RESUMO
OBJECTIVE: To present a case report of bilateral posterior tarsal tunnel syndrome (PTTS) caused by an accessory flexor digitorum longus (AFDL), including the surgical technique and a review of the literature. MATERIALS AND METHODS: Twenty-nine year old male diagnosed with bilateral PTTS, refractory to conservative management, with 53 points on the preoperative AOFAS score. MR of both ankles showed an AFDL within the tarsal tunnel, in close relationship to the posterior tibial nerve. Bilateral tarsal tunnel decompression and AFDL resection was performed. RESULTS: There were no post-operative complications. At 6 months after surgery, the patient had no pain and had 87 points on the AOFAS score. DISCUSSION: The PTTS is an entrapment neuropathy of the posterior tibial nerve or one of its terminal branches. A rare cause is the presence of an AFDL, and its resection is associated with good clinical results. Careful scar tissue resection and neurolysis is recommended. Knowing the normal pathway and anatomical variability of the posterior tibial nerve and its branches is essential to avoid iatrogenic injury. In our case report, MR and intraoperative findings identified a bilateral FDLA in close relationship to the common flexor digitorum, an unusual finding, with few reports in current literature. CONCLUSIONS: Careful tarsal tunnel decompression and AFDL resection in our patient with bilateral symptomatic PTTS has good clinical results and no complications, particularly when diagnosed and treated early.
Assuntos
Descompressão Cirúrgica/métodos , Procedimentos Ortopédicos/métodos , Síndrome do Túnel do Tarso/cirurgia , Adulto , Humanos , Masculino , Músculo Esquelético/cirurgia , Síndrome do Túnel do Tarso/diagnóstico , Síndrome do Túnel do Tarso/etiologiaRESUMO
Fast-flow perfusion and flash photolysis of caged compounds were used to study the activation kinetics of L-type calcium current (ICa) in frog cardiac myocytes. Rapid exposure to isoproterenol (Iso) for 1 s or approximately 1 min produced similar kinetics of increase in ICa with an initial lag period of approximately 3 s, followed by a monophasic rise in current with a half-time of approximately 20 s. Epinephrine, as well as caged Iso, produced increases with similar kinetics. The fact that ICa increased significantly even after short Iso applications suggests that agonist binding to the receptor is rapid and that the increase in ICa is independent of free agonist. To dissect the kinetic contributions of various steps in the cAMP-phosphorylation cascade, the kinetics of the responses to caged cAMP and caged GTP gamma S and fast perfusion of forskolin, acetylcholine, and propranolol were compared. The response to caged cAMP exhibited no lag period, but otherwise increased at a rate similar to that produced by Iso and reached a peak at approximately 40 s after flash photolysis. This suggests that the lag period itself is due to a step before cAMP accumulation, but that activation of protein kinase and phosphorylation of the calcium channel are relatively slow. A lag period was also observed when ICa was stimulated by flash photolysis of caged GTP gamma S and when adenylyl cyclase was activated directly by rapid perfusion with forskolin. The lag period observed with forskolin may be due to slow binding of forskolin. The lag period was not due to the time required for cAMP to reach a threshold concentration, because a similar lag was observed in response to Iso in cells having ICa previously stimulated submaximally by internal perfusion with a low concentration of cAMP. These results suggest that the lag period can be attributed to a step associated with activation of adenylyl cyclase and cAMP accumulation.
Assuntos
Canais de Cálcio/fisiologia , AMP Cíclico/biossíntese , Miocárdio/metabolismo , Receptores Adrenérgicos beta/fisiologia , Acetilcolina/farmacologia , Animais , Colforsina/farmacologia , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Epinefrina/farmacologia , Proteínas de Ligação ao GTP/biossíntese , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Coração/efeitos dos fármacos , Coração/fisiologia , Temperatura Alta , Técnicas In Vitro , Isoproterenol/farmacologia , Cinética , Miocárdio/citologia , Fotólise , Propranolol/farmacologia , Rana catesbeiana , Rana esculenta , Sistema Nervoso Simpático/fisiologiaRESUMO
The hypothalamic GHRH neurons secrete pulses of GHRH to generate episodic GH secretion, but little is known about the mechanisms involved. We have made transgenic mice expressing enhanced green fluorescent protein (eGFP) specifically targeted to the secretory vesicles in GHRH neurons. GHRH cells transported eGFP from cell bodies in the arcuate nucleus to extensively arborized varicose fiber terminals in the median eminence. Patch clamp recordings from visually identified GHRH cells in mature animals showed spontaneous action potentials, often firing in short bursts up to 10 Hz. GHRH neurons received frequent synaptic inputs, as demonstrated by the recording of abundant inward postsynaptic currents, but spikes were followed by large after-hyperpolarizations, which limited their firing rate. Because many GHRH neurons lie close to the ventral hypothalamic surface, this was examined by wide-field binocular epifluorescence stereomicroscopy. This approach revealed an extensive horizontal network of GHRH cells at low power and individual fiber projections at higher power in the intact brain. It also showed the dense terminal projections of the GHRH cell population in the intact median eminence. This model will enable us to characterize the properties of individual GHRH neurons and their structural and functional connections with other neurons and to study directly the role of the GHRH neuronal network in generating episodic secretion of GH.
Assuntos
Núcleo Arqueado do Hipotálamo/citologia , Hormônio Liberador de Hormônio do Crescimento/genética , Neurônios/fisiologia , Potenciais de Ação/fisiologia , Animais , Núcleo Arqueado do Hipotálamo/fisiologia , Mapeamento Encefálico/métodos , Proteínas de Fluorescência Verde , Hibridização In Situ , Indicadores e Reagentes/metabolismo , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , Vias Neurais , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/fisiologia , RNA Mensageiro/análiseRESUMO
1. To investigate the participation of guanylyl cyclase in the muscarinic regulation of the cardiac L-type calcium current (ICa), we examined the effects of three guanylyl cyclase inhibitors, 1H-[1,2,4]oxidiazo-lo[4,3-a]quinoxaline-1-one (ODQ), 6-anilino-5,8-quinolinedione (LY 83583), and methylene blue (MBlue), on the beta-adrenoceptor; muscarinic receptor and nitric oxide (NO) regulation of ICa and on the muscarinic activated potassium current I(K,ACh), in frog atrial and ventricular myocytes. 2. ODQ (10 microM) and LY 83583 (30 microM) antagonized the inhibitory effect of an NO-donor (S-nitroso-N-acetylpenicillamine, SNAP, 1 microM) on the isoprenaline (Iso)-stimulated ICa which was consistent with their inhibitory action on guanylyl cyclase. However, MBlue (30 microM) had no effect under similar conditions. 3. In the absence of SNAP, LY 83583 (30 microM) potentiated the stimulations of ICa by either Iso (20 nM), forskolin (0.2 microM) or intracellular cyclic AMP (5-10 microM). ODQ (10 microM) had no effect under these conditions, while MBlue (30 microM) inhibited the Iso-stimulated ICa. 4. LY 83583 and MBlue, but not ODQ, reduced the inhibitory effect of up to 10 microM acetylcholine (ACh) on ICa. 5. MBlue, but not LY 83583 and ODQ, antagonized the activation of I(K,ACh) by ACh in the presence of intracellular GTP, and this inhibition was weakened when I(K,ACh) was activated by intracellular GTPgammaS. 6. The potentiating effect of LY 83583 on Iso-stimulated ICa was absent in the presence of either DL-dithiothreitol (DTT, 100 microM) or a combination of superoxide dismutase (150 u ml(-1)) and catalase (100 u ml(-1)). 7. All together, our data demonstrate that, among the three compounds tested, only ODQ acts in a manner which is consistent with its inhibitory action on the NO-sensitive guanylyl cyclase. The two other compounds produced severe side effects which may involve superoxide anion generation in the case of LY 83583 and alteration of beta-adrenoceptor and muscarinic receptor-coupling mechanisms in the case of M Blue.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Guanilato Ciclase/antagonistas & inibidores , Coração/efeitos dos fármacos , Canais de Potássio/efeitos dos fármacos , Aminoquinolinas/farmacologia , Animais , Canais de Cálcio Tipo L , AMP Cíclico/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Azul de Metileno/farmacologia , Óxido Nítrico/fisiologia , Nitroprussiato/farmacologia , Rana esculenta , Receptores Adrenérgicos beta/fisiologia , Receptores Muscarínicos/fisiologia , Superóxidos/metabolismoRESUMO
OBJECTIVES: Colchicine poisoning may be lethal and a decrease in cardiac function has been reported in several case reports, but the precise cardiotoxicity of colchicine remains unknown. DESIGN: The experimental in vitro study assessed the intrinsic contractility of left ventricular papillary muscle in rats, 24 h after administration of intraperitoneal colchicine or saline. RESULTS: The administration of colchicine (2 or 4 mg.kg-1) in adult Wistar rats markedly impaired intrinsic myocardial contractility, as shown by a decrease in maximum shortening velocity (-32 and -61%, respectively), active isometric force (-47 and -65%, respectively), and peak power output (-57 and -69%, respectively) of left ventricular papillary muscle. Colchicine impaired isotonic relaxation and load dependence of relaxation, suggesting a decrease in sarcoplasmic reticulum function. Conversely, colchicine significantly accelerated isometric relaxation, suggesting a decrease in calcium myofilament sensitivity. Myothermal economy was markedly impaired only in some rats (3/10 in each group), in which the negative inotropic effect of colchicine appeared to be more particularly pronounced. CONCLUSION: The results indicate that the administration of high doses of colchicine induced intrinsic cardiotoxic effects. Due to its amplitude, such cardiotoxic action may participate in the fatal outcome of acute colchicine poisoning.
Assuntos
Colchicina/intoxicação , Contração Miocárdica/efeitos dos fármacos , Função Ventricular Esquerda/efeitos dos fármacos , Doença Aguda , Animais , Débito Cardíaco/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Metabolismo Energético/efeitos dos fármacos , Injeções Intraperitoneais , Contração Miocárdica/fisiologia , Músculos Papilares/efeitos dos fármacos , Intoxicação/complicações , Ratos , Ratos Wistar , Retículo Sarcoplasmático/efeitos dos fármacos , Função Ventricular Esquerda/fisiologiaRESUMO
A fast perfusion system was used to analyze the kinetics of the response of L-type calcium current (ICa) to rapid exposures to beta-adrenergic or dihydropyridine agonists in whole-cell patch-clamped frog ventricular myocytes. The perfusion system was based on the lateral motion of an array of plastic capillary tubes from which solutions flowed at a velocity of approximately 5 cm/s. Movement from one capillary to the adjacent one occurred in < 20 ms and complete exchange of extracellular solution was achieved in < 50 ms as demonstrated by the block of ICa by fastflow application of Cd during a depolarizing pulse. Fastflow applications of increasing concentrations of isoprenaline (Iso) led to a dose-dependent stimulation of ICa at [Iso] > 1 nM. The response of ICa to Iso always started after a delay of several seconds. The delay duration decreased as [Iso] increased, and was typically approximately 3 s at 10 microM Iso. The rising phase of ICa increase was monophasic and independent of [Iso] > 100 nM. For short applications of Iso (8.8 s), half maximal and maximal stimulation of ICa occurred approximately 20 s and approximately 40 s after the beginning of Iso application, respectively. When Iso was applied during a depolarizing pulse (with Ba as the charge carrier), IBa never increased during that pulse. The kinetics of the ICa response to Iso were not affected by varying the voltage clamp protocols or the ionic composition of intracellular and extracellular solutions. In comparison with the effects of Iso, the stimulatory effect of the dihydropyridine agonist (-)Bay K 8644 on ICa was approximately 15 times faster: delay, half-time to maximal and time to maximal responses were 15 times shorter with (-)Bay K 8644 than with Iso. It is concluded that frog ventricular myocytes respond slowly to a quick application of beta-adrenergic agonists.
Assuntos
Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Cálcio/fisiologia , Isoproterenol/farmacologia , Função Ventricular , Animais , Canais de Cálcio/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Rana catesbeiana , Rana esculenta , Fatores de TempoRESUMO
Muscarinic agonists regulate the L-type calcium current in isolated cardiac myocytes. The second messengers pathways involved in this regulation are discussed briefly, with particular emphasis on the involvement of cAMP and cGMP pathways.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Coração/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Animais , AMP Cíclico/biossíntese , GMP Cíclico/biossíntese , Humanos , Óxido Nítrico/fisiologiaRESUMO
The effects of extracellular application of cicletanine on the voltage-sensitive calcium current (ICa) was studied in isolated cells from frog ventricle. Myocytes were isolated by enzymatic dissociation and ICa was measured using the whole-cell configuration of the patch-clamp technique modified to permit intracellular perfusion with various substances. Cicletanine (10 to 100 microM) had no effect on control ICa. However, when ICa was enhanced by superfusion of the cell with saturating doses of beta-adrenergic agonist (isoprenaline, 2 microM) or by intracellular perfusion with maximal doses of cAMP (20 microM), cicletanine exerted a dual effect on ICa. At 10 microM, cicletanine generally induced a transient or sustained stimulation of ICa (5 to 40%), while 100 microM of the drug generally reduced ICa. The effects of cicletanine were reversible and not voltage-dependent. These results suggest that cicletanine affects ICa by acting on a mechanism occurring after cAMP synthesis, by enhancing cAMP concentration (e.g. through an inhibition of cAMP phosphodiesterase) or facilitating cAMP-dependent phosphorylation of the Ca channels.
Assuntos
Anti-Hipertensivos/farmacologia , Cálcio/metabolismo , Diuréticos/farmacologia , Canais Iônicos/efeitos dos fármacos , Miocárdio/metabolismo , Piridinas , Animais , AMP Cíclico/metabolismo , Condutividade Elétrica , Canais Iônicos/metabolismo , Miocárdio/citologia , Perfusão , Rana esculenta , Fatores de TempoAssuntos
Gentamicinas/administração & dosagem , Adolescente , Adulto , Idoso , Feminino , Gentamicinas/metabolismo , Humanos , Cinética , Masculino , Pessoa de Meia-IdadeRESUMO
Objetivo. Describir un caso de síndrome de túnel del tarso posterior (STTP) bilateral causado por un tendón flexor digitorum longus accesorio (FDLA), la técnica de resección quirúrgica y una revisión de la literatura. Materiales y métodos. Reportamos el caso de un paciente varón de 29 años con diagnóstico de STTP bilateral, refractario al manejo conservador con una puntuación AOFAS de 53 puntos. Se solicitó una RM de ambos tobillos encontrándose la presencia del músculo FDLA dentro del túnel tarsiano, en íntima relación con el nervio tibial posterior. Se realiza una descompresión bilateral del túnel tarsiano resecando el músculo FDLA que producía un conflicto de espacio con el nervio tibial posterior. Resultados. El paciente no presentó complicaciones postoperatorias. A los 6 meses de cirugía, presentaba una puntuación final AOFAS de retropié de 87 puntos. Discusión. El STTP consiste en una neuropatía por atrapamiento del nervio tibial posterior o una de sus ramas terminales. Una de sus causas es la presencia FDLA, y su resección está asociada a buenos resultados clínicos. Se recomienda la neurólisis del tejido cicatricial y adherencias alrededor del nervio. Conocer la anatomía normal y su variabilidad para liberar el nervio tibial posterior y sus ramas es fundamental para evitar lesiones iatrogénicas. En nuestro caso clínico, la RM identificó un FDLA bilateral, que al ser resecado se encontraba en íntima relación con el flexor digitorum común, hallazgo poco común en la literatura. Conclusiones. La descompresión cuidadosa del túnel del tarso en un paciente con STTP bilateral sintomático por un FDLA se asocia a buenos resultados, particularmente en aquellos pacientes con diagnóstico y tratamiento precoz. Nivel de evidencia. IV (AU)
Objective. To present a case report of bilateral posterior tarsal tunnel syndrome (PTTS) caused by an accessory flexor digitorum longus (AFDL), including the surgical technique and a review of the literature. Materials and methods. Twenty-nine year old male diagnosed with bilateral PTTS, refractory to conservative management, with 53 points on the preoperative AOFAS score. MR of both ankles showed an AFDL within the tarsal tunnel, in close relationship to the posterior tibial nerve. Bilateral tarsal tunnel decompression and AFDL resection was performed. Results. There were no post-operative complications. At 6 months after surgery, the patient had no pain and had 87 points on the AOFAS score. Discussion. The PTTS is an entrapment neuropathy of the posterior tibial nerve or one of its terminal branches. A rare cause is the presence of an AFDL, and its resection is associated with good clinical results. Careful scar tissue resection and neurolysis is recommended. Knowing the normal pathway and anatomical variability of the posterior tibial nerve and its branches is essential to avoid iatrogenic injury. In our case report, MR and intraoperative findings identified a bilateral FDLA in close relationship to the common flexor digitorum, an unusual finding, with few reports in current literature. Conclusions. Careful tarsal tunnel decompression and AFDL resection in our patient with bilateral symptomatic PTTS has good clinical results and no complications, particularly when diagnosed and treated early (AU)
Assuntos
Humanos , Masculino , Adulto , Síndrome do Túnel do Tarso/complicações , Síndrome do Túnel do Tarso/cirurgia , Síndrome do Túnel do Tarso , Descompressão Cirúrgica/métodos , Ossos do Tarso/cirurgia , Espectroscopia de Ressonância Magnética/métodos , Nervo Tibial/lesões , Nervo Tibial/patologia , Complicações Pós-Operatórias/patologia , Tornozelo/cirurgia , TornozeloRESUMO
Early studies in whole heart indicated that cGMP antagonized the positive inotropic effects of catecholamines and cAMP. Since the L-type Ca2+ channel current (ICa) plays a predominant role in the initiation and development of cardiac electrical and contractile activities, regulation of ICa by cGMP pathways has received much attention over the last ten years. Patch-clamp measurements of ICa in isolated cardiac myocytes reveal at least three different cGMP effectors that may participate to different degrees in different animal species and cardiac tissues in the regulation of ICa by cGMP. In frog ventricular myocytes, cGMP inhibits ICa by stimulation of a cGMP-stimulated cAMP phosphodiesterase (PDE2), whereas in rat ventricular myocytes, cGMP predominantly inhibits ICa via a mechanism involving activation of a cGMP-dependent protein kinase (cGMP-PK). In guinea pig, frog and human cardiomyocytes, cGMP can also stimulate ICa via an inhibition of a cGMP-inhibited cAMP phosphodiesterase (PDE3). This effect is most predominant in human atrial myocytes and appears readily during an activation of the soluble guanylate cyclase activity by low concentrations of nitric oxide (NO)-donors. Biochemical characterization of the endogenous phosphodiesterases and cGMP-PK in purified cardiac myocytes provide further evidence in support of these mechanisms of cGMP action on ICa. However, the regulation of cGMP levels by a variety of agents is not always consistent with their effects on contractility. In particular, the participation of cGMP and NO pathways in the regulation of cardiac ICa and contractility by acetylcholine is still questionable.
Assuntos
Canais de Cálcio/metabolismo , GMP Cíclico/metabolismo , Miocárdio/metabolismo , Óxido Nítrico/metabolismo , Animais , GMP Cíclico/fisiologia , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Coração/fisiologia , Humanos , Óxido Nítrico/fisiologia , Inibidores de Fosfodiesterase/metabolismo , Diester Fosfórico Hidrolases/metabolismo , RatosRESUMO
A new and inexpensive system allowing rapid and synchronized changes of solutions around a membrane patch or a cell under voltage-clamp conditions is described. Four plastic capillary tubings (OD 640 microns; ID 430 microns) were glued together horizontally and attached to a coil of a commercially available loudspeaker. Servo-control of the position of the coil allowed the mouth of any of the capillaries to be positioned near the pipette tip within 6 ms. A high flow speed of the test solution was crucial to achieve rapid solution exchange. At a flow speed of 5 cm/s, complete exchange of the external environment of a frog ventricular cell was achieved within 20-30 ms. The time course of solution change was found to be 3-5 times faster at the tip of an open patch pipette. To preserve the physical integrity of the cell, the cell was usually perfused by a control capillary at a slow velocity (0.2-0.4 cm/s) and test solutions flowing out of adjacent capillaries at high velocity (4-5 cm/s) were applied to the cell only for short periods. Determination of the three-dimensional contamination profile around the mouth of the control capillary allowed the optimal conditions for the use of the system to be established and possible sources of contamination to be avoided between adjacent capillaries with unmatched flow speeds. Successive and multiple changes in external solutions could be easily synchronized with voltage-clamp depolarizations to examine the time course of the effect of drugs on voltage-operated ion channels.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Membrana Celular/fisiologia , Eletrofisiologia/métodos , Soluções , Animais , Potássio/fisiologiaRESUMO
1. The role of the cGMP pathway in the modulation of the cardiac L-type Ca2+ current (ICa,L) by nitric oxide (NO) was examined in rat ventricular myocytes. 2. The NO donors DEANO, SIN-1, SNP, SNAP and GSNO had no significant effects on basal ICa,L. However, DEANO (100 microM) inhibited ICa,L after the current had been previously stimulated by either isoprenaline (Iso, 1-10 nM), a beta-adrenergic agonist, or isobutylmethyl-xanthine (IBMX, 10-80 microM), a wide spectrum phosphodiesterase (PDE) inhibitor. 3. The anti-adrenergic effect of DEANO on ICa,L was not mimicked by other NO donors (SIN-1, SNAP and SPNO). 4. The NO-sensitive guanylyl cyclase inhibitor ODQ (10 microM), antagonized the inhibitory effect of DEANO on ICa,L. Likewise, inhibitors of the cGMP-dependent protein kinase (cG-PK), Rp-8-chloro-phenylthio-cGMP (10 microM) and KT5823 (0.1 and 0.3 microM), also abolished the inhibitory effect of DEANO on Iso (1-10 nM)-stimulated ICa,L. 5. Intracellular dialysis with exogenous cAMP (10-100 microM) blunted the inhibitory effect of DEANO (10 and 100 microM) on ICa,L. SNAP and SNP also had no effect on the cAMP-stimulated ICa,L. 6. Pre-treatment of the myocytes with pertussis toxin (0.5 microg ml-1, 4-6 h at 37 degrees C) eliminated the inhibitory effect of DEANO (100 microM) on ICa,L, in the presence of either Iso (0.01 and 1 nM) or IBMX (10-80 microM). 7. These results demonstrate that DEANO produces anti-adrenergic effects in rat ventricular myocytes. This effect of DEANO occurs in a cGMP-dependent manner, and involves activation of cG-PK and regulation of a pertussis toxin-sensitive G protein.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Proteínas de Ligação ao GTP/fisiologia , Coração/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Animais , Eletrofisiologia , Guanilato Ciclase/antagonistas & inibidores , Ventrículos do Coração/citologia , Ventrículos do Coração/efeitos dos fármacos , Técnicas In Vitro , Masculino , Potenciais da Membrana/fisiologia , Microdiálise , Miocárdio/citologia , Técnicas de Patch-Clamp , Ratos , Ratos WistarRESUMO
Recently, an inhibitor of adenosine deaminase, erythro-9-(2-hydroxyl-3-nonyl)adenine (EHNA), was shown to selectively block the activity of purified cGMP-stimulated phosphodiesterase (PDE) (cGS-PDE, or PDE2) in human and porcine heart [J. Mol. Cell. Cardiol. 24 (Suppl. V):102 (1992)]. Because cGS-PDE was found to mediate the cGMP-induced inhibition of L-type Ca2+ current (Ica) in frog ventricular cells, we tested the effects of EHNA in this preparation. Ica was measured using the whole-cell patch-clamp technique and a perfusing pipette. EHNA (0.3-30 microM) had no significant effect on either basal Ica or isoprenaline (1 nM)- or cAMP (10 microM)-elevated Ica. However, EHNA dose-dependently (IC50 approximately 3 microM) reversed the inhibitory effect of cGMP on cAMP-stimulated Ica. EHNA (30 microM) also blocked the inhibitory effect of NO donors, such as sodium nitroprusside (1 mM) and 3-morpholinosydnonimine (30 microM), on isoprenaline-stimulated Ica. In addition, EHNA dose-dependently (IC50 approximately 4-5 microM) inhibited the cGMP-induced stimulation of PDE activity in frog ventricle particulate fraction, as well as purified soluble cGS-PDE. However, EHNA (up to 30 microM) did not modify the activities of three other purified soluble PDE isoforms. Moreover, EHNA did not change the Ka (40 nM) for cGMP activation of cGS-PDE, which suggests that EHNA does not inhibit cGS-PDE by displacing cGMP from the allosteric regulator site. Because adenosine did not mimic the effects of EHNA on Ica or PDE activity, it is unlikely that the effects of EHNA are due to adenosine deaminase inhibition. We conclude that EHNA acts primarily to inhibit cGS-PDE in intact cardiac myocytes. This compound should be useful in evaluating the physiological role of cGS-PDE in various tissues.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/antagonistas & inibidores , Adenina/análogos & derivados , Miocárdio/enzimologia , Adenina/farmacologia , Adenosina Desaminase/metabolismo , Animais , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , GMP Cíclico/metabolismo , Miocárdio/citologia , Óxido Nítrico/metabolismo , Rana esculentaRESUMO
1. We report opposite inotropic effects of NO donors in frog cardiac fibres. The negative effect, elicited by either 3-morpholino-sydnonimine (SIN-1) or S-nitroso-N-acetyl-penicillamine (SNAP), involved cyclic GMP (cGMP) production. However, SIN-1, unlike SNAP, could elicit a positive effect, in a superoxide dismutase (SOD)-sensitive manner. SIN-1, unlike SNAP, can release both NO and superoxide anion, the precursors of peroxynitrite (OONO-). The role of these messengers was examined. 2. Catalase did not reduce the positive inotropic effect of SIN-1. Thus, a conversion of superoxide anion into hydrogen peroxide was not involved in this effect. In addition, catalase did not modify the negative effects of SIN-1 plus SOD, or SNAP plus SOD. 3. LY 83583, a superoxide anion generator, elicited a positive inotropic effect, like SIN-1. The effect of LY 83583 was additive to the negative effects of SIN-1 or SNAP, and to the positive effect of SIN-1. Thus, superoxide anion generation, per se, did not account for the positive effect of SIN-1. 4. Authentic peroxynitrite (OONO-), but not mock-OONO- (negative control plus decomposed OONO-), exerted a dramatic positive inotropic effect in cardiac fibres. The effect of OONO- was larger in atrial fibres, as compared with ventricular fibres. 5. The positive effect of OONO- was not additive with that of SIN-1, suggesting a common mechanism of action. In contrast, the effects of either OONO- or SIN-1 were additive with the negative inotropic effect of SNAP. Furthermore, the effect of OONO-, like that of SIN-1, was not antagonized by 1H-[1,2,4]xidiazolo[4, 3-a]quinoxaline-1-one (ODQ; 10 microM), the guanylyl cyclase inhibitor. 6. The positive inotropic effects of SIN-1 and OONO- were not modified by hydroxyl radical scavengers, such as dimethyl-thio-urea (DMTU; 10 mM). 7. The positive inotropic effect of SIN-1 (100 microM) was abolished in sodium-free solutions, a treatment that eliminates the activity of the sodium-calcium exchanger. In contrast, the effect of SIN-1 was unchanged by a potassium channel inhibitor (tetraethyl-ammonium, 20 mM), or a sodium-potassium pump inhibitor (ouabain 10 microM). 8. We conclude that OONO- is a positive inotropic agent in frog cardiac fibres. The generation of OONO- accounts for the positive inotropic effect of SIN-1. OONO- itself was responsible for the positive inotropic effect, and appeared to modulate the activity of the sodium-calcium exchanger.
Assuntos
Fibras Musculares Esqueléticas/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Miocárdio/citologia , Nitratos/farmacologia , Oxidantes/farmacologia , Aminoquinolinas/farmacologia , Animais , Função Atrial , Catalase/farmacologia , GMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Guanilato Ciclase/antagonistas & inibidores , Átrios do Coração/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Radical Hidroxila/metabolismo , Molsidomina/análogos & derivados , Molsidomina/farmacologia , Fibras Musculares Esqueléticas/enzimologia , Doadores de Óxido Nítrico/farmacologia , Oxidiazóis/farmacologia , Quinoxalinas/farmacologia , Rana esculenta , Sódio/farmacologia , Trocador de Sódio e Cálcio/metabolismo , Função VentricularRESUMO
1. The cardiac effects of the NO donors sodium nitroprusside (SNP), S-nitroso-N-acetyl-penicillamine (SNAP) and 3-morpholino-sydnonimine (SIN-1) were studied in frog fibres to evaluate the contribution of cyclic GMP-dependent mechanisms. 2. SNP and SNAP (0.1-100 microM) reduced the force of contraction in a concentration-dependent manner in atrial and ventricular fibres. This effect was associated with a reduction in the time to peak (TTP) and the time for half-relaxation of contraction (T). 3. SIN-1 (100 microM) also reduced the force of contraction in two-thirds of the atrial fibres. However, it exerted a positive inotropic effect in the remaining atrial fibres, as well as in most ventricular fibres. 4. The guanylyl cyclase inhibitor 1H-[1,2,4]oxidiazolo[4,3-a]quinoxaline-1-one (ODQ, 10 microM) antagonized the negative inotropic effects of SIN-1 (50 microM) and SNAP (25 microM) but had no effect on the positive inotropic response to SIN-1 (100 microM). 5. In the presence of SIN-1, superoxide dismutase (SOD, 50-200 U ml-1) either potentiated the negative inotropic effect or turned the positive inotropic effect of the drug into a negative effect. SOD had no effects when applied alone or in the presence of SNAP. 6. 6-Anilino-5,8-quinolinedione (LY 83583, 3-30 microM), a superoxide anion generator also known as a cyclic GMP-lowering agent, exerted a positive inotropic effect, which was antagonized by SOD (200-370 U ml-1) but not by ODQ (10 microM). 7. We conclude that SNP, SNAP and SIN-1 exert cyclic GMP-dependent negative inotropic effects, which are attributed to the generation of NO. In addition, SIN-1 and LY 83583 exert cyclic GMP-independent positive inotropic effects, which require the generation of superoxide anion.
Assuntos
Coração/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Aminoquinolinas/farmacologia , Animais , GMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Guanilato Ciclase/antagonistas & inibidores , Meia-Vida , Átrios do Coração/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Técnicas In Vitro , Molsidomina/análogos & derivados , Molsidomina/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Nitroprussiato/farmacologia , Penicilamina/análogos & derivados , Penicilamina/farmacologia , Rana esculenta , S-Nitroso-N-AcetilpenicilaminaRESUMO
The positive inotropic effect of the sympathetic nervous system on the heart is partly mediated by an increase in the voltage-gated Ca2+ current (ICa). This increase is generally attributed to beta-adrenergic receptor-stimulated cyclic AMP-dependent phosphorylation of the Ca2+ channel. It has been suggested that cAMP-dependent phosphorylation cannot explain all the effects of beta-adrenergic agonists on ICa and that a parallel membrane-delimited pathway involving the 'direct' action of the G protein Gs also stimulates ICa. A precedent exists for such a membrane-delimited pathway in the activation of a K+ channel by acetylcholine in heart. A membrane-delimited pathway for stimulation of ICa might be important in rapid beat-to-beat regulation of contraction by the sympathetic nervous system, because isoproterenol may produce a biphasic increase in ICa with the rapid phase (tau = 150 ms) putatively mediated by the direct pathway and the slow phase (tau = 35 s) by cAMP-dependent phosphorylation. Here we report that in frog, rat, and guinea pig ventricular myocytes ICa increases slowly and monophasically in response to isoproterenol. The increase is completely blocked by inhibitors of cAMP-dependent phosphorylation. Furthermore, the time course of the increase in ICa closely parallels the increase in contractile force produced by sympathetic nerve stimulation. These data refute earlier suggestions that regulation of Ca2+ channels by the sympathetic nervous system involves or requires a direct G-protein pathway.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/fisiologia , AMP Cíclico/fisiologia , Coração/fisiologia , Fosfoproteínas/fisiologia , Sistema Nervoso Simpático/fisiologia , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Proteínas de Ligação ao GTP/fisiologia , Cobaias , Ativação do Canal Iônico/efeitos dos fármacos , Isoproterenol/farmacologia , Contração Miocárdica , Fosforilação , Inibidores de Proteínas Quinases , RatosRESUMO
Regulation of cardiac contraction by neurotransmitters and hormones is often correlated with regulation of the L-type Ca2(+)-channel current (ICa) through the opposite actions of two second messengers, cyclic AMP and cyclic GMP. While cyclic AMP stimulation of ICa is mediated by the activation of cyclic AMP-dependent protein kinase, inhibition of ICa by cyclic GMP in frog heart is largely mediated by activation of cyclic AMP phosphodiesterase. The present patch-clamp study reveals that, in rat ventricular cells, cyclic GMP can also regulate ICa via activation of endogenous cyclic GMP-dependent protein kinase (cGMP-PK). Indeed, the effect of cyclic GMP on ICa was mimicked by intracellular perfusion with the proteolytic active fragment of purified cGMP-PK. Moreover, cGMP-PK immunoreactivity was detected in pure rat ventricular myocytes by using a specific polyclonal antibody. These results demonstrate a dual mechanism for the inhibitory action of cyclic GMP in heart, as well as a physiological role for cGMP-PK in the control of mammalian heart function.
Assuntos
Canais de Cálcio/fisiologia , AMP Cíclico/farmacologia , GMP Cíclico/farmacologia , Coração/fisiologia , Proteínas Quinases/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Canais de Cálcio/efeitos dos fármacos , Células Cultivadas , Ventrículos do Coração/enzimologia , Cinética , Masculino , Miocárdio/enzimologia , Fragmentos de Peptídeos/farmacologia , Ratos , Ratos Endogâmicos , Função VentricularRESUMO
Glucagon exerts positive inotropic and chronotropic effects in the heart. Like its glycogenolytic effect in liver cells, the cardiac effects of glucagon are often correlated with adenylyl cyclase stimulation. Therefore, cyclic AMP-dependent phosphorylation of L-type Ca2+ channels might be involved in the inotropic effect of glucagon. There have been no reports, however, of the effects of glucagon on the cardiac Ca2+ current (ICa). Also, the physiological effects of glucagon could involve mechanisms other than stimulation of adenylyl cyclase. Here we show that glucagon enhances ICa in frog and rat ventricular myocytes. The effect of glucagon in rats resulted from a stimulation of adenylyl cyclase. In frogs, however, the effect of glucagon on ICa was smaller and occurred at a concentration tenfold lower than in rats, and adenylyl cyclase was not modified. In addition, cAMP potentiated the effect of glucagon on ICa in frog ventricle, which correlated with the observed inhibition by glucagon of low-Km cAMP phosphodiesterase activity. Therefore, this is an example of a hormone that affects cardiac function in a similar way to a variety of synthetic cardiotonic compounds, such as milrinone and Ro-20-1724. Inhibition of phosphodiesterase activity by glucagon may be essential in animals in which glucagon increases cardiac contractility but does not effectively stimulate adenylyl cyclase.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Adenilil Ciclases/metabolismo , Canais de Cálcio/fisiologia , Glucagon/farmacologia , Coração/fisiologia , Acetilcolina/farmacologia , Animais , AMP Cíclico/biossíntese , AMP Cíclico/farmacologia , Sinergismo Farmacológico , Condutividade Elétrica , Ativação Enzimática/efeitos dos fármacos , Isoproterenol/farmacologia , Masculino , Rana esculenta , Ratos , Ratos EndogâmicosRESUMO
The effects of the nitric oxide (NO) donor 3-morpholino-sydnonimine (SIN-1) on the L-type Ca2+ current (ICa) were examined in frog ventricular myocytes under basal and phosphorylated conditions. SIN-1 was found to exert insignificant effects on basal ICa but to induce a biphasic action on stimulated ICa. Indeed, in the nanomolar range of concentrations (0.1-10 nM), SIN-1 induced a pronounced (approximately 40%) stimulation of ICa elevated by a non-maximal concentration of forskolin (0.3 microM). However, the stimulatory effects of SIN-1 on ICa were not additive with those of maximal concentrations (10 microM) of forskolin or intracellular cAMP. In contrast, at higher concentrations (100 nM to 1 mM), SIN-1 strongly reduced ICa (by up to 85%) which had been previously stimulated by cAMP, forskolin, or isoprenaline. All the effects of SIN-1 appeared to be mediated by the liberation of NO since they were suppressed by methylene blue and LY83583 and were not mimicked by SIN-1C, a metabolite of SIN-1. The stimulatory or inhibitory effects of SIN-1 were absent, respectively, in the presence of milrinone (10 microM) or when the hydrolysis-resistant cAMP analog 8-bromo-cAMP was used instead of cAMP to stimulate ICa. In addition to its effects on ICa, SIN-1 induced a dose-dependent stimulation of guanylyl cyclase activity in the cytosolic and membrane fractions of frog ventricle. The membrane form of guanylyl cyclase displayed a higher sensitivity to SIN-1 than the cytosolic form, which correlated with SIN-1 sensitivity of ICa. Our data suggest that the activatory and inhibitory effects of NO donors on ICa result from an inhibition of the cGMP-inhibited cAMP-phosphodiesterase and an activation of the cGMP-stimulated cAMP-phosphodiesterase, respectively, both linked to the activation of guanylyl cyclase, possibly a membrane form of the enzyme.