Effect of Amlodipine on Isolated Coronary Tone PDF

Lab 3 : Effect of Amlodipine on the isolated coronary tone: Smooth muscle cells are the major constituent of the blood vessel wall. Vascular smooth muscle contractility is determined by the free intracellular Ca2+ and Ca2+ sensitivity of contractile proteins. The entry of Ca2+ from the extracellula...

Lab 3 : Effect of Amlodipine on the isolated coronary tone: Smooth muscle cells are the major constituent of the blood vessel wall. Vascular smooth muscle contractility is determined by the free intracellular Ca2+ and Ca2+ sensitivity of contractile proteins. The entry of Ca2+ from the extracellular space, through Ca2+ channels, as well as Ca2+ release from intracellular stores results in Ca2+ binding to calmodulin, the complex in turn activates myosin light chain kinase (MLCK) which is the key event in smooth muscle contraction. Mechanism of contration: Role of K+ channel: Opening of K+ channels in the cell membrane increases K+ efflux with membrane potential hyperpolarization. This effect is followed by the closure of voltage- dependent Ca2+ channels, with subsequent reduction in Ca2+ entry and vasodilation. Closure of K+ channels has the opposite effect i.e. K+ channels inhibition causes membrane depolarization and vasoconstriction. Thus, the presence of a physiological or pharmacological agent that alters membrane potential. Figure 1. Role of Ca2+ channel: Voltage-gated Ca2+ channels (VDCCs) play a central role in the regulation of vascular tone and blood pressure regulation. Activation of Ca2+ channels by membrane depolarization, leads to Ca2+ influx, Ca2+ elevation, and vasoconstriction. Whereas membrane potential hyperpolarization closes voltage-gated Ca2+ channels with subsequent vasodilatation. Figure 1. Figure 1: Smooth muscle cell explain the role of K+ and Ca2+ channels. Method: Bovine coronary arteries were used to characterize the effects of amlodipine. Figure 2. 1. The coronary segments were obtained and transported to the laboratory in a tube containing Krebs buffer gassed with a mixture of 95% O2 and 5% CO2. 2. The artery was cleaned of fat and connective tissues. 3. Each segment ring was suspended between two metal hooks in organ baths. The upper hook was connected to a force transducer for tension recording while the other was connected to a fixed hook. Each bath was connected to a thermostat to maintain the temperature at 37oC and constantly gassed with air. 4. Tissues were initially pre-tensioned to 8 gm and then left to relax to baseline for approximately 30 minutes. 5. Two consecutive KCl responses with bath concentration of 60 mM were obtained for standardization. After about 10 min the tissue was washed from KCl with Kreb, and then left to relax to baseline, the process was then repeated with a second KCl addition, then washed and left to relax. 6. third KCl the tissue contracted with KCl and treated with amlodipine, and left for about 1 hour. Compare the effect of amlodipne with the segment treated with the ethanol (solvent control). Note: High concentration KCl (60mM) in the bath inhibit K+ leak, change membrane potential (depolarization) causing activation of voltage gated Ca2+ channel with subsequent Ca2+ influx and contraction. Note: Agonists such as endothelin, thromboxane and angiotensins act on Gq proteins, activate second messengers, binds to the endoplasmic reticulum, causing release of Ca2+ into the cytosol. With subsequent sustained contraction. Figure 2: Tissue Bath representing a ring of tissue placed in the double-wall jacket. Krebs Solution: Sodium Chloride: 118 mM about 6.9 gm in 1L. Potassium Chloride: 4.8 mM about 0.36 gm in 1L. Magnesium Sulphate: 1.1 mM about 0.29 gm in 1L. Sodium hydrogen carbonate: 25mM about 25 gm in 1L. Potassium dihydrogen phosphate: 1.2 mM about 1.2 gm in 1L. Glucose: 12 mM about 12 gm in 1L. Calcium chloride: 1.25mM about 1.25 gm in 1L. Figure shows the effect of amlodipine on the contracted coronary vessels.