Human Biochemistry, Volume 2 - Murray R. 1993

Biochemistry of Intracellular and Intercellular Communication
Hormones Regulating Calcium Metabolism
Hormones Involved in Calcium Homeostasis — Calcitriol [1,25-(OH)2-D3]

General provisions on The Role of calcitriol in Calcium Homeostasis

A. Historical Background. Rickets—a childhood disease characterized by impaired skeletal mineralization and severe, disfiguring bone deformities—was widespread in North America and Western Europe at the beginning of the century. A series of studies suggested that rickets was caused by a dietary component deficiency. After it was discovered that rickets could be prevented by adding cod Liver oil to the diet, but that vitamin A was not its active component, this rickets-preventing factor was designated as the fat-soluble vitamin D. Around the same time, it was shown that ultraviolet irradiation (artificial or sunlight) also prevents The Development of the disease. Subsequently, an adult equivalent of rickets was identified, namely Osteomalacia. This disease, characterized by impaired bone mineralization, also responded to vitamin D Treatment. Data showing that vitamin D treatment of patients with liver or Kidney damage did not produce the expected effect played a key role in the advancement of further research. Over the past 50 years, The Structure of Vitamin D and its MECHANISM OF ACTION have been studied, with progress being especially rapid over the past decade.

B. Role in homeostasis. The primary Biological Role of calcitriol is The stimulation of calcium and phosphate Absorption in the intestine. Calcitriol is the only hormone that promotes calcium transport against the concentration gradient existing across the intestinal Cell membrane. Because calcitriol production is very tightly regulated (Fig. 47.4), it is evident that a delicate mechanism exists to maintain the Ca²⁺ level in ECF despite significant fluctuations in dietary calcium content. This mechanism maintains the concentrations of calcium and phosphate necessary for The formation of hydroxyapatite crystals deposited in bone Collagen fibrils. In vitamin D (calcitriol) deficiency, the formation of new bone is slowed and bone remodeling is impaired. PTH, which acts on bone Cells, participates primarily in The regulation of these processes, but small concentrations of calcitriol are also required. Calcitriol is also capable of enhancing the action of PTH on renal calcium reabsorption.

Biochemistry

A. Biosynthesis. Calcitriol is a hormone in every sense. It is produced through a complex sequence of enzymatic reactions that involves the Blood transport of precursor molecules to various Tissues (Fig. 47.4). Subsequently, the active compound—calcitriol—is transported to other Organs, where it activates specific biological processes via a mechanism similar to that of Steroid Hormones.

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Fig. 47.4. Formation and hydroxylation of vitamin D₃. 25-Hydroxylation occurs in the liver, and hydroxylation at other positions occurs in the Kidneys. Formation of 25,26-(OH)₂-D₃ and 1,25,26-(OH)₃-D₃ is quite likely. The formulas of 7-dehydrocholesterol, vitamin D₃, and 1,25-(OH)₂-D₃ (calcitriol) are shown. (Reproduced, with permission, from Ganong W. F. Review of Medical Physiology, 13th ed. Appleton and Lange, 1987.)

1. Skin. Small amounts of vitamin D are found in foods (fat, fish liver, egg yolk), but the bulk of the vitamin D used in calcitriol synthesis is formed in the Malpighian layer of the epidermis from 7-dehydrocholesterol via a non-enzymatic, ultraviolet light-dependent photolysis reaction. Process activity is directly dependent on radiation intensity and inversely dependent on the degree of skin pigmentation. With age, the 7-dehydrocholesterol content in the epidermis decreases, which may be directly related to the development of negative calcium balance in the elderly.

2. Liver. A specific transport protein, termed D-binding protein, binds vitamin D₃ and its metabolites and carries D₃ from the skin or intestine to the liver, where it undergoes 25-hydroxylation, the first obligatory step in calcitriol formation. 25-Hydroxylation occurs in The Endoplasmic reticulum in a reaction involving magnesium, NADPH, molecular oxygen, and an unidentified cytoplasmic factor. Two Enzymes participate in the reaction: NADPH-dependent cytochrome P-450 reductase and cytochrome P-450. The reaction is unregulated; it proceeds not only in the liver but also (at a low rate) in the kidneys and intestines. The reaction product, 25-OH-D₃, enters the Blood Plasma (constituting the major form of vitamin D present in the blood) and is transported to the kidneys via the D-binding protein.

3. Kidneys. 25-OH-D₃ is a weak agonist; to exhibit full biological activity, this compound must be modified by C-1 hydroxylation. This occurs in the Mitochondria of the proximal convoluted renal tubules via a complex monooxygenase reaction involving NADPH, Mg²⁺, molecular oxygen, and at least three enzymes: 1) renal ferredoxin reductase (a flavoprotein), 2) renal ferredoxin (an iron-containing sulfoprotein), and 3) cytochrome P-450. This system produces 1,25-(OH)₂-D₃, the most active of the natural vitamin D metabolites.

4. Other tissues. The Placenta contains 1α-hydroxylase, which appears to play an important role as a source of extrarenal calcitriol. The activity of this enzyme is also detected in other tissues, including bone, but the physiological Significance of the enzyme in these tissues is minimal, judging by the fact that in non-pregnant animals after nephrectomy, calcitriol levels are very low.

B. REGULATION OF METABOLISM and synthesis. Like other steroid hormones, calcitriol is subject to strict feedback regulation (Fig. 47.4 and Table 47.1). In intact animals, low dietary calcium and hypocalcemia cause a significant increase in 1α-hydroxylase activity. This effect is mediated by PTH, which is also released in response to hypocalcemia. The role of PTH in this process is not yet clear, but it has been established that it stimulates 1α-hydroxylase activity in both vitamin D-deficient animals and animals receiving vitamin D. Dietary phosphorus deficiency and hypophosphatemia also induce 1α-hydroxylase activity, but appear to be weaker stimuli than hypocalcemia.

Calcitriol is an important regulator of its own production. Elevated calcitriol levels inhibit renal 1α-hydroxylase and activate 24-hydroxylase synthesis, leading to the formation of a byproduct, 24,25-(OH)₂-D₃, which appears to lack biological activity. Estrogens, progesterones, and androgens significantly increase 1α-hydroxylase levels in laying (ovulating) birds. The role of these hormones (along with Insulin, Growth Hormone, and prolactin) in calcitriol synthesis in mammals remains unclear.

Table 47.1. Regulation of renal 1α-hydroxylase

The sterol backbone that forms The basis of calcitriol can undergo modifications in alternative metabolic pathways, specifically being hydroxylated at positions 1, 23, 24, 25, and 26 to form various lactones. Over 20 metabolites have been discovered, but biological activity has not been unequivocally proven for any of them.

Mechanism of Action

The cellular action of calcitriol is analogous to that of other steroid hormones (Fig. 47.5). Studies using radioactive calcitriol have shown that it accumulates in the nuclei of intestinal villus and crypt cells, as well as osteoblasts and distal renal tubular cells. In addition, it has been found in the nuclei of cells not previously suspected of being calcitriol target cells, namely Cells of the epidermal Malpighian layer and pancreatic islets of Langerhans, certain Brain cells, and certain cells of the pituitary, Ovaries, Testes, placenta, Uterus, Mammary Glands, Thymus, and myeloid progenitor cells. Calcitriol binding has also been detected in parathyroid cells, which is extremely interesting as it indicates the potential involvement of calcitriol in the regulation of PTH metabolism.

A. Calcitriol receptor. A protein with a Molecular Weight of 90,000–100,000 present in intestinal cells binds calcitriol with high affinity and low capacity. Binding is saturable, specific, and reversible. Thus, this protein meets the basic criteria characterizing a receptor; it has been found in many of the tissues listed above. When physiological salt concentrations are used in the assay, the majority of the unoccupied receptor is found in The Nucleus in a Chromatin-bound state. This is similar to the localization of the receptors for progesterone and T₃, if not all steroid hormones. It remains unclear whether preliminary activation of the calcitriol-receptor complex is required for chromatin binding, as occurs with typical steroid-receptor complexes.

B. Calcitriol-dependent Gene products. As has been known for A number of years, alterations in transport processes in intestinal cells in response to calcitriol addition require RNA and Protein Synthesis. Studies demonstrating the nuclear binding of calcitriol receptors to chromatin suggested that calcitriol stimulates gene Transcription and the formation of specific mRNAs. Indeed, one such example has been identified, namely the induction of mRNA encoding calcium-binding protein (CaBP).

Fig. 47.5. Calcitriol (C) Functions similarly to other steroid hormones. It induces gene products that facilitate calcium Transport from the intestinal lumen into the extracellular fluid. CaBP — calcium-binding protein.

Several cytosolic Proteins bind Ca²⁺ with high affinity. Some of these belong to the group of calcitriol-dependent proteins. This group includes several proteins that differ in molecular weight, antigenicity, and tissue origin (intestine, skin, bone). Of these proteins, intestinal CaBP has been best studied. In vitamin D-deficient rats, CaBP is virtually absent in such cells; overall, CaBP concentration correlates strongly with The amount of nuclear-localized calcitriol.

C. Effect of calcitriol on the intestinal mucosa. The transport of Ca²⁺ and PO₄^3- across the intestinal mucosa requires 1) uptake and transfer across the brush border and microvillar membrane, 2) Transport Across the mucosal cell membrane, and 3) extrusion across the basolateral membrane into the ECF. It is clear that calcitriol activates one or more of these steps, but the specific mechanism of its action has not been established. It was hypothesized that CaBP is directly involved in this process, but it was subsequently shown that Ca²⁺ transport occurs 1–2 hours after calcitriol administration, i.e., long before the increase in CaBP concentration in response to calcitriol. It is likely that CaBP, by binding Ca²⁺, protects mucosal cells during periods of Active Transport of this ion. Some researchers continue to search for proteins that may be involved in Ca²⁺ transport, while others believe that this process, especially the initial increase in Ca²⁺ flux, may be mediated by Changes in membrane potential. The role of polyphosphoinositide metabolites is also being discussed.

D. Effect of calcitriol on other tissues. Much less is known about the action of calcitriol on other tissues. Its nuclear receptors have been identified in bone cells, and it has been shown that the calcitriol-induced increase in Ca²⁺ concentration is coupled with RNA and Protein synthesis. However, the gene products putatively induced by calcitriol have not been identified; nor is the mechanism linking calcitriol and PTH in their action on bone cells known.

A fascinating indication of the role of calcitriol in Cell Differentiation comes from studies demonstrating that this hormone promotes The conversion of promyelocytic leukemia cells into macrophages. Since osteoclasts are believed to be either closely related to macrophages or directly derived from them, it is quite likely that calcitriol participates in this process by promoting bone cell differentiation.

Pathophysiology

Rickets is a childhood disease characterized by low plasma calcium and phosphate levels and impaired bone mineralization, leading to skeletal deformities. Most commonly, rickets is caused by Vitamin D deficiency. There are two types of hereditary vitamin D-dependent rickets. Type I is caused by an autosomal recessive gene that determines the impaired conversion of 25-OH-D₃ into calcitriol. Type II represents an autosomal recessive defect apparently characterized by the absence of calcitriol receptors.

In adults, vitamin D deficiency causes osteomalacia. This condition is marked by a decrease in both the absorption of calcium and phosphate and the levels of these ions in the ECF. As a result, osteoid mineralization and Bone Formation are impaired; this inadequate bone mineralization leads to structural weakness. When a significant portion of the renal parenchyma is damaged by a pathological process or lost, calcitriol production declines, leading to a corresponding decrease in calcium absorption. The subsequent hypocalcemia triggers a compensatory increase in PTH secretion, which acts on Bone tissue to increase Ca²⁺ levels in the ECF. This is accompanied by intense bone remodeling and structural changes, giving rise to the symptoms of the disorder known as Renal osteodystrophy. Timely, early-stage treatment with vitamin D helps mitigate the severity of the disease.