Systematic Control Of Calcium And Homeostasis Maintenance Biology Essay

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Calcium, in addition to its role in maintaining bones and teeth, extracellular calcium ions in the plasma (Caex) also plays a pivotal role in maintaining blood pressure, glucose metabolism and muscle contraction. The Caex balance is mediated by calcium sensing receptor (CaR) in parathyroid glands, kidney and bones. CaR is the first G-protein coupled receptor identified which responds only to Caex and binds to Ca2+ at low affinity. As Ca2+ is the physiological agonist of the receptor, one of the mechanisms of varying parathyroid hormone secretion without affecting Caex is by influencing CaR. Calcimimetics and calcilytics do so. Calcimimetics are ligands which interact with CaR and increase its affinity for Ca2+. Cinacalcet is the first calcimimetic drug released into the market. This essay addresses the role CaR plays in monitoring Caex, the effect of calcimimetic, cinacalcet on CaR and inturn on Caex and, the potential role of CaR beyond its role in maintain Caex. (155)

Introduction

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Cacium is required for the maintenance of bones and teeth. The body also requires extracellular calcium ions in the plasma (Caex) for maintenance of blood pressure, glucose metabolism and homeostasis, transmission of nerve impulses and muscle contraction. Caex is controlled by a homeostatic system which includes parathyroid gland, kidneys, bones and intestine. The Caex in a normal individual oscillates between 1 and 1.5mM#1. An imbalance in Caex is mainly caused by chronic kidney disease (CKD) and progressive renal failure. CKD is associated with phosphate retention and impaired formation of vitamin D or calcitriol which leads to hypocalcemia, increased parathyroid hormone (PTH) secretion and hyperplasia of parathyroid gland#2. Any abnormal alternation in PTH and calcitonin secretion causes an imbalance in calcium metabolism which leads to a range of disorders as listed in table 1.

Disease

Cause

Symptoms and diagnosis

Treatment

Primary hyperparathyroidism (PHPT)

Increased plasma Ca2+ levels. No medical cause found

Mild to moderate PHPT is asymptomatic. Accelerated PHPT patients show confusion, visual disturbances, vomiting and nausea. Can be diagnosed by checking the blood pressure

Lifestyle modification

Secondary Hyperparathyroidism (SHPT)

Reduced plasma Ca2+ levels. High blood pressure because of CKD or tumors

Increased cardiovascular mortality. Renal osteodystrophy, loss of bone mineral density (BMD), bone pain and fracture. Diagnosed by measuring the blood pressure

Vit D sterols, Ca and P are traditional medical therapies.

Hypocalcemia

Increased secretion of Parathyroid hormone (PTH) due to phosphate retention, impaired formation of active vitamin D or calcitriol

Red or purple spots appear which later become rashes

Provide calcium supplements, phosphate binders and vitamin D analogues

Hypercalcemia

Hyperparathyroidism, hyperthyroidism, adrenocortical insufficiency and phaechromocytoma

Characterized by reduced responsiveness of parathyroid and kidney to calcium and by PTH-dependent hypercalcemia

Calcium salts, thiazide diuretics, vitamin A and vitamin D analogues

Table 1: Disorders caused by an imbalance in the extracellular calcium

Therefore, regulation of systemic calcium is crucial for the human body and this is controlled by parathyroid hormone. PTH in the parafollicular cells of the thyroid secretes calcitonin. Calcitonin lowers plasma calcium and phosphorus concentrations, calcium absorption from the gut and; increases urinary calcium and phosphate excretion. PTH secretions and Caex have an inverse sigmoidal relationship i.e. a decrease in Caex levels leads to an increase in PTH secretion which stimulates the Ca2+ release by the kidney and bone and vice versa. PTH directly acts on the kidney to enable Ca2+ conservation by stimulating the synthesis of 1-α-hydroxylase in the renal proximal tubule thereby increasing calcitonin production. Thus normocalcemia is restored. The initial event of stimulus secretion coupling in parathyroid cells is controlled by calcium sensing receptor (CaR). CaR is the pivotal regulator mechanism as it controls PTH secretion, calcium excretion by the kidney, bone remodeling and Ca2+ reabsorption by the gastrointestinal tract.

CaR's role in maintaining systemic calcium

The cloning of CaR from bovine parathyroid by expression cloning in Xenopus Laevis oocytes has established the molecular mechanisms involved in Caex homeostasis by PTH#3. CaR subsequently isolated from other organs like kidney, pancreatic islets and brain has provided insight into the action of CaR and Caex in these cell types. CaR, a cationic binding receptor identified by Dr. Brown, found in the surface tissues of parathyroid glands is directly involved in maintaining systemic calcium#1. It continuously regulates the PTH secretion by monitoring the calcium ion concentrations in the extracellular fluid#2.

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CaR belongs to family C of G-protein coupled receptors and calcium is the physiological agonist of the receptor. It has seven transmembrane domains, an exoplasmic N-terminal domain which is essential for ion sensing and binding ligands and a cytoplasmic loop which interacts with the G-proteins. The carboxy terminus of CaR determines receptor processing, cell-surface expression and signal transduction#1. The extracellular domain of CaR responds only to Ca2+ and binds to Ca2+ at low affinity. Unlike other members of the G-proteins, CaR's binding to Ca2+ is cooperative suggesting that CaR has many binding sites and the binding of Ca2+ at one site increases the affinity for Ca2+ at another site. The Hill coefficient lies in the range of 3-5, depending upon the assay used. CaR is already 50% active at physiological Caex and this is demonstrated in human parathyroid cells where the PTH secretion is suppressed by 50% when Caex is 1.2mM. CaR also exhibits little desensitization when subjected to prolonged exposure to Caex. #4

To date only a single CaR isoform has been isolated which activates phospholipases C, A2 and D#3. The activation of PLC is a direct G protein-mediated process. However, the activation of PLA and PLD by an increase in Caex is indirect. This involves the CaR-activated transduction pathways involving protein kinase C (PKC). Bai et al. showed that in bovine cells an increase in Caex led to the phosphorylation of PKC which made CaR less sensitive to Ca2+ resulting in a decrease in mobilizing intracellular Ca2+ (Cain) and vice versa. This suggests that PKC has inhibitory effects of Ca2+ mobilization. Riccardi suggested that CaR also mediates cross talks with other biological pathways to maintain systemic calcium concentration#1. For example, a depression in Caex leads to a depression of hormone- induced cAMP accumulation and production of arachidonic acid and its metabolites. This is sensed by CaR, and the Ca2+ in the intracellular store is mobilized and these mechanisms are shown in figure 1.

http://www3.interscience.wiley.com/cgi-bin/fulltext/122602387/nf2

Figure 1: A schematic diagram of the cell signaling mechanism of a CaR receptor on a parathyroid cell surface #2

Positional cloning has suggested that individuals heterozygous for inherited mutations in the extracellular CaR gene show hypercalcemia. Those who are homozygous for such mutations present with neonatal severe hyperparathyroidism (NSHPT) and have marked parathyroid hypercellularity#5. Mice with reduced CaR expression in the parathyroid glands, exhibited an altered PTH secretory response to serum calcium levels suggesting that the number of CaR on the parathyroid cell surface contributes importantly to the setting of Caex#6. Also, mice with inactive CaR show marked hypercalcemia due to severe hyperparathyroidism.

Therefore, drugs which influence the activity of CaR must be used to treat disorders due to Caex imbalance. Drugs traditionally used such as calcium supplements, phosphate binders and vitamin D leads to the worsening of hypercalcemia and hyperphosphatemia in the long term. This increases the risk for soft-tissue and vascular calcification without reducing the levels of PTH#6. Therefore hydrophobic, synthetic positive modulators of CaR, 'calcimimetic', and negative modulators, 'calcilytics' have been developed to cure disorders caused by failing kidneys.

Calcimimetics and calcilytics

One of the very few ways of modulating PTH secretion without changing the Caex is by using calcimimetics and calcilytics. Calcimimetics are ligands which interact with the transmembrane domains of the receptor of CaR and increase its affinity for Ca2+. The description of these two drugs is presented in the table 2.

Calcimimetics

Calcilytics

Effect on CaR

Activates parathyroid cell CaR

Block parathyroid cell CaR

Effect on PTH levels

Lower circulating levels of PTH

Stimulates PTH secretion

Treats

Treatment of primary and secondary hyperparathyroidism

Anabolic therapy for osteoporosis

Key characteristics

Type I calcimimetic

Agonists which increase the sensitivity of CaR by activating Caex. Examples: inorganic and organic polycations

Type II calcimimetics

Allosteric activators which include certain L-amino acids and phenylalkylamines. Phenylalkylamines interact with the membrane-spanning segments of the CaR and enhance signal transduction.

Calcilytics like type II calcimimetics are allosteric inactivators. They dampen the sensitivity of the Ca2+ receptor to Caex. They do not affect PTH secretion at very low or high levels of extracellular Ca2+ but they do stimulate secretion of PTH under normal calcemic conditions.

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Table 2: Table showing some key characteristics of calcilytic and calcimimetic.The first type II calcimimetic drug that was evaluated as a potential drug was the phenylalkylamine NPS R-568. It significantly decreased PTH levels in SHPT patients by activating parathyroid CaR. However, this drug had many adverse side effects.

Cinacalcet was produced because it is efficient like NPS R-568 and has improved bioavailability and metabolic properties. Cinacalcet, a stereoselective type II calcimimetic agent which belongs to the structural class of phenylalkylamines, is the first allosteric modulator of a G-protein released into the market in January 2005#7.

Cinacalcet

Cinacalcet selectively activates the parathyroid CaR and inhibits PTH secretion without increasing the calcium-phosphorus product (ca x ph). An experiment conducted by Edward on wild type human embryonic kidney (HEK 293) cells showed that cinacalcet induces an increase in intracellular Ca (Cain) in HEK 293 cells which expresses the CaR but not the wild type HEK 293 which does not have CaR's. It is presumed to induce conformational change which reduces the threshold for CaR activation by the Ca2+. Although the wild type HEK 293 cells express receptors for ATP, bradykinin and thrombin that are coupled to the mobilization of Cain , cinacalcet does not act on these receptors and it does not affect the activity of receptors which are structurally homolgous to CaR#8. Its molecular structure is presented in figure 2.

Cinacalcet hydrochloride, N-((1R)-1-(1-Naphthyl)ethyl)-3-(3-(trifluoromethyl)phenyl)propan-1-amine hydrochloride, CAS #: 364782-34-3

Figure 2: Cinacalcet HCl or N-[(1R)-1-(1-naphthyl) ethyl]-3-[3-(trifluoromethyl) phenyl] propan-1-amine #2

The inhibitory effects of cinacalcet on PTH secretion are evident in the experiment conducted by Edward. He incubated bovine PTH in a buffer containing varying amounts of Caex with or without 10 or 100mM of cinacalcet. The results for the experiment are presented in figure 3. In the control a decrease in the concentration of Caex from 0.1 to 2mM decreased PTH secretion by 80%. However, in the presence of cinacalcet HCl, the concentration response curve for Caex shown in figure 3 shifted to the left, but the magnitude of the secretory response obtained at low or high concentrations of Caex was not altered#8. It can be derived that cinacalcet causes an increase in Cain by inhibition of PTH secretion within a certain Caex range. E. F. Nemeth stated that cinacalcet does not affect cellular responses at Caex concentrations below 0.1mM and above 3mM#9. As cinacalcet has suppressive effects on bovine PTH, it suggests that it also has inhibitory effects on human PTH even at low Caex levels.

Figure 3: Effect of different concentrations of cinacalcet on PTH secretion and Caex. The graph clearly shows the sigmoid relationship of between PTH secretion and Caex. The addition of cinacalcet pushes the curve to the left showing the inhibitory effects cinacalcet has on PTH secreation. #8

Edward also showed that cinacalcet produces a concentration dependent shift in the potency of Ca2+ to stimulate calcitonin release shown in figure 4. He showed that the cells release calcitonin in response to increasing concentrations of Caex and that cinacalcet induced potentiation of Ca2+ stimulated calcitonin release. However, cinacalcet did not stimulate calcitonin release in the absence of added Ca2+ and did not increase the maximal response to Ca2+. Hence cinacalcet has been approved by the U.S. Food and Drug Administration for the treatment of SHPT in patients with chronic kidney disease on dialysis and for the treatment of hypercalcemia in patients with parathyroid carcinoma as it substantially lowers serum calcium concentration and improves ca x ph homeostasis #6.

Figure 4: Effect of cinacalcet HCl on calcitonin secretion. The figure shows that there is a linear increase in calcitonin secretion by cinacalcet HCl only in the presence of Ca2+ #8

The suppression in PTH secretion and stimulation of calcitonin by cinacalcet occurs in a dose dependent manner. In vitro studies showed that an increase in calcitonin release occurred over the same concentration range of cinacalcet HCl associated with increases in Cain and reduction in PTH secretion. However, in vivo studies revealed that the potency of cinacalcet to reduce the plasma level of PTH was considerably greater than its ability to increase calcitonin levels, even though the nucleotide sequence of the coding region of the CaR is identical in parathyroid cell and thyroid C cells. This is because cinacalcet is showing conditional efficacy and it demonstrates that the same receptor genotype can have different pharmacological phenotypes depending on the cellular environment and explains the predominant effect of PTH rather than calcitonin in vivo.

Cinacalcet HCl can significantly reduce or maintain reduction in PTH while simultaneously lowering calcium, phosphorus, and ca x ph; and in patients taking vitamin D at baseline and cinacalcet HCl had significant mean reductions in vitamin dose. The results derived from an experiment conducted by NHS where patients with end stage renal disease on dialysis were treated with cinacalcet are presented in table 3.

Percentage in 741 patients who were given 300pg/mL of cinacalcet

Patients receiving cinacalcet who achieved primary end point

43%

Patients on cinacalcet who had more than a 30% reduction in PTH levels from baseline

64%

Reduction in mean PTH levels during efficacy assessment phase

43%

Reduction in mean serum calcium, phosphorus and ca x ph product levels

6.8%, 8.4% and 14.6% respectively

Table 3: Effect of 300pg/mL cinacalcet on 741 patients with end stage renal disease on dialysis

J.V Torregrosa et al. showed that kidney transplant patients who received 60mg/d of cinacalcet exhibited increased PTH concentrations, hypercalcemia and hypophosphatemia while those who received 30mg/d of cinacalcet remained stable and had no hypercalcemia or hypophosphatemia. Patients with controlled SHPT undergoing dialysis with low-dose cinacalcet therapy have less severe hyperplasia, normal parathyroid function and recovered easily after kidney transplant. In contrast, patients who require higher dosage of cinacalcet usually have more severe SHPT that is more difficult to control after kidney transplant. Therefore, in the case of patients with SHPT undergoing dialysis and receiving cinacalcet 60mg/d or more, treatment should be continued after kidney transplant to avert serious hypercalcemia or hypophosphatemia#10.

Advantages of cinacalcet

Disadvantages of cinacalcet

Acts allosterically on its target and might have some therapeutic advantages

Ineffective at minimal or maximal levels of the physiological ligands

Prevents the development of parathyroid gland hyperplasia

Causes nausea and vomiting

Reduces serum calcium and PTH levels in patients with mild PHPT and in patients with parathyroid cancer.

Causes transient episodes of hypercalcemia is a some people

Table 4: Advantages and disadvantages of cinacalcet

Therefore, cinacalcet is used in SHPT patients undergoing dialysis as it influences PTH and calcitonin secretion by targeting CaR to bring about normocalcemia. Cinacalcet is also used in studying the function of CaR in different sites in the body. However, before the prevalence of cinacalcet the long-term effects of cinacalcet on pathological parathyroid tumors should be determined in animal models.

Initially CaR was thought to be found in parathyroid glands only, however subsequent research has identified it different cells such as kidney, intestine, bones and pancreatic cells. CaR in different cells has different functions suggesting that it has other important roles apart from maintaining systemic calcium. CaR in the kidney directly influences Ca2+ to modulate body fluid and calcium homeostasis. The CaR expressed thoughout the gastrointestinal tract regulates gastric acid secretion and modulates fluid transport in the colon. In the bone, CaR is expressed in osteoblasts where it responds to fluctuations in Caex within the physiological range by activating signaling pathways which led to prolifearation, differentiation and ultimately culminate in formation of new bone.

Expression of CaR in bones

Apart from maintaining calcium homeostasis, osteoclastic bones directly inhibit resorption and stimulate proliferation and chemotaxis of osteoblasts due to an increase in Caex. Osteoblasts obtained from CaR-null mice to sense cations and amino acids suggested that a different molecule other than the parathyroid CaR could be involved in osteoblast calcium sensing#11. It was then identified that another family GPCRC6A senses Caex at levels higher than in extracellular fluids. Comparison of GPRC6A with CaR revealed conservation of both calcium and calcimimetic binding sites. Osteocalcin, a calcium-binding protein which is highly expressed in bone, dose-dependently stimulated GPRC6. An activity in the presence of calcium but inhibited the calcium-dependent activation of CaR. These results suggested that GPCRC6A could be an osteoblast specific calcium sensor however, at present these receptors are classified as a bona fide CaR#11.

CaR in osteoblast is thought to play a key role in regulating bone turnover by stimulating the proliferation and migration of cells to sites of bone resorption as a result of local release of Caex. CaR on the surface of these tissues monitor the Caex and it is a target for the changes of Caex in the microenvironment thereby acting as a 'growth factor' in various cells residing in the bone marrow#11. The signaling pathway in the osteoblast on CaR activation involving PLC is similar to parathyroid CaR.

Monocytes-macrophages can fuse with each other and differentiate into mature functional osteoclast under specific culture conditions. The existence of the CaR expression in monocytes-macrophages raises the possibility that CaR expression could persist throughout the differentiation of monocytes-macrophages to mature osteoclast. This shows the involvement of CaR in physiological responses of Caex in the skeletal microenvironment.

Exposing the osteoclasts to millimolar levels of Ca2+ results in dramatic cell retraction followed by a profound inhibition of bone resorption. Osteoclasts first attach to mineralized surfaces and actively resorb bone, releasing calcium into the extracellular environment. The release of high concentration of Ca2+ due to resorbing activity of the osteoclasts in turn inhibits bone resorption and/or increased osteoclast retraction and detached from the bone#11.The mechanism of this feedback inhibition by calcium on osteoclast activity is not fully understood.

High Ca2+ has been shown to inhibit osteoclast like cell formation by presumably acting on the CaR present in osteoclast precursor cells. CaR expression has been reported in mature bone resorbing osteoclast isolated from rabbits as well. Evidence on CaR's role in both osteoclast differentiation and osteoclast apoptosis has been provided. CaR is also expressed in cells that are not involved in Ca2+ homeostasis such as oligodendrocytes, pancreatic cells where it is associated with diverse functions including cell proliferation and the regulation of secretion.

Expression of CaR in pancreatic cells

The islets of langerhans in the pancreatic cells are small clusters of endocrine cells that are scattered throughout the exocrine pancreas. The islet contains β-cells which secrete insulin and α-cells which secrete glucagon. The cells in the islets are joined together by gap-junction uncouplers. A homotypic interaction between the cells by autocrine and paracrine signals regulates glucose homeostasis. The loss of β-cells or failure of their secretory function results in diabetes. Isidora et al. stated that activation of CaR in the islets enhanced secretion of insulin and glucagon suggesting an important function for CaR in regulating islet secretory function#12.

CaR regulates islet function by acting as an intra-islet mechanism of homotypic communication between adjacent cells and this is done by monitoring Caex. There is a continuous change in the Caex concentration because of the influx/efflux pathways across the plasma membrane. The space between cells in the pancreatic islets is small and therefore large changes in Ca2+ occur in the microenvironment immediately surrounding cells#13. The accumulation of calcium in the extracellular region activates the CaR in neighboring cells and facilitates cellular co-operation. Figure 5 shows the extrusion of Ca2+ from stimulated cells, recruitment of Ca2+ by neighboring cells and amplification and integration of the signal to cause a tissue-wide response.

Figure 1

Figure 5: An increase in glucose increases insulin secretion in one cell which stimulates insulin secretion in other neighboring cells which express CaR.#13

An activation of CaR using calcimimetics lead to an influx of Ca2+ in the cell. This leads to a transient increase in insulin secretion. However, calcimimetic activation of CaR in autosomal-dominant hypocalcaemia causes hypocalcaemia of varying severity without hypoglycaemia. The reason for the decrease in insulin production is perhaps because of a decrease in Ca2+ influx leads to glucose-stimulated closure of ATP sensitive potassium channels on the β-cells.

The CaR is now known to be expressed in cells types which are not involved in systemic calcium homeostasis suggesting that its prime function in these cells is something other than the detection of circulation. One alternative function for the CaR may be to detect and respond to localized rather than systemic changes in Caex and the CaR has been implicated in the detection of Ca concentration in pancreatic juice and the intestinal lumen and as a mechanism through which neuronal cells are influenced by the electrical activity of their near neighbour via local changes in Caex#12. Therefore, more research must be conducted to decipher the role of CaR in monitoring Caex.

CaR activation has been associated with increased proliferation in a variety of cell types such as osteoblasts. The role played by CaR in cell proliferation in primary islets was shown by Isidora et al. He showed that reduced levels of CaR expression in insulin-expressing MIN6 cells did not affect their proliferation capacity when the cells were grown as monolayers. But, the CaR-depleted cells showed significantly reduced rates of proliferation when configured as pseudoislets. This suggests that the signaling through the CaR is involved in the regulation of cell proliferation where the cell are sufficiently close to communicate by localized release of an endogenous CaR activator but not where the cells are anatomically separate i.e. monolayers#12.

Conclusion

Caex is necessary to maintain blood pressure, glucose metabolism and muscle contraction. An imbalance in calcium homeostasis results in various disorders like SHPT, PHPT etc. Initially patients with SHPT were given vitamin D however; research has suggested that it has very little effect on the patient. Therefore, calcimimetic drugs such as cinacalcet are used. It interacts with CaR and increases its affinity for Ca2+. CaR is G-protein coupled receptor which responds only to Caex and binds to Ca2+ at low affinity. Apart from the pivotal role played by CaR in calcium homeostasis, it acts as metabolic sensors in different tissues. Recent research into CaR has suggested the role in plays in osteoblast proliferation and in cell to cell communication in islets of langerhans. However, the true potential of CaR is yet to be discovered. Therefore, the study CaR in CaR mutants and wild-types using cinacalcet HCl form an exciting area of research. (2982)