Neurovascular anatomy of the embryonic quail hindlimb.

Blood vessel and nerve development in the vertebrate embryo possess certain similarities in pattern and molecular guidance cues. To study the specific influence of shared guidance molecules on nervous and vascular development, an understanding of the normal neurovascular anatomy must be in place. The present study documents the pattern of nervous and vascular development in the Japanese quail hindlimb using immunohistochemistry and fluorescently labeled intravital injection combined with confocal and epifluorescent microscopy. The developmental patterns of major nerves and blood vessels of embryonic hindlimbs between stages E2.75 (HH18) and E6.0 (HH29) are described. By E2.75, the dorsal aortae have begun to fuse into a single vessel at the level of the hindlimb, and have completely fused by E3 (HH20). The posterior cardinal vein is formed at the level of the hindlimb by E3, as is the main artery of the early hindlimb, the ischiadic artery, as an offshoot of the dorsal aorta. Our data suggest that eight spinal segments, versus seven as reported by others (Tanaka and Landmesser,1986a; Tyrrell et al.,1990), contribute to innervation of the quail hindlimb. Lumbosacral neurites reach the plexus region by E3.5 (HH21 & 22), pause for approximately 24 hr, and then enter the hindlimb along with the ischiadic and crural arteries through shared foramina in the pelvic anlage. The degree of anterior-posterior spatial congruency between major nerves and blood vessels of the quail hindlimb was found to be highest medial to the pelvic girdle precursor, versus in the hindlimb proper.

N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine inhibits ligand binding to certain G protein-coupled receptors.

N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) is used widely in biological systems to chelate certain heavy metals, particularly Zn2+. Here we show that TPEN inhibits ligand binding to certain G protein-coupled receptors and is an antagonist at muscarinic receptors. In intact human neuroblastoma SH-SY5Y cells, the binding of the muscarinic receptor ligand [N-methyl-3H]scopolamine methyl chloride was inhibited by TPEN (Ki approximately 26 microM), as was muscarinic receptor agonist-induced inositol 1,4,5-trisphosphate formation (Ki approximately 26 microM). This antagonism was not due to metal ion chelation, indicating that it resulted from a direct interaction of TPEN with muscarinic receptors. Examination of the effects of TPEN on other receptors in SH-SY5Y cell membrane preparations showed that the binding of the nonpeptide opioid receptor ligand [15,16-3H]diprenorphine was strongly inhibited, whereas binding of [125I]vasoactive intestinal polypeptide was not. This pattern of selectivity was also seen in AR4-2J rat pancreatoma cell membranes, in which TPEN inhibited ligand binding to muscarinic receptors, but not that to cholecystokinin receptors. In conclusion, these data show that TPEN inhibits ligand binding to certain G protein-coupled receptors and exhibits selectivity towards those receptors whose transmembrane helices form the predominant site for ligand interaction. TPEN may have widespread antagonistic activity towards G protein-coupled receptors of this kind.