In this classic experiment by Folkow (1948), the hind limb of an anaesthetised cat was isolated surgically, and blood flow through the limb was measured in response to stimulation of the sympathetic nerves from L3 to L5. This smoke drum trace records changes in limb arterial pressure and blood flow in response to sympathetic stimulation. Under control conditions sympathetic nerve stimulation causes a fall in blood pressure and an increase in flow—that is, vasodilatation (left panel). In the presence of atropine, a muscarinic receptor antagonist, the fall in blood pressure is abolished, and blood flow decreases, indicating a degree of vasoconstriction (right panel) (reproduced with the kind permission of Blackwell Publishing).²

The famous experiment that first described the existence of an endothelium derived relaxing factor. In these experiments, rings of rabbit aorta were suspended in organ chambers and made to contract with noradrenaline (NA). When the contraction was stable, acetylcholine (ACh) was added cumulatively (1 × 10⁻⁸ M, 1 × 10⁻⁷ M, etc). (A) In “unrubbed” rings—that is, rings of aorta in which the endothelium was intact, ACh caused a concentration dependent relaxation. (B) In aortic rings where the endothelium had been deliberately removed by rubbing the vessel lumen, ACh had no effect (or sometimes caused a contraction) (reproduced with the kind permission of the authors and the Nature Publishing Group).5

This rather complex set up helped to establish the diffusibility and the transient nature of endothelium dependent relaxing factor (EDRF). When the ring of canine coronary artery was sitting under the metal pipe (direct superfusion), acetylcholine (ACh) added to the superfusing mixture caused the coronary artery to contract because it had no endothelium. When the superfusing mixture passed through a femoral artery with endothelium, the ACh stimulated the release of an EDRF, which then caused relaxation of the underlying coronary artery. This part of the experiment established that released EDRF could travel a short distance, and thus could diffuse from the endothelial cell to the smooth muscle. To show the transient nature of EDRF, the superfusate from the femoral artery was passed through a series of pipes to increase the transit time before it reached the coronary artery (see right of figure; times are 8, 30, 60, and 120 seconds). The longer the transit time, the smaller the relaxation. The effect of increasing transit time could be partially offset by infusing superoxide dismutase at site 2 or site 3. This experiment indicated that EDRF was an unstable molecule that could be rendered inactive by superoxide ion (reproduced with the kind permission of the authors and the American Physiological Society).22

Working hypothesis by Pearce and colleagues for the role of nitric oxide (NO) in mitochondrial respiration. Under normal conditions, protons (4H⁺) and electrons from the citric acid cycle are passed along the electron transport chain, from coenzyme Q to cytochromes b, c₁, c, and a + a₃ (4c²⁺), generating energy in the process (oxidative phosphorylation). At the end of the chain the electrons and protons combine with oxygen to form water. Cytochrome c oxidase catalyses the final transfer of electrons from cytochrome c to cytochromes a + a₃ (the fast reaction shown at the bottom of the diagram). NO has a high affinity for cytochrome c oxidase, and will bind to this mitochondrial enzyme more readily than does molecular oxygen (O₂). However, molecular oxygen can diffuse into the active site where the NO is bound, and may gain an electron from one of the metal ion cofactors present (the authors suggest CuB). This results in the formation of superoxide—that is, O₂⁻. The superoxide immediately reacts with the NO, forming peroxynitrite, which then further oxidises the metal ion cofactor to produce water and nitrite ion. Because all intermediates are enzyme associated, no superoxide or peroxynitrite is released into free solution. Thus, under normal physiological conditions, cytochrome c oxidase suppresses peroxynitrite formation by scavenging available NO and preventing it from reacting with superoxide in free solution (the slow reaction shown on the right of the diagram). Because the overall reaction is slow, and the affinity of NO for cytochrome oxidase is high, normal mitochondrial respiration is inhibited. Although this interpretation of the mitochondrial/NO interaction is intriguing, other proposed inhibitory reaction cycles (for example, mechanisms described in: Torres and colleagues64 and Guiffre and colleagues65) are at least equally plausible. Indeed, there may be more than one mechanism operative, depending on whether NO or O₂ is the first to enter the active site (reproduced with the kind permission of the authors and the American Society for Chemistry and Molecular Biology).63

Outline of the classic “organ” or “tissue” chamber set up on which much of the important work in 20th century pharmacology was carried out, including the elucidation of the identity of nitric oxide.

Similar experiments to those depicted in fig 3 ▶ were also carried out using rabbit aorta. In all experiments, a stable contraction is reached with phenylephrine (PE) before the relaxation in response to acetylcholine (ACh) is examined. (A) Vessel ring with endothelium (+EC) relaxes in response to ACh. (B) This vessel ring also has an intact endothelium but the relaxation to ACh is abolished by the presence of haemoglobin (HB). (C) Control response in vessel with endothelium. (D) This vessel has an intact endothelium, but in the presence of the inhibitor of guanylyl cyclase, methylene blue (MB), the relaxation to ACh is abolished and converted into a contraction (reproduced with the kind permission of the authors and the American Society of Pharmacology and Experimental Therapeutics).20

The sequence of deductive reasoning that led to the discovery of nitric oxide. EDRF, endothelium dependent relaxing factor.

Main actions of nitric oxide (NO) and reactive nitrogen species (RNS) on mitochondria. NO specifically and reversibly inhibits cytochrome oxidase (complex IV); RNS inactivate multiple respiratory complexes (I, II, IV), the ATP synthetase (ATPase), aconitase, and Mn superoxide dismutase (MnSOD). RNS activate the permeability transition pore (PTP). Activation of PTP may lead to cytochrome c release either as a result of swelling induced rupture of the outer membrane, or through specific pores. Cytochrome c release may also be induced by the insertion of proapoptotic BH₃ proteins into the outer membrane (reproduced with the kind permission of the authors and Taylor and Francis, Inc, http://www.routledge-ny.com).62 Ant, ATP/ADP translocator; CP, cyclophilin D; CrK, creatinine phosphokinase; VDAC, voltage dependent anion channel.