Aims Connexins form gap-junctions (GJs) that directly connect cells, thereby coordinating vascular cell function and controlling vessel diameter and blood flow. GJs are composed of two hemichannels contributed by each of the connecting cells. Hemichannels also exist as non-junctional channels that, when open, lead to the entry/loss of ions and the escape of ATP. Here we investigated cross-talk between hemichannels and Ca2+/purinergic signalling in controlling blood vessel contraction. We hypothesized that hemichannel Ca2+ entry and ATP release contributes to smooth muscle cell (SMC) Ca2+ dynamics, thereby influencing vessel contractility. We applied several peptide modulators of hemichannel function and inhibitors of Ca2+ and ATP signalling to investigate their influence on SMC Ca2+ dynamics and vessel contractility. Methods and results Confocal Ca2+ imaging studies on small mesenteric arteries (SMAs) from rat demonstrated that norepinephrine-induced SMC Ca2+  oscillations were inhibited by blocking IP3 receptors with xestospongin-C and by interfering with hemichannel function, most notably by the specific Cx43 hemichannel blocking peptide TAT-L2 and by TAT-CT9 that promotes Cx43 hemichannel opening. Evidence for hemichannel involvement in SMC function was supported by the fact that TAT-CT9 significantly increased SMC resting cytoplasmic Ca2+ concentration, indicating it facilitated Ca2+ entry, and by the observation that norepinephrine-triggered vessel ATP release was blocked by TAT-L2. Myograph tension measurements on isolated SMAs showed significant inhibition of norepinephrine-triggered contractility by the ATP receptor antagonist suramin, but the strongest effect was observed with TAT-L2 that gave ∼80% inhibition at 37 °C. TAT-L2 inhibition of vessel contraction was significantly reduced in conditional Cx43 knockout animals, indicating the effect was Cx43 hemichannel-dependent. Computational modelling suggested these results could be explained by the opening of a single hemichannel per SMC. Conclusions These results indicate that Cx43 hemichannels contribute to SMC Ca2+ dynamics and contractility, by facilitating Ca2+ entry, ATP release, and purinergic signalling.

Key points Using recombinant DNA technology, the present study provides the first strong and direct evidence indicating that β‐alanine is an efficient substrate for the mammalian transaminating enzymes 4‐aminobutyrate‐2‐oxoglutarate transaminase and alanine‐glyoxylate transaminase. The concentration of carnosine and anserine in murine skeletal and heart muscle depends on circulating availability of β‐alanine, which is in turn controlled by degradation of β‐alanine in liver and kidney. Chronic oral β‐alanine supplementation is a popular ergogenic strategy in sports because it can increase the intracellular carnosine concentration and subsequently improve the performance of high‐intensity exercises. The present study can partly explain why the β‐alanine supplementation protocol is so inefficient, by demonstrating that exogenous β‐alanine can be effectively routed toward oxidation. AbstractThe metabolic fate of orally ingested β‐alanine is largely unknown. Chronic β‐alanine supplementation is becoming increasingly popular for improving high‐intensity exercise performance because it is the rate‐limiting precursor of the dipeptide carnosine (β‐alanyl‐l‐histidine) in muscle. However, only a small fraction (3–6%) of the ingested β‐alanine is used for carnosine synthesis. Thus, the present study aimed to investigate the putative contribution of two β‐alanine transamination enzymes, namely 4‐aminobutyrate‐2‐oxoglutarate transaminase (GABA‐T) and alanine‐glyoxylate transaminase (AGXT2), to the homeostasis of carnosine and its methylated analogue anserine. We found that, when transfected into HEK293T cells, recombinant mouse and human GABA‐T and AGXT2 are able to transaminate β‐alanine efficiently. The reaction catalysed by GABA‐T is inhibited by vigabatrin, whereas both GABA‐T and AGXT2 activity is inhibited by aminooxyacetic acid (AOA). Both GABA‐T and AGXT2 are highly expressed in the mouse liver and kidney and the administration of the inhibitors effectively reduced their enzyme activity in liver (GABA‐T for vigabatrin; GABA‐T and AGXT2 for AOA). In vivo, injection of AOA in C57BL/6 mice placed on β‐alanine (0.1% w/v in drinking water) for 2 weeks lead to a 3‐fold increase in circulating β‐alanine levels and to significantly higher levels of carnosine and anserine in skeletal muscle and heart. By contrast, specific inhibition of GABA‐T by vigabatrin did not affect carnosine and anserine levels in either tissue. Collectively, these data demonstrate that homeostasis of carnosine and anserine in mammalian skeletal muscle and heart is controlled by circulating β‐alanine levels, which are suppressed by hepatic and renal β‐alanine transamination upon oral β‐alanine intake.

ABSTRACTIntroduction: The contribution of reduced testosterone levels to tail suspension (TS)‐induced muscle atrophy remains equivocal. The molecular mechanism by which testosterone regulates muscle mass during TS has not been investigated. Methods: Effects of TS on serum testosterone levels, muscle mass, and expression of muscle atrophy‐ and hypertrophy‐inducing targets were measured in soleus (SOL) and extensor digitorum longus (EDL) muscles after testosterone administration during 1, 5, and 14 days of TS in male mice. Results: TS produced an increase followed by a transient drop in testosterone levels. Muscle atrophy was associated with downregulation of Igf1 and upregulation of Mstn, Redd1, Atrogin‐1, and MuRF1 mRNA with clear differences in Igf1, Mstn, and MAFbx/Atrogin‐1 gene expression between SOL and EDL. Testosterone supplementation did not affect muscle mass or protein expression levels during TS. Conclusions The known anabolic effects of testosterone are not sufficient to ameliorate loss of muscle mass during TS. Muscle Nerve 52: 278–288, 2015

Background: Propofol is a short-acting intravenous anesthetic agent. In rare conditions, a life-threatening complication known as propofol infusion syndrome can occur. The pathophysiologic mechanism is still unknown. Some studies suggested that propofol acts as uncoupling agent, others suggested that it inhibits complex I or complex IV, or causes increased oxidation of cytochrome c and cytochrome aa3, or inhibits mitochondrial fatty acid metabolism. Although the exact site of interaction is not known, most hypotheses point to the direction of the mitochondria. Methods: Eight rats were ventilated and sedated with propofol up to 20 h. Sequential biopsy specimens were taken from liver and skeletal muscle and used for determination of respiratory chain activities and propofol concentration. Activities were also measured in skeletal muscle from a patient who died of propofol infusion syndrome. Results: In rats, authors detected a decrease in complex II+III activity starting at low tissue concentration of propofol (20 to 25 µM), further declining at higher concentrations. Before starting anesthesia, the complex II+III/citrate synthase activity ratio in liver was 0.46 (0.25) and in skeletal muscle 0.23 (0.05) (mean [SD]). After 20 h of anesthesia, the ratios declined to 0.17 (0.03) and 0.12 (0.02), respectively. When measured individually, the activities of complexes II and III remained normal. Skeletal muscle from one patient taken in the acute phase of propofol infusion syndrome also shows a selective decrease in complex II+III activity (z-score: −2.96). Conclusion: Propofol impedes the electron flow through the respiratory chain and coenzyme Q is the main site of interaction with propofol.

PURPOSE:Enhanced carnosine levels have been shown to be ergogenic for high-intensity exercise performances, although the role of carnosine in the control of muscle function is poorly understood. Therefore, the aim of this study was to investigate the effect of long-term supplementation with increasing doses of carnosine and beta-alanine on muscle carnosine, anserine, and taurine levels and on in vitro contractility and fatigue in mice. METHODS:Male Naval Medical Research Institute mice (n = 66) were control fed or supplemented with either carnosine (0.1%, 0.5%, or 1.8%) or beta-alanine (0.6 or 1.2%) in their drinking water for 8-12 wk. Soleus and extensor digitorum longus (EDL) were tested for in vitro contractile properties, and carnosine, anserine, and taurine content were measured in EDL and tibialis anterior by high-performance liquid chromatography. RESULTS:Only supplementation with 1.8% carnosine and 1.2% beta-alanine resulted in markedly higher carnosine (up to +160%) and anserine levels (up to +46%) compared with control mice. Beta-alanine supplementation (1.2%) resulted in increased fatigue resistance in the beginning of the fatigue protocol in soleus (+2%-4%) and a marked leftward shift of the force-frequency relation in EDL (10%-31% higher relative forces). CONCLUSION:Comparable with humans, beta-alanine availability seems to be the rate-limiting step for synthesis of muscle histidine-containing dipeptides in mice. Moreover, muscle histidine-containing dipeptides loading in mice moderately and muscle dependently affects excitation-contraction coupling and fatigue.

Cannabinoids have potent vasodilatory actions in a variety of vascular preparations. Their mechanism of action, however, is complex. Apart from acting on vascular smooth muscle or endothelial cannabinoid receptors, several studies point to the activation of type 1 vanilloid (TRPV1) receptors on primary afferent perivascular nerves, stimulating the release of calcitonin gene-related peptide (CGRP). In the present study, the direct influence of the cannabinoid methanandamide and the neuropeptide CGRP on the membrane potassium ion (K(+)) currents of rat mesenteric myocytes was explored. Methanandamide (10 μM) decreased outward K(+) currents, an effect similar to that observed in smooth muscle cells from the rat aorta. Conversely, CGRP (10 nM) significantly increased whole-cell K(+) currents and this effect was abolished by preexposure to tetraethylammonium chloride (1 mM) or iberiotoxin (100 nM), inhibitors of large-conductance calcium-dependent K (BK(Ca)) channels but not by glibenclamide (10 μM), an inhibitor of ATP-dependent K channels. In the presence of the CGRP receptor antagonist CGRP(8-37) (100 nM), the adenylyl cyclase inhibitor SQ22536 (100 μM), or the protein kinase A inhibitor Rp-cAMPS (10 μM), CGRP had no effect. These findings show that methanandamide does not increase membrane K(+) currents in smooth muscle cells of small mesenteric arteries, supporting an indirect mechanism for the reported hyperpolarizing influence in this vessel. Moreover, CGRP acts directly on these smooth muscle cells by increasing BK(Ca) channel activity in a CGRP receptor and cyclic adenosine monophosphate-dependent way. Collectively, these data indicate that methanandamide relaxes and hyperpolarizes intact mesenteric vessels by releasing CGRP from perivascular nerves.

The dipeptide carnosine is found in high concentrations in mammalian skeletal muscles. The loading of muscle carnosine through beta-alanine supplementation has been shown to improve performance during high-intensity exercises in humans. Two possible ergogenic mechanisms have been proposed: 1/ improved calcium sensitivity of contractile apparatus and 2/ suppression of exercise-induced acidosis. PURPOSE: To investigate the effect of oral carnosine supplementation in drinking water on the muscle carnosine concentration and on muscle contractility in mice. METHODS: 22 male NMRI mice were either control or carnosine-supplemented (80mM in drinking water) during 8 weeks. Intact soleus and extensor digitorum longus (= EDL) were tested for in vitro contractile properties through electrical stimulation inducing repeated tetanic contractions and carnosine, anserine and taurine content was measured by means of HPLC. RESULTS: The EDL carnosine and anserine concentrations were respectively 68 and 39% higher in carnosine supplemented mice (CAR: 7 mmol/kg dw, ANS: 9,8 mmol/kg dw) compared to controls (CAR: 4,2 mmol/kg dw, ANS: 7,1 mmol/kg dw), whereas the taurine content was unchanged (both 120 mmol/kg dw). The loading of carnosine was less obvious in soleus (+ 21%, p > 0.05). The tetanic force at the beginning of the fatigue protocol was non-significantly higher in carnosine supplemented mice (SOL: 148 mN, EDL: 95 mN) compared to controls (SOL: 117 mN, EDL: 86 mN, p > 0.05). The resistance to fatigue was not affected by carnosine supplementation. However, the rate of force development (N/s) was significantly faster at the end of a fatigue protocol in the EDL of carnosine supplemented mice. Furthermore, the rate of force decline (N/s) in soleus was significantly faster at the beginning of the fatigue protocol in carnosine-fed mice versus control. CONCLUSION: It can be concluded that high levels of dietary carnosine result in enhanced carnosine and anserine concentrations in EDL muscle, but not significantly in soleus. This increase in histidine-containing dipeptides did not result in significantly enhanced force, but improved the kinetic properties of repeated tetanic contractions.

The present study reports of an endothelium-dependent and NO- and prostanoid-independent relaxation in isolated choroidal arteries, and evaluates the hypothesis of an endothelium-derived hyperpolarising factor (EDHF) playing a role in the choroidal circulation. Choroidal arteries were isolated from bovine eyes and mounted in a small vessel wire-myograph for isometric tension recording. Concentration-response curves for acetylcholine (0.1 nM–10 μM) were constructed in isolated choroidal arteries contracted with 10 μM norepinephrine. Acetylcholine induced a concentration-dependent relaxation in the choroidal arteries. The presence of the NO-synthase inhibitor L-NA and the cyclo-oxygenase inhibitor indomethacin only had a limited effect on this relaxation. All further experiments were performed in the presence of L-NA and indomethacin, in order to study the NO- and prostanoid-independent part of the acetylcholine-relaxations. Both removal of the vascular endothelium or the presence of an increased K+ concentration in the organ bath abolished the NO- and prostanoid-independent part of the acetylcholine-relaxations. The presence of TEA, a rather non-specific K+ channel blocker, significantly reduced the acetylcholine-relaxations. Simultaneous application of apamin (an inhibitor of small-conductance Ca2+-activated K+ channels) and charybdotoxin (an inhibitor of intermediate- and large-conductance Ca2+-activated K+ channels) abolished the acetylcholine-induced relaxation and even resulted in a concentration-dependent contraction. Transmembrane potential recordings in isolated choroidal arteries revealed a clear membrane hyperpolarisation in the vascular smooth muscle cells of isolated choroidal arteries. It was therefore concluded that the acetylcholine-induced relaxation of choroidal arteries in the presence of NO-synthase and cyclo-oxygenase inhibitors is mediated by an endothelium-derived hyperpolarising factor. This EDHF seems to be of more importance than endothelium-derived NO or prostanoids.

In the present study, the relaxant effect of the cannabinoid methanandamide was explored in rat gastric arteries. Since in some vessels cannabinoids have been shown to release calcitonin gene-related peptide (CGRP) from perivascular nerves, the influence of methanandamide was compared with that of exogenous CGRP. Methanandamide and CGRP elicited concentration-dependent, endothelium-independent relaxations. Methanandamide-induced relaxations were unaffected by the CB1 receptor antagonist AM251, the CB2 receptor antagonists AM630 and SR144528, and combined pre-exposure to AM251 and SR144528. Pre-exposure to O-1918, an antagonist of a novel nonCB1/nonCB2 cannabinoid receptor, did not influence the relaxations to methanandamide. Capsaicin or capsazepine treatment slightly inhibited methanandamide-induced relaxations. Preincubation with 30 mmol/L extracellular K+ or 3 mmol/L TEA had no significant effect on the responses elicited by methanandamide, but reduced CGRP-induced relaxations. Relaxation to 10(-5) mol/L methanandamide was significantly blunted by Bay K8644 and by preincubation with nifedipine. Furthermore, 10(-5) mol/L methanandamide significantly inhibited CaCl2-induced contractions in norepinephrine-stimulated vessels previously depleted of intra- and extracellular Ca2+. Finally, preincubation with 10(-5) mol/L methanandamide almost completely abolished high K+-induced contractions. These findings suggest that the vasorelaxant action of methanandamide in rat gastric arteries is not mediated by stimulation of known cannabinoid receptors and only partly related to stimulation of TRPV1 receptors on perivascular nerves. At high concentrations, methanandamide might induce relaxation by reducing calcium entry into the smooth muscle cells.

In rat small mesenteric arteries, the influence of modulation of basal smooth muscle K+ efflux on the mechanism of endothelium-dependent hyperpolarization was investigated. The membrane potentials of the vascular smooth muscle cells were measured using conventional microelectrode techniques. Incubation of resting arteries with the gap junction uncoupler carbenoxolone (20 micro M) decreased the endothelium-dependent hyperpolarization elicited by a submaximal concentration of acetylcholine (3 micro M) to about 65% of the control. In the presence of Ba2+ (200 micro M), which depolarized the membrane potential by 10 mV, the acetylcholine-induced membrane potential response was doubled in magnitude, reaching values not different from control. Moreover, the hyperpolarization was more resistant to carbenoxolone in these conditions. Finally, both in the absence and in the presence of carbenoxolone, the combined application of Ba2+ and ouabain (0.5 mM) did not abolish the acetylcholine response. These results suggest that gap junctional coupling plays a role in endothelium-dependent hyperpolarization of smooth muscle cells of resting rat small mesenteric arteries. Additionally, these findings show that the hyperpolarization does not rely on activation of inward rectifying K+ channels. Although a minor contribution of Na-K pumping cannot be excluded, the Ba2+ experiments show that the membrane electrical response is mediated by activation of a Ba2+-resistant K+ conductance.