Molecular and physiological properties of tyrosine hydroxylase induced by reserpine in rat adrenal gland

Newly synthesized tyrosine hydroxylase (TH) induced by reserpine was compared with the enzyme in control rats in terms of the molecular and physiological properties. When repeated doses of reserpine were given at daily intervals for three days, the enzyme activity measured in homogenates of the adrenal glands was increased 3-fold. Furthermore, when TH in the adrenal glands from both control and reserpine-treated rats was purified, both total activity of the enzyme and the enzyme protein content purified from reserpine-treated rats were also about 3-fold higher than those of the control rats. The two purified enzymes revealed similar properties; a single subunit with a Mr of 60,000 was observed by SDS polyacrylamide gel electrophoresis, and the Km value for a pterin cofactor, 6-methyl-tetrahydropterin was about 300 microM. In contrast, in situ TH activity measured under physiological conditions at pH 7.2 in adrenal tissue slices was elevated 6-fold by reserpine pretreatment for 3 days, and was stimulated by carbachol (0.1 mM) and elevated K+ (52 mM) in a roughly proportional rather than additive way relative to slices from untreated rats. These results indicate that newly synthesized TH induced by reserpine in rat adrenal gland had similar properties as the enzyme in control rats and that reserpine increased not only the amount of TH molecules but also the in situ activity of TH. Since reserpine also increases the biosynthesis of tetrahydrobiopterin as demonstrated by Viveros and co-workers, this 6-fold increase in in situ TH activity may depend both upon the 3-fold increase in the amount of enzyme molecules and upon the increase of the physiologically available tetrahydrobiopterin in the adrenal gland.     

Cyclic AMP-dependent protein kinase is not involved in the in vivo activation of tyrosine hydroxylase in the adrenal gland after decapitation

Tyrosine hydroxylase is activated in the adrenal gland in vivo after acute stresses, such as decapitation or electroconvulsive shock. In nonstressed animals that are anesthetized with pentobarbital prior to surgical removal of the adrenals, approximately 5-10% of the enzyme molecules are in the activated form, whereas in stressed animals, approximately 40-50% of the enzyme molecules are in the activated form. In the present study, we have tested the hypothesis that the stress-induced activation of the adrenal enzyme in vivo is due to the phosphorylation of the enzyme by cyclic AMP-dependent protein kinase. Soluble adrenal tyrosine hydroxylase prepared from either stressed or nonstressed rats is incubated in vitro with [gamma-32P]ATP and purified cyclic AMP-dependent protein kinase under optimal conditions for the phosphorylation of the enzyme. Using this assay, we have measured the number of vacant sites remaining on the enzyme, which are available for in vitro phosphorylation by cyclic AMP-dependent protein kinase. These studies suggest that the initial, in vitro rate of phosphorylation of tyrosine hydroxylase isolated from stressed rats is less than the initial rate of phosphorylation of the enzyme isolated from nonstressed rats. However, there is no significant difference in the final level of 32P phosphorylation of tyrosine hydroxylase isolated from either stressed or nonstressed rats. We conclude that, even though phosphorylation of tyrosine hydroxylase by cyclic AMP-dependent protein kinase leads to the activation of the enzyme under in vitro conditions, this mechanism cannot account for the activation of the enzyme in vivo in the adrenal gland following decapitation.     

An immunochemical study of the induction of tyrosine hydroxylase in rat adrenal glands

Tyrosine hydroxylase-specific protein is increased in rat adrenal medulla by immobilization stress, cold exposure, and 6-hydroxydopamine administration. The kinetic properties of the induced enzyme are identical to those of the control enzyme.     

Central angiotensin II increases biosynthesis of tyrosine hydroxylase in the rat adrenal medulla

Angiotensin II acting centrally contributes to the regulation of blood pressure and water intake and stimulates the release of catecholamines from the adrenal medulla. We hypothesized that the central angiotensin II is one mediator of biosynthesis of catecholamines in the adrenal medulla. Rats were administered i.c.v. angiotensin II or saline, and TH mRNA and protein levels in adrenal medulla were measured 1 or 3 h later. Angiotensin II did not change TH mRNA or protein 1 h later. However, by 3 h, angiotensin II increased TH mRNA and protein levels. Centrally administered angiotensin II elevates TH mRNA expression and protein levels in the adrenal medulla. In conclusion, one component of central angiotensin II elevation of blood pressure may be the result of increased catecholamine synthesis in the adrenal gland and elevated TH synthesis represents one underlying mechanism.     

Perinatal evolution and hormonal control of adrenal tyrosine hydroxylase activity in the rat.

The evolution of adrenal tyrosine hydroxylase activity has been measured in the rat fetus from 18 1/2 days of gestation until 24 h after birth. This activity increases gradually in the fetal adrenals with a sudden and transient increase between 0 and 6 h postpartum. It is suggested that a nervous mechanism related to the stress of birth is responsible for this increase. Fetal decapitation reduces adrenal tyrosine hydroxylase activity at term. This reduction can be partially prevented by administering adrenocorticotropic hormone (ACTH) to the decapitated fetus; cortisol administration has no effect. The results indicate that ACTH has a direct action on adrenal tyrosine hydroxylase in the fetus as it does in the adult.     

Intracellular localization of tyrosine hydroxylase immunoreactivity in the rat adrenal chromaffin and pheochromocytoma cells

The localization of tyrosine hydroxylase (TH) immunoreactivity in rat adrenal chromaffin and pheochromocytoma (PC12) cells was investigated by immunoelectron microscopy using monoclonal and polyclonal antisera against TH purified from rat adrenal medulla. Strong TH immunoreactivity was found uniformly in the granules of the adrenaline cells; the immunoreactivity was visible mainly within the periphery, but not in the clear space of the granules of the noradrenaline cells. In the PC12 cells, strong TH immunoreactivity was also observed uniformly in the granules. In addition, TH immunoreactivity was seen in the cytoplasm, the ribosomes attached to the endoplasmic reticulum and the free ribosomes of both the rat adrenal chromaffin and PC12 cells. These results suggest that TH may be localized in the granules, cytoplasm and ribosomes of rat adrenal chromaffin and PC12 cells.     

A rapid and simplified assay method for tyrosine hydroxylase

Tyrosine hydroxylase can be measured by release of tritiated water from labeled tyrosine, and the assay method has now been modified to allow recovery of 3H2O from the reaction mixture in a much more rapid and less tedious manner than previously possible. In the new method, the tyrosine hydroxylase reaction is stopped with sodium carbonate, pH 11.6. At this pH the tritium in 3H2O, but not other 3H species, is extracted into an organic scintillant containing 25% isoamyl alcohol, toluene, 2,5-diphenyloxazole, and p-bis-[2-(5-phenyloxazolyl)]benzene. The selective extraction occurs by means of exchange of tritium in 3H2O with the hydroxyl proton of isoamyl alcohol. It is the [3H]isoamyl alcohol that is then extracted into the scintillant and quantified by liquid scintillation spectrometry. Although the organic extraction method is somewhat less sensitive than the more frequently used ion-exchange method for isolating the 3H2O formed in the tyrosine hydroxylase reaction, it is much more rapid, as well as cost effective, since the enzyme reaction, extraction, and counting are carried out within the same vial.     

Characterization of tyrosine hydroxylase from bovine adrenal medulla

Tyrosine hydroxylase purified to apparent homogeneity from the soluble fraction of bovine adrenal medulla had an apparent Mr of about 280,000 by Bio-Gel A-1.5m chromatography, and gave a single band with a Mr of 60,000 by sodium dodesyl sulfate polyacrylamide gel electrophoresis. The enzyme is considered to be composed of four identical subunits. Isoelectric point of purified enzyme was pH 6.0. The amino acid composition of the enzyme was characterized by fairly high contents of glutamic acid and alanine residues. The N-terminal amino acid was determined to be glutamic acid.     

Regulation of tyrosine hydroxylase from human pheochromocytoma, bovine adrenal and rat striatum.

Soluble tyrosine hydroxylase from human pheochromocytoma, bovine adrenal medulla and rat striatum can be activated by Mg²⁺, ATP and cyclic AMP. In pheochromocytoma, this activation is due to a decreased Km for the pterin cofactor, whereas in adrenal medulla, it is a result of an increase in the Vmax. Norepinephrine increases the Km for pterin cofactor for tyrosine hydroxylase from both of these tissues. The Ki for norepinephrine is not altered by the presence of Mg²⁺, ATP and cyclic AMP with enzyme from pheochromocytoma or adrenal medulla. On the other hand, striatal tyrosine hydroxylase shows a two-fold increase in the Ki for dopamine after exposure to Mg²⁺, ATP and cyclic AMP.     

Influence of hypothyroidism on the ontogenic development of tyrosine hydroxylase induction in the adrenal glands of the rat

The ontogenic development of the transsynaptic induction of adrenal tyrosine hydroxylase (TH), evoked by reserpine and nicotine was studied in control and hypothyroid young rats, aged 3-52 days. The enzymatic induction was measured as an increase in the enzyme activity, since this increase was shown to be impaired either by an inhibitor of RNA synthesis or by a ganglionic blocker. In the control animals, TH induction elicited by reserpine increases between 3 and 32 days of age. In the hypothyroid rats, the enzymatic induction is impaired up to 32 days; at 52 days the induction is similar in both groups of animals. When nicotine is used as a stimulating agent, hypothyroidism still impairs the enzymatic induction at 5 and 21 days, indicating that at least one of the mechanisms inhibited by hypothyroidism is localized in the adrenal chromaffin cells. The present results, taken together with previous findings dealing with adrenal epinephrine secretion, show that the thyroid hormones play a crucial role in the responses of the adrenal medulla to a stimulation in the developing rat, while they have no effect in the adult.     

Phenylalanine as substrate for tyrosine hydroxylase in bovine adrenal chromaffin cells.

Incubation of bovine chromaffin cells with L-[14C]phenylalanine resulted in label accumulation in catecholamines at about 30% of the rate seen with L-tyrosine as precursor. Studies with purified tyrosine hydroxylase (EC 1.14.16.2) showed that the enzyme catalysed the hydroxylation of L-phenylalanine first to L-p-tyrosine and then to 3,4-dihydroxyphenylalanine (DOPA). No evidence for a significant involvement of an L-m-tyrosine intermediate in DOPA formation was found.     

Purification and immunochemical characterization of human adrenal tyrosine hydroxylase

Tyrosine hydroxylase (TH) was purified from the soluble fraction of human adrenal glands. The enzyme in human adrenal glands that was purified to apparent homogeneity had an apparent M_r of about 280,000. Sodium dodecyl sulfate (SDS) gel electrophoresis gave a single band with a M_r of 60,000 similar to the M_r of bovine adrenal enzyme. The enzyme is considered to be composed of four identical subunits. The specific activity of the final preparation was approximately 310 nmol 3,4-dihydroxyphenylalanine (DOPA) formed/min/mg protein. The use of the “Western Blot” method showed that human adrenal TH did not aggregate as rapidly as bovine adrenal TH.     

The adrenal gland and progesterone stimulates testicular steroidogenesis in the rat in vivo

Administration of pharmacological doses of glucocorticoid to male rats in vivo suppresses adrenal steroidogenesis and inhibits testicular steroidogenesis by inhibiting the anterior pituitary secretion of LH. In contrast, administration of ACTH to these pharmacologically-suppressed rats stimulates the adrenal secretion of progesterone and testicular steroidogenesis. The mechanism by which ACTH increases testicular steroidogenesis is dependent on the presence of the adrenal gland and is reproduced by the administration of progesterone. The conclusion from these data is that the adrenal gland has an important role in generating external signals that modulate the hypothalamic-pituitary-gonadal axis in male rats. The adrenal secretion of glucocorticoid acts as a negative signal to testicular steroidogenesis whereas progesterone acts as a positive signal. The adrenal secretion of progesterone and its conversion to testosterone by steroidogenic enzymes in the cytoplasm of the Leydig cell may provide an alternative pathway for testosterone biosynthesis and may account for the increased plasma testosterone levels during the acute phase of stress and mating.     

Immunocytochemical localization of tyrosine hydroxylase, dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase in the adrenal glands of the frog and rat by a peroxidase-antiperoxidase method.

The subcellular localizations of tryrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) in the adrenal glands of the frog and rat have been examined by a peroxidase-antiperoxidase (PAP) method. TH was localized in the ground substance of the adrenaline-containing cells and noradrenaline-containing cells, but not in the nucleus or in the mitochondria. TH was also located on the outside of the membrane of the chromaffin granules. DBH was observed only inside the granules. PNMT was found not only in the ground substance but also on the membrane of some adrenaline-containing granules. Cortical lipid cells of the frog adrenals did not show TH-, DBH-, and PNMT-reactions. The negative reactions to TH-, DBH-, and PNMT-antiserum exhibited by the summer cells of the frog adrenals prove that they belong to the cortical cells.     

An improved method for tyrosine hydroxylase immunolabeling of catecholamine cells in whole mounted rat retina

We developed a rapid and efficient method to achieve good penetration of anti-tyrosine hydroxylase (TH) antiserum into whole mounted rat retinas. The retinas were defatted before incubation in the primary antibody. This operation was thought to increase membrane permeability, facilitating penetration of antibodies. As a result, all TH-positive cells in the entire retina were labeled. Thus, it becomes possible to study the morphology, the distribution, and the counting of TH immunoreactive cells in whole retinas.