Inhibition Of Adenylate Cyclase Research Articles

PGE2 mediates hemocyte-spreading behavior by activating aquaporin via cAMP and rearranging actin cytoskeleton via Ca2+

Spreading behavior of hemocytes (= insect blood cells) is essential for cellular immune responses against various microbial pathogens. It is activated by prostaglandin E2 (PGE2) via its membrane receptor associated with secondary messenger, cAMP, in insects. This study observed an increase of calcium ion (Ca2+) level after an acute increase of cAMP induced by PGE2 treatment and clarified the intracellular signals underlying the hemocyte-spreading behavior. Inhibition of Ca2+ flux significantly impaired the hemocyte-spreading and subsequent cellular immune response, phagocytosis. The up-regulation of intracellular Ca2+ in response to PGE2 was dependent on cAMP because RNA interference (RNAi) of PGE2 receptor expression or inhibiting adenylate cyclase prevented Ca2+ mobilization. The up-regulation of Ca2+ was induced by inositol triphosphate (IP3) via its specific IP3 receptor. Furthermore, inhibition of ryanodine receptor impaired Ca2+ mobilization, suggesting Ca2+-induced Ca2+ release. However, the effective spreading behavior of hemocytes was dependent on both secondary messengers. Ca2+ signal stimulated by cAMP was required for activating small G proteins because RNAi treatments of small G proteins such as Rac1, RhoA, and Cdc42 failed to stimulate hemocyte-spreading. In contrast, aquaporin was activated by cAMP. Its activity was necessary for changing cell volume during hemocyte-spreading. These results indicate that PGE2 mediates hemocyte-spreading via cAMP signal to activate aquaporin and via Ca2+ signal to activate actin cytoskeletal rearrangement.

Nucleobindin-derived nesfatin-1 and nesfatin-1-like peptide stimulate pro-opiomelanocortin synthesis in murine AtT-20 corticotrophs through the cAMP/PKA/CREB signaling pathway

Nucleobindin (NUCB)-derived peptides, nesfatin-1 (NES-1) and nesfatin-1-like peptide (NLP) have several physiological roles in vertebrates. While NES-1 is implicated in stress, whether NUCB1/NLP and NUCB2/NES-1 have any effect on proopiomelanocortin (POMC) remains unknown. The main aim of this study was to determine if NES-1 and/or NLP affect POMC synthesis in mouse corticotrophs. Immunocytochemistry was employed to target NUCB colocalization with POMC in immortalized mouse tumoral corticotrophs (AtT-20 cells). The ability of NES-1 and NLP to modulate POMC mRNA and protein in AtT-20 cells was assessed by qPCR and Western blot, respectively. Moreover, cell-signaling molecules mediating the effect of NES-1 and NLP on POMC synthesis in mouse tumoral corticotrophs were studied using pharmacological blockers. Mouse tumoral corticotrophs showed immunoreactivity for both NUCB1/NLP and NUCB2/NES-1. Both NES-1 and NLP exerted a stimulatory effect on POMC transcript abundance and protein expression in a dose- and time-dependent manner. This effect was comparable to corticotropin-releasing factor (CRF, positive control) stimulation of POMC. Incubation of mouse tumoral corticotrophs with NES-1 or NLP upregulated the phosphorylation of protein kinase A (PKA) and cAMP-response element-binding protein (CREB). The stimulatory effect of these peptides on POMC transcript abundance and protein expression was blocked by the PKA inhibitor, H89, and an adenylate cyclase inhibitor, 2′,3′-dideoxyadenosine (DDA). These pharmacological studies indicate that NES-1 and NLP act through the cAMP/PKA/CREB cellular pathway to stimulate POMC synthesis. Our results provide molecular evidence to support a stimulatory role for nucleobindin-derived peptides on POMC synthesis from corticotrophs. Collectively, this research indicates that corticotrophs produce NUCBs, and the encoded peptides NES-1 and NLP could elicit a direct action to stimulate the pituitary stress hormone. This stimulatory effect is mediated by an uncharacterized G protein-coupled receptor (GPCR) that utilizes the cAMP/PKA/CREB pathway.

Early Effects of the Soluble Amyloid β25-35 Peptide in Rat Cortical Neurons: Modulation of Signal Transduction Mediated by Adenosine and Group I Metabotropic Glutamate Receptors.

The amyloid β peptide (Aβ) is a central player in the neuropathology of Alzheimer’s disease (AD). The alteration of Aβ homeostasis may impact the fine-tuning of cell signaling from the very beginning of the disease, when amyloid plaque is not deposited yet. For this reason, primary culture of rat cortical neurons was exposed to Aβ25-35, a non-oligomerizable form of Aβ. Cell viability, metabotropic glutamate receptors (mGluR) and adenosine receptors (AR) expression and signalling were assessed. Aβ25-35 increased mGluR density and affinity, mainly due to a higher gene expression and protein presence of Group I mGluR (mGluR1 and mGluR5) in the membrane of cortical neurons. Intriguingly, the main effector of group I mGluR, the phospholipase C β1 isoform, was less responsive. Also, the inhibitory action of group II and group III mGluR on adenylate cyclase (AC) activity was unaltered or increased, respectively. Interestingly, pre-treatment of cortical neurons with an antagonist of group I mGluR reduced the Aβ25-35-induced cell death. Besides, Aβ25-35 increased the density of A1R and A2AR, along with an increase in their gene expression. However, while A1R-mediated AC inhibition was increased, the A2AR-mediated stimulation of AC remained unchanged. Therefore, one of the early events that takes place after Aβ25-35 exposure is the up-regulation of adenosine A1R, A2AR, and group I mGluR, and the different impacts on their corresponding signaling pathways. These results emphasize the importance of deciphering the early events and the possible involvement of metabotropic glutamate and adenosine receptors in AD physiopathology.

Inhibition of Adenylate Cyclase of Regeneration-Competent Cells of Nervous Tissue: a Novel Approach for the Treatment of Alcoholic Encephalopathy

Researching new pharmacological targets and developing highly effective drugs to treat ethanol-induced encephalopathy is an urgent task of neuroscience and pharmacology. The work aimed to study the neuroprotective and neuroregenerative effects of adenylate cyclase (AC) inhibitors in modeling alcoholic encephalopathy in experimental animals. The morphofunctional repair of the brain and the functioning of progenitors (neural stem cells (NSC) and neuronal-committed progenitors (NCP)) and neuroglial cells (astrocytes, oligodendrocytes, microglial cells) of the subventricular zone of the cerebral hemispheres (SVZ) have been investigated. A significant correction of brain morphological changes, disorders of exploratory behavior, and conditioned reflex activity in laboratory animals under the influence of the AС inhibitor were revealed. Increased proliferation of both types of progenitor cells and accelerated differentiation of the NSC were observed when AC inhibitors were administered to mice with neurodegeneration. Improved neurotrophin secretion by astrocytes and microglia was also identified. The findings show the promise of developing a novel approach to treating alcohol-induced encephalopathy with AC inhibitors.

Cholinergic regulation of cardiac pacemaker activity by L-type Cav1.3 channels

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Fondation Recherche Médicale Introduction The cholinergic regulation of heart rate (HR) is mediated by acetylcholine (ACh)-dependent activation of M2-receptors (M2R). Activated M2R promote release of the βγ-subunit of G-proteins to directly gate GIRK1/4 channels (underlying the cardiac IKACh current), while αi-subunits inhibit adenylate cyclase (AC) activity. AC inhibition reduces the intracellular concentration of cAMP, decreasing the activity of ion channels involved in pacemaking, including "funny" f-(HCN4) and L-type Cav1.3 calcium channels. Purpose To determine the role of L-type Cav1.3 channels in cholinergic regulation of heart rate. Methods We recorded the frequency of activation and position of pacemaker leading site in ex vivo sinus nodes and the HR of isolated Langendorff perfused hearts of mice at baseline or during ACh perfusion. We used control wild type (WT) mice, and five genetically modified mouse models: Cav1.3 knockout (KO, ablated Cav1.3-mediated L-type current), GIRK4KO (ablated IKACh current), HCN4-CNBD (selective deletion of cAMP-dependent regulation of HCN4), GIRK4KO/HCN4-CNBD and GIRK4KO/Cav1.3KO. We performed in vivo telemetric recordings of heart rate (HR) in WT and GIRK4KO/Cav1.3KO animals. Results Data from optical mapping experiments showed that, under basal conditions, perfusion of 3 μM ACh significantly reduced the frequency of action potentials in WT (44%), HCN4-CNBD (38%), Cav1.3KO (65%) and GIRK4KO (8%) isolated mouse sinus node tissues. ACh application did not significantly affect the frequency of action potentials recorded in tissue from GIRK4KO/HCN4-CNBD and GIRK4KO/Cav1.3KO animals. Furthermore, in all sinus nodes tested, regardless of genotype, ACh shifted the pacemaker leading site from its normal position by at least 0.7 mm. Upon stimulation of the β-adrenergic pathway by Isoproterenol, to reproduce conditions of accentuated antagonism, 3µM ACh reduced HR in isolated hearts from WT (43.8%), HCN4-CNBD (38.7%), Cav1.3KO (25,4%), GIRK4KO (16.9%) and GIRK4KO/HCN4-CNBD (16.4%) mice. No significant HR reduction was recorded in hearts from GIRK4KO/Cav1.3KO animals. In vivo data indicate that HR reduction induced by combined injection of Hexamethonium ( a Nicotinic acetylcholine receptor blocker) with Carbamoylcholine (CCH, M2 receptor agonist) or with 2-Chloro-N6-Cyclopentyladenosine (CCPA, A1 receptor agonist) is higher in WT than in GIRK4KO/Cav1.3KO animals (68% vs 48% CCH, and 79% vs 62% CCPA, respectively). Conclusion Our data indicate that L-type Cav1.3 channels are involved in cholinergic regulation of heart rate in mice. In addition, when the intracellular concentration of cAMP is elevated (i.e. under conditions of accentuated antagonism), cholinergic regulation of sinus node pacemaking is reliant on Cav1.3 and KACh channels.

Carbon monoxide activates large-conductance calcium-activated potassium channels of human cardiac fibroblasts through various mechanisms.

Carbon monoxide (CO) is a cardioprotectant and potential cardiovascular therapeutic agent. Human cardiac fibroblasts (HCFs) are important determinants of myocardial structure and function. Large-conductance Ca2+-activated K+ (BK) channel is a potential therapeutic target for cardiovascular disease. We investigated whether CO modulates BK channels and the signaling pathways in HCFs using whole-cell mode patch-clamp recordings. CO-releasing molecules (CORMs; CORM-2 and CORM-3) significantly increased the amplitudes of BK currents (IBK). The CO-induced stimulating effects on IBK were blocked by pre-treatment with specific nitric oxide synthase (NOS) blockers (L-NG-monomethyl arginine citrate and L-NG-nitroarginine methyl ester). 8-bromo-cyclic GMP increased IBK. KT5823 (inhibits PKG) or ODQ (inhibits soluble guanylate cyclase) blocked the CO-stimulating effect on IBK. Moreover, 8-bromo-cyclic AMP also increased IBK, and pre-treatment with KT5720 (inhibits PKA) or SQ22536 (inhibits adenylate cyclase) blocked the CO effect. Pre-treatment with N-ethylmaleimide (a thiol-alkylating reagent) also blocked the CO effect on IBK, and DL-dithiothreitol (a reducing agent) reversed the CO effect. These data suggest that CO activates IBK through NO via the NOS and through the PKG, PKA, and S-nitrosylation pathways.

Calmodulin Regulates Ciliary Beats in the Human Nasal Mucosa Through Adenylate/Guanylate Cyclases and Protein Kinases A/G

Background: The ciliary beat of the airway epithelium, including the sinonasal epithelium, has a significant role in frontline defense and is thought to be controlled by the level of intracellular Ca²⁺. Involvement of calmodulin and adenylate/guanylate cyclases in the regulation of ciliary beats has been reported, and here we investigated the interrelation between these components of the ciliary beat regulatory pathway. Methods: The inferior turbinates were collected from 29 patients with chronic hypertrophic rhinitis/rhinosinusitis during endoscopic sinonasal surgery. The turbinate mucosa was cut into thin strips, and mucociliary movement was observed under a phase-contrast light microscope equipped with a high-speed digital video camera. Results: The ciliary beat frequency (CBF) was significantly increased by stimulation with 100 μM CALP3 (calmodulin agonist), which was completely suppressed by adding 100 µM SQ22536 (adenylate cyclase inhibitor) and 10 µM ODQ (guanylate cyclase inhibitor) together and by adding 1 µM KT5720 (protein kinase A inhibitor) and 1 µM KT5823 (protein kinase G inhibitor) together. The CBF was significantly increased by stimulation with 10 µM forskolin (adenylate cyclase activator) and 10 µM BAY41-2272 (guanylate cyclase activator) and by stimulation with 100 µM 8-bromo-cAMP (cAMP analog) and 100 µM 8-bromo-cGMP (cGMP analog), which was not changed by adding 1 µM calmidazolium (calmodulin antagonist). Conclusions: These results confirmed that the regulatory pathway of ciliary beats in the human nasal mucosa involves calmodulin, adenylate/guanylate cyclases, and protein kinases A/G and indicate that adenylate/guanylate cyclases and protein kinases A/G act downstream of calmodulin, but not vice versa, and that these cyclases relay calmodulin signaling.

Discovery of Selective Pituitary Adenylate Cyclase 1 Receptor (PAC1R) Antagonist Peptides Potent in a Maxadilan/PACAP38-Induced Increase in Blood Flow Pharmacodynamic Model.

Inhibition of the pituitary adenylate cyclase 1 receptor (PAC1R) is a novel mechanism that could be used for abortive treatment of acute migraine. Our research began with comparative analysis of known PAC1R ligand scaffolds, PACAP38 and Maxadilan, which resulted in the selection of des(24-42) Maxadilan, 6, as a starting point. C-terminal modifications of 6 improved the peptide metabolic stability in vitro and in vivo. SAR investigations identified synergistic combinations of amino acid replacements that significantly increased the in vitro PAC1R inhibitory activity of the analogs to the pM IC90 range. Our modifications further enabled deletion of up to six residues without impacting potency, thus improving peptide ligand binding efficiency. Analogs 17 and 18 exhibited robust in vivo efficacy in the rat Maxadilan-induced increase in blood flow (MIIBF) pharmacodynamic model at 0.3 mg/kg subcutaneous dosing. The first cocrystal structure of a PAC1R antagonist peptide (18) with PAC1R extracellular domain is reported.

Aversive operant conditioning alters the phototactic orientation of the marbled crayfish.

Aversive learning was applied to affect the phototactic behaviour of the marbled crayfish. Animals initially showed negative phototaxis to white light and positive taxis to blue light. Using an aversive learning paradigm, we investigated the plasticity of innate behaviour following operant conditioning. The initial rate of choosing a blue-lit exit was analysed by a dual choice experiment between blue-lit and white-lit exits in pre-test conditions. During training, electrical shocks were applied to the animals when they oriented to the blue-lit exit. Memory tests were given to analyse the orientation rate to the blue-lit exit in trials 1 and 24 h after training and these rates were compared with the pre-test. In general, animals avoided the blue-lit exit in the memory tests. When training was carried out three times, the long-term memory was retained for at least 48 h, although a single bout of training was also enough to form a long-term memory. Cooling animals at 4°C or injection of cycloheximide immediately after training altered the formation of long-term memory, but had no effect on short-term memory formation. Administration of the adenylate cyclase inhibitor SQ22536, the PKA inhibitor H89 or the CREB inhibitor KG-501 immediately after training also blocked the formation of long-term memory, but had no effect on short-term memory formation. Thus, our pharmacological behavioural analyses showed that new protein synthesis was necessary to form long-term memories and that the cAMP/PKA/CREB pathway is the main signal cascade for long-term memory formation in the marbled crayfish.

Caffeine promotes angiogenesis through modulating endothelial mitochondrial dynamics.

Caffeine induces multiple vascular effects. In this study we investigated the angiogenic effect of physiological concentrations of caffeine with focus on endothelial cell behaviors (migration and proliferation) during angiogenesis and its mitochondrial and bioenergetic mechanisms. We showed that caffeine (10-50 μM) significantly enhanced angiogenesis in vitro, evidenced by concentration-dependent increases in tube formation, and migration of human umbilical vein endothelial cells (HUVECs) without affecting cell proliferation. Caffeine (50 μM) enhanced endothelial migration via activation of cAMP/PKA/AMPK signaling pathway, which was mimicked by cAMP analog 8-Br-cAMP, and blocked by PKA inhibitor H89, adenylate cyclase inhibitor SQ22536 or AMPK inhibitor compound C. Furthermore, caffeine (50 μM) induced significant mitochondrial shortening through theincreased phosphorylation of mitochondrial fission protein dynamin-related protein 1 (Drp1) in HUVECs, which increased its activity to regulate mitochondrial fission. Pharmacological blockade of Drp1 by Mdivi-1 (10 μM) or disturbance of mitochondrial fission by Drp1 silencing markedly suppressed caffeine-induced lamellipodia formation and endothelial cell migration. Moreover, we showed that caffeine-induced mitochondrial fission led to accumulation of more mitochondria in lamellipodia regions and augmentation of mitochondrial energetics, both of which were necessary for cell migration. In a mouse model of hindlimb ischemia, administration of caffeine (0.05% in 200 mL drinking water daily, for 14 days) significantly promoted angiogenesis and perfusion as well as activation of endothelial AMPK signaling in the ischemic hindlimb. Taken together, caffeine induces mitochondrial fission through cAMP/PKA/AMPK signaling pathway. Mitochondrial fission is an integral process in caffeine-induced endothelial cell migration by altering mitochondrial distribution and energetics.

Kynurenine Relaxes Arteries of Normotensive Women and Those With Preeclampsia.

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The thrombin receptor links brain derived neurotrophic factor to neuron cholesterol production, resiliency and repair after spinal cord injury

Despite concerted efforts to identify CNS regeneration strategies, an incomplete understanding of how the needed molecular machinery is regulated limits progress. Here we use models of lateral compression and FEJOTA clip contusion-compression spinal cord injury (SCI) to identify the thrombin receptor (Protease Activated Receptor 1 (PAR1)) as an integral facet of this machine with roles in regulating neurite growth through a growth factor- and cholesterol-dependent mechanism. Functional recovery and signs of neural repair, including expression of cholesterol biosynthesis machinery and markers of axonal and synaptic integrity, were all increased after SCI in PAR1 knockout female mice, while PTEN was decreased. Notably, PAR1 differentially regulated HMGCS1, a gene encoding a rate-limiting enzyme in cholesterol production, across the neuronal and astroglial compartments of the intact versus injured spinal cord. Pharmacologic inhibition of cortical neuron PAR1 using vorapaxar in vitro also decreased PTEN and promoted neurite outgrowth in a cholesterol dependent manner, including that driven by suboptimal brain derived neurotrophic factor (BDNF). Pharmacologic inhibition of PAR1 also augmented BDNF-driven HMGCS1 and cholesterol production by murine cortical neurons and by human SH-SY5Y and iPSC-derived neurons. The link between PAR1, cholesterol and BDNF was further highlighted by demonstrating that the deleterious effects of PAR1 over-activation are overcome by supplementing cultures with BDNF, cholesterol or by blocking an inhibitor of adenylate cyclase, Gαi. These findings document PAR1-linked neurotrophic coupling mechanisms that regulate neuronal cholesterol metabolism as an important component of the machinery regulating CNS repair and point to new strategies to enhance neural resiliency after injury.

Absence of metabotropic glutamate receptor homolog(s) accelerates acetylcholine neurotransmission in Caenorhabditis elegans

Glutamate (Glu) and Acetylcholine (ACh), are excitatory neurotransmitters, acting through ionotropic (iR) and metabotropic receptors (mR). Importantly, both neurotransmitters and their signalling are impaired in the prevalent neurodegenerative disease-Alzheimer disease (AD). Glu and its signalling cascade’s influence on ACh-neurotransmission (NT) are sparsely understood. The mGluRs coupled to G-protein signalling acting through PI3K cascade (GrpI) or inhibition of adenylate cyclase–cAMP cascade (GrpII and GrpIII) brings about long-lasting structural/functional changes. These complexities are challenging to decipher. Here, we report that human/mouse mGluRs when compared with their Caenorhabditis elegans homologs, MGL-1-3 showed overall of homology of ∼31–39 %. Phylogeneitc analysis revealed homology of MGL-2 to GrpI, MGL-3 with Grp1 &II and GRM6 of GrpIII and MGL-1, a low homology that falls between GrpI & GrpII. Then, alteration of ACh-NT in C. elegans loss-of-function mutants of mgl-1, mgl-2, mgl-3, PI3K (age-1) and iGluR (NMDA)(nmr-1) was estimated by well-established acute aldicarb (Ald), that increases ACh at synapse, and levamisole (Lev) (postsynaptic activation of levamisole sensitive iAChR) induced time-dependent paralysis assays. Surprisingly, all of them were hypersensitive to Ald and Lev compared to wildtype (in percentage), namely, mgl-1 -17, 54; mgl-2 - 7.2, 24; mgl-3 –52, 64; age-1 – 27, 32; nmr-1- 24, 48; respectively. Of the three, mgl-3 contributes to maximal overall acceleration of ACh-NT. Adenylate cyclase, acy-1 gain-of-function mutant showed less hypersensitivity, Ald – 7% and Lev- 25 %. Together, Glu receptors and signalling cascades are altering ACh-NT permanently, thus establishing the interplay between them thereby provide potential drug targets to be considered for AD.

Triiodothyronine stimulates steroid and VEGF production in murine Leydig cells via cAMP-PKA pathway.

Thyroid hormones affect testicular development as well as functions like spermatogenesis and steroidogenesis, thereby influencing male fertility. Our group earlier showed that the stimulatory role of the thyroid hormone, T3 , on the production of vascular endothelial growth factor (VEGF) by murine Leydig cells is mediated by steroids and hypoxia-inducible factor-1 (HIF-1α). The current study further defines the signalling pathway(s) utilised by T3 to stimulate the production of steroids, VEGF and HIF-1α in mouse Leydig tumour cell line (MLTC-1). Specific inhibitors for different signalling molecules were used to study the role of cyclic AMP (cAMP), and its downstream mediators. Expression of VEGF and HIF-1α mRNA were measured by quantitative RT-PCR; VEGF secretion by ELISA; steroid secretion by radioimmunoassay and HIF-1α protein levels by western blotting. Inhibitors of adenylate cyclase (AC), protein kinase A (PKA), sarcoma kinase (SrcK), phosphoinositide 3-kinase (PI3K) and MAP kinase kinase (MEK1/2) abolished the T3 -induced increase in VEGF mRNA and protein levels. The same signalling molecules also mediated the increased production of steroids and HIF-1α protein in response to T3 . Therefore, it was concluded that T3 stimulates steroid secretion and HIF-1α protein in MLTC-1 cells through the AC-cAMP-PKA-PI3K-MEK pathway, which in turn stimulate VEGF production.

Taurochenodeoxycholic acid mediates cAMP-PKA-CREB signaling pathway.

Taurochenodeoxycholic acid (TCDCA) is one of the main effective components of bile acid, playing critical roles in apoptosis and immune responses through the TGR5 receptor. In this study, we reveal the interaction between TCDCA and TGR5 receptor in TGR5-knockdown H1299 cells and the regulation of inflammation via the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-cAMP response element binding (CREB) signal pathway in NR8383 macrophages. In TGR5-knockdown H1299 cells, TCDCA significantly activated cAMP level via TGR5 receptor, indicating TCDCA can bind to TGR5; in NR8383 macrophages TCDCA increased cAMP content compared to treatment with the adenylate cyclase (AC) inhibitor SQ22536. Moreover, activated cAMP can significantly enhance gene expression and protein levels of its downstream proteins PKA and CREB compared with groups of inhibitors. Additionally, TCDCA decreased tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, IL-8 and IL-12 through nuclear factor kappa light chain enhancer of activated B cells (NF-κB) activity. PKA and CREB are primary regulators of anti-inflammatory and immune response. Our results thus demonstrate TCDCA plays an essential anti-inflammatory role via the signaling pathway of cAMP-PKA-CREB induced by TGR5 receptor.

Cholinergic regulation of cardiac pacemaker activity by l-type cav1.3 channels

Abstract Introduction The cholinergic regulation of heart rate (HR) is mediated by acetylcholine (ACh)-dependent activation of M2-receptors (M2R). Activated M2R promote release of the βγ-subunit of G-proteins to directly gate GIRK1/4 channels (underlying the cardiac IKACh current), while αi-subunits inhibit adenylate cyclase (AC) activity. AC inhibition reduces the intracellular concentration of cAMP, decreasing the activity of ion channels involved in pacemaking, including “funny” f-(HCN4) and L-type Cav1.3 calcium channels. Purpose To determine the importance of L-type Cav1.3 channels in the cholinergic regulation of heart rate. Methods We recorded the frequency and the position of the pacemaker leading site in ex vivo sinus nodes and the HR of isolated Langendorff perfused hearts of mice in control or during ACh perfusion. We used control wild type (WT) mice, and five genetically modified mouse models: Cav1.3 knockout (KO, ablated Cav1.3-mediated L-type current), GIRK4KO (ablated IKACh current), HCN4-CNBD (selective deletion of cAMP-dependent regulation of HCN4), GIRK4KO/HCN4-CNBD and GIRK4KO/Cav1.3KO. Results Data from optical mapping experiments showed that, under basal conditions, perfusion of 3 μM ACh significantly reduced the frequency of action potentials in WT (44%), HCN4-CNBD (38%), Cav1.3KO (65%) and GIRK4KO (8%) isolated mouse sinus node tissues. ACh application did not significantly affect the frequency of action potentials recorded in tissue from GIRK4KO/HCN4-CNBD and GIRK4KO/Cav1.3KO animals. Furthermore, in all the sinus node tissues tested, regardless of the genotypes, ACh shifted the pacemaker leading site from its normal position by at least 0.7 mm. Upon stimulation of the β-adrenergic pathway by Isoproterenol, to reproduce conditions of accentuated antagonism, 3μM ACh reduced HR in isolated hearts from WT (43.8%), HCN4-CNBD (38.7%), Cav1.3KO (25,4%), GIRK4KO (16.9%) and GIRK4KO/HCN4-CNBD (16.4%) mice. No significant HR reduction was recorded in hearts from GIRK4KO/Cav1.3KO animals. Conclusion Our data indicate that L-type Cav1.3 channels are involved in cholinergic regulation of heart rate in mice. In addition, when the intracellular concentration of cAMP is elevated (i.e. under conditions of accentuated antagonism), the cholinergic regulation of sinus node pacemaking is predominantly ensured by Cav1.3 and KACh channels. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): “Fondation pour la recherche medicale” FRM

A comparative study of intravenous combination of Dexmedetomidine and ketamine versus fentanyl and ketamine as sedative agents in children undergoing interventional Cardiac Catheterization procedures

Fentanyl is a highly selective mu-agonist (or MOP agonist); the MOP receptor appears to be specifically involved in the mediation of analgesia. Opioids appear to exert their effects by interacting with presynaptic Gi-protein receptors, leading to hyperpolarization of the cell membrane by increasing K+ conductance. Inhibition of adenylate cyclase, leading to reduced production of cAMP, and closure of voltage-sensitive calcium channels also occur. After institutional scientific and ethical committee approval, a written informed consent was obtained from parents. 70 paediatric patients, American Society of Anesthesiologists (ASA) physical status II and III, aged between 4 to 12 yrs with acyanotic congenital heart disease admitted for elective interventional cardiac catheterization procedures are included in this study. There is significant difference in the mean duration of sedation attained by group D and group F both statistically and clinically. The sedation time does include the procedure duration and duration from end of procedure to attainment of the steward score of 7.

Progesterone increases blood glucose via hepatic progesterone receptor membrane component 1 under limited or impaired action of insulin

Hepatic gluconeogenesis is the main pathway for blood glucose maintenance activated during fasting. Retardation of insulin action, such as in diabetes mellitus, activates gluconeogenesis during the fed state. While the role of progesterone (P4) in diabetes is controversial, the P4 receptor, progesterone receptor membrane component 1 (PGRMC1), is known to stimulate pancreatic insulin secretion. We investigated the role of P4, via hepatic PGRMC1, during gluconeogenesis. The PGRMC1 binding chemical, AG-205, induced PGRMC1 monomer (25 kDa) abundance, and increased PEPCK expression and glucose production in parallel with cyclic AMP (cAMP) induction in Hep3B cells. PGRMC1-mediated cyclic AMP was inhibited by an adenylate cyclase inhibitor (MDL-12,330A). PEPCK suppression in Pgrmc1 KO hepatocyte was not observed after treatment of MDL-12,330A. PGRMC1 knockdown or overexpression systems in Hep3B cells confirmed that PGRMC1 mediates PEPCK expression via phosphorylation of cAMP-response element binding protein (CREB). CREB phosphorylation and PEPCK expression in primary hepatocytes were greater than that in PGRMC1 knock-out hepatocytes. Progesterone increased PGRMC1 expression, which induced cAMP and PEPCK induction and glucose production. In vivo, P4 suppressed gluconeogenesis following plasma insulin induction under normal conditions in a mouse model. However, P4 increased blood glucose via gluconeogenesis in parallel with increases in PGRMC1 and PEPCK expression in mice in both insulin-deficient and insulin-resistant conditions. We conclude that P4 increases hepatic glucose production via PGRMC1, which may exacerbate hyperglycaemia in diabetes where insulin action is limited.

Cholinergic regulation of heart rate: Functional importance of l-type cav1.3 channels

The cholinergic regulation of heart rate (HR) is mediated by acetylcholine (ACh)-dependent activation of M2-receptors (M2R). Activated M2R promote release of the βγ-subunit of G-proteins to directly gate GIRK1/4 channels (underlying IKACh), while αi-subunits inhibit adenylate cyclase (AC) activity. AC inhibition reduces the intracellular concentration of cAMP, decreasing the activity of several ion channels involved in pacemaking, including “funny” f-(HCN4) and L-type Cav1.3 calcium channels. To determine the importance of Cav1.3, GIRK1/4 and HCN4 in cholinergic regulation of HR. We recorded the HR of isolated Langendorff perfused hearts and the pacemaker leading site in ex vivo sinus nodes of mice in control or during ACh perfusion. We used control wild-type (WT) mice, and five genetically modified mouse models: Cav1.3KO (no Cav1.3-mediated L-type current), Girk4KO (no IKACh), HCN4-CNBD (no cAMP-dependent regulation of HCN4), Girk4KO/HCN4-CNBD and Girk4KO/Cav1.3KO. Perfusion of 1 μM ACh significantly reduced HR in hearts from control (18%), Girk4KO (12%) and HCN4-CNBD (23%) animals, but not in hearts from in Cav1.3KO, Girk4KO/HCN4-CNBD and Girk4KO/Cav1.3 mice. 3 μM ACh significantly reduced HR in all the strains tested except for Girk4KO/Cav1.3KO animals. Preliminary results obtained from optical mapping experiments show ACh-induced shift of pacemaker leading site in WT and HCN4-CNBD mice. Girk4KO/Cav1.3KO mice show a larger shift of the leading pacemaker site than WT and HCN4-CNBD mice, to regions outside the sinus node area. Our data indicate that Cav1.3 channels play an important role in the cholinergic regulation of heart rate. Indeed, concomitant ablation of GIRK1/4 and Cav1.3 channels nearly abolished regulation in isolated murine hearts. Preliminary optical mapping results may indicate that this concomitant ablation increases pacemaker shift in cholinergic stimulation.

Prostaglandylinositol cyclic phosphate, the natural antagonist of cyclic AMP.

While searching for a counterpart to cyclic AMP, a new compound was found to inhibit adenylate cyclase. It was identified as prostaglandyl-(15-4')-myo-inositol (1':2'-cyclic)-phosphate (cyclic PIP). The substrates for its biosynthesis are prostaglandin E (PGE) and the novel inositol phosphate, guanosine diphospho-4-myo-inositol 1:2-cyclic phosphate (n-IP). The basic regulatory properties of cyclic PIP are to inhibit dose-dependently protein kinase A (PKA) and to seven-fold activate protein ser/thr phosphatase holoenzyme. These regulations occur as rapidly as the activation of PKA by cyclic AMP. Such regulatory properties are essential for the meticulous regulation of the equilibrium between the phospho- and de-phospho-form of interconvertible enzymes. The synthesis of cyclic PIP is stimulated by insulin and noradrenaline (α-receptor action). The insulin-stimulated cyclic PIP synthase is active in a tyrosine-phosphorylated state. A comparable characterization of the adrenaline-stimulated cyclic PIP synthase is still incomplete. In streptozotocin-diabetic rats, the hormonal stimulation of cyclic PIP synthesis decreases with time. Cyclic PIP synthesis is activated by biguanides as metformin two to four-fold and by antihypertensive drugs two-fold. Inhibition of cyclic PIP synthesis leads to a metabolic state as observed in early-stage type-2 diabetes. In summary, all living cells synthesize cyclic PIP, which switches on anabolism, whereas cyclic AMP triggers catabolism.