Nitroso-sulfide coupled signaling triggers specific vasoactive effects in the intrarenal arteries of patients with arterial hypertension.

In normotensive conditions, it has been confirmed that S-nitrosothiols (RSNO), can interact with hydrogen sulfide (H2S) and create new substances with specific vasoactive effects. This interaction could also represent a new regulator signaling pathway in conditions of hypertension. Until now, these effects were studied only in normotensive rats, and they have not been carried out in humans yet. We investigated the vasoactive effects of the products of the H2S/S-nitrosoglutathione (S/GSNO) interaction in lobar arteries (LA) isolated from the nephrectomized kidneys of patients suffering from arterial hypertension and in renal arteries (RA) of spontaneously hypertensive rats (SHR). The changes in the isometric tension of pre-contracted arteries were evaluated. Acetylcholine-induced vasorelaxation of LA was reduced compared to the effect induced by an NO donor, sodium nitroprusside suggesting an endothelium dysfunction. While 1 µmol/L Na2S had a minimal effect on the vascular tone, the concentration 20 µmol/L evoked a slight vasorelaxation. GSNO at 0.1 µmol/L induced vasorelaxation, which was less pronounced compared to the effect induced by 1 µmol/L. The S/GSNO products (final concentration 0.1 µmol/L) prepared as the mixture of GSNO (0.1 µmol/L) + Na2S (1 µmol/L) induced a higher vasorelaxation compared to GSNO (0.1 µmol/L) alone only in the 5th minute and without the differences in the speed. On the other hand, the S/GSNO products (final concentration 1 µmol/L) prepared as the mixture of GSNO (1 µmol/L) + Na2S (10 µmol/L) induced a higher and faster vasorelaxation compared to the effect induced by GSNO (1 µmol/L) alone. In RA of SHR this S/GSNO products induced similar vasorelaxation (higher and faster than GSNO) with involvement of HNO (partially) and cGMP as mediators. However, the products of the H2S/NO donor (DEA NONOate) manifested differently than S/GSNO indicating the unique interaction between GSNO and H2S. In this study, we confirmed for the first time that specific vasoactive effects of coupled nitroso-sulfide signaling were also triggered in human arterial tissue. We suggest that in hypertension, H2S in interaction with GSNO regulated a vasoconstrictor-induced increase in arterial tone towards a stronger vasorelaxation compared to GSNO alone or H2S alone.

The adaptive role of nitric oxide and hydrogen sulphide in vasoactive responses of thoracic aorta is triggered already in young spontaneously hypertensive rats.

The aorta plays an important role in blood pressure control so the early determination of its vasoactive properties could predict pathological changes in hypertension. The aim of study was to compare vasoactive properties and geometry of thoracic aorta (TA) and the participation of two vasoactive transmitters, nitric oxide (NO) and hydrogen sulphide (H2S), in TA tone regulation in young Wistar rats (WR) and spontaneously hypertensive rats (SHR). Four-weeks-old WR and SHR were used. Systolic blood pressure (sBP) was measured by plethysmography. The vasoactivity of TA was evaluated by changes in isometric tension. For morphological study the geometry of TA was measured using light microscopy. Decomposition of NO donor (nitrosoglutathione, GSNO) induced by H2S donor (Na2S) was studied by UV-VIS spectroscopy. In SHR the sBP was not increased in spite of cardiac hypertrophy compared to WR. Vasoconstriction to noradrenaline (NA) was decreased in SHR compared to WR which correlated with arterial wall hypotrophy. Acetylcholine (Ach)-induced vasorelaxation was increased and NO component participated in vasorelaxation and basal tone regulation significantly more in SHR. Na2S induced biphasic effect in both experimental groups, however, the shift towards vasorelaxation was demonstrated in SHR. Pretreatment with NO-synthase inhibitor, NG-nitro-L-arginine methylester (L-NAME), diminished the contractile part of vasoactive Na2S effects in both strains, moreover, an increased sensitivity in behalf of vasorelaxation was observed in SHR. Pretreatment with Na2S did not affect Ach-induced vasorelaxation in WR, but an inhibition was demonstrated in SHR. On the other hand, pretreatment with Na2S increased the release of NO from GSNO which corresponded with increased GSNO-induced vasorelaxation in both groups. However, this effect was stronger in SHR. The study showed that TA of prehypertensive SHR disposed by decreased contractility and strengthened endothelium-regulated vasorelaxant mechanisms involving of NO and H2S interaction which could serve as adaptive mechanisms in the adulthood.

Captopril partially decreases the effect of H(2)S on rat blood pressure and inhibits H(2)S-induced nitric oxide release from S-nitrosoglutathione.

We studied the effects of the H(2)S donor Na(2)S on the mean arterial blood pressure (MAP) and heart and breathing rates of anesthetized Wistar rats in the presence and absence of captopril. Bolus administration of Na(2)S (1-4 micromol/kg) into the right jugular vein transiently decreased heart and increased breathing rates; at 8-30 micromol/kg, Na(2)S had a biphasic effect, transiently decreasing and increasing MAP, while transiently decreasing heart rate and increasing and decreasing breathing rate. These results may indicate independent mechanisms by which H(2)S influences MAP and heart and breathing rates. The effect of Na(2)S in decreasing MAP was less pronounced in the presence of captopril (2 micromol/l), which may indicate that the renin-angiotensin system is partially involved in the Na(2)S effect. Captopril decreased H(2)S-induced NO release from S-nitrosoglutathione, which may be related to some biological activities of H(2)S. These results contribute to the understanding of the effects of H(2)S on the cardiovascular system.

Sulphide signalling potentiates apoptosis through the up-regulation of IP3 receptor types 1 and 2.

AIM: To investigate an interaction between the calcium and sulphide signalling pathways, particularly effects of the slow H2 S release donor morpholin-4-ium-4-methoxyphenyl-(morpholino)-phosphinodithioate (GYY4137) on the expression of inositol 1,4,5-trisphosphate receptors (IP3 R) with the possible impact on the apoptosis induction in HeLa cells. METHODS: Gene expression, Western blot analysis, apoptosis determination by Annexin-V-FLUOS and drop in mitochondrial membrane potential by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC1) and immunofluorescence were used to determine differences in control and GYY4137-treated HeLa cells. RESULTS: In HeLa cell line, GYY4137 (10 µm) up-regulated expression of the IP3 R1 and IP3 R2, but not IP3 R3 on both mRNA and protein levels. Concurrently, cytosolic calcium increased and reticular calcium was depleted in concentration-dependent manner, partially by the involvement of IP3 R. Depletion of calcium from reticulum was accompanied by increase in endoplasmic reticulum (ER) stress markers, such as X-box, CHOP and ATF4, thus pointing to the development of ER stress due to GYY4137 treatment. Also, GYY4137 treatment of HeLa cells increased the number of apoptotic cells. CONCLUSION: These results suggest an involvement of H2 S in both IP3 -induced calcium signalling and induction of apoptosis, possibly through the activation of ER stress.

Blood pressure-independent hypotrophy of the heart, kidneys and conduit arteries after 7-nitroindazole administration to Wistar rats from the prenatal period to adulthood.

The aim of this study was to investigate the long-term effects of 7-nitroindazole on the heart, kidneys, thoracic aorta, and carotid arteries from the progeny of mothers that had been treated with 7-nitroindazole (7NI) (10 mg/kg/day in drinking water) during gestation and nursing. The offspring were also treated with 7NI (10 mg/kg/day in drinking water) until 10 weeks of age. Mean arterial pressure (BP) was measured by tail-cuff plethysmography starting at 4 weeks of age. After perfusion fixation with glutaraldehyde at 120 mmHg, the heart and kidneys were weighed and the thoracic aorta and carotid arteries were processed for morphological investigation. The BP and body weight of treated rats did not differ from age-matched control rats during the course of the experiment. In the experimental group, at the end of the experiment, the heart weight/body weight and kidney weight/body weight ratios were decreased. In addition, the wall thickness (intima + media), cross sectional area (intima + media), and wall thickness/inner diameter ratio were significantly decreased in both the thoracic aorta and carotid arteries without a change in the inner vessel diameter. Circumferential wall tension was increased in both arteries. The data clearly indicate that long-term inhibition of neuronal nitric oxide (NO) synthase with the specific inhibitor 7NI evokes BP-independent hypotrophy of the heart, kidneys, and conduit arterial walls in normotensive Wistar rats.

Effects of chronic neuronal nitric oxide-synthase inhibition on arterial function and structure in spontaneously hypertensive rats.

While the effect of chronic non-specific NO-synthase inhibition in the cardiovascular system has been recognized under normotensive and hypertensive conditions, there are no data relating the long-term inhibition of neuronal NO-synthase (nNOS) in essential hypertension. The aim of this study was to investigate the long-term effect of nNOS inhibitor 7-nitroindazole (7-NI) administration on arterial function and structure in spontaneously hypertensive rats (SHR). Ten weeks old SHR were divided in two groups: control group and group administered 7-NI (10 mg/kg/day) for six weeks in drinking water. Systolic blood pressure (SBP) was measured using the plethysmographic method. The vasoactivity of isolated thoracic aorta (TA) and mesenteric artery (MA) was recorded via changes in isometric tension, and the geometry of both arteries was measured using light microscopy. Chronic treatment with 7-NI did not affect either SBP or heart/body weight ratio. Acetylcholine-induced relaxation of both arteries was unchanged after 7-NI. 7-NI administration did not affect the sensitivity and contraction to exogenous noradrenaline in TA, whereas both parameters were augmented in MA. The contractile response of MA induced by transmural nerve stimulation (endogenous noradrenaline) was unaffected after 7-NI. The mass of TA wall was unchanged, whereas hypertrophy was observed in MA after 7-NI. In summary, although SBP and endothelial function were not changed after chronic nNOS inhibition, the contractile and structural properties of TA and MA were affected differently. The data suggest that nNOS triggers original and tissue-specific regulatory pathways in essential hypertension.

Modulation of the sodium-calcium exchanger in the rat kidney by different sequential stressors.

Stress, if exaggerated, modulates a variety of metabolic pathways and results in development of serious health consequences. The cell membrane sodium-calcium exchanger (NCX) is a major calcium extrusion system and is also modulated by stress. It has been shown previously that mRNA, protein levels and activity of the type 1 NCX (NCX1) in the left ventricle of the rat heart are increased by stressors, such as immobilization or hypoxia. In this study we investigated whether exposure to a subsequent different stressor can affect gene expression, protein level and activity of the NCX1 in rat kidney compared to exposure to only one type of stressor. In these experiments, we used immobilization and cold as the model stressors.We found that cold exposure at 4 degrees C for 24 h, when applied after immobilization repeated seven times, completely abolished the immobilization-induced increase in NCX mRNA level and after 7 days cold exposure the increases in NCX1 protein and activity in rat kidney were also abolished. Permanently increased NCX1 expression can result in imbalance of cellular calcium homeostasis and thus contribute to kidney dysfunction. Based on our results, we conclude that exposure to a cold stressor can have a protective effect on the kidney in rats exposed previously to repeated immobilization stress. This might be explained by differential stimulation of sympathetic neural and adrenal medullary responses by these different stressors.

Uranyl acetate modulates gene expression and protein levels of the type 2, but not type 1 inositol 1,4,5-trisphosphate receptors in mouse kidney.

Nephrotoxic effect of uranium is already well documented. Nevertheless, little is known about the effect of uranium on calcium homeostasis and calcium transport systems. Calcium released from endoplasmic reticulum through special calcium release channels--inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs)--serves as a main source of cytosolic calcium signaling in the majority of cell types. To contribute to understanding mechanism of toxicity of the uranyl acetate (UA), we focused on modulation of the gene expression, protein levels and activity of IP3 receptor's intracellular calcium channels by UA in mouse kidney. We have found that UA did not affect mRNA and protein levels of the type 1 IP3Rs, but increased mRNA and also protein levels of the type 2 IP3 receptors in kidney. Nevertheless, IP3-induced calcium release was decreased by addition of UA. We assume that decreased activity of IP3 receptors due to the acute exposure to UA results in feedback, which triggers activation of IP3R2 expression. Thus, inhibition of calcium release and increased levels of the type 2 IP3 receptors might participate, at least partially, in UA-induced nephrotoxicity.

Potassium-chloride promiscuous channels in mitochondrial membranes.

Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and channel currents were measured in 250/50 mmol/l KCl cis/trans solutions. The channel currents measured from -40 to +40 mV had various linear voltage-current relationships and K(+)/Cl(-) permeability ratios at distinct voltage ranges. The channels possessed K(+)-Cl(-) promiscuous property. Depending on voltage, membrane permeability suddenly switched from K(+) over Cl(-) to Cl(-) over K(+) and back. The channels had Cl(-)/K(+) > 1 permeability at potentials around 0 mV and the permeability was switched to K(+)/Cl(-) > 1 at more negative and positive potentials. The chloride channel blocker, 5-nitro-2-(phenylpropylamino)-benzoate (NPPB, 5 x 10(-5) mol/l), influenced properties of the promiscuous channels - it activated potassium conductance of the channels.

IP3 type 1 receptors in the heart: their predominance in atrial walls with ganglion cells.

Previously we have shown that inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are abundantly expressed in the atria of rat hearts. Since arrangement of atria is very heterogeneous, in this work we focused on the precise localization of IP3 receptors in the left atrium, where the gene expression of the type 1 IP3R was the highest. The mRNA levels of the IP3 type 1 receptors in the left atrium, left ventricle and myocytes were determined using real-time polymerase chain reaction and Taqman probe. For precise localization, immunohistochemistry with the antibody against type 1 IP3Rs was performed. The mRNA of type 1 IP3 receptor was more than three times higher in the left atrium than in the left ventricle, as determined by real-time PCR. Expression of the type 1 IP3 receptor mRNA was higher in the atria, especially in parts containing cardiac ganglion cells. The atrial auricles, which are particularly free of ganglion cells, and the ventricles (wall of the right and left ventricle and ventricular septum) contained four to five times less IP3 receptors than atrial samples with ganglia. IP3R type 1 immunoreactivity detected by a confocal microscope attributed the most condensed signal on ganglionic cells, although light immunoreactivity was also seen in cardiomyocytes. These results show that type 1IP3 receptors predominate in intrinsic neuronal ganglia of cardiac atria.

Quinine inhibits mitochondrial ATP-regulated potassium channel from bovine heart.

The mitochondrial ATP-regulated potassium (mitoK(ATP) channel has been suggested as trigger and effector in myocardial ischemic preconditioning. However, molecular and pharmacological properties of the mitoK(ATP) channel remain unclear. In the present study, single-channel activity was measured after reconstitution of the inner mitochondrial membrane from bovine ventricular myocardium into bilayer lipid membrane. After incorporation, a potassium-selective current was recorded with mean conductance of 103 +/- 9 pS in symmetrical 150 mM KCl. Single-channel activity of this reconstituted protein showed properties of the mitoK(ATP) channel: it was blocked by 500 microM ATP/Mg, activated by the potassium-channel opener diazoxide at 30 microM, inhibited by 50 microM glibenclamide or 150 microM 5-hydroxydecanoic acid, and was not affected by the plasma membrane ATP-regulated potassium-channel blocker HMR1098 at 100 microM. We observed that the mitoK(ATP) channel was blocked by quinine in the micromolar concentration range. The inhibition by quinine was additionally verified with the use of 86Rb+ flux experiments and submitochondrial particles. Quinine inhibited binding of the sulfonylurea derivative [3H]glibenclamide to the inner mitochondrial membrane. We conclude that quinine inhibits the cardiac mitoK(ATP) channel by acting on the mitochondrial sulfonylurea receptor.

Involvement of L-arginine-nitric oxide system in the response of isolated trachea to reactive oxygen species.

The aim of this study was to investigate the effect of reactive oxygen species (ROS), generated by electrolysis of Krebs-Henseleit solution (GE-KHS), on isolated guinea pig trachea and to assess the possible involvement of nitric oxide (NO) in the observed effects. The isolated trachea was superfused in GE-KHS, generating H2O2 and hypochlorous acid (HOCl), both of which slowly increased in the organ bath and reached final stable concentrations of 42 and 63 microM, respectively, at the rate of 20 ml/min(-1), and 261 and 245 microM, respectively, at the rate of 5 ml/min(-1). ROS GE-KHS-induced relaxation of tracheal rings was preceded by a small transient contraction in 40% and 65% of experiments when tracheal rings were superfused at the rate of 20 ml/min(-1) and 5 ml/min(-1), respectively. Removal of tracheal epithelium abolished the relaxation of the trachea induced by ROS GE-KHS and unmasked or potentiated trachealis contraction. The ROS GE-KHS-induced changes in trachealis tension were accompanied by an increase in thiobarbituric acid reactive substances (TBARS) and a decrease in nonprotein (NP) thiols in the trachea. These changes were inhibited by treatment with the antioxidant N-acetylcysteine (100 microM). Pretreatment of tracheal rings with the inhibitor of NO synthase (NOS) N(omega)-nitro-L-arginine (L-NOARG; 100 microM) for 20 min prior to exposure to ROS GE-KHS decreased the ROS GE-KHS-induced relaxation. When L-NOARG (100 microM) was present in the superfusing solution, not only 20 min before but also during superfusion with ROS GE-KHS, the evoked trachealis relaxation was reduced in the first 15 min but was enhanced in the 30th min. This late enhancement of relaxation was accompanied by a 12-fold increase in nitric oxide metabolites (NO(x)). ROS GE-KHS-induced elevation of TBARS levels in the trachea was decreased to 63% by pretreatment with L-NOARG (100 microM). Elevation of TBARS levels induced by incubation of brain liposomes with a hydroxyl radical generating system was decreased to 90% by L-NOARG (10, 100 microM), while the antioxidant stobadine (100 microM) nearly completely inhibited the evoked lipid peroxidation. In comparison with Trolox, L-NOARG exerted a slight scavenging effect on the 1,1-diphenyl-2-picrylhydrazyl radical. The presence of L-arginine and D-arginine in the superfusion fluid for 15-20 min before and during exposure of the trachea to ROS GE-KHS inhibited trachealis relaxation. Results indicate that epithelium derived NO may participate in the response of guinea pig trachea to ROS GE-KHS. The presence of L-NOARG in the bathing fluid during superfusion with ROS GE-KHS gave rise to NO(x), with relaxing activity. L- and D-arginine induced an inhibition of the relaxatory response to ROS GE-KHS and partially prevented a ROS-induced decrease in NP thiols. The involvement of the small antioxidant effects of L-NOARG and L- and D-arginine in the above mentioned actions of L-NOARG and L-arginine requires additional investigation.

The inositol 1,4,5-trisphosphate receptor--transcriptional regulation and modulation by phosphorylation.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular membrane calcium channels essential for the release of calcium from intracellular stores. They form pathways for Ca2+ release that amplifies signals transmitted via plasma membrane receptors and so mediate a large array of calcium-regulated signal transduction events, from control of gene expression, through regulation of cell proliferation and other functions, to cell death. Molecular properties, expression and regulation of IP3Rs have been recently reviewed by others and thus these aspects will be mentioned only briefly in this article. This minireview will focus on the transcriptional regulation of IP3Rs and their modulation by phosphorylation.

Coupled gating between individual cardiac ryanodine calcium release channels.

In order to study interactions between ryanodine receptor calcium release (RyR2) channels during excitation-contraction coupling in cardiac muscle, we used bilayer lipid membrane (BLM) and improved the method of cardiac sarcoplasmic vesicle fusion into BLM. We increased fusion gradient for the vesicles, used chloride ions for fusion up to concentration of 1.2 mol/l and fused the vesicles by adding them directly to the forming BLM. Under these conditions, increased probability of fusion of vesicles containing 2-7 ryanodine channels into BLM was observed. Interestingly about 10% of the channels did not gate into BLM independently, but their gating was coupled. At 53 mmol/l calcium solution, two coupled gating channels had double conductance (191 +/- 15 pS) in comparison with the noncoupled channels (93 +/- 10 pS). Activities of the coupled channels were decreased by 5 micromol/l ryanodine and inhibited by 10 micromol/l ruthenium red similarly as single RyR2 channels. We suppose that cardiac sarcoplasmic vesicles contain single as well as coupled RyR2 channels.

Distribution of neuronal and non-neuronal spliced variants of type 1 IP(3)-receptor in rat hypothalamus and brain stem.

In the nervous system, inositol 1,4,5-trisphosphate (IP(3)) is one of the second messengers produced by PI hydrolysis and triggers IP(3)-receptor (IP(3)R) mediated calcium release from intracellular pools. Throughout the brain, the type 1 IP(3)R is predominantly expressed and its mRNA is widely distributed. Alternative splicing of IP(3)R1 (SI and SII) occurs in two distinct regions. SI splicing in the middle of the ligand binding domain may alter the IP(3) binding activity, while SII splicing probably affects the protein kinase A phosphorylation sites and kinetics. Selective use of IP(3)-receptor subtypes may permit a tissue specific and developmentally specific expression of functionally distinct channels. The present work was focused on detection of the alternatively spliced mRNA of type 1 IP(3)-receptor in individual brain structures and nuclei. Using RT-PCR we detected neuronal (535bp) and non-neuronal (410bp) forms. We identified both spliced variants in the majority of brain structures, except in the cerebellum and medulla. In the cerebellum, the neuronal form of type 1 IP(3)R was found exclusively, while in the medulla, the non-neuronal form was much more abundant. Nevertheless, Western blot analysis and hybridization with specific antibody against IP(3)R revealed no qualitative, but only quantitative differences. Similarly, IP(3) dependent calcium release did not show any differences between the cerebellum and pons. These results demonstrate the distribution of alternatively spliced S2 variants of type 1 IP(3)R in selected brain structures and nuclei. The physiological relevance of these two forms remains to be elucidated by further studies.

Effect of ethanol on tracheal potassium channels reconstituted into bilayer lipid membranes.

We examined the effect of ethanol on single potassium channels derived from plasma membranes of bovine tracheal smooth muscles. The observed potassium channels had a conductance of 296 +/- 31 pS (mean +/- S.D.) in symmetrical 250 mmol/l KCl solutions, and exhibited a voltage- and Ca2+-dependence similar to BKCa channels. Ethanol at 50, 100 and 200 mM concentrations increased the probability of open potassium channels to 112 +/- 5, 127 +/- 7 and 121 +/- 13% (mean +/- S.E.M.), respectively. It is suggested that increased activity of the BKCa channels by ethanol hyperpolarizes the plasma membrane and thus may contribute to relaxation of tracheal smooth muscle.

Coupled gating between cardiac calcium release channels (ryanodine receptors).

Excitation-contraction coupling in heart muscle requires the activation of Ca(2+)-release channels/type 2 ryanodine receptors (RyR2s) by Ca(2+) influx. RyR2s are arranged on the sarcoplasmic reticular membrane in closely packed arrays such that their large cytoplasmic domains contact one another. We now show that multiple RyR2s can be isolated under conditions such that they remain physically coupled to one another. When these coupled channels are examined in planar lipid bilayers, multiple channels exhibit simultaneous gating, termed "coupled gating." Removal of the regulatory subunit, the FK506 binding protein (FKBP12.6), functionally but not physically uncouples multiple RyR2 channels. Coupled gating between RyR2 channels may be an important regulatory mechanism in excitation-contraction coupling as well as in other signaling pathways involving intracellular Ca(2+) release.

Effect of nicotinic acid adenine dinucleotide phosphate on ryanodine calcium release channel in heart.

Nicotinic acid adenine dinucleotide phosphate (NAADP), a molecule derived from nicotinamide adenine dinucleotide phosphate (NADP+), is a recently identified nucleotide that activates Ca2+ release from intracellular stores in invertebrate eggs and in mammalian cells. NAADP could function as an intracellular messenger for mobilizing internal Ca2+ stores, however the targets and nature of NAADP-induced Ca2+ release are unknown. We report here that NAADP (3-10 microM) induces Ca2+ release from rat heart microsomes and that NAADP (1-10 microM) activates single ryanodine receptor/calcium release channels (RyR2) from dog heart incorporated into bilayer lipid membranes. The results indicate that NAADP may play a role in cardiac excitation-contraction coupling by acting on RyR2 channels.