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Nerve growth factor increases sodium channel expression in pancreatic ß cells: implications for insulin secretion¹

Department of Biophysics, Institute for Cell Physiology, Universidad Nacional Autónoma de México, UNAM, Mexico D.F., 04510 Mexico

²Correspondence: AP 70–253, Ciudad Universitaria, Mexico D.F., 04510 Mexico. E-mail: mhiriart{at}ifisiol.unam.mx

SPECIFIC AIMS

The purpose of this study is to understand the cellular mechanisms involved in the up-regulation by nerve growth factor (NGF) of sodium currents in adult rat pancreatic ß cells and the effects of Na⁺ current increase on electrical activity and insulin secretion.

PRINCIPAL FINDINGS

1. Na⁺ current density in ß cells treated for 5 days with NGF increases 97% compared with control cells.

NGF-induced increase in Na⁺ current density is dependent on protein synthesis and mRNA transcription, because exposure to either cycloheximide or actinomycin D prevents the rise of sodium current density in NGF-treated ß cells (Fig. 1 A).

View larger version (26K): [in this window] [in a new window]   Figure 1. NGF increases sodium channel expression in pancreatic ß cells. A) Sodium current I-V relationship of control (circles), NGF- (triangles), NGF + CHX- (diamonds), and NGF + ActD-treated cells (squares). Insert: Representative recordings elicited by a depolarizing pulse to +10 mV for 15 ms from different experimental groups of cells. B) Activation (left panel) and steady-state inactivation (right panel) of the Na+ current from the experimental groups (same symbols as in panel A). CHX and ActD both inhibited the increase in Na current promoted by NGF. There were no significant changes in steady-state inactivation or voltage-dependent activation of the Na+ currents. *P < 0.01 vs. all groups. C) RT-PCR amplification products of the NaV1.3 and GAPDH genes from control (C), NGF- (N), dbcAMP- (A), and NGF+dbcAMP-treated cells (NA). D) Normalized NaV1.3 mRNA levels from control, NGF-, dbcAMP-, and NGF + dbcAMP-treated cells (NA). In the NGF- and NGF+dbcAMP-treated groups, there is a two- to threefold increase in mRNA levels. *P < 0.01 vs. control and dbcAMP, n = 4 independent experiments.

2. Similar Na channels are expressed in control and NGF-treated cells (Fig. 1B, C ).

3. NGF increases by nearly twofold steady-state levels of mRNA coding for type III Na channel subunit (NaV 1.3, Fig. 1C, D ).

4. NGF-treated cells show an increased electrical activity, reaching higher levels of depolarization and firing action potentials for longer periods (Fig. 2 A).

View larger version (26K): [in this window] [in a new window]   Figure 2. Sodium currents play an important role in ß cell electrical activity and insulin secretion. A) Perforated-patch recording of electrical activity from control cell in response to stimulation with 20.6 mM glucose and a 2 nA current for 12 s from control (left panel) and NGF-treated cells (right panel). B) Electrical activity of NGF-treated ß cell in response to 20.6 mM glucose and a stimulating pulse of 2 nA for 1 s before (left panel) and after (right panel) exposure to the Na+ channel blocker TTX. C) TTX decreases insulin secretion stimulated by 5.6 and 15.6 mM glucose in NGF-treated cells (right portion), but only affects insulin secretion stimulated by 15.6 mM glucose in control cells (left portion). Open bars: 5.6 mM glucose, black bars: 5.6 mM glucose + TTX, diagonal hatched bars: 15.6 mM glucose, cross-hatched bars: 15.6 mM glucose + TTX. *P < 0.05 vs. TTX, P < 0.05 vs. control, P < 0.05 vs. 5.6 mM glucose.

5. Sodium current is an important contributor to rat ß cell action potentials, because tetrodotoxin (TTX), a Na⁺ channel blocker, partially inhibits the electrical activity (Fig. 2B ).

6. Insulin secretion in single ß cells treated for 5 days with NGF are nearly 130% higher than their respective controls.

7. Increased Na⁺ current is partially responsible for this rise in insulin secretion (Fig. 2C ) and is necessary to achieve high insulin output rates.

DISCUSSION

The results show that NGF-treatment induces an increase in Na channel expression in pancreatic ß cells. To the best of our knowledge, this is the first demonstration that this growth factor modulates ion channel expression in an endoderm-derived cell type.

Sodium currents play a major role during action potential firing in rat ß cells, an issue that has been disputed for a long time, because the electrical activity of these cells is decreased by TTX (Fig. 2B ). These currents also contribute to the amount of insulin secreted by single rat ß cells in control and NGF-treated cells, as TTX decreases insulin secretion in both groups and erases the differences in secretion levels observed between control and NGF-treated cells (Fig. 2C ).

Pancreatic ß cells secrete NGF, and this growth factor has been shown to induce an increase in insulin secretion through an autocrine loop. The rise in insulin secretion could be partially explained by the NGF-increased Na⁺ current because it can lead to stronger depolarizations that increase calcium entry and exocytosis.

NGF autocrine modulation can lead to short-term increases in ionic currents, as observed for Ca²⁺ currents, or to long-term effects on ionic channel expression, similar to the ones observed in this work for Na channels (Fig. 1C, D ). These two effects may contribute to maintain an adequate number of active channels in the plasma membrane of ß cells necessary to accomplish an electrical activity able to sustain high insulin secretion rates.

When the NGF–autocrine loop is disturbed, a reduction in active channels could ensue that would lead to deteriorated electrical activity and lower insulin secretion levels (Fig. 3 ) and, in such scenario, to the onset of diabetes mellitus.

View larger version (33K): [in this window] [in a new window]   Figure 3. NGF modulation could be necessary to maintain an adequate expression of ionic channels in ß cells. NGF exerts trophic effects on different levels of ß cell physiology either through increased expression, as occurs with Na channels and insulin, or post-translational modulation, as observed with Ca2+ currents (15). Exposure to this growth factor leads to stronger depolarizations and higher insulin secretion rates (right portion). Such modulation could be exerted through an autocrine loop within the pancreatic islets, as ß cells secrete and are sensitive to NGF (15). If this autocrine loop is disturbed, ß cells are no longer able to sustain adequate insulin and NGF outputs, which can be related to the onset of diabetes mellitus and/or peripheral neuropathy (left portion).

These observations may contribute to a better understanding of the physiopathology of diabetes mellitus, where serum NGF levels are diminished.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0934fje; to cite this article, use FASEB J. (April 23, 2002) 10.1096/fj.01-0934fje.

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