Membrane peptidases in the pig choroid plexus and on other cell surfaces in contact with the cerebrospinal fluid

Abstract

A comprehensive survey of 11 peptidases, all of which are markers for renal microvillar membranes, has been made in membrane fractions prepared from pig choroid plexus. Two fractionation schemes were explored, both depending on a MgCl2-precipitation step, the preferred one having advantages in speed and yield of the activities. The specific activities of the peptidases in the choroid-plexus membranes were, with the exception of carboxypeptidase M, lower than in renal microvillar membranes: those of aminopeptidase N, peptidyl dipeptidase A ('angiotensin-converting enzyme') and gamma-glutamyltransferase were 3-5-fold lower, those of aminopeptidase A and endopeptidase-24.11 were 12-15 fold lower, and those of dipeptidyl peptidase IV and aminopeptidase W were 50-70-fold lower. Carboxypeptidase M had a similar activity in both membranes. Alkaline phosphatase and (Na+ + K+)-activated ATPase were more active in the choroid-plexus membranes. No activity for microsomal dipeptidase, aminopeptidase P and carboxypeptidase P could be detected. Six of the peptidases and (Na+ + K+)-activated ATPase were also studied by immunoperoxidase histochemistry at light- and electron-microscopic levels. Endopeptidase-24.11 and (Na+ + K+)-activated ATPase were uniquely located on the brush border, and the other two peptidases appeared to be much more abundant on the endothelial lining of microvessels. Dipeptidyl peptidase IV and aminopeptidase W were also detected in microvasculature. Pial membranes associated with the brain and spinal cord also stained positively for endopeptidase-24.11, aminopeptidase N and peptidyl dipeptidase A. The immunohistochemical studies indicated the subcellular fractionation did not discriminate between membranes derived from epithelial cells (i.e. microvilli) and those from endothelial cells. The possible significance of these studies in relation to neuropeptide metabolism and the control of cerebrospinal fluid production is discussed.

Great Increase in Antinociceptive Potency of [Leu5]Enkephalin After Peptidase Inhibition

“…Additionally, endopeptidase-24.11, which is inhibited by phosphoramidon, is indicated to play a key role in regulating the concentrations of neuropeptides, including enkephalins, in cerebrospinal fluid (18). Three reports (1,17,18) suggest that the hydrolysis of LE administered i.t.v. is largely prevented in the presence of the three PIs: amastatin, captopril, and phosphoramidon.…”

“…Low molecular weight opioid peptides such as ME and dyn (1-7) are shown to be hydrolyzed in cerebrospinal fluid mainly by aminopeptidase M that is inhibited by amastatin, and the other peptidase activities are suggested to be low in cerebrospinal fluid (17). Additionally, endopeptidase-24.11, which is inhibited by phosphoramidon, is indicated to play a key role in regulating the concentrations of neuropeptides, including enkephalins, in cerebrospinal fluid (18). Three reports (1,17,18) suggest that the hydrolysis of LE administered i.t.v.…”

The Enhancing Effects of Peptidase Inhibitors on the Antinociceptive Action of [Met5]Enkephalin-Arg6-Phe7 in Rats

“…Additionally, endopeptidase-24.11, which is inhibited by phosphoramidon, is indicated to play a key role in regulating the concentrations of neuropeptides, including enkephalins, in cerebrospinal fluid (11). Three reports (1,10,11) suggested that the hydrolysis of ME-RF administered intra-thirdventricularly is largely prevented in the presence of the three PIs: amastatin, captopril, and phosphoramidon. Therefore, the antinociceptive potency of ME-RF shown in the present study probably largely reflects the real potency of ME-RF itself.…”

“…Low molecular weight opioid peptides such as [Met 5 ]enkephalin (ME) and dynorphin A (1-7) are shown to be hydrolyzed in cerebrospinal fluid mainly by aminopeptidase M that is inhibited by amastatin, and the other peptidase activities are suggested to be low in cerebrospinal fluid (10). Additionally, endopeptidase-24.11, which is inhibited by phosphoramidon, is indicated to play a key role in regulating the concentrations of neuropeptides, including enkephalins, in cerebrospinal fluid (11). Three reports (1,10,11) suggested that the hydrolysis of ME-RF administered intra-thirdventricularly is largely prevented in the presence of the three PIs: amastatin, captopril, and phosphoramidon.…”

Hydrolysis of Atrial and Brain Natriuretic Peptides by the Human Astrocytoma Clone D384 and the Neuroblastoma Line SH-SY5Y

“…Amastatin and enkephalin analogues were from Sigma Chemical Co (St. Louis, Mo., USA). All other pepti dase assay materials and inhibitors were obtained as noted by Bourne et al [26] and Hooper and Turner [27], The reagents used for immunohistochemistry were obtained as described previously [28], Materials for cell culture were purchased from sources described by Lyall et al [23] and Vaughan and Ball [29]. D384 cells were obtained as described pre viously [23,24] and SH-SY5Y cells [25] were generously provided by Dr J.L.…”