To examine their role in insulin secretion, actin filaments (AFs) were disrupted by Clostridium botulinum C2 toxin that ADP-ribosylates G-actin. Ribosylation also prevents polymerization of G-actin to F-actin and inhibits AF assembly by capping the fast-growing end of F-actin. Pretreatment of HIT-T15 cells with the toxin inhibited stimulated insulin secretion in a time-and dose-dependent manner. The toxin did not affect cellular insulin content or nonstimulated secretion. In static incubation, toxin treatment caused 45-50% inhibition of secretion induced by nutrients alone (10 mM glucose + 5 mM glutamine + 5 mM leucine) or combined with bombesin (phospholipase C-activator) and 20% reduction of that potentiated by forskolin (stimulator of adenylyl cyclase). In perifusion, the stimulated secretion during the first phase was marginally diminished, whereas the second phase was inhibited by -80%. Pretreatment of HIT cells with wartmannin, a myosin light chain kinase inhibitor, caused a similar pattern of inhibition of the biphasic insulin release as C2 toxin. Nutrient metabolism and bombesin-evoked rise in cytosolic free Ca2+ were not affected by C2 toxin, indicating that nutrient recognition and the coupling between receptor activation and second messenger generation was not changed. In the toxin-treated cells, the AF web beneath the plasma membrane and the diffuse cytoplasmic F-actin fibers disappeared, as shown both by staining with an antibody against G-and F-actin and by staining F-actin with fluorescent phallacidin. C2 toxin dose-dependently reduced cellular F-actin content. Stimulation of insulin secretion was not associated with changes in F-actin content and organization. Treatment of cells with cytochalasin E and B, which shorten AFs, inhibited the stimulated insulin release by 30-50% although differing in their effects on F-actin content. In contrast to HIT-T15 cells, insulin secretion was potentiated in isolated rat islets after disruption of microfilaments with C2 toxin, most notably during the first phase. This effect was, however, diminished, and the second phase became slightly inhibited when the islets were degranulated. These results indicate an important role for AFs in insulin secretion. In the poorly granulated HIT-T15 cells actin-myosin interactions may participate in the recruitment of secretory granules to the releasable pool. In native islet 3-cells the predominant function of AFs appears to be the limitation of the access of granules to the plasma membrane. INTRODUCTIONThe actin filaments (AFs)1 are among the components of the cytoskeleton that, in addition to maintaining cell shape, participate in contraction, mitosis, migration, endocytosis, and secretion (Korn, 1982;Howell and Tyhurst, 1986;Pollard and Cooper, 1986;Stossel, 1989;Schelling et al., 1992;Burgoyne and Morgan, 1993). AFs are formed by polymerization of G-actin to F-actin (Korn, 1982;Pollard and Cooper, 1986). The extent of actin polymerization is controlled by the free G-actin concentration, the actin-binding proteins, and co...
Using a recirculation procedure to perfuse anaesthetised rat jejunum, E. coli STa enterotoxin can be shown to inhibit net fluid absorption profoundly, while not causing net fluid secretion, provided fluid measurement is by mass or volume. This observation contradicts many reports of STa causing secretion, implying that the recovered volume technique in the anaesthetised animal over a period of some hours cannot detect secretion because of conjectured or unspecified flaws. Experiments are presented here confirming the viability of the perfusion protocol used in this laboratory but also demonstrate that if secretion were to be occurring, the recovered volume protocol would detect it. It will only return a negative finding, if secretion does not occur. To this end, the effect of two secretory toxins on intestinal fluid movement in a closed loop preparation JAMMR, 28(2): 1-11, 2018; Article no.JAMMR.44118 2 were studied to demonstrate that the anaesthetic, intestinal preparation or perfusion duration did not hinder the demonstration of net secretion when the intestine was exposed to E. coli LT and C. difficile toxin A.. It is evident that STa itself only reduces net absorption but can appear to be secretory if driving forces such as luminal osmotic pressure or capillary hydrostatic pressure through vasodilatation are introduced, as was likely to have occurred with pithing and theophylline. The recognition that STa is a non-secretory enterotoxin necessarily falsifies several alternative methods that claim to demonstrate secretion. Since STa is not secretory many other substances identified by these methods need also not be secretory and alternative explanations must be found to explain their action. The importance of recognising that action on the small intestine cannot be attributed to a secretory mechanism within the enterocyte adds further weight to the concept that where net secretion does occur, the likely mechanism for it is a combination of increased vasodilatation together with increased hydraulic conductivity. Original Research Article
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