Leandro Bueno Bergantin

Activating cAMP/PKA signaling in skeletal muscle suppresses the ubiquitin-proteasome-dependent proteolysis: implications for sympathetic regulation

Although we have recently demonstrated that plasma catecholamines induce antiproteolytic effects on skeletal muscle (Graça FA, Gonçalves DAP, Silveira WA, Lira EC, Chaves VE, Zanon NM, Garófalo MAR, Kettelhut IC, Navegantes LCC. Am J Physiol Endocrinol Metab. 305: E1483-E1494, 2013), the role of the muscle sympathetic innervation and, more specifically, norepinephrine (NE) in regulating the ubiquitin (Ub)-proteasome system (UPS) remains unknown. Based on previous findings that chemical sympathectomy acutely reduces UPS activity, we hypothesized that muscle NE depletion induces adrenergic supersensitivity in rat skeletal muscles. We report that surgical sympathetic denervation (SDEN), a condition in which only muscle NE from both hindlimbs is depleted, transiently reduced the overall proteolysis and the UPS activity (∼25%) in both soleus and extensor digitorum longus muscles. This antiproteolytic response was accompanied by increased activity of adenylyl cyclase (112%), levels of cyclic adenosine monophosphate (cAMP; 191%), and the serine phosphorylation of cAMP response element-binding protein (32%). In extensor digitorum longus from normal rats, NE (10(-4) M) in vitro increased the levels of cAMP (115%) and the serine phosphorylation of both cAMP response element-binding protein (2.7-fold) and forkhead box class O1 transcription factor. Similar effects were observed in C2C12 cells incubated with forskolin (10 μM). In parallel, NE significantly reduced the basal UPS (21%) activity and the mRNA levels of atrophy-related Ub-ligases. Similar responses were observed in isolated muscles exposed to 6-BNZ-cAMP (500 μM), a specific PKA activator. The phosphorylation levels of Akt were not altered by SDEN, NE, forskolin or 6-BNZ-cAMP. Our results demonstrate that SDEN induces muscle adrenergic supersensitivity for cAMP leading to the suppression of UPS, and that the suppressive effects of NE on UPS activity and expression of Ub-ligases can be mediated by the activation of cAMP/PKA signaling, with the inhibition of forkhead box class O1 transcription factor.

Pharmacological implications of the Ca2+/cAMP signaling interaction: from risk for antihypertensive therapy to potential beneficial for neurological and psychiatric disorders

In this review, we discussed pharmacological implications of the Ca2+/cAMP signaling interaction in the antihypertensive and neurological/psychiatric disorders therapies. Since 1975, several clinical studies have reported that acute and chronic administration of L‐type voltage‐activated Ca2+ channels (VACCs) blockers, such as nifedipine, produces reduction in peripheral vascular resistance and arterial pressure associated with an increase in plasma noradrenaline levels and heart rate, typical of sympathetic hyperactivity. Despite this sympathetic hyperactivity has been initially attributed to adjust reflex of arterial pressure, the cellular and molecular mechanisms involved in this apparent sympathomimetic effect of the L‐type VACCs blockers remained unclear for decades. In addition, experimental studies using isolated tissues richly innervated by sympathetic nerves (to exclude the influence of adjusting reflex) showed that neurogenic responses were completely inhibited by L‐type VACCs blockers in concentrations above 1 μmol/L, but paradoxically potentiated in concentrations below 1 μmol/L. During almost four decades, these enigmatic phenomena remained unclear. In 2013, we discovered that this paradoxical increase in sympathetic activity produced by L‐type VACCs blocker is due to interaction of the Ca2+/cAMP signaling pathways. Then, the pharmacological manipulation of the Ca2+/cAMP interaction produced by combination of the L‐type VACCs blockers used in the antihypertensive therapy, and cAMP accumulating compounds used in the antidepressive therapy, could represent a potential cardiovascular risk for hypertensive patients due to increase in sympathetic hyperactivity. In contrast, this pharmacological manipulation could be a new therapeutic strategy for increasing neurotransmission in psychiatric disorders, and producing neuroprotection in the neurodegenerative diseases.

Autophagy and Alzheimer’s Disease: From Molecular Mechanisms to Therapeutic Implications

Alzheimer’s disease (AD) is the most common cause of progressive dementia in the elderly. It is characterized by a progressive and irreversible loss of cognitive abilities and formation of senile plaques, composed mainly of amyloid β (Aβ), and neurofibrillary tangles (NFTs), composed of tau protein, in the hippocampus and cortex of afflicted humans. In brains of AD patients the metabolism of Aβ is dysregulated, which leads to the accumulation and aggregation of Aβ. Metabolism of Aβ and tau proteins is crucially influenced by autophagy. Autophagy is a lysosome-dependent, homeostatic process, in which organelles and proteins are degraded and recycled into energy. Thus, dysfunction of autophagy is suggested to lead to the accretion of noxious proteins in the AD brain. In the present review, we describe the process of autophagy and its importance in AD. Additionally, we discuss mechanisms and genes linking autophagy and AD, i.e., the mTOR pathway, neuroinflammation, endocannabinoid system, ATG7, BCL2, BECN1, CDK5, CLU, CTSD, FOXO1, GFAP, ITPR1, MAPT, PSEN1, SNCA, UBQLN1, and UCHL1. We also present pharmacological agents acting via modulation of autophagy that may show promise in AD therapy. This review updates our knowledge on autophagy mechanisms proposing novel therapeutic targets for the treatment of AD.

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

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 β-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.

Advances for the pharmacotherapy of depression - Presenting the rising star: Ca2+/camp signaling interaction

The pharmacotherapy for treating depression is clearly limited. The classical pharmacotherapy that exists nowadays was developed decades ago. Indeed, depression is characterized by reduction of neurotransmitters release in central nervous system, mainly serotonin and catecholamines. Besides, since 70 ́s several clinical studies have reported that administration of Ca2+ channel blockers (CCBs) produces reduction in vascular resistance associated with an increase in sympathetic hyperactivity, originated from increase of neurotransmitter release. In 2010 ́s, we discovered that this paradoxical sympathetic hyperactivity produced by CCBs is mediated by Ca2+/cAMP signalling interaction (Ca2+/cAMP interaction) acting by increasing neurotransmitter release. Considering this interaction has been postulated to be universally-operated in neuroendocrine cells, the pharmacological handling of the Ca2+/cAMP interaction is a clear rising star for increasing neurotransmitter release, including serotoninergic and monoaminergic neurotransmission in depression. This mini review compiles our previous results published in 7 cited international papers, herein discussing novel strategies to treat depression, throughout our discovery entitled “calcium paradox” phenomenon due to interaction of Ca2+/cAMP intracellular signalling pathways. Correspondence to: Department of Pharmacology – Universidade Federal de São Paulo – Escola Paulista de Medicina, Laboratory of Autonomic and Cardiovascular Pharmacology – 55 11 5576-4973, Rua Pedro de Toledo, 669 – Vila Clementino, São Paulo – SP, Brazil, CEP: 04039-032, E-mail: leanbio39@ yahoo.com.br Received: January 20, 2017; Accepted: April 24, 2017; Published: April 27, 2017 Introduction The treatment of depression by current pharmacotherapy is clearly restricted. The classical pharmacotherapy that exists in the present was developed decades ago. Depression is a psychiatric disease resulting mainly by dysfunction of monoaminergic neurotransmission in central nervous system [1,2]. Depression is a severe global illness, becoming more and more common each decade. Because of specific symptoms, it is considered as a leading cause of disability all over the world with a high death factor due to suicides. There are many antidepressants used in the pharmacotherapy, but still more than one-third of patients do not respond to the current therapy [2]. In addition to the monoaminergic hypothesis of depression, there are also many other approaches connected with the pathophysiology of this disease, including hypothalamic-pituitary-adrenal axis dysregulation, dopaminergic, cholinergic, glutamatergic or GABAergic neurotransmission [2]. Complementing these hypotheses, the neurotrophic one is also emerging. The preclinical and clinical data, and imaging studies, support a neurotrophic hypothesis of depression, and antidepressant responses [3,4]. This hypothesis proposes that depression results from decreased neurotrophic support, leading to neuronal atrophy, decreased hippocampal neurogenesis and loss of glia. It is proposed that antidepressant treatment blocks, or reverses, this neurotrophic factor deficit, and thereby reverses the atrophy and cell loss [3,4]. Nevertheless, it can be unambiguously stated that serotonergic, noradrenergic and dopaminergic systems (monoaminergic neurotransmission) are clearly associated with pathogenesis of depression, and are therefore considered as valuable targets in patients’ treatment. In this mini review, we discuss novel strategies to treat depression, throughout our recent discovery entitled “calcium paradox” phenomenon due to interaction of Ca2+/cAMP intracellular signalling pathways [1,5-10]. Current hypothesis for depression and its pharmacotherapy Depression is a debilitating psychiatric condition that causes a significant problem on human health. There is still a deficiency of a clear comprehension of the neuropathological changes associated with this illness, and the efficacy of antidepressants is very limited [11]. Research into antidepressant therapies has derived from observations in human trials and animal models after the first monoaminergic hypothesis emerged decades ago. In addition, glutamatergic modulators, such as ketamine also have become the forefront of antidepressant exploration, especially for treatment-resistant depression and suicidal ideation [11]. The glutamatergic hypothesis of depression is not novel, however other NMDA receptor modulators do not seem to share the rapid and sustained effects of ketamine, suggesting that a unique combination of intracellular targets might be involved in its effect [11,12]. Nonetheless, the monoamine hypothesis of depression continues to lead the field and clinical trials, which postulates that an imbalance in monoaminergic neurotransmission is causally related to the clinical features of depression [12]. Antidepressants influence serotonin whose mainly goal consist at raising serotonin concentrations, thereby increasing serotonergic transmission at the level of the synapse, for example by inhibiting the serotonin transporter. However, the serotonin system is multifaceted. Different serotonin receptor subtypes turn the serotonergic system into a complex neurochemical arrangement that Bergantin LB (2017) Advances for the pharmacotherapy of depression Presenting the rising star: Ca2+/camp signaling interaction J Syst Integr Neurosci, 2017 doi: 10.15761/JSIN.1000161 Volume 3(3): 2-5 influences diverse neurotransmitters in various brain regions. Classical antidepressants, as well as other psychopharmacological agents have various crucial effects on serotonin receptors. Researchers aim to provide a useful characterization of serotonin receptor subtypes in the treatment of depression. Clarifying the mode of action and the interplay of serotonin receptors with pharmacological agents should help elucidate antidepressant mechanisms and typical side effects to better understanding. In addition, clinical medicine featured the novel antidepressants vortioxetine, vilazodone and milnacipran/ levomilnacipran with regard to their serotonin receptor targets such as the 5-HT1A, 5-HT3 and 5-HT7, which may account for their specific effects on certain symptoms of depression as well as a characteristic side-effect profile [12]. The combination of novel ideas added to improvements on the discoveries may lead to advances in antidepressant research field with the promise of finding pharmaceuticals more effective. Thus, new visions for more efficient pharmacological treatments of depression are clearly needed. Presenting the rising star: Ca2+/cAMP signalling interaction Although the cellular and molecular mechanisms involved in the synergistic actions of cAMP on the exocytosis of neurotransmitter and hormones remain under debate, the evidences suggest that this intracellular messenger can participate in the regulation of exocytosis due to its modulatory action on the intracellular Ca2+ signals. Indeed, this concept was initially resulted from the study of cat adrenal gland perfused with acetylcholine executed by Douglas and Rubin in the 1960s [13]. The discovery that increase in the cytosolic Ca2+ concentration ([Ca2+] c) was a basic requirement for exocytosis in adrenal catecholaminergic cells was made by Baker and Knight in 1970 ́s [14]. In addition, some studies showed that cAMP rises transmitter release at many synapses in autonomic nervous system of vertebrate, including sympathetic and parasympathetic ganglion neurons [15]. The hypothesis for an interaction between the intracellular signalling pathways mediated by Ca2+ and cAMP (Ca2+/cAMP interaction) has been extensively studied in many cells and tissues. Generally, this interaction results in synergistic effects on cell functions [1,5,6,16,17] and occurs at the level of adenylyl cyclases (ACs) or phosphodiesterases (PDEs) (Figure 1). The Ca2+/cAMP interaction has particularly been extensively studied at the Ca2+ channels [e.g.: ryanodine receptors (RyR)] of the endoplasmic reticulum (ER) [1,5,6,16,17]. Phosphorylation of RyR by protein kinase A (PKA), and also inositol trisphosphate receptor (IP3R) at submaximal IP3 concentrations, may increase the open probability of ER Ca 2+ stores, amplifying Ca2+-induced Ca2+ release (CICR) mechanism and cellular responses [1,5,6] (Figure 1). Recent evidences suggest that Ca2+/ cAMP interaction participates in exocytosis regulation of neurons and neuroendocrine cells [1,5,6]. Then, dysfunctions of cellular homeostasis of Ca2+ and/or cAMP in these cells could result in the dysregulation of Ca2+/cAMP interaction and exocytotic response, or could be a novel therapeutic target for medicines (Figure 1). Since 1975, nifedipine and verapamil, classical L-type Ca2+ channel blockers (CCBs) have been reported to produce reduction in peripheral vascular resistance and arterial pressure associated with an increase in plasma noradrenaline levels and heart rate, typical signals of sympathetic hyperactivity [18]. However, the cellular and molecular mechanisms involved in this apparent sympathomimetic effect of the L-type CCBs remained unclear for decades. In addition, experimental studies using isolated tissues richly innervated by sympathetic nerves showed that neurogenic responses were completely inhibited by L-type CCBs in high concentrations (>1 μmol/L), but paradoxically potentiated in concentrations below 1 μmol/L [19-21]. During almost four decades, these enigmatic phenomena named by us as “calcium paradox” remained unclear. In 2013, we discovered that this paradoxical increase in sympathetic activity produced by L-type CCBs is due to Ca2+/cAMP interaction [5]. Then, the pharmacological manipulation of the Ca2+/ cAMP interaction produced by combination of the L-type CCBs used in the antihypertensive therapy, and cAMP accumulating compounds used in the anti-depressive therapy such as rolipram, could represent a potential cardiovascular risk for hyperte