| Abstract |
Normal adrenal chromaffin cells and their tumors, the pheochromocytomas, express the genes and secrete the products of several neuropeptides and cytokines which are considered regulators of the hypothalamus-pituitary-adrenal stress axis. Indeed, they produce corticotropin-releasing hormone (CRH), opioids, and the cytokines IL-1b and IL-6. Aim of the present work was to study the paracrine role(s) of these factors on several parameters of pheochromocytomas and compare them to normal adrenal chromaffin cells. We have studied the following parameters; (a) synthesis and secretion of catecholamines, (b) proliferation rates, and ( c) production of other neuropeptides. For this study we used two pheochromocytoma models: the PC12 rat pheochromocytoma cells and the KAT45 human pheochromocytoma cells and compared them to primary cultures of normal rat adrenal chromaffin cells. It is now well established that endogenous opioids participate in local regulatory loops within the central and peripheral nervous systems regulating the release of catecholamines from neighbouring cells via presynaptic opioid receptors. Opioids also, suppress the release of adrenal catecholamines in an autocrine / paracrine manner. Our results regarding this part of our experiments are as follows: the specific kappa opioid agonists U69593, U50488 and dynorphin Α inhibit the release of catecholamines from PC12 cells in a time- and dose dependent manner under basal and nicotine-induced conditions. DADLE (a mu and delta specific opioid agonist) and DAGO (synthetic mu opioid agonist) are less effective in suppressing catecholamines release from PC12 cells. The suppressive effect of all opioid agonists was blocked by naloxone suggesting that conventional opioid receptors were involved. Naloxone, also stimulate the release of catecholamines from PC12 cells in a dose-dependent manner which suggests that their secretion of catecholamines is under tonic inhibition by locally produced opioids. Several laboratories have shown that opioids affect the proliferation rate of normal and tumoral cells. Delta and mu opioids are the main opioid effectors of cell proliferation. In most instances their effect is inhibitory. Our results show that the kappa opioid agonists Dynorphin Α, U69593 και U50488 suppress basal proliferation of PC12 and KAT45 cells in a dose-dependent manner. DAGO, DADLE and DSLET don't alter the proliferation rate of PC12 cells, but suppress (only in high doses) the proliferation rate of KAT45 cells. The effect of kappa opioids is blocked by naloxone and the selective kappa opioid antagonist nor-binaltorphimine. Furthermore, both opioid antagonists have a strong stimulatory effect on the proliferation rate of both cell lines, suggesting that the proliferation of PC12 and KAT45 cells is under tonic inhibition by locally produced kappa opioids. Finally, the kappa opioid agonist U69593 inhibited the EGF-induced proliferation of PC12 cells, a finding suggesting that the effect of kappa opioisd may involve the suppression of EGF-dependent oncogenes. Several lines of evidence suggest that the adrenal medulla of several species and some human pheochromocytomas produce CRH. In the central nervous system (CNS) there is a close anatomical proximity between CRH and catecholaminergic neurons a finding suggesting that CRH affect the release of catecholamines. There is evidence indicate that CRH plays a similar role in the adrenal medulla since the latter contains CRH receptors. Our results are as follows: normal adrenal chromaffin cells and PC12 and KAT45 cells contain and secrete comparable amounts of immunoreactive (IR)-CRH under basal conditions and following K+-induced depolarization, nicotine, IL-1b and dexamethasone. The physicochemical characteristics of the IR-CRH in the cells and the media were identical to the putative CRH peptide on both sieve chromatography and RP-HPLC. Synthetic CRH induced the production of catecholamines from the three cell types in a dose- and time-dependent manner. This effect was abolished by the antagonist ahCRH. Synthetic CRH stimulates the production of ACTH and β-endorphin from KAT45 cells. CRH, also, inhibits the proliferation rate of KAT45 cells. Exposure of PC12 cells to NGF (for one week) resulted in their neuronal differentiation and the stimulation of their production of CRH by thirty times and of dopamine by ten times compared to parallel controls. This effect of NGF was abolished by ahCRH. Finally, it is now known that the adrenal medulla of several species and some pheochromocytomas contain and produce intreleukin-1 and -6. In CNS there is a close anatomical proximity between IL-1β and -6 and CRH. Il-1β induces the production of hypothalamic CRH. IL-6 is also a potent inducer of ACTH and CRH. Our results show that: KAT45 cells produce IL-1β and IL-6. CRH induces the production of IL-1β from KAT45 cells, which in turn stimulates the production of catecholamines. This effect is blocked by IL-1ra. rhIL-1β induces the production of CRH from KAT45 cells. CRH inhibit the production of IL-6 from KAT45 cells in a dose-dependent manner and this phenomenon is blocked by CRH antagonist, ahCRH. rhIL-6 has no effect on catecholamine secretion. In conclusion, our data suggest that there two regulatory loops within the normal and tumoral adrenal medulla regulating the release of catecholamines. CRH is the main effector both loops. In the first, CRH inhibits catecholamines release via the induction of the production of opioids which affect also the proliferation rate of the cells. In the second, CRH stimulates the production of catecholamines via the stimulation of the production of IL-1β. The interaction between these two loops appears to be important for the regulation of several chromaffin cell functions.
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