Norepinephrine exerts powerful influences on the metabolic, neuroprotective and immunoregulatory functions of astrocytes. Until recently, all effects of norepinephrine were believed to be mediated by receptors localized exclusively to the plasma membrane. However, recent studies in cardiomyocytes have identified adrenergic receptors localized to intracellular membranes, including Golgi and inner nuclear membranes, and have shown that norepinephrine can access these receptors via transporter-mediated uptake. We recently identified a high-capacity norepinephrine transporter, organic cation transporter 3 (OCT3), densely localized to outer nuclear membranes in astrocytes, suggesting that adrenergic signaling may also occur at the inner nuclear membrane in these cells. Here, we used immunofluorescence and western blot to show that β1 -adrenergic receptors are localized to astrocyte inner nuclear membranes; that key adrenergic signaling partners are present in astrocyte nuclei; and that OCT3 and other catecholamine transporters are localized to astrocyte plasma and nuclear membranes. To test the functionality of nuclear membrane β1 -adrenergic receptors, we monitored real-time protein kinase A (PKA) activity in astrocyte nuclei using a fluorescent biosensor. Treatment of astrocytes with norepinephrine induced rapid increases in PKA activity in the nuclear compartment. Pretreatment of astrocytes with inhibitors of catecholamine uptake blocked rapid norepinephrine-induced increases in nuclear PKA activity. These studies, the first to document functional adrenergic receptors at the nuclear membrane in any central nervous system cell, reveal a novel mechanism by which norepinephrine may directly influence nuclear processes. This mechanism may contribute to previously described neuroprotective, metabolic and immunoregulatory actions of norepinephrine.

A sharp increase in mitochondrial Ca2+ marks the activation of brown adipose tissue (BAT) thermogenesis, yet the mechanisms preventing Ca2+ deleterious effects are poorly understood. Here, we show that adrenergic stimulation of BAT activates a PKA-dependent mitochondrial Ca2+ extrusion via the mitochondrial Na+/Ca2+ exchanger, NCLX. Adrenergic stimulation of NCLX-null brown adipocytes (BA) induces a profound mitochondrial Ca2+ overload and impaired uncoupled respiration. Core body temperature, PET imaging of glucose uptake and VO2 measurements confirm a thermogenic defect in NCLX-null mice. We show that Ca2+ overload induced by adrenergic stimulation of NCLX-null BAT, triggers the mitochondrial permeability transition pore (mPTP) opening, leading to a remarkable mitochondrial swelling and cell death. Treatment with mPTP inhibitors rescue mitochondrial function and thermogenesis in NCLX-null BAT, while calcium overload persists. Our findings identify a key pathway through which BA evade apoptosis during adrenergic stimulation of uncoupling. NCLX deletion transforms the adrenergic pathway responsible for thermogenesis activation into a death pathway.

Infarct size and brain edema following ischemia/reperfusion are reduced by inhibitors of the Na+, K+, 2Cl-, and water cotransporter NKCC1 and by β1-adrenoceptor antagonists. NKCC1 is a secondary active transporter, mainly localized in astrocytes, driven by transmembrane Na+/K+ gradients generated by the Na+,K+-ATPase. The astrocytic Na+,K+-ATPase is stimulated by small increases in extracellular K+ concentration and by the β-adrenergic agonist isoproterenol. Larger K+ increases, as occurring during ischemia, also stimulate NKCC1, creating cell swelling. This study showed no edema after 3 hr medial cerebral artery occlusion but pronounced edema after 8 hr reperfusion. The edema was abolished by inhibitors of specifically β1-adrenergic pathways, indicating failure of K+-mediated, but not β1-adrenoceptor-mediated, stimulation of Na+,K+-ATPase/NKCC1 transport during reoxygenation. Ninety percent reduction of extracellular Ca2+ concentration occurs in ischemia. Ca2+ omission abolished K+ uptake in normoxic cultures of astrocytes after addition of 5 mM KCl. A large decrease in ouabain potency on K+ uptake in cultured astrocytes was also demonstrated in Ca2+-depleted media, and endogenous ouabains are needed for astrocytic K+ uptake. Thus, among the ionic changes induced by ischemia, the decrease in extracellular Ca2+ causes failure of the high-K+-stimulated Na+,K+-ATPase/NKCC1 ion/water uptake, making β1-adrenergic activation the only stimulus and its inhibition effective against edema.

New data on the pharmacology of tricyclic antidepressants (TCAs), their affinities for human cloned CNS receptors and their cytochrome P450 enzyme inhibition profiles, allow improved deductions concerning their effects and interactions and indicate which of the TCAs are the most useful. The relative toxicity of TCAs continues to be more precisely defined, as do TCA interactions with selective serotonin reuptake inhibitors (SSRIs). TCA interactions with monoamine oxidase inhibitors (MAOIs) have been, historically, an uncertain and difficult question, but are now well understood, although this is not reflected in the literature. The data indicate that nortriptyline and desipramine have the most pharmacologically desirable characteristics as noradrenaline reuptake inhibitors (NRIs), and as drugs with few interactions that are also safe when coadministered with either MAOIs or SSRIs. Clomipramine is the only available antidepressant drug that has good evidence of clinically relevant serotonin and noradrenaline reuptake inhibition (SNRI). These data assist drug selection for monotherapy and combination therapy and predict reliably how and why pharmacodynamic and pharmacokinetic interactions occur. In comparison, two newer drugs proposed to have SNRI properties, duloxetine and venlafaxine, may have insufficient NRI potency to be effective SNRIs. Combinations such as sertraline and nortriptyline may therefore offer advantages over drugs like venlafaxine that have fixed ratios of SRI/NRI effects that are not ideal. However, no TCA/SSRI combination is sufficiently safe to be universally applicable without expert knowledge. Standard texts (e.g. the British National Formulary) and treatment guidelines would benefit by taking account of these new data and understandings.

We propose the possibility of 5-hydroxytryptamine (5-HT)1A receptor involvement in mild serotonin toxicity. A 64-year-old woman who experienced hallucinations was treated with perospirone (8 mg/day). She also complained of depressed mood and was prescribed paroxetine (10 mg/day). She exhibited finger tremors, sweating, coarse shivering, hyperactive knee jerks, vomiting, diarrhea, tachycardia, and psychomotor agitation. After the discontinuation of paroxetine and perospirone, the symptoms disappeared. Another 81-year-old woman, who experienced delusions, was treated with perospirone (8 mg/day). Depressive symptoms appeared and paroxetine (10 mg/day) was added. She exhibited tachycardia, finger tremors, anxiety, agitation, and hyperactive knee jerks. The symptoms disappeared after the cessation of paroxetine and perospirone. Recently, the effectiveness of coadministrating 5-HT1A agonistic psychotropics with selective serotonin reuptake inhibitors (SSRIs) has been reported, and SSRIs with 5-HT1A agonistic activity have been newly approved in the treatment of depression. Perospirone is a serotonin-dopamine antagonist and agonistic on the 5-HT1A receptors. Animal studies have indicated that mild serotonin excess induces low body temperature through 5-HT1A, whereas severe serotonin excess induces high body temperature through 5-HT2A activation. Therefore, it could be hypothesized that mild serotonin excess induces side effects through 5-HT1A, and severe serotonin excess induces lethal side effects with hyperthermia through 5-HT2A. Serotonin toxicity via a low dose of paroxetine that is coadministered with perospirone, which acts agonistically on the 5-HT1A receptor and antagonistically on the 5-HT2A receptor, clearly indicated 5-HT1A receptor involvement in mild serotonin toxicity. Careful measures should be adopted to avoid serotonin toxicity following the combined use of SSRIs and 5-HT1A agonists.

Although pruritus is the critical symptom of atopic dermatitis that profoundly affect the patients' quality of life, controlling and management of prurirtus still remains as unmet needs mainly due to the distinctive multifactorial pathogenesis of pruritus in atopic dermatitis. Based on the distinct feature of atopic dermatitis that psychological state of patients substantially influence on the intensity of pruritus, various psychotropic drugs have been used in clinic to relieve pruritus of atopic dermatitis patients. Only several psychotropic drugs were reported to show real antipruritic effects in atopic dermatitis patients including naltrexone, doxepin, trimipramine, bupropion, tandospirone, paroxetine and fluvoxamine. However, the precise mechanisms of antipruritic effect of these psychotropic drugs are still unclear. In human skin, serotonin receptors and serotonin transporter protein are expressed on skin cells such as keratinocytes, melanocytes, dermal fibroblasts, mast cells, T cells, natural killer cells, langerhans cells, and sensory nerve endings. It is noteworthy that serotonergic drugs, as well as serotonin itself, showed immune-modulating effect. Fenfluramine, fluoxetine and 2, 5-dimethoxy-4-iodoamphetamine significantly decreased lymphocyte proliferation. It is still questionable whether these serotonergic drugs exert the immunosuppressive effects via serotonin receptor or serotonin transporter. All these clinical and experimental reports suggest the possibility that antipruritic effects of selective serotonin reuptake inhibitors in atopic dermatitis patients might be at least partly due to their suppressive effect on T cells. Further studies should be conducted to elucidate the precise mechanism of neuroimmunological interaction in pruritus of atopic dermatitis.

Tinnitus, phantom sound perception, is a worldwide highly prevalent disorder for which no clear underlying pathology has been established and for which no approved drug is on the market. Thus, there is an urgent need for new approaches to understand this condition. We used a network pharmacology side-effect analysis to search for genes that are involved in tinnitus generation. We analyzed a network of 1,313 drug-target pairs, based on 275 compounds that elicit tinnitus as side effect and their targets reported in databases, and used a quantitative score to identify emergent significant targets that were more common than expected at random. Cyclooxigenase 1 and 2 were significant, which validates our approach, since salicylate is a known tinnitus generator. More importantly, we predict previously unknown tinnitus-related targets. The present results have important implications toward understanding tinnitus pathophysiology and might pave the way toward the design of novel pharmacotherapies.CPT Pharmacometrics Syst. Pharmacol. (2014) 3, e97; doi:10.1038/psp.2013.75; published online 29 January 2014.

The analysis of blood samples from mice treated with the PDE4 inhibitor Roflumilast revealed an unexpected reduction in serum potassium levels, while sodium and chloride levels were unaffected. Treatment with several structurally distinct PAN-PDE4 inhibitors, including Roflumilast, Rolipram, RS25344, and YM976 dose-dependently reduced serum potassium levels, indicating the effect is a class-characteristic property. PDE4 inhibition also induces hypothermia and hypokinesia in mice. However, while general anesthesia abrogates these effects of PDE4 inhibitors, potassium levels decrease to similar extents in both awake as well as in fully anesthetized mice. This suggests that the hypokalemic effects of PDE4 inhibitors occur independently of hypothermia and hypokinesia. PDE4 inhibition reduces serum potassium within 15 min of treatment, consistent with a rapid transcellular shift of potassium. Catecholamines promote the uptake of potassium into the cell via increased cAMP signaling. PDE4 appears to modulate these adrenoceptor-mediated effects, as PDE4 inhibition has no additional effects on serum potassium in the presence of saturating doses of the β-adrenoceptor agonist Isoprenaline or the α2-blocker Yohimbine, and is partially blocked by pre-treatment with the β-blocker Propranolol. Together, these data suggest that PDE4 inhibitors reduce serum potassium levels by modulating the adrenergic regulation of cellular potassium uptake.

Since the α1-adrenergic antagonist prazosin (PRZ) was introduced into medicine as a treatment for hypertension and benign prostate hyperplasia, several studies have shown that PRZ induces apoptosis in various cell types and interferes with endocytotic trafficking. Because PRZ is also able to induce apoptosis in malignant cells, its cytotoxicity is a focus of interest in cancer research. Besides inducing apoptosis, PRZ was shown to serve as a substrate for an amine uptake mechanism originally discovered in neurones called transport-P. In line with our hypothesis that transport-P is an endocytotic mechanism also present in non-neuronal tissue and linked to the cytotoxicity of PRZ, we tested the uptake of QAPB, a fluorescent derivative of PRZ, in cancer cell lines in the presence of inhibitors of transport-P and endocytosis. Early endosomes and lysosomes were visualised by expression of RAB5-RFP and LAMP1-RFP, respectively; growth and viability of cells in the presence of PRZ and uptake inhibitors were also tested. Cancer cells showed co-localisation of QAPB with RAB5 and LAMP1 positive vesicles as well as tubulation of lysosomes. The uptake of QAPB was sensitive to transport-P inhibitors bafilomycin A1 (inhibits v-ATPase) and the antidepressant desipramine. Endocytosis inhibitors pitstop(®) 2 (general inhibitor of endocytosis), dynasore (dynamin inhibitor) and methyl-β-cyclodextrin (cholesterol chelator) inhibited the uptake of QAPB. Bafilomycin A1 and methyl-β-cyclodextrin but not desipramine were able to preserve growth and viability of cells in the presence of PRZ. In summary, we confirmed the hypothesis that the cellular uptake of QAPB/PRZ represents an endocytotic mechanism equivalent to transport-P. Endocytosis of QAPB/PRZ depends on a proton gradient, dynamin and cholesterol, and results in reorganisation of the LAMP1 positive endolysosomal system. Finally, the link seen between the cellular uptake of PRZ and cell death implies a still unknown pro-apoptotic membrane protein with affinity towards PRZ.

Background: Lower survival rates for many cancer types correlate with changes in nuclear size/scaling in a tumor-type/tissue-specific manner. Hypothesizing that such changes might confer an advantage to tumor cells, we aimed at the identification of commercially available compounds to guide further mechanistic studies. We therefore screened for Food and Drug Administration (FDA)/European Medicines Agency (EMA)-approved compounds that reverse the direction of characteristic tumor nuclear size changes in PC3, HCT116, and H1299 cell lines reflecting, respectively, prostate adenocarcinoma, colonic adenocarcinoma, and small-cell squamous lung cancer. Results: We found distinct, largely nonoverlapping sets of compounds that rectify nuclear size changes for each tumor cell line. Several classes of compounds including, e.g., serotonin uptake inhibitors, cyclo-oxygenase inhibitors, β-adrenergic receptor agonists, and Na+/K+ ATPase inhibitors, displayed coherent nuclear size phenotypes focused on a particular cell line or across cell lines and treatment conditions. Several compounds from classes far afield from current chemotherapy regimens were also identified. Seven nuclear size-rectifying compounds selected for further investigation all inhibited cell migration and/or invasion. Conclusions: Our study provides (a) proof of concept that nuclear size might be a valuable target to reduce cell migration/invasion in cancer treatment and (b) the most thorough collection of tool compounds to date reversing nuclear size changes specific to individual cancer-type cell lines. Although these compounds still need to be tested in primary cancer cells, the cell line-specific nuclear size and migration/invasion responses to particular drug classes suggest that cancer type-specific nuclear size rectifiers may help reduce metastatic spread.

Tyrosine-kinase inhibitors (TKIs) have revolutionized cancer therapy in recent years. Although more targeted than conventional chemotherapy, TKIs exhibit substantial cardiotoxicity, often manifesting as hypertension or heart failure. Here, we assessed myocyte intrinsic cardiotoxic effects of the TKI sorafenib and investigated underlying alterations of myocyte calcium homeostasis. We found that sorafenib reversibly decreased developed force in auxotonically contracting human myocardia (3 µM: -25 ± 4%, 10 µM: -29 ± 7%, 30 µM: -43 ± 12%, p < 0.01), reduced peak cytosolic calcium concentrations in isolated cardiomyocytes (10 µM: 52 ± 8.1% of baseline, p < 0.001), and slowed cytosolic calcium removal kinetics (RT50, RT10, Tau, p < 0.05). Beta-adrenergic stimulation induced augmentation of calcium transient (CaT) amplitude was attenuated in sorafenib-treated cells (2.7 ± 0.3-fold vs. 3.6 ± 0.2-fold in controls, p < 0.001). Sarcoplasmic reticulum (SR) calcium content was reduced to 67 ± 4% (p < 0.01), and SR calcium re-uptake slowed (p < 0.05). Sorafenib significantly reduced serine 16 phosphorylation of phospholamban (PLN, p < 0.05), while PLN threonine 17 and CaMKII (T286) phosphorylation were not altered. Our data demonstrate that sorafenib acutely impairs cardiac contractility by reducing S16 PLN phosphorylation, leading to reduced SR calcium content, CaT amplitude, and slowed cytosolic calcium removal. These results indicate myocyte intrinsic cardiotoxicity irrespective of effects on the vasculature and chronic cardiac remodeling.

Drug repurposing, identifying novel indications for drugs, bypasses common drug development pitfalls to ultimately deliver therapies to patients faster. However, most repurposing discoveries have been led by anecdotal observations (e.g. Viagra) or experimental-based repurposing screens, which are costly, time-consuming, and imprecise. Recently, more systematic computational approaches have been proposed, however these rely on utilizing the information from the diseases a drug is already approved to treat. This inherently limits the algorithms, making them unusable for investigational molecules. Here, we present a computational approach to drug repurposing, CATNIP, that requires only biological and chemical information of a molecule. CATNIP is trained with 2,576 diverse small molecules and uses 16 different drug similarity features, such as structural, target, or pathway based similarity. This model obtains significant predictive power (AUC = 0.841). Using our model, we created a repurposing network to identify broad scale repurposing opportunities between drug types. By exploiting this network, we identified literature-supported repurposing candidates, such as the use of systemic hormonal preparations for the treatment of respiratory illnesses. Furthermore, we demonstrated that we can use our approach to identify novel uses for defined drug classes. We found that adrenergic uptake inhibitors, specifically amitriptyline and trimipramine, could be potential therapies for Parkinson's disease. Additionally, using CATNIP, we predicted the kinase inhibitor, vandetanib, as a possible treatment for Type 2 Diabetes. Overall, this systematic approach to drug repurposing lays the groundwork to streamline future drug development efforts.

Ca leak from the sarcoplasmic reticulum through the ryanodine receptor (RyR) reduces the amplitude of the Ca transient and slows its rate of decay. In the presence of β-adrenergic stimulation, RyR-mediated Ca leak produces a biphasic decay of the Ca transient with a fast early phase and a slow late phase. Two forms of Ca leak have been studied, Ca-sensitising (induced by caffeine) and non-sensitising (induced by ryanodine) and both induce biphasic decay of the Ca transient. Only Ca-sensitising leak can be reversed by traditional RyR inhibitors such as tetracaine. Ca leak can also induce Ca waves. At low levels of leak, waves occur. As leak is increased, first biphasic decay and then slowed monophasic decay is seen. The level of leak has major effects on the shape of the Ca transient. In heart failure, a reduction in Ca transient amplitude and contractile dysfunction can by caused by Ca leak through the sarcoplasmic reticulum (SR) Ca channel (ryanodine receptor, RyR) and/or decreased activity of the SR Ca ATPase (SERCA). We have characterised the effects of two forms of Ca leak (Ca-sensitising and non-sensitising) on calcium cycling and compared with those of SERCA inhibition. We measured [Ca(2+)]i with fluo-3 in voltage-clamped rat ventricular myocytes. Increasing SR leak with either caffeine (to sensitise the RyR to Ca activation) or ryanodine (non-sensitising) had similar effects to SERCA inhibition: decreased systolic [Ca(2+)]i , increased diastolic [Ca(2+)]i and slowed decay. However, in the presence of isoproterenol, leak produced a biphasic decay of the Ca transient in the majority of cells while SERCA inhibition produced monophasic decay. Tetracaine reversed the effects of caffeine but not of ryanodine. When caffeine (1 mmol l(-1)) was added to a cell which displayed Ca waves, the wave frequency initially increased before waves disappeared and biphasic decay developed. Eventually (at higher caffeine concentrations), the biphasic decay was replaced by slow decay. We conclude that, in the presence of adrenergic stimulation, Ca leak can produce biphasic decay; the slow phase results from the leak opposing Ca uptake by SERCA. The degree of leak determines whether decay of Ca waves, biphasic or monophasic, occurs.

Endocytosis mediates the cellular uptake of micronutrients and cell surface proteins. Fast Endophilin-mediated endocytosis, FEME, is not constitutively active but triggered upon receptor activation. High levels of growth factors induce spontaneous FEME, which can be suppressed upon serum starvation. This suggested a role for protein kinases in this growth factor receptor-mediated regulation. Using chemical and genetic inhibition, we find that Cdk5 and GSK3β are negative regulators of FEME. They antagonize the binding of Endophilin to Dynamin-1 and to CRMP4, a Plexin A1 adaptor. This control is required for proper axon elongation, branching and growth cone formation in hippocampal neurons. The kinases also block the recruitment of Dynein onto FEME carriers by Bin1. As GSK3β binds to Endophilin, it imposes a local regulation of FEME. Thus, Cdk5 and GSK3β are key regulators of FEME, licensing cells for rapid uptake by the pathway only when their activity is low.