Potential binding sites for relaxin in pregnant rabbits.

The purpose of this study was to identify tissues in the day 25 pregnant rabbit that bind relaxin. First, the clearance half-life of relaxin from the rabbit (n = 6) was determined by injecting 10 g porcine relaxin via the marginal ear vein. One-milliliter blood samples were collected via a cannula in the central ear artery. Samples were collected at 10- and 5-min pre-relaxin and at 1-min intervals post-relaxin injection, and the relaxin concentrations determined by radioimmunoassay. The clearance half-life was 4 min. Next, pregnant rabbits were infused with [125I]-relaxin. Control rabbits (n = 3) received 10 g radio-inert relaxin via the marginal ear vein in order to saturate endogenous receptors. Ten minutes later, 10 ng [125I]-relaxin was similarly injected. Treated rabbits (n = 3) received only [125I]-relaxin. After allowing sufficient time for clearance (24 min), a 1-mL blood sample was removed via the central ear vein. Rabbits were euthanized, tissues of maternal and fetal origin excised, and cpm/mg of tissue divided by cpm/mL of blood was determined. Differences in uptake of [125I]-relaxin between control and treated animals, using the Student paired t test, were found for the uterus (P < .05), uterine cervix (P < .03), and mammary gland (P < .05). These data suggest potential rabbit tissues with the LGR-7 receptor.

Is relaxin a calcium transporter/buffer?

Relaxin inhibits smooth muscle contractions by binding Ca2+ and making it unavailable to the contractile elements. Rat uterus sarcoplasmic reticulum (SR) was isolated, incubated with 45Ca in the presence and absence of porcine relaxin, and filtered. In the presence of relaxin, only 4% of the 45Ca was on the filter with the SR as compared to that measured on the filter with SR alone or in the presence of proteins of similar isoelectric point (cytochrome C) and size (insulin). A Sephadex G-25 column was equilibrated with buffer containing 45Ca, and 0.5 mL fractions were collected. Each fraction contained 240-250 K cpm. Relaxin was diluted in the buffer, added to the top of the column, and eluted. A peak of 45Ca (>600 K cpm) was found with the eluted relaxin. No peak of 45Ca was associated with cytochrome C or insulin under the same conditions. The eluted fractions containing 45Ca and relaxin were combined. A sample was incubated with rabbit anti-porcine relaxin serum and goat anti-rabbit serum and centrifuged, and 45Ca in the pellet and supernatant was determined. Ninety-eight percent of the 45Ca was found in the supernatant. Thus, 45Ca was not tightly bound to relaxin or the antibodies knocked the 45Ca off. Is relaxin a calcium transporter or buffer?

Immunohistochemical localization of relaxin-like factor/insulin-like peptide-3 in the bovine corpus luteum.

Relaxin-like factor/insulin-like peptide (INSL)-3 is highly expressed in the bovine corpus luteum throughout the estrous cycle and pregnancy. Demonstration of translation of the relaxin-like factor message was previously shown for the follicle but not the corpus luteum. In this study, relaxin-like factor mRNA was highly expressed in the corpus luteum on days 7 and 14 of pregnancy. Tissues were fixed in 10% neutral buffered formalin, and utilizing two different antibodies to relaxin-like factor, W3 rabbit anti-bovine and 2-8F mouse anti-bovine, relaxin-like factor was localized in fibroblast-like cells. Staining was also observed in the Leydig cells of bovine testicular tissue. No staining was observed in small and large steroidogenic luteal cells, indicating a nonsteroidogenic source of luteal relaxin-like factor. Definitive cell type identification is currently being determined via electron microscopy.