Delineation of high and low affinity 45Ca incorporation and of 45Ca efflux in canine aortic smooth muscle microsomes

Specific Alterations in Binding and Uptake of High and Low Affinity Ca⁺⁺ in Canine Aortic Microsomes

“…The effect of Sr++ on 45Ca efflux from canine aortic microsomes was similar to that of Ca++ [Kutsky et al, 1980]. The influence of neomycin and gentamicin on 45Ca efflux from canine aortic microsomes preincubated for 1 h in 0.03 mM Ca++ was examined in a similar manner.…”

“…Strips were subsequently incubated for 60 min in the experimental solutions containing 45Ca; n = 6. 2 Uptakes were calculated as the product of the extracellular Ca" concentration (approximately 0.031 or 3.001 including 0.001 m M for added 45Ca) and the difference between the 4SCa tissue/medium ratio and the extracel lular ( l4C-sucrose) space of 0.41 ml/g [as reported previously by Kutsky et al, 1980). 5 The level of significance (p) is calculated for the difference (unpaired) between control values and those obtained in the presence of Sr" .…”

“…30 mM Tris and 5.0 mM sodium azide. The diluted microsomes were incubated at 37 °C and aliquots were Millipore-filtered at 2-min intervals for 32 min [Kutsky et al, 1980]. Agents under investigation were added to the medium after 20 min of washout.…”

“…The tissue space value (a tissue/mcdium ratio ex pressed in terms of milliliters per gram of tissue) is obtained by dividing the amount of radioisotope taken up per gram of tissue (wet weight) by the concentration of radioisotope per milliliter of incubation solution. The 45Ca space values were converted to uptake units (moles per gram of muscle) after subtraction of the extracellular (l4C-sucrose) space of 0.41 ml/g [Kutsky et al, 1980) by multiplying them by the total concen tration of Ca" in the 45Ca incubation solution. This was estimated as the concentration of added Ca" plus a maximum of 1.0 jxM Ca" from contaminants (from other reagents) and from the high specific activity 45Ca solution.…”

“…This provides a new parameter for delineating the actions of agonists and antagonists on tension develop ment and Ca++ movements in vascular smooth muscle. This approach, in combina tion with washes in high (80.8 m M) lan thanum solutions at low (0.5 °C) temperature (which remove superficial Ca++ and block essentially all Ca++ influx and efflux), demon strates that norepinephrine and K+ have dif ferent effects on Ca++ bound at either high or low affinity Ca++ binding sites [Karaki and Weiss, 1979], A microsomal membrane fraction from canine aortic smooth muscle has been char acterized [Kursky and Goodman, 1978], and Scatchard-coordinate plots indicate that two Ca++ uptake components, similar to those found in the whole tissue, are also present in this canine aortic microsomal fraction [Kutsky et al, 1980], This type of analysis has revealed a similar separation of Ca++ uptake components in cardiac sarcolemma [Morcos and Jacobson, 1979], bovine carotid media intimal microsomes [Klinneret al, 1978] and guinea pig ileal microsomes [Godfraind et al, 1976]. We have shown that, in 0.03 m M Ca++ (a concentration at which high affinity Ca++ uptake predominates).…”