Quantal secretion from adrenal medulla: all-or-none release of storage vesicle content.

Neurogenic secretion of catecholamines from the adrenal medulla in rabbits, induced by administration of insulin, caused decreases in both the dopamine-beta-hydroxylase activity and the catecholamine content of the storage vesicle fraction. After sedimentation through a sucrose density gradient, the storage vesicles obtained from insulin-treated animals had the same density and the same ratio of dopamine-beta-hydroxylase to catecholamine as did vesicles from untreated animals. These and other data indicate that neurogenic secretion from the adrenal medulla occurs by an all-or-none release from the storage vesicles.

Secretion of newly taken-up ascorbic acid by adrenomedullary chromaffin cells.

Primary cultures of bovine adrenomedullary cells accumulate carbon-14-labeled ascorbic acid through a saturable and energy-dependent process. The newly taken-up ascorbate is released concomitantly with catecholamines upon stimulation of chromaffin cell secretion. The release of ascorbate is Ca2+-dependent and mediated through activation of nicotinic receptors. These results indicate that exogenous ascorbate taken up into chromaffin cells is incorporated in situ into a secretable compartment, probably the catecholamine-containing chromaffin vesicles.

Biopterin cofactor biosynthesis: independent regulation of GTP cyclohydrolase in adrenal medulla and cortex.

Guanosine triphosphate cyclohydrolase, the enzyme that is apparently rate-limiting in biopterin biosynthesis, is increased in adrenal cortex and medulla of rats treated with insulin or reserpine. Denervation and hypophysectomy block the increase in medullary and cortical enzyme activity, respectively, whereas cycloheximide presents the increase in both tissues. These results provide evidence for induction and regulation of guanosine triphosphate cyclohydrolase.

Hormonal regulation of guanosine triphosphate cyclohydrolase activity and biopterin levels in the rat adrenal cortex.

The regulation of GTP-cyclohydrolase (GTP-CH) activity and tetrahydrobiopterin (BH4) levels in the adrenal cortex were studied in intact and hypophysectomized rats. Treatment with a single dose of reserpine (5 mg/kg) or insulin-induced hypoglycemia (4 h) elevated adrenocortical BH4 3-fold by 10 h; BH4 levels remained elevated after 24 h and returned to control levels by 48-72 h. GTP-CH was significantly increased 24 h after hypoglycemic shock, and the increased enzyme activity preceded the changes in BH4 levels after reserpine treatment. Two and a half hours of stress by immobilization also increased GTP-CH activity and BH4 levels in the adrenal cortex. The activities of sepiapterin reductase and dihydrofolate reductase, putative enzymes in the biosynthetic pathway from GTP to BH4, were not increased by reserpine. Both reserpine and insulin increased the apparent maximum velocity for GTP, with no increase in the affinity of the enzyme for its substrate, further suggesting that the experimental treatments induce the synthesis of GTP-CH. Hypophysectomy completely blocked the reserpine-dependent increase in both cortical GTP-CH activity and BH4 content. The administration of purified porcine ACTH to intact and hypophysectomized rats elevated adrenocortical GTP-CH activity and cofactor levels. Synthetic ACTH-(1-24) also enhanced the enzyme activity and BH4 levels in the adrenal cortex. Thus, pituitary control of adrenal cortical GTP-CH synthesis and biopterin levels appears to be mediated through the secretion of ACTH. The changes in enzyme activity and cofactor levels after activation of the hypothalamo-hypophyseal axis or ACTH administration suggest that BH4, a cofactor for certain monooxygenases, has some function, as yet unknown, in the adaptive changes of the adrenal cortex in response to stress.

Regulation of tetrahydrobiopterin biosynthesis in cultured adrenal cortical tumor cells by adrenocorticotropin and adenosine 3',5'-cyclic monophosphate.

Y-1 adrenal cortical tumor cells in culture, which contain substantial amounts of tetrahydrobiopterin [6R-(L-erythro-1',2'-dihydroxypropyl)5,6,7,8-tetrahydropterin] (BH4) and GTP cyclohydrolase (GTP-CH), were used to study the regulation of BH4 biosynthesis by ACTH and cAMP. ACTH produced a dose-dependent increase in steroidogenesis, BH4 levels and GTP-CH activity. Maximal stimulation of BH4 biosynthesis occurred at the same concentration of ACTH that caused maximal stimulation of steroidogenesis. ACTH-(1-24) was more potent than ACTH-(1-39). The stimulation of BH4 biosynthesis by ACTH was dependent on cell density, being greater at lower cell densities, but was independent of time in culture. The lack of stimulation by ACTH at higher cell densities was due to an increase in the specific activity of GTP-CH in the control cells as density increased. This increase may be due in part to the increased release of steroids, since exogenous steroids added to low density cultures also resulted in an increase in the specific activity of the enzyme. Addition of steroids had no effect on ACTH-dependent stimulation of BH4 biosynthesis at low cell densities. (Bu)2cAMP, 8-bromo-cAMP, and forskolin all produced time- and dose-dependent increases in BH4 levels, GTP-CH activity, and steroidogenesis. Maximum increases in GTP-CH and BH4 occurred at concentrations similar to those required for maximal stimulation of steroidogenesis. In the Kin-8 mutant of Y-1 cells, which has a type 1 cAMP-dependent protein kinase with an altered regulatory subunit, ACTH was unable to increase BH4 levels or GTP-CH activity at a concentration that produced maximal stimulation of BH4 and steroid biosynthesis in the parent Y-1 line. These studies indicate that Y-1 cells in culture are useful for studying the regulation of BH4 biosynthesis in the adrenal cortex.

Multicompartmental secretion of ascorbate and its dual role in dopamine beta-hydroxylation.

The neurobiological functions of ascorbate have both intra- and extracellular sites of action. Intracellularly, it participates predominantly in enzymic and transport reactions for neurotransmitter and hormone biosynthesis. Ascorbate is the cofactor for the dopamine beta-hydroxylase and peptidylglycine alpha-amidating monooxygenase systems, which catalyze the synthesis of norepinephrine and a variety of alpha-amidated peptides, respectively. The localization of these enzymes within the neurotransmitter- or hormone-containing storage vesicle requires a system for the constant regeneration of ascorbate to the reduced form. In fact, ascorbate participates in its own regeneration as a component of the vesicular electron-transport system. In addition to the roles of ascorbate in messenger synthesis, it is secreted from cells from different subcellular compartments. The extracellular role(s) of ascorbate are still unknown, although its interaction with and modification of plasma membrane proteins suggests some modulatory function.

Opioid peptides and noradrenaline co-exist in large dense-cored vesicles from sympathetic nerve.

The possibility that opioid peptides and noradrenaline co-exist not only in the desheathed bundle of bovine splenic nerve which contains approximately 98% sympathetic C-fibers, but also in the population of large dense-cored noradrenergic vesicles from these fibers, has been investigated. The primary fraction of large dense-cored vesicles which can be prepared at about 85% purity has been further subjected to density gradient and fractional centrifugation procedures, including D2O-loading and unloading on modified second gradients, in an attempt to separate any minor population of particles which potentially could contain opioid peptides and contaminate the large dense-cored vesicle fraction. Measurement of opioid peptides, noradrenaline, dopamine and dopamine beta-hydroxylase activity supports the conclusion that opioid peptides are stored in the primary population of large dense-cord vesicles per se, rather than in a minor population of contaminating particles from cells other than sympathetic C-fibers. This conclusion has implications for exocytotic release and the physiological role of the opioid peptides intra- and extra-neuronally. Nerve vesicle opioid peptides have a size less than 5000 daltons, in contrast to the high proportion of large peptides containing enkephalin sequences in the bovine adrenal medulla.

Opioid peptide synthesis in bovine and human adrenal chromaffin cells.

Addition of inhibitors of chromaffin vesicle catecholamine uptake, reserpine or tetrabenazine, to cultures of bovine adrenal chromaffin cells led to a depletion of cellular catecholamine stores and to an increased opioid peptide content. Addition of monoamine oxidase inhibitors, catecholamines or catecholamine receptor agonists to the cultures produced similar increases in the opioid peptide content without altering catecholamine stores, suggesting that an extravesicular catecholamine pool influences chromaffin cell opioid peptide content. This elevated peptide content resulted from increased synthesis by the cells and led to enhanced stimulus-evoked secretion of opioid peptides. Dissociated cells obtained from a human pheochromocytoma tumor also increased their opioid peptide content following treatment with reserpine or a secretagogue. The results obtained with cultured chromaffin and pheochromocytoma cells suggest that a mechanism for replenishing and increasing adrenal medullary opioid peptide stores and secretory capacity may function in vivo. The observed regulation of opioid peptide stores may also be important in the clinical pathology of opioid peptide containing tumors.

Coordinate and differential regulation of phenylethanolamine N-methyltransferase, tyrosine hydroxylase and proenkephalin mRNAs by neural and hormonal mechanisms in cultured bovine adrenal medullary cells.

Primary cultures of bovine adrenal medullary cells (AM) in a chemically defined media were used to examine the role of neural and hormonal factors in the expression of proenkephalin A (pEK), phenylethanolamine N-methyltransferase (PNMT) and tyrosine hydroxylase (TH) genes. Acetylcholine or nicotine reduced cellular content of catecholamines by 30% and increased the relative abundance of pEK, TH, and PNMT mRNAs. The increases produced by acetylcholine were +129%, +147%, and +43% for pEK, TH, and PNMT mRNA, respectively. The kinetics of increases produced by nicotine were different for the 3 mRNAs, with pEK and TH showing enhanced levels over 48 h incubation, while PNMT showed increase during the initial 18 h (+90%) followed by decline to control levels at 48 h. 8-Br cAMP and forskolin elicited a similar pattern of changes as nicotine, suggesting that cyclic AMP may be involved in the mediation of the nicotinic effects. To examine the role of depletion of cellular catecholamines in the regulation of mRNA levels, cells were exposed to tetrabenazine or reserpine. Decreases in cellular catecholamine contents were accompanied by increases in TH and pEK mRNA levels, while the expression of PNMT gene exhibited a transient 4-fold increase and then profound inhibition (60-95%) over a 48-h period. The tetrabenazine effect on TH and pEK mRNA was reduced by alpha-amanitin, suggesting transcriptionally-mediated regulation. Inductions of pEK but not TH or PNMT mRNAs were inhibited by cycloheximide. Hormonal regulation of TH, PNMT, and pEK mRNAs was examined by incubation of cells with dexamethasone. Low concentrations of dexamethasone (0.1, 10 nM) were effective to increase PNMT (+35%, +90%) and pEK (+27%, 45%) mRNA levels. TH mRNA was not affected by similar concentrations of dexamethasone, however, there was a 45% increase at 1 microM. Dexamethasone-elicited increases in PNMT mRNA levels were observed at 48 h and persisted up to 7 days, suggesting that hormonal mechanisms may be distinct from those mediating effects of nicotine, cAMP or tetrabenazine. Taken together, these results indicate that (1) the level of TH, PNMT, and pEK mRNAs are regulated by direct neural (acetylcholine) and hormonal (glucocorticoid) inputs to adrenal medullary cells; (2) effects of acetylcholine could be mediated by cyclic AMP and alterations in catecholamine content; and (3) expression of individual genes is regulated differentially. Such differential regulation of TH, PNMT, and pEK mRNAs may contribute to the long-term selective control of hormonal output from adrenomedullary cells.

Stimulation of retinal dopamine biosynthesis in vivo by exogenous tetrahydrobiopterin: relationship to tyrosine hydroxylase activation.

Intravitreal injection of tetrahydrobiopterin (BH4), the cofactor for tyrosine hydroxylase (TH), increases 3,4-dihydroxyphenylalanine (DOPA) accumulation in retinas of dark-adapted rats, as does exposure to light. In contrast, BH4 had no significant effect on DOPA accumulation in retinas of light-exposed rats. The levels of endogenous retinal BH4 and the uptake of injected BH4 into the retinal tissue were not affected by light exposure. These data indicate that TH is not saturated with endogenous BH4 in the retinas of dark-adapted rats. In addition, the observations support the interpretation that the decrease in apparent Km of TH for the cofactor in response to light exposure is of sufficient magnitude to allow near saturation of TH by endogenous BH4 and, thus, is causally related to the increase of dopamine biosynthesis in response to short-term photic stimulation.

Roles of the pituitary-adrenocortical axis in control of the native and cryptic enkephalin levels and proenkephalin mRNA in the sympathoadrenal system of the rat.

The effects of hypophysectomy (HPX) and dexamethasone (DEX) on the levels of Met5-enkephalin (ME), ME precursors, and the abundance of proenkephalin (pEK) mRNA, were examined in the adrenal medulla (AM) and superior cervical ganglia (SCG). To assess possible changes in enkephalin processing, both cryptic (after trypsin and carboxypeptidase B digestions) and native (without enzyme digestions) ME-like immunoreactivity (ME-LI) was measured. Three weeks after HPX the proportion of pEK mRNA to the total RNA content in the AM was not significantly changed when compared to sham-operated (SO) animals. Total (native + cryptic) ME-LI was decreased by 45% in the AM of HPX rats. This decrease was paralleled by a 58% depletion of AM proteins. Cryptic ME-LI was also reduced by 43%. In contrast, native ME-LI was not altered after HPX, indicating enhanced processing of ME precursors. Treatment with DEX (5 daily injections--1 mg/kg, i.p.) increased the relative abundance of pEK mRNA (+27%) and total ME-LI in the AM of HPX group, but not in SO group. Native ME-LI, cryptic ME-LI, and their ratio were not significantly affected by DEX in the AM of HPX or SO rats. In SCG, the relative abundance of pEK mRNA decreased by 25% after hypophysectomy. Total and cryptic ME-LI in the SCG of HPX rats were not changed when compared to SO rats. In contrast, HPX reduced native ME-LI suggesting decreased processing of ME precursors. Similarly, as in AM, DEX produced increase in the SCG pEK mRNA only in HPX (+68%) and not in the SO rats. In SCG, DEX produced decreases in total ME-LI which could be attributed to an increased enkephalin release. An overall reduction of cryptic ME-LI was also observed after DEX, whereas native ME-LI remained unchanged suggesting increased processing of enkephalins. Our findings indicate that the pituitary adrenocortical axis controls the relative proportions of ME to its precursors, and that this control involves both glucocorticoid-dependent (SCG) and glucocorticoid-independent (AM) mechanisms. In contrast, our studies do not suggest specific control of pEK synthesis by the pituitary adrenocortical axis. The pituitary adrenocortical axis may also influence the relative contents of ME and catecholamines in the AM and SCG. The ratio of ME/catecholamines increased after HPX (AM and SCG) and after DEX (SCG). Such regulation may contribute to the control of co-transmitter output in the sympathoadrenal system.

Regulation of tyrosine hydroxylase and phenylethanolamine N-methyltransferase mRNA levels in the sympathoadrenal system by the pituitary-adrenocortical axis.

The pituitary-adrenocortical axis plays a complex role in the regulation of the levels of enzymes of the catecholamine biosynthetic pathway. In this report we have explored molecular mechanisms of these regulations, by examining the effects of hypophysectomy (HPX) and dexamethasone (DEX) on tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) mRNA levels in the adrenal medulla (AM) and superior cervical ganglia (SCG). Three weeks after hypophysectomy weights (-48%), total RNA (-49%), and DNA (-22%) contents in AM were significantly reduced, when compared to sham-operated animals (SO). In SCG decreases in weight (-23%) and in the ratio of RNA/DNA (-25%) were also found. TH mRNA contents paralleled decreases in total RNA levels and no significant change in the relative abundance of TH mRNA was found. When HPX rats were injected for 5 days with DEX (1 mg/kg, i.p.), TH mRNA levels in the SCG (+51%) and in the AM (+74%) were significantly increased when compared to saline-treated HPX animals. DEX given to SO rats increased TH mRNA in SCG (+49%); a 27% increase in TH mRNA in the AM was also observed. The relative abundance of PNMT mRNA in the AM was reduced after hypophysectomy (-64%). This decrease was completely reversed by DEX. In contrast, DEX did not affect PNMT mRNA levels in the AM of SO rats. PNMT mRNA was not detected in SCG of saline- or DEX-treated rats. In conclusion, our findings suggest that the pituitary-adrenocortical axis is involved in the regulation of the steady-state levels of TH and PNMT mRNAs. This regulation involves: (1) induction of TH mRNA contents in AM and SCG by increased plasma glucocorticoid levels; and (2) maintenance of the steady-state levels of PNMT mRNA in AM by glucocorticoid-dependent mechanisms.

Potentiation by reserpine and tetrabenazine of brain catecholamine depletions by MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) in the mouse; evidence for subcellular sequestration as basis for cellular resistance to the toxicant.

Administration to mice of the neurotoxicant MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) decreased striatal dopamine and, to a lesser extent, hippocampal noradrenaline levels when measured 2 weeks after the last dose of MPTP. Reserpine and tetrabenazine, inhibitors of catecholamine vesicular transporter, potentiated the catecholamine depletions produced by MPTP in the hippocampus and striatum, respectively. These results are compatible with our hypothesis that sequestration of the toxic MPTP metabolite MPP+ (1-methyl-4-phenylpyridinium) in the catecholamine storage vesicle retards the catecholaminergic toxicity of MPTP.

Substance P and opiate-like peptides in human adrenal medulla.

Opiate-like peptides (OLP), substance P (SP) and catecholamines (CA) were measured in 15 human adrenal medullae. Two groups of subjects were investigated. Group a consisted of subjects who died after traffic accidents and Group b consisted of subjects with other causes of death. OLP levels in Group a were only about 13%, and SP and CA levels about 50% of Group b. It is suggested that these differences might be due to massive premortal adrenal medullary discharge rather than post mortem degradation.

Opioid peptides and catecholamines in human pheochromocytoma.

Opioid peptides (OP) and catecholamines (CA) were measured in twelve human pheochromocytomas (PHEO). In all tumors the CA concentrations were much higher than those OP (range: 300-85,000 fold higher). Large intertumor variability in the levels of both substances was encountered (mean +/- S.E.M. = CA: 44.9 +/- 7.7 mumoles/g; OP: 38.1 +/- 17.1 nmoles/g; range = CA: 10.1-93.1 mumoles/g; OP: 0.7-181 nmoles/g). Norepinephrine (NE) was the main CA in seven of the 12 tumors. In four of these PHEO, NE accounted for 85% or more of the total CA. These "noradrenergic PHEO" derived from the right adrenals, were of smaller size (36 +/- 15g), had the lowest levels of OP (1.1 +/- 0.3 nmoles/g) and CA (28 +/- 10 mumoles/g), produced moderate to severe sustained hypertension (MBP: 160 +/- 11 mmHg) and the most severe and persistent clinical manifestations. Epinephrine (EPI) was the main CA in five of the 12 tumors. These PHEO had intermediate levels of OP (12 +/- 3 nmoles/g), and four of them were of left adrenal origin. Patients bearing these tumors were generally normotensive (MBP: 103 +/- 4 mmHg) and asymptomatic, with occasional paroxysmal crisis. The highest levels of OP (132 +/- 24 nmoles/g) were found in two tumors of extra-adrenal location and in one of right adrenal origin. The proportion of NE and EPI ranged between 60-80% and 20-40% respectively, of total tumor CA. The two extra-adrenal PHEO were the largest of this series (180 and 245g). These patients had mild hypertension (MBP: 118 +/- 7 mmHg) of sustained or paroxysmal course, and frequent symptomatic episodes. Differences in the synthesis, storage, metabolism and release of CA and OP in PHEO probably account for their variable tumoral content, as well as for the clinical heterogeneity produced by these tumors. It remains to be seen whether OP can contribute to the clinical manifestations of patients with pheochromocytomas.

Differences in the reserpine-sensitive storage in vivo of 1-methyl-4-phenylpyridinium in rats and mice may explain differences in catecholamine toxicity to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Administration of reserpine, an inhibitor of vesicular catecholamine storage, differentially reduced the accumulation of MPP+ formed from MPTP in rats and mice. The effects were most pronounced in the adrenal gland for either species. In rats, reserpine decreased striatal and hippocampal MPP+ levels while in mice reserpine did not affect the disposition of MPP+ in the striatum but decreased hippocampal MPP+. The data suggest that mice may be more sensitive to the toxicant because less striatal MPP+ appears to be stored in the reserpine-sensitive storage vesicle.