Ole Saetrum Opgaard

Positive inotropy mediated via CGRP receptors in isolated human myocardial trabeculae

Isometric contractile force were studied on isolated human myocardial trabeculae that were paced at 1.0 Hz in tissue baths. Alpha calcitonin gene-related peptide (alpha-CGRP) had a potent positive inotropic effect in most trabeculae from both the right atrium and left ventricle, and this effect was partially antagonized by the CGRP(1) receptor antagonist alpha-CGRP-(8-37) (10(-6) M). Amylin and the CGRP(2) receptor agonist [Cys(acetylmethoxy)(2, 7)]CGRP had a positive inotropic effect in some trabeculae, whereas adrenomedullin had no inotropic effect. Using reverse transcriptase-polymerase chain reaction (PCR) mRNAs encoding the human calcitonin receptor-like receptor and the receptor associated modifying proteins (RAMPs) RAMP1, RAMP2, and RAMP3 were detected in human myocardial trabeculae from both the right atrium and left ventricle. In conclusion, functional CGRP(1) and CGRP(2) receptors may mediate a positive inotropic effect at both the atrial and ventricular level of the human heart. mRNAs for calcitonin receptor-like receptor and specific RAMPs further support the presence of CGRP receptors.

Localization of endothelin immunoreactivity and demonstration of constrictory endothelin-A receptors in human coronary arteries and veins

Summary: Strong immunoreactivity for endothelins (ETs) was observed in the endothelium of both human epicardial coronary arteries and veins. The contractile responses to ET-1, ET-2, and ET-3 were investigated in isolated circular human coronary vessel segments. ET-1, ET-2, and ET-3 induced significantly higher maximum contraction (measured in percentage of contraction induced by 60 mM potassium) and more potent responses in veins as compared with arteries. ET-1 as compared with ET-2 induced equal maximum contraction and equipotent responses both when tested in arteries and veins, whereas ET-3 induced lower maximum contraction and less potent responses in both vessel types. FR 139317, a selective ETA receptor antagonist, significantly decreased the potency of ET-1 and ET-2 responses in both human coronary arteries and veins, but the maximum effect obtained did not change significantly. The existence of ET immunoreactivity (IR) in endothelial cells from both human coronary arteries and veins indicates that ETs may be released endogenously. The effect of the selective ETA receptor antagonist FR 139317 indicates that the contraction induced by ET-1 and ET-2 in both arteries and veins is mediated by ETA receptors.

Peptide-containing nerve fibres in guinea-pig coronary arteries: Immunohistochemistry, ultrastructure and vasomotility

The peptidergic innervation of guinea-pig coronary arteries was investigated by means of immunohistochemical, ultrastructural and in vitro pharmacological techniques. A network of nerves was demonstrated in all major epicardial arteries by means of an antiserum to the neuronal marker protein gene product 9.5. The majority of nerve fibres possessed neuropeptide Y (NPY) and tyrosine hydroxylase (TH) immunoreactivity, the number and distribution of nerves immunoreactive for NPY being similar to that of nerves containing TH immunoreactivity. Numerous nerve fibres displaying immunoreactivity for substance P, neuropeptide K and calcitonin gene-related peptide (CGRP) were also found. In double-stained preparations substance P immunoreactivity was co-localized with CGRP and with neuropeptide K immunoreactivities in the same varicose nerve fibres. Ultrastructural studies revealed the presence of numerous axon varicosities at the adventitial-medial border. NPY immunoreactivity was localized in large granular vesicles in nerve varicosities which also contained numerous small granular vesicles. Large granular vesicle-containing nerves also displayed immunoreactivity for dopamine-beta-hydroxylase. With an in vitro method, the vasomotor responses to perivascular peptides were characterized in epicardial and intramyocardial arteries. In epicardial arteries neither noradrenaline nor NPY elicited a contractile response. Only in some intramyocardial arteries was an NPY-mediated contraction demonstrated. No potentiating effect of noradrenaline and NPY was observed in either epicardial or intramyocardial arterial segments. In contrast, CGRP, substance P and vasoactive intestinal peptide (VIP) all produced a concentration-dependent relaxation of both epicardial and intramyocardial arteries. These results suggest that peptide-containing nerves associated with guinea-pig coronary arteries may predominantly be involved in mediating vasodilation.

Positive inotropy of calcitonin gene-related peptide and amylin on porcine isolated myocardium

Isolated porcine myocardial trabeculae from right atria and left ventricles were paced at 1.5 Hz in tissue baths, and changes in isometric contractile force upon exposure to agonist were studied. Alpha calcitonin gene-related peptide (alpha-CGRP) increased contractile force in nearly half of the trabeculae, whereas the selective CGRP(2) receptor agonist [Cys(acetylmethoxy)(2,7)]-CGRP had effect in only a few. Preincubation with the CGRP(1) receptor antagonist alpha-CGRP-(8-37) (10(-6) M) almost completely blocked positive inotropic responses to alpha-CGRP. Amylin had weak positive inotropic effects in some atrial, but not in ventricular trabeculae. Adrenomedullin did not affect contractility in either atrial or ventricular trabeculae. In conclusion, these results suggest that alpha-CGRP has a positive inotropic effect that can be mediated by both CGRP(1) and CGRP(2) receptors. Amylin seems to have a potential positive inotropic effect on atrial tissue, whereas no direct effect of adrenomedullin could be measured.

Positive inotropic responses mediated by endothelin ETA and ETB receptors in human myocardial trabeculae

The aim of the present study was to determine possible inotropic effects mediated by endothelin ET(A) and ET(B) receptors in human myocardial trabeculae from the right atrium and the left ventricle. Isolated trabeculae from human hearts were paced at 1.0 Hz in tissue baths, and changes in isometric contractile force upon exposure to agonist were studied. Endothelin-1 (ET-1) and ET-3 had a strong positive inotropic effect in all trabeculae. ET-1 was significantly more potent than ET-3 in both atrial (P < 0.001) and ventricular (P < 0.05) trabeculae. Preincubation with the ET(A) receptor antagonist FR139317 (1 microM) decreased significantly (P < 0.005) the potency of ET- I in both atrial and ventricular trabeculae, without any significant changes in Emax (maximum effect obtained with an agonist). The ET(B) receptor agonist IRL 1620 had a positive inotropic effect only in some trabeculae, and the ET(B) receptor antagonist BQ 788 (1 microM) almost completely blocked this effect. These results suggest that both ET(A) and ET(B) receptors mediate positive inotropic responses at both the atrial and ventricular level in the human heart.

Vasoactive intestinal peptide has a direct positive inotropic effect on isolated human myocardial trabeculae

The aim of the present study was to assess the inotropic effects of vasoactive intestinal peptide (VIP) on isolated myocardial trabeculae from the right atrium and the left ventricle of human hearts. Furthermore, using reverse transcriptase-PCR, we wanted to determine the presence of mRNAs encoding the three cloned human VIP receptors, VPAC(1), VPAC(2) and PAC(1). The trabeculae were paced at 1.0 Hz in tissue baths, and changes in isometric contractile force upon exposure to agonist were studied. VIP had a potent positive inotropic effect in some of the atrial and ventricular trabeculae tested. This effect was almost completely blocked by the VIP-receptor antagonist VIP-(6-28). mRNAs encoding the human VPAC(1), VPAC(2) and PAC(1) receptors were detected in human myocardial trabeculae from both the right atrium and the left ventricle. In conclusion, VIP has a direct positive inotropic effect in both the atria and the ventricles of the human heart. The presence of mRNAs for the VPAC(1), VPAC(2) and PAC(1) receptors suggest that VIP may mediate its effect via these receptors.

Neuropeptide Y modulates the action of vasodilator agents in guinea-pig epicardial coronary arteries

Recently, we have demonstrated that guinea-pig epicardial coronary arteries are supplied by numerous nerve fibres containing neuropeptide Y (NPY) immunoreactivity. However, examination of vasomotor responses revealed that NPY did not elicit a contractile response in these arteries. In contrast, acetylcholine (ACh), calcitonin gene-related peptide (CGRP), substance P and vasoactive intestinal polypeptide (VIP) all relaxed precontracted arteries. In the present study, we have used histochemical, immunohistochemical and in vitro pharmacological techniques, in order to further investigate the possible role of NPY in guinea-pig epicardial coronary arteries. A double-immunofluorescence staining technique revealed that CGRP and substance P were co-localized in nerve fibres distinct from those displaying NPY immunoreactivity. Furthermore, using a method combining immunofluorescence and histochemical techniques, we observed that putative cholinergic nerve fibres (identified by their acetylcholinesterase content) and NPY-immunoreactive nerve fibres are two different nerve populations. An in vitro pharmacological method demonstrated that NPY markedly inhibited the relaxant responses mediated by ACh, VIP, substance P and isoprenaline but had no effect on CGRP. These results suggest that NPY-containing nerves associated with guinea-pig epicardial coronary arteries may be predominantly involved in modulating the action of vasodilator agents.

Endothelin-A and -B receptors in human coronary arteries and veins

Using reverse transcriptase-polymerase chain reaction, products corresponding to mRNA encoding endothelin-A and -B (ETA and ETB) receptors were demonstrated in human coronary arteries and veins with intact endothelium and in endothelium-denuded human coronary arteries. Vasomotor responses were studied on isolated segments of human epicardial coronary arteries and veins at resting tension and after precontraction with U46619. In both arteries and veins, endothelin-1 (ET) induced strong and potent contractions, and preincubation with different concentrations of the non-selective ETA/ETB receptor antagonist PD 145065 caused a rightward shift of the concentration-response curves without significantly changing maximum responses (pA2 value 6.7 arteries, 7.4 veins). The ETB receptor agonist IRL 1620 induced no contraction of arteries or veins at resting tension, but induced weak relaxation of all arteries and most precontracted veins, the relaxation being endothelium-dependent in arteries. ET at low concentrations induced weak relaxations of most precontracted arteries, but no veins. In conclusion, mRNA encoding ETA and ETB receptors is present in human coronary arteries and veins, ETA receptors mediating contraction and ETB receptors mediating relaxation. In arteries, mRNA for both receptor types was detected in the media, but ETB receptor-mediated relaxation was endothelium-dependent.

Endocardial expression and functional characterization of endothelin-1

Endothelin1 (ET1), a 21 amino acid peptide exerts a wide range of biological activities including vasoconstriction, mitogenesis and inotropic effects on the heart. In this study, we examined whether endocardial endothelial cells express ET1 and evaluated its functional properties. Using immunofluorescence localization method, we demonstrated cytoplasmic staining of ET1 in the human endocardial endothelial cells from the right atrium and left ventricle. Employing reverse transcriptase polymerase chain reaction (RTPCR) expression of ET1 mRNA and its receptors ETA and ETB mRNAs were found in human myocardial as well as in endocardial endothelial cells. Biological activity of endocardial endothelial cells derived ET1 was established as the conditioned media obtained from cultured porcine endocardial endothelial cells induced a slowly developing, strong and longlasting contraction of circular rat aortic segments, with similar characteristics to that obtained with exogenous ET1. Furthermore, the selective endothelinA receptor antagonist, FR139317, blocked the conditioned media induced contractions. Our results suggest that endocardial endothelial cells express and release biologically active ET1 which could play a pivotal role in the regulation of myocardial contractility as well as a circulatory peptide may further act in other peripheral target organs.

Influence of Ageing on Vasomotor Responses of Human Epicardial Coronary Arteries

Vasomotor responses to various agonists were studied on isolated circular segments of human epicardial coronary arteries from three different age groups; 23-38 years, 40-58 years and 63-86 years. Noradrenaline had no or only weak contractile effect on coronary arteries from younger patients but induced contraction of all artery segments from older patients. The Emax value was significantly (P<0.0001) higher in arteries from the oldest group compared to each of the two younger age groups, whereas the potency was similar in all three groups. Linear regression analysis of noradrenaline-induced contraction in individual patients revealed a significantly positive age-correlation (correlation coefficient 0.67, P<0.0001). Contraction induced by endothelin-1 and relaxation induced by substance P, calcitonin gene-related peptide and vasoactive intestinal peptide on arteries precontracted with U46619 showed no significant differences in maximum responses and potencies between the three age groups, and no significant linear age-correlation. These data demonstrate a large variability in contractile responses to noradrenaline with contractions seen mostly in coronary arteries from older patients. It thus seems that sympathetic activation could contribute to coronary ischaemia in some patients.