ABSTRACT
The presence of VIP/PACAP receptors was investigated on the human erythroleukemic cell line HEL. Specific binding of [125I]-PACAP or [125I]-VIP on HEL cells or membranes was very low and did not allow to perform competition curves. At 37 degrees C PACAP transiently increased cAMP levels in the presence of the non-specific phosphodiesterase inhibitor IBMX, suggesting rapid desensitization. Kinetic studies revealed that optimal conditions to measure the EC(50) of PACAP(1-27) were 10 min at 20 degrees C. Under those conditions, PACAP-related peptides increased cAMP levels with EC(50) in agreement with the pharmacological profile of the VPAC(1) receptor subtype: PACAP = VIP > [K(15), R(16,) L(27)]VIP(1-7)/GRF(8-27) = [R(16)]ChSn (two VPAC(1) agonists) >> helodermin = secretin. RO 25-1553, a selective activator of VPAC(2) receptor was inactive at 1 microM. Dose-response curves of VPAC(1) agonist molecules (PACAP, VIP, [K(15), R(16), L(27)]VIP(1-7)/GRF(8-27), [R(16)]ChSn) were shifted to the right by the VPAC(1) receptor antagonist [AcHis(1), D-Phe(2), Lys(15), Leu(17)]VIP(3-7)/GRF(8-27), with a K(i) of 3 +/- 1 nM (n = 3). The presence of VPAC(1) receptor mRNA was confirmed by RT-PCR. Preincubation with PACAP or PMA showed that VPAC(1) receptors underwent homologous and heterologous desensitization. This study provides the first evidence for the expression of functional VPAC(1) receptors undergoing rapid desensitization in HEL cells.
Subject(s)
Leukemia, Erythroblastic, Acute/metabolism , Receptors, Pituitary Hormone/metabolism , Receptors, Vasoactive Intestinal Peptide/metabolism , Base Sequence , Cyclic AMP/metabolism , DNA Primers , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Humans , Leukemia, Erythroblastic, Acute/pathology , Neuropeptides/pharmacology , Pituitary Adenylate Cyclase-Activating Polypeptide , Protein Kinase Inhibitors , RNA, Messenger/genetics , Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide , Receptors, Pituitary Hormone/genetics , Receptors, Vasoactive Intestinal Peptide/genetics , Temperature , Tumor Cells, CulturedABSTRACT
Cricetomys gambianus are rodents living in savanna and follow area. They can live with restricted drinking water eating fresh food. Therefore their kidney may have some adaptive mechanisms for ion/water homeostasis compared to usual laboratory rats. In this study we have looked for calbindin, an intracellular calcium binding protein previously found in distal convoluted tubules from all mammalian species that have been studied and able to increase, in vitro, Ca2+ reabsorption. We have shown by using in situ hybridization, immunoblotting and immunohistochemistry that calbindin was expressed in three different portions of the distal nephron of the African giant rat. Calbindin was found in distal convoluted tubules, in cortical collecting tubules and in outer medullary collecting ducts. By contrast, in laboratory rat, calbindin was only found in distal convoluted tubules and undetectable in medullary collecting ducts. Thick ascending limb of Henle's loop were calbindin negative as shown by double immunolabelling using anti-uromucoid (Tamm-Horsfall protein). As previously shown in laboratory rat and rabbit, transcellular Ca2+ movement seems to be facilitated by calbindin in renal tubules segments predominantly actively transporting Ca2+, it may be suggested that in African giant rat, outer medullary collecting ducts may also actively transport Ca2+. As calretinin, another intracellular calcium binding protein highly homologous to calbindin but whose function is still conjectural has been suspected to be expressed in kidney, we have looked and not found any calretinin in both adult rat species.
