G-protein ßγ subunits in vasorelaxing and anti-endothelinergic effects of calcitonin gene-related peptide.

BACKGROUND AND PURPOSE: Calcitonin gene-related peptide (CGRP) has been proposed to relax vascular smooth muscle cells (VSMC) via cAMP and can promote dissociation of endothelin-1 (ET-1) from ET(A) receptors. The latter is not mimicked by other stimuli of adenylate cyclases. Therefore, we evaluated the involvement of G-protein ßγ subunits (Gßγ) in the arterial effects of CGRP receptor stimulation. EXPERIMENTAL APPROACH: To test the hypothesis that instead of α subunits of G-proteins (Gαs), Gßγ mediates the effects of CGRP receptor activation, we used (i) rat isolated mesenteric resistance arteries (MRA), (ii) pharmacological modulators of cyclic nucleotides; and (iii) low molecular weight inhibitors of the functions of Gßγ, gallein and M119. To validate these tools with respect to CGRP receptor function, we performed organ bath studies with rat isolated MRA, radioligand binding on membranes from CHO cells expressing human CGRP receptors and cAMP production assays in rat cultured VSMC. KEY RESULTS: In isolated arteries contracted with K(+) or ET-1, IBMX (PDE inhibitor) increased sodium nitroprusside (SNP)- and isoprenaline (ISO)- but not CGRP-induced relaxations. While fluorescein (negative control) was without effects, gallein increased binding of [(125) I]-CGRP in the absence and presence of GTPγS. Gallein also increased CGRP-induced cAMP production in VSMC. Despite these stimulating effects, gallein and M119 selectively inhibited the relaxing and anti-endothelinergic effects of CGRP in isolated arteries while not altering contractile responses to K(+) or ET-1 or relaxing responses to ISO or SNP. CONCLUSION AND IMPLICATIONS: Activated CGRP receptors induce cyclic nucleotide-independent relaxation of VSMC and terminate arterial effects of ET-1 via Gßγ.

LPS differentially affects vasoconstrictor responses: a potential role for RGS16?

The profound hypotension in septic shock patients is difficult to treat as it is accompanied by depressed constrictor responses to alpha1-adrenoceptor agonists. Bacterial lipopolysaccharide (LPS) is the main trigger for most of the cardiovascular alterations occurring in septic shock. In this study we investigated the effects of LPS exposure on vascular contractility in general and the role of Regulator of G protein Signalling (RGS) proteins in the LPS-induced vascular alterations. Exposure of rat aortic rings to various LPS concentrations (3, 10, 30 microg/ml) for 22 hours differentially affected agonist-induced contractile responses at four distinct G-protein coupled receptors (alpha1-adrenoceptors, angiotensin II, serotonin and endothelin-1 receptors). While the endothelin-1-induced contraction was unaffected by LPS pre-treatment, phenylephrine- and angiotensin II-induced contraction were significantly reduced whereas serotonin-induced contraction was significantly enhanced. Concomitantly, LPS treatment increased the RGS16 mRNA expression both in aortic rings and cultured vascular smooth muscle cells (VSMCs) but not that of RGS2, RGS3, RGS4 or RGS5. The significant increase in RGS16 mRNA expression in VSMCs by LPS was time- and concentration-dependent but independent of increased inducible NO synthase (iNOS) activity. The changes in RGS16 mRNA might contribute to the differential regulation of the contractile responses to vasoconstrictors upon LPS exposure.

Different response patterns of several ligands at the sphingosine-1-phosphate receptor subtype 3 (S1P(3)).

BACKGROUND AND PURPOSE: Recently, some ligands targeting the sphingosine-1-phosphate receptor subtype 3 (S1P(3)) have become available. The characterization of these compounds was mainly based on one functional read-out system, although S1P(3) receptors are known to activate different signal transduction pathways. Therefore, this study pharmacologically characterizes these compounds using different assays. EXPERIMENTAL APPROACH: Using CHO-FlpIn cells expressing the human S1P(3) receptor the potencies and maximal effects of S1P, FTY720-P, VPC23019, VPC23153 and VPC24191 were determined in three different assays [inhibition of cAMP accumulation, elevation of intracellular calcium concentrations ([Ca(2+)](i)) and S1P(3) receptor internalization]. KEY RESULTS: All compounds tested inhibited forskolin-induced cAMP accumulation, increased [Ca(2+)](i) and induced S1P(3) receptor internalization but with different potencies and maximal effects. S1P was the most potent compound in all assays followed by FTY720-P. The VPC compounds were generally less potent than S1P and FTY720-P. Regarding the maximal effects, all compounds except VPC23153, behaved as full agonists in the cAMP accumulation assay. In the calcium assay, FTY720-P, VPC23019 and VPC24191 displayed partial and VPC23153 weak partial agonist activity, relative to S1P. Interestingly, treatment with the G(i) inactivator Pertussis toxin, did not affect S1P-induced [Ca(2+)](i) elevations but inhibited those in response to the other compounds, by about 50%. CONCLUSIONS AND IMPLICATIONS: This study demonstrated differential response patterns at the S1P(3) receptor for a range of ligands. These differences could indicate the presence of functional selectivity at this receptor as FTY720-P and the VPC compounds seemed to signal predominantly via G(i)- whereas S1P activated G(i) and G(q)-coupled pathways.

BML-241 fails to display selective antagonism at the sphingosine-1-phosphate receptor, S1P(3).

BACKGROUND AND PURPOSE: The thiazolidine carboxylic acid, BML-241, has been proposed as a lead compound in development of selective antagonists at the sphingosine-1-phosphate receptor (S1P3), based on its inhibition of the rise in intracellular calcium concentrations ([Ca2+]i) in HeLa cells overexpressing S1P receptors. We have studied the antagonistic properties of BML-241 for the S1P(3) receptor in more detail. EXPERIMENTAL APPROACH: Chinese hamster ovary (CHO) cells stably transfected with the S1P3, S1P2 or alpha(1A)-adrenoceptors were used to investigate the effect of BML-241 on increases in [Ca2+]i mediated via different receptors. CHO-K1 cells were used to study ATP-induced [Ca2+]i elevations. Effects on S1P3 -mediated inhibition of forskolin-induced cAMP accumulation and on binding to alpha(1A)-adrenoceptors were also investigated. In addition, the effect of BML-241 on contractions of rat mesenteric artery induced by phenylephrine was studied in an organ bath. KEY RESULTS: High concentrations of BML-241 (10 microM) inhibited the rise in [Ca2+]i induced by S1P3 and S1P2 receptor stimulation; lower concentrations were ineffective. This high concentration of BML-241 also inhibited [Ca2+]i increases via P2 (nucleotide) receptor or alpha(1A)-adrenoceptor stimulation. Moreover, BML-241 (10 microM) inhibited alpha(1)-adrenoceptor-mediated contraction of rat mesenteric artery but did not displace [3H]-prazosin from alpha(1A)-adrenoceptors in concentrations up to 100 microM. BML-241 (10 microM) did not affect the S1P3 -mediated decrease of forskolin-induced cAMP accumulation. CONCLUSIONS AND IMPLICATIONS: We conclude that BML-241 is a low potency, non-selective inhibitor of increases in [Ca2+]i, rather than a specific antagonist at the S1P3 receptor.

Recombinant Semliki Forest virus for over-expression and pharmacological characterisation of the histamine H(2) receptor in mammalian cells.

We describe the use of recombinant Semliki Forest virus (SFV) vectors for efficient expression of the rat histamine H(2) (rH(2)) receptor in COS-7 (African green monkey kidney cells) cells. Recombinant SFV-infected COS-7 cells express the histamine rH(2) receptor in a time-dependent fashion with a maximum expression level of 50 pmol mg(-1) after 40 h. SFV-mediated histamine rH(2) receptor expression shows similar pharmacological properties as the receptor expressed transiently or stably in mammalian cells. In addition, we demonstrate the pharmacological and functional characterisation of the D(115)N mutated histamine rH(2) receptor. It has been shown that the D(115)N mutation renders the receptor constitutively active and structurally unstable. The rapid onset of and high maximal expression levels obtained from SFV-infected COS-7 cells enabled us to characterise this mutant receptor. We prove that recombinant SFV vectors are powerful tools for heterologous expression of G-protein-coupled receptors and that one can achieve both the high-level gene expression described for baculovirus-infected insect cells and the use of mammalian cells as hosts.

The effect of mutations in the DRY motif on the constitutive activity and structural instability of the histamine H(2) receptor.

In previous studies we showed that the wild-type histamine H(2) receptor stably expressed in Chinese hamster ovary cells is constitutively active. Because constitutive activity of the H(2) receptor is already found at low expression levels (300 fmol/mg protein) this receptor is a relatively unique member of the G-protein-coupled receptor (GPCR) family and a useful tool for studying GPCR activation. In this study the role of the highly conserved DRY motif in activation of the H(2) receptor was investigated. Mutation of the aspartate 115 residue in this motif resulted in H(2) receptors with high constitutive activity, increased agonist affinity, and increased signaling properties. In addition, the mutant receptors were shown to be highly structurally instable. Mutation of the arginine 116 residue in the DRY motif resulted also in a highly structurally instable receptor; expression of the receptor could only be detected after stabilization with either an agonist or inverse agonist. Moreover, the agonist affinity at the Arg-116 mutant receptors was increased, whereas the signal transduction properties of these receptors were decreased. We conclude that the Arg-116 mutant receptors can adopt an active conformation but have a decreased ability to couple to or activate the G(s)-protein. This study examines the pivotal role of the aspartate and arginine residues of the DRY motif in GPCR function. Disruption of receptor stabilizing constraints by mutation in the DRY motif leads to the formation of active GPCR conformations, but concomitantly to GPCR instability.

Agonist-independent regulation of constitutively active G-protein-coupled receptors.

G-protein-coupled receptors constitute one of the largest protein super-families in mammals. Since the cloning of the encoding genes, these important drug targets have been subjected to thorough biochemical and pharmacological studies. It has become clear that G-protein-coupled receptors not only transmit signals after stimulation by agonists but can also spontaneously couple to signal-transduction pathways. Recent findings show that constitutively active G-protein-coupled receptors can also be regulated in an agonist-independent manner, which has important implications for the interpretation of the actions of (inverse) agonists and the results of site-directed-mutagenesis studies.

Histamine inhibits the production of interleukin-12 through interaction with H2 receptors.

IL-12 is essential for T helper 1 (Th1) development and inhibits the induction of Th2 responses. Atopic diseases, which are characterized by Th2 responses, are associated with the overproduction of histamine. Here we present evidence that histamine, at physiological concentrations, strongly inhibits human IL-12 p40 and p70 mRNA and protein production by human monocytes. The use of specific histamine receptor antagonists reveals that this inhibition is mediated via the H2 receptor and induction of intracellular cAMP. The inhibition of IL-12 production is independent of IL-10 and IFN-gamma. The observation that histamine strongly reduces the production of the Th1-inducing cytokine IL-12 implies a positive feedback mechanism for the development of Th2 responses in atopic patients.

Constitutive activity and structural instability of the wild-type human H2 receptor.

Stable expression of the human H2 receptor in Chinese hamster ovary cells resulted in an increase in basal cyclic AMP (cAMP) production, which was inhibited by the inverse agonists cimetidine, famotidine, and ranitidine with potencies similar to those found for the rat H2 receptor. Burimamide, a neutral antagonist at the rat H2 receptor, behaved as a weak partial agonist at the human H2 receptor. Burimamide competitively antagonized both the histamine-induced increase in cAMP and the cimetidine-induced reduction of the basal cAMP level with apparent K(B) values that were similar to its H2 receptor affinity. Investigation of the modulation of receptor expression after long-term drug treatment revealed that at low concentrations histamine induced a significant reduction in H2 receptor expression, whereas at high concentrations receptor expression was slightly increased. The partial agonist burimamide induced, like inverse agonists, an up-regulation of the human H2 receptor after prolonged treatment. These findings suggest a structural instability of the constitutively active human H2 receptor in transfected Chinese hamster ovary cells. Occupation of the H2 receptor by any ligand reduces the instability, thus resulting in higher cellular expression levels.

From the H(2) receptor gene to reclassification of the H(2) receptor antagonists.

From previous studies it is known that long-term stimulation of the histamine H(2) receptor results in receptor downregulation. Two different pathways are involved in the downregulation process of the H(2) receptor: a cAMP-dependent and cAMP-independent agonist-dependent pathway. Recently, it became evident that in the absence of an agonist the H(2) receptor expressed in CHO cells already stimulate cAMP production, also referred to as spontaneous activity. The spontaneous activity can be inhibited by several H(2) antagonists, previously thought to act as competitive antagonists, and these antagonists are referred to as inverse agonists. Some antagonists, e.g. burimamide, are not able to inhibit the spontaneous activity and are referred to as neutral antagonists. Inverse agonism appears to be the mechanistic basis of upregulation. Only inverse agonists and not neutral antagonists induce receptor upregulation after long-term treatment as these compounds inhibit the spontaneous receptor activity and thus the basal receptor downregulation. Moreover it might also explain previously reported observations after long-term treatment of gastric ulcers, such as intragastric hyperacidity.

Modulation of forskolin-mediated adenylyl cyclase activation by constitutively active G(S)-coupled receptors.

In transfected CHO cells constitutively active histamine H2 receptors not only increase the basal cAMP level, but also enhance forskolin-induced cAMP production. The increased forskolin response was inhibited by inverse H2 agonists with potencies similar to those determined at basal levels. The modulation of the forskolin response was also observed after H2 receptor expression in HEK-293 and Sf9 cells or TSH receptor expression in COS-7 cells. The enhancement of forskolin-induced cAMP production seems to be a general characteristic of constitutively active G(S)-coupled receptors and can be very useful to study inverse agonism at wild-type receptors.

Inverse agonism of histamine H2 antagonist accounts for upregulation of spontaneously active histamine H2 receptors.

Histamine H2 receptors transfected in Chinese hamster ovary (CHO) cells are time- and dose-dependently upregulated upon exposure to the H2 antagonists cimetidine and ranitidine. This effect appears to be H2 receptor-mediated as no change in receptor density was observed after H1 or H3 antagonist treatment or after incubation with the structural analogue of cimetidine, VUF 8299, which has no H2 antagonistic effects. By using transfected CHO cells expressing different densities of wild-type H2 receptors or an uncoupled H2Leu124Ala receptor, the histamine H2 receptor was found to display considerable agonist-independent H2 receptor activity. Cimetidine and ranitidine, which both induce H2 receptor upregulation, actually functioned as inverse agonists in those cell lines displaying spontaneous agonist-independent H2 receptor activity. Burimamide, on the other hand, was shown to act as a neutral antagonist and did as expected not induce H2 receptor upregulation after long-term exposure. The displayed inverse agonism of H2 antagonists appears to be a mechanistic basis for the observed H2 antagonist-induced H2 receptor upregulation in transfected CHO cells. These observations shed new light on the pharmacological classification of the H2 antagonists and may offer a plausible explanation for the observed development of tolerance after prolonged clinical use.

Two distinct pathways for histamine H2 receptor down-regulation. H2 Leu124 --> Ala receptor mutant provides evidence for a cAMP-independent action of H2 agonists.

Pretreatment of Chinese hamster ovary cells expressing the histamine H2 receptor (CHOrH2 cells) with histamine resulted in a time-dependent (t1/2 approximately 7 h) and dose-dependent (EC50=18 nM) H2 receptor down-regulation measured as [125I]iodoaminopotentidine binding (44+/-10% down-regulation). Pretreatment of CHOrH2 cells with cholera toxin or forskolin also led to H2 receptor down-regulation. Forskolin time-dependently (t1/2 approximately 7 h) and dose-dependently (EC50 = 0.3 microM) induced H2 receptor down-regulation. Both histamine and forskolin induced rapid down-regulation of H2 receptor mRNA levels, probably caused by mRNA destabilization. Recently, Moro et al. (Moro, O. Lameh, J., Hogger, P., and Sadée, W. (1993) J. Biol. Chem. 268, 22273-22276) showed that hydrophobic amino acids in a conserved G-protein-coupled receptor motif in the second intracellular loop are implicated in G-protein coupling. To uncouple the H2 receptor from the Gs-protein, we introduced the Leu124 --> Ala mutation in the second intracellular loop of the H2 receptor. The H2 Leu124 --> Ala mutant showed altered agonist-binding parameters, attenuated histamine-induced cAMP production, and was down-regulated by concentrations of histamine that did not give rise to cAMP production. Taken together, in CHOrH2 cells, H2 receptor down-regulation appears to be induced by two distinct pathways, a cAMP-dependent and cAMP-independent pathway.

Visualization of agonist-induced internalization of histamine H2 receptors.

Histamine H2 receptors were tagged at the N-terminus with the eight amino acid Flag epitope to allow the immunological identification of the receptor peptide with the monoclonal anti-Flag M2 antibody. The introduction of the epitope did not modify the binding of several H2 ligands to the H2 receptor, nor the ability of histamine to stimulate the H2 receptor mediated cAMP production in HEK-293 cells. Western blots revealed a major protein band of 57 +/- 1 kDa, whereas a second band of 31 +/- 1 kDa was probably the result of a proteolytic breakdown of the 57 kDa band. Immunofluorescence measurements of stably transfected HEK-293 cells revealed the presence of anti-Flag-immunoreactivity in the plasma membrane. This immunoreactivity completely disappeared after a one hour treatment with histamine. The receptor internalization was reversible and blocked by the endocytosis inhibitor phenylarsine oxide. Forskolin did not induce H2 receptor internalization, indicating that histamine causes H2 receptor internalization via a cAMP-independent pathway.