A Heart Attack, Backward — Part 3: Pressure, flow, and mouth chemistry

Most scientists show their results. Giles Brindley dropped his trousers.

In 1983, at a Urodynamics Society meeting in Las Vegas, Brindley gave a lecture on drug-induced erections. According to urologist Laurence Klotz, who was in the audience, Brindley had injected himself with a vasoactive drug in his hotel room before the talk. He arrived ready to present the evidence personally.

Slides apparently weren't enough.

Brindley lowered his trousers and underwear to demonstrate the resulting erection. Klotz's published recollection describes an audience whose evening had taken a considerable turn from the usual conference program. Klotz, 2005

A memorable presentation. A difficult one to follow after the coffee break.

The useful lesson for us is less theatrical: changing the behavior of smooth muscle can change what blood does in a tissue. That principle reaches well beyond erectile function. It gives us a way into the living, responsive vessel wall we need to understand this week.

Brindley's demonstration belongs to the history of erectile-dysfunction treatment. The identification of nitric oxide as an endothelial signal came through separate experiments, which we will get to shortly.

For now, everyone can keep their trousers on. We are going back inside an artery.

Last week we stayed inside the artery wall.

We found retained particles, cells carrying cholesterol, material that had not been cleared, and areas of mineralization. We also saw why the amount of plaque and the size of the channel are different measurements.

Now let the blood move again.

The wall we have been studying is alive. Its cells receive signals, make signals, and change how tightly the vessel contracts. To understand circulation, we need to follow that activity as well as the material accumulating in the wall.

Following one of those signals will take us from an artery to the tongue, through saliva, and back into the circulation. Before we make that trip, we need to separate a few things that are easy to collapse into one story.

If you missed it, Part 2 looks inside the plaque.

A blood pressure cuff tells us about pressure in an artery. Flow describes the volume of blood moving through a region over time. A pressure reading alone cannot tell us how much blood is reaching a particular piece of heart muscle.

Blood moves down a pressure gradient. The resistance along the route helps determine how much gets through. Small arteries and arterioles are especially important here: their surrounding smooth muscle can contract or relax, changing the resistance encountered by the moving blood.

Imagine following blood from a larger artery into its smaller branches. The channel has a structure, but its caliber also depends on what the muscle in the wall is doing. At a given pressure difference, a change in resistance can change flow. In a living person, pressure, pumping, and resistance can all change together.

That is why the familiar clogged-pipe picture only takes us so far. Last week's plaque described disease within the wall. This week's question is how that wall participates in controlling the circulation from moment to moment.

In 1980, Robert Furchgott and John Zawadzki investigated an inconsistency in experiments with isolated blood vessels. Acetylcholine could relax one preparation of rabbit aorta, yet fail to relax another.

The difference lay in how the tissue had been handled.

Rubbing the inner surface during preparation removed the endothelial lining and eliminated the relaxation response. When that surface was preserved, acetylcholine could still produce relaxation. The smooth muscle's response depended on cells lining the vessel releasing a substance that acted on it. Furchgott and Zawadzki, 1980

The lining had been easy to damage and easy to underestimate. A thin layer of cells was participating in the behavior of the much thicker muscle beside it.

The unknown signal became known as endothelium-derived relaxing factor, or EDRF. The name described where it came from and what it did. Identifying the molecule was the next problem.

In 1987, Palmer, Ferrige, and Moncada measured nitric oxide released by cultured endothelial cells. The amount and activity could account for the relaxing factor. NO and EDRF also behaved similarly when the researchers tested their instability and responses to substances that altered their effects. Palmer and colleagues, 1987

That same year, Ignarro and colleagues reported matching biological and chemical properties in preparations of arteries and veins. Both NO and EDRF increased cyclic GMP, an intracellular messenger associated with the relaxation response. Ignarro and colleagues, 1987

The signal was nitric oxide: one nitrogen atom joined to one oxygen atom.

For our walk backward through a heart attack, the important discovery is the conversation between neighboring cells. The endothelium can influence the muscle around it through a small, short-lived molecule. A vessel's behavior depends partly on whether that message is made, survives, and reaches its target.

Endothelial cells make NO from the amino acid L-arginine. In 1988, experiments with cultured porcine endothelial cells traced labeled nitrogen from L-arginine into the NO produced by those cells. That helped establish the starting material for the reaction. Palmer and colleagues, 1988

The enzyme responsible in the endothelium is endothelial nitric oxide synthase, usually abbreviated eNOS. Molecular studies subsequently identified and characterized this enzyme. Its name is a useful guide: it synthesizes nitric oxide. Lamas and colleagues, 1992

The reaction uses oxygen and produces citrulline alongside NO. This is an enzyme-dependent route, with cellular machinery doing the chemical work. White and Marletta, 1992

NO can diffuse into adjacent vascular smooth muscle. There it activates soluble guanylyl cyclase, an enzyme that produces cyclic GMP, or cGMP. This carries the signal forward inside the muscle cell toward relaxation. Earlier laboratory work had shown that NO could activate guanylyl cyclase and increase cGMP in tissue preparations. Arnold and colleagues, 1977

Hold the sequence in your mind: endothelial cell, NO, smooth muscle, relaxation.

That sequence explains a change in vascular tone. It does not describe removing lipid from a plaque or dissolving a clot already obstructing an artery.

Figure 1. Endothelial NO can signal adjacent smooth muscle to relax through soluble guanylyl cyclase and cGMP. A change in vascular tone can change resistance and flow; it does not show plaque removal or clot dissolution. Simplified schematic, not to scale.

A tissue preparation can reveal a mechanism. To understand its contribution to circulation, we also need experiments in people.

In 1989, Vallance, Collier, and Moncada infused an inhibitor of NO synthesis into the artery supplying healthy volunteers' forearms. Resting forearm blood flow fell. The response to acetylcholine was also reduced. Vallance and colleagues, 1989

The researchers had changed a chemical pathway without waiting for a plaque to grow. The vessels changed their behavior, and the amount of blood passing through the region changed with it.

This gives us a second timescale to place beside the history of the plaque. The wall can remodel over years. Its muscle can also respond to signals during an experiment. Both belong in our picture of circulation, but they answer different questions.

We have now followed one route to NO inside the vessel wall. Keep it separate as we move to another route that involves the mouth.

Three similar names appear in this part of the story: nitrate, nitrite, and nitric oxide. They describe different molecules, and the steps between them matter.

Nitrate is present in foods including leafy vegetables and beetroot. The body also generates nitrate through the metabolism of NO. Some circulating nitrate is taken up by the salivary glands and concentrated in saliva, bringing it into contact with the oral microbial community. Kapil and colleagues, 2013

Certain oral bacteria can convert nitrate into nitrite. Bacteria on the tongue participate in this chemistry. Once swallowed, nitrite becomes available for further reactions along the digestive route and, after absorption, in blood and tissues. Webb and colleagues, 2008

Saliva therefore does more than wet the tissues we examine. It also carries material from the circulation into a place where microbial activity can change it before it is swallowed again.

Calling that activity useful does not make every organism or every biofilm beneficial. The question here concerns a particular chemical function. It cannot be answered simply by counting all the bacteria in a mouth.

Nitrite is not a finished package of NO delivered unchanged to every organ. Its conversion depends on the chemistry around it.

In the acidic stomach, swallowed nitrite can contribute to NO formation. Human experiments detected substantial gastric NO production and showed that suppressing stomach acidity sharply reduced it. These experiments helped establish a chemical route operating in a very different environment from the endothelial cell. Lundberg and colleagues, 1994

Circulating nitrite can also participate in NO formation. In human forearm experiments, infused nitrite increased blood flow even when NO synthase was inhibited. Accompanying laboratory work showed that deoxygenated hemoglobin could reduce nitrite to NO, connecting nitrite bioactivation with oxygen availability. Cosby and colleagues, 2003

Now we can see why the two routes deserve separate arrows in a drawing. In one, eNOS makes NO from L-arginine using oxygen. In the other, nitrate can be processed through oral bacterial nitrite production, with subsequent NO generation influenced by conditions such as acidity and low oxygen.

They can contribute to related biology through different chemistry. Neither arrow means that the mouth produces all the body's NO.

Figure 2. The endothelial L-arginine/eNOS route and the nitrate–nitrite route involve different chemistry. Oral bacteria perform an important nitrate-to-nitrite step; subsequent NO formation depends on the local environment. Neither pathway means that the mouth supplies all the body's NO.

A useful way to test a pathway is to interrupt one step while leaving the starting material available.

Govoni and colleagues did this in a crossover experiment involving seven healthy volunteers. After a nitrate dose, a chlorhexidine-containing antibacterial rinse suppressed the conversion of nitrate to nitrite in saliva and markedly reduced the rise in circulating nitrite. Nitrate itself still accumulated in saliva and plasma. Govoni and colleagues, 2008

The material had arrived. Its processing had changed.

Webb and colleagues used a different interruption. After beetroot juice, healthy volunteers showed increased circulating nitrite and reduced blood pressure. In a separate small crossover experiment, having participants spit out their saliva prevented those responses. The swallowed material was an important part of the route. Webb and colleagues, 2008

These experiments connect a mouth-level process to measurable physiology. Their strength is the intervention: something in the route was deliberately changed, and the investigators measured what followed.

Their limits are just as important. An acute experiment in a few healthy volunteers does not tell us what happens to cardiovascular events over years.

A later study followed 19 healthy volunteers through a control week and then a week using a 0.2% chlorhexidine rinse twice daily. Oral nitrate-reducing activity and circulating nitrite fell, while blood pressure rose by a few millimeters of mercury. Everyone received the periods in the same order, which limits what the design can exclude. Kapil and colleagues, 2013

Other experiments have produced different results. In a randomized, double-blind crossover study of 17 healthy young women on a low-nitrate diet, three days of chlorhexidine-containing rinse reduced oral nitrate conversion without changing plasma nitrite or 24-hour blood pressure. Sundqvist and colleagues, 2016

The participants, diet, formulation, and exposure belong with each finding.

In a randomized-order experiment involving 12 healthy men, Woessner and colleagues compared water with three rinse formulations after beetroot juice. The nitrite responses differed between the water/essential-oil conditions and the cetylpyridinium chloride/chlorhexidine conditions. Participants repeatedly rinsed during a four-hour experiment. Woessner and colleagues, 2016

That is a reason to ask which product was tested, at what concentration, how often, and against what comparison. The single word mouthwash loses information we need.

Oral benefits also belong in the assessment. Human experimental studies have shown that chlorhexidine can suppress dental plaque formation and gingivitis. Those outcomes matter when choosing a treatment for an actual patient. Löe and Schiott, 1970

The nitrate experiments do not establish that all rinses cause cardiovascular harm, or that switching to a particular product prevents heart attacks. A prescribed course should be discussed with the treating clinician before changing it.

We began this series with a clot, then examined the plaque beneath it. This week we added a living vessel wall, a local signaling pathway, and oral chemistry that can contribute to another route to NO.

The map is becoming more complete. It is also becoming more important to keep each kind of evidence in its place.

A change in salivary nitrite measures chemistry. A change in blood pressure measures physiology. A reduction in heart attacks is a clinical outcome that requires its own evidence. We cannot substitute the first two for the third.

For a hygienist, the practical gain is a better conversation: ask what a rinse is intended to accomplish, recognize that formulation and duration matter, and understand why an oral chemical pathway interests vascular researchers. An oral examination cannot reveal a patient's coronary plaque or whole-body NO status.

The periodontal question still needs its own chapter. Next week we will examine where periodontal disease enters the story, what associations can show, and what treatment studies can establish.

What do your patients ask about gum health and their heart? Please leave out names and identifying patient details.

— Thad

For the next team huddle: Trace both routes to NO. Then explain why relaxing vascular smooth muscle, changing salivary nitrite, and preventing a heart attack are three different claims.

The four-part series:

1. Start with the clot.

2. What was in the artery before the clot?

3. Pressure, flow, and mouth chemistry. This letter.

4. Where does periodontal disease enter? Forthcoming.

If this helped, pass it to a hygienist you work with.

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