development, design of the sample mounting hardware and sensors interfaced to the National Ignition Facility's diagnostics insertion system, and system integration into the facility. This paper focuses on the X-ray Transport and Radiation Response Assessment (XTRRA) test cassettes built for these experiments. The test cassette is designed to position six samples at three predetermined distances from the source, each known to within ±1% accuracy. Built-in calorimeters give in situ measurements of the x-ray environment along the sample lines of sight. The measured accuracy of sample responses as well as planned modifications to the XTRRA cassette is discussed.

4.
Mech Ageing Dev ; 160: 28-31, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27751771

RESUMO

Reactive oxygen and nitrogen species (ROS/RNS) have been widely implicated in the ageing process and various approaches exist for monitoring these species in biological tissues. These approaches at present are limited to monitoring either a single pro-oxidant species or total pro-oxidant levels and therefore provide limited insight into the range of pro-oxidant species and their relative proportions in the ageing process. We have utilised a sensor that allows us to simultaneously monitor hydrogen peroxide, peroxynitrite, nitric oxide and nitrite. Using CNS homogenates from the pond snail, Lymnaea, we were able to show that levels of these ROS/RNS increased between young and old CNS homogenates and were different in various aged CNS regions.


Assuntos
Envelhecimento/metabolismo , Sistema Nervoso Central/metabolismo , Técnicas Eletroquímicas/métodos , Lymnaea/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais
5.
Neurogastroenterol Motil ; 25(7): e469-77, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23634828

RESUMO

BACKGROUND: The innervation of the mouse internal anal sphincter (IAS) has been little studied, and how it changes during aging has not previously been investigated. The aim of this study was therefore to characterize the distribution and density of subtypes of nerve fibers in the IAS and underlying mucosa in 3-, 12- to 13-, 18- and 24- to 25-month-old male C57BL/6 mice. METHODS: Nerve fibers were immunolabeled with antibodies against protein gene product 9.5 (PGP9.5), neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP), and calretinin (CR). Immunoreactivity in nerve fibers in the circular muscle and mucosa was quantified using Image J software. KEY RESULTS: In young adult (3 month) mice, nNOS-immunoreactive (IR) nerve fibers were densely distributed in the circular muscle, but relatively few in the mucosa; VIP-IR nerve fibers were abundant in the circular muscle and common in the mucosa; SP-IR nerve fibers were common in circular muscle and mucosa; CGRP- and CR-IR nerve fibers were dense in mucosa and sparse in circular muscle. The density of PGP9.5 immunoreactivity (IRY) was not significantly reduced with age, but a significant reduction in nNOS-IRY and SP-IRY with age was found in the IAS circular muscle. Neuronal nitric oxide synthase-, VIP-, and SP-IRY in the anal mucosa were significantly reduced with age. CGRP-IRY in both circular muscle and mucosa was increased in 18-month-old animals. CONCLUSIONS & INFERENCES: The density of immunoreactivity of markers for some types of IAS nerve fibers decreases during aging, which may contribute to age-related ano-rectal dysfunction.


Assuntos
Envelhecimento/fisiologia , Canal Anal/inervação , Fibras Nervosas/metabolismo , Animais , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Nervosas/química
6.
Rev Sci Instrum ; 84(4): 043506, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23635195

RESUMO

The neutron spectrum produced by deuterium-tritium (DT) inertial confinement fusion implosions contains a wealth of information about implosion performance including the DT yield, ion-temperature, and areal-density. The Magnetic Recoil Spectrometer (MRS) has been used at both the OMEGA laser facility and the National Ignition Facility (NIF) to measure the absolute neutron spectrum from 3 to 30 MeV at OMEGA and 3 to 36 MeV at the NIF. These measurements have been used to diagnose the performance of cryogenic target implosions to unprecedented accuracy. Interpretation of MRS data requires a detailed understanding of the MRS response and background. This paper describes ab initio characterization of the system involving Monte Carlo simulations of the MRS response in addition to the commission experiments for in situ calibration of the systems on OMEGA and the NIF.

7.
Neurogastroenterol Motil ; 25(7): e495-e505, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23517051

RESUMO

BACKGROUND Age-associated myenteric neuronal loss has been described in several species. In some studies,cholinergic neurons have been reported to be selectively vulnerable, whereas nitrergic neurons are spared. Aging of the mouse enteric nervous system(ENS) and the subtypes of mouse myenteric neurons that may be lost have been little studied. We therefore investigated changes in the numbers of total neurons and two neuronal subpopulations in the mouse distal colon during aging. METHODS Wholemount preparations from 3­4-, 12­13-, 18­19-, and 24­25-month-old C57BL/6 mice were double immunolabeled with HuC/D antibody to identify the total neuronal population and antisera to either calbindin or neuronal nitric oxide synthase (nNOS) to identify myenteric neuronal subpopulations. Samples were analyzed by confocal microscopy. New procedures were employed to ensure unbiased counting and to correct for changes in gut dimensions with age and stretch during sample preparation. The density of nerve fibers in the tertiary plexus was also studied. KEY RESULTS No significant change in numbers of total neurons or of either subpopulation with age was measured, but because of gut growth, the density of myenteric neurons decreased between 3­4 and 12­13 months. The density of nNOS-immunoreactive nerve fibers in the tertiary plexus increased significantly with age, up to 18­19 months. Numerous swollen processes of CB and nNOS-immunoreactive neurons were observed in 18­19- and 24­25-month-old animals. Conclusions &Inferences These results indicate that aging does not result in a loss of myenteric neurons in mouse distal colon at the ages studied, although neurodegenerative changes, which may impact on neuronal function, do occur.


Assuntos
Colo/inervação , Plexo Mientérico/citologia , Envelhecimento , Animais , Contagem de Células , Colo/citologia , Colo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plexo Mientérico/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo
8.
Rev Sci Instrum ; 81(10): 10D319, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21033845

RESUMO

The first 3 of 18 neutron time-of-flight (nTOF) channels have been installed at the National Ignition Facility (NIF). The role of these detectors includes yield, temperature, and bang time measurements. This article focuses on nTOF data analysis and quality of results obtained for the first set of experiments to use all 192 NIF beams. Targets produced up to 2×10(10) 2.45 MeV neutrons for initial testing of the nTOF detectors. Differences in neutron scattering at the OMEGA laser facility where the detectors were calibrated and at NIF result in different response functions at the two facilities. Monte Carlo modeling shows this difference. The nTOF performance on these early experiments indicates that the nTOF system with its full complement of detectors should perform well in future measurements of yield, temperature, and bang time.

9.
Rev Sci Instrum ; 81(10): 10D325, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21033848

RESUMO

The National Ignition Facility (NIF) successfully completed its first inertial confinement fusion (ICF) campaign in 2009. A neutron time-of-flight (nTOF) system was part of the nuclear diagnostics used in this campaign. The nTOF technique has been used for decades on ICF facilities to infer the ion temperature of hot deuterium (D(2)) and deuterium-tritium (DT) plasmas based on the temporal Doppler broadening of the primary neutron peak. Once calibrated for absolute neutron sensitivity, the nTOF detectors can be used to measure the yield with high accuracy. The NIF nTOF system is designed to measure neutron yield and ion temperature over 11 orders of magnitude (from 10(8) to 10(19)), neutron bang time in DT implosions between 10(12) and 10(16), and to infer areal density for DT yields above 10(12). During the 2009 campaign, the three most sensitive neutron time-of-flight detectors were installed and used to measure the primary neutron yield and ion temperature from 25 high-convergence implosions using D(2) fuel. The OMEGA yield calibration of these detectors was successfully transferred to the NIF.

10.
Proc Inst Mech Eng H ; 223(7): 839-47, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19908423

RESUMO

This paper investigates a new metacarpophalangeal joint design aimed at treating patients with moderate to severe forms of arthritis affecting the index, long, ring, and little fingers. Current small joint arthroplasty designs, including those for the metacarpophalangeal joint of the hand, have had limited success owing to mechanical failures and can be divided into two main families: single-piece elastomer implants and surface articulating implants. The design proposed in this study involves combining the principles of a surface weight-bearing articulating implant with those of a spanning elastomeric implant. The design consists of metacarpal and proximal phalangeal articulating housings and a central flexible spanning elastomer rod that maintains the alignment of the metacarpal and proximal phalangeal components. A preliminary finite element analysis was used to investigate the stresses in the design, the wear of the articulating bearing surfaces for different material combinations, and the stresses between the central elastomer rod and the articulating housings. This preliminary analysis shows that the design should withstand the probable loading conditions experienced within the human body and that the wear rates of the articulating surfaces and the central elastomer are acceptable.


Assuntos
Desenho Assistido por Computador , Prótese Articular , Articulação Metacarpofalângica/fisiopatologia , Articulação Metacarpofalângica/cirurgia , Análise de Falha de Equipamento , Estudos de Viabilidade , Humanos , Desenho de Prótese
11.
BMC Genomics ; 10: 451, 2009 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-19775440

RESUMO

BACKGROUND: The freshwater snail Lymnaea stagnalis (L. stagnalis) has served as a successful model for studies in the field of Neuroscience. However, a serious drawback in the molecular analysis of the nervous system of L. stagnalis has been the lack of large-scale genomic or neuronal transcriptome information, thereby limiting the use of this unique model. RESULTS: In this study, we report 7,712 distinct EST sequences (median length: 847 nucleotides) of a normalized L. stagnalis central nervous system (CNS) cDNA library, resulting in the largest collection of L. stagnalis neuronal transcriptome data currently available. Approximately 42% of the cDNAs can be translated into more than 100 consecutive amino acids, indicating the high quality of the library. The annotated sequences contribute 12% of the predicted transcriptome size of 20,000. Surprisingly, approximately 37% of the L. stagnalis sequences only have a tBLASTx hit in the EST library of another snail species Aplysia californica (A. californica) even using a low stringency e-value cutoff at 0.01. Using the same cutoff, approximately 67% of the cDNAs have a BLAST hit in the NCBI non-redundant protein and nucleotide sequence databases (nr and nt), suggesting that one third of the sequences may be unique to L. stagnalis. Finally, using the same cutoff (0.01), more than half of the cDNA sequences (54%) do not have a hit in nematode, fruitfly or human genome data, suggesting that the L. stagnalis transcriptome is significantly different from these species as well. The cDNA sequences are enriched in the following gene ontology functional categories: protein binding, hydrolase, transferase, and catalytic enzymes. CONCLUSION: This study provides novel molecular insights into the transcriptome of an important molluscan model organism. Our findings will contribute to functional analyses in neurobiology, and comparative evolutionary biology. The L. stagnalis CNS EST database is available at http://www.Lymnaea.org/.


Assuntos
Sistema Nervoso Central/metabolismo , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Lymnaea/genética , Sequência de Aminoácidos , Animais , Aplysia/genética , Biomphalaria/genética , Mapeamento Cromossômico , Hibridização Genômica Comparativa , Biologia Computacional , Biblioteca Gênica , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência
12.
Ann Biomed Eng ; 37(11): 2266-87, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19657741

RESUMO

The mechanics of arteries result from the properties of the soft tissue constituents and the interaction of the wall layers, predominantly media and adventitia. This concept was adopted in this study for the design of a tissue regenerative vascular graft. To achieve the desired structural properties of the graft, most importantly a diametric compliance of 6%/100 mmHg, finite element methods and genetic algorithms were used in an integrated approach to identify the mechanical properties of an adventitial fabric layer that were required to optimally complement an intimal/medial polyurethane layer with interconnected porosity of three different size classes. The models predicted a compliance of 16.0, 19.2, and 31.5%/100 mmHg for the non-reinforced grafts and 5.3, 5.5, and 6.0%/100 mmHg for the fabric-reinforced grafts. The latter, featuring fabrics manufactured according to the required non-linear mechanical characteristics numerically predicted, exhibited an in vitro compliance of 2.1 +/- 0.8, 3.0 +/- 2.4, and 4.0 +/- 0.7% /100 mmHg. The combination of finite element methods and genetic algorithms was shown to be able to successfully optimize the mechanical design of the composite graft. The method offers potential for the application to alternative concepts of modular vascular grafts and the incorporation of tissue ingrowth and biodegradation.


Assuntos
Artérias/fisiologia , Artérias/transplante , Bioprótese , Prótese Vascular , Modelos Cardiovasculares , Têxteis , Transplantes , Algoritmos , Simulação por Computador , Desenho Assistido por Computador , Análise de Elementos Finitos , Modelos Genéticos , Desenho de Prótese/métodos
13.
Neurobiol Aging ; 27(12): 1892-901, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16289475

RESUMO

This study examined whether electrophysiological changes in the endogenous properties and connectivity of the modulatory serotonergic cerebral giant cells (CGCs) contributed to the age-related changes in feeding behavior of the pond snail, Lymnaea. With increasing age there was a decrease in spontaneous CGC firing rates and decreased excitability of the CGCs to both chemosensory stimulation (0.05M sucrose applied to the lips) and direct intracellular current injection. These changes could be accounted for by a decrease in the input resistance of the neuron and an increase in the amplitude and the duration of the after-hyperpolarization. Decreases were also seen in the % of CGC pairs that were electrically coupled causing asynchronous firing. Together these changes would tend to reduce the ability of the CGCs to gate and control the frequency of the feeding behavior. Part of the ability of the CGCs to gate and frequency control the feeding network is to provide a background level of excitation to the feeding motor neurons. Recordings from B1 and B4 motor neurons showed an age-related hyperpolarization of the resting membrane potential consistent with a deficit in CGC function. Increases were seen in the strength of the evoked CGC-->B1 connection, however, this increase failed to compensate for the deficits in CGC excitability. In summary, age-related changes in the properties of the CGCs were consistent with them contributing to the age-related changes in feeding behavior seen in Lymnaea.


Assuntos
Envelhecimento/fisiologia , Encéfalo/fisiologia , Comportamento Alimentar/fisiologia , Lymnaea/fisiologia , Neurônios Motores/fisiologia , Animais , Técnicas In Vitro
14.
Neurobiol Aging ; 27(12): 1880-91, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16280186

RESUMO

This study used behavioral and electrophysiological techniques to examine age-related changes in the feeding behavior and chemosensory processing in the pond snail, Lymnaea stagnalis. Increasing age was associated with a 50% decrease in long-term food consumption. Analysis of short-term sucrose-evoked feeding bouts showed an age-related increase in the number of animals that failed to respond to the stimulus. Of the animals that did respond increasing age was associated with a decrease in the number of sucrose-evoked bites and a increase in the duration of the swallow phase. These changes were observed with both 0.01 and 0.05M sucrose stimuli but were not seen when 0.1M sucrose was used as the stimulus. Electrophysiological analysis of the chemosensory pathway in semi-intact lip-CNS preparations failed to demonstrate a significant change in the neuronal information entering the cerebral ganglia from the lips via the median lip nerve, but did demonstrate an age-related deficit in the neuronal output from the cerebral ganglia. This deficit was also dependent on the sucrose concentration and mirrored the concentration-dependent changes in feeding behavior. In summary, aging appeared to affect central but not peripheral processing of chemosensory information and suggests that this deficit contributes to the age-related changes in feeding behavior.


Assuntos
Envelhecimento/fisiologia , Células Quimiorreceptoras/fisiologia , Comportamento Alimentar/fisiologia , Lymnaea/fisiologia , Processamento de Proteína Pós-Traducional , Animais , Células Quimiorreceptoras/metabolismo , Ingestão de Alimentos/fisiologia , Rede Nervosa/fisiologia
15.
Biogerontology ; 2(2): 85-97, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11708380

RESUMO

Ageing can have profound effects on the post-mitotic organ of behaviour, the brain. As yet the precise causes of these deleterious effects are unknown. However, clear insights into the putative mechanisms and consequences of ageing in the CNS have been achieved through the use of invertebrate models. It is now clear that ageing alters the endogenous properties of neurones, their morphology, the efficacy of the connections that the neurones make with their targets and may even lead to neurone loss. While the precise mechanisms underlying these changes are presently unclear clues from post-mitotic organisms such as C. elegans have provided putative targets which are currently being investigated. It is clear to date that the age-induced changes in CNS function observed in invertebrates are conserved in mammalian species and that further work on invertebrates may provide informative insights in to the mechanisms of neuronal ageing.


Assuntos
Envelhecimento/fisiologia , Sistema Nervoso Central/fisiologia , Animais , Astrócitos/fisiologia , Comportamento , Cálcio/fisiologia , Contagem de Células , Divisão Celular , Pareamento Cromossômico/fisiologia , Ingestão de Energia , Junções Comunicantes/fisiologia , Humanos , Invertebrados , Longevidade/fisiologia , Modelos Biológicos , Neurônios/fisiologia , Transdução de Sinais/fisiologia
16.
J Neurophysiol ; 82(5): 2415-27, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10561415

RESUMO

We have used a combination of current-clamp and voltage-clamp techniques to characterize the electrophysiological properties of enzymatically dissociated Lymnaea heart ventricle cells. Dissociated ventricular muscle cells had average resting membrane potentials of -55 +/- 5 mV. When hyperpolarized to potentials between -70 and -63 mV, ventricle cells were capable of firing repetitive action potentials (8.5 +/- 1.2 spikes/min) that failed to overshoot 0 mV. The action potentials were either simple spikes or more complex spike/plateau events. The latter were always accompanied by strong contractions of the muscle cell. The waveform of the action potentials were shown to be dependent on the presence of extracellular Ca(2+) and K(+) ions. With the use of the single-electrode voltage-clamp technique, two types of voltage-gated K(+) currents were identified that could be separated by differences in their voltage sensitivity and time-dependent kinetics. The first current activated between -50 and -40 mV. It was relatively fast to activate (time-to-peak; 13.7 +/- 0.7 ms at +40 mV) and inactivated by 53.3 +/- 4.9% during a maintained 200-ms depolarization. It was fully available for activation below -80 mV and was completely inactivated by holding potentials more positive than -40 mV. It was completely blocked by 5 mM 4-aminopyridine (4-AP) and by concentrations of tetraethylammonium chloride (TEA) >10 mM. These properties characterize this current as a member of the A-type family of voltage-dependent K(+) currents. The second voltage-gated K(+) current activated at more depolarized potentials (-30 to -20 mV). It activated slower than the A-type current (time-to-peak; 74.1 +/- 3.9 ms at +40 mV) and showed little inactivation (6.2 +/- 2.1%) during a maintained 200-ms depolarization. The current was fully available for activation below -80 mV with a proportion of the current still available for activation at potentials as positive as 0 mV. The current was completely blocked by 1-3 mM TEA. These properties characterize this current as a member of the delayed rectifier family of voltage-dependent K(+) currents. The slow activation rates and relatively depolarized activation thresholds of the two K(+) currents are suggestive that their main role is to contribute to the repolarization phase of the action potential.


Assuntos
Coração/fisiologia , Canais de Potássio/fisiologia , 4-Aminopiridina/farmacologia , Animais , Cádmio/farmacologia , Cálcio/fisiologia , Células Cultivadas , Ventrículos do Coração , Ativação do Canal Iônico/fisiologia , Lymnaea , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Técnicas de Patch-Clamp , Potássio/fisiologia , Canais de Potássio/efeitos dos fármacos , Tetraetilamônio/farmacologia
17.
J Neurophysiol ; 82(5): 2428-40, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10561416

RESUMO

The single-electrode voltage-clamp technique was used to characterize voltage-gated Ca(2+) currents in dissociated Lymnaea heart ventricular cells. In the presence of 30 mM tetraethylammonium (TEA), two distinct Ca(2+) currents could be identified. The first current activated between -70 and -60 mV. It was fully available for activation at potentials more negative than -80 mV. The current was fast to activate and inactivate. The inactivation of the current was voltage dependent. The current was larger when it was carried by Ca(2+) compared with Ba(2+), although changing the permeant ion had no observable effect on the kinetics of the evoked currents. The current was blocked by Co(2+) and La(3+) (1 mM) but was particularly sensitive to Ni(2+) ions ( approximately 50% block with 100 microM Ni(2+)) and insensitive to low doses of the dihydropyridine Ca(2+) channel antagonist, nifedipine. All these properties classify this current as a member of the low-voltage-activated (LVA) T-type family of Ca(2+) currents. The activation threshold of the current (-70 mV) suggests that it has a role in pacemaking and action potential generation. Muscle contractions were first seen at -50 mV, indicating that this current might supply some of the Ca(2+) necessary for excitation-contraction coupling. The second, a high-voltage-activated (HVA) current, activated at potentials between -40 and -30 mV and was fully available for activation at potentials more negative than -60 mV. This current was also fast to activate and with Ca(2+) as the permeant ion, inactivated completely during the 200-ms voltage step. Substitution of Ba(2+) for Ca(2+) increased the amplitude of the current and significantly slowed the rate of inactivation. The inactivation of this current appeared to be current rather than voltage dependent. This current was blocked by Co(2+) and La(3+) ions (1 mM) but was sensitive to micromolar concentrations of nifedipine ( approximately 50% block 10 microM nifedipine) that were ineffective at blocking the LVA current. These properties characterize this current as a L-type Ca(2+) current. The voltage sensitivity of this current suggests that it is also important in generating the spontaneous action potentials, and in providing some of the Ca(2+) necessary for excitation-contraction coupling. These data provide the first detailed description of the voltage-dependent Ca(2+) currents present in the heart muscle cells of an invertebrate and indicate that pacemaking in the molluscan heart has some similarities with that of the mammalian heart.


Assuntos
Canais de Cálcio Tipo L/fisiologia , Canais de Cálcio Tipo T/fisiologia , Coração/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Bário/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo T/efeitos dos fármacos , Células Cultivadas , Cobalto/farmacologia , Ventrículos do Coração , Lantânio/farmacologia , Lymnaea , Potenciais da Membrana/efeitos dos fármacos , Tetraetilamônio/farmacologia
18.
J Exp Biol ; 202(Pt 19): 2581-93, 1999 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-10482718

RESUMO

This paper examines the importance of the calcium-mobilizing inositol phosphate pathway in mediating the effects of FMRFamide and its gene-related neuropeptides on the myogenic heart beat of the pond snail Lymnaea stagnalis. These peptides are encoded on a single exon of the FMRFamide gene and mediate diverse physiological effects in the isolated heart. The rate of production of inositol-1,4, 5-trisphosphate [Ins(1,4,5)P(3)] and inositol-1,3,4, 5-tetrakisphosphate [Ins(1,3,4,5)P(4)], measured using an HPLC method, were both significantly elevated in a concentration-dependent manner by FMRFamide (and were also elevated by FLRFamide). The threshold for increasing inositol phosphate production was low (100 pmol l(-1)) with a peak response occurring at 1 micromol l(-1) FMRFamide. The shape of the dose-response curve for FMRFamide-induced elevation of heart-beat frequency, obtained in pharmacological experiments on the isolated whole heart, was similar to that for stimulation of inositol phosphate levels in homogenized heart tissue. FMRFamide and Ins(1,4,5)P(3) produced similar effects on the rate of heart beat in permeabilized whole hearts. In addition, the phospholipase C inhibitor, neomycin (2.5 mmol l(-)(1)), blocked the stimulatory effects of FMRFamide on Ins(1, 4,5)P(3) production in heart homogenate, and attenuated the excitatory effects of this neuropeptide in the isolated heart. The 'isoleucine' pentapeptides, EFLRIamide and pQFYRIamide, also encoded by the FMRFamide gene, produced no significant effects on inositol phosphate production when applied alone or in combination with FMRFamide. These results suggested that FMRFamide (and FLRFamide), but not EFLRIamide and pQFYRIamide, mediated their main effects on heart beat via the inositol phosphate pathway. The fifth peptide, SEQPDVDDYLRDVVLQSEEPLY ('SEEPLY') had no effect when applied alone but appeared to modulate the effects of FMRFamide by delaying the time-to-peak of the Ins(1,4,5)P(3) response from 5 s to 20 s by an unknown mechanism.


Assuntos
FMRFamida/genética , Inositol 1,4,5-Trifosfato/metabolismo , Fosfatos de Inositol/metabolismo , Lymnaea/fisiologia , Neuropeptídeos/farmacologia , Sistemas do Segundo Mensageiro , Sequência de Aminoácidos , Animais , Cardiotônicos/farmacologia , Cromatografia Líquida de Alta Pressão , Éxons , FMRFamida/farmacologia , Coração/efeitos dos fármacos , Coração/fisiologia , Frequência Cardíaca/efeitos dos fármacos , Inositol 1,4,5-Trifosfato/farmacologia , Dados de Sequência Molecular , Neuropeptídeos/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Transdução de Sinais
19.
J Exp Biol ; 202(Pt 19): 2595-607, 1999 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-10482719

RESUMO

We have used a combination of biochemical and pharmacological techniques to investigate the role of the cyclic nucleotides, 3', 5'-cyclic adenosine monophosphate (cyclic AMP) and 3',5'-cyclic guanosine monophosphate (cyclic GMP), in mediating the cardioregulatory effects of FMRFamide and other neuropeptides encoded on exon II of the FMRFamide gene of Lymnaea stagnalis. The 'isoleucine' peptides (EFLRIamide and pQFYRIamide) produced complex biphasic effects on the frequency, force of contraction and tonus of the isolated heart of L. stagnalis, which were dependent on adenylate cyclase (AC) activity of the heart tissue. At a control rate of cyclic AMP production of less than or equal to 10 pmoles min(-)(1 )mg(-)(1) protein, the 'isoleucine' peptides produced a significant increase in AC activity in heart membrane preparations. This suggested that the enhanced AC activity is responsible for the stimulatory effects of the 'isoleucine' peptides on frequency and force of contraction of heart beat. This excitation sometimes followed an initial 'inhibitory phase' where the frequency of beat, force of contraction and tonus of the heart were reduced by the 'isoleucine' peptides. Hearts that showed the inhibitory phase of the 'isoleucine' response, but characteristically lacked the delayed excitatory phase, were found to have high levels of membrane AC activity (breve)10 pmoles min(-)(1 )mg(-)(1) protein in controls. Application of the 'isoleucine' peptides to membrane homogenate preparation from these hearts failed to increase AC activity. The addition of FMRFamide produced significant increases in the rate of cyclic AMP production in the heart membrane preparations, which could account, at least in part, for the cardioexcitatory effects of this peptide in the isolated whole heart. A membrane-permeable cyclic AMP analogue (8-bromo-cyclic AMP) and an AC activator (forskolin) were also cardioexcitatory. The peptide SEEPLY had no effects on the beat properties of the isolated heart and did not alter AC activity. The activity of the membrane-bound (particulate) guanylate cyclase (GC) was not significantly affected by any of the peptides.


Assuntos
AMP Cíclico/fisiologia , FMRFamida/genética , Lymnaea/fisiologia , Neuropeptídeos/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Adenilil Ciclases/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Colforsina/farmacologia , AMP Cíclico/biossíntese , GMP Cíclico/fisiologia , FMRFamida/farmacologia , Coração/efeitos dos fármacos , Coração/fisiologia , Frequência Cardíaca , Isoleucina , Dados de Sequência Molecular , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/fisiologia , Neuropeptídeos/genética , Sistemas do Segundo Mensageiro , Transdução de Sinais
20.
J Neurophysiol ; 81(4): 1818-26, 1999 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10200216

RESUMO

The cell-attached, patch-clamp technique was used to investigate the modulatory role of the neuropeptide SEQPDVDDYLRDVVLQSEEPLY ("SEEPLY") on FMRFamide-activated Ca2+ channels in isolated Lymnaea heart ventricular cells. Both SEEPLY and FMRFamide are encoded on the same neuropeptide gene and are coexpressed in a pair of excitatory motor neurons that innervate the heart. FMRFamide applied alone was capable of significantly increasing the P(open) time of a Ca2+ channel in isolated heart muscle cells. However, SEEPLY applied alone did not significantly alter the basal level of Ca2+ channel activity in the same cells. Repeated applications of FMRFamide (15 s every min) resulted in a progressive reduction in the number of Ca2+ channel openings and the overall P(open) time of the channel. The fifth successive 15-s application of FMRFamide failed to cause the Ca2+ channels to open in the majority of cells tested. When FMRFamide and SEEPLY were repeatedly applied together (2-min applications every 4 min) the FMRFamide-activated Ca2+ channels continued to respond after the fifth application of the two peptides. Indeed channel activity was seen to continue after repeated 2-min applications of FMRFamide and SEEPLY for as long as the patch lasted (

Assuntos
Canais de Cálcio/metabolismo , FMRFamida/genética , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Regulação para Baixo/genética , FMRFamida/farmacologia , Expressão Gênica/fisiologia , Lymnaea , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Dados de Sequência Molecular , Miocárdio/química , Miocárdio/citologia , Técnicas de Patch-Clamp , Regulação para Cima/genética
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