Biomedical subjects

T Kuwaki

Publications and source records attributed to T Kuwaki.

At least 37 records · Page 2Linked to original sources

PubMed1999

Elevation of blood pressure by genetic and pharmacological disruption of the ETB receptor in mice.

Exogenously administered endothelin (ET) elicits both pressor and depressor responses through the ETA and/or the ETB receptor on vascular smooth muscle cells and ETB on endothelial cells. To test whether ETB has pressor or depressor effects under basal physiological conditions, we determined arterial blood pressure (BP) in ETB-deficient mice obtained by crossing inbred mice heterozygous for targeted disruption of the ETB gene with mice homozygous for the piebald (s) mutation of the ETB gene (ETBs/s). F1 ETB-/s and ETB+/s progeny share an identical genetic background but have ETB levels that are approximately (1)/(8) and (5)/(8), respectively, of wild-type mice (ETB+/+). BP in ETB-/s mice was significantly higher, by approximately 20 mmHg, than that in ETB+/s or ETB+/+ mice. Immunoreactive ET-1 concentration in plasma as well as respiratory parameters was not different between ETB-/s and ETB+/s mice. A selective ETB antagonist, BQ-788, increased BP in ETB+/s and ETB+/+ but not in ETB-/s mice. Pretreatment with indomethacin, but not with NG-monomethyl-L-arginine, can attenuate the observed pressor response to BQ-788. The selective ETA antagonist BQ-123 did not ameliorate the increased BP in ETB-/s mice. Moreover, BP in mice heterozygous for targeted disruption of the ETA gene was not different from that in wild-type controls. These results suggest that endogenous ET elicits a depressor effect through ETB under basal conditions, in part through tonic production of prostaglandins, and not through secondary mechanisms involving respiratory control or clearance of circulating ET.

Animals↗

PubMed1999

Responses of blood pressure and catecholamine metabolism to high salt loading in endothelin-1 knockout mice.

The molecular mechanism responsible for salt sensitivity is poorly understood. Mice heterozygous for the null mutation of the endothelin-1 (ET-1) gene, Edn1, may be a potential tool for studying this mechanism, because they have elevated blood pressure and disturbances in central sympathetic nerve regulation. In the present study, we used this mouse model to examine the degree to which ET-1 contributes to the responses of blood pressure and catecholamine metabolism to high salt loading. Male Edn1+/- heterozygous mice and Edn1+/+ wild-type littermates were given either a high salt (8%) or a normal salt (0.7%) diet for 4 wk. During the normal diet, renal ET-1 levels in Edn1+/- mice were approximately 50% lower than ET-1 levels in wild-type mice, whereas the high salt diet decreased renal ET-1 levels by about 50% in both Edn1+/- and wild-type mice. The high salt diet significantly increased urinary sodium excretion and fractional excretion of sodium (FENa) but did not affect circulating plasma volume, serum electrolytes, creatinine clearance, or systemic blood pressure. In addition, urinary norepinephrine and normetanephrine excretion were significantly increased, indicating that salt loading can increase sympathetic nerve activity in normal mice. These responses to salt loading did not differ between Edn1+/- mice and their wild-type littermates. We conclude that physiological changes in ET-1 production do not affect the responses of blood pressure and catecholamine metabolism to salt loading, although the renal ET-1 content is decreased by salt loading.

Animals↗

PubMed1998

Renal sympathetic nerve activity in mice: comparison between mice and rats and between normal and endothelin-1 deficient mice.

Recently generated knockout mice with disrupted genes encoding endothelin (ET)-1 showed an elevation of arterial blood pressure (AP) and supplied an evidence for intrinsic ET-1 as one of the physiological regulators of systemic AP. Little is yet known, however, why deficiency of ET-1, which was originally found as a potent vasoconstrictor, led to higher AP in these mice. To address this apparent paradox, we first developed a method to measure renal sympathetic nerve activity (RSNA) in mice using rats as reference and successively compared it between normal and ET-1 deficient mice. RSNA was successfully recorded in urethane-anesthetized and artificially ventilated mice by a slight modification of the method used for rats. At basal condition, mean AP (MAP) and RSNA in ET-1 deficient mice (105+/-2 mmHg and 9.71+/-1.49 muVs, n=20) were significantly higher than those in wild-type mice (96+/-2 mmHg and 5. 07+/-0.70 muVs, n=25). Basal heart rate (HR) and baroreflex-control of HR was not significantly different between the two. On the other hand, resting RSNA, RSNA range, and maximum RSNA were significantly greater in ET-1 deficient mice, and thus MAP-RSNA relationship was upwards reset. Hypoxia-induced increase in RSNA was not different between ET-1 deficient (73.4+/-9.4%) and wild-type mice (91.2+/-12.0%), while hypercapnia-induced one was significantly attenuated in ET-1 deficient mice (18.8+/-3.6 vs. 39.1+/-5.2% at 10% CO2). These results indicate that endogenous ET-1 participates in the central chemoreception of CO2 and reflex control of the RSNA. Baroreceptor resetting and normally preserved hypoxia-induced chemoreflex may explain a part of the elevation of AP in ET-1 deficient mice.

Animals↗

PubMed1998

Impaired anaphylactic responses with intact sensitivity to endotoxin in mice lacking a platelet-activating factor receptor.

Platelet-activating factor (PAF) is a potent phospholipid mediator with diverse biological activities in addition to its well-known ability to stimulate platelet aggregation. Pharmacologic studies had suggested a role for PAF in pregnancy, neuronal cell migration, anaphylaxis, and endotoxic shock. Here we show that disruption of the PAF receptor gene in mice caused a marked reduction in systemic anaphylactic symptoms. Unexpectedly, however, the PAF receptor-deficient mice developed normally, were fertile, and remained sensitive to bacterial endotoxin. These mutant mice clearly show that PAF plays a dominant role in eliciting anaphylaxis, but that it is not essential for reproduction, brain development, or endotoxic shock.

Anaphylaxis↗

PubMed1998

Quantitative analysis of thrombopoietin receptors on human megakaryocytes.

Thrombopoietin (TPO), or c-MPL ligand, is the primary regulator of megakaryocyte and platelet production. TPO receptors expressed on human megakaryocytes derived from peripheral blood (PB) and cord blood (CB) progenitors cultured in the presence of TPO have now been analyzed quantitatively. Like those on human PB platelets, TPO receptors on the cultured megakaryocytes exhibited a molecular mass of approximately 80 kDa. Various characteristics of PB- and CB-derived megakaryocytes indicated that the former were more mature than the latter. Both PB- and CB-derived megakaryocytes expressed a single class of high-affinity TPO receptors, with 1933 +/- 772 (n = 3) and 184 +/- 48 (n = 4) sites per cell, respectively. These data indicate that the number of TPO receptors on human megakaryocytes increases with cell maturation.

Blood Platelets↗

PubMed1998

Hypertension, hypertriglyceridemia, and impaired endothelium-dependent vascular relaxation in mice lacking insulin receptor substrate-1.

Insulin resistance is often associated with atherosclerotic diseases in subjects with obesity and impaired glucose tolerance. This study examined the effects of insulin resistance on coronary risk factors in IRS-1 deficient mice, a nonobese animal model of insulin resistance. Blood pressure and plasma triglyceride levels were significantly higher in IRS-1 deficient mice than in normal mice. Impaired endothelium-dependent vascular relaxation was also observed in IRS-1 deficient mice. Furthermore, lipoprotein lipase activity was lower than in normal mice, suggesting impaired lipolysis to be involved in the increase in plasma triglyceride levels under insulin-resistant conditions. Thus, insulin resistance plays an important role in the clustering of coronary risk factors which may accelerate the progression of atherosclerosis in subjects with insulin resistance.

Animals↗

PubMed1998

Neutralization of biological activity and inhibition of receptor binding by antibodies against human thrombopoietin.

Thrombopoietin (TPO) is a recently isolated cytokine that primarily regulates megakaryocytopoiesis and thrombopoiesis. We recently reported the development of a variety of antibodies (Abs) to synthetic peptides of human (h)TPO and to recombinant human TPO (rhTPO). In this study, we characterized the Abs and mapped immunologically distinct areas of the molecule. Among the five different antipeptide polyclonal Abs, only one, raised against synthetic peptide D8 to Q28, neutralized the TPO-dependent growth of FDCP-2 cells expressing human Mpl (FDCP-hMpl5 cells). One out of seven anti-rhTPO monoclonal Abs, designated as TN1, also showed neutralizing activity. TN1 was found to be specifically reactive with two proteolytic fragments, residues S1 to R117 and A60 to K122 of hTPO, indicating that the epitope(s) of TN1 is localized in residues A60 to R117 of the molecule. These two neutralizing Abs inhibited the binding of biotinylated rhTPO to FDCP-hMpl5 cells. On the other hand, the other Abs, which reacted with five polypeptides of S47 to D62, L108 to A126, N172 to A190, S262 to T284, and P306 to G332 of hTPO, did not show either the neutralizing activity or the ability to inhibit the binding of biotinylated rhTPO to the cell surface hMpl. These findings indicate that two regions, residues D8 to Q28 and A60 to R117 of hTPO, may contain the domains associated with its receptor, C-Mpl. These Abs characterized here are valuable for studying the structural analysis and the biological function of hTPO mediated by its receptor.

Antibodies↗

PubMed1998

Successful treatment of pustulosis palmaris et plantaris with granulocyte colony stimulating factor in a patient with hereditary neutropenia.

A 26-year-old female with hereditary neutropenia had Pustulosis Palmaris et Plantaris. Drug administration did not improve her symptoms. Following administration of granulocyte colony stimulating factor (G-CSF; filgrastim), her neutrophil count increased from 300 to 58,000/microliters, and her dermatosis improved. Pustulosis Palmaris et Plantaris has been described as a representative second lesion of focal infection. Neutropenia may be one cause of Pustulosis Palmaris et Plantaris due to refractory focal infection. In the present case, increase in neutrophils with G-CSF may have improved focal infection, resulting in improvement in refractory Pustulosis Palmaris et Plantaris.

Adult↗

PubMed1998

Plasma thrombopoietin (TPO) levels and expression of TPO receptor on platelets in patients with myelodysplastic syndromes.

Data on endogenous thrombopoietin (TPO) levels and their regulation in myelodysplastic syndromes (MDS) are sparse. We examined the plasma TPO level of 85 MDS patients by a sensitive enzyme immunoassay and the platelet expression of TPO receptor (TPO-R) protein, which metabolizes endogenous TPO, in 19 MDS patients with an equilibrium binding assay using 125I-TPO. The MDS patients had higher plasma TPO levels (7.0 +/- 9.3 fmol/ml) than 52 normal subjects (P < 0.0001). Refractory anaemia (RA) patients (n = 39) had higher plasma TPO levels than patients (n = 28) with RA with excess blasts (RAEB) or RAEB in transformation (RAEB-t) (P = 0.0002), irrespective of similar platelet counts in these groups. The plasma TPO level correlated inversely with the platelet count in RA patients (P = 0.0027) but not in RAEB and RAEB-t patients (P = 0.7865). These data suggest that the physiological pathway for TPO production and metabolism is conserved, at least partially, in RA, but deranged in RAEB/RAEB-t. The number of TPO-R per platelet was significantly smaller in 19 MDS patients (17.5 +/- 13.3) than in normals (P = 0.0014), but similar between RA patients and patients with RAEB and RAEB-t. Further, the bone marrow megakaryocyte count, determined in 31 MDS patients, was quite similar between RA patients and patients with RAEB or RAEB-t. Thus, in addition to thrombocytopenia, a reduced platelet TPO-R number may contribute to elevated plasma TPO levels in MDS, and a regulatory pathway for circulating TPO other than platelet TPO-R and marrow megakaryocytes, such as blasts expressing TPO-R, may operate in RAEB/RAEB-t.

Blood Platelets↗

PubMed1998

Mobilization of primitive haemopoietic progenitor cells and stem cells with long-term repopulating ability into peripheral blood in mice by pegylated recombinant human megakaryocyte growth and development factor.

We investigated in detail the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on peripheral blood progenitor cell (PBPC) mobilization in male BDF1 mice. Treatment with PEG-rHuMGDF for 5 d stimulated a striking expansion of the circulating levels of multiple types of colony-forming units in culture (CFU-c), including CFU-granulocyte-macrophage, CFU-megakaryocyte, burst-forming units-erythroid, and multipotent CFU-c, and primitive day-12 CFU-spleen. All of these progenitors were mobilized into the peripheral blood (PB) with similar kinetics; their numbers peaked after the cessation of treatment and then declined earlier than platelet numbers peaked. The maximal increase in any of the four CFU-c in the PB was attained with at least 300 microg/kg/d of PEG-rHuMGDF, whereas peripheral platelet counts plateaued at 30 microg/kg/d. Adoptive transfer with PB from PEG-rHuMGDF-treated donor mice resulted in greater survival of lethally irradiated recipients. The majority of the recipients that survived at 187 d after transplantation with PEG-rHuMGDF-mobilized PB showed significant donor engraftment at the progenitor cell level. The combined administration of appropriate doses of PEG-rHuMGDF and recombinant human granulocyte colony-stimulating factor induced a synergistic increase in the circulating levels of the four CFU-c compared to either factor alone. These results indicate that PEG-rHuMGDF as a single agent can mobilize a full spectrum of PBPCs in mice.

Animals↗

PubMed1998

Systemic and renal response to salt loading in endothelin-1 knockout mice.

Endothelin-1 (ET-1) knockout mice demonstrate elevated blood pressure, which may be associated with disturbance in central cardiorespiratory regulation. In this study we examined responses to salt loading in ET-1 knockout mice to investigate whether ET-1 is involved in the pathophysiology of salt-sensitive hypertension. Male Edn1+/- heterozygous mice and their wild-type littermates were fed either a high NaCl (8%) or a normal (0.2%) diet for 4 weeks. Systemic blood pressure and tissue ET-1 levels were measured as well as several parameters relating to sodium handling and volume homeostasis. On normal diet, renal ET-1 levels of Edn1+/- mice were about 50% of those of wild-type mice. A high-salt diet caused a significant decrease in renal ET-1 levels by about 50% in both groups. Urine volume, urinary sodium excretion, and FENa in mice on the 8% NaCl diet were significantly higher than those in mice on the 0.2% NaCl diet, whereas there were no differences in circulating plasma volume, serum electrocytes, and creatinine clearance. These responses were similar in Edn1+/- and wild-type mice. Although systemic blood pressure was significantly higher in Edn1+/- mice than in the wild-type, the effect of salt loading on blood pressure was not significant in either Edn1+/- or wild-type mice. We conclude that changes in ET-1 production within a physiologic range do not affect salt sensitivity, although renal ET-1 content is decreased by salt loading.

Animals↗

PubMed1998

Development of an analysis system for 24-hour blood pressure and heart rate variability in the rat.

We have developed a system to evaluate 24-h changes of heart rate (HR) and blood pressure variability (BPV) in free moving rat. As a sensor to monitor electrocardiogram, we used the telemetry system. Analog data were sent to an analog to digital converter directly. The data were digitized at a sampling frequency of 1250 Hz and digitally stored on a computer. RR-intervals (difference of successive RR in electrocardiogram) were calculated using custom designed programs. Circadian change of the blood pressure and heart rate variability were calculated. We suggest that the system is a powerful tool in the evaluation of circadian changes of hemodynamics and autonomic nervous function in free moving rats.

Algorithms↗

PubMed1998

Cranial and cardiac neural crest defects in endothelin-A receptor-deficient mice.

Neural crest cells arise in the dorsal aspect of the neural tube and migrate extensively to differentiate into a variety of neural and non-neural tissues. While interactions between neural crest cells and their local environments are required for the proper development of these tissues, little information is available about the molecular nature of the cell-cell interactions in cephalic neural crest development. Here we demonstrate that mice deficient for one type of endothelin receptor, ETA, mimic the human conditions collectively termed CATCH 22 or velocardiofacial syndrome, which include severe craniofacial deformities and defects in the cardiovascular outflow tract. We show that ETA receptor mRNA is expressed by the neural crest-derived ectomesenchymal cells of pharyngeal arches and cardiac outflow tissues, whereas ET-1 ligand mRNA is expressed by arch epithelium, paraxial mesoderm-derived arch core and the arch vessel endothelium. This suggests that paracrine interaction between neural crest-derived cells and both ectoderm and mesoderm is essential in forming the skeleton and connective tissue of the head. Further, we find that pharyngeal arch expression of goosecoid is absent in ETA receptor-deficient mice, placing the transcription factor as one of the possible downstream signals triggered by activation of the ETA receptor. These observations define a novel genetic pathway for inductive communication between cephalic neural crest cells and their environmental counterparts.

Animals↗

PubMed1997

Physiological role of brain endothelin in the central autonomic control: from neuron to knockout mouse.

Although endothelin (ET) was discovered as a potent vascular endothelium-derived constricting peptide, its presumed physiological and pathophysiological roles are now considered much more diverse than originally though. Endothelin in the brain is thought to be deeply involved in the central autonomic control and consequent cardiorespiratory homeostasis, possibly as a neuromodulator or a hormone that functions locally in an autocrine/paracrine manner or widely through delivery by the cerebrospinal fluid (CSF). This notion is based on the following lines of evidence. (1) Mature ET, its precursors, converting enzymes, and receptors all are detected at strategic sites in the central nervous system (CNS), especially those controlling the autonomic functions. (2) The ET is present in the CSF at concentrations higher than in the plasma. (3) There is a topographical correspondence of ET and its receptors in the CNS. (4) The ET is released by primary cultures of hypothalamic neurons. (5) When ET binds to its receptors, intracellular calcium channels. (6) An intracerebroventricular or topical application of ET to CNS sites elicits a pattern of cardiorespiratory changes accompanied by responses of vasomotor and respiratory neurons. (7) Recently generated knockout mice with disrupted genes encoding ET-1 exhibited, along with malformations in a subset of the tissues of neural crest cell lineage, cardiorespiratory abnormalities including elevation of arterial pressure, sympathetic overactivity, and impairment of the respiratory reflex. Definitive evidence is expected from thorough analyses of knockout mice by applying conventional experimental methods.

Amino Acid Sequence↗

PubMed1997

Differential central modulation of the baroreflex by salt loading in normotensive and spontaneously hypertensive rats.

In salt-sensitive hypertensive animal models and human subjects compared with their salt-resistant counterparts, sympathetic activity is abnormally enhanced during a high salt diet. We examined whether salt loading differentially modulates the arterial baroreceptor reflex (ABR), a major control mechanism of arterial pressure and sympathetic vasomotor activity, in young normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Six-week-old WKY and SHR were fed a normal (0.66%) or high (8.00%) salt diet for 4 weeks. After the diet regimen, baseline levels of mean arterial pressure (MAP), renal sympathetic nerve activity (RSNA), and the overall and central properties of the ABR were compared among the four groups of rats under halothane anesthesia. In WKY, a high salt diet did not affect baseline arterial pressure and RSNA but potentiated the ABR, as evidenced by an increase in the maximal slope of MAP-RSNA and MAP-heart rate relationships. In SHR, by contrast, salt loading accelerated hypertension and sympathetic overactivity and impaired the ABR. Salt-induced modulation of the ABR was associated with that of the central property, since reflex inhibition of RSNA by stimulation of the aortic depressor nerve was augmented in WKY and attenuated in SHR. These results suggest that differential modulation of the central mechanism subserving the baroreflex control of sympathetic activity at least partly accounts for the difference in salt sensitivity between WKY and SHR.

Animals↗

PubMed1997

Determination of ventilatory volume in mice by whole body plethysmography.

We examined the reliability of whole-body plethysmography (WBP) for determining the ventilatory volume in mice. The degree of agreement of tidal volume (VT) determined consecutively by WBP with that by direct plethysmography (DP) or pneumotachography (PT) was analyzed using urethane-anesthetized young male 129/Sv mice. Between WBP and DP, the mean difference of VT was 0.000 ml with a 95% confidence interval of -0.003 to 0.002, whereas that between WBP and PT was 0.000 ml (-0.004 to 0.003). The limits of agreement, or mean +/- 2SD of the difference, were -0.008 and 0.008 ml in WBP vs. DP, and -0.011 and 0.010 ml in WBP vs. PT. These values were 5.1-6.6% of the average VT (0.157 ml in WBP and DP, and 0.165 ml in WBP and PT). Thus, VT can be determined by WBP without any bias and with a systemic error of less than 7% of that measured by the other direct methods examined. Through applying WBP to conscious 129/Sv and 129/SvJ-ICR hybrid mice, we further studied the effects of differences in mouse strain, age and sex on respiratory rate (f) and minute volume (V = VT.f), and their reflex changes in responses to systemic hypoxia and hypercapnia. V was significantly greater in young (10-16 weeks old) male 129/Sv than in young male 129/SvJ-ICR hybrids (139 +/- 11 and 73 +/- 8 ml/min/100 g, respectively), while the pattern of reflex responses was similar. In aged (32-44 weeks old) male 129/Sv, the basal level of V and its reflex increases were all diminished as compared to those in their young counter parts. However, we were unable to find significant differences between aged male and female 129/Sv mice. In conclusion, WBP is a useful and reliable method to determine ventilatory parameters in conscious and anesthetized mice. Furthermore, there are considerable strain- and age-related differences in baseline ventilation and reflex responses to hypoxia and hypercapnia.

Age Factors↗

PubMed1997

[Central autonomic control in human hypertension].

Sympathetic nerve fibers innervating the heart and blood vessels exhibit tonic activity which originates from vasomotor neurons in the rostral ventrolateral medulla (RVLM). Whether sympathetic nerve activity (SNA) is elevated in humans with essential hypertension is still unsettled. Recording of muscle SNA (MSNA) by the microneurographic method mostly points to elevated SNA in hypertensives including borderline and mildly hypertensive patients. Exaggerated MSNA is most conspicuous in the accelerated hypertensives. Power spectral analyses of heart rate and blood pressure variability provide controversial results on this issue. Nevertheless, most studies demonstrate altered sympathetic cardiovascular control in hypertensive subjects. In young borderline hypertensives, the sympathetic response to mental stress or passive tilting is exaggerated, whereas in elderly subjects it is attenuated.

Blood Pressure↗

PubMed1996

Effects of recombinant human granulocyte colony-stimulating factor on the growth potential of two murine myeloid leukemias.

We investigated the in vitro and in vivo effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the proliferation of two murine leukemic cell lines. The rhG-CSF stimulated leukemic colony formation of the promyelocytic leukemic cell line L-8801 in methylcellulose culture and increased the number of L-8801 cells in liquid culture. However, rhG-CSF treatment prolonged the median survival period of mice implanted with L-8801 cells and the emergence of the leukemic blast cells in peripheral blood. Meanwhile, rhG-CSF had no influence on that of the megakaryoblastic leukemic cells L-8057 and failed to prolong the median survival period of L-8057 leukemic mice. Receptor binding analysis revealed that L-8801 cells expressed a G-CSF receptor (Kd=125 pM, 479 binding sites/cell) and L-8057 cells had no G-CSF receptors. Then, we examined the growth potential of these cells. The median survival period was longer for mice implanted with L-8801 cells cultured with rhG-CSF for 72 h in vitro than for cells grown without rhG-CSF. Furthermore, the median survival period of mice implanted with spleen cells from L-8801 leukemic mice treated with rhG-CSF was prolonged compared with those from leukemic mice without rhG-CSF. In contrast, there was no effect of rhG-CSF on the growth potential of the spleen from L-8057 leukemic mice. The results of our present study demonstrate that rhG-CSF reduced the growth of L-8801 leukemic cells in vitro and in vivo mediated through G-CSF receptors, thereby suppressing the development of leukemia.

Animals↗