In our last meeting we introduced one of the major cellular signal tranduction pathways. We saw how a hormone signal could be transduced into increased intracellular cAMP concentrations, which in turn activates enzymes involved in some cellular responses to the hormone. The main idea is recognizing that a signal, rather than a hormone per se, leads to a cellular response. cAMP is a universal intracellular messenger. Changing concentrations of cAMP are translated into changes in enzyme activities, and the enzyme activities produce the cellular response.

Most cells express multiple signal transduction pathways. In addition to the cAMP pathway, nearly every cell expresses a Ca⁺⁺ signal transduction pathway. As we saw for cAMP, Ca⁺⁺ is another universal intracellular messenger. The cellular actions of these two intracellular signals are fairly similar. Both messengers act by binding to specific proteins, and the binding activates the proteins by causing them to undergo conformational changes. Very often, multiple intracellular messengers interact with each other, and the cross-talk among signal transduction pathways influences the intensity of a cellular response to hormones or other extracellular signals. For an example, a diuretic hormone known as AVP increases fluid secretion in mammalian kidneys. At the same time, certain prostaglandins attenuate the intensity of fluid secretion. Hence, the overall secretory response to the hormone is the outcome of multiple signal transduction pathways.

An important condition must be met to allow Ca⁺⁺ to serve as an intracellular second messenger. Ca⁺⁺ must be present in very low concentrations, typically something like 10^-7 M, in the cytosolic fraction of cells. Most eucaryotic cells elaborate a sophisticated system of mechanisms to maintain these low Ca⁺⁺ concentrations. Most cells have a Ca⁺⁺-ATPase in the plasma membrane, and this enzyme serves as a Ca⁺⁺ pump, moving Ca⁺⁺ ions against a chemical gradient from the cytosol to the extracellular spaces at the expense of ATP. The ions are pumped against a gradient because Ca⁺⁺ is present in substantial concentrations, around 10^-3M, in extracellular spaces. Some cells, such as muscle cells, have an additional Ca⁺⁺ pump in their plasma membranes. This is known as a Na⁺-driven Ca⁺⁺ antiport, or exchanger. This antiport works by coupling the inward transport of Na⁺⁺ with the efflux of Ca⁺⁺. The antiport is particularly important when Ca⁺⁺ concentrations are elevated by frequent stimulation of muscle cells. Both pumps maintain low intracellular Ca⁺⁺ concentrations by pumping Ca⁺⁺ ions into the extracellular spaces.

Whereas the Ca⁺⁺ concentrations in the cytosolic compartment of cells are very low, there is a substantial amount of Ca⁺⁺ in eucaryotic cells. Most of the Ca⁺⁺ is segregated in a specialized compartment called a calcium-sequestering compartment. The membranes of this intracellular compartment also have a Ca⁺⁺-ATPase that pumps Ca⁺⁺ ions into the compartment. The plasma membranes and the membranes of the calcium-sequestering compartment have Ca⁺⁺ channels that are briefly opened by transduction of extracellular signals. Ca⁺⁺ ions rapidly move down their chemical gradients, into the cytosolic compartment, creating a dramatic, local increase in Ca⁺⁺ concentrations. The brief spike in intracellular Ca⁺⁺ activates proteins in the Ca⁺⁺ signal transduction pathways.

Let us turn, now, to the mechanism of releasing Ca⁺⁺ from the intracellular compartment. The mechanism involves phospholipid metabolism. The plasma membranes of eucaryotic cells are comprised of lipids, mainly phospholipids, and proteins. Here is a typical cell membrane, showing a bilayer of phospholipids. With respect to bulk quantities, phosphatidylcholine and phosphatidylethanolamine are the main components of biomembranes. Minor amounts of many other lipids are also present in membranes. One of these quantitatively minor lipids is known as phosphatidylinositol (PI). PI is just a garden-variety, off-the-shelf phospholipid with a single molecule of a sugar, specifically inositol, in the polar head group. PI usually makes up less than 10% of cell membrane lipids.

Three forms of PI are present in biomembranes. An enzyme known as PI-kinase can add a phosphate to carbon-4 of the inositol moiety, creating phosphatidylinositol 4-phosphate (PIP). Then another enzyme, known as PIP-kinase can add a phosphate to carbon-5 of the inositol moiety, creating phosphatidylinositol 4,5-bisphosphate (PIP₂). PI, PIP and PIP₂ are the three forms of PI in cell membranes. PIP₂ is present in very low amounts, typically less than 10% of all PI forms, and less than 1% of membrane lipids. As usual in cell signaling, despite its low concentration, the PIP₂ is the most important part of our story.

Compared to our understanding of the cAMP pathway, the details of the Ca⁺⁺ signaling pathway are not as well understood. It is thought that the Ca⁺⁺ signal transduction mechanism begins with interaction of an extracellular signal with a cell surface receptor. As we saw in the cAMP pathway, the receptor undergoes a change in conformation, and interacts with a G-protein, known as the Gp protein. The Gp protein then activates an enzyme known as phospholipase C (PLC). The particular PLC is is specific to PIs. This enzyme cleaves PIP₂ into two products, a diacylglycerol (DAG) and inositol triphosphate (IP₃). The inositol has three phosphates, two from the PI kinase and PIP kinase actions on carbons 4 and 5, and the third from the phosphodiester linkage to the glycerol backbone of the PI.

IP₃ is a water soluble, small molecule. It is biologically inactivated by action of a specific phosphatase that removes one phosphate. As an active messenger, IP₃ interacts with Ca⁺⁺ channels in the calcium-sequestering compartment. This interaction allows a rapid and brief increase in cytosolic Ca⁺⁺ concentrations. The Ca⁺⁺ ions activate target proteins, and thereby translate an extracellular hormone signal into a specific cellular response.

Ca⁺⁺ does not directly activate target proteins in cells. Free Ca⁺⁺ ions are bound by a very specific Ca⁺⁺ binding protein known as calmodulin. Calmodulin in a ubiquitous protein, found in all eucaryotic cells analyzed for it. As we have seen before, Ca⁺⁺ binding induces a conformational change in calmodulin. The Ca⁺⁺-calmodulin complex is now able to activate proteins by binding to the target. The target protein undergoes a conformational change and becomes activated. This is represented in a figure.

The overall physiology can be summarized like this: an extracellular messenger binds to a specific receptor on a cell surface. The receptor changes conformation, and interacts with a Gp protein. The Gp protein activates a PLC, which splits PIP₂ into DAG and IP₃. The IP₃ interacts with Ca⁺⁺ channels on the membrane of the calcium-sequestering compartment. Ca⁺⁺ flows into the cytosolic compartment where it interacts with calmodulin. The Ca⁺⁺-calmodulin complex then activates target proteins.

The story is not quite complete because the PLC step actually activates two signaling pathways. The DAG formed by hydrolysis of the IP₃ may express two roles in cell signaling. For one, arachidonic acid may be hydrolyzed from the DAG. The free arachidonic acid is taken into biosynthesis of prostaglandins and other eicosanoids. As we mentioned before, eicosanoids can modulate cellular responses to hormones. For just a bit more detail on eicosanoid actions, most eicosanoids are thought to act through specific cell surface receptors. The receptors act through G proteins to modify the cAMP and C-kinase pathways.

DAG activates a separate pathway known as the Protein kinase C, or C-kinase, pathway. Protein kinase C binds DAG, which causes a slight conformational change. In its changes form, protein kinase C expresses increased affinity for Ca⁺⁺, which then binds to protein kinase C. When bound to DAG and Ca⁺⁺, the active protein kinase C transfers a phosphate from ATP to specific serene or threonine components of target proteins. A common example is the phosphorylation and activation of the plasma membrane Na⁺-H⁺ exchanger. This exchanger regulates intracellular pH. For an example closer to our interest, AKH also acts through specific cell surface receptors in fat body of the migratory locust. AKH stimulates increased intracellular cAMP, release of IP₃, and increased intracellular Ca⁺⁺ concentrations. These changes in intracellular messengers are responsible for release of sugar residues from cellular glycogen stores and for mobilization of fat body lipids.

The role of IP₃ in releasing Ca⁺⁺ from intracellular stores, and the role of Ca⁺⁺ as an intracellular second messenger are basic biological actions in cellular physiology. Michael Berridge, now a leading administrator in the Medical Research Council in England, is credited with discovering the importance of the IP₃ signal transduction pathway. Used the sheep blowfly salivary gland as his research model. The isolated salivary gland secretes saliva in response to 5-hydroxytryptamine, another biogenic amine. Fluid secretion in the isolated glands is mediated by increased intracellular cAMP concentrations and by the IP₃ pathway.