Pip

Phosphorylation of substrates

Endoplasmic reticulum

Store-operated 0 0

Endoplasmic reticulum

M FIGURE 13-29 IP3/DAG pathway and the elevation of cytosolic Ca2+.

This pathway can be triggered by ligand binding to certain G protein-coupled receptors and several other receptor types, leading to activation of phospholipase C. Cleavage of PIP2 by phospholipase C yields IP3 and DAG (step hi). After diffusing through the cytosol, IP3 interacts with and opens Ca2+ channels in the membrane of the endoplasmic reticulum (step |2|), causing release of stored Ca2+ ions into the cytosol (step |3|). One of various cellular responses induced by a rise in cytosolic Ca2+ is recruitment of protein kinase C (PKC) to the plasma membrane (step |4|), where it is activated by DAG (step |5|). The activated kinase can phosphorylate various cellular enzymes and receptors, thereby altering their activity (step |6|). As endoplasmic reticulum Ca2+ stores are depleted, the IP3-gated Ca2+ channels bind to and open store-operated TRP Ca2+ channels in the plasma membrane, allowing influx of extracellular Ca2+ (step |7|). [Adapted from J. W. Putney, 1999, Proc. Natl. Acad. Sci. USA 96:14669.]

lated. For example, stimulation of hormone-secreting cells in the pituitary by luteinizing hormone-releasing hormone (LHRH) causes rapid, repeated spikes in the cytosolic Ca2+ level; each spike is associated with a burst in secretion of luteinizing hormone (LH). The purpose of the fluctuations of Ca2 + , rather than a sustained rise in cytosolic Ca2 + , is not understood. One possibility is that a sustained rise in Ca2 + may be toxic to cells.

Diacylglycerol (DAG) Activates Protein Kinase C, Which Regulates Many Other Proteins

After its formation by hydrolysis of PIP2 or other phospho-inositides, DAG remains associated with the plasma membrane. The principal function of DAG is to activate a family of protein kinases collectively termed protein kinase C (PKC). In the absence of hormone stimulation, protein kinase C is present as a soluble cytosolic protein that is catalytically inactive. A rise in the cytosolic Ca2+ level causes protein kinase C to bind to the cytosolic leaflet of the plasma membrane, where the membrane-associated DAG can activate it. Thus activation of protein kinase C depends on an increase of both Ca2+ ions and DAG, suggesting an interaction between the two branches of the IP3/DAG pathway (see Figure 13-29).

The activation of protein kinase C in different cells results in a varied array of cellular responses, indicating that it plays a key role in many aspects of cellular growth and metabolism. In liver cells, for instance, protein kinase C helps regulate glycogen metabolism by phosphorylating and thus inhibiting glycogen synthase. Protein kinase C also phospho-rylates various transcription factors; depending on the cell type; these induce synthesis of mRNAs that trigger cell proliferation.

Ca2+/Calmodulin Complex Mediates Many Cellular Responses to External Signals

Ligand binding to several types of receptors, in addition to G protein-coupled receptors, can activate a phospholipase C isoform, leading to an IP3-mediated increase in the cytosolic level of free Ca2 + . Such localized increases in cytosolic Ca2 + in specific cell types are critical to its function as a second messenger. For example, acetylcholine stimulation of G proteincoupled receptors in secretory cells of the pancreas and parotid gland induces an IP3-mediated rise in Ca2+ that triggers the fusion of secretory vesicles with the plasma membrane and release of their contents into the extracellular space. In blood platelets, the rise in Ca2+ induced by thrombin stimulation triggers a conformational change in these cell fragments leading to their aggregation, an important step in plugging holes in blood vessels. Secretion of insulin from pancreatic p cells also is triggered by Ca2 + , although the increase in Ca2+ occurs by a different mechanism (see Figure 15-7).

A small cytosolic protein called calmodulin, which is ubiquitous in eukaryotic cells, functions as a multipurpose switch protein that mediates many cellular effects of Ca2 +

ions. Binding of Ca2+ to four sites on calmodulin yields a complex that interacts with and modulates the activity of many enzymes and other proteins (see Figure 3-28). Because Ca2+ binds to calmodulin in a cooperative fashion, a small change in the level of cytosolic Ca2+ leads to a large change in the level of active calmodulin. One well-studied enzyme activated by the Ca2+/calmodulin complex is myosin light-chain kinase, which regulates the activity of myosin in muscle cells (Chapter 19). Another is cAMP phosphodiesterase, the enzyme that degrades cAMP to 5'-AMP and terminates its effects. This reaction thus links Ca2+ and cAMP, one of many examples in which two second messengers interact to fine-tune certain aspects of cell regulation.

In certain cells, the rise in cytosolic Ca2+ following receptor signaling via PLC-generated IP3 leads to activation of specific transcription factors. In some cases, Ca2+/calmod-ulin activates protein kinases that, in turn, phosphorylate transcription factors, thereby modifying their activity and regulating gene expression. In other cases, Ca2+/calmodulin activates a phosphatase that removes phosphate groups from a transcription factor. An important example of this mechanism involves T cells of the immune system in which Ca2 + ions enhance the activity of an essential transcription factor, NFAT (nuclear factor of activated T cells). In unstimulated cells, phosphorylated NFAT is located in the cytosol. Following receptor stimulation and elevation of cytosolic Ca2 + , the Ca2+/calmodulin complex binds to and activates cal-cineurin, a protein-serine phosphatase. Activated calcineurin then dephosphorylates key phosphate residues on cytosolic NFAT, exposing a nuclear localization sequence that allows NFAT to move into the nucleus and stimulate expression of genes essential for activation of T cells.

The Ca2+/calmodulin complex also plays a key role in controlling the diameter of blood vessels and thus their ability to deliver oxygen to tissues. This pathway involves a novel signaling molecule and provides another example of cGMP functioning as a second messenger.

Signal-Induced Relaxation of Vascular Smooth Muscle Is Mediated by cGMP-Activated Protein Kinase G

Nitroglycerin has been used for over a century as a treatment for the intense chest pain of angina. It was known to slowly decompose in the body to nitric oxide (NO), which causes relaxation of the smooth muscle cells surrounding the blood vessels that "feed" the heart muscle itself, thereby increasing the diameter of the blood vessels and increasing the flow of oxygen-bearing blood to the heart muscle. One of the most intriguing discoveries in modern medicine is that NO, a toxic gas found in car exhaust, is in fact a natural signaling molecule. I

Definitive evidence for the role of NO in inducing relaxation of smooth muscle came from a set of experiments in which acetylcholine was added to experimental preparations of the smooth muscle cells that surround blood vessels. Direct application of acetylcholine to these cells caused them to contract, the expected effect of acetylcholine on these muscle cells. But addition of acetylcholine to the lumen of small isolated blood vessels caused the underlying smooth muscles to relax, not contract. Subsequent studies showed that in response to acetylcholine the endothelial cells that line the lumen of blood vessels were releasing some substance that in turn triggered muscle cell relaxation. That substance turned out to be NO.

We now know that endothelial cells contain a G0 protein-coupled receptor that binds acetylcholine and activates phospholipase C, leading to an increase in the level of

► FIGURE 13-30 Regulation of contractility of arterial smooth muscle by nitric oxide (NO) and cGMR Nitric oxide is synthesized in endothelial cells in response to acetylcholine and the subsequent elevation in cytosolic Ca2+. NO diffuses locally through tissues and activates an intracellular NO receptor with guanylyl cyclase activity in nearby smooth muscle cells. The resulting rise in cGMP leads to activation of protein kinase G (PKG), relaxation of the muscle, and thus vasodilation. The cell-surface receptor for atrial natriuretic factor (ANF) also has intrinsic guanylyl cyclase activity (not shown); stimulation of this receptor on smooth muscle cells also leads to increased cGMP and subsequent muscle relaxation. PPj = pyrophosphate. [See C. S. Lowenstein et al., 1994, Ann. Intern. Med. 120:227.]

Lumen of blood vessel

Endothelial cells

Smooth muscle cells

Smooth muscle cells

Acetylcholine 1

Acetylcholine GPCR

Phospholipase r

Ca2+/Calmodulin

NO synthase

Arginine + o2 Citrulline + No!

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