Adrenergically activated Ca2+ increases in brown fat cells: effects of Ca2+, K+, and K channel block

Abstract

We measured intracellular calcium concentration ([Ca2+]i) during adrenergic stimulation using fura-2 ratio imaging of individual cultured neonatal rat brown fat cells. One micromolar norepinephrine (NE) increased [Ca2+]i from an average resting value of 105 nM to 555 nM in approximately 30 s. [Ca2+]i remained elevated as long as NE was present but returned to resting levels within 2-3 min after NE removal. The response was half maximal at approximately 50 nM NE and was primarily alpha-adrenergic. The sustained, but not the initial, increase in [Ca2+]i required extracellular calcium. Cells stimulated in high-K media had [Ca2+]i responses like those in 0 Ca2+, suggesting that depolarization abrogates calcium influx. Parallel perforated-patch recordings showed that the increase in [Ca2+]i activates a calcium-activated K conductance. Blocking K channels with moderate concentrations of tetraethylammonium (TEA) had only small effects on NE-induced changes in [Ca2+]i, but high concentrations of TEA significantly reduced the response. We conclude that cytoplasmic calcium is modulated by fluxes from both intracellular and extracellular sources and that K channels may not be required for normal short-term [Ca2+]i responses to hormone.

Multiple P2 Receptors Contribute to a Transient Increase in Intracellular Ca2+ Concentration in Atp‐Stimulated Rat Brown Adipocytes

“…Concentration-dependent inhibition data were collected for the purinoceptor antagonist suramin or PPADS and the inhibition curves were computed by fitting the mean values of the responses at each concentration with the equation: Figure 1A demonstrates the representative [Ca 2+ ] i response to extracellular ATP in a rat brown adipocyte previously stimulated with noradrenaline in the standard bath solution. As expected, 1 mM noradrenaline evoked a sustained elevation in [Ca 2+ ] i of 1580 ± 145 nM above a resting level of 54 ± 4 nM (n = 10), which has been suggested to arise initially from the InsP 3 -mediated Ca 2+ release from the endoplasmic reticulum Ca 2+ stores via a-adrenoceptor stimulation, followed by the influx of extracellular Ca 2+ presumably through the store-operated pathway (Nånberg & Putney, 1986;Schimmel et al 1986;Lee et al 1993;Omatsu-Kanbe & Matsuura, 1999 Figure 1B illustrates that the cell responds to 100 nM thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca 2+ pump which normally depletes the stores (Thastrup et al 1990), leading to a gradual increase in [Ca 2+ ] i with a mean amplitude of 1455 ± 177 nM (n = 10), remaining stable at around the peak level. As described previously (Omatsu-Kanbe & Matsuura, 1999) (Ehrlich et al 1986;Naik et al 1991;Chen & Lo, 1991;Saito et al 1999).…”

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

“…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. In brown adipose tissue, nitric oxide is apparently released during adrenergic stimulation, although it is not known from which cells in the tissue the nitric oxide is released (Nagashima, Ohinata & Kuroshima, 1994); the brown fat cell itself would, of course, be a candidate.…”

β-Adrenergic potentiation of endoplasmic reticulum Ca2+ release in brown fat cells

“…The second hypothesis proposes that isoproterenol has sufficient activity at ␣ 1 -adrenoceptors to elicit cytosolic Ca 2ϩ increases (4,16). This "dirty drug" hypothesis is supported by evidence that norepinephrineinduced Ca 2ϩ increases can be completely blocked by the ␣-adrenergic antagonist phentolamine (16) and that isoproterenol-induced Ca 2ϩ increases can be blocked by the ␣ 1 -adrenergic antagonist prazosin (4).…”

“…Other adrenergic receptor subtypes are also present on mature adipocytes and include ␣ 1 -adrenoceptors coupled to inositol 1,4,5-trisphosphate (IP 3 ) production and release of Ca 2ϩ from intracellular stores (16,22,25,39,41). Increased cytosolic Ca 2ϩ affects both the acute thermogenic response (42) and the long-term capacity of BAT to produce heat (8,27).…”

“…Previous investigations of Ca 2ϩ signaling in brown adipocytes have noted cytosolic Ca 2ϩ increases in response to not only ␣-adrenergic stimulation but also the ␤-adrenergic agonist isoproterenol (4,16,41). Because the ␤-adrenergic pathway is mediated by changes in cAMP levels, it is not immediately clear how this signaling pathway also affects cytosolic Ca 2ϩ levels.…”