Leszek Szadujkis-Szadurski

Role of nitric oxide and cGMP in the modulation of vascular contraction induced by angiotensin II and Bay K8644 during ischemia/reperfusion

Vascular smooth muscle tone changes under the influence of numerous contracting and relaxing factors. The purpose of the present study was to determine the modulating effect of ischemia and reperfusion (I/R) on contraction triggered by angiotensin II (ANG II) and Bay K8644 as well as to investigate the importance of nitric oxide (NO) and cGMP in these reactions. Experiments were performed on isolated and perfused Wistar rat tail arteries. The contraction triggered by ANG II and Bay K8644 with the use of intracellular (in calcium-free physiological salt solution; FPSS) and extracellular (in physiological salt solution; PSS) pools of calcium ions after I/R and in the presence of sodium nitroprusside (SNP), 8Br-cGMP, an endothelial NO synthase (NOSe) inhibitor (L-NG-nitroarginine methyl ester; L-NAME) or ODQ [an inhibitor of soluble guanylyl cyclase (GC)] was evaluated. ANG II triggered contraction in FPSS and PSS, but Bay K8644 only in PSS. Ischemia reduced and reperfusion intensified the response of the artery to ANG II, but did not change the action of Bay K8644. SNP and 8Br-cGMP reduced the response of the vessels to ANG II and did not change the modulating effect of ischemia, but reduced the intensifying action of reperfusion on contraction caused by the presence of ANG II. SNP lowered the action of Bay K8644 in PSS. In PSS, L-NAME and ODQ intensified the action of ANG II, eliminating the reducing effect of ischemia on the contraction caused by ANG II, but did not influence the intensifying reaction caused by reperfusion. L-NAME and ODQ did not influence the action of Bay K8644. I/R modulated the contraction of arteries triggered by ANG II, but did not influence the response to Bay K8644. The intra- and extracellular pools of calcium ions mediate the action of ANG II, but Bay K8644 stimulated contraction only with participation of calcium ions flowing into the cell. Control of the vascular smooth muscle tone associated with the action of NO and cGMP is subject to modulation under conditions of I/R.

Modulating activity of M1 receptor to the reaction of ileal smooth muscle.

BACKGROUND The subject of the study was determination of the effect of drugs on ileal smooth muscle contraction induced by activation of M(1) type muscarinic receptors. Drugs that have an effect on muscarinic receptors are divided to agonists, with close ties to the receptor and high internal activity and antagonists, with no internal activity. Conducted experiments tested interactions between a broad-spectrum agonist of muscarinic receptors, carbachol and a selective muscarinic receptor antagonist of M(1) type, pirenzepine. MATERIAL/METHODS Testing was conducted on tissues isolated from rats intestine. Male Wistar rats with weight between 220 g and 360 g were anesthetized by intraperitoneal injection of urethane (120 mg/kg). Concentration-effect curves were determined with the use of cumulated concentration method, in accordance with the van Rossum method (1963) in Kenakin modification (2006). RESULTS The purpose of the study was determination of concentration-effect curves for carbachol. This curve was compared with the curve of receptor occupation depending on concentration of this drug. Based on concentration-effect curves, the average value of EC(50) was calculated for carbachol, amounting to 2.44×10(-6) [M/l]. CONCLUSIONS The results confirmed that atropine is effective in stopping contractions caused by carbachol, meeting the conditions of competitive antagonists. Atropine caused the shift of curves for carbachol to the right. Pirenzepine, selectively blocking muscarinic receptors of M(1) type gave similar results. It was proved that in the preparation of gastric fundus smooth muscle, M(1) type receptors occur not only presynaptically, but also postsynaptically.

The Role of the Endothelium in the Regulation of Vascular Smooth Muscle Cell Contractions Induced by Angiotensin II after Ischemia and Reperfusion

In this study, we investigated smooth muscle cell contractions induced by angiotensin II (ANG II) in blood vessels with or without endothelium after ischemia/reperfusion. Experiments were done on tail arteries, with or without endothelium, of perfused male Wistar rats. We analyzed the influence of ANG II on vascular contractions in the presence of sodium nitroprusside or 8Br-cGMP and after ischemia/reperfusion using classical pharmacometric methods. Vascular contractions induced by ANG II were decreased by sodium nitroprusside and 8BrcGMP. Reductions in maximal response and increases in EC50 values after ischemia were observed only in vessels with endothelium. After reperfusion, increases in maximal response and decreases in EC50 values were observed. Decreases in ANG II induced vascular contractions on caused by ischemia involves the presence of endothelium, synthesis of nitric oxide and activation of cGMP. The increase in the reaction to ANG II that is induced by reperfusion is independent of the endothelium.

Influence of celecoxib on the vasodilating properties of human mesenteric arteries constricted with endothelin-1.

The mitogenic and vasoconstrictive properties of the vascular system are attributed to endothelin-1 (ET-1). ET-1 serum concentration increases in a number of pathological conditions, particularly in those associated with blood vessel constriction. ET-1 is also associated with the underlying pathomechanisms of primary pulmonary hypertension, arterial hypertension and eclampsia. The aim of this study was to compare the vasodilating properties of selected phosphodiesterase (PDE) inhibitors and celecoxib in human mesenteric arteries constricted with ET-1, and investigate the role of the endothelium in relaxation. Perfused human mesenteric arteries were collected and stored under the same conditions as organs for transplantation. The mesenteric arteries (with and without the endothelium) were constricted by the addition of ET-1 and treated with one of the following: sildenafil (PDE5 inhibitor), zaprinast (PDE5 and 6 inhibitor), rolipram (PDE4 inhibitor) and celecoxib [cyclooxygenase-2 (COX-2) inhibitor]. Based on the observed changes of the perfusion pressure, concentration response curves (CRCs) were prepared for the respective inhibitors and the EC50 (concentration causing an effect equal to half of the maximum effect), pD2 (negative common logarithm of EC50) and relative potency (RP) were calculated. The results suggested that all the inhibitors triggered a concentration-dependent decrease in the perfusion pressure in isolated human superior mesenteric arteries with endothelium constricted by the addition of ET-1. In the arteries without endothelium, CRCs for celecoxib and rolipram were shifted to the right without a significant decrease in the maximum dilating effect. Moreover, CRCs for sildenafil and zaprinast were shifted to the right with a simultaneous significant decrease in the maximum dilating effect and with an increased inclination angle in reference to the concentration axis. In the presence of the endothelium, all of the evaluated PDE inhibitors, as well as celecoxib, reduced the reactivity of the mesenteric arteries caused by ET-1. Sildenafil indicated the lowest efficacy in the presence of the endothelium, but showed a higher potency compared to that of the other compounds. Removing the endothelium significantly reduced the vasodilating efficacy of PDE5 and 6 inhibitors and a statistically significant influence on the vasodilating efficacy of PDE4 inhibitor and celecoxib was observed. The high vasorelaxing efficacy of celecoxib at the background of the PDE inhibitors was observed, not only in the presence, but also in the absence of the endothelium and may be evidence for the relaxation induced by this COX-2 inhibitor in the cAMP- and cGMP-dependent pathways.

Role of endothelium, acetylocholine and calcium ions in Bay K8644- and KCl-induced contraction

The aim of this study was to establish the involvement of acetylcholine (Ach) and calcium ions in modulating contractions induced by Bay K8644 (an agonist of calcium channels located in the cell membrane) and KCl (at depolarizing concentrations), and also to examine the importance of the vascular endothelium in the activity of Bay K8644. The study was performed on perfused Wistar rat tail arteries. Contraction induced by Bay K8644 with the participation of intracellular (in calcium‑free physiological salt solution, FPSS) and extracellular (in physiological salt solution, PSS, following the emptying of the cellular Ca2+ stores) pools of Ca2+ and the addition of nitro-L-arginine (L‑NNA; nitric oxide synthase inhibitor) or 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ; an inhibitor of soluble guanylyl cyclase) was studied. In addition, the effect of Ach on the contraction response was analyzed and the results were compared with the depolarizing action of KCl. The effects of 8Br‑cGMP on the artery contraction induced by Bay K8644 prior to and following removal of the endothelium were compared. Bay K8644 and KCl in PSS induced vascular contraction, which was reduced with the addition of Ach. The spasmolytic Ach action did not occur in the presence of L‑NNA and ODQ. 8Br‑cGMP reduced the contraction of arterial walls (with and without endothelium) induced by Bay K8644. The increase in vascular tone induced by Bay K8644 and KCl was independent of the intracellular calcium ion pool. The relaxant effect of Ach on the responses stimulated by Bay K8644 and KCl indicated the participation of nitric oxide in modulating the reactivity of the arteries to the factors examined, resulting in an influx of Ca2+ into the cell.

Rho-kinase inhibitor reduces hypersensitivity to ANG II in human mesenteric arteries retrieved and conserved under the same conditions as transplanted organs.

Rho-kinase and GTP-ase Rho are important regulators of vascular tone and blood pressure. The aim of this study was to investigate the role of Rho-kinase in artery reactions induced by angiotensin II (ANG II) and the effects of ischemia-reperfusion injury as well as the function of intra- and extracellular calcium in these reactions. Experiments were performed on mesenteric superior arteries procured from cadaveric organ donors and conserved under the same conditions as transplanted kidneys. The vascular contraction in reaction to ANG II was measured in the presence of Rho-kinase inhibitor Y-27632, after ischemia and reperfusion, in Ca2+ and Ca2+-free solution. The maximal response to ANG II was reduced after ischemia, while an increase was observed after reperfusion. Vascular contraction induced by ANG II was decreased by Y-27632. Y-27632 reduced vascular contraction after reperfusion, both in Ca2+ and Ca2+-free solution. Reperfusion augments vascular contraction in reaction to ANG II. The Rho-kinase inhibitor Y-27632 reduces the hypersensitivity to ANG II after reperfusion mediated by both intra- and extracellular calcium. These results confirm the role of Rho-kinase in receptor-independent function of ANG II and in reperfusion-induced hypersensitivity.

Activity of guanylyl cyclase activators on the reaction of tracheal smooth muscle contraction.

INTRODUCTION The subject of the study compare the influences of YC-1 guanylyl cyclase activator with ODQ guanylyl cyclase inhibitor on the tracheal smooth muscle contraction induced by carbachol. The study specified the influence of increasing concentrations of soluble guanylyl cyclase activators YC-1 and 8Br cGMP on the reaction of tracheal smooth muscle contraction released by carbachol. The author also examined the effect of increasing concentrations of soluble guanylyl cyclase inhibitor ODQ on the concentration-effect curves for carbachol. MATERIAL/METHODS Testing was conducted on an isolated trachea of both sexes of Wistar rats with weight ranging between 350 g and 450 g. Tracheas were prepared in accordance with the Akcasu (1959) method in Szadujkis-Szadurski (1996) modification. Concentration-effect curves were determined with the use of cumulated concentration method, in accordance with the van Rossum method (1963) in Kenakin (2006) modification. RESULTS According to conducted testing, activation of soluble guanylyl cyclase with the use of YC-1 and 8Br cGMP caused reduced reaction of the tracheal smooth muscle with carbachol on average to 80%. Comparing concentration-effect curves for carbachol before and after the use of 8Br cGMP, similar results were obtained for those released by YC-1. On the other hand, increasing concentrations of guanylyl cyclase inhibitor - ODQ cause shift of curves to the left, decrease of EC(50) value and an increase of maximum reaction to carbachol. CONCLUSIONS Carbachol, depending on concentration, causes tracheal smooth muscle contraction. According to testing, we can confirm that activation of guanylyl cyclase leads to reduction of the reaction of tracheal smooth muscle to carbachol on average up to 80%

Role of calcium and calmodulin in reaction of gastric fundus contraction.

BACKGROUND The subject of this study is determination of the influence of calmodulin and calcium on gastric fundus smooth muscle contraction. During experiments, the author tested the influence of a serotonin receptor agonist, serotonin (5-HT), causing smooth muscle contraction. MATERIAL/METHODS Testing was conducted on tissues isolated from rats stomach. Male Wistar rats with weight between 220 g and 360 g were anesthetized by intraperitoneal injection of urethane (120 mg/kg). The stomach was dissected, and later the gastric fundus was isolated. Tissue was placed in a dish for insulated organs with 20 ml in capacity, filled with Krebs fluid. Results contained in the study are average values ± SE. In order to determine statistical significance, the principles of receptor theory were used (Kenakin modification). RESULTS According to conducted tests, we can deduce that 8 Br cGMP stops the reaction of gastric fundus smooth muscle contraction induced by serotonin. The use of 8Br-cGMP in the range of concentrations between 10 and 300 µM leads to reduction of maximum effect from 100% to 46%. Similar changes were obtained after the use of guanylate cyclase activator (CG) - YC-1. Curves for the contractile activity of serotonin along with an increase of concentration YC-1 are shifted to the right, and the maximum effect of reaction decreases. Increasing concentrations of flunarizine, a calmodulin antagonist, in a concentration-dependent way blocks binding between calcium and calmodulin, and at the same time leads to the shift of concentration-effect curves for serotonin to the right and a decrease of maximum reaction. Increasing concentrations of ODQ, a guanylate cyclase inhibitor lead to statistically significant shift of the curves to the left, decrease of EC(50) value and simultaneous increase of maximum reaction to serotonin. CONCLUSIONS According to conducted testing, serotonin causes gastric fundus smooth muscle contraction dependent on concentration. Reaction of contraction induced by serotonin is stopped by a calmodulin antagonist, flunarizine. In addition, experiments confirmed participation of cyclical nucleotides in blocking reaction of gastric fundus contraction.

Role of M1 receptor in regulation of gastric fundus smooth muscle contraction

BACKGROUND The subject of this study is determination of the influence of drugs on gastric fundus smooth muscle contraction induced by activation of muscarinic receptors M1. Experiments tested interactions between a receptor agonist, carbachol and muscarinic receptor antagonists, atropine and pirenzepine. MATERIAL/METHODS Testing was conducted on tissues isolated from rats stomach. Male Wistar rats with weight between 220 g and 360 g were anesthetized by intraperitoneal injection of urethane (120 mg/kg). The stomach was dissected, and later the gastric fundus was isolated. Tissue was placed in a dish for insulated organs with 20 ml in capacity, filled with Krebs fluid. Results contained in the study are average values ± SE. In order to determine statistical significance, the principles of receptor theory were used (Kenakin modification). RESULTS According to tests, carbachol, in concentrations ranging between 10(-8) M to 10(-4) M, in a dosage-dependent way induces gastric fundus smooth muscle contraction. Presented results indicate that carbachol meets the conditions posed to full agonists. On the other hand, atropine, a non-selective muscarinic receptor antagonist, causes a concentration-dependent shift of concentration-effect curve (for carbachol) to the right, maintaining maximum reaction. According to analysis of the curve determined, we can deduce that atropine meets the conditions posed to competitive antagonists. The use of pirenzepine, a competitive receptor agonist M1, causes shift of concentration-effect curve (for carbachol) to the right, maintaining maximum reaction. CONCLUSIONS From the testing conducted on the preparation of the gastric fundus we can deduce that atropine causes shift of concentration-effect curves for carbachol to the right. A similar effect is released by pirenzepine, selectively blocking muscarinic receptors of M1 type. The results indicate that in the preparation of the gastric fundus smooth muscle, M1 type receptors occur also postsynaptically.