ABSTRACT
As well as substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) have recently been found in the superficial dorsal horn of the spinal cord; NKA originating mainly in fine primary afferents. We have investigated the effects of these tachykinins and a range of analogues on somatosensory responses of single identified dorsal horn neurons, when applied ionophoretically to the region of the substantia gelatinosa. Behavioural reflex tests of thermal nociception were carried out in parallel. The role of NK-1, NK-2 and NK-3 receptors was addressed. NK-1-selective agonists attenuated the non-nociceptive responses of identified multireceptive spinocervical tract (SCT) neurons. Of the endogenous tachykinins, both SP and NKB (a weak NK-1 agonist) showed this effect. No role for NK-3 receptors was identified in our experiments. NK-2-selective agonists (including NKA) caused a unique and selective facilitation of thermal nociceptive responses. NKA also reduced reflex response latency in tail-flick and hot plate tests. NKA as a primary afferent transmitter may thus be involved in mediating or facilitating the expression of thermal nociceptive inputs in the substantia gelatinosa. NKA and SP could be considered as acting in concert in the superficial dorsal horn in an effectively pro-nociceptive modulatory role. Evidence from receptor-selective antagonists supports that obtained with agonists for the roles of particular NK receptors in somatosensory processing. NK-2, but not NK-1 or NK-3 antagonists attenuated endogenous thermal nociceptive responses, supporting the hypothesis that an NK-2 agonist (such as NKA) may normally participate in expression of thermal nociception in the superficial dorsal horn. Behavioural experiments showing increased response latencies with a putative NK-2 selective antagonist further supported the involvement of NK-2 receptors in thermal nociception.
Subject(s)
Neurokinin A/pharmacology , Neurons/physiology , Receptors, Neurotransmitter/physiology , Spinal Cord/physiology , Substance P/pharmacology , Animals , Axons/physiology , Bombesin/pharmacology , Cats , Evoked Potentials, Somatosensory/drug effects , Glutamates/pharmacology , Glutamic Acid , Hot Temperature , Kinetics , Neurons/drug effects , Pain/physiopathology , Receptors, Neurokinin-2 , Receptors, Neurotransmitter/drug effects , Spinal Cord/drug effects , Spinal Cord/physiopathology , Substance P/analogs & derivativesABSTRACT
An antiserum raised to the extracellular domain (residues 556-566) of the Alzheimer amyloid precursor protein recognized 70 and 88 kDa proteins in Western blots of rat, Alzheimer, Down's syndrome and control human brain separated by SDS-PAGE. The 70 kDa protein band was resolved into 5 spots by two-dimensional electrophoresis. The findings provide further evidence that a 70 kDa protein is a metabolite of the amyloid precursor protein and reveal an 88 kDa protein which was reduced in 3 out of 6 brains with Alzheimer pathology.
Subject(s)
Amyloid/metabolism , Brain/metabolism , Nerve Tissue Proteins/immunology , Protein Precursors/metabolism , Alzheimer Disease/metabolism , Amyloid/immunology , Amyloid beta-Protein Precursor , Blotting, Western , Down Syndrome/metabolism , Electrophoresis, Gel, Two-Dimensional , Extracellular Space/metabolism , Humans , Molecular Weight , Nerve Tissue Proteins/metabolism , Peptide Fragments/metabolism , Protein Precursors/immunologyABSTRACT
A number of novel luteinizing hormone releasing hormone (LHRH) analogues incorporating biotin together with potential covalent attachment sites have been synthesized. Those based on the des-Gly10-[D-Lys6]-LHRH ethylamide peptide backbone resulted in the most useful characteristics of binding to the LHRH receptor in rat anterior pituitary gland membranes. Of these, des-Gly10-[biotinyl-aminoethylglycyl-D-Lys6]-LHRH ethylamide (XBAL) gave the best specific: non-specific binding ratio, with 44 +/- 6% (+/- S.E.M.) of total binding being specific with a Kd of 131 +/- 16 pM (+/- S.E.M., n = 4) as determined by Scatchard analysis. Two methods have been used to covalently crosslink these analogues with the LHRH receptor; photoaffinity labelling and the use of homobifunctional N-hydroxysuccinimide ester crosslinkers. The photoaffinity analogues gave poor specific: non-specific binding ratios. Of the chemical crosslinkers tested, ethylene glycolbis(succinimidylsuccinate) (EGS) was found to be the most efficient at covalently linking the 125I-XBAL bound to the LHRH receptor site. At an EGS concentration of 5 mM, 23 +/- 3% (+/- S.E.M.) of the specific binding of 125I-XBAL was covalently crosslinked.
Subject(s)
Receptors, LHRH , Affinity Labels , Animals , Cross-Linking Reagents , Gonadotropin-Releasing Hormone/analogs & derivatives , Ligands , Male , Membranes/metabolism , Pituitary Gland, Anterior/metabolism , Rats , Rats, Inbred StrainsABSTRACT
A rabbit antiserum to the C-terminus of the putative brain amyloid precursor was used to probe Western blots of tissue proteins separated by SDS-PAGE. The antiserum specifically labelled a protein of approx. 70 kDa in the Tris buffer-soluble fraction of brain samples from rat, Alzheimer subjects, cases of young and old Down's syndrome, and age-matched controls. The 70 kDa protein was present in low concentrations in human liver and kidney, and was undetectable in human skeletal muscle. The 70 kDa protein may be a metabolite of the amyloid precursor.
