Membrane responses to norepinephrine in cultured brown fat cells.

Abstract

We used the "perforated-patch" technique (Horn, R., and A. Malty, 1988. Journal of General Physiology. 92:145-159) to examine the effects of adrenergic agonists on the membrane potentials and membrane currents in isolated cultured brown fat cells from neonatal rats. In contrast to our previous results using traditional whole-ceU patch clamp, 1-23-d cultured brown fat cells clamped with the perforated patch consistently showed vigorous membrane responses to both aand/~-adrenergic agonists, suggesting that cytoplasmic components essential for the thermogenic response are lost in whole-cell experiments. The membrane responses to adrenergic stimulation varied from cell to cell but were consistent for a given cell. Responses to bath-applied norepinephrine in voltage-clamped cells had three possible components: (a) a fast transient inward current, (b) a slower outward current carried by K § that often oscillated in amplitude, and (c) a sustained inward current largely by Na § The fast inward and outward currents were activated by a-adrenergic agonists while the slow inward current was mediated by /~-adrenergic agonists. Oscillating outward currents were the most frequently seen response to norepinephrine stimulation. Activation of this current, termed IreNE, was independent of voltage and seemed to be carried by Ca~+-activated K channels since the current oscillated in amplitude at constant membrane potential and gradually decreased when the cells were bathed with calcium-free external solution. IK.N~ had a novel pharmacology in that it could be blocked by 4-aminopyridine, tetraethylammonium, apamin, and charybdotoxin. Both ImNE and the voltage-gated K channels also present in brown fat (Lucero, M. T., and P. A. Pappone, 1989a. Journal of General Physiology. 93:451-472) may play a role in maintaining cellular homeostasis in the face of the high metabolic activity involved in thermogenesis.

Cannabinoids inhibit N-type calcium channels in neuroblastoma-glioma cells.

“…NG108-15 cells (passages [16][17][18][19][20][21][22][23][24][25][26][27][28] were grown by standard techniques on polylysine-coated coverslip fragments (14,15). Four to seven days prior to recording, differentiation was induced by decreasing the serum content of the medium to 1% and by adding 100 ,uM IBMX and 10 ,uM prostaglandin E1.…”

Modulation of calcium‐activated non‐selective cation channel activity by nitric oxide in rat brown adipose tissue.

“…Physiological implications in brown fat cells A full understanding of the role that calcium-activated non-selective cation channels play in the physiology of the brown fat cell has not yet been reached. It is likely that non-selective cation channel activity is the background (Siemen & Reuhl, 1987) for the late and long-lasting depolarization observed after noradrenaline stimulation (Girardier et al 1968;Lucero & Pappone, 1990) and thus may explain the noradrenaline-stimulated Na+ influx (Connolly, NAnberg & Nedergaard, 1986). As depolarization leads to a decrease in the driving force for calcium and thus to a decrease in the cytosolic Ca2+ level (Lee, Nuccitelli & Pappone, 1993), a blockade of the non-selective cation channels by nitric oxide would enable the cell to maintain a high Ca2+ level during adrenergic stimulation.…”

“…Since then, non-selective cation channels have been described in a wide variety of species and cells (Partridge & Swandulla, 1988;Siemen & Hescheler, 1993), including brown fat cells (Siemen & Reuhl, 1987). The function of this channel in brown adipose tissue is not well known but it may explain (Siemen & Reuhl, 1987) the longlasting depolarization observed during adrenergic stimulation of the tissue (Girardier, Seydoux & Clausen, 1968;Lucero & Pappone, 1990). Recently, the presence of a critical sulfhydryl group on the cytosolic side of the non-selective cation channel was suggested.…”

Calcium current activated by muscarinic receptors and thapsigargin in neuronal cells.

“…The pipette solution contained (in millimolar): 16 KCI or CsCI, 70 K2SO4 or Cs2SO4, 5 MgSO 4, l0 HEPES, and 100 sucrose to ~320 mosM at pH 7.2. After filtering the pipette solution, 0.05% Pluronic acid F-127 (Molecular Probes Inc., Eugene, OR) was added to increase the solubility of nystatin (Lucero and Pappone, 1990). The calculated reversal potential for C1-= -60 mV and for K § = -85 mV.…”

“…Whole cell voltage clamp was done using the nystatin perforated-patch technique (Hamill, Marry, Neher, Sakmann, and Sigworth, 1981;Horn and Marty, 1988;Lucero and Pappone, 1990). Nystatin was added to filtered pipette solution from a DMSO stock (2.5 mg nystatin in 50 I~l DMSO) to a final concentration of 100-200 Ixg/ml.…”