Mark A Hollywood | PubMed Health

PubMed2005

Pacemaker activity in urethral interstitial cells is not dependent on capacitative calcium entry.

The aim of the present study was to investigate the properties and role of capacitative Ca(2+) entry (CCE) in interstitial cells (IC) isolated from the rabbit urethra. Ca(2+) entry in IC was larger in cells with depleted intracellular Ca(2+) stores compared with controls, consistent with influx via a CCE pathway. The nonselective Ca(2+) entry blockers Gd(3+) (10 microM), La(3+) (10 microM), and Ni(2+) (100 microM) reduced CCE by 67% (n = 14), 65% (n = 11), and 55% (n = 9), respectively. These agents did not inhibit Ca(2+) entry when stores were not depleted. Conversely, CCE in IC was resistant to SKF-96365 (10 microM), wortmannin (10 microM), and nifedipine (1 microM). Spontaneous transient inward currents were recorded from IC voltage-clamped at -60 mV. These events were not significantly affected by Gd(3+) (10 microM) or La(3+) (10 microM) and were only slightly decreased in amplitude by 100 microM Ni(2+). The results from this study demonstrate that freshly dispersed IC from the rabbit urethra possess a CCE pathway. However, influx via this pathway does not appear to contribute to spontaneous activity in these cells.

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PubMedDate not supplied

Optimizing low-light microscopy with back-illuminated electron multiplying charge-coupled device: enhanced sensitivity, speed, and resolution.

The back-illuminated electron multiplying charge-coupled device (EMCCD) camera is having a profound influence on the field of low-light dynamic cellular microscopy, combining highest possible photon collection efficiency with the ability to virtually eliminate the readout noise detection limit. We report here the use of this camera, in 512 x 512 frame-transfer chip format at 10-MHz pixel readout speed, in optimizing a demanding ultra-low-light intracellular calcium flux microscopy setup. The arrangement employed includes a spinning confocal Nipkow disk, which, while facilitating the need to both generate images at very rapid frame rates and minimize background photons, yields very weak signals. The challenge for the camera lies not just in detecting as many of these scarce photons as possible, but also in operating at a frame rate that meets the temporal resolution requirements of many low-light microscopy approaches, a particular demand of smooth muscle calcium flux microscopy. Results presented illustrate both the significant sensitivity improvement offered by this technology over the previous standard in ultra-low-light CCD detection, the GenIII+intensified charge-coupled device (ICCD), and also portray the advanced temporal and spatial resolution capabilities of the EMCCD.

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