Regulation Of Adenylate Cyclase Research Articles

Carbonic anhydrase and soluble adenylate cyclase regulation of cystic fibrosis cellular phenotypes.

Several aspects of the cell biology of cystic fibrosis (CF) epithelial cells are altered including impaired lipid regulation, disrupted intracellular transport, and impaired microtubule regulation. It is unclear how the loss of cystic fibrosis transmembrane conductance regulator (CFTR) function leads to these differences. It is hypothesized that the loss of CFTR function leads to altered regulation of carbonic anhydrase (CA) activity resulting in cellular phenotypic changes. In this study, it is demonstrated that CA2 protein expression is reduced in CF model cells, primary mouse nasal epithelial (MNE) cells, excised MNE tissue, and primary human nasal epithelial cells (P < 0.05). This corresponds to a decrease in CA2 RNA expression measured by qPCR as well as an overall reduction in CA activity in primary CF MNEs. The addition of CFTR-inhibitor-172 to WT MNE cells for ≥24 h mimics the significantly lower protein expression of CA2 in CF cells. Treatment of CF cells with l-phenylalanine (L-Phe), an activator of CA activity, restores endosomal transport through an effect on microtubule regulation in a manner dependent on soluble adenylate cyclase (sAC). This effect can be blocked with the CA2-selective inhibitor dorzolamide. These data suggest that the loss of CFTR function leads to the decreased expression of CA2 resulting in the downstream cell signaling alterations observed in CF.

Oxytocin Downregulates the CaV1.2 L-Type Ca2+ Channel via Gi/cAMP/PKA/CREB Signaling Pathway in Cardiomyocytes.

Oxytocin (OT) and its receptor (OTR) are expressed in the heart and are involved in the physiological cardiovascular functional system. Although it is known that OT/OTR signaling is cardioprotective by reducing the inflammatory response and improving cardiovascular function, the role of OT in the cardiac electrical excitation modulation has not been clarified. This study investigates the molecular mechanism of the action of OT on cardiomyocyte membrane excitation focusing on the L-type Ca2+ channel. Our methodology uses molecular biological methods and a patch-clamp technique on rat cardiomyocytes with OT, combined with several signal inhibitors and/or activators. Our results show that long-term treatment of OT significantly decreases the expression of Cav1.2 mRNA, and reduces the L-type Ca2+ channel current (ICa.L) in cardiomyocytes. OT downregulates the phosphorylated component of a transcription factor adenosine-3′,5′-cyclic monophosphate (cAMP) response element binding protein (CREB), whose action is blocked by OTR antagonist and pertussis toxin, a specific inhibitor of the inhibitory GTP-binding regulators of adenylate cyclase, Gi. On the other hand, the upregulation of Cav1.2 mRNA expression by isoproterenol is halted by OT. Furthermore, inhibition of phospholipase C (PLC) was without effect on the OT action to downregulate Cav1.2 mRNA—which suggests a signal pathway of Gi/protein kinase A (PKA)/CREB mediated by OT/OTR. These findings indicate novel signaling pathways of OT contributing to a downregulation of the Cav1.2-L-type Ca2+ channel in cardiomyocytes.

C-Peptide and Insulin during Combined Intranasal Administration Improve the Metabolic Parameters and Activity of the Adenylate Cyclase System in the Hypothalamus, Myocardium, and Epididymal Fat of Rats with Type 2 Diabetes

C-peptide, a product of proinsulin proteolysis, regulates physiological and biochemical processes by increasing the bioavailability of insulin due to the formation of complexes and also by binding to C-peptide receptors. Under insulin deficiency in severe forms of type 2 diabetes mellitus (DM), the levels of C-peptide and insulin in the brain, the target of their action, are decreased. This deficit can be compensated by intranasally administered C-peptide (IACP) and insulin (IAI). It is assumed that the administration of IACP and IAI in type 2 diabetes will lead to the normalization of the activity of the adenylate cyclase signal system (ACSS) in the hypothalamus and on the periphery and will provide, thereby, a therapeutic effect of IACP and IAI. The aim of the work was to study the effect of a 9-day treatment of 4-month-old male rats with the neonatal model of type 2 diabetes with IACP (at a daily dose of 10 μg per animal) and IAI (at a daily dose of 20 μg per animal) under their combined and separate administration on metabolic and hormonal indicators and the activity of ACSS in the hypothalamus, myocardium and epididymal fat (EF). The treatment of diabetic rats with IAI and IACP + IAI weakened hyperglycemia and insulin resistance, improved lipid metabolism and decreased atherogenic index. In the hypothalamus, the regulation of adenylate cyclase (AC) activity by MC4-melanocortin and D2-dopamine receptor agonists weakened in type 2 diabetes was restored. In the myocardium and EF, the restoration of stimulating effects of β1/β2- and β3-adrenoreceptor agonists on the AC activity were noted, which indicates an improvement in hormonal regulation of the cardiovascular system and lipolytic processes in the adipose tissue. The combined use of IACP + IAI was more effective than the IAI monotherapy, which evidences an increase in the regulatory effects of insulin in the presence of C-peptide. The treatment of diabetic rats with IACP was not very effective. The obtained results suggest the prospects for the combined use of equimolar amounts of IACP and IAI for the correction of metabolic and functional disorders in severe forms of type 2 diabetes.

Sir2D, a Sirtuin family protein, regulates adenylate cyclase A expression through interaction with the MybB transcription factor early in Dictyostelium development upon starvation

Sirtuin interacts with many regulatory proteins involved in energy homeostasis, DNA repair, cell survival, and lifespan extension. We investigated the functional roles of Sir2D during early Dictyostelium development upon starvation. We found that ectopic expression of Sir2D accelerated development among three Sirtuins containing highly homologous catalytic domain sequences to mouse Sirt1. Sir2D expression upregulated adenylate cyclase A (aca) mRNA expression 2, 4 and 6 h after starvation. We have previously reported that nicotinamide, a Sirt1 inhibitor, treatment delayed the development and decreased the expression of aca at 4 h after starvation. Sir2D expressing cells showed resistance against the nicotinamide effect. RNAi-mediated Sir2D knockdown cells were generated, and their development was also delayed. Aca expression was decreased 4 h after starvation. Sir2D expression restored the developmental impairment of Sir2D knockdown cells. The induction of aca upon starvation starts with transcriptional activation of MybB. The ectopic expression of MybB accelerated the development and increased the expression of aca 2 and 4 h after starvation but did not restore the phenotype of Sir2D knockdown cells. Sir2D expression had no effects on MybB-null mutant cells during early development. Thus, MybB is necessary for the upregulation of aca by Sir2D, and Sir2D is necessary for the full induction of aca after 4 h by MybB. MybB was coimmunoprecipitated with Sir2D, suggesting an interaction between MybB and Sir2D. These results suggest that Sir2D regulates aca expression through interaction with the MybB transcription factor early in Dictyostelium development upon starvation.

Forskolin As a Neuroprotector and Modulator of Glutamate-Evoked Ca2+ Entry into Cerebellar Neurons

The analysis of neuronal viability of cerebellar neurons in primary culture during long-lasting (240 min) treatment with glutamate revealed that forskolin (1 μM), an adenylate cyclase activator, prevents apoptosis and necrosis of cells, thus exhibiting neuroprotective properties. As the cytotoxic action of glutamate causes an increase of the intracellular Ca2+ concentration, we further studied forskolin influence on glutamate-evoked Ca2+ responses of neurons. A short-time pretreatment with 1 μM forskolin significantly decreases Ca2+ entry into neurons during glutamate action. The obtained results demonstrate an important role of adenylate cyclase and cAMP in regulation of the Ca2+ entry into neurons and intracellular signaling pathways preventing neuronal death of cerebellar neurons in excytotoxic stress.

Comparative transcriptomic and proteomic analysis of Arthrobacter sp. CGMCC 3584 responding to dissolved oxygen for cAMP production

Arthrobacter sp. CGMCC 3584 is able to produce high yields of extracellular cyclic adenosine monophosphate (cAMP), which plays a vital role in the field of treatment of disease and animal food, during aerobic fermentation. However, the molecular basis of cAMP production in Arthrobacter species is rarely explored. Here, for the first time, we report the comparative transcriptomic and proteomic study of Arthrobacter cells to elucidate the higher productivity of cAMP under high oxygen supply. We finally obtained 14.1% and 19.3% of the Arthrobacter genome genes which were up-regulated and down-regulated notably, respectively, with high oxygen supply, and identified 54 differently expressed proteins. Our results revealed that high oxygen supply had two major effects on metabolism: inhibition of glycolysis, pyruvate metabolism, nitrogen metabolism, and amino acid metabolism (histidine, branched-chain amino acids and glutamate metabolism); enhancement of the tricarboxylic acid cycle and purine metabolism. We also found that regulation of adenylate cyclase and phosphodiesterase was not significant under high oxygen supply, suggesting efficient cAMP export might be important in cAMP production. These findings may contribute to further understanding of capacities of Arthrobacter species and would be highly useful in genetic regulation for desirable production.

Protein Expression Protocol for the Catalytic Domain of an Adenylate Cyclase Membrane-Anchored by a Vibrio Quorum Sensing Receptor.

The direct regulation of a mycobacterial adenylate cyclase (Rv1625c) via exchange of its membrane anchor by the quorum sensing receptor CqsS (Vibrio harveyi) has recently been reported ( Beltz et al., 2016 ). This protocol describes the expression and membrane preparation for these chimeric proteins.

Spatial Memory and the Control of Adenylate Cyclase by Serotonin and Dopamine in the Brain in Rats with Streptozotocin Diabetes

One of the complication of type 1 diabetes mellitus (DM1) is cognitive deficit, developing as a result of neurodegenerative changes in the brain. The aims of the present work were to study learning and spatial memory in rats with streptozotocin DM1 of different durations (1.5 and 6 months) and to investigate the activity of the adenylate cyclase signal system (ACSS) in the brain, this system being sensitive to serotonin and dopamine receptor agonists. Rats with DM1 for 1.5 months showed no impairment to spatial memory in a Morris water maze. When the duration of DM1 was increased to six months, spatial memory and learning ability decreased. Impaired regulation of adenylate cyclase by types 1 and 6 serotonin receptor antagonists and type 2 dopamine receptors was seen at both durations of DM1, indicating that these are primary in the development of cognitive deficit. Impairments to the ACSS can be regarded as key factors in the etiology and pathogenesis of cognitive dysfunctions in DM1. It is hypothesized that cognitive deficit develops only at the late stages of DM1, and results from impairments to the serotonin and dopamine signal systems of the brain.

Adenosine A2A receptor signaling attenuates LPS-induced pro-inflammatory cytokine formation of mouse macrophages by inducing the expression of DUSP1

Adenosine is known to reduce inflammation by suppressing the activity of most immune cells. Previous studies have shown that lipopolysaccharide (LPS) stimulated mouse macrophages produce adenosine, and the adenosine A2A receptor (A2AR) signaling activated in an autocrine manner attenuates LPS-induced pro-inflammatory cytokine formation. It has been suggested that A2AR signaling inhibits LPS-induced pro-inflammatory cytokine production through a unique cAMP-dependent, but PKA- and Epac-independent signaling pathway. However, the mechanism of inhibition was not identified so far. Here we report that LPS stimulation enhances A2AR expression in mouse bone marrow derived macrophages, and loss of A2ARs results in enhanced LPS-induced pro-inflammatory response. Loss of A2ARs in A2AR null macrophages did not alter the LPS-induced NF-κB activation, but an enhanced basal and LPS-induced phosphorylation of MAP kinases (especially that of JNKs) was detected in A2AR null cells. A2AR signaling did not alter the LPS-induced phosphorylation of their upstream kinases, but by regulating adenylate cyclase activity it enhanced the expression of dual specific phosphatase (DUSP)1, a negative regulator of MAP kinases. As a result, lower basal and LPS-induced DUSP1 mRNA and protein levels can be detected in A2AR null macrophages. Silencing of DUSP1 mRNA expression resulted in higher basal and LPS-induced JNK phosphorylation and LPS-induced pro-inflammatory cytokine formation in wild type macrophages, but had no effect on that in A2AR null cells. Our data indicate that A2AR signaling regulates both basal and LPS-induced DUSP1 levels in macrophages via activating the adenylate cyclase pathway.

Synergy Between Gαz Deficiency and GLP-1 Analog Treatment in Preserving Functional β-Cell Mass in Experimental Diabetes.

A defining characteristic of type 1 diabetes mellitus (T1DM) pathophysiology is pancreatic β-cell death and dysfunction, resulting in insufficient insulin secretion to properly control blood glucose levels. Treatments that promote β-cell replication and survival, thus reversing the loss of β-cell mass, while also preserving β-cell function, could lead to a real cure for T1DM. The α-subunit of the heterotrimeric Gz protein, Gαz, is a tonic negative regulator of adenylate cyclase and downstream cAMP production. cAMP is one of a few identified signaling molecules that can simultaneously have a positive impact on pancreatic islet β-cell proliferation, survival, and function. The purpose of our study was to determine whether mice lacking Gαz might be protected, at least partially, from β-cell loss and dysfunction after streptozotocin treatment. We also aimed to determine whether Gαz might act in concert with an activator of the cAMP-stimulatory glucagon-like peptide 1 receptor, exendin-4 (Ex4). Without Ex4 treatment, Gαz-null mice still developed hyperglycemia, albeit delayed. The same finding held true for wild-type mice treated with Ex4. With Ex4 treatment, Gαz-null mice were protected from developing severe hyperglycemia. Immunohistological studies performed on pancreas sections and in vitro apoptosis, cytotoxicity, and survival assays demonstrated a clear effect of Gαz signaling on pancreatic β-cell replication and death; β-cell function was also improved in Gαz-null islets. These data support our hypothesis that a combination of therapies targeting both stimulatory and inhibitory pathways will be more effective than either alone at protecting, preserving, and possibly regenerating β-cell mass and function in T1DM.

Buprenorphine-elicited alteration of adenylate cyclase activity in human embryonic kidney 293 cells coexpressing κ-, μ-opioid and nociceptin receptors.

Buprenorphine, a maintenance drug for heroin addicts, exerts its pharmacological function via κ- (KOP), μ-opioid (MOP) and nociceptin/opioid receptor-like 1 (NOP) receptors. Previously, we investigated its effects in an in vitro model expressing human MOP and NOP receptors individually or simultaneously (MOP, NOP, and MOP+NOP) in human embryonic kidney 293 cells. Here, we expanded this cell model by expressing human KOP, MOP and NOP receptors individually or simultaneously (KOP, KOP+MOP, KOP+NOP and KOP+MOP+NOP). Radioligand binding with tritium-labelled diprenorphine confirmed the expression of KOP receptors. Immunoblotting and immunocytochemistry indicated that the expressed KOP, MOP and NOP receptors are N-linked glycoproteins and colocalized in cytoplasmic compartments. Acute application of the opioid receptor agonists— U-69593, DAMGO and nociceptin— inhibited adenylate cyclase (AC) activity in cells expressing KOP, MOP and NOP receptors respectively. Buprenorphine, when applied acutely, inhibited AC activity to ~90% in cells expressing KOP+MOP+NOP receptors. Chronic exposure to buprenorphine induced concentration-dependent AC superactivation in cells expressing KOP+NOP receptors, and the level of this superactivation was even higher in KOP+MOP+NOP-expressing cells. Our study demonstrated that MOP receptor could enhance AC regulation in the presence of coexpressed KOP and NOP receptors, and NOP receptor is essential for concentration-dependent AC superactivation elicited by chronic buprenorphine exposure.

Hyaluronic acid as capacitation inductor: metabolic changes and membrane-associated adenylate cyclase regulation.

The aim of this research was to study the effect of hyaluronic acid on bovine cryopreserved spermatozoa compared with heparin as regards the variation of capacitation induction, cellular oxidative metabolism and intracellular signal induced by membrane-associated adenylate cyclase to propose hyaluronic acid as a capacitation inductor. Heparin or hyaluronic acid and lysophosphatidylcholine were used to induce sperm capacitation and acrosome reaction, respectively. 2',5'-dideoxyadenosine was used as a membrane-associated adenylate cyclase inhibitor. The highest percentages of capacitated spermatozoa and live spermatozoa with acrosome integrity were obtained by incubating sperm for 60 min using 1000 μg/ml hyaluronic acid. In these conditions, capacitation induced by hyaluronic acid was lower compared with heparin; nonetheless both glycosaminoglycans promote intracellular changes that allow true acrosome reaction in vitro induced by lysophosphatidylcholine in bovine spermatozoa. Oxygen consumption in heparin-capacitated spermatozoa was significantly higher than in hyaluronic acid-treated spermatozoa. With all treatments, mitochondrial coupling was observed when a specific uncoupler of the respiratory chain was added. The inhibition of membrane-associated adenylate cyclase significantly blocked capacitation induction produced by hyaluronic acid, maintaining a basal sperm oxygen uptake in contrast to heparin effect in which both sperm parameters were inhibited, suggesting that the membrane-associated adenylate cyclase activation is involved in the intracellular signal mechanisms induced by both capacitation inductors, but only regulates mitochondrial oxidative phosphorylation in heparin-capacitated spermatozoa.

Dynamics of adenylate cyclase regulation via heterotrimeric G-proteins

A wide variety of G-protein-coupled receptors either activate or inhibit ACs (adenylate cyclases), thereby regulating cellular cAMP levels and consequently inducing proper physiological responses. Stimulatory and inhibitory G-proteins interact directly with ACs, whereas G(q)-coupled receptors exert their effects primarily via Ca2+. Using the FRET-based cAMP sensor Epac1 (exchange protein directly activated by cAMP 1)-cAMPS (adenosine 3',5'-cyclic monophosphorothioate), we studied cAMP levels in single living VSMCs (vascular smooth muscle cells) or HUVECs (human umbilical vein endothelial cells) with subsecond temporal resolution. Stimulation of purinergic (VSMCs) or thrombin (HUVECs) receptors rapidly decreased cAMP levels in the presence of the β-adrenergic agonist isoprenaline via a rise in Ca2+ and subsequent inhibition of AC5 and AC6. Specifically in HUVECs, we observed that, in the continuous presence of thrombin, cAMP levels climbed slowly after the initial decline with a delay of a little less than 1 min. The underlying mechanism includes phospholipase A2 activity and cyclo-oxygenase-mediated synthesis of prostaglandins. We studied further the dynamics of the inhibition of ACs via G(i)-proteins utilizing FRET imaging to resolve interactions between fluorescently labelled G(i)-proteins and AC5. FRET between Gα(i1) and AC5 developed at much lower concentration of agonist compared with the overall G(i)-protein activity. We found the dissociation of Gα(i1) subunits and AC5 to occur slower than the G(i)-protein deactivation. This led us to the conclusion that AC5, by binding active Gα(i1), interferes with G-protein deactivation and reassembly and thereby might sensitize its own regulation.

Effect of peptides corresponding to extracellular domains of serotonin 1B/1D receptors and melanocortin 3 and 4 receptors on hormonal regulation of adenylate cyclase in rat brain.

The ligand-recognizing part of G protein-coupled receptors consists of their extracellular loops and N-terminal domain. Identification of these sites is essential for receptor mapping and for the development and testing of new hormone system regulators. The peptides corresponding by their structure to extracellular loop 2 of serotonin 1B/1D receptor (peptide 1), extracellular loop 3 of melanocortin 3 receptor (peptide 2), and N-terminal domain of melanocortin 4 (peptide 3) were synthesized by the solid-phase method. In synaptosomal membranes isolated from rat brain, peptide 1 (10(-5)-10(-4) M) attenuated the effects of 5-nonyloxytryptamine (selective agonist of serotonin 1B/1D receptor) and to a lesser extent serotonin and 5-methoxy-N,N-dimethyltryptamine acting on all the subtypes of serotonin receptor 1. Peptide 2 (10(-5)-10(-4) M) significantly reduced the adenylate cyclase-stimulating effect of γ-melanocyte-stimulating hormone (agonist of melanocortin receptor 3), but had no effect on the adenylate cyclase effect of THIQ (agonist melanocortin receptor 4). Peptide 3 reduced the adenylate cyclase-stimulating effects of THIQ and α-melanocyte-stimulating hormone (non-selective agonist of melanocortin receptors 3 and 4), but did not modulate the effect of γ-melanocyte-stimulating hormone. The effect of peptide 3 was weaker: it was observed at peptide 3 concentration of 10(-4) M. Peptides 1-3 did no change the adenylate cyclase-modulating effects of hormones acting through non-homologous receptors. Thus, the synthesized peptides specifically inhibited the regulatory effects of hormones acting through homologous receptors. This suggests that the corresponding extracellular domains are involved in ligand recognition and binding and determine functional activity of the receptor.

Pharmacological Profiles of Oligomerized μ-Opioid Receptors

Opioids are widely prescribed pain relievers with multiple side effects and potential complications. They produce analgesia via G-protein-protein coupled receptors: μ-, δ-, κ-opioid and opioid receptor-like 1 receptors. Bivalent ligands targeted to the oligomerized opioid receptors might be the key to developing analgesics without undesired side effects and obtaining effective treatment for opioid addicts. In this review we will update the biological effects of μ-opioids on homo- or hetero-oligomerized μ-opioid receptor and discuss potential mechanisms through which bivalent ligands exert beneficial effects, including adenylate cyclase regulation and receptor-mediated signaling pathways.

Roles of Protein Kinase A and Adenylate Cyclase in Light-Modulated Cellulase Regulation in Trichoderma reesei

The cyclic AMP (cAMP) pathway represents a central signaling cascade with crucial functions in all organisms. Previous studies of Trichoderma reesei (anamorph of Hypocrea jecorina) suggested a function of cAMP signaling in regulation of cellulase gene expression. We were therefore interested in how the crucial components of this pathway, adenylate cyclase (ACY1) and cAMP-dependent protein kinase A (PKA), would affect cellulase gene expression. We found that both ACY1 and PKA catalytic subunit 1 (PKAC1) are involved in regulation of vegetative growth but are not essential for sexual development. Interestingly, our results showed considerably increased transcript abundance of cellulase genes in darkness compared to light (light responsiveness) upon growth on lactose. This effect is strongly enhanced in mutant strains lacking PKAC1 or ACY1. Comparison to the wild type showed that ACY1 has a consistently positive effect on cellulase gene expression in light and darkness, while PKAC1 influences transcript levels of cellulase genes positively in light but negatively in darkness. A function of PKAC1 in light-modulated cellulase gene regulation is also reflected by altered complex formation within the cel6a/cbh2 promoter in light and darkness and in the absence of pkac1. Analysis of transcript levels of cellulase regulator genes indicates that the regulatory output of the cAMP pathway may be established via adjustment of XYR1 abundance. Consequently, both adenylate cyclase and protein kinase A are involved in light-modulated cellulase gene expression in T. reesei and have a dampening effect on the light responsiveness of this process.

Systems analysis of GLP-1 receptor signaling in pancreatic β-cells

Glucagon-like peptide-1 (GLP-1) elevates intracellular concentration of cAMP ([cAMP]) and facilitates glucose-dependent insulin secretion in pancreatic β-cells. There has been much evidence to suggest that multiple key players such as the GLP-1 receptor, G(s) protein, adenylate cyclase (AC), phosphodiesterase (PDE), and intracellular Ca(2+) concentration ([Ca(2+)]) are involved in the regulation of [cAMP]. However, because of complex interactions among these signaling factors, the kinetics of the reaction cascade as well as the activities of ACs and PDEs have not been determined in pancreatic β-cells. We have constructed a minimal mathematical model of GLP-1 receptor signal transduction based on experimental findings obtained mostly in β-cells and insulinoma cell lines. By fitting this theoretical reaction scheme to key experimental records of the GLP-1 response, the parameters determining individual reaction steps were estimated. The model reconstructed satisfactorily the dynamic changes in [cAMP] and predicted the activities of cAMP effectors, protein kinase A (PKA), and cAMP-regulated guanine nucleotide exchange factor [cAMP-GEF or exchange protein directly activated by cAMP (Epac)] during GLP-1 stimulation. The simulations also predicted the presence of two sequential desensitization steps of the GLP1 receptor that occur with fast and very slow reaction rates. The cross talk between glucose- and GLP-1-dependent signal cascades for cAMP synthesis was well reconstructed by integrating the direct regulation of AC and PDE by [Ca(2+)]. To examine robustness of the signaling system in controlling [cAMP], magnitudes of AC and PDE activities were compared in the presence or absence of GLP-1 and/or the PDE inhibitor IBMX.(1).

Yohimbine does not affect opioid receptor activation but prevents adenylate cyclase regulation by morphine in NG108-15 cells

Aims Yohimbine has been shown to modulate the pharmacological actions of opioid drugs in a way that could be of potential therapeutic interest. This work tries to study if this interaction involves the impairment of opioid receptor activation at the cellular level by studying the effects of morphine and yohimbine on NG108-15 neuroblastoma x glioma hybrid cells. Main methods [ 35S]GTPγS binding assays were performed to study δ-opioid and α 2B-adrenoceptor activation by opioid and adrenoceptor agonists in the presence and absence of yohimbine. The effect of morphine was also studied after 6 h pre-incubations with morphine, yohimbine and combinations of these drugs taking into account previous results showing an interaction between both drugs in these conditions. Forskolin-induced cAMP accumulation was also studied by immunoassay in cells incubated with morphine for 6 h in the presence and absence of naloxone and yohimbine. Key findings Yohimbine behaved as a competitive antagonist/inverse agonist on α 2B-adrenoceptors but did not modify G-protein activation by morphine, either in acute conditions or after 6 h of incubation. However, morphine-induced inhibition of cAMP accumulation was prevented both by naloxone and yohimbine when these drugs were present in the incubation medium. Significance Yohimbine seems to desensitise adenylate cyclase to the inhibitory effect of opioid-activated G proteins. This cellular effect could underlie the antagonistic actions of yohimbine on many pharmacological effects of the opioid both in vitro and in vivo.

Photo-dynamics of the BLUF domain containing soluble adenylate cyclase (nPAC) from the amoeboflagellate Naegleria gruberi NEG-M strain

The amoeboflagellate Naegleria gruberi NEG-M comprises a BLUF ( blue light sensor using flavin) regulated adenylate cyclase (nPAC). The nPAC gene was expressed heterologously in Escherichia coli and the photo-dynamics of the nPAC protein was studied by optical absorption and fluorescence spectroscopy. Blue-light exposure of nPAC caused a typical BLUF-type photo-cycle behavior (spectral absorption red-shift, fluorescence quenching, absorption and fluorescence recovery in the dark). Additionally, time-delayed reversible photo-induced one-electron reduction of fully oxidized flavin (Fl ox) to semi-reduced flavin (FlH ) occurred. Furthermore, photo-excitation of FlH caused irreversible electron transfer to fully reduced anionic flavin (FlH −). A photo-induced electron transfer from Tyr or Trp to flavin (Tyr + –Fl − or Trp + –Fl − radical ion-pair formation) is thought to cause H-bond restructuring responsible for BLUF-type photo-cycling and permanent protein re-conformation enabling photo-induced flavin reduction by proton transfer. Some photo-degradation of Fl ox to lumichrome was observed. A model of the photo-dynamics of nPAC is developed.

PPARγ-Dependent Regulation of Adenylate Cyclase 6 Amplifies the Stimulatory Effect of cAMP on Renin Gene Expression

The second messenger cAMP plays an important role in the regulation of renin gene expression. Nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) is known to stimulate renin gene transcription acting through PPARγ-binding sequences in renin promoter. We show now that activation of PPARγ by unsaturated fatty acids or thiazolidinediones drastically augments the cAMP-dependent increase of renin mRNA in the human renin-producing cell line Calu-6. The underlying mechanism involves potentiation of agonist-induced cAMP increase and up-regulation of adenylate cyclase 6 (AC6) gene expression. We identified a palindromic element with a 3-bp spacer (Pal3) in AC6 intron 1 (AC6Pal3). AC6Pal3 bound PPARγ and mediated trans-activation by PPARγ agonist. AC6 knockdown decreased basal renin mRNA level and attenuated the maximal PPARγ-dependent stimulation of the cAMP-induced renin gene expression. AC6Pal3 decoy oligonucleotide abrogated the PPARγ-dependent potentiation of cAMP-induced renin gene expression. Treatment of mice with PPARγ agonist increased AC6 mRNA kidney levels. Our data suggest that in addition to its direct effect on renin gene transcription, PPARγ "sensitizes" renin gene to cAMP via trans-activation of AC6 gene. AC6 has been identified as PPARγ target gene with a functional Pal3 sequence.