Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P.

We evaluated the effects of nitric oxide (NO) generators and endogenous production of NO elicited by substance P (SP) in the angiogenesis process. Angiogenesis was monitored in the rabbit cornea in vivo and in vitro by measuring the growth and migration of endothelial cells isolated from coronary postcapillary venules. The angiogenesis promoted in the rabbit cornea by [Sar9]-SP-sulfone, a stable and selective agonist for the tachykinin NK1 receptor, and by prostaglandin E1 (PGE1), was potentiated by sodium nitroprusside (SNP). Conversely, the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME), given systemically, inhibited angiogenesis elicited by [Sar9]-SP-sulfone and by PGE1. Endothelial cells exposed to SNP exhibited an increase in thymidine incorporation and in total cell number. Exposure of the cells to NO generating drugs, such as SNP, isosorbide dinitrate, and glyceryl trinitrate, produced a dose-dependent increase in endothelial cell migration. Capillary endothelial cell proliferation and migration produced by SP were abolished by pretreatment with the NO synthase inhibitors N omega-mono-methyl-L-arginine (L-NMMA), N omega-nitro-L-arginine (L-NNA), and L-NAME. Exposure of the cells to SP activated the calcium-dependent NO synthase. Angiogenesis and endothelial cell growth and migration induced by basic fibroblast growth factor were not affected by NO synthase inhibitors. These data indicate that NO production induced by vasoactive agents, such as SP, functions as an autocrine regulator of the microvascular events necessary for neovascularization and mediates angiogenesis.

Gene expression of endothelin-1, endothelin-converting enzyme-1, and endothelin receptors in human epididymis.

We have previously reported the presence of endothelin-1 (ET-1) and its receptors in the human testis. In the present study we extended our investigations to human epididymis. The rationale of our study originated from the fact that sperm appear to be immotile during their transit through the epididymis. Hence, it is conceivable that specific factors, unknown to date, are present in this organ, capable of inducing smooth muscle contractions, thus forcing sperm transport. In this paper it is shown that ET-1 messenger ribonucleic acid and protein are readily detectable in the epithelial compartment of the human epididymis, and that ET-converting enzyme-1, which converts the precursor pro-ET-1 into the active peptide ET-1, is expressed in the epididymis, thus indicating an active processing of the prohormone. In addition, two classes of ET receptors were characterized and located in the muscle cells of the epididymis. These receptors correspond, in terms of affinity constants and capacity, to the ETA and ETB receptors previously characterized. These receptors mediate the contractile activity of the epididymis in vitro, thus suggesting that ET-1 can be responsible of sperm progression through this organ, acting via a paracrine mode of action.

Identification, characterization, and biological activity of endothelin receptors in human ovary.

We previously reported the expression of endothelin-1 (ET-1) in granulosa cells (GCs) of the human ovary and the presence of ET-1-like immunoreactivity in human follicular fluid obtained from women in an in vitro fertilization program. In follicular fluid, but not in plasma, the levels of ET-1-like immunoreactivity were higher in gonadotropin-stimulated vs. spontaneous cycles, suggesting hormonal regulation of follicular ET-1. To identify and characterize ET receptors in human ovary, we performed autoradiography, radioligand binding, and functional studies. Mathematical analysis of families of self- and cross-competition curves among [125I]ET-1, [125I]ET-3, and selective analogs indicates that human ovary expresses both subtypes of ET receptors, i.e. ETA and ETB receptors. However, the concentration of the ETB site was 100-fold lower than that of the ETA one. By using [125I]ET-1, we demonstrated that the density of binding sites in human ovary is not affected by the hormonal milieu (similar concentrations in normal cycling, postmenopausal, and combined oral contraceptive-treated women). In situ binding studies indicate that the majority of ETA and ETB receptors are expressed in the blood vessels of the ovary. In particular, ETA receptors are abundant in the ovulatory follicles and localized in the theca interna, in close proximity to the granulosa layer. Few GCs of the ovulatory follicle were specifically labeled. Conversely, in the rat ovary, used as a control, ETB receptors were predominantly expressed and localized in GCs. Accordingly, ETB receptors negatively regulated estrogen accumulation in rat GCs. In human granulosa-luteal cells, neither ET-1 (unselective ligand) nor ET-3 or sarafotoxin 6c (ETB ligands) affected estrogen or progesterone secretion. ET-1 was 2.5-fold more potent than noradrenaline in eliciting contraction of ovarian artery, acting through the ETA receptor. Our results indicate that in human ovary, at variance with rat ovary, the endothelin system is primarily involved in the regulation of ovarian blood flow and not steroidogenesis.

Different effects of prostaglandins on adrenergic neurotransmission in atrial and ventricular preparations.

1. The effects of prostaglandin E2 (PGE2) and iloprost on the cardiac response to adrenergic nerve stimulation in guinea-pig atrial and ventricular preparations have been studied. 2. In guinea-pig isolated atria both PGE2 (0.1-10 nM) and iloprost (0.1-3 microM) concentration-dependently reduced the cardiac response to adrenergic nerve stimulation. 3. The inhibition of cyclo-oxygenase by indomethacin and acetylsalicylic acid potentiated the response to nerve stimulation in the atrial preparations. 4. Arachidonic acid (1-10 microM) reduced the response to nerve stimulation in atria. This effect was prevented by indomethacin and acetylsalicylic acid. 5. In guinea-pig ventricles PGE2 and iloprost were found to be effective at higher concentrations than in atrial preparations: arachidonic acid, indomethacin or acetylsalicylic acid did not modify the cardiac response to adrenergic nerve stimulation. 6. These results suggest a different modulator role for endogenous prostaglandins in atrial and ventricular tissue.

Effect of verapamil and diltiazem on calcium-dependent electrical activity in cardiac Purkinje fibres.

The effects of verapamil and diltiazem on normal action potentials, abnormal automaticity at depolarized membrane potential and oscillatory afterpotentials were compared in sheep cardiac Purkinje fibres. Concentrations of verapamil and diltiazem exerting the same action on abnormal automaticity due to slow action potentials, caused different effects on action potential characteristics and on oscillatory afterpotentials. Diltiazem significantly shortened action potential duration whereas verapamil slightly lengthened it (NS). Diltiazem appeared to be more effective than verapamil in preventing the development of oscillatory afterpotentials induced by barium or by strophanthidin. In 50% of barium-treated preparations, verapamil caused the appearance of spontaneous activity due to enhanced normal diastolic depolarization, while diltiazem had no such effect. The observed differences were explained in terms of the different effects of the two drugs on currents other than the slow inward current, since diltiazem was more potent than verapamil in depressing Vmax.

ATP modulates the efferent function of capsaicin-sensitive neurones in guinea-pig isolated atria.

1. The effect of adenosine triphosphate (ATP) and its stable analogues, alpha, beta-methylene-ATP and beta, gamma-methylene-ATP, on the efferent function of capsaicin-sensitive non-adrenergic, non-cholinergic (NANC) nerves was tested in guinea-pig isolated atria. 2. Transmural nerve stimulation of atria isolated from reserpine-pretreated guinea-pigs, in the presence of 1 microM atropine and 0.3 microM CGP 20712A, induced a transient positive inotropic effect attributable to calcitonin gene-related peptide (CGRP) release from NANC nerve endings. 3. ATP (1-30 microM) concentration-dependently reduced the cardiac response to transmural nerve stimulation, without affecting the inotropic response to 10 nM exogenous CGRP. The inhibitory effect of ATP was competitively antagonized by the P1-purinoceptor antagonist, 8-phenyltheophylline (8-PT, 1 microM), but was unaffected by the P2-purinoceptor antagonist, suramin (100 microM). 4. beta, gamma-methylene-ATP in the same concentration range as ATP, inhibited the cardiac response to transmural nerve stimulation. The inhibitory effect of beta, gamma-methylene ATP was antagonized by 1 microM 8-PT. The desensitizing agonist for P2-purinoceptors, alpha, beta-methylene ATP did not induce any inhibitory effect either on the cardiac response to transmural nerve stimulation or on the inhibitory effect curve for ATP. 5. The inhibitory effect of ATP on the NANC neurotransmission was inconsistently modified in the presence of 10 microM alpha, beta-methylene-adenosine diphosphate, an inhibitor of the 5'-ectonucleotidases. 6. These results demonstrate that ATP modulates the efferent function of cardiac NANC nerve endings through prejunctional inhibitory receptors belonging to the P1 type. The metabolic conversion of ATP to adenosine does not seem to be a pre-requisite for the ATP agonist activity.

Prejunctional prostanoid receptors on cardiac adrenergic terminals belong to the EP3 subtype.

1. The effects of prostaglandin E2 (PGE2) and of several synthetic prostanoids on the cardiac response to sympathetic nerve stimulation in guinea-pig atria have been evaluated. 2. PGE2 (0.01-100 nM), sulprostone (0.01-100 nM) and misoprostol (0.1-100 nM), but not butaprost (0.1-100 nM), dose-dependently reduced the increase in cardiac contractility induced by electrical field stimulation of sympathetic terminals. 3. The EP1 antagonist AH6809 (1 microM) did not modify the inhibition of cardiac response induced by PGE2, sulprostone and misoprostol. 4. In preparations preloaded with [3H]-noradrenaline, tritium overflow induced by electrical field stimulation was greatly and significantly reduced by 100 nM PGE2 and by 100 nM sulprostone. 5. These results indicate that PGE2 and other synthetic prostanoids reduce noradrenaline release from cardiac adrenergic nerve terminals acting on prejunctional inhibitory receptors belonging to the EP3 subtype.

Blockade of adenosine receptors unmasks a stimulatory effect of ATP on cardiac contractility.

1. The effects of ATP, alpha,beta-methylene ATP and beta,gamma-methylene ATP on the contractile tension of guinea-pig isolated left atria were evaluated. 2. ATP (1-100 microM) produced a concentration-dependent negative inotropic effect; this response was converted to a positive inotropic effect in the presence of the antagonist of adenosine A1 receptors, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 0.1 microM), and in the presence of 8-phenyltheophylline (10 microM), an antagonist of A1 and A2 receptors. 3. The positive inotropic effect of ATP was antagonized by the P2 receptor antagonist, suramin (500 microM). Reactive blue 2 (30-500 microM), a putative P2y receptor antagonist, concentration-dependently reduced and finally abolished the effect of ATP. 4. In the presence of 8-phenyltheophylline, the stable analogues of ATP, alpha,beta-methylene ATP and beta,gamma-methylene ATP (1-30 microM), produced a concentration-dependent increase in atrial contractility of a lesser degree than that induced by ATP. 5. The results suggest that when inhibitory adenosine receptors are blocked, ATP produces a positive inotropic effect, probably mediated by P2y receptor stimulation.

Alpha-adrenoceptor modulation of the efferent function of capsaicin-sensitive sensory neurones in guinea-pig isolated atria.

1. Transmural nerve stimulation of guinea-pig atria, obtained from animals pretreated with reserpine (5 mg kg-1, i.p.), in the presence of atropine 1 microM and of the beta-adrenoceptor blocker CGP 20712A 1 microM, induced a positive inotropic effect which was reduced by the calcitonin gene-related peptide (CGRP) antagonist hCGRP-(8-37) and abolished by pretreatment with capsaicin 1 microM. 2. Noradrenaline concentration-dependently (0.01-10 microM) reduced the increase in cardiac contractility induced by transmural nerve stimulation. The inhibitory effect of noradrenaline was antagonized by yohimbine (0.5-1 microM), in a dose-dependent manner. Prazosin (0.5-1 microM) antagonized the effect of noradrenaline and this effect was independent of concentration. 3. In the presence of yohimbine, the lower part of the inhibitory-response curve for noradrenaline was slightly but significantly shifted by prazosin. A similar degree of antagonism was observed in the presence of 1 microM phenoxybenzamine. 4. The selective alpha 2 agonists BHT 920 and clonidine reduced, in the same concentration-range (0.01-1 microM), the cardiac response to transmural nerve stimulation in a yohimbine-sensitive fashion. 5. Phenylephrine (0.1-100 microM) and methoxamine (1-300 microM) also induced an inhibitory effect on transmural nerve stimulation. The effect of phenylephrine was antagonized by yohimbine (1 microM) more efficiently than by prazosin (0.5 microM). 6. These results are in keeping with the presence of inhibitory prejunctional alpha 2-adrenoceptors on cardiac sensory nerve endings which modulate the efferent function of capsaicin-sensitive neurones.

Vasorelaxant effects induced by the antiangiogenic drug linomide in aortic and saphenous vein preparations of the rabbit.

1. Linomide (N-phenylmethyl-1,2-dihydro-4-hydroxyl-1-methyl-2-oxoquinoline-3-carb oxa mide) inhibits vascular proliferation and has been proposed as an antiangiogenic drug. We have investigated the vascular effect of linomide in rabbit aortic and saphenous vein ring preparations and in rat cultured vascular smooth muscle cells (VSMCs). 2. Linomide (25-300 micrograms ml-1) did not alter the basal tone of the preparations. The drug induced a concentration-dependent relaxant effect in aortic rings with endothelium, preconstricted by noradrenaline (NA), 5-hydroxytryptamine (5-HT) and by the thromboxane mimetic U46619. 3. The degree of relaxation induced by linomide was significantly reduced by exposure to the cyclooxygenase inhibitors indomethacin (3 microM) and acetylsalicylic acid (500 microM), and was not influenced by pretreatment with the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (100 microM) in aortic rings with endothelium, preconstricted with NA. 4. Endothelium removal significantly reduced the relaxant response to linomide in aortic ring preparations. 5. A concentration-dependent relaxant response was observed also in rabbit saphenous vein preparations deprived of endothelium and preconstricted either by NA or U46619. The degree of relaxation obtained in a high potassium solution was consistently smaller than that observed in NA-pretreated venous preparations. 6. The vasorelaxant effect of linomide was consistently blunted by the adenylate cyclase inhibitor SQ 22536 (50 microM), both in intact aortic rings and in those deprived of endothelium. 7. In rat cultured vascular smooth muscle cells, linomide (100-200 micrograms ml-1) induced a significant increase in cyclic AMP levels, which was blocked by exposure to 50 microM SQ 22536. 8. In endothelium-deprived aortic ring preparations, the linomide-induced relaxant effect was greatly reduced in high potassium medium (KCl = 25 mM). Pretreatment with the ATP potassium channel inhibitor glibenclamide (3 microM) significantly reduced the linomide-induced relaxation. 9. The results show that linomide possesses a vasorelaxant effect which is attributable to both endothelium-dependent and -independent properties. While the former component of the drug's activity is apparently due to the release of a prostanoid from endothelial cells, the endothelium-independent mechanism involved in linomide relaxation is linked to cyclic AMP accumulation and to ATP-sensitive potassium channel activation in VSMCs.

Electrophysiological mechanisms for the antiarrhythmic action of mexiletine on digitalis-, reperfusion- and reoxygenation-induced arrhythmias.

The antiarrhythmic potency of mexiletine was evaluated on three groups of guinea-pig isolated hearts. Arrhythmias were induced (a) with digitalis intoxication, (b) with hypoxia followed by reoxygenation and (c) with ischaemia followed by reperfusion. Mexiletine 10 microM was found to be very effective against all three types of arrhythmias in all three groups. The electrophysiological effects of mexiletine were then studied on sheep cardiac Purkinje fibres manifesting oscillatory afterpotentials and triggered automaticity induced by barium or strophanthidin. Mexiletine 10 microM consistently decreased the amplitude of oscillatory afterpotentials and blocked subsequent triggered activity in sheep Purkinje fibres. In contrast, mexiletine 10 microM had no significant effect on Vmax in normal, barium- and strophanthidin-treated preparations. The results are discussed in relation to the mechanisms of antiarrhythmic action of mexiletine.

Electrophysiological mechanism for the antiarrhythmic action of propafenone: a comparison with mexiletine.

1. The antiarrhythmic potency of propafenone was evaluated in the guinea-pig isolated heart; arrhythmias were induced with (a) digitalis intoxication and (b) hypoxia followed by reoxygenation. 2. Propafenone, 0.5 microM, was found to be the minimal but effective antiarrhythmic concentration. The antiarrhythmic activity of propafenone developed slower than that of 10 microM mexiletine, which was the lowest effective concentration under the same experimental conditions. 3. The electrophysiological effects of propafenone were then studied on sheep cardiac Purkinje fibres (manifesting oscillatory afterpotentials and triggered automaticity induced by barium or strophanthidin) and compared with those of 10 microM mexiletine. 4. Both 0.5 microM propafenone and 10 microM mexiletine consistently blocked triggered activity in sheep Purkinje fibres. The onset of the effect of propafenone was slower than that of mexiletine. 5. Unlike mexiletine, propafenone did not reduce the amplitude of oscillatory afterpotentials. 6. In contrast, propafenone significantly reduced Vmax in barium- and strophanthidin-treated preparations. 7. It is concluded that the antiarrhythmic action of propafenone on digitalis- and reoxygenation-induced arrhythmias is probably due to an electrophysiological mechanism different from that of mexiletine. Mexiletine, by reducing the amplitude of oscillatory afterpotentials, prevents the attainment of the threshold; propafenone, by reducing the excitability of the cell, increases the threshold and consequently an oscillatory afterpotential of the same amplitude will not generate arrhythmias.

Nitric oxide is not involved in the effects induced by non-adrenergic non-cholinergic stimulation and calcitonin gene-related peptide in the rat mesenteric vascular bed.

The mechanism involved in the effects induced by the activation of perineural non-adrenergic non-cholinergic (NANC) nerves or by exogenous calcitonin gene-related peptide (CGRP) was investigated in the rat mesenteric vascular bed (MVB) perfused with Kreb's solution containing methoxamine and guanethidine. The activation of NANC terminals of the tissue was carried out by means of electrical field stimulation (EFS). An increase in the perfusion pressure of the preparations was observed in the presence of two inhibitors of nitric oxide synthase: NG-monomethyl-L-arginine (L-NMMA) (100 microM) and NG-nitro-L-arginine methyl ester (L-NAME) (100 microM). However L-NMMA and L-NAME did not modify the relaxant effect induced by EFS and exogenous CGRP. Furthermore the relaxant effect induced by EFS and exogenous CGRP was not affected by the removal of endothelium from the preparations. These results provide evidence that the vasodilation induced by NANC stimulation or by exogenous CGRP in MVB does not involve NO production.

Positive inotropic effects of CGRP and isoprenaline: analogies and differences.

In guinea-pig isolated left atria, electrically stimulated at 1 Hz, isoprenaline and calcitonin gene-related peptide (CGRP) induced a positive inotropic effect in the same concentration range (0.3-100 nM). The increase in contractile tension induced by both agonists was associated with a reduction in time to peak tension and relaxation time. However CGRP was more active than isoprenaline in reducing the time to peak; this effect was more evident when the bath temperature was reduced from 30 degrees to 24 degrees C. The positive inotropic effects of isoprenaline and CGRP were potentiated by forskolin (30 nM), a direct activator of adenylcyclase; on the other hand, cholera toxin (1 microgram/ml), which irreversibly ribosylates Gs protein, did not modify the effect of CGRP, while antagonizing the concentration-response curve for isoprenaline. It is concluded that the increase in atrial contractile tension produced by isoprenaline and CGRP are linked to the adenylcyclase system in a different manner.

Modulation by adrenergic transmitters of the efferent function of capsaicin-sensitive nerves in cardiac tissue.

In atrial preparations obtained from reserpine-pre-treated guinea-pigs, incubated in the presence of 1 microM atropine plus 1 microM CGP 20712A (a beta 1 blocking drug), a positive inotropic effect due to CGRP release from capsaicin-sensitive sensory neurons was induced by electrical field stimulation (EFS). This response was concentration-dependently reduced by noradrenaline (0.01-3 microM), neuropeptide Y (NPY, 3-300 nM) and adenosine triphosphate (ATP, 1-30 microM). On the other hand, the overflow of [3H]-noradrenaline from sympathetic nerve terminals induced by EFS in isolated atria obtained from normal untreated animals was not modified in 10 nM calcitonin gene-related peptide (CGRP). Substance P (SP) and neurokinin A (NKA), at concentrations ranging from 0.01 to 1 microM did not affect the cardiac response to field stimulation of adrenergic terminals of atrial tissue. These findings demonstrate that all the co-transmitters stored in adrenergic nerve terminals have a modulatory role on the efferent function of cardiac capsaicin-sensitive sensory neurons, while cardiac adrenergic neurotransmission is not influenced by the peptidergic transmitters released from sensory neurons.

Effect of the non-peptide blocker (+/-) CP 96,345 on the cellular mechanism involved in the response to NK1 receptor stimulation in human skin fibroblasts.

The effect of the selective non-peptide antagonist for NK1 receptors (+/-)CP 96,345 on cellular transduction mechanisms elicited by the NK1 selective agonist [Sar9]-substance P-sulfone ([Sar9]-SP) was investigated in a stabilized culture of human skin fibroblasts (HF) and compared to the effects of two peptide antagonists, FK 888 and GR 82, 334. The exposure of the cells to [Sar9]-SP (100 nM) produced an early increase in inositol 1,4,5-trisphosphate (IP3) level, which peaked after 6 s, and a later rise in cellular inositol 1-phosphate (IP1) content which reached the maximum level in 15 min. The cAMP level was not significantly modified. The increase in IP1 was greatly reduced, at approximately the same extent by the 10 min pretreatment with a concentration of (+/-)CP 96,345 (100 nM) 10 times smaller than that of FK 888 and GR 82,334 (1 microM). The cytosolic Ca2+ mobilization in response to the NK1 agonist was monitored both by spectrofluorimetric and single-cell image analysis determinations on adherent cells loaded with the Ca(2+)-sensitive fluorescent indicators Fura-2/AM and Indo-1, respectively. [Sar9]-SP (100 nM) produced a rapid increase in the intracellular Ca2+ level in Fura-2/AM loaded cells. Cytosolic Ca2+ mobilization, measured by single-cell image analysis, indicated a concentration-dependent increase in both the ratio and in the number of cells responding to [Sar9]-SP. Either the non-peptide or the peptide selective NK1 receptor antagonists inhibited the increase in Ca2+ level in both the assays. In the spectrofluorimetric experiments the antagonizing effects of (+/-)CP 96,345 (1-100 nM), FK 888 (10 nM-1 microM) and GR 82,334 (10 nM-1 microM) were concentration-dependent. Moreover, the non-peptide antagonist was more potent than the two peptide antagonists, producing an 82.5% inhibition of Ca2+ mobilization at a concentration (10 nM) at which FK 888 and GR 82,334 decreased the response by only 62.3 and 60%, respectively. Stimulation of phosphatidylinositol turnover and calcium mobilization were also induced by 10 nM bradykinin; these effects were influenced neither by the previous administration of the NK1 receptor agonist nor by the three antagonists tested. These results demonstrate that the cellular transduction mechanisms induced in human skin fibroblasts by NK1 receptor stimulation are specifically and effectively antagonized by (+/-)CP 96,345, and that this non-peptide antagonist is more potent than the two peptide antagonists tested.

Effects of opioid drugs on capsaicin-sensitive neurones in guinea-pig atria.

Transmural nerve stimulation of isolated guinea-pig atria in the presence of atropine induced a biphasic positive inotropic effect but only a slow increase in contractility (NANC response) in atria obtained from 6-hydroxy-dopamine-pretreated animals. The latter effect disappeared after exposure of the preparations to capsaicin. The effects of some opioid peptides were investigated on NANC responses. [D-Ala2,D-Leu5]enkephalin (DADLE) and [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAGO, 0.1-10 microM) inhibited the cardiac response to transmural nerve stimulation in a dose-dependent and naloxone-sensitive manner. Dynorphin-(1-13) and morphine, at 10-fold higher concentrations (1-10 microM), reduced the response in a naloxone-sensitive manner. Naloxone alone however did not affect the response. Opioid peptides were not able to reduce the positive inotropic effect induced by calcitonin gene-related peptide (CGRP), or the increase in cardiac contractility produced by capsaicin. These results suggest that opioid receptors exert a modulatory role on peripheral terminals of capsaicin-sensitive sensory nerves.

Characterization of opioid receptors modulating the function of capsaicin-sensitive neurons in guinea-pig atria.

Transmural nerve stimulation of isolated atria, obtained from reserpine-pretreated guinea-pigs, in the presence of atropine and the beta 1-adrenoceptor-blocking drug CGP 20712A, induced a positive inotropic effect. [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAGO), [D-Ala2,D-Leu5]enkephalin (DADLE), morphine and dynorphin dose dependently reduced the cardiac response to transmural nerve stimulation. The delta receptor selective agonist [D-Pen2,D-Pen5]enkephalin (DPDPE), and the kappa receptor agonist, U50488, were unable modify the response. The inhibitory effect of all the active opioid agonists was antagonized by naloxone but not by the selective delta and kappa opioid receptor antagonists, ICI 174.864 and MR 2266. These results suggest the presence on sensory nerve terminals of inhibitory opioid receptors belonging to the mu, but not to the delta and kappa subtypes.

Effects of thromboxane agonists on cardiac adrenergic neurotransmission.

The effects of thromboxane B2 (TxB2) and of two thromboxane mimetics, dl-(9,11), (11,12)-dimethano-TxA2 (ONO 11006) and 9,11-dideoxy-11,9-epoxymethano prostaglandin F2 alpha (U46619) on the cardiac response to adrenergic nerve stimulation in isolated guinea-pig atria were evaluated. All the agonists dose dependently reduced the positive inotropic effect induced by field stimulation, U46619 being the most active. The inhibitory effect of U46619 was reduced by the thromboxane receptor antagonists, sulotroban and AH 23848B. U46619 did not significantly reduce the positive inotropic effect induced by exogenous noradrenaline. However U46619 was unable to modify the tritium overflow induced by field stimulation in preparations preloaded with [3H]noradrenaline. In addition to this influence on adrenergic neurotransmission, U46619 also had a direct positive inotropic effect on cardiac contractility, which was antagonized by AH 23848B. These results indicate that U46619 reduces the cardiac response to sympathetic nerve stimulation and that is also has a direct stimulatory effect on cardiac muscle.

The inhibitory effect of opioid and alpha 2-adrenoceptor agonists on cardiac sensory neurones is pertussis toxin-insensitive.

The role of pertussis toxin-sensitive G proteins on the alpha 2-adrenoceptor and mu-opioid receptor-mediated inhibition of the efferent function of capsaicin-sensitive neurones was investigated in guinea-pig atria pretreated with guanethidine. In the presence of atropine, CGP 20712A (2-hydroxy-5-(2-[hydroxy-3-(4-[(1-methyl- 4-trifluormethyl)1H-imidazol-2-yl]-phenoxy)propyl]aminoethoxyl+ ++)-benzamide) and prazosin, [D-Ala2,NMe-Phe4,Gly5-ol]enkephalin (DAGO, 0.1-3 microM) and 2-amino-6-allyl-5,6,7,8-tetrahydro-4H-thiazolo(4,5-d)azepine (BHT 920, 0.01-1 microM) reduced the positive inotropic effect induced by transmural stimulation of preparations obtained from control and from pertussis toxin-treated animals. These results suggest that pertussis toxin-sensitive G proteins are not involved in the inhibitory regulation of the efferent function of capsaicin-sensitive nerve terminals in cardiac tissue induced by alpha 2 and opioid receptor stimulation.