Cooperation between interleukin-5 and the chemokine eotaxin to induce eosinophil accumulation in vivo.

Experiments were designed to study the effect of systemically administered IL-5 on local eosinophil accumulation induced by the intradermal injection of the chemokine eotaxin in the guinea pig. Intravenous interleukin-5 (IL-5) stimulated a rapid and dramatic increase in the numbers of accumulating eosinophils induced by i.d.-injected eotaxin and, for comparison, leukotriene B4. The numbers of locally accumulating eosinophils correlated directly with a rapid increase in circulating eosinophils: circulating eosinophil numbers were 13-fold higher 1 h after intravenous IL-5 (18.3 pmol/kg). This increase in circulating cells corresponded with a reduction in the number of displaceable eosinophils recovered after flushing out the femur bone marrow cavity. Intradermal IL-5, at the doses tested, did not induce significant eosinophil accumulation. We propose that these experiments simulate important early features of the tissue response to local allergen exposure in a sensitized individual, with eosinophil chemoattractant chemokines having an important local role in eosinophil recruitment from blood microvessels, and IL-5 facilitating this process by acting remotely as a hormone to stimulate the release into the circulation of a rapidly mobilizable pool of bone marrow eosinophils. This action of IL-5 would be complementary to the other established activities of IL-5 that operate over a longer time course.

Kinetics of eotaxin generation and its relationship to eosinophil accumulation in allergic airways disease: analysis in a guinea pig model in vivo.

Challenge of the airways of sensitized guinea pigs with aerosolized ovalbumin resulted in an early phase of microvascular protein leakage and a delayed phase of eosinophil accumulation in the airway lumen, as measured using bronchoalveolar lavage (BAL). Immunoreactive eotaxin levels rose in airway tissue and BAL fluid to a peak at 6 h falling to low levels by 12 h. Eosinophil numbers in the tissue correlated with eotaxin levels until 6 h but eosinophils persisted until the last measurement time point at 24 h. In contrast, few eosinophils appeared in BAL over the first 12 h, major trafficking through the airway epithelium occurring at 12-24 h when eotaxin levels were low. Constitutive eotaxin was present in BAL fluid. Both constitutive and allergen-induced eosinophil chemoattractant activity in BAL fluid was neutralized by an antibody to eotaxin. Allergen-induced eotaxin appeared to be mainly in airway epithelium and macrophages, as detected by immunostaining. Allergen challenge of the lung resulted in a rapid release of bone marrow eosinophils into the blood. An antibody to IL-5 suppressed bone marrow eosinophil release and lung eosinophilia, without affecting lung eotaxin levels. Thus, IL-5 and eotaxin appear to cooperate in mediating a rapid transfer of eosinophils from the bone marrow to the lung in response to allergen challenge.

Hypoxemia modifies circulating and exudate neutrophil number and functional responses in carrageenin-induced pleurisy in the rat.

To assess the effect of hypoxemia on the responses of polymorphonuclear neutrophils (PMN) during an inflammatory response, rats were maintained in a low F1O2 atmosphere (9% O2) or room air for 12 h before intrathoracic injection of carrageenin or intradermal injections of agonists. After 4 h, hypoxemic rats had 50% more circulating PMN in blood and 25% less PMN in pleural exudate, whereas the number of PMN in skin biopsies did not differ from controls. Following hypoxemia, basal adhesion of blood PMN to serum-coated plastic wells was unchanged, whereas fMLP-stimulated adhesion was 50% greater. In contrast, basal adhesion of exudate PMN was 72% greater. In hypoxemic rats, exudate PMN produced 64% more PMA-stimulated superoxide than blood PMN; furthermore, blood and exudate PMN produced 4.5- and 2-fold more LPS-stimulated nitric oxide than controls, respectively. These results show that a moderate level of hypoxemia may trigger mechanisms that will interfere with PMN emigration yet prime these cells for enhanced responses upon stimulation.

Metabolic disposition of leukotriene B4 (LTB4) and oxidation-resistant analogues of LTB4 in conscious rabbits.

1. The kinetics of leukotriene B4 (LTB4), after single i.v. injections of doses of 0.1 to 1 micrograms kg-1, were investigated in conscious rabbits and compared with those of the omega- and beta-oxidation resistant bioactive analogues, 20, 20, 20-trifluoro-LTB4 (20-F3-LTB4) and 3-thio-LTB4, respectively. 2. Immunoreactive LTB4 (IR-LTB4) elimination was first-order, as shown by a constant systemic clearance (ClLTB4) and a proportional increase in the area under the curve (AUC) of the plasma concentration versus time curve over the dose-range studied. Our results showed a good correlation between observed steady-state plasma concentrations (Css) of IR-LTB4 after continuous infusion of LTB4 and those predicted by using the mean estimated ClLTB4 of 93 +/- 4 ml min-1 kg-1, further confirming the linearity of IR-LTB4 elimination. 3. The half-life (t1/2) or IR-LTB4 increased from 0.47 +/- 0.02 to 0.63 +/- 0.04 min as a consequence of a change in the apparent volume of distribution (Vd) from 72 +/- 5 to 109 +/- 13 ml kg-1, for the 0.1 and 1 micrograms kg-1 doses injected, respectively. 4. Single i.v. injections of [3H]-LTB4 (4.7 ng kg-1) were administered, and the decay of plasma [3H]-LTB4 following h.p.l.c. purification was used to estimate the kinetic parameters. The kinetic parameters of [3H]-LTB4 were characterized by a mean systemic clearance (Cl) of 96 +/- 11 ml min-1 kg-1, a t1/2 of 0.53 +/- 0.03 min, and an apparent Vd of 85 +/- 9 ml kg-1, similar to the parameters obtained after LTB4 boluses. 5. The disposition of LTB4 analogues, whether resistant to Omega- or to Beta-oxidation in vitro, did not differ significantly from the disposition of the LTB4 molecule. The half-lives of 20-F3-LTB4 and 3-thio-LTB4 in the circulation were 0.52 +/- 0.07 min and 0.70 +/- 0.11 min, respectively.6. In summary, our results showed that LTB4, as well as Omega-oxidation- and Beta-oxidation-resistant analogues were cleared very rapidly from the rabbit circulation and indicate that in situ, metabolism in blood is not a rate-limiting factor for the elimination of LTB4.

Endogenous atrial natriuretic factor after endopeptidase 24.11 inhibition by Thiorphan.

The neutral endopeptidase 24.11 (NEP) was shown to degrade atrial natriuretic factor (ANF) in kidney membranes. An infusion of Thiorphan (25 micrograms/min/kg x 90 min), a specific NEP inhibitor, induced an increase in plasma ANF (65 +/- 26 to 163 +/- 52 pg/ml), plasma renin activity and in norepinephrine concentrations at 50 minutes of infusion in conscious rabbits. The increase in plasma ANF was accompanied by a gradual decrease in renal blood flow, despite maintenance of a stable mean arterial pressure. In conclusion, Thiorphan infusion produced an increase in endogenous plasma ANF. However, it may also have affected other hormonal systems which may have contributed to the overall dynamic and hormonal profile documented.

Permeability of the peptidic GH secretagogues hexarelin and EP 51389, across rat jejunum.

The intestinal permeability of hexarelin and EP 51389, two growth hormone releasing hexa- and tri- peptide analogues, was assessed in vitro with side-by-side diffusion chambers in the apical-to-basolateral (AP-to-BL) and in the basolateral-to-apical (BL-to-AP) direction using excised rat jejunal segments. The effect of EP 51389 on P-glycoprotein (P-gp) was evaluated by rhodamine 123 accumulation on monolayers of CH(R)C5 cells with increasing concentrations of EP 51389. Hexarelin and EP 51389 permeability were found to be < 1%. Permeability coefficients (P(app)) were 18.87 +/- 2.86 (x10(-7) cm/s) and 5.87 +/- 0.45 (x10(-7) cm/s) for hexarelin and EP 51389, respectively. Bidirectional studies revealed that hexarelin transport was similar in both directions. EDTA did not influence hexarelin permeability. Permeability was predominantly secretory for EP 51389 as P(app) in the BL-to-AP direction [32.56 +/- 6.11 (x10(-7) cm/s)] was greater than AP-to-BL. Confirming involvement of a secretory transport system, chlorpromazine inhibited EP 51389 transport across the jejunum. EP 51389 inhibited P-gp in a dose dependent manner resulting in the intracellular accumulation of rhodamine in CH(R)C5 cells. These results suggest that: 1) the intestinal permeability of hexarelin and EP 51389 is poor; 2) the passage of hexarelin is mainly via a transcellular passive pathway since the contribution of paracellular permeability to the overall permeability is rather low; 3) P-gp may act as a potential barrier for the intestinal absorption of EP 51389.

Radioimmunoassay of meterelin and pharmacokinetics after single injection and implant administration in dogs.

A sensitive and specific radioimmunoassay for a novel luteinizing-hormone-releasing-hormone (LHRH) agonist, [2-Me-D-Trp6, DesGly10]LHRH ethylamide (meterelin), was developed for documenting the pharmacokinetic parameters of this peptide following its intravenous (iv) and subcutaneous (sc) administration in dogs. The assay was also used for monitoring meterelin in plasma following its release from d,l-lactide-glycolide implants in the same species. Rabbit antisera generated against [DespyroGlu1] meterelin and conjugated to bovine serum albumin with glutaraldehyde showed high specificity, whereas crossreactivity to LHRH and its fragments and to analogs with substitutions at residues 6 and 10 was found insignificant. The assay was validated in terms of accuracy (recovery range, 94.0-105.4%), in terms of precision (intra- and interassay variations of 10.0-12.4% and 8.6-11.3%, respectively), and in terms of sensitivity (minimum detectable dose of 2.7 pg/assay). Following iv acute administration, a biexponential decline of plasma meterelin levels was observed, with distribution and elimination half-lives of 5.9 +/- 2.5 and 106 +/- 22 min, respectively. After sc acute administration, the elimination half-life was in the range of 103 to 173 min. The systemic clearance (CLT) ranged from 1.6 to 2.6 mL/min/kg, and the volume of distribution at steady state (Vdss) varied from 285 to 438 mL/kg. The elimination half-life (T1/2 beta), Vdss, and ClT were not significantly different after both routes of administration over the 1-100-microgram/kg dose range of peptide studied. Castrate levels of testosterone were attained 10 days after sc administration of the implant, lasted for up to 247 days, and were well correlated with plasma levels of meterelin.

Furosemide dynamics in conscious rabbits: modulation by angiotensin II.

The aim of this study was to investigate the effects of an infusion of angiotensin II (50 ng/kg/min) on furosemide pharmacodynamics and kinetics in the conscious rabbit. The protocol included a 90-minute phase to estimate the glomerular filtration rate and the renal plasma flow, followed by a 60-minute phase where 5 mg/kg (n = 12) or 10 mg/kg (n = 9) of furosemide were administered. During the pre-furosemide phase, compared to control rabbits, angiotensin II increased natriuresis and diuresis. In the presence of angiotensin II, the furosemide-induced natriuresis decreased, that is, it was 174 +/- 14 versus 95 +/- 25 mumol/min (p < 0.05) and 187 +/- 17 versus 89 +/- 21 mumol/min (p < 0.05) for the 5 and the 10 mg/kg doses, respectively. The infusion of angiotensin II decreased renal plasma flow without modifying the glomerular filtration rate, thus the filtration fraction was increased. Angiotensin II increased the area under the furosemide plasma concentrations as a function of time since it decreased its systemic clearance. However, furosemide urinary excretion rate was not altered and its renal clearance decreased slightly without reaching statistical significance. It is concluded that angiotensin II decreases the response to furosemide and the mechanism underlying this effect is related to the pharmacodynamics rather than the kinetics of the diuretic.

In vivo desensitization to leukotriene B4 (LTB4) in the rabbit. Inhibition of LTB4-induced neutropenia during intravenous infusion of LTB4.

Bolus injections of leukotriene B4 (LTB4) at 30-min intervals repeatedly induced similar profound and reversible neutropenias. In contrast, after a 30-min infusion of LTB4, the neutropenia induced by bolus injections of LTB4 was inhibited in a dose-dependent manner; a threshold inhibition was seen at the infusion rate of 10 ng LTB4/min/kg, whereas almost complete inhibition was observed at 50 ng LTB4/min/kg. Steady state arterial plasma concentrations of LTB4 increased proportionally to LTB4 infusion rate, ranging from 0.15 +/- 0.01 nM (control) to 2.80 +/- 0.16 nM (100 ng/min/kg). Extending the infusion period of LTB4 up to 330 min did not result in an enhanced inhibition of the neutropenia in response to bolus injections of LTB4. Reversibility of the desensitization was shown by an almost complete recovery of the neutropenic response within 30 min after cessation of the infusion. The desensitization achieved towards LTB4 showed some specificity, inasmuch as a profound and reversible neutropenia was observed in response to a bolus of either FMLP or C5a under conditions in which sensitivity to LTB4 was lost. These findings suggest that a specific desensitization to LTB4 is feasible in vivo and may constitute a useful approach, in addition to the use of 5-lipoxygenase inhibitors and LTB4 antagonists, to delineate the significance of LTB4 as a mediator of inflammation.

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)

Kinetics and disposition of hexarelin, a peptidic growth hormone secretagogue, in rats.

To document the disposition of hexarelin, a peptidyl growth hormone secretagogue, male Sprague-Dawley rats received a 5-microg/kg bolus i.v. dose or three single s.c. doses of 5, 10, and 50 microg/kg. To assess hexarelin tissue distribution and excretion, rats were given 1 microg/kg of [(3)H]hexarelin (9.4 Ci/mmol). Metabolism of [(3)H]hexarelin was assessed in bile duct-exteriorized rats given 50 microg/kg where radiolabeled hexarelin biliary and urinary excretion was quantified. After its i.v. injection, hexarelin displayed a half-life of 75.9 +/- 9.3 min, a systemic clearance of 7.6 +/- 0.7 ml/min/kg, and a volume of distribution at steady state of 744 +/- 81 ml/kg. After s.c. administration, the area under the curve (477-3826 pmol.min/ml) estimated with increasing doses confirmed the absence of hexarelin accumulation. Clearance/F (12-15 ml/min/kg) and volume of distribution/F (1208-1222 ml/kg) were dose independent. Hexarelin bioavailability given s.c. was 64%. The highest radioactivity levels were detected in the kidney, liver, and duodenum. The pattern of hexarelin excretion was similar after i.v. or s.c. administrations. Total radioactivity in bile, urine, and feces corresponded to 60, 22, and 10% of the dose, respectively. Of the radioactivity excreted in bile and urine, 90 and 71% was unchanged hexarelin, respectively. These results suggest that: 1) the kinetics of hexarelin appear to be first order up to 50 microg/kg; 2) hexarelin is rapidly absorbed after s.c. administration; 3) biliary excretion is the primary route of hexarelin elimination; and 4) the high recovery of unchanged peptide in bile and urine demonstrates hexarelin stability toward proteolytic enzymes.

Reverse-phase high-performance liquid chromatography analysis of arachidonic acid metabolites in plasma after stimulation of whole blood ex vivo.

Following the stimulation of heparinized blood ex vivo, aliquots of plasma were denatured with organic solvents containing the internal standards prostaglandin (PG) B2 and 19-hydroxy-PGB2. Precipitated material was removed by centrifugation and the supernatants were directly analyzed by reverse-phase HPLC with on-line extraction and uv detection. Stimulation of blood with A23187 lead to the formation of both leukocyte and platelet arachidonic acid metabolites as the 5-lipoxygenase products leukotriene (LT) B4, 5-hydroxy-eicosatetraenoic acid (5-HETE), 20-hydroxy-LTB4 and 20-carboxy-LTB4, the 12-lipoxygenase product 12-HETE, and the cyclooxygenase product 12-hydroxy-heptadecatrienoic acid (HHTrE) were detected in plasma; in some plasma samples LTC4 and/or LTE4 were also detected. Stimulation of blood with zymosan lead to the synthesis of LTB4 and 5-HETE as major products and of 12-HETE. Recoveries of 20-hydroxy-LTB4, LTB4, 5-HETE, 12-HETE, and HHTrE added to plasma were high (> or = 90%) while those of LTC4 and LTE4 were lower (50-70%); however, washing of the precipitated protein pellet resulted in > 90% recoveries for all metabolites including the cysteinyl-leukotrienes. Levels of arachidonic acid metabolites in native plasma samples stored at -20 degrees C were stable for at least 28 days, while significant loss of material was observed over the same period of time in denatured plasma samples. Finally, we made the critical observation that the capacity for A23187- (but not zymosan-, ionomycin-, or LPS and FMLP-) induced arachidonic acid metabolite synthesis in blood decreased by 80% within 1 h of blood collection.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatic extraction of hexarelin, a new peptidic GH secretagogue, in the isolated perfused rat liver.

PURPOSE: To assess the hepatic extraction of hexarelin (HEX), a novel peptidyl GH secretagogue, in the isolated perfused rat model and document the in vitro binding of HEX to plasma proteins using plasma from rats, dogs, pigs, and humans. METHODS: Rat liver was perfused in situ using a recirculating system. The recirculating perfusate consisted of a Krebs Henseleit buffer containing 20% (v/v) prewashed bovine red blood cells, 1% albumin, and lg/L dextrose. Three HEX concentrations of 5, 50, and 500 ng/ml were examined. In vitro plasma binding was determined by the ultrafiltration method. RESULTS: The disappearance rate constant (K), half-life (t1/2), clearance (Cl), and hepatic extraction ratio (E) were: K = 0.013-0.014 min-1, t1/2 = 45-55 min, Cl = 0.345-0.401 ml/min/g liver, and E = 19-21% for the different concentrations of HEX. A linear increase in AUC (270-24334 min pmol/ml) was observed with increasing concentrations. Binding of HEX to plasma proteins of rats, dogs, pigs, and humans was 68.7 +/- 0.8%, 78.7 +/- 0.6%, 67.3 +/- 0.7%, and 65.2 +/- 0.6% respectively. Plasma binding was concentration-independent in the range between 0.003-3 microM for the four species examined. CONCLUSIONS: These results show that 1) the hepatic extraction of HEX is low, 2) the hepatic clearance is concentration independent up to 500 ng HEX/ml of perfusate, and 3) the plasma protein binding of HEX is significant over the dose range studied. HEX exhibits a low hepatic extraction ratio, allowing us to predict that its hepatic clearance may be limited upon HEX protein binding.

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.

Role of 5-lipoxygenase products in the local accumulation of neutrophils in dermal inflammation in the rabbit.

Studies were undertaken to define the role of 5-lipoxygenase (5-LO) products and, in particular, of leukotriene (LT) B4 in the polymorphonuclear leukocyte (PMN) emigration process using a rabbit model of dermal inflammation. Our results show that i.v. administration to rabbits of MK-0591, a compound that inhibits LT biosynthesis in blood and tissues when administered in vivo, significantly reduced 51Cr-labeled PMN accumulation in response to intradermally injected chemotactic agonists, including IL-8, FMLP, C5a, and LTB4 itself. In addition, pretreatment of the labeled PMN with MK-0591 ex vivo before their injection in recipient animals was equally effective in reducing 51Cr-labeled PMN emigration to dermal inflammatory sites. These results support a role for de novo synthesis of 5-LO metabolites by PMN for their chemotactic response to inflammatory mediators. Other studies demonstrated that elevated intravascular concentration of LTB4 interferes with PMN extravasation inasmuch as a continuous i.v. infusion of LTB4, in the range of 5-300 ng/min/kg, dose-dependently inhibited extravascular PMN accumulation to acute inflammatory skin sites elicited by the chemoattractants LTB4, FMLP, C5a, and IL-8 and by TNF-alpha, IL-1beta, and LPS; such phenomena may constitute a natural protective mechanism from massive tissue invasion by activated PMN in specific pathologic conditions such as ischemia (and reperfusion). These studies demonstrate additional functions of 5-LO products in the regulation of PMN trafficking, distinct from the well-characterized chemotactic activity of LTB4 present in the extravascular compartment.

Role of the endopeptidase 24.11 in the disposition and metabolism of endogenous atrial natriuretic factor in the rabbit.

The neutral endopeptidase 24.11 (NEP) has been shown to inactivate the atrial natriuretic factor (ANF) by opening the ring structure. To document the role of NEP in the metabolic fate of ANF in vivo, the effects of an infusion of thiorphan (25 micrograms/min/kg), a specific NEP inhibitor, on the kinetics and metabolism of endogenous ANF were studied in conscious rabbits. A bolus of [125I]ANF(99-126) was injected 50 min after the beginning of the infusion of thiorphan. Plasma samples containing the radioactive peptides were separated by reverse-phase HPLC. The parent compound could be separated from at least two other minor metabolites, corresponding to the elution position of [125I]ANF(99-105/106-126), the inactive ring-opened metabolite, and of [125I]ANF(103-126), an N-truncated analog. The generation of the N-truncated metabolite was increased by thiorphan. Thiorphan also induced an increase in plasma ANF (29%) that was closely associated with a 32% reduction in the systemic clearance of [125I]ANF(99-126), whereas no modification in the estimated secretion rate was detected. These results support a role for NEP in the regulation of endogenous ANF plasma levels. These results also suggest that specific inhibition of NEP may result in an increase in the apparent activity of alternative metabolic pathways.

Human RANTES acts as a receptor antagonist for guinea pig eotaxin in vitro and in vivo.

The guinea pig C-C chemokine, eotaxin, is a potent and selective eosinophil chemoattractant in guinea pig airways and skin in vivo, and stimulates both guinea pig and human eosinophils in vitro. The human C-C chemokine RANTES (30% homology with guinea pig eotaxin) stimulates human eosinophils in vitro, but does not stimulate guinea pig eosinophils, even though these cells bind 125I-RANTES. Similar concentrations of eotaxin and unlabeled RANTES competitively inhibit the binding of 125I-RANTES to guinea pig eosinophils, suggesting that eotaxin and RANTES share a common binding site on these cells. In the present study, we investigated the possibility that human RANTES, binding to a putative eotaxin receptor on guinea pig eosinophils, might block functional responses to eotaxin. When fura-2-loaded cells were first exposed to RANTES, which failed to elevate the intracellular calcium concentration, the response to a subsequent challenge with eotaxin was inhibited in a dose-dependent manner. Inhibition was also demonstrated when the two chemokines were added simultaneously. Another human C-C chemokine, MCP-3 (52% homology with guinea pig eotaxin), had similar inhibitory effects on the eotaxin-induced activation of guinea pig eosinophils in vitro. RANTES inhibited (111)In-eosinophil accumulation in response to intradermal eotaxin in vivo. In contrast, RANTES had no significant effect on responses to leukotriene B4 in vitro or in vivo. Thus, these experiments in the guinea pig demonstrate that human RANTES is the first prototypic antagonist of an eotaxin receptor.

Analysis of albuterol in human plasma based on immunoaffinity chromatographic clean-up combined with high-performance liquid chromatography with fluorimetric detection.

A method combining immunoaffinity chromatography with high-performance liquid chromatography was developed for the determination of albuterol in human plasma. The immunoaffinity chromatography, based on the specific interaction of albuterol with the immobilized antibody raised against it, was used as a clean-up step. Albuterol eluted from this immunochemical solid-phase clean-up step was analysed by reversed-phase high-performance liquid chromatography with fluorimetric detection. The performance of the assay was validated on six normal volunteers after a 4-mg oral dose of albuterol, which gave a peak plasma concentration in the range 6.67-15.31 ng/ml at 3-4 h after the dose. Plasma levels (0.79-1.56 ng/ml) of albuterol could be detected up to 24 h after the dose.

Radioimmunoassay for albuterol using a monoclonal antibody: application for direct quantification in horse urine.

A monoclonal antibody was synthesized in mouse against the O-(3-carboxypropionyl) derivative of albuterol linked to bovine serum albumin. Isotyping of this material revealed the IgG1 class characterized by an affinity constant of 1.03 nM-1 and a density of sites of 0.55 nM. This antibody was found specific as its cross-reactivity to structurally related molecules was less than 1% except for clenbuterol (75%). A radioimmunoassay was set up with culture supernatant (final dilution 1/1000) and [3H] albuterol. The calibration curve was characterized by a maximum binding of 28%, an ED50 of 1.15 pmol per tube, the detection limit was 28.8 fmol/tube and the linearity of the response was up to 39.8 pmol/tube. This RIA method has been used for direct quantitation of albuterol in horse urine without any clean-up or extraction step.

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.