Passive heat therapy in sedentary humans increases skeletal muscle capillarization and eNOS content but not mitochondrial density or GLUT4 content.

Passive heat therapy (PHT) has been proposed as an alternative intervention to moderate-intensity continuous training (MICT) in individuals who are unable or unwilling to exercise. This study aimed to make the first comparison of the effect of PHT and MICT on 1) skeletal muscle capillarization and endothelial-specific endothelial nitric oxide synthase (eNOS) content and 2) mitochondrial density, glucose transporter 4 (GLUT4), and intramuscular triglyceride (IMTG) content. Twenty young sedentary males (21 ± 1 yr, body mass index 25 ± 1 kg/m2) were allocated to either 6 wk of PHT (n = 10; 40-50 min at 40°C in a heat chamber, 3×/wk) or MICT (n = 10; time-matched cycling at ~65% V̇o2peak). Muscle biopsies were taken from the vastus lateralis muscle before and after training. Immunofluorescence microscopy was used to assess changes in skeletal muscle mitochondrial density (mitochondrial marker cytochrome c oxidase subunit 4), GLUT4, and IMTG content, capillarization, and endothelial-specific eNOS content. V̇o2peak and whole body insulin sensitivity were also assessed. PHT and MICT both increased capillary density (PHT 21%; MICT 12%), capillary-fiber perimeter exchange index (PHT 15%; MICT 12%) (P < 0.05), and endothelial-specific eNOS content (PHT 8%; MICT 12%) (P < 0.05). However, unlike MICT (mitochondrial density 40%; GLUT4 14%; IMTG content 70%) (P < 0.05), PHT did not increase mitochondrial density (11%, P = 0.443), GLUT4 (7%, P = 0.217), or IMTG content (1%, P = 0.957). Both interventions improved aerobic capacity (PHT 5%; MICT 7%) and whole body insulin sensitivity (PHT 15%; MICT 36%) (P < 0.05). Six-week PHT in young sedentary males increases skeletal muscle capillarization and eNOS content to a similar extent as MICT; however, unlike MICT, PHT does not affect skeletal muscle mitochondrial density, GLUT4, or IMTG content. NEW & NOTEWORTHY The effect of 6-wk passive heat therapy (PHT) compared with moderate-intensity continuous training (MICT) was investigated in young sedentary males. PHT induced similar increases in skeletal muscle capillarization and endothelial-specific endothelial nitric oxide synthase content to MICT. Unlike MICT, PHT did not improve skeletal muscle mitochondrial density, glucose transporter 4, or intramuscular triglyceride content. These microvascular adaptations were paralleled by improvements in V̇o2peak and insulin sensitivity, suggesting that microvascular adaptations may contribute to functional improvements following PHT.

Waon therapy upregulates Hsp90 and leads to angiogenesis through the Akt-endothelial nitric oxide synthase pathway in mouse hindlimb ischemia.

BACKGROUND: Thermal therapy, namely Waon therapy, has previously been reported to regulate nitric oxide (NO) and endothelial NO synthase (eNOS) and augment ischemia-induced angiogenesis in mice and improve limb ischemia in patients with peripheral artery disease. The aim of this study was to clarify the precise mechanism by which Waon therapy augments angiogenesis in mice with hindlimb ischemia. METHODS AND RESULTS: Unilateral hindlimb ischemia was induced in apolipoprotein E-deficient mice and Waon therapy was performed for 5 weeks. Heat shock protein 90 (Hsp90), phosphorylated-Akt, and phosphorylated-eNOS were detected in arterial endothelial cells of ischemic hindlimbs and all were upregulated by Waon therapy compared to controls. Waon therapy also increased serum concentrations of nitrite and nitrate. Capillary density and the ischemic limb/normal side blood perfusion ratio monitored by laser Doppler perfusion imaging in the Waon therapy group were significantly increased beyond those in the control group. The effect of Waon therapy on angiogenesis through the activation of the Hsp90/Akt/eNOS pathway was attenuated by the administration of a Hsp90 inhibitor. CONCLUSIONS: It is suggested that Waon therapy upregulates Hsp90, which contributes to the activation of the Akt/eNOS/NO pathway, and induces angiogenesis in mice with hindlimb ischemia.

The P2Y purinoceptor in rat brain microvascular endothelial cells couple to inhibition of adenylate cyclase.

1. B10 cells, a clonal line of rat brain capillary endothelial cells, exhibit a single P2 purinoceptor, activation of which leads to increases in free intracellular calcium. In the current study the identity of this P2Y receptor was determined by its binding parameters for a range of purinoceptor ligands and by its complementary DNA (cDNA) sequence. The signal transduction mechanism activated by this receptor was also investigated. 2. The radioligand [35S]-dATP alpha S bound with high affinity (Kd = 9.8 nM) to the P2Y purinoceptor expressed on B10 cells, which was found to be extremely abundant (Bmax = 22.5 pmol mg-1 protein). The calculated Ki values of a range of P2 purinoceptor agonists which competitively displaced binding of [35S]-dATP alpha S led to the rank order of affinity: dATP alpha S (Ki 3.4 nM) > 2-chloroATP (2-ClATP) (13 nM), ATP (22 nM) > ATP gamma S (43 nM) > 2-methylthioATP (2-MeSATP) (88 nM) > ADP (368 nM) > > UTP, L-beta,gamma-methyleneATP (both > 10,000 nM). The P2 purinoceptor antagonists, Reactive blue 2 and suramin, were also able to displace binding, with Ki values of 833 and 1358 nM respectively. In contrast pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium (PPADS) was able to displace only 20% of [35S]-dATP alpha S binding at a concentration of 100 microM. 3. 2-ClATP (EC50 = 0.22 microM), 2-MeSATP (0.54 microM), ADP (7.9 microM) and ATP (a partial agonist), but not UTP, inhibited the cyclic AMP formation stimulated by cholera toxin, in a manner that was prevented by pertussis toxin. The purinoceptor antagonist, PPADS, was found to be inactive at a concentration of 100 microM. 4. A P2Y receptor cDNA was derived from mRNA from B10 cells and from C6-2B, a rat glioma cell line known to possess a P2Y receptor that is coupled to the inhibition of adenylate cyclase. Sequence analysis of the entire coding region revealed that both were 100% identical to the rat P2Y1 purinoceptor cDNA. No other P2Y-type receptor mRNA could be detected in B10 cells. Exactly the same sequence was isolated from rat brain cortical astrocytes, where 2-MeSATP has been shown to increase phospholipase C activity. 5. Since the receptor responsible for the transduction shares with the aforementioned binding site significant pharmacological features, including a strong activity of 2-MeSATP (characteristic of P2Y1 receptors alone among all known P2Y purinoceptors) and an unusual insensitivity to PPADS, and since abundant mRNA is present of the P2Y1 receptor but not of any other type resembling the known P2Y receptors, it is concluded that a P2Y1 receptor on rat brain microvascular endothelial cells can account for all of the observations. This single P2Y1 receptor, therefore, appears to couple in different native cell types to either adenylate cyclase inhibition or to phospholipase C activation.

Effect of long-term treatment with the dihydropyridine-type calcium channel blocker darodipine (PY 108-068) on the cerebral capillary network in aged rats.

The effects of treatment with the dihydropyridine Ca+2 antagonist darodipine (PY 108-068) on age-related changes in the cerebral capillary network was studied using alkaline phosphatase histochemistry with quantitative image analysis. The investigation was performed on male Wistar rats of 12 months (adult reference group) and 27 months. The 27-month-old rats consisted of two groups, the first of control untreated animals and the second of rats receiving an oral dose of 5 mg/kg/day of darodipine from the 21st to the 27th month. The cerebral areas examined included the frontal cortex, the occipital cortex, Ammon's horn of the hippocampus, and the dentate gyrus. The number and the average length of alkaline phosphatase-positive capillaries were decreased in old rats, when compared with adult rats. The intercapillary distance, which is considered as a sensitive parameter for capillary density was increased in aged rats in comparison to adult rats. The capillary diameter was increased slightly or unchanged in old rats. The Ammon's horn and the frontal cortex were the cerebral areas most affected by age-dependent changes of the capillary network. Treatment with darodipine increased the number and the average length of alkaline phosphatase-reactive capillaries and reduced the intercapillary distance and the diameter of cerebral capillaries in old rats. The pericapillary microenvironment of the Ammon's horn was the most sensitive to treatment with darodipine. The above results showed that treatment with darodipine is capable of counteracting some microvascular changes occurring in the brain of aged rats. This suggests that the blockade of dihydropyridine-type Ca2+ channels has a positive effect on the brain microvascular system and may counteract the impairment of pericapillary microenvironment occurring with aging.

Glutamine stimulates growth in rat cerebral endothelial cell culture.

Endothelial cells were isolated from rat cerebral cortices using combined enzymatic digestions and Percoll gradient centrifugation. Primary cultures were subsequently grown on collagen-covered dishes in a medium containing 20% fetal calf serum and 0.6 mmol glutamine. The majority of cultures became confluent by day 7 or 8, but some could not reach confluence. The cells were fusiform in shape and exhibited immunoreactivity to factor VIII-related antigen and binding to the lectin Griffonia simplicifolia. Exposure of cultures to media containing 2.6 mmol glutamine resulted in accelerated growth (in cultures were confluent at days 3-4) and change in culture morphology, namely the formation of circular, cell-free areas. However, this treatment did not restore gamma-glutamyl transpeptidase activity that was lost during cultivation. As for other amino acids, asparagine was less potent, glycine and phenylalanine failed to mimic the glutamine effect. In summary, glutamine stimulates growth of cerebral endothelial cells in vitro and so it may supplement for other growth factors in the culture media.

Attenuation of phosphoinositidase activity and phosphatidylinositol bisphosphate level of bovine retinal capillary pericytes in high glucose.

Both phosphoinositidase (PIase) and individual species of inositol phospholipid (IPL) of bovine retinal capillary pericytes (BRCP) were quantitatively determined. When glucose in growth medium was increased from 5- to 15- or 30 mM, PIase activity was attenuated to 82% or 55%, respectively. In contrast, when glucose (5-, 15-, 30 mM) was added to an enzyme extract from cells grown in the standard growth medium (5 mM glucose, 0.04 mM myo-inositol) the PIase activity was not changed, indicating that the reduced PIase activity was not due to the direct effect of glucose. When IPLs from BRCP were analysed by HPLC and TLC, we observed reduction of the total and newly formed IPLs including the substrate of PIase. Phosphatidylinositol bisphosphate (PIP2). Reduced levels of IPLs were associated with a decrease in myo-inositol and an increase in sorbitol. The changes in IPL metabolism were reversed by adding either free myo-inositol or AL1576, an aldose reductase inhibitor (ARI), to the high-glucose medium. However, the addition of myo-inositol to the growth medium with a standard concentration of glucose only caused a marked increase in phosphatidylinositol, but not in PIP or PIP2, while the supplement of AL1576 in the standard medium did not cause any changes in IPL formation. These findings suggest that the alteration in IPL metabolism in BRCP may be related to insufficient myo-inositol or activated sorbitol pathway under high-glucose conditions. Further explanation of the role of the altered hydrolysis of PIP2 triggered by PIase may provide clues to understanding of the mechanism of decreased pericyte viability in the presence of high glucose concentrations.

Monoamine oxidase activities in human brain microvessels.

Microvessels can be easily isolated from human brain samples obtained at autopsy. Human frontal cortex MAO type A and B activities are similar in microvessel and microvessel-free preparations. In microvessels, enzyme activities and the ratio of MAO type A to type B vary among the areas studied and could selectively regulate the passage of certain amines though the blood vessel wall.

Regional distribution of neurohumoral responsive adenylate cyclase in rabbit brain microvessels.

Neurohumoral activation of adenylate cyclase was evaluated in microvessel fractions isolated from different regions of the rabbit brain. The greatest degree of enzyme by norepinephrine, dopamine and histamine was observed in microvessels from the brain stem, hippocampus and hypothalamus. The least degree of enzyme stimulation occurred in the thalamic preparation while the cerebellar microvessels were sensitive to norepinephrine alone.

Regional distribution of membrane-bound gamma-glutamyl transpeptidase activity in mouse brain. Comparison with rabbit brain.

Activity of membrane-bound gamma-glutamyl transpeptidase (gamma-GTP) was examined in various regions of mouse brain, in capillaries of the cerebral cortex and in telencephalic choroid plexuses. The level of activity in the capillaries was double and that of the choroid plexus nine times that of the gamma-GTP activity found in the brain, septum, hippocampus, hypothalamus, thalamus, cerebellum, frontal cortex, pons, medulla oblongata, and amygdala. Histochemically the gamma-GTP activity was demonstrated in the surface membranes of choroidal cells and in the endothelium of small capillaries. The activities of gamma-GTP of cerebral cortex, choroid plexus, and capillaries from rabbit were 5--17 times greater than those from corresponding areas of mouse brain. While 30 mM methionine stimulated (in vitro) the enzyme from mouse brain, no such effect was observed with the enzyme activity from rabbit brain. The gamma-GTP activity from the capillaries of cerebral cortex of both mouse and rabbit was not affected by the presence of methionine. These findings suggest existence of differences in the specificity of gamma-GTP activity in these two species.

[Electron-cytochemical study of ATP-ase activity in the brain after death]. / Elektronno-tsitokhimicheskoe issledovanie ATF-aznoi aktivnosti v golovnom mozge posle smerti

The lead method was applied to determine the localization of the ATP-asic activity in the rat and human brains at different periods after death. This activity was revealed in the cytoplasm of the cells, chromatin and the nucleolus, and also in the synaptic terminals. In the vascular capillaries the product of reaction was localized in the basal layer and on the cytomembrane of the endothelial cells. The results obtained pointed to a good preservation of the ATP-asic activity in the postmortem brain.