Inositol trisphosphate and diacylglycerol

IP₃ and diacylglycerol are produced through hydrolysis of inositol phospholipids (phosphoinositides), which are mainly located in the inner half of the plasma membrane. The inositol phospholipid known as phosphatidyl bisphosphate (PIP₂) is the most important member of this class of lipids in terms of cell signalling, even though it accounts for less than 10% of the total inositol lipids and less than 1% of all the phospholipids in the cell membrane. The breakdown of PIP₂ starts with the binding of a signalling molecule to its receptor in the plasma membrane. The activated receptor stimulates a guanine-nucleotide-binding protein (G protein) known as G_q, which in turn activates an inositide-specific phospholipase C called phospholipase C-β. This enzyme cleaves PIP₂ into two products (Figure 5.3):

These two molecules act as mediators for separate signalling pathways.

Figure 5.3

Phospholipase C signalling pathway

IP₃ is small and water-soluble. It diffuses into the cytosol, where it binds to and opens IP₃-gated Ca²⁺ release channels in the endoplasmic reticulum (Figure 5.3) or, in muscle cells, ryanodine receptors in the sarcoplasmic reticulum. In fact, in many cell types both forms of Ca²⁺ receptors are present. To end the Ca²⁺ response, Ca²⁺ is pumped back out of the cytosol and IP₃ is broken down by phosphatases within the cell.

Some of the IP₃ is also phosphorylated to form inositol tetrakisphosphate (IP₄), which may promote the refilling of the intracellular Ca²⁺ stores and/or mediate slower or longer-lived responses within the cell.

Diacylglycerol remains attached to the plasma membrane and has two potential fates. First, it can be cleaved to give arachidonic acid, which can be used as a precursor in the synthesis of eicosanoids. Second, and more importantly, it can act as a second messenger. The initial rise in Ca²⁺ brought about by IP₃ causes an enzyme called protein kinase C (C because it is Ca²⁺-dependent) to move from the cytosol to the plasma membrane, where it is activated by diacylglycerol (Figure 5.3).

Protein kinase C is a serine/threonine protein kinase that regulates the function of other proteins by phosphorylating their serine and/or threonine residues. In addition, protein kinase C can alter the transcription of specific genes. In one pathway, the enzyme catalyses the phosphorylation of mitogen-activated protein (MAP) kinase, which in turn phosphorylates and activates the transcription factor Elk-1. Together with another protein (the serum response factor), Elk-1 binds to the serum response element, a short DNA sequence in the promoter region of the target gene. This leads to transcription of the gene. In another pathway, activation of protein kinase C results in the release of nuclear factor kappa B, which moves into the nucleus also to activate the transcription of specific genes.

As diacylglycerol is rapidly metabolised, sustained activation of protein kinase C for longer-term responses depends on a second wave of diacylglycerol production. This time, diacylglycerol is released by phospholipase-mediated cleavage of phosphatidylcholine, the major phospholipid in the cell.

Eicosanoids

Eicosanoids are signalling molecules that are continuously made in the plasma membrane of all mammalian tissues. They are synthesised from 20-carbon fatty acid chains (mainly arachidonic acid), which in turn are cleaved from membrane phospholipids by phospholipases. There are four major groups of eicosanoids:

The first three are collectively known as prostanoids. The generation of prostanoids is catalysed by the enzyme prostaglandin H₂ synthase, also known as cyclooxygenase (COX), whereas formation of leucotrienes occurs via a lipoxygenase pathway.

Detailed information on the prostaglandin synthetic pathway is provided in Chapter 6. In the current context, it is important to note that there are two main forms of COX, known as COX-1 and COX-2. These two enzymes have similar functions but are the products of different genes. In general, COX-1 is found in tissues that produce prostaglandins constantly, such as the stomach mucosa, whereas COX-2 is only expressed at sites of inflammation and so is inducible. A third type of COX, COX-3, is formed by alternative splicing of the gene encoding COX-1.

Prostaglandins and leucotrienes are important stimulators of the myometrium during labour (see Chapter 6 for a discussion of the regulation of myometrial contractility). The increase in prostaglandin levels during labour is thought to be caused by induction of COX-2 in fetal membranes. Bacterial products and proinflammatory cytokines can also increase expression of COX-2 and hence prostaglandin synthesis. In addition, high concentrations of prostanoids have been reported during menstruation, in particular in conditions associated with painful menstruation, such as menorrhagia, dysmenorrhoea and endometriosis.

The synthetic pathways of eicosanoids can be targeted by therapeutic drugs. For example, corticosteroid drugs such as cortisone inhibit phospholipase and are used to treat inflammatory conditions such as arthritis. Non-steroidal anti-inflammatory drugs (NSAIDs), including aspirin and ibuprofen, block the first oxidation step of arachidonic acid that is catalysed by COX.

Older NSAIDs such as indometacin inhibit both COX-1 and COX-2. Newer NSAIDs are selective for COX-2. In general, the more COX-2-selective an NSAID is, the better its adverse effect profile. For example, COX-2-selective inhibitors are just as effective in the treatment of menstrual pain as NSAIDs but have fewer gastrointestinal adverse effects.

Aspirin inhibits both COX-1 and COX-2 (it is actually much more active against COX-1 than COX-2; hence its poor adverse effect profile). Unlike most NSAIDs, which are competitive antagonists, aspirin functions by permanently acetylating the active site of the COX enzyme. It can be used at a low dose to inhibit platelet thromboxane synthesis with little effect upon vascular endothelial prostacyclin synthesis. This may be of value in thromboprophylaxis and in the management of pre-eclampsia.

Low doses of aspirin permanently disable platelet COX as the platelets pass through the hepatic portal system. Since the platelet has no nucleus, it cannot synthesise a new supply of COX and so platelet thromboxane synthesis is permanently inhibited. Most of the aspirin is then inactivated within the liver. The small amount of aspirin that then passes into the general circulation may acetylate vascular endothelial COX but, since these cells have a nucleus, they can synthesise a new supply of COX and maintain prostacyclin synthesis.

Nitric oxide and carbon monoxide

Most signalling molecules are hydrophilic molecules but some are small enough to pass straight into the cell where they can directly exert their effects. One such example is the gas nitric oxide (NO). NO is produced from l-arginine by the enzyme NO synthase in the presence of co-factors and O₂; the by-product is l-citrulline. There are three main forms of this enzyme:

These enzyme isoforms are the products of different genes that share about 50–60% sequence homology. Many cells express more than one form of NO synthase.

eNOS was first described in endothelial cells. It is constitutively expressed and is dependent on Ca²⁺ and calmodulin. NO has a very short half-life (5–10 seconds) and is converted to nitrates and nitrites in the blood. Endothelial cells on blood vessels release NO in response to increased shear stress or agents such as acetylcholine. The released NO diffuses to the underlying smooth muscle, where it reacts with iron in the active site of the enzyme guanylate cyclase to produce the intracellular mediator cGMP. The effects of guanylate cyclase are rapid and result in muscle relaxation. Thus, continual release of NO from blood vessels is one of the main mechanisms for keeping blood pressure at its normal level.

In pregnancy, blood pressure falls and it is thought that the associated vasodilation is partly mediated by increased NO release. NO is also important in the regulation of blood flow within the placenta and eNOS is expressed on the entire syncytiotrophoblast surface, just as it is expressed on blood vessels. Placental eNOS is thought to inhibit the aggregation of neutrophils and platelets present in maternal blood in the intervillous space.

The expression of iNOS is induced in response to inflammatory signals such as bacterial cell-wall products, which include lipopolysaccharides and cytokines such as interferon gamma or tumour necrosis factor alpha. It is induced in activated macrophages and neutrophils. NO released from these cells helps them to kill invading microorganisms. The activity of iNOS is independent of Ca²⁺ and calmodulin.

bNOS was first described in the brain. Like eNOS, it is constitutively expressed and is dependent on Ca²⁺ and calmodulin for activity.

NO is released by many types of nerve cell to convey signals to neighbouring cells. For example, NO released by autonomic nerves in the penis causes local blood-vessel dilatation resulting in penile erection. In reproductive biology, NO has also been implicated in the control of myometrial quiescence, in the onset of labour and in cervical ripening, although the evidence for some of these functions is certainly not conclusive.

The effects of NO on blood vessels also explain the efficacy of glyceryl trinitrate in the treatment of angina, for which it has been used for nearly 100 years. Glyceryl trinitrate is converted to NO, which relaxes blood vessels in the heart. Glyceryl trinitrate has also been used in an attempt to prevent preterm labour by relaxing myometrial smooth muscle and to ripen the cervix.

Carbon monoxide (CO) is another gas that stimulates guanylate cyclase. CO is produced by the enzymes haem oxygenase 1 and 2. Haem oxygenase 1 is inducible, while haem oxygenase 2 is constitutively expressed. It is now known that CO, like NO, is important in maintaining vasodilatation in the placenta.

Calcium

Cells maintain low concentrations of free Ca²⁺ (100 nmol/l) despite much higher concentrations in the extracellular fluid (10 mmol/l) and endoplasmic reticulum. Increases in intracellular Ca²⁺ concentrations are one way in which extracellular signals are transmitted across the plasma membrane. When Ca²⁺ channels are transiently opened in the plasma or endoplasmic reticulum membranes, intracellular Ca²⁺ concentrations rise to about 50 micromoles/l and activate Ca²⁺-responsive proteins in the cell. Resting Ca²⁺ concentrations are kept very low by several means. Ca²⁺-ATPases in the plasma membrane pump Ca²⁺ out of the cell. Nerve and muscle cells, which use Ca²⁺ to a greater extent for signalling, have an additional Ca²⁺ pump (Na⁺,Ca²⁺-exchanger) in the plasma membrane, which couples Na⁺ influx to Ca²⁺ efflux.

In the endoplasmic reticulum, there is yet another Ca²⁺-ATPase that takes up Ca²⁺ from the cytosol. Mitochondria can also pump Ca²⁺ inside; a low-affinity, high-capacity Ca²⁺ pump in the inner mitochondrial membrane uses the electrochemical gradient generated across the membrane during electron transport in oxidative phosphorylation. This pump only operates when Ca²⁺ levels are extremely high, usually as a consequence of cell damage. Ca²⁺-binding proteins in the cytoplasm and in the endoplasmic reticulum offer additional Ca²⁺ buffering capacity.

Calmodulin is a Ca²⁺-binding protein found in all eukaryotic cells. It mediates many Ca²⁺-regulated pro-cesses and undergoes a conformational change when bound to Ca²⁺. When this happens, the Ca²⁺–calmodulin complex can bind to various target proteins and alter their activity. Among the Ca²⁺–calmodulin targets are various enzymes (such as eNOS and bNOS) and membrane transport proteins. Most effects of Ca²⁺–calmodulin are, however, indirect and are mediated by Ca²⁺–calmodulin-dependent protein kinases; an example for such an enzyme is myosin light chain kinase, which activates smooth muscle contraction.

Two pathways of Ca²⁺ signalling have been well defined. One is used mainly by excitable cells. When nerve-cell membranes are depolarised by an action potential, voltage-gated Ca²⁺ channels open and Ca²⁺ enters the cell, leading to the secretion of neurotransmitters. In the other pathway, binding of extracellular signalling molecules to cell-surface receptors ultimately leads to the opening of channels in the endoplasmic reticulum and a rise in the intracellular Ca²⁺ concentration. The opening of these channels is brought about by the intermediate molecule IP₃.