Stimulation Of Adenylyl Cyclase Research Articles

Role of Orai1 and Ca2+-stimulated adenylyl cyclase 8 in the adverse cardiac remodeling after myocardial infarction

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Agencia Estatal de Investigación. Background It is well established that abnormalities in the regulation of the intracellular concentration of Ca2+ ([Ca2+]i) occurs in diseased heart such as under acute myocardial infarction (AMI). Recent studies have demonstrated that proteins related to the Store Operated Calcium Entry (SOCE), such as Orai1/2/3 and TRPC channels, play a role in the adverse cardiac remodelling. Previously, we demonstrated that the expression of Orai1 increased in heart subjected to ischemia and reperfusion (I/R) syndrome. However, the signalling pathway implicated in Orai1 regulation has not been addressed. Objective The aim of this study was to examine the molecular mechanism and signalling pathways involved in the of Orai1 upregulation after I/R in cardiomyocytes. Materials and Methods The experiments were performed in adult (ARVM) and neonatal rat ventricular cardiomyocytes (NRVM), in a rat model of myocardial I/R and in ventricular biopsies of patients with ischemic heart failure. Ca2+ studies were realized using microfluorimetric technique in FURA-2AM loaded cardiomyocytes. Immunofluorescence was used to investigate CREB activation using a specific phospho-CREB antibody and Orai1 and AC8 protein expression were analysed by Western Blot. This study was performed in accordance with the recommendations of the Royal Decree 53/2013 in agreement to the Directive 2010/63/EU of the European Parliament and approved by the local Ethics Committee on human Research and the Animal Research Committee. Results We found that under I/R diastolic level of [Ca2+]i was significantly increased in cardiac myocytes isolated from I/R hearts, which was inhibited by SOCE blockers. Moreover, we found that Orai1 and Ca2+-sensitive adenylyl cyclase 8 (AC8) are overexpressed in the heart isolated from I/R rat model, as compared to sham. Similarly, we found significant increase in the expression of Orai1 and AC8 in ventricle biopsies of post-infarction patients with HF. We also observed that I/R stimulated cAMP production, involving Orai1 and AC8 activation in rats, indicating that Ca2+ entry through Orai1 stimulated AC8. Moreover, I/R phosphorylated cAMP response element-binding protein (CREB) through PKA activation, but not via EPAC2 or ERK1/2. Interestingly, we found that CREB facilitated I/R-induced Orai1 upregulation and related exacerbated SOCE by its activation of Orai1 promoter under I/R. Finally, the intramyocardial administration of AAV9 holding AC8 shRNA decreased the expression of AC8, Orai1 and CREB, which restored diastolic [Ca2+]i. Conclusions Our results show that the activation of Orai1/AC8/CREB axis plays a key role in the I/R-induced Orai1 upregulation, which participated in diastolic [Ca2+]i mishandling related to the adverse cardiac remodelling.

The potential of caffeine contained in the coffee to modulate the immune system of the COVID-19 sufferers: A review

Background: The COVID-19 pandemic is a worldwide epidemic caused by the SARS-CoV-2 virus, initially detected in Wuhan City, Hubei, China in December 2019. This presents an unparalleled difficulty in identifying efficacious medications for treatment. Despite numerous clinical trials employing various medicines, there remains a dearth of targeted therapies for COVID-19. The progression of COVID-19 clinical symptoms, for example a severe pneumonia, arespiratory distress syndrome (ARDS), and multiorgan failure, is a result of an exaggerated immune response characterized by increased inflammation, oxidation, and cytokine storm. Presently, coffee beans contain caffeine, which is a highly consumed chemical. Caffeine exerts beneficial benefits on the human body, influencing multiple systems such as the immunological system, central nervous system, digestive system, and respiratory system. The impact of caffeine is contingent upon the quantity and composition of the product in which it is present. Objective: Analyze the potential of caffeine as an immunomodulator in COVID-19 sufferers. Discussion: Caffeine functions as an agonist of TAS2R receptors and an antagonist of adenosine receptors. The immunomodulatory properties of caffeine can assist in diminishing the severity of SARS-CoV-2 infection by stimulating adenylyl cyclase, which converts ATP into cAMP. The heightened level of cAMP stimulates the intiation of protein kinase A (PKA), which subsequently suppresses the secretion of pro-inflammatory cytokines. Furthermore, caffeine has the ability to suppress the generation of reactive oxygen species (ROS), hence reducing the unleash of pro-inflammatory cytokines. Conclusion: Caffeine provides health benefits with its immunomodulating properties to COVID-19 patients and can be regarded as a supplementary treatment in patients with COVID-19.

SAT019 Ligand-independent Signaling And Migration Of Breast Cancer Cells Overexpressing Membrane Androgen Receptor, ZIP9

Abstract Disclosure: P. Thomas: None. The zinc transporter ZIP9 also functions as a membrane androgen receptor (mAR) that mediates androgen-dependent rapid signaling and pro- or anti-apoptotic, proliferative, and migratory responses in fish, rodent, and human cells. Testosterone activation of ZIP9 in two triple-negative breast cancer cell lines, MDA-MB-468 cells and MDA-MB-231 cells transfected with ZIP9 (231-ZIP9), induces G-protein-dependent second messenger signaling and increases in intracellular free zinc. However, the relative importance of these two signaling pathways in the regulation of pro- and anti-tumorigenic responses in these cell lines is unclear because both androgen signaling and zinc status influence tumorigenesis in breast tissues. The effects of overexpression of ZIP9 on free zinc status, G protein- and zinc-dependent regulation of migration, and migration marker expression in 231-ZIP9 cells in the absence of testosterone were compared to those observed in MDA-MB-231 cells transfected with vector alone (231-vect). Migration and invasion of 231-ZIP9 cells were significantly increased after 24-48 hr culture in serum-free media compared to 231-vect cells which was accompanied by a significant increase in free zinc levels. Treatment of 231-vect cells with a zinc ionophore also increased migration, whereas migration of 231-ZIP9 cells was attenuated by treatment with an intracellular zinc chelator, TPEN. Expression of the migration markers, MYL9 and CYR61, was increased in 231-ZIP9 after 24-48 hr culture, whereas expression of apoptotic markers was decreased compared to 231-vect controls. Proximity Ligation Analysis revealed that ZIP9 is closely associated with a stimulatory G protein (Gs) in 231-ZIP9 cells which is consistent with the finding that treatment with the adenylyl cyclase (AC) stimulator forskolin, further increased migration marker expression and migration compared to vehicle controls, whereas the PKA inhibitor, H-89, attenuated these responses as well as the increase in free zinc concentrations. Knockdown of ZIP9 expression in MDA-MB-468 cells which express relatively high levels of ZIP9 with siRNA decreased cell migration and invasion as well as free zinc levels, confirming a role of unliganded ZIP9 in migration in another breast cancer cell line. Treatment of 231-ZIP9 cells with 100 nM testosterone further stimulated CYR61 and MYL9 expression and cell migration which were blocked by co-treatment with a Gs inhibitor and TPEN. The results indicate that high ZIP9 expression in breast cancer cells causes androgen-independent increased cell migration and invasion which in 231-ZIP9 cells is through Gs/mAC/PKA-mediated increases in intracellular zinc concentrations and upregulation of MYL9 and CYR-61 expression. Furthermore, the results suggest that testosterone-dependent signaling through ZIP9 acts through the same pathways to further enhance signaling and migration. Presentation: Saturday, June 17, 2023

A preliminary study of phosphodiesterases and adenylyl cyclase signaling pathway on red blood cell deformability of sickle cell patients.

Sickle cell disease (SCD) is an inherited hemoglobinopathy characterized by chronic anemia, intravascular hemolysis, and the occurrence of vaso-occlusive crises due to the mechanical obstruction of the microcirculation by poorly deformable red blood cells (RBCs). RBC deformability is a key factor in the pathogenesis of SCD, and is affected by various factors. In this study, we investigated the effects of adenylyl cyclase (AC) signaling pathway modulation and different phosphodiesterase (PDE) modulatory molecules on the deformability and mechanical stress responses of RBC from SCD patients (HbSS genotype) by applying 5Pa shear stress with an ektacytometer (LORRCA). We evaluated RBC deformability before and after the application of shear stress. AC stimulation with Forskolin had distinct effects on RBC deformability depending on the application of 5Pa shear stress. RBC deformability was increased by Forskolin before shear stress application but decreased after 5Pa shear stress. AC inhibition with SQ22536 and protein kinase A (PKA) inhibition with H89 increased RBC deformability before and after the shear stress application. Non-selective PDE inhibition with Pentoxifylline increased RBC deformability. However, modulation of the different PDE types had distinct effects on RBC deformability, with PDE1 inhibition by Vinpocetine increasing deformability while PDE4 inhibition by Rolipram decreased RBC deformability after the shear stress application. The effects of the drugs varied greatly between patients suggesting some could benefit from one drug while others not. Developing drugs targeting the AC signaling pathway could have clinical applications for SCD, but more researches with larger patient cohorts are needed to identify the differences in the responses of sickle RBCs.

Oxidized soluble guanylyl cyclase causes erectile dysfunction in alcoholic mice.

Alcohol abuse has been associated with erectile dysfunction (ED) but the molecular mechanisms implicated are unknown. This study analyses the role of alterations in soluble guanylyl cyclase (sGC) in ED. ED was analysed in adult male C57BL/6J mice subjected to the Chronic Intermittent Ethanol (CIE) paradigm. Erectile function was assessed in anesthetized mice in vivo by evaluating intracavernosal pressure (ICP) and in vitro in isolated mice corpora cavernosa (CC) mounted in a myograph. Protein expression and reactive oxygen species were analysed by Western blot and dihydroethidium staining, respectively. In CIE mice, we observed a significant decrease in the relaxant response of the CC to stimulation of NO release from nitrergic nerves by electrical field stimulation, to NO release from endothelial cells by acetylcholine, to the PDE5 inhibitor sildenafil, and to the sGC stimulator riociguat. Conversely, the response to the sGC activator cinaciguat, whose action is independent on the oxidation state of sGC, was significantly enhanced in these CC. The responses to the adenylyl cyclase stimulation with forskolin were unchanged. We found an increase in reactive oxygen species in the CC from CIE mice as well as an increase in CYP2E1 and NOX2 protein expression. In vivo pre-treatment with tempol prevented alcohol-induced erectile dysfunction. Our results demonstrate that alcoholic mice show ED in vitro and in vivo due to an alteration of the redox state of sGC and suggest that sGC activators may be effective in ED associated with alcoholism.

Activity of cAMP-Dependent Protein Kinase is Reduced in Protein-Energy Malnourished Rats

Glucagon decreases glutathione synthesis in hepatocytes from well-nourished rats. However, in hepatocytes from malnourished rats, glucagon does not inhibit glutathione synthesis, suggesting a desensitization of cAMP-mediated signal transduction. We investigated the mechanism for this desensitization of cAMP-mediated responsiveness in malnourished rats by comparing the signal transduction pathways in rats fed very low protein diets (0.5 g protein/100 g diet) with those of rats fed diets adequate in protein (15 g protein/100 g diet) for 2 wk. Glucagon receptor and forskolin-stimulated cAMP production were greater in hepatocytes from malnourished rats. Stimulation of adenylyl cyclase with forskolin, guanine nucleotides or manganese in hepatic membranes was also enhanced after malnutrition. Moreover, quantity of the stimulatory guanine nucleotide regulatory protein was 70–80% greater in hepatocytes from malnourished rats but the inhibitory guanine nucleotide regulatory protein was not different. These results suggested that desensitization of cAMP-mediated signal transduction after malnutrition occurred at a site distal to cAMP production. Maximal activity of cAMP-dependent protein kinase was 60% lower in liver homogenates from malnourished rats compared with controls. This difference in activity was confined to the cytosolic compartment, with no difference in activity observed in the particulate fraction. Lower activity of cAMP-dependent protein kinase in the cytosol of malnourished rats was associated with a 43% reduction in the quantity of regulatory subunit type I, with no difference in the regulatory subunit type II. These data indicate that desensitization of cAMP signal transduction in rat liver after malnutrition is due to a decrease in the quantity and activity of cAMP-dependent protein kinase.

Thyroid-stimulating hormone receptor signaling restores skeletal muscle stem cell regeneration in rats with muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe and progressive myopathy leading to motor and cardiorespiratory impairment. We analyzed samples from patients with DMD and a preclinical rat model of severe DMD and determined that compromised repair capacity of muscle stem cells in DMD is associated with early and progressive muscle stem cell senescence. We also found that extraocular muscles (EOMs), which are spared by the disease in patients, contain muscle stem cells with long-lasting regenerative potential. Using single-cell transcriptomics analysis of muscles from a rat model of DMD, we identified the gene encoding thyroid-stimulating hormone receptor (Tshr) as highly expressed in EOM stem cells. Further, TSHR activity was involved in preventing senescence. Forskolin, which activates signaling downstream of TSHR, was found to reduce senescence of skeletal muscle stem cells, increase stem cell regenerative potential, and promote myogenesis, thereby improving muscle function in DMD rats. These findings indicate that stimulation of adenylyl cyclase leads to muscle repair in DMD, potentially providing a therapeutic approach for patients with the disease.

Emergence of nociceptive functionality and opioid signaling in human induced pluripotent stem cell-derived sensory neurons.

Induced pluripotent stem cells (iPSCs) have enabled the generation of various difficult-to-access cell types such as human nociceptors. A key challenge associated with human iPSC-derived nociceptors (hiPSCdNs) is their prolonged functional maturation. While numerous studies have addressed the expression of classic neuronal markers and ion channels in hiPSCdNs, the temporal development of key signaling cascades regulating nociceptor activity has remained largely unexplored. In this study, we used an immunocytochemical high-content imaging approach alongside electrophysiological staging to assess metabotropic and ionotropic signaling of large scale-generated hiPSCdNs across 70 days of in vitro differentiation. During this period, the resting membrane potential became more hyperpolarized, while rheobase, action potential peak amplitude, and membrane capacitance increased. After 70 days, hiPSCdNs exhibited robust physiological responses induced by GABA, pH shift, ATP, and capsaicin. Direct activation of protein kinase A type II (PKA-II) through adenylyl cyclase stimulation with forskolin resulted in PKA-II activation at all time points. Depolarization-induced activation of PKA-II emerged after 35 days of differentiation. However, effective inhibition of forskolin-induced PKA-II activation by opioid receptor agonists required 70 days of in vitro differentiation. Our results identify a pronounced time difference between early expression of functionally important ion channels and emergence of regulatory metabotropic sensitizing and desensitizing signaling only at advanced stages of in vitro cultivation, suggesting an independent regulation of ionotropic and metabotropic signaling. These data are relevant for devising future studies into the development and regulation of human nociceptor function and for defining time windows suitable for hiPSCdN-based drug discovery.

Ca2+-stimulated adenylyl cyclases as therapeutic targets for psychiatric and neurodevelopmental disorders.

As the main secondary messengers, cyclic AMP (cAMP) and Ca2+ trigger intracellular signal transduction cascade and, in turn, regulate many aspects of cellular function in developing and mature neurons. The group I adenylyl cyclase (ADCY, also known as AC) isoforms, including ADCY1, 3, and 8 (also known as AC1, AC3, and AC8), are stimulated by Ca2+ and thus functionally positioned to integrate cAMP and Ca2+ signaling. Emerging lines of evidence have suggested the association of the Ca2+-stimulated ADCYs with bipolar disorder, schizophrenia, major depressive disorder, post-traumatic stress disorder, and autism. In this review, we discuss the molecular and cellular features as well as the physiological functions of ADCY1, 3, and 8. We further discuss the recent therapeutic development to target the Ca2+-stimulated ADCYs for potential treatments of psychiatric and neurodevelopmental disorders.

Preferential Expression of Ca2+-Stimulable Adenylyl Cyclase III in the Supraventricular Area, including Arrhythmogenic Pulmonary Vein of the Rat Heart.

Ectopic excitability in pulmonary veins (PVs) is the major cause of atrial fibrillation. We previously reported that the inositol trisphosphate receptor in rat PV cardiomyocytes cooperates with the Na+-Ca2+ exchanger to provoke ectopic automaticity in response to norepinephrine. Here, we focused on adenylyl cyclase (AC) as another effector of norepinephrine stimulation. RT-PCR, immunohistochemistry, and Western blotting revealed that the abundant expression of Ca2+-stimulable AC3 was restricted to the supraventricular area, including the PVs. All the other AC isotypes hardly displayed any region-specific expressions. Immunostaining of isolated cardiomyocytes showed an enriched expression of AC3 along the t-tubules in PV myocytes. The cAMP-dependent response of L-type Ca2+ currents in the PV and LA cells is strengthened by the 0.1 mM intracellular Ca2+ condition, unlike in the ventricular cells. The norepinephrine-induced automaticity of PV cardiomyocytes was reversibly suppressed by 100 µM SQ22536, an adenine-like AC inhibitor. These findings suggest that the specific expression of AC3 along t-tubules may contribute to arrhythmogenic automaticity in rat PV cardiomyocytes.

Interaction of the preferential D3 agonist (+)PHNO with dopamine D3-D2 receptor heterodimers and diverse classes of monoamine receptor: relevance for PET imaging

(+)-4-Propyl-9-hydroxynaphthoxazine ((+)PHNO) is a high affinity, preferential dopamine D3 versus D2 agonist employed in view of its high specificity and excellent signal-to-noise ratio as a radiotracer for positron emission tomography (PET) imaging. Surprisingly, its profile at other classes of monoamine receptor remains undocumented. In addition to hD3 and hD2L receptors, (+)PHNO revealed high affinity at hD4.4 but not hD1 or hD5 receptors. It also revealed significant affinity for several other G protein-coupled monoaminergic receptors, in particular h5-HT1A and h5-HT7. (+)PHNO behaved as a full agonist at hD4.4 and h5-HT1A receptors with potencies comparable to its actions at hD3 and hD2L receptors, and with less potency at 5-HT7 receptors. In binding assays with membranes derived from cells co-expressing hD3 and hD2L receptors and labeled with [3H]Nemonapride or [3H]Spiperone, the proportion of high affinity binding sites recognized by (+)PHNO was higher than an equivalent mixture of membranes from cells expressing hD3or hD2L receptors, suggesting that (+)PHNO promotes formation of hD3-hD2L heterodimers. Further, in cells co-expressing hD3 and hD2L receptors, (+)PHNO showed higher efficacy for inhibiting forskolin stimulated adenylyl cyclase and inducing adenylyl cyclase super-sensitization than in cells transfected with only hD2L receptors. In conclusion, (+)PHNO is a potent agonist at hD4.4, h5-HT1A and h5-HT7 as well as hD3 and hD2L receptors, and it potently activates dopamine hD3-hD2L heterodimers. These interactions should be considered when interpreting PET studies with [11C](+)PHNO and may be relevant to its functional and potential clinical properties in Parkinson's disease and other disorders.

Identification of Peptide Inhibitors of Gαs that Block its Effector Binding Site

Heterotrimeric G proteins (Gαβγ) are molecular switches that alternate between “on” (GTP‐bound) and “off” (GDP‐bound) states to control the flow of signals from the extracellular milieu to the cytoplasm. Gαs is a prototypical Gα subunit of heterotrimeric G proteins. When bound to GTP, Gαs promotes the synthesis of cAMP in the cytosol through stimulation of the enzymatic activity of adenylyl cyclases. Dysregulation of this mechanism leads to human diseases. For example, naturally occurring mutations like R201C lock Gαs in an active conformation that leads to signaling hyperactivity in cancer. Despite the critical role of Gαs in signaling and its involvement in disease, there are currently no chemical or peptide tools to specifically inhibit its signaling activity. We reasoned that peptides that bind to active Gαs and block the effector binding site would inhibit its ability to signal through adenylyl cyclases. For this, we screened peptides from a phage display library and identified two unrelated peptides that bound to active Gαs. These peptides bound efficiently to GTP‐bound Gαs or to the constitutively active Gαs R201C mutant, but not to inactive GDP‐bound Gαs. Fluorescence polarization experiments revealed an equilibrium dissociation constant (KD) of approximately 1 μM for the binding of these peptides to Gαs‐GTP. Mutations in the adenylyl cyclase binding region of Gαs greatly reduced binding of these two peptides, indicating that they may work as competitive inhibitors of adenylyl cyclase stimulation. Consistently, both peptides blocked Gas‐mediated stimulation of adenylyl cyclase activity in vitrowith μM potency, but had no effect on Gαs‐independent stimulation of adenylyl cyclase activity by forskolin. Protein‐protein binding experiments revealed that one of the two peptides bound to Gαs but not to Gα subunits of other G protein families, whereas the second peptide bound to Gαi‐GTP in addition to Gαs‐GTP. In summary, we have identified a peptide inhibitor specific for Gαs‐GTP that acts by binding to its effector binding site and precluding adenylyl cyclase stimulation. The peptide identified here could be leveraged as a genetically‐encoded probe or derivatized as a cell‐penetrating peptide to inhibit Gαs‐mediated cell signaling.

Repurposing Probenecid for the Treatment of Heart Failure (Re-Prosper-HF): a study protocol for a randomized placebo-controlled clinical trial

BackgroundImproving contractility in heart failure with reduced ejection fraction (HFrEF) has resurfaced as a potential treatment goal. Inotropic therapy is now better understood through its underlying mechanism as opposed to the observed effect of increasing contractility. Calcitropes are a subgroup of inotropes that largely depend on the stimulation of adenylyl cyclase to transform ATP into cyclic adenosine monophosphate (cAMP). At least two clinically relevant calcitropes—istaroxime and probenecid—improve contractility through an increase in systolic intracellular calcium without activating cAMP production.Probenecid, which has been safely used clinically for decades in non-cardiac conditions, has recently been identified as an agonist of the transient receptor potential vanilloid 2 channel. Translational studies have shown that it improves calcium cycling and contractility without activating noxious pathways associated with cAMP-dependent calcitropes and can improve cardiac function in patients with HFrEF.MethodsThe Re-Prosper-HF study (Repurposing Probenecid for the Treatment of Heart Failure with Reduced Ejection Fraction) is a three-site double-blinded randomized-controlled trial that will test the hypothesis that probenecid can improve cardiac function in patients with HFrEF. Up to 120 patients will be randomized in this double-blind, placebo-controlled study that will assess whether oral probenecid administered at 1 g orally twice per day for 180 days in patients with NYHA II-III HFrEF improves systolic function (aim 1), functional status (aim 2), and self-reported health status (aim 3).DiscussionFindings from this study will provide data informing its use for improving symptomatology in patients with HFrEF as well as exploratory data for outcomes such as hospital admission rates.Trial tegistrationThe Re-Prosper HF Study (Re-Prosper HF) is registered on ClinicalTrials.gov with the identifier as NCT04551222. Registered on 9 September 2020.

A novel peripheral biomarker for depression and antidepressant response.

In contrast to healthy controls, the heterotrimeric G protein, Gsalpha (Gsα) is ensconced predominantly in lipid rafts in subjects with major depressive disorder (MDD) resulting in impaired stimulation of adenylyl cyclase. In this small proof-of-concept study, we examined the hypothesis that translocation of Gsα from lipid rafts toward a more facile activation of adenylyl cyclase is a biomarker for clinical response to antidepressants.There were 49 subjects with MDD (HamD17 score ≥15) and 59 healthy controls at the screen visit. The AlphaScreen (PerkinElmer) assay measured both basal activity and prostaglandin E1 (PGE1) stimulation of Gsα-adenylyl cyclase to assess the extent of coupling of Gsα with adenylyl cyclase.At screen, platelet samples obtained from MDD subjects revealed significantly lower PGE1 activation of adenylyl cyclase activity than controls (p =0.02). Subsequently, 19 consenting MDD subjects completed a 6-week open label antidepressant treatment trial. The 11 antidepressant responders (HamD17 improvement ≥50% from screen) revealed significant increase in PGE1-stimulated adenylyl cyclase compared to non-responders (p=0.05) with an effect size of 0.83 for the PGE1/Gsα lipid-raft biomarker. PGE1 stimulation increased by ≥30% from screen assessment in 8 responders (72.7%) and 2 non-responders (25.0%) [Fisher exact= 0.07] with a positive predictive value for response of 80.0%.In this small, pilot study, increased PGE1 stimulated adenylyl cyclase was associated with antidepressant response in MDD subjects. These data suggest that a simple, high-throughput-capable assay for depression and antidepressant response can be developed. Future studies are needed to evaluate the utility of this biomarker for the treatment of MDD.

Photochemical reaction to increase melanogenesis using Buddleja officinalis and blue light-emitting diode irradiation in B16F10

Recently, the incidence of vitiligo has increased because of stresses induced by external environment. Ultraviolet (UV) light therapy is the most commonly used method of treating the disease; however, UV light therapy requires a long treatment period, and prolonged exposure to UV radiation has side effects. The purpose of the present study was to investigate the effects of natural products and LED irradiation (LED-IR) on the synthesis of melanin. It was not possible to effectively increase intracellular melanin production through individual applications of Buddleja officinalis (BO), which is a natural substance selected through screening, or blue light irradiation (Blue-IR). However, when used in combination, these two agents stimulated adenylyl cyclase (AC) and melanin production was induced in the stimulated cells via the CREB/MITF/TYR pathway. Furthermore, the combined treatment with BO and Blue-IR generated low levels of cellular reactive oxygen species (ROS) and induced p38 phosphorylation, which in turn activated MITF in ROS-stimulated synthetic melanocytes, resulting in the promotion of melanogenic pathways other than the CREB/MITF/TYR pathway. In addition, this treatment combination effected melanin transport. These results suggested that the combined therapies can be used to treat melanin-deficiency skin diseases such as vitiligo.

Defining a Role for G-Protein Coupled Receptor/cAMP/CRE-Binding Protein Signaling in Hair Follicle Stem Cell Activation

Manipulation of adrenergic signaling has been shown experimentally and clinically to affect hair follicle growth. In this study, we provide direct evidence that canonical cAMP/CRE-binding protein signaling through adrenergic receptors can regulate hair follicle stem cell (HFSC) activation and hair cycle. We found that CRE-binding protein activation is regulated through the hair cycle and coincides with HFSC activation. Both isoproterenol and procaterol, agonists of adrenergic receptors, show the capacity to activate the hair cycle in mice. Furthermore, deletion of ADRB2 receptor, which is thought to mediate sympathetic nervous system regulation of HFSCs, was sufficient to block HFSC activation. Downstream, stimulation of adenylyl cyclase with forskolin or inhibition of phosphodiesterase to increase cAMP accumulation or direct application of cAMP was each sufficient to promote HFSC activation and accelerate initiation of hair cycle. Genetic induction of a Designer Receptors Exclusively Activated by Designer Drug allele showed that G-protein coupled receptor/GαS stimulation, specifically in HFSCs, promoted the activation of the hair cycle. Finally, we provide evidence that G-protein coupled receptor/CRE-binding protein signaling can potentially act on HFSCs by promoting glycolytic metabolism, which was previously shown to stimulate HFSC activation. Together, these data provide mechanistic insights into the role of sympathetic innervation on HFSC function.

Ectopically Expressed Olfactory GPCRs OR51E1/2: Ligand Specificity, Signal Transduction, and Cytostatic and Cytotoxic Activity

Olfactory receptors (ORs) are the largest family of G protein-coupled receptors: there are ~380 ORs in humans and ~1,000 in mice. This family was originally cloned from the nasal epithelium, but several years ago, expression of many OR genes was detected in the airways, gut, blood vessels, brain, and other organs. These GPCRs are referred to as ectopic ORs (eORs). Understanding eOR function has lagged behind that of other GPCRs, and the majority of ORs remain to be orphan receptors. One reason for this delay is that the expression of functionally active ORs has not been technically possible for many years. In contrast to the most other Class A GPCRs, which can be produced using standard methods such as transient transfection, stability and trafficking ORs requires special chaperones and/or modification with unique signal peptides. Difficulties with detection and functional analysis of both endogenous and overexpressed eORs caused many controversies, and overall, this vast and potentially interesting family of GPCRs has been grossly understudied. Here, we investigated two eORs, OR51E1 and OR51E2, which due to their marked upregulation in prostate cancer are also known as “prostate-specific G protein-coupled receptors” (PSGR2 and PSGR, respectively). To investigate their signaling and effects on proliferation and survival we developed an inducible expression system in a prostate cancer cell line LNCaP. The robust expression and trafficking of ORs allowed us to analyze their function using cAMP, Ca2+, protein phosphorylation and other assays. Consistent with previous studies, OR51E1 stimulated adenylyl cyclase in response to treatment by short- to medium-chain organic acids (C3-C9) an some branched aliphatic acids. OR51E2 responded to acetate and propionate, but not to the longer chain organic acids. Other putative ligands mentioned in the literature did not have activity. Stimulation of LNCaP cells with butyrate inhibited their growth, and the knockdown of the endogenous OR51E1 negated this cytostatic effect. Interestingly, induced overexpression of OR51E1 or OR51E2 suppressed LNCaP cell proliferation. Overexpression of OR2AT4, beta2-adrenergic receptor or treatment of cells with forskolin did not suppress cell proliferation, indicating that rise in cAMP is not sufficient to induce cytostasis. These findings indicate that like other GPCRs, eORs can signal via more than one G protein pathway. Overexpression of OR51E1 caused upregulation of cytostatic and cell death markers including p27, p21, and p53, strongly increased annexin V staining and stimulated ERK1/2. OR51E1 had no effect on proliferation of HEK293 cells, indicating that cytotoxicity of OR51E1/2 is specific for LNCaP cells. Elucidating this mechanism will contribute to understanding etiology of prostate cancer and may illuminated these two and possibly other eORs as future therapeutic targets.

Inhibition of PACAP/PAC1/VPAC2 signaling impairs the consolidation of social recognition memory and nitric oxide prevents this deficit

Social recognition memory (SRM) forms the basis of social relationships of animals. It is essential for social interaction and adaptive behavior, reproduction and species survival. Evidence demonstrates that social deficits of psychiatric disorders such as autism and schizophrenia are caused by alterations in SRM processing by the hippocampus and amygdala. Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and its receptors PAC1, VPAC1 and VPAC2 are highly expressed in these regions. PACAP is a pleiotropic neuropeptide that modulates synaptic function and plasticity and is thought to be involved in social behavior. PACAP signaling also stimulates the nitric oxide (NO) production and targets outcomes to synapses. In the present work, we investigate the effect of the infusion of PACAP-38 (endogenous neuropeptide and potent stimulator of adenylyl cyclase), PACAP 6–38 (PAC1/VPAC2 receptors antagonist) and S-Nitroso-N-acetyl-DL-penicillamine (SNAP, NO donor) in the CA1 region of the hippocampus and in the basolateral amygdala (BLA) on the consolidation of SRM. For this, male Wistar rats with cannulae implanted in CA1 or in BLA were subjected to a social discrimination paradigm, which is based on the natural ability of rodents to investigate unfamiliar conspecifics more than familiar one. In the sample phase (acquisition), animals were exposed to a juvenile conspecific for 1 h. Immediately, 60 or 150 min after, animals received one of different pharmacological treatments. Twenty-four hours later, they were submitted to a 5 min retention test in the presence of the previously presented juvenile (familiar) and a novel juvenile. Animals that received infusions of PACAP 6–38 (40 pg/side) into CA1 immediately after the sample phase or into BLA immediately or 60 min after the sample phase were unable to recognize the familiar juvenile during the retention test. This impairment was abolished by the coinfusion of PACAP 6–38 plus SNAP (5 μg/side). These results show that the blockade of PACAP/PAC1/VPAC2 signaling in the CA1 and BLA during a restricted post-acquisition time window impairs the consolidation of SRM and that the SNAP is able to abolish this deficit. Findings like this could potentially be used in the future to influence studies of psychiatric disorders involving social behavior.

Ectopically expressed olfactory receptors OR51E1 and OR51E2 suppress proliferation and promote cell death in a prostate cancer cell line

Olfactory receptors (ORs), the largest family of G protein–coupled receptors, are expressed in the nasal epithelium where they mediate the sense of smell. However, ORs are also found in other non-nasal tissues, but the role of these ectopic ORs in cell signaling, proliferation, and survival is not well understood. Here, using an inducible expression system in the lymph node carcinoma of the prostate (LNCaP) cell line, we investigated two ectopic ORs, OR51E1 and OR51E2, which have been shown to be upregulated in prostate cancer. We found that, consistent with previous studies, OR51E1 stimulated adenylyl cyclase in response to treatment by short-chain to medium-chain organic acids (C3–C9) but not by acetate. OR51E2 responded to acetate and propionate but not to the longer chain organic acids. Stimulation of LNCaP cells with butyrate inhibited their growth, and the knockdown of the endogenous OR51E1 negated this cytostatic effect. Most significantly, overexpression of OR51E1 or OR51E2 suppressed LNCaP cell proliferation. Overexpression of another ectopic OR OR2AT4, β2-adrenergic receptor, or treatment of cells with forskolin did not suppress cell proliferation, indicating that a rise in cAMP is not sufficient to induce cytostasis. Overexpression of OR51E1 caused an upregulation of cytostatic and cell death markers including p27, p21, and p53, strongly increased annexin V staining, and stimulated extracellular signal–regulated protein kinases 1 and 2. Overexpression and/or activation of OR51E1 did not affect human embryonic kidney 293 cell proliferation, indicating that cytotoxicity of OR51E1/OR51E2 is specific for LNCaP cells. Together, our results further our understanding of prostate cancer etiology and suggest that ectopic ORs may be useful therapeutic targets.

Comparative Study of the Steroidogenic Effects of Human Chorionic Gonadotropin and Thieno[2,3-D]pyrimidine-Based Allosteric Agonist of Luteinizing Hormone Receptor in Young Adult, Aging and Diabetic Male Rats.

Low-molecular-weight agonists of luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptor (LHCGR), which interact with LHCGR transmembrane allosteric site and, in comparison with gonadotropins, more selectively activate intracellular effectors, are currently being developed. Meanwhile, their effects on testicular steroidogenesis have not been studied. The purpose of this work is to perform a comparative study of the effects of 5-amino-N-tert-butyl-4-(3-(1-methylpyrazole-4-carboxamido)phenyl)-2-(methylthio)thieno[2,3-d] pyrimidine-6-carboxamide (TP4/2), a LHCGR allosteric agonist developed by us, and hCG on adenylyl cyclase activity in rat testicular membranes, testosterone levels, testicular steroidogenesis and spermatogenesis in young (four-month-old), aging (18-month-old) and diabetic male Wistar rats. Type 1 diabetes was caused by a single streptozotocin (50 mg/kg) injection. TP4/2 (20 mg/kg/day) and hCG (20 IU/rat/day) were administered for 5 days. TP4/2 was less effective in adenylyl cyclase stimulation and ability to activate steroidogenesis when administered once into rats. On the 3rd–5th day, TP4/2 and hCG steroidogenic effects in young adult, aging and diabetic rats were comparable. Unlike hCG, TP4/2 did not inhibit LHCGR gene expression and did not hyperstimulate the testicular steroidogenesis system, moderately increasing steroidogenic proteins gene expression and testosterone production. In aging and diabetic testes, TP4/2 improved spermatogenesis. Thus, during five-day administration, TP4/2 steadily stimulates testicular steroidogenesis, and can be used to prevent androgen deficiency in aging and diabetes.