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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Scaramello
Christianne B V
CB
Departamento de Farmacologia Básica e Clínica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro 21941-590, Brazil.
Cunha
Valéria M N
VM
Rodriguez
Juliane B R
JB
Noël
François
F
D000818:Animals; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D027682:Cation Transport Proteins; D006706:Homeostasis; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D008297:Male; D053499:Plasma Membrane Calcium-Transporting ATPases; D051381:Rats; D012433:Ryanodine / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D013347:Subcellular Fractions / Q000737:chemistry* / Q000378:metabolism*; D014649:Vas Deferens / Q000166:cytology* / Q000378:metabolism*
D027682:Cation Transport Proteins; D012433:Ryanodine; D015544:Inositol 1,4,5-Trisphosphate; D053499:Plasma Membrane Calcium-Transporting ATPases; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium