Role of serotonergic input in the regulation of the beta-adrenergic receptor-coupled adenylate cyclase system.

The action of desipramine on the norepinephrine-sensitive adenylate cyclase system and the density of beta-adrenergic receptors in rat cortex was studied after selective lesioning of serotonergic neurons with 5,7-dihydroxytryptamine. In animals with lesions desipramine failed to reduce the density of beta-adrenoceptors but decreased the response of adenosine 3',5'-monophosphate to isoproterenol and norepinephrine to the same degree as in animals without lesions. The results demonstrate a functional linkage between serotonergic and noradrenergic systems in the rat cortex, with beta-adrenergic receptors and neurohormonal sensitivity of the adenosine 3',5'-monophosphate-generating system being under separate regulatory control.

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.

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.

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.

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.

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.

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.

Regulation and function of noradrenaline receptor systems in brain. Psychopharmacological aspects.

Central beta-adrenoceptors are coupled to adenylate cyclase in a stimulatory manner. However, there is only circumstantial evidence that alpha 2-adrenoceptors in brain are coupled to adenylate cyclase in an inhibitory manner. The desensitization of the beta-adrenoceptor system induced by antidepressants seems to be a common action of clinically effective antidepressants. alpha 2-Adrenoceptor subsensitivity, if it occurs following administration of some antidepressants, contributes to the development of down-regulation of beta-adrenoceptors. The occupancy of adrenoceptors by noradrenaline (NA) is a prerequisite for both desensitization of the system and the reduction in the number of beta-adrenoceptors while serotonin (5-HT) is co-required with NA for the regulation of the density of beta-adrenoceptors. The decrease in the number of beta-adrenoceptors induced by antidepressants is rapidly reversible following inhibition of 5-HT synthesis by p-chlorophenylalanine. Since beta-adrenoceptor-coupled adenylate cyclase systems function as kinetic amplification systems, small changes in the NA signal transfer are amplified or deamplified respectively. beta-Adrenoceptors may also subserve a critical role in neuronal membranes by determining the sensitivity of other membrane receptor systems.

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.

Affinity of 10-(4-methylpiperazino)dibenz[b,f]oxepins for clozapine and spiroperidol binding sites in rat brain.

10-(4-Methylpiperazino)dibenz[b,f]oxepins were prepared and evaluated as potential antipsychotic agents using specific clozapine [8-chloro-11-(4-methylpiperazino)-5H-dibenzo[b,e][1,4]diazepine] binding sites in rat forebrain that are noncholinergic and nondopaminergic in nature and from which [3H]clozapine is displaced by known antipsychotic agents. [3H]Clozapine binding in the presence of atropine represents nonmuscarinic binding, while binding in the absence of atropine represents muscarinic (cholinergic) plus nonmuscarinic binding. The relative affinity for dopamine binding sites was determined by displacement of [3H]spiroperidol from binding sites in rat caudate nuclei. Thus, clozapine, its 2-chloro isomer, its dechloro analogue, and their 5H-dibenzo[a,d]cycloheptene and dibenz[b,f]oxepine analogues have about the same relative affinity for the nonmuscarinic clozapine binding sites. At the spiroperidol (dopaminergic) sites, both the nature of the tricyclic system and the presence of a chlorine atom on the tricyclic system have a substantial effect on the binding affinity. Within each series, shift or a chlorine atom from the position distal to the piperazino group to the proximal position increases the binding affinity by a factor of about nine, but removal of the chlorine atom substantially decreases the binding affinity. Nevertheless, 10-(4-methylpiperazino)dibenz[b,f]oxepin has a threefold greater affinity for the dopaminergic binding sites than does clozapine itself.

Synthesis of clozapine analogues and their affinity for clozapine and spiroperidol binding sites in rat brain.

Analogues of clozapine, some prepared by a novel, shorter synthesis than those described previously, were evaluated as potential antipsychotic agents using clozapine binding sites in rat forebrain that are nonmuscarinic and nondopaminergic in nature and from which [3H]clozapine is displaced by known antipsychotic agents. The binding of clozapine to muscarinic sites is inhibited in the presence of atropine. Displacement of [3H]clozapine by an analogue of clozapine in the presence of atropine represents nonmuscarinic binding, while displacement in the absence of atropine represents muscarinic (cholinergic) plus nonmuscarinic binding. The relative affinity of the analogues for dopamine binding sites was determined by their ability to displace [3H]spiroperidol from binding sites in rat caudate nuclei. To the extent which binding affinity for nonmuscarinic clozapine sites in rat forebrain reflects the antipsychotic potential of a particular drug, dibenzo-5H-cycloheptene analogues of clozapine are as effective as clozapine itself. Strong binding to nonmuscarinic clozapine sites is not dependent on the presence of a chlorine atom on th tricyclic system. One or both of the nitrogen atoms in the dibenzo-5H-[1,4]diazepine ring of clozapine appear to be necessary for the strong inhibition of clozapine binding to spiroperidol sites in rat caudate nuclei. Anticholinergic activity is substantially higher for clozapine and its dibenz[1,4]oxazepine analogue than for its benzo-5H-cycloheptene analogue.

Serotonin-norepinephrine receptor interactions in the brain: implications for the pharmacology and pathophysiology of affective disorders.

When chronically administered, most clinically effective antidepressant treatments (pharmacotherapy and ECT) reduce the sensitivity of the norepinephrine-sensitive adenylate cyclase in brain which, in turn, is associated with a down-regulation of the beta-adrenoceptor subpopulation. Because this norepinephrine receptor system is linked to an amplifier system, small changes in the number of receptors or in the accumulation of the second messenger cyclic AMP will be amplified. Results of the studies discussed in this paper demonstrate that an intact serotonergic neuronal input is required for the proper functioning of beta-adrenoceptors and for the down-regulation of the density of these receptors by antidepressant treatments. Under conditions of impaired serotonergic activity, beta-adrenoceptors display profound decreases in agonist but not in antagonist affinity. The changes are reminiscent of "uncoupled" receptors. While beta-adrenoceptors are coupled in a stimulatory fashion to adenylate cyclase, resulting in the formation of the second messenger cyclic AMP, serotonin (5-HT) receptors are linked to phosphatidylinositol hydrolysis (5-HT2 receptors in cortex, 5-HT1C receptors in choroid plexus) generating two second messengers, diacylglycerol and inositol-1,4,5-trisphosphate. The final common pathway of aminergic receptor activation seems to be protein-kinase-mediated protein phosphorylation leading to changes in cellular activity. Evidence is presented suggesting that the delayed down-regulation of the linked 5-HT/norepinephrine beta-adrenoceptor system by antidepressant treatment reflects a therapeutically relevant biochemical action and prompts the generation of the "5-HT/norepinephrine link hypothesis" of affective disorders.

Update on neuroreceptor mechanisms and their implication for the pharmacotherapy of affective disorders.

Unlike "acceptors" (binding sites), receptors for neurohormones display the dual function of recognition and biologic response via coupling to effector systems that generate second messengers or to ion channels that modify the passage of specific ions. The demonstration that membrane receptors for norepinephrine are coupled in a stimulatory (beta 1, beta 2) or inhibitory (alpha 2) way via nucleotide regulatory proteins to adenylate cyclase, thus increasing or decreasing the formation of the second messenger cyclic AMP, is discussed. Serotonin receptors are linked to phosphatidylinositol hydrolysis generating two second messengers, diacylglycerol and inositol triphosphate. Pharmacologically, serotonin (5-HT) receptors that are linked to phosphatidylinositol hydrolysis are of the 5-HT2 type in the cerebral cortex and of the 5-HT1c type in the choroid plexus. The final common pathway of signal transduction appears to be the protein-kinase-mediated phosphorylation of cellular proteins. Glucocorticoid receptors have been found to be located in the cytoplasma or nuclei of aminergic cell bodies and may exert their effects by modifying the genomic expression of the respective neurons. The two aminergic receptor systems are biomolecularly linked, with glucocorticoids exerting a modulatory role. The implications of central receptor research for the pharmacotherapy and the pathophysiology of affective disorders are reviewed.

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.

Effect of selective monoamine oxidase inhibition by clorgyline and deprenyl on the norepinephrine receptor-coupled adenylate cyclase system in rat cortex.

The consequences of selective monoamine oxidase (MAO) inhibition on the norepinephrine(NE)-sensitive adenylate cyclase system were determined in slices of rat cerebral cortex. The chronic administration of clorgyline, which selectively inhibited the activity of MAO-A, caused a significant decrease in the responsiveness of the noradrenergic cyclic AMP-generating system. The noradrenergic subsensitivity was accompanied by a significant decrease in the density of beta-adrenoceptors, as measured by 3H-dihydroalprenolol (DHA) binding, without altering the Kd value. However, selective inhibition of MAO-B by deprenyl did not alter the sensitivity of the cyclic AMP-generating system to NE or the specific DHA binding. The basal levels of cyclic AMP in the cortex were unaltered by the drugs. Since inhibition of MAO-A, but not MAO-B, increases the availability of NE, the results support the hypothesis that a persistent NE-receptor interaction is one of the prerequisites for the in vivo densitization of the NE-sensitive adenylate cyclase and the concomitant down-regulation of the number of beta-adrenoceptors in brain.

Desensitization by antidepressants of central norepinephrine receptor systems coupled to adenylate cyclase.

The experimental results discussed in this paper provide evidence that antidepressant-induced attenuation of the NE receptor-coupled adenylate cyclase system in brain and the down-regulation of its beta adrenoceptor subpopulation result in a net deamplification of the NE signal. The desensitization of the NE receptor system requires an unhindered occupancy of the receptor by the agonist NE. Following adrenalectomy, the non-beta population of NE receptors coupled to adenylate cyclase shows an enhanced response to NE without changes in the activity of adenylate cyclase or phosphodiesterase. This supersensitivity to NE can be prevented by corticosterone. The synaptic availability of 5HT is co-required for the down-regulation by DMI-like drugs of the density of beta adrenoceptors. Moreover, beta adrenoceptors from tissue deprived of serotonergic neuronal input display a marked decrease in agonist affinity as determined from competition binding of (-)-isoproterenol for [3H]dihydroalprenolol. Using NE as an agonist, competition binding curves with membrane preparations from cortical tissue lacking 5HT input show low affinity binding of the receptor for NE that cannot be further modified by guanine nucleotides. The reduction in beta adrenoceptor agonist affinity following reduction of the synaptic availability of 5HT is accentuated by chronic administration of DMI or zimelidine. The new experimental data on the biomolecular linkage between serotonergic and noradrenergic neurons, expressed functionally at the level of NE receptors, provide the scientific basis for a "serotonin-norepinephrine link hypothesis" of affective disorders. The pursuit of studies on the molecular mechanisms of the action of steroid hormones on central NE receptor systems and on mechanisms underlying the functional 5HT-NE linkage and its modification by antidepressants should generate a deeper understanding of neuronal signal processing in brain.

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.

An evaluation of the use of intraventricularly administered [3H]5-hydroxytryptamine as a marker for endogenous brain 5-hydroxytryptamine.

Following the intraventricular injection of a small amount of [3H]5-hydroxytryptamine ([3H]5-HT), the amount of radioactivity in telencephalic structures on the injected side was 6--7 times larger than that in corresponding areas on the opposite side. Moreover, a multiphasic disappearance of [3H]5-HT from whole brain or midbrain was found after the intraventricular injection of the labeled amine. However, after the intraventricular injection of [3H]tryptophan, the levels of [3H]5-HT in midbrain declined in a monophasic manner. A significant portion of the labeled amine derived from intraventricularly administered [3H]5-HT was resistant to the depleting effect of Ro4-1284 or to that elicited by destruction of the midbrain raphe nuclei, both of which caused an almost complete loss of endogenous 5-HT and labeled 5-HT formed from tryptophan. It thus appears that the intraventricular injection of [3H]5-HT leads to the formation of artifactual pools which are not present if the amine is synthesized in vivo. Studies with 6-hydroxydopamine suggested, however, that uptake of [3H]5-HT into adrenergic neurons did not occur to any great extent.

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.

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.