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ATP released from damaged cells or by controlled secretion could be an important factor in the formation or remodeling of bone. In a variety of other tissues ATP has been shown to control cellular processes by acting on P2-purinoceptors and activating the calcium signaling pathway. Here we demonstrate for the first time that extracellular ATP increases the intracellular free calcium [Ca2+]i concentration in normal human osteoblasts and in SaOS-2 cells, a human osteosarcoma-derived cell line, but not in ROS 17/2.8 cells. The ATP-induced increase in [Ca2+]i was dose dependent, and the concentrations of ATP required were similar to those reported to regulate cellular functions in other cell types. Although ATP is metabolized rapidly by bone cells, the effects on [Ca2+]i appeared to be mediated directly by ATP rather than one of its metabolites. Adenosine 3-thiotriphosphate, a nonhydrolyzable analog of ATP, induced similar changes in [Ca2+]i. This indicates that P2-purinoceptors are present on osteoblast-like cells and that extracellular ATP from various sources might be an important factor in the regulation of osteoblast functions.

Summary A prospective study of 128 adult cardiac surgical patients was undertaken in order to quantify net magnesium loss and its relationship to serum magnesium levels and postoperative problems, particularly arrhythmias. Peri-operative magnesium flux on the first day was calculated from the administered magnesium (in cardioplegia solution and intravenous infusion) and urinary magnesium loss. Magnesium input ranged from 24 mmol to 40 mmol, resulting in a net magnesium gain in 94% of patients. Hypomagnesaemia, identified in 34% of patients pre-operatively and 30% of patients postoperatively, had no significant correlation with the measured peri-operative magnesium flux or the electrocardiograph corrected-QT interval. Fifty-three patients developed postoperative arrhythmias, but there was no significant correlation with the serum total magnesium concentration, or with the peri-operative change in serum magnesium level, magnesium flux, or QT interval. The data suggest that serum total magnesium is not a useful measurement upon which to base preventive or therapeutic measures in cardiac surgical patients.

Chronic electrical stimulation of skeletal muscle at 10 Hz induces fast-to-slow fiber type transformation. Does a lower aggregate amount of activity lead to a less complete transformation, or does it produce the same transformation over a longer time course? We examined this question by subjecting adult rabbit tibialis anterior and extensor digitorum longus muscles to continuous stimulation at 2.5 Hz for 2-12 wk. Most of the fibers acquired the histochemical and immunocytochemical characteristics of type 2A, not type 1, fibers. There was a corresponding rise in oxidative activity, but this was accompanied by a marked decline in anaerobic glycolysis. The activities of hexokinase and 3-oxoacid CoA-transferase stopped increasing after 2 wk, glutamate oxaloacetate transaminase after 4 wk, and beta-hydroxyacyl-CoA dehydrogenase after 6 wk of stimulation. Succinate dehydrogenase, citrate synthase, lactate dehydrogenase, and creatine phosphokinase continued to change up to 12 wk of stimulation. Changes in enzyme activity were not as rapid or as marked as those observed for stimulation at 10 Hz, and none showed the typical two-phase response of oxidative enzyme activities to stimulation at 10 Hz. The latter may therefore be dependent on induction of type 1 myosin isoforms.

We studied changes in the mechanical properties and myosin isoform composition of rabbit tibialis anterior muscles that were subjected to continuous stimulation at 2.5 Hz for up to 12 wk. The effects of stimulation at 2.5 Hz were less profound than those observed for the same duration of stimulation at 10 Hz (12). Stimulation at 10 Hz for 12 wk induced complete transformation to a slow-contracting muscle homogeneous in slow myosin isoforms; stimulation for the same period at 2.5 Hz resulted in moderate changes in contractile speed and a very small increase in the synthesis of slow myosin isoforms. On the other hand, the fatigue resistance of muscles stimulated at 2.5 Hz was as great, in both isometric and dynamic fatigue tests, as that of the muscles stimulated at 10 Hz. Thus entire fast skeletal muscles can be transformed to a state in which fast myosin isoforms continue to be synthesized, but the oxidative capacity is sufficient to support sustained working at a higher power output than that associated with slow muscle.

Fast muscles of the rat hind limb were stimulated continuously at 10 or 20 Hz for periods of 55–61 days by means of an implantable neuromuscular stimulator. Gel electrophoresis clearly demonstrated the presence in stimulated muscles of slow myosin light and heavy chains, although fast isoforms were still present in all cases. Thus, contrary to previous reports, induction of slow myosin isoforms does occur in this, as in other, mammalian species. The time course of the response to stimulation appears to be more extended than that seen in the rabbit.

It is well recognized that skeletal muscle is incapable of maintaining the continuous power output needed for significant circulatory assistance unless it is rendered fatigue resistant by electrical stimulation. Most studies using conditioning patterns related to the pumping rate of the natural heart have suggested that such patterns can produce complete transformation to a slow phenotype. Such a transformation has two undesirable accompaniments: a reduction in the contractile speed of the muscle and an extreme reduction in its power-generating capacity. We have investigated the chronic effect on rabbit muscle of intermittent high frequency contractions either alone or overlaid on a continuous background of 10 Hz. If the amount of stimulation were the overriding stimulus for transformation, then the rate and/or extent of transformation should, if anything, be greater with the additional activation. If, on the other hand, components of the transformation could be modulated independently by the pattern of impulses, then the reduction in power output might be avoided to some extent by a pattern somewhat analogous to a strength-training exercise regime. Our results suggest that high force contractions may indeed preserve power output without compromising the acquisition of fatigue resistance.

The beta subunits of voltage-dependent calcium channels, exert marked regulatory effects on the biophysical and pharmacological properties of this diverse group of ion channels. However, little is known about the comparative neuronal expression of the four classes of beta genes in the CNS. In the current investigation we have closely mapped the distribution of beta1, beta2, beta3 and beta4 subunits in the human cerebellum by both in situ messenger RNA hybridization and protein immunohistochemistry. To our knowledge, these studies represent the first experiments in any species in which the detailed localization of each beta protein has been comparatively mapped in a neuroanatomically-based investigation. The data indicate that all four classes of beta subunits are found in the cerebellum and suggest that in certain neuronal populations they may each be expressed within the same cell. Novel immunohistochemical results further exemplify that the beta voltage-dependent calcium channel subunits are regionally distributed in a highly specific manner and studies of Purkinje cells indicate that this may occur at the subcellular level. Preliminary indication of the subunit composition of certain native voltage-dependent calcium channels is suggested by the observation that the distribution of the beta3 subunit in the cerebellar cortex is identical to that of alpha(1E). Our cumulative data are consistent with the emerging view that different native alpha1/beta subunit associations occur in the CNS.

Rabbit tibialis anterior muscles were stimulated continuously at 10 Hz for periods ranging from 2 min to 96 h and were analyzed for energy reserves and metabolic intermediates. Glycogen, ATP and phosphocreatine fell rapidly during the first 5 min of stimulation. Glycogen continued to fall to very low levels, whereas ATP and phosphocreatine rose, reaching 70% of control by 1 h, despite ongoing stimulation. After 2 h, glycogen also increased, regaining control levels in 4 days. Glucose rose to 4.5 times control in 30 min and still exceeded 2.5 times control at 24 h. In the first 2 min, glycolytic intermediates, glucose 6-phosphate (G-6-P), fructose 1,6-bisphosphate, lactate, and pyruvate more than doubled and then returned to control levels or below. Malate and 3-glycerophosphate rose 600 and 200%, respectively. Both of these compounds participate in shuttling reducing equivalents from cytoplasm into mitochondria. Citrate and alpha-ketoglutarate underwent much more modest changes. Glucose 1,6-bisphosphate (G-1,6-P2) fell to one-third of control by 2 h and then rose dramatically at 4 h. At 4 days it was still twice control. The 6-phosphogluconate (6PG) doubled at 2 min, then rose to 12 times control at 2 h, fell somewhat, and peaked at 16 times control at 24 h. Aspartate and alanine both exhibited a biphasic rise in concentration, whereas glutamate fell to 30% in 15 min and rose slowly after 4 h. The rise in glucose was interpreted to be the consequence of rapid glycogenolysis together with inhibition of hexokinase by G-1,6-P2 and elevated G-6-P. Paradoxically, glycogen resynthesis apparently occurred when the glycogen synthase stimulator, G-6-P, was very low, and the glycolysis stimulator, G-1,6-P2, was high. Although G-1,6-P2 is an inhibitor of 6PG dehydrogenase, the timing of the changes in G-1,6-P2 and 6PG levels suggests that the accumulation of 6PG was initiated by some other influence.

A prospective study of 128 adult cardiac surgical patients was undertaken in order to quantify net magnesium loss and its relationship to serum magnesium levels and postoperative problems, particularly arrhythmias. Peri-operative magnesium flux on the first day was calculated from the administered magnesium (in cardioplegia solution and intravenous infusion) and urinary magnesium loss. Magnesium input ranged from 24 mmol to 40 mmol, resulting in a net magnesium gain in 94% of patients. Hypomagnesaemia, identified in 34% of patients pre-operatively and 30% of patients postoperatively, had no significant correlation with the measured peri-operative magnesium flux or the electrocardiograph corrected-QT interval. Fifty-three patients developed postoperative arrhythmias, but there was no significant correlation with the serum total magnesium concentration, or with the peri-operative change in serum magnesium level, magnesium flux, or QT interval. The data suggest that serum total magnesium is not a useful measurement upon which to base preventive or therapeutic measures in cardiac surgical patients.

In response to patterns of chronic electrical stimulation that increase its overall level of use, mammalian skeletal muscle becomes highly resistant to fatigue. The metabolic basis for this adaptation is well documented in the rabbit, but up to now it has not been possible to identify analogous changes in the dog. In this study, canine latissimus dorsi muscles were stimulated in situ for 2, 6 and 12 mo. Marked increases in fatigue resistance were consistently demonstrated. Citrate synthase and succinic dehydrogenase, conventionally used as markers of oxidative metabolism, did not increase in activity, but enzymes involved in major pathways supplying substrates to the tricarboxylic acid cycle increased up to threefold. Stimulation elevated the volume fraction of mitochondria 1.5-fold and that of lipid droplets 4.5-fold. After 6 mo of stimulation, mean fiber diameter had decreased by 30% and the area occupied by nonmuscle tissue had increased by 11%; these changes showed no further progression at 12 mo. Thus stimulated muscle becomes stably adapted to an increase in use, but the metabolic strategies for achieving increased fatigue resistance vary between species.

Immunogold labelling was used to study the distribution of newly synthesized slow muscle myosin (SM) at the ultrastructural level as it replaced fast muscle myosin (FM) in rabbit muscles undergoing stimulation-induced type transformation. Control fast muscle was labelled strongly with antibody to FM and control slow muscle with antibody to SM; label was confined to the A-band. Well-defined differences in the distribution of label within the A-band suggested that the monoclonal antibodies used corresponded to epitopes on different parts of the myosin molecule; this was confirmed by Western blots of subfragments prepared from FM and SM. After 4 weeks of continuous stimulation at 10 Hz, fibres of the tibialis anterior muscle reacted with antibodies to both isoforms; after 6 weeks, labelling was obtained only with antibody to SM. After a 7-week period of stimulation and 3 further weeks of recovery, fibres again reacted with both antibodies. In all positively-labelled sections, the distribution of gold particles was characteristic of the antibody and independent of the origin or history of the fibres. This observation supports the conclusion that newly synthesized myosin is capable of being incorporated throughout the length and cross-section of the A-band.

Treatment of ‘Dewar hexafluorobenzene’(DHFB, hexafluorobicyclo[2,2,0]hexa-2,5-diene) with the anions [Re(CO)5]– or [(π-C5H5)Fe(CO)2]– leads mainly to a replacement of a vinylic fluorine by the anionic group with retention of the bicyclo[2,2,0]-structure. The reactionof DHFB with enneacarbonyldi-iron is complex; the two compounds (DHFB)Fe(CO)4 and (DHFB)[Fe(CO)4]2 have been characterised. Tetrakis(triphenylphosphine)platinum, acetylacetonatobis(ethylene)rhodium, or chlorocarbonylbis(diphenylmethylphosphine)iridium react with DHFB to give the complexes (Ph3P)2Pt(DHFB), (C2H4) acac Rh(DHFB), and (Ph2PCH3)2(CO)ClIr(DHFB) in all of which the Dewar structure is retained. Reaction of the platinum derivative with iodine regenerates DHFB. Hydridopentacarbonylrhenium adds rapidly to both double bonds of DHFB to give C6H2F6[Re(CO)5]2. The reaction of pentacarbonyltrimethyltinmanganese with DHFB is complex; decafluorodiphenyl and C6F6HMn(CO)5 have been identified as products.

Seventy-three patients with diabetic neuropathy have been followed for periods ranging from two to four and three-quarter years. Symptoms of neuropathy tended to improve though some patients thought that numbness became more severe. Of the signs of neuropathy, weakness and hyperalgesia or hyperesthesia usually improved, impaired pain sensation deteriorated twice as often as it improved, and ankle jerks, vibration sense at the ankles, and position sense in the big toes usually remained the same. Impairment of these last three signs occurred as frequently in a random group of diabetic patients as in the present selected series and was therefore not a feature of symptomatic neuropathy.

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Thirteen diabetic patients with chronic diarrhea have been studied clinically and subjected to jejunal biopsy. Eleven conformed to the accepted clinical picture of “diabetic diarrhea,” all showing evidence of gross neuropathy and a normal jejunal mucosa was found in every case. The other two were found to have a flat jejunal biopsy; in one celiac disease had been diagnosed at the age of two, but in the other there was no remote history of gastrointestinal disorder. The diagnosis of “diabetic diarrhea” is one of exclusion and there is no single feature which can be regarded as diagnostic. The nature of the diarrhea is uncertain but the response in some patients to antibiotics suggests an abnormal colonization of bacteria in the intestine though this cannot be demonstrated. The association of diarrhea with diabetic neuropathy is strong.

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The compounds trans-(Ph3P)2Ir(CO)Cl and trans-(Ph2PMe)2Ir(CO)Cl rapidly undergo oxidative-addition reactions with trifluoroacetyl cyanide affording the crystalline complexes L2Ir(CO)Cl[CF3COCN](L = Ph3P or Ph2PMe). The related rhodium complex (Ph2PMe)2Rh(CO)Cl[CF3COCN] is obtained from trans-(Ph2PMe)2Rh(CO)Cl. Spectroscopic studies suggest that the complexes are best depicted as trifluoroacetyl cyanide adducts of iridium(III) and rhodium(III) in which both the carbon and oxygen atoms of the carbonyl group of the ligand are bonded to the metal atoms. Moreover the phosphine ligands have a trans-configuration in the iridium adducts, whereas they have a relative cis-configuration in the rhodium complex.

Treatment of hexafluorobuta-1,3-diene with the anions [π-C5H5Fe(CO)2]– and [Re(CO)5]– affords the perfluorobutadienyl complexes cis-CF2:CF·CF:Fe·CF(CO)2(π-C5H5) and trans-CF2:CF·CF:CF·Re(CO)5, respectively. The corresponding pentacarbonylmanganese complex cannot be obtained by this route; however, u.v. irradiation of hexafluorobuta-1,3-diene and Me3Sn·Mn(CO)5 gives trimethyltin fluoride and cis-CF2:CF·CF:CF·Mn(CO)5. In contrast, irradiation of hexafluoro-1,3-butadiene and Me3Ge·Mn(CO)5 gives trans-CF2:CF·CF:CF·Mn(CO)5, and the complex Me3Ge·Fe2·CF2·CF:CF·Mn(CO)5 corresponding to an initial 1,4-addition reaction. The i.r., 19F and 1H n.m.r. spectra of the compounds are described.

Hexafluoroazomethane reacts with trans-chloro-carbonylbis(methyldiphenylphosphine)iridium(I) to yield stable nitrene complexes, cis- and trans-(Ph2PMe)2Ir(CO)(Cl)(NCF3).