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.

Expression pattern of type 3 adenylyl cyclase in rodent dorsal root ganglion and its primary afferent terminals.

cAMP (Cyclic Adenosine monophosphate), one of the most highly studied second messengers, is regulated by a family of adenylyl cyclase (AC) enzymes. Type 3 adenylyl cyclase (abbreviated as AC3), a subtype of adenylyl cyclase, is reported to be expressed in cilia in the olfactory and central nervous system and plays an important role in many physiological functions such as olfaction, development. However, expression of AC3 in the dorsal root ganglion (DRG) is not reported. In the present study, using immunohistochemical method, we discovered that AC3 immunoreactivity (IR) is predominantly expressed in the cytoplasm of small to medium sized DRG neurons. Double labelling revealed that the majority of AC3 IR are colocalized with CGRP (a peptidergic nociceptor marker), rarely with NF200 (a myelinated neuronal marker) or IB4 (a nonpeptidergic nociceptor marker). Furthermore, dense AC3 IR exists in the superficial dorsal horn, especially in laminaⅠand dorsal part of lamina II, where CGRP-positive DRG neurons terminate. The expression pattern of AC3 is similar between C57/BL6 J mouse and Sprague Dawley rat. For instance, AC3 is primarily expressed in the cell bodies of small to medium sized DRG neurons and the majority of AC3 IR is also in CGRP-containing neurons in rat. Taken together, our data suggest that AC3 is primarily expressed in the small to medium sized cell bodies and central terminals of CGRP-positive DRG neurons, implying AC3 enzyme might potentially function in nociception.

Correlation between Activity and Domain Complementation in Adenylyl Cyclase Demonstrated with a Novel Fluorescence Resonance Energy Transfer Sensor.

Adenylyl cyclase (AC) activity relies on multiple effectors acting through distinct binding sites. Crystal structures have revealed the location of these sites, and biochemical studies have explored the kinetics of ACs, but the interplay between conformation and activity remains incompletely understood. Here, we describe a novel fluorescence resonance energy transfer (FRET) sensor that functions both as a soluble cyclase and a reporter of complementation within the catalytic domain. We report a strong linear correlation between catalytic domain complementation and cyclase activity upon stimulation with forskolin and Gαs. Exploiting this, we dissect the mechanism of action of a series of forskolin analogs and a P-site inhibitor, 2'-d3'-AMP. Finally, we demonstrate that this sensor is functional in live cells, wherein it reports forskolin-stimulated activity of AC.

Pituitary Adenylate Cyclase Activating Peptide (1-38) and its analog (Acetyl-[Ala15, Ala20] PACAP 38-polyamide) reverse methacholine airway hyperresponsiveness in rats

The aim of this study was to investigate both functionally and structurally bronchodilator effects of Pituitary adenylate cyclase activating peptide (PACAP38) and acetyl-[Ala15, Ala20] PACAP38-polyamide, a potent PACAP38 analog, in rats challenged by methacholine (MeCh). Male Wistar rats were divided randomly into five groups. Groups 1 and 2 inhaled respectively aerosols of saline or increasing doses of MeCh (0.5, 1, 2.12, 4.25, 8.5, 17, 34 and 68mg/L). The other groups received terbutaline (Terb) (250 µg/rat) (10-6 M), PACAP38 (50 µg/rat) (0.1 mM) or PACAP38 analog (50 µg/rat) associated to MeCh from the dose of 4.25 mg/L. Total lung resistances (RL) were recorded before and 2 min after MeCh administration by pneumomultitest equipment. MeCh administration induced a significant and a dose-dependent increase (p<0.05) of RL compared to control rats. Terb, PACAP38 and PACAP38 analog reversed significantly the MeCh-induced bronchial constriction, smooth muscle (SM) layer thickness and bronchial lumen mucus abundance. PACAP38 analog prevents effectively bronchial smooth muscle layer thickness, mucus hypersecretion and lumen decrease. Therefore, it may constitute a potent therapeutic bronchodilator.

O objetivo deste estudo foi investigar funcionalmente e estruturalmente efeito broncodilatador do peptídeo ativador da adenilato ciclase pituitária (PACAP1-38) e da acetil-[Ala15, Ala20]PACAP 38-poliamida, potente análogo do PACAP-38, nos ratos desafiados pelo metacolina (MeCh). Ratos Wistar machos foram aleatoriamente divididos em cinco grupos. Grupos 1 e 2, inalando aerossóis de solução salina ou doses crescentes de MeCh (0,5, 1, 2,12, 4,25, 8,5, 17, 34 e 68 mg/L). Os outros grupos recebendo terbutalina (Terb) (250 µg/rato) (10-6M), PACAP-38 (50 µg/rato) (0.1 mM) ou análogo do PACAP-38 (50 µg/rato) associados a MeCh na dose de 4,25 mg/L. A resistência pulmonar total (RL) foi registrada antes e 2 min após a administração de Mech pelo equipamento pneumomultiteste. A administração MeCh induziu aumento significativo e dose dependente (p<0,05) de RL em comparação com ratos do grupo controle. Terb e PACAP1-38 e análogo do PACAP-38 reverteram, significativamente, a constrição brônquica induzida por Mech, a espessura do músculo liso (SM) e abundância de muco do lume brônquico. O análogo PACAP-38 do mesmo modo que a Terb impediu a responsividade brônquica a MeCh e pode se constituir em um importante regulador no desenvolvimento da doença inflamatório pulmonar. Contudo, o uso do peptídeo nativo para aplicações terapêuticas é limitado por sua baixa estabilidade metabólica. Consequentemente, o análogo metabolicamente estável representa ferramenta promissora no tratamento de doenças pulmonares inflamatórias.

Soluble adenylyl cyclase antibody (R21) as a diagnostic adjunct in the evaluation of lentigo maligna margins during slow Mohs surgery.

Margin-controlled staged excision (slow Mohs) has emerged as a preferred method for the treatment of lentigo maligna (LM). The interpretation of margins for LM is one of the most challenging tasks faced by a dermatopathologist. R21 is a mouse monoclonal antibody against soluble adenylyl cyclase (sAC), overexpressed in the nuclei of LM but not in native melanocytes. The objective of this study was to validate the use of sAC immunohistochemistry in histological assessment of slow Mohs surgery margins for LM. Seventeen randomly selected cases of patients who underwent slow Mohs surgery for LM at Lahey Clinic, Burlington, MA, were studied. Ninety-nine margins were stained with R21 and microphthalmia transcription factor antibodies and reevaluated blindly by 2 observers. Sixteen of 17 lesions expressed sAC. In all cases, observers agreed on interpretation of R21 stains. In 85 (86%) margins, there was concordance between routine sections and R21 stains. In 14 margins (14%), the results were discrepant. In 2 margins, R21 identified foci of LM missed on routine sections. In 8 margins, atypical melanocytes, interpreted as positive in routine sections, were negative for R21 questioning the accuracy of the original interpretation. Microphthalmia transcription factor stained nuclei of melanocytes in all margins. We found significant correlation between assessment of margins by sAC immunohistochemistry and routine histology. Evaluation of sAC expression using R21 antibody is a useful diagnostic adjunct in the evaluation of margins of LM during slow Mohs surgery.

Soluble adenylyl cyclase antibody profile as a diagnostic adjunct in the assessment of pigmented lesions.

OBJECTIVE: To investigate the usefulness of a novel marker for melanocytic proliferations. DESIGN: Using a novel monoclonal antibody against soluble adenylyl cyclase (sAC), various benign and malignant melanocytic proliferations were immunostained. SETTING: Weill Medical College of Cornell University dermatopathology laboratory. MAIN OUTCOME MEASURES: The results were qualitative, not quantifiable. RESULTS: The sAC immunostaining produced distinctive patterns that paralleled melanomagenesis. At one pole of the spectrum were benign nevi, including atypical nevi of special sites and recurrent nevi showing a distinct pattern of dotlike Golgi staining, while at the opposite pole was melanoma, in which many cells demonstrated an intense pannuclear expression pattern, often accompanied by loss of the Golgi expression pattern. Melanomas of lentigo maligna and acral lentiginous subtypes exhibited the most striking pannuclear expression, while nodular melanomas showed the least, although with supervening enhanced diffuse cytoplasmic expression. Loss of the Golgi expression pattern was a feature of malignant melanoma. CONCLUSION: The sAC expression pattern is complex but seems discriminatory, with distinctive and variable staining patterns according to the nature of the lesion biopsied.

Neuroanatomical distribution and neurochemical characterization of cells expressing adenylyl cyclase isoforms in mouse and rat brain.

Transmembrane adenylyl cyclases (Adcy) are involved in the regulation of multiple brain processes such as synaptic plasticity, learning and memory. They synthesize intracellular cyclic adenosine monophosphate (cAMP) following activation by G-protein coupled receptors. We examined the neuroanatomical distribution of the nine Adcy isoforms in rat and mouse brain by in situ hybridization, as well as their location in glutamatergic, GABAergic and cholinergic neurons in several mouse brain areas by double in situ hybridization. The Adcys are widely distributed throughout the brain in both rat and mouse, being especially abundant in cortex, hippocampus, thalamic nuclei, the olfactory system and the granular layer of the cerebellum. Double-labeling experiments showed that Adcy isoforms are differently expressed in glutamatergic, GABAergic and cholinergic neuronal cell populations. We report the neuroanatomical distribution of the nine known Adcy isoforms in rat and mouse brain and their cellular localization.

Assembly of a beta2-adrenergic receptor--GluR1 signalling complex for localized cAMP signalling.

Central noradrenergic signalling mediates arousal and facilitates learning through unknown molecular mechanisms. Here, we show that the beta(2)-adrenergic receptor (beta(2)AR), the trimeric G(s) protein, adenylyl cyclase, and PKA form a signalling complex with the AMPA-type glutamate receptor subunit GluR1, which is linked to the beta(2)AR through stargazin and PSD-95 and their homologues. Only GluR1 associated with the beta(2)AR is phosphorylated by PKA on beta(2)AR stimulation. Peptides that interfere with the beta(2)AR-GluR1 association prevent this phosphorylation of GluR1. This phosphorylation increases GluR1 surface expression at postsynaptic sites and amplitudes of EPSCs and mEPSCs in prefrontal cortex slices. Assembly of all proteins involved in the classic beta(2)AR-cAMP cascade into a supramolecular signalling complex and thus allows highly localized and selective regulation of one of its major target proteins.

Label-free fluorescent real-time monitoring of adenylyl cyclase.

In cellular signaling, adenylyl cyclase plays a key role in the hydrolysis of ATP to cyclic AMP and pyrophosphate. Using a synthetic fluorescent chemosensor (PyDPA) which binds strongly to the pyrophosphate group, we have developed a label-free fluorescent real-time detection system for adenylyl cyclase. This assay would be the first adenylyl cyclase assay based on chemosensing the production of pyrophosphate.

beta(1)-Adrenergic receptor vs adenylyl cyclase 6 expression in cardiac myocytes: differences in transgene localization and intracellular signaling.

Adenylyl cyclase type 6 (AC6) and the beta(1) adrenergic receptor (beta(1)AR) are pivotal proteins in transmembrane betaAR-signaling in cardiac myocytes. Increased expression of AC6 has beneficial effects on the heart, but increased beta(1)AR expression has marked deleterious effects. Why do these two elements of the betaAR pathway have such different effects? Using adenovirus-mediated gene transfer of the two transgenes in neonatal rat cardiac myocytes, we assessed cellular distribution and performed selected biochemical assays. beta(1)AR was found predominantly in the plasma membrane. In contrast, AC6 was found in the plasma membrane but also was associated with the nuclear envelope, sarcoplasmic reticulum, mitochondria, and cytoplasm. Increased beta(1)AR, but not AC6, increased follistatin expression, p38 phosphorylation, phosphatidylserine translocation to the PM, and apoptosis. In contrast, increased AC6, but not beta(1)AR, inhibited PHLPP2 activity, activated PI3K and Akt, and increased p70S6 kinase phosphorylation and Bcl-2 expression; apoptosis was unchanged. The distribution of AC6 to multiple cellular compartments appears to enable interactions with other proteins (e.g., PHLPP2) and activates cardioprotective signaling (PI3K/Akt). In contrast, beta(1)AR, confined to the plasma membrane, increased phosphatidylserine translocation and apoptosis. These data provide a potential underlying mechanism for the beneficial vs deleterious effects of these two related betaAR-signaling elements.

Interactions between angiotensin II and arginine vasopressin in water homeostasis.

Mice deficient in the angiotensin II type 1a (AT(1a)) receptor demonstrate a vasopressin-resistant nephrogenic diabetes insipidus. These knockout mice exhibit a threefold increase in 24-h urine excretion. Neither 2 weeks of exogenous vasopressin nor 5 days of fluid restriction reversed this polyuric state. This nephrogenic diabetes insipidus was associated with reductions in adenylyl cyclase protein and in the phosphorylated mitogen-activated protein kinase extracellular signal-regulated kinase 1/2. The results support an important interaction between vasopressin and angiotensin II in maximal urinary concentration.

Functional expression of the olfactory signaling system in the kidney.

Olfactory-like chemosensory signaling occurs outside of the olfactory epithelium. We find that major components of olfaction, including olfactory receptors (ORs), olfactory-related adenylate cyclase (AC3) and the olfactory G protein (G(olf)), are expressed in the kidney. AC3 and G(olf) colocalize in renal tubules and in macula densa (MD) cells which modulate glomerular filtration rate (GFR). GFR is significantly reduced in AC3(-/-) mice, suggesting that AC3 participates in GFR regulation. Although tubuloglomerular feedback is normal in these animals, they exhibit significantly reduced plasma renin levels despite up-regulation of COX-2 expression and nNOS activity in the MD. Furthermore, at least one member of the renal repertoire of ORs is expressed in a MD cell line. Thus, key components of olfaction are expressed in the renal distal nephron and may play a sensory role in the MD to modulate both renin secretion and GFR.

Rapid, semi-automated, and inexpensive radioimmunoassay of cAMP: application in GPCR-mediated adenylate cyclase assays.

Cyclic AMP (cAMP) is an important signal transduction second messenger that is commonly used as a functional mirror on the actions of G protein-coupled receptors that can activate or inhibit adenylate cyclases. A radioimmunoassay for cAMP with femtomole sensitivity was first reported by Steiner more than 30 years ago, and there have been several subsequent modifications that have improved this assay in various ways. Here we describe additional improvement to existing methods that markedly improve speed and reduce cost without sacrificing sensitivity, and is also adaptable to analysis of cGMP. The primary antibody is coupled directly to magnetic beads that are then separated from unbound marker using filtration on microplates. This eliminates the need for a secondary antibody, and markedly increases throughput. In addition, we report a simple, reproducible, and inexpensive method to make the radiomarker used for this assay. Although still requiring the use of radioactivity, the resulting method retains a high degree of accuracy and precision, and is suitable for low-cost high throughput screening. Use of aspects of this method can also improve throughput in other radioimmunoassays.

Reciprocal regulation of dopamine D1 and D3 receptor function and trafficking by heterodimerization.

Colocalization of dopamine D1 (D1R) and D3 receptors (D3R) in specific neuronal populations suggests that their functional cross-talk might involve direct interactions. Here we report that the D1R coimmunoprecipitates with the D3R from striatal protein preparations, suggesting that they are clustered together in this region. Using bioluminescence resonance energy transfer (BRET(2)), we further suggest the existence of a physical interaction between D1R and D3R. Tagged D1R and D3R cotransfected in human embryonic kidney (HEK) 293 cells generated a significant BRET(2) signal that was insensitive to agonist stimulation, suggesting that they form a constitutive heterodimer. D1R and D3R regulate adenylyl cyclase (AC) in opposite ways. In HEK 293 cells coexpressing D1R and D3R, dopamine stimulated AC with higher potency and displaced [3H]R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH23390) binding with higher affinity than in cells expressing the D1R. In HEK 293 cells individually expressing D1R or D3R, agonist stimulation induces internalization of D1R but not of D3R. Heterodimerization with D3R abolishes agonist-induced D1R cytoplasmic sequestration induced by selective D1R agonists and enables internalization of the D1R/D3R complex in response to the paired stimulation of both D1R and D3R. This mechanism involves beta-arrestin binding because it was blocked by mutant beta-arrestinV53D. These data suggest that as a result of dimerization, the D3R is switched to the desensitization mechanisms typical of the D1R. These data give a novel insight into how D1R and D3R may function in an integrated way, providing a molecular mechanism by which to converge D1R- and D3R-related dysfunctions.

Derlin-1 and p97/valosin-containing protein mediate the endoplasmic reticulum-associated degradation of human V2 vasopressin receptors.

The endoplasmic reticulum-associated degradation (ERAD), the main quality control pathway of the cell, is crucial for the elimination of unfolded or misfolded proteins. Several diseases are associated with the retention of misfolded proteins in the early secretory pathway. Among them is X-linked nephrogenic diabetes insipidus, caused by mutations in the gene encoding the V2 vasopressin receptor (V2R). We studied the degradation pathways of three intracellularly retained V2R mutants with different misfolded domains in human embryonic kidney 293 cells. At steady state, the wild-type V2R and the complex-glycosylated mutant G201D were partially located in lysosomes, whereas core-glycosylated mutants L62P and V226E were excluded from this compartment. In pulse-chase experiments, proteasomal inhibition stabilized the nonglycosylated and core-glycosylated forms of all studied receptors. In addition, all mutants and the wild-type receptor were found to be polyubiquitinylated. Nonglycosylated and core-glycosylated receptor forms were located in cytosolic and membrane fractions, respectively, confirming the deglycosylation and retrotranslocation of ERAD substrates to the cytosol. Distinct Derlin-1-dependent and -independent ERAD pathways have been proposed for proteins with different misfolded domains (cytosolic, extracellular, and membrane) in yeast. Here, we show for the first time that V2R mutants with different misfolded domains are able to coprecipitate the ERAD components p97/valosin-containing protein, Derlin-1 and the 26S proteasome regulatory subunit 7. Our results demonstrate the presence of a Derlin-1-mediated ERAD pathway degrading wild-type and disease-causing V2R mutants with different misfolded domains in a mammalian system.

Molecular analysis of the interaction of Bordetella pertussis adenylyl cyclase with fluorescent nucleotides.

The calmodulin (CaM)-dependent adenylyl cyclase (AC) toxin from Bordetella pertussis (CyaA) substantially contributes to the pathogenesis of whooping cough. Thus, potent and selective CyaA inhibitors may be valuable drugs for prophylaxis of this disease. We examined the interactions of fluorescent 2',3'-N-methylanthraniloyl (MANT)-, anthraniloyl- and trinitrophenyl (TNP)-substituted nucleotides with CyaA. Compared with mammalian AC isoforms and Bacillus anthracis AC toxin edema factor, nucleotides inhibited catalysis by CyaA less potently. Introduction of the MANT substituent resulted in 5- to 170-fold increased potency of nucleotides. K(i) values of 3'MANT-2'd-ATP and 2'MANT-3'd-ATP in the AC activity assay using Mn(2+) were 220 and 340 nM, respectively. Natural nucleoside 5'-triphosphates, guanine-, hypoxanthine- and pyrimidine-MANT- and TNP nucleotides and di-MANT nucleotides inhibited CyaA, too. MANT nucleotide binding to CyaA generated fluorescence resonance energy transfer (FRET) from tryptophans Trp69 and Trp242 and multiple tyrosine residues, yielding K(d) values of 300 nM for 3'MANT-2'd-ATP and 400 nM for 2'MANT-3'd-ATP. Fluorescence experiments and docking approaches indicate that the MANT- and TNP groups interact with Phe306. Increases of FRET and direct fluorescence with MANT nucleotides were strictly CaM-dependent, whereas TNP nucleotide fluorescence upon binding to CyaA increased in the absence of CaM and was actually reduced by CaM. In contrast to low-affinity MANT nucleotides, even low-affinity TNP nucleotides generated strong fluorescence increases upon binding to CyaA. We conclude that the catalytic site of CyaA possesses substantial conformational freedom to accommodate structurally diverse ligands and that certain ligands bind to CyaA even in the absence of CaM, facilitating future inhibitor design.

Fluorescence-based adenylyl cyclase assay adaptable to high throughput screening.

The second messenger cAMP has been implicated in numerous cellular processes such as glycogen metabolism, muscle contraction, learning and memory, and differentiation and development. Genetic evidence suggests that the enzyme that produces cAMP, adenylyl cyclase (AC), may be involved in pathogenesis in many of these cellular processes. In addition, these data suggest that membrane-bound ACs may be valuable targets for therapeutics to treat pathogenesis of these processes. The development of a robust real-time adenylyl cyclase assay that can be scalable to high-throughput screening could help in the development of novel therapeutics. Here we report a novel fluorescence-based cyclase assay using Bodipy FL GTPgammaS (BGTPgammaS). The fluorescence of the Bodipy moiety of BGTPgammaS was dramatically enhanced by incubation with the minimal catalytic core of wild-type-AC (wt-AC) and a mutant with decreased purine selectivity (mut-AC), in an AC activation-dependent manner. No increase in fluorescence was observed using Bodipy FL ATPgammaS (BATPgammaS) as substrate for either wt-AC or mut-AC. Using BGTPgammaS, forskolin, Gsalpha.GTPgammaS and the divalent cation Mn(2+) potently enhanced the rate of fluorescence increase in a concentration-dependent manner. The fluorescence enhancement of the Bodipy moiety was inhibited by known inhibitors of AC such as 2'deoxy,3'AMP and 2',5'-dideoxy-3'ATP. Furthermore, the fluorescence assay is adaptable to 96-well and 384-well multiplate format and is thus applicable to high throughput screening methodologies.