Differential coupling of the P2Y1 receptor to Galpha14 and Galphaq/11 proteins during the development of the rat salivary gland.
In rat submandibular gland (SMG), the P2Y1 receptor (P2Y1R) mediates increases in the intracellular calcium concentration, [Ca2+]i that diminish as the animal ages from 1 to 4-6 weeks. However, P2Y1R mRNA levels do not change with age, suggesting that the age-dependent decrease in the [Ca2+]i response to P2Y1R agonists may be due to alterations in the activity of a component of the P2Y1R signalling pathway.


OBJECTIVE
To assess whether the decrease in P2Y1R-mediated intracellular calcium signalling in SMG cells as rats age is due to a decrease in P2Y1R coupling to G proteins or to a decrease in the expression of a cognate G protein.


METHODS
SMG cells were isolated from Sprague-Dawley rats. P2Y1R function was assessed by measuring 2-MeSADP-induced increases in [Ca2+]i and ERK1/2 activation. P2Y(1)R-mediated activation of G proteins was determined by the [35S]GTPgammaS binding assay. Gq protein expression was determined by RT-PCR, Northern, and Western analysis.


RESULTS
In SMG cells from 1-week-old rats, two bands (52 and 42kDa) were detected using anti-Galpha14 antibody, whereas in SMG cells from 4- to 6-week-old rats only the 42 kDa band was detected. Furthermore, 2-MeSADP-induced GTPgamma35S binding to Galpha14 and Galphaq/11 decreases in SMG cells from 4- to 6-week-old rats as compared to 1-week-old rats.


CONCLUSIONS
These findings suggest that the age-dependent decrease in P2Y1R-mediated intracellular calcium signalling in rat SMG cells is due to a loss of 52 kDa Galpha14 and indicate the differential coupling of the P2Y1R to Galpha14 and Galphaq/11 as the gland develops.
Baker
Olga J
OJ
Department of Biochemistry, University of Missouri-Columbia, 540E Life Sciences Center, 1201 Rollins Road, Columbia, MO 65211-7310, USA.
Camden
Jean M
JM
Ratchford
Ann M
AM
Seye
Cheikh I
CI
Erb
Laurie
L
Weisman
Gary A
GA
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D015152:Blotting, Northern / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002217:Carbachol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D018721:Muscarinic Agonists / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding / Q000187:drug effects; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D051381:Rats; D017207:Rats, Sprague-Dawley; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058472:Receptors, Purinergic P2Y1; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012469:Salivary Glands / Q000254:growth & development* / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology
D044385:GTP-Binding Protein alpha Subunits; D018721:Muscarinic Agonists; C111914:N(6)-methyl-2'-deoxyadenosine 3',5'-diphosphate; C546384:P2ry1 protein, rat; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D013873:Thionucleotides; C002480:methylthio-ADP; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000244:Adenosine Diphosphate; D002217:Carbachol; D004492:Edetic Acid; D048049:Extracellular Signal-Regulated MAP Kinases; D002118:Calcium