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J C Fong

Publications and source records attributed to J C Fong.

At least 19 recordsLinked to original sources
PubMed2001

Endothelin-1 increases glucose transporter glut1 mRNA accumulation in 3T3-L1 adipocytes by a mitogen-activated protein kinase-dependent pathway.

The mechanism of enhancing glucose transport by prolonged endothelin-1 (ET-1) treatment of 3T3-L1 adipocytes was examined. Western and Northern blot analyses indicated that ET-1 increased the amount of both GLUT1 protein and mRNA. The degradation rate of GLUT1 mRNA as measured in the presence of actinomycin D, nevertheless, was not significantly altered by ET-1. Whereas various inhibitors for distinct signalling pathways were tested, only the mitogen-activated protein kinase (MAPK) kinase inhibitor, PD98059, was found to decrease significantly the enhancing effect of ET-1. Similar extent of inhibition was observed in cells pretreated with pertussis toxin (PT). Immunoblot analysis revealed that ET-1 may stimulate a transient phosphorylation of p42/p44 MAPK and both PT and PD98059 inhibited this stimulation. In addition, the effect of ET-1 on GLUT1 mRNA accumulation was inhibited by PD98059 and cycloheximide, implying that a trans-activation was involved. Taken together, these results suggest that ET-1 may induce GLUT1 gene expression by a MAPK-dependent mechanism.

3T3 Cells↗
PubMed2000

Effects of crude drugs on glucose uptake in 3T3-L1 adipocytes.

In this study, various water-extracted crude drugs from Radix Asparagi, Radix Ginseng, Radix Scutellariae, Cortex Lycii Radicis, Cortex Phellodendri and Radix Ophiopogonis were investigated in their effects on [3H]-2-deoxyglucose uptake in 3T3-L1 adipocytes. Following treatment of cells with various crude drugs for 60 mim, the basal [3H]-2-deoxyglucose uptake in cultured 3T3-L1 cells was changed by Radix Asparagi from 140 pmole/min/mg protein of control to 513 (0.1 mg/ml), 201 (1 mg/ml) and 97 (10 mg/ml). Glucose uptake was changed to 324 (0.1 mg/ml), 146 (1 mg/ml) and 46 (10 mg/ml) with Radix Ginseng. In the presence of Radix Scutellariae, glucose uptake was changed to 215 (0.1 mg/ml), 213 (1 mg/ml) and 34 (10 mg/ml). In the presence of Cortex Lycii Radicis, glucose uptake was 230 (0.1 mg/ml), 188 (1 mg/ml) and 38 (10 mg/ml). In the case of Cortex Phellodendri and Radix Ophiopogonis, uptake was changed to 142 (0.1 mg/ml), 132 (1 mg/ml), 24 (10 mg/ml) and 489 (0.1 mg/ml), 374 (1 mg/ml), 344 (10 mg/ml), respectively. In insulin-stimulated cells, the [3H]-2-deoxyglucose uptake was changed by Radix Asparagi from 570 pmole/min/mg protein of the control to 816 (0.1 mg/ml), 674 (1 mg/ml) and 532 (10 mg/ml). After incubation with Radix Ginseng, the glucose uptake was changed to 254 (0.1 mg/mi), 123 (1 mg/mi) to 76 (10 mg/mi). In the presence of Radix Scutellariae, the glucose uptake was changed to 315 (0.1 mg/ml), 265 (1 mg/ml) and 33 (10 mg/ml). After incubation of Cortex Lycii Radicis, the uptake activity was changed to 281 (0.1 mg/ml), 248 (1 mg/ml) and 37 (10 mg/ml). In the case of Cortex Phellodendri and Radix Ophiopogonis, the activity of glucose uptake was measured as 747 (0.1 mg/ml), 523 (1 mg/ml), 33 (10 mg/ml) and 753 (0.1 mg/ml), 740 (1 mg/ml), and 421 (10 mg/ml), respectively. These results indicate that the water-extracted materials of Radix Asparagi and Radix Ophiopogonis increase the glucose uptake in basal and insulin-stimulated 3T3-L1 adipocytes.

3T3 Cells↗
PubMed1999

Cyclosporin A induces a biphasic increase in KCl-induced calcium influx in GH3 pituitary cells.

The role of calcineurin in modulation of calcium channel activity was examined in GH3 pituitary cells by using its selective inhibitor cyclosporin A. While cyclosporin A had little effect on basal activity, it induced a biphasic increase in K+-induced 45Ca2+ influx. Cyclosporin A rapidly increased K+-induced 45Ca2+ influx to approximately 140% of control in 1 h and the increment maintained at this magnitude for 1-8 h. Thereafter, K+-induced 45Ca2+ influx gradually increased further to approximately 220% after 24 h exposure to this compound. In the presence of anisomycin, however, the increase occurred at the latter phase was abolished. In addition, the increased calcium influx in cyclosporin A-pretreated cells had a similar sensitivity to KCl and verapamil as in untreated cells. Measurement of intracellular Ca2+ level by Fura-2 analysis indicated that [Ca2+]i increase induced by high K+ or vasoactive intestinal peptide was similarly augmented in cyclosporin A-pretreated cells. Thus the results of this study suggest that calcineurin may play a tonic control on L-type Ca2+ channel, and inhibition of this enzyme may induce a subsequently protein synthesis-dependent higher channel activity.

Animals↗
PubMed1999

Synergistic effect of arachidonic acid and cyclic AMP on glucose transport in 3T3-L1 adipocytes.

The combined effect of arachidonic acid and cAMP on glucose transport was examined in 3T3-L1 adipocytes. In cells pre-treated with arachidonic acid and increasing concentrations of 8-bromo cAMP for 8 h, although either agent alone enhanced glucose uptake, the simultaneous presence of both agents dramatically increased 2-deoxyglucose uptake in a synergistic fashion. Insulin-stimulated glucose transport, on the other hand, was only slightly affected. The synergistic effect of these two agents was abolished in the presence of cycloheximide. Immunoblot analysis revealed that the contents of ubiquitous glucose transporter (GLUT1) in total cellular and plasma membranes were similarly augmented in cells pre-treated with both arachidonic acid and 8-bromo cAMP, to a greater extent than the additive effect of each agent alone. The content of GLUT4, on the other hand, was not altered under the same experimental conditions. In cells pre-treated with 4beta-phorbol 12beta-myristate 13alpha-acetate (PMA) for 24 h to down-regulate protein kinase C (PKC), the subsequent synergistic effect of arachidonic acid and 8-bromo cAMP was greatly inhibited. In addition, pre-treatment with both PMA and 8-bromo cAMP enhanced glucose transport in a similarly synergistic fashion. Thus the present study seems to indicate that arachidonic acid may act with cAMP in a synergistic way to increase glucose transport by a PKC-dependent mechanism. The increased activity may be accounted for by increased GLUT1 synthesis.

3T3 Cells↗
PubMed1998

4-Bromocrotonic acid enhances basal but inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes.

Inhibitors of fatty acid oxidation, 2-bromopalmitic acid (Br-C16) and 4-bromocrotonic acid (Br-C4) were examined for their effect on glucose transport in 3T3-L1 adipocytes. Whereas Br-C16 was without effect, Br-C4 augmented basal but inhibited insulin-stimulated 2-deoxyglucose uptake in a dose- and time-dependent manner. Immunoblot analysis indicated that following Br-C4 pretreatment, the content of GLUT1 in plasma membranes was increased whereas insulin-induced translocation of GLUT4 was greatly eliminated. The total cellular amount of GLUT1 or GLUT4, on the other hand, was not altered. Thus these results seem to suggest that Br-C4 has opposite effect on basal and insulin-stimulated glucose transport by a mechanism other than its inhibition of fatty acid oxidation. The translocation processes for both GLUT1 and GLUT4 transporters appears to be altered.

3T3 Cells↗
PubMed1998

Arachidonic acid and protein synthesis inhibitor act synergistically to suppress insulin-stimulated glucose transport in 3T3-L1 adipocytes.

The effect of arachidonic acid (AA) on insulin-stimulated glucose transport by 3T3-L1 adipocytes was examined in the presence of cycloheximide. We found that AA acted synergistically with cycloheximide to suppress insulin-stimulated glucose transport, although it alone was without effect. Similar phenomena were observed while protein synthesis inhibitors other than cycloheximide were employed. Immunoblot analysis indicated that the increase in plasma membranes of the insulin-regulated glucose transporter (GLUT4) in response to insulin was decreased in cells pretreated with cycloheximide for a prolonged time, while total amount of GLUT4 was not altered. Simultaneous presence of AA with cycloheximide had no further effect on the amount of GLUT4 in either total or plasma membranes. Thus the present study suggests that AA in the presence of a protein synthesis inhibitor seems to decrease the intrinsic activity of GLUT4.

3T3 Cells↗
PubMed1997

Differential inhibition of lipolysis by 2-bromopalmitic acid and 4-bromocrotonic acid in 3T3-L1 adipocytes.

Two inhibitors of fatty acid oxidation, 2-bromopalmitic acid (Br-C16) and 4-bromocrotonic acid (Br-C4) were examined for their effect on lipolysis in 3T3-L1 adipocytes. Both agents inhibited in a dose-dependent manner the rate of oxidation of exogenously added [1-14C]palmitate with similar time-courses, reaching a plateau at 3-9 h. While Br-C16 at 50 microM and 100 microM inhibited palmitate oxidation by approximately 40% and 60%, respectively, pretreatment with both concentrations inhibited lipolysis in washed cells in an almost identical manner. The magnitude of inhibition increased with time of pretreatment. On the other hand, like inhibition of fatty acid oxidation, inhibition of lipolysis by Br-C4 pretreatment was dose-dependent with maximal inhibition reached after 3 h pretreatment. The finding that isoproterenol- and dibutyryl cAMP-stimulated lipolysis were similarly suppressed by either Br-C4 or Br-C16 pretreatment, suggesting that a step distal to cAMP formation was involved. In addition, while the inhibitory effect of Br-C16 was not significantly influenced, the inhibition of lipolysis caused by Br-C4 was attenuated by pretreating cells with crotonic acid, octanoate, or palmitate. The longer chain-length of the fatty acids the cells were exposed, the stronger attenuation of the inhibition caused by Br-C4 was observed. Moreover, whereas pretreatment with Br-C16 was without effect, pretreatment with Br-C4 significantly decreased hormone-sensitive lipase (HSL) activity in cell extracts, albeit to an extent much smaller than its inhibitory effect on lipolysis. In conclusion, these results indicate that irreversible inhibition of lipolysis by Br-C16 or Br-C4 cannot be attributed to their effect on fatty acid oxidation. Some factor capable of modulating HSL activity seems to be involved.

3T3 Cells↗
PubMed1997

Effects of antiglaucoma drugs and corticosteroids on the cellular glucose uptake in cultured porcine corneal endothelial cells.

In this study, various commercially used antiglaucoma drugs and corticosteroids were investigated in their effects on porcine corneal endothelial cells especially in cellular glucose uptake. Cellular glucose uptake directly affects the pumping efficiency in corneal endothelial cells. Following the cells' treatment with various antiglaucoma eyedrops for 100 min, the 3H-2-deoxyglucose uptake in cultured porcine corneal endothelial cells was affected by betaxolol from 3.1% (1.6 mM), 181% (0.16 mM) to 158% (0.016 mM), by timolol from 93% (0.79 mM), 227% (0.079 mM) to 151% (0.0079 mM), by carteolol from 141% (3.4 mM), 180% (0.34 mM) to 97% (0.034 mM), by levobunolol from 80% (1.5 mM), 98% (0.15 mM) to 90% (0.015 mM), by dipivefrin from 116% (0.2 mM), 176% (0.02 mM) to 108% (0.002 mM) and by pilocarpine from 115% (9.6 mM), 210% (0.96 mM) to 210% (0.096 mM) when the cells were compared with a control medium. In the presence of various corticosteroids, the glucose uptake in corneal endothelial cells was affected by fluorometholone from 160% (0.26 mM), 139% (0.026 mM) to 107% (0.0026 mM), by dexamethasone from 85% (0.25 mM), 117% (0.025 mM) to 109% (0.0025 mM) and by betamethasone from 95% (0.25 mM), 96% (0.025 mM) to 99% (0.0025 mM). These results show that the commercial eyedrops of antiglaucoma drugs and corticosteroids will not decrease the cellular glucose uptake in cultured porcine corneal endothelial cells except when incubated with high concentrations of betaxolol, levobunolol and dexamethasone.

Adrenal Cortex Hormones↗
PubMed1997

Alteration of glucose uptake in cultured human corneal endothelial cells grown in high glucose media via cAMP-dependent pathway.

In this study, cultured human corneal endothelial cells were incubated in media containing various concentrations of glucose at 5 mM, 10 mM, and 25 mM for 2 days. Then, the cellular 2-deoxyglucose uptake and cAMP concentration of cultured human corneal endothelial cells were measured. The results indicated that the activity of cellular glucose uptake of nmole/min/mg protein was decreased gradually from 0.18 (5 mM), 0.10 (10 mM), 0.07 (20 mM) to 0.06 (25 mM) after 2 days incubation with a high concentration of glucose. The glucose uptake in insulin-treated human corneal endothelial cells also exhibited a similar declining effect in high glucose media from 0.30 (5 mM), 0.11 (10 mM), 0.08 (20 mM) to 0.05 (25 mM). The cAMP concentration in human corneal endothelial cells was measured in the presence of high glucose media. It was indicated that the cAMP concentrations of pmole/well in both insulin-treated and non-insulin treated cells were also decreased after increasing the glucose concentration in the media from 73 (5 mM) to 20 (25 mM) and 101 (5 mM) respectively. The cAMP concentration in insulin-treated cells was less than in non-insulin treated cells. This decreasing effect was significantly reversed by the addition of 1 mM dibutyryl-cAMP to the cells for 1 hour in both groups. These results suggest that the diabetic state may decrease the 2-deoxyglucose uptake in human corneal endothelial cells via cAMP-dependent pathway.

Animals↗
PubMed1996

Arachidonic acid stimulates the intrinsic activity of ubiquitous glucose transporter (GLUT1) in 3T3-L1 adipocytes by a protein kinase C-independent mechanism.

Exposure of adipocytes to arachidonic acid rapidly enhanced basal 2-deoxyglucose uptake, reaching maximal effect at approximately 8 hr. Insulin-stimulated 2-deoxyglucose uptake was not altered over the experimental period. While the short-term (2-h exposure) effect of arachidonic acid was negligibly influenced by cycloheximide, the enhancement of glucose transport by long-term (8-h) exposure to arachidonic acid was markedly decreased by the simultaneous presence of protein-synthesis inhibitors, implying that the short-term and long-term effects of arachidonic acid may involve distinct mechanisms. Immunoblot analysis revealed that 8-h but not 2-h exposure to arachidonic acid increased the content of the ubiquitous glucose transporter (GLUT1) in both total cellular and plasma membranes. The insulin-responsive glucose transporter (GLUT4), on the other hand, was not affected. Following 2-h exposure to arachidonic acid, kinetic studies indicated that the apparent Vmax of basal 2-deoxyglucose uptake was more than doubled, while the apparent Km for 2-deoxyglucose remained unchanged. Protein kinase C (PKC) depletion by pretreating cells with 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) for 24 h had little influence on the subsequent enhancing effect of arachidonic acid on 2-deoxyglucose uptake. In addition, PMA was able to stimulate 2-deoxyglucose uptake in arachidonic-acid-pretreated cells with similar increments as in non-treated cells. Thus, our data seem to suggest that arachidonic acid may enhance the intrinsic activity of GLUT1 by a PKC-independent mechanism.

3T3 Cells↗
PubMed1994

Endothelin-1 inhibits insulin-stimulated glucose uptake in isolated rat adipocytes.

A single class of high affinity endothelin-1 (ET-1) binding sites with an apparent Kd of 350 pM and a binding capacity of 69,000 sites/cell was found in isolated rat adipocytes. Whereas ET-1 had no effect on basal glucose uptake, it inhibited insulin-stimulated glucose uptake in both a dose- and time-dependent manner. Insulin binding, however, was not altered. Thus, the present study demonstrates the presence of ET-1 receptor in isolated rat adipocytes and the inhibition of insulin-stimulated glucose uptake by ET-1 via a post-receptor mechanism.

Adipocytes↗
PubMed1994

Enhanced cyclic AMP responses in GH3 pituitary cells pretreated with muscarinic receptor agonists.

The effect of prolonged activation of cholinergic receptors on the subsequent cAMP responses in GH3 pituitary cells was examined. Whereas acetylcholine pretreatment had no effect on basal cAMP levels, it transiently enhanced vasoactive intestinal peptide-stimulated cAMP accumulation in the cells and this effect was abolished by atropine. Inclusion of eserine in the incubation medium, however, sustained and reinforced the enhancing effect of acetylcholine on forskolin-stimulated cAMP synthesis over 24 h. Similar enhancing effect was observed by oxotremorine or carbachol pretreatment. Furthermore, cAMP production in response to forskolin in the absence or presence of 3-isobutyl-1-methylxanthine was similarly augmented by acetylcholine pretreatment. The enhancement of forskolin-stimulated cAMP accumulation was arrested by pertussis toxin whether it was added before or during the oxotremorine pretreatment. Although cholera toxin pretreatment increased greatly the forskolin-stimulated cAMP levels, concomitant addition of oxotremorine was able to augment the response further in GH3 cells. In addition, cholera toxin- or pertussis toxin-catalysed ADP-ribosylation of G proteins as well as immunoreactive Gs alpha or Gi alpha levels in membranes obtained from oxotremorine-pretreated cells were not different from those from non-treated cells. Thus, the present study has demonstrated that continuous muscarinic receptor activation leads to increased adenylate cyclase activity in GH3 cells. Although a functional Gi/Go seems to be required for the development of this enhanced activity, neither Gi/Go nor Gs appears to be altered.

Acetylcholine↗
PubMed1993

Correlation between prolactin secretion and Gs protein expression during sustained cholera-toxin stimulation.

We have studied the chronic effect of cholera toxin (CTX) on prolactin synthesis and secretion in GH3 pituitary-tumour cells. Time-course analysis showed that prolactin secretion increased with time of CTX exposure, reached a peak at 3 h, and decreased thereafter. Prolactin synthesis was also shown to be stimulated by CTX. The basic and forskolin-stimulated cyclic AMP levels of the CTX-treated cells followed a biphasic time response similar to that of prolactin secretion. Exposure of cells to CTX for more than 3 h abolished the subsequent CTX-catalysed ADP-ribosylation in vitro. Moreover, a significant decrease in the pertussis-toxin-catalysed ADP-ribosylation was found after cells were exposed to CTX for longer than 6 h. Western-blot analysis indicated that the amount of Gs alpha (alpha-subunit of Gs) protein increased within 3 h, followed by a gradual decrease to 50% of the control level at 24 h. The accumulation of Gs alpha mRNA increased within 6 h of CTX exposure, and decreased thereafter to 40% of the basal level at 48 h. Our findings that prolonged treatment of CTX induced similar patterns of time responses in Gs alpha protein expression, cyclic AMP production and prolactin secretion indicate that CTX-induced changes in Gs alpha protein levels may be responsible for the cellular response leading to prolactin secretion.

Adenosine Diphosphate Ribose↗
PubMed1993

Multiple mechanisms for desensitization of A2a adenosine receptor-mediated cAMP elevation in rat pheochromocytoma PC12 cells.

To understand the regulation of A2a adenosine receptor (A2a-R) response, we examined the molecular mechanisms underlying the desensitization of A2a response in rat pheochromocytoma PC12 cells, which possess an A2a-R identical with the A2a receptor we recently cloned from rat brain. Prolonged exposure of PC12 cells to adenosine agonists significantly inhibited the response of the cells to subsequent stimulation with an A2a-selective adenosine agonist (CGS21680). No significant change in the number of binding sites and affinity for CGS21680 was observed in desensitized cells, nor did we find any significant change in the transcript level of A2a-R in cells pretreated with adenosine agonists. However, the basal adenylyl cyclase activity and the cyclase activities stimulated by adenosine agonists, by GTP gamma S, and by forskolin were reduced in desensitized cells. Prolonged exposure of PC12 cells to dibutyryl-cAMP did not significantly change either the basal or the adenosine agonist-evoked adenylyl cyclase activity. Therefore, elevation of cellular cAMP content is by itself not sufficient to produce the observed reductions of adenylyl cyclase activity with A2a desensitization. Inhibition of adenylyl cyclase activity in desensitized cells occurred after short-term (30 min) incubation with CGS21680 and could be blocked by the adenosine antagonist 8-cyclopentyl-1,3-dipropylxanthine. Gs alpha protein levels did not significantly change after a 30-min exposure to CGS21680. In contrast, long-term exposure (12-20 hr) of PC12 cells to adenosine agonists resulted in a slight further reduction of adenylyl cyclase activity and a consistent decline in the Gs alpha protein level. In addition, long-term incubation with adenosine agonists or with forskolin-enhanced phosphodiesterase (PDE) activity in the cytosolic and membrane fractions by 57 +/- 9% and 53 +/- 18%, respectively. Hydrolysis of cAMP was significantly faster in agonist-desensitized cells than in control cells. PDE might therefore play an important role in desensitization of the A2a response in PC12 cells. Polymerase chain reaction-based analysis of the mRNA for A2a-R and A2b-R indicated that both A2a-R and A2b-R were present in PC12 cells; the A2b response was also diminished in A2a-desensitized cells. Our data suggest that inhibition of adenylyl cyclase after short-term agonist treatment, down-regulation of Gs alpha protein level after long-term agonist treatment, and activation of PDE after long-term agonist treatment account for desensitization of the A2a-mediated response in PC12 cells.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1991

Enhanced lipolysis in 3T3-L1 adipocytes following prolonged exposure to tolbutamide.

The effect of tolbutamide on lipolysis was examined in 3T3-L1 adipocytes. Whereas lipolysis was reversibly inhibited by tolbutamide, prolonged treatment with this agent dose-dependently increased both basal and isoproterenol-stimulated lipolysis in washed adipocytes. The latter effect of tolbutamide was not accompanied with altered cAMP levels in the cells and was abolished in the presence of cycloheximide. Moreover, the lipolytic responses induced by isobutylmethylxanthine, forskolin and dibutyryl cAMP were also augmented by prolonged treatment of adipocytes with tolbutamide. Thus, it appears that development of enhanced lipolysis in 3T3-L1 adipocytes following prolonged exposure to tolbutamide requires continuous protein synthesis and probably involves a step distal to cAMP production.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Somatostatin inhibits pepsinogen secretion via a cyclic AMP-independent pathway.

Frog esophageal mucosa contains peptide glands which release pepsinogen in response to a variety of secretagogues and serves as a model to examine the inhibitory action of somatostatin. The pepsinogen secretion in response to bethanechol was inhibited by somatostatin in a noncompetitive fashion. The maximal response induced by bethanechol was reduced and the EC50 for bethanechol was increased in the presence of somatostatin. On the other hand, somatostatin showed essentially no effect on pepsinogen release evoked by ionophore A23187, dibutyryl cAMP or by forskolin in the presence of atropine. Atropine was included in the incubation mixture to eliminate the effect of acetylcholine released by forskolin from the intrinsic cholinergic neurons also present in the mucosa. Somatostatin did not exert any significant effect on the basal or the forskolin-stimulated cAMP accumulation in the mucosa, nor the basal or the forskolin-stimulated adenylate cyclase activity in the membranes of the peptic cells isolated from the mucosa. Thus, these results seem to suggest that somatostatin inhibits pepsinogen secretion from frog esophageal mucosa by a cAMP-independent pathway.

Adenylyl Cyclases↗
PubMed1991

Potentiation of thyrotropin-releasing hormone-stimulated prolactin mRNA levels in GH3 cells by acetylcholine.

We have examined the effect of acetylcholine (ACh) pretreatment on the thyrotropin-releasing hormone (TRH) induced prolactin gene expression in GH3 cells, a rat pituitary tumor cell line. Prolonged exposure (greater than 6 h) to ACh enhanced the TRH-induced prolactin mRNA accumulation in a time- and concentration-dependent manner while ACh by itself did not affect the basal prolactin mRNA levels appreciably. Maximal augmentation of the TRH-induced prolactin mRNA accumulation was obtained when cells were pretreated with 10(-5) M ACh for 24 h. The activation was mimicked by carbachol and oxotremorine and was blocked by the simultaneous presence of atropine. Preincubation of GH3 cells with pertussis toxin abolished the augmenting effect of ACh. These results indicate that prolonged exposure to muscarinic receptor agonists may enhance the TRH-stimulated prolactin mRNA expression and a pertussis toxin sensitive G-protein may be involved.

Acetylcholine↗