Dynamic insulin secretion from perifused rat pancreatic islets

Abstract

Insulin secretion from isolated pancreatic islets of 8- to 12-day-old rats was investigated in a dynamic in vitro (perifusion) system. The aims of the study were (i) to describe a carefully controlled in vitro method to study the mechanism of insulin secretion and to analyse the effects and dynamic interactions of bioactive compounds on isolated rat pancreatic islets, (ii) to validate the method by comparing fundamental data on the functions of the islets obtained with this method to those collected with other techniques; and (iii) to find novel features of the control of insulin secretion. The method was carefully designed to maintain the functional capacity of the explanted cells. A functional standardization system was elaborated consisting of (i) analysis of the changes in the basal hormone secretion of the cells; (ii) evaluating responses to a standard, specific stimuli (50 mM glucose for 3 min); (iii) determining the alteration of the momentary size of the hormone pool with responses to KCl; and (iv) direct determination of the total intracellular hormone content from the extract of the column. The technique provides accurate quantitative data on the dynamic responses to biologically active compounds that act directly on the pancreatic islets. The islets maintained their full responsiveness for up to 7 days, and responses as close as in 1-min intervals could be distinguished. A linear dose-response relationship was found on the glucose-induced insulin release in case of 3-min stimulation with 4 and 500 mM of glucose (lin-log graph). Utilizing this method, we showed that no desensitization to glucose-induced insulin release can be observed if the responsiveness of the cells is properly maintained and the parameters of the stimulation are carefully designed. Exposure of the explanted islets to 10 microM acetylcholine or 30 mM arginine (Arg) induced a transitory elevation of insulin release similar in shape to that experienced after glucose stimulation. Norepinephrine (NE), dopamine (DA) and somatostatin (SS) did not induce any detectable alteration on the basal insulin secretion of the islets. However, 100 nM SS given together with 50 mM glucose, 30 mM Arg or 10 microM acetylcholine significantly reduced the insulin-releasing effect of these substances (by 75.5, 71.5 and 72.5%, respectively). At the same time, SS did not alter the insulin response of the islets to 100 mM elevation of K+ concentration. SS also inhibited glucose-induced insulin release in a dose-dependent way (ED50 = 22 nM). A similar dose-dependent inhibitory effect on glucose-induced insulin release was found with NE (ED50 = 89 nM) and DA (ED50 = 2.2 microM). gamma-Aminobutyric acid (GABA) did not influence insulin release under similar circumstances.

‘TRPing’ Synaptic Ribbon Function in the Rat Pineal Gland: Neuroendocrine Regulation Involves the Capsaicin Receptor TRPV1

“…NE elicited a clear augmentation of melatonin secretion into the perifusion fluid. Such a response was observed previously [21,22], and is also in accord with in vivo studies showing that melatonin synthesis increases upon activation of the sympathetic system [39]. …”

“…This part of the present study was conducted according to previous protocols [21,22,23,28,29]. For each experiment, pineal glands from 6 rats were taken out, immediately bisected and packed into a 6.6-mm glass column together with Sephadex G-10.…”

“…After we found out that kinesin and TRPV1 are colocalized in rat pinealocytes (suggesting that the receptor molecule is part of SR), we studied the functionality of this receptor in the pineal gland. One approach was to investigate intact parts of rat pineal glands in a perifusion system [20,21,22,23] and to test whether melatonin secretion responds to application of capsaicin (the prototypically naturally occurring vanilloid) and/or the competitive TRPV1 antagonist capsazepine. The second functional approach was to utilize calcium imaging to test whether the exogenous or endogenous TRPV1 ligands, capsaicin or AEA (an endocannabinoid that also binds to TRPV1) [24,25] may activate TRPV1 and thereby increase free intracellular calcium in isolated rat pinealocytes in culture.…”

‘Classical’ and ‘new’ diabetogens—comparison of their effects on isolated rat pancreatic islets in vitro

“…Perifusion. Approximately 300 isolated islets supplemented with Sephadex G-10 were placed into a glass column of a perifusion system (detailed description in Csernus et al [30]) and continuously perifused with culture medium supplemented with 2.22 g/l sodium hydrogen carbonate, 1.75 g/l BSA, 80 mg/l gentamycin, and 3 mmol/l glucose. After passing the cells, the medium was collected in fractions (intervals lasting 3 or 30 min).…”

Apoptosis in Cultured Rat Islets of Langerhans and Occurrence of Bcl-2, Bak, Bax, Fas and Fas Ligand

“…Male BDE rats (Harlan Winkelmann GmbH, Borchens, Germany) were kept under a 12-hour light/dark cycle with free access to water and food and lights on at 6.00 a.m. The islets were isolated by collagenase digestion as recommended by various authors [Lernmark, 1974;Jindal et al, 1996;Dickinson et al, 1997;Emilsson et al, 1997;Flodstrom and Eizirik, 1997;Heald et al, 1997;Mendola et al, 1997;Miley et al, 1997;Asada et al, 1998;Bouaziz et al, 1998;Csernus et al, 1998;Petrik et al, 1998;Peschke et al, 1998]. To summarize, after cervical dislocation between 8.00 and 9.00 a.m. followed by exsanguination, the pancreata were distended with 6 ml of Hanks' balanced salt solution containing HEPES, 10 mmol/l, and 2 mg/ml collagenase (Type CLS IV, Biochrom KG, Berlin, Germany), excised and incubated in a water bath at 37°C.…”