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The accumulation of polymorphonuclear leukocytes (PMN) in synovial fluid is a common feature of rheumatoid arthritis (RA). We studied the chemotactic response of PMN obtained from the synovial fluid and from the peripheral blood of patients with RA using a modified Boyden's method, in which interleukin-8 (IL-8) or N-formyl-methionyl-leucyl-phenylalanine (FMLP) was used as a chemotactic agent. The IL-8-induced response of peripheral blood PMN from 15 patients with RA did not differ from that of 15 healthy controls. A decreased chemotactic response to IL-8 was, however, observed in PMN from the synovial fluid of 12 patients with RA compared with peripheral blood cells of the same individual. This defective chemotactic ability of PMN was inversely correlated with the number of infiltrating cells in the synovial fluid. We also obtained similar results with FMLP. These results indicate that the chemotactic ability of PMN may be reduced after migrating to the synovial fluid.

The activity of serum type IV collagen-degrading enzyme was measured in 18 patients with hepatocellular carcinoma (HCC). The enzyme activity was significantly higher, in HCC patients with a tumor thrombus in the portal vein than in healthy controls, liver cirrhosis patients and HCC patients without a tumor thrombus. Moreover, the activity in HCC patients with lung metastasis tended to be higher than that in HCC patients without lung metastasis. The activity of serum type IV collagen-degrading enzyme did not correlate with tumor size, serum alpha-fetoprotein level, or macroscopic classification of tumor growth. These results suggest that the activity of serum type IV collagen-degrading enzyme represents the metastatic potential or the ongoing metastatic activity of HCC. The enzyme is a useful serum marker of metastasis from HCC.

Type IV collagen-degrading enzyme activity was detected in human serum. Serum was preincubated with 4-aminophenylmercuric acetate and trypsin to activate the enzyme prior to assay. Type IV collagen, purified from human placentas and radiolabeled with [1-14C] acetic anhydride, was used as the substrate. The enzyme activity was measured at pH 7.5 and inhibited by treatment with ethylenediaminetetraacetic acid or heat. The assay of type IV collagen-degrading enzyme in human serum might be useful for estimating the degradation of type IV collagen.

The stability of recombinant human superoxide dismutase (r-hSOD) in buffer solutions was studied in solutions at various pH and temperatures. Additionally, we studied the effects of incubation with proteases, serum and two types of hypothermic perfusates. R-hSOD was stable in the pH range of 6-11 and at temperatures up to 80 degrees C for 30 min. R-hSOD activity was not affected by incubation with trypsin, aminopeptidase M or serum for 2 h. R-hSOD activity determined at various temperatures (4-37 degrees C) did not vary remarkably. R-hSOD in hypothermic perfusates was stable at 4-37 degrees C for 24 h.

In vitro release of atrial natriuretic peptide (ANP) from atria was examined by ANP radioimmunoassay. Isolated right rat atria were incubated in Krebs-Ringer bicarbonate buffer, and test substances were added to the incubation medium. The fluid was assayed for rat ANP by a radioimmunoassay method recently developed in our laboratory. We produced an antiserum to human ANP(99-216) (alpha-hANP(1-28)) which showed a good cross-reactivity of 63% with rat ANP(99-126) (alpha-rANP(1-28)) and was useful for measuring rat ANP concentrations of the medium. Application of the medium to a reverse phase high performance liquid chromatography (HPLC) system resulted in a single peak of immunoreactive rat ANP corresponding to a small molecular weight synthetic rat ANP of 28 amino acid residues. Catecholamines (epinephrine, norepinephrine and isoproterenol) reduced the basal secretion of ANP, whereas acetylcholine stimulated the release of ANP. Forskolin and dibutyryl cyclic AMP did not affect the release of ANP. These results suggest the possibility that the regulation of ANP release may be partially associated with adrenergic and cholinergic mechanisms.

Ether and restraint stress-induced peripheral plasma corticotropin releasing hormone (CRH), arginine vasopressin (AVP), oxytocin (OXY) and adrenocorticotropin (ACTH) levels were measured by radioimmunoassays. Plasma CRH, AVP, OXY and ACTH rose to approximately twice the level of control rats 2 min after the onset of a 1-min exposure to ether. Plasma CRH rose further 5 min after the onset of ether stress, while plasma AVP and OXY returned to the baseline levels at 5 min. Plasma CRH, OXY and ACTH showed significant elevation 2 min after the onset of restraint stress, while plasma AVP did not show a significant change. Plasma OXY and ACTH rose further 5 min after the onset of restraint stress, whereas plasma CRH returned to baseline levels. CRH and OXY concentrations in the hypothalamic median eminence decreased 5 min after the onset of ether exposure and restraint, while the AVP concentration did not differ from control levels. The results, including the discrepancy between plasma CRH and ACTH 5 min after stress, suggest that CRH in the peripheral plasma is derived from both hypothalamic and extrahypothalamic tissues. The levels of stress-induced CRH in the peripheral plasma were sufficient to stimulate ACTH release. These results suggest that ether and restraint stress elevate plasma CRH shortly after the onset of the stress, and that this elevation in the plasma CRH level is at least partly responsible for stress-induced ACTH secretion.

Some patients with rheumatoid arthritis (RA) as well as those with other collagen diseases are positive for antinuclear antibody (ANA). We investigated the frequency of positivity for ANA in 104 patients with RA and evaluated the clinical features and laboratory data in the ANA-positive and -negative groups. The presence of ANA in sera was studied by indirect immunofluorescence using HEp-2 cells as the antigen substrate. Sera with a positive fluorescence at a dilution of 1:20 were considered to be positive for ANA. Of the 104 patients, 39 (37.5%) were positive for ANA. The staining pattern in the positive cases varied, but most were speckled (64.1%) and homogeneous (48.7%). A small number showed a nucleolar (20.5%) or a centromere (10.3%) pattern. None showed a shaggy pattern. The ANA titer was lower in RA patients compared with those with other collagen-related diseases such as systemic lupus erythematosus or progressive systematic sclerosis. None of the patients positive for ANA with either a nucleolar or centromere staining pattern had progressive systemic sclerosis or the CREST syndrome. One patient each had Raynaud's phenomenon and pulmonary fibrosis. There was no correlation between ANA positivity and indicators of joint inflammation. The prevalence of ANA positivity in patients with advanced or prolonged disease was higher than those with early stages or short durations. There was no correlation with drug therapy.

We examined 8 normal subjects and 16 patients with non-functioning pituitary tumors with a combined anterior pituitary test to evaluate the clinical usefulness of the test. Diagnoses included 9 of chromophobe adenoma, 3 of craniopharyngioma, 2 of Rathke's cleft cyst, and 1 each of intrasellar cyst and tuberculum sella meningioma. All subjects received hypothalamic releasing hormones: 1 micrograms/kg corticotropin releasing hormone (CRH), 1 micrograms/kg growth hormone releasing hormone (GRH), 500 micrograms thyrotropin-releasing hormone (TRH), 100 micrograms luteinizing hormone releasing hormone (LH-RH), and a relatively small dose (5 mU/kg) of lysine vasopressin (LVP). In the normal subjects, the addition of LVP potentiated the secretion of adenocorticotropic hormone (ACTH) induced by CRH, but had no significant effect on the secretion of other anterior pituitary hormones. In the combined test with 5 releasing hormones, the plasma ACTH and cortisol responses were not impaired in the majority of the patients before pituitary surgery. Serum thyroid-stimulating hormone (TSH), prolactin (PRL) and follicle-stimulating hormone (FSH) responses were not impaired in 82%, 70% and 67% of the patients, respectively, while the serum LH and GH responses were impaired in 67% and 73% of the patients, respectively. Following pituitary surgery, responses of these hormones to combined testing were similarly impaired in more than 75% of the patients. These results indicate that plasma ACTH, cortisol and serum TSH responses are fairly good before pituitary surgery but are impaired significantly after surgery. No subjects experienced any serious adverse effects related to the testing. These results suggest that combined testing with hypothalamic hormones is a convenient and useful method for evaluating pituitary function.

The effects of salt loading and adrenalectomy on arginine vasopressin (AVP) mRNA levels in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) of the hypothalamus were studied by semiquantitative in situ hybridization histochemistry, using a synthetic oligonucleotide probe and a computer-assisted image analysis system. Salt loading (2% NaCl) for 7 days produced marked increases in AVP mRNA levels in the magnocellular neurons of the PVN, SON, and accessory nuclei. Adrenalectomy caused an increase in AVP mRNA expression in the magnocellular part of the PVN and the expansion of hybridization signals into its medial parvocellular region, where the cell bodies of corticotropin-releasing hormone (CRH) neurons are located. No apparent alteration of AVP mRNA levels was observed in the SON following adrenalectomy. These results indicate that hyperosmotic stimulation and the loss of circulating glucocorticoids had differential effects on AVP gene expression in the PVN and SON, and that the magnocellular PVN and SON neurons responded in different manners to the loss of feedback signals.

We made posterior hypothalamic knife cuts in rats to transect the fibers of the medial forebrain bundle (MFB) at the level of the mammillary body. The role of the MFB in the baroreflex and hemorrhage-induced hormonal responses was then examined in the unanesthetized, freely moving condition. The slopes for the relationship between changes in pulse interval and mean arterial pressure (MAP) in the posterior-cut group were significantly steeper than those in the sham-cut group both when there were phenylephrine-induced increases in MAP (1.13 +/- 0.07 vs 0.86 +/- 0.10 msec/mmHg) and nitroprusside-induced decreases in MAP (1.16 +/- 0.10 vs 0.77 +/- 0.05 msec/mmHg). This result indicates that posterior cuts elevated baroreflex sensitivity when MAP was increased or decreased. The resting MAP was not changed, but the resting heart rate (HR) was lowered by the posterior cuts. Furthermore, the posterior cuts augmented hypotensive hemorrhage-induced bradycardia. Hypotensive hemorrhage (16-17 ml/kg) caused elevation of the plasma catecholamine, ACTH and vasopressin (AVP) levels, but the posterior cuts attenuated these hormonal responses. These results indicate that the fibers in the MFB have a tonic inhibitory effect on the baroreflex in the resting condition, and play a stimulatory role in hemorrhage-induced catecholamine, ACTH and AVP responses.

To elucidate the effect of the arginine vasopressin (AVP) system in vivo, especially V1 and V2 activity, on blood pressure, we measured the acute changes in blood pressure and heart rate after AVP, OPC-21,268 (a V1 receptor antagonist), and OPC-31,260 (a V2 receptor antagonist) were injected intravenously in anesthetized spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats at the age of 15 weeks. Compared with the control period, single injection of AVP 5 ng/kg significantly increased systolic blood pressure in WKY rats without a concomitant increase in heart rate, but there was no significant increase in blood pressure in SHR. In contrast, single injection of either OPC-21,268 3 mg/kg or OPC-31,260 3 mg/kg did not affect blood pressure or heart rate in either SHR or WKY rats. Injection of AVP after the administration of OPC-31,260 induced a greater increase in blood pressure in SHR than in WKY rats, whereas injection of AVP after the administration of OPC-21,268 did not induce any clear increase in blood pressure in SHR or WKY rats. These results suggest that SHR have enhanced pressor activity mediated by V1 receptors and that this increase may be due to an increase in their number. In conclusion, enhancement of V1 activity may contribute to the development of high blood pressure in SHR.

We recently reported that stimulation of the arginine vasopressin (AVP) V1-receptor enhanced the pressor response in spontaneously hypertensive rats (SHR). In the present study, we investigated acute changes in systolic blood pressure (SBP) and heart rate (HR) after intravenous injections of AVP, OPC-21268 (a V1-receptor antagonist), and OPC-31260 (a V2-receptor antagonist), in anesthetized DOCA-salt hypertensive rats (DOCA) and age-matched sham-operated Wistar rats (control) to determine whether the pressor effect is specific to SHR or is present in other hypertensive animal models. SBP increased significantly in DOCA rats 9 min after injection of AVP 5 ng/kg without a concomitant increase in HR. Neither OPC-21268 3mg/kg nor OPC-31260 3mg/kg caused significant changes in SBP or HR. SBP tended to increase when AVP was administered after injection of OPC-31260. HR increased significantly 15 min after the combined treatment with OPC-31260 and AVP in DOCA rats compared with control rats. SBP did not change significantly when AVP was administered after injection of OPC-21268 in DOCA or control rats, but HR decreased significantly from 1 to 4 min after injection of AVP in DOCA rats. Our results suggest that V1-receptor stimulation does not enhance the pressor response in the DOCA rat, which is a model of volume-dependent hypertension, suggesting that the AVP system, especially V1-receptor, is not as important in the development or maintenance of hypertension in DOCA rats as in SHR.

Plasma immunoreactive CRF measured by radioimmunoassay decreased rapidly after intravenous injection of synthetic ovine corticotropin releasing factor (CRF) and showed a bi-exponential decay curve in five macaca fuscatas. Half lives of plasma immunoreactive CRF were 5.8 +/- 1.4 (Mean +/- SEM) min for the fast component and 38.3 +/- 2.4 min for the slow component. A bolus injection of 5 micrograms/kg CRF significantly increased the plasma cortisol level. CRF at 5 micrograms/kg induced a delayed response of ACTH and cortisol. Arginine vasopressin (AVP) at 0.5 micrograms/kg induced a slight increase in plasma ACTH and cortisol, but AVP at 0.1 micrograms/kg evoked no significant increase. When 0.5 micrograms/kg CRF and 0.1 micrograms/kg AVP were administered simultaneously, significant ACTH and cortisol responses occurred. The results indicate that CRF and AVP act synergistically to stimulate ACTH secretion in vivo.

The effects of angiotensin II, catecholamines and glucocorticoid on CRF-induced ACTH release were examined using rat anterior pituitary cells in monolayer culture. Synthetic ovine CRF induced a significant ACTH release in this system. Angiotensin II produced an additive effect on CRF-induced ACTH release. The ACTH releasing activity of CRF was potentiated by epinephrine and norepinephrine. Dopamine itself at 0.03-30 ng/ml did not show any significant effect on ACTH release, but it inhibited CRF-induced ACTH release. Corticosterone at 10(-7) and 10(-6)M inhibited CRF-induced ACTH release. These results indicate that angiotensin II, catecholamines and glucocorticoid modulate ACTH release at the pituitary level.