RESISTANCE BLOOD VESSELS

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9

RESISTANCE BLOOD VESSELS

Chapter objectives

After studying this chapter you should be able to:

1. Identify the arterioles as the main population of blood vessels posing a high resistance to blood flow and therefore the main locus for regulation of the peripheral circulation.

2. Describe the mechanism of contraction in vascular smooth muscle and explain the sources of Ca++ ions needed to trigger contraction.

3. Explain the beneficial and unwanted side effects of the use of calcium channel blocking drugs.

4. Discuss the role played by chemical mediators released from the endothelial cells, particularly nitric oxide and endothelins, in the control of peripheral blood vessel diameter.

5. Explain the therapeutic uses of drugs which act as exogenous sources of nitric oxide.

6. Outline the role of ‘local metabolites’ such as CO2, H+, K+ and adenosine in matching the distribution of blood flow to the metabolic needs of a tissue.

7. Understand the role played by local hormones such as histamine, serotonin and bradykinin in the pathological responses to injury.

8. Describe the components of the renin-angiotensin system and the physiological actions of angiotensin II.

9. Discuss the therapeutic uses of drugs which oppose the actions of angiotensin II.

10. Explain the role played by catecholamine hormones adrenaline (epinephrine) and noradrenaline (norepinephrine) in cardiovascular regulation.

11. Outline the role played by the autonomic nervous system in the control of the peripheral circulation.

12. Describe the characteristics of circulatory regulation in the brain, skin, kidneys and lungs.

13. Outline the characteristics of Raynaud’s disease.

Introduction

In this chapter the location of the major resistance blood vessels, the arterioles, will be identified and the properties of vascular smooth muscle, the means by which the diameter of these vessels is modified, will be described. This will provide a basis for understanding the local control mechanisms which normally allow an appropriate distribution of blood flow around the body. In many cases this means matching blood flow to the metabolic needs of tissues but in other cases a further aspect of organ function, such as glomerular filtration in the kidney, must be catered for. There are therefore variations in the ways in which blood flow to different organs of the body is regulated and a series of ‘special circulations’, such as the brain and muscle, will be discussed individually.

Failure to match blood flow to metabolic need will lead to loss of function of a tissue, to the development of pain and subsequently to tissue death. The case history of a lady with a problem in the regulation of the blood flow to her fingers is introduced in Case 9.1:1.

Case 9.1   Resistance blood vessels: 1

A lady with blue fingers

Sheila Duxworth is a 27-year-old lady who has always ‘felt the cold’ but over the last 2 years has had increasing trouble with painful fingers and hands particularly during cold weather. She sought advice from her GP who elicited a history of recurrent episodes of mild pain and numbness followed by quite severe pain lasting for up to an hour. Episodes were clearly associated with exposure of the hands to cold. Close questioning revealed that her hands became blue then white and then red and throbbing. The latter phase was particularly associated with pain.

The GP told Sheila that the problem was caused by an acute reduction in blood flow to her hands.

The GP carried out a visual examination of the capillary beds in Sheila’s fingers in order to try to detect signs of a connective tissue disease such as scleroderma. She also made an appointment for Sheila to have an X-ray of her neck region to check for compression of the blood vessels supplying the arm. A blood sample was taken so that aspects of Sheila’s immune system could be investigated.

Consideration of this presentation leads to the following questions:

Resistance to blood flow

Where the blood flows to when it leaves the aorta depends on the relative resistance to flow in each part of the circulation. In each case, the blood flows through a series of blood vessels, arteries, arterioles, capillaries, venules and veins. The structure of the walls of all these vessels is described in Chapter 1 (see Figs 1.7, 1.8).

As noted in Chapter 8, there has to be a pressure gradient to achieve blood flow. Mean pressure in the arterial tree is typically close to 100 mm Hg and pressure in the right atrium is about 0 mm Hg (i.e. close to atmospheric pressure). Figure 9.1 shows the pressure drop going round the systemic circulation and it can be seen that the population of blood vessels through which there is the largest drop in pressure is the arterioles. These vessels, therefore, must be the segment of the circulation that have the highest resistance to blood flow. This concept is important because it means that by regulating the arterioles, we can:

The product of the cardiac output and the peripheral resistance to blood flow determines the arterial blood pressure (see Chapter 10). As the arterial pressure provides the driving force to perfuse tissues, physiological control systems act to keep arterial pressure relatively constant from moment to moment and from day to day. Indeed, sustained raised arterial pressure can cause serious damage to many parts of the body (see Chapter 10). The consequence of a fall in arterial pressure is often poor brain blood flow which results in syncope (fainting).

Adjustment of the arteriolar resistance is achieved by altering the state of contraction of vascular smooth muscle. The mechanisms of smooth muscle contraction are now described.

Vascular smooth muscle

Smooth muscle is located in the walls of the hollow structures of the body including blood vessels, airways, gut and bladder. The cells are spindle-shaped with a central nucleus and this is the first way in which smooth muscle cells differ from skeletal or cardiac muscle cells (see Chapter 6).

Source of Ca++ for smooth muscle contraction

As with the other two types of muscle, contraction of smooth muscle is triggered by a rise in intracellular [Ca++]. The source of the calcium is, however, different in the three types of muscle. The calcium involved in skeletal muscle contraction is stored intracellularly. It is released from the sarcoplasmic reticulum and is pumped back into these stores during muscle relaxation. In cardiac muscle, most of the calcium used in contraction derives from intracellular stores but some enters the cardiac muscle cell down a concentration gradient from the extracellular fluid via plasma membrane calcium ion channels (see page 20).

In smooth muscle, much of the increase in [Ca++] which generates contraction comes from transmembrane flux through calcium channels. A component of the rise in [Ca++] is contributed by release from intracellular stores but smooth muscle does not have a structure equivalent to sarcoplasmic reticulum.

Two broad groups of stimulus-contraction coupling mechanism can be identified. In ‘electromechanical coupling’ depolarization of the smooth muscle cell is followed by opening of L-type voltage-gated calcium channels. The consequent rise in intracellular [Ca++] leads to further release of Ca++ from intracellular stores (calcium-induced calcium release—CICR). This mechanism predominates in the major vascular resistance vessels which have an internal diameter less than 0.5 mm. In ‘pharmacomechanical coupling’ there is no change in membrane potential but the binding of a hormone or drug to a receptor leads to an increase in intracellular [Ca++] either via a G-protein coupled activation of the inositol phosphate pathway and release of Ca++ from intracellular stores or by the opening of receptor-operated calcium channels. These mechanisms are discussed below in relation to the action of specific vasoactive mediators.

The membrane potential of vascular smooth muscle studied in vitro is close to −60 mV but in vivo it is only about −40 mV. This is because the pressure inside blood vessels stretches the smooth muscle and this stretch leads to the opening of a population of ion channels which result in partial depolarization of the cell and hence partial contraction of the smooth muscle. This is the basis for what has long been known as the ‘Bayliss Effect’. Basically, if you stretch vascular smooth muscle it responds by contracting. An advantage of having partially contracted vascular smooth muscle is that physiological mediators (locally released chemicals, hormones or neurotransmitters) can either cause further contraction or relaxation of smooth muscle as appropriate. Some physiological mediators (see section ‘Metabolite control of local blood flow’ on p. 104) act via a population of ATP-sensitive K+ channels. A decrease in [ATP] inside the smooth muscle cell increases the probability that this population of K+ channels will be open. This leads to hyperpolarization and hence relaxation of smooth muscle.

Relaxation of smooth muscle requires that intracellular [Ca++] is reduced. This can be achieved either by pumping the calcium back into intracellular stores or by expelling it outside the cell (see page 20 for a description of the equivalent mechanisms in cardiac muscle).

The use of different sources of calcium for contraction in the three types of muscle is illustrated by the pharmacological effects of calcium channel blocking drugs. Drugs such as nifedipine, diltiazem and verapamil will, to varying degrees, reduce heart rate and the contractility of the heart (see Chapter 5). These drugs may also be used to achieve peripheral vasodilatation as part of antihypertensive therapy (see Chapter 10). Their side effects are fairly predictable. These include facial flushing, headache and dizziness as a result of their effects on vascular smooth muscle but also constipation is a common side effect because of the effects of calcium channel blocking drugs on gut smooth muscle. Calcium channel blocking drugs have no effect on skeletal muscle function because all the calcium needed for contraction is stored within the sarcoplasmic reticulum.

The calcium channel blocking drug nifedipine was tried as therapy for the patient with Raynaud’s disease described in Case 9.1:2, the aim being to cause vasodilation and improve the blood flow to the fingers.

The total calcium concentration in the extracellular fluid is normally in the range 2.1–2.6 mmol/L. Just over half of this calcium is bound to protein (particularly albumin) and so is not able to enter cells through calcium ion channels. The remaining, ionized, [Ca++] is about 1.1 mmol/L. The relative amounts of ionized and bound calcium depend partly on acid–base status. Hydrogen ions displace calcium ions from anionic binding sites on albumin and therefore acidosis will increase the proportion of Ca++ which is in the ionized form. Routine clinical measurements of plasma calcium usually refer to ‘total calcium’. This may be reported along with a ‘corrected’ measurement which means that allowance has been made for variations in the [albumin].

Contraction of smooth muscle

The contractile mechanism for smooth muscle is different to the two other types of muscle. In skeletal and cardiac muscle the contractile proteins, actin and myosin, are arranged in parallel layers and this is the origin of the striated (striped) appearance when these muscles are viewed under the polarized light microscope. Contraction of striated muscle (see Chapter 2) is initiated by the binding of Ca++ to the control protein troponin. This has the effect of moving another protein, tropomyosin, out of a groove on the bundle of actin filaments. Formation of a ‘cross-bridge’ is then achieved by the myosin head having access to a binding site on the actin filament. Muscle contraction takes place with the hydrolysis of ATP to provide the energy.

Smooth muscle does have actin and myosin as contractile proteins but does not have troponin. The Ca++ released into the cytosol of smooth muscle cells binds to the protein calmodulin. The calcium–calmodulin complex activates the enzyme myosin light chain kinase and this promotes phosphorylation of the myosin filament. Once this has been achieved, interaction between actin and myosin phosphate generates contraction of the smooth muscle cell. Figure 9.2 summarizes the events associated with cross-bridge formation and hence contraction of smooth muscle. When intracellular [Ca++] decreases, myosin is dephosphorylated by myosin light chain phosphatase. Even when dephosphorylated myosin can retain its interaction with actin. These attachments are called latch-bridges. They only detach slowly and so they maintain a level of muscle tension with little consumption of ATP.

There are several broad types of mechanism which contribute to the overall regulation of intracellular [Ca++]. These mechanisms are illustrated in Figures 9.3 and 9.4. Some vasoconstrictor agents such as noradrenaline (norepinephrine) act through more than one mechanism:

• Vasoconstrictor hormones such as noradrenaline, angiotensin II, endothelins, vasopressin and thromboxane A2 bind to G-protein coupled receptors. Subsequent generation of the second messenger inositol trisphosphate (IP3) leads to the opening of channels in intracellular calcium stores and release of Ca++ (Fig. 9.3).

• Vasoconstrictors also lead to membrane depolarization by several mechanisms. These include opening of ligand gated ion channels in the plasma membrane which permits influx of Na+ and Ca++ accompanied by inhibition of K+ channels (Fig. 9.3).

• Intracellular [Ca++] also depends on the Ca++ removal mechanisms. These include pumping Ca++ back into intracellular stores and active extrusion of Ca++ across the plasma membrane both of which involve Ca-ATPase enzymes. There is also a Na+/Ca++ antiport exchanger. Entry of Na+ into the cell down its concentration gradient is coupled to extrusion of Ca++ against its concentration gradient. The low intracellular [Na+] is of course maintained by the sodium pump (Na+/K+ ATPase) (Fig. 9.4).

• Vasodilator agents act via production of either cAMP (e.g. adenosine, prostacyclin, β-adrenoceptor agonists) or cGMP (nitric oxide, atrial natriuretic peptide) as second messengers. Both cAMP and cGMP activate protein kinases and hence lead to protein phosphorylation. A reduction in plasma [Ca++] may then be secondary to cell hyperpolarization following opening of K+ channels. The hyperpolarization closes Ca++ channels. An alternative mechanism for vasodilatation is the activation of Ca++ pumps leading to either extrusion of Ca++ from the cell or sequestration of Ca++ into intracellular stores (Fig. 9.4).

Smooth muscle contracts more slowly than skeletal or cardiac muscle and has less than one third of the myosin content. However, it generates a comparable force per unit cross-sectional area to skeletal muscle. Furthermore, smooth muscle can contract to only 25% of its resting length. Smooth muscle does not fatigue and maintains tension with a low energy cost which is only 1% of the equivalent amount of ATP needed to contract skeletal muscle. It can, if necessary, contract using ATP generated anaerobically in the glycolytic pathway.

Inappropriate spasm of vascular smooth muscle is the diagnosis suggested in the case study in Case 9.1:2.

Local control of vascular smooth muscle

Endothelial factors in the control of local blood flow

The adult human circulation consists of about 60 000 miles of tubing (see Chapter 1). It is lined by a thin monolayer of endothelial cells. These cells not only provide a barrier between the blood and the other cells of the body (see Chapter 11), but they are also the source of a range of vasoactive agents which cause relaxation or contraction of underlying blood vessel smooth muscle (Figs 9.5, 9.6). One of these compounds, before it was chemically identified, was initially named endothelium-derived relaxing factor (EDRF). It is now thought that most, but not necessarily all, of the vascular effects of EDRF can be attributed to nitric oxide. Other factors produced by the endothelium and which also affect vascular smooth muscle contraction have been identified (see p. 103).

Nitric oxide (NO)

NO is synthesized from the amino acid l-arginine by the action of nitric oxide synthase (NOS) enzymes (Fig. 9.5). The terminology for these enzymes is a little confusing as it reflects the original site of discovery rather than current opinion of their site of importance. Endothelial NOS (eNOS) and neuronal NOS (nNOS) are both constitutively expressed in a wide range of cells, including many cell types in the cardiovascular system. These enzymes generate NO continuously. Inducible NOS (iNOS) is synthesized by cells exposed to inflammatory cytokines such as tumour necrosis factor alpha (TNFα), interleukin 1β (IL1β) and interferon alpha (IFNα). A range of other cytokines have the opposite effect and suppress iNOS expression. Overall the balance of local cytokines determines the expression of iNOS and the rate of NO production as required. NO produced in this way in macrophages has cytotoxic actions. Excessive production of NO by iNOS also occurs in some forms of septic shock and will lead to peripheral vasodilatation and a fall in arterial blood pressure (see Chapter 14). It is assumed that iNOS generated NO does not contribute to the normal physiological control of blood vessel diameter.

Analogues of l-arginine which act as inhibitors of NOS enzymes have been developed. Much has been learnt in experimental studies about the physiological roles of NO using these inhibitors. They are also being evaluated with regard to their potential clinical uses.

NO generated constitutively by eNOS and nNOS is part of moment to moment normal vascular control mechanisms. As the physiological half-life of NO is short (a few seconds), it must be generated continuously and is available to contribute to short-term changes in blood vessel diameter. The main site of action of NO is on large diameter arterioles. The single most important trigger for NO production in relation to circulatory control is increased shear stress on the blood vessel wall (Fig. 9.5). If blood flow velocity in an artery increases, this leads to increased NOS activity in the endothelial cells and hence vessel dilatation. The existence of flow dependent vasodilatation was first reported by Schretzenmayr in 1923 but the mechanisms involved only became apparent when NO was recognized as an important vasoactive compound in 1986. NO synthesis can also be triggered by blood borne agonists such as bradykinin, acetylcholine and thrombin acting via receptors on the endothelial cell (Fig. 9.5). This has applied importance, for example, when considering the action of the angiotensin converting enzyme inhibitor (ACEI) group of drugs (see Chapter 10). The ACE enzyme is responsible for the inactivation of bradykinin as well as for the activation of angiotensin II. ACEI drugs, therefore, lead to increased bradykinin concentration and so increased NO synthesis. Part of the antihypertensive action of ACEI drugs is, therefore, related to NO induced vasodilatation.

NO produced in endothelial cells diffuses out of these cells and through the plasma membrane of underlying smooth muscle cells. Within smooth muscle NO binds to the haem part of guanylate cyclase and thereby increases the rate of production of cyclic GMP. In smooth muscle cells this mediator, via activation of protein kinase G, leads to a reduction in intracellular [Ca++] and hence smooth muscle relaxation (Fig. 9.5).

Endothelial dysfunction is important in many disease mechanisms including diabetes, hypertension (see Chapter 10) and atherosclerosis. This dysfunction is not, however, simply explained by alterations in the rate of NO production by endothelial cells. The superoxide anion O2 inactivates NO and forms the peroxynitrite anion (ONOO). This highly reactive anion has many different adverse effects on cell function. Countering the effects of superoxide anion is the strategic basis for many new approaches to therapy for chronic vascular disease. In the absence of NO, superoxide anion leads to the production of hydroxyl anion (OH) which can also have damaging effects.

Drugs which are sources of NO

The group of drugs known as organic nitrates or nitrovasodilators have been in clinical use since the nineteenth century. They either spontaneously release NO (e.g. sodium nitroprusside) or are enzymically degraded and release NO (e.g. glyceryl trinitrate (GTN), amyl nitrate, isosorbide mononitrate and isosorbide dinitrate). These drugs therefore mimic the effects of endogenous NO. Nitric oxide and the nitrosothiols which are produced by the interaction of NO with sulphydryl compounds such as glutathione activate guanylate cyclase (see above) and lead to vasodilatation. The rationale for the use of these drugs in the management of angina is further discussed in Chapter 5 but the fundamentals are as follows. The drugs have their actions on three particular populations of blood vessels.

The combined effect of nitrovasodilators working on capacitance vessels and arterial resistance vessels is a reduction in work done by the heart and, hence, a drop in cardiac oxygen demand.

3. Coronary arteries are often, mistakenly, thought to be the major site of action of nitrovasodilator drugs. In an ischaemic heart these vessels may well be nearly maximally dilated as a result of the accumulation of local metabolites (see p. 104). There is therefore little remaining vasoconstriction which could be reversed by nitric oxide donor drugs. However, nitrovasodilators may improve blood supply within the heart by actions on collateral blood vessels and can also be useful in the relief of coronary artery spasm (see Chapter 5).

As mentioned above, NO has a very short half-life and nitrovasodilator drugs are also subject to extensive first pass metabolism in the liver. These factors severely limit the duration of action of the drugs. The first pass metabolism effects can be reduced by using appropriate drug administration routes. These are sublingual (under the tongue), buccal (tablets held between the inside of the cheek and the gum) and transdermal (steady absorption from a skin patch for up to about 24 hours). Intravenous administration of GTN may be used to obtain acute effects of the drug.

The side effects of nitrovasodilator drugs are fairly predictable. Effects on the venous capacitance vessels will lead to pooling of blood in the veins and hence to postural hypotension, dizziness, fainting (syncope) and a reflex increase in heart rate (see Chapter 10). Arterial dilatation leads to headache as a result of dilatation of cerebral blood vessels and raised intracranial pressure (see p. 108) and to increased skin blood flow, which appears particularly as facial flushing.

Tolerance, a reduction in effect with sustained high plasma levels of the drugs, is also a problem with nitrovasodilator drugs. Possible mechanisms for tolerance include an increase in superoxide anion production within smooth muscle cells and a reduction in the availability of thiol groups to produce nitrosothiols. There are also other probable effects which contribute to the development of tolerance. The problems posed by tolerance can be reduced by adopting appropriate dosing strategies.

Other endothelium derived relaxing factors

The chemical structure and precise physiological role of endothelium derived hyperpolarizing factor (EDHF) is as yet unknown. It is thought to cause smooth muscle relaxation by promoting the opening of K+ channels (Fig. 9.4).

Prostacyclin (prostaglandin I2, PGI2) is released from endothelial cells and is a potent inhibitor of platelet aggregation. PGI2 is also a vasodilator and acts by increasing the production of cyclic AMP and hence activating protein kinase A (PKA) in smooth muscle cells (Fig. 9.5).

Endothelins

The endothelins are a group of peptides which were first discovered in 1988. There are three endothelins designated ET-1, ET-2 and ET-3. Of these three compounds, ET-1 is considered the most important in humans and it has its action predominantly on the ETA type receptor but it also has actions on the ETB receptor. Interaction of ET-1 with ETB receptor on the endothelial cell leads to increased NO production (a vasodilator), but the dominant action of ET-1 is vasoconstriction following binding to an ETA receptor on vascular smooth muscle (Fig. 9.6).

The initial product of gene expression is a preproendothelin molecule with 212 amino acids. This is enzymatically cleaved to form a 38 amino acid peptide—‘Big endothelin 1’. Final processing of this peptide to the main biologically active compound ET-1 involves endothelin-converting enzyme (ECE).

ET-1 is the most potent naturally occurring pressor agent known and, as the name implies, endothelin gene expression does occur in the vascular endothelium. However, this is not the only site of synthesis. Vascular effects of ET-1 are widespread and are thought to contribute to normal cardiovascular regulation with the coronary, kidney and brain blood vessels being particularly sensitive. An interesting historical footnote is that structural analogues of ET-1, the sarafotoxin peptides, occur in the venom of a snake, the Israeli burrowing asp. In 30 BC, Queen Cleopatra of Egypt died after being bitten by this snake. We now know that the cause of her death was likely to be intense coronary vasoconstriction following interaction of the snake venom sarafotoxin with ETA type receptors.

In addition to a role in normal vascular control, there is much interest in the involvement of endothelins in disease mechanisms. Sustained endothelin induced vasoconstriction has been implicated in the mechanisms of essential hypertension, congestive heart failure and chronic renal failure amongst others. Intermittent vasoconstriction produced by endothelins is thought to occur in a range of conditions including unstable angina, acute renal failure, subarachnoid haemorrhage, Raynaud’s disease and migraine. In addition to the intrinsic vasoconstrictor effects, endothelins augment the effects of other vasoconstrictors such as angiotensin II, noradrenaline (norepinephrine) and serotonin (Fig. 9.6).

The roles of endothelins in these disease mechanisms are not confined to vasoconstrictor effects. ET-1 has effects on gene expression and protein synthesis, and the outcomes of this include smooth muscle and cardiac myocyte hypertrophy. Effects on fibroblasts lead to increased deposition of fibrotic proteins such as collagen. ET-1 is also a co-mitogen leading to an increased rate of cell division in some tissues.

At the time of writing, there are no specific drugs targeting the endothelin system in routine clinical use. However, there are many compounds in various stages of development by the drug companies. These include selective ETA and ETB receptor antagonists, non-selective (mixed) ETA /ETB blockers and endothelin converting enzyme (ECE) blocking drugs. These drugs are potentially useful as antihypertensive agents and also in the management of heart failure, pulmonary hypertension and renal failure.

Other endothelium derived constricting factors

Endothelial cells can synthesize the vasoconstrictor prostanoids thromboxane A2 and prostaglandin H2 (Fig. 9.6). Other contributions of endothelial cells to blood pressure elevation include the production of superoxide anions which inhibit the dilator actions of nitric oxide (see p. 102) and the activation of the physiologically inert peptide angiotensin I to the potent pressor agent angiotensin II. The enzyme involved, angiotensin-converting enzyme (ACE), is present on the surface of the endothelial cells (see p. 106).

The bottom line on the role played by the endothelial cells in circulatory control is that they produce a mixture of compounds with a number of actions including both dilator or constrictor effects. The endothelium plays an important role in both physiological and pathological mechanisms involved in circulatory control.

Metabolite control of local blood flow

A major rationale for endothelial involvement in vascular control mechanisms (described above) appears to be to adjust arterial and arteriolar diameter to match changes in blood flow velocity. Increased flow velocity leads to increased shear stress between blood and the endothelial wall. This is an important factor regulating production of both NO and ET-1. The equivalent rationale for metabolite control of blood vessel size is that it enables matching of blood flow to the metabolic needs of a tissue. A local increase in metabolic rate will lead to an accumulation of metabolites which will in turn cause vasodilatation. The principle of metabolite induced vasodilatation is outlined in Figure 9.7. Metabolite effects, therefore, provide the major mechanism for regulating the distribution of blood flow. Metabolite-based regulation appears to be particularly important both in tissues, such as the brain, which require a fairly constant blood flow and in tissues in which metabolic needs may fluctuate widely, such as the heart or skeletal muscle. The kidney, which has excretion and fluid volume regulation as its main functions, has a high blood flow compared to the tissue’s metabolic needs. Metabolites therefore play little role in blood flow regulation in the kidney. The major target vessels for metabolite based control are arterioles and precapillary sphincters. The main site of metabolite effects is on small, rather than large, arterioles.

What are metabolites? Common examples are CO2, H+, adenosine and K+. Local changes in the osmolarity of the interstitial fluid also contribute to blood flow regulation. It is perhaps surprising that oxygen concentration does not directly figure in this context but distribution of blood flow is normally regulated by the accumulation of waste products rather than by changes in oxygen availability. Under hypoxic conditions the low O2 levels can have some effect by opening ATP-sensitive K+ channels and hence causing vasodilatation but this is not relevant to normal physiological circumstances. Release of vasodilator prostaglandins is also linked to Po2-dependent processes. Increased production of CO2 in a tissue leads to increased H+ production from the dissociation of carbonic acid.

It is thought that H+, rather than CO2 directly, is the effective agent as, in isolated tissues, dilute HCl infusion produces similar vasodilator responses. Increased [H+] ions work through opening ATP-sensitive K+ channels on smooth muscle and, following hyperpolarization of the smooth muscle cell, relaxation of smooth muscle leads to increased blood flow.

Adenosine is a potent vasodilator in skeletal and cardiac muscle cells. It is formed in the muscle cells either by the complete dephosphorylation of ATP to adenosine or in a parallel pathway which involves the intermediate formation of S-adenosyl methionine from ATP (see Fig 5.5). It should be remembered that the normal [ATP] inside a muscle cell is of the order of 5×10−3 mol/L whereas [adenosine] outside the myocyte is about 1×10−8 mol/L, a 500 000-fold difference. Significant accumulation of adenosine, therefore, only requires the use of a minute proportion of the available ATP. Adenosine, a non-polar molecule, can cross myocyte membranes. The action of adenosine as a dilator is partly mediated by ATP-sensitive K+ channels on the endothelial cells which increase NO production and partly by similar actions on K+ channels leading to hyperpolarization of the underlying smooth muscle.

K+ ions leave skeletal and cardiac muscle cells and also neurons during the repolarization phase of action potentials. Changes in extracellular [K+] contribute to the initiation of the cardiovascular responses to exercise (see Chapter 13).

In summary, accumulation of ‘metabolites’ in a tissue leads to vasodilatation. In terms of tissue selectivity, in the brain CO2/H+ levels are particularly important, whereas in skeletal and cardiac muscle adenosine and K+ are of greater significance. Metabolite control is discussed in more detail in relation to specific tissues later in this chapter. There is still much to be discovered in this area.

Hormonal control of blood vessel diameter

Mechanisms concerned with hormonal control of vascular smooth muscle can be grouped under two headings.

Local hormones

Histamine

Histamine has widespread physiological actions which are mediated via three types of receptor. It is involved as a neurotransmitter in the central nervous system (action on H3 receptors) and in the control of gastric acid secretion (action on H2 receptors). The vascular actions of histamine, which form part of the inflammatory responses to trauma and allergic responses, are mediated by the H1 receptor type. Histamine is synthesized and stored in mast cells, particularly in those tissues which come into contact with the outside world (skin, lungs and gut). It is also found in basophils where it again forms part of tissue defence mechanisms.

The major vascular responses to H1 receptor stimulation include a transient increase in capillary permeability which can lead to oedema (see Chapter 11). If this occurs to a substantial extent, it can lead to a reduction in circulating blood volume and, consequently, hypotension. This may be accompanied by histamine induced arteriole and capillary dilatation which will also contribute to blood pressure lowering. In severe allergic reactions, such as an anaphylactic reaction to a bee sting, these responses may be life threatening. Treatment includes the rapid use of antihistamine drugs, together with glucocorticoids as anti-inflammatory agents and adrenaline (epinephrine) as a vasoconstrictor (see p. 107). Actions of neurally released histamine in the skin contribute to the weal and flare response which is part of local allergic responses.

Antihistamine drugs (H1 receptor antagonists) suppress most of the vascular effects of histamine. Some of the older drugs in this class, such as chlorphenamine (chlorpheniramine) and promethazine, have sedative side effects. Newer antihistamines, such as terfenadine, do not cause marked sedation.

Bradykinin

The nine amino acid peptide bradykinin causes dilatation of arterioles and an increase in venule permeability. It is generated by the enzyme kallikrein from kininogen during inflammatory responses. Bradykinin also binds to receptors on the endothelial cells and increases the production of nitric oxide (see p. 102). Interest in this area has been stimulated by the development of angiotensin converting enzyme (ACE) inhibitor drugs (see p. 107). ACE inactivates bradykinin and so ACE inhibitor drugs increase the physiological half-life of bradykinin. Bradykinin is also the most potent autocoid in pain responses, an action shared by histamine (acting on H3 receptors) and by serotonin.

Systemic hormones

Renin-angiotensin system

Renin is a systemic hormone secreted by the kidney. It is an enzyme which generates angiotensin I as shown in Figure 9.8.

Angiotensin I (Ang I), is physiologically inert but it is the precursor of angiotensin II (Ang II) which is produced as a result of the actions of angiotensin converting enzyme (ACE). This enzyme is mainly bound to vascular endothelial cells. There is also evidence of locally generated Ang II, which has actions as a paracrine hormone within a number of tissues in the body including blood vessel walls. The main physiological actions of Ang II on AT1 receptors can be summarized as follows:

The full range of responses to a second receptor type, the AT2 receptor, is not yet clear but it appears to oppose the pressor actions of Ang II mediated by the AT1 receptor.

Although Ang II is still regarded as the dominant peptide mediating the actions of the renin-angiotensin system, there is now considerable interest in related peptides. These are designated Ang III, Ang IV and Ang (1–7).

Ang II has an important role in pathological events as well. It can promote cell hypertrophy and hyperplasia and also promotes fibrotic changes in many tissues. An example is in the series of changes referred to as ‘remodelling of the heart’ during progressive cardiac failure (see Chapter 6).

Pharmacological blockade of the renin-angiotensin system

Blocking drugs for the renin-angiotensin system were initially conceived as antihypertensive agents, which act by reducing peripheral resistance, and as drugs for the management of congestive heart failure. In the latter case, the beneficial effects include reduction in fluid retention by reducing aldosterone synthesis (reduced preload on the heart) and reduction in arterial blood pressure (reduced afterload on the heart). However, more recently, the value of ‘organ protective’ effects of renin blockade have become recognized. Renin system blockade reduces the rate of progression of the fibrotic changes associated with the development of chronic renal failure, for example in diabetic patients, as well as the fibrotic changes in heart failure.

What drugs are available to block the renin angiotensin system? β-adrenoceptor blocking drugs (see p. 106) provided the earliest, but rather non-specific, form of renin blockade. Renin secretion from the juxtaglomerular cells in the kidney is partly regulated by circulating catecholamines and by sympathetic nerves acting through β-adrenoceptors. However, beta-blockers have many other components to their pharmacological spectrum of activity.

Angiotensin converting enzyme inhibitors (ACEI) were developed during the 1980s. There are now over 30 drugs in this class available internationally. The first drug clinically available was captopril and the second was enalapril. All of the drug names in this class end in -pril. They not only block the formation of the active hormone Ang II but also block the breakdown of bradykinin to inactive peptides (see p. 106).

The antihypertensive actions of ACEI therefore include blockade of the direct vasoconstrictor effects of Ang II and a modest diuretic action mediated by reduced aldosterone production. The drugs are also described as being sympatholytic as Ang II potentiation of the sympathetic nervous system is blocked. A further component of the antihypertensive action may be linked to increased levels of the vasodilator bradykinin. Certainly, increased [bradykinin] is the basis for the major side effect of these drugs, a dry cough in 10–30% of patients. Bradykinin is also an agonist on endothelial cells for the production of nitric oxide (Fig. 9.5). This is also thought to contribute to the hypotensive action of these drugs.

In the 1990s non-peptide angiotensin receptor blocking (ARB) drugs became available. These act on the AT1 receptor and have a similar profile of action to ACEI except that bradykinin metabolism is not affected and the actions of Ang II on the AT2 receptor are not blocked. The first clinically available drug in this class was losartan and this was followed by valsartan and a number of other drugs, all with names ending in -sartan.

Renin inhibitor drugs which block the enzymatic actions of renin have been developed and are now entering clinical use.

Adrenal medullary hormones

The human adrenal medulla produces a mixture of catecholamine hormones which is approximately 80% adrenaline (also known as epinephrine) and 20% noradrenaline (also known as norepinephrine). These two hormones are also released from sympathetic nerve endings although in this case the relative proportions are reversed and noradrenaline is the major component. The effects of the neuronally released hormones are much more significant in the control of circulatory function under normal physiological conditions than the effects of the hormones from the adrenal medulla. Chromaffin cell tumours, phaeochromocytomas, which are often, but not necessarily, in the adrenal medulla, secrete excessive amounts of adrenaline and noradrenaline which have marked effects on the cardiovascular system.

Receptors for catecholamines were originally divided into two types, α and β, classified on the basis of agonist potency as indicated in Figure 9.9. Subsequently, following the development of selective antagonist drugs, the receptor types were subdivided into α1 and α2, β1, β2 and β3. Sometimes further subdivisions of this basic classification based on pharmacological and gene cloning studies are used.

The main receptor type on the heart is the β1 receptor. Stimulation leads to an increase in the force and rate of cardiac contraction (see p. 45). The dominant catecholamine receptor on blood vessels is the α1 receptor and this mediates vasoconstriction. In some parts of the peripheral circulation postsynaptic α2 receptors also exist on vascular smooth muscle and stimulation leads to vasoconstriction. Presynaptic α2 receptors modulate the release of neurotransmitters into the synaptic cleft. Thus, a rise in transmitter concentration in the synapse stimulates presynaptic α2 receptors and shuts off further transmitter release. Blood vessels also have a limited distribution of β1 and β2 receptors which, when activated, lead to vasodilatation.

The dominant effects of excessive adrenal medulla activity or of exogenous adrenaline (epinephrine) are inotropic and chronotropic actions on the heart (β1 receptor effects) and vasoconstriction on peripheral blood vessels (α1 receptor effect). This is the basis for the use of drugs such as adrenaline in emergency situations involving circulatory collapse.

Autonomic nervous system and peripheral circulation control

The autonomic nervous system has two branches, the sympathetic (SNS) and parasympathetic (PNS) nervous systems. Various attempts have been made to define the difference between SNS and PNS on either a functional or a chemical (neurotransmitter) basis. The only satisfactory definition, however, has an anatomical basis determined by where the nerves enter or leave the central nervous system.

Sympathetic nerves pass through the roots of spinal cord thoracic (T) and lumbar (L) segments. Specifically, this involves segments T1 to L2 and the SNS can be described as thoracolumbar in origin. The nerves emerging from the spinal cord (preganglionic nerves) are relatively short and form a synapse in a ganglion (a collection of nerve cell bodies outside the central nervous system). The postganglionic nerves are relatively long and run to the tissue being supplied.

Parasympathetic nerves originate in or enter the cranial (brain) segments III, VII, IX and X and the sacral segments (S2–S4) of the spinal cord. The PNS can, therefore, be described as being craniosacral in origin. Each of the branches of the autonomic nervous system has both sensory (afferent) and motor (efferent) functions.

The dominant vascular response to SNS activation is vasoconstriction mediated by α1 receptors. Skeletal muscle and coronary blood vessels have a limited distribution of β1 and β2 receptors which exert a vasodilator effect but this is a minor response compared to α1-mediated vasoconstriction even in these tissues. The existence of a sympathetic cholinergic nerve supply to blood vessels in skeletal muscle has been demonstrated in some experimental animals but is still a matter for conjecture in humans.

There is no parasympathetic nerve supply to most of the peripheral circulation as it is confined to erectile and secretory tissues. Activation of a PNS supply to blood vessels leads to vasodilatation as part of the erectile response in the genitalia. PNS induced vasodilatation also occurs in the pancreas and salivary glands as part of their secretory functions. In the pancreas vasoactive intestinal polypeptide (VIP) is a major neurotransmitter for the parasympathetic nerve supply.

In summary, the major characteristics of the nerve supply to blood vessels are that they are sympathetic in origin releasing noradrenaline (norepinephrine) onto α1 receptors and resulting in vasoconstriction. Regulation of these nerves is further discussed in relation to blood pressure regulation in Chapter 10. A possible role for sympathetically induced vasoconstriction in patients with Raynaud’s disease is discussed in the case history in Case 9.1:3.

Case 9.1   Resistance blood vessels: 3

Problems with the calcium channel blocking drug

Sheila, discussed in Case 9.1:1 and 9.1:2, returned to her GP after two weeks of taking the prescribed nifedipine and told her that there was some good news but mainly bad news. The good news was that Sheila had not had problems with her blue, white and red fingers but the bad news was a series of other problems. Her face was always flushed and she seemed to have a continuous headache. On several occasions, she had felt dizzy and had to sit down. This had forced her to give up taking the nifedipine 3 days previously and the side effects had now disappeared.

This part of the history raises the following questions:

Special circulations

The gross distribution of blood flow to the various parts of the body is discussed in Chapter 13. The regulation of some specific vascular beds is discussed here. Coronary blood flow regulation is described in Chapter 5.

Brain (cerebral) circulation

The brain receives about 15% of resting cardiac output, for the textbook 70 kg person a flow rate of about 750 mL/min. This is a relatively high flow rate as the brain, which typically weighs 1.4 kg in a textbook adult, only represents about 2% of body weight. Flow rate is substantially higher to the grey matter (mainly cell bodies) than to the white matter (mainly nerve axons) of the brain. The brain has a high oxygen consumption rate and a high heat generation rate. Interruption of the blood supply to the brain results in loss of consciousness within a few seconds and permanent damage within a few minutes.

A detailed discussion of the vascular anatomy of the brain is outside the scope of this book but is fundamental to the diagnosis and management of cerebrovascular problems. Two pairs of blood vessels, the basilar and internal carotid arteries, enter the cranium and anastomose beneath the optic chiasma to form the circle of Willis. The brain is supplied by branches from the circle of Willis, the anterior, middle and posterior cerebral arteries. Arterioles within the brain are quite short and so much of the vascular control occurs at the level of the small arteries.

Brain blood flow is autoregulated (Fig. 9.10) such that flow is kept fairly constant at about 55 mL/min/100 g tissue and this is independent of fluctuations in mean arterial pressure across the range 60–175 mm Hg. Although the brain has a rich sympathetic nerve supply, stimulation of these nerves makes little difference to autoregulation. However, the autoregulation mechanism is very sensitive to the Pco2 of arterial blood. If a subject hyperventilates and therefore reduces arterial Pco2, then brain blood flow will decrease substantially. This is the reason behind the feeling of ‘light headedness’ following a period of voluntary hyperventilation. The autoregulation response is abolished by hypercapnia (high Pco2) and brain blood flow then increases in proportion to arterial pressure (Fig. 9.10). As the brain is encased in the cranium and cannot expand, retention of CO2, as in chronic obstructive pulmonary disease, results in cerebral vasodilatation and raised intracranial pressure. The patient may therefore complain of headache. Increased metabolic activity in specific parts of the brain will lead to local increases in blood flow (functional hyperaemia).

As humans have an erect posture, the brain is above the level of the heart. Mean cerebral artery pressure is therefore typically of the order of 77 mm Hg. Figure 9.10 shows that if arterial pressure falls below about 50 mm Hg the autoregulation mechanism fails. If the perfusion pressure of the brain falls below about 40 mm Hg then syncope (fainting) occurs. Such a fall in blood pressure may occur as a result of a sudden drop in cardiac output (e.g. because of venous pooling of blood in the limbs) or because of excessive peripheral vasodilatation (e.g. because of the effects of a high ambient temperature). However, when fainting occurs in response to psychological stress associated with fear, pain or shock, the mechanisms are less well understood. In the period immediately before a faint the subject becomes pale and sweats profusely. They tend to hyperventilate, leading to hypocapnia, and then often yawn. Loss of consciousness follows a sudden increase in vagal outflow leading to a slowing of heart rate (bradycardia). This is accompanied by dilatation of peripheral vascular vessels, particularly in skeletal muscle. This is attributed to an inhibition of sympathetic vasoconstriction outflow which originates in the hypothalamus. This series of events is sometimes referred to as a ‘vasovagal attack’.

Skeletal muscle blood flow

Under resting conditions, skeletal muscle receives about 20% of the cardiac output even though muscle accounts for 50% of body weight. Only a relatively small proportion, about one third, of capillaries are being fully perfused at any one time in resting muscle. Access of blood to the remaining capillaries is limited by the closure of precapillary sphincters. These sphincters have no nerve supply but are sensitive to changes in local metabolite concentration and so will open during exercise. Terminal arterioles are also dilated by metabolite accumulation. During vigorous exercise, muscle blood flow can increase more than 20-fold and may account for 80–90% of the increased cardiac output. This can mean an increase in muscle blood flow from about 1 L/min at rest to 20–22 L/min in intense exercise for the textbook person (see Chapter 13).

There is a rich sympathetic vasoconstrictor nerve supply to blood vessels in skeletal muscles. Under resting conditions, this maintains muscle blood flow at a relatively low level. Cutting the sympathetic nerves to a resting muscle leads to a doubling of blood flow. During exercise, the sympathetic vasoconstrictor nerves continue to be active but their effects on vascular tone are opposed by local accumulation of metabolites. Interstitial [K+] is particularly important as a metabolite in skeletal muscle. During muscle action potentials, K+ ions leave the muscle and local interstitial fluid [K+] may rise from a resting value of about 4 mmol/L to as high as 9 mmol/L at the start of exercise before blood flow has fully increased. A concurrent rise in local osmolarity of up to 10% and a rise in inorganic [phosphate] also contribute to vascular regulation. The magnitude of any adenosine-mediated effects is related to the extent of local tissue hypoxia.

Skin (cutaneous) blood flow

Unlike tissues such as skeletal and cardiac muscle, the metabolic requirements of the skin for oxygen are fairly constant. Under resting conditions the skin, which weighs about 2 kg in the textbook person, has a blood flow of 200 mL/min or about 4% of cardiac output. Skin blood flow is regulated particularly by sympathetic vasoconstrictor nerves which are more profuse in some areas of the skin than in others. The skin of the hands and feet is more richly supplied than the trunk and limbs.

A major feature of the cutaneous circulation is its role in body temperature regulation. Heat loss is promoted by increasing the blood flow through capillary loops which run close to the surface of the skin. Shunting of blood towards or away from these capillary loops is achieved by opening and closing arteriovenous anastomoses, thick wall coiled vessels which link arterioles and veins in the skin. These anastomoses, together with cutaneous arterioles and veins, are controlled by the sympathetic nerve supply acting through α1 receptors. Central control of these nerves originates in the hypothalamus, the location of the body’s thermostat. Aspects of the regulation of skin blood flow in a person with Raynaud’s disease are described in Box 9.1 in this chapter.

Box 9.1   Raynaud’s syndrome

Skin blood flow is partially regulated by temperature; exposure to cold leads initially to vasoconstriction. In 1862 Raynaud described abnormalities of this mechanism, an exaggerated reaction which he attributed to overactivity of the sympathetic nervous system. This explanation still exists as a possibility today and some patients have been successfully treated by cutting the sympathetic nerve supply to the arms. However, the problem does not necessarily permanently resolve following sympathectomy and this treatment is now seldom used. Roles for other vasoconstrictor mechanisms, such as the endothelins, have also been proposed. It is suggested that at least part of the problem is not the result of an exaggerated initial response to cold but a failure to recover normally from cold exposure. Usually, skin blood returns to appropriate levels quite rapidly after cessation of cold exposure, but in patients with Raynaud’s syndrome there is a delay.

Raynaud’s syndrome is about ten times as common in women as in men. As in the case of Sheila in the previous case history boxes, the phenomenon may be exaggerated by stress, which also leads to activation of the sympathetic nervous system.

In some patients Raynaud’s phenomena are an early feature of the autoimmune disease scleroderma. This is a problem for which the aetiology is unclear but it is associated with the excessive deposition of collagen and mucopolysaccharide in various parts of the body including the face. Later this may spread to the arms, legs and trunk. Within the cardiovascular system there is intimal fibrosis in small and medium-sized arteries hence leading to diminished skin blood supply. Of the order of 10% of patients with Raynaud’s phenomena will develop overt scleroderma but about 90% of patients with scleroderma have Raynaud’s syndrome.

The colour changes which occurred in Sheila’s hands following exposure to cold (blue, white and then red) can be explained as follows. The initial blue phase represents peripheral cyanosis associated with inappropriate vasoconstriction and sluggish blood flow. There is excessive deoxygenation of the available haemoglobin leaving higher than normal concentrations of deoxyhaemoglobin and hence cyanosis (see Chapter 1). Normally this is followed by metabolite-induced vasodilatation (red phase). In people displaying Raynaud’s syndrome however there is a phase of intense vasoconstriction (white phase) in which there is insufficient blood flow to even give discernable cyanosis. Ultimately when the vasospasm is released, metabolite induced vasodilatation does occur (red phase) but there may also have been sufficient accumulation of metabolites to cause a pain response, perhaps mediated by the excessive release of pain mediating neurotransmitters.

With regard to therapy for mild cases of Raynaud’s syndrome, treatment can be targeted at minimizing the problem by avoiding the cold, stopping smoking and dressing appropriately. This may include using gloves with a built-in heating mechanism. Use of a mixed α12-adrenoceptor antagonist may be helpful. It is vitally important to avoid tissue necrosis leading to ulceration and gangrene.

Kidney (renal) blood flow

Under resting conditions renal blood flow, at about 20–25% of cardiac output, is high compared to the size of the kidneys which only account for about 0.5% of body weight. Flow is autoregulated and is controlled particularly in relation to the need to maintain glomerular filtration rate (GFR).

The renal vasculature has an intense sympathetic vasoconstrictor nerve supply. At times of activation of the sympathetic nervous system, as in exercise (see Chapter 13) and during circulatory shock (see Chapter 14), renal blood flow is reduced substantially below resting levels. If this period of reduced blood flow is prolonged during circulatory shock situations and GFR is reduced, this may lead to pathological changes within the nephron (acute tubular necrosis—ATN) which can compromise patient survival.

Renal blood flow is not determined by metabolite effects. Indeed, with such a high resting blood flow rate, it is difficult to see how such a mechanism could function effectively.

Splanchnic blood supply

Under resting conditions the splanchnic blood supply (gastrointestinal tract and liver) receives about 24% of the cardiac output. The venous drainage from most of the gastrointestinal (GI) tract enters the hepatic portal vein and this supplies 70% of the hepatic blood flow. The remaining 30% is provided by the hepatic artery. During exercise and other situations when the baroreceptor reflex is activated, sympathetically mediated constriction of the veins and venules displaces blood from the splanchnic beds so that more blood volume is available for use in other parts of the circulation. This constriction which raises the local resistance to flow also contributes to the maintenance of arterial blood pressure.

The liver has a major role in drug metabolism and orally administered drugs which are absorbed in the gastrointestinal tract will be taken in the hepatic portal vein to the liver and subjected to ‘first pass metabolism’. An anatomical point of pharmacological significance is that the venous drainage from the extreme ends of the GI tract, the buccal cavity and the anal canal, does not enter the hepatic portal vein. Drugs absorbed in these two regions of the GI tract are not therefore subject to first pass metabolism.

Lung blood flow

The lungs normally receive a blood flow which is the same as the cardiac output. During exercise, when there is an increase in cardiac output, lung vascular resistance must fall otherwise there would be a marked rise in pulmonary artery pressure. The fall in resistance is largely achieved by passive dilatation in response to any tendency to an increase in pressure. There is a sympathetic vasoconstrictor nerve supply to the lungs but the effects of increased sympathetic activity during exercise appear to be swamped by the passive dilatation effects.

An interesting aspect of the control of the lung circulation is hypoxic vasoconstriction. In regions of the lung which are poorly ventilated and fall below a threshold level of Po2, vasoconstriction occurs. The oxygen sensor mechanism is located within the pulmonary smooth muscle, possibly in the mitochondria. A signal produced in this way activates a population of K+ channels and, via a change in membrane potential, increases Ca++ entry and causes constriction of local pulmonary vascular smooth muscle.

Further reading

Born, G. V. R., Schwartz, C. J. Vascular Endothelium. Physiology, Pathology and Therapeutic Opportunities. Stuggart: Schattauer; 1997.

Braddock, M., Schwachtgen, J. -L., Houston, P., et al. Fluid shear stress modulation of gene expression in endothelial cells. News Physiol. Sci.. 1998; 13:241–246.

Hobbs, A. J., Higgs, A., Moncada, S. Inhibition of nitric oxide synthase as a potential therapeutic target. Annu. Rev. Pharmacol. Ther.. 1999; 39:191–220.

Kelm, M. Flow-mediated dilatation in human circulation: diagnostic and therapeutic aspects. Am. J. Physiol.. 2002; 282:H1–H5.

Ledoux, J., Werner, M. E., Brayden, J. E., Nelson, M. T. Calcium activated potassium channels and the regulation of vascular tone. Physiology. 2006; 21:69–79.

Levick, J. R. An Introduction to Cardiovascular Physiology, fifth ed. New York: Arnold; 2009.

Pohl, U., de Wit, C. A unique role of NO in the control of blood flow. NIPS: News Physiol. Sci.. 1999; 14:74–80.

Tomita T., Bolton T. B., Bolton T. B., eds. Smooth Muscle Excitation. London: Academic Press, 1996.

Waller, D. G., Renwick, A. G., Hillier, K. Medical Pharmacology and Therapeutics, third ed. Edinburgh: WB Saunders; 2009.

Wolfe, J. H. N. ABC of Vascular Diseases. London: BMJ Books; 1992.