Aims/hypothesis The molecular basis of the exocytosis of secretory insulin-containing granules (SGs) during biphasic glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells remains unclear. Syntaxin (SYN)-1A and SYN-4 have been shown to mediate insulin exocytosis. The insulinsecretory function of SYN-3, which is particularly abundant in SGs, is unclear. Methods Mouse pancreatic islets and INS-1 cells were treated with adenovirus carrying Syn-3 (also known as Stx3) or small interfering RNA targeting Syn-3 in order to examine insulin secretion by radioimmunoassay. The localisation and distribution of insulin granules were examined by confocal and electron microscopy. Dynamic single-granule fusion events were assessed using total internal reflection fluorescence microscopy (TIRFM). Results Depletion of endogenous SYN-3 inhibited insulin release. TIRFM showed no change in the number or fusion competence of previously docked SGs but, instead, a marked reduction in the recruitment of newcomer SGs and their subsequent exocytotic fusion during biphasic GSIS. Conversely, overexpression of Syn-3 enhanced both phases of GSIS, owing to the increase in newcomer SGs and, remarkably, to increased SG-SG fusion, which was confirmed by electron microscopy. Conclusions/interpretation In insulin secretion, SYN-3 plays a role in the mediation of newcomer SG exocytosis and SG-SG fusion that contributes to biphasic GSIS.
When proximal tubules are immersed in hypotonic medium, they quickly swell to a peak volume. In a second, slower phase, termed volume regulatory decrease (VRD), they shrink as K, anion, and water leave the cells. We investigated the role of the cytoskeleton during this biphasic hypotonic volume regulatory response. Isolated, collapsed rabbit proximal convoluted tubules (PCT) were crimped tightly between two pipettes, and their volume was assessed optically. PCT volume increased to a peak 70-80% above baseline on sudden immersion in dilute medium (150 mosmol/kgH2O). After completing VRD, control tubules had regulated their volume 73 +/- 2% back toward baseline. Tubules exposed to the microtubule inhibitor vincristine (5 microM) regulated 75 +/- 2%. Tubules exposed to the microfilament inhibitor cytochalasin B (50 microM) regulated less (57 +/- 5%), and tubules exposed to both inhibitors regulated only 39 +/- 3% (P less than 0.01 vs. control). Hypotonic VRD was unimpaired in PCT loaded with NaCl by prior exposure to ouabain but was significantly reduced by cytochalasin B. We conclude that VRD is not cation specific and that intact microtubules and microfilaments play a synergistic role in the VRD of rabbit PCT in hyposmotic medium.
The state of the endothelial cell (EC) determines the nature of its control of vascular smooth muscle cell (vSMC) biology. Conditioned medium from postconfluent ECs inhibits vSMC proliferation, whereas subconfluent conditioned medium from the same ECs has a stimulatory effect. We and others have identified confluent endothelial cells' production of heparan sulfate proteoglycans (HSPG) as critical to vSMC growth control. The question that arises is whether the stimulation that is observed with subconfluent cells is from (1) aberrant HSPG production, (2) elaboration of noninhibitory species of HSPG, or (3) production of other factors, such as mitogens, which counteract the inhibitory HSPG to stimulate vSMCs. We studied the relative effects of conditioned medium produced by both subconfluent and postconfluent EC cultures on vSMC growth. Conditioned medium was fractionated into nonproteoglycan (non-PG) and proteoglycan (PG) components by anion-exchange chromatography. The PG fractionation profile and the antiproliferative activity of the HSPGs isolated from both subconfluent and postconfluent EC-conditioned media were similar. However, the HSPG fraction alone could not approach the inhibitory potential of unfractionated conditioned medium from postconfluent EC cultures. Non-PG proteins produced by the endothelial cultures had no effect on vSMC growth on their own. Yet, when they were mixed together with HSPG fractions, from either subconfluent or postconfluent EC cultures, the full growth effects were returned. Non-PG protein fractions from postconfluent cultures with HSPG fractions gave maximal inhibition of vSMC growth, whereas non-PG protein fractions from subconfluent EC cultures with HSPG fractions produced the maximal stimulation. Thus, whereas the net stimulatory or inhibitory effect on vSMC growth of EC-conditioned medium is density dependent, this effect does not result from a difference in the antiproliferative heparan sulfate component but rather from non-PG proteins that interact with the heparan sulfates.
The relative importance of heparin-like compounds in mediating vascular repair is unclear. We investigated how protamine, a chelator of heparin, affected endothelial cell inhibition of vascular smooth muscle cell growth and intimal hyperplasia. The 52% ( P < 0.001) reduction in smooth muscle cell proliferation produced by postconfluent endothelial cell-conditioned medium was entirely reversed by pretreatment of medium with heparinase and heparitinase and was inhibited in a dose-dependent fashion by the coadministration of protamine. Pretreatment of conditioned medium with heparinase and heparitinase largely prevented protamine’s mitogenic activity, suggesting that protamine affects growth by interacting with heparin-like compounds. Perivascular implantation of polymer-engrafted endothelial cells reduced neointima formation in denuded rat carotid arteries by 92% ( P < 0.001) and cell proliferation by 81% ( P < 0.001). Coadministration of protamine abolished the inhibitory potential of the cell implants, resulting in a nearly twofold exacerbation of intimal hyperplasia compared with controls ( P < 0.001). Thus heparin-like molecules are essential to the biochemical regulation of vascular repair provided by endothelial cells, and the continued routine clinical use of heparin chelators, like protamine, may be questionable.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.