Stephen J. Hill

G protein-coupled-receptor cross-talk: the fine-tuning of multiple receptor-signalling pathways

Signalling via the large family of G protein-coupled receptors (GPCRs) can lead to many cellular responses, ranging from regulation of intracellular levels of cAMP to stimulation of gene transcription. Members of this receptor family have been grouped into different categories dependent on the particular G protein subtypes that they predominantly interact with. Thus, receptors that couple to GS proteins will stimulate adenylate cyclase in many cells, while Gq/11-coupled receptors can mobilize intracellular Ca2+ via activation of phospholipase C. There is accumulating evidence, however, that activation of one particular signalling pathway by a GPCR can amplify intracellular signalling within a parallel but separate pathway. In this article Lisa Selbie and Stephen Hill review some of the evidence for these synergistic interactions and suggest that they may have an important role in finetuning signals from multiple receptor signalling pathways.

G-protein-coupled receptors: past, present and future

The G‐protein‐coupled receptor (GPCR) family represents the largest and most versatile group of cell surface receptors. Drugs active at these receptors have therapeutic actions across a wide range of human diseases ranging from allergic rhinitis to pain, hypertension and schizophrenia. This review provides a brief historical overview of the properties and signalling characteristics of this important family of receptors.

Increases in intracellular calcium via activation of an endogenous P2‐purinoceptor in cultured CHO‐K1 cells

1 Increases in intracellular calcium ([Ca2+]i) were measured in Chinese hamster cultured ovary cells (clone, CHO‐K1), by use of the fluorescent, calcium‐sensitive dye, fura‐2. 2 Addition of both ATP and UTP elicited rapid increases in [Ca2+]i due to mobilization from intracellular stores and calcium entry across the plasma membrane. 3 Omission of calcium from the extracellular medium and pre‐incubation with the inorganic calcium channel blocker, nickel (Ni2+) prevented the calcium entry components of the responses. 4 Investigation of the concentration‐response relationships of various analogues of ATP suggests the presence of a purinoceptor which cannot be characterized as P2X or P2Y. In addition, there appears to be a sub‐population of P2Y‐purinoceptors which do not cross‐react with the ‘nucleotide’ receptor population. 5 Cross‐desensitization and additivity experiments suggest that both ATP and UTP activate the same receptor. 6 Pre‐incubation with the tumour‐promoting agent, β‐phorbol‐12,13 dibutyrate (PDBu), caused a reduction in the increases in [Ca2+]i, suggesting a role for protein kinase C in feedback inhibition of purinoceptor responses in this cell line. 7 In summary, we present evidence for the existence of an endogenous P2U‐purinoceptor (or ‘nucleotide receptor’) which is linked to increases in [Ca2+]i in CHO‐K1 cells.

Reporter-gene systems for the study of G-protein-coupled receptors

Reporter-gene assays offer an alternative to biochemical assays for following signal transduction pathways from receptors at the cell surface to nuclear gene transcription in living cells. Specific reporter-gene systems are now available for the study of ligand activity at G alpha(i/o), G alpha(s) and G alpha(q) G-protein-coupled receptors. In recent years reporter genes have been applied in academia and industry to the study of ligand efficacy and affinity in recombinant and primary cell lines using a variety of colour, fluorescent or luminescent read-outs.

Expression of Muscarinic M sub 3 -Receptors Coupled to Inositol Phospholipid Hydrolysis in Human Detrusor Cultured Smooth Muscle Cells

AbstractPurpose: To investigate the effect of muscarinic receptor agonists and antagonists on the accumulation of inositol phosphates in cultures of human detrusor smooth muscle cells.Materials and Methods: Primary explant culture was used to derive smooth muscle cell lines from small bladder biopsies. The cells were loaded with [sup 3 H]-myoinositol, stimulated with muscarinic agonists, and the accumulation of [sup 3 H]-inositol phosphates was measured by liquid scintillation counting.Results: Carbachol (EC50 8.3 micromolar), methacholine (EC50, 7.5 micromolar), oxotremorine (EC50 2.5 micromolar) and pilocarpine (EC sub 50 8.3 micromolar) produced concentration-dependent rises in the accumulation of total [sup 3 H]-inositol phosphates. M1 (pirenzepine), M sub 2 (methoctramine) and M3 (4-DAMP and pf-HHSiD) muscarinic receptor antagonists significantly antagonized the response induced by a submaximal concentration of carbachol (100 micromolar). The apparent pA2 values were atropine (9.4), 4-DAMP (9.2), pfH...

β -Adrenoceptor stimulation inhibits histamine-stimulated inositol phospholipid hydrolysis in bovine tracheal smooth muscle

1 Histamine and carbachol produced concentration‐related increases in the accumulation of 3Hinositol phosphates in slices of bovine tracheal smooth muscle. 2 Noradrenaline alone produced a small stimulation of 3H‐inositol phosphate accumulation which was inhibited by the α‐adrenoceptor antagonist phentolamine. In contrast, when noradrenaline (0.1 mm) was added simultaneously with histamine it significantly reduced the inositol phosphate response to high (≥0.1mm) concentrations of histamine. However, noradrenaline had no inhibitory effect on the carbachol‐induced inositol phosphate response. 3 The non‐selective β‐agonist isoprenaline (IC50 = 0.08 μm) and the β2‐selective agonist salbutamol (IC50 = 0.29 μm) both produced a dose‐related inhibition of the inositol phosphate response to 0.1 mM histamine. The inhibitory effect of salbutamol was antagonized by propranolol (KA = 2.4 × 109M−1) and the β2‐selective adrenoceptor antagonist ICI 118551 (KA = 1.7 × 109 M−1). 4 The accumulation of 3H‐inositol phosphates induced by histamine increased steadily over a 40 min period after an initial lag period of 3–4 min. Following the simultaneous addition of histamine and salbutamol there was a further delay of 3–4 min before the appearance of the inhibitory effect of salbutamol. 5 The effect of histamine on inositol phosphate accumulation was accompanied by a stimulation of [3H]‐inositol incorporation into membrane phospholipids which was reduced by the presence of salbutamol. However, when histamine was used to stimulate maximally [3H]‐inositol incorporation during the prelabelling period, salbutamol produced a marked inhibition of histamine‐stimulated 3H‐inositol phosphate accumulation under conditions in which there was no change in the level of incorporation.

Adenosine A2B‐receptor‐mediated cyclic AMP accumulation in primary rat astrocytes

1 The effects of adenosine receptor agonists and antagonists on the accumulation of cyclic AMP have been investigated in primary cultures of rat astrocytes. 2 Adenosine A2‐receptor stimulation caused a concentration‐dependent increase in the accumulation of [3H]‐cyclic AMP in cells prelabelled with [3H]‐adenine. The rank order of agonist potencies was 5′‐N‐ethylcarboxamidoadenosine (NECA; EC50 = 1 μm) > adenosine (EC50 = 5 μm) > 2‐chloroadenosine (EC50 = 20 μm) >> CGS 21680 (EC50 > 10 μm). The presence of 0.5 μm dipyridamole, an adenosine uptake blocker, had no effect on the potency of adenosine. 3 The response to 10 μm NECA was antagonized in a concentration‐dependent manner by the non‐selective adenosine receptor antagonists, xanthine amine congener (apparent KD = 12 nM), PD 115,199 (apparent KD = 134 nM) and 8‐phenyltheophylline (apparent KD = 126 nM). However, the A1‐receptor‐selective antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine, had no significant effect on the responses to NECA or 2‐chloroadenosine at concentrations up to 1 μm. 4 Stimulation of A1‐receptors with the selective agonist, N6‐cyclopentyladenosine, did not alter the basal accumulation of [3H]‐cyclic AMP but inhibited a forskolin‐mediated elevation of [3H]‐cyclic AMP accumulation by a maximal value of 42%. This inhibition was fully reversed in the presence of 0.1 μm, 8‐cyclopentyl‐1,3‐dipropylxanthine. 5 The time course for NECA‐mediated [3H]‐cyclic AMP accumulation was investigated. The results suggest that there is a substantial efflux of cyclic AMP from the cells in addition to the rapid and sustained elevation of intracellular cyclic AMP (5 fold over basal) which was also observed. 6 These data indicate that rat astrocytes in primary culture express an A2B‐adenosine receptor coupled positively to adenylyl cyclase. Furthermore, the presence of A1‐receptors negatively coupled to adenylyl cyclase appears to have no significant effect on the A2B‐receptor‐mediated cyclic AMP responses to NECA and 2‐chloroadenosine.

Inhibition of histamine‐stimulated inositol phospholipid hydrolysis by agents which increase cyclic AMP levels in bovine tracheal smooth muscle

1 The effect on histamine‐stimulated [3H]‐inositol phosphate accumulation of a range of agents which increase the accumulation, or mimic the actions, of cyclic AMP has been investigated in bovine tracheal smooth muscle. 2 Salbutamol (1 μm), forskolin (1 μm) and vasoactive intestinal peptide (VIP, 1 μm) inhibited the inositol phosphate response to 0.1 mm histamine and increased the accumulation of [3H]‐cyclic AMP in [3H]‐adenine‐labelled slices of bovine tracheal smooth muscle. The effect on inositol phospholipid hydrolysis was mimicked by the membrane permeant analogues of cyclic AMP, dibutryl‐cyclic AMP (1 mm) and 8‐bromo‐cyclic AMP (1 mm). 3 In contrast to salbutamol, which was equally effective at producing the two effects, forskolin produced large increases in [3H]‐cyclic AMP accumulation (EC50 = 1.2 μm) at much higher concentrations than those required for inhibition of histamine‐stimulated [3H]‐inositol phosphate accumulation (EC50 = 0.09 μm). However, significant increases in [3H]‐cyclic AMP accumulation, of similar magnitude to those obtained with salbutamol and VIP, were observed over the concentration range appropriate for inhibition of the inositol phosphate response to histamine. 4 In the presence of histamine (0.1 mm), isobutylmethylxanthine (IBMX, 1 mm) and rolipram (0.1 mm) both significantly (P < 0.05) elevated tissue [3H]‐cyclic AMP levels. IBMX, rolipram and (to a lesser extent) SKF 94120 significantly (P < 0.05) reduced histamine‐stimulated [3H]‐inositol phosphate accumulation by 81%, 68% and 20%, respectively. M&B 22948 was without a significant effect on either [3H]‐cyclic AMP or histamine‐induced [3H]‐inositol phosphate accumulation. 5 Both rolipram and forskolin reduced the increase in incorporation of [3H]‐inositol into membrane phospholipids which followed stimulation with histamine. However, a significant inhibition of [3H]‐inositol phosphate accumulation could be demonstrated under conditions in which there was no change in the level of [3H]‐inositol incorporation.

The growth response of Escherichia coli to neurotransmitters and related catecholamine drugs requires a functional enterobactin biosynthesis and uptake system

ABSTRACT The neurotransmitter norepinephrine (NE) stimulates the growth of low inocula of Escherichia coli in a minimal medium (SAPI) supplemented with serum (SAPI+serum) and induces the production of an “autoinducer” (AI) which, in turn, promotes E. coli growth in the absence of NE. Given the importance of NE, epinephrine, and their corresponding adrenergic agonists and antagonists in clinical medicine, we sought to investigate the molecular basis for these observations. Using a variety of NE precursors, metabolites, and therapeutic agents, we demonstrated that their ability to stimulate E. coli growth in SAPI+serum is dependent on the presence of a catechol (1,2-dihydroxybenzene) moiety with maximal activity requiring a two-carbon substituent incorporating a terminal primary amine. Serum contains the iron-binding glycoprotein, transferrin, and when SAPI+serum was supplemented with sufficient Fe3+ to saturate transferrin, growth inhibition was relieved. Other metal cations, including Mg2+, Ca2+, and Zn2+, had no effect. These data suggested that the stimulation of E. coli growth by NE in SAPI+serum may involve the catecholate siderophore, enterobactin, a cyclic triester of 2,3-dihydroxybenzoylserine. Consistent with this hypothesis, E. coli strains with mutations in ferrienterobactin transport (fepA or tonB) or enterobactin biosynthesis (entA) did not respond to NE. Furthermore, NE induced expression of the ferrienterobactin receptor, FepA, during growth in SAPI+serum. The enterobactin degradation product, 2,3-dihydroxybenzoylserine (DBS) was as effective as NE in stimulating the growth of E. coli and mutations in fepA or tonB abolished the DBS-dependent growth stimulation. In contrast to NE, however, DBS stimulated the growth of the entA mutant. Moreover, after growth in an iron-limited M9 medium in the absence of NE, ethyl acetate extracts of the E. coli entA+ parent but not of the entA mutant contained AI, i.e., stimulated the growth of E. coli in SAPI+serum. Taken together, these data show that when low numbers of E. coli are inoculated into SAPI+serum, NE, DBS, and related catecholamines induce the enterobactin iron uptake system. This, in turn, facilitates iron sequestration from transferrin and indicates that the AI present in NE-conditioned SAPI+serum medium is enterobactin and its DBS breakdown products.

Quantitative profiling of nucleotides and related phosphate-containing metabolites in cultured mammalian cells by liquid chromatography tandem electrospray mass spectrometry.

A method has been developed for the quantitative profiling of over twenty nucleotides and related phosphorylated species using ion-pair reversed-phase liquid chromatography hyphenated to negative ion tandem electrospray mass spectrometry. The influence of mobile phase pH and ion-pairing agent concentration were assessed to optimise separation and peak shapes. Full quantitative analysis was obtained for the nucleotides by reference to structurally related calibration standards. The developed method was applied to profile changes in nucleotides and related compounds in monolayer cultured Chinese hamster ovary (CHO) cells expressing the beta(2) adrenoceptor when exposed to pharmacological stimuli. These experiments demonstrate the potential of the LC-MS/MS method to detect changes in nucleotide drug targets as well as the simultaneous monitoring of levels of other nucleotides.