Distinct intracellular calcium responses of individual cultured human keratinocytes to air pressure changes.

BACKGROUND: We previously showed that application of hydraulic pressure to cultured human keratinocytes induced elevation of intracellular calcium concentration ([Ca(2+) ]i ), but the absolute value of the pressure could not be determined. PURPOSE: To evaluate the effect of the absolute value of pressure on keratinocytes and other skin cells. METHODS: In the present work, we examined the effect of changes in absolute pressure level by observing the [Ca(2+) ]i responses of cultured human keratinocytes and other cells cultured at the bottom of a hermetically sealed plastic flask as the air pressure in the flask was increased gradually, held stable, and then decreased abruptly, using the Ca(2+) -indicator fura-2. RESULTS: We found that the [Ca(2+) ]i of differentiated keratinocytes was changed significantly in each phase, whereas undifferentiated keratinocytes and other cells derived from skin or dorsal root ganglion showed no response. Removal of calcium from the medium blocked the increase in [Ca(2+) ]i in differentiated keratinocytes. The [Ca(2+) ]i responses of individual differentiated keratinocytes in the increasing, stable and decreasing phases of pressure change varied from cell to cell. The threshold of air-pressure increase from the original level for inducing [Ca(2+) ]i response was 5 - 20 hPa. CONCLUSION: These results suggest that epidermal keratinocytes might contain a sensory system that detects changes of external pressure on the skin.

Phosphodiesterase inhibitors block the acceleration of skin permeability barrier repair by red light.

We previously demonstrated that exposure to red light (550-670 nm) accelerates epidermal permeability barrier recovery after barrier disruption. Furthermore, we showed that photosensitive proteins, originally found in retina, are also expressed in epidermis. In retina, transducin and phosphodiesterase 6 play key roles in signal transmission. In this study, we evaluate the role of phosphodiesterese 6 in the acceleration by red light of epidermal permeability barrier recovery. Immunohistochemical study and reverse transcription-PCR assays confirmed the expression of both transducin and phosphodiesterase 6 in epidermal keratinocytes. Topical application of 3-isobutyl-1-methylxanthine, a non-specific phosphodiesterase inhibitor, blocked the acceleration of the barrier recovery by red light. Topical application of zaprinast, a specific inhibitor of phosphodiesterases 5 and 6, also blocked the acceleration, whereas T0156, a specific inhibitor of phosphodiesterase 5, had no effect. Red light exposure reduced the epidermal hyperplasia induced by barrier disruption under low humidity, and the effect was blocked by pretreatment with zaprinast. Our results indicate phosphodiesterase 6 is involved in the recovery-accelerating effect of red light on the disrupted epidermal permeability barrier.

Calcium ion propagation in cultured keratinocytes and other cells in skin in response to hydraulic pressure stimulation.

We have previously suggested that a variety of environmental factors might be first sensed by epidermal keratinocytes, which represent the frontier of the body. To further examine this idea, in the present study, we examined the intracellular calcium responses of cultured keratinocytes to external hydraulic pressure. First, we compared the responses of undifferentiated and differentiated keratinocytes with those of fibroblasts, vascular endothelial cells (VEC), and lymphatic endothelial cells. Elevation of intracellular calcium was observed after application of pressure to keratinocytes, fibroblasts, and VEC. The calcium propagation extended over a larger area and continued for a longer period of time in differentiated keratinocytes, as compared with the other cells. The response of the keratinocytes was dramatically reduced when the cells were incubated in medium without calcium. Application of a non-selective transient receptor potential (TRP) channel blocker also attenuated the calcium response. These results suggest that differentiated keratinocytes are sensitive to external pressure and that TRP might be involved in the mechanism of their response.

Neuronal nitric oxide synthase in epidermis is involved in cutaneous circulatory response to mechanical stimulation.

The source of nitric oxide (NO) in the cutaneous circulation remains controversial. We hypothesized that epidermis might generate NO in response to mechanical stimulation. In hairless mouse (HR-1) skin organ culture, mechanical stimulation resulted in NO release, which declined within 30 minutes after cessation. A similar NO release occurred in a reconstructed skin model containing only keratinocytes and fibroblasts and was suppressed after detachment of the epidermal layer. Moreover, the stimulation-induced NO release was significantly lower in skin organ culture from neuronal NO synthase knockout (nNOS-KO) mice, compared with wild-type (WT) mice. Mechanical stimulation of skin organ cultures from HR-1, nNOS-KO, endothelial NOS-KO (eNOS-KO), and WT mice caused an enlargement of cutaneous lymphatic vessels. The enlargement was significantly lower after detachment of the epidermal layer than in normal skin samples and was significantly lower for nNOS-KO than for WT mice. Skin blood flow in nNOS-KO mice after stimulation was significantly lower than in WT mice. eNOS-KO mice also showed lower responses than WT mice, and the difference was similar to that in the case of nNOS-KO mice. These results are consistent with the idea that NO generated by epidermal nNOS has a significant role in the cutaneous circulatory response to mechanical stimulation.

Mechanical-stimulation-evoked calcium waves in proliferating and differentiated human keratinocytes.

Calcium dynamics in the epidermis play a crucial role in barrier homeostasis and keratinocyte differentiation. We have recently suggested that the electro-physiological responses of the keratinocyte represent the frontier of the skin sensory system for environmental stimuli. In the present study, we have evaluated the responses of proliferating and differentiated human keratinocytes to mechanical stress by measuring the intracellular calcium level. Before differentiation, mechanical stress induces a calcium wave over a limited area; this is completely blocked by apyrase, which degrades ATP. In the case of differentiated keratinocytes, the calcium wave propagates over a larger area. Application of apyrase does not completely inhibit this wave. Thus, in differentiated cells, the induction of calcium waves might involve not only ATP, but also another factor. Immunohistochemical studies indicate that connexins 26 and 43, both components of gap junctions, are expressed in the cell membrane of differentiated keratinocytes. Application of octanol or carbenxolone, which block gap junctions, significantly reduces calcium wave propagation in differentiated keratinocytes. Thus, signaling via gap junctions might be involved in the induction of calcium waves in response to mechanical stress at the upper layer of the epidermis.

Expressions of rod and cone photoreceptor-like proteins in human epidermis.

Previous reports have suggested the existence of photoreceptors for visible radiation at the surface of the human body. Rhodopsin is a well-known photosensitive protein found in the rod cells of the retina and detects light/dark contrast. Cone opsins are also photosensitive receptors in the cone cells of the retina and detect colour. Here, we describe immunochemical studies using anti-rhodopsin and anti-opsin antibodies on human skin. Both mouse retina and human epidermis showed clear immunoreactivity with each antibody. Interestingly, immunoreactivity against longer-wavelength opsin antibody was observed in the basal layer of the epidermis, while immunoreactivity against rhodopsin and shorter-wavelength opsin was observed in the upper layer. PCR analysis confirmed the expression of rhodopsin-like and opsin-like genes in human retina and the skin. These results suggest that a series of proteins, which play a crucial role in visual perception, are expressed in human epidermis.

Calcium ion gradients and dynamics in cultured skin slices of rat hindpaw in response to stimulation with ATP.

Ionotropic receptors, originally found in the brain, were recently also identified in epidermal keratinocytes. Moreover, concentration gradients and movement of calcium are crucial in epidermal homeostasis. Thus, imaging of calcium in the living epidermis is expected to provide insight into epidermal physiology and pathology. Here we describe the imaging of calcium dynamics in the living epidermis of cultured skin slices. The basal calcium concentration was highest in the upper layer of the epidermis. The increase of intracellular calcium in response to adenosine triphosphate (ATP) varied in each layer of epidermis, and was greater at the bottom than in the uppermost layer. Further, the extent of elevation of intracellular calcium in response to ATP in cultured keratinocytes varied depending on the level of differentiation. These results suggest that the response to stimulation of keratinocytes in cultured skin slices varies depending upon the location (depth) within the epidermis.

Mulberry leaf powder prevents atherosclerosis in apolipoprotein E-deficient mice.

Mulberry is commonly used to feed silkworms. Here we examined whether a dietary intake of mulberry leaf (ML) could affect atherogenesis in vivo and in vitro. Apolipoprotein E-deficient mice were fed either normal chow (control group) or a diet containing 1% ML powder (ML group) from 6 weeks of age. The mice were sacrificed after 12 weeks. The susceptibility of plasma lipoprotein to oxidation was assessed using diene formation. A significant increase in the lag time of lipoprotein oxidation was detected in the ML group compared with the control group. Furthermore, the ML group showed a 40% reduction in atherosclerotic lesion size in the aortae compared with the control. We also examined the direct anti-oxidative activity of ML in vitro. Aqueous extract of ML had a strong scavenging effect on 1,1-diphenyl-2-picrylhydrazyl and inhibited lipoprotein oxidation. These results confirm that ML contains anti-oxidative substances that might help prevent atherosclerosis.

Topical application of neuronal nitric oxide synthase inhibitor accelerates cutaneous barrier recovery and prevents epidermal hyperplasia induced by barrier disruption.

The effect of nitric oxide (NO) on skin barrier recovery rate was evaluated in hairless mouse. Topical application of an NO synthase (NOS) inhibitor and a neuronal nitric oxide synthase (nNOS) inhibitor accelerated the barrier recovery after tape stripping, whereas application of an inducible NOS (iNOS) inhibitor had no effect. After tape stripping, the barrier recovery in nNOS-/- mice was significantly faster than in wild type. Topical application of the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) delayed the barrier recovery in hairless mice. Immediately after barrier disruption on skin organ culture, NO release from the skin was significantly increased. The increase was blocked by nNOS inhibitor, but not by iNOS inhibitor. Topical application of the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) accelerated the barrier recovery, whereas SIN-1 chloride, a guanylyl cyclase activator, delayed the barrier recovery. In cultured human keratinocytes, SNAP increased the intracellular calcium concentration. The increase was blocked by ODQ, but not by the calcium channel-blocker nifedipine. In calcium-free medium, SNAP increased the intracellular calcium concentration. Topical application of both nNOS inhibitor and ODQ also reduced the epidermal hyperplasia induced by barrier disruption under low environmental humidity. These results suggest that NO plays an important signaling role in cutaneous barrier homeostasis and in epidermal hyperplasia induced by barrier disruption.

Epidermal keratinocytes as the forefront of the sensory system.

Various sensors that respond to physical or chemical environmental factors have been identified in the peripheral nervous system. Some of them, which respond to mechanical stress, osmotic pressure, temperature and chemical stimuli (such as pH), are also expressed in epidermal keratinocytes. Neurotransmitters and their receptors, as well as receptors that regulate the neuroendocrine system of the skin, are also present in keratinocytes. Thus, broadly speaking, epidermal keratinocytes appear to be equipped with sensing systems similar to those of the peripheral and central nervous systems. It had long been considered that only nerve C-terminals in the epidermis play a role in skin surface perception. However, building on earlier work on skin receptors and new findings introduced here, we present in this review a novel hypothesis of skin sensory perception, i.e. first, keratinocytes recognize various environmental factors, and then the information is processed and conveyed to the nervous system.

Influence of inflated lung pressure on lung mechanical properties during cold storage in rats.

The degree of lung inflation pressure during cold storage has a deep connection with lung functions after reperfusion. However, the change of the lung mechanical properties during cold storage at the different lung inflation pressure is unknown. We investigated the change of the mechanical properties both at the organ and the tissue level under different inflated lung pressure soon after the cold storage. In group I, the lungs were not preserved. In group II, the lungs were preserved in deflated condition. In groups III and IV, the lungs were inflated with room air at pressures of 14 and 25 cm H2O. After 24-hour storage, the input impedance (Z) was measured by a computer-controlled small animal ventilator (n = 8 each group). All data were analyzed using a homogeneous linear model, which includes airway resistance (R(aw)), tissue elastance (H), and tissue damping (G). Hysteresivity (eta) was calculated as G/H. After Z measurement, the tissue elasticity (Eqs) obtained from the quasi-static stress-strain curve was compared. We also analyzed the biochemical changes of surfactant in bronchoalveolar lavage (BAL) fluid. R(aw )was significantly lower in groups III and IV than in group I (p < 0.01). Hysteresivity (eta) was significantly lower in group IV than in groups I and III (p < 0.05). Eqs was significantly higher in groups III and IV than in groups I and II (p < 0.01). The total protein level in the BAL fluid of the preserved groups was significantly higher compared with group I (p < 0.01). We conclude that hyperinflated storage would deteriorate lung tissue mechanical properties.

Glycine ameliorates lung reperfusion injury after cold preservation in an ex vivo rat lung model.

BACKGROUND: The role of glycine has not been investigated in lung ischemia-reperfusion injury after cold preservation. Furthermore, the role of apoptosis after reperfusion following cold preservation has not been fully understood. METHODS: Lewis rats were divided into three groups (n=6 each). In the GLY(-) and GLY(+) groups, isolated lungs were preserved for 15 hr at 4 degrees C after a pulmonary artery (PA) flush using our previously developed preservation solution (ET-K; extracellular-type trehalose containing Kyoto), with or without the addition of glycine (5 mM). In the Fresh group, isolated lungs were reperfused immediately after a PA flush with ET-K. They were reperfused for 60 min with an ex vivo perfusion model. Pulmonary function, oxidative stress, apoptosis, and tumor necrosis factor (TNF)-alpha expression were assessed after reperfusion. RESULTS: Shunt fraction and peak inspiratory pressure after reperfusion in the GLY(-) group were significantly higher than those in the GLY(+) and Fresh groups. Oxidative damage and apoptosis in the alveolar epithelial cells of the GLY(-) group, assessed by immunohistochemical staining and quantification of 8-hydroxy-2'-deoxyguanosine and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling method, were significantly higher than those of the GLY(+) and Fresh groups. There were correlations among shunt fraction, oxidative damage, and apoptosis. There was no expression of TNF-alpha messenger RNA in all groups evaluated by the reverse transcription-polymerase chain reaction. CONCLUSIONS: Glycine attenuates ischemia/reperfusion injury after cold preservation by reducing oxidative damage and suppressing apoptosis independent of TNF-alpha in this model. The suppression of apoptosis might ameliorate lung function after reperfusion.