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:

1. How is blood flow to peripheral vessels regulated?

2. What is the explanation for the sequence of colour changes in the fingers?

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:

Fig. 9.1 Pressure drop during passage of blood around the circulation. The smooth line shows the fall in mean blood pressure starting from about 100 mm Hg in the aorta to close to zero in the right atrium. Blood flow in the aorta and arteries is pulsatile but this is damped out in the arterioles. The largest pressure drop occurs in the arterioles, the site of the major resistance to blood flow.

• control the distribution of blood flow from the arteries

• regulate arterial blood pressure.

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.

Case 9.1 Resistance blood vessels: 2

A possible diagnosis and treatment

Sheila, who has always felt the cold, returned to her GP to hear the results of the blood tests and X-ray examination all of which failed to provide any positive information.

The GP was unable to offer any specific treatment but advised Sheila to give up smoking and suggested that she should try a course of the calcium channel blocking drug, nifedipine.

This part of the history raises the following questions:

1. What are the mechanisms involved in vascular smooth muscle contraction and how are calcium ions involved? What would be the pharmacological actions of a calcium channel blocking drug?

2. What role is played by the nerve supply to blood vessels? How might this be linked to stress?

3. Why does the blood supply to the fingers increase above normal levels once the vascular spasm has ended? This is the basis for the redness and throbbing felt by Sheila in her 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.

Fig. 9.2 Events leading to the contraction of vascular smooth muscle. Unlike skeletal muscles, the key event following the formation of a calcium–calmodulin complex is the phosphorylation of myosin as the trigger for cross-bridge formation.

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:

Fig. 9.3 Main pathways leading to a rise in intracellular [Ca⁺⁺]. These pathways occur in vascular smooth muscle as part of vasoconstrictor mechanisms. A rise in cytosolic [Ca⁺⁺] results in further release of Ca⁺⁺ from intracellular stores. This is called calcium-induced calcium release (CICR). The second messenger inositol trisphosphate (IP₃) acts via a receptor on the intracellular [Ca⁺⁺] stores.

Fig. 9.4 Main pathways leading to a decrease in intracellular [Ca⁺⁺] as part of vasodilatation mechanisms.

• 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).

Fig. 9.5 Endothelin dependent vasodilator mechanisms. The dominant effect on NO production is probably shear stress produced by the interaction of blood flow with endothelial cells. Other agonists, in addition to bradykinin and ACh, may work through endothelial cell receptor mechanisms. Abbreviations are defined in the text.

Fig. 9.6 Endothelin dependent vasoconstrictor mechanisms. Interaction of ET-1 with the ET_A receptor on smooth muscle leads to vasoconstriction but binding of ET-1 to the ET_B receptor leads to synthesis of NO and hence vasodilatation. The vasoconstrictor response is dominant. Superoxide anion destroys NO but also generates toxic species such as peroxynitrite and hydroxyl anions.

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.

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