Tadeusz Malinski

Nitric Oxide Measured by a Porphyrinic Micro Sensor in Rat Brain after Transient Middle Cerebral Artery Occlusion

We measured, in vivo, the local concentration of nitric oxide (NO) in cerebral tissue, during and after transient middle cerebral artery occlusion in the rat (n = 8). Baseline concentration of NO was <10−8 M; upon initiation of ischemia, NO concentration increased to ∼10−6 M and then declined. Reperfusion likewise stimulated an increase in NO concentration to above baseline level. Administration of N-nitro-l-arginine methyl ester (n = 4), an inhibitor of nitric oxide synthase, before onset of ischemia, maintained NO at basal levels. Our data indicate that large increases in NO occur at onset of ischemia, which may affect tissue response to an ischemic insult.

Direct In Situ Measurement of Nitric Oxide in Mesenteric Resistance Arteries Increased Decomposition by Superoxide in Hypertension

The endothelium plays a critical role in maintaining vascular tone by releasing vasoconstrictor and vasodilator substances. Endothelium-derived nitric oxide is a vasodilator that can be rapidly inactivated by superoxide (reaction rate constant, K = 3.6 x 10(9) L/mol per second). The measurement of nitric oxide concentration in biological systems is a challenging analytic problem because nitric oxide is also rapidly inactivated by Fe(II), Fe(III), and O2, all of which are found in great abundance in biological systems. To date, no currently used instrumental technique has been suitable for direct in situ measurement of NO in isolated resistance arteries. We designed the present study to perform for the first time direct in situ measurements of NO in rat mesenteric resistance arteries and to delineate the effects of hypertension on the release of NO and/or its interaction with superoxide. We describe here an adaptation of the recently published design of a porphyrinic sensor for direct in vitro measurement of NO in a single cell. The most significant advantage of this modified porphyrinic microsensor is that its small size makes it ideal for NO measurement in resistance arteries with an internal diameter of 200 microns or less. Small segments of the third-order branch of the mesenteric artery were isolated from normotensive Wistar-Kyoto rats and stroke-prone spontaneously hypertensive rats and placed in an organ chamber filled with Hanks balanced salt solution buffer (2 mL, 37 degrees C). The tip of the porphyrinic microsensor was inserted into the lumen of an isolated vascular ring, and NO release was monitored in situ after maximal stimulation of NO synthase with the receptor-independent agonist calcium ionophore A23187 (10 mumol/L). Maximal surface concentration of NO measured after A23187 administration was significantly smaller in 15-week-old hypertensive rats (0.28 +/- 0.03 mumol/L, n = 10) than in age-matched normotensive rats (0.38 +/- 0.03 mumol/L, n = 10, P < .03). However, in the presence of the superoxide scavenger superoxide dismutase (100 U/mL), the peak NO level from the hypertensive rats was 0.37 +/- 0.04 mumol/L (n = 10), which was comparable to that observed for the normotensive rats in the absence and presence of superoxide dismutase. In summary, our results demonstrate that in rat mesenteric resistance arteries hypertension is associated with increased NO decomposition by superoxide, whereas NO release remains unaffected. This may be important in the pathogenesis of hypertension and its cardiovascular complications.

Interactions among ACE, kinins and NO

Time for primary review 22 days. nnAngiotensin converting enzyme (ACE) is a transmembrane zinc metallopeptidase that cleaves carboxy-terminal dipeptides from several peptides and is expressed in great amounts in vascular endothelial cells [1,2]. A soluble form of the enzyme is found in plasma which is presumably derived from the membrane-bound form by proteolytic cleavage [3]. ACE plays a major role in the regulation of the vascular tone by converting the biological inactive decapeptide angiotensin I (ANG I) into the vasoconstrictor and proliferative octapeptide angiotensin II (ANG II). In a similar manner, ACE inactivates the vasodilatory nonapeptide bradykinin (BK), which derives from a number of different sources [4].nnEndothelium-derived or exogenously added BK exerts its vasodilatory action through stimulation of endothelial B2 kinin receptors thereby causing the synthesis and release of vasodilator substances such as endothelium-derived hyperpolarizing factor (EDHF) [5], prostacyclin and nitric oxide (NO) [6]. Many of the effects of NO on platelets [7], smooth muscle cells [8], and cardiac myocytes [9,10] are mediated by activation of soluble guanylyl cyclase to synthesize cyclic GMP. The biological function of soluble guanylyl cyclase and NO/cyclic GMP in endothelial cells is not yet completely understood. One function of endothelial cyclic GMP may be a negative feed-back mechanism to turn off further NO synthesis [11,12]. Changes in the synthesis of ACE, BK and NO are associated with a number of cardiovascular conditions including hypertension, atherosclerosis or coronary heart disease. ACE inhibitors are able to treat these diseases by both, accumulation of endothelium-derived kinins and the inhibition of ANG II [13,14].nnThe separate effects of ACE, kinins as well as NO on the cardiovascular system have been thoroughly investigated and described. Since only a small amount of information is available concerning the physiological/pathophysiological significance of … nn* Corresponding author. Tel.: +49-69-305-6868, fax: +49-69-305-81252 wolfgang.linz{at}hmrag.com

Nitric oxide changes in the rat brain after transient middle cerebral artery occlusion

Using a porphyritic microsensor, we measured the cortical NO concentration within ischemic tissue during 2 h of middle cerebral artery (MCA) occlusion and 1 h of reperfusion in the rat (n = 36). Local cerebral blood flow was simultaneously measured by laser Doppler flowmetry to verify MCA occlusion and reperfusion. Baseline concentration of NO was < 10(-8) M. The maximum concentrations of NO during MCA occlusion and reperfusion were, respectively, 1.47 +/- 0.45 microM and 0.54 +/- 0.24 microM. Administration of N-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase, prior to ischemia, significantly (p < 0.05) reduced NO release to 0.04 +/- 0.02 microM during MCA occlusion and completely inhibited NO release during 1 h of reperfusion. Administration of L-arginine 30 min after administration of L-NAME restored NO release (3.45 +/- 1.14 microM) during MCA occlusion; however, administration of L-arginine did not overcome the effect of L-NAME on mean arterial blood pressure. Our data indicate that NO is released in the brain after the onset of ischemia and NO levels can be modulated by administration of NO substrate and NO antagonists.

Measurements of nitric oxide in biological materials using a porphyrinic microsensor

Abstract The application and optimization of a microsensor for in situ measurements of nitric oxide (NO) is biological systems are described. The sensor (diameter 0.5–0.8 μm), exhibiting a response time better than 10 ms and a detection limit of 10 nM, consists of several layers of p-type semiconducting polymeric porphyrin and cation exchanger (Nafion) deposited on a thermally-sharpened carbon fiber. The sensor has been applied to studies of NO release from a single endothelial cell in a pulmonary artery, as well as for the determination of NO in blood.

Simultaneous Measurements of Ca2+ and Nitric Oxide in Bradykinin-Stimulated Vascular Endothelial Cells

The production of endothelium-derived relaxing factor (EDRF), known to be nitric oxide (NO), is triggered by a rise in the cytoplasmic calcium concentration ([Ca2+]i) subsequent to receptor binding of vasoactive agonists. In vascular endothelial cells, NO is synthesized from L-arginine by the Ca2+/calmodulin-dependent NO synthase. In this study, we report the first simultaneous measurements of [Ca2+]i and [NO] at the level of single endothelial cells. In cultured bovine aortic endothelial cells, extracellular application of bradykinin (BK, 10 to 20 mumol/L) caused transient (sometimes oscillatory) increase in [Ca2+]i, which was measured with the fluorescent Ca2+ indicator fura 2 and fluorescence imaging microscopy. BK caused an increase in [Ca2+]i, primarily through release from intracellular stores. Under identical experimental conditions, BK caused a transient increase in [NO], which was measured by application of a porphyrinic NO microsensor. [NO] peaked at approximately 0.5 mumol/L. Simultaneous measurements of [Ca2+]i and [NO] in BK-stimulated endothelial cells revealed that a transient increase in [Ca2+]i was rapidly followed by an increase in [NO] that outlasted the [Ca2+]i transient.

HMG-CoA reductase inhibition improves endothelial cell function and inhibits smooth muscle cell proliferation in human saphenous veins ☆

OBJECTIVESnThis study examined effects of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitor cerivastatin on human saphenous vein (SV), endothelial cells (EC) and smooth muscle cells (SMC).nnnBACKGROUNDnVenous bypass graft failure involves EC dysfunction and SMC proliferation. Substances that improve EC function and inhibit SMC proliferation would be of clinical relevance.nnnMETHODSnBoth EC and SMC were isolated from SV. Endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) production were analyzed by immunoblotting and porphyrinic microsensor. The SMC proliferation was assayed by 3H-thymidine incorporation. Protein kinases and cell cycle regulators were analyzed by immunoblotting.nnnRESULTSnCerivastatin (10(-9) to 10(-6) mol/liter) enhanced eNOS protein expression and NO release (about two-fold) in EC in response to Ca2+ ionophore (10(-6) mol/liter). This was fully abrogated by the HMG-CoA product mevanolate (2 x 10(-4) mol/liter). In SMC, platelet-derived growth factor (5 ng/ml) enhanced 3H-thymidine incorporation (298 +/- 23%, n = 4), activated cyclin-dependent kinase (Cdk2), phosphorylated Rb and down-regulated p27Kip1 (but not p21CiP1). Cerivastatin reduced the 3H-thymidine incorporation (164 +/- 11%, p < 0.01), inhibited Cdk2 activation and Rb phosphorylation, but did not prevent p27Kip1 down-regulation, nor p42mapk and p70S6K activation. Mevalonate abrogated the effects of cerivastatin on Cdk2 and Rb but only partially rescued the 3H-thymidine incorporation (from 164 +/- 11% to 211 +/- 13%, n = 4, p < 0.01).nnnCONCLUSIONSnIn humans, SVEC inhibition of HMG-CoA/mevalonate pathway contributes to the enhanced eNOS expression and NO release by cerivastatin, whereas in SMC, inhibition of this pathway only partially explains cerivastatin-induced cell growth arrest. Inhibition of mechanisms other than p42mapk and p70S6K or Cdk2 are also involved. These effects of cerivastatin could be important in treating venous bypass graft disease.

Direct electrochemical measurement of nitric oxide in vascular endothelium.

The endothelium plays a critical role in maintaining vascular tone by releasing vasoconstrictor and vasodilator substances. Endothelium-derived nitric oxide (NO) is a vasodilator rapidly inactivated by superoxide and by Fe(II) and Fe(III), all found in significant quantities in biological systems. Thus due to the short life of NO in tissue (t1/2 = 3-6 s), in situ quantification of NO is a challenging problem. We designed the present study to perform direct measurements of nitric oxide using the electrochemical porphyrinic sensor. The most significant advantages of this sensor is small size (0.5-8 microm), rapid response time (0.1-1 ms), and low detection limit (10(-9) mol l(-1)). The porphyrinic sensor was used for in vitro and in vivo measurements of NO in an isolated single cell or tissue. Effects of hypertension, endotoxemia, and ischemia/reperfusion on the release of NO and/or its interaction with superoxide (O2-) were delineated. In the single endothelial cell (rabbit endocardium), NO concentration was highest at the cell membrane (950 +/- 50 nmol l(-1)), decreasing exponentially with distance from cell, and becoming undetectable at distances beyond 50 microm. The endothelium of spontaneously hypertensive rats (SHR) released 35% less NO (580 +/- 30 nmol l(-1)) than that of normotensive rats (920 +/- 50 nmol l(-1)), due to the higher production of O2- in SHR rats. Endothelial NO synthase (eNOS) generated NO (140 +/- 20 nmol l(-1)) in lung during the acute phase (first 10-15 min) of endotoxemia, followed by production of NO by inducible NOS. High production of O2- was observed during the entire period of endotoxemia. Ischemia (lower limb of rabbit) caused a significant increase of NO peaking at 15 min and decreasing thereafter, also due to O2- production.

Late Treatment With Ramipril Increases Survival in Old Spontaneously Hypertensive Rats

Spontaneously hypertensive rats (SHR) begin to die from cardiovascular complications at approximately 15 months of age. We tested whether chronic ACE-inhibitor treatment would extend the lifespan of such old animals. We also studied cardiac hypertrophy and function, endothelial function and expression, and activity of NO synthase (eNOS). One hundred 15-month-old SHR were randomized into 3 groups, control (n=10), placebo-treated (n=45), and ramipril-treated with an antihypertensive dose of 1 mg. kg(-1). d(-1) in drinking water (n=45). Ex vivo experiments were performed after 15 months (control) and 21 months, when approximately 80% of the placebo group had died. Late treatment with ramipril significantly extended lifespan of the animals from 21 to 30 months. Fully established cardiac hypertrophy, observed in placebo-treated animals and in controls, was significantly reversed by ramipril treatment. In isolated working hearts, a significantly improved function associated with increased cardiac eNOS expression was seen versus placebo and control hearts. Endothelial dysfunction in isolated aortic rings from control and placebo-treated SHR was significantly improved by ACE inhibition and associated with enhanced NO release. Late treatment of SHR with the ACE inhibitor ramipril extended lifespan from 21 to 30 months, which is comparable to the lifespan of untreated normotensive Wistar-Kyoto rats. This lifespan extension, probably due to blood pressure reduction, correlated with increased eNOS expression and activity followed by a regression of left ventricular hypertrophy and cardiac and vascular dysfunction.

Determination of nitric oxide saturated (stock) solution by chronoamperometry on a porphyrine microelectrode

Abstract A porphyrinic-based microelectrode was applied for the determination of nitric oxide (NO) saturated solution by absolute chronoamperometry. This method is based upon the separation of the current component limited by linear diffusion from that limited by the cylindrical contribution to the total flux of the determined substance to the cylindrical electrode. Precision and accuracy were tested and compared with the UV/VIS Griess method. The concentration of saturated NO solution was 1.74±0.03 mmol−1.