DIFFERENT MODULATORY EFFECTS OF AMMONIUM IONS ON ANGIOTENSIN VASCULAR ACTIONS IN ISOLATED RAT AORTIC AND RENAL ARTERIES

In the present study, we were interested in the vascular effects of angiotensin II on perfused rings of the rat thoracic aorta and renal artery. Our results demonstrated different modulator alterations of these preparations induced by ammonium ions. Unlike the aortic rings, which exhibited only a reduction of angiotensin-induced contractility by NH4Cl, the renal artery preparations showed both activation of vasoconstriction and inhibition of vasorelaxation in the ring precontracted with phenylephrine or noradrenalin. These results are interpreted as a modulation by the ammonium ions of vascular reactions induced by the stimulation of the vasoconstrictor AT1 receptor on the one side and AT2 vasodilator receptors on the other. The potentiation of renal vasoconstriction accompanied by the reduction of angiotensin vasodilation by NH4Cl suggests the possibility of involvement from the blood flow and renal vascular tonus disturbances induced by ammonium ions during hyperammonemia of various causes.


INTRODUCTION
Ammonia (NH 3 ) is the simplest nonionic molecule of any nitrogenous compound that has an essential role in many biological processes.Being a weak base, NH 3 is formed by the natural deamination of endogenous molecules and occurs in micro-molecular concentrations in human blood as result of its diffusion across the cell membranes.As part of the nitrogen cycle, NH 3 fulfils the role of a component for amino acids, nucleotide and protein synthesis.Having many endogenous and exogenous origins, most of the NH 3 is detoxified in mammals, mainly by its conversion to urea in the liver with participation of the five urea cycle enzymes (Visek, 1984).
In the renal tubular cells, NH 3 is protonated and converted to ionic form (NH 4 + ) by reaction with se-creted H + ions.The resulting ammonium ion rises within cells by trapping H + because of the more acid intracellular environment and highly permeable membranes.Under such circumstances, NH 3 equilibrates between the extra-and intracellular spaces, inducing a large variety of biochemical and neuropathological alterations.
In the biological fluids, NH 3 exists as such or predominantly as ammonium ions (NH 4 + ), depending on the pH of solutions.The effects of NH 3 /NH 4 + in biological systems are as irritants, and they are responsible for some of the toxic actions that interface with energy metabolism, particularly in the brain.Elevated blood NH 4 + /NH 3 has been linked to hepatic coma, decreased neural excitability, epileptic seizures, convulsions induced by high-pressure oxygen breathing and fatigue states (Mutch and Banister, 1983).Ammonium ions inhibit synaptic transmission by postsynaptic action, producing an inhibitory effect on glutamate-induced firing (Fan et al., 1990).Chronic hyperammonemia intensifies neuronal apoptosis and increases the serotonin turnover inducing the alterations of sleep patterns seen in hepatic encephalopathy (Szerb and Butterworth, 1992).
Several electrophysiological and biochemical mechanisms have been proposed to explain the deleterious effects of NH 4 + /NH 3 on central nervous system (CNS) functions, beginning with normal processes of uptake, storage or release of neurotransmitters, and finishing with the disruption of neuronastrocyte trafficking of amino acids or neuroamines in the brain (Butterworth, 1998).
Acute ammonia toxicity is mediated through an increased accumulation of nitric oxide which combines with free radicals to form a highly toxic compound, peroxynitrite (Konopacka, 2006).A hydrogen bond formed between the superoxide anion and ammonium ion accelerates electron transfer from the radical anion to oxygen.The production of reactive nitrogen intermediates protein tyrosine nitration alters astrocyte function and contributes to ammonia neurotoxicity (Schliess et al., 2002).
Hyperammonemia exerts also a calcium channel blocking action that enhances the effects of central nervous system depression and of certain opioid analgesics (Kuta et al., 1984).
A number of investigators have reported the influences of hyperammonemia on hormones of metabolisms.A decreased glucose tolerance and an inhibitory action on the pancreatic beta cells were found (Barej and Harmeyer, 1979).Experiments on dairy cattle provided evidence that the increased production of ammonia reduced fertility and reproduction by lowering the plasma LH, progesterone and steroid hormone (Jordan and Swanson, 1979).
The relationship between hyperammonemia and the hormonal renin-angiotensin-system has been predominantly studied at renal level.NH 4 Cl admin-istration is associated with metabolic acidosis and with the increased plasma renin-angiotensin and aldosterone secretion (Schambelan et al., 1987, Györke et al., 1991).Infusion of NH 4 Cl for 60 min suppressed plasma renin activity (Kisch et al., 1976).
While angiotensin II (Ang II) has potent effects on renal ammonia production and secretion rates by proximal tubule segments (Chobanian and Julin, 1991), potentiated by NH 4 Cl acidosis (Nagami et al., 2004), the effect of ammonium ions on the vascular smooth muscle in general and the renal artery in particular, are still under investigations.Using helical strips of isolated rabbit aortas, Furtado et al. (1987) have signaled that a brief exposure to NH 4 Cl did not affect the resting aortic tension but modified the responsiveness of the precontracted strips induced by norepinephrine.Ammonium ions cause relaxation of isolated large canine arteries, independent of the presence of the endothelium (Feletou et al., 1989), and amplify the K + -induced contraction of the rat aorta by facilitating transmembrane Ca ++ influx (Tanaka et al., 1996).On the other hand, the vascular tone and spontaneous twitch contractions of rat portal isolated vein were augmented by ammonium chloride and abolished in calcium-free solution or in the presence of 1µM nifedipine (Wakabayashi et al., 1992).
The influence of ammonium ions on the renal vascular actions of angiotensin peptides have been studied less.The aim of the present work was to determine their comparative actions on the contractility of rat aorta and renal arteries induced by angiotensin II.

MATERIALS AND METHODS
The subjects (n=24) were experimentally naive, male Wistar rats, weighing approximately 200-250 g at the beginning of the experiment.The animals were housed in a temperature-and light-controlled room (at 22°C and a 12-h cycle starting at 08:00 h), and were fed and allowed to drink water ad libitum.Rats were treated in accordance with the guidelines of animal bioethics from the Act on Animal Experimen-tation and Animal Health and Welfare Act of Romania, and all procedures complied with the European Communities Council Directive of 24 November 1986 (86/609/EEC) (Hogas et al., 2011, Hritcu et al., 2011).This study was approved by the local Ethics Committee and efforts were made to minimize animal suffering and to reduce the number of animals used.
The renal arteries were collected from extrarenal and intrarenal territories using the same technical procedure.The rings were stretched with 2 g of preload and left to balance for 60 min before the beginning of the experiment.The presence of the functional endothelium was checked pharmacologically with carbachol (10 -6 M) administration, which releases the relaxant endothelial NO on the precontracted rings with phenylephrine (10 -6 M).The isometric tension of the vascular smooth muscle was continuously recorded with a force transducer (Experimetria, Budapest, Hungary) connected to a computer data acquisition system.The reactivity of the preparations was tested by electric and pharmacological stimulation using angiotensin II (10 -7 -10 -5 M), phenylephrine (10 -7 -10 -6 M) and noradrenaline (10 -7 M).In order to avoid tachyphylaxis, the administration of Ang II was repeatedly washed-out and readministered only after more than 60 min.The vascular reactivity to Ang II was examined on resting rings and rings that were precontracted with phenylephrine or noradrenaline.
Electrical field stimulation (EFS) was produced using an Exp-MCU stimulator (Experimetria, Budapest, Hungary) with a wave pulse of 10 volts and duration of 0.5 msec at frequency ranging for 4-20 Hz in increments of 4 Hz.After equilibration, a fre-quency response curve was performed as control for each administration.The vascular effects of the ammonium ions on the electrical and pharmacological stimulation were recorded before and after short exposure (10 min) of the preparations to NH 4 Cl in various doses (5-50µM).
Data acquisition was made using an analogdigital convertor (National Instruments NiDaq 12DD713, USA).
The experiments were performed in series, and the results are presented as representative individual traces, tables and percentile graphs.

Data analysis
The changes in contractility on the vascular smooth muscle were statistically analyzed using one-way analysis of variance (one-way ANOVA).The results were expressed as the mean ± SEM.Post hoc analyses were performed using Tukey's honestly significant difference test in order to compare the different doses of NH 4 Cl + Ang II used.F values for which P<0.05 were regarded as statistically significant (Padurariu et al., 2011).

RESULTS
In the first series of experiments, the reactivity of rings from the thoracic aorta and renal artery, both normal and without endothelium, was tested.The stimulation was performed with either EFS or pharmacologically, using phenylephrine, noradrenaline and angiotensin II.The vasoconstrictor reactions were more intense for the renal arteries, accompanied by slow spontaneous variations of the basal vascular tonus.The enhanced responses were observed both for electrical and pharmacological stimulations.
Unlike the vascular reactions of the thoracic aorta, which were exclusively vasoconstrictor indifferent to the nature of the stimulus, the renal artery demonstrated biphasic responses for angiotensin II.Besides its well-known vasoconstrictive action angiotensin II induced a relaxation in the rings of renal artery which were precontracted with phenylephrine or noradrenalin.
The reactivity of renal and aortic rings was studied comparatively under the influence of short time exposure to ammonium ions.The preparations, incubated for 10 min with NH 4 Cl in variable amounts (0.5 -50 µM), demonstrated different reactions in the renal arteries for angiotensin II.While the vasoconstriction of the aortic rings induced by angiotensin II was strongly inhibited by NH 4 Cl, the reactivity of the renal artery preparations increased significantly in the presence of ammonium ions (Fig. 1).
Additionally, regarding the renal artery, we demonstrated a significant increase of the contraction process in the NH 4 Cl 5 μm + Ang II group (F(1.10)=7,p<0.02), as well as the NH 4 Cl 25 μm + Ang II group (F(1.10)=7,p<0.02) and NH 4 Cl 50 μm + Ang II group (F(1.10)=117,p<0.0001), in comparison with the control Ang II group (Fig. 2B).However, in this case the post-hoc analysis showed significant differ-  The vasodilating effects produced by angiotensin II on rings that were precontracted with phenylephrine or noradrenalin were significantly inhibited by NH 4 Cl and intensified on the precontracted ring (Fig. 3).The pH of the solution was kept within a variation range of 7.38 -7.42.

DISCUSSION
Our results are in agreement with previous literature data (Furtado et al., 1987, Feletou et al., 1989, Tanaka et al., 1996).They offer an important contribution to the study of the different modulated properties of ammonium ions within the thoracic aorta and renal artery of the rat.The mechanisms through which these effects might occur seems to be very complex, beginning with the reactivity of the specific receptors and the accompanying electro-chemical membrane reactions, and ending with cellular metabolic reactions.
Even if both vascular structures are conductance vessels, with different densities and sensitivities of receptor formations, the alpha-adrenergic receptors tested with phenylephrine or noradrenalin demonstrated no significant changes of reactivity in the presence of ammonium ions, while angiotensin receptors were largely influenced in their presence, mainly in what concerns the renal artery rings.
The comparative study of the effects of NH 4 Cl on the contractility of aortic or renal artery rings in the presence of angiotensin II demonstrated the inhibition of its vasoconstrictive action in the thoracic aorta and a significant potentiation of the contractility of the renal arteries.Most of the previous studies have demonstrated that the vascular effects of angiotensin II are mainly constrictive due to the stimulation of AT 1 receptors and protein kinase C, the activation of IP 3 and cytosolic calcium ion release, accompanied by detectable vasodilating properties (Timmermans et al., 1993).The vasodilating actions of angiotensin II rely on the stimulation of the AT 2 receptors (Toda and Miyazaki, 1981) that activate the synthesis and release of NO-cGMP, prostaglandins and plasma kinins (Israel et al., 2000, Katada andMajima, 2002).While AT 1 receptors interact with G 5 proteins induc- ing phosphorylation of tyrosine kinase and activation of nitrogen-activated protein kinase (Touyz and Berry, 2002), AT 2 receptors are involved through G i proteins in the activation of dephosphorylating phosphatases (Nouet and Nahmias, 2000).As the vasorelaxation induced by angiotensin II was obvious in our experiments only in the renal vessels precontracted with phenylephrine or noradrenalin, this has also allowed the demonstration of the modulator properties of NH 4 Cl on the AT 2 receptors in the renal artery.Unlike the vasoconstriction that was potentiated by NH 4 Cl, its vasodilating effects, previously indicated by Tirapelli et al. (2006) in the isolated rat carotid and Grbovic et al. (2008) in the isolated renal artery, were strongly inhibited in the presence of the ammonium ions.
The functional significance of these changes of renal vascular reactivity by the excess ammonium ions is insufficiently established.The potentiation of the constriction of the renal artery produced by an increased reactivity of the AT 1 receptors, accompanied by the inhibition of the relaxation induced by the AT 2 receptors, might contribute to the faulty renal blood flow in hyperammonemia of various causes.
The above findings provide further evidence for different modulated properties of ammonium ions in the thoracic aorta and renal artery in the rat.Although promising, these studies require further replication and new investigations using more advanced techniques and methods.

Fig. 1 .
Fig. 1.Influence of ammonium ions on vasoconstrictor effects of angiotensin II on rat thoracic aorta (A) and renal artery (B).

Fig. 3 .
Fig. 3. Different modulatory properties of NH4Cl upon the vascular effects of angiotensin II in the rat isolated renal artery (A) and thoracic aorta (B).