Abstract
ABSTRACTParathyroid hormone (PTH) stimulates adenylyl cyclase through type 1 PTH receptors (PTH1R) and potentiates the Ca2+ signals evoked by carbachol, which stimulates formation of inositol 1,4,5-trisphosphate (IP3). We confirmed that in HEK cells expressing PTH1R, acute stimulation with PTH(1-34) potentiated carbachol-evoked Ca2+ release. This was mediated by locally delivered cyclic AMP (cAMP), but unaffected by inhibition of protein kinase A (PKA), exchange proteins activated by cAMP, cAMP phosphodiesterases (PDEs) or substantial inhibition of adenylyl cyclase. Sustained stimulation with PTH(1-34) causes internalization of PTH1R–adenylyl cyclase signalling complexes, but the consequences for delivery of cAMP to IP3R within cAMP signalling junctions are unknown. Here, we show that sustained stimulation with PTH(1-34) or with PTH analogues that do not evoke receptor internalization reduced the potentiated Ca2+ signals and attenuated carbachol-evoked increases in cytosolic IP3. Similar results were obtained after sustained stimulation with NKH477 to directly activate adenylyl cyclase, or with the membrane-permeant analogue of cAMP, 8-Br-cAMP. These responses were independent of PKA and unaffected by substantial inhibition of adenylyl cyclase. During prolonged stimulation with PTH(1-34), hyperactive cAMP signalling junctions, within which cAMP is delivered directly and at saturating concentrations to its targets, mediate sensitization of IP3R and a more slowly developing inhibition of IP3 accumulation.
Highlights
Parathyroid hormone (PTH) is the major endocrine regulator of plasma Ca2+ and phosphate concentrations and, with PTH-related peptide (PTHrP), it regulates bone remodelling (Potts and Gardella, 2007)
At the highest concentrations used (.300 nM), PTH(1-34) alone evoked small (,40 nM) increases in [Ca2+]c (Short and Taylor, 2000) (Fig. 1F) that were unaffected by inhibition of adenylyl cyclase, cyclic nucleotide phosphodiesterases (PDEs), protein kinase A (PKA) or EPACs
Signalling from PTH1R to Ca2+ signals through adenylyl cyclase–IP3R junctions PTH(1-34) potentiates carbachol-evoked Ca2+ release by increasing the sensitivity of IP3R (Fig. 1A)
Summary
Parathyroid hormone (PTH) is the major endocrine regulator of plasma Ca2+ and phosphate concentrations and, with PTH-related peptide (PTHrP), it regulates bone remodelling (Potts and Gardella, 2007). Many effects of PTH and PTHrP are mediated by type 1 PTH receptors (PTH1Rs), which are G-protein-coupled receptors (GPCRs) (Mahon, 2012; Mannstadt et al, 1999). *Present address: Metabolic and Structural Biology Department, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, 226015, India.
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