NUCLEOSIDE DIPHOSPHATE KINASE ASSOCIATED WITH RAT PANCREATIC MEMBRANES REGULATES CCK RECEPTOR AFFINITY

We have previously demonstrated in permeabilized rat pancreatic acini that the existence of two affinity states of the pancreatic cholecystokinin (CCK) receptor seen in intact cells depends on the presence of ATP. In the present study, we demonstrate that this effect of ATP is mediated by the enzyme nucleoside diphosphate kinase (NDPK). Northern blot hybridization analysis demonstrated NDPK mRNA in pancreas. Furthermore, pancreatic membranes possessed NDPK activity, which transferred high-energy phosphate groups to [8-H-3]GDP. This enzyme also utilized UTP and ITP as a source of gamma-phosphate for GTP formation while guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was formed in the presence of adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S). However, adenylyl (beta,gamma-methylene)-diphosphate (AMP-PCP) did not serve as a substrate for NDPK. Analysis of I-125-Bolton-Hunter-labeled CCK octapeptide ([I-125]BH-CCK-8) binding data in the absence of nucleotides was consistent with a single affinity state with dissociation constant (K-d) equal to 80 pM and maximal binding equal to 50.8 fmol/mg. ATP, UTP, ITP, ATP gamma S, and GTP gamma S all induced two CCK binding affinity states, which in the presence of 1 mM ATP were K-d = 74 pM for high-affinity sites and K-d = 4.3 nM for low-affinity sites; AMP-PCP did not induce two affinity states. GDP at 10 mu M had no effect on CCK binding but potentiated the effect of ATP. GTP gamma S, in addition to inducing high- and low-affinity states, also elicited a significant concentration-dependent reduction in the total number of measurable CCK receptors. Rate constants for dissociation of bound [I-125]BH-CCK-8 indicated that GTP gamma S-induced dissociation occurred about seven times as fast as ATP, suggesting that the effect of ATP was dependent on an enzymatically catalyzed reaction. The protein kinase inhibitors H-7 and staurosporine altered neither the activity of NDPK nor the ability of ATP to induce two CCK binding affinity states. It can be concluded that non-guanine base nucleotides act via NDPK to alter CCK receptor affinity. In addition, GTP gamma S elicits the disappearance of measurable CCK receptors, presumably by reducing receptor affinity to a new state in which affinity is too low to be distinguished from nonspecific binding.