Basics of muscle contraction

Control of intracellular Ca²⁺ - principal mechanism that initiates contraction and relaxation in smooth and striated muscle

Regulatory pathways:

striated muscle-Ca²⁺ activates contraction by binding to thin filament associated protein, troponin

smooth muscle-Ca²⁺ binds to calmodulin, which then associates with the catalytic subunit of myosin light chain kinase-phosphorylates serine 19 on the regulatory light chain of myosin. Phosphorylation of Ser19 allows the myosin ATPase to be activated by actin and the muscle to contract.

Basics of muscle contraction

Calcium regulation is vital

In smooth muscle, the cytosolic free Ca²⁺ concentration is ~ 0.1 mM in basal state; ~ 10,000 times lower than that present in the extracellular space (mM)

Activation of cells induces an increase in cytosolic concentration up to ~1-10 mM.

Ca²⁺ diffuses in cell much more slowly than predicted from its small volume; Ca²⁺ atom migrate 0.1-0.5 mm, lasting only ~ 50 ms before being bound.

Ca²⁺ used by different vasoactive agents comes from extracellular and/or intracellular space.

Intracellular Ca²⁺ is localized in the mitochondria and SR

Location is most important

Slide 4

Slide 5

"The degree of interaction is..."

The degree of interaction is determined by the net level of phosphorylation of the 20 kDa regulatory light chains of myosin II (rMLC).

MLC is regulated by MLC kinase (MLCK) and MLC phosphatase (MLCP or PP1M).

The extent of the rMLC phosphorylation and the amplitude of force production depends on the balance of the activities of MLCK and MLCP.

Under certain conditions, force is also regulated independent of the changes in rMLC phosphorylation levels perhaps by thin filament associated proteins (caldesmon and calponin), which can be phosphorylated by MAP kinase and/or other kinases.

Thin filament associated proteins might modulate the effect of rMLC phosphorylation, which is alone sufficient to initiate and maintain contraction.

MLCP is a trimer comprising a 130 kD regulatory myosin binding subunit (MBS), a 37 kD catalytic subunit (PP1c), and a 20 kD protein of uncertain function (M20).

"Well-established that cAMP and cGMP..."

Well-established that cAMP and cGMP decreases Ca²⁺ sensitivity of contraction in both intact and permeabilized smooth muscle.

In vitro, PKA phosphorylates MLCK at two sites; site A decreases affinity of MLCK for Ca²⁺/calmodulin complex.

However, agents that elevate PKA have negligible effects on phosphorylation of site A and Ca²⁺ activation of MLCK; suggests that cAMP/PKA desensitizes smooth muscle by an alternate mechanism.

Phosphorylation of MLCK by PKG has no effect on activity.

Endogenous nitric oxide and related nitrovasodilators regulate blood pressure by activation of soluble guanylate cyclase, elevation of cGMP, activation of cGMP dependent kinase (cGKIa or PKG). cGMP-mediated vascular smooth muscle cell relaxation is characterized by a reduction in intracellular calcium concentration and activation of PP1M, which reduces the sensitivity of the contractile apparatus to intracellular calcium.

The mechanism by which cGMP increases PP1M activity and myosin light chain dephosphorylation was elucidated in a series of experiments published by Surks et al.

Slide 8

Ion channels in smooth muscle

Slide 10


	Control of intracellular Ca²⁺ - principal mechanism that initiates contraction and relaxation in smooth and striated muscle
	Regulatory pathways:
	striated muscle-Ca²⁺ activates contraction by binding to thin filament associated protein, troponin
	smooth muscle-Ca²⁺ binds to calmodulin, which then associates with the catalytic subunit of myosin light chain kinase-phosphorylates serine 19 on the regulatory light chain of myosin. Phosphorylation of Ser19 allows the myosin ATPase to be activated by actin and the muscle to contract.


	Calcium regulation is vital
	In smooth muscle, the cytosolic free Ca²⁺ concentration is ~ 0.1 mM in basal state; ~ 10,000 times lower than that present in the extracellular space (mM)
	Activation of cells induces an increase in cytosolic concentration up to ~1-10 mM.
	Ca²⁺ diffuses in cell much more slowly than predicted from its small volume; Ca²⁺ atom migrate 0.1-0.5 mm, lasting only ~ 50 ms before being bound.
	Ca²⁺ used by different vasoactive agents comes from extracellular and/or intracellular space.
	Intracellular Ca²⁺ is localized in the mitochondria and SR
	Location is most important


	The degree of interaction is determined by the net level of phosphorylation of the 20 kDa regulatory light chains of myosin II (rMLC).
	MLC is regulated by MLC kinase (MLCK) and MLC phosphatase (MLCP or PP1M).
	The extent of the rMLC phosphorylation and the amplitude of force production depends on the balance of the activities of MLCK and MLCP.
	Under certain conditions, force is also regulated independent of the changes in rMLC phosphorylation levels perhaps by thin filament associated proteins (caldesmon and calponin), which can be phosphorylated by MAP kinase and/or other kinases.
	Thin filament associated proteins might modulate the effect of rMLC phosphorylation, which is alone sufficient to initiate and maintain contraction.
	MLCP is a trimer comprising a 130 kD regulatory myosin binding subunit (MBS), a 37 kD catalytic subunit (PP1c), and a 20 kD protein of uncertain function (M20).


	Well-established that cAMP and cGMP decreases Ca²⁺ sensitivity of contraction in both intact and permeabilized smooth muscle.
	In vitro, PKA phosphorylates MLCK at two sites; site A decreases affinity of MLCK for Ca²⁺/calmodulin complex.
	However, agents that elevate PKA have negligible effects on phosphorylation of site A and Ca²⁺ activation of MLCK; suggests that cAMP/PKA desensitizes smooth muscle by an alternate mechanism.
	Phosphorylation of MLCK by PKG has no effect on activity.
	Endogenous nitric oxide and related nitrovasodilators regulate blood pressure by activation of soluble guanylate cyclase, elevation of cGMP, activation of cGMP dependent kinase (cGKIa or PKG). cGMP-mediated vascular smooth muscle cell relaxation is characterized by a reduction in intracellular calcium concentration and activation of PP1M, which reduces the sensitivity of the contractile apparatus to intracellular calcium.
	The mechanism by which cGMP increases PP1M activity and myosin light chain dephosphorylation was elucidated in a series of experiments published by Surks et al.