Ca2+ is a highly versatile intracellular signal that operates over a wide temporal range to regulate many different cellular processes. An extensive Ca2+-signalling toolkit is used to assemble signalling systems with very different spatial and temporal dynamics. Rapid highly localized Ca2+ spikes regulate fast responses, whereas slower responses are controlled by repetitive global Ca2+ transients or intracellular Ca2+ waves. Ca2+ has a direct role in controlling the expression patterns of its signalling systems that are constantly being remodelled in both health and disease.

/pdf/calcium-signalling-dynamics-homeostasis-and-remodelling-2oni038yeg.pdf

Calcium signalling: dynamics, homeostasis and remodelling

The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in PCR. The recent introduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of transcription. This review discusses the technical aspects involved, contrasts conventional and kinetic RT-PCR methods for quantitating gene expression and compares the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).

/pdf/absolute-quantification-of-mrna-using-real-time-reverse-2kyvkvy0jn.pdf

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays.

Thapsigargin, a tumor-promoting sesquiterpene lactone, discharges intracellular Ca2+ in rat hepatocytes, as it does in many vertebrate cell types. It appears to act intracellularly, as incubation of isolated rat liver microsomes with thapsigargin induces a rapid, dose-dependent release of stored Ca2+. The thapsigargin-releasable pool of microsomal Ca2+ includes the pools sensitive to inositol 1,4,5-trisphosphate and GTP. Thapsigargin pretreatment of microsomes blocks subsequent loading with 45Ca2+, suggesting that its target is the ATP-dependent Ca2+ pump of endoplasmic reticulum. This hypothesis is strongly supported by the demonstration that thapsigargin causes a rapid inhibition of the Ca2(+)-activated ATPase activity of rat liver microsomes, with an identical dose dependence to that seen in whole cell or isolated microsome Ca2+ discharge. The inhibition of the endoplasmic reticulum isoform of the Ca2(+)-ATPase is highly selective, as thapsigargin has little or no effect on the Ca2(+)-ATPases of hepatocyte or erythrocyte plasma membrane or of cardiac or skeletal muscle sarcoplasmic reticulum. These results suggest that thapsigargin increases the concentration of cytosolic free Ca2+ in sensitive cells by an acute and highly specific arrest of the endoplasmic reticulum Ca2+ pump, followed by a rapid Ca2+ leak from at least two pharmacologically distinct Ca2+ stores. The implications of this mechanism of action for the application of thapsigargin in the analysis of Ca2+ homeostasis and possible forms of Ca2+ control are discussed.

https://www.pnas.org/content/pnas/87/7/2466.full.pdf

Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase.

In electrically nonexcitable cells, Ca2+ influx is essential for regulating a host of kinetically distinct processes involving exocytosis, enzyme control, gene regulation, cell growth and prolifera...

Store-operated calcium channels.

Mammalian skeletal muscle comprises different fiber types, whose identity is first established during embryonic development by intrinsic myogenic control mechanisms and is later modulated by neural and hormonal factors. The relative proportion of the different fiber types varies strikingly between species, and in humans shows significant variability between individuals. Myosin heavy chain isoforms, whose complete inventory and expression pattern are now available, provide a useful marker for fiber types, both for the four major forms present in trunk and limb muscles and the minor forms present in head and neck muscles. However, muscle fiber diversity involves all functional muscle cell compartments, including membrane excitation, excitation-contraction coupling, contractile machinery, cytoskeleton scaffold, and energy supply systems. Variations within each compartment are limited by the need of matching fiber type properties between different compartments. Nerve activity is a major control mechanism of the fiber type profile, and multiple signaling pathways are implicated in activity-dependent changes of muscle fibers. The characterization of these pathways is raising increasing interest in clinical medicine, given the potentially beneficial effects of muscle fiber type switching in the prevention and treatment of metabolic diseases.

Fiber types in mammalian skeletal muscles.

Molecular physiology of the SERCA and SPCA pumps

A contraction of the rabbit ear artery can be induced by depolarizing the cells with a K-rich solution if Ca is present. 10(-9)-10(-6) M noradrenaline and 10(-8)-10(-7) M histamine cause a contraction of this tissue without modifying the membrane potential. If the histamine concentration exceeds 10(-7) M some depolarization of the membrane also occurs. Both noradrenaline and histamine also induce a contraction in Ca-free medium, even if La is present. None of these stimuli produces action potentials or fluctuations of the membrane potential. Besides these tonic contractions, the ear artery can also produce phasic contractions when 10 mM TEA is added to the medium. Such contractions are caused by the appearance of action potentials which are Ca dependent and which are similar to those appearing in visceral smooth muscle. A study of 45Ca fluxes has revealed that K depolarization and noradrenaline cause only a small increase in 45Ca uptake by the cells, while noradrenaline also releases cellular Ca, even in Ca-free medium. A comparison of tension development and 45Ca release induced by noradrenaline in Ca-free medium suggests that Ca extrusion could be very efficient in the rabbit ear artery and that it could play a direct role in its relaxation.

https://rupress.org/jgp/article-pdf/70/2/129/1246551/129.pdf

Electro- and pharmacomechanical coupling in the smooth muscle cells of the rabbit ear artery

Quantitative PCR: theoretical considerations with practical implications.

Abnormal intracellular ca(2+)homeostasis and disease.

Phospholamban of isolated sarcoplasmic reticulum of cardiac and smooth muscle is phosphorylated by cyclic GMP-dependent protein kinase (G-kinase). Concomitantly, the affinity of the Ca2+ pump for Ca2+ is increased. These effects are very similar to those seen with cyclic AMP-dependent protein kinase (A-kinase). The phosphate incorporation into phospholamban and the stimulatory effects of both kinases on the Ca2+ pump are not additive, suggesting that G-kinase phosphorylates the same serine residue as A-kinase. A possible physiological role for phosphorylation of phospholamban by G-kinase is discussed.

/pdf/cyclic-gmp-dependent-protein-kinase-phosphorylates-2w2tdwmekv.pdf

Luc Raeymaekers

Papers

Molecular physiology of the SERCA and SPCA pumps

Electro- and pharmacomechanical coupling in the smooth muscle cells of the rabbit ear artery

Quantitative PCR: theoretical considerations with practical implications.

Abnormal intracellular ca(2+)homeostasis and disease.

Cyclic GMP-dependent protein kinase phosphorylates phospholamban in isolated sarcoplasmic reticulum from cardiac and smooth muscle.