November 6, 1992;
Recoverin in pineal organs and retinae of various vertebrate species including man.
is a recently discovered 26 kDa calcium-binding protein, which activates guanylate cyclase in retinal photoreceptors when the intracellular concentration of free calcium drops upon photoexcitation. In this study we examined the distribution of recoverin
in retinae and pineal organs of Xenopus laevis larvae, 1-day-old chicken, adult pigeon, albino rat, sheep and man by means of immunocytochemistry. Recoverin
immunoreaction was found in all species investigated except for the chicken. In the retina
immunoreaction was restricted to photoreceptors; all other cell types were immunonegative. In the pineal organ
, the recoverin
immunoreaction labeled ''pinealocytes of the sensory line'', i.e. classical pineal photoreceptors of Xenopus laevis larvae, modified pineal photoreceptors of pigeon, and pinealocytes of mammals. The number of recoverin
immunoreactive pinealocytes varied considerably among species of mammals: very few cells were stained in the rat pineal organ
, whereas in rabbit, sheep and man, numerous pinealocytes were found to be recoverin
-immunoreactive. No immunocytochemical staining was observed after preabsorption of the recoverin
antibody with the recombinant protein. Immunoblotting experiments showed that the immunoreaction is due to a protein of 26 kDa in both retina
and pineal tissue
. Thus, recoverin
appears to belong to the family of proteins which are expressed in both retina
and pineal organ
and are highly conserved in the course of phylogeny. Recoverin
may be involved in phototransduction in the directly light-sensitive pineal organs of poikilothermic vertebrates and birds. However, the functional role of recoverin
in the mammalian pineal organ
, which is not photosensitive, remains unknown.
[+] show captions
Fig. 1. Immunocytochemical demonstration of recoverin in retinae of various vertebrate species, a,b: larvae of Xenopus iaevis (stage 61 according
to Nieuwkoop and Faber21). c: adult pigeon, d: adult albino rat. e: sheep, f: man. PE, pigment epithelium (black color is due to endogenous
pigment; *, outer segments; arrowheads, outer nuclear layer; IN, inner nuclear layer, OT, optic tract. Note that the recoverin immunoreaction is
concentrated in the perikarya of photoreceptor cells (outer nuclear layer). Magnification: a,c-f × 400; b × 630. For further details see Materials
Fig. 2. Immunocytochemical demonstration of recoverin in the pineal organ of various vertebrates, a,b: larvae of Xenopus laevis (stage 61
according to Nieuwkoop and Faber 21 }, frontal sections. Arrowheads, recoverin immunoreactive perikarya of pineal photoreceptors cells; double
arrows, meningeal melanophores; Ill, third ventricle, c: pigeon. Recoverin immunoreaction occurs mainly in structures (*) protruding into the
lumen of the pineal follicles and resembling irregular outer segments of modified pineal photoreceptors, d,e: albino rat. Recoverin immunoreaction
is restricted to scattered pinealocytes (arrowheads) mainly located in the periphery of the pineal organ (PO). Surrounding brain regions, e.g.
the occipital cortex (PC) and the superior colliculi (SC) are immunonegative. Some pinealocytes display recoverin-immunoreactive processes
(arrows). f: rabbit. Recoverin-immunoreactive pinealocytes are more numerous than in the rat, but are also found mainly in the periphery of the
pineal organ. Strongly labeled cells can be distinguished from weakly stained or immunonegative elements. Arrows, recoverin immunoreactive
processes of pinealocytes. Magnification: a x 200; b x 630; c,e × 400; d × 50; f × 1,800. For further details see Materials and Methods
Fig. 3. Immunocytochemical demonstration of recoverin in the pineal organ of sheep (a,b) and man (c,d). Note that the intensity of the recoverin
immunoreaction varies markedly from cell to cell. CT, septa of connective tissue. For further details see Materials and l~,~ethods. Magnification: a
x 200: b,d × 240, c x 100.
Fig. 4. Comparison of recoverin immunoreactivity on Western blots
of homogenates from pineal glands of different species. Unless
otherwise indicated a sample of homogenate containing 80 /~g of
protein was applied to each lane: (1) rat; (2) rabbit; (3) sheep; (4)
bovine; (5) human; (6) chicken (50/~g). M r is indicated on the left.
Autoradiographic exposure was 14 h. For further details see Materials
Fig. 5. Western blot analysis of recoverin in human brain, human
retina, bovine retina and individual human pineal glands. Samples of
homogenates containing 60 p,g of protein were applied to each lane
as follows: (1) human brain; (2) human pineal gland pool A; (3)
bovine pineal gland; (4) human retina; (5) bovine retina; (6) human
pineal gland pool B from samples in lanes 7-41; (7-11) individual
human pineal glands. The autoradiographic exposure time was 7 h.
For further details see Materials and Methods.
Fig. 6. Immunorcactivity corresponding to recoverin is blocked by
preincubation of the antiserum with recombinant recoverin. Western
blot analysis of two different human pineal pools of seven glands was
performed. Strips containing 40 ~zg total protein were cut from each
of the two 'curtain' Western blots. Strips i and 2 were obtained from
one blot, and strips 3 and 4 were taken from the second blot. Strips i
and 3 were incubated overnight with recoverin antiserum (1: 1,000)
and strips 2 and 4 were incubated with recoverin a~tiserum (1 : 1,000)
preabsorbed ( <24 h)with 17 ~tg/ml recombir~ant recoverin peptide.
The location of recoverin is indicated by the arrow. The autoradiographic
exposure time was 24 h. For fur|her details see Materials and