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Biomedical subjects

R Ishitani

Publications and source records attributed to R Ishitani.

At least 37 records · Page 2Linked to original sources
PubMed1990

Presence of specific binding sites for [3H]sarcophytol-A in cultured human skin fibroblasts.

The presence of specific binding sites for [3H]sarcophytol-A in human skin fibroblasts was examined using biochemical and morphological methods. The displacement studies clearly revealed that high (KD = 31.0 nM) and low (KD = 6.05 microM) affinity sites were present in the intact cells. Moreover, autoradiographic studies using light microscopy revealed that the specific binding sites may exist in both the cytoplasm and the nuclei.

Antineoplastic Agents↗
PubMed1990

Effects of protein-modifying reagents on brain tryptamine binding sites: possible involvement of a thiol group in temperature-induced high-affinity [3H]tryptamine binding sites.

To investigate the biochemical nature of temperature-induced high-affinity [3H]tryptamine binding sites, we subjected whole rat brain synaptic membranes to treatment with various protein-modifying reagents and examined the subsequent [3H]tryptamine binding properties of the membranes. Pretreatment of the membrane preparations with NEM, NBS, PCMB, PAPMA and MA, but not with iodoacetamide, DTT, glutathione and cysteine, reduced the [3H]tryptamine binding. In addition, to at least approx. 10(-4) M, the inactivation properties of NEM, PCMB, PAPMA and MA, except for NBS, were temperature-dependent. Furthermore, it was revealed that the Scatchard plot of [3H]tryptamine binding in membranes pretreated with these thiol reagents conformed to a curved line, as well as in the case of the control membranes. Nonlinear regression analysis of these data showed that NEM decreased the Bmax values of both the high and low affinity binding sites with no significant alteration in the KD values, whereas PCMB, PAPMA and MA increased only the KD value of the high affinity sites, accompanying the decrease of the Bmax values of both sites. These results indicate that the temperature-induced high-affinity [3H]tryptamine binding molecule(s) is a thiol protein.

Animals↗
PubMed1989

Existence of parathyroid hormone binding sites on murine hemopoietic blast cells.

We demonstrated that 125I-labeled human parathyroid hormone (1-34;8,18-Nle,34-Tyr)[[125I]hPTH(1-34)] bound specifically to hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor. Half-maximal inhibition of binding was achieved at concentrations of unlabeled hPTH(1-34) of about 5 x 10(-9)M. Insulin and hPTH(39-68) did not compete for PTH binding sites. Specific binding of hPTH(1-34) was detected in neither macrophages nor multinucleated cells (MNC's). Furthermore, treatment of hemopoietic blast cells with hPTH(1-34) stimulated MNC formation, and the range of concentrations (10(-10)-10(-8)M) over which hPTH(1-34) caused these effects was similar to that which inhibited the binding of [125I]hPTH(1-34). These findings suggest the presence of a PTH receptor on osteoclast precursors and the direct effect of PTH on them, resulting in osteoclast-mediated bone resorption.

Animals↗
PubMed1988

Glutaraldehyde pretreatment blocks temperature-induced high-affinity [3H]tryptamine binding.

The effect of glutaraldehyde (and Azure A) on temperature-sensitive high-affinity [3H]tryptamine binding was investigated in rat brain synaptic plasma membranes. In the 0.01-0.1% concentration range, the glutaraldehyde pretreatment preferentially inhibited only the above-mentioned portion of the binding, whereas the posttreatment of this reagent had no effect. On the other hand, in cases of pretreatment or posttreatment, a concentration of glutaraldehyde as high as 0.1% was inactive on the basal [3H]ligand binding capacity of the membranes (i.e., temperature-independent binding). Furthermore, it was revealed that the Scatchard plot of [3H]tryptamine binding in membranes pretreated with glutaraldehyde (0.05%) conformed to a straight line, as did a similar plot of temperature-independent binding. And, it was interesting to find that the binding parameters (KD and Bmax values) of both samples corresponded closely to each other. On the contrary, in all concentrations, Azure A affected nonspecifically both the temperature-dependent and the independent [3H]tryptamine binding to the same degree, regardless of whether or not there was pretreatment or posttreatment. All these observations clearly demonstrate that an appropriate concentration (0.01-0.1%) of glutaraldehyde pretreatment specifically blocks the temperature-induced allosteric modifications of high-affinity [3H]tryptamine binding sites.

Aldehydes↗
PubMed1987

[3H]tryptamine binding to reconstituted fraction of acidic lipids.

The possible involvement of sulphatides (CS), phosphatidylserine (PS) and phosphatidylinositol (PI) in [3H]tryptamine binding to various reconstituted fractions of these acidic lipids was examined by Sephadex LH20 column chromatography. The results indicated that each of the four systems, PS, PS-CS, PS-PI and PS-CS-PI, had the same binding capacity for [3H]tryptamine, whereas other systems (CS, PI and CS-PI systems) had no binding capacity. Furthermore, competitive inhibition experiments revealed that among these four reconstituted systems, the PS-CS system exhibited the highest affinity for 5-methoxytryptamine. Kinetic studies suggested that at least two binding components (or sites) are implicated in the binding of [3H]tryptamine to the reconstituted system of PS and CS with apparent KD values of 3 and 10 nM. Displacement studies with various compounds indicated that only tryptamine and 5-methoxytryptamine inhibited the [3H]tryptamine binding to this fraction, while other indoleamine analogues and neurotransmitters had no effect. In addition, we subjected whole rat brain synaptic plasma membranes to treatment with several kinds of lipid-modifying reagents and examined the [3H]tryptamine binding capacities of the membranes by a radioreceptor-binding assay. [3H]Tryptamine binding was decreased by treatment with Azure A and phospholipase A2, while phospholipase D had no effect. All these observations led to the inference that PS and CS may be involved in the tryptamine binding activities as recognition sites.

Animals↗
PubMed1986

Temperature-sensitive high affinity [3H]tryptamine binding sites in rat brain.

The influence of prior incubation on [3H]tryptamine binding was investigated in rat brain synaptic plasma membranes. A 55 min preincubation of the membranes at 37 degrees C induced an approx. 2.4-fold increase in the specific binding of [3H]ligand to the subsequently washed preparations and this phenomenon was quite temperature-dependent. On the other hand, the proportion of nonspecific binding sites was significantly decreased by 70% of the original sites within 20 min of the start of preincubation. Pargyline, ascorbic acid, EGTA, metal ions (Ca2+, Mg2+, Na+) and guanine nucleotides, included in the preincubation buffer, were all inactive on the stimulation of [3H]tryptamine binding, while the pretreatment of membranes with glutaraldehyde antagonized the augmentation of this binding. Furthermore, it was revealed that the Scatchard plot of the [3H]tryptamine binding preincubated at 0 degree C conformed to a straight line (KD = 33.1 nM, Bmax = 543 fmoles/mg protein), whereas a curvilinear Scatchard plot was obtained at 37 degrees C preincubation. Nonlinear regression analysis of the latter resulted in apparent KD (nM) & Bmax (fmoles/mg protein) values of 0.45 & 102.7 and 33.7 & 603.4 for the high and low affinity sites, respectively. All these observations lead to the inference that the preincubation-induced increase in [3H]tryptamine binding (i.e., nearly high affinity proportion of sites) may occur as a result of temperature-sensitive interconvertible conformational changes.

Animals↗
PubMed1985

Implication of acidic lipids in 5-hydroxytryptamine receptor mechanisms.

To establish the possible involvement of acidic lipids in 5-HT receptor mechanisms, we subjected whole rat brain synaptic plasma membranes to treatment with several kinds of lipid-modifying reagents and examined the [3H]5-HT and [3H]spiperone binding properties of the membranes. [3H]5-HT binding was decreased by treatment with Azure A, while [3H]spiperone binding was not altered. Similarly, prior treatment with arylsulphatase reduced the former binding, but had no effect on the latter binding. On the other hand, neither [3H]ligand binding was sensitive to phospholipases C and D. In contrast, prior treatment with phospholipase A2 (unheated) drastically decreased the [3H]5-HT binding and also affected the [3H]spiperone binding to some extent. Chelation of Ca2+ by EGTA (5 mM) prior to incubation of membranes with the unheated phospholipase A2 did not completely prevent the inhibitory effect of this enzyme on [3H]5-HT binding, while in the heated enzyme (at 100 degrees C for 10 min) EGTA exhibited this preventive effect perfectly. Furthermore, it was an interesting find that at least a low concentration of the heated phospholipase A2 (0.01 U) had no effect on the [3H]spiperone binding, as contrasted with the case of [3H]5-HT binding. In addition, the reduction of [3H]5-HT binding capacity in membranes treated with phospholipase A2 (heated and unheated) was restored only slightly by treatment with BSA (1%). Scatchard analysis of the [3H]5-HT binding showed that Azure A and phospholipase A2 (heated) decreased the Bmax values with no significant alteration in the KD values, whereas arylsulphatase increased only the KD value. All these observations infer that certain acidic lipids may play a role as the recognition site(s) or modulator(s) of 5-HT1 receptor molecules.

Animals↗
PubMed1985

[3H]5-hydroxytryptamine binding to reconstituted fraction with sulphatides, phosphatidylserine and phosphatidylinositol.

As a first step toward the examination of the involvement of sulphatides, phosphatidylserine and phosphatidylinositol in 5-HT receptor mechanisms, we performed [3H]5-HT binding experiments on the various reconstituted fractions with these acidic lipids. A binding assay of [3H]5-HT to these fractions was carried out by Sephadex LH20 column chromatography. Among various reconstituted fractions, only the reconstitution system with the three acidic lipids exhibited a saturable [3H]5-HT binding capacity, whereas no binding was seen with [3H]-spiperone. When the binding of [3H]5-HT to this fraction was plotted as a function of the ligand concentration, a multiple binding mode with three classes of binding components (or sites) was observed. Furthermore, the double reciprocal plot indicated that this reconstitution system had three apparent KD values of 4.7, 15 and 59 nM. The displacement studies with various compounds indicated that only a few 5-HT agonists (5-methoxytryptamine and tryptamine) and neurotransmitters (DA and ACh) inhibited the [3H]5-HT binding to this fraction, but 5-HT antagonists, LSD analogues and neuroleptics had no effect. Moreover, GTP, GDP and Gpp(NH)p clearly inhibited the [3H]5-HT binding in spite of their weak potencies, while GMP did not have any effect.

Animals↗
PubMed1984

Selective labelling of high affinity 5-hydroxytryptamine1 receptors in whole rat brain.

In synaptic membranes prepared from whole rat brain, cinanserin produced a relatively shallow inhibition curve of [3H] 5-HT binding with Hill slope of 0.68, as compared with that observed in the [3H]spiperone binding (Hill slope = 0.99). Furthermore, nonlinear regression analysis demonstrated that the former inhibition curve is best fitted by a two-site model. Regional and Scatchard analyses both clearly revealed that, in rat brain, at least two populations of binding sites were labelled by [3H]5-HT; however, high affinity binding sites (K(D) = 1.90 nM, Bmax = 0.188 pmoles/mg protein) could apparently be differentiated from the others by the action of 5 nM cinanserin.

Animals↗
PubMed1983

Butanol extracts from myelin fragments: tryptamine binding to lipid fractions.

To investigate the nature of the tryptamine binding components that originated from myelin butanol extracts (i.e., myelin proteolipids), the lipid mixtures obtained from these extracts were further fractionated by silicic acid column chromatography, and binding assays of 14C-tryptamine to those fractions were carried out by Sephadex LH20 column chromatography. Among several lipid fractions, only the F-C fraction retained the tryptamine binding properties of the original myelin butanol extracts, i.e., binding capacity, chromatographic profile and interaction with indoleamine analogues and other neurotransmitters. Since the quantitative TLC analysis indicated that this fraction contained a considerable amount of phosphatidylserine (PS) and phosphatidylinositol (PI), recombination experiments with these two acidic lipids were planned. The recombination system with PI did not show a tryptamine binding capacity, while the PS fraction possessed a tryptamine binding capacity similar to that of the myelin butanol extracts. However, displacement studies revealed that the recombinant fraction with PS alone did not display the complete regeneration of the specificity which had been observed in the myelin extracts. All these observations infer that the tryptamine binding components from myelin proteolipids are lipid in nature, and its binding entity is mainly PS. However, some specifically organized constitution with PS and other lipid molecule(s) may be necessary to regenerate the original tryptamine binding properties.

Animals↗
PubMed1982

Butanol extracts from myelin fragments: identification of 5-hydroxytryptamine binding components.

To investigate the nature of 5-HT binding components originated from myelin butanol extracts (i.e. proteolipids), the recombinate experiments with acidic lipids were planned. Binding assay of 14C . 5-Ht to recombinates was carried out by Sephadex lh20 column chromatography. Among several recombinate fractions, recombinate of sulphatides, phosphatidylserine and phosphatidylinositol showed the same elution profile and binding capacity for 14C . 5-HT with those of the myelin extracts. The displacement studies with other neurotransmitters revealed that the recombinate of these three acidic lipids completely regenerated the specificity of the original myelin proteolipids. Namely, tryptamine, DA and ACh inhibited the 5-HT binding but NA had no effect. A double reciprocal plot of 5-HT binding represented a multiple binding mode, not a straight line. This observation suggested that, at least, three binding components (or sites) are implicated in the binding of 14C . 5-HT to this recombinate system having 3 Kds of 2.2 x 10(-7), 4.1 x 10(-7) and 4.2 x 10(-6) m. All these observations infer that the 5-HT binding components of myelin proteolipids are mainly three acidic lipids in nature.

Animals↗