UV second harmonic generation at 266 nm in He+ implanted beta-BaB2O4 optical waveguides.

We report on the second harmonic generation of deep UV light in beta -BaB(2)O(4) (BBO) waveguides pumped by a frequency-doubled continuous-wave Nd:YAG laser. An output power of 0.32 mW at 266 nm has been achieved for an internal pump power of 670 mW. Optical channel waveguides in BBO crystals were produced by He(+) ion implantation, lithographic masking and ion etching. The linear and nonlinear optical properties and the power handling capability of these waveguides are presented.

Noradrenergic antidepressants: does chronic treatment increase or decrease nuclear CREB-P?

Chronic administration of noradrenergic antidepressants causes a desensitization of the beta adrenoceptor coupled adenylate cyclase system. In the present studies, we attempted to answer the question of whether or not this deamplification is reflected beyond the second messenger system. Nuclear CREB-P was determined in frontal cortex of rats following acute and chronic administration of desipramine (DMI) or reboxetine and in human fibroblasts following incubation for 48 hours with DMI, reboxetine or venlafaxine. Nuclear CREB-P in the frontal cortex was significantly decreased following chronic administration of DMI or reboxetine. Moreover, incubation of human fibroblasts with DMI or reboxetine, but not with venlafaxine, caused a highly significant reduction in nuclear CREB-P suggesting that the noradrenergic antidepressants exert direct effects beyond beta adrenoceptors. The results are consistent with the view that chronic treatment with antidepressants causes a net deamplification of the norepinephrine mediated signal transduction cascade which might "normalize" the increased noradrenergic activity evident in major depression.

Detection of an mRNA polymorphism by differential display.

Differential Display (DD) technology was utilized to compare programs of gene expression in primary cultures of human skin fibroblasts from normal volunteers and patients diagnosed with melancholic depression. Polymorphic transcripts of a single gene differing by one tandem repeat sequence of four nucleotides (TGAT) in the 3' noncoding region were detected.

Cross-talk between PKA and PKC in human fibroblasts: what are the pharmacotherapeutic implications?

BACKGROUND: The present study was designed to confirm or refute in human fibroblasts the hypothesized cross-talk elicited via neurotransmitter transduction cascades at the level of protein kinase mediated phosphorylation of the nuclear transcription factor CREB. METHODS: Human fibroblasts from normal control subjects were subcultured and incubated at confluency after five growth passages with isoproterenol (stimulation of PKA mediated phosphorylation) and/or phorbol 12-myristate 13-acetate (PMA) (stimulation of PKC mediated phosphorylation) followed by the determination of nuclear CREB-P by immunoblotting, enhanced chemiluminescence and quantitation of the autoradiograms by laser densitometry. RESULTS: Using the nuclear transcription factor CREB as a target, both the activation of the cyclic AMP-PKA pathway by isoproterenol and the activation of the PKC pathway by PMA caused phosphorylation of nuclear CREB. This phosphorylation is additive in nature and appears to occur at the same molecular site, serine133 of CREB. CONCLUSIONS: The present results in human fibroblasts demonstrate that the hypothesized cross-talk at the level of protein kinase mediated phosphorylation of transcription factors is no longer hypothetical. Since it is the phosphorylation of nuclear CREB that determines its dimerization and transcriptional activation of programs of CRE containing genes, the results suggest that this convergence of the neurotransmitter signals may be the critical mechanism in gene expression following the administration of antidepressant drugs that affect noradrenergic, serotonergic or both transduction cascades. The results may also provide a rationale for the apparent superior clinical efficacy of dual uptake inhibitors.

Human fibroblasts as a relevant model to study signal transduction in affective disorders.

BACKGROUND: Previous studies have demonstrated a blunted beta adrenoceptor-linked protein kinase A (PKA) response in the 900xg supernatant fraction of human fibroblasts cultured from patients with major depression. RESULTS: Results of the present studies demonstrate a significant reduction in the B(max) value of [3H]cyclic AMP binding to the regulatory subunit of PKA in the supernatant fraction of fibroblasts from patients with major depression with no change in the K(d) values. The data are consistent with the previous observation that the maximal stimulation of PKA by cyclic AMP is reduced without a change in the EC(50) value. The blunted beta adrenoceptor-mediated PKA response in fibroblasts from patients with major depression is reflected in a significant reduction in the isoproterenol-stimulated phosphorylation of the nuclear transcription factor CREB. Both, the isoproterenol-mediated phosphorylation of nuclear CREB and the activation of the stably transfected luciferase reporter gene, pAD neo2-C12-BGL, were inhibited by the beta(2) adrenoceptor antagonist ICI 118551, thus indicating that the gene activating action of isoproterenol in human fibroblasts is mediated via the beta(2) adrenoceptor cascade. The low EC(50) value of 1 nM isoproterenol for activation of gene expression in stably transfected human fibroblasts appears to be a reflection of the amplification mechanism occurring via the beta adrenoceptor-cyclic AMP-PKA-CREB transduction cascade. CONCLUSIONS: The results support the notion that human fibroblasts represent a relevant model for studying processes of signal transduction in patients with affective disorders.

Lack of beta adrenoceptor desensitization in brain following the dual noradrenaline and serotonin reuptake inhibitor venlafaxine.

Venlafaxine, a dual amine reuptake inhibitor, was utilized to delineate the role of the individual aminergic components of the 'serotonin/noradrenaline link' in modifying receptor-linked second messenger cascades. Venlafaxine (20 mg/kg i.p. bid for 10 days) failed to alter in normal animals either the density of beta adrenoceptors or the response of the beta adrenoceptor-coupled adenylate cyclase system to noradrenaline but significantly decreased the cyclic AMP response to noradrenaline in the brain of rats with selective depletion of brain serotonin by p-chlorophenylalanine. The studies provide evidence for a cross-talk between noradrenergic and serotonergic receptor cascades at the level of mechanisms involved in the desensitization of the beta adrenoceptor-coupled adenylate cyclase system.

Increased synaptic availability of norepinephrine following desipramine is not essential for increases in GR mRNA. Short communication.

Male Sprague-Dawley rats were treated for 7 days with the norepinephrine (NE) uptake inhibitors desipramine (DMI) or (+)-oxaprotiline or the inactive (-)-enantiomer of oxaprotiline. DMI, as previously reported, significantly increased hippocampal glucocorticoid receptor (GR) mRNA while the equipotent NE uptake inhibitor (+)-oxaprotiline like the inactive (-)-oxaprotiline did not alter hippocampal levels of GR mRNA. The results indicate that an increase in the synaptic availability of NE as a consequence of uptake inhibition is not responsible for the action of DMI on GR gene expression.

Beta-adrenoceptor-linked protein kinase A (PKA) activity in human fibroblasts from normal subjects and from patients with major depression.

Human fibroblasts from normal subjects and from patients with major depression are cultured and their beta-adrenoreceptor-cyclic AMP-protein kinase A (PKA) system characterized. The results indicate that the beta-adrenoreceptor-mediated activation of PKA in the 900 g supernatant fraction of human fibroblasts is mediated via beta-adrenoreceptors. The activation of PKA by isoproterenol is very rapid with maximal stimulation occurring at 5 seconds. The time course of PKA activation by isoproterenol in fibroblasts from patients with major depression is identical to that in fibroblasts from normal subjects but the magnitude of activation is significantly reduced in fibroblasts from patients with major depression. Dose-response curves on cyclic AMP mediated activation of PKA confirmed the previously reported reduction in activation of PKA in patients with major depression but demonstrated that this reduction occurs without a change in the EC50 values of cyclic AMP (approximately 20 nmol/L). The blunted beta-adrenoceptor-linked PKA responses in patients with major depression occur without a change in the expression of the PKA catalytic subunit C alpha. The studies suggest that the beta-adrenoceptor-coupled adenylate cyclase PKA system in human fibroblasts may represent a valid model to explore possible abnormalities in the fine tuning of the beta-adrenergic transduction cascade in patients with affective disorders.

cAMP-dependent protein kinase activity in major depression.

OBJECTIVE: The author's intent was to evaluate the activity of the beta-adrenoceptor-linked, cAMP-dependent protein kinase (protein kinase A) in patients with major depression compared with a group of nondepressed volunteer subjects. METHOD: Skin fibroblast samples were obtained by 2-mm punch biopsy from 12 patients (11 were women) who had major depression diagnosed according to the Structured Clinical Interview for DSM-III-R and from 10 nondepressed volunteers (seven were women). Fibroblasts were cultured in Dulbecco's modified Eagle medium. Baseline and cAMP-stimulated activities of protein kinase A were determined in both particulate and supernatant fractions (900g). Linkage of the finding to beta adrenergic receptor function was evaluated by determination of protein kinase A activity after incubation of the confluent cultures for 30 minutes with 10 microM isoproterenol. RESULTS: There were significant differences between groups in the baseline and cAMP-stimulated phosphorylation in the supernatant fraction. Moreover, the attenuated protein kinase A response was accompanied by a blunted isoproterenol response. CONCLUSIONS: Patients with depression exhibit significantly less activity of beta-adrenoceptor-linked protein kinase A than do normal subjects. The reductions in protein kinase A activity support the significance of beta-receptor-mediated events in depression.

Norepinephrine-independent regulation of GRII mRNA in vivo by a tricyclic antidepressant.

Desipramine (DMI), a tricyclic antidepressant drug used in the treatment of depression, has been shown to increase steady-state levels of glucocorticoid receptor type II (GRII) mRNA in vitro and in vivo. To determine whether this effect is secondary to norepinephrine (NE) reuptake inhibition i.e., increases in synaptic NE induced by DMI, GRII mRNA levels were assayed in rat hippocampus following neurotoxic lesioning of NE neurons with DSP4. Chronic DMI treatment significantly increased GRII mRNA levels to the same degree in lesioned and non-lesioned animals. In contrast to DMI, the non-tricyclic antidepressant fluoxetine had no effect on GRII mRNA. These results provide evidence which demonstrates that a tricyclic antidepressant can regulate steady-state mRNA levels in vivo by a mechanism which is independent of its effects on synaptic monoamine levels.

The 'serotonin/norepinephrine link' beyond the beta adrenoceptor.

C6 rat glioma cells were utilized as a model system to probe the 'serotonin/norepinephrine link' at the level of preproenkephalin (PPE) gene expression. The beta adrenoceptor mediated increase in PPE mRNA was attenuated by the selective beta 1 adrenoceptor antagonist metoprolol which blocked the isoproterenol induced cyclic AMP generation by 97%. The subtype nonspecific antagonist propranolol blocked both the isoproterenol induced increase in cyclic AMP and the increase in the PPE mRNA steady-state levels. Serotonin (5-HT) had no effect on the density of beta adrenoceptors or their down-regulation by isoproterenol and did not alter the PPE gene expression in the absence of the beta signal. However, 5-HT significantly deamplified the beta signal mediated enhancement of the PPE mRNA thus indicating that the aminergic link occurs beyond the beta adrenoceptor.

The beta-adrenoceptor-coupled adenylate cyclase system in rat C6 glioma cells. Deamplification by isoproterenol and oxaprotiline.

The beta-adrenoceptor-coupled adenylate cyclase system in rat C6 glioma cells displays many characteristics observed in brain tissue: using nonlinear regression analysis of agonist competition binding curves, we demonstrated that the bulk of beta-adrenoceptors show high nanomolar affinity for isoproterenol; like in brain tissue, Gpp(NH)p does not shift agonist competition binding curves to the right; and the agonist isoproterenol rapidly downregulates the number of beta-adrenoceptors and deamplifies the norepinephrine signal. However, unlike in brain tissue, where (-)-oxaprotiline fails to decrease the number of beta-adrenoceptors and to desensitize the cyclic adenosine monophosphate generating system, it desensitizes the beta-adrenoceptor-coupled adenylate cyclase system in C6 glioma cells. Determination of the relative steady-state levels of beta-adrenoceptor messenger ribonucleic acid (mRNA) by Northern blot analysis showed a twofold increase in the steady-state levels of the mRNA at 30 minutes following exposure to (-)-isoproterenol or (-)-oxaprotiline. At 48 hours, basal values of mRNA were observed at a time when beta-adrenoceptors were maximally decreased. Further experiments on transcriptional activation, and mRNA stability and translation will be required to unravel the complexity of agonist-dependent and agonist-independent regulation of beta-adrenoceptor density and function.

Ethidium bromide fluorescence of 28S ribosomal RNA can be used to normalize samples in northern or dot blots when analyzing small drug-induced changes in specific mRNA.

Quantitative analysis of Northern blots is frequently accomplished with the aid of an internal standard. Most common is probing for an additional message the steady-state levels of which do not change in response to the experimental conditions and the signal of which is sufficiently removed from that of the target gene after gel electrophoresis. However, this strategy is not always feasible. When total RNA is immobilized on nylon, 28S ribosomal RNA on the blot can be used as an internal standard and quantitated by scanning the negative photograph of the blotted RNA stained with ethidium bromide. This procedure can also be used for RNA dot blots. The method is quick, reliable, will work with laser or white-light densitometers, and can serve as a universal internal standard, eliminating the need for additional probes.

Dose-dependent down-regulation of beta-adrenoceptors by isoproterenol in rat C6 glioma cells.

Nano- and micromolar isoproterenol concentrations were compared by studying cyclic AMP, beta-adrenoceptor density and beta 1-adrenoceptor mRNA in rat C6 glioma cells. 1 microM isoproterenol significantly changed all parameters at 15-30 min. The beta 1-antagonist metoprolol attenuated the response. No effects of nanomolar isoproterenol on these early changes were observed, although the density of beta-adrenoceptors was significantly reduced beginning at 12 h. The results indicate a different process for beta-adrenoceptor desensitization in C6 cells following physiologically low agonist concentrations.

Characterization of the inducible serotonin-sensitive dihydroalprenolol binding sites with low affinity for isoproterenol.

The previous findings that the inducible [3H]-dihydroalprenolol (DHA) binding sites with low affinity for isoproterenol (RL) could be regulated by serotonin (5-HT) in vitro and by 5-hydroxytryptophan and the 5-HT uptake inhibitor fluoxetine in vivo, prompted the present pharmacologic characterization of these receptor sites, using nonlinear regression analysis of competition binding curves. If isoproterenol was used as the displacing agent, lesioning with 5,7-dihydroxytryptamine selectively increased [3H]-DHA binding sites with low micromolar affinity. By contrast, if 5-HT was used as the displacing agent, the receptor population with high agonist affinity showed a fourfold increase whereas the density of [3H]-DHA sites with low micromolar affinity for 5-HT was not altered. Neither the 5-HT1A agonist, 8-OH-DPAT, nor mianserin, a 5-HT2 and 5-HT1C antagonist, altered the induced RL receptor population, whereas the selective 5-HT1B agonist CGS-12066B reduced the increase in the RL receptor population with a potency equal to that of 5-HT. These results strengthen the notion that the [3H]-DHA sites with low agonist affinity for isoproterenol represent 5-HT1B receptors induced following a reduction of serotonergic neuronal function.

Dual aminergic regulation of central beta adrenoceptors. Effect of "atypical" antidepressants and 5-hydroxytryptophan.

Nonlinear regression analysis of agonist competition binding curves reveals that the [3H]-dihydroalprenolol-labeled receptor population with low affinity for isoproterenol is increased by p-chlorophenylalanine (PCPA) and this increase is abolished by 5-hydroxytryptophan (5-HTP) in vivo. Desipramine (DMI) decreased the beta adrenoceptor population with high agonist affinity to the same degree in PCPA-treated animals as in control animals, thus explaining the reported discrepancy between beta adrenoceptor number and responsiveness of the beta adrenoceptor-coupled adenylate cyclase system. Mianserin also selectively reduced the beta adrenoceptor population with high agonist affinity in membrane preparations of normal animals, whereas fluoxetine selectively abolished the upregulation of the low affinity sites in reserpinized animals and had no effect on either receptor population from brain of normal animals. The results emphasize the importance of nonlinear regression analysis of agonist competition binding for the interpretation of drug action and encourage the pursuit of the molecular neurobiology of the serotonin (5-HT)/norepinephrine (NE) link in brain.

New perspectives on the molecular pharmacology of affective disorders.

Research with antidepressants has emphasized the importance of a delayed deamplification of the linked serotonin (5HT)/norepinephrine (NE) receptor coupled adenylate cyclase system in brain. The basic phenomena of regulation of receptor number and function of the beta adrenoceptor linked adenylate cyclase system in brain are well established, with NE regulating beta adrenoceptors in the high agonist affinity conformation (linked to adenylate cyclase and down-regulated by antidepressants), and with 5HT regulating those receptors in the low agonist affinity conformation. The biochemical effector systems of NE and 5HT are discussed and it is concluded that the final common pathway of signal transduction is protein kinase mediated phosphorylation of cellular proteins. Glucocorticoid receptors are located in the perikarya of aminergic cell bodies and may exert their effects by modifying the genomic expression of the diffusely projecting stress-responsive monoamine systems. The molecular neurobiology of beta adrenoceptors, with its implication for genetic and immunologic investigations, is briefly discussed and further research on stimulus-transcription coupling and regulation of gene expression in brain is suggested as an exciting new direction in central receptor research relevant to the psychopharmacology of affective and other disorders.

Effects of thyroid alterations and carbamazepine on cortical beta-adrenergic receptors in the rat.

The effect of alteration in thyroid status on beta adrenergic receptors in the cortex of the rat was assessed. Normal animals were treated with large doses of thyroxine (T4) and triiodothyronine (T3) and thyroidectomized animals were treated with physiological replacement doses of T4 and T3 in order to assess the possible differential effects of these hormones. In addition, a group of rats was treated with a diet of carbamazepine (an anticonvulsant also used in the treatment of manic-depressive illness), which has been shown to reduce peripheral levels of thyroid hormone in humans. The intended manipulations of the thyroid were achieved by the various treatments with thyroid hormone, and carbamazepine-diet-treated animals had significantly lower plasma T4 levels as compared with controls. No significant alteration in the density or affinity of beta-adrenergic receptors in the cortex was noted with major, short-term alterations in thyroid status or with treatment with carbamazepine. It is concluded that even marked, but relatively short-term, changes in thyroid status do not necessarily affect beta-receptors in the cerebral cortex and that carbamazepine may represent an exception to the general proposition that antidepressant agents decrease the number of beta-receptors.

The serotonin/norepinephrine-link in brain. II. Role of serotonin in the regulation of beta adrenoceptors in the low agonist affinity conformation.

The present studies were undertaken to characterize further the role of serotonin (5-HT) in the regulation of the norepinephrine (NE) beta adrenoceptor coupled adenylate cyclase system in the rat cortex. Although 5-HT in vitro did not influence maximum binding and Kd values of [3H]dihydroalprenolol binding or the IC50 value for isoproterenol as estimated from competition binding curves in cortical tissue from control animals, 5-HT abolished the increase in beta adrenoceptor number and the marked elevation of the IC50 value for isoproterenol in cortical membrane preparations after selective lesions with 5,7-dihydroxytryptamine (5,7-DHT). Nonlinear regression analysis of competition binding curves revealed that the increase in the maximum binding of beta adrenoceptors after 5,7-DHT is due exclusively to an increase in beta adrenoceptors in the agonist low affinity conformation and that it is this receptor population that is reduced by nanomolar concentrations of 5-HT. The increase in the density of beta adrenoceptors in the low affinity conformation occurred approximately 11 days after the lesions and remained elevated throughout the experimental period of 28 days. Ritanserin in a dose that virtually abolished 5-HT2 receptor binding in cortex did not mimic the effect of 5,7-DHT.(ABSTRACT TRUNCATED AT 250 WORDS)