Human GH has two disulfide bridges linking Cys-53 to Cys-165 and Cys-182 to Cys-189. Although absence of the first disulfide bridge has been shown to affect the bioactivity of GH in transgenic mice, little is known of the importance of this bridge in mediating the GH/GH-receptor (GHR) interaction in humans. However, we have identified a missense mutation (G705C) in the GH1 gene of a Serbian patient. This mutation was found in the homozygous state and leads to the absence of the disulfide bridge Cys-53 to Cys-165. To study the impact of this mutation in vitro, GHR binding and Janus kinase (Jak)2/signal transducer and activator of transcription (Stat)5 activation experiments were performed, in which it was observed that at physiological concentrations (3-50 ng/ml) both GHR binding and Jak2/Stat5 signaling pathway activation were significantly reduced in the mutant GH-C53S, compared with wild-type (wt)-GH. Higher concentrations (400 ng/ml) were required for this mutant to elicit responses similar to wt-GH. These results demonstrate that the absence of the disulfide bridge Cys-53 to Cys-165 affects the binding affinity of GH for the GHR and subsequently the potency of GH to activate the Jak2/Stat5 signaling pathway. In conclusion, we have demonstrated that GH-C53S is a bioinactive GH at the physiological range and that the disulfide bridge Cys-53 to Cys-163 is required for mediating the biological effects of GH.

Despite almost forty years of widespread use, the mode of action of antidepressant drugs is still largely unknown. There is agreement that these drugs interact with central neurotransmission. Common findings are acute inhibitory actions on reuptake mechanisms for norepinephrine (NE) and for serotonin (5-HT) at presynaptic axons and chronic adaptive effects on neurotransmitter receptors on postsynaptic membranes. In particular, beta-adrenoceptor downregulation has been observed after chronic treatment with most antidepressants in vivo and in cell culture systems. We studied the effectiveness of Ze 117 (St. John's wort) extract (Hypericum perforatum) on NE- and 5-HT-uptake into rat brain slices. Potency and efficacy of the Ze 117 extract were compared with those of tricyclic (TCA) and selective serotonin reuptake inhibitor (SSRI)-type antidepressants. A dose-dependent inhibition was seen on NE and 5-HT uptake into brain slices. The Ze 117 extract was more selective for the uptake of NE than for that of 5-HT. The maximal extent of uptake inhibition by Ze 117 extract was comparable to that of imipramine (IMI), desipramine (DMI) or fluvoxamine for 5-HT, but lower for NE transport, than that of the synthetic antidepressants. Chronic exposure (8 days) of confluent C6-cell cultures to Ze 117 extract resulted in a dose-dependent beta-adrenoceptor downregulation equal to that induced by DMI, a potent TCA. None of these effects could be achieved with either hypericin or hyperforin alone in a relevant dose range. Our results indicate that the St. John's wort extract Ze 117 contains active, but as yet unknown antidepressant principles with effects comparable to those of TCAs.

Reduction in surface ␤ 1 -adrenoceptor (␤1AR) density is thought to play a critical role in mediating the therapeutic long term effects of antidepressants. Since antidepressants are neither agonists nor antagonists for G protein-coupled receptors, receptor density must be regulated through processes independent of direct receptor activation. Endocytosis and recycling of the ␤1AR fused to green fluorescent protein at its carboxylterminus (␤1AR-GFP) were analyzed by confocal fluorescence microscopy of live cells and complementary ligand binding studies. In stably transfected C6 glioblastoma cells, ␤1AR-GFP displayed identical ligand-binding isotherms and adenylyl cyclase activation as native ␤1AR. Upon exposure to isoproterenol, a fraction of ␤1AR-GFP (10 -15%) internalized rapidly and colocalized with endocytosed transferrin receptors in an early endosomal compartment in the perinuclear region. Chronic treatment with the tricyclic antidepressant desipramine (DMI) did not affect internalization characteristics of ␤1AR-GFP when challenged with isoproterenol. However, internalized receptors were not able to recycle back to the cell surface in DMI-treated cells, whereas recycling of transferrin receptors was not affected. Endocytosed receptors were absent from structures that stained with fluorescently labeled dextran, and inhibition of lysosomal protease activity did not restore receptor recycling, indicating that ␤1AR-GFP did not immediately enter the lysosomal compartment. The data suggest a new mode of drug action causing a "switch" of receptor fate from a fast recycling pathway to a slowly exchanging perinuclear compartment. Antidepressant-induced reduction of receptor surface expression may thus be caused by modulation of receptor trafficking routes.Chronic treatment with most classes of antidepressants leads to a reduction in the number of central postsynaptic ␤-adrenoceptors (␤AR) 1 in vivo and of surface ␤AR density in cell cultures (1-4). This effect is reported to be specific for the ␤ 1 -adrenoceptor subtype (5, 6). ␤AR down-regulation is accompanied by decreased receptor-stimulated cAMP formation (7). The two major effects at the molecular level become apparent in vivo after 10 -20 days of drug administration and coincide with the onset of clinical antidepressant response in humans. Therefore ␤AR down-regulation and diminished cAMP response to catecholamines may relate to the therapeutic action of antidepressants.It has been proposed that the reduction in the number of functional ␤AR could be a regulatory response to the enhanced presence of norepinephrine in the synaptic cleft after acute inhibition of norepinephrine reuptake or of monoamine oxidase activity by antidepressants (1, 8). Some clinically effective antidepressants, however, neither influence norepinephrine reuptake nor inhibit monoamine oxidase activity but still cause a ␤AR down-regulation. Furthermore, this model fails to explain the observed time lag between the rapid drug-induced increase in intrasynaptic neurotransmitter concentrations ...

The mode of action of antidepressants is still a matter of debate. Acute inhibition of neurotransmitter reuptake in central neuronal synapses, followed by a down-regulation of central postsynaptic beta-adrenoceptor (beta-AR) numbers were consistently observed in vivo, while a reduction in surface beta-AR density was found in cell cultures. Effects of the tricyclic antidepressant desipramine (DMI) were abolished by vitamin E (alpha-TOC) in vitro as well as in vivo. Alpha-TOC interfered with antidepressant-induced changes of cellular plasma membrane properties and with recycling of beta-AR. St. John's wort (SJW) extract reduced beta-AR numbers in cultured cells to a similar extent as DMI or the selective serotonin re-uptake inhibitor fluoxetine. We chronically co-exposed cell cultures to SJW extract and to alpha-TOC. Receptor down-regulation following exposure to the plant extract was inhibited in the presence of alpha-TOC suggesting a mode of action of SJW extract comparable to that of synthetic antidepressants. Inhibition of cell proliferation by the plant extract was also significantly reduced by alpha-TOC.