Expression of serine protease inhibitors in epidermal keratinocytes is increased by calcium but not 1,25-dihydroxyvitamin D3 or retinoic acid.

BACKGROUND: In human skin, the serine proteases kallikrein-related peptidase (KLK)5 and KLK7 degrade corneodesmosome proteins, leading to desquamation. Serine protease activity of the skin is tightly regulated by the interplay between such proteases and serine protease inhibitors, including lymphoepithelial Kazal-type related inhibitor (LEKTI), encoded by SPINK5; secretory leucocyte peptidase inhibitor (SLPI); and elafin. Expression of KLK5 and KLK7 is controlled and upregulated by stimulants such as calcium, 1,25-dihydroxyvitamin D3 [1,25(OH)2 VD3 ] and retinoic acid (RA). OBJECTIVES: To understand the effect of calcium, 1,25(OH)2 VD3 and RA on the expression of serine protease inhibitors in epidermal keratinocytes. METHODS: We stimulated normal human epidermal keratinocytes (NHEKs) with high calcium, 1,25(OH)2 VD3 or RA, and then analysed the expression of serine protease inhibitors using quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay and immunocytofluorescence. We also analysed trypsin- and chymotrypsin-like serine protease activities in stimulated NHEKs. RESULTS: High calcium, but not 1,25(OH)2 VD3 or RA, significantly induced the expression of LEKTI, SLPI and elafin at both transcript and protein levels in NHEKs. These inductions were time- and dose-dependent. The activities of trypsin- and chymotrypsin-like serine proteases were significantly up- and downregulated by high calcium, respectively, in NHEKs. CONCLUSIONS: High calcium, but not 1,25(OH)2 VD3 or RA, increases the expression of serine protease inhibitors in epidermal keratinocytes. Our findings contribute to the understanding of the mechanisms by which serine protease activities are regulated by serine proteases and related inhibitors in epidermal keratinocytes.

Mechanical behavior of in-situ alloyed Ti6Al4V(ELI)-3 at.% Cu lattice structures manufactured by laser powder bed fusion and designed for implant applications.

In the present study, cellular lattice structures for implant applications are reported for the first-time incorporating copper directly by in-situ alloying in the laser powder bed fusion process. The aim to incorporate 3 at.% Cu into Ti6Al4V(ELI) is selected for improved antibacterial properties while maintaining appropriate mechanical properties. Previously, topologically optimized Ti6Al4V(ELI) lattice structures were successfully designed, manufactured and studied for implant applications. The development of a new alloy produced by in-situ alloying of elemental powder mixture of Ti6Al4V(ELI) and pure Cu powders was used here for the production of identical lattice structures with improved antibacterial properties. One of the same as-designed CAD models was used for the manufacturing of these lattices compared to previous work on pure Ti6Al4V(ELI) lattices, making direct comparison of mechanical properties possible. Similar manufacturability highlights the applicability of this alloying technique to other lattice designs. Microstructural characterization was performed by optical and electron microscopies, as well as microCT. Mechanical characterization was performed by means of compression tests and hardness measurements. Results showed that in-situ alloying with copper leads to the formation of localized Cu-rich regions, refinement of martensitic phase and the formation of CuTi2 intermetallic precipitates, which increased the hardness and strength of the material. Deviations in wall thickness between the as-designed and as-manufactured lattices led to anisotropy of the mechanical properties of the lattices. Higher compressive strength values were obtained when thicker walls were oriented along the loading direction. Nevertheless, alloying with Cu had a higher impact on the compressive strength of lattice structure than the wall thickness deviations. The direct in-situ alloying of copper in Ti6Al4V(ELI) is a promising route for direct manufacturing of antibacterial implants.

Isolation and characterization of 6-hydroxymethylpterin as the Crithidia growth-promoting factor from spinach chloroplasts.

2-Amino-4-hydroxypteridine compounds were isolated as the Crithidia factor from spinach chloroplasts by DEAE-Sephadex A-50, DEAE-cellulose, Sephadex G-25, and thin-layer chromatography. One of the compounds was characterized as 6-hydroxymethylpterin by gas-liquid chromatography-mass spectrometry and by comparison with authentic specimen.

Effects of portal infusion of hypotonic- and hypertonic solutions on neuronal activity in the rat dorsal motor nucleus of the vagus.

The present experiment was designed to elucidate the characteristics of the response of neurons in the dorsal motor nucleus of the vagus (DMV) to stimulation of the hepatoportal area by hypotonic as well as hypertonic solutions. Responses of 81 neurons that exhibited an antidromic response to electrical stimulation of the ventral gastric vagus were recorded in the left DMV in urethane-chloralose anesthetized rats. The effects on these 81 neurons of portal infusion of hypertonic saline (3.6% NaCl) and of pure water were examined. The discharge rates of 16 neurons increased in response both to portal infusion of hypertonic saline and to that of water. Portal infusion of 0.9% NaCl produced no changes in firing rates. Their discharge rates of seven neurons increased in response to portal infusion of hypertonic saline but not to that of water. The other 58 neurons did not respond to these stimuli. Jugular infusion of water produced no response. Therefore, the responses to portal infusion of water appear to be derived from activation of the hepatoportal receptors. These results indicate that a certain fraction of DMV neurons respond similarly to portal infusions of hypertonic and hypotonic saline. It is possible that there exist some reflex arcs that mediate a similar response to both an increase and a decrease in portal blood osmolarity (or Na+ concentration), namely, a suppression of absorption.

Effect of portal infusion of hypertonic saline on neurons in the dorsal motor nucleus of the vagus in the rat.

Effects of hepatoportal osmo-receptive (or sodium-receptive) afferents on neurons within the dorsal motor nucleus of the vagus (DMV) were investigated electrophysiologically in urethane-chloralose anesthetized rats. Responses of 56 spontaneously active neurons to antidromic stimulation of the ventral trunk of the subdiaphragmatic vagus were recorded in the left DMV. Among them, 35 neurons were inhibited by electrical stimulation of the hepatic branch of the vagus nerve (inhibitory neurons), except two neurons that were slightly excited. Effects of portal infusion of 3.6% NaCl were examined on 26 inhibitory neurons. Sixteen neurons increased their discharge rates and one neuron decreased its discharge rate in response to portal infusion of hypertonic saline. Thirty-five right DMV neurons responded to electrical stimulation of the dorsal trunk of the subdiaphragmatic vagus were inhibited by electrical stimulation of the hepatic branch of the vagus. Four neurons were excited by this stimulation. Relatively smaller number of neurons (5 out of 22 inhibitory neurons) increased their discharge rates in response to portal infusion of hypertonic saline. In conclusion, the response of DMV neuron observed in this experiment was characterized by increasing the frequency of spike discharges in response to portal infusion of hypertonic saline. However, these neurons were inhibited by electrical stimulation of the hepatic branch of the vagus nerve. These results suggest that the hepatoportal osmo-receptive afferents may be conveyed to the DMV via inhibitory synapses.

Differences in the intrinsic membrane characteristics of parabrachial nucleus neurons processing gustatory and visceral information.

Whole-cell current-clamp recordings were made from neurons in the rat parabrachial nucleus (PBN) in three rostro-caudal brain slices. During recording the neurons were located in one of four quadrants of the PBN. Successful recordings were obtained from neurons in three of these quadrants termed the dorsolateral (DL), dorsomedial (DM) and ventromedial (VM) quadrants. Recordings were made of the intrinsic membrane properties and repetitive discharge characteristics of 58 neurons in the DL, 60 neurons in the DM, and 54 neurons in the VM-quadrants. The input resistance of the neurons in the DL quadrant was significantly lower and the membrane time constant significantly shorter than that of the neurons in the DM- and VM-quadrants. The mean action potential duration of the VM-quadrant neurons was significantly longer than that of both DL- and DM-quadrant neurons. The discharge frequency in response to a 1500 ms 100 pA current pulse of the DL quadrant neurons was significantly lower than that of the neurons in the other two quadrants. The latency of action potential initiation following a 100 pA depolarizing current pulse was significantly longer for DL quadrant neurons compared to neurons in the other two quadrants. Neurons were divided into groups based on their response to a long depolarizing current pulse immediately preceded by a hyperpolarizing current pulse. In all three rostro-caudal slices of the PBN, the largest populations of neurons were in Group II and Group III. The results demonstrate that neurons in different locations in the PBN have different membrane and repetitive discharge properties. These different PBN locations receive inputs from the visceral and gustatory regions of the NST. It is possible therefore that the differences in properties of the PBN neurons may relate to the type of sensory information that they process.

Effects of GABA on neurons of the gustatory and visceral zones of the parabrachial nucleus in rats.

Response characteristics of neurons in the gustatory and visceral zone of the parabrachial nucleus (PBN) to gamma-aminobutyric acid (GABA) were examined using whole cell recordings in brain slices of the rat. Based on the recording site, neurons were divided into three groups: neurons in the dorsolateral quadrant of the PBN (DL-neurons), neurons in the dorsomedial quadrant of the PBN (DM-neurons) and neurons in the ventromedial quadrant of the PBN (VM-neurons). Recordings were made from 44 DL-, 43 DM-, 39 VM-neurons. Superfusion of GABA resulted in a concentration-dependent reduction in input resistance in 67.5% of the neurons in the PBN (73.1% of the DL-, 62.5% of the DM-, 66.7% of the VM-neurons). No obvious difference of the concentration-response curve was found among three groups. The mean reversal potential of the GABA effect was about -74 mV and no significant differences were observed among three groups of neurons. The GABA response was partly or completely blocked by the GABAA antagonist bicuculline in all neurons tested. Superfusion of the GABAA agonist muscimol resulted in a decrease of the input resistance in all neurons tested. It was concluded that GABA functions as an inhibitory neurotransmitter in both gustatory and visceral part of the PBN, mediated in part, by GABAA receptors.

Identification of ST2A1 as a rat brain neurosteroid sulfotransferase mRNA.

A hydroxysteroid sulfotransferase (ST2A1) was identified as a form mediating neurosteroid sulfation in rat brain. The sole expression among known rat ST2A forms was indicated by brain RT-PCR. All nucleotide sequences of seven ST2A cDNA clones isolated from brain matched completely with that of hepatic ST2A1. The recombinant ST2A1 protein mediated neurosteroid sulfation. These data strongly suggest a functional role of ST2A1 as a neurosteroid sulfotransferase in rat brain.

Chemosensitive neurons within the area postrema of the rat.

It is demonstrated that some neurons within the area postrema, if not all, are responsive to glucose or sodium ions applied topically by means of microelectro-osmotic techniques. Glucose-responsive neurons displayed a marked decrease in the discharge rate in response to the topical application of glucose. Two different types of sodium-responsive neurons were observed; one was characterized by increasing the frequency of the discharges responding to microiontophoretic application of Na+, while the other showed the opposite response by decreasing the discharge rate in response to the same stimulation. They may serve enteroceptors in response to changes in the glucose or sodium concentrations of cerebrospinal fluid (CSF) or blood.

Effects of hepatic vagotomy on suppression of water intake induced by hepatic portal infusion of water in water-deprived rats.

We investigated the effects of hepatic vagotomy on the suppression of water intake induced by hepatic portal infusion of water by 24-h water-deprived rats. In sham-operated rats, water intake of the portal infusion group was significantly less than that of the jugular infusion group during and after the infusion of deionized water for 3.5 h at a rate of 52 microliters/min. This result reconfirmed our previous findings. On the other hand, in hepatic-vagotomized rats, the suppression during the portal infusion of water was not observed, but the suppression after portal infusion was observed. It is concluded that the suppression, at least during the portal infusion of water, was mediated by hepatoportal osmo-receptive (or sodium-receptive) afferent signals contained in the hepatic branch of the vagus nerve.

Convergence of hepatoportal glucose-sensitive afferent signals to glucose-sensitive units within the nucleus of the solitary tract.

Units which are activated by ascending impulses from the liver within the nucleus of the solitary tract (NTS) were identified by electrical stimulation delivered to the hepatic branch of the vagus. Responses of descending units were eliminated by a collision test. The units which showed decreased firing rates during portal infusion of isotonic glucose solution were also glucose-sensitive so that they showed decreased firing rates during topical application of glucose by means of micro-electro-osmotic techniques. It is concluded that glucose-sensitive neurons exist within the NTS and also that they are functionally linked with hepatoportal glucose-sensitive afferent units.

Response of neurons in the solitary tract nucleus, area postrema and lateral parabrachial nucleus to gastric load of hypertonic saline.

c-Fos was induced in the solitary tract nucleus (NTS), area postrema (AP) and lateral parabrachial nucleus (LPB) by the intragastric injection of hypertonic saline. The effect was bilateral and most dramatic in the NTS caudal to the facial nucleus and the AP, the areas receiving dense vagal afferent projection from the subdiaphragmatic structures. At least a part of these Fos-LI is considered to have been induced by the activation of the visceral osmoreceptors.

A direct hepatic osmoreceptive afferent projection from nucleus tractus solitarius to dorsal hypothalamus.

Thirty units that responded antidromically to electrical stimulation of the zona incerta (ZI) or the dorsal portion of hypothalamus were recorded in the nucleus tractus solitarii (NTS). These units were analyzed in relation to hepatoportal afferent inputs. Electrical stimulation of the hepatic branch of the vagus nerve facilitated six units (facilitatory units) and suppressed 10 units (suppressed units). Effect of the portal infusion of hypertonic saline was examined on six facilitatory and eight suppressed units. One facilitatory unit and one suppressed unit increased their discharge rates in response to portal infusion. Four facilitatory units and one suppressed unit decreased their discharge rates in response to the same stimulation. Increased or decreased discharge rates in response to portal infusion of hypertonic saline were observed in units that responded antidromically to electrical stimulation of the paraventricular hypothalamic nucleus, the lateral hypothalamic area, or the ZI. It is concluded that hepatoportal osmoreceptive signals are conveyed to the hypothalamus or the ZI directly from the NTS.

A hepatoportal osmoreceptive afferent projection from nucleus tractus solitarius to caudal ventrolateral medulla.

This study was performed to examine projection of nucleus tractus solitarii (NTS) neurons to the caudal ventrolateral medulla (cVLM) in rats. One hundred and seven neurons that responded antidromically to electrical stimulation of the cVLM were recorded within the NTS. Electrical stimulation of the hepatic branch of the vagus nerve (hepatic vagus) elicited facilitation on 62 neurons (facilitatory neurons) and suppression on 6 neurons (suppressed neurons). Effect of portal infusion of hypertonic saline was examined on 44 facilitatory and 4 suppressed neurons. Twelve facilitatory and 2 suppressed neurons showed a decrease in the discharge rate. One suppressed neuron showed an increase in the discharge rate. It is concluded that hepatoportal osmoreceptive signals are conveyed from the NTS to the cVLM. The responses are mostly characterized by the decrease in the discharge rate by portal infusion of hypertonic saline.

Chemosensitivity of neurons in the dorsal motor nucleus of the vagus that responded to portal infusion of hypertonic saline in rats.

Neural responses in the dorsal motor nucleus of the vagus (DMV) to topical administrations of sodium and portal infusions of hypertonic saline were investigated electrophysiologically by using multibarrel electrodes in anesthetized rats. Of 102 neurons that showed antidromic response to electrical stimulation of the ventral gastric vagus or the accessory celiac vagus, 51 neurons increased and 13 neurons decreased their discharge rates in response to the electrophoretic administration of sodium. The other 38 neurons did not respond to this stimulation. The portal infusion of hypertonic saline elicited neural responses of some DMV neurons whose axons are involved into either the ventral gastric or the accessory celiac vagus. Further, effects of the topical administration and the portal infusion of hypertonic saline were examined on 33 neurons. Typical response was characterized by an increase in discharge rate responding to both of the portal infusion and the topical administration. In conclusion, the DMV neurons receiving the afferent inputs from hepatoportal osmoreceptors may have an enteroceptor function detecting the change in osmotic pressure of their environment.

Convergence of gastric and hepatic information in brain stem neurons of the rat.

Convergence of gastric and hepatic information in the nucleus of the solitary tract (NTS) was investigated by single-pulse electrical stimulation of the hepatic and the gastric branches of the vagus. Facilitation induced by both stimuli occurred most often in responses of neurons in the NTS, indicating convergence of information there. In a second experiment using gastric distension and portal infusion of glucose to analyze such convergence, activation by gastric distension and suppression by portal glucose was the most prominent combination of responses to both stimuli. This confirmed the results of electrical stimulation, because distension increases the firing rate of gastric afferents and glucose infusion decreases the firing rate of hepatic afferents. Analysis of the responses to varying degrees of gastric distension revealed that some NTS neurons are activated by weak distension, but inhibited by strong distensions.

Chemosensitive neurons in the area postrema of the rat and their possible functions.

The present study is an attempt to demonstrate chemosensitive neurons within the area postrema (AP) electrophysiologically. Three types of chemosensitive neurons were identified: 1) glucose-responsive neurons that may participate in control of blood glucose and satiation, 2) sodium (osmotic pressure)-responsive neurons that may contribute to control of sodium and water balance of the body fluid and may be involved in salt appetite, 3) nausea-related neurons which respond to excess distension of stomach and LiCl as well. They may play a role in formation of conditioned taste aversion.

Effect of hepatic portal infusion of water on water intake by water-deprived rats.

To determine whether or not hepatoportal osmoreceptive (or sodium-receptive) signals participate in the control of drinking, we examined the effects of portal infusion of water, 0.9% saline, and 1.8% saline on water intake by water-deprived rats. Infusion was started 0.5 h prior to the end of the water deprivation period for 3.5 h at a rate of 52 microliters/min through either a portal or a jugular catheter. After 24-h water deprivation, water intake was measured successively for 24 h without food. As a result of the water infusion tests, water intake of the portal infusion group was significantly less than that of the jugular infusion group during and after the infusion. Portal infusion of neither 0.9% nor 1.8% saline affected the water intake compared to similar infusion into the jugular vein. It is concluded that hypotonic stimulation of the hepatoportal osmoreceptor suppresses water intake in water-deprived rats. On the contrary, isotonic or hypertonic stimulation does not produce any change of water intake.

Abdominal vagi mediate c-Fos expression induced by X-ray irradiation in the nucleus tractus solitarii of the rat.

The mechanism of induction of emesis by X-ray irradiation remains largely unknown. The purpose of the present research was to clarify the neuronal basis of the induction of nausea induced by X-ray irradiation analyzing c-Fos expression in the nucleus tractus solitarii (NTS) as a marker of cellular excitation. We confirmed that the dose of X-ray irradiation (4 Gy) used for the present research could actually induce nausea by preliminary measurement of kaolin intake. Induction of c-Fos immunoreactivity in the NTS was observed in the animals that received X-ray irradiation of the whole body. The mean number of c-Fos positive cells in the animals that received irradiation was significantly larger than that in the non-irradiated animals. Partial exposure of the abdomen to X-rays showed significantly greater c-Fos expression than that of the head. These results indicated the presence of a certain route for transmitting information from the periphery toward the central nervous system by X-ray irradiation. The number of c-Fos positive cells induced by X-ray irradiation in animals vagotomized at the subdiaphragmatic level was lower than that in sham-operated animals. Animals receiving a serotonin subtype three (5-HT3, 5-hydroxytryptamine) receptor antagonist (tropisetron, ICS 205-930, 3-tropanyl-indole-3-carboxylate) showed a significant reduction in c-Fos protein expression compared to animals receiving a vehicle. These results strongly suggested that X-ray irradiation activates 5-HT3 receptors on the terminals of the abdominal vagal nerves to excite the afferent pathway, thereby inducing emesis.

Role of parabrachial nucleus in submandibular salivary secretion induced by bitter taste stimulation in rats.

When rats lick a bitter taste solution such as quinine-hydrochloride, they secrete profuse amounts of saliva. The salivation has a higher flow rate than that induced by other qualities of taste stimulation: sweet, salty, and sour. The present study is aimed to clarify the neural mechanism of the quinine-evoked salivation by means of behavioral, neuroanatomical, and electrophysiological experiments. Behaviorally, submandibular salivary secretion and rejection behavior (gaping) were observed in normal rats, as well as in rats chronically decerebrated at the precollicular level. In chronically decerebrate rats, these quinine-evoked reactions were strongly suppressed by destruction of the medial part of the parabrachial nucleus, including the so-called taste area, and ventral part of the parabrachial nucleus, including the pontine reticular formation. Neuroanatomical study using a retrograde tracer, Fluoro-gold, revealed that the neurons sending their axons to the superior salivatory nucleus, parasympathetic secretory center, were located mainly in the pontine reticular formation ventral to the parabrachial nucleus, not in the parabrachial taste area. Extracellular neural activity was recorded from the parabrachial region in decerebrate rats, and responsiveness to taste stimulation, jaw movements, and electrical stimulation of the superior salivatory nucleus was examined. Neurons responsive to both taste stimulation and antidromic stimulation of the superior salivatory nucleus were found in the pontine reticular formation ventral to the parabrachial nucleus, which responded well to quinine and HCl taste stimuli. Neurons in the parabrachial taste area could respond to four qualities of taste stimulation, but not to antidromic stimulation of the salivary center. These results suggest that aversive taste information from the parabrachial taste area reaches the salivary secretory center via the reticular formation ventral to the parabrachial nucleus.