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Evidence That Additional Mechanisms to Cyclic GMP Mediate the Decrease in Intracellular Calcium and Relaxation of Rabbit Aortic Smooth Muscle to Nitric Oxide

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Evidence That Additional Mechanisms to Cyclic GMP Mediate the Decrease in Intracellular Calcium and Relaxation of Rabbit Aortic Smooth Muscle to Nitric Oxide British Journal of Pharmacology (1998) 125, 1695 ± 1707 ã 1998 Stockton Press All rights reserved 0007±1188/98 $12.00 http://www.stockton-press.co.uk/bjp Evidence that additional mechanisms to cyclic GMP mediate the decrease in intracellular calcium and relaxation of rabbit aortic smooth muscle to nitric oxide 1Robert M. Weisbrod, 1Mark C. Griswold, 1Mohammad Yaghoubi, 2Padmini Komalavilas, 2Thomas M. Lincoln & 1,3Richard A. Cohen 1Vascular Medicine and Biology, Evans Department of Clinical Research, Whitaker Cardiovascular Institute, Department of Medicine, Boston University Medical Center, Boston, Massachusetts and 2Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, U.S.A. 1 The role of cyclic GMP in the ability of nitric oxide (NO) to decrease intracellular free calcium 2+ concentration [Ca ]i and divalent cation in¯ux was studied in rabbit aortic smooth muscle cells in primary culture. In cells stimulated with angiotensin II (AII, 1077 M), NO (10710 ±1076 M) increased 2+ cyclic GMP levels measured by radioimmunoassay and decreased [Ca ]i and cation in¯ux as indicated by fura-2 ¯uorimetry. 2 Zaprinast (1074 M), increased NO-stimulated levels of cyclic GMP by 3 ± 20 fold. Although the 2+ phosphodiesterase inhibitor lowered the level of [Ca ]i reached after administration of NO, the 2+ initial decreases in [Ca ]i initiated by NO were not signi®cantly dierent in magnitude or duration from those that occurred in the absence of zaprinast. 3 The guanylyl cyclase inhibitor, H-(1,2,4) oxadiazolo(4,3-a) quinoxallin-1-one (ODQ, 1075 M), blocked cyclic GMP accumulation and activation of protein kinase G, as measured by back phosphorylation of the inositol trisphosphate receptor. ODQ and Rp-8-Br-cyclic GMPS, a protein 710 78 2+ kinase G inhibitor, decreased the eects of NO, 10 ±10 M, but the decrease in [Ca ]i or cation in¯ux caused by higher concentrations of NO (1077 ±1076 M) were unaected. Relaxation of intact rabbit aorta rings to NO (1077 ±1075 M) also persisted in the presence of ODQ without a signi®cant increase in cyclic GMP. Rp-8-Br-cyclic GMPS blocked the decreases in cation in¯ux caused by a cell permeable cyclic GMP analog, but ODQ and/or the protein kinase G inhibitor had no signi®cant eect on the decrease caused by NO. 4 Although inhibitors of cyclic GMP, protein kinase G and phosphodiesterase can be shown to 2+ aect the decrease in [Ca ]i and cation in¯ux via protein kinase G, these studies indicate that when these mechanisms are blocked, cyclic GMP-independent mechanisms also contribute signi®cantly to 2+ the decrease in [Ca ]i and smooth muscle relaxation to NO. Keywords: Nitric oxide; smooth muscle; calcium; cyclic GMP; protein kinase G Introduction Endothelium-dependent vasodilation is mediated by nitric contractile tone of blood vessels is largely dependent on 2+ oxide (NO), which after being synthesized by endothelial cell [Ca ]i, there may be other means by which NO mediates NO synthase diuses to smooth muscle cells. NO is thought to relaxation. These include cyclic GMP-dependent decrease in mediate relaxation primarily by activating guanylyl cyclase to smooth muscle contractile protein sensitivity to Ca2+ produce cyclic GMP from GTP (Lincoln et al., 1996; (McDaniel et al., 1992), as well as cyclic GMP-dependent Waldman & Murad, 1987; Ignarro & Kadowitz, 1985). Cyclic (Robertson et al., 1993; Taniguchi et al., 1993; Archer et al., GMP activates cyclic GMP-dependent protein kinase (protein 1994) and cyclic GMP-independent activation of K+ ion kinase G) which is thought to have several actions which could channels (Bolotina et al., 1994) or Na+/K+ ATPase (Gupta et mediate relaxation (Lincoln et al., 1996). These actions include al., 1994), which could mediate hyperpolarization and 2+ 2+ a decrease in intracellular calcium concentration [Ca ]i relaxation by inhibiting voltage-dependent Ca in¯ux. mediated by (1) inhibition of phospholipase C (Hirata et al., Although each of these cyclic GMP-independent mechanisms 1990; Rapoport, 1986), (2) inhibition of inositol trisphosphate has been proposed, their relative contribution to NO-induced 2+ 2+ (IP3) receptor mediated Ca release from intracellular Ca vasodilation with respect to cyclic GMP-dependent mechan- stores (Koga et al., 1994; Komalavilas & Lincoln, 1994), (3) isms has been controversial (Moro et al., 1996). inhibition of Ca2+ in¯ux through ion channels (Quignard et The purpose of the current studies was to assess the role of al., 1997; Blatter & Wier, 1994), and (4) stimulation of Ca2+ cyclic GMP in mediating the Ca2+ inhibitory and relaxant removal from the cytoplasm by Na+/Ca2+ exchange (Fur- eects of NO in vascular smooth muscle. In these studies the ukawa et al., 1991), or plasma membrane (Furukawa et al., increases in cyclic GMP stimulated by NO were manipulated 1988) and sarcoplasmic reticulum Ca2+ ATPase transporters by (1) inhibiting the breakdown of cyclic GMP with a (Cornwell et al., 1991; Raeymaekers et al., 1988). Although phosphodiesterase inhibitor, zaprinast (Schoeter et al., 1988), (2) inhibiting guanylyl cyclase with H-(1,2,4) oxadiazo- 3 lo(4,3-a) quinoxallin-1-one (ODQ) (Olson et al., 1997; Brunner Author for correspondence at: Vascular Biology Unit, Boston Medical Center, R408, 80 E. Concord Street, Boston, MA 02118, et al., 1996; Garthwaite et al., 1995), and (3) inhibiting protein U.S.A. kinase G activation with a cell permeable, non-hydrolyzable, 1696 R.M. Weisbrod et al Cyclic GMP-independent actions of NO inactive cyclic GMP analogue, Rp-8-Br-cyclic GMPS (Butt et levels (maximum) or without Ca2+ (minimum), respectively, R al., 1990; Corbin et al., 1986). Because of the possibility that is the sample ratio of 340/380 nm ¯uorescence, and Sf2 and Sb2 cyclic GMP levels may not accurately re¯ect protein kinase G are the raw 380 nm ¯uorescence values for minimum and activation, protein kinase G activity stimulated by NO was maximum standards, respectively. Rmin,Rmax,Sf2, and Sb2 assayed by back phosphorylation of the IP3 receptor values were determined using external standard solutions (Komalavilas & Lincoln, 1994, 1996). These ®ndings provide consisting of PSS containing 50 nM of the pentapotassium salt 2+ evidence for mechanisms additional to those via cyclic GMP of fura-2 with either no [Ca ]i (EGTA-buered, `minimum') 2+ 2+ and protein kinase G by which NO inhibits increases in [Ca ]i or 1.2 mM [Ca ]i (`maximum'). Background auto¯uorescence in vascular smooth muscle. was subtracted at the end of each experimental run by adding 1 mM ionomycin and 20 mM MnCl2 to quench all fura-2 2+ ¯uorescence. [Ca ]i is expressed as the concentration (nM) Methods estimated by the above equation. The duration of each experimental run was 20 min. Cells Cell culture were allowed to equilibrate for 6 min before 0.1 mM 2+ angiotensin II (AII) was added to increase [Ca ]i. Other Strips were aseptically removed from the medial section of pharmacological compounds were present for at least 30 min rabbit thoracic aorta, dispersed with collagenase (4 mg ml71) before AII was added. In analysing the response to AII, the 71 2+ and elastase (1 mg ml ), and grown in media M199 with 20% maximal rise in [Ca ]i during the ®rst 10 s (peak), and the heat-inactivated fetal bovine serum as previously described level achieved 30 s after the addition of AII (plateau) were (Weisbrod et al., 1997). Cells were seeded into 24-well plates quanti®ed. Logarithmic increases in NO or SNAP concentra- for cyclic GMP studies, and onto 9622 mm #1 glass tion were added beginning 30 s following the increase in 2+ 2+ coverslips for [Ca ]i studies. Upon reaching con¯uence, cells [Ca ]i caused by AII, and each subsequent concentration was 2+ were growth arrested by placing them in M199 with 1% serum added after [Ca ]i reached a new stable level. Data from these 2+ for 3 ± 4 days before experiments. All experiments were done experiments were analysed by (1) subtracting basal [Ca ]i 2+ on primary cultures which stain uniformly for smooth muscle levels and calculating the maximal percent decrease in [Ca ]i a-actin (Weisbrod et al., 1997). caused by each concentration of NO or SNAP, and (2) 2+ measuring the duration(s) of the transient reduction in [Ca ]i Cyclic GMP measurements to each concentration of NO starting from the initial decrease 2+ until the [Ca ]i level rose to reach a value that equalled the 2+ Cells were equilibrated for 60 min in HEPES buered (pH 7.4) mean of the trough [Ca ]i value and the new stable level 2+ physiological salt solution (PSS) of the following composition which followed the response. In addition, because [Ca ]i was (mM): NaCl 119, NaHEPES 20, KCl 4.6, CaCl2 1.2, MgSO4 1.0, not constant after AII, in some experiments (Figures 5 and 7) 2+ Na2HPO4 0.15, KH2PO4 0.4 and NaHCO3 5.0, supplemented the average value of [Ca ]i over a 10 min period following the with 0.1% albumin. At the appropriate time, PSS from addition of NO (1076 M) was compared to values in unstimulated cells (basal) or from NO-stimulated cells was experiments in which NO was not added. These average immediately removed, and 200 ml of 0.1 N HCl was added to the values were obtained with an integration algorithm and are 2+ cells and allowed to remain for 1 h to extract cyclic GMP. Cyclic reported as a percentage decrease in the [Ca ]i measured at GMP in the HCl was assayed with a radioimmunoassay kit the time at which NO was added. (Research Biochemicals Inc., Stoughton, MA, U.S.A.).
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