Injection Of Lactic Acid Research Articles

Role played by acid-sensitive ion channels in evoking the exercise pressor reflex

The exercise pressor reflex arises from contracting skeletal muscle and is believed to play a role in evoking the cardiovascular responses to static exercise, effects that include increases in arterial pressure and heart rate. This reflex is believed to be evoked by the metabolic and mechanical stimulation of thin fiber muscle afferents. Lactic acid is known to be an important metabolic stimulus evoking the reflex. Until recently, the only antagonist for acid-sensitive ion channels (ASICs), the receptors to lactic acid, was amiloride, a substance that is also a potent antagonist for both epithelial sodium channels as well as voltage-gated sodium channels. Recently, a second compound, A-317567, has been shown to be an effective and selective antagonist to ASICs in vitro. Consequently, we measured the pressor responses to the static contraction of the triceps surae muscles in decerebrate cats before and after a popliteal arterial injection of A-317567 (10 mM solution; 0.5 ml). We found that this ASIC antagonist significantly attenuated by half (P<0.05) the pressor responses to both contraction and to lactic acid injection into the popliteal artery. In contrast, A-317567 had no effect on the pressor responses to tendon stretch, a pure mechanical stimulus, and to a popliteal arterial injection of capsaicin, which stimulated transient receptor potential vanilloid type 1 channels. We conclude that ASICs on thin fiber muscle afferents play a substantial role in evoking the metabolic component of the exercise pressor reflex.

An evaluation of non-surgical castration by single intratesticular injection of lactic acid in adult mubende goats (capra hircus)

On a utilisé quarante chèvres (5 groupes de 8) pour évaluer les effets de l\'injection intratesticulaire d\'acide lactique pour la castration. Le 1er groupe (groupe-témoin) a reçu une injection intratesticulaire d\'eau isotonique, tandis que le 2ème groupe était castré à l\'aide de la chirurgie. Les 3è, 4è et 5è groupes ont reçu chacun une seule injection bilatérale de 88% d\'acide lactique à raison de 1, 2 et 3 ml/10kg de poids vif, respectivement. Les chèvres ont fait l\'objet de contrôle quotidien pour observer les réactions de stress telles que les changements d\'appétit, la démarche et le comportement. Le sang était prélevé chaque semaine pour l\'hématologie, le test de cortisol dans le plasma et le prolan A (FSH). On a également effectué une évaluation du sperme et des tests d\'enzymes de tissu testiculaire: la glutamyl-transptidase (GTP) et la déshydrogénase de l\'acide lactique (LDH). Les traitements par la chirurgie et l\'acide lactique ont considérablement augmenté (p

Acid-sensing ion and epithelial sodium channels do not contribute to the mechanoreceptor component of the exercise pressor reflex

Amiloride, injected into the popliteal artery, has been reported to attenuate the reflex pressor response to static contraction of the triceps surae muscles. Both mechanical and metabolic stimuli arising in contracting skeletal muscle are believed to evoke this effect, which has been named the exercise pressor reflex. Amiloride blocks both acid-sensing ion channels, as well as epithelial sodium channels. Nevertheless, amiloride is thought to block the metabolic stimulus to the reflex, because this agent has been shown to attenuate the reflex pressor response to injection of lactic acid into the arterial supply of skeletal muscle. The possibility exists, however, that amiloride may also block mechanical stimuli evoking the exercise pressor reflex. The mechanical component of the reflex can be assessed by measuring renal sympathetic nerve activity during the first 2-5 s of contraction. During this period of time, the sudden tension developed by contraction onset briskly discharges mechanoreceptors, whereas it has little effect on the discharge of metaboreceptors. We, therefore, examined the effect of amiloride (0.5 microg/kg) injected into the popliteal artery on the renal sympathetic and pressor responses to static contraction of the triceps surae muscles in decerebrated cats. We found that amiloride significantly attenuated the pressor and renal sympathetic responses to contraction; for the latter variable, the attenuation started 10 s after the onset of contraction. Our findings lead us to conclude that acid-sensing ion channels and epithelial sodium channels play little, if any, role in evoking the mechanical component of the exercise pressor reflex.

Sensing tension: epithelial sodium channel/acid-sensing ion channel proteins in cardiovascular homeostasis.

The epithelial sodium (Na+) channel (ENaC) plays a critical role in blood pressure regulation by controlling renal salt and water reabsorption. Channel overactivity can lead to severe hypertension and underactivity to salt wasting and hypotension.1 In addition to their role in salt/water homeostasis, recent studies suggest that ENaC proteins, and their relatives, the acid-sensing ion channel (ASIC) proteins, may play more ubiquitous roles in cardiovascular regulation than considered previously. Recent evidence suggests that ENaC/ASIC proteins may act as mechanosensors and chemosensors in the cardiovascular system. ENaC/ASIC proteins are expressed in mechanosensing and chemosensing tissues, such as vascular smooth muscle cells (VSMCs), carotid body glomus cells, and sensory neurons innervating arterial baroreceptors, heart, and skeletal muscle. Disruption of the ENaC/ASIC channels alters myogenic constriction, arterial chemoreceptor and baroreceptor responses, and acid-induced responses in heart and skeletal muscle. This brief review summarizes the evidence supporting a role for ENaC and ASIC proteins in diverse systems of cardiovascular mechanosensing and chemosensing. Together, these studies suggest that ENaC/ASIC proteins contribute to cardiovascular homeostasis by mediating neural and local regulatory mechanisms. ENaC and ASIC proteins are members of a protein family termed the degenerin (DEG)/ENaC/ASIC family. Members of this family are expressed in a wide range of species (nematode Caenorhabditis elegans , Drosophila , and mammals) and participate in diverse biological functions, including neurodegeneration, acid sensation, taste, learning and memory, proprioception, Na+/water transport, and mechanosensation. All of the members of the DEG/ENaC/ASIC family share a highly conserved structure: intracellular N and C termini and 2 membrane-spanning domains separated by a large extracellular domain. Most DEG/ENaC/ASIC proteins form amiloride sensitive, nonvoltage, gated cation channels.1,2 ### C elegans DEGs Members were first identified in the nematode, where a chemically induced mutation caused a subset of neurons to swell and lyse. This phenotype led to the first …

Respiratory and cardiovascular responses evoked by tibialis anterior muscle afferent fibers in rats

The muscle metaboreflex is thought to be one of the neural mechanisms involved in the cardiovascular and respiratory adjustments to muscular activity. The afferent arm of the reflex is composed of thinly myelinated group III and unmyelinated group IV sensitive fibers. Such reflex arc had been extensively described in cats, dogs, rabbits and humans. However, results obtained in rats are controversial and the role of the afferent fibers from the tibialis anterior skeletal muscle has never been shown. The purpose of the present experiments was to study the responses of both respiratory and cardiovascular systems following activation of the metabosensitive fibers originating from tibialis anterior muscle in non decerebrated and non vagotomized barbituric anesthetized adult rats. Mean arterial blood pressure, mean arterial blood flow, heart rate and phrenic nerve activity (frequency and amplitude) were monitored during electrically induced fatigue or after intramuscular injection of potassium chloride or lactic acid (specific stimuli of the group III and IV afferent fibers). The experiments were performed under normal condition, then after regional circulatory occlusion, which isolated and maintained the neural drive and abolished humoral communication and after section of the peroneal nerve innervating the tibialis anterior muscle. We showed that cardiorespiratory parameters were increased significantly in response to stimuli under normal conditions and after venous outflow occlusion excluding any participation of central chemoception. No change was observed after nerve section. Our data indicate that changes occurring in rat hindlimb muscle such as the tibialis anterior are sufficient to regulate the cardiorespiratory function via metabosensitive fiber activation.

Blockade of acid sensing ion channels attenuates the exercise pressor reflex in cats

Although thin fibre muscle afferents possess acid sensing ion channels (ASICs), their contribution to the exercise pressor reflex is not known. This lack of information is partly attributable to the fact that there is no known selective in vivo antagonist for ASICs. Although amiloride has been shown to antagonize ASICs, it also has been shown to antagonize voltage-gated sodium channels, thereby impairing impulse conduction in sensory nerves. Our aim was to test the hypothesis that lactic acid accumulation in exercising muscle acted on ASICs located on thin fibre muscle afferents to evoke the metabolic component of the exercise pressor reflex. To test this hypothesis, we determined in decerebrate cats if amiloride attenuated the pressor and cardioaccelerator responses to static contraction, to tendon stretch and to arterial injections of lactic acid and capsaicin. We found a dose of amiloride (0.5 microg kg(-1); i.a.) that attenuated the pressor and cardioaccelerator responses to both contraction and lactic acid injection, but had no effect on the responses to stretch and capsaicin. A higher dose of amiloride (5 microg kg(-1), i.a.) not only blocked the pressor and cardioaccelerator responses to lactic acid and contraction, but also attenuated the responses to stretch and to capsaicin, manoeuvers in which ASICs probably play no significant role. In addition, we found that the low dose of amiloride (0.5 microg kg(-1)) had no effect on the responses of muscle spindles to tendon stretch and to succinylcholine, whereas the high dose (5 microg kg(-1)) attenuated the responses to both. Our data suggest the low dose of amiloride used in our experiments selectively blocked ASICs, whereas the high dose blocked ASICs and impulse conduction in muscle afferents. We conclude that ASICs play a role in the metabolic component of the exercise pressor reflex.

Lactic Acid, Acid-Sensing Ion Channels and the Exercise Pressor Reflex

Lactic acid is one of the products of contraction that is thought to be a metabolic stimulus that evokes reflex increases in arterial pressure, heart rate and ventilation, otherwise known as the exercise pressor reflex. For example, injection of lactic acid into the arterial supply of skeletal muscle has been shown to evoke reflex increases in arterial pressure, heart rate and ventilation that approximate those occurring during static exercise; whereas neutral controls failed to evoke any reflex cardiovascular effects. Moreover, injection of lactic acid into the arterial supply of skeletal muscle has been shown to increase the discharge of the group III and IV muscle afferents that comprise the afferent arm of this reflex. To demonstrate lactic acid contributes to the elicitation of the exercise pressor reflex, we needed to show that blockade of a specific receptor to the metabolite prevents or attenuates the expression of the reflex. This demonstration has been difficult to achieve for lactic acid because its receptor on group III and IV afferents was not clearly identified. Lactic acid is thought to activate both the TRPV 1 receptor as well as the acid sensing ion channel (ASIC). Recently, the TRPV 1 receptor was shown to play little if any role in evoking the metabolic component of the exercise pressor reflex. Although this finding pointed towards the ASIC receptor, the list of tested selective antagonists is small. One candidate is amiloride, which has been shown to block ASICs in vitro. Unfortunately, amiloride has also been shown to antagonize voltage-gated sodium channels (ENaC), suggesting it may not be an ideal antagonist to investigate the role of lactic acid in vivo. Another candidate is A-317567, a compound shown in vitro to be selective for ASICs in general. A-317567 does not appear in block ENaC at doses that effective antagonize ASICs but the in vitro kinetics are somewhat complex. In the experiments to be reported, we have used amiloride and A-317567 to determine whether lactic acid plays a role in the exercise pressor reflex. Finding a suitable selective antagonist for ASIC would enable us to examine the role played by this channel and by lactic acid in the generation of the metabolic component of the exercise pressor reflex.

Evidence that A‐317567 is a selective ASIC antagonist for thin fiber muscle afferents in decerebrate cats

Lactic acid is widely hypothesized to accumulate in contracting muscle to activate the thin fiber muscle afferents causing the exercise pressor reflex. Testing this hypothesis has been difficult because of the lack of a selective ASIC antagonist. We, therefore, examined if A-317567 attenuated the pressor and cardioaccelerator responses to popliteal arterial injections of lactic acid (0.2 to 0.5 ml, 24mM) and capsaicin (2–5 μg) as well as to tendon stretch. In decerebrate cats we found that A-317567 (10 mM; 0.5 ml) blocked the pressor-cardioaccelerator responses to lactic acid injection but had no effect on the pressor-cardioaccelerator responses to either capsaicin injection or tendon stretch. For example, the pressor response to lactic acid injection averaged 28 ± 8 mmHg before A-317567 and 4 ± 5 mmHg 10 minutes afterwards (p = 0.027; n = 4). In contrast, the pressor response to capsaicin injection averaged 33 ± 8 mm Hg before the ASIC antagonist and 33 ± 7 mm Hg afterwards (p = 0.78; n = 4). A dose of 0.5 ml of 1 mM A-317567 had no effect of the pressor and cardioaccelerator responses to arterial injections of lactic acid and capsaicin. Our data suggest that A-317567 is a specific blocker of ASIC channels and may be a useful tool to examine the role played by these channels in the exercise pressor reflex.

(628): Effects of pain and analgesia on intracranial self-stimulation (ICSS) in rats

The effective management of pain continues to be major clinical problem. Current preclinical models of pain focus on the measurement of pain-evoked behavior, which may yield false positives. Our laboratory has focused on methods development for assays of pain-suppressed behavior, which may complement existing assays. The present study compared the effects of IP lactic acid injections and morphine pretreatments on the writhing response (a pain-evoked behavior) and intracranial self-stimulation (ICSS; a behavior that may be suppressed by pain). For the ICSS procedure, Sprague Dawley rats implanted with electrodes in the lateral hypothalamus were tested on a FR1 schedule of reinforcement to respond for electrical stimulation. Response rates were measured across a descending series of 15 current frequencies, and rate-frequency curves were determined. Acid-induced writhing was measured over 60 min in a separate group of animals. Both writhing and ICSS were evaluated under baseline conditions and after treatment with lactic acid (0.32-3.2 %, IP) and/or morphine (1.0-10 mg/kg, IP). Lactic acid produced time- and concentration-dependent increases in writhing, with peak writhes occurring at 20 min. and subsiding by 60 min. Lactic acid also produced rightward shifts in the ICSS curve rate-frequency curves and increased ICSS thresholds. However, lactic acid was more potent in evoking writhing than in suppressing ICSS. Morphine dose-dependently reduced acid-evoked writhing, and effects of morphine on acid-suppressed ICSS are being evaluated now. ICSS may be an especially useful assay for evaluation of pain-suppressed behavior, because it is often used to assess reward threshold and “hedonic state” in rodents. Accordingly, ICSS may be useful for exploring interactions between pain and depression.

The Role of ASIC Channels in the Exercise Pressor Reflex

In vivo, H+ activate thin fiber muscle afferents involved in the exercise pressor reflex; however, the receptor for this effect is unknown. We hypothesized that the hydrogen ions associated with lactic acid accumulation in the exercising muscles act on ASIC channels on thin fiber muscle afferents. To test this hypothesis we examined if amiloride, an ASIC antagonist, attenuated the reflex pressor and cardioaccelerator responses to static contraction, stretch of the calcaneal tendon, as well as popliteal arterial injections of lactic acid and capsaicin. We established a dose of amiloride that blocked the reflex pressor response to static contraction and lactic acid injection but had no effect on the reflex pressor responses to stretch and capsaicin injection. Specifically, in four decerebrate cats we examined the pressor and cardioaccelerator responses to static contraction, stretch of the calcaneal tendon, and popliteal artery injections of lactic acid (0.5 ml, 24 mM; i.a) and capsaicin (2-5 μg; i.a.) before and after popliteal arterial injection of amiloride. The pressor responses to static contraction averaged 28 ± 4 mmHg before amiloride and 5 ± 2 mmHg after (0.5 μg/kg; i.a.; P = 0.09; n=4). Likewise, the pressor response to lactic acid injections averaged 30 ± 4 mmHg before amiloride and 12 ± 3 mmHg after (P = 0.06; n = 4). The pressor responses to tendon stretch and capsaicin were unchanged by amiloride. The heart rate responses were similar to those of blood pressure. In three separate decerebrate cats a high dose of amiloride (5 μg/kg) abolished all cardiovascular responses to static contraction, stretch of the calcaneal tendon, and injections of lactic acid and capsaicin. These data suggest ASIC channels may play a role in the exercise pressor reflex.

Eccentric exercise alters muscle sensory motor control through the release of inflammatory mediators

Following downhill exercise, muscle damage and local inflammatory reactions, induced by lengthening contractions, are observed and voluntary muscle activation decreases. The hypothesis that feedback carried by the group IV muscle afferents could be involved has often been raised but never measured in vivo in these conditions. In this experiment, we tested the response of the group IV muscle afferents from the lower limb to injections of KCl and lactic acid in non-exercising rats and at 1, 2, and 8 days after one running session (−13°, 16 m/min). At days 1 and 2, the baseline discharge of the group IV afferents increased, but further activation by test agents was absent. After 8 days, the afferent response was equivalent to the control response. Pretreatment with betamethasone before exercise abolished the effects of downhill exercise. In non-exercising rats, arachidonic acid evoked group IV afferent discharge and suppressed their further response to another stimulus. These results demonstrate that exhaustive downhill running highly activates, for at least 2 days, the sensory feedback carried by group IV afferents through the local release of inflammatory mediators. Such an altered sensori-motor control, accompanying the post-eccentric inflammatory syndrome, could play a key role in deterioration of muscle performance and of its voluntary activation.

L-3[C-11] lactic acid injection in canines for cardiac PET studies: in vivo metabolites determination

L-3[C-11] lactic acid injection in canines for cardiac PET studies: in vivo metabolites determination

Activation of dopamine D2-like receptors attenuates pulmonary C-fiber hypersensitivity in rats.

This study was performed to determine whether activation of dopamine D2-like receptors inhibits the hyperresponsiveness of pulmonary C fibers induced by inflammatory mediators such as prostaglandin E2 (PGE2). In anesthetized, open-chest rats, constant infusion of PGE2 (1.5-4.5 microg/kg per minute, 2 minutes) significantly enhanced the C-fiber response to capsaicin injection. At 20 minutes after pretreatment with quinpirole (3 mg/kg, intravenous), a D2-like receptor agonist, the hyperresponsiveness to capsaicin of the same C fibers induced by PGE2 infusion was markedly attenuated, and this inhibitory effect lasted for more than 90 minutes. The effect of quinpirole was dose dependent and was antagonized by pretreatment with domperidone (5 mg/kg, intravenous), a D2-like receptor antagonist, administrated 10 minutes before the quinpirole injection. In a separate series of experiments, C-fiber responses to injections of phenyl biguanide and lactic acid and to constant-pressure lung inflation were augmented by PGE2; these potentiating responses were also significantly reduced by quinpirole. Furthermore, the effect of quinpirole was equally effective in inhibiting the increase in excitability of pulmonary C fibers induced by alveolar hypercapnia or constant infusion of adenosine. In conclusion, these results clearly show that activation of the dopamine D2-like receptors attenuates the hyperresponsiveness of pulmonary C fibers to both chemical stimuli and lung inflation.

Comparison between the effects of chronic and acute hypoxaemia on muscle afferent activities from the tibialis anterior muscle

The reflex loops initiated by the activation of muscle afferents are altered by a reduction of the oxygen supply. This has been shown in different mammalian species under experimental conditions of acute or chronic hypoxemia. In the present study in rats, we compared the effects of acute and chronic hypoxemia on the activity of afferents from the tibialis anterior muscle to investigate the existence of possible adaptive mechanisms to hypoxaemia. The activity in groups I and II (mechanosensitive) and groups III and IV (mechano- and mostly chemosensitive) muscle afferents was recorded under conditions of normoxaemia and acute and chronic hypoxemia. Chronic hypoxaemia was achieved by exposing the rats for 45 consecutive days to a gas mixture containing 10% oxygen in nitrogen, whereas acute hypoxaemia was limited to a 1-h exposure to a 10% oxygen mixture. Different test agents: muscle stimulation (MS), arterial injection of KCl or lactic acid and tendon vibrations, were used to activate muscle afferents. Both acute and chronic hypoxaemia suppressed the post-MS activation of groups III and IV muscle afferents and significantly depressed the response of these afferents to KCl and lactic acid. The pattern of response of mechanosensitive afferents to mechanical tendon vibration was similar under the three conditions but chronic hypoxemia significantly reduced the response to 10-50 Hz vibrations. We conclude that inhibitory effects on muscle metabosensitive afferents occurred in the first minutes following acute hypoxemia and was prolonged with chronic hypoxemia. Our data also suggest that chronic hypoxemia depresses mechanosensitive muscle afferents. This may explain the observation that sensorimotor control of skeletal muscles is often markedly impaired in hypoxemic humans.

Effects of chronic hypoxemia on the afferent nerve activities from skeletal muscle.

An acute reduction of the oxygen supply to contracting muscles not only affects their metabolism but also modifies their sensorimotor control through changes in afferent discharge of the group I and group III-IV nerve fibers, the latter playing a pivotal role in the protective mechanisms against muscle fatigue. The effects of chronic hypoxemia on the muscle sensitivity are totally unknown. In the present study, group I fibers (mechanosensory afferents) and group III-IV fibers (mechanosensory and chemosensory afferents) from the anterior tibial muscle were recorded in normoxemic and chronic hypoxemic rats. Hypoxemic rats breathed for 45 d a gas mixture containing 9.5 to 10% O(2) in N(2). The data were compared with those obtained in normoxemic animals of the same age. To activate the different muscle afferents, we used different test agents, including electrically induced fatigue (EIF), KCl, lactic acid injections, as well as tendon vibrations. The conduction velocity of all nerve fibers was significantly (p < 0.01) higher in hypoxemic rats than in the normoxemic group. Chronic hypoxemia significantly depressed the response of the group III-IV muscle afferents to KCl injections and even abolished their response to lactic acid and EIF. However, the response to tendon vibrations of the group I afferents was similar in hypoxemic and normoxemic rats. These results suggest that chronic hypoxemia markedly alters the chemosensitivity of the group III-IV muscle afferents, which may explain the higher fatigability of hypoxemic subjects.

Tenderization of beef by lactic acid injected at different times post mortem

The potential to tenderize beef muscles by the injection of lactic acid (0.5 M, 10% w/w) was studied using the pectoralis profundus muscle from cull cows. The injection was performed either 1 h (pre rigor) or 24 h (post rigor) post mortem, and the meat was stored for 2 or 14 days post mortem. Both treatments caused a rapid pH drop to around 5.0 within 4 h of injection. Other effects were: (1) an accelerated release of lysosomal enzymes into the cytosol; (2) a greater degradation of myosin heavy chains; (3) ultrastructural alterations of the myofibrils which included a general weakening or rupture in the M-lines and, to a lesser extent, in the I-bands; (4) a decreased heat stability of perimysial collagen indicated by a lower insoluble collagen content, lower differential scanning calorimetry transition temperature, and lower transition temperatures in isometric tension tests on muscle strips. The lactic acid injections improved significantly the textural traits of the meat (shear value, tensile strength, sensory scores) at 2 days post mortem with little further improvement when storage was extended to 14 days post mortem. Changes in texture were of similar amplitude at both post mortem injection times. The tenderization mechanisms of lactic acid injection are discussed.

Prostaglandin E(2) enhances chemical and mechanical sensitivities of pulmonary C fibers in the rat.

It has been recently reported that pulmonary reflex responses to injection or inhalation challenge of capsaicin are enhanced by exogenous Prostaglandin E(2) (PGE(2)). The present study was carried out to determine whether PGE(2) enhances the stimulatory effects of chemical stimulants and lung inflation on vagal pulmonary C fibers, and if so, whether the excitabilities of other types of lung afferents are also augmented by PGE(2). In anesthetized, open-chest rats, administration of PGE(2) (1.5 microgram/kg/min for 2 min) did not significantly change the baseline activity of vagal pulmonary C fibers, but it markedly enhanced the stimulatory effects of both low (0.25 microgram/kg) and high doses (0.5 microgram/kg) of capsaicin on these fibers. Similarly, potentiating effects of PGE(2) were found on the pulmonary C-fiber responses to injections of lactic acid and adenosine, although considerable variability existed in the degrees of potentiation between the different stimulants. Furthermore, PGE(2) infusion also significantly enhanced the C-fiber response to constant-pressure lung inflation (tracheal pressure [Pt] = 30 cm H(2)O). In contrast, PGE(2) did not alter the responses of either slowly adapting pulmonary receptors or rapidly adapting pulmonary receptors to lung inflation. In summary, these results show that the sensitivity of pulmonary C-fiber afferents to both mechanical and chemical stimuli is enhanced by PGE(2), suggesting that endogenous release of this autocoid may play a part in the airway irritation and dyspneic sensation associated with airway inflammation.

Relationship between proteolytic changes and tenderness in prerigor lactic acid marinated beef

A meat tenderising procedure involving injection of a lactic acid solution into prerigor muscle was investigated using beef M pectoralis profundus. The distribution of lysosomal enzymes in subcellular fractions, densities of myofibrillar protein bands after SDS-PAGE and shear force were measured in non-injected, 0.5 M and 1.0 M lactic-acid-injected samples during a 21 days ageing period. The activities of cathepsin Ba L and b-glucuronidase in the soluble fraction increased with level of lactic acid and with time post-mortem (P< 0.001). Lactic acid and storage decreased densities of SDS- PAGE bands migrating at the position of myosin heavy chain (MHC) and a-actinin and increased densities of a 150 kDa band (P< 0.01). SDS-PAGE of isolated perimysium cleaved with CNBr showed proteolytic cleavage of collagen after prolonged storage. Lactic acid injection significantly reduced shear force (P< 0.001). The cathepsin Ba L activity in the soluble fraction correlated to shear force (r =ˇ0.8), the degradation of MHC and a-actinin (r =ˇ0.88 and ˇ0.90) and the generation of the 150 kDa fragment (r = 0.90) but not to the generation of a 31 kDa fragment (r = 0.05). A major part of the tenderness improvement after lactic acid injection was complete at 24h post-mortem, and was therefore due to a rapid process, eg pH-induced swelling of the muscle structure. The data on enzyme activities and protein degradation, however, suggested that the action of lysosomal cathepsins also contributed to textural changes. # 1999 Society of Chemical Industry

Mechanisms of capsaicin- and lactic acid-induced bronchoconstriction in the newborn dog.

1. Capsaicin activation of the pulmonary C fibre vanilloid receptor (VR1) evokes the pulmonary chemoreflex and reflex bronchoconstriction. Among potential endogenous ligands of C fibre afferents, lactic acid has been suggested as a promising candidate. We tested the hypotheses that (a) lactic acid behaves as a stimulant of C fibre receptors in the newborn dog to cause reflex bronchoconstriction, and (b) lactic acid causes reflex bronchoconstriction via the same pulmonary C fibre receptor mechanism as capsaicin using the competitive capsaicin/VR1 receptor antagonist capsazepine. 2. Right heart injection of lactic acid caused a significant increase (47 +/- 8.0 %) in lung resistance (RL) that was atropine sensitive (reduced by 75 %; P < 0.05), consistent with reflex activation of muscarinic efferents by stimulation of C fibre afferents. 3. Infusion of the competitive capsaicin antagonist capsazepine caused an 80 % reduction (P < 0.01) in the control bronchoconstrictor response (41 +/- 8.5 % increase in RL) to right heart injections of capsaicin. The effects of capsazepine are consistent with reversible blockade of the VR1 receptor to abolish C fibre-mediated reflex bronchoconstriction. 4. Lactic acid-evoked increases in RL were unaffected by VR1 blockade with capsazepine, consistent with a separate lactic acid-induced reflex mechanism. 5. We conclude that (a) putative stimulation of C fibres with lactic acid causes reflex bronchoconstriction in the newborn dog, (b) capsazepine reversibly antagonizes reflex bronchoconstriction elicited by right heart injection of capsaicin, presumably by attenuating capsaicin-induced activation of the C fibre 'capsaicin' receptor (VR1), and (c) capsazepine resistance of lactic acid-induced bronchoconstriction indicates that lactic acid evokes reflex bronchoconstriction by a separate mechanism, possibly via the acid-sensing ionic channel.

Study of the pharmacological effect of the bile salt, sodium scymnol sulfate, from Rhizoprionodon acutus. III. Protective effect of scymnol against vascular endothelial cell damage in a rat peripheral arterial occlusion model.

The prophylactic action of scymnol in a rat peripheral arterial occlusion model, involving injection of 5% lactic acid into the femoral artery, was investigated. Increases in serum lactate dehydrogenase (LDH), glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) activities and in plasma levels of thrombin and antithrombin III complex (TAT) were observed in this model 3 h after injection of lactic acid. Changes in LDH activity were characterized by increases in isozymes LDH4 and LDH5 and an elevated LDH4/LDH5 ratio. The ratio of the LDH4 to LDH5 increments was similar to that seen in a rat endothelial cell culture. Oral preadministration of scymnol had a preventive effect on the development of lesions in this model. It significantly reduced the LDH4 and LDH5 activity, the LDH4/LDH5 ratio and the TAT levels dose-dependently over the range 1, 3 or 10 mg/kg, compared with the values in control rats. However, its administration after lactic acid injection, or to sham-operated rats was ineffective, even at a dose of 10 mg/kg. The effects of scymnol were also compared with those of ticlopidine and argatroban. The findings show that scymnol may be useful in preventing thrombotic peripheral arterial occlusive disorders and that it potently protects endothelial cells against lactic acidosis in this model.