Lab 3: Amlodipine and Coronary Tone Effects

Study Notes

Smooth muscle cells are the primary component in blood vessel walls.
Contractility is determined by intracellular Ca²⁺ and Ca²⁺ sensitivity of contractile proteins.
Ca²⁺ from the extracellular space enters through Ca²⁺ channels and is released from intracellular stores.
Ca²⁺ binds to calmodulin, which activates myosin light chain kinase (MLCK).
MLCK is crucial for smooth muscle contraction.

Role of K⁺ channels: Opening increases K⁺ efflux, causing membrane hyperpolarization.
This leads to closure of voltage-dependent Ca²⁺ channels, reducing Ca²⁺ entry, and vasodilation.
Inhibition of K⁺ channels causes membrane depolarization and vasoconstriction.
Physiological or pharmacological agents can alter membrane potential.
Role of Ca²⁺ channels: Voltage-gated Ca²⁺ channels (VDCCs) regulate vascular tone and blood pressure.
Activation by membrane depolarization leads to Ca²⁺ influx, increased Ca²⁺ levels, and vasoconstriction.
Hyperpolarization closes VDCCs, resulting in vasodilation.

Bovine coronary arteries were used to study amlodipine effects.
Arteries were obtained in Krebs buffer (95% O₂ and 5% CO₂).
Tissues were cleaned of fat and connective tissue.
Segments were suspended in organ baths, pre-tensioned to 8 gm, and allowed to relax for 30 minutes.
Two KCl responses (60 mM) were used for standardization.
The third KCl response was used to test amlodipine.
Tissues were treated with amlodipine and compared to an ethanol control.
High KCl concentration (60mM) inhibits K⁺ leak, changes membrane potential (depolarization), causing voltage-gated Ca²⁺ channel activation. Leading to Ca²⁺ influx and contraction.
Agonists like endothelin, thromboxane, and angiotensins trigger Gq protein activation, and second messenger release of Ca²⁺ into the cytosol. This leads to sustained contraction.

Figure 1: Illustrates the role of K⁺ and Ca²⁺ channels in smooth muscle cells.
Figure 2: Depicts the tissue bath setup.
Figure 3 Presentation of experimental data showing effect of amlodipine on contracted vessels. Krebs' solution's composition with concentrations.