The most effective therapy of human prolactinomas is represented by dopamine D-2 receptor agonists; there is, however, a population of nonresponder patients who require surgical intervention. In the present study, we report that prolactinomas totally resistant to pharmacological therapy have a high potential of both growing in soft agar and forming tumors in nude mice and lack D-2 receptors for dopamine. These tumors express the receptors for nerve growth factor (NGF) and are sensitive to its differentiating activity. After exposure to NGF for 4 days, prolactinoma cells decreased their proliferation rate, lost their capability to form colonies in soft agar, lost their tumorigenic activity in nude mice, and reexpressed the lactotroph-specific D-2 receptor protein inhibiting prolactin release. These effects were permanent after NGF withdrawal and were reproducible in vivo in nude mice transplanted with the tumors. NGF in fact remarkably and lastingly depressed tumor growth and induced expression of D-2 receptors when injected intravenously once a day for 5 days into prolactinoma-bearing nude mice. These data suggest that NGF may induce a long-lasting switch of gene expression in human prolactinomas, modifying their transforming phenotype and reverting them to more differentiated, less malignant, dopamine-sensitive lactotroph-like cells. The possibility thus arises that short-term treatment with NGF may restore the refractory patients to conventional pharmacological therapy with D-2 agonists.
Na+-dependent binding of 3H-Cocaine is located on dopaminergic terminals and is related to the dopamine uptake system. Studies on senescent rats indicate a decrease in 3H-Cocaine binding density in the striatum. In the same tissue an increase in the Km for dopamine uptake is observed. These changes might be relevant in the age-dependent alterations in dopaminergic function.
We summarize here our data showing that various phenotypical characteristics distinguish prolactinoma cell lines obtained from responder and nonresponder patients, as defined by their responses to bromocriptine administration. Nonresponder cell lines have a higher degree of malignancy than responder cells and do not express D2 receptors for dopamine. Both cell lines express NGF receptors. Exposure of the most malignant nonresponder cell lines to NGF, both in vitro and when transplanted in vivo in nude mice, results in their differentiation into the responder phenotype reexpressing D2 receptors. Sequential administration of NGF and bromocriptine thus may be a promising therapy for patients refractory to bromocriptine.
Measuring adenylate cyclase activity (AC) as a biochemical index of dopamine (DA) receptor function, we obtained evidence for the presence in rabbit vasculature of both D1 receptors associated with stimulation of AC and of D2 receptors coupled with AC in an inhibitory way. The cAMP generating system in rabbit mesenteric artery was stimulated by DA and several DA agonists, an effect antagonized by the D1-receptor blocker SCH 23390 and by other neuroleptic drugs. When activation of D1 sites was impeded by SCH 23390, DA, (-)apomorphine, and (-)NPA inhibited cAMP formation. In addition, selective D2 agonists inhibited basal AC activity even when there was no D1-receptor blockade. The relative order of potency of various neuroleptics in antagonizing bromocriptine-induced inhibition of AC confirmed the D2 nature of these binding sites. Inhibition of AC activity elicited by bromocriptine remained unchanged after chemical sympathectomy, suggesting that vascular D2 receptors inhibiting AC activity are located postsynaptically in the arterial wall.
Mutations in the presenilin 1 and presenilin 2 genes account for the majority of familial Alzheimer disease (FAD) cases in which a genetic defect was detected. The majority of presenilin mutations result in increased production/aggregation of amyloid beta–peptide (Abeta), the main constituent of senile plaques. Several experimental evidences suggest that cystatin C exerts a protective role in the brain favoring stem cell proliferation and interfering with Aβ formation and deposition. In sporadic AD brain, cystatin C protein is accumulated intracellularly in neurons more susceptible to neurodegeneration. The aim of this study was to investigate the interplay between presenilin 2 and cystatin C taking in consideration the role played by the recently described presenilin 2 T122R (PS2 T122R) mutation. Mouse primary cortical neurons were transfected by using pcDNA3 void vector, pcDNA3 PS2 wt/T122R constructs. Media and cells lysates were analyzed by Western blot. Proteins trafficking was investigated using confocal imaging. Inducible PS2–wt, –T122R expression system: the transactivator (t–TA)–expressing HeLa Tet–Off cell line was transiently transfected with pMAR void vector, PS2 wt/T122R. In order to modulate PS2 protein expression, increasing doses of doxycyline were used. In neurons, the over–expression of PS2 T122R resulted in an intracellular accumulation and in an extracellular reduction of native and glycosylated cystatin C. Experiments performed in the tetracycline–inducible PS2 expression system demonstrated that the effect of PS2 T122R on this phenotype is tightly related to the dose of the over–expressed mutant protein. Confocal imaging showed that the effect of PS2 T122R mutation was a massive recruitment of cystatin C into the neuronal processes, in the presence of an intact cytoskeletal structure. Taken together, our data suggest that the reduced extracellular levels of cystatin C could be a consequence of the massive recruitment of cystatin C in the intracellular compartment, mostly in the axons, probably occurring through an increase of its endocytotic reuptake. We might speculate that cystatin C could act to protect cells from damage caused by a misregulation of axonal transport, an early and critical step for the initiation of neuronal pathology.
