THE AUSTRALIAN NATIONAL UNIVERSITY

1. THE AUSTRALIAN NATIONAL UNIVERSITY Calcium and Phosphate HomeostasisChristian StrickerAssociate Professor for Systems PhysiologyANUMS/JCSMR - ANUChristian.Stricker@anu.edu.auhttp://stricker.jcsmr.anu.edu.au/Ca&Pi.pptx

3. Aims The students should • appreciate the physiological role of extracellular Ca2+; • know the principles of storage of Ca2+ in different compartments; • be able to explain the major factors determining extracellular Ca2+; • recognize principles involved in Ca2+ and Pi homeostasis via hormones; • understand how extracellular Ca2+ in sensed and signalled; and • be able to distinguish differences between regulation of Ca2+ and Pi.

4. Contents • Calcium (Ca2+) • Notion of free and bound Ca2+ • Extracellular versus intracellular Ca2+ • Ca2+ exchanges (uptake→storage→excretion) • Regulation of Ca2+ • Short-term: pH and its consequences • Long-term • Nature of the Ca2+-sensor • Hormonal control of Ca2+homeostasis • Phosphate (Pi) • Free and bound • Regulation of Pi

5. Total & “Free” Extracellular Ca2+ • Non-filterable = protein bound (“albumin”) = cannot permeate through filter pores = non-diffusible = biologically not available (on normal time scale). • Filterable = can permeate filter pores = diffusible; only some Ca2+ can be excreted. • The absolute numbers might vary by a few percentages depending on source.

6. Extra- and Intracellular Ca2+

7. Ca2+ Balance • Homeostasis is result of • uptake (absorption), • deposition and resorption (bone), • secretion and excretion, and • exchange between ICF ↔ ISF. • Ca2+taken up in proximal part of small bowel (duodenum > jejunum). • Excretion primarily via urine (kidney). • Constant exchange between ICF and ISF as well as ISF and bone (no net change).

8. Exchange between ECF and ICF • Influx into cell via • Ion channels • Voltage-dependent, non-selective cation, store-operated and ligand-gated ion channels • Transporters (reversed transport in pathology) • Outflow via transporters • Ca-ATPase • Na/Ca-exchanger (3 Na+against 1 Ca2+) • Sequestration within cell • Smooth endoplasmic reticulum • Mitochondria • Calcicosomes • Fixed and soluble buffers (Ca2+-binding proteins)

9. Ca2+ Deposition and Resorption • Deposition/accretion via osteoblasts into bone • Resorption via osteoclasts from bone • Tightly controlled by hormones • Guarantees a rapidly exchangeable pool (4 g out of recently made-up bone) • Bone • Inorganic minerals 70% • Hydroxyapatite • Organic part (matrix) 30% • Collagen

10. Dietary Uptake • Demand: 1 g / d 25 mmol / d • Demand dependent on age and gravidity • Children: 1.2 g / d • Adolescent: 1.0 g / d • Pregnancy: 1.5 - 2.5 g / d • Lactation: 1.5 - 2.5 g / d • Resorption: 25 - 40%**demand limited; • also depends on Ca2+-complexes formed/presented in GI tract (oxalic acid, phytin) • pH • Source: • Predominantly dairy products (milk, cheese, etc.)

11. Mechanism of Dietary Uptake Boron/Boulpaep, 2003 • Two pathways: Paracellular and transcellular. • Paracellular: Uptake from ISF into blood via diffusion through vessel fenestrations. • Transcellular: • Requires a luminal Ca2+ channel and intracellular binding to a Ca2+-binding protein. • Rate limiting step is into ISF; Ca-ATPase and/or Na/Ca-exchanger. • Calcitriol (“vitamin D”) can speed up resorption rate (via protein expression).

12. Excretion of Ca2+ • Kidney is organ for Ca2+ net-excretion. • Only 65% of Ca2+ can be filtered (“protein free” fraction). • 98% of filtered Ca2+resorbed. • 2% of Ca2+excreted. • Mechanism of resorption is • paracellular • TAL (driven by voltage). • transcellular • Ca-ATPase • Na/Ca-exchanger • Resorption is hormonally controlled (see later).

13. Calcium Regulation • Short-term: pH (faster than regulatory hormonal changes can act…) and its consequences. • Long-term • Ca2+sensing on cell membrane. • hormonal feedback-loops for Ca2+ homeostasis.

14. Short-Term Regulation pH and Its Consequences

15. pH and [Ca2+]: Competition • Acidosis → total Ca2+↑: Ca2+ unbinds not only from plasma, but also from protein on endothelial membrane/sub-endothelial space (significant volume). • pH also affects solubility products (Ca-phosphates, -carbonates, etc).

16. Effects of [Ca2+] Change • György’s formula for serum electrolytes: • Describes qualitatively excitability of neuromuscular system; i.e. tendency for tetanic reactions (cramps).(tetanus = steady muscle contraction without distinct twitching) • Examples: • [K+]↑(hyperkalaemia): tendency for tetanus since excitability increases (depolarisation of membrane potential). • [Ca2+]↓(hypocalcaemia): tendency for tetanus since excitability increases. • [H+]↓(alkalosis): tendency for tetanus since excitability increased (respiratory; i.e. during hyperventilation; double whammy since [Ca2+] also drops…).

17. Long-Term Regulation Sensor of Extracellular [Ca2+] Hormonal Ca2+ Homeostasis

18. Ca2+- Sensor/Receptor (FREQ) • Gene on chromosome 9: Frequenin-like protein (FREQ) or NCS-1; GPCR – dimer. • Binds to a host of adaptor proteins. • Transduction pathways (incomplete) • Phospholipases (C, A2, D): DAG, IP3 • Adenylyl cyclase (inhibits) • MAPKinase • Regulated processes • Secretion (hormones - relevant here…) • Synaptic plasticity, memory formation • Proliferation, differentiation, apoptosis • Gene expression • Diseases • Rod and cone diseases (phosphorylation of rhodopsin) • Up-regulated in bipolar disease and schizophrenia/autism http://www.liv.ac.uk/physiology/ncs/index.html

19. Parathyroid Hormone: [Ca2+]↑ • Peptide (84 AA) from parathyroid gland • Secretion “threshold”: < 1.5 mM(i.e. normally, very little PTH is secreted). • Ca-sensor transduction: via phospholipase C→IP3 production AND adenylylcyclase • Target organs: • Bone: osteoclasts (resorption) • Kidney: accelerates vitamin D synthesis • Indirectly: Gut, kidney (reabsorption, loss of phosphate…) Harper et al. 1979

20. Calcitriol: [Ca2+]↑ • Lipophilic, steroid-like hormone • Synthesis involves 3 steps: • Skin: Calciol • Liver: 25-OH-cholecalciferol • Kidney: Calcitriol • Target organs: • Primary: gut (uptake) • Secondary: bone, kidney, placenta, breasts, hair follicles, skin, etc. • Dosage: 5 µg/d adults; 10 µg/d children. • Deficiencies: • Inadequate dietary intake. • Reduced absorption. • Insufficient sun light exposure (England 19th century; kids/adults in front of computers, hospital beds, etc…). • Reduced 1α-OH-ation (kidney disease). Despopoulos & Silbernagl 2003

21. Calcitonin: [Ca2+]↓ • Peptide hormone (32 AA) • Secreted from C-cells in the thyroid gland. • Secretion directly proportional to [Ca2+] (secreted when [Ca2+] normal). • Ca-sensor transduction: via AC→cAMP. • Target organs: • Bone: inhibits osteoclast activity. • Kidney: converts vit. D precursor to 24,25-(OH)2-cholecalciferol (inactive) → Ca2+ absorption↓. Harper et al. 1979

22. Ca2+- Regulation Overview

23. Phosphate (HPO42-; Pi) • Largest amount intracellularly: ~ 75 - 90 mM; ~ 200 g. • Extracellularly, mostly as HPO42-; H2PO4- (Pi; 25%; pH!) • Largest buffer in urine (some Pi always excreted). • Reference range (plasma): 0.8 – 1.5 mM • 85 – 90% filterable (Pi): 50% ionised; 50% complexed (Ca, Mg). • 10 – 15% protein bound (as phosphorylation products). • Less well regulated than Ca2+ (concentrations vary largely after food intake). • Despite Ca/Pisupersaturation, no precipitation in tissue (pyrophosphate is one of many inhibitors of precipitation). • Regulation tightly linked to Ca2+ because • of bone (large deposit): hydroxyapatite, and • Pi regulation is linked to PTH, calcitriol and calcitonin (little!).

24. PiBalance • Pi homeostasis is result of • amount of Pi in the body (bones); and • distribution between ICF and ECF. • Under normal conditions, Pi absorption = excretion (ss). • Constant exchange between ICF and ECF as well as ECF and bone (no net change). • Mostly from dairy products. • Absorbed in proximal small bowel (duodenum > jejunum) by Na/Pisymporter. • Excretion primarily via urine.

25. Targets of Pi Regulation • Regulation by maximal renal reabsorption capacity. • Pi excess: rate of renal excretion↑; and • Pi shortage: rate of renal excretion↓. • Volume expansion response renal excretion↑ and vice versa. • Primary targets (very similar to Ca2+) • Kidney (80% reabsorbed in PT) • Under normal conditions, Pi transport is saturated and matched to absorption: if Pi↑, more is lost than absorbed; if Pi↓, more Pi is retained. • Low levels of Pi, alkalosis and hypercalcaemia cause insertion of Na/Pi symporter into apical membrane → reabsorption↑. • High levels of Pi, acidosis and hypocalcaemia cause removal of Na/Pi symporter from apical membrane → reabsorption↓. • Bone • Resorption at the level of osteoclast (PTH, calcitriol) • Formation at the level of osteoblast (calcitonin) • Gut • Enterocyte in duodenum/jejunum (calcitriol)

26. Pi Homeostasis • PTH (most important): [Pi]↓ • increases bone resorption • increases renal filtration • Calcitriol: [Pi]↑ • increases gut absorption • increases bone resorption • increases renal reabsorption • Calcitonin (transient; least important): [Pi]↓ • increases bone formation • increases renal filtration • Other hormones: • growth hormone: Pi↑ in children • glucocorticoids: Pi excretion↑ • Not in line with Ca2+homeostasis (more complex).

27. Take-Home Messages • Ca2+ is central to extra- and intracellular signalling. • [Ca2+]edetermines excitability. • [Ca2+] is sensed via FREQ (NCS-1), which requires adaptor protein(s) for transduction. • Short-term, pH determines [Ca2+] and [Pi]. • Hormones regulate [Ca2+] and [Pi]: • PTH: [Ca2+]↑ and [Pi]↓. • Calcitriol(vit. D): [Ca2+]↑ and [Pi]↑ via uptake↑ and also [Pi]↑ via renal reabsorption↑. • Calcitonin: [Ca2+]↓and [Pi]↓.

28. MCQ John Mak, a 58 year-old male, was diagnosed with prostate cancer and multiple osteolytic lesions causing markedly increased serum calcium. Which of the following statements best describes the accompanying blood Pi concentration? • Normal independent of calcium concentration • Elevated dependent on calcium concentration • Lowered dependent on calcium concentration • Lowered independent of calcium concentration • Elevated independent of calcium concentration

29. That’s it folks…

30. MCQ John Mak, a 58 year-old male, was diagnosed with prostate cancer and multiple osteolytic lesions causing markedly increased serum calcium. Which of the following statements best describes the accompanying blood Pi concentration? • Normal independent of calcium concentration • Elevated dependent on calcium concentration • Lowered dependent on calcium concentration • Lowered independent of calcium concentration • Elevated independent of calcium concentration