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 diffi cult 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 refl ex 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 chromaffi n 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 effi cient (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. Mini Review


INTRODUCTION
Depression is a psychiatric disease resulting mainly by dysfunction of serotoninergic and monoaminergic neurotransmission in central nervous system (CNS) [1,2]. Depression is a severe global illness, becoming more and more common each decade. Because of speci ic 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 therapy, but still more than one-third of patients do not respond to the current therapy [2]. The heterogeneous nature of the illness and its complex, and unclear etiology, may be responsible for treatment dif iculties. Next to the main monoaminergic hypothesis of depression, there are also many other approaches connected with the pathophysiology of this disease, including hypothalamic-pituitaryadrenal axis dysregulation, dopaminergic, cholinergic, glutamatergic or GABA-ergic neurotransmission [2]. Nevertheless, it can be unambiguously stated that serotonergic, noradrenergic and dopaminergic systems (monoaminergic neurotransmission) are precisely associated with pathogenesis of depression, and should be therefore considered as valuable targets in patients' treatment. In this review, we discuss novel strategies to treat depression, throughout our recent discovery entitled "calcium paradox" phenomenon due to interaction between the intracellular signalling pathways mediated by Ca 2+ and cAMP (Ca 2+ /cAMP signaling interaction) [1,[3][4][5][6][7][8].

CURRENT THERAPY TO TREAT DEPRESSION
Depression is an incapacitating psychiatric condition that causes a signi icant problem on individuals and society. There is still a lack of a clear understanding of the neuropathological changes associated with this illness, and the ef icacy of antidepressants is still far from the best [9]. Research into antidepressant therapies has derived from observations in human trials and animal models after the irst monoaminergic hypothesis emerged (about six decades ago). However, glutamatergic modulators, such as ketamine also have become the forefront of antidepressant exploration, especially for treatment-resistant depression and suicidal ideation [9]. 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 [9]. Interestingly, in lammation can impact the glutamatergic system enhancing excitotoxicity and decreasing neuroplasticity. The points of convergence between the in lammatory and glutamatergic hypotheses of depression are not completely established, especially regarding the effects of fast-acting antidepressants [9,10].
Nonetheless, the monoamine hypothesis of depression continues to dominate the ield and clinical trials, which postulates that an imbalance in monoaminergic neurotransmission is causally related to the clinical features of depression [10]. Anti-depressants in luence serotonin whose main goal consist to raise 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 in luences 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 speci ic effects on certain symptoms of depression as well as a characteristic side-effect pro ile [10].
The combination of novel ideas added to improvements on the discoveries may lead to advances in antidepressant research with the promise of inding compounds that are both effective, and fast-acting, including in patients who have tried other therapies with limited success. In conclusion, new insights for more ef icient pharmacological treatments of depression are clearly needed.

CA 2+ /CAMP SIGNALING INTERACTION: A BRIEF REVIEW
Several experiments initiated decades ago, using catecholaminergic cells, have originated the concept of stimulus-secretion coupling to elucidate neurotransmitter release and hormone secretion. This concept has been initially resulted from the study of cat adrenal gland perfused with acetylcholine executed by Douglas and Rubin in the 1960s [11]. The discovery that increase in the cytosolic Ca 2+ concentration ([Ca 2+ ]c) was a basic requirement for exocytosis in adrenal catecholaminergic cells was made by Baker and Knight in 1970´s [12]. In addition, some studies showed that cAMP raises transmitter release at many synapses in autonomic nervous system of vertebrate, including sympathetic and parasympathetic ganglion neurons [13]. Although the cellular and molecular mechanisms involved in these synergistic actions of cAMP on the exocytosis of neurotransmitter and hormones remain uncertain, the evidences suggest that this intracellular messenger can participate in ine regulation of exocytosis due to its modulatory action on the intracellular Ca 2+ signals.
In fact, the hypothesis for the Ca 2+ /cAMP signaling interaction has been extensively studied in many cells and tissues. Generally, this interaction results in synergistic effects on cell functions [1,[3][4][5][6][7][8]14,15] and occurs at the level of adenylyl cyclases (AC) or phosphodiesterases (PDE) (Figure 1). The Ca 2+ /cAMP signaling interaction has particularly been extensively studied at the Ca 2+ channels [e.g.: ryanodine receptors (RyR)] of the endoplasmic reticulum (ER) [1,[3][4][5][6][7][8]14,15]. Phosphorylation of RyR by protein kinase A (PKA), and also inositol trisphosphate receptor (IP 3 R) at submaximal IP 3 concentrations, may increase the open probability of ER Ca 2+ stores, amplifying Ca 2+ -induced Ca 2+ release (CICR) mechanism and cellular responses ( Figure 1). Indeed, the concept of the complex cAMP-IP 3 R interaction, which may mediate the synergistic action of Ca 2+ and cAMP signalling, is now emerging [16]. Recent data suggest that IRBIT (IP3 receptors binding protein release with IP3) may become central-stage in the mechanism mediating the synergism between cAMP and Ca 2+ signalling pathways by functioning as a "third messenger", which favours the crosstalk between IP 3 Rs and other proteins. Another central component is the classical PKA phosphorylation of IP 3 Rs. For this, IP 3 Rs, IRBIT, PKA and the effector proteins have to be assembled into microdomains to allow the ef iciency of IRBIT. In resting cells, when cellular IP 3 levels are low, IRBIT is bound to IP 3 Rs; thus IP 3 Rs work to buffer the availability of free IRBIT [16]. Increases in cAMP levels may lead to dissociation of IRBIT from IP 3 receptors, and its translocation to effector proteins, located either at intracellular organelles and/ or the plasma membrane; in this manner, IRBIT functions as a "third messenger" that transmits the information carried out by the second messenger's cAMP and IP 3 . At the same time, IRBIT integrates, and synergizes, the activity of the cAMP and Ca 2+ signalling systems, providing a molecular mechanism for the synergistic action between them.
Recent evidences suggest that Ca 2+ /cAMP signaling interaction participates in exocytosis regulation of neurons and neuroendocrine cells [1,[3][4][5][6][7][8]. Then, dysfunctions of cellular homeostasis of Ca 2+ and/or cAMP in these cells could result in the dysregulation of Ca 2+ /cAMP signaling interaction and exocytotic response, or could be a novel therapeutic target for medicines ( Figure 1).

PARADOXICAL EFFECTS OF CCBS AND THEIR PLEIOTROPIC EFFECTS IN DEPRESSION
Since 1975, several clinical studies have been reporting that acute and chronic administration of L-type Ca 2+ channel blockers (CCBs), such as nifedipine and verapamil, produces reduction in peripheral vascular resistance and arterial pressure associated with an increase in plasma noradrenaline levels and heart rate, typical signals of sympathetic hyperactivity [17]. 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 [18][19][20]. 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 Ca 2+ /cAMP signaling interaction [3]. Then, the pharmacological manipulation of the Ca 2+ /cAMP signaling interaction produced by combination of the L-type CCBs used in the antihypertensive therapy, and cAMP accumulating compounds used in the antidepressive therapy such as rolipram, 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 the psychiatric disorders, such as depression.
In addition, several studies have been demonstrating pleiotropic effects of CCBs. CCBs, like nifedipine, genuinely potentiate the effect of tricyclic and atypical antidepressants [21]. However, the molecular mechanisms involved in these pleiotropic effects remain under debate. In fact, apart from its classical functions, CCBs are described to have bene iciary roles on the cognitive pro ile of the aged population and individuals with hypertension, diabetes, Parkinson's disease, and Alzheimer's disease [22][23][24][25]. Different mechanisms have been proposed, but the exact mechanisms of antidepressant effects and cognitive improvement are still uncertain.
In contrast to adverse effects produced by combination of L-type CCBs with cAMPenhancer compounds in the cardiovascular diseases, the pharmacological implications of the Ca 2+ /cAMP signaling interaction produced by this drug combination could be used to enhance neurotransmission [3][4][5][6][7][8]. Recent studies have showed that chronic treatment with rolipram, together with typical antidepressants has been successful in the reduction of depression symptoms due to potentiation of these antidepressants effects [26]. Considering our model in which increment of [cAMP]c stimulates Ca 2+ release from ER (Figure 1), it may be plausible that the therapeutic use of the PDE inhibitor rolipram [27,28], in combination with low doses of verapamil to potentiate neurotransmission (Figure 1) in the areas of central nervous system involved in neurological/psychiatric disorders in which neurotransmission is reduced, including depression. This new pharmacological strategy for the treatment of psychiatric disorders could increase the therapeutic ef icacy and reduce the adverse effects of the medicines currently used for treating depression. Considering that CCBs genuinely exhibit cognitive-enhancing abilities and reduce the risk of psychiatric disorders like depression [29][30][31] and that the mechanisms involved in these pleiotropic effects are largely unknown. Then, whether Ca 2+ /cAMP signaling interaction is involved in such effects deserves special attention.
In addition, considering [Ca 2+ ]c elevation could contribute to both: negatively to neuroprotective effects and positively to exocytosis, it may be plausible the therapeutic use of the PDE inhibitors [26,27] for antidepressant purposes. Then, pharmacological interference of the Ca 2+ /cAMP signaling interaction produced by combination of L-type CCBs and cAMP-enhancer compounds could enhance antidepressant response and reduce clinical symptoms of psychiatric disorders. Thus, the association of currently medicines could enhance antidepressant treatments. For example: the association of typical antidepressants with CCBs or rolipram could dramatically improve typical antidepressant medicines, mainly by reducing their adverse effects and increasing their effectiveness. This new pharmacological strategy could be alternatively used for treatment of the symptoms of psychiatric disorders, including depression. Finally, special attention should be given in hypertensive patients, when treating depression by interfering on Ca 2+ /cAMP signaling interaction, considering this new therapy could make hypertension worse.

CONCLUSION
The diagnosis of psychiatric disorders like depression relies critically on collaborative history of patients. In addition, emerging therapies may supplement clinical assessment in the next years. Although pharmacological therapies have been largely unsuccessful in curing depression, targeting potential risk factors aiming to decrease incidence of this psychiatric disorder is an important public health edge. Finally, novel strategies to treat depression, throughout our recent discovery entitled "calcium paradox" phenomenon due to Ca 2+ /cAMP signaling interaction, could greatly contribute to enhance therapeutic strategies for increasing serotoninergic and monoaminergic neurotransmission. Thus, the association of typical antidepressants with cAMP-enhancer compounds such as rolipram could dramatically improve antidepressant therapies, mainly by reducing adverse effects and improving effectiveness of these typical antidepressants.