Modifications of intracellular calcium release channels and calcium mobilization following 70% hepatectomy.

The aim of this study was to investigate the properties of ryanodine and IP3 receptors in regenerating liver following 70% hepatectomy, and to evaluate the hepatic Ca2+ distribution and mobilization during this process. Specific [3H]ryanodine and [3H]IP3 binding to hepatic smooth endoplasmic reticulum membranes, as well as subcellular Ca2+ determination by atomic absorption flame photometry and Ca2+ mobilization by INDO-1 AM spectrofluorescence in hepatocytes, was performed in regenerating livers after surgical 70% hepatectomy. Incorporation of 14C amino acids into proteins and of 32P into phospholipids was done in subcellular fractions. Ryanodine receptor Kd presented a dramatic increase after 12 h of surgery and remained high up to 2 days of treatment. IP3 receptor Bmax showed a significant augmentation starting at 6 h after hepatectomy and returning to normal values after 1 week. Cytosolic total calcium content decreased from 12 h until 4 days after hepatectomy whereas the microsomal and mitochondrial total calcium increased at 1 and 2-4 days of liver regeneration, which coincided with the differential turnover of proteins and phospholipids in these fractions. ATP-induced Ca2+ transients in hepatocytes of 24-h-hepatectomized rats confirmed the altered sensitivity of the ryanodine receptor toward its ligand, since 10 times more ryanodine was necessary to alter the ATP-induced Ca2+ transient. The data support the notion that the calcium release channels are targets of mechanisms of metabolic control during the proliferative response following 70% hepatectomy and might be part of the modified intracellular Ca2+ dynamics during liver regeneration.

Ryanodine receptor binding constants in skeletal muscle, heart, brain and liver of the Mexican volcano mouse (Neotomodon alstoni alstoni; Rodentia:Cricetidae). Comparison with five other rodent species.

Equilibrium [3H]ryanodine binding assay was applied to total membrane fractions of six rodent species, including the Mexican volcano mouse Neotomodon alstoni alstoni, Wistar rat Rattus norvegicus albinus, golden hamster Mesocritus auratus, gerbil Meriones unguiculatus, guinea-pig Cavia porcellus, and ground squirrel Spermophillus mexicanus. The organs selected for this study were: skeletal muscle, heart, brain and liver. The constants derived from Scatchard analysis show slight variations in their Kd, ranging from 3 to 15 nM, except in the gerbil's skeletal muscle (38 nM) and the hamster's brain (27 nM). Remarkably, the Bmax calculated in guinea-pig muscle was as high as that reported for the rabbit fast twitch muscle (4.6 pmol/mg of protein) using the same membrane fraction preparation. For all the other skeletal muscles, Bmax was similar to the corresponding heart Bmax values (from 0.5 to 1 pmol/mg of protein). Gerbil cardiac Bmax was the highest (1.1 pmol/mg of protein). The ground squirrel was the rodent with more cerebral ryanodine binding sites (0.26 pmol/mg of protein), whereas the rat and the volcano mouse showed the lowest values (0.12 pmol/mg of protein). The richest sources of hepatic ryanodine receptor were the guinea-pig and rat livers (approximately equal to 0.35 pmol/mg of protein), whereas the lowest Bmax corresponded to the hamster liver (0.018 pmol/mg of protein). These results allow us to detect the similarities and differences of the ryanodine receptor binding constants from four different tissues of some of the rodents most widely used as biomedical laboratory animals.

Properties of the ryanodine-sensitive release channels that underlie caffeine-induced Ca2+ mobilization from intracellular stores in mammalian sympathetic neurons.

The most compelling evidence for a functional role of caffeine-sensitive intracellular Ca2+ reservoirs in nerve cells derives from experiments on peripheral neurons. However, the properties of their ryanodine receptor calcium release channels have not been studied. This work combines single-cell fura-2 microfluorometry, [3H]ryanodine binding and recording of Ca2+ release channels to examine calcium release from these intracellular stores in rat sympathetic neurons from the superior cervical ganglion. Intracellular Ca2+ measurements showed that these cells possess caffeine-sensitive intracellular Ca2+ stores capable of releasing the equivalent of 40% of the calcium that enters through voltage-gated calcium channels. The efficiency of caffeine in releasing Ca2+ showed a complex dependence on [Ca2+]i. Transient elevations of [Ca2+]i by 50-500 nM were facilitatory, but they became less facilitatory or depressing when [Ca2+]i reached higher levels. The caffeine-induced Ca2+ release and its dependence on [Ca2+]i was further examined by [3H]ryanodine binding to ganglionic microsomal membranes. These membranes showed a high-affinity binding site for ryanodine with a dissociation constant (KD = 10 nM) similar to that previously reported for brain microsomes. However, the density of [3H]ryanodine binding sites (Bmax = 2.06 pmol/mg protein) was at least three-fold larger than the highest reported for brain tissue. [3H]Ryanodine binding showed a sigmoidal dependence on [Ca2+] in the range 0.1-10 microM that was further increased by caffeine. Caffeine-dependent enhancement of [3H]ryanodine binding increased and then decreased as [Ca2+] rose, with an optimum at [Ca2+] between 100 and 500 nM and a 50% decrease between 1 and 10 microM. At 100 microM [Ca2+], caffeine and ATP enhanced [3H]ryanodine binding by 35 and 170% respectively, while binding was reduced by > 90% with ruthenium red and MgCl2. High-conductance (240 pS) Ca2+ release channels present in ganglionic microsomal membranes were incorporated into planar phospholipid bilayers. These channels were activated by caffeine and by micromolar concentrations of Ca2+ from the cytosolic side, and were blocked by Mg2+ and ruthenium red. Ryanodine (2 microM) slowed channel gating and elicited a long-lasting subconductance state while 10 mM ryanodine closed the channel with infrequent opening to the subconductance level. These results show that the properties of the ryanodine receptor/Ca2+ release channels present in mammalian peripheral neurons can account for the properties of caffeine-induced Ca2+ release. Our data also suggest that the release of Ca2+ by caffeine has a bell-shaped dependence on Ca2+ in the physiological range of cytoplasmic [Ca2+].