Accumulation of Enkephalin, Proenkephalin mRNA, and Neuropeptide Y in Immunologically Denervated Rat Adrenal Glands: Evidence for Divergent Peptide Regulation

Abstract: To investigate transsynaptic effects on peptides of adrenal chromaffin cells in the rat, presynaptic sympathetic terminals were destroyed by intravenous injection of monoclonal antibodies to acetylcholinesterase. At several times thereafter, neuropeptide Y (NPY)-like immunoreactivity (NPY-IR) and methionine-enkephalin-like immunoreactivity (Met-Enk-IR) were measured by radioimmunoassay. Within 2 days of antibody injection, adrenal Met-Enk-IR increased five- to 10-fold and NPY-IR increased 50%. These effects were accompanied by large increases in proenkephalin A mRNA assayed by polymerase chain reaction. The peptide responses could reflect either an acute activation, as presynaptic terminals degenerated, or a chronic synaptic inactivation after terminal degeneration. To test the possibilities, muscarinic and nicotinic receptors were inhibited by repeated injection of atropine (1 mg/kg) and chlorisondamine (5 mg/kg). Measurements of urinary free catecholamine excretion showed that this treatment prevented the paroxysmal release of norepinephrine and reduced the release of epinephrine that normally followed injection of acetylcholinesterase antibodies. When the drugs were given alone for 2 or 4 days, adrenal Met-Enk-IR increased modestly and NPY-IR remained steady or declined. When given together with acetylcholinesterase antibodies, the cholinergic antagonists blocked the increase of NPY-IR but not Met-Enk-IR. Adding naloxone (1 mg/kg) to the treatment regimen enhanced the blockade of epinephrine excretion and largely prevented the antibody-induced increase in Met-Enk-IR. These findings indicate that adrenal NPY and enkephalin are not regulated identically. Adrenal NPY behaves as though controlled by transsynaptic cholinergic input. On the other hand, adrenal enkephalin may be regulated by additional or different mechanisms, possibly involving peptidergic transmission or synaptic inactivation.     

Regulation of Phenylethanolamine N-Methyltransferase Gene Expression by Imidazoline Receptors in Adrenal Chromaffin Cells

Abstract: As adrenal medullary chromaffin cells express imidazoline binding sites in the absence of α₂-adrenergic receptors, these cells provide an ideal system in which to determine whether imidazolines can influence catecholamine gene expression through nonadrenergic receptors. This study evaluates the ability of clonidine and related drugs to regulate expression of the gene for the epinephrine-synthesizing enzyme phenylethanolamine N -methyltransferase (PNMT) in the rat adrenal gland and in bovine adrenal chromaffin cell cultures. In vivo, PNMT and tyrosine hydroxylase (TH) mRNA levels increase in rat adrenal medulla after a single injection of clonidine. Clonidine also dose-dependently stimulates PNMT mRNA expression in vitro in primary cultures of bovine chromaffin cells, with a threshold dose of 0.1 μ M . Other putative imidazoline receptor agonists, including cimetidine, rilmenidine, and imidazole-4-acetic acid, likewise enhance PNMT mRNA production showing relative potencies that correlate with their binding affinities at chromaffin cell I₁-imidazoline binding sites. The effects of clonidine on PNMT mRNA appear to be distinct from and additive with those exerted by nicotine. Moreover, neither nicotinic antagonists nor calcium channel blockers, which attenuate nicotine's influence on PNMT mRNA production, diminish clonidine's effects on PNMT mRNA. Although 100 μ M clonidine diminishes nicotine-stimulated release of epinephrine and norepinephrine in chromaffin cells, this effect appears unrelated to stimulation of imidazoline receptor subtypes. This is the first report to link imidazoline receptors to neurotransmitter gene expression.     

Induction of Adrenal Tyrosine Hydroxylase mRNA by Single Immobilization Stress Occurs Even After Splanchnic Transection and in the Presence of Cholinergic Antagonists

Abstract: Immobilization (IMO) stress elevates plasma catecholamines and increases tyrosine hydroxylase (TH) gene expression in rat adrenals. This study examined the mechanism(s) of IMO-induced changes in adrenal TH mRNA levels. Innervation of the adrenal medulla is predominantly cholinergic and splanchnicotomy as well as nicotinic receptor antagonists prevent the cold-induced rise in TH mRNA levels. In this study, the IMO-induced rise in plasma catecholamines, but not TH mRNA levels, was reduced by the antagonist chlorisondamine. Muscarinic antagonist atropine also did not prevent the IMO stress-elicited rise in TH mRNA. Furthermore, denervation of the adrenals by unilateral splanchnicotomy did not block the IMO-induced rise in TH mRNA but completely prevented the induction of neuropeptide Y mRNA. These results suggest that (1) the large increase in adrenal TH gene expression elicited by a single IMO stress is not regulated via cholinergic receptors or splanchnic innervation, and (2) there is a dissociation between regulatory mechanisms of catecholamine secretion and elevation of TH gene expression in the adrenal medulla of rats during IMO stress.     

Muscarinic receptors on bovine chromaffin cells mediate a rise in cytosolic calcium that is independent of extracellular calcium

Although the mechanism by which nicotinic receptors on adrenal chromaffin cells regulate catecholamine secretion is reasonably well understood, that of the muscarinic receptors remains obscure. The effects of both acetylcholine and specific muscarinic agonists on cytosolic free calcium in isolated bovine adrenal chromaffin cells have been measured using the fluorescent probe Quin-2. Acetylcholine (0.1 mM) evokes a large increase in cytosolic free calcium from resting levels near 100 nM into the microM range, most of which is blocked by hexamethonium (0.5 mM) or removal of extracellular calcium. A small component of the acetylcholine-evoked rise in cytosolic free calcium (approximately 50-100 nM) is independent of extracellular calcium and is unaffected by 0.5 mM hexamethonium, but is totally blocked by 0.5 microM atropine. The muscarinic nature of this component is further confirmed by the fact that the muscarinic agonists, muscarine (0.1 mM) and methacholine (0.3 mM), stimulate a 50-100 nM rise in chromaffin cell cytosolic calcium which is blocked by 0.5 microM atropine and is largely independent of extracellular calcium. These results suggest that muscarinic receptors regulate cytosolic calcium in chromaffin cells by a new mechanism different from that of nicotinic receptors, a mechanism utilizing an intracellular calcium source. The small size of the muscarinic-induced rise in cytosolic calcium in the bovine chromaffin cell would explain why no secretion is evoked by muscarinic agonists in this species.     

Cold-induced increases in phenylethanolamine N-methyltransferase (PNMT) mRNA are mediated by non-cholinergic mechanisms in the rat adrenal gland

Previously, we reported that cold stress induces a rapid increase in adrenomedullary PNMT mRNA levels, followed by concomitant increases in PNMT immunoreactivity (10). In the present study, the extracellular signals mediating this adaptive response to stress were investigated using northern analysis and RNA slot-blot hybridization. Although adrenal denervation significantly diminished cold-induced increments in adrenomedullary PNMT mRNA levels, it did not completely abolish the cold stress response. In contrast to these results, splanchnectomy completely inhibited cold-induced increments in TH mRNAs in the same tissue samples. These findings indicate that the effects of cold exposure on PNMT mRNA levels are mediated by both neural and non-neural mechanisms, and that adrenal PNMT and TH are differentially regulated in response to cold stress. Surprisingly, the neural component of the PNMT stress response could not be attenuated by peripheral administration of chlorisondamine, a powerful nicotinic ganglionic blocking agent. In contrast, chlorisondamine was effective in inhibiting sympathetic neural activity, as judged by the drug's ability to completely block increases in blood pressure, heart rate, and plasma catecholamines resulting from spinal cord stimulation in pithed rats. The administration of atropine, a muscarinic receptor antagonist, also failed to inhibit cold-induced alterations in adrenal PNMT mRNA. These results suggest that the trans-synaptic induction of adrenal PNMT mRNA involves a non-cholinergic component, and that cold-induced increases in PNMT mRNA are not coupled to acetylcholine-mediated adrenal catecholamine release.     

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.     

Activation of Tyrosine Hydroxylase in the Superior Cervical Ganglion by Nicotinic and Muscarinic Agonists

Both dimethylphenylpiperazinium (DMPP), a nicotinic agonist, and bethanechol, a muscarinic agonist, increase 3,4-dihydroxyphenylalanine (DOPA) synthesis in the superior cervical ganglion of the rat. DMPP causes approximately a fivefold increase in DOPA accumulation in intact ganglia whereas bethanechol causes about a two-fold increase in DOPA accumulation. These effects are additive with each other and with the increase in DOPA accumulation produced by 8-bromo cyclic AMP. The action of DMPP is dependent on extracellular Ca2+ while the actions of bethanechol and 8-bromo cyclic AMP are not dependent on extracellular Ca2+. Cholinergic agonists and cyclic nucleotides produce a stable activation of tyrosine hydroxylase (TH) in the ganglion. The activation of TH by nicotinic and muscarinic agonists can be detected after 5 min of incubation of the ganglia with these agents. The nicotinic response disappears after 30 min of incubation, whereas the muscarinic response persists for at least 30 min. The Ca2+ dependence of the TH activation produced by these agents is similar to the Ca2+ dependence of their effects on DOPA accumulation in intact ganglia. These data are consistent with the hypothesis that nicotinic agonists, muscarinic agonists, and cyclic AMP analogues increase TH activity by three distinct mechanisms. The activation of TH presumably underlies the increase in DOPA synthesis produced by these agents.     

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

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

Cholinergic Stimulation of Inositol Phosphate Formation in Bovine Adrenal Chromaffin Cells: Distinct Nicotinic and Muscarinic Mechanisms

The ability of cholinergic agonists to activate phospholipase C in bovine adrenal chromaffin cells was examined by assaying the production of inositol phosphates in cells prelabeled with [3H]inositol. We found that both nicotinic and muscarinic agonists increased the accumulation of [3H]inositol phosphates (mainly inositol monophosphate) and that the effects mediated by the two types of receptors were independent of each other. The production of inositol phosphates by nicotinic stimulation required extracellular Ca2+ and was maximal at 0.2 mM Ca2+. Increasing extracellular Ca2+ from 0.22 to 2.2 mM increased the sensitivity of inositol phosphates formation to stimulation by submaximal concentrations of 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) but did not enhance the response to muscarine. Elevated K+ also stimulated Ca2+-dependent [3H]inositol phosphate production, presumably by a non-receptor-mediated mechanism. The Ca2+ channel antagonists D600 and nifedipine inhibited the effects of DMPP and elevated K+ to a greater extent than that of muscarine. Ca2+ (0.3-10 microM) directly stimulated the release of inositol phosphates from digitonin-permeabilized cells that had been prelabeled with [3H]inositol. Thus, cholinergic stimulation of bovine adrenal chromaffin cells results in the activation of phospholipase C by distinct muscarinic and nicotinic mechanisms. Nicotinic receptor stimulation and elevated K+ probably increased the accumulation of inositol phosphates through Ca2+ influx and a rise in cytosolic Ca2+. Because Ba2+ caused catecholamine secretion but did not enhance the formation of inositol phosphates, phospholipase C activation is not required for exocytosis. However, diglyceride and myo-inositol 1,4,5-trisphosphate produced during cholinergic stimulation of chromaffin cells may modulate secretion and other cellular processes by activating protein kinase C and/or releasing Ca2+ from intracellular stores.     

Selective induction of tyrosine hydroxylase by cell-cell contact in bovine adrenal chromaffin cells is mimicked by plasma membranes

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.     

Leptin directly stimulates catecholamine secretion and synthesis in cultured porcine adrenal medullary chromaffin cells.

Leptin, a protein encoded by the ob gene, is an adipose tissue-derived signaling factor involved in body weight homeostasis. The hypothalamus is a major site of central action for leptin. However, mounting evidence indicates expression of leptin receptor mRNA in various peripheral organs including the adrenal medulla. Therefore, we investigated the effects of leptin on catecholamine secretion and synthesis in cultured porcine adrenal medullary chromaffin cells. We initially confirmed the expression of leptin receptor (Ob-Rb) mRNA in cultured porcine adrenal medullary cells. Murine recombinant leptin (>==50 nM) strongly induced the release of both epinephrine (E) and norepinephrine (NE) from chromaffin cells. Removal of external Ca(2+) significantly suppressed these effects. Also, leptin (>==1 nM) enhanced nicotine-induced increases in E- and NE. Leptin (1, 10, 100 nM) significantly increased tyrosine hydroxylase (TH) (a rate-limiting enzyme in the biosynthesis of catecholamine) mRNA levels in a concentration-dependent manner. Furthermore, leptin (1, 10, 100 nM) significantly induced increases in cAMP levels, suggesting that the stimulatory effects on TH mRNA are mediated, at least in part, by the cAMP/protein kinase A pathway. These results indicate that leptin directly stimulates catecholamine release and synthesis, which in turn may potentiate the anti-obesity effects of leptin.     

PACAP stimulates the sustained phosphorylation of tyrosine hydroxylase at serine 40

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine synthesis. Its activity is controlled by PACAP, acutely by phosphorylation at Ser40 and chronically by protein synthesis. Using bovine adrenal chromaffin cells we found that PACAP, acting via the continuous activation of PACAP 1 receptors, sustained the phosphorylation of TH at Ser40 and led to TH activation for up to 24 h in the absence of TH protein synthesis. The sustained phosphorylation of TH at Ser40 was not mediated by hierarchical phosphorylation of TH at either Ser19 or Ser31. PACAP caused sustained activation of PKA, but did not sustain activation of other protein kinases including ERK, p38 kinase, PKC, MAPKAPK2 and MSK1. The PKA inhibitor H89 substantially inhibited the acute and the sustained phosphorylation of TH mediated by PACAP. PACAP also inhibited the activity of PP2A and PP2C at 24 h. PACAP therefore sustained TH phosphorylation at Ser40 for 24 h by sustaining the activation of PKA and causing inactivation of Ser40 phosphatases. The PKA activator 8-CPT-6Phe-cAMP also caused sustained phosphorylation of TH at Ser40 that was inhibited by the PKA inhibitor H89. Using cyclic AMP agonist pairs we found that sustained phosphorylation of TH was due to both the RI and the RII isotypes of PKA. The sustained activation of TH that occurred as a result of TH phosphorylation at Ser40 could maintain the synthesis of catecholamines without the need for further stimulus of the adrenal cells or increased TH protein synthesis.     

Sustained phosphorylation of tyrosine hydroxylase at serine 40: a novel mechanism for maintenance of catecholamine synthesis

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine synthesis. Its activity is known to be controlled acutely (minutes) by phosphorylation and chronically (days) by protein synthesis. Using bovine adrenal chromaffin cells we found that nicotine, acting via nicotinic receptors, sustained the phosphorylation of TH at Ser40 for up to 48 h. Nicotine also induced sustained activation of TH, which for the first 24 h was completely independent of TH protein synthesis, and the phosphorylation of TH at Ser31. Imipramine did not inhibit the acute phosphorylation of TH at Ser40 or TH activation induced by nicotine, but did inhibit the sustained responses to nicotine seen at 24 h. The protein kinase(s) responsible for TH phosphorylation at Ser40 switched from being protein kinase C (PKC) independent in the acute phase to PKC dependent in the sustained phase. Sustained phosphorylation and activation of TH were also observed with histamine and angiotensin II. Sustained phosphorylation of TH at Ser40 provides a novel mechanism for increasing TH activity and this leads to increased catecholamine synthesis. Sustained phosphorylation of TH may be a selective target for drugs or pathology in neurons that contain TH and synthesize dopamine, noradrenaline or adrenaline.