Cell Electrophysiology PDF

PHYSIOLOGY: LE1 | TRANS 03 CELL 3: CELL ELECTROPHYSIOLOGY CARINA C. GOMEZ, M.D, FPSP | 08/07/2024 3 OUTLINE IONIC COMPOSITION AND GRADIEN...

PHYSIOLOGY: LE1 | TRANS 03 CELL 3: CELL ELECTROPHYSIOLOGY CARINA C. GOMEZ, M.D, FPSP | 08/07/2024 3 OUTLINE IONIC COMPOSITION AND GRADIENT I. Cell Membrane II. A. Review Ionic Composition and Gradient distribution of ions in and out of the cell? 👩‍⚕️ QUESTION: What are the reasons why there is unequal A. Na+/K+ Pump III. Membrane Voltage Potential ANSWER: A. Ion channels Selective permeability of the cell membrane B. Resting Membrane Potential Activity of Na+/K+ pump. C. Action Potential To maintain the ionic and voltage gradient with the use of D. Periods of Action Potential ATP IV. Refractory Periods To drive transport of other ions and molecules in and out of V. Review Questions the cell VI. References Composition inside the cell is different from outside the cell. ○ Greater concentration: SUMMARY OF ABBREVIATIONS Inside the cell: potassium, magnesium, EG Electrical Gradient phosphate, sulfate, amino acid CG Concentration Gradient Outside the cell: Na+, Ca2+, Cl-, HCO3- mV Millivolt (bicarbonate) RMP Resting Membrane Potential Na+/K+ Pump Sodium Potassium Pump or ATPase NICE-TO-KNOW: AP Action Potential Major extracellular cation: Na+ LP Local Potential Major intracellular cation: K+ LEGENDS USEFUL MNEMONICS: PISO ❗ Must know 👩‍⚕️ Lecturer 📖 Book 🖥 Presentation 📝 Old Trans Potassium In, Sodium Out A. Na+/K+ PUMP LEARNING OBJECTIVES At the end of the lecture, the student should be able to: Electrogenic pump (transport charged molecules across the ✔ Describe the cell membrane. ✔ Describe protein channels. ○ ❗ membrane) 🖥 Maintains ionic & voltage gradients → drive transport of ions in/out of the cell: ✔ Differentiate between leak and gated channels and characterize each. Ionic gradient ✔ Enumerate the different types of membrane potentials. Na+, K+, and other ions ✔ Differentiate between local and action potential. Voltage gradient ✔ Discuss the ionic events that happen during the generation of Negative charge inside the vicinity of the local and action potential. membrane ✔ Describe the sodium-potassium pump. Positive charge outside the vicinity of the ✔ Give the phases of action potential. membrane ✔ Discuss refractory period. USEFUL MNEMONICS: NOKIA 321 ✔ Give the two types of excitable cells. 3 Na+ Out, 2 K+ In, 1 ATP used CELL MEMBRANE ❗REVIEW CELL MEMBRANE Almost made up entirely of proteins & lipids ↓ SELECTIVE PERMEABILITY Lipid bilayer ↓ UNEQUAL ION DISTRIBUTION Inside of cell: more (-) Outside of cell: more (+) ↓ IONIC GRADIENT ↓ ELECTRICAL ACTIVITY OF THE CELL leak channels. [Guyton & Hall, 14th Ed.] 📖 Figure 1. Functional characteristics of the Na+-K+ pump and the K+ Electrophysiology Secondary active transport mechanisms: needs ionic gradient, primarily Na+ ○ Cotransport ○ Counter transport ○ Ionic transport. LE 1 Trans 1 TG: BC, SC, LC, SC, KD, DL, CM, BM, NN, AS, RS, CT Page 1 of 7 [Refer to Figure 2.] 📝 Na⁺-K⁺ pump: 3 Na⁺ ⇄ 2 K⁺ (NOKIA 321) A. ION CHANNELS ○ ↓ Na+ concentration inside the cell → Na⁺ moves into Usually integral/intrinsic proteins → conduct ions the cell to restore gradient → kinetic energy (fuel) is ○ Recognize and select specific ions because some are stored → amino acid will move into the cell via stored energy ○ ❗ selective than others Selectively permeable: Size: Smaller = ↑ permeable Bigger = ↓ permeable ○ Shape: Flex & slender = ↑ permeable Globular = ↓ permeable ○ Electrical Charge: Positive = ↑ permeable Negative = ↓ permeable ○ Chemical bonds Open and close in response to specific stimuli 📖 Responsible for generating local or action potential Found in all cells, especially in excitable cells ○ Neurons ○ Muscle cells 📖 Gating: Ion channels fluctuate between an open and closed state 📖 Selectivity: nature of the ions that pass through the channels 📖 Figure 2. Cell model depicting how cellular gradients and the Channel conductance: # of ions that pass through the membrane potential are established. [Berne & Levy, 6th Ed.] channel ○ Expressed in picosiemens (pS) MEMBRANE VOLTAGE POTENTIAL Table 1. Concentration of some ions inside and outside ❗ Voltage at which there is 0 net ion flow into/out of the cell Membrane voltage potential: movement of K⁺ inside = movement of K⁺ outside = 0 net movement mammalian spinal motor neurons Ion Con. (mmol/L of Con. (mmol/L of Equilibrium H2O) Inside cell H2O) Outside cell Potential (mV) Na+ 15.0 150.0 +60 K+ 150.0 5.5 -90 Cl+ 9.0 125.0 -70 ❗Resting Membrane Potential (RMP) = -70 mV B. TYPES OF MEMBRANE CHANNELS RESTING OR LEAK CHANNELS (NON-GATED) Channels that are already open even at rest ○ stimuli is not needed to open it Generates resting membrane potential ○ K+ leak channels (more abundant) Figure 4. Diffusion potentials. [Guyton & Hall, 14th Ed.] 📖 ○ Na+ channels GATED CHANNELS Driven by the Concentration Gradient: high conc. → low ○ Na⁺ moves into the cell because there is more Na⁺ Closed or open at rest outside the cell than inside ○ Stimuli is required for closing/opening ○ K⁺ moves outside the cell because there is more K⁺ Voltage-gated inside the cell than outside (PISO) ○ Stimuli: voltage or electrical changes ○ Most abundant Electrical Gradient: vicinity inside the cell is (-); vicinity Ligand-gated outside the cell is (+) due to the activity of Na⁺/K⁺ pump ○ Stimuli: chemical substances (for activation) ○ Na+ moves into the cell because the inside of the cell is ○ e.g. Acetylcholine (Ach) more negatively charged relative to the outside Mechanically- gated Once the membrane potential = +60mV (positive ○ Mechanical inside of the cell) → entry of Na+ will be repelled ○ Needs to be stretched by pressure or touch. At +60mV: 0 net movement of Na⁺ across the Phosphorylated-gated channels membrane ○ Phosphate is needed to open (not necessarily from ATP, ○ K⁺ moves out of the cell due to the concentration could also be from other sources) gradient; moves into the cell due to the voltage gradient VOLTAGE-GATED NA+ CHANNELS As K⁺ continues to move out of the cell → membrane potential reaches -90mV → K+ will be More abundant than K+ channels. pulled inside the cell 2 gates of Na+ channel. At -90mV: 0 net movement of K⁺ across the ○ Activation (M-gate): Faces outside the cell membrane (extracellular) MEMORY AID: Na+/K+ equilibrium potential ○ Inactivation (H-gate) - Present inside the cell Sixty = Sodium (intracellular) Potassium = 90 (inverted P) LE 1 Trans 1 TG: BC, SC, LC, SC, KD, DL, CM, BM, NN, AS, RS, CT Page 2 of 7 ❗ REMEMBER Resting Stage (-90mV) REASONS FOR THE GENERATION OF THE RESTING MEMBRANE POTENTIAL ○ Activation Gate (M gate): Close ○ Inactivation Gate (H gate): Open Ion concentration gradient: Activated State (-90mV to +35mV) ○ Na+ conc. higher outside the cell ○ Activated Gate (M gate): Open ○ K+ conc. higher inside the cell. ○ Inactivation Gate (H gate): Open Membrane permeability: ○ Na+ moves into the Cell ○ Cell membrane: more permeable to K+ than to Na+ Inactivated State (+35mV to 90mV, delayed) K+ can leak out of the cell. ○ Activation Gate (M gate): Open Na+-K+ pump: ○ Inactivation Gate (H gate): Close ○ Active transport ○ Pumps 3 Na+ out of the cell and 2 K+ in VOLTAGE-GATED K+ CHANNELS Contributes to the overall negative intracellular charge. Only have activation gates (M-gate) → inside the cell (intracellular). Table 2. Major ions inside & outside the cell ○ Resting Stage (-90mV): Gate is closed Inside the Cell Outside the Cell ○ Slow Activation (+35 to -90mV): Gate is open Major cation K+ Na+ K+ channels will only open if: Na+ gates are closed Major anion A- Cl- NOTE: Negativity of the membrane (-70 mV, -90 mV, -95 mV) K+ channel opening is almost simultaneous with the ○ ↑ negativity = ↑ magnitude of RMP. closing of the Na+ channel 👩‍⚕️ QUESTION: Who is responsible for the negativity/magnitude of resting membrane potential? ANSWER: glucose potassium efflux 👩‍⚕️ Table 3. Permeability of ions Ions Permeability Na+ ✔ ❌ K+ ✔ Cl- A- (negatively charged proteins & Phosphates) ❌ NERNST EQUATION 📖 EQUATION VS. GOLDMAN-HODGKIN-KATZ Nernst Equation Calculates the equilibrium potential for a single ion across a membrane Consider only one ion type and its concentration gradient Figure 4. Goldman-Hodgkin-Katz equation Goldman-Hodgkin-Katz Equation Calculates the membrane potential considering multiple ions Takes into account the permeability of the membrane to different ions Provides a more accurate representation of the actual membrane potential. Figure 3. Three types of gated ion channels. Figure 5. Goldman-Hodgkin-Katz equation B. RESTING MEMBRANE POTENTIAL POTENTIAL DIFFERENCE ACROSS THE MEMBRANE AKA transmembrane potential or steady potential Electrical potential difference across the plasma membrane Vicinity or lining of the cell: when not being stimulated. ○ Inside: (-) charge ○ Inside of the cell: more negative ○ Outside: (+) charge ○ Outside of the cell: more positive Due to activity of leak/resting channels. ○ Stimuli not required → channels open at rest LE 1 Trans 1 TG: BC, SC, LC, SC, KD, DL, CM, BM, NN, AS, RS, CT Page 3 of 7  Resting Membrane Potential (Resting State) Occurs before the AP begins Neurons’ RMP is typically around -70mv, maintained by Na+/K+ pump & selective permeability to ions Threshold Stimulus (Firing Level) Threshold level: -55mV Threshold is the level of MP wherein there is a 50/50 chance of generating an AP. ❗ ALL-OR-NONE LAW No AP occurs if the stimulus is subthreshold in magnitude strength above the threshold intensity📖 AP has a constant amplitude and form at any stimulus ○ If threshold level is reached → there is an AP ○ If threshold level is not reached → there is no occurrence of AP ❗ Weakest stimulus that can generate AP👩‍⚕️ THRESHOLD INTENSITY Example: Figure 6. Resting Membrane Potential Resting Membrane Potential of Excitable Cells Figure 8. Graph on threshold intensity Table 4. RMP of Different Tissues ○ 2V & 4V are not the threshold intensity because AP Resting Membrane Potential is not produced → subthreshold Nerve -70 mV ○ 8V & 10V reached the threshold level but they are not Skeletal -90 mV the threshold intensity because although there is an -95 mV (ventricular & atrial muscle fiber) AP, these values are not the weakest → maximal & Cardiac -60 mV (SA node & AV node) supramaximal intensity Smooth -90 mV ○ 6V → weakest stimulus that generated an AP (threshold intensity) C. ACTION POTENTIAL Depolarization along the nerve fiber membrane 📖 Rapid changes in the membrane potential that spread rapidly The initial phase where the membrane potential becomes with other excitable cells (nerve and muscle cells) 👩‍⚕️ Process allowing neurons to communicate with each other and Activation of nerve cells is triggered by various stimuli that can more positive due to the influx of Na+ ions through voltage-gated channels An AP is initiated when a stimulus causes the MP to become initiate an AP. less negative (depolarize), reaching a threshold (usually Some common types of stimuli: around -55mv). ○ Electrical ○ In nerve cells → +35mV = cue for the inactivation gate ○ Chemical (ligand) to close ○ Mechanical Periods of Action Potential QUESTION: “Why is there a peak value of +35mV?” 👩‍⚕️ ANSWER: In nerve cells, +35mV is the sign that the gate will close ○ Na+ cannot enter ○ Gated channel is re-opened when the value becomes more negative. Rising Phase (Peak) Threshold → voltage-gated Na+ channels open → Na+ ions flow into the cell → further depolarization ○ Overshoot: reversed direction of the electrical gradient for Na+ channels 📝 limits further influx of Na+ Figure 7. Periods of Action Potential LE 1 Trans 1 TG: BC, SC, LC, SC, KD, DL, CM, BM, NN, AS, RS, CT Page 4 of 7  Repolarization LOCAL POTENTIAL & ACTION POTENTIAL Rapid efflux of K+ through voltage-gated K+ channels → re-establishes the normal negative RMP → MP returns to AP is more frequently used than LP 👩‍⚕️ are both observed in the nerve and muscle cells 👩‍⚕️ Electrical responses used in dissemination of information and resting potential Table 5. Local Potential VS Action Potential Hyperpolarization Local Potential Action Potential This occurs when MP temporarily becomes more negative Amplitude Small Large than the RMP Intensity Threshold, maximal, Subthreshold Back to Resting State Level supramaximal Stimulus Intensity dependent Intensity independent Na+/K+ pump & leak channels help restore the MP back to its Summation Present None resting state All-or-none The neurons are in refractory period None Present Law ○ Less responsive to new stimuli Type of Passive, Active Propagated Propagation non-propagated HOW IS AN ACTION POTENTIAL PRODUCED? STRENGTH-DURATION CURVE of threshold stimulus ○ 🖥 Relationship between strength and the duration of application Weak stimuli → longer ○ Strong stimuli → shorter D. EQUILIBRIUM POTENTIAL Electrochemical gradient (electro-chemical potential difference) to move across a membrane 📖 Quantitates the driving force acting on a molecule to cause it No net movement of a specific ion across the membrane despite concentration difference REFRACTORY PERIODS Period wherein no action potential is produced nor generated even if the threshold intensity is used Ensures that conduction is unidirectional 📖 A. ABSOLUTE REFRACTORY PERIOD No action potential produced even with strongest stimulus intensity From firing level → 1⁄3 of repolarization Figure 9. Steps involved in the production of action potential Rationale: ○ Inactivation gates are still close/are already closed ○ Present in action potential and not in local responses Additional Information: The cell is totally unresponsive to whatever type of stimuli. ⇨ Why? Because the conditions for the voltage-gated channels are not met (closed Na+ activation/ M gate) ⇨ When the charge of the cells is near RMP/ hyperpolarized cell, the cell is more excitable ⇨ After hyperpolarization state/ subnormal period/ hyperpolarized cells, the cell is less excitable ⇨ The number of Na+ channels required to produce an action potential cannot be met. B. RELATIVE REFRACTORY PERIOD AP is produced if stimulus intensity is above/greater than threshold potential Starts after 1⁄3 of repolarization → end of hyperpolarization Rationale: ○ Some Na+ channels (H-gate) can be opened using strong-intensity stimulus → depolarization within the cell Figure 10. Periods of action potential Figure 11. H-gate LE 1 Trans 1 TG: BC, SC, LC, SC, KD, DL, CM, BM, NN, AS, RS, CT Page 5 of 7  👩‍⚕️ A.1. REFERENCES 1. PhD, J. H. E., & MSc., M. M. H. E. (2020). Guyton and Hall Textbook of Medical Physiology (Guyton Physiology) (14th ed.). Elsevier. 2. 2. B. (2022). Ganong’s Review Medical Physiology 26e. Mc Graw Hill Education (Uk). 3. Powerpoint: Gomez, C. (2024). Cell Electrophysiology 4. 5. Previous Trans: 2022 REVIEW QUESTIONS 1. This protein is responsible for generating the resting membrane potential. A. Na+/K+ ATPase B. K+ leaky channels A.2. C. Na+-glucose cotransport D. Na+-H+ exchange RATIONALE ANSWER: B K+ leaky channels are primarily responsible for generating the RMP in cells. These channels allow potassium ions to move out of the cell, leading to a negative charge inside the cell relative to the outside. The Na+/K+ ATPase is only responsible for maintaining the RMP. 2. Which of the following factors can contribute to the termination of an action potential? A. Decrease sodium permeability. B. Increased potassium permeability. C. Continuous influx of calcium ions. D. Opening of ligand-gated sodium channels. RATIONALE ANSWER: B During the termination of an action potential, the increase in potassium permeability allows K+ ions to flow out of the Figure 12. Absolute refractory vs. relative refractory neuron, repolarizing the membrane and bringing the QUESTION: “Do all cells produce action potential?” 👩‍⚕️ membrane potential back toward its resting state. 3. The Goldman-Hodgkin-Katz Equation differs from the ANSWER: Nernst Equation primarily by: NO, only excitable cells can produce action potential A. Considering only one ion species 1. Nerve cells B. Ignoring the effect of temperature on ion movement 2. Muscle Cell C. Accounting for the permeability of of multiple ion ○ Skeletal Muscles species ○ Cardiac Muscles D. Predicting the equilibrium potential for a single ion ○ Smooth Muscles 📝 TYPES OF LOCAL POTENTIAL 4. During the relative refractory period of an action potential: Electronic Potential A. No stimulus, regardless of strength, can initiate ○ Generated along the nerve from positive to negative pole another action potential Generator Potential B. A stronger-than-usual stimulus can initiate another ○ Generated in the sensory receptors (nerve) action potential Synaptic Potential C. It is easier to generate another action potential as ○ Generated in synapses (between two neurons or the membrane is hyperpolarized neurons to muscle cells) D. Sodium channels are open and the membrane is ○ Skeletal Muscles - End plate potential (between nerve depolarized. and skeletal muscle) ○ Smooth Muscles 5. During which phase of the action potential are Excitatory postsynaptic potential (EPSP) voltage-gated potassium channels fully open, allowing Excitatory neurotransmitters potassium ions to leave the cell? Inhibitory postsynaptic potential (IPSP) A. Resting state Inhibitory neurotransmitters B. Depolarization C. Repolarization D. Hyperpolarization RATIONALE ANSWER: C During the repolarization phase, voltage-gated potassium channels open, allowing K+ ions to exit the LE 1 Trans 1 TG: BC, SC, LC, SC, KD, DL, CM, BM, NN, AS, RS, CT Page 6 of 7  neuron. This outflow of potassium ions causes the MP to return toward the resting level. Depolarization (B) involves the opening of sodium channels, while hyperpolarization (D) occurs as potassium channels remain open slightly longer than needed. The resting state (A) is when the neuron is not actively generating an AP. 6. What primarily causes the depolarization phase of an action potential? A. Inflow of sodium ions B. Outflow of sodium ions C. Outflow of potassium ions D. Inflow of potassium ions RATIONALE ANSWER: A The rapid depolarization phase is primarily caused by the inflow of sodium ions through voltage-gated sodium channels. This influx makes the inside of the neuron more positive. Potassium ions outflow (C) during repolarization. Answers: B, B, C, B, C, A REFERENCES 5. Font size: 9, Font: Arial – numbered accordingly 6. Aligned to the left 7. For Books: Author, A. (Year). Title of work (1st ed). Place of publication: Publisher 8. Online Book: Author, A. (Year). Title of article. Retrieved from URL. 9. Website with an author: Author, A. (Year). Title of article. Retrieved from URL. 10. Website without an author: Article title (Year). Title of work (1st ed). Retrieved from URL. 11. Journal Article: Author, A. (Publication Year). Article Title. Periodical Title, Volume (Issue), pp-pp. 12. Powerpoint: Lecturer, A. (Year). Title of powerpoint [lecture powerpoint]. 13. Previous Trans: Year & Section lecture transcription. LE 1 Trans 1 TG: BC, SC, LC, SC, KD, DL, CM, BM, NN, AS, RS, CT Page 7 of 7  PHYSIOLOGY: LE1 | TRANS 04 CELL 4: CELL COMMUNICATION CARINA C. GOMEZ, M.D, FPSP | 08/07/2024 OUTLINE Gene regulatory proteins Cell cycle proteins I. Cell Communication V.Signal Transduction II. Forms of Cell A. Catalytic Cell signaling or cell communication Communication Receptor-Linked ○ Cell signaling pathways ensure that cellular response to A. Gap Junctions Signal Transduction external messenger is: (SWAT) B. Contact-dependent Pathway Specific (binding to a specific receptor) Signaling or B. Nuclear Receptor Well-coordinated Juxtacrine Linked Signal Amplified response C. Chemical Signaling Transduction Tightly regulated (no over increase or deficiency) III. Principles of Cell Pathway Communication VI. Review Question FORMS OF CELL COMMUNICATION IV. Signaling Molecules Three forms of cellular communication: A. Receptors ○ Gap junctions B. Types of Membrane ○ Contact-dependent signaling (juxtacrine) Receptors ○ Chemical signaling C. Types of Intracellular Receptors Autocrine Paracrine SUMMARY OF ABBREVIATIONS Neurocrine ABBREV N/A Endocrine Neuroendocrine LEGENDS A. GAP JUNCTIONS ❗ Must know 👩‍⚕️ Lecturer 📖 Book 🖥 Presentation 📝 Old Trans Specialized connection between two adjacent cells ○ Forms tunnels/connexons (composed of connexins) LEARNING OBJECTIVES Allow diffusion of molecules between two adjacent cells At the end of the lecture, the student should be able to: ✔Give the importance of cell communication. ○ 👩‍⚕️ Usually inorganic ions, small molecules, and current flow Observed in excitable cells ✔ Enumerate the factors that affect the speed of cellular response to extracellular signals ○ Neurons ✔ Describe the three forms of cell communication. ○ Glial cells ✔ Explain the mechanism on how cells communicate via ○ Cardiac (common) extracellular messengers (receptors, second messengers, target Intercalated disc proteins, signaling pathways, cellular responses). ○ Unitary smooth muscle cells (common) ✔ Enumerate the factors that affect the speed of cellular response Syncytial smooth muscle to extracellular signals. Regulated by Ca++, H+, and cAMP ✔ Discuss the mechanism of signal transduction as to: Couple cells both electrically chemically and metabolically extracellular and intracellular. More permeable than membrane channels Communication: short distances CELL COMMUNICATION NICE-TO-KNOW: Mouse studies demonstrated that connexin deletions lead the efficiency of our body function 👩‍⚕️ We have 300 trillion of cells that must communicate to ensure Cell communication is essential for and to: to electrophysiological defects in the heart and predisposition to sudden cardiac death, female sterility, abnormal bone development, abnormal growth in the liver, ○ Coordination and integration of cellular functions cataracts, hearing loss, and a host of other abnormalities. ○ Homeostatic responses Cell to cell communication is provided by external signals: ○ Chemical messengers - most common B. CONTACT-DEPENDENT (JUXTACRINE) Includes the ff: Membrane-bound Hormones Physical cell contact Neurotransmitters For development and immune response Metabolites (ex. somatomidines) ○ Ex. antigen-presenting cells (APC) – antigen-antibody Odorant & growth factors recognition Small molecules (ions, gases, nucleotides, amino acids) C. CHEMICAL SIGNALING ○ Physical signals - transduced to chemical signals Most common type of cellular communication Thermal, mechanical, and light stimuli 📖 Cells communicate by releasing signaling molecules (e.g., hormones, neurotransmitters) surface, in the cytoplasm, or nucleus. ○ 📖 Chemical messengers bind to protein receptors on the cell Triggers intracellular changes → physiological effects ○ Molecules bind to receptors in the plasma membrane, Downregulation cytoplasm, or nucleus ○ ○ Binding activates or inactivates intracellular messengers Key proteins involved: kinases, phosphatases, and G ○ neurotransmitters are in excess. Upregulation 📖 Active receptors decrease when hormones or proteins Ligands should bind with receptors then are converted to ion channels and other transport proteins metabolic enzymes ○ chemical messengers. 📖 Active receptors increase when there is a deficiency of Cytoskeletal proteins LE 1 Trans 1 TG: BC, SC, LC, SC, KD, DL, CM, BM, NN, AS, RS, CT Page 1 of 9  Signalling cell should release these chemicals to start PRINCIPLES OF CELL COMMUNICATION communication NEUROCRINE AKA synaptic signaling ○ Occurs in synapses Signaling cells ○ Released by axon terminals of neurons into synaptic junctions and act locally to control nerve cell functions👩‍⚕️ axons and the release of neurotransmitters at synapses. ○ Affects neighboring cells, even at long distance 👩‍⚕️ Involves electrical signals (action potential) traveling through Neuron-to-cell Neuron-to-neuron Neuron-to-endocrine cell Signaling molecules affect the function of another neuron or cells in immediate vicinity but distant from neuron cell body Synapse → axons → electrical signals (AP) → neurotransmitters released → distant cells ENDOCRINE affect distant target cells (another location in the body) Widely distributed throughout the body. 📝 Hormones from endocrine cells are released into the blood to Occurs over long distances. Hormones → bloodstream → distant cells NEUROENDOCRINE Figure 1: Overview on How Cells Communicate Release of substance by neurons, which reach the target Cells release extracellular signaling molecules (hormones, cells via the bloodstream neurotransmitters, etc.) body 📖 Influence the function of target cells at another location in the Occurs over long distances ↓ Molecules bind to receptors in the plasma membrane, cytoplasm, or nucleus Endocrine signals are either neurotransmitters or hormones ↓ ○ Ex: epinephrine & norepinephrine, antidiuretic hormone, Signal transduction activates or inactivates intracellular oxytocin messengers Neurons → hormone → bloodstream → distant cells ↓ PARACRINE Receptors interact with signaling proteins (kinases, phosphatases, G proteins) Signaling molecules are released by signaling cells to affect ↓ other cells of different type (same tissue) Signaling proteins regulate target proteins (ion channels, Signal molecules are rapidly inactivated by extracellular transport proteins, enzymes, cytoskeletal proteins, gene enzymes, taken up by target cells (endocytosis), or regulators, cell cycle proteins) immobilized by the extracellular matrix. ↓ ○ Ex: Histamine from ECL (enterochromaffin-like cells) Cellular response affects parietal cells to secrete HCl. Short duration and distance. CELL COMMUNICATION AUTOCRINE Signaling molecules frequently have different effects on its Signaling molecules are released and affect the same cell or target cell. other cells of the same type. Speed of cellular responses on extracellular signals depends ○ Ex: Insulin from pancreatic beta cells affect beta cells via on the following; 👩‍⚕️ diffusion. ○ Mechanism of delivery Short duration due to rapid enzyme degradation. Secreted to extracellular fluid and affects the function of the ○ signaling (seconds to minutes) Changes on cellular activity 👩‍⚕️ Ex: neurocrine (milliseconds) faster than endocrine same cells that produced them. Ex: changes in protein activity (milliseconds) faster gene expression and de novo synthesis of proteins (hours to days) ○ Ability of the molecule to reach a particular cell Ex. electrical signal is faster than travel via blood ○ Expression of cognate receptor ○ Cytoplasmic signaling molecule Ex. gene transcription (slow) Antagonism by constitutive and regulated feedback mechanisms ○ Minimize the response and provide tight regulatory control over these signaling pathways LE 1 Trans 1 TG: BC, SC, LC, SC, KD, DL, CM, BM, NN, AS, RS, CT Page 2 of 9  SIGNALING EVENTS INITIATED BY MEMBRANE Catecholamines (epinephrine, norepinephrine) (exocytosis) ASSOCIATED RECEPTORS Steroid hormones (estrogen, progesterone, calcitriol, Step I - Recognition of the signal by its receptor aldosterone, and retinoids) (diffusion) Step II - Transduction Iodothyronines (T3 and T4) (diffusion) Step III - Transmission Eicosanoids (prostaglandins, leukotrienes, thromboxane, Step IV - Modulation prostacyclin) (exocytosis) Step V - Response Small molecules (amino acids, nucleotides, ions) Step VI - Termination Gases (nitric oxide, carbon dioxide) (diffusion) ION-CHANNEL LINKED RECEPTOR SIGNALING MOLECULES Signaling events initiated by membrane associated receptors Extracellular signaling molecule (AKA ligand or first ❗ AKA ligand-gated ion channels or ionotropic ❗ Receptor linked to an ion channel Once the signaling molecule binds to the receptor of the messenger) ion channel, it will open and close the ion channel. ○ Binds with a receptor with high affinity and specificity Mediate direct and rapid synaptic signaling between ○ Produces response depending or the expression of electrically excitable cells receptors It affects the ionic permeability of the membrane and ○ Released via: membrane potential Exocytosis – proteins (water-soluble compounds) ○ Open = Increase permeability Diffusion – lipid-soluble compounds ○ Close = decrease permeability ○ Secretion is cell type specific Example: Nicotinic Acetylcholine receptor which is present CLASSES OF SIGNALING MOLECULES (LIGANDS) in the skeletal muscle is the one being utilized by Acetylcholine Signal molecules are released by signaling cells to affect 2 ATP Receptors other cells of different type ○ P2X (Ion-Channel). P2Y (G-Protein) Produce response depending on expression of receptor ○ Hormones and neurotransmitters ○ Peptides and proteins (insulin, ADH, prolactin, TSH, oxytocin) ○ Catecholamines (epinephrine and NE) ○ Steroid hormones (estrogen, calcitriol, progesterone and aldosterone, retinoids) ○ Iodothyronines (T3 and T4) Figure 2. Ligand-Ion Gated Channel ○ Eicosanoids (prostaglandin, leukotrienes, thromboxanes and prostacyclins) G-PROTEIN COUPLED RECEPTOR ○ Small molecules (amino acids, nucleotides and ions) AKA Metabotropic Receptor/ Seven Helix Receptor ○ Gases (nitric oxide and carbon dioxide) A. RECEPTORS 👩‍⚕️ Serpentine Receptor 👩‍⚕️ Most abundant Muscarinic Structure where the ligand binds Regulates the activity of two proteins Acts as signal transducers ○ Both affects the ion channel and enzyme ○ End point: cellular response Largest category of plasma membrane proteins Requirement: Stimulation of G-Protein causes: ○ Converts all forms of stimuli to a cellular response ○ Ion channel: change ionic membrane permeability and Types/Classes: membrane potential ○ Membrane receptors ○ Enzyme: activates or inhibits target proteins that For substances that cannot cross the membrane (water-soluble) regulate signaling pathways 📝 Seven transmembrane proteins ( segments that loop in and out of the cell membrane) Proteins Catecholamines Basic Components: ○ Intracellular Receptors ○ Receptors - where the ligand binds For lipid-soluble substances ○ G-protein complex - α, β, and γ subunit ○ Membrane bound enzyme or ion channel B. TYPES OF MEMBRANE RECEPTORS Adenylate cyclase Phospholipase (A2, C and D) AKA plasma membrane receptors Guanylate cyclase Requirement: Phosphodiesterase ○ Extracellular signaling molecule (Ligand/ First ○ Heterotrimeric G proteins - “tri” = 3 subunits (α, β, and γ) ❗ Messenger) Individual messengers (or ligands) typically bind to a plasma membrane receptor to initiate Substances that can bind to these proteins: Peptides Odorants 📖 intracellular changes that lead to physiological changes ( Ganong) Binds with a receptor with high affinity and high Cytokines specificity. Produces response depending on the receptor's expression. Released via exocytosis (neurocrine, endocrine) or diffusion (autocrine, paracrine) (small) Secretions are cell type specific Examples: Hormones and Neurotransmitter ○ Peptides and Proteins (Insulin, ADH, Prolactin, TSH, Figure 3. G-Protein-Coupled Receptor Oxytocin) (exocytosis) LE 1 Trans 1 TG: BC, SC, LC, SC, KD, DL, CM, BM, NN, AS, RS, CT Page 3 of 9 ENZYME-LINKED RECEPTOR AKA catalytic receptors ❗ MUST-KNOW: ALZHEIMER’S DISEASE Progressive neurodegenerative disease characterized Affects only the enzyme by the formation of amyloid plaques Functions as an enzyme Manifestation: progressive memory loss ○ Associated with or regulate an enzyme Mechanism: Most of these enzymes are protein kinases or associated Amyloid-β protein precursor with protein kinases (phosphorylation; phosphatases = (RIP) dephosphorylate) ↓ Cause phosphorylation of specific proteins that change Amyloid-β protein protein activity (accumulates) Seven transmembrane protein ↓ Formation of amyloid plaques ↓ Neurodegenerative disease C. TYPES OF INTRACELLULAR RECEPTORS AKA nuclear receptors These receptors are for the fat-soluble signaling molecule (substances that can cross the membrane) that can passively Figure 4. Enzyme-Linked Receptor enter the plasma membrane, which gives a signal to the nucleus to respond REGULATED INTRAMEMBRANE PROTEOLYSIS (RIP) ○ Steroid hormones Pag nagbind sa kanya, nagkakaroon ng proteolysis ○ Retinoids ○ Breakdown of proteins into smaller polypeptide chains or amino acid fragments Involves membrane proteins that do not fit the classic ○ ○ 👩‍⚕️ Vitamin D Sex Hormones Some are located in the cytoplasm and enter the nucleus definition of receptors after binding (for cortisol and aldosterone) ○ What is the classic definition of receptors? Some are located in the nucleus (thyroid hormone) Usually binds with a ligand Example: Transduce signal Affect the receptor It only has a receptor-like function ○ Recognize extracellular signaling molecules ○ Act as transducer Facilitated by membrane of protein known as metalloproteinase-disintegrin family Figure 7. Steroid Hormone Stimulate the Transcription of Early-Response Genes and Late Response Table 1. Intracellular vs Membrane Receptors Solubility Membrane Permeability Intracellular Lipid-soluble Can cross membrane Figure 5. Regulated Intramembrane Proteolysis (RIP) Membrane Water-soluble Cannot cross membrane Example RECEPTOR ACTIVATION When a signaling molecule (ligand) binds with the receptor 🡪 the receptor is activated 🡪 there should be conformational changes in the receptor 🡪 the transduction will happen because they serve as their transducers CHARACTERISTICS OF MESSENGER-RECEPTORS INTERACTION Specificity - (lock and key Interaction) e.g., Insulin molecule will only bind with Insulin receptor High Affinity - Substance should bind immediately with the receptor because some are easily inactivated by enzymes Figure 6. Sterol Regulatory Element-Binding Protein LE 1 Trans 1 TG: BC, SC, LC, SC, KD, DL, CM, BM, NN, AS, RS, CT Page 4 of 9  Saturation - Degree to which receptors are occupied 🡪 other substances will not be able to bind anymore (Vmax), plateau (Acetylcholine = mabilis ang effect kasi madaling sirain) Competition - Ability of different molecules very similar in structure to combine with the same receptor; compete for the same receptor REGULATION OF MESSENGER-RECEPTOR INTERACTIONS Down-regulation ○ AKA desensitization or adaptation ○ Caused by prolonged exposure to hormone ○ Receptor undergoes endocytosis ○ Cellular decrease in the number of receptors ○ Reduces or terminates the response Up-regulation ○ AKA sensitization ○ Cellular increase in the number of receptors ○ Amplify or integrate signals SIGNAL TRANSDUCTION Figure 8. G-Protein Coupled Signal Transduction Pathway A process by which a stimulus is transformed into a Uses intracellular signaling molecules (2nd messengers) response Types of pathways Act as molecular switches (active - inactive or vise versa) ○ pathways initiated by extracellular receptors (Via IC ○ Examples: signaling pathway cAMP (adenylate cyclase) ○ pathways initiated by intracellular receptors (Via cGMP (guanylate cyclase) regulation of gene expression) Calcium Pathways initiated by extracellular receptors DAG (PLC) ○ Relay signals via intracellular signaling pathway IP3 (PLC) ○ Uses intracellular signaling molecules (second messengers = only formed in the G-Protein) Monomeric G-protein has 5 families 1. Ras- cell division, proliferation and death A. ION CHANNEL-LINKED SIGNAL TRANSDUCTION 2. Rho- actin cytoskeletal organization, cell cycle progression PATHWAY and gene expression Signal transduction → membrane potential → polarization or 3. Rab - intravesicular transport and trafficking proteins hyperpolarization 4. Ran - nucleocytoplasmic transport of RNA Receptor itself constitutes an ion channel 5. Arf - vesicular transport Receptor activation causes channel opening (or closing) and 📝 subsequent entry (or nonentry) of ions Can cause changes in membrane potential and membrane Adenylate cyclase Catalyzes conversion of cytosolic ATP to cAMP 📝 permeability 👩‍⚕️ Typical ligand: Neurotransmitter Usually observed in Muscle Activated when ligand binds to G protein (alpha s), -TSH, ACTH, LH, glucagon, PTH, epinephrine - ↑ cAMP Inhibited when it binds G (alpha i) - catecholamines - ↓ cAMP B. G-PROTEIN COUPLED SIGNAL TRANSDUCTION cAMP activates PKA PATHWAY G-protein complex: 1000 receptors ○ α: 16 ○ β: 5 📝 ○ γ: 11 Pag wala pang activation the G-protein complex is linked 📝 to a receptor via GDP (Guanosine diphosphate) Pag nag-bind na ang receptor the G-protein will dissociate the receptor because na-activate na iyong receptor o once nag-attached to the receptor there would be transduction and 📝 activation and the GDP will become GTP Add phosphate to GDP and becomes GTP, hihiwalay na si alpha para i-activate yung enzyme or channel Figure 9. Adenylate cyclase pathway LE 1 Trans 1 TG: BC, SC, LC, SC, KD, DL, CM, BM, NN, AS, RS, CT Page 5 of 9  Phosphodiesterase EPOXYGENASE PATHWAY Catalyzes conversion of systolic cGMP to GMP Epoxygenase enzyme catalyzes the release of Activated when ligand binds to G protein (alpha t), - ↓ cAMP hydroxyeicosatetranoic acid (HETE) and cis- epoxyeicosatetranoic acid (EET) from ARA ○ Both of which increase the release of calcium from the endoplasmic reticulum and promotes cell proliferation Calcium as an ubiquitous messenger Cellular concentration kept extremely low (relative to ECF) by active processes Calcium binding is key to the ff. Intermediary proteins: ○ Calmodulin (particular on smooth muscle cells) Once the calcium binds it will activate another kinase (myosin light chain kinase) cAMP (protein kinase A) DAG (protein kinase C) cGMP (protein kinase G) ○ Troponin (particularly on skeletal and cardiac cells) To others, causing structural alteration upon binding Figure 10. Phosphodiesterase pathway INTRACELLULAR TARGET PROTEINS Phospholipase C REVIEW NOTE: Catalyzes conversion of cytosolic inositol diphosphate (IP2) Extracellular signal transduction depends on the type of to IP3 and DAG membrane receptor where the ligand binds. Under the G Examples: vasopressin receptor in liver and acetylcholine PROTEIN signal transduction pathway: when a ligand receptors in smooth muscle binds with the GPCR, the protein complex will dissociate activated when ligand binds to G protein (alpha q) and will either activate or inhibit downstream target ↑ DAG - V PKC proteins. ↑ IP3 - (+)SR - ↑ Ca release Phospholipase A2 (PLA2) pathway Table 2. Summary: Second Messengers Enzyme that release arachidonic acid from membrane G Protein phospholipids Membrane enzyme 2nd messenger Target proteins ɑ-subunit arachidonic released from cells ---- (+) Protein kinase ○ Regulates neighboring cells Gs (ɑs) (+) Adenylate cyclase ↑ cAMP A (PKA) ○ Stimulates inflammation catalyzes the release of arachidonic acid (ARA) from Gi (ɑi) (-) Adenylate cyclase ↓ cAMP (-) PKA phospholipids ↓ cGMP Gt (ɑt) (+) Phosphodiesterase PKG ○ ARA released from PLA2 activity either (cGMP→GMP) ARA will now be acted upon by either of these 3 ↑ Diacylglycerol enzymes: (+) PKC (DAG) Cyclooxygenase Gq (ɑq) (+) Phospholipase C Lipoxygenase ↑ Inositol ↑ Release of triphosphate Epoxygenase Ca++ from SR (IP3) CYCLOOXYGENASE (COX) PATHWAY G (+) Phospholipase A2 ↑ Arachidonic acid Cyclooxygenase (COX) enzyme catalyzes the release of Ca++ Ca++ Calmodulin MLCK the following products from ARA: Ubiquitous ○ Thromboxane - mediates platelet aggregation and vasoconstriction PROTEIN KINASE - any enzyme that adds a phosphate ○ Prostacyclin - inhibits platelet aggregation and group (phosphorylation) to other proteins, causing a change vasodilation in the activity or conformation of the protein ○ Prostaglandin - mediates platelet aggregation, PROTEIN PHOSPHATASES - catalyze removal of airway constriction and induces inflammation phosphates from proteins (dephosphorylation) Cyclooxygenase is the inhibitory target of drugs like SECOND MESSENGERS - serve as molecular aspirin and mefenamic acid, hence their categorization switches—turning on or turning off the activities of enzymes. as COX inhibitors G PROTEIN ACTIVATION - may increase or decrease cellular levels of second messengers LIPOXYGENASE (LOX) PATHWAY Lipoxygenase (LOX) enzyme catalyzes the release of QUESTION: So how do second messengers initiate cellular leukotrienes from ARA responses? ○ Leukotriene - participates in allergic and ANSWER: inflammatory response (i.e., asthma, rheumatoid Activation of second messenger-dependent kinases arthritis, and inflammatory bowel disease) Activation and closure of ion channels LE 1 Trans 1 TG: BC, SC, LC, SC, KD, DL, CM, BM, NN, AS, RS, CT Page 6 of 9  Examples of membrane-bound proteins regulated by RECEPTOR THREONINE/ SERINE CYCLASE G-protein receptors Activated by TGF-B (Transforming growth factor) Adenylate cyclase Guanylate cyclase Phospholipase C Phospholipase A2 Ion Channels 2nd messenger-dependent kinases cAMP-dependent protein kinase (PKA) cGMP-dependent protein kinase (PKG) DAG (Protein Kinase C) Ca++/calmodulin-dependent protein kinase (MLCK) ○ CALMODULIN - found in all cells and has 4 Ca++ binding sites SIGNAL TRANSDUCTION A. CATALYTIC RECEPTOR-LINKED SIGNAL TRANSDUCTION PATHWAY RECEPTOR GUANYLYL CYCLASE Activated by ANP (atrial natriuretic peptide) and NO (nitric oxide) RECEPTOR TYROSINE KINASES Activated by Insulin, Epidermal/Nerve Growth Factor and; Platelet Derived Growth Factor Figure 11. Activation of tyrosine-kinase NGF Figure 12. Activation of tyrosine-kinase insulin LE 1 Trans 1 TG: BC, SC, LC, SC, KD, DL, CM, BM, NN, AS, RS, CT Page 7 of 9  B. NUCLEAR RECEPTOR LINKED SIGNAL TRANSDUCTION PATHWAY Two subfamilies (structure and mechanism of action) ○ Receptors that bind steroid hormones (glucocorticoid and mineralocorticoid) ○ Receptors that bind retinoic acid, vitamin D, thyroid hormones, and steroids (estrogen and progesterone) TYROSINE KINASE-ASSOCIATED RECEPTOR Activated by Cytokine, Erythropoietin, Prolactin, Growth Hormone, Interleukins No enzymatic activity but is associated tyrosine Kinase Belongs to Src and Janus Family Sometimes known as the JAK/STAT pathway REFERENCES 1. PhD, J. H. E., & MSc., M. M. H. E. (2020). Guyton and Hall Textbook of Medical Physiology (Guyton Physiology) (14th ed.). Elsevier. 2. B. (2022). Ganong’s Review Medical Physiology 26e. Mc Graw Hill Education (Uk). 3. Md, B. K. M., PhD, & PhD, B. S. A. (2017). Berne & Levy Physiology (7th ed.). Elsevier. 4. Powerpoint: Gomez, C. (2022). Cell Electrophysiology 5. Previous Trans: 2021 REVIEW QUESTIONS 1. Which type of cellular communication involves signaling molecules being released into the bloodstream to affect distant target cells?