1. Endothelial cell activation is correlated with increased cytosolic Ca¥ concentration, often monitored with cytoplasmic Ca¥ dyes, such as fura_2 and Calcium Green-1. We tested the hypothesis that during weak stimulation of porcine coronary artery endothelial cells, focal, subplasmalemmal Ca¥ elevations occur which are controlled by cell membrane Na¤-Ca¥ exchange near mitochondrial membrane and superficial endoplasmic reticulum (SER). 2. Bulk Ca¥ concentration ([Ca¥]b) was monitored using fura_2 or Calcium Green-1 and subplasmalemmal Ca¥ concentration ([Ca¥]sp) was determined with FFP-18. The distribution of the SER network was estimated using laser scanning and deconvolution microscopy. 1 nmol l¢ Bk or 10 mmol l¢ NaF yielded focal [Ca¥] elevation in the subplasmalemmal region with no increase in the perinuclear area. 6. Treatment with 10 ìmol l¢ nocodazole caused the SER to collapse and unmasked Ca¥ release in response to 1 nmol l¢ Bk and 10 mmol l¢ NaF, similar to low-Na¤ conditions, while the effect of thapsigargin was not changed. 7. These data show that in endothelial cells, focal, subplasmalemmal Ca¥ elevations in response to small or slow IP× formation occur due to vectorial Ca¥ release from the SER towards the plasmalemma followed by Ca¥ extrusion by Na¤-Ca¥ exchange. While these local Ca¥ elevations are not detectable with Ca¥ dyes for the determination of [Ca¥]b, prevention of Ca¥ extrusion or SER disruption yields increases in [Ca¥]b partially due to CICR. 8. All of the data support our hypothesis that in weakly stimulated endothelial cells, intracellular Ca¥ release and [Ca¥] elevation are limited to the subplasmalemmal region. We propose that the SER co-operates with associated parts of the plasma membrane to control Ca¥ homeostasis, Ca¥ distribution and Ca¥ entry. The existence of such a subplasmalemmal Ca¥ control unit (SCCU) needs to be considered in discussions of Ca¥ signalling, especially when cytoplasmic Ca¥ dyes, such as fura_2 or Calcium Green-1, are used.

The phenotypic dedifferentiation of vascular smooth muscle cells (SMCs) is an early event associated with cell culturing and vascular injury. The purpose of this study was to evaluate the SMC phenotype underlying the functional responsiveness of SMCs to nucleotides in organ culture. Porcine coronary arteries were either used fresh, cold stored (5°C) 4 days, or organ cultured (37°C) 4 days. Fura-2 digital imaging of single SMCs was used to measure the myoplasmic calcium (Ca_m) response to 10 µM of the following nucleotide receptor agonists: UTP, UDP, ATP, ADP, and 2-MeSATP. In contrast to the nucleotides UDP, ATP, ADP, and 2-MeSATP, the Ca_m response increased 10-fold and the number of cells that responded to UTP increased 5-fold in SMCs from organ culture compared to SMCs from fresh or cold-stored arteries. Simultaneous imaging of Ca_m, DNA content, and SR distribution in SMCs from organ culture indicated that the UTP-induced Ca_m increase occurred exclusively in SMCs that had a dedifferentiated cell phenotype. Three-dimensional image reconstruction of the nucleus and sarcoplasmic reticulum (SR) revealed a novel transnuclear SR distribution that intertwined with the nucleus in fresh SMCs, while in SMCs from organ culture the SR was predominantly perinuclear and cytoplasmic. This study demonstrates that the functional up-regulation of UTP-sensitive receptors and the disappearance of the transnuclear SR distribution are novel features of dedifferentiated coronary SMCs.

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A new, simple classroom technique helps cell biology students understand principles of Michaelis-Menten enzyme kinetics. A student mimics the enzyme and the student's hand represents the enzyme's active site. The catalytic event is the transfer of marbles (substrate molecules) by hand from one plastic container to another. As predicted, increases in marble concentration increase the number of marbles transferred per unit time (initial rate, V 0 ) until the turnover number becomes rate limiting and V 0 approaches the maximum velocity (V max ), as described by the Michaelis-Menten equation. With this demonstration, students visualize an important concept: the turnover number is constant and independent of marble concentration. A student assessment of this exercise showed that it helped students visualize the turnover number and V max but not K m , the marble concentration at which V 0 is one-half V max . To address the concept of K m , we use supplemental laboratory and lecture exercises. This exercise with plastic containers and marbles is equally suited to demonstrate the kinetics of carrier-mediated membrane transport. We conclude that this exercise helps students visualize the turnover number and V max and gives students important insights into the kinetic parameters used to characterize the catalytic activity of enzymes and membrane transporters.