Cellular physiology

General Principles:

Body fluid compartments –

• Extracellular fluid (ECF) = 1/3 total body water, ~20% body weight
  • Interstitial fluid “bathes cells” (15% body weight)
  • Plasma (5% body weight) + cellular components (3% body weight)  = total blood volume (8% body weight)
• Intracellular fluid (ICF) = 2/3 total body water, 40% body weight

Body fluid volumes –

• Plasma volume = 5% x body weight (70kg adult) = 3500mL
• Total blood volume = plasma volume x (100/(100-haematocrit))
• ECF volume = difficult to measure, ill defined
  • Transcellular fluids = CSF, joint/ eye fluids
• Interstitial volume = ECF – plasma volume

Intracellular fluid volume = TBW – ECF

Units for measuring concentration of solutes

• Moles = gram molecular weight of a substance = 6×10²³ molecules(Avogadro’s no)
• Equivalent (1 Eq) = 1 mol of ionised substance divided  by its valence
• Water is ideal solvent for physiological reactions

pH

• Negative log of [H+]
• pH of water at 25 degrees = H+ and OH- are present in equal numbers, is 7.0
• Each pH unit less than 7.0 -> [H+] increased ten fold

Buffers

• Maintain ECF/ ICF pH = substance which can bind/ release H+ in solution
• Carbonic acid (H2CO3), blood and cellular proteins

Diffusion

• Process by which gas or substance in solution expands to fill available volume
• Particles dissolved in solvent are in continuous random movement
• Will move from area of higher concentration -> lower concentration
• Fick’s law of diffusion:
  • Magnitude of diffusing tendency directly proportional to cross sectional area and concentration gradient
  • J = -DA (Change in c / Change in x)
  • J (net area of diffusion), D (diffusion coefficient), A (area), concentration gradient (Change in c/ Change in x) which will be a negative value

Osmosis

• Diffusion of solvent molecules (eg. water) into region of higher solute concentration (eg. glucose) to which the membrane is impermeable
• Pressure necessary to prevent solvent migration = osmotic pressure
• Concentration of osmotically active particles = osmoles
• Osmolarity is number of osmoles/ L
  • Affected by volume of solute in solution and temperature
• Osmolality is number of osmoles/ kg of solvent

Tonicity

• Osmolality of a solution relative to plasma
• Isotonic = same osmolality, Hypertonic = greater, Hypotonic = lesser
• Solutions can change their tonicity, if solutes diffuse/ are metabolised
  • 0.9% NaCl remains isotonic as no net movement of particles
  • 5% glucose is isotonic -> hypotonic, as glucose is metabolised
• Substances contributing to osmolality = 285 mmol/kg
• Normal serum osmolality = Na+, Cl-, HCO3-, proteins, glucose
• Osmolality (msom/L) = 2[Na+] mEq/L + 0.055 [glucose] mg/dL + 0.36 [BUN] mg/dL
• Cells swell when exposed to extracellular hypotonicity
  • Swelling activates channels = increased efflux of K+, Cl- and organic osmolytes = water follows = cell returns to normal
• Cells shrink when exposed to extracellular hypertonicity
• Non ionic diffusion = Molecules of undissociated substance diffuse from one side of membrane to the other, then dissociate

Donnan Effect

• In the presence of a non-diffusible ion, the diffusible ions distribute themselves so that at equilibrium, their concentration ratios are equal – Gibbs Donnan Equation:
  • [K+_x] [Cl-_x] = [K+_y] [Cl-_y]
• For any pair of cations and anions of same valence
• Three effects in body:
  • Na+/K+ ATPase pumps ions out of cells (3 x Na+ OUT for 3x K+ IN), preventing rupture = normal cell volume
  • Asymmetric ion distribution across membrane = electrical difference
  • Plasma proteins > interstitial proteins, Donnan effect on ion movement across capillary wall

Forces acting on ions

• Concentration gradient
• Electrical gradient
• Equilibrium = influx and efflux are equal
• Equilibrium potential = membrane potential at which this equilibrium exists
• Nernst Equation: E_cl = 61.5 log [Cl_i-]/[Cl_o-] at 37 degrees
• Eg. Cl – has equilibrium potential of -70mV, Na + has equilibrium potential of +60mV (moving inward)

Parts of eukaryotic cells

Structure and function of DNA and RNA

• DNA = two long nucleotide chains containing bases adenine (A), guanine (G), thymine (T) and cytosine (C), bound together by hydrogen bonding  (A-T, C-G) with deoxyribose
• Double helical structure, 3×10⁹ base pairs
• “Genetic blueprint” -> message transcribed to ribosomes via RNA -> proteins
• RNA = single stranded, uracil (U) in place of thymine with ribose
• Mutations = alterations in base DNA sequence

DNA polymorphism

• Exons = protein coding portions of genes (3% human gene)
• Introns = “junk DNA” (97%)
• DNA has structural variability for each individual -> restriction fragment length polymorphism (RFLP) is in effect a DNA fingerprint -> can be replicated by PCR

Mitosis = somatic cell division process

• Two DNA chains separate, each serving as a template for new complementary chain
• DNA polymerase catalyses reaction
• Daughter cell has same DNA as parent cell

Telomeres = ends of chromosomes

• Reverse transcriptase synthesises short repeats of DNA, characterise ends (telomeres) of DNA
• High telomerase activity -> multiply more eg. Cancer cells

Meiosis = reduction division of germ cells

• One of each pair of chromosomes ends up inside mature germ cell
• Sperm unites with ovum = complete set
• “Ploidy” = number of chromosomes in cells -> euploid are normal resting diploid cells, tetraploid is just before division and aneuploidy is haploid/ multiple of haploid chromosomes (cancer cells)

Cell cycle

Initiate mitosis and cell division, four phases: G1 – S – G2 – M

1. G1: cell increases in size and duplicates cellular contents
2. S: DNA replication
3. G2: more growth, organelles and proteins develop for cell division
4. M: Mitosis and cytokinesis (cell separation)

• Check points to ensure DNA is correct
• Regulated by proteins called cyclins and cyclin dependent kinases

Protein synthesis: Transcription and translation

• mRNA strand formed from DNA strand = transcription, catalysed by RNA polymerase from 5’ to 3’ end
• Post transcriptional processing
• mRNA moves to cytoplasm and dictates formation of polypeptide chain of protein = translation (in ribosomes)
  • Gene made up of:
    • Exons – segments dictate formation of proteins
    • Introns – segments not translated, eliminated by spliceosomes or self splicing
    • TATA sequence = site where RNA polymerase binds, to ensure transcription starts at correct point
    • CAAT sequence = basal promotor region
  • RNA transcript capped by 7-methylguanosine triphosphate to 5’ end -> needed for binding to ribosome at 40S subunit site
  • Amino acids in cytoplasm are activated with enzyme + adenosine monophosphate ->combines with transfer RNA
  • tRNA- amino acid complex attaches to polypeptide chain at 60S subunit site
  • Genetic code made up of triplets, each standing for amino acid
  • Starts with AUG triplet = methionine
• Post translation modification: protein folding, secretion via exocytosis

Apoptosis

• Programmed cell death
• Activation of caspases -> DNA fragmentation, chromatin and cytoplasmic condensation and membrane bleb formation -> cell breakup
• Triggered by different stimuli:
  • Fas (membrane protein) from natural killer cells and T lymphocytes
  • TNF
• Mitochondria: cytochrome C release + smac/ DIABLO protein -> facilitate caspase activation

Molecular medicine

• CF – defectively regulated Cl- channel
• Huntington’s – unstable trinucleotide repeats
• Cancer – oncogenes + proto-oncogenes causing somatic mutation, including of tumour suppressor genes such as p53 on Chromosome 17

Transport across cell membranes

• Exocytosis = vesicles containing material for export bond to cell membrane via v-SNARE/t-SNARE arrangement -> area of fusion breaks down -> contents of vesicles expelled
  • Ca2+ dependent process
• Endocytosis = reverse exocytosis

• Phagocytosis (cell eating)
  • Bacteria/ dead tissue/ microscopic material engulfed by cell
  • Makes contact with cell membrane -> invaginates and is pinched off -> engulfed material inside vacuole inside cell
  • Pinocytosis (cell drinking)
    • Similar process to above
    • Substances ingested are in solution and not visible under microscope
  • Clathrin mediated: protein accumulates and surround vesicle ->  dynamin pinches off vesicle ->  early to late endosome ->  lysosome

• Rafts = areas of cell membrane rich in cholesterol and shingolipids, precursors of caveolae = depressions infiltrated with protein called caveolin

Membrane permeability and transport proteins: ways in which substances are transported

• Ion channels – open or gated (voltage vs. ligand which can be internal or external)
  • Epithelial sodium channels (ENaCs) eg. In kidney, are inhibited by amiloride
  • Cl- channels
  • Subject to channelopathies
• Carriers – bind ions/ molecules and change configuration
• When molecules move down chemical or electrical gradients, no energy is required = facilitated diffusion
• When molecules move against their gradients = active transport
• Carrier molecules are ATP-ases = enzymes that catalyse hydrolysis of ATP
• Na+/K+ ATP-ase:
  • Catalyses hydrolysis of ATP to ADP, uses energy to move 3x Na+ OUT of cell and 2x K+ INTO cell

• Na+ binds to alpha subunit, ATP binds too and is converted to ADP – causing configuration change -> Na+ pumped out, K+ binds extracellularly -> dephosphorylating alpha subunit -> previous conformation, releases K+ into cytoplasm
  • Regulated by cAMP and diacylglycerol (DAG), inhibited by ouabain
  • Increased by thyroid hormone, insulin and aldosterone
  • Decreased by dopamine
• Uniports (1 substance) vs symports (binding of >1 substance, transferred together) vs antiports (exchange 1 substance for another)
• Secondary active transport = active transport of Na+ coupled with other substances
  • Intestinal mucosal cells, symport for Na+ and glucose
  • Cardiac myocytes, antiport for intracellular Ca2+ for extracellular Na+

The Capillary Wall

• Separates plasma from interstitial fluid
• Relies on pressure difference for filtration
• Impermeable to colloid proteins, cannot pass through junctions in large quantities
• Oncotic pressure = plasma colloid osmotic pressure ~ 25mmHg (HOLDS IN)
• Hydrostatic pressure = opposes oncotic pressure (PUSHES OUT)
• Transcytosis = proteins transported out of capillaries via vesicles into cells

Intracellular Communication

• Chemical messengers = amines, amino acids, steroids, polypeptides, lipids, nucleotides
• 5 general types:
  • Neural  – neurotransmitter released at synaptic junction and act on post synaptic cell
  • Endocrine – hormones reach cells via circulating blood
  • Paracrine/ autocrine – cell products diffuse into ECF to affect itself/ neighbouring cells
  • Gap junctions – directly from cell to cell
  • Juxtacrine – express proteins (eg. TGFa) extracellularly to provide anchor to other cells

• Radioimmunoassay = technique to measure messengers in body fluids, naturally occurring ligand competes with radioactive ligand to bind to ligand Ab (if natural ligand > radioactive ligand, latter will bind less to the Ab)
• Receptors:
  • Hormone/ neurotransmitter present in excess, number of R decreases (down regulation) – receptor mediated endocytosis
  • Presence of deficiency, number of R increases (up regulation)
  •  EXCEPTION: Angiotensin II actions on adrenal cortex = increases R on adrenal
• Mechanisms:
  • First messengers = extracellular ligands -> initiate release of second messengers via membrane R and GTP- binding proteins
  • Second messengers = intracellular ligands -> altering cell function via activation of protein kinase -> catalyse phosphorylation

• Intracellular Ca2+:
  • Regulates proliferation, neural signalling, contraction, secretion, fertilisation
  • Free Ca2+ in cytoplasm ~100 nmol/L, 12 000x in interstitial fluid
  • Inward concentration and electrical gradient
  • Stored in ER and mitochondria -> mobilised via ligand gated channels -> icreased cytoplasmic Ca2+ binds to calcium binding proteins (CaBP) -> activates protein kinases -> physiologic effects
  • Ca2+ enters cells via voltage/ligand gated channels/ SOCCs
  • Transported out of cell by Ca2+/H+ ATP-ase (1 Ca2+ for 2 H+) and Na+/Ca2+ antiport (1 Ca2+ for 3 Na+)
• Calcium binding proteins:
  • Troponin (contraction of skeletal muscle)
  • Calmodulin -> can activate 5 different kinases:
    • Myosin light chain kinase -> smooth muscle contraction
    • Calcineurin -> inactive Ca2+ channels (activating T cells, inhibited by immunosuppressants)
  • Calbindin (synaptic function)
• G proteins
  • Nucleotide regulatory proteins that bind GTP, guanosine analog of ATP
  • Signal reaches G protein -> nucleotide exchange GDP for GTP via GTP-ase -> effect
  • Small G proteins – cellular function, rate of vesicle traffic
  • Larger heterotrimeric G proteins – intracellular formation of second messengers/ couple R to ion channels
  • Serpentine R = G protein coupled receptors (span membrane 7 times)
• Second messengers:
  • Inositol triphosphate (IP3) – triggers release Ca2+ from ER
  • Diacylglycerol (DAG) – activates protein kinase C
  • Cyclic AMP (cAMP) – formed from ATP by action of adenylyl cyclase -> activates protein kinase A -> catalyses phosphorylation of proteins, is metabolised by phosphodiesterase.
    • Cholera and pertussis toxin – increased adenylyl cyclase activity
  • Cyclic GMP (cGMP) – formation catalysed by guanylyl cyclase, important for vision in rods/ cones
  • Phosphatases – remove phosphate groups, associated with tyrosine kinase

• Growth factors
  • Multiplication/ cell development = nerve growth factor, insulin like growth factor (IGF-1), activins + inhibins, epidermal growth factor (EGF)
    • Ligand binds to R -> tyrosine kinase autophosphorylates -> production of ras proto-oncogene + MAP kinases -> transcription factors in nucleus
  • Cytokines = produced by macrophages/ lymphocytes to regulate immune system
  • Colony stimulating factors = regulate proliferation/ maturation and RBC + WBC
    • Initiate tyrosine kinase activity in cytoplasm (Janus tyrosine kinases = JAKs) -> activate STAT proteins (signal transducer and activator of transcription)

Last Updated on July 14, 2021 by Andrew Crofton