Receptors linked to changes in intracellular calcium concentration[Ca 2+] i in a human erythroleukemia cell line and in non-transformed human erythroid precursor cells

Abstract

One of the major mechanisms by which hormones elevate intracellular Ca2+levels is by generating the second messenger inositol 1,4,5-trisphosphate (InsP3), which activates a Ca2+channel (InsP3receptor) located in the endoplasmic reticulum (ER). This study undertakes to identify the InsP3receptor subtypes (isoforms) in heart and aorta and to characterize their functional properties. The InsP3receptor isoforms were identified from rat heart and aorta tissues using both reverse-transcriptase polymerase chain reaction (RT-PCR) to assess the presence of mRNA for the different isoforms and immunochemistry using InsP3receptor isoform-specific antibodies. Functional studies included ligand binding experiments using [3H]InsP3and InsP3-induced Ca2+release studies using Fluo-3 as the Ca2+sensing dye. All three isoforms of the InsP3receptor were identified using RT-PCR and immunochemical analyses. [3H]InsP3binding studies using microsomes derived from these tissues showed that heart had a 3-fold lower abundance of InsP3receptors than aorta, while both have considerably lower abundance than the well characterized cerebellar microsomes. The affinity of the InsP3binding to the receptor was also different in the three tissues. In cerebellum the Kdwas 60 nM, while aorta had a much higher Kdof 220 nM. Heart microsomes, appeared to show two classes of binding affinity with Kds of 150 nM and 60 nM. Furthermore, the effects of free [Ca2+] on [3H]InsP3binding levels were also different for the three tissues. InsP3binding to both cerebellar and aorta microsomes decreased by 90% and 60%, respectively, above 30 nM free [Ca2+], while InsP3binding to heart was relatively insensitive to changes in [Ca2+]. At maximal InsP3concentrations, aorta microsomes were able to release about 5% of the accumulated Ca2+, compared to 25% by cerebellar microsomes. Heart microsomes, however, showed only very little InsP3-induced Ca2+release ( <0.5%). The EC50concentration for InsP3-induced Ca2+release was 1.2 μ M for aorta while that for cerebellum was 0.3μ M. Known agonists of the cerebellar InsP3receptor such as 3-deoxy InsP3and adenophostin A were also able to mobilize Ca2+from aorta microsomes. In addition, the competitive antagonist heparin and the non-competitive antagonists of the cerebellar InsP3receptor, tetracaine and tetrahexylammonium chloride, were also able to block InsP3-induced Ca2+release from aorta microsomes.