PubMed · 12234760
The source did not provide an abstract. Follow the original record for more information.
Explore connections, maps & timelines
Evangelia Kranias. 2002. Introduction.. https://doi.org/10.1006/jmcc.2002.2036
Cite the original work for its findings. Save a collection to share your selection of sources.
KEEP EXPLORING
The patterning of cardiac myocytes on a micron scale ( approximately 5 microm) was achieved by microcontact printing of fibronectin onto a hydrophobically pretreated glass substrate. The patterned cardiac myocytes conjugated with each other by forming a gap junction, as judged from the synchronized Ca(2+) transition over the pattern, and thus simultaneously contracted. The dynamic change of the Ca(2+) concentration within the patterned tissue was analyzed quantitatively during successive contraction and relaxation using a Nipkow-type high-speed confocal microscope.
Translocation of protein kinase C (PKC) alpha, beta II, delta and epsilon fused to enhanced green fluorescent protein (EGFP) was studied in living neuroblastoma cells by confocal microscopy. Exposure to carbachol elicited transient translocation of PKC alpha-EGFP and beta II-EGFP in most of the cells, PKC delta-EGFP in a few cells and induced sustained translocation of PKC epsilon-EGFP. To monitor levels of Ca(2+) and diacylglycerol and the translocation of PKC in the same cell, the Ca(2+)-sensitive C2 domain, diacylglycerol-sensitive C1 domains and full-length PKC were fused to red, cyan and yellow fluorescent proteins respectively. PKC alpha was translocated a few seconds after the C2 domain, which represents an increase in Ca(2+). This delay was insensitive to removal of the pseudosubstrate in PKC alpha, but the isolated regulatory domain translocated simultaneously with the C2 domain. Translocation of PKC epsilon coincided with the increase in diacylglycerol. Ionomycin induced translocation of PKC alpha and the C2 domain, whereas 1,2-dioctanoylglycerol caused translocation of the C1 domains and PKC epsilon, but not PKC alpha. Experiments with individual C1 domains showed that treatment with carbachol or phorbol 12,13-dibutyrate elicited translocation of PKC alpha C1a, PKC epsilon C1a and PKC epsilon C1b, whereas PKC alpha C1b was largely insensitive to these agents. In contrast with full-length PKC alpha, the regulatory domain of PKC alpha and pseudosubstrate-devoid PKC alpha responded to the carbachol-stimulated increase in diacylglycerol.
The release of Ca(2+) from intracellular stores via InsP(3) receptors shows anomalous kinetics. Successive additions of low concentrations of InsP(3) cause successive rapid transients of Ca(2+) release. These quantal responses have been ascribed to all-or-none release from stores with differing sensitivities to InsP(3) or, alternatively, to a steady-state mechanism where complex kinetic properties of the InsP(3) receptor allow partial emptying of all the stores. We present here an adaptive model of the InsP(3) receptor that can show either pattern, depending on the imposed experimental conditions. The model proposes two interconvertible conformational states of the receptor: one state binds InsP(3) rapidly, but with low affinity, whereas the other state binds slowly, but with high affinity. The model shows repetitive increments of Ca(2+) release in the absence of a Ca(2+) gradient, but more pronounced incremental behaviour when released Ca(2+) builds up at the mouth of the channel. The sensitivity to Ins P (3) is critically dependent on the density of InsP(3) receptors, so that different stores can respond to different concentration ranges of Ins P (3). Since the model generates very high Hill coefficients (h approximately 7), it allows all-or-none release of Ca(2+) from stores of differing receptor density, but questions the validity of the use of h values as a guide to the number of InsP(3) molecules needed to open the channel. The model presents a mechanism for terminating Ca(2+) release in the presence of positive feedback from released Ca(2+), thereby providing an explanation of why elementary Ca(2+) signals ('blips' and 'puffs') do not inevitably turn into regenerative waves.