Previous studies have indicated that activation ofthe CCK-B receptor on GH3 rat pituitary tumor cells leads to Ca2+ mobilization in association with stimulation of PI turnover and IP3 production (7). The present findings are consistent with these studies and show that human functionless pituitary tumors cells also possess biologically active CCK-B receptors. Moreover, using reverse transcription followed by restriction digestion, we have demonstrated that functionless human pituitary tumors express CCK-B receptor mRNA but not CCK-A receptor. Taken together, these data correlate with previous findings that suggest GH3 cells express a single class of high-affinity binding sites for radiolabeled CCK-8 with a Kd of48 pM. The binding sites had high affinity for CCK-8 and CCK-B antagonists, but low affinity for CCK-A antagonists, indicating that the binding sites are for CCK-B receptors (4).

Figure 2. Agonist induced increase in PPI turnover in human pituitary tumor cells in a dose dependant manner after 6.0-h incubations. Mean values of all triplicate results obtained for each dosage are presented. Results are shown as number of cells per flask The bars represent the mean ± SD.

The role of IPs as intracellular second messengers is well established, with activation of cell surface receptors, promoting hydrolysis of membrane-bound PI 4,5-bisphosphate to generate IP3 and diacylglycerol. The former mobilizes Ca2+ from intracellular stores, and the latter can activates the PKC enzyme family, bringing about protein phosphorylation. Many other IPs have been identified although their precise roles are still under investigation. In the present study, we have examined CCK-B receptor signal transduction. The results suggest that PI turnover is not a signaling mechanism exclusive to CCK-A receptors and demonstrate that CCK-B receptors on cells extracted from human pituitary adenomas are part ofthe family ofreceptors which employ IPs in cell signaling (2).

With respect to desensitization of CCK-B receptors in GH3 cells, it has been reported that they show both homologous and heterologous desensitization. The initial exposure to CCK-8 reduced the responsiveness to the second exposure to CCK-8, leading to the conclusion that desensitization of the CCK-B receptors is mediated by some intracellular messengers (3). The fact that a supramaximal decline in response was consistently observed in agonist dose/response curves at high concentrations suggests that it may be desensitization. Some recent studies have shown evidence that PI hydrolysis is biphasic with a faster initial rate during the first 30 sec of agonist exposure, and with a subsequent rate reduction afterwards, indicating a possible mediation by rapid receptor phosphorylation. Evidence suggests that CCK receptors undergo rapid but partial desensitization through phosphorylation in a concentration-dependent manner in response to agonists (8). In addition, Li suppresses the supply of free inositol for PI resynthesis, which may result in a stimulus-dependent desensitization of the agonist effects (8). It is possible that similar desensitization mechanisms are present in human functionless pituitary tumor cells, although further confirmation studies are required.

The role of CCK-B receptors in pituitary cell function remains to be determined. However, there is considerable evidence that CCK receptors are coupled to a mitogenic response (5). In addition, PI hydrolysis is coupled to hormone secretion (9, 10), and thus it is conceivable that CCK may modulate the effects of hypothalamic releasing and inhibiting factors. These two observations together with the present findings raise the possibility that CCK may play a role in human pituitary tumorigenesis.

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