A G protein is involved in the angiotensin AT2 receptor inhibition of the T-type calcium current in non-differentiated NG108-15 cells

In non-differentiated NG108-15 cells, both angiotensin II (Ang II) (100 nM) and CGP 42112 (100 nM) decreased the T-type calcium current amplitude by 24 +/- 2% and 21 +/- 3%, respectively. cGMP is not a mediator of the Ang II effect, since loading of cells with 50 microM cGMP did not prevent the inhibitory effects of Ang II. The effects of Ang II involves a non-identified GTPase activity since incubation with GDP beta S (3 mM) completely reversed the inhibitory effect of Ang II while GTP gamma S mimicked its effect. However, Ang II binding was not affected by GTP gamma S, and the effect of Ang II was not modified in pertussis toxin-treated cells. The inhibitory effect of Ang II on the T-type Ca2+ current involves a phosphotyrosine phosphatase activity since sodium orthovanadate prevented the effects of Ang II, although microcystin-LR, a selective Ser/Thr phosphatase 1 and 2A inhibitor, did not modify the effect of Ang II. These results provide the first evidence of a modulation of membrane conductance by Ang II through the AT2 receptor and demonstrate the involvement of a phosphotyrosine phosphatase and a G protein in the AT2 transduction mechanism in NG108-15 cells. Moreover, our data suggest that phosphotyrosine phosphatase activation is proximal to receptor occupation, since sodium orthovanadate inhibits both GTPase activity and T-type current blockage induced by Ang II or CGP 42112, while GTP gamma S inhibition of the T-type calcium current is not impaired.     

2-Arachidonoylglycerol, a putative endogenous cannabinoid receptor ligand, induces rapid, transient elevation of intracellular free Ca2+ in neuroblastoma x glioma hybrid NG108-15 cells

The hypothesis that tea drinking may protect against coronary heart disease (CHD) through effects on clotting as measured by plasma fibrinogen, tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) was tested in 65 healthy volunteers (31 men and 34 women; aged 20-74 years) in a randomized, blind, placebo-controlled, crossover study lasting 10 weeks (run-in phase 2 weeks, tea and placebo phases 4 weeks). During the placebo phase, intakes of milk, sugar, water and caffeine were matched to those in the tea phase during which 6 mugs of tea were drunk daily. Compliance with tea intake was measured by marking tea bags with p-aminobenzoic acid and measuring recovery in 24-hour urine collections. The mean +/- SD fibrinogen level, PAI-1 activity and tPA antigen level at baseline of 2.91 +/- 0.81 g/l, 7.9 +/- 5.3 U/ml and 4.76 +/- 2.17 ng/ml, respectively, were in the normal range. No significant differences in these variables between the run-in, tea or placebo phases were observed. The putative protective effect of tea against development of CHD is not mediated through effects of black tea on fibrinogen, tPA or PAI-1.     

Differential coupling of alpha1-, alpha2-, and beta-adrenergic receptors to mitogen-activated protein kinase pathways and differentiation in transfected PC12 cells

Three adrenergic receptor families that selectively activate three different G proteins (alpha1/Gq/11, alpha2/Gi, and beta/Gs) were used to study mitogen-activated protein kinase (MAPK) activation and differentiation in PC12 cells. PC12 cells were stably transfected with alpha1A-, alpha2A-, or beta1-adrenergic receptors (ARs) in an inducible expression vector, and subclones were characterized. Norepinephrine stimulated inositol phosphate formation in alpha1A-transfected cells, inhibited cyclic adenosine 3'5'-monophosphate (cAMP) formation in alpha2A-transfected cells, and stimulated cAMP formation in beta1-transfected cells. Nerve growth factor activated extracellular signal-regulated kinases (ERKs) in all cell lines; however, norepinephrine activated ERKs only in alpha1A- and beta1-transfected cells but not in alpha2A-transfected cells. Norepinephrine also activated c-Jun NH2-terminal kinase and p38 MAPK in alpha1A-transfected cells but not in beta1- or alpha2A-transfected cells. Norepinephrine caused differentiation of PC12 cells expressing alpha1A-ARs but not those expressing beta1- or alpha2A-ARs. However, norepinephrine acted synergistically with nerve growth factor in promoting differentiation of cells expressing beta1-ARs. Whereas ERKs are activated by Gi- but not Gs-linked receptors in many fibroblastic cell lines, we observed the opposite in PC12 cells. The results show that activation of the different G protein signaling pathways has different effects on MAPKs and differentiation in PC12 cells, with Gq signaling pathways activating all three major MAPK pathways.     

Negative regulation of MAP kinase by diacylglycerol-dependent mechanisms via G protein-coupled receptors in rat basophilic RBL-2H3 (ml) cells

Carbachol and 5'-(N-ethylcarboxamido)-adenosine (NECA), stimulants of G protein-coupled receptors, induce MAP kinase activation in the muscarinic ml receptor-transfected mast cell line, RBL-2H3 (ml) cells. The phospholipase C inhibitor neomycin and the phosphatidate phosphohydrolase inhibitor propranolol augmented MAP kinase activation induced by carbachol and NECA without affecting the antigen-induced MAP kinase activation. Furthermore, the duration of MAP kinase activation induced by carbachol or NECA was also prolonged by neomycin and propranolol. The specific protein kinase C inhibitor Ro 31-8425 enhanced the carbachol- or NECA-induced MAP kinase activation. These findings suggest that the MAP kinase activation mediated by the G protein-coupled receptors is negatively regulated by diacylglycerol and activated protein kinase C(s).     

D2 dopamine receptors stimulate mitogenesis through pertussis toxin-sensitive G proteins and Ras-involved ERK and SAP/JNK pathways in rat C6-D2L glioma cells

Dopamine D2 receptors are members of the G protein-coupled receptor superfamily and are expressed on both neurons and astrocytes. Using rat C6 glioma cells stably expressing the rat D2L receptor, we show here that dopamine (DA) can activate both the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) pathways through a mechanism involving D2 receptor-G protein complexes and the Ras GTP-binding protein. Agonist binding to D2 receptors rapidly activated both kinases within 5 min, reached a maximum between 10 and 15 min, and then gradually decreased by 60 min. Maximal activation of both kinases occurred with 100 nM DA, which produced a ninefold enhancement of ERK activity and a threefold enhancement of JNK activity. DA-induced kinase activation was prevented by either (+)-butaclamol, a selective D2 receptor antagonist, or pertussis toxin, an uncoupler of G proteins from receptors, but not by (-)-butaclamol, the inactive isomer of (+)-butaclamol. Cotransfection of RasN17, a dominant negative Ras mutant, prevented DA-induced activation of both ERK and JNK. PD098059, a specific MEK1 inhibitor, also blocked ERK activation by DA. Transfection of SEK1 (K --> R) vector, a dominant negative SEK1 mutant, specifically prevented DA-induced JNK activation and subsequent c-Jun phosphorylation without effect on ERK activation. Furthermore, stimulation of D2 receptors promoted [3H]thymidine incorporation with a pattern similar to that for kinase activation. DA mitogenesis was tightly linked to Ras-dependent mitogen-activated protein kinase (MAPK) and JNK pathways. Transfection with RasN17 and application of PD098059 blocked DA-induced DNA synthesis. Transfection with Flag delta169, a dominant negative c-Jun mutant, also prevented stimulation of [3H]thymidine incorporation by DA. The demonstration of D2 receptor-stimulated MAPK pathways may help to understand dopaminergic physiological functions in the CNS.     

S 18126 ([2-[4-(2,3-dihydrobenzo[1,4]dioxin-6-yl)piperazin-1-yl methyl]indan-2-yl]), a potent, selective and competitive antagonist at dopamine D4 receptors: an in vitro and in vivo comparison with L 745,870 (3-(4-[4-chlorophenyl]piperazin-1-yl)methyl-1H-pyrrolo[2, 3b]pyridine) and raclopride

The novel benzoindane S 18126 possessed > 100-fold higher affinity at cloned, human (h) D4 (Ki = 2.4 nM) vs. hD2 (738 nM), hD3 (2840 nM), hD1 (> 3000 nM) and hD5 (> 3000 nM) receptors and about 50 other sites, except sigma1 receptors (1.6 nM). L 745,870 similarly showed selectivity for hD4 (2.5 nM) vs. hD2 (905 nM) and hD3 (> 3000 nM) receptors. In contrast, raclopride displayed low affinity at hD4 (> 3000 nM) vs. hD2 (1.1 nM) and hD3 receptors (1.4 nM). Stimulation of [35S]-GTPgammaS binding at hD4 receptors by dopamine (DA) was blocked by S 18126 and L 745,870 with Kb values of 2.2 and 1.0 nM, respectively, whereas raclopride (> 1000 nM) was inactive. In contrast, raclopride inhibited stimulation of [35S]-GTPgammaS binding at hD2 sites by DA with a Kb of 1.4 nM, whereas S 18126 (> 1000 nM) and L 745,870 (> 1000 nM) were inactive. As concerns presynaptic dopaminergic receptors, raclopride (0.01-0.05 mg/kg s.c. ) markedly enhanced DA synthesis in mesocortical, mesolimbic and nigrostriatal dopaminergic pathways. In contrast, even high doses (2. 5-40.0 mg/kg s.c.) of S 18126 and L 745,870 were only weakly active. Similarly, raclopride (0.016 mg/kg i.v.) abolished inhibition of the firing rate of ventrotegmental dopaminergic neurons by apomorphine, whereas even high doses (0.5 mg/kg i.v.) of S 18126 and L 745,870 were only weakly active. As regards postsynaptic dopaminergic receptors, raclopride potently (0.01-0.3 mg/kg s.c.) reduced rotation elicited by quinpirole in rats with unilateral lesions of the substantia nigra, antagonized induction of hypothermia by PD 128, 907, blocked amphetamine-induced hyperlocomotion and was effective in six further models of potential antipsychotic activity. In contrast, S 18126 and L 745,870 were only weakly active in these models (5.0-> 40.0 mg/kg s.c.). In six models of extrapyramidal and motor symptoms, such as induction of catalepsy, raclopride was likewise potently active (0.01-2.0 mg/kg s.c.) whereas S 18126 and L 745,870 were only weakly active (10.0-80.0 mg/kg s.c.). In freely moving rats, raclopride (0.16 mg/kg s.c.) increased levels of DA by + 55% in dialysates of the frontal cortex. However, it also increased levels of DA in the accumbens and striatum by 70% and 75%, respectively. In contrast to raclopride, at a dose of 0.16 mg/kg s.c. , neither S 18126 nor L 745,870 modified frontal cortex levels of DA. However, at a high dose (40.0 mg/kg s.c.), S 18126 increased dialysate levels of DA (+ 85%) and noradrenaline (+ 100%), but not serotonin (+ 10%), in frontal cortex without affecting DA levels in accumbens (+ 10%) and striatum (+ 10%). In conclusion, S 18126 and L 745,870 behave as potent and selective antagonists of cloned, hD4 vs. other dopaminergic receptor types in vitro. However, their in vivo effects at high doses probably reflect residual antagonist actions at D2 (or D3) receptors. Selective blockade of D4 receptors was thus associated neither with a modification of dopaminergic transmission nor with antipsychotic (antiproductive) or extrapyramidal properties. The functional effects of selective D4 receptor blockade remain to be established.