Physiologically relevant measurements of nitric oxide in cardiovascular research using electrochemical microsensors

Nitric oxide (NO) plays an important role in the regulation of blood flow. Pharmacological tools and a series of other techniques have been developed for studying the NO/L-arginine pathway, but it has proved difficult to make a quantitative link between effect and tissue NO concentration. NO microsensors have been applied with success for the measurement of NO in suspensions of mitochondria and cells, such as platelets and leukocytes, and in cell cultures, which together with other interventions or measurements are particularly useful for the examination of cell signalling related to the NO/L-arginine pathway. In isolated vascular segments, studies using the NO microsensor have defined the relationship between NO concentration and relaxation and revealed residual NO release in the presence of NO synthase inhibitors. Moreover, simultaneous measurements of NO concentration and vasorelaxation in isometric preparations have shown that agonist-induced relaxation is L-arginine dependent and NO release is reduced in hypertension. By placing NO microsensors in catheters, it is possible to measure NO in the living animal and man. This approach has been applied for the measurements of NO concentration in relation to increases in flow, erection, in conditions of hypoxia, and in endotoxemia. However, further methodological development of NO microsensors is necessary to avoid the influence of changes in temperature, pH and oxygen on the measurements.     

Elevated pressure selectively blunts flow-evoked vasodilatation in rat mesenteric small arteries

BACKGROUND AND PURPOSE: The present study investigated mechanisms underlying impaired endothelium-dependent vasodilatation elicited by elevating the intraluminal pressure in rat mesenteric small arteries. EXPERIMENTAL APPROACH: Arterial segments (internal diameter 316+/-2 microm, n=86) were mounted in a pressure myograph. The effect of elevating pressure from 50 to 120 mmHg for 1 h before resetting it to 50 mmHg was studied on endothelium-dependent vasodilatation. KEY RESULTS: In arteries constricted with U46619 in the presence of indomethacin, shear stress generated by flow, evoked vasodilatation that was abolished by an inhibitor of nitric oxide (NO) synthase, asymmetric dimethylarginine (1 mM), whereas acetylcholine-induced vasodilatation was unchanged. After elevation of intraluminal pressure for 1 h and then resetting it to 50 mmHg, vasodilatation induced by shear stress and the NO donor, S-nitrosopenicillamine was inhibited, while vasodilatation induced by a guanylyl cyclase activator, BAY 412272, and acetylcholine was unaltered. Superoxide levels sensitive to polyethylene glycol superoxide dismutase were increased in segments exposed to elevated pressure. A superoxide scavenger, tempol (300 microM), a general endothelin receptor antagonist, SB 217242 and the selective ET(A) receptor antagonist, BQ 123 preserved shear stress-evoked vasodilatation. CONCLUSIONS AND IMPLICATIONS: The present study shows that transient exposure to an elevated intraluminal pressure selectively inhibits flow-evoked NO-mediated vasodilatation, probably through activation of endothelin receptors and increased formation of superoxide. In contrast, elevation of pressure did not affect the acetylcholine-evoked endothelium-derived hyperpolarizing factor type vasodilatation in mesenteric small arteries.     

Cohabitation with a sick cage mate: consequences on behavior and on ehrlich tumor growth

The present study analyzed the effects of cohabitation for 11 days with a sick cage mate on behavior and Ehrlich tumor growth in mice. Pairs of female mice were divided into one control and one experimental group. One mouse of each control pair was kept undisturbed and called 'healthy companion' (HC). One animal of each experimental pair of mice was inoculated (i.p.) with 5 x 10(6) Ehrlich tumor cells, and the other, the object of this study, was called 'sick companion' (SC). The SC mice presented: (1) increased activity in an open field, (2) increased number of entries and of movements within the plus-maze open arms, (3) similar levels of plus-maze closed-arm exploration, (4) a decrease in the exploratory activity in a hole board, (5) a decrease in the number of white but not red blood cells, and (6) similar corticosterone serum levels. Eleven days after cohabitation with a conspecific, HC and SC mice were injected with 5 x10(6) Ehrlich tumor cells. Results showed that SC animals presented decreased resistance to the ascitic form of the Ehrlich tumor. The observed data provide experimental evidence that psychosocial stress induced by cohabitation with a sick cage mate changed at the same time some behavioral and physiological parameters, and decreased resistance to Ehrlich tumor. These data are discussed in the light of a possible neuroimmune system interaction.     

Role for Tyrosine Kinases in Contraction of Rat Penile Small Arteries

IntroductionThe devasting effect of cancer and treatment thereof contribute to sexual dysfunction. Recently, a series of tyrosine kinase inhibitors have been approved either as add-on or for targeted treatment of cancer. However, tyrosine kinases are not only important for cell growth and proliferation, but also in regulation of vascular tone.AimThe present study investigated whether tyrosine kinases contribute to contractility in rat penile arteries, and addressed whether they are involved in calcium entry and/or related to the RhoA/Rho-kinase pathway.MethodsSegments of the rat dorsal penile artery were mounted in microvascular myographs for simultaneous measurements of intracellular calcium concentration ([Ca²⁺]_i) and tension, and tyrosine kinase activity, and phosphorylation of 20-kDa myosin light chain (MLC₂₀) was measured in dorsal penile artery homogenates.Main Outcome MeasuresIn vitro evidence for contractility and changes in intracellular Ca²⁺ in small penile arteries.ResultsSodium vanadate (Na₃VO₄, 1 mM), a tyrosine phosphatase inhibitor, increased [Ca²⁺]_i and tension. A l-type calcium channel blocker, nifedipine (1 µM), markedly reduced Na₃VO₄-evoked increases in [Ca²⁺]_i and tension. A thromboxane analog, U46619, increased TK activity. In contrast to the inactive analogue, genistein, a general TK inhibitor, concentration-dependently reduced both U46619-evoked contraction, and [Ca²⁺]_i. U46619-induced contraction was markedly inhibited by tyrphostin A23 and bis-tyrphostin, whereas there was no effect of the tyrosine kinase c-Src inhibitor, herbimycin A. Tyrphostin A23 suppressed U46619-mediated phosphorylation of MLC₂₀.ConclusionsThis study suggests that activation of tyrosine kinases is involved in contraction of rat penile smooth muscle probably by regulation of calcium entry through l-type calcium channels. These findings may have implications for the selections of novel add on anticancer treatments, e.g., inhibitors of tyrosine kinases, and for novel approaches to treat erectile dysfunction. Villalba N, Kun A, Stankevicius E, and Simonsen U. Role for tyrosine kinases in contraction of rat penile small arteries.     

KRAS,NRAS and BRAF mutations in colorectal cancer and melanoma

Cancers are the group of diseases, which arise because of the uncontrolled behavior of some of the genes in our cells. There are possibilities of gene amplifications, overexpressions, deletions and other anomalies which might lead to the development and spread of cancer. One of the most dangerous ways to the cancers is the mutations of the genes. The mutated genes can start unstoppable proliferation of cells, their uncontrolled motility, protection from apoptosis, the DNA mutation enhancement as well as other anomalies, leading to the cancer. This review focuses on the genes, which are frequently mutated in various cancers and are known to be important in the advance and progression of colorectal cancer and melanoma, namely KRAS, NRAS and BRAF.     

Time Attributable to Obesity in Surgery: A Multi-specialty Report on Day-of-Surgery Resource Utilization from 189,264 Cases

Background

Obesity presents a unique challenge in caring for surgical patients and has been shown to adversely affect outcomes for several operative procedures. However, quantitative data on surgical resource utilization attributable to obesity are scarce. The aim of this study was to quantify day-of-surgery resource utilization by degree of obesity.

Methods

Patients undergoing one of 14 common surgical procedures at our multicenter institution between 2008 and 2017 were identified from our operating room management databank. Multiple-variable regression analysis (MVRA) was performed to quantify the independent effect of body mass index (BMI) category on day-of-surgery resource utilization variables including procedure time, non-operative OR time, PACU time, number of unique staff and number of supplies used. Trends in mean BMI were examined for each procedure studied.

Results

MVRA of the 189,264 cases in the database revealed consistently significant (p?<?0.05) stepwise increase in procedure time by BMI category for all procedures studied. Non-operative OR time was also significantly prolonged, though to a lesser degree. There was no significant impact on number of unique staff, supplies utilized or PACU time by BMI category. Procedures most impacted by BMI category in terms of resource utilization were ventral hernia repair, laminectomy and hysterectomy.

Conclusion

Our study quantified day-of-surgery resource utilization for 14 major surgical procedures by BMI category. The need for additional resources to accommodate patients in higher BMI groups was consistent across all procedures studied and was primarily reflected by lengthened operative times.

     

NS11021, a novel opener of large-conductance Ca2+-activated K+ channels,enhances erectile responses in rats

Background and purpose:

Large-conductance Ca²⁺-activated K⁺ channels (BK_Ca), located on the arterial and corporal smooth muscle, are potential targets for treatment of erectile dysfunction (ED). This study investigated whether NS11021 (1-(3,5-Bis-trifluoromethyl-phenyl)-3-[4-bromo-2-(1H-tetrazol-5-yl)-phenyl]-thiourea), a novel opener of BK_Ca channels, relaxes erectile tissue in vitro and enhances erectile responses in intact rats. The effects were compared with sildenafil, an inhibitor of phosphodiesterase type 5.

Experimental approach:

Patch clamp was used to record whole cell current in rat isolated corpus cavernosum smooth muscle cells (SMCs) and human umbilical vein endothelial cells (HUVECs). Isometric tension was measured in intracavernous arterial rings and corpus cavernosum strips isolated from rats and men, and simultaneous measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and tension were performed in intracavernous arteries. Erectile response was measured in anaesthetized rats.

Key results:

In patch clamp recordings, NS11021 increased currents sensitive to the selective BK_Ca channel blocker, iberiotoxin (IbTX) in SMCs, but did not modulate K⁺ current in HUVECs. NS11021 reduced [Ca²⁺]_i and tension in penile arteries. IbTX inhibited the vasorelaxation induced by NS11021 and sildenafil in human erectile tissue. NS11021 and sildenafil but not vehicle increased erectile responses in anaesthetized rats, an effect which was abolished after pretreatment with tetraethylammonium.

Conclusions and implications:

NS11021 leads to relaxation of both intracavernous arteries and corpus cavernosum strips primarily through opening of BK_Ca channels. It is also effective in facilitating erectile responses in anaesthetized rats. These results suggest a potential for use of BK_Ca openers in the treatment of ED.     

Blunted acetylcholine relaxation and nitric oxide release in arteries from renal hypertensive rats

OBJECTIVE : Investigation of the effect of hypertension on endothelium-dependent relaxation and release of nitric oxide (NO) in normotensive and renal hypertensive rats. DESIGN AND METHODS : Sprague-Dawley rats were randomly allocated into two groups: uninephrectomized controls and one-kidney one-clip (Goldblatt hypertension) hypertensive rats, a non-renin dependent model of hypertension. After 10 weeks and in the presence of the cyclooxygenase inhibitor indomethacin, simultaneous measurements of the NO concentration, measured with a NO-specific microelectrode and endothelium-dependent relaxation were performed in isolated rat superior mesenteric arteries. RESULTS : Addition of the NO scavenger, oxyhaemoglobin, showed that basal NO concentration was unaltered in arterial segments from hypertensive rats. In norepinephrine-contracted arteries, acetylcholine increased the NO concentration and caused relaxations, and both parameters were significantly reduced in renal hypertensive arteries. Relaxations induced by the NO donor, S-nitroso-N-acetylpenicillamine were reduced. The superoxide scavenger, superoxide dismutase, and the NO synthase substrate, l-arginine, did not change the increase in NO concentration or acetylcholine relaxation in arteries from normotensive or renal hypertensive animals. In contrast, the NO synthase inhibitor, asymmetric dimethyl l-arginine, reduced the NO concentration and acetylcholine relaxation, while these responses were abolished in the presence of oxyhaemoglobin. CONCLUSIONS : This study provides direct evidence that reduced endothelium-dependent relaxations in the superior mesenteric artery from renal hypertensive rats is due, at least in part, to diminished NO release. The reduced NO release and relaxation persist in the presence of excess of substrate for NO synthase.