C2006/F2402 '10 Outline for Lecture 24 -- (c) 2010 D. Mowshowitz -- Lecture updated 04/26/10
Handouts From last time: 23C -- Stress Response & TK receptors
New this time: 24A -- Basic
Processes in Kidney Tubule
24B -- Kidney Structure
Recent NPR story on oxytocin: When the 'Trust Hormone' is out of balance. http://www.npr.org/templates/story/story.php?storyId=126141922
I. Circuits, cont.
A. Lactation -- done last time; see handout 23A.
B. Stress response (Handout 23C.)
1. Phase one -- Nerve (Sympathetic) activity stimulates target organs (that are not glands) → Direct response of heart, liver, lungs, etc.
2. Phase two -- Nerve (Sympathetic activity) → activation of glands
a. Stimulate pancreas → glucagon release stimulated; insulin release inhibited → secretion of glucagon → additional stimulation of some of same target organs
b. Stimulate adrenal medulla → release of epinephrine → stimulation of same targets as sympathetic nerve activity & some additional targets -- hormones can reach where nerves can't go.
3. Phase three -- stimulate HT/AP axis to produce cortisol
HT in brain → releases CRH → AP → releases ACTH → adrenal cortex → produces cortisol → target organs → stimulation of breakdown of fats & protein for energy (sparing glucose for brain); inhibition of immune system.
Note that each additional phase is slower but involves additional degrees of amplification due to second messengers, transcription, etc.
II. Signaling with RTK's. How do RTK's Work?
A. Importance of Catalytic Receptors
1. What are they? Catalytic Receptors are surface receptors whose intracellular domain is an enzymatic domain (or binds to one)
2. How do they work? Ligand binding activates the enzymatic domain. The active enzyme modifies a cellular protein, which binds to or modifies other proteins, etc.
3. Types: The enzymatic domain is usually a kinase, often a tyrosine kinase. We will stick to receptor tyrosine kinases & TK linked receptors.
4. What signaling molecules use TK receptors?
- Many growth factors (such as EGF) and other paracrines, autocrines and juxtacrines act through TK receptors.
- Insulin, PL, and GH, but not most other endocrines, act through TK receptors. (See Sadava 15.6)
5. Results: Activation of TK receptors often leads to changes in TF's and transcription. (Note this not the usual case with G-linked receptors -- their end target is usually not a TF.)
B. Important Properties of Receptor TKs -- See handout 23C & chart below.
1. Receptor is usually a single pass protein
2. Ligand binding usually leads to dimerization of receptors (Sadava fig. 15.6 or Becker 14-17). Why does it matter that TK receptor monomers (or any protein) must dimerize in order to act?
a. Function: If 1/2 the receptors (1/2 the monomers) are abnormal, most of the dimers that form are abnormal.
b. Inheritance: "lack of function" mutations in TK receptors are often dominant. (For an example see Becker figs. 14-20 & 14-21 (14-19 & 14-20). Dimers form, but they are inactive. See below for more details.
3. Dimerization activates a TK in cytoplasmic domain of receptor or in separate TK (that binds to receptor).
4. Active kinase auto-phosphorylates itself. TK adds phosphates (from ATP) to its own tyrosines -- each subunit phosphorylates the others.
5. Other proteins are activated by binding to phosphorylated TK's. (Sadava fig. 15.10 (15.9), or Becker 14-18)
a. Direct Effect: Some proteins can be activated by binding directly to TK.
- Example? PLC. Ligand binding to TK-linked receptor can activate a type of PLC → IP3 & DAG → etc.
- This means that the IP3 pathway can be activated by both types of receptors -- GPCRs and TKs.
- The cAMP pathway (as far as we know) can only be activated by GPCRs.
b. Indirect Effect: Some proteins are activated by binding indirectly -- these proteins bind to "adapter proteins" that are bound to the TK. How it happens:
(1). Adapter proteins bind directly to the phosphorylated TK (to the phosphorylated tyrosines)
(2). Additional proteins bind to the adapters
(3). Binding to adapters → activation of the additional "recruited" proteins.
(4). Most famous (infamous?) example is the small G protein ras. Why does anyone care about ras?
(a). Overactive form of ras (stuck in the activated 'on' form) can cause cancer. Implies normal ras involved in control of cell cycle. (More on ras in lect. 25.)
(b). Large % (about 30%) of tumors have overactive ras.
c. SH2 domains. Proteins that bind directly to TK have certain types of domains -- usually called SH2 binding domains.
d. Recruitment. Note that the initial target protein(s) to be activated come to or are "recruited by" the TK; this is the opposite of the situation with most 2nd messengers where the messenger diffuses throughout the cytoplasm and "seeks out" the target protein to be activated. The recruitment method may be important in localizing the response to a particular part of the cell.
C. A human example of TK receptor signaling: FGF (Fibroblast growth factor) and FGF Receptor. Significance of dimerization. See Becker figs.14-20 & 14-21 (14-19 & 14-20).
1. FGFR is a TK receptor
2. FGF & FGFR needed for proper development as described in Becker chap.14. Failure of signal transmission (or premature transmission) causes developmental abnormalities. See Becker fig. 14-20 (10-20).
3. Achondroplasia (a type of dwarfism), is due to a defective FGF receptor.
4. Achondroplasia is dominant. In a heterozygote for achondroplasia, 1/2 the FGF receptor (monomers) are defective, therefore dimers that form are defective.
a. Example #1: In the example shown in Becker & on handout, the cytoplasmic domain of the defective receptors is missing. Dimers form, but most dimers are never activated -- the two monomers can not phosphorylate each other. (Fig. 14-19) Therefore the signaling is badly disrupted. (In the example shown in Fig. 14-20, the FGF is needed to turn on formation of certain embryonic tissues, so the mutant fails to form these tissues. This type of mutation is called a 'dominant negative' as explained below.)
b. (FYI) Example #2: In most cases of human achondroplasia, the molecular explanation is different. In these cases, the mutation is in the transmembrane domain of FGF3 Receptor and dimers form, but act abnormally. (In these cases, the FGF signal is needed to turn on bone differentiation and turn off cell growth. Mutant dimers signal prematurely, so differentiation starts -- and cell growth stops -- before bones are long enough. This type of mutation is called a 'gain of function' mutation, because it works when it shouldn't.)
5. Significance of a 'Dominant Negative'
a. Recessive (ordinary) negative mutations. 'Negative' mutations (those that produce inactive protein) are usually NOT dominant. Most negative or "lack of function" alleles (or mutations) are recessive. If there is a mixture of normal and abnormal protein in the heterozygote, the normal, active, protein usually works (in spite of the presence of abnormal, inactive, protein). So usually, overall, there is NO lack of function in the heterozygote.
b. 'Dominant negative' mutations. Sometimes negative mutations are dominant. How does this occur? There is a mixture of normal and abnormal protein present, as usual. What's unusual is that the abnormal, inactive, protein 'gets in the way' and interferes with the working of the normal, active, protein. So overall, there is a lack of function in the heterozygote.
c. Definition: An abnormal allele like the one that produces the FGF receptor without a cytoplasmic domain is called a "dominant negative mutation." A dominant negative allele (or mutation) makes an inactive protein that disrupts function even in the presence of a normal allele (and normal protein). 'Dominant negative' means that the heterozygote is negative for function, not that it doesn't produce any protein.
d. Implications: What does the existence of a dominant negative mutation imply? It indicates that the gene involved probably codes for a protein that must polymerize in order to act.
D. How do GPLR's and RTK's Compare? -- See table below for reference for comparison of basic features of TK or TK linked receptors and G protein linked receptors. For TK's, see Sadava figs. 15.6 & 15. 10 (15.9) or Becker fig. 14-17 for structure and Becker 14-18 for signaling pathway.
|Properties of Two main types of Cell Surface Receptors|
Type of Receptor
|Property of Receptor||G protein Linked Receptor||Tyrosine Kinase or TK Linked Receptor (RTK)|
|Structure||Multipass (7X) transmembrane protein#||Single pass transmembrane protein##|
|What happens on ligand binding||Change in conformation||Usually aggregation (dimers)##|
|Receptor activates (binds to)||G protein||Self (other subunit) or separate TK|
|Nucleotide role in activation||Replace GDP with GTP||Add phosphate from ATP to tyr side chains|
|Activated TK or G protein subunit* binds to:||adenyl cyclase or
phospholipase C or
ligand gated channels
|Many dif. proteins, usually protein with SH2 domains**
Can bind a PLC → IP3 etc.
|Which 2nd messengers used?||cAMP or IP3 etc.||IP3, etc. (if any); not cAMP. Often does not use a 2nd messenger.|
|Receptors for||Epinephrine, ADH, glucagon (most endocrines)||Insulin and most GF's such as EGF (many paracrines & autocrines)|
|Usually modify||Enzymes, not TF's||Transcription Factors|
|Affect Transcription?||Not usually||Often|
|How contact target?||Broadcast||Recruitment|
# See Becker Fig. 14-4.
## See Becker fig. 14-17 or Sadava 15.6 & 15.3.
*Either part, alpha or beta + gamma may be activator/inhibitor; G proteins can also be inhibitory
**SH2 = sarc homology 2 domain
Review problems 6-1 & 6-3
E. Signaling Pathways all interrelate
1. Different 2nd messengers can influence the same enzyme/pathway. See problem 6-20.
2. Each signaling system can affect the others -- For example, Ca++ levels can affect kinases/phosphatases and phosphorylations can affect Ca++ transport proteins (& therefore Ca++ levels). See an advanced text if you are interested in the details.
3. The same signal (same activated TK receptor) may trigger more than one signaling pathway. For example, EGF can trigger both the IP3 pathway and the ras pathway. (Becker fig. 14-18).
IV. Intro to Kidney Function (Handout 24A). See also Sadava Sect. 51.4. & 51.5.
Here's an article from the LA Times on a recent artificial kidney.
A. Overall Function -- what does the kidney do?
1. Function: Controls water loss and determines what other specific components (including protons & other ions) will be excreted (lost in urine) and what will be retained.
2. What processes occur in kidney?
tubular secretion (addition of substances to the filtrate) = secretion into the tubule
tubular reabsorption (removal of substances from filtrate) = reabsorption from the tubule
control of volume of urine
B. Details of Basic Processes
1. Basic set up→ glomerular capillaries → efferent arteriole → peritubular capillaries → venule → vein (back to heart)
a. Capillaries: Artery (from heart) → afferent arteriole
b. Filtration: Material moves from glomerular capillaries into tubule.
c. Secretion & Reabsorption: Materials moves between inside of tubule and inside of peritubular capillaries (surrounding kidney tubule).
2. The 4 Basic Processes
(1). Occurs in glomerulus
(2). About 20% of blood liquid (plasma) enters Bowman's capsule = filtrate
(3). Filtrate contains no large proteins or cells
b. Tubular (selective) secretion: Material is added to the filtrate.
(1): Terminology: Secretion is NOT the same as excretion.
Secretion = extruded by the cells into extracellular space (into filtrate, lumen, etc.).
Excretion = carried out of body in urine or feces.
(2). Result of filtration: filtrate carries high concentrations of certain dissolved materials (secreted by cells lining the lumen) -- removes waste, toxins from circulation.
c. Tubular (selective) reabsorption: Material is removed from the filtrate.
(1). Result of reabsorption: Filtrate does NOT carry certain materials (which are selectively reabsorbed) -- conserves valuable materials; returns them to circulation.
(2). Aldosterone affects Na+ reabsorption (& K+ secretion). Details below.
d. Volume Control:
(1). Water loss is adjusted at the end of the tubule using ADH. (See below.)
(2). Urine can be more -- or less -- concentrated than the plasma. Concentration and/or volume can be varied to suit need.
(3). Water loss or conservation in tubule controls volume of body fluids (not just urine volume). Controls volume of plasma, extra cellular fluid, etc. (& blood pressure).
3. How does tubular secretion/reabsorption occur? Structure of cells lining tubules -- see handout 24A bottom or Sadava fig. 51.12 (for a different example).
a. Tubules are lined by layer of polarized epithelial cells (similar to those lining intestine)
b. Materials must cross epithelial cells to enter or exit lumen of tubules.
c. Interstitial fluid separates epithelial cells and peritubular capillaries.
d. Epithelial cells have different proteins/channels/transporters on their two surfaces -- the apical or luminal surface (facing lumen) and basolateral surface (facing interstitial fluid and capillaries).
e. Cells in different parts of the tubule have different transporters/channels on their luminal surface.
f. All cells in tubule that absorb Na+ have the Na+/K+ pump on their basolateral surface. Other transporters may vary.
g. What cells transport (& in which direction) depends primarily on which transport proteins are on the luminal surface. Depending on transporters, cells can secrete materials into lumen or reabsorb material from lumen.
h. Cells lining tubule do actual secretion/reabsorption but peritubular capillaries remove reabsorbed material or provide material to be secreted. Therefore (as shown on handout 24A, top left):
(1). Result of tubular reabsorption = net transfer from filtrate to capillary.
(2). Result of tubular secretion = net transfer from capillary to filtrate.
Try problem 12-3.
3. Example of reabsorption -- see 24A upper right. (Fig. 14-18). How Na+ is reabsorbed.
Q: How could K+ be secreted? What would you have to add/remove from the diagram?
4. Role of Hormones
a. Overall -- Hormones cause water and/or some remaining Na+ to be removed (reabsorbed from filtrate) at end of tubule
aldosterone affects Na+ reabsorption (& K+ secretion)
ADH affects water reabsorption
b. Role of aldosterone in Na+ reabsorption
(1). Promotes reabsorption of Na+
(2). Stimulates virtually all steps of reabsorption -- all steps shown in 24A, upper right.
c. Role/Mech. of action of ADH
(1). Filtrate entering end of tubule is at minimum osmolarity (Osmolarity = concentration of dissolved particles; see below)
(2). ADH (using cAMP) controls insertion of water channels/pores into membranes of cells lining end part of tubule.
Qs: Where are the water channels that are regulated by ADH?
Are they in the luminal membrane, BL membrane, or both?
Are they inserted or removed in response to hormone?
Why are cells in only some areas of tubule responsive to ADH (or aldosterone)?
(3). Water flows out water channels (if in membrane) because of salt gradient in interstitial fluid surrounding tubule
(4). Diabetes insipidus -- result of no ADH or no response to ADH
d. Questions to think about: Where are the receptors for ADH? Aldosterone? Which hormone elicits a faster response?
See problems 12-8 to 12-10. (See below for location of cells affected by each hormone.)
V. Kidney Structure
A. Overall structure -- see handout 24B or Sadava fig. 51.9. Alternatively, see Kimball's biology pages.
1. Kidney has medulla (inner part) and cortex (outer)
2. Functional unit = nephron (Sadava 51.7 )
3. Visible unit (in medulla) = Renal Pyramid = bottoms of many nephrons
4. Tops of nephrons in cortex
B. Structure of Nephron -- see handout 24B or Sadava fig. 51.7 & 51.9 . For EM pictures see Sadava 51.8. (We may do the parts as we need them, but all are summarized here.)
1. Nephron itself -- parts in cortex
a. Bowman's capsule
b. proximal (convoluted) tubule
c. distal (convoluted) tubule
2. In medulla
a. Loop of Henle
b. Collecting duct (shared by many nephrons)
3. Capillaries (discussed last time)
a. 2 sets in series
(a). form glomerulus inside Bowman's capsule
(b). function in filtration
(a). surround tubules
(b). fyi: part in medulla (surrounding loop of Henle) is called the vasa recta
(c). function in secretion & reabsorption
b. How capillaries connected. Circulation goes as follows:
Artery (from heart) → afferent arteriole → glomerular capillaries → efferent arteriole → peritubular capillaries → venule → vein (back to heart)
VI. Kidney Function, revisited.
A. Function of Nephron -- Let's follow some liquid through.
1. Filtration in glomerulus
2. Reabsorption & secretion (of most substances) occurs in proximal tubule
3. Loop of Henley -- overall picture of state of filtrate
a. Definition: Osmolarity (Osm) = total solute concentration = concentration of dissolved particles = osmol/liter. (One osmol = 1 mole of solute particles.)
Examples: 1M solution of glucose = 1 Osm; 1M solution of NaCl = 2 Osm.
b. Events in Loop
(1). Descending: Osmolarity increases as filtrate descends due to loss of water
(2). Ascending: Osmolarity decreases as filtrate ascends due to loss of salt; reaches min. value less than that of blood. Therefore can excrete urine that is hypo-osmotic (less concentrated) than blood.
(3). Overall: Net effect of going through countercurrent loop -- less volume, less total salt to excrete (even if filtrate and blood are iso-osmotic when done).
4. Distal Tubule & Collecting Ducts
a. Control of removal from filtrate (reabsorption) of remaining Na+ (Role of aldosterone.)
b. Volume Control -- occurs in collecting ducts. (Role of ADH.)
B. Details for Proximal Tubule
1. Many substances removed from lumen by secondary act. transport
a. examples: glucose and amino acids
b. AA etc. cross apical/luminal surface of epithelial cell by Na+ co-transport -- therefore a lot of Na+ removed from filtrate (along with glucose, AA, etc.)
c. Exit basolateral side of cells into intersit. fluid by facilitated diffusion
c. Process is similar to absorption in cells lining intestine
2. Na+/K+ pump on basolateral side keeps internal Na+ low.
3. Water follows salt.
4. Secretion of most materials (except K+) occurs here -- toxins etc. transported to filtrate
C. Details of Transport Events in Loop of Henley (See Sadava 51.10)
1. Water permeability. Luminal cell membranes in descending loop and lower part of ascending loop are permeable to water.
2. Generating the Na+ gradient in the medulla.
a. Luminal cell membranes in rest of ascending are impermeable to water and pump NaCl from lumen to interstitial fluid.
b. NaCl pumped out from ascending loop accumulates in medulla, forming a gradient of increasing osmolarity (outside the tubule) as reach bottom of loop = core of medulla.
3. Water loss: Filtrate from proximal tubule loses water as it descends into medulla → NaCl stays in tubule → high concentration NaCl in tubule→ to be removed in ascending. (Na+ not pumped out of these cells on BL side.)
4. Escalator Effect: If NaCl diffuses into descending loop, it is carried around and pumped out in ascending = escalator effect.
5. Why called countercurrent? Because flow in two sides of loop is in opposite directions -- physically and with respect to osmolarity.
a. First leg (descending) of loop removes water → higher osmolarity in filtrate as it proceeds (on the way down).
b. Second leg (ascending) removes salt → lower osmolarity in filtrate as it proceeds (on the way up).
c. Net effect is higher osmolarity toward the bottom on both legs.
d. Why doesn't flow in peritubular capillaries (vasa recta) wash out the salt gradient in the medulla? Because capillaries exit out the top of the nephron, carrying a low amount of salt.
See problems 12-1 to 12-3.
D. Distal Tubule and Collecting Ducts -- A few more Details
a. Reminder: Filtrate entering distal tubule is at minimum osmolarity
b. This is the only part of the tubule affected by ADH and aldosterone
(1). Events in distal convoluted tubule (& first part of coll. ducts) depend on aldosterone
(2). Events in collecting duct (volume control) depend on ADH
c. Hormones cause water and/or remaining Na+ to be removed (reabsorbed from filtrate)
(1). aldosterone affects Na+ reabsorption (& K+ secretion) -- See handout 24B, top right.
(2). ADH affects water reabsorption
2. Importance of aldosterone (in water/Na+ balance)
a. Promotes reabsorption of Na+; water follows (not necessarily in same part of tubule).
b. Amount of Na+ reabsorbed due to aldosterone is small % of total, but adds up; affects blood pressure.
c. Aldosterone promotes K+ secretion -- this may be of major importance, but we are focusing on role of hormone in Na+ balance.
3. Importance of ADH. Controls water retention in body. Osmolarity of filtrate will increase (and volume decrease) in collecting duct if ADH (vasopressin) present and water removed. (See above for mechanism.)
4. Where do the hormones come from? What triggers their production/release?
a. ADH produced by HT (& released in PP) primarily in response to high osmolarity of blood.
b. Aldosterone produced by adrenal cortex in response to inadequate blood flow through kidney, not primarily in response to ACTH.
See problems 12-8 to 12-13 & 12-15.
Next Time: Last Lecture! How Kidney Function & Blood Pressure are Regulated; Cancer & Control of Cell Growth