Inhibition Of Adenylyl Cyclase Activity Research Articles

Adenosine A1 Receptor Protein Levels and Activity Is Increased in the Cerebral Cortex in Creutzfeldt-Jakob Disease and in Bovine Spongiform Encephalopathy-Infected Bovine-PrP Mice

Prion diseases are characterized by neuronal loss, astrocytic gliosis, spongiform change, and abnormal protease-resistant prion protein (PrP) deposition. Creutzfeldt-Jakob disease (CJD) is the most prevalent human prion disease, whereas scrapie and bovine spongiform encephalopathy (BSE) are the most common animal prion diseases. Several candidates have been proposed as mediators of degeneration in prion diseases, one of them glutamate. Recent studies have shown reduced metabotropic glutamate receptor/phospholipase C signaling in the cerebral cortex in CJD, suggesting that this important neuromodulator and neuroprotector pathway is attenuated in CJD. Adenosine is involved in the regulation of different metabolic processes under physiological and pathologic conditions. Adenosine function is mediated by adenosine receptors, which are categorized into 4 types: A1, A2A, A2B, and A3. A1Rs are G-protein-coupled receptors that induce the inhibition of adenylyl cyclase activity. The most dramatic inhibitory actions of adenosine receptors are on the glutamatergic system. For these reasons, we examined the levels of A1Rs in the frontal cortex of 12 patients with CJD and 6 age-matched controls and in BSE-infected bovine-PrP transgenic mice (BoPrP-Tg110 mice) at different postincubation times to address modifications in A1Rs with disease progression. A significant increase in the protein levels of A1Rs was found in the cerebral cortex in CJD and in the murine BSE model at advanced stages of the disease and coincidental with the appearance of PrP expression. In addition, the activity of A1Rs was analyzed by in vitro assays with isolated membranes of the frontal cortex in CJD. Increased activity of the receptor, as revealed by the decreased forskolin-stimulated cAMP production in response to the A1R agonists cyclohexyl adenosine and cyclopentyl adenosine, was observed in CJD cases when compared with controls. Finally, mRNA A1R levels were similar in CJD and control cases, thus suggesting abnormal A1R turnover or dysregulation of raft-associated signaling pathways in CJD. These results show, for the first time, sensitization of A1Rs in prion diseases.

Muscarinic Acetylcholine Receptors

Muscarinic acetylcholine receptors mediate diverse physiological functions. At present, five receptor subtypes (M(1) - M(5)) have been identified. The odd-numbered receptors (M(1), M(3), and M(5)) are preferentially coupled to G(q/11) and activate phospholipase C, which initiates the phosphatidylinositol trisphosphate cascade leading to intracellular Ca(2+) mobilization and activation of protein kinase C. On the other hand, the even-numbered receptors (M(2) and M(4)) are coupled to G(i/o), and inhibit adenylyl cyclase activity. They also activate G protein-gated potassium channels, which leads to hyperpolarization of the plasma membrane in different excitable cells. Individual members of the family are expressed in an overlapping fashion in various tissues and cell types. Recent gene targeting approaches have unraveled the specific function of these muscarinic receptor subtypes, which were not able to be fully elucidated with pharmacological approaches because of the non-selective effects of the available ligands. Based on these findings, muscarinic receptors have been emerging as an important therapeutic target for various diseases, including dry mouth, incontinence and chronic obstructive pulmonary disease. Here we review the latest advances in the structural and functional characterization of muscarinic acetylcholine receptors and the pharmaceutical development of muscarinic receptor ligands.

Decreased Glucagon Responsiveness by Bile Acids: A Role for Protein Kinase Cα and Glucagon Receptor Phosphorylation

Dihydroxy bile acids like chenodeoxycholic acid (CDCA) induce heterologous glucagon receptor desensitization. We previously demonstrated that protein kinase C (PKC) was activated by certain bile acids and mediated the CDCA-induced decrease in glucagon responsiveness. The aim of the present study was to explore the role of PKC in the phosphorylation and desensitization of the glucagon receptor by CDCA. Desensitization was evaluated by measuring adenylyl cyclase activity. Receptor phosphorylation was assayed by metabolic labeling with [gamma-(32)P] ATP. Protein kinase C (PKC) translocation and activation was visualized by fluorescence microscopy. CDCA decreased cAMP production induced by glucagon in a dose-dependent manner without affecting cAMP synthesis through stimulation of either stimulatory GTP-binding protein (Gs) by NaF or adenylyl cyclase by forskolin. The CDCA-induced inhibition of adenylyl cyclase activity was potentiated by the phosphatase inhibitor, okadaic acid. The desensitizing effect of CDCA was bile acid-specific and was significantly reduced in the presence of PKC inhibitors and after PKC down-regulation by phorbol 12-myristate 13-acetate. CDCA increased glucagon receptor phosphorylation more than 3-fold at concentrations as low as 25 mum. Furthermore, CDCA significantly stimulated human recombinant PKCalpha autophosphorylation in vitro, as well as PKCalpha translocation to the plasma membrane and phosphorylation in vivo at concentrations as low as 25 mum. CDCA also stimulated PKCdelta translocation to the perinuclear region. Activated PKCalpha, PKCzeta, and to a lesser extent, PKCdelta, phosphorylated the glucagon receptor in vitro. This study demonstrates that certain bile acids, such as CDCA, stimulate phosphorylation and heterologous desensitization of the glucagon receptor, involving at least PKCalpha activation.

Optimized conditions for measuring lipolysis in murine primary adipocytes

The current literature on lipolysis in murine primary adipocytes is rife with experiments performed under conditions not optimized for reproducible and reliable results. Here, we present conditions for optimizing the measurement of lipolysis in murine adipocytes. We demonstrate that adenosine management is of paramount importance in evaluating the lipolytic response under basal and stimulated conditions. Also, adipocyte concentrations in the 10,000-15,000 cells per milliliter range produce a greater increase in stimulated lipolysis than higher concentrations, and the response is further enhanced by agitating the cells.

Nitric oxide modulates Gi-protein expression and adenylyl cyclase signaling in vascular smooth muscle cells

We have previously shown that treatment of rats with the nitric oxide (NO) synthase inhibitor N 6-nitro-L-arginine methyl ester for 4 weeks resulted in the augmentation of blood pressure and enhanced levels of Giα proteins. The present studies were undertaken to investigate if NO can modulate the expression of Gi proteins and associated adenylyl cyclase signaling. A10 vascular smooth muscle cells (VSMC) and primary cultured cells from aorta of Sprague-Dawley rats were used for these studies. The cells were treated with S-nitroso- N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP) for 24 h and the expression of Giα proteins was determined by immunobloting techniques. Adenylyl cyclase activity was determined by measuring [ 32P]cAMP formation for [α- 32P]ATP. Treatment of cells with SNAP (100 μM) or SNP (0.5 mM) decreased the expression of Giα-2 and Giα-3 by about 25–40% without affecting the levels of Gsα proteins. The decreased expression of Giα proteins was reflected in decreased Gi functions (receptor-independent and -dependent) as demonstrated by decreased or attenuated forskolin-stimulated adenylyl cyclase activity by GTPγS and inhibition of adenylyl cyclase activity by angiotensin II and C-ANP 4–23, a ring-deleted analog of atrial natriuretic peptide (ANP) that specifically interacts with natriuretic peptide receptor-C (NPR-C) in SNAP-treated cells. The SNAP-induced decreased expression of Giα-2 and Giα-3 proteins was not blocked by 1 H[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylyl cyclase, or KT5823, an inhibitor of protein kinase G, but was restored toward control levels by uric acid, a scavenger of peroxynitrite and Mn(111)tetralis (benzoic acid porphyrin) MnTBAP, a peroxynitrite scavenger and a superoxide dismutase mimetic agent that inhibits the production of peroxynitrite, suggesting that NO-mediated decreased expression of Giα protein was cGMP-independent and may be attributed to increased levels of peroxynitrite. In addition, Gsα-mediated stimulation of adenylyl cyclase by GTPγS, isoproterenol, and forskolin was significantly augmented in SNAP-treated cells. These results indicate that NO decreased the expression of Giα protein and associated functions in VSMC by cGMP-independent mechanisms. From these studies, it can be suggested that NO-induced decreased levels of Gi proteins and resultant increased levels of cAMP may be an additional mechanism through which NO regulates blood pressure.

Functional expression of the GABAB receptor in human airway smooth muscle

gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system and exerts its actions via both ionotropic (GABA(A)/GABA(C)) and metabotropic (GABA(B)) receptors (R). In addition to their location on neurons, GABA and functional GABA(B) receptors have been detected in nonneuronal cells in peripheral tissue. Although the GABA(B)R has been shown to function as a prejunctional inhibitory receptor on parasympathetic nerves in the lung, the expression and functional coupling of GABA(B) receptors to G(i) in airway smooth muscle itself have never been described. We detected the mRNA encoding multiple-splice variants of the GABA(B)R1 and GABA(B)R2 in total RNA isolated from native human and guinea pig airway smooth muscle and from RNA isolated from cultured human airway smooth muscle (HASM) cells. Immunoblots identified the GABA(B)R1 and GABA(B)R2 proteins in human native and cultured airway smooth muscle. The GABA(B)R1 protein was immunohistochemically localized to airway smooth muscle in guinea pig tracheal rings. Baclofen, a GABA(B)R agonist, elicited a concentration-dependent stimulation of [(35)S]GTPgammaS binding in HASM homogenates that was abrogated by the GABA(B)R antagonist CGP-35348. Baclofen also inhibited adenylyl cyclase activity and induced ERK phosphorylation in HASM. Another GABA(B)R agonist, SKF-97541, mimicked while pertussis toxin blocked baclofen's effect on ERK phosphorylation, implicating G(i) protein coupling. Functional GABA(B) receptors are expressed in HASM. GABA may modulate an uncharacterized signaling cascade via GABA(B) receptors coupled to the G(i) protein in airway smooth muscle.

Functional expression of the P2Y 14 receptor in human neutrophils

Previous studies using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis have shown that the P2Y 14 receptor is expressed at high levels in human neutrophils. Therefore the primary aim of this study was to determine whether the P2Y 14 receptor is functionally expressed in human neutrophils. In agreement with previous studies RT-PCR analysis detected the expression of P2Y 14 receptor mRNA in human neutrophils. UDP-glucose (IC 50 = 1 μM) induced a small but significant inhibition ( circa 30%) of forskolin-stimulated cAMP accumulation suggesting functional coupling of endogenously expressed P2Y 14 receptors to the inhibition of adenylyl cyclase activity in human neutrophils. In contrast, the other putative P2Y 14 receptor agonists UDP-galactose and UDP-glucuronic acid (at concentrations up to 100 μM) had no significant effect, whereas 100 μM UDP- N-acetylglucosamine-induced a small but significant inhibition of forskolin-stimulated cAMP accumulation (20% inhibition). UDP-galactose, UDP-glucuronic acid and UDP- N-acetylglucosamine behaved as partial agonists by blocking UDP-glucose mediated inhibition of forskolin-induced cAMP accumulation. Treatment of neutrophils with pertussis toxin (G i/o blocker) abolished the inhibitory effects of UDP-glucose on forskolin-stimulated cAMP accumulation. UDP-glucose (100 μM) also induced a modest increase in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, whereas the other sugar nucleotides had no effect on ERK1/2 activation. Finally, UDP-glucose and related sugar nucleotides had no significant effect on N-formyl-methionyl-leucyl-phenylalanine-induced elastase release from neutrophils. In summary, although we have shown that the P2Y 14 receptor is functionally expressed in human neutrophils (coupling to inhibition of forskolin-induced cAMP and ERK1/2 activation) it does not modulate neutrophil degranulation (assessed by monitoring elastase release). Clearly further studies are required in order to establish the functional role of the P2Y 14 receptor expressed in human neutrophils.

Adenylyl cyclase type II activity is regulated by two different mechanisms: Implications for acute and chronic opioid exposure

Acute and chronic activation of opioid receptors differentially regulate the activity of the various adenylyl cyclase (AC) isoforms. In several AC isoforms (I, V, VI and VIII) acute opioid activation (by agonists such as morphine) leads to AC inhibition, while prolonged opioid activation leads to increase in AC activity, a phenomenon known as AC sensitization or superactivation. In several other AC isoforms (II, IV and VII), acute opioid activation leads to AC stimulation, while chronic opioid exposure inhibits AC activity, in a process, which in analogy to the term “superactivation” is referred to as “superinhibition”. AC-II is highly regulated by multiple and independent biochemical stimuli, including G βγ, G αs and PKC activation. We investigated the regulation of AC-II by G αs and by PKC under conditions of acute and chronic exposure to opioid agonists in COS-7 transfected cells. We found that acute opioid exposure led to an increase in AC-II activity by either G αs or PKC stimulation. This effect seems to be regulated by G βγ subunits, in both activation pathways, as the increase in AC-II activity was abolished by pertussis toxin treatment and by G βγ scavengers. On the other hand, while chronic opioid exposure led to a decrease in AC-II activity (“superinhibition”) upon stimulation of the G αs pathway, this superinhibition was not observed when the opioid treated cells were stimulated via PKC activation.

Short- and Long-Term Regulation of Adenylyl Cyclase Activity by δ-Opioid Receptor Are Mediated by Gαi2in Neuroblastoma N2A Cells

Activation of the opioid receptor results in short-term inhibition of intracellular cAMP levels followed by receptor desensitization and subsequent increase of cAMP above the control level (adenylyl cyclase superactivation). Using adenovirus to deliver pertussis toxin-insensitive mutants of the alpha-subunits of G(i/o) that are expressed in neuroblastoma Neuro2A cells (Galpha(i2), Galpha(i3), and Galpha(o)), we examined the identities of the G proteins involved in the short- and long-term action of the delta-opioid receptor (DOR). Pertussis toxin pretreatment completely abolished the ability of [d-Pen(2), d-Pen(5)]-enkephalin (DPDPE) to inhibit forskolin-stimulated intracellular cAMP production. Expression of the C352L mutant of Galpha(i2), and not the C351L mutants of Galpha(i3) or Galpha(o), rescued the short-term effect of DPDPE after pertussis toxin treatment. The ability of Galpha(i2) in mediating DOR inhibition of adenylyl cyclase activity was also reflected in the ability of Galpha(i2), not Galpha(i3) or Galpha(o), to coimmunoprecipitate with DOR. Coincidently, after long-term DPDPE treatment, pertussis toxin treatment eliminated the antagonist naloxone-induced superactivation of adenylyl cyclase activity. Again, only the C352L mutant of Galpha(i2) restored the adenylyl cyclase superactivation after pertussis toxin treatment. More importantly, the C352L mutant of Galpha(i2) remained associated with DOR after long-term agonist and pertussis toxin treatment whereas the wild-type Galpha(i2) did not. These data suggest that Galpha(i2) serves as the signaling molecule in both DOR-mediated short- and long-term regulation of adenylyl cyclase activity.

Lack of interaction between prostaglandin E 2 receptor subtypes in regulating adenylyl cyclase activity in cultured rat dorsal root ganglion cells

The hyperalgesic response to prostaglandin E 2 (PGE 2) is thought to be mediated by activation of the cAMP/protein kinase A pathway in primary sensory neurones. The aim of this study was to investigate the relative contribution of different PGE 2 (EP) receptor subtypes to the overall activity of adenylyl cyclase in adult rat isolated dorsal root ganglion (DRG) cells, in vitro. PGE 2 and the prostanoid EP 4 receptor agonist ONO-AE1-329 increased [ 3H]cAMP production with EC 50 values of 500 nM and 70 nM, respectively, and showed similar efficacies. No combination of prostanoid EP 1, EP 2, EP 3 or EP 4 receptor selective agonists produced synergistic increases in [ 3H]cAMP. The prostacyclin mimetic cicaprost increased [ 3H]cAMP production with an EC 50 value of 42 nM and produced a significantly greater maximal response compared with PGE 2. No evidence for prostanoid EP 3 receptor-dependent inhibition of adenylyl cyclase activity could be obtained to account for the relatively weak effect of PGE 2 compared with prostacyclin receptor agonists. Interestingly, sulprostone (prostanoid EP 3/EP 1 receptor agonist) caused a Rho-kinase-dependent retraction of neurites, suggesting an alternative role for prostanoid EP 3 receptors in DRG cells. In conclusion, PGE 2 mediated increases in adenylyl cyclase activity in primary sensory neurones is likely to be mediated by activation of prostanoid EP 4 receptors, and is not under inhibitory control by prostanoid EP 3 receptors.

Heterotrimeric G‐protein serves as scaffold for Mu‐opioid receptor mediated signal transduction in lipid rafts

Mu-opioid receptor (MOR) belongs to the G-protein coupled receptors, which couples specifically to Gi/Go heterotrimeric G proteins. Our recent studies suggest that Gi2 not only is responsible for opioid agonist inhibition of adenylyl cyclase activity, but also performs as a scaffold to recruit the receptor and post-receptor signaling molecules to form a signaling complex for adenylyl cyclase superactivation after chronic agonist treatment. Our current results show that in neuroblastoma Neuro2A-MOR (N2AMOR) cells, MOR and Gi2 are both located in lipid rafts fractions, the plasma membrane microdomains that are rich in sphingolipid and cholesterol. Using co-immunoprecipitation and confocal fluorescent microscopy techniques, MOR and Gi2 are colocalized and interacted with each other in lipid rafts. Disruption of lipid rafts by methyl-beta-cyclodextrin causes the loss of colocalization of mu-receptors and Gi2. More importantly, disruption of lipid rafts also causes the loss of MOR activities, including the inhibition of agonist-induced intracellular cAMP level and mobilization of the intracellular Ca2+. Furthermore these activities can be restored after adding back cholesterol. Taken together, these observations suggest that G-protein scaffolding and lipid raft localization are essential for the receptor to regulate the signal transduction precisely and efficiently. (Research supported by NIDA grants DA0166774 and DA007339)

Rat NPFF 1 receptor-mediated signaling: functional comparison of neuropeptide FF (NPFF), FMRFamide and PFR(Tic)amide

Neuropeptide FF (NPFF) participates in many physiological functions associated with opioids in the mammalian CNS. We established a pheochromocytoma PC-12 cell line clone stably expressing rat NPFF 1 receptors. Both NPFF and FMRFamide activated NPFF 1 receptors to couple with Gi/o protein and inhibited adenylyl cyclase activity in PC-12/rNPFF 1 cells, but there were no effects on MAPKs (ERK1/2 and p38 MAPK) or PI3K/Akt pathway. FMRFamide also inhibited DARPP-32/Thr34 phosphorylation in the presence of forskolin. Similarly, PFR(Tic)amide, a ‘super-agonist’ of NPFF receptors, inhibited the production of cAMP and slightly decreased DARPP-32/Thr34 phosphorylation in PC-12/rNPFF 1 cells. Intracerebroventricular injections of PFR(Tic)amide blocked behavioral sensitization of locomotor activity to amphetamine, and intrathecal injection of PFR(Tic)amide caused a dose-dependent antinociception in vivo in rats. Thus, “over-activation” of NPFF receptors by PFR(Tic)amide induced different bio-effects from those induced by NPFF in vivo.

Detection of Adenylyl Cyclase Activity Using a Fluorescent ATP Substrate and Capillary Electrophoresis

A capillary electrophoresis laser-induced fluorescence (CE-LIF) assay was developed for detection of adenylyl cyclase (AC) activity using BODIPY FL ATP (BATP) as substrate. In the assay, BATP was incubated with AC and the resulting mixture of BATP and enzyme product (BODIPY cyclic AMP, BcAMP) separated in 5 min by CE-LIF. Substrate depletion and product accumulation were simultaneously monitored during the course of the reaction. The rate of product formation depended upon the presence of AC activators forskolin or Galpha(s)-GTPgammaS as evidenced by a more rapid BATP turnover to BcAMP compared to basal levels. The CE-LIF assay detected EC50 values for forskolin and Galpha(s)-GTPgammaS of 27 +/- 6 microM and 317 +/- 56 nM, respectively. These EC50 values compared well to those previously reported using [alpha-32P]ATP as substrate. When AC was concurrently activated with 2.5 microM forskolin and 25 nM Galpha(s)-GTPgammaS, the amount of BcAMP formed was 3.4 times higher than the additive amounts of each activator alone indicating a positively cooperative activation by these compounds in agreement with previous assays using radiolabeled substrate. Inhibition of AC activity was also demonstrated using the AC inhibitor 2'-(or-3')-O-(N-methylanthraniloyl) guanosine 5'-triphosphate with an IC50 of 9 +/- 6 nM. The use of a fluorescent substrate combined with CE separation has enabled development of a rapid and robust method for detection of AC activity that is an attractive alternative to the AC assay using radioactive nucleotide and column chromatography. In addition, the assay has potential for high-throughput screening of drugs that act at AC.

Agonist activity of naloxone benzoylhydrazone at recombinant and native opioid receptors

1. In the present study, we examined the pharmacological activity of the putative kappa3-opioid receptor agonist naloxone benzoylhydrazone (NalBzoH) at recombinant human opioid receptors individually expressed in Chinese hamster ovary (CHO) cells and native opioid receptors present in rat striatum. 2. At the mu-opioid receptor (MOR), NalBzoH stimulated guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding (pEC50=8.59) and inhibited cyclic AMP accumulation (pEC50=8.74) with maximal effects (Emax) corresponding to 55 and 65% of those obtained with the MOR agonist DAMGO, respectively. The MOR antagonist CTAP blocked the stimulatory effects of NalBzoH and DAMGO with similar potencies. 3. At the kappa-opioid receptor (KOR), NalBzoH stimulated [35S]GTPgammaS binding (pEC50=9.70) and inhibited cyclic AMP formation (pEC50=9.45) as effectively as the selective KOR agonist (-)-U-50,488. The NalBzoH effect was blocked by the KOR antagonist nor-binaltorphimine (nor-BNI) (pKi=10.30). 4. In CHO cells expressing the delta-opioid receptor (DOR), NalBzoH increased [35S]GTPgammaS binding (pEC50=8.49) and inhibited cyclic AMP formation (pEC50=8.61) almost as effectively as the DOR agonist DPDPE. Naltrindole (NTI), a selective DOR antagonist, completely blocked the response to NalBzoH (pKi of 10.40). 5. In CHO cells expressing the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), NalBzoH failed to exert agonist effects and antagonized the agonist-induced receptor activation. 6. When compared to other opioid receptor ligands, NalBzoH showed an efficacy that was lower than that of morphine at MOR, but higher at KOR and DOR. 7. In rat striatum, NalBzoH enhanced [35S]GTPgammaS binding and inhibited adenylyl cyclase activity. These effects were antagonized by either CTAP, nor-BNI or NTI, each antagonist blocking a fraction of the NalBzoH response. 8. These data demonstrate that NalBzoH displays agonist activity at MOR, DOR and KOR expressed either in a heterologous cell system or in a native environment.

Interleukin-1β-induced apoptosis through adenylyl cyclase and ERK1/2 inhibition in primary cultured thyroid cells

The programmed cell death plays a crucial role in the regulation of numerous physiological and pathological phenomena. In this study, we show that interleukin-1 β (IL-1β) induces an early production of endogenous ceramides via N-sphingomyelinase (N-Smase) as well as an inhibition of adenylyl cyclase activity in pig thyroid cells. This effect is followed by a down-regulation of the extracellular signal-regulated protein kinase (ERK1/2) phosphorylation, an activation of caspase-3, and ends by setting up the programmed cell death. The permeable exogenous C 2-ceramide reproduces IL-1β effects on: (i) inhibition of adenylyl cyclase activity, (ii) down-regulation of ERK1/2 phosphorylation, (iii) activation of caspase-3, and (iv) apoptosis in pig thyroid cells. Cell treatment with a PKA inhibitor down-regulates ERK1/2 phosphorylation. Furthermore, inhibition of ERK1/2 signaling pathway by U-0126 enhances caspase-3 activity and sets up programmed cell death. Both IL-1β and exogenous C 2-ceramide effects are reproduced by U-0126 so illustrating the implication of ERK1/2 down-regulation in both caspase-3 activation and apoptosis induction. Our study shows for the first time that endogenous ceramides are important second messengers in IL-1β-induced apoptosis in pig thyroid cells through inhibition of adenylyl cyclase and ERK1/2 activities.

Functional expression of the P2Y14 receptor in murine T‐lymphocytes

Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis has previously shown that the P2Y(14) receptor is expressed in peripheral immune cells including lymphocytes. Although in transfected cells the P2Y(14) receptor couples to pertussis toxin-sensitive G(i/o) protein, the functional coupling of endogenously expressed P2Y(14) receptors to the inhibition of adenylyl cyclase activity has not been reported. Therefore, the primary aim of this study was to determine whether the P2Y(14) receptor is functionally expressed in murine spleen-derived T- and B-lymphocyte-enriched populations. RT-PCR analysis detected the expression of P2Y(14) receptor mRNA in whole spleen and isolated T- and B-lymphocytes. In T cells, UDP-glucose (EC(50) = 335 nM) induced a small but significant inhibition (circa 20%) of forskolin-stimulated cAMP accumulation, suggesting functional coupling of endogenously expressed P2Y(14) receptors to the inhibition of adenylyl cyclase activity. In contrast, the other putative P2Y(14) receptor agonists UDP-galactose, UDP-glucuronic acid and UDP-N-acetylglucosamine had no significant effect alone but behaved as partial agonists by blocking UDP-glucose responses. In B cells, UDP-glucose (100 microM) had no significant effect on forskolin-stimulated cAMP accumulation. Treatment of T cells with pertussis toxin (G(i/o) blocker) abolished the inhibitory effects of UDP-glucose on forskolin-stimulated cAMP accumulation. T-cell proliferation in response to anti-CD3 monoclonal antibody (1 microg ml(-1)) was significantly inhibited by UDP-glucose (59% inhibition; p[IC(50)] = 5.9 +/- 0.3), UDP-N-acetylglucosamine (37%; 6.1 +/- 0.3), UDP-galactose (56%; 8.2 +/- 0.2) and UDP-glucuronic acid (49%; 6.3 +/- 0.2). Interleukin-2- (5 ng ml(-1)) induced T-cell proliferation was also significantly inhibited by all four agonists. In summary, we have shown that the P2Y(14) receptor appears to be functionally expressed in murine spleen-derived T-lymphocytes. These observations suggest that UDP-glucose and related sugar nucleotides presumably via the P2Y(14) receptor may play an important role in modulating immune function.

Design, Synthesis, and Preliminary Pharmacological Evaluation of a Set of Small Molecules That Directly Activate Gi Proteins

Heterotrimeric G proteins play a pivotal role in the communication of cells with the environment. G proteins are stimulated by cell surface receptors (GPCR) that catalyze the exchange of GDP, bound to Galpha subunit, with GTP and can per se be the target of drugs. Based on the structure of two nonpeptidic modulators of Gi proteins, a series of new molecules characterized by a long hydrophobic chain and at least two nitrogen atoms protonated at physiological pH was designed. The compounds were tested for their ability to stimulate binding of GTPgammaS to recombinant Gi proteins. Gi activation properties were also evaluated by inhibition of adenylyl cyclase activity in intact lymphocytes. Most compounds were able to stimulate GTPgammaS binding and to inhibit cAMP production at micromolar doses. Among the active compounds, 34 showed good efficacy and was the most potent compound studied, particularly on alpha(o) subtype; its regioisomer, 36, was the most efficacious one. Compound 7 showed also an interesting profile as it showed selectivity toward the alpha(o) subtype, in both efficacy and potency. Some of the compounds synthesized and found to be active may be useful leads to develop more potent and selective Gi protein modulators.

The Endocannabinoid Noladin Ether Acts as a Full Agonist at Human CB2 Cannabinoid Receptors

Noladin ether (NE) is a putative endogenously occurring cannabinoid demonstrating agonist activity at CB1 receptors. Because of reported selective affinity for CB1 receptors, the pharmacological actions of NE at CB2 receptors have not been examined. Therefore, the purpose of this study was to characterize the binding and functional properties of NE at human CB2 receptors stably expressed in Chinese hamster ovary (CHO) cells as well as in HL-60 cells, which express CB2 receptors endogenously. Surprisingly, in transfected CHO cells, NE exhibits a relatively high nanomolar affinity for CB2 receptors (K(i) = 480 nM), comparable to that observed for the endocannabinoid 2-arachidonoyl glycerol (2-AG) (K(i) = 1016 nM). Furthermore, NE activates G proteins and inhibits the intracellular effector adenylyl cyclase with equivalent efficacy relative to the full cannabinoid agonists 2-AG and CP 55,940 (CP) [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol]. The rank order of potency for G protein activation and effector regulation by the three agonists is similar to their apparent affinity for CB2 receptors; CP > NE > or = 2-AG. Regulation of adenylyl cyclase activity by all agonists is inhibited by pertussis toxin pretreatment or by coincubation with AM630 [6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)-methanone], a CB2 antagonist. Chronic treatment with NE or CP results in CB2 receptor desensitization and down-regulation. All agonists also inhibit adenylyl cyclase activity in HL-60 cells. Together, these data indicate that NE acts as a full agonist at human CB2 receptors and thus might have important physiological functions at peripheral cannabinoid receptors.

Different pharmacological profile of two closely related endocannabinoid ester analogs

The pharmacological and neuroprotective properties of two ester analogs of the endocannabinoids, arachidonoylethyleneglycol (AA-EG) and α,α,-dimethyl arachidonoylethyleneglycol (DMA-EG), were investigated. We examined the interaction of both compounds with cannabinoid receptors (CB 1 and CB 2) and their efficacy in functional assays. In competition binding assays, AA-EG and DMA-EG had low potency to displace the CB 1/CB 2 agonist [ 3H]CP-55,940 in membrane preparations expressing rodent or human receptors. Binding data correlate with low efficacy of both compounds as regards to inhibition of adenylyl cyclase activity. It was also shown that DMA-EG resists hydrolysis by rat brain membranes while AA-EG undergo complete splitting under these conditions. In the cannabinoid tetrad, AA-EG induced hypomotility, analgesia, catalepsy and decreased rectal temperature indicating cannabimimetic activity. By contrast, DMA-EG was completely inactive in the same models. DMA-EG and AA-EG potently protected rat cortical neurons in culture against oxygen deprivation at nanomolar concentrations. In glutamate-induced damage, the compounds were less active protecting neurons at micromolar concentrations. The data obtained indicate that the ester endocannabinoid template can be used for the development of new compounds with potent biological activity lacking some of the undesirable behavioral side effects.

Natriuretic peptide receptor-C signaling and regulation

The natriuretic peptides (NP) are a family of three polypeptide hormones termed atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). ANP regulates a variety of physiological parameters by interacting with its receptors present on the plasma membrane. These are of three subtypes NPR-A, NPR-B, and NPR-C. NPR-A and NPR-B are guanylyl cyclase receptors, whereas NPR-C is non-guanylyl cyclase receptor and is coupled to adenylyl cyclase inhibition or phospholipase C activation through inhibitory guanine nucleotide regulatory protein (Gi). ANP, BNP, CNP, as well as C-ANP 4–23, a ring deleted peptide that specifically interacts with NPR-C receptor inhibit adenylyl cyclase activity through Gi protein. Unlike other G-protein-coupled receptors, NPR-C receptors have a single transmembrane domain and a short cytoplasmic domain of 37 amino acids, which has a structural specificity like those of other single transmembrane domain receptors. A 37 amino acid cytoplasmic peptide is sufficient to inhibit adenylyl cyclase activity with an apparent Ki similar to that of ANP 99–126 or C-ANP 4–23. In addition, C-ANP 4–23 also stimulates phosphatidyl inositol (PI) turnover in vascular smooth muscle cells (VSMC) which is attenuated by dbcAMP and cAMP-stimulatory agonists, suggesting that NPR-C receptor-mediated inhibition of adenylyl cyclase and resultant decreased levels of cAMP may be responsible for NPR-C-mediated stimulation of PI turnover. Furthermore, the activation of NPR-C receptor by C-ANP 4–23 and CNP inhibits the mitogen-activated protein kinase activity stimulated by endothelin-3, platelet-derived growth factor, phorbol-12 myristate 13-acetate, suggesting that NPR-C receptor might also be coupled to other signal transduction system or that there may be an interaction of the NPR-C receptor and some other signaling pathways. In this review article, NPR-C receptor coupling to different signaling pathways and their regulation will be discussed.