Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 May;166(1):297-308.
doi: 10.1111/j.1476-5381.2011.01774.x.

G-protein βγ subunits in vasorelaxing and anti-endothelinergic effects of calcitonin gene-related peptide

M J P M T Meens  1 , N J A Mattheij, P B van Loenen, L J A Spijkers, P Lemkens, J Nelissen, M G Compeer, A E Alewijnse, J G R De Mey
Affiliations

G-protein βγ subunits in vasorelaxing and anti-endothelinergic effects of calcitonin gene-related peptide

M J P M T Meens et al. Br J Pharmacol. 2012 May.

Abstract

Background and purpose: Calcitonin gene-related peptide (CGRP) has been proposed to relax vascular smooth muscle cells (VSMC) via cAMP and can promote dissociation of endothelin-1 (ET-1) from ET(A) receptors. The latter is not mimicked by other stimuli of adenylate cyclases. Therefore, we evaluated the involvement of G-protein βγ subunits (Gβγ) in the arterial effects of CGRP receptor stimulation.

Experimental approach: To test the hypothesis that instead of α subunits of G-proteins (Gαs), Gβγ mediates the effects of CGRP receptor activation, we used (i) rat isolated mesenteric resistance arteries (MRA), (ii) pharmacological modulators of cyclic nucleotides; and (iii) low molecular weight inhibitors of the functions of Gβγ, gallein and M119. To validate these tools with respect to CGRP receptor function, we performed organ bath studies with rat isolated MRA, radioligand binding on membranes from CHO cells expressing human CGRP receptors and cAMP production assays in rat cultured VSMC.

Key results: In isolated arteries contracted with K(+) or ET-1, IBMX (PDE inhibitor) increased sodium nitroprusside (SNP)- and isoprenaline (ISO)- but not CGRP-induced relaxations. While fluorescein (negative control) was without effects, gallein increased binding of [(125) I]-CGRP in the absence and presence of GTPγS. Gallein also increased CGRP-induced cAMP production in VSMC. Despite these stimulating effects, gallein and M119 selectively inhibited the relaxing and anti-endothelinergic effects of CGRP in isolated arteries while not altering contractile responses to K(+) or ET-1 or relaxing responses to ISO or SNP.

Conclusion and implications: Activated CGRP receptors induce cyclic nucleotide-independent relaxation of VSMC and terminate arterial effects of ET-1 via Gβγ.

PubMed Disclaimer

Figures

Figure 1
Figure 1
In rat MRA, inhibition of soluble guanylyl cyclase inhibits relaxations induced by SNP but does not inhibit the relaxing and anti-endothelinergic effects of CGRP. (A) Schematic trace illustrating a contraction induced by ET-1, which was terminated by CGRP. The anti-endothelinergic effect of CGRP was determined at ‘X’ 8 min after removal (‘Wash’) of all vasoactive compounds and was compared with a time control experiment conducted in parallel. (B) ET-1-induced contractions were not affected by the presence of ODQ. (C) Long-lasting contractile effect of ET-1 assessed 8 min after removal of all vasoactive compounds from the organ baths. Transient exposure to CGRP reduced the long-lasting ET-1-induced contractions (11 ± 3% vs. 99 ± 6% of K+max. P < 0.001). This anti-endothelinergic effect of CGRP was not altered in the presence of ODQ. (D) K+-induced contractions were increased in the presence of ODQ (Emax 136 ± 5 vs. 86 ± 7% of K+max. P < 0.001). (E) SNP-induced relaxations during 40 mM K+-induced contractions were abolished in the presence of ODQ indicating full inhibition of soluble guanylyl cyclase (Emax−7 ± 3 vs. 54 ± 9% relaxation. P < 0.001). (F) CGRP-induced relaxations during K+-induced contractions were not altered in the presence of ODQ. Data are expressed as % K+max or as % reduction of the pre-existing contraction and are shown as mean ± SEM (n= 4–6). **P < 0.01, ***P < 0.001 versus control.
Figure 2
Figure 2
In rat MRA, inhibition of PDEs increases relaxations induced by (i) SNP or (ii) ISO but does not increase relaxing and anti-endothelinergic effects of CGRP. (A) K+-induced contractions were increased in the presence of IBMX (Emax 10 ± 3 vs. 20 ± 3%.of NAmax. P < 0.05). (B) ET-1-induced contractions were not altered in the presence of IBMX. (C) During 40 mM K+-induced contractions, SNP induced relaxations more potently in the presence of IBMX (EC50 27 ± 0.2 vs. 92 ± 0.09 nM. P < 0.05). (D) During 40 mM K+-induced contractions, ISO induced relaxations more potently and with bigger amplitude in the presence of IBMX (EC50 0.10 ± 0.03 vs. 0.26 ± 0.1 µM. P < 0.05. Emax 72 ± 2 vs. 36 ± 5% relaxation. P < 0.001). (E) During 40 mM K+-induced contractions, CGRP-induced relaxations were not altered by IBMX. (F) During 32 nM ET-1-induced contractions, CGRP-induced relaxations were not altered by IBMX. Data are expressed as % NAmax or as % reduction of the pre-existing contraction and are shown as mean ± SEM (n= 6). *P < 0.05, ***P < 0.001 versus control.
Figure 3
Figure 3
The presence of gallein acutely increases the binding of [125I]-CGRP to membrane fragments from CHO cells expressing human CGRP receptors. (A) Displacement of 0.5 nM [125I]-CGRP by unlabelled CGRP in the absence and presence of 10 µM GTPγS. (B) Gallein markedly increased the binding of 0.5 nM [125I]-CGRP in the absence and presence (red line) of 10 µM GTPγS. (C) Fluorescein did not affect binding of 0.5 nM [125I]-CGRP in the absence and presence of 10 µM GTPγS. Data are shown as mean ± SEM and are the means of three experiments in duplicate.
Figure 4
Figure 4
The presence of gallein increases binding of [125I]-CGRP. Saturation binding experiment performed (i) in the absence of GTPγS or gallein (control), (ii) in the presence of GTPγS (10 µM), (iii) in the presence of gallein (100 µM) and (iv) in the presence of gallein (100 µM) and GTPγS (10 µM). Data are shown as mean ± SEM and are the means of three experiments in duplicate. ***P < 0.001 control versus gallein.
Figure 5
Figure 5
The presence of gallein increases cAMP production in mesenteric artery smooth muscle cells. (A) The presence of gallein but not fluorescein increases ISO-induced cAMP production (Emax 192 ± 22 vs. 100 ± 3%. P < 0.001). (B) The presence of gallein but not fluorescein increases CGRP-induced cAMP production (Emax 216 ± 33 vs. 100 ± 2%. P < 0.001). Data are expressed as % of maximal agonist-induced cAMP accumulation in the absence of gallein/fluorescein and are shown as mean ± SEM (n= 6/7). ***P < 0.001 versus control.
Figure 6
Figure 6
In rat MRA, an inhibitor of Gβγ subunits reduces the relaxing effects of CGRP. (A) K+-induced contractions were not altered by the presence of gallein. (B) ET-1-induced contractions were not affected by the presence of gallein. (C) During 40 mM K+-induced contractions, SNP-induced relaxations were not altered by the presence of gallein. (D) During 40 mM K+-induced contractions, ISO-induced relaxations were not affected by the presence of gallein. (E) During 40 mM K+-induced contractions, the presence of gallein concentration-dependently inhibited CGRP-induced relaxations [Emax 26 ± 12 (in the presence of 30 µM gallein) or −6 ± 10 (in the presence of 100 µM gallein) vs. 74 ± 3 (control) % relaxation; P < 0.001 for both conditions]. (F) During 32 nM ET-1-induced contractions, the presence of gallein concentration-dependently inhibited CGRP-induced relaxations [Emax 47 ± 13 (in the presence of 30 µM gallein) or 45 ± 13 (in the presence of 100 µM gallein) vs. 88 ± 4 (control) % relaxation; P < 0.05 for both conditions]. Data are expressed as % K+max or as % reduction of the pre-existing contraction and are shown as mean ± SEM (n= 5/6). *P < 0.05 30 µM gallein versus control; ***P < 0.05 100 µM gallein versus control; #P < 0.05 100 µM gallein versus control; ###P < 0.05 30 µM gallein versus control.
Figure 7
Figure 7
The presence of gallein concentration-dependently inhibits CGRP-induced reduction of long-lasting ET-1-induced contractions. (A) Schematic trace illustrating the experimental protocol. WO: washout; removal of all vasoactive peptides. Data were collected at ‘X’. (B) Average wall tension at ‘X’ for time control, control and for experiments performed in presence of increasing concentrations of gallein [162 ± 23 (time control) or 170 ± 53 (100 µM gallein) vs. 21 ± 8% (control) of K+max respectively]. Data are expressed as % K+max and are shown as mean ± SEM (n= 5/6). *P < 0.05, **P < 0.01 versus control.
Figure 8
Figure 8
The presence of fluorescein (100 µM), a gallein-like molecule that does not bind Gβγ subunits, does not affect vasomotor function. (A) K+-induced contractions in the presence or absence of 100 µM fluorescein. (B) ET-1-induced contractions in the presence or absence of fluorescein. (C) Relaxations induced by SNP during 40 mM K+-induced contractions in the presence or absence of fluorescein. (D) Relaxations induced by ISO during 40 mM K+-induced contractions in the presence or absence of fluorescein. (E) Relaxations induced by CGRP during 40 mM K+-induced contractions in the presence or absence of fluorescein. (F) Relaxations induced by CGRP during 32 nM ET-1-induced contractions in the presence or absence of fluorescein. Data are expressed as % K+max or as % reduction of the pre-existing contraction and are shown as mean ± SEM (n= 6).
Figure 9
Figure 9
The presence of M119, a gallein-like inhibitor of Gβγ subunits, concentration-dependently inhibits CGRP-induced relaxations during K+-induced contractions (Emax 5 ± 4 vs. 67 ± 3% relaxation in the presence and absence of 100 µM M119, respectively; P < 0.001). Data are expressed as % reduction of the pre-existing contraction and are shown as mean ± SEM (n= 4–8). ***P < 0.001.
Figure 10
Figure 10
Scheme illustrating interactions of Gα and Gβγ with arterial smooth muscle CGRP receptors and downstream targets. (1) Activated CGRP receptors function as guanine nucleotide exchange factor and cause conversion of GDP bound to Gα to GTP. Consequently, the heterotrimeric Gαβγ dissociates into Gα and Gβγ, which can both interact with intracellular targets. (2) Gα activates AC, which produces cAMP, but, possibly due to abundantly expressed PDEs, this does not seem to be involved in the relaxation of VSM in our setting. (3) In contrast, activated Gβγ causes both the relaxing and the ‘anti-endothelinergic’ effects induced by activation of CGRP receptors. (4) Ultimately, the intrinsic GTPase activity of Gα converts GTP into GDP allowing Gαβγ to reassemble.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alexander SPH, Mathie A, Peters JA. Guide to Receptors and Channels (GRAC), 5th Edition. Br J Pharmacol. 2011;164(Suppl. 1):S1–S324. - PMC - PubMed
    1. Bagnato A, Spinella F, Rosano L. The endothelin axis in cancer: the promise and the challenges of molecularly targeted therapy. Can J Physiol Pharmacol. 2008;86:473–484. - PubMed
    1. Bayewitch ML, Avidor-Reiss T, Levy R, Pfeuffer T, Nevo I, Simonds WF, et al. Differential modulation of adenylyl cyclases I and II by various G beta subunits. J Biol Chem. 1998a;273:2273–2276. - PubMed
    1. Bayewitch ML, Avidor-Reiss T, Levy R, Pfeuffer T, Nevo I, Simonds WF, et al. Inhibition of adenylyl cyclase isoforms V and VI by various Gbetagamma subunits. FASEB J. 1998b;12:1019–1025. - PubMed
    1. Beavo JA. cGMP inhibition of heart phosphodiesterase: is it clinically relevant? J Clin Invest. 1995;95:445. - PMC - PubMed

Publication types

MeSH terms