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; andbe able to distinguish differences between regulation of Ca2+ and Pi.
4 Contents Calcium (Ca2+) Phosphate (Pi) Notion of free and bound Ca2+ Extracellular versus intracellular Ca2+Ca2+ exchanges (uptake → storage → excretion)Regulation of Ca2+Short-term: pH and its consequencesLong-termNature of the Ca2+-sensorHormonal control of Ca2+ homeostasisPhosphate (Pi)Free and boundRegulation 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.
7 Ca2+ Balance Homeostasis is result of uptake (absorption),deposition and resorption (bone),secretion and excretion, andexchange 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 viaIon channelsVoltage-dependent, non-selective cation, store-operated and ligand-gated ion channelsTransporters (reversed transport in pathology)Outflow via transportersCa-ATPaseNa/Ca-exchanger (3 Na+ against 1 Ca2+)Sequestration within cellSmooth endoplasmic reticulumMitochondriaCalcicosomesFixed and soluble buffers (Ca2+-binding proteins)
9 Ca2+ Deposition and Resorption Deposition/accretion via osteoblasts into boneResorption via osteoclasts from boneTightly controlled by hormonesGuarantees a rapidly exchangeable pool (4 g out of recently made-up bone)BoneInorganic minerals %HydroxyapatiteOrganic part (matrix) %Collagen
10 Dietary Uptake Demand: 1 g / d 25 mmol / d Demand dependent on age and gravidityChildren: g / dAdolescent: g / dPregnancy: g / dLactation: g / dResorption: %* *demand limited;also depends on Ca2+-complexes formed/presented in GI tract (oxalic acid, phytin)pHSource:Predominantly dairy products (milk, cheese, etc.)
11 Mechanism of Dietary Uptake Boron/Boulpaep, 2003Two 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 isparacellularTAL (driven by voltage).transcellularCa-ATPaseNa/Ca-exchangerResorption is hormonally controlled (see later).
13 Calcium RegulationShort-term: pH (faster than regulatory hormonal changes can act…) and its consequences.Long-termCa2+ sensing on cell membrane.hormonal feedback-loops for Ca2+ homeostasis.
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 Sensor of Extracellular [Ca2+] Hormonal Ca2+ Homeostasis Long-Term RegulationSensor 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, IP3Adenylyl cyclase (inhibits)MAPKinaseRegulated processesSecretion (hormones - relevant here…)Synaptic plasticity, memory formationProliferation, differentiation, apoptosisGene expressionDiseasesRod and cone diseases (phosphorylation of rhodopsin)Up-regulated in bipolar disease and schizophrenia/autism
19 Parathyroid Hormone: [Ca2+]↑ Peptide (84 AA) from parathyroid glandSecretion “threshold”: < 1.5 mM (i.e. normally, very little PTH is secreted).Ca-sensor transduction: via phospholipase C → IP3 production AND adenylyl cyclaseTarget organs:Bone: osteoclasts (resorption)Kidney: accelerates vitamin D synthesisIndirectly: Gut, kidney (reabsorption, loss of phosphate…)Harper et al. 1979
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
23 Phosphate (HPO42-; Pi)Largest amount intracellularly: ~ 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 mM85 – 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/Pi supersaturation, no precipitation in tissue (pyrophosphate is one of many inhibitors of precipitation).Regulation tightly linked to Ca2+ becauseof bone (large deposit): hydroxyapatite, andPi regulation is linked to PTH, calcitriol and calcitonin (little!).
24 Pi Balance Pi homeostasis is result of Mostly from dairy products. amount of Pi in the body (bones); anddistribution 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/Pi symporter.Excretion primarily via urine.
25 Targets of Pi Regulation Regulation by maximal renal reabsorption capacity.Pi excess: rate of renal excretion↑; andPi 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↓.BoneResorption at the level of osteoclast (PTH, calcitriol)Formation at the level of osteoblast (calcitonin)GutEnterocyte in duodenum/jejunum (calcitriol)
26 Pi Homeostasis PTH (most important): [Pi]↓ Calcitriol: [Pi]↑ increases bone resorptionincreases renal filtrationCalcitriol: [Pi]↑increases gut absorptionincreases renal reabsorptionCalcitonin (transient; least important): [Pi]↓increases bone formationOther hormones:growth hormone: Pi↑ in childrenglucocorticoids: Pi excretion↑Not in line with Ca2+ homeostasis (more complex).
27 Take-Home MessagesCa2+ is central to extra- and intracellular signalling.[Ca2+]e determines 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 MCQJohn 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 concentrationElevated dependent on calcium concentrationLowered dependent on calcium concentrationLowered independent of calcium concentrationElevated independent of calcium concentration
30 MCQJohn 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 concentrationElevated dependent on calcium concentrationLowered dependent on calcium concentrationLowered independent of calcium concentrationElevated independent of calcium concentration