Nα-Boc-Nω-(methoxybenzenesulfonyl)-L-arginine

The effect of Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) on voltage-gated ion channels is widely studied through the use of specific CaMK II blockers such as 2-[N-(2-hydroxyethyl)]-N-(4methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN-93). The present study demonstrates that KN-93 is a direct extracellular blocker of a wide range of cloned Kv channels from a number of different subfamilies. In all channels tested, the effect of 1 microM KN-93 was independent of CaMK II because 1 microM2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, phosphate (KN-92), an inactive analog of KN-93, caused similar inhibition of currents. In addition, dialysis of cells with 10 microM CaMK II inhibitory peptide fragment 281-301 (CIP) had no effect on current kinetics and did not prevent the inhibitory effect of KN-93. The IC(50) for block of the Kv1.5 channel (used as an example to determine the nature of KN-93 block) was 307 +/- 12 nM. KN-93 blocked open channels with little voltage dependence that did not alter the V(1/2) of channel activation. Removal of P/C-type inactivation by mutation of arginine 487 to valine in the outer pore region of Kv1.5 (R487V) greatly reduced KN-93 block, whereas enhancement of inactivation induced by mutation of threonine 462 to cysteine (T462C) increased the potency of KN-93 by 4-fold. This suggested that KN-93 acted through promotion and stabilization of C-type inactivation. Importantly, KN-93 was ineffective as a blocker when applied intracellularly, suggesting that CaMK II-independent effects of KN-93 on Kv channels can be circumvented by intracellular application of KN-93.

Whereas arginine vasopressin binds to its receptor subtypes V(1)R and V(2)R with equal affinity of approximately 2 nM, nonpeptide antagonists interact differently with vasopressin receptor subtypes. The V(2)R antagonist binding site was mapped by site-directed mutagenesis at six selected amino acid positions, K100D, A110W, M120V, L175Y, R202S, and F307I, predicted to be involved in antagonist binding differences between V(2) R and V(1)R. These mutations did not alter the affinity for arginine vasopressin. However, the affinity for six nonpeptide receptor antagonists SR121463B [1-[4-(N-tert-butylcarbamoyl)-2-methoxybenzenesulfonyl]-5-ethoxy-3-spiro-[4[(2 morpholinoethoxy)cy-clohexane]indoline-2-one, phosphate monohydrate cis-isomer], SR49059 [(2S)1-[(2R3S)-(5-chloro-3-(2 chlorophenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2-carboxamide], SSR149415 [(2S,4R)-1-[5-chloro-1-[(2,4-dimethoxyphenyl)sulfonyl]-3-(2-methoxyphenyl)-2-oxo-2,3-dihydro-1H-indol-3-yl]-4-hydroxy-N,N-dimethyl-2pyrrolidine carboxamide, isomer(-)], OPC21268 [1-[1-[4-(3-acetylaminopropoxy)benzoyl]-4-piperidyl]-3,4-dihydro-2(1H)-quinolinone], OPC41061 [(+/-)-4'-[(7-chloro-2,3,4,5-tetrahydro-5-hydroxy-1H-1-benzazepin-1-yl)carbonyl]-o-tolu-m-toluidide], and OPC31260, [(+/-)-5-dimethylamino-1-[4-(2-methylbenzoylamino)benzoyl]-1,2, 3,4,5-tetrahydro-1H-benzazepine monohydrochloride], was altered to varying degrees, resulting in differences up to 6000-fold. Replacement of the small alanine for the bulky tryptophan in position 110 resulted in a reduced affinity for all six antagonists. In contrast, replacement of the large methionine for the smaller valine in position 120 caused a dramatic increase in affinity, up to a K(i) of 7 fM for OPC31260. Molecular modeling revealed that the binding sites for arginine vasopressin and the nonpeptide antagonists are partially overlapping. Whereas arginine vasopressin binds on the extracellular surface of V(2) R, the nonpeptide antagonists penetrate deeper into the transmembrane region of the receptor, in particular OPC21268. The mutagenesis data point to significant differences in the shape of the V(1)R and V(2)R antagonist binding pockets. The most important factor determining the specificity of nonpeptide antagonists seems to be the shape of the binding pocket on the receptor.

Arginine vasopressin (AVP) plays an important role in renal hemodynamic alterations, water retention, and cardiac remodeling in congestive heart failure (CHF). The present study evaluated the acute and chronic effects of vasopressin V(1a) receptor subtype (V(1a)) and vasopressin V(2) receptor subtype (V(2)) antagonists on renal function and cardiac hypertrophy in rats with CHF. The effects of acute administration of SR 49059 [(2S)1-[(2R,3S)-5-chloro-3-(2-chlorophenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2-carboxamide)] (0.1 mg/kg) and SR 121463B (1-[4-(N-tert-butylcarbamoyl)-2-methoxybenzenesulfonyl]-5-ethoxy-3-spiro-[4-(2-morpholinoethoxy)cyclohexane]indol-2-one, fumarate; equatorial isomer) (0.3 mg/kg), V(1a) and V(2) antagonists, respectively, on renal function, and of chronic treatment (3.0 mg/kg/day for 7 or 28 days, via osmotic minipumps or p.o.), on water excretion and cardiac hypertrophy were studied in rats with aortocaval fistula and control rats. CHF induction increased plasma AVP (12.8 +/- 2.5 versus 32.2 +/- 8.3 pg/ml, p < 0.05). Intravenous bolus injection of SR 121463B to controls produced dramatic diuretic response (from 5.5 +/- 0.8 to 86.3 +/- 21.9 microl/min; p < 0.01). In contrast, administration of SR 49059 did not affect urine flow. Likewise, administration of SR 121463B, but not SR 49059, to rats with CHF significantly increased urinary flow rate from 20.8 +/- 6.4 to 91.6 +/- 26.5 microl/min (p < 0.01). The diuretic effects of SR 121463B were associated with a significant decline in urinary osmolality and insignificant change of Na+ excretion. In line with its acute effects, chronic administration of SR 121463B to CHF rats increased daily urinary volume 2 to 5-fold throughout the treatment period. Both SR 121463B and SR 49059 significantly reduced heart weight in CHF rats when administered for 4 weeks, but not 1 week. These results suggest that V(2) and V(1a) antagonists improve water balance and cardiac hypertrophy in CHF and might be beneficial for the treatment of water retention and cardiac remodeling in CHF.