Inhibition Of Cyclic AMP Accumulation Research Articles

Comparison of the function of two novel human dopamine D2 receptor variants identifies a likely mechanism for their pathogenicity.

Two recently discovered DRD2 mutations, c.634A > T, p.Ile212Phe and c.1121 T > G, p.Met374Arg, cause hyperkinetic movement disorders that have overlapping features but apparently differ in severity. The two known carriers of the Met374Arg variant had early childhood disease onset and more severe motor, cognitive, and neuropsychiatric deficits than any known carriers of the Ile212Phe variant, whose symptoms were first apparent in adolescence. Here, we evaluated if differences in the function of the two variants in cultured cells could explain differing pathogenicity. Both variants were expressed less abundantly than the wild type receptor and exhibited loss of agonist-induced arrestin binding, but differences in expression and arrestin binding between the variants were minor. Basal and agonist-induced activation of heterotrimeric Gi/o/z proteins, however, showed clear differences; agonists were generally more potent at Met374Arg than at the Ile212Phe or wild type variants. Furthermore, all Gα subtypes tested were constitutively activated more by Met374Arg than by Ile212Phe. Met374Arg produced greater constitutive inhibition of cyclic AMP accumulation than Ile212Phe or the wild type D2 receptor. Met374Arg and Ile212Phe were more sensitive to thermal inactivation than the wild type D2 receptor, as reported for other constitutively active receptors, but Ile212Phe was affected more than Met374Arg. Additional pharmacological characterization suggested that the mutations differentially affect the shape of the agonist binding pocket and the potency of dopamine, norepinephrine, and tyramine. Molecular dynamics simulations provided a structural rationale for enhanced constitutive activation and agonist potency. Enhanced constitutive and agonist-induced G protein-mediated signaling likely contributes to the pathogenicity of these novel variants.

Site-specific O-glycosylation of N-terminal serine residues by polypeptide GalNAc-transferase 2 modulates human δ-opioid receptor turnover at the plasma membrane

G protein-coupled receptors (GPCRs) are an important protein family of signalling receptors that govern a wide variety of physiological functions. The capacity to transmit extracellular signals and the extent of cellular response are largely determined by the amount of functional receptors at the cell surface that is subject to complex and fine-tuned regulation. Here, we demonstrate that the cell surface expression level of an inhibitory GPCR, the human δ-opioid receptor (hδOR) involved in pain and mood regulation, is modulated by site-specific N-acetylgalactosamine (GalNAc) -type O-glycosylation. Importantly, we identified one out of the 20 polypeptide GalNAc-transferase isoforms, GalNAc-T2, as the specific regulator of O-glycosylation of Ser6, Ser25 and Ser29 in the N-terminal ectodomain of the receptor. This was demonstrated by in vitro glycosylation assays using peptides corresponding to the hδOR N-terminus, Vicia villosa lectin affinity purification of receptors expressed in HEK293 SimpleCells capable of synthesizing only truncated O-glycans, GalNAc-T edited cell line model systems, and site-directed mutagenesis of the putative O-glycosylation sites. Interestingly, a single-nucleotide polymorphism, at residue 27 (F27C), was found to alter O-glycosylation of the receptor in efficiency as well as in glycosite usage. Furthermore, flow cytometry and cell surface biotinylation assays using O-glycan deficient CHO-ldlD cells revealed that the absence of O-glycans results in decreased receptor levels at the plasma membrane due to enhanced turnover. In addition, mutation of the identified O-glycosylation sites led to a decrease in the number of ligand-binding competent receptors and impaired agonist-mediated inhibition of cyclic AMP accumulation in HEK293 cells. Thus, site-specific O-glycosylation by a selected GalNAc-T isoform can increase the stability of a GPCR, in a process that modulates the constitutive turnover and steady-state levels of functional receptors at the cell surface.

Structure, Pharmacology and Roles in Physiology of the P2Y12 Receptor.

P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular nucleotides. The platelet ADP-receptor which has been denominated P2Y12 receptor is an important target in pharmacotherapy. The receptor couples to Gαi2 mediating an inhibition of cyclic AMP accumulation and additional downstream events including the activation of phosphatidylinositol-3-kinase and Rap1b proteins. The nucleoside analogue ticagrelor and active metabolites of the thienopyridine compounds ticlopidine, clopidogrel and prasugrel block P2Y12 receptors and, thereby, inhibit ADP-induced platelet aggregation. These drugs are used for the prevention and therapy of cardiovascular events such as acute coronary syndromes or stroke. The recently published three-dimensional crystal structures of the human P2Y12 receptor in complex with agonists and antagonists will facilitate the development of novel therapeutic agents with reduced adverse effects. P2Y12 receptors are also expressed on vascular smooth muscle cells and may be involved in the pathophysiology of atherogenesis. P2Y12 receptors on microglial cells operate as sensors for adenine nucleotides released during brain injury. A recent study indicated the involvement of microglial P2Y12 receptors in the activity-dependent neuronal plasticity. Interestingly, there is evidence for changes in P2Y12 receptor expression in CNS pathologies including Alzheimer's diseases and multiple sclerosis. P2Y12 receptors may also be involved in systemic immune modulating responses and the susceptibility to develop bronchial asthma.

Modulation of primary cilia length by melanin-concentrating hormone receptor 1

Melanin-concentrating hormone (MCH) receptor 1 (MCHR1) is a class A G-protein-coupled receptor (GPCR). The MCH-MCHR1 system has been implicated in the regulation of feeding, emotional processing, and sleep in rodents. Recent work revealed that MCHR1 is selectively expressed in neuronal primary cilia of the central nervous system. Cilia have various chemosensory functions in many types of cell, and ciliary dysfunction is associated with ciliopathies such as polycystic kidney disease and obesity. Although dynamic modulation of neuronal cilia length is observed in obese mice, the functional interaction of neuronal ciliary GPCR and its endogenous ligand has not yet been elucidated. We report here that MCH treatment significantly reduced cilia length in hTERT-RPE1 cells (hRPE1 cells) transfected with MCHR1. Quantitative analyses indicated that MCH-induced cilia shortening progressed in a dose-dependent manner with an EC50 lower than 1nM when cells were treated for 6h. Although the assembly and disassembly of primary cilia are tightly coupled to the cell cycle, cell cycle reentry was not a determinant of MCH-induced cilia shortening. We confirmed that MCH elicited receptor internalization, Ca2+ mobilization, ERK and Akt phosphorylation, and inhibition of cyclic AMP accumulation in MCHR1-expressing hRPE1 cells. Among these diverse pathways, we revealed that Gi/o-dependent Akt phosphorylation was an important component in the initial stage of MCH-induced cilia length shortening. Furthermore, induction of fewer cilia by Kif3A siRNA treatment significantly decreased the MCH-mediated phosphorylation of Akt, indicating the functional importance of the MCHR1-Akt pathway in primary cilia. Taken together, the present data suggest that the MCH-MCHR1 axis may modulate the sensitivity of cells to external environments by controlling the cilia length. Therefore, further characterization of MCHR1 as a ciliary GPCR will provide a potential molecular mechanism to link cilia length control with obesity.

Novel fluorescent antagonist as a molecular probe in A3 adenosine receptor binding assays using flow cytometry

The physiological role of the A3 adenosine receptor (AR) was explored in cardiac ischaemia, inflammatory diseases and cancer. We report a new fluorophore-conjugated human (h) A3AR antagonist for application to cell-based assays in ligand discovery and for receptor imaging. Fluorescent pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-ylamine (pyrazolo-triazolo-pyrimidine, PTP) and triazolo[1,5-c]quinazolin-5-yl)amine (triazolo-quinazoline, TQ) AR antagonists were compared. A chain-extended and click-conjugated Alexa Fluor-488 TQ derivative (MRS5449) displayed a radioligand binding Ki value of 6.4±2.5nM in hA3AR-expressing CHO cell membranes. MRS5449 antagonized hA3AR agonist-induced inhibition of cyclic AMP accumulation in a concentration-dependent manner (KB=4.8nM). Using flow cytometry (FCM), MRS5449 saturated hA3ARs with very high specific-to-nonspecific binding ratio with an equilibrium binding constant 5.15nM, comparable to the Kd value of 6.65nM calculated from kinetic experiments. Ki values of known AR antagonists in inhibition of MRS5449 binding in whole cell FCM were consistent with radioligand binding in membranes, but agonist binding was 5-20 fold weaker than obtained with agonist radioligand [125I]I-AB-MECA. Further binding analysis of MRS5549 suggested multiple agonist binding states of the A3AR. Molecular docking predicted binding modes of these fluorescent antagonists. Thus, MRS5449 is a useful tool for hA3AR characterization.

Quantification of Gi-Mediated Inhibition of Adenylyl Cyclase Activity Reveals That UDP Is a Potent Agonist of the Human P2Y14 Receptor

The P2Y14 receptor was initially identified as a G protein-coupled receptor activated by UDP-glucose and other nucleotide sugars. We have developed several cell lines that stably express the human P2Y14 receptor, allowing facile examination of its coupling to native Gi family G proteins and their associated downstream signaling pathways (J Pharmacol Exp Ther 330:162-168, 2009). In the current study, we examined P2Y14 receptor-dependent inhibition of cyclic AMP accumulation in human embryonic kidney (HEK) 293, C6 glioma, and Chinese hamster ovary (CHO) cells stably expressing this receptor. Not only was the human P2Y14 receptor activated by UDP-glucose, but it also was activated by UDP. The apparent efficacies of UDP and UDP-glucose were similar, and the EC50 values (74, 33, and 29 nM) for UDP-dependent activation of the P2Y14 receptor in HEK293, CHO, and C6 glioma cells, respectively, were similar to the EC50 values (323, 132, and 72 nM) observed for UDP-glucose. UDP and UDP-glucose also stimulated extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in P2Y14 receptor-expressing HEK293 cells but not in wild-type HEK293 cells. A series of analogs of UDP were potent P2Y14 receptor agonists, but the naturally occurring nucleoside diphosphates, CDP, GDP, and ADP exhibited agonist potencies over 100-fold less than that observed with UDP. Two UDP analogs were identified that selectively activate the P2Y14 receptor over the UDP-activated P2Y6 receptor, and these molecules stimulated phosphorylation of ERK1/2 in differentiated human HL-60 promyeloleukemia cells, which natively express the P2Y14 receptor but had no effect in wild-type HL-60 cells, which do not express the receptor. We conclude that UDP is an important cognate agonist of the human P2Y14 receptor.

S.19.02 Preclinical and clinical pharmacology of H3-receptor inverse agonists

Melanin-concentrating hormone (MCH) receptor 1 (MCHR1) is a class A G-protein-coupled receptor (GPCR). The MCH-MCHR1 system has been implicated in the regulation of feeding, emotional processing, and sleep in rodents. Recent work revealed that MCHR1 is selectively expressed in neuronal primary cilia of the central nervous system. Cilia have various chemosensory functions in many types of cell, and ciliary dysfunction is associated with ciliopathies such as polycystic kidney disease and obesity. Although dynamic modulation of neuronal cilia length is observed in obese mice, the functional interaction of neuronal ciliary GPCR and its endogenous ligand has not yet been elucidated. We report here that MCH treatment significantly reduced cilia length in hTERT-RPE1 cells (hRPE1 cells) transfected with MCHR1. Quantitative analyses indicated that MCH-induced cilia shortening progressed in a dose-dependent manner with an EC50 lower than 1 nM when cells were treated for 6 h. Although the assembly and disassembly of primary cilia are tightly coupled to the cell cycle, cell cycle reentry was not a determinant of MCH-induced cilia shortening. We confirmed that MCH elicited receptor internalization, Ca2 + mobilization, ERK and Akt phosphorylation, and inhibition of cyclic AMP accumulation in MCHR1-expressing hRPE1 cells. Among these diverse pathways, we revealed that Gi/o-dependent Akt phosphorylation was an important component in the initial stage of MCH-induced cilia length shortening. Furthermore, induction of fewer cilia by Kif3A siRNA treatment significantly decreased the MCH-mediated phosphorylation of Akt, indicating the functional importance of the MCHR1-Akt pathway in primary cilia. Taken together, the present data suggest that the MCH-MCHR1 axis may modulate the sensitivity of cells to external environments by controlling the cilia length. Therefore, further characterization of MCHR1 as a ciliary GPCR will provide a potential molecular mechanism to link cilia length control with obesity.

The role of conserved transmembrane domain residues in the activation of the dopamine D2 receptor

The interaction between two highly conserved amino acid residues in second (TM2) and seventh (TM7) transmembrane domains (aspartate and asparagine respectively) of GPCRs plays an important role in receptor activation and coupling specificity to different signal transduction pathways. Although single mutations of each conserved residue can selectively alter receptor signaling, reciprocal mutations in TM2 and TM7 appear to partially restore function in a receptor dependent manner. For example, reciprocal mutations in alpha2a‐adrenergic and 5HT2a serotonin receptors restores receptor function, but reciprocal mutation in the CB1cannabinoid receptor does not. We have characterized the role of the two conserved residues in the activation and signaling of the D2 dopamine receptor. We report that the D80N mutation in TM2 of the D2 receptor ablates receptor‐dependent activation of Kir 3.0 channels and significantly blunts receptor‐dependent inhibition of cyclic AMP accumulation. In contrast to the corresponding mutant (D164N) in the CB1 receptor, the D80N mutation also ablated receptor‐dependent phosphorylation of ERK1/2. We also report that the reciprocal mutant in D2 receptor restores receptor‐dependent phosphorylation of ERK1/2 and activation of Kir3.0 channels indicating a receptor specific effect of the conserved residues. This work is supported by NIP grant from the AACP.

Interactions of the Dopamine D2 Receptor with the Calcium‐Binding Protein S100B

S100B is a calcium‐binding protein with numerous regulatory activities in the mammalian brain. Using a bacterial two‐hybrid assay, we have identified a novel interaction between S100B and the third cytoplasmic loop of the dopamine D2 receptor (D2‐IC3). The binding of S100B to D2‐IC3 was confirmed using a polyHis pull‐down assay. The binding of heterologous S100B to full‐length D2 receptor in HEK293 cells, and of endogenous S100B to endogenous D2 receptor in the rat neostriatum, was demonstrated by co‐precipitation. A putative S100B binding motif is near the N‐terminus of D2‐IC3. Although S100B is considered a glial protein, S100B expression was detected in microtubule‐associated protein‐2 (MAP2) expressing neurons in neostriatal cultures. Expression of S100B in HEK293 cells also stably expressing the D2 receptor significantly increased D2 receptor stimulation of extracellular signal‐regulated kinases (ERKs), while causing little change in NGF‐stimulated activation of ERKs, and also potentiated D2 receptor inhibition of cyclic AMP accumulation. Taken together, these fundings suggest that the interaction of S100B with the D2 dopamine receptor enhances D2 receptor signaling. (MH045372 and VA Merit Review)

Extracellular Calcium as a Candidate Mediator of Prostate Cancer Skeletal Metastasis

Prostate cancer almost exclusively metastasizes to skeletal sites, indicating that the bone provides a favorable microenvironment for its localization and progression. A natural yet understudied factor in bone that could facilitate tumor localization is elevated extracellular calcium ([Ca2+]o). The present study found that elevated [Ca2+]o (2.5 mmol/L) enhanced proliferation of skeletal metastatic prostate cell lines (PC-3 and C4-2B), but not the nonskeletal metastatic, epithelial-derived prostate cell line LNCaP. The proliferative effect of elevated [Ca2+]o was associated with higher expression of the calcium-sensing receptor (CaSR), a heterotrimeric G-protein-coupled receptor that is the predominant cell-surface sensor for [Ca2+]o. Knockdown of the CaSR via RNA interference reduced cell proliferation in vitro and metastatic progression in vivo. CaSR signaling in PC-3 cells was evaluated by measuring the elevated [Ca2+]o-dependent inhibition of cyclic AMP accumulation, induced by either prostaglandin E2 or forskolin. Elevated [Ca2+]o stabilized expression of cyclin D1, a protein required for cell cycle transition. Furthermore, elevated [Ca2+]o triggered activation of the Akt signaling pathway and enhanced PC-3 cell attachment. Both pertussis toxin (a G-protein inhibitor) and LY294002 (an inhibitor of Akt signaling) reduced cell attachment. These data suggest that elevated [Ca2+]o following increased bone remodeling could facilitate metastatic localization of prostate cancer via the CaSR and the Akt signaling pathway. Taken together, [Ca2+]o is a candidate mediator of prostate cancer bone metastasis.

Intracerebroventricular (ICV) microinjection of a selective kappa opioid agonist increases inducible cAMP element repressor (ICER) expression in the supraoptic nucleus of conscious rats.

Central and peripheral administration of kappa (κ) opioid agonists, U-50488H (U-50), in conscious rats produces a water free diuresis via central inhibition of vasopressin (AVP). Activation of κ receptors has been associated with the inhibition of cyclic AMP accumulation, which may stimulate the expression of endogenous transcriptions factors involved in suppression of cyclic AMP (i.e., ICER). The goal of this study is to examine central κ-mediated changes in ICER staining in the two CNS sites involved in AVP secretion, the paraventricular nuclei (PVN) and supraoptic nucleus (SON). Urine samples were collected during control (15-min) and after ICV vehicle (VEH, n=8) or U-50 (10 μg; n= 8). 3 additional urine samples (15-min) were collected after the ICV injection. The forebrain was processed for ICER using polyclonal anti-ICER antibody raised in rabbit. In VEH injected rats, renal excretion of water or sodium was not altered. U-50 produced a rapid and marked increase in urine flow and decrease in urinary sodium excretion rate. In addition, ICV U-50 produced significant increases in ICER staining in the SON (VEH, 15 ± 8; U-50 33 ± 9; P < .05) without increasing ICER in the PVN (VEH, 26 ± 13; U-50, 22 ± 10; P < .05). In the SON, double labeling shows that 80% of the ICER positive cells were AVP neurons. These results indicate that 1) ICV U-50 injections are associated with differential changes in ICER expression in SON versus PVN magnocellular neurons 2) the diuretic effects of U-50 may be mediated by the inhibition of SON AVP secreting neurons. Supported by NIH HL62579 & DK57822.

N-terminally Truncated Variant of the Mouse GAIP/RGS19 Lacks Selectivity of Full-length GAIP/RGS19 Protein in Regulating ORL1 Receptor Signaling

The regulators of G protein signaling (RGS) are a family of proteins with conserved RGS domains and play essential roles in regulating G protein-mediated signal transduction and physiological events. GAIP/RGS19 (G alpha interacting protein, also classified as RGS19), a member of the RGS family, has been shown to negatively regulate the signaling of many G protein-coupled receptors, including the opioid receptors. Two GAIP/RGS19 mRNA variants, resulted from an alternative splicing of exon 2 of the GAIP/RGS19 gene, were identified in multiple mouse tissues. One of the transcripts consists of a complete set of exons and encodes a full-length GAIP/RGS19 protein, and the other does not have exon 2 and therefore encodes an N-terminal 22 residue truncated short GAIP/RGS19 protein. When co-expressed with either the opioid-receptor-like (ORL1) receptor or one of the mu, delta, and kappa opioid receptors, by transfecting dual-expression plasmids into COS-7 cells, the full-length GAIP/RGS19 was more effective than the N-terminally truncated variant and was more selective in regulating the ORL1 receptor signaling than in regulating the mu, delta, and kappa opioid receptors, as measured by the effectiveness to increase the agonist-stimulated GTPase activity and to reverse the agonist-induced inhibition of cyclic AMP accumulation. In the same assays, the N-terminally truncated GAIP/RGS19 did not distinguish ORL1 from the mu, delta, and kappa opioid receptors. In contrast, co-expression of RGS4 with either ORL1 or opioid receptors showed the selectivity of RGS4 for regulating opioid receptors was mu > kappa > delta > ORL1, an order completely different from that of GAIP/RGS19. The results suggest that GAIP/RGS19 prefers regulating ORL1 receptor signaling over other opioid receptors, and that the N-terminal domain of GAIP/RGS19 plays a crucial role in its receptor preference.

D2 dopamine receptors modulate Galpha-subunit coupling of the CB1 cannabinoid receptor.

CB(1) cannabinoid (CB(1)) and D(2) dopamine (D(2)) receptors are known to couple to the G protein Galpha(i/o). It has been reported that concurrent activation of D(2) receptors and CB(1) receptors, in primary striatal neuronal culture, promotes functional CB(1) receptor coupling to Galpha(s) resulting in elevations in intracellular cyclic AMP levels. We now report that in the absence of D(2) receptors, acute activation of CB(1) receptors inhibits cyclic AMP accumulation, whereas the presence of D(2) receptors promotes CB(1)-stimulated cAMP accumulation, presumably through Galpha(s). This Galpha(s) subunit switching was not prevented by pertussis toxin treatment and occurred in the presence and absence of D(2) receptor activation. Thus, coexpression of the D(2) receptor with the CB(1) receptor was sufficient to switch the coupling of the CB(1) receptors from Galpha(i/o) to Galpha(s). Persistent activation of D(2) receptors resulted in heterologous sensitization of adenylate cyclase to subsequent stimulation by forskolin, whereas the persistent activation of CB(1) receptors did not. Additional studies in human embryonic kidney cells cotransfected with D(2) and CB(1) receptors revealed that persistent activation (18 h) of D(2) receptors induced a switch of CB(1) receptor coupling from Galpha(s) to Galpha(i/o). This D(2) receptor-induced effect allowed for CB(1) receptor-mediated inhibition of cyclic AMP accumulation. The present studies suggest D(2) receptors may have a significant modulatory role in determining the G protein coupling specificity of CB(1) receptors.

Sensitization of adenylate cyclase by short-term activation of 5-HT1A receptors

Long-term (18 h) activation of 5-HT1A receptors alters 5-HT1A receptor-G protein coupling and leads to heterologous sensitization of adenylate cyclase. In contrast, the effects of short-term (2 h) 5-HT1A receptor activation on subsequent adenylate cyclase activity have not been determined. The present study examined and characterized 5-HT1A receptor-induced heterologous sensitization following short-term activation in CHO-5-HT1A cells. Short-term activation of 5-HT1A receptors with full agonists, as well as the partial agonist, buspirone, markedly enhanced subsequent forskolin-stimulated cyclic AMP accumulation. This heterologous sensitization was evident after 30 min treatment with 5HT and appeared to be near maximal following 2 h agonist treatment. Sensitization was characterized by a dose-dependent increase in forskolin-stimulated cyclic AMP accumulation and was prevented by WAY 100635 or by pertussis toxin treatment. The ability of the 5-HT1A agonists to induce heterologous sensitization was not significantly altered by agents shown previously to modulate 5-HT1A-mediated inhibition of cyclic AMP accumulation.

Integrity of extracellular loop 1 of the human cannabinoid receptor 1 is critical for high-affinity binding of the ligand CP 55,940 but not SR 141716A

Like other G-protein coupled receptors with hydrophobic ligands, the human cannabinoid receptor 1 (CB1) is thought to bind its ligands within the transmembrane region of the receptor. However, for some of these receptors the extracellular loops (ECs) have also been shown to play a role in ligand recognition and selectivity. We have taken a mutagenesis approach to examine the role of the amino terminus, EC1, and EC3 of CB1 in ligand binding. Eight mutant receptors, each with a dipeptide insertion, were constructed, expressed, and evaluated for binding to the cannabinoid ligands (−)- cis-3[2-hydroxy-4-(1′,1′-dimethylheptyl)phenyl]- trans-4-(3-hydroxypropyl)cyclohexanol (CP 55,940) and N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H-pyrazole-3-carboxamide hydrochloride (SR 141716A). Mutants with insertions in the membrane distal region of the amino terminus or EC3 maintained affinity for both ligands. Those with insertions in the membrane proximal region of the amino terminus or EC1 exhibited a loss of affinity for CP 55,940 while retaining wild-type affinity for SR 141716A. Representative mutants were analyzed for agonist-induced inhibition of cyclic AMP accumulation, and the results indicated that G-protein coupling remained intact. Alanine substitution mutants were made to address whether it was the perturbation of the overall structure of the region or the displacement of particular side chains that was responsible for the loss of CP 55,940 binding. We conclude that a structurally intact EC1, but not the comparably short EC3, is essential for high-affinity CP 55,940 binding.

Subtype-specific regulation of the expression and function of muscarinic acetylcholine receptors in embryonic chicken retinal cells.

We examined the effect of long-term agonist exposure on muscarinic acetylcholine receptor expression and function in embryonic chicken retinal cells. Long-term carbachol exposure induced a time- and concentration-dependent decrease in M2, M3 and M4 muscarinic receptor numbers. Kinetic analyses revealed a first-order process with similar rate constants for all three subtypes. Both the maximal decrease and the agonist potency for regulation of M3 were significantly higher than those for M2 and M4. Upon agonist removal, M2 and M4 numbers returned to control values, but M3 recovery after 24 h was no higher than 40%. Agonist treatment did not alter the levels of receptor mRNAs. Receptor inactivation with a covalent alkylating antagonist demonstrated that the partial M3 protein recovery was not due to a decreased intrinsic basal rate of synthesis, suggesting that it is induced by agonist treatment. Prolonged carbachol exposure induced concomitant decreases in muscarinic-mediated inhibition of cyclic AMP accumulation which were completely reversed after agonist removal. Sustained receptor activation also promoted significant decreases in muscarinic receptor-stimulated phosphoinositide turnover, which were only partially reversed after agonist removal. These data demonstrate subtype-specific regulation of the expression and function of muscarinic receptors in the retina.

Identification of potent non-peptide somatostatin antagonists with sst(3) selectivity.

Using a solution-phase parallel synthesis strategy, a series of non-peptide somatostatin analogues were prepared, and their binding affinities to the five human somatostatin receptor subtypes (sst(1-5)) were determined. Imidazolyl derivatives 2 were found to bind with moderate affinity but with high selectivity to the sst(3) receptor subtype. Further modifications of these structures led to a more potent class of ligands, the tetrahydro-beta-carboline derivatives 4. Among these, compounds 4k (BN81644) and 4n (BN81674) bind selectively and with high affinity to the sst(3) receptor subtype (K(i) = 0.64 and 0.92 nM, respectively). Furthermore, 4k and 4n reverse the inhibition of cyclic AMP accumulation induced by 1 nM somatostatin via sst(3) receptors, with IC(50) = 2.7 and 0.84 nM, respectively. The most potent compound 4n was shown to be a competitive antagonist of human sst(3) receptors by increasing the EC(50) of SRIF-14-mediated inhibition of cAMP accumulation with a K(B) of 2.8 nM (where K(B) is the concentration of antagonist that shifts the agonist dose-response 2-fold). These new derivatives are, to our knowledge, the first potent and highly selective non-peptide human sst(3) antagonists known and, as such, are useful tools for investigating the physiological role of sst(3) receptors.

Characteristics of the Ca(2+)-dependent inhibition of cyclic AMP accumulation by histamine and thapsigargin in human U373 MG astrocytoma cells.

1. Histamine, acting on H(1)-receptors, caused a Ca(2+)-dependent inhibition of forskolin- and isoprenaline-induced cyclic AMP accumulation in monolayers of human U373 MG cells (IC(50) 1.3+/-0.3 microM, maximum inhibition 66+/-3%). The inhibition was not reversed by the protein kinase inhibitor K-252A. 2. Thapsigargin also inhibited cyclic AMP accumulation (IC(50) 6.0+/-0.3 nM, maximum inhibition 72+/-1%). In the absence of extracellular Ca(2+) 5 microM thapsigargin caused only a 12+/-2% inhibition of cyclic AMP accumulation. 3. The inhibitory effect of 100 nM thapsigargin on forskolin-stimulated cyclic AMP accumulation was blocked by La(3+) (best-fit maximum inhibition 81+/-4%, IC(50) 125+/-8 nM). In contrast, the inhibitory action of 10 microM histamine was much less sensitive to reversal by 1 microM La(3+) (33+/-5% reversal, compared with 78+/-6% reversal of the inhibition by thapsigargin measured concurrently). However, in the presence of both thapsigargin and histamine the inhibition of cyclic AMP accumulation was reversed by 1 microM La(3+) to the same extent as the inhibition by thapsigargin alone. 4.++Thapsigargin (5 microM)+1 microM La(3+) caused only a 20+/-1% inhibition of histamine-stimulated phosphoinositide hydrolysis. 5. There was no indication from measurement of intracellular Ca(2+) of any persistent La(3+)-insensitive Ca(2+) entry component activated by histamine. 6. The results provide evidence that Ca(2+) entry is required for the inhibition by histamine and thapsigargin of drug-induced cyclic AMP accumulation in U373 MG astrocytoma cells. The differential sensitivity of the inhibitory action of the two agents to block by La(3+) suggests that more than one pathway of Ca(2+) entry is involved.

Differential A(1)-adenosine receptor reserve for inhibition of cyclic AMP accumulation and G-protein activation in DDT(1) MF-2 cells.

1. The A(1)-adenosine receptor (A(1)AdoR) reserve for N(6)-cyclopentyladenosine (CPA) mediated inhibition of (-)isoprenaline stimulated cyclic AMP accumulation and stimulation of [(35)S]-guanosine-5'-O-(thiotriphosphate) (GTPgammaS) binding, a measure of guanine nucleotide binding protein (G-protein) activation, was determined in DDT(1) MF-2 cells. 2. Inactivation of the A(1)AdoRs with the chemoreactive ligand 8-cyclopentyl-3-[3-[[4-(fluorosulphonyl)benzoyl]oxy]propyl]-1-p ropylx anthine (FSCPX) caused a progressive rightward shift of the concentration-response curves for CPA to inhibit cyclic AMP accumulation, with a maximum of 10 fold increase in the EC(50) value. In contrast, inactivation of A(1)AdoR's caused only a 1.7 fold rightward shift in the CPA concentration-response for stimulation of [(35)S]-GTPgammaS binding. 3. The A(1)AdoR occupancy-response relationship for CPA inhibition of cyclic AMP accumulation was hyperbolic with 43% receptor occupancy required to elicit the maximal response, i.e. a 57% A(1)AdoR reserve. In contrast, the A(1)AdoR occupancy-response relationship for CPA mediated stimulation of [(35)S]-GTPgammaS binding was linear indicating little or no receptor reserve for G-protein activation. The relationship between CPA stimulation of [(35)S]-GTPgammaS binding and cyclic AMP inhibition was also hyperbolic with 44% G-protein activation sufficient to cause maximal inhibition. 4. The data suggest that the A(1)AdoR reserve for CPA mediated inhibition of cyclic AMP accumulation occurs at the level of G-protein interaction with adenylyl cyclase. However, each A(1)AdoR appears to activate a constant fraction of the total G-protein population suggesting signal amplification at the receptor-G-protein level which may also contribute to the receptor reserve for CPA.

Pertussis toxin treatment prevents 5‐HT5a receptor‐mediated inhibition of cyclic AMP accumulation in rat C6 glioma cells

Pertussis toxin treatment prevents 5‐HT5a receptor‐mediated inhibition of cyclic AMP accumulation in rat C6 glioma cells