Adenylate Cyclase Activator Forskolin Research Articles

Endocrine-Disruptive Effects of Adenylate Cyclase Activator Forskolin: In Vitro and In Vivo Evidence

Endocrine-Disruptive Effects of Adenylate Cyclase Activator Forskolin: In Vitro and In Vivo Evidence

Hormonally and chemically defined expansion conditions for organoids of biliary tree Stem Cells

Wholly defined ex vivo expansion conditions for biliary tree stem cell (BTSC) organoids were established, consisting of a defined proliferative medium (DPM) used in combination with soft hyaluronan hydrogels. The DPM consisted of commercially available Kubota's Medium (KM), to which a set of small molecules, particular paracrine signals, and heparan sulfate (HS) were added. The small molecules used were DNA methyltransferase inhibitor (RG108), TGF- β Type I receptor inhibitor (A83-01), adenylate cyclase activator (Forskolin), and L-type Ca2+ channel agonist (Bay K8644). A key paracrine signal proved to be R-spondin 1 (RSPO1), a secreted protein that activates Wnts. Soluble hyaluronans, 0.05 % sodium hyaluronate, were used with DPM to expand monolayer cultures. Expansion of organoids was achieved by using DPM in combination with embedding organoids in Matrigel that was replaced with a defined thiol-hyaluronan triggered with PEGDA to form a hydrogel with a rheology [G*] of less than 100 Pa. The combination is called the BTSC-Expansion-Glycogel-System (BEX-gel system) for expanding BTSCs as a monolayer or as organoids. The BTSC organoids were expanded more than 3000-fold ex vivo in the BEX-gel system within 70 days while maintaining phenotypic traits indicative of stem/progenitors. Stem-cell-patch grafting of expanded BTSC organoids was performed on the livers of Fah-/- mice with tyrosinemia and resulted in the rescue of the mice and restoration of their normal liver functions. The BEX-gel system for BTSC organoid expansion provides a strategy to generate sufficient numbers of organoids for the therapeutic treatments of liver diseases.

PPARγ activation by lipolysis-generated ligands is required for cAMP dependent UCP1 induction in human thermogenic adipocytes.

The uncoupling protein 1 (UCP1) is induced in brown or "beige" adipocytes through catecholamine-induced cAMP signaling, which activates diverse transcription factors. UCP1 expression can also be enhanced by PPARγ agonists such as rosiglitazone (Rsg). However, it is unclear whether this upregulation results from de-novo differentiation of beige adipocytes from progenitor cells, or from the induction of UCP1 in pre-existing adipocytes. To explore this, we employed human adipocytes differentiated from progenitor cells and examined their acute response to Rsg, to the adenylate-cyclase activator forskolin (Fsk), or to both simultaneously. Adipocytes generated from primary human progenitor cells were differentiated without exposure to PPARγ agonists, and treated for 3, 6 or 78 hours to Fsk, to Rsg, or to both simultaneously. Bulk RNASeq, RNAScope, RT-PCR, CRISPR-Cas9 mediated knockout, oxygen consumption and western blotting were used to assess cellular responses. UCP1 mRNA expression was induced within 3 hours of exposure to either Rsg or Fsk, indicating that Rsg's effect is independent on additional adipocyte differentiation. Although Rsg and Fsk induced distinct overall transcriptional responses, both induced genes associated with calcium metabolism, lipid droplet assembly, and mitochondrial remodeling, denoting core features of human adipocyte beiging. Unexpectedly, we found that Fsk-induced UCP1 expression was reduced by approximately 80% following CRISPR-Cas9-mediated knockout of PNPLA2 , the gene encoding the triglyceride lipase ATGL. As anticipated, ATGL knockout suppressed lipolysis; however, the associated suppression of UCP1 induction indicates that maximal cAMP-mediated UCP1 induction requires products of ATGL-catalyzed lipolysis. Supporting this, we observed that the reduction in Fsk-stimulated UCP1 induction caused by ATGL knockout was reversed by Rsg, implying that the role of lipolysis in this process is to generate natural PPARγ agonists. UCP1 transcription is known to be stimulated by transcription factors activated downstream of cAMP-dependent protein kinases. Here we demonstrate that UCP1 transcription can also be acutely induced through PPARγ-activation. Moreover, both pathways are activated in human adipocytes in response to cAMP, synergistically inducing UCP1 expression. The stimulation of PPARγ in response to cAMP occurs as a result of the production of natural PPARγ activating ligands through ATGL-mediated lipolysis.

Intestinal barrier function in the naked mole-rat: an emergent model for gastrointestinal insights.

The intestinal barrier plays a crucial role in homeostasis by both facilitating the absorption of nutrients and fluids and providing a tight shield to prevent the invasion by either pathogen or commensal microorganisms. Intestinal barrier malfunction is associated with systemic inflammation, oxidative stress, and decreased insulin sensitivity, which may lead to the dysregulation of other tissues. Therefore, a deeper understanding of physiological aspects related to an enhanced barrier function is of significant scientific and clinical relevance. The naked mole-rat has many unusual biological features, including attenuated colonic neuron sensitivity to acid and bradykinin and resistance to chemical-induced intestinal damage. However, insight into their intestinal barrier physiology is scarce. Here, we observed notable macroscopic and microscopic differences in intestinal tissue structure between naked mole-rats and mice. Moreover, naked mole-rats showed increased number of larger goblet cells and elevated mucus content. In measuring gut permeability, naked mole-rats showed reduced permeability compared with mice, measured as transepithelial electrical resistance, especially in ileum. Furthermore, intestinal ion secretion induced by serotonin, bradykinin, histamine, and capsaicin was significantly reduced in naked mole-rats compared with mice, despite the expression of receptors for all these agonists. In addition, naked mole-rats exhibited reduced prosecretory responses to the nonselective adenylate cyclase activator forskolin. Collectively, these findings indicate that naked mole-rats possess a robust and hard-to-penetrate gastrointestinal barrier that is resistant to environmental and endogenous irritants. Naked mole-rats may therefore provide valuable insights into the physiology of the intestinal barrier and set the stage for the development of innovative and effective therapies.NEW & NOTEWORTHY This is the first study to characterize the intestinal function of naked mole-rats. We found that these animals show a robust gut tissue structure, displaying thicker intestinal layers, longer villi, and larger crypts. Naked mole-rats showed more and larger goblet cells, with increased mucus content. Intestinal permeability, especially in the ileum, was substantially lower than that of mice. Finally, naked mole-rats showed reduced intestinal anion secretion in response to serotonin, bradykinin, histamine, capsaicin, and forskolin.

Pharmacological Evidence That Dictyostelium Differentiation-Inducing Factor 1 Promotes Glucose Uptake Partly via an Increase in Intracellular cAMP Content in Mouse 3T3-L1 Cells

Differentiation-inducing factor 1 (DIF-1) isolated from the cellular slime mold Dictyostelium discoideum can inhibit mammalian calmodulin-dependent cAMP/cGMP phosphodiesterase (PDE1) in vitro. DIF-1 also promotes glucose uptake, at least in part, via a mitochondria- and AMPK-dependent pathway in mouse 3T3-L1 fibroblast cells, but the mechanism underlying this effect has not been fully elucidated. In this study, we investigated the effects of DIF-1 on intracellular cAMP and cGMP levels, as well as the effects that DIF-1 and several compounds that increase cAMP and cGMP levels have on glucose uptake in confluent 3T3-L1 cells. DIF-1 at 20 μM (a concentration that promotes glucose uptake) increased the level of intracellular cAMP by about 20% but did not affect the level of intracellular cGMP. Neither the PDE1 inhibitor 8-methoxymethyl-3-isobutyl-1-methylxanthine at 10–200 μM nor the broad-range PDE inhibitor 3-isobutyl-1-methylxanthine at 40–400 μM had any marked effects on glucose uptake. The membrane-permeable cAMP analog 8-bromo-cAMP at 200–1000 μM significantly promoted glucose uptake (by 20–25%), whereas the membrane-permeable cGMP analog 8-bromo-cGMP at 3–100 μM did not affect glucose uptake. The adenylate cyclase activator forskolin at 1–10 μM promoted glucose uptake by 20–30%. Thus, DIF-1 may promote glucose uptake by 3T3-L1 cells, at least in part, via an increase in intracellular cAMP level.

Reproductive toxicity of bisphenol A, at environmentally relevant concentrations, on ovarian redox balance, maturational response, and intra-oocyte signalling events in Labeo bata

Bisphenol A (BPA) is a widely used plastic monomer that potentially interferes with ovarian neuroendocrine, endocrine, and autocrine/paracrine factors, causing reproductive dysfunction. However, the influence of BPA on redox balance, estrogen receptor (ER) expression vis-à-vis meiotic cell cycle progression, and intra-oocyte signalling events has not been extensively investigated. The present study examines the impact of BPA on reproductive toxicity in female Labeo bata (Order Cypriniformes, Family Cyprinidae), a freshwater teleost preferred as a food fish in the Indian subcontinent. Our results show that while ovarian weight (gonadosomatic index, GSI) and dynamics of follicular growth undergo pronounced changes during the annual reproductive cycle, chronic BPA exposure at environmentally relevant concentrations promotes follicular atresia concomitant with reduced GSI during the spawning phase, the highest response being observed due to low-dose (0.1 μg/L, 0.438 nM) BPA exposure in vivo. Furthermore, BPA perturbation of ovarian StAR expression and ERα/ERβ homeostasis corroborates with elevated oxidative stress in BPA-treated ovary, FG follicles, and follicular cells. A sharp increase in ROS accumulation and nitric oxide (NO) levels in BPA-treated full-grown (FG) follicles coupled with loss of redox balance, elevated follicular cell death, and activation of apoptotic markers (caspase -8, -9, -3, Bax) indicate poor oocyte health and reproductive toxicity. Importantly, maturational steroid (MIS, 17,20β-P)-induced cyclin B-p34cdc2 activation and elevated GVBD (germinal vesicle breakdown) response require protein kinase A (PKA) inhibition and participation of Mos/MAPK- and cdc25-mediated signalling events. While the adenylate cyclase activator forskolin (FK) abrogates, priming with a PKA inhibitor (H89) promotes the meiotic G2-M1 transition, confirming the role of PKA in meiotic cell cycle progression in this species. Furthermore, the negative influence of BPA priming on 17,20β-P-induced oocyte maturation involves elevated PKAc phosphorylation (activation) and significant alteration in Mos/MAPK signalling, indicating derailed meiotic maturational competence and disrupted oocyte quality.

Influence of obesity on the tone of bronchial smooth muscles in rats

Background. Overweight and obesity are key factors for the occurrence of many morphofunctional disorders in organs and tissues, including bronchopulmonary system.The aim. To study the influence of metabolic disorders that occur against the background of obesity on the state of the airways tone in rats.Materials and methods. Obesity in male Wistar rats was induced using a high-fat and high-carbohydrate diet. In animals, body weight and fat mass were measured, and the heart-lung complex was extracted. In blood serum, the levels of glucose, insulin, leptin, triglycerides, and cholesterol were assessed. Bronchoalveolar lavage fluid was obtained by an open method, in which the concentration of protein, interleukin (IL) 6 and IL-10 was determined. The contractile activity of the isolated bronchial smooth muscle segments was studied using mechanographic method. The effect of acetylcholine (10–7–10–4 M), indomethacin (10–5 M), and forskolin (10–7–10–5 M) on the changes in the tone of airway smooth muscles was assessed.Results. High-fat and high-carbohydrate diet caused an increase in body weight, visceral obesity, hyperglycemia, insulin resistance, leptinemia, dyslipidemia in rats of the experimental group. In the bronchoalveolar lavage fluid of experimental animals, an increase in the content of protein and IL-6 was found, which positively correlated with the level of leptin and the fat mass. In obese rats, the contractile responses of bronchial smooth muscle segments increased in response to the effect of the cholinergic agent acetylcholine. The bronchoconstrictor effect of acetylcholine was reduced by the cyclooxygenase inhibitor indomethacin. In turn, the adenylate cyclase activator forskolin caused relaxation of the airway segments smooth muscles in rats of both groups, which was more pronounced in the experimental group.Conclusion. The obtained results indicate that the change in the reactivity of the respiratory tract can be the cause of bronchospastic conditions in obesity and of the inflammatory reaction in the respiratory system induced by obesity.

Forskolin versus cAMP-Induced Decidualization and Survival of Endometrial Stromal Cells of Endometriosis Patients.

Impairment of decidualization of eutopic human endometrial stromal cells (hESCs) may cause an increase in cell survival of endometrial tissue in the peritoneal cavity constituting a precondition for endometriosis development. Decidualization is a physiological process involving progesterone action and cAMP signaling. We here evaluated the effect of 8-Br-cAMP, the adenylate cyclase activator forskolin and of the progestin progesterone and medroxyprogesterone acetate (MPA) alone and in combination on decidualization induction using prolactin ELISA, and on cell size, cell granularity, and cell survival via flow cytometry in hESCs of patients with and without endometriosis. While progestins alone did not induce functional decidualization in hESCs, 8-Br-cAMP and forskolin induced decidualization in hESCs from both cohorts, whereas the induction of FOXO1 transcription and prolactin secretion by forskolin was significantly lower than by 8-Br-cAMP. 8-Br-cAMP- and forskolin-induced prolactin secretion was significantly enhanced by MPA, but not by progesterone. Decidualization entailed a decrease in cell size and in cell granularity. In general, hESCs from women with mild (ASRM I/II) as well as severe (ASRM III/IV) endometriosis in trend displayed a higher granularity, whereas mainly hESCs from severe endometriosis showed a stronger resistance to the induction of cell death after decidualization induction. In both cohorts, the amount of the decidual marker protein prolactin rather exhibited an anti-proportional correlation to cell death induction during six day treatment. This study contributes to widen our understanding of the connection of decidualization and cell death in endometriosis.

Increase of c-FOS promoter transcriptional activity by the dual leucine zipper kinase

The dual leucine zipper kinase (DLK) and the ubiquitously expressed transcription factor c-FOS have important roles in beta-cell proliferation and function. Some studies in neuronal cells suggest that DLK can influence c-FOS expression. Given that c-FOS is mainly regulated at the transcriptional level, the effect of DLK on c-FOS promoter activity was investigated in the beta-cell line HIT. The methods used in this study are the following: Luciferase reporter gene assays, immunoblot analysis, CRISPR-Cas9-mediated genome editing, and real-time quantitative PCR. In the beta-cell line HIT, overexpressed DLK increased c-FOS promoter activity twofold. Using 5′-,3′-promoter deletions, the promoter regions from − 348 to − 339 base pairs (bp) and from a − 284 to − 53 bp conferred basal activity, whereas the promoter region from − 711 to − 348 bp and from − 53 to + 48 bp mediated DLK responsiveness. Mutation of the cAMP response element within the promoter prevented the stimulatory effect of DLK. Treatment of HIT cells with KCl and the adenylate cyclase activator forskolin increased c-FOS promoter transcriptional activity ninefold. Since the transcriptional activity of those promoter fragments activated by KCl and forskolin was decreased by DLK, DLK might interfere with KCl/forskolin-induced signaling. In a newly generated, genome-edited HIT cell line lacking catalytically active DLK, c-Fos mRNA levels were reduced by 80% compared to the wild-type cell line. DLK increased c-FOS promoter activity but decreased stimulated transcriptional activity, suggesting that DLK fine-tunes c-FOS promoter-dependent gene transcription. Moreover, at least in HIT cells, DLK is required for FOS mRNA expression.

Lithium rescues dendritic abnormalities in Ank3 deficiency models through the synergic effects of GSK3β and cyclic AMP signaling pathways.

Bipolar disorder (BD) is a highly heritable mood disorder with intermittent episodes of mania and depression. Lithium is the first-in-line medication to treat BD, but it is only effective in a subset of individuals. Large-scale human genomic studies have repeatedly linked the ANK3 gene (encoding ankyrin-G, AnkG) to BD. Ank3 knockout mouse models mimic BD behavioral features and respond positively to lithium treatment. We investigated cellular phenotypes associated with BD, including dendritic arborization of pyramidal neurons and spine morphology in two models: (1) a conditional knockout mouse model which disrupts Ank3 expression in adult forebrain pyramidal neurons, and (2) an AnkG knockdown model in cortical neuron cultures. We observed a decrease in dendrite complexity and a reduction of dendritic spine number in both models, reminiscent of reports in BD. We showed that lithium treatment corrected dendrite and spine deficits in vitro and in vivo. We targeted two signaling pathways known to be affected by lithium using a highly selective GSK3β inhibitor (CHIR99021) and an adenylate cyclase activator (forskolin). In our cortical neuron culture model, CHIR99021 rescues the spine morphology defects caused by AnkG knockdown, whereas forskolin rescued the dendrite complexity deficit. Interestingly, a synergistic action of both drugs was required to rescue dendrite and spine density defects in AnkG knockdown neurons. Altogether, our results suggest that dendritic abnormalities observed in loss of function ANK3 variants and BD patients may be rescued by lithium treatment. Additionally, drugs selectively targeting GSK3β and cAMP pathways could be beneficial in BD.

A1 Adenosine Receptor Activation Inhibits P2X3 Receptor-Mediated ATP Currents in Rat Dorsal Root Ganglion Neurons.

Purinergic signaling is involved in multiple pain processes. P2X3 receptor is a key target in pain therapeutics, while A1 adenosine receptor signaling plays a role in analgesia. However, it remains unclear whether there is a link between them in pain. The present results showed that the A1 adenosine receptor agonist N6-cyclopentyladenosine (CPA) concentration dependently suppressed P2X3 receptor-mediated and α,β-methylene-ATP (α,β-meATP)-evoked inward currents in rat dorsal root ganglion (DRG) neurons. CPA significantly decreased the maximal current response to α,β-meATP, as shown a downward shift of the concentration-response curve for α,β-meATP. CPA suppressed ATP currents in a voltage-independent manner. Inhibition of ATP currents by CPA was completely prevented by the A1 adenosine receptor antagonist KW-3902, and disappeared after the intracellular dialysis of either the Gi/o protein inhibitor pertussis toxin, the adenylate cyclase activator forskolin, or the cAMP analog 8-Br-cAMP. Moreover, CPA suppressed the membrane potential depolarization and action potential bursts, which were induced by α,β-meATP in DRG neurons. Finally, CPA relieved α,β-meATP-induced nociceptive behaviors in rats by activating peripheral A1 adenosine receptors. These results indicated that CPA inhibited the activity of P2X3 receptors in rat primary sensory neurons by activating A1 adenosine receptors and its downstream cAMP signaling pathway, revealing a novel peripheral mechanism underlying its analgesic effect.

Можливе значення аденілатциклазного сигнального шляху в синтезі оксиду азоту мітохондріями міометрія

NO synthase activity (mtNOS) in uterine smooth muscle mitochondria under the action of the cAMP/protein kinase A signaling system modulators was studied. The experiments were performed on isolated mitochondria from rat myometrium using the NO-sensitive fluorescent probe DAF-FM-DA. NO synthesis in mitochondria was increased by adenylate cyclase activators NaHCO3 (30 mM) and forskolin (10 μM), as well as phosphodiesterase inhibitor caffeine (1 mM). The addition of ATP (0.5-5 mM) caused a slight increase in nitric oxide synthesis. The effect of ATP was enhanced in the presence of NaHCO3 and caffeine. The intensity of NO formation in mitochondria decreased by approximately 50 % in the case of inhibition of adenylate cyclase activity by the compound KH7 (25 μM). In the presence of the protein kinase A inhibitor PKI (10 nM) NO synthesis in mitochondria was also significantly reduced. When the constitutive NO-synthase inhibitor L-NAME (100 μM) was introduced into the incubation medium, the stimulating effect of the studied compounds on NO synthesis in mitochondria was not observed. These data suggests a possible dependence of mtNOS function on the activity of the cAMP/protein kinase A signaling system in smooth muscle mitochondria.

Adenylate cyclase type 9 antagonizes cAMP accumulation and regulates endothelial signalling involved in atheroprotection.

The adenylate cyclase type 9 (ADCY9) gene appears to determine atherosclerotic outcomes in patients treated with dalcetrapib. In mice, we recently demonstrated that Adcy9 inactivation potentiates endothelial function and inhibits atherogenesis. The objective of this study was to characterize the contribution of ADCY9 to the regulation of endothelial signalling pathways involved in atherosclerosis. We show that ADCY9 is expressed in the endothelium of mouse aorta and femoral arteries. We demonstrate that ADCY9 inactivation in cultured endothelial cells paradoxically increases cAMP accumulation in response to the adenylate cyclase activators forskolin and vasoactive intestinal peptide (VIP). Reciprocally, ADCY9 overexpression decreases cAMP production. Using mouse femoral artery arteriography, we show that Adcy9 inactivation potentiates VIP-induced endothelial-dependent vasodilation. Moreover, Adcy9 inactivation reduces mouse atheroma endothelial permeability in different vascular beds. ADCY9 overexpression reduces forskolin-induced phosphorylation of Ser157-vasodilator-stimulated phosphoprotein (VASP) and worsens thrombin-induced fall of RAP1 activity, both leading to increased endothelial permeability. ADCY9 inactivation in thrombin-stimulated human coronary artery endothelial cells results in cAMP accumulation, increases p-Ser157-VASP, and inhibits endothelial permeability. MLC2 phosphorylation and actin stress fibre increases in response to thrombin were reduced by ADCY9 inactivation, suggesting actin cytoskeleton regulation. Finally, using the Miles assay, we demonstrate that Adcy9 regulates thrombin-induced endothelial permeability in vivo in normal and atherosclerotic animals. Adcy9 is expressed in endothelial cells and regulates local cAMP and endothelial functions including permeability relevant to atherogenesis.

Ser14‐Rpn6/PSMD11 Phosphorylation Mediates the Activation of 26S Proteasomes by cAMP and Protects against Cardiac Proteotoxic Stress in Mice

BackgroundMeans to enhance proteasome (PSM) functioning are highly sought. In cultured cells, cAMP‐dependent protein kinase (PKA) was shown to activate 26S PSMs by phosphorylating Ser14 of Rpn6/PSMD11 (a key PSM subunit), but this remains to be established in animals. Hence, we sought to test the hypothesis that Ser14‐Rpn6 phosphorylation (pS14‐Rpn6) mediates the activation of PSMs by PKA and reduces proteotoxicity in vivo (Figure 1).Methods and ResultsWe generated via CRSPR/Cas9 gene editing two mouse models, in which the codon for Ser14 in the Rpn6 gene was mutated to encode either Alanine (S14A) for blocking pS14‐Rpn6 or Aspartic acid (S14D) for mimicking this phosphorylation. Treatment with adenylate cyclase activator forskolin (5mg/kg, i.p.) increased cardiac pS14‐Rpn6 (2.52±0.08 vs. 1.0, p<0.001) and PSM activities (292.35±45.75 vs. 100, p<0.005) in wild type mice, but these were abolished by co‐treatment of PKA inhibitor H89 (15mg/kg). Forskolin did not show these effects in S14A mice, demonstrating that pS14‐Rpn6 is required for PKA to activate the PSM. Cross‐breeding transgenic GFPdgn (a proven substrate of the ubiquitin‐proteasome system [UPS]) into S14D mice revealed that S14D decreased cardiac GFPdgn proteins (0.81±0.08 vs. 1.0±0.08, p<0.05) without altering GFPdgn mRNA levels, indicating that S14D suffices to enhance UPS performance. Basal myocardial PSM activities were significantly higher in S14D mice compared to WT mice (1.52±0.38 vs. 1.0±0.16, p<0.005). To determine the effect of increasing pS14‐Rpn6 on proteotoxicity, we cross‐bred S14D into the CryAB‐R120G mice (R120G), a classic model of cardiac proteotoxicity. We detected that myocardial total (0.76±0.07 vs. 1.0±0.19, p<0.005) and insoluble (0.80±0.12 vs. 1.0±0.14, p<0.01) CryAB‐R120G protein levels were lower in S14D::R120G compared to WT::R120G mice (n=4 males+4 females/group), suggesting that PSM activation by PKA decreases the accumulation and aggregation of misfolded proteins in vivo. Echocardiography further revealed that S14D significantly attenuated the decrease of left ventricular (LV) ejection fraction (51.80±11.65 vs. 40.20±13.88 %, p<0.01), stroke volume (38.44±7.30 vs. 28.65±5.69 μl, p<0.005) and cardiac output (17.45±3.09 vs. 12.77±2.46 ml/min, p<0.005) of R120G mice at 6 months of age, which indicates that genetic mimicry of pS14‐Rpn6 ameliorates cardiac proteotoxicity.ConclusionsWe have established for the first time in vivo that pS14‐Rpn6 is required for activation of 26S PSMs by PKA and provides the first genetic evidence that PKA‐induced PSM activation protects against proteotoxicity in animals.

The Adenylate Cyclase Activator Forskolin Potentiates the Positive Inotropic Effect of the Phosphodiesterase Inhibitor Milrinone But Not of the Calcium Sensitizer Levosimendan nor of Its Hemodynamically Active Metabolites: An Apparent Conundrum.

OR-1855 and OR-1896 are 2 hemodynamically active metabolites of the inodilator levosimendan, with calcium sensitizing activity, but their mechanism of action is still not fully understood. It has been previously reported that the positive inotropic effect of levosimendan is not potentiated by the adenylate cyclase activator forskolin, whereas forskolin does potentiate the effects of the phosphodiesterase (PDE) inhibitor milrinone. To ascertain whether the active metabolites follow the same pattern of levosimendan, the positive inotropic effects of OR- 1855 and OR-1896 were studied in guinea-pig-isolated papillary muscle in the presence and absence of forskolin. OR-1855 and OR-1896 were also tested as inhibitors of PDE-III and PDE-IV. Our results show that 0.1 µM forskolin did not potentiate the positive inotropic effect of OR-1855 or OR-1896, as in the case of the parent compound levosimendan. As in previous studies, the positive inotropic effect of milrinone was markedly potentiated in the presence of forskolin. From these data, we propose an explanation for the divergent behavior of the calcium sensitizing drugs and PDE inhibitors.

History-dependent dopamine release increases cAMP levels in most basal amygdala glutamatergic neurons to control learning.

Dopaminergic inputs to basal amygdala (BA) instruct learning of motivational salience. This learning depends on intracellular plasticity signals such as cyclic adenosine monophosphate (cAMP), which is regulated by activation of dopamine receptors. We examine the dynamics of dopamine release and downstream signaling during multiple salient events occurring within tens of seconds. We perform real-time tracking and manipulation of cAMP in BA neurons invitro and invivo. Optogenetically evoked release of dopamine drives proportional increases in cAMP in almost all BA glutamatergic neurons, suggesting widespread actions of dopamine across neurons preferring positive or negative valence. This cAMP response decreases across trials with short intertrial intervals owing to depression of dopamine release. No such depression is evident when photostimulating cAMP production directly. cAMP and protein kinase A responses to repeated appetitive or aversive stimuli also exhibit pronounced depression. Thus, history-dependent dynamics of dopamine and cAMP may regulate learning of temporally clustered, salient stimuli.

Suppression of ASIC activity by the activation of A1 adenosine receptors in rat primary sensory neurons

Peripheral A1 adenosine receptor signaling has been shown to have analgesic effects in a variety of pain conditions. However, it is not yet fully elucidated for the precise molecular mechanisms. Acid sensing ion channels (ASICs) are expressed predominantly in nociceptive sensory neurons responding to protons. Given that both A1 adenosine receptors and ASICs are present in dorsal root ganglia (DRG) neurons, we therefore investigated whether there was a cross-talk between the two types of receptors. Herein, electrophysiological recordings showed that the A1 adenosine receptor agonist N6-cyclopentyladenosine (CPA) suppressed acid-induced currents and action potentials, which were mediated by ASICs, in rat DRG neurons. CPA inhibited the maximum response to protons, as shown a downward shift of concentration-response curve for protons. The CPA-induced suppression of ASIC currents was blocked by the A1 adenosine receptor antagonist KW-3902 and also prevented by intracellular application of the Gi/o-protein inhibitor pertussis toxin, the adenylate cyclase activator forskolin, and the cAMP analog 8-Br-cAMP. Finally, intraplantar pretreatment of CPA dose-dependently relieved acid-induced nociceptive responses in rats through peripheral A1 adenosine receptors. These results suggested that CPA suppressed ASICs via A1 adenosine receptors and intracellular Gi/o-proteins and cAMP signaling cascades in rat DRG neurons, which was a novel potential mechanism underlying analgesia of peripheral A1 adenosine receptors.

Suppression of P2X3 receptor-mediated currents by the activation of α2A -adrenergic receptors in rat dorsal root ganglion neurons.

AimsThe α2‐adrenergic receptor (α2‐AR) agonists have been shown to be effective in the treatment of various pain. For example, dexmedetomidine (DEX), a selective α2A‐AR agonist, can be used for peripheral analgesia. However, it is not yet fully elucidated for the precise molecular mechanisms. P2X3 receptor is a major receptor processing nociceptive information in primary sensory neurons. Herein, we show that a functional interaction of α2A‐ARs and P2X3 receptors in dorsal root ganglia (DRG) neurons could contribute to peripheral analgesia of DEX.MethodsElectrophysiological recordings were carried out on rat DRG neurons, and nociceptive behavior was quantified in rats.ResultsThe activation of α2A‐ARs by DEX suppressed P2X3 receptor‐mediated and α,β‐methylene‐ATP (α,β‐meATP)‐evoked inward currents in a concentration‐dependent and voltage‐independent manner. Pre‐application of DEX shifted the α,β‐meATP concentration‐response curve downwards, with a decrease of 50.43 ± 4.75% in the maximal current response of P2X3 receptors to α,β‐meATP in the presence of DEX. Suppression of α,β‐meATP‐evoked currents by DEX was blocked by the α2A‐AR antagonist BRL44408 and prevented by intracellular application of the Gi/o protein inhibitor pertussis toxin, the adenylate cyclase activator forskolin, and the cAMP analog 8‐Br‐cAMP. DEX also suppressed α,β‐meATP‐evoked action potentials through α2A‐ARs in rat DRG neurons. Finally, the activation of peripheral α2A‐ARs by DEX had an analgesic effect on the α,β‐meATP‐induced nociception.ConclusionsThese results suggested that activation of α2A‐ARs by DEX suppressed P2X3 receptor‐mediated electrophysiological and behavioral activity via a Gi/o proteins and cAMP signaling pathway, which was a novel potential mechanism underlying analgesia of peripheral α2A‐AR agonists.

Role of Membrane Lipid Rafts in MRP4 (ABCC4) Dependent Regulation of the cAMP Pathway in Blood Platelets.

Platelet cytosolic cyclic adenosine monophosphate (cAMP) levels are balanced by synthesis, degradation, and efflux. Efflux can occur via multidrug resistant protein-4 (MRP4; ABCC4) present on dense granule and/or plasma membranes. As lipid rafts have been shown to interfere on cAMP homeostasis, we evaluated the relationships between the distribution and activity of MRP4 in lipid rafts and cAMP efflux. Platelet activation and cAMP homeostasis were analyzed in human and wild-type or MRP4-deleted mouse platelets in the presence of methyl-β-cyclodextrin (MßCD) to disrupt lipid rafts, and of activators of the cAMP signalling pathways. Human platelet MRP4 and effector proteins of the cAMP pathway were analyzed by immunoblots in lipid rafts isolated by differential centrifugation. MßCD dose dependently inhibited human and mouse platelet aggregation without affecting per se cAMP levels. An additive inhibitory effect existed between the adenylate cyclase (AC) activator forskolin and MßCD that was accompanied by an overincrease of cAMP, and which was significantly enhanced upon MRP4 deletion. Finally, an efflux of cAMP out of resting platelets incubated with prostaglandin E1 (PGE1) was observed that was partly dependent on MRP4. Lipid rafts contained a small fraction (≈15%) of MRP4 and most of the inhibitory G-protein Gi, whereas Gs protein, AC3, and phosphodiesterases PDE2 and PDE3A were all present as only trace amounts. Our results are in favour of part of MRP4 present at the platelet surface, including in lipid rafts. Lipid raft integrity is necessary for cAMP signalling regulation, although MRP4 and most players of cAMP homeostasis are essentially located outside rafts.

Opposing functions of α- and β-adrenoceptors in the formation of processes by cultured astrocytes

Astrocytes are glial cells with numerous fine processes which are important for the functions of the central nervous system. The activation of β-adrenoceptors induces process formation of astrocytes via cyclic AMP (cAMP) signaling. However, the role of α-adrenoceptors in the astrocyte morphology has not been elucidated. Here, we examined it by using cultured astrocytes from neonatal rat spinal cords and cortices. Exposure of these cells to noradrenaline and the β-adrenoceptor agonist isoproterenol increased intracellular cAMP levels and induced the formation of processes. Noradrenaline-induced process formation was enhanced with the α1-adrenoceptor antagonist prazosin and α2-adrenoceptor antagonist atipamezole. Atipamezole also enhanced noradrenaline-induced cAMP elevation. Isoproterenol-induced process formation was not inhibited by the α1-adrenoceptor agonist phenylephrine but was inhibited by the α2-adrenoceptor agonist dexmedetomidine. Dexmedetomidine also inhibited process formation induced by the adenylate cyclase activator forskolin and the membrane-permeable cAMP analog dibutyryl-cAMP. Moreover, dexmedetomidine inhibited cAMP-independent process formation induced by adenosine or the Rho-associated kinase inhibitor Y27632. In the presence of propranolol, noradrenaline inhibited Y27632-induced process formation, which was abolished by prazosin or atipamezole. These results demonstrate that α-adrenoceptors inhibit both cAMP-dependent and -independent astrocytic process formation.