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PHYSIOLOGY WEEK 2: MUSCLES AND MEMBRANES 1/24/00

W. Crone (303 FTZ, 629-7439, cronewil@hvcc.edu, http://www.hvcc.edu/academ/faculty/crone/index.html)

Cormack: Chs. 4, 5; Guyton: Chs. 4-9; Michael and Rovick: Units 1-3; van Wysberghe and Cooley: Cases 5,6

possible web sites: http://pb010.anes.ucla.edu (the nerve impulse and simulations)

http://muscle.ucsd.edu/musintro/ (introduction to muscle physiology)

link to Dr. Paul Spannbauer (HVCC Biology) handout on membrane potentials

MEMBRANES:

Phospholipid bilayer and fluid-mosaic model. The phosphoglycerides are amphipathic, meaning that one end is soluble in water (often phosphatidyl choline) and the other less so (diglyceride tails). In the watery world of ECF and ICF, these phospholipids will form a bilayer so that the hydrophilic heads"protect" the hydrophobic tails. Much of a membrane is protein, e.g., receptors, ion channels, etc. The uncharged hydrophobic portions of proteins will remain the inner portion of the bilayer, and the charged hydrophilic portions protruding into the ECF/ICF.

There are many functions of proteins in membranes. For example:

1. pumps (transporting ions across the membrane)
2. carriers (transporting substances down electrochemical gradients by facilitated diffusion)
3. enzymes, to catalyse reactions at the surfaces of the membrane
4. glycoproteins that function in immune recognition
5. ion channels are instrinsic proteins that can open a gate to different ions given a particular stimulus. Two general types of ion channels:

voltage-gated ion channels open when the membrane potential exceeds a threshold value, e.g., those involved along the axon of nerves

ligand-gated (chemically gated) channels open when bound by a specific agonist, e.g., a neurotransmitter

6. receptors (bind neurotransmitters, hormones, to start physiologic changes in cells) Receptors are proteins that have their numbers increase and decrease in response to changing conditions. For example, if a hormone is in excess, # of active receptors decreases (down regulation), e.g., desensitization, and if hormone is low, up regulation of receptors.

Also cholesterol is a membrane component, as it is a large, flat planar molecule and it seems to affect liquidity of the membrane. In all, we see a lot of lateral movement in the"sea" of the bilayer, but there is little exchange of protein and/or lipid to either side of the membrane.

Ways to cross membranes:

1. Simple diffusion or random movement in response to a concentration gradient, e.g., O₂ from lungs to blood stream.
2. Facilitated diffusion through protein channels or carrier molecules in the membrane, e.g., for amino acids after digestion.

Diffusion: process by which molecules in solution flow down their concentration gradient. Diffusion is rapid over short distances, e.g., 0.000456 sec for 1 mm (vs. 5+ days for 1 cm). Movements of molecules through a solution by diffusion is called a flux, not a flow, since the liquid as a whole does not move (bulk flow of fluid with a pressure gradient, would be something more like the contractions of the heart pumping blood).¹

3. Filtration, a forcible filtering of small molecules through a membrane with (hydrostatic) water pressure, e.g., glomerular filtration in kidneys.

4. Active transport, requiring energy to take in materials against a concentration gradient, e.g., the sodium pump, or Ca²⁺ pumps to keep intracellular [Ca²⁺] levels low.
5. Osmosis, passage of water from area of higher concentration (right? fewer particles of solute?) to one of lower concentration through a selectively permeable membrane, e.g., effects on cell size.

The pressure necessary to stop osmosis is the osmotic pressure of a solution. Osmotic pressure is determined by the number of particles in solution, hence the osmole or 1 gmw per kilogram of solvent. Normal body osmolality is about 300 mOsm (although we usually end up measuring osmolarity per liter of solution).³

6. Endocytosis, bulk movement of materials inward, e.g., a macrophage eating; and exocytosis, the packaging of materials for secretion, e.g., delivering neurotransmitters to the synapse.

THE SODIUM PUMP:

Primary active transport involves direct use of metabolic energy in the form of ion pumps (ATPases). The Na⁺ - K⁺ ATPase catalyses ATP à ADP + P_inorganic, hence using energy to remove 3 Na⁺ from cell and take 2 K⁺ into cell, or a coupling ratio of 3/2. The sodium pump is found all over the body, accounting for much of"basal metabolism."

But, since Na⁺ and K⁺ are small ions, they are able to diffuse back through the membrane though multiple"leak channels." The membrane permeability of K⁺ is almost 100X that of Na⁺. But, the ICF has a lot of (-) charged proteins and phosphates, so we have not only the issue of chemical gradients but electrical gradients as well!

This pumping can have several major effects:³

1. steep sodium gradient can allow for cotransport of items, e.g., glucose
2. assists in maintaining osmotic balance of the cell--"water follows the sodium"
3. allows for regulation of basal metabolism rate (by thyroid hormone)
4. helps to set up a membrane potential for excitable nerve and muscle cells

A resting membrane potential exists across the plasma membrane, with the inside of the cell negative relative to the exterior. This process involves relatively few cells along the margins of the membranes. The Nerst equation helps to give the value for the electrical difference necessary for a particular ion to be at equilibrium. Putting in numbers for this equation for body temp, E_x = 61/z log₁₀ [X_o]/[X_i]. Hence, the equlibrium potential will be different for each ion because of the equation, e.g., Na⁺ and K⁺ are present at different [ICF] and [ECF]. Since the membrane is most permeable to K⁺, the resting potential for most cells is near to the equilibrium potential for K⁺. So, the combination of anions from proteins and phosphates within the cell, the sodium pump, and the differential permeability of the membrane to K⁺ and Na⁺ all combine to form a negative membrane potential, about -90mV for nerve cells.

action potentials: In your typical excitable cell--nerve or muscle--gates in channels open transiently and then close due to changes in the potential across the (plasma or sarcolemmal) membrane. Typically, the sodium channel is closed. When the membrane potential is suddenly less negative, e.g., with an excitation wave, the sodium channel opens up and sodium ions rush in to depolarize the cell membrane. When the membrane potential is positive enough, additional potassium channels open up to allow potassium to rush out, repolarizing the cell. After the additional K⁺ channels close, the action of the sodium pump can assist to reset the resting potential, as overall very few ions are involved in this membrane surface activity.

The action potential is all or none: once the stimulus suddenly increases to a certain (-65 to -60 mV) threshold, the above events will occur. When the Na⁺ gates are open, there is an absolute refractory period and the gates cannot respond to a subsequent stimulus. When the (voltage-gated) K⁺ channels are open, a very strong stimulus can create a depolarization, so that the membrane is in a relative refractory period.

A neuron conducts its impulse along its axon. The myelin sheath is formed by Schwann cells wrapping about 1 mm around an axon, leaving exposed axon in the gaps known as the nodes of Ranvier. In an unmyelinated axon, every stretch of membrane containing Na⁺ and K⁺ channels can produce an action potential that is conducted without decrement. In other words, the action potential is repeated down the length of the axon. In contrast, in a myelinated axon, the nodes of Ranvier allow the action potential allow for the cable property of the axon to spread the charge over their 1 mm separation, and let the action potential reoccur at the nodes of Ranvier, or saltatory conduction. Since cable conduction is faster than the action potential, this has the effect of speeding up the rate of conduction.

When the action potential reaches the synapse at the end of the axons, vesicles of neurotransmitters are released via exocytosis. At the presynaptic neuron, the action potential will open up voltage-gated Ca²⁺ channels. The influx of Ca²⁺ activates calmodulin, which turns on protein kinase to phosphorylate synapsins that allow the release of neurotransmitters.² After use, neurotransmitters are either:

1. enzymatically inactivated: acetylcholine broken down by acetylcholinesterase
2. reuptaken by presynaptic neurons: norepinephrine (and then inactivated by monoamine oxidase [MAO]).

MUSCLE STRUCTURE AND FUNCTION:

Striated skeletal muscle fibers (cells) contain many myofibrils that push the rest of the cytoplasm, including the nuclei, to the periphery. The sarcolemma (plasma membrane) surrounds the muscle fiber.

Sarcomere: distance between (and including) two Z lines (discs) in a myofibril. The sarcomeres contain the active filaments actin and myosin necessary for the force needed for the pumping action. Both remain constant in length, so that the actin slides past the myosin and the sarcomere shortens, as seen by the decreasing distance between Z lines during contraction.

The strength of contraction depends on the presence of available intracellular Ca²⁺ (which has several mechanisms to control it, depending on the muscle type). Our understanding of muscle contraction is that it works by the filaments of actin and myosin sliding over each other. Heads of myosin form cross bridges to actin and then, with the expenditure of ATP, will ratchet along the length of the actin. The role of Ca²⁺ is to open up the binding sites on the actin. Ca²⁺ binds to the protein troponin. The Ca²⁺-troponin complex interacts with tropomyosin to unblock active sites and allow the myosin head to continue to ratchet along.

When the nerve stimulation and muscle contraction is over, Ca²⁺ is reaccumulated (and concentrated with the assistance of calsequestrin) in a specially modified smooth endoplasmic reticulum called the sarcoplasmic reticulum (SR), thus allowing the reestablishment of troponin/tropomyosin inhibition.

Intracellular [Ca²⁺] needs to increase past its normal level of 10^-7 M in order for troponin binding, and hence muscle contraction, to occur, and below that for muscle relaxation. Therefore, to keep cytoplasmic Ca²⁺ levels low, the SR actively accumulates Ca²⁺ (i.e., needing ATP expenditure), particularly in expanded terminal cisternae. These terminal cisternae are in close approximation to T tubules derived from the sarcolemma. Therefore, when an action potential is generated at the neuromuscular junction, it is conducted along the sarcolemma to the T tubules. Excitation-contraction coupling causes the release of Ca²⁺ into the cytoplasm, so as long as there is an action potential, Ca²⁺ release and hence muscle contraction will occur.

This general strategy is modified in the two other types of muscles:

Specializations of smooth muscle: no striations, as they do not contain the sarcomeres, and overall, smaller cells. Instead, the actin filaments are very long, radiating from dense bodies (the equivalent of the Z lines). This means that smooth muscle can contract when it's stretched--think of a full bladder between highway stops. The SR of smooth muscle is less extensive than that of skeletal muscle, so that extracellular Ca²⁺ entering through the cell membrane through voltage-gated Ca²⁺ channels (sometimes known as Ca²⁺ - Na⁺ channels) is necessary to keep contractions going. These contractions are slow compared to skeletal muscle, with slow racheting of the myosin and actin, slow pumping of the Ca²⁺ out of the smooth muscle cytoplasm, and even slow onset of the action potential because it is more dependent on the Ca²⁺ channels than scarce Na⁺ channels.³

Instead of troponin, in smooth muscles the Ca²⁺ binds to calmodulin. The Ca²⁺-calmodulin complex activates myosin light-chain kinase (MLCK). MLCK then phosphorylates the myosin cross bridges before they reach over to the actin filaments.² Smooth muscle contractions are slow and sustained in comparison to skeletal muscle with this slow cycling of cross bridges.

Specializations of cardiac muscle: Striated, reflecting the presence of sarcomeres in the myofibril. Attached to other muscle cells at the ends by gap junctions in the intercalated discs. Branched, to assist in lateral spread of electrical activity.

CARDIAC MUSCLE:

Remember how to affect membrane potentials:

Action potentials in ventricular, atrial, and Purkinje cardiac tissue. Note that this action potential is >150 milliseconds, much longer than the 1-2 msec action potentials of nerves:⁴

phase 0: rise in action potential due to rapidly increasing Na⁺ current by opening up voltage-gated Na⁺ channels. These channels are blocked by Class IA antiarrhythmics (quinidine) and Class IB antiarrhythmics (lidocaine).⁴

phase 1: slight repolarization of membrane with those Na⁺ channels shutting and some K⁺ leaking (overall pretty low K⁺ permeability).

phase 2: plateau phase potential remains positive with little change, due to outward flow of K⁺ as above, and inward flow of Ca²⁺ from calcium channels"cancelling" each other. These are channels sensitive to calcium-channel blockers, e.g., verapamil (Class IV antiarrhythmics).⁴

phase 3: repolarization, with closing of Ca²⁺ channels and opening of voltage-gated K⁺ channels, so K⁺ flows out unopposed. Class III antiarrhythmics, e.g., bretylium, block these channels, and so prolong depolarization.⁴

phase 4: resting (diastolic) membrane potential

phase 0 - partway through phase 3: absolute refractory period, with the sarcolemmal membrane inexcitable. Following in phase 3 is the relative refractory period, with decreased excitability.

When the action potential repolarizes, the SR takes up Ca²⁺ from the cytosol via a calcium pump on the SR, or excess Ca²⁺ is exchanged with Na⁺ out of the cell at a Ca²⁺/Na⁺ exchanger.

In cardiac muscle, the amount of Ca²⁺ entering from extracellular space in phase 2 (the plateau) helps to trigger the release of Ca²⁺ from the intracellular stores of Ca²⁺ in the SR. Unlike skeletal muscle, the Ca²⁺ stored in cardiac muscle SR isn't sufficient.--there is a need for the extracellular Ca²⁺ coming in from large T tubules to have an effect. The more depolarization, the more voltage-gated Ca²⁺ channels will open up, and hence the stronger the contraction.

Therefore, events that raise cytosolic Ca²⁺ increase contractile force, and vice-versa.

1) catecholamines (norepinephrine) increase Ca²⁺ into the cell by opening up more of the appropriate Ca²⁺ channels via the cAMP second-messenger system. Class II antiarrythmics are beta blockers.

2) one could decrease the Na⁺ gradient across the sarcolemma and so lose less intracellular Ca²⁺ by the Na⁺/Ca²⁺ exchange mechanism. Digitalis compounds work by inhibiting the cardiac sodium pump to lead to this effect.⁴

Anyhow, at the end of systole (contraction), the Ca²⁺ influx across the sarcolemma ceases and the SR is no longer stimulated to release Ca²⁺. Ca²⁺ is then actively pumped into the SR and T tubules. Ca²⁺ is then not bound by troponin, tropomyosin goes back to blocking the sites, and relaxation (diastole) occurs.

The SA (sino(u)atrial) node acts as the major pacemaker of the heart with a higher discharge rate than that of the AV node or the Purkinje fibers. Pacemaker cells are distinctive in that their action potentials show diastolic depolarization or pacemaker potentials, such that the potential rises gradually until it reaches a threshold potential from the effects of slow-opening Ca²⁺ channels. At this point, about -40mV, calcium channels open, leading to depolarization. There is no plateauing at the SA node.³ The"resting potential" in the SA node is only -55mV, at which point the slower calcium-sodium channels can set off the action potential. The natural leakiness of the SA node cell membranes to Na⁺ help to create this autodepolarization.³

1. JJ Bray et al., eds, Lecture Notes on Human Physiology, 3rd ed. (Blackwell Scientific, Oxford, 1994), p. 393.
2. SI Fox, Human Physiology, 6th ed. (WCB McGraw-Hill, Boston, 1999),pp. 138, 166-167, 354.
3. AC Guyton and JE Hall, Textbook of Medical Physiology, 9^th ed. (WB Saunders, Philadelphia, 1996), pp. 51, 97-98, 122-123.
4. JM Olson, Clinical Pharmacology Made Ridiculously Simple (Medmaster, Miami, 1997), pp. 76-79.

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