Ghrelin receptor-knockout mice display alterations in circadian rhythms of activity and feeding under constant lighting conditions.

Ghrelin is an orexigenic hormone produced by the stomach. Ghrelin, however, may also be a modulator of the circadian system given that ghrelin receptors are expressed in the master clock, the suprachiasmatic nucleus (SCN) and several outputs of this region. To investigate this, we performed analyses of running wheel activity and neuronal activation in wild type (WT) and growth hormone secretagogue receptor-knockout (GHSR-KO) mice under various lighting conditions. GHSR-KO and WT mice were maintained under constant dark (DD) or constant light (LL) with ad libitum access to food before being placed on a schedule of temporally restricted access to food (4 h/day) for 2 weeks. There were no differences between KO and WT mice in free-running period under DD, but GHSR-KO mice required more days to develop a high level of food anticipatory activity, and this was lower than that observed in WT mice. Under LL, GHSR-KO mice showed greater activity overall, lengthening of their circadian period, and more resistance to the disorganisational effects of LL. Furthermore, GHSR-KO mice showed greater activity overall, and greater activity in anticipation of a scheduled meal under LL. These behavioral effects were not correlated with changes in the circadian expression of the Fos, Per1 or Per2 proteins under any lighting conditions. These results suggest that the ghrelin receptor plays a role in modulating the activity of the circadian system under normal conditions and under restricted feeding schedules, but does so through mechanisms that remain to be determined.

Activation of Ca(2+)-dependent protein kinase II during repeated contractions in single muscle fibres from mouse is dependent on the frequency of sarcoplasmic reticulum Ca(2+) release.

AIM: To investigate the importance and contribution of calmodulin-dependent protein kinase II (CaMKII) activity on sarcoplasmic reticulum (SR) Ca(2+)-release in response to different work intensities in single, intact muscle fibres. METHODS: CaMKII activity was blocked in single muscle fibres using either the inhibitory peptide AC3-I or the pharmacological inhibitor KN-93. The effect on tetanic force production and [Ca(2+)](i) was determined during work of different intensities. The activity of CaMKII was assessed by mathematical modelling. RESULTS: Using a standard protocol to induce fatigue (50x 70 Hz, 350 ms duration, every 2 s) the number of stimuli needed to induce fatigue was decreased from 47 +/- 3 contractions in control to 33 +/- 3 with AC3-I. KN-93 was a more potent inhibitor, decreasing the number of contractions needed to induce fatigue to 15 +/- 3. Tetanic [Ca(2+)](i) was 100 +/- 11%, 97 +/- 11% and 67 +/- 11% at the end of stimulation in control, AC3-I and KN-93 respectively. A similar inhibition was obtained using a high intensity protocol (20x 70 Hz, 200 ms duration, every 300 ms). However, using a long interval protocol (25x 70 Hz, 350 ms duration, every 5 s) no change was observed in either tetanic [Ca(2+)](i) or force when inhibiting CaMKII. A mathematical model used to investigate the activation pattern of CaMKII suggests that there is a threshold of active CaMKII that has to be surpassed in order for CaMKII to affect SR Ca(2+) release. CONCLUSION: Our results show that CaMKII is crucial for maintaining proper SR Ca(2+) release and that this is regulated in a work intensity manner.

The effects of the myosin-II inhibitor N-benzyl-p-toluene sulphonamide on fatigue in mouse single intact toe muscle fibres.

AIM: This study determined whether fatigue in skeletal muscle is primarily due to the repeated elevations of myoplasmic free calcium concentration ([Ca(2+)](i)) or to metabolite accumulation. METHODS: We examined the effects of N-benzyl-p-toluene sulphonamide (BTS) which is a potent and specific inhibitor of fast muscle myosin-II on the development of fatigue in mouse flexor digitorum brevis (FDB) muscle fibres. Single intact FDB fibres were micro-injected with indo-1 to monitor changes in [Ca(2+)](i) and stimulated repeatedly for a maximum of 150 tetani or until force declined to 40%. RESULTS: BTS markedly reduced tetanic force but had no effect on the tetanic [Ca(2+)](i) transients. When fatigue was induced in the presence of BTS, the reduction in [Ca(2+)](i) and force transients occurred much more slowly than in the absence of BTS. The extent of force depression was similar after induction of fatigue in fibres exposed to Tyrode only or to BTS and force recovered to the same extent. CONCLUSION: The results suggest that the decrease in tetanic [Ca(2+)](i) and force caused during fatigue are due mainly to accumulated metabolic changes.

Effects of a myosin-II inhibitor (N-benzyl-p-toluene sulphonamide, BTS) on contractile characteristics of intact fast-twitch mammalian muscle fibres.

We have examined the effects of N-benzyl-p-toluene sulphonamide (BTS), a potent and specific inhibitor of fast muscle myosin-II, using small bundles of intact fibres or single fibres from rat foot muscle. BTS decreased tetanic tension reversibly in a concentration-dependent manner with half-maximal inhibition at approximately approximately 2 microM at 20 degrees C. The inhibition of tension with 10 microM BTS was marked at the three temperatures examined (10, 20 and 30 degrees C), but greatest at 10 degrees C. BTS decreased active muscle stiffness to a lesser extent than tetanic tension indicating that not all of the tension inhibition was due to a reduced number of attached cross-bridges. BTS-induced inhibition of active tension was not accompanied by any change in the free myoplasmic Ca2+ transients. The potency and specificity of BTS make it a very suitable myosin inhibitor for intact mammalian fast muscle and should be a useful tool for the examination of outstanding questions in muscle contraction.

Prolonged force increase following a high-frequency burst is not due to a sustained elevation of [Ca2+]i.

A brief high-frequency burst of action potentials results in a sustained force increase in skeletal muscle. The present study investigates whether this force potentiation is the result of a sustained increase of the free myoplasmic [Ca2+] ([Ca2+]i). Single fibers from mouse flexor brevis muscles were stimulated with three impulses at 150 Hz (triplet) at the start of a 350-ms tetanus or in the middle of a 700-ms tetanus; the stimulation frequency of the rest of the tetanus ranged from 20 to 60 Hz. After the triplet, force was significantly (P < 0.05) increased between 17 and 20% when the triplet was given at the start of the tetanus and between 5 and 18% when the triplet was given in the middle (n = 7). However, during this potentiation, [Ca2+]i was not consistently increased. Hence, the increased force following a high-frequency burst is likely due to changes in the myofibrillar properties.

Neuromuscular junction disassembly and muscle fatigue in mice lacking neurotrophin-4.

Neurotrophin-4 (NT-4) is produced by slow muscle fibers in an activity-dependent manner and promotes growth and remodeling of adult motorneuron innervation. However, both muscle fibers and motor neurons express NT-4 receptors, suggesting bidirectional NT-4 signaling at the neuromuscular junction. Mice lacking NT-4 displayed enlarged and fragmented neuromuscular junctions with disassembled postsynaptic acetylcholine receptor (AChR) clusters, reduced AChR binding, and acetylcholinesterase activity. Electromyographic responses, posttetanic potentiation, and action potential amplitude were also significantly reduced in muscle fibers from NT-4 knock-out mice. Slow-twitch soleus muscles from these mice fatigued twice as rapidly as those from wild-type mice during repeated tetanic stimulation. Thus, muscle-derived NT-4 is required for maintenance of postsynaptic AChR regions, normal muscular electrophysiological responses, and resistance to muscle fatigue. This neurotrophin may therefore be a key component of an activity-dependent feedback mechanism regulating maintenance of neuromuscular connections and muscular performance.

The role of Ca2+ and calmodulin in insulin signalling in mammalian skeletal muscle.

The role of Ca2+ in mediating effects of insulin on skeletal muscle has been widely debated. It is believed that in skeletal muscle Ca2+ has a permissive role, necessary but not of prime importance in mediating the stimulatory actions of insulin. In this review, we present evidence that insulin causes a localized increase in the concentration of Ca2+. Specifically, insulin induces a rise in near-membrane Ca2+ but not the bulk Ca2+ in the myoplasm. The rise in near-membrane Ca2+ is because of an influx through channels that can be blocked by L-type Ca2+ channel inhibitors. Calcium appears to exert some of its subsequent effects via calmodulin-dependent processes as calmodulin inhibitors block the translocation of glucose transporters and other enzymes as well as the insulin-stimulated increase in glucose transport.

Contraction and intracellular Ca(2+) handling in isolated skeletal muscle of rats with congestive heart failure.

A decreased exercise tolerance is a common symptom in patients with congestive heart failure (CHF). This decrease has been suggested to be partly due to altered skeletal muscle function. Therefore, we have studied contractile function and cytoplasmic free Ca(2+) concentration ([Ca(2+)](i), measured with the fluorescent dye indo 1) in isolated muscles from rats in which CHF was induced by ligation of the left coronary artery. The results show no major changes of the contractile function and [Ca(2+)](i) handling in unfatigued intact fast-twitch fibers isolated from flexor digitorum brevis muscles of CHF rats, but these fibers were markedly more susceptible to damage during microdissection. Furthermore, CHF fibers displayed a marked increase of baseline [Ca(2+)](i) during fatigue. Isolated slow-twitch soleus muscles of CHF rats displayed slower twitch contraction and tetanic relaxation than did muscles from sham-operated rats; the slowing of relaxation became more pronounced during fatigue in CHF muscles. Immunoblot analyses of sarcoplasmic reticulum proteins and sarcolemma Na(+),K(+)-ATPase showed no difference in flexor digitorum brevis muscles of sham-operated versus CHF rats. In conclusion, functional impairments can be observed in limb muscle isolated from rats with CHF. These impairments seem to mainly involve structures surrounding the muscle cells and sarcoplasmic reticulum Ca(2+) pumps, the dysfunction of which becomes obvious during fatigue.

Melphalan and purine analog-containing preparative regimens: reduced-intensity conditioning for patients with hematologic malignancies undergoing allogeneic progenitor cell transplantation.

A reduced-intensity preparative regimen consisting of melphalan and a purine analog was evaluated for allogeneic transplantation in 86 patients who had a variety of hematologic malignancies and were considered poor candidates for conventional myeloablative therapies because of age or comorbidity. Seventy-eight patients received fludarabine 25 mg/m(2) daily for 5 days in combination with melphalan 180 mg/m(2) (n = 66) or 140 mg/m(2) (n = 12). Eight patients received cladribine 12 mg/m(2) continuous infusion for 5 days with melphalan 180 mg/m(2). The median age was 52 years (range, 22-70 years). Disease status at transplantation was either first remission or first chronic phase in 7 patients, untreated first relapse or subsequent remission in 16 patients, and refractory leukemia or transformed chronic myelogenous leukemia in 63 patients. Nonrelapse mortality rates on day 100 were 37.4% for the fludarabine/melphalan combination and 87.5% for the cladribine/melphalan combination. The median percentage of donor cells at 1 month in 75 patients was 100% (range, 0%-100%). The probability of grade 2-4 and 3-4 acute graft-versus-host disease was 0.49 (95% CI, 0.38-0.60) and 0.29 (95% CI, 0.18-0.41), respectively. Disease-free survival at 1 year was 57% for patients in first remission or chronic phase and 49% for patients with untreated first relapse or in a second or later remission. On multivariate analysis the strongest predictor for disease-free survival was a good or intermediate risk category. In summary, fludarabine/melphalan combinations are feasible in older patients with associated comorbidities, and long-term disease control can be achieved with reduced-intensity conditioning in this population.

Changes in mitochondrial Ca2+ detected with Rhod-2 in single frog and mouse skeletal muscle fibres during and after repeated tetanic contractions.

The present study investigated mitochondrial Ca2+ uptake and release in intact living skeletal muscle fibres subjected to bouts of repetitive activity. Confocal microscopy was used in conjunction with the Ca2+-sensitive dye Rhod-2 to monitor changes in mitochondrial Ca2+ in single Xenopus or mouse muscle fibres. A marked increase in the mitochondrial Ca2+ occurred in Xenopus fibres after 10 tetani applied at 4 s intervals. The mitochondrial Ca2+ continued to increase with increasing number of tetani. After the end of tetanic stimulation, mitochondrial Ca2+ declined to 50% of the maximal increase within 10 min and thereafter took up to 60 min to return to its original value. Depolarization of the mitochondria with FCCP greatly attenuated the rise in the mitochondrial Ca2+ evoked by repetitive tetanic stimulation. In addition, FCCP slowed the rate of decay of the tetanic Ca2+ transient which in turn led to an elevation of resting cytosolic Ca2+. Accumulation of Ca2+ in the mitochondria was accompanied by a modest mitochondrial depolarization. In contrast to the situation in Xenopus fibres, mitochondria in mouse toe muscle fibres did not show any change in the mitochondrial Ca2+ during repetitive stimulation and FCCP had no effect on the rate of decay of the tetanic Ca2+ transient. It is concluded that in Xenopus fibres, mitochondria play a role in the regulation of cytosolic Ca2+ and contribute to the relaxation of tetanic Ca2+ transients. In contrast to their important role in Xenopus fibres, mitochondria in mouse fast-twitch skeletal fibres play little role in Ca2+ homeostasis.

Functional significance of Ca2+ in long-lasting fatigue of skeletal muscle.

Repeated activation of skeletal muscle causes fatigue, which involves a reduced ability to produce force and slowed contraction regarding both the speed of shortening and relaxation. One important component in skeletal muscle fatigue is a reduced sarcoplasmic reticulum (SR) Ca2+ release. In the present review we will describe different types of fatigue-induced inhibition of SR Ca2+ release. We will focus on a type of long-lasting failure of SR Ca2+ release which is called low-frequency fatigue, because this type of fatigue may be involved in the muscle dysfunction and chronic pain experienced by computer workers. Paradoxically it appears that the Ca2+ released from the SR, which is required for contraction, may actually be responsible for the failure of SR Ca2+ release during low-frequency fatigue. We will also discuss the relationship between gross morphological changes in muscle fibres and long-lasting failure of SR Ca2+ release. Finally, a model linking muscle cell dysfunction and muscle pain is proposed.

Vacuole formation in fatigued skeletal muscle fibres from frog and mouse: effects of extracellular lactate.

Isolated, living muscle fibres from either Xenopus or mouse were observed in a confocal microscope and t-tubules were visualized with sulforhodamine B. Observations were made before and after fatiguing stimulation. In addition, experiments were performed on fibres observed in an ordinary light microscope with dark-field illumination. In Xenopus fibres, recovering after fatigue, t-tubules started to show dilatations 2-5 min post-fatigue. These swellings increased in size over the next 10-20 min to form vacuoles. After 2-3 h of recovery the appearance of the fibres was again normal and force production, which had been markedly depressed 10-40 min post-fatigue, was close to control. Vacuoles were not observed in mouse fibres, fatigued with the same protocol and allowed to recover. In Xenopus fibres, fatigued in normal Ringer solution and allowed to recover in Ringer solution with 30-50 mM L-lactate substituting for chloride (lactate-Ringer), the number and size of vacuoles were markedly reduced. Also, force recovery was significantly faster. Replacement of chloride by methyl sulphate or glucuronate had no effect on vacuolation. Resting Xenopus fibres exposed to 50 mM lactate-Ringer and transferred to normal Ringer solution displayed vacuoles within 5-10 min, but to a smaller extent than after fatigue. Vacuolation was not associated with marked force reduction. Mouse fibres, fatigued in 50 mM lactate-Tyrode (L-lactate substituting for chloride in Tyrode solution) and recovering in normal Tyrode solution, displayed vacuoles for a limited period post-fatigue. Vacuolation had no effect on force production. The results are consistent with the view that lactate, formed during fatigue, is transported into the t-tubules where it attracts water and causes t-tubule swelling and vacuolation. This vacuolation may be counteracted in vivo due to a gradual extracellular accumulation of lactate during fatigue.

Granisetron (Kytril) plus dexamethasone for antiemetic control in bone marrow transplant patients receiving highly emetogenic chemotherapy with or without total body irradiation.

This prospective trial evaluated the efficacy and toxicity of granisetron for antiemetic control in patients receiving high-dose cyclophosphamide (CY)-containing regimens with/without TBI for bone marrow (BM) or peripheral blood stem cell (PBSC) transplantation or PBSC mobilization. Granisetron 1 mg i.v. plus dexamethasone 10 mg i. v. were administered daily 30 min before chemotherapy or radiation for a median of 5 days. Response was defined as the number of emetic episodes per 24 h: complete response, 0 and no emetic rescue; major response, 1-2; minor response, 3-5; failure, >5. One hundred patients were enrolled. Ninety-eight received CY-containing regimens and 26 of these additionally received TBI (12 Gy divided over 4 days). Response was complete on 216 (47%) of a total 456 patient days, major on 222 (49%), minor on 14 (3%), and failure on 4 (1%). Mean number of emetic episodes per patient per day and breakthrough medication required per patient per day was 0.24 (range 0-8) and 0. 40 (range 0-8), respectively. Adverse effects were minimal, with headache (20%) reported most frequently. Based on these results, granisetron plus dexamethasone is an effective and well-tolerated antiemetic regimen in BMT/PBSCT recipients conditioned with high-dose chemotherapy with/without TBI. Bone Marrow Transplantation (2000) 25, 1279-1283.

Frog skeletal muscle fibers recovering from fatigue have reduced charge movement.

Following prolonged exercise, muscle force production is often impaired. One possible cause of this force deficit is impaired intracellular activation. We have used single skeletal muscle fibers from the lumbrical muscle of Xenopus laevis to study the effects of fatigue on excitation-contraction coupling. Fatigue was induced in 13 intact fibers. Five fibers recovered in normal Ringer only (fatigued-only fibers). The remaining eight fibers were subjected to a brief hypotonic treatment (F-H fibers) that is known to prolong the effects of fatigue. Intramembrane charge movement, changes in intracellular calcium concentration ([Ca2+]i) and force transients were measured in a single Vaseline gap chamber under voltage clamp. In F-H fibers, membrane capacitance was reduced. Confocal microscopy showed that this was not due to closure of the transverse tubules. The amount of normalized intramembrane charge was reduced from 21.0 +/- 2.8 nC/microF (n = 10) in rested fibers to 12.2 +/- 1.1 nC/microF in F-H fibers. However, the voltage dependence of intramembrane charge movement was unchanged. In F-H fibers, force production was virtually abolished. This was the consequence of the greatly reduced [Ca2+]i accompanying a depolarizing pulse. In recovering fatigued-only fibers, while the maximal available charge was not significantly smaller (18.3 +/- 1.1 nC/ microF), both calcium and force were reduced, albeit to a lesser extent than in F-H fibers. The data are consistent with a model where fatigue reduces the number of voltage sensors in the t-tubules and, in addition, alters the coupling between the remaining functional voltage sensors and the calcium channels of the sarcoplasmic reticulum.

Dimethylsulfoniopropionate and methanethiol are important precursors of methionine and protein-sulfur in marine bacterioplankton.

Organic sulfur compounds are present in all aquatic systems, but their use as sources of sulfur for bacteria is generally not considered important because of the high sulfate concentrations in natural waters. This study investigated whether dimethylsulfoniopropionate (DMSP), an algal osmolyte that is abundant and rapidly cycled in seawater, is used as a source of sulfur by bacterioplankton. Natural populations of bacterioplankton from subtropical and temperate marine waters rapidly incorporated 15 to 40% of the sulfur from tracer-level additions of [(35)S]DMSP into a macromolecule fraction. Tests with proteinase K and chloramphenicol showed that the sulfur from DMSP was incorporated into proteins, and analysis of protein hydrolysis products by high-pressure liquid chromatography showed that methionine was the major labeled amino acid produced from [(35)S]DMSP. Bacterial strains isolated from coastal seawater and belonging to the alpha-subdivision of the division Proteobacteria incorporated DMSP sulfur into protein only if they were capable of degrading DMSP to methanethiol (MeSH), whereas MeSH was rapidly incorporated into macromolecules by all tested strains and by natural bacterioplankton. These findings indicate that the demethylation/demethiolation pathway of DMSP degradation is important for sulfur assimilation and that MeSH is a key intermediate in the pathway leading to protein sulfur. Incorporation of sulfur from DMSP and MeSH by natural populations was inhibited by nanomolar levels of other reduced sulfur compounds including sulfide, methionine, homocysteine, cysteine, and cystathionine. In addition, propargylglycine and vinylglycine were potent inhibitors of incorporation of sulfur from DMSP and MeSH, suggesting involvement of the enzyme cystathionine gamma-synthetase in sulfur assimilation by natural populations. Experiments with [methyl-(3)H]MeSH and [(35)S]MeSH showed that the entire methiol group of MeSH was efficiently incorporated into methionine, a reaction consistent with activity of cystathionine gamma-synthetase. Field data from the Gulf of Mexico indicated that natural turnover of DMSP supplied a major fraction of the sulfur required for bacterial growth in surface waters. Our study highlights a remarkable adaptation by marine bacteria: they exploit nanomolar levels of reduced sulfur in apparent preference to sulfate, which is present at 10(6)- to 10(7)-fold higher concentrations.

Toxicity of single daily dose gentamicin in stem cell transplantation.

To determine the safety of single daily dose (SDD) gentamicin in recipients of stem cell transplantation (SCT), we evaluated all adult patients at MD Anderson Cancer Center who received SDD gentamicin for treatment of febrile neutropenia. Thirty-three patients received gentamicin 5 mg/kg i.v. every 24 h. Mean duration of therapy was 7 days (range 3-32 days). All patients received vancomycin and 17 received cisplatinum. All patients had normal renal function prior to therapy. Serum gentamicin levels were monitored only when renal function deteriorated. The incidence of nephrotoxicity and clinically significant ototoxicity was 3% and 12%, respectively. All four patients who developed ototoxicity had normal renal function before and during therapy. The mean duration of gentamicin therapy was significantly longer in patients who developed ototoxicity, 20 days vs 9 days (P = 0.001). Patients treated with SDD gentamicin for >10 days were more likely to develop ototoxicity (P = 0.045). Single daily dosing of gentamicin was associated with clinically significant ototoxicity in 12% of our patients. A larger randomized EORTC trial evaluating SDD vs MDD amikacin failed to detect a difference in ototoxicity. However, the median duration of therapy was only 8 days. The increased incidence of ototoxicity in our study may be due to prolonged therapy, type of aminoglycoside used, concomitant ototoxic agents, small sample size, or a combination of the above.

Vacuole formation in fatigued single muscle fibres from frog and mouse.

Force recovery from fatigue in skeletal muscle may be very slow. Gross morphological changes with vacuole formation in muscle cells during the recovery period have been reported and it has been suggested that this is the cause of the delayed force recovery. To study this we have used confocal microscopy of isolated, living muscle fibres from Xenopus and mouse to visualise transverse tubules (t-tubules) and mitochondria and to relate possible fatigue-induced morphological changes in these to force depression. T-tubules were stained with either RH414 or sulforhodamine B and mitochondrial staining was with either rhodamine 123 or DiOC6(3). Fatigue was produced by repeated, short tetanic contractions. Xenopus fibres displayed a marked vacuolation which started to develop about 2 min after fatiguing stimulation, reached a maximum after about 30 min, and then receded in about 2 h. Vacuoles were never seen during fatiguing stimulation. The vacuoles developed from localised swellings of t-tubules and were mostly located in rows of mitochondria. Mitochondrial staining, however, showed no obvious alterations of mitochondrial structure. There was no clear correlation between the presence of vacuoles and force depression; for instance, some fibres showed massive vacuole formation at a time when force had recovered almost fully. Vacuole formation was not reduced by cyclosporin A, which inhibits opening of the non-specific pore in the mitochondrial inner membrane. In mouse fibres there was no vacuole formation or obvious changes in mitochondrial structure after fatigue, but still these fibres showed a marked force depression at low stimulation frequencies ('low-frequency fatigue'). Vacuoles could be produced in mouse fibres by glycerol treatment and these vacuoles were not associated with any force decline. In conclusion, vacuoles originating from the t-tubular system develop after fatigue in Xenopus but not in mouse fibres. These vacuoles are not the cause of the delayed force recovery after fatigue.

Antiemetic efficacy of granisetron plus dexamethasone in bone marrow transplant patients receiving chemotherapy and total body irradiation.

Few trials exist regarding the antiemetic efficacy of granisetron in bone marrow transplant (BMT) recipients conditioned with high-dose chemotherapy and total body irradiation (TBI). In this single-center, open-label, prospective, trial, the antiemetic efficacy and safety of granisetron plus dexamethasone were evaluated in 26 patients conditioned with cyclophosphamide-containing regimens (the majority receiving 60 mg/kg per day on 2 consecutive days), and TBI (12 Gy divided over 4 days). Daily intravenous doses of granisetron 1 mg plus dexamethasone 10 mg were given 30 min prior to chemotherapy or radiation, and continued for 24 h after the last conditioning treatment for a median of 6 days (range 3-9). Emetic control was defined by the number of emetic episodes occurring within a 24 h period, or the requirement for rescue medication for nausea or vomiting. A total of 25 patients completed 186 evaluable treatment days. Response (emetic control by treatment days) was complete in 50% of patients, major in 48%, minor in 2%, and there were no failures. Adverse effects were minor, with diarrhea (15%), headache (14%), and constipation (11%) reported most often. Based on these results, the antiemetic regimen of granisetron plus dexamethasone appears effective and well tolerated during BMT conditioning with high-dose cyclophosphamide and TBI.

Insulin increases near-membrane but not global Ca2+ in isolated skeletal muscle.

It has long been debated whether changes in Ca2+ are involved in insulin-stimulated glucose uptake in skeletal muscle. We have now investigated the effect of insulin on the global free myoplasmic Ca2+ concentration and the near-membrane free Ca2+ concentration ([Ca2+]mem) in intact, single skeletal muscle fibers from mice by using fluorescent Ca2+ indicators. Insulin has no effect on the global free myoplasmic Ca2+ concentration. However, insulin increases [Ca2+]mem by approximately 70% and the half-maximal increase in [Ca2+]mem occurs at an insulin concentration of 110 microunits per ml. The increase in [Ca2+]mem by insulin persists when sarcoplasmic reticulum Ca2+ release is inhibited but is lost by perfusing the fiber with a low Ca2+ medium or by addition of L-type Ca2+ channel inhibitors. Thus, insulin appears to stimulate Ca2+ entry into muscle cells via L-type Ca2+ channels. Wortmannin, which inhibits insulin-mediated activation of glucose transport in isolated skeletal muscle, also inhibits the insulin-mediated increase in [Ca2+]mem. These data demonstrate a new facet of insulin signaling and indicate that insulin-mediated increases in [Ca2+]mem in skeletal muscle may underlie important actions of the hormone.