Ultrastructure of thymus and liver following adrenalectomy and replacement adrenal hormone therapy in rats

Prior research in this laboratory has shown that dexamethasone, aldosterone, and epinephrine interact in regulating the activity of ornithine decarboxylase (EC 4.1.1.17, ODC) in rat thymus and liver. The three primary adrenal hormones were administered alone and in various combinations to adrenalectomized rats. Liver and thymus samples were removed, prepared for electron microscopy, and morphometric analysis of thymic micrographs was performed. It was found that both corticosteroids induced thymic lympholysis and that concurrent administration of epinephrine 'rescued' the lymphocytes. Observations of liver micrographs indicated that changes in liver glycogen deposition vary in response to the hormone treatment regimen. The liver response to a combination of the glucocorticoid and catecholamine was different from the response to the mineralocorticoid and catecholamine, which indicated that the liver response to the two steroids may be mediated via different mechanisms. Evidence is provided to support the conclusion that the influence of the adrenal gland on rat thymus and liver is not restricted to glucocorticoids but may also involve mineralocorticoids and catecholamines.     

Desensitization of ornithine decarboxylase activity to norepinephrine in the testis of rat

Injection of norepinephrine (NE) at a dose of 10 micrograms per testis caused the testis refractory in terms of ornithine decarboxylase (ODC) activity at 24 h. This desensitization was found to be both time and dose dependent. Injection with follicle stimulating hormone, luteinizing hormone, prostaglandin F2 alpha, cyclic AMP or epinephrine to norepinephrine desensitized testis caused stimulation of ODC activity. This indicates that the refractoriness caused by norepinephrine is specific to this agent alone.     

Effect of catecholamines on ornithine decarboxylase activity in the testis of immature rat

Intratesticular injection of epinephrine and norepinephrine caused stimulation of ornithine decarboxylase (ODC) activity in the testis of immature rat. The effect of epinephrine was time and dose dependant. The minimal effective dose for epinephrine was found to be 100 pg and optimal stimulation was observed with 500 ng of the drug. Maximal stimulation of ODC occurred at 2 h after the treatment and reduced significantly at 4 h reaching to control levels at 6 h. Simultaneous injection of epinephrine with dibutyryl cAMP, luteinizing hormone, follicle stimulating hormone or prostaglandin E₂ caused additional stimulation of the enzyme activity. Injection of epinephrine to norepinephrine treated animals caused additional effect. Both epinephrine and norepinephrine were found to stimulate the enzyme activity in leydig cell and seminiferous tubule fractions. These results suggest that catecholamines are also involved in the regulation of ODC activity in the testis of rat.     

Ornithine decarboxylase induction and polyamine levels in the kidney of estradiol-treated castrated male rats

Ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), and thymidine kinase (TK) activities and polyamine concentrations on the kidneys of male castrated rats were studied following sc injection of estradiol. Estradiol caused an 11-fold increase in ODC activity 24 hours after administration. SAMDC activity doubled but TK activity decreased by two-thirds 2 days after estradiol treatment. The concentrations of polyamines, especially putrescine, showed sharp elevations 2 days following estradiol treatment, 1 day after the peak of ODC activity. The increase in ODC activity was suppressed by cycloheximide and by actinomycin D. Estradiol and diethylstilbestrol (DES), but not progesterone increased ODC activity. Estradiol suppressed ODC activities of liver, thymus, adrenal glands, testes and prostate. A specific estradiol-binding protein was demonstrated in the rat kidney. The dissociation constant (Kd) was 1.64 × 10^?10 M and numbers of binding sites were 31 fmoles/mg protein. Correlation between the binding of estradiol to the cytosol protein and elevation of ODC by estradiol was observed.     

Inhibition of the rat adrenal ornithine decarboxylase activity by immobilization stress and/or dexamethasone

The effects of immobilization stress and/or dexamethasone (DEX) on the adrenal ornithine decarboxylase (ODC) activities of sham-operated and adrenal-medulloectomized (enucleated) male Sprague-Dawley rats were investigated. On day 11 after surgery, rats were injected with saline or DEX (1 mg/kg), 3 h before the time of sacrifice (0600 h or 1800 h). Four groups, from sham-operated and enucleated rats (ENU) treated with saline or DEX were subjected to immobilization stress for 1 h prior to sacrifice. Groups of rats from stress-sham-DEX, non stress-sham-DEX, stress-sham, non stress-sham, stress-ENU-DEX, non stress-ENU-DEX, stress-ENU, and non stress-ENU were sacrificed at 0600 h or 1800 h on day 11 after surgery. Adrenal glands were excised and later analyzed for ODC activities. Results indicated that DEX and/or immobilization stress inhibited ODC activities (p < 0.05) in normal and regenerating adrenal glands at 1800 h and ODC activity varies diurnally, the activity being greater at 1800 h than at 0600 hours (p < 0.001).     

Location of renal ornithine decarboxylase activity

Renal ornithine decarboxylase (ODC) activity was found to be more prevalent in the medulla of the normal rat kidney than in the cortex. When renal ODC activity was stimulated by ethanol, growth hormone, ACTH, or corticosterone, proportional increases were observed in both medulla and cortex. After hypophysectomy, ODC activities fell equally in both areas of the kidney. The administration of cycloheximide, which is known to cause a rebound increase after six hours in overall renal ODC activity, was followed by an increase of medullary ODC activity while cortical activity remained suppressed.     

Regulation of ODC activity in the thymus and liver of rats by adrenal hormones

The activity of L-ornithine decarboxylase (EC 4.1.1.17, ODC) has become a useful indicator of hormone responsiveness. Various regimens of dexamethasone, aldosterone and epinephrine, alone or in combination, were administered to adrenalectomized rats either in acute or chronic doses. In addition, adrenalectomized rats, which were chronically treated with aldosterone and epinephrine, were given a single injection of 50 micrograms dexamethasone and sacrificed at various time intervals after hormone treatment. Hepatic and thymic ODC activity was measured. The expected dexamethasone effect, an increase in hepatic and a decrease in thymic ODC, was observed. This study also revealed that aldosterone induced similar responses in these tissues. Epinephrine had the opposite effect since chronic administration of dexamethasone or aldosterone with epinephrine resulted in control levels of ODC. Furthermore, when aldosterone and epinephrine were chronically administered to adrenalectomized rats, to study the acute effects of dexamethasone on rat thymus and liver, the time course of the response in each tissue was found to be distinct. The influence of the adrenal gland on rat thymus and liver is not restricted only to glucocorticoids, but may also involve other hormones which it secretes.     

Purification and characterization of antizyme inhibitor of ornithine decarboxylase from rat liver

A protein inhibiting a protein inhibitor (antizyme) to ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) (ODC), antizyme inhibitor, was purified from the liver cytosol of thioacetamide-treated rats by procedures including antizyme affinity chromatography. Overall purification was roughly estimated to be about 17,000,000-fold and recovery was about 2.4%. The purified preparation showed one major protein band and a faint band corresponding in mobility to molecular weights of 51,000 and 53,500, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Judging from the ornithine decarboxylase activity of the final preparation, the faint band may be ornithine decarboxylase. The apparent molecular weight of antizyme inhibitor estimated by gel filtration on Sephacryl S-200 was approx. 62,000, indicating that antizyme inhibitor may be composed of a single polypeptide chain. In order to examine the question of whether antizyme inhibitor is a protein derived from ornithine decarboxylase, an inactive ornithine decarboxylase, in an immunotitration study and analysis of the binding to antizyme were investigated. The results indicate that antizyme inhibitor may be a protein distinct from ornithine decarboxylase.     

The in vivo stimulation of rat liver ornithine decarboxylase activity by dibutyryl cyclic adenosine 3',5'-monophosphate, theophylline and dexamethasone

The hepatic ornithine decarboxylase (ODC) activity of normal rats was stimulated more than 7-fold 3 hours after a single intraperitoneal injection of dibutyryl cyclic adenosine 3′,5′-monophosphate (dibu-cAMP). The 3-hour ODC activity was also stimulated by single injections of either theophylline or dexamethasone (10- and 21-fold, respectively). The simultaneous administration of actinomycin D with either dibu-cAMP, theophylline or dexamethasone reduced the 3-hour ODC activity by 91, 62 and 58 percent, respectively. When actinomycin D was given one hour after dibu-cAMP, no inhibition of ODC activity was observed.     

Glucocorticoid suppression of the sympathetic nervous system and adrenal medulla

Studies were performed to evaluate the effects of glucocorticoids on the activity of the sympathetic nervous system and adrenal medulla. Plasma concentrations of norepinephrine and epinephrine were measured in rats in which endogenous glucocorticoids were removed by bilateral adrenalectomy and in rats to which exogenous glucocorticoids were administered. In intact rats, dexamethasone (2.5, 25 or 250 micrograms) pretreatment suppressed ether vapor-induced elevations of norepinephrine and epinephrine concentrations in plasma. Corticosterone (3 mg/kg), similar to dexamethasone, attenuated the elevation of plasma concentrations of norepinephrine and epinephrine in rats exposed to ether vapor. Glucocorticoids did not alter the elevation of plasma catecholamines stimulated by intracerebroventricular injections of corticotropin-releasing factor or calcitonin gene-related peptide, thus demonstrating functional integrity of the sympathetic nervous system and adrenal medulla. Adrenalectomy resulted in elevation of basal plasma norepinephrine levels and accentuation of ether vapor-induced elevations of plasma norepinephrine concentrations in rats. Dexamethasone (25 ug) administration blunted the effects of adrenalectomy on both basal and ether vapor-stimulated levels of plasma norepinephrine. It is concluded that glucocorticoids acting at as yet undefined sites may be involved in the regulation of sympathetic nervous system and adrenal medullary function.     

Effect of Oxotremorine on Ornithine Decarboxylase Activity of the Adrenal Gland in Rat

Abstract: In this work we have studied the mechanism for the increase of adrenal ODC (ornithine decarboxylase, EC 4.1.1.17) activity provoked by oxotremorine, a muscarinic agonist. 1. Oxotremorine increased medullary ODC activity maximally at 2 h. Cortical enzyme responded much more slowly. 2. Blockade of peripheral muscarinic receptors with methylatropine partially reduced the response to oxotremorine in the medulla, but not cortex. 3. Hy-pophysectomy abolished the cortical, but not the medullary, responses to oxotremorine. Methylatropine reduced the effect of oxotremorine on medullary ODC in hypophysectomized rats. 4. In unilaterally splanchnicotomized rats oxotremorine caused an increase of ODC activity of the denervated adrenal gland relative to control value; activities in both medulla and cortex were significantly lower than those observed in the innervated gland. Evidence was obtained for a compensatory increase of ODC activity of the adrenal cortex (but not medulla) on the intact side of unilaterally operated rats. 5. Surgical intervention, in the form of a sham operation for transection of the spinal cord, leads to an increase of ODC activity in both parts of the adrenal gland. Transection of the cord attenuates these increases. 6. The additional increase of medullary ODC activity owing to the administration of oxotremorine to sham-operated rats is partially reduced in the adrenal medulla by muscarinic blockade, and completely in the cortex. This effect of methylatropine in regard to cortical ODC activity was not apparent in the other experiments with intact or unilaterally splanchnicotomized (unoperated side) rats. The results with unilaterally splanchnicotomized rats and those with transected spinal cord suggest that oxotremorine-induced modifications of adrenal ODC activity are centrally mediated, above the level of origin of the splanchnic nerves in the spinal cord (T_8–10). Experiments with hypophysectomized rats show that the response of the adrenal cortex to oxotremorine is entirely mediated by the hypophysis.     

Inhibition of epinephrine and gonadotropic hormone induced ornithine decarboxylase activity by phenoxybenzamine in the testis of immature rat

The effect of α and β adrenergic receptor blockers on epinephrine and gonadotropic hormone induced ornithine decarboxylase (ODC) activity in the testis of immature rats was studied. Intratesticular injection with phenoxybenzamine at 15 min before treatment with epinephrine or gonadotropic hormones blocked ODC activity. Similar injection with propranolol or practolol had no effect on ODC activity. These results show that α adrenergic receptors are involved in the action of epinephrine and gonadotropic hormones in the testis.     

Dexamethasone increases both catecholamines and methionine-enkephalin in cultured bovine adrenal chromaffin cells and human extramedullary pheochromocytoma cells

The effect of dexamethasone on dispersed cells in primary monolayer culture from bovine adrenal medulla and human extramedullary pheochromocytoma was examined by estimating the level of catecholamines (CAs) and Methionine-enkephalin (Met-enk) in the medium and cells. In cultured bovine adrenal chromaffin cells, dexamethasone caused significant increase in Met-enk levels 18 hours after administration. There was no release of Met-enk and CAs in the medium 10 min after administration, although nicotine did cause a significant release of Met-enk and CAs. A dose response increase in the level of CAs and Met-enk in bovine adrenal chromaffin cells was obtained with doses varying between 0 and 10(-6)M dexamethasone 18 hours after administration. In cultured human extramedullary pheochromocytoma cells, dexamethasone significantly increased the levels of norepinephrine and Met-enk in a dose dependent manner 24 hours after administration. These results suggest that dexamethasone does not act as a secretagogue but may be related to the synthesis of Met-enk and CAs.     

Evaluation of plasma norepinephrine as an index of sympathetic neuron function in the conscious, unrestrained rat.

Measurement of plasma norepinephrine and epinephrine concentrations in the conscious, unrestrained rat yielded values of 138±10 and 55±8 pg/ml, respectively. Ganglionic blockade reduced basal norepinephrine levels without affecting plasma epinephrine levels. Adrenal demedullation reduced plasma epinephrine to undetectable levels (<20 pg/ml) and gave rise to an apparent compensatory increase in plasma norepinephrine levels. Adrenal demedullation in combination with ganglionic blockade reduced plasma norepinephrine to the same level as did ganglionic blockade alone. These observations indicated that the plasma epinephrine was of adrenal origin. Furthermore, under these experimental conditions, the results suggested that the major portion of the plasma norepinephrine was of neuronal origin. When specific destruction of the sympathetic nerve terminals without alteration of adrenal medullary function was accomplished with 6-hydroxydopamine, a fivefold increase in plasma epinephrine concentration was observed at 24 hours. Plasma norepinephrine levels at 24 hours were not significantly altered from the control levels by the 6-hydroxydopamine suggesting that the rodent adrenal medulla was capable of secreting substantial amounts of norepinephrine under these conditions. It was concluded that plasma norepinephrine concentrations reflect both sympathetic neuronal and adrenomedullary activity. However, in the absence of changes in plasma epinephrine, plasma norepinephrine appears to be an index of sympathetic neuron function.     

Is cyclic AMP the regulator of hepatic ornithine decarboxylase activity?

The role of cyclic AMP in the regulation of hepatic ornithine decarboxylase (ODC) activity in the rat was studied in the whole animal and in the perfused organ. Dibutyryl cyclic AMP or butyrate given to intact rats increased ODC activity; this increase was abolished by hypophysectomy 1 h prior to administering ether compound. Administration of 1 mg 1-methyl-3-isobutylxanthine (MIX) to intact rats increased ODC activity within 4 hours whereas hypophysectomy 1 h before treatment prevented this increase. No change in hepatic cyclic AMP content was seen in either intact or hypophysectomized rats following MIX. Perfusion with 0.5 mM dibutyryl cyclic AMP decreased ODC activity in isolated livers whereas perfusion with 0.5 mM 8-bromocyclic GMP produced a small increase in ODC activity. These data suggest that the effect of dibutyryl cyclic AMP in intact animals may be a property of the butyrate and that this action as well as the action of MIX may be mediated through the permissive effect of pituitary and/or adrenal hormones. The normal hepatocyte does not increase its ornithine decarboxylase activity after direct exposure to dibutyryl cyclic AMP.     

Effect of dexamethasone on the activity and expression of ornithine decarboxylase in rat liver and thymus

A single intraperitoneal injection of the synthetic glucocorticoid dexamethasone into rats resulted in a marked stimulation (more than 60-fold) of hepatic ornithine decarboxylase (ODC) at 4 h after the injection, whereas the enzyme activity in thymus was almost totally (about 95%) depressed at the same time. The stimulation of ODC activity in liver was in all likelihood attributable to a greatly enhanced accumulation of mRNA species for the enzyme as revealed by Northern blot and dot-blot hybridization analyses. ODC activity in thymus, in response to dexamethasone, was only 5% of that found in control animals, but this decrease was apparently not accompanied by similar reductions of the levels of ODC message, which was in fact decreased only by 50% at the maximum. In addition to two mRNA species (2.1 and 2.6 kilobases; kb), typical to mouse cells, rat tissues seemed to contain a third hybridizable message for ODC, smaller (1.6 kb) than the above-mentioned species and not seen in samples obtained from mouse or human cells. Interestingly, these smaller poly(A)+ RNA sequences, hybridizable with cDNA complementary to mouse ODC mRNA, were apparently constitutively expressed, as the treatment with glucocorticoid altered the amount of these sequences only slightly.     

Ornithine Decarboxylase Induction by Glucocorticoids in Brain and Liver of Adrenalectomized Rats

Abstract: Ornithine decarboxylase (ODC), the rate-limiting enzyme in the biosynthesis of polyamines, was measured in the brain and the liver of adrenalectomized rats after an acute S.C. treatment with glucocorticoids. The effects of corticosterone and dexamethasone were compared in three brain areas, the cerebral cortex, hippocampus, and cerebellum. These structures have similar concentrations of cytosolic glucocorticoid receptor, as measured by an in vitro exchange assay using a specific glucocorticoid ligand, [³H]RU 26988, but contain different amounts of mineralocorticoid receptor. Corticosterone and dexamethasone increased ODC activity in the liver and brain areas in a dose dependent manner, dexamethasone being more active than corticosterone in all tissues. Moreover, estradiol, progesterone, and testosterone were inactive. Aldosterone, at high doses, increased brain ODC activity. Glucocorticoids, selected for their weak binding, or lack of binding to the mineralocorticoid receptor, were tested and found to be highly active in inducing brain and liver ODC, thus showing that ODC induction by steroids is specific for glucocorticoids. These results are among the first to suggest biochemically a central action of glucocorticoids following an acute treatment and confirm that the brain is a glucocorticoid target organ.     

Alterations in cardiovascular responsiveness and adrenoceptor binding during catecholamine infusion hypertension in rats.

Chronic continuous infusion of norepinephrine in rats causes alterations in biochemical and physiologic responses of the cardiovascular system and in cardiovascular adrenoceptor number. The response of cardiac and aortic ornithine decarboxylase (ODC) activity to stimulation by norepinephrine was decreased in rats receiving norepinephrine infusion. These responses are due to stimulation of beta- and alpha-adrenergic receptors, respectively. Additionally, there was reduced stimulation of aortic ODC activity by angiotensin II and vasopressin. The cardiac ODC response to angiotensin II was decreased, but the response to vasopressin was not affected. The decreased ODC response is accompanied by decreased pressor responses to the alpha-adrenergic agonist phenylephrine. Decreased numbers of alpha- and beta-adrenoceptor binding sites (as measured by the binding of [3H]prazosin and [125I]pindolol) might mediate, in part, the altered responses to adrenergic agonists. The decreased cardiovascular responsiveness measured in these animals after several days of norepinephrine infusion hypertension contrasts with the increased responses found in most other forms of hypertension. This provides a useful model in which to examine the consequences of prolonged adrenergic receptor stimulation.     

Mirex induces ornithine decarboxylase in female rat liver

Ornithine decarboxylase, the rate-limiting enzyme in polyamine synthesis, was significantly induced in female rat liver following oral administration of the pesticide mirex. After dual oral exposure (120 mg/kg of mirex; 21 and 4 hr prior to sacrifice), ornithine decarboxylase activity in rat liver cytosol was 70-fold higher than control values. A single oral dose of mirex (180 mg/kg) induced hepatic ornithine decarboxylase activity 55-fold over controls. After a single oral dose of mirex the maximal induction of ODC activity occurred at 36 hr. Mirex is an unusually potent and long-lasting inducer of rat hepatic ornithine decarboxylase activity.