Inhibition Of Adenylate Cyclase Activity Research Articles

Reduction in Hippocampal Amyloid-β Peptide (Aβ) Content during Glycine-Proline-Glutamate (Gly-Pro-Glu) Co-Administration Is Associated with Changes in Inflammation and Insulin-like Growth Factor (IGF)-I Signaling.

Alzheimer's disease (AD) is characterized by the deposition in the brain of senile plaques composed of amyloid-β peptides (Aβs) that increase inflammation. An endogenous peptide derived from the insulin-like growth factor (IGF)-I, glycine-proline-glutamate (GPE), has IGF-I-sensitizing and neuroprotective actions. Here, we examined the effects of GPE on Aβ levels and hippocampal inflammation generated by the intracerebroventricular infusion of Aβ25-35 for 2 weeks (300 pmol/day) in ovariectomized rats and the signaling-related pathways and levels of Aβ-degrading enzymes associated with these GPE-related effects. GPE prevented the Aβ-induced increase in the phosphorylation of p38 mitogen-activated protein kinase and the reduction in activation of signal transducer and activator of transcription 3, insulin receptor substrate-1, and Akt, as well as on interleukin (IL)-2 and IL-13 levels in the hippocampus. The functionality of somatostatin, measured as the percentage of inhibition of adenylate cyclase activity and the levels of insulin-degrading enzyme, was also preserved by GPE co-treatment. These findings indicate that GPE co-administration may protect from Aβ insult by changing hippocampal cytokine content and somatostatin functionality through regulation of leptin- and IGF-I-signaling pathways that could influence the reduction in Aβ levels through modulation of levels and/or activity of Aβ proteases.

Anti-craving Property of Chlorpromazine in Pentazocine addiction among sickle cell disease patients: A Case Series

Background: Opioids are useful in the management of vaso-occlusive crisis among Sickle Cell Disease patients; however, there is a need to search for drugs that can ameliorate the withdrawal symptoms without causing further dependence. Case presentations: Three patients living with Sickle cell disease abusing Pentazocine were managed with Tab Chlorpromazine 100mg twice daily for three weeks and had a significant reduction in the level of craving and also experienced improved sleep. Chlorpromazine demonstrates a high affinity for dopamine (DA) receptors and acts as a receptor antagonist by inhibiting adenylate cyclase activity. Conclusion: Chlorpromazine was found to be useful in the management of cravings associated with Pentazocine addiction.

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].

Fluorene-9-bisphenol regulates steroidogenic hormone synthesis in H295R cells through the AC/cAMP/PKA signaling pathway

Fluorene-9-bisphenol (BHPF), which has been used as a substitute for bisphenol A (BPA) in consumer goods and industrial products, can be detected in environmental media and human urine. BHPF has been reported to have endocrine-disrupting effects, whereas deleterious effects on steroidogenesis in H295R cells and underlying mechanisms are still unclear. Here, we investigated effects of BHPF on steroidogenesis using human adrenocortical carcinoma cells (H295R). Cytotoxicity was initially assessed and half-maximal inhibitory concentration (IC50) was determined based on proliferation of cells. Responses of four steroid hormones, aldosterone, cortisol, testosterone and 17β-estradiol (E2), and ten critical genes, StAR, HMGR, CYP11A1, CYP11B1, CYP11B1, HSD3B2, CYP21, CYP17, 17β-HSD, and CYP19, involved in steroidogenesis after exposure to non-cytotoxic concentrations of BHPF were determined in the presence or absence of 100 μM dbcAMP. Adenylate cyclase (AC) activity, intracellular concentrations of cAMP, PKA activity and amounts of steroidogenic factor-1 (SF-1) gene and expressions of proteins were determined to elucidate underlying mechanisms of effects on steroidogenesis. BHPF was cytotoxic to H295R cells in a dose- and time-dependent manner. Effects on production of hormones results demonstrated that exposure to greater concentrations of BHPF inhibited productions of aldosterone, cortisol, testosterone and E2 by down-regulation of steroidogenic genes. Inhibition of AC activity, intercellular cAMP content and PKA activity after exposure to BHPF implied that the AC/cAMP/PKA signaling pathway was involved in BHPF-induced suppression of steroidogenesis in H295R cells. Additionally, BHPF inhibited steroidogenesis and expressions of steroidogenic genes via decreasing expression of SF-1 protein, both in basal and dbcAMP-induced treatment. These results contributed to understanding molecular mechanisms of BHPF-induced effects on steroidogenesis and advancing the comprehensive risk assessment of BPs.

Можливе значення аденілатциклазного сигнального шляху в синтезі оксиду азоту мітохондріями міометрія

NO synthase activity (mtNOS) in uterine smooth muscle mitochondria under the action of the cAMP/protein kinase A signaling system modulators was studied. The experiments were performed on isolated mitochondria from rat myometrium using the NO-sensitive fluorescent probe DAF-FM-DA. NO synthesis in mitochondria was increased by adenylate cyclase activators NaHCO3 (30 mM) and forskolin (10 μM), as well as phosphodiesterase inhibitor caffeine (1 mM). The addition of ATP (0.5-5 mM) caused a slight increase in nitric oxide synthesis. The effect of ATP was enhanced in the presence of NaHCO3 and caffeine. The intensity of NO formation in mitochondria decreased by approximately 50 % in the case of inhibition of adenylate cyclase activity by the compound KH7 (25 μM). In the presence of the protein kinase A inhibitor PKI (10 nM) NO synthesis in mitochondria was also significantly reduced. When the constitutive NO-synthase inhibitor L-NAME (100 μM) was introduced into the incubation medium, the stimulating effect of the studied compounds on NO synthesis in mitochondria was not observed. These data suggests a possible dependence of mtNOS function on the activity of the cAMP/protein kinase A signaling system in smooth muscle mitochondria.

Hyperthermia-induced seizures during neonatal period alter the functionality of A1 and A2A receptors in the cerebellum and evoke fine motor impairment and gait disturbances in adult rats

Febrile seizures (FS) are one of the most common types of convulsive disorder of early childhood and they can be classified into simple and prolonged depending on the duration. Nowadays, simple FS have a good prognosis but there is controversy about the outcome of prolonged FS. In a previous work using an animal model of prolonged FS, we showed that hiperthermia-induced seizures (HIS) evoked fine motor coordination impairment and gait disturbances in adolescent rats in a process in which seemed to be involved modulation of the cerebellar adenosinergic system. The aim of the present work was to verify whether the effect was maintained in adulthood. To this end, neonatal rats (PD 12) were exposed to HIS and after 48 days (PD 60) they were assayed on balance beam and footprint tests. Animals were sacrificed 53 days after HIS and adenosine A1 and A2A receptor signalling pathways were studied in cerebellar plasma membranes by using radioligand binding assays and by measuring the activities of 5´-nucleotidase and adenylyl cyclase. Results obtained revealed that adult rats exposed to HIS showed gait disturbances and motor impairments. Besides, animals exposed to hyperthermic insult showed an increase in adenosine A2A receptor functionality and 5´-nucleotidase activity. Surprisingly, the functionality of the adenosine A1 receptor resulted significantly changed causing stimulation instead of inhibition of adenylate cyclase activity. These results showed that the effect of prolonged FS at the early age also persist in adulthood suggesting on must pay attention to FS in children.

. Effect of fluoxetine on the inhibition of adenylate cyclase activity in foskolin-stimulated MLTC-1 leydig cells

Fluoxetine (FLX), a widely used antidepressant primarily acting as a selective serotonin reuptake inhibitor, has been shown to exhibit other mechanisms of action in various cell types. Cyclic adenosine monophosphate (cAMP) is a second messenger used for intracellular signal induction. Cyclic AMP is a nucleotide synthesized within the cell from adenosine triphosphate by the adenylyl cyclase enzyme, and is inactivated enzymatically to 5′AMP by hydroxylation with a group of enzymes called phosphodiesterase. The aim of this study was to determine the effects of FLX on MLTC-1 Leydig cells on intracellular cyclic AMP response to forskolin (FSK). MLTC-1 cells were incubated at 37°C in media supplemented with or without different doses of FLX (0, 0.156, 0.3125, 0.625, 1.25, 2.5, 5 and 10 µM). We then looked for how the concentration of FLX for a short-time (2 hours) and a long-time (24 hours) affects the concentration of intracellular cyclic AMP response to FSK and ATP levels on MLTC-1 cells. Our results show that FLX decreased the intracellular cAMP response to FSK depending on FLX concentration. FLX decreased significantly cAMP levels only at 10 µM after 2 hours of incubation but after 24 hours of incubation FLX caused an effect on cAMP levels at 5 µM and at 10 µM. Moreover, as expected, FLX also caused a decline of steroidogenesis, which is under the control of cAMP and ATP levels in the cells. Taken together, these findings demonstrate that the inhibition of cAMP synthesis by FLX is dose-dependent, and that FLX also inhibited hormone-induced steroidogenesis in MLTC-1 cells.

Efforts Toward GPR88 Deorphanization: Challenges and Alternative Approaches to Troublesome Cell‐Based Second Messenger Assays

G protein‐coupled receptors (GPCR) comprise a family of over 800 integral membrane proteins involved in signal transduction and serve as the primary cellular sensors for chemical stimuli. About 35% of current drugs act on GPCR targets. In addition, of the 300 agents in current clinical trials, greater than 20% target novel GPCRs for which there are no approved drugs (orphan GPCRs). Ligand discovery and deorphanization efforts seeking to assign function to understudied GPCRs are not trivial. Notoriously complex in nature, GPCR‐dependent signaling centers around receptor‐ligand interactions that serve to stabilize conformational access to intracellular signaling partners, G proteins. In GPCR signaling, receptors and heterotrimeric G proteins (made of the Gα and Gβγ subunits) work together to transmit signals via downstream effectors and distinct pathways. The measurement of GPCR mediated second messengers such as cyclic AMP (cAMP), intracellular calcium (Ca2+) mobilization, and ERK activation (MAP kinase) are commonly used as tools to detect ligand induced GPCR activation. However, experimental pathway elucidation is complicated by the tendencies for pathway crosstalk, receptor desensitization, and receptor‐G protein interaction promiscuity. Cell‐based assays that monitor second messenger responses are often used as screening tools for potential GPCR agonists and antagonists, however, commonly used cell‐lines such as HEK‐293, express many endogenous GPCRs that can complicate data analysis. Many classes of Gα subunits exist: Gαs and Gα i/o G‐proteins modulate cyclic AMP (cAMP) through stimulation or inhibition of adenylate cyclase while Gαq subunits activate phospholipase C and a subsequent rapid release of intracellular Ca2+. Specifically, the orphan receptor, GPR88, has been linked with psychiatric diseases including schizophrenia and bipolar disorder. Lacking a validated endogenous agonist, the GPR88 signaling pathway remains poorly understood, however, in vivo knockout experiments and in vitro preclinical studies with small molecule synthetic agonists indicate that GPR88 couples to Gαi/o G‐proteins to inhibit adenylate cyclase activity and reduce intracellular cAMP in cell‐based assays. In this work, we describe our challenges in the study of the GPR88 signaling pathway using second messenger cell‐based assays and explain our decision to move to a real‐time NanoLuc‐based GPCR/G protein complementation assay.Support or Funding InformationNIH grant 1 R15 MH109034

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.

The Mechanisms Involved in Morphine Addiction: An Overview.

Opioid use disorder is classified as a chronic recurrent disease of the central nervous system (CNS) which leads to personality disorders, co-morbidities and premature death. It develops as a result of long-term administration of various abused substances, along with morphine. The pharmacological action of morphine is associated with its stimulation of opioid receptors. Opioid receptors are a group of G protein-coupled receptors and activation of these receptors by ligands induces significant molecular changes inside the cell, such as an inhibition of adenylate cyclase activity, activation of potassium channels and reductions of calcium conductance. Recent data indicate that other signalling pathways also may be involved in morphine activity. Among these are phospholipase C, mitogen-activated kinases (MAP kinases) or β-arrestin. The present review focuses on major mechanisms which currently are considered as essential in morphine activity and dependence and may be important for further studies.

Pyrimidinyl Biphenylureas Act as Allosteric Modulators to Activate Cannabinoid Receptor 1 and Initiate β-Arrestin-Dependent Responses.

Cannabinoid receptor 1 (CB1) is a G-protein-coupled receptor that is abundant in the central nervous system. It binds several compounds in its orthosteric site, including the endocannabinoids, arachidonoyl ethanolamide (anandamide) and 2-arachidonoyl glycerol, and the plant-derived Δ9-tetrahydrocannabinol, one of the main psychoactive components of marijuana. It primarily couples to Gi/o proteins to inhibit adenylate cyclase activity and typically induces downstream signaling that is Gi-dependent. Since this receptor is implicated in several maladies, such as obesity, pain, and neurodegenerative disorders, there is interest in developing therapeutics that selectively target this receptor. Allosteric modulators of CB1 offer one new approach that has tremendous therapeutic potential. Here, we reveal receptor- and cellular-level properties consistent with receptor activation by a series of pyrimidinyl biphenylureas (LDK1285, LDK1288, LDK1305, and PSNCBAM1), including promoting binding of the agonist CP55940 with positive cooperativity and inhibiting binding of the inverse agonist SR141716A with negative cooperativity, demonstrated via radioligand binding studies. Consistent with these findings, the allosteric modulators induced cellular internalization of the receptor and recruitment of β-arrestin 2 in human embryonic kidney cell line 293 cells monitored with confocal and total internal reflective fluorescence microscopy, respectively. These allosteric modulators, however, caused G-protein-independent but β-arrestin 1-dependent phosphorylation of the downstream kinases extracellular signal-regulated kinase 1/2, mitogen-activated protein kinase, and Src, shown by immunoblotting studies. These results are consistent with the involvement of β-arrestin and suggest that these allosteric modulators induce biased signaling.

The T2238C Human Atrial Natriuretic Peptide Molecular Variant and the Risk of Cardiovascular Diseases.

Atrial natriuretic peptide (ANP) is a cardiac hormone which plays important functions to maintain cardio-renal homeostasis. The peptide structure is highly conserved among species. However, a few gene variants are known to fall within the human ANP gene. The variant rs5065 (T2238C) exerts the most substantial effects. The T to C transition at the 2238 position of the gene (13–23% allele frequency in the general population) leads to the production of a 30-, instead of 28-, amino-acid-long α-carboxy-terminal peptide. In vitro, CC2238/αANP increases the levels of reactive oxygen species and causes endothelial damage, vascular smooth muscle cells contraction, and increased platelet aggregation. These effects are achieved through the deregulated activation of type C natriuretic peptide receptor, the consequent inhibition of adenylate cyclase activity, and the activation of Giα proteins. In vivo, endothelial dysfunction and increased platelet aggregation are present in human subjects carrying the C2238/αANP allele variant. Several studies documented an increased risk of stroke and of myocardial infarction in C2238/αANP carriers. Recently, an incomplete response to antiplatelet therapy in ischemic heart disease patients carrying the C2238/αANP variant and undergoing percutaneous coronary revascularization has been reported. In summary, the overall evidence supports the concept that T2238C/ANP is a cardiovascular genetic risk factor that needs to be taken into account in daily clinical practice.

Somatostatin and dopamine receptor regulation of pituitary somatotroph adenomas.

Somatostatin and dopamine receptors are expressed in normal and tumoral somatotroph cells. Upon receptor stimulation, somatostatin and the somatostatin receptor ligands octreotide, lanreotide, and pasireotide, and to a lesser extent, dopamine and the dopamine analogs bromocriptine and cabergoline, suppress growth hormone (GH) secretion from a GH-secreting pituitary somatotroph adenoma. Somatostatin and dopamine receptors are Gαi-protein coupled that inhibit adenylate cyclase activity and cAMP production and reduce intracellular calcium concentration and calcium flux oscillations. Although their main action on somatotroph cells is acute inhibition of GH secretion, they also may inhibit GH production and possibly somatotroph proliferation. These receptors have been reported to create complexes that exhibit functions distinct from that of receptor monomers. Somatostatin suppression of GH is mediated mainly by somatostatin receptor subtype 2 and to a lesser extent by SST5. Human somatostatin receptor subtype 5 has also been shown to harbor mutations associated with GH levels, somatotroph tumor behavior, and somatostatin receptor ligand (SRL) responsiveness. Reviewing current knowledge of somatostatin and dopamine receptor expression and signaling in normal and tumoral somatotroph cells offers insights into mechanisms underlying SRL and dopamine agonist effectiveness in patients with acromegaly.

Agonist‐induced activation of histamine H3 receptor signals to extracellular signal‐regulated kinases 1 and 2 through PKC‐, PLD‐, and EGFR‐dependent mechanisms

The histamine H3 receptor (H3R), abundantly expressed in the central and the peripheral nervous system, has been recognized as a promising target for the treatment of various important CNS diseases including narcolepsy, Alzheimer's disease, and attention deficit hyperactivity disorder. The H3R acts via Gi/o -proteins to inhibit adenylate cyclase activity and modulate MAPK activity. However, the underlying molecular mechanisms for H3R mediation of the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) remain to be elucidated. In this study, using HEK293 cells stably expressing human H3R and mouse primary cortical neurons endogenously expressing mouse H3R, we found that the H3R-mediated activation of ERK1/2 was significantly blocked by both the pertussis toxin and the MEK1/2 inhibitor U0126. Upon stimulation by H3R agonist histamine or imetit, H3R was shown to rapidly induce ERK1/2 phosphorylation via PLC/PKC-, PLDs-, and epidermal growth factor receptor (EGFR) transactivation-dependent pathways. Furthermore, it was also indicated that while the βγ-subunits play a key role in H3R-activated ERK1/2 phosphorylation, β-arrestins were not required for ERK1/2 activation. In addition, when the cultured mouse cortical neurons were exposed to oxygen and glucose deprivation conditions (OGD), imetit exhibited neuroprotective properties through the H3R. Treatment of cells with the inhibitor UO126 abolished these protective effects. This suggests a possible neuroprotective role of the H3R-mediated ERK1/2 pathway under hypoxia conditions. These observations may provide new insights into the pharmacological effects and the physiological functions modulated by the H3R-mediated activation of ERK1/2. Histamine H3 receptors are abundantly expressed in the brain and play important roles in various CNS physiological functions. However, the underlying mechanisms for H3R-induced activation of extracellular signal-regulated kinase (ERK)1/2 remain largely unknown. Here, we provide evidence that upon activation by an agonist, H3Rs trigger ERK1/2 activation via phospholipase C/protein kinase C (PLC/PKC)-, phospholipase D (PLD)s-, and matrix metallopeptidase/epidermal growth factor receptor (MMP/EGFR) transactivation-dependent pathways. Moreover, we demonstrate that H3Rs exhibit a neuroprotective effect on the cultured mouse cortical neurons under hypoxia conditions through the ERK1/2 pathway.

Partial Adenosine A1 Agonist in Heart Failure.

Adenosine exerts a variety of physiological effects by binding to cell surface G-protein-coupled receptor subtypes, namely, A1, A2a, A2b, and A3. The central physiological role of adenosine is to preclude tissue injury and promote repair in response to stress. In the heart, adenosine acts as a cytoprotective modulator, linking cardiac function to metabolic demand predominantly via activation of adenosine A1 receptors (A1Rs), which leads to inhibition of adenylate cyclase activity, modulation of protein kinase C, and opening of ATP-sensitive potassium channels. Activation of myocardial adenosine A1Rs has been shown to modulate a variety of pathologies associated with ischemic cardiac injury, including arrhythmogenesis, coronary and ventricular dysfunction, apoptosis, mitochondrial dysfunction, and ventricular remodeling. Partial A1R agonists are agents that are likely to elicit favorable pharmacological responses in heart failure (HF) without giving rise to the undesirable cardiac and extra-cardiac effects observed with full A1R agonism. Preclinical data have shown that partial adenosine A1R agonists protect and improve cardiac function at doses that do not result in undesirable effects on heart rate, atrioventricular conduction, and blood pressure, suggesting that these compounds may constitute a valuable new therapy for chronic HF. Neladenoson bialanate (BAY1067197) is the first oral partial and highly selective A1R agonist that has entered clinical development for the treatment of HF. This review provides an overview of adenosine A1R-mediated signaling in the heart, summarizes the results from preclinical and clinical studies of partial A1R agonists in HF, and discusses the potential benefits of these drugs in the clinical setting.

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

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

A role for cAMP-driven transactivation of EGFR in cancer aggressiveness – Therapeutic implications

In many common cancers, production of cAMP boosts cancer proliferation, survival, and aggressiveness, reflecting the fact that, through mechanisms that require further clarification, cAMP can promote tyrosine phosphorylation, notably transactivation of the epidermal growth factor receptor (EGFR). Hormones which activate adenylate cyclase in many cancers include PGE2 – often produced by cox-2 activity within tumors – and adrenergic hormones, acting on beta2 receptors. NSAID cyclooxygenase inhibitors, including low-dose aspirin, clearly reduce risk for many adenocarcinomas, but the impact of cox-2 inhibitors in clinical cancer therapy remains somewhat equivocal. There is increasing evidence that increased sympathetic drive, often reflecting psychic stress or tobacco usage, increases risk for, and promotes the aggressiveness of, many cancers. The non-specific beta antagonist propranolol shows cancer-retardant activity in pre-clinical rodent studies, especially in stressed animals, and a limited amount of epidemiology concludes that concurrent propranolol usage is associated with superior prognosis in breast cancer, ovarian cancer, and melanoma. Epidemiology correlating increased resting heart rate with increased total cancer mortality can be interpreted as compelling evidence that increased sympathetic drive encourages the onset and progression of common cancers. Conversely, hormones which inhibit adenylate cyclase activity in cancers may have potential for cancer control; GABA, which can be administered as a well-tolerated nutraceutical, has potential in this regard. Combination regimens intended to down-regulate cancer cAMP levels, perhaps used in conjunction with EGFR inhibitors, may have considerable potential for suppressing the contribution of cAMP/EGFR to cancer aggressiveness. This model also predicts that certain other hormones which activate adenylate cylase in various tissue may play a yet-unsuspected role in cancer induction and spread.

An Immunological Fingerprint Differentiates Muscular Lymphatics from Arteries and Veins

The principal function of the lymphatic system is to transport lymph from the interstitium to the nodes and then from the nodes to the blood. In doing so lymphatics play important roles in fluid homeostasis, macromolecular/antigen transport and immune cell trafficking. To better understand the genes that contribute to their unique physiology, we compared the transcriptional profile of muscular lymphatics (prenodal mesenteric microlymphatics and large, postnodal thoracic duct) to axillary and mesenteric arteries and veins isolated from rats. Clustering of the differentially expressed genes demonstrated that the lymph versus blood vessel differences were more profound than between blood vessels, particularly the microvessels. Gene ontology functional category analysis indicated that microlymphatics were enriched in antigen processing/presentation, IgE receptor signaling, catabolic processes, translation and ribosome; while they were diminished in oxygen transport, regulation of cell proliferation, glycolysis and inhibition of adenylate cyclase activity by G-proteins. We evaluated the differentially expressed microarray genes/products by qPCR and/or immunofluorescence. Immunofluorescence documented that multiple MHC class II antigen presentation proteins were highly expressed by an antigen-presenting cell (APC) type found resident within the lymphatic wall. These APCs also expressed CD86, a co-stimulatory protein necessary for T-cell activation. We evaluated the distribution and phenotype of APCs within the pre and postnodal lymphatic network. This study documents a novel population of APCs resident within the walls of muscular, prenodal lymphatics that indicates novel roles in antigen sampling and immune responses. In conclusion, these prenodal lymphatics exhibit a unique profile that distinguishes them from blood vessels and highlights the role of the lymphatic system as an immunovascular system linking the parenchymal interstitium, lymph nodes and the blood.

The stereoisomer (+)-naloxone potentiates G-protein coupling and feeding associated with stimulation of mu opioid receptors in the parabrachial nucleus

Classically, opioids produce their effects by activating Gi-proteins that inhibit adenylate cyclase activity. Previous studies proposed that mu-opioid receptors can also stimulate adenylate cyclase due to an initial transient coupling to Gs-proteins. Treatment with ultra-low doses of the nonselective opioid antagonist (-)-naloxone or its inactive enantiomer (+)-naloxone blocks this excitatory effect and enhances Gi-coupling. Previously we reported that infusion of the mu-opioid receptor agonist [D-Ala2, N-Me-Phe4, Glycinol5]-Enkephalin (DAMGO) into the mu-opioid receptor expressing lateral parabrachial nucleus increases feeding. Pretreatment with (-)-naloxone blocks this effect. We used this parabrachial circuit as a model to assess cellular actions of ultra-low doses of (-)-naloxone and (+)-naloxone in modifying the effects of DAMGO. Our results showed that an ultra-low concentration of (-)-naloxone (0.001 nM) and several concentrations of (+)-naloxone (0.01-10 nM) enhanced DAMGO-stimulated guanosine-5'-0-(γ-thio)-triphosphate incorporation in parabrachial sections in vitro. Further, we analyzed the relevance of these effects in vivo. In the present study, we show that (+)-naloxone can potentiate DAMGO-induced feeding at doses at which (-)-naloxone was an antagonist. These results implicated (+)-naloxone as a novel tool for studying mu-opioid receptor functions and suggest that (+)-naloxone may have therapeutic value to enhance clinical actions of opiate drugs.

Tacrine-induced tachyphylaxis in gastric smooth muscles

Abstract Tacrine is a medication applied in cases of mild to moderate dementia in Alzheimer’s disease. By blocking acetylcholinesterase activity the drug increases the concentration of acetylcholine, whose effects influence the functions of different organs and systems of the body. The effect of tacrine on smooth muscle preparations isolated from rat stomach was studied by isometric registration of muscle contractility. Our investigations found a specific significant systematic decrease in the strength of consecutive tacrine-induced contractions of smooth muscle preparations, a phenomenon known as tachyphylaxis. The tacrineinduced tachyphylaxis was significantly inhibited by SQ22536 (inhibitor of adenylate cyclase activity), by blockers of nitric oxide synthase and KT5823 (inhibitor of protein kinase G). The process was not influenced by cyclopiazonic acid (specific blocker of sarco/endoplasmic reticulum Ca2+-ATPase,) and atropine (blocker of M-cholinergic receptors). We hypothesize that the overlapping and different time-development of the two opposing processes: smooth muscle contraction caused by acetylcholinesterase inhibition and tacrine-induced relaxation influenced by synthesis of nitric oxide, results in tachyphylaxis.