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Previous studies reported inconsistent findings on autophagy activation in skeletal muscles after acute exercise. In this study, we investigated the effect of a single bout of exhaustive treadmill exercise on AMPK and autophagy activations in mice gastrocnemius muscle in vivo. Male ICR/CD-1 mice were randomly divided into the control and exercise groups. The later was subjected to a single bout of exhaustive treadmill exercise. Changes of AMPK, phosphorylation of AMPK Thr172 (pAMPK Thr172 ), and autophagy markers including Beclin1, LC3II/LC3I and p62 mRNA and protein expressions in gastrocnemius muscle at different times (0, 6, 12, 24 h) after the exercise were analysed by quantitative real-time PCR and western blot. Our results demonstrated that a single bout of exhaustive treadmill exercise significantly induced AMPK content and AMPK activity at 0, 6 and 12 h after the exercise, and changed the expressions of autophagy markers at different time points in the recovery period, respectively. Moreover, we observed positive correlations between expressions of LC3II/LC3I ratio and pAMPK Thr172 or AMPK, and a negative correlation between expressions of p62 and AMPK or pAMPK Thr172 . In conclusion, a single bout of exhaustive treadmill exercise in mice caused a prolonged activation of AMPK and improved autophagy in the gastrocnemius muscle. The regulation of autophagic markers were related to enhanced AMPK activity. The findings indicate that acute exercise enhanced AMPK-related autophagy activation may be the underlying molecular mechanism that regulates cellular energy metabolism during exercise., (© 2022 John Wiley & Sons Australia, Ltd.)

This study aimed to elucidate the role of transcription factor EB (TFEB) in protecting C2C12 myotubes against palmitate (PA)-induced insulin resistance (IR) and explored its mechanism associated with autophagy. PA treatment significantly decreased insulin sensitivity in myotubes and downregulated TFEB protein expression. TFEB overexpression significantly reversed the PA-suppressed glucose transporter 4 (GLUT4) protein expression and improved intracellular glucose uptake and consumption, and also alleviated the decrease of autophagy markers induced by PA. The effect of TFEB overexpression on GLUT4 was also abolished by the autophagy inhibitor 3-MA. In addition, AMPKɑ2-DN inhibited or abolished the effects of TFEB overexpression on upregulation of GLUT4 and PA-induced decrease of autophagy marker expressions. Taken together, our data demonstrated that upregulation of TFEB improved PA-induced IR in C2C12 myotubes by enhancing autophagy and upregulating AMPK activity. TFEB, as a critical regulator of glucose homeostasis in skeletal muscle cells, may be a potential therapeutic target for IR and Type 2 diabetes., (© 2021 John Wiley & Sons Australia, Ltd.)

1. The effects of copper chelators were investigated on the relaxant actions of the S-nitrosothiols S-nitrosoglutathione (GSNO) and S-nitroso-N-acetyl-d,l-penicillamine (SNAP), the non-S-nitrosothiol nitric oxide (NO) donor sodium nitroprusside (SNP), free radical NO (NO.) and the nitrergic neurotransmitter in rat isolated anococcygeus muscle. 2. Cumulative additions of GSNO (0.01-100 micro mol/L), SNAP (0.001-10 micro mol/L), SNP (0.001-1 micro mol/L) and NO. (0.5-5 micro mol/L) and electrical field stimulation (EFS; 1-5 Hz, 10 s) of nitrergic nerves in preparations precontracted with guanethidine (10-30 micro mol/L) and clonidine (0.01-0.3 micro mol/L) produced concentration-dependent relaxations. 3. The Cu[I] chelator neocuproine (10-30 micro mol/L) produced concentration-dependent inhibitions of the relaxations to GSNO and SNAP. At 30 micro mol/L, neocuprinone had no effect on relaxations to SNP (0.001-1 micro mol/L), NO. (0.5-5 micro mol/L) or EFS (1-5 Hz, 10 s). 4. The Cu[II] chelator cuprizone (30 micro mol/L) slightly and significantly enhanced relaxations to GSNO and NO., but had no effect on relaxations to SNAP, SNP or EFS. 5. In conclusion, the results indicate that Cu[I], but not Cu[II], may be involved in the relaxant actions of GSNO and SNAP in the rat anococcygeus muscle.

1. The effects of endogenous and exogenous acetylcholine (ACh) on nitrergic relaxations elicited by electrical field stimulation (EFS) were studied in guinea-pig endothelium-denuded basilar artery preparations precontracted with 1 micromol/L prostaglandin F(2 alpha) and a possible role of K+ channels in mediating the effects was investigated. 2. Acetylcholine (3 micromol/L) and physostigmine (10 micromol/L) produced small, yet statistically significant, inhibitions of EFS-induced nitrergic relaxations, while atropine (1 micromol/L) slightly enhanced the nitrergic response. The ACh-induced inhibition was atropine sensitive. Acetylcholine or atropine did not affect relaxations induced by sodium nitroprusside. 3. The inhibition of nitrergic relaxations by 3 micromol/L ACh was prevented by the K+ channel blockers tetraethylammonium and 4-aminopyridine, but was not changed by iberiotoxin, apamin or glibenclamide. 4. Neither vasoactive intestinal polypeptide nor the alpha2-adrenoceptor agonists noradrenaline and clonidine modulated nitrergic neurotransmission in the guinea-pig basilar artery. 5. The findings show that ACh acts on prejunctional muscarinic receptors of nitrergic nerves to inhibit nitrergic neurotransmission. It is suggested that endogenous ACh may have this effect; however, the physiological significance of this prejunctional modulation is not clear due to the relatively small effect produced. The prejunctional inhibitory action of ACh may involve opening of neuronal K+ channels.

1. The effects of endogenous and exogenous acetylcholine (ACh) on nitrergic relaxations elicited by electrical field stimulation (EFS) were studied in guinea-pig endothelium-denuded basilar artery preparations precontracted with 1 mumol/L prostaglandin F2 alpha and a possible role of K+ channels in mediating the effects was investigated. 2. Acetylcholine (3 mmol/L) and physostigmine (10 mumol/L) produced small, yet statistically significant, inhibitions of EFS-induced nitrergic relaxations, while atropine (1 mumol/L) slightly enhanced the nitrergic response. The ACh-induced inhibition was atropine sensitive. Acetylcholine or atropine did not affect relaxations induced by sodium nitroprusside. 3. The inhibition of nitrergic relaxations by 3 mumol/L ACh was prevented by the K+ channel blockers tetraethylammonium and 4-aminopyridine, but was not changed by iberiotoxin, apamin or glibenclamide. 4. Neither vasoactive intestinal polypeptide nor the alpha 2-adrenoceptor agonists noradrenaline and clonidine modulated nitrergic neurotransmission in the guinea-pig basilar artery. 5. The findings show that ACh acts on prejunctional muscarinic receptors of nitrergic nerves to inhibit nitrergic neurotransmission. It is suggested that endogenous ACh may have this effect; however, the physiological significance of this prejunctional modulation is not clear due to the relatively small effect produced. The prejunctional inhibitory action of ACh may involve opening of neuronal K+ channels.

1. The effects of ethacrynic acid were studied on relaxations elicited by nitric oxide (NO), the NO-donors sodium nitroprusside (SNP) and glyceryl trinitrate (GTN), nitrergic nerve stimulation and the NO-independent agent papaverine in isolated preparations of rat anococcygeus muscles. 2. Ethacrynic acid (100 mumol/L) produced complete relaxation of partially contracted anococcygeus muscles, but the tone recovered after the ethacrynic acid was washed out. Following exposure to ethacrynic acid, the relaxant responses to NO, SNP, GTN and nitrergic nerve stimulation were abolished or markedly reduced; however, the response to papaverine was only slightly reduced. 3. The presence of 3 mmol/L L-cysteine during the period of exposure to ethacrynic acid prevented the inhibition of the relaxing effects of SNP, GTN and nitrergic nerve stimulation almost completely, but did not affect the slight reduction in responses to papaverine. 4. The addition of L-cysteine (3 mmol/L) after incubation with ethacrynic acid did not significantly affect the inhibited responses to SNP and GTN; however, the inhibited responses to nitrergic nerve stimulation were slightly but significantly increased. 5. The results suggest that endogenous sulphydryl groups are required for the actions of NO, NO-donating drugs and the nitrergic transmitter in the rat anococcygeus muscle and possibly for the synthesis or release of the nitrergic transmitter.

1. The pharmacological actions of the oxidized and reduced forms of nicotinamide-adenosine dinucleotide (NAD, NADH) and nicotinamide-adenosine dinucleotide phosphate (NADP, NADPH) were studied on rat isolated anococcygeus muscles. 2. The actions of the two nucleotides were different, but there were no apparent qualitative differences between the oxidized and reduced forms of each. 3. In fully relaxed anococcygeus muscles, NADP(H) produced transient contractions that were subject to desensitization, but NAD(H) had no effect. 4. NADP(H) slightly enhanced contractions elicited by noradrenergic nerve stimulation. In contrast, noradrenergic contractions were inhibited by NAD(H). NADH reduced the stimulation-induced release of noradrenaline, but enhanced contractions elicited by exogenous noradrenaline. 5. In anococcygeus muscles partly contracted with guanethidine, NAD(H) produced a further sustained increase in tone; in contrast, NADP(H) mainly produced transient relaxations to which there was immediate desensitization. 6. Relaxations of anococcygeus muscle elicited by nitrergic nerve stimulation were not affected by NAD. In contrast, NADP(H) reduced them. 7. The actions of NAD(H) were generally the same as those of adenosine and can be attributed to activation of P1-purinoceptors since they were blocked by the selective antagonist 8-sulphophenyltheophylline. 8. The actions of NADP resembled those of the P2-purinoceptor agonist ATP to some extent, but there were some differences. As suggested by others, NADP may act on a unique receptor.

1. The effects of hydroxocobalamin (Vitamin B12a) on relaxations produced by nitric oxide (NO), some NO-donating compounds and nitrergic nerve stimulation in isolated preparations of the rat anococcygeus muscle were compared with the effects of haemoglobin. 2. Hydroxocobalamin (30 mumol/L) significantly reduced relaxations induced by NO (0.1-3 mumol/L) and sodium nitroprusside (SNP; 0.01-0.3 mumol/L) but did not affect relaxations induced by glyceryl trinitrate (GTN; 0.01-1 mumol/L), S-nitrosocysteine (0.1-0.3 mumol/L) or stimulation of nitrergic nerves. A higher concentration of hydroxocobalamin (100 mumol/L) slightly reduced nitrergic nerve stimulation-induced relaxations. 3. Haemoglobin (10 mumol/L) blocked relaxation induced by NO and reduced relaxations induced by SNP, GTN, S-nitrosocysteine and nitrergic nerve stimulation. 4. When nitrergic nerve stimulation-induced relaxations had been partially reduced by the NO synthase inhibitor L-NAME (5-10 mumol/L), hydroxocobalamin had only a weak and transient inhibitory effect. 5. Noradrenergic contractions induced by field stimulation were not affected by hydroxocobalamin (30 mumol/L), but were enhanced by haemoglobin (10 mumol/L). 6. The results suggest that the transmitter released from nitrergic nerves in anococcygeus muscles resembles NO-releasing compounds such as S-nitrosocysteine and GTN but not SNP or free NO.

1. Relaxations of strips of rat gastric fundus were elicited with nicotine (100 mumol/L), nitric oxide (NO; 30 mumol/L), sodium nitroprusside (SNP; 100 nmol/L) and vasoactive intestinal polypeptide (VIP; 1 nmol/L). 2. Methylene blue (30 mumol/L), an inhibitor of soluble guanylate cyclase, reduced relaxations elicited by NO and nicotine, but not those elicited by VIP. 3. Chymotrypsin (1 U/mL) abolished VIP-induced relaxations and reduced nicotine-induced relaxations, but had no effect on SNP-induced relaxations. 4. NG-nitro-L-arginine methyl ester (L-NAME; 100 mumol/L), an inhibitor of NO synthase, reduced relaxations elicited by nicotine, but not those elicited by SNP or VIP. 5. When nicotine-induced relaxations had been reduced by either L-NAME or chymotrypsin, the addition of the other agent produced a greater reduction. However, the relaxations were not abolished. 6. Nicotine-induced relaxations were abolished by tetrodotoxin (1 mumol/L) or hexamethonium (100 mumol/L), indicating that they were due to activation of neuronal nicotinic receptors. Their reduction by methylene blue and L-NAME indicates that an NO-like mediator was involved. Their reduction by chymotrypsin indicates that a VIP-like peptide was involved. However, since they were not abolished by a combination of L-NAME and chymotrypsin, it appears that at least one more as yet unidentified mediator may be involved.

1. The effects of diphenylene iodonium (DPI), an inhibitor of reduced nicotinamide adenine dinucleotide phosphate-dependent oxidases (which generate superoxide anions), were studied on nitric oxide (NO)-mediated responses in isolated preparations of the rat aorta and anococcygeus muscle. 2. In aortic rings, the endothelium-dependent relaxant action of acetylcholine was reduced by DPI (0.3-10 mumol/L) in a concentration-dependent manner and abolished by the NO synthase (NOS) inhibitor L-nitro-NG-arginine methylester (L-NAME; 100 mumol/L). Relaxations induced by sodium nitroprusside (SNP) or NO were not affected by DPI or L-NAME. 3. In anococcygeus muscles, DPI (0.3-10 mumol/L) as well as L-NAME (5-100 mumol/L) produced concentration-dependent reductions of relaxations produced by nitrergic nerve stimulation. Relaxations induced by NO and SNP were not affected by either DPI or L-NAME. L-Arginine (1 mmol/L) prevented the reduction of nitrergic relaxations by L-NAME but not by DPI. 4. Contractions of anococcygeus muscles elicited by exogenous noradrenaline (1 mumol/L) were not affected or were inhibited by DPI (0.3-10 mumol/L), but the contractions elicited by noradrenergic nerve stimulation were significantly enhanced by DPI and L-NAME. When noradrenergic contractions had already been maximally enhanced by L-NAME (100 mumol/L), DPI produced no further enhancement. L-Arginine (1 mmol/L) prevented the enhancement of noradrenergic contractions by L-NAME but not by DPI. 5. The efflux of radioactivity induced by field stimulation from anococcygeus muscles previously incubated with [3H]-noradrenaline was not affected by either DPI or L-NAME.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Argininosuccinic acid (ASA), a naturally occurring NG derivative of arginine, and the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) were compared for their ability to reduce responses to nitric oxide (NO) derived from endothelial cells (aorta) and nitrergic nerves (anococcygeus muscle). 2. In isolated rings of rat aorta, endothelium-dependent relaxation responses to acetylcholine were abolished by L-NAME (0.1 mmol/L) and were reduced by ASA (0.1 and 0.3 mmol/L). Relaxations induced by sodium nitroprusside (SNP) were not affected by L-NAME but were reduced by ASA. 3. In rat isolated anococcygeus muscles, relaxations elicited by nitrergic nerve stimulation at 1 Hz were abolished by L-NAME (0.1 mmol/L) but were only slightly reduced by ASA (1 mmol/L). The effect of ASA was not sustained. L-Arginine (1 mmol/L) prevented the effect of L-NAME but not that of ASA. Neither ASA or L-NAME inhibited SNP-induced relaxation in the anococcygeus muscle. 4. The results suggest that ASA inhibits NOS but this does not totally account for its effects in reducing NO-mediated relaxations produced by the endothelium-dependent vasodilator acetylcholine in rat aortic rings and stimulation of nitrergic nerves in the rat anococcygeus muscle.

1. Nicotine (10 mumol/L) produced rapidly developing but transient contractions of anococcygeus muscle isolated from rats. The magnitude of the response varied considerably between preparations. Tachyphylaxis occurred, such that no response was elicited by the same or a larger concentration in the continued presence of 10 mumol/L nicotine. 2. Contractions produced by nicotine were not affected by atropine, but were abolished by Hexamethonium and the alpha-adrenoceptor antagonists prazosin and phentolamine. Contractions were absent in the anococcygeus muscles of rats pretreated with reserpine. 3. The alpha 2-adrenoceptor agonist UK14304, or guanethidine, raised the tone of the anococcygeus muscle, and converted responses to field stimulation and nicotine to relaxations. Nicotine-induced relaxations were more pronounced in the presence of UK14304 than guanethidine. 4. Relaxations produced by nicotine (1-18 mumol/L) were transient, and tachyphylaxis occurred. When precautions were taken to avoid tachyphylaxis, concentration-response curves could be constructed. The relaxations elicited by nicotine were abolished or greatly reduced by hexamethonium, tetrodotoxin or omega-conotoxin GVIA. 5. The nitric oxide synthase inhibitor L-NG-nitroarginine methyl ester (90 mumol/L) enhanced contractile responses to field stimulation and nicotine, and markedly reduced relaxations elicited by field stimulation and nicotine in the presence of UK14304. These relaxations were restored by L-arginine (270 mumol/L). 6. The results suggest that nicotine acts on nicotinic receptors of noradrenergic and nitrergic nerve terminals in the rat anococcygeus muscle, resulting in the release of noradrenaline and nitric oxide respectively.

1. The present study was carried out to determine whether rilmenidine, a recently introduced antihypertensive agent which acts on alpha 2-adrenoceptors, has partial agonist activity on prejunctional alpha 2-adrenoceptors in guinea-pig atria. 2. Isolated preparations of guinea-pig atria were incubated with [3H]-noradrenaline and the efflux of radioactivity induced by stimulation of intramural sympathetic nerves was used as an index of release of transmitter noradrenaline. 3. Rilmenidine (1 mumol/l) inhibited noradrenaline release evoked by short trains (five, 20 and 50 pulses) of sympathetic nerve stimulation and this inhibitory effect of rilmenidine was antagonized by the alpha 2-adrenoceptor antagonists, idazoxan (0.1 and 0.3 mumol/l) and rauwolscine (0.3 mumol/l) whereas it was not affected by the alpha 1-adrenoceptor antagonist prazosin (0.1 mumol/l). 4. On the other hand, rilmenidine (1 mumol/l) enhanced noradrenaline release evoked by long trains (150 and 300 pulses) of stimulation and this effect was also abolished by idazoxan (0.1 mumol/l). 5. These findings suggest that the effects of rilmenidine on transmitter release depend on the degree of auto-inhibition: when the concentration of noradrenaline in the biophase of the prejunctional alpha 2-adrenoceptors is low, rilmenidine acts as an agonist, but when the concentration is high it acts as an antagonist. Thus, rilmenidine, like clonidine, is a partial agonist on prejunctional alpha 2-adrenoceptors in guinea-pig atria.

1. The nitric oxide (NO) synthesis inhibitor NG-monomethyl-L-arginine (L-NMMA), but not D-NMMA, inhibited the NANC-mediated relaxations of the rat anococcygeus muscle, but did not affect the relaxation produced by sodium nitroprusside. 2. The inhibitory effect of L-NMMA was reversed by L-arginine but not by D-arginine, and prior exposure to L-arginine blocked the effect of L-NMMA. 3. The noradrenergically mediated contractions of the anococcygeus elicited by field stimulation were slightly enhanced by L-NMMA, but the response to noradrenaline was not affected. 4. The results suggest that NANC transmission in the rat anococcygeus muscle involves the generation of NO from arginine.