Discovery of adrenomedullin in rat ischemic cortex and evidence for its role in exacerbating focal brain ischemic damage.

Focal brain ischemia is the most common event leading to stroke in humans. To understand the molecular mechanisms associated with brain ischemia, we applied the technique of mRNA differential display and isolated a gene that encodes a recently discovered peptide, adrenomedullin (AM), which is a member of the calcitonin gene-related peptide (CGRP) family. Using the rat focal stroke model of middle cerebral artery occlusion (MCAO), we determined that AM mRNA expression was significantly increased in the ischemic cortex up to 17.4-fold at 3 h post-MCAO (P < 0.05) and 21.7-fold at 6 h post-MCAO (P < 0.05) and remained elevated for up to 15 days (9.6-fold increase; P < 0.05). Immunohistochemical studies localized AM to ischemic neuronal processes, and radioligand (125I-labeled CGRP) displacement revealed high-affinity (IC50 = 80.3 nmol) binding of AM to CGRP receptors in brain cortex. The cerebrovascular function of AM was studied using synthetic AM microinjected onto rat pial vessels using a cranial window or applied to canine basilar arteries in vitro. AM, applied abluminally, produced dose-dependent relaxation of preconstricted pial vessels (P < 0.05). Intracerebroventricular (but not systemic) AM administration at a high dose (8 nmol), prior to and after MCAO, increased the degree of focal ischemic injury (P < 0.05). The ischemia-induced expression of both AM mRNA and peptide in ischemic cortical neurons, the demonstration of the direct vasodilating effects of the peptide on cerebral vessels, and the ability of AM to exacerbate ischemic brain damage suggests that AM plays a significant role in focal ischemic brain injury.

Inhibitors of human heart chymase based on a peptide library.

We have synthesized two sets of noncleavable peptide-inhibitor libraries to map the S and S' subsites of human heart chymase. Human heart chymase is a chymotrypsin-like enzyme that converts angiotensin I to angiotensin II. The first library consists of peptides with 3-fluorobenzylpyruvamides in the P1 position. (Amino acid residues of substrates numbered P1, P2, etc., are toward the N-terminal direction, and P'1, P'2, etc., are toward the C-terminal direction from the scissile bond.) The P'1 and P'2 positions were varied to contain each one of the 20 naturally occurring amino acids and P'3 was kept constant as an arginine. The second library consists of peptides with phenylalanine keto-amides at P1, glycine in P'1, and benzyloxycarbonyl (Z)-isoleucine in P4. The P2 and P3 positions were varied to contain each of the naturally occurring amino acids, except for cysteine and methionine. The peptides of both libraries are attached to a solid support (pins). The peptides are evaluated by immersing the pins in a solution of the target enzyme and evaluating the amount of enzyme absorbed. The pins with the best inhibitors will absorb most enzyme. The libraries select the best and worst inhibitors within each group of peptides and provide an approximate ranking of the remaining peptides according to Ki. Through this library, we determined that Z-Ile-Glu-Pro-Phe-CO2Me and (F)-Phe-CO-Glu-Asp-ArgOMe should be the best inhibitors of chymase in this collection of peptide inhibitors. We synthesized the peptides and found Ki values were 1 nM and 1 microM, respectively. The corresponding Ki values for chymotrypsin were 10 nM and 100 microM. The use of libraries of inhibitors has advantages over the classical method of synthesis of potential inhibitors in solution: the libraries are reusable, the same libraries can be used with a variety of different serine proteases, and the method allows the screening of hundreds of compounds in short periods of time.

Correlation of peptide specificity and IgG subclass with pathogenic and nonpathogenic autoantibodies in pemphigus vulgaris: a model for autoimmunity.

Pemphigus vulgaris (PV) is a rare, potentially fatal, autoimmune disease that affects the skin and mucous membranes. The PV antigen (PVA) has been characterized as desmoglein 3. PV patients carry HLA-DR4- or HLA-DR6-bearing extended haplotypes. We recently demonstrated that patients with active disease have high titers of PV autoantibodies of the IgG1 and IgG4 subclasses. Patients in remission, healthy unaffected relatives, and some MHC-matched normal individuals have low levels of PV autoantibodies, which are IgG1 only. Furthermore, intraperitoneal injection of IgG from patients with active disease caused clinical disease in mice, but IgG from patients in remission, healthy relatives, or MHC-matched normal individuals did not. We prepared 12 peptides of 30 amino acids each (peptides Bos 1-12) spanning the extracellular domain of PVA. Patients with active disease recognize peptides Bos 1 and Bos 6 with high titers of IgG1 and IgG4 autoantibodies. Patients in remission have IgG1 autoantibodies to peptide Bos 1 only, in statistically significantly lower titers (P < 0.01). They no longer have IgG4 subclass autoantibodies to peptide Bos 6. Healthy relatives and normal unrelated individuals have low levels of only IgG1 autoantibodies that recognize only Bos 1. In vitro studies indicate that Bos 6-specific IgG and, to a lesser extent, Bos 1-specific IgG can cause acantholysis. Our data suggest that Bos 6-specific IgG4 is probably the main acantholytic autoantibody, while Bos 1-specific IgG4 may act as a facilitator or enhancer of the process. In this study we illustrate some of the paradigms that demonstrate the interactions between the MHC, subclass of autoantibodies, and peptide specificities of the autoantibodies in the autoimmune process. Thus, PV provides an important model to study the pathogenesis of autoimmunity.