Regulated Exocytosis

All mammalian cells display constitutive secretion, which is thought to be regulated at the stage of the budding of microvesicles from the trans-Golgi network (Burgess and Kelly, 1987). In addition, the specialized secretory cells exhibit regulated secretion. The docking of secretory vesicles on, and their fusion with, the plasma membrane are processes that appear to share many features with intracellular vesicle fusion (Scheller, 1995). The control over regulated exocytosis is exerted through second messenger pathways, in particular those implicating Ca2+ and cAMP. stimulation of insulin Glucose and leucine stimulate insulin secretion by raising the con-

secretion centration of cytosolic Ca2+ ([Ca2+]|) in the (3-cell and by providing metabolic coupling factors (Gembal et a/., 1993) that may amplify the action of Ca2+ on insulin exocytosis. The [Ca2+], rise is due to Ca2+ influx through voltage-sensitive Ca2+ channels, mainly of the L-type, which open as a consequence of membrane depolarization (Bokvist etal., 1995). Glucose and leucine cause membrane depolarization by closure of ATP-sensitive K+ channels subsequent to the stimulation of oxidative metabolism and an increase in the ATP/ADP ratio in the cytosol (Ashcroft and Rorsman, 1989). The precise nature of the coupling factors that enhance the action of Ca2+ in nutrient-evoked exocytosis remains obscure. Of the potentiating agents of insulin secretion, acetylcholine and cholecystokinin stimulate phospholipase C, thereby promoting Ca2+ mobilization (and influx) and activation of protein kinase C (Wollheim and Regazzi, 1990). GLP-1 and GIP, on the other hand, generate cAMP and activate protein kinase A, as a consequence of the stimulation of adenylyl cyclase (Widmann et a/.,

1994). In electrically permeabilized insulin-secreting cells, the influence of soluble second messengers on exocytosis can be studied directly, since it is possible to dialyze such cells with respect to nucleotides and ions while cytosolic proteins are retained. In the presence of ATP, Ca2+ stimulates insulin exocytosis with an EC50 of approximately 1.6(xM (Vallar etal., 1987, Ullrich etal., 1990). This is in close agreement with the value for Ca2+-stimulated exocytosis in patch-clamped mouse P-cells obtained using the capacitance method (Bokvist et a/.,

1995). In contrast to, cAMP is unable to stimulate exocytosis on its own, but potentiates Ca2+-induced exocytosis (Vallar etal., 1987). This is consistent with the role of cAMP-generating hormones as potentiators of insulin secretion.

The mechanism by which epinephrine and other neurohormones inhibit insulin secretion involves pertussis toxin-sensitive G-proteins. These hormones exert multiple actions, all contributing to the marked reduction of stimulated insulin secretion (Ullrich and Wollheim, 1988, Lang et a/., 1993). They thus inhibit adenylyl cyclase activity, hyperpo-larize the membrane potential by increasing K+ conductance, and promote closure of voltage-sensitive channels. However, we have clearly demonstrated by various approaches in permeabilized cells that an overriding inhibitory influence on exocytosis is directly exerted by the activation of G, and G0 proteins (Lang et a/., 1995).

It has been suggested that stimulators of exocytosis cause a remodeling of the microfilamentous cell web, which could act as a barrier, interfering with the access of the secretory granules to the plasma membrane (Burgoyne, 1990). There is, however, no evidence in insulin-secreting cells that Ca2+ and/or cAMP are capable of changing the arrangement of actin filaments. We found that drastic reduction of F-actin following treatment of HIT-T15 cells or rat pancreatic islets with C. botulinum C2 exotoxin mainly affects the recruitment of secretory granules to the plasma membrane (Li et a/., 1994). It is unlikely, therefore, that stimulators or inhibitors of insulin exocytosis act primarily on the composition of the cytoskeleton.

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