ABSTRACT
Intracerebroventricular (i.c.v.) administration of tachykinin NK(1) receptor agonists induces tapping of the hind legs in gerbils, so-called gerbil foot tapping, which is thought to reflect a fear-related response. The aim of the present study was to examine how ligands selective for NK(1), NK(2) and NK(3) receptors affect the gerbil foot tap response. Agonists selective for NK receptor subtypes were administered i.c.v. and the gerbil foot tap response was monitored. The effect of systemically administered antagonists was also studied. The interaction of ligands with gerbil NK(1) receptors was evaluated using autoradiography on gerbil brain slices with [(3)H]-Sar,Met(O(2))-substance P or [(3)H]GR205171 as radioligand. The effects of ligands on NK(1) and NK(3) receptor-mediated increases in intracellular calcium in vitro were studied in Chinese hamster ovary cells expressing the cloned gerbil receptors. The selective NK(1) receptor agonist ASMSP and the selective NK(3) receptor agonist senktide induced dose-dependent increases in gerbil foot tapping with similar potency. The maximal effect of senktide was approximately 40% of the maximal response evoked by ASMSP. The effects of ASMSP and senktide were blocked by administration of the selective NK(1) receptor antagonist CP99,994 (10 micromol/kg s.c.). The effects of senktide, but not ASMSP, were blocked by administration of the selective NK(3) receptor antagonist SB223412 (50 micromol/kg i.p.). Senktide did not displace NK(1) receptor radioligand binding and was >1000-fold less potent than ASMSP at activating gerbil NK(1) receptors. The selective NK(3) receptor agonist senktide evokes fear-related gerbil foot tapping, an effect which probably involves indirect enhancement of NK(1) receptor signalling.
Subject(s)
Behavior, Animal/drug effects , Neurokinin-1 Receptor Antagonists , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/pharmacology , Receptors, Neurokinin-3/antagonists & inhibitors , Substance P/analogs & derivatives , Animals , Autoradiography , Brain/metabolism , CHO Cells , Calcium/metabolism , Cloning, Molecular , Cricetinae , Cricetulus , Dose-Response Relationship, Drug , Foot , Gerbillinae , Injections, Intraventricular , Male , Neurokinin A/analogs & derivatives , Neurokinin A/pharmacology , Piperidines/pharmacology , Quinolines/pharmacology , Receptors, Neurokinin-2/antagonists & inhibitors , Substance P/antagonists & inhibitors , Substance P/pharmacologyABSTRACT
The Galalpha1-3Gal (alphaGal) antigen is considered the main xenoantigen in the pig to human species combination but other porcine antigens have to be considered such as the swine lymphocyte antigen (SLA), the blood group A/O and the Hanganutziu-Deicher (H-D) antigens. The H-D antigens are N-glycolyl-neuraminic acid (NeuGc) terminated gangliosides that are widely distributed in mammalian species but absent in humans. Upon exposure to a vascularized pig organ, the human recipient can be immunized by direct interaction with the pig tissue or/and by transfer of tissue/cells from the organ into the recipient. In the present work, we describe the release of cells from porcine kidneys upon perfusion and the expression of glycolipid based alphaGal, blood group A/O and H-D antigens in pig lymphocytes. Pig kidneys were flushed with 20 ml of NaCl or Lidocain containing 5000 U heparin, and thereafter perfused with 3000-ml perfusion solution and the cells released were counted and examined microscopically. Neutral glycolipid and ganglioside fractions were extracted from purified pig lymphocytes. The extracted components were characterized by thin layer chromatography, degradation and mass spectrometry. The expression of alphaGal and H-D epitopes on cells released from pig kidneys and purified pig lymphocytes were studied by immune electron microscopy. A total amount of about 300 x 106 leukocytes, mainly lymphocytes were released in the perfusate from the kidneys, of which about 100 x 106 cells were eluated in the 600 to 2400 ml perfusate fraction. Immunelectron microscopical analysis with Griffonia simplicifolia isolectin B4 showed staining of pig leukocytes and other cells, morphologically similar to endothelial cells, released in the perfusate. The purified porcine lymphocytes contained 930 microg neutral glycolipid (4.2 microg/mg cell protein) of which 95% was glycolipids with one to four sugar residues. Immunostaining of the neutral glycolipid fractions revealed alphaGal terminated compounds migrating in the five and 10 to 12 sugar regions and blood group A compounds in the six and eight sugar regions. Two major gangliosides NeuGc-GM3 and NeuGc-GD3 were found in the pig lymphocytes. In a patient extracorporeally xenoperfused with a pig kidney, an increased staining of both alphaGal terminated structures as well as the H-D reactive gangliosides were found in the post-perfusion serum samples. In summary, leukocytes, mainly lymphocytes are released from pig kidneys during perfusion which may contribute to immunization of human xenograft recipients.
