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Differential mechanisms of Ca(2+) release from vascular smooth muscle cell microsomes
Ahad N K Yusufi1, Jingfei Cheng, Michael A Thompson
1Renal Pathophysiology Laboratory, Department of Laboratory Medicine and Pathology, Mayo Clinic, Mayo Medical School, Rochester, Minnesota 55905, USA.
Abstract:
The release of Ca(2+) from intracellular stores is a fundamental element of signaling pathways involved in regulation of vascular tone, proliferation, apoptosis, and gene expression. Studies of sea urchin eggs have led to the identification of three functionally distinct Ca(2+) signaling pathways triggered by IP3, cADPR, and NAADP. The coexistence and functional relevance of these distinct intracellular Ca(2+) release systems has only been described in a few mammalian cell types. The purpose of this study was to determine whether the IP3, cADPR, and NAADP Ca(2+) release systems coexist in smooth muscle cells (SMC) and to determine the specificity of these intracellular Ca(2+) release pathways. Microsomes were prepared from rat aortic SMC (VSMC) and were loaded with 45Ca(2+). cADPR, NAADP, and IP3 induced Ca(2+) release from VSMC microsomes in a dose-dependent fashion. Heparin blocked only IP3-mediated Ca(2+) release, whereas the ryanodine channel inhibitors 8-Br-cADPR and ruthenium red blocked only cADPR-induced Ca(2+) release. Nifedipine, an L-type Ca(2+) channel blocker, inhibited NAADP elicited Ca(2+) release, but had no effect on IP3- or cADPR-mediated Ca(2+) release. An increase in pH from 7.2 to 8.2 inhibited cADPR-mediated Ca(2+) release, but had no effect on IP3- or NAADP-induced Ca(2+) release. By RT-PCR, VSMC expressed ryanodine receptor types 1, 2, and 3. Ca(2+)-dependent binding of [3H]-ryanodine to VSMC microsomes was enhanced by the ryanodine receptor agonists 4-chloro-methyl-phenol (CMP) and caffeine, but was inhibited by ruthenium red and cADPR. We conclude that VSMC possess at least three functionally distinct pathways that promote intracellular Ca(2+) release. IP3-, cADPR-, and NAADP-induced intracellular Ca(2+) release may play a critical role in the maladaptive responses of VSMC to environmental stimuli that are characteristically associated with hypertension and/or atherogenesis.
Insights
Vascular smooth muscle cells utilize three distinct pathways for intracellular calcium release: inositol trisphosphate (IP3), cyclic ADP-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP). These pathways are crucial for regulating vascular tone and may contribute to hypertension and atherosclerosis.
Area of Science:
- Cellular Biology
- Physiology
- Biochemistry
Background:
- Intracellular calcium (Ca2+) release is vital for cellular signaling, regulating vascular tone, proliferation, apoptosis, and gene expression.
- Distinct Ca2+ signaling pathways (IP3, cADPR, NAADP) identified in sea urchin eggs, but their coexistence and function in mammalian cells are less understood.
Purpose of the Study:
- To investigate the presence and specificity of IP3, cADPR, and NAADP Ca2+ release systems in vascular smooth muscle cells (VSMC).
Main Methods:
- Microsomes from rat aortic VSMC were loaded with 45Ca2+ to measure Ca2+ release.
- Specific inhibitors (heparin, 8-Br-cADPR, ruthenium red, nifedipine) and pH changes were used to differentiate pathways.
- RT-PCR was employed to detect ryanodine receptor expression.
Main Results:
- IP3, cADPR, and NAADP all induced dose-dependent Ca2+ release from VSMC microsomes.
- Pathway specificity was confirmed using selective inhibitors and pH manipulation.
- VSMC expressed ryanodine receptor types 1, 2, and 3, with Ca2+-dependent binding modulated by agonists and inhibitors.
Conclusions:
- VSMC possess at least three distinct functional pathways for intracellular Ca2+ release: IP3, cADPR, and NAADP.
- These pathways likely play a significant role in VSMC maladaptive responses associated with hypertension and atherogenesis.