Characterization of subcellular fractions and distribution profiles of transport components involved in Ca(2+) homeostasis in rat vas deferens

The sarcoplasmic reticulum present in eukaryotic cells contains Ca(2+) pumps (SERCA type) that accumulate Ca(2+) from the cytosol and Ca(2+) channels, such as ryanodine receptors and inositol 1,4,5-trisphosphate receptors, that release Ca(2+) from the lumen of this organelle. The use of a preparatio...

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Published in:Journal of pharmacological and toxicological methods 2002-03, Vol.47 (2), p.93-98
Main Authors: Scaramello, Christianne B V, Cunha, Valéria M N, Rodriguez, Juliane B R, Noël, François
Format: Article
Language:English
Subjects:
Animals
Biological Transport, Active
Calcium - metabolism
Calcium-Transporting ATPases - metabolism
Cation Transport Proteins
Homeostasis
In Vitro Techniques
Inositol 1,4,5-Trisphosphate - metabolism
Male
Plasma Membrane Calcium-Transporting ATPases
Rats
Ryanodine - metabolism
Sarcoplasmic Reticulum - enzymology
Sarcoplasmic Reticulum Calcium-Transporting ATPases
Subcellular Fractions - chemistry
Subcellular Fractions - metabolism
Vas Deferens - cytology
Vas Deferens - metabolism
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Summary:The sarcoplasmic reticulum present in eukaryotic cells contains Ca(2+) pumps (SERCA type) that accumulate Ca(2+) from the cytosol and Ca(2+) channels, such as ryanodine receptors and inositol 1,4,5-trisphosphate receptors, that release Ca(2+) from the lumen of this organelle. The use of a preparation rich in sarcoplasmic reticulum vesicles and poorly contaminated with plasmalemmal vesicles would be a prerequisite for studies of Ca(2+) efflux through ryanodine and inositol 1,4,5-trisphosphate receptors, so the present work was aimed to characterize the distribution profiles of various markers of sarcoplasmic reticulum and plasma membrane among fractions obtained from rat vas deferens. Oxalate-dependent Ca(2+) uptake, thapsigargin-sensitive (Ca(2+)-Mg(2+)) ATPase activity and binding of [3H]ryanodine and [3H]inositol 1,4,5-trisphosphate were measured in the nuclear, mitochondrial, and microsomal fractions obtained by differential centrifugation of rat vas deferens homogenate. The recovery of the thapsigargin-resistant (Ca(2+)-Mg(2+)) ATPase activity, supposed to label the plasma membrane, was the same among nuclear, mitochondrial, and microsomal fractions, whereas the recovery of the thapsigargin-sensitive (Ca(2+)-Mg(2+)) activity, oxalate-dependent Ca(2+) uptake, and [3H]inositol 1,4,5-trisphosphate binding, used as sarcoplasmic reticulum markers, was higher in nuclear fraction than in the others. The recovery profiles of the four sarcoplasmic reticulum markers, including [3H]ryanodine binding, were statistically the same among the different subcellular fractions. Caffeine, an agonist of ryanodine receptors, induced the release of 17% of Ca(2+) taken up by the vesicles present in the nuclear fraction but had no effect in microsomes. Although this nuclear fraction is less purified in sarcoplasmic reticulum markers than the microsomal fraction, it is more suitable for studying Ca(2+) release through ryanodine receptors, primarily because it is less contaminated with vesicles from the plasma membrane which are able to take up Ca(2+) but are insensitive to caffeine.
ISSN:1056-8719
1873-488X
DOI:10.1016/S1056-8719(02)00205-8