- Email: [email protected]
The autoradiographic localization of neurokinin binding sites in human urinary bladder
NEUROPEI’TIDES:
237
FUNCTION AND PHARhlA COLOGY
Glaxo Group Research, Ware, UK, and *Glaxo Institute of Molecular Biology, Geneva, Switzerland Using the potent, selective tetrapeptide NIG receptor antagonist radioligand [“HI-GR100679 (cyclohexylC0. Gly.Ala.D-Trp.Phe.NMe$, we have character&d human ileum NIG receptors stably transfected into Chinese hamster ovary cells (CHO/T) and examined the localization of NK, receptors in rat brain. [3H]-GR100679 bound (KD 0.69 t&l) to a single population ofbinding sites (Bmax 45 000 sites per cell) in CHO/T cells. Neurokinin agonists inhibited binding with rank order of potency consistent with interaction at NIG receptors. Antagonists competed for binding sites with rank order ofpotency GR94800 (PhCO.Ala.Ala.D-Trp.Phe.DPro.Pro.Nle.NHz; pKi 9.93) > f SR48968 @Ki 9.88) > GR100679 @Ki 8.91) > MEN10376 @Ki 8.57) > MEN10207 @Ki 8.07) > L-659,877 (PKi 7.29) = R396 (pKi 7.20) > > CP-96,345 @Ki 5.23). There was excellent correlation (r = 0.96) between atBnities in CHOA’ cells and rabbit pulmonary artery (RPA)I but poor correlation (r = 0.70) between affinities in CHO/T cells and hamster trachea.’ Specific [3H]-GR100679 (1.5 nM) binding was observed in neonate ( 1O-l 2 d) rat brain in a limited number of areas, particularly thalamic nuclei, deeper layers of frontal cortex and hippocampus. High levels of nonspecific binding precluded identification of NIG receptors in adult. We have used [3H]-GR100679 to demonstrate NK2 receptors in neonate rat brain and show that the human ileum NKz receptor closely resembles the putative MGA subtype, as defined by antagonist potencies in RPA. 1. Maggi, C. A., Patacchini, R., Rovero, P. and Giachetti, A. (In Press). Tachykinin receptors and tachykinin receptor antagonists. J. Autonom. Pharmacol.
P118 Characterization and Autoradiographic Localization of Tachykinin NK-2 Receptors in Human Urinary Bladder E. Burcher, X.-P. Zeng and K. H. Moore* School of Physiology and Pharmacology, University of New South Wales, Sydney, NSW 2033 and *Department of Obstetrics and Gynaecology, The St George Hospital, Kogarah, NSW 22 17, Australia Tachykinin peptides are potent in contracting the urinary bladder of many species. In this study, we have employed radioligand binding, autoradiographic and functional studies to investigate tachykinin receptors in the human bladder, using selective agonists and the highly selective NK-2 receptor antagonists SR48968, MDL29913 and MENl0207. Bladders were obtained at cystectomy. Membranes from the detrusor muscle were finally suspended in incubation buffer containing 50 mM Tris, 0.02% BSA, 3 mM MnClz and peptidase inhibitors.
Specific binding of the NK-2 selective mdioligand [1251][Lys5, Tyr(L)‘, MeLeu9,Nle10]-NKA(4-10) (50 PM) was saturable, and appeared to bind to two sites. Nonspecific binding was defined by 1 pM cold [Lys5,Tyr(Iz)‘, MeLeu9,NIe10]-NKA(4-10). Specific binding was inhibited by NKA = [Lys5,MeLeu9,Nle*o]-NKA(4-10) = neuropeptide y 2 SR48968 > [Lys5,Tyr(I~)‘,MeLeu9,Nle10]NKA(bIO) > MEN10207 > MDL29913 > > [Sar9, Met(O#‘]-SP and set&tide, indicating binding to NK-2 receptors. In the presence of phosphoramidon (10 pM), isolated strips of detrusor muscle were contracted by NKA, [Lys5,MeLeu9,Nle10]-NKA(4-10), neuropeptide y and [Lys5,Tyr(12)‘,MeLeu9, Nle*“]-NKA(4-10), with pD2 values 8.4,8.4,8.1 and 7.2, respectively. [Sat+‘,Met(O2)“]SP and set&tide were ineffective contractile agents, indicating an absence of NK-1 and NK-3 receptors in this tissue. In autoradiographic studies, binding sites for [lz51][Lys5,Tyr(I~)7,MeLeu9,Nleio]-NKA(&l 0) were localized over the detrusor smooth muscle.
P119 The Autoradiographic Localization of Neurokinin Binding Sites in Human Urinary Bladder A. J. Nirnmo, P. 0. Andersson*, C. R. Chapplet and J. R. Carstairs Department of Chemistry and Biochemistry, James Cook University, Townsville 448 11, Australia, *Pfizer Central Research, Sandwich, Kent CT 13 9NJ, UK and TDepartment of Urology, Royal Hallamshire Hospital, Sheffield, UK The aim of the present study was to characterize and localize the binding of [1251]BoltonHunter-labelled substance P ([YIBHSP) and [1251]iodohistidyl neurokinin A ([1251]NKA)to human bladder tissue sections. Biopsy samples of human bladder were obtained at surgery for urinary carcinoma. Tissue sections, 20 m thick, were cut on a cryostat and thaw-mounted onto microscope slides. Specificity of radioligand binding was determined by competition studies. For autoradiography, tissue sections were incubated with either [‘*‘I]BHSP or [129]NKA (0.1 nM) for 90 min at 25°C. Autoradiograms were generated by apposing sections to coverslips coated with autoradiographic emulsion, Sections incubated with [1251]BHSP, showed intense labelling of the vascular endotbelium of small arterioles, capillaries and venules. These NKI receptors may be involved in mediating substance P-induced neurogenic plasma extravasation in the bladder. Sections incubated with [1251]NKA showed specific labelling of the visceral smooth muscle and submucosa. In the rat bladder, we have shown the submucosal labelling to be associated with afferent nerve terminals. In the human bladder, only NK2 receptors were associated with the visceral smooth muscle, correlating functional studies which have shown that neurokinin-induced contractions are mediated by N& receptors.
237
FUNCTION AND PHARhlA COLOGY
Glaxo Group Research, Ware, UK, and *Glaxo Institute of Molecular Biology, Geneva, Switzerland Using the potent, selective tetrapeptide NIG receptor antagonist radioligand [“HI-GR100679 (cyclohexylC0. Gly.Ala.D-Trp.Phe.NMe$, we have character&d human ileum NIG receptors stably transfected into Chinese hamster ovary cells (CHO/T) and examined the localization of NK, receptors in rat brain. [3H]-GR100679 bound (KD 0.69 t&l) to a single population ofbinding sites (Bmax 45 000 sites per cell) in CHO/T cells. Neurokinin agonists inhibited binding with rank order of potency consistent with interaction at NIG receptors. Antagonists competed for binding sites with rank order ofpotency GR94800 (PhCO.Ala.Ala.D-Trp.Phe.DPro.Pro.Nle.NHz; pKi 9.93) > f SR48968 @Ki 9.88) > GR100679 @Ki 8.91) > MEN10376 @Ki 8.57) > MEN10207 @Ki 8.07) > L-659,877 (PKi 7.29) = R396 (pKi 7.20) > > CP-96,345 @Ki 5.23). There was excellent correlation (r = 0.96) between atBnities in CHOA’ cells and rabbit pulmonary artery (RPA)I but poor correlation (r = 0.70) between affinities in CHO/T cells and hamster trachea.’ Specific [3H]-GR100679 (1.5 nM) binding was observed in neonate ( 1O-l 2 d) rat brain in a limited number of areas, particularly thalamic nuclei, deeper layers of frontal cortex and hippocampus. High levels of nonspecific binding precluded identification of NIG receptors in adult. We have used [3H]-GR100679 to demonstrate NK2 receptors in neonate rat brain and show that the human ileum NKz receptor closely resembles the putative MGA subtype, as defined by antagonist potencies in RPA. 1. Maggi, C. A., Patacchini, R., Rovero, P. and Giachetti, A. (In Press). Tachykinin receptors and tachykinin receptor antagonists. J. Autonom. Pharmacol.
P118 Characterization and Autoradiographic Localization of Tachykinin NK-2 Receptors in Human Urinary Bladder E. Burcher, X.-P. Zeng and K. H. Moore* School of Physiology and Pharmacology, University of New South Wales, Sydney, NSW 2033 and *Department of Obstetrics and Gynaecology, The St George Hospital, Kogarah, NSW 22 17, Australia Tachykinin peptides are potent in contracting the urinary bladder of many species. In this study, we have employed radioligand binding, autoradiographic and functional studies to investigate tachykinin receptors in the human bladder, using selective agonists and the highly selective NK-2 receptor antagonists SR48968, MDL29913 and MENl0207. Bladders were obtained at cystectomy. Membranes from the detrusor muscle were finally suspended in incubation buffer containing 50 mM Tris, 0.02% BSA, 3 mM MnClz and peptidase inhibitors.
Specific binding of the NK-2 selective mdioligand [1251][Lys5, Tyr(L)‘, MeLeu9,Nle10]-NKA(4-10) (50 PM) was saturable, and appeared to bind to two sites. Nonspecific binding was defined by 1 pM cold [Lys5,Tyr(Iz)‘, MeLeu9,NIe10]-NKA(4-10). Specific binding was inhibited by NKA = [Lys5,MeLeu9,Nle*o]-NKA(4-10) = neuropeptide y 2 SR48968 > [Lys5,Tyr(I~)‘,MeLeu9,Nle10]NKA(bIO) > MEN10207 > MDL29913 > > [Sar9, Met(O#‘]-SP and set&tide, indicating binding to NK-2 receptors. In the presence of phosphoramidon (10 pM), isolated strips of detrusor muscle were contracted by NKA, [Lys5,MeLeu9,Nle10]-NKA(4-10), neuropeptide y and [Lys5,Tyr(12)‘,MeLeu9, Nle*“]-NKA(4-10), with pD2 values 8.4,8.4,8.1 and 7.2, respectively. [Sat+‘,Met(O2)“]SP and set&tide were ineffective contractile agents, indicating an absence of NK-1 and NK-3 receptors in this tissue. In autoradiographic studies, binding sites for [lz51][Lys5,Tyr(I~)7,MeLeu9,Nleio]-NKA(&l 0) were localized over the detrusor smooth muscle.
P119 The Autoradiographic Localization of Neurokinin Binding Sites in Human Urinary Bladder A. J. Nirnmo, P. 0. Andersson*, C. R. Chapplet and J. R. Carstairs Department of Chemistry and Biochemistry, James Cook University, Townsville 448 11, Australia, *Pfizer Central Research, Sandwich, Kent CT 13 9NJ, UK and TDepartment of Urology, Royal Hallamshire Hospital, Sheffield, UK The aim of the present study was to characterize and localize the binding of [1251]BoltonHunter-labelled substance P ([YIBHSP) and [1251]iodohistidyl neurokinin A ([1251]NKA)to human bladder tissue sections. Biopsy samples of human bladder were obtained at surgery for urinary carcinoma. Tissue sections, 20 m thick, were cut on a cryostat and thaw-mounted onto microscope slides. Specificity of radioligand binding was determined by competition studies. For autoradiography, tissue sections were incubated with either [‘*‘I]BHSP or [129]NKA (0.1 nM) for 90 min at 25°C. Autoradiograms were generated by apposing sections to coverslips coated with autoradiographic emulsion, Sections incubated with [1251]BHSP, showed intense labelling of the vascular endotbelium of small arterioles, capillaries and venules. These NKI receptors may be involved in mediating substance P-induced neurogenic plasma extravasation in the bladder. Sections incubated with [1251]NKA showed specific labelling of the visceral smooth muscle and submucosa. In the rat bladder, we have shown the submucosal labelling to be associated with afferent nerve terminals. In the human bladder, only NK2 receptors were associated with the visceral smooth muscle, correlating functional studies which have shown that neurokinin-induced contractions are mediated by N& receptors.