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1.
Figure 6

Figure 6. From: S-Nitrosothiols modulate G protein-coupled receptor signaling in a reversible and highly receptor-specific manner.

GSNO accelerates the rate of M2/M4 AChR-stimulated [35S]GTPγS binding to rat forebrain membranes but has no effect on basal guanine nucleotide exchange rate. Membranes were preincubated for 30 min in control conditions or in the presence of 0.5 mM GSNO, and time-response for basal and CCh-stimulated (10-4 M] [35S]GTPγS binding was determined, as detailed in the Methods section. Values represent specific binding (mean ± SD of duplicate determinations) from one representative experiment that was replicated three times with similar outcome.

Tarja Kokkola, et al. BMC Cell Biol. 2005;6:21-21.
2.
Figure 5

Figure 5. From: S-Nitrosothiols modulate G protein-coupled receptor signaling in a reversible and highly receptor-specific manner.

The P2Y12 receptor is not a direct target of RSNO action. Human P2Y12 receptor was stably expressed in CHO cells and agonist-stimulated G protein activity was determined in control conditions and in membranes pretreated for 30 min with 0.5 mM GSNO or CysNO, as detailed in the Methods section. There were no statistical differences in the agonist potency [log (EC50): control -9.20 ± 0.09; GSNO -9.14 ± 0.07; CysNO -9.27 ± 0.14) or efficacy [Emax (% basal): control 182 ± 3 %; GSNO 180 ± 2; CysNO 179 ± 5%]. Values are mean ± SE from three independent experiments performed in duplicate.

Tarja Kokkola, et al. BMC Cell Biol. 2005;6:21-21.
3.
Figure 7

Figure 7. From: S-Nitrosothiols modulate G protein-coupled receptor signaling in a reversible and highly receptor-specific manner.

GSNO increases the number of M2/M4 AChR interacting high-affinity [35S]GTPγS binding sites in rat forebrain membranes. Membranes were preincubated for 30 min in control conditions or in the presence of 0.5 mM GSNO, and incubated thereafter for 90 min with 0.15 nM [35S]GTPγS, 10-5 M GDP and indicated concentrations of unlabeled GTPγS in the presence and absence of CCh (10-4 M), as detailed in the Methods section. Statistical comparison of one- versus two-site competition curves (nonlinear regression) indicated that the one-site model best described GTPγS displacement in agonist-stimulated conditions. Note that GSNO significantly increased the number of high-affinity [35S]GTPγS binding sites in CCh-treated membranes (CCh-control 1.49 ± 0.02; CCh-GSNO 1.75 ± 0.02 pmol/mg protein, P < 0.001, unpaired T-test). There were no statistical differences in the potency for GTPγS in displacing radioligand in any condition [log (EC50): basal-control -8.10 ± 0.07; basal-GSNO -8.11 ± 0.12; CCh-control -8.06 ± 0.06; CCh-GSNO -8.10 ± 0.04). Values represent specific binding (mean ± SE) from three independent experiments performed in duplicate.

Tarja Kokkola, et al. BMC Cell Biol. 2005;6:21-21.
4.
Figure 2

Figure 2. From: S-Nitrosothiols modulate G protein-coupled receptor signaling in a reversible and highly receptor-specific manner.

GSNO modulates GPCR signaling in a dose-dependent manner. [35S]GTPγS autoradiography was conducted using a 3-step protocol with DPCPX (10-6 M) present throughout steps 2 and 3, as detailed in the Methods section. GSNO was present at the indicated concentrations for 60 min during the GDP loading (step 2). Carbachol (CCh, 10-4 M), 2MeSADP (10-6 M) or LPA (5 × 10-5 M in 0.1% fatty acid free BSA) were present in step 3. Note dose-dependent amplification of CCh-stimulated G protein activity, most evident at this coronal plane in the cerebral cortex (Cx), the striatum (Str), and the thalamus (Thal). Note also dose-related attenuation of 2MeSADP- and LPA-stimulated responses, especially in the white matter regions, including the corpus callosum (cc), the fimbria of the hippocampus (fi), and the striatal white matter (Sw). Scale bar = 2 mm. For quantitative data on selected brain regions, see Supplementary Figs. 1 and 2 in additional file 1.

Tarja Kokkola, et al. BMC Cell Biol. 2005;6:21-21.
5.
Figure 4

Figure 4. From: S-Nitrosothiols modulate G protein-coupled receptor signaling in a reversible and highly receptor-specific manner.

GSNO only marginally affect opiate related receptor (ORL1), μ opioid receptor (MOR) and adenosine A1 (Ado A1) receptors signaling in brain sections. [35S]GTPγS autoradiography was conducted using a 3-step protocol with adenosine deaminase (ADA, 1 U/ml) present throughout steps 2 and 3, as detailed in the Methods section. Where indicated, GSNO (1 mM) was present for 60 min during the GDP loading (step 2). Protease inhibitor cocktail was included in step 2 for brain sections used for testing ORL1 and MOR responses. Receptor agonists nociceptin (ORL1), DAMGO (MOR), and 2-chloroadenosine (2ClAdo) (adenosine A1 receptor) were present at submaximal concentrations during step 3. Note wide distribution of nociceptin-responsive brain regions, including the cerebral cortex (Cx). Note also robust response to DAMGO in the MOR-enriched striatal patches (Sp), as well as the relatively GSNO-resistant, and widely distributed adenosine A1 receptor-dependent signal throughout the sagittal plane. Scale bar = 2 mm. For quantitative data on selected brain regions, see Supplementary Table 2 in additional file 1.

Tarja Kokkola, et al. BMC Cell Biol. 2005;6:21-21.
6.
Figure 8

Figure 8. From: S-Nitrosothiols modulate G protein-coupled receptor signaling in a reversible and highly receptor-specific manner.

GSNO-evoked potentiation of muscarinic signaling is preserved under heterologus expression but the effect diminishes with increasing constitutive activity. The human M4 (hM4) receptor was stably transfected into CHO cells and wild type (WT) or mutant (C133S) cell lines with differential G protein activation capacity were compared with that in rat forebrain membranes. CCh-stimulated G protein activity was determined in control conditions and in membranes pretreated for 30 min with 0.5 mM GSNO, as detailed in the Methods section. Note that cell lines WT-C2, WT-E5 and C133S-H2 have maximal responses comparable to that in native brain tissue. Note also robust G protein activation in WT-A1 cell line, as well as its constitutive activity, as evidenced by the ability of the inverse agonist atropine to inhibit basal G protein activity in this cell line. Potency and efficacy values are summarized in Tables 1 (rat brain) and 2 (hM4 clones). Values are mean ± SE from at least three independent experiments performed in duplicate.

Tarja Kokkola, et al. BMC Cell Biol. 2005;6:21-21.
7.
Figure 3

Figure 3. From: S-Nitrosothiols modulate G protein-coupled receptor signaling in a reversible and highly receptor-specific manner.

S-nitrosocysteine (CysNO) mimics the effects of GSNO in modulating GPCR signaling, whereas sodium nitroprusside (SNP) and 8-bromo cyclic GMP (8Br-cGMP) do not. [35S]GTPγS autoradiography was conducted using a 3-step protocol with DPCPX (10-6 M) present throughout steps 2 and 3, as detailed in the Methods section. Where indicated, CysNO (1 mM), SNP (0.5 mM), or 8Br-cGMP (0.25 mM) were present for 60 min during the GDP loading (step 2). Carbachol (10-4 M), 2MeSADP (10-6 M), or LPA (5 × 10-5 M in 0.1% fatty acid free BSA) were present in step 3. Note amplification of CCh-stimulated G protein activity by CySNO, most evident at this coronal plane in the cerebral cortex (Cx), the thalamus (Thal), including the superficial gray layer of the superior colliculus (SuG). Note also CysNO-dependent attenuation of 2MeSADP- and LPA-stimulated responses, most evident in the corpus callosum (cc) and the fimbria of the hippocampus (fi). Scale bar = 2 mm. For quantitative data on selected brain regions, see Supplementary Table 1 in additional file 1.

Tarja Kokkola, et al. BMC Cell Biol. 2005;6:21-21.
8.
Figure 1

Figure 1. From: S-Nitrosothiols modulate G protein-coupled receptor signaling in a reversible and highly receptor-specific manner.

S-nitrosoglutathione (GSNO) reversibly modulates basal and receptor-dependent G protein activity in rat brain cryostat sections. [35S]GTPγS autoradiography of sagittal brain sections was conducted using a 3-step protocol with DPCPX (10-6 M) present throughout steps 2 and 3, as detailed in the Methods section. Where indicated, GSNO (0.5 mM) was present for 60 min during the GDP loading (step 2). When used, DTT (1 mM) or GSH (1 mM) were present during the [35S]GTPγS labeling (step 3). The muscarinic agonist, carbachol (CCh, 10-4 M), the P2Y receptor agonist 2-methylthio-ADP (2MeSADP, 10-5 M) or lysophosphatidic acid (LPA, 5 × 10-5 M in 0.1% fatty acid free BSA) were present in step 3. In the control panel (left), the anatomical loci where receptor agonists typically activate G proteins are indicated. Note GSNO-dependent overall increase in basal G protein activity, as well as robust amplification of CCh-stimulated G protein activity in several gray matter regions visible at this sagittal plane, most notably the brain stem (bs) nuclei, the striatum (Str), and the superficial gray layer of the superior colliculus (SuG). Note also clear attenuation of 2MeSADP-stimulated responses in all brain regions, and blunting of LPA-stimulated responses, especially in the white matter areas, including the corpus callosum (cc), the fimbria of the hippocampus (fi) and the cerebellar white matter (Cbw). Scale bar = 2 mm. For quantitative data on selected brain regions, see Supplementary Figs. 1 and 2 in additional file 1.

Tarja Kokkola, et al. BMC Cell Biol. 2005;6:21-21.

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