Calcitonin gene-related peptide (CGRP) is a neuropeptide widely distributed in the autonomic nerve terminals supplying the cardiovascular system (1-4). The discovery of specific binding sites in both myocardium and vascular smooth muscle (5-7) and the presence of the peptide in the plasma (8,9) would suggest the possibility of a physiologic role of CGRP in the control of cardiovascular functions.
Furthermore, CGRP was demonstrated to increase coronary flow when administered by intracoronary infusion in patients affected by variant angina (10). However, clear-cut evidence of the mechanism of action of this peptide is not available. An interference with the cyclic nucleotide system, producing an increase in the second-messenger concentrations in the intracellular space and the mobilization of endothelial factors, has been supposed to mediate the physiopharmacologic activities of CGRP (11,12); some studies also indicated that the response of the peptide is due to an endothelium-independent mechanism (13).
We investigated the effects of human CGRP on isolated rabbit hearts to evaluate the mechanisms responsible for the vasodilatory actions of the peptide on the coronary region with simultaneous monitoring of the effects on left ventricular pressure (LVP) and heart rate (HR). To investigate further the mechanism of CGRP vasodilatory action, we evaluated human internal mammary artery (IMA) reactivity to excitatory drugs acting with different mechanisms and the inhibitory response of CGRP in comparison with commonly used vasodilatatory agents.
These experiments were performed by using the excess segments of the IMA pedicle obtained for myocardial revascularization of patients affected by coronary obstruction according to the previously used procedure (14).
Rabbit heart preparations
New Zealand albino rabbits of both sexes weighing 2-2.5 kg were used. The animals were killed by cervical dislocation. The hearts were removed quickly and placed in an ice-cold Ringer-Locke solution, oxygenated with 100% O2, and containing in millimoles per liter: NaCl, 136.9; KCl, 2.68; MgCl, 0.99; CaCl2, 1.7; NaHPO4, 0.42; NaHCO3, 3.93; and glucose, 5.55 (pH 7.4).
A previously described procedure was followed (15). After removal of the pericardium and surrounding tissues, the hearts were perfused with Ringer-Locke solution according to the nonrecirculating Langendorff technique (16).
The perfusion fluid was continuously gassed with 100% O2, maintained at 37°C, and delivered to the aortic inflow cannula at a constant rate of 22-24 ml/min by using a peristaltic pump (Gilson, Miniplus HP2HF). The perfusion pressure was measured by a Statham transducer connected to the sidearm of the perfusion cannula.
Because retrograde flow (coronary flow) was kept constant during the experiment, coronary perfusion pressure (CPP) represented a direct measure of the coronary resistances. A fluid-filled balloon connected to a pressure transducer was inserted into the left ventricular cavity through an opening in the left ventricular atrium, thus obtaining an isovolumically beating preparation (17).
Both LVP and CPP were recorded simultaneously by using a polygraph (OTE Biomedica; C6B). Except for experiments in which chronotropic effects were evaluated, the hearts were electrically paced to exclude LVP variations depending on HR changes. Rectangular pulses (1-ms width at 0.5 V up to threshold stimulation) were applied to the preparation via two platinum electrodes, one connected to the metal inflow cannula and the other implanted directly in the ventricular apex.
The frequency of stimulation was 10% greater than the basal HR. The hearts were left to equilibrate for 30 min before the administration of drugs, which were added to the perfusion fluid and allowed to act until the maximal effect was obtained (5-15 min). CGRP was dissolved in a Ringer-Locke solution containing serum bovine albumin (0.12%) and administered in the perfusion fluid, by using the cumulative-doses method (18). Forskolin (10−8M) was left to act for 30 min before the administration of vasopressin (10−9M). In another series of experiments, the isolated rabbit hearts were perfused with indomethacin (1.4 × 10−5M), a cyclooxygenase inhibitor, added to the perfusion fluid 30 min before the administration of vasopressin (10−9M) and methoxamine (10−5M).
In a limited series of experiments (n = 5), CGRP was administered by pulse injection directly into the coronary inflow cannula, dissolved in 0.3 ml of Ringer solution.
Human IMA preparations
The excess segments of IMA were obtained from 30 male patients (age range, 40-68 years) and immediately put in Krebs-Henseleit solution gassed with 95% O2 and 5% CO2 of the following composition in millimoles per liter: NaCl, 113; KCl, 4.7; CaCl, 1.9; MgSO4, 1.2; KH2PO4, 1.2; and glucose, 11.5. Thereafter, the vascular wall was separated from the surrounding tissues and spirally cut to obtain 2-cm-long and 3-mm-wide strips. The strips were set up in isolated organ baths (5 ml) containing Krebs-Henseleit solution at 37°C and gassed with 95% O2 and 5% CO2 (pH 7.4). Preparations were suspended under an isometric passive stretch of 1 g and left to equilibrate for 120 min. Tension was measured by means of an isometric transducer connected to a pen-writing recorder (Unirecord 7050; Basile, Milan, Italy). Spasmogenic compounds, KCl (90 mM), noradrenalin (10−5M), serotonin (10−6M), and angiotensin II (10−6M) were administered directly into the organ bath. When contractions reached a plateau (usually 5-10 min after administration), CGRP or verapamil or nitroglycerin was added according to the cumulative-doses method (18).
Integrity of the endothelial structure was assessed pharmacologically by using the acetylcholine test, and the inhibitory effect of CGRP was evaluated in preparations with endothelial integrity and also after ablation of endothelium (19).
Data were expressed as mean ± SEM of six to eight experiments. Inhibitory responses of CGRP on CCP were calculated as percentage decrease of the basal value, and those on the coronary spasm, as a percentage of inhibition on the plateau of CPP increase induced by different spasmogenic compounds.
The inhibitory effect of CGRP on the IMA segments pre-contracted with different spasmogenic agents was calculated as percentage reduction of the maximal contractile force increase (plateau), taken as 100.
Analysis of variance was applied when comparison of data was requested, and statistical significance was defined for a probability value <5% (p < 0.05).
The following drugs were used: human CGRP (Peninsula Labs, Belmont, CA, U.S.A.), methoxamine, serotonin, noradrenaline, PGF2α, forskolin, acetylcholine hydrochloride, angiotensin II, L-arginine, NG-monomethyl-L-arginine (Sigma Chemical, St. Louis, MO, U.S.A.), Bay K 8644, nifedipine (Bayer, Leverkusen, FRG), vasopressin (Sandoz, Basel, Switzerland), nitroglycerin (Simes, Milan, Italy), indomethacin (Chiesi, Italy), and KCl and other reagents for buffer solution (Bracco, Milan, Italy).
Isolated heart preparations
After the stabilization period, the hearts used as controls showed a mean basal LVP of 51.3 ± 4.2 mm Hg and a mean basal CPP of 62.2 ± 4.8 mm Hg. Mean basal HR was 135.5 ± 8.1 beats/min.
In these pilot experiments, spontaneous changes in basal LVP and CPP were <5% after 120 min, whereas a 13.6 ± 0.8% reduction was measured for HR.
CGRP (10−10-10−7M) administered in the perfusion fluid did not substantially modify LVP and HR values, whereas CPP was slightly reduced from 65.2 ± 4.1 to 54.4 ± 6.3 mm Hg (Fig. 1).
When administered by pulse injection into the aortic cannula, CGRP showed a more consistent decrease in CPP starting from a concentration of 10−8M (from 75.7 ± 3.5 to 64.6 ± 4.4 mm Hg) and reaching the maximal inhibitory effect at 10−7M (from 75.3 ± 5.2 to 50.6 ± 7.5 mm Hg), as shown in Fig. 2. LVP was increased only at the higher concentration (from 80.8 ± 2.7 to 95.3 ± 3.4 mm Hg; Fig. 2).
Vasopressin (10−9M), methoxamine (10−5M), and PGF2α (10−7M) induced a coronary spasm (from 71.6 ± 5.4 to 137.7 ± 10.4 mm Hg, n = 8; from 68.2 ± 6.4 to 108.5 ± 8.2 mm Hg, n = 7; and from 50 ± 7.9 to 140 ± 10.2 mm Hg, n = 7, respectively). Bay K 8644 (10−7M) strongly enhanced LVP (from 56.3 ± 6.2 to 78.9 ± 8.4 mm Hg), CPP (from 67.3 ± 7.8 to 150 ± 12.5 mm Hg), and HR (from 142 ± 13 to 191 ± 23 beats/min; n = 6). CGRP produced a consistent vasodilatory effect on coronary spasm induced by Bay K 8644, PGF2α(Fig. 3), vasopressin, and methoxamine, starting from a concentration of 10−11M and reaching the maximal inhibitory effect at 10−8-10−7M. This effect was similar in extent on the different spasmogens used (Fig. 4).
In Fig. 5, the vasodilatory effect of CGRP is compared with those obtained with nifedipine and nitroglycerin. All these compounds were able to inhibit the vasopressin-induced coronary spasm, CGRP being the most potent. However, nifedipine and nitroglycerin produced a complete inhibition of coronary spasm, whereas CGRP was only partially active.
The adenylate cyclase activator forskolin (10−8M) did not modify the inhibitory effect of CGRP. At the same concentration, forskolin showed a sustained inotropic and chronotropic activity (not shown). At a lower concentration of forskolin (10−9M), we did not observe any influence on CGRP vasodilatory effect.
The cyclooxygenase inhibitor, indomethacin (1.4 × 10−5M), did not modify the spasmolytic action of CGRP on vasopressin- and methoxamine-induced coronary spasm (not shown).
Internal mammary artery segments
Figure 6 shows the effects of CGRP on IMA segments precontracted with four different spasmogenic agents. Noradrenaline (10−5M) induced a tonic contraction of 0.45 ± 0.07 g; serotonin (10−6M), of 1.8 ± 0.2 g; KCl (90 mM), of 0.48 ± 0.03 g; and angiotensin II (10−6M), of 0.30 ± 0.2 g (14).
CGRP significantly inhibited the contractions induced by the spasmogenic compounds starting at 10−11M and reaching the maximal inhibitor effect at 10−7M. The contractions of noradrenaline and angiotensin II were completely antagonized, whereas the contractions induced by serotonin and KCl were reduced by 70 and 55%, respectively.
When the endothelium was removed, the relaxant effect of CGRP observed on the IMA segments precontracted with noradrenaline (10−5M) was suppressed (Fig. 7, right). The same preparations were previously tested by acetylcholine, showing a similar lack of relaxation after the endothelial ablation (Fig. 7, left).
Figure 8 represents the effects of CGRP (10−7M) on contractions induced by noradrenaline on IMA segments in basal condition and in the presence of L-arginine or a L-arginine analog, (L-NMMA) or both. L-NMMA added to the bath 30 min before the contraction induced by noradrenaline (10−5M) abolished the vasorelaxant effect of CGRP. In the same way, the addition of L-arginine alone did not significantly potentiate the endothelium-dependent relaxation of the CGRP. However, L-arginine partially reversed the inhibitory effect of the L-NMMA by 90%, and this result was statistically significant (p < 0.01). L-NMMA alone did not induce a significant effect on basal tension.
CGRP has been shown to induce a wide range of cardiovascular effects in different isolated preparations and in humans (1,20,21). With cardiac functions, CGRP has been demonstrated to produce a positive inotropic and chronotropic effect in isolated guinea pig atria and in rat hearts (22-24). Furthermore, the peptide is able to increase coronary flow (10) and more generally to inhibit vascular tone (25,26).
Whereas chronotropic and inotropic effects have been related to an activation of adenylate cyclase, with a consequent increase in cyclic adenosine monophosphate (AMP) levels, vascular effects seem to be independent of this mechanism (13); some authors hypothesized that a part of the relaxation produced by CGRP results from activation of adenosine triphosphate (ATP)-sensitive potassium channels in smooth-muscle cells (27).
Our data show that in rabbit hearts, CGRP inhibits coronary smooth-muscle tone; however, the effects on chronotropism and inotropism were scanty and detectable only with very high doses of peptide directly injected into the coronary circulation.
The adenylate-cyclase activator forskolin (28) was unable to increase the spasmolytic activity of CGRP on precontracted coronary smooth muscle and thus seems to exclude an effect directly involving the adenylate- cyclase system. Furthermore, the results obtained in the rabbit hearts perfused by a cyclooxygenase inhibitor, indomethacin, suggested that the role of prostaglandin system is not relevant in the coronary vasodilation of CGRP.
In an attempt to investigate the mechanism responsible for coronary vasodilation, we tested the ability of CGRP to antagonize the increase of coronary resistances induced by drugs having different mechanisms of action: vasopressin, methoxamine, PGF2α, and Bay K 8644 (29). The inhibitory effect of CGRP was virtually equivalent on all stimulants, thus suggesting a nonspecific effect. In particular, the antagonistic effect on Bay K 8644, known to activate calcium influx through potential-dependent calcium channels (30,31), might suggest an interference with calcium availability or with intracellular calcium modulators.
In this context, an interaction with the cyclic guanosine monophosphate (GMP) system, possibly mediated by endothelial factors as suggested by other studies, should be taken into account (12).
The experiments performed on the segments of human IMA confirmed the spasmolytic action of CGRP observed on the coronary region of the rabbit. In the experiments on isolated human vessels, as on the rabbit coronary arteries, the inhibitory effect was nonspecific on different spasmogenic compounds, in particular on stimuli acting with specific receptors such as noradrenaline, serotonin, and angiotensin II (32). CGRP induced a partial effect on KCl in comparison with receptor-dependent agonists; these findings suggest that CGRP affects preferentially the mobilization of intracellular calcium rather than the influx of calcium from the extracellular milieu as stimulated by KCl. In addition, these results suggest that the contractions induced by a K+ depolarization can be observed without the interference of endothelial factors (33). Moreover, in endothelial cultured cells, an inhibitory effect of the depolarizing K+ solution on EDRF formation has been demonstrated, probably the result of a significant reduction in the bradykinin-induced release of EDRF (34).
The experiments performed for evaluating the role of endothelial function have shown that the spasmolytic action of CGRP on the contractions induced by noradrenaline disappeared when the endothelium was ablated. The same response was obtained with acetylcholine. The observation that the relaxation of isolated preparations of mammary arteries by CGRP was strictly dependent on the presence of endothelial cells supported further investigations. In particular, we verified whether the vasodilating effect of CGRP was correlated to the synthesis of the most studied endothelial factor, nitric oxide (NO).
It is well known that the synthesis of NO begins by L-arginine, and in particular, the interaction of an agonist with its receptor leads to an increase in intracellular Ca2+, which stimulates NO synthase, resulting in the formation of NO and citrulline (35,36).
The enzyme is inhibited by an analog of L-arginine, L-NMMA. The NO thus formed activates soluble guanylate cyclase responsible for the cGMP accumulation, with consequent relaxation of vascular smooth muscle (37). The experiments performed on IMA segments incubated with L-NMMA confirmed that the action of CGRP may be related to synthesis of NO.
The results obtained on different experimental models have shown that the vasodilation induced by CGRP can be verified through different mechanisms of action with regard to the vascular bed studied. In particular in some arterial preparations, such as rat aorta and porcine iliac artery, the relaxant effect of CGRP is dependent on intact endothelium (11,12,25,38); in other vascular preparations, the peptide acts through an endothelium-independent mechanism (13,39).
Moreover, the positive inotropic effect of CGRP, shown in heart preparations of guinea pigs and rats, seems to be mediated through the formation of cyclic AMP (22,23).
The demonstration in segments of human IMA that the vasodilation of CGRP is strictly dependent on synthesis of NO has particular importance for the knowledge of the mechanism of action of the peptide and for the clinical/surgical implications of coronary disease. It is well known that the use of IMA for myocardial revascularization has been strongly recommended for a better expectation of long-term patency compared with the preparation of saphenous vein (40).
Our results contribute to the knowledge of the mechanisms of the regulation of vascular tone, and in particular, the fact that endogenous factors can modulate endothelial function suggests the importance of surgical manipulation of IMA pedicle during coronary bypass surgery. Moreover, the pharmacologic therapy of patients who have undergone coronary surgery must take into consideration the role of factors that act through the endothelial mechanism. It is also necessary to promote research into the mechanisms of the protection of endothelial structures.
Acknowledgment: We are grateful to Ornella Bonometti for secretarial assistance.
1. Gennari C, Fisher JA. Cardiovascular action of calcitonin gene-related peptide in humans. Calcif Tissue Int
2. Mulderry PK, Ghatei MA, Rodrigo GJ, et al. Calcitonin gene-related peptide in cardiovascular tissues of the rat. Neuroscience
3. Kawasaki H, Nuki C, Saito A, Takasaki K. Role of calcitonin gene-related peptide-containing nerves in the vascular adrenergic neurotransmission. J Pharmacol Exp Ther
4. Del Bianco E, Perretti F, Tramontana M, Manzini S, Geppetti P. Calcitonin gene-related peptide in rat arterial and venous vessels: sensitivity to capsaicin, bradykinin and FMLP. Agents Actions
5. Sigrist S, Franco-Cereceda A, Muff R, Henke H, Lundberg JM, Fisher JA. Specific receptors and cardiovascular effects of calcitonin gene-related peptide. Endocrinology
6. Wimalawansa SJ, MacIntyre I. Calcitonin gene-related peptide and its specific binding sites in the cardiovascular system of the rat. Int J Cardiol
7. Han SP, Naes L, Westfall TC. Inhibition of periarterial nerve stimulation-induced vasodilation of the mesenteric arterial bed by CGRP (8-37) and CGRP receptor desensitization. Biochem Biophys Res Commun
8. Mair J, Lechleitner P, Langle T, Wiedermann C, Diensti F. Plasma CGRP in acute myocardial infarction. Lancet
9. Girgis SI, MacDonald DW, Stevenson JC, et al. Calcitonin gene-related peptide: potent vasodilator and major product of calcitonin gene. Lancet
10. McEwav JR, Larkin S, Davies G, et al. Calcitonin gene-related peptide: a potent vasodilator of human epicardial coronary arteries. Circulation
11. Schini-Kerth VB, Fisslthaler B, Busse R. CGRP enhances induction of NO synthase in vascular smooth muscle cells via a cAMP-dependent mechanism. Am J Physiol
12. Gray DW, Marshall I. Human α-calcitonin gene-related peptide stimulates adenylate cyclase and guanylate cyclase and relaxes rat thoracic aorta by releasing nitric oxide
. Br J Pharmacol
13. Greenberg B, Rhoden K, Barnes P. Calcitonin gene-related peptide (CGRP) is a potent non-endothelium-dependent inhibitor of coronary vasomotor tone. Br J Pharmacol
14. Raddino R, Gargano M, Pelà G, Alfieri O, Visioli O. Vascular reactivity of internal mammary artery used for myocardial revascularization. In: Strano A, Novo S, eds. Advances in vascular pathology.
Elsevier Science Publisher, 1990:395-9.
15. Raddino R, Poli E, Pelà G, Manca C. Action of steroid sex hormones on the isolated rabbit heart. Pharmacology
16. Broadley KJ. The Langendorff heart preparation: reappraisal of its role as a research and teaching model for coronary vasoactive drugs. J Pharmacol Methods
17. Fallen EL, Elliot WC, Gorlin R. Apparatus for the study of ventricular function and metabolism in the isolated perfused rabbit heart. J Appl Physiol
18. Van Rossum JM. Cumulative dose-response curves: techniques for making the dose-response curves in isolated organs and the evaluation of drug parameters. Arch Int Pharmacodyn Ther
19. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature
20. Marshall I, Al Kazwini SJ, Roberts PM, Shepperson NB, Adams M, Craig RK. Cardiovascular effect of human and rat CGRP compared in the rat and other species. Eur J Pharmacol
21. Franco-Cereceda A, Gennari C, Nami R, et al. Cardiovascular effects of calcitonin gene-related peptide I and II in man. Circulation
22. Ishikawa T, Okamura N, Saito A, Masaki T, Goto K. Positive inotropic effect of calcitonin gene-related peptide mediated by cyclic AMP in guinea pig heart. Circ Res
23. Asimakis GK, Dipette DJ, Conti VR, Holland OB, Zwischenberger JB. Hemodynamic action of calcitonin gene-related peptide in the isolated rat heart. Life Sci
24. Bell D, McDermott BJ. Calcitonin gene-related peptide stimulates a positive contractile response in rat ventricular cardiomyocytes. J Cardiovasc Pharmacol
25. Brain SD, Williams TJ, Tippins JR, Morris HR, MacIntyre I. Calcitonin gene-related peptide is a potent vasodilator. Nature
26. Kawasaki H, Takasaki K, Saito S, Goto K. Calcitonin gene-related peptide acts as a novel vasodilator neurotransmitter in mesenteric resistance vessels of the rat. Nature
27. Nelson MT, Huang Y, Brayden JE, Hescheler J, Standen NS. Arterial dilations in response to calcitonin gene-related peptide involve activation of K+
28. Bristow MR, Ginsburg R, Strosberg A, Montgomery W, Minobe W. Pharmacology and inotropic potential of forskolin in human heart. J Clin Invest
29. Raddino R, Pelà G, Poli E, Mascaro F, Manca C, Visioli O. Different effects of captopril and other angiotensin correcting enzyme inhibitors on cardiovascular preparations. Pharmacol Res
30. Schramm M, Thomas G, Towart R, Franckowiak G. Novel dihydropiridines with positive inotropic action through activation of calcium channel. Nature
31. Raddino R, Poli E, Pasini E, Ferrari R. Effects of the novel calcium channel blocker, anipamil, on the isolated rabbit heart: comparison with verapamil and gallopamil. Naunyn Schmiedebergs Arch Pharmacol
32. Bolton TB. Mechanisms of action of transmitters and other substances on smooth muscle. Physiol Rev
33. Takeda K, Schini V, Stoeckel H. Voltage-activated potassium, but not calcium currents in cultured bovine aortic endothelial cells. Pflugers Arch
34. Luckoff A, Busse R, Winter I, Bassenge E. Characterization of vascular relaxant factor released from cultured endothelial cells. Hypertension
35. Griffith TM, Hughesedwards DM, Lewis MJ, Newby AC, Henderson AH. The nature of endothelium-derived vascular relaxant factor. Nature
36. Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide
release accounts for the biological activity of endothelium-derived relaxing factor. Nature
37. Murad F, Rapoport RM, Fiscus R. Role of cyclic-GMP in relaxation of vascular smooth muscle. J Cardiovasc Pharmacol
38. Samuelsson UE, Jernbeck J. Calcitonin gene-related peptide relaxes porcine arteries via one endothelium-dependent and one endothelium-independent mechanism. Acta Physiol Scand
39. Amerini S, Mantelli L, Ledda F. Nitric oxide
is not involved in the effects induced by non-adrenergic, non-cholinergic stimulation in the rat mesenteric vascular bed. Neuropeptides
40. Luscher TF, Diederich D, Siebenmann R, et al. Difference between endothelium-dependent relaxation in arterial and in venous coronary bypass grafts. N Engl J Med