Calcium regulation

• Overview of uses of calcium in body

  Intracellular - Excitation-contraction coupling, synaptic transmission, intracellular second messenger in many processes (cell division, muscle contraction, cell motility, membrane trafficking, exocytosis, activation of SNARE proteins)

  Extracellular - blood clotting (platelet aggregation), mechanical strength bone
• What levels are calcium held at in the body?

  Calcium concentration very tightly regulated - free plasma calcium (available) is 1.2 mM, total plasma calcium is 2.4 mM - 50% free calcium, 40% reversibly bound to protein, 10% bound to citrate and phosphate

  Roughly 1:10,000 ratio of intracellular:extracellular calcium

  99% of calcium stored in the skeleton (hydroxyapatite bound to collagen), 1% in the ECF, 0.1% in the ICF

  25 moles (1kg) of calcium stored
• Effects of hypocalcaemia, and one potential cause

  When <1.2 mM total plasma calcium, can be due to secondary hyperparathyroidism due to chronic kidney disease

  Very dangerous

  Decrease in the threshold potential at the NMJ (increase in Nav1.4 activity in skeletal muscle, hypersensitive), leading to nerve hyperexcitability, rapid firing of action potentials, muscle tetany, as well as reduced calcium for muscle relaxation - extremely dangerous, can lead to asphyxiation if closure of vocal cords,
• Causes and effects of hypercalcaemia

  Can be due to primary hyperparathyroidism due to multiple endocrine neoplasia

  General repression of nervous system - decreased excitability of nerves - calcium salts fairly insoluble so urinary stones and tissue calcification, presents as fractured bones, renal stones, phychic moans, and abdominal groans - dangerous in the long term

  Secretion of calcium in urine causes polyuria, so dehydration, exacerbates problem

  >3 mM causes renal failure, >4 mM cardiac failure
• Effect of protein concentration on plasma calcium

  Multiple myeloma = tumour of plasma cells in bone marrow, causes excess myeloma protein (abnormal immunoglobulin fragment, not normal serum albumin), so hypocalcaemia

  Nephrotic syndrome (final stage diabetes) - loss of protein via damaged glomeruli, so hypercalcaemia, so fall in plasma colloidal osmotic pressure, reduced fluid retention in capillaries (reduced reabsorption), stimulation of renin-angiotensin-aldosterone system, upregulation of sodium and water, generalised peripheral oedema created
• Effect of pH on plasma calcium?

  Alkalosis - increase negative charge of plasma proteins, more calcium bound to protein, hypocalcaemia, hypocalcaemic tetany caused

  Acidosis - decrease negative charge on plasma proteins, less calcium bound to protein, hypercalcaemia

  Plasma proteins are attracted to but not bound to calcium in the same way as calmodulin or calsequestrin (bind calcium ions covalently)
• Draw diagram showing calcium turnover and the various hormones involved

  Hormones pull in different organ systems to help with maintenance:

  
• Role of PTH in calcium regulation?

  Parathyroid hormone mobilises free calcium, principal control of plasma calcium - guards against hypocalcaemia - released when drop in calcium, acts rapidly (restores calcium concentration within an hour), half life of a few minutes

  Drives calcium homeostasis in two ways - activates VitD3 (hydroxylates at first position), which has long lasting effect (around for a few days), also drives reabsorption of calcium in the kidney, activates osteoclasts, PTH sensitive calcium pumps inserted in the bone so can mobilise free calcium stored in bone fluid
• Role of Calcitonin in calcium regulation?

  Reduces plasma calcium concentration, helping with hypercalcaemia
• List the 4 main hormones influencing calcium levels

  Vitamin D (and its metabolites), parathyroid hormone, calcitonin, FGF-23 (for phosphate homeostasis)
• Overview of bone as a calcium store

  Major store of body calcium as hydroxyapatite, but also stored in bone as exchangeable non-crystalline calcium salts, and as calcium bone fluid (both of these can be rapidly mobilised in hypocalcaemia)

  Dynamic tissue - bone cells (osteoblasts form, osteoclasts absorb, osteocytes regulate) play role in formation, maintenance, remodelling of bone

  Bone formation stimulate by mechanical stress (exercise), loss caused by disuse (illness, immobilisation, space etc)
• Describe the osteoblast and clast feedback loops

  Osteoclast maturation and differentiation driven by RANK activation by RANKL (RANK ligand) - made by osteoblasts, binds to receptor and activates clasts

  Intermittent and chronic activation of bone turnover by PTH - PTH activates osteoblasts for normal bone turnover, directly regulating calcium

  Hyperparathyroidism causes chronic activation PTH receptor, overactivation of osteoclasts, outstrips activity of osteoblasts so more bone excavated, osteoporosis caused
• Normal bone turnover rates?

  Occurs throughout life, mechanism not well understood

  Reabsorption via osteoclasts within 3 weeks, formation by osteoblasts 3-4 months

  5-10% of skeleton replaced a year due to remodelling, entire skeleton replaced ~every 10 years

  Don't know how a site in need of remodelling is identified
• Osteoporosis causes?

  When activity of clasts outstrips that of blasts (more bone excavation)

  Can be caused by deficits of activity of oestrogen and testosterone on osteoblasts and precursors (decrease in osteoblastic activity)
• Mechanism of coupling between bone reabsorption and formation?

  New bone at bone resorption sites in each BRC, in bone matrix TGF-beta1 and IGF1 act as primary coupling factors (released following clast bone reabsorption), induce blast migration so new bone formation and resorption spatiotemporally coupled (matricellular signalling)

  RANK-RANKL mediates communication, induces clast progenitor differentiation

  Sema4D from clasts regulates blast differentiation, production of Sema4D stimulated by increase in osteoblastic RANKL, Sema4D inhibits blast differentiation - negative feedback loop
• Effects of PTH on bone remodelling

  Intermittent PTH essential for normal bone turnover, activates clasts (too much means osteoporosis), stimulates blast proliferation - osteoblasts express PTH1R, triggers RANKL release, which activates clasts by binding to the RANK receptor

  PTH1R activation also stimulates blasts to reorganises to allow calcium ion efflux from the matrix, and access to clasts
• Effect of PTH on calcium in the kidney

  Increases calcium reabsorption in the distal tubule via insertion of active calcium transporters
• Pathology of PTH

  Deficiency of PTH can be due to damage in thyroid surgery, receptor deficit etc - causes low plasma calcium and tetany

  Excess PTH from parathyroid tumours - causes bone destruction, high plasma calcium, urinary stones, slow CNS
• VitD overview?

  Made by the skin in the sun, changes 7-dehydrocholesterol to cholecalciferol, hydroxylated at the 25th position in the liver, pre-VitD3 stored in liver, when needed it travels to the kidney and is hydroxylated on the first position to activate under the control of PTH

  Active VitD3 increases the absorption of calcium in the gut - binds to receptors in the nucleus of cells, drive transcription of calcium binding proteins within an hour - transcellular reabsorption system via TRPv6 and PMCA (very little absorbed paracellularly)
• Vitamin 1,25 D3 pathology

  Rickets caused either by lack of VitD3 (renal rickets when renal failure, or osteomalcia when lack of minerals) or due to VitD3 resistance (renal stones, VotD3 poisoning), causes normal bone turnover but lack of calcium to calcify the collagen in bones, making bone flexible
• Effect of VitD3 on PTH

  Inhibits PTH synthesis - increased serum calcium inhibits PTH secretion, activation of CaSR by increase in calcium concentration causes generation of AA metabolites, inhibit PTH release, also causes increased expression of VDR, increasing cell's sensitivity to negative feedback from VitD - means VitD suppresses synthesis of PTH - negative feedback loop
• How is calcium sensed?

  GPCR located in the parathyroid gland, distal tubule of kidney, gut enterocytes, osteoblasts

  Increased plasma calcium activates GPCR, inhibits adenylate cyclase (Gi pathway, reduces cAMP) and stimulates phospholipase C (Gq pathway, via IP3), inhibiting PTH secretion via two different pathways - activation of receptor prevents hormone release

  Fall in plasma calcium thus causes secretion of PTH as no activation PLC, reduced inhibition of AC so high cAMP
• What are calcimimetics?

  Positive allosteric modulators of CaSR, increase sensitivity to extracellular calcium, used to treat hyperparathyroidism
• What is only hormone in body that exocytosis of is inhibited by external calcium, internal calcium independent

  PTH - everything else is exocytosed in response to increase in intracellular calcium

  Due to calcium ion dependent synthesis of AA inhibiting PTH release
• Role of calcitonin in calcium regulation?

  Calcitonin is a peptide hormone released from C cells in the thyroid following raised plasma calcium, involved in the prevention of hypercalcaemia and excess bone breakdown

  cAMP-linked receptors

  Inhibits the clast breakdown of bone, increases blast activity, with the greatest effect in the young (when resorption rapid)

  In the gut it helps to control the rise in calcium following meals by inhibiting absorption

  It has a very brief half life

  Opposite effects to PTH
• What hormones affect calcium during lactation in a mother?

  Prolactin - natural contraceptive during lactation

  PTHrP - mobilises maternal calcium for the baby, stimulates calcium uptake via VitD3

  Calcitonin - protects against excessive calcium loss

  GnRH, FSH, and LH all act to control the release of these hormones
• Overview phosphate homeostasis

  Maintenance of phosphate poorly understood, not strictly regulated (serum phosphate lowers blood VitD3, VitD3 increases renal retention phosphate)

  0.2mM plasma (varies with age, sex, diet, pH), 10% protein bound

  85% in skeleton as hydroxyapatite, can be released

  X-linked phosphataemia = phosphate wasting syndrome

  FGF23 secreted by osteocytes in hypercalcaemia, inhibits VitD3 in the kidney and thyroid, explains effect of serum phosphate on VitD3, mutated in autosomal dominant hypophosphatemic rickets, kidney loses phosphate, inhibits PTH transcription
• Draw feedback loops for VitD regulation

  VitD regulation by different pathways

  
• Relationship between VitD3, PTH, and FGF23

  Inverse relationship between phosphate and 1,25VitD3

  FGF23 and PTH both promote phosphate excretion by kidney

  VitD3 enhances reabsorption of phosphate in the kidneys

  VitD3 main role is to promote mineralisation of new bone, so increases calcium and phosphate absorption

  PTH acts to prevent hypocalcaemia, so promotes phosphate loss
• Causes of secondary hyperparathyroidism

  Declining kidney function, impaired phosphate excretion, failure to bioactivate VitD (calcitriol)
• Draw diagram of regulation of calcium and phosphate by PTH, VitD, and FGF23

  Overview of mechanisms:

  
• Difference between osteoporosis, rickets, osteomalacia, PrimHyperPara, SecHyperPara?

  Osteoporosis + PrimHyperPara - decrease bone formation (reduced blast activity), well calcified bone

  VitD deficiency + Osteocmalcia + Rickets - normal bone turnover but bone matrix cannot calcify

  Osteomalacia + Osteoporosis (=SecHyperPara) - impaired bone turnover and calcification