Search

Pretreatment of rats with dexamethasone (2.5 mumol/kg, a dose which blocks the release of ACTH from the pituitary gland) abolished the reserpine mediated increase in cAMP and the increase in the cAMP/cGMP ratio in the adrenal medulla. In contrast, the reserpine-mediated induction of tyrosine hydroxylase (TH) remained unchanged. Hypophysectomy had a similar effect to dexamethasone treatment. Since changes in cAMP and changes in the cAMP/cGMP ratio are not indispensible prerequisities for the subsequent induction of TH, a causal relationship between the two phenomena seems to be excluded.

1. Isolated superior cervical ganglia of the rat were incubated for 2--30 min (37 degrees C) in Krebs' solution or tissue culture medium (BGJb) containing 22Na and then washed for 30 min in ice-cold 22Na-free Krebs' solution (to clear extracellular space). The radioactivity remaining in the ganglia was taken as a measure of 22Na influx into the intracellular compartment of the ganglion. 2. Addition of cholinomimetics (100 microM nicotine or 100 microM carbachol) to the incubation led to an increase in 22Na influx. This increase reached maximal values after 10 min of incubation; it was more pronounced after incubation in Krebs' solution than in BGJb medium. 3. While chlorisondamine (3 microM) or dopamine (100 microM) greatly reduced the carbachol-induced 22Na influx, tetrodotoxin (2 microM) did not have any effect. 4. In ganglia obtained from animals treated with 6-hydroxydopamine in the early postnatal phase (resulting in an extensive destruction of peripheral sympathetic neurons) neither carbachol (100 microM) nor nicotine (100 microM) produced an increase in 22Na influx demonstrating that the intraneuronal compartment is responsible for this enhanced influx. 5. The effects of dopamine, chlorisondamine and tetrodotoxin on the carbachol-induced 22Na uptake into superior cervical ganglia are similar to their effects on carbachol-mediated induction of tyrosine hydroxylase in superior cervical ganglia kept in tissue culture (Thoenen and Otten 1977b). It is concluded that the induction of tyrosine hydroxylase via nicotinic receptors is closely linked to the enhanced sodium influx into the adrenergic neurons mediated by the same receptors.

We have established a continous cell line (G1) in which the tyrosine hydroxylase specific activity is increased as much as 50-100-fold in response to dexamethasone. This response is specific for the glucocorticoid class of steroid hormones; it is elicited by dexamethasone, corticosterone, and triamcinolone, but not by estradiol, testosterone, progesterone, or deoxycorticosterone acetate. The increase in tyrosine hydroxylase specific activity is likely to be due to the increased synthesis of new enzyme protein rather than an activation of existing protein molecules, inasmuch as this increase is completely blocked by cycloheximide.

We have studied the regulation of tyrosine hydroxylase by cell-cell contact in primary cultures of bovine adrenal chromaffin cells. Preparation of dissociated chromaffin cells from bovine adrenal medullae or the harvesting of cultured cells resulted in a rapid decrease of the specific mRNA(TH) (defined as the amount of mRNA(TH) (where TH represents tyrosine hydroxylase) per microgram of total RNA). The decrease in mRNA(TH) levels appears to be stimulated by the loss of cell contact, as it occurs much more rapidly than would be expected from the turnover rate of mRNA(TH) in cultured cells. Similarly an enhanced rate of tyrosine hydroxylase enzyme degradation was observed in chromaffin cells when brought into low contact cultures. In contrast to the decrease in mRNA(TH) levels, however, the decrease in tyrosine hydroxylase enzyme levels was not so rapid and could be prevented in high contact cultures. The mRNA(TH) increased 4-fold in high contact cultures relative to dissociated cells within 1 day after plating. Similarly the rate of synthesis of tyrosine hydroxylase enzyme molecules was maximal after 1 day, although the increase in the absolute amount of tyrosine hydroxylase occurred only slowly. The increase in specific mRNA(TH) by cell contact was inhibited by alpha-amanitin, indicating that cell contact evokes an increase in tyrosine hydroxylase levels by increasing the transcription of mRNA(TH), in addition to inhibiting the degradation of tyrosine hydroxylase molecules.

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.

The trans-synaptic induction of tyrosine hydroxylase [tyrosine 3-monooxygenase; EC 1.14.16.2, L-tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating)] in adrenal medulla and sympathetic ganglia by short-term (1-2 hr) cold stress (4 degrees) exhibits a circadian rhythm which seems to be causally related to the diurnal changes in adrenal glucocorticoid synthesis. In induction is maximal during the morning hours, when plasma corticoid concentrations (reflecting corticoid synthesis in the adrenal cortex) are minimal. In contrast, initiation of tyrosine hydroxylase induction in sympathetic ganglia is only possible in the afternoon. These observations suggest that tyrosine hydroxylase inducibility in the adrenal medulla is optimal during periods of low corticoid synthesis (the adrenal medulla is exposed to excessively high corticoid concentrations directly originating from the adjacent cortex), whereas in sympathetic ganglia an induction is only possible during the period of high plasma corticoid concentrations. This assumption is supported by the observation that in the first postnatal weeks, when the pituitary--adrenocortical system is not yet operating and plasma corticoid concentrations are low, initiation of tyrosine hydroxylase induction in the adrenal medulla is possible at any time of the day, whereas in sympathetic ganglia it is not possible at all. However, after administration of glycocorticoids initiation of tyrosine hydroxylase induction by short-term cold stress is also possible in newborn animals and in adults during the morning hours. The importance of glucocorticoids as modulators for the initiation of trans-synaptic tyrosine hydroxylase induction can also be deduced from the observation that in sympathetic ganglia kept in organ cultures and induction of the hydroxylase by cholinomimetics is only possible when glycocorticoids are added to the culture medium.

As a first step towards the identification and purification of the molecule(s) that are involved in cell contact-mediated tyrosine hydroxylase (TH) induction in cultures of bovine adrenal chromaffin cells, we have prepared plasma membranes (PM) from bovine adrenal medulla and tested their ability to mimick cell contact-mediated TH induction in low density chromaffin cultures. PM indeed induced TH in a manner similar to that observed in high density cultures. The maximal TH induction reached by PM corresponded to 69% of that of high density cultures, and half-maximal TH induction was obtained with 12 micrograms of PM per ml of medium. The induction of TH by PM was blocked by alpha-amanitin as observed in high density cultures. Since acetylcholinesterase was neither induced in high density nor in PM-treated low density cultures, an induction of TH as a result of a general increase in protein synthesis was excluded. The cell contact molecule(s) appear to be intrinsic membrane proteins. They were not removed by high or low salt extraction, but solubilized by 50 mM octylglucoside. They were resistant to 0.1% trypsin and heat denaturation but inactivated by 0.01% chymotrypsin. PM isolated from the adrenal cortex, kidney, and liver also induced TH in low density chromaffin cell cultures, although to a smaller extent than PM of the adrenal medulla. In contrast, muscle and erythrocyte PM were inactive. This shows that the cell contact molecule(s) are not restricted to the adrenal medulla, but are also present in some other but not all tissues.

It has previously been shown that cell density increases the specific enzyme activity of tyrosine hydroxylase (TH) in cultures of PC12 cells. The difference in TH activity was shown to be due to cell-cell contact rather than to diffusible factors released by the PC12 cells (C. Lucas, D. Edgar, and H. Thoenen (1979) Exp. Cell Res. 121, 79-86). We have extended these studies and demonstrated that TH is regulated at the transcriptional level. Cells were harvested from confluent high-density cultures and replated at either low (2 x 10(4) cells/cm2) or high density (5 x 10(5) cells/cm2). At low density, the mRNATH and TH enzyme activity decreased to low levels within several hours. The decrease in TH activity resulting from the loss of cell-cell contact appears to be an active process, as it occurs much more rapidly than would be expected from the turnover rate of the protein in cells cultured continuously at high density. In contrast to low-density cultures the mRNATH and TH activity levels in replated high-density cultures decreased only slightly and then increased four fold at the mRNATH and five fold at the TH activity levels as compared to low-density cultures 2 days after replating. The increase in mRNATH preceded the increase in TH activity by 1 day. Since alpha-amanitin inhibited the increase in TH levels, we conclude that cell-cell contact regulates TH in PC12 cells at the transcriptional level.

Exposure of rat sympathetic ganglia to 3 microgram/ml of 2.5 S nerve growth factor (NGF) resulted in a 100% increase in tyrosine hydroxylase activity within 48 h. Pulselabeling of proteins with [3H]leucine, followed by immunoprecipitation with antibodies to tyrosine hydorxylase and isolation of the precipitated enzyme by gel electrophoresis, demonstrated that the increase in tyrosine hydroxylase activity was due to enhanced de novo synthesis. The incorporation of [3H]leucine into tyrosine hydroxylase was increased by 150% compared to a 17% increase in total protein synthesis, which was not statistically significant. The fact that the half-life of pulse-labeled tyrosine hydroxylase was the same for NGF-treated and control organ cultures of superior cervical ganglia excludes the possibility that enhanced tyrosine hydroxylase labeling by NGF is due to decreased degradation. We conclude that, without modulatory factors which play a role in vivo, NGF can enhance the synthesis of tyrosine hydroxylase in sympathetic ganglia in vitro, provided organ culture conditions which permit optimal survival of adrenergic neurons are selected.

The previously obtained cDNAs coding for bovine tyrosine hydroxylase (TH) mRNA (mRNATH) were further analyzed, and the entire nucleotide sequence was determined. The mRNATH consists of 1,706 nucleotides with an open reading frame for 491 amino acids, which corresponds to a calculated molecular weight of 55,011. The predicted amino acid sequence of bovine TH is compared with that of rat TH and shows a similarity of 66% in the amino terminal (amino acids 1-157) and 91% in the carboxy terminal (amino acids 158-491) region of the TH protein molecule. The carboxy terminal region has been shown to make up the catalytic site of TH and, therefore, is conserved to a greater extent in different species than the amino terminal region, which has been shown to be mainly responsible for the regulation of the catalytic activity of TH. Three of the four serine residues (Ser 8, 19, and 40) that have been shown to be substrates for various protein kinases in rat TH are also present in bovine TH and are located near the amino terminal end of the molecule. The amino acids from position 60 to position 66 of rat TH are not present in bovine TH, resulting in the absence of a predicted hydrophobic region as compared with rat TH. This difference could result in an altered degree of regulation by posttranslational phosphorylation and also association to cell organelle membranes of bovine TH as compared with rat TH.

Selective, highly efficient uptake of [125I]NGF by nerve terminals followed by retrograde axonal transport, and specific induction of tyrosine hydroxylase by NGF are well known phenomena in peripheral adrenergic neurons of adult rats. In the present study these parameters were used in order to detect possible interactions of NGF with central catecholaminergic neurons. No selective retrograde transport of [125I]NGF could be detected by light microscopic autoradiography from the caudate nucleus to the dopaminergic neurons in the substantia nigra or from the hippocampus to the noradrenergic nerve cells of the locus coeruleus. Biochemically, no change in tyrosine hydroxylase activity could be observed for up to 3 days after injection of either NGF, anti-NGF antibodies, or control proteins close to the nerve cell bodies in the substantia nigra or the locus coeruleus. These data suggest a fundamental difference between central and peripheral adrenergic neurons with regard to their responsiveness of NGF.

To decide whether adenosine 3':5'-cyclic monophosphate (cyclic AMP) plays a role as a second messenger in the trans-synaptic induction of tyrosine 3-monooxygenase (EC 1.14.16.2), it is desirable to discriminate between neuronal and extraneuronal changes in cyclic AMP concentration. Treatment of newborn rats with nerve growth factor antiserum or 6-hydroxydopamine, leading to destruction of 61-85% of the adrenergic nerve cell bodies in the superior cervical ganglion, led to a decrease in cyclic AMP of only 16-28%. This observation demonstrates that a relatively small portion of cyclic AMP is localized in the adrenergic neurons. However, administration of isoproterenol produced an increase (12-fold) in cyclic AMP only in this neuronal pool. Neither single nor repeated injections of isoproterenol led to induction of tyrosine monoxygenase. This, together with previous observations that experimental conditions leading to induction of the enzyme do not produce significant increases of cyclic AMP in the whole ganglion, is taken as an indication that cyclic AMP is not acting as a second messenger in the trans-synaptic induction of tyrosine monooxygenase in the rat superior cervical ganglion. In the rat adrenal medulla, treatment with reserpine led to both a shortlasting (60-90 min) increase in cyclic AMP and a subsequent induction of tyrosine monooxygenase. However, the increase in cyclic AMP was almost completely prevented (40 compared to 320%) by pretreatment of the rats with propranolol while the induction of tyrosine monooxygenase was not diminished. This observation also argues against an exclusive key-function of cyclic AMP in trans-synaptic induction of tyrosine monooxygenase in the adrenal medulla.

It was the aim of the present study to elucidate the mechanisms involved in specific tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) induction by potassium depolarization and cholinomimetics in rat superior cervical ganglia kept in organ culture. The effect of high (54 mM) potassium concentration on intact ganglia seems to result in a dual action: a) a specific induction of TH and DBH via release of acetylcholine from preganglionic cholinergic nerve terminals. b) a non-specific effect on terminal adrenergic neurons resulting in a general increase of protein synthesis as indicated by the increase in DOPA decarboxylase (DDC) and monoamine oxidase (MAO) activities. In decentralized superior cervical ganglia potassium depolarization failed to produce the specific TH and DBH induction although a small increase in DDC activity persisted. Carbamylcholine, acetylcholine and nicotine at concentrations of 10(-4) M elicited a selective induction of TH and DBH both in intact and decentralized ganglia via nicotinic receptor stimulation. Bethanechol, predominantly stimulating muscarinic receptors had no significant effect on TH activity. A 4 h pulse of 10(-4) M carbamylcholine produced optimal induction of DBH and TH 24 h and 48 h later respectively. Longer exposure to carbamylcholine resulted in a significantly smaller rise in TH activity.

We have previously shown that primary cultures of calf chromaffin cells respond to nerve growth factor (NGF) treatment with a selective induction of tyrosine hydroxylase (TH), which takes 48 h to be manifested. In the present study, we report that short exposure of calf chromaffin cells to NGF (5-60 min) results in TH activation, which involves a change in the Vmax of the enzyme with no change in the number of enzyme molecules, similar to an effect that has been previously reported in PC12 cells. This activation is markedly potentiated when the chromaffin cells are plated on a laminin substrate, such that after 5 min of NGF exposure, there is an approximately fourfold increase in the TH activity. Both short-term activation and long-term TH induction brought about by NGF treatment are blocked by 5'-deoxy-5'-methylthioadenosine and other drugs that act as S-adenosylhomocysteine (SAH) hydrolase inhibitors to block methylation by end-product inhibition. These drugs did not inhibit cyclic AMP-mediated TH activation or increases in the levels of TH. However, measurements of the degree of blockade of methylation in cells treated with these drugs, taken together with conceptual information regarding the nonregulatory nature of methylation in eukaryotic cells, were not consistent with inhibition of methylation as the crucial effect of the drugs to block the effects of NGF. Nonetheless, since SAH hydrolase inhibitors selectively inhibited NGF-mediated effects, and not comparable effects triggered by other stimuli, these compounds provide useful tools in future studies of the biochemical signalling mechanism of NGF.

Concomitant daily treatment of newborn rats for a 2-week to 1-month period with 10 mug/g of body weight of nerve growth factor and 100 mug/g of body weight of 6-hydroxydopamine produces in the cell bodies of adrenergic neurons the characteristic effects of the growth factor but in the nerve terminals the characteristic effects of 6-hydroxydopamine. The dual opposite effects result in a striking volume increase of sympathetic ganglia which far exceeds that produced by nerve growth factor alone. The selective induction of tyrosine hydroxylase [L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] in these chemically axotomized adrenergic neurons is even more pronounced than that produced by nerve growth factor alone in intact neurons.

Administration of NGF to newborn and adult rats elicits a selective increase in TH and DBH both in sympathetic ganglia and adrenal medulla. This effect does not depend on intact preganglionic cholinergic fibers. The augmented enzyme activity results from enhanced enzyme synthesis since it can be abolished by cycloheximide and NGF has been shown to enhance the incorporation of [3H]leucine into DBH molecules. The responsiveness of the adrenal medulla to NGF is also supported by light and electron microscopic autoradiograms which show that intravenously injected 125I-NGF is accumulated with high selectivity in adrenal chromaffin as compared to adjacent adrenal cortical cells. In spite of the many similarities between the response of the adrenergic neurons and adrenal chromaffin cells to NGF, there are also two distinct differences. (a) In newborn rats the ratio between the TH increase effected by a single and 10 subsequent daily injections of NGF is 1:2 in the adrenal medulla and 1:7 in the superior cervical ganglia. (b) If adrenal medullae are transferred to organ culture after intravenous injection of NGF, maximal TH response is initiated 60-90 min after NGF administration. In superior cervical ganglia only a half-maximal response is initiated at that time. After a stationary phase a second increase starts after about 6 h to reach the maximum after 12 h. The biphasic time course of the initiation of TH induction by NGF in sympathetic ganglia is in agreement with the time course of 125I-NGF accumulation after intravenous injection27 reflecting the moiety of NGF reaching the cell bodies of the adrenergic neurons directly by the blood stream (initial accumulation) and by retrograde axonal transport (second phase).

The specific activity of tyrosine hydroxylase (TH) in bovine adrenal chromaffin cells can be controlled by changing cell density. Chromaffin cells initially plated at low density (2-3 X 10(4) cells/cm2), and subsequently replated at a 10-fold higher density showed a sixfold increase in specific TH activity within 48 h, resulting from enhanced synthesis (increased number of TH molecules as demonstrated by immunotitration and blockade by cycloheximide) rather than activation. The density-mediated TH induction was blocked by inhibitors of both messenger RNA synthesis (alpha-amanitin) and processing (9-beta-arabinofuranosyladenine), indicating a transcriptional level of regulation. Medium conditioned by high density replated cells could not mimic the effect of high density plating itself, thus direct cell contact, rather than a diffusible factor, is responsible for the density-mediated TH induction. Since neither acetylcholinesterase nor lactate dehydrogenase specific activities were increased by high cell density, it can be concluded that the contact-mediated induction of TH is rather specific, and not the result of a general process of enzyme induction.