A cDNA encoding guinea-pig uterine substance P (SP) receptor has been isolated using the homology screening approach. Northern blot analysis reveals that the corresponding mRNA, of approx. 4.8 kb, is expressed in all tissues tested, but predominantly in the uteri of non-pregnant animals; during pregnancy its expression is reduced. The guinea-pig SP receptor was expressed in COS-7 cells and demonstrated relative Iigand affinity in the order: SP » neurokinin A > neurokinin B.Substance P (SP) belongs to the tachykinin family of peptides which includes, in addition to SP, the peptides substance K (neurokinin A) and neuromedin K (neurokinin B). The tachykinins share a common carboxylterminal sequence and give rise to similar spectra of biological activity. SP was the first of these neuropeptides tobe discovered, and is the best characterized. It causes smooth muscle contraction and vasodilation, and plays a role in neuron excitation, pain ttansmission and netve regeneration [1,2]. An important target organ for SP is the uterus. lrnmunocytochemical studies have shown that numerous SP-positive neurons innervate the myometrium and uterine arteries [3][4][5]. SP, in combination with other neuropeptides, plays an essential role in the regulation of myometrial activity and, consequently, in the maintenance of pregnancy and parturition.SP exerts its effect via the SP receptor (so called . NK-1 receptor ), a G-protein-coupled receptor which utilises the phosphatidyl inositol second messenger system.cDNAs corresponding to the rat [6,7] and human [8] SP receptors have recently been cloned and characterized. Here we report the molecular cloning of the guinea-pig SP receptor cDNA and its expression in COS cells. The aim of our study was the isolation of cDNAs corresponding to neuropepÜde receptors expressed in the guinea-pig uterus. These are G-protein-coupled receptors that share common structural features and have consetved amino acid sequences, particularly within the transmembrane domains. Therefore, we chose the homology screening approach to analyse guinea-pig uterine cDNA library constructed in ,\gtlO bacteriophage. A degenerate oligonucleotide (5'-GG2A~CCAGCAGA
Congenital nephrogenic diabetes insipidus (NDI) is an X-linked inherited disorder characterized by renal resistance to the antidiuretic hormonal action of vasopressin. This study describes the molecular basis of nephrogenic diabetes insipidus in a dog family. Kidney membranes prepared from NDI-affected male huskies were examined for vasopressin binding and response. Compared to membranes from unaffected canines, those from the kidney inner medulla of NDI-dogs possessed normal V2-receptor numbers, but with 10-fold lower affinity for [Arg8] vasopressin (AVP). Adenylate cyclase stimulation by AVP in contrast to that by forskolin or GTP-analogues was similarly reduced in a dose responsive manner. The NDI-affected dogs showed antidiuretic responses to very high doses of V2-specific agonists, consistent with their possessing V2-receptors of lower affinity. Prolonged treatment with V2-agonists, 1-deamino [D-Arg8] VP (dDAVP) and 1-deamino [Val4, Sar7] AVP (dVSAVP), rendered the NDI-affected dogs near normal in terms of water intake and urine osmolality.
The photoreactive analogue of vasopressin, [1-(3-mercapto)propionic acid, 8-(N6-4-azidophenyl-amidino)lysine] vasopressin (apa-LVP) could be used to elicit stimulation of cAMP production in LLC-PK renal epithelial cells, detectable up to 24 h after photoactivation by flash photolysis. This is in contrast to cells treated with vasopressin, or apa-LVP without photoactivation, where cAMP synthesis is down regulated within 4 h. The prolonged stimulation of cAMP production induced by photoactivation of apa-LVP was demonstrated to be cytotoxic to LLC-PK1 cells, whereas the vasopressin receptor negative LLC-PK1 mutant M18 was resistant to the cytotoxic effect. A selection strategy was developed for mutants resistant to this long-term stimulation of cAMP production, whereby multiple cycles of treatment with apa-LVP and photoactivation were used. Mutants so selected were then characterized using a novel screening system for detection of the production of urokinase-type plasminogen activator in response to cAMP agonists. One mutant was examined and found to be impaired in hormonal responsiveness, whereby hormone and forskolin stimulated cAMP-mediated responses were markedly reduced. It exhibited resistance to the long-term stimulation of cAMP production elicited by apa-LVP and photoactivation. This implies that apa-LVP can be used to select for novel mutants specifically impaired in cAMP metabolism and in particular down-regulation of cAMP response.
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