C3. Vascular Smooth Muscle- detailed

Question 2

Headings pneumonic

Answer 2

CMPLENMNN

Question 3

Headings (list)

Answer 3

Introduction

Calcium sparks & contraction

Myogenic tone

Pressure-sensitivity

L-type calcium channels, regional heterogeneity, BKCa channels

Endothelial dependent vasodilation

Negative feedback and calcium signalling

Metabolic control of VSMC contraction

Neural control of VSMC contraction

Neurovascular coupling and HPV

Conclusion

Question 4

Calcium sparks & contraction subheadings (list)

Answer 4

Calcium sparks promote membrane hyperpolarisation

Nelson 1995

Sodium, chloride and potassium

Question 5

(Calcium sparks) Calcium sparks promote membrane hyperpolarisation

Answer 5

● Whilst calcium is widely known as one of the most important ions in VSMC contraction, other ions such as chloride, sodium and potassium ion channels are increasingly recognised.

● As mentioned, calcium is thought to promote VSMC calcium contraction, however, there is also evidence to suggest that calcium can promote relaxation.

● Subsurface Ca2+ sparks promote membrane hyperpolarization through activation of BKCa causing spontaneous transient outward currents in arterial smooth muscle myocytes.

● Hyperpolarization reduces Ca2+ influx via voltage-operated/dependent channels (VDCCs) relaxing arterial smooth muscle.

Question 6

(Calcium sparks) Nelson 1995

Answer 6

● This was demonstrated by Nelson et al in the Cheng laboratory that discovered calcium sparks in 1995.

● The authors measured calcium events in VSMCs isolated from cerebral resistance arteries, and showed elementary calcium sparks by using the calcium indicator Fluo-3.

● The authors then used voltage clamp and showed that outward transient currents that were attenuated by inhibitors of local sparks.

● The relevance of this was demonstrated in pressurised arteries where calcium spark inhibition constricted arteries.

Question 7

(Calcium sparks) Sodium, chloride and potassium

Answer 7

● Sodium channels may also be important in promoting vascular smooth muscle contraction, and it is now thought that Na+ influx drives reverse mode NCX, Ca2+ entry and Cl-Ca activation to cause depolarisation.

● Chloride levels in vascular smooth muscle cells are elevated due to active accumulation via Cl−/HCO3 − exchange and Na+-K+-2Cl− cotransport.

● Therefore, opening of Cl- channels can result in a depolarising chloride ion efflux activating voltage gated calcium channels.

● The ensuing rise in the cytosolic Ca2+ concentration ([Ca2+] i enhances force development by activating myosin light chain kinase via an increased formation of Ca2+-calmodulin.

Question 8

Myogenic tone subheadings (list)

Answer 8

Radial force stretches VSMCs

Autoregulation

Integrins

Colinas 2015

Question 9

(Myogenic tone) Radial force stretches VSMCs

Answer 9

● In a resistance artery or arteriole, increased luminal pressure results in a radial force that increases tangential wall stress (tension).

● The increased wall stress passively dilates the vessel and “stretches” (induces strain in) the smooth muscle cells in the vessel wall.

● The discovery of the myogenic mechanism is attributed to Bayliss in the early 1900s, who observed changes in vascular tone in response to increased intraluminal pressure.

● These two processes, the myogenic response and myogenic tone, participate in the blood flow autoregulation in organs, such as the brain, heart, kidney, eye buffering organ blood flow and capillary pressure in the face of changes in blood pressure.

Question 10

(Myogenic tone) Autoregulation

Answer 10

● Myogenic tone offers a resting level of smooth muscle contractile activity such that resistance arteries and arterioles can both dilate and constrict around their resting diameters, maintaining cardiovascular homeostasis.

● Furthermore, this autoregulation is a protective mechanism to protect capillaries from high haemodynamic pressure that may cause rupturing.

● This myogenic tone appears to be initiated by the smooth muscle cell layer, given the response persists in the presence of endothelial denudation by mechanical abrasion with hair.

● However, the exact mechanism of myogenic tone remains relatively elusive.

Question 11

(Myogenic tone) Integrins

Answer 11

● Recently, interest has focused on the integrin family of adhesion molecules as possible “transducers” of changes in vascular smooth muscle tension.

● The integrins are a large family of cell-cell adhesion receptors comprising at least 16 α and 8 β subunits that can heterodimerize to produce more than 20 transmembrane receptors.

● The integrins can recognize ligands of the extracellular matrix and transmit extracellular stimuli into intracellular signalling events.

Question 12

(Myogenic tone) Colinas 2015

Answer 12

● Colinas et al in 2015 demonstrated that alpha5 integrin-mediated cellular signalling contributed to the myogenic response of cerebral resistance arteries.

● The authors used function-blocking antibodies against these integrins.

● The authors demonstrated in pressurised arteries using phosphorylation-specific antibodies in Western blotting that intravascular pressure induced FAK and SFK phosphorylation.

● The authors then demonstrated that integrin-blocking antibodies prevented myogenic constriction and inhibited the phosphorylation of FAK and SFK.

Question 13

Pressure-sensitivity subheadings (list)

Answer 13

Transient receptor potential channels (TRPC6 and TRPM4)

Gonzales 2010

Schleifenbaum 2014

Question 14

(Pressure) Transient receptor potential channels (TRPC6 and TRPM4)

Answer 14

● Pressure myography studies have repeatedly shown that pressure-induced myogenic tone involves vascular smooth muscle membrane depolarization, activation of VGCCs, and an increase in intracellular Ca2+.

● The transient receptor potential channels are a well-known pressure-sensitive set of ion channels that cause this depolarisation.

● Therefore, it was proposed that these ion channels may play a role in myogenic tone, particularly TRPC6 and TRPM4.

Question 15

(Pressure) Gonzales 2010

Answer 15

● Indeed, Gonzales et al in 2010 used a TRPM4 inhibitor, 9-phenanthrol, and observed in patch-clamped isolated smooth muscle cells, that inhibition of TRPM4 resulted in hyperpolarisation of the smooth muscle membrane.

● Furthermore, when the authors elevated the pressure in an isolated cerebral artery to 70mmHg and observed the development of myogenic tone.

● It was thus shown that 9-phenanthrol inhibited the development of myogenic tone in a dose-dependent manner, increasing the diameter of the pressurised arterioles.

● Whilst there are concerns about the specificity of these TRP inhibitors, the authors attempted to show that it does not interact with other proteins.

Question 16

(Pressure) Schleifenbaum 2014

Answer 16

● Schleifenbaum et al in 2014 used transgenic mice deficient for either the AT1 receptor or angiotensinogen.

● The authors then isolated mesenteric and renal interlobular arteries, before pressurising them to physiological pressures.

● The authors then performed stepwise increases in pressure and showed that AT1a receptors were responsible for myogenic decreases in vessel diameter.

● These findings were then replicated in the renal circuits.

Question 17

L-type calcium channels, regional heterogeneity, BKCa channels subheadings (list)

Answer 17

Kotecha & Hill 2005

Jackson 2017 and regional heterogeneity

Large-conductance Ca2+-activated K+ (BKCa) channels

Question 18

(L-type) Kotecha & Hill 2005

Answer 18

● L-type calcium channels have long been thought to be important in myogenic tone.

● L-type VGCCs composed of CaV1.2 α-pore-forming subunits contribute substantially to pressure-induced myogenic tone that is observed in pressurized resistance arteries and arterioles studied ex vivo.

● Indeed Kotecha & Hill in 2005 demonstrated that pharmacological inhibition of these channels was sufficient to ablate myogenic tone.

● The authors used pressurised skeletal muscle arterioles and were able to demonstrate that step-wise pressure increases depolarised smooth muscle cells and promoted vasoconstriction.

Question 19

(L-type) Jackson 2017 and regional heterogeneity

Answer 19

● What remains unclear is what determines which of these putative mechanosensitive elements are expressed in a particular blood vessel and how this expression is controlled under different physiological and pathological conditions.

● Figure 3

● Jackson et al in 2017 evaluated some of the heterogeneity in myogenic tone mechanisms using pressurised hamster arterioles taken from the cremaster or cheek-pouch.

● The authors used pharmacological inhibition of Phopholipase C with U73122 and IP3Rs with another small molecule inhibitor.

Question 20

(L-type) Large-conductance Ca2+-activated K+ (BKCa) channels

Answer 20

● Membrane depolarization in cells that express VGCCs is inherently a positive feedback process that would lead to depolarization approaching the Nernst equilibrium potential for Ca2+ (approximately +60 mV) and maximal vasoconstriction if it were not for negative feedback mechanisms that limit membrane depolarization and the activity of VGCCs.

● In VSMCs, this negative feedback is provided by large-conductance Ca2+-activated K+ (BKCa) channels and several voltage-gated K+ (KV) channel family members including KV1.5, 2.1, and 7.X channels.

Question 21

Endothelial dependent vasodilation subheadings (list)

Answer 21

Szekeres 2004 and nitric oxide

PGI2 binding to VSMCs

Garland & McPherson 1992

ACh production at the NJM, EDHF potassium

Edwards 1998

Conducted vasodilation

Question 22

(Endothelium) Szekeres 2004 and nitric oxide

Answer 22

● Whilst the process of myogenic tone is independent of the endothelium, the endothelium itself is important in modulating the myogenic response.

● Nitric oxide is one of the most important modulators of vascular smooth muscle cell contraction. NO is synthesised by endothelial nitric oxide synthase from L-arginine and converted to L-citrulline.

● This was demonstrated by Szekeres et al in 2004, who studied intramural coronary arterioles.

● The authors pressurised these arteries and performed stepwise increases in intraluminal pressure from 0-40mmHg which elicited increases in diameter.

Question 23

(Endothelium) PGI2 binding to VSMCs

Answer 23

● Nitric oxide appears to be particularly important in modulating the myogenic response in the coronary vasculature.

● However, there is evidence to suggest that PGI2 binding to the smooth muscle cell IP receptor activates adenylate cyclase which induces the synthesis of cyclic adenosine monophosphate (cAMP).

● cAMP then activates protein kinase A, which allows relaxation of the smooth muscle.

Question 24

(Endothelium) Garland & McPherson 1992

Answer 24

● Garland & McPherson in 1992 demonstrated that whilst nitric oxide is important in small mesenteric artery hyperpolarisation and relaxation, it is not the dominant response to acetylcholine.

● The authors isolated the small arteries and mounted them on a wire myograph, where there were intracellular patch clamp experiments taken using glass microelectrodes.

● The authors showed that whilst acetylcholine induced smooth muscle hyperpolarisation, this was not attenuated by L-NNA, but was attenuated by gilbenclamide a potassium channel blocker.

● The authors therefore concluded that NO was not important.

Question 25

(Endothelium) ACh production at the NJM, EDHF potassium

Answer 25

● It has also been suggested that spillover of acetylcholine production at the neuromuscular junction is capable of activating distal arterioles, which in turn promotes retrograde conducted vasodilation increasing blood flow to the muscle. ● The putative endothelium-derived hyperpolarising factor remains relatively disputed, although one school of thought states that endothelial cells calcium sensitive potassium channels cause an efflux of potassium, which activates inwardly rectifying potassium channels in smooth muscle cells causing hyperpolarisation.

Question 26

(Endothelium) Edwards 1998

Answer 26

● Edwards et al in 1998 showed this by demonstrating that 5mM increases in extracellular potassium induced similar hyperpolarisations in hepatic artery smooth muscle cells to ACh, which were abolished by apamin. ● Furthermore, given the hyperpolarisation sensitivity to barium the authors were able to conclude this was via Kir channels. ● The authors then repeated these findings in small mesenteric arteries. ● The authors suggested that the calcium channels involved were likely small conductance channels, as charybdotoxin can’t inhibit the hyperpolarisation.

Question 27

(Endothelium) Conducted vasodilation

Answer 27

● Conducted vasodilation is also important in endothelial cell physiology. ● In brief, CVD entails the initiation of SMC relaxation from a discrete location within the resistance network and the ensuing spread of vasodilation along the vessel wall via intercellular communication through gap junctions. ● The transmitted signal is hyperpolarisation. ● As hyperpolarization travels, it is transmitted into surrounding smooth muscle cells through myoendothelial coupling to promote relaxation.

Question 28

Negative feedback and calcium signalling subheadings (list)

Answer 28

Heterocellular (myoendothelial) gap junctions Dora-Garland group 2017

Question 29

(Negative feedback) Heterocellular (myoendothelial) gap junctions

Answer 29

● Heterocellular (myoendothelial) gap junctions enable electrical coupling and Ca2+ signaling and between respective cell layers of the microvessel wall. ● In mouse mesenteric arteries, Ca2+ “pulsars” were found to be localized in membrane domains of ECs that project through the internal elastic lamina (IEL) to adjacent SMC and co-localized with intermediate-conductance Ca2+-sensitive potassium channels (IKCa), suggesting that myoendothelial junctions form spatially-restricted intercellular signaling domains in the vessel wall. ● The signalling between the endothelium and smooth muscle cell layers is now thought to be biredctional. ● With hyperpolarisation transmitted through myoendothelial gap junctions to relax smooth muscle, as well as calcium now known to cause the release of endothelial-derived relaxing factors.

Question 30

(Negative feedback) Dora-Garland group 2017

Answer 30

● Dora-Garland group in 2017 used rat cremasteric arterioles and showed that direct activation of smooth muscle LTCCs activated calcium events in endothelial cells, but not in endothelial cell tubes. ● Simultaneous imaging of both the internal elastic lamina with a secondary antibody shown to bind to elastin and the calcium indicator OGB1 showed these events coincided. ● Furthermore, the authors demonstrated these endothelial cell calcium events remained localised in the presence of IP3R blockade by heparin, but in the absence of heparin formed calcium waves. ● The authors then showed that the vasoconstriction induced to BayK was only temporary and followed by subsequent vasodilation.

Question 31

Metabolic control of VSMC contraction subheadings (list)

Answer 31

Metabolic substances and adenosine Groucher 1990 Zhao 2020

Question 32

(Metabolic) Metabolic substances and adenosine

Answer 32

● The metabolic control of skeletal muscle arterioles is important to match perfusion to skeletal muscle metabolism. ● During enhanced metabolic demand, and depending on the actual conditions, tissue cells release several substances that apparently act as vasodilatory mediators, including increased pCO2, lactate, K+, adenosine, H+, inorganic phosphate and reactive oxygen species. ● Adenosine is mainly generated from AMP that is released from the skeletal muscle fibres and dephosphorylated by ecto 5′nucleotidase bound to the sarcolemma. ● This adenosine is a potent vasodilator in both the coronary and skeletal muscle vasculature by acting on extraluminal A2A receptors on the vascular smooth muscle.

Question 33

(Metabolic) Groucher 1990

Answer 33

● In a seminal study in 1990, Groucher et al used the adenosine receptor antagonist, 8-phenyltheophylline, which had lower phosphodiesterase interactions than its predecessors. ● The authors exposed the right gracilis muscle in anaesthetised cats and stimulated contraction with electrical impulses to the obturator nerve. ● The authors measured blood flow with an electromagnetic flow probe and measured the blood flow for 30 minutes following exercise. ● It was shown that gracilis muscle blood flow increased during exercise. However, this increase in blood flow was decreased over 40%, suggesting adenosine is a significant in metabolic regulation of vascular tone.

Question 34

(Metabolic) Zhao 2020

Answer 34

● Another adenosine-based molecule, ATP, is also important in metabolic regulation of vascular tone. ● Zhao et al in 2020 showed the importance of ATP-sensitive potassium channels in cardiac myocytes. ● The authors used a mouse papillary muscle preparation where the small septal artery was cannulated and pressurised. ● The authors showed that pinacidil application to this preparation induced vasodilation in the artery through opening of KATP channels.

Question 35

Neural control of VSMC contraction subheadings (list)

Answer 35

Neurotransmitters Channels and co-transmitters Heart and disease

Question 36

(Neural control) Neurotransmitters

Answer 36

● The autonomic nervous system (ANS) is involved in mediating the behaviour of the endothelial function. In the anatomical view of ANS, endothelial cells (ECs) do not receive direct SNS innervation due to the long distances. ● However, Neurotransmitters released from varicosities in the perivascular plexus via autonomic neuroeffector junctions can reach endothelial receptors and regulate endothelial function. ● Stimulation of the perivascular ANS can induce the release of vasoactive mediators, such as norepinephrine (NE), adenosine triphosphate (ATP) and Neuropeptide Y (NPY) that cause vasoconstriction, and acetylcholine (ACh) and calcitonin gene-related peptide (CGRP), which cause vasodilation.

Question 37

(Neural control) Channels and co-transmitters

Answer 37

● The cholinergic nerve endings innervate the muscular and endothelial layers of blood vessels. ● It has been shown that the M3 AChRs on the endothelium mediate vasorelaxation by regulating the release of NO in arteries. ● In contrast, NE is a predominant endogenous neurotransmitter released mainly from the nerve terminals of sympathetic nerve. ● Activation of smooth muscle α1-adrenergic receptors contributes to smooth muscle cells contraction, resulting in vasoconstriction.

Question 38

(Neural control) Heart and disease

Answer 38

● Neural control is vascular bed-specific; in the heart, circulating adrenaline promotes vasodilation to meet increased metabolic demand. ● In disease, upregulation of VSMC ion channels (e.g. Cav1.2 in hypertension) and reduced myoendothelial gap junctions (e.g. in T2D) impair vasodilatory mechanisms, contributing to microvascular dysfunction.

Question 39

Neurovascular coupling and HPV subheadings (list)

Answer 39

Acetylcholine production at the NMJ K+ ion concentration and astrocytes Filosa 2006 Hypoxic pulmonary vasoconstriction

Question 40

(Neuro and HPV) Acetylcholine production at the NMJ

Answer 40

● Neurovascular coupling describes the relationship between local neural activity and changes in cerebral blood flow, ensuring adequate oxygen and nutrient delivery to active brain regions ● The metabolic control of vasodilation and constriction is particularly important in the cerebral vasculature, where metabolic control of small-resistance arteries is important in neurovascular coupling. ● Elevation of external K+ depolarizes most cells. ● Counter-intuitively, however, small elevations of external K+ cause a striking hyperpolarization of SM cells in cerebral and parenchymal arteries.

Question 41

(Neuro and HPV) K+ ion concentration and astrocytes

Answer 41

● To exert an effect on the vascular wall KIR channel, K+ ions must be concentrated within an appropriate range in the restricted extracellular space between the SM and the overlying astrocytic end-feet. ● However, there is now mounting evidence that astrocytes act as intermediaries in this process, with the release of K+ from the endfeet in response to intracellular signalling cascades evoked by neuronal activity serving as a key mechanism of neurovascular coupling.

Question 42

(Neuro and HPV) Filosa 2006

Answer 42

● Filosa et al. in 2006 were some of the first researchers to demonstrate this first in isolated cerebral arterial smooth muscle cells, where elevation of extracellular potassium induced membrane hyperpolarisation in whole-cell patch clamp recordings, that was sensitive to the Kir inhibitor barium. ● The authors then verified these findings in intact cerebral arteries the authors then studied the response of elevating extracellular potassium to 10mM on arteriolar diameter in brain slices. ● This elicited a potent vasodilation which was blocked by barium ions. ● The physiological source of this potassium was hypothesised to be the astrocytes and the authors used the BKca channel inhibitor TEA on electrically stimulated vasodilation, and the authors were able to demonstrate that this reduced electrical-stimulation induced vasodilation significantly.

Question 43

(Neuro and HPV) Hypoxic pulmonary vasoconstriction

Answer 43

● The mechanism of this vasodilation seems to be the retrograde spread of hyperpolarization from capillaries to arterioles. ● Unlike other organs, decreased partial pressures of oxygen cause vasoconstriction in the pulmonary vasculature. ● This is a protective mechanism to redistribute blood away from poorly-oxygenated areas and to optimise ventilation perfusion matching. ● The molecular mechanisms of HPV are still debated, however, there is evidence to implicate TRPC6 channels, potassium channels and reactive oxygen species.

Answer 44

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