Nitrogen: The unsung hero of vascular physiology

THE NERVES AND VESSELS

Our nitric oxide journey must start with understanding the link between nerves and blood vessels. In the 19th century, the French physiologist Dr. Claude Bernard² performed experiments by severing the cervical sympathetic ganglion in rabbits, which resulted in increased “calorification” (heat production) and vasodilation (widening of visible vessels in the thin skin of an albino rabbit’s ear). This was the first clear demonstration of neural regulation of blood vessel physiology.³ Decades would pass, during which there was much controversy and arguing among neurologists, before we understood how the nerves influence vasodilation or vasoconstriction—was it electricity or some kind of neurotransmitter?

The hormone adrenaline was discovered in 1894 and found to mimic the sympathetic nervous system, triggering vasoconstriction and elevating blood pressure.⁴ Acetylcholine, a neurotransmitter, was later discovered to be a potent vasodilator, but the mystery of its full involvement in physiology took much longer to unravel because acetylcholine was difficult to detect in tissues. Acetylcholine is tightly regulated, so it is rapidly broken down by acetylcholinesterase on release from the nerve. The breakthrough came by using eserine, an extract from the Calabar bean and a known neurotoxin, which inhibited acetylcholinesterase and prevented acetylcholine from breaking down.⁴ Once acetylcholine could be measured, its function as a key mediator of the parasympathetic nervous system, including lowering blood pressure by vasodilation, was quickly recognized.

NITRIC OXIDE’S LINK TO ACETYLCHOLINE

In 1976, Furchgott and Zawadzski⁵ used a bioassay and tissue culture to understand the mechanism of how acetylcholine interacts with vascular smooth muscle to cause relaxation. Metal probes inserted into the lumen of a rabbit aorta measured the force exerted from the smooth muscle contracting or dilating against the probes when the aorta was exposed to various substances such as histamine, serotonin, angiotensin, and acetylcholine. The 2 doctors recognized a problem: when the isolated rabbit aorta was exposed to acetylcholine, no relaxation occurred. In the process of preparing the tissue, filter paper was used to rub the endothelial cells off the lumen of the aorta to allow acetylcholine direct access to the smooth muscle. They repeated the experiment multiple times until finally trying the experiment without rubbing away the endothelial lining, and voilà! The rabbit aorta dilated on contact with acetylcholine.⁵

The discovery that endothelial cells were important to vasodilation was critical. It would later be found that acetylcholine activates the formation of nitric oxide, as a gas, within endothelial cells, which is then diffused out of the cells and into the neighboring smooth muscle, triggering additional second messengers (eg, cyclic guanosine monophosphate and cyclic adenosine monophosphate) and smooth muscle relaxation.⁶ This was a major achievement in understanding vascular physiology.

THERAPEUTIC USE OF NITROGEN AND TROUBLE WITH TACHYPHYLAXIS

As our understanding of nitric oxide’s role in vasodilation evolved, treatment of hypertension was the obvious medical application, but there was a catch. Nitrogen-based compounds were used therapeutically long before we knew the role nitric oxide played in vasodilation. In the middle 19th century, nitroglycerin (which gets broken down to nitric oxide) began to be used in patients with anginal chest pain.⁷ It’s not clear why nitroglycerin was chosen to treat angina, but it’s possibly because nitroglycerin ingestion caused tachycardia and thus had a clear physiologic effect on the heart.⁶ However, a major limitation of therapeutic nitrogen was recognized very early: tachyphylaxis. In the early days of treating angina, a doctor noted his patient’s chest pain responded to inhaling 5 to 10 drops of nitrite from a cloth, but efficacy waned with continued use, and the patient required increased doses to have the same response.⁷ It became clear that, if nitrogen compounds were given continuously, patients rapidly developed a tolerance.

At the dawn of the 20th century, workers in trinitrotoluene factories were also aware that constant exposure to nitrate-containing compounds led to tachyphylaxis.⁷ Workers often complained of headaches and a racing heart on Monday, and their symptoms would slowly resolve over the course of the week. Nitrate tolerance is short-lived, so after a day or 2 off on the weekends, symptoms would start again on Monday. This phenomenon was referred to as Monday disease.⁷ It became practice for some workers to take home pieces of nitrate over the weekend to rub on their skin until returning to work on Monday, continuing the exposure and preventing the headaches they experienced when returning to work.^7–9

CIRCUMVENTING TACHYPHYLAXIS AND THE BREAKTHROUGH

Like most things in medicine, overcoming nitrogen tolerance is complicated because nitric oxide doesn’t act alone. Nitric oxide stimulates smooth muscle relaxation and vasodilation, but a series of second messengers are also triggered once nitric oxide diffuses into the smooth muscle cell, leading to decreased calcium levels and smooth muscle relaxation.⁶ Given that tachyphylaxis develops in response to exogenous nitric oxide, could the second messengers, instead of nitric oxide, be manipulated to increase vasodilation and bypass tachyphylaxis?

In the middle 1980s, Pfizer’s cardiovascular research division was looking for a novel target to treat hypertension and chose phosphodiesterase type 5 (PDE5).⁶ PDE5 breaks down the second messengers responding to nitric oxide, decreasing the vasodilatory response. The goal was to inhibit PDE5, thus allowing the continuation of smooth muscle response to nitric oxide. Sildenafil was developed with hopes of treating hypertension and angina through PDE5 inhibition. The results of the initial trials are widely known in the medical world—men on the PDE5 inhibitor noted the development of erections.⁶ The pursuit of sildenafil as a treatment for angina or hypertension was sidelined, and it became a blockbuster medication to treat erectile dysfunction.

THE PULMONARY ARTERIAL HYPERTENSION CONNECTION

Research on sildenafil provided evidence that not all vascular physiology is the same. The effect of PDE5 inhibitors on lowering peripheral blood pressure was modest, but certain tissues, such as the corpus cavernosum of the penis, have a profound response to the drug.⁶ Further research explored the role of PDE5 in vascular territories throughout the body. Using a combination of animal models and human tissue, a particularly high expression of PDE5 was found in lung tissue.^6,10 Nitric oxide turns out to be an important regulator of oxygenation and blood flow (ventilation-perfusion matching) in the pulmonary vessels. As alveoli are aerated and expand, vascular endothelial cells are stretched and release nitric oxide, leading to vasodilation and increased blood flow to the well-oxygenated alveoli.⁹ With a clearer sense of the roles nitric oxide and PDE5 play in pulmonary physiology, attention turned once again to treating pulmonary arterial hypertension with sildenafil.

Experiments with a chronically hypoxic rodent model demonstrated that treatment with a PDE5 inhibitor protected the mice from developing pulmonary hypertension.⁶ Soon, multiple case reports were published on the efficacy of PDE5 inhibition in patients with pulmonary arterial hypertension, resulting in the SUPER-1 (Sildenafil Use in Pulmonary Hypertension) trial in 2002¹¹ that showed improvements in the 6-minute walk as well as pulmonary hemodynamics. Based on these favorable outcomes, the US Food and Drug Administration approved sildenafil in 2005 for the treatment of pulmonary arterial hypertension. Multiple medications are now approved to treat pulmonary arterial hypertension, including 2 PDE5 inhibitors.

DISCLOSURES

Dr. Brown has disclosed consulting and teaching and speaking for Amgen and Chemocentryx.