Abstract
The ryanodine receptor has been mainly regarded as the Ca2+ release channel from sarcoplasmic reticulum controlling skeletal and cardiac muscle contraction. However, many studies have shown that it is widely expressed, with functions not restricted to muscular contraction. This study examined whether ryanodine receptor plays a role in calcium signaling in the liver. RT-PCR analysis of isolated hepatocytes showed expression of a truncated type 1 ryanodine receptor, but no type 2 or type 3 message was detected. We also detected binding sites for [3H]ryanodine in the microsomal cellular fraction and in permeabilized hepatocytes. This binding was displaced by caffeine and dantrolene, but not by ruthenium red, heparin or cyclic ADP-Ribose. Ryanodine, by itself, did not trigger Ca2+ oscillations in either primary cultured hepatocytes or hepatocytes within the intact perfused rat liver. In both preparations, however, ryanodine significantly increased the frequency of the cytosolic free [Ca2+] oscillations evoked by an alpha1 adrenergic receptor agonist. Experiments in permeabilized hepatocytes showed that both ryanodine and cyclic ADP-ribose evoked a slow Ca2+ leak from intracellular stores and were able to increase the Ca2+-released response to a subthreshold dose of inositol 1,4,5-trisphosphate. Our findings suggest the presence of a novel truncated form of the type 1 ryanodine receptor in rat hepatocytes. Ryanodine modulates the pattern of cytosolic free [Ca2+] oscillations by increasing oscillation frequency. We propose that the Ca2+ released from ryanodine receptors on the endoplasmic reticulum provides an increased pool of Ca2+ for positive feedback on inositol 1,4,5-trisphosphate receptors.
Highlights
As with the IP3R family, the ryanodine receptor (RyR) family consists of three isoforms: RyR1 is commonly associated with skeletal muscle cells, RyR2 with cardiac muscle cells and RyR3 is widely distributed with low expression and unclear physiological role [4]
We propose that RyRs play an active role in the Ca2ϩ signaling of hepatocytes, creating local Ca2ϩ microdomains that enhance the responsiveness of neighboring IP3Rs through Ca2ϩ-positive feedback
[3H]Ryanodine Binding in Hepatic Microsomes and Permeabilized Hepatocytes—We examined the binding of [3H]ryanodine to microsomes prepared from rat liver to verify the expression of RyRs at the protein level
Summary
Hepatocytes are polarized epithelial cells where Ca2ϩ signaling plays an essential role in the control of metabolism and function. Hormones, such as vasopressin, angiotensin II, and ␣-adrenergic agonists, mobilize internal Ca2ϩ stores by stimulating the production of the second messenger IP3. On one hand there is evidence for [3H]ryanodine binding to microsomal fractions [13, 14] and Ca2ϩ release from internal stores induced by ryanodine treatment [15, 16], but on the other failure to detect RyR message [17, 18], decrease in frequency of [Ca2ϩ]c oscillations after ryanodine challenge [19] and insensitivity to uncaging of cADPR [18] are reported.
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