Steven P. Wilson

The acetylcholine receptor of the adrenal medulla.

Abstract— The acetylcholine receptor of the bovine adrenal medulla was studied by specific binding of [1251]α‐bungarotoxin to membrane fractions and by perfusion of the isolated gland. The subcellular distribution of the acetylcholine receptor paralleled the distribution of the plasma membrane markers, acetylcholinesterase and calciumstimulated ATPase. The dissociation constant for the binding of α‐bungarotoxin to a purified plasma membrane fraction was calculated from Scatchard plots to be 1.6 nM, with a concentration of 190 fmol of binding sites/mg of membrane protein. Correcting for recovery, this corresponds to 0.9 pmol acetylcholine receptor/g adrenal medulla. In decreasing order of effectiveness, d‐tubocurarine, nicotine, acetylcholine, carbamylcholine, acetate plus choline, decamethonium, atropine and hexamethonium inhibited binding of α‐bungarotoxin.

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.

The adrenal chromaffin cell as a model to study the co-secretion of enkephalins and catecholamines.

The enkephalins, endogenous opioid pentapeptides first discovered in brain, are present in high concentrations in the adrenal medulla chromaffin cell. The enkephalins and other peptides containing enkephalin sequences are stored with catecholamines in the secretory organelles (chromaffin vesicles); these peptides are apparently incorporated into the vesicles at the time of their biosynthesis as opposed to later accumulation, as is the case with catecholamines. The enkephalins, catecholamines and other soluble components of the vesicle are co-secreted by the process of exocytosis. Regulatory mechanisms, apparently triggered by a critical catecholamine pool, control the synthesis of enkephalins. These mechanisms allow for rapid recovery of enkephalin content after secretion. These findings have been extended from the chromaffin cell to the ontogenically related sympathetic neurons and pheochromocytoma tumors. Secreted enkephalins and related peptides reach ubiquitous opiate receptors through the synaptic gap or the circulation and may modulate a number of important systemic functions. The co-storage and co-secretion of adrenomedullary opioid peptides and catecholamines is only one of a growing number of examples of co-existence of multiple messengers in single neuronal or endocrine cell types. Co-secreted multiple messengers may act in a co-ordinated fashion to produce integrated organismal responses.

Regulation of Guanosine Triphosphate Cyclohydrolase and Tetrahydrobiopterin Levels and the Role of the Cofactor in Tyrosine Hydroxylation in Primary Cultures of Adrenomedullary Chromaffin Cells

Abstract: Selective modification of the tetrahydrobiopterin levels in cultured chromaffin cells were followed by changes in the rate of tyrosine hydroxylation. Addition of sepiapterin, an intermediate on the salvage pathway for tetrahydrobiopterin synthesis, rapidly increased intracellular levels of tetrahydrobiopterin and elevated the rate of tyrosine hydroxylation in the intact cell. Tyrosine hydroxylation was also enhanced when tetrahydrobiopterin was directly added to the incubation medium of intact cells. When the cultured chromaffin cells were treated for 72 h with N‐acetylserotonin, an inhibitor of sepiapterin reductase, tetrahydrobiopterin content and the rate of tyrosine hydroxylation were decreased. Addition of sepiapterin or N‐acetylserotonin had no consistent effect on total extractable tyrosine hydroxylase activity or on catecholamine content in the cultured chromaffin cells. Three‐day treatment of chromaffin cell cultures with compounds that increase levels of cyclic AMP (forskolin, cholera toxin, theophylline, dibutyryl‐ and 8‐bromo cyclic AMP) increased total extractable tyrosine hydroxylase activity and GTP‐cyclohydrolase, the rate‐limiting enzyme in the biosynthesis of tetrahydrobiopterin. Tetrahydrobiopterin levels and intact cell tyrosine hydroxylation were markedly increased after 8‐bromo cyclic AMP. The increase in GTP‐cyclohydrolase and tetrahydrobiopterin induced by 8‐bromo cyclic AMP was blocked by the protein synthesis inhibitor cycloheximide. Agents that deplete cellular catecholamines (reserpine, tetrabenazine, and brocresine) increased both total tyrosine hydroxylase and GTP‐cyclohydrolase activities, although treating the cultures with reserpine or tetrabenazine resulted in no change in cellular levels of cyclic AMP. Brocresine and tetrabenazine increased tetrahydrobiopterin levels, but the addition of reserpine to the cultures decreased catecholamine and tetrahydrobiopterin content and resulted in a decreased rate of intact cell tyrosine hydroxylation in spite of the increased activity of the total extractable enzyme. These data indicate that in cultured chromaffin cells GTP‐cyclohydrolase activity like tyrosine hydroxylase activity is regulated by both cyclic AMP‐dependent and cyclic AMP‐independent mechanisms and that the intracellular level of tetrahydrobiopterin is one of the many factors that control the rate of tyrosine hydroxylation.

Isolation and characterization of plasma membranes from the adrenal medulla.

Abstract– The isolation of a plasma membrane fraction from the bovine adrenal medulla and its characterization are described. The plasma membranes are enriched 13‐fold in AChE, a plasma membrane marker, and represent 0.7% of the homogenate membrane protein. The yield of these membranes is typically 10‐12% by the criterion of the percentage of total membrane bound AChE in the homogenate. The membranes were characterized as to their polypeptide, phospholipid and cholesterol content and compared with chromaffin vesicle, mitochondrial and microsomal membranes by these parameters.

Relationship Between the Regulation of Enkephalin-Containing Peptide and Dopamine β-Hydroxylase Levels in Cultured Adrenal Chromaffin Cells

Abstract: Adrenal medullary chromaffin cells were maintained under conditions known to increase their cellular levels of enkephalin‐containing peptides and the effects of these treatments on another chromaffin vesicle component, dopamine β‐hydroxylase, were examined. Catecholamine‐depleting drugs, such as tetrabenazine, and cyclic nucleotide‐elevating drugs, including forskolin, 8bromo‐cyclic AMP, and theophylline, increase chromaffin cell enkephalin‐containing peptide levels but fail to increase dopamine β‐hydroxylase. In contrast, insulin treatment increases the levels of both enkephalin‐containing peptides and dopamine β‐hydroxylase. The increased amounts of enkephalin‐containing peptides produced by tetrabenazine and by insulin are stored in subcellular particles with properties identical to chromaffin vesicles on density‐gradient centrifugation. These results suggest that following insulin treatment chromaffin cells synthesize new chromaffin vesicles with a full complement of enkephalin‐containing peptides, but that after treatment with catecholamine‐depleting or cyclic nucleotide‐related agents enkephalin‐containing peptides can be inserted into preexisting vesicles or that new vesicles, made as a part of the normal turnover of cellular components, contain elevated peptide levels.

Different subcellular storage sites for decidual- and pituitary-derived prolactin: possible explanation for differences in regulation

The intracellular sites of storage for human decidual- and pituitary-derived prolactins have been determined by measuring the hormones in subcellular fractions following differential and density gradient centrifugations. In decidual tissue, 78 +/- 4% (mean +/- SE, n = 5) of the prolactin was detected in the post-microsomal supernatant, while 98% of the prolactin in the pituitary was detected in mitochondrial and microsomal particulate fractions. Following centrifugation on sucrose density gradients, decidual prolactin was distributed diffusely throughout the gradients, while greater than 90% of the pituitary prolactin sedimented as a single peak with a mean density of 1.22 g/cm3. These studies indicate that, unlike pituitary prolactin, decidual prolactin is not stored in secretory granules. Differences in the intracellular storage sites of decidual and pituitary prolactins may explain observed differences in the regulation of prolactin secretion by decidual and pituitary tissues.

Turnover and Storage of Newly Synthesized Adenine Nucleotides in Bovine Adrenal Medullary Cell Cultures

Abstract: The adenine nucleotide stores of cultured adrenal medullary cells were radiolabeled by incubating the cells with 32Pi and [3H]adenosine and the turnover, subcellular distribution, and secretion of the nucleotides were examined. ATP represented 84–88% of the labeled adenine nucleotides, ADP 11–13%, and AMP 1–3%. The turnover of 32P‐adenine nucleotides and 3H‐nucleotides was biphasic and virtually identical; there was an initial fast phase with a t½ of 3.5–4.5 h and a slow phase with a half‐life varying from 7 to 17 days, depending upon the particular cell preparation. The t½ of the slow phase for labeled adenine nucleotides was the same as that for the turnover of labeled catecholamines. The subcellular distribution of labeled adenine nucleotides provides evidence that there are at least two pools of adenine nucleotides which make up the component with the long half‐life. One pool, which contains the bulk of endogenous nucleotides (75% of the total), is present within the chromaffin vesicles; the subcellular localization of the second pool has not been identified. The studies also show that [3H]ATP and [32P]ATP are distributed differently within the cell; 3 days after labeling 75% of the [32P]ATP was present in chromaffin vesicles while only 35% of the [3H]ATP was present in chromaffin vesicles. Evidence for two pools of ATP with long half‐lives and for the differential distribution of [32P]ATP and [3H]ATP was also obtained from secretion studies. Stimulation of cell cultures with nicotine or scorpion venom 24 h after labeling with [3H]adenosine and 32Pi released relatively twice as much catecholamine as 32P‐labeled compounds and relatively three times as much catecholamine as 3H‐labeled compounds.

Purification of peptides derived from proenkephalin A on Bio-Rex 70

The weakly acidic, cation-exchange resin Bio-Rex 70 was used at a low pH for chromatography of enkephalins and other peptides containing enkephalin sequences. Recoveries of various peptide standards and peptides in adrenal chromaffin cell extracts were greater than 85%. Catecholamines and other substances such as density gradient media are not retained by the resin under the conditions employed. A significant purification of enkephalins and larger peptides containing enkephalin sequences with respect to total protein was also obtained by this method. This chromatographic technique should be useful where a low-cost, preliminary purification of samples containing peptides derived from proenkephalin A is required and may be adaptable to other peptides as well.