Cortisol-binding protein in the cytosol of rat carrageenin granuloma.

Cortisol-binding protein was prepared and partially purified by (NH4)2SO4 fractionation and DEAE-cellulose column chromatography from 105,000 g supernatant fraction of cytoplasm in rat carrageenin granuloma, which is assumed to be one of the most appropriate experimental models of inflammation. The cortisol-binding protein in the inflammatous tissue, although similar to transcortine, was not transcortine itself. The binding protein was eluted at 0.12 M NaCl by DEAE-cellulose column chromatography with a shallow salt gradient. Sedimentation constant and dissociation constant of the binding protein were 4-5 S and 1.0 X 10(-9) M, respectively. Optimum pH for binding to cortisol was 8.0. Binding ability of the binding protein to cortisol was very sensitive to pronase E and trypsin but resistant to RNase. Specificity of the protein for binding other steroids revealed that 17beta-estradiol did not bind to the protein, while androstenedione and testosterone had one sixth as much affinity to the binding protein as that of cortisol. There was good a correlation between the amount of the binding protein in the inflammatory tissue and anti-inflammatory effect of cortisol. Namely, the maximum cortisol-binding ability was seen on a 5 day old granuloma which is the so called 'steroid sensitive stage'. Thereafter, the binding ability decreased with the increasing stage of granuloma.     

Mediators of the inflammation induced in the rat paw by carrageenin

1. The time course of oedema formation in rats caused by injection of carrageenin into the paw was followed for 5.5 hours. Intact or adrenalectomized rats which had previously been injected with ellagic acid or saliva to reduce considerably the concentration of blood kininogens, or with methysergide to antagonize 5-hydroxytryptamine (5-HT) showed a reduced inflammatory response. It was concluded that kinins and 5-HT contributed significantly to oedema formation during this period.2. Mepyramine alone had no effect on oedema formation, but in combination with ellagic acid treatment, with or without methysergide, it caused a reduction suggesting that histamine played a minor role in oedema formation during the first 3 hours.3. Vascular permeability studies indicated that injection of ellagic acid did not interfere with the normal responses in skin to intradermal injections of histamine, 5-HT, bradykinin or compound 48/80. Mepyramine and methysergide, at the doses used in the carrageenin experiments, completely antagonized histamine and 5-HT, respectively, and did not affect the skin responses of bradykinin.4. Treatment in vivo with ellagic acid or rat saliva was equally effective in reducing plasma kininogen concentrations by an amount equivalent to more than 10 times the quantity of substrate 1 measured by Gautvik & Rugstad (1967).5. Rat saliva, but not ellagic acid, lowered complement levels by approximately 20%.     

Arachidonic acid, an in vivo inhibitor of carrageenin induced granulomas in the rat

Arachidonic acid (AA) injected locally into carrageenin/sponge granulomas, but not if given orally, inhibited granuloma growth. Granuloma macrophage (M0) infiltration was inhibited, and prostaglandin E₂ (PGE₂) synthesis (ng/100 mg granuloma dry weight) stimulated, by AA treatment. M0 adenosine 3′,5′-cyclic monophosphate (cAMP) levels and granuloma exudate volume were not affected. Granuloma M0s incubated in vitro with arachidonic acid synthesised thromboxane B₂ (Txb₂), 6-ketoprostaglandin F₁ (6-ketoPGF₁), and preferentially, PGE₂. The AA inhibition of granuloma growth was possibly mediated via the synthesis of PGE₂.     

Leucocyte migration and lysosomal enzymes release in rat carrageenin pleurisy.

The time course of rat carrageenin pleurisy has been studied. The inflammatory reaction is characterized by exudate formation and massive leucocyte emigration into the pleural space both reaching peak values at 24 hours. Moreover betaglucuronidase, acid phosphatase and lactic dehydrogenase have been assayed in the exudate. The activity of lysosomal enzymes parallels the severity of the inflammatory response, while that of cytoplasmic enzyme lactic dehydrogenase resulted unmodified. Treatment of animals with indomethacin, phenylbutazone, aspirin and flufenamic acid inhibited both exudate formation and leucocyte emigration. In contrast none of these drugs was able to reduce lysosomal enzyme release.     

Rebound of rat carrageenin granuloma following cessation of anti-inflammatory steroid therapy

Glucocorticoid therapy induced rapid involution of chronic granulomatous inflammation in rats by subcutaneous injection of carrageenin. Hydrocortisone acetate injected into the granuloma pouch at doses higher than 3 mg/kg/day for 3 days caused maximum involution. After withdrawal of the corticoid therapy, rebound of the granulomatous inflammation took place resulting in rapid recovery of the wet weight and total content of tissue DNA and non-collagen proteins. A dose of 3 mg/kg/day was optimal for observing this rebound phenomenon. In order to investigate metabolic aspects of the rebound phenomenon minced granuloma was incubated in vitro with [³H] thymidine or [³H] proline. The rate of incorporation of the labeled precursor into non-collagen protein was elevated near to the normal level by 24 hr after the interruption of the corticoid treatment. A second step in the course of the recovery was a rapid increase in the incorporation of labeled thymidine into DNA which was attained by 48 hr after the last injection of the corticoid. The rate of recovery of the total amount of non-collagen protein, however, was rather slow compared with that of DNA which reached the control level 3 days after the withdrawal of the corticoid therapy. The total non-collagen protein of the granuloma reached almost complete recovery 1 day later. These results suggest that the synthesis of some fractions of the granuloma proteins which involve proteins essential for DNA synthesis was activated before the reactivation of the synthesis of DNA and some other proteins. Recovery of collagen synthesis was not complete until 4 days after the cessation of the corticoid treatment. Consequently, the total amount of collagen was still lower than that of the control on the last day of the experiment.     

Some properties of the denervated anterior gracilis muscle of the rat

1. Microdrops of acetylcholine (ACh) were topically applied to denervated, mammalian skeletal muscle in vivo, and ACh-evoked depolarization and contracture were recorded simultaneously.2. Contracture tension was directly proportional to the degree of ACh-elicited depolarization.3. Atropine and (+)-tubocurarine increased the amount of ACh required to produce a given amount of depolarization, but these anticholinergic agents did not alter the relationship between the degree of ACh-evoked depolarization and contracture tension.4. Topically applied catecholamines did not produce either depolarization or contracture, despite the fact that parenterally administered catecholamines elicited both responses.     

Some properties of 5-hydroxytryptamine receptors in the hindquarters of the rat.

1 The rat hindquarter preparation, as described, responds with reproducible vasoconstriction to noradrenaline and tryptamines. 2 The receptors involved in these responses are distinct. 3 Evidence of heterogeneity of tryptamine receptors was not obtained. 4 The 5-hydroxytryptamine (5-HT) antagonists, methysergide and cyproheptadine, although very potent, displayed antagonism of a non-competitive type whereas a series of phenothiazines and phentolamine displayed competitive antagonism against 5-HT. 5 For the phenothiazines the order of increasing potency was promazine less than chlorpromazine less than triflupromazine.     

Accumulation of blood platelets in carrageenin rat paw oedema. Possible role in the inflammatory process

The accumulation of blood platelets in the carrageenin-induced paw oedema in rats was studied, using 51Cr-labeled platelets. A maximum in the accumulation was seen after 4 hours, followed by a decline after 5--6 hours. During the first 6 hours of the oedema formation, change in blood volume were small. A comparison was made with the accumulation of both 125J-albumin as a measure of extravasation and 125J-fibrinogen as an indication of blood clotting. When platelet aggregation was measured during the first 6 hours of the oedema formation, the lag time between the addition of collagen and the beginning of the aggregation was increased. No change in platelet serotonin was seen. These data support the idea that platelets participate in the inflammatory process.     

Some pharmacodynamic effects of the babesicidal agents quinuronium and amicarbalide

The intravenous injection of a therapeutic dose of quinuronium methylsulphate (1 mg/kg) causes a fall in blood pressure in sheep, which is partly prevented by mepyramine and abolished by atropine. Larger doses of quinuronium cause more marked hypotension and inhibition of respiratory movement, which are not affected by atropine. Quinuronium strongly increases the amplitude of contraction of the isolated rabbit heart. This effect is not antagonized by atropine or mepyramine. Contractions of plain muscle in the guinea-pig and sheep, and hypersecretion of gastric acid in the rat and of saliva in the sheep were all produced by quinuronium. The responses to acetylcholine were potentiated by quinuronium, an effect which was abolished by atropine. Amicarbalide isethionate by comparison was weakly active. The drug causes no change in blood pressure, smooth muscle contraction or salivary secretion, but stimulates gastric secretion and partially inhibits the actions of acetylcholine in these preparations.     

Effects of cortisol and tetrahydrocortisol on the cloned fibroblast derived from rat carrageenin granuloma

A fibroblast clone SM-Cl was established by cloning cultured fibroblasts derived from a rat carrageenin granuloma. The cloned fibroblasts were found to produce and secrete large amounts of acidic glycosaminoglycans (AGAG) and collagen into the medium, and the AGAG were identified with chondroitin 4-sulfate and hyaluronic acid by 2-dimensional electrophoresis and enzymatic digestion. The cells were exposed to 10^?6M cortisol or 10^?4M tetrahydrocortisol for 2 days during their stationary phase. The amounts of AGAG and collagen secreted into the medium, the protein and RNA contents of the cells, the amounts of free amino acids per 10⁹ cells, the intracellular concentration of each free amino acid, and the distribution ratio (ratio of intracellular concentration to that in the medium) of each free amino acid of the cells were compared with those of control cells. The production of intracellular substances was markedly inhibited by cortisol, i.e., by 75 per cent for AGAG and by 50 per cent for collagen. Both the amount of each amino acid per 10⁹ cells and the distribution ratios were markedly depressed by cortisol treatment for all the determined amino acids except serine. The cloned fibroblasts were then studied with regard to the rate of uptake of 2-amino[1-¹⁴C]isobutyric acid (AIB) from the medium by control and steroid-treated cells. Cortisol treatment decreased [¹⁴C]AIB uptake of the cell markedly from the beginning up to 90 min of incubation. Tetrahydrocortisol, one of the main metabolites of cortisol, exerted no effect even at a concentration as high as 10^?4 M.Incorporation of [³H]thymidine into DNA of the cells in the growing phase was affected very slightly by the drugs.     

Physiological pharmacokinetic and pharmacodynamic model of physostigmine in the rat.

A physiological model for physostigmine disposition was developed in the rat which incorporated anatomical, physiological, and biochemical parameters, i.e. tissue volume, plasma flow rates, drug metabolism, and tissue-to-plasma partition coefficients. Predicted concentrations of physostigmine in different tissue compartments were consistent with the experimental observations in the rat following an iv dose. Part of this study also compared the time course changes in measured effect, as percentage change in cholinesterase activity in brain, and related these changes to the plasma or brain drug level in either a combined pharmacokinetic-pharmacodynamic (plasma physostigmine-effect relationship) or a dynamic model (brain physostigmine-effect relationship). Fitting the time course of the effect in a pharmacokinetic-pharmacodynamic model required an effect compartment with the equilibration rate constant between it and the plasma compartment. Both models help to understand whether the cholinesterase activity is homogeneous or heterogenous in the brain.     

Pharmacokinetic and pharmacodynamic properties of inhaled ciclesonide

Inhaled corticosteroids are recommended first-line therapy for persistent asthma of all severities; however, oropharyngeal and systemic adverse events can be a concern. Inhaled corticosteroids exert their therapeutic and adverse effects by interacting with glucocorticoid receptors within and outside the lungs, respectively. Ciclesonide is a novel inhaled corticosteroid that possesses a unique pharmacokinetic and pharmacodynamic profile. Ciclesonide is inactive itself and converted to its pharmacologically active metabolite, desisobutyryl-ciclesonide, in the target organ, the lungs. Pulmonary activation combined with low oral deposition may minimize oropharyngeal adverse events, and low oral bioavailability, rapid clearance, and high protein binding may reduce systemic exposure. In addition, high pulmonary deposition due to the highly respirable particles, combined with the potential for prolonged lung retention via lipid conjugation, provides for effective therapeutic action.     

Therapeutic implications of beta-adrenergic receptor pharmacodynamic properties

In the last decades new pharmacodynamic properties of beta-adrenoceptors have been discovered that could greatly impact in the use of beta-adrenergic agents in the clinical practice. Concepts such as multiple binding sites, constitutive activity, polymorphism and intracellular signaling of betaadrenoceptors may contribute in the discovery of more efficacious pharmacological agents for treatment of heart failure and asthma. beta-Adrenoceptors show a relative high constitutive activity in both cardiac and pulmonar tissues. Most beta-blockers exert an inverse agonist action that could contribute to their beneficial effects in the treatment of heart failure. Recently, the existence of multiple affinity sites has been described for beta1-adrenoceptor. It was proposed that beta-blockers that show agonist properties at the beta1L-adrenoceptors binding site may exert neutral or harmful effects when used for treatment of heart failure. Considering the cardiac effect of beta1L-adrenoceptors, activation of the low-affinity state of beta1-adrenoceptor could be deleterous in cardiovascular pharmacology. The ability of beta2-adrenoceptor to couple to Gs or Gi-protein gives the possibility that different agonists can activate different signaling cascades. Full beta2-adrenoceptor agonists would be highly useful for improvement bronchodilatation in the acute treatment of asthma. Polymorphic variants of beta-adrenoceptors have profound impact in the understanding of normal physiology and pathophysiology. Genotypic characterization of patients could improve selection of patients during beta-adrenergic pharmacotherapies. The aim of the present review is to describe new insights in pharmacological and biochemical properties of beta-adrenoceptors and their impact on the use of beta-adrenergic agents in the treatment of cardiovascular and respiratory diseases.     

Species differences in pharmacokinetic and pharmacodynamic properties of nebicapone

The present study was designed to characterize pharmacodynamic and pharmacokinetic properties of nebicapone in rats and mice. Upon oral administration of nebicapone the extent of mouse liver catechol-O-methyltransferase (COMT) inhibition is half that in the rat. Nebicapone was rapidly absorbed reaching plasma C_max within 30 min and being completely eliminated by 8 h. Nebicapone was metabolized mainly by glucuronidation and methylation in both species, but rat had an extra major metabolite, resulting from sulphation. Administration of nebicapone by the intraperitoneal route significantly increased compound AUC in the rat while in the mouse a significant increase in AUC of metabolites was observed. These results show that nebicapone exhibited marked species differences in bioavailability and metabolic profile. Evaluation of COMT activity in rat and mice liver homogenates revealed that both had similar methylation efficiencies (K_cat values, respectively 7.3 and 6.4 min⁻¹), but rat had twice active enzyme units as the mouse (molar equivalency respectively 150 and 83). Furthermore, nebicapone inhibited rat liver COMT with a lower K_i than mouse liver COMT (respectively 0.2 nM vs. 1.2 nM). In conclusion, the results from the present study show that mice and rats respond differently to COMT inhibition by nebicapone. The more pronounced inhibitory effects of nebicapone in the rat may be related to an enhanced oral availability and less pronounced metabolism of nebicapone in this specie, but also concerned with the predominant expression of S-COMT over MB-COMT, the latter of which is less sensitive to inhibition by nebicapone than the former.     

Animal-to-human extrapolation of the pharmacokinetic and pharmacodynamic properties of buprenorphine

OBJECTIVES: This investigation describes the interspecies scaling of the pharmacokinetics and pharmacodynamics of buprenorphine. METHODS: Data on the time course of the antinociceptive and respiratory depressant effects of buprenorphine in rats and in humans were simultaneously analysed on the basis of a mechanism-based pharmacokinetic-pharmacodynamic model. RESULTS: An allometric three-compartment pharmacokinetic model described the time course of the concentration in plasma. The value of the allometric coefficient for clearance was 35.2 mL/min (relative standard error [RSE] = 5.6%) and the value of the allometric exponent was 0.76 (RSE 5.61%). A combined biophase distribution-receptor association/dissociation model with a linear transduction function described hysteresis between plasma concentration and effect. The values of the drug-specific pharmacodynamic parameters were identical in rats and in humans. For the respiratory depressant effect, the values of the second-order rate constant of receptor association (k(on)) and the first-order rate constant of receptor dissociation (k(off)) were 0.23 mL/ng/min (RSE = 15.8%) and 0.014 min(-1) (RSE = 27.7%), respectively, and the value of the equilibrium dissociation constant (K(diss)) was 0.13 nmol/L. The value of the intrinsic activity alpha was 0.52 (RSE = 3.4%). For the antinociceptive effect, the values of the k(on) and k(off) were 0.015 mL/ng/min (RSE = 18.3%) and 0.053 min(-1) (RSE = 23.1%), respectively. The value of the K(diss) was 7.5 nmol/L. An allometric equation described the scaling of the system-specific parameter, the first-order distribution rate constant (k(e0)). The value of the allometric coefficient for the k(e0) was 0.0303 min(-1) (RSE = 11.3%) and the value of the exponent was -0.28 (RSE = 9.6%). CONCLUSIONS: The different values of the drug-specific pharmacodynamic parameters are consistent with the different opioid mu receptor subtypes involved in the antinociceptive and respiratory depressant effects.     

Relationship of pharmacokinetic and pharmacodynamic properties of the organic nitrates

Glyceryl trinitrate (nitroglycerin), isosorbide dinitrate and isosorbide mononitrate are, in various formulations, available for clinical use. The pharmacokinetics of glyceryl trinitrate are complex and only 1% of the drug administered orally can be detected in the plasma due to a pronounced arteriovenous concentration gradient, hydrolysis in the blood, and rapid denitration in the liver. There is a poor and usually transient correlation between plasma concentrations and therapeutic effects, due in part to the complex pharmacokinetics of glyceryl trinitrate, but primarily due to development of tolerance during continuous administration, either via the intravenous or cutaneous route. Isosorbide dinitrate kinetics are complicated by its extensive metabolism into 2- and 5-mononitrates, which are pharmacologically active, and which also accumulate more than the parent drug during long term treatment. These facts, combined with development of tolerance during long term therapy, preclude the establishment of a concentration-response relationship. Isosorbide-5-mononitrate has ideal and dose-linear kinetics and is nearly 100% bioavailable after oral administration. However, tolerance develops during long term therapy, and therefore a relationship between plasma concentrations and clinical effects cannot be established. On the basis of available data, plasma concentrations of various nitrates do not reliably predict clinical effects.