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Modifications of VIP interaction with intestinal epithelial cells and hepatic plasma membranes from partially hepatectomized rats
$44 BINDING SITES AND RESPONSE OF RAT PANCREATIC ACINI TO HELODERMIN ( H d ) . J. WlNAND T J . - P . DEHAYEI P. ROBBERECHT AND J. CHRISTOPHE Department of Biochemistry and N u t r i t i o n 9 Medical School, Universit~ L i b r a de Bruxelles, Brussels, Belgium. Helodermin (Hd) is a new peptide of the VIP/PHI/secretin family isolated from the venom of Heloderma suspectum. In rat pancreatic membranes, Hd stimulates adenylste cyclase through secretin (Sn) receptors : this stimulation is inhibited not only by helodermin-(7-35) but also, like that of Sn, by secretin-(4-27) and secretin-(7-27). In intact pancreatic acini, Hd increases cAMP 12-fold (ECSO 56 nM). Hd in the 1 to lO0 nM range induces the phosphorylation of 3 particulate proteins (21,25 and 33K). Hd also stimulates amylase secretion in s biphasic pattern: a slight 2-fold increase above 1 nM and a further 4-fold increase above I00 nM. Similar results are obtained with Sn while only the first modest increase in amylase secretion is obtained with VIP and PHI. The 4 peptides potentiate the secretory effect of
CCK-81251-Hd binds to rat pancreatic acini. Competition curves for Hd and PHI suggest binding to one population of sites (Kd of 20 nM and 560 nM, respectively). The curve for VIP could be best fitted with 85~ of the sites being occupied with a Kd of 70 nM. The competition curve for Sn is best fitted by considering the coexistence of two sites (15 ~ with a Kd of 2 nM, and 85% with a Kd of 180 nM). Taken together, these results suggest that Hd binds to three populations of Sn receptors: l/ high affinity receptors responsible for the slight increase in cAMP when occupied by Sn at a i nM concentration, 2/ a low affinity-high capacity population of Sn receptors also able to bind Hd, VIP and PHI, responsible for most of the cAMP increase and protein phosphorylation, for slight amylase release (per se) but also for the potentiation of the secretory response to CCK-8 with all four peptides of the Sn family; 3/ very low affinity Sn receptors provoking most of the emeiocytosis at high concentrations of Sn and Hd.
MODIFICATIONS OF VIP INTERACTION WITH INTESTINAL EPITHELIAL CELLS AND HEPATIC PLASMA MEMBRANES FROM PARTIALLY HEPATECTOMIZED RATS Juan C. Prieto~ Jos~ L. Diaz-Ju~rez~ Eduardo Arilla Departamento de Bioqulmica, Facultad de Medicina, Alcal~ de Henares-Madrid, Spain VIP plasma levels appear to increase in cirrhosis (Henriksen et al.,Scand. J. Gastroenterol., 15, 787, 1980) and other liver diseases (Hunt et al., Arch. Intern. Med., 139, 994, 1979), probably due to compromised hepatic elimination.The present study was undertaken in order to observe possible modifications of VlP interaction at the membrane level with both isolated cells of small (jejuno-ileal villous and crypt cells) and large (colonic epithelial cells) intestine, as well as hepatic plasma membranes, three days after excision of 70% of the liver. Male Wistar rats (180-200 g) were used, methods of cell and membrane isolation and of VlP binding and stimulation of cAMP being those previously described (Prieto et al., Acta End~ crinol., 96, i00, 1981). The binding capacity, but not the affinity, of VlP receptors (either of high and low affinity) in jejuno-ileal (from both villous and crypt origin) as well as in colonic epithelial cells increased as a consequence of the surgical manipulation. Accordingly, VlP was equally potent but showed a higher efficiency in stimulating cAMP in intestinal epithelial cells of hepatectomized rats, as compared with sham-operated animals. These results could be a consequence of increased plasma levels of VIP thus indicating an up-regulation of VIP receptors by the circulating concentration of the neuropeptide. However, it should be noted that VIP is locally released by nerve endings at the site of action, the plasma concentration of VIP reflecting only a dynamic state of continuous release of VIP from the neurons and ineffective postsynaptic elimination (Rosselin et al., Mol. Cell. Endocrinol., 27, 243, 1982). A very different pattern was observed at the hepatic level since the affinity, but not the binding capacity, of VIP receptors (either of high and low affinity) decreased in the hepatectomized group. This last result could support a role of VIP in the mechanisms of growth of the regenerating liver but it could be also a consequence of the adaptive response.
CCK-81251-Hd binds to rat pancreatic acini. Competition curves for Hd and PHI suggest binding to one population of sites (Kd of 20 nM and 560 nM, respectively). The curve for VIP could be best fitted with 85~ of the sites being occupied with a Kd of 70 nM. The competition curve for Sn is best fitted by considering the coexistence of two sites (15 ~ with a Kd of 2 nM, and 85% with a Kd of 180 nM). Taken together, these results suggest that Hd binds to three populations of Sn receptors: l/ high affinity receptors responsible for the slight increase in cAMP when occupied by Sn at a i nM concentration, 2/ a low affinity-high capacity population of Sn receptors also able to bind Hd, VIP and PHI, responsible for most of the cAMP increase and protein phosphorylation, for slight amylase release (per se) but also for the potentiation of the secretory response to CCK-8 with all four peptides of the Sn family; 3/ very low affinity Sn receptors provoking most of the emeiocytosis at high concentrations of Sn and Hd.
MODIFICATIONS OF VIP INTERACTION WITH INTESTINAL EPITHELIAL CELLS AND HEPATIC PLASMA MEMBRANES FROM PARTIALLY HEPATECTOMIZED RATS Juan C. Prieto~ Jos~ L. Diaz-Ju~rez~ Eduardo Arilla Departamento de Bioqulmica, Facultad de Medicina, Alcal~ de Henares-Madrid, Spain VIP plasma levels appear to increase in cirrhosis (Henriksen et al.,Scand. J. Gastroenterol., 15, 787, 1980) and other liver diseases (Hunt et al., Arch. Intern. Med., 139, 994, 1979), probably due to compromised hepatic elimination.The present study was undertaken in order to observe possible modifications of VlP interaction at the membrane level with both isolated cells of small (jejuno-ileal villous and crypt cells) and large (colonic epithelial cells) intestine, as well as hepatic plasma membranes, three days after excision of 70% of the liver. Male Wistar rats (180-200 g) were used, methods of cell and membrane isolation and of VlP binding and stimulation of cAMP being those previously described (Prieto et al., Acta End~ crinol., 96, i00, 1981). The binding capacity, but not the affinity, of VlP receptors (either of high and low affinity) in jejuno-ileal (from both villous and crypt origin) as well as in colonic epithelial cells increased as a consequence of the surgical manipulation. Accordingly, VlP was equally potent but showed a higher efficiency in stimulating cAMP in intestinal epithelial cells of hepatectomized rats, as compared with sham-operated animals. These results could be a consequence of increased plasma levels of VIP thus indicating an up-regulation of VIP receptors by the circulating concentration of the neuropeptide. However, it should be noted that VIP is locally released by nerve endings at the site of action, the plasma concentration of VIP reflecting only a dynamic state of continuous release of VIP from the neurons and ineffective postsynaptic elimination (Rosselin et al., Mol. Cell. Endocrinol., 27, 243, 1982). A very different pattern was observed at the hepatic level since the affinity, but not the binding capacity, of VIP receptors (either of high and low affinity) decreased in the hepatectomized group. This last result could support a role of VIP in the mechanisms of growth of the regenerating liver but it could be also a consequence of the adaptive response.