Adrenomedullin (porcine)

Adrenomedullin (ADM) is an evolutionarily conserved multifunctional peptide hormone that regulates implantation, embryo spacing, and placentation in humans and rodents. However, the potential roles of ADM in implantation and placentation in pigs, as a litter-bearing species, are not known. This study determined abundances of ADM in uterine luminal fluid, and the patterns of expression of ADM and its receptor components (CALCRL, RAMP2, RAMP3, and ACKR3) in uteri from cyclic and pregnant gilts, as well as conceptuses (embryonic/fetus and its extra-embryonic membranes) during the peri-implantation period of pregnancy. Total recoverable ADM was greater in the uterine fluid of pregnant compared with cyclic gilts between Days 10 and 16 post-estrus and was from uterine luminal epithelial (LE) and conceptus trophectoderm (Tr) cells. Uterine expression of CALCRL, RAMP2, and ACKR3 were affected by day (P < 0.05), pregnant status (P < 0.01) and/or day x status (P < 0.05). Within porcine conceptuses, the expression of CALCRL, RAMP2, and ACKR3 increased between Days 10 and 16 of pregnancy. Using an established porcine trophectoderm (pTr1) cell line, it was determined that 10-7 M ADM stimulated proliferation of pTr1 cells (P < 0.05) at 48 h, and increased phosphorylated mechanistic target of rapamycin (p-MTOR) and 4E binding protein 1 (p-4EBP1) by 6.1- and 4.9-fold (P < 0.0001), respectively. These novel results indicate a significant role for ADM in uterine receptivity for implantation and conceptus growth and development in pigs. They also provide a framework for future studies of ADM signaling to affect proliferation and migration of Tr cells, spacing of blastocysts, implantation, and placentation in pigs.

Purpose: To investigate adrenomedullin-induced relaxation in isolated porcine ciliary arteries. Methods: In a myograph system (isometric force measurement), precontracted vessels ( approximately 0.1 micro M U46619; thromboxane A(2) analogue or approximately 10 nM endothelin-1) were exposed, in a cumulative manner, to increasing concentrations of adrenomedullin (1 nM to 1 micro M) in the presence or absence of different drugs. Some experiments were conducted in vessels with nonfunctional (intentionally mechanically damaged) endothelium. Results: Adrenomedullin evoked marked relaxation [maximum relaxation (Rel(max)): 85.5 +/- 3.0%; negative log M concentration inducing 50% of Rel(max) (pD(2)): 7.4 +/- 0.1] in comparison to time-controls (Rel(max): 19.2 +/-4.8%; P < 0.001). Relaxation was inhibited by 3 micro M CGRP[8-37] (CGRP(1) receptor antagonist; Rel(max): 27.2% +/- 5.3%; P < 0.001) but not by 3 micro M adrenomedullin[22-52] (presumed adrenomedullin receptor antagonist; P = 0.75). Adrenomedullin-induced relaxation was less pronounced in nonfunctional endothelium vessels (Rel(max): 67.6% +/- 3.1%; pD(2): 6.9 +/- 0.1; P < 0.01). In vessels with functional endothelium, relaxation was not significantly influenced by 0.1 mM N(G)-nitro-L-arginine methyl ester (L-NAME; a nitric oxide synthesis inhibitor), 10 micro M indomethacin (a cyclooxygenase inhibitor), or 10 micro M 17-octadecynoic acid (a cytochrome P(450) inhibitor). In contrast, relaxation was significantly inhibited by either 10 mM tetraethylammonium (nonselective potassium channel inhibitor; P < 0.01) or 50 nM apamin (small conductance potassium channel inhibitor), together with 50 nM charybdotoxin (large and intermediate potassium channel inhibitor; P < 0.01). In the presence of these potassium channel inhibitors, the amount of relaxation was not significantly different (P > 0.50) from that observed in vessels with nonfunctional endothelium. Conclusions: In isolated porcine ciliary arteries, adrenomedullin induces relaxation that involves CGRP(1) receptors and is in part endothelium dependent. Endothelium-dependent relaxation was blocked by some potassium channel inhibitors, suggesting the possible release of an endothelium-derived hyperpolarizing factor (EDHF).

Using a sensitive radioimmunoassay which recognizes the carboxy-terminal region of porcine adrenomedullin, the distribution of immunoreactive adrenomedullin was widely distributed in porcine tissue, and its concentration was highest in adrenal medulla (126 +/- 32 fmol/mg wet tissue; mean +/- SD). Relatively high concentrations of immunoreactive adrenomedullin were found in the right atrium, lung, duodenum, and pituitary gland. The molecular form of immunoreactive adrenomedullin in all porcine tissues was found to be almost wholly porcine adrenomedullin itself by analysis with reverse-phase high-performance liquid chromatography coupled with radioimmunoassay. Minor molecular forms of immunoreactive adrenomedullin were found in the porcine duodenum. These were isolated and identified as adrenomedullin [26-52] and adrenomedullin [34-52].