Cellular physiology pharmacology relevant to anaesthesia

This chapter is divided into four main sections: a description of receptors (including second messengers); representation of drug-receptor interaction (receptor pharmacology); intracellular Ca2+ as a vital signalling molecule; and, finally, potential targets(s) for anaesthetic action are discussed.

RECEPTORS

Receptors recognize specific small signalling molecules to produce a biological effect. In the unbound state, a receptor is functionally silent. Excluding the intracellular receptor for inositol(l,4,5) triphosphate and the ryanodine receptor (discussed below) there are four main classes of receptor: G-protein coupled (GPCR), ligand-gated ion channels, tyrosine kinase coupled and intracellular steroid receptors. It has been estimated that 60% of the drugs routinely given to patients target the GPCR.

G-PROTEIN COUPLED

G-protein coupled receptors have been, and continue to be, the subject of intense research. Some examples are shown in Table 2.1. As the name suggests, the signal produced upon activation is transduced by a guanine nucleotide binding (or G) protein. The receptor spans the plasma membrane seven times with the N-ter-minus extracellular and the C-terminus intracellular (Fig. 2.1). Once activated, the receptor undergoes a conformational change such that the G-protein is able (in most circumstances) to interact with the third intracellular loop and the C-terminus.

G-proteins

These proteins form the link between receptor and effector to generate a second messenger. As the name suggests, G-proteins bind guanine nucleotides (guanosine triphosphate-GTP and guanosine diphosphate-GDP). The protein is composed of three subunits, a, P and y, with the a subunit defining the G-protein (Fig. 2.1). For example, there are inhibitory G-proteins (Gj/G0) and stimulatory G-proteins (Gs and Gq). In the past, G-protein activity was defined using two bacterial toxins: pertussis toxin, which inhibits the action of the Gi/G0 class of G-protein, and cholera toxin, which persistently activates the Gs class of G-proteins. However, the use of specific antibodies has made G-protein classification more precise. The a subunit is extremely important in the control of signalling via GPCRs; it is the site of GTP/GDP binding and is also an intrinsic GTPase. When an agonist binds to a GPCR, the G-protein can then interact with the C-terminus and the third intracellular loop of the (activated) receptor. In the inactive state, the G-protein has GDP bound to the a subunit. Following activation with agonist, GDP is exchanged for GTP and the G-protein is activated. Ga dissociates from Gp/y and activates an appropriate effector molecule. As the a subunit displays GTPase activity, there is a marked tendency to cleave GTP to GDP and hence inactivate the G-protein. In essence, the Ga subunit acts as an 'off switch.

Table 2.1 Examples of G-protein couplcd receptors (GPCRs) and their characteristics

Receptor

G-protein

Effector

Second messenger

«] - Adrenoceptor

Gq

PLC

Increased Ins{ 1,4,5)1\;/Ca-*

c12 ■ Ad re n oceptor

Gi/o

Adcnvlvl cyclase

Decreased cAMP

vscc;

Decreased Ca-* influx

K+ channel

Increased K> efflux

f>- Adrenoceptor

G¿

Adcnylyl cyclase

Increased cAMP

|i, i> and k opioid1

G¡/0

Adcnvlvl cvclase

Decreased cAMP

VSCC "

Decreased Ca2* influx

K* channel

Increased K> efflux

MgluR

Class I

G,

PLC

Increased Ins( 1,4,5 )Ps/CaJ*

Class Il/in

' Gi/o

Adenvlvt cvclase

Decreased cAMP

Brady kin in

G,

PLC '

Increased Ins( 1,4,5)Pj/Ca2+

^Similar cficcts can be observed for muscarinic m2/4 and cannabinoid.

MgluR, metaborrophic glutamate receptor: PLC, phosphoüpase C; VSCC, volt age-sensitive calcium channel.

^Similar cficcts can be observed for muscarinic m2/4 and cannabinoid.

MgluR, metaborrophic glutamate receptor: PLC, phosphoüpase C; VSCC, volt age-sensitive calcium channel.

Receptor

Schematic representation of G-protein receptor-effector coupling. The seven transmembrane spanning G-protein coupled receptor (GPCR) is depicted coupling to an effector enzyme (e.g. adenylyl cyclase, phospholipase C) via a guanine nucleotide binding (G) protein. The G-protein comprises three subunits, a, (5 and y. aGTP interacts with the effector enzyme to produce a second messenger. These second messengers modulate cellular activity (see text for details).

cAMP lnsP3

Schematic representation of G-protein receptor-effector coupling. The seven transmembrane spanning G-protein coupled receptor (GPCR) is depicted coupling to an effector enzyme (e.g. adenylyl cyclase, phospholipase C) via a guanine nucleotide binding (G) protein. The G-protein comprises three subunits, a, (5 and y. aGTP interacts with the effector enzyme to produce a second messenger. These second messengers modulate cellular activity (see text for details).

When aGTP is converted to aGDP, the a subunit can re-associate with the P/y subunits and the G-protein is then free to interact with another receptor. One activated receptor may interact with multiple G-proteins.

Effectors

In this chapter, adenylyl cyclase, phospholipase C and ion channels (Ca2+ and K+) are considered as examples of effector enzymes. Guanylyl cyclase, the target for nitric oxide, is not discussed in detail. Adenylyl cyclase is the enzyme responsible for the conversion of intracellular adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP). The enzyme is variably sensitive to G-protein a subunits, Ca2+ and G-protein p/y subunits. Adenylyl cyclase is activated by the Gs class of G-proteins (e.g. by norepinephrine acting at p-adrenoceptors) to increase the formation of cAMP, or inhibited by the Gj class of G-proteins (e.g. by norepinephrine acting at (^-adrenoceptors) to reduce cAMP formation (Fig. 2.2). Phospholipase C is the plasma membrane-bound enzyme responsible for the conversion of the membrane phosphoinositide, PIP2 (phosphatidyl inositol 4,5 bisphosphate), to two second messengers, inositol( 1,4,5 triphosphate [Ins(l,4,5)P3] and diacylglycerol (DAG). In common with adenylyl cyclase, the enzyme is variably sensitive to Ga subunits of the Gq class (e.g. norepinephrine acting at aj-adrenoceptors) of G-proteins, Ca2+ and G-protein p/y subunits. Members of the G;/G0 G-protein coupled class of receptors (e.g. opioid, C12-adrenoceptor, cannabinoid) are capable of closing voltage-sensitive Ca2+ channels and activating K+ channels (inward rectifiers -Kjr) to enhance an efflux of K+, resulting in membrane hyperpo-larization. This ion channel interaction is, as for the events described above, mediated by Ga subunits. One activated G-pro-tein can potentially interact with multiple effector molecules.

Control of cyclic adenosine monophosphate (cAMP) formation. Stimulatory GPCRs (RJ and inhibitory GPCRs (Rj) couple to adenylyl cyclase via stimulatory G-proteins (Gs) and inhibitory G-proteins (Gj), respectively. Activation of Gs activates adenylyl cyclase to increase the formation of cAMP. Activation of Gj inhibits adenylyl cyclase to inhibit the formation of cAMP. cAMP activates protein kinase A (PKA) and the cAMP signal is terminated by a phosphodiesterase (PDE).

Control of cyclic adenosine monophosphate (cAMP) formation. Stimulatory GPCRs (RJ and inhibitory GPCRs (Rj) couple to adenylyl cyclase via stimulatory G-proteins (Gs) and inhibitory G-proteins (Gj), respectively. Activation of Gs activates adenylyl cyclase to increase the formation of cAMP. Activation of Gj inhibits adenylyl cyclase to inhibit the formation of cAMP. cAMP activates protein kinase A (PKA) and the cAMP signal is terminated by a phosphodiesterase (PDE).

Second messengers

Adenylyl cyclase and phospholipase C are responsible for the generation of cAMP and Ins(l,4,5)P3/DAG, respectively. The action of cAMP is usually mediated by a cytosolic enzyme, protein kinase A, although this is not always the case. cAMP-sensitive target sites include elements of the contractile apparatus in muscle and metabolic enzymes. In addition, the If, K+ channel, which is involved in repolarization after action potential firing, is sensitive to cAMP. Prostaglandins increase and opioids decrease cAMP in addition to being pronociceptive and antinociceptive, respectively. It is thought that activation of Ih by increased cAMP in nociceptive neurones leads to more rapid repolarization, and hence an increased rate of firing may be involved in the pronociceptive actions of prostaglandins. Conversely, opioids reduce cAMP, thereby reducing Ih activity and delaying repolarization, and hence decreasing nociceptive transmission. However, with opioids, the antinociceptive action also involves closure of voltage-sensitive calcium channels (VSCCs) and activation of K;r. Activated adenylyl cyclase is capable of producing many molecules of cAMP.

The discovery that Ins( 1,4,5 )P3 releases Ca2+ from the endoplasmic reticulum provided the link between agonist-stimulated phosphoinositide turnover and agonist-stimulated increases in intracellular Ca2+. Ins(l,4,5)P3 binds to an intracellular receptor located on the endoplasmic reticulum membrane and occupation leads to a release of intracellular stored Ca2+ and a rise in cytosolic free Ca2+ (see below).

Table 2.2

Examples of ligand-gated ion channels

Receptor

Ligand

Ion and direction of flow

Nicotinic

Acetylcholine

Na+ inward

GABAa

GABA

CI" inward

Glvcinc

Glycine

CI" inward

NMDA

Glutamate

Ca^+ inward

AMPA

Glutamate

Na* inward

SHTjj

Serotonin

Ca^+ inward

The fact that one activated receptor can interact with many G-proteins, and in turn many effectors, to generate many molecules of second messenger allows this system to amplify receptor input.

The actions of cAMP are terminated by phosphodiesterase activity converting cAMP to the inactive 5'-AMP. cAMP-dependent phosphodiesterase is inhibited by methylxanthines such as caffeine. Termination of the Ins(I,4,5)P3 signal is more complex but still involves metabolism. Ins(l,4,5)P3 can be phosphorylated (3-kinase) to Ins(l,3,4,5)P4, which may have some biological activity. Ins(l,3,4,5)P4 and Ins(l,4,5)P3 are both dephosphorylated by a 5-phosphatase to yield Ins(l,3,4)P3 and Ins(l,4)P2, respectively, both of which are inactive. Further dephosphorylation reactions yield inositol which is then re-incorporated into the membrane pool of phosphoinositides. Various stages in the dephosphorylation pathway are inhibited by Li+, and it is believed that interruption of the phosphoinositide cycle underlies the antimanic action of this monovalent cation.

LIGAND-GATED ION CHANNELS

This class of receptor is found on the plasma membrane and is composed of four or five subunits in various combinations depending on the particular receptor. The protein doubles as both a receptor and an ion channel with distinct structural motifs encoding the ligand binding site, ion channel pore and modulatory site(s). When the receptor is activated by the appropriate agonist, a range of mono- and divalent ions flow along their concentration gradient either into or out of the cell (Table 2.2, Fig. 2.3A). This class of receptor is very important from the anaesthetic viewpoint in that the nicotinic acetylcholine receptor is the target for neuromuscular relaxants, the W-methyl-D-aspartate (NMDA) receptor is the target for ketamine, and the GABAa receptor is a major target for a range of inhalation and intravenous general anaesthetic agents (excluding ketamine).

TYROSINE KINASE COUPLED

In this class of receptor, the protein is a monomer that spans the plasma membrane only once. The extracellular domain binds the ligand and the intracellular domain possesses tyrosine kinase activity. Agonists for this receptor class include insulin and growth factors (Fig. 2.3B). When activated, the intracellular domain autophosphorylates, the receptor dimerizes and then activates a range of intracellular target proteins. These include enzymes/ transporters involved in glucose metabolism (insulin) and phos-pholipase C.

Ligand binding

Extracellular

III!

Intracellular

Extracellular

III!

Intracellular

Functional receptor formed from four or five subunits

Ligand binding

Extracellular

Intracellular

Ligand binding

Extracellular

Intracellular

Receptor dimerizes

Tyrosine kinase

Ligand (steroid) binding

DNA binding

Schematic representation of the structure of non-G-protein coupled receptors. A. A single subunit of a ligand-gated ion channel. A functional channel results from the combination of four or five of these subunits. B. A tyrosine kinase receptor. When activated, these receptors usually dimerize. C. A steroid receptor. Unlike in (A) and (B), the receptor is not located on the plasma membrane and resides in the cytoplasm. Upon activation, the receptor translocates to the nucleus and initiates gene transcription (see text for details).

INTRACELLULAR STEROID RECEPTORS

These monomeric receptors are not found on the plasma membrane. Examples of agonists for this class of receptor include sex hormones, thyroid hormones and adrenal hormones. The receptor possesses a ligand binding domain, a catalytic domain and a set of 'zinc fingers'. In the inactive (no ligand bound) state, the receptor is located in the cytosol of the cell. When the receptor is activated, it translocates to the nucleus and via the 'zinc fingers' binds to DNA and initiates gene transcription and new protein synthesis^Fig. 2.3C). Responses produced via activation of this receptor are slow compared with the other three receptor types.

Table 2,3 Commonly used pharmacological terms to describe drug-receptor interaction

Agonists A)

Agii [list

Agonist potency

Agonist effita<\

bull agonist Partial agonist

A ligand that binds to a reccptor to produce a functional response. "Fhis results from an increase in the proportion of active receptors The ability to produce a response expressed in terms of concentration of the agonist. This should be defined further with: I;( -q. concentration of agonist producing 50% maximal response pECäO' -log 10 of EC so

There are many definitions for tins term. In its simplest form, ¡t relates to the size of a response. For example, an agonist tli.it produces a maximum response of 100" has higher efficacy than an agonist in the same tissue/system that produces a maximum response of 50%

An agonist that produces a 'maximum' response in a particular tissue/cellular system. Typically, full agonists produce this ar low lev els of receptor occupancv

An agonist that produces a lower than maximum (than a full agonist 1 response. Even at full receptor occupancy, a full response cannot be elicited

(A) Agonist concent ration-response curve

LOg.0 Concentration

Full agonist has higher efficacy than partial agonist. In this example both have the same potency (pEC;o).

Antagonists (see Fig. B i Antagonist Antagonist potency

Competitive antagonist pA2

Non-competitive antagonist

A drug that reduces the activity of another drug (usually an agonist 1 1CS0; concentration of an antagonist that reduces a specified response bv 50% plC;H: -log hi of IC50

The inhibitory effect of-the antagonist can he o\crcomc by increasing the concentration of the agonist. The effect is competitive or surmountable

-log H) of the concentration of an antagonist that makes it necessarv to double the agonist concentration in order to elicit the original response

The inhibitory effect of the antagonist Cannot be overcome by increasing the concentration of the agonisi

(B) Effect of antagonist on agonist concentration -response curve

(B) Effect of antagonist on agonist concentration -response curve

Log ..j Concentration

DRUG-RECEPTOR INTERACTION

The interaction of a drug with a receptor usually displays three main characteristics. This interaction is specific, dose-related and saturable. Drug-receptor interactions are often defined in terms of IC50 and EC50 (expression of potency) and these terms are obtained from a dose-response curve (see Table 2.3 for a description of some of the more commonly encountered basic pharmacological terms). A dose-response curve describes the administration of a dose of drug to a patient or animal. A concentration-response curve describes the incubation of an isolated tissue or a cell preparation with various concentrations of various drugs. Ignoring allosterism, drugs used in anaesthetic practice can be simply classified as agonists or antagonists.

Agonists

A drug that binds to a receptor to produce a functional response is an agonist, and the ability to produce a functional response is termed 'efficacy'. Efficacy depends on receptor numbers and type of coupling. An agonist that produces a maximum possible response is termed a full agonist. An agonist that produces a maximum response in the same tissue that is lower than that of a full agonist is termed a partial agonist. Antagonists have no efficacy. Agonists may have very different potencies but equal efficacies and vice versa. Potency and efficacy are not interchangeable, e.g. a high-potency drug may have lower efficacy than a low-potency drug (Table 2.3).

Relationship between receptor occupation and response and receptor reserve

In general, full agonists elicit maximal responses at low levels of receptor occupancy. Partial agonists cannot elicit a full response even when the entire receptor complement is occupied. If a full response is observed at low occupancy, this system is said to have a receptor reserve (this is commonly found for drugs that elicit smooth muscle contraction, relaxation or cardiac stimulation). These spare receptors are not hidden away; there are simply more than are needed.

Antagonists

There are two main classes of antagonist (see Table 2.3):

Competitive. For this class of antagonists, the effect is surmountable, i.e. increasing the agonist concentration will overcome the antagonist effect. This is the most common type of antagonism. Examples include propranolol antagonism of the effects of isoprenaline or atropine antagonism of methacholine in the heart.

Non-competitive. For this class, the effect cannot be overcome, i.e. increasing the agonist concentration does not overcome the antagonist effect. In tissues with a receptor reserve, low concentrations of non-competitive antagonists appear competitive (because the maximum response declines only when the receptor reserve is gone, i.e. when using a higher concentration). Non-competitive antagonism is sometimes termed irreversible. The concentration-response curves look similar but the explanation is different. Irreversible antagonists are usually experimental drugs that bind to and modify a receptor. In contrast, non-competitive block usually occurs at a site distal to the ligand binding site on the receptor. For example, ketamine inhibits NMDA receptor activity by occupying the ion channel pore - it does not alter binding.

Mixed agonist-antagonist

Where receptor subtypes exist, the potential for mixed agonist-antagonist behaviour exists, e.g. nalorphine: 6-agonist and ^-antagonist; pentazocine: p-antagonist and S/k-partial agonist.

A plethora of physiological responses are dependent on Ca2+, including muscle contraction, neurotransmission and cell division. Altered Ca2+ homeostasis is involved in the pathophysiology of malignant hyperthermia and underlies neuronal death resulting from ischaemic episodes.

GRADIENT MAINTENANCE

Intracellular Ca2+ concentration is maintained at approximately 100 nM in the presence of an enormous concentration gradient, with extracellular Ca2+ being around 1 mM. This concentration gradient is maintained by three main mechanisms, i.e. extrusion, sequestration and binding. Ca2+ is extruded across the plasma membrane utilizing the Ca2+-ATPase enzyme (PMCA) which, as the name suggests, is an energy-requiring process. Ca2+ also leaves in exchange for Na+ via the Na+-Ca2+ co-transporter. At face value, this does not appear to be energy-requiring. However, for activity there needs to be a concentration gradient for Na+ (in the inward direction) and this is set up via the Na+/K+-ATPase system. Therefore, Ca2+ entering the cell down its concentration gradient will be actively pumped out and/or exchanged for Na+ (entering down its concentration gradient) (Fig. 2.4). In addition, Ca2+ may be

Ca3+

Schematic representation of the regulation of intracellular Ca2+ concentrations ([Ca2+],). Extracellular Ca2+ concentration is approximately 1 mM. As a result of plasma membrane Ca2+-ATPase (PMCA) and antiporter activity coupled with sequestration into intracellular organelles, [Ca2+]j is maintained at approximately 100 nM. Typically, agonist stimulation or depolarization increases [Ca2+]i up to 1 pM. This increase can arise from extracellular sources via plasma membrane Ca2+ channels. These may be voltage-sensitive (VSCCs) or receptor-operated (ROCCs). Additionally, [Ca2+]j may increase via release from intracellular stores. Gq-coupled GPCRs activate phospholipase C to produce Ins(l,4,5)P3 and diacylglycerol (DAG). Ins(l,4,5)P3 activates an Ins(l,4,5)P3 receptor (IP3R) on the endoplasmic reticulum (ER) to release stored Ca2+. DAG activates protein kinase C (PKC). Ca2+ can also be released from ryanodine-sensitive stores via activation of the ryanodine receptor (RyanR) via increased Ca2+. Ca2+ entering via the plasma membrane or released from intracellular stores is either pumped back across the membrane and/or resequestered into the intracellular stores (see text for details).

Schematic representation of the regulation of intracellular Ca2+ concentrations ([Ca2+],). Extracellular Ca2+ concentration is approximately 1 mM. As a result of plasma membrane Ca2+-ATPase (PMCA) and antiporter activity coupled with sequestration into intracellular organelles, [Ca2+]j is maintained at approximately 100 nM. Typically, agonist stimulation or depolarization increases [Ca2+]i up to 1 pM. This increase can arise from extracellular sources via plasma membrane Ca2+ channels. These may be voltage-sensitive (VSCCs) or receptor-operated (ROCCs). Additionally, [Ca2+]j may increase via release from intracellular stores. Gq-coupled GPCRs activate phospholipase C to produce Ins(l,4,5)P3 and diacylglycerol (DAG). Ins(l,4,5)P3 activates an Ins(l,4,5)P3 receptor (IP3R) on the endoplasmic reticulum (ER) to release stored Ca2+. DAG activates protein kinase C (PKC). Ca2+ can also be released from ryanodine-sensitive stores via activation of the ryanodine receptor (RyanR) via increased Ca2+. Ca2+ entering via the plasma membrane or released from intracellular stores is either pumped back across the membrane and/or resequestered into the intracellular stores (see text for details).

Gq-coupled R

Ca2+ vscc

Extracellular ~1mM

Extracellular Ca2+ -1 mM

Gq-coupled R

Table 2.4 Classification of voltage-sensitive Ca2+ channels including predominant location(s) and some examples of functions ascribed to their activity

L

N

P/Q

R

T

Specific inhibitor

DHPs

üi-CgTx

id Aga-IVA3

None

None

HVA/LVA

HVA

HVA

HVA

NVA

LVA

Location

Heart

Neurona!

Neuronal

Neuronal

Heart

Function

Contraction

Release

Release

Release

Pacemaker

Anaesthetic interaction

Volatile

Sensitive

Sensitive

Controversial

Unknown''

Sensitive

Intravenous

Sensitive

Sensitive

Controversial

Unknown

Controversial

channels blocked by low concentrations of Agatoxin and Q-channels blocked by high concentrations, b Unaware of anv studies.

N'T, neurotransmitter; DHPs, dihydropyridines; m-CgTx, tu-conotoxin GVIA/VIIA; w-Aga-IVA, lo-Agatoxin-IVA; LVA/HVA, low/high voltage-activated.

channels blocked by low concentrations of Agatoxin and Q-channels blocked by high concentrations, b Unaware of anv studies.

N'T, neurotransmitter; DHPs, dihydropyridines; m-CgTx, tu-conotoxin GVIA/VIIA; w-Aga-IVA, lo-Agatoxin-IVA; LVA/HVA, low/high voltage-activated.

sequestered into intracellular organelles including mitochondria and the endo/sarcoplasmic reticulum. The former store is described as non-releasable, whereas the latter store(s) are releasable (see below). Ca2+ is also bound to intracellular proteins. Intracellular Ca2+ concentration may be elevated by opening membrane Ca2+ channels or releasing Ca2+ from intracellular (releasable) storage sites.

ELEVATING INTRACELLULAR CALCIUM Calcium entry

Ca2+ can enter the cell across the plasma membrane via two classes of Ca2+ channels: voltage-sensitive and receptor-operated. Classification of the predominant voltage-sensitive Ca2+ channel classes is noted in Table 2.4. Ca2+ can also enter through a receptor-operated Ca2+ channel, of which a good example would be the glutamate NMDA receptor (a ligand-gated ion channel - see above). When glutamate binds to the NMDA receptor, the channel opens and Ca2+ flows into the cell. Other types of less obvious receptor-operated Ca2+ channels are those that are, for example, opened by increased concentrations of the intracellular second messenger Ins(l,4,5)P3 (see below) or Ca2+ itself. These clearly require receptor activation for the production of the Ins( 1,4,5 )P3 or Ca2+ signal (Fig. 2.4). These types of receptor-operated Ca2+ channels can be found in a variety of neurones and cells of the immune system.

Release of sequestered calcium

Activation of the Gq coupled class of GPCR stimulates the formation of the inositol polyphosphate second messenger Ins( 1,4,5 )P3 which releases Ca2+ from the endoplasmic reticulum. Ca2+ itself also acts as a co-factor, further enhancing release. Ins(l,4,5)P3 activates an intracellular receptor, which is distinct from steroid receptors, located on the endoplasmic reticulum membrane. This receptor is also an intrinsic Ca2+ channel and Ca2+ flows down its concentration from the endoplasmic reticulum lumen to the cytoplasm. The stores are also equipped with a Ca2+-ATPase to enable them to refill. An additional intracellular Ca2+-channel receptor is the ryanodine receptor, which responds to increased [Ca2+]; to release its store contents (Fig. 2.4). There is currently much controversy as to whether a natural 'Ins(l,4,5)P3-like' activator for this receptor is present. One candidate is cyclic ADP ribose. This channel also interacts with the classic dihydropyridine receptor (L-channel) and is involved in excitation-contraction coupling. Moreover, it is thought that a mutation in the ryanodine receptor is present in malignant hyperthermia-susceptible patients, increasing the sensitivity of the receptor to triggering agents like halothane.

CALCIUM-SENSITIVE TARGETS

In order that the increase in intracellular [Ca2+]j can be translated into a physiological response, cells need to express Ca2+-sensitive target proteins. These interactions may be direct, e.g. by a direct Ca2+ interaction such as with protein kinase C or phospholipase C. However, a large number of Ca2+-sensitive targets require the activation of calmodulin. Calmodulin binds four molecules of Ca2+ and then goes on to activate a range of proteins. For example, the plasma membrane Ca2+-ATPase is sensitive to calmodulin where its activity is increased. This is a feedback mechanism to limit rises in [Ca2+];. In this model, Ca2+ rises and activates calmodulin, and the Ca2+-calmodulin complex activates the PMCA to pump Ca2+ out of the cell and lower [Ca2+]j.

MECHAN I SM(S) OI: ANAESTHESIA

Anaesthesia has been practised for over a century. Despite this, little is known regarding the target site(s) for anaesthetic agents. Over the years, many have searched for a single anaesthetic target site, with limited success. If there was a single anaesthetic target, then all anaesthetic agents would behave in essentially the same manner - this is clearly not the case. Early theories relied on the observed correlation between (volatile) anaesthetic potency and lipid (olive oil) solubility (Fig. 2.5). In this correlation, agents with high lipid solubility were more potent anaesthetic agents than those with lower lipid solubility. This led many authors to suggest that membrane lipids were the target for anaesthetic agents. However, lipids per se do not modify cellular activity. This led to the thought that it was the lipid surrounding integral membrane proteins that was important, with the anaesthetic modulating protein activity indirectly as a consequence of lipid interaction. In support of a specific protein target site for anaesthetic agents, there is the observation that some anaesthetics exist as stereoisomers and that one isomer is in general more potent than the other. This is

Correlation between anaesthetic potency, expressed as minimum alveolar concentration (MAC), and lipid solubility, expressed as oil:gas partition coefficient.

Oihgas partition coefficient

Table 2.5 Characteristics of GABA receptors-1

GABAAb

GABAr

Receptor type

LG IC

GPCR

Effector

Cl- influx

K+efflux

Close VSCC

Location

Postsynaptic

Presynaptic

Action

Inhibitory

Inhibit or v

Agonist

Endogenous

GABA

GABA

Pharmacological

Muscimol

Baclofen

Antagonist

Bicuculline

Phaclof'en

aThere is good evidence for GABAc receptors, which are similar to GABAa.

hGABAA receptor action is potentiated by benzodiazepines. [,GIC, ligand-gated ion channel.

aThere is good evidence for GABAc receptors, which are similar to GABAa.

hGABAA receptor action is potentiated by benzodiazepines. [,GIC, ligand-gated ion channel.

Correlation between anaesthetic potency, expressed as minimum alveolar concentration (MAC), and lipid solubility, expressed as oil:gas partition coefficient.

the case for isoflurane [S(+) more potent than R(-)], barbiturates (in general S more potent than R) and ketamine [S(+) more potent than R(-)]. The literature reveals three potential (protein) target sites: GABAa receptors, VSCCs and excitatory amino acid transmission/receptors.

GABAa receptors

GABA receptors are classified into A and B subtypes (with a third type C suggested) (Table 2.5), and are activated by the major inhibitory transmitter in the brain, y-aminobutyric acid (GABA). GABAa receptors are hetero-oligomeric protein ligand-gated ion channels and GABAg receptors are GPCRs. GABAa receptors comprise five subunits assembled in various combinations. GABAa receptor subunits used to assemble a functional channel arise from a number of families (a, p, 6 and y) and there are several genes encoding these subunits. This clearly allows much diversity in the make-up of individual GABAa receptors and can make comparative studies of GABAa receptor function difficult.

Indeed, it has been suggested that there may be in excess of 100 000 possible permutations in a functional channel, although in nature only a tiny number of these permutations exist. The GABAa receptor has been championed by many authorities as the major and unifying anaesthetic target site. With the exception of ketamine, all anaesthetic agents tested to date appear to interact with the GABAa receptor at clinically relevant concentrations. The effect produced is to potentiate GABAA-mediated Cl~ influx, leading to hyperpolarization. This effect on Ch conductance requires the presence of GABA.

Voltage-sensitive calcium channels

Voltage-sensitive Ca2+ channels are involved in the control of neurotransmitter release. If we accept that anaesthetic agents inhibit neurotransmission without affecting axonal conduction, then blockade of VSCCs represents a logical target site for anaesthetic agents (Table 2.4).

Several in vivo studies have suggested that the L-channel may contribute to the mechanism of anaesthesia. However, with the exception of nimodipine, passage through the blood-brain barrier of L-channel blockers is poor, making clinical studies difficult. The

Table 2,6 Characteristics of glutamate rcceptors

lonotrophic

Metabotrophic

NMDA

AMPA

Kainate

Receptor type

LGIC

LGIC

LGIC

GPCR

Main effector

Ca2+

Na+

Na+

PLC (+AC)

Location

Postsynaptic3

Postsynaptic-1

Postsynaptic*

Presynaptic

Postsynaptic

Agonist

Endogenous

Glutamate

Glutamate

Glutamate

Glutamate

Pharmacological

NMDA

AMPA

>

Various'1

Antagonist

D-AP5

CNQX

CNQX

Various'1

PLC, pliospholipase C (the

activation of PLC leads to die formation of Ins( 1,4,5 )P,i and release of stored intracellular Ca3+); AC, adenvlvl cvclase;

AMPA, a-amino-3-hydroxy-S-methyiisox azol e-4-propionic acid; NMDA, N-methyl-D - aspartate (the NMDA receptor channel is blocked by Mg2+,

dizoclipine (MK 801 ) and ketamine); D-AP5, 2-amino-5-phospli«pentan«ic acid; CNQX, 6-cyano

7-nitroquinoxaline-2,3-dione.

»May be some evidence for

presynaptic action.

^Depends on subtype, eight I mGIuRl -8) identified.

minimum alveolar concentration (MAC) for halothane in dogs is reduced by verapamil, and verapamil, flunarizine and nitrendipine augmented the general anaesthetic potencies of ethanol and pentobarbital. In addition, a range of intravenous anaesthetic agents have been shown to bind to L-channels, although no functional correlates were made in these studies. It is not possible to determine whether these actions at L-channels contribute to the anaesthetic state and it should be remembered that L-channels are not normally involved in neurotransmitter release. However, L-VSCCs are found in the heart and these anaesthetic actions at L-VSCCs may explain some of the cardiovascular side-effects of anaesthesia. In the absence of any antagonists for use in humans/whole animals, it is difficult to determine a role for N- and P-VSCCs (and others) in anaesthesia, although in electrophysiological studies, a range of anaesthetic agents are capable of inhibiting Ca2+ influx through N- and P-VSCCs, although the latter remain controversial. T-channel block is unlikely to be of any significance to anaesthesia. The major problem for the acceptance of VSCC block as a target for anaesthesia can be found by comparing the concentration-response curve for anaesthetic block of VSCCs with the dose-response curve for determination of MAC. In general, the latter curve lies to the left of the former, indicating that clinically relevant/achievable concentrations have little or no effect on VSCC activity. However, it should be borne in mind that the comparison is very artificial in that the dose of anaesthetic required to reduce movement to surgical stimulation in 50% of a population of individuals is compared with the electrophysiological measurement of single channel currents. In addition, as very small amounts of Ca2+ influx are capable of supporting neurotransmitter release, the question of how much inhibition of influx is functionally relevant remains unresolved.

Excitatory transmission

In contrast to a potentiation of GABA-ergic inhibition, it would also be advantageous to depress excitatory transmission. Indeed, glutamate (the major excitatory transmitter in the mammalian CNS) release from a variety of preparations is inhibited by a range of intravenous and volatile anaesthetic agents. This inhibition could be via an action on glutamate receptors on glutamatergic neurones, inhibition of VSCC activity on glutamatergic neurones or secondary to enhanced GABA-ergic input into glutamatergic-synapses. The most well known anaesthetic target in the glutamate receptor family is the NMDA receptor. This receptor is a ligand-gated ion channel (Table 2.6) and is under the modulatory control of a number of different agents, including glycine, Mg2+, Zn2+ and polyamines. The dissociative anaesthetic ketamine is a non-competitive antagonist at the NMDA receptor. From the discussion in previous sections, the non-competitive block results from an interaction at a site other than the primary ligand (glutamate) binding site. Ketamine 'sits' in the ion channel pore and prevents the influx of Ca2+, thus depressing glutamatergic (excitatory) transmission.

It is likely that anaesthesia results from an interaction of the three target groups described above and further research is needed to define the extent of interaction at each site for different classes of anaesthetic agents.

FURTHER READING

Barritt G J 1994 Communication within animal cells. Oxford Science

Publications, Oxford Calvey T N, Williams N E 1997 Principles and practice of pharmacology for anaesthetists, 3rd edn. Blackwell Scientific Publications, Oxford Franks N P, Lieb W R 1994 Molecular and cellular mechanisms of anaesthesia. Nature 367: 607-614 Hudspith M J 1997 Glutamate: a role in normal brain function, anaesthesia, analgesia and CNS injury. British Journal of Anaesthesia 78:731-747

International Union of Pharmacology (IUPHAR) 1998 The IUPHAR compendium of receptor characterization and classification. IUPHAR Media, London

Mehta A K, Ticku M K 1999 An update on GABAa receptors. Brain

Research. Brain Research Reviews 29: 196-217 Rang H P, Dale M M, Ritter J M 1999 Pharmacology, 4th edn.

Churchill Livingstone, London Tanelian D L, Kosek P, Mody I, Maclver B 1993 The role of the GABAa receptor/chloride channel complex in anesthesia. Anesthesiology 78: 757-776

Various 1993 British Journal of Anaesthesia (Postgraduate issue) 71(1): 1-163

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