Experimental autoimmune cystitis: further characterization and serum autoantibodies.

Previously, we described an animal model for interstitial cystitis (IC), experimental autoimmune cystitis (EAC) [Luber-Narod et al. Urol Res 24:367]. Further characterization of animals with EAC indicates that peak and mean urinary frequency are elevated compared with sham-injected controls and that the disease progresses with at least two cycles of exacerbations and remissions. We had shown evidence suggesting EAC to be autoimmune in nature. In this paper, we identify serum autoantibodies from 9/10 EAC animals which bind to a protein specific to rat bladder with a relative molecular weight of 12-kDa. Such autoantibodies are absent in 12/13 normal and sham-injected animals as well as animals which fail to develop EAC despite disease induction. These findings suggest that EAC is a reproducible model of cyclical increases of urinary frequency, and that a 12-kDa antigen is the target of autoantibodies which correlate with those elevations. Identification of this target antigen may explain the pathogenesis of increased urinary frequency in these animals and potentially in IC as well.

Evaluation of the in vitro Activity of Two Fourth-Generation Cephalosporins Against Bacterial Samples Isolated From Patients in Several Brazilian Hospitals.

Cefpirome and cefepime are two fourth-generation cephalosporins recently introduced in Brazil. They have a very similar range of in vitro antimicrobial activity, but some differences have been noticed. The goal of this study was to compare the in vitro activity of cefpirome and cefepime against bacterial samples isolated in Brazilian hospitals. We studied 931 samples taken from hospitalized patients between April and June, 1998. The minimum inhibitory concentration (MIC) was determined by the Etest method. The potency of cefpirome was similar to that of cefepime, except against enterococci and coagulase-negative staphylococci, where cefpirome proved 2-fold more potent. The MIC(90) for cefepime were inferior to cefpirome in response to Klebsiella pneumoniae (MIC(90), 24 and 96µg/mL, respectively), Pseudomonas aeruginosa (MIC(90), 48 and 128µg/mL, respectively), and other Gram-negative organisms (MIC(90), 64 and 256µg/mL, respectively). Despite the fact that cefpirome presented a slightly broader range of action against Gram-positive bacteria (90% sensitive vs. 78% sensitive to cefepime), and that cefepime presented an equally broad range against Gram-negative bacteria (74% sensitive vs. 65% sensitive to cefpirome), these differences were not considered clinically significant because the sensitivity differed in MIC by less than 2 dilutions. Only 16 (1.7%) of the 931 samples tested showed a significant difference in sensitivity. This study suggests that, except for Acinetobacter sp. and P. aeruginosa, laboratories may routinely test only cefpirome and apply the same category result to cefepime. Since category discrepancies are very rare and cefpirome is slightly less active than cefepime against Enterobacteriaceae, isolates susceptible to cefepime will certanly also be susceptible to cefpirome. To optimize the treatment of severely infected patients, especially where species such as Acinetobacter sp and P. aeruginosa are involved, we recommend that both cephalosporins be tested by using the same susceptibility test method to determine the MIC.

Identification of methionine as the site of covalent attachment of a p-benzoyl-phenylalanine-containing analogue of substance P on the substance P (NK-1) receptor.

Previously we have been able to restrict the site of covalent attachment of a photolabile and radiolabeled derivative of substance P (SP), p-benzoylphenylalanine8-SP (Bpa8-SP), to residues 178-183 located on the second extracellular loop (E2) of the SP (NK-1) receptor (Boyd, N. D., Kage, R., Dumas, J. J., Krause, J. E., and Leeman, S. E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 433-437). To ascertain the specific amino acid in this sequence that serves as the site of covalent attachment for 125I-Bolton-Hunter reagent (BH)-Bpa8-SP, we have employed here a novel solid-phase approach to cyanogen bromide cleavage of the photolabeled receptor followed by mass spectrometric analysis of a purified labeled fragment. SP receptors on transfected Chinese hamster ovary cells were photolabeled with isotopically diluted 125I-BH-Bpa8-SP. A membrane preparation of the photolabeled receptors was adsorbed onto C-18-derivatized silica gel and cleaved with cyanogen bromide. A single radiolabeled fragment containing 63% of the photoincorporated radioactivity was generated and purified by high performance liquid chromatography. Mass spectrometric analysis identified a single molecular ion with a molecular mass of 1751.4 +/- 2, establishing that upon irradiation the bound photoligand forms a covalent link with the methyl group of a methionine residue at the peptide binding site. In view of our previous findings, this methionine is Met-181 on the primary sequence of the SP receptor.

Endogenous substance P mediates cold water stress-induced increase in interleukin-6 secretion from peritoneal macrophages.

Previous studies from this laboratory had shown that exposure of mice to cold water stress leads to an increase in the secretion of interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF alpha) from their peritoneal macrophages. We now report that the secretion of IL-6 from peritoneal macrophages is also increased after cold water stress and that the peptide substance P (SP) participates in this stress-induced response. The stress paradigm involved subjecting male C57BL/6J mice to 5 min swim tests in 10 +/- 2 degrees C water twice daily for 4 d. Cold water stress augments the lipopolysaccharide-induced IL-6 secretion from peritoneal macrophages, elevates immunoreactive SP (iSP) in the peritoneal wash fluid, and reduces iSP in certain peritoneum-containing tissues or organs (i.e., diaphragm, abdominal wall, ileum, and rectum). The 10 d stress time studies indicate that increased IL-6 secretion is positively related to elevated iSP in the peritoneal wash fluid and inversely related to reduced iSP in certain peritoneum-containing tissues. Pretreatment with capsaicin, which depletes SP in the sensory nerve endings, eliminates stress-control differences in the peritoneal wash fluid and in certain peritoneal tissues. Moreover, RP67,580, a specific SP antagonist, eliminates the cold water stress-induced augmentation of IL-6 secretion from peritoneal macrophages. These results suggest that cold water stress promotes the release of SP from peritoneal tissues into the peritoneal cavity, where it participates in the cold water stress-induced macrophage functional alterations.

The peptide binding site of the substance P (NK-1) receptor localized by a photoreactive analogue of substance P: presence of a disulfide bond.

Substance P (SP) is a neuropeptide that mediates multiple physiological responses including transmission of painful stimuli and inflammation via an interaction with a receptor of known primary sequence. To identify the regions of the SP receptor, also termed the NK-1 receptor, involved in peptide recognition, we are using analogues of SP containing the photoreactive amino acid p-benzoyl-L-phenylalanine (Bpa). In the present study, we used radioiodinated Bpa8-SP to covalently label with high efficiency the rat SP receptor expressed in a transfected mammalian cell line. To identify the amino acid residue that serves as the site of covalent attachment, a membrane preparation of labeled receptor was subjected to partial enzymatic cleavage by trypsin. A major digestion product of 22 kDa was identified. Upon reduction with 2-mercaptoethanol the mass of this product decreased to 14 kDa. The 22-kDa tryptic fragment was purified in excellent yield by preparative SDS/PAGE under nonreducing conditions. Subcleavage with Staphylococcus aureus V8 protease and endoproteinase ArgC yielded fragments of 8.2 and 9.0 kDa, respectively. Upon reductive cleavage, the V8 protease fragment decreased to 3.0 kDa while the endoproteinase ArgC fragment decreased to 3.2 kDa. Taking into consideration enzyme specificity, molecular size, determination of the presence or absence of N-glycosylation sites, and recognition by antibodies to specific sequences of the SP receptor, the V8 protease fragment is Thr-173 to Glu-183, while the endoproteinase ArgC fragment is Val-178 to Arg-190. These two fragments share the common sequence Val-Val-Cys-Met-Ile-Glu (residues 178-183). The site of covalent attachment of radioiodinated Bpa8-SP is thus restricted to a residue within this overlap sequence. The data presented here also establish that the cysteine residue in this sequence Cys-180, which is positioned in the middle of the second extracellular loop, participates in a disulfide bond that links the first and second extracellular loops of the receptor.

Substance P and stress-induced changes in macrophages.

The present paper further links nervous-endocrine-immune systems by describing influences of SP on the immune system, and more specifically, on macrophage function. We have discussed how macrophages are important to immune responses in that much of cellular and humoral responses depend on macrophage function. Macrophages are sensitive to stress in that cold-water stress causes increased cytokine production, either spontaneously (IL-1), or after induction with LPS (IL-6, TNF alpha). Increased cytokine levels (IL-1, IL-6) may induce acute phase reactants in the liver, which is presumably the mechanism operative in the studies indicating increases in acute phase reactants after certain stressors in animals. SP is a likely candidate to affect immune function. Previous data show that macrophages from various species have receptors for and respond to SP in vitro. SP stimulates phagocytic and chemotactic capacity, as well as increased cytokine, PGE2, and thromboxane B2 production. SP is also involved in neurogenic inflammation and is likely to be involved in the pathogenesis of several inflammatory diseases. Present data indicate SP's involvement in macrophage responses to stress. We have shown that stress induced differential SP receptor binding to peritoneal macrophages, although the precise nature of binding differences has not yet been clearly elucidated. Stress also induces more immunoreactive SP in the peritoneal fluid that bathes the peritoneal macrophages. We hypothesize that the two events, altered SP binding and concomitant increased ligand, are causally related. In addition to other correlational data showing concomitant increased SP binding plus ligand concentrations, there is more direct evidence that SP ligand may induce SP receptor expression since the SP antagonist, CP-96,345, prevents the induction of SP receptor mRNA in the staphylococcal toxin A-induced gastroenteritis (C. Pothoulakis and S. E. Leeman, personal communication). Further supporting our notion for a causal relationship we have found the elimination of SP in vivo (via capsaicin pretreatment) reduced SP binding, as has been previously reported. We have also examined the role of SP on stress-induced altered macrophage function in vitro. SP greatly enhanced the LPS-induced macrophage TNF alpha production from stressed animals; in contrast, it produced relatively little effect on macrophages from control animals. Capsaicin pretreatment diminished the enhanced cytokine production in response to stress, such that levels of TNF alpha and IL-6 approximated those of control mice. Taken together, past and present data suggest that (1) stress may initiate, or at least contribute to, an inflammatory response, and that (2) SP is involved in the macrophage stress response. SP has long been known to be involved in inflammatory processes; our data further suggest its role in mediating stress-induced cytokine alterations.

Characterization of the substance P (NK-1) receptor in tunicamycin-treated transfected cells using a photoaffinity analogue of substance P.

Chinese hamster ovary cells expressing the N-glycosylated substance P (NK-1) receptor were treated with the glycosylation inhibitor tunicamycin and photolabeled with 125I-Bolton-Hunter-p-benzoyl-L-phenylalanine8-substance P. Two radioactive proteins of M(r) 80,000 and 46,000, representing the glycosylated and nonglycosylated substance P (NK-1) receptor, respectively, were observed. The IC50 for the inhibition of photolabeling of both receptor forms was 0.3 +/- 0.1 nM for substance P and 30 +/- 5 nM for neurokinin A (substance K). Thus, glycosylation of the substance P (NK-1) receptor has no detectable effect on the affinity of the substance P (NK-1) receptor for substance P or neurokinin A (substance K).

Substance P enhances the secretion of tumor necrosis factor-alpha from neuroglial cells stimulated with lipopolysaccharide.

The neuropeptide, substance P (SP), can stimulate secretion of TNF-alpha from macrophages. Neuroglia have SP receptors and subserve various macrophage-like functions in the central nervous system. We investigated whether SP stimulates secretion of TNF-alpha from primary cultures of neuroglial cells containing both astrocytes (approximately 90%) and microglia (approximately 10%). SP alone had no effect; however in the presence of LPS (10 ng/ml), SP (1 to 10 nM) caused a dose-dependent increase in TNF-alpha secretion above the level measured in response to LPS alone. The effective doses of SP correlated with 125I-labeled Bolton Hunter-conjugated SP binding (Kd 0.2 nM) to these cultures. Incubation with LPS did not change the number or affinity of SP-binding sites. In cultures enriched for microglia (> 99% pure), LPS stimulated the secretion of TNF-alpha but SP caused no enhancement. Microglia have no detectable 125I-labeled Bolton-Hunter-conjugated SP binding sites in the presence or absence of LPS. These results indicate that the action of SP is mediated through astrocytes. We investigated whether IL-1 mediates the SP enhancement of TNF-alpha secretion. Addition of IL-1-neutralizing antisera to mixed cultures stimulated with both LPS and 10 nM SP decreased TNF-alpha secretion to the level observed with LPS alone. LPS alone stimulated the secretion of IL-1 in a dose-dependent manner in the primary cultures, and this LPS-mediated IL-1 secretion was enhanced by SP. This enhancement was not observed in microglial cultures. SP may therefore play a role in neuropathologies in which these cytokines have been implicated.

Biochemical characterization of two different forms of the substance P receptor in rat submaxillary gland.

Studies were designed to examine the basis for the difference in molecular weights of the two proteins detected in membrane preparations of rat submaxillary glands after photolabeling with a radioactive analogue of substance P, 125I-p-benzoyl-L-phenylalanine8-substance P. When the two proteins were separated and individually digested with endoglycosidase F, the relative molecular weight of each protein was reduced by approximately 10,000, indicating that the extent of glycosylation of both proteins is the same. To test whether the difference in their molecular weights can be attributed to a difference in the lengths of the two proteins, photolabeled membranes were treated with carboxypeptidase Y before solubilization to remove from each photolabeled protein the carboxy-terminal portion that extends beyond the membrane. Only one, albeit diffuse, band was now observed that on subsequent deglycosylation with endoglycosidase F was more clearly seen to be a single band, indicating that differing lengths of peptide chains were cleaved from the two proteins. These results permit the interpretation that the difference in the two forms of the substance P receptor present in rat submaxillary glands is due to differences in the length of their carboxy termini.

Rabbit neuromedin U-25: lack of conservation of a posttranslational processing site.

The rabbit small intestine contains neuromedin U-like immunoreactivity (22 pmol/g wet tissue weight) that was resolved into a single major molecular form by reversed-phase HPLC. The primary structure of the peptide was established as: Phe-Pro-Val-Asp-Glu-Glu-Phe-Gln-Ser-Pro10-Phe-Gly-Ser-Arg-Ser-Arg- Gly-Tyr-Phe- Leu20-Phe-Arg-Pro-Arg-Asn.NH2. In rabbit neuromedin U, the Arg16-Arg17 dibasic residue processing site that is found in pig and dog neuromedin U-25 is replaced by Arg-Gly, but this potential monobasic processing site is not utilized by cleavage enzyme(s) in the intestine.

Quantitation and characterization of peptides from the C-terminal flanking region of rat and bovine preprotachykinins.

Sequence analysis of cDNAs has shown that the biosynthetic precursors of substance P (alpha-, beta-, and gamma-preprotachykinins) contain a common amino acid sequence in the C-terminal flanking region that has not been conserved between species. Antisera have been raised against the synthetic peptide Tyr-Glu-Arg-Ser-Ala-Met-Gln-Asn-Tyr-Glu, which represents rat beta-preprotachykinin-(117-126)-peptide, and used in radioimmunoassays. Antiserum R50 reacted strongly with C-flanking peptides in extracts of rat and bovine tissues whereas antiserum GP-4 reacted only with the rat peptides. The primary structure of the predominant molecular form of preprotachykinin C-flanking peptide in an extract of bovine corpus striatum was established as: Ala-Leu-Asn-Ser-Val5-Ala-Tyr-Glu-Arg-Ser10-Val-Met-Gln-Asp-Tyr1 5-Glu. This sequence represents beta-preprotachykinin-(111-126)-peptide which is equivalent to gamma-preprotachykinin-(96-111)-peptide. A C-flanking peptide with similar chromatographic properties was identified in extracts of rat brain and gut together with a second immunoreactive component that may represent a fragment or a posttranslationally modified variant. A peptide corresponding to the 37-amino-acid residue C-flanking peptide derived from alpha-preprotachykinin was not detected in the extracts as expected from the known low abundance of alpha-preprotachykinin mRNA in rat brain and gut.

Neurokinin B in a human pheochromocytoma measured with a specific radioimmunoassay.

An N-terminally directed antiserum to neurokinin B was raised in rabbits using an immunogen prepared by coupling the free-SH group of neurokinin B extended from its C-terminus by a cysteine residue (NKB-Cys) to an -NH2 group on human serum albumin using a heterobifunctional cross-linking reagent. In radioimmunoassay with 125I-Bolton-Hunter-labelled NKB-Cys as tracer, the antiserum showed no cross-reactivity with other tachykinins. An extract of a human pheochromocytoma, previously shown to contain peptides derived from preprotachykinin A, contained NKB-LI (13 pmol/g wet weight). The retention time of tumor neurokinin on reversed-phase HPLC was the same as that of synthetic neurokinin B. Peptides with the retention times of substance P, neurokinin A, neurokinin A (3-10)-peptide and neuropeptide K were also identified in the tumor extract. NKB-LI was not detected in extracts of a further nine pheochromocytomas or in five carcinoid tumors that expressed the preprotachykinin A gene.

Post-translational processing of preprotachykinins. Isolation of protachykinin-(1-37)-peptide from human adrenal-medullary phaeochromocytoma tissue.

The biosynthetic precursors of the mammalian tachykinins, alpha-, beta-and gamma-preprotachykinins, contain a common N-terminal region of 74 amino acids. A polyclonal antiserum was raised against a synthetic peptide representing N-tyrosylated beta-preprotachykinin-(48-56)-peptide as predicted from the nucleotide sequence of cloned DNA complementary to human beta-preprotachykinin mRNA. By using this antiserum in radioimmunoassay, a single immunoreactive peptide was identified in an extract of a human pheochromocytoma that produced substance P and neurokinin A. Partial microsequencing and determination of the amino acid composition of the peptide indicated identity with preprotachykinin-(20-56)-peptide. Thus the data demonstrate that the Ala19-Glu20 bond in preprotachykinin is the site of cleavage of the signal peptide.

Measurement and partial characterization of the C-terminal flanking peptides from bovine preprotachykinins in extracts of brain and gut.

Sequence analysis of bovine cDNAs has shown that the biosynthetic precursors of the tachykinins (alpha-, beta- and gamma-preprotachykinins) contain a common amino acid sequence [AYERSVMQDYERRRK] in the C-terminal flanking region. Using an antiserum raised against the synthetic peptide [YERSVMQDYE] in a specific radioimmunoassay, preprotachykinin C-terminal flanking peptide (C-PPT)-like immunoreactivity was measured in extracts of bovine corpus striatum, cerebral cortex and small intestine in concentrations that were equimolar with substance P. Consistent with the presence of two amino acid substitutions in the C-terminal flanking region of human and rat preprotachykinins, the antiserum did not detect immunoreactivity in extracts of human and rat tissues. Chromatographic analysis of the extracts identified two major immunoreactive components. It is proposed that they represent the 16-amino acid residue C-terminal flanking peptide derived from beta- and gamma-preprotachykinins and the 37-amino acid residue C-terminal flanking peptide derived from alpha-preprotachykinin. Treatment of tissue extracts with carboxypeptidase B did not result in a change in C-PPT-like immunoreactivity or in a change in chromatographic properties of the C-terminal flanking peptides suggesting that the C-terminal basic tetrapeptide (RRRK) had already been removed from the primary transcript of the preprotachykinin mRNAs by the action of endogenous processing enzymes.

Neuropeptide-gamma: a peptide isolated from rabbit intestine that is derived from gamma-preprotachykinin.

The neurokinin A-like immunoreactivity in an extract of rabbit small intestine was resolved into two molecular forms by gel permeation chromatography. These components were purified to apparent homogeneity by reverse-phase HPLC. The primary structure of the larger component was established as the following: Asp-Ala-Gly-His-Gly-Gln-Ile-Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser-Phe-Val- Gly-Leu - Met.NH2. This amino acid sequence represents residues (72-92) of gamma-preprotachykinin, as predicted from the nucleotide sequence of a cloned cDNA from the rat. The peptide, termed neuropeptide-gamma, lacks residues (3-17) of neuropeptide K, and this segment is specified exactly by exon 4 in the preprotachykinin gene. The smaller form of neurokinin A-like immunoreactivity was identical to neurokinin A. Neuropeptide K was not present in the extract, demonstrating that the pathways of post-translational processing of beta- and gamma-preprotachykinins in the rabbit gut are different.