From Plant to Patient: Thapsigargin, a Tool for Understanding …
81
muscles. In contrast, 1 had no significant effect on the right atrium [102]. This might
be explained by a higher concentration of SERCA in the cardiac muscle, requiring a
higher dose of 1 [103, 104].
8.3 Effect on Cells
Based on the idea that the thapsigargin-triggered skin irritation was caused by the
secretion of histamine from mast cells, thapsigargin (1) was investigated for its
ability to provoke the release of mediators from rat mast cells. An efficient release of
histamine was observed if the mast cells were incubated with submicromolar concentrations of 1 in a Ca
2+ -containing medium. No release was observed in the absence of
Ca
2+ in the extracellular medium [105]. Subsequent experiments confirmed that 1 not
only releases histamine from mast cells but also provokes secretions of mediators
from a broad spectrum of cells belonging to the immune system [106]. Thapsigargin (1) has been shown to induce histidine decarboxylase expression in murine
macrophage RAW 264.7 cells affording a higher content of histamine in the cells
[107]. The discovery that 1 was a cytotoxin initiated all the work for developing it
into a potential drug [108].
9 Molecular Pharmacology of Thapsigargin
9.1 Calcium Homeostasis
In a resting cell, the cytosolic Ca
2+ concentration is about 60–100 nM, the concentration in the endoplasmic/sarcoplasmic reticulum (ER/SR) 100–600 μM, and the
concentration in the extracellular medium approximately 1 mM [109–112]. Calcium
ions are present either as free ions or complexed with calcium-binding proteins [113,
114]. The steady-state with several orders of magnitude difference between the Ca
2+
concentrations in the ER, the extracellular environment and the cytosol are essential
for the survival of the cells and for enabling the cellular response through calcium
signaling coupled to external stimuli (Fig. 8) [109–111]. The concentration gradients are mainly maintained by Ca
2+ ATPases, ion exchangers and release of Ca
2+
mediated by calcium channels. Channels allow the Ca
2+ facilitated down gradient
diffusion through membranes [110]. The ATPases enable Ca
2+ to be pumped against
the concentration gradient into the ER/SR, the Golgi apparatus, or to the external
medium by the use of energy obtained by hydrolysis of ATP to ADP [115–117].
After stimulation of the cell by the interaction of an agonist with a G-protein coupled
receptor (GPCR) a cascade, the reaction occurs [112, 114, 118]. The activation of
the receptor causes protein lipase C to cleave phosphatidyl-inositol-4,5-bisphosphate
(PIP 2 ) to inositol trisphosphate (IP 3 ) and diacylglycerol (DAG) [118, 119]. IP 3 opens
81
muscles. In contrast, 1 had no significant effect on the right atrium [102]. This might
be explained by a higher concentration of SERCA in the cardiac muscle, requiring a
higher dose of 1 [103, 104].
8.3 Effect on Cells
Based on the idea that the thapsigargin-triggered skin irritation was caused by the
secretion of histamine from mast cells, thapsigargin (1) was investigated for its
ability to provoke the release of mediators from rat mast cells. An efficient release of
histamine was observed if the mast cells were incubated with submicromolar concentrations of 1 in a Ca
2+ -containing medium. No release was observed in the absence of
Ca
2+ in the extracellular medium [105]. Subsequent experiments confirmed that 1 not
only releases histamine from mast cells but also provokes secretions of mediators
from a broad spectrum of cells belonging to the immune system [106]. Thapsigargin (1) has been shown to induce histidine decarboxylase expression in murine
macrophage RAW 264.7 cells affording a higher content of histamine in the cells
[107]. The discovery that 1 was a cytotoxin initiated all the work for developing it
into a potential drug [108].
9 Molecular Pharmacology of Thapsigargin
9.1 Calcium Homeostasis
In a resting cell, the cytosolic Ca
2+ concentration is about 60–100 nM, the concentration in the endoplasmic/sarcoplasmic reticulum (ER/SR) 100–600 μM, and the
concentration in the extracellular medium approximately 1 mM [109–112]. Calcium
ions are present either as free ions or complexed with calcium-binding proteins [113,
114]. The steady-state with several orders of magnitude difference between the Ca
2+
concentrations in the ER, the extracellular environment and the cytosol are essential
for the survival of the cells and for enabling the cellular response through calcium
signaling coupled to external stimuli (Fig. 8) [109–111]. The concentration gradients are mainly maintained by Ca
2+ ATPases, ion exchangers and release of Ca
2+
mediated by calcium channels. Channels allow the Ca
2+ facilitated down gradient
diffusion through membranes [110]. The ATPases enable Ca
2+ to be pumped against
the concentration gradient into the ER/SR, the Golgi apparatus, or to the external
medium by the use of energy obtained by hydrolysis of ATP to ADP [115–117].
After stimulation of the cell by the interaction of an agonist with a G-protein coupled
receptor (GPCR) a cascade, the reaction occurs [112, 114, 118]. The activation of
the receptor causes protein lipase C to cleave phosphatidyl-inositol-4,5-bisphosphate
(PIP 2 ) to inositol trisphosphate (IP 3 ) and diacylglycerol (DAG) [118, 119]. IP 3 opens