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In a previous study, we reported the presence of endothelin-1 and endothelin receptors in the human testis. We have now extended our investigations to the human epididymis. Since sperm appear to be immotile during their transit through the epididymis, it is conceivable that specific local factors promote smooth muscle contraction, enabling sperm transport. In this paper, we show that endothelin-1 mRNA and protein are readily detectable in the epithelial compartment of the human epididymis, and that endothelin converting enzyme- 1, which converts the precursor pro-endothelin-1 into active endothelin-1, is expressed in the epididymis, consistent with active processing of the prohormone. In addition, two classes of endothelin receptors were characterized and located in the muscle cells of the epididymis. These receptors correspond, in terms of affinity constants and capacity, to the previously characterized endothelinA and endothelinB receptor. These receptors appear to be biologically active and mediate the contractile activity of the epididymis in vitro. Our data suggest that endothelin-1, via a paracrine mode of action, may be responsible for sperm progression through this organ.

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.

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...

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.

We investigated vasodilator responses to acetylcholine (ACh) in isolated mesenteric vascular bed preparations (preconstricted with methoxamine) of young (2 months) and old (18 months) normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). ACh produced a similar dose-dependent vasorelaxant effect in preparations from both 2-month old normotensive and hypertensive rats. This vasodilator response to ACh decreased with age, especially in hypertensive animals. In preparations from young WKY, the vasorelaxant effect of ACh was not affected by 100 microM NG-nitro-L-arginine methyl ester (L-NAME), and was only slightly reduced by 500 microM L-NAME. The K+ channel blocker tetraethylammonium (TEA 2.5-10 mM) concentration-dependently antagonized the ACh-induced vasodilation in the same preparations. In preparations obtained from aged WKY animals, as well as in those from young and aged SHR animals, ACh-induced vasodilation was significantly and concentration-dependently reduced by 100 and 500 microM L-NAME. On the other hand, TEA induced a lesser antagonistic effect than that observed in young normotensive animals. In preparations preconstricted with 80 mM KCl, ACh caused vasodilation that was weaker in preparations from young WKY than in those from aged WKY; on the contrary, ACh was more effective in young than in aged SHR. These results confirm that the vasodilating response to ACh decreases with age and hypertension and suggest that the main mechanism responsible for the effect of ACh in vessels of young normotensive animals consists of activation of K+ channels. In preparations from old normotensive, as well as in those from young and old hypertensive animals, ACh induces vasorelaxation mainly through nitric oxide (NO) release.

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.

We investigated the influence of aging and hypertension on the vasorelaxant effect of calcitonin gene-related peptide (CGRP), examining the responses to stimulation of perivascular vasodilatory nerves and to administration of the peptide in isolated mesenteric vascular bed preparations of young (aged 2-3 months) and old (aged 18 months) normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). We used preparations preconstricted by perfusion with 100 microM methoxamine with addition of 5 microM guanethidine. The stimulation-induced vasorelaxation in the preparations of young SHR animals was significantly lower than that in those of age-matched WKY rats. Moreover, the vasodilator response to stimulation displayed an age-dependent decline in vascular beds of normotensive animals. The degree of the relaxant response to CGRP (0.01-1 microM) did not differ significantly between vascular preparations of normotensive and hypertensive rats; but was significantly reduced in preparations of both SHR and WKY rats aged 18 months as compared with those of young animals. An age-dependent decrement in the vascular reactivity, qualitatively similar to that observed with CGRP, was also detected with two other vasodilators, i.e., the endothelium-dependent vasodilator acetylcholine (ACh 0.1-100 microM) and the directly acting nitrovasodilator sodium nitroprusside (SNP, 1-10 microM). We conclude that the vascular sensitivity to CGRP, as well as that to other vasodilator agents acting by different mechanisms, decreases with age in both normotensive and genetically hypertensive rats.

The cardiac response to field stimulation of adrenergic nerve terminals in isolated atrial preparations from adult (6-month-old) normotensive Wistar Kyoto (WKY) and spontaneously hypertensive (SH) rats was enhanced in comparison to that observed in the atrial tissue of young (2-month-old) animals of both strains; the increase in the sympathetic response was significantly higher in preparations from SHR than in those from age-matched WKY rats. The sensitivity of cardiac adrenergic neurotransmission to the prejunctional inhibitory effects exerted by exogenously administered prostaglandin E2 (0.1 nM-1 microM) and iloprost (0.1-10 microM) did not show any strain-dependent difference in preparations from both young and adult rats. Moreover, acetylsalicylic acid (500 microM) induced a similar degree of potentiation of the response to sympathetic stimulation in atrial tissues of young WKY and SH animals; however, the effect of the cyclo-oxygenase inhibitor was completely missing in preparations from adult rats of both strains. Finally, arachidonic acid (10 microM) inhibited the adrenergic response to a greater extent in preparations from young and adult SH rats than in those from age-matched normotensive rats. The results of the study indicate that, at least in cardiac preparations, changes in the modulatory role of endogenous prostaglandins occur as age-dependent processes and, therefore, may not be indicative of possible differences in the role of prostaglandins between hypertensive and normotensive animals. The possible significance of the dissimilar response to arachidonic acid, detected as the only difference between preparations from SH and WKY rats, is discussed.

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.

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 μM) and NG-nitro-L-arginine methyl ester (L-NAME) (100 μM). 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.

The negative inotropic effect of adenosine (1-100 microM) was abolished in isolated guinea-pig atria obtained from pertussis toxin-pretreated guinea pigs electrically driven at 4 Hz. However, the inhibitory effect of the same concentrations of adenosine on the cardiac response to stimulation of non-adrenergic non-cholinergic (NANC), capsaicin-sensitive sensory nerves, was not modified by the toxin. These results suggest that, while pertussis toxin-sensitive G proteins are involved in the negative inotropic effect of adenosine, they do not mediate the inhibitory effect of adenosine on cardiac NANC neurotransmission.

The presence of beta 2-adrenoceptors in the sheep ventricular myocardium was assessed by the radioligand binding technique and functional studies. In membrane preparations, the competition curve between [3H]-dihydroalprenolol and the selective beta 1-antagonist CGP 20712A (0.1 nM-1 mM) was clearly biphasic, and revealed the presence of two different binding sites showing an affinity (pKD) for CGP 20712A of 9.5 +/- 0.9 and 4.5 +/- 0.4, respectively. The relative proportion of beta 1:beta 2 adrenoceptors was about 70:30 in both the right and left ventricle. In ventricular trabeculae driven at 1Hz, isoprenaline (1-300 nM) caused a dose-dependent increase in the force of contraction, the maximum effect being 298 +/- 26 mg, associated with reduction of time to peak tension (t1, clinotropic effect) and relaxation time (t2, 298 +/- 26 mg, associated with reduction of time to peak tension (t1, clinotropic effect) and relaxation time (t2, lusitropic effect). The inotropic dose-response curve for isoprenaline was significantly shifted to the right by pretreatment of the preparations with 0.1 microM CGP 20712A or with the selective beta 2-antagonist ICI 118551 (50 nM). In the presence of CGP 20712A (0.1 microM), isoprenaline, up to a concentration of 10 microM, did not affect either t1 or t2; on the other hand, pretreatment of the preparations with ICI 118551 (50 nM) fully antagonized the clinotropic but not the lusitropic effect of isoprenaline. In the presence of CGP 20712A procaterol (0.01-10 microM), a beta 2-adrenoceptor agonist, induced a positive intropic effect which was not associated with any significant modifications in t1 or t2. This effect was completely abolished by ICI 118551 (50 nM). The positive inotropic action of isoprenaline (1 microM) was associated with a significant decrease in action potential duration measured at -60 mV (220 +/- 8 and 193 +/- 10 ms in the absence and presence of isoprenaline, respectively; P less than 0.05). In the presence of CGP 20712A (0.1 microM) alone, isoprenaline (1 microM) still induced a significant increase in contractility but the action potential profile was only slightly affected. The effects of isoprenaline were fully antagonized by the simultaneous presence of CGP 20712A and ICI 118551 (10 nM). It is concluded that both beta 1- and beta 2-adrenoceptors appear to coexist in sheep ventricular myocardium where their stimulation mediates a positive inotropic effect. However, their functional role on the relaxation phase of the twitch may be different.

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.

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.

We used intracellular microelectrodes to study the effects of histamine on both normal and abnormal automaticity in sheep Purkinje fibers. Histamine, dimaprit and 4-methylhistamine caused a similar reduction of the action potential duration in driven Purkinje fibers. Histamine (10-6 M) induced spontaneous activity in p previously quiescent preparations more often than did equimolar concentrations of dimaprit and 4-methylhistamine. The effects of histamine on automaticity were enhanced in the presence of barium In fact histamine, at concentrations which were unable to induce automaticity in normal preparations, induced it in the presence of barium. In Purkinje fibers manifesting barium-induced automatic activity, histamine (10-7--10-6M) significantly increased the average number of spontaneous action potentials and shortened the time of their appearance. In the same range of concentrations, histamine dose-dependently increased the iological manifestation of calcium overload. Histamine (10-6--10-4M) increased the OAP amplitude of strophanthidin -treated Purkinje fibers, eventually inducing triggered extrabeats. All these previously described effects were selectively blocked by cimetidine (10-5 M). It is concluded that histamine may induce cardiac arrhythmias under conditions of calcium overload and that this effect may be due to induction or enhancement of oscillatory afterpotentials.

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.

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.

Substance P (SP) is associated with lymphatic tissue and is a putative mediator of inflammation. The lymph pump is one of the major "safety factors" preventing edema and its activity is altered by inflammatory mediators. The impact of SP on lymphatics was studied in the rat mesentery.Rats were prepared for in situ lymphatic observation using intravital videomicroscopic techniques. Sections of the small intestine and mesentery were exteriorized and superfused. Lymphatic diameters were measured and pumping activity was determined from the lymphatic diameter tracing. Lymph pump parameters evaluated included diastolic diameter, systolic diameter, contraction frequency, stroke volume, and lymph pump flow. After a control period, the tissues were exposed to SP (10(-9), 10(-8), 10(-7), 10(-6) M). SP reduced both diameters and increased contraction frequency in a concentration-dependent manner. SP (1.0 microM) produced the following changes (% of control): reductions in diastolic diameter (50%), systolic diameter (55%), and stroke volume (63%); a large increase in contraction frequency (661%); and a modest increase in lymph pump flow (44%). SP also stimulated vessels that were not contracting phasically to develop typical contraction patterns.Although SP produced a tonic constriction of the lymphatics, lymph pump flow was maintained or slightly elevated via an increase in contraction frequency. SP also induced pumping activity in lymphatics that were previously quiescent, thereby further stimulating flow. These effects may serve to minimize the formation of edema in face of the inflammatory edemagenic conditions produced by SP.

The effect of ATP in human and rabbit corpus cavernosum (CC) smooth muscle was investigated. Strips of human CC were vertically mounted in an organ bath and the tonic tension was recorded. ATP (0.1-3 mM) induced a concentration-dependent relaxant effect, with a pD2 value of 3.01+/-0.3. The purine-induced relaxation was not affected by L-NAME (100 microM). In rabbit CC, ATP also induced a concentration-dependent relaxation, which was not influenced by L-NAME or by indomethacin (3 microM), with a pD2 value of 3.1 +/-0.4. The ATP-induced relaxant effect in rabbit CC was increased by both the inhibitor of adenosine reuptake, dipyridamole (3 microM) and by the inhibitor of adenosine deaminase, EHNA (0.3 microM). Moreover CGS 15943 (3 microM), an A2a adenosine antagonist, reduced the ATP-induced relaxation. UTP was not able to produce relaxation. The two ATP analogues 2-methylthioATP and alpha,beta-methylene ATP were able to induce relaxation in rabbit CC, with the following order of potency: 2-methylthioATPATPalpha,beta-methylene ATP thus suggesting a role for P2y receptors. However, reactive blue (500 microM), an unspecific P2y antagonist, did not modify the ATP relaxant response. The inhibition of phospholipase C by U73122 (3 microM) and of the endoplasmic reticulum Ca2+ATPase by thapsigargin (1 microM) did not modify the ATP-induced relaxation. The P2x specific antagonist PPADS (30 microM) and suramine (500 microM) were not able to modify the ATP relaxation either in the absence or presence of CGS 15943 (3 microM). These results confirm that ATP acts as a potent and NO-independent relaxant agent of human and rabbit CC. Our findings also show that the ATP effect is partially attributable to the metabolic breakdown of ATP to adenosine, which acts through A2a receptor stimulation, but is also due to a direct stimulation of P2 receptors that are different from the classical P2y and P2X receptor subtypes for ATP.

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.

We investigated the influence of the Ca(2+)-ATPase inhibitor thapsigargin (TG) on the vasorelaxant response to different endothelium-dependent and endothelium-independent relaxing agents in an isolated thoracic aorta preparation of the rabbit, precontracted by norepinephrine (NE). Pretreatment with 100 microM L-arginine methyl ester (L-NAME) an inhibitor of nitric oxide (NO) synthesis, completely prevented acetylcholine (ACh)-induced relaxation; the inactive stereoisomer D-NAME did not modify the effect of ACh. The exposure of the preparations to 1 microM TG induced a slowly developing slight increase in the basal tension during 30-min contact. The same concentration of TG also slightly reduced the response to the subsequent administration of NE. The antagonist effect of TG on the ACh response was concentration dependent in the range between 0.1 and 10 microM. A 30-min pretreatment with 1 microM TG appeared to be sufficient to induce a consistent antagonism of the ACh (0.01-10 microM) concentration-relaxant effect curve, since an increase to 60 min did not produce a further significant increment in the degree of the antagonist effect. The concentration-dependent relaxation induced by substance P (SP 0.1-3 nM) was also significantly antagonized by 1 microM TG. The effect of the calcium ionophore A23187 (0.01-1 microM) was reduced by the Ca(2+)-ATPase inhibitor only at the higher concentrations tested (0.3-1 microM). However, a 30-min contact time with 1 microM TG was completely ineffective in antagonizing the concentration-relaxant response curves to the two nitrovasodilators sodium nitroprusside (SNP 0.1-100 microM) and nitroglycerin (NTG 1-300 nM) and to the cyclic GMP analogue 8-Bromo-cyclic GMP (3-100 microM). The effects of the beta-adrenoceptor agonist isoprenaline (ISO 0.1-10 microM) and of the direct adenylate cyclase activator forskolin (FK 0.01-10 microM) were also completely unaffected by 1 microM TG. These results demonstrate that TG affects the response to agents that induce an endothelium-dependent relaxation through receptor-dependent calcium mobilization. However, they do not support the hypothesis that sarcoplasmic pump activity is essential for the development of a vasorelaxant response to endothelium-independent agents.

Fibroblast migration is an important component of the tissue response during the repair process, and substance P (SP) has been shown to exert trophic effects. In the present study, cell migration was evaluated as the distance travelled by adherent human skin fibroblasts (HF) at 96 h and by the number of individual cells moving across a filter within 5 h. In control conditions (1% calf serum) adherent fibroblasts moved from the starting line by approximately 700 microns. The addition of SP (10(-11)-10(-7) M) increased HF mobilisation in a concentration-dependent manner, with maximal activity at 10(-8) M (50% increase in migration over control). Migration of individual HF in suspension was also promoted by SP in a concentration-dependent manner, with an EC50 of 2.2 x 10(-9) M. The response produced by the maximally effective concentration of SP was equal to 65 and 90% of the effect elicited by 100 ng/ml Platelet-Derived Growth Factor A/B (PDGF A/B) on adherent and individual cells respectively. The synthetic NK1 receptor agonist [Sar9]SP-sulphone (10(-11)-10(-6) M) reproduced the SP effect. The NK2 and NK3 receptor agonists [beta Ala8]NKA(4-10) and [MePhe7]NKB were devoid of any effect. The effect of SP was antagonised by two selective antagonists of NK1 receptors, namely (+/-) CP 96,345 (10(-10)-10(-8) M) and FK 888 (10(-9)-10(-7) M), while the NK2 receptor antagonist MEN 10627 (10(-8)-10(-7) M) was not effective. Our data indicate that SP is a potent effector of fibroblast migration and the NK1 receptor is responsible for this effect. These observations further support the specific role of the NK1 receptor in mediating the trophic function of SP at the cutaneous level.

The role of the vascular endothelium in the response to a vasoconstrictor agent acting through a non-receptorial mechanism, such as KCI, was tested in the isolated mesenteric vascular bed of the rat. It was confirmed that the vasoconstrictor response evoked by stimulation of sympathetic terminals was unaffected by 100μ m N G -nitro- d -arginine methyl ester ( d -NAME), but was significantly potentiated by 100 μ m N G -nitro- l -arginine methyl ester ( l -NAME) and by removal of endothelium. Responses to exogenous noradrenaline (1–100 μ m were also enhanced by treatment with 100 μ m N G -monomethyl- l -arginine ( l -NMMA) and with l -NAME, but not with d -NAME. The potentiating effect of NO synthesis inhibitors was reversed by 1 m m l -arginine. Moreover, the noradrenaline-induced vasoconstriction was significantly increased by endothelium-deprivation. Potassium chloride (80 m m ) induced a vasoconstrictor response which was not modified by pretreatment with prazosin (0.1 μ m ) and yohimbine (0.1 μ m . The response to KCl was unaffected by d -NAME (100 μ m ) but the l -stereoisomer induced a significant increase in the perfusion pressure. In endothelium-denuded preparations the vasoconstrictor response to KCI was greater than in control conditions and was quantitatively similar to that observed in l -NAME-treated preparations. The responses to electrical field stimulation, noradrenaline and KCl in endothelium-denuded preparations were not modified by l -NAME. The results suggest that an increase in vascular tone, per se , may represent a trigger for the release of endothelium-derived relaxing factor from endothelial cells.

The vascular response to bradykinin was investigated in mesenteric vascular bed preparations preconstricted with methoxamine, obtained from 2- and 18-month old normotensive (WKY) and spontaneously hypertensive (SHR) rats. In preparations from young normotensive rats bradykinin (1 nm-10 microM) produced an endothelium-dependent vasorelaxant effect which was greatly reduced by the B2 receptor antagonist Ac-D-Arg[Hyp3,D-Phe7,Leu8]-bradykinin (1 microM), and was unaffected by the B1 receptor antagonist des-Arg9,[Leu8]-bradykinin (1 microM). The degree of vasodilation was similar in preparations from age-matched SHR rats. In vessels obtained from old animals bradykinin induced an endothelium-independent vasoconstrictor response; this effect was more pronounced in preparations from SHR than in those from WKY rats. The vasoconstriction was unaffected by both B1 and B2 receptor antagonists, and was abolished by 3 microM indomethacin. We conclude that the vasorelaxant effect of bradykinin in vessels of young animals is due to stimulation of B2 receptors. This vasodilating response can be converted by aging to a vasoconstriction and is probably due to the release of a prostanoid product; moreover it is more pronounced in spontaneously hypertensive animals.

Rat hearts made hypoxic for 20 min by perfusion with 95% N2/5% CO2 and reoxygenated for 20 min in a Langerdorff apparatus showed a dose-dependent reduction of lactate dehydrogenase release when incubated with ganglioside GM1 (0.1–10 mu;M). The decline of contractile force during hypoxia was also reduced dose dependently in the presence of GM1. Similar effects were observed in hearts obtained from animals treated i.p. with 40 mg/kg GM1 for 14 days. The levels of Na+, K+-ATPase in ventricular tissue were also reduced after hypoxia-reoxygenation and the reduction was prevented in hearts from GM1-treated animals. GM1 (1–30mu;M) reduced the functional response to field stimulation of adrenergic nerve terminals in isolated atria. Rat atria made hypoxic in glucose-free media maintained normal stores of tissue noradrenaline in the presence of 1 mu;M GM1. In the rabbit, GM1 (40 mg/kg i.p. for 4 days) reduced the alterations of the ST segment of the ECG during acute occlusion of the left descending and circumflex coronaries artery. In conclusion, ganglioside GM1 reduces some effects of hypoxia-reoxygenation in the heart, through still unknown mechanisms.

The vasodilating effect of substance P (SP) at the microvascular level is endothelium-dependent. In the present study we evaluated whether SP activates nitric oxide (NO) production by venular endothelial cell. We evaluated NO activation by measuring cyclic GMP levels in cultured endothelial cells isolated from coronary postcapillary venules of bovine origin (CVEC). Our results indicate that 5 min exposure of CVEC to 10 nM SP doubled basal cyclic GMP levels. Cell treatment with the NO synthase inhibitor L-NMMA reduced the basal levels of cyclic GMP and abolished the effect of SP but did not modify the increase in cyclic GMP in response to exogenous NO. These data indicate that a) microvascular endothelium responds in an autocrine fashion to NO with increased cyclic GMP levels, b) SP activates cyclic GMP pathway through NO production.

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.

The role of pertussis toxin-sensitive G proteins on the α 2 -adrenoceptor and μ-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-Ala 2 ,NMe-Phe 4 ,Gly 5 -ol]enkephalin (DAGO, 0.1−3 μM) and 2-amino-6-allyl-5,6,7,8-tetrahydro-4H-thiazolo(4,5-d)azepine (BHT 920, 0.001−1 μM) 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 α 2 and opioid receptor stimulation.

The effects of thromboxane B 2 (TxB 2 ) and of two thromboxane mimetics, dl-(9,11), (11,12)-dimethano-TxA 2 (ONO 11006) and 9,11-dideoxy-11,9-epoxymethano prostaglandin F 2α (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 [ 3 H]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.

We have previously shown that endogenous adenosine inhibits non-adrenergic, non-cholinergic (NANC) neurotransmission in isolated guinea-pig atria. In the present study the effect of adenosine analogues, such as N6 cyclopentyladenosine (CPA), 5' N-ethylcarboxamide adenosine (NECA), 2 chloroadenosine (2-CADO), R- and S-phenylisopropyladenosine (R- and S-PIA) on the cardiac response to transmural nerve stimulation has been tested in order to characterize the subtype of adenosine receptor involved in the inhibitory control of NANC neurotransmission. The effect of the adenosine antagonist 8-phenyltheophylline (8-PT) was then tested against CPA and NECA. The prototypical A-1 selective agonist CPA was the most active agonist, reducing the response to the stimulation of NANC nerves with an IC50 value of 2.8 nM; R-PIA, NECA and 2-CADO showed IC50 values of 9.5, 13.7 and 35 nM respectively. S-PIA was the least active agonist, showing an IC50 value (306 nM) about 30-fold greater than that of R-PIA (9.5 nM). None of the agonists tested was able to modify cardiac response to exogenous CGRP. Furthermore, 8-PT competitively antagonized the effect of CPA and NECA with very close pA2 values (6.77 +/- 0.01 and 6.63 +/- 0.08 respectively). From these findings we concluded that prejunctional inhibitory adenosine receptors on capsaicin sensitive sensory nerves of cardiac tissue belong to the A-1 subtype.

Transmural stimulation of non-adrenergic, non-cholinergic sensory nerves in guinea-pig atria, isolated from reserpine-pretreated animals, in the presence of atropine and the beta-adrenoceptor-blocking drug CGP 20712A, induced a positive inotropic effect. Adenosine (0.1-10 microM) concentration-dependently reduced the cardiac response to transmural nerve stimulation, without modifying the response to exogenous calcitonin gene-related peptide; the inhibitory effect of adenosine was antagonized by 1 microM 8-phenyltheophylline. Moreover, the cardiac response to field stimulation was enhanced by 8-phenyltheophylline (0.1, 1 microM) and by adenosine deaminase (1 microgram/ml), but was reduced by dipyridamole (1 microM). These findings indicate the presence of inhibitory adenosine receptors on cardiac sensory nerves and suggest a modulatory effect of endogenous adenosine on cardiac non-adrenergic, non-cholinergic neurotransmission.