Characteristics of substance P transport across the blood-brain barrier.

Substance P (SP; NH3(+)-Arg(+)-Pro-Lys(+)-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) belongs to a group of neurokinins that are widely distributed in the central nervous system and peripheral nervous system. The biological effects mediated by SP in the central nervous system include regulation of affective behavior, emesis, and nociception. Many of these actions are believed to be the result of the binding of SP to the neurokinin-1 (NK-1) receptor and subsequent transport across the blood-brain barrier (BBB). The objective of the study was to investigate the involvement of the NK-1 receptor in the permeation of SP across the BBB. Transport of 3H SP (1-13 nM) was investigated using BBMEC monolayers grown on polycarbonate membranes mounted on a Side-bi-Side diffusion apparatus. 3H SP samples were analyzed by scintillation spectrometry. Liquid chromatography-tandem mass spectrometry was used to monitor the transport at higher concentrations (micromolar). SP transport across BBMEC monolayers was found to be saturable (Km = 8.57 +/- 1.59 nM, Vmax = 0.017 +/- 0.005 pmol min(-1) mg(-1) protein) in the concentration range of 0-13 nM. Significant (p < 0.05) decline in 3H SP permeation was observed in the presence of unlabeled SP and at 4 degrees C, indicating that the transport process is carrier-mediated. High-performance liquid chromatography analysis showed no significant metabolism of 3H SP in either the donor or receiver chambers. 3H SP transport was inhibited by 2-11 SP (p < 0.05) but not by any other fragments, indicating that both the C- and N-terminal regions are essential for molecular recognition by the receptor. Endocytic inhibitors (chloroquine, phenylarsine oxide, monensin, and brefeldin) did not inhibit SP transport, suggesting the involvement of a nonendocytic mechanism in SP permeation. Pro(9) SP, a high-affinity substrate for the NK-1 major subtype receptor, significantly (p < 0.05) inhibited the transport of SP. However, Sar(9)Met(O2)(11) SP, a high-affinity substrate for the NK-1 minor subtype receptor, septide, and neurokinin A, inhibitors of NK-1 and neurokinin-2 (NK-2) receptors, respectively, did not produce any inhibition of SP transport. Western blot analysis confirmed the presence of the NK-1 receptor in BBMEC monolayers. The above results provide functional and molecular evidence for the existence of a carrier-mediated mechanism in the transport of SP across the BBB. The effects of specific inhibitors and the results of Western blot analyses demonstrate the involvement of the NK-1 receptor in the transport of SP across the BBB.