A specific binding moiety for 1,25-dihydroxyvitamin D₃in basal lateral membranes of carp enterocytes

Abstract

Carp (Cyprinus carpio), a freshwater fish that lives in a low-calcium environment, and Atlantic cod (Gadus morhua), a seawater fish that lives in a high-calcium environment, were studied for the presence of a novel membrane binding protein ("receptor") for the vitamin D metabolite, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. Basal lateral membranes from enterocytes of either species were prepared and analyzed for specific saturable binding. Membranes from carp revealed a dissociation constant of 1.23 nM with a maximal binding capacity of 212 fmol/mg protein. In comparison, membranes from Atlantic cod enterocytes revealed very low and nonsignificant levels of specific binding. The [(3)H]1,25(OH)(2)D(3) binding activity in carp was further characterized for protein dependence, detergent extractability, and competition for binding with the metabolites 25(OH)D(3) and 24R,25(OH)(2)D(3). Finally, introduction of 1,25(OH)(2)D(3) to isolated carp enterocytes enhanced protein kinase C activity within 5 min, whereas intracellular Ca(2+) concentrations were unaffected by a range of 1,25(OH)(2)D(3) concentrations, as judged by fura 2 fluorescence. Thus the binding moiety may be a putative plasma membrane receptor for vitamin D, because it is functionally coupled to at least one signal transduction pathway.

Putative basal lateral membrane receptors for 24,25‐dihydroxyvitamin D3 in carp and Atlantic cod enterocytes: Characterization of binding and effects on intracellular calcium regulation

“…Data are expressed as meanAESEM (n6±10) and the level of signi®cance was set at P`0.05. Our data demonstrate that membrane receptors for 24,25(OH) 2 D 3 in both Atlantic cod and carp could be described as associated with the membrane rather than as an integral protein, as previously reported for the 1,25(OH) 2 D 3 plasma membrane receptor in both chicken [Nemere et al, 1994] and carp [Nemere et al, 2000] [Kato et al, 1998], plasma vitamin D binding protein in ®sh [Hay and Watson, 1977] and DBP in birds and mammals [Hay and Watson, 1977;Kato et al, 1998] Kim et al [1996] reported that the classic cytosolic/ nuclear vitamin D receptor could be associated with cellular acceptance sites in ROS 17/2.8 cells. Putative cytosolic/nuclear receptors for 24R,25(OH) 2 D 3 have been demonstrated in several tissues from different animals [Corvol et al, 1980;Merke and Norman, 1981;So Èmjen et al, 1982aSo Èmjen et al, ,b, 1986, and there are several lines of evidence that 24R,25(OH) 2 D 3 induces transcription [So Èmjen et al, 1986] and protein synthesis [So Èmjen et al, 1984[So Èmjen et al, , 1986.…”

“…Basal lateral membranes from ®sh was prepared by differential centrifugation and sucrose gradient centrifugation as previously described by Nemere et al [2000].…”

“…No long-term effects have been reported for 24,25(OH) 2 D 3 [Sundell et al, 1996]. In the short-term calcium regulation (affecting homeostasis within seconds to minutes), 1,25(OH) 2 D 3 and 24R,25(OH) 2 D 3 have different actions on intestinal Ca 2 -uptake in marine ®sh [Sundell and Bjo Èrnsson, 1990;Larsson et al, 1995;Nemere et al, 2000] compared to freshwater living ®sh [Chartier et al, 1979;Flik et al, 1982;Nemere et al, 2000]. In the marine Atlantic cod (Gadus morhua), 24R,25(OH) 2 D 3 decreases intestinal Ca 2 -in¯ux, whereas 1,25(OH) 2 D 3 is without effect [Sundell and Bjo Èrnsson, 1990;Larsson et al, 1995].…”

Membrane actions of vitamin D metabolites 1α,25(OH)2D3 and 24R,25(OH)2D3 are retained in growth plate cartilage cells from vitamin D receptor knockout mice

“…Rapid membrane-dependent responses to 1a,25(OH) 2 D 3 have been reported in chick intestinal epithelium [Cancela et al, 1988;Nemere and Norman, 1988;de Boland and Norman, 1998], fish enterocytes [Nemere et al, 2000], lymphocytes [Norman, 2000], and osteoblasts [Farach-Carson and Ridall, 1998;Baran et al, 2000;Boyan et al, 2002]. There is evidence that the 1,25-nVDR may associate with plasma membranes [Kim et al, 1996] and in doing so it could contribute to the rapid response.…”

The vitamin D receptor and its ligand 1α,25-dihydroxyvitamin D3 in Atlantic salmon (Salmo salar)

“…As mentioned in the considerations above, rapid responses to calcitriol exist: within minutes after administration, calcitriol increases intestinal calcium transport and activates protein kinase C in FW fish (Nemere et al 2000) and mammals (Boyan et al 1999, Wali et al 2003. Larsson et al (2003) conducted binding studies to enterocyte membrane receptors for calcitriol in rainbow trout (Oncorhynchus mykiss), a close relative of salmon.…”