INTRODUCTION

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AFTER THE RELEASE of an endothelium-derived relaxing factor (EDRF) in response to acetylcholine was demonstrated by Furchgott and Zawadzki (6), the role of the endothelium shifted from that of a passive lining to a dynamic regulator of vascular tone. One EDRF has since been demonstrated to be nitric oxide (NO) derived from L-arginine by the enzyme NO synthase (NOS) (14), which diffuses into vascular smooth muscle and stimulates guanylyl cyclase resulting in the generation of cGMP. L-Arginine analogs such as N^G-monomethyl-L-arginine (L-NMMA) have proven useful in defining the physiology of the NO system as they competitively inhibit NOS, thereby attenuating the generation of NO (15). Other EDRFs have since been identified, including C-type natriuretic peptide (CNP) (16-19). In contrast to NO, CNP causes relaxation through activation of natriuretic peptide type B receptors on vascular smooth muscle cells, which are linked to particulate guanylyl cyclase receptors and generation of cGMP (1, 9, 10). HS-142-1 is a nonpeptide inhibitor to the natriuretic peptide receptors that reduces CNP-stimulated cGMP production (20, 21). In addition to direct stimulation of guanylate cyclase, both NO and CNP also activate potassium channels, thereby acting as endothelium-derived hyperpolarizing factors (EDHF) (2-4, 20).

On the basis of reports that acetylcholine and bradykinin may release factors in addition to NO, the objective of the present study was to investigate the potential synergistic release of NO and CNP with endothelial stimulation by acetylcholine and bradykinin.

	MATERIALS AND METHODS

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Organ chamber protocol. After anesthesia (pentobarbital sodium, 30 mg/kg iv) was administered, hearts were removed from adult mongrel dogs (male and female, weight ~25 kg) and placed into chilled modified Krebs-Ringer bicarbonate buffer (composition in mM: 118.3 NaCl, 4.7 KCl, 2.5 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, 25.0 NaHCO₃, 0.026 CaEDTA, and 11.1 glucose; control solution). The left circumflex artery was carefully dissected free of connective tissue, removed, and cut into 4-mm rings. One-half of the rings were denuded of endothelium by inserting a pair of blunt forceps into the lumen, gently rolling over a Krebs-Ringer wetted paper, and rinsing with control solution. Rings were suspended in parallel as pairs, with and without endothelium, between a fixed clip and a clip attached to a pressure transducer (UC-2; Gould, Glen Burnie, MD) in eight 25-ml tissue baths filled with control solution aerated with 95% O₂-5% CO₂ and maintained at 37°C. Changes in isometric force were recorded on an eight-channel recorder (model 7418A recorder; Hewlett-Packard, Palo Alto, CA). Each ring was progressively stretched to its optimal length on a passive tension-active tension curve determined by the force of contraction generated by 20 mM KCl. Maximal force generated to 60 mM KCl was then determined, and vessels were washed and allowed to stabilize before determination of final baseline tension.

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Reagents. The following drugs were used: acetylcholine, indomethacin, and PGF₂ (Sigma Chemical, St. Louis, MO), L-NMMA (CalBiochem-Novabiochem, La Jolla, CA), CNP and bradykinin (Phoenix Pharmaceutical, Mountain View, CA), and HS-142-1 (a gift from Kyowa Hakko Kogyo, Tokyo, Japan). Drugs were prepared daily and stored at 4°C until use. Concentrations reported are final concentration in the organ bath.

Preparation of NO. A gas bulb fitted with a silicon rubber injection septum was filled with NO from a cylinder (Union Carbide, Chicago, IL). From this bulb 10, 100, and 1,000 µl were removed with a microsyringe and injected into three glass bulbs containing 100 ml of distilled water previously purged of oxygen by infusing with helium for 3 h. Resulting NO stock solutions were 4 × 10⁶, 4 × 10⁵, and 4 × 10⁴ M as described by Palmer et al. (14).

Statistical analyses. For all studies, n equals the number of dogs from which rings were harvested. Values are expressed as means ± SE. All comparisons were performed by unpaired t-test or one-way ANOVA followed by Bonferroni's post hoc test as appropriate. Significance is defined at P < 0.05. Concentration producing 50% of the contraction to PGF₂ was calculated from individual concentration-response curves, and the average was expressed as negative log molar.

	RESULTS

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Effects of inhibitors. None of the inhibitors caused significant contractions of rings without endothelium (data not shown, n = 10-12 per group). In rings with endothelium L-NMMA caused statistically significant increase in tension (Table 2). The combination of HS-142-1 plus L-NMMA did not cause greater increase in tension than L-NMMA alone (Table 2).

View this table: [in this window] [in a new window]   Table 2.   Tension generated by inhibitors and PGF2 in rings with endothelium

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Relaxations to CNP and NO. CNP caused concentration-dependent relaxations of rings with and without endothelium (Fig. 1). HS-142-1 but not L-NMMA attenuated relaxations to CNP in rings with and without endothelium. Relaxations to CNP in the presence of HS-142-1 plus L-NMMA were not different from those obtained with HS-142-1 alone. Exogenous NO caused comparable concentration-dependent relaxations of rings with and without endothelium. Neither HS-142-1, L-NMMA nor combined L-NMMA plus HS-142-1 affected NO-mediated relaxations (Fig. 2).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Cumulative concentration-response curves to C-type natriuretic peptide (CNP) in canine coronary arteries with endothelium (n = 10) in absence (control) and presence of HS-142-1, NG-monomethyl-L-arginine (L-NMMA), or combined HS-142-1 and L-NMMA. Experiments were conducted in the presence of indomethacin (105 M). Data are shown as a percent change in tension from contraction to prostaglandin F2 (PGF2) and are presented as means ± SE. * Statistical significance in maximal relaxation vs. control and L-NMMA-treated groups.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Concentration-response curves to nitric oxide in canine coronary arteries with endothelium (n = 4) in absence (control) and presence of HS-142-1, L-NMMA, or combination of HS-142-1 and L-NMMA. Experiments were conducted in the presence of indomethacin (105 M). Data are shown as percent change in tension from contraction to PGF2 and are presented as means ± SE.

Relaxations to acetylcholine and bradykinin. Acetylcholine caused concentration-dependent relaxations only in rings with endothelium. Relaxations to acetylcholine were not attenuated by incubation with HS-142-1 alone. Incubation of rings with L-NMMA shifted the dose-response curve to the right. Coincubation of the rings with L-NMMA plus HS-142-1 significantly inhibited relaxations to acetylcholine compared with incubation with either inhibitor alone (Fig. 3, Table 3).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Concentration-response curves to acetylcholine in canine coronary arteries with endothelium (n = 7) in absence (control) and presence of HS-142-1, L-NMMA, and L-NMMA plus HS-142-1. Experiments were conducted in presence of indomethacin (105 M). Data are shown as percent change in tension from contraction to PGF2 and are presented as means ± SE. * Statistical significance in concentration causing 50% relaxation vs. control and HS-142-1 treated. # Statistical significance vs. L-NMMA treated (also see Table 2).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Concentration-response curves to bradykinin in canine coronary arteries with endothelium (n = 7) in absence (control) and presence of HS-142-1, L-NMMA, and L-NMMA plus HS-142-1. Experiments were conducted in presence of indomethacin (105 M). Data are shown as percent change in tension from contraction to PGF2 and are presented as means ± SE. * Statistical significance in concentration causing 50% relaxation vs. control and HS-142-1 treated. # Statistical significance vs. L-NMMA treated (also see Table 2).

	DISCUSSION

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The current studies confirm that CNP directly stimulated vascular smooth muscle as it relaxed isolated coronary arteries without endothelium, and relaxations were unaffected by inhibition of NO production with L-NMMA in either rings with or without endothelium (19). The current study also confirms that CNP mediates relaxation via the natriuretic peptide receptors as HS-142-1 attenuated the actions of CNP.

The results also support that NO and not CNP may be tonically released from canine coronary arterial endothelial cells as L-NMMA and not HS-142-1 significantly increased basal tension.

Most importantly, the current study extends previous reports to support the concept that endothelium-dependent relaxations to acetylcholine and bradykinin may be mediated in part via release of CNP. This conclusion is based on the marked attenuation of acetylcholine and bradykinin relaxations by coinhibition of NO generation with L-NMMA and natriuretic peptide receptor antagonism with HS-142-1. Expressing results as a percent change in tension from contractions to PGF₂ is a conservative estimate of the inhibition as total active tension (response to inhibitors plus the prostanoid) would be greater than with that of the prostanoid alone. Furthermore, the greater inhibition of relaxations by the combined inhibitors is not due to greater contraction because neither the contractions to PGF₂ nor total active tension (prostaglandin plus inhibitors) was different with HS-142-1 plus L-NMMA than with L-NMMA alone.

Several mechanisms may explain the current findings. The most direct would be that HS-142-1 acts as a nonspecific guanylyl cyclase inhibitor of both particulate and soluble guanylyl cyclases. This does not appear to be the case, since HS-142-1 alone does not significantly inhibit acetylcholine- or bradykinin-induced relaxations. Furthermore, HS-142-1 alone or in combination with L-NMMA did not inhibit relaxations to exogenous NO. An alternative explanation would be that CNP is released from the endothelium in response to acetylcholine or bradykinin at the same time as NO despite the fact that HS-142-1 alone did not significantly inhibit relaxations to either agonist. This later observation may reflect either the greater effect of NO compared with CNP in stimulating guanylyl cyclase and inducing relaxation, or that acetylcholine and bradykinin release a relatively small amount of CNP compared with the amount of NO generated. Indeed, masking of the particulate guanylyl cyclase activity would occur secondary to the relative efficacy or amount released. This concept would be consistent with studies that have recently demonstrated that inhibition of endogenous NOS activity augments the affect of ANP in both relaxation of isolated renal glomeruli and in generation of cGMP (12).

Previous studies have documented that both CNP and NO hyperpolarize vascular smooth muscle in addition to activating guanylyl cyclases (2, 4, 11, 13, 20). Specifically, high K⁺ buffers inhibit CNP- and NO-induced relaxations, suggesting that both agents act as EDHF. It has also been shown that inhibition of EDHF activity by high K⁺ buffer attenuates relaxations to acetylcholine to a greater extent than inhibition of NO alone (5, 8). These previous studies are consistent with the current observation of enhanced attenuation of acetylcholine- and bradykinin-induced relaxations in the presence of combined HS-142-1 and L-NMMA and does not eliminate the possibility for simultaneous release of other endothelium-derived factors. The current studies provide additional support for a dual mechanism for activation of cGMP pathways responsible for the control of vascular tone. These two pathways now include the NO system involving soluble guanylyl cyclase and the natriuretic peptide system involving particulate guanylyl cyclase.

In summary, results of the present study confirm that relaxations to CNP in canine coronary arteries do not require the endothelium, are independent of NO, and are inhibited by the natriuretic peptide receptor inhibitor HS-142-1. Furthermore, a novel interaction between the NO and the natriuretic peptide system is demonstrated by the increased attenuation of endothelium-dependent relaxations to acetylcholine and bradykinin when both NOS and natriuretic peptide receptors are inhibited. These data provide support for the concept of release of multiple endothelium-derived factors, including CNP in response to acetylcholine- and bradykinin-receptor stimulation of endothelial cells. In addition, they provide the basis for further investigations into cross-talk among endothelium-derived factors in the regulation of vascular tone.