Jörg Hüser | PubMed Health

PubMed2005

A cell-based cGMP assay useful for ultra-high-throughput screening and identification of modulators of the nitric oxide/cGMP pathway.

We have established a rapid, homogeneous, cell-based, and highly sensitive assay for guanosine 3'-5'-cyclic monophosphate (cGMP) that is suitable for fully automated ultra-high-throughput screening. In this assay system, cGMP production is monitored in living cells via Ca2+ influx through the olfactory cyclic nucleotide-gated cation channel CNGA2, acting as the intracellular cGMP sensor. A stably transfected Chinese hamster ovary (CHO) cell line was generated recombinantly expressing soluble guanylate cyclase, CNGA2, and aequorin as a luminescence indicator for the intracellular calcium concentration. This cell line was used to screen more than 900,000 compounds in an automated ultra-high-throughput screening assay using 1536-well microtiter plates. In this way, we have been able to identify BAY 58-2667, a member of a new class of amino dicarboxylic acids that directly activate soluble guanylate cyclase. The assay system allows the real-time cGMP detection within living cells and makes it possible to screen for activators and inhibitors of enzymes involved in the nitric oxide/cGMP pathway.

Animals↗

PubMed2003

Local calcium gradients during excitation-contraction coupling and alternans in atrial myocytes.

Subcellular Ca(2+) signalling during normal excitation-contraction (E-C) coupling and during Ca(2+) alternans was studied in atrial myocytes using fast confocal microscopy and measurement of Ca(2+) currents (I(Ca)). Ca(2+) alternans, a beat-to-beat alternation in the amplitude of the [Ca(2+)](i) transient, causes electromechanical alternans, which has been implicated in the generation of cardiac fibrillation and sudden cardiac death. Cat atrial myocytes lack transverse tubules and contain sarcoplasmic reticulum (SR) of the junctional (j-SR) and non-junctional (nj-SR) types, both of which have ryanodine-receptor calcium release channels. During E-C coupling, Ca(2+) entering through voltage-gated membrane Ca(2+) channels (I(Ca)) triggers Ca(2+) release at discrete peripheral j-SR release sites. The discrete Ca(2+) spark-like increases of [Ca(2+)](i) then fuse into a peripheral 'ring' of elevated [Ca(2+)](i), followed by propagation (via calcium-induced Ca(2+) release, CICR) to the cell centre, resulting in contraction. Interrupting I(Ca) instantaneously terminates j-SR Ca(2+) release, whereas nj-SR Ca(2+) release continues. Increasing the stimulation frequency or inhibition of glycolysis elicits Ca(2+) alternans. The spatiotemporal [Ca(2+)](i) pattern during alternans shows marked subcellular heterogeneities including longitudinal and transverse gradients of [Ca(2+)](i) and neighbouring subcellular regions alternating out of phase. Moreover, focal inhibition of glycolysis causes spatially restricted Ca(2+) alternans, further emphasising the local character of this phenomenon. When two adjacent regions within a myocyte alternate out of phase, delayed propagating Ca(2+) waves develop at their border. In conclusion, the results demonstrate that (1) during normal E-C coupling the atrial [Ca(2+)](i) transient is the result of the spatiotemporal summation of Ca(2+) release from individual release sites of the peripheral j-SR and the central nj-SR, activated in a centripetal fashion by CICR via I(Ca) and Ca(2+) release from j-SR, respectively, (2) Ca(2+) alternans is caused by subcellular alterations of SR Ca(2+) release mediated, at least in part, by local inhibition of energy metabolism, and (3) the generation of arrhythmogenic Ca(2+) waves resulting from heterogeneities in subcellular Ca(2+) alternans may constitute a novel mechanism for the development of cardiac dysrhythmias.

Animals↗

PubMed1998

Capacitative Calcium Entry.

Capacitative Ca(2+) entry is a recently discovered Ca(2+) entry pathway that is activated on depletion of intracellular Ca(2+) stores, providing an avenue for store refilling. Despite recent progress in elucidating the capacitative Ca(2+) entry pathway, the mysteries of its molecular identity, its biophysical properties, and the store depletion signal remain.

Journal Article↗