Regulated secretion of neurotrophins by metabotropic glutamate group I (mGluRI) and Trk receptor activation is mediated via phospholipase C signalling pathways.

Neurotrophins (NTs) play an essential role in modulating activity-dependent neuronal plasticity. In this context, the site and extent of NT secretion are of crucial importance. Here, we demonstrate that the activation of phospolipase C (PLC) and the subsequent mobilization of Ca(2+) from intracellular stores are essential for NT secretion initiated by both Trk and glutamate receptor activation. Mutational analysis of tyrosine residues, highly conserved in the cytoplasmic domain of all Trk receptors, revealed that the activation of PLC-gamma in cultured hippocampal neurons and nnr5 cells is necessary to mobilize Ca(2+) from intracellular stores, the key mechanism for regulated NT secretion. A similar signalling mechanism has been identified for glutamate-mediated NT secretion-which in part depends on the activation of PLC via metabotropic receptors-leading to the mobilization of Ca(2+) from internal stores by inositol trisphosphate. Thus, PLC-mediated signal transduction pathways are the common mechanisms for both Trk- and mGluRI-mediated NT secretion.

Are there differences between the secretion characteristics of NGF and BDNF? Implications for the modulatory role of neurotrophins in activity-dependent neuronal plasticity.

In previous experiments the activity-dependent secretion of nerve growth factor (NGF) from native hippocampal slices and from NGF-cDNA transfected hippocampal neurons showed unusual characteristics [Blochl and Thoenen (1995) Eur J Neurosci 7:1220-1228; (1996) Mol Cell Neurosci 7:173-190]. In both hippocampal slices and cultured hippocampal neurons the activity-dependent secretion proved to be independent of extracellular calcium, but dependent on the release of calcium from intracellular stores. Under different experimental conditions, Goodman et al. [(1996) Mol Cell Neurosci 7:222-238] reported that the high potassium-mediated secretion of brain-derived neurotrophic factor (BDNF) from hippocampal cultures was dependent on extracellular calcium. Mowla et al. [(1997) Proc 27th Annu Meet Soc Neurosci New Orleans 875.10] reported on even further-reaching differences between NGF and BDNF secretion, namely, that in hippocampal neurons and in pituitary cell lines NGF was secreted exclusively according to the constitutive pathway, whereas BDNF was exclusively sorted according to the activity-dependent regulated pathway. In view of the crucial importance of such potential differences between the processing, sorting, and secretory mechanisms of different neurotrophins for their modulatory roles in activity-dependent neuronal plasticity, a thorough analysis under comparable experimental conditions was mandatory. We demonstrate that in native hippocampal slices and adenoviral-transduced hippocampal neurons there are no differences between NGF and BDNF with respect to the subcellular distribution and mechanism of secretion; that the activity-dependent secretion of both NGF and BDNF is dependent on intact intracellular calcium stores; and that the differences between our own observations and those of Goodman et al. (ibid.) regarding the dependence on extracellular calcium do not reflect differences between NGF and BDNF sorting and secretion, but reflect the differing experimental conditions used.

Neurotrophin release by neurotrophins: implications for activity-dependent neuronal plasticity.

Neurotrophins, secreted in an activity-dependent manner, are thought to be involved in the activity-dependent refinement of synaptic connections. Here we demonstrate that in hippocampal neurons and the rat pheochromocytoma cell line PC12 application of exogenous neurotrophins induces secretion of neurotrophins, an effect that is mediated by the activation of tyrosine kinase neurotrophin receptors (Trks). Like activity-dependent secretion of neurotrophins, neurotrophin-induced neurotrophin secretion requires mobilization of calcium from intracellular stores. Because neurotrophins are likely to be released from both dendrites and axons, neurotrophin-induced neurotrophin release represents a potential positive feedback mechanism, contributing to the reinforcement and stabilization of synaptic connections.

p75(NGFR) and TrkA receptors collaborate to rapidly activate a p75(NGFR)-associated protein kinase.

The role of the low affinity nerve growth factor receptor (p75(NGFR)) in NGF-mediated signaling is not yet understood. Here we show by co-immunoprecipitation that NGF activates a protein kinase that is directly associated with p75(NGFR) in dorsal root ganglion (DRG) cells and PC12 cells in culture. Two proteins of 120 and 104 kDa constitute the majority of this activity. In PC12 cells, TrkA activation was necessary to elicit p75(NGFR)-associated kinase activity. Although NGF binding to p75(NGFR) was not necessary for kinase activation, it accelerated the activation of the kinase at low NGF concentrations. Deletion analysis showed that a 43 amino acid region in the cytoplasmic domain of p75(NGFR) was responsible for this effect. These findings show that p75(NGFR) accelerates TrkA-mediated signaling and, in addition, demonstrate that p75NGFR and TrkA collaborate to activate a previously undescribed p75(NGFR)-associated protein kinase.

Transphosphorylation of the neurotrophin Trk receptors.

The potential for the activation of one Trk receptor by ligand binding to another Trk receptor was explored by determining if transphosphorylation on tyrosine residues can occur between receptors. For most of these experiments, functional chimeric receptors were used that contained the extracellular domain of the human type 2 tumor necrosis factor receptor and the transmembrane and cytoplasmic domains of rat TrkA, TrkB, or TrkC and that, when activated by the tumor necrosis factor, mediated the nerve growth factor-like biological activities in PC12 cells. Cotransfection experiments in COS-7 cells and fibroblasts showed that despite the presence of different extracellular regions, intermolecular transphosphorylation of homologous cytoplasmic domains occurred between TrkA or TrkB and their cognate chimeras. Heterologous transphosphorylation between TrkB and TrkC kinase domains was also observed when one partner was a chimeric receptor; however, TrkA did not transphosphorylate the TrkB or TrkC kinase domains of chimeric receptors or act as a transphosphorylation substrate for these two receptors. The failure of TrkA to take part in transphosphorylation reactions with TrkB and TrkC was confirmed using the natural receptors. Trk receptor transphosphorylation occurs in the two non-neuronal cell types, but TrkA is excluded from these reactions.

Estrogen regulation of prodynorphin gene expression in the rat adenohypophysis: effect of the antiestrogen tamoxifen.

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.

Design, synthesis, and biological activity of methoctramine-related tetraamines bearing an 11-acetyl-5,11-dihydro-6H-pyrido[2,3-b][1,4] benzodiazepin-6-one moiety: structural requirements for optimum occupancy of muscarinic receptor subtypes as revealed by symmetrical and unsymmetrical polyamines.

Tetraamines 5-13 and diamines 14-17 as well as monoamine 18 were synthesized, and their biological profiles at muscarinic receptor subtypes were assessed by functional experiments in isolated guinea pig left atrium (M2) and ileum (M3) and by binding assays in rat cortex (M1), heart (M2), and submaxillary gland (M3) homogenates and NG 108-15 cells (M4). An appropriate number and type of substituents on the terminal nitrogens of a tetraamine backbone afforded compounds, such as tripitramine (8) and dipitramine (6), which are endowed with different affinity and selectivity profiles. Tripitramine, a nonsymmetrical tetraamine, resulted in the most potent and the most selective M2 muscarinic receptor antagonist so far available (pA2 = 9.75 +/- 0.02; pKi = 9.54 +/- 0.08). However, it failed to discriminate between M1 and M4 muscarinic receptor subtypes (selectivity ratio: M2/M3, 1600-2200; M2/M1, 81; M2/M4, 41; M1/M3, 28; M4/M3, 55; M4/M1, 2). Dipitramine, another nonsymmetrical tetraamine bearing two substituents on the same terminal nitrogen, displayed the highest affinity for M1 muscarinic receptors (pKi = 8.60 +/- 0.15) and was able to differentiate, unlike 8, all four muscarinic receptor subtypes investigated (selectivity ratio: M1/M2, 5; M1/M3, 2700; M1/M4, 76; M2/M3, 260-520; M2/M4, 15; M4/M3, 35). The results are discussed in terms of a possible mode of interaction of tetraamines with muscarinic receptor subtypes.

Inhibition of proopiomelanocortin expression by an oligodeoxynucleotide complementary to beta-endorphin mRNA.

Gene expression in mammalian cells can be suppressed by oligonucleotides complementary to the target mRNA. This strategy was explored as a means of arresting translation of the prohormone precursor proopiomelanocortin (POMC), used as a model system of peptide messengers that are synthesized and released from endocrine and neuronal cells. The synthesis of the POMC-derived peptides adrenocorticotropin (ACTH) and beta-endorphin (beta-END) was markedly reduced by an oligodeoxynucleotide (ODN) complementary to a region of beta-END mRNA in AtT-20 cells, which retain many of the differentiated phenotypes of corticotrophs; this treatment did not affect the steady-state levels of POMC mRNA. Antisense ODN was stable in cell culture medium for 24 h, and cellular uptake was low (approximately 2.5% of the added ODN); however, the intracellular levels of the ODN were sufficient to form a ribonuclease-resistant duplex with complementary cellular mRNA. Addition of ODN to the cell culture did not affect the cellular levels of chromogranin A-(264-314)/pancreastatin or cell viability and proliferation, as evidenced by bromodeoxyuridine incorporation and ornithine decarboxylase activity. Microinfusion of the antisense ODN in the rat hypothalamic arcuate nucleus, where the majority of POMC-positive brain perikarya are located, significantly reduced ACTH- and beta-END-immunopositive neurons, and antisense ODN-treated rats showed substantially less of the grooming behavior usually observed in a novel environment.

Chimeric tumor necrosis factor-TrkA receptors reveal that ligand-dependent activation of the TrkA tyrosine kinase is sufficient for differentiation and survival of PC12 cells.

To elucidate the function of the two nerve growth factor (NGF) receptors, p75NGFR and p140trk, chimeric molecules were constructed of tumor necrosis factor (TNF) and NGF receptors. Rat PC12 pheochromocytoma cells transiently transfected with TNF-p140trk chimeras, which contain the extracellular domain of TNF receptor and the transmembrane and cytoplasmic domains of p140trk, showed TNF-dependent neuronal differentiation and cell survival. The activity of TNF-p140trk chimeras was completely blocked by the tyrosine kinase inhibitor K252a, and TNF was unable to induce neurite elongation in PC12 cells transfected with a tyrosine kinase-defective chimeric receptor. The TNF-p75NGFR chimeras, which contain the cytoplasmic domain of p75NGFR, were nonfunctional. Our results suggest that p140trk may function as ligand-activated homodimers and that ligand-mediated activation of the cytoplasmic domain of p140trk alone is sufficient for inducing a neuronal phenotype.

Prodynorphin mRNA expression in adult cultured rat ventricular cardiac myocytes.

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.

Prodynorphin mRNA is synthesized in adult cultured rat ventricular cardiomyocytes.

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.

Relationship between plasma atrial natriuretic factor and opioid peptide levels in healthy subjects and in patients with acute congestive heart failure.

We evaluated plasma atrial natriuretic factor (ANF), beta-endorphin, met-enkephalin, dynorphin and noradrenaline levels in 20 healthy subjects and 20 acute congestive heart failure (CHF) patients. In all acute CHF patients plasma values of these hormones were higher than in healthy subjects. The hormonal pattern differed in patients with the more severe acute CHF (group 1) from patients with less severe acute CHF (group 2) (ANF 53.8 +/- 1.0 vs 34.6 +/- 1.5 pg.ml-1, noradrenaline 563.8 +/- 13.4 vs 202.4 +/- 10.6 pg.ml-1, met-enkephalin 41.0 +/- 3.2 vs 17.0 +/- 1.6 fmol.ml-1, dynorphin 46.8 +/- 3.7 vs 25.2 +/- 2.0 fmol.ml-1, P < 0.01; beta-endorphin 50.6 +/- 5.2 vs 41.8 +/- 4.1 fmol.ml-1,ns). Administration of an opioid antagonist (naloxone, 8 mg i.v.) did not modify ANF or noradrenaline concentration in healthy subjects. In group 1 naloxone administration significantly raised ANF (68.0 +/- 1.4 pg.ml-1), noradrenaline (776.6 +/- 18.7 pg.ml-1), blood pressure and heart rate, whereas in group 2 it significantly decreased ANF values (21.9 +/- 0.5 pg.ml-1) and did not modify the other parameters. Our findings suggest that the opioid system affects ANF release in acute CHF. In patients with severe CHF opioid peptides may attenuate ANF secretion reducing noradrenergic stimulation. On the other hand, when CHF is less severe and the sympathetic activity is moderate, opioid peptides may directly stimulate ANF secretion.

Immunoreactive dynorphin A-like material in extracted human hypothalamic-hypophysial plasma.

Immunoreactive dynorphin A-like material (ir-dyn A) in human plasma was measured by a validated radioimmunoassay. In peripheral plasma extracts mean concentrations between 20 and 40 fmol/ml were determined in volunteers and in patients with pituitary adenomas. In this latter group superimposable levels were detected three days before and during transsphenoidal microsurgery. Interestingly, ir-dyn A levels evaluated in extracts of hypothalamic-hypophysial plasma obtained during surgery, just after tumor removal, were 4-5 times higher than in peripheral plasma. Reverse-phase high performance liquid chromatography (rp-HPLC) of extracts of peripheral plasma samples revealed two immunoreactive peaks. The major form had the same retention time of dyn A-(1-32); whereas a second, more lipophilic, peak eluted later and was not further characterized. In contrast, rp-HPLC analysis of extracts of plasma collected from the suprapituitary region displayed only one peak eluting in the position of synthetic dyn A-(1-17). The presence of dyn-related peptides in hypothalamic-hypophysial plasma supports the hypothesis that they may play a part in the regulation of hypothalamic and/or pituitary functions in humans.

Dynorphin B-like immunoreactivity in gastroduodenal biopsy specimens from gallstone patients.

Dynorphin B-like immunoreactivity (ir-dyn B) was measured by a validated radio-immunoassay in gastroduodenal biopsy specimens from control and gallstone patients. Levels were significantly lower in acetic acid extracts of specimens of the transverse portion of the duodenum from gallstone patients. Gel permeation chromatography showed that almost all ir-dyn B in duodenal samples corresponded to a molecular form co-eluting with authentic dyn B. Duodenal extracts from gallstone patients had less of this form. Reverse-phase high performance liquid chromatography of the pooled gel chromatography fractions showed up a molecular form with the same retention time as synthetic dyn B which was significantly less in fractions from duodenal extracts of gallstone patients. These results indicate the occurrence of dyn B in the human gastrointestinal tract; however, at this stage of our understanding, no causal relationship can be demonstrated with functional alterations of the biliary tree.

Plasma endogenous opioid levels in acute myocardial infarction patients, with and without pain.

Plasma levels of beta-endorphin, met-enkephalin and dynorphin were assessed in acute myocardial infarction (AMI) patients, with and without pain (group I: no pain, N = 12; group II: severe pain, N = 16). Plasma opioid peptide concentration was measured on admission to hospital (between 1 and 3 h after the myocardial infarction onset), at 7, 12, 24 h and at 2, 3 and 4 days. A transient increase in plasma beta-endorphin levels was found in AMI patients with severe pain, the levels normalizing within 12-18 h when pain had ceased. No changes in beta-endorphin concentration were observed in AMI patients without pain. Compared with healthy subjects, low levels of met-enkephalin were found in both groups of AMI patients throughout the study. Low levels of dynorphin were observed in patients with no pain while in the other patients initial low levels of dynorphin normalized when pain ceased. Blood pressure, heart rate and central venous pressure values were normal and did not correlate with plasma opioid levels. The results suggest that endogenous opioids do not affect pain in the early phase of myocardial infarction. The rise in beta-endorphin concentration observed in patients with severe pain seems to be induced by pain stress.

Dynorphin A-(1-17) and dynorphin B are released from in vitro superfused rat hypothalami. Effects of depolarizing agents and ovariectomy.

We measured the release of immunoreactive (ir) dynorphin (dyn) A-(1-17) and dyn B from the rat hypothalamus by an in vitro superfusion technique. The system was validated on the basis of the recovery and stability of radiolabeled peptides added to the superfused hypothalami. These were detected as authentic peptides by reverse-phase high-performance liquid chromatography (rp-HPLC) only in the presence of a cocktail of peptidase inhibitors added to the superfusion medium. We observed spontaneous release of ir-dyn B, evaluated by a validated radioimmunoassay in the superfusates, that was increased by potassium and veratridine depolarization. It was calcium-dependent and tetrodotoxin-sensitive. We could not evaluate ir-dyn A-(1-17) directly in the superfusates, because the peptidase inhibitors added to the medium significantly altered the tracer-antibody reaction. To obviate this problem, pooled superfusate samples were purified on C18 cartridges and assayed by rp-HPLC. Rp-HPLC analysis of superfusates revealed two molecular forms with the same retention time as authentic dyn A-(1-17) and dyn B which were four times higher in K(+)-stimulated fractions. We could not detect dyn A-(1-32), comprising dyn A-(1-17) and dyn B, even though this peptide is recognized by the antibodies used in this study and is detected in acetic acid extracts of the rat hypothalamus. The spontaneous and K(+)-evoked release of ir-dyn A-(1-17) and ir-dyn B were significantly higher in 2-week ovariectomized rats, in parallel with the increase of their content in the anterior hypothalamus preoptic area.

Heterogeneity of immunoreactive dynorphin B-like material in human, rat, rabbit and guinea-pig heart.

Immunoreactive dynorphin B-like material (ir-dyn B) was detected in acetic acid extracts of human atrial specimens and of rat, rabbit and guinea-pig atria and ventricles by a validated radioimmunoassay. Levels were high in rabbit atrium (66.76 +/- 7.04 pmol/g) but lower and superimposable in human and rat atria (28.18 +/- 3.20 and 30.22 +/- 2.45 pmol/g, respectively). Gel permeation chromatography revealed ir-dyn B eluting close to column exclusion and in forms with an apparently higher molecular weight than authentic dyn B in human and rat samples. In contrast, almost all the immunoreactivity from rabbit and guinea-pig acetic extracts eluted as a single peak in the region of standard dyn B. Reverse-phase high performance liquid chromatography of the pooled gel chromatography fractions of this peak showed up a molecular form with the same retention time as authentic dyn B and a second minor peak of unknown immunoreactive material eluting three fractions earlier. Digestion with carboxypeptidase B excluded the hypothesis that this latter could be dyn B-Arg14. Therefore, it might be a metabolite of endogenous dyn B recognized by the antibody used in this study.

Effects of sodium naproxen eye drops on rabbit ocular inflammation induced by sodium arachidonate.

Sodium naproxen, a reversible competitive inhibitor of cyclooxygenase, is widely used as an anti-inflammatory agent in clinical practice. The purpose of this study was to determine whether eye drops containing 0.5% (w/v) sodium naproxen reduce a number of inflammatory responses produced by sodium arachidonate in the rabbit's eye. Sodium naproxen eye drops successfully reduced the primary signs of ocular inflammation elicited by 0.5% sodium arachidonate on conjunctiva and iris. However, the drug was less effective in reducing conjunctival inflammation induced by 1% sodium arachidonate. Sodium naproxen treatment significantly reduced the levels of prostaglandin E2 (PGE2), polymorphonuclear leukocytes and protein concentration in aqueous humor samples obtained from the eyes of rabbits treated with 0.5% sodium arachidonate whereas aqueous humor levels of leukotriene B4(LTB4) were not found significantly different from control rabbits. Interestingly, PGE2 as well as LTB 4 "de novo" production by corneas and lenses obtained from rabbits sacrificed 2 h after arachidonate and incubation "in vitro" for 20 min were significantly higher in samples taken from controls than in tissues obtained from the eyes treated with sodium naproxen eye drops. Finally, this drug treatment significantly antagonized the rise in intraocular pressure induced by 0.5% sodium arachidonate. Present data suggest that sodium naproxen may be employed topically to prevent ocular inflammatory reactions where the arachidonic acid cascade is activated.