In vivo and in vitro evidence of blood-brain barrier transport of a novel cationic arginine-vasopressin fragment 4-9 analog.
The blood-brain barrier (BBB) transport and metabolism of a novel arginine-vasopressin fragment 4-9 [AVP(4-9), isoelectric point; (pI) = 9.2] analog, that is, cationic AVP(4-9) (C-AVP(4-9), PI = 9.8), were examined in vivo and in vitro. At 45 min after an i.v. administration to mice, the cerebrum-to-plasma concentration ratios of (35)S-labeled AVP(4-9) and (125)I-labeled C-AVP(4-9) were 0.103 and 0.330 ml/g cerebrum, respectively, and the BBB permeation clearances were 1.47 x 10(-4) and 3.10 x 10(-4) ml/min/g cerebrum, respectively. In the in vitro study using mouse brain capillary endothelial cells immortalized by SV40 infection (MBEC4), the acid-resistant binding values of (35)S-labeled AVP(4-9) and (125)I-labeled C-AVP(4-9) to MBEC4 at 120 min were 0.93 and 1.95 microliter/mg protein (as the cell/medium ratios), respectively. (35)S-labeled AVP(4-9) showed two-phase saturable acid-resistant binding, and its half-saturation constants (K(D)) were 3.8 nM (high affinity) and 45.7 microM (low affinity). (125)I-labeled C-AVP(4-9) showed single-phase saturable acid-resistant binding, with a K(D) value of 16.4 microM. The acid-resistant binding of (125)I-labeled C-AVP(4-9) was significantly dependent on temperature and medium osmolarity. The acid-resistant binding of (125)I-labeled C-AVP(4-9) was inhibited by dancylcadaverine, phenylarsine oxide (endocytosis inhibitors), 2,4-dinitrophenol (a metabolic inhibitor), and AVP(4-9), poly(L-lysine), and protamine (cationic substances), but not by poly(L-glutamic acid) (an anionic peptide) and the V(1) and V(2) vasopressin receptor antagonists. In addition, the conversion of C-AVP(4-9) to AVP(4-9) in the cerebral homogenate was confirmed by HPLC and mass spectrometry. The present results demonstrate that C-AVP(4-9) is transported through the BBB more effectively than AVP(4-9), via absorptive-mediated endocytosis, and that C-AVP(4-9) is converted to the neuroactive parent peptide, AVP(4-9), in the cerebrum.
Yakult Central Institute for Microbiological Research, Kunitachi-shi, Tokyo, Japan.
S
S
Tanabe
Y
Y
Shimohigashi
Y
Y
Nakayama
T
T
Makino
T
T
Fujita
T
T
Nose
G
G
Tsujimoto
T
T
Yokokura
M
M
Naito
T
T
Tsuruo
T
T
Terasaki
D000818:Animals; D001127:Arginine Vasopressin / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics*; D001711:Biotransformation; D001812:Blood-Brain Barrier; D001921:Brain / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D002196:Capillaries / Q000378:metabolism / Q000502:physiology; D002412:Cations; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004705:Endocytosis / Q000187:drug effects; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics*; D000085822:Prolyl Oligopeptidases; D011485:Protein Binding; D012077:Renal Artery / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D013696:Temperature
D002412:Cations; D010446:Peptide Fragments; D001127:Arginine Vasopressin; C045881:argipressin (4-9); D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases