Síndrome del túnel del tarso posterior bilateral por músculo flexor digitorum longus accesorio; reporte de un caso y descripción de técnica quirúrgica / Bilateral posterior tarsal tunnel syndrome caused by accessory flexor digitorum longus; case report and surgical technique

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)

Bilateral posterior tarsal tunnel syndrome caused by accessory flexor digitorum longus; case report and surgical technique. / Síndrome del túnel del tarso posterior bilateral por músculo flexor digitorum longus accesorio; reporte de un caso y descripción de técnica quirúrgica.

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.

Entre a psicanálise e a educação: diálogos e experiências / Between psychoanalysis and education: dialogues and experiences

Os autores apresentam a evolução de um trabalho inovador que um grupo de psicanalistas da Sociedade Psicanalítica de Porto Alegre (SPPA) está realizando com as Escolas de Educação Infantil Conveniadas da Secretaria Municipal de Educação (SMED). Discutem o modelo de trabalho, com suas principais características , formas de abordagem e a evolução do mesmo ao longo dos quatro anos em que as atividades vêm sendo desenvolvidas. Destacam a construção de um modelo de interação entre psicanalistas e educadores diferente daquele da consultoria escolar tradicional. Portem da hipótese de que intervenção primária na infância pode ser realizada também capacitando os profissionais que se ocupam das crianças nos seus primeiros anos. Tratando-se de comunidades que apresentam alto grau de vulnerabilidade social, tem sido fundamental a discussão permanente com um consultor da área do serviço social com larga experiência no trabalho com estas comunidades. A ênfase tem sido dada na discussão em pequenos grupos para que vivências possam ser compartilhadas com o propósito de criar um espaço de reflexão.

The authors present the evolution of an innovative work which is being developed by a group of psychoanalysts from the Porto Alegre Psychoanalytical Society (SPPA) among the staff of nursery schools associated with the City Department of Education (SMED). The type of work, its main characteristics, approaches and evolution throughout the four years of implementation are discussed. The construction of a different model of interaction between psychoanalysts and educators is emphasized. They start with the assumption that the primary intervention in childhood can be accomplished by training the professionals who deal with children in their first years. Ongoing discussions with Social Service consultant who has long experience in working with communities with high level of social vulnerability have been essential to this process. Discussions in small groups that enable the sharing of experience have been strengthened to make possible the construction of a space for reflection.

Entre a psicanálise e a educação: diálogos e experiências / Between psychoanalysis and education: dialogues and experiences

Os autores apresentam a evolução de um trabalho inovador que um grupo de psicanalistas da Sociedade Psicanalítica de Porto Alegre (SPPA) está realizando com as Escolas de Educação Infantil Conveniadas da Secretaria Municipal de Educação (SMED). Discutem o modelo de trabalho, com suas principais características , formas de abordagem e a evolução do mesmo ao longo dos quatro anos em que as atividades vêm sendo desenvolvidas. Destacam a construção de um modelo de interação entre psicanalistas e educadores diferente daquele da consultoria escolar tradicional. Portem da hipótese de que intervenção primária na infância pode ser realizada também capacitando os profissionais que se ocupam das crianças nos seus primeiros anos. Tratando-se de comunidades que apresentam alto grau de vulnerabilidade social, tem sido fundamental a discussão permanente com um consultor da área do serviço social com larga experiência no trabalho com estas comunidades. A ênfase tem sido dada na discussão em pequenos grupos para que vivências possam ser compartilhadas com o propósito de criar um espaço de reflexão. (AU)

The authors present the evolution of an innovative work which is being developed by a group of psychoanalysts from the Porto Alegre Psychoanalytical Society (SPPA) among the staff of nursery schools associated with the City Department of Education (SMED). The type of work, its main characteristics, approaches and evolution throughout the four years of implementation are discussed. The construction of a different model of interaction between psychoanalysts and educators is emphasized. They start with the assumption that the primary intervention in childhood can be accomplished by training the professionals who deal with children in their first years. Ongoing discussions with Social Service consultant who has long experience in working with communities with high level of social vulnerability have been essential to this process. Discussions in small groups that enable the sharing of experience have been strengthened to make possible the construction of a space for reflection. (AU)

Rotura del tendón tibial anterior / Tibialis anterior rupture

The tibialis anterior muscle is the main ankle extensor. Its tendon ruptures are very infrecuent, and there are only a few case reports about them in literature. Treatment in these patients must be tailored to their functional activity, being only those who are inactive candidates to orthopedic treatment. In most cases treatment is surgical, by termino-terminal repair, augmentation techniques with extensor hallucis longus (EHL) or tibialis anterior tendon allografting, among other techniques, depending mainly on the type of lesion and the time of evolution. In this case report we present a male 60 year old patient with End Stage Chronic Kidney Disease on Hemodialysis, who after a minor trauma in left foot while on plantar flexion presents acute pain in ankle’s medial aspect and step page gait. In the Emergency Department evaluation he was unable to stand in his heels, despite an adequate foot dorsiflexion. On Magnetic Resonance Imaging a rupture of the distal part of the tibialis anterior tendon was shown. Two weeks later a surgical termino-terminal repair with ethibond was performed, followed by 9 weeks of inmobilization and then rehabilitation. Twelve weeks after the surgical procedure the patient regain his normal activities, without gait limitation.

El músculo tibial anterior es el principal extensor del tobillo. Las roturas de su tendón son muy poco frecuentes y existen en la literatura sólo casos aislados que reportan su existencia. El tratamiento en estos pacientes debe basarse en la actividad funcional de estos, siendo sólo los pacientes inactivos candidatos al tratamiento ortopédico con órtesis. En la mayoría de los casos el tratamiento es quirúrgico, indicándose reparación término-terminal, técnicas de transferencia tendínea con extensor hallucis longus (EHL) o la utilización de aloinjerto entre otras técnicas, dependiendo del tipo de rotura y tiempo de evolución. En este reporte se relata el caso de un hombre de 60 años, con antecedentes de insuficiencia renal crónica, en diálisis, quien al sufrió un trauma menor en flexión plantar del pie izquierdo, presentó dolor agudo en la región medial del tobillo y posteriorincapacidad de extender el tobillo al realizar la marcha. Fue evaluado en el servicio de urgencia destacando la imposibilidad de mantenerse en talones, pese a tener una dorsiflexión adecuada al examen físico. Se solicitó una Resonancia Magnética (RM), donde se evidenció una rotura completa en la región distal del tendón tibial anterior. Se operó a las 2 semanas, realizándose una reparación término-terminal con sutura no reabsorbible. El post operatorio se manejó con inmovilización por 9 semanas y posterior rehabilitación. El paciente no presentó complicaciones, logrando reintegrarse a sus actividades laborales a las 12 semanas.

Transferencia de tibial posterior con procedimiento de "Bridle" modificado en pie paralítico fláccido / Posterior tibial transfer with modified

El pie paralítico fláccido tiene como deformidades más frecuentes el equino, varo y marcha en "steppage". Existen múltiples tratamientos quirúrgicos. Recientemente, Rodríguez describe una modificación del procedimiento de "Bridle", en el cual el tibial posterior es transferido al dorso del pie, con inserción ósea en la cuña media y tenodesado con el peroneo longus y el tibial anterior, a modo de "rienda" balanceada con tracción en tres puntos. Objetivos: Evaluar el resultado funcional de pacientes con pie paralítico fláccido flexible y semiflexible operados con procedimiento de "Bridle" modificado. Material y método: Evaluación retrospectiva de pacientes con pie paralítico fláccido tratados con técnica de "Bridle" modificado en el Hospital Clínico PUC entre agosto/2000 y abril/2004. Resultados:6 pacientes, 4 hombres y 2 mujeres, con 6 pies operados. Edad promedio de 27,6 (18-37) años. Seguimiento promedio de 26 meses. AOFAS pre/post cirugía: 32/76. Satisfacción completa en todos los pacientes (escala Kenneth-Johnson). Cinco pacientes regresaron a trabajar. Retorno parcial a actividad deportiva en 3 de 4 pacientes. Complicaciones locales en 1 caso; sin complicaciones sistémicas. Conclusión: existen pocas publicaciones sobre este procedimiento; nuestros resultados son comparables con la literatura. El procedimiento de "Bridle" modificado permite obtener un pie funcional, estable y plantígrado, con satisfacción completa en todos los pacientes.

Growth hormone-releasing hormone (GHRH) neurons in GHRH-enhanced green fluorescent protein transgenic mice: a ventral hypothalamic network.

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.

G protein-mediated inhibitory effect of a nitric oxide donor on the L-type Ca2+ current in rat ventricular myocytes.

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.

Peroxynitrite is a positive inotropic agent in atrial and ventricular fibres of the frog heart.

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.

Sequential changes in autonomic regulation of cardiac myocytes after in vivo endotoxin injection in rat.

We report that in vivo injection of endotoxin (EDTX, 6 mg. kg(-)(1)) induces cardiovascular alterations in rats that closely mimic the clinical situation, as assessed by in vivo hemodynamic measurements in anesthetized and conscious, chronically instrumented animals. The patch-clamp technique was used to characterize the L-type calcium current (I(Ca)) and its autonomic regulation in isolated cardiac myocytes. The density of I(Ca) progressively decreased at 12 and 36 h after EDTX injection. However, the dihydropyridine (+/-)Bay K 8644 (100 nM) enhanced I(Ca) to levels similar to those in control and EDTX-treated myocytes. In addition, the net stimulatory effect of a beta-adrenergic agonist (isoproterenol) on I(Ca) was increased 12 h after EDTX injection. This change in the beta-adrenergic effect declined 24 h later. The potentiation in the beta-adrenergic stimulation of I(Ca) was mimicked by L858051 (10 microM), a direct activator of adenylyl cyclase, but not by IBMX (200 microM), a phosphodiesterase inhibitor. Besides, the antiadrenergic effect of acetylcholine on I(Ca) was unchanged 12 h after EDTX injection, but increased 36 h after EDTX injection. These results support the hypothesis that time-dependent changes in the adenylyl cyclase pathway in cardiac myocytes may contribute, via the autonomic regulation of I(Ca), to the severity of myocardial dysfunction during sepsis.

Biological effects of C-type natriuretic peptide in human myofibroblastic hepatic stellate cells.

During chronic liver diseases, hepatic stellate cells (HSC) acquire a myofibroblastic phenotype, proliferate, and synthetize fibrosis components. Myofibroblastic HSC (mHSC) also participate to the regulation of intrahepatic blood flow, because of their contractile properties. Here, we examined whether human mHSC express natriuretic peptide receptors (NPR). Only NPR-B mRNA was identified, which was functional as demonstrated in binding studies and by increased cGMP levels in response to C-type natriuretic peptide (CNP). CNP inhibited mHSC proliferation, an effect blocked by the protein kinase G inhibitor 8-(4 chlorophenylthio)-cGMP and by the NPR antagonist HS-142-1 and reproduced by analogs of cGMP. Growth inhibition was associated with a reduction of extracellular signal-regulated kinase and c-Jun N-terminal kinase and with a blockade of AP-1 DNA binding. CNP and cGMP analogs also blunted mHSC contraction elicited by thrombin, by suppressing calcium influx. The relaxing properties of CNP were mediated by a blockade of store-operated calcium channels, as demonstrated using a calcium-free/calcium readdition protocol. These results constitute the first evidence for a hepatic effect of CNP and identify mHSC as a target cell. Activation of NPR-B by CNP in human mHSC leads to inhibition of both growth and contraction. These data suggest that during chronic liver diseases, CNP may counteract both liver fibrogenesis and associated portal hypertension.

Positive and negative inotropic effects of NO donors in atrial and ventricular fibres of the frog heart.

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.

Methylene blue is a muscarinic antagonist in cardiac myocytes.

We studied the mechanism of action of methylene blue (Mblue), a putative guanylyl cyclase inhibitor, on the L-type calcium current (ICa) and the muscarinic activated K+ current (IK,ACh) in rat ventricular and atrial myocytes, respectively, and on the binding of [3H]quinuclidinyl benzylate in rat ventricular membranes. Superfusion, but not internal dialysis, with 30 microM Mblue antagonized the inhibitory effect of acetylcholine (ACh, 1 microM) on beta-adrenergic stimulation of ICa with isoprenaline (Iso, 10 nM or 1 microM). However, Mblue had no effect on the basal ICa or on the stimulation of ICa by Iso in the absence of ACh. The activation of IK,ACh by 3 microM ACh was also antagonized by Mblue in a dose-dependent manner. In contrast, Mblue had no effect on the activation of IK,ACh by either guanosine-5'-O-(3-thio)triphosphate or guanosine-5'-(beta,gamma-imido)triphosphate. Chlorpromazine (CPZ), a piperazine derivative like Mblue, also inhibited the muscarinic activation of IK,ACh in a dose-dependent manner. The specific binding of [3H]QNB, a muscarinic ligand, to rat ventricular membranes was displaced in a dose-dependent manner by Mblue and CPZ. The piperazine derivatives behaved like competitive antagonists of [3H]QNB binding, exhibiting equilibrium dissociation constant (Ki) values of 187 nM for Mblue and 366 nM for CPZ. In conclusion, Mblue exerts antimuscarinic effects on ICa and IK,ACh in rat cardiac myocytes that are best explained by the binding of Mblue to the M2 subtype of muscarinic receptors. This property probably contributes to the antimuscarinic effect of the putative guanylyl cyclase inhibitor reported in previous studies.

A comparative study of the effects of three guanylyl cyclase inhibitors on the L-type Ca2+ and muscarinic K+ currents in frog cardiac myocytes.

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.

Muscarinic regulation of the L-type calcium current in isolated cardiac myocytes.

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.

Binding constants determined from Ca2+ current responses to rapid applications and washouts of nifedipine in frog cardiac myocytes.

1. A fast perfusion system was used to analyse the kinetics of the response of L-type calcium current (ICa) to rapid applications and washouts of the dihydropyridine antagonist nifedipine in whole-cell patch-clamped frog ventricular myocytes. 2. Both the inhibition of ICa induced by nifedipine and the recovery from inhibition upon washout of the drug behaved as mono-exponential functions of time. 3. During application or washout of 100 nM nifedipine, only the peak amplitude of ICa varied but not its time course of activation or inactivation. 4. The rate constant of the onset of ICa inhibition increased with the concentration of nifedipine. However, the time course of the recovery from inhibition was independent of drug concentration. 5. Both rate constants were strongly sensitive to the holding potential but insensitive to the test potential. 6. Using simple rate equations and a one-binding-site analysis it was possible to determine the rate constants for association (k1) and dissociation (k-1) and the equilibrium dissociation constant (KD) of the reaction between nifedipine and Ca2+ channels. KD values for nifedipine were identical to IC50 values obtained from classical steady-state experiments. 7. With depolarized holding potentials, KD decreased strongly due to a large reduction in k-1 and a modest increase in k1. Assuming that these changes result from the distribution of Ca2+ channels between resting and inactivated states, a low-affinity binding to the resting state (R) and a high-affinity binding to the inactivated state (I) were obtained with the binding constants: k1R = 1.0 x 10(6) M-1 S-1, k-1R = 0.077 S-1, and KDR = 77 nM for the resting state; k1I = 4.47 x 10(6) M-1 S-1, k-1I = 7.7 x 10(-4) S-1, and KDI = 0.17 nM for the inactivated state. 8. Rapid application/washout experiments provide a unique way to determine, in an intact cell and in a relatively short period (2-4 min), the binding rate constants and the KD value of the reaction between a dihydropyridine antagonist and the Ca2+ channels.

Nitric oxide synthase does not participate in negative inotropic effect of acetylcholine in frog heart.

In the heart, the parasympathetic neurotransmitter acetylcholine (ACh) reduces the force of contraction. Although the effect of ACh can be partly explained by an inhibition of adenylyl cyclase, some of the effects of ACh may also be mediated via stimulation of nitric oxide synthase (NOS) and production of guanosine 3', 5'-cycle monophosphate (cGMP). NOS inhibitors can prevent the negative chronotropic effect of ACh on spontaneously beating cardiomyocytes and suppress the inhibition of the L-type calcium current (ICa) by ACh in sinoatrial myocytes. This pathway may be relevant not only to the chronotropic effect of ACh but also to its inotropic effect, because ACh, NO, and cGMP regulate the force of contraction and ICa in the cardiac ventricle. Here we report the effects of L-arginine (L-Arg), the substrate of NOS, and NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine (L-NNA), two NOS inhibitors, on muscarinic effects in the cardiac ventricle. We found that L-Arg, L-NMMA, and L-NNA have no effect on the muscarinic inhibition of ICa in isolated frog myocytes. In addition, these compounds have no significant effects on basal ICa or beta-adrenergic stimulation of ICa. L-Arg and its analogues did not change the negative inotropic effect of ACh in frog ventricular fibers. Basal active tension and the positive inotropic effect of isoproterenol, a beta-adrenergic agonist, also were unaffected. We conclude that NOS in not involved in muscarinic inhibition of ICa in isolated from ventricular myocytes or the negative inotropic effect of ACh in the frog ventricle.

[Regulation of cardiac calcium current by cGMP/NO route]. / Régulation du courant calcique cardiaque par la voie du GMPc/NO.

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.

Regulation of myocardial calcium channels by cyclic AMP metabolism.

Hormonal regulation of cardiac inotropism is often correlated with modification of the L-type Ca-channel current. Among several regulatory pathways that control Ca-channel activity, the best described one is the cAMP cascade. Cyclic AMP-dependent phosphorylation of the Ca-channel results in an increase of the mean open probability of the individual Ca-channels and, thus, of the macroscopic Ca current. Modulation of cAMP concentration can take place at the level of adenylyl cyclases or cAMP phosphodiesterases. Of major interest is the fact that the activity of two different forms of phosphodiesterases is controlled by the level of intracellular cGMP. Thus, cAMP metabolism is intimately associated with cGMP metabolism, and both determine the degree of cAMP-dependent phosphorylation of cardiac Ca-channels. This brief discussion will focus on these two levels of control and their relative importance in the cAMP-dependent regulation of myocardial Ca-channels.

Erythro-9-(2-hydroxy-3-nonyl)adenine inhibits cyclic GMP-stimulated phosphodiesterase in isolated cardiac myocytes.

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.