Role of Ca2+ and protein kinase C in the receptor-mediated activation of Na+/H+ exchange in isolated liver cells.

This work aimed to study the relationship between agonist-induced changes in cytosolic free calcium levels, protein kinase C (PKC) activity and intracellular pH in isolated liver cells. We observed that, like alpha1-adrenergic agonists, the Ca2+-mobilizing vasoactive peptides vasopressin and angiotensin II produced an extracellular-Na+-dependent, 5-(N-ethyl-N-isopropyl)amiloride-sensitive, intracellular alkalinization, indicative of Na+/H+ antiporter activation. Blocking the agonist-induced increase in the intracellular Ca2+ concentration using the calcium chelator bis-(o-aminophenoxy)ethane-N,N,N', N'-tetra-acetic acid (BAPTA) prevented all types of receptor-mediated intracellular alkalinization. Thus activation of the Na+/H+ exchanger by either alpha1-adrenergic agonists or vasoactive peptides relies on the mobilization of intracellular Ca2+. In contrast, only the alpha1-adrenergic-agonist-induced alkalinization was dependent on extracellular Ca2+. Even though alpha1-adrenergic as well as vasoactive peptide agonists stimulated protein kinase C (PKC) activity in isolated liver cells, only the alpha1-adrenoreceptor-mediated intracellular alkalinization was dependent on PKC. According to these observations, Ca2+-mobilizing agonists appear to activate the Na+/H+ exchanger by at least two different mechanisms: (1) the alpha1-adrenoreceptor-mediated activation that is dependent on extracellular Ca2+ and PKC; and (2) vasoactive-peptide-induced alkalinization that is independent of extracellular Ca2+ and PKC. The alpha1-adrenoreceptor-mediated, PKC-sensitive, activation of the Na+/H+ exchanger seems to be responsible for the distinct ability of these receptors to elicit the sustained stimulation of hepatic functions.

Identification of amidated forms of GLP-1 in rat tissues using a highly sensitive radioimmunoassay.

The development of a sensitive radioimmunoassay (RIA) for C-terminally amidated forms of glucagon-like peptide-1 (GLP-1) is described. Rabbits immunized with GLP-1(7-36)amide conjugated to bovine serum albumin with glutaraldehyde produced antisera containing high-affinity antibodies directed against an epitope that included the free amidated C-terminus of the peptide. These antisera could be used in a sensitive RIA (detection limit 0.1 fmol/tube) that measured GLP-1(7-36)amide and GLP-1(1-36)amide equally. Total concentrations of amidated GLP-1 immunoreactivity in extracts of rat hypothalamus, pancreas and intestine were determined by RIA, and resolved into GLP-1(7-36)amide, GLP-1(1-36)amide and unidentified cross-reacting substances by HPLC. Whereas only GLP-1(7-36)amide could be identified in the hypothalamus, in amounts that represented 55-94% of total glucagon-like immunoreactivity (GLI), the pancreas produced chiefly GLP-1(1-36)amide, representing 0.8-3.4% of total GLI, and only trace or undetectable amounts of GLP-1(7-36)amide (0-0.36% of total GLI). This argues against any role of intrapancreatic GLP-1(7-36)amide in the secretion of insulin. In the terminal ileum total amidated GLP-1 immunoreactivity represented 27-73% of total GLI, and in five of six specimens only GLP-1(7-36)amide could be identified on HPLC, in amounts representing 13-17% of total GLI. Only one specimen of terminal ileum contained HPLC-identified GLP-1(1-36)amide (13% of total GLI) in addition to GLP-1(7-36)amide (31% of total GLI). Acid-ethanol extraction of peptide-free rat plasma with added GLP-1(7-36)amide gave recoveries of 91+/-SEM 2% in the range 20-200 pmol/l. Basal plasma amidated GLP-1 in six unanaesthetized rats was 4.1+/-1.1 pmol/l and rose to a maximum of 15.4+/-3.0 pmol/l 10 min after intragastric glucose 1 g/kg, illustrating the modest level of plasma responses of amidated forms of GLP-1.

Effect of lithium on plasma glucose, insulin and glucagon in normal and streptozotocin-diabetic rats: role of glucagon in the hyperglycaemic response.

1. Lithium salts, used in the treatment of affective disorders, may have adverse effects on glucose tolerance in man, and suppress glucose-stimulated insulin secretion in rats. 2. To study the interaction of these effects with pre-existing diabetes mellitus, plasma glucose and insulin responses to lithium chloride were measured in male Wistar rats made diabetic with intraperitoneal streptozotocin, and in normal controls. 3. In both normal and diabetic anaesthetized rats, intravenous lithium (4 mEq kg-1) caused a rise in plasma glucose. In absolute terms, the rise was greater in diabetic (5.2 mmol l-1) than in normal rats (2.3 mmol l-1). 4. Plasma insulin concentrations were reduced by lithium in normal rats, but the low insulin concentrations measured in the diabetic rats were not significantly changed. 5. After intravenous glucose (0.5 g kg-1), lithium-treated diabetic rats showed a second rise in plasma glucose at 60-90 min without any insulin response, while normal rats showed typically reduced insulin responses and initial glucose disappearance rates. 6. Intravenous glucose reduced plasma glucagon concentrations to a greater extent in normal than in diabetic rats, but lithium induced an equal rise in plasma glucagon in both groups, with a time-course similar to that of the hyperglycaemic effect. 7. The hyperglycaemic action of lithium is greater in the hypoinsulinaemic diabetic rats and appears to involve a stimulation of glucagon secretion in both normal and diabetic animals.

Insulin components released by the normal and subtotal degranulated perifused islets of Langerhans of the rat.

The insulin components released from normal and tolbutamide treated islets of the rat were studied in a perifusion system. The treated islets showed diminished quantities of insulin secreted as compared to the controls and an increase in the proportions of "big insulin" secreted during the second phase of this release. On the other hand, the main component of insulin released during the first phase was "little insulin". These studies confirm that the subtotally degranulated islets are less efficient than the controls in producing insulin release.