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      Signal Crosstalk between Calcium-Sensing and Prostanoid Receptors and its Role in Parathyroid Hormone Secretion

      Albar, Halah

      [Thesis]. Manchester, UK: The University of Manchester; 2018.

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      Abstract

      The calcium-sensing receptor (CaR) maintains extracellular calcium Ca2+o homeostasis by suppressing parathyroid hormone (PTH) secretion. However, the underlying cellular driving force for tonic PTH secretion remains unclear. It is believed that agonists that generate intracellular cyclic-AMP levels may increase PTH secretion and therefore bidirectional crosstalk between CaR signalling and cAMP levels could be of physiological importance. In order to identify possible contributors to parathyroid cAMP accumulation I first confirmed the gene and protein expression of EP4R (prostanoid) and CALCRL (calcitonin receptor-like receptor) in bovine PT gland by using RT-PCR and western blotting respectively. Also, I confirmed the gene expression of the PGE synthase enzyme required for the PGE2 synthesis in bovine PT gland by RT-PCR. And I further confirmed the gene expression of the CALCRL’s associated components particularly the RAMPs and adrenomedullin. Next, I investigated the influence of cAMP on CaR signalling as determined first by Ca2+i mobilisation. Cyclic AMP-generating agonists including PGE2, isoprenaline and histamine enhanced CaR-induced Ca2+i mobilisation at subthreshold Ca2+o concentrations. Indeed, PGE2 and isoprenaline lowered the EC50 for Ca2+o-induced Ca2+i mobilisation (2.5 ± 0.1 for PGE2 and 2.7 ± 0.2 for isoprenaline vs. 4.0 ± 0.3 mM for control, P<0.01) in CaR-HEK cells using Fura2-based microfluorometry. In addition, PGE2 significantly enhanced the CaR-mediated phosphorylation of extracellular signal-regulated kinase (ERK) but tended to inhibit P70 S6 kinase in CaR-HEK cells exposed to moderate Ca2+o concentrations. PGE2 specifically enhanced ERK1/2 phosphorylation via activation of EP4R as this effect was inhibited with the selective EP4R antagonist L161982 (p<0.01). Then, cotreatment with three Gαs-agonists at subthreshold concentrations significantly enhanced CaR-induced Ca2+i mobilisation in the presence of 2 mM Ca2+o whereas each was without effect on its own. This raises the possibility that CaR signalling could be modulated by modest physiological stimuli acting through a variety of Gαs-coupled GPCRs. Then using a FRET-based cAMP reporter (based on Epac) it was shown that PGE2 stimulates cAMP accumulation in CaR-HEK cells while high Ca2+o- or R568-mediated CaR activation lowers cAMP levels via a pertussis toxin-sensitive signalling pathway. Furthermore, the CaR-induced decrease in intracellular cAMP levels was not altered by PKC inhibition, despite this intervention increasing CaR-induced Ca2+i mobilisation. Finally, there is evidence that CaR-inhibiting calcilytic drugs might treat asthma by antagonising the effects of cationic compounds on CaR activation in airway hyper responsiveness disease. Therefore, here I first showed that increasing Ca2+o concentration in human bronchial smooth muscle cells (BSMCs) permits the β2AR agonist salbutamol to increase Ca2+i mobilisation and inhibit ERK1/2 phosphorylation. However, neither effect could be inhibited by calcilytic cotreatment suggesting that the Ca2+o effect does not occur via CaR activation in these BSMCs derived from a non-asthmatic donor. The major finding of the present study is that there is significant crosstalk between CaR and Gαs-coupled GPCR signalling pathways with modest concentrations of cAMP-generating agonists potentiating CaR-induced Ca2+i mobilisation and CaR stimulation attenuating enhanced cAMP levels. This may be of greatest relevance in vivo where endogenous agonists of Gαs-coupled GPCRs may be more abundant than in the simple salt solutions used in vitro. Together, this work provides novel insights into the possible driving force for PTH secretion as well as the CaR-mediated regulation of secretion.

      Bibliographic metadata

      Type of resource:
      Content type:
      Form of thesis:
      Type of submission:
      Degree type:
      Doctor of Philosophy
      Degree programme:
      PhD Physiology 4yr (DEG)
      Publication date:
      Location:
      Manchester, UK
      Total pages:
      204
      Abstract:
      The calcium-sensing receptor (CaR) maintains extracellular calcium Ca2+o homeostasis by suppressing parathyroid hormone (PTH) secretion. However, the underlying cellular driving force for tonic PTH secretion remains unclear. It is believed that agonists that generate intracellular cyclic-AMP levels may increase PTH secretion and therefore bidirectional crosstalk between CaR signalling and cAMP levels could be of physiological importance. In order to identify possible contributors to parathyroid cAMP accumulation I first confirmed the gene and protein expression of EP4R (prostanoid) and CALCRL (calcitonin receptor-like receptor) in bovine PT gland by using RT-PCR and western blotting respectively. Also, I confirmed the gene expression of the PGE synthase enzyme required for the PGE2 synthesis in bovine PT gland by RT-PCR. And I further confirmed the gene expression of the CALCRL’s associated components particularly the RAMPs and adrenomedullin. Next, I investigated the influence of cAMP on CaR signalling as determined first by Ca2+i mobilisation. Cyclic AMP-generating agonists including PGE2, isoprenaline and histamine enhanced CaR-induced Ca2+i mobilisation at subthreshold Ca2+o concentrations. Indeed, PGE2 and isoprenaline lowered the EC50 for Ca2+o-induced Ca2+i mobilisation (2.5 ± 0.1 for PGE2 and 2.7 ± 0.2 for isoprenaline vs. 4.0 ± 0.3 mM for control, P<0.01) in CaR-HEK cells using Fura2-based microfluorometry. In addition, PGE2 significantly enhanced the CaR-mediated phosphorylation of extracellular signal-regulated kinase (ERK) but tended to inhibit P70 S6 kinase in CaR-HEK cells exposed to moderate Ca2+o concentrations. PGE2 specifically enhanced ERK1/2 phosphorylation via activation of EP4R as this effect was inhibited with the selective EP4R antagonist L161982 (p<0.01). Then, cotreatment with three Gαs-agonists at subthreshold concentrations significantly enhanced CaR-induced Ca2+i mobilisation in the presence of 2 mM Ca2+o whereas each was without effect on its own. This raises the possibility that CaR signalling could be modulated by modest physiological stimuli acting through a variety of Gαs-coupled GPCRs. Then using a FRET-based cAMP reporter (based on Epac) it was shown that PGE2 stimulates cAMP accumulation in CaR-HEK cells while high Ca2+o- or R568-mediated CaR activation lowers cAMP levels via a pertussis toxin-sensitive signalling pathway. Furthermore, the CaR-induced decrease in intracellular cAMP levels was not altered by PKC inhibition, despite this intervention increasing CaR-induced Ca2+i mobilisation. Finally, there is evidence that CaR-inhibiting calcilytic drugs might treat asthma by antagonising the effects of cationic compounds on CaR activation in airway hyper responsiveness disease. Therefore, here I first showed that increasing Ca2+o concentration in human bronchial smooth muscle cells (BSMCs) permits the β2AR agonist salbutamol to increase Ca2+i mobilisation and inhibit ERK1/2 phosphorylation. However, neither effect could be inhibited by calcilytic cotreatment suggesting that the Ca2+o effect does not occur via CaR activation in these BSMCs derived from a non-asthmatic donor. The major finding of the present study is that there is significant crosstalk between CaR and Gαs-coupled GPCR signalling pathways with modest concentrations of cAMP-generating agonists potentiating CaR-induced Ca2+i mobilisation and CaR stimulation attenuating enhanced cAMP levels. This may be of greatest relevance in vivo where endogenous agonists of Gαs-coupled GPCRs may be more abundant than in the simple salt solutions used in vitro. Together, this work provides novel insights into the possible driving force for PTH secretion as well as the CaR-mediated regulation of secretion.
      Thesis main supervisor(s):
      Thesis co-supervisor(s):
      Language:
      en

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        Record metadata

        Manchester eScholar ID:
        uk-ac-man-scw:314453
        Created by:
        Albar, Halah
        Created:
        7th May, 2018, 14:53:37
        Last modified by:
        Albar, Halah
        Last modified:
        3rd June, 2019, 11:00:52

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