Muscle Tissue Homogenates Research Articles

Muscle Distribution of the Neuromuscular Blocker gallamine Using Microdialysis

Measurement of drug concentrations in target tissue has the potential to provide insight into the pharmacokinetics and pharmacodynamics of a drug. In this study, the distribution of the neuromuscular blocker, gallamine, into muscle tissue was investigated in urethane‐anesthetized rats after an intravenous bolus dose (6 mg/kg). Microdialysis sampling was used to continuously determine gallamine concentrations in muscle interstitial fluid (MIF). In vivo microdialysis recovery of gallamine was determined as the relative loss of gallamine from the perfusate into muscle tissue after perfusion with gallamine (2 μg/mL). Recovery was determined in each rat before the pharmacokinetic studies. Terminal muscle sampling followed by homogenization was also performed to examine gallamine distribution within muscle tissue. All samples were assayed for gallamine using a validated high‐performance liquid chromatography assay. Gallamine was rapidly distributed into MIF with a MIF–plasma partition coefficient of 0.9 ± 0.1 (n = 6). By contrast, the estimated gallamine concentration in muscle tissue homogenate was only 23 ± 5% (n = 5) of the concentration in MIF as estimated by microdialysis sampling at the terminal sampling time. These findings suggest that gallamine is not distributed uniformly within muscle but selectively distributes into MIF. Simulations using a hybrid physiologically based pharmacokinetic model which describes uptake of drug only into the interstitial space showed good agreement between predicted and observed concentration data obtained from microdialysis sampling, supporting the findings that gallamine selectively distributes into MIF. These studies demonstrate microdialysis combined with conventional terminal tissue sampling provides valuable information on intra‐tissue drug distribution. © 2002 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91: 769–775, 2002

Application of a high-performance liquid chromatographic assay for the neuromuscular blocker gallamine to analysis of rat plasma, muscle and microdialysate samples

A reliable high-performance liquid chromatographic method has been validated for determination of gallamine in rat plasma, muscle tissue and microdialysate samples. A C 18 reversed-phase column with mobile phase of methanol and water containing 12.5 m M tetrabutyl ammonium (TBA) hydrogen sulphate (22:78, v/v) was used. The flow-rate was 1 ml/min with UV detection at 229 nm. Sample preparation involved protein precipitation with acetonitrile for plasma and muscle tissue homogenate samples. Microdialysate samples were injected into the HPLC system without any sample preparation. Intra-day and inter-day accuracy and precision of the assay were <13%. The limit of quantification was 1 μg/ml for plasma, 1.6 μg/g for muscle tissue and 0.5 μg/ml for microdialysate samples. The assay was applied successfully to analysis of samples obtained from a pharmacokinetic study in rats using the microdialysis technique.

Gustatory Responses of Ghost Crab Ocypode quadrata to Seawater Extracts and Chemical Fractions of Natural Stimuli

The efficacy of seawater-extracted fresh and decomposing blue crab (Callinectes sapidus) claw muscle homogenates as stimulants of feeding behavior by the ghost crab (Ocypode quadrata) was tested with cheliped flexion as a bioassay. Stimulatory components of extracts were heat-stable and <1 kDa. Fresh seawater extracts of muscle tissue homogenate elicited the most responses and decreased in efficacy with decomposition. Ultrafiltrates <1 kDa also became less stimulating with increasing decay of the homogenate. When ultrafiltrates were extracted with ethyl ether, the aqueous phase elicited the most responses. To some degree, active components were soluble in ether. Ion-exchange chromatography of the aqueous phase yielded eluates containing neutral and acidic compounds, which, following a peak in activity, became less stimulatory over time. In contrast, eluates containing amphoteric and basic compounds remained highly effective throughout bacterial degradation. However, their activity was significantly suppressed when they were mixed with neutral and acidic compounds isolated from the same samples. Mixture suppression may function as a mechanism ensuring the consumption of high-quality foods. The ability of O. quadrata to respond to both fresh and decomposing tissues contributes to this species' flexibility in foraging strategies and its success as an inhabitant of sandy beaches.

Increased angiotensin-converting enzyme activity in the left ventricle after infarction.

An increase in angiotensin-converting enzyme (ACE) activity has been observed in the heart after myocardial infarction (MI). Since most studies have been conducted in chronically infarcted individuals exhibiting variable degrees of heart failure, the present study was designed to determine ACE activity in an earlier phase of MI, before heart failure development. MI was produced in 3-month old male Wistar rats by ligation of the anterior branches of the left coronary artery, control rats underwent sham surgery and the animals were studied 7 or 15 days later. Hemodynamic data obtained for the anesthetized animals showed normal values of arterial blood pressure and of end-diastolic pressure in the right and left ventricular cavities of MI rats. Right and left ventricular (RV, LV) muscle and scar tissue homogenates were prepared to determine ACE activity in vitro by measuring the velocity of His-Leu release from the synthetic substrate Hyp-His-Leu. ACE activity was corrected to the tissue wet weight and is reported as nmol His-Leu g-1 min-1. No significant change in ACE activity in the RV homogenates was demonstrable. A small nonsignificant increase of ACE activity (11 +/- 9%; P > 0.05) was observed 7 days after MI in the surviving left ventricular muscle. Two weeks after surgery, however, ACE activity was 46 +/- 11% (P < 0.05) higher in infarcted rats compared to sham-operated rats. The highest ACE activity was demonstrable in the scar tissue homogenate. In rats studied two weeks after surgery, ACE activity in the LV muscle increased from 105 +/- 7 nmol His-Leu g-1 min-1 in control hearts to 153 +/- 11 nmol His-Leu g-1 min-1 (P < 0.05) in the remaining LV muscle of MI rats and to 1051 +/- 208 nmol His-Leu g-1 min-1 (P < 0.001) in the fibrous scar. These data indicate that ACE activity increased in the heart after infarction before heart failure was demonstrable by hemodynamic measurements. Since the blood vessels of the scar drain to the remaining LV myocardium, the high ACE activity present in the fibrous scar may increase the angiotensin II concentration and decrease bradykinin in the cardiac tissues surrounding the infarcted area. The increased angiotensin II in the fibrous scar may contribute to the reactive fibrosis and hypertrophy in the left ventricular muscle surviving infarction.

Antagonistic properties of [Phe 3,Leu 13]porcine motilin

We describe the antagonistic properties due to the replacement of Pro 3 by phenylalanine in porcine motilin. The analogue, [Phe 3,Leu 13]porcine motilin (OHM-11526), displaces iodinated [Nle 13]porcine motilin bound to a homogenate of rabbit antral smooth muscle tissue. The dissociation constant (p K d) was 9.26 ± 0.04, versus 9.11 ± 0.01 for motilin and 8.24 ± 0.06 for ANQ-11125, the (1–14) fragment of OHM-11526. The Hill coefficient was close to one and Schild plot analysis confirmed the competitive nature of the interaction. In the tissue bath OHM-11526 was unable to induce contractions of segments of rabbit duodenum. At a concentration of 10 −6 M, OHM-11526 inhibited the effect of maximally effective doses of porcine motilin and of the erythromycin derivative, EM-523, but was without effect on contractions induced by acetylcholine, substance P and serotonin. Increasing doses of OHM-11526 shifted the dose-response curves of motilin and EM-523 to the right, but caused a depression of the maximal response as well. From the motilin curves, and assuming a dual competitive and non-competitive interaction, the pA 2 was 7.79 ± 0.08, the pD′ 2 6.91 ± 0.08. The EM-523 curves yielded comparable data (pA 2 = 8.10 ± 0.12 and pD′ 2 = 7.06 ± 0.13). OHM-11526 also blocked the motilin responses observed with smooth muscle strips from the rabbit and human antrum. However, in a preparation of the chicken small intestine, OHM-11526 was a full agonist with a potency (pD 2 = 6.84) comparable to that of porcine motilin (pD 2 = 6.71). Our data confirm the interaction of motilides with the motilin receptor. Due to its increased affinity for the motilin receptor, OHM-11526 will be a valuable tool for studying the physiology of motilin and the pharmacology of motilin and motilides.

A Method for Isolation and Partial Purification of Trichinella spiralis Nurse Cells

Invasion of vertebrate muscle cells by larvae of Trichinella spiralis is accompanied by redifferentiation of the host myofiber into a novel structure called the nurse cell. The nurse cell protects and nurtures the enclosed parasite during its long stay in host muscle. It is anatomically independent of the surrounding uninfected muscle cells and can be isolated from host tissue by mechanical or enzymatic means. Current methods employed for this purpose have yielded only small numbers of nurse cells. An apparatus designed to isolate large numbers of nurse cells and a method for removal of all free larvae and most host muscle debris is described. Homogenization and trypsin digestion of muscle tissue was followed by passage of muscle/parasite suspensions maintained at 37 C through a jacketed glass column fitted with a 40-mesh stainless steel screen at the top and a Nitex screen with 150-microns-diameter pores at the bottom. Nurse cells were retained by the Nitex screen. Density gradient centrifugation using Percoll removed all free larvae and most contaminating muscle debris from nurse cell suspensions. The large quantities of nurse cells made available by this method will allow evaluation of the molecular biology, nutrition, biochemistry, and metabolism of the enclosed parasite and of the Trichinella-modified host muscle cell.

Myosin light chain phosphatase activity in ragweed pollen-sensitized canine tracheal smooth muscle.

We have reported that myosin light chain phosphorylation is increased in contracting airway smooth muscle from hyperresponsive, ragweed pollen-sensitized dogs. This alteration is manifest physiologically in smooth muscle tissue from sensitized animals as it demonstrates faster shortening velocity and increased shortening capacity. One of the mechanisms underlying the defect is increased myosin light chain kinase activity; it is not known whether modulation of myosin phosphatase activity contributes to enhanced myosin light chain phosphorylation in sensitized canine smooth muscle. We describe a myosin phosphatase assay that we have used to compare the enzyme's activity in crude tracheal smooth muscle tissue homogenates from control and sensitized airway smooth muscle. Twenty kilodalton myosin light chain phosphorylation was initiated with Mg(2+)-ATP, and maximum levels were reached within 40 s; peak phosphorylation levels were stable for at least 3 min. The relative stoichiometry of 20 kD myosin light chain phosphorylation was estimated by chemiluminescent immunoblot assay. Smooth muscle phosphatase activity was estimated by the rate of decline in peak light chain phosphorylation, while myosin light chain kinase was inhibited indirectly with trifluoperazine, with EGTA, or directly by a synthetic peptide inhibitor. Okadaic acid, an inhibitor of phosphatase activity, curbed the decline in light chain phosphorylation seen after myosin light chain kinase inhibition, indicating that the light chain dephosphorylation observed was the result of smooth muscle phosphatase activity. Addition of okadaic acid to the samples led to a 30 to 40% increase in the peak myosin light chain phosphorylation attained for all samples. This indicates that similar populations of phosphatases were present in the homogenates of both control and sensitized tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of the 70‐kDa heat‐shock protein from catfish liver: Immunological comparison of the protein in different fish species and its potential use as a stress indicator

AbstractThe heat‐shock protein or stress‐70 family was isolated from catfish liver. The homogeneity of the purified protein was analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Fish subjected to whole‐body hyperthermia contained the constitutive and the heat‐inducible stress‐70 with approximate molecular weights of 70 and 68 kDa, respectively. The final purification product from livers of catfish raised under normal temperature was only the constitutive stress‐70. Western blot analysis with rabbit antiserum prepared against purified catfish (Ictalurus punctatus) liver stress‐70 showed that the antibody cross‐reacted with liver, muscle, and gill tissue homogenates of fathead minnows (Pimephales promelas), red shiners (Cyprinella lutrensis), black bass (Micropterus salmoides), and bluegill (Lepomis macrochirus), with various intensities suggesting that stress‐70s from different tissues of various fish species share common antigenic determinants of the protein. This substantiates that the antigen/antibody approach of stress‐70 is useful as a stress indicator and, consequently, as a potential biomarker for water quality.

The Motilin Antagonist ANQ-11125 Blocks Motilide-Induced Contractions in Vitro in the Rabbit

Studies on the physiological role of motilin, and more recently, on the relationship between motilin and erythromycin A, have been hampered by the lack of antagonists. We now have discovered such a compound. ANQ-11125 displaces motilin bound to an homogenate of rabbit antral smooth muscle tissue. The dissociation constant (pKd) was 8.16 ± 0.10. However, ANQ-11125 did not induce contractions of segments of rabbit duodenum, except at high concentrations. In the presence of 1 μM ANQ-11125 the dose response curves of erythromycin-A, De(N-methyl)-N-ethyl-8,9 anhydroerythromycin A 6,9-hemiacetal and motilin were shifted about one log unit to the right, but the responses to ACh and Substance P were unaffected. Schild-analysis showed the competitive nature of the interaction and allowed the calculation of the pA2 : 7.03 ± 0.05 (motilin curves) and 7.55 ± 0.06 (EM-523 curves). This is the first report of a motilin antagonist. Its properties definitively prove that motilides are motilin agonists.

Purine metabolic pathways in rat hindlimb perfusion model during ischemia and reperfusion.

The perfused rat hindlimb preparation was used with a blood cell-free perfusate to investigate alterations in the purine nucleotide metabolism, flow rate, perfusion pressure, and venous excretion in response to ischemia and ischemia followed by reperfusion in skeletal muscle. The development of a physical hindrance during postischemic reperfusion, indicated by an increase in reperfusion pressure and a decrease in flow rate, coincided with a 90% decrease in phosphocreatine and a 50-70% reduction in total adenine nucleotide pool. The reflow impairment could not be explained by blood cell plugging of the capillaries. Washout of several metabolites was demonstrated during reperfusion. Hypoxanthine accumulated intracellularly during ischemia, and a substantial amount of uric acid was excreted into the venous effluent during reperfusion. The experimental data were fitted into a computer simulation model of the purine pathways. The model indicated that AMP deaminase was the predominant enzymatic pathway for the AMP degradation. It was demonstrated that ATP preferably accumulated as inosine-5'-monophosphate during ischemia and that xanthine oxidase was undetectable in skeletal muscle tissue homogenates. However, vascular endothelial cell xanthine oxidase activity responsible for a free radical-induced reperfusion injury could not be excluded.

Effect of erythromycin and of octreotide on motilin receptor density in the rabbit

Recent studies have shown that erythromycin lactobionate (EMLB) acts as a motilin agonist and is able to accelerate gastric emptying in diabetic gastroparesis. Using the rabbit as a model, we have studied the changes in motilin receptor density induced by EMLB (a motilin agonist) and octreotide (a somatostatin analogue and an inhibitor of motilin secretion). Binding studies were performed with antral smooth muscle tissue homogenates using iodinated nor-leucine 13-porcine-motilin, and binding parameters were obtained from computerized fits to displacement curves. The contractile capacity towards motilin (10 −7 M) and EMLB (10 −5 M) was measured isotonically on duodenal segments and the response was expressed relative to the maximum obtained with ACh (10 −4 M). The first hours after the last i.v. administrations of EMLB (50mg/day given on 3 consecutive days), motilin binding was completely abolished to 0.02 ± 0.006 fmol/mg protein, compared to the control group ( 0.64 ± 0.12 fmol/mg protein). The effect was dose-related: total doses of 17.5 mg, 87.5 mg, 175 mg EMLB reduced motilin binding and contractility towards motilin and EMLB to respectively 95 ± 10, 82 ± 5%; 36 ± 9, 38 ± 9%; 3 ± 1, 24 ± 2% of the control values. The effect was also long lasting: binding was still reduced to 60% of the control value 48 h after the highest dose. In contrast, octreotide induced a marked but short lasting upregulation. After 3 daily s.c. injections of 5 μg, B max rose to 13.6 ± 1.9 fmol/mg protein ( P < 0.05). It was already obtained 1 h after 3 × 2.5 μ g/24 h . The changes in receptor-density were not related to changes in affinity. We conclude that motilin receptors can be regulated by EMLB and octreotide presumably because one compound minicks hypermotilinemia, the other one induces hypomotilinemia.

Development of motilin receptors and of motilin- and erythromycin-induced contractility in rabbits

The development of the motilin receptor was studied through contraction and binding studies of groups of three rabbits aged between 2 and 289 days. The contractility of small intestinal smooth muscle strips was measured isotonically. The aborally decreasing gradient in response to motilin, known to exist in adult rabbits, was already present at day 8 (maximum contractile responses expressed as a percent of the maximal response to acetylcholine were 77% ± 9%, 34% ± 10%, and 25% ± 8% for duodenal, jejunal, and ileal strips, respectively). Throughout the observation period, the doses of motilin and its agonist erythromycin that were required to induce 50% of the response remained constant and were not significantly different from doses required for adults (their negative logarithms were 8.55 ± 0.35 and 5.70 ± 0.25, respectively). The correlation between the maximum contractile response toward motilin and erythromycin was almost perfect (r2 = 0.82). Binding studies with iodinated norleucine13-porcine motilin were performed using antral smooth muscle tissue homogenates. The maximum number of binding sites increased rapidly after 8 days (3.3 ± 0.4 fmol/mg protein) and reached a peak at 21 days (20.7 ± 1.4 fmol/mg protein), but decreased at that point toward the adult value (40 days, 10.6 ± 1.3; 289 days, 9.8 ± 1.1 fmol/mg protein). The dissociation constant, however, remained unchanged. The peak value of receptor density occurred at about the time that the rate of increase of the length of the intestine and of the weight of the antrum were at maximum levels (at 18 and 27 days, respectively). Motilin receptors are expressed early postnatally, and the regional gradient in sensitivity towards motilin is also established soon after birth. If applicable to humans, an early response to erythromycin may have therapeutic value.

Structure‐Activity Relation of Erythromycin‐Related Macrolides in Inducing Contractions and in Displacing Bound Motilin in Rabbit Duodenum

ERYTHROMYCIN A appears to be a motilin agonist, although motilin (a peptide) and erythromycin A (a 14‐member macrolide antibiotic carrying two sugar substituents) are structurally totally unrelated. To obtain information concerning the structural requirements for erythromycin's interaction with the motilin receptor, we studied 60 derivatives and seven fragments of erythromycin, and six macrolides with a 16‐member ring structure. For every compound the ability to displace motilin bound to a crude homogenate of rabbit antral smooth muscle tissue and the ability to induce contractions in rabbit duodenal segments were determined. For both parameters dose‐response curves were obtained, and the negative logarithms of the concentrations inhibiting 50% of the binding (pIC‐50) or inducing 50% of the maximum response to a maximal contractile dose of acetylcholine (pEC‐50) were determined. All macrolides with a 16‐member ring structure were inactive in both types of experiments. In all erythromycin derivatives variations of the ring structure had a marked effect. For both parameters the order of potency was enol ether &gt; pseudo–enol ether &gt; parent ring &gt; pseudo‐hemiketal &gt; anhydro. The two sugars attached to the ring were important too, because removing either or both of them resulted in an inactive compound. Modifications of the dimethylamino group of the desosamine sugar affect the potency, while the orientation of the cladinose moiety seems to be important too. For all active compounds, both parameters studied were well correlated (r= 0.80, p &lt; 0.001). Our results support the concept that erythomycin‐like macrolides are motilin agonists. The structural requirements of these “motilides” involve the ring structure, especially the part that can be transformed into an enol ether, and both attached sugars. Potent derivatives may prove to be useful as gastrokinetic agents with a very specific target zone.

Comparison of motilin binding to crude homogenates of human and canine gastrointestinal smooth muscle tissue

Pharmacological studies indicate that in man and in rabbit, but not in dog, motilin has a direct influence upon gastrointestinal smooth muscle. In accordance with this hypothesis we have presented direct biochemical evidence for the presence of motilin receptors on rabbit smooth muscle tissue. We have now extended our studies to human and canine tissue. Tissue homogenates were studied in binding experiments with iodinated porcine [Leu 13]motilin and iodinated canine motilin. It was ascertained that the iodination procedure had little effect on the biological activity of the porcine analogue. In the human antrum specific binding of the iodinated porcine analogue was only found in the smooth muscle layer. It was absent in mucosal or serosal preparations. At 30°C and pH 8.0, binding was maximal after 60 min of incubation, and was reversed by the addition of unlabeled porcine motilin. Binding was enhanced in the presence of calcium and magnesium ions. At a concentration of 10 mM MgCl 2, binding was 220% of the binding observed in its absence. Displacement studies with synthetic porcine [Leu 13]motilin or synthetic natural porcine motilin indicated a dissociation constant ( K d) of 3.6 ± 1.6 nM and a maximal binding capacity ( B max) of 77 ± 9 fmol per mg protein. Canine motilin displaced iodinated porcine motilin with an apparent K d of 2.2 ± 0.9 nM. Compared to antral binding, receptor density decreased aborally and orally, and was absent in jejunum and ileum. In dog specific binding could not be demonstrated in antral and duodenal tissue, neither with labeled porcine nor with labeled canine motilin. However, labeled canine motilin was equipotent to labeled porcine motilin in binding studies with human tissue: the dissociation constant was 0.9 ± 0.6 nM. The present studies therefore demonstrate the existence of a specific motilin receptor in the antroduodenal region of the human gut. Apparently, such receptors are not present in the canine gut. Our data support the hypothesis that in the human gastrointestinal tract, the gastroduodenal area is motilin's target region.

Zinc can activate cellular acidic alpha-D-glucosidase activity.

In an attempt to elucidate the effect of metallic ions and EDTA on acidic alpha-D-glucosidase activity, we measured acidic alpha-D-glucosidase activity from either lymphocyte and muscle tissue homogenates or intact cells after incubation with metallic ions. The results showed that this enzyme activity was strongly inhibited by Ag+, Hg2+, and Fe3+ in either lymphocyte or muscle tissue homogenates. There was no effect of Zn2+, Cu2+, and Cd2+. However, intact cells, either lymphocyte or muscle cells, after incubation with Zn2+ for 1 or 2 hr, showed enhanced enzyme activity and suppression in the other metallic ion groups, especially in Ag+, Hg2+, and Fe3+. Since deficiency of this enzyme can cause type II glycogen storage disease (Pompe's disease), the more we understand the character of this enzyme, the more we can improve our enzymatic therapy.

Direct binding studies on ileal and cardiac muscarinic receptors.

1 Functional studies have indicated that muscarinic receptors in cardiac tissue differ from those in the ileum. In the present study ileal and cardiac muscarinic receptors identified by [3H]-N-methyl scopolamine ([3H]-NMS) were characterized and the selectivity of currently available ileal and atrial selective antagonists determined. 2 In terms of the current functional classification of muscarinic receptors both ileal and cardiac muscarinic receptors were of the M2 subtype based upon their low affinity for pirenzepine. 3 Cyclohexylphenyl(2-piperidinoethyl)silanol (CPPS), a highly ileal selective antagonist in functional studies, was unable to distinguish between ileal and atrial muscarinic receptors identified in binding studies. Furthermore, although AF-DX 116 and dicyclomine were able to differentiate atrial and ileal muscarinic receptors, neither compound was more than 2 fold selective. These data indicate that it is not possible to subclassify ileal and atrial muscarinic receptors using direct ligand binding studies with these antagonists. 4 In circular ileal smooth muscle, apparent heterogeneity of the M2 muscarinic receptor population was observed. Thus AF-DX 116 identified two populations of sites with affinities differing by 30 fold. These two populations of M2 muscarinic receptors may represent the typical M2 muscarinic receptors identified in cardiac tissue and the more recently discovered 'gland type' M2 muscarinic receptors. 5 The circular ileal smooth muscle tissue homogenate was able to decrease dramatically the apparent affinity of adiphenine. This activity, which appeared to result from a phenylmethylsulphonylfluoride (PMSF) sensitive protease effect, should be considered when conducting studies using this tissue preparation and compounds of similar structure to adiphenine.

Evidence for reduced catabolism by the antiglucocorticoid RU 38486 in acutely uremic rats.

Previous studies suggested that increased blood levels of, or increased tissue sensitivity to, glucocorticoids may contribute to catabolism in acute uremia. To examine this possibility we determined urea nitrogen (urea-N) appearance, plasma levels of Nt-methylhistidine and the activity of the alkaline myofibrillar proteinase in acutely uremic rats with and without treatment with RU 38486, a selective antiglucocorticoid. Forty-eight hours after bilateral nephrectomy, the rats had markedly elevated serum levels of urea-N, creatinine, potassium and phosphorus. In uremic rats receiving RU 38486, comparable levels of serum creatinine were found, but the serum levels of urea-N (221 +/- 4 vs. 259 +/- 5 mg/dl) and phosphorus (6.5 +/- 0.3 vs. 8.5 +/- 0.4 mmol/l) were significantly decreased as compared to uremic animals without RU 38486. In comparison to sham-operated rats, urea-N appearance (net urea production) was increased by 56% 48 h after bilateral nephrectomy. This increment was almost completely reversed in uremic animals receiving the antiglucocorticoid. In untreated uremic rats, plasma levels of Nt-methylhistidine were 10.3 +/- 0.9 microgram/dl, whereas the administration of RU 38486 caused a significant decline in the levels of this amino acid (7.6 +/- 0.5 microgram/dl). This reduction in Nt-methylhistidine was associated with a concomitant decrease of myofibrillar proteinase activity in muscle tissue homogenates. Compared to sham-operated animals, this proteinase activity was increased by 30% in uremic rats, but was normal in those given RU 38486. Taken together, these data support the view that in acute uremia accelerated ureagenesis occurs, while enhanced muscle protein breakdown, owing to an increment in myofibrillar proteinase activity, provides the necessary amino acid precursors.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of Drug Distribution and Binding Competition by Two-Chamber and Multi-Chamber Distribution Dialysis

Binding competition between blood and tissue, a determinant of drug distribution, can be simulated and quantitated in vitro by distribution dialysis. In a study with a standardized two-chamber system, six model drugs, selected according to their ratio of plasma to tissue binding, were allowed to distribute between blood and eight tissue homogenates of rats. The tissue:blood concentration ratios were 1 for antipyrine, <1 for salicylic acid and phenylbutazone, slightly >1 for pentobarbital and thiopental, and » 1 for imipramine. Comparable values of tissue:blood ratios were obtained in rats in vivo. A modified dialysis system was developed which allows the simultaneous distribution of a drug between blood and four tissue homogenates. This multichamber system was used for homogenates of liver, lung, muscle, and adipose tissue. The drugs tested reached a first apparent distribution equilibrium after 2.5 to 4 h, comparable with the distribution in the twochamber system, as a result of tissue:blood binding competition. In the following hours there was a redistribution as a result of binding competition among individual tissues, except in the case of pentobarbital. A minor redistribution from muscle to adipose tissue was observed with phenylbutazone, and from muscle to liver with imipramine. There was a considerable redistribution from all tissues, including blood, to adipose tissue with thiopental. In a sequential mode of operation this multichamber system was used to simulate the influence of the different perfusion rates of individual tissues by adding the homogenates of muscle and/or adipose tissue several hours after the other tissues. This modification showed that adipose tissue rapidly took up large amounts of thiopental by redistribution, whereas the uptake of imipramine was considerably reduced. Two-chamber distribution between adipose and other tissues confirmed the strong binding competition by nonadipose tissues in the case of imipramine, and its lack in the case of thiopental. It is concluded that drug distribution in vivo is largely determined by binding competition between tissue and blood as well as among individual tissues. Distribution dialysis is proposed as a useful in vitro model for the simulation and prediction of drug distribution.

Comparative effects of polymyxin B and compound [formula omitted] on histamine metabolism in rat muscle and gastric tissue

Polymyxin B, administered in vivo , increased histidine decarboxylase (HDC) activity and histamine (HM) concentrations in muscle tissue homogenates and supernatants. When administered in vitro it increased HDC activity and HM concentrations in both muscle and gastric tissue. The stimulatory effect on muscle was similar to that obtained with compound 48 80 , but 48 80 , unlike polymyxin B, did not affect gastric tissue. In vitro additions of alphafluoromethylhistidine inhibited both in vivo and in vitro stimulatory effects of polymyxin B. The results of these studies show that the action of compound 48 80 and of polymyxin B are similar, and that both affect HM synthesis in a manner that requires further elucidation.

Fatty Acid-Binding Protein Activities in Bovine Muscle, Liver and Adipose Tissue

Subcutaneous adipose tissue, sternomandibularis muscle and liver were obtained from steers immediately postmortem. Muscle strips and adipose tissue snips were incubated with 0.75 mM [1-14C]palmitate and 5 mM glucose. Muscle strips esterified palmitate at the rate of 2.5 nmol/min per gram tissue, which was 30% of the rate observed for adipose tissue. Fatty acid-binding protein activity was measured in 104,000 × g supernatant fractions of liver, muscle and adipose tissue homogenates. Muscle and adipose tissue fractions bound 840 and 140 pmol [1-14C]palmitoyl-CoA per gram tissue, respectively. Fatty acid-binding protein activity was greater in adipose tissue than in muscle when data were expressed per milligram protein (35 vs. 13 pmol palmitoyl-CoA bound per milligram of soluble protein, respectively). Fatty acid binding-protein activity was correlated with the rate of palmitate esterification within each tissue. Liver contained the highest fatty acid-binding protein activity (13,000 pmol palmitoyl-CoA bound per gram tissue and 215 pmol palmitoyl-CoA bound per milligrams soluble protein).