Frantisek Kristek

Endothelium-dependent relaxation in rabbit aorta after cold storage

The extent of preservation of endothelial, smooth muscle and neurogenic function following cold storage was studied in rabbit thoracic aorta. Relaxation responses to acetylcholine and sodium nitroprusside were compared between fresh aortic rings and rings that had been stored in a refrigerator for 2-8 days at 4 degrees C. In fresh aortic rings, the addition of acetylcholine to precontracted vessels resulted in dose-dependent relaxation. The magnitude of relaxation was gradually decreased after 4-8 days of cold storage. Relaxation in response to sodium nitroprusside did not change. Following cold storage contractions of aortic rings in response to noradrenaline and phenylephrine were not reduced. Contractile responses induced by transmural nerve stimulation were gradually attenuated with the length of cold storage. Electron microscopy after 4 days showed partial damage of endothelial cells (slightly vacuolized mitochondria). After 8 days, endothelial cells were destroyed; only membranous material was present. The structure of smooth muscle cells was only partially changed even after 8 days. Sympathetic nerve endings on the 4th day were partially, but on the 8th day completely destroyed. These results suggest that after cold storage of rabbit aorta, the gradual reduction of endothelium-dependent relaxation is probably caused by a decrease in production of the endothelium-derived relaxing factor due to the destruction of endothelial cells.

Myoendothelial Relations in the Conduit Coronary Artery of the Dog and Rabbit

This paper describes the myoendothelial relations in ramus interventricularis anterior and its branches in the dog and rabbit. Endothelial cells (ECs) are separated from smooth muscle cells (SMs) by fenestrated internal elastic lamina (IEL). The number of fenestrations increases towards the periphery; the first- and second-order branches have only patches of elastin in some places. ECs and SMs emit protrusions of various shapes into the fenestrae. The distance between EC and SM protrusions varies. Close appositions were also found. The diffusion of autacoids, or transmitters, as well as the electrical and biochemical mechanism of transmission seem to be involved in the transfer of information from ECs to SMs and vice versa. Biomechanical transfer (for example shear stress), via EC-SM junctions is also considered.

Effects of AP39, a novel triphenylphosphonium derivatised anethole dithiolethione hydrogen sulfide donor, on rat haemodynamic parameters and chloride and calcium Cav3 and RyR2 channels.

H2S donor molecules have the potential to be viable therapeutic agents. The aim of this current study was (i) to investigate the effects of a novel triphenylphosphonium derivatised dithiolethione (AP39), in the presence and absence of reduced nitric oxide bioavailability and (ii) to determine the effects of AP39 on myocardial membrane channels; CaV3, RyR2 and Cl(-). Normotensive, L-NAME- or phenylephrine-treated rats were administered Na2S, AP39 or control compounds (AP219 and ADT-OH) (0.25-1 µmol kg(-1)i.v.) and haemodynamic parameters measured. The involvement of membrane channels T-type Ca(2+) channels CaV3.1, CaV3.2 and CaV3.3 as well as Ca(2+) ryanodine (RyR2) and Cl(-) single channels derived from rat heart sarcoplasmic reticulum were also investigated. In anaesthetised Wistar rats, AP39 (0.25-1 µmol kg(-1) i.v) transiently decreased blood pressure, heart rate and pulse wave velocity, whereas AP219 and ADT-OH and Na2S had no significant effect. In L-NAME treated rats, AP39 significantly lowered systolic blood pressure for a prolonged period, decreased heart rate and arterial stiffness. In electrophysiological studies, AP39 significantly inhibited Ca(2+) current through all three CaV3 channels. AP39 decreased RyR2 channels activity and increased conductance and mean open time of Cl(-) channels. This study suggests that AP39 may offer a novel therapeutic opportunity in conditions whereby (•)NO and H2S bioavailability are deficient such as hypertension, and that CaV3, RyR2 and Cl(-) cardiac membrane channels might be involved in its biological actions.

The role of arterial smooth muscle in vasorelaxation

The concept of endothelium-derived relaxing factor (EDRF) implies that nitric oxide (NO) produced by NO synthase (NOS) in the endothelium in response to vasorelaxants such as acetylcholine (ACh) acts on the underlying vascular smooth muscle cells (VSMC) inducing vascular relaxation. The EDRF concept was derived from experiments on denuded blood vessel strips and, in frames of this concept, VSMC were regarded as passive recipients of NO from endothelial cells. However, it was later found that VSMC express NOS by themselves, but the principal question remained unanswered, is the NO generation by VSMC physiologically relevant? We hypothesized that the destruction of the vascular wall anatomical integrity by rubbing off the endothelial layer might increase vascular superoxides that, in turn, reduced the NO bioactivity as a relaxing factor. To test our hypothesis, we examined ACh-induced vasorelaxation under protection against oxidative stress and found that superoxide scavengers restored vasodilatory responses to ACh in endothelium-deprived blood vessels. These findings imply that VSMC can release NO in amounts sufficient to account for the vasorelaxatory response and challenge the concept of the obligatory role of endothelial cells in the relaxation of arterial smooth muscle.

Effect of chronic nNOS inhibition on blood pressure, vasoactivity, and arterial wall structure in Wistar rats.

While the unequivocal pattern of endothelial nitric oxide (NO) synthase (eNOS) inhibition in cardiovascular control has been recognised, the role of NO produced by neuronal NOS (nNOS) remains unclear. The purpose of the present study was to describe the cardiovascular effects of NO production interference by inhibition of nNOS with 7-nitroindazole (7-NI). Wistar rats (10 weeks old) were used: control and experimental rats were administered 7-NI 10 mg/kg b.w./day in drinking water for 6 weeks. Systolic blood pressure (BP) was measured by the tail-cuff plethysmographic method. Isolated thoracic aortas (TAs) were used to study vasomotor activity of the conduit artery in vitro. The BP response of anaesthetised animals was used to follow the cardiovascular-integrated response in vivo. Geometry of the TA was measured after perfusion fixation (120 mm Hg) by light microscopy. Expression of eNOS was measured in the TA by immunoblot analysis. Although 6 weeks of nNOS inhibition did not alter systolic BP, the heart/body weight ratio was decreased. Relaxation of the TA in response to acetylcholine (10(-9)-10(-5)mol/L) was moderately inhibited. However, no difference in the BP hypotensive response after acetylcholine (0.1, 1, 10 microg) was observed. The contraction of TA in response to noradrenaline (10(-10)-10(-5)mol/L), and the BP pressor response to noradrenaline (0.1, 1 microg) was attenuated. The inner diameter of the TA was increased, and the wall thickness, wall cross-sectional area, and wall thickness/inner diameter ratio were decreased. The expression of eNOS in the TA was increased. In summary, cardiac and TA wall hypotrophy, underlined by decreased contractile efficiency, were observed. The results suggested that two constitutive forms of NOS (nNOS, eNOS) likely participate in regulation of cardiovascular tone by different mechanisms.

Pentaerythrityl tetranitrate attenuates structural changes in conduit arteries evoked by long-term NO-synthase inhibition

The aim of the study was to determine whether the pentaerythrityl tetranitrate (PETN), a tolerance devoid exogenous NO donor could prevent morphological changes in the cardiovascular system evoked by long‐term NO‐synthase inhibition. Three groups of 10‐week‐old Wistar rats were used: (1) controls, (2) treated by L‐NG‐nitroarginine methyl ester (L‐NAME) in water (50 mg kg−1), and (3) treated by L‐NAME (50 mg kg−1 in water)+PETN (2×50 mg kg−1, using gavage). Blood pressure (BP) was measured by the tail plethysmographic method. After sacrificing the animals were perfused (120 mmHg) by glutaraldehyde fixative and processed according to standard electron microscopy procedure. Wall thickness (WT), cross sectional area (CSA), inner diameter (ID) of thoracic aorta (TA), carotid (CA) and septal branch of the left descending coronary artery (RS) were measured in light microscopy. After 6 weeks, the BP was increased to 172±1.7 mmHg (P<0.01) in the L‐NAME group, compared to 127±1.4 mmHg in controls. In L‐NAME+PETN‐treated rats, BP was 163±0.9 mmHg (P<0.01), and significantly lower (P<0.01) in comparison to L‐NAME‐treated rats. Heart weight and heart/body weight ratio was not significantly changed. In L‐NAME‐treated rats, both WT and CSA were increased in all three arteries (P<0.01). ID was increased only in TA (P<0.01). Wall/diameter ratio (WD) was increased in TA (P<0.01) and CA (P<0.01). In L‐NAME+PETN treated rats, WT was found to be increased only in TA (P<0.01). In comparison to the L‐NAME treated group, WT was decreased in TA (P<0.01), in CA (P<0.01), in RS (P<0.05). CSA was increased only in TA (P<0.01), yet in comparison to the L‐NAME group it was decreased in CA (P<0.01). ID was increased in comparison to both control and L‐NAME treated rats only in TA (P<0.01). WD did not differ from the control value. In comparison to L‐NAME‐treated rats, it was decreased in both TA and CA (P<0.01). These data suggest that the changes in the cardiovascular system evoked by long‐term NO‐synthase inhibition were attenuated by simultaneous administration of the exogenous donor of nitric oxide–PETN.

Perinatally administered losartan augments renal ACE2 expression but not cardiac or renal Mas receptor in spontaneously hypertensive rats

Since the identification of the alternative angiotensin converting enzyme (ACE)2/Ang‐(1‐7)/Mas receptor axis, renin‐angiotensin system (RAS) is a new complex target for a pharmacological intervention. We investigated the expression of RAS components in the heart and kidney during the development of hypertension and its perinatal treatment with losartan in young spontaneously hypertensive rats (SHR). Expressions of RAS genes were studied by the RT‐PCR in the left ventricle and kidney of rats: normotensive Wistar, untreated SHR, SHR treated with losartan since perinatal period until week 9 of age (20 mg/kg/day) and SHR treated with losartan only until week 4 of age and discontinued until week 9. In the hypertrophied left ventricle of SHR, cardiac expressions of Ace and Mas were decreased while those of AT1 receptor (Agtr1a) and Ace2 were unchanged. Continuous losartan administration reduced LV weight (0.43 ± 0.02; P < 0.05 versus SHR) but did not influence altered cardiac RAS expression. Increased blood pressure in SHR (149 ± 2 in SHR versus 109 ± 2 mmHg in Wistar; P < 0.05) was associated with a lower renal expressions of renin, Agtr1a and Mas and with an increase in ACE2. Continuous losartan administration lowered blood pressure to control levels (105 ± 3 mmHg; P < 0.05 versus SHR), however, only renal renin and ACE2 were significantly up‐regulated (for both P < 0.05 versus SHR). Conclusively, prevention of hypertension and LV hypertrophy development by losartan was unrelated to cardiac or renal expression of Mas. Increased renal Ace2, and its further increase by losartan suggests the influence of locally generated Ang‐(1‐7) in organ response to the developing hypertension in SHRs.

Effects of PPARγ Agonist Pioglitazone on Redox-Sensitive Cellular Signaling in Young Spontaneously Hypertensive Rats

PPARγ receptor plays an important role in oxidative stress response. Its agonists can influence vascular contractility in experimental hypertension. Our study was focused on the effects of a PPARγ agonist pioglitazone (PIO) on blood pressure regulation, vasoactivity of vessels, and redox-sensitive signaling at the central (brainstem, BS) and peripheral (left ventricle, LV) levels in young prehypertensive rats. 5-week-old SHR were treated either with PIO (10 mg/kg/day, 2 weeks) or with saline using gastric gavage. Administration of PIO significantly slowed down blood pressure increase and improved lipid profile and aortic relaxation after insulin stimulation. A significant increase in PPARγ expression was found only in BS, not in LV. PIO treatment did not influence NOS changes, but had tissue-dependent effect on SOD regulation and increased SOD activity, observed in LV. The treatment with PIO differentially affected also the levels of other intracellular signaling components: Akt kinase increased in the the BS, while β-catenin level was down-regulated in the BS and up-regulated in the LV. We found that the lowering of blood pressure in young SHR can be connected with insulin sensitivity of vessels and that β-catenin and SOD levels are important agents mediating PIO effects in the BS and LV.

Beneficial effect of pentaerythrityl tetranitrate on functional and morphological changes in the rat thoracic aorta evoked by long-term nitric oxide synthase inhibition

The present study examined whether pentaerythrityl tetranitrate (PETN), a tolerance-devoid exogenous donor of nitric oxide (NO), could attenuate functional and morphological changes in the rat thoracic aorta evoked by 6-week NO synthase inhibition by NG-nitro-L-arginine methyl ester (L-NAME). Systolic blood pressure in L-NAME + PETN-treated rats (163 +/- 1 mm Hg) was significantly lower than in L-NAME-treated rats (172 +/- 2 mm Hg) but was still higher than in age-matched controls (126 +/- 2 mm Hg). Six weeks of treatment of rats with L-NAME significantly inhibited endothelium-dependent relaxation of the isolated thoracic aorta induced by acetylcholine. The inhibitory effect of L-NAME was entirely reversed by the simultaneous treatment with PETN. The enhancing effect of L-NAME on noradrenaline-induced contraction was antagonised by long-term treatment with PETN. Wall thickness, cross-sectional area and wall/diameter ratio of the thoracic aorta in L-NAME-treated rats were markedly increased. In the L-NAME + PETN-treated rats, the increment of these parameters was significantly lower. The results suggest that PETN administered to rats during development of NO-deficient hypertension prevented functional impairment and at the same time reduced structural changes in the thoracic aorta induced by long-term inhibition of NO synthase.

Lipids modulate H2S/HS− induced NO release from S-nitrosoglutathione

Recently we observed that a gas messenger H(2)S/HS(-) released NO from S-nitrosoglutathione (Ondrias et al., Pflugers Arch. 457 (2008) 271-279). However, the effect of biological compounds on the release is not known. Measuring the NO oxidation product, which is nitrite, by the Griess reaction, we report that unsaturated fatty acid-linoleic acid and lipids having unsaturated fatty acids: asolectin, dioleoylphosphocholine and dioleoylphosphoserine depressed the H(2)S/HS(-) induced NO release from S-nitrosoglutathione. On the other hand, a depression effect of the saturated fatty acid-myristic acid and lipids having saturated fatty acids, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine was less pronounced. The inhibition effect increased with the decreasing gel-to-liquid phase transitions temperature of the fatty acids and lipids. We suggest that lipid composition of biological membranes modulates NO release from nitrosoglutathione induced by H(2)S/HS(-), assuming that a reaction of H(2)S/HS(-) with unsaturated bonds of fatty acids may be partially responsible for the effect.