Receptor isoforms mediate opposing proliferative effects through gbetagamma-activated p38 or Akt pathways.

The opposing effects on proliferation mediated by G-protein-coupled receptor isoforms differing in their COOH termini could be correlated with the abilities of the receptors to differentially activate p38, implicated in apoptotic events, or phosphatidylinositol 3-kinase (PI 3-K), which provides a source of survival signals. These contrasting growth responses of the somatostatin sst(2) receptor isoforms, which couple to identical Galpha subunit pools (Galpha(i3) > Galpha(i2) >> Galpha(0)), were both inhibited following betagamma sequestration. The sst(2(a)) receptor-mediated ATF-2 activation and inhibition of proliferation induced by basic fibroblast growth factor (bFGF) were dependent on prolonged phosphorylation of p38. In contrast, cell proliferation and the associated transient phosphorylation of Akt and p70(rsk) induced by sst(2(b)) receptors were blocked by the PI 3-K inhibitor LY 294002. Stimulation with bFGF alone had no effect on the activity of either p38 or Akt but markedly enhanced p38 phosphorylation mediated by sst(2(a)) receptors, suggesting that a complex interplay exists between the transduction cascades activated by these distinct receptor types. In addition, although all receptors mediated a sustained activation of extracellular signal-regulated kinases (ERK1 and ERK2), induction of the tumor suppressor p21(cip1) was detected only following amplification of ERK and p38 phosphorylation by concomitant bFGF and sst(2(a)) receptor activation. Expression of constitutively active Akt in the presence of a p38 inhibitor enabled a proliferative response to be detected in sst(2(a)) receptor-expressing cells. These findings demonstrate that the duration of activation and a critical balance between the mitogen-activated protein kinase and PI 3-K pathways are important for controlling cell proliferation and that the COOH termini of the sst(2) receptor isoforms may determine the selection of appropriate betagamma-pairings necessary for interaction with distinct kinase cascades.

Adenosine A3 receptors mediate hypotension in the angiotensin II-supported circulation of the pithed rat.

The cardiovascular effects of N6-2-(4-aminophenyl)ethyladenosine (APNEA), which when radiolabelled with 125I shows high affinity for the newly described adenosine A3 receptor, have been investigated in the angiotensin II-supported circulation of the pithed rat. APNEA induces hypotensive responses which are unaffected by high doses (20-40 mg kg-1) of the broad spectrum, adenosine receptor antagonist, 8-(p-sulphophenyl)theophylline (8-SPT). 8-SPT-resistant falls in blood pressure are also seen, in the absence of bradycardia, with 5'-N-ethylcarboxamidoadenosine (NECA) and the R- and S-enantiomers of N6-phenylisopropyladenosine (PIA). Xanthine insensitivity, high potencies of APNEA, NECA and R-PIA, and an enantiomeric selectivity favouring R- over S-PIA are distinguishing features of the adenosine A3 receptor. We suggest that hypotension in the pithed rat may be a functional correlate of this site.

Pharmacological characterization of type 1alpha metabotropic glutamate receptor-stimulated [35S]-GTPgammaS binding.

1. The activation of G proteins by type 1alpha metabotropic glutamate receptors (mGluRs) in membranes from recombinant baby hamster kidney cells expressing the cloned rat mGluR1alpha receptor has been studied by use of a [35S]-guanosine 5'-[gamma-thio]triphosphate ([35S]-GTPgammaS) binding assay. 2. L-Glutamate increased the rate of [35S]-GTPgammaS binding in a concentration-dependent manner (-logEC50 (M) 5.25 +/- 0.07), with an optimal (62.4 +/- 1.6%) increase over basal binding being observed following 60 min incubation at 30 degrees C with 70 pM [35S]-GTPgammaS, 1 microM GDP, 10 mM MgCl2, 100 mM NaCl and 100 microg membrane protein ml(-1). The L-glutamate (100 microM)-stimulated increase in [35S]-GTPgammaS binding was totally prevented in the presence of the group I mGluR antagonist (S)-4-carboxy-3-hydroxyphenylglycine (300 microM). 3. Quantitative analysis of the affinity and number of G proteins activated by a maximally effective concentration of L-glutamate revealed an equilibrium dissociation constant (K(D)) for [35S]-GTPgammaS binding of 0.76 +/- 0.20 nM and a maximal number of GTPgammaS-liganded G proteins (Bmax) of 361 +/- 30 fmol mg(-1) protein. 4. Metabotropic glutamate receptor agonists, quisqualate (-logEC50 (M) 6.74 +/- 0.06), 1S,3R-ACPD (4.64 +/- 0.08) and (S)-3,5-dihydroxyphenylglycine (5.16 +/- 0.23) also increased [35S]-GTPgammaS binding in a concentration-dependent manner, with the latter two agents behaving as partial agonists. 5. (+)-alpha-Methylcarboxyphenylglycine (300 microM) caused a parallel rightward shift of the L-glutamate concentration-effect curve for [35S]-GTPgammaS binding, allowing an antagonist equilibrium dissociation constant (K(D)) of 34.0 +/- 7.8 microM to be calculated for this mGluR antagonist. 6. Pretreatment of BHK-mGluR1alpha cells with a concentration of pertussis toxin (PTX) shown to be maximally effective (100 ng ml(-1), 24 h) before membrane preparation resulted in a marked decrease in agonist-stimulated [35S]-GTPgammaS binding (by 66.0 +/- 0.9%), and an altered concentration-effect relationship for agonist-stimulated [35S]-GTPgammaS binding by the residual PTX-insensitive G-protein population. 7. The modulation of [35S]-GTPgammaS binding by agonists and antagonists in membranes from recombinant cells provides an excellent system in which to study mGluR interactions with PTX-sensitive and -insensitive G proteins.

Activation of adenylate cyclase by human recombinant sst5 receptors expressed in CHO-K1 cells and involvement of Galphas proteins.

1. The coupling of the human somatostatin sst5 receptor recombinantly expressed in Chinese hamster ovary (CHO-K1) cells to adenylate cyclase was investigated using receptor selective ligands. 2. Forskolin (10 microM)-stimulated adenosine 3': 5'-cyclic monophosphate (cyclic AMP) accumulation was inhibited by somatostatin-14 and a number of receptor-selective agonists with a rank order of agonist potency typical of the sst5 receptor. L-362,855 and BIM-23056 behaved as full agonists. At higher somatostatin-14 concentrations there was sub-maximal inhibition resulting in a bell-shaped concentration-effect relationship. Pertussis toxin (PTx; 100 ng ml(-1), 18 h) pre-treatment abolished agonist-mediated inhibition of cyclic AMP accumulation and markedly enhanced stimulation of cyclic AMP at higher agonist concentrations. 3. The concentration of prostaglandin E2 (PGE2) in the incubation media was raised 14 fold by 1 microM somatostatin-14 but was insufficient to stimulate adenylate cyclase activity via endogenous prostanoid receptors. 4. Pre-treatment with cholera toxin (ChTx; 20 microg ml(-1), 18 h) markedly inhibited sst5 receptor-mediated increases in cyclic AMP formation in intact cells. Somatostatin-14-stimulated cyclic AMP accumulation was also observed in sst5 receptor containing CHO-K1 membranes and was inhibited by the synthetic peptide Galphasacetyl-354-372-amide (100 microM) by 65.9+/-3.5%, implicating a Galphas protein involvement in this response. 5. Activation of Galphas proteins by somatostatin-14 could be demonstrated with [35S]-guanosine 5'-[gamma-thio]triphosphate ([35S]-GTPgammaS) binding and subsequent immunoprecipitation of 35S labelled Galphas proteins with anti-Galphas serum. 6. These data show that the sst5 receptor is very efficiently coupled in a negative manner to adenylate cyclase. However, at higher agonist concentrations the receptor can also mediate activation of adenylate cyclase by a mechanism apparently involving Galphas protein activation.

Molecular cloning and functional characterization of a rat somatostatin sst2(b) receptor splice variant.

1. The mouse somatostatin (SRIF) sst2 receptor exists in two splice variants, sst2(a) and sst2(b), which differ in their intracellular carboxy-termini only. The murine sst2(b) receptor was reported to be less prone to agonist-induced desensitization as compared with the sst2(a) receptor. To determine whether a sst2(b) splice variant with similar functional characteristics exists in the rat, we have isolated a cDNA fragment from rat gastric mucosa encoding a sst2(b) receptor and expressed the full-length protein in CHO-K1 cells for functional characterization. 2. This study provides the first evidence for the occurrence in the rat of the sst2(b) receptor, which has a 15 amino acid carboxy-terminus differing in composition to the 38 amino acid C-terminus of the rat sst2(a) receptor. 3. In CHO-K1 cells expressing rat recombinant sst2(a) or sst2(b) receptors, SRIF caused concentration-dependent increases in extracellular acidification rates (EAR) with pEC50 values of 9.0 and 9.9, respectively. Pre-treatment with pertussis toxin (Ptx) caused a rightward displacement of the SRIF concentration-effect curves with pEC50 values of 8.3 (sst2(a) and 8.4 (sst2(b)). 4. SRIF (3 pM-3 nM) also caused concentration-dependent inhibition of forskolin-stimulated cyclic AMP formation in CHO-sst2(a) cells (pIC50 10.5) and CHO-sst2(b) cells (pIC50 10.4). The degree of inhibition was less with higher concentrations of SRIF resulting in bell-shaped concentration-effect curves. Following pre-treatment with Ptx, the inhibitory effect of SRIF was abolished and SRIF caused only increases in cyclic AMP formation. 5. Both the SRIF-induced increases in EAR and inhibition of cyclic AMP formation were susceptible to agonist-induced desensitization, but this was less apparent following pre-treatment with Ptx. 6. This demonstrates that the operational characteristics of the recombinant rat sst2(a) and sst2(b) receptors are broadly similar. Both isoforms couple to Ptx-sensitive as well as -insensitive G proteins and are equally prone to agonist-induced desensitization.

Activity of adenosine diphosphates and triphosphates on a P2Y(T) -type receptor in brain capillary endothelial cells.

1. A P2Y (nucleotide) receptor activity in a clonal population (B10) of rat brain capillary endothelial cells is coupled to inhibition of adenylyl cyclase and has functional similarities to the P2Y(T) (previously designated 'P2T') receptor for ADP of blood platelets. However, the only P2Y receptor which was detectable in a previous study of B10 cells by mRNA analysis was the P2Y(1) receptor, which elsewhere shows no transduction via cyclic nucleotides. We have sought here to clarify these issues. 2. The inhibition of forskolin-stimulated adenylyl cyclase induced by purified nucleotides was measured on B10 cells. The EC(50) value for 2-methylthioADP (2-MeSADP) was 2.2 nM and, surprisingly, 2-MeSATP was an almost equally strong agonist (EC(50)=3.5 nM). ATP and 2-ClATP were weak partial agonists (EC(50)=26 microM and 10 microM respectively) and under appropriate conditions could antagonise the activity on 2-MeSADP. 3. A known selective antagonist of the platelet P2Y(T) receptor, 2-propylthioadenosine-5'-(beta,gamma)-difluoromethylene) triphosphonate (AR-C 66096), was a competitive antagonist of this B10 cell receptor, with pK(B)=7.6. That ligand is inactive at the P2Y(1) receptor in the same cells. Conversely, the competitive P2Y(1) receptor antagonists, the 3', 5'- and 2', 5'-adenosine bis-monophosphates, are, instead, weak agonists at the adenylyl cyclase-inhibitory receptor. 4. The inhibition of adenylyl cyclase by 2-MeSADP was completely abolished by pertussis toxin. 5. In summary, these brain endothelial cells possess a P2Y(T)-type receptor in addition to the P2Y(1) receptor. The two have similarities in agonist profiles but are clearly distinguishable by antagonists and by their second messenger activations. The possible relationships between the B10 and platelet P2Y(T) receptors are discussed.

Effect of pertussis toxin treatment on the putative adenosine A3 receptor-mediated hypotensive response in the rat.

The effect of pertussis toxin pretreatment on the putative A3 receptor-mediated hypotensive response to N6-2-(4-aminophenyl)ethyladenosine (APNEA) has been investigated in the anaesthetised rat. Pertussis toxin pretreatment essentially abolished the bradycardia induced by the prototype A1 receptor agonist, N6-cyclopentyladenosine, whereas the fall in blood pressure induced by the selective A2A receptor agonist, CGS 21680 was enhanced. Pertussis toxin substantially reduced the hypotensive response to APNEA. In this respect, the mechanism of action resembles that of the cloned A3 receptor which when expressed in CHO cells couples negatively to adenylate cyclase by a pertussis toxin-sensitive mechanism. The data provide further evidence that adenosine A3 receptors mediate the hypotensive response to APNEA in the rat.

The cardiovascular effects of selective adenosine A1 and A2 receptor agonists in the pithed rat: no role for glibenclamide-sensitive potassium channels.

The cardiovascular effects of N6-cyclopentyladenosine (CPA), a selective adenosine A1 receptor agonist and 2-[p-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21,680), a selective A2 receptor agonist have been investigated in the pithed rat with blood pressure raised to normal levels with angiotensin II. Cumulative intravenous administration of CPA, 0.3-10 micrograms/kg, induced dose-related falls in blood pressure and heart rate; over the same dose range CGS 21,680 induced hypotension but no bradycardia. Pretreatment with a maximally effective dose of the A1/A2 receptor antagonist 8-(p-sulphophenyl) theophylline (8-SPT) blocked the bradycardiac effects of CPA (92-fold) more effectively than its hypotensive activity (5.1-fold); the vasodepressor effects of CGS 21,680 were blocked 19-fold by 8-SPT. Glibenclamide, a blocker of ATP-sensitive potassium (K+ATP) channels, administered intravenously at 20 mg/kg markedly attenuated the vasodepressor effects of the potassium channel opener, (-)-(3S,4R)-3,4-dihydro-3-hydroxy-2,2-dimethyl-4-(3-oxo-cyclopent- 1-enyloxy)-2H-1-benzopyran-6-carbonitrile (SDZ PCO 400). In contrast, neither the hypotensive nor the bradycardic effects of CPA nor the fall in blood pressure following CGS 21,680 was significantly affected by pretreatment with glibenclamide. These results indicate that a significant component of the blood pressure fall induced by CPA and CGS 21,680 in the pithed rat with blood pressure supported by angiotensin II occurs by a mechanism which is insensitive to 8-SPT and unlikely, therefore, to be mediated by A1 or A2 receptors. Moreover, in contrast to the prevailing literature, the cardiovascular effects arising from adenosine receptor activation in this model are not mediated by glibenclamide-sensitive, K+ATP channels.

Ultrastructure of human erythrocyte GLUT1.

This study was undertaken to examine GLUT1 quaternary structure. Independent but complementary methodologies were used to investigate the influence of membrane-solubilizing detergents on GLUT1/lipid/detergent micelle hydrodynamic radii. Hydrodynamic size analysis and electron microscopy of GLUT1/lipid/detergent micelles and freeze-fracture electron microscopy of GLUT1 proteoliposomes support the hypothesis that the glucose transporter is a multimeric (probably tetrameric) complex of GLUT1 proteins. GLUT1 forms a multimeric complex in octyl glucoside that dissociates upon addition of reductant. Some detergents (e.g., CHAPS and dodecyl maltoside) promote the dissociation of GLUT1 oligomers into smaller aggregation states (dimers or monomers). These complexes do not reassemble as larger oligomers when dissociating detergents are subsequently replaced with nondissociating detergents such as octyl glucoside or cholic acid. When dissociating detergents are replaced with lipids, the resulting proteoliposomes catalyze protein-mediated sugar transport, and the subsequent addition of solubilizing, nondissociating detergents generates higher (tetrameric) GLUT1 aggregation states. These findings suggest that some detergents stabilize while others destabilize GLUT1 quaternary structure. GLUT1 does not appear to exchange rapidly between protein/lipid/detergent micelles but is able to self-associate in the plane of the lipid bilayer.

Enhanced type 1alpha metabotropic glutamate receptor-stimulated phosphoinositide signaling after pertussis toxin treatment.

The regulation of phosphoinositide hydrolysis by the type 1alpha metabotropic glutamate receptor (mGluR1alpha) was investigated in stably transfected baby hamster kidney (BHK) cells. Incubation of the cells with L-glutamate, quisqualate, and 1-aminocyclopentane-1S, 3R-dicarboxylic acid resulted in a marked accumulation of [3H]inositol monophosphate (InsP1) and inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] mass in a time- and concentration-dependent manner. Pretreatment of BHK-mGluR1alpha cells with pertussis toxin [ 100 ng/ml, 24 hr] led to a dramatic 12-16-fold increase in the accumulation of [3H]InsP1 and a 2-fold increase in Ins(1,4,5)P3 in the absence of added agonist. Although only very low levels (/=75%, and the EC50 shifted leftward by 65-fold [-log EC50 values (molar), 7.26 +/- 0.23 versus 5.45 +/- 0.07; n = 4) in PTX-treated compared with control cells. In contrast, antagonist effects on agonist-stimulated [3H]InsP1 responses were similar in control and PTX-treated BHK-mGluR1alpha cells. These changes in the concentration-effect curves for mGluR agonists are consistent with a model in which the receptor associates with PTX-sensitive inhibitory (Gi/o) and PTX-insensitive stimulatory (Gq/11) G proteins that can each influence PIC activity. The present observations are consistent with a dual regulation of mGluR1alpha-mediated PIC activity that could be fundamental in controlling the output of phosphoinositide-derived messengers.

Adenosine A(1) receptor-mediated inhibition of protein kinase A-induced calcitonin gene-related peptide release from rat trigeminal neurons.

Calcitonin gene-related peptide (CGRP), a potent vasodilator, has been implicated in the pathogenesis of migraine. Its release from adult rat trigeminal neurons in culture was shown to be markedly increased by the activation of adenylate cyclase with forskolin. Modulation of this secretion was investigated by a number of agents with known inhibitory effects on cAMP generation mediated via receptor coupling to G(i/o) proteins. Significantly, forskolin-stimulated CGRP release could be closely correlated with the phosphorylation of the protein kinase A (PKA) substrate cyclic AMP response element-binding protein (CREB). Forskolin-stimulated CGRP release could be potently and effectively inhibited by the adenosine A(1) receptor-selective agonist GR79236X (pIC(50) = 7.7 +/- 0.1, maximal inhibition 65 +/- 2.5% at 300 nM), whereas the A(2A) (CGS21680) and the A(3) (2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide) receptor-selective agonists were without effect. GR79236X-mediated inhibition was abolished by the A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. Immunocytochemical studies and Western analysis revealed the presence of adenosine A(1) receptors on trigeminal neurons. However, despite the additional detection of 5-hydroxytryptamine (5-HT)(1B) receptors on these cells, the clinically effective antimigraine 5-HT(1B/1D) agonist sumatriptan did not inhibit forskolin-stimulated CGRP release nor did it show any effect on the concomitant CREB phosphorylation. In contrast, the mu-opioid agonist fentanyl elicited a 74 +/- 4% reduction in CGRP levels. Forskolin-stimulated CGRP release and CREB phosphorylation could be mimicked by incubation of the cells with chlorophenylthio-cAMP and blocked by pretreatment with the PKA inhibitor myrPKI(14-22). Taken together, the present data confirm the PKA-dependence of forskolin-stimulated CGRP release and suggest that A(1) adenosine agonists may warrant further investigation in models of migraine and neurogenic inflammation.