Glucagon stimulation of ruthenium red-insensitive calcium ion transport in developing rat liver

Abstract

The maturation of glucagon-stimulated Ruthenium Red-insensitive Ca2+-transport activity was determined in livers of rats ranging in age from 5 days preterm to 10 weeks of adult life. Previous indications are that this activity is confined to vesicles derived mainly from the endoplasmic reticulum. Perinatal-rat liver contains near-adult values of Ruthenium Red-insensitive Ca2+-transport activity, and exhibits large transient increases in the rate of this activity at two stages of development, immediately after birth, and at 2-5 days after birth. The administration of glucagon to foetal rats, at developmental stages after 19.5 days of gestation (2.5 days before birth), results in a large stable increase (greater than 100%) of Ca2+-transport activity in a subsequently isolated 'heavy' microsomal fraction. That this fraction was enriched in vesicles derived from the rough endoplasmic reticulum was indicated by both an electron-microscopic examination and a marker-enzyme analysis of the subcellular fractions. The administration of glucagon into newborn animals only hours old does not enhance further the initial rate of Ca2+-transport activity, and from day 1 to 10 weeks after birth the administration of the hormone results in the moderate enhancement of Ca2+ transport. Experiments with cyclic AMP and inhibitors of phosphodiesterase activity suggest that cyclic AMP plays a key role in the enhancement by glucagon of Ruthenium Red-insensitive Ca2+ transport, and arguments are presented that this transport system has an important metabolic role in the redistribution of intracellular Ca2+ in liver tissue.

A procedure for the rapid isolation from rat liver of plasma membrane vesicles exhibiting Ca2+-transport and Ca2+-ATPase activities

“…lysosomes and peroxisomes, and flattened cisternal structures originating from the Golgi apparatus also are present although in substantially lesser proportions. Vesicles derived from the rough endoplasmic reticulum and which are readily identified in microsomal preparations obtained by using buffers identical with those adopted in this study (Reinhart & Bygrave, 1981) seldom are encountered in fraction Al (Fig. 2, arrow).…”

“…The following marker enzymes were assayed as described: cytochrome oxidase, EC 1.3.99. 1 (a mitochondrial marker), Reinhart & Bygrave (1981); NADPH-cytochrome c reductase, EC 1.6.99.2 (an endoplasmic reticulum marker), Dallner et al (1966); catalase, EC 1.11.1.6 (a peroxisomal marker), Luck (1965); UDP-galactose: N-acetylglucosamine galactosyltransferase, EC 2.4.1.38 (a marker for Golgi apparatus), Bretz & Staubli (1977); alkaline phosphatase, EC 3.1.3.1 (a plasma membrane marker), Pekarthy et al (1972); acid phosphatase, EC 3.1.3.2 (a lysosomal marker), Baudhuin (1974); 5'-nucleotidase, EC 3.1.3.5, and glucose 6-phosphatase, EC 3.1.3.9 (markers for plasma membranes and endoplasmic reticulum, respectively), Reinhart & Bygrave (1981); ouabain-sensitive (Na++K+)-ATPase, EC 3.6.1.3 (a plasma membrane marker), Seiler & Fleischer (1982). [3H]Ouabain binding was determined as described by Mansier et al (1983).…”

Calcium: its modulation in liver by cross-talk between the actions of glucagon and calcium-mobilizing agonists

“…A large body of work showed, for instance, that administration of glucagon to intact rats will enhance the ability of the subsequently isolated microsomes to accumulate Ca21 in vitro (e.g. Bygrave and Tranter, 1978;Reinhart and Bygrave, 1981;Andia-Waltenbaugh et al, 1981). With mitochondria too, it was established that prior glucagon administration to the intact rat led to an increased ability of the subsequently isolated organelle to retain Ca2+ (e.g.…”

Modulation of the bilayer thickness of exocytic pathway membranes by membrane proteins rather than cholesterol

“…Apical and basolateral plasma membrane vesicles were purified as in Meier et al (23). Apical and basolateral membrane fractions were routinely assayed for 5Ј-nucleotidase activity (28). Total protein concentration in all samples was determined according to Lowry et al (29).…”