Smooth Muscle and the Myogenic Response

Push harder on the inside of a small artery and its muscle pushes back. A garden hose does the opposite: raise the pressure and it bulges. The muscle in an arterial wall answers a rise in pressure by contracting, and that answer is built into the muscle rather than wired in from the brain. The same machinery runs the gut, airways and bladder.

Force without sarcomeres

A smooth muscle cell is a spindle, a few micrometres across and roughly 30 to 200 micrometres long, with one nucleus, and it has no stripes, because its actin and myosin are not arranged in sarcomeres. Skeletal muscle gets its length-tension curve from that regular overlap; smooth muscle has no such register.

Its thin filaments are anchored instead in dense bodies, protein clusters in the cytoplasm, and in dense plaques on the inside of the membrane. Myosin filaments run among them at an angle, and intermediate filaments tie the dense bodies together, so when myosin pulls, the whole cell shortens and bulges. Because the filaments are not stacked in sarcomeres, the cell can develop force over a much wider range of lengths than a sarcomere can, which matters for an organ that fills.

Smooth muscle has no troponin, so calcium cannot act on the thin filament as it does in skeletal and cardiac muscle. The switch sits on myosin: calcium binds calmodulin, calcium-calmodulin activates myosin light-chain kinase (MLCK), and MLCK puts a phosphate on the 20-kDa regulatory light chain, LC20, after which the motor can cycle on actin. Muscle and movement gave this switch its first look, and calcium signaling covers calmodulin and the stores.

Calcium arrives by two routes. Depolarization opens L-type channels (CaV1.2) and calcium flows in down its electrochemical gradient: electromechanical coupling. Or an agonist on a Gq-coupled receptor makes IP3, which opens release channels on the sarcoplasmic reticulum (phospholipase C, IP3, DAG and PKC). That route can contract the cell with little change in membrane potential: pharmacomechanical coupling.

Set the three types side by side. Skeletal muscle links a voltage sensor in the T tubule mechanically to the release channel of the sarcoplasmic reticulum, so it needs no calcium from outside, and calcium acts through troponin. Cardiac muscle needs the calcium entering during the action potential, which triggers a larger release from the store (cardiac electrophysiology and the ECG), and it uses troponin too. Smooth muscle draws on both sources and switches the motor itself; its cross-bridges cycle more slowly, so it generally shortens slowly and, above all in tonic smooth muscle, holds force cheaply.

Calcium sets the switch; sensitivity sets the gain

MLCK is only half the story. Myosin light-chain phosphatase (MLCP) takes the phosphate off LC20, and it works the whole time; in permeabilized rabbit arterial strips, where the bath sets calcium inside the cells, that removal can be followed directly. Force therefore follows the balance of kinase and phosphatase activity. Calcium sets the kinase, so anything that changes the phosphatase changes force at the same calcium.

That is what calcium sensitization means: more force, or more phosphorylation, at an unchanged calcium concentration.

The clearest early evidence came from the Somlyo laboratory. In alpha-toxin-permeabilized rabbit portal vein, with calcium and ATP removed and MLCK blocked by ML-9 so only the phosphatase could act, Kitazawa, Masuo and Somlyo found that GTP-gamma-S, which keeps G proteins active, slowed dephosphorylation and relaxation to about half the control rates: a G protein can raise force by slowing the phosphatase.

The route is now drawn through RhoA, a GTPase switch (GTPase switches covers the cycle and its exchange factors, and integrins and Rho GTPases the same kinase elsewhere), and Rho kinase (ROCK), which phosphorylates MYPT1, the targeting subunit of MLCP, and inhibits the phosphatase. Protein kinase C adds a second route, through CPI-17, a protein that inhibits MLCP when it is itself phosphorylated.

The opposite arrow runs through nitric oxide. Nitric oxide from endothelium activates soluble guanylate cyclase, a reaction shown with purified bovine enzyme to depend on its heme group, and the cGMP that follows activates protein kinase G (nitric oxide and cGMP). In muscle the result is both a lower calcium and a higher phosphatase activity: in permeabilized rabbit artery with calcium clamped, the analog 8-bromo-cGMP relaxed the muscle, which the authors read as indirect activation of the phosphatase.

Latch: holding force cheaply

Record LC20 phosphorylation and force together in a stimulated arterial strip. Phosphorylation rises first, then falls back toward low values while force stays near its peak. Something holds the load after the phosphates come off, and that state is called latch.

Hai and Murphy proposed a kinetic model for this in 1988. Myosin heads move among four states: phosphorylated or not, attached or not. Kinase and phosphatase act on heads whether or not they are bound to actin, and a head that loses its phosphate while attached does not release at once. It becomes a latch bridge, whose slow detachment is the rate-limiting step, so force per head stays high while cycling rate, and ATP cost, fall. It is a model fitted to phosphorylation and stress data, not a structure anyone has imaged.

Two things often said about latch are wrong. It is not a state without ATP: phosphorylated cross-bridges keep cycling, latch bridges let go slowly, and calcium pumps keep working, so latch is economy, not shutdown. Nor is it rigor, the stiff state of a muscle whose ATP has run out.

In 2022 Hammell and colleagues tested the prediction on a few purified myosin molecules: myosin purified from pig stomach fundus (tonic) and chicken gizzard (phasic) pulled on actin in a laser trap with 200 micromolar MgATP present, and MLCP was injected at plateau force. Force was held for a median of 32 seconds (interquartile range 27 to 48) for the tonic myosin and 21 seconds (15 to 27) for the phasic myosin, five assays each. In the parallel motility assay, the midpoint of the loss of actin movement after injection, an indirect clock for dephosphorylation, came at about 27 seconds for the tonic and 26 seconds for the phasic myosin, so the authors read the tonic hold as force kept during dephosphorylation and the phasic hold less convincingly. Two limits: purified non-human proteins, not an intact vessel, and dephosphorylation followed in a parallel motility assay rather than on the trapped heads.

One unit or many

In single-unit (visceral) smooth muscle, gap junctions couple neighbouring cells electrically, so excitation spreads and the cells contract as one mass; the gut and uterus are the usual examples. Bladder and arterial muscle sit between the two types: their cells are less tightly coupled, and in arteries the endothelium carries much of the electrical spread. Some of these tissues also carry pacemaker activity, and the gut’s slow waves belong to gut motility and secretion.

In multi-unit smooth muscle, cells have little electrical coupling and each is driven by its own nerve terminals, allowing finely graded control; the iris and the piloerector muscles are the usual examples.

A hollow organ that fills is stretched steadily, and visceral smooth muscle can relax after being stretched, a behaviour called stress relaxation: tension falls back after the stretch, so pressure inside rises much less during filling than in a stiff container. An artery does the opposite when transmural pressure rises: it contracts.

Receptors, not nerves in general, set the direction, so “sympathetic input contracts smooth muscle” is wrong as a rule. Norepinephrine on alpha-1 adrenoceptors contracted isolated rat aorta and mesenteric resistance vessels, mobilizing calcium from inside and outside the cell. In isolated human detrusor strips, noradrenaline produced concentration-dependent relaxation, which the authors suggested was mediated mainly through beta-3 adrenoceptors. Beta receptors classically signal through cAMP and protein kinase A, though in rat bladder strips cAMP played only a minor part in relaxation by the beta agonist isoproterenol (cAMP and PKA), and the receptor families are mapped in the hypothalamus and the autonomic nervous system. Name the receptor and the preparation, and the contradictions go away.

Arterioles answer pressure

Bayliss’s 1902 paper in the Journal of Physiology began from a puzzling curve. He had enclosed a rabbit hind leg in a volume recorder, cut the nerves running to the limb, and recorded arterial pressure and limb volume together, lowering systemic pressure by stimulating the central end of the depressor nerve. He then repeated the effect in dogs and cats, raising pressure by stimulating the splanchnic nerves or by asphyxia and lowering it by compressing the aorta, and tried excised arteries, most clearly a dog carotid removed after death. A passive tube with its nerves cut should simply follow pressure. His account, in a paper whose footnote points to a preliminary report of March 1901, was that the muscular coat contracts when stretched and relaxes when tension falls, independently of the central nervous system. Limb volume sums arteries, veins and tissue, so the limb tracings did not say which vessels responded; the excised carotid showed that an artery can respond without the rest. Bayliss appears again, with Starling, in signals and distance.

The modern version is a pressure myograph and a mouse. Chennupati, Wirth and colleagues deleted G-protein alpha subunits in smooth muscle of adult mice, mounted mesenteric arteries, and raised intraluminal pressure in 20 mmHg steps from 20 to 140 mmHg, reading active diameter in calcium-containing and passive diameter in calcium-free solution. Control arteries peaked at about 80 to 120 mmHg. Arteries lacking G12 and G13 showed no active myogenic constriction and strongly reduced RhoA activity, yet calcium rose with pressure as normal; a Rho kinase inhibitor blocked the response in controls; arteries lacking Gq and G11 behaved like controls; cerebral arteries kept about half their response. Two requirements, then, in this bed: a calcium rise (which L-type plus TRP channel blockers together cut by about half) and Rho-dependent sensitization. Keep one conflict open. In 2022 Cui and colleagues found that deleting the angiotensin II type 1a receptor in smooth muscle left about 35 percent of wild-type constriction in mouse mesenteric arteries, which still constricted to a depolarizing potassium solution, and that a Gq/11 inhibitor abolished the response in the beds they tested. Deletion and inhibition, and different vessel orders and beds, are not interchangeable, so the upstream wiring is unsettled.

Why does a 7 percent change in radius matter? Treat an arteriole as a rigid cylinder of fixed length carrying steady laminar flow of a Newtonian fluid, assumptions blood and arterioles both violate, and Poiseuille’s law applies (blood and vessels).

Q = (pi x delta P x r^4) / (8 x viscosity x length), so Q2 / Q1 = (delta P2 / delta P1) x (r2 / r1)^4

Take made-up but convenient numbers: delta P1 = 60 mmHg across the segment, r1 = 100 micrometres, downstream and outside pressures fixed, so a rise in inlet pressure raises both the driving gradient and the transmural pressure that stretches the wall. Raise the gradient to 80 mmHg. If the vessel were passive and distended by 5 percent, Q2 / Q1 = (80 / 60) x 1.05^4 = 1.62, a 62 percent rise in flow. For flow to stay constant instead, (r2 / r1)^4 = 60 / 80 = 0.75, so r2 = 100 x 0.75^(1/4) = 93.1 micrometres, a 6.9 percent narrowing. Resistance scales as 1 / r^4, so R2 / R1 = 1 / 0.75 = 1.33, the same one-third increase as the pressure gradient, which is what Q = delta P / R requires. Overshoot and flow falls: at r2 = 90 micrometres, Q2 / Q1 = 1.333 x 0.6561 = 0.875, 12.5 percent below baseline. Tone is a separate quantity: 200 micrometres passive and 160 active at one pressure give tone = 100 x (200 - 160) / 200 = 20 percent of diameter, while flow at the same gradient would be 0.8^4 = 0.41 of passive.

Pressure difference across the arteriole, ΔP 80 mmHg (baseline 60) Radius, r 105 micrometres (baseline 100) radius that holds baseline flow (the lesson’s what-if)

Flow relative to baseline, Q / Q₁

Resistance relative to baseline, R / R₁

Change in radius from 100 micrometres

In this sketch: idealized rigid tube with Poiseuille flow. The tube has fixed length and viscosity, steady laminar flow of a Newtonian fluid, and downstream and outside pressures held fixed, so the pressure difference is the only driver; blood and arterioles both violate these assumptions, so the numbers are a sketch of the logic, not a prediction for a real vessel. The baselines (60 mmHg, 100 micrometres) are the lesson’s made-up but convenient numbers; the slider ranges are made up for the sketch; the toggle simply sets whatever radius keeps flow at baseline.

Rule to keep: flow scales with the fourth power of radius, so a small change in radius can cancel a large change in pressure. Holding flow while the difference rises from 60 to 120 mmHg takes only a 16 percent fall in radius.

Holding flow steady across a range of pressures is autoregulation, produced by several mechanisms together; its metabolic and endothelial parts belong to resistance, compliance and local flow, and the renal version, with tubuloglomerular feedback, to glomerular filtration. Three inputs act on the same kinase-phosphatase balance: pressure raises calcium and sensitization, endothelial nitric oxide lowers both, sympathetic alpha-1 input raises both. Net diameter reflects their combined effect.

Use that to reason about raised pressure. In hypertensive rodents small arteries often show more myogenic tone at a given pressure, and in people with essential hypertension small arteries show inward remodelling of the wall (how much myogenic tone contributes in people is less settled), so the lumen is narrower at the same transmural pressure; by the fourth-power law that raises segment resistance steeply, and higher resistance at a given cardiac output holds mean arterial pressure up. Practise the directions: tone up, lumen down, resistance up. A fall in pressure runs the loop the other way: after blood loss, pressure in a small artery falls, wall tension falls, the muscle tends to relax and lower segment resistance, while the baroreflex raises sympathetic outflow (blood pressure regulation) and alpha-1 input pulls the diameter back down.

Design problem

Does nitric oxide relax arterial smooth muscle by lowering calcium, by lowering calcium sensitivity, or both? You have endothelium-denuded rabbit femoral arteries, a nitric oxide donor, an alpha-1 agonist, the calcium dye Fura-2, alpha-toxin to permeabilize strips, and antibodies reporting LC20, MYPT1 and CPI-17 phosphorylation. Design the experiments.

One way to do it

Start with intact, denuded strips, confirming the denudation (acetylcholine no longer relaxes them). Contract them with the alpha-1 agonist, then add the donor while recording force and Fura-2 together. A calcium-force curve in the same strips says what a given fall in calcium should cost in force; more relaxation than that points to lost sensitivity too.

Then clamp calcium: in alpha-toxin-permeabilized strips the bath sets calcium, so if the donor or cGMP still relaxes them, desensitization is part of the mechanism at fixed calcium. Lee, Li and Kitazawa used that design in 1997; 8-bromo-cGMP, a cGMP analog, relaxed the muscle, which they read as indirect activation of the phosphatase.

To ask whether the phosphatase speeds up, reverse the 1991 logic: phosphorylate LC20, remove calcium and ATP, block MLCK, and follow dephosphorylation with and without cGMP. Faster dephosphorylation means more phosphatase activity. Follow MYPT1 and CPI-17 too; the 2009 study found CPI-17 phosphorylation at a minimum within 15 seconds of the donor in this preparation, in parallel with the fall in calcium, which the authors read as a consequence of suppressed calcium release.

Controls: time-matched strips with no donor, a guanylate cyclase inhibitor to confirm the cGMP route, the same starting force each time. Limits: a donor on a denuded artery is not endogenous nitric oxide in a flowing arteriole, rabbit is not human, and permeabilization removes ion gradients and voltage control, though receptors and G proteins remain coupled. Expect both arrows at once.