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Tris (Hydroxymethyl) Aminomethane (THAM) Induced Stimulation of Insulin Release by Islets of Langerhans Previously Isolated from Rat Pancreas
Abstract
Islets isolated from rat pancreas were studied in regard to release of immunoreactive insulin (IRI) as influenced by changes in glucose concentration and pH and type of buffer in the incubation media. The pH of the media was varied from 7.4 to 9.0. For phosphate and bicarbonate buffered media, change in pH did not result in significant change in IRI release rates. For media containing the pH buffer and chelating compound THAM (Tris [hydroxymethyl] aminomethane), IRI release increased significantly with increase in either THAM concentration or pH. Since increase in pH results in increase in that fraction of THAM which is in the unionized, therefore presumably more readily penetrating form, it is postulated that THAM molecules penetrate and act at intracellular loci to induce insulin release, possibly by chelating Zn complexed with insulin in beta cell granules.
... On the other hand it may itself activate secretion , or perhaps act by increasing intracellular pH. Isotonic Tris solutions were found to promote insulin release from pancreatic islets (Fedynskyi & Beck, 1970) and a larger effect was seen at pH 7-8 than at pH 7*4. This suggested that it is the entry of un-ionized Tris, which increases in concentration as pH rises, that is responsible for the response. ...
45Ca efflux and uptake, net Ca movements, and catecholamine secretion were studied in thin slices of bovine adrenal medulla. There was a slow component of 45Ca wash-out which is attributed to efflux of intracellular Ca. This efflux was strongly influenced by cations in the external solution, being reduced by 50% when Mg replaced Ca, and the residual efflux being reduced by 80% when choline replaced Na. None of the substitutes tested, choline, K, Li, Tris or sucrose, could replace Na in maintaining 45Ca efflux into Ca-free solution. The Na-dependent Ca efflux showed sigmoidal activation by Na, indicating a requirement for the co-operative action of two or more Na ions in the extrusion of each Ca.
... In vitro, Tris is known to be toxic to cells in culture, 2 to cause release of specific membrane-associated proteins from Escherichia coli, suggesting that it may react uniquely with cell membranes,' and to specifically increase secretion of insulin by pancreatic islet cells in cul-ture. 24 In such situations, it is possible that the action of Tris might be related to a disruption of the Golgi or other subcellular organelles in cells at large. Such data reinforce the need to investigate more closely the interaction of Tris with cells and organs in vitro and in vivo. ...
Tris(hydroxymethyl)aminomethane (Tris, Tromethamine, THAM) and other non-amphoteric amines were previously reported to inhibit the conversion of proparathyroid hormone to parathyroid hormone in bovine parathyroid glands incubated in vitro. This inhibition correlated with a striking dilation of the Golgi complex. This work has now been extended to normal, hyperplastic, and adenomatous parathyroid glands from human subjects. The tissues were incubated for up to 3 hours with 3H-leucine in physiologic solutions (control) or in the same solutions containing 50 mM Tris. In one case, diethylamine also was tested. Electron microscopy revealed that the amines produced a dilation of the Golgi complex and swelling of vesicles, predominantly in the region of the Golgi zone. Other organelles were normal in appearance. During the same period, Tris reduced by sixfold the ratio of the parathyroid hormone to proparathyroid hormone, from a control value of 2:1 to 1:3. It was apparent that Tris exerted the same biochemical and morphologic actions in human parathyroid tissues as it was previously shown to do in bovine glands. These studies support the concept that the Golgi zone is that region in the parathyroid gland in which proparathyroid hormone to parathyroid hormone conversion is initiated and that Tris inhibits this conversion through disruption of the converting site.
... In these experiments [Na]o was reduced from 142 to 39 mm by replacement of all extracellular NaCl with choline chloride or LiCl. Tris-Cl proved an unsatisfactory Na+ substitute, causing irreversible membrane potential changes; it has also a variable effect per se on insulin release (Broeckaert, 1970; Fedynskyi & Beck, 1970). The immediate effect of replacing NaCl with choline chloride was a small increase in islet cell membrane potential. ...
1. Responses of the membrane electrical characteristics of mouse pancreatic islet cells to ionic environmental changes have been used to assess the role of [Na]0 and [K]0 in the control of membrane potential, i.e. by electrodiffusion or via an electrogenic sodium pump. Islet cell electrical properties were measured in vitro with intracellular glass micro-electrodes. 2. Substitution of LiCl for extracellular NaCl did not change the islet cell membrane potential significantly in low (2.8 mM) glucose solutions, but readmission of NaCl caused a transient hyperpolarization (membrane potential maximum: -70 mV) in high glucose; when choline chloride was substituted for NaCl no hyperpolarization was observed on NaCl re-admission. 3. Superfusion with K-free solution gave no marked change in membrane potential during 30 min incubation in either low (2-8 mM) or high (28 mM) glucose concentrations but longer periods of exposure to K-free solutions caused progressive depolarization. 4. Readmission of K+ induced a transient hyperpolarization of up to 30 mV magnitude and 10 min duration in the presence of high (28 mM) but not low glucose (2-8 mM) concentrations. At the level of maximum hyperpolarization the membrane potential reached -60 mV, the electrical activity induced by the high glucose concentration being concurrently completely blocked. Replacement of [Cl]0 by isethionate accentuated these effects. 5. Ouabain, 10(-3) M, or a decrease in temperature from 37 to 7 degrees C depolarized the islet cells and blocked the transient hyperpolarization induced by readmission of K+. 6. Diphenylhydantoin, 1-5 times 10(-4) M, caused a significant hyperpolarization of the islet cells in low glucose (2-8 mM) and inhibited the electrical activity induced by high glucose (28 mM) or tolbutamide (0-7 mM). 7. It is concluded from these results that both an electrogenic and ionic component contribute to the membrane potential of the mouse pancreatic islet cell although electrodiffusional control normally predominates; acceleration of the Na-K exchange pump by diphenylhydantoin inhibits glucose-induced electrical activity. These findings are discussed in relation to the permeability characteristics of the islet cell membrane and the mechanism of insulin release.
In the course of work concerned with the inhibition of small intestinal carbohydrate digesting enzymes, experiments were performed on rats and two healthy volunteers using tris as a sucrase inhibitor. The following results were obtained: (1) Tris does not lower the blood glucose in fasting rats after oral or subcutaneous doses up to 500 mg/kg, when administered as neutral solution (pH 7.0). (2) Tris reduces reduces the glycemia in rats and human subjects after a sucrose load. In addition, the insulinemia caused by administration of sucrose is reduced in man. This smoothing effect on both curves is dose-dependent. A delay of gastric emptying by tris could be excluded. (3) After a glucose or matose load in rats, tris has no effect on the blood sugar curve. (4) The marked smoothing effect of tris is after sucrose loading is probably caused by its well-known in vitro inhibitory effect on intestinal sucrase activity of pigs and humans.
The effects of a continuous infusion of tris(hydroxymethyl)aminomethane (THAM) on pH, base excess, p50, serum osmolality, and plasma drug concentration during respiratory acidosis were studied in newborn piglets. Measurements were made during three experimental periods: (1) control period with normal blood gases; (2) hypercapnia period, and (3) hypercapnia plus THAM period (THAM infusion: 1.65 mmol/kg/h). pH decreased and paCO2 increased between control period (7.40 +/- 0.05 and 45 +/- 3 mm Hg) and hypercapnia period (7.24 +/- 0.06 and 59 +/- 2 mm Hg; p < 0.001; mean +/- SD). pH returned to baseline (7.37 +/- 0.04) during the hypercapnia plus THAM period, while paCO2 remained elevated (63 +/- 4 mm Hg). p50 increased from 30.7 +/- 5.9 to 38.3 +/- 4.7 (p < 0.05) during hypercapnia and decreased with hypercapnia plus THAM. THAM concentration and base excess increased with time and were linearly related. Serum osmolality was unchanged during the THAM infusion. We conclude that continuous infusion of THAM is effective in normalizing pH during respiratory acidosis in the piglet.
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