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Cardiovascular Physiology
Neuro-Control of the Heart and Vasculature
1. What is the role of the autonomic nervous system?
It provides for the moment to moment control of the heart and the circulatory system.
2. What is the effect of activation of the autonomic nervous system?
Activation of the sympathetic nervous system:
  • Increases heart rate
  • Increases the speed of conduction
  • Decreases the refractory period
  • Increases contractility
  • Increases the ability of the heart to relax and fill

Activation of the parasympathetic system:

  • Decreases heart rate
  • Decreases contractility
  • Reduces the speed of conduction
  • Increases the refractory period
  • Inhibits the release of and therefore effect of sympathetic action at the level of the heart
3. What is the messenger of the SNS and PNS:
For the SNS it is epinephrine and norepinephrine
  • The source of Epinephrine is mainly from the secretion of the adrenal gland
  • The source of Norepinephrine is mainly from the nerve terminals and the adrenal gland

For the PNS it is acetylcholine

4. How does the SNS mediate its action?
The catecholamines act on specific receptors called adrenergic receptors that are located on the cell surface of the target organ.

  • Types of adrenergic receptors:
    • Alpha 1: mainly located on blood vessels; stimulation causes vasoconstriction (arterial vasoconstriction or venoconstriction). They are mainly located post-synaptically.
    • Alpha 2: mainly located pre-synaptically on the nerve terminal and activation inhibits the release of norepinephrine. Thus they reduce the effect of Alpha 1 activity (negative feedback on norepinephrine). However, distal to the synaptic cleft alpha 2 receptors promote vasoconstriction. Vasoconstriction is the dominant effect.
    • Beta 1: mainly located on the heart; stimulation causes increase in heart rate, contractility, relaxation, etc
    • Beta 2: mainly located on the arteries (mainly coronary and skeletal muscle); stimulation causes vasodilation; no specific innervation to these receptors thus stimulated by circulating catecholamines.
    5. How does the PNS mediate its action?
  • Acetylcholine acts on specific receptors called cholinergic receptors
  • There are two main types of cholinergic receptors: muscarinic and nicotinic receptors. Only muscarinic receptors concern us with activation of cardiovascular activity.
  • Types of muscarinic receptors:
    • M2: located on the heart; stimulation causes decreased heart rate, contractility, etc. Acetylcholine acts on the M2 receptor.
    • M3: located on the arterial tree; stimulation causes vasodilation. Nitric oxide acts via the M3 receptor activation.
    6. How do Beta receptors work?
    Activation of a beta receptor by epinephrine or norepinephrine results in activation of the cytosolic messenger cAMP which promotes the opening of the Ca channel during the action potential
  • The activated beta receptor (a transmembrane protein) induces a series of changes to the membrane bound G protein complex (G proteins are classified as stimulatory (Gs) or inhibitory (Gi); in the case of beta receptor activation a Gs is involved).
  • The activated Gs protein itself activates the transmembrane enzyme system called adenyl cyclase (also called adenylate cyclase). The triple combination of beta receptor, G protein complex, and adenyl cyclase is termed the beta-adrenergic system.
  • Activation of adenylate cyclase produces intracellular cAMP from ATP. Cyclic AMP is the second messenger of the beta-adrenergic system.
  • Cyclic AMP induces the activation of a number of third intracellular messengers called protein kinases, specifically protein kinase A (PKA).
  • Protein kinases phosphorylate various important proteins and enzymes.
    • PKA phosphorylates the Ca channel protein (L type) enhancing Ca entry into the cell during Phase 2 of the action potential. This starts the process of Ca induced Ca release and contraction.
    • PKA also phosphorylates phospholamban, releasing the inhibition of SERCA, resulting in Ca uptake by the SR and induction/promotion of diastole and relaxation.
  • Phosphodiesterases within the cytosol are responsible for the degradation of cAMP promoting the "turning off" of beta stimulation.
  • Beta2 receptors on the vascular smooth muscle wall are activated mainly by epinephrine.

    • As with Beta receptors on the heart, Beta2 vascular receptors result in the release of cytosolic cAMP.
    • Cyclic AMP inhibits the myosin light chain kinase, a protein that enables actin-myosin interaction, hence Beta 2 stimulation promotes vasodilation.
  • PKA, activated by cAMP, is responsible for phosphorylating several proteins involved in contraction and relaxation:
    • Phosphorylates the L type Ca channels causing an increase in Ca flux into the cell during phase 2 of the action potential.
    • Phosphorylates the ryanodine receptor causing an increase in Ca released from the SR.
    • Phosphorylates phospholamban releasing its inhibition of the activity of SERCA causing an increase in Ca uptake in the SR.
    • Phosphorylates Tn-I thereby decreasing the sensitivity of Tn-C for Ca thus enhancing relaxation.
  • 7. How do Alpha receptors work?
  • Alpha receptors mainly function on the vascular smooth muscle wall and are mainly stimulated by norepinephrine.
  • Principally we are concerned with Alpha 1 receptors. Alpha 2 receptors are presynaptic inhibiting norepinephrine release from the nerve terminal promoting vasodilation and postsynaptic promoting vasoconstriction.
  • Alpha 1 receptor activation on vascular smooth muscle activates a G protein which itself activates phospholipase C (PLC)
  • PLC splits phosphatidylinositol into inositol triphosphate (IP3) and diacylglycerol (DAG)
  • IP3 promotes the release of Ca from the SR secondary to Ca induced Ca release
  • The increase in cytosolic Ca binds to the protein calmodulin. The Ca-calmodulin complex activates myosin light chain kinase, which enables the interaction of actin and myosin.
  • DAG activates a protein kinase C that promotes/augments contraction
  • In addition to activating the phosphatidylinositol system, Alpha 1 receptor activation directly opens the Ca channel to promote vasoconstriction.
  • 8. How do Muscarinic receptors work?
  • M2 receptors are activated by acetylcholine release from vagal stimulation
    • The activated M2 receptor activates a G protein (Gi), which binds to adenylate cyclase and inhibits its activation. This results in less cAMP being produced and thus less Ca influx into the cell at the time of the next action potential.
    • The activated Gi protein activates a K channel (KACH) that promotes the loss of K and causes resting membrane potential to become more negative (hyperpolarize) at the end of Phase 3 of the action potential of the SA node thus slowing the heart rate (Phase 4 takes longer to reach threshold and fire).
    • In addition to inhibiting adenylate cyclase, M2 activation may activate a guanylate cyclase enzyme system to increase the level of cGMP in the cell, which reduces the flow of Ca across the L type Ca channel during Phase 2 of the action potential thus mediating a reduction in contractility.
    • M2 receptors are also located on the pre-synaptic nerve terminal and function to inhibit the release of norepinephrine from the nerve terminal thus reducing the activity of the sympathetic system.

  • M3 receptor activation is unclear. Acetylcholine probably activates this receptor. It appears to result in the activation of nitric oxide within the membrane resulting in the activation of adenylate-guanylate cyclase and increase in cGMP within the vascular smooth muscle cell. Cyclic GMP inhibits the myosin light chain kinase, which is responsible for activation of the myosin head and cross bridge formation.
    • Cyclic GMP may also inhibit Ca entry into the smooth muscle cell thus promoting vasodilation
    9. Discuss the regulation of G protein coupled receptors:
    Beta receptor activation is used as a model to describe G protein coupled receptor activation

    Beta receptor (G protein coupled receptor [GPCR]) is activated by epinephrine or norepinephrine

    • The GPCR also called the 7 transmembrane receptor
    • Conformational change to heterotrimeric Gs protein
      • Gby subunits are separated
      • Ga binds to receptor-ligand complex causing a second messenger response
        • 4 functional subtypes of Ga are described from 16 heterotrimeric Gs proteins:
          • Gai - this subunit is involved in M2 receptor activation
          • Gas - this subunit is involved in beta adrenergic receptor activation
          • Gaq - this subunit is involved in alpha adrenergic, angiotensin II receptor activation
          • Ga12 - role of this subunit is unknown
    • Promotes the production of cAMP

    Activation of GPCR by its ligand initiates the process of receptor desensitization (the GPCR is phosphorylated)

    • This process arrests G protein signaling
    • Requires/involves two families of proteins:
      1. G protein-coupled receptor serine/threonine kinases (GRKs) {also known as B-ark [Beta agonist receptor kinase]}
        • There are 7 GRKs
        • GRK2 and GRK3 are most involved in Beta GPCR regulation
          • They are not membrane proteins but cytosolic and must be translocated to the membrane to work
            • This translocation requires G protein activation which liberates Gby dimers that attract the GRK2 and GRK3
            • GRK2 and GRK3 must bind to Gby dimmers for phosphorylation of the receptor
      2. Arrestins
        • There are 4
        • Arrestin 2 and 3 (formerly Beta arrestin 1 and 2)
        • Action of Arrestin 2 and 3:
          • Bind to the phosphorylated receptor that blocks further G protein-initiated signaling through a steric mechanism
          • Also increase the rate of degradation of cAMP by recruiting phosphodiesterases (PDs) to the receptor thus placing the PDs in close proximity to the sites of generation of cAMP.
            • Thus beta arrestins act to desensitize cAMP formation and PKA activation by both impeding the rate of its synthesis and enhance the rate of its degradation.
          • Also induces the process of internalization
            • They bring activated receptors to calthrin-coated pits for endocytosis, a process required for receptor recycling and degradation.
            • They bind to other proteins that assist receptor internalization
        • Ubiquitination of arrestin
          • Ubiquitination involves the attachment of ubiquitin (a 76 aa residue containing protein)
          • Previously thought to tag proteins for proteasomal destruction (kiss of death)
          • Ubiquitination is now believed to be an important process such that the attachment of ubiquitin mediates novel outcomes including protein trafficking and signal transduction
            • Beta arrestins are ubiquitinated by Mdm2, an E3 ubiquitin ligase
            • This is required for the endocytic functions
          • The beta arrestin must be interacting with a stimulated receptor to be ubiquitinated.
          • Ubiquitination is not required for receptor internalization but is required for targeting the internalized receptors to lysosomes for degradation.
          • Signaling leads to various cell survival and anti-apoptotic effects, some forms of chemotaxis, effects on cardiac contractility
          • Cellular signal transduction involves highly coordinated cascades of events. The number of possible downstream targets for any given member of a signaling network is vast, and to maintain integrity and specificity of signaling, cells employ molecular scaffolds. These are large chaperone complexes that hold together specific members of a signaling network to give them preferential access to one another, thus ensuring the fidelity of a particular signaling response. Thus in addition to their classic roles in desensitization and internalization, beta arrestins can also act as signaling scaffolds for many pathways and in particular those of the MAPKs (mitogen-activated protein kinases).
            • The downstream effectors of MAPKs control many cellular functions, including cell cycle progression, transcriptional regulation, and apoptosis.
            • ERKs are a specific MAPK
          • Beta arrestins are involved in nuclear function - transcriptional regulation
          • Beta arrestins may be activated without activation of a G protein receptor
      • GRK2 and GRK3 can also reduce Gq-coupled receptor activity (alpha receptor IP3 system)
        • GRK2 and GRK3 bind to activated Gaq subunit and sequester it preventing its coupling to downstream effectors
      • Binding of GPCR to ligand causes a conformational change - this enables binding of GRKs which results in phosphorylation of residues on the intracellular loops and carboxyl terminus of GPCR
      • Binding of GRK 2 and 3 to GPCR, without receptor phosphorylation, may also be sufficient to suppress signaling
      • Phosphorylation of the GPCR residues promotes the high-affinity binding of the arrestin family of proteins (there are 4) to the GPCR which prohibits further coupling to G proteins. Can cause an 80% reduction of receptor signaling.

    Phosphorylation of GPCR can also occur by PKA and PKC and c-Src causing receptor desensitization.

      • This is a feedback mechanism whereby the agonist-stimulated GPCR generates a second messenger (like cAMP) that activates a kinase (like PKA) that decreases the activity of the receptor (the GPCR) and ultimately attenuates the production of the second messenger.

    Regulation of GRKs

      • Other kinases phosphorylate GRKs modulating their activity
        • Site of phosphorylation affects GRKs activity
          • Site is dependant on the type of kinase
      • Basal state GRK2 exists in a phosphorylated state in the cytosol that is the inactive form
        • Phosphorylated by Erk1/2 (extracellular signal-regulated kinases)
          • Phosphorylates GRK2 at Ser670
            • Causes a marked reduction in activity of GRK2
            • Causes a marked decrease in ability to bind to Gby subunits
          • Erk1/2 are activated by Erk1/2 mitogen-activated protein kinase/s
        • Agonist-occupied GPCR and Gby subunits promote GRK2 and Erk1 rapid association
      • PKA and PKC enhances GRK2 activity
      • The non receptor tyrosine kinase c-Src phosphorylates GRK2
        • This phosphorylation is dependent on the ability of beta-arrestin to bind to c-Src and recruit c-Src to the GPCR
        • Tyrosine phosphorylation affects GRK2 activity in two ways
          • Rapidly and transiently increases GRK2 activity
            • Enhances phosphorylation of GPCR and desensitization
          • Promotes degradation of GRK2 by ubiquitin/proteosome pathway
            • Reduce GRK2 protein levels

    The scheme:

      • GPCR activation → splits the heterotrimeric G protein separating off the Ga subunit
        • Phosphorylated GRK2 is dephosphorylated by a phosphatase → dephosphorylated GRK2 (active) is recruited to the plasma membrane → GRK2 binds to receptor and Gby subunits → GRK2 binds Erk1 or 2 → phosphorylates GRK2 on Ser670 → deactivates GRK2 → promotes release of GRK2 from plasma membrane and back into the cytosol in its inhibited ("off") phosphorylated state.
        • Phosphorylated GRK2 promotes the interaction of beta arrestins with the phosphorylated receptor leading in turn to desensitization of further G protein signaling by steric exclusion by the beta arrestin.

    Other Regulation of GPCR activity

      • Activated GPCR activates a second messenger:
        1. cAMP → activates PKA
        2. Diacylglycerol → activates PKC
        3. IP3 → activates calcium/calmodulin-activated kinases to mobilize Ca
      • Two of these kinases (PKA and PKC) directly phosphorylate GPCR (desensitize it)
      • PKA augments GRK2 activity
        • Increases its affinity for Gby dimer
          • This promotes recruiting GRK2 to the plasma membrane and to complex with the activated receptor substrate
      • PKC augments GRK2 activity
        • Increases activity of GRK2 by promoting its translocation to the plasma membrane