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T Fuwa

Publications and source records attributed to T Fuwa.

At least 37 records · Page 2Linked to original sources
PubMed1993

Effect of absorption promoters on subcutaneous absorption of human epidermal growth factor in rats.

Subcutaneous administration of human epidermal growth factor (hEGF) to rats gave a significantly smaller value of area under the curve (AUC) of concentration in plasma of immunoreactive hEGF versus time than intravenous administration, probably because the slow entry rate into the blood circulation and consequently the enzymic degradation of hEGF at the injection site. In the present study, absorption promoters such as sodium caprate, N-acylamino acids, disodium ethylenediamine-tetraacetate (EDTA), and sodium glycocholate were used because they were expected to inhibit the enzymic degradation of hEGF at the injection site and to facilitate the entry of hEGF into the blood circulation. Coadministration of an absorption promoter with hEGF significantly increased the entry rate and AUC value of immunoreactive hEGF compared with the case without the absorption promoter. The enzymic degradation of hEGF in the supernatant of the rat subcutaneous tissue homogenates and in the buffer solution containing leucine aminopeptidase or protease was markedly inhibited by the presence of the absorption promoters except EDTA. On the other hand, only EDTA increased the initial entry rate of FITC-dextran (M(r), 4000), which is not metabolized at the injection site, although all absorption promoters including EDTA markedly increased the extravasation of Evans blue. Thus, the increased subcutaneous bioavailability of hEGF in the presence of absorption promoters (except EDTA) was mainly attributed to the inhibitory effect of absorption promoters against the enzymic degradation of hEGF at the subcutaneous tissues.

Adjuvants, Pharmaceutic↗
PubMed1993

Systemic effects of epidermal growth factor (EGF) ointment containing protease inhibitor or gelatin in rats with burns or open wounds.

The systemic effects of epidermal growth factor (EGF) ointment containing nafamostat (NM), gabexate, or gelatin was studied in rats with burns or open wounds. At 1 d after burn, plasma epinephrine, cortisol, and glutamic-oxalacetic transaminase (GOT) levels were elevated, but treatment with EGF plus NM (EGF+NM) ointment significantly suppressed the increase in these levels. Further, there was no loss of body weight in the open wound model following treatment with EGF+NM ointment, while loss of body weight occurred in animals in which EGF ointments without NM were applied. Increases in plasma epinephrine 1 d after open wound formation were also suppressed by the application of EGF+NM ointment. Treatment with EGF ointment containing gabexate (GX) or gelatin (GL) ameliorated changes in body weight that occurred after open wound formation, while loss of body weight in animals with open wounds occurred following the application of ointment base, EGF ointment, GX ointment, or GL ointment. The present study thus indicates that the topical application of EGF ointment containing a protease inhibitor has ameliorative systemic effects.

Animals↗
PubMed1992

Existence of two pathways for the endocytosis of epidermal growth factor by rat liver: phenylarsine oxide-sensitive and -insensitive pathways.

The effect of phenylarsine oxide (PAO) on the internalization rate of epidermal growth factor (EGF) was investigated using perfused rat liver and isolated rat hepatocytes. In perfused liver, a tracer concentration of 125I-EGF alone or with excess unlabeled EGF (20 nM) was perfused and the internalization rate constants (kint) were measured. In the absence of PAO, kint values did not differ significantly for either dose condition. However, with the addition of PAO to the perfusate, the kint value dropped to 4% of that of the control at the low concentration of EGF, while dropping to only 40% of that of the control at the high concentration of EGF. These results suggest the existence of a PAO-insensitive internalization pathway having a kint value comparable with that of the other pathway. Similar EGF concentration-dependent inhibition of 125I-EGF internalization caused by PAO was ascertained using isolated rat hepatocytes. PAO also decreased the cellular ATP content in isolated hepatocytes. However, when we lowered the cellular ATP content with rotenone, the cell-surface binding and internalization of EGF were comparable with the control levels. We concluded that there exist dual pathways for the internalization of EGF and that excess doses of EGF lead to EGF internalization not only through a PAO-sensitive pathway but also through a PAO-insensitive pathway, whereas at a tracer dose of EGF, the internalization occurs mainly via the PAO-sensitive pathway.

Adenosine Triphosphate↗
PubMed1991

Analysis of the forelimb crossed extension reflex in thalamic cats during stepping.

Forelimb crossed extension reflexes were examined in 22 thalamic cats. These reflexes were elicited either by backward passive movement or by repetitive electrical stimulation of cutaneous and joint afferent nerves in the contralateral forelimb. Single stimulation of the superficial radial nerve evoked two types of reflex responses--early (ER) and late (LR)--from the triceps brachii muscle on the contralateral side. The latencies were about 7 and 16-25 ms, corresponding to the propriospinal (PSR) and spino-bulbo-spinal (SBS) reflexes of the ipsilateral flexor, respectively. Repetitive stimulation of the superficial radial nerve evoked the LR but not the ER. The crossed extension reflex and LR were abolished by lesions of the dorsolateral funiculus of the cervical cord on the side opposite to the recording. The tonic EMG activity, crossed extension reflex and LR in the extensor on the side of lesions were abolished by lesions of the ventrolateral funiculus of the cervical cord. During forelimb stepping, the amplitudes of both ER and LR fluctuated depending on the phase of the step cycle. The ER appeared during a narrow period in the early phase of the stance, whereas the LR was observed during a wide period from the middle of the swing to the middle of the stance. Both responses were absent from the middle of the stance to the middle of the swing. These observations suggest that forelimb crossed extension reflexes involve both spinal and supraspinal (SBS) loop mechanisms, and that these are utilized during stepping, with the latter mechanism in particular playing an important part in the extension phase of the forelimb forward movement.

Afferent Pathways↗
PubMed1991

Comparison of the methods for determining cell-surface and intracellular receptors for epidermal growth factor in the rat liver.

We compared methods for determining the distribution of epidermal growth factor (EGF) receptors between the cell surface and the cell interior in the rat liver. Incubation of isolated hepatocytes with 100 nM EGF for 20 min at 37 degrees C remarkably decreased the cell-surface EGF receptor density (internalization of receptors). The detergent Brij 35 was previously reported to permit assay of the intracellular latent EGF receptors in liver homogenates, but in the present investigation, Brij 35 lowered the affinity of EGF for the receptor depending on the detergent concentration, and the appearance of latent receptors was not observed. In contrast, permeabilization of the cells with digitonin, followed by an acid-washing procedure, increased the EGF binding capacity to close to the control level. Hence, the EGF receptors, internalized together with EGF molecules, were not degraded for at least 20 min, and the digitonin method is suitable for quantifying the intracellular EGF receptors. The binding capacities of the digitonin-treated and untreated control cells showed no difference upon digitonin treatment, suggesting that the bulk of EGF receptors exists on the cell surface. Further, cell-surface EGF receptor density was determined after the i.v. administration of EGF (300 micrograms/kg) to rats. Isolated hepatocytes prepared 30 min after the administration of EGF showed little binding for EGF on the cell surface, while the cell-surface EGF receptor density recovered to close to control values in cells prepared after 3 hr.

Animals↗
PubMed1991

Kinetic analysis of receptor-mediated endocytosis of epidermal growth factor by isolated rat hepatocytes.

The interaction of epidermal growth factor (EGF) with cell surface receptors and their subsequent endocytosis in isolated rat hepatocytes were analyzed by measuring changes in the concentrations of cell surface-bound, internalized, and degraded EGF. The kinetic model proposed by Wiley and Cunningham (Cell 25: 433-440, 1981) and Gex-Fabry and Delisi [Am. J. Physiol. 247 (Regulatory Integrative Comp. Physiol. 16): R768-R779, 1984] was basically utilized for the model analysis. The following kinetic parameters were obtained: association and dissociation rate constants for EGF-receptor interaction, internalization rate constant for EGF-receptor complex (kappa e), internalization rate constant for free receptor (kappa t), sequestration rate constant (kappa s) of the complex from shallow (exchangeable) to deep (nonexchangeable) membraneous compartment, intracellular degradation rate constant and initial cell-surface receptor density. The kappa s value, which was obtained by analyzing the time profiles of EGF association with cells, was approximately 5-10 times larger than the kappa e value determined by directly measuring internalized EGF with the acid-washing technique. This suggests the necessary presence of deep (nonexchanging) compartment of the complex in the plasma membrane. The calculated kappa e value is at least several times larger than the kappa t value, yielding the kinetic basis for the occurrence of receptor downregulation induced by excess EGF. We conclude that, in the overall receptor-mediated processing of EGF after bound to the cell surface receptors, the dissociation process is rapid [half-time (t1/2) less than 1 min], the degradation process is much slower (t1/2 approximately equal to 3 h), and the receptor internalization process is intermediate (t1/2 approximately equal to 6-7 min). In addition, two pools for EGF-receptor complex in the plasma membrane seem to be present, although their identification cannot be made.

Animals↗
PubMed1991

Improvement in wound healing by epidermal growth factor (EGF) ointment. II. Effect of protease inhibitor, nafamostat, on stabilization and efficacy of EGF in burn.

The healing effect of human epidermal growth factor (hEGF) on second degree burn was studied in rats. No improvement in wound healing was found on topical application of EGF alone to burn sites, but an ointment containing EGF and nafamostat mesilate (NM), a protease inhibitor, accelerated the healing rate of burns. The dry weight of the granulation tissue on the wound site in the group treated with EGF plus NM ointment did not change, although that in other groups decreased. After treatment with EGF ointment containing NM, the content of uronic acid, as an index of acid mucopolysaccharide, at 3 d after burn rapidly increased and had recovered to nearly normal levels at 7 d after burn. However, the uronic acid content in the other groups (control, EGF alone, and NM alone) showed a higher value at 7 d than at 3 d. When compared with the control values significant increases in hydroxyproline, as an index of collagen, in the wound site were observed at 7 d after treatment with EGF ointment containing NM. The degradation of [125I]EGF in burned tissue homogenate decreased significantly in a concentration-dependent manner in the presence of NM. Body weights did not change after treatment with EGF plus NM ointment, although the body weights of other treatment groups decreased after burn, suggesting that EGF ointment containing the protease inhibitor, NM, alleviated the effects of burn shock. These findings indicate that the stabilization of EGF at the wound site is an important factor for the expression of its healing effects.

Animals↗
PubMed1991

Absorption, biliary excretion, and metabolism of a new cholelitholytic agent, ursodeoxycholyl N-carboxymethylglycine and its esters in rats.

Intestinal absorption, biliary excretion and metabolism of a calcium gallstone dissolving agent, [11,12-3H]ursodeoxycholyl-N-carboxymethylglycine (UDC-CMG) and its monoethyl, diethyl and dipivaloyloxyethyl esters (UDC-CMG-Et, UDC-CMG-Et2 and UDC-CMG-PV2) were studied in bile duct cannulated rats. Biliary recovery of [3H]-labeled UDC-CMG, UDC-CMG-Et and UDC-CMG-Et2 after intraduodenal administration were 65%, 80%, 98%, respectively. Radio-thin layer chromatography analysis of the bile revealed that UDC-CMG didn't undergo any biotransformation during administration and excretion. About 80% and 20% of radioactivity recovered in the bile was identified as UDC-CMG-Et and UDC-CMG, respectively, after intraduodenal administrations of [3H]UDC-CMG-Et2 and [3H]UDC-CMG-Et. The administered intact UDC-CMG-Et2 was not found in the bile. Intraduodenally administered [3H]UDC-CMG-PV2 was rapidly recovered in the bile. The total recovery rate was 78% within a 24 h period. More than 80% of the radioactivity recovered in the bile was found as UDC-CMG. Lesser amounts of the monopivaloyloxyethyl ester of UDC-CMG were also found, but intact UDC-CMG-PV2 was not detected in the bile as in the case of UDC-CMG-Et2. Among the esters of UDC-CMG investigated in the present studies, only UDC-CMG-PV2 was excreted in the bile mainly as the perhydrolyzed form, UDC-CMG. These results suggest the usefulness of UDC-CMG-PV2 as the pro-drug in calcium gallstone dissolution therapy.

Animals↗
PubMed1991

Enhanced rectal and nasal absorption of human epidermal growth factor by combined use of the absorption promoter and the synthetic polymer in rats.

Previously, the presence of sodium carboxymethylcellulose (CMC Na) in addition to an absorption promoter, sodium caprate (C10 Na), in the dosing solution was found to be necessary for the enhancement of the rectal absorption of human epidermal growth factor (hEGF). In the present study, other synthetic polymers and absorption promoters were examined for their ability to enhance the rectal and nasal absorption of hEGF in rats. The effect of polymers in combined use with 100 mM C10 Na on the rectal absorption of hEGF was in the following order: 1% methylcellulose 1% hydroxypropylmethylcellulose 0.1% polyacrylic acid 1% CMC Na. Other absorption promoters such as N-lauroyl-alanine (C12-A) and dihydroxy-bile salts also enhanced the rectal absorption of hEGF in combined use with CMC Na. In order to confirm the increased rectal absorption of hEGF, the disappearance of hEGF from the rectal loop was examined. When hEGF in a 1% CMC Na solution (200 ug/kg) was administered in the rectal loop, the disappearance percent of hEGF during 60 min was 13.9% of the dose, although hEGF was not detected in the plasma. The presence of promoters such as 10 mM C10 Na or 15 mM C12-A in 1% CMC Na increased the disappearance percent to about 50% in a dosing range of hEGF from 100 to 500 ug/kg. On the other hand, a markedly enhanced nasal absorption of hEGF by 100 mM C10 Na was observed even in the absence of any polymer in a dising solution. However, addition of CMC Na into the dosing solution accelerated the rate of nasal absorption of hEGF in early phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrylic Resins↗
PubMed1990

Nuclear-magnetic-resonance studies of human epidermal growth factor.

The 1H-NMR spectra of native human epidermal growth factor (EGF) and a derivative lacking the final five residues have been assigned by two-dimensional methods, enabling their structures to be compared. The same structural features are observed for each protein, although the final five residues of native human EGF interact with residues earlier in the sequence. Comparison of the resonance shifts of human, rat and mouse EGF and human transforming growth factor alpha (TGF alpha) enables shifts characteristic of the EGF conformation to be identified, providing standards by which the structures of related proteins may be assessed.

Amino Acid Sequence↗
PubMed1990

Crossed forelimb extension produced in thalamic cats by injection of putative transmitter substances into the paralemniscal pontine reticular formation.

To analyze the descending pathways of the paralemniscal pontine reticular formation (PLRF), a technique was used for the selective activation of cell bodies by localized injection of putative neurotransmitters in the PLRF. When a small amount (less than 0.1 microliter) of 0.1 M glutamate was injected into the PLRF unilaterally in thalamic cats, the forelimb contralateral (c-forelimb) to the injection was extended, and occasionally the ipsilateral forelimb was flexed. These responses were similar to those obtained by electrical stimulation of the PLRF, but were relatively weaker. Unit spikes of PLRF neurons were increased in frequency following administration of glutamate. The latent periods and durations of increases in spike frequency varied depending on the concentration and quantity of the glutamate solution, and were roughly similar to those of the extensor EMG in the c-forelimb. Since the firing of PLRF neurons preceded the EMG with 11 ms latency, the unit spike of PLRF neurons could be used as a triggering signal to observe a spike triggered averaged EMG response in the extensor muscle of the c-forelimb. Results similar to those with glutamate were observed upon administration of quisqualate, kainate and aspartate. The most effective compound was quisqualate. Application to the PLRF of 1-naphthylacetyl spermine (1-NA-Spm), an analogue of the natural spider toxin JSTX-3 and an antagonist of glutamate, suppressed both the PLRF neuron activity and the extensor EMG of the c-forelimb. These observations suggest that extensor muscles of the forelimb are excited by the contralateral PLRF, perhaps via the crossed reticulospinal tract from the PLRF. PLRF neurons may be activated by glutamate (quisqualate) receptors.

Animals↗
PubMed1990

Possible mechanism of gastric mucosal protection by epidermal growth factor in rats.

The mechanism of the protection by human epidermal growth factor (hEGF) against the gastric mucosal lesions induced by acidified ethanol was studied in rats. At different times following the subcutaneous administration of hEGF (30 micrograms/kg), intragastric acidified ethanol (EtOH: 0.125 M HC1 = 50:50 v/v%) was administered to induce an experimental gastric mucosal lesion. Mean length of the lesion in the gastric mucosa was used as a lesion index. Extravasation of intravenously injected Evans blue into the gastric wall and gastric contents was used as an indicator of vascular permeability. Pretreatment with hEGF decreased both the gastric mucosal lesions and the increase of vascular permeability caused by acidified ethanol with similar time profiles relative to pretreatment with hEGF. Maximal protective actions of hEGF occurred about 10 to 30 min after the observed peak plasma concentration of hEGF. Indomethacin and N-ethylmaleimide, but not iodoacetamide, blocked the protective action of hEGF, indicating that endogenous prostaglandins and/or sulfhydryls may participate in the protective action of hEGF. The content of endogenous nonprotein sulfhydryls in the gastric mucosa decreased markedly after acidified ethanol. However, pretreated hEGF did not restore the sulfhydryl contents. Thus, it seemed that endogenous prostaglandins, but not sulfhydryls, are the probable mediators for protection against gastric mucosal injury caused by acidified ethanol.

Animals↗
PubMed1990

Protective effect of human epidermal growth factor against the experimental gastric mucosal lesions in rats.

The protective effect of human epidermal growth factor (hEGF) on the gastric mucosal lesions in rats was examined in relation to the immunoreactive concentration of plasma. Human EGF (30 micrograms/kg) was administered intravenously, intraperitoneally or subcutaneously. At different times following the administration of hEGF, rats received acidified ethanol solution to induce an experimental gastric mucosal lesion. Sum of lesion length in the gastric mucosa was used as a lesion index. Human EGF administered parenterally markedly decreased the gastric mucosal lesions in 10 min after administration of necrotizing solution, and 10 to 30 min delay was observed in the development of maximal protective action. Profiles of protective potency against the hEGF dose administered intraperitoneally or subcutaneously 30 min before administration of necrotizing solution revealed that the effective dose of hEGF (ED50) was about 5.2 and 2.6 micrograms/kg, for intraperitoneal and subcutaneous administrations, respectively.

Animals↗
PubMed1990

Comparison between spino-bulbo-spinal and propriospinal reflexes in thalamic cats during stepping.

To analyze changes in the excitability of both the spinal cord and brainstem in thalamic cats stepping on a moving treadmill, we examined the cutaneous propriospinal (PSR) and spino-bulbo-spinal (SBS) reflex responses in 20 adult cats. Tracheal cannulation, spinal transection at the T10 segment, and decerebration at the stereotaxic A12 level were performed under ether anesthesia. Immediately after decerebration, the ether was withdrawn. The head was fixed in a stereotaxic device, the T2 spinal process clamped to a metal frame, and the lumbar region suspended by a hammock, with bilateral forelimb contact on the floor of a treadmill. Electrical stimulation was applied to the superficial radial nerve with a cuff electrode, and two reflex responses (PSR and SBS) were recorded from the biceps brachii muscle in the same forelimb. Shortly before the appearance of forelimb stepping, both PSR and SBS reflex responses were elevated in amplitude. During forelimb stepping, the amplitudes of PSR and SBS reflex responses fluctuated depending on the phase of the step cycle. The PSR response was enhanced in the early phase of the swing, whereas the SBS response was elevated during a wider period from the beginning of the stance to the middle of the swing. The SBS response was completely absent in the late phase of the swing. This period corresponded to the transfer from flexion to extension and the appearance of the EMG of the triceps brachii muscle of the same forelimb. The fluctuation of the SBS response during stepping may be produced at the brainstem level, and not the spinal cord level, because the PSR response was enhanced only during narrow periods. The generation of locomotion thus seems to result in an enhancement of excitability of reflex pathways in the spinal cord and particularly in the brainstem.

Action Potentials↗
PubMed1990

Improvement in wound healing by epidermal growth factor (EGF) ointment. I. Effect of nafamostat, gabexate, or gelatin on stabilization and efficacy of EGF.

The healing effect of human epidermal growth factor (hEGF) on open wounds was studied in rats. No improvement in wound healing was found by topical application of EGF alone to open wound sites. We found an ointment containing EGF and a protease inhibitor, nafamostat mesilate or gabexate mesilate, or gelatin accelerated the healing rate of open wounds. Significant increases in the dry weight of the wound site granulation tissue, uronic acid (as an index of acid mucopolysaccharide) and hydroxyproline (as an index of collagen) were observed by treatment with EGF ointment containing nafamostat compared with the controls. The effects of the protease inhibitor on wound healing were dose dependent. Nafamostat was more efficient than gabexate or gelatin on wound healing. The degradation of 125I-EGF in wound tissue homogenate was significantly decreased in the presence of a protease inhibitor, such as nafamostat or gabexate, or gelatin. These findings indicate that the stabilization of EGF at the wound site is an important factor in permitting the expression of its healing effects and suggest that the ointment containing EGF and a stabilizing agent would be a suitable dosage form for acceleration of wound repair.

Animals↗
PubMed1990

Epidermal growth factor prolongs survival time of tumor-bearing mice.

We observed that human epidermal growth factor (hEGF) alone prolonged the survival time of mice bearing various murine syngeneic tumors as well as athymic nude mice bearing human xenografts. No changes in the subcutaneous solid tumor mass volume were observed. Prolongation of survival time by hEGF was observed in mice bearing murine epidermoid carcinoma (BSC) and human gastric carcinoma (KATO III), but not in murine epidermoid carcinoma (KLN205) or human epidermoid carcinoma (A431). Human tumor cells such as A431, KATO III, and murine tumor cells, KLN205, BSC had roughly 2 X 10(6), 3 X 10(4), 1.3 X 10(3) and 1 X 10(3) EGF receptors/cell, respectively. Although KLN205 and BSC tumor cells maintained nearly the same number of EGF receptors, the effects of hEGF were very different. Although A431 tumor cells had nearly 100 times more receptors than KATO III cells, the prolongation of survival time of mice bearing A431 by hEGF was no better than that of mice bearing KATO III. Accordingly, it appears that this prolongation of survival time by hEGF is independent of the number of EGF receptors on tumor cells. In addition, hEGF was shown to inhibit experimental pulmonary metastasis of murine BSC tumor, but was ineffective with murine KLN205 tumor. These results suggest that prolongation of survival time by hEGF may result from the inhibition of tumor cell metastasis and EGF may play a role in preventing the metastasis of certain malignant neoplasms unrelated to its effects through the EGF receptor on tumor cells.

Adenocarcinoma↗
PubMed1990

Kinetic analysis of the downregulation of epidermal growth factor receptors in rats in vivo.

We previously clarified the specific binding sites for epidermal growth factor (EGF) in several organs in rats based on in vivo kinetic analysis (D. C. Kim, Y. Sugiyama, H. Sato, T. Fuwa, T. Iga, and M. Hanano. J. Pharm. Sci. 77: 200-207, 1988). In the present study, we have determined the extent of the receptor downregulation and the recovery rate of the available receptors for EGF in several organs in vivo. At the specified times (30 min-24 h) after intravenous administration of excess unlabeled EGF (300 micrograms/kg), the early-phase (less than 3 min) uptake clearances (k1) of the tracer amount of 125I-EGF, which are proportional to the cell-surface available receptor densities, were determined in the liver, kidney, duodenum, jejunum, ileum, stomach, and spleen. As the result, the k1 value in each organ at 30 min after intravenous administration of unlabeled EGF was lowered close to the receptor-independent clearance value, indicating that the cell-surface receptors were almost completely downregulated, and thereafter, the k1 value showed gradual recovery to the control level. Furthermore, the recovery half-lives showed interorgan differences, namely the half-life (20 min) in the liver was much shorter than those (2-4.5 h) in other organs. These results were considered to reflect the processes of the recycling of internalized EGF receptors to the cell-surface or recruitment of new receptors. It was concluded that the recovery rate of the downregulated receptors in the liver, which is most responsible for the plasma clearance of EGF, is much faster than those in other organs.

Animals↗