Lecture 7 Physiology of the Vasculature I

Question 1

What are the different types of blood vessels

Answer 1

Large arteries medium-sized arteries arterioles capillaries venules medium-sized veins and large veins

Question 2

Outline the structure of the vascular wall

Answer 2

The innermost layer surrounds the lumen of the vessel and is called the tunica intima. This consists of a single cell layer of endothelial cells attached to the basement membrane. The next layer is the tunica media this layer contains the vascular smooth muscle as well as elastic tissue. The relative proportion of elastic tissue and vascular smooth muscle varies between different types of vessels. The outermost layer is the tunica adventitia or tunica externa. This consists of a protective layer made up of fibrous connective tissue and extracellular matrix proteins such as collagen

Question 3

Below is a histological staining of a blood vessel label the endothelium vascular smooth muscle and fibrous connective tissue layers on the diagram

Answer 3

See completed labels

Question 4

What are the two main ways that the endothelium and adjacent VSMCs communicate

Answer 4

Gap junctions and via the release of mediators from the endothelium that act on the vascular smooth muscle

Question 5

What is the role of the glycocalyx

Answer 5

Under normal conditions where the endothelium lining the blood vessels is healthy there is a layer on top known as the glycocalyx. This consists of carbohydrate chains attached to the cell membranes of the endothelial cells. The glycocalyx as an anti-coagulant effect by preventing circulating cells from binding to the endothelial layer

Question 6

What happens to the glycocalyx in disease and what is the effect of this

Answer 6

Activated/Dysfunctional endothelium in disease will shed the glycocalyx. This exposes cell adhesion molecules (CAMs) that allow immune system cells such as monocytes neutrophils and platelets to bind to the endothelium

Question 7

Glycocalyx shedding is a phenomenon that only happens in disease T or F

Answer 7

F - Glycocalyx shedding is a normal response tissue injury infection and inflammation

Question 8

Other than disease what other factors can cause glycocalyx shedding

Answer 8

Glycocalyx shedding can also occur at disturbed blood flow. This is where eddies and turbulent flow causes loss of glycocalyx

Question 9

Outline the healthy endothelial cell signalling that goes on in the blood vessels

Answer 9

In healthy endothelium neurotransmitters such as acetylcholine histamine 5-HT and bradykinin bind to receptors and increase Ca2+. A rise in intracellular Ca2+ activates endothelial nitric oxide synthase (eNOS) which then converts arginine into NO and citrulline. NO diffuses out of the endothelium and acts on adjacent smooth muscle cells where it causes relaxation

Question 10

High shear blood flow also increases Ca2+ and activates eNOS T or F

Answer 10

T

Question 11

Outline what happens in activated endothelium during disease

Answer 11

Activated endothelium have shed their glycocalyx and then IL-1 thrombin and endotoxins activate the receptors of the endothelial cells leading to the production of endothelin 1 (ET-1). Meanwhile there is an increased upregulation of ROS ICAM-1 VCAM1 IL-8 and COX2. All these pathway act to cause vascular disease

Question 12

Explain the role of Ca2+ and its downstream targets in the contraction of VSMCs

Answer 12

Activation of Ca2+ channels in the plasma membrane of the endothelium leads to an influx of Ca2+ ions. This rise in intracellular Ca2+ activates calmodulin. Calmodulin in the presence of Ca2+ activates MLCK which then phosphorylates inactive myosin allowing it to bind actin. Once phosphorylated and bound to actin myosin can mediate the contraction of the smooth muscle

Question 13

What intrinsic mechanism in smooth muscle accounts for its relaxation

Answer 13

Myosin light chain phosphatase (MLCP) causes the relaxation of VSMCs by dephosphorylating myosin. This enzyme is constrictively active in the vasculature meaning that it will trigger relaxation as soon as the contractile stimuli has passed

Question 14

What are the key differences between skeletal muscle and VSMCs

Answer 14

In contrast to skeletal muscle myosin in vascular smooth muscle needs to be phosphorylated in order to bind actin. The other key difference is that myosin light chain phosphatase (MLCP) is constitutively active in VSMCs and hence as Ca2+ levels fall there is a natural relaxation

Question 15

How else can Ca2+ levels be raised inside vascular smooth muscle cells in order to trigger contraction

Answer 15

Receptors that activate IP3 can also lead to VSMC contraction via acting on IP3R and RyR1 receptors to cause store release of Ca2+

Question 16

What are the two classes of contractile stimuli in the VSMCs

Answer 16

GPCRs that are GαQ coupled these activate PLC-γ and increase IP3 levels which feeds in to Ca2+. The other class are Ca2+ channels which act to directly increase intracellular Ca2+

Question 17

Give some examples of contractile stimuli that raise IP3 levels

Answer 17

Endothelin A/B receptors the thromboxane prostanoid receptor (TP-R) the AT-1 (angiotensin II) receptor the histamine receptor as well as the αARs

Question 18

Give some examples of contractile stimuli that directly raise Ca2+ levels

Answer 18

L-type voltage-gated Ca2+ channels ligand-gated cation channels such as P2X (ATP-activated) and TRP channels as well as store operated Ca2+ channels like Orai1

Question 19

What are the three key players in VSMC relaxation and how do the broadly act

Answer 19

cGMP – produced from the activation of guanylate cyclase and causes a reduction in Ca2+ levels. cAMP – produced from the activation of adenylate cyclase and causes a reduction of Ca2+ levels. K+ channels – activation leads to K+ efflux and a hyperpolarisation of the membrane potential which in turn acts to decrease Ca2+ levels

Question 20

What is the universal mechanism by which VSMC stimuli cause relaxation

Answer 20

All three mechanisms feed in to reduce Ca2+ levels which decreases the activity of MLCK activity. This means that MLCP activity now predominates due to its constitutive acitivity and can dephosphorylate myosin leading to its dissociation from actin and subsequent relaxation

Question 21

What are the four classes of relaxile stimuli

Answer 21

NO from the endothelium GPCRs that are GαS coupled K+ channels and PDEs

Question 22

Broadly how does NO cause vascular relaxation

Answer 22

NO from the endothelium activates guanylate cyclase which then increases the levels of cGMP. cGMP then activates PKG which phosphorylates and increases the activity of MLCP. Dephosphorylation of myosin by MLCP causes its dissociation from actin and thus relaxation of the VSMCs.

Question 23

Broadly how do some GPCRs cause vascular relaxation

Answer 23

GPCRs that are coupled to GαS subunits stimulate adenylate cyclase and increase the activity of cAMP. cAMP then causes activation of PKA which also phosphorylates MLCP to increase its activity. In addition raised cAMP levels also cause a reduction in Ca2+ levels which decreases the activity of MLCK

Question 24

Broadly how do some K+ channels cause vascular relaxation

Answer 24

K+ channel activation leads to K+ efflux and a hyperpolarisation of the membrane potential which in turn acts to decrease Ca2+ levels. This can occur through their direct activation through changes in the membrane potential or indirect activation through β-adrenoceptor agonist binding which activates the βγ subunit of the G-protein which can in turn activate GIRKs

Question 25

Broadly how can regulation of PDEs cause vascular relaxation

Answer 25

PDEs hydrolyse cAMP and cGMP. Hence by inhibiting these enzymes you can reduce cAMP and cGMP hydrolysis this potentiating their activity and leading to relaxation

Question 26

What are the 4 pathways by which the endothelium regulates the vascular smooth muscle and what is the effect of each pathway

Answer 26

NO release – relaxation prostanoid synthesis – relaxation or contraction endothelin release – contraction angiotensin II – contraction

Question 27

What are the two effects of raising cAMP in vascular smooth muscle cells

Answer 27

Increased cAMP levels results in a decrease in Ca2+ levels which decreases the activity of MLCK. Meanwhile elevated cAMP also activates PKA. PKA phosphorylates MLCP and increases its activity. This switches the balance of these two proteins so that MLCP dominates and dephosphorylates myosin

Question 28

NO is an important regulator of blood pressure and regional blood flow T or F

Answer 28

T

Question 29

Where is eNOS usually found and what is the significance of this

Answer 29

eNOS is held within caveolae which acts to localise the enzyme to clusters of nearby receptors

Question 30

NO is produce by NOS in the endothelium in response to what stimulus

Answer 30

Elevated Ca2+ levels in the endothelial cells

Question 31

What is the effect of NO once release from the endothelium

Answer 31

It diffuses to the VSMCs and acts on guanylate cyclase to increase the production of cGMP. This acts to decrease Ca2+ levels in the vascular smooth muscle cells

Question 32

What is the effect of smoking on the NO pathway

Answer 32

eNOS activity is impaired in smoking. This there can lead to hypertension due to a failure to produce NO to dilate blood vessels

Question 33

What is the effect of diabetes on the NO pathway

Answer 33

Sustained high blood glucose levels leads to internalisation of the insulin-receptors. This results in a decreased activation of Akt which is another kinase required to phosphorylate and activate eNOS. Hence there is decrease activation of eNOS decreased NO synthesis and potential hypertension

Question 34

What is the effect of high levels of LDL on the NO pathway

Answer 34

Oxidised LDL (oxLDL) depletes cholesterol from the caveolae which in turn results in a loss of eNOS. This can also lead to hypertension

Question 35

How is the precursor to prostanoids made in the endothelial cells how can this pathway be stimulated

Answer 35

The COX enzymes convert arachidonic acid to PGH2 the precursor to prostanoid pathway components. COX2 activated by rise in intracellular Ca2+ or ROS levels in the endothelium

Question 36

PGH2 can be processed to thromboxane A2 before it is released by the endothelium cells what is the effect of TxA2 on the VSMCs and how does this occur

Answer 36

TxA2 released from the endothelium acts on the TP-R receptor which is a GαQ receptor that couples to PLC-γ and increases IP3 levels. IP3 causes Ca2+ release and subsequent constriction

Question 37

PGH2 can be processed to PGE2 before it is released by the endothelium cells what is the effect of PGE2 on the VSMCs and how does this occur

Answer 37

PGE2 released from the endothelium acts on the TP-R receptors 1-4 which are either GαS or Gα­I coupled receptors. The GαS coupled EP-R receptors stimulate adenylate cyclase and increase cAMP levels which acts to decrease Ca2+ levels and cause relaxation. Meanwhile the GαI coupled EP-R receptors inhibit adenylate cyclase and decrease cAMP levels which acts to decrease Ca2+ levels and cause contraction

Question 38

PGH2 can be processed to PGI2 before it is released by the endothelium cells what is the effect of PGI2 on the VSMCs and how does this occur

Answer 38

PGI2 released from the endothelium acts on the IP-R receptor which is a GαS receptor that leads to the simulation of adenylate cyclase and an increase in cAMP levels. This in turn leads to a decrease in Ca2+ levels and subsequent relaxation. In addition the rise in cAMP levels also lead to activation of PKA which then phosphorylates MLCP increasing its activity in promoting relaxation

Question 39

What are the three prostanoids that can be formed from PGH2 and how do their effects of the VSMCs differ

Answer 39

TxA2 – causes contraction via IP3 pathway. PGE2 – can cause contraction via the inhibition of adenylate cyclase or relaxation by stimulating it. PGI2 – causes relaxation by stimulating adenylate cyclase and PKA activity

Question 40

Outline how the endothelin pathway causes vasoconstriction

Answer 40

The precursor to endothelin-1 produced in the endothelial cells is called big endothelin. Endothelin converting enzyme (ECE) in the endothelium then cleave big endothelin to endothelin-1 (ET-1). ET-1 is then released by the endothelium where is acts on the ETA or ETB receptors on the membrane of the VSMCs. These receptors are GαQ-coupled GPCRs that activate PLC-γ and increase IP3 levels. IP3 then causes Ca2+ release from the SR by acting on the IP3R and RyR1 receptors. This then causes contraction of the vascular smooth muscle

Question 41

What factors cause an upregulation of big endothelin

Answer 41

IL-1 Thrombin Glucose OxLDL Insulin Angiotensin II Cortisol Adrenaline and Hypoxia

Question 42

Outline the negative feedback mechanisms that exists in the endothelin pathway

Answer 42

The endothelial cells themselves express the ETB receptors. Hence ET-1 released by the endothelium acts back on its own receptors to cause an increase in Ca2+. Increases in Ca2+ in endothelial cells activates eNOS producing NO that actually inhibits ECE. Hence there is a negative feedback loop that acts on the endothelial cells to transiently downregulate ET-1. There is also a negative feedback on the vascular smooth muscle cells as the NO released from the endothelium can then act on the VSMCs to promote relaxation hence opposing the effect of ET-1

Question 43

Outline how the angiotensin II pathway causes vasoconstriction

Answer 43

Angiotensin II is produced the conversion of circulating angiotensin I to angiotensin II by ACE enzymes. Angiotensin II acts on the AT-1 receptors expressed by the VSMCs. These AT-1 receptors are GαQ-coupled GPCRs that activate PLC-γ and increase IP3 levels. This hence causes contraction by causes Ca2+ release from the SR

Question 44

Describe the long term effects of the angiotensin II pathway

Answer 44

The AT-1 receptor also activates the MAPK pathway which accounts for the long-term changes in the VSMCs. This is mediate through alterations in gene expression and protein synthesis and results in a more long-term contractile response