Quantitative and functional characterization of muscarinic receptor subtypes in insulin-secreting cell lines and rat pancreatic islets

• Published in: Diabetes (New York, N.Y.) Vol. 49; no. 3; pp. 392 - 398
• Main Authors: Iismaa, T P, Kerr, E A, Wilson, J R, Carpenter, L, Sims, N, Biden, T J
• Format: Journal Article
• Language: English
• Published: United States American Diabetes Association 01-03-2000
• Subjects: Animals; Carbachol - pharmacology; Cells, Cultured; Cholinergic Agonists - pharmacology; Hydrolysis; Immunohistochemistry; Insulin - metabolism; Insulin Secretion; Islands of Langerhans; Islets of Langerhans; Islets of Langerhans - drug effects; Islets of Langerhans - metabolism; Male; Muscarinic antagonists; Muscarinic Antagonists - metabolism; Muscarinic receptors; Phosphatidylinositol 4,5-Diphosphate - metabolism; Physiological aspects; Polymerase chain reaction; Protein Isoforms - genetics; Protein Isoforms - metabolism; Protein Isoforms - physiology; Quinuclidinyl Benzilate - metabolism; Rats; Rats, Wistar; Receptors, Muscarinic - genetics; Receptors, Muscarinic - metabolism; Receptors, Muscarinic - physiology; Transfection
• ISSN: 0012-1797; 1939-327X
• DOI: 10.2337/diabetes.49.3.392

Quantitative and functional characterization of muscarinic receptor subtypes in insulin-secreting cell lines and rat pancreatic
islets.
T P Iismaa ,
E A Kerr ,
J R Wilson ,
L Carpenter ,
N Sims and
T J Biden
Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, Australia.
Abstract
Expression of muscarinic receptors in rat islets, RINm5F cells, and INS-1 cells was established by reverse transcriptase-polymerase
chain reaction (RT-PCR) and quantified by RNase protection. Both methods indicated that m3 and m1 receptors were expressed
approximately equally in the various cellular preparations and to a much greater extent than the m5 subtype. However, the
cell lines, especially RINm5F cells, expressed less of a given receptor subtype than did islets. Immunohistochemistry indicated
that m3 receptors were expressed throughout the islet core. Binding studies using the radiolabeled muscarinic receptor antagonist
QNB demonstrated a maximal binding capacity of INS-1 cells of 23.0+/-2.9 fmol/mg protein. Functional analyses were undertaken
using INS-1 cells stably transfected with either m1 or m3 receptor cDNAs. Overexpression of either receptor did not affect
basal responses but markedly enhanced maximal responses to the muscarinic receptor agonist carbachol. Although maximal hydrolysis
of phosphatidylinositol 4,5-bisphosphate (Ptd InsP2) was twofold greater in m1-transfectants as compared with m3-transfectants,
cell lines overexpressing either receptor gave essentially equivalent secretory responses to a full range of carbachol doses.
The results demonstrate that both m1 and m3 muscarinic receptors are well expressed in pancreatic beta-cells, functionally
linked to signaling pathways, and capable of initiating insulin secretion with equal potencies.