A 38-amino-acid peptide isolated from the venom of the green mamba snake (Dendroaspis angusticeps) shares structural homology with the family of natriuretic peptides (1). This peptide also shares biologic properties with other known natriuretic peptides including natriuresis, relaxation of isolated aortic rings, and increases in cyclic guanosine monophosphate (cGMP) in cultured vascular endothelial cells and myocytes. Therefore it has been designated D-type natriuretic peptide or DNP. Like atrial natriuretic peptide (ANP), DNP is a ligand for the natriuretic peptide A receptor (NPRA), which contains a particulate guanylate cyclase domain and the NPRC receptor or clearance receptor (1). Preliminary experiments from our laboratory show that DNP causes relaxation of coronary arteries from experimental animals, but the mechanisms mediating these relaxations are not defined (2).
C-type natriuretic peptide (CNP), another member of the natriuretic peptide family of endothelial origin, also relaxes coronary arterial smooth muscle. CNP stimulates NPRB and NPRC receptors (3,4), and relaxations to this peptide are mediated through activation of particulate guanylate cyclase and hyperpolarization by activation of potassium channels (5-7). Relaxations to CNP are reduced in the presence of the endothelium (3), but CNP itself may be released as an endothelium-derived relaxing factor simultaneous with nitric oxide (8). How relaxations to DNP are modulated by the endothelium is not clear.
Pharmacologic and genetic manipulation of endogenous natriuretic peptide systems in experimental animals emphasize the importance of these peptides in integration of neurohumoral control of sodium balance and arterial pressure (9-15). In humans, a Dendroaspis natriuretic peptide-like immunoreactive substance increases both in plasma and in atrial myocytes of patients with congestive heart failure (16). The defined sequence of this DNP-like immunoreactive substance in humans remains to be defined. However, elucidating pharmacologic mechanisms of Dendroaspis-derived peptide in coronary arteries of experimental animals provides a background on which to study how similar peptides once defined in humans might participate in either limiting or facilitating progression of cardiovascular disease. Therefore experiments were designed to determine how the endothelium affects responses of canine coronary arteries to DNP and to determine mechanisms by which DNP directly relaxes canine coronary arterial smooth muscle.
Left circumflex coronary arteries were removed from anesthetized (30 mg/kg sodium pentobarbital, intravenously) adult, male mongrel dogs. Arteries were cleaned of adventitia, cut into rings, and suspended for the measurement of isometric force in organ chambers filled with modified Krebs-Ringer bicarbonate solution (control solution; millimolar composition: NaCl, 118.3; KCl, 4.7; MgSO4, 1.2; KH2PO4, 1.2; CaCl2, 2.5; NaHCO3, 25; dextrose, 11.1; and CaEDTA, 0.026) at 37°C and bubbled with 95% O2/5% CO2. In some rings, the endothelium was removed deliberately.
After a 15-min equilibration period, each ring was placed at the optimal point on the length-tension curve by determining the active tension developed to 20 mM KCl at different levels of passive tension. After a second equilibration period, rings were either untreated or incubated with one of the following inhibitors for 30 min: C-ANP (10−6M, an inhibitor of natriuretic peptide clearance receptors); indomethacin (10−5M, an inhibitor of cyclooxygenase); HS-142-1 (10−5M, an inhibitor of particulate guanylate cyclase), NG-monomethyl-L-arginine (L-NMMA, 10−4M, an inhibitor of nitric oxide production); 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 10−5M, an inhibitor of soluble guanylate cyclase), or tetraethylammonium chloride (TEA, 10−3 or 10−2M, a nonselective inhibitor of potassium channels). Rings then were contracted with either prostaglandin F2α (PGF2α, 2 × 10−6M), endothelin-1 (10−7M), or KCl (40 or 60 mM). Once contractions plateaued, concentration-response curves were obtained to DNP (10−10−10−7M). Responses in the absence and presence of inhibitors were studied in parallel, and only one response to DNP was obtained per ring.
Drugs and chemicals
All drugs were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.) except the following: DNP and endothelin-1, Phoenix Pharmaceuticals Inc., Mountain View, CA, U.S.A.; and HS-142-1, Kuowa Hakko Kogyo, Tokyo, Japan. Unless specified, drugs were prepared daily in distilled water and kept on ice until use. ODQ was dissolved in dimethyl sulfoxide and further diluted in distilled water (final bath concentration, 10−5M). All drug concentrations are expressed as the final molar (M) concentration in the organ chamber.
Results are expressed as mean ± SEM. "n" represents the number of dogs from which arteries were taken. Relaxations are expressed as percentage change in tension from contractions to either PGF2α, endothelin-1, or KCl. Concentrations causing 50% of the maximal response (EC50) were calculated for individual response curves, and the mean values compared by analysis of variance. One-way analysis of variance also was used to compare maximal relaxations or areas under the concentration-response curves. When the F value was significant, a Scheffé's test was used for post hoc analysis of the means. The α level was set at 0.05 for all tests.
Effect of endothelium
DNP caused concentration-dependent relaxations of coronary arterial rings with and without endothelium contracted with PGF2α. Relaxations of rings with endothelium were significantly greater than those of rings without endothelium (Fig. 1). Relaxations of rings with endothelium were reduced by the inhibitor of natriuretic clearance receptors, C-ANP, but not by L-NMMA or indomethacin (Fig. 2). In the presence of C-ANP, relaxations of rings with and without endothelium were comparable. The inhibitor of particulate guanylate cyclase, HS-142-1, also significantly inhibited relaxations to DNP in rings with endothelium (Fig. 3). In the presence of HS-142-1, relaxations to DNP in rings with endothelium were similar to those of rings without endothelium in the absence of the inhibitor.
Mechanism of relaxations to DNP in smooth muscle
In rings without endothelium contracted with PGF2α, maximal relaxations to DNP were reduced from 60.8 ± 8.9% to 47.4 ± 7.1% by C-ANP (paired t test, p < 0.05). Relaxations to DNP were not altered by indomethacin or L-NMMA in rings without endothelium (data not shown; n = 4-6/group). HS-142-1 also significantly inhibited relaxations to DNP in rings without endothelium (Fig. 3).
In rings without endothelium, relaxations to DNP were significantly reduced when rings were contracted with KCl (either 40 mM, not shown, n = 3; or 60 mM;Fig. 4) compared with matched contractions with either PGF2α or endothelin-1. However, the nonselective inhibitor of potassium channels, TEA, did not alter relaxations to DNP in rings contracted with PGF2α(Fig. 5). Neither indomethacin nor an inhibitor of soluble guanylate cyclase, ODQ, significantly reduced relaxations to DNP in rings without endothelium contracted with PGF2α(Fig. 5).
Results of this study indicate that like ANP and CNP, DNP directly relaxes coronary arterial smooth muscle from an experimental animal (dog). These results also identify that unlike ANP and CNP, to which relaxations either are not affected or are reduced by the endothelium (1,6,17-20), relaxations to DNP are augmented in the presence of endothelial cells. The endothelial component of the DNP response probably does not involve nitric oxide or inhibitory prostanoids, as relaxations of rings with endothelium are not inhibited by either L-NMMA or indomethacin, respectively. The endothelial component of the response probably does involve release of endothelium-derived relaxing factors or inhibition of endothelium-derived contracting factors associated with activation of natriuretic peptide clearance receptors (C-receptors). This conclusion is supported by the observation that C-ANP, which inhibits the clearance receptors, shifted the concentration-response curve of relaxations to DNP in rings with endothelium to levels comparable to relaxations in rings without endothelium. The mediator(s) of this response remains to be determined but could involve release of another natriuretic peptide, for example, CNP, from endothelial cells (8). This new finding of an endothelial component of the response to DNP may be important. A DNP-like immunoreactive substance is elevated in plasma and atrial myocytes of humans with heart failure (16). Whether this DNP-like immunoreactive substance in humans represents a fourth member of the natriuretic peptides in humans awaits definitive sequencing of the peptide. Changes in endothelial function may participate in vascular adaptations in diseases such as congestive heart failure, atherosclerosis, hypertension, and diabetes. If a homologous peptide is identified in humans, changes in its production and associated endothelial function may be important in understanding vascular changes in these diseases.
Activation of the natriuretic peptide clearance receptors also mediates in part relaxations of the smooth muscle to DNP. This conclusion is supported by the observation that relaxations to DNP are reduced further by C-ANP when the endothelium is removed. This suggests that the endothelium may represent a diffusion or functional barrier for C-ANP to reach the smooth muscle. There may be differences in the number of endothelial compared with smooth muscle clearance receptors or differences in affinity of the clearance receptors for DNP in the two types of cells.
As with other natriuretic peptides, relaxations to DNP involve activation of receptors coupled to particulate and not soluble guanylate cyclase, as relaxations in both rings with and without endothelium were inhibited by HS-142-1, but not by ODQ (1,6).
Although relaxations to DNP are reduced when the smooth muscle is depolarized, relaxations were not inhibited by the nonselective inhibitor of potassium channels. Therefore this indirect evidence suggests that unlike CNP (5-7), DNP may not directly hyperpolarize smooth muscle cells. However, indirect activation of potassium channels subsequent to changes in cGMP cannot be ruled out at this time (21).
Acknowledgment: These studies were supported in part by grants from the National Institutes of Health HL07111-24 and the Mayo Foundation.
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