Dissociation of p75 receptors and nerve growth factor neurotrophic effects: lack of p75 immunoreactivity in striatum following physical trauma, excitotoxicity and NGF administration.

While the function and regulation of the low affinity (p75) nerve growth factor (NGF) receptor in the central nervous system (CNS) remains a mystery, one of the more intriguing observations involves its response to injury in the adult rat striatum. Following mechanical injury to the striatum, a re-expression of striatal p75 receptors and mRNA purportedly occurs (apparently mediated by elevations in NGF), thus reversing the natural loss of these phenotypic markers that is known to occur during development. This observation has important implications for understanding both the regulation of NGF neurotrophic activity and the role of the p75 receptor, for it implies that the presence of this receptor may be required for NGF trophic activity in the CNS. In an effort to gain a greater understanding of the function and regulation of the low affinity p75 NGF receptor, we performed a series of experiments to study the injury-induced, re-expression phenomenon in the striatum. In the first experiment, we duplicated the mechanical, cannula-induced injury used in the original study. In a follow-up study, we exacerbated that injury by infusing quinolinic acid directly into the striatum. In a third study, the mechanical injury was complemented with chronic striatal infusions of NGF. In a final study, we examined striatal tissue from rats who had been protected from striatal quinolinic acid neurotoxicity by administration of NGF. In no instance was the re-expression of p75 striatal receptors observed, despite positive controls for (a) effective neural trauma, confirmed by histologic and immunocytochemical methods, (b) effective antibody staining, confirmed by appropriate basal forebrain p75 immunoreactivity, and (c) effective biological activity of exogenous NGF, confirmed by hypertrophy of choline acetyltransferase (ChAT)-positive striatal neurons and protection of ChAT-positive striatal neurons against excitotoxicity. At least two important conclusions can be drawn from these studies: (1) the presence or induction of low affinity p75 receptors is not necessary, while the presence of constitutive high affinity tropomyosin related kinase (trk) NGF receptors seem sufficient for NGF trophic activity in the CNS, and (2) the variables necessary to induce re-expression of p75 striatal receptors in adult rats have not yet been elucidated and are apparently complex.

The bradykinin analog RMP-7 increases intracellular free calcium levels in rat brain microvascular endothelial cells.

The vasoactive peptide bradykinin is believed to cause increased vascular permeability by the activation of B2 receptors on the vascular endothelium. A bradykinin analog, H-Arg-Pro-Hyp-Gly-Thi-Ser-Pro-4-Me-Tyr(psi CH2NH)-Arg-OH (RMP-7), was designed and it was proposed that it might increase cerebrovascular permeability by activating B2 receptors on brain microvasculature. In this report, the effects of RMP-7 and related peptides on bradykinin receptor-induced calcium signaling were examined in rat brain microvascular endothelial (RBME) cultures. RBME cells are responsive to bradykinin and exhibit specific [3H]-bradykinin binding, with Scatchard analysis indicating a major class of sites with a Kd of 3.9 +/- 1.4 nM and a minor class of higher affinity sites (Kd approximately 33 pM). RMP-7 displaces a significant component of specific [3H]-bradykinin binding from RBME cells; RMP-10, and RMP-7 diastereomer with a D-amino acid substitution in the number 9 position, does not. [3H]-bradykinin binding to RBME cells is not displaced by desArg9-bradykinin, which indicates that neither class of sites is a B1 bradykinin receptor. RMP-7 induces an increase in intracellular free calcium levels in RBME cells. The time course, magnitude and concentration dependence of RMP-7-induced calcium signaling is similar to that induced by bradykinin in RBME and other endothelial cells. Compared with RMP-7, RMP-10 is at least 1000 times less potent. However, the level of contamination by RMP-7 in the RMP-12 preparation accounts for its activity, an indication that both the RMP-10 and RMP-12 diastereomers are essentially inactive.(ABSTRACT TRUNCATED AT 250 WORDS)

Stereospecific synthesis of peptidyl alpha-keto amides as inhibitors of calpain.

Peptidyl alpha-keto amides have been synthesized and tested as inhibitors of the cysteine protease calpain. A stereospecific synthesis was devised in which Cbz-dipeptidyl-alpha-hydroxy amides were oxidized with TEMPO/hypochlorite to the corresponding alpha-keto amides. This oxidation was accomplished in good yields and without epimerization of the chiral center adjacent to the ketone. The potent inhibition of porcine calpain I by the L,L diastereomers, combined with the poor inhibition by the L,D diastereomers, established the requirement for the all-L stereochemistry of the active inhibitor. The early lead inhibitors were very hydrophobic and, therefore, poorly soluble in aqueous solutions. Using the stereospecific route, new compounds were prepared with polar groups at the C- and N-termini. These modifications resulted in more soluble inhibitors that were still potent inhibitors of calpain. Studies of the stability of these alpha-keto amides showed that absolute stereochemistry can be maintained in acidic and unbuffered environments but general base-catalyzed epimerization of the chiral center adjacent to the ketone occurred rapidly. The alpha-hydroxy precursors were inactive as inhibitors of calpain, which supports the hypothesis that the alpha-keto compounds reversibly form an enzyme-bound tetrahedral species that results from the nucleophilic addition of the catalytic thiol of calpain to the electrophilic ketone of the inhibitor.