Acute stress-induced facilitation of the hypothalamic-pituitary-adrenal axis: evidence for the roles of stressor duration and serotonin.

Stress-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis stimulates the release of both facilitatory and inhibitory components. We proposed that the transient removal of the inhibitory component, corticosterone, during a stressor would leave the HPA axis in a state of hyper-responsiveness (facilitated state). Consistent with this expectation, we have previously observed that aminoglutethimide (AG)-induced removal of corticosterone during an immobilization stressor resulted in the hypersecretion of both ACTH and corticosterone to a subsequent stressor. In the present study we determined the effect of stressor duration on the magnitude of facilitation. AG plus a 10-min immobilization (IMM(10)) stress on day 1 resulted in facilitation of the HPA axis. This was reflected in higher ACTH and corticosterone responses to an injection stress on day 2 as compared to appropriate control rats. AG plus a 60-min immobilization (IMM(60)) stress on day 1 resulted in significantly greater facilitation as compared to the AG+IMM(10) pretreatment. It is apparent that facilitation of the HPA axis is dependent on the duration of stress. Stress can alter plasma corticosterone-binding globulin levels and AG administration can cause accumulation of the corticosterone biosynthetic precursor, adrenal cholesterol. In order to rule out these peripheral reasons for the hypersecretion of ACTH and corticosterone in our paradigm, we measured the plasma free fraction of corticosterone and adrenal mitochondrial cholesterol levels on day 2 after different pretreatments on day 1. AG+IMM(60) pretreatment caused a significant increase in the plasma free fraction of corticosterone. Hypersecretion of ACTH and corticosterone in this group, despite an enhanced feedback signal, suggests central loci for the origin of facilitation. Also, AG treatment on day 1 did not result in accumulation of free or esterified adrenal cholesterol levels on day 2, and therefore cannot account for the hypersecretion of corticosterone. In our final study we attempted to determine if serotonin released during the first stressor is partially responsible for stress-induced facilitation of the HPA axis. We administered 8-hydroxy-2-(di-n-propylamino)tetralin (DPAT), a 5HT(1A) agonist, either alone or in conjunction with stress and examined the effects of these pretreatments on the magnitude of facilitation. Interestingly, DPAT administered in lieu of stress produced facilitation similar in magnitude to that produced by IMM(10). DPAT administered in conjunction with IMM(10) augmented stress-induced facilitation. Our results suggest that stress-induced facilitation of the HPA axis is associated with the release of serotonin during stress.

Evaluation of possible reasons for the low phenylalanine ammonia lyase activity in cellulose nitrate membrane microcapsules.

Microencapsulated phenylalanine ammonia lyase (PAL) exhibits a marked reduction in activity compared to the activity of the free enzyme in pH 8.5 Tris buffer. The purpose of this investigation was to evaluate the contribution of incomplete entrapment, the internal environment of cellulose nitrate membrane microcapsules, the diffusional barrier of the membrane and the microcapsulation process to the low activity of encapsulated PAL. A solution of PAL and 10% w/v hemoglobin was incorporated into cellulose nitrate membrane microcapsules. Hemoglobin incorporation was used as a surrogate marker of PAL entrapment. Using 14C hemoglobin, the encapsulation efficiency was determined to be 70% and suggested that incomplete entrapment might partially account for the low activity of encapsulated PAL. The effect of the internal environment of the microcapsule (10% hemoglobin solution) on PAL activity was evaluated by comparing enzyme activity in 10% w/v hemoglobin solution and pH 8.5 Tris buffer. Similar K(M) and V(max) values of PAL in the two media indicated that the internal environment of the microcapsule did not contribute to the reduction in activity of the encapsulated enzyme. The contribution of a membrane diffusional barrier was determined by breaking the putative barrier and measuring PAL activity in intact and broken microcapsules. Similar activity of PAL in these two conditions is evidence for the lack of a diffusional barrier. The effect of the microencapsulation process on PAL activity was evaluated by comparing K(M) and V(max) of free and encapsulated PAL. Similar K(M) values in these two media suggested that the process did not affect the conformation of PAL. However, encapsulated PAL had a 50% lower V(max) value compared to free PAL, which showed that the microencapsulation process deactivated a substantial proportion of the enzyme.

Acute-stress-induced facilitation of the hypothalamic-pituitary-adrenal axis.

It has been hypothesized that the hypothalamic-pituitary-adrenal (HPA) axis responds to a stressor by secreting facilitatory and inhibitory factors. During a stressor, the relative magnitude of secretion of these factors determines the responsiveness of the HPA axis to a subsequent stressor. Previous studies have suggested that corticosterone (B) secreted during the first stressor is an inhibitory factor. We hypothesized that the transient removal of the inhibitory factor, B, during the first stressor would result in the secretion of only facilitatory factors. This would cause the HPA axis to exist in a state of hyperresponsiveness, and to hypersecrete corticotropin (ACTH) and B in response to a second stressor. Therefore, our primary objective was to demonstrate stress-induced facilitation of the HPA axis response to a subsequent stressor. Male Sprague-Dawley rats were subjected to a 1-hour physical immobilization stressor (IMM) or administered a single dose of ACTH on day 1. B response during these treatments was markedly but transiently attenuated with an 100 mg/kg i.p. dose of aminoglutethimide (AG). Twenty-four hours later, rats were subjected to an intraperitoneal saline injection stressor. B and ACTH levels were measured 15 min after the injection stressor. Rats treated with AG plus IMM on day 1 hypersecreted B and ACTH after the injection stressor on day 2. These results suggest that immobilization stress induces facilitation of both pituitary and adrenal responses. Exogenous administration of ACTH- to AG-pretreated rats on day 1, in lieu of immobilization stress, did not affect the responsiveness of the HPA axis on day 2. This suggests that ACTH secreted during the first stressor does not play an important role in acute-stress-induced facilitation.

The effect of acute, chronic and chronic intermittent stress on the central noradrenergic system.

The objective of this investigation was to examine the immediate and long term effects of acute, chronic and chronic intermittent stress on the central noradrenergic system of rats. Male Sprague-Dawley rats were subjected to one hour of physical immobilization stress either as a single exposure, or as 14 exposures applied either on consecutive days, or randomly over 60 days. Animals were sacrificed immediately, 6 h and 24 h following the last stressor. Levels of norepinephrine (NE) and 3-methoxy-4-hydroxyphenylethylene-glycol sulfate (MHPG-sulfate) were measured in the hypothalamus, hippocampus, cerebral cortex and locus coeruleus region and beta-adrenergic receptor (BAR) density was determined in the cortex. Immediately after acute stress, a significant reduction in hypothalamic NE levels and marked increases in MHPG-sulfate levels in all four brain regions were observed. In contrast immediately after the last stressor of a chronic or chronic intermittent stress regimen, no change in NE concentration was observed while levels of MHPG-sulfate in the four brain regions showed a smaller increase than that observed after an acute stressor. Acute stress induced changes normalized within 6 h while chronic and chronic intermittently stressed animals had altered NE or MHPG-sulfate levels in certain brain regions for up to 6-24 h. Cortical BAR binding parameters remained unchanged after all stress paradigms.

Analysis of corticosterone in rat plasma by high-performance liquid chromatography.

A sensitive and specific high-performance liquid chromatographic assay for the determination of corticosterone in rat plasma using dexamethasone as the internal standard is reported. Rat plasma (0.5 ml) is extracted with methylene chloride, washed with 0.1 M sodium hydroxide and then with water. The extract is analyzed by HPLC on a C18 column with ultraviolet absorbance detection at 254 nm. Pooled rat plasma was treated with activated decolorizing carbon to remove endogenous corticosterone, and was then used to prepare standards for the assay. Using 0.5 ml plasma for extraction, the detection limit of the assay is 10 ng/ml. The standard curve is linear over the concentration range 10-500 ng/ml. The recovery of corticosterone after extraction was independent of concentration and ranged from 87 to 95%. The coefficient of variation for intra-day and inter-day precision ranged from 2.4 to 7.4% and 2.1 to 8.7%, respectively. In addition, for concentrations ranging from 10 to 500 ng/ml the accuracy is within 5% of the spiked standards. The assay was utilized to examine the circadian rhythm of plasma corticosterone, and to examine the effect of immobilization stress on corticosterone levels in rats.

Design of a desipramine dosing regimen for the rapid induction and maintenance of maximal cortical beta-adrenoceptor downregulation.

Chronic administration of desipramine to rats causes a gradual reduction in cortical beta-adrenoceptor density. We examined the relationship between the duration of treatment with desipramine, and the rate and intensity of cortical beta-adrenoceptor downregulation. Male Sprague-Dawley rats were administered a 3.75 mg/kg/12 hr dose of desipramine for 4, 8 or 16 days. After 4 and 8 days of treatment, cortical beta-adrenoceptor density was reduced by 14 and 26% respectively. After 16 days of treatment, cortical beta-adrenoceptor density was maximally reduced by 36%. In our next series of experiments, we tested the hypothesis that the dose of desipramine required to rapidly induce maximal beta-adrenoceptor downregulation was higher than the dose required to maintain maximal beta-adrenoceptor downregulation. Initially, cortical beta-adrenoceptors were rapidly, and maximally downregulated with a four day, 10 mg/kg/12 hr induction regimen of desipramine. Trough, steady-state brain/cortical concentrations of desipramine plus desmethyldesipramine at the end of this regimen were approx 4000 ng/gm. Subsequently, maintenance desipramine regimens of 3.75 mg/kg/12 hr and 1.87 mg/kg/12 hr or vehicle were initiated for the next four days. Inspite of a 20-fold drop in brain/cortical concentrations of desipramine plus its metabolite, the 3.75 mg/kg maintenance regimen sustained maximal cortical beta-adrenoceptor downregulation. The 1.87 mg/kg maintenance regimen did result in a marked (25%) but non-significant recovery in the density of beta-adrenoceptors. Animals administered a vehicle maintenance regimen showed a large (50%) and statistically significant recovery of cortical beta-adrenoceptor density.

The pharmacodynamics of desipramine and desmethyldesipramine in rats.

The interrelationships between dose, drug and metabolite levels in the brain, cortex and blood and the magnitude of cortical beta adrenergic receptor downregulation were studied in rats after chronic i.p. administration of desipramine and its demethylated metabolite, desmethyldesipramine. Desipramine and desmethyldesipramine were distributed extensively into the brain and cortex with mean tissue to blood concentration ratios of approximately 10 to 1 and 14 to 1, respectively. Increasing doses of desipramine and desmethyldesipramine resulted in greater than linear increases in the corresponding steady-state trough concentrations of these drugs in brain, cortex and blood. Both drugs caused dose-dependent decreases in cortical beta adrenergic receptor density. The higher doses of desipramine and the highest dose of desmethyldesipramine used in this study caused maximal downregulation of beta adrenergic receptors. No changes were observed in the Kd of cortical beta adrenergic receptors after the administration of the two drugs. The ED50 for desipramine was determined to be 5.10 mg/kg, whereas the ED50 for desmethyldesipramine was 7.71 mg/kg. Nonlinear least-squares fitting of cortical concentration-effect data to the pharmacodynamic Emax model equation after the separate administration of desipramine and desmethyldesipramine generated Emax estimates for desipramine and desmethyldesipramine of approximately 36 and 29%, respectively, and EC50 estimates of 365 and 467 ng/g, respectively. Desmethyldesipramine is capable of maximally downregulating cortical beta adrenergic receptors and could account, in part, for the effect observed after desipramine administration.

Effect of miconazole on warfarin disposition in rabbits.

To elucidate the mechanism underlying the reported potentiation of warfarin anticoagulant action after initiation of miconazole therapy, the effects of acute and chronic miconazole administration on warfarin disposition were examined in six adult New Zealand male rabbits. The rabbits received a 3.5 mg/kg iv dose of warfarin either alone, 1 hr after a single 100 mg/kg ip miconazole dose, or on day 5 of a 6-day 50 mg/kg/12 hr ip miconazole dosing regimen. Acute miconazole administration decreased the elimination rate constant of warfarin, but other warfarin disposition parameters were not altered. Chronic miconazole administration caused a 47% increase in warfarin plasma-free fraction (probably caused by competitive or noncompetitive protein binding displacement by miconazole metabolites) and a 42% decrease in warfarin intrinsic clearance (probably caused by a miconazole-induced inhibition in warfarin metabolism). As a consequence of these quantitatively similar but opposite changes, the total body clearance of warfarin (a low clearance drug) was marginally decreased. A significant decrease in the elimination rate constant and an increase in the tissue-free fraction of warfarin were also observed during chronic miconazole treatment. These results suggest that chronic miconazole administration should not significantly affect the steady-state plasma concentrations of total warfarin, but should increase the steady-state plasma concentrations of free warfarin. The expected increases in the steady-state plasma concentrations of free, pharmacologically active warfarin may account for the reported potentiation of the pharmacological action of warfarin when coadministered with chronic miconazole. Measurement of total plasma concentrations, and estimation of total body clearance might be misleading, and inadequate in identifying certain drug interactions involving low clearance drugs.

Increased carbamazepine plasma concentrations after fluoxetine coadministration.

The interaction between fluoxetine and carbamazepine was investigated in six normal, healthy male volunteers (aged 23 to 40 years). Subjects were given carbamazepine, 400 mg every morning, for 3 weeks. Venous carbamazepine blood samples were obtained at baseline and 1, 2, 4, 6, 8, 10, 12, and 24 hours after the morning dose. Fluoxetine, 20 mg every morning, was then coadministered with carbamazepine for 7 days. Venous carbamazepine blood samples were again obtained as described. Carbamazepine and carbamazepine-10,11-epoxide (CBZE) were assayed by HPLC. Addition of fluoxetine resulted in a significant increase in the area under the concentration-time curve of carbamazepine (105.93 +/- 18.05 micrograms/ml.hr versus 134.97 +/- 12.15 micrograms/ml.hr; t = 3.284; df = 5; p = 0.022) and CBZE (11.6 +/- 1.93 micrograms/ml.hr versus 15.2 +/- 2.4 micrograms/ml.hr; t = 2.805; df = 5; p = 0.038). Both oral and intrinsic clearance of carbamazepine was decreased significantly on fluoxetine addition (3.87 +/- 0.68 L/hr versus 2.98 +/- 0.26 L/hr; t = 3.025; df = 5; p = 0.029 and 17.90 +/- 4.9 L/hr versus 11.92 +/- 1.4 L/hr; t = 3.037; df = 5; p = 0.029, respectively). No significant changes were determined for fraction of absorbed dose, volume of distribution, absorption rate constant, and elimination rate constant. These findings suggest that fluoxetine can inhibit the metabolism of carbamazepine. Careful monitoring of patients is recommended when these two drugs are coadministered.

Pharmacokinetics of the cyclosporine-ketoconazole interaction in dogs.

Numerous clinical reports have documented an increase in trough blood concentrations of cyclosporine in transplant recipients treated concomitantly with ketoconazole. The objective of this study was to elucidate the mechanism(s) underlying the cyclosporine-ketoconazole interaction using a choledochoureterostomy dog model. Five male beagle dogs received a 4 mg/kg, i.v. bolus dose of cyclosporine either alone or on day seven of a 10-day, 13 mg/kg/day, oral dosing regimen of ketoconazole. Blood samples were collected prior to and at predetermined times for 60 hrs after the cyclosporine dose, while the bile/urine mixture was collected quantitatively for 96 hours after the cyclosporine dose. Ketoconazole decreased the systemic clearance of cyclosporine from 7.0 ml/min/kg to 2.5 ml/min/kg. The terminal disposition rate constant was also decreased significantly from 0.0794 to 0.0354 hrs-1. Ketoconazole caused no significant changes in cyclosporine steady state volume of distribution, or plasma unbound fraction. Ketoconazole did not significantly alter the excretion of cyclosporine and various cyclosporine metabolites in the bile/urine mixture. Inhibition of hepatic drug metabolizing enzymes appears to be the primary reason for the ketoconazole induced elevation in cyclosporine concentration.

Evaluation of mono- and dibenzoyl esters of dopamine as potential pro-drugs for dopamine in the central nervous system.

In this study, two ester pro-drugs of dopamine (DA) were synthesized and evaluated. These derivatives were the monobenzoyl (MBDA) and dibenzoyl (DBDA) esters of DA. MBDA was 300-fold and DBDA was 20,000-fold more lipophilic than DA itself. The half-lives of hydrolysis for MBDA and DBDA at physiologic pH and temperature were 15 and 420 min respectively. These compounds were radiolabelled and their uptake into brain measured. 14C-DBDA penetrated the brain rapidly; 0.28% of the dose injected was taken up per gram of brain tissue at 5 min. However DBDA did not produce measurable increases in DA levels in the brain. 14C-MBDA was found not to penetrate the brain. However, when MBDA was administered intracerebroventricularly (i.c.v.) to rats, it caused DOPAC levels to increase significantly both in the striatum and in the rest of the brain. The increase in the amount of DOPAC measured in the striatum was 3 to 10-fold greater than that seen in the rest of the brain. In rats that were pretreated with the MAO inhibitor, pargyline, MBDA given i.c.v. caused increases in DA levels in both the striatum and in the rest of the brain. The increased DA levels in striatum were considerably greater than those seen in the rest of the brain. From these results, it is inferred that MBDA is being hydrolyzed in vivo in the brain to form DA which is then taken up into dopaminergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

The action of sodium deoxycholate on Escherichia coli.

Sodium deoxycholate is used in a number of bacteriological media for the isolation and classification of gram-negative bacteria from food and the environment. Initial experiments to study the effect of deoxycholate on the growth parameters of Escherichia coli showed an increase in the lag time constant and generation time and a decrease in the growth rate constant and total cell yield of this microorganism. Cell fractionation studies indicated that sodium deoxycholate at levels used in bacteriological media interferes with the incorporation of [U-14C]glucose into the cold-trichloroacetic acid-soluble, ethanol-soluble, and trypsin-soluble cellular fractions of E. coli. Finally, sodium deoxycholate interfered with the flagellation and motility of Proteus mirabilis and E. coli. It would appear then that further improvement of the deoxycholate medium may be in order.

Effects of chronic administration of bronchodilators on microshock in sensitized guinea-pigs.

In conscious guinea-pigs isoprenaline, adrenaline and salbutamol protected the animals against egg albumin and histamine microshock in a dose-dependent manner. Chronic aerosol pretreatment three times daily with adrenaline or isoprenaline, but not with salbutamol, enhanced the acute dose protection against egg albumin microshock, but there was no enhancement when the bronchodilators were given six times daily. Aminophylline when administered three times daily enhanced the protective effect of an acute dose of adrenaline. With histamine microshock, desensitization occurred to adrenaline, when given three and six times daily, and to isoprenaline, when given six times daily, but not to salbutamol. Cross-desensitization could be induced to adrenaline by isoprenaline but not by salbutamol. The data indicate that, depending on the experimental conditions, enhancement of or desensitization to the effects of bronchodilators could be shown in conscious guinea-pigs.

Responses of isolated human internal anal sphincter to drugs and electrical field stimulation.

The effects of drugs and electrical field stimulation on muscle strips from the human internal anal sphincter have been examined to provide information about the receptors and nerves that might be involved in the relaxation of the muscle in vivo. Acetylcholine and bethanechol usually relaxed muscle strips; this effect was abolished by hyoscine and antagonized to a varying degree by tetrodotoxin. Hexamethonium in concentrations sufficient to block relaxations to 1,1-dimethyl-4-phenylpiperazinium iodide or nicotine had no effect on relaxations due to acetylcholine, thereby indicating that acetylcholine was acting on muscarinic receptors. The nerves stimulated by acetylcholine released an unknown transmitter. Both 1,1-dimethyl-4-phenylpiperazinium iodide and nicotine relaxed muscle strips, possibly by releasing an adrenergic neurotransmitter which, because the responses to nicotinic receptor-stimulation were blocked by propranolol, stimulated beta-adrenergic inhibitory receptors. Sphincter muscle was also relaxed by electrical field stimulation of intrinsic nerves; this response was blocked by tetrodotoxin but unaffected by hexamethonium, hyposcine, or propranolol. The nerves responding to electrical field stimulation were therefore post-ganglionic, noncholinergic, and nonadrenergic. Compounds discounted as possible neurotransmitters of the noncholinergic, nonadrenergic inhibitory nerves were prostaglandin E2 and F2 alpha, histamine, 5-hydroxytryptamine, and dopamine. Some evidence allows vasoactive intestinal peptide and adenosine triphosphate to be considered as possible neurotransmitters; this could not be confirmed because selective antagonists are not yet available.