Biomedical subjects

James L Rae

Publications and source records attributed to James L Rae.

3 recordsLinked to original sources

PubMed2002

Sodium current in human jejunal circular smooth muscle cells.

BACKGROUND & AIMS: Sodium channels are key regulators of neuronal and muscle excitability. However, sodium channels have not been definitively identified in gastrointestinal smooth muscle. The aim of the present study was to determine if a Na(+) current is present in human jejunal circular smooth muscle cells. METHODS: Currents were recorded from freshly dissociated cells using patch-clamp techniques. Complementary DNA (cDNA) libraries constructed from the dissociated cells were screened to determine if a message for alpha subunits of Na(+) channels was expressed. Smooth muscle cells were also collected using laser-capture microdissection and screened. RESULTS: A tetrodotoxin-insensitive Na(+) channel was present in 80% of cells patch-clamped. Initial activation was at -65 mV with peak inward current at -30 mV. Steady-state inactivation and activation curves revealed a window current between -75 and -60 mV. The Na(+) current was blocked by lidocaine and internal and external QX314. A cDNA highly homologous to SCN5A, the alpha subunit of the cardiac Na(+) channel, was present in the cDNA libraries constructed from dissociated cells and from smooth muscle cells collected using laser-capture microdissection. CONCLUSIONS: Human jejunal circular smooth muscle cells express a tetrodotoxin-insensitive Na(+) channel, probably SCN5A. Whether SCN5A plays a role in the pathophysiology of human gut dysmotilities remains to be determined.

Anesthetics, Local↗

PubMed2002

alpha(1C) (Ca(V)1.2) L-type calcium channel mediates mechanosensitive calcium regulation.

Smooth muscle exhibits mechanosensitivity independent of neural input, suggesting that mechanosensitive pathways reside within smooth muscle cells. The native L-type calcium current recorded from human intestinal smooth muscle is modulated by stretch. To define mechanosensitive mechanisms involved in the regulation of smooth muscle calcium entry, we cloned the alpha(1C) L-type calcium channel subunit (Ca(V)1.2) from human intestinal smooth muscle and expressed the channel in a heterologous system. This channel subunit retained mechanosensitivity when expressed alone or coexpressed with a beta(2) calcium channel subunit in HEK-293 or Chinese hamster ovary cells. The heterologously expressed human cardiac alpha(1C) splice form also demonstrated mechanosensitivity. Inhibition of kinase signaling did not affect mechanosensitivity of the native channel. Truncation of the alpha(1C) COOH terminus, which contains an inhibitory domain and a proline-rich domain thought to mediate mechanosensitive signaling from integrins, did not disrupt mechanosensitivity of the expressed channel. These data demonstrate mechanical regulation of calcium entry through molecularly identified L-type calcium channels in mammalian cells and suggest that the mechanosensitivity resides within the pore forming alpha(1C)-subunit.

Animals↗

PubMed2001

Single-cell electroporation.

Using modified patch-clamp methodology, we demonstrated that it is possible to insert genes or other compounds routinely into single cells by electroporation. When the cell is indented by a small-tipped microelectrode, a voltage of 10 V or less in the pipette is divided by the pipette resistance and the series resistance of the cleft between the pipette tip and the cell surface. The voltage at the cell membrane can be high enough to cause localized dielectric breakdown of the membrane and create pores that allow compounds in the pipette to enter the cell. Rectangular pulses from 20 micros to more than 300 ms are effective, as are frequencies from DC to 5 kHz. The most significant parameter was the total time for which the voltage was applied. Pipette voltages of 2-10 V were required, with larger genes requiring larger voltages. With optimal parameters, transfection rates in excess of 80% were also possible routinely. This approach offers an effective alternative to intracellular pressure injection and iontophoresis for placing genes, drugs, and other compounds in cells. Because of the small size of the electrode tips, substances can be inserted in cells from almost any location on their surfaces. In addition, the small tips electroporated only a limited area and so did little cell damage.

Contrast Media↗