Human Biochemistry, Volume 2 - Murray R. 1993

Biochemistry of Intra- and Intercellular Communication
Action of Hormones
Mechanism of Action of Group II Hormones (Peptide Hormones) - Calcium- and Phosphoinositide-Mediated Hormone Action

Ionized calcium serves as a vital regulator of diverse biological processes, such as Muscle contraction, stimulus-secretion coupling, Blood Coagulation cascades, The activity of numerous Enzymes, and Cell membrane excitability. It also acts as an intracellular second messenger for several Hormones.

Calcium METABOLISM

The concentration of extracellular calcium (Ca²⁺) is 5 mmol/L and is strictly regulated (see Ch. 47). The intracellular concentration of free Calcium Ions is much lower, at 0.1–10 µmol/L, whereas The amount of Ca²⁺ bound to intracellular Organelles (Mitochondria and Endoplasmic reticulum) ranges from 1 to 20 µmol/L. Despite this 5,000- to 10,000-fold concentration gradient and a transmembrane electrical gradient that favors Ca²⁺ entry, calcium influx into Cells is tightly restricted. Changes in cytosolic Ca²⁺ concentration occur via three mechanisms. Certain hormones (Class II.B) increase membrane permeability to Ca²⁺, thereby enhancing calcium entry into The Cell. This can be mediated by a high-capacity, low-affinity Na⁺/Ca²⁺ exchange mechanism. There is also an ATPase-dependent Ca²⁺/2H⁺ pump that extrudes Ca²⁺ from the cell in exchange for H⁺. This mechanism features high affinity for Ca²⁺ but low capacity, and it is presumably responsible for the fine-tuning of cytosolic Ca²⁺ levels. Finally, both the mobilization of Ca²⁺ from mitochondria and The endoplasmic reticulum, and the sequestration of Ca²⁺ into these organelles, are possible.

Current concepts regarding The Role of Ca²⁺ as an intracellular messenger in hormone action are based on two key observations. First, researchers have successfully quantified rapid changes in intracellular Ca²⁺ concentration—fluctuations that align with the role of an intracellular messenger. These data were obtained using various Methods, including fluorescent Ca²⁺ chelators such as Quin 2 and Fura 2. These compounds enable the quantitative assessment of rapid submicromolar changes in Ca²⁺ concentration. The second crucial observation linking Ca²⁺ to hormonal effects was the identification of the intracellular targets of this ion: the discovery of a Ca²⁺-dependent regulator of phosphodiesterase activity provided the foundation for understanding how Ca²⁺ and cAMP interact within the cell.

Calmodulin

This calcium-dependent regulatory protein is named calmodulin; it has a Molecular Weight of 17,000 and is homologous in Structure and function to the muscle protein troponin C. Calmodulin contains four Ca²⁺-binding sites. Binding of Ca²⁺ to all four sites leads to a marked conformational change in the protein, with a large portion of the molecule adopting an a-helical structure. These conformational transitions presumably determine calmodulin's ability to activate or inactivate specific enzymes. The interaction of calcium ions with calmodulin (and the corresponding alteration in its activity) is fundamentally similar to the binding of cAMP to protein kinase, which triggers the activation of that enzyme. Calmodulin frequently serves as one of the numerous subunits of complex Proteins and typically participates in regulating the activity of various Kinases, as well as enzymes involved in the Synthesis and degradation of Cyclic NUCLEOTIDES. A list of certain enzymes directly or indirectly regulated by Ca²⁺ (presumably via calmodulin) is provided in Table 44.5.

The Ca²⁺-calmodulin complex exerts regulatory control not only over enzyme activity and ion transport, but also over the function of many structural elements within the cell. The latter include the smooth muscle Actomyosin complex, which is under ß-adrenergic control, as well as microfilaments in non-contractile cells that mediate processes such as cell motility, shape changes, mitosis, granule release, and endocytosis.

Table 44.5. Enzymes regulated by the calcium-calmodulin complex

Calcium as a Mediator of Hormone Action

The role of ionized calcium in hormone action is supported by the following observations: The Effect of many hormones 1) is abolished in calcium-free media or upon depletion of intracellular Ca²⁺ stores; 2) can be mimicked by agents that elevate cytosolic Ca²⁺ concentration, such as the Ca²⁺ ionophore A23187; and 3) is coupled with transmembrane Ca²⁺ transport. These phenomena have been studied in considerable detail in pituitary cells, smooth muscle, Salivary Glands, and platelets; the mechanism regulating hepatic Glycogen Metabolism by vasopressin and a-adrenergic catechols has been investigated most thoroughly. This mechanism is schematically illustrated in Figures 19.5 and 19.7.

The addition of a₁-agonists or vasopressin to isolated hepatocytes induces a 3-fold increase in cytosolic Ca²⁺ content (from 0.2 to 0.6 µmol/L) within just a few seconds. This surge precedes a comparable rise in phosphorylase activity; investigation of each effect has demonstrated that they occur at comparable hormone concentrations. a₁-Antagonists inhibit the rise in cytosolic Ca²⁺, whereas hormone withdrawal leads to a rapid decline in both cytosolic Ca²⁺ concentration and phosphorylase a levels. Initially, Ca²⁺ is evidently mobilized from cellular organelles, and the Ca²⁺ stored within them appears sufficient to manifest the immediate hormonal response. Sustained action requires either influx of extracellular Ca²⁺ or inhibition of its efflux via the Ca²⁺ pump—a process dependent on a simultaneous rise in cAMP concentration.

Phosphorylase activation occurs through The conversion of phosphorylase b to phosphorylase a, catalyzed by the enzyme phosphorylase b kinase. This enzyme contains calmodulin (as its d-subunit), and its activity increases as Ca²⁺ concentration rises within the 0.1–1 µmol/L range—that is, within the exact limits by which cytosolic calcium levels increase in the Liver in the presence of the hormone. The Link Between Ca²⁺ and phosphorylase activation is definitive.

The regulation of a series of key metabolic enzymes is achieved via Ca²⁺, phosphorylation, or both mechanisms simultaneously; these include glycogen synthase, glycerol-3-phosphate dehydrogenase, pyruvate dehydrogenase, pyruvate kinase, and pyruvate carboxylase. It remains unclear whether calmodulin participates directly in this regulation or if the primary role belongs to recently discovered protein kinases (either Ca²⁺/calmodulin-dependent or Ca²⁺/phospholipid-dependent).

The Role of Phosphoinositide Turnover Products in Ca²⁺-Dependent Hormone Action

Clearly, communication between the hormone receptor on The Plasma Membrane and intracellular Ca²⁺ stores must be mediated by a specific signal. The most likely candidates for this signaling role are the products of phosphoinositide metabolism. Phosphatidylinositol 4,5-bisphosphate is hydrolyzed by phospholipase C to yield Inositol 1,4,5-trisphosphate and diacylglycerol (Fig. 44.5). In hepatocytes, this reaction is observed within seconds after the addition of vasopressin or epinephrine. As demonstrated in various membrane and whole-organelle preparations, myo-inositol-P₃ at concentrations of 0.1–0.4 µmol/L triggers a very rapid release of Ca²⁺. Attempts to mimic the hormonal effect using this compound (an important step in establishing their interrelationship) have been only partially successful, likely because it is difficult to achieve intracellular penetration of inositol-P₃, and it undergoes rapid intracellular Hydrolysis. Another phosphoinositide hydrolysis product, 1,2-diacylglycerol, activates the Ca²⁺-phospholipid-dependent protein kinase by increasing the enzyme's affinity (lowering the K_m) for Ca²⁺. The precise role of this process in the action of Ca²⁺-dependent hormones is currently under investigation.

The action of steroidogenic agents—including ACTH and cAMP in the adrenal cortex; angiotensin II, K⁺, serotonin, ACTH, and dibutyryl-cAMP in the adrenal zona glomerulosa; LH in the Ovaries; and LH and cAMP in Leydig cells (Testes)—is coupled with elevated concentrations of phosphatidic acid, phosphoinositol, and polyphosphoinositides in the respective target Tissues.

Several additional Examples can be cited. For instance, 5–10 seconds after adding TRH to pituitary cells, phosphoinositide Cleavage by phospholipase C increases markedly; this elevates intracellular levels of inositol di- and trisphosphates, resulting in the mobilization of intracellular calcium. This sequence activates a Ca²⁺-dependent protein kinase, which in turn phosphorylates A number of proteins (one of which presumably participates in TSH release). Calcium apparently also serves as an intracellular mediator of GnRH action on LH release. Calmodulin is also believed to be involved in this process.

The role of calcium and polyphosphoinositide cleavage products in hormone action is illustrated in Fig. 44.5. As the scheme shows, phosphoinositide hydrolysis products act as second messengers, whereas Ca²⁺ Functions essentially as a third messenger. The scheme could be further supplemented by the fact that a G-protein likely participates in coupling membrane events with Ca²⁺ release. Such an intricate network of intracellular messengers is evidently not a unique phenomenon.