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Biomedical subjects

James E Krause

Publications and source records attributed to James E Krause.

8 recordsLinked to original sources
PubMed2007

Leukocyte effects of C5a-receptor blockade during simulated extracorporeal circulation.

BACKGROUND: Distinct pathways of leukocyte activation during simulated cardiopulmonary bypass are mediated by the complement C5a anaphylatoxin. We hypothesized that a human C5a receptor antagonist would specifically inhibit the inflammatory response of neutrophils to simulated extracorporeal circulation, while preserving the C5b-9 pathway for innate immunity. METHODS: An in vitro extracorporeal circuit recirculated fresh heparinized whole blood through a membrane oxygenator with and without addition of a small molecule human C5a receptor antagonist. Samples were periodically drawn over 90 minutes for complement and leukocyte activation studies. RESULTS: Addition of the C5a receptor antagonist to simulated extracorporeal circulation abrogated both neutrophil CD11b upregulation and interleukin 8 release (p < 0.01 for both), despite full generation of C3a and C5b-9; however, elastase release from neutrophils was unaffected. Although C5a receptor blockade only trended toward inhibiting monocyte CD11b upregulation (p = 0.09), circuit clearance of both monocytes (p = 0.04) and neutrophils (p = 0.01) was significantly decreased. In addition, the C5a receptor antagonist completely blocked both neutrophil-platelet and monocyte-platelet conjugate formation (p < 0.001 for both), without affecting platelet P-selectin expression. CONCLUSIONS: C5a receptor blockade during simulated extracorporeal circulation completely blocked neutrophil beta2 integrin upregulation and induction of plasma interleukin 8, suggesting an acute downregulatory effect on neutrophil chemotaxis-related pathways, while preserving terminal complement generation and neutrophil elastase release. Inhibition of leukocyte-platelet conjugate formation suggests a novel function for leukocyte adhesive receptors, possibly related to preservation of elastase generation.

Blood Platelets↗
PubMed2006

Neurokinin A and neurokinin B in the human retina.

Very recently, the authors found levels of neurokinin (NK) A-like immunoreactivities in the human retina which were more than five times higher than those of substance P (SP). The present study aimed to find out how many of these immunoreactivities can be attributed to NKA and NKB and then the exact distribution pattern of both NKA and NKB was evaluated in the human retina and compared with that of SP. For this purpose, NKA-like immunoreactivities were characterized in the human retina by reversed phase HPLC followed by radioimmunoassay using the K12 antibody which recognizes both NKA and NKB. Furthermore, the retinae from both a 22- and 70-year-old donor were processed for double-immunofluorescence NKA/SP and NKB/SP. The results showed that NKA contributes to approximately two thirds and NKB to approximately one third of the immunoreactivities measured with the K12 antibody. NKA was found to be localized in sparse amacrine cells in the proximal inner nuclear layer, in displaced amacrine cells in the ganglion cell layer with processes ramifying in stratum 3 of the inner plexiform layer and also in sparse ganglion cells. By contrast, staining for NKB was only observed in ganglion cells and in the nerve fiber layer. Double-immunofluorescence revealed cellular colocalization of NKA with SP and also of NKB with SP. Thus, the levels of NKA and NKB are more than three and two times higher than those of SP, respectively. Whereas the distribution pattern of NKA is typical for neuropeptides, the localization of NKB exclusively in ganglion cells is atypical and unique.

Chromatography, High Pressure Liquid↗
PubMed2005

Molecular characterization of the gerbil C5a receptor and identification of a transmembrane domain V amino acid that is crucial for small molecule antagonist interaction.

Anaphylatoxin C5a is a potent inflammatory mediator associated with pathogenesis and progression of several inflammation-associated disorders. Small molecule C5a receptor (C5aR) antagonist development is hampered by species-specific receptor biology and the associated inability to use standard rat and mouse in vivo models. Gerbil is one rodent species reportedly responsive to small molecule C5aR antagonists with human C5aR affinity. We report the identification of the gerbil C5aR cDNA using a degenerate primer PCR cloning strategy. The nucleotide sequence revealed an open reading frame encoding a 347-amino acid protein. The cloned receptor (expressed in Sf9 cells) bound recombinant human C5a with nanomolar affinity. Alignment of the gerbil C5aR sequence with those from other species showed that a Trp residue in transmembrane domain V is the only transmembrane domain amino acid unique to small molecule C5aR antagonist-responsive species (i.e. gerbil, human, and non-human primate). Site-directed mutagenesis was used to generate human and mouse C5aRs with a residue exchange of this Trp residue. Mutation of Trp to Leu in human C5aR completely eliminated small molecule antagonist-receptor interaction. In contrast, mutation of Leu to Trp in mouse C5aR enabled small molecule antagonist-receptor interaction. This crucial Trp residue is located deeper within transmembrane domain V than residues reportedly involved in C5a- and cyclic peptide C5a antagonist-receptor interaction, suggesting a novel interaction site(s) for small molecule antagonists. These data provide insight into the basis for small molecule antagonist species selectivity and further define sites critical for C5aR activation and function.

Amino Acid Sequence↗
PubMed2005

The P2X3 antagonist P1, P5-di[inosine-5'] pentaphosphate binds to the desensitized state of the receptor in rat dorsal root ganglion neurons.

P2X3 purinergic receptors are predominantly expressed in dorsal root ganglion (DRG) neurons and play an important role in pain sensation. P2X3-specific antagonists are currently being sought to ameliorate pain in several indications. Understanding how antagonists interact with the P2X3 receptor can aid in the discovery and development of P2X3-specific antagonists. We studied the activity of the noncompetitive antagonist P1, P5-di[inosine-5'] pentaphosphate (IP5I) at the P2X3 receptor, compared with the well studied competitive antagonist TNP-ATP, using a whole-cell voltage-clamp technique in dissociated rat DRG neurons. IP5I blocked alphabeta-methylene ATP (alphabeta-meATP)-evoked P2X3 responses in a concentration-dependent manner (IC50 = 0.6 +/- 0.1 microM). IP5I effectively inhibited P2X3 currents when pre-exposed to desensitized but not unbound receptors. Furthermore, IP5I equally blocked 1 and 10 microM alphabeta-meATP-evoked currents and had no effect on the desensitization rate constant of these currents. This supports the action of IP5I as a noncompetitive antagonist that interacts with the desensitized state of the P2X3 receptor. In contrast, TNP-ATP inhibited the current evoked by 1 microM alphabeta-meATP significantly more than the one evoked by 10 microM alphabeta-meATP. It also significantly slowed down the desensitization rate constant of the current. These results suggest that TNP-ATP acts as a competitive antagonist and competes with alphabeta-meATP at the P2X3 agonist binding site. These findings may help to explain why IP5I acts selectively at the fast-desensitizing P2X1 and P2X3 subtypes of the P2X purinoceptor, while having much less potency at slow-desensitizing P2X2 and P2X(2/3) subtypes that lack the fast desensitized conformational state.

Adenosine Triphosphate↗
PubMed2005

Transient receptor potential ion channels as targets for the discovery of pain therapeutics.

A subset of transient receptor potential (TRP) channels exhibits activity that is highly sensitive to temperature changes and is expressed in sensory tissues, such as nociceptors and skin. Some of these thermosensitive TRP channels, such as TRPV1, TRPV4 and TRPA1, are activated or sensitized by molecules generated by inflammation and/or cell damage. TRPV1, also known as the capsaicin receptor, is particularly important in mediating hyperalgesic responses in inflammatory pain states, as demonstrated by research in knockout animals and with small-molecule antagonists. It is anticipated that TRPV1 antagonists, and perhaps antagonists at other thermosensitive TRP channels, will provide new therapeutic options with which to treat clinical pain.

Analgesics↗
PubMed2004

Yohimbine inhibits firing activities of rat dorsal root ganglion neurons by blocking Na+ channels and vanilloid VR1 receptors.

Yohimbine, an indole alkaloid, is a natural alpha(2)-adrenoceptor antagonist and is frequently used to assess the mechanism of a drug's effect on alpha-adrenoceptors. Recently, several studies showed that yohimbine exhibited analgesic effects in in vivo animal models. However, the underlying mechanism is not known. We investigated the effects of yohimbine on Na(+) channels and vanilloid VR1 receptors in dorsal root ganglion cells. We found that yohimbine inhibited tetrodotoxin-sensitive Na(+) channels (Na(V)1.2), the tetrodotoxin-resistant Na(+) channels, including both slow inactivating (Na(V)1.8) and persistent (Na(V)1.9) Na(+) channels, and capsaicin-sensitive vanilloid VR1 receptors. Action potential firing activities of dorsal root ganglion neurons evoked by current injection or capsaicin were eliminated by yohimbine. The blocking effects of yohimbine on nociceptive-related ion channels and firing activities of dorsal root ganglion neurons may underlie the ionic mechanism of yohimbine's analgesic effects observed in in vivo studies.

Action Potentials↗
PubMed2003

C5L2, a nonsignaling C5A binding protein.

C5a anaphylatoxin, a potent inflammatory mediator, is known to act through a specific G protein coupled receptor. However, some of the complex effects of C5a in vivo may not be explained solely by the deletion of the known receptor. Here, we show that an orphan receptor, identified as C5L2, is a high affinity C5a binding protein. Unlike the previously described C5aR, C5L2 is obligately uncoupled from heterotrimeric G proteins, in part by virtue of an amino acid alteration in the so-called DRY sequence at the end of the third transmembrane segment. Both human and murine C5L2 bear a leucine for arginine replacement at this site. C5L2, when transfected into several cell types, is weakly phosphorylated in transfected cells following binding of C5a but does not induce significant activation of MAP kinases, mediate calcium flux, or stimulate chemotaxis. Bone marrow cells from wild type respond robustly to C5a with induction and suppression of a number of inflammation related genes. In contrast, C5a receptor deficient mice, which bear C5L2 alone, do not respond to C5a with changes in gene transcription by microarray analyses. Biophysical properties of the C5L2, including slow ligand on and off rates, absence of internalization, and relatively high affinity for the C5a des Arg metabolite, suggest that this receptor may serve to modulate C5a biological functions in vivo. Finally, in contrast to previous reports, we find absolutely no interaction of C5L2 with other anaphylatoxins C3a and C4a.

Amino Acid Sequence↗