In vitro simultaneous measurements of relaxation and nitric oxide concentration in rat superior mesenteric artery - PubMed (original) (raw)

In vitro simultaneous measurements of relaxation and nitric oxide concentration in rat superior mesenteric artery

U Simonsen et al. J Physiol. 1999.

Abstract

1. The relationship between nitric oxide (NO) concentration measured with an NO-specific microelectrode and endothelium-dependent relaxation was investigated in isolated rat superior mesenteric artery contracted with 1 microM noradrenaline. 2. Acetylcholine (10 microM) induced endothelium-dependent simultaneous increases in luminal NO concentration of 21 +/- 6 nM, and relaxations with pD2 values and maximum of 6.95 +/- 0.32 and 97.5 +/- 0.7 % (n = 7), respectively. An inhibitor of NO synthase, N G-nitro-L-arginine (L-NOARG, 100 microM) inhibited the relaxations and increases in NO concentration induced by acetylcholine. 3. Oxyhaemoglobin (10 microM) reversed the relaxations and increases in NO concentrations induced by acetylcholine, S-nitroso-N-acetylpenicillamine (SNAP) and S-morpholino-sydnonimine (SIN-1), but not the relaxations induced with forskolin. Oxyhaemoglobin also decreased the NO concentration below baseline level. 4. In the presence of L-NOARG (100 microM), a small relaxation to acetylcholine (10 microM) of noradrenaline-contracted segments was still seen; oxyhaemogobin inhibited this relaxation and decreased the NO concentration by 14 +/- 4 nM (n = 4). 5. The NO concentration-relaxation relationship for acetylcholine resembled that for SNAP and SIN-1 more than for authentic NO. Thus while 7-17 nM NO induced half-maximal relaxations in response to SNAP or SIN-1, 378 +/- 129 nM NO (n = 4) was needed for half-maximal relaxation to authentic NO. 6. The present study provides direct evidence that the relaxation of the rat superior mesenteric artery with the endothelium-dependent vasodilator acetylcholine is correlated to the endogeneous release of NO. The study also suggests that NO mediates the L-NOARG-resistant relaxations in this artery, and that there is a basal NO release.

PubMed Disclaimer

Figures

Figure 1

Figure 1. Calibration of NO-sensitive microelectrode

A, representative recording of the NO microelectrode calibration performed in a glass vial containing PSS at 37 °C with constant stirring. The vertical bars below the trace indicate injection of increasing concentrations of NO. The horizontal bar indicates time. B, linear regression analysis of the relationship between amount of NO added and output of the electrode for one electrode tip (ISONOP30), _r_2 = 0.9928, 1 pA = 3.0 nM. The inset shows the relationship for the lowest concentrations with this electrode. C, linear regression analyses of relationship for authentic NO (○) and chemical titration (•) versus current output for a membrane-type electrode, showing that both calibrations give the same result.

Figure 2

Figure 2. Increases in measured NO concentrations are independent of force measurements

Simultaneous measurements of changes in isometric force (upper traces) and NO concentration (lower traces) in an endothelium-intact segment of rat superior mesenteric artery. A, acetylcholine (ACh, 3 μM) was added with the preparation at resting tension; B, ACh was added with the preparation contracted with 1 μM noradrenaline (NA). W, washout. The traces are representative of 4 experiments.

Figure 3

Figure 3. The effect of mechanical endothelial cell removal on simultaneously obtained relaxations and increases in NO concentration

Simultaneous measurements of force (upper traces) and NO concentration (lower traces) in an endothelium-intact (+E) segment of rat superior mesenteric artery contracted with 0.5 μM noradrenaline (NA) and relaxed with either 10 μM acetylcholine (ACh) (A), or 10 μM SNAP (B), and the lack of relaxation to ACh, but relaxation and increases in NO induced by SNAP in the same segment after mechanical endothelial cell removal (-E) (C). The traces are representative of 4 experiments. W, washout.

Figure 4

Figure 4. L-NOARG (100 μM) inhibits simultaneously measured relaxations and NO concentrations

Average relaxations (circles) and increases in NO concentration (squares) induced by increasing concentrations of ACh in rat superior mesenteric arterial segments in the absence (open symbols) and the presence (filled symbols) of the nitric oxide synthase inhibitor, L-NOARG (100 μM). Relaxations are relative to the initial contraction induced by noradrenaline (NA, 0.5-1 μM) and NO concentrations are given as increases above the level in the absence of acetylcholine. The points are means ±

s.e.m.

of 6 experiments. First concentration of acetylcholine causing significant relaxation or increase in NO concentration: *, P < 0.05.

Figure 5

Figure 5. Oxyhaemoglobin reverses relaxations and increases in NO concentration

Records showing simultaneous measurements of force (upper traces) and NO concentration (lower traces) in segments of the superior mesenteric artery. The vessels were contracted with 1 μM noradrenaline (NA) and relaxed with either 10 μM ACh (A), 10 μM SIN-1 (B), or 1 μM forskolin (C), and when a plateau was reached, oxyhaemoglobin (OxyHb, 10 μM) was added. The average results are summarized in Table 1.

Figure 6

Figure 6. Concentration-dependent relaxations and increases in NO concentration induced by exogenously added NO

Graphs showing average relaxations (○) and increases in NO concentrations (□) with increasing concentrations of SNAP (A), SIN-1 (B) and dissolved authentic NO (C) in the rat superior mesenteric artery precontracted with noradrenaline (NA, 0.5-1 μM). NO concentrations are given as increases above the level in the absence of NO donor. The results are means ±

s.e.m.

of 4-6 experiments. First concentration of NO donor causing significant relaxation or increase in NO concentration: *,P < 0.05.

Figure 7

Figure 7. Time relationship for simultaneously measured relaxations and increases in NO

Averaged relaxations (○) and increases in NO (□) at various times after the addition of 10 μM SNAP (A), 10 μM SIN-1 (B), and 50 nmol l−1 min−1 infused NO (C) in rat superior mesenteric arteries contracted with 0.5 μM noradrenaline (NA). The NO infusion (C) was stopped after 10 min (arrow), and this was followed by a fall in NO concentration and recovery of the noradrenaline-induced contraction. NO concentrations are given as increases above the level in the absence of acetylcholine. Each point represents the mean ±

s.e.m.

of 4-6 preparations.

Figure 8

Figure 8. Time relationship for simultaneously measured relaxations and endogenous NO concentrations

A, average of simultaneously obtained force (○) and NO concentration (□) curves for acetylcholine (10 μM) in segments of the rat mesenteric superior artery contracted with noradrenaline (0.5 μM). B, the same results as A with relaxation plotted against NO concentration. The results are means of 6 experiments and horizontal and vertical bars represent

s.e.m.

Similar articles

Cited by

References

    1. Bauer JA, Fung H-L. Photochemical generation of nitric oxide from nitro-containing compounds: possible relation to vascular photorelaxation phenomena. Life Sciences. 1993;54:PL1–4. - PubMed
    1. Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and the ugly. American Journal of Physiology. 1996;271:C1424–1437. - PubMed
    1. Bolotina VM, Najibi S, Palacino JJ, Pagano PJ, Cohen RA. Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature. 1994;368:850–853. - PubMed
    1. Broderick MP, Taha Z. Nitric oxide detection using a popular electrochemical sensor. World Precision Instruments, Satellite Symposium, 4th IBRO World Congress of Neuroscience, Kyoto, Japan. 1995:1–10.
    1. Butler AR, Rhodes P. Chemistry, analysis, and biological roles of S-nitrosothiols. Analytical Biochemistry. 1997;249:1–9. <10.1006/abio.1997.2129>. - DOI - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources