Purinergic responses in HT29 colonic epithelial cells are mediated by G protein α -subunits

Cummins, M.; O’Mullane, Lauren M; Barden, Julian A.; Cook, David I.; Poronnik, Philip

doi:10.1054/ceca.2000.0120

Cited by 11 publications

(28 citation statements)

References 27 publications

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“…[61][62][63][64] Fori nstance, the free Gβγ released during P2Y 2 Rs timulation is involved in activation of PLCb 61 and Gp rotein-activated inwardly rectifying K + channels. 65 Our owns tudies in human colonic epithelial (HT29) cells suggested that free Gβγ,r eleased during M 3 muscrinic receptor stimulation, mediates mobilization of Ca 2+ from intracellular stores, 66 and that free Gβγ,r eleased during P2Y 2 Rs timulation, regulates membrane Ca 2+ influx (unpublished data). One of our most surprising findings is the discovery that the mechanisms by which P2Y 2 R signalling regulates the activity of ENaC involves the βγ subunits of G proteins.…”

Section: G-protein βγ Subunits Play a Role In The P2y 2 R Inhibition mentioning

confidence: 91%

Purinergic regulation of the epithelial Na⁺ channel

O’Mullane

Cook

Dinudom

2009

Clin Exp Pharma Physio

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Summary The epithelial Na+ channel (ENaC) is a major conductive pathway that transports Na + across the apical membrane of the distal nephron, the respiratory tract, the distal colon and the ducts of exocrine glands. ENaC is regulated by hormonal and humoral factors, among which are extracellular nucleotides that can be available from the epithelial cells themselves.2. Extracellular nucleotides, via the P2Y 2 receptors (P2Y 2 Rs) at the basolateral and apical membrane of epithelia, trigger signalling systems that cause inhibition of the activity of ENaC and activation of Ca 2+ -dependent Cl − secretion.3. Recent data from our laboratory suggest that stimulation of the P2Y 2 Rs at the basolateral membrane inhibits activity of ENaC by a signalling mechanism that involves Gβγ subunits freed from a PTX-sensitive G protein and phospholipase C-β4. A similar signalling mechanism is also partially responsible for inhibition of ENaC during activation of apical P2Y 2 Rs 4. Stimulation of apical P2Y 2 Rs also stimulates an additional signalling mechanism that inhibits ENaC, involving the activated Gα subunit of a PTX-insensitive G protein, and activation of an unidentified PLC. The effect of this PTX-insensitive system requires the activity of the basolateral Na + /K + /2Cl − co-transporter.

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Section: G-protein βγ Subunits Play a Role In The P2y 2 R Inhibition mentioning

confidence: 91%

Purinergic regulation of the epithelial Na⁺ channel

O’Mullane

Cook

Dinudom

2009

Clin Exp Pharma Physio

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“…Infection of HT29 cells with the G aq antisense virus led to a 58% suppression of the Ca 2+ response to carbachol and a comparable suppression of the endogenous G aq protein [21]. It was not clear from this study, however, whether the incomplete inhibition of the Ca 2+ signal was due to incomplete suppression of endogenous G aq or was due to another G q family isoform, such as G a11 , mediating the residual response [21]. In the current study we have addressed this issue by infecting the cells with adenoviruses expressing antisense G aq and/or antisense G a11 to determine the relative contributions of these isoforms to M 3 -receptortriggered Ca 2+ signalling in HT29 cells.…”

Section: Introductionmentioning

confidence: 95%

“…To overcome this limitation, we have developed an adenovirus-based method for highly efficient expression of antisense and other constructs directed against G protein a-subunits in cultured epithelial cells [19,20,21], and have used it to study Ca 2+ signalling in the human colonic carcinoma cell line HT29 [20,21,22].…”

Section: Introductionmentioning

confidence: 99%

“…These experiments were, however, unable to distinguish whether the muscarinic signal was due to G q , to G 11 or to both, because the dominant negative G aq subunit could potentially interfere with G a11 -mediated signalling. In a subsequent study, we therefore used an adenovirus expressing antisense directed against G aq in order to determine the extent to which suppression of the endogenous levels of G aq resulted in an inhibition of M 3 -receptor-triggered Ca 2+ signalling [21]. Infection of HT29 cells with the G aq antisense virus led to a 58% suppression of the Ca 2+ response to carbachol and a comparable suppression of the endogenous G aq protein [21].…”

Section: Introductionmentioning

confidence: 99%

“…In a subsequent study, we therefore used an adenovirus expressing antisense directed against G aq in order to determine the extent to which suppression of the endogenous levels of G aq resulted in an inhibition of M 3 -receptor-triggered Ca 2+ signalling [21]. Infection of HT29 cells with the G aq antisense virus led to a 58% suppression of the Ca 2+ response to carbachol and a comparable suppression of the endogenous G aq protein [21]. It was not clear from this study, however, whether the incomplete inhibition of the Ca 2+ signal was due to incomplete suppression of endogenous G aq or was due to another G q family isoform, such as G a11 , mediating the residual response [21].…”

Section: Introductionmentioning

confidence: 99%

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Antisense co-suppression of G α q and G α 11 demonstrates that both isoforms mediate M 3 -receptor-activated Ca 2+ signalling in intact epithelial cells

Cummins

O’Mullane

Barden

et al. 2002

Pfl�gers Archiv European Journal of Physiology

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We used replication-deficient adenoviruses overexpressing antisense against G(q) class alpha-subunits to determine the roles of G(q) and G(11) in mediating M(3)-receptor-coupled Ca(2+) mobilization in intact HT29 human colonic carcinoma epithelial cells. Western blot analysis and confocal microscopy showed that the viruses expressing antisense directed against the alpha-subunits of G(q) or G(11) produced isoform-specific reductions in the levels of these alpha-subunits. Fura-2 was used to measure changes in the Ca(2+) response following activation of the M(3) receptors by carbachol. The G(alpha)(q) antisense virus suppressed the peak Ca(2+) response by 70%, whereas the G(alpha)(11) antisense virus reduced it by 34%. We then used co-infection with both viruses to determine the effect of concomitant suppression of both G(alpha)(q) and G(alpha)(11). Overexpression of antisense to both alpha-subunits reduced by approximately 50% the levels of both G(alpha)(q) and G(alpha)(11). It also almost completely inhibited the Ca(2+) response to carbachol. These data show that both G(q) and G(11) are involved in mediating the action of the M(3) receptor on cytosolic Ca(2+) in HT29 cells. Furthermore, they suggest that the coupling of the M(3) receptor to these G proteins is specific, in that G(alpha)(q) cannot substitute for G(alpha)(11), and vice versa.

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Purinergic signalling and cancer

Burnstock

Virgilio

2013

Purinergic Signalling

267

265

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Receptors for extracellular nucleotides are widely expressed by mammalian cells. They mediate a large array of responses ranging from growth stimulation to apoptosis, from chemotaxis to cell differentiation and from nociception to cytokine release, as well as neurotransmission. Pharma industry is involved in the development and clinical testing of drugs selectively targeting the different P1 nucleoside and P2 nucleotide receptor subtypes. As described in detail in the present review, P2 receptors are expressed by all tumours, in some cases to a very high level. Activation or inhibition of selected P2 receptor subtypes brings about cancer cell death or growth inhibition. The field has been largely neglected by current research in oncology, yet the evidence presented in this review, most of which is based on in vitro studies, although with a limited amount from in vivo experiments and human studies, warrants further efforts to explore the therapeutic potential of purinoceptor targeting in cancer.

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Purinergic responses in HT29 colonic epithelial cells are mediated by G protein α -subunits

Cited by 11 publications

References 27 publications

Purinergic regulation of the epithelial Na⁺ channel

Purinergic regulation of the epithelial Na⁺ channel

Antisense co-suppression of G α q and G α 11 demonstrates that both isoforms mediate M 3 -receptor-activated Ca 2+ signalling in intact epithelial cells

Purinergic signalling and cancer

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Purinergic responses in HT29 colonic epithelial cells are mediated by G protein α -subunits

Cited by 11 publications

References 27 publications

Purinergic regulation of the epithelial Na+ channel

Purinergic regulation of the epithelial Na+ channel

Antisense co-suppression of G α q and G α 11 demonstrates that both isoforms mediate M 3 -receptor-activated Ca 2+ signalling in intact epithelial cells

Purinergic signalling and cancer

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Purinergic regulation of the epithelial Na⁺ channel

Purinergic regulation of the epithelial Na⁺ channel