Visualization of nitric oxide formation in cell cultures and living tissue

Abstract

We have visualized nitric oxide (NO) released from cell cultures and living tissue. NO was visualized by a reaction with luminol and hydrogen peroxide to yield photons which were counted using a microscope coupled to a photon counting camera. Murine macrophages were activated with interferon-gamma (IFN-gamma) and endotoxin (LPS). Cultured endothelial cells were stimulated with bradykinin, and neurones in the guinea-pig myenteric plexus and the rabbit hypogastric nerve trunk were electrically stimulated. There was a marked increase in photons emitted from the cultured cells as well as from the living tissues during stimulation. The stimulation-induced photon emission was markedly reduced by inhibition of nitric oxide synthase (NOS); removal of L-arginine from the medium also decreased photon counts. The present method allowed integration times in the order of minutes to improve signal-to-noise ratio. However, the high sensitivity of this method also makes it possible to generate an image in seconds, allowing the production of real time films. Photon emission was enhanced under conditions known to increase NO production, and diminished in the presence of NO inhibitors. Thus, this method has demonstrated specificity for the L-arginine:NO pathway from a wide range of biological sources such as cultured cells and living tissues, and has the potential for real time imaging of NO formation, with high temporal and spatial resolution.

Specific Visualization of Nitric Oxide in the Vasculature with Two-Photon Microscopy Using a Copper Based Fluorescent Probe

“…Next, the reaction of HCAECs to the addition of either H 2 O 2 (150 µM) or Ach (10 µM) as NO generator was tested at 45min after stimulation, both without and in the presence of L- N G -nitroarginine methyl ester (L-NAME), a widely used nitric oxide synthesis inhibitor [7,17,19,20,23]. Based on past experience, we used low-mid passage HCAECs (passage <6) for cellular studies because cells cultured at high passage number (above passage 9) lose their ability to respond to ACh-induced NO synthesis.…”

“…of NO complicate real time detection [7,8,9]. Hence, little is known about the time course and diffusion profile of endogenously produced NO.…”

“…We used fluorescent probe-based imaging methods to study NO dynamics. The high sensitivity, spatial resolution, and experimental feasibility make fluorescent-based methods the preferred imaging modality [6,7,8]. An added advantage of this strategy is that structural and functional imaging can be executed simultaneously [5,10].…”

The Distribution of Primary Nitric Oxide Synthase- and Parvalbumin- Immunoreactive Afferents in the Dorsal Funiculus of the Lumbosacral Spinal Cord in a Dog

“…This assumption is based on the results obtained using the luminol chemiluminiscence method allowing for visualization of NO formation (Wiklund et al, 1997(Wiklund et al, , 1999. Notwithstanding that our present study is aimed mainly at a detailed description of the distribution pattern in CI-CV in the DF of lumbosacral segments, it may be linked with a recent study dealing with the monosynaptic Ia-motoneuron pathway in the dog (Maršala et al, 2005), which described the nitrergic afferent limb of the stretch reflex circuit, and disclosed proprioceptive nNOS-IR terminal arbors and Ia boutons in terminal regions detected previously by tract-tracing methods in lamina VI and VII (Maxwell and Bannatyne, 1983;Fyffe and Light, 1984), Clarke's column (Walmsley et al, 1985;Nicol and Walmsley, 1991), and lamina IX (Ishizuka et al, 1979;Hongo et al, 1987;Pierce and Mendell, 1993).…”

Activation of the nitric oxide response in gilthead seabream after experimental infection with Photobacterium damselae subsp. piscicida

“…Two NOS isoforms predominantly expressed in neurones (nNOS) and endothelial cells (eNOS) are constitutively expressed enzymes which generate transient and limited amounts of NO required to maintain the vascular tone [2], retrograde synaptic signalling in neurons [3e5] and tissue homeostasis [6]. In contrast, a third inducible enzyme (iNOS) is regulated at the transcriptional and transductional level in macrophages and a variety of non-immunocompetent cells and once expressed generates large and sustained amounts of NO involved in tissue remodelling, inflammation and host-defence [7,8].…”