Negative Intrinsic Activity Research Articles

Ligands Raise the Constraint That Limits Constitutive Activation in G Protein-coupled Opioid Receptors

Using a cell-free bioluminescence resonance energy transfer strategy we compared the levels of spontaneous and ligand-induced receptor-G protein coupling in δ (DOP) and μ (MOP) opioid receptors. In this assay GDP can suppress spontaneous coupling, thus allowing its quantification. The level of constitutive activity was 4-5 times greater at the DOP than at the MOP receptor. A series of opioid analogues with a common peptidomimetic scaffold displayed remarkable inversions of efficacy in the two receptors. Agonists that enhanced coupling above the low intrinsic level of the MOP receptor were inverse agonists in reducing the greater level of constitutive coupling of the DOP receptor. Yet the intrinsic activities of such ligands are identical when scaled over the GDP base line of both receptors. This pattern is in conflict with the predictions of the ternary complex model and the "two state" extensions. According to this theory, the order of spontaneous and ligand-induced coupling cannot be reversed if a shift of the equilibrium between active and inactive forms raises constitutive activation in one receptor type. We propose that constitutive activation results from a lessened intrinsic barrier that restrains spontaneous coupling. Any ligand, regardless of its efficacy, must enhance this constraint to stabilize the ligand-bound complexed form.

Serotonin2C ligands exhibiting full negative and positive intrinsic activity elicit purposeless oral movements in rats: distinct effects of agonists and inverse agonists in a rat model of Parkinson's disease

This study examined in naive or hemiparkinsonian rats the effect of various serotonin 2C (5-HT(2C)) receptor ligands differing in their intrinsic activity at 5-HT(2C) receptors on purposeless oral movements, a motor response integrated in the basal ganglia. Intraperitoneal administration of a non-selective [meta-chlorophenylpiperazine (m-CPP) 0.1-3 mg/kg], preferential [S-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine, Ro60-0175, 0.1-3 mg/kg] or selective [(7bR,10aR)-1,2,3,4,8,9,10,10a-octahydro-7bH-cyclopenta-[b][1,4]diazepino[6,7,1hi]indole, WAY163909, 0.3-10 mg/kg] 5-HT(2C) agonists enhanced oral bouts in naive rats. The 5-HT(2C) inverse agonists SB206553 [1-20 mg/kg; 5-methyl-1-(3-pyridylcarbamoyl)-1,2,3,5-tetrahydropyrrolo[2,3-f]indole] and S32006 [1-20 mg/kg; N-pyridin-3-yl-1,2-dihydro-3H-benzo[e]indole-3-carboxamide], but not the 5-HT(2C) antagonist SB243213 [1-10 mg/kg; 5-methyl-1-[[2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-6-trifluoromethylindoline], likewise dose-dependently enhanced oral movements. The effects induced by preferential 5-HT(2C) agonists and inverse agonists, but not by the cholinomimetic drug pilocarpine (5 mg/kg), were abolished by SB243213 underpinning its specificity. S32006-induced oral bouts was unaffected by the 5,7-dihydroxytryptamine lesions of 5-HT neurons. Nigrostriatal dopaminergic lesions potentiated oral effects induced by the agonists Ro60-0175 (3 mg/kg) and WAY163909 (1 mg/kg), but not by the inverse agonist SB206553 (10 mg/kg). The effect of Ro60-0175 in dopamine-lesioned rats was suppressed by SB243213. These data show that 5-HT(2C) agonists and full inverse agonists (but not neutral antagonists) perturb oral activity in rodents, paralleling studies of common antidepressant, anxiolytic and antipsychotic properties. The differential sensitivity of their actions to depletion of dopamine suggests recruitment of different contrasting neural mechanisms in the basal ganglia.

Inverse agonism and its therapeutic significance

A large number of G-protein-coupled receptors (GPCRs) show varying degrees of basal or constitutive activity. This constitutive activity is usually minimal in natural receptors but is markedly observed in wild type and mutated (naturally or induced) receptors. According to conventional two-state drug receptor interaction model, binding of a ligand may initiate activity (agonist with varying degrees of positive intrinsic activity) or prevent the effect of an agonist (antagonist with zero intrinsic activity). Inverse agonists bind with the constitutively active receptors, stabilize them, and thus reduce the activity (negative intrinsic activity). Receptors of many classes (α-and β-adrenergic, histaminergic, GABAergic, serotoninergic, opiate, and angiotensin receptors) have shown basal activity in suitable in vitro models. Several drugs that have been conventionally classified as antagonists (β-blockers, antihistaminics) have shown inverse agonist effects on corresponding constitutively active receptors. Nearly all H1 and H2 antihistaminics (antagonists) have been shown to be inverse agonists. Among the β-blockers, carvedilol and bucindolol demonstrate low level of inverse agonism as compared to propranolol and nadolol. Several antipsychotic drugs (D2 receptors antagonist), antihypertensive (AT1 receptor antagonists), antiserotoninergic drugs and opioid antagonists have significant inverse agonistic activity that contributes partly or wholly to their therapeutic value. Inverse agonism may also help explain the underlying mechanism of beneficial effects of carvedilol in congestive failure, naloxone-induced withdrawal syndrome in opioid dependence, clozapine in psychosis, and candesartan in cardiac hypertrophy. Understanding inverse agonisms has paved a way for newer drug development. It is now possible to develop agents, which have only desired therapeutic value and are devoid of unwanted adverse effect. Pimavanserin (ACP-103), a highly selective 5-HT2A inverse agonist, attenuates psychosis in patients with Parkinson's disease with psychosis and is devoid of extrapyramidal side effects. This dissociation is also evident from the development of anxioselective benzodiazepines devoid of habit-forming potential. Hemopressin is a peptide ligand that acts as an antagonist as well as inverse agonist. This agent acts as an antinociceptive agent in different in vivo models of pain. Treatment of obesity by drugs having inverse agonist activity at CB1/2 receptors is also underway. An exciting development is evaluation of β-blockers in chronic bronchial asthma—a condition akin to congestive heart failure where β-blockade has become the standard mode of therapy. Synthesis and evaluation of selective agents is underway. Therefore, inverse agonism is an important aspect of drug–receptor interaction and has immense untapped therapeutic potential.

Itraconazole, a Commonly Used Antifungal that Inhibits Hedgehog Pathway Activity and Cancer Growth

In a screen of drugs previously tested in humans we identified itraconazole, a systemic antifungal, as a potent antagonist of the Hedgehog (Hh) signaling pathway that acts by a mechanism distinct from its inhibitory effect on fungal sterol biosynthesis. Systemically administered itraconazole, like other Hh pathway antagonists, can suppress Hh pathway activity and the growth of medulloblastoma in a mouse allograft model and does so at serum levels comparable to those in patients undergoing antifungal therapy. Mechanistically, itraconazole appears to act on the essential Hh pathway component Smoothened (SMO) by a mechanism distinct from that of cyclopamine and other known SMO antagonists, and prevents the ciliary accumulation of SMO normally caused by Hh stimulation.

β1-Adrenoceptors in Rat Anterior Pituitary May Be Constitutively Active. Inverse Agonism of CGP 20712A on Basal 3′,5′-Cyclic Adenosine 5′-Monophosphate Levels

Catecholamines directly stimulate GH, ACTH, and prolactin secretion from rat anterior pituitary through the beta(2)-adrenoceptor (AR). We recently showed that gonadotrophs express the beta(1)-AR and that glucocorticoids drastically increase its mRNA expression level. The present investigation explores whether beta(1)-ARs are functionally coupled to adenylate cyclase. In anterior pituitary cell aggregates, the highly selective beta(1)-AR antagonists CGP 20712A and ICI 89,406-8a attenuated isoproterenol-stimulated cAMP accumulation, but no agonist action of norepinephrine could be detected. Remarkably, CGP 20712A inhibited basal cAMP levels by its own for at least 50%, an action that tended to be more effective in dexamethasone-supplemented medium. The latter effect was abolished by the beta-AR antagonist carvedilol, but not by other beta-AR antagonists. Pretreatment with pertussis toxin abolished the action of CGP 20712A on basal cAMP. CGP 20712A also attenuated isoproterenol-induced cAMP accumulation in the gonadotroph cell lines alphaT3-1 and LbetaT2, but not in the somatotroph precursor cell line GHFT and the folliculo-stellate cell line TtT/GF. However, in LbetaT2 cells CGP 20712A did not inhibit basal cAMP levels by its own. The present data suggest that beta(1)-AR in the anterior pituitary is positively coupled to adenylyl cyclase but is constitutively active in a pertussis toxin-sensitive manner. CGP 20712A may act as an inverse agonist with approximately 50% negative intrinsic activity, suggesting that the beta(1)-AR significantly contributes to basal adenylate cyclase activity in the pituitary.

Reanalysis of constitutively active rat and human 5-HT7(a) receptors in HEK-293F cells demonstrates lack of silent properties for reported neutral antagonists

The present study reinvestigated a series of 5-HT receptor antagonists at both constitutively active rat and human 5-HT7(a) receptors in HEK-293F cells using the cAMP signalling pathway as a functional read-out. Both rat and human 5-HT7(a) receptors were expressed in similar amounts ([3H]-LSD binding: 1.0 to 1.1 pmol/mg protein). Attenuation of basal cAMP formation by the inverse agonist SB-691673 (1 microM) was slightly larger by the human 5-HT7(a) (-73+/-3 %) than rat 5-HT7(a) receptor (-62+/-3 %). The 5-HT receptor antagonists investigated here displayed systematically inverse agonism. While methiothepin and SB-269970 displayed similar negative intrinsic activity to SB-691673 at the rat 5-HT7(a) receptor, the compounds SB-258719, mesulergine and metergoline displayed some lower negative intrinsic activity (between -38 and -49%). Inverse agonist properties were observed with potencies fitting with their respective binding pIC50 values and pKB values as estimated from antagonist studies with 5-HT. With the exception of SB-258719 and mesulergine, which remained a partial inverse agonist at the human 5-HT7(a) receptor, the other compounds behaved with a similar Emax value to the full inverse agonist SB-691673. In conclusion, none of the 5-HT receptor antagonists investigated displayed silent properties at the rat or human 5-HT7(a) receptor, when these are expressed in a system allowing detection of constitutive activity. They appear to be partial to full inverse agonists, further illustrating that an antagonist is preferentially an inverse agonist when investigated under constitutively active receptor conditions.

Histamine, récepteurs de l’histamine et anti-histaminiques : données récentes

Histamine was discovered at the beginning of the 20th century. It has been recently shown that immunocompetent cells (T lymphocyte, dendritic cell) are capable of histamine synthesis and that histamine can regulate the Th1/Th2 balance. This immune regulation by histamine is related to its activity as a potent polarizing factor for dendritic cells, giving rise to DC2 dendritic cells that are involved in the differentiation of T helper cells towards Th2 phenotype. The majority of histamine is metabolized by N-methyltransferase. A common polymorphism of this enzyme that is associated with decreased enzyme activity has been associated with asthma. Histamine binds to four receptor types that are expressed on different types of cells and that mediate the numerous actions of histamine. H 1-receptors exhibit constitutive activity that is inhibited by several H 1-receptor antagonists, which therefore display a negative intrinsic activity called inverse agonist activity. In addition, the cerebral P-glycoprotein-mediated efflux of recent H 1-receptor antagonists may explain the lack of nervous system side effects of second-generation anti-histamines.

Typical antipsychotics exhibit inverse agonist activity at rat dopamine D1-like receptors expressed in Sf9 cells

The baculovirus system has been used to express the rat dopamine D1 receptors in Spodoptera frugiperda (Sf9) cells. A panel of typical antipsychotics including, α-flupenthixol, fluphenazine and thioridizine were found to inhibit dopamine-dependent stimulation of adenylyl cyclase. However, these compounds were also found to inhibit adenylyl cyclase activity in the absence of agonist in Sf9 cells expressing dopamine D1-like receptors. Therefore, these nonselective dopamine receptor compounds displayed negative intrinsic or inverse agonist activity. None of the compounds tested were neutral antagonists.

The putative «silent» 5-HT 1A receptor antagonist, WAY 100635, has inverse agonist properties at cloned human 5-HT 1A receptors

Agonist binding to G protein-coupled receptors induces the formation of a receptor–G protein complex and subsequent guanosine 5′-diphosphate/guanosine 5′-triphosphate (GDP/GTP) exchange. Some receptors, however, form receptor–G protein complexes and promote GDP/GTP exchange even when not occupied by agonists. Such receptors preferentially activate pertussis toxin-sensitive G proteins (i.e., G i/G o), and the interactions of receptors and G proteins are affected by monovalent cations (most notably Na +), both in the occupied and unoccupied state. We investigated the effects of Na + on the intrinsic activity of 5-hydroxytryptamine 1A (5-HT 1A) receptor ligands, measured as maximal effect ( E MAX), using guanosine 5′-0-(3-[ 35S]thio)-triphosphate ([ 35S]GTPγS) binding to membranes prepared from human epithelioid carcinoma (HeLa) cells, expressing 500 fmol/mg protein of cloned human 5-HT 1A receptor (HA7 cells). A decrease of the NaCl concentration decreased the maximal effect of serotonin, increased basal [ 35S]GTPγS binding, and increased the negative intrinsic activity of spiperone and N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]- N-(2-pyridinyl)cyclohexanecarboxamide (WAY 100635). This ability of WAY 100635 to decrease basal [ 35S]GTPγS binding was antagonized by (s)- N- tert-butyl-3-(4-(2-methoxyphenyl)piperazine-1-yl)-2-phenylpropanamide ((s)-WAY 100135) (p A 2=7.77). Further, WAY 100635 was able to antagonize carboxamidotryptamine (5-CT)-stimulated [ 35S]GTPγS binding with a p A 2 of 9.9, in standard NaCl conditions, and of 9.7, in the absence of NaCl. Changes in membrane concentration did not affect the ability of WAY 100635 to decrease [ 35S]GTPγS binding. WAY 100635 did not affect basal [ 35S]GTPγS binding to membranes from untransfected HeLa cells. Pertussis toxin (200 ng/ml) prevented WAY 100635 and spiperone to decrease [ 35S]GTPγS binding, showing that their effects were mediated by G proteins of the G i/G o family. In conclusion, the constitutive and stimulated activity of human 5-HT 1A receptors expressed in HA7 cells is sodium-dependent, which allowed to confirm the 5-HT 1A inverse agonist properties of spiperone, and to show that WAY 100635 is an inverse agonist at 5-HT 1A receptors that inhibits basal [ 35S]GTPγS binding to a lesser extent than spiperone. [ 35S]GTPγS binding to membranes from HA7 cells under low NaCl conditions appears to be especially suitable to evidence and pharmacologically analyze the inverse agonist properties of 5-HT 1A receptor ligands.

Inverse agonism at G protein-coupled receptors: (patho)physiological relevance and implications for drug discovery.

Inverse agonism at G protein-coupled receptors: (patho)physiological relevance and implications for drug discovery.

Constitutive activity of G protein coupled receptors and drug action

Publisher Summary A new concept in G protein coupled receptor (GPCR)-mediated drug action is the occurrence of constitutive activity of GPCRs in the absence of agonist stimulation. Originally reported for the δ-opioid receptor in NG108-15 cells, this GPCR activity has now been described for a variety of GPCRs. Concomitant with the notion of agonist-independent signaling it has been realized that the constitutive GPCR activity can be inhibited by some antagonists (inverse agonists), but not by all antagonists (neutral antagonists) introducing the need for a pharmacological reclassification of GPCR antagonists. Moreover, for agonists and inverse agonist, a full spectrum of (negative) intrinsic activity from -1–0 can be observed. Neutral antagonists—that is, compounds with actually no intrinsic activity—are quite rare. Nevertheless, for some GPCRs, ligands with virtually no (negative) intrinsic activity are found. For example, the H 2 receptor has been identified as a ligand with very low intrinsic activity. Burimamide is able to block the effects of both an agonist (histamine) and an inverse agonist (cimetidine) on the cAMP levels in CHO cells expressing the H 2 receptor, with apparent pK B values that match its PK A value.

SB-236057, a selective 5-HT 1B receptor inverse agonist, blocks the 5-HT human terminal autoreceptor

A novel compound, SB-236057 (1′-ethyl-5-(2′-methyl-4′-(5-methyl-1,3,4-oxadiazol-2-yl)biphenyl-4-carbonyl)-2,3,6,7-tetrahydrospiro[furo[2,3-f]indole-3,4′-piperidine]) has been shown to have high affinity for human 5-hydroxytryptamine 1B (5-HT 1B) receptors (p K i=8.2) and displays over 75 or more-fold selectivity for the human 5-HT 1B receptor over other 5-HT receptors, including the human 5-HT 1D receptor, and a range of other receptors, ion channels and enzymes. In functional studies using [ 35 S ]GTPγS binding, SB-236057 displayed negative intrinsic activity (pEC 50=8.0) at human 5-HT 1B receptors stably expressed in Chinese Hamster Ovary (CHO) cells and caused a rightward shift of agonist concentration response curves consistent with competitive antagonism (p A 2=8.9). SB-236057 potentiated [ 3 H ]5-HT release from electrically stimulated guinea pig or human cortical slices. SB-236057 also abolished the inhibitory effect of exogenously superfused 5-HT on electrically-stimulated release from slices of the guinea pig cortex. These studies using SB-236057 confirm that, in both the guinea pig and human cerebral cortex, the terminal 5-HT autoreceptor is of the 5-HT 1B subtype.

SB-224289--a novel selective (human) 5-HT1B receptor antagonist with negative intrinsic activity.

1. Human 5-HT1B (h5-HT1B) and human 5-HT1D (h5-HT1D) receptors show remarkably similar pharmacology with few compounds discriminating the receptors. We report here on a novel compound, SB-224289 (1'-Methyl-5-[[2'-methyl-4'-(5-methyl- 1,2,4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl]-2,3,6,7-tetrahydro spiro [furo [2,3-f]indole-3,4'-piperidine] oxalate), which has high affinity for h5-HT1B receptors (pK1=8.16+/-0.06) and displays over 75 fold selectivity for the h5-HT1B receptor over all other 5-HT receptors including the h5-HT1D receptor and all other receptors tested thus far. 2. Functional activity of SB-224289 was measured in a [15S]GTPgammaS binding assay on recombinant h5-HT1B and h5-HT1D receptors expressed in Chinese Hamster Ovary (CHO) cells. SB-224289 displayed negative intrinsic activity at both receptors with higher potency at h5-HT1B receptors. SB-224289 caused a rightward shift of agonist concentration response curves consistent with competitive antagonism and generated affinities comparable with those obtained from competition radioligand receptor binding studies. 3. SB-224289 potentiated [3H]5-HT release from electrically stimulated guinea-pig cerebral cortical slices to the same extent as as the non-selective 5-HT1 antagonist methiothepin. SB-224289 also fully reversed the inhibitory effect of exogenously superfused 5-HT on electrically stimulated release. 4. Using SB-224289 as a tool compound, we confirm that in guinea-pig cerebral cortex the terminal 5-HT autoreceptor is of the 5-HT1B subtype.

The effect of beta-blocker on chronic atrial fibrilation : the difference between positive and negative intrinsic sympathomimetic activity

The effect of beta-blocker on chronic atrial fibrilation : the difference between positive and negative intrinsic sympathomimetic activity

GR127935 acts as a partial agonist at recombinant human 5-HT 1Dα and 5-HT 1Dβ receptors

In this study we have investigated the functional activity of GR127935 (2-methyl-4-(5-methyl-1,2,4 oxadiazol-3-yl)-biphenyl-[4-carboxylic acid 4-methoxy-3-(4-methyl-piperazine-1-yl)-phenyl]-amide) at human 5-HT 1Dα and 5-HT 1Dβ receptors which have been expressed in a Chinese Hamster Ovary (CHO) cell line. Using [ 35S]GTPγS binding to cell membranes as a measure of receptor-G protein coupling, GR127935 showed partial agonist activity in both 5-HT 1Dα and 5-HT 1Dβ receptor expressing cells ( E max: 29 and 31% above basal control; pEC 50: 8.6 and 9.7, respectively). GR127935 also acted as a potent antagonist at the 5-HT 1Dα (app. pA 2 8.5) and 5-HT 1Dβ (app. pA 2 9.1) receptors. From studies measuring cAMP accumulation in cultured CHO cells GR127935 also displayed partial agonism, as well as acting as a potent antagonist at the 5-HT 1Dα receptors which stimulate cAMP levels and 5-HT 1Dβ receptors which inhibit cAMP levels (app. pA 2 8.6 and 9.7, respectively). The 5H̄T 1-like receptor antagonist methiothepin showed negative intrinsic activity at both receptors in the [ 35S]GTPγS binding assay only. From studies using the receptor alkylating agent EEDQ (N-ethoxycarbonyl-2-methoxy-1,2-dihydroquinoline) the 5-HT 1Dα cell line displayed a lack of receptor reserve but it was evident in the 5-HT 1Dβ cell line. In previous studies we have also shown that agonist stimulation of 5-HT 1Dα receptors increases cAMP levels which may be due to high receptor expression. Further investigation using up to 1 μM EEDQ to reduce 5-HT 1Dα receptor number did not reveal an underlying inhibitory adenylyl cyclase response. In conclusion, GR127935 acts as a partial agonist, aswell as a potent antagonist, at the human 5-HT 1Dα and 5-HT 1Dβ receptors when expressed in CHO cells.

Specific delta-opioid antagonists exert an agonist-independent inhibitory effect, similar to the agonist, on the release of GnRH in vitro.

1. In in vitro studies with adult male rats we have recently shown that the delta-opioid agonist DTLET inhibits the release of the Gonadotropin-Releasing Hormone (GnRH) from hypothalamic fragments containing the arcuate nucleus and the median eminence. This effect is receptor mediated and eicosanoid dependent (Gerozissis et al., 1993). 2. In the present study we report that the delta-opioid antagonists with negative intrinsic activity, Diallyl-G and ICI 174864, applied under the same experimental conditions (30 min static incubations at 37 degrees C, in a potassium rich milieu), in the absence of the agonist DTLET, also exert a similar to the agonist inhibitory effect on the release of GnRH. 3. The dose-dependent inhibitory effect of Diallyl-G on GnRH release is reversed by increasing concentrations of DTLET. The mu and delta opioid antagonist, naloxone is without effect in the absence of DTLET. However, naloxone acts as an antagonist on the Diallyl-G-induced inhibition of GnRH release. 4. Diallyl-G also inhibits the release of prostaglandin E2 (PGE2). In the presence of indomethacin or nordihydroguaiaretic acid, Diallyl-G is ineffective to further inhibit the release of GnRH. These latter observations taken together with the results of eicosanoid estimation suggest that PGE2 but not leukotrienes participate in the agonist-independent effects of Diallyl-G on GnRH release. 5. Therefore these results support the hypothesis that delta-opioid antagonists with negative intrinsic activity exert agonist-independent biological responses similar to those of the agonists.

A lack of supersensitivity to opioid receptor agonists following chronic spinal opioid receptor antagonist administration in the rat

1. 1. Male Sprague-Dawley rats were chronically tested with intrathecal (i.t.) receptor selective opioid antagonists to determine if antinociceptive supersensitivity developed to selective i.t. opioid receptor agonists. 2. 2. A subcutaneously implanted osmotic minipump was used to deliver the μ-opioid receptor antagonist CTOP (0.3 nmol) or the b-opioid receptor antagonist naltrindole (5.5 nmol) for 7 days. 3. 3. Following a 24 hr washout period, rats received a single i.t. dose (ED 50) of either DAMPGO (for CTOP-treated animals) or DPDPE (for naltrindole-treated animals) and the antinociceptive effects of the agents were tested on the tail-flick test. 4. 4. Our findings revealed that chronic spinal treatment with selective opioid receptor antagonists did not induce an antinociceptive supersensitivity to selective opioid receptor agonists. 5. 5. Perhaps this lack of supersensitivity is reflective of difficulties inherent to opioid receptor antagonists that do not possess negative intrinsic activity .

Agonist induced constitutive activation of the μ opioid receptor by phosphorylation

We previously proposed that constitutive μ opioid receptor activation during prolonged agonist stimulation represents a key step in narcotic tolerance and dependence. Morphine pretreatment of SH-SY5Y cells was shown to lead to a gradual conversion of μ receptors to a constitutively active state, μ ∗, detectable with an antagonist having negative intrinsic activity (naloxone) (1). μ ∗ formation was blocked and reversed by the general protein kinase inhibitor H7, and further, H7 rapidly reversed acute morphine induced tolerance and dependence in mice (1). These results suggest that constitutive μ receptor activation occurs via phosphorylation. To test these hypothesis, we transfected the cloned μ opioid receptor (2) into HEK 293 cells. Morphine pretreatment caused extensive formation of μ ∗, as determined with cAMP measurements using naloxone and the neutral antagonist CTAP. H7 inhibited μ ∗ formation, whereas the phosphatase inhibitor calyculin A enhanced it. μ receptor phosphorylation, measured by immunoprecipitating an epitope tagged μ receptor following 32P labeling, paralleled the activity state of the receptor. These combined results strongly support the hypothesis that agonist induced phosphorylation of the μ receptor leads to its constitutive activation, rather than the commonly assumed desensitization.

Antagonists of bradykinin that stabilize a G-protein-uncoupled state of the B2 receptor act as inverse agonists in rat myometrial cells

Several B2 bradykinin (BK) receptor-specific antagonists including HOE140, NPC17731, and NPC567 exhibited negative intrinsic activity, which was observed as a decrease in basal phosphoinositide hydrolysis in primary cultures of rat myometrial cells, and this response was opposite to that elicited by the agonist BK. The order of potency of the antagonists in attenuating basal activity was essentially the same as that in competing both [3H]BK and [3H]NPC17731 for binding to B2 receptors on both intact rat myometrial cells and bovine myometrial membranes. We previously proposed a three-state model for the binding of agonists to G-protein-coupled B2 receptors in bovine myometrial membranes (Leeb-Lundberg, L. M. F. and Mathis, S. A. (1990) J. Biol. Chem. 265, 9621-9627). This model was based on the ability of BK to promote the sequential formation of three receptor binding states where formation of the third, equilibrium state was blocked by Gpp(NH)p (guanyl-5'-yl imidodiphosphate) identifying it as the G-protein-coupled state of the receptor. Here, we show that, in contrast to BK, these antagonists bound preferentially to a G-protein-uncoupled state of the receptor. These results indicate that B2 receptor antagonists that stabilize a G-protein-uncoupled state of the receptor act as inverse agonists. Furthermore, these results provide strong evidence that endogenous G-protein-coupled receptors exhibit spontaneous activity in their natural environment in the absence of agonist occupancy.

Unoccupied β-adrenoceptor-induced adenylyl cyclase stimulation in turkey erythrocyte membranes

The influence of β-adrenoceptor ligands and a β-adrenoceptor-derived peptide was studied on stimulation of adenylyl cyclase in membrane preparations of turkey erythrocytes. In the absence of receptor ligands, the hydrolysis-resistant GTP analogs, guanosine 5′-[γ-thio]triphosphate and guanosine 5′-[β,γ-imino]triphosphate, caused a slow, time-dependent increase in adenylyl cyclase activity, which was accelerated and potentiated by the additional presence of the agonist, isoproterenol. In contrast, the β-adrenoceptor antagonists, propranolol and pindolol, almost completely prevented stimulation of adenylyl cyclase by the GTP analogs alone, i.e. in the absence of an agonist, in a concentration-dependent and stereo-selective manner. This antagonist action was mimicked by a peptide corresponding to the second cytoplasmic loop of the turkey erythrocyte β-adrenoceptor. On the other hand, GTP analog-preactivated cyclase activity and enzyme stimulation by fluoride and forskolin were not or only slightly β-adrenoceptors can cause significant G s protein and subsequent adenylyl cyclase activation, and that this unoccupied receptor action can be blocked by β-adrenoceptor antagonists, thus exhibiting by themselves a negative intrinsic activity.