Studies of the genetic basis of type 2 diabetes suggest that variation in the calpain-10 gene affects susceptibility to this common disorder, raising the possibility that calpain-sensitive pathways may play a role in regulating insulin secretion and/or action. Calpains are ubiquitously expressed cysteine proteases that are thought to regulate a variety of normal cellular functions. Here, we report that short-term (4-h) exposure to the cell-permeable calpain inhibitors calpain inhibitor II and E-64-d increases the insulin secretory response to glucose in mouse pancreatic islets. This dose-dependent effect is observed at glucose concentrations above 8 mmol/l. This effect was also seen with other calpain inhibitors with different mechanisms of action but not with cathepsin inhibitors or other protease inhibitors. Enhancement of insulin secretion with short-term exposure to calpain inhibitors is not mediated by increased responses in intracellular Ca2+ or increased glucose metabolism in islets but by accelerated exocytosis of insulin granules. In muscle strips and adipocytes, exposure to both calpain inhibitor II and E-64-d reduced insulin-mediated glucose transport. Incorporation of glucose into glycogen in muscle also was reduced. These results are consistent with a role for calpains in the regulation of insulin secretion and insulin action.
The antidepressant-sensitive serotonin transporter (SERT) is a key regulator of serotonin (5-HT) signaling and availability in the CNS. The robust expression of SERT in adrenal chromaffin cells (ACCs), which comprise the neuroendocrine arm of the sympathetic nervous system, is less well understood. ACCs synthesize and secrete catecholamines (epinephrine, norepinephrine) which mediate the physiological response to stress. They do not synthesize 5-HT, but do accumulate small amounts through SERT-mediated uptake (5-HT content is <0.15% of the epinephrine content). We hypothesized that the chromaffin cells utilize this 5-HT for autocrine / paracrine control of the sympathoadrenal stress response. Consistent with this hypothesis, we previously reported that 5-HT1A receptors inhibit catecholamine secretion by reducing the number of secretory vesicles that fuse with the plasma. The objective of the current study was to investigate an additional, receptor-independent mechanism by which SERT controls adrenal catecholamine secretion. We used carbon fiber amperometry to analyze catecholamine secretion from ACCs that were isolated from either wild type mice, global SERT-knockout mice (SERT-/- ), or sympathoadrenal system-specific SERT knockout mice (SERTΔTH ). Transmitter release from individual vesicle fusion events can be resolved as amperometric spikes. There was no difference in the number or time-course of amperometric spikes evoked by 30mM KCl. The charge of the spikes is directly proportional to the amount of transmitter released by a single vesicle (i.e. quantal size). Spike charge was significantly smaller (~35%) and spike duration (half-width) was significantly shorter in SERT-knockout cells compared to matched controls. This was surprising given that HPLC analysis revealed no change in the catecholamine content of adrenal glands isolated from knockout mice compared to wild-type controls; the 5-HT content was significantly reduced but this accounted for <0.15% of the total monoamine content. Changes in calcium entry can modulate the quantal size of catecholamine release in ACCs, but patch-clamp recording revealed there was no significant difference in voltage-gated calcium channel currents in SERT knockout cells and ratiometric calcium imaging found no difference in KCl-evoked calcium entry. The decrease in quantal size was mimicked in wild-type cells by treating for >24 hours with escitalopram (an SSRI antidepressant which blocks SERT), or by depleting extracellular 5-HT from the culture medium for >24 hours (use of dialyzed serum in the culture media). In contrast, acute (minutes) or short-term (<6-8 hrs) treatment with escitalopram or 5-HT depletion had no effect. Adrenal chromaffin cells lack the rate limiting enzyme for 5-HT synthesis (tryptophan hydroxylase), but if the product of this enzyme (5-hydroxytryptophan; 5-HTP) is provided it can be converted into 5-HT. Treating ACCs with 5-HTP for 1-3 hrs had no effect but 24 hr treatment rescued the reduced spike charge seen in SERT knockout cells. Together, our data suggest that, following SERT-mediated uptake, intracellular 5-HT modulates the kinetics and thus fraction of secretory vesicle content that is released during a fusion event.
The adrenal gland contains resident macrophages, some of which lie adjacent to the catecholamine producing chromaffin cells. Because macrophages release a variety of secretory products, it is possible that paracrine signaling between these two cell types exists. Of particular interest is the potential paracrine modulation of voltage-gated calcium channels (I(Ca)), which are the main calcium influx pathway triggering catecholamine release from chromaffin cells. We report that prostaglandin E(2) (PGE(2)), one of the main signals produced by macrophages, inhibited I(Ca) in cultured bovine adrenal chromaffin cells. The inhibition is rapid, robust, and voltage dependent; the activation kinetics are slowed and inhibition is largely reversed by a large depolarizing prepulse, suggesting that the inhibition is mediated by a direct G-protein betagamma subunit interaction with the calcium channels. About half of the response to PGE(2) was sensitive to pertussis toxin (PTX) incubation, suggesting both PTX-sensitive and -insensitive G proteins were involved. We show that activation of macrophages by endotoxin rapidly (within minutes) releases a signal that inhibits I(Ca) in chromaffin cells. The inhibition is voltage dependent and partially PTX sensitive. PGE(2) is not responsible for this inhibition as blocking cyclooxygenase with ibuprofen did not prevent the production of the inhibitory signal by the macrophages. Nor did blocking the lipoxygenase pathway with nordihydroguaiaretic acid alter production of the inhibitory signal. Our results suggest that macrophages may modulate I(Ca) and catecholamine secretion by releasing PGE(2) and other chemical signal(s).
G-protein-coupled receptors (GPCR) play important roles in controlling neurotransmitter and hormone release. Inhibition of voltage-gated Ca(2+) channels (Ca(2+) channels) by G protein betagamma subunits (Gbetagamma) is one prominent mechanism, but there is evidence for additional effects distinct from those on calcium entry. However, relatively few studies have investigated the Ca(2+)-channel-independent effects of Gbetagamma on transmitter release, so the impact of this mechanism remains unclear. We used carbon fiber amperometry to analyze catecholamine release from individual vesicles in bovine adrenal chromaffin cells, a widely used neurosecretory model. To bypass the effects of Gbetagamma on Ca(2+) entry, we stimulated secretion using ionomycin (a Ca(2+) ionophore) or direct intracellular application of Ca(2+) through a patch pipette. Activation of endogenous GPCR or transient transfection with exogenous Gbetagamma significantly reduced the number of amperometric spikes (the number of vesicular fusion events). The charge ("quantal size") and amplitude of the amperometric spikes were also significantly reduced by GPCR/Gbetagamma. We conclude that independent from effects on calcium entry, Gbetagamma can regulate both the number of vesicles that undergo exocytosis and the amount of catecholamine released per fusion event. We discuss possible mechanisms by which Gbetagamma might exert these novel effects including interaction with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex.
Inhibition of presynaptic voltage-gated calcium channels by direct G-protein betagamma subunit binding is a widespread mechanism that regulates neurotransmitter release. Voltage-dependent relief of this inhibition (facilitation), most likely to be due to dissociation of the G-protein from the channel, may occur during bursts of action potentials. In this paper we compare the facilitation of N- and P/Q-type Ca(2+) channels during short trains of action potential-like waveforms (APWs) using both native channels in adrenal chromaffin cells and heterologously expressed channels in tsA201 cells. While both N- and P/Q-type Ca(2+) channels exhibit facilitation that is dependent on the frequency of the APW train, there are important quantitative differences. Approximately 20 % of the voltage-dependent inhibition of N-type I(Ca) was reversed during a train while greater than 40 % of the inhibition of P/Q-type I(Ca) was relieved. Changing the duration or amplitude of the APW dramatically affected the facilitation of N-type channels but had little effect on the facilitation of P/Q-type channels. Since the ratio of N-type to P/Q-type Ca(2+) channels varies widely between synapses, differential facilitation may contribute to the fine tuning of synaptic transmission, thereby increasing the computational repertoire of neurons.
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