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Protein kinase C and the calcium-sensing receptor in human G cells
PROTEIN
KINASE
C AND THE CALCIUM-SENSING
RECEPTOR
IN HUMAN G CELLS.
Buchan, Alison M.J.; Squires, Paul; Ring, Mark; Meloche, Mark University of British Columbia, Vancouver, Canada Human antral G cells express the calcium-sensing receptor (CaR) and respond to extracellular calcium by releasing gastrin. The receptor on G cells is relatively insensitive to serum calcium requiring levels * 2mM. Human G cells in short term culture were used to determine both the sequence of the receptor expressed and the signal transduction pathways involved. Gastrin cells were cultured for 48h, RNA collected and sequence analysis performed by RT-PCR using overlapping primer pairs covering the entire coding sequence of the CaR. For release experiments cells were incubated with 1 - 3.5mM calcium in the presence or absence of antagonists of a variety of signaling pathways for 1 h at 37oC, supernatants and cell extracts were collected and gastrin levels quantified by RIA. Intracellular calcium levels were measured using Fura-2AM. Analysis of the mRNA encoding the CaR in G cells demonstrated no changes to the sequence previously identified in parathyroid cells. Inhibition of phospholipase C with O.luM U73122 inhibited calcium-stimulated gastrin release and the rise in intracellular calcium levels by 60% (p
QUANTIFICATION ZUCKER RATS
OF OREXIN A lMMUNOREACTlVlTY
AND PREPRO-OREXIN
mRNA IN THE CNS OF
Taheri, Shahrad; Gardiner, James V; Ghatei, Mohammad A; Bloom, Stephen R Imperial College School of Medicine, London, United Kingdom Orexins (A and B) are neuropeptides synthesised from prepro-orexin in the dorsolateral hypothalamus. They were originally named because injection of orexins into the cerebral ventricle of rats resulted in a significant increase in food intake and prepro-orexin mRNA was upregulated with fasting. Orexin neurons have been reported to have leptin receptors, suggesting regulation of orexins by leptin. Using a sensitive and specific radioimmunoassay for orexin A and northern blotting for prepro-orexin mRNA, we studied the content and distribution of orexin A immunoreactivity and prepro-orexin mRNA content in the CNS of obese and lean Zucker rats. Obese Zucker rats have defective leptin receptor action resulting in hyperphagia, obesity and insulin resistance. In both obese and lean Zucker rats, highest orexin A immunoreactivity was detected in the hypothalamus, brainstem, thalamus, the colliculi and the septum. lmmunoreactive orexin A content of hypothalamic regions of obese Zucker rats (in fmol/mg protein) was: Lateral 353.7 +/- 43.4, Posterior 350.4 +I- 37, Ventromedial 622 +/- 47.1, Anterior 399 +/- 43). Despite significantly greater body weight and epididymal fat pad weight, there was no significant difference in orexin A immunoreactivity in any brain or hypothalamic region of obese Zucker rats compared to their lean controls. However, immunoreactivity for melanin-concentrating hormone (MCH), a lateral hypothalamic orexigenic neuropeptide, was higher in the lateral hypothalamic area (obese Vs lean, 9.26 +/- 1.34 pmol/mg protein Vs. 5.76 +/- 0.63 pmol/mg protein, PcO.01) and the ventromedial hypothalamic area (obese Vs lean, 9.91 +I- 0.72 pmol/mg protein Vs. 7.89 +/0.49 pmol/mg protein, P
KINASE
C AND THE CALCIUM-SENSING
RECEPTOR
IN HUMAN G CELLS.
Buchan, Alison M.J.; Squires, Paul; Ring, Mark; Meloche, Mark University of British Columbia, Vancouver, Canada Human antral G cells express the calcium-sensing receptor (CaR) and respond to extracellular calcium by releasing gastrin. The receptor on G cells is relatively insensitive to serum calcium requiring levels * 2mM. Human G cells in short term culture were used to determine both the sequence of the receptor expressed and the signal transduction pathways involved. Gastrin cells were cultured for 48h, RNA collected and sequence analysis performed by RT-PCR using overlapping primer pairs covering the entire coding sequence of the CaR. For release experiments cells were incubated with 1 - 3.5mM calcium in the presence or absence of antagonists of a variety of signaling pathways for 1 h at 37oC, supernatants and cell extracts were collected and gastrin levels quantified by RIA. Intracellular calcium levels were measured using Fura-2AM. Analysis of the mRNA encoding the CaR in G cells demonstrated no changes to the sequence previously identified in parathyroid cells. Inhibition of phospholipase C with O.luM U73122 inhibited calcium-stimulated gastrin release and the rise in intracellular calcium levels by 60% (p
QUANTIFICATION ZUCKER RATS
OF OREXIN A lMMUNOREACTlVlTY
AND PREPRO-OREXIN
mRNA IN THE CNS OF
Taheri, Shahrad; Gardiner, James V; Ghatei, Mohammad A; Bloom, Stephen R Imperial College School of Medicine, London, United Kingdom Orexins (A and B) are neuropeptides synthesised from prepro-orexin in the dorsolateral hypothalamus. They were originally named because injection of orexins into the cerebral ventricle of rats resulted in a significant increase in food intake and prepro-orexin mRNA was upregulated with fasting. Orexin neurons have been reported to have leptin receptors, suggesting regulation of orexins by leptin. Using a sensitive and specific radioimmunoassay for orexin A and northern blotting for prepro-orexin mRNA, we studied the content and distribution of orexin A immunoreactivity and prepro-orexin mRNA content in the CNS of obese and lean Zucker rats. Obese Zucker rats have defective leptin receptor action resulting in hyperphagia, obesity and insulin resistance. In both obese and lean Zucker rats, highest orexin A immunoreactivity was detected in the hypothalamus, brainstem, thalamus, the colliculi and the septum. lmmunoreactive orexin A content of hypothalamic regions of obese Zucker rats (in fmol/mg protein) was: Lateral 353.7 +/- 43.4, Posterior 350.4 +I- 37, Ventromedial 622 +/- 47.1, Anterior 399 +/- 43). Despite significantly greater body weight and epididymal fat pad weight, there was no significant difference in orexin A immunoreactivity in any brain or hypothalamic region of obese Zucker rats compared to their lean controls. However, immunoreactivity for melanin-concentrating hormone (MCH), a lateral hypothalamic orexigenic neuropeptide, was higher in the lateral hypothalamic area (obese Vs lean, 9.26 +/- 1.34 pmol/mg protein Vs. 5.76 +/- 0.63 pmol/mg protein, PcO.01) and the ventromedial hypothalamic area (obese Vs lean, 9.91 +I- 0.72 pmol/mg protein Vs. 7.89 +/0.49 pmol/mg protein, P