| Abstract |
Neuropeptide Y (NPY) and peptide YY (PYY) are potent antisecretory
agonists in the gastrointestinal tract, attenuating electrogenic chloride
and fluid secretion in vitro and in vivo in various species. Human
adenocarcinoma cell lines provide model systems in which to investigate
the mechanisms by which epithelial Y receptors modulate anion transport,
in the absence of other mucosal cell types. Two of these (HT-29 and HCA-7
Colony 1) do not possess PYY receptors and were consequently stably
transfected with the cDNA encoding the Y1 subtype, endogenously expressed
in human colonic mucosa. Both wild type epithelia and the Y1 clones
responded to a similar range of secretory and antisecretory agents when
voltage-clamped in Ussing chambers; in addition, PYY transiently reduced
both basal and cAMP-stimulated short-circuit current in Y1 epithelial
layers. These antisecretory responses were inhibited by the Y1 antagonists
BIBP 3226 and GR231118. Surprisingly, PYY and [Leu31, Pro34] (Pro34) PYY
were 10-20 fold more potent than NPY or Pro34NPY in both HT-29 and Colony
1 clones, despite each agonist displaying equivalent binding affinities.
It is suggested that metabolism by membrane surface peptidases may
underlie this selectivity, and that this may influence responses at native
mucosal Y1 receptors. The Colony 1 Y1 clone was also compared with those
transfected with Y1 receptor cDNAs containing single amino acid mutations.
The Y1(S255E) receptor was apparently down-regulated, with associated
consequences for PYY antisecretory responses; phosphorylation of Ser255 in
the third intracellular loop (imitated by Glu) may therefore be important
in desensitisation. Substitution of Cys337, potentially palmitoylated, did
not alter receptor expression or the functional potency of PYY. However
Y1(C337S) mediated responses remained sustained at high agonist
concentration, indicating that Y1 receptor depalmitoylation may inhibit
its inactivation. Epithelial expression of receptor mutants may therefore
be valuable in assessing their functional properties, particularly
regarding desensitisation.
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