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SAN BEDA COLLEGE OF MEDICINE Cellular Transport and Signaling BY Julianne Lopez, MD-MBA

SAN BEDA COLLEGE OF MEDICINE of 6 Female Reproductive Physiology BY Julianne Lopez, MD-MBA

Cellular Transport and Signaling by Julianne Lopez, MD-‐MBA [email protected] 09178520849

Cellular Transport and Signaling EDUCATIONAL OBJECTIVES At the end of the 4-‐hour lecture, the future Bedan Doctor must be able to: • Illustrate the fluid-‐mosaic model of cell membranes • Describe the types of membrane transport.

o Differentiate between active and passive transport. o Describe features of the types of passive transport and

give their mechanisms. ! Simple diffusion ! Facilitated diffusion ! Osmosis

o Name and give features of the types of active transport. ! Primary active transport ! Secondary active transport

o Explain, using specific examples, the difference between primary and secondary active transport.

o Explain the importance and characteristics of carrier-‐mediated transport. ! Stereospecificity ! Competition ! Saturation

• Describe the modes of cell communication and signaling. o Describe the mechanisms of cellular communication

and regulation. ! Endocrine ! Neurocrine ! Paracrine ! Autocrine ! Juxtacrine

o Discuss the role of receptor proteins in cell signaling. o Name the types of signal transduction pathways.

! G-‐Protein mediated ! Second Messenger – dependent ion channels ! Second Messenger – dependent protein kinases

• Calmodulin dependent protein kinases • Cyclic AMP – dependent protein kinases • Atrial Natriuretic Peptide Receptor – guanylyl

cyclases

Cellular Membranes Cellular membranes (structure and composition) -‐ also called plasma membrane -‐ a thin, pliable, elastic structure that envelops the cell -‐ 7.5 to 10 nm thick -‐ composition:

FLUID MOSAIC MODEL:

-‐ primarily composed of a ___________ and ___________ -‐ phospholipid BILAYER

-‐ Mosaic " within the phospholipid bilayer are many

different types of embedded proteins and cholesterol molecules

-‐ Fluid "the embedded molecules can move sideways throughout the membrane, meaning the membrane is not solid, but more like fluid o Fluidity of the membrane is determined by

temperature and lipid composition ! Temperature: the higher the temperature, the

more fluid the membrane ! Lipid composition: presence of unsaturated

fatty acyl chains (double bond " “kink”) increases membrane fuidity

LIPID component of cell membrane: -‐ lipid bilayer -‐ composed of phospholipid molecules (major lipids of the

plasma membrane) -‐ phospholipids = ______________ backbone (hydrophilic,

charged, polar head) + 2 ___________ (hydrophobic, uncharged, nonpolar tail) o because it has a hydrophilic and hydrophobic end, a

phospholipid molecule is considered ___________ -‐ impermeable to water soluble substances

(_________________________________) -‐ permeable to lipid soluble substances

(_________________________________) -‐ cholesterol molecules is a critical component of the bilayer

o also amphipathic o contribute to the fluidity of the membrane o prevents the hydrophobic chains from packing too

closely together o stabilize the membrane at normal body temperature

-‐ glycolipids

o 2 fatty acyl chains linked to polar head groups that consist of carbohydrates

o also amphiphatic

55% 25%

13% 4% 3% protein

phospholipids

cholesterol

other lipids

carbohydrates



Protein Component of Cell membranes -‐ may either be integral or peripheral Integral membrane proteins -‐ embedded in, and anchored to, the cell membrane by

hydrophobic interactions -‐ some integral proteins are ______________________ (span the

lipid bilayer one or more times), thus are in contact with both ECF and ICF

-‐ some integral proteins are embedded but do not span it -‐ examples: ligand-‐binding receptors (for hormones or

neurotransmitters), transport proteins (i.e. Na+-‐K+ ATPase), pores, ion channels, G protein-‐coupled receptors

Peripheral membrane proteins -‐ are not embedded in the membrane -‐ are not covalently bound to cell membrane components

(loosely attached by _________________________________) -‐ attached to either the intracellular or extracellular side of

the cell membrane -‐ example: ankyrin – a peripheral membrane protein that

anchors the sytoskeleton of red blood cells to an integral membrane transport protein, the CL-‐HCO3 exchanger

Transport Across Membranes General Overview:

Notes:

Simple diffusion:

-‐ process by which molecules move spontaneously from an

area of high concentration to one of low concentration -‐ occurs as a result of random thermal motion of molecules

-‐ only form of transport that is NOT carrier-‐mediated -‐ occurs down an electrochemical gradient (“_________________”)

(meaning no energy expenditure), therefore, does NOT require metabolic energy and is considered PASSIVE

-‐ can be measured using the following equation (assuming a nonelectrolyte it is uncharged):

J = -‐PA (C1 – C2)

Where: J = flux (flow) (mmol/sec) P = permeability (cm/sec)

A = area (cm2) C1= concentration1 (mmol/L) C2= concentration2 (mmol/L)

-‐ Concentration Gradient (C1 – C2)

o The __________________________________ across the membrane is the DRIVING FORCE for net diffusion

o The larger the difference in solute concentration

between the 2, the greater the driving force, greater net diffusion

o What happens if 2 solutions are equal? Answer:

-‐ Permeability: describes the ease with which a solute diffuses through a membrane

P = KD Δx

Where:

transport across

membranes

simple diffusion

carrier-‐mediated transport

facilitated diffusion

active transport

primary secondary



P = permeability K = partition coefficient D = Diffusion coefficient X = membrane thickness

Factors affecting permeability:

Partition coefficient (K) o by definition, describes the solubility of a solute in oil

relative to its solubility in water ! the greater the relative solubility in oil, the higher

the partition coefficient, the more easily the solute can dissolve in the cell membrane’s lipid bilayer

o Predict: ! Non polar solutes: soluble in oil " _________________

_________________ ! Polar solutes: insoluble in oil

__________________________________ o Can be measured by adding the solute to a mixture of

olive oil and water then measuring its concentration in the oil phase relative to its concentration in water

K = concentration in olive oil concentration in water

Diffusion coefficient (D) o it is defined by the stokes-‐einstein equation

D = KT 6πrη

Where:

D = diffusion coefficient K = Boltzmann’s constant

o it correlates _________________with the molecular radius of the solute and viscosity of the medium ! Predict:

Small solutes " _________________diffusion coefficients " __________________________________ Viscous solutions " _________________ diffusion coefficients " __________________________________

Membrane thickness (x) o the thicker the cell membrane, the greater the distance

the solute must diffuse and the lower the rate of diffusion, therefore, membrane thickness and rate of diffusion is _________________________________________________

-‐ Surface Area (A) o The greater the SA, the _________________ the rate of

diffusion, therefore, it is _________________ proportional to one another

Diffusion of electrolytes -‐ implications:

1. The potential difference across the membrane will alter the rate of diffusion of a charged solute / electrolyte

2. When a charged solute diffuses down a concentration gradient (from higher concentration to lower concentration) that diffusion can itself generate a potential difference across a membrane called __________________________________

Carrier Mediated Transport -‐ facilitated diffusion, primary active transport and secondary

active transport all involve integral membrane proteins and are considered carrier-‐mediated

-‐ characteristics of carrier-‐mediated transport: 1. Saturation o based on the concept that carrier proteins have a

limited number of binding sites for the solute

o ex: glucose transport in the proximal tubule of kidney 2. Stereospecific o binding sites for solute on the transport proteins are

stereospecific o can distinguish between isomers o in contrast, simple diffusion, does not distinguish

between isomers 3. Competition o can recognize, bind, and transport chemically related

solutes o ex: D-‐glucose vs D-‐galactose (competitive inhibitor of

D-‐glucose) Facilitated Diffusion: -‐ characteristics:

o occurs down an electrochemical gradient (downhill), similar to simple diffusion

o no input of metabolic energy, therefore is passive

o at low solute concentration " more rapid than simple

diffusion o at high solute concentration " carriers can become

saturated and facilitated diffusion will level off (the rate of diffusion approaches a maximum, Vmax. It cannot rise greater than Vmax level)

o is carrier-‐mediated, therefore, exhibits stereospecificity, saturation and competition

-‐ example: GLUT 4 transporter o responsible for the insulin-‐mediated transport of D-‐

glucose from the bloodstream to the skeletal muscles and adipose tissue

o competitive inhibitors: D-‐galactose, 3-‐O-‐methyl glucose, phlorizin

o L-‐glucose (non-‐physiologic stereoisomer) is NOT recognized by GLUT-‐4

Primary Active transport: -‐ moved against an electrochemical potential gradient (uphill)

" solute is moved from an area of low concentration to an area of high contentration

-‐ there is input of metabolic energy in the form of ATP -‐ when directly coupled to the transport process " primary

active transport -‐ examples:



__________________________________ o main function: maintaining concentration gradients for

Na and K across cell membranes (goal: low intracellular Na and high intracellular K)

o present in the membranes of ALL cells o 3 Na+ out (ICF " ECF) & 2 K+ in (ECF " ICF) o because of this stoichiometry, more positive charge is

pumped out of the cell than is pumped into the cell (making ICF more negative) ! sub-‐units:

• alpha sub-‐units o contains ATPase activity and binding

sites of transported ions • beta-‐sub units

o has 2 states: ! E1 state: binding site for Na and K face the ICF;

enzyme has high affinity for Na ! E2 state: binding site for Na and K face the ECF;

enzyme has high affinity for K ! The cycling between the 2 states is powered by

ATP hydrolysis

check p. 9 for words ★ Clinical correlation: _________________________________ (i.e. ouabain / digitalis) are a class of drugs that inhibits Na+-‐K+ ATPase by binding to the E2 ~ P form near the K binding site on ECF side (preventing conversion back to E1) Result: _________________ intracellular Na and _________________ intracellular K __________________________________

o Main function: extrude 1 Ca2+ (for 1 ATP) from the cell (ICF " ECF) against an electrochemical gradient, which is responsible for maintaining a low intracellular Ca2+

o Present in the membrane of MOST cells o SERCA (___________________________________________________)

! Is a variant of Ca2+ ATPase found in sarcoplasmic reticulum of muscles and endoplasmic reticulum of other cells

! Function: 2 ions Ca2+ (for 1 ATP) from ICF to the interior of sarcoplasmic or endoplasmic reticulum

__________________________________

o Found in the _________________ of gastric mucosa ! Pumps H+ from ICF of pariental cells into the

lumen of stomach " acidifies gastric contents o Also found in the ______________________________ of renal

collecting duct

★ Clinical correlation: ___________________________________________________, an inhibitor of gastric H+–K+ ATPase can be used therapeutically to reduce the secretion of H+ in the treatment of peptic ulcer disease Secondary Active Transport -‐ transport of 2 or more solutes is coupled

o one of the solutes, usually _________________moves _________________ its electrochemical gradient, and the other solute moves _________________ its electrochemical gradient

o downhill movement of Na+ provides the energy for the uphill movement of the other solute

o indirect input of metabolic energy Physio Pearl: Inhibition of _________________ (i.e. by treatment of _________________), diminished the transport of Na+ from ICF to ECF, causing the intracellular Na+ concentration to increase " _________________ the size of the transmembrane gradient Implication: indirectly, all secondary active transport processes are diminished by inhibitors of the Na+-‐K+ ATPase because their energy source, the Na+ gradient, is diminished. -‐ 2 types of secondary active transport:

o Cotransport or symport o Countertransport, antiport or exchange

Cotransport -‐ form of secondary active transport in which all solutes are

transported in the_________________ direction across the cell membrane

-‐ examples:

__________________________________

o found in the absorbing epithelia of small intestine and

renal proximal tubule o both solutes are required for transport

__________________________________ -‐ present in the luminal membrane of epithelial cells of the

thick ascending limb of the Loop of Henle -‐ Countertransport (antiport or exchange) -‐ form of active transport in which solutes move in

_________________ direction across the cell membrane -‐ example:

__________________________________ __________________________________ Type of transport

Active or Passive

Carrier-‐Mediated

Uses metabolic energy

Dependent on Na+ gradient

Simple diffusion Passive; downhill

No No No

Facilitated diffusion

Passive; downhill

Yes No No

Primary Active Transport

Active; uphill

Yes Yes; direct No

Cotransport Secondary Active*

Yes Yes; indirect

Yes (solutes move in SAME direction as Na across cell



membrane) Countertransport Secondary

Active* Yes Yes;

indirect Yes (solutes move in OPPOSITE direction as Na across cell membrane)

*Na+ is transported downhill and one or more solutes are transported uphill

Osmosis -‐ The process of net movement of water through a selective

membrane caused by a concentration

Basic Concepts in Osmosis SOLUTION -‐ homogeneous mixture composed of two or more substances

o homogenous means that components and properties of the mixture are uniform throughout its entire volume

-‐ solute is the substance dissolved -‐ solvent is the substance that dissolves the solute OSMOLARITY -‐ concentration of all osmotically active particles (osmoles)

per liter of solution (osmol/L) -‐ colligative property that can be measured by freezing point

depression

OSMOLALITY -‐ concentration of all osmotically active particles (osmoles)

per kilogram of solvent (osmol/kg) -‐ determines osmotic pressure between solutions ISOSMOTIC -‐ two solutions that have the same osmolarity HYPEROSMOTIC -‐ solution with the higher osmolarity HYPOSMOTIC -‐ solution with the lower osmolarity

Osmotic Pressure -‐ The exact amount of pressure required to stop osmosis -‐ pressure which needs to be applied to a solution to prevent

the inward flow of water across a semipermeable membrane -‐ calculated using van’t Hoff’s law or Morse law physiologic implications: -‐ osmotic pressure is HIGHER with higher osmolality -‐ osmotic pressure is HIGHER with higher temperature -‐ the higher the osmotic pressure of a solution, the greater the

tendency for water to flow into the solution TONICITY -‐ measure of the osmotic pressure of two solutions separated

by a semipermeable membrane -‐ influenced only by solutes that cannot cross the membrane

Cell Communication and Signaling Signaling Pathways

! multiple, hierarchical steps ! amplification of the hormone-‐receptor binding event ! activation of multiple pathways and regulation of

multiple cellular functions ! antagonism by constitutive and regulated feedback

mechanisms, Signaling Molecules

! peptides and proteins Iinsulin) ! catecholamines (epinephrine and norepinephrine) ! steroid hormones (aldosterone, estrogen) ! iodothyronines ! eicosanoids (prostaglandins, leukotrienes,

thromboxanes, and prostacyclins) ! small molecules

– amino acids, nucleotides, ions (e.g., Ca++), and gases, such as nitric oxide (NO) and carbon dioxide (CO2

Mechanisms of Cellular Communication

! endocrine ! neurocrine ! paracrine ! autocrine ! juxtacrine

Endocrine Signaling ! transport of hormones along the blood stream to a

distant target organ ! EXAMPLES:

– thyroid hormone – insulin – glucagon

Neurocrine Signaling ! also called synaptic transmission ! transport of neuro-‐transmitters from a presynaptic cell

to a postsynaptic cell ! EXAMPLE:

– neuromuscular junction Paracrine Signaling

! release and diffusion of local hormones with regulatory action on neighboring target cells

! EXAMPLE: – testosterone in spermatogenesis – retinoic acid on retina

Autocrine Signaling ! a cell secretes hormones or chemical messengers that

binds to the same cell ! EXAMPLE:

– IL-‐1 on monocytes Juxtacrine Signaling

! also called contact-‐dependent signaling ! transmitted via oligosaccharide, lipid or protein

components of a cell membrane ! occurs between adjacent cells linked by gap junctions

A SOLVENT (water) undergoes osmosis from an area of low solute concentration to an area of high solute concentration. A SOLUTE undergoes diffusion from an area of high solute concentration to an area of low solute concentration.

OSMOLARITY ≠ TONICITY OSMOLARITY accounts for all solutes.

TONICITY accounts for only non-permeating solutes



Receptors ! Signal transducers ! Membrane receptors ! Nuclear receptors

Plasma Membrane Receptors

! Ion-‐channel linked ! G-‐protein coupled receptors ! Catalytic receptors ! Transmembrane receptors

Nuclear Receptors ! Long biological half-‐life ! Diffuse across the plasma membrane ! Bind to nuclear receptors ! hormone-‐receptor complex binds to DNA and regulates

the transcription of specific genes ! Early primary response -‐-‐" gene activation to stimulate

other genes " biological effect Signal Transduction

! Second messengers ! Involves small molecules in complicated networks

within the cell ! Signal amplification ! Molecular switches

Types of Signal Transduction Pathways ! g proteins ! ion channels ! protein kinases

– calmodulin-‐dependent protein kinases – camp-‐dependent protein kinases – anp-‐guanylyl cyclases

Ion Channel Linked Signal Transduction Pathway ! mediate direct and rapid synaptic signaling between

electrically excitable cells ! neurotransmitters bind to the receptors and either

open or close the ion channel ! Chemical "electrical signal" response ! Examples: Voltage gated-‐channels in NMJ, ryanodine

receptor, Arachidonic acid, caffeine G Protein-‐Coupled Signal Transduction Pathways

! Heterotimeric complexes -‐ α, β, and γ subunits ! are linked with more than 1000 diferent receptors ! family of integral transmembrane proteins that possess

seven transmembrane domains ! EXAMPLES:

! adrenergic receptors ! chemokine receptors ! TSH receptors

! Active ! GTP with alpha subunit ! Activation via guanine nucleotide exchange

factors (GEFs) [facilitates the dissociation of GDP and binding of GTP]

! Inactive ! GDP ! Inactivation via GTPase-‐accelerating

proteins (GAPS) and RGS proteins (regulation of G protein signaling),

! Desensitization and endocytic removal (β-‐arrestins )

! Signals ! adenylyl cyclase, phosphodiesterases, and

phospholipases Catalytic Receptors

! Many, many, many types! ! Receptor gunaylyl cyclases

– Atrial natriuretic peptide and NO ! Threonine/serine kinases

– Transforming growth factor Beta ! Tyrosine kinases

– insulin Protein Kinases

! kinase is an enzyme that modifies other proteins by phosphorylation

! phosphorylation usually results in a functional change of the target protein

– changing enzyme activity – modifying cellular location – associating with other proteins

Calmodulin-‐dependent Protein Kinases

! calcium (Ca2+) binding causes conformational alterations in calmodulin

– binds to and regulates other signaling proteins (cAMP phosphodiesterase)

– binds to calmodulin-‐dependent protein kinases

• phosphorylates specific serine and threonine residues in many proteins (myosin light-‐chain kinase)

! EXAMPLE: muscle cells cGMP-‐dependent Kinases

! binding of ANP causes dimerization and activation of guanylyl cyclase, which metabolizes GTP to cGMP

! cGMP activates cGMP-‐dependent protein kinases – phosphorylates proteins on specific serine and

threonine residues – in the kidney, ANP inhibits sodium and water

reabsorption by the collecting duct ! EXAMPLES: ANP, NO

SOURCES:

1. Guyton & Hall Textbook of Medical Physiology 12th Edition by Hall, John &, Guyton, Arthur C. , , Published in Philadelphia, Pensylvania: Saunders/Elsevier, 2011

2. Berne & Levy Physiology 6th Edition bby Berne, Robert M., 1918-‐2001., Koeppen, Bruce M., Published: Philadelphia : Mosby/Elsevier, 2008

3. BRS Physiology 5th Edition by Linda Constanzo, 2011, Published: Lippincott and Williams & Wilkins

4. Harper’s Illustrated Biochemistry 27th Edition by Murray, Robert K. by Lange

5. Basic and Clinical Pharmacology 11th Edition by by Katzung, Bertram G. , Published: New York : McGraw-‐Hill Medical, 2009

6. Various Internet Websites

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