Rapid down regulation of beta adrenergic receptors by combining antidepressant drugs with forced swim: a model of antidepressant-induced neural adaptation

The hypothesis that behavioral responses to antidepressant drugs in the forced swim test are related to a rapid neural adaptation produced by the combination of drug treatment and swim stress was explored. As a measure of adaptation, brain beta adrenergic receptors were assayed using [3H]dihydroalprenolol [( 3H]DHA) binding to brain membranes from rats that were processed in the forced swim test. The combination of swim stress and imipramine treatment antagonized immobility induced by forced swimming and resulted in a reduction in [3H] DHA binding to membranes from forebrain preparations which did not include the corpus striatum. Administration of antidepressant drugs from other chemical classes, including pargyline, iprindole and nomifensine, also reduced immobility induced by the forced swim and produced a reduction in [3H]DHA binding to forebrain membranes. In homogenates of the corpus striatum, [3H]DHA binding was not altered by swim stress combined with antidepressant drug treatment. Chlordiazepoxide was without an effect on immobility or beta receptor binding when combined with forced swim. Even though atropine and amphetamine exhibited a positive activity in the forced swim test, they did not reduce [3H]DHA binding. Therefore, by combining behavioral and neurochemical analysis of animals processed in the forced swim test, it may be possible to differentiate, with greater confidence, potential antidepressant drugs from "false positives." The present studies support the hypothesis that antidepressant drug action in the forced swim test involves a rapid neural adaptation as reflected by the down regulation of beta adrenergic receptors. Thus, this behavioral paradigm may serve as a model of adaptive mechanisms induced by antidepressant drugs.     

Behavioral effects of beta adrenergic agonists and antidepressant drugs after down-regulation of beta-2 adrenergic receptors by clenbuterol

Acute treatment with the centrally active beta-2 adrenergic agonist clenbuterol reduced response rate and increased reinforcement rate of rats responding under a differential-reinforcement-of-low-rate (DRL) 72-sec schedule in a dose-dependent manner (ED50 value of about 0.1 mg/kg). With repeated treatment, rapid tolerance developed to this effect of clenbuterol. Redetermination of the dose-response function for clenbuterol, following 2 weeks of repeated daily administration, showed that clenbuterol no longer affected DRL behavior at doses up to 3 mg/kg. Interestingly, tolerance developed to clenbuterol even when it was administered after each daily session. This suggests that behavioral factors did not contribute, in an appreciable manner, to the development of tolerance to clenbuterol, and that neuropharmacological changes were sufficient for tolerance development. Such an interpretation is supported by the finding that the density of beta-2 adrenergic receptors in the cerebral cortices and cerebella of rats receiving the same repeated-treatment regimen was reduced with a time course similar to the loss of behavioral responsiveness. The effects of two additional beta-2 selective agonists, SOM-1122 and zinterol, on DRL behavior also were attenuated after repeated treatment with clenbuterol. By contrast, the effects of the beta-1 selective agonists dobutamine and prenalterol and the antidepressants desipramine, phenelzine and fluoxetine on DRL behavior were unaltered after repeated treatment with clenbuterol. These findings suggest functional independence of the beta adrenergic receptor subtypes and further suggest that, consistent with neuropharmacological data, the behavioral effects of the antidepressants do not depend on functionally responsive beta-2 adrenergic receptors.     

Desensitization of adenylate cyclase and down regulation of beta adrenergic receptors after in vivo administration of beta agonist

In vitro incubation of cells with catecholamines leads to both down regulation of beta adrenergic receptor number and desensitization of agonist-stimulated adenylate cyclase activity. These same parameters, down regulation of beta adrenergic receptor number and desensitization of adenylate cyclase activity were assessed in rat lung membranes after in vivo administration of metaproterenol, a beta-2 selective agonist. In vivo treatment with metaproterenol leads to: 1) reduced beta adrenergic receptor number; 2) reduced isoproterenol-stimulated adenylate cyclase activity; 3) unaffected NaF or 5'-guanylylimidodiphosphate-stimulated adenylate cyclase activity; and 4) reduced affinity of the receptor for isoproterenol similar to the affinity observed in the presence of 5'-guanylylimidodiphosphate. The date suggest that in vivo metaproterenol administration results in an uncoupled receptor-adenylate cyclase complex. The effects of in vivo administration of the glucocorticoid, methylprednisolone, to metaproterenol-pretreated animals were also assessed. Glucocorticoid treatment was associated with 1) increased beta adrenergic receptor number in rats in which the receptors have been down regulated, 2) increased isoproterenol responsiveness in agonist-desensitized rats and 3) no effect on agonist affinity in desensitized animals. These data suggest that the restoration of agonist responsiveness by glucocorticoids in the catecholamine refractive state is not simply a reversal of receptor down regulation or adenylate cyclase desensitization.     

Selective regulation of beta-2 adrenergic receptors by the chronic administration of the lipophilic beta adrenergic receptor agonist clenbuterol: an autoradiographic study

Many antidepressant drugs, when administered chronically to rats, have been shown to produce decreases in the density of beta adrenergic receptors in the central nervous system. The centrally active beta adrenergic receptor agonist clenbuterol is currently being evaluated clinically as an antidepressant. The chronic administration of this drug to rats resulted in a large decrease in the density of beta adrenergic receptors in some areas of the rat brain but not in others. Thus, autoradiographic studies revealed that the total density of beta adrenergic receptors in the molecular layer of the cerebellum, but not in layers 1 to 3 or layer 4 of the cerebral cortex, was decreased. To examine whether this regional selectivity occurred because of differences in plasticity of cerebellum and cortex or because cerebellum contains mainly beta-2 adrenergic receptors and cortex contains mainly beta-1 adrenergic receptors, separate analyses of the subtypes of beta adrenergic receptors were performed in each area. These experiments indicated that the decrease in receptor density was entirely specific for beta-2 adrenergic receptors, whereas the density of beta-1 receptors was unchanged. Thus, even in layers 1 to 3 and layer 4 of the cerebral cortex, beta-2 receptor density was decreased, with no change in beta-1 receptor density. Using the autoradiographic assay for ligand binding, it was shown that clenbuterol has equal affinity for beta-1 and beta-2 adrenergic receptors, indicating that the selective effect of this drug was not due to a selective affinity for beta-2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in the density of beta adrenergic receptors in rat lymphocytes, heart and lung after chronic treatment with propranolol

Abrupt withdrawal after the chronic administration of propranolol results in clinical syndromes that suggest adrenergic hypersensitivity. Furthermore, propranolol administration has been shown to lead to an increase in the density of beta adrenergic receptors on human lymphocytes. The present studies were designed to assess the relevance of changes measured in lymphocytes to changes that may occur in solid tissues. Direct measurement of the density and properties of beta adrenergic receptors in membrane fragments was performed in vitro using the radioligand [125I]iodohydroxybenzylpindolol. Chronic infusion of propranolol by s.c. implanted osmotic minipumps generated sustained plasma concentrations of propranolol sufficient to cause chronic blockade of beta adrenergic receptors. Infusion of propranolol for 7 days resulted in significant increases in the density of beta adrenergic receptors in rat ventricles, lungs and lymphocytes. A computer-assisted graphic analysis of results obtained in studies with drugs selective for beta-1 or beta-2 receptors revealed increases in the densities of both beta-1 an beta-2 adrenergic receptors. These results are consistent with the hypothesis that change in beta adrenergic receptors on lymphocytes are qualitatively similar to alterations in beta adrenergic receptors in solid tissues not routinely accessible in humans. Increases in the densities of beta-1 and/or beta-2 adrenergic receptors in solid tissues may be related to some of the untoward effects observed in humans after abrupt discontinuation of propranolol administration.     

Effects of antidepressant drugs injected into the amygdala on behavioral responses of rats in the forced swim test

Experiments were conducted to determine if the actions of antidepressant drugs in a pharmacological screen would be localized to specific brain regions. Rats were infused in discrete brain regions with drugs of different pharmacological properties and processed in the forced swim test. Infusion of imipramine and pargyline into the amygdala produced behavioral responses similar to i.p. injections of the drugs. The regions of the amygdala from which positive responses could be elicited were highly selective. From cannula placements and diffusion studies with autoradiography it appears that a locus of action of imipramine and pargyline is confined to the central, basolateral and/or lateral nuclear regions of the amygdala. Behavioral responses of rats in which imipramine was infused into the anterior amygdala or caudate-putamen did not differ from saline controls. When infused into regions of the amygdala in which imipramine was active, iprindole, an "atypical" antidepressant, did not produce a behavioral response in the forced swim test. Two "false positives" in the forced swim test, atropine and amphetamine, were also not active when infused into the amygdala. These results indicate that the amygdala is a critical site of action for certain antidepressant drugs in the forced swim test, and that behavioral activation in the test induced by iprindole, amphetamine and atropine involve brain regions other than the amygdala.     

Rapid decrease in rat brain beta adrenergic receptor binding during combined antidepressant alpha-2 antagonist treatment

The delay in onset of the therapeutic effect of antidepressants is believed to be due to a progressive decrease in the density of central beta adrenergic receptors. The changes in beta adrenergic receptor density could result from an increase in the synaptic concentration of norepinephrine, which is secondary to a decrease in the sensitivity of the alpha-2 adrenergic receptor which normally inhibits release and the firing of the locus ceruleus. We have observed an acceleration of beta receptor desensitization with combined administration of antidepressants and alpha-2 adrenergic antagonists. After one day of administration of desipramine (DMI) with phenoxybenzamine, there was a marked decrease in beta adrenergic receptor density. One day of treatment with DMI alone had no significant effect on beta receptor density. Rapid desensitization occurs not only in rat limbic cortex, but also in hippocampus and mesencephalon. Furthermore, combination therapy of DMI with yohimbine or dihydroergotamine, both alpha-2 adrenergic blockers, is similar to DMI-phenoxybenzamine treatment. Combined administration with prazosin, an alpha-1 antagonist, had no effect on antidepressant-induced desensitization. Combined administration of alpha-2 antagonists accelerated beta receptor desensitization by amitriptyline, mianserin, iprindole, tranylcypromine and pargyline. These observations suggest that pharmacological blockade of alpha-2 adrenergic receptors enhances antidepressant-induced decreases in central nervous system beta-adrenergic receptor density.     

Kinetics of competitive drug action at 5-hydroxytryptamine2 receptors in isolated rabbit aorta

The kinetics of agonist and antagonist interactions with the 5-hydroxytryptamine2 receptor were studied in the isolated rabbit aorta by following the antagonist-induced decrease in the steady-state response to an agonist. A model describing the competitive drug-receptor interactions was fitted to the data and yielded estimates of the association and dissociation rate constants of the agonist and the antagonist. A high concentration of the agonist ([agonist] much greater than KA) was used to reduce the influence of antagonist diffusion to the receptor upon the onset of antagonism. The effect of a diffusion barrier was evaluated by comparing the kinetics of drug competition in the absence and in the presence of the adventitia. The rate constants of the high-affinity antagonists spiperone, methysergide or ketanserin were similar in the absence and in the presence of the adventitia. In contrast, the rate constants of the low affinity antagonist 5-methoxygramine were reduced almost 5-fold in the presence of the adventitia. This observation may be explained by the large partition coefficients of the high-affinity antagonists as compared to the relatively low partition coefficient of 5-methoxygramine. The ratios of the estimated rate constants (k-x/kx) are in good agreement with the dissociation constants of the drugs determined with steady-state methods. In addition the results suggest that the association rate constant is a primary determinant of drug affinity for the receptor. The kinetic rate constants of the high-affinity antagonists measured in this preparation are similar to those previously reported in high-affinity binding studies. We conclude that the kinetic parameters obtained in our experiments reflect primarily the molecular interactions of these drugs with the receptor.     

Presynaptic modulation of beta adrenergic receptors in rat cerebral cortex after treatment with antidepressants.

Treatment with desmethylimipramine (DMI), a tricyclic antidepressant, for 7 to 21 days resulted in a 35 to 45% decrease in the accumulation of adenosine cyclic 3':5'-monophosphate (cAMP) in response to a maximally effective concentration of (-)-isoproterenol (ISO) in rat cerebral cortical slices. The EC50 for ISO-stimulated cAMP accumulation was not affected by DMI administration. The diminution in responsiveness to catecholamines was accompanied by a 35 to 40% decrease in the density of beta adrenergic receptors as measured by the binding of [125I]iodohydroxybenzylpindolol. Decreases in ISO-sensitive cAMP accumulation and in beta adrenergic receptor density were temporally correlated, maximal decreases being observed within 5 to 7 days. Within 7 days after cessation of chronic DMI treatment ISO-stimulated cAMP accumulation and beta adrenergic receptor density returned to normal. The role of presynaptic nerve terminals in mediating these phenomena was also investigated. Treatment of newborn rats with 6--hydroxydopamine inhibited the development of noradrenergic nerve terminals in the cerebral cortex and blocked the effects of DMI on cortical cAMP accumulation and on beta adrenergic receptor density. The administration of the beta adrenergic receptor antagonist propranolol led to increases in maximal ISO-stimulated cAMP accumulations and beta adrenergic receptor density in the rat cerebral cortex. This increase was not affected by the simultaneous administration of propranolol and DMI. Thus, the effect of DMI appears to be mediated through an action of norepinephrine at beta adrenergic receptors. Chronic treatment with two other clinically effective antidepressants, pargyline and iprindole, led to effects similar to those observed with DMI administration. Pretreatment of neonates with 6-hydroxydopamine blocked the effect of iprindole on beta adrenergic receptors. Preincubation of cortical membranes with guanosinetriphosphate before determination of the density of beta adrenergic receptors had no effect on the decreased number of receptors had no effect on the decreased number of receptors seen in DMI-treated animals. These experiments suggest that antidepressants, acting presynaptically, increase the concentration of transmitter at noradrenergic synapses and induce a compensatory decrease in the density of beta adrenergic receptors.     

Interaction of beta adrenergic agonists and antagonists with brain beta adrenergic receptors in vivo

There is interest in knowing whether beta adrenergic antagonists or agonists, when administered systemically, can enter the brain to interact with central beta adrenergic receptors. To study this, the reduction in the radioactive content in the brain of rats after administration of (-)-[125I]iodopindolol (IPIN) by systemically administered beta agonists or antagonists was measured. Previous studies show that after the i.v. administration of IPIN the binding in vivo to various areas of the central nervous system has the characteristics expected of binding to beta adrenergic receptors. Of the antagonists tested, pindolol and butylpindolol showed potent interactions with beta receptors in both cortex and cerebellum whereas atenolol and practolol did not interact at doses up to 30 mg/kg. CGP-12177 showed moderate potency in inhibiting IPIN binding in vivo. We have shown previously that propranolol and alprenolol inhibit IPIN binding with high potency in cortex and cerebellum. At high doses, butoxamine, a beta-2 antagonist, reduced the binding of IPIN in the cerebellum but not in the cortex. Of the agonists tested, clenbuterol and prenalterol caused a significant dose-dependent reduction of the binding of IPIN, with clenbuterol being more potent. Isoproterenol, salbutamol, salmefamol and dobutamine had no effect. With the exception of CGP-12177, the affinity of the drugs for central beta adrenergic receptors measured in vitro was correlated significantly with their ability to inhibit IPIN binding in vivo whereas their degree of lipophilicity was not correlated significantly with potency in vivo. The inhibition of IPIN binding in vivo from brain areas can be used to evaluate whether drugs penetrate into brain and interact with central beta adrenergic receptors.     

Pharmacological characterization of 5-hydroxytryptamine2 and 5-hydroxytryptamine3 receptors in rat dorsal root ganglion cells

5-Hydroxytryptamine (5-HT) depolarized 87% of the rat dorsal root ganglion cells recorded. 5-HT increased the input resistance (Rin) in 50%, decreased Rin in 41% and produced both responses in 9% of the responding cells. When 5-HT increased the Rin, the response was mimicked by the 5-HT2 agonists alpha-methyl-5-HT, (+/-)-1-(2,5-dimethoxy-4 iodophenyl)-2-aminopropane HCl, quipazine and MK 212 (6-chloro-1-[1-piperazinyl]-pyrazine), but not by 2-methyl-5-HT or carboxamidotryptamine. The response to 5-HT was antagonized by ketanserin, spiperone and methiothepin. The unsurmountable blockade induced by higher concentrations of ketanserin was not explained by pseudo-irreversible antagonism or multiple receptor subtypes, but could result from a two-state receptor model or multiple subtypes of the 5-HT2 receptor. This conclusion is supported by the partial agonist action of DOI. Cells responding to 5-HT with depolarization and decreased Rin responded similarly to 2-methyl-5-HT and phenylbiguanide, but not to alpha-methyl-5-HT or carboxyamidotryptamine. This response was surmountably blocked by ICS 205-930 (3-tropanyl-indole-3-carboxylate) (pA2 = 10.3) and MDL 72222 (3-tropanyl-3,5-dichlorobenzoate)(pA2 = 7.8). The arylpiperazines, quipazine and MK 212, antagonized the action of 2-methyl-5-HT with IC50 values of 8 and 4 nM, respectively. These data indicate that 5-HT2 receptors mediate the increased Rin and 5-HT3 receptors mediate the decreased Rin.     

Antidepressant-like effects of kappa-opioid receptor antagonists in the forced swim test in rats

We showed previously that cAMP response element-binding protein (CREB) within the nucleus accumbens (NAc) of rats regulates immobility in the forced swim test (FST), an assay used to study depression. Because CREB regulates expression of dynorphin (which acts at kappa-opioid receptors) in NAc neurons, these findings raised the possibility that kappa-receptors mediate immobility behaviors in the FST. Here, we report that i.c.v. administration of the kappa-antagonist nor-binaltorphimine dose dependently decreased immobility in the FST, suggesting that it has antidepressant-like effects. Implicating a specific effect at kappa-receptors, similar antidepressant-like effects were seen after treatment with either of two novel, structurally dissimilar kappa-antagonists: 5'-guanidinonaltrindole, which was effective after i.c.v. but not systemic treatment, and 5'-acetamidinoethylnaltrindole (ANTI), which was potent and effective after systemic treatment. The behavioral effects of the kappa-antagonists resembled those of tricyclic antidepressants (desipramine) and selective serotonin reuptake inhibitors (fluoxetine and citalopram). Conversely, systemic administration of the kappa-agonist [5alpha,7alpha,8beta]-N-methyl-N-[7-[1-pyrrolidinyl]-1-oxaspiro[4.5]dec8-yl]-benzenacetamide (U-69593) dose dependently increased immobility in the FST, consistent with prodepressant-like effects. The effects of the kappa-ligands in the FST were not correlated with nonspecific effects on locomotor activity. Furthermore, the most potent and effective kappa-antagonist (ANTI) did not affect the rewarding impact of lateral hypothalamic brain stimulation at a dose with strong antidepressant-like effects. These findings are consistent with the hypothesis that CREB-mediated induction of dynorphin in the NAc "triggers" immobility behavior in the FST. Furthermore, they raise the possibility that kappa-antagonists may have efficacy as antidepressants, but lack stimulant or reward-related effects.     

Potentiation of responses to adrenergic nerve stimulation in isolated rat atria during chronic tricyclic antidepressant administration

Several weeks of tricyclic antidepressant administration are required to effectively reverse depression. To determine whether there are adaptive changes in andrenergic nerve function which correspond to the clinical onset of antidepressant action, the endogenous norepinephrine content, [3H]norepinephrine uptake and retention, responses to exogenous norepinephrine and the release of norepinephrine during field stimulation were studied using left atrial strips isolated from rats treated with either acutely or chronically with tricyclic antidepressants. Desipramine, nortriptyline, chlorimipramine and iprindole were administered to rats, 10 mg/kg i.p., twice daily. After 14 days of drug administration, the responses to field stimulation were potentiated markedly by all four tricyclics. In contrast, 1 day of tricyclic treatment had only slight potentiating effects. When phenoxybenzamine, 10(-7) M, was added to the organ bath in order to block the inhibitory presynaptic alpha receptor, the responses of control atria and atria from rats treated for 1 day with desipramine were potentiated but those of atria treated for 21 days with desipramine were not potentiated. The development of presynaptic alpha receptor subsensitivity during chronic tricyclic administration would explain these findings. Other possible explanations were also investigated. The uptake and retention of [3H]norepinephrine was markedly inhibited to a similar degree of either 1 or 14 days of desipramine or nortriptyline administration. One day of chlorimipramine treatment decreased the amount of [3H]norepinephrine taken up and retained by left atrial strips, and after 14 days of treatment decreased the amount further. In contrast, neither 1 nor 14 days of iprindole administration had any effect on the uptake and retention of [3H]norepinephrine. These data indicate that the potentiation of the responses to field stimulation cannot be explained by the inhibition of norepinephrine uptake. The inotropic response to exogenous norepinephrine was not altered by any duration of administration of any of the four tricyclics studied. Furthermore, the endogenous norepinephrine content of atria did not change after as many as 21 days of desipramine administration. The present results indicate that the potentiation of the effects of adrenergic nerve transmission during chronic tricyclic administration is the result of an increase in norepinephrine release which occurs due to the development of presynaptic alpha receptor subsensitivity. The time course of development of presynaptic receptor subsensitivity corresponds well with the onset of clinical activity of these drugs.     

Labeling in vivo of beta adrenergic receptors in the central nervous system of the rat after administration of [125I] iodopindolol

The amount of radioactivity in vivo in the central nervous system (CNS) of the rat has been studied after tail-vein injections of (-)- [125I] iodopindolol (IPIN). The content of radioactivity in cortex and cerebellum 1 to 4 hr after IPIN administration was significantly reduced in rats pretreated with I-propranolol (1 mg/kg) given i.v. 5 min before IPIN; only a small effect of I-propranolol was seen in brainstem and spinal cord. The maximum reduction in radioactivity caused by I-propranolol was approximately the same in cortex and cerebellum (about 60-65%) and occurred 2 hr after IPIN administration. I-Propranolol was approximately 1500-fold more potent than d-propranolol in reducing radioactivity. Pretreatment of rats with other lipophilic drugs that act at beta receptors was able to reduce the binding of IPIN in vivo; in contrast, pretreatment of rats with drugs which do not have direct agonist or antagonist activity at beta adrenergic receptors (desmethylimipramine, metergoline, diazepam, fluoxetine, phentolamine and haloperidol) had no effect. Experiments using ICI 118, 551, a beta-2 antagonist and betaxolol, a beta-1 antagonist, indicated that the majority of radioactivity in the cortex in vivo was bound specifically to the beta-1 subtype of the receptor whereas in the cerebellum the majority of specific binding was to the beta-2-subtype. When the specific binding of IPIN to beta adrenergic receptors was measured in vitro in seven regions of the CNS, at a ligand concentration of 30 pM, a high correlation was found with the I-propranolol displaceable radioactivity measured in vivo (r = 0.97, P less than .001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of beta adrenergic antagonists with isolated rat alveolar type II pneumocytes. I. Analysis, characterization and regulation of specific beta adrenergic receptors

Binding of beta adrenergic receptors (BAR) in membranes of freshly isolated type II pulmonary epithelial cells to the radioligand [125I]iodocyanopindolol was saturable, steresospecific and of high affinity. Optimal conditions for the assay of BAR on type II pneumocyte membranes are presented. Type II pneumocyte BAR are primarily of the beta-2 subtype, as indicated by inhibition of subtype-specific antagonists, ICI 118,551 and betaxolol. Maximum binding and Kd were measured (Kd, 4-20 pM; maximum binding, 30-50 fmol/mg of protein) and used to identify factors which alter BAR. The number of BAR on type II pneumocytes doubled after 42-hr culture in the presence of dexamethasone. Ligand-receptor interactions were of similar affinity to those in membrane particulates from whole lung, but maximum binding was reduced 3- to 4-fold. Less than 5% of total pulmonary BAR can be accounted for by those expressed on freshly isolated type II pneumocyte membranes. Other cell types thus probably account for the majority of specific beta adrenergic binding sites in the lung as a whole.     

Inhibition of adrenergic neurotransmission in canine tibial artery after exposure to 5-hydroxytryptamine in vitro

Blood vessels may be exposed to 5-hydroxytryptamine when platelets aggregate and release vasoactive substances at sites of damage or disease. The functional consequences were studied of exposing the dog tibial artery for 2 hr to 5-hydroxytryptamine (10(-6) M) in vitro. During the exposure, unmetabolized 5-hydroxytryptamine was accumulated by the cocaine-sensitive amine uptake mechanism of tibial artery adrenergic nerves. After exposure to [3H]-5-hydroxytryptamine, transmural electrical stimulation caused the release of the tritiated indoleamine which was blocked by tetrodotoxin. After a 1-hr washout of rings of tibial artery exposed previously to 5-hydroxytryptamine, contractions in response to transmural electrical stimulation were depressed, whereas the response to exogenously added norepinephrine was unaffected. That the decreased response to electrical stimulation after exposure to 5-hydroxytryptamine was due to decreased release of norepinephrine from adrenergic nerves was demonstrated in strips of the artery preincubated in [3H]norepinephrine. The inhibition of [3H]norepinephrine release after exposure to 5-hydroxytryptamine, was blocked by the serotonergic antagonist, methiothepin, but not by the alpha adrenergic antagonist, phentolamine, suggesting that serotonergic receptors mediate the inhibition. The inhibition of [3H]norepinephrine release also was prevented by blocking adrenergic neuronal uptake with cocaine before exposure to 5-hydroxytryptamine. These results suggest that 5-hydroxytryptamine is accumulated and released by tibial artery adrenergic nerves as a cotransmitter. In so doing, the indoleamine inhibits adrenergic neurotransmission in the tibial artery by its action at prejunctional serotonergic receptors.     

Neural adaptation in imipramine-treated rats processed in forced swim test: assessment of time course, handling, rat strain and amine uptake

The intent of the present series of experiments was to better understand the events that produce a rapid adaptation of beta adrenergic and serotonin-2 (5-HT2) receptors when imipramine treatment and forced swim are combined in Sprague-Dawley rats. Beta adrenergic and 5-HT2 receptors were evaluated at specific stages of the forced swim test with and without imipramine treatment. Rapid changes in receptor binding were observed in saline-treated rats during specific stages of the test. The changes observed during forced swim could not be attributed to the transport-novelty that occurs during forced swim. Binding for both monoamine receptors was reduced in hippocampus and frontal cortex before the test swim in imipramine-treated rats as they were 10 min, 3 hr and 24 hr after the test swim. The increase in corticosterone induced by the second forced swim was not altered by imipramine, indicating that imipramine was not interfering with this measure of the stress response. In the Fisher-344 rat strain, imipramine did not produce a behavioral change during the test swim. In contrast to this lack of a behavioral change in the Fischer-344 rats, beta adrenergic and 5-HT2 receptor down-regulation was facilitated in this rat strain, similar to that found in imipramine-treated Sprague-Dawley rats subjected to swim. This latter finding suggests that beta adrenergic or 5-HT2 receptor adaptation alone is insufficient to cause an imipramine-induced behavioral change in the swim test. Studies with specific norepinephrine- and serotonin-uptake inhibitors, nisoxetine and fluoxetine, respectively, indicate that the behavioral effects of imipramine in the forced swim test are dependent upon norepinephrine uptake inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of serotonin2 (5-HT2) receptors labeled with [3H]spiroperidol by chronic treatment with the antidepressant amitriptyline

Recently, we reported that chronic administration of several antidepressants of different classes produced larger reductions in numbers of serotonin2 (5-HT2) receptors in rat brain labeled by [3H[spiroperidol than in beta adrenergic receptors. In the present study, we examine detailed properties of 5-HT2 receptor regulation by chronic treatment with amitriptyline. Chronic but not acute treatment with the tricyclic antidepressant amitriptyline reduces binding to 5-HT2 receptors by [3H]spiroperidol and beta adrenergic receptor binding of [3H]dihydroalprenolol in brain membranes. The decrease is time-dependent, gradually reversible and represents a change in the number of binding sites with no alteration in drug affinities for 5-HT2 receptors. The effect can be observed at daily doses of 2.5 mg/kg, similar to clinically effective doses in humans. At all doses and time intervals, the decrease in 5-HT2 receptors is more marked than the concurrent change in total beta adrenergic receptor binding. The properties of 5-HT2 receptor reduction after chronic antidepressant treatment indicate that this alteration could be associated with therapeutic response.     

Mutual-effect amplification of contractile responses elicited by simultaneous activation of alpha-1 adrenergic and 5-hydroxytryptamine2 receptors in isolated rat aorta.

5-Hydroxytryptamine (5-HT), and the selective alpha-1 agonists phenylephrine (PE) and oxymetazoline (OXY), were used to study the effects of simultaneous coactivation of the 5-HT2 and alpha-1 adrenergic receptors, respectively, on the contractile responses of isolated rat aortic rings. Dissociation constants (KA) were determined for each of the agonists at their respective receptor subtypes. The KA values for PE and OXY at the alpha-1 receptor were 316 nM and 1.82 microM, respectively, while the KA for 5-HT at the 5-HT2 receptor was 478 nM. Concentration-response curves for each agonist were analyzed by the Black and Leff operational model of pharmacological agonism to determine efficacy (tau) and slope factor values. The estimated tau for PE (16.02) was much greater than the tau for either OXY (4.15) or 5-HT (2.95), which had similar efficacies. Using a previously described drug concentration paradigm, a mutual-effect amplification of the 5-HT-induced contractile response was observed with mixtures of 5-HT and PE, whereas mixtures of OXY and 5-HT elicited a mutual-effect amplification of the observed response to OXY alone. In both cases, the theoretical concentration-response curve constructed using the Poch and Holzmann method of equiactive substitution demonstrated that mutual-effect amplification was largely the result of simple additivity of agonist effects. In addition, estimates of tau and slope factor determined from the Black and Leff equation were substituted into the Leff model of mutual-effect amplification and used to accurately predict the location of the concentration-response curves elicited by mixtures of 5-HT and PE, as well as mixtures of OXY and 5-HT. This represents the first time a mathematical model has been used to accurately predict the outcome of coactivation of the alpha-1 and 5-HT2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding and functional characteristics of beta adrenergic receptors in the intact neutrophil

Beta adrenergic receptors have been previously characterized in human neutrophil sonicates. In the present study the intact neutrophil has been assessed for the number and affinity of beta adrenergic binding sites by using the antagonist DNA. Agonist and antagonist potencies, characterized by their effect on DHA binding and cyclic AMP accumulation, are compared with agonist inhibition of lysosomal enzyme (beta glucuronidase) release. Criteria for beta adrenergic receptor identification were successfully demonstrated. At 30 degrees C, beta adrenergic binding was rapid (t 1/2 2 min) and reversible (t 1/2 9 min). Receptor binding was saturable, revealing approximately 900 high-affinity receptors per neutrophil with DHA concentrations of 0.1 to 10 nM. By utilizing both equilibrium and kinetic techniques, the KD was determined to be approximately 0.6 nM. Agonists and antagonists competed for DHA binding in a manner consistent with their effect on cyclic AMP generation. Rank order potency was suggestive of a beta-2 receptor: isoproterenol greater than epinephrine greater than norepinephrine. Stereoselectivity was shown by the greater potency of L-propranolol compared to the D isomer. A high degree of receptor-adenylate cyclase coupling efficiency was suggested by the observation that with only 1% receptor occupancy isoproterenol stimulated 50% maximal cyclic AMP generation. Finally, there was an excellent correlation between the isoproterenol concentration which resulted in 50% of maximal inhibition of beta glucuronidase release (Ki) and that causing 50% maximal cyclic AMP stimulation (Kact), suggestive of a close relationship between beta adrenergic-induced adenylate cyclase activation and beta adrenergic regulation of neutrophil lysosomal enzyme release. The data presented suggest that the use of the intact neutrophil for study of the beta adrenergic receptor is feasible and may provide information which is considerably more closely related to modulation of physiological function by neurohormones than is possible with disrupted cell preparations.