Biomedical subjects

H Ong

Publications and source records attributed to H Ong.

At least 73 records · Page 4Linked to original sources

PubMed1989

Bidimensional reversed-phase high-pressure liquid chromatography analysis of cultured cell neuropeptides: application to atrial natriuretic factor.

We report here a one-step procedure for extraction and analysis of neuropeptides in chromaffin cell culture media and acid extracts using reversed-phase HPLC. The bidimensional HPLC system consists of a precolumn connected to a six-port switching valve which is on-line with an analytical column. The direct injection of the biological samples onto the precolumn previously equilibrated with 15% acetonitrile allows the elimination of interfering substances. The samples purified on the precolumn can then be eluted onto the analytical column via the switching valve for neuropeptide separation. This trace-enrichment system allows a minimum of sample handling, both saving time and reducing possibilities of loss and contamination. This method has been applied to monitor the precursor and mature forms of atrial natriuretic factor from chromaffin cell secretion media and cell content extracts. The recovery of atrial natriuretic factor is in the range of 80-100%. This procedure could be applied to the study of the precursor-product relationship of any neuropeptide, e.g., from radiolabeled extracts of pulse-chase experiments performed on cultured chromaffin cells.

Animals↗

PubMed1989

Release of atrial natriuretic factor (ANF) induced by acute airway obstruction.

The aim of this study was to measure the effects of an increase in negative intrathoracic pressure on the release of ANF. With the subjects seated comfortably, 3 control blood samples were obtained over 30 minutes. Eight subjects then breathed for 30 min. through an inspiratory resistance in such a way that maximal inspiratory pleural pressures were between -30 to -40 cmH2O. Three blood samples were withdrawn after 20, 25, and 30 min., with the subject still breathing against the artificial resistance. Plasma concentrations of ANF were analysed by RIA. They measured: control value 24.6 +/- 3.7 pg ANF/mL (X +/- SE); with resistance 37.1 +/- 8.1 pg/mL (p less than or equal to .05). These results suggest that ANF could be released during an asthma attack.

Adult↗

PubMed1989

Diffusion of rat atrial natriuretic factor in thermoreversible poloxamer gels.

Concentrated poloxamer 407 solutions display a negative thermorheological behaviour, resulting in a higher viscosity at body temperature than below. This could be exploited for various controlled delivery applications including the nasal delivery of peptides. The rheology of the gel and the diffusion of ANF were studied as a function of temperature. A theory is proposed, according to which a temperature rise creates an intermicellar network resulting in a viscosity increase. The diffusion of the drug appears limited only by the presence of micelles which lengthens the diffusion pathways and is not significantly influenced by the formation of this network.

Atrial Natriuretic Factor↗

PubMed1989

Highly sensitive and rapid radioimmunoassay for aldosterone in plasma and cell culture medium.

A radioimmunoassay for aldosterone was developed using a sensitive and specific antibody and 125Iodinated-aldosterone. This assay could be used for direct determination of aldosterone in cell culture medium or after extraction of aldosterone from plasma by solid phase procedure using C18 Sep-pak cartridges. The very low cross-reactivity of the antibody with cortisol and corticosterone (0.005% and 0.04% respectively) would allow the direct determination of aldosterone in cell culture medium without any prior extraction step. Since the incubation is performed at room temperature for 1 h and then, at 4 degrees C for 15 min, the results can be obtained in less than 3 h. The assay was linear from 0.5 fmol/tube to 1500 fmol/tube with an ED50 at 30 fmol/tube. The accuracy of the assay estimated using spiked plasma samples with a known amount of aldosterone give a coefficient of correlation of 0.97 (n = 10) between the aldosterone concentrations found and expected levels. The within-assay variability for plasma aldosterone varied from 4.7 to 11.1% and the between-assay variability ranged from 13.9 to 14.2%. The coefficient of correlation between plasma aldosterone measured by this new assay or by a current assay was 0.8 (n = 43). In summary, the combination of a shortened incubation time with a simple solid phase extraction for aldosterone in serum samples represents the major advantage of the present assay over the current methodology which usually requires a chromatographic separation of the mineralocorticoid prior to radioimmunoassay. Therefore this assay would be useful in experimental studies as well as in clinical laboratory.

Aldosterone↗

PubMed1989

Aldosterone secretion inhibitory factor: a novel neuropeptide in bovine chromaffin cells.

We report the identification and the primary structure of a novel neuroendocrine peptide which inhibits aldosterone secretion. The peptide was isolated from 1.5 x 10(10) cultured bovine chromaffin cells by reversed phase and ion exchange HPLC. It is chromatographically and structurally distinct from atrial natriuretic factor (ANF), which we formely identified in chromaffin tissue. This new Aldosterone Secretion Inhibitory Factor (ASIF) is predominant in cultured chromaffin cells and cross-reacts with ANF receptor sites involved in the inhibition of aldosterone production in zona glomerulosa cells. The sequence of this 35-amino acid peptide includes the shorter fragment isolated from porcine brain confirming its neuropeptide character. ASIF might be involved in concert with ANF in the paracrine regulation of aldosterone secretion and the presence of both peptides in cultured chromaffin cells indicates that this system can be used as a model for studying natriuretic neuropeptide production.

Adrenal Glands↗

PubMed1989

Purification and primary structure of pro-aldosterone secretion inhibitory factor from bovine adrenal chromaffin cells.

This report documents the purification and the complete primary structure of bovine aldosterone secretion inhibitory factor precursor (pro-ASIF). ASIF-(1-103) contains at position 69-103 of its carboxy-terminal end the formely identified 35-amino acid biologically active form, hence confirming the endogenous character of ASIF in the adrenal medulla. Compared to atrial natriuretic factor (ANF)-related peptide precursors, bovine ASIF displays 65% homology at the carboxy-terminal while the remaining amino-terminal part shows much more variability. Bovine pro-ASIF exhibits 73% homology with porcine pro-brain natriuretic peptide (BNP), a situation reminiscent of the relationship of pro-ANF in various species. When ANF- and BNP-related COOH-termini of bovine, porcine, human, rat, and chicken are compared, it appears that bovine ASIF and porcine BNP are closely related and belong to the same family which however appears to be much more heterogenous than the ANF-related family. These results strongly suggest that bovine ASIF is encoded by a precursor gene similar to the gene of BNP but different from the one encoding ANF.

Adrenal Medulla↗

PubMed1989

Disposition and dynamics of atrial natriuretic factor in conscious rabbits.

The kinetics and dynamics of atrial natriuretic factor (ANF), rat ANF(99-126), were documented in conscious rabbits. The kinetics of immunoreactive ANF (IR-ANF) were first-order after a bolus of up to 300 ng/kg. Compared to the bolus, prolonged infusion of ANF produced a significant reduction in the systemic clearance of IR-ANF, from 132 to 59 and 70 ml/min/kg (P less than .0001) for the 81 +/- 8 ng/min/kg (x140 min) and 126 +/- 6 ng/min/kg (x480 min) infusion rates, respectively; however, the distribution of ANF was not affected. During the infusion of ANF, there was a marked decrease in mean arterial pressure and renal plasma flow, whereas the effects on renal excretion of water and sodium parameters were minimal. The change in hemodynamics was accompanied by a rebound increase in plasma renin activity. It is concluded that the kinetics of IR-ANF are zero-order after a prolonged infusion, secondary to a fall in the systemic clearance of ANF and that in this animal model, and at the doses used, this peptide only elicited hemodynamic effects, possibly related to the activation of counter-regulatory mechanisms.

Animals↗

PubMed1989

Distinct properties of atrial natriuretic factor receptor subpopulations in epithelial and fibroblast cell lines.

We have characterized two atrial natriuretic factor (ANF) receptor subtypes, designated ANF-R1 and ANF-R2, in two established cell lines that express exclusively one receptor subtype. The ANF-R1 receptor is selectively expressed by the kidney epithelial cell line LLC-PK1. It is a 130-kDa protein that has a much higher affinity for the biologically active forms of ANF than for its metabolites. The binding of ANF to this subtype is potentiated by amiloride and by divalent cations. The activation of the ANF-R1 receptor leads to an accumulation of cyclic GMP that is only partially inhibited by methylene blue. The ANF-R2 receptor, which is expressed selectively by the fibroblast cell line NIH-3T3, is a 130-kDa protein composed of two disulfide-linked subunits of 64-kDa. Activation of this subtype by saturating concentrations of ANF does not appear to elicit cyclic GMP production. However, supraphysiological concentrations of ANF induce a nonsaturable accumulation of cyclic GMP with an apparent ED50 in the high micromolar range. In contrast to the ANF-R1 subtype, the stimulation of cyclic GMP production is completely abolished by methylene blue. This subtype recognizes the active forms of ANF as well as its metabolites, and the binding is insensitive to amiloride and is decreased by divalent cations. These two cell lines can serve as models for studying the differential regulatory properties of ANF-R1 and ANF-R2 subtypes. In addition, we have also characterized the two ANF receptor subtypes in rat kidney glomeruli, where they show the same structure and pharmacological characteristics as in the two model cell lines.

Amiloride↗

PubMed1988

Atrial natriuretic factor R1 receptor from bovine adrenal zona glomerulosa: purification, characterization, and modulation by amiloride.

The atrial natriuretic factor (ANF) R1 receptor from bovine adrenal zona glomerulosa was solubilized with Triton X-100 and purified 13,000-fold, to apparent homogeneity, by sequential affinity chromatography on ANF-agarose and steric exclusion high-performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the purified receptor preparation in the absence or presence of dithiothreitol revealed a single protein band of Mr 130,000. Affinity cross-linking of 125I-ANF to the purified receptor resulted in the labeling of the Mr 130,000 band. The purified receptor bound ANF with a specific activity of 6.8 nmol/mg of protein, corresponding to a stoichiometry of 0.9 mol of ANF bound/mol of Mr 130,000 polypeptide. Starting with 500 g of adrenal zona glomerulosa tissue, we obtained more than 500 pmol of purified receptor with an overall yield of 9%. The purified receptor showed a typical ANF-R1 pharmacological specificity similar to that of the membrane-bound receptor. The homogeneous Mr 130,000 receptor protein displayed high guanylate cyclase activity [1.4 mumol of cyclic GMP formed min-1 (mg of protein)-1] which was not stimulated by ANF. This finding supports the notion that the ANF binding and the guanylate cyclase activities are intrinsic components of the same polypeptide. Finally, the purified ANF-R1 receptor retained its sensitivity to modulation by amiloride, suggesting the presence of an allosteric binding site for amiloride on the receptor protein.

Amiloride↗

PubMed1988

The amino acid sequences of frog heart atrial natriuretic-like peptide and mammalian ANF are closely related.

Despite few studies conducted in non-mammalian species, there has been a number of reports pertaining to the occurrence of a natriuretic-like substance in lower organisms. Thus, an immunoreactive substance reacting with antibodies directed against mammalian atrial natriuretic factor has previously been detected both in heart atria and ventricles of a chordate, the frog. This substance was isolated and purified from frog heart atria and its amino acid sequence established. The sequence, Ala-Pro-Arg-Ser-Ser-Asp-Cys-Phe-Gly-Ser-Arg-Ile-Asp-Arg-Ile-Gly-Ala-Gln- Ser-Gly - Met-Gly-Cys-Gly-Arg-(Phe), is highly homologous to known mammalian ANF sequences. However, when aligned with the complete mammalian ANF precursor sequence at positions 121 to 151, it exhibits a single amino acid insertion at position 129 and other substitutions at positions 121, 125, 133, 135, 144, 147 and 148. Some evidence is also presented concerning the occurrence of uncleaved frog pronatriodilatin, the precursor form of ANF. This study represents the first report pertaining to the structure of a non-mammalian ANF and its precursor.

Amino Acid Sequence↗

PubMed1988

Secretion and biosynthesis of atrial natriuretic factor by cultured adrenal chromaffin cells.

In our previous work, the existence of the precursor and mature forms of atrial natriuretic factor (ANF) within the bovine chromaffin granules has been reported. To confirm the endogenous character of these peptides, we demonstrate that nicotinic activation and depolarization by KCl increase their co-secretion from cultured chromaffin cells. The increase of intracellular levels of these atrial peptides by phorbol ester is potentiated by addition of forskolin. The release of ANF and their de novo synthesis within the cultured chromaffin cells emphasize the usefulness of this model in the study of the mechanisms of release and storage of these peptides in the neuronal tissues.

Adrenal Medulla↗

PubMed1988

A highly specific radioreceptor assay for the active circulating form of atrial natriuretic factor in human plasma.

This highly sensitive radioreceptor assay (RRA) for the active circulating form of atrial natriuretic factor (ANF), fragment 99-126, in human plasma requires 125I-labeled ANF, bovine zona-glomerulosa membrane receptors, and amiloride HCl. The amiloride elicits an increase in the binding affinity of ANF to its receptors. ANF is extracted from human plasma with "Sep-Pak" cyano cartridges. After a 90-min incubation at 25 degrees C, bound and free fractions are separated by filtration. The ANF concentration that inhibits receptor binding of the radioligand by 50% is 12.4 fmol of unlabeled ANF per tube. The minimum detectable concentration is 0.2 fmol per tube. Using ANF-supplemented plasma samples, there was a good correlation (r = 0.99) between ANF concentrations found and those expected. Only the active circulating form of ANF fully cross-reacts in this assay, which confirms its high selectivity. Results correlated strongly (r = 0.93) for clinical samples tested by RRA and RIA.

Adrenal Cortex↗

PubMed1988

Characterization of the effect of nicotine on vasopressin and atrial natriuretic factor in the rabbit.

The effects of nicotine on the secretion of arginine vasopressin (AVP) and of the atrial natriuretic factor (ANF) were examined in conscious rabbits. Nicotine was shown to produce significant increases in plasma AVP from 1.7 +/- 0.4 to 75.3 +/- 35.1 pg/ml (P less than .05) and in plasma ANF levels from 39 +/- 11 to 121 +/- 52 pg/ml (P less than .05) within 5 min of an i.v. dose of 0.5 mg/kg. These nicotine-induced stimulations could not be inhibited by muscarinic (atropine), dopaminergic [(+/-)-sulpiride], alpha (phenoxy-benzamine) or beta adrenergic (propranolol) blockers or by a rapid infusion of fluids. Trimetaphan was ineffective in blocking the stimulation of AVP secretion but completely abolished the nicotine-induced secretion of ANF. The more lipophilic ganglionic blocker, mecamylamine, blocked the stimulation of the secretion of both peptides. The effect of nicotine on AVP production was confirmed in vitro using the rat hypothalamo-neurohypophysial system preparation where nicotine increased AVP secretion in a dose-dependent manner. This in vitro stimulation was blocked by the ganglionic blocker hexamethonium. The increase in ANF plasma concentrations was probably due to a primary response to nicotine, for although exogenous AVP (1 microgram i.v.) increased ANF levels by a factor of 3 (P less than .05), the AVP antagonist [-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid)-2- (O-methyl)tyrosine]arginine vasopressin did not prevent the nicotine-induced increase in ANF. Thus, nicotine or its effects appear to stimulate the secretion of ANF and not AVP. It was concluded that nicotine stimulates the secretion of AVP by activating central nicotinic projections to the hypothalamus.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Effect of moderate hypoxemia on atrial natriuretic factor and arginine vasopressin in normal man.

The object of this study was to assess the effect of moderate acute hypoxemia on plasma concentrations of atrial natriuretic factor (ANF), arginine vasopressin (AVP), plasma renin activity (PRA) and urinary excretion of prostaglandin E2 (UPGE2V). Eight volunteers were exposed for 2 hours to a gas mixture containing 10% O2, 4.5% CO2 and 85.5% N2. Hypoxia increased diastolic blood pressure and free water clearance. Hypoxia did not change the AVP, PRA or UPG2V, although increased ANF from 17.7 +/- 3.4 pg/mL to 27.2 +/- 1.7 pg/mL (p less than 0.005) at 120 minutes. ANF changes were closely associated with the rise in blood pressure.

Adult↗

PubMed1987

Bovine adrenal chromaffin granules are a site of synthesis of atrial natriuretic factor.

We have previously reported the existence of a peptide factor in the adrenal medulla which inhibits aldosterone secretion in cultured bovine zona glomerulosa cells. The acid extracts of chromaffin granules from bovine adrenal medulla were purified by a four step high performance liquid chromatography procedure. Two active fractions exhibited sequence homology with bovine atrial natriuretic factor ANF (Ser99-Tyr126) and its polypeptide precursor (Asn1-Tyr126). The occurrence of both precursor and mature forms of ANF within chromaffin granules indicates the endogenous character of ANF in the adrenal medulla and suggests the potential usefulness of cultured adrenal chromaffin cells for investigating the synthesis, maturation and secretion of atrial peptides.

Adrenal Medulla↗

PubMed1987

Functional heterogeneity of atrial natriuretic factor receptor in bovine adrenal zona glomerulosa is explained by an amiloride-sensitive high affinity molecular complex.

The effects of amiloride on the molecular characteristics of the atrial natriuretic factor (ANF) receptor from bovine adrenal zona glomerulosa were studied by computer modeling of competitive binding data, by affinity labeling experiments, and by steric exclusion high performance liquid chromatography of solubilized receptor. The order of potency of a series of truncated ANF analogs in competing for 125I-ANF binding to bovine adrenal zona glomerulosa membranes was the same as that obtained for inhibition of aldosterone secretion. Deletion of amino acids at the COOH-terminal end drastically reduced the affinities of the peptides. Computer analysis of competition curves revealed that all ANF analogs tested show similar binding characteristics: shallow competition curves, discrimination of varying proportions of high and low affinity binding states, and sensitivity to amiloride which increases the proportion of the high affinity binding component. These results from binding studies are suggestive of potential heterogeneity of ANF binding sites. In contrast, results from affinity cross-linking experiments are consistent with the notion of a single receptor protein. Incubation of membranes with increasing concentrations of 125I-ANF-(99-126) up to 3 nM resulted in the labeling of a single band of Mr 130,000. The ability of ANF analogs to compete for the labeling of the Mr 130,000 band by 125I-ANF-(99-126) agreed well with their potency as inhibitors of 125I-ANF binding to intact membranes. Addition of amiloride caused a dose-dependent increase in the labeling of the Mr 130,000 band. A single Mr 130,000 band was also labeled in bovine aorta and LLC-PK1 cell membranes. In order to further investigate the molecular basis for the apparent heterogeneity of ANF binding we have prelabeled the membrane receptor with 125I-ANF-(99-126) prior to solubilization with octyl-beta-D-glucoside and chromatography on a Superose 6 steric exclusion column. The elution profile of the prelabeled receptor consistently showed two peaks of radioactivity with mean Stokes radii of 70 and 50 A. When amiloride was added to the incubation medium, the elution profile consisted almost exclusively of the 70-A peak. Quantitative analysis of the chromatographic profiles revealed that amiloride increases by 2-3 times the area of the 70-A peak. We conclude that the 70-A form represents a ternary complex of the receptor with an amiloride-sensitive effector protein.

Adrenal Cortex↗

PubMed1987

The effect of cyclophosphamide on arginine vasopressin and the atrial natriuretic factor.

The effect of cyclophosphamide (CTX) on the renal handling of water and sodium and on plasma concentrations of vasopressin (AVP) and of the atrial natriuretic factor (ANF) was studied in the rabbit. When compared to controls, animals receiving CTX (100 mg/kg IV) showed an increase in the diuresis and natriuresis. In addition, ANF plasma concentrations increased by 84% between 4 and 8 hours after CTX, while AVP plasma concentrations remained unchanged. It is concluded that in the rabbit CTX has a diuretic and a natriuretic effect that may, at least in part, be related to the increase in ANF plasma concentrations.

Animals↗

PubMed1987

Lidocaine and indocyanine green kinetics: effect of hypoxemia and/or hypercapnia.

Patients with premature ventricular contractions are also frequently hypoxemic (HO) and/or hypercapnic (HC). The aims of the present study were to document the effect of HO and/or HC on the kinetics of lidocaine, a first choice drug for the i.v. treatment of premature ventricular contractions, and on indocyanine green (ICG), another flow-dependent drug. For this purpose, five groups of rabbits were used: a control group received 2.5 mg/kg i.v. of ICG followed, 30 min later, by 7.5 mg/kg of lidocaine as a bolus. Four other groups received lidocaine as an infusion (261 micrograms/min/kg) for 255 min and ICG (249 micrograms/min/kg) for 8 min, the latter beginning 180 min after the start of the lidocaine infusion. The controls and one test group receiving the lidocaine infusion were breathing air (PaO2 = 89 +/- 2 (S.E.M.) and PaCO2 = 20 +/- 1 mm Hg); the second test group had HO (PaO2 = 51 +/- 1 mm Hg), a third group HC (PaO2 = 68 +/- 1 mm Hg) and the fourth HO combined with HC (HOHC) (PaO2 = 53 +/- 1 and PaCO2 = 61 +/- 2 mm Hg). Multiple blood samples were drawn. Lidocaine and its metabolites and ICG were assayed by high-performance liquid chromatography. Lidocaine volume of distribution was not affected by HO and/or HC. Compared to control values, the clearance of infused lidocaine (CIL) tended to decrease from 65 +/- 2 to 53 +/- 3 ml/min/kg. In animals with HO, HC and HOHC, CIL was 49 +/- 6, 45 +/- 5 and 46 +/- 3 ml/min/kg, respectively, these values not being significantly different from CIL.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗