Adenylate Cyclase Activity Research Articles

Regulation of the firing activity by PKA-PKC-Src family kinases in cultured neurons of hypothalamic arcuate nucleus.

The cAMP‐dependent protein kinase A family (PKAs), protein kinase C family (PKCs), and Src family kinases (SFKs) are found to play important roles in pain hypersensitivity. However, more detailed investigations are still needed in order to understand the mechanisms underlying the actions of PKAs, PKCs, and SFKs. Neurons in the hypothalamic arcuate nucleus (ARC) are found to be involved in the regulation of pain hypersensitivity. Here we report that the action potential (AP) firing activity of ARC neurons in culture was up‐regulated by application of the adenylate cyclase activator forskolin or the PKC activator PMA, and that the forskolin or PMA application‐induced up‐regulation of AP firing activity could be blocked by pre‐application of the SFK inhibitor PP2. SFK activation also up‐regulated the AP firing activity and this effect could be prevented by pre‐application of the inhibitors of PKCs, but not of PKAs. Furthermore, we identified that forskolin or PMA application caused increases in the phosphorylation not only in PKAs at T197 or PKCs at S660 and PKCα/βII at T638/641, but also in SFKs at Y416. The forskolin or PMA application‐induced increase in the phosphorylation of PKAs or PKCs was not affected by pre‐treatment with PP2. The regulations of the SFK and AP firing activities by PKCs were independent upon the translocation of either PKCα or PKCβII. Thus, it is demonstrated that PKAs may act as an upstream factor(s) to enhance SFKs while PKCs and SFKs interact reciprocally, and thereby up‐regulate the AP firing activity in hypothalamic ARC neurons.

The role of CAMP-dependent signaling systems in regulation of electrical and contractile properties of smooth muscles of the ureter in hypoxia in guinea pigs

Aim. To study the features of regulating the electrical activity and mechanical tension of smooth muscle cells (SMCs) of the guinea pig ureter as modulated by cyclic adenosine monophosphate (cAMP) in hypoxia. Materials and methods. The effects of isoprenaline (100 μM), forskolin (1 μM), 3-isobutyl-1-methylxanthine (IBMX, 100 μM) and tetraethylammonium chloride (TEA, 5 μM) on the contractile and electrical properties of isolated smooth muscle segments of the guinea pig ureter in normoxia and hypoxia were measured by the double sucrose bridge. Hypoxic conditions were created by placing the SMCs in Krebs solution containing (10.0 ± 0.2) vol. % O2. Results. It was found that an increase in the intracellular cAMP level caused by isoprenaline, the β-adrenergic receptor agonist, and activation of adenylate cyclase by forskolin, an inhibitor of phosphodiesterase IBMX, caused a decrease in the electrical and constrictor properties of the SMCs in the guinea pig ureter. The decrease in the level of oxygen in the perfusion solution resulted in the increase in the action potential amplitude and contraction of smooth muscles from the ureter. With an increase in the intracellular cAMP level, the activating effect of hypoxia on smooth muscle segments decreased. Inhibition of potassium conductivity of the ureteral SMCs with TEA in normoxia suppressed the cAMP-dependent processes induced by forskolin, whereas in hypoxia it caused the potentiation of an activating effect on the electrical activity and contractions of smooth muscle segments. Conclusion. Thus, the results suggest the involvement of cAMP-dependent signaling system in the influence of hypoxia on the electrical and contractile properties of ureteral SMCs. Modification of the intracellular cAMP level reduced the stimulatory effect of hypoxia on the smooth muscle strips of the ureter caused by increase in the ionic conductivity of the membrane and contributed to their adaptation to environmental conditions.

Peptides come to the rescue of pancreatic β cells

Insulin and glucagon are well-known peptide hormones that keep glucose levels within a healthy range in the body. But they are only part of a complex network that controls concentrations of this ubiquitous sugar in blood and tissues. Other molecules regulate glucose by controlling insulin secretion from the pancreas or protecting pancreatic β cells against stresses that lead to cellular dysfunction or cell death (1). One of these protective regulators is glucagon-like peptide 1 (GLP-1), a 30-amino-acid-long peptide produced in specialized epithelial cells of the intestine, called L cells, and also in the brain and other organs and tissues (2). GLP-1 belongs to a group of peptides that mediate the “incretin effect,” an endocrine response to glucose arising from food digestion in the intestines (2, 3). This response helps regulate food intake and the fate of dietary glucose. Specifically, GLP-1 is released from the intestinal cells when food is ingested and then binds to and activates the GLP-1 receptor (GLP-1R), a G protein–coupled receptor on many cell types, including β cells in which GLP-1R signaling stimulates insulin synthesis and secretion (3). Notably, the incretin effect stimulates insulin secretion from pancreatic β cells more strongly than exposure to glucose alone. An article published in the Journal of Biological Chemistry (4), recognized as a Classic here, added to our understanding of the incretin effect by showing that GLP-1R signaling protects β cells from cell death (Fig. 1). This finding was significant for preventing or managing type 2 diabetes, in which β-cell apoptosis occurs (5) and may contribute to insufficient pancreatic insulin production (6). Open in a separate window Figure 1. Li et al. (4) have shown that binding of the receptor agonist exendin-4 to GLP-1R on pancreatic β cells protects the cells from cellular injury and cytokine-induced apoptosis and thereby preserves glucose homeostasis in mice. Binding of GLP-1 to its cognate receptor on pancreatic β cells up-regulates intracellular cAMP levels, in turn reducing streptozotocin-induced β-cell death. Images of exendin-4 and GLP-1R (with GLP-1 bound) are from Ref. 7; image of cAMP is from Wikimedia, used under Creative Commons.

Rare, functional, somatic variants in gene families linked to cancer genes: GPCR signaling as a paradigm

Oncodriver genes are usually identified when mutations recur in multiple tumours. Different drivers often converge in the activation or repression of key cancer-relevant pathways. However, as many pathways contain multiple members of the same gene family, individual mutations might be overlooked, as each family member would necessarily have a lower mutation frequency and thus not identified as significant in any one-gene-at-a-time analysis. Here, we looked for mutated, functional sequence positions in gene families that were mutually exclusive (in patients) with another gene in the same pathway, which identified both known and new candidate oncodrivers. For instance, many inactivating mutations in multiple G-protein (particularly Gi/o) coupled receptors, are mutually exclusive with Gαs oncogenic activating mutations, both of which ultimately enhance cAMP signalling. By integrating transcriptomics and interaction data, we show that the Gs pathway is upregulated in multiple cancer types, even those lacking known GNAS activating mutations. This suggests that cancer cells may develop alternative strategies to activate adenylate cyclase signalling in multiple cancer types. Our study provides a mechanistic interpretation for several rare somatic mutations in multi-gene oncodrivers, and offers possible explanations for known and potential off-label cancer treatments, suggesting new therapeutic opportunities.

A Dual GLP-1/GIP Receptor Agonist Does Not Antagonize Glucagon at Its Receptor but May Act as a Biased Agonist at the GLP-1 Receptor.

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are important regulators of metabolism, making their receptors (GLP-1R and GIPR) attractive targets in the treatment of type 2 diabetes mellitus (T2DM). GLP-1R agonists are used clinically to treat T2DM but the use of GIPR agonists remains controversial. Recent studies suggest that simultaneous activation of GLP-1R and GIPR with a single peptide provides superior glycemic control with fewer adverse effects than activation of GLP-1R alone. We investigated the signaling properties of a recently reported dual-incretin receptor agonist (P18). GLP-1R, GIPR, and the closely related glucagon receptor (GCGR) were expressed in HEK-293 cells. Activation of adenylate cyclase via Gαs was monitored using a luciferase-linked reporter gene (CRE-Luc) assay. Arrestin recruitment was monitored using a bioluminescence resonance energy transfer (BRET) assay. GLP-1, GIP, and glucagon displayed exquisite selectivity for their receptors in the CRE-Luc assay. P18 activated GLP-1R with similar potency to GLP-1 and GIPR with higher potency than GIP. Interestingly, P18 was less effective than GLP-1 at recruiting arrestin to GLP-1R and was inactive at GCGR. These data suggest that P18 can act as both a dual-incretin receptor agonist, and as a G protein-biased agonist at GLP-1R.

Cardiovascular effects of intravenous colforsin in normal and acute respiratory acidosis canine models: A dose-response study

In acidosis, catecholamines are attenuated, and higher doses are often required to improve cardiovascular function. Colforsin activates adenylate cyclase in cardiomyocytes without beta-adrenoceptor. Here, six beagles were administered colforsin or dobutamine four times during eucapnia (partial pressure of arterial carbon dioxide 35–40 mm Hg; normal) and hypercapnia (ibid 90–110 mm Hg; acidosis) conditions. The latter was induced by CO2 inhalation. Anesthesia was induced with propofol and maintained with isoflurane. Cardiovascular function was measured by thermodilution and a Swan-Ganz catheter at baseline and 60 min after 0.3 μg/kg/min (low), 0.6 μg/kg/min (middle), and 1.2 μg/kg/min (high) colforsin administration. The median pH was 7.38 [range 7.33–7.42] and 7.01 [range 6.96–7.08] at baseline in the Normal and Acidosis conditions, respectively. Endogenous adrenaline and noradrenaline levels at baseline were significantly (P < 0.05) higher in the Acidosis than in the Normal condition. Colforsin induced cardiovascular effects similar to those caused by dobutamine. Colforsin increased cardiac output in the Normal condition (baseline: 3.9 ± 0.2 L/kg/m2 [mean ± standard error], low: 5.2 ± 0.4 L/kg/min2, middle: 7.0 ± 0.4 L/kg/m2, high: 9.4 ± 0.2 L/kg/m2; P < 0.001) and Acidosis condition (baseline: 6.1 ± 0.3 L/kg/m2, low: 6.2 ± 0.2 L/kg/m2, middle: 7.2 ± 0.2 L/kg/m2, high: 8.3 ± 0.2 L/kg/m2; P < 0.001). Colforsin significantly increased heart rate and decreased systemic vascular resistance compared to values at baseline. Both drugs increased pulmonary artery pressure, but colforsin (high: 13.3 ± 0.6 mmHg in Normal and 20.1 ± 0.2 mmHg in Acidosis) may have lower clinical impact on the pulmonary artery than dobutamine (high: 19.7 ± 0.6 in Normal and 26.7 ± 0.5 in Acidosis). Interaction between both drugs and experimental conditions was observed in terms of cardiovascular function, which were similarly attenuated with colforsin and dobutamine under acute respiratory acidosis.

EPIDEMIOLOGICAL AND CLINICAL-PATHOGENETIC ASPECTS OF DIFFERENTIAL DIAGNOSIS OF ACUTE INTESTINAL INFECTIONS IN ADULTS

The incidence of acute intestinal infections in the Russian Federation remains high and does not tend to decrease. The General susceptibility of the population, the variety of pathogens of infectious diseases accompanied by diarrhea syndrome, necessitates the improvement of clinical diagnosis and differential diagnosis in the treatment of patients with diarrhea at the primary stage of medical care. The article presents the etiological and epidemiological aspects of the most common in the Russian Federation acute intestinal infectious diseases accompanied by diarrhea syndrome: food toxicoinfections, viral gastroenteritis, salmonellosis, shigellosis, campylobacteriosis, pseudomembranous colitis. The sources of infection and pathways of pathogens are characterized. The pathogenesis of the main types of diarrhea caused by various pathogens of intestinal infections is described. It is shown that the action of enterotoxins of pathogens that activate adenylate cyclase contributes to the development of secretory type of diarrhea; the place of localization of the pathological process is the small intestine. Pathogens exhibiting high invasive activity contribute to the development of inflammatory type of diarrhea; the pathological process is localized in the colon. With direct damage to enterocytes of the small intestine by pathogens of viral gastroenteritis, an osmotic type of diarrhea is formed, associated with a violation of the digesting and absorption function of the small intestine. Presented distinctive clinical criteria of three types of diarrhea: color, consistency of the stool, the presence of pathological impurities in it. The differences of abdominal syndrome in the analyzed nosology: the nature and localization of abdominal pain. The presented differential diagnostic signs of diarrhea and abdominal syndrome allow for early diagnosis of infectious disease before laboratory confirmation and contribute to the improvement of the algorithm of medical care in acute intestinal infections accompanied by diarrhea syndrome.

Clinically Advanced p38 Inhibitors Suppress DUX4 Expression in Cellular and Animal Models of Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is characterized by misexpression of the double homeobox 4 (DUX4) developmental transcription factor in mature skeletal muscle, where it is responsible for muscle degeneration. Preventing expression of DUX4 mRNA is a disease-modifying therapeutic strategy with the potential to halt or reverse the course of disease. We previously reported that agonists of the β-2 adrenergic receptor suppress DUX4 expression by activating adenylate cyclase to increase cAMP levels. Efforts to further explore this signaling pathway led to the identification of p38 mitogen-activated protein kinase as a major regulator of DUX4 expression. In vitro experiments demonstrate that clinically advanced p38 inhibitors suppress DUX4 expression in FSHD type 1 and 2 myoblasts and differentiating myocytes in vitro with exquisite potency. Individual small interfering RNA-mediated knockdown of either p38α or p38β suppresses DUX4 expression, demonstrating that each kinase isoform plays a distinct requisite role in activating DUX4 Finally, p38 inhibitors effectively suppress DUX4 expression in a mouse xenograft model of human FSHD gene regulation. These data support the repurposing of existing clinical p38 inhibitors as potential therapeutics for FSHD. The surprise finding that p38α and p38β isoforms each independently contribute to DUX4 expression offers a unique opportunity to explore the utility of p38 isoform-selective inhibitors to balance efficacy and safety in skeletal muscle. We propose p38 inhibition as a disease-modifying therapeutic strategy for FSHD. SIGNIFICANCE STATEMENT: Facioscapulohumeral muscular dystrophy (FSHD) currently has no treatment options. This work provides evidence that repurposing a clinically advanced p38 inhibitor may provide the first disease-modifying drug for FSHD by suppressing toxic DUX4 expression, the root cause of muscle degeneration in this disease.

Eicosanoid mediation of cannabinoid actions.

Interactions between cannabinoids and eicosanoids have been observed for the last several decades and account for a variety of cannabinoid actions. These were seen both in vitro and in vivo and may provide a molecular basis for these actions. Some of the topics included in this review are; effects on adenylate cyclase activity, alteration of behavioral responses, reduction of pain sensation, reduction and resolution of inflammation, hypotensive and vasorelaxant responses, anti-cancer and anti-metastatic activities, reduction of intraocular pressure and others. The most widely studied cannabinoids so far are tetrahydrocannabinol and cannabidiol. However, synthetic agents such as CP55,940, ajulemic acid, JWH-133 and WIN-55,212-2 were also investigated for interaction with eicosanoids. The endocannabinoids anandamide and 2-arachidonoylglycerol have been examined as well. Among the eicosanoids mediating cannabinoid actions are PGE2, 15-deoxy-Δ12,14-prostaglandin-J2, lipoxin A4, lipoxin B4, and leukotriene B4. Enzyme activities involved include monoacylglycerylipase, adenylatecyclase, phospholipase A2, cyclooxygenases-1, 2 and 5, lipoxygenases-12 and 15. Receptors involved include CB1, CB2 and the EP3 and EP3 prostanoid receptors. While not all cannabinoid activities can be accounted for, many are best explained by eicosanoid participation. The recent surge in interest in "medical marijuana" makes understanding mechanisms of cannabinoid actions particularly important.

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.

Modification of the first constant domain of heavy chain enabled effective folding of functional anti-forskolin antigen-binding fragment for sensitive quantitative analysis.

The assembly between heavy and light chains is a critical step of immunoglobulin (Ig) and fragment antigen-binding (Fab) antibody expression and of their binding activity. The genes encoding Fab were obtained from hybridoma cells secreting monoclonal antibody (MAb, IgG2b) against adenylate cyclase activator forskolin (FOR). The subclass of the first constant domain of heavy chain (CH 1) of IgG2b was modified to IgG1 via overlap extension polymerase chain reaction and expressed via Escherichia coli bacterial system. Since both Fabs (IgG2b and IgG1) were expressed as inclusion bodies, functional analysis was performed after in vitro refolding via stepwise dialysis. The result indicated that the folding efficiency between VH -CH 1 and VL -CL was improved by the CH 1 modification from IgG2b to IgG1 subclass, although their specificity for FOR was not altered. Effective folding of IgG1 was also observed when they were expressed in the hemolymph of silkworm larvae using the Bombyx mori nuclear polyhedrosis virus bacmid system. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was then developed for the determination of FOR using effectively prepared Fab IgG1. The sensitivity of FOR determination was in the range of 3.91-62.5 ng/mL with less than 9% relative standard deviation, implying the sensitive and reliable analysis of developed icELISA. In addition, high accuracy of the icELISA was supported by the results of spiked-and-recovery tests, ranging from 100.2 to 102.3%. Therefore, Fab could be utilized reliably for icELISA instead of the more expensive MAb. Collectively, this approach improved productivity of Fab and reduced the cost of antibody production.

Neurally mediated syncope diagnosis based on adenylate cyclase activity in Japanese patients.

The study aims to clarify the mechanism in patients with neurally mediated syncope (NMS), focusing on the adenylate cyclase (AC) activity level in lymphocytes. This study included 40 subjects: 22 healthy volunteers and 18 NMS patients. We investigated the changes in AC activity that occur during of syncope at rest and during the head-up tilt (HUT) test. We obtained 8 mL of blood at rest time and four times during the HUT test. Then, we measured the AC activity and the test reagent was added to the lymphocytes (10,000) and reacted for 30 min at room temperature. We were able to determine the standard value of AC activity when adrenaline (AD) and isoproterenol (IP) were added to lymphocytes. The results of our study showed one of the causes of NMS has a difference in AC activity level and classification of the patients into two different types of NMS was possible: either the vasodepressor type (VT) or mixed type (MT). At rest time, VT patients showed significantly higher AC activity (AD; 100 μM: p = 0.005, IP; 50 μM: p = 0.02) and MT patients showed significantly lower AC activity (AD; 10 μM: p = 0.02, IP; 50 μM: p = 0.004) than the average AC activity in healthy volunteers. Moreover, VT patients had significantly higher AC activity than healthy volunteers at the four points of the HUT test. MT patients had significantly lower AC activity (AD: p = 0.04 and IP: p = 0.04) than healthy volunteers at the rest time of HUT. Our study showed a significant difference in AC activities between NMS patients and healthy volunteers at rest. Therefore, a detailed NMS diagnosis can be made by examining AC activity levels in blood taken at rest time.

Pathophysiological Mechanisms in Migraine and the Identification of New Therapeutic Targets.

Migraine is a strongly disabling disease characterized by a unilateral throbbing headache lasting for up to 72h for each individual attack. There have been many theories on the pathophysiology of migraine throughout the years. Currently, the neurovascular theory dominates, suggesting clear involvement of the trigeminovascular system. The most recent data show that a migraine attack most likely originates in the hypothalamus and activates the trigeminal nucleus caudalis (TNC). Although the mechanisms are unknown, activation of the TNC leads to peripheral release of calcitonin gene-related protein (CGRP), most likely from C-fibers. During the past year monoclonal antibodies against CGRP or the CGRP receptor have emerged as the most promising targets for migraine therapy, and at the same time established the strong involvement of CGRP in the pathophysiology of migraine. The viewpoint presented here focuses further on the activation of the CGRP receptor on the sensory Aδ-fiber, leading to the sensation of pain. The CGRP receptor activates adenylate cyclase, which leads to an increase in cyclic adenosine monophosphate (cAMP). We hypothesize that cAMP activates the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, triggering an action potential sensed as pain. The mechanisms behind migraine pain on a molecular level, particularly their importance to cAMP, provide clues to potential new anti-migraine targets. In this article we focus on the development of targets related to the CGRP system, and further include novel targets such as the pituitary adenylate cyclase-activating peptide (PACAP) system, the serotonin 5-HT1F receptor, purinergic receptors, HCN channels, adenosine triphosphate-sensitive potassium channels (KATP), and the glutaminergic system.

Luteal peptides and their genes as important markers of ovarian differentiation

Secreted peptide hormones and components of the steroidogenic machinery are molecules that are expressed usually in high amounts and in a time- and cell-specific fashion within the cells that give rise to the bovine corpus luteum. They thus serve as useful markers for the events occurring within the nuclei of these cells that result in differentiation and the expression of the specific luteal phenotype. We have studied the bovine genes of three such luteal products: oxytocin, the new relaxin-like factor (RLF), and the steroidogenic acute regulatory protein (StAR). The oxytocin gene is expressed in the granulosal cells of the preovulatory follicle and in the large luteal cells of the immediately resulting early corpus luteum. The RLF gene is a major thecal cell product in antral and atretic follicles. It is also transcribed in luteal cells, but only in the mid- to late ovarian cycle and in pregnancy, following a temporal pattern of expression very similar to that of relaxin in pigs. The StAR gene appears to be upregulated only in the mid- to late ovarian cycle, several days after the increase in steroidogenic enzymes associated with luteinization and progesterone production. All three genes make use of the transcription factor SF-1 (Ad4BP) and, although they all respond to LH activation of adenylate cyclase, none utilize CRE-linked systems. Specific transcriptional activation must involve other factors to encode the information for the widely diverse temporal and cellular patterns of gene expression for these three genes.

MOLECULAR BASES FOR HIGH (HF) AND LOW FREQUENCY (LF) BANDS: A NEW APPROACH TO HEART RATE VARIABILITY ANALYSIS

Heart Rate Variability (HRV) in Frequency‐domain estimate the distribution into low‐frequency (LF: 0.04–0.15 Hz) and high‐frequency (HF 0.15–0.4 Hz) bands. On the other hand, when analyzing norepinephrine and acetylcholine cell signalling time kinetics, the summation of such times once converted to frequencies in Hertz, match the previously mentioned LF and HF values.Materials and MethodsGoogle Scholar databases and National Library of Medicine, were searched for articles published from 2000 to 2019 related to the time kinetics involved in cell signaling, with the following combination of terms: 1) “Title (TI) = (FRET) AND TI = (BRET) OR ‘Activation time B1 adrenergic’ OR ‘Activation time M2 muscarinic’ OR ‘Gs Protein Activation’ OR ‘Gi Protein Activation’ ‘Time Kinetics’ OR ‘Adenylate Cyclase’ ‘Activation HCN4’ OR ‘Nodal Pace Marker’ OR ‘In Vitro Model Cells')]”. The inclusion criteria for each study included the ligand‐receptor interaction and time related signalling cascade involved in the depolarization or hyperpolarization of cells. Both database searches yielded 7 studies matching the inclusion criteria. Once all the times of cell signalling cascade were found or estimated for all receptors, such times were summed, they were converted to Hertz using the inverse relationship between time and frequency.RESULTSDatabase searches yielded 7 studies matching the inclusion criteria. For B1‐Adrenergic Receptor cell signalling cascade the estimated times were: Ligand‐Receptor‐Activation (0.045 s), Receptor Gs protein interaction (0.044 s), Gs protein activation (0.369 s), Adenylate Cyclase activation (28.8s), cAMP mediated activation of HCN4 (0.461 s), for a total of 29.76 seconds, yielding 0.0336 Hz.For M2‐Cholinergic Receptor the estimated times were: Ligand‐Receptor activation (0.750 s), Receptor Gi protein interaction (12 ns**), Gi protein activation (1 s), and Gi mediated Adenylate Cyclase inactivation (3 s), for a total of 4.75 seconds, yielding 0.211 Hz.DISCUSSIONB1‐Adrenergic Receptor cell signaling summed 29.76 seconds, yielding 0.0336 Hertz, a very close value to the 0.04 Hz of LF band. On the other hand, M2 Cholinergic receptor cell signalling time was 4.75 s, yielding 0.211 Hz, a value within the HF band range of 0.15 to 0.4 Hz. Such results coincide with the possibility of direct correlation of biochemical cell signalling and HF and LF bands spectrums. However due to the scarcity of biochemical studies, it is important to mention that during the estimation process there was two issues worth mentioning. First, B1 Adrenergic Receptor Gs Adenylate cyclase activation was roughly estimated. Second, for M2 Cholinergic Gi mediated AC inhibition, a A2‐Adrenergic receptor model was used. Despite the previously mentioned limitations, the current study results suggest the possibility that LF and HF band spectrum could be refined into smaller range of Hertz segments, which might correspond to the summation of time kinetics involved in cell signalling cascade related to the different adrenergic or cholinergic receptor subtypes.Support or Funding InformationPROSEIMThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NF1 dissociates cell type specific contributions toward reward valuation and motor control

Neurofibromin 1 (NF1) is a large multidomain signaling molecule that is a major Ras regulator but also acts as a positive regulator of cAMP levels by mediating G protein‐coupled receptor (GPCR)‐dependent activation of adenylate cyclase. Mutations in the NF1 gene cause Neurofibromatosis type 1, a genetic disorder characterized by multiple benign and malignant tumors with prominent neuropsychiatric symptoms that include learning and attention deficits, as well as motor impairments. Recently, we found that NF1 is a direct effector of GPCR signaling via Gβγ subunits in the striatum. However, it remains unclear the signaling contribution of NF1 in striatal‐mediator behaviors. Here we demonstrate the impact of intracellular signaling pathways commonly targeted by neurotransmitter inputs that impact medium spiny neurons (MSN) activity can be dissociated in a cell‐specific manner with distinct contribution to motor learning and reward‐related behaviors. We genetically ablated NF1 with cellular resolution demonstrating the diverging behavioral and signaling specific manner in which NF1 contributes to the acquisition and consolidating of learning a motor skill, as well as its regulation of morphine reward.Support or Funding InformationThis work was supported by the NIH and by the Canadian Institutes of Health Research (CIHR) Fellowship.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanisms of G Protein‐Selectivity in Muscarinic Acetylcholine Receptor Family

Muscarinic acetylcholine receptors are integral membrane proteins that regulate physiological activity. Acetylcholine is a neurotransmitter that binds to the extracellular side of these G protein coupled receptors (GPCRs) activating the Gα subunit of the trimeric G protein inside the cell. There are 5 different muscarinic receptor subtypes (M1–M5), where the same ligand acetylcholine causes M2 and M4 receptors to preferentially couple to the Gαi subunit and the receptors M1, M3, and M5 to preferentially couple to the Gαq subunit. This selectivity of Gα subunits mediates different secondary messenger pathways in the cell: Gαi being responsible for deactivating adenyl cyclase activity and Gαq being responsible for activating phospholipase C. We are using computational biophysical methods to probe the mechanisms of G protein selectivity of this muscarinic receptor family. We have built structural models for the M1 and M2 receptors coupled to Gαi or Gαq proteins and used molecular dynamics simulations to obtain relaxed structures of these complexes, so that the thermodynamic analysis of the receptor G protein interactions can be conducted. The analyses are beginning to show that thermodynamically, some complexes are more stable than others in agreement with corresponding biochemical experiments. In addition, some receptor ‐ G protein interactions may be kinetically controlled. Overall, these studies are beginning to provide us with the missing mechanistic insight into the G protein selectivity of the muscarinic receptors.Support or Funding InformationWe acknowledge the CSUPERB New Investigator Grant. We thank the RISE (GM063787) program for their student support.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Notch signaling facilitates hepatitis B virus covalently closed circular DNA transcription via cAMP response element-binding protein with E3 ubiquitin ligase-modulation

Notch1 is regulated by E3 ubiquitin ligases, with proteasomal degradation of the Notch intracellular domain affecting the transcription of target genes. cAMP response element-binding protein (CREB) mediates the transcription of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA). We assessed the relationship between HBV cccDNA and Notch signaling activities. HBV cccDNA levels and relative gene expression were evaluated in HBV-replicating cells treated with Jagged1 shRNA and a γ-secretase inhibitor. The effects of these factors in surgically resected clinical samples were also assessed. Notch inhibition suppressed HBV cccDNA and CREB-related expression but increased ITCH and NUMB levels. Proteasome inhibitor augmented HBV cccDNA, restored Notch and CREB expression, and inhibited ITCH and NUMB function. Increased HBV cccDNA was observed after ITCH and NUMB blockage, even after treatment with the adenylate cyclase activator forskolin; protein kinase A (PKA) inhibitor had the opposite effect. Notch activation and E3 ligase inactivation were observed in HBV-positive cells in clinical liver tissue. Collectively, these findings reveal that Notch signaling activity facilitates HBV cccDNA transcription via CREB to trigger the downstream PKA-phospho-CREB cascade and is regulated by E3 ubiquitin ligase-modulation of the Notch intracellular domain.

Hypercapnia Alters Alveolar Epithelial Repair by a pH-Dependent and Adenylate Cyclase-Mediated Mechanism

Lung cell injury and repair is a hallmark of the acute respiratory distress syndrome (ARDS). Lung protective mechanical ventilation strategies in these patients may lead to hypercapnia (HC). Although HC has been explored in the clinical context of ARDS, its effect upon alveolar epithelial cell (AEC) wounding and repair remains poorly understood. We have previously reported that HC alters the likelihood of AEC repair by a pH-sensitive but otherwise unknown mechanism. Adenylate cyclase (AC) is an attractive candidate as a putative AEC CO2 sensor and effector as it is bicarbonate sensitive and controls key mediators of AEC repair. The effect of HC on AC activity and plasma membrane (PM) wound repair was measured in AEC type 1 exposed to normocapnia (NC, 40 Torr) or HC (80 Torr), ± tromethamine (THAM) or sodium bicarbonate (HCO3) ± AC probes in a micropuncture model of AEC injury relevant to ARDS. Intracellular pH and AC activity were measured and correlated with repair. HC decreased intracellular pH 0.56, cAMP by 37%, and absolute PM repair rate by 26%. Buffering or pharmacologic manipulation of AC reduced or reversed the effects of HC on AC activity (THAM 103%, HCO3 113% of NC cAMP, ns; Forskolin 168%, p < 0.05) and PM repair (THAM 87%, HCO3 108% of NC likelihood to repair, ns; Forskolin 160%, p < 0.01). These findings suggest AC to be a putative AEC CO2 sensor and modulator of AEC repair, and may have implications for future pharmacologic targeting of downstream messengers of the AC-cAMP axis in experimental models of ARDS.

The PACAP-derived peptide MPAPO facilitates corneal wound healing by promoting corneal epithelial cell proliferation and trigeminal ganglion cell axon regeneration.

It is well known that the cornea plays an important role in providing protection to the eye, but it is fragile and vulnerable. To clarify the biological effects and molecular mechanisms of the pituitary adenylate cyclase activating polypeptide (PACAP)-derived peptide MPAPO (named MPAPO) to promote corneal wound healing, we applied a mechanical method to establish a corneal injury model and analyzed the repair effects of MPAPO on corneal injury. MPAPO significantly promoted corneal wound repair in C57BL/6 mice. In addition, we established injury models of epithelial cells and trigeminal ganglion cells with H2O2. The results show that when the concentration of MPAPO is 1 μM, it can significantly promote the repair of injured corneal epithelial cells and the regeneration of trigeminal ganglion cell axons. MPAPO repairs epithelial cells through the promotion of GSK3β phosphorylation by binding to PAC1 and activating AKT. β-catenin escapes the phosphorylation of GSK3β and enters the nucleus to promote the expression of cyclin D1, accelerate cell cycle progression and promote cell proliferation. MPAPO promotes axonal regeneration by binding to the PAC1 receptor and activating adenylate cyclase activity, followed by the cAMP activation of protein kinase A activity and the promotion of CREB phosphorylation. Phosphorylated CREB promotes Bcl2 expression and axonal regeneration. In conclusion, our data support the role of MPAPO to facilitate corneal wound healing by promoting corneal epithelial cell proliferation and trigeminal ganglion cell axon regeneration.