Biomedical subjects

S Rees

Publications and source records attributed to S Rees.

At least 55 records · Page 3Linked to original sources

PubMed1998

Human metabotropic glutamate receptor 2 couples to the MAP kinase cascade in chinese hamster ovary cells.

We have examined the functional coupling of the human metabotropic glutamate receptor type 2 (mGluR2) with the regulation of the mitogen activated protein kinase (MAP kinase) signal transduction cascade. We demonstrated that L-glutamate stimulation of the human mGluR2 receptor transiently expressed in chinese hamster ovary (CHO) cells leads to a rapid increase in the activity of p42/p44 MAP kinase (also known as the extracellular signal regulated kinases, ERK1 and ERK2). Activation of p42/p44 MAP kinase has been demonstrated in a peptide phosphorylation assay and through the demonstration of a shift in electrophoretic mobility of p42 MAP kinase following activation. In both assay systems L-glutamate stimulation of MAP kinase was inhibited by pertussis toxin and by the MEK (MAP/ERK activating kinase) inhibitor PD 98059. We conclude that L-glutamate stimulation of the mGluR2 receptor in CHO cells mediated regulation of p42/p44 MAP kinase following the activation of pertussis toxin-sensitive G alpha(i) G-proteins via a distinct protein kinase signalling pathway that utilizes MEK.

Animals↗

PubMed1998

Immunohistochemical localisation of mGluR7 protein in the rodent and human cerebellar cortex using subtype specific antibodies.

Metabotropic glutamate receptors (mGluRs) are a heterogeneous family of G protein coupled receptors that are linked to multiple second messenger systems to regulate neuronal excitability and synaptic transmission. To characterise the protein expression of the two mGluR7 receptor splice variants in human and rat cerebellar cortex, antibodies specific to mGluR7 were generated. Antibodies were raised against a glutathione-S-transferase fusion protein containing amino acid residues located in the extracellular domain common to both the human and rat mGluR7 splice variants. These antibodies specifically detected human mGluR7a in mammalian cells transfected with this receptor. In agreement with mGluR7 in situ hybridisation studies, immunohistochemistry performed at the light microscope level revealed that mGluR7 protein expression occurred most prominently in a particular population of nerve cells common to both the human and rat, located within the cerebellar cortex of gray matter contained within transverse folia. Moreover, strong mGluR7-like immunoreactivity was seen in Purkinje cell body cytoplasm of the Purkinje cell layer. In the most superficial cerebellar cortical layer, the molecular layer, immunostaining was observed in Purkinje cell associated proximal and distal dendritic trees. No detectable labelling was evident in intrinsic deep cerebellar nuclei known to contain GABAergic terminals of projecting Purkinje cell axons. These data are suggestive of a post-synaptic location of mGluR7 in this central nervous system structure. In the rodent, additional non-Purkinje cells thought to represent inhibitory interneurones were labelled at all levels in the molecular layer. mGluR7-like immunoreactivity was not associated with glial cells.

Animals↗

PubMed1998

Quantitative analysis of a cysteine351glycine mutation in the G protein Gi1alpha: effect on alpha2A-adrenoceptor-Gi1alpha fusion protein activation.

Fusion proteins were constructed between the porcine alpha2A-adrenoceptor and either wild-type (Cys351) or a pertussis toxin-resistant (Gly351) form of the G protein Gi1alpha. Addition of adrenaline to membranes expressing the fusion proteins resulted in concentration-dependent stimulation of their high affinity GTPase activity. The alpha2A-adrenoceptor-wild type Gi1alpha fusion protein produced substantially higher maximal stimulation of GTPase activity in response to adrenaline than that containing Gly351 Gi1alpha. Treatment of the fusion proteins as agonist-regulated enzymes allowed measurement of Vmax and turnover number for adrenaline-stimulation of the GTPase activity of each fusion construct. The turnover number of the alpha2A-adrenoceptor-Cys351 Gly Gi1alpha fusion protein was only 44'S, of that for the alpha2A-adrenoceptor-wild type Gi1alpha fusion protein. These data provide the first direct quantitative evaluation of the effects of a mutation of a G protein on the capacity of an agonist-occupied receptor to activate the mutant.

Animals↗

PubMed1998

Physiological and induced neuronal death are not affected in NSE-bax transgenic mice.

Bax, a family member of the survival protein Bcl-2, is expressed in the nervous system during development and throughout adulthood. Bax deficiency has been demonstrated to prevent developmental and trophic factor deprivation-induced neuronal death. To further clarify the role of Bax in naturally occurring neuronal death and in neuronal death following apoptotic stimuli, we generated several lines of transgenic mice expressing the human Bax protein specifically in neurons, under the control of the neuron-specific enolase promoter. Transgene expression was first detected around E10.5 and E12.5, depending on the transgenic line. The total number of ganglion cells in the retina and of pyramidal cells in the hippocampus, both expressing the transgene, was similar in control and transgenic mice. In addition, in our model system, Bax overexpression did not appear to influence the in vitro survival of sensory neurons isolated from dorsal root ganglia after nerve grwoth factor (NFG) deprivation or the apoptotic death of motor neurons following axotomy.

Animals↗

PubMed1998

Agonist occupation of an alpha2A-adrenoreceptor-Gi1alpha fusion protein results in activation of both receptor-linked and endogenous Gi proteins. Comparisons of their contributions to GTPase activity and signal transduction and analysis of receptor-G protein activation stoichiometry.

A fusion protein between a pertussis toxin-resistant (C351G) mutant of the alpha subunit of the G protein Gi1 and the porcine alpha2A-adrenoreceptor was stably expressed in Rat 1 fibroblasts. Agonists caused stimulation of high affinity GTPase activity, which was partially prevented by pertussis toxin treatment, demonstrating that the toxin-resistant component of the GTPase activity was derived from the receptor-fused G protein and the remainder from endogenous Gialpha. Half-maximal stimulation of the GTPase activity of endogenous Gi was achieved with lower concentrations of agonist. Although the Km for GTP of the fusion protein-linked Gi was lower than for the endogenous G protein, Vmax measurements demonstrated that adrenaline activated some 5 mol of endogenous Gi/mol of fusion protein-linked Gi. The isolated alpha2A-adrenoreceptor could activate Gs; however, the fusion protein did not. Compared with adrenaline, the efficacy of a range of partial agonists to stimulate endogenous Gialpha was greater than for the fusion protein-constrained C351G Gi1alpha. alpha2A-Adrenoreceptor agonists could stimulate both p44 mitogen-activated protein kinase and p70 S6 kinase and inhibit forskolin-amplified adenylyl cyclase activity in untreated alpha2A-adrenoreceptor-C351G Gi1alpha fusion protein-expressing cells, but these signals were abolished following pertussis toxin treatment. These results demonstrate conclusively, and for the first time, that agonist occupancy of a receptor-G protein fusion protein can result in activation of G proteins other than that physically linked to the receptor. This was selective between G protein classes. Analysis of the contributions of fusion protein-linked and endogenous G proteins to agonist-stimulated GTPase activity provided a direct and original measure of receptor-G protein activation stoichiometry.

Adrenergic alpha-2 Receptor Agonists↗

PubMed1998

Structural studies of Impatiens balsamina antimicrobial protein (Ib-AMP1).

Structural studies of Ib-AMP1, a small antimicrobial peptide derived from the seeds of Impatiens balsamina have been performed using circular dichroism (CD) and two-dimensional proton nuclear magnetic resonance (1H NMR). This 20-residue peptide is highly basic with five arginine residues and contains four cysteines which form two intramolecular disulfide bonds. CD results reveal that the peptide may include a beta-turn but do not show evidence for either helical or beta-sheet structure over a range of temperature and pH. Structural information from NMR was obtained in the form of proton-proton internuclear distances inferred from NOEs and dihedral angle restraints from spin-spin coupling constants, which were used for distance geometry calculations. Owing to the difficulty in obtaining the correct disulfide connectivities by chemical methods, three separate calculations were performed; with no disulfides and with the two possible alternate disulfide connectivities. Results from distance geometry calculations reveal that although the peptide is small, the cysteines constrain part of it to adopt a well-defined main chain conformation. From residue 6 to 20, the backbone is well defined, whilst the N-terminal region, residues 1-5, has very few constraints and appears to be very flexible. In the defined core region, there are three beta-turns at residues 9-12, 10-13, and 12-15. The side chains show no strong interactions in the NMR spectra and are therefore thought to adopt multiple conformations. Superposition of the structures generated shows that the peptide has two hydrophilic patches which are at opposite ends of the molecule separated by a large hydrophobic patch. Little is known about the mode of action of this protein, but it is thought to interact with a membrane-bound receptor, and possible sites of interaction are discussed. The structures determined are compared with those of the alpha-conotoxins, which are also highly basic proteins with similar disulfide connectivities.

Amino Acid Sequence↗

PubMed1998

Depletion of nitric oxide synthase-containing neurons in the diabetic retina: reversal by aminoguanidine.

A close association of neuronal nitric oxide synthase-immunoreactive (nNOS-IR) neurons with the retinal vasculature has been reported and it is proposed that activation of these neurons could be the mechanism by which retinal blood flow and metabolism are linked. Further, advanced glycation end products (AGEs) have previously been shown to be increased in the diabetic retina and aminoguanidine (AG), an inhibitor of advanced glycation, has been shown to attenuate the development of AGE accumulation as well as the progression of experimental diabetic retinopathy. This study examined the effects of short (1 and 3 weeks) and long term (32 weeks) diabetes on nNOS-containing neurons of the retina using NADPH diaphorase (NADPHd) histochemistry. In addition, the effect of aminoguanidine (an inhibitor of advanced glycation and NOS) and NG-nitro-L-arginine methyl ester (L-NAME) (a non-selective NOS inhibitor) on retinal nNOS-containing neurons was examined in short and long term control and diabetic rats. In a separate study, the effect of 2,3 diamino-phenazine (NN0028) (an inhibitor of advanced glycation, but not NOS) was examined in short term (3 weeks) diabetic rats. The number of NADPHd-positive neurons per retina was reduced after one week of diabetes and remained decreased in long term diabetic rats, an effect not observed in diabetic rats rendered euglycaemic by intensified insulin treatment. Treatment of diabetic animals with aminoguanidine or NN0028 prevented the depletion in the nNOS-containing neuron number, an effect not reproduced by L-NAME. These studies suggest that the action of AG in restoring the number of nNOS-containing retinal neurons is mediated by the inhibition of AGE formation. The depletion of nNOS-containing neurons may contribute to alterations in the autoregulation of blood flow which occurs in diabetes.

Animals↗

PubMed1998

Fetal brain injury following prolonged hypoxemia and placental insufficiency: a review.

It is well-established that severe, acute episodes of hypoxemia can damage the brain before birth, but the effects of more sustained hypoxemia are less well understood. We have used fetal fetal sheep in a series of studies aimed at determining the effects of prolonged hypoxemia, induced by placental insufficiency of differing severity and duration, on fetal brain structure. Restriction of placental, and hence fetal, growth by carunclectomy caused impaired development of neural processes and connections in the hippocampus, cerebellum, and visual cortex; neuronal migration and neuronal numbers did not appear to be affected. Twenty days of placental insufficiency during late gestation induced by umbilicoplacental embolisation also caused abnormalities in brain structure; the cerebellum, which develops late in gestation, was particularly affected. In the cortex, there was evidence of white matter lesions, an increase in the size of capillaries and a proliferation of astroglia. We also examined the effects of shorter periods of hypoxemia (6-12 hr) near mid-gestation on brain structure; fetuses were allowed to recover for 7 or 35 days after the hypoxemic challenge. The major changes were mild focal damage in the cortical white matter, a reduction in the number of Purkinje cells, a delay in the growth of neural processes in the cerebellum and proliferation of blood vessels. The hippocampus was also affected, in particular the areal density of pyramidal cells was reduced. The use of several classes of pharmacological agents with the potential to protect neurons from hypoxemic injury is discussed in relation to the developing brain.

Animals↗

PubMed1998

Mammalian expression of transmembrane receptors for pharmaceutical applications.

Three mammalian expression systems suitable for expressing recombinant receptors have been described. Each is suited to a different aspect of the study of receptors and their behaviour. IRES-based vectors are ideal for creating stable mammalian cell lines suitable for screening receptors using a signalling readout. Unlike traditional vectors they result in almost 100% of cell lines generated expressing a particular receptor, thus increasing the efficiency of cell line generation and increasing the chance of higher expression-level cell lines being generated. They may also be utilized to express more than one protein of interest, for example it is possible to co-express a particular receptor with a particular signalling protein or trafficking protein from a single RNA, thus ensuring that both are expressed simultaneously in the same cell. The ecdysone-inducible expression system is ideal for studying receptor signalling and behaviour. It is possible to alter receptor expression levels in an identical cellular background thus making it possible to study phenomena such as constitutive receptor activity in the absence of agonist. The SFV expression system is ideal for expressing receptors at high levels of a mammalian cell. It is thus a good system for purifying receptors for structural analysis and for providing material for binding assays. All of the expression systems described above have been demonstrated to express seven-transmembrane receptors with the expected pharmacological and functional profile.

Animals↗

PubMed1998

A controlled study of gold contact hypersensitivity.

1203 patients attending for routine patch testing at 3 hospitals and 105 volunteers were tested with 0.5% and 0.05% gold sodium thiosulfate (GST). 38 patients (3.2%) and 5 volunteers (4.8%) had positive patch tests to GST. There were no significant differences between volunteers and patients with respect to age, sex, atopy or exposure to gold in dental restorations, jewellery or through occupation. There were no significant differences in prevalence of GST hypersensitivity in the 3 hospitals, or between patients and controls. This is the 1st controlled study of hypersensitivity to GST, and suggests that routine patch testing to gold is of limited clinical benefit.

Adolescent↗

PubMed1998

Calcium- and hydroxyapatite-binding properties of glucuronic acid-rich and iduronic acid-rich glycosaminoglycans and proteoglycans.

This study describes the interaction of a small chondroitin sulphate proteoglycan and the glycosaminoglycans chondroitin 4-sulphate, dermatan sulphate and heparan sulphate with hydroxyapatite. All macromolecules possessed a high affinity, with the iduronic acid-rich dermatan sulphate and heparan sulphate displaying higher adsorption maxima than the glucuronic acid-rich chondroitin 4-sulphate. At similar concentrations, dermatan sulphate produced a 30% inhibition of hydroxyapatite-induced crystal growth, whilst chondroitin 4-sulphate yielded 50% inhibition. Estimation of the calcium binding capacity of these glycosaminoglycans using equilibrium dialysis indicated that chondroitin 4-sulphate bound five times more calcium than dermatan sulphate at a calcium concentration similar to that of serum. The data indicate a possible important role for chondroitin 4-sulphate in dentinogenesis where it is the dominant glycosaminoglycan, since it could act as a capture point for calcium ions during mineralisation, with the leucine-rich domain of its parent proteoglycan acting as anchor points to type I collagen.

Adsorption↗

PubMed1998

Selective activation of a chimeric Gi1/Gs G protein alpha subunit by the human IP prostanoid receptor: analysis using agonist stimulation of high affinity GTPase activity and [35S]guanosine-5'-O-(3-thio)triphosphate binding.

A FLAG-tagged form of the human IP prostanoid receptor was expressed stably in HEK 293 cells. This bound [3H]iloprost with high affinity and stimulated cAMP production when exposed to agonist. Iloprost produced weak stimulation of GTPase activity and [35S]guanosine-5'-O-(3-thio)triphosphate binding in membranes of these cells. Pretreatment of cells with pertussis toxin did not modify iloprost-mediated stimulation, but this was blocked by cholera toxin. The effects of iloprost were not increased by coexpression of either Gsalpha or Gi1alpha. In contrast, coexpression of a chimeric G protein alpha subunit in which the carboxyl-terminal six amino acids of Gi1alpha were altered to those of Gsalpha resulted in robust stimulation by iloprost. Because the chimeric G protein alpha subunit (Gi1/Gs6alpha) is not a substrate for either pertussis or cholera toxin, pretreatment of cells coexpressing the IP prostanoid receptor and Gi1/Gs6alpha with a mixture of these toxins resulted in resolution of the signal derived from activation of the chimeric G protein. Agonist-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate binding and GTPase activity assays are the most commonly used strategies to examine interactions between G protein-coupled receptors and G proteins. These usually are not appropriate for receptors such as the IP prostanoid receptor that interact with G proteins with low rates of guanine nucleotide exchange and hydrolysis. Chimeric G proteins such as Gi1/Gs6alpha that allow appropriate receptor contacts to be converted to the higher nucleotide turnover rates typical of the Gi family G proteins can overcome this and offer a novel means to examine agonist function at such receptors.

Cell Line↗

PubMed1998

Effects of chronic placental insufficiency on brain development in fetal sheep.

Clinical evidence has linked intrauterine compromise such as fetal hypoxemia to poor neurologic outcome in the newborn. In this study we examined the effects of inducing chronic fetal hypoxemia by impairment of placental function on brain development in fetal sheep. Placental insufficiency was induced from 120 to 140 d of gestation (term = 145-148 d) by injection of microspheres into the umbilical circulation in five fetal sheep. Fetal partial pressure of oxygen, PaO2, was reduced from 24.1 +/- 0.5 mm Hg before embolization to 14.8 +/- 0.4 mm Hg after embolization (p < 0.05). In another three fetuses a similar level of hypoxemia (PaO2, 13.8 +/- 0.4 mm Hg) occurred spontaneously. At 140 d of gestation the fetal brains were perfused with fixatives and compared with five control fetuses for the assessment of structural and immunohistochemical alterations. Hypoxemic fetuses demonstrated severe gliosis in the cerebral cortex and reduced myelination of subcortical white matter as visualized by glial fibrillary acidic protein and myelin basic protein staining, respectively (p < 0.05). White matter lesions were observed in two fetuses. The diameter of cerebral capillaries was increased in hypoxemic fetuses (p < 0.05), but there was no change in the number of nitric oxide synthase immunoreactive cells. Growth of neuronal processes was affected in the cerebellum, where there was also a reduction in the number of Purkinje neurons (p < 0.05). These results show that a prolonged period of placental insufficiency, resulting in moderate fetal hypoxemia during the last third of gestation, can affect neurodevelopmental processes that occur late in gestation such as myelination and growth of the cerebellum. This prenatal damage could affect neural connectivity and have functional consequences after birth.

Animals↗

PubMed1998

A novel and efficient method for the stable expression of heteromeric ion channels in mammalian cells.

The 5' untranslated leader sequence from the encephalomyocarditis virus was used to engineer bicistronic or tricistronic expression vectors encoding two subunits (P2X2 and P2X3) of an ATP-gated cation channel. Human embryonic kidney (293) and chinese hamster ovary (CHO-K1) cells were transfected with the vector, and stable cell lines were generated by single cell subcloning. Selection was made in a 96-well format on the basis of a sustained increase in intracellular calcium (fluorescence of Fluo3-loaded cells) evoked by the ATP analog alpha beta methylene ATP. A high proportion of transformants expressed heteromeric receptors containing both P2X2 and P2X3 subunits, as evidenced by a nondesensitizing current in response to alpha beta methylene ATP. The method is fast and simple and could be generally useful for the stable expression of heteromultimeric channel proteins.

Adenosine Triphosphate↗

PubMed1997

Ageing has a differential effect on nitric oxide synthase-containing and catecholaminergic amacrine cells in the human and rat retina.

In this study, we assessed the effects of normal ageing on the number, distribution, and somal area of nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-positive (NADPHd+) and tyrosine hydroxylase-immunoreactive (TH-IR) amacrine cells in human and rat retina. By using a double-labelling immunohistochemical technique, we have shown that these two enzymes are located in separate amacrine cell populations in the human retina. In normal human retinas from organ donors, we have shown that there was no change in the number, somal area, or retinal distribution of NADPHd+ neurons over an age range of 19-89 years. In contrast, there was a significant decrease (P < 0.05) of 52% in the total number of TH-IR neurons in the group aged 65-89 years compared with the group aged 19-64 years. CA1 and CA2 TH-IR neurons were reduced by 44% and 55%, respectively. In young (3 months) and old (2 years) rats, the number of NADPHd+ neurons did not decrease with ageing, but the number of TH-IR neurons was significantly reduced by 21% (P < 0.05). In a companion study on monkey retina, we have shown that a postmortem delay of 12.5 hours between death and fixation results in a decrease of 33% in the number of both NADPHd+ and TH-IR neurons in the retina compared with the number in retinas fixed immediately after death. The findings of this study on the two subsets of amacrine cells, therefore, are likely to demonstrate the consequences of ageing in the retina and might contribute to visual impairment in the elderly.

Adult↗

PubMed1997

The vulnerability of the fetal sheep brain to hypoxemia at mid-gestation.

Our aim was to test the hypothesis that a brief episode of hypoxemia near mid-gestation in fetal sheep will result in damage to the fetal brain with the extent and type of damage in any particular region being related to the developmental processes occurring at the time of the insult. Hypoxemia was induced, sufficient to reduce arterial O2 content by approximately 50%, by restricting utero-placental blood flow in 14 chronically catheterised fetuses for 6 h or 12 h at 84 days of gestation (term 145-8 days). Age-matched fetuses (n = 14; 4 operated and 10 unoperated) were used as controls. Fetuses were killed 7 days after being exposed to hypoxemia, and brains removed for histological analysis at the light and ultrastructural levels. Body weights of hypoxemic fetuses did not differ significantly from controls but brain weights were significantly reduced both in absolute terms and when expressed in relation to body weight (P < 0.05). Most fetuses exposed to hypoxemia sustained no gross brain damage. However, in one hypoxemic fetus from a multiple pregnancy there was extensive leucomalacia in the cortical white matter; mild focal damage was seen in another 8 hypoxemic fetuses. In the cerebral cortex (frontal lobe) the surface folding index was significantly reduced (P < 0.05) in hypoxemic fetuses compared to controls suggesting that gyral formation had been delayed. In these fetuses there were also degenerating neurons in the deeper cortical layers. In the hippocampus of hypoxemic fetuses there was a delay (P < 0.05), compared to controls, in the migration of cells from the germinal layer to the pyramidal layer in the CA1 region, and decreases (P < 0.05) in the density (area1) of neurons in the pyramidal layer and in the width of stratum oriens. In the cerebellum of hypoxemic fetuses there was a decrease (P < 0.05), compared to controls, in the density (area1) of mitotic bodies in the external granule cell layer. However, there were no significant differences in the number of pyknotic cells in this layer, in the density of Purkinje cells, in their somal area, or in the width of the external granule cell or molecular layers. There was an increase (P < 0.05) in the proportion of the brain parenchyma occupied by blood vessels in both the hippocampus and cortex of hypoxemic fetuses compared to controls. This study has shown that an hypoxemic insult near mid-gestation can result, one week later, in white matter damage and in neuronal death in the hippocampus and to a lesser extent in the cerebral cortex and cerebellum. It can also retard neuronal migration and the growth of neural processes in the hippocampus where development is well established at this age. Such brain damage could result in less than optimal neuronal connectivity and could affect function postnatally.

Animals↗

PubMed1997

Localization of mGluR4 protein in the rat cerebral cortex and hippocampus.

The cellular distribution of the rat metabotropic glutamate receptor type 4 (mGluR4) was examined in the adult rat cerebral cortex and hippocampus. Antibodies were raised against amino acid residues located in the extracellular amino terminal domain that is common to both the mGluR4a and mGluR4b splice variants, and used for an immunohistochemical investigation. The affinity-purified antibodies on immunoblot analysis specifically detected mGluR4 protein in transfected mammalian cells, showing no cross-reactivity with other members of the mGluR family. At the light microscope level intense mGluR4-like immunoreactivity was detected in the CA2 region of Ammon's horn in the hippocampus. In the cerebral cortex numerous non-pyramidal cells were strongly immunolabelled.

Animals↗

PubMed1997

A bioluminescent assay for agonist activity at potentially any G-protein-coupled receptor.

Transient expression of apoaequorin in Chinese hamster ovary (CHO) cells and reconstitution with the co-factor coelenterazine resulted in a large, concentration-dependent agonist-mediated luminescent response following cotransfection with the endothelin ETA, angiotensin ATII, thyrotropin-releasing hormone (TRH), and neurokinin NK1 receptors, all of which interact pre-dominantly with the G alpha q-like phosphoinositidase-linked G-proteins. A substantially greater luminescence was obtained with mitochondrially targeted apoaequorin compared to cytoplasmically expressed apoaequorin. To generate a system amenable for the study of agonist activity at virtually any G-protein-coupled receptor the alpha subunit of the receptor promiscuous G-protein G alpha 16 was either transiently or stably expressed in CHO cells together with apoaequorin. In cells expressing G alpha 16, but not in its absence, agonists at a series of receptors which normally interact with either G alpha s or G alpha i were now able to cause a luminescent response from mitochondrially targeted apoaequorin. In the case of the A1 adenosine receptor, this response was clearly a result of activation of G alpha 16 and not a consequence of the release of the G alpha i-associated beta/gamma complex, as the luminescent response was unaffected by pertussis toxin treatment of the cells, whereas agonist-mediated inhibition of adenylyl cyclase activity was attenuated. These studies describe the use of coexpressed apoaequorin as a reporter for G-protein-coupled receptor-mediated calcium signaling. Furthermore, coexpression of G alpha 16 and apoaequorin provides a basis for a generic mammalian cell microplate assay for the assessment of agonist action at virtually any G-protein-coupled receptor, including orphan receptors for which the physiological signal transduction mechanism may be unknown.

Adenosine-5'-(N-ethylcarboxamide)↗