Emanuel J. Diliberto

Secretion of Catecholamines from Individual Adrenal Medullary Chromaffin Cells

Abstract: Catecholamine secretion has been measured with electrochemical techniques from isolated, single adrenal medullary chromaffin cells with carbon‐fiber microelectrodes. The electrode tip, which is of similar dimensions to the cell, is placed adjacent to the cell to enable the measurement of local secretion. Secretion is caused by exposing the cell to nanoliter volumes of solution containing nicotinic receptor agonists or depolarizing agents. The identification of secreted substances is made with cyclic voltammetry at both bare electrodes and electrodes coated with a perfluorinated cationexchange polymer. Catecholamine secretion is induced by nicotine (10–500 μM), carbamylcholine (1 mM), and K+ (60 mM). All agents that induce secretion lead to a broad envelope of secreted catecholamines on which sharp concentration spikes are superimposed. The concentration spikes can be monitored with a time resolution of tens of milliseconds when the electrodes are used in the amperometric mode. Release induced by nicotine and K+ is inhibited by Cd2+ (0.5 mM), and hexamethonium selectively blocks the nicotineinduced secretion. The actions of nicotine are found to continue for a longer period of time than those of the other secretagogues tested.

Tissue, Subcellular, and Submitochondrial Distributions of Semidehydroascorbate Reductase: Possible Role of Semidehydroascorbate Reductase in Cofactor Regeneration

Abstract: The immediate product of ascorbate oxidation coupled to dopamine‐β‐hydroxylation is not dehydroascorbate, as previously thought, but rather semidehydroascorbate. For this reason, the possible participation of the enzyme semidehydroascorbate reductase (SDR) in cofactor regeneration was investigated. In the adrenal medulla, the primary subcellular localization of this reductase was shown to be in the mitochondria. Submitochondrial fractionation studies indicated that SDR is an outer membrane protein. Thus, although dopamine‐β‐hydroxylase and SDR have different subcellular localizations, a physiological role for SDR in β‐hydroxylation still appears plausible through reduction of cytosolic semidehydroascorbate. The specific activities of SDR in various rat and guinea pig tissues appear to parallel their ascorbate contents, suggesting a similar participation of SDR in ascorbate metabolism in other tissues.

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.

Reflex Splanchnic Nerve Stimulation Increases Levels of Carboxypeptidase E mRNA and Enzymatic Activity in the Rat Adrenal Medulla

Carboxypeptidase E (CPE; EC 3.4.17.10) is a carboxypeptidase B‐like enzyme involved with the biosynthesis of numerous peptide hormones and neurotransmitters, including the enkephalins. Reflex splanchnic stimulation of the rat adrenal medulla, which has previously been found to substantially increase enkephalin mRNA and enkephalin peptide levels, was examined for an influence on CPE mRNA and enzymatic activity. Several hours after insulin‐induced reflex splanchnic stimulation, the levels of CPE activity in rat adrenal medulla are reduced to 40–60% of control. CPE activity returns to the control level 2 days after the treatment and then continues to increase, reaching ˜200% of control 1 week after the treatment. The time course of the changes in CPE activity is different from those of the changes in epinephrine levels and the previously reported changes in enkephalin peptide levels. CPE mRNA is also influenced by the insulin shock, with levels increasing to 155% of the control level after 6 h and 170% after 2 days. The time course of the change in CPE mRNA levels is similar to that previously found for proenkephalin mRNA. However, the magnitude of the change is much different: Proenkephalin mRNA has been reported to increase by 1,600%. The changes in CPE mRNA and enzymatic activity are consistent with the proposal that CPE is not a rate‐limiting enzyme in the biosynthesis of enkephalin.

Characterization of Cellular Transport, Subcellular Distribution, and Secretion of the Neurotoxicant 1‐Methyl‐4‐Phenylpyridinium in Bovine Adrenomedullary Cell Cultures

Abstract Cultures of bovine adrenomedullary chromaffin cells accumulated l‐[methyl‐3H]methyl‐4‐phenylpyridinium ([3H]MPP+) in a time‐ and concentration‐dependent manner with an apparent Km of 0.7 μM and a Vmax of 3 pmol/min/106 cells. The uptake was sodium dependent and sensitive to inhibitors of the cell‐surface catecholamine transporter. At low concentrations of MPP+, the subcellular distribution was identical to that of endogenous catecholamines in the catecholamine‐containing chromaffin vesicles. However, at a higher concentration of MPP+, a larger proportion of the toxicant was recovered in the cytosolic fraction, with less in the chromaffin vesicle fractions. When cells were prelabeled with [3H]MPP+, at 1 and 300 μM, and then permeabilized with digitonin in the absence of Ca2+, there was a proportionally greater release of MPP+ from the cells labeled at the higher concentration of the toxicant. In the presence of Ca2+, cell permeabilization induced a time‐dependent secretion of catecholamines and a parallel secretion of MPP+. Under these conditions, the secretion of endogenous catecholamines was unaffected by the presence of MPP+. When the permeabilization studies were carried out in the presence of tetrabenazine, a massive release of MPP+ was observed in the absence of Ca2+ and was not further increased by Ca2+. In intact cells prelabeled with 300 μM [3H]MPP+, the secretagogues nicotine and veratridine elicited a Ca2+‐dependent secretion of catecholamines and MPP+ from the cells in similar proportions to their cellular contents. Barium‐induced release of both species was independent of external Ca2+. Collectively, these data reveal that MPP+ is a novel substrate for both the cellular and vesicular catecholamine transporters and that, in intact cells, vesicular transport may become limiting relative to the cell‐surface transporter, leading to the cytosolic accumulation of the toxicant.

Newly synthesized dopamine as the precursor for norepinephrine synthesis in bovine adrenomedullary chromaffin cells.

The precursor pool of dopamine for norepinephrine synthesis was investigated in cultured bovine adrenomedullary chromaffin cells incubated with [14C]tyrosine. Under conditions where the intracellular [14C]tyrosine specific activity was constant and [14C]dopamine synthesis was maximal, [14C]dopamine and [14C]norepinephrine accumulated over time, and the total intracellular dopamine content more than doubled within 120 min. When [14C]norepinephrine synthesis was calculated at different times based on the specific activity of [14C]dopamine, this rate was approximately equal to the rate of [l4C]dopamine synthesis and was, thus, inconsistent with the observed dopamine accumulation. However, the rate of [14C]norepinephrine synthesis based on the [l4C]tyrosine specific activity accounted for the dopamine accumulation, an observation suggesting that newly synthesized dopamine, i.e., dopamine with a specific activity equivalent to that of its precursor, [14C]tyrosine, is preferentially utilized for norepinephrine synthesis. Further studies showed that the subcellular distribution of [14C]dopamine was identical to that of norepinephrine and epinephrine and that the accumulated [14C]dopamine could be converted to norepinephrine within the chromaffin vesicle if dopamine uptake was blocked. Taken together, these results suggest that a small intravesicular dopamine pool, rapidly replenished by newly synthesized dopamine, serves as the substrate for dopamine β‐hydroxylase. Several mechanisms to account for this observation are discussed.

A novel tetradecapeptide isolated from bovine adrenal medulla chromaffin vesicles with strong homology to an internal sequence coded by the rat 1B1075 (Preprosecretogranin III) gene.

A novel tetradecapeptide, PheProLysProAlaGlySerGlnAspLysProLeuHisAsn, was isolated from boiling water extracts of bovine adrenal medulla chromaffin vesicles. The primary structure of the peptide was characterized by amino acid analysis, fast atom bombardment mass spectrometry, and gas-phase sequencing. The synthetic and native peptides comigrated on reversed-phase high-performance liquid chromatography, supporting the proposed sequence. This peptide shares 86% homology to residues 67-80 of the recently reported rat 1B1075 gene product secretogranin III and probably represents a processed product derived from the bovine equivalent.

Nicotinic Regulation of Adrenomedullary Opioid Peptide Synthesis and Secretion: A Model to Study Monoamine Neuropeptide Cotransmission

The adrenal medulla chromaffin cell synthesizes and stores in various subcellular compartments a number of proteins, peptides, nucleotides, and other small molecules to be secreted by Ca2+-dependent, nicotine receptor-mediated mechanism(s). Of these proteins and peptides, the enkephalins and other proenkephalin-derived opioid peptides are found in all species examined in substantial amounts, where they are costored with the catecholamines in the chromaffin vesicles. Splanchnic nerve stimulation, nicotine, and other secretagogues induce the cosecretion of these opioid peptides with the amines and other soluble components of these vesicles by the process of exocytosis. Regulatory mechanisms triggered by activation of nicotinic receptors, depolarization, and catecholamine depletion that involve cAMP-dependent and -independent mechanisms control the synthesis of enkephalins at the transcriptional, translational, and peptide processing levels. These mechanisms allow for rapid recovery of the opioid peptide content after secretion and for long-term modulation of the relative proportions and amounts in which catecholamines and enkephalins are costored and cosecreted. Opioid peptides secreted from the adrenal medulla reach ubiquitous opiate receptors throughout the organism and may modulate a number of important systemic functions including behavioral responses to stress. Enkephalins and norepinephrine also coexist in postganglionic sympathetic neurons, and some of the effects of nicotine administration may result from peripheral opiatergic responses through its powerful activation of the sympathetic system. The costorage and cosecretion of opioid peptides and catecholamines is only one of a growing number of examples of coexistence of multiple chemical messengers in single neurons or endocrine cells. This new principle of cotransmission is drastically changing our concepts and understanding of synaptic and endocrine function. Fast and slow dynamic changes in the ratios in which cotransmitters are stored and released and, thus, coact at the effector sites illustrate an unsuspected degree of synaptic plasticity. The exploration of the short-and long-term effects of chronic use of tobacco needs to take into consideration not only the effect of nicotine on classical transmitters but particularly how the biochemistry and function of these central and peripheral cotransmitter systems are being modified.

Initial characterization of multiple endopeptidases in bovine adrenal chromaffin vesicles

All regulatory peptides are synthesized as large, inactive precursor proproteins that must undergo specific endoproteolytic processing to yield bioactive peptides. In most cases, enzymatic release of the biologically active peptides occurs by endoproteolytic cleavage at doublets of basic amino acid residues that precede and/or follow that particular sequence [1,2]. The catecholamine-containing chromaffin vesicles of the adrenal medulla are enriched in a great variety of regulatory peptides (i.e. enkephalins, neurotensin, neuropeptide Y, etc.) and thus are good sources for the isolation and characterization of peptide processing enzymes [3,4,5]. To isolate putative endopeptidases of the thioland serineprotease families, the dialysed lysate of purified bovine chromaffin vesicles was consecutively fractionated through p-chloro-mercuribenzoateagarose (PCMB-agarose), p-aminobenzamidineagarose (p-ABZ-agarose) and soybean trypsin inhibitor-agarose (STI-agarose) affinity columns. Three intermediate proenkephalin precursor peptides (BAM12P, BAM22P and amidorphin) were used as substrates for the assay of endopeptidase activities. These peptides contain pairs of basic amino acids, Arg-Arg, Lys-Arg and Lys-Lys, which have putative cleavage sites for the endopeptidases. Degradation peptide fragments were separated by reverse phase HPLC and identified by FAB mass spectrometry, amino acid analysis and sequence analysis, which was performed with an Applied Biosynthesis model 477a protein sequencer using Edman degradation chemistry. The fraction retained and eluted from PCMB-agarose affinity chromatography hydrolyzed the Arg-Arg sequence of BAM12P, resulting in the generation of Met-enkephalin and Met-enkephalin-Arg at pH 5.7. However, this enzyme preparation was unable to hydrolyze amidorphin at the Lys-Lys pair of basic residues. This activity was inhibited by PCMB and E64, indicating that a thiol protease is involved. The dialysate fraction that was not retained by the PCMB-agarose column was subsequently retained and eluted from the p-ABZagarose affinity column. This dialysate fraction contained enzyme activity which cleaved at the Lys-Arg of BAM22P and at the Lys-Lys of amidorphin at pH 7.4. BAM12P was however a poor substrate for this fraction. This activity was not inhibited by ST1, which is indicative of a non-trypsin-like endopeptidase. Additionally, a separate endopeptidase cleaving at Glu-Trp of BAM22P, resulting in the generation of BAM12P, was also found in this preparation. The dialysate fraction not retained in the first two columns but retained and eluted from the STI-agarose affinity column had an enzyme activity capable of hydrolyzing amidorphin at the carboxy side of Lys-Lys. This activity was completely inhibited by STI which is indicative of a trypsin-like endopeptidase. BAM12P however was poorly cleaved by this preparation. This study demonstrates that a variety of different endopeptidase activities is found in soluble lysates of adrenal medulla chromaffin vesicles. A multiplicity of peptide processing enzymes with different specificities suggests the possibility that modification of a particular processing enzyme may result in specific changes in the cocktail of regulatory peptides.