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531 Stimulation of Soluble Guanylate Cyclase by an Acetylcholine-Induced Endothelium-Derived Factor from Rabbit and Canine Arteries Ulrich Forstermann, Alexander Miilsch, Eycke Bohme, and Rudi Busse From the Department of Pharmacology, University of Freiburg, Freiburg, F.R.C., the Department of Pharmacology, University of Heidelberg, Heidelberg, F.R.C., and the Department of Applied Physiology, University of Freiburg, Freiburg, F.R.G. Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 SUMMARY. The present study was designed to investigate the hypothesis that, during acetylcholine-induced endothelium-dependent relaxation, a factors) is released from endothelial cells which directly activates soluble guanylate cyclase. We attempted to determine what similarities or differences existed between this factor and endothelium-derived relaxing factor. The study was performed on segments of rabbit aorta and canine femoral artery. Purified soluble guanylate cyclase was injected into the lumen of these vascular segments, together with its substrate, for intraluminal incubation of the enzyme. In endothelium-intact vascular segments, the activity of guanylate cyclase was enhanced over control values obtained by incubation in test tubes. The stimulation was further increased by acetylcholine in concentrations which caused relaxation of the vascular segments. The stimulating principle could not be transferred from the vessel lumen to an external solution of guanylate cyclase, indicating a short life-time. Removal of the endothelium prevented formation and release of the guanylate cyclase stimulating factors). Atropine, mepacrine, or nordihydroguaiaretic acid, which inhibit acetylcholine-induced endothelium-dependent relaxations, also inhibited acetylcholine-induced endothelium-mediated activation of guanylate cyclase. The results support the hypothesis that acetylcholine-induced endotheliumderived relaxing factor increases cyclic guanosine monophosphate levels of vascular smooth muscle by a stimulation of soluble guanylate cyclase. {Circ Res 58: 531-538, 1986) RELAXATION of several isolated blood vessel preparations by acetylcholine (ACh) and numerous other agents is dependent upon the presence of an intact endothelium (Furchgott and Zawadzki, 1980; Chand and Alrura, 1981; Furchgott et al., 1981; DeMey et al., 1982; Furchgott, 1983, 1984; Furchgott et al., 1984). Such agents induce the formation and release of an unknown factor (or factors) which relaxes the subjacent smooth muscle. It has been shown that the ACh-induced endothelium-derived relaxing factor (EDRF) is a humoral agent with a short half life in the range of seconds (Griffith et al., 1984; Forstermann et al., 1984). The chemical structure of EDRF and its mechanism of action at the molecular level are still unknown. In blood vessel preparations with intact endothelium, relaxations elicited by muscarinic agonists, and some other agents were found to be associated with increased levels of cyclic guanosine monophosphate (cGMP) in smooth muscle cells. In addition, in vessels with the endothelium removed or damaged, this increase in cGMP levels was reduced or abolished (Holzmann, 1982; Diamond and Chu, 1983; Furchgott and Jothianandan, 1983; Rapoport and Murad, 1983a, 1983b; Furchgott et al., 1984; Ignarro et al., 1984; Miller et al., 1984; Rapoport et al., 1984). It has been proposed that the increase of smooth muscle cGMP levels by endothelium-dependent vasodilators is mediated through activation of guanylate cyclase (GC) (Furchgott et al., 1981; Holzmann, 1982; Rapoport and Murad, 1983a; Ignarro et al., 1984). Relaxation of a variety of blood vessel preparations by nitric oxide (NO) containing vasodilators like organic nitrates and sodium nitroprusside (SNP) is also associated with an increase of smooth muscle cell cGMP levels (for review, see Rapoport and Murad, 1983b; Ignarro and Kadowitz, 1985). These vasodilators, or their metabolites, activate soluble GC by interacting directly with the enzyme (Mittal and Murad, 1982; Craven and DeRubertis, 1983; Bohme et al., 1984; Ignarro and Kadowitz, 1985). These findings raise the possibility that EDRF may also be a direct activator of soluble GC. We report here that endothelial cells generate a factors) that activates soluble guanylate cyclase, probably in a direct manner. The formation and/or release of this factor(s) is stimulated by ACh. ACh-induced soluble GC-stimulating factors) and EDRF share several common properties with respect to stability, formation, and/or release. Methods Preparation and Perfusion of Arterial Segments Rabbits (2.5-3.5 kg) were killed by decapitation. The descending thoracic aorta and a femoral artery were dis- 532 Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 sected out. Mongrel dogs (25-35 kg) were anesthetized with sodium pentobarbital, and the femoral artery and larger side branches were excised. Arterial segments of a length of 3 cm (rabbit aorta and canine femoral artery) or 1 cm (rabbit femoral arteries and side branches of canine femoral arteries) were prepared free of connective tissue. Any damage of the endothelium was avoided during the preparation of the vascular segments. In some experiments, the endothelium was removed by gently rubbing the intimal surface with a rough steel cannula or by inverting the segments and carefully removing the endothelium with a razor blade. Both procedures proved to be equieffective as indicated by the complete failure of ACh to induce relaxation and confirmed by light microscopy after silver staining (Poole et al., 1958). Contractile responses to norepinephrine (NE) were not affected by either rubbing procedure. After ligation of the side branches, each segment was cannulated with two L-shaped stainless steel cannulas and fixed with ligatures. The segment was stretched to its approximate in situ length, and the vertical parts of the cannulas were fixed in a plastic frame at this distance. This device containing the segment was then placed in a special organ bath so that the top ends of the cannulas protruded beyond the surface (cf. Busse et al., 1983,1984). The bath contained oxygenated Tyrode's solution (Poj > 460 mm Hg, pH 7.4, 37°C) as the extraluminal medium. The proximal end of the inflow cannula was connected to a perfusion pump and the segment was perfused (0.66 ml/min) with modified Tyrode's solution (Na+, 144.0 ITIM; Ca++, 1.6 mM; Mg++, 3.0 mM; Cl", 145.0 ITIM; HCO3", 11.9 ITIM; H2PO«~, 0.35 ITIM; CaNa2EDTA, 0.025 nw; glucose, 11.2 mM; glutathione, 0.5 ITIM; bovine 7-globulin, 0.1 mg/ ml; P02 = 140 mm Hg). The pH of the solution was adjusted to 7.4 at 37°C by gassing with 5% CO^ 20% O2, and 75% N2. Glutathione and bovine 7-globulin are essential for the determination of the soluble GC activity described below. Glutathione protects soluble GC from oxidative inactivation. Bovine 7-globulin prevents adsorption of the purified GC to different surfaces, e.g., test tubes. To assess mechanical responsiveness, vascular segments were perfused with modified Tyrode's solution containing NE (0.1 ^M) until a contraction plateau had been reached. Then ACh (0.01-2 MM) was added to induce relaxation. To test the effects of drugs in modified Tyrode's solution, vascular segments were perfused for 60 minutes with mepacrine (30 ^M), nordihydroguaiaretic acid (20 HM), or indomethacin (20 JZM), respectively, before exposure to NE and ACh. In some experiments sodium nitroprusside (SNP, 1 nM to 1 J/M) was added instead of ACh. Measurement of Smooth Muscle Tone The experiments were performed with endotheliumintact rabbit aortas and canine femoral arteries as the 'donor' vessels for EDRF. For bioassay of EDRF released into its lumen, the 'donor' vessel was perfused in series with endothelium-denuded 'detector' vessel (rabbit femoral artery or side branch of canine femoral artery) as described by Forstermann et al. (1984). The tone of the vascular segments was measured by continuously recording the external diameters at their midpoints using photoelectric devices (Busse et al., 1983, 1984). For bioassay of EDRF, the mechanical responses of 'donor' and 'detector' segments were recorded simultaneously. Circulation Research/Vol. 58, No. 4, April 1986 Purification of Soluble Guanylate Cyclase from Bovine Lung Soluble, heme-containing GC was purified from bovine lung to apparent homogeneity according to the method described by Gerzer et al. (1981), but with some modifications. We used Sepharose CL-6B containing 1 jtmol Cibacron blue F3G-A/ml swollen gel prepared according to Heyns and De Moor (1974). It allows elution of GC under low ionic strength conditions with a yield of about 50%. The properties of the isolated enzyme with an approximative molecular weight of 150,000 daltons were described briefly (Mulsch and Bohme, 1984). Determination of the Activity of Soluble Guanylate Cyclase The incubation procedure to determine the activity of purified soluble GC was carried out in test tubes as well as in the lumen of vascular segments. Purified soluble GC (10-100 nM), with and without drugs added, was preincubated in test tubes for 3 minutes at 37°C and pH 7.4 in modified Tyrode's solution under 5% CO2/ 20% O2 and 75% N2 or in a triethanolamine/HCl-buffer (50 HIM) containing glutathione (0.5 ITIM), MgCl2 (3 mM), and bovine 7-globulin (0.1 mg/ml). All concentrations given are final concentrations at the respective step. The incubations were started by addition of [a-32P)]GTP (0.1 mM, 0.2 jtCi). If the effect of SNP on the activity of GC was to be evaluated SNP was added together with [a-32P]GTP. After 3 minutes the reactions were stopped by addition of zinc acetate (50 ITIM) and subsequently sodium carbonate (55 mM). Isolation of cGMP and calculation of guanylate cyclase activity were performed as described (Schultz and Bohme, 1984). Control experiments for the determination of non-enzymatically formed cGMP were performed in the absence of guanylate cyclase. For the determination of recovery of cGMP during the incubation procedure, cyclic [3H]GMP (0.05 mM, 20 nCi) was used. Incubation in Test Tubes Initially, we investigated whether the effluent of endothelium-intact rabbit aortic segments stimulates purified soluble GC in a test tube. Modified Tyrode's solution containing NE (0.1 /IM) with and without ACh (2 ^M) was injected into the lumen of rabbit aorta segments with intact endothelium, preincubated for 0.5 and 3 minutes, and transferred within 5 or 60 seconds to test rubes containing purified soluble GC. Subsequently, the incubations were started by addition of [a"P]GTP (0.1 mM, 0.5 iiCi). The reactions were stopped after 25 or 180 seconds, as described above. In control experiments preincubations of NE with and without ACh were performed in test tubes instead of vascular segments. Incubations of Guanylate Cyclase Injected into the Lumen of Vascular Segments To circumvent technical difficulties arising from the short half life of ACh-induced EDRF, we determined the activity of purified soluble GC after injecting it into the lumen of vascular segments. The segment was at first perfused and tested for mechanical responsiveness to ACh, as described above. Then the perfusion was stopped Forstermann et a/./Endothelial Relaxing Factor and Guanylate Cydase Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 and the perfusion system was disconnected from the top ends of the L-shaped steel cannulas while the arterial segment remained in place in the organ bath. The remaining inrraluminal medium was cleared from the segment by quickly removing the device containing the arterial segment from the organ bath, turning it sideways, and passing a gentle stream of air through the lumen. The segment was promptly returned into the organ bath. Then a preincubated (3 minutes, 37°C) purified soluble GC (34170 nM) in modified Tyrode's solution was mixed with [a-32P]GTP (0.1 ITIM, 0.5 nCi) and cyclic [3H]GMP (0.05 ITIM, 20 nCi) and injected within 20 seconds into the lumen of the vascular segments. The mixture contained cyclic [3H]GMP to determine the recovery of cGMP. The ends of both cannulas were closed with plugs and the incubation was allowed to proceed for a specified time. The cannulas were then reopened, and the intraluminal incubation mixture was rapidly transferred from the vascular lumen into the zinc acetate solution by gently blowing air through the segment as described above. Following this, the vascular segment was reconnected to the perfusion system. The isolation and determination of the cyclic [ PJGMP formed and calculation of GC activity was performed as described (Schultz and Bohme, 1984). Control experiments were performed in test tubes instead of vascular segments under comparable conditions. Blank values were obtained by omitting purified soluble GC from the reaction mixture and were the same in test tubes and isolated vascular segments. After the first intraluminal determination of soluble GC activity, the vascular segment was perfused for at least 16 minutes to wash out residual GC-containing solution prior to the next incubation procedure. A maximum of five runs was performed on each segment. In the experiments in which the effects of drugs on EDRF formation and/or release were to be evaluated, the activity of GC injected into the lumen of vascular segments was first determined in the presence of NE (0.1 ^M) and then in the presence of both NE (0.1 /tM) and ACh (2 MM). Subsequently, the segments were perfused for 60 minutes with modified Tyrode's solution containing the drug under study. Then, the determinations of GC activity were repeated in the presence or absence of the drug. GC activities are expressed as percent of test tube controls (mean ± SE). Differences were tested for significance by Student's Ntest. Significance was accepted at the 0.05 level of probability. Materials Norepinephrine-HCl, acetylcholine-HCl, mepacrineHC1, nordihydroguaiaretic acid, indomethacin, and glutathione were purchased from Sigma. Atropine sulfate was obtained from Drobena, and bovine 7-globulin was from Serva. Other materials for the purification of soluble GC and determination of enzyme activity were obtained as previously described (Gerzer et al., 1981). Stock solutions of drugs were prepared immediately before use in different solvents, and were then diluted with modified Tyrode's solution or triethanolamine/HCl buffer. Nordihydroguaiaretic acid was dissolved in modified Tyrode's solution (37°C) and indomethacin in 25% (vol/vol) ethanol, 0.675% (wt/vol) NaCl, and 0.75% (wt/vol) NaHCO3; all other drugs were dissolved in twice-distilled water. 533 Results Relaxation of Vascular Segments by Acetylcholine and Sodium Nitroprusside ACh (0.01-2 HM) induced relaxations of precontracted (NE, 0.1 HM), endothelium-intact rabbit aortas, and canine femoral arteries in modified Tyrode's solution. Similarly, the ACh-induced release of EDRF into the lumen of endothelium-intact 'donor' rabbit aortic segments and the transfer of this EDRF to endothelium-denuded 'detector* rabbit femoral segments could be demonstrated in modified Tyrode's solution (Fig. 1). GTP (0.1 ITIM) and cGMP (0.05 mM) had no effect on ACh-induced relaxation and intraluminal release of EDRF (data not shown). Furthermore, ACh-induced relaxations of 'donor" as well as 'detector' vascular segments were abolished in the presence of atropine (2 /XM), after pretreatment of the 'donor' segments for 60 minutes with mepacrine (30 ^M) or nordihydroguaiaretic acid (20 JIM), and after removal of the endothelium from the 'donor' segments. Thus, the arteries showed identical mechanical response in modified as in unmodified Tyrode's solution (cf. Forstermann et al., 1984). 0.1 uM Norepinephrine [0.05 |2uM ACh I 3860 -, 1480-1 FIGURE 1. Representative diameter recordings (D) of an endotheliumintact rabbit aortic segment ("donor" segment, upper panel) and of an endothelium-denuded rabbit femoral segment ("detector" segment, lower panel) perfused in series with modified Tyrode's solution. The effluent from the endothelium-intact vascular segment was subsequently perfused through the endothelium-denuded segment as described previously (Forstermann et al, 1984). Both vascular segments were precontracted by perfusion with norepinephrine (NE, 0.1 pu). Addition of acetylcholine (ACh, 0.05 and 2 yiM) induced relaxation of the endothelium-intact vascular segment and the release of EDRF from the "donor" segment which relaxed the endothelium-denuded "detector" segment. The constituents of modified Tyrode's solution, glutathione (0.5 mM), MgCl2 (3 mM), and bovine -/-globulin (0.1 mg/ ml), as well as GTP (0 1 mM) and cGMP (0.05 mM), did not affect ACh-induced formation and/or release of EDRF. Circulation Research/Vol. 58, No. 4, April 1986 534 The NO containing vasodilator SNP in a concentration of 10 nM caused the same degree of relaxation of precontracted (NE, 0.1 /XM) endothelium-free rabbit femoral arteries as did the effluent of endothelium-intact arteries after stimulation with ACh (2 Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 Activity of Purified Soluble Guanylate Cyclase The activity of soluble, heme-containing GC purified from bovine lung was significantly inhibited with modified Tyrode's solution instead of the triethanolamine/HCl buffer described in methods (Table 1). Basal GC activity determined in modified Tyrode's solution was increased about 2-fold by omission of NaCl (134 mM)or CaCl2 (1.6 ITIM), respectively. In the absence of NaCl and CaCl2, the enzyme activity was comparable to that determined in triethanolamine/HCl buffer. Correspondingly, GC activity determined in triethanolamine/HCl buffer was reduced to about 30% by the addition of NaCl or CaCl2. The enzyme activity in the presence of NaCl and CaCl2 was comparable to that measured in modified Tyrode's solution (Table 1). Also, the stimulation of soluble GC by SNP was reduced in modified Tyrode's solution. Maximal stimulation achieved with SNP was about 60-fold in triethanolamine/HCl buffer, but only about 20fold in modified Tyrode's solution, both at about 20 HM SNP. The SNP concentration for half-maximum stimulation was about 2 HM, independent of the buffer used (data not shown). At 1 HM SNP the extent of stimulation was about 20-fold in triethanolamine/HCl buffer and about 8-fold in modified Tyrode's solution (Table 1). Addition of NaCl (134 mM), CaCl2 (1.6 ITIM), or both to the triethanolamine/ HC1 buffer reduced enzyme activities measured in the presence of SNP and reduced the extent of TABLE 1 Inhibition of Purified Soluble Guanylate Cyclase by Some Constituents of Tyrode's Solution Guanylate cyclase activity (nmol/mg protein per min) Buffer system Without SNP With SNP (1 MM) added Triethanolamine/HCl + CaCl2 + NaCl + CaCl2 + NaCl 67.0 ± 10.0 25 1 ±4.7 20 1 ±4.2 9.4 ± 24 1340 ± 136 377 ±51.2 261 ±42.0 65.8 ± 8 5 Tyrode's solution - CaCl2 - NaCl - CaClj - NaCl 10.0 ± 2 2 20 0 ±3.0 24.1 ± 2.8 68.3 ± 6.5 80.0 ±9.8 240 ± 43.1 313 ±49 4 1230 ± 125 The activity of soluble, heme-containing guanylate cyclase purified from bovine lung was determined in the presence and absence of sodium nitroprusside (SNP, 1 MM) Incubations were carried out in triethanolamine/HCl (5 IT\M) buffer or modified Tyrode's solution, as described in Methods. CaClj (1.6 ITIM) and NaCl (134 mM) were added (+) to or omitted (—) from the incubation medium Data given are mean ± SE of three experiments performed in triplicates. stimulation. Correspondingly, enzyme activities and the extent of stimulation were increased by omission of NaCl, CaCl2, or both, from the modified Tyrode's solution (Table 1). Basal activity of GC in modified Tyrode's solution was 10.0 ± 2.2 nmol/mg per min. NE, ACh, atropine, mepacrine, nordihydroguaiaretic acid, and indomethacin, in the concentrations used, did not affect basal GC activity or stimulation of the enzyme by SNP when determined in modified Tyrode's solution. In contrast, nordihydroguaiaretic acid (20 HM) and indomethacin (20 ^M) caused inhibition (about 20%) of GC when determined in triethanolamine/HCl buffer. ACh-Induced Stimulation of Purified Soluble Guanylate Cyclase In our first attempts to demonstrate activation of soluble GC by an ACh-induced stimulating factor, we transferred the effluent from the lumen of endothelium-intact rabbit aortic segments into test tubes containing purified soluble GC. There was no significant effect on GC activity under the conditions described in Methods. Therefore, the purified soluble GC was injected into the lumen of rabbit aortic segments, where the incubations for the determination of the enzyme activity were carried out. There was a significant increase in GC activity (164 ± 12%; n = 12) in the absence of ACh and NE, in comparison to control experiments performed in test tubes (100%) (Fig. 2a). In the presence of ACh (2 ^M), enzyme activity was about 4.4-fold higher than in control experiments (Fig. 2c). The effect was dependent on the concentration of ACh (Fig. 2, a-c), but independent of the presence of NE (Fig. 2, d-f). The effects described were independent of enzyme concentrations used (10-100 nM, data not shown). SNP (1 ^M) enhanced the activity of soluble GC about 5-fold in the arterial lumen (Fig. 2g), and about 8-fold in test tubes (Table 1). This difference probably results from diffusive loss of SNP into vessel walls and the surrounding medium during the incubation period. The activity of GC injected into canine femoral segments was increased to 189 ± 39% (n = 6) of control experiments. The activity was further increased to 341 ± 44% (n = 6) in the presence of NE (0.1 fiM) and ACh (2 HM). Formation of cGMP by purified soluble GC was approximately proportional to the incubation time (at least up to 720 seconds) in test tubes and in rabbit aortic segments (Fig. 3). Also, the AChinduced stimulation of GC remained nearly constant between 160 and 720 seconds, as indicated by the approximately constant relation between stimulated and control values (Fig. 3). Inhibition of ACh-Induced Stimulation of Purified Soluble Guanylate Cyclase The activity of purified soluble GC injected into the lumen of endothelium-denuded rabbit aortic Forstermann et a/./Endothelial Relaxing Factor and Guanylate Cyclase 500 535 300-i NE * ACh (aorta) 400 8 ? — 250 300 N JS u © O I 200- 200 150- c 1001OO0 J Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 NE [|jM] ACh [yM] SNP[uM] - — 0.05 - — 2 - 01 — - 01 Q05 - 01 2 - a 1 FIGURE 2. Stimulation of soluble guanylate cyclase injected into the lumen of rabbit aortic segments. Purified soluble guanylate cyclase (CQ in modified Tyrode's solution was injected, together with its substrate, into the lumen of vascular segments disconnected from the perfuswn. The enzyme activity was determined intraluminaily in the absence and presence of ACh, NE, and ACh with NE, as indicated. CC activities obtained by incubation of the enzyme in modified Tyrode's solution in test tubes were taken as control activities (100%). The control value for the enzyme activity in the presence of sodium nitroprusside (SNP) was obtained by incubation of the enzyme in test tubes without SNP. The incubation time was always 3 minutes. Enzyme activities are given as mean ± SE of seven or more experiments. CC activity obtained by intralummal incubation without additions was significantly greater than that obtained by incubation of the enzyme m test tubes + = Significantly greater than column a (P < 0.01). * = Significantly greater than column d (P < 0.01) z 50- 2 4 6 8 X) 12 incubation time [min] FIGURE 3. Time-dependent formation of cGMP by soluble guanylate cyclase. Purified soluble GC was incubated in modified Tyrode's solution in test tubes ( ) and intraluminaily in rabbit aortic segments ( ) for various times. NE (0.1 ^M) alone (open symbols), or NE (0.1 IIM) ACh (2 HM) (closed symbols) were added to the incubation medium. Each point represents means ± SE of four to six experiments. + = Significantly greater than in test tubes at the same incubation time (+ = P < 0.05; ++ = P < 0.01). * = Significantly greater than with NE alone in the vascular segment at the same incubation time C = P < 0.05, " = P < 0.01). stimulation of GC was not affected by these drugs (Fig. 4, c-e). segments was not significantly different from test tube controls (Fig. 4a). In addition, the ACh-induced stimulation of GC was abolished in these segments (Fig. 4a). The addition of atropine (2 ^M) significantly reduced ACh-induced stimulation of GC in endothelium-intact segments to 163 ± 20% (n = 8) of control values, whereas basal (ACh-independent) enzyme stimulation due to endothelial cells was not significantly altered (Fig. 4b). Pretreatment of endothelium-intact rabbit aortic segments for 60 minutes with mepacrine (30 MM) or nordihydroguaiaretic acid (20 MM) significantly reduced ACh-induced stimulation of GC to 198 ± 17% (n = 6) or 221 ± 10% (n = 6) of control values, respectively (Fig. 4, c and d). These effects are independent of the presence of mepacrine or nordihydroguaiaretic acid, during the inrraluminal incubation of soluble GC (data not shown). Pretreatment of the vascular segments with indomethacin (20 ^M) did not significantly alter ACh-induced GC stimulation (Fig. 4e). The AChindependent and basal endothelium-dependent Discussion To demonstrate the proposed stimulatory effect of an ACh-induced endothelial factor on soluble GC, it was necessary to use a medium which does not disturb the integrity and responsiveness of the endothelial cell membrane with respect to the formation and release of endothelial relaxing factoids). On the other hand, this medium must allow the determination of the activity of a purified soluble GC. To meet the first demand, we had to use an extracellular medium like Tyrode's solution; however, additions such as glutathione, 7-gIobulin, GTP, cGMP, and additional MgC^ were necessary to achieve a sufficient GC activity and recovery of the cGMP formed. The constituents of Tyrode's solution NaCl and CaCl2 reduced significantly the activity of the purified soluble, heme-containing GC, and, to a greater degree, the enzyme stimulation by SNP. The effect of NaCl is probably caused by the increased ionic strength of the buffer. Effects of Ca++ on soluble GC have been described by several Circulation Research/Vol. 58, No. 4, April 1986 536 400 -i Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 -Endo Atrop Mepa NDGA Indo a b C d e FIGURE 4. Effect of different expenmental conditions on the stimulation of soluble guanylate cyclase injected into the lumen of rabbit aortic segments. The vascular segments were disconnected from the perfusion system; purified soluble GC was injected together with its substrate into the lumen. The enzyme activity was determined in the presence of NE (0.1 \IM); incubation time was 3 minutes. After a wash-out period (perfusion), the enzyme activity was determined again in the presence of NE (0.1 fiM) and ACh (2 ^M). The determinations of enzyme activities were repeated after the following treatments of the vascular segments: (a) removal of the endothelium (-Endo), (b) addition of atropine (Atrop, 2 HM), (C) perfusion of the segments for 60 minutes with mepacrine (Mepa, 30 HM), (d) with nordihydroguaiaretic acid (NDGA, 20 fiin), (e) with indomethacin (Indo, 20 fiM). Each column represents the mean ± SE of at least four experiments. GC activities determined in the lumen of the vascular segments are given as percent of the enzyme activities obtained in parallel test tube incubations with the respective additions. Plusses = significantly greater or smaller than the first column of the same group, i.e., NE alone (+ = P < 0.05; ++ = P < 0.005). Asterisks = significantly smaller than the second column of the same group, i.e., NE and ACh (* = P < 0.05; ** = P < 0.005). n.s. = not significantly different. authors. So far, these studies show conflicting results, probably because different enzyme preparations were used. Inhibitory effects of Ca on the stimulation of soluble GC by NO-containing compounds but not on the basal enzyme activity have been described in more detail, e.g., by Gruetter et al. (1980). The effect of Ca++ as well as ionic strength on the purified, soluble, heme-containing guanylate cyclase certainly deserves further investigation. If purified soluble GC was injected, together with its substrate, into the lumen of endothelium-intact rabbit aortic or canine femoral arterial segments, a significant stimulation of the enzyme was observed in the absence of ACh. Since removal of endothelium reduced the enzyme activities to values obtained in the test tubes, this points to a basal release of a GC-stimulating factors) from endothelial cells. Similarly, a basal release of ERDF has recently been demonstrated by Griffith et al. (1984). Furthermore, it has been shown that removal of endothelium decreases cGMP levels in the subjacent smooth muscle cells (Holzmann, 1982; Diamond and Chu, 1983; Rapoport and Murad, 1983a, 1983b; Furchgott et al., 1984; Miller et al., 1984). The activity of the purified soluble GC injected into the lumen of vascular segments was further stimulated by ACh in a concentration-dependent manner. This stimulation was prevented by removal of endothelium or by the addition of atropine. These results indicate that ACh-induced stimulation of soluble GC is endothelium dependent and is mediated by muscarinic receptors. The same has been shown for ACh-induced formation and/or release of EDRF (Furchgott and Zawadzki, 1980). The extent of the ACh-induced stimulation of soluble GC was independent of the incubation time. The stimulatory factor(s) obviously reached a steady state level, suggesting a short life time as described for the ACh-induced EDRF. Similarly, we were unable to demonstrate any significant stimulation of soluble GC in test tubes by the effluent of endothelium-intact rabbit aortic segments treated with ACh. This further indicates that the stimulating activity does not accumulate in the vascular lumen, and is obviously chemically unstable, as has been reported for EDRF (Griffith et al., 1984; Forstermann et al., 1984). ACh-induced stimulation of soluble GC was inhibited after pretreatment of endothelium-intact rabbit aortic segments with mepacrine or nordihydroguaiaretic acid at concentrations that also inhibited the ACh-induced formation and/or release of EDRF (Furchgott and Zawadzki, 1980; Chand and Alrura, 1981; Furchgott, 1983, 1984; Forstermann et al., 1984; Forstermann and Neufang, 1984). The blockade of the ACh-induced GC stimulation was Forstermann et a/./Endothelial Relaxing Factor and Guanylate Cyclase Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 evident, even though these drugs were absent during the actual incubation. Thus, inhibition takes place at the level of the endothelium, and is not caused by an interaction of these drugs with soluble GC itself. The ineffectiveness of indomethadn to inhibit ACh-induced stimulation of soluble GC implies that the formation and/or release of the stimulating principle is insensitive to indomethacin, at least in the concentration used, as shown for EDRF (Furchgott and Zawadzki, 1980; Furchgott, 1983, 1984; Forstermann and Neufang, 1984). It has previously been reported that platelet membranes can release fatty acids that modulate GC activity (Gerzer et al., 1983). Arachidonic acid or linoleic acid are known to stimulate basal activity of soluble GC (Bohme et al., 1983; Gerzer et al., 1983; Wolin and Ignarro, 1983). However, it is unlikely that these fatty acids, or any of their relatively stable metabolites, are responsible for the activation of GC observed here, since the GC stimulating factors) obviously has a short life time. The extent of ACh-induced increase in GC activity shown here would not account for the increase of cGMP levels in rabbit aorta (Diamond and Chu, 1983; Furchgott and Jothianandan, 1983; Furchgott et al., 1984). However, the intracellular medium is different from the intraluminal medium in which the determination of soluble GC was performed. The basal enzyme activity, and especially the stimulation by SNP, were reduced in modified TyTode's solution. Therefore, it is likely that the effect of the ACh-induced stimulatory factor(s) on GC is reduced as well. Furthermore, the release of ACh-induced EDRF seems to be greater in the abluminal than in the intraluminal direction (Busse et al., 1985). In conclusion, the results presented demonstrate that endothelial cells, of at least two types of arteries (rabbit aorta and canine femoral artery), produce a labile factor(s) which stimulates soluble GC. The formation and release of this factor is increased by ACh. The factor may be identical with the AChinduced EDRF. This assumption is supported by correlations between known properties of EDRF and those found for the GC-stimulating factors). Thus, stimulation of GC, at least of the soluble enzyme form, could be responsible for the increase in cGMP levels observed in vascular smooth muscle cells during endothelium-dependent relaxation. We would like to acknowledge the excellent technical assistance of Cornelia Kellner. This work was presented in part at the Spring Meeting of the German Pharmacological Society, March 1985 (Mulsch et al., 1985) The work was supported by the Deutsche Forschungsgemeinschaft (Grants Schu 2/14-5 and Bu 436/2-2). Dr. Forstermann was affiliated with the Department of Pharmacology at the University of Freiburg, Drs Mulsch and Bohme with the Department of Pharmacology at the University of Heidelberg, and Dr. Busse with the Department of Applied Physiology at the University of Freiburg. 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U Förstermann, A Mülsch, E Böhme and R Busse Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 Circ Res. 1986;58:531-538 doi: 10.1161/01.RES.58.4.531 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1986 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/58/4/531 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. 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