Evidence for a specific uptake and retention mechanism for 25-hydroxyvitamin D (25OHD) in skeletal muscle cells.

Little is known about the mechanism for the prolonged residence time of 25-hydroxyvitamin D (25OHD) in blood. Several lines of evidence led us to propose that skeletal muscle could function as the site of an extravascular pool of 25OHD. In vitro studies investigated the capacity of differentiated C2 murine muscle cells to take up and release 25OHD, in comparison with other cell types and the involvement of the membrane protein megalin in these mechanisms. When C2 cells are differentiated into myotubes, the time-dependent uptake of labeled 25OHD is 2-3 times higher than in undifferentiated myoblasts or nonmuscle osteoblastic MG63 cells (P < .001). During in vitro release experiments (after 25OHD uptake), myotubes released only 32% ± 6% stored 25OHD after 4 hours, whereas this figure was 60% ± 2% for osteoblasts (P < .01). Using immunofluorescence, C2 myotubes and primary rat muscle fibers were, for the first time, shown to express megalin and cubilin, endocytotic receptors for the vitamin D binding protein (DBP), which binds nearly all 25OHD in the blood. DBP has a high affinity for actin in skeletal muscle. A time-dependent uptake of Alexafluor-488-labeled DBP into mature muscle cells was observed by confocal microscopy. Incubation of C2 myotubes (for 24 hours) with receptor-associated protein, a megalin inhibitor, led to a 40% decrease in 25OHD uptake (P < .01). These data support the proposal that 25OHD, after uptake into mature muscle cells, is held there by DBP, which has been internalized via membrane megalin and is retained by binding to actin.

Competing carboxylases: circadian and metabolic regulation of Rubisco in C3 and CAM Mesembryanthemum crystallinum L.

The temporal co-ordination of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPc) activities by Mesembryanthemum crystallinum L. in C(3) and crassulacean acid metabolism (CAM) modes was investigated under conventional light-dark (LD) and continuous light (LL) conditions. When C(3) , net CO(2) assimilation rate increased during each subjective night under LL with maximum carboxylation unrelated to Rubisco activation state. The CAM circadian rhythm of CO(2) uptake was more pronounced, with CO(2) assimilation rate maximal towards the end of each subjective night. In vivo and in vitro techniques were integrated to map carboxylase enzyme regulation to the framework provided by CAM LL gas exchange activity. Rubisco was activated in vitro throughout each subjective dark period and consistently deactivated at each subjective dawn, similar to that observed at true dawn in constitutive CAM species. Instantaneous carbon isotope discrimination showed in vivo carboxylase co-dominance during the CAM subjective night, initially by Rubisco and latterly C(4) (PEPc), despite both enzymes seemingly activated in vitro. The circadian rhythm in titratable acidity accumulation was progressively damped over successive subjective nights, but maintenance of PEPc carboxylation capacity ensures that CAM plants do not become progressively more 'C(3) -like' with time under LL.

Do the competition rules of synchronized swimming encourage undesirable levels of hypoxia?

Recent anecdotal reports that some synchronized swimmers have become dizzy or disorientated towards the end of their performance, and in the worst cases fainted underwater, have caused concern. However, the rules of synchronized swimming encourage slow performance of compulsory figures, and an analysis of the competition placings and duration of underwater sequences showed that the highest rankings were gained by slowly performed compulsory figures and free programmes containing a long underwater sequence. The combination of breath-holding and the vigorous exercise involved suggests that some of the symptoms complained of by the swimmers might be due to hypoxia. We therefore studied the alveolar gas tensions in nine members of the Great Britain National Squad immediately following the performance of set figures and the initial underwater sequence of their free routine in a swimming-bath. All were cyanosed after the underwater sequences of the free routine and reported being mildly confused. The mean(s.d.) alveolar PO2 at this stage was 5.07(1.1) KPa, while three girls had an alveolar PO2 below 4 KPa, the lowest being 3.67 KPa. These gas tensions suggest that potentially dangerous levels of hypoxia may develop during competitive synchronized swimming and that prolonged underwent sequences should not be encouraged.

Variations in performance in simple muscle tests at different phases of the menstrual cycle.

The effect of the menstrual cycle on the performance of women in sporting activities is a very confused subject. In this study, performances in simple muscle tests--the handgrip and standing long jump, were studied at three phases of the menstrual cycle--menstrual (day 1-4), follicular (day 12-14) and luteal (day 19-21). Within subject paired "t" testing showed that in the handgrip test, performance was significantly superior during the menstrual phase than those during both the follicular and luteal phases. In the standing long jump test, performance was again superior during the menstrual phase, although not significantly with respect to the luteal phase. This finding is discussed in terms of the reported effects of the menstrual cycle on sporting performance, the variation in the types of exercise, and the possible role of the female sex hormones.

The relationship of lean limb volume to performance in the handgrip and standing long jump tests in boys and girls, aged 11.6-13.2 years.

Twenty-three girls and 19 boys performed the handgrip and standing long jump (SLJ) tests. Their total forearm and leg volumes were calculated from circumference and length measurements and the lean volumes (bone + muscle) were calculated by making allowance for skinfold thickness. Although the boys were older than the girls (12.8 and 12.4 years), there was no significant difference in their heights or body masses. The absolute performances of the boys were superior to those of the girls in both tests (handgrip 234 and 205 N and SLJ 1.53 and 1.34 m), but when jumping performance was expressed as distance x body mass, there was no significant difference. In both tests, performance in terms of unit lean limb volume showed no significant gender difference. When performance was related to lean limb volume, both boys and girls showed a linear relationship in the two tests, with no significant difference between them. This absence of a gender difference contrasts with the results of a previous study on young adults and comparison shows that the relationships between lean limb volume and performance in the two tests for both boys and girls lie just below those of the young, adult females. The difference between the girls and the young adult females was just significant in the handgrip (p less than 0.05), but not significant in the SLJ (p greater than 0.25), whereas the differences between the boys and young adult males were significant (p less than 0.01) in both tests. Thus it would appear that a gender difference in the performance of skeletal muscle develops during adolescence and possible contributory factors are discussed.

Gender difference in the relationship of performance in the handgrip and standing long jump tests to lean limb volume in young adults.

Groups of young, adult males and females performed the handgrip and standing long jump tests. Their total forearm and leg volumes were calculated from a series of circumference and length measurements, and the lean volumes (bone + muscle) calculated by taking the skinfold thickness into consideration. In the handgrip, the mean female performance was 298 N compared with 496 N for the males. In the standing long jump, mean performance expressed as distance x body mass was 87.3 kg.m for females compared with 137.7 kg.m for males. These superior performances of males could simply reflect their greater muscle mass, as the mean lean volumes of female and male limbs respectively were 0.54 l and 0.89 l for forearms, and 11.82 l and 14.82 l for the two legs. However, when the performances of males and females were grouped by lean limb volume, it was found that while in both tests there were linear relationships, males and females did not share a common line. In both tests the male relationship was at a higher level than the female; therefore, for a given lean volume, the male performance was significantly superior to that of the female. The gender difference found in this study has not been seen in other studies in which the performance of skeletal muscle has been related to the cross-sectional area of the active muscles and the possible reasons for the differences are considered.

Erythrocyte uptake by the dog's spleen during splenic venous pressure elevation.

The spleens of chloralose-urethane anaesthetized dogs were isolated, placed in a plethysmograph and perfused at constant arterial pressure from a cannulated femoral artery. Splenic venous pressure (PV) was elevated by between 2.5 and 20 cm H2O: it caused pressure-dependent increases in spleen volume of up to 98 +/- 12 ml/100 g spleen weight. The increase in spleen volume could be separated into an initial phase associated with passive distension of capacitance vessels (Phase-1) followed by a more continuous increase in spleen volume (Phase-2). We used techniques designed to distinguish these components of the volume response to PV elevation, and found Phase-2 rates of increase in spleen volume of 5--10 ml min-1 mm Hg-1 100 g-1 expressed in terms of PV elevation and terminal, drained and trimmed spleen weight. Splenic venous haematocrit determination showed selective sequestration of RBCs during the Phase-2 volume response to PV elevation which was related to the extent of the PV elevation. Both the Phase-2 volume response and the haematocrit changes were reduced by splenic nerve stimulation or i.a. infusions of adrenaline but were unaffected by i.a. infusions of bradykinin.

The differential effect of cooling on the responses of splenic capsular and vascular smooth muscle to nerve stimulation and noradrenaline.

The responses of the capsular and vascular smooth muscle of the dog's spleen to splenic nerve stimulation and to infused noradrenaline have been studied in the isolated, blood-perfused preparation at 37 degrees C, at 27 degrees C and again after rewarming to 37 degrees C. It was found that cooling per se had no effect on perfusion pressure but reduced splenic arterial blood flow, and caused no appreciable alteration in spleen volume. The increase in splenic flow resistance in response to nerve stimulation and noradrenaline was significantly greater at 27 degrees C than at 37 degrees C, but the concomitant reduction in spleen volume was significantly reduced. The enhanced effect of splenic nerve stimulation and noradrenaline on splenic flow resistance is discussed in terms of the relative contributions of an increased smooth muscle response and increased blood viscosity. The different effects of cooling on the responses of splenic vascular and capsular smooth muscle to nerve stimulation and noradrenaline are discussed in the context of the effect of cooling on other vascular and non-vascular smooth muscle.

The responses of the isolated, blood-perfused spleen of the dog to angiotensin, oxytocin and vasopressin.

1 The responses of the smooth muscle of the capsule and blood vessels of the isolated, blood-perfused spleen of the dog to angiotensin, oxytocin and vasopressin have been investigated and compared to the actions of the catecholamines, adrenaline and noradrenaline.2 Increasing doses of each of the three polypeptides cause graded increases in splenic vascular resistance and reductions in spleen volume.3 Doses of the polypeptides which evoked increases in splenic vascular resistance not significantly different from increases produced by chosen doses of each catecholamine caused significantly smaller reductions in spleen volume.4 The time-course of action of the polypeptides on the splenic vascular smooth muscle is different since the time to 50% recovery from vasopressin is highly significantly longer than that for equieffective doses of either angiotensin or oxytocin.5 Phenoxybenzamine, in a dose which almost blocked the actions of the catecholamines, increased the responses of the vascular and capsular smooth muscle to oxytocin, vasopressin and angiotensin. This increase was not observed with another alpha-adrenoceptor blocking agent, phentolamine.6 The significant species variation in the responses of the smooth muscle of the spleen to polypeptides and catecholamines are discussed and the results are considered in the context of the possible physiological roles of the polypeptides in haemorrhage.

Frequency-dependent differences in the responses of the capsular and vascular smooth muscle of the spleen of the dog to sympathetic nerve stimulation.

1. The responses of the capsular and vascular smooth muscle to splenic nerve stimulation have been studied simultaneously in the isolated blood-perfused dog's spleen.2. Low frequencies of splenic nerve stimulation (below 1.0 Hz) caused pronounced contraction of the splenic capsule but little or no constriction of the splenic vascular bed.3. Splenic contraction reached a maximum at stimulation frequencies of 1-2 Hz; maximum vasoconstriction occurred at frequencies of 7-10 Hz.4. The separation of responses of the capsular and vascular smooth muscle was mimicked by close arterial infusions of either adrenaline or noradrenaline.5. The maximum responses of the splenic vascular smooth muscle to nerve stimulation, adrenaline and noradrenaline were not significantly different.6. The maximum reduction in spleen volume to sympathetic nerve stimulation was significantly greater than the maximum response to close arterial noradrenaline.7. The maximum contractions of the spleen to adrenaline and noradrenaline were not significantly different. At concentrations producing submaximal responses adrenaline was more potent than noradrenaline.8. It is suggested that the frequency dependent separation of smooth muscle responses to sympathetic nerve stimulation is due to a different sensitivity of the capsular and vascular smooth muscle to the chemical transmitter noradrenaline.9. The results are discussed in the context of the function of the dog's spleen.

Responses of the isolated, perfused human spleen to sympathetic nerve stimulation, catecholamines and polypeptides.

1. The responses of the smooth muscle of the capsule and blood vessels of the isolated, perfused human spleen to sympathetic nerve stimulation, adrenaline, noradrenaline, angiotensin, oxytocin, vasopressin, isoprenaline and acetylcholine have been investigated and compared with those of dog spleen.2. Stimulation of the postganglionic sympathetic nerves to the human spleen at frequencies of 3-10 Hz evoked graded vasoconstriction but very small changes in spleen volume.3. The injection of adrenaline and noradrenaline in doses of 0.25-25 mug to the human spleen produced graded increases in splenic vascular resistance with very small decreases in spleen volume.4. Administration of the alpha-adrenoceptor blocking drug phenoxybenzamine completely abolished or considerably reduced the vascular responses of the human spleen to sympathetic nerve stimulation or the injection of noradrenaline.5. The vascular action of adrenaline was often reversed to elicit a vasodilatation after phenoxybenzamine suggesting the presence of beta-adrenoceptors in the vascular bed. This was confirmed by the administration of isoprenaline which induced a marked reduction in vascular resistance of the human spleen.6. The polypeptides angiotensin and vasopressin induced a marked vasoconstriction in the human spleen without changes in the spleen volume. These effects were uninfluenced by the administration of phenoxybenzamine.7. The polypeptide oxytocin caused a slight vasodilatation in the human spleen, an effect almost exactly mimicked by the preservative chlorobutanol.8. Preliminary experiments suggest that noradrenaline is the transmitter released by the postganglionic nerves to the human spleen.9. These results provide direct evidence that the normal human spleen, unlike that of the dog, does not have a reservoir function. It is suggested that contractions of the enlarged human spleen may occur in various pathological conditions.

Actions of prostaglandin F2-alpha on the splenic vascular and capsular smooth muscle in the dog.

1. The actions of prostaglandin F(2alpha), which is known to be released by the spleen on sympathetic nerve stimulation, have been investigated in the isolated blood perfused spleen of the dog.2. Low arterial concentrations of F(2alpha) (less than 10 mug/100 ml) caused marked reductions in splenic vascular resistance.3. High arterial concentrations of F(2alpha) (above 10 mug/100 ml) caused increases in splenic vascular resistance.4. Little action on splenic volume was observed by any arterial concentration of F(2alpha).5. The reductions in splenic vascular resistance caused by low arterial concentrations of F(2alpha) were reversed by blocking doses of phenoxybenzamine.6. No appreciable interaction between F(2alpha) and the splenic responses to sympathetic nerve stimulation, adrenaline and noradrenaline was observed.7. The role of the prostaglandins released by the spleen is discussed.