Biomedical subjects

K Chihara

Publications and source records attributed to K Chihara.

At least 163 records · Page 9Linked to original sources

PubMed1996

The 69-84 amino acid region of the parathyroid hormone molecule is essential for the interaction of the hormone with the binding sites with carboxyl-terminal specificity.

We evaluated the competitive inhibitory effect of intact PTH, the amino-terminal PTH(1-34) fragment, and a series of truncated carboxyl-terminal PTH fragments on the binding of internally 35S-labeled human PTH(1-84) ([35S]hPTH(1-84)) to osteoblastic cells (ROS 17/2.8), in order to identify the minimum and critical elements within the PTH molecule for the interaction with the binding sites specific for the carboxyl-terminal region of the hormone. When the amino-terminal region of the PTH molecule was truncated stepwise, hPTH(35-84), hPTH(53-84) and hPTH(69-84), but not hPTH(70-84), significantly inhibited the [35S]hPTH(1-84) binding. On the other hand, the simple deletion of the carboxyl-terminal glutamine at position 84 of hPTH(53-84) [hPTH(53-83)] resulted in blunting the inhibitory effect of the peptide on the [35S]hPTH(1-84) binding. Furthermore, hPTH(35-84), hPTH(53-84) and hPTH(69-84), but not hPTH(70-84) nor hPTH(53-83), augmented the inhibitory effect of the amino-terminal PTH fragment [hPTH(1-34)] on the [35S]hPTH(1-84) binding. Of special interest was that the combination of hPTH(1-34) and hPTH(35-84) reproduced the inhibitory effect of unlabeled hPTH(1-84) on the [35S]hPTH(1-84) binding, on an equimolar basis. The 69-84 region of the PTH molecule thus appears to be crucial for binding to the carboxyl-terminal specific binding sites for PTH in osteoblasts. The interaction of the amino-terminal and carboxyl-terminal regions of a PTH molecule with their own respective binding sites seemed to occur in a fairly independent manner.

Alkaline Phosphatase↗

PubMed1996

Estrogen blocks parathyroid hormone (PTH)-stimulated osteoclast-like cell formation by selectively affecting PTH-responsive cyclic adenosine monophosphate pathway.

Several lines of evidence have previously indicated that estrogen inhibits PTH-induced bone resorption in vivo and in vitro. However, its mechanism remains unknown. Therefore, the present study was performed to investigate the effect of estrogen on PTH-stimulated osteoclast-like cell formation and clarify its mechanism. 17 beta-estradiol (17 beta-E2) significantly antagonized osteoclast-like cell formation stimulated by 10(-8) M human (h) PTH-(1-34) as well as 10(-8) M hPTH-related peptide (PTHrP)-(1-34)in osteoblast-containing mouse bone cell cultures. The conditioned medium derived from osteoblastic SaOS-2 cells or MC3T3-E1 cells pretreated with both PTH-(1-34) (10(-8)M) and 17 beta-E2(10(-8)M) stimulated osteoclast-like cell formation from hemopoietic blast cells more weakly than conditioned medium from cells pretreated with PTH-(1-34) alone. Moreover, 10(-8) M 17 beta-E2 significantly blocked the formation of osteoclast-like cells stimulated by 10(-8) M hPTH-(1-34) in spleen cell cultures derived from 5-fluorouracil-pretreated mice. On the other hand, 10(-8) M 17 beta-E2 significantly inhibited osteoclast-like cell formation stimulated by dbcAMP (10(-4)M) and Sp-cAMPS (10(-4)M), as well as forskolin (10(-5)M) in mouse bone cell cultures. In contrast, 10(-8)M 17 beta-E2 did not affect PMA (10(-7)M)-, A23187 (10(-7)M)-, or BAYK-8644 (5 x 10(-6) M)-stimulated osteoclast-like cell formation. In conclusion, the present study demonstrated that estrogen inhibits PTH-stimulated osteoclast-like cell formation by directly acting on hemopoietic blast cells as well as by indirectly acting on them via osteoblasts. The inhibitory effects of estrogen on PTH-stimulated osteoclast-like cell formation seemed to be mediated through blocking the cAMP-dependent protein kinase pathway but not by blocking calcium/protein kinase C.

Animals↗

PubMed1996

Novel ipriflavone receptors coupled to calcium influx regulate osteoclast differentiation and function.

Ipriflavone (7-isopropoxyisoflavone) is an effective antiresorptive agent used to treat osteoporosis. However, the mechanism of its action on osteoclasts and their precursor cells is not well understood. To determine whether the mechanism involves direct effects on osteoclasts or their precursors, we examined the effects of ipriflavone on cytosolic free calcium ([Ca2+]i) in osteoclasts and their precursors and measured specific binding of 3H-labeled ipriflavone. Highly purified chicken osteoclast precursors, which spontaneously differentiate into multinucleated osteoclasts in 3-6 days, were loaded with fura-2, and the subcellular [Ca2+]i distribution was monitored by videoimaging. Ipriflavone induced a rapid increase in [Ca2+]i followed by a sustained elevation [EC50 = 5 x 10(-7) M, 263 +/- 74 nM (SE) (n = 8) above basal levels, by 10(-6) M ipriflavone, sustained phase]. The responses were the same in differentiated chicken osteoclasts and isolated rabbit osteoclasts. An influx of extracellular Ca2+ is likely to be responsible for the ipriflavone-induced change in [Ca2+]i because the response was abolished by 0.5 mM LaCl3, or by Ca-free medium containing EGTA. Moreover, high [Ca2+]i levels were detected adjacent to the cell membrane after ipriflavone addition. Ipriflavone induced Ca influx mainly through dihydropyridine-insensitive Ca2+ channels, because nicardipine (10(-7)M) and verapamil (10(-7)M) had no effects on ipriflavone-induced [Ca2+]i responses. [3H]Ipriflavone binding studies indicated the presence of specific ipriflavone binding sites (two classes), both in precursor cells [dissociation constant (Kd), 7.60 x 10(-8)M, 2.67 x 10(-6)M] and in mature osteoclasts (Kd, 4.98 x 10(-8)M, 3.70 x 10(-6)M). Specific ipriflavone binding was not displaced by various modulators of avian osteoclast function, such as estradiol (10(-8)M) or retinoic acid (10(-6)M), indicating that ipriflavone receptors differ from the receptors for these Ca-regulating hormones. The fusion of osteoclast precursor cells was significantly inhibited by ipriflavone, which led to dose-dependent inhibition of bone resorption and tartrate-resistant acid phosphatase activity. Novel specific ipriflavone receptors that are coupled to Ca2+ influx were demonstrated in osteoclasts and their precursor cells. These ipriflavone receptors may provide a mechanism to regulate osteoclast differentiation and function.

Acid Phosphatase↗

PubMed1996

Short stature caused by a mutant growth hormone with an antagonistic effect.

The molecular basis of biologically inactive GH remained unclear until recently. We have very recently reported a child with short stature and a mutant GH caused by a single missense mutation in the GH-1 gene, which itself cannot transduce the GH-signal to the cells but can blunt the action of wild-type GH by virtue of its greater affinity for the GH binding protein (GHBP)/GH receptor. Briefly the clinical features of the patient are: At the age of 4.9 years his height was 81.7 cm (-6.1 SD) and bone age was 2 years. The patient's serum insulin-like growth factor-1 (IGF-1) concentration was 34 ng/ml. The basal serum GH concentration ranged from 7.0 to 14.0 ng/ml and peak concentrations after insulin hypoglycemia, arginine and L-dopa were 38.0, 15.0 and 35.0 ng/ml, respectively. A heterozygous single base substitution was identified in the GH-1 gene of the proband, predicted to convert codon 77 from arginine to cysteine. Isoelectric focusing revealed the presence of an abnormal GH peak in addition to a normal GH peak. The affinity of expressed mutant GH to GHBP was approximately 6 times higher than that of wild-type GH. The mutant GH not only failed to stimulate tyrosine phosphorylation by itself, but it also inhibited the activity of wild-type GH when added simultaneously even in a one tenth dose of wild-type GH. The child whom we reported is therefore the first case of short stature caused by mutant GH with an antagonistic effect.

Age Determination by Skeleton↗

PubMed1996

No correlation of growth hormone receptor gene mutation P561T with body height.

Analysis of the growth hormone receptor (GHR) gene in GH insensitivity syndrome revealed various mutations, mainly in the gene encoding the extracellular domain of GHR. On the other hand, the mutation of the gene encoding the cytoplasmic domain of GHR was not found. We have reported, in the cytoplasmic domain of a GHR gene, mutation P561T in a patient with Noonan syndrome who showed a blunted insulin-like growth factor I (IGF-I) response to an acute injection of GH. However, her mother possessing the same mutation had no growth failure. To clarify the significance of the GHR gene mutation P561T, 96 volunteers (41 males aged 21-80 years; 55 females, aged 20-80 years, were tested for the presence of this mutation. By the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, three of the 41 males and 11 of the 55 females examined revealed heterozygous missense mutation P561T. The body height (cm) was 168 +/- 5.3 (mean +/- SD) in three males with the mutation and 164.1 +/- 5.8 in 38 males without the mutation. The difference between them was not statistically significant. The body height in 11 females with the mutation was 152.6 +/- 5.4, which did not differ significantly from 151.3 +/- 6.2 in 44 females without the mutation. These findings suggest that the heterozygous missense mutation P561T in the cytoplasmic domain of GHR does not play a significant role in determining the final body height.

Adult↗

PubMed1996

[Growth hormone prevents the steroid myopathy in rats].

To clarify whether growth hormone possesses the counteracting effect on steroid myopathy, we examined the influence of simultaneous administration of recombinant human growth hormone (rhGH) with glucocorticoid on the diameter of muscle fibers in rats. Female Sprague-Dawley rats weighing 120-140 g were divided into four groups. A group treated with glucocorticoid alone was subcutaneously injected with 5 mg/kg/day triamcinolone, a GH-treated group with 10 IU/kg/day rhGH alone, a steroid-GH-group with the same doses of glucocorticoid and rhGH. The control rats were injected with the vehicle alone. After 14 days of treatment, each rat was anesthetized with ether and subjected to muscle biopsy of extensor digitorum longus (EDL) and soleus. Administration of rhGH alone failed to affect the diameter of muscle fibers in either EDL or soleus. Glucocorticoid treatment caused a significant reduction in the diameter of muscle fibers in the EDL, compared with the control. In the type II muscle fibers of the EDL, simultaneous administration of rhGH attenuated glucocorticoid-induced muscle atrophy significantly. We concluded that GH administration would at least partially prevents steroid myopathy.

Animals↗

PubMed1995

Neurokinin receptor antagonists inhibit the binding of growth hormone-releasing peptide to EP-1 human neuroblastoma cells.

KP-102, a second generation growth hormone-releasing peptide, specifically bound to the human neuroblastoma cell line, EP-1, cultured in vitro with a Kd value comparable to that in cell membranes of rat pituitary and hypothalamus. By crosslinking study, the molecular size of the KP-102-receptor complex was found to be approximately 80kDa. Substance P analogue, as well as nonpeptidyl antagonists of the neurokinin receptor, competed with KP-102 in its binding to EP-1 cells, although neither substance P nor substance K affected the binding. These results suggest that KP-102 binds to a receptor-like molecule resembling neurokinin receptor.

Alprostadil↗

PubMed1995

Cholecystokinin-B/gastrin receptor: a novel molecular probe for human small cell lung cancer.

The brain-gut hormones, gastrin and cholecystokinin, have a trophic effect on the gastrointestinal mucosa in vivo and promote the growth of several neoplastic cell lines. In this study, cholecystokinin-B/gastrin receptor has been demonstrated to provide a novel molecular marker for the diagnosis of small cell lung cancer by using biopsy specimens. Physiological expression of the receptor mRNA is detectable in particular areas of the human brain, stomach, and pancreas but not in the lung. The receptor mRNA was detected selectively in all small cell lung cancer (10 cases) with a RT-PCR assay. By contrast, it was detectable in only 1 of 13 squamous cell carcinomas or 21 adenocarcinomas of the lung. Thus, the cholecystokinin-B/gastrin receptor could be an attractive therapeutic target for small cell lung cancer.

Adenocarcinoma↗

PubMed1995

EDA-containing fibronectin is synthesized from rheumatoid synovial fibroblast-like cells.

OBJECTIVE: To identify the cells that synthesize EDA-containing fibronectin (FN) and examine the role of EDA+FN in the pathogenesis of rheumatoid joint lesions. METHODS: Localization of EDA+FN and c-Fos protein in rheumatoid joints was studied immunohistochemically by utilizing antibodies for EDA+FN and c-Fos. Expression of EDA+FN was studied by immunoelectron microscopy and in situ hybridization. The amount of EDA+FN was measured by enzyme-linked immunosorbent assay. RESULTS: EDA+FN was specifically localized in the synovial lining layer of synovium with active rheumatoid arthritis (RA) (n = 17), but not in that with osteoarthritis (n = 4) or with inactive fibrous RA (n = 2). EDA+FN messenger RNA was localized in the synovial lining layer. EDA+FN was immunoelectron microscopically localized in the synovial lining fibroblast-like (type B) cells. EDA+FN was also detected at the cartilage-pannus junction and on the surface of RA cartilage. Double staining showed that EDA+FN colocalized with c-Fos protein in the rheumatoid synovial lining layer. Quantification of EDA+FN showed that it was highly concentrated in rheumatoid synovial fluids. CONCLUSION: EDA+FN is synthesized by the synovial lining fibroblast-like (type B) cells in situ in rheumatoid synovium, and appears to be expressed in association with activated or transformed states of synovium.

Animals↗

PubMed1995

Stimulatory effect of bone morphogenetic protein-2 on osteoclast-like cell formation and bone-resorbing activity.

Although the action of bone morphogenetic protein (BMP) on osteoblast differentiation has been extensively investigated, its effect on osteoclast differentiation remains unknown. In the present study, in vitro effects of BMP-2 on osteoclast-like cell formation and bone resorption were examined. BMP-2 (1-100 ng/ml) significantly stimulated bone resorption by preexistent osteoclast-like cells in mouse bone cell cultures containing stromal cells, whereas it did not affect the bone-resorbing activity of isolated rabbit osteoclast-like cells. When BMP-2 was added to unfractionated bone cells after degeneration of preexistent osteoclast-like cells, BMP-2 dose-dependently stimulated osteoclast-like formation at a minimal effective concentration of 10 pg/ml. BMP-2 also enhanced the osteoclast-like cell formation induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Moreover, osteoclast-like cells newly formed by BMP-2 from unfractionated bone cells possessed the ability to form pits on dentine slices. Because these results indicated that BMP-2 directly or indirectly stimulated osteoclast differentiation and activity, we next examined the direct effect of BMP-2 on osteoclast precursors in the absence of stromal cells using hemopoietic blast cells derived from spleen cells. The mRNA for BMP-2/4 receptor was detected in hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor (GM-CSF) as well as osteoblastic MC3T3-E1 cells and MC3T3-G2/PA6 stromal cells by RNase protection assay. BMP-2 dose-dependently stimulated osteoclast-like cell formation from hemopoietic blast cells supported by GM-CSF at a minimal effective concentration of 10 pg/ml. BMP-2 also enhanced 1,25(OH)2D3-induced osteoclast-like formation from hemopoietic blast cells. The present data are the first to indicate that BMP-2 stimulates bone resorption through both direct stimulation of osteoclast formation and activation of mature osteoclasts, possibly via stomal cells, in vitro.

3T3 Cells↗

PubMed1995

Competition between c-fos and 1,25(OH)2 vitamin D3 in the transcriptional control of type I collagen synthesis in MC3T3-E1 osteoblastic cells.

Interaction between c-fos and 1,25(OH)2 vitamin D3 (VD) on the type I collagen synthesis was studied. VD inhibited collagen synthesis and type I collagen mRNA expression in MC3T3-E1 osteoblastic cells. In contrast, VD reversed the inhibition of collagen synthesis and mRNA expression of the c-fos transfectants that overexpressed c-fos gene to a comparable level as those of the control transfectants. The gel shift assay showed the vitamin D receptor (VDR) complex binding to vitamin D responsive element (VDRE) was inhibited under constitutively expressed c-fos gene, suggesting that c-fos gene product, c-Fos, may inhibit the binding of VDR complex to VDRE by making a c-Fos-VDR complex. The result suggests the existence of a fine tuning between c-fos and VD in the bone metabolism which may be relevant to the pathogenesis of rheumatoid bone lesion.

3T3 Cells↗

PubMed1995

Retinoic acid induces osteoclast-like cell formation by directly acting on hemopoietic blast cells and stimulates osteopontin mRNA expression in isolated osteoclasts.

Although retinoic acid (RA) has been considered to be a bone-resorbing agent both in vivo and in vitro, its mechanism remains still unclear. The present study was performed to examine the effect of RA on osteoclast-like cell formation in the presence or absence of osteoblasts and to study whether RA would affect osteopontin mRNA expression in isolated rabbit osteoclasts. RA (10(-8) and 10(-6) M) significantly stimulated the formation of osteoclast-like cell in osteoblast-containing mouse bone cell cultures. Also, RA caused a stimulation of osteoclast-like cell formation from hemopoietic blast cells supported by granulocyte macrophage-colony stimulating factor (GM-CSF) in mouse spleen cell cultures. However, RA did not affect blast cell number in these cultures and significantly inhibited GM-CSF-stimulated proliferation of hemopoietic blast cells. On the other hand, RA stimulated the bone-resorbing activity of mature osteoclasts in mouse bone cell cultures. Moreover, RA caused a stimulation of osteopontin mRNA expression in isolated rabbit osteoclasts. The present study demonstrated for the first time that RA stimulated osteoclast-like cell formation, presumably through directly acting on the hemopoietic blast cells, and that RA stimulated osteopontin mRNA expression in isolated rabbit osteoclasts.

Animals↗

PubMed1995

Effect of fasting and growth hormone (GH) administration on GH receptor (GHR) messenger ribonucleic acid (mRNA) and GH-binding protein (GHBP) mRNA levels in male rats.

To elucidate whether GHR and GHBP are coordinately regulated or not, we studied the effect of fasting with or without GH administration on the GHR and GHBP mRNAs in the liver as well as in extrahepatic tissues in rats. Tissues were collected from 7-week-old male rats by decapitation 1,3, and 7 days after the start of fasting. Liver GHR mRNA levels were not affected 1 day after the start of fasting but progressively decreased for the subsequent 3 and 7 days of fasting as compared with those in control rats fed ad libitum. In contrast, liver GHBP mRNA levels significantly rose after 1 day fasting, returned to the control level after 3 days and further reduced after 7 days of fasting. Changes in GHBP mRNA level after fasting were different among the tissues. A transient increase in GHBP mRNA levels was observed in muscle and heart as well as liver, while the GHBP mRNA levels in fat tissues did not change throughout 7 days of fasting. Next, bovine GH(bGH) was administered ip to the fasted rats and control fed rats for either 1 day(100 micrograms [corrected], tid) or 5 days(150 micrograms [corrected], daily). In fed rats, liver GHR mRNA level was significantly increased by 1 day bGH treatment, but after 5 days treatment with bGH it was not different from the level in saline-injected control. Accordingly, net increment of plasma IGF-I was 296.0 ng/ml with 1 day bGH treatment and 234.2 ng/ml with bGH administration for 5 days. In fasted rats, liver GHR mRNA level did not changed after 1 day treatment with bGH, but markedly decreased 5 days after bGH administration. Net increment of plasma IGF-I was slightly reduced to 284 ng/ml with 1 day treatment with bGH, and markedly decreased to 37.0 with bGH administration for 5 days. The effect of GH administration on liver GHBP mRNA level was virtually absent in either fasting or fed state. These findings suggest that GHR and GHBP mRNAs in the liver are expressed in different ways and that expression of GHBP mRNA is differently regulated among tissues.

Animals↗

PubMed1995

Increases in cortical neuropeptide Y and somatostatin concentrations following haloperidol-depot treatment in rats.

The concentrations of neuropeptide Y (NPY) and somatostatin (SS) have been said to be altered in the brain and cerebrospinal fluid of schizophrenic patients. This alteration could result from the neuroleptic treatment. Therefore, it is of interest to evaluate effects of long-term treatment with neuroleptics on the peptide concentrations in the brain. Haloperidol (HPD) is one of the most frequently used neuroleptics for the treatment of schizophrenia. We determined regional brain levels of NPY and SS following HPD administration in the rat. A single intraperitoneal injection of HPD, at a dose of 1 mg/kg, did not affect peptide levels in the brain regions studied. Four weeks after an intramuscular deposit injection of HPD decanoate, 50 mg/kg, NPY concentrations were increased in a number of areas of the cerebral cortex. SS content was also significantly increased in the lateral prefrontal cortex and anterior cingulate cortex. Both peptide levels were decreased in the striatum. These results suggest that the reduction found in these peptides' levels in the cerebral cortex of the brain from schizophrenic patients may not be the consequence of HPD treatment and that these peptides' levels might be increased in the cerebral cortex of the brain of schizophrenic patients following the treatment with HPD.

Animals↗

PubMed1995

Possible discrimination of Gitelman's syndrome from Bartter's syndrome by renal clearance study: report of two cases.

We observed two patients who had hypokalemic metabolic alkalosis as well as hypomagnesemia and hypocalciuria with elevated serum renin levels. In renal clearance studies in our patients using furosemide or thiazide, urine volume and chloride clearance (CCI) were increased after furosemide administration but not after thiazide administration. Furthermore, the distal fractional chloride reabsorption [CH2O/(CH2O + CCI)] was dramatically decreased by furosemide administration in our patients, whereas thiazide had little effect on it, suggesting the presence of a defect in the distal tubule rather than in the thick ascending loop of Henle. These findings are compatible with the concept of Gitelman's syndrome, a variant form of Bartter's syndrome.

Adult↗

PubMed1995

Successful treatment of hyperphosphatemic tumoral calcinosis with long-term acetazolamide.

We describe a patient with tumoral calcinosis, in which acetazolamide (ACZ) was, for the first time, tested for its therapeutic efficacy. The 19-year-old Japanese man had been suffering from multiple recurrent calcific masses with tenderness around the finger, knee, and toe joints since 10 months of age. Radiographs revealed several calcific subcutaneous masses around the finger joints, and calcific myelitis around the right knee joint and in the calvarium. The patient had hyperphosphatemia with elevated maximal threshold of renal phosphate excretion in the presence of normal kidney function and normocalcemia, suggesting a reduced ability to excrete phosphorus in the urine. A delay of disappearance of orally administered phosphate from the blood stream was found. A serum parathyroid hormone (PTH) level was normal, and responses to PTH and ACZ were also normal regarding the induction of phosphaturia. Since the masses tended to recur easily despite repeated surgical resections, we started medical treatment with phosphorus deprivation by oral aluminum hydroxide. However, the drug alone had no effect on hyperphosphatemia or calcific lesions, and ACZ was added in expectation of making the patient's phosphorus balance negative by its phosphaturic effect. Fourteen years of administration of the two drugs apparently improved the patient's symptoms, the biochemical findings, and the calcific lesions on radiographs. Thus, ACZ appeared to be useful for tumoral calcinosis resistant to phosphorus deprivation by aluminum hydroxide alone.

Acetazolamide↗

PubMed1995

Effect of retinoic acid on the proliferation and alkaline phosphatase activity of osteoblastic MC3T3-E1 cells by modulating the release of local regulators from monocytes.

There has been some evidence suggesting an important role of mononuclear cells at bone remodeling sites in the coupling of bone formation to bone resorption. Since cells of the monocyte-macrophage lineage produce important local regulators of bone remodeling, we examined effects of human monocytes-conditioned medium (CM) treated with retinoic acid on [3H] thymidine incorporation (TdR) and alkaline phosphatase (ALP) activity of osteoblastic MC3T3-E1 cells. Treatment of MC3T3-E1 cells with retinoic acid (10(-8) to 10(-6) M) caused an inhibition of TdR in a dose-dependent manner and an inhibition of ALP activity at 10(-6) M. Conditioned medium from monocytes untreated with retinoic acid caused a stimulation of TdR and an inhibition of ALP activity in these cells. In contrast, treatment of monocytes with retinoic acid (10(-8) or 10(-6) M) abolished both stimulation of DNA synthesis and inhibition of ALP activity induced by CM. The present study suggested that retinoic acid modulated osteoblast proliferation and ALP activity not only directly but also indirectly, presumably through modulating the release of local regulators as to bone remodeling from monocytes.

Alkaline Phosphatase↗

PubMed1995

Hydrolysis of a carboxy-terminal fragment of parathyroid hormone-related protein by rat kidney: evidence for a crucial role of meprin.

Although parathyroid hormone-related protein (PTHrP) is known to be secreted into the circulation as heterogeneous forms, with its N-terminal and C-terminal fragments as well as the intact form, the fate of these molecules in the plasma has not been fully understood. As for a C-terminal fragment, the kidney seems to be physiologically important in its metabolism, because it is elevated in circulating plasma in patients with chronic renal failure. In this study, we examined the mechanism by which a C-terminal fragment of PTHrP was metabolized by rat kidney and by other rat organs in vitro. When human (h) PTHrP-(109-141) was incubated for 2 h with an extract of rat kidney, it was almost completely hydrolyzed. This hydrolysis was readily blocked by the additions of o-phenanthrolline and dithiothreitol, indicating the participation in this process of a metallo-protease possessing disulfide bonds. This hydrolytic activity showed a meprin-like character, with being sensitive to actinonin but not to phosphoramidon. Furthermore, the participation of meprin itself in this process was directly confirmed by the experiment comparing degradation products of the peptide by the microvillar membranes of rat kidney with those by a purified rat meprin, in which a large part of the metabolites by rat kidney membranes corresponded to those by rat meprin. Among other rat organs examined, the extracts of the small intestine pancreas, spleen and urinary bladder exerted remarkable hydrolytic activities for hPTHrP-(109-141).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗