Proceedings of The Physiological Society

University of Cambridge (2008) Proc Physiol Soc 11, C112

Oral Communications

Ca2+ influx pathways in white adipocytes from rat

P. A. Smith1, P. Pulbutr1, S. L. Chan1

1. School of Biomedical Sciences, University of Nottingham, Nottingham, United Kingdom.


Ca2+ is an important signaling molecule for many cell types. Changes in its intracellular concentration may involve alteration in Ca2+ flux across the plasma membrane or/and Ca2+ mobilization from intracellular Ca2+ stores. Abnormal handling of intracellular Ca2+ in white adipocytes, a major target for insulin, is thought to be associated with insulin resistance and diabetes mellitus. However, since little is known about the Ca2+ pathways and their related functions in white adipocytes, we have explored this area with Ca2+ imaging technology. White adipocytes were isolated from rat epididymal fat pads by collagenase digestion and were attached to glass coverslips. Adipocytes were imaged at 32°C with constant perifusion of a HEPES-buffered salt solution. Changes in intracellular Ca2+ were monitored as changes in the fluorescence intensity of the Ca2+-sensitive dye, Fluo-4. Fluorescence intensity is presented as the mean ± SEM percentage of control value with n, the number of experiments. Unpaired Students T-test or ANOVA were used to determine statistical significance relative to control. Oxytocin (1 μM), but not insulin, elicited a transient increase in fluorescence (282 ± 34%, n=21), which then decayed back down to a plateau level(123 ± 5 %, n=21) which was sustained for the duration of the peptide addition. Omission of extracellular Ca2+ did not affect the peak response, but decreased the plateau level to value that was sub-basal (83 ± 22%, n=6; p <0.05). 2-APB (75 μM), a selective blocker of store-operated Ca2+ channels, did not affect the peak response to oxytocin but abolished the plateau phase (104 ± 6%, n=10; p<0.05). Elevation of extracellular K+ from 5 to 50 mM, by isosmotic substitution with Na+, increased intracellular Ca2+ with a peak fluorescence intensity of 207 ± 17% (n=21). This response had an obligatory requirement for extracellular Ca2+ and was significantly attenuated by either 20 μM nifedipine (124 + 4%, n=17; p < 0.05) or 20 μM (149 ± 9%, n=12; p < 0.05), inhibitors of the L-type voltage-gated Ca2+ channel. Reduction of extracellular Na+ from 145 to 98 mM, by isosmotic replacement with N-Methyl-D-glucamine H+, also increased intracellular Ca2+ (130 ± 4%, n=11; p < 0.05); an effect abolished by 10 μM KB-R 7343, a putative Na+/ Ca2+ exchange inhibitor (p < 0.05). In summary, oxytocin, but not insulin, can increase intracellular Ca2+ in rat adipocytes via mobilization of Ca2+ from internal stores and influx through store-operated Ca2+ channels. Furthermore, the results of this study suggest that rat white adipocytes possess functional L-type Ca2+ channels and that they can also undergo reverse mode Na+/ Ca2+ exchange. The roles of these Ca2+ pathways in adipocyte physiology remain to be established.

Where applicable, experiments conform with Society ethical requirements