Enhancement of neurite outgrowth-promoting activity by heparin derivatives in sodium chlorate-treated explant cultures of rat central neurons.
Periodate-oxidized/borohydride-reduced 2-O-desulfated heparin (OR2DSH) was prepared using intact heparin from pig intestine as the starting material. Successive treatments of the heparin by oxidation with sodium periodate and reduction with sodium borohydride yielded periodate-oxidized/borohydride-reduced heparin (OR-heparin). Subsequent 2-O-desulfation of OR-heparin, according to a previously established method, yielded OR2DSH. Digestion of OR2DSH with heparitinases generated unsaturated disaccharides, comprising 86.5% DeltaDiHS-(6,N)S (DeltaUA1-->4GlcNS(6S)) and 13.5% DeltaDiHS-NS (DeltaUA1-->4GlcNS), as well as undigested oligosaccharides in which uronate moieties were derivatized by the cleavage of the covalent bond between the C-2 and C-3 positions by periodate-oxidation. The molecular mass of OR2DSH was determined to be 11 kDa, which is almost the same as those of other heparin derivatives such as 2-O-desulfated heparin (2DSH), 6-O-desulfated heparin (6DSH) and N-desulfated N-reacetylated heparin (NDSNAc-heparin). The ability of OR2DSH to enhance neurite outgrowth-promoting activity was evaluated using the explant culture of neocortical tissue from rat embryo in which endogenous heparan sulfate at the cell surface lost substantial numbers of sulfate groups by the action of 40 micro M sodium chlorate. The maximum activity of OR2DSH (29.7%) was achieved at 10 micro g/ml, and those of OR-heparin (21.7%), 2DSH (18.7%) and intact heparin (16.3%) were 100 micro g/ml, whereas that of NDSNAc-heparin (16.5%) was 1,000 micro g/ml. Completely 6-O-desulfated heparin (100:6DSH) exhibited very weak activity (3.3%) at 1,000 micro g/ml. These results suggest that the potency of OR2DSH to enhance neurite outgrowth-promoting activity is exerted synergetically by two different components in OR2DSH, i.e., the IdoA alpha1-->4GlcNS(6S) unit, which contains 6-O- and 2-N-sulfate groups, and the uronate moiety in which the covalent bond between C-2 and C-3 is cleaved, although the mode of action remains to be clarified.
Usuki
Seigo
S
Central Research Laboratories, Seikagaku Corporation, 3-1253 Tateno, Higashiyamato, Tokyo 207-0021.
Kariya
Yutaka
Y
Rösner
Harald
H
Ishihara
Masayuki
M
Sakamoto
Takashi
T
Masuda
Hiroyuki
H
Kyogashima
Mamoru
M
D000818:Animals; D001894:Borohydrides / Q000737:chemistry; D002240:Carbohydrate Sequence; D002455:Cell Division / Q000187:drug effects; D046508:Culture Techniques; D005260:Female; D006493:Heparin / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology*; D006497:Heparan Sulfate / Q000737:chemistry; D008969:Molecular Sequence Data; D016501:Neurites / Q000187:drug effects* / Q000502:physiology; D010504:Periodic Acid / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar
D001894:Borohydrides; D006493:Heparin; D006497:Heparan Sulfate; D010504:Periodic Acid; C025364:sodium borohydride; C009288:metaperiodate