Desensitisation of native and recombinant human urotensin-II receptors.
Human urotensin-II (U-II) is an 11-amino-acid cyclic peptide that activates a G(q)-coupled receptor named UT. Little is known about the desensitisation profile of this receptor as peptide binding is essentially irreversible. In the present study, we have examined the effects of U-II and carbachol on Ca(2+) signalling in SJCRH30 rhabdomyosarcoma (receptor density, B(max) approximately 0.1 pmol/mg protein) and human embroynic kidney (HEK)(hUT) (B(max) approximately 1.4 pmol/mg protein) cells expressing native and recombinant UT, respectively. In SJCRH30, HEK(hUT) and human peripheral blood mononuclear cells induced to express native UT by treatment with 2 microg/ml lipopolysaccharide (LPS), we have measured the effects of U-II treatment on UT mRNA. In SJCRH30 cells, primary stimulation with carbachol (250 microM) did not affect a secondary challenge with U-II (1 microM) and primary challenge with U-II did not affect a secondary challenge with carbachol. In contrast, in HEK(hUT) cells, primary stimulation with carbachol (250 microM) reduced a secondary challenge to U-II (1 microM) by 84% and primary challenge with U-II reduced a secondary challenge to carbachol by 76%. Pre-treatment of SJCRH30 cells with U-II reduced UT mRNA after 6 h and this returned to basal after 24 h. In recombinant HEK(hUT) cells, UT mRNA expression increased following a 6 h treatment with 1 microM U-II. U-II treatment of naïve un-stimulated peripheral blood mononuclear cells was without effect. However, when UT expression is up-regulated following 15 h of LPS treatment, a 6 h U-II challenge reduced UT mRNA by 66%. In summary, we report differences in the desensitisation profiles of native and recombinant U-II receptors. Design and interpretation of functional experiments are hampered by irreversibility of U-II binding.
Batuwangala
Madura S
MS
Division of Anaesthesia, Critical Care and Pain Management, University Department of Cardiovascular Sciences (Pharmacology and Therapeutics Group), University of Leicester, Leicester, LE1 5WW, UK.
Calo
Girolamo
G
Guerrini
Remo
R
Ng
Leong L
LL
McDonald
John
J
Lambert
David G
DG
D000328:Adult; D020013:Calcium Signaling / Q000187:drug effects; D002217:Carbachol / Q000494:pharmacology*; D002460:Cell Line; D045744:Cell Line, Tumor; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008070:Lipopolysaccharides; D008875:Middle Aged; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012208:Rhabdomyosarcoma / Q000378:metabolism; D014579:Urotensins / Q000494:pharmacology*
D008070:Lipopolysaccharides; D012333:RNA, Messenger; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; C121697:UTS2R protein, human; D014579:Urotensins; D002217:Carbachol; C029677:urotensin II
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