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Alzheimer’s Disease (AD) is a neurodegenerative condition characterized both by the presence of tau protein neurofibrillary tangles and amyloid beta (Aβ) containing extracellular “plaques”. The cleavage of amyloid precursor protein (APP) yields several Aβ peptides. Although Aβ toxicity to neurons has been described extensively, its effects on other components of the neurovasculature such as vascular smooth muscle cells have been less well characterized. AD is now also recognized as a neurovascular disease characterized by cerebral microbleeds and disturbances in autoregulation. AD is also a neuroinflammatory condition in which several proinflammatory cytokines are elevated and may contribute to the intensification of AD severity. Cerebral autoregulation (the mechanism by which brain blood flow is maintained despite changes in perfusion pressure) is extremely tightly controlled in the brain and shows disturbances in AD. The failure of autoregulation in AD may make the brain susceptible to cerebral microbleeds through a reduced capacity to limit blood flow when pressure is increased. Conversely, reduced vasodilation during low flow might could also exacerbate tissue hypoxia. Currently, whether and how Aβ peptides and inflammatory cytokines depress brain smooth muscle cell tonic contraction is not known, but could reveal important targets in the preservation of autoregulation which is disturbed in AD. We used a collagen gel contractility assay to evaluate the influence of Aβ25-35, Aβ1-40 and Aβ1-42 peptides and inflammatory cytokines on the tonic contractility of human brain vascular smooth muscle cells (HBVSMC) as an in vitro model of cerebral autoregulation. We found that 5 and 10 μM Aβ1-42 significantly depressed HBVSM contractility, while Aβ1-40 5–20 μM had no effect on contractility. Conversely, Aβ25-35 (1–50 μM) increased contractility. Interestingly, the inflammatory cytokines TNF-α (20 ng/mL), IL-1β (20 ng/mL) and IFN-γ (1000 U/mL) also depressed HBVSM tonic contractility alone and in combination. These data suggest that both the inflammatory milieu in AD as well as the abundance of Aβ peptides may promote autoregulatory failure and increase brain susceptibility to dysregulated perfusion and microbleeds which are an important and devastating characteristic of AD.

The future of awake bruxism assessment will incorporate physiological data, possibly electromyography (EMG) of the temporal muscles. But up to now, temporal muscle contraction patterns in awake bruxism have not been characterized to demonstrate clinical utility. The present study aimed to perform surface EMG evaluations of people assessed for awake bruxism to identify possible different subtypes. A 2-year active search for people with awake bruxism in three regions of the country resulted in a total of 303 participants (223 women, 38 ± 13 years, mean and SD). Their inclusion was confirmed through non-instrumental approaches for awake bruxism: self-reported questionnaire and clinical exam, performed by three experienced and calibrated dentists (Kappa = 0.75). Also, 77 age- and sex-matched healthy controls were recruited (49 women, 36 ± 14 years). Temporalis surface EMG was performed with a portable device (Myobox; NeuroUp, Brazil). EMG signals were sent to a computer via Bluetooth 4.0 at a sampling rate of 1,000 Hz. Digital signal processing was performed using the commercial neuroUP software, transformed in RMS and then normalized for peak detection (EMG peaks/min), in a 10 min session. Cluster analysis revealed three distinct subtypes of awake bruxism: phasic, tonic, and intermediate. Individuals with a predominance of EMG peaks/min were classified as the “phasic” subtype (16.8%). Those with the highest EMG rest power were classified as the “tonic” subtype (32.3%). There was also an “intermediate” subtype (50.8%), when both variables remained low. Characterization of awake bruxism physiology is important for future establishment of instrumental assessment protocols and treatment strategies.

Alzheimer's Disease (AD) is a neurodegenerative condition characterized both by the presence of tau protein neurofibrillary tangles and amyloid beta (Aβ) containing extracellular "plaques". The cleavage of amyloid precursor protein (APP) yields several Aβ peptides. Although Aβ toxicity to neurons has been described extensively, its effects on other components of the neurovasculature such as vascular smooth muscle cells have been less well characterized. AD is now also recognized as a neurovascular disease characterized by cerebral microbleeds and disturbances in autoregulation. AD is also a neuroinflammatory condition in which several proinflammatory cytokines are elevated and may contribute to the intensification of AD severity. Cerebral autoregulation (the mechanism by which brain blood flow is maintained despite changes in perfusion pressure) is extremely tightly controlled in the brain and shows disturbances in AD. The failure of autoregulation in AD may make the brain susceptible to cerebral microbleeds through a reduced capacity to limit blood flow when pressure is increased. Conversely, reduced vasodilation during low flow might could also exacerbate tissue hypoxia. Currently, whether and how Aβ peptides and inflammatory cytokines depress brain smooth muscle cell tonic contraction is not known, but could reveal important targets in the preservation of autoregulation which is disturbed in AD. We used a collagen gel contractility assay to evaluate the influence of Aβ25-35, Aβ1-40 and Aβ1-42 peptides and inflammatory cytokines on the tonic contractility of human brain vascular smooth muscle cells (HBVSMC) as an in vitro model of cerebral autoregulation. We found that 5 and 10 μM Aβ1-42 significantly depressed HBVSM contractility, while Aβ1-40 5–20 μM had no effect on contractility. Conversely, Aβ25-35 (1–50 μM) increased contractility. Interestingly, the inflammatory cytokines TNF-α (20 ng/mL), IL-1β (20 ng/mL) and IFN-γ (1000 U/mL) also depressed HBVSM tonic contractility alone and in combination. These data suggest that both the inflammatory milieu in AD as well as the abundance of Aβ peptides may promote autoregulatory failure and increase brain susceptibility to dysregulated perfusion and microbleeds which are an important and devastating characteristic of AD. [ABSTRACT FROM AUTHOR]

The future of awake bruxism assessment will incorporate physiological data, possibly electromyography (EMG) of the temporal muscles. But up to now, temporal muscle contraction patterns in awake bruxism have not been characterized to demonstrate clinical utility. The present study aimed to perform surface EMG evaluations of people assessed for awake bruxism to identify possible different subtypes. A 2-year active search for people with awake bruxism in three regions of the country resulted in a total of 303 participants (223 women, 38 ± 13 years, mean and SD). Their inclusion was confirmed through non-instrumental approaches for awake bruxism: self-reported questionnaire and clinical exam, performed by three experienced and calibrated dentists (Kappa = 0.75). Also, 77 age- and sex-matched healthy controls were recruited (49 women, 36 ± 14 years). Temporalis surface EMG was performed with a portable device (Myobox; NeuroUp, Brazil). EMG signals were sent to a computer via Bluetooth 4.0 at a sampling rate of 1,000 Hz. Digital signal processing was performed using the commercial neuroUP software, transformed in RMS and then normalized for peak detection (EMG peaks/min), in a 10 min session. Cluster analysis revealed three distinct subtypes of awake bruxism: phasic, tonic, and intermediate. Individuals with a predominance of EMG peaks/min were classified as the "phasic" subtype (16.8%). Those with the highest EMG rest power were classified as the "tonic" subtype (32.3%). There was also an "intermediate" subtype (50.8%), when both variables remained low. Characterization of awake bruxism physiology is important for future establishment of instrumental assessment protocols and treatment strategies. [ABSTRACT FROM AUTHOR]

Diarrhea is a gastrointestinal symptom associated with systemic anaphylaxis and could be induced by increased colonic motility. We determined colonic motility and expulsion by measuring the intracolonic pressure (ICP) and expelled fluid weight in anesthetized rats during anaphylactic hypotension. Substantial systemic hypotension occurred in every sensitized rat after antigen injection. One min after antigen injection, ICP began to increase and remained elevated for 5 min, which was revealed to represent tonic contraction by the video-recording procedure, and was accompanied by increased colonic fluid expulsion. Parasympathectomy composed of subdiaphragmatic vagotomy combined with pelvic nerve transection reduced the duration of the tonic contraction, but not expelled colonic fluid. Furthermore, denervation of afferent parasympathetic nerves produced essentially the same effect as parasympathectomy. Sympathectomy did not significantly change any parameters. In conclusion, the colonic motility during anaphylactic hypotension is characterized by 5-min lasting tonic contraction which is associated with increased colonic fluid expulsion and is involved by parasympathetic nerves, especially their afferents, but not sympathetic nerves, in anesthetized rats. [ABSTRACT FROM AUTHOR]

Up to 40% of patients with gastroesophageal reflux disease (GERD) suffer from proton pump inhibitor refractory GERD but clinically the medications to strengthen the lower esophageal sphincter (LES) to avoid irritating reflux are few in number. This study aimed to examine whether Salvia miltiorrhiza (SM) extracts induce tonic contraction of rat LES ex vivo and elucidate the underlying mechanisms. To investigate the mechanism underlying the SM extract-induced contractile effects, rats were pretreated with atropine (a muscarinic receptor antagonist), tetrodotoxin (a sodium channel blocker), nifedipine (a calcium channel blocker), and Ca2+-free Krebs-Henseleit solution with ethylene glycol tetraacetic acid (EGTA), followed by administration of cumulative dosages of SM extracts. SM extracts induced dose-related tonic contraction of the LES, which was unaffected by tetrodotoxin, atropine, or nifedipine. However, the SM extract-induced LES contraction was significantly inhibited by Ca2+-free Krebs-Henseleit solution with EGTA. Next, SM extracts significantly induce extracellular Ca2+ entry into primary LES cells in addition to intracellular Ca2+ release and in a dose-response manner. Confocal fluorescence microscopy showed that the SM extracts consistently induced significant extracellular Ca2+ influx into primary LES cells in a time-dependent manner. In conclusion, SM extracts could induce tonic contraction of LES mainly through the extracellular Ca2+ influx pathway.

Up to 40% of patients with gastroesophageal reflux disease (GERD) suffer from proton pump inhibitor refractory GERD but clinically the medications to strengthen the lower esophageal sphincter (LES) to avoid irritating reflux are few in number. This study aimed to examine whether Salvia miltiorrhiza (SM) extracts induce tonic contraction of rat LES ex vivo and elucidate the underlying mechanisms. To investigate the mechanism underlying the SM extract-induced contractile effects, rats were pretreated with atropine (a muscarinic receptor antagonist), tetrodotoxin (a sodium channel blocker), nifedipine (a calcium channel blocker), and Ca2+-free Krebs-Henseleit solution with ethylene glycol tetraacetic acid (EGTA), followed by administration of cumulative dosages of SM extracts. SM extracts induced dose-related tonic contraction of the LES, which was unaffected by tetrodotoxin, atropine, or nifedipine. However, the SM extract-induced LES contraction was significantly inhibited by Ca2+-free Krebs-Henseleit solution with EGTA. Next, SM extracts significantly induce extracellular Ca2+ entry into primary LES cells in addition to intracellular Ca2+ release and in a dose-response manner. Confocal fluorescence microscopy showed that the SM extracts consistently induced significant extracellular Ca2+ influx into primary LES cells in a time-dependent manner. In conclusion, SM extracts could induce tonic contraction of LES mainly through the extracellular Ca2+ influx pathway. [ABSTRACT FROM AUTHOR]

The role of SMA and SMB smooth muscle myosin heavy chain (MHC) isoforms in tonic and phasic contractions was studied in phasic (longitudinal ileum and stomach circular antrum) and tonic (stomach circular fundus) smooth muscle tissues of SMB knockout mice. Knocking out the SMB MHC gene eliminated SMB MHC protein expression and resulted in upregulation of the SMA MHC protein without altering the total MHC protein level. Switching from SMB to SMA MHC protein expression decreased the rate of the force transient and increased the sustained tonic force in SMB(-/-) ileum and antrum with high potassium (KPSS) but not with carbachol (CCh) stimulation. The increased tonic contraction under the depolarized condition was not through changes in second messenger signaling pathways (PKC/CPI-17 or Rho/ROCK signaling pathway) or LC20 phosphorylation. Biochemical analyses showed that the expression of contractile regulatory proteins (MLCK, MLCP, PKCδ, and CPI-17) did not change significantly in tissues tested except for PKCα protein expression being significantly decreased in the SMB(-/-) antrum. However, specifically activating PKCα with phorbol dibutyrate (PDBu) was not significantly different in knockout and wild-type tissues, with total force being a fraction of the force generation with KPSS or CCh stimulation in SMB(-/-) ileum and antrum. Taken together, these data show removing the SMB MHC protein expression with a compensatory increase in the SMA MHC protein results in enhanced sustained KPSS-induced tonic contraction with a reduced rate of force generation in these phasic tissues. [ABSTRACT FROM AUTHOR]

Abstract: Ethnopharmacological relevance: Danshen, root of Salvia miltiorrhiza (SM), has been traditionally used in Chinese medicine for the treatment of heart, abdomen, gurgling in the intestines, and relieving fullness. However, the effects of SM on intestine have rarely been done to date. Aim of the study: To investigate the contraction effect of SM on isolated rat ileum and its mechanisms involved. Materials and methods: The isometric contractions of ileum segments were investigated in organ baths for spontaneous activity and response to ethanolic extracts of SM. To determine the contraction mechanism caused by SM extracts, atropine (a muscarinic receptor antagonist), tetrodotoxin (TTX, a sodium channel blocker), nifedipine (a calcium channel blocker), Ca2+ free Krebs solution with EGTA, or trifluoperazine (TFP, a calmodulin blocker) was administered and its response to cumulative dosages of SM extracts were examined. The effect of SM extracts on Ca2+ signaling in the intestinal epithelial cell-6 (IEC-6) was examined using fura-2 as a Ca2+ indicator. Results: SM extracts caused dose-dependent tonic contraction on rat ileum in ex vivo organ bath studies. The contraction induced by SM extracts was not inhibited by atropine, TTX, nifedipine, or in Ca2+ free solution. However, the ileal contractions induced by SM extracts were significantly inhibited by TFP in a dose-dependent manner. In IEC-6 cells, the SM extracts induced extracellular Ca2+ entry and massive intracellular Ca2+ release in Ca2+-contained medium, and induced intracellular Ca2+ release in Ca2+-free medium. Conclusion: These results demonstrate that SM extracts cause ileal contraction through the Ca2+-calmodulin pathway. [Copyright &y& Elsevier]

Muscular lymphatics use both phasic and tonic contractions to transport lymph for conducting their vital functions. The molecular mechanisms regulating lymphatic muscle contractions are not well understood. Based on the well-established finding that the phosphorylation of myosin light chain 20 (MLC20) plays an essential role in blood vessel smooth muscle contraction, we investigated if phosphorylated MLC20 (pMLC20) would modulate the tonic and/or phasic contractions of lymphatic muscle. The effects of ML-7, a MLC kinase inhibitor (1-10 were tested on the contractile parameters of isolated and cannulated rat mesenteric lymphatics during their responses to the known modulators, pressure (1-5 cmH20) and substance P (SP; 10-7 M). Immunohistochemical and Western blot analyses of pMLC20 were also performed on isolated lymphatics. The results showed that 1) increasing pressure decreased both the lymphatic tonic contraction strength and pMLC20-to-MLC20 ratio; 2) SP increased both the tonic contraction strength and phosphorylation of MLC20 3) ML-7 decreased both the lymphatic tonic contraction strength and pMLC20-to-MLC20 ratio; and 4) the increase in lymphatic phasic contraction frequency in response to increasing pressure was diminished by ML-7; however, the phasic contraction amplitude was not significantly altered by ML-7 either in the absence or presence of SP. These data provide the first evidence that tonic contraction strength and phasic contraction amplitude of the lymphatics can be differentially regulated, whereby the increase in MLC20 phosphorylation produces an activation in the tonic contraction without significant changes in the phasic contraction amplitude. Thus, tonic contraction of rat mesenteric lymphatics appears to be MLC kinase dependent. [ABSTRACT FROM AUTHOR]

Conclusion: The position of a subject's eyes during vestibular evoked myogenic potential (VEMP) recording significantly alters the magnitude of the response. This change is largely due to an alteration in the tonicity of the sternocleidomastoid muscle (SCM) caused by variations in the position of the eye. However, even if electromyographic (EMG) normalization is conducted effects of eye position remain. Objective: To determine if eye position has a significant effect on the magnitude of the VEMP. Subjects and methods: VEMPs were collected from 32 ears measured on 16 healthy subjects. The recordings were made unilaterally using the head turn method. The acoustic stimuli were 500 Hz air-conduction short tone bursts. VEMPs were measured in three recording conditions: (i) eyes in the same direction as head turn, (ii) eyes straight ahead, (iii) eyes in the opposite direction to head turn. Results: All 32 ears tested showed a VEMP response with eyes in all three positions. Repeated measures analysis of variance (RM-ANOVA) verified an overall significant effect of eye position (p<0.001). Post hoc paired t tests revealed statistically significant differences between the eyes opposite and the other two conditions (p<0.001). Normalization of the VEMP magnitude using pre-stimulus EMG reduced the effect; however, some variability remained. [ABSTRACT FROM AUTHOR]

Abstract: It is difficult to measure gastrointestinal smooth muscle (SM) tone except in sphincter regions. Since tone affects the biomechanical properties, the aim of the present study was to evaluate intestinal SM tone by studying the morphometry and biomechanical properties with and without muscle tone. Circumferential rings of 0.8–1mm in width were cut from the rat duodenum, jejunum and ileum. Sectors were obtained by cutting the rings opposite to the mesentery. The rings and the sectors were immersed in physiological Krebs solution in order to maintain the tone and into Krebs solution without Ca++ and with EGTA to abolish the tone. The circumferences, area, the circularity and residual strain of the mucosal and serosal surfaces, opening angle, and opening angle tone/non-tone ratio were measured or computed. The tone affects the opening angle and residual strain in the intestinal sectors. The opening angle in the tissue sectors with tone was smaller (P<0.05) than those without tone in all three segments. The opening angle tone/non-tone ratio was 0.40±0.05, 0.43±0.06 and 0.36±0.11 for duodenum, jejunum and ileum, respectively, and did not differ among the three intestinal segments. The residual strain between sectors with and without SM tone differed in duodenal and jejunal mucosa and in the serosa of all three segments (P<0.05). The intestinal rings with tone showed axial variation for luminal area (P<0.001), for wall area (P<0.05), and for the mucosal and serosal residual strains (P<0.05). In conclusion, the intestinal mechanical properties are affected by intestinal SM tone. The tone can be evaluated by measuring the opening angle and residual strains of sectors in intestinal segments with and without SM tone. [Copyright &y& Elsevier]

Frog atrial muscle strips were placed in a double sucrose gap chamber and perfusion of the central node was arranged as to allow rapid changes of external Ca or Na concentration during long-lasting (15s) depolarizing clamps. When the superfusing fluid was suddenly switched, the intercellular space inside the fibre bundle equilibrated with a time constant in the order of 1 s. A fast change of perfusate during clamp was followed by a delayed change of tonic (sustained) contraction. When [Ca] was increased from 0.25 to 4 mM, tension rose in a sigmoid manner and the level reached at the end of the clamp was almost identical with the steady control in 4 mM-Ca-Ringer. A similar tension increase occurred upon a reduction of [Na] from 100 to 25% of normal. At a given depolarization time course and height of the tension responses followed the ratio of [Ca]/[Na]. Transient tension responses are interpreted in terms of a sudden perturbation of a transmembrane Ca−Na exchange system leading to a depression of Ca pumping activity. [ABSTRACT FROM AUTHOR]

The role of extracellular Ca in the toniccontractile response to muscarinic receptor stimulation was investigated in isolated detrusor smooth muscle from the pig urinary bladder. Carbachol (10 −10 M) produced a concentration-dependent contractile response in isolated pig detrusor smooth muscle strips consisting of an initial phasic component followed by a tonic component. During the plateau of the tonic contractions induced by carbachol at the submaximal concentration of 10 M, the inhibiting effects of atropine, EGTA, nifedipine (a voltage-dependent calcium channel antagonist), H-7 [a protein kinase C (PKC) inhibitor] and YM 934 (a potassium channel opener) on the contractions were evaluated. Atropine (10 −3 × 10 M) concentration-dependently inhibited the tonic contractions induced by carbachol. In the same experimental conditions, EGTA (4 mM) and nifedipine (10 −3 × 10 M) depressed the tonic contractions in a concentration-dependent manner as did H-7 (10 −3 × 10 M) and YM934 (10 -10 M). However, H-7 (10-3 × 10 M) and YM934 (10 M) were very weak in inhibiting the contractions induced by KCl (50 mM) in isolated pig detrusor smooth muscle strips. These results suggest that the tonic-contractile response induced by carbachol in pig detrusor smooth muscle strips is dependent mainly on depolarization of the cell membranes and an influx of extracellular Ca, and also suggest that this depolarizing response may be due to inactivation of ATP-sensitive potassium channels through muscarinic activation of PKC. [ABSTRACT FROM AUTHOR]

The purpose of this investigation was to determine whether α-adrenoceptor subtypes (co)exist in the rat portal vein and, if so, whether they could be functionally associated with the phasic and tonic types of contraction as a response to α-adrenoceptor stimulation by phenylephrine. A low Ca concentration (0.9 mmol/l) in the Tyrode solution enabled us to quantify changes both in the phasic myogenic activity and in the basal tone of the rat portal vein preparation very precisely. We used both competitive and non-competitive α-adrenoceptor antagonists which have been employed successfully by other investigators to discriminate between α-adrenoceptor subtypes in vascular and other tissues. Schild analysis showed that the competitive α-adrenoceptor antagonists prazosin, phentolamine, yohimbine, corynanthine, idazoxan, rauwolscine and 5-methyl-urapidil could not distinguish between the phasic and tonic responses to phenylephrine and/or different α-adrenoceptor subtypes in the rat portal vein. However, when we compared our pA values with those found to be representative indicators according to subclassifications based on the use of selective antagonists in different tissues, the α-adrenoceptors in the rat portal vein appeared to belong to the α- or α-subtype. This subclassification was not in accordance with the data obtained with the irreversible α-cadrenoceptor antagonist chloroethylclonidine. However, the validity of this alkylating agent as a tool for receptor classification was restricted, at least in the rat portal vein, by its effects on receptor reserve. In contrast to the competitive α-adrenoceptor antagonists, the irreversible α-adrenoceptor antagonists phenoxybenzamine and chloroethylclonidine could indeed discriminate between the phasic and tonic types of contraction in response to α-adrenoceptor stimulation by phenylephrine, indicating two different receptor reserves for phenylephrine for the two types of responses. In conclusion, both the phasic and tonic types of contraction elicited by phenylephrine in the longitudinal smooth muscle of the rat portal vein appear to be mediated by one particular α-adrenoceptor subtype as defined by Schild analysis with selective, competitive α-adrenoceptor antagonists. However, using the method of receptor alkylation with phenoxybenzamine, two different affinity constants for the two types of responses could be calculated for phenylephrine. This may reflect the involvement of two different subtypes of α-adrenoceptors or more probably, the existence of only one α-adrenoceptor subtype, which is coupled with two different intrinsic efficacies to the effector pathways mediating the phasic and tonic responses, respectively. [ABSTRACT FROM AUTHOR]

Properties of the contraction produced by PGF in the guinea-pig taenia coli were compared to those produced by ACh. Prostaglandin (PG) F (3 × 10 M) and acetylcholine (ACh, 10 M) induced an initial transient contraction (phasic contraction) and a subsequent late contraction (tonic contraction). Both phasic and tonic contractions produced by PGF or ACh were abolished in Ca-free Krebs solution containing 0.5 mM EGTA. The tonic contractions caused by PGF and ACh were markedly suppressed by α-[3-[[2-(3,4-dimethoxy-phenyl)-ethyl]methylamino]-propyl]-3,4,5-trimethoxy-α-(1-methylethyl) benzeneacetonitrile hydrochloride (D600, > 10 M) as well as nifedipine (5 × 10 M), a Ca-antagonist. However, the phasic contraction produced by PGF, but not by ACh, was greatly inhibited by Mn (> 10 M). Furthermore, the phasic contraction caused by PGF was abolished in 18 mM K Krebs solution with D600 (2 × 10 M), whereas that induced by ACh and the tonic contractions produced by PGF as well as by ACh were unaffected in this high K solution without D600. Membrane potentials of the tissue in normal (K, 5.9 mM) and 18 mM K Krebs solution containing D600 were about −55 mV and −43 mV, respectively. In a fluorescence study which used Fura-2 an intracellular free Ca indicator in the presence of D600, PGF and ACh increased fluorescence intensity in the tissue, which coupled with the magnitude of contractions. Both the enhanced fluorescence intensity and tension development evoked by PGF, but not by ACh, were markedly decreased in 18 mM K Krebs solution. It is, thus, suggested that the phasic and tonic contractions evoked by PGF, unlike those by ACh, may be mediated via certain different Ca channels from extracellular Ca-source. [ABSTRACT FROM AUTHOR]

The contribution of anaerobic metabolism in normoxic and hypoxic anterior byssus retractor muscles (ABRM) to ATP production during tonic and repeated phasic contractions under isotonic conditions was estimated by analysing changes in the levels of ATP, phospho- l-arginine, l-arginine, octopine, alanopine, strombine, l- and d-lactate, succinate, l-aspartate and l- and d-alanine. [ABSTRACT FROM AUTHOR]