Rat Diaphragm Research Articles

Intra- and extracellular measurement of reactive oxygen species produced during heat stress in diaphragm muscle.

Skeletal muscles are exposed to increased temperatures during intense exercise, particularly in high environmental temperatures. We hypothesized that heat may directly stimulate the reactive oxygen species (ROS) formation in diaphragm (one kind of skeletal muscle) and thus potentially play a role in contractile and metabolic activity. Laser scan confocal microscopy was used to study the conversion of hydroethidine (a probe for intracellular ROS) to ethidium (ET) in mouse diaphragm. During a 30-min period, heat (42 degrees C) increased ET fluorescence by 24 +/- 4%, whereas in control (37 degrees C), fluorescence decreased by 8 +/- 1% compared with baseline (P < 0.001). The superoxide scavenger Tiron (10 mM) abolished the rise in intracellular fluorescence, whereas extracellular superoxide dismutase (SOD; 5,000 U/ml) had no significant effect. Reduction of oxidized cytochrome c was used to detect extracellular ROS in rat diaphragm. After 45 min, 53 +/- 7 nmol cytochrome c. g dry wt(-1). ml(-1) were reduced in heat compared with 22 +/- 13 nmol. g(-1). ml(-1) in controls (P < 0.001). SOD decreased cytochrome c reduction in heat to control levels. The results suggest that heat stress stimulates intracellular and extracellular superoxide production, which may contribute to the physiological responses to severe exercise or the pathology of heat shock.

Donepezil dose-dependently inhibits acetylcholinesterase activity in various areas and in the presynaptic cholinergic and the postsynaptic cholinoceptive enzyme-positive structures in the human and rat brain

In the symptomatic treatment of mild to moderately severe dementia associated with Alzheimer’s disease, donepezil (E2020) has been introduced for the inhibition of acetylcholinesterase activity in the human brain. However, there is no morphological evidence as to how this chemical agent affects the acetylcholinesterase-positive structures in the various areas of the human and the rat CNS. This study demonstrates by histochemical means that donepezil exerts a dose-dependent inhibitory effect in vitro on acetylcholinesterase activity. The most sensitive areas were the cortex and the hippocampal formation. Within the different layers of the cortex, the cholinoceptive acetylcholinesterase-positive postsynaptic pyramidal cell bodies were more sensitive than the presynaptic cholinergic axonal processes. In the cortex, the cell body staining was already abolished by even 2×10 −8 M donepezil, whereas the axonal staining could be eliminated only by at least 5×10 −8 M donepezil. In the hippocampus, the axonal acetylcholinesterase reaction end-product was eliminated by 5×10 −7 M donepezil. The most resistant region was the putamen, where the staining intensity was moderately reduced by 1×10 −6 M donepezil. In the rat brain, the postsynaptic cholinoceptive and presynaptic cholinergic structures were inhibited by nearly the same dose of donepezil as in the human brain. These histochemical results provide the first morphological evidence that, under in vitro circumstances, donepezil is not a general acetylcholinesterase inhibitor in the CNS, but rather selectively affects the different brain areas and, within these, the cholinoceptive and cholinergic structures. The acetylcholinesterase staining in the nerve fibers (innervating the intracerebral blood vessels of the human brain and the extracerebral blood vessels of the rat brain) and at the neuromuscular junction in the diaphragm and gastrocnemius muscle of rat, was also inhibited dose dependently by donepezil. It is concluded that donepezil may be a valuable tool with which to influence both the pre- and the postsynaptic acetylcholinesterase-positive structures in the human and rat central and peripheral nervous systems.

Hyperpolarizing effect of acetylcholine in the skeletal muscle with different types of muscle fibers

Acetylcholine (ACh) is continuously released from motor nerve endings in a non-quantal form (as individual molecules). This is believed to play an important role in maintaining the rest membrane potential (RMP) of skeletal muscle fibers [1-4]. According to several reports, the nonquantal ACh, as well as exogenous ACh at a comparable concentration (50-100 nmol), produces a hyperpolarizing effect that increases the muscle fiber RMP value by several millivolts [36]. The cell-membranous mechanism of the hyperpolarizing effect of ACh in the skeletal muscle has not yet established [3-7]. Nothing has also been known about development of this effect in muscle fibers differing by their functional characteristics. Experiments were carried out on isolated phrenico-diaphragmal preparations of male white rats. Rat diaphragm is characterized by a wide range of motor units, from slow to fast ones. Three main types of muscle fibers were found in this muscle: white (23-33%), red (50-60%), and intermediate ones (15-18%) [8]. We used a flowing saline aerated with carbogen (95% 02 + 5% CO2) of the following composit ion (mmol/ l) : 137 NaC1, 5KCI , 2 CaCI2, 2 MgC12, 24 NaHCO3, 1 NaH2PO 4, 11 glucose; pH 7.4-7.6, temperature 28~ The RMP value was measured in the extrasynaptic area of muscle fibers (usually in layers 1-3), using the standard microelectrode technique. The measurements began 1 h after extirpation of the muscle; each time the RMP was recorded in 25-35 fibers. ACh or its non-hydrolisable analog carbacholine (100 nmol) were added to the solution for 15 rain after 60 min of RMP recording, then this solution was replaced by the initial one. To inhibit acetylcholinesterase (ACHE) activity, armin (0.4 pmol), an organophosphorus inhibitor of ACHE, was added to the solution for the whole time of the experiment. In each series of experiments, results of the RMP measurements in 4-7 muscles were summarized. The statistical significance of differences was estimated by Student's criterion. Values in the text and figure are presented as the means and their errors. In experiments with inhibition of AChE (5 muscles) after 15 min of ACh action, the RMP was observed to increase from the initial value of -75.5 + 0.5 mV (156 fibers) to -79.6 + 0.5 mV (151 fiber), i.e., the hyperpolarization of the muscle membrane was 4.1 + 0.5 mV (p < 0.01). After removing ACh from the solution, the RMP value continued to rise and reached its maximum, -81.7 + 0.6 mV (139 fibers) after 15 min of washing out (hyperpolarization was as high as 6.2 + 0.6 mV). Then the RMP value slightly decreased but still significantly exceeded the initial value for 1.5 h after removal of ACh from the solution, i.e., the hyperpolarization was of a trace character (see figure). Before addition of ACh, the histogram of the RMP value distribution had a char-

Denervation alters myosin heavy chain expression and contractility of developing rat diaphragm muscle.

We hypothesized that unilateral denervation (DNV) of the rat diaphragm muscle (Dia(m)) in neonates at postnatal day 7 (D-7) alters normal transitions of myosin heavy chain (MHC) isoform expression and thereby affects postnatal changes in maximum specific force (P(o)) and maximum unloaded shortening velocity (V(o)). The relative expression of different MHC isoforms was analyzed electrophoretically. With DNV at D-7, expression of MHC(neo) in the Dia(m) persisted, and emergence of MHC(2X) and MHC(2B) was delayed. By D-21 and D-28, relative expression of MHC(2A) and MHC(2B) was reduced in DNV compared with control (CTL) animals. Expression of MHC(neo) also reappeared in adult Dia(m) by 2-3 wk after DNV, and relative expression of MHC(2B) was reduced. At each age, P(o) was reduced and V(o) was slowed by DNV, compared with CTL. In CTL Dia(m), postnatal changes in P(o) and V(o) were associated with an increase in fast MHC isoform expression. In DNV Dia(m), no such association existed. We conclude that, in the Dia(m), DNV induces alterations in both MHC isoform expression and contractile properties, which are not necessarily causally linked.

Maximum specific force depends on myosin heavy chain content in rat diaphragm muscle fibers.

In the present study, myosin heavy chain (MHC) content per half sarcomere, an estimate of the number of cross bridges available for force generation, was determined in rat diaphragm muscle (Dia(m)) fibers expressing different MHC isoforms. We hypothesize that fiber-type differences in maximum specific force [force per cross-sectional area (CSA)] reflect the number of cross bridges present per CSA. Studies were performed on single, Triton X-100-permeabilized rat Dia(m) fibers. Maximum specific force was determined by activation of single Dia(m) fibers in the presence of a high-calcium solution (pCa, -log Ca(2+) concentration of 4.0). SDS-PAGE and Western blot analyses were used to determine MHC isoform composition and MHC content per half sarcomere. Differences in maximum specific force across fast MHC isoforms were eliminated when controlled for half-sarcomere MHC content. However, the force produced by slow fibers remained below that of fast fibers when normalized for the number of cross bridges available. On the basis of these results, the lower force produced by slow fibers may be due to less force per cross bridge compared with fast fibers.

Carbachol and acetylcholine delay the early postdenervation depolarization of muscle fibres through M1-cholinergic receptors.

The resting membrane potential (RMP) of denervated muscle fibres of rat diaphragm muscle is depolarized by approximately 8-10 mV during the first 3 h after nerve section and this early postdenervation depolarization is reduced substantially by the presence of 5x10(-8) M acetylcholine (ACh) or carbachol (CB). The muscarinic antagonist atropine (Atr; 5x10(-9) to 5x10(-6) M) reduced the effect of CB in a dose-dependent manner (K(i)=7x10(-8) M) and increased the rate of the early postdenervation depolarization. In lower doses (5x10(-7) M), Atr acted only in the presence of an allosteric stabilizator hexamethylene-bis-[dimethyl-(3-phtalimidopropyl)ammonium] (W-84). Also pirenzepine, a specific inhibitor of the M1 subtype of muscarinic receptor, blocked the action of CB in a dose-dependent manner with an apparent inhibition constant K(i)=1x10(-7) microM. DAMP, a specific M3 antagonist, was without effect on the muscle hyperpolarization induced by CB. CB also hyperpolarized the membrane potentials of muscles which were denervated for 1-3 days. It is concluded that ACh and CB protect the muscle fibres from early depolarization through M1-cholinergic receptors on the muscle membrane. These particular receptors can apparently mediate the 'trophic', non-impulse regulation of RMP in skeletal muscles when they are activated by acetylcholine released non-quantally.

The effects of equinatoxin II on respiration — possible mechanism of the toxin lethality

In rat an intravenous application (i.v.) of a lethal dose of the equinatoxin II (EqT II) provoked a respiratory arrest. It is not known whether the respiratory arrest is a result of a direct actions of the toxin on lung tissue, on neuromuscular junctions or the on the central nervous system. The influences of the toxin on the neuromuscular transmission and on muscular contraction were studied in isolated rat diaphragm. The effect of EqT II on end plate potentials of m. cutaneus pectoris was measured in the frog Rana esculenta. To monitor equinatoxin II effects on the central nervous system of a rat, the toxin was injected directly into the forth brain ventricle. The respiratory arrest was not the result of the toxin action on the neuromuscular junctions and peripheral nerves. The cessation of respiratory activity was most probably a result of equinatoxin II direct actions on lung tissue and on brain microcirculation.

Interaction of nitric oxide and reactive oxygen species on rat diaphragm contractility.

Growing evidence indicates that reactive oxygen species (ROS) as well as nitric oxide (NO) have a profound influence on contractile function of skeletal muscle possibly through modulation of excitation-contraction coupling. We hypothesized that if NO and xanthine oxidase (XO) interact at key sites in excitation-contraction coupling, the effects of XO with nitric oxide synthase (NOS) inhibitors and NO donors on contractile function of the unfatigued diaphragm would not be additive. Diaphragm fibre bundles were extracted from 4-month Fischer-344 rats and placed in Krebs solution bubbled with 95% O2, 5% CO2. Baseline twitch tension, tension at 20 Hz (low-frequency), and maximal tetanic tension (Po) at 120 Hz were then measured (PRE). In Experiment 1 diaphragm fibre bundles were exposed to Krebs with 200 microM hypoxanthine as a control (CON); 0.02 U mL-1 XO + 200 microM hypoxanthine; 1 mM of the NOS inhibitor N-nitro-L-arginine (L-NNA) or L-NNA + XO. Five minutes were allowed for equilibration, and a second set of contractile measures was taken (POST). In Experiment 2 we exposed diaphragm fibre bundles to one of the following four solutions: CON, XO, 100 microM of the NO donor sodium nitroprusside (SNP) and XO + SNP, and evaluated contractile function as described above. In Experiment 3 we tested to determine if peroxynitrite production from the reaction of superoxide anion and NO affected the above results for SNP using 30 microM ebselen as a peroxynitrite quencher. Xanthine oxidase resulted in a significant potentiation of diaphragm twitch tension and tension at 20 Hz (+29%) without affecting Po. L-NNA also significantly increased 20 Hz tension but did not alter Po. However, the combination of XO + L-NNA did not further increase low-frequency contractility. Sodium nitroprusside alone did not affect diaphragm contractility, but did attenuate XO-induced potentiation in the XO + SNP group. Ebselen did not alter the impact of SNP on XO in the diaphragm. These data support the hypothesis that XO and NO interact or compete at similar sites of action that modulate contractility of the unfatigued diaphragm.

Differences in apoptotic frequency in the quadriceps muscle and the diaphragm of rats with chronic heart failure: Relation to iNOS‐expression and aerobic metabolism

Differences in apoptotic frequency in the quadriceps muscle and the diaphragm of rats with chronic heart failure: Relation to iNOS‐expression and aerobic metabolism

Acute Effects of an Insect Repellent, N,N-Diethyl-m-toluamide, on Cholinesterase Inhibition Induced by Pyridostigmine Bromide in Rats

Acute lethal interactions have been previously described between a cholinesterase (ChE) inhibitor, pyridostigmine bromide (PB), and the insect repellent, N,N-diethyl-m-toluamide (DEET). The mechanism of toxic interaction between these agents is unknown. Alterations in membrane permeability caused by DEET could facilitate or enhance absorption, or alter the distribution of peripherally restricted PB, causing increased inhibition of ChE at a given dose. Studies were conducted to investigate PB-induced ChE inhibition in the presence of DEET. Rats received ip injections of PB (1, 2, or 3 mg/kg), DEET (200 mg/kg), or PB + DEET at doses that potentiated acute lethality. ChE activity was measured in heart, diaphragm, blood, whole brain, or specific brain areas using a modified spectrophotometric assay. DEET did not alter PB-induced inhibition of ChE activity in rat diaphragm, heart, or blood. Administration of DEET alone had no effect on ChE activity. PB alone did not inhibit ChE in whole brain, but PB (3 mg/kg) + DEET (200 mg/kg) caused significant inhibition of whole brain ChE activity to approximately 60% of controls. In specific brain areas, (cortex, cerebellum, medulla, hypothalamus, hippocampus, midbrain, and striatum) PB alone did not inhibit ChE activity. PB (3 mg/kg) + DEET (200 mg/kg) reduced ChE activity to approximately 65–75% of controls in each brain area, but those results were not statistically significant. In conclusion, DEET did not alter PB-induced inhibition of ChE activity in the periphery. While DEET may have facilitated the access of PB into the CNS at high doses, it is doubtful that the resulting minor reduction in ChE activity would have resulted in death. It is unlikely that the lethal interaction between PB and DEET is mediated through a cholinergic effect resulting from increased inhibition of ChE.

Induction of the ATP-sensitive potassium (uK(ATP)-1) channel by endotoxemia.

The effect of sepsis on the ubiquitously expressed ATP-sensitive potassium (uK(ATP)-1) channel expression was measured in Sprague-Dawley rat diaphragms. Rats were treated with either 0.5 ml saline or 20 mg/Kg E. coli lipopolysaccharides and sacrificed at 3, 6, 12, 24, or 48 h later. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that channel mRNA expression was increased at 3 h and continued to rise up to 48 h. Western blotting analysis showed a approximately 9-fold increase in channel protein expression 24 h after sepsis. Our results demonstrate that sepsis upregulates the uK(ATP)-1 channel.

Corticosteroids decrease mRNA levels of SERCA pumps, whereas they increase sarcolipin mRNA in the rat diaphragm.

1. In order to explore the potential role of the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA)-type pumps and of their modulators phospholamban (PLB) and sarcolipin (SLN) in the functional alterations of the diaphragm induced by corticosteroid treatment, expression of SERCA, PLB and SLN was assessed by RT-PCR in the diaphragm of rats treated daily for 5 days either with triamcinolone (80 mg kg-1, n = 8) or with saline (control; 0.6 ml, n = 8). 2. Triamcinolone treatment reduced the normalised overall amount of all SERCA mRNA in diaphragm by 70 % compared to controls (P < 0.05). This reduction was accounted for by a relatively larger decrease in the SERCA1 mRNA (-69 %, P < 0.05) whilst the decrease in SERCA2 mRNA (-49 %, P = 0.09) did not reach statistical significance. As a result the relative proportion of SERCA2 mRNA was increased from 43 +/- 7 % in control diaphragm to 52 +/- 4 % after triamcinolone treatment (P < 0.05). 3. Only the adult isoform of SERCA1 (i.e. SERCA1a) mRNA was found in the diaphragm of the 15-week-old control rats. Furthermore, triamcinolone treatment resulted in reduced levels of SERCA2a (-40 %, P < 0.05) and increased levels of SLN mRNA (+100 %, P < 0.05), while the decrease in PLB mRNA (-31 %, P = 0.277) did not reach statistical significance. SERCA1b, SERCA2b and SERCA3 mRNA levels fell below the detection limit in the diaphragm of both control and triamcinolone-treated rats. 4. Compared to control diaphragm, control rat heart showed a relatively high PLB/(SERCA1 + SERCA2) mRNA ratio of 7.88 while this ratio amounted only to 0.16 in control extensor digitorum longus (EDL) muscle. Remarkably, the SLN/(SERCA1 + SERCA2) mRNA ratio in normal cardiac muscle (0.96) was nearly the same as in diaphragm, but in EDL it amounted to only 0.05 that in diaphragm. This indicates the very low expression of SLN in rat EDL. 5. These data reveal that considerable alterations in SERCA mRNA levels accompany the functional changes seen in diaphragm after corticosteroid treatment. The relatively larger decrease in SERCA1 mRNA is in agreement with the selective type II fibre atrophy previously observed in the diaphragm of triamcinolone-treated rats, but the magnitude of SERCA alterations is more pronounced than expected on the basis of the structural changes in the diaphragm. The increase in SLN mRNA levels may represent a compensatory mechanism.

Effects of protein kinase A inhibition on rat diaphragm force generation

Although protein kinases are known to play a role in modulating a variety of intracellular functions, the direct effect of inhibition of these enzymes on skeletal muscle force production has not been studied. The purpose of the present study was to examine this issue by determining the effects produced on diaphragm force generation by two protein kinase inhibitors: (a) H7, an inhibitor of both cAMP-dependent protein kinase (PKA) and of protein kinase C, and (b) H89, a selective inhibitor of PKA. Experiments ( n=15) were performed using isolated, arterially perfused, electrically stimulated rat diaphragms. Perfusate temperature was adjusted to maintain muscle temperature at 27°C and arterial pressure was kept at 150 Torr. Animals were divided into three groups: (a) a control group perfused with Krebs-Henselheit solution equilibrated with 95% O 2/5% CO 2, (b) a group in which H7 (2 μM) was added to the perfusate, and (c) a group perfused with solution containing H89 (4 μM). In all three groups, we assessed diaphragm twitch kinetics, force-frequency relationships and in vitro fatiguability. We found that both H7 and H89 administration slowed twitch relaxation, augmented force generation in response to low frequency stimulation, and increased the rate of development of fatigue. Specifically, for control, H7 and H89 groups, respectively, we found: (a) 1/2 relaxation time averaged 64±2 S.E.M., 87±6 and 90±2 ms, P<0.003, (b) force production during 10-Hz stimulation averaged 12.6±1.1, 20.1±2.3, and 20.3±2.1 N/cm 2, P<0.035, and (c) force fell to 14.3±2.0, 9.5±0.5 and 8.7±0.2% of its initial value after 20 min of fatiguing stimulation, P<0.035. These data show that it is possible to produce large increases in low frequency skeletal muscle force generation by directly inhibiting PKA. We speculate that it may be possible to pharmacologically augment respiratory muscle force and pressure generation in clinical medicine by administration of PKA inhibitors.

Effect of nitric oxide and NO synthase inhibition on nonquantal acetylcholine release in the rat diaphragm.

After anticholinesterase treatment, the postsynaptic muscle membrane is depolarized by about 5 mV due to nonquantal release of acetylcholine (ACh) from the motor nerve terminal. This can be demonstrated by the hyperpolarization produced by the addition of curare (H-effect). The magnitude of the H-effect was decreased significantly to 3 mV when the nitric oxide (NO) donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) were applied to the muscle, or when NO production was elevated by adding L-arginine, but not D-arginine, as a substrate. The H-effect was increased to 8-9 mV by inhibition of NO synthase by L-nitroarginine methylester (L-NAME), or by guanylyl cyclase inhibition by methylene blue and 1H-[1,2,4]oxidiazolo[4,3-a]quinoxalin-1-one (ODQ). ODQ increased the H-effect to 7.3 +/- 0.2 mV and diminished the SNP-induced decrease of the H-effect when applied together with SNP. The effects of NO donors and L-arginine were eliminated by adding reduced haemoglobin, an extracellular NO scavenger. The present results, together with earlier evidence for the presence of NO synthase in muscle fibres, indicate that nonquantal release of ACh is modulated by NO production in the postsynaptic cell.

THE MYONEURAL EFFECTS OF LITHIUM CHLORIDE ON THE NERVE–MUSCLE PREPARATIONS OF RATS. ROLE OF ADENOSINE TRIPHOSPHATE-SENSITIVE POTASSIUM CHANNELS

The potential effects of lithium chloride on the neuromuscular transmission and muscular contraction were studied using in vitro and in vivo nerve–muscle preparations of rats. Addition of lithium chloride to the fluid bathing the isolated rat diaphragm produced a concentration-dependent inhibition of diaphragmatic contractions elicited by either indirect or direct electrical stimulation. The threshold concentrations were 1 mmol l−1and 3 mmol l−1, respectively. Similarly, the intravenous administration of lithium chloride as bolus injections, produced a dose-dependent progressive inhibition of the indirectly- and directly-induced gastrocnemius muscle contractions during the 2-h period of investigation. The indirectly-induced contractions were much more sensitive to the inhibitory effect of lithium chloride than directly-induced contractions. Also, lithium chloride was found to be capable of enhancing the paralysis of the indirectly stimulated rat diaphragm in vitro and gastrocnemius muscle in vivo induced by either pipecuronium or succinylcholine. The combination of lithium chloride and pipecuronium led to a synergistic inhibition of the neuromuscular transmission, while the combination of lithium chloride and succinylcholine led to additive inhibition. Pretreatment with lithium chloride at the threshold concentrations enhanced the inhibitory effects of verapamil on diaphragmatic contractions elicited either indirectly or directly. The inhibitory effects of verapamil on the indirectly- and directly-induced rat gastrocnemius muscle contractions were potentiated by lithium chloride administration to rats. Glibenclamide was found to be capable of inhibiting the relaxant effects of lithium chloride on the indirectly- and directly-elicited contractions of rat diaphragm in vitro and rat gastrocnemius muscle in vivo, in a concentration- and dose-dependent manner, respectively. Doubling the concentration of magnesium in the bathing fluid potentiated the inhibitory effects of the threshold concentrations of lithium chloride on the diaphragmatic contractions induced either indirectly or directly. Pretreatment with 4-aminopyridine or barium chloride inhibited the relaxant effects of lithium chloride on the indirectly- and directly-elicited diaphragmatic contractions. The inhibitory effects of diazoxide on the indirectly-evoked contractions of rat diaphragm in vitro and rat gastrocnemius muscle in vivo were potentiated by lithium chloride. Pretreatment with glibenclamide inhibited markedly the combined effects of lithium chloride and diazoxide on the contractions of the diaphragm and gastrocnemius muscles induced indirectly. Additionally, the intravenous administration of lithium chloride into rats as bolus injections produced a dose-dependent progressive increase in plasma potassium level and a dose-dependent progressive decrease in the intracellular levels of adenosine triphosphate in the sciatic nerve and gastrocnemius muscle. It is concluded that lithium chloride, via activation of adenosine triphosphate- sensitive potassium channels, acts presynaptically to inhibit the neuromuscular transmission and acts at the muscle membrane to inhibit the muscular contraction.

Catchlike property of rat diaphragm: subsequent train frequency effects in variable-train stimulation.

A high-frequency burst of pulses at the onset of a subtetanic train of stimulation allows skeletal muscle to hold force at a higher level than expected from the extra pulses alone because of the catchlike property of muscle. The present study tested the hypothesis that the presence and degree of force increase induced by a high-frequency burst are strongly modulated by the subsequent train frequency. Rat diaphragm muscle strips (studied in vitro at 37 degrees C) underwent two-, three-, or four-pulse bursts [interpulse interval (IPI) of 5 or 10 ms] at the onset of 10- to 50-Hz subtetanic trains. Force was quantified during the train with respect to its peak value (F(peak)), mean value (F(mean)), and force-time integral (F(area)), and it was compared with that produced during subtetanic trains of an equal number of pulses without preceding pulse bursts (Diff-F(peak), Diff-F(mean), Diff-F(area)). F(peak) and F(mean) increased with two-, three-, and four-pulse bursts, and Diff-F(peak) and Diff-F(mean) increased progressively with decreasing frequency of the subtetanic train. F(area), the best reflection of catchlike force augmentation, was increased mainly by the four-pulse bursts with an IPI of 10 ms, and Diff-F(area) was maximal at subsequent train frequencies of 15-25 Hz. The use of incorrect patterns of burst stimulation could also precipitate F(area) decreases, which were observed with the four-pulse, 5-ms IPI paradigm. The time required to reach 80% of maximal force (T(80%)) became shorter for each of the pulse burst stimulation patterns, with maximal reduction of Diff-T(80%) occurring at a subsequent train frequency of 20 Hz in all cases. These data indicate that extra-pulse burst stimulation paradigms need to incorporate the optimal combinations of extra-pulse number, IPI, and the frequency of the subsequent subtetanic train to take greatest advantage of the catchlike property of muscle.

Expression of Glut-4 and Glut-1 transporters in rat diaphragm muscle

Glucose transporters (Gluts) are a family of membrane proteins responsible for the transport of glucose across cellular membranes. Generally, alterations of Gluts expression in limb skeletal muscle have been reported. However, the changes of Glut isoforms in respiratory muscle which contracts with a duty cycle have rarely been studied. This study was performed to evaluate at the light microscopy level the expression of Glut-4 and Glut-1 transporters in normal and denervated diaphragm by immunohistochemistry method with specific Gluts antibodies. The results showed Glut-4 immunoreactivity in both the cell periphery and the interior of myocytes. Glut-1 was also present in the cell border and in the interior of myocytes in control diaphragm. However, Glut-4 staining was stronger than Glut-1 staining in control diaphragm. In denervated hemidiaphragm, the Glut-4 immunolabelling decreased and Glut-1 increased. These data indicated that (1) Glut-4 and Glut-1 transporters were observed in diaphragm; and (2) there were alterations in the expression of both glucose transporters after denervation. These alterations in Glut isoforms after denervation may be associated with the removal of innervation itself, and/or may partly result from passive stretch imposed by inspiratory activation of the contralateral side.

Contractile and fatigue properties of the rat diaphragm musculature during the perinatal period.

The following two hypotheses regarding diaphragm contractile properties in the perinatal rat were tested. First, there is a major transformation of contractile and fatigue properties during the period between the inception of inspiratory drive transmission in utero and birth. Second, the diaphragm muscle properties develop to functionally match changes occurring in phrenic motoneuron electrophysiological properties. Muscle force recordings and intracellular recordings of end-plate potentials were measured by using phrenic nerve-diaphragm muscle in vitro preparations isolated from rats on embryonic day 18 and postnatal days 0-1. The following age-dependent changes occurred: 1) twitch contraction and half relaxation times decreased approximately two- and threefold, respectively; 2) the tetanic force levels increased approximately fivefold; 3) the ratio of peak twitch force to maximum tetanic force decreased 2.3-fold; 4) the range of forces generated by the diaphragm in response to graded nerve stimulation increased approximately twofold; 5) the force-frequency curve was shifted to the right; and 6) the propensity for neuromuscular transmission failure decreased. In conclusion, the diaphragm contractile and phrenic motoneuron repetitive firing properties develop in concert so that the full range of potential diaphragm force recruitment can be utilized and problems associated with diaphragm fatigue are minimized.

Detrimental Effects of Short-term Glucocorticoid Use on the Rat Diaphragm

The purpose of this study was to determine the effect of short-term, high doses of glucocorticoids on both body and diaphragm weights as well as contractile characteristics of the rat diaphragm. Adult, female Sprague-Dawley rats were divided into 2 groups: a control group (n=16) and a prednisolone group (n=16). The prednisolone group received prednisolone at a dosage of 5 mg/kg, and the control group received sham saline injections for 5 days. Animals were weighed prior to and after completion of the drug injection period. At the completion of the drug injection period, the animals were sacrificed, and the diaphragm, soleus, and extensor digitorum longus muscles were removed and weighed. A small strip of the costal diaphragm was connected to a force transducer, and the following contractile characteristics were measured: maximal specific isometric tetanic tension, peak isometric twitch specific tension, one-half relaxation time, and time to peak tension. Both body and diaphragm weights decreased by 15% in the prednisolone group as compared with the control group. Maximal specific isometric tetanic tension was reduced 13% in the prednisolone group as compared with the control group. There was no difference in any twitch contractile characteristics between the 2 groups. These data support the hypothesis that glucocorticoid treatment over a 5-day period results in a decrease in specific tension as well as diaphragm and body weight. These results may have implications for the treatment of patients receiving high doses of glucocorticoids for acute medical conditions.

Nandrolone decanoate does not enhance training effects but increases IGF-I mRNA in rat diaphragm.

To examine whether concomitant anabolic steroid treatment combined with training might enhance previously observed training effects (A. Bisschop, G. Gayan-Ramirez, H. Rollier, R. Gosselink, R. Dom, V. de Bock, and M. Decramer. Am. J. Respir. Crit. Care Med. 155: 1583-1589, 1997) and whether insulin-like growth factor I (IGF-I) was involved in these changes, male and female rats were submitted to inspiratory muscle training (IMT) for 8 wk (30 min/day, 5 times/wk) and were compared with untrained controls. During the last 5 wk of training, trained rats were divided to receive weekly either low-dose (LD; 1.5 mg/kg) or high-dose (HD; 7.5 mg/kg) nandrolone decanoate or saline for the IMT and control rats. In both sexes, diaphragm muscle mass and contractile properties were unchanged with treatment. In males, HD resulted in decreased diaphragm type I cross-sectional area (-15%; P < 0.05, HD vs. IMT), whereas no changes were observed in females. Finally, an increase in IGF-I mRNA levels was present in HD male (+73%; P < 0.05, HD vs. IMT) and female treated rats [LD (+58%) and HD (+96%) vs. IMT; P < 0.001]. We conclude that administration of nandrolone decanoate did not enhance the previously observed training effects in rat diaphragm, although it increased the IGF-I mRNA expression levels.