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Activity-dependent secretion of brain-derived neurotrophic factor (BDNF) is thought to enhance synaptic plasticity, but the mechanisms controlling extracellular availability and clearance of secreted BDNF are poorly understood. We show that BDNF is secreted in its precursor form (pro-BDNF) and is then cleared from the extracellular space through rapid uptake by nearby astrocytes after theta-burst stimulation in layer II/III of cortical slices, a paradigm resulting in long-term potentiation of synaptic transmission. Internalization of pro-BDNF occurs via the formation of a complex with the pan-neurotrophin receptor p75 and subsequent clathrin-dependent endocytosis. Fluorescence-tagged pro-BDNF and real-time total internal reflection fluorescence microscopy in cultured astrocytes is used to monitor single endocytic vesicles in response to the neurotransmitter glutamate. We find that endocytosed pro-BDNF is routed into a fast recycling pathway for subsequent soluble NSF attachment protein receptor-dependent secretion. Thus, astrocytes contain an endocytic compartment competent for pro-BDNF recycling, suggesting a specialized form of bidirectional communication between neurons and glia.

Prodynorphin (Prodyn)-derived peptides are synthesized in a subset of gonadotrope cells and released concomitantly with LH and FSH, and their levels in the rat adenohypophysis are influenced by the gonadal steroid environment. In several hormonal systems, factors that affect peptide levels may modulate the transcription of messenger RNA (mRNA) encoding for the target gene. Therefore, the present study was designed to investigate the effects of gonadal ablation and estrogen replacement on changes in steady state levels of anterior pituitary Prodyn mRNA and on the transcription rate in the adult female rat. The antiestrogen tamoxifen was employed for further exploring the relationships between estrogens and dynorphin (dyn)-related peptides. Adopting a solution hybridization-ribonuclease protection assay, steady state levels of Prodyn mRNA doubled in 2-week ovariectomized (OVX) rats, in parallel with a 3-fold increase in immunoreactive dyn-A-(1-17)-like material (irdyn-A). Estradiol (E2) replacement through sc SILASTIC implants for 1, 3, 7, and 14 days, which produces serum E2 levels between 25-35 pg/ml, restored in a time-dependent manner mRNA and peptide concentrations to values in sham-OVX rats. A significant decrease was observed after 3 days, and after 7 days, the effect was maximal. Tamoxifen (250 micrograms/kg.day, sc) administered simultaneously antagonized the action of E2 on Prodyn gene expression. Tamoxifen administered without E2 for 7 or 14 days significantly raised anterior pituitary levels of Prodyn mRNA and ir-dyn-A. To establish whether E2 and tamoxifen exert their effects on adenohypophyseal Prodyn mRNA by influencing the transcriptional activity of this gene, an in vitro transcriptional elongation assay was performed on nuclei from the anterior pituitary. The transcriptional rate of the Prodyn gene was significantly increased in 2-week OVX rats. Prodyn mRNA synthesis was suppressed in OVX rats exposed to E2, an effect antagonized by tamoxifen administered concomitantly. The antiestrogen administered alone for 14 days further elevated the transcriptional rate of Prodyn mRNA induced by gonadal ablation. In conclusion, E2 down-regulated the synthesis of Prodyn-derived peptides in adenohypophyseal cells. The antiestrogen tamoxifen antagonized the effect of E2 and, when chronically administered to OVX rats, further elevated the postcastrational rise in Prodyn gene expression.

Prodynorphin mRNA was synthesized both in rat atrial and ventricular tissue, as well as in adult cultured rat ventricular cardiac myocytes. In the cultured cells, the content of prodynorphin mRNA did not differ from that detected in the original ventricle, indicating that the myocardial cell is an important source for prodynorphin mRNA in the rat ventricular tissue. This study demonstrated the presence of immunoreactive dynorphin B-like material in the cultured cardiomyocytes. Gel permeation chromatography analysis of this material revealed the presence of forms with an apparently higher molecular weight than authentic dynorphin B.

In the myocardial cell, stimulation of the K opioid receptor is involved in the modulation of cytosolic calcium and pH homeostasis, as well as in the regulation of myofilament responsiveness to calcium. In the present study, we found that prodynorphin mRNA, which encodes for the synthesis of a common precursor of opioid peptides interacting with K sites, is synthesized both in atrial and ventricular tissue of the rat heart. In adult cultured rat ventricular cardiomyocytes, the level of prodynorphin mRNA did not differ from that detected in the original ventricular tissue. This finding indicates that the myocardial cell is a source for prodynorphin gene expression and has the potential for an intrinsic synthesis of dynorphin-related peptides.