Pharmacology and Regulation of the Ion Channels of the Brown Adipocyte Plasma Membrane

Abstract

Brown adipose tissue is responsible for regulatory nonshivering thermogenesis in most mammals of small body size. Sympathetic nerve endings throughout the tissue allow neuronal control. After stimulation by their transmitter noradrenaline, the adipocyte shows a biphasic response of its membrane potential. An early brief depolarisation and a late long-lasting depolarisation are separated by a hyperpolarisation about 1 min after the stimulus (1, 2). The underlying ion channels have at least partly been identified by single channel analysis. A nonselective channel for mono-valent cations probably accounts for the tetrodotoxin(TfX)-insensitive sodium influx (3, 4, for review see 5). Part of the potassium efflux is mediated by a voltage-dependent K-channel (6, 7). Another major contribution to the potassium efflux derives from a Ca2+-activated K-channel first demonstrated by flux measurements (8) and later shown by whole-cell patch clamp experiments (9). Recently a further K-channel was shown that is voltage-insensitive (7). There are indications that further ion channels contribute to the response. In this paper we will focus on pharmacological mechanisms which can modify the nonselective cation channel as well as some of the K-channels. Methods Mature brown adipocytes were prepared from the interscapular tissue of 3-5 week old male Sprague-Dawley rats. Cells were isolated by collagenase treatment (5mglmI) and grown in Dulbecco's Modified Eagle's Medium, supplemented by 10% Newborn Calf serum, 4 nM insulin, 50 IU/mI penicillin and 50 1lg/m1 streptomycin. Culture dishes were kept in an incubator at 37 ·C, 8% C02 in air and 100% humidity. We used the patch-clamp technique to study single channel currents in excised inside-out and outside-out patches. Details of the methods in general are given by Hamill et al. (10). Pipettes were made from borosilicate glass (Clark, Pangbourne, England). Currents were recorded by a L/M-EPC7 patch-clamp amplifier (List, Darmstadt, Germany) and stored on video tape or on computer. For data acquisition and analysis we used the P-CLAMP software (Axon instruments, Foster City, CA, USA).