Sarco‐endoplasmic reticulum Ca²⁺ ATPase (SERCA) inhibitors identify a novel calcium pool in the central nervous system

Abstract

The endoplasmic reticulum (ER) is comprised of subcompartments with distinct functional roles in protein synthesis, protein glycosylation, Ca 2+ sequestration and cell survival (Verma et al. 1990b;Takei et al. 1992;Mattson et al. 2000;Paschen and Frandsen 2001). Disrupted ER Ca 2+ handling may alter protein synthesis, protein folding, and glycosylation and is implicated in both acute and chronic neurological disorders (Mattson et al. 2000;Paschen and Frandsen 2001). The ER utilizes a family of high affinity P-type calcium pumps known as sarco-endoplasmic reticulum Ca 2+ ATPases (SERCA; East 2000). ER-accumulated Ca 2+ can also be released into the cytoplasm in response to second messengers via specific receptor channels, such as inositol-1,4,5-trisphosphate receptors (IP 3 R) and ryanodine receptors (RyR) (Berridge and Irvine 1989;Iino 1999). The expression patterns of SERCAs, IP 3 R and RyR display a high degree of regional heterogeneity within the brain (Verma et al. 1990b(Verma et al. , 1992 and even within individual brain cells (Takei et al. 1992;Berridge 1998). These observations suggest that brain ER Ca 2+ handling properties may be highly specialized in different brain regions.All SERCA isoforms are potently and selectively inhibited by thapsigargin (TG) (Lytton et al. 1991). Although a TG-resistant (TG-R) Ca 2+ -sequestering ER subcompartment has also been identified in several cell lines, it remains poorly understood (Foskett and Wong 1991;Ghosh et al. 1991;Tanaka and Tashjian 1993;Hardy et al. 1995;Waldron et al. 1995;Darby et al. 1996; Galione 1996, Pizzo et al. 1997). The TG-R store is three orders of magnitude less sensitive to inhibition by TG (Waldron et al. 1995) (Hirono et al. 1999;Salvador and Mata 1998;Pizzo et al. 1997). In fact, TG resistance has been proposed by some investigators to simply reflect the luminal filling state of all ER rather than representing a distinct ER compartment (Wells and Abercrombie 1998).In the present study we have utilized ATP-and Mg 2+ -dependent 45 Ca2+ uptake in rat brain microsomes, tissue sections, and digitonin-permeabilized brain-derived cell lines to study the TG-R Ca 2+ buffering compartment. We have also evaluated the contribution of this compartment to cytoplasmic Ca 2+ buffering via calcium imaging studies in non-permeabilized cells. We show that the TG-R pool is primarily expressed in nervous tissue, is markedly enriched in brainstem and spinal cord, is loaded by a novel Mg 2+ -dependent P-type Ca 2+ ATPase activity and displays unique anion permeabilities. (Chaudhary et al. 2001) was synthesized at the Uniformed Services University bio-instrumentation core facility. All other reagents used were of the highest grade available. Methods and materials Materials Cell culturesAll cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in a humidified incubator at 37°C in a 5% CO 2 atmosphere under ATCC recommended conditions. Cerebellar granule cells were cultured from cerebella of 8-day-old Wistar rat pup...

Regulation of Cellular Calcium in Vestibular Supporting Cells by Otopetrin 1

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Secretory pathway Ca2+-ATPase (SPCA1) Ca2+ pumps, not SERCAs, regulate complex [Ca2+]i signals in human spermatozoa

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