From Cells to Whole Animals:

1
From Cells to Whole Animals Responses to
Extracellular Osmolarity Changes or Why the Beer
Comes Out So Quickly Myles Akabas
2
Initial Cellular Responses to Osmolarity Changes
Difference in water concentration across the cell
membrane is the driving force for net water
movement.
3
How Do Differences in Osmotic Pressure Make Water
Move? or The Mysterious Osmotic Force Demystified
Vant Hoff Equation for Osmotic Pressure ( ? ) ?
RTcsolute
There is no mysterious force, osmotic pressure.
It is simply water moving down its concentration
gradient.
100 mM NaCl
10 mM NaCl
55.54 M H2O
55.45 M H2O
H2O
water moves from low osmotic pressure to high
osmotic pressure water moves from high
hydrostatic pressure to low hydrostatic pressure
4
Bacterial Responses to Hypotonic Stresses
The Problem How to survive an environmental
change from 300 mOsm/l into water (0
mOsm/l)? ?? 6 atm The Risk Cell membrane
will rupture and cell death will result. The
Solution Mechanosensitive channels that open
rapidly and release small solutes (proline,
sugars, salts) and small proteins (thioredoxin,
etc.). These channels must be tightly closed to
prevent spontaneous depolarization of the cell
membrane but must react within seconds to
tension in the membrane to avoid cell
rupture. Bacteria express 3 distinct types of
mechanosensitive channels MscL, MscS,
MscM High resolution structures are available
for MscL, MscS No known eukaryotic homologues of
these proteins
5
Science (1998) 2822220-2226
6
Science (1998) 2822220-2226
7
Science (1998) 2822220-2226
8
Cell Volume is a Tightly Regulated Parameter
Cells regulate their size and volume. Within an
organ similar types of cells will have similar
size. Mechanisms by which cells determine their
size (volume) are poorly understood.
Alberts et al. Molecular Biology of the Cell, 3rd
Ed., Fig. 20-4
9
Cells evolved to regulate their own size/volume.
In multicellular organisms cells assume
extracellular osmolarity will be constant.
Given a constant extracellular osmolarity, the
major determinant of cell volume is the number of
osmotically active particles within the cell.
UNKNOWN How do cells determine their size/volume
as they grow and know when to stop growing?
This is a distinct issue from detecting volume
changes.
10
Regulatory Volume Decrease in Eukaryotic
Cells Response to Hypotonic Osmotic Shock
Okada, Y. et al. (2001) J. Physiol. 5323-16
11
Mechanisms of Regulatory Volume Decrease Vary
Among Different Cells
P2Y2 Receptor
ATP
Ca2
ATP
?
Swelling- Induced ATP release
Anions
K
Volume- Sensitive Anion Channel (VSOC)
Sensory mechanisms by which cells detect swelling
are unclear. ? Membrane tension ? Interactions
with cytoskeleton
12
Two Important Issues in Regulatory Volume Decrease

• How do cells detect cell swelling? What are the
  sensory mechanisms?
• How is the efflux mechanism regulated to turn it
  on and off at the
• appropriate times?
• second messenger systems
• interactions with sensory systems

13
Regulatory Volume Increase Response to Cell
Shrinkage By Hypertonic Solutions Mechanisms
Differ Among Cells
2Cl-
Na Cl-
Na K
Cl-
Na
H
HCO3-
14
Commonly Used Intravenous Solutions
Normal body osmolarity 290 300 mOsm/l
Normal Saline NS 0.9 NaCl (0.9 g/100 ml)
154 mM NaCl 5 Dextrose D5W 278 mOsm/l ½
Normal Saline 0.45 NaCl 77 mM NaCl Lactated
Ringers Solution (in mEq/l) 130 Na, 4 K, 2.7
Ca2, 109 Cl-, 28 lactate, 273
mOsm/l, pH 6.5
15
Effect of Osmolarity Changes on Water
Distribution Between Body Compartments
Normal Body Osmolarity 290 300 mOsm Total Body
Water (TBW) 2/3 Body Weight (2/3)70 kg 46
L
Intracellular Volume (2/3 TBW) 31 L
Cell Membrane
Extracellular Volume (1/3 TBW) 15 L
Vascular Endothelium
Interstitial Volume (2/3 ECV) 10 L
Plasma Volume (1/3 ECV) 5 L
16
Differences in Permeability Properties of the
Cell Membrane and the Vascular Endothelium
Cell Membrane Permeable to water urea Impe
rmeable to NaCl sugars proteins
Vascular Endothelium Permeable
to water urea NaCl sugars Impermeable
to proteins
17
What happens if we administer 3 liters of Normal
Saline (Isotonic) (0.9 NaCl) to a 70 kg person?
Where will the volume go?
18
What happens if we administer 3 liters of Normal
Saline (isotonic) (0.9 NaCl) to a 70 kg person?
Where will the volume go?
extracellular volume overload
Does the body osmolarity change?
19
What happens if we administer 3 liters of water
to a 70 kg person? Into which body compartments
will the water go? What is the final osmolarity
of body fluids?
20
What happens if we administer 3 liters of water
to a 70 kg person? Into which body compartments
will the water go? What is the final osmolarity
of body fluids?
total body volume overload
(Osmfinal)(Volumefinal) (Osminitial)(Volumeini
tial)
(Osmfinal) (Osminitial)(Volumeinitial)/(Volumef
inal) (300 mOsm/L)46 L/49
L 282 mOsm/L
21
What happens if a 70 kg person runs a marathon on
a hot day, does not drink and looses 3 liters of
sweat? From which body compartments will the
fluid come? What happens to total body osmolarity?
22
What happens if a 70 kg person runs a marathon on
a hot day, does not drink and looses 3 liters of
sweat? From which body compartments will the
fluid come? What happens to total body osmolarity?
29 L
2 L
9.33 L
0.67 L
4.67 L
0.33 L
Final
total body volume depletion
(Osmfinal)(Volumefinal) (Osminitial)(Volumeini
tial)
(Osmfinal) (Osminitial)(Volumeinitial)/(Volumef
inal) (300 mOsm/L)46 L/43
L 321 mOsm/L
23
How to Maintain Water Balance
Water sources Water losses
Water sources Metabolic water (200-400 ml/day)
CHO H2O CO2 100 g fat 100 ml 100 g
CHO 60 ml 100 g protein 45 ml Solid
Food (800-1000 ml/day) Fluid Intake (500-1500
ml/day)
Water losses Insensible Respiratory (500-1000
ml/day) variable depends on activity,
temp. Stool (50-200 ml/day) up to 10-15 L/day
with secretory diarrhea Sweat (up to 10 L/day)
depends on temp., humidity, activity Urine (600
ml/day minimum) depends on intake and losses
24
How does the Body Respond to Changes in
Osmolarity? Where are the Osmoreceptor Cells?
Hypothalamus Supraoptic and Paraventricular
Nuclei Magnocellular Neurosecretory Cells axons
extend into the posterior pituitary, no
Blood-Brain Barrier secrete Anti-Diuretic Hormone
(ADH), Vasopressin
Berne Levy Physiology 3rd Ed, p 759
25
How does the Body Respond to Changes in
Osmolarity? Where are the Osmoreceptor Cells?
Hypothalamus Supraoptic and Paraventricular
Nuclei Magnocellular Neurosecretory Cells axons
extend into the posterior pituitary Posterior
Pituitary no blood brain barrier fenestrated
capillaries allow secreted peptide hormones
to enter circulation secrete Anti-Diuretic
Hormone (ADH), Vasopressin
Brenner Rector, The Kidney 3rd Ed. p. 390
26
Structure of Anti-Diuretic Hormone (ADH),
Vasopressin
Brenner Rector, The Kidney 3rd Ed. p. 389
27
ADH is Synthesized as a PreProHormone and
Matures by Proteolytic Cleavage
Goodman Gilman Pharmacological Basis of
Theraputics 9th Ed. p. 719
28
Patch Clamp Recording A Brief Digression on How
to Observe Single Molecules in Action
open
closed
29
Patch Clamp Recording from Hypothalamic
Osmoreceptor Cells
Bourque Oliet (1997) Annu. Rev. Physiol.
59601-619
30
Relationship Between Plasma Osmolarity and Plasma
ADH
Brenner Rector, The Kidney 3rd Ed. p. 392
31
Blood Volume Also Affects ADH Secretion
Blood Volume (or Pressure), but only if gt 10
decrease
Baroreceptors located in left atria, pulmonary
vessels aortic arch, carotid sinus Information
integrated in Nucleus Tractus Solitarius (NTS) in
brain stem
Berne Levy Physiology 3rd Ed, p 760-761
32
Other Factors That Affect ADH Secretion
Stimulate ADH secretion nausea angiotensin
II nicotine Inhibit ADH secretion atrial
natriuretic peptide (ANP, ANF) ethanol, this is
why you only borrow the beer for a short time
33
Effects of ADH/Vasopressin Mediated by Two
Classes of Receptors
Receptors are members of the 7 membrane-spanning
segment, G-protein coupled receptor gene
superfamily Vasopressin V1 Receptors widely
expressed vascular smooth muscle
cells neurons in central nervous system
coupled to regulation of intracellular calcium
concentration mediate vasoconstrictor and
thirst-inducing effects of ADH Vasopressin V2
Receptors principal cells in kidney collecting
duct activate adenylate cyclase and increase
cAMP mediate water reabsorption and urine
concentrating effects
34
Systemic Effects of ADH Secretion

• Kidney
• ?water perm. collecting duct
• ?urea permeability of inner medullary collecting
  duct
• ?NaCl reabsorption by ascending limb of loop of
  Henle
• ?water reabsorption
• ?urine osmolarity
• ?urine volume
• Vascular system
• ?vasoconstriction
• ?vascular volume
• ?blood pressure
• Brain
• ?thirst

?medullary osmotic gradient
Berne Levy Physiology 3rd Ed, p 759
35
ADH Mediated Effects in the Collecting Duct
Principal Cells
Goodman Gilman Pharmacological Basis of
Theraputics 9th Ed. p. 723
36
Tubular Water Reabsorption in the Absence of
ADH How to Make Large Volumes of Dilute Urine
70 of volume 126 L/day
Filtered 180 L/day
300 mOsm
50 mOsm
140 mM NaCl
Cortex 300 mOsm
NaCl
H2O
20 of volume 36 L/day
Medulla 1200 mOsm 300 mM NaCl 600 mM Urea
NaCl
1200 mOsm, 560 mM NaCl
18 L/day, 25-50 mOsm
37
Tubular Water Reabsorption in the Presence of
ADH How to Make Small Volumes of Concentrated
Urine
NaCl
70 of volume 126 L/day
Filtered 180 L/day
300 mOsm
H2O
50 mOsm
8 of volume 15 L/day
140 mM NaCl
Cortex 300 mOsm
300 mOsm
3 L
1.5 of volume 2.3 L/day
H2O
20 of volume 36 L/day
urea
Medulla 1200 mOsm 300 mM NaCl 600 mM Urea
NaCl
1200 mOsm, 560 mM NaCl
0.6 L/day, 1200 mOsm
38
Other Issues
Countercurrent multiplier Vasa recta provide
nutrients and O2 to medullary cells role in
removal of reabsorbed H2O and NaCl from the
medulla How is the medullary osmotic gradient
first formed? role of the ascending limb of the
loop of Henle and active NaCl transport Medullary
osmotic gradient is not static magnitude
changes ADH angiotensin II stimulate increase
in medullary osmotic gradient by increasing
NaCl reabsorption in ascending limb
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Glomerular Filtration Rate Creatinine Clearance
Rate of filtration is the most important
determinant of renal function Urine output is not
a measure of renal function no normal urine
output Urine output water intake minus water
losses Clinical measure of GFR endogenous
substance freely filtered and then neither
secreted or reabsorbed Amount filtered Amount
excreted Creatinine produced by non-enzymatic
hydrolysis of creatine in muscle amount produced
daily proportional to muscle mass GFRCrplasma
UVCrurine GFR UVCrurine/Crplasma