Elevation of RNA Coding for Tyrosine Hydroxylase in Rat Adrenal Gland by Reserpine Treatment and Exposure to Cold

When rats are treated daily with reserpine or maintained at 4 degrees C, the level of a specific RNA coding for tyrosine hydroxylase is elevated in the adrenal gland. The increase in this specific RNA temporally precedes and is quantitatively equal to the increase in adrenal tyrosine hydroxylase enzyme activity elicited by these treatments. These results suggest that prolonged stress may lead to changes in the levels of specific RNA species in the adrenal gland.     

Vasoactive intestinal polypeptide induces tyrosine hydroxylase in PC12 cells.

Physiological stress induces tyrosine hydroxylase, the rate-limiting enzyme for catecholamine biosynthesis, via trans-synaptic mechanisms within the adrenal medulla. Previous studies have implicated cAMP as a second messenger capable of inducing tyrosine hydroxylase; however, it is unclear whether any receptor coupled to adenylate cyclase mediates tyrosine hydroxylase induction. Recently, vasoactive intestinal polypeptide, whose receptor is coupled to adenylate cyclase in many tissues, has been shown to meet many of the criteria for a neuromodulator within the adrenal medulla. We therefore undertook a series of studies to determine whether vasoactive intestinal polypeptide may induce tyrosine hydroxylase in PC12 cells, a cell line derived from rat adrenal medulla. Here we report that vasoactive intestinal polypeptide produces a transient, time- and concentration-dependent increase in tyrosine hydroxylase mRNA levels which is followed by a stable increase in tyrosine hydroxylase protein. The increase in tyrosine hydroxylase mRNA does not occur in a mutant PC12 cell line deficient in cAMP-dependent protein kinase activity, indicating that the effect of vasoactive intestinal polypeptide is mediated through the cAMP second messenger pathway. This is the first report demonstrating that a neuromodulator which acts on an adenylate cyclase-coupled receptor can induce tyrosine hydroxylase.     

Laminin increases both levels and activity of tyrosine hydroxylase in calf adrenal chromaffin cells

We have investigated the effects of substrate-bound laminin on levels of enzymes of the catecholamine biosynthetic pathway in primary cultures of calf adrenal chromaffin cells. Laminin increases the levels of the enzymes tyrosine hydroxylase, dopamine-beta-hydroxylase, and phenylethanolamine-N-methyl-transferase. This effect is selective, in that levels of other enzymes (lactate dehydrogenase, aromatic amino acid decarboxylase, and acetylcholinesterase) are not increased. The effect of laminin can be blocked by antibodies directed against a fragment of the heparin-binding domain of the molecule, whereas antibodies directed against other fragments do not block the increase in tyrosine hydroxylase. Thus the laminin domain involved in enzyme regulation in chromaffin cells is apparently the same as that previously implicated in laminin's interactions with neurons to potentiate survival and stimulate neurite outgrowth (Edgar, D., R. Timpl, and H. Thoenen, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:1463-1468). The increase in chromaffin cell tyrosine hydroxylase levels is preceded by an activation of the enzyme in which the Vmax (but not the Km) is altered. The effects of laminin appear to be developmentally regulated, since neither activation nor increased levels of tyrosine hydroxylase occur in adult adrenal chromaffin cells exposed to laminin.     

Effects of melanin on tyrosine hydroxylase and phenylalanine hydroxylase.

Melanin inhibited rat liver phenylalanine hydroxylase, but activated tyrosine hydroxylase from rat brain (caudate nucleus), rat adrenal glands, and bovine adrenal medulla. Activation of tyrosine hydroxylase by melanin was demonstrated with the extensively dialyzed enzyme and in suboptimal concentrations of the substrate (tyrosine) and the cofactor (6-methyltetrahydropterin). Tyrosine hydroxylase from rat brain was activated by melanin more markedly than that from rat adrenal glands. Purified and extensively dialyzed bovine adrenal tyrosine hydroxylase had two Km values with 6-methyltetrahydropterin, depending upon its concentrations, but the melanin-activated tyrosine hydroxylase had a single Km value and showed the classical Michaelis-Menten kinetics.     

Effects of substance P on the long-term regulation of tyrosine hydroxylase activity and catecholamine levels in cultured adrenal chromaffin cells

Acetylcholine, released from splanchnic nerve terminals innervating adrenal chromaffin cells, is known to increase synthesis of adrenal tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis. The neuropeptide substance P is also present in the splanchnic nerve innervating the adrenal medulla, and this study examined whether substance P has any long-term effects on tyrosine hydroxylase activity and catecholamine levels in cultures of adult bovine adrenal chromaffin cells. When cultures were incubated for 3 days with substance P and carbachol, a cholinergic agonist, substance P (10(-6) M, and greater) completely inhibited the increase in tyrosine hydroxylase activity normally induced by carbachol. Long-term stimulation with carbachol also depleted endogenous catecholamines from the cells and substance P prevented this carbachol-induced depletion of catecholamine content. Substance P by itself, in the absence of carbachol, had only a slight effect on tyrosine hydroxylase activity. 8-Bromoadenosine 3':5'-cyclic monophosphate, an analogue of adenosine 3':5'-cyclic monophosphate, also increases tyrosine hydroxylase activity in chromaffin cells; however, substance P had no effect on the increase in tyrosine hydroxylase activity induced by this analogue. These results indicate that substance P's effects are relatively specific for the carbachol-induced increased in tyrosine hydroxylase activity and that the primary site of action of substance P is not a site common to the mechanism of tyrosine hydroxylase induction by carbachol and 8-bromoadenosine 3':5'-cyclic monophosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of electron transport in the regulation of the lyase activity of C21 side-chain cleavage P-450 from porcine adrenal and testicular microsomes

The C21 side-chain cleavage enzymes from porcine adrenal and testicular microsomes have been purified and shown to resemble each other very closely (Nakajin, S., Shinoda, M., Hanui, M., Shively, J.E., and Hall, P.F. (1984) J. Biol. Chem. 259, 3971-3976). We have investigated the reason for the low levels of lyase activity shown by adrenal microsomes as compared to testicular microsomes. Competition for substrate with 21-hydroxylase in adrenal microsomes was excluded by studies showing that antibodies to 21-hydroxylase do not increase lyase activity in spite of almost complete inhibition of 21-hydroxylation. Reconstitution of the purified testicular enzyme in lipids extracted from adrenal and testicular microsomes excluded a specific effect of lipids on lyase activity. On the other hand, addition of porcine hepatic P-450 reductase to microsomes from adrenal and testis increased the activity of lyase relative to hydroxylase. The same effect is seen when reductase is added to the pure enzymes. As the concentration of reductase increases, lyase activity increases relative to hydroxylase until the rates of the activities become almost equal. Vmax is the same for both activities (hydroxylase and lyase) of the two enzymes (6.3-6.5 nmol/min/nmol of P-450). Km for reductase is approximately the same for the hydroxylase activities (0.4-0.6 microM) and for the lyase activities (1.7-2.0 microM) of the two enzymes. Antibodies to reductase, when added to testicular microsomes, inhibit both activities, but inhibition of lyase is greater than that of hydroxylase. The enzyme activity of reductase in testicular microsomes is 3-4 times higher than that of adrenal microsomes (0.29 and 0.08 nmol/min/mg of protein, respectively). These findings may account for the greater activity of lyase in testicular as opposed to adrenal microsomes.     

Quantitation of tyrosine hydroxylase, protein levels: spot immunolabeling with an affinity-purified antibody

Tyrosine hydroxylase was purified from bovine adrenal chromaffin cells and rat pheochromocytoma using a rapid (less than 2 days) procedure performed at room temperature. Rabbits were immunized with purified enzyme that was denatured with sodium dodecylsulfate, and antibodies to tyrosine hydroxylase were affinity-purified from immune sera. A Western blot procedure using the affinity-purified antibodies and 125I-protein A demonstrated a selective labeling of a single Mr approximately 62,000 band in samples from a number of different tissues. The relative lack of background 125I-protein A binding permitted the development of a quantitative spot immunolabeling procedure for tyrosine hydroxylase protein. The sensitivity of the assay is 1-2 ng of enzyme. Essentially identical standard curves were obtained with tyrosine hydroxylase purified from rat pheochromocytoma, rat corpus striatum, and bovine adrenal medulla. An extract of PC 12 cells (clonal rat pheochromocytoma cells) was calibrated against purified rat pheochromocytoma tyrosine hydroxylase and used as an external standard against which levels of tyrosine hydroxylase in PC12 cells and other tissue were quantified. With this procedure, qualitative assessment of tyrosine hydroxylase protein levels can be obtained in a few hours and quantitative assessment can be obtained in less than a day.     

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.     

ORGAN CULTURE OF THE RAT ADRENAL MEDULLA: A MODEL SYSTEM FOR THE STUDY OF TRANS-SYNAPTIC ENZYME INDUCTION

—It was the aim of the present study to develop organ culture conditions for the rat adrenal medulla which are representative for the in vivo situation. This is a prerequisite for studying the complex processes involved in trans-synaptic enzyme induction. The processes of trans-synaptic enzyme induction initiated in vivo by injecting 5 mg/kg of reserpine 2 h prior to the removal of the adrenal medulla, continued in this culture system and final levels of tyrosine hydroxylase were comparable to those seen in vivo. That these culture conditions are representative for the in vivo induction is also supported by the fact that transection of the splanchnic fibres supplying the adrenal medulla or administration of actinomycin D prior to reserpine abolished the rise in tyrosine hydroxylase activity not only in vivo, but also in culture. The findings that high concentrations (0·29 mm ) of corticosterone in the culture medium inhibited the increase in tyrosine hydroxylase activity caused by reserpine support the hypothesis that glucocorticoids act as modulatory agents in trans-synaptic enzyme induction. This inhibition was exhibited only when corticosterone was added at the initiation of the culture period. If added 2 or 4 h after the beginning of the culture period there was little or no effect on the subsequent increase of tyrosine hydroxylase.     

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.     

The rapid activation of tyrosine hydroxylase by the subcutaneous injection of formaldehyde

Ten minutes following the subcutaneous injection of formaldehyde, there is a 2-fold activation of adrenal medulla tyrosine hydroxylase. Enzyme activation appears to involve a shift of a fraction of the low affinity form of the enzyme to a higher affinity form. In the nonstessed rat approximately 29% of adrenal TH is in the activated (low Km) form. Following formaldehyde injection, approximately 54% of the enzyme is in the activated (low Km) form. The addition of cyclic AMP, ATP and Mg⁺⁺, in the presence of endogenous cyclic AMP-dependent protein kinase, to the enzyme obtained from adrenals of either the stressed or nonstressed rats increased the activity of both enzyme preparations to the same level. These data indicate that acute stress activates adrenal tyrosine hydroxylase in the rat and that a cyclic AMP-dependent protein phosphorylating system mediates the activation.     

Bovine adrenal tyrosine hydroxylase: purification and properties.

Bovine adrenal tyrosine hydroxylase has been obtained in a form that is 85 to 90% pure. Sodium dodecyl sulfate-gel electrophoresis and density gradient centrifugation studies have established that the subunit molecular weight of the chymotrypsin-solubilized enzyme is 34,000. The presence of iron in the purified enzyme (0.50 to 0.75 mol of iron/mol of enzyme) has been established. Crude particulate tyrosine hydroxylase can be activated by the phospholipid, phosphatidyl-L-serine, or by exposure to enzymatic phosphorylating conditions. Both forms of activation lower the Km of the enzyme for its 2-amino-4-hydroxypteridine cofactor. By contrast, tyrosine hydroxylase that has been solubilized by chymotrypsin cannot be activated by either of these methods.     

Interaction of tyrosine hydroxylase with tubulin

Bovine adrenal medulla tyrosine hydroxylase associates with microtubules during tubulin assembly. Limited proteolytic digestion of tyrosine hydroxylase does not affect the enzymatic activity but prevents its association with tubulin. A possible interpretation is that an ionic interaction occurs between microtubules and a negatively charged region of the enzyme which is removed by the protease treatment. Tyrosine hydroxylase is able to induce purified tubulin assembly as do the microtubule associated proteins; however, the association induced by tyrosine hydroxylase corresponds to the formation of aggregates or organized structures different from microtubules. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and electron microscopy of proteins obtained from bovine adrenal medulla show the presence of tubulin in this tissue.     

Regulation of dopamine beta-hydroxylase in rat adrenal glands.

Rat adrenal gland levels of dopamine beta-hydroxylase are subject to dual control. Activation of the splanchnic nerves to the adrenal medulla by reserpine induces the synthesis of dopamine beta-hydroxylase without altering the rate of enzyme degradation. In contrast, hypophysectomy causes a decline in steady state dopamine beta-hydroxylase levels by first accelerating the rate of degradation, then by slowing the rate of enzyme synthesis as well. Adrenocorticotropic hormone administration partially reversed the effect of hypophysectomy on dopamin beta-hydroxylase degradation. These findings suggest that the trans-synaptic factors controlling dopamine beta-hydroxylase induction act by a different mechanism (enzyme synthesis) than the hormonal controls regulating steady state levels (enzyme degradation). Thus, active inhibition of enzyme degradation may be an important control in maintenance of steady state enzyme levels.     

Central Regulation of Adrenal Tyrosine Hydroxylase: Effect of Induction on Catecholamine Levels in the Adrenal Medulla and Plasma

The effect of induction of adrenal tyrosine hydroxylase (TH) by various centrally acting drugs on catecholamine levels in adrenal and plasma was investigated in rats. All the drugs tested, namely oxotremorine, Piribedil, B-HT 920, and HA-966, produced significant increases in adrenal dopamine content and plasma epinephrine level. Denervation of the adrenal abolished the increase in adrenal dopamine as it did the induction of tyrosine hydroxylase. The results suggest that the induced increase of adrenal TH activity, as mediated by certain drugs, results in an elevation of the plasma epinephrine level and that the adrenal dopamine content is a better indicator of the catecholamine-synthesizing capacity of the adrenal medulla than are the other catecholamines.     

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.     

Interaction of tyrosine hydroxylase with ribonucleic acid and purification with DNA-cellulose or poly(A)-sepharose affinity chromatography

Tyrosine hydroxylase in bovine adrenal medulla was activated up to fourfold by incubation with low concentrations (15 micrograms/ml) of ribonucleic acids. At higher RNA concentrations, enzyme activity was inhibited. This interaction with RNA was exploited with the use of poly(A)-Sepharose and DNA-cellulose to effect a rapid purification of stable tyrosine hydroxylase from rat brain and bovine adrenal medulla in high yield (up to 58%). With the purified rat brain enzyme, RNA acted as an uncompetitive inhibitor, a concentration of 15 micrograms/ml lowering the Vmax of tyrosine hydroxylase from 1050 to 569 nmol min-1 mg-1 and lowering the Km for tyrosine from 6.1 to 3.6 microM. With the natural cofactor, tetrahydrobiopterin (BH4), two Km values were obtained, indicating the presence of two forms of the enzyme. Both Km values were decreased only slightly by RNA. The purified brain and adrenal enzymes both contained about 0.07 mol of phosphate/63,000-Da subunit; in both cases, cyclic AMP-dependent protein kinase catalyzed the incorporation of an additional 0.8 mol of phosphate/subunit. The purified enzyme also contains ribonucleic acid, which comprises about 10% of the total mass and appears to be important for full activity.     

GLUCOCORTICOIDS AS A REGULATORY FACTOR FOR BRAIN TRYPTOPHAN HYDROXYLASE

—The normal developmental rise of tryptophan hydroxylase levels in neonatal rat brain was blocked by adrenalectomy. Similarly, adrenalectomy prevented the rescrpine-induced elevation of tryptophan hydroxylase activity in brain stem of adult mice. In both cases, the effects of adrenalectomy could be reversed by replacement injections of corticosterone. Repeated injections of corticosterone (5 mg/kg daily) in fact induced a rise of brain tryptophan hydroxylase levels in neonatal brain. However, neither adrenalectomy nor repeated injections of large doses of the hormone (20 mg/kg, daily) was found to be effective in affecting the normal enzyme levels in adult brain. Apparent K_m of the enzyme for substrate was unchanged by corticosterone in vivo or in vitro. These results indicate that glucocorticoids have a significant role in the regulation of brain tryptophan hydroxylase: possibly as an inducing signal during neonatal development and as a permissive factor at adult age.