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Publisher Summary This chapter describes experimental procedures used for the measurement of inositol polyphosphate accumulation in cultured adrenal chromaffin cells and N1E-115 neuroblastoma cells by high-performance liquid chromatography (HPLC). High-performance liquid chromatography offers high-resolution separations of inositol polyphosphates, including isomers of InsP 3 , InsP 4 , and InsP 5 , by a strong anion-exchange column using a gradient of ammonium phosphate. Radioactivity in HPLC effluents is quantitated by liquid scintillation counting in an on-line radioactivity flow detector equipped with a computer-aided analytical program, which offers a rapid method of quantitatively detecting [ 3 H] inositol polyphosphates. Changes in the radioactivity of [3H] inositol phosphates reflect changes in the amount there of only when cells are radiolabeled to isotopic equilibrium. [ 3 H]Inositol taken up into cells is incorporated into phosphatidylinositol, which is eventually phosphorylated to phosphatidylinositol 4, 5-bisphosphate. This compound is the major precursor of most [ 3 H] inositol polyphosphates. Stimulus-induced formation of [3H]inositol phosphates, including [3H]Ins-(1,4,5)P 3 , [3H]Ins(l,3,4,6)P 4 , [3H]Ins P 5 , and [3H]InsP 6 , can be detected in cultured neuronal cells. High-performance liquid chromatography combined with an online radioactivity flow detector offers a powerful tool for simultaneously separating and radiochemically quantitating many [3H] inositol phosphates from intact cells.

The chromaffin cell grafts in the Parkinsonism models is used primarily as the living depots of catecholamines. Because of their phenotypic plasticity, however, chromaffin cells have considerably more potential as donor tissue than has been utilized heretofore. These cells can be maintained in their normal chromaffin phenotype by the addition of corticosteroids, and they can grow neurites when the steroid is removed and nerve growth factor (NGF) or fibroblast growth factor (FGF) is added. Moreover, when the appropriate conditioned medium (CM) is supplied along with NGF, the efficiency of conversion of neonatal chromaffin cells into sympathetic neurons is enhanced and chromaffin cells taken from adult rats can also be converted. In addition, the chromaffin cell-derived neurons can be converted to the cholinergic phenotype when an instructive differentiation factor purified from cultured heart cells is added. These converted, cholinergic sympathetic neurons can form functional cholinergic synapses.

Publisher Summary This chapter discusses the biosynthesis of enkephalins by adrenal chromaffin cells. Immunohistochemical studies have shown that bovine adrenal chromaffin cells contain immunoreactive enkephalin. Leu- and Met-enkephalin represent only a part of this immune-reactivity. These cells contain a large amount of a newly characterized enkephalin: met-enkephalin-Arg-Phe. The concentration of this new compound is higher than that of met-enkephalin. The chromaffin granules contain several large Mr peptides containing the aminoacid sequences of the enkephalins. The steady state levels of the enkephalins and their precursors in primary monolayer bovine adrenal chromaffin cells may indicate that they actively synthesize these molecules de-novo.

SGC (Small Granule Chromaffin) cells in the mouse adrenal medulla characterized by the smallness of the secretory granules and by the aggregations of synaptic-type vesicles may represent a third type of chromaffin cell.

Adrenal chromaffin granules in an isoosmotic medium can be induced to release their contents, including epinephrine, in an all or none fashion. Such release is based on osmotic lysis of the granules and is regulated by Mg-ATP and permeant anions. In this paper, we examine the mechanism of this release as it relates to membrane sites involving Mg-ATP and anions. We conclude that osmotic lysis of chromaffin granules in an isoosmotic medium is the result of electrogenic pumping of protons into the granule via a membrane Mg-ATPase and subsequent anion uptake via a selective transport site. Studies involving ATP-independent osmotic release mechanisms and anion transport blocking drugs further suggest that these two granule membrane sites may be physically as well as functionally coupled.

Publisher Summary Implants of both chromaffin and glomus cells reverse amphetamine-induced rotational behavior and appear viable in the unilateral 6-hydroxydopamine (6-OHDA) rodent model. This chapter describes the potential of several alternative sources of donor tissue for neural transplantation, and emphasizes upon two members of the sympathoadrenal cell lineage, the adrenal chromaffin and carotid body glomus cells, as implants in the unilateral 6-hydroxydopamine (6-OHDA) rodent model. The particular focus of the present study is on the fine structure of these paraneuronal grafts 30 days post-implantation into the denervated striatum. Once implanted, the chromaffin cells demonstrate a plasticity of their dense-core vesicles, with the average diameter decreasing in size when compared to normal adrenal medullary cells. However, the study raises some important questions that need to be addressed, such as (1) will these paraneuronal cells survive and continue to exert positive behavioral effects in long-term experiments, and (2) what is the critical mass of implanted tissue needed to achieve functional recovery, and where in the brain are the optimal sites for ransplantation of these cells. The chapter indicates that answers to these questions are essential for designing rationale clinical approaches for the treatment of parkinsonism, and to encourage the acceleration of transplantation research in appropriate animal models.

S Our autoradiographic study in the mouse using tritiated leucine, tyrosine, dopa, dopamine and noradrenaline demonstrated that each chromaffin cell possessed special trapping and concentrating systems for catecholamines in addition to those for amino acids. The systems for catecholamines were, under the control of pituitary ACTH, better developed in the chromaffin cells in the periphery of the adrenal medulla than in those in the center of the adrenal medulla.

Publisher Summary This chapter discusses a study analyzing two mechanisms of catecholamine storage in adrenal chromaffin granules. The chapter highlights that two different processes seem to be involved in the storage of CA in adrenal chromaffin granules. The amines are apparently pumped into the organelles against a considerable concentration gradient by Mg2+/ATP-dependent mechanism operating at the level of the granular membrane and involving ATP-ase. Experiments show that the maximum NA concentration reached in isolated granular membranes amounts to 5 mM, but the CA concentration in intact chromaffin granules was calculated to be approximately 500 mM. The build-up of this high concentration is most likely because of the existence of the second mechanism, that is, the intra-granular interaction of the CA with nucleotides such as ATP and also with chromogranins. The storage of ATP in the adrenal chromaffin granules is shown to precede that of CA in the course of the ontogenetic development of rabbits and rats. Therefore, the amines transported into the organelles by the membrane pump can undergo intra-molecular bonding with the nucleotide.

Publisher Summary This chapter Presents the results concerning the innervation of both extra-adrenal chromaffin tissue (EACT) and intra-adrenal chromaffin tissue (IACT) correlated with the differentiation of these tissues. The first formaldehyde-induced fluorescence (FIF) appeared in EACT of a 7-week-old fetus. The light-microscopic studies indicate that at this stage of development only phaeochromoblasts—an intermediate cell type between primitive sympathetic cell (PSC) and mature chromaffin cell (CC)—could be found. Axons and some occasional axon terminals were found in EACT. However, no typical synaptic contacts could be found in any age-group studied, whereas synapses were regularly found in adrenal medullary chromaffin tissue after the 12th week. Similar findings were made in rabbit chromaffin tissue. The present results support the opinion of research that EACT probably lack a functional innervation, at least during the first two-thirds of fetal development. Although the possibility of endocrine-active EACT has been neglected by several authors, it is apparent that it possesses features indicating this kind of function. The EACT is known to contain relatively larger amounts of catecholamines than the adrenal chromaffin tissue, the amine being predominantly noradrenaline.