Christopher J. Pazoles

The chromaffin granule and possible mechanisms of exocytosis.

Publisher Summary This chapter discusses the chromaffin granule and possible mechanisms of exocytosis. Chromaffin granules and chromaffin cells have been subjects of biological and chemical investigation for over a quarter of a century, and fundamental, seminal information has been obtained about the general process of secretion from cells. Chromaffin granules play several roles in adrenal medullary cells, some of which may be amenable to biochemical analysis. This view presumes that chromaffin granules are assembled in the Golgi apparatus, by analogy to studies on other cells such as those of the pancreas, though in fact studies on the ultrastructural biochemistry of chromaffin-granule assembly are in a relatively primitive stateThis chapter describes current information on the biochemical basis of exocytosis as viewed from the vantage point of the chromaffin granule. It presents a general view of exocytosis and included granule assembly, the mechanism of calcium action, the release event, and recovery of the granule membrane after secretion.

Mechanism of Calcium Action and Release of Vesicle-Bound Hormones during Exocytosis

Publisher Summary The term exocytosis is primarily an anatomic word—it is a term used to describe the release of zymogen granule contents from exocrine cells. The data supporting the concept have been mainly ultrastructural. This chapter describes the mechanism of calcium action and the release of vesicle-bound hormones during exocytosis. Calcium plays a critical role in regulating exocytosis both in the adrenal medulla and in a number of other systems. In particular, an increase in intracellular free calcium concentration often seems to be a prerequisite for secretion. Calcium might mediate the initial interaction between the storage organelle and the plasma membrane just prior to secretion. Calcium penetrates the presynaptic nerve terminal and elicits secretion over a time interval that is too short to allow calcium to diffuse more than a short distance from the plasma membrane. The chapter describes the investigations that have been made into the process of membrane breakage or fission that results in the release of secretory vesicle contents outside the cell.

A simple and novel method for radiometric analysis of glucose utilization by adrenal chromaffin cells

Abstract Glucose utilization by cells and tissues can be followed by measuring the release of [ 3 H]H 2 O from added d -[5- 3 H]glucose, and we have developed a method whereby the whole reaction and assay can be performed in a single scintillation vial. The basic principle behind our new assay is that the released tritiated hydrogen ion in water can be quantitatively exchanged with the hydroxyl proton of simple alcohols such as isoamyl alcohol. The radiolabeled alcohol can then be extracted into an organic solvent to which 2,5-diphenyloxazole and p -bis[2-(5-phenyloxazoyl)]benzene have been previously added. Using this new assay we studied isolated chromaffin cells and found them to utilize glucose at a linear rate for at least 30 min. The assay was precise and reproducible enough to allow detailed analysis of various inhibitors of glycolysis and of oxidative phosphorylation. The new method is simple and rapid, can be done in open test tubes, requires no complex equipment, and is intrinsically highly accurate.

Evidence for direct coupling of proton and anion transport in chromaffin granules.

Chromaffin granules isolated from the bovine adrenal medulla undergo osmotic lysis when exposed to MgATP and permeant anions such as chloride. Lysis occurs as a consequence of ATPase-driven proton uptake and the concomitant uptake of permeant anions via a transport site having pharmacological similarities to the anion transport site of erythrocytes.’, These similarities are based on observations that drugs such as SITS (4-acetamido-4’-isothiocyanostilbene-2,2’-disulfonate), pyridoxyl phosphate, and probenecid inhibit granule lysis by competing with chloride in the same concentration ranges in which they block anion transport in erythrocytes.2-5 In addition, we have directly shown that SITS inhibits chloride uptake into granules.2 In the erythrocyte, the membrane protein responsible for anion transport, and the site of SITS and pyridoxyl phosphate action, has been shown to be a 100,000-dalton protein, Band 3.6 We found the situation in the chromaffin granule to be more complex when we discovered that these compounds also inhibit the granule ATPase activity. The inhibition of ATPase activity occurred over the same concentration ranges previously shown to inhibit lysis, and kinetic analysis of such inhibition by SITS revealed the same mechanism (competitive with respect to chloride) and Ki (35 pM) as for inhibition of granule lysis. This raised the possibility that these compounds had multiple sites of action on the granule membrane, including the ATPase as well as anion transport sites. Alternatively, their effects on one of these processes could simply have been due to electrical coupling between proton and anion transport. The latter appeared to receive support from our finding that the ATPase activity of intact granules was activated by permeant anions (Cl-, Br-) but not impermeant ones (isethionate, phosphate). However, the differential effects of anions and inhibitors on the ATPase were observed with water-lysed and Lubrol WX-solubilized granules as well, suggesting that they acted directly on the ATPase rather than indirectly by virtue of an electrical coupling. Surprisingly, this seemingly direct effect was not necessarily linked to ATP hydrolysis. Anions and anion transport inhibitors still acted when MgATP was omitted from the granule lysis reaction and an artificial inward proton gradient put in its place. This was done by suspending granules in a medium of low pH (less than 5.5-6.0), a condition under which protons were observed by 31P-NMR to enter the granules in a chloride-dependent manner. Results with other ATP-independent mechanisms of granule lysis, such as exposure to ammonia, nigericin/K+, valinomycinl K+, or nonactin/K+, showed that anion transport inhibitors were effective at blocking lysis only when the mechanism involved co-transport of both anions and protons. Finally, measurements of the granule transmembrane electrical potential further demon-

Chapter 20 Role of Ions and Intracellular Proteins in Exocytosis

Publisher Summary This chapter describes a new protein called synexin that induces Ca 2+ -dependent formation of pentalaminar complexes among secretory granule membranes. Synexin has proved distinct from other more conventional candidates for Ca 2+ -effect mediators, such as calmodulin, actomyosin, and tubulin. The chapter also describes the process of membrane breakage or fission that finally results in the release of secretory vesicle contents. Studies comparing the granule lysis reaction with predicted behavior of secreting chromaffin and other cells are briefly reviewed in the chapter. The biochemical data supporting the exocytosis hypothesis are that the storage organelles from different cells can be isolated and therefore shown to be discrete objects and when secretion occurs, the entire organelle contents ranging from small molecules to large proteins can be found in the extracellular medium. Calcium plays a critical role in regulating exocytosis in a number of systems.

Identification and subcellular localization of catalase activity in bovine adrenal medulla and cortex

Abstract Catalase activity was detected in homogenates of bovine adrenal cortex and medulla. Analysis by equilibrium density centrifugation in isoosmotic metrizamide-sucrose gradients revealed that 70% of the medullary catalase activity was soluble while most of the remainder was found in a particulate form with a density of 1.175 g/ml. This was distinct from the densities of lysosomes, mitochondria, and chromaffin granules. Catalase activity in adrenal cortex was primarily (90%) soluble with only 6% being particulate, with a density of 1.185 g/ml. d -Amino acid, uric acid, and α-hydroxyacid oxidase activities, often associated with peroxisomes in other tissues, were absent from homogenates and catalase-containing gradient fractions from either cortex or medulla. There was an indication that some catalase activity was associated with chromaffin granules on the basis of density gradient analysis of both medullary homogenates and crude granule preparations. When granule fractions were subjected to osmotic shock, catalase activity distributed between soluble and sedimentable fractions differently from epinephrine and dopamine β-hydroxylase activity. The sedimentable catalase activity remained associated with chromaffin granule membranes upon isopycnic centrifugation. We concluded that catalase activity in both adrenal cortex and medulla was largely cytoplasmic, but that both tissues contained at least some catalase in dense organelles. Catalase activity which may be associated with chromaffin granules represents a small fraction of the total activity in the medulla.

EVIDENCE IN SUPPORT OF A CHEMIOSMOTIC MECHANISM FOR EXOCYTOSIS FROM PLATELETS, PARATHYROID AND CHROMAFFIN CELLS

ABSTRACT A chemiosmotic mechanism is the basis of ATP, Cl–-induced lysis of chromaffin granules, and we have now shown that the granule model successfully predicts the secretory properties of human platelets, bovine parathyroid cells, and bovine chromaffin cells. Like the granule lysis system, secretion from these cells requires specific anions in the medium, is inhibited by anion transport blocking drugs and proton ionophores, and is suppressed by elevated osmotic strength. We suggest that secretory granules fused to plasma membranes in secreting cells may experience net solute uptake and subsequently undergo local, outwardly directed osmotic lysis, or exocytosis.

SYNEXIN: AN ADRENAL MEDULLARY PROTEIN THAT MAY BE AN INTRACELLULAR RECEPTOR FOR Ca2+ IN THE PROCESS OF EXOCYTOSIS

ABSTRACT Adrenal medullary tissue contains a soluble protein of M.W. 47,000 which specifically binds Ca2+ and, in the presence of Ca2+, induces the fusion of the outer leaflets of chromaffin granule membranes. We have named this protein synexin, from the Greek word “synexis” which means “a meeting”. We suggest synexin may be an intracellular receptor for Ca2+ in the process of exocytosis, acting to promote fusion between secretory vesicles and the plasma membrane, or, in the case of compound exocytosis, between vesicles.

IDENTIFICATION OF ANION TRANSPORT SITES IN CHROMAFFIN GRANULES AND THEIR INVOLVEMENT IN AN OSMOTIC MECHANISM FOR EPINEPHRINE RELEASE

ABSTRACT 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.