Desensitisation of calcitonin gene-related peptide responsiveness but not adrenomedullin responsiveness in vascular smooth muscle cells.

Adrenomedullin (ADM) and calcitonin gene-related peptide (CGRP) are distantly related peptides. Both act through G protein-coupled receptors on vascular smooth muscle cells to increase intracellular cAMP concentrations, causing vasorelaxation. Recent evidence suggests that both peptides bind to a common heptahelical receptor, with specificity for each peptide being determined by a receptor activity modifying protein (RAMP). This hypothesis predicts that each peptide should desensitise the cellular response to subsequent stimulation by the other. We have studied the patterns of desensitisation of ADM/CGRP receptors in rat aortic vascular smooth muscle cells. Cells were incubated for 20 min in either serum free medium (SFM), alone (control) or in SFM containing vasoactive agonist (e.g. ADM 10(-8) M, CGRP 10(-7) M, angiotensin II 10(-9) M or isoproterenol 10(-6) M). Cells were then washed and incubated for a further 20 min in SFM containing a second agonist and 1 mM isobutyryl methyl xanthine. Cells were harvested and assayed for cAMP. Pre-exposure of cells to CGRP, isoproterenol, angiotensin II or ADM, decreased cAMP generation in response to subsequent stimulation with CGRP by 84% (+/-5), 66% (+/-18), 45% (+/-5) and 60% (+/-10) respectively (mean+/-s.d.). Pre-incubation of cells with 100 nM H-89, a protein kinase A (PKA) inhibitor, abolished the desensitisation of CGRP by itself, implying that this desensitisation was mediated through PKA. In contrast, there was no attenuation of the cAMP response to stimulation with ADM by pre-exposure to ADM and all other agonists tested. Identical results were seen with or without PKA inhibition by H-89. These results indicate that the ADM receptor does not desensitise over this time period in RAVSMCs, in contrast to the CGRP receptor, which is desensitised by pre-exposure to CGRP and other vaso-active agonists. These data also suggest that ADM and CGRP act through separate receptors in these cells.

Desensitization of CGRP and adrenomedullin receptors in SK-N-MC cells: implications for the RAMP hypothesis.

Recent evidence (1) suggests that the related peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) bind to the same heptahelical transmembrane receptor, with receptor specificity being determined by a receptor associated modifying protein (RAMP). If correct, this hypothesis would predict that each peptide should desensitize the cellular response to subsequent stimulation by itself or the other peptide. We have therefore studied the patterns of desensitization of these receptors in SK-N-MC cells. SK-N-MC cells were stimulated for 20 minutes in either serum free medium alone (control) or SFM containing AM 10(-8) M or CGRP 10(-7) M. Cells were then incubated for a further 20 minutes in SFM containing a second agonist and 1 mM isobutyryl methylxanthine (IBMX), before harvesting and assay for cAMP. Pre-exposure of cells to CGRP or AM decreased cAMP generation in response to subsequent stimulation with CGRP by 58% (+/-14) and 42% (+/-14) (SD) respectively. Pre-incubation of cells with 100 nM H-89 abolished this effect, indicating that desensitization was mediated through PKA. In contrast, there was no attenuation of the cAMP response to stimulation with AM by pre-exposure to AM or CGRP. These results suggest that CGRP and AM receptors exhibit different patterns of desensitization in SK-N-MC cells: a finding with significant implications for the RAMP hypothesis.

Endogenous polypeptide-chain length and partial sequence of aspartate transcarbamoylase from wheat, characterised by immunochemical and cDNA methods.

Aspartate transcarbamoylase (ATCase) is purified from wheat germ as a monofunctional trimer of 36 kDa chains. The possibility that this may be a proteolytic fragment of a large endogenous complex in which ATCase is covalently fused to other pyrimidine-pathway enzymes (such as exists in animals or fungi) was tested. Examination of a rabbit antiserum raised against the purified enzyme confirmed the presence of anti-(wheat ATCase) antibodies by several independent methods. Extracts of wheat seedlings prepared under non-proteolysing conditions were challenged by the antiserum, and in some cases by purified anti-(36 kDa ATCase) antibodies, using immunoblotting techniques. The 36 kDa species was the dominant immunopositive polypeptide. However, the extract also contained small amounts of two larger (45 and 55 kDa) immunopositive polypeptides, as well as traces of polypeptides smaller than 36 kDa, which were assumed to be minor proteolytic products. Neither of the 45 or 55 kDa polypeptides is large enough to also incorporate a carbamoyl phosphate synthetase or dihydroorotase of the sizes found in other organisms. They may be targeted precursors of ATCase with intact leader sequences. A screen of a wheat cDNA expression library by the anti-(ATCase) serum yielded a single positive clone which was shown, by DNA sequencing, to be a concatemer of two cDNAs, one of which encoded a partial ATCase. Northern analysis using this clone as probe identified two transcripts of about 1.3 and 1.0 kbp, but showed no evidence of a transcript of 2 kbp or greater which would be required to encode a bifunctional polypeptide. These results confirm that wheat ATCase is not translationally fused to dihydroorotase or carbamoylphosphate synthetase, as it is in animals and fungi. The deduced amino-acid sequence of the partial wheat ATCase is compared with the catalytic chain of the ATCase from Escherichia coli and with other ATCases.

Identification of a novel vertebrate homeobox gene expressed in haematopoietic cells.

This paper describes the characterisation of a novel chicken homeobox gene, Prh, whose encoded homeodomain sequence differs significantly from those of other factors which have been described. As expected, a portion of the encoded protein, containing the homeodomain, is capable of sequence-specific DNA-binding. Outside the homeodomain, Prh, possesses an N-terminal region extremely rich in proline residues and a C-terminal acidic portion, either of which may function as transcription regulatory domains. Since, among the chicken tissues tested, its transcription is restricted to haematopoietic cells, lung and liver, it may function in tissue-specific patterns of gene regulation. Human and murine Prh homologues have also been identified; so it is likely that such genes are a general feature of vertebrate genomes.

The application of aqueous two-phase systems to oligosaccharide synthesis by alpha-mannosidase catalysed glycosyl transfer reactions.

Aqueous two-phase systems formed from PEG and dextran have been applied to the synthesis of oligosaccharide by Jack bean alpha-mannosidase in reverse. Whilst rates of synthesis and percentage yields were similar in two-phase systems and one-phase aqueous buffer systems, a ten-fold increase in yield of product per unit of enzyme was seen. In addition, the use of aqueous two-phase systems offers potential process advantages over one-phase systems for the synthesis of oligosaccharides.