Mechanisms of exocytosis

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MECHANISMS OF EXOCYTOSIS Alan M. Poisner Department of Pharmacology, University of Kansas Medical Center, Kansas 66103, U.S,A. Exocytosis implies release of stored secretory product to the cell exterior without a preliminary discharge into the cytoplasm and without loss of cytoplasmic constituents. Evidence for exocytosis can be obtained from electron microscopic studies but these are not entirely satisfactory due to problems in sampling and interpretation and to limitations in quantitative estimates. On the other hand, chemical approaches to studying exocytosis yield indirect evidence but are amenable to quantitative analysis. The present review will discuss factors thought to be involved in exocytosis in the adrenal medulla. Catecholamine release by means other than exocytosis Some agents can cause catecholamine discharge without a concomitant or parallel release of other granule constituents or may cause the release of cytoplasmic markers. Drugs which cause the release of catecholamines without parallel release of chromogranin or dopamine B-hydroxylase (DBO) include tyramine and reserpine. Leakage of a cytoplasmic marker (LDH), which suggests loss of cellular integrity, accompanies catecholamine release evoked by elevated pH. Chemical evidence for exocytosis A prime piece of evidence that exocytosis is occurring is the finding that catecholamines and ATP (and metabolites) are secreted in a fixed ratio (about 4:1) which is the same ratio found in isolated chromaffin granules. This type of evidence has been reported for secretion evoked by acetylcholine or by splanchnic nerve stimulation. Cholinergic stimulation also causes the release of chromogranin and DBO without increasing the release of LDH or PNMT. All of these methods of stimulation require extracellular calcium and thus there seems to be a common link of calcium in exocytosis. Recent evidence suggests that exocytosis can occur in the absence of extracellular calcium. Aminophylline and caffeine can evoke catecholamine release in calcium-free medium and evidence has been presented that intracellular calcium may support this discharge. Aminophylline-induced catecholamine release from bovine adrenal glands in calcium-free medium is accompanied by ATP release which parallels hormone secretion. The ratio of catecholamines: ATP found in six tests was 5.00 ± 0.56, which can be compared to the ratio reported for chromaffin granules, 4.8.

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The fusion reaction One s t e p in e x o c y t o s i s , well i l l u s t r a t e d in s e c r e t i o n from the e x o c r i n e p a n c r e a s and the anterior p i t u i t a r y gland, is the fusion reaction. T h i s p r o c e s s in which the granule membrane b e c o m e s t r a n s i e n t l y fused with the s u r f a c e membrane i s not well understood. C l u e s to the mechanism of fusion of b i o l o g i c a l membranes have come from s t u d i e s on n o n - s e c r e t o r y t i s s u e s , l ~ y s o l e c i t h i n h a s been shown to c a u s e fusion of e r y t h r o c y t e s and it h a s been s u g g e s t e d that granule l y s o l e c i t h i n may promote c a t e c h o l a m i n e s e c r e t i o n by a c t i o n on the granule a n d / o r s u r f a c e membrane. F u s i o n of e r y t h r o c y t e s h a s a l s o been found to be promoted by c a l c i u m ions. Since c a l c i u m is required for s e c r e t o r y a c t i v i t y and b i n d s to s e c r e t o r y g r a n u l e s , it is p o s s i b l e that one role of calcium may be a s a p a r t i c i p a n t in the fusion reaction. It h a s p r e v i o u s l y been s u g g e s t e d that calcium along with A T P and membrane A T P a s e can promote fusion of the chromaffin granule membrane with the s u r f a c e membrane. After c o n s i d e r i n g t h e s e and other t h e o r i e s of fusion of b i o l o g i c a l membranes, P o s t e and A l l i s o n p r o p o s e d that e m p h a s i s should be Uplaced on the role of c a l c i u m , A T P and m e m b r a n e - a s s o c i a t e d A T P a s e enzyme s y s t e m s in r e g u l a t i n g the membrane c h a n g e s r e s p o n s i b l e for f u s i o n " . T h e fact that c a l c i u m , A T P and A T P a s e may be c r i t i c a l for t h i s s t e p in e x o c y t o s i s d o e s not e x c l u d e the p o s s i b i l i t y that t h e s e s u b s t a n c e s may a l s o act ,it other s t e p s in the d i s c h a r g e p r o c e s s . F u s i o n may a l s o occtu between g r a n u l e s a s a s t e p in hormone r e l e a s e .

Small molecules in exocytosis: culcium, A T P arut cyclic AMP T h e c r i t i c a l role of c a l c i u m in c a t e c h o l a m i n e r e l e a s e is well documented. E l e c t r o p h y s i o l o g i c a l and c h e m i c a l m e a s u r e m e n t s have shown that c a l c i u m influx a c c o m p a n i e s some forms of adrenal medullary s t i m u l a t i o n and e x t r a c e l l u l a r calcium (or a rapidly e q u i l i b r a t i n g c e l l u l a r pool) is c r i t i c a l for s e c r e t i o n . Some s e c r e t o g o g u e s can u t i l i z e i n t r a c e l l u l a r c a l c i u m . Many c e l l o r g a n e l l e s in the adrenal medulla can bind c a l c i u m j u s t a s in other t i s s u e s . T h u s the p o t e n t i a l s i t e s for c a l c i u m i n t e r a c t i o n in e x o c y t o s i s are multiple. Although some c a l c i u m binding s i t e s may s e r v e in r e g u l a t i n g c y t o p l a s m i c c o n c e n t r a t i o n s (and thereby i n d i r e c t l y affect s e c r e t i o n ) , other s i t e s may be d i r e c t l y involved in the terminal d i s c h a r g e p r o c e s s . It h a s been p r o p o s e d that a s i m u l t a n e o u s binding of granule and p l a s m a membrane by c a l c i u m i s a required s t e p in i n i t i a t i n g fusion and d i s c h a r g e . A T P (or a high*energy intermediate) has been i m p l i c a t e d in c a t e c h o l a m i n e r e l e a s e b e c a u s e of s t u d i e s with m e t a b o l i c i n h i b i t o r s . A T P can a l s o c a u s e the r e l e a s e of c a t e c h o l a m i n e s from i s o l a t e d chromaffin g r a n u l e s . Since A T P s e e m s

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to be required for intracellular movement in s e v e r a l types of c e l l s , this nucleotide may also f a c i l i t a t e movement of chromaffin granules to the c e l l surface. F i n a l l y , as mentioned earlier, AT1a could a l s o act in the final fusion and discharge process. ATP a l s o s e r v e s as the precursor for c y c l i c AMP, which could play a role in catecholamine r e l e a s e . Three of the criteria for second messenger s t a t u s have been met for c y c l i c AMP in adrenal medullary secretion: (1) c y c l i c AMP is present in the adrenal medulla (in high concentrations) and its level i n c r e a s e s when secretion is stimulated in vivo, (2) c y c l i c AMP i t s e l f can initiate catecholamine r e l e a s e from the adrenal medulla, and (3) p h o s p h o d i e s t e r a s e inhibitors which raise l e v e l s of c y c l i c AMP a l s o c a u s e catecholamine release. The means by which c y c l i c AMP could initiate e x o c y t o s i s include mobilization of c e l l calcium and activation of protein k i n a s e s . K i n a s e s may phosphorylate c r i t i c a l cell c o n s t i t u e n t s , such as secretory granules, plasma membrane or microtubules.

Macromolecules in exocytosis: contractile protein and microtubule protein The s i m i l a r i t i e s between secretion and contraction have led to the concept that one (or more) s t e p s in the discharge p r o c e s s involved a contractile event. Support for this view is provided by the finding that chromaffin granules p o s s e s s an A T P a s e with c h a r a c t e r i s t i c s similar to actomyosin. Following this line, one role of calcium could be to bind to an inhibitory complex (analogous to the troponin-tropomyosin system) and thereby permit an ATP-actomyosin interaction which c a u s e s hormone r e l e a s e . Contractile protein in the surface membrane may also contribute to the final discharge. Microtubule protein e x i s t s in the adrenal medulla in various forms. It is present as formed microtubules, as subunits not v i s u a l i z e d by electron microscopy, and p o s s i b l y as membrane-bound protein. Since antimitotic agents block catecholamine r e l e a s e , it is p o s s i b l e that some secretory phenomena u t i l i z e microtubules in intracellular transport prior to granule secretion. Other types of s e c r e t o g o g u e s may be little affected or potentiated by antimitotic drugs. This either reflects a s i t e of action other than microtubule protein or i n d i c a t e s that secretion can proceed by different mechanisms depending on the secretogogue. Vinblastine can c a u s e r e l e a s e of catecholamines from the intact adrenal gland and from i s o l a t e d chromaffin granules. This could be related to its interaction with microtubule- or actin- like protein in the granules. There is evidence that vinblastine may act at a calcium binding site. There are reasons to s u s p e c t an interaction between contractile protein and microtubule protein as in other c e l l s . Another agent which blocks catecholamine r e l e a s e is c y t o c h a l a s i n B. Its inhibitory actions, however, are not equivalent on all types of secretogogues.

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s i n c e c y t o c h a l a s i n B can act on microfilaments, it has been suggested that inhibition by this agent could represent block of c o n t r a c t i l e protein. However, this agent has many other s i t e s of action as well. The present view of e x o c y t o s i s , which is almost entirely s p e c u l a t i v e , reflects the complexity which has developed with the advent of recent information on c e l l u l a r physiology. Just as smooth muscle contraction induced by a variety of agents is no longer explained by a single simple mechanism, so e x o c y t o s i s from the adrenal medulla could prove to involve a variety of pathways.

HYPOTHALAMIC RELEASING HORMONES AND CATECHOLAMINES'. A NEW INTERFACE Arthur J. Prange, Ian C. Wilson, George R. Breese, Nicholas P. Plotnikoff, P a t r i c i o P. Lara, Morris A. Lipton Department of Psychiatry, School of Medicine, University of North Carolina, North Carolina 27514, U,S,A, Pyroglutamyl.histadyl~proline amide, thyrotropin r e l e a s i n g hormone (TRIt), a s u b s t a n c e of hypothalamic origin, is a potent r e l e a s e r of prolactin and thyrotropin (TSH). In 1972 our group first reported that TRH, when injected intravenously, had an immediate though temporary antidepressant action. In a double-blind, placebo-controlled, cross-over study ten women with unipolar d e p r e s s i o n were asked to mark a 100 mm line, one end of which was identified as "happy," the other end as " d e p r e s s e d . " Each score was measured as millimeters from the "happy" end and this was taken as analogous to degree of depression. After injection of 600 #g TRH at 9 a.m., patients were s u b s t a n t i a l l y improved by noon but tended to r e l a p s e later in the day. Saline injection had l i t t l e effect. The Hamilton Rating Scale for Depression was used daily. It showed that patients were s i g n i f i c a n t l y improved for three days after TRH but as a group had relapsed by the sixth day. The a n t i d e p r e s s a n t property of TRH was immediately confirmed by Itil and quickly by Kastin e t al. and by van der Vis~Melsen and Wiener. Following our c l i n i c a l study we turned to a population of age-matched normal women. We were motivated by two c o n s i d e r a t i o n s . F i r s t , we recognized that the full spectrum of behavioral activity of TRH might not be apparent in a d e p r e s s e d population; second, we needed to generate control data pertaining to TSH response to TRH. In normal women the pattern of s u b j e c t i v e response to TRH was found to be quite regular. Two to three hours after injection s u b j e c t s experienced a s e n s e of tranquility or p e a c e f u l n e s s . They often felt s l e e p y but rarely slept. When they did s l e e p , they were e a s i l y awakened. Four or five