Effect of melatonin on neurogenic vasoreactivity: formation and modulation of the vessel response

The effect of melatonin (0.1 microM) on the contractile response to electrical field stimulation (EFS) of the juvenile rat tail artery segment was studied. The spontaneous decrease in reactivity of the segment observed in the course of our experiments was accompanied with a melatonin-evoked increase in the reactivity which was proportional to this decrease and was not connected with a sensitization of the segment to the substance. Perfusion of the segment with an acidic solution leads to a more pronounced inhibition of the response as well as to a greater melatonin-evoked protentiation of the response. Noradrenaline-evoked response was not augmented by melatonin. The results suggest that the potentiating effect of melatonin on the EFS-evoked response of the juvenile rat tail artery depends on degree of the change in the artery reactivity and was not due to change in sensitivity of postjunctional membrane to noradrenaline.     

Effects of exogenic noradrenaline and melatonin on the neurogenic vasoreactivity

The effect of 0.1 microM noradrenaline and melatonin on the response to electrical field stimulation (EFS) of the juvenile rat artery segment was studied. Noradrenaline like melatonin was shown to potentiate the EFS-evoked constriction decreased in the course of experiments or in the acidic solution (pH 6.6), and this potentiation was proportional to the extent of the preceding decrease of the constriction. The effect of these substances was additive. The results suggest that noradrenaline as well as melatonin can serve as a means to restore the diminished neurogenic reactivity of blood vessels.     

Restoring effect of noradrenalne on diminished neurogenic vasoreactivity

The effect of 0.01-1.0 microM noradrenali on response to electrical field stimulation (EFS) of the juvenile rat tail artery segment was studied. Noradrenali was shown to potentiate the EFS-evoked constriction decreased in the course of experiments or in the acidic solution (pH 6.6) and this potentiation was proportional to the extent of the preceding decrease of the constriction. The more decreased was the EFS-evoked constriction the higher was the noradrenali concentration which produced the maximal potentiation and the wider was the potentiative noradrenali concentration range. The potentiative effect of noradrenali was not prevented by the NO synthase inhibitor NG-nitro-L-arginine. The results suggest that noradrenali can restore the diminished neurogenic reactivity of blood vessels, and this effect is not connected with the change in the NO production.     

The effect of noradrenaline on the neurogenic vasoreactivity at various frequencies of the electrical field stimulation

The effect of 0.03-10.0 microM noradrenaline on the response to electrical field stimulation (EFS) of the juvenile rat tail artery segment was studied. At frequencies of the EFS equal to 10 or 40 Hz, noradrenaline was shown to cause much more pronounced potentiation or--at higher concentration--much less pronounced inhibition of the EFS-evoked constriction in arteries characterized by spontaneous decrease in the constriction value in the course of experiments as compared with arteries which were not characterized by such a decrease. At frequencies of the EFS equal to 3 or 5 Hz, the value and/or direction of the change in the neurogenic vasoconstriction in the presence of noradrenaline depends on the presence of the spontaneous decrease in the constriction evoked by EFS at 10 Hz, rather than at 3 or 5 Hz. It is concluded that the character of the change in the neurogenic vasoreactivity is a factor of a great importance for the prediction of the further change in the reactivity in the presence of noradrenaline.     

Modulation of [H]noradrenaline release by endothelin-1 in the rat tail artery

The effect of endothelin-1 (ET-1) on the increase in perfusion pressure and the release of noradrenaline produced by electrical field stimulation were examined in isolated perfused/superfused rat tail arteries. ET-1 (1–30 nM) increased, in an identical concentration-dependent manner, the basal perfusion pressure and the stimulation-evoked tritum overflow, whereas the basal outflow of noradrenaline was not changed by the peptide. These results show that, besides its postjunctional vasoconstrictor effect, ET-1 exerts in the rat tail artery a prejunctional action which might be involved in the modulation of stimulation-evoked noradrenaline release from postganglionic nerves.     

Effects of extracellular pH on the response of the isolated rat mesenteric artery to electrical field stimulation

In experiments on isolated segments of the rat mesenteric artery, effects of changes in solution pH on the response of the segments to noradrenaline (10 microM) or electrical field stimulation (EFS) were studied. The pH 7.8 solution slightly increased (from 0.48 +/- 0.07 mN at pH 7.4 to 0.67 +/- 0.12 mN or by 41%). while the pH 7.0 and 6.6 solutions significantly decreased (to 0.16 +/- 0.05 and 0.08 +/- 0.04 mN or by 66 and 83%, respectively) the EFS-evoked response of the vessel prestretched to the value corresponding to the intravascular pressure of about 100 mm Hg. A pH shift either to the alkaline or acidic range did not change the resting tension (15.65 +/- 0.74 mN at pH 7.4) of the vessel without precontraction. The pH 6.6 solution reduced the response to noradrenaline twofold. Dilation produced by EFS of noradrenaline-precontracted segment was inhibited and the constrictor responses appeared in the pH 6.6 solution. In the vessel pretreated with N(G)-nitro-L-arginine (100 microM), the acidification of the solution (pH 6.6) inhibited the response of the vascular segment to EFS to a lower extent and did not change its response to noradrenaline. The data obtained demonstrate an inhibitory effect of acidosis on reactivity of the rat mesenteric artery as well as a modification of this effect under a high initial tone of the vessel studied.     

The neurogenic constrictor response of isolated rat saphenous artery is reduced after 2-week tail suspension

Under real or simulated microgravity conditions the control of arterial vascular tone is greatly disturbed. The low arterial vessel reactivity to sympathetic influences may be the cause of an increase in flow in hind limb skeletal muscles in tail-suspended (TS) rats. Our previous experiments with constant pressure perfusion of rat hind limb demonstrated the reduced vasoconstrictor responses to sympathetic nerve stimulation in TS rats. Responses to exogenous noradrenaline depended on the perfusion conditions. It is known that the vessels of various branching orders noticeably differ in nerve density and in sensitivity to vasoconstrictor agonists. So under neurogenic or exogenous noradrenaline influences the vascular resistance may be increased at different levels of vascular bed, thus making the data analysis seriously complicated. This uncertainty may be overcome by investigation of a single vessel isolated from hind limb vascular bed. The saphenous artery, a resistance artery with dense innervation, is a very convenient object for this purpose. Thus, this study was aimed at comparing the effects of 2-week tail suspension upon the constrictor responses of isolated saphenous artery to neurogenic and exogenous noradrenaline stimuli in rats.     

Inactivation of G(i) proteins by pertussis toxin diminishes the effectiveness of adrenergic stimuli in conduit arteries from spontaneously hypertensive rats

Treatment with pertussis toxin (PTX) which eliminates the activity of G(i) proteins effectively reduces blood pressure (BP) and vascular resistance in spontaneously hypertensive rats (SHR). In this study we have compared the functional characteristics of isolated arteries from SHR with and without PTX-treatment (10 microg/kg i.v., 48 h before the experiment). Rings of thoracic aorta, superior mesenteric artery and main pulmonary artery were studied under isometric conditions to measure the reactivity of these vessels to receptor agonists and to transmural electrical stimuli. We have found that the treatment of SHR with PTX had no effect on endothelium-dependent relaxation of thoracic aorta induced by acetylcholine. In PTX-treated SHR, the maximum contraction of mesenteric artery to exogenous noradrenaline was reduced and the dose-response curve to cumulative concentration of noradrenaline was shifted to the right. Similarly, a reduction in the magnitude of neurogenic contractions elicited by electrical stimulation of perivascular nerves was observed in the mesenteric artery from PTX-treated SHR. PTX treatment of SHR also abolished the potentiating effect of angiotensin II on neurogenic contractions of the main pulmonary artery. These results indicate that PTX treatment markedly diminishes the effectiveness of adrenergic stimuli in vasculature of SHR. This could importantly affect BP regulation in genetic hypertension.     

Fast and Local Electrochemical Monitoring of Noradrenaline Release from Sympathetic Terminals in Isolated Rat Tail Artery

Abstract: Noradrenaline release from sympathetic nerve terminals was evoked by electrical nerve stimulation of an isolated segment of rat tail artery. This release was recorded by a carbon fiber electrode combined with differential pulse amperometry. The active part of the electrode (one carbon fiber 8 μm in diameter and 50 μm in length) was placed in close contact with the arterial surface. The oxidation current appearing at +120 mV and corresponding to the local noradrenaline concentration at the electrode surface was recorded every 0.5 s. No oxidation current was detected under resting conditions, but electrical stimulation evoked an immediate increase in this current. This response was suppressed when tetrodotoxin was added to the perfusion medium and was enhanced when noradrenaline reuptake was inhibited by cocaine. The amplitude of the response was increased with increasing stimulation frequencies (2–25 Hz) and train lengths (1–16 pulses). Finally, the time resolution of the method (0.5 s) was good enough to show that noradrenaline release precedes the postsynaptic response, i.e., the electrically evoked contraction of the artery.     

Aging impairs neurogenic contraction in guinea pig urinary bladder: role of oxidative stress and melatonin

The incidence of urinary bladder disturbances increases with age, and free radical accumulation has been proposed as a causal factor. Here we investigated the association between changes in bladder neuromuscular function and oxidative stress in aging and the possible benefits of melatonin treatment. Neuromuscular function was assessed by electrical field stimulation (EFS) of isolated guinea pig detrusor strips from adult and aged female guinea pigs. A group of adult and aged animals were treated with 2.5 mg x kg(-1) x day(-1) melatonin for 28 days. Neurotransmitter blockers were used to dissect pharmacologically the EFS-elicited contractile response. EFS induced a neurogenic and frequency-dependent contraction that was impaired by aging. This impairment is in part related to a decrease in detrusor myogenic contractility. Age also decreased the sensitivity of the contraction to pharmacological blockade of purinergic and sensitive fibers but increased the effect of blockade of nitrergic and adrenergic nerves. The density of cholinergic and nitrergic nerves remained unaltered, but aging modified afferent fibers. These changes were associated with an increased level of markers for oxidative stress. Melatonin treatment normalized oxidative levels and counteracted the aging-associated changes in bladder neuromuscular function. In conclusion, these results show that aging modifies neurogenic contraction and the functional profile of the urinary bladder plexus and simultaneously increases the oxidative damage to the organ. Melatonin reduces oxidative stress and improves the age-induced changes in bladder neuromuscular function, which could be of importance in reducing the impact of age-related bladder disorders.     

Blood vessels of the rat tail: a histological re-examination with respect to blood vessel puncture methods

The rat tail vascularization is histologically re-examined especially with respect to blood sampling and vascular-injection methods. The terminal third of the tail is recommended for blood vessel puncturing. In this segment, the arteries and veins are most prominent, since the structures of the musculo-skeletal system diminish towards the tip of the tail. In addition to the commonly-known blood vessels (one ventral artery, two lateral veins), there is a dorsal vein in the rat tail that is well suited for puncture and cannulation.     

Neurogenic contractions of the rat tail artery under isobaric conditions: effect of transmural pressure and function of the endothelium]

In stimulation of the rat nerve with a modulated sine pattern, an increase in the modulating frequency from 0.03 to 0.15 Hz diminished the latency between the stimulating signals and changes in the vessel resistance as well as the amplitude of the flow oscillations, but did not affect tonic contractions of the vessel. A reduction of transmural pressure from 80 to 40 mm Hg increased both the tonic and the phasic components of the vessel contraction. Following the endothelium removal no change in the response latency occurred. The data obtained suggest that, during a rhythmic neurogenic influence, the vascular endothelium may work as an "amplifier" of the vessel's phasic contractions.     

Inhibitory effect of hemicholinium-3 on presynaptic nicotinic acetylcholine receptors located on the terminal region of myenteric motoneurons

Previously we have demonstrated the presence of presynaptic nicotinic acetylcholine receptors on the terminals of myenteric neurons in Auerbach's plexus of guinea-pig ileum. During these studies we observed, that the presence of hemicholinium-3, an inhibitor of the high affinity choline uptake significantly influences the contraction of the longitudinal muscle strip preparation. Our aim was to investigate the neurochemical background of this effect and quantitatively characterize the action of HC-3. We studied the effect of HC-3 on epibatidine- and electrical stimulation-evoked contraction and release of [3H]acetylcholine from the guinea-pig longitudinal muscle strip preparation. We found that in the presence of tetrodotoxin, when the contribution of somatodendritic nicotinic acetylcholine receptors to the response was prevented due to the inhibition of axonal conduction, HC-3 inhibited the epibatidine-evoked contraction and [3H]acetylcholine release in the submicromolar range (IC50 = 897 nM and IC50 = 693 nM, respectively), whereas the electrical stimulation-evoked contraction was not affected by HC-3, and the release of [3H]acetylcholine was apparently enhanced. Our data indicate that HC-3 inhibits the presynaptic nicotinic acetylcholine receptors of myenteric neurons. Since these receptors play an important role in the regulation of cholinergic neurotransmission in the enteric nervous system, the use of HC-3 in [3H]acetylcholine release experiments might bias the interpretation of data.     

Hyperthermia-induced vasoconstriction of the carotid artery, a possible causative factor of heatstroke.

Clinical and experimental studies indicate that hyperthermia can cause heatstroke with cerebral ischemia and brain damage. However, no study has examined the direct effects of heating carotid artery smooth muscle and tested the hypothesis that hyperthermia induces arterial vasoconstriction and, thereby, decreases cerebral blood flow. We recorded isometric tension of rabbit carotid artery strips in organ baths during stepwise temperature elevation. The heating responses were tested at basal tone, in norepinephrine- and KCl-precontracted vessels, and after electrical field stimulation. Stepwise heating from 37 degrees C to 47 degrees C induced reproducible graded contraction proportional to temperature. The responses could be elicited at basal tone and in precontracted vessels. Heating decreased the contractile responses to norepinephrine and electrical field stimulation but increased contraction to KCl. These responses were not eliminated by pretreatment with the neuronal blocker tetrodotoxin. Our results demonstrate that heating carotid artery preparations above 37 degrees C (normothermia) induced a reversible graded vasoconstriction proportional to temperature. In vivo this reaction may lead to a decrease in cerebral blood flow and cerebral ischemia with brain damage as in heatstroke. The heating-induced contraction is not mediated by a neurogenic process but is due to altered transcellular Ca2+ transport. Cooling, in particular of the neck area, therefore, should be used in the treatment of heatstroke.     

褪黑素改善内毒素血症大鼠血管反应性

观察褪黑素(melatonin,MT)对脂多糖(lipopolysaccharide,LPS)诱导的体循环和肺循环血管反应性失调的影响,并探讨可能的作用机制。实验分为溶剂对照组、LPS组、LPS+MT组和MT组。制备离体胸主动脉环和肺动脉环,应用血管张力检测技术检测各组血管环对苯肾上腺素(phenylephrine,PE)和乙酰胆碱(acetylcholine,ACh)的反应性并绘制累积剂量反应曲线;制备各组血管组织匀浆,测定丙二醛(malondialhyde,MDA)和超氧化物歧化酶(superoxidedismutes,SOD)含量变化。结果显示:与对照组相比,LPS6h后胸主动脉对PE的收缩反应减弱(P<0.01),对PE(1×10–8~1×10–5mol/L)累积剂量反应曲线下移;而肺动脉对ACh的舒张反应显著下降(P<0.01),对ACh(1×10–8~1×10–5mol/L)累积剂量反应曲线下移。加用MT可显著改善LPS诱导的胸主动脉对缩血管剂PE的低反应性,同时可逆转LPS对肺动脉舒张反应的抑制,LPS+MT组胸主动脉对PE的累积剂量反应曲线和肺动脉对ACh的累积剂量反应曲线位于对照组和LPS组之间;MT还可对抗LPS导致的脂质过氧化,使MDA含量减少,提高抗氧化酶SOD的活性。上述结果提示,MT可改善内毒素血症大鼠的血管反应性失调,抗氧化途径可能是其发挥保护作用的机制之一。     

The effect of inhibition of endothelial cell cyclooxygenase on arterial thrombosis

Arterial platelet thrombosis was induced in vivo in a branch of the mesenteric artery of the white Wistar rat by topical superfusion by adenosine diphosphate following local de-endothelialization of the dissected segment by means of a small electrical current. Detection of the thrombotic phenomena was performed by projection of the arterial segment onto a set of light sensitive elements, which allows the registration of several discriminating parameters. The addition of arachidonic acid to the superfusing mixture results in an increase in thromboformation; this increasing effect can be blocked completely by prior inhibition of the vessel wall cyclooxygenase activity by inhibitors such as flurbiprofen or indomethacin. We therefore concluded that the arachidonate cascade, triggered within the endothelial cells, is involved in platelet-vessel wall interaction leading ultimately to local thrombosis.     

Dependence of the elastic characteristics and contractile activity of the rat tail artery on the transmural difference of potentials

Change in the diffuse portion of the double electric layer at the blood-vascular wall border under the effect of thorium nitric oxide and heparin affects the tone and contractile ability of the rat tail artery: thorium diminishes the vessel rigidity and the amplitude of its contractile response, whereas heparin augments the rigidity as well as the amplitude of the response. Thorium nitric oxide also diminishes rigidity of the vessel connective tissue skeleton. The difference of potentials between the vessel lumen and its wall seems to be capable of affecting the contractile apparatus of the smooth muscle cells by means of activation of potential-sensitive ionic channels.     

The effect of inhibition of endothelial cell cyclooxygenase on arterial thrombosis

Arterial platelet thrombosis was induced in vivo in a branch of the mesenteric artery of the white Wistar rat by topical superfusion by adenosine diphosphate following local de-endothelialization of the dissected segment by means of a small electrical current. Detection of the thrombotic phenomena was performed by projection of the arterial segment onto a set of light sensitive elements, wich allows the registration of several discriminating parameters. The addition of arachidonic acid to the superfusing mixture results in an increase in thromboformation; this increasing effect can be blocked completely by prior inhibition od the vessel wall cyclooxygenase activity by inhibitors such as flurbiprofen or indomethacin. We therefore concluded that the arachidonate cascade, triggered within the endothelial cells, is involved in platelet-vessel wall interaction leading ultimately to local thrombosis.     

Peroxynitrite inhibits relaxation and induces pulmonary artery muscle contraction in the newborn rat

Nitric oxide (NO) reacts with superoxide anion to form the peroxynitrite anion (ONOO-), a molecule with pulmonary vasodilator properties in the adult rat. The purpose of this study was to compare the effects of ONOO- on intrapulmonary arteries from the newborn (days 4-7), juvenile (day 14), and adult rat. Following thromboxane A2 (TXA2) analogue (U46619) prestimulation, newborn vessels were more sensitive to ONOO- -induced muscle contraction, compared to both the juvenile and the adult vessels. Peroxynitrite-induced contraction in newborn vessels was abrogated by ibuprofen, an endothelin B-receptor blocker (A-192621), or a rho-kinase-specific inhibitor (Y27632) (all p < 0.01). Following KCl stimulation and TXA2 receptor blockade, ONOO- induced NO-dependent muscle relaxation in newborn vessels via stimulation of the endothelial and inducible nitric oxide synthases. However, in the presence of ONOO-, the pulmonary artery relaxation response to endothelium-dependent stimulation was significantly reduced (p < 0.01). Finally, newborn but not adult pulmonary arteries exposed to ONOO- showed a 10-fold increase in 8-isoprostane production, a possible mediator of ONOO- -induced contraction. We conclude that exposure to ONOO- results in a unique response in newborn intrapulmonary arteries characterized by increased 8-isoprostane generation, which we believe is responsible for its vasoconstrictor effect. This unique response potentially renders the newborn more susceptible to ONOO- -induced pulmonary hypertension than older animals.     

Rho kinase inhibition partly weakens myogenic reactivity in rat small arteries by changing calcium sensitivity

The hypothesis that Rho kinase is involved in myogenic reactivity was investigated in pressurized rat tail small arteries using videomicroscopic diameter determination and calcium fluorimetry. The potent Rho kinase inhibitor Y-27632 reversibly increased vessel diameter at 80 mmHg without changing the intracellular calcium concentration ([Ca](i)) shifting the relationship between diameter change and [Ca](i) to higher calcium levels. Neither endothelium removal nor inhibition of neural transmission affected the Y-27632-induced effect. Y-27632 at 3 x 10(-6) mol/l attenuated the myogenic response in the pressure range from 10 to 120 mmHg, shifting the relationship between vessel tone and [Ca](i) to higher calcium levels. In addition, the Y-27632-induced shift of the relationship between vessel tone and [Ca](i) was larger at 80 than at 10 mmHg. These results suggest that smooth muscle cell Rho kinase in rat tail small arteries 1) is in an active state partly determining the level of the myogenic tone, and 2) alters the strength of the myogenic response by changing calcium sensitivity, probably caused by the pressure-induced activation of the kinase.