Assessing physiological concentrations of endogenous substances in situ by inducing calcium oscillations in vitro. Case of liver.
Abstract
AIM: To identify the physiological concentration ranges of norepinephrine (NE), vasopressin (VP), and ATP in the rat liver.
METHODS: Rat hepatocytes were isolated by collagenase perfusion. Isolated cells were loaded with the fluorescent calcium indicator Fura-2 acetoxymethyl ester (Fura-2 AM). The effects of different concentrations of norepinephrine, vasopressin, and ATP on intracellular calcium concentration ([Ca2+]i) increases in the freshly isolated rat hepatocytes were investigated. [Ca2+]i was measured by microfluorometry and recorded as fluorescence ratios (F340/F380).
RESULTS: NE, VP, and ATP induced increases in [Ca2+]i in a concentration-dependent manner. At lower concentrations, [Ca2+]i tended to show an oscillatory increase; with increasing concentrations, [Ca2+]i in more cells tended to show phasic or plateau increases. NE, VP, and ATP concentrations likely to induce an oscillatory [Ca2+]i response were 100 - 500 nmol/L, 50 - 100 pmol/L, and < 1 micromol/L respectively.
CONCLUSION: Physiological concentrations of NE, VP, and ATP are 100 - 500 nmol/L, 50 - 100 pmol/L, and < 1 micromol/L respectively in the rat liver.
Keywords:
METHODS: Rat hepatocytes were isolated by collagenase perfusion. Isolated cells were loaded with the fluorescent calcium indicator Fura-2 acetoxymethyl ester (Fura-2 AM). The effects of different concentrations of norepinephrine, vasopressin, and ATP on intracellular calcium concentration ([Ca2+]i) increases in the freshly isolated rat hepatocytes were investigated. [Ca2+]i was measured by microfluorometry and recorded as fluorescence ratios (F340/F380).
RESULTS: NE, VP, and ATP induced increases in [Ca2+]i in a concentration-dependent manner. At lower concentrations, [Ca2+]i tended to show an oscillatory increase; with increasing concentrations, [Ca2+]i in more cells tended to show phasic or plateau increases. NE, VP, and ATP concentrations likely to induce an oscillatory [Ca2+]i response were 100 - 500 nmol/L, 50 - 100 pmol/L, and < 1 micromol/L respectively.
CONCLUSION: Physiological concentrations of NE, VP, and ATP are 100 - 500 nmol/L, 50 - 100 pmol/L, and < 1 micromol/L respectively in the rat liver.