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.     

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)     

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.     

ACTH stimulation of adrenal epinephrine and norepinephrine release

Epinephrine (E) and norepinephrine (NE) levels were measured simultaneously in the adrenal veins of 6 patients before and after stimulation with 0.25 mg beta 1-24 ACTH. In 1 patient with Cushing's syndrome, E and NE were also measured before and 30 min after dexamethasone. There was a significant increase in NE and E secretion (p less than 0.002) from both adrenal glands after ACTH stimulation. In the patient with Cushing's syndrome, there was also a slight increase in plasma E levels after dexamethasone. It is postulated that ACTH stimulated NE and E secretion by augmenting blood flow through the adrenals and by induction of tyrosine hydroxylase and dopamine beta-hydroxylase, although a direct effect of ACTH on NE and E secretion cannot be excluded. It is also possible that the increase in adrenal catecholamine secretion after ACTH may be due to ACTH augmentation of catecholamine secretion by endogenous opioids such as beta-endorphin.     

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.     

Activation of Tyrosine Hydroxylase by Nicotine in Rat Adrenal Gland

The administration of nicotine activates tyrosine hydroxylase in the rat adrenal gland. This activation is apparently maximal 25 min after a single subcutaneous injection of nicotine at 2.3 mg/kg. Repeated injections of nicotine (seven injections once every 30 min) are associated with a persistent activation of adrenal tyrosine hydroxylase for at least 3 h. The nicotinic receptor antagonist hexamethonium does not significantly inhibit the nicotine-mediated activation of tyrosine hydroxylase in innervated adrenal glands. However, hexamethonium completely blocks the activation of adrenal tyrosine hydroxylase by nicotine in denervated adrenal glands. Furthermore, even though a single injection of nicotine activates tyrosine hydroxylase in both innervated and denervated adrenal glands, repeated injections of nicotine do not activate tyrosine hydroxylase in denervated adrenal glands. Our results suggest that the systemic administration of nicotine activates adrenal tyrosine hydroxylase by two mechanisms: (1) via direct interaction with adrenal chromaffin cell nicotinic receptors; and (2) via stimulation of the CNS leading to the release from the splanchnic nerve of substances that interact with adrenal chromaffin cell receptors other than the nicotinic receptor.     

Retinol activates tyrosine hydroxylase acutely by increasing the phosphorylation of serine40 and then serine31 in bovine adrenal chromaffin cells

Tyrosine hydroxylase is the rate-limiting enzyme in the biosynthesis of the catecholamines. It has been reported that retinol (vitamin A) modulates tyrosine hydroxylase activity by increasing its expression through the activation of the nuclear retinoid receptors. In this study, we observed that retinol also leads to an acute activation of tyrosine hydroxylase in bovine adrenal chromaffin cells and this was shown to occur via two distinct non-genomic mechanisms. In the first mechanism, retinol induced an influx in extracellular calcium, activation of protein kinase C and serine40 phosphorylation, leading to tyrosine hydroxylase activation within 15 min. This effect then declined over time. The retinol-induced rise in intracellular calcium then led to a second slower mechanism; this involved an increase in reactive oxygen species, activation of extracellular signal-regulated kinase 1/2 and serine31 phosphorylation and the maintenance of tyrosine hydroxylase activation for up to 2 h. No effects were observed with retinoic acid. These results show that retinol activates tyrosine hydroxylase via two sequential non-genomic mechanisms, which have not previously been characterized. These mechanisms are likely to operate in vivo to facilitate the stress response, especially when vitamin supplements are taken or when retinol is used as a therapeutic agent.     

Functional cerebral activity of an analogue of serotonin formed in situ

Reduction of the serotonin content of the brain of rats (specifically in the medial raphe nucleus) by various means results in spontaneous increase of adrenal tyrosine hydroxylase activity. This neurally mediated induction is attenuated by appropriate administration of the serotonin precursor 5-hydroxytryptophan to the animals, along with carbidopa (Quik and Sourkes, J. Neurochem.28, 137, 1977). In the present work adrenal tyrosine hydroxylase was induced by giving rats either the neurotoxin 5,7-dihydroxytryptamine (injected into the cerebral ventricles) or the monoamine depletor reserpine (given intraperitoneally). Other rats received alpha-methyltryptophan. This amino acid causes a marked decline of the serotonin content of the brain, but gives rise to relatively large amounts of alpha-methylserotonin in that organ (Roberge et al., Neuropharmacology11, 197, 1972). Alpha-methyltryptophan had no effect on adrenal tyrosine hydroxylase activity but, when it was given with dihydroxytryptamine or reserpine, it prevented the induction of adrenal tyrosine hydroxylase that otherwise occurred. The results are discussed in relation to the effect of alpha-methyltryptophan on the content of indoles (tryptophan, serotonin, 5-hydroxyindoleacetic acid, alpha-methyltryptophan, alpha-methylserotonin) in the plasma and brain, as detected by HPLC. It is concluded that alpha-methylserotonin can functionally replace cerebral serotonin, at least in relation to the transneuronal regulation of adrenal tyrosine hydroxylase activity.     

Plasma and adrenal aldosterone levels in premature mice at birth and during neonatal development

The concentrations of aldosterone in the plasma and adrenal glands, the concentrations of sodium and potassium in the plasma and the hematocrit were estimated from birth to day 6 after birth in premature mice removed by Caesarean section on day 19 of pregnancy in comparison with newborn mice delivered spontaneously vaginally on day 20 of pregnancy. In premature mice, the plasma aldosterone concentrations increased twice: at birth after reanimation, then at 6 h after birth. The first increase at birth resulted probably from ACTH stimulation. Several factors could be involved in the peak at 6 h after birth : ACTH stimulation, the decrease in the level of sodium in the plasma and the increase in the hematocrit due to kidney immaturity of premature mice. The results suggest that the renin-angiotensin-aldosterone system is able to respond to stimulations in the first 6 h after birth in premature mice. The rise in the level of plasma aldosterone which has been found at birth in newborns delivered spontaneously vaginally on day 20 of pregnancy (control animals) did not result from variations of plasma electrolytes, plasma volume and ACTH ; this rise has been induced by labor of the parturition which caused the aldosterone release from adrenal glands.     

In situ phosphorylation of tyrosine hydroxylase in chromaffin cells: Localization to soluble compartments

The present studies investigated the subcellular distribution of acetylcholine's effects upon the phosphorylation of tyrosine hydroxylase in isolated purified bovine adrenal chromaffin cells. After labeling the intact chromaffin cells with ³²P_i, over 90% of the [³²P]tyrosine hydroxylase was found in soluble fractions. Stimulation of the cells with acetylcholine, the natural secretagogue of chromaffin cells, increased the phosphorylation of tyrosine hydroxylase and over 90% of the increase was associated with soluble tyrosine hydroxylase. Homogenates and subcellular fractions from chromaffin cells were also prepared and phosphorylated in vitro in an attempt to optimize detection of tyrosine hydroxylase phosphorylation. In chromaffin cell homogenates, both 8-bromo-cyclic AMP and calcium increased ³²P incorporation into tyrosine hydroxylase, and again over 90% of the increase was observed in soluble fractions. In the particulate fraction, phosphorylation of a band which comigrated with tyrosine hydroxylase in electrophoresis was occasionally detected but only with very long autoradiographic exposures.Tyrosine hydroxylase enzymatic activity in the isolated purified chromaffin cells was also found to be associated predominantly (approx 90%) with soluble fractions. In contrast, a large portion (40–50%) of the tyrosine hydroxylase activity from crude bovine adrenal medullae was associated with the particulate fraction.The data indicate that although tyrosine hydroxylase (and possibly kinases) can associate with particulate fractions when isolated from crude bovine adrenal medullae, the enzyme is predominantly soluble when isolated from the isolated cells. Further, the effects of acetylcholine on the isolated chromaffin cells are predominantly associated with this soluble tyrosine hydroxylase and its attendant kinases.     

MINIMUM DURATION OF TRANS-SYNAPTIC STIMULATION REQUIRED FOR THE INDUCTION OF TYROSINE HYDROXYLASE BY RESERPINE IN THE RAT SUPERIOR CERVICAL GANGLION

—The period during which trans-synaptic stimulation is required by the rat superior cervical ganglion for induction of tyrosine hydroxylase by reserpine has been studied. Ganglia were decentralized on one side at various times before or after an injection of reserpine. The tyrosine hydroxylase activity of the denervated and control ganglia was assayed 72 h after drug treatment. When decentralization was performed 8 h after an injection of reserpine the increase in tyrosine hydroxylase activity was blocked in the denervated ganglia. Decentralization 12 h after reserpine treatment or later had no effect on the enzyme induction. The actual increase in tyrosine hydroxylase activity occurred between 24 and 48 h after injection of reserpine.     

Co-localization of vasoactive intestinal peptide (VIP) and enkephalins in chromaffin cells of the adrenal gland of amphibia. Stimulation of corticosteroid production by VIP

Recent studies have shown that biologically active peptides and monoaminergic neurotransmitters coexist in certain neuronal cell populations. Using the immunofluorescence technique, we have examined the localization of enkephalins, vasoactive intestinal peptide (VIP) and tyrosine hydroxylase in the adrenal gland of the frog Rana ridibunda. Most chromaffin cells which stained for tyrosine hydroxylase contained VIP-like immunoreactivity, whereas methionine- (Met-) and leucine- (Leu-) enkephalin-like immunoreactivity was detected in about 40% of the cells revealed by the anti-tyrosine hydroxylase serum. No VIP- or enkephalin-like immunoreactive nerve fibres were observed. Since in the frog, the chromaffin cells are in close contact with the adrenocortical (interrenal) tissue, a possible action of VIP and opiates on corticosteroidogenesis has been investigated. At doses 10(-6) and 10(-5) M, 20-min infusions of synthetic porcine or chicken VIP elicited a significant increase in corticosterone and aldosterone production by perifused frog adrenals, in a dose-dependent manner. As compared to ACTH, VIP was several orders of magnitude less effective in stimulating corticosteroid production. Morphine, Met- and Leu-enkephalins (10(-5) M) had no effect on spontaneous secretion of corticosteroids. In addition, Met- and Leu-enkephalins (10(-5) M) did not alter the production of corticosterone induced by ACTH. THese results suggest that VIP contained in the chromaffin cells of the frog adrenal gland may exert a local action in stimulating corticosteroid production by the interrenal tissue.     

Effect of vasopressin on the induction of adrenal tyrosine hydroxylase and phenylethanolamine N-methyltransferase

We have assessed the effect of arginine vasopressin (AVP) on adrenal tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) activities. Both enzymes show marked increases after systemic administration of AVP in the range of 66 and 100 micrograms/day. To determine whether the pituitary gland plays a role in these inductions, the effect of AVP (66 micrograms per day, given divided into 3 doses for 4 days) on the adrenal enzymes was studied in hypophysectomized rats. These animals showed induction of TH but not PNMT. This indicates that a pituitary factor(s) mediates the increase in PNMT caused by AVP. Adrenal TH activity was measured after the injection of AVP (1 or 2 micrograms per rat) into the lateral ventricle: there was a statistically significant increase in TH. TH was not induced in the denervated adrenal gland of rats administered AVP systemically. These findings suggest that AVP may act centrally to induce the enzyme. The continuous s.c. infusion of AVP by osmotic minipump at the rate of 1 microgram/day for 6 days led to a striking increase in adrenal TH activity. However, PNMT did not increase significantly. It can be concluded that different mechanisms are involved in the induction of adrenal TH and PNMT caused by AVP. A neural mechanism is involved in TH induction, whereas PNMT induction requires release of a pituitary factor, presumably ACTH, but innervation of the adrenal is not needed for it. Moreover, the inductions of these two enzymes are differentially sensitive to the concentration of circulating AVP.     

Elevated renal norepinephrine in proANP gene-disrupted mice is associated with increased tyrosine hydroxylase expression in sympathetic ganglia

The sympatholytic properties of atrial natriuretic peptide (ANP) contribute to its vasodilatory and natriuretic effects. High circulating catecholamine levels, along with renal dysfunction, present in proANP gene-disrupted (-/-) mice are thought to contribute to the hypertension characteristic of this model. To further understand the mechanism by which the absence of ANP leads to stimulation of sympathetic activity we measured tyrosine hydroxylase expression in mice with and without ANP. The adrenal and prevertebral ganglionic expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine production, was significantly increased in ANP -/- mice. ANP's sympatholytic properties include the depression of ganglionic and adrenal TH expression and catecholamine production. Thus, these results suggest that the absence of ANP's sympatholytic effects is not completely compensated for in ANP -/- mice. In addition, mice devoid of ANP display an increase in renal sympathetic nerve activity from birth through to adulthood which may lead to structural and/or functional changes in the early postnatal kidney that contribute to the hypertensive phenotype of ANP -/- mice. The over-activation of the sympathetic nervous system in mice lacking ANP confirms the important role of this peptide in the modulation of sympathetic nerve activity and its contribution to blood pressure homeostasis.     

The effects of enalapril maleate and cold stress exposure on tyrosine hydroxylase activity in some rat tissues

Enalapril is a highly specific and competitive inhibitor of angiotensin-I converting enzyme (ACE) and thus belongs to the category of ACE inhibitors. The beneficial effects of ACE inhibitors appear to result primarily from the suppression of the plasma renin-angiotensin-aldesterone system. This study was designed to detect the effects of enalapril maleate and cold stress on tyrosine hydroxylase (TH) activity in adrenal medulla, heart and hypothalamus in rat. In cold stress treatment (exposed to 8 degrees C cold for 48 h) TH activity was found to be raised significantly (p < 0.05) in adrenal medulla, hypothalamus and heart tissues. In the adrenal medulla, hypothalamus and heart tissues, TH activity of enalapril maleate treated rats (10 mg kg(-1) body weight) group was not raised significantly (p > 0.05). Following intraperitoneal injection of enalapril maleate (10 mg kg(-1) body weight) the rats were exposed to 8 degrees C cold for 48 h. After cold stress and enalapril maleate treatment no statistically significant change in tyrosine hydroxylase activity was detected in adrenal medulla, hypothalamus or heart (p > 0.05). The results of our studies show that enalapril maleate blocks the effect of cold stress on the regulation of TH activity.     

Activation of Tyrosine Hydroxylase in PC12 Cells by the Cyclic GMP and Cyclic AMP Second Messenger Systems

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is subject to regulation by a variety of agents. Previous workers have found that cyclic AMP-dependent protein kinase and calcium-stimulated protein kinases activate tyrosine hydroxylase. We wanted to determine whether cyclic GMP might also be involved in the regulation of tyrosine hydroxylase activity. We found that treatment of rat PC12 cells with sodium nitroprusside (an activator of guanylate cyclase), 8-bromocyclic GMP, forskolin (an activator of adenylate cyclase), and 8-bromocyclic AMP all produced an increase in tyrosine hydroxylase activity measured in vitro or an increased conversion of [14C]tyrosine to labeled catecholamine in situ. Sodium nitroprusside also increased the relative synthesis of cyclic GMP in these cells. In the presence of MgATP, both cyclic GMP and cyclic AMP increased tyrosine hydroxylase activity in PC12 cell extracts. The heat-stable cyclic AMP-dependent protein kinase inhibitor failed to attenuate the activation produced in the presence of cyclic GMP. It eliminated the activation produced in the presence of cyclic AMP. Sodium nitroprusside also increased tyrosine hydroxylase activity in vitro in rat corpus striatal synaptosomes and bovine adrenal chromaffin cells. In all cases, the cyclic AMP-dependent activation of tyrosine hydroxylase was greater than that of the cyclic GMP-dependent second messenger system. These results indicate that both cyclic GMP and cyclic AMP and their cognate protein kinases activate tyrosine hydroxylase activity in PC12 cells.     

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.     

Bovine Tyrosine Hydroxylase Gene-Promoter Regions Involved in Basal and Angiotensin II-Stimulated Expression in Nontransformed Adrenal Medullary Cells

Abstract: The tyrosine hydroxylase gene is expressed specifically in catecholaminergic cells, and its activity is regulated by afferent stimuli. To characterize molecular mechanisms underlying those regulations, we have constructed chimeric genes consisting of bovine tyrosine hydroxylase gene promoters (wild-type or deletion mutants) and a luciferase reporter gene. The basal expression of these genes and their regulation by angiotensin II were examined in cultured bovine adrenal medullary cells. Luciferase activity was normalized to the amount of transfected plasmid DNA. A pTHgoodLUC plasmid containing the -428/+21-bp fragment of the tyrosine hydroxylase gene promoter expressed luciferase activity at severalfold higher levels than the promoterless pOLUC plasmid. Deletion of the -194/-54-bp promoter fragment containing POU/Oct, SP1, and other putative regulatory elements increased luciferase expression fivefold. An additional deletion further upstream (-269/-194 bp), including a 12-O-tetradecanoylphorbol 13-acetate (TPA)-responsive element (TRE)-like site, reduced promoter activity. These results indicate the presence of negatively and positively acting regions in the bovine tyrosine hydroxylase gene promoter controlling basal promoter activity in adrenal medullary cells. Angiotensin II stimulated the expression of endogenous tyrosine hydroxylase gene and pTHgood-LUC approximately threefold without affecting the expression of pOLUC. A comparable threefold stimulation was observed following the deletion of the -194/-54-bp promoter region, despite the increase in basal promoter activity. Additional deletion of the -269/-194-bp promoter fragment reduced stimulation by angiotensin II to 1.5-fold. These results indicate that the angiotensin II receptor-responsive element is located in the -269/-194-bp promoter region containing the TRE-like site. Additional angiotensin II-responsive site(s) may be present outside this region. Gel mobility shift assays demonstrated constitutive and angiotensin II-induced protein binding to the tyrosine hydroxylase gene promoter. Some DNA-protein complexes were displaced with c-Fos antibodies. The results suggest that c-Fos-related antigens support basal promoter activity and mediate activation of tyrosine hydroxylase by angiotensin II receptor.     

Induction of 5-aminolevulinate synthase by activators of steroid biosynthesis

Different cytochromes P450 are involved in steroid biosynthesis. These cytochromes have heme as the prosthetic group. We previously reported that ACTH, an activator of glucocorticoid biosynthesis in adrenal, requires heme biosynthesis for a maximal response. In the present study, we investigated the effect of ACTH, and the effect of two activators of the adrenal mineralocorticoid synthesis, endothelin-1 and low sodium diet on 5-aminolevulinate-synthase (ALA-s) mRNA. ALA-s is the rate-limiting enzyme in heme biosynthesis. It was found that infusion of rats with ACTH for 1 h caused an increase of adrenal ALA-s mRNA and activity accompanied by an increase in plasma corticosterone. CYP21, a cytochrome involved in the synthesis of both corticosterone and aldosterone, was not modified at the RNA level in adrenal glands by 1 h of ACTH infusion. Consistently, infusion of endothelin-1 for 1 h increased ALA-s mRNA and aldosterone content in adrenal gland without modifying CYP21 mRNA levels. To study if ALA-s is also regulated by the main physiological stimuli that increase adrenal mineralocorticoid secretion, we fed rats with low salt diet for 2 or 15 days. Low salt diet treatment increased adrenal gland ALA-s mRNA levels. On the other hand, the rapid stimulation of ALA-s mRNA by ACTH which acts through cyclic AMP was confirmed in H295R human adrenocortical cells, the only human adrenal cell line that has a steroid secretion pattern and regulation similar to primary cultures of adrenal cells. Our findings suggest that the acute activation of adrenal steroidogenic cytochromes by trophic hormones involves an increase in heme biosynthesis which will favor the production of active cytochromes.