Andy K. Lee

Mechanism underlying corticotropin-releasing hormone (CRH) triggered cytosolic Ca2+ rise in identified rat corticotrophs.

1 The patch‐clamp technique was used in conjunction with the fluorescent Ca2+ indicator indo‐1 to measure simultaneously cytosolic Ca2+ concentration ([Ca2+]i) and membrane potential in single rat corticotrophs identified with the reverse haemolytic plaque assay. 2 Application of the adrenocorticotropin (ACTH) secretagogue, corticotropin‐releasing hormone (CRH), triggered a sustained [Ca2+]i elevation and membrane depolarization. 3 The CRH action was mediated via the cAMP‐dependent protein kinase cascade. Both the CRH‐induced depolarization and [Ca2+]i elevation could be mimicked by extracellular application of the adenylate cyclase activator forskolin or the membrane‐permeable cAMP analogue, 8‐(4‐chlorophenylthio)‐adenosine‐3’,5’‐cyclic monophosphate (8‐CPT‐cAMP). Intracellular adenosine cyclic 3’,5’‐(Rp)‐phosphothioate (Rp‐cAMPS), a protein kinase A inhibitor, abolished the CRH effects. 4 Voltage‐clamp studies suggest that the CRH‐triggered depolarization was due to the reduction of background K+ conductances. The CRH‐sensitive current was Ca2+ independent and was insensitive to the K+ channel blockers tetraethylammonium (TEA) or 4‐amino‐pyridine (4‐AP), but could be partially inhibited by Ba2+. 5 The CRH‐triggered steady‐state depolarization stimulated extracellular Ca2+ entry via voltage‐gated Ca2+ channels and raised [Ca2+]i. CRH failed to stimulate [Ca2+]i rise in cells that were voltage clamped at their resting potential. Removal of extracellular Ca2+ or inhibition of Ca2+ channels by Ni2+ abolished the [Ca2+]i rise. 6 Voltage‐clamp studies of voltage‐gated Ca2+ channels using Ba2+ as charge carrier show that ∼90% of the channels were available for activation at the resting potential. CRH did not enhance the voltage‐gated Ca2+ channels.

Autocrine and paracrine actions of ATP in rat carotid body

Carotid bodies are peripheral chemoreceptors that detect lowering of arterial blood O(2) level. The carotid body comprises clusters of glomus (type I) cells surrounded by glial-like sustentacular (type II) cells. Hypoxia triggers depolarization and cytosolic [Ca(2+)] ([Ca(2+)](i)) elevation in glomus cells, resulting in the release of multiple transmitters, including ATP. While ATP has been shown to be an important excitatory transmitter in the stimulation of carotid sinus nerve, there is considerable evidence that ATP exerts autocrine and paracrine actions in carotid body. ATP acting via P2Y(1) receptors, causes hyperpolarization in glomus cells and inhibits the hypoxia-mediated [Ca(2+)](i) rise. In contrast, adenosine (an ATP metabolite) triggers depolarization and [Ca(2+)](i) rise in glomus cells via A(2A) receptors. We suggest that during prolonged hypoxia, the negative and positive feedback actions of ATP and adenosine may result in an oscillatory Ca(2+) signal in glomus cells. Such mechanisms may allow cyclic release of transmitters from glomus cells during prolonged hypoxia without causing cellular damage from a persistent [Ca(2+)](i) rise. ATP also stimulates intracellular Ca(2+) release in sustentacular cells via P2Y(2) receptors. The autocine and paracrine actions of ATP suggest that ATP has important roles in coordinating chemosensory transmission in the carotid body.

Voltage‐gated Ca2+ channels and intracellular Ca2+ release regulate exocytosis in identified rat corticotrophs

1 The patch clamp technique was used in conjunction with a fluorescent Ca2+ indicator (indo‐1, or indo‐1FF) to measure simultaneously cytosolic Ca2+ concentration ([Ca2+]i) and exocytosis (changes in membrane capacitance) in single, identified rat corticotrophs. 2 Exocytosis could be stimulated by extracellular Ca2+ entry (via voltage‐gated Ca2+ channels). A train of depolarizations could exhaust the pool of readily releasable granules and the pool replenished with a time constant of 42 s (at 22–25 °C). 3 Recordings from cells with 0.5 mm intracellular cAMP showed that the amplitude of the depolarization‐triggered exocytosis, the Ca2+ sensitivity of exocytosis, as well as the rate of replenishment of the readily releasable pool, were similar to the controls. 4 Exocytosis could also be stimulated by intracellular Ca2+ release from the inositol 1,4,5‐trisphosphate (IP3)‐sensitive store (via flash photolysis of caged IP3). At comparable [Ca2+]i, extracellular Ca2+ entry and intracellular Ca2+ release had similar efficacy in triggering exocytosis. 5 The rate of exocytosis triggered via depolarization or intracellular Ca2+ release was much faster than that triggered via uniform elevation of [Ca2+]i (Ca2+ dialysis or flash photolysis of caged Ca2+). 6 The above findings suggest that both intracellular Ca2+ release and voltage‐gated extracellular Ca2+ entry generate a spatial Ca2+ gradient, such that the local [Ca2+] near the exocytic sites was ∼3‐fold higher than the mean cytosolic [Ca2+]. However, neither cAMP nor the spatial Ca2+ gradient generated during depolarization could account for the high efficacy of corticotropin‐releasing hormone (CRH) in stimulating adrenocorticotropic hormone (ACTH) secretion from corticotrophs.

Ca2+ signaling and exocytosis in pituitary corticotropes

The secretion of adrenocorticotrophin (ACTH) from corticotropes is a key component in the endocrine response to stress. The resting potential of corticotropes is set by the basal activities of TWIK-related K(+) (TREK)-1 channel. Corticotrophin-releasing hormone (CRH), the major ACTH secretagogue, closes the background TREK-1 channels via the cAMP-dependent pathway, resulting in depolarization and a sustained rise in cytosolic [Ca(2+)] ([Ca(2+)](i)). By contrast, arginine vasopressin and norepinephrine evoke Ca(2+) release from the inositol trisphosphate (IP(3))-sensitive store, resulting in the activation of small conductance Ca(2+)-activated K(+) channels and hyperpolarization. Following [Ca(2+)](i) rise, cytosolic Ca(2+) is taken into the mitochondria via the uniporter. Mitochondrial inhibition slows the decay of the Ca(2+) signal and enhances the depolarization-triggered exocytotic response. Both voltage-gated Ca(2+) channel activation and intracellular Ca(2+) release generate spatial Ca(2+) gradients near the exocytic sites such that the local [Ca(2+)] is ~3-fold higher than the average [Ca(2+)](i). The stimulation of mitochondrial metabolism during the agonist-induced Ca(2+) signal and the robust endocytosis following stimulated exocytosis enable corticotropes to maintain sustained secretion during the diurnal ACTH surge. Arachidonic acid (AA) which is generated during CRH stimulation activates TREK-1 channels and causes hyperpolarization. Thus, corticotropes may regulate ACTH release via an autocrine feedback mechanism.

Arachidonic acid stimulates extracellular Ca2+ entry in rat pancreatic β cells via activation of the noncapacitative arachidonate-regulated Ca2+ (ARC) channels

Arachidonic acid (AA) is generated in the pancreatic islets during glucose stimulation. We investigated whether AA activated extracellular Ca(2+) entry in rat pancreatic beta cells via a pathway that was independent of the activation of voltage-gated Ca(2+) channels. The AA triggered [Ca(2+)](i) rise did not involve activation of GPR40 receptors or AA metabolism. When cells were voltage clamped at -70mV, the AA-mediated intracellular Ca(2+) release was accompanied by extracellular Ca(2+) entry. AA accelerated the rate of Mn(2+) quench of indo-1 fluorescence (near the Ca(2+)-independent wavelength of indo-1), reflecting the activation of a Ca(2+)-permeable pathway. The AA-mediated acceleration of Mn(2+) quench was inhibited by La(3+) but not by 2-APB (a blocker of capacitative Ca(2+) entry), suggesting the involvement of arachidonate-regulated Ca(2+) (ARC) channels. Consistent with this, intracellular application of the charged membrane-impermeant analog of AA, arachidonyl-coenzyme A (ACoA) triggered extracellular Ca(2+) entry, as well as the activation of a La(3+)-sensitive small inward current (1.7pA/pF) at -70mV. Our results indicate that the activation of ARC channels by intracellular AA triggers extracellular Ca(2+) entry. This action may contribute to the effects of AA on Ca(2+) signals and insulin secretion in rat beta cells.

Cholesterol Elevation Impairs Glucose-Stimulated Ca2+ Signaling in Mouse Pancreatic β-Cells

Recent studies have demonstrated that cholesterol elevation in pancreatic islets is associated with a reduction in glucose-stimulated insulin secretion, but the underlying cellular mechanisms remain elusive. Here, we show that cholesterol enrichment dramatically reduced the proportion of mouse β-cells that exhibited a Ca(2+) signal when stimulated by high glucose. When cholesterol-enriched β-cells were challenged with tolbutamide, there was a decrease in the amplitude of the Ca(2+) signal, and it was associated with a reduction in the cell current density of voltage-gated Ca(2+) channels (VGCC). Although the cell current densities of the ATP-dependent K(+) channels and the delayed rectifier K(+) channels were also reduced in the cholesterol-enriched β-cells, glucose evoked only a small depolarization in these cells. In cholesterol-enriched cells, the glucose-mediated increase in cellular ATP content was dramatically reduced, and this was related to a decrease in glucose uptake via glucose transporter 2 and an impairment of mitochondrial metabolism. Thus, cholesterol enrichment impaired glucose-stimulated Ca(2+) signaling in β-cells via two mechanisms: a decrease in the current density of VGCC and a reduction in glucose-stimulated mitochondrial ATP production, which in turn led to a smaller glucose-evoked depolarization. The decrease in VGCC-mediated extracellular Ca(2+) influx in cholesterol-enriched β-cells was associated with a reduction in the amount of exocytosis. Our findings suggest that defect in glucose-stimulated Ca(2+) signaling is an important mechanism underlying the impairment of glucose-stimulated insulin secretion in islets with elevated cholesterol level.

Endocytosis in identified rat corticotrophs

We used the patch‐clamp technique, in conjunction with membrane capacitance measurement, fluorescence measurement of intracellular calcium concentration ([Ca2+]i), and flash photolysis of caged Ca2+ to study exo‐ and endocytosis in identified rat corticotrophs. Exocytosis stimulated by depolarization pulses was typically followed by a ‘slow’ endocytosis that retrieved the membrane with a time constant of ∼6 s. The efficiency (the endocytosis/exocytosis amplitude ratio) of ‘slow’ endocytosis was ∼1.2 at [Ca2+]i < 3 μm and increased to ∼1.6 at [Ca2+]i > 3 μm. Whole‐cell dialysis through a patch pipette did not affect the kinetics and the efficiency of ‘slow’ endocytosis, but the amplitude of exocytosis was reduced. ‘Slow’ endocytosis did not require sustained [Ca2+]i elevation and its kinetics was only weakly [Ca2+]i dependent. Our results suggest that ‘slow’ endocytosis involves a Ca2+ sensor with a high Ca2+ affinity (∼500 nm). At high [Ca2+]i (> 10 μm), the ‘slow’ endocytosis was frequently preceded by a ‘fast’ endocytosis that comprised multiple steps of rapid decrease in membrane capacitance. Neither calmodulin nor calcineurin appeared to be the Ca2+ sensor for endocytosis because the two forms of endocytosis were not affected by the calmodulin inhibitor calmidazolium (500 μm) or the calcineurin inhibitors cyclosporin A (1 μm) and calcineurin autoinhibitory peptide (1 mg ml−1). Ba2+, a poor activator of calmodulin, could support both forms of endocytosis but slowed the kinetics of ‘slow’ endocytosis ∼2‐fold. Non‐hydrolysable analogues of GTP (GDP‐β‐S) and ATP (ATP‐γ‐S) also failed to inhibit either form of endocytosis, indicating that neither GTP nor ATP was essential for endocytosis. We suggest that the high Ca2+ affinity of ‘slow’ endocytosis may be important for maintaining continuous cycles of exocytosis‐endocytosis during sustained adrenocorticotropin secretion in corticotrophs.

Arginine Vasopressin Potentiates the Stimulatory Action of CRH on Pituitary Corticotropes via a Protein Kinase C–Dependent Reduction of the Background TREK-1 Current

The hypothalamic hormone arginine vasopressin (AVP) potentiates the stimulatory action of CRH on ACTH secretion from pituitary corticotropes, but the underlying mechanism is elusive. Using the perforated patch-clamp technique to monitor membrane potentials in mouse corticotropes, we found that AVP triggered a transient hyperpolarization that was followed by a sustained depolarization. The hyperpolarization was caused by intracellular Ca(2+) release that in turn activated the small conductance Ca(2+)-activated K(+) (SK) channels. The depolarization was due to the suppression of background TWIK-related K(+) (TREK)-1 channels. Direct activation of protein kinase C (PKC) reduced the TREK-1 current, whereas PKC inhibition attenuated the AVP-mediated reduction of the TREK-1 current, implicating the involvement of PKC. The addition of CRH (which stimulates the protein kinase A pathway) in the presence of AVP, or vice versa, resulted in further suppression of the TREK-1 current. In corticotropes with buffered cytosolic Ca(2+) concentration ([Ca(2+)]i), AVP evoked a sustained depolarization, and the coapplication of AVP and CRH caused a larger depolarization than that evoked by AVP or CRH alone. In cells with minimal perturbation of [Ca(2+)]i and background TREK-1 channels, CRH evoked a sustained depolarization that was superimposed with action potentials, and the subsequent coapplication of AVP and CRH triggered a transient hyperpolarization that was followed by a larger depolarization. In summary, AVP and CRH have additive effects on the suppression of the TREK-1 current, resulting in a more robust depolarization in corticotropes. We suggest that this mechanism contributes to the potentiating action of AVP on CRH-evoked ACTH secretion.

Arachidonic acid mobilizes Ca2+ from the endoplasmic reticulum and an acidic store in rat pancreatic β cells

In rat pancreatic β cells, arachidonic acid (AA) triggered intracellular Ca(2+) release. This effect could be mimicked by eicosatetraynoic acid, indicating that AA metabolism is not required. The AA-mediated Ca(2+) signal was not affected by inhibition of ryanodine receptors or emptying of ryanodine-sensitive store but was reduced by ∼70% following the disruption of acidic stores (treatment with bafilomycin A1 or glycyl-phenylalanyl-β-naphthylamide (GPN)). The action of AA did not involve TRPM2 channels or NAADP receptors because intracellular dialysis of adenosine diphosphoribose (ADPR; an activator of TRPM2 channels) or NAADP did not affect the AA response. In contrast, stimulation of IP(3) receptors via intracellular dialysis of adenophostin A, or exogenous application of ATP largely abolished the AA-mediated Ca(2+) signal. Intracellular dialysis of heparin abolished the ATP-mediated Ca(2+) signal but not the AA response, suggesting that the action of AA did not involve the IP(3)-binding site. Treatment with the SERCA pump inhibitor, thapsigargin, reduced the amplitude of the AA-mediated Ca(2+) signal by ∼70%. Overall, our finding suggests that AA mobilizes Ca(2+) from the endoplasmic reticulum as well as an acidic store and both stores could be depleted by IP(3) receptor agonist. The possibility of secretory granules as targets of AA is discussed.

Influence of Cholesterol on Cellular Signaling and Fusion Pore Kinetics

Cholesterol is an important lipid component of cellular membranes. Recent studies have shown that changes in cellular cholesterol level can affect cellular functions. Here, we summarize our recent findings on the impact of cholesterol on the glucose-stimulated Ca2+ signaling in rat pancreatic β cells and the fusion pore kinetics of large dense core granules in rat chromaffin cells. In mouse pancreatic β cells, pharmacological elevation of cellular cholesterol (but not cholesterol extraction) reduced the current density of the delayed rectifier K+ channels, the ATP-dependent K+ channels, and voltage-gated Ca2+ channels. Importantly, cholesterol enrichment impaired glucose-stimulated Ca2+ signaling in mouse pancreatic β cells via a suppression of voltage-gated Ca2+ channels and a decrease in mitochondrial ATP production, which in turn led to a reduction in the glucose-evoked depolarization. In rat chromaffin cells, we found that the persistence of the semi-stable fusion pore was increased by cholesterol enrichment, and acute cholesterol extraction from the cytosolic side of the cell destabilized the semi-stable fusion pore. Overall, our findings highlight the importance of cholesterol in the regulation of cellular signaling and exocytosis.