Molecular and Cellular Physiology of Vascular Smooth Muscle Cells

Explain the mechanism by which KCl induces vascular contraction

Collapses the k + gradient causing calcium to go in and k to come out The KCl-induced contraction and Ca2+ oscillations were abolished in the absence of extracellular Ca2+ and presence of the voltage-dependent Ca2+ channel blockers, nifediphine and NiCl2, suggesting that KCl-induced effects require an influx of Ca2+

KCl causes VSMC contraction

Collapses the k + gradient causing calcium to go in and k to come out The KCl-induced contraction and Ca2+ oscillations were abolished in the absence of extracellular Ca2+ and presence of the voltage-dependent Ca2+ channel blockers, nifediphine and NiCl2, suggesting that KCl-induced effects require an influx of Ca2+

Vascular Smooth Muscle Contraction high cytosoloc calcium (what happens)

Four Ca2+ ions bind to calmodulin molecule (protein closely related to troponin C) and, then, the Ca2+-calmodulin complex activates an enzyme known as myosin light chain kinase (MLCK), which phosphorylates the regulatory light chain associated with a motor head region of myosin molecules. ATP hydrolysis = ADP + Pi Phosphorylation of the light chain alters the conformation of the myosin head that greatly increases its ATPase activity. Therefore, an increase in cellular Ca2+ concentration regulates ATP hydrolysis in smooth muscle cells and, thus, initiates the cross-bridge formation. Unlike myosin light chain kinase, which is only active when cytoplasmic Ca2+ is elevated, myosin light chain phosphatase is always active.

Source of intracellular Ca2+ ions:

Source of intracellular Ca2+ ions: •Ca2+-induced Ca2+-release mechanism •Inositol 1,4,5-triphosphate (IP3)-mediated Ca2+ release

KCl causes VSMC contraction pathway

kcl causes depolarization and calcium to enter the cell then la la la

gap junction

A type of intercellular junction in animals that allows the passage of materials between cells.

Action potential causes depolarization Voltage gated channels open when threshold is reached Na fast to open K slow to open An increase in Ca2+ concentration during depolarization phase activates Ca2+-dependent-K+-channels which lead to the outward K+-current and progressive hyperpolarization phase of the wave.

Action potential causes depolarization Voltage gated channels open when threshold is reached Na fast to open K slow to open An increase in Ca2+ concentration during depolarization phase activates Ca2+-dependent-K+-channels which lead to the outward K+-current and progressive hyperpolarization phase of the wave.

alpha one pathway

Alpha 1 - Gq - Phospholipase C - IP3 inositoltriphosphate

alpha 2 pathway

Alpha 2 - Gi - Adenylcyclase - cAMP

beta 2 pathway

Beta 2 receptor - adenylate cyclase -cyclic AMP - PKA - relaxation

Structure of VSMC Cytoskeletal filaments 1. Thin filaments : Actin 2. Thick filaments: Myosin 3. Intermediate filaments: Desmin and Vimentin. Support the shape of the smooth muscle cells and hold the dense bodies in place

Cytoskeletal filaments 1. Thin filaments : Actin 2. Thick filaments: Myosin 3. Intermediate filaments: Desmin and Vimentin. Support the shape of the smooth muscle cells and hold the dense bodies in place

Identify contractile proteins from VSMC and describe the source and movement of calcium ions on VSMC.

Cytoskeletal filaments 1. Thin filaments : Actin 2. Thick filaments: Myosin 3. Intermediate filaments: Desmin and Vimentin. Support the shape of the smooth muscle cells and hold the dense bodies in place Dense bodies Located in the cytoplasm and at the cell membrane

Cholinergic nerve ending where? non adrengic nerve ending where?

Endothelium Smooth muscle

Epinephrine and norepinephrine are released by the blank blank and blank blank respectively. They are the flight/fight hormones that are released when the body is under extreme stress. Structure: Epinephrine and Norepinephrine are the same except that epinephrine has a blankyl group. Epinephrine is release during activity and then causes blank. NOR causes greater blank than EPI because it does not include compensatory vasodilation via blank receptors on blood vessels supplying skeletal muscle.

Epinephrine and norepinephrine are released by the adrenal medulla and nervous system respectively. They are the flight/fight hormones that are released when the body is under extreme stress. Structure: Epinephrine and Norepinephrine are the same except that epinephrine has a methyl group.Epinephrine is release during activity and then causes vasodilation. NOR causes greater vasoconstriction than EPI because it does not include compensatory vasodilation via B2 receptors on blood vessels supplying skeletal muscle.

Smooth muscle contraction is terminated by which of the following: A. Dephosphorylation of myosin light chain kinase B. Dephosphorylation of myosin light chain C. Efflux of calcium ions across the plasma membrane D. Inhibition of myosin light chain phosphatase E. Spontaneous inward current of sodium ions

Incorrect - Dephosphorylation of myosin light chain kinase increases its activity to phosphorylate the regulatory light chain and stimulate smooth muscle contraction b. Correct - Dephosphorylation of the regulatory light chain by myosin light chain phosphatase causes relaxation of smooth muscle c. Correct - Efflux of calcium ions across the plasma membrane decreases cellular concentration of calcium ions that inhibits the cross-bridge formation and causes smooth muscle relaxation d. Incorrect - Inhibition of myosin light chain phosphatase causes increased phosphorylation of the regulatory light chains that stimulate smooth muscle contraction e. Incorrect - Spontaneous inward current of sodium ions changes the membrane voltage that opens voltage-gated calcium channels in sarcolemma causing smooth muscle contraction

The drug sodium nitroprusside relaxes vascular smooth muscle via activation of protein kinase G (PKG). The action of which local metabolite, neurotransmitter or circulating hormone is mimicked by this drug? A. Norepinephrine B. Epinephrine C. Acetylcholine D. Nitric oxide E. Endothelin

Incorrect - Norepinephrine stimulates vascular smooth muscle contraction acting via G(alpha)q protein and phospholipase C which stimulate the hydrolysis of phosphatidyl inositol 4,5- bisphosphate with a release of inositol 1,4,5-triphosphate b. Incorrect - Epinephrine relaxes vascular smooth muscle cells acting via adenylyl cyclase which forms the second messenger cyclic AMP that activates protein kinase A c. Incorrect - Acetylcholine stimulates vascular smooth muscle contraction acting via G(alpha)q protein and phospholipase C which stimulate the hydrolysis of phosphatidyl inositol 4,5- bisphosphate with a release of inositol 1,4,5-triphosphate d. Correct - Sodium nitroprusside relax vascular smooth muscle via activation of protein kinase G by mimicking the action of nitric oxide and causing a similar chain of the intracellular events e. Incorrect - Endothelin stimulates vascular smooth muscle contraction acting via G(alpha)q protein and phospholipase C which stimulate the hydrolysis of phosphatidyl inositol 4,5- bisphosphate with a release of inositol 1,4,5-triphosphate

Smooth muscle cells that exhibit rhythmical contraction in the absence of external stimuli also necessarily exhibits which of the following: A. Slow voltage-sensitive calcium channels B. Intrinsic pacemaker wave activity C. Higher resting cytosolic calcium concentration D. Hyperpolarized membrane potential E. Action potentials with plateaus

Incorrect - The presence of slow voltage-sensitive calcium channels does not play a necessary role in rhythmical smooth muscle contraction b. Correct - Clusters of specialized (self-excitable) smooth muscle cells within a smooth muscle functional syncytium can generate spontaneous electrical activity and they are able to undergo spontaneous depolarization and generate action potentials without any external stimulation. Such self-excitable smooth muscle cells have always displayed the pacemaker potentials analogous to that of the heart c. Incorrect - Higher resting cytosolic calcium concentration would cause a sustained tonic smooth muscle contraction d. Incorrect - Hyperpolarized membrane potential would cause a sustained smooth muscle relaxation e. Incorrect - Action potentials with plateaus results in the prolonged contraction as a result of external stimuli

Neural Control of VSMC Contraction Neurotransmitter released by: •Sympathetic Fiber: Norepinephrine alpha 1, alpha 2 and beta 2 adrenergic receptors •Parasympathetic Fiber: Acetylcholine Muscarinic receptors

Neurotransmitter released by: •Sympathetic Fiber: Norepinephrine alpha 1, alpha 2 and beta 2 adrenergic receptors •Parasympathetic Fiber: Acetylcholine Muscarinic receptors Norepinephrine opens calcium channels Calcium enters and is then able to form with calmodulin which lets the muscle contract

Arrangement of Contractile Proteins

Sliding of thin actin filaments over thick myosin filaments causes the shortening of the contractile units and, as a result, the whole cell shortens and bulges out between the points where the thin filaments are attached to the dense bodies on the plasma membrane

Ca++ Removal from VSMC 3 ways

Smooth muscle cell remove cytosolic Ca2+ by: 1. Na+/Ca2+ exchanger and Ca2+ pump in the plasmatic membrane 2. Ca2+-ATPase in sarcoplasmic reticulum (SERCA) 3. Ca2+-binding proteins, such as calsequestrin and calreticulin, help to store Ca2+ ions in the sarcoplasmic reticulum. The main source of calcium in smooth muscle is extracellular IP3-gated calcium channel in SR release Calcium to cytoplasm

Binding of epinephrine to what type of adrenergic receptors will cause the smooth muscle cells of the arterioles to accumulate cAMP and dilate? A. Alpha-1 adrenergic receptors B. Beta-1 adrenergic receptors C. Alpha-2 adrenergic receptors D. Beta-2 adrenergic receptors E. Beta-3 adrenergic receptors

a. Incorrect - Alpha-1 adrenergic receptors on vascular smooth muscle cells act via G(alpha)q protein and activate phospholipase C (PLC). PLC then hydrolyzes phosphatidyl inositol 4,5- bisphosphate (PIP2) to inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). The accumulation of IP3 induces the release of Ca2+ ions from SR into the cytoplasm and, therefore, stimulates smooth muscle cell contraction, and hence, vasoconstriction. b. Incorrect - Beta-1 adrenergic receptors are located on the cardiac myocytes and their stimulation increases cardiac contractility c. Incorrect - Alpha-2 adrenergic receptors are predominantly located on the presynaptic terminals and their stimulation inhibits neurotransmitter release d. Correct - Binding of epinephrine to beta-2 adrenergic receptors on vascular smooth muscle cells activates the adenylate cyclase, which forms the second messenger cyclic AMP (cAMP). cAMP then activates cAMP-dependent protein kinase A (PKA). PKA phosphorylates two membrane proteins: 1) phosphorylation of Ca2+ channels in the sarcolemma causes the channels to close, stopping the influx of Ca2+ and 2) phosphorylation of the Ca2+-ATPase in the sarcoplasmic reticulum increases its activity and, thereby, increases the removal of Ca2+ from the cytoplasm. The effect of both actions is reduction in cytoplasmic Ca2+ ions. At the same time, PKA phosphorylates myosin light chain kinase reducing its catalytic effectiveness that causes a decrease in myosin light chain phosphorylation. The overall effect of all these actions is smooth muscle cell relaxation and, therefore, vasodilation. e. Incorrect - Beta-3 adrenergic receptors are located on the fat cells and they participate in termogenesis

What initiates the formation of cross-bridges in smooth muscle? A. Binding of ATP to myosin heads B. Release of inorganic phosphate from the myosin heads C. Binding of calcium ions to troponin C D. Phosphorylation of the regulatory myosin light chain E. Release of ADP from myosin heads

a. Incorrect - Binding of ATP to myosin head reduces the affinity of myosin for actin, causing the myosin head to release from the actin filament b. Incorrect - The release of inorganic phosphate from the myosin head triggers the power stroke c. Incorrect - Binding of calcium ions to troponin C initiates cross-bridge cycling in skeletal muscle d. Correct - Phosphorylation of the regulatory light chain alters the conformation of the myosin head, which greatly increases its ATPase activity and allows it to hydrolyze an ATP molecule. This action initiates the formation of cross-bridges e. Incorrect - The release of ADP from the myosin head leaves the actin-myosin complex in a rigid state in skeletal muscle

What is the quality of multi-unit smooth muscle cells? A. Cells are electrically connected via gap junctions B. Cells function in syncytial fashion C. Electrical stimulation of one cell leads to stimulation of adjacent cells through gap junctions D. Cells are not electrically connected via gap junctions E. Electrical stimulation of the cells results in wave of contraction

a. Incorrect - Cells are electrically connected via gap junctions in single-unit smooth muscle b. Incorrect - Cells function in syncytial fashion in single-unit smooth muscle c. Incorrect - Electrical stimulation of one cell leads to stimulation of adjacent cells through gap junctions in single-unit smooth muscle d. Correct - Cells in multi-unit smooth muscle are not electrically connected via gap junctions. Each cell operates independently of the others and often innervated by a single nerve ending. e. Incorrect - Electrical stimulation of the cells results in wave of contraction in single-unit smooth muscle

A 32-year-old male is diagnosed with primary hypertension. His physician recommended a new drug for hypertension that acts by decreasing vascular smooth muscle contractile activity without affecting ventricular contractility. Which of the following is the most likely site of action for new drug? A. Protein kinase A B. Calmodulin C. Tropomyosin D. Beta1-adrenergic receptors E. Troponin

b

Shortly after bypass surgery the reactivity of a patient's coronary vessels was tested by intracoronary administration of several vasoactive agents. Which of the following substances elicited an increase in coronary vessel contraction? A. Nitric oxide B. Endothelin C. Prostacyclin D. Adenosine E. Histamine

b

Which component is necessary for activation of myosin light chain kinase? A. Ca2+- troponin complex B. Myosin light chain phosphatase C. Ca2+- calmodulin complex D. Troponin-tropomyosin complex E. ADP Source: Dr. Dedkov,

c

myosin and actin

contractile proteins because they shorten a muscle fiber

dense bodies in smooth muscle

cytoplasmic structure to which thin filaments of a smooth muscle fiber are anchored

What ion channels does the second messenger inositol triphosphate (IP3) open when it increases contractile force of smooth muscle cells? A. Potassium B. Sodium C. Chloride D. Calcium E. Magnesium

d

What is the main action of myosin light chain kinase in smooth muscle? A. Phosphorylation of calmodulin B. Phosphorylation of ADP to ATP C. ATP hydrolysis D. Phosphorylation of the regulatory myosin light chain E. Dephosphorylation of the regulatory myosin light chain

d

A 57-year-old woman is undergoing a femoral popliteal bypass for her peripheral vascular disease. The vascular surgeon wishes to induce a localized arteriolar constriction to help control hemostasis. Which of the following agents will cause systemic vasoconstriction? A. Nitric oxide B. Atrial natriuretic peptide C. beta-2 adrenergic agonist D. Adenosine E. Angiotensin II

e

What is the main action of myosin light chain phosphotase in smooth muscle? A. Phosphorylation of calmodulin B. Dephosphorylation of the myosin light chain kinase C. Phosphorylation of ADP to ATP D. ATP hydrolysis E. Dephosphorylation of the regulatory myosin light chain

e

Vascular Smooth Muscle Contraction

one more time to see it

Caveolae

pouchlike infoldings that sequester bits of extracellular fluid containing a high concentration of Ca2+ close to the membrane; when calcium channels open here Ca2+ influx occurs rapidly

Prostacyclin causes VSMC relaxation

process Activates adenylate cyclase (atp0--> camp) activates protein kinases-> vasodilation

Vascular Smooth Muscle Relaxation After a contraction-activating stimulus (electrical or chemical) has subsided, Ca2+ ions must be removed from the cytoplasm for muscle relaxation to occur. •Extracellular extrusion of Ca2+ by Ca2+ pump and Na+/Ca2+ exchanger •Ca2+ re-uptake in sarcoplasmic reticulum by Ca2+-ATPase (SERCA) •Intracellular Ca2+ concentration falls to resting level •Formation of Ca2+-calmodulin complexes decreases •MLCK activity decreases •MLC phosphatase dephosphorylates MLC on myosin heads •Myosin ATPase activity is reduced •Termination of cross-bridge cycling

same thing as last few cards

dense bodies

the structure that allows smooth muscle to generate intracellular contractile tension; analogous to z-line

Multiunit §Respond to blank blank. §blank, but no action potential generation. §Hormones, such as blank blank, can directly stimulates smooth muscle contraction.

§Respond to neurogenic stimulus. §Depolarization, but no action potential generation. §Hormones, such as ET-1 and angiotensin II, can directly stimulates smooth muscle contraction.

Single-Unit §Small vessels: almost no iblank §Self blank §Two major types of spontaneous depolarization are displayed by self-excitable smooth muscle cells: blank blank

§Small vessels: almost no innervation. §Self excitable §Two major types of spontaneous depolarization are displayed by self-excitable smooth muscle cells: 1.Pacemaker potential 2.Slow waves

Type of Smooth Muscle Multi vs single unit

•Multiunit Smooth Muscle (individual innervated, neurogenic, fine control): large blood vessels, iris/eyes •Single-Unit Smooth Muscle (self-excitatory, myogenic, gross control): small blood vessels

calcium channel blockers

•Nifedipine •Amlodipine •Verapamil All L-type calcium blocker stop vasoconstricion

Basic Characteristics of VSMC •Not blank •Not organized in blank •Trblankn is absent •Native contractile phenotype •Dblank bodies instead of Z disks

•Not striated •Not organized in sarcomeres •Troponin is absent •Native contractile phenotype •Dense bodies instead of Z disks

myogenic tone •Small caliber blood vessels constrict and relax in response to changes in intraluminal pressure. • inc in intraluminal pressure cause blank •¯dec in intraluminal pressure cause blank (talking about gfr e tal)

•Small caliber blood vessels constrict and relax in response to changes in intraluminal pressure. • intraluminal pressure cause vasoconstriction •¯ intraluminal pressure cause vasodilation

Action Potential in Smooth Muscle Cells Resting Potential : -50 to -60mV •Spontaneous depolarization in smooth muscle is the result of opening of voltage-gated blank). •Membrane potential is not blank. •The change in membrane potential from rest to threshold is the blank potential. (tipo o coraçao) •In blank blank cells, the membrane potential cycles between depolarization and hyperpolarization. •Not every slow wave depolarization is strong enough to reach threshold. •If depolarization reaches a threshold, a burst of action potentials occurs. •Gastrointestinal blank

•Spontaneous depolarization in smooth muscle is the result of opening of voltage-gated Ca++ channels (inward Ca++ current). •Membrane potential is not stable. •The change in membrane potential from rest to threshold is the pacemaker potential. •In slow wave cells, the membrane potential cycles between depolarization and hyperpolarization. •Not every slow wave depolarization is strong enough to reach threshold. •If depolarization reaches a threshold, a burst of action potentials occurs. •Gastrointestinal motility

Source of extracellular Ca2+ ions :

•Voltage-gated L-type Ca2+ channels •Voltage-independent Ca2+ channels (store-operated, ligand-gated, and "leak" channels)