Romina Vargiu

Cardiac effects of trace amines: pharmacological characterization of trace amine-associated receptors

Trace amine-associated receptors, a novel class of G-protein coupled receptors which respond to trace amines but not to classical biogenic amines, have been found to be expressed in heart. Therefore, we investigated the cardiac effects of the trace amines p-tyramine, beta-phenylethylamine, octopamine, and tryptamine. Isolated rat hearts were perfused in the presence of trace amines, monitoring the hemodynamic variables. In addition, radioligand binding experiments with [3H]-p-tyramine and [125I]-3-iodothyronamine were performed in rat ventricular tissue. Octopamine, beta-phenylethylamine, and tryptamine produced a dose-dependent negative inotropic effect as shown by reduced cardiac output (IC(50)=109 microM, 159 microM, and 242 microM, respectively). In the same preparation a similar effect was produced by thyronamine and 3-iodothyronamine, with IC(50)=94 microM and 27 microM, respectively. The negative inotropic effect of octopamine was confirmed in a papillary muscle preparation. All trace amines except tryptamine increased the heart rate, but this action could be attributed to their sympathomimetic properties, since it was abolished by propranolol. The negative inotropic effect of trace amines was significantly increased by the tyrosine kinase inhibitor genistein. Specific and saturable binding of [(3)H]-p-tyramine and [125I]-3-iodothyronamine was observed in ventricular tissue. While [3H]-p-tyramine was displaced by 3-iodothyronamine, [(125)I]-3-iodothyronamine was not displaced by p-tyramine. In conclusion, trace amines and thyronamines are negative inotropic agents. Their effect appears to be mediated by a subtype of trace amine-associated receptor which is characterized by the rank of potency: 3-iodothyronamine > thyronamine = octopamine = beta-phenylethylamine, while tryptamine and p-tyramine are significantly less active.

Inhibition of peristaltic activity by cannabinoids in the isolated distal colon of mouse

The effects of the cannabinoid receptor agonist Win 55,212-2 and of the competitive cannabinoid receptor antagonist SR 141716A on the electrically-evoked peristalsis of isolated distal colon of mouse were studied. Intraluminal pressure, longitudinal displacement, ejected fluid volume and changes in morphology of external intestinal wall were simultaneously recorded in the pre-drug period and in presence of Win 55,212-2 alone or in combination with SR 141716A. In the pre-drug period (control), peristaltic activity was characterised by regular, monophasic waves and the intraluminal content propelled towards anterograde (oro-aboral) direction with a propulsion velocity of 1.25 +/- 0.1 mm x s(-1). Pressure and shortening waves showed a peak amplitude of 2.44 +/- 0.32 kPa and 1.8 +/- 0.72 mm, respectively. The mean amount of fluid volume ejected during each contraction was 80 +/- 12.6 microl. The addition of Win 55,212-2 [10(-7)-10(-4) M] to the organ bath determined a dose-related attenuation of peristaltic activity consequent to the decrease of circular and longitudinal muscle strength. The decrease of contractile activity was followed by dose-dependent decrease of the amount of fluid ejected during peristalsis. The effects of Win 55,212-2 [10(-7)-10(-5) M] were prevented by SR 141716A, indicating the presence of cannabinoid CB1 receptors in the mouse distal colon. SR 141716A alone enhanced both tonic and phasic motor activities in the colonic longitudinal smooth muscle, suggesting that CB1 receptor antagonists could act either through antagonising the effect of endogenous CB1 receptor agonist or by an agonist effect on these receptors. The present results further support the hypothesis that cannabinoids perform a neuromodulatory role in various tracts of gastrointestinal system and first demonstrate their action also in the distal colon of rodents.

Functional role of inducible nitric oxide synthase on mouse colonic motility

A possible functional role of inducible isoform of nitric oxide synthase (iNOS) was explored in vitro on the motility of mouse distal colon. Using an isotonic - non-isovolumic technique, peristaltic activity and video images of the external wall of colonic segments were recorded before and after addition to the medium of Aminoguanidine (AG) and N-(3-(aminomethyl)benzyl) acetamidine (W1400) [10(-7) M-10(-4) M], two iNOS inhibitors. AG and W1400 induced an hyperexcitability of visceral smooth muscle characterised by an increase of basal tone and spontaneous phasic activity. As a consequence of these effects, the peristaltic activity declined and disappeared at the highest concentrations. These findings indicated a removal of inhibitory action performed by NO synthesised by iNOS in the colonic segment. The implications of results are discussed in term of tonic relaxation of intestinal smooth muscle to allow intraluminal content accommodation.

Mechanical properties of smooth muscle portal vein in normal and dystrophin-deficient (mdx) mice

Mechanical properties of the vascular smooth muscle from normal and dystrophin‐deficient (mdx) mice were examined. Changes in resting and developed tensions in response to stretch were recorded in isolated portal vein. The vascular segments were elongated in 5% increments of the ‘in situ’ length (Lr) up to 1·30Lr. The resting length‐tension curves in male mdxmice were similar to normal mice, while a marked decrease in the slope of the curve was noted in female mdx mice. These findings were not affected by atropine, phentolamine, tetrodotoxin or [Ca2+] in the surrounding media. At Lr, the tension of isolated portal vein was characterized by spontaneous synchronized uniform force waves in normal mouse. In contrast, in mdxmouse portal veins an irregular motor pattern characterized by desynchronized force waves with a decrease of amplitude and an increase in frequency was recorded. Extension of the length of the portal vein segment did not increase the spontaneous phasic activity developed in female mdx mice although this was noted with male mdx mice and normal mice. Experiments with chemical depolarizing agents indicated that spontaneous myogenic excitation activated the great majority of vascular smooth muscle cells in normal mouse portal vein, whereas in mdx mice only a reduced number of these cells were excited suggesting that in the mdx mouse the intercellular electronic coupling is altered. In conclusion this study provides the first description of the mechanical activities of portal vein longitudinal muscle and shows that in mdx mice the motor activity is severely disrupted.

Enhancement of shortening velocity, power, and acto-myosin crossbridge (CB) kinetics following long-term treatment with propionyl-L-carnitine, coenzyme Q10, and omega-3 fatty acids in BIO TO-2 cardiomyopathic Syrian hamsters papillary muscle.

Impaired functions of myocardial muscle cells in human and animals, is a primary defect associated with idiopathic dilated cardiomyopathy (DCM). The pathophysiological mechanisms implicated in the DCM are yet to be clarified and an effective therapy is still not available. The BIO TO‐2 cardiomyopathic Syrian Hamsters (CMSHs) represent an animal model of idiopathic DCM. The aim of this study was to investigate the effect of long‐term treatment (2 months) with propionyl‐L‐carnitine (PLC), coenzyme Q10, omega‐3 fatty acids and a combination of these three agents (formulation HS12607) on mechanical properties and acto‐myosin crossbridges (CBs) kinetics of left ventricular (LV) papillary muscle from control and treated 10 month old BIO TO‐2 CMSHs. Isometric and isotonic contractile properties of isolated papillary muscle from control and treated CMSHs were investigated, and acto‐myosin CB number, force and kinetics were calculated using Huxleys equations. Mechanical parameter values were higher in treated than in control hamsters, particularly when substances were administered together in a coformulation (HS12607). Compared to control, HS12607‐treated papillary muscles showed a significant increase of maximum peak isometric tension (Po) (30.06 ± 4.91 vs. 19.74 ± 5.00 mN/mm2), maximum extent of muscle shortening (0.13 ± 0.03 vs. 0.07 ± 0.02 L/Lmax), maximum unloaded shortening velocity (1.18 ± 0.24 vs. 0.53 ± 0.13 L/Lmax s−1) and maximum peak of power output (5.52 ± 1.61 vs. 1.58 ± 0.83). The curvature of the hyperbolic force‐velocity relationships did not differ between control and treated hamsters. When compared to controls, acto‐myosin CB number increased in treated hamsters [(6.67 ± 1.91) 1010/mm2 vs. (3.55 ± 2.08) 1010/mm2], whereas the unitary force of single CB was similar in control and treated animals. The peak value of the rate constant for CB attachment (f1) and detachment (g2) was higher in treated animals when compared to control. (93.87 ± 25.82 vs.47.28 ± 10.88 s−1 and 214.40 ± 44.64 vs. 95.56 ± 23.49 s−1, respectively). In conclusion, the present study illustrates that supplementation with PLC, CoQ10 and omega‐3 fatty acids improved motor parameters, energetic, and CB kinetics of BIO TO‐2 CMSH papillary muscle indicating that these naturally occurring substances may be a valid adjuvant to conventional therapy in DCM.

Enhancement of muscular performance by a coformulation of propionyl-l-carnitine, coenzyme Q10, nicotinamide, riboflavin and pantothenic acid in the rat

A coformulation of essential factors, i.e. propionyl-L-carnitine (PLC), coenzyme Q10 (CoQ10), nicotinamide (NAM), riboflavin and pantothenic acid, was administered orally to Wistar rats for 7 weeks and its efficacy was tested through in vivo and in vitro techniques in improving motor functions of striated, cardiac and smooth musculature of the rat. In vivo experiments showed that long-term supplementation significantly improved horizontal locomotor activity by about 19% in male and 26% in female rats. Maximum values of shortening velocity, work and power were significantly increased (P<.05) in papillary muscle isolated from treated rats. A positive inotropic effect was also observed on colonic smooth muscle strips upon treatment. Work was the most affected parameter and it increased by 160% in smooth muscle from treated animals. The present results indicate that supplementation with the combination of the above mentioned substances elicits positive functional changes on motor performance of skeletal, cardiac and smooth muscle of the rat.

Biphasic effects of NMDA on the motility of the rat portal vein.

The effect of NMDA on the motility of the rat portal vein was studied in an isolated preparation. NMDA induced a concentration‐dependent (10−7–10−4 M) increase of the contraction frequency (maximum increase, 148±6% of control at NMDA 10−4 M). The NMDA‐induced excitatory response was prevented by the competitive NMDA receptor antagonists (±)‐2‐Amino‐5‐phosphonopentanoic acid (AP‐5, 5×10−4 M) or (RS)‐3‐(2‐carboxypiperazine‐4‐yl) propyl‐1‐phosphonic acid (CPP, 10−4 M). Tetrodotoxin (TTX, 10−6 M) or atropine (10−4 M) abolished the NMDA‐induced increase of the portal vein motility and reversed the excitatory effect to a concentration‐dependent inhibition (maximum inhibition, 52±8 and 29±7% of controls, respectively, at NMDA 10−3 M). Removal of the endothelium abolished the NMDA‐induced inhibitory response. Sodium nitroprusside concentration‐dependently (10−7–10−5 M) inhibited the portal vein motility, while L‐NG‐nitro‐arginine methyl ester (L‐NAME, 10−4 M) reversed the inhibitory effect of NMDA (in the presence of TTX), restoring the portal vein spontaneous activity to control values. These results show that NMDA modulates the portal vein motility in a biphasic manner: via indirect activation, through prejunctional NMDA receptors presumably located on intrinsic excitatory neuronal afferences, or via direct inhibition, through endothelial NMDA receptors activating the nitric oxide pathway. Overall these findings support the hypothesis of the existence of a peripheral glutamatergic innervation modulating the contractile activity of the rat portal vein.

Human Ejaculatory Duct: Parameters of Smooth Muscle Motor Activity and Modulatory Role of Autonomic Drugs

The contractile behaviour and effects of several autonomic drugs on the motor activity of human isolated ejaculatory ducts were investigated. Ejaculatory ducts exhibited spontaneous contractions characterised by an amplitude of 2.35 ± 0.28 mN, a duration of 62.9 ± 3.72 s and a frequency of 0.64 ± 0.014 waves min‐1. Acetylcholine (10‐5‐10‐4 m) induced a slight increase in basal tone and in the frequency of the contraction waves. These effects were suppressed by atropine (10‐4 m). Noradrenaline (norepinephrine) increased the basal tone and frequency of spontaneous contractions in a dose‐dependent manner. These responses were competitively inhibited by HEAT, a selective a1‐adrenoceptor antagonist. These preliminary functional findings, indicating the presence of spontaneous motor activity of human ejaculatory ducts and its possible control by adrenergic agonists, suggests a physiological role for human ejaculatory duct in the propulsion of semen from the seminal vesicle towards the urethra.

Origin of motion in the human ureter: mechanics, energetics and kinetics of the myosin molecular motors

Background Ureteral peristalsis is the result of coordinated mechanical motor performance of longitudinal and circular smooth muscle layer of the ureter wall. The main aim of this study was to characterize in smooth muscle of proximal segments of human ureter, the mechanical properties at level of muscle tissue and at level of myosin molecular motors. Methods Ureteral samples were collected from 15 patients, who underwent nephrectomy for renal cancer. Smooth muscle strips longitudinally and circularly oriented from proximal segments of human ureter were used for the in vitro experiments. Mechanical indices including the maximum unloaded shortening velocity (Vmax), and the maximum isometric tension (P0) normalized per cross-sectional area, were determined in vitro determined in electrically evoked contractions of longitudinal and circular smooth muscle strips. Myosin cross-bridge (CB) number per mm2 (Ψ) the elementary force per single CB (Ψ) and kinetic parameters were calculated in muscle strips, using Huxleys equations adapted to nonsarcomeric muscles. Results Longitudinal smooth muscle strips exhibited a significantly (p<0.05) faster Vmax (63%) and a higher P0 (40%), if compared to circular strips. Moreover, longitudinal muscle strips showed a significantly higher unitary force (Ψ) per CB. However, no significant differences were observed in CB number, the attachment (f1) and the detachment (g2) rate constants between longitudinal and circular muscle strips. Conclusions The main result obtained in the present work documents that the mechanical, energetic and unitary forces per CB of longitudinal layer of proximal ureter are better compared to the circular one; these preliminary findings suggested, unlike intestinal smooth muscle, a major role of longitudinal smooth muscle layer in the ureter peristalsis.

Cardiovascular remodelling in female diabetic rats

Diabetic cardiomyopathy involves both cardiac and large vessels alterations in their biochemical and biomechanical properties. Part of these dysfunctions is due to ROS overproduction and advanced glycated end-products (AGEs) synthesis caused by high blood glucose concentrations (1). Epidemiological studies usually ignore sexgender outcomes of diabetes that has higher cardiovascular risk in women than in men (2). The aim of the present study was to assess the effects of diabetes on aorta, portal vein and myocardium morphology in females Wistar rats. Diabetes was induced by a single dose of streptozotocin 65 mg/kg, and, after 4 and half months, we evaluated the cardiovascular remodelling by light and transmission electron microscopy (TEM). Paraformaldehyde fixed samples of aorta and portal vein were stained with Masson Trichrome method (for collagen fibers), Weigert’s stain (for elastic fibers), Hematoxylin and Eosin (for nuclei), and underwent to morphometric analysis. TEM samples were prepared accordingly to common protocols. Morphometric analysis performed on diabetic aortas showed a reduction of tunica media thickness, but the internal diameter width or the lumen cross-area was unchanged compared to controls. The number of smooth muscle cells increased in tunica media of diabetic aortas. The main change observed in diabetic portal veins was a reduction of the area occupied by elastic fibers in tunica adventitia. TEM observations of papillary muscles did not reveal any changes in the sarcomere lengths across the two experimental groups. These results display slight differences on what was reported in male rats (3) and account for a different development of diabetes in female subjects.