Characterization of binding sites for VIP-related peptides and activation of adenylate cyclase in developing pancreas.
HPLC-purified 125I-labeled vasoactive intestinal peptide (VIP) bound in a specific, saturable, and reversible manner to pancreatic plasma membranes isolated from newborn calves, from milk-fed calves at 28 and 119 days, and from weaned calves at 119 days. A series of VIP analogues, including pituitary adenylate cyclase-activating polypeptide (PACAP), displaced 125I-VIP binding and activated adenylate cyclase in the same order of relative potency: PACAP-38 greater than helodermin greater than VIP, PACAP-27 greater than PHM (human peptide with NH2-terminal histidine and COOH-terminal methionine amide). At maximally effective concentrations, these five peptides produced the same two- to threefold increase of adenylate cyclase activity in pancreatic membranes from newborn and 28-day-old calves, and fourfold in ruminant or preruminant animals at 119 days. The activation constant for PACAP-38 ranged from 0.1 to 0.34 nM throughout the postnatal development. Helospectin I and II were three times less potent than VIP in inhibiting 125I-VIP binding. At concentrations up to 0.1 microM, secretin, rat and human growth hormone-releasing factors, glucagon, oxyntomodulin, the truncated form of glucagon-like peptide-1 lacking the 6 NH2-terminal amino acid sequence (TGLP-1), GLP-2, gastric inhibitory peptide, gastrin, CCK, and insulin had no effect on binding. Scatchard plots from 28- and 119-day-old calves were compatible with the presence of two classes of 125I-VIP binding sites: one with a high affinity for VIP and a low binding capacity (Kd = 0.11-0.4 nM, Bmax = 66-174 fmol/mg protein) and the other with a low affinity and high binding capacity. At birth, only one class of binding sites was observed (Kd = 0.4 nM, Bmax = 858 fmol/mg protein). The covalently cross-linked PACAP-preferring 125I-VIP binding site is a glycoprotein of 55 kDa with higher sensitivity to PACAP vs. helodermin and VIP. Our results suggest that calf pancreatic functions might be regulated at an early stage of postnatal development by PACAP receptors linked to cAMP generation.
Le Meuth
V
V
Institut National de la Santé et de la Recherche Médicale, U55, Hôpital Saint-Antoine, Paris, France.
Farjaudon
N
N
Bawab
W
W
Chastre
E
E
Rosselin
G
G
Guilloteau
P
P
Gespach
C
C
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism; D000375:Aging; D000818:Animals; D000831:Animals, Newborn; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D004789:Enzyme Activation; D007700:Kinetics; D008297:Male; D010179:Pancreas / Q000254:growth & development* / Q000378:metabolism; D011964:Receptors, Gastrointestinal Hormone / Q000302:isolation & purification / Q000378:metabolism*; D018005:Receptors, Vasoactive Intestinal Peptide; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism* / Q000494:pharmacology
D000345:Affinity Labels; D011964:Receptors, Gastrointestinal Hormone; D018005:Receptors, Vasoactive Intestinal Peptide; D014660:Vasoactive Intestinal Peptide; D000262:Adenylyl Cyclases