Distinct regional localization of neurocalcin, a Ca2+-binding protein, in the bovine adrenal gland

Abstract

Neurocalcin (molecular weight 23,000 and 24,000) is a Ca(2+)-binding protein with three putative Ca(2+)-binding domains and is present in large amounts in nervous tissues. Neurocalcin isoproteins separated by C18 reverse-phase column chromatography are insoluble in buffer solution and it is impossible to determine the dissociation constant of neurocalcin with Ca2+. To overcome this difficulty, recombinant neurocalcin was synthesized, based on one of the cDNAs of the neurocalcin isoproteins. Stoichiometric titration experiments, using recombinant neurocalcin, indicated that this protein bound 2 mol Ca2+/mol protein and that the apparent dissociation constant for Ca2+ was 2.2 mumol/l, suggesting that neurocalcin plays a physiological role in cellular function. Immunoblotting showed that neurocalcin is present in the bovine adrenal gland in addition to the nervous tissues. Neurocalcin, identified by immunoblotting, was purified from the bovine adrenal gland. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of neurocalcin from the bovine brain showed 23 kDa and 24 kDa double bands, while SDS-PAGE of neurocalcin from the adrenal gland showed a single band of apparently 24 kDa, suggesting that the expression of neurocalcin isoproteins differs from tissue to tissue. The content of neurocalcin in the adrenal gland was 10 micrograms protein/100 g wet tissue. Immunohistochemical analysis showed the occurrence of neurocalcin in zona glomerulosa and adrenal medulla but not in zona fasciculata or zona reticularis. The restricted localization of neurocalcin in the adrenal gland suggests that a similar Ca2+signal pathway may be present in zona glomerulosa a nd the adrenal medulla.

Heterogeneous distribution of neurocalcin-immunoreactive nerve terminals in the mouse adrenal medulla

“…Chromaffin cells were negative in reaction. In the adrenal cortex, the zona glomerulosa showed intense immunoreactivity, as reported by Nakano et al (1993) (Fig. 1A).…”

“…Nakano et al (1993) demonstrated neurocalcin-immunoreactivity in all chromaffin cells of the bovine adrenal gland, but they did not show immunoreactivity of the nerve fibers and terminals. Thus, this study was the first to demonstrate immunoreactivity in the nerve terminals in the adrenal medulla.…”

“…Previous immunohistochemical studies for neurocalcin have demonstrated a specific distribution in neurons of the central and peripheral nervous system (Nakano et al 1992;Bastianelli et al 1993;Okazaki et al 1994;Iino et al 1995a,b;Junttilla et al 1995). In the bovine adrenal gland, it was shown that neurocalcin-like immunoreactivity occurred in cortical cells of the glomerular layer and in all chromaffin cells of the medulla (Nakano et al 1993). However, our preliminary study using the mouse adrenal medulla revealed that neurocalcin-like immunoreactivity was negative in the chromaffin cells and restricted to neural elements, such as nerve fibers and nerve terminals.…”

Intracellular neuronal calcium sensor proteins: a family of EF-hand calcium-binding proteins in search of a function

“…Several observa- Nef 1996) Rat brain: hippocampus: pyramidal cells in CA1; cerebellum: Purkinje cells, basket cell terminals on Purkinje cells (Schaad et al 1996) Rat, bovine and chick retina: ganglion cells, bipolar cells (De Raad et al 1995) Mouse brain: widely distributed in peripheral and central nervous system, highest in hippocampus CA1-CA3, dentate gyrus; ventral horn spinal cord, dorsal root ganglia; high in cortex; low in putamen; cerebellar cortex; astroglial cells in hippocampus and spinal cord (Olafsson et al 1997) r-flup Transcripts widely distributed in the rat brain: cortex, hippocampus, cerebellum, thalamic nuclei (Lindemeier et al 1996) (Boekhoff et al 1997; Fig. 2) Rat, bovine and chick retina: amacrine, bipolar and ganglion cells (De Raad et al 1995) Gerbil hippocampus: pyramidal cells in CA1, CA3, hilus neurons, granule cells of dentate gyrus (Lenz et al 1996b) At the ultrastructural level, VILIP-1 is found both presynaptically, e.g., in parallel and climbing fiber terminals in the cerebellum (Lenz et al 1996a), and postsynaptically, e.g., at hippocampal shaft and spine synapses (Lenz et al 1996b) VILIP-2/NVP-2 Rat brain: all regions except cerebellum and pons (Kajimoto et al 1993 , 1994a) (Grant et al 1995 in situ hybridization and lacZ expression under the control of hippocalcin promoter) Rat retina: ganglion cells (Bastianelli et al 1995b) Mouse brain: hippocampus: pyramidal cells; cerebellum: Purkinje cells; cortex: pyramidal neurons layer II-VI (Sugisaki et al 1994) neurocalcins (α, β, γ1, γ2, δ) Note, antibodies against "neurocalcin" may cross-react with a number of different NCS proteins (see Martinez-Guijarro et al 1998;Kato et al 1998) Bovine cerebrum, cerebellum, brainstem, spinal cord, retina (Nakano et al 1992 b ) Bovine retina: amacrine and ganglion cells Bovine adrenal gland (Nakano et al 1993) Bovine cerebellum: granule cells and molecular layer, weakly in Purkinje cells; cortex: pyramidal cells (Hidaka and Okazaki 1993) Bovine inner ear (Iino et al 1995) Bovine cranial motoneurons (Junttila et al 1995) Mouse adrenal gland (Iino et al 1997) Rat hippocampus: GABAergic interneurons (Martinez-Guijarro et al 1998) Rat retina: amacrine and ganglion cells (Bastianelli et al 1995b) Rat olfactory bulb …”

Atrial natriuretic factor receptor guanylate cyclase, ANF-RGC, transduces two independent signals, ANF and Ca2+

“…Because earlier studies from our laboratory and others’ had shown that NCδ is expressed in the adrenocortical zona glomerulosa (Nakano et al, 1993; Duda et al, 2012a) and that the adrenal gland also contains functional ANF-RGC transduction system (Takayanagi et al, 1987; Sharma et al, 1989; Duda et al, 1991; Rondeau et al, 1995) the adrenal gland appeared to be the tissue of choice for testing whether the ANF-RGC NCδ-Ca 2+ signal transduction system is operational and of physiological significance there.…”