Changes in capillary luminal diameter in rat soleus muscle after hind-limb suspension.

This study examined the time course change of the capillary luminal diameter and the number of capillaries in the rat soleus muscle during hind-limb suspension. Male Wistar rats were divided into 1 and 3 weeks of hind-limb suspension (HS) groups (HS-1 and HS-3). The HS groups were compared with age-matched control groups. All morphometric parameters with respect to capillary and muscle fibre cross-sectional area were determined in perfusion-fixed soleus muscles. After 1 and 3 weeks of hind-limb suspension, the mean muscle fibre cross-sectional area was significantly decreased in HS-1 (-32.0%) and HS-3 (-59.3%) compared with age-matched control groups. Despite a lower capillary-to-fibre ratio (HS-1, -19.3%; HS-3, -21.2%), the capillary density was unchanged in HS-1 and significantly increased in HS-3 compared with age-matched control groups. The mean capillary luminal diameter was significantly smaller in HS-1 (-19.9%) and HS-3 (-21.9%) than in the age-matched control groups. The capillary-to-fibre perimeter ratio which indicates the capillary surface area available for gas exchange between blood and tissue did not significantly differ between control groups and HS groups. In conclusion, the morphometrical adaptations in rat soleus with the suspension involved changes in both the capillary luminal diameter and number of capillaries, and the change in capillary surface area was proportional to the degree of muscle atrophy in HS groups.

Physiological and anatomical organization of multiwhisker response interactions in the barrel cortex of rats.

To understand the physiological properties and anatomical organization of the spatiotemporal interaction of the responses to multiwhisker stimulation in neurons of the rat barrel cortex, single-unit recordings of 114 neurons were performed across all layers (layer II/III, n = 39; IV, n = 33; V/VI, n = 42) of the posteromedial barrel subfield of the primary somatosensory cortex of anesthetized rats. Two neighboring principal and adjacent whiskers (PW and AW, respectively) in the same row were deflected rostrally or caudally at varying interstimulus intervals (ISIs). In 37% of the neurons, the response to the combined stimulus was significantly larger than the sum of the responses to stimulation of the individual whiskers. In instances in which response facilitation was observed, selectivity was noted for the combination (75%) of the PW with a particular AW or for a particular direction (60%) of whisker deflection. The direction bias of the responses to multiwhisker stimulation was well correlated with that of the sum of the responses to single whisker stimulation (r = 0.83; p < 0.001). The pattern and magnitude of the response interaction in the neurons of the superficial layers were closely related to the location of the recorded cell in the barrel columns. Multiwhisker stimulation at short ISIs (

Temporal characteristics of response integration evoked by multiple whisker stimulations in the barrel cortex of rats.

We investigated the responses of 114 cells in the barrel cortex of rats to describe the temporal characteristics of excitatory interactions among neurons serving two vibrissae. To examine these interactions, the principal whisker and one adjacent whisker in the same row were stimulated simultaneously or serially at various interstimulus intervals (ISIs). In 37% of the cells tested, combined stimulation of two whiskers exhibited response facilitation; the response to the combined stimulus was larger than the sum of the responses to stimulation of the individual whiskers. The occurrence and magnitude of the facilitation were strongly dependent on the ISI. The ISI capable of producing facilitation for a particular cell was tuned to a narrow range (mean +/- SD, 5.3 +/- 2.3 msec). The ISI that evoked the maximal facilitation was 1.3 +/- 1.3, 3.4 +/- 2.3, and 2.8 +/- 4.5 msec for neurons in layers II/III, IV, and V/VI, respectively. These ISIs corresponded to the difference in latencies between the responses to the individual stimulations of the principal and adjacent whiskers. A significant response facilitation was observed in the regular-spiking cells but not in the fast-spiking cells. When the ISI was longer than the range that evoked facilitation, a suppression of the response to the second whisker stimulation was observed. Facilitation was observed predominantly in layer II/III cells (69%) and to a lesser extent in cells of layers IV (15%) and V/VI (24%). Our results suggest that, in the barrel cortex, the temporal relationships among tactile stimuli are coded by facilitatory and inhibitory interactions among neurons located in neighboring barrel columns.

Mitogenic action of adenosine on osteoblast-like cells, MC3T3-E1.

The purpose of this study was to investigate the mechanisms by which adenosine stimulates proliferation of osteoblast-like cells, MC3T3-E1. Adenosine by itself induces the stimulation of cell proliferation and accentuates the mitogenecity of PDGFs (AA and BB homodimers) for the cells. 8-Cyclopentyl-1,3-dimethylxanthine (CPX), a nonselective adenosine receptor antagonist, partially inhibited adenosine-induced DNA synthesis in a competitive manner, suggesting that the mitogenic action of adenosine is, at least in part, mediated by xanthine-sensitive receptors. In pertussis-toxin (PTX)-pretreated cells, adenosine- but not PDGF-BB-stimulated DNA synthesis was partially inhibited, and CPX did not exert a further inhibitory effect, suggesting an involvement of PTX-sensitive G-protein downstream of CPX-sensitive receptor. When adenosine uptake was prevented with dipyridamole, the stimulation of proliferation by adenosine was not decreased at all, indicating that the CPX-insensitive part of adenosine action is not associated with the uptake of adenosine and subsequent incorporation into the nucleotide pool. Adenosine did not influence the basal level or the PDGF-BB-induced increase in [Ca2+]i. Since it is known that the cAMP pathway acts in inhibiting osteoblast proliferation, the mitogenic action of adenosine would be dependent on neither the cAMP pathway nor the phospholipase C/Ca2+ pathway. It has been concluded that adenosine exerts a mitogenic effect via two pathways at least, one mediated by xanthine-sensitive receptor and PTX-sensitive G-protein and the other through an unknown xanthine- and PTX-insensitive process.

Effects of different intensity endurance training on the capillary network in rat skeletal muscle.

Effects of low- and high-intensity endurance training on the capillary luminal diameter and number were studied morphometrically in the rat plantaris muscle. Male Wistar-Imamichi rats were divided into three groups: sedentary control group (Cont, n = 9), low-intensity (running speed of 20 m/min) training group (T-20, n = 8) and high-intensity (running speed of 40 m/min) training group (T-40, n = 7). Rats in both training groups were subjected to each treadmill running program for 60 min/day, 5 days/week for 9 weeks. After 9 weeks of training, citrate synthase activity significantly increased in T-40 compared with Cont, but did not change in T-20. All morphometric parameters with respect to capillary and muscle fiber area were determined in the perfusion-fixed plantaris muscle. The mean muscle fiber areas in both T-20 and T-40 were similar to that in Cont. The capillary-to-fiber ratios were significantly higher in T-20 (2.28 +/- 0.06) and T-40 (2.29 +/- 0.06) than in Cont (2.00 +/- 0.07). The number of capillaries with a small luminal diameter (2-4 microns) was significantly higher in T-20 than in Cont. In contrast, T-40 had a significantly higher number of capillaries with a large luminal diameter (8-10 microns) compared with Cont. This study indicates that endurance training induces changes in the capillary luminal diameter as well as capillary number, and that the adaptive response of the capillary luminal diameter to endurance training depends on the training intensity.

Morphological adaptation of capillary network in compensatory hypertrophied rat plantaris muscle.

The aim of this study was to examine the morphological adaptation of the capillary network in hypertrophied plantaris muscles by examining both capillary numbers and luminal circumferences. Hypertrophy of the plantaris muscle was induced by myectomy of the gastrocnemius muscle. This hypertrophy was characterised by increases in muscle mass and fibre cross-sectional area. All capillary parameters were determined using morphometric methods in perfusion-fixed plantaris muscle. Increased capillary-to-fibre ratio was observed in the overloaded plantaris muscle while no change was observed in the capillary luminal circumference. No differences were observed in the capillary density and the capillary-to-fibre perimeter ratio of the normal and the hypertrophied plantaris muscle. These results indicated that chronic overload-induced neocapillarization, but not enlargement of capillary luminal circumference, contributed to the prevention of decreases in the capillary-to-fibre perimeter ratio in the plantaris muscle in the hypertrophied process.

Muscle contractile activity modulates GLUT4 protein content in the absence of insulin.

We examined whether muscle contractile activity directly modulates GLUT4 protein content in rat skeletal muscle without the participation of insulin action or via amplified insulin action. To attain this purpose, the effects of increased, by training, or eliminated, by denervation, muscle contractile activity on muscle GLUT4 protein concentration were investigated in severely insulin-deficient diabetic rats. For the first set of experiments, insulin-deficient diabetic rats (induced by injection of 80 mg/kg B.W. streptozotocin) were trained for three weeks by treadmill running (90 min/day, 19 m/min, 10%, 6 days/week). GLUT4 protein concentration in soleus muscle was increased by 48% (p < 0.01) as compared with diabetic sedentary animals. For the second set of experiments, rats were injected with streptozotocin (100 mg/kg). The muscles innervated by the sciatic nerve of one leg were denervated four days after injection of streptozotocin. Three days after denervation, soleus muscles in both legs were excised. Insulin deficiency decreased GLUT4 protein concentration in innervated soleus muscle. In insulin-deficient diabetic rats, denervation also decreased soleus GLUT4 protein concentration by 50% (p < 0.01) as compared with the contralateral innervated muscle. Furthermore, the effects of insulin-deficiency and denervation on GLUT4 protein concentration were additive. These results provide evidence that muscle contractile activity directly modulates skeletal muscle GLUT4 protein concentration independent of insulin action.

ATP and adenosine act as a mitogen for osteoblast-like cells (MC3T3-E1).

Extracellular ATP, and to a lesser extent adenosine, an ATP metabolite, stimulated cell proliferation in osteoblast-like cells (MC3T3-E1). ATP increased cytosolic Ca2+ due to Ca2+ mobilization from intracellular storage in the same concentration range of the nucleotide as that effective for DNA synthesis, suggesting the mediation of the phospholipase C/Ca2+ system in the mitogenic action. Since adenosine induced no Ca2+ mobilization, P2-purinergic receptor appears to be associated with ATP actions. The growth-promoting effect of ATP was not inhibited by H7, a protein kinase C inhibitor, and indomethacin, a cyclooxygenase inhibitor, indicating no involvement of activation of protein kinase C and production of prostaglandins in ATP-induced mitogenic signals. Either ATP or adenosine remarkably and synergistically potentiated platelet derived growth factor-induced DNA synthesis. These findings suggest that extracellular ATP and adenosine may play a physiological role in the regulation of bone formation.

Creatine kinase release from regenerated muscles after eccentric contractions in rats.

The purpose of this study was to test the hypothesis that an increase in plasma creatine kinase (CK) activity after eccentric contractions (ECC) would be attenuated in regenerated muscle fibres. Adult male Wistar rats (aged 12-14 weeks) were randomly assigned to a treatment group (n = 14) or a control group (n = 10). In the treatment group, 1.2% barium chloride solution (BaCl2) was injected into the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles to induce degeneration and subsequent regeneration. The same amount of isotonic saline solution was injected into TA and EDL for the control group. Histological observation showed that approximately 50% of the fibres in the transverse sections of both muscles underwent necrosis 2 days after BaCl2 injection. The CK activity increased about tenfold at 2-4 h after BaCl2 injection. At 4 weeks after BaCl2 injection, when the regeneration process was almost complete, the TA and EDL of anaesthetized rats from both groups were subjected to ECC in which maximal dorsiflexion was caused by nerve electrical stimulation and the flexed foot was forcibly extended by a lever arm connected to a motor. This action was performed in 2 sets of 30 repetitions. Maximal isometric torque of the dorsiflexors decreased to about 15% (P < 0.01) of the pre-ECC value immediately after the exercise. Blood samples were collected before and 2, 4, 12, 24, 48 h after ECC. The CK activity increased significantly (P < 0.01) and peaked at 2-4 h after ECC, and there was no significant difference in the amount of CK increase between the treatment [1007 (SEM 120) IU.l-1] and the control [1064 (SEM 120) IU.l-1] group. Contrary to the hypothesis, CK release after ECC was not attenuated in muscle regenerated from BaCl2-induced myonecrosis.

Permissive stimulation of Ca(2+)-induced phospholipase A2 by an adenosine receptor agonist in a pertussis toxin-sensitive manner in FRTL-5 thyroid cells: a new 'cross-talk' mechanism in Ca2+ signalling.

We have described the pertussis toxin (PTX)-sensitive potentiation of P2-purinergic agonist-induced phospholipase C activation, Ca2+ mobilization and arachidonic acid release by an adenosine receptor agonist, N6-(L-2-phenylisopropyl)adenosine (PIA), which alone cannot influence any of these cellular activities [Okajima, Sato, Nazarea, Sho and Kondo (1989) J. Biol. Chem. 264, 13029-13037]. In the present study we have found that arachidonic acid release was associated with lysophosphatidylcholine production, and conclude that arachidonic acid is produced by phospholipase A2 in FRTL-5 thyroid cells. This led us to assume that PIA augments P2-purinergic arachidonic acid release by increasing [Ca2+]i which, in turn, activates Ca(2+)-sensitive phospholipase A2. The arachidonic acid-releasing response to PIA was, however, always considerably higher (3.1-fold increase) than the Ca2+ response (1.3-fold increase) to the adenosine derivative. In addition, arachidonic acid release induced by the [Ca2+]i increase caused by thapsigargin, an endoplasmic-reticulum Ca(2+)-ATPase inhibitor, or calcium ionophores was also potentiated by PIA without any effect on [Ca2+]i and phospholipase C activity. This action of PIA was also PTX-sensitive, but not affected by the forskolin- or cholera toxin-induced increase in the cellular cyclic AMP (cAMP), suggesting that a PTX-sensitive G-protein(s) and not cAMP mediates the PIA-induced potentiation of Ca(2+)-generated phospholipase A2 activation. Although acute phorbol ester activation of protein kinase C induced arachidonic acid release, P2-purinergic and alpha 1-adrenergic stimulation of arachidonic acid release was markedly increased by the protein kinase C down-regulation caused by the phorbol ester. This suggests a suppressive role for protein kinase C in the agonist-induced activation of arachidonic acid release. We conclude that PIA (and perhaps any of the G1-activating agonists) augments an agonist (maybe any of the Ca(2+)-mobilizing agents)-induced arachidonic acid release by activation of Ca(2+)-dependent phospholipase A2 in addition to enhancement of agonist-induced phospholipase C followed by an increase in [Ca2+]i.

A single species of A1 adenosine receptor expressed in Chinese hamster ovary cells not only inhibits cAMP accumulation but also stimulates phospholipase C and arachidonate release.

Chinese hamster ovary cells were transfected with both A1 adenosine receptor and muscarinic type 3 acetylcholine receptor cDNAs. The muscarinic receptor agonist carbachol stimulated phospholipase C activity, resulting in Ca2+ mobilization and arachidonate release. N6-Cyclopentyladenosine (CPA), an A1 receptor agonist, did not activate Ca(2+)-related signal transduction systems by itself but instead inhibited cAMP accumulation. In the presence of carbachol, however, the A1 receptor agonist enhanced muscarinic receptor agonist-induced phospholipase C/Ca2+ responses. In addition, the arachidonate release caused by Ca2+ ionophores or thapsigargin was also amplified by CPA, without a change in phospholipase C activity. Thus, CPA augments Ca(2+)-mediated phospholipase A2 activation in addition to and separate from its ability to amplify phospholipase C-mediated Ca2+ mobilization. Because the permissive actions of CPA on phospholipase C and phospholipase A2 activation were each reversed by pertussis toxin treatment, in a manner similar to that of the CPA-induced inhibition of cAMP accumulation, we conclude that a single species of A1 receptor expressed in Chinese hamster ovary cells can couple to multiple signal transduction systems stemming from phospholipase C stimulation, phospholipase A2-mediated and Ca(2+)-dependent arachidonate release, and inhibition of cAMP accumulation. A pertussis toxin-sensitive G protein (or proteins) mediates the permissive actions of the A1 receptor in the stimulation of phospholipase C- and phospholipase A2-mediated arachidonate release.

Physical exercise increases bone mineral density in postmenopausal women.

To examine whether physical exercise is beneficial in preventing postmenopausal osteoporosis, we measured bone mineral density (BMD) in three distinct groups of healthy postmenopausal Japanese women aged 49-61 yrs: 11 volleyball players (V) and 5 joggers (J), and 9 controls (C) who had not been participating in regular physical activity. BMD was measured at the lumbar spine (L2-L4) and proximal femur using dual energy X-ray absorptiometry, and at the radius using single X-ray photon absorptiometry. Serum levels of estradiol (E2), parathyroid hormone (PTH) and calcitonin were also measured by radioimmunoassay. Osteocalcin was determined by enzyme immunoassay. BMD in the lumbar spine was greater in the V and J groups than in the C group (P < 0.01). The J group had a significantly lower PTH level than the C group. In contrast to weight-bearing bones, we found no significant differences in BMD at the radius among the three groups. BMD at the distal radius was negatively correlated with years after menopause in both the V group and the J group significantly. These results indicate that regular physical exercise has a positive effect on the maintenance of bone mineral in postmenopausal women and that the protective action is localized in skeletal sites used predominantly for the sport without opposing the negative regulation caused by estrogen deficiency in systemic bones.