The lumbrical muscle: a novel in situ system to evaluate adult skeletal muscle proteolysis and anticatabolic drugs for therapeutic purposes

Bergantin LB, Figueiredo LB, Godinho RO. the lumbrical muscle: a novel in situ system to evaluate adult skeletal muscle proteolysis and anticatabolic drugs for therapeutic purposes. J Appl Physiol 111: 1710-1718, 2011. First published September 15, 2011; doi:10.1152/japplphysiol.00586.2011.-The molecular regulation of skeletal muscle proteolysis and the pharmacological screening of anticatabolic drugs have been addressed by measuring tyrosine release from prepubertal rat skeletal muscles, which are thin enough to allow adequate in vitro diffusion of oxygen and substrates. However, the use of muscle at accelerated prepubertal growth has limited the analysis of adult muscle proteolysis or that associated with aging and neurodegenerative diseases. Here we established the adult rat lumbrical muscle (4/hindpaw; 8/rat) as a new in situ experimental model for dynamic measurement of skeletal muscle proteolysis. By incubating lumbrical muscles attached to their individual metatarsal bones in Tyrode solution, we showed that the muscle proteolysis rate of adult and aged rats (3-4 to 24 mo old) is 45-25% of that in prepubertal animals (1 mo old), which makes questionable the usual extrapolation of proteolysis from prepubertal to adult/senile muscles. While acute mechanical injury or 1- to 7-day denervation increased tyrosine release from adult lumbrical muscle by up to 60%, it was reduced by 20-28% after 2-h incubation with beta-adrenoceptor agonists, forskolin or phosphodiesterase inhibitor IBMX. Using inhibitors of 26S-proteasome (MG132), lysosome (methylamine), or calpain (E64/leupeptin) systems, we showed that ubiquitin-proteasome is accountable for 40-50% of total lumbrical proteolysis of adult, middle-aged, and aged rats. in conclusion, the lumbrical model allows the analysis of muscle proteolysis rate from prepubertal to senile rats. By permitting eight simultaneous matched measurements per rat, the new model improves similar protocols performed in paired extensor digitorum longus (EDL) muscles from prepubertal rats, optimizing the pharmacological screening of drugs for anticatabolic purposes.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Div Cellular Pharmacol, Dept Pharmacol, Escola Paulista Med, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Div Cellular Pharmacol, Dept Pharmacol, Escola Paulista Med, BR-04044020 São Paulo, BrazilFAPESP: 05/59006-1FAPESP: 08/55988-2CNPq: 304602/2008-6Web of Scienc

Heighpubs Journal of Clinical Hypertension Insights for Antihypertensive pharmacotherapy from the"Calcium Paradox" due to Ca 2+ /camp Interaction

ABSTRACT Several experimental studies performed since 1975, using smooth muscles richly innervated by sympathetic nerves to exclude the autonomic infl uence of adjusting refl ex (rodent vas deferens), showed that L-type voltageactivated Ca 2+ channels (VACC) blockers completely inhibited neurogenic contractions induced by electrical fi eld stimulation (EFS) in high concentrations (>10 -6 M), but paradoxically increased these EFS-contractions in low concentrations (<10 -6 M), suggesting that other mechanisms than only autonomic adjusting refl ex are involved in these paradoxical effects. In 2013, we showed that these paradoxical effects of L-type VACC blockers, named by us "calcium paradox" phenomenon, were potentiated by drugs which increase cytosolic cAMP concentration ([cAMP] c-enhancers), such as rolipram, IBMX and forskolin, indicating that this sympathetic hyperactivity druginduced is due to interaction of the Ca 2+ /cAMP intracellular signaling pathways (Ca 2+ /cAMP interaction). Then, the pharmacological manipulation of this interaction produced by combination of the L-type VACC blockers used in the antihypertensive therapy, and [cAMP] c-enhancers used in the antidepressive therapy, could represent a potential cardiovascular risk for hypertensive patients due to sympathetic hyperactivity. Then, we discussed the role of Ca 2+ /cAMP interaction for antihypertensive pharmacotherapy

Journal of Neuroscience and Neurological Disorders The "Calcium Paradox" Due To Ca 2+ / Camp Interaction: New Insights for the Neuroscience Field

ABSTRACT In the cardiovascular fi eld, tachycardia and increment of catecholamine plasma levels (sympathetic hyperactivity) have been reported by hypertensive patients that use L-type Ca 2+ channel blockers (CCBs) since 70´s. Our discovery of the involvement of interaction between the intracellular signalling pathways mediated by Ca 2+ and cAMP (Ca 2+ /cAMP interaction) revealed that this phenomenon (sympathetic hyperactivity) was resulting of increase of transmitter release from sympathetic neurons stimulated by CCBs due to its interference on the Ca 2+ /cAMP interaction. In the neuroscience fi eld, this discovery has produced new paths for the understanding of the cellular and molecular mechanisms involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer´s and Parkinson's diseases. In this way, novel journeys for the development of new pharmacological strategies more effective for the treatment of neurodegenerative diseases may be initiated

Novo modelo para o paradoxo de cálcio na transmissão simpática de músculos lisos: papel da via amp cíclico

Está bem estabelecido que a redução do influxo de Ca2+ através de canais de Ca2+ voltagem-dependentes do tipo L (VDCC), ou aumento da concentração citosólica de AMPc ([AMPc]c), inibem a atividade contrátil do músculo liso em resposta a transmissores liberados dos nervos simpáticos. Objetivos: Surpreendentemente, neste trabalho observamos que a administração simultânea de bloqueador VDCC (verapamil) e incrementadores de [AMPc]c (rolipram, IBMX e forscolina) potencializou a atividade contrátil de músculo liso de ducto deferente de rato, ao invés de apresentar uma forte inibição. Estes resultados, incluindo o seu papel na transmissão simpática, podem ser considerados como um "paradoxo de cálcio". Métodos/Resultados: Por outro lado, esta potencialização foi atenuada pela redução de [AMPc]c via inibição da adenilil ciclase (SQ 22536) ou pelo esgotamento de armazenagem de Ca2+ no retículo endoplasmático através do bloqueio da recaptação de Ca2+ (tapsigargina). Além disso, a concentração citosólica de Ca2+ ([Ca2+]c) foi avaliada por microscopia de fluorescência em fatias de adrenais, a qual foi significativamente reduzida por verapamil ou por rolipram. Em contraste, a incubação simultânea destes fármacos em fatias adrenais aumentou significativamente [Ca2+ ]c. Este efeito foi atenuado pela tapsigargina. Discussão: Assim, uma redução de [Ca2+ ]c devido ao bloqueio do influxo de Ca2+ através de VDCC poderia estimular a atividade da adenilil ciclase, aumentando [AMPc]c que, em resposta, estimularia a liberação de Ca2+ do retículo endoplasmático, resultando na liberação de transmissores em nervos simpáticos, e incrementando assim a contração. Conclusões: Em conjunto, nossos resultados sugerem que a interação entre as vias de sinalização celular do Ca2+ e AMPc poderia ser importante na regulação da liberação de transmissores a partir de terminações nervosas, e disfunções dessa interação induzidas por tratamentos farmacológicos poderiam estar envolvidas em efeitos adversos do uso combinado de antihipertensivos e antidepressivos.Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

The “Calcium Paradox” Due To Ca2+/Camp Interaction: New Insights for the Neuroscience Field

In the cardiovascular field, tachycardia and increment of catecholamine plasma levels (sympathetic hyperactivity) have been reported by hypertensive patients that use L-type Ca2+ channel blockers (CCBs) since 70´s. Our discovery of the involvement of interaction between the intracellular signalling pathways mediated by Ca2+ and cAMP (Ca2+/cAMP interaction) revealed that this phenomenon (sympathetic hyperactivity) was resulting of increase of transmitter release from sympathetic neurons stimulated by CCBs due to its interference on the Ca2+/cAMP interaction. In the neuroscience field, this discovery has produced new paths for the understanding of the cellular and molecular mechanisms involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer´s and Parkinson’s diseases. In this way, novel journeys for the development of new pharmacological strategies more effective for the treatment of neurodegenerative diseases may be initiated

Novel Challenges for the Therapeutics of Depression: Pharmacological Modulation of Interaction between the Intracellular Signaling Pathways Mediated by Ca2+ and cAMP

Depression is a psychiatric disease resulting mainly by dysfunction of serotoninergic and monoaminergic neurotransmission in central nervous system (CNS). Due to the multifaceted nature of depression and our limited understanding on its etiology, depression is difficult to be treated with currently available pharmaceuticals. Then, new therapeutic strategies for depression have been proposed. Since 1975, several clinical studies have reported that L-type Ca2+ channel blockers (CCBs), used in anti-hypertensive therapy, produce increase of plasma catecholamine levels and tachycardia, typical symptoms of sympathetic hyperactivity. Despite these adverse effects of CCBs have been initially attributed to adjust reflex of arterial pressure, during almost four decades these enigmatic phenomena remained unclear. In 2013, we discovered that this paradoxical sympathetic hyperactivity produced by CCBs results from the increase of catecholamines release from sympathetic nerves, and adrenal chromaffin cells, due to its modulatory action on the interaction between intracellular signaling pathways mediated by Ca2+ and cAMP (Ca2+/cAMP signalling interaction). Then, the pharmacological modulation of this interaction by combined use of L-type CCBs, and cAMP-enhancer compounds, could be a more efficient (and safer) therapeutic strategy to produce increase of serotoninergic and monoaminergic neurotransmission in the CNS due to enhance of serotonin and monoamines release, thus attenuating clinical symptoms of depression in humans.&nbsp