Direct versus indirect actions of ghrelin on hypothalamic NPY neurons

<div><p>Objectives</p><p>Assess direct versus indirect action(s) of ghrelin on hypothalamic NPY neurons.</p><p>Materials and methods</p><p>Electrophysiology was used to measure ion channel activity in NPY-GFP neurons in slice preparations. Ca²⁺ imaging was used to monitor ghrelin activation of isolated NPY GFP-labeled neurons. Immunohistochemistry was used to localize Trpm4, SUR1 and Kir6.2 in the hypothalamus.</p><p>Results</p><p>Acylated ghrelin depolarized the membrane potential (MP) of NPY-GFP neurons in brain slices. Depolarization resulted from a decreased input resistance (IR) in ~70% of neurons (15/22) or an increased IR in the remainder (7/22), consistent with the opening or closing of ion channels, respectively. Although tetrodotoxin (TTX) blockade of presynaptic action potentials reduced ghrelin-induced changes in MP and IR, ghrelin still significantly depolarized the MP and decreased IR in TTX-treated neurons, suggesting that ghrelin directly opens cation channel(s) in NPY neurons. In isolated NPY-GFP neurons, ghrelin produced a sustained rise of [Ca²⁺]_c, with an EC₅₀ ~110 pM. Pharmacologic studies confirmed that the direct action of ghrelin was through occupation of the growth hormone secretagogue receptor, GHS-R, and demonstrated the importance of the adenylate cyclase/cAMP/protein kinase A (PKA) and phospholipase C/inositol triphosphate (PLC/IP₃) pathways as activators of 5' AMP-activated protein kinase (AMPK). Activation of isolated neurons was not affected by CNQX or TTX, but reducing [Na⁺]_o suppressed activation, suggesting a role for Na⁺-permeable cation channels. SUR1 and two channel partners, Kir6.2 and Trpm4, were identified immunologically in NPY-GFP neurons <i>in situ</i>. The actions of SUR1 and Trpm4 modulators were informative: like ghrelin, diazoxide, a SUR1 agonist, elevated [Ca²⁺]_c and glibenclamide, a SUR1 antagonist, partially suppressed ghrelin action, while 9-phenanthrol and flufenamic acid, selective Trpm4 antagonists, blocked ghrelin actions on isolated neurons. Ghrelin activation was unaffected by nifedipine and ω-conotoxin, inhibitors of L- and N-type Ca²⁺ channels, respectively, while Ni²⁺, mibefradil, and TTA-P2 completely or partially inhibited ghrelin action, implicating T-type Ca²⁺ channels. Activation was also sensitive to a spider toxin, SNX-482, at concentrations selective for R-type Ca²⁺ channels. Nanomolar concentrations of GABA markedly inhibited ghrelin-activation of isolated NPY-GFP neurons, consistent with chronic suppression of ghrelin action <i>in vivo</i>.</p><p>Conclusions</p><p>NPY neurons express all the molecular machinery needed to respond directly to ghrelin. Consistent with recent studies, ghrelin stimulates presynaptic inputs that activate NPY-GFP neurons <i>in situ</i>. Ghrelin can also directly activate a depolarizing conductance. Results with isolated NPY-GFP neurons suggest the ghrelin-activated, depolarizing current is a Na⁺ conductance with the pharmacologic properties of SUR1/Trpm4 non-selective cation channels. In the isolated neuron model, the opening of SUR1/Trpm4 channels activates T- and SNX482-sensitive R-type voltage dependent Ca²⁺ channels, which could contribute to NPY neuronal activity <i>in situ</i>.</p></div

NYP-expressing neurons of the arcuate nucleus express subunits of both SUR1-Kir6.2 (K_ATP) and SUR1-Trpm4 channels.

<p>Coronal sections of the hypothalamus from an NPY-GFP mouse, showing NPY-GFP neurons (green) immunolabeled for SUR1(A), or Kir6.2 (B) or Trpm4 (C), as indicated (red); merged images show co-expression of GFP and all three channel subunits by NPY neurons (yellow) DAPI nuclear labeling shown in blue. The images (low power view, <i>upper panel</i>s, and high power view, <i>lower panels</i>) shown are representative of findings in 2 NPY-GFP mice.</p

AMPK and Ca²⁺/calmodulin (CaM)-dependent protein kinase kinases (CaMKK2) contribute to the ghrelin activation of NPY neurons.

<p>(A) AICAR (400 μM), an AMPK activator, mimics the effect of ghrelin. (B) Compound-C (30 μM), an inhibitor of AMPK, completely blocks the effect of oligomycin. (C) STO-609 (1 μM), a CAMKK2 inhibitor, partially reverses the effect of ghrelin. Ghrelin was applied at 100 pM (red bars). Traces are mean values from 5–20 cells.</p

Ghrelin directly activates NPY-GFP neurons.

<p>(A) Representative field of isolated neurons from NPY-GFP mice. (B) Same field after addition of ghrelin. (C) Merged image of fields (A) and (B). Arrows point to non-NPY cells. (D) Average values (n = 4) of Relative [Ca²⁺]_c after sequential addition of indicated concentrations of acylated ghrelin. (E) Ghrelin dose-response curve. (F) Ghrelin induced [Ca²⁺]_c increases are blocked by the GHS-R antagonist [D-lys3]-GHRP-6 (50 μM). (G) CNQX (2 μM), an AMPA receptor antagonist, did not affect ghrelin action. Ghrelin (200 pM; red bars) was applied in F and G. Traces are mean values from 6–12 neurons.</p

Ghrelin stimulation of GHS-R in isolated NPY-GFP neurons activates both adenylate cyclase (AC)-PKA and PLC-IP₃ signaling pathways.

<p>(A) The adenylate cyclase agonist, Forskolin (10 μM), mimics the effect of ghrelin. (B) The PKA inhibitor, H89 (10 μM) inhibits the effect of ghrelin. (C) U73122 (1 μM), an irreversible PLC inhibitor, reduced ghrelin action. (D) Thapsigargin (0.5 μM), an inhibitor of SERCA, the ER Ca²⁺ pump, did not inhibit ghrelin induced [Ca²⁺]_c increases. Ghrelin was applied at 100 pM (red bars). Traces are mean values from 5–20 cells.</p

K_ATP channel modulators have SUR1 dependent effects on isolated NPY-GFP neurons.

<p>(A) Diazoxide (200 μM) did not affect ghrelin action. (B) When applied alone, diazoxide (200 μM) mimics the effect of ghrelin, while glibenclamide (1μM) reduced the stimulatory effect of ghrelin. (C-D) Neither diazoxide (200 μM) nor glibenclamide (1μM) affected ghrelin action in SUR1^-/- mice lacking SUR1. Ghrelin was applied at 200 pM as shown (red bars). (E) Comparison of the percentages of all ghrelin-activated cells in preparations of neurons isolated from SUR1^-/- versus NPY-GFP mice shows the mean values are not significantly different, p > 0.7, using an unpaired, two-tailed t-test. About two-thirds of the activated neurons in NPY-GFP mouse preparations were GFP-positive. Values are the means ± S.D. from four NPY-GFP and six SUR1^-/- preparations totaling more than 1000 cells.</p

Antagonists of Trpm4 channels inhibit ghrelin action on isolated NPY-GFP neurons.

<p>(A) 9-phenanthrol (50 μM) and (B) flufenamic acid (100 μM) potently block the effects of ghrelin (200 pM; red bars).</p

Blockade of T-type and R-type voltage dependent channels reduces the effect of ghrelin.

<p>(A) Ni²⁺ (100 μM), an inhibitor of T and R-type channels, and 5 μM mibefradil, an inhibitor of T and L type channels, inhibit the effect of ghrelin. (B) TTA-P2 (1 μM), a selective inhibitor of T-type channels, partially inhibited ghrelin action. (C) SNX482 (50 nM), an inhibitor of R-type channels, partially inhibits ghrelin induced [Ca²⁺]_c increases. Ghrelin was applied at 100 pM (red bars).</p

Extracellular Na⁺ influx is required to sustain ghrelin activation.

<p>(A) Tetrodotoxin (TTX; 1 μM), an inhibitor of voltage-dependent Na⁺ channels, did not affect ghrelin induced [Ca²⁺]_c increases. (B) The substitution of choline for Na⁺ rapidly and reversibly blocked the action of ghrelin. Ghrelin was applied at 200 pM (red bars). The traces are the mean values from 5–20 neurons.</p

GABA inhibits the stimulatory effects of acylated ghrelin (100 pM) in a dose-dependent manner.

<p>The trace is the mean from 12 neurons.</p