Author + information
- Received December 18, 1995
- Revision received April 4, 1996
- Accepted May 7, 1996
- Published online October 1, 1996.
- R. SCOTT WRIGHT*
- ↵*Address for correspondence: Dr. R. Scott Wright, Division of Cardiovascular Diseases, Mayo Clinic, 200 First Street, SW, Mail Stop SMH 1-251, Rochester, Minnesota 55905.
- CHEOL H. KIMa
- LAWRENCE L. AARHUS,
- JOHN C. BURNETT Jr. and
- WAYNE L. MILLER
Objectives. We tested the hypothesis that C-type natriuretic peptide (CNP) mediates coronary vasodilation through activation of cyclic guanosine monophosphate (cGMP) by way of particulate guanylate cyclase.
Background. CNP has known peripheral vasodilator properties, and preliminary data have suggested that it can function as a coronary vasodilator.
Methods. The actions of CNP were studied in instrumented dogs and in organ chamber rings in the presence and absence of a known antagonist to particulate guanylate cyclase, HS-142-1. Additionally, the actions of HS-142-1 were tested on acetylcholine-mediated coronary vasodilation, and immunohistochemical staining was utilized to localize the presence of CNP in the coronary endothelium.
Results. CNP relaxed isolated coronary arteries with (mean ± SEM 45.9 ± 7%*) and without (72.0 ± 7%*†) an endothelium (*p < 0.05 for CNP effect alone, †p < 0.05 for endothelium vs. no endothelium with CNP). Intracoronary infusions increased coronary blood flow (baseline, 64.6 ± 5.1 ml/min; CNP-5, 79.9 ± 6.1*; CNP-20, 103.3 ± 13.6* [*p < 0.05 vs. baseline value]) and reduced coronary vascular resistance (baseline, 1.6 ± 0.3 mm Hg/ml per min; CNP-5, 1.4 ± 0.3*; CNP-20, 1.2 ± 0.3*). Intracoronary injections increased coronary blood flow (Δ baseline coronary flow, 30 ± 9* ml/min [*p < 0.05]). HS-142-1 significantly attenuated these increases (Δ coronary flow, 30 ± 9* ml/min [CNP] to 14 ± 6† [CNP + HS-142-1] [†p < 0.05 CNP vs. CNP + HS-142-1]) and the relaxation of organ chamber rings (56 ± 7% [CNP] to 18 ± 6%† [HS-142-1 + CNP]). Finally, CNP was localized to the coronary endothelium and smooth muscle by immunohistochemical staining.
Conclusions. CNP functions as a coronary vasodilator through activation of cGMP by way of particulate guanylate cyclase. CNP-mediated coronary vasodilation is attenuated by intracoronary HS-142-1. Intracoronary HS-142-1 does not affect acetylcholine-mediated coronary vasodilation. These observations support a role for exogenous CNP as a potent coronary vasodilator.
C-type natriuretic peptide (CNP) is a recently described 22-amino acid vasoactive peptide . CNP represents the third member of a family of structurally homologous but genetically distinct peptides that includes atrial (ANP) and brain (BNP) natriuretic peptides . It relaxes isolated peripheral arteries and veins  and reduces cardiac preload and arterial pressure in the absence of any change in renal sodium excretion [4, 5]. It also inhibits serum-mediated vascular smooth muscle proliferation . Although CNP shares many of the actions of ANP, the major distinction between the two peptides is that CNP does not act as a mediator for natriuresis. The lack of CNP-mediated natriuresis occurs in part because CNP and ANP bind to different receptors . CNP is the ligand for a particulate guanylate cyclase-linked receptor known as the NPR-B receptor [2, 7]. The NPR-B receptor is genetically distinct from the receptor (NPR-A) for ANP and BNP. The NPR-B receptor is preferentially expressed in cardiac tissue and vascular smooth muscle, whereas the NPR-A receptor is highly expressed in the kidney . Recently, Stingo et al.  and Suga et al.  reported that endothelial cells synthesize, store and release CNP, demonstrating that CNP is of endothelial cell origin . In addition, Wei et al.  have demonstrated the presence of CNP in the human heart and microcirculation.
Studies  have emphasized the importance of endothelial cell-derived vasodilators like nitric oxide, endothelial cell hyperpolarization factor and prostacyclin as potent coronary vasodilators. The localization of the CNP receptor to cardiac tissue and vascular smooth muscle and the observation that CNP is of endothelial cell origin suggest a potential role for CNP as a vasodilator for the coronary circulation. We  have reported preliminary data suggesting that CNP functions as a coronary vasodilator.
The current investigation was undertaken with several objectives. We sought to determine 1) whether CNP relaxes preconstricted epicardial canine coronary arteries, 2) whether CNP functions as a coronary vasodilator with associated alterations in cardiac function, 3) how the coronary nitric oxide and particulate guanylate cyclase systems affect CNP-mediated coronary vasodilation, and 4) whether CNP can be localized to the coronary endothelium by immunohistochemical staining.
Studies were conducted in isolated coronary arteries obtained from normal dogs for the organ chamber preparations and in anesthetized dogs for the in vivo protocols.
Organ chamber protocol. Rings of epicardial coronary arteries (left circumflex and left anterior descending) were obtained from five normal mongrel dogs and suspended in organ chambers filled with aerated (95% oxygen, 5% carbon dioxide), modified Krebs-Ringer bicarbonate solution of the following composition: (in millimol/liter 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3, 0.026 CaNa2EDTA and 11.1 dextrose control solution) at 37°C. In half of the vessel rings, the endothelium was removed by gently rubbing the intimal surface with a cotton swab wetted with control solution. Each ring was stretched to the optimal point on its length-tension curve as determined by the maximal tension developed to potassium chloride (20 millimol/liter) at each level of stretch. The functional presence of endothelium was determined at the beginning of the experiment by a relaxation response to acetylcholine (10−6 mol/liter) during a contraction to PGF2α (2 × 10−6 mol/liter) at optimal length. To study responses to CNP, the rings were contracted with PGF2α (2 × 10−6 mol/liter). A dose-response curve to cumulative concentrations of CNP (10−10 mol/liter to 3 × 10−6 mol/liter) was obtained, and final results were obtained utilizing 10−7 mol/liter CNP. For five studies involving the particulate guanylate system, a novel inhibitor, HS-142-1, was utilized at a concentration of 10−5 mol/liter HS-142-1 together with 10−7 mol/liter CNP. The following drugs were used: acetylcholine chloride (Sigma Chemical Corporation), human CNP-22 (Peninsula Laboratories), potassium chloride (Sigma) and PGF2α (Sigma). HS-142-1 was provided by two of the investigators (M.K., Y.M.). All drugs were dissolved in distilled water immediately before the study, and the concentrations reported represent the final molar concentrations in the organ chamber.
In vivo protocol. The first in vivo study was conducted in six male mongrel dogs weighing between 17 and 22 kg. Before the day of study, the dogs were fasted overnight but allowed free access to tap water. They were anesthetized with sodium pentobarbital (30 mg/kg body weight intravenously) and ventilated with room air and supplemental oxygen at 4 liters/min (Harvard Respiratory). After induction of anesthesia, each dog underwent a left thoracotomy to expose the anterior wall of the left ventricle. One centimeter of the proximal left circumflex coronary artery was carefully dissected free of surrounding tissue. A calibrated electromagnetic flow probe was positioned on the proximal circumflex coronary artery and connected to a flow meter (model FM 5010, Carolina Medical Electronics, Inc.) for measurement of coronary blood flow. At least 30 minutes of time was allowed to elapse to ensure stability of the preparation and consistency of baseline coronary flow. All measurements with the flow probe were validated by an occlusive zero measurement at the beginning of the procedure, and actual flow values were compared with standardized values used to calibrate the flow probe. Any animal preparation not meeting these criteria was excluded from study and the experiment terminated at that time.
Proximal to the flow meter, a small needle was carefully positioned in the artery to allow infusion of CNP or saline solution to the coronary circulation. Each dog also underwent placement of polyethylene catheters into the left femoral artery and vein. The femoral artery catheter was connected to a fluid-filled Statham transducer for measurement of arterial pressure. A left ventricular (LV) catheter was advanced from the right femoral artery into the LV and connected to a Statham fluid-filled transducer to measure LV pressure and allow calculation of its first derivative, dP/dt. A 6F pigtail LV catheter was used in the first series of experiments and a high fidelity Millar pressure transducer LV catheter in all subsequent experiments. A balloon-tipped, flow-directed pulmonary artery catheter was advanced through the left external jugular vein for measurement of right heart pressures and for calculation of cardiac output by using the thermodilution technique (American Edwards Laboratories, Inc.). Cardiac output was recorded as the average of four measurements. Continuous recordings of coronary blood flow, mean arterial pressure, right atrial and pulmonary artery pressure and LV end-diastolic pressure were performed on a model 2200 Gould strip recorder.
After surgical instrumentation, each dog underwent an equilibration period to establish and ensure stability of the preparation. Each animal received a systemic intravenous infusion of normal saline solution at 2 ml/min and an intracoronary infusion of normal saline solution at 1 ml/min during the equilibration period. The intravenous saline infusion served to replace insensitive and urinary fluid losses and the intracoronary infusion served as a vehicle control during the baseline period to prevent confounding of coronary flow data by an intracoronary infusion. The equilibration period was followed by a baseline period for measurement of cardiac hemodynamic data, mean arterial pressure, coronary blood flow and dP/dt and by two successive 45-min periods during which intracoronary CNP was administered in two stepwise doses, 5 and 20 ng/kg per min. The intracoronary infusion rate of the peptide was constant at 1 ml/min during both periods of drug infusion. Coronary blood flow was continuously recorded during each period, and cardiac hemodynamic variables were measured at the midpoint and end of each infusion period. The 20-ng/kg per min infusion was started immediately after completion of the 5-ng/kg per min infusion. The infusion doses were based on data from previous studies [3, 4] that demonstrated potent in vivo responses to CNP. No dog had any cardiac rhythm abnormalities during the intracoronary CNP infusion.
HS-142-1 protocol. In a second series of experiments, we tested the actions of intracoronary HS-142-1 on CNP-mediated coronary vasodilation. Because of the need for a continuous intracoronary HS-142-1 infusion, we utilized an intracoronary bolus (100 ng/kg) of CNP rather than an intracoronary infusion as in the first series of experiments. The dose of CNP was based on data from previous studies with CNP [3, 9] modified by preliminary pilot data that demonstrated efficacy. The dose of intracoronary HS-142-1 (0.25 mg/kg) was based on previous work that demonstrated excellent blockade of the natriuretic peptide-linked particulate guanylate cyclase system . Studies were conducted in six mongrel dogs with use of the open thoracotamy method described earlier. Basal coronary blood flow did not significantly change in the presence of intracoronary HS-142-1 (33 ± 4 ml/min before vs. 40 ± 2 ml/min with HS-142-1, p = NS). In addition to intracoronary CNP, we administered an intracoronary bolus of acetylcholine (1 ng/kg) to serve as a control agent and to exclude a nonspecific inhibitory action by HS-142-1 on coronary vasomotor function. All hemodynamic measurements were made as described before. At least 15 min elapsed between periods to allow complete washout of the tested peptide or substance. This interval was considered adequate because the half-life of CNP and HS-142-1 is generally believed to be ≤2 min in vivo.
l-NMMA protocol. In a third series of experiments, intracoronary NG-monomethyl-l-arginine (l-NMMA) was infused at a dose (10 μg/kg per min) previously demonstrated to inhibit intracoronary nitric oxide synthase and confirmed in pilot work to attenuate acetylcholine-mediated coronary vasodilation. Coronary flow and cardiac hemodynamic variables were measured, as before, in five mongrel dogs. Coronary artery diameter was measured by piezoelectric crystals placed distal to the infusion catheter in the isolated coronary artery preparations. The intracoronary l-NMMA infusion was followed by intracoronary infusion of l-arginine (1 mg/kg per min) to reverse the effects of l-NMMA on the coronary nitric oxide system. Again, ≥15 min was allowed to elapse between periods.
Immunohistochemical protocol. Epicardial coronary arteries were obtained from seven dogs. They were prepared as previously described , and half of the vessels underwent endothelial denudation. All tissue specimens were fixed in 10% buffered formalin, embedded in paraffin, sectioned at 6 μm intervals and stained for CNP, as previously described, with CNP antibody obtained from Peninsula Laboratories. Normal rabbit serum was used for control. There was no cross-reactivity between the CNP antibody and ANP and BNP. The slides were independently reviewed and interpreted by two investigators. Slide magnification is at ×1,000.
Statistical analysis. Data are expressed as mean value ± SEM. Data were analyzed and considered significant with a two-tailed p value < 0.05. Paired t testing was utilized to compare individual intervention periods with control values and repeated measures analysis of variance; the Fisher least significance test was utilized for multiple comparisons in the in vivo studies. Paired and unpaired t testing, corrected for multiple comparisons, was utilized for analysis of the in vitro experiments.
In vitro responses.Fig. 1Fig. 2 illustrate CNP-mediated relaxation of the isolated precontracted epicardial coronary arteries in the organ chamber preparation. A representative dose-response curve utilizing a single-vessel preparation with and without the endothelium is illustrated in Fig. 1. The response in five arterial rings to the maximal CNP dose is illustrated in Fig. 2. CNP mediated potent relaxation in the isolated epicardial coronary arteries in the presence (45.9 ± 7%, p < 0.05) and absence (72.0 ± 7%, p < 0.05) of the intact endothelium. In addition, the vasorelaxing action of CNP was independent of and actually enhanced (p < 0.05) by removal of the endothelium. Table 1 reports the dose-response and median effective dose ED50 data for CNP-mediated relaxation. CNP mediated minimal vasorelaxation at low concentrations (−) log mol/liter of −10 and −9.0) and tended to mediate its actions at the −8.0 and −7.0 −log mol/liter doses. The ED50 of CNP-mediated vasorelaxation was 7.2 (−)log mol/liter and 7.4 (−)log mol/liter for vessels with and without an intact endothelium, respectively. There was no statistical difference for the ED50 values between vessels with and without an intact endothelium.
In vivo responses.Fig. 3 and Table 2 report the hemodynamic data from the in vivo CNP administration. Intracoronary CNP increased coronary blood flow and reduced coronary vascular resistance at both infusion doses. CNP infusion did not alter cardiac output, atrial filling pressures, coronary perfusion pressure or systemic vascular resistance, but it was associated with a small but significant increase in cardiac contractility (as assessed by an increase in dP/dt) at the low dose. CNP appeared to increase contractility at the high dose, but the p value was slightly nonsignificant (p < 0.08) when corrected for multiple comparisons. Most likely, this finding represents a type II statistical error rather than a lack of effect by CNP. CNP was measured in arterial plasma and in plasma obtained from the coronary sinus of each dog before infusion of peptide and at the maximal infusion dose. Preinfusion values of CNP were comparable in the coronary sinus (6.9 ± 0.8 ng/dl) and descending aorta (8.8 ± 1.9 ng/dl, p = NS), demonstrating no cardiac stepup of CNP as exists for ANP. Postinfusion CNP values were at least 10-fold higher in each sample site, demonstrating the pharmacologic effects of exogenous CNP.
Modulation of coronary particulate guanylate cyclase system.Fig. 4 illustrates the action of intracoronary HS-142-1 on CNP and acetylcholine-mediated coronary vasodilation. CNP mediated potent increases in coronary blood flow, which was significantly attenuated in the presence of intracoronary HS-142-1 (p < 0.05 vs. CNP bolus before HS-142-1). During the recovery period, CNP-mediated coronary vasodilation was restored. Acetylcholine administration resulted in potent coronary vasodilation that was not attenuated by the presence of intracoronary HS-142-1. In addition, treatment with intracoronary HS-142-1 was not associated with any significant change in cardiac hemodynamic variables (Table 3) or basal coronary blood flow (33 ± 4 ml/min baseline vs. 40 ± 2 ml/min HS-142-1, [ = NS). The lack of attenuation of acetylcholine-mediated coronary vasodilation by HS-142-1 and the lack of an effect of HS-142-1 on basal coronary flow establish the lack of a generalized toxic effect of HS-142-1 on coronary tone and coronary flow reserve.
We tested the action of HS-142-1 on CNP-mediated relaxation of epicardial coronary vessels. Fig. 5 illustrates these results. CNP mediated potent relaxation of epicardial coronary vessels in the presence and absence of endothelium, further supporting an endothelial cell-independent action of CNP. In the presence of HS-142-1, there was significant attenuation of CNP-mediated vasorelaxation of isolated precontracted epicardial coronary arteries in the presence (56 ± 7% vs. 18 ± 6%*) and absence (61 ± 6% vs. 25 ± 7%*) of endothelium (*p < 0.05 CNP alone vs. CNP + HS-142-1).
Modulation of coronary nitric oxide synthase system.Table 4 summarizes the modulating effects of the coronary nitric oxide synthase system on CNP-mediated coronary vasodilation. Infusion of intracoronary l-NMMA did not affect CNP-mediated coronary vasodilation. In addition, intracoronary l-NMMA did not alter basal coronary flow, coronary diameter, cardiac output or systemic vascular resistance. There was a nonsignificant trend for coronary vascular resistance to increase with intracoronary l-NMMA infusion. Basal cardiac contractility decreased slightly but significantly with intracoronary l-NMMA.
Fig. 6, A through D, illustrates the localization of CNP in the coronary circulation. CNP is localized in the smooth muscle and endothelium (panels A and B) of a control dog. In a representative artery taken from a dog with intracoronary infusion of CNP, there is more intense staining of CNP in the coronary smooth muscle (panel C). A nonimmune control vessel stained with normal rabbit serum was negative for CNP (panel D).
The coronary circulation is a complex circulatory unit regulated by several endogenous vasodilator and vasoconstrictive mechanisms . Recent studies  have emphasized the importance of endothelial cell-derived substances like nitric oxide, endothelial cell hyperpolarization factor and prostacyclin as potent coronary vasodilators. The localization of the CNP receptor to cardiac tissue and vascular smooth muscle suggests a potential role for CNP as a vasodilator for the coronary circulation. Our hypothesis that CNP would function as a coronary vasodilator was confirmed by the data from this study. We also found that CNP appears to act through activation of particulate guanylate cyclase, linking the coronary vasodilation of CNP to the second messenger cyclic guanosine monophosphate (cGMP).
Intracoronary actions. Intracoronary infusion and bolus injections of CNP were associated with increased coronary blood flow and reduced coronary vascular resistance. The mechanism by which CNP mediated coronary vasodilation involved activation of particulate guanylate cyclase, as was demonstrated by use of an NPR-B inhibitor, HS-142-1. Previous studies  have demonstrated the presence of the NPR-B receptor subtype in cardiac tissue and vascular smooth muscle. Other coronary vasodilators, including nitric oxide , sodium nitroprusside  and isosorbide dinitrate , mediate coronary vasodilation through activation of cGMP by way of soluble guanylate cyclase. Specific evidence for a direct mechanistic link between coronary vasodilation and activation of particulate guanylate cyclase by the NPR-B receptor comes from the current observations with HS-142-1. HS-142-1 is a known inhibitor of the particulate guanylate cyclase subunit, which is internally linked with the NPR-A and NPR-B receptors .
In the present study, intracoronary administration of HS-142-1 selectively attenuated CNP-mediated coronary vasodilation without altering acetylcholine-mediated coronary vasodilation. This observation suggests that a mechanism of CNP-mediated coronary vasodilation is indeed through a cGMP-linked response secondary to activation of particulate guanylate cyclase by way of the NPR-B receptor, and it extends earlier observations  that demonstrated CNP-mediated production of cGMP in vascular smooth muscle. The lack of an effect of HS-142-1 on acetylcholine-mediated coronary vasodilation also supports a specific action by HS-142-1 on inhibiting cGMP production from activation of particulate guanylate cyclase by CNP rather than through a generalized inhibition of cGMP within the coronary circulation. Acetylcholine stimulates cGMP production through activation of soluble guanylate cyclase by way of nitric oxide , and this pathway remained intact in the presence of HS-142-1. The current findings thus demonstrate that CNP mediates its vasodilation through activation of the particulate guanylate cyclase system. Inhibition of activation of the particulate system did not alter the ability of nitric oxide to mediate coronary vasodilation through activation of the soluble guanylate cyclase system. Thus, the current investigation supports a role for at least two separate pathways for cGMP-mediated coronary vasodilation through activation of the soluble and particulate guanylate cyclase-dependent systems.
CNP appears to mediate coronary vasodilation predominantly at the level of the coronary resistance vessels, as was demonstrated by the lack of a significant change in epicardial coronary diameter during CNP-mediated increases in coronary blood flow and CNP-mediated decreases in coronary vascular resistance. This observation suggests a predominant CNP action in the nonepicardial resistance vessels, and it is consistent with the long-standing recognition that the coronary resistance vessels account for the major autoregulatory capacity within the coronary circulation. The dose of CNP used to achieve this effect was clearly pharmacologic, as was demonstrated by our measuring of plasma levels before and after infusion of CNP. We were unable to clearly ascertain the role of endogenous CNP in mediating coronary vasodilation because of the lack of specificity of HS-142-1 in blocking the NPR-B receptor alone. HS-142-1 is a nonspecific antagonist to both the NPR-A and NPR-B receptors. To fully test the role of endogenous CNP on basal coronary flow, one would need a specific antagonist to the NPR-B receptor to exclude any countermodulatory roles of ANP and BNP on basal coronary flow through activation of the NPR-A receptor. Additionally, the inconsistencies within basal coronary flow values reflected in Tables 3 and 4 when compared with values in Fig. 3 simply reflect the variability of different animal preparations during testing at different stages of this study. What is most reassuring in these data is the consistency of CNP in augmenting basal coronary flow despite such variability. This finding serves to reinforce our observations about CNP as a potent exogenous coronary vasodilator.
Organ chamber observations. Of additional significance is the finding that CNP also mediated potent coronary vasorelaxing actions in the organ chamber in isolated epicardial coronary arteries. In the isolated organ chamber rings, we demonstrated that CNP mediated potent relaxation of epicardial coronary arteries whether or not the endothelium was intact. Indeed, in the initial set of experiments, the actions of CNP were enhanced in de-endothelialized rings. This enhancement was not observed in the second series of control rings in the HS-142-1 study, but the important observation is that CNP plays a direct role as a coronary vasodilator that is independent of the presence of a functioning endothelium. The coronary vasodilator properties of CNP appear importantly linked to the production of cGMP by way of particulate guanylate cyclase in the organ chamber experiments because preincubation with HS-142-1 significantly attenuated CNP-mediated vasorelaxation. This finding confirms the observation of a particulate guanylate cyclase-linked mechanism for CNP-mediated coronary vasodilation in the intracoronary infusion experiments.
Impact of coronary nitric oxide synthase system. Blockade of the coronary nitric oxide synthase system with l-NMMA did not affect the coronary vasodilation of CNP. This finding is not surprising because CNP binds to a receptor that generates cGMP by way of particulate guanylate cyclase, whereas the coronary nitric oxide system works through the soluble guanylate cyclase system. However, both the particulate and soluble guanylate cyclase systems function through generation of intracellular cGMP, and both are potent vasodilators. The data from the current investigation support their independent mechanisms of action. Blockade of the intracoronary nitric oxide synthase system did alter the response of cardiac contractility to CNP. Intracoronary l-NMMA reduced cardiac contractility (dP/dt) and attenuated the ability of CNP to significantly increase dP/dt. Perhaps CNP acts in a paracrine manner to alter contractility or simply stimulates greater flow and sheer stress, which then act through nitric oxide formation to alter contractility. The increase in contractility could also be a direct action of intracoronary CNP on ventricular muscle, or it may be indirectly related to a generalized increase in coronary blood flow, as with the Gregg phenomenon [21, 22]. The increase in contractility could also be secondary to CNP activation of an intermediate substance that augments contractility. Recently, Paulis et al.  demonstrated that the coronary endothelium alters cardiac contractility in a paracrine manner, and Hare et al.  demonstrated that nitric oxide may contribute to the beta-adrenergic hyporesponsiveness observed in advanced heart failure. Our data suggest an interaction between CNP and the coronary nitric oxide synthase system with regard to control of myocardial contractility.
Implications. The present investigation demonstrates that CNP functions as a coronary vasodilator independent of an intact vascular endothelium. The in vitro observations that CNP mediates coronary relaxation in the presence and absence of an intact endothelium and that administration of HS-142-1 attenuates CNP-mediated coronary relaxation in both conditions support a role for CNP as a direct coronary smooth muscle vasodilator. In addition, the current investigation demonstrates that a functional endothelium is not essential for the action of CNP. This observation confirms previous reports  of enhanced CNP vasorelaxation in peripheral vessels without an endothelium and extends those findings by demonstrating potent CNP-mediated, endothelial cell-independent vasorelaxation in the coronary vasculature. This distinction separates CNP from the coronary nitric oxide system, which requires a normally functioning endothelium to allow generation of nitric oxide [24, 25].
The demonstration that CNP mediates its actions independent of a functional endothelium is consistent with recent work establishing endothelial cell synthesis and secretion of CNP [9–11] and identifying the receptor for CNP in vascular smooth muscle . The current observations therefore support a hypothesis that CNP is secreted by coronary endothelium and acts through a paracrine mechanism on the coronary smooth muscle, much like endothelin and nitric oxide [16, 25]. Our observations also support a role for exogenously administered CNP as a coronary vasodilator whose actions resemble those of nitrovasodilators in the coronary circulation [20, 26]. In addition, we have demonstrated localization of CNP to coronary endothelium and smooth muscle by immunohistochemical staining. The localization of CNP to the coronary circulation by this method does not definitively distinguish between local synthesis of CNP and binding of circulating CNP, but it does establish the presence of CNP in the coronary circulation and supports a role for CNP as a paracine mediator within the coronary circulation.
Summary. In summary, the current investigation demonstrates that CNP is a potent in vivo and in vitro coronary vasodilator. It also demonstrates that the in vitro actions of CNP are independent of a functional endothelium. CNP mediates its coronary vasodilation through activation of particulate guanylate cyclase and is independent of the coronary nitric oxide synthase system. CNP may have a role as an exogenous coronary vasodilator.
We appreciate the technical assistance of Virginia M. Miller, PhD with the organ chamber experiments and of Roland R. Brandt, MD with the sonomicrometric studies.
A.1 Abbreviations and Acronyms
ANP = atrial natriuretic peptide
BNP = brain natriuretic peptide
cGMP = cyclic guanosine monophosphate
CNP = C-type natriuretic peptide
dP/dt = first derivative of left ventricular pressure
ED50 = median effective dose
lNMMA = NG-monomethyl-l-arginine
LV = left ventricular
↵1 This study was supported in part by grants from the American Heart Association, Minnesota Affiliate, Minneapolis, Minnesota; the National Heart, Lung, and Blood Institute (Grant HL-033643), National Institutes of Health, Bethesda, Maryland; and the Mayo Foundation, Rochester, Minnesota.
- Received December 18, 1995.
- Revision received April 4, 1996.
- Accepted May 7, 1996.
- THE AMERICAN COLLEGE OF CARDIOLOGY
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