Endogenous cystinyl aminopeptidase in Chinese hamster ovary cells: characterization by [125I]Ang IV binding and catalytic activity.
ABSTRACT The angiotensin II C-terminal hexapeptide fragment angiotensin IV (Ang IV) exerts central and cardiovascular effects. Cystinyl aminopeptidase (EC 18.104.22.168), a membrane-associated zinc-dependent metallopeptidase of the M1 family, has recently been found to display high affinity for Ang IV and it was proposed to represent the AT4 receptor. We present evidence for the presence of endogenous cystinyl aminopeptidase in membranes from Chinese hamster ovary (CHO-K1) cells by binding studies with [125I]Ang IV and by measuring the cleavage of L-leucine-p-nitroanilide. The equilibrium dissociation constant of [125I]Ang IV in saturation binding studies (KD= 0.90 nM) was similar to the value (KD= 0.70 nM) calculated from the association and dissociation rates. Binding was displaced with high potency by the "AT4 receptor" ligands (Ang IV > divalinal1-Ang IV approximately LVV-hemorphin-7 approximately LVV-hemorphin-6 > Ang (3-7) > Ang III > Ang (4-8)) but not by AT1/AT2 receptor antagonists. Enzymatic activity in CHO-K1 cell membranes was competitively inhibited upto 94% by Ang IV and other "AT4 receptor" ligands (Ang IV > Ang III approximately divalinal1-Ang IV approximately Ang (3-7) approximately LVV-hemorphin-7 > Ang (4-8) approximately LVV-hemorphin-6). High affinity binding of [125I]Ang IV required the presence of metal chelators and the ligands such as Ang IV and LVV-hemorphin-7 displayed higher potency in the binding studies as in the enzyme assay. This difference in potency varied from one peptide to another. These pharmacological properties match those previously reported for the recombinantly-expressed human cystinyl aminopeptidase in embryonal kidney cells.
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ABSTRACT: We have characterized a specific binding site for angiotensin IV on bovine aortic endothelial cell membranes. Pseudo-equilibrium studies at 37°C for 2 h have shown that this binding site recognizes angiotensin IV with a high affinity (Kd=0.71; average of two experiments that yielded values of 0.71 and 0.72 nM). The binding site is saturable and relatively abundant with a maximal binding capacity of 0.59 pmol/mg protein (average of two experiments that yielded values of 0.39 and 0.78 pmol/mg of protein). Non-equilibrium kinetic analyses at 37°C revealed a calculated Kd of 59 pM (average of two experiments that yielded values of 67 and 50 pM). The binding site is pharmacologically distinct from the classic angiontensin receptors AT1 or AT2. An analysis of specificity showed that the binding site displays a high affinity for angiotensin IV, low affinities for angiotensin II, [Sar1, Val5, Ala8]angiotensin II and does not recognize L-158,809 (5,7-dimethyl-2-ethyl-3-[(2′-(H-tetrazol-5-yl)[1,1′-biphenyl]-4-yl)methyl]-3H-imidazo [4,5-β]pyridine H2O) and PD 123319 (1-[4-(dimethylamino)3-methylphenyl]methyl-5-(diphenylacety)4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid). A few unrelated hormones (bradykinin, [Arg8]vasopressin, endothelin-1, atrial natriuretic factor, isoproterenol and adrenocorticotropic hormone) were unable to inhibit any 125I-angiotensin IV binding. The affinities of different structural analogues of angiotensin IV revealed that the N-terminal position is critical for receptor recognition and the C-terminal proline is also important. GTPγS and polyvinyl sulfate did not affect the binding, suggesting that the receptor is not coupled to a G-protein. The divalent cations Mg2+ and Ca2+ were shown to diminish the binding of 125I-angiotensin IV. Cross-linking of 125I-angiotensin IV to bovine aortic endothelial cell membranes in the presence of disuccinimidyl suberate, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a major band of 186±12 kDa. The presence in high concentration of this angiotensin binding site an aortic endothelial cells suggests the existence of a novel mechanism involved in the control of vascular tone or vascular permeability.European Journal of Pharmacology 11/1995; · 2.59 Impact Factor
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ABSTRACT: Angiotensin IV (Val Tyr Ile His Pro Phe), administered centrally, increases memory retrieval and induces c-fos expression in the hippocampus and piriform cortex. Angiotensin IV binds to a high affinity site that is quite distinct in pharmacology and distribution from the angiotensin II AT1 and AT2 receptors and is known as the AT4 receptor. These observations suggest that the AT4 receptor may have multiple central effects. The present study uses in vitro receptor autoradiography, and employs [125I]angiotensin IV to map AT4 receptors in the macaca fascicularis brain. The distribution of the AT4 receptor is remarkable in that its distribution extends throughout several neural systems. Most striking is its localization in motor nuclei and motor associated regions. These include the ventral horn spinal motor neurons, all cranial motor nuclei including the oculomotor, abducens, facial and hypoglossal nuclei, and the dorsal motor nucleus of the vagus. Receptors are also present in the vestibular, reticular and inferior olivary nuclei, the granular layer of the cerebellum, and the Betz cells of the motor cortex. Moderate AT4 receptor density is seen in all cerebellar nuclei, ventral thalamic nuclei and the substantia nigra pars compacta, with lower receptor density observed in the caudate nucleus and putamen. Abundant AT4 receptors are also found in areas associated with cholinergic nuclei and their projections, including the nucleus basalis of Meynert, ventral limb of the diagonal band and the hippocampus, somatic motor nuclei and autonomic preganglionic motor nuclei. AT4 receptors are also observed in sensory regions, with moderate levels in spinal trigeminal, gracile, cuneate and thalamic ventral posterior nuclei, and the somatosensory cortex. The abundance of the AT4 receptor in motor and cholinergic neurons, and to a lesser extent, in sensory neurons, suggests multiple roles for the AT4 receptor in the primate brain.Brain Research 04/1996; 712(2):307-24. · 2.88 Impact Factor
- Journal of Pharmacology and Experimental Therapeutics - J PHARMACOL EXP THER. 01/2003; 305(1):205-211.