Increase In Adenylate Cyclase Activity Research Articles

Onset Mechanisms and Prognosis of Neurally Mediated Syncope

Neurally mediated syncope (NMS) is associated with a sudden loss of consciousness. However, the diagnostic tools and measures for prognosis management are limited. To overcome these limitations, the differences in the binding energies of Giα-protein-coupled receptors to the Glu9 and Glu12 residues on the α2B-AR gene were elucidated through the analysis of α2B-AR gene polymorphism. The suppression of the activity of adenylate cyclase (AC), which is involved in vasoconstriction, may be related to the onset of NMS. The head-up tilt (HUT) test results indicated differences in systolic blood pressure (SBP) and AC activity between patients with vasodepressor (VT)-NMS and healthy volunteers. Patients with VT-NMS had increased AC activity and decreased SBP. Conversely, in healthy volunteers, no changes in AC activity or SBP were found. These findings suggest that a high SBP and elevated AC activity at rest are likely to cause syncope. A high incidence of cardiovascular events is found in patients with negative HUT test results, highlighting the importance of investigating the cause of syncope in cases where the HUT test results are negative. Overall, our results may provide a means of assessing the risk of NMS development within healthy populations and underscore the importance of subsequent treatments for NMS.

P2Y receptors in GtoPdb v.2023.1

P2Y receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on P2Y Receptors [3, 5, 189]) are activated by the endogenous ligands ATP, ADP, UTP, UDP, UDP-glucose and adenosine. The eight mammalian P2Y receptors are activated by distinct nucleotides: P2Y1, P2Y11, P2Y12 and P2Y13 are activated by adenosine-nucleotides; P2Y2, P2Y4 can be activated by both adenosine and uridine nucleotides, with some species-specific differences; P2Y6 is mainly activated by UDP; P2Y14 is preferentially activated by sugar-uracil nucleotides. The missing numbers in the receptor nomenclature refer either to non-mammalian orthologs or receptors having some sequence homology to P2Y receptors but for which there is no functional evidence of responsiveness to nucleotides [380]. Based on their G protein coupling P2Y receptors can be divided into two subfamilies: P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11 receptors couple via Gq proteins to stimulate phospholipase C followed by increases in inositol phosphates and mobilization of Ca2+ from intracellular stores. P2Y11 receptors couple in addition to Gs proteins followed by increased adenylate cyclase activity. In contrast, P2Y12, P2Y13, and P2Y14 receptors signal primarily through activation of Gi proteins and inhibition of adenylate cyclase activity or control of ion channel activity [380]. Clinically used drugs acting on these receptors include the dinucleoside polyphosphate diquafosol, agonist of the P2Y2 receptor subtype, approved in Japan and South Korea for the management of dry eye disease [238], and the P2Y12 receptor antagonists prasugrel, ticagrelor and cangrelor, all approved as antiplatelet drugs [52, 320].

The IgG3 subclass of β1-adrenergic receptor autoantibodies is an endogenous biaser of β1AR signaling.

Dysregulation of immune responses has been linked to the generation of immunoglobulin G (IgG) autoantibodies that target human β1ARs and contribute to deleterious cardiac outcomes. Given the benefits of β-blockers observed in patients harboring the IgG3 subclass of autoantibodies, we investigated the role of these autoantibodies in human β1AR function. Serum and purified IgG3(+) autoantibodies from patients with onset of cardiomyopathy were tested using human embryonic kidney (HEK) 293 cells expressing human β1ARs. Unexpectedly, pretreatment of cells with IgG3(+) serum or purified IgG3(+) autoantibodies impaired dobutamine-mediated adenylate cyclase (AC) activity and cyclic adenosine monophosphate (cAMP) generation while enhancing biased β-arrestin recruitment and Extracellular Regulated Kinase (ERK) activation. In contrast, the β-blocker metoprolol increased AC activity and cAMP in the presence of IgG3(+) serum or IgG3(+) autoantibodies. Because IgG3(+) autoantibodies are specific to human β1ARs, non–failing human hearts were used as an endogenous system to determine their ability to bias β1AR signaling. Consistently, metoprolol increased AC activity, reflecting the ability of the IgG3(+) autoantibodies to bias β-blocker toward G-protein coupling. Importantly, IgG3(+) autoantibodies are specific toward β1AR as they did not alter β2AR signaling. Thus, IgG3(+) autoantibody biases β-blocker toward G-protein coupling while impairing agonist-mediated G-protein activation but promoting G-protein–independent ERK activation. This phenomenon may underlie the beneficial outcomes observed in patients harboring IgG3(+) β1AR autoantibodies.

Role of PKCζ-NADPH oxidase signaling axis in PKCα-mediated Giα2 phosphorylation for inhibition of adenylate cyclase activity by angiotensin II in pulmonary artery smooth muscle cells.

We sought to determine the mechanism by which angiotensin II (AngII) inhibits isoproterenol induced increase in adenylate cyclase (AC) activity and cyclic adenosine monophosphate (cAMP) production in bovine pulmonary artery smooth muscle cells (BPASMCs). Treatment with AngII stimulates protein kinase C-ζ (PKC-ζ), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and PKC-α activities, and also inhibits isoproterenol induced increase in AC activity and cAMP production in the cells. Pertussis toxin pretreatment eliminates AngII caused inhibition of isoproterenol induced increase in AC activity without a discernible change in PKC-ζ, NADPH oxidase, and PKC-α activities. Treatment of the cells with AngII increases α2 isoform of Gi (Giα2) phosphorylation; while pretreatment with chemical and genetic inhibitors of PKC-ζ and NADPH oxidase attenuate AngII induced increase in PKC-α activity and Giα2 phosphorylation, and also reverse AngII caused inhibition of isoproterenol induced increase in AC activity. Pretreatment of the cells with chemical and genetic inhibitors of PKC-α attenuate AngII induced increase in Giα2 phosphorylation and inhibits isoproterenol induced increase in AC activity without a discernible change in PKC-ζ and NADPH oxidase activities. Overall, PKCζ-NADPH oxidase-PKCα signaling axis plays a crucial role in Giα2 phosphorylation resulting in AngII-mediated inhibition of isoproterenol induced increase in AC activity in BPASMCs.

Trauma Leads to a Rise in Intracellular cAMP and Adenylate Cyclase Activity in Rats

Background: Severe trauma and hemorrhage leads to an acute coagulopathy as exemplified by a decrease in clotting firmness and platelet aggregation. Because platelets contribute 70-80% of clot strength, evaluating the intracellular mechanisms that regulate aggregation in platelets may lead to strategies to mitigate the development of coagulopathy. It is known that platelet aggregation can be inhibited by a rise in intracellular cyclic adenosine monophosphate (cAMP) or cyclic guanosine monophosphate (cGMP). Changes in cAMP/cGMP would necessitate a change in activity of those enzymes that either synthesize (adenylate cyclase/ guanylate cyclase) or breakdown (phosphodiesterases) these 2nd messengers.Objective: Determine if the intracellular levels of cAMP and cGMP in platelets rise in rats subjected to polytrauma and hemorrhage. And if so, if there is a change in cyclase or phosphodiesterase activity.Methods: Polytrauma was induced in isoflurane anesthetized Sprague-Dawley rats (n=10 per group) by damage to the small intestines, right and medial lobes of the liver, the right leg skeletal muscle, and by fracturing the right femur. 40% hemorrhage was then performed immediately after trauma. Rats were euthanized at 4hrs. Platelet intracellular intermediates were measured in whole blood before, and 0.5, 1, 2 and 4hrs after trauma. Blood samples were taken before, and 0.5, 2 and 4 hours after trauma. Platelet rich plasma (PRP) was generated by centrifugation of whole blood at 200g for 10min, no brakes. Cyclic AMP, cGMP, AMP and GMP were extracted from 100ul of PRP after adding 1ml of EtOH, 10mM ammonium formate, with 10ug/ml cGMP-Br as an internal control. IP3 and IP1 (metabolic breakdown product of IP3) were extracted from another 100ul PRP by addition of 50% EtOH, 500mM Formic Acid with 10ug/ml ATP-C13 as an internal control. Samples were centrifuged at 20K g for 10min, and supernatant dried. All Samples were brought up in 200ul of 0.1% formic acid for analysis by LC-MS/MS. Cyclase and phosphodiesterases were isolated from 100ul of rat PRP after sonication to lyse cells, centrifugation to release intracellular constituents, and Spin Column (Zeba, ThermoFisher) removal of small molecular weight constituents. Cyclase activity was measured for 15min after addition of either ATP or GTP and measuring cAMP or cGMP with inhibition of PDE activity (IBMX 1uM). Phosphodiesterase was measured by addition of cAMP or cGMP and measuring AMP or GMP. Data was generated by subtracting measurement from control.Results: Trauma and hemorrhage led to a significant rise in the intracellular cAMP and a fall in cGMP. Adenylate cyclase activity in platelets also significantly increase after trauma. However, trauma had no effect on guanylate cyclase activity. Phosphodiesterase activity was elevated after trauma for both conversion of cAMP and cGMP to AMP and GMP, but neither was significant.Conclusion: Trauma and hemorrhage leads to coagulopathy and platelet dysfunction. The platelet dysfunction is likely due to a rise in the intracellular cAMP, but not to cGMP as it fell precipitously after trauma. The rise in cAMP is likely due to an increase in adenylate cyclase activity induced by trauma. Although there was a rise in PDE activity, it was not significant, but may play a role in blunting the rise and cAMP, and contribute to the fall in cGMP. This study was funded by the US Army MRMC and conducted in compliance with the Animal Welfare Act, the implementing of Animal Welfare Regulations, and the principles of the Guide for the Care and Use of Laboratory Animals. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Computational Approach for Understanding the Interactions between Graphene Oxide and Nucleoside Diphosphate Kinase with Implications for Heart Failure.

During a heart failure, an increased content and activity of nucleoside diphosphate kinase (NDPK) in the sarcolemmal membrane is responsible for suppressing the formation of the second messenger cyclic adenosine monophosphate (cAMP)—a key component required for calcium ion homeostasis for the proper systolic and diastolic functions. Typically, this increased NDPK content lets the surplus NDPK react with a mutated G protein in the beta-adrenergic signal transduction pathway, thereby inhibiting cAMP synthesis. Thus, it is thus that inhibition of NDPK may cause a substantial increase in adenylate cyclase activity, which in turn may be a potential therapy for end-stage heart failure patients. However, there is little information available about the molecular events at the interface of NDPK and any prospective molecule that may potentially influence its reactive site (His118). Here we report a novel computational approach for understanding the interactions between graphene oxide (GO) and NDPK. Using molecular dynamics, it is found that GO interacts favorably with the His118 residue of NDPK to potentially prevent its binding with adenosine triphosphate (ATP), which otherwise would trigger the phosphorylation of the mutated G protein. Therefore, this will result in an increase in cAMP levels during heart failure.

Stimulation of gastric acid secretion by rabbit parietal cell A2Badenosine receptor activation

Adenosine modulates different functional activities in many cells of the gastrointestinal tract; some of them are believed to be mediated by interaction with its four G protein-coupled receptors. The renewed interest in the adenosine A2B receptor (A2BR) subtype can be traced by studies in which the introduction of new genetic and chemical tools has widened the pharmacological and structural knowledge of this receptor as well as its potential therapeutic use in cancer and inflammation- or hypoxia-related pathologies. In the acid-secreting parietal cells of the gastric mucosa, the use of various radioligands for adenosine receptors suggested the presence of the A2 adenosine receptor subtype(s) on the cell surface. Recently, we confirmed A2BR expression in native, nontransformed parietal cells at rest by using flow cytometry and confocal microscopy. In this study, we show that A2BR is functional in primary rabbit gastric parietal cells, as indicated by the fact that agonist binding to A2BR increased adenylate cyclase activity and acid production. In addition, both acid production and radioligand binding of adenosine analogs to isolated cell membranes were potently blocked by selective A2BR antagonists, whereas ligands for A1, A2A, and A3 adenosine receptors failed to abolish activation. We conclude that rabbit gastric parietal cells possess functional A2BR proteins that are coupled to Gs and stimulate HCl production upon activation. Whether adenosine- and A2BR-mediated functional responses play a role in human gastric pathophysiology is yet to be elucidated.

A new knock-in mouse model of l-DOPA-responsive dystonia.

Abnormal dopamine neurotransmission is associated with many different genetic and acquired dystonic disorders. For instance, mutations in genes critical for the synthesis of dopamine, including GCH1 and TH cause l-DOPA-responsive dystonia. Despite evidence that implicates abnormal dopamine neurotransmission in dystonia, the precise nature of the pre- and postsynaptic defects that result in dystonia are not known. To better understand these defects, we generated a knock-in mouse model of l-DOPA-responsive dystonia (DRD) mice that recapitulates the human p.381Q>K TH mutation (c.1141C>A). Mice homozygous for this mutation displayed the core features of the human disorder, including reduced TH activity, dystonia that worsened throughout the course of the active phase, and improvement in the dystonia in response to both l-DOPA and trihexyphenidyl. Although the gross anatomy of the nigrostriatal dopaminergic neurons was normal in DRD mice, the microstructure of striatal synapses was affected whereby the ratio of axo-spinous to axo-dendritic corticostriatal synaptic contacts was reduced. Microinjection of l-DOPA directly into the striatum ameliorated the dystonic movements but cerebellar microinjections of l-DOPA had no effect. Surprisingly, the striatal dopamine concentration was reduced to ∼1% of normal, a concentration more typically associated with akinesia, suggesting that (mal)adaptive postsynaptic responses may also play a role in the development of dystonia. Administration of D1- or D2-like dopamine receptor agonists to enhance dopamine signalling reduced the dystonic movements, whereas administration of D1- or D2-like dopamine receptor antagonists to further reduce dopamine signalling worsened the dystonia, suggesting that both receptors mediate the abnormal movements. Further, D1-dopamine receptors were supersensitive; adenylate cyclase activity, locomotor activity and stereotypy were exaggerated in DRD mice in response to the D1-dopamine receptor agonist SKF 81297. D2-dopamine receptors exhibited a change in the valence in DRD mice with an increase in adenylate cyclase activity and blunted behavioural responses after challenge with the D2-dopamine receptor agonist quinpirole. Together, our findings suggest that the development of dystonia may depend on a reduction in dopamine in combination with specific abnormal receptor responses.

Perfluorooctyl Iodide Stimulates Steroidogenesis in H295R Cells via a Cyclic Adenosine Monophosphate Signaling Pathway.

Perfluorinated iodine alkanes (PFIs) are used widely in the organic fluorine industry. Increased production of PFIs has caused environmental health concerns. To evaluate the potential endocrine-disrupting effect of PFIs, we investigated the effects of perfluorooctyl iodide (PFOI) on steroidogenesis in human adrenocortical carcinoma cells (H295R). Levels of aldosterone, cortisol, 17β-estradiol, and testosterone were measured in H295R culture medium upon treatment with perfluorooctanoic acid (PFOA) and PFIs. Expression of 10 steroidogenic genes (StAR, HMGR, CYP11A1, 3βHSD2, 17βHSD, CYP17, CYP21, CYP11B1, CYP11B2, and CYP19) was measured by real-time polymerase chain reaction. Levels of cyclic adenosine monophosphate (cAMP) and adenylate cyclase (AC) activity were measured to understand the underlying mechanism of steroidogenic perturbations. Levels of production of aldosterone, cortisol, and 17β-estradiol were elevated significantly, and the level of testosterone generation decreased upon treatment with 100 μM PFOI. Similar to the effect induced by forskolin (AC activator), expression of all 10 genes involved in the synthesis of steroid hormones was upregulated significantly upon exposure to 100 μM PFOI. PFOA had no effect on steroid hormone production or steroidogenic gene expression even though it is highly structurally similar with PFOI. Therefore, the terminal -CF2I group in PFOI could be a critical factor for mediation of steroidogenesis. PFOI increased AC activity and cAMP levels in H295R cells, which implied an underlying mechanism for the disturbance of steroidogenesis. These data suggest that PFOI may act as an AC activator, thereby stimulating steroidogenesis by activating a cAMP signaling pathway.

McCune Albright syndrome

Inheritance MAS is not inherited. It is a sporadic genetic disorder, caused by a mutation in the GNAS gene encoding the alpha subunit of the stimulatory G protein (GSalpha), cyclic AMP protein kinase dependent cellular signaling pathway involving G protein coupled receptors. An arginine or occasionally serine, leucine or glycine to histidine transposition at residue 201 attenuates GTpase activity. This results in constitutive activation of adenylyl cyclase. The somatic mutation occurs in early embryogenesis, resulting in widespread tissue distribution of abnormalities. The post zygotic mutation is responsible for the mosaic pattern of tissue distribution and the extreme variability of clinical changes, varying from multi system disease to almost unrecognized disorders with single organ involvement. Increased adenylate cyclase activity in bone from clinical fibrous dysplastic lesions is caused by expression of Cfos proto-oncogene in bone. Recent identification of increased fibroblast growth factor (FGF23) activity in fibrous dysplastic lesions has linked the bony abnormalities to a recognized marker of bone metabolism.

Lithium inhibition of the adenosine-induced increase of adenylate cyclase activity.

Lithium inhibition of the adenosine-induced increase of adenylate cyclase activity.

R2D2 for C4Eo: an ‘alliance’ of PGD2 receptors is required for LTC4 production by human eosinophils

Eosinophils play important roles in limiting parasitic infection and in allergic inflammation in the asthmatic airways. Activation of eosinophils by diverse stimuli, including prostaglandin D2 (PD2), leads to leukotriene C4 (LTC4) synthesis that contributes to the expulsion of parasites and to epithelial injury in allergic inflammation. Mesquita‐Santos et al. in this issue of the journal describe a collaboration between the two PGD2 receptors, DP1 and DP2[also known as CRTH2 (chemoattractant receptor‐homologous molecule expressed on Th2 lymphocytes)] that is required to trigger LTC4 synthesis. DP1 receptors coupled to Gαs increase adenylate cyclase activity and cAMP/ protein kinase A–dependent formation of lipid bodies, and DP2 receptors coupled to Gαi increase calcium. Each of these signals is required for LTC4 production. These observations lead to consideration of the effects of other stimuli for eosinophil cAMP, such as the β2‐adrenoceptor agonists, which inhibit rather than enhance LTC4 production.LINKED ARTICLE This article is a commentary on Mesquita‐Santos et al., pp. 1674–1685 of this issue. To view this paper visit http://dx.doi.org/10.1111/j.1476‐5381.2010.01086.x

A mechanism by which the osmotic laxative magnesium sulphate increases the intestinal aquaporin 3 expression in HT-29 cells

Aims We have suggested that an osmotic laxative, magnesium sulphate (MgSO 4), may act as a cathartic in a very rational manner by increasing the aquaporin 3 (AQP3) expression level and by changing osmotic pressure in the colon. In this study, we examined the mechanism by which MgSO 4 increases the intestinal AQP3 expression level by using the human colon cancer HT-29 cell line. Main methods After the addition of MgSO 4 to HT-29 cells, the expression levels of AQP3 mRNA and protein were measured using real-time RT-PCR and western blotting, respectively. The intracellular Mg 2+ concentration, adenylate cyclase (AC) activity and protein kinase A (PKA) activity were also determined. The phosphorylated cAMP response element-binding protein (CREB) expression levels were determined by western blotting. Key findings The AQP3 mRNA expression level started to increase significantly at 1 h after MgSO 4 addition and peaked at 9 h, at a level 3 times as high as the control levels. The AQP3 protein expression level started to increase 6 h after the addition and reached a level almost twice as high as the control levels by hour 12. In the HT-29 cells treated with MgSO 4, there was a 1.4-fold increase in the intracellular Mg 2+ concentration, a 1.5-fold increase in AC activity, a 1.6-fold increase in PKA activity, and a significant increase in phosphorylation of the CREB. Significance These results suggest that the AC activation caused by an increase in the intracellular Mg 2+ concentration may trigger CREB phosphorylation through PKA activation and promote AQP3 gene transcription.

The significance of GPR119 agonists as a future treatment for type 2 diabetes

GPR119 is a G protein-coupled receptor that is expressed on only a limited number of tissues, including pancreatic β-cells and enteroendocrine cells in the small intestine, and that appears to be involved in the regulation of metabolic homeostasis. The protein was originally defined as an orphan receptor, but it has subsequently been shown to bind a variety of lipid-derived ligands, as well as a range of small synthetic molecules. There is still debate as to the identity of its principal endogenous ligand, but certain lysophospholipids species, various fatty acyl-ethanolamides and N-oleoyldopamine have all been proposed as potential agonists. GPR119 is coupled to the signal transducer Gαs and activation of the receptor leads to increased adenylate cyclase activity via Gαs and a rise in intracellular cAMP. This then potentiates glucose-induced insulin secretion or promotes the release of intestinal incretin hormones, according to cell type. Both mechanisms ultimately lead to a rise in insulin secretion (either directly or indirectly) and improved glucose control. Thus, GPR119 may represent an important new therapeutic target for the design of insulin secretagogues able to promote improvements in blood glucose control in patients with type 2 diabetes. Accordingly, a range of lead compounds are in development as potential therapeutic agents.

Paracrine stimulation of vascular smooth muscle proliferation by diadenosine polyphosphates released from proximal tubule epithelial cells

The purinergic receptor system plays an important role in the regulation of both vascular and tubular functions within the kidney; however, the release of purinergic agonists other than ATP by renal tissue is not known. In this investigation, we determine if kidney tissue is a source of diadenosine polyphosphates, which have high affinity for the P(2X) and P(2Y) receptors. Both diadenosine pentaphosphate and hexaphosphate were identified by matrix-assisted laser desorption ionization-mass spectrometry in extracts purified from both whole porcine kidney and from cloned cells of the LLC-PK1 cell line. Both polyphosphates in nanomolar concentrations were found to significantly stimulate the proliferation of vascular smooth muscle cells derived from rat thoracic aortas. The purinergic-receptor antagonist, suramin, did not significantly affect the growth-stimulatory properties of the polyphosphates. The growth stimulation of vascular smooth muscle cells by platelet-derived growth factor was potentiated by both diadenosine polyphosphates. We conclude that diadenosine polyphosphates are endogenous purinergic agonists of the kidney that have physiologic and pathophysiologic relevance. These epithelial cell metabolic products have vasoregulatory properties while linking the energy supply and tubular function.

Interplay of Ca2+ and cAMP Signaling in the Insulin-secreting MIN6 β-Cell Line

Ca2+ and cAMP are important second messengers that regulate multiple cellular processes. Although previous studies have suggested direct interactions between Ca2+ and cAMP signaling pathways, the underlying mechanisms remain unresolved. In particular, direct evidence for Ca2+-regulated cAMP production in living cells is incomplete. Genetically encoded fluorescence resonance energy transfer-based biosensors have made possible real-time imaging of spatial and temporal gradients of intracellular cAMP concentration in single living cells. Here, we used confocal microscopy, fluorescence resonance energy transfer, and insulin-secreting MIN6 cells expressing Epac1-camps, a biosynthetic unimolecular cAMP indicator, to better understand the role of intracellular Ca2+ in cAMP production. We report that depolarization with high external K+, tolbutamide, or glucose caused a rapid increase in cAMP that was dependent on extracellular Ca2+ and inhibited by nitrendipine, a Ca2+ channel blocker, or 2',5'-dideoxyadenosine, a P-site antagonist of transmembrane adenylate cyclases. Stimulation of MIN6 cells with glucose in the presence of tetraethylammonium chloride generated concomitant Ca2+ and cAMP oscillations that were abolished in the absence of extracellular Ca2+ and blocked by 2',5'-dideoxyadenosine or 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase. Simultaneous measurements of Ca2+ and cAMP concentrations with Fura-2 and Epac1-camps, respectively, revealed a close temporal and causal interrelationship between the increases in cytoplasmic Ca2+ and cAMP levels following membrane depolarization. These findings indicate highly coordinated interplay between Ca2+ and cAMP signaling in electrically excitable endocrine cells and suggest that Ca2+-dependent cAMP oscillations are derived from an increase in adenylate cyclase activity and periodic activation and inactivation of cAMP-hydrolyzing phosphodiesterase.

Mutations in the carboxy-terminus of the third intracellular loop of the human recombinant VPAC 1 receptor impair VIP-stimulated [Ca 2+] i increase but not adenylate cyclase stimulation

The vasoactive intestinal polypeptide (VIP) VPAC 1 receptor is preferentially coupled to Gαs protein that stimulates adenylate cyclase activity and also to Gαq and Gαi proteins that stimulate the inositol phosphate/calcium pathway. Previous studies indicated the importance of the third intracellular loop of the receptor for G protein coupling. By site-directed mutation of the human recombinant receptor expressed in Chinese hamster ovary cells, we identified two domains in this loop that contain clusters of basic residues conserved in most of the G-protein-coupled seven transmembrane domains receptors. We found that mutations in the proximal domain (K 322) reduced the capability of VIP to increase adenylate cyclase activity without any change in the calcium response, whereas mutations in the distal part of the loop (R 338, L 339, R 341) markedly reduced the calcium increase and Gαi coupling but only weakly the adenylate cyclase activity. Thus, the interaction of different G proteins with the VPAC 1 receptor involves different receptor sub-domains.

Effects of Chronic Hypogonadism on Corticosterone Secretion and Cyclic AMP Production in Male Rat Adrenocortical Cells

Aim: To determine the secretion of corticosterone (CCS) both in vivo and in vitro during different intervals after orchidectomy in male rats. Methods: Three- and 12-month-old rats had been orchidectomized 0, 3, 6, or 9 months before decapitation. Results: Orchidectomy increased the concentrations of plasma CCS, the basal release of CCS, and the adenosine 3′, 5′-cyclic monophosphate (cAMP) production in rat zona fasciculata reticularis (ZFR) cells. The forskolin/3-isobutyl-l-methylxanthine-stimulated releases of CCS and cAMP production by ZFR cells were higher in rats with chronic hypogonadism. The CCS release from ZFR cells of orchidectomized rat was not altered by 8-bromo-cAMP treatment. Orchidectomy enhanced the stimulatory effect of deoxycorticosterone on CCS release in ZFR cells. Conclusion: These results suggest that orchidectomy-related increases of CCS secretion in rats are associated with an increase of adenylate cyclase activity, cAMP generation, and 11-β-hydroxylase activity in ZFR cells.

Elucidation of signaling properties of vasopressin receptor-related receptor 1 by using the chimeric receptor approach.

The identification of endogenous or surrogate ligands for orphan G protein-coupled receptors (GPCRs) represents one of the most important tasks in GPCR biology and pharmacology. The challenge lies in choosing an appropriate assay in the absence of ways to activate the receptor of interest. We investigated the signaling pathway for an orphan GPCR referred to here as vasopressin receptor-related receptor 1 (VRR1) by generating a chimeric receptor, V1a/VRR1. The engineered construct contained vasopressin V1a receptor with all three intracellular loops and C terminus replaced by those of VRR1. The chimera behaved like a typical GPCR when transiently and stably expressed in mammalian cell lines based on radioligand binding and receptor internalization studies. Upon arginine vasopressin stimulation, this chimeric receptor induced robust calcium mobilization and increase of adenylate cyclase activity. The observed signaling activities are through the activation of the chimera instead of endogenously expressed receptors, as single amino acid changes in the second transmembrane regions of the chimera drastically reduced receptor efficacy and potency. Our results suggest that VRR1 has dual signaling properties in coupling to both G(q) and G(S) pathways. Analysis of native VRR1 receptor signaling pathway by using a recently identified ligand for VRR1 confirmed this conclusion and therefore validated the utility of the chimeric receptor approach for signaling pathway identification.

Effects of adenosine on bacterial lipopolysaccharide- and interleukin 1-induced nitric oxide release from equine articular chondrocytes.

To determine whether adenosine influences the in vitro release of nitric oxide (NO) from differentiated primary equine articular chondrocytes. Articular cartilage harvested from the metacarpophalangeal and metatarsophalangeal joints of 11 horses (3 to 11 years old) without history or clinical signs of joint disease. Chondrocytes were isolated, plated at a high density (10(5) cells/well), and treated with adenosine, the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), bradykinin, or other agents that modify secondary messenger pathways alone or in combination with bacterial lipopolysaccharide (LPS) or recombinant human interleukin-1alpha (rhIL-1alpha). Nitric oxide release was measured indirectly by use of the Griess reaction and was expressed as micromol of nitrite in the supernatant/microg of protein in the cell layer. Inducible nitric oxide synthase (iNOS) activity was determined by measuring the conversion of radiolabeled arginine to radiolabeled citrulline. Treatment of chondrocytes with adenosine alone had no significant effect on NO release. However, adenosine and NECA inhibited LPS- and rhIL-1alpha-induced NO release. This response was mimicked by forskolin, which acts to increase adenylate cyclase activity, but not by the calcium ionophore A23187 Treatment of chondrocytes with phorbol myristate acetate, which acts to increase protein kinase C activity, potentiated LPS-induced NO release. Adenosine treatment also significantly inhibited the LPS-induced increase in iNOS activity. Adenosine and the nonspecific adenosine receptor agonist NECA inhibited inflammatory mediator-induced release of NO from equine articular chondrocytes. Modulation of adenosine receptor-mediated pathways may offer novel methods for treatment of inflammation in horses with joint disease.