Heterogeneity of rat FSH by chromatofocusing: studies on in-vitro bioactivity of pituitary FSH forms and effect of neuraminidase treatment

1985 ◽  
Vol 105 (1) ◽  
pp. 17-27 ◽  
Author(s):  
W. F. P. Blum ◽  
G. Riegelbauer ◽  
D. Gupta
Keyword(s):  
Apparent Molecular Weight ◽  
Maximum Response ◽  
Dependent Manner ◽  
In Vitro Bioactivity ◽  
Response Curves ◽  
Neuraminidase Treatment ◽  
Rat Pituitary ◽  
Exclusion Chromatography ◽  
Reference Preparation

ABSTRACT This study concerned the resolution of rat pituitary FSH utilizing chromatofocusing. Among the 11 components resolved and positively identified, ten had apparent isoelectric points (pI) between 3·1 and 5·1. Approximately 1% of pituitary FSH eluted at pH 9·4. Treatment with varying amounts of neuraminidase followed by refocusing generated FSH components of higher pI values. Treatment with other glycosidases did not alter the elution characteristics in chromato-focusing, while exclusion chromatography established an inverse relationship between apparent molecular weight and pi. Dose–response curves of various FSH components and of the reference preparation in the current radioimmunoassay system were parallel to each other. A study of their in-vitro bioactivity, utilizing granulosa cells which produce a plasminogen activator due to FSH in a dose-dependent manner, provided the following evidence: increased acidity of the components led to (1) an increase of maximum response and (2) an increase of the dose necessary for half-maximum response. Considering the observed alterations in the hetereogeneity of FSH with changing physiological states of the animal, it is concluded that qualitative changes of the FSH molecule are perhaps involved in a modulatory role in the biopotencies of the hormone. J. Endocr. (1985) 105, 17–27

In vitro evidence of a role for inhibin in female rabbits

1984 ◽  
Vol 246 (3) ◽  
pp. E243-E248
Author(s):  
A. L. Goodman
Keyword(s):  
Granulosa Cells ◽  
Pituitary Cells ◽  
Dependent Manner ◽  
C Cells ◽  
Rat Pituitary ◽  
Reference Preparation ◽  
Lh Release ◽  
Rat Pituitary Cells ◽  
I Cells

To examine a regulatory role for inhibin in female rabbits, an in vitro bioassay for inhibin activity was modified to use cultured rabbit pituitary cells and charcoal-extracted porcine follicular fluid (pFFx) as a reference preparation. pFFx inhibited follicle-stimulating hormone (FSH) release in a dose-dependent manner in cultures from both intact (I) and castrate (C) does at doses that also inhibited FSH release by cultured rat pituitary cells. Basal FSH release by I cells was inhibited greater than 10% by 0.02% (vol/vol) and greater than 90% by greater than or equal to 0.2% pFFx, whereas in C cells maximal inhibition of FSH release plateaued at only approximately 75%. FSH secretion was restored after removal of pFFx in day 2 media. Luteinizing hormone (LH) release by C cells was not inhibited at any dose of pFFx, but in I cells LH was progressively inhibited to approximately 60% of control levels during day 2 (but not day 1). Charcoal-extracted media (0.25-1%) in which 5 X 10(5) rabbit granulosa cells had been earlier cultured for 72 h produced a parallel inhibition of FSH release. The present findings demonstrate that 1) rabbit pituitary cells are responsive to inhibin, i.e., pFFx preferentially inhibited FSH secretion in a direct, graded, and reversible manner and 2) rabbit follicular granulosa cells secrete an inhibin-like substance.

A comparison of preparations of highly purified human pituitary luteinizing hormone: differences in the luteinizing hormone potencies as determined by in vivo bioassays, in vitro bioassay and immunoassay

1982 ◽  
Vol 101 (3) ◽  
pp. 339-347 ◽  
Author(s):  
P. L. Storring ◽  
A. A. Zaidi ◽  
Y. G. Mistry ◽  
Monica Lindberg ◽  
Bridget E. Stenning ◽  
...  
Keyword(s):  
Luteinizing Hormone ◽  
In Vitro Bioactivity ◽  
In Vitro Bioassay ◽  
Response Curves ◽  
Human Pituitary ◽  
International Reference ◽  
Reference Preparation ◽  
In Vivo Bioassay

Abstract. The LH potencies of 12 preparations of highly purified human pituitary LH, from 6 laboratories, were estimated by 2 in vivo bioassays and an in vitro bioassay in terms of the International Reference Preparation of Human Pituitary Gonadotrophins (FSH and LH) for Bioassay (coded 69/104); and by immunoassay in terms of the International Reference Preparation of Human Pituitary Luteinizing Hormone for Immunoassay (IRP; coded 68/40). The LH potencies varied between preparations, including the IRP (68/40), from 864 to 5740 IU/mg by seminal vesicle weight gain (SVW) assay; from 1510 to 11500 IU/mg by ovarian ascorbate depletion (OAAD) assay; from 4490 to 14500 IU/mg by in vitro (testicular interstitial-cell testosterone production) bioassay; and from 2030 to 9180 IU/mg by immunoassay. Estimates of protein content were based on the assumption that the absorbance of LH at 280 nm (A 1% 1 cm) was 6.0. The LH potency of most preparations was highest by in vitro bioassay and lowest by SVW assay. The correlation between activities determined by SVW and OAAD assays was more marked than that between estimates by OAAD assay and in vitro bioassay; there was no correlation between estimates by SVW assay and in vitro bioassay. The slopes of the log dose-response curves of preparations in the OAAD assay were positively correlated with their potencies by OAAD assay and negatively correlated with the slopes of their log dose-response curves in the SVW assay. The qualitative differences between preparations are considered to be a reflection of the heterogeneity of LH and of its modification by different purification procedures. The present data, together with the different patterns of heterogeneity found in some of these preparations by isoelectric focusing in a separate study, suggest that the more basic molecular forms of LH, which are preferentially purified during the isolation of LH free from FSH and TSH, have shorter plasma survival times than the more acidic forms. The LH immunoreactivities of all preparations were significantly correlated with their potencies estimated by each of the in vivo bioassays but not with those estimated by in vitro bioassay. The ratios of in vitro bioactivity (in terms of IRP (68/40)): immunoreactivity varied between preparations from 0.53–1.5. The FSH content of each preparation was less than 2% (w/w) by bioassay and immunoassay. Most preparations were more potent by in vitro bioassay than by in vivo bioassay, which contrasted with, and complemented, findings for purified FSH preparations. This indicated that, as in the case of LH, the more basic molecular species of FSH are associated with lower ratios of in vivo: in vitro bioactivity than are the more acidic species. This study provides the most comprehensive comparison available of the activities of purified preparations of LH isolated from frozen and acetone-dried human pituitary glands in different experienced laboratories. These data are needed for selecting material for an international reference preparation of LH for immunoassay on the basis of high LH potency by in vivo bioassay, recommended by the WHO as a criterion for the identity of the hormone and for its freedom from contaminants. The consequences of the heterogeneity of LH are considered for the purification of the reference material and for the suitability of the latter for the various types of specimens which require LH assays.

Production and utilization of monoclonal antibodies to human/rat corticotrophin-releasing factor-41

1990 ◽  
Vol 5 (2) ◽  
pp. 159-166 ◽  
Author(s):  
N. G. N. Milton ◽  
E. W. Hillhouse ◽  
S. A. Nicholson ◽  
C. H. Self ◽  
A. M. McGregor
Keyword(s):  
Monoclonal Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Corticotrophin Releasing Factor ◽  
Tissue Extracts ◽  
Rat Pituitary ◽  
Hypothalamic Tissue ◽  
Dose Dependent

ABSTRACT Murine monoclonal antibodies against human/rat corticotrophin-releasing factor-41 (CRF-41) were produced and characterized for use in the immunological and biological characterization of CRF-41. Spleen cells from BALB/c mice immunized with CRF-41 conjugated to bovine γ-globulin were fused with a BALB/c-derived non-secretor X-63 myeloma line. Hybridomas were selected for CRF antibody production by enzyme-linked immunosorbent assay, and positive hybridomas cloned twice. Three monoclonal antibodies were obtained (KCHMB001, KCHMB002 and KCHMB003) and characterized as IgG1, IgG1 and IgG2a isotypes respectively, with affinity constants for rat CRF-41 of 30, 53 and 34 nmol/l respectively. All three monoclonal antibodies recognize an epitope contained between residues 34 and 41 of the human/rat sequence. The antibodies were able to neutralize the ACTH-releasing activity of rat CRF-41, applied to rat pituitary fragments in vitro, in a dose-dependent manner. Isoelectric focusing showed that KCHMB 003 detected bands of synthetic rat CRF-41 and rat [Met(O)21,38]-CRF-41 at pH 7·1 and 6·8 respectively. Use of KCHMB003 in a two-site enzyme-amplified immunoassay showed that this antibody recognizes both synthetic rat CRF-41 and immunoreactive CRF-41 in rat hypothalamic tissue extracts.

Effects of ω-hydroxylase product on distal human pulmonary arteries

2008 ◽  
Vol 294 (3) ◽  
pp. H1435-H1443 ◽  
Author(s):  
Caroline Morin ◽  
Christelle Guibert ◽  
Marco Sirois ◽  
Vincent Echave ◽  
Marcio M. Gomes ◽  
...  
Keyword(s):  
Pulmonary Arteries ◽  
Rho Kinase ◽  
Dependent Manner ◽  
Response Curves ◽  
Inhibitory Protein ◽  
Concentration Dependent Manner ◽  
Intracellular Process ◽  
Free Arachidonic Acid ◽  
Kinase Pathway

The aim of the present study was to provide a mechanistic insight into how 20-hydroxyeicosatetraenoic acid (20-HETE) relaxes distal human pulmonary arteries (HPAs). This compound is produced by ω-hydroxylase from free arachidonic acid. Tension measurements, performed on either fresh or 1 day-cultured pulmonary arteries, revealed that the contractile responses to 1 μM 5-hydroxytryptamine were largely relaxed by 20-HETE in a concentration-dependent manner (0.01–10 μM). Iberiotoxin pretreatments (10 nM) partially decreased 20-HETE-induced relaxations. However, 10 μM indomethacin and 3 μM SC-560 pretreatments significantly reduced the relaxations to 20-HETE in these tissues. The relaxing responses induced by the eicosanoid were likely related to a reduced Ca2+ sensitivity of the myofilaments since free Ca2+ concentration ([Ca2+])-response curves performed on β-escin-permeabilized cultured explants were shifted toward higher [Ca2+]. 20-HETE also abolished the tonic responses induced by phorbol-ester-dibutyrate (a PKC-sensitizing agent). Western blot analyses, using two specific primary antibodies against the PKC-potentiated inhibitory protein CPI-17 and its PKC-dependent phosphorylated isoform pCPI-17, confirmed that 20-HETE interferes with this intracellular process. We also investigated the effect of 20-HETE on the activation of Rho-kinase pathway-induced Ca2+ sensitivity. The data demonstrated that 20-HETE decreased U-46619-induced Ca2+ sensitivity on arteries. Hence, this observation was correlated with an increased staining of p116Rip, a RhoA-binding protein. Together, these results strongly suggest that the 20-hydroxyarachidonic acid derivative is a potent modulator of tone in HPAs in vitro.

scholarly journals Molecular forms of N-CAM and its RNA in developing and denervated skeletal muscle.

1986 ◽  
Vol 102 (3) ◽  
pp. 731-739 ◽  
Author(s):  
J Covault ◽  
J P Merlie ◽  
C Goridis ◽  
J R Sanes
Keyword(s):  
Primary Cultures ◽  
Apparent Molecular Weight ◽  
Fold Increase ◽  
Nucleic Acid Hybridization ◽  
Molecular Forms ◽  
Neuraminidase Treatment ◽  
Adult Muscle ◽  
Neural Cell Adhesion

The neural cell adhesion molecule (N-CAM) is present in both embryonic and perinatal muscle, but its distribution changes as myoblasts form myotubes and axons establish synapses (Covault, J., and J. R. Sanes, 1986, J. Cell Biol., 102:716-730). Levels of N-CAM decline postnatally but increase when adult muscle is denervated or paralyzed (Covault, J., and J. R. Sanes, 1985, Proc. Natl. Acad. Sci. USA., 82:4544-4548). To determine the molecular forms of N-CAM and N-CAM-related RNA during these different periods we used immunoblotting and nucleic acid hybridization techniques to analyze N-CAM and its RNA in developing, cultured, adult, and denervated adult muscle. As muscles develop, the extent of sialylation of muscle N-CAM decreases, and a 140-kD desialo form of N-CAM (generated by neuraminidase treatment) is replaced by a 125-kD form. This change in the apparent molecular weight of desialo N-CAM is paralleled by a change in N-CAM RNA: early embryonic muscles express a 6.7-kb RNA species which hybridizes with N-CAM cDNA, whereas in neonatal muscle this form is largely replaced by 5.2- and 2.9-kb species. Similar transitions in the desialo form of N-CAM, but not in extent of sialylation, accompany differentiation in primary cultures of embryonic muscle and in cultures of the clonal muscle cell lines C2 and BC3H-1. Both in vivo and in vitro, a 140-kD desialo form of N-CAM and a 6.7-kb N-CAM RNA are apparently associated with myoblasts, whereas a 125-kD desialo form and 5.2- and 2.9-kb RNAs are associated with myotubes and myofibers. After denervation of adult muscle, a approximately 12-15-fold increase in the levels of N-CAM is accompanied by a approximately 30-50-fold increase in N-CAM RNA, suggesting that N-CAM expression is regulated at a pretranslational level. Forms of N-CAM and its RNA in denervated muscle are similar to those seen in perinatal myofibers.

In vitro myometrial inhibition by the partitioned aqueous fraction of Anthocleista djalonensis leaves

2010 ◽  
Vol 88 (9) ◽  
pp. 880-887
Author(s):  
Enitome Evi Bafor ◽  
Lucky Osaro Okunrobo
Keyword(s):  
Uterine Contraction ◽  
Calcium Influx ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Response Curves ◽  
Concentration Dependent Manner ◽  
Aqueous Fraction ◽  
Uterine Contractions ◽  
Β Adrenergic Receptors

This study investigated the effect on the uterus of the aqueous fraction of the partitioned methanol crude extract of the leaves of Anthocleista djalonensis (AD) and the possible mechanism of AD activity. AD inhibited the concentration–response curves induced by oxytocin and CaCl2 on the rat uterus in vitro and significantly reduced the EC50 in a concentration-dependent manner (p < 0.05). A similar effect was observed with salbutamol and verapamil on the concentration–response curves obtained for oxytocin and CaCl2. The inhibitory effect of AD was not attenuated in the presence of propranolol. AD, salbutamol, and verapamil also produced a concentration-dependent relaxation on K+-induced sustained uterine contraction. In Ca2+-free medium, AD and salbutamol similarly inhibited oxytocin-induced contraction, but verapamil failed to produce this effect. The present results suggest that AD, being a mixture of phytochemicals, probably exerts inhibitory activity on in vitro uterine contractions of the nonpregnant, diethylstilboestrol-treated rat by multiple mechanisms that do not involve interaction with β-adrenergic receptors and do not solely depend on inhibition of calcium influx.

Heterogeneity of rat FSH by chromatofocusing: studies on serum FSH, hormone released in vitro and metabolic clearance rates of its various forms [ill]

1985 ◽  
Vol 105 (1) ◽  
pp. 29-37 ◽  
Author(s):  
W. F. P. Blum ◽  
D. Gupta
Keyword(s):  
Molecular Size ◽  
Male Rats ◽  
Metabolic Clearance ◽  
Clearance Rates ◽  
Charged Species ◽  
Rat Pituitary ◽  
Isoelectric Points ◽  
Exclusion Chromatography ◽  
Pituitary Glands

ABSTRACT Rat pituitary FSH was fractionated by chromatofocusing between pH 6 and 3. Ten components were resolved having apparent isoelectric points between 3·1 and 5·1. A comparative study of pituitary FSH and FSH secreted in vitro by quartered pituitary glands in the presence and in the absence of gonadotrophin-releasing hormone (GnRH) revealed similar patterns of charged species of intracellular and released FSH. Although GnRH increased FSH secretion about fourfold, no influence on the pattern of charged species was observed. Utilizing exclusion chromatography and chromatofocusing, pituitary FSH was compared to serum FSH which had been extracted by immunoaffinity chromatography. The results demonstrate for serum FSH a larger molecular size and a relative shift to more acidic components. Metabolic clearance rates of eight FSH components separated by chromato-focusing were measured in adult male rats. Half-lives varied between 13 min and several hours. A correlation existed between decrease of isoelectric points and decrease of metabolic clearance rates. These findings suggest that (1) all hypophysial FSH components are secreted into the circulation at similar rates and (2) the more acidic FSH components which appear to contain increased sialic acid, have a longer circulatory half-life and are more abundant in serum. It is concluded that sialylation may be involved in modulating serum FSH levels. J. Endocr. (1985) 105, 29–37

Neural and myogenic effects of leukotrienes C4, D4, and E4 on canine bronchial smooth muscle

1994 ◽  
Vol 266 (4) ◽  
pp. L414-L425 ◽  
Author(s):  
A. Abela ◽  
E. E. Daniel
Keyword(s):  
Smooth Muscle ◽  
Receptor Antagonist ◽  
Contractile Activity ◽  
Neuromuscular Control ◽  
Dependent Manner ◽  
Gamma Glutamyl Transpeptidase ◽  
Response Curves ◽  
Bronchial Smooth Muscle ◽  
Concentration Dependent Manner

The leukotrienes (LTs), referred to as the slow-reacting substance of anaphylaxis (SRS-A), are reported to have little or no activity in the canine airway. The objective of this study was to determine whether LTC4, LTD4, and LTE4 (10(-10)-10(-7) M) play a role in neuromuscular control of third- to fifth-order canine bronchi. In the presence of 1 microM indomethacin (Indo), canine bronchial smooth muscle contracted and was depolarized in a concentration-dependent manner by LTC4 or LTD4 but not by LTE4. LTC4 and LTD4 concentration-response curves were not significantly affected when conducted in the presence of any of the following: 10(-7) M propranolol (beta-adrenoceptor antagonist), 10(-6) M chlorpheniramine (H1-receptor antagonist), 10(-6) M ketanserin (nonselective 5-hydroxytryptamine receptor antagonist), 10(-7) M atropine (muscarinic receptor antagonist), and 10(-6) M tetrodotoxin (sodium channel blocker). LTC4 and LTD4 also potentiated electrical field-stimulated (EFS) excitatory junction potentials (EJPs), suggesting a possible prejunctional enhancement of acetylcholine release. In the absence of Indo, no postjunctional responses to LTC4 and LTD4 occurred. Endogenous prostaglandin E2 (PGE2) and 6-keto-PGF1 alpha (a stable metabolite of PGI2) levels from canine bronchi were significantly reduced by Indo. In the presence of Indo, addition of > or = 10(-8) M of PGE2 suppressed contractions to LTC4 and LTD4. These data suggest that the decrease in PGE2 and PGI2 production by Indo is sufficient to unmask the excitatory postjunctional actions of LTC4 and LTD4 on bronchial smooth muscle. Serine borate (45 mM; an inhibitor of gamma-glutamyl transpeptidase, which prevents the conversion of LTC4 to LTD4) increased selectively the contractile activity of LTC4. L-Cysteine (3 mM; an inhibitor of an aminopeptidase, which prevents the conversion of LTD4 to LTE4) enhanced the contractile responses to LTD4. Serine borate increased the amplitude and duration of EFS contractions and potentiated the amplitude of EFS EJPs; the last effects were prevented by nordihydroguaiaretic acid. These and other studies suggest that LTs are synthesized by canine bronchi and have receptors on canine bronchial smooth muscle but that contractions to LTC4 and LTD4 in the canine airway are usually not observed because of the presence of inhibitory prostanoids (PGE2 and PGI2). We suggest that decreases in PGE2 and PGI2 in models of airway disease in combination with increases in LTC4, LTD4, and thromboxane A2 may contribute to airway hyperresponsiveness in vitro.

Differential sensitivities of the sphincter of Oddi and gallbladder to cholecystokinin in the guinea pig: their role in transsphincteric bile flow

1992 ◽  
Vol 70 (10) ◽  
pp. 1336-1341 ◽  
Author(s):  
Karen Harrington ◽  
Arieh Bomzon ◽  
Keith A. Sharkey ◽  
Joseph S. Davison ◽  
Eldon A. Shaffer
Keyword(s):  
Guinea Pig ◽  
Circular Muscle ◽  
Sphincter Of Oddi ◽  
Dependent Manner ◽  
Field Stimulation ◽  
Response Curves ◽  
Gallbladder Contraction ◽  
Concentration Dependent Manner ◽  
Cholinergic Mechanism

Cholecystokinin (CCK) is considered to simply contract the gallbladder and relax the sphincter of Oddi with meals. In this study, we examined this hypothesis by investigating the action of CCK on the sphincter of Oddi and gallbladder of the guinea pig. The experimental design used an in vitro preparation of the sphincter of Oddi to measure contraction of the circular muscle. CCK increased tone in both the gallbladder and the sphincter of Oddi in a concentration-dependent manner. The normalized concentration–response curves for CCK, however, revealed that the gallbladder had a greater sensitivity to CCK (ED50 7 nM) than the sphincter of Oddi (ED50 22 nM; p < 0.01). Conversely, the sphincter was more sensitive to bethanechol than was the gallbladder. When the sphincter of Oddi was stimulated maximally with CCK in the presence of atropine (10−6 M) or tetrodotoxin (10−6 M), the contractile response was significantly reduced (p < 0.05) although not abolished. Conversely, atropine completely abolished the responses to bethanechol (10−3 M) and transmural field stimulation (70 V, 10 Hz, 1 ms, for 20 s). Transmural field stimulation of the sphincter that had been precontracted with CCK (26 nM) caused a transient, initial relaxation followed by contraction. Pretreatment with atropine augmented the duration of this relaxation, which could be completely abolished by tetrodotoxin. Thus, CCK contracts the sphincter of Oddi in the guinea pig by a direct (myogenic) and a neural (likely cholinergic) mechanism. Relaxation of the sphincter of Oddi also occurs in the guinea pig via noncholinergic inhibitory nerves. Duodenal delivery of bile is expedited by CCK, which induces gallbladder contraction at low, near-physiological levels without stimulating the sphincter. Under other conditions, sphincter of Oddi relaxation may facilitate transsphincteric flow. In contrast, increased cholinergic tone may help prevent duodenal reflux into the biliary system.Key words: cholecystokinin, bethanechol, enteric nervous system, gallbladder function, sphincter of Oddi.

scholarly journals Variability in the Apparent Molecular Weight of Inhibin

1985 ◽  
Vol 38 (3) ◽  
pp. 327 ◽  
Author(s):  
H WG Baker ◽  
LW Eddie ◽  
B Hudson ◽  
HD Niall
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulfate ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Polyacrylamide Gel ◽  
Pituitary Cells ◽  
Response Curves

An in vitro bioassay based on suppression of GnRH-stimulated FSH secretion by pituitary cells in culture was used to monitor inhibin activity after dialysis, gel filtration or polyacrylamide gel electrophoresis of protein preparations from a variety of gonadal secretions and extracts under native and dissociating conditions. The suggestion that inhibin is a peptide of molecular weight less than 5000 was not confirmed. Although some fractions of low molecular weight suppressed FSH secretion, the amount of activity was low and the dose response curves were not parallel with a standard preparation of inhibin. Under most conditions, inhibin eluted with an apparent molecular weight of about 90 000. However, gel filtration of rete testis fluid protein in I M acetic acid resulted in elution of inhibin activity with a lower apparent molecular weight and with polyacrylamide gel electrophoresis in o� 1% (w/v) sodium dodecylsulfate, the apparent molecular weight was 30 000. It is concluded that inhibin is a protein which tends to aggregate and coelute with larger molecules.

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