Tobacco-specific N-nitrosamines NNN and NNK levels in cigarette brands between 2000 and 2014.

The evolution of the levels of tobacco-specific N-nitrosamines (TSNA), N-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in mainstream (MS) cigarette smoke is investigated based on smoke and tobacco chemistry data of cigarette brands sold by Philip Morris International (PMI) between 2000 and 2014. A total of 315 cigarette samples representing a wide range of product and design characteristics manufactured by PMI between 2008 and 2014 were analyzed and compared to a previously published dataset of PMI brands manufactured in 2000. The data indicate that there is a substantial reduction of NNN and NNK levels in tobacco fillers and MS cigarette smoke per mg of tar and per mg of nicotine using Health Canada Intense (HCI) machine-smoking regime. This observed reduction in NNN and NNK levels in MS cigarette smoke is also supported by the downward trend observed on NNN and NNK levels in USA flue-cured Virginia and Burley tobacco lots from 2000 to 2014 crops, reflecting effectiveness of measures taken on curing and agricultural practices designed to minimize TSNA formation in tobacco.

Cerebral glucose transporters expression and spatial learning in the K-ATP Kir6.2(-/-) knockout mice.

K-ATP channels formed of the Sur and Kir subunits are widely distributed in the brain. Sur1-Kir6.2 is the most common combination of K-ATP channel subunits in the brain and Kir6.2 plays an important role in glucose metabolism through pancreatic insulin secretion or hypothalamic glucose sensing. K-ATP channels have also been reported to play a role in memory processing. Therefore, the aim of the present experiment is to assess the gene and protein expression of GLUT1, GLUT3 and GLUT4 in various brain regions of Kir6.2(-/-) K-ATP knockout mice and to test their working memory performance. GLUT4 was measured using two antibodies, one recognizing an intracellular epitope and the other, an extracellular epitope. Relative to their corresponding wild type, semi-quantitative immunohistochemistry showed that GLUT4 protein expression as measured by a GLUT4 antibody recognizing an extracellular epitope was increased in the Kir6.2(-/-) K-ATP mice. However, there was only a small increase in GLUT4 labeling using the GLUT4 antibody recognizing the intracellular epitope. These results suggest a compensatory higher GLUT4 inclusion at the cellular neuronal membrane in the cerebral cortex, hippocampus and cerebellum of the Kir6.2(-/-) K-ATP knockout mice. However, there was no change in GLUT4 gene expression assessed by TaqMan PCR except for a decrease in the cerebellum of these mice. Working memory performance of the Kir6.2(-/-) K-ATP mice was disrupted at age of 12 weeks but not at 5 weeks. The mild glucose intolerance that is observed in the Kir6.2 knockout mice is unlikely to have created the memory deficits observed. Rather, in light of the effects of K-ATP channel modulators on memory, the memory deficits in the Kir6.2(-/-) K-ATP mice are more likely due to the absence of the Kir6.2 and possible disruption of the GLUT4 activity in the brain.

Treadmill running causes significant fiber damage in skeletal muscle of KATP channel-deficient mice.

Although it has been suggested that the ATP-sensitive K(+) (K(ATP)) channel protects muscle against function impairment, most studies have so far given little evidence for significant perturbation in the integrity and function of skeletal muscle fibers from inactive mice that lack K(ATP) channel activity in their cell membrane. The objective was, therefore, to test the hypothesis that K(ATP) channel-deficient skeletal muscle fibers become damaged when mice are subjected to stress. Wild-type and K(ATP) channel-deficient mice (Kir6.2(-/-) mice) were subjected to 4-5 wk of treadmill running at either 20 m/min with 0 degrees inclination or at 24 m/min with 20 degrees uphill inclination. Muscles of all wild-type mice and of nonexercised Kir6.2(-/-) mice had very few fibers with internal nuclei. After 4-5 wk of treadmill running, there was little evidence for connective tissues and mononucleated cells in Kir6.2(-/-) hindlimb muscles, whereas the number of fibers with internal nuclei, which appear when damaged fibers are regenerated by satellite cells, was significantly higher in Kir6.2(-/-) than wild-type mice. Between 5% and 25% of the total number of fibers in Kir6.2(-/-) extensor digitum longus, plantaris, and tibialis muscles had internal nuclei, and most of such fibers were type IIB fibers. Contrary to hindlimb muscles, diaphragms of Kir6.2(-/-) mice that had run at 24 m/min had few fibers with internal nuclei, but mild to severe fiber damage was observed. In conclusion, the study provides for the first time evidence 1) that the K(ATP) channels of skeletal muscle are essential to prevent fiber damage, and thus muscle dysfunction; and 2) that the extent of fiber damage is greater and the capacity of fiber regeneration is less in Kir6.2(-/-) diaphragm muscles compared with hindlimb muscles.

KATP channels depress force by reducing action potential amplitude in mouse EDL and soleus muscle.

Although ATP-sensitive K+ (KATP) channel openers depress force, channel blockers have no effect. Furthermore, the effects of channel openers on single action potentials are quite small. These facts raise questions as to whether 1) channel openers reduce force via an activation of KATP channels or via some nonspecific effects and 2) the reduction in force by KATP channels operates by changes in amplitude and duration of the action potential. To answer the first question we tested the hypothesis that pinacidil, a channel opener, does not affect force during fatigue in muscles of Kir6.2-/- mice that have no cell membrane KATP channel activity. When wild-type extensor digitorum longus (EDL) and soleus muscles were stimulated to fatigue with one tetanus per second, pinacidil increased the rate at which force decreased, prevented a rise in resting tension, and improved force recovery. Pinacidil had none of these effects in Kir6.2-/- muscles. To answer the second question, we tested the hypothesis that the effects of KATP channels on membrane excitability are greater during action potential trains than on single action potentials, especially during metabolic stress such as fatigue. During fatigue, M wave areas of control soleus remained constant for 90 s, suggesting no change in action potential amplitude for half of the fatigue period. In the presence of pinacidil, the decrease in M wave areas became significant within 30 s, during which time the rate of fatigue also became significantly faster compared with control muscles. It is therefore concluded that, once activated, KATP channels depress force and that this depression involves a reduction in action potential amplitude.

Modulation of force development by Na+, K+, Na+ K+ pump and KATP channel during muscular activity.

Extracellular K+ concentration increases during exercise and especially during fatigue development. It has been proposed that K+ is an important factor in the etiology of skeletal muscle fatigue because it suppresses membrane excitability and eventually force development. Based on the effect of K+, it has then been proposed the Na+ K+ pump reduces increases in extracellular K+ concentration while the ATP-sensitive K+ channel (KATP channel) allows for rapid increases in extracellular K+ to suppress force development when ATP levels start to fall or when the levels of metabolic end-products become high. However, recent studies have now demonstrated that an increase in extracellular K+ concentration can be advantageous to muscle during exercise because it not only stimulates vasodilation and the exercise pressor reflex, but it also potentiates force development when the Na+ concentration gradient is maintained. A new hypothesis is therefore proposed in which the Na+ K+ pump is important in maintaining the Na+ concentration gradient (and not the K+ concentration gradient as previously suggested), while the activation of KATP channels is important to increase the K+ efflux and extracellular concentration. This situation then optimizes the development of force during exercise. Another hypothesis is proposed in which more KATP channels are activated while the activity of the Na+ K+ pump is reduced when ATP levels start to decrease to allow for an accumulation of intracellular Na+ and further increases in extracellular K+ concentration. These concentration changes then reduce membrane excitability and force development (i.e., fatigue) to protect muscle against large ATP depletion and function impairment.

Denervation enhances the physiological effects of the K(ATP) channel during fatigue in EDL and soleus muscle.

The objective was to determine whether denervation reduces or enhances the physiological effects of the K(ATP) channel during fatigue in mouse extensor digitorum longus (EDL) and soleus muscle. For this, we measured the effects of 100 microM of pinacidil, a channel opener, and of 10 microM of glibenclamide, a channel blocker, in denervated muscles and compared the data to those observed in innervated muscles from the study of Matar et al. (Matar W, Nosek TM, Wong D, and Renaud JM. Pinacidil suppresses contractility and preserves energy but glibenclamide has no effect during fatigue in skeletal muscle. Am J Physiol Cell Physiol 278: C404-C416, 2000). Pinacidil increased the (86)Rb(+) fractional loss during fatigue, and this effect was 2.6- to 3.4-fold greater in denervated than innervated muscle. Pinacidil also increased the rate of fatigue; for EDL the effect was 2.5-fold greater in denervated than innervated muscle, whereas for soleus the difference was 8.6-fold. A major effect of glibenclamide was an increase in resting tension during fatigue, which was for the EDL and soleus muscle 2.7- and 1.9-fold greater, respectively, in denervated than innervated muscle. A second major effect of glibenclamide was a reduced capacity to recover force after fatigue, an effect observed only in denervated muscle. We therefore suggest that the physiological effects of the K(ATP) channel are enhanced after denervation.

A K(ATP) channel deficiency affects resting tension, not contractile force, during fatigue in skeletal muscle.

The objective of this study was to determine how an ATP-sensitive K(+) (K(ATP)) channel deficiency affects the contractile and fatigue characteristics of extensor digitorum longus (EDL) and soleus muscle of 2- to 3-mo-old and 1-yr-old mice. K(ATP) channel-deficient mice were obtained by disrupting the Kir6.2 gene that encodes for the protein forming the pore of the channel. At 2-3 mo of age, the force-frequency curve, the twitch, and the tetanic force of EDL and soleus muscle of K(ATP) channel-deficient mice were not significantly different from those in wild-type mice. However, the tetanic force and maximum rate of force development decreased with aging to a greater extent in EDL and soleus muscle of K(ATP) channel-deficient mice (24-40%) than in muscle of wild-type mice (7-17%). During fatigue, the K(ATP) channel deficiency had no effect on the decrease in tetanic force in EDL and soleus muscle, whereas it caused a significantly greater increase in resting tension when compared with muscle of wild-type mice. The recovery of tetanic force after fatigue was not affected by the deficiency in 2- to 3-mo-old mice, whereas in 1-yr-old mice, force recovery was significantly less in muscle of K(ATP) channel-deficient than wild-type mice. It is suggested that the major function of the K(ATP) channel during fatigue is to reduce the development of a resting tension and not to contribute to the decrease in force. It is also suggested that the K(ATP) channel plays an important role in protecting muscle function in older mice.

Mini- and full-length dystrophin gene transfer induces the recovery of nitric oxide synthase at the sarcolemma of mdx4cv skeletal muscle fibers.

In normal skeletal muscle fibers, dystrophin accumulates at the cytoplasmic face of the sarcolemma where it associates with dystrophin-associated proteins (DAPs). Several studies have recently shown that the neuronal isoform of nitric oxide synthase (nNOS) is also located at the sarcolemma, and that this membrane localization is mediated through interactions of nNOS with one of the DAPs, namely alpha 1-syntrophin. Since the lack of dystrophin in muscle fibers from Duchenne muscular dystrophy patients and mdx mice is accompanied by an absence of sarcolemmal nNOS, we examined in the present study, whether dystrophin gene replacement would lead to the restoration of nNOS at its appropriate subcellular location. To this end, tibialis anterior muscles from mdx4cv mice were directly injected with plasmid DNA encoding either full-length (pRSV-dys) or mini-(pRSV-dyB; lacking exons 17-48) dystrophin. For these experiments, we chose to study 10-week-old mdx4cv mice since at this developmental stage, muscles from these mice have already undergone several cycles of degeneration-regeneration. Immunofluorescence experiments performed on serial cross-sections revealed that approximately 50% of the dystrophin-positive fibers also exhibited significant levels of nNOS at their sarcolemma 2 weeks following gene transfer with pRSV-dys. Similar results were obtained with pRSV-dyB indicating that exons 17-48 of the dystrophin gene are not essential for the correct localization of nNOS in skeletal muscle fibers. Taken together with the recent demonstration that dystrophin gene transfer leads to significant physiological benefits our results suggest that dystrophin gene therapy using full-length or truncated dystrophin, also induces a rapid recovery of biochemical functions.

Blocking ATP-sensitive K+ channel during metabolic inhibition impairs muscle contractility.

The objectives of this study were to determine the metabolic conditions in which ATP-sensitive K+ channels (K+(ATP) channels) contribute to a decrease in force. Sartorius muscles of the frog Rana pipiens were subjected to a 60-min metabolic inhibition by exposing them to cyanide (2 mM) and iodoacetate (1 mM). Muscles were exposed to glibenclamide (100 microM) to block K+ATP channels either 60 min before or 8 or 18 min into metabolic inhibition. Resting potentials, action potentials, and membrane conductance were measured using intracellular microelectrodes. Tetanic and resting tension were measured with a force transducer. ATP, ADP, and phosphocreatine (PCr) were measured by high-pressure liquid chromatography. Glibenclamide completely blocked the shortening of action potential but only partially blocked the increase in membrane conductance. When glibenclamide was added 60 min before metabolic inhibition, the decrease in tetanic force was faster than in control muscle (no glibenclamide). This faster decrease in tetanic force was associated with significant membrane depolarizations, greater increases in resting tension, greater depletions of ATP and PCr contents, and greater increases in ADP content. Addition of glibenclamide 8 min into metabolic inhibition caused an increase in tetanic force followed by a faster decrease compared with control. Addition of glibenclamide 18 min into metabolic inhibition had no effect on the tetanic force compared with control muscles. The data indicate that K+ATP channels 1) were activated during metabolic inhibition and 2) contributed to the decrease in tetanic force but also 3) had a myoprotective effect protecting skeletal muscle against muscle function impairment.

Mini-dystrophin gene transfer in mdx4cv diaphragm muscle fibers increases sarcolemmal stability.

To date, all dystrophin gene transfer studies have been performed on mdx hindlimb skeletal muscles which in comparison to the severe deficits seen in muscles from patients afflicted with Duchenne muscular dystrophy (DMD), exhibit only modest morphological and functional changes. Since the mdx diaphragm muscle presents the same pathophysiological alterations characteristic of DMD muscles, we therefore injected recombinant plasmid DNA encoding the dystrophin mini-gene (pRSVdy-B) into diaphragm muscles of 10-week-old mdx4cv mice and examined the physiological consequences of dystrophin expression in a muscle that has undergone a phase of massive degeneration and regeneration. Immunoperoxidase and immunofluorescence experiments revealed that 1 and 3 weeks following gene transfer, approximately 17% of the fibers in a bundle of diaphragm muscle expressed dystrophin at the sarcolemma. Most importantly, this level of dystrophin expression was sufficient to protect all fibers present within these diaphragm muscle bundles from the damaging effects of repetitive lengthening contractions. In addition, dystrophin expression partially restored the ability of transduced mdx4cv muscle bundles to generate isometric tetanic tension following lengthening contractions. These results show that mini-dystrophin expression leads to rapid and significant functional improvements in diaphragm muscles of mdx4cv mice. Although these data provide encouraging results for future therapeutic strategies aimed at curing DMD, additional work will none the less be necessary to determine the full impact of dystrophin gene replacement. In this context, it is clear from the data presented here that the diaphragm muscle of the mdx mouse is an invaluable model system to address this critical issue.

Modulation of muscle contractility during fatigue and recovery by ATP sensitive potassium channel.

The activity of ATP-sensitive potassium channels of skeletal muscle is controlled by changes in the bioenergetic state of the cell. These channels are inactive in unfatigued muscle and become activated during fatigue. It has been postulated that ATP-sensitive potassium channels shorten the action potential duration, increase the potassium efflux and contribute to the decrease in force during fatigue. Although blocking ATP-sensitive potassium channels during fatigue prolongs the action potential duration and decreases the potassium efflux as expected, it does not affect the rate of fatigue development as observed from the decrease in tetanic force. Even though such results are not consistent with the hypothesis that ATP-sensitive potassium channels contribute to the decrease in force during fatigue, a reduced capacity of skeletal muscles to recover their tetanic force following fatigue is also observed when ATP-sensitive potassium channels are blocked during fatigue, suggesting that these channels have a myoprotective effect. It is thus possible that removing this myoprotection during fatigue results in deleterious effects which counteract the expected slower decrease in force. However ATP-sensitive potassium channel openers also fall to affect the rate of fatigue development. Therefore, the results obtained so far do not support the hypothesis that ATP-sensitive potassium channels contribute to the decrease in force during fatigue.

Effect of tolbutamide on the rate of fatigue and recovery in frog sartorius muscle.

The goal of this study was to determine how blocking ATP-sensitive K+ channels with tolbutamide affects the excitability and contractility of intact frog sartorius muscle during fatigue development. Fatigue was elicited with one tetanic contraction every sec for 3 min. During fatigue the resting potential decreased by 10 mV although the action potential overshoot remained constant. The addition of 2 mmol.liter-1 tolbutamide 60 min before fatigue did not modify the effect of fatigue on the resting potential and action potential overshoot. During fatigue development the half-repolarization time of control muscles increased by 0.26 msec in control muscles, although it increased by 0.77 msec in the presence of 2 mmol.liter-1 tolbutamide; the difference was significant. The decrease in force during fatigue development was not affected by 2 mmol.liter-1 tolbutamide (added 60 min before fatigue), whereas the recovery of force after fatigue was slower in tolbutamide- exposed muscles than in control muscles. Addition of 2 mmol.liter-1 tolbutamide after 5 min of recovery reduced the recovery rate of the resting potential and half-repolarization time, but did not affect the recovery of tetanic force during the first 40 min. Our results are consistent with the hypothesis that ATP-sensitive K+ channels are activated during fatigue development and that they contribute to the repolarization phase of action potentials, but they do not support the hypothesis that ATP-sensitive K+ channels contribute to the decrease in force.

Na+ and K+ effect on contractility of frog sartorius muscle: implication for the mechanism of fatigue.

Although a decrease in extracellular Na+ and an increase in K+ concentration are believed to contribute to the decrease in force during fatigue, the force of unfatigued muscle decreases only with quite large changes in Na+ and K+ concentration. The objective of this study was to determine whether concomitant and smaller changes in Na+ and K+ concentration have greater effects on muscle contractility than individual changes. At 3 mM K+, a large decrease in Na+ from 120 to 60 mM had no effect on the twitch force, while the tetanic force decreased by 31.2%. At 120 mM Na+, an increase in K+ from 3 to 9 mM potentiated the twitch force by 41.1%, had no effect on the tetanic force at 7 mM, and decreased the tetanic force by 40.4% at 9 mM; both the twitch force and tetanic force were completely abolished at 11 mM K+. The potentiation of the twitch force between 3 and 9 mM K+ was less at 60, 80, and 100 mM than at 120 mM Na+. A reduction in Na+ concentration also reduced the K+ concentration at which the twitch force and tetanic force decreased and were completely abolished. It is shown that the combined effects of Na+ and K+ on the twitch and tetanic contractions were greater than the sum of their individual effects. Furthermore, it is proposed that neither Na+ nor K+ alone can be considered as an important factor in the decrease in force during fatigue, whereas together they are important for the tetanic contraction, but not for the twitch contraction.

The effect of K+ on the recovery of the twitch and tetanic force following fatigue in the sartorius muscle of the frog, Rana pipiens.

The goal of this study was to investigate how an increase in the extracellular K+ (K0+) concentration immediately after fatigue affects the recovery of the resting potential, the twitch and tetanic contraction of frog sartorius muscle to further understand the role of K+ in the mechanism of fatigue. Resting potentials were measured with conventional microelectrodes. Twitch and tetanic contractions were elicited by field stimulation. All muscles were fatigued with tetanic contractions at a rate of one contraction per second for 3 min while being exposed to 3 mmole l-1 K0+. During fatigue development the resting potential decreased by 16 mV (control group and pH0 7.2, extracellular pH), while the decrease in the twitch force was 32.8%, compared to 79.3% for the tetanic force, and 84.6% for the maximum rate of force development of the tetanus. Fatigued muscles were also unable to maintain a plateau phase during a tetanus: force declined by 14.8% during this phase. During the recovery period under control conditions (3 mmole l-1 K0+), all four parameters returned to their pre-fatigue values, the recovery of the plateau phase was the fastest (10 min), while that of the twitch force was the slowest (80 min). When K0+ was increased to 7.5 or 9.5 mmole l-1 immediately after fatigue, the recovery rate of the tetanic force and plateau phase was reduced. The maximum rate of force development of the tetanus, however, recovered at a faster rate than control muscles. The recovery of the twitch force was also increased above that of control when K0+ was increased to 9.0 mmole l-1 (a concentration which maximally potentiates the twitch force of unfatigued muscle). Frog sartorius muscles were also tested at pH0 6.4, a pH0 which inhibits force recovery. At that pH0 the effects of K0+ were similar to those observed at pH0 7.2. It is concluded that the role of K+ in muscle fatigue is more complex and may not involve just a contribution to the decrease in force during fatigue development, but may also contribute to an increase in force development under some conditions.

An ATP-sensitive potassium channel blocker decreases diaphragmatic circulation in anesthetized dogs.

The goal of this study was to determine whether in the dog ATP-sensitive K+ channels blocked with glibenclamide affect diaphragmatic blood flow [phrenic arterial blood flow (Qpa)] during both spontaneous breathing at rest and increased diaphragmatic activity. A control group (no glibenclamide; n = 4) and an experimental group (50 mg/kg of glibenclamide; n = 5) were studied. During spontaneous breathing at rest, Qpa was 15.0 ml.min-1 x 100 g-1 and decreased by 5% in the presence of glibenclamide. Diaphragmatic pacing (30 min-1) generated by phrenic nerve pacing produced an initial diaphragmatic tension-time index of 0.25 in both groups. A 50% decay in transdiaphragmatic pressure was reached at 165 s in the experimental group compared with 421 s in the control group. Diaphragmatic pacing increased Qpa by 46% in the experimental group and 65% in the control group, yielding a 63% greater vascular resistance in the experimental group. Phrenic vein K+ content at rest was unchanged by the presence of glibenclamide, being 3.6 +/- 0.16 mmol/l compared with 3.5 +/- 0.19 mmol/l in the control group. Phrenic nerve pacing in the control group produced a 13% increase in phrenic vein K+ content, whereas in the experimental group a 16% decrease was observed. We suggest that ATP-sensitive K+ channels play an important role in the modulation of Qpa.

The effect of glibenclamide on frog skeletal muscle: evidence for K+ATP channel activation during fatigue.

1. The purpose of this study was to determine whether ATP-sensitive K+ (K+ATP) channels are activated and contribute to the decrease in force during fatigue development in the sartorius muscle of the frog, Rana pipiens. Tetanic force (elicited by field stimulation), action potential and membrane conductance (using conventional microelectrodes), were measured in the presence and absence of glibenclamide, a K+ATP channel antagonist. Experiments were performed in bicarbonate-buffered solutions at pH 7.2. 2. In unfatigued muscle 100 mumol l-1 glibenclamide had no effect on the resting potential, the overshoot, the half-depolarization time or the maximum rate of depolarization of action potentials, while the mean half-repolarization time increased by 19 +/- 4% (+/- S.E.M.) and the maximum rate of repolarization decreased by 17 +/- 5%. 3. Fatigue was elicited using 100 ms tetanic contractions every 1 s for 3 min. In the absence of glibenclamide the mean half-repolarization time increased from 0.57 +/- 0.05 to 0.89 +/- 0.05 ms during fatigue. The mean half-repolarization times after fatigue, when muscle fibres were exposed to 100 mumol l-1 glibenclamide either 60 min prior to fatigue or 60 s before the end of fatigue, were 1.16 +/- 0.08 and 1.17 +/- 0.07 ms respectively. Application of 100 mumol l-1 glibenclamide after 5 min of recovery did not increase the half-repolarization time, but decreased the rate of recovery compared to control values. 4. In unfatigued muscles, 100 mumol l-1 glibenclamide did not affect the tetanic contraction. In the absence of glibenclamide, the mean tetanic force after fatigue was 11.0 +/- 0.9% of prefatigue values. Application of 100 mumol l-1 glibenclamide 60 min before fatigue increased the rate of fatigue development as the mean tetanic force was 4.8 +/- 0.8% after 3 min of stimulation. The addition of 100 mumol l-1 glibenclamide 60 s before the end of fatigue had no effect on tetanic force during this time compared to control. 5. In the absence of glibenclamide, muscles recovered 90.1 +/- 1.6% of their tetanic force after 100 min. Addition of 100 mumol l-1 glibenclamide 60 min prior to fatigue significantly reduced the capacity of muscles to recover their tetanic force: after 100 min of recovery tetanic force was only 47.3 +/- 9.4% of the pre-fatigue value.(ABSTRACT TRUNCATED AT 400 WORDS)

Tolbutamide, but not glyburide, affects the excitability and contractility of unfatigued frog sartorius muscle.

The goal of this study was to characterize the effects of tolbutamide and glyburide, two known KATP channel blockers, on intact, unfatigued sartorius muscle fibres of the frog, Rana pipiens. Tetanic contractions were elicited by field stimulation with 200 ms long train of pulses (0.5 ms, 6 V, 140 Hz). Resting and action potentials were measured using conventional microelectrodes. At pHo 7.2 (extracellular pH), the tetanic force was unaffected by 0.5 mM and 1.0 mM tolbutamide, but at 2.0 mM it decreased by 15.5 +/- 1.0%. The effect of tolbutamide on the tetanic force was significantly greater at pHo 6.4: all three tolbutamide concentrations caused a significant decrease in tetanic force, being 62.3 +/- 9.4% at 2 mM. In the presence of tolbutamide a large number of fibres became unexcitable at pHo 6.4, but not at pHo 7.2. Glyburide at 10 microM, on the other hand, caused a 5-7% decrease in tetanic force at both pHo 6.4 and 7.2, but no further decreases in tetanic force were observed when the glyburide concentration was increased up to 100 microM. Unlike tolbutamide, glyburide did not affect the excitability of muscle fibres, but significantly prolonged the repolarization phase of action potentials, especially at pHo 6.4. We suggest that several of the tolbutamide effects reported in this study cannot be accounted for by a direct effect on KATP channels, and that the large decrease in membrane excitability and muscle contractility in the presence of tolbutamide must seriously be taken into consideration when this channel blocker is used to study the physiological role of KATP channels in intact muscle fibres.

Force-length properties and functional demands of cat gastrocnemius, soleus and plantaris muscles.

The purpose of this study was to measure isometric force-length properties of cat soleus, gastrocnemius and plantaris muscle-tendon units, and to relate these properties to the functional demands of these muscles during everyday locomotor activities. Isometric force-length properties were determined using an in situ preparation, where forces were measured using buckle-type tendon transducers, and muscle-tendon unit lengths were quantified through ankle and knee joint configurations. Functional demands of the muscles were assessed using direct muscle force measurements in freely moving animals. Force-length properties and functional demands were determined for soleus, gastrocnemius and plantaris muscles simultaneously in each animal. The results suggest that isometric force-length properties of cat soleus, gastrocnemius and plantaris muscles, as well as the region of the force-length relation that is used during everyday locomotor tasks, match the functional demands.

Effects of K+ on the twitch and tetanic contraction in the sartorius muscle of the frog, Rana pipiens. Implication for fatigue in vivo.

The effects of increasing the extracellular K+ concentration on the capacity to generate action potentials and to contract were tested on unfatigued muscle fibers isolated from frog sartorius muscle. The goal of this study was to investigate further the role of K+ in muscle fatigue by testing whether an increased extracellular K+ concentration in unfatigued muscle fibers causes a decrease in force similar to the decrease observed during fatigue. Resting and action potentials were measured with conventional microelectrodes. Twitch and tetanic force was elicited by field stimulation. At pHo (extracellular pH) 7.8 and 3 mmol K+.L-1 (control), the mean resting potential was -86.6 +/- 1.7 mV (mean +/- SEM) and the mean overshoot of the action potential was 5.6 +/- 2.5 mV. An increased K+ concentration from 3 to 8.0 mmol.L-1 depolarized the sarcolemma to -72.2 +/- 1.4 mV, abolished the overshoot as the peak potential during an action potential was -12.0 +/- 3.9 mV, potentiated the twitch force by 48.0 +/- 5.7%, but did not affect the tetanic force (maximum force) and the ability to maintain a constant force during the plateau phase of a tetanus. An increase to 10 mmol K+.L-1 depolarized the sarcolemma to -70.1 +/- 1.7 mV and caused large decreases in twitch (31.6 +/- 26.1%) and tetanic (74.6 +/- 12.1%) force. Between 3 and 9 mmol K+.L-1, the effects of K+ at pHo 7.2 (a pHo mimicking the change in interstitial pH during fatigue) and 6.4 (a pHo known to inhibit force recovery following fatigue) on resting and action potentials as well as on the twitch and tetanic force were similar to those at pHo 7.8. Above 9 mmol K+.L-1 significant differences were found in the effect of K+ between pHo 7.8 and 7.2 or 6.4. In general, the decrease in peak action potential and twitch and tetanic force occurred at higher K+ concentrations as the pHo was more acidic. The results obtained in this study do not support the hypothesis that an accumulation of K+ at the surface of the sarcolemma is sufficiently large to suppress force development during fatigue. The possibility that the K+ concentration in the T tubules reaches the critical K+ concentration necessary to cause a failure of the excitation-contraction coupling mechanism is discussed.