Multiple Gi protein subtypes regulate a single effector mechanism.

alpha 2-Adrenergic receptor (alpha 2-AR) responses are mediated by the pertussis toxin-sensitive guanine nucleotide-binding protein (G protein) Gi. Because all three known Gi subtypes are inactivated by pertussis toxin, it has been difficult to determine which of the subtypes are involved in alpha 2-AR responses. In order to investigate alpha 2-AR/Gi coupling, we performed binding and adenylyl cyclase experiments in membranes from CHO-K1 cells transfected with the human alpha 2A-AR. Antisera directed against the carboxyl-terminal region of the Gi1/Gi2 or the Gi3 proteins were used to determine which subtypes were important for high affinity agonist binding and inhibition of adenylyl cyclase. The CHO-K1 cell membranes exhibited immunoreactivity at an apparent molecular mass of 40-41 kDa for both Gi1/Gi2 and Gi3 antisera. Western blot analysis, using purified bovine brain G proteins for comparison, demonstrated that the transfected CHO-K1 cells possess Gi2 and Gi3. High affinity guanosine 5'-(beta,gamma-imido) triphosphate-sensitive binding of the alpha 2-AR agonists [3H]bromoxidine and p-[125I]iodoclonidine ([125I]PIC) was reduced by 30-50% by either the Gi1/Gi2 or Gi3 antiserum. Bromoxidine (1 microM) and PIC (1 microM) inhibited membrane adenylyl cyclase by 34 and 27%, respectively. Gi3 antiserum reduced the inhibition by 26% and 67% for bromoxidine and PIC, respectively. The Gi1/Gi2 antiserum reduced the inhibition by 56% and 63% for bromoxidine and PIC, respectively. Furthermore, when both antisera were used together, there was a complete reversal of alpha 2-AR-mediated inhibition. These observations provide evidence of alpha 2A-AR coupling to at least two subtypes of Gi proteins and the first evidence of functional involvement of Gi3 in the inhibition of adenylyl cyclase.     

Differential inactivation and G protein reconstitution of subtypes of [3H]5-hydroxytryptamine binding sites in brain

The sulfhydryl reagents p-chloromercuribenzoate and N-ethylmaleimide (NEM) inactivate high affinity [3H]serotonin [( 3H]5-HT) binding to bovine and rat brain membranes in a concentration-dependent manner. In both species, 15-25% of total specific high affinity [3H]5-HT binding is relatively insensitive to NEM. This study examines the NEM sensitivity of the various high affinity [3H]5-HT binding subtypes, using selective ligands, tissues, and pharmacological masks to study each subtype. Reconstitution of NEM-inactivated binding by addition of GTP-binding proteins (G proteins, Gi and Go) is also described. Agonist binding to 5-HT1A, 5-HT1B, and 5-HT1D sites in rat brain and to 5-HT1A and 5-HT1D sites in bovine brain is sensitive to NEM. Binding of [3H]dihydroergotamine and [125I]iodocyanopindolol, both of which are weak partial agonists to 5-HT1B sites is relatively insensitive to NEM. Binding of [3H]5-HT to 5-HT1C sites in rat and bovine brain and choroid plexus is relatively insensitive to NEM. In the presence of spiperone to mask binding of 5-HT2 sites, binding of antagonist [( 3H]mesulergine) to 5-HT1C sites is also insensitive to NEM. Likewise, binding of the agonist [3H]4-bromo-2,5-dimethoxyphenylisopropylamine and of the antagonist [3H]ketanserin to 5-HT2 sites is not inhibited by NEM treatment of membranes. These findings suggest that agonist binding to 5-HT1A, 5-HT1B, and 5-HT1D sites is sensitive to NEM alkylation. Binding of neither agonist nor antagonist to 5-HT1C and 5-HT2 sites is sensitive to NEM. Inability of high concentrations of a variety of ligands to protect the sensitive binding sites against NEM inactivation indicates that the critical sulfhydryl group(s) are not located in the ligand binding domain of the NEM-sensitive binding sites. When membranes are treated with NEM, displacement of [125I]iodocyanopindolol by 5-HT is no longer sensitive to 5'-guanylyl imidodiphosphate (Gpp(NH)p). Gpp(NH)p sensitivity of agonist displacement of 5-HT1B binding to NEM-treated membranes is restored by addition of purified guanine nucleotide binding proteins (Gi plus Go). In addition, NEM-inactivated binding to 5-HT1A and 5-HT1D sites can be restored by addition of Gi plus Go. These data suggest that NEM exerts its effects on 5-HT1A, 5-HT1B, and 5-HT1D binding sites by inactivating the G protein(s) associated with the 5-HT receptor subtypes.     

Cerebral muscarinic acetylcholine receptors interact with three kinds of GTP-binding proteins in a reconstitution system of purified components

A new GTP-binding protein, which serves as a substrate for pertussis toxin, was prepared from porcine brain. The new G protein was separated from other GTP-binding proteins, Gi and Go, by an anion-exchange column chromatography. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the alpha subunit of the new G protein was between those of alpha subunits of Gi and Go. Evidence that the alpha subunit is not a proteolytic fragment of the alpha subunit is not a proteolytic fragment of the alpha subunit of Gi or Go was provided by experiments involving partial hydrolysis of these G proteins with thermolysin and their interaction with an antibody raised against the amino terminal peptide of the alpha subunit of Gi. In addition, the gamma subunit of the new G protein was indicated to be different from the gamma subunits of Gi and Go, because the latter were found to be phosphorylated by protein kinase C but the former was not. GTP-sensitive high affinity binding of muscarinic receptors with acetylcholine was observed when muscarinic receptors purified from porcine cerebrum were reconstituted in phospholipid vesicles with the new G protein as well as with Gi or Go. The proportion of the high affinity sites increased with the concentrations of the G proteins, the potency of the new G protein being similar to that of Gi but a little lower than that of Go. This GTP-sensitive high affinity binding was not observed when each G protein was pretreated with pertussis toxin and then reconstituted with muscarinic receptors. Acetylcholine accelerated the dissociation of [3H]GDP from the new G protein as well as from Gi and Go, which were reconstituted with muscarinic receptors. These results indicate that muscarinic receptors interact with at least the above three kinds of G proteins, in a pertussis toxin-sensitive manner.     

Molecular interaction of the human alpha 2-C10-adrenergic receptor, when expressed in Rat-1 fibroblasts, with multiple pertussis toxin-sensitive guanine nucleotide-binding proteins: studies with site-directed antisera.

DNA encoding the human alpha 2-C-10-adrenergic receptor was transfected into Rat-1 fibroblasts by CaPO4 precipitation, and clones expressing the receptor were isolated and expanded. One clone (1C) expressing high levels of the receptor was studied in order to determine the contacts between this receptor and guanine nucleotide-binding proteins (G proteins) mediating second messenger signaling. The alpha 2-adrenergic agonist UK 14304 stimulated high affinity GTPase activity in membranes from these cells. Incubation of these membranes with Protein A-purified fractions from an antiserum able to identify the carboxyl-terminal decapeptide common to Gi1 alpha and Gi2 alpha was partially able to prevent agonist stimulation of high affinity GTPase activity. Similar results were produced with an antiserum that identifies the carboxyl-terminal decapeptide of Gi3 alpha. In contrast, equivalent fractions of antisera that identify the carboxyl-terminal decapeptides of Go alpha and Gs alpha did not inhibit receptor stimulation of high affinity GTPase activity. Coincubation of the membranes from the cells with Protein A-purified fractions from the anti-Gi1 alpha + Gi2 alpha antiserum and the anti-Gi3 alpha antiserum produced greater inhibition of UK14304-stimulated GTPase activity than did either of the two antisera in isolation. These data show direct interaction of the human alpha 2-C10-adrenergic receptor, when expressed in this clone of Rat-1 fibroblasts, with multiple pertussis toxin-sensitive G proteins and demonstrate that a single receptor has the physical capacity to interact functionally with more than a single pertussis toxin-sensitive G protein in a native membrane. Furthermore, because the two antisera were able to inhibit receptor stimulation of high affinity GTPase activity to similar degrees, the G protein pools identified by these antisera must contribute similar amounts of the total receptor activation of pertussis toxin-sensitive G proteins in these cells.     

Pertussis toxin inhibits norepinephrine-stimulated inositol phosphate formation in primary brain cell cultures

Norepinephrine (NE) increased formation of [3H]inositol phosphates ( [3H]InsPs) in primary cultures of neuronal and glial cells from 1-day-old rat brain. This response appeared to be mediated by alpha 1-adrenergic receptors, because prazosin was 40-fold more potent than yohimbine in blocking it. Pretreatment with pertussis toxin (PTX) dose-dependently decreased this response by 70-80%. The IC50 for PTX (7 ng/ml) was similar to that for blocking of alpha 2-adrenergic receptor-mediated decreases in cyclic AMP accumulation in the same cells. PTX pretreatment caused only a small, not statistically significant, inhibition of the [3H]InsP response to the muscarinic cholinergic receptor agonist carbachol in these cells. Radioligand binding studies showed that both neuronal and glial cultures contained mixed populations of alpha 1a- and alpha 1b-adrenergic receptor subtypes. Selective inactivation of the alpha 1b population by chloroethylclonidine reduced NE-stimulated [3H]InsP formation by 25 +/- 6%. Pretreatment with both PTX and chloroethylclonidine caused additive decreases (90 +/- 3%) in the NE response. NE-stimulated [3H]InsP formation was partially dependent on extracellular calcium, because it was decreased 64 +/- 6% by removal of calcium and 56 +/- 13% by addition of 1 mM CdCl2, although it was not affected by 1 microM nifedipine. These results suggest that NE stimulates [3H]InsP formation in neuronal and glial cultures through a pertussis toxin-sensitive guanine nucleotide-binding protein. This response appears to be mediated primarily by the alpha 1a subtype and may be subsequent to calcium influx.     

Regulation of the avidity of ternary complexes containing the human 5-HT(1A) receptor by mutation of a receptor contact site on the interacting G protein alpha subunit

1 Fusion proteins were constructed between the human 5-HT(1A) receptor and pertussis toxin-resistant forms of both G(i1)alpha and G(o1)alpha mutated at residue(351) from cysteine to either glycine or isoleucine. Each of these was expressed stably in HEK293 cells. 2 Increasing concentrations of GDP inhibited binding of the agonist [(3)H]-8-OH-DPAT but not the antagonist [(3)H]-MPPF to each construct. 3 The IC(50) for GDP was greater for constructs containing isoleucine at residue(351) of the G proteins compared to those with glycine at this position. 4 The G protein antagonist suramin had similar effects to GDP on the binding of [(3)H]-8-OH-DPAT. 5 The proportion of 5-HT(1A) receptor binding sites detected by [(3)H]-MPPF that displayed high affinity for 8-OH-DPAT was significantly greater when the interacting G protein contained isoleucine rather than glycine at residue(351). 6 The 5-HT(1A) receptor displayed similar avidity of interaction with G(i1)alpha and G(o1)alpha. 7 These results indicate that a higher avidity ternary complex is formed between 8-OH-DPAT, the 5-HT(1A) receptor and G proteins when isoleucine rather than glycine is located at residue(351) of the interacting G protein.     

Identification of three separate guanine nucleotide-binding proteins that interact with the delta-opioid receptor in NG108-15 neuroblastoma x glioma hybrid cells.

Five separate guanine nucleotide-binding proteins (G proteins) were immunologically identified in membranes from neuroblastoma x glioma NG108-15 hybrid cells. These alpha subunit proteins were Gi2 alpha, two isoforms of Gi3 alpha, and two isoforms of Go alpha. The G proteins that interacted with delta-opioid receptors in these membranes were identified using cholera toxin (CTX)-induced ADP-ribosylation and antisera selective for various G protein alpha subunits. In the presence of delta-opioid agonists, CTX induced the incorporation of [32P]ADP-ribose into three pertussis toxin substrates. Using antisera generated against peptide sequences from G alpha subunits, these three pertussis toxin substrates were identified as Gi2 alpha, Go2 alpha, and one isoform of Gi3 alpha, which has yet to be identified. This CTX-induced labeling was demonstrated to be mediated via the delta-opioid receptor in these hybrid cells by the observation that delta agonists D-Ala2-D-Leu5-enkephalin (DA-DLE) and D-Pen2-D-Pen5-enkephalin, as well as the nonselective agonists etorphine and bremazocine, were active, but the mu agonist PL017 and the kappa agonist U-50-488H did not show this activity. This incorporation into all three substrates induced by DADLE was dose dependent, with EC50 (95% confidence interval) values ranging from 12 (3-52) to 183 (65-520) nM, which compared with the Kd value of 10 +/- 1.5 nM for this agonist, a dose that produces maximal inhibition of adenylate cyclase activity. Furthermore, pretreatment of the cells with pertussis toxin or treatment of the membranes with the antagonist naloxone blocked the incorporation induced by DADLE. Incorporation of [32P]ADP-ribose into all three substrates decreased 35-83% in membranes in which the receptors had been down-regulated by chronic treatment of the cells with DADLE. Thus, a single opioid receptor type can interact with three separate G proteins.     

Agonist and antagonist binding to alpha 2-adrenergic receptors in purified membranes from human platelets. Implications of receptor-inhibitory nucleotide-binding protein stoichiometry

The agonist- and antagonist-binding properties of the alpha 2-adrenergic receptor in a purified plasma membrane preparation from human platelets were determined both by direct binding of radiolabeled ligands and by competition with the labeled alpha 2-antagonist, [3H] yohimbine. Binding of [3H]yohimbine was characterized by a single high affinity binding site (Kd = 6.2 +/- 1.4 nM, Bmax = 507 +/- 53 fmol/mg). In direct binding studies, the imidazoline full alpha 2-agonist, [3H]-5-bromo-6-N(2-4,5-dihydroimidazolyl)quinoxaline ([3H] UK 14,304), bound to only one quantifiable high affinity site (Kd = 0.88 +/- 0.17 nM), representing 65 +/- 6% of the number of [3H]yohimbine sites. Binding of the partial agonist [3H]-p-aminoclonidine (PAC) showed nonlinear Scatchard plots. Analysis according to a model of multiple independent binding sites showed the data to be consistent with two sites (Kd1 = 0.62 +/- 0.18 nM and Kd2 = 7.9 +/- 1.4 nM). The high affinity site corresponded to 15 +/- 6% and the low affinity site corresponded to 39 +/- 6% of the number of [3H]yohimbine sites. Competition for binding of the alpha 2-antagonist, [3H]yohimbine, with nonradiolabeled ligands revealed a single affinity for yohimbine. In contrast, competition for [3H]yohimbine binding by the full agonist UK 14,304 and epinephrine is best fit by a model with two independent binding sites. The partial agonist PAC was best characterized by a model with three distinct binding sites. The full agonists UK 14,304 and epinephrine inhibited adenylate cyclase approximately 30%, whereas PAC produced only 12% inhibition. The inhibitory guanine nucleotide-binding protein (Ni) with Mr 40,700 was the sole pertussis toxin substrate in the purified membranes. It was quantitated by pertussis toxin-catalyzed [32P]ADP ribosylation in cholate extracts. There is a 20- to 100-fold excess of Ni over alpha 2-adrenergic receptors. Comparisons made between the experimental data for agonist binding and theoretical predictions of the simple ternary complex model suggest that there is compartmentalization of Ni and/or that the alpha 2 receptors are heterogeneous.     

Identification of drug receptors in porcine dental pulp by the radioligand binding assay

1. The acetylcholine (ACh), histamine and serotonin (5-HT) receptors in porcine dental pulp were characterized by the radioligand binding assay. 2. For [3H]nicotine binding site, Kd was 8.06 +/- 1.65 nM and Bmax was 270.83 +/- 32.68 fmol/mg protein. 3. For [3H]QNB binding site, Kd was 1.04 +/- 0.14 nM and Bmax was 24.83 +/- 3.09 fmol/mg protein. 4. For [3H]histamine binding site, Kd was 1.22 +/- 0.1 nM and Bmax was 283.15 +/- 33.1 fmol/mg protein. 5. For [3H]5-HT binding site, Kd was 1.41 +/- 0.1 nM and Bmax was 53.1 +/- 3.4 fmol/mg protein. 6. These findings indicate that the specific receptors for ACh, histamine and 5-HT are present in the porcine dental pulp, and that the ACh receptor is predominantly nicotinic.     

Primary structure and functional characterization of a human 5-HT1D-type serotonin receptor.

We describe the nucleic acid sequence encoding a human 5-hydroxytryptamine1D (5-HT1D) serotonin receptor and some of the functional characteristics of the gene product. The receptor gene was isolated by hybridization to a probe based on a canine thyroid cDNA (called RDC4) previously isolated by others and believed to encode a heretofore undetermined member of the guanine nucleotide-binding protein (G protein)-linked receptor family. The human clone we isolated, called MA6A, contains an apparently intronless open reading frame encoding a 377-amino acid polypeptide with the seven hydrophobic domains characteristic of G protein-linked receptors. The MA6A deduced amino acid sequence is 88% identical to that for RDC4 and 43% identical to that for the human 5-HT1A receptor. Expression of the human gene product in transfected cell lines results in the appearance of saturable high affinity 5-HT1D-type [3H]5-HT binding. The expressed receptor exhibits features indicative of coupling to Gi proteins, i.e., robust inhibition of forskolin-stimulated cAMP accumulation and formation of a pertussis toxin-sensitive high agonist affinity binding state. These findings may help clarify several ambiguities in the classification and action of serotonin receptor subtypes.     

Alpha 1-adrenergic receptor-linked guanine nucleotide-binding protein in muscle and kidney epithelial cells

We have studied the interaction of guanine nucleotides with alpha 1-adrenergic receptors of two cloned cell lines, the Madin Darby canine kidney (MDCK-D1) cells and BC3H-1 muscle cells. Although guanylylimidodiphosphate, Gpp(NH)p, had no effect on the affinity or the total number of [3H]prazosin-binding sites in membranes prepared from these cells, the nucleotide decreased the apparent affinity of the agonists (-)-epinephrine and (-)-norepinephrine in competing for [3H]prazosin-binding sites in both cell types. A maximal effect of Gpp(NH)p occurred at 10 microM. Guanine nucleotides were significantly more effective in shifting agonist affinity for the alpha 1 receptor than adenine nucleotides, and Mg2+ was required to observe a maximal effect. Binding of agonist to alpha 1-adrenergic receptors activated phosphatidylinositol (PI) hydrolysis in both cell types but had no effect on membrane adenylate cyclase activity. Incubation of MDCK cells for 19 hr with 100 ng/ml pertussis toxin, which eliminated the ability of pertussis toxin added to membranes to ADP-ribosylate 39-41-KDa substrate(s), failed to alter binding of agonists to alpha 1-adrenergic receptors, the ability of Gpp(NH)p to regulate agonist binding to these receptors, or epinephrine-stimulated PI hydrolysis and prostaglandin E2 production. Incubation of BC3H1 cells with pertussis toxin had no effect on the ability of epinephrine to stimulate PI turnover. These results show that binding of agonists to alpha 1-adrenergic receptors in mammalian kidney and muscle cells is regulated by guanine nucleotides, presumably by interaction with a guanine nucleotide-binding (G) protein. The failure of the G-protein to regulate adenylate cyclase activity and the lack of effect of pertussis toxin to alter receptor-mediated binding or functional activity suggests that a G-protein other than Gs, Gi, or Go interacts with alpha 1-adrenergic receptors in kidney and smooth muscle.     

Coupling of an endogenous 5-HT1B-like receptor to increases in intracellular calcium through a pertussis toxin-sensitive mechanism in CHO-K1 cells.

1. Chinese hamster ovary cells (CHO-K1) express an endogenous 5-hydroxytryptamine (5-HT)1B-like receptor that is negatively coupled to adenylyl cyclase through a pertussis toxin (PTX)-sensitive mechanism. Furthermore, the human adenosine A1 receptor when expressed in CHO-K1 cells (CHO-A1) has been shown to mobilize intracellular Ca2+ through a PTX-sensitive mechanism. Therefore the aim of this investigation was to determine whether the endogenous 5-HT1B-like receptor was able to stimulate increases in intracellular free [Ca2+] ([Ca2+]i) in CHO-A1 cells. 2. In agreement with previous studies using CHO cells, 5-hydroxytryptamine (5-HT) elicited a concentration-dependent inhibition of forskolin-stimulated [3H]-cyclic AMP production in CHO-A1 cells (p[EC50] = 7.73 +/- 0.13). 5-HT (1 microM) inhibited 47 +/- 5% of the [3H]-cyclic AMP accumulation induced by 3 microM forskolin. Forskolin stimulated [3H]-cyclic AMP accumulation was also inhibited by the 5-HT1 receptor agonists (p[EC50] values) 5-carboxyamidotryptamine (5-CT; 8.07 +/- 0.08), RU 24969 (8.12 +/- 0.33) and sumatriptan (5.80 +/- 0.31). 3. 5-HT elicited a concentration-dependent increase in [Ca2+]i in CHO-A1 cells (p[EC50] = 8.07 +/- 0.05). In the presence of 2 mM extracellular Ca2+, 5-HT (1 microM) increased [Ca2+]i from 174 +/- 17 nM to 376 +/- 22 nM. The 5-HT1 receptor agonists (p[EC50] values), 5-carboxyamidotryptamine (5-CT; 7.9 +/- 0.02), RU 24969 (8.1 +/- 0.07) and sumatriptan (5.9 +/- 0.11) all elicited concentration-dependent increases in [Ca2+]i. Similar maximal increases in [Ca2+]i were obtained with each agonist. The selective 5-HT1A receptor agonist, 8-OH-DPAT (10 microM) did not stimulate increases in [Ca2+]i. 5-HT (100 microM) and 5-CT (10 microM) did not stimulate a measurable increase in [3H]-inositol phosphate accumulation in CHO-A1 cells. 4. 5-HT (1 microM)-mediated increases in [Ca2+]i were insensitive to the 5-HT receptor antagonist, ritanserin (5-HT2; 100 nM), ketanserin (5-HT2; 100 nM), LY-278,584 (5-HT3; 1 microM) and WAY 100635 (5-HT1A; 1 microM). The response to 5-HT (100 nM) was antagonized by the non-selective 5-HT1 antagonist, methiothepin (pKb = 8.90 +/- 0.09) and the 5-HT1D antagonist GR 127935 (pKb = 10.44 +/- 0.06). 5. Pretreatment with PTX (200 ng ml-1 for 4 h) completely attenuated the Ca2+ response to 100 microM 5-HT. 6. In untransfected CHO-K1 cells, 5-HT (1 microM), RU 24969 (1 microM), and 5-CT (1 microM) elicited increases in [Ca2+]i similar to those observed in CHO-A1 cells. 7. These data demonstrate that in CHO-K1 cells the endogenously expressed 5-HT1B-like receptor couples to the phospholipase C/Ca2+ signalling pathway through a PTX-sensitive pathway, suggesting the involvement of Gi/Go protein(s).     

3H]rauwolscine behaves as an agonist for the 5-HT1A receptors in human frontal cortex membranes.

The alpha 2 adrenergic antagonist [3H]rauwolscine binds with comparable nanomolar affinity to alpha 2 adrenoceptors and the nonadrenergic 5-HT1A receptors sites in human frontal cortex membranes. Addition of 0.5 mM GTP into the incubation medium produces a significant decrease in the amount of [3H]rauwolscine binding sites (Bmax = 230 +/- 16 and 115 +/- 11 fmol/mg protein in the absence and presence of GTP, respectively). The affinity for [3H]rauwolscine remains unchanged (i.e. KD = 40 +/- 0.9 nM and 4.1 +/- 1 nM). This effect of GTP can be attributed to decreased binding of the radioligand to the 5-HT1A receptors. GTP decreases binding of [3H]rauwolscine to nearly the same level as the one corresponding to the alpha 2 adrenoceptors in membranes from both the human frontal cortex and hippocampus. The venom of the marine cone snail, Conus tessulatus, preferentially inhibits [3H]rauwolscine binding to 5-HT1A receptors as compared with the alpha 2 adrenoceptors. Following complete masking of the 5-HT1A receptors by this venom. GTP no longer affects the saturation binding characteristics of [3H]rauwolscine for the remaining alpha 2 adrenoceptors. Nucleotides decrease the binding of [3H]rauwolscine to the 5-HT1A receptors with an order of potencies (i.e. GTP gamma S greater than GPP(NH)P much greater than GDP greater than GTP much greater than ATP) that is typical for nucleotide-mediated receptor-G protein dissociation. This suggests that [3H]rauwolscine is a 5-HT1A receptor agonist and this conclusion is compatible with earlier functional studies, indicating that rauwolscine (as well as yohimbine) has agonistic properties at the level of 5-HT autoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

5-HT1B and 5-HT2 serotonin binding sites in cultured Wistar-Kyoto rat aortic smooth muscle cells

We studied [3H]serotonin [( 3H]5-HT) binding on cultured arterial smooth muscle cells from rat aorta. We found a high and low affinity binding site. Binding to the higher affinity site could be displaced by drugs in an order corresponding most closely with the 5-HT1B subtype, and high affinity [125I]iodo-cyanopindolol binding was also found. We found evidence for a 5-HT2 subtype using [3H]ketanserin binding, with similar results whether specific binding was determined using unlabelled ketanserin, methysergide or mianserin.     

Identification and distribution of 5-HT3 recognition sites in the rat gastrointestinal tract.

1. Tritiated derivatives of the potent and selective 5-HT3 receptor antagonists GR65630 and LY278584 were used to identify 5-HT3 recognition sites in the rat gastrointestinal tract. 2. Binding studies were carried out in homogenates of the rat oesophagus, the cardia, fundus, body and antrum of the stomach, regions of the small intestine, caecum and large intestine. The specific binding of a single concentration of GR65630 (0.5 nM) defined by granisetron (10 microM) in these areas indicated that the density of 5-HT3 recognition sites varied from 2.4 +/- 1.0 to 10.1 +/- 1.0 fmol mg-1 protein. 3. Saturable binding of [3H]-GR65630 could only be demonstrated in the terminal regions of the small intestine (Bmax in the range of 13.83 +/- 4.54-21.19 +/- 0.89 fmol mg-1 protein; mean +/- s.e. mean) and of high affinity (Kd in the range of 0.42 +/- 0.18-0.79 +/- 0.24 nM). Use of [3H]-LY278584 revealed a similar binding density (Bmax 19.54 +/- 0.26 fmol mg-1 protein) and affinity (Kd 1.04 +/- 0.07 nM) in the terminal small intestine. 4. Binding of [3H]-GR65630 and [3H]-LY278584 to the terminal region of the small intestine was inhibited by 5-HT3 receptor ligands ondansetron and S-zacopride (and 5-hydroxytryptamine), but not by 5-HT1, 5-HT2, catecholamine, gamma-aminobutyric acid and opioid receptor ligands. 5. These data demonstrate that there are regional variations in the density of 5-HT3 recognition sites within the rat gastrointestinal tract. Such data are relevant to the potential use of 5-HT3 receptor ligands to modify secretory and contraction responses in the gastrointestinal system.     

Inhibitory GTP-binding regulatory protein Gi3 can couple angiotensin II receptors to inhibition of adenylyl cyclase in hepatocytes

Angiotensin II can inhibit hormone-stimulated adenylyl cyclase in intact hepatocytes or in hepatic membrane preparations. Because the response can be blocked by pertussis toxin, the object of the present study was to determine which of the known variants of Gi can couple angiotensin II receptors to inhibition of adenylyl cyclase. The potential candidates were identified by probing RNA isolated from rat hepatocytes with cDNAs specific for the alpha subunits of known toxin-sensitive guanine nucleotide-binding regulatory proteins (G proteins). Hepatocytes contained no detectable RNA for the Go or Gi1 alpha subunits and similar levels of RNA coding for the Gi2 and Gi3 alpha subunits. To determine whether Gi3 could couple angiotensin receptors to inhibition of cyclase, membranes were prepared from hepatocytes whose G proteins were fully ADP-ribosylated with pertussis toxin, and the Gi3 holoprotein purified from rabbit liver was reconstituted into the membranes. The nature of the Gi3 reconstituted into the membrane was assessed by immunoblotting with antibodies specific for the Gi alpha subunits. Reconstitution of 6-10 pmol of Gi3/mg of membrane protein into the toxin-treated membranes restored the ability of 10 nM angiotensin II to inhibit adenylyl cyclase. Because pertussis toxin has nonspecific effects, an assay was developed to measure the interaction of the angiotensin receptor with reconstituted G proteins in normal membranes. In the presence of Mg2+, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) caused a reduction of the affinity of the angiotensin II receptor for 125I-angiotensin II that was stable to washing and the detergents used to reconstitute G proteins into the membranes. Using this protocol to activate G proteins and "uncouple" receptors, the ability of the GDP-liganded form of Gi to restore high affinity binding was examined. Reconstitution of about 10-15 pmol of oligomeric Gi3/mg of membrane protein restored both the high affinity state of the angiotensin II receptor and the ability of GTP gamma S to shift the affinity to a lower state. The same shift in receptor affinity could be accomplished by reconstituting the Gi3 alpha subunit, resolved free of beta gamma subunits, into the membranes. Reconstitution of up to 50 pmol of Gs/mg of membrane protein had no effect on angiotensin II receptor affinity. The results suggest that a major form of Gi in hepatocytes is Gi3 and that it can couple angiotensin receptors to inhibition of adenylyl cyclase.     

Differential activation of Gq/11 and Gi(3) proteins at 5-hydroxytryptamine(2C) receptors revealed by antibody capture assays: influence of receptor reserve and relationship to agonist-directed trafficking

As determined by a guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding assay, which does not distinguish G protein subtypes, 5-hydroxytryptamine (5-HT) and 2(S)- 1-(6-chloro-5-fluoro-1H-indol-1-yl)-2-propanamine fumarate (Ro600175) behaved as full agonists at human 5-HT(2C) (h5-HT(2C)) receptors (VSV isoform) stably expressed in Chinese hamster ovary (CHO) cells, whereas 1-2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI), d-lysergic acid diethylamide (LSD), and lisuride exhibited partial agonist properties. After treatment with pertussis toxin to uncouple 5-HT(2C) receptors from Gi/Go but not Gq/11, DOI and LSD were as efficacious as 5-HT and Ro600175 in stimulating [(35)S]GTPgammaS binding, whereas lisuride still exhibited low efficacy (40%). Correspondingly, in a scintillation proximity assay employing specific antibodies against Gq/11, 5-HT, Ro600175, DOI, and LSD behaved as high-efficacy agonists, whereas lisuride showed efficacy of 36%. In contrast, when employing a specific antibody recognizing Gi(3), DOI and LSD were less efficacious (80 and 30%, respectively) than 5-HT and Ro600175, and lisuride was inactive. Agonist actions were specifically mediated by h5-HT(2C) receptors inasmuch as the selective 5-HT(2C) antagonist SB242,084 blocked [(35)S]GTPgammaS binding at both Gq/11 and Gi(3). Agonist potency for stimulation of Gi(3) was ~6- to 8-fold less than for Gq/11, indicating that the latter was preferentially engaged by h5-HT(2C) receptors. Inactivation of h5-HT(2C) receptors with the alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline did not modify the efficacy of 5-HT, Ro600175, and DOI at Gq/11, whereas their efficacies were substantially reduced at Gi(3), indicating a greater receptor reserve for the former. Finally, the preferential activation of Gq/11 versus Gi(3) by DOI, LSD, and lisuride was diminished in the presence of lower receptor number. In conclusion, h5-HT(2C) receptors couple to both Gq/11 and Gi(3) in CHO cells, and efficacy for G protein subtype activation is both ligand- and receptor reserve-dependent.     

The inhibitory effect of opioid and alpha 2-adrenoceptor agonists on cardiac sensory neurones is pertussis toxin-insensitive.

The role of pertussis toxin-sensitive G proteins on the alpha 2-adrenoceptor and mu-opioid receptor-mediated inhibition of the efferent function of capsaicin-sensitive neurones was investigated in guinea-pig atria pretreated with guanethidine. In the presence of atropine, CGP 20712A (2-hydroxy-5-(2-[hydroxy-3-(4-[(1-methyl- 4-trifluormethyl)1H-imidazol-2-yl]-phenoxy)propyl]aminoethoxyl+ ++)-benzamide) and prazosin, [D-Ala2,NMe-Phe4,Gly5-ol]enkephalin (DAGO, 0.1-3 microM) and 2-amino-6-allyl-5,6,7,8-tetrahydro-4H-thiazolo(4,5-d)azepine (BHT 920, 0.01-1 microM) reduced the positive inotropic effect induced by transmural stimulation of preparations obtained from control and from pertussis toxin-treated animals. These results suggest that pertussis toxin-sensitive G proteins are not involved in the inhibitory regulation of the efferent function of capsaicin-sensitive nerve terminals in cardiac tissue induced by alpha 2 and opioid receptor stimulation.     

Enhanced delta-opioid receptor-mediated antinociception in mu-opioid receptor-deficient mice

This study examined the binding of serotonin receptor antagonists at the 5-HT(2A) and 5-HT(2C) receptors of the rabbit's cerebral cortex. The 5-HT(2A) receptor was characterized by the binding of [3H]MDL 100,907 (R(+)-alpha-(2, 3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine-methan ol) to cortical membranes and the 5-HT(2C) receptor by the binding of [3H]mesulergine in the presence of the selective 5-HT(2A) receptor ligand spiperone. Both [3H]MDL 100,907 and [3H]mesulergine demonstrated high affinity binding to single sites in rabbit membranes. Based on Scatchard plots of [3H]MDL 100,907 binding, the mean B(max) was 8.5+/-0.7 fmol/mg tissue and the mean K(d) was 33. 1+/-3.5 pM. For [3H]mesulergine binding the mean B(max) was 3.70+/-0. 58 fmol/mg tissue and the mean K(d) was 0.35+/-0.05 nM. Binding of [3H]MDL 100,907 to the 5-HT(2A) receptor and of [3H]mesulergine to the 5-HT(2C) receptor was confirmed by displacement studies with highly selective 5-HT(2A) and 5-HT(2C) receptor ligands. The pharmacological profile of these ligands in rabbits correlated highly with published values for 5-HT(2A) (r=0.91, P<0.001) and 5-HT(2C) (r=0.94, P<0.001) receptors in humans. There was also a high correlation between the profiles for human and rat 5-HT(2C) receptor (r=0.92, P<0.001), but not for 5-HT(2A) receptors (r=0.53, P>0.10). It was concluded that the rabbit provides an appropriate animal model for studies attempting to predict the pharmacology of human 5-HT(2A) and 5-HT(2C) receptors.