Bio-inspired low frictional surfaces having micro-dimple arrays prepared with honeycomb patterned porous films as wet etching masks.

Some kinds of snakes have micro-dimple arrays on their skins and show low frictional properties. Cost-effective and simple preparation methods of surfaces having micro-dimple arrays without burrs have been required. In this study, micro-dimple arrays were successfully prepared on aluminum plates and pipes by using honeycomb patterned porous films as wet etching masks. Resulting surfaces having 5 and 8 µm dimple diameters show low frictional coefficients compared with polished surfaces at a fluid lubrication regime.

Robust platforms for creating organic-inorganic nanocomposite microspheres: decorating polymer microspheres containing mussel-inspired adhesion layers with inorganic nanoparticles.

We describe a method for creating robust and stable core-shell polymer microspheres decorated with inorganic (IO) nanoparticles (NPs) by a self-organization process and heterocoagulation using a mussel-inspired polymer adhesive layer between the IO NPs and the microspheres.

Chloride conductance determining membrane potential of rabbit articular chondrocytes.

Membrane conductance of cultured rabbit articular chondrocytes was characterized by means of the patch-clamp technique. The resting membrane potential of the articular chondrocytes was about -42 mV. The membrane potential shifted in accordance with the prediction by the Nernst equation for Cl- when intracellular and extracellular concentrations of Cl- were changed. On the other hand, change in extracellular concentration of K+ produced no shift in the membrane potential of chondrocytes. The Cl- channel blocker 4-acetamido-4'-isothiocyanatostilbene-2'2-disulfonic acid (SITS) depolarized the membrane potential. These findings suggest that the membrane potential of the chondrocytes is determined mainly by Cl- conductance. Using the cell-attached patch-clamp method, a large unitary conductance of 217 pS was observed in the articular chondrocytes. The unitary current was reversibly blocked by SITS. Therefore, the unitary current was carried by Cl-. The Cl- channel showed voltage-dependent activation and the channels exhibited long-lasting openings. Therefore, the membrane potential of rabbit cultured articular chondrocytes was mainly determined by the activities of the large-conductance and voltage-dependent Cl- channels.

Decrease in the Ca2+-activated K+ current of pulmonary arterial smooth muscle in pulmonary hypertension rats.

Pulmonary hypertension exhibits acute elevation of vascular tone and hyperreactivity of pulmonary vasculature, which are closely related to patient mortality. In the present study, we investigated the characteristics of membrane currents of isolated pulmonary artery smooth muscle cells taken from rats with monocrotaline-induced pulmonary hypertension. Male Wistar rats were given a single subcutaneous injection of monocrotaline or saline, and then sacrificed between 18 to 21 days after the injection. The membrane currents in the smooth muscle cells from both groups of rats were compared using the whole-cell patch clamp technique. With 0.1 mM EGTA in the pipette, the densities of outward currents in monocrotaline-injected rats were smaller than those in control rats. When EGTA in patch pipettes was increased to 10 mM, the densities of the outward currents in monocrotaline-injected rats were equal to those of control rats. The Ca2+-activated K+ channel blockers (TEA, iberiotoxin) and nisoldipine were less effective on the outward currents of monocrotaline-injected rats. In the current clamp mode, a depolarization of membrane potential induced by 4-aminopyridine was greater in monocrotaline-injected rats than in control rats because of the reduced activity of the Ca2+-activated K+ channels. The Ca2+-activated K+ channels were decreased in pulmonary hypertension. The reduced activity of the currents may be related to the vascular hyperreactivity in pulmonary hypertension.

Alternation of inwardly rectifying background K+ channel during development of rat fetal cardiomyocytes.

The resting membrane potential of rat ventricular myocytes dramatically hyperpolarizes in the late phase of the fetal period. In order to investigate the mechanisms of this hyperpolarization, we examined the electrophysiological properties and molecular structure of the inwardly rectifying background K+ channels of rat fetal ventricular myocytes. In a patch-clamp experiment the whole-cell current of the inwardly rectifying background K+ channel increased 12-fold from between 12 and 18 days after impregnation. In the single channel recording, the large-conductance (35 pS) channels were mainly observed in the 18-day fetal ventricular myocytes. In the 12-day cells, the large-conductance channel was not observed although the low-conductance channels (11 and 16 pS) were infrequently observed. These data of single channel recordings suggested that channel proteins conducting the inwardly rectifying background K+ current were altered during the fetal development. Therefore, we compared the expression of Kir 2.1 mRNA and Kir 2.2 mRNA between 12 days and 18 days using the RT-PCR method, in order to investigate the possible molecular regulation which contributes to the electrophysiological changes. During the fetal period, the expression of Kir 2.2 mRNA increased tremendously (17 times), whereas the increase in the expression of Kir 2.1 mRNA (two times) was not so great. These results show that hyperpolarization in the late fetal period seems to be mainly due to the dramatic increase in expression of Kir 2.2 mRNA rather than expression of Kir 2.1 mRNA.

Characteristics of current fluctuations originating from activities of inward-rectifier K+ channels in guinea-pig heart cells.

Although outward current through inward-rectifier K+ channels has been observed in the whole-cell mode of the patch-clamp technique, no outward unitary current in single-channel studies has been recorded with the physiological ionic conditions. Hence, the relationship between single-channel activities and the inward rectification of the whole-cell current has been poorly understood. Therefore, characteristics of inward-rectifier K+ channels in guinea-pig ventricular myocytes were assessed by the noise analysis of the K+ current using the whole-cell patch clamp method. Partial blockade of the inward-rectifier K+ current by Ba2+ was used to obtain different levels of mean current and current fluctuation as needed for variance-to-mean analysis. The plot of variance of current fluctuation against mean currents was well fitted by theoretical parabolic curves, and the unitary conductance, the open probability, and the density of functional channels were deduced. The unitary conductance of the inward-rectifier K+ channel exhibited an inward-rectification, although the channel open probability and the density of functional channels were not much different at various holding potentials used. The unitary conductance was not changed when the intrapipette concentration of Mg2+ was reduced, but tended to be smaller when the pipette contained high Mg2+ concentration. Spermine also tended to reduce the outward unitary conductances, although the reduction was not statistically significant. These results suggest that the inward rectification in the whole-cell current was due to the inward-rectifying property of the unitary conductance of the K+ channels. Inward rectification of the unitary conductance may be caused by blocking of the channels by both Mg2+ and polyamines.

Vascular reactivity to endothelium-derived relaxing factor in human umbilical artery at term pregnancy.

It has been reported that human umbilical artery (HUA) at term pregnancy released endothelium-derived relaxing factor (EDRF), using a superfusion bioassay system. However, other reports showed that endothelium-dependent relaxation was not observed in isometric tension studies using HUA ring with intact endothelium. Thus, we intended to clarify whether vascular smooth muscle of HUA at term is sensitive to EDRF. HUA was obtained after normal vaginal delivery or cesarean section at term. Isometric tension studies were performed in normal Krebs solution, using HUA rings or strips, which were prepared in calcium-free Krebs solution. Sodium nitroprusside (SNP), a nitric oxide (NO) donor drug, relaxed HUA rings precontracted with 0.1 microM 5-hydroxytryptamine (5HT) in a dose-dependent manner (1 nM-10 microM). Histamine, substance P, carbachol, or the calcium ionophore A23187, which are considered to be EDRF-releasing agents, did not relax the HUA rings. By immunohistochemical study, it was confirmed that endothelial cells were present in the luminal surface of the HUA rings after the isometric tension recording. In a co-axial bioassay system involving HUA strips denuded of endothelium and rabbit aorta with intact endothelium, HUA strips precontracted with 0.1 microM 5HT were relaxed in response to 1 microM SNP but not 1 microM carbachol, which released EDRF from the endothelium of rabbit aorta. These findings suggest that HUA at term is sensitive to NO but not EDRF.

Morphological and pharmacological characterization of guinea-pig prostatic smooth muscle cells in vitro.

BACKGROUND: Prostatic smooth muscle is thought to play a major role in the pathogenesis of bladder outlet obstruction in patients with benign prostatic hypertrophy. However, the physiology of prostatic smooth muscle cells remains largely unknown, in part due to the lack of a suitable model system. We therefore sought to establish an in vitro culture of guinea pig prostatic smooth muscle cells. METHODS: Immature guinea pig prostate was treated by enzymatic digestion and the cells obtained were used to initiate the primary culture. After 3 to 4 passages, cultured smooth muscle cells were examined morphologically by immunocytochemistry and electron microscopy. The contractile properties of cultured smooth muscle cells were also examined. RESULTS: The cultured prostatic cells demonstrated hill and valley morphology, which is a hallmark of smooth muscle cells in vitro, and stained positively for desmin. In addition, electron microscopic examination of ultrastructural morphology revealed myofilaments. Confluent cultures of prostatic smooth muscle cells showed a clear, dose-dependent contractile response to phenylephrine. Furthermore, contraction of the prostatic smooth muscle cells by 10(-6) mol/L phenylephrine was completely inhibited by pretreatment with 10(-6) mol/L terazosin. CONCLUSIONS: An in vitro culture of prostatic smooth muscle cells was established. This culture is likely to provide a powerful tool for elucidating the physiology and pathophysiology of prostatic smooth muscle.

Chromatographic screening of growth inhibitors for Cladosporium herbarum in the extracts of calcareous red algae.

The thin-layer chromatographic screening of growth inhibitors for an imperfect fungus was carried out in two types of calcareous red algae, Corallina pilulifera (Corallinaceae) and Lithothamnium pacificum (Corallinaceae) and Crathromorchum circumscriptum (Corallinaceae). Several substances having positive activities on Cladosporium herbarum were detected in the alcoholic extracts of the marine plants. The observed differences among the substances can be attributed to the morphological characteristics of the sea weeds.

Voltage-gated ionic channels in cultured rabbit articular chondrocytes.

The membrane properties of cultured cells of rabbit articular chondrocytes were studied using the whole-cell patch clamp technique. The average cell capacitance was 37.9 +/- 9.0 pF (n = 13), and the cell resting potential was -41.0 +/- 7.0 mV (n = 11). We were unable to induce an action potential by applying a depolarizing current. Upon step depolarization, under voltage clamp conditions, one kind of inward and two kinds of outward currents were elicited. The inward current was initially observed at around -30 mV, peaked at 0 mV, and reversed at around +90 mV. Tetrodotoxin (TTX; 1 microM) was shown to completely block this inward current. At steady state, the inward current was half-inactivated at -51 mV, with a slope factor of 6.3 mV. Two outward currents were determined from measurements of activation threshold, reversal potential, and pharmacological responses. One was observed at around -30 mV, and its amplitude increased with membrane depolarization. Extracellularly applied 4-aminopyridine (4 AP) (1 mM) and tetraethyl ammonium chloride (TEA) (5 mM) blocked this current. The other outward current was observed at around +10 mV, and its direction reversed at a potential close to that predicted by the Nernst equation for a Cl- selective channel. This current fluctuated markedly, and the fluctuation did not decline throughout the 100 ms of the step pulse. Extracellularly applied 4-acetamido-4'-isothiocyanostilbenezene-2,2-disulfonic acid (SITS) (0.25 mM) blocked this current, but the same dose of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) had little effect. These results suggest the presence of TTX-sensitive Na+, 4-AP- and TEA-sensitive K+, and SITS-sensitive Cl- channels in rabbit articular chondrocyte membrane. The functional significance of these channels is discussed.

Unitary current through the inward rectifier K+ channel cloned from rabbit heart--comparison with the native K+ channel.

Recently, we have cloned a cDNA for a putative cardiac inward rectifier K+ channel (RBHIK1) from rabbit cardiac muscles. However, it's single channel characteristics have remained unknown. Therefore, we investigated the single channel characteristics of the RBHIK1 channel expressed in Xenopus oocytes by the cell attached patch clamp configuration, and compared them with those of the native Ik1 channel of the freshly-isolated ventricular myocytes under similar temperature conditions. In patch clamp experiments with 145 mmol/l K+ in the pipette at room temperature (20-22 degrees C), both the RBHIK1 currents and the native Ik1 showed a strong inward rectifying property. The single channel conductance of the RBHIK1 channel was 17.8 +/- 0.47 pS (n = 4), and that of the native Ik1 channel was 23.5 +/- 0.29 pS (n = 5). The activities of the cloned channel were sensitive to the putative K+ channel blockers (TEA, Cs+ and Ba2+). The open and closed time histograms at -140 mV could be fitted by a single exponential both in the RBHIK1 channel and the native Ik1 channel. Although the closed-time histogram of the native Ik1 channel was fitted by a sum of two exponential curves, that of the RBHIK1 channel was fitted by a single exponential curve. The sublevel corresponding to two-thirds of the unitary current was observed both in the RBHIK1 channel and the native Ik1, but it was more frequently detected in the RBHIK1 channel. Amplitude histogram constructed at -140mV in the RBHIK1 channel exhibited three peaks, which indicated closed, full-open, and 2/3 sublevel state, respectively. Unitary current was calculated to be 2.5 pA and sublevel of the unitary current was 1.68 pA. These characterization in the single channel activities of the RBHIK1 channel will help to study the molecular regulation of the Ik1 channel in cardiac cells.

Voltage-dependent suppression of calcium current by caffeine in single smooth muscle cells of the guinea-pig urinary bladder.

The suppressive action of caffeine on L-type Ca current (Ica) in smooth muscle cells of the guinea-pig urinary bladder was investigated using the whole-cell patch clamp technique. Caffeine (5-30 mM) suppressed Ica, the effect having two phases: a rapid and transient suppression of Ica, which was followed by a sustained suppression. When intracellular Ca2+ was strongly buffered by the Ca2+ chelator EGTA (20 mM) or BAPTA (5 mM) in the patch pipette, the transient suppression of Ica was abolished, whereas the sustained effect remained. Similarly, inclusion of both 10 mM procaine and 1 mg/ml heparin in the patch pipette blocked the transient suppression of Ica, but did not block the sustained effect. The degree of the sustained effect of caffeine on Ica was dose-dependent with a kd of 20 mM. Application of the cyclic AMP analogue, 8-bromo-cyclic AMP (100 microM) or forskolin (10 microM) to the bath failed to mimick the sustained suppression of Ica, suggesting that inhibition of phosphodiesterase activity was not involved in the caffeine action. The steady-state activation curve remained unchanged by 10 mM caffeine but the steady-state inactivation curve was significantly shifted in the negative direction by 15.6 mV in 1.8 mM Ca2+ solution or by 10 mV in 1.8 mM Ba2+ solution. From these results it appears that caffeine inhibits L-type Ica via two mechanisms: (1) it releases Ca2+ from an internal store causing a transient Ca2+ -mediated inactivation of the Ca channel; (2) it inhibits Ca channel via a mechanism that does not require such a Ca2+ release. It is possible that caffeine suppresses Ica through a preferential binding to the inactivated state of L-type Ca channel.

Muscarinic suppression of Ca2+ current in smooth muscle cells of the guinea-pig urinary bladder.

The suppressive action of carbachol (CCh) on the Ca2+ current (ICa) in smooth muscle cells of the guinea-pig urinary bladder was investigated using the whole-cell patch clamp technique. Bath application of 10 microM CCh reduced the amplitude of ICa by 92 +/- 3.8% (n = 9). Adding 1 microM atropine to the bath completely blocked the action of CCh, indicating that the suppressive action of CCh on ICa is mediated by the activation of muscarinic receptors. Intracellular perfusion of the non-hydrolysable GTP analogue, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 200 microM) mimicked the effects of CCh. Sustained suppression of ICa was observed when GTP gamma S was present in the cytoplasm. Intracellular perfusion of inositol 1,4,5-trisphosphate (InsP 3; 20 microM) also suppressed ICa; its effect was not sustained but transient. The protein kinase C activator, phorbol 12,13-dibutyrate (PDBu), however, could not mimic the effects of CCh on ICa. When intracellular Ca2+ was strongly buffered by the Ca2+ chelator EGTA (20 mM) in the patch pipette, the sustained suppression of ICa was abolished. Inclusion of 3 mg/ml heparin, a blocker of InsP3-induced Ca2+ release, in the patch pipette reduced the degree of sustained ICa suppression by 43.2 +/- 1.9% (n = 7). Adding thapsigargin (TG), a sarcoplasmic reticulum Ca2+-ATPase inhibitor, to a wash solution reduced the recovery of ICa by about 50%, suggesting that approximately half of the ICa suppression induced by CCh is due to Ca2+ release from TG-sensitive internal Ca2+ stores. From these results it appears that CCh suppresses ICa via two independent mechanisms: (1) Ca(2+)-mediated inactivation of the Ca2+ channel, which is caused by Ca2+ release from InsP3- and TG-sensitive internal stores, and (2) a GTP-binding protein-mediated mechanism, which requires intracellular Ca2+.

[The effect of local transient cooling on anodermal blood flow].

To clarify the regulatory mechanism of blood flow of anodermal mucosal layers responding to the localized and transient coolong stimulation (around -4 degrees C), we examined the change of anodermal blood flow with use of Laser Doppler Flowmeter (Periflux, Perimed). The cooling stimulation was applied to anoderm by the insertion of chilled cold stick (Poscool, Maruho) into the anus of 11 healthy male volunteer, aged 20-25 years. Experimental results obtained are as follow; 1) The anodermal blood flow of healthy subjects are variant in degree and widely distributed from 30 to 80 Perfusion Unit (PU). 2) After cooling for five minutes by the insertion of a frozen Poscool, anodermal blood flow were increased in 8 cases out of eleven (72.7%). The changes corresponds to be 1.4 to 5.2 times. 3) Eight cases are divided into two groups on the basis of the time course after cooling: the one is "delayed responding group" (there is a delay 10-40 minutes before the blood flow is increasing) and the other is "rapid responding group" (the blood flow is immediately increasing). These results indicate that the anodermal blood flow, which are exposed to cooling, of healthy subjects is regulated not only by nervous mechanism but by humoral mechanism. Then, based on the Lewis reaction (cold vasodilatation), it is suggested that the short time cooling of anoderm should be appreciated as a conservative therapy of hemorrhoids and anal fissures.

Properties of Ca(2+)-mediated inactivation of L-type Ca channel in smooth muscle cells of the guinea-pig urinary bladder.

The properties of Ca(2+)-mediated inactivation as revealed by a conventional double-pulse protocol were examined by using the whole-cell patch clamp technique. A U-shaped relationship between the conditioning potential and the Ca2+ current (ICa) inactivation was observed, with a maximum inactivation of 52 +/- 4% (n = 5) at 10 mV with 0.5 mM EGTA in the patch pipettes. The maximum inactivation was reduced significantly, to 31 +/- 5.7% (n = 12) and 32 +/- 7.0% (n = 5), when a high concentration of EGTA (20 mM) or a more efficient Ca2+ chelator, BAPTA, was included in the patch pipettes, respectively. The same double-pulse protocol was applied under conditions where the stored Ca2+ was depleted by using caffeine or the stored Ca2+ release function was blocked by using ryanodine or procaine and heparin. No significant difference in the maximum ICa inactivation before (45%) and after (50%) application of 10 mM caffeine was observed. The maximum ICa inactivations of 48 +/- 3.2% (n = 4) and 52 +/- 8.4% (n = 6) were still observed after treatment of the cell with ryanodine (20 microM) or loading 10 mM procaine and 1 mg/mL heparin in the patch pipettes, respectively. These results suggest that Ca2+ mobilization from an internal Ca2+ store is not essential for the Ca(2+)-mediated inactivation observed in the double-pulse experiment, rather influx of Ca2+ through a voltage-dependent Ca channel seems to be important for ICa inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization and transcriptional regulation of the modABCD genes for molybdenum transport in Escherichia coli.

The mod genes coding for molybdenum transport system were isolated from Escherichia coli by means of complementation of the tor mutation that causes no synthesis of some molybdenum-containing enzymes. The nucleotide sequence of 3048-base pair showed four open reading frames including the previously reported modC gene. The genes were named modA, modB, modC, and modD. Promoter analysis by lacZ assay and primer extension showed the genes modA and modB individually have their own promoters. No transcriptional regulation of the two genes were observed in the presence of molybdate, nitrate, and oxygen in E. coli strain DH5 alpha.

Effect of cytochalasin B on intestinal smooth muscle cells.

The inhibitory effect of cytochalasin B on contraction of smooth muscle cells isolated from guinea-pig taenia coli was investigated. Cytochalasin B (10-70 microM) inhibited the high K+ (70 mM)-induced contraction in a dose-dependent manner, and the maximum and the half-maximum effects were obtained at 50 and 15 microM, respectively. Cytochalasin B (70 microM) decreased ATPase activity in skinned guinea-pig taenia coli. However, cytochalasin B (50 microM) had no significant effect on the voltage-dependent Ca2+ currents, the passive membrane properties or the membrane potential. Cytochalasin B also had no effect on the phosphorylation of 20 kDa myosin light chain induced by high K+ and cytosolic Ca2+ levels. These results suggest that the inhibition of contraction by cytochalasin B may be due to its effects on actin of microfilaments and contractile filaments of guinea-pig taenia coli smooth muscle cells.