CVPR 03-26-14 08-10am Regulation of the CV system - Proenza Flashcards
G Protein-Coupled Receptors (GPCRs) – defn.
7-transmembrane-spanning (7TM) integral membrane proteins that transduce ligand binding to intracellular signaling; some of the most prevalent drug targets (beta blockers, angiotensin II blockers)
Cardiovascular GPCRs include…
α & β adrenergic receptors, ACh receptors, endothelin receptors, adenosine receptors, angiotensin II receptors.
GPCR activation scheme
Agonist binds receptor –> GTP replaces GDP on α subunit of heterotrimeric G protein –> dissociation of α and βγ G protein subunits –> Both α and βγ can be active signals
GPCR deactivation:
Auto dephosphorylation of GTP to GDP by α subunit permits reassociation with βγ…..Rebinding of G protein to receptor causes inactivation.
Families of G proteins involved in cardiovascular function:
Gs and Gi/o proteins
Gs & Gi/o are stimulatory & inhibitory, respectively, for cAMP production by adenylate cyclase
Gq protein
Its activation increases intracellular Ca2+ via activation of phospholipase C (PLC) and Protein Kinase C (PKC)
α1 adrenergic receptor - G protein, signaling pathway, effects
Gq —- Pathway: PLC, PKC –> increases intracellular Ca2+ —> vasoconstriction
β adrenergic receptor - G protein, signaling pathway, effects
Gs (β1 and β2)— Pathway: Simulates adenylate cyclase, increases cAMP —> In heart, increases chronotropy, inotropy, lusitropy, dromotropy…..In skeletal muscle vascular beds, vasodilation.
Muscarinic receptor - G protein, signaling pathway, effects
Gi/o —- Pathway: Inhibits adenylate cyclase –> decreases cAMP; Releases βγ subunits….. Effect: Decreased chronotropy
Regulation of Inotropy
Autonomic; both sympathetic & parasympathetic
Sympathetic regulation of inotropy
cAMP signaling, Phospholamban (PLB), phosphorylation of L-type Ca2+ channels & RyRs by PKA, phosphorylation of TN-I
Process to Increase of cAMP
Sympathetic neurons innervate the heart, release norepinephrine –> binds β adrenergic receptors to increase cAMP
Phosphodiesterases - what it is & action
= counterpart to adenylate cyclase; Breakdown cAMP (and cGMP) —> help to establish intracellular signaling microdomains and specificity of signaling
Protein Kinase A (PKA) - what it is & action
cAMP-dependent protein kinase; Major effector for cAMP signaling in heart; Phosphorylates target proteins (its counterpart is phosphatases that dephosphorylate these targets) —> changes protein function by changing conformation & charge.
Phospholamban (PLB)
PLB is an inhibitor of SERCA (which removes Ca2+ from cytosol following contraction by pumping it back into the SR)
Un-inhibiting SERCA
Phosphorylation of PLB by PKA causes it to dissociate from SERCA, thereby relieving the inhibition and increasing Ca2+ reuptake rate.
Two effects of Faster Ca2+ reuptake has on cardiac performance:
1) directly increases “lusitropy” (relaxability) – and 2) increases inotropy by increasing SR Ca2+ load.
Lusitropy
the ability of the heart to relax
Inotropy
Alteration of the the force or energy of muscular contractions….Negatively inotropic agents weaken the force of muscular contractions, while Positively inotropic agents increase the strength of muscular contraction
L-type Ca2+ channels (LTCCs) - action
On the plasma membrane; Activated by depolarization —> influx of Ca2+ —> triggers larger Ca2+ release from SR via ryanodine receptors (Ca2+-induced Ca2+ release (CICR))
PKA & L-type Ca2+ channels
Phosphorylation of L-type Ca2+ channels
by PKA slows inactivation –> increases magnitude of L-type Ca2+ current —> this increase in “trigger Ca2+” elicits a larger release of Ca2+ from the SR, thereby increasing inotropy.
Troponin I (TnI) - action
The inhibitory unit of the troponin complex (TnC-TnI-TnT); Along w/tropomyosin, inhibits the interaction btwn actin & myosin in the absence of Ca2+.
Phosphorylation of Troponin I (TnI)
TnI is phosphorylated by multiple kinases, including PKA…..Phosphorylation of TnI decreases the Ca2+ sensitivity of TnC —> would expect to decrease inotropy (counter to the sympathetic effect), BUT it rather results in faster dissociation of Ca2+ from TnC, thereby increasing lusitropy, which allows the heart to fill more quickly (increases lusitropy; no effect on inotropy?). This is particularly important at higher heart rates.
Parasympathetic regulation of inotropy
Parasympathetic innervation of the ventricle is sparse, thus there is little parasympathetic control of inotropy.
Basal autonomic tone and “intrinsic” heart rate
Resting heart rate in humans is normally 60-80 bpm; However, both divisions of the autonomic nervous system have basal activity that influences the resting HR…..Normally the parasympathetic tone at rest is greater than the sympathetic tone (but there still is basal sympathetic activity at rest). Intrinsic heart rate is revealed by block of both sympathetic and parasympathetic tone (heart rate w/out nervous regulation).
Block of M2 muscarinic acetylcholine receptors with atropine - effect on HR
increases HR by inhibiting tonic parasympathetic activity
Block of β adrenergic receptors w/ propanolol - effect on HR
decreases HR by inhibiting tonic sympathetic activity
Molecular targets for sympathetic stimulation of chronotoropy
- Hyperpolarization-activated cyclic nucleotide-gated channels (HCNs)…..2. L-type Ca2+ channels…..3. Ryanodine receptors and Sodium-Calcium exchanger
Hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) - Action
HCN channels are highly expressed in SA node myocytes and produce the cardiac funny current (If), which is an inward (depolarizing) current at diastolic potentials —> promotes excitability & spontaneous APs
Sympathetic regulation of Hyperpolarization-activation cyclic nucleotide-gated channels (HCNs)
Sympathetic stimulation of SA cells causes an increase in activity of HCNs via cAMP binding (shifts voltage dependence of activation —> channels more likely to open —> more inward current to speed the rate of diastolic depolarization)
β adrenergic stimulation of L-type Ca2+ channels
β adrenergic stimulation increases L-type Ca2+ current —> net inward (depolarizing) current –> contributes to sympathetic increase in HR via increased excitability and all spontaneous APs, since nodal cells use “slow” Ca2+ APs….. Activity increased further by sympathetic stimulation (phosphorylation by PKA)
Sympathetic stimulation of Ryanodine receptors and Sodium-Calcium exchanger
Sympathetic stimulation increases SR Ca2+ load via PKA phosphorylation of L-type Ca2+ channels, RyRs, and phospholamban —-> increases spontaneous Ca2+ release rate & contributes to diastolic depolarization via the sodium-calcium exchanger, NCX (Ca2+ out, 3 Na+ in, generating a net inward current) –> removes Ca2+ from cytoplasm & promotes excitability / spontaneous APs
Parasympathetic regulation of pacemaking is mediated by…
…release of acetylcholine (ACh) from vagal nerve endings in the SA node; ACh activates M2 muscarinic ACh receptors coupled to Gi/o heterotrimeric G protein —> activation of Gi/o releases two signals: the Gαi/o subunit and the Gβγ subunit complex.
Molecular targets for parasympathetic inhibition of chronotropy
GIRKs, HCNs, L-type Ca2+ channels, and ryanodine receptors
GIRKs (G-protein coupled inwardly-rectifying K+) and Chronotropy
The Gβγ subunit complex binds directly to GIRK channels to activate the IKACh current - IKACh stabilizes the membrane potential near the K+ equilibrium potential, thereby dampening excitation & slowing spontaneous firing frequency = appears to be primary mechanism for parasympathetic slowing of HR
HCNs, L-type Ca2+ channels, and ryanodine receptors
The Gαi/o subunit inhibits adenylate cyclase, thus reducing intracellular cAMP levels —> opposite effect to sympathetic stimulation of pacemaking: reduction in inward current via HCN channels, L-type Ca2+ channels, and RyR-NCX = these mechanisms appear to play a secondary role in parasympathetic regulation of HR
Vascular smooth muscle cells (VSMCs) – characteristics
small mononucleate cells, electrically coupled via gap junctions.
Why smooth (vs. striated)?
Myofilaments are not arranged in sarcomeres in smooth muscle.
Ca2+ role in VSMCs (Vascular Smooth Muscle Cells)
Ca2+ release from SR not essential for contraction in VSMCs…..However, Ca2+ reuptake mechanisms are similar (SERCA and PLB are present) in VSMC & striated muscle.
Contraction in VSMCs vs. striated muscle
Rate of contraction is slower in VSMCs and contraction is sustained & tonic (vs. short duration in cardiac muscle).
Review of striated muscle contraction mechanism
At rest, troponin I is bound to actin. Troponin T recruits tropomyosin, which blocks myosin binding site on actin. An action potential (required) triggers Ca2+ release from SR via excitation-contraction coupling —> Ca2+ binds to troponin C —> rearrangement of troponin complex & tropomyosin that uncovers the myosin binding site on actin THIN filament —-> permits cross bridge cycling to occur……Contraction is halted by removal of Ca2+.
Smooth muscle contraction - ways to initiate
Can be initiated by mechanical (stretching, via myogenic response), electrical, or chemical stimuli [ rather than requiring AP]
Vascular Smooth muscle differences from Skeletal
Mononuclear; No sarcomeres; No troponin or tropomyosin; Different contraction mechanism (thick rather than thin filament regulation); APs not required
Electrical means to initiate smooth muscle contraction
Electrical depolarization can elicit contraction via activation of L-type Ca2+ channels…. Different from striated muscle in that APs are not required; graded potentials are sufficient, and strength of contraction is proportional to stimulus intensity
Chemical means to initiate smooth muscle contraction
Chemical stimulation by several neural & hormonal regulators (eg: norepi, angiotensin II, vasopressin, endothelin, and thromboxane A2) can directly activate contraction
Contraction of VSMCs depends on…
…phosphorylation of the myosin head as the essential step, rather than Ca2+ directly activating contraction (although it is indirectly Ca2+-dependent).
Ca2+ regulation of smooth vs. striated muscle contraction
Ca2+ regulation of SMOOTH muscle contraction is via myosin THICK filaments, whereas Ca2+ regulation of STRIATED muscle contraction is via actin thin THIN filaments. (Remember, smooth muscle does not have the Ca2+-sensitive troponin complex or tropomyosin).
How Calcium gets into the cytoplasm during VSMC activation
mainly from SR but also via voltage-gated Ca2+ channels on surface membrane
Calmodulin (CaM)
a ubiquitous intracellular Ca2+ binding protein
Steps in VSMC activation
Trigger (mechanical, chemical, electrical) –> Ca2+ enters cytoplasm —> Ca2+ binds Calmodulin (CaM) —> Ca2+-CaM binds to Myosin Light Chain Kinase (MLCK) to activate it. —> Activated MLCK phosphorylates the light chain of myosin (myosin head) —> cross bridge cycling.
cAMP’s effect on vascular smooth muscle cells
cAMP causes relaxation of VSMC (in contrast to its effect on cardiac myocytes, where it promotes contraction via PKA)…in VSMCs, PKA phosphorylates myosin light chain kinase to inhibit its activity, and thus reduce VSMC contraction
Type(s) of Autonomic Regulation of the Vasculature
Primarily sympathetic innervation of the vasculature (relatively little parasympathetic innervation)
Sympathetic stimulation in vasculature generally causes…
Vasoconstriction; Contraction of VSMCs, independent of membrane depolarization.
α1 adrenergic receptors
GPCRs coupled to the Gq heterotrimeric G protein
Phospholipase C (PLC) - what it is & what it produces
An enzyme activated by Gαq of the α1 adrenergic receptors, to produce diacylglycerol (DAG) and inositol trisphosphate (IP3).
Inositol trisphosphate (IP3) - action
Produced by PLC; Activates IP3 receptors on the SR of VSMCs (intracellular Ca2+ release channels, like RyRs) —> opens IP3Rs causing Ca2+ release from the SR into the cytoplasm —> VSMC contraction & thus vasoconstriction.
Protein kinase C (PKC)
Ca2+-dependent protein kinase; Phosphorylates many targets in VSMCs, including L-type Ca2+ channels (LTCC), which are activated —> Inward current through LTCCs in turn activates additional intracellular Ca2+ release (Ca2+-induced Ca2+ release)