Effect of gram-negative bacterial lipopolysaccharide-derived polysaccharides, glycolipids, and lipopolysaccharides on rabbit and human platelets in vitro.

The in vitro effect of gram-negative bacterial LPS-derived polysaccharide (PS), glycolipid (GL), and lipopolysaccharide (LPS) was investigated both on rabbit and human platelets. Rabbit platelets aggregated when they were treated with either GL or LPS, but no aggregation occurred when PS was used. No aggregation occurred when human platelets were treated with LPS, PS, or GL. However, when either human or rabbit platelets were treated with LPS-antibody complexes (LPS-ab), aggregation took place. Guinea-pig serum inhibited the aggregation caused by LPS-ab, but had no effect on rabbit platelet aggregation caused by LPS or GL alone. The factor(s) in guinea-pig serum that inhibited aggregation was heat-stable. These results suggest that there may be two different mechanisms involved in rabbit platelet aggregation by endotoxin in vitro. Using human platelets, only one mechanism was observed.

Kinetics of aggregation of human platelets.

A study of the aggregation kinetics of human platelets using an electronic counting device is reported. The experimental data were analyzed quantitatively by a physical model, which assumed that the initial disappearance rate of single platelets versus time fitted a second-order type of aggregation with respect to platelet number. The mechanism of aggregation was surface barrier controlled. Thus, the aggregation rate constants in different adenosine diphosphate concentrations (1.5-9.0 microgram) were 10-100 times greater than the rate constant (6.6325 x 10(-12) cm3/sec) for a diffusion-controlled mechanism, were smaller in the surface-barrier-controlled process and ranged from 0.00741 to 0.0467. The extent of aggregation was indicated by the calculation of a sticking probability constant as determined by the barrier. Adenosine diphosphate induced a rapid aggregating effect. Prostaglandin E1 produced the most drastic deaggregating effect as compared to dinoprostone (prostaglandin E2) and dinoprost (prostaglandin F2alpha). Aspirin completely blocked the aggregating effect of arachidonic acid.

Effects of cytochalasin, colchicine, and ethylenediaminetetraacetic acid on linoleic acid transport across rat jejunal enterocytes.

The involvement and the site of interference of the cytoskeleton in the transport of linoleic acid across the rat jejunum was investigated by administration of microfilamentous and microtubular altering agents such as cytochalasin, colchicine, and ethylenediaminetetraacetic acid (EDTA). An isolated jejunal segment was perfused with a buffer containing labeled linoleic acid, and portal blood and perfusate samples were collected concomitantly at 5-min intervals and assayed for their radioactivity. At the end of the perfusion, the amount of radioactivity retained in the intestine was also determined. The results were analyzed by using a three-compartment physical model that allows the determination of mucosal and serosal permeability coefficients, from which changes in the permeability of the mucosal and serosal membranes were assessed. Cytochalasin decreased the permeability of the mucosal membrane to linoleic acid, but not that of the serosal membrane. The administration of colchicine, EDTA, or cytochalasin + colchicine increased the permeability of the serosal membrane but did not affect the mucosal membrane.

Differential absorption of vitamin D3 and 1,25-dihydroxyvitamin D3 by intestinal jejunal cells of the rat.

An intestinal perfusion technique is reported for the study of the differential absorption of vitamin D3 and its active metabolite, 1,25-dihydroxyvitamin D3, through intact jejunal segments of rats. Samples of introduced and collected perfusates, intestinal homogenates, and portal blood were assayed for [14C]vitamin D3 or [3H]1 alpha,25-dihydroxyvitamin D3 content at specified time intervals in control rats and in rats injected ip with cycloheximide (3 mg/kg body weight). Vitamin D3 uptake from the perfusates in cycloheximide-treated groups did not differ from controls. However, an approximately 2-fold increase of vitamin D3 retention in the perfused intestinal segments was observed after cycloheximide treatment. A 0.25-fold decrease was observed in the uptake of 1,25-dihydroxyvitamin D3 from the perfusates after cycloheximide treatment, and an approximately 2.5-fold increase in its intestinal retention was noted. An increase in the active metabolite concentration was observed in the portal venous system 75 min after initiation of perfusion, with no detectable amounts being recorded prior to the first hour. The results suggest that intracellular binding proteins may be involved in the transport of labeled vitamin D3 and labeled 1,25-dihydroxyvitamin D3 through rat enterocytes. Furthermore, vitamin D3 may have been more readily channeled through an esterification process than 1,25-dihydroxyvitamin D3 prior to their appearance in the portal venous system.

Agglutination kinetics of enzymatically treated normal and diabetic rat hepatocytes.

The effect of trypsin and neuraminidase treatments on concanavalin A-induced agglutination of viable hepatocytes from normal and diabetic rats are reported. Trypsin (1.0 microgram/mL) treatment resulted in a increased rate of hepatocyte agglutination in both normal and diabetic cells in the presence of 100 micrograms/mL of concanavalin A. However, neuraminidase treatment resulted in a decrease in the rate of cytoagglutination in the normal cells and an increase in the rate in the diabetic counterpart. The results suggest that trypsin may have caused the removal of a surface protein and/or split a peptide bond on the agglutinin receptors resulting in identical receptor exposure and clustering in normal and diabetic cells. The neuraminidase data suggest that the arrangements of the neuraminic acid moieties on the receptors in normal cell membranes were different from those in the diabetic cells, eliminating the possible effect of changes in the surface charge density. In conclusion, normal cells carry numerous clustered (possibly some in the "cryptic" state) agglutinin receptors in the cell membrane as compared with cells from diabetic rats.

Sodium-dependent retention of prostaglandins in rat ileum.

An intestinal perfusion technique is reported to study the steady-state ileal mucosal retention of labeled prostaglandins E1, E2, and F2 alpha in rats. Ileal homogenates were analyzed for [3H]prostaglandin E1, [14C]prostaglandin E2, and [14C]prostaglandin F2 alpha after intestinal segments were perfused with Krebs improved Ringer buffers containing sodium or depleted from sodium and replaced with choline, and buffers containing 1 mM ouabain, 2 mM amiloride, or 50 nM tetrodotoxin. Prostaglandin ileal mucosal uptake was also studied after iv injection of tetrodotoxin (20 micrograms/kg body weight). Prostaglandin concentration-dependent transport studies support passive uptake mechanisms for prostaglandin E1 and E2. Physiological concentrations of sodium increased labeled prostaglandins E1, E2, and F2 alpha ileal mucosal uptake and decreased n-octanol-buffer partition coefficients. Unlike sodium, potassium showed no effect on labeled prostaglandin ileal transport. Ouabain, amiloride, and tetrodotoxin in the perfusates did not significantly alter prostaglandin mucosal uptake. However, injecting tetrodotoxin into rats caused a drastic increase of prostaglandin uptake through the ileal mucosa. Therefore, the role of sodium on labeled prostaglandin E1, E2, and F2 alpha ileal mucosal transport can be postulated to be controlled by either one or both of the following mechanisms: a pH-partition passive transport mechanism, and/or a sodium channel-dependent pathway whereby prostaglandin permeation possibly proceeds via a sodium-prostaglandin ion-pair mechanism which is controlled by a gating phenomenon.

Effects of verapamil, ouabain, and ethacrynic acid on calcium retention in perfused rat kidneys.

A Sprague-Dawley rat kidney perfusion technique was used in situ to study the effect of verapamil, ouabain, and ethacrynic acid on renal calcium retention. The technique involves perfusion of the kidneys via the abdominal aorta and then through the left and right renal arteries and dorsal aorta. Verapamil (1 mM) in Krebs-improved Ringer solution increased calcium retention in the kidneys by approximately 117% compared with controls. With Na-free Krebs-improved Ringer solution, calcium retention increased by only 92%. However, in Krebs-improved Ringer solutions containing 59 and 122 mequivalents of Na, calcium retention in the kidney increased by 46 and 43%, respectively, compared with controls. With Krebs-improved Ringer solution containing 15 mM ouabain, calcium retention in the kidney decreased by 29.5%, whereas with 15 mM ouabain plus 1 mM ethacrynic acid in the perfusate, the effect on calcium retention in the kidney was insignificant compared with controls. These results suggest that two sodium-dependent calcium-transporting systems exist at the peritubular side of the kidney tubules: (1) a Na(+)-Ca+2 countertransport system sensitive to verapamil and (2) a Na(+)-Ca+2 cotransport system sensitive to the intracellular concentration of sodium.

Quantitative estimation of particulate matter in pharmaceutical preparations intended for intravenous administration.

A fast, reproducible, economical, and dependable automated counter method is recommended for the quality control of pharmaceutical preparations intended for intravenous administration. The USP gives no specifications on the limitations of particulate matter in intravenous products, while the BP specifies limitations on intravenous solutions of more than 500-ml volume. Out of 15 different marketed products, few passed the BP specifications. The microscopically identifiable particles included starch, cellulose fibers, glass, rubber, lacquer flakes, carbon black, and metal shavings. The proposed quality control method introduces a modification to the BP specifications. The method includes a standard log-log plot obtainable from a least-squares line fit to the data of the marketed products showing minimal particle contamination. The standard plot is compared to the experimental data obtained from any other product and, accordingly, it is decided whether the product is acceptable or not.

Calcium-prostaglandin aggregation and its effect on prostaglandin uptake by isolated rabbit intestine.

A quantitative estimate of the role of calcium ions on the lipid--water partition coefficients of prostaglandin E1 and dinoprost suggested the possibility of prostaglandin molecules and calcium ions aggregating in a 14:1 ratio to produce a lipid-soluble aggregate. The aggregation is postulated to be a characteristic of prostaglandin molecules as compared to simple fatty acids, e.g., 1-octanoic acid, which, in the presence of calcium, behave differently than the prostaglandins. The uptake of prostaglandins by the mucosal surface of the rabbit intestine increased in the presence of calcium. For example, at 25 mM calcium, prostaglandin E1 was transported at approximately twice the rate as in the system containing no calcium. The uptake rate of dinoprost was estimated to be three times faster with 10 mM calcium than in the absence of calcium. Therefore, it is proposed that a carrier-mediated diffusion process, for both the prostaglandin molecules and calciumi ions, takes place in the uptake mechanism. Diffusion coefficients ranging from 0.48 X 10(-5) to 7.19 X 10(-5) cm2/sec and permeability coefficients ranging from 1.04 X 10(-2) to 15.6 X 10(-2) cm/sec were estimated for all systems studied.

System approach to the transepithelial transport across rat jejunal enterocytes: effect of cytochalasin B, colchicine, and ethylenediaminetetraacetic acid on butyric acid transport.

A three-compartment physical model is devised for transepithelial passive transport across intestinal cells. The mathematical equations derived from the model allow the simultaneous and quantitative measurements, in the form of permeability coefficients, of solute transport across both the luminal-serosal and serosal-blood barriers. The proposed model is used to study the involvement of the cytoskeleton in butyric acid absorption by the rat jejunum. Alterations in cytoskeletal functions are introduced by the administration of microfilamentous and microtubular altering agents such as cytochalasin, colchicine, or EDTA. An isolated jejunal segment perfused with a buffer containing labeled butyric acid was homogenized at the end of the experiment and assayed for its butyric acid content. During the perfusion, portal blood samples, as well as perfusate samples collected 10 cm distal to the perfusion site were drawn at 5-min intervals and assayed for their radioactivity. Cytochalasin was found to decrease the permeability of the mucosal membrane to butyric acid and to increase that of the serosal membrane. Colchicine did not have any effect either on the mucosal or on the serosal side. Cytochalasin and colchicine, when given in the same experiment, increased the permeability of the serosal membrane to butyric acid, but were without any effect on the mucosal barrier. Also, EDTA had no effect on the mucosal side, but decreased significantly the permeability of the serosal membrane to the fatty acid.

Effect of treatment with phospholipase A2, colchicine, and beta-galactosidase on agglutination of rat hepatocytes.

The effects of phospholipase A2, colchicine, and beta-galactosidase on concanavalin A-induced agglutination of viable hepatocytes isolated from normal and diabetic rats are reported. Phospholipase A2 (0.92 microgram/mL), colchicine (400 micrograms/mL), and beta-galactosidase (300 micrograms/mL) treatments caused a significant increase in the agglutination rate of hepatocytes. These findings suggest that phospholipase A2 treatment resulted in the unshielding of lectin receptors. Colchicine treatment seemed to release those receptors from cellular restraints which tend to separate and/or direct them. The promoting effect of beta-galactosidase could be attributed to a decrease in repulsive forces due to a reduction in net negative charge density after removal of N-acetylneuraminic acid residues. Normal rat hepatocytes seem to be richer in galactosides, phospholipids, and the microfilament-microtubule network than their diabetic counterparts.

Adsorption isotherms of ouabain on hepatocytes from normal and diabetic (streptozotocin-induced) rats.

A cell surface adsorption isotherm approach is investigated with normal and diabetic (streptozotocin-induced) rat hepatocytes utilizing mathematical modeling. Freshly prepared monodispersed viable rat hepatocytes in Ca(2+)- and Mg(2+)-free phosphate buffer are obtained by collagenase perfusion and used in this study. [3H]ouabain is used as a ligand that specifically binds with the alpha 1 and alpha 2 isoforms of the alpha-protein subunit of the hepatocyte-membrane-incorporated Na-K-ATPase. The model that fits the experimental data assumes the presence of multiple receptors on the cell surface, and only when a specific fraction of the total number of one receptor have effectively reacted will the other receptor initiate reaction with the ligand. The results suggest the existence of two receptors, in normal and diabetic hepatocytes, interacting with ouabain and having different equilibrium constants. The alpha 2 isoform interacts more strongly with ouabain than the alpha 1 isoform in both types of cells. The alpha 1 isoform of the diabetic hepatocytes has stronger affinity with the glycoside than the alpha 1 isoform of the normal hepatocytes, while alpha 2 of the diabetics shows weaker affinity than alpha 2 of the normal hepatocytes. Therefore, the alpha 1 and alpha 2 isoforms of Na-K-ATPase in hepatocyte-cell-membrane have different affinities for ouabain and have been conformationally and/or structurally altered in chronic diabetes.

Angiotensin II delivery and binding at the microvascular endothelium and cardiac myocyte surfaces in perfused rat hearts.

Peptide delivery toward its targets in an intact organ is equally as important as its routing from the systemic circulation to cell surface receptor sites. A physical model pertinent to a heart perfusion technique in Sprague-Dawley rats is presented describing reversible binding of angiotensin II and/or antagonist (DUP 753, losartan) with the microvascular endothelial receptor subtypes as well as with the cardiac myocyte receptor subtypes that are exposed to the perfusate by CHAPS-treatment. Analysis of the collected effluents are curve-fitted with a conservation equation and a first-order Bessel function. The results suggest that angiotensin II delivery and binding to the pool of receptor subtypes both at the level of the microvascular endothelium and cardiac myocyte sites differ marginally in binding affinities. The findings postulate that angiotensin II can have access to the myocyte site in an intact heart by an endothelial angiotensin II-receptor-internalization process. In addition, considering that the AT1- and AT2-receptor subtypes are present in equal proportions and have equal binding affinities with angiotensin II, the results of the 3H-DUP 753 binding indicated approximately 3-3.5 times higher affinity to the AT1-receptors subtype than angiotensin II at both the endothelial and myocyte sites. In the presence of losartan, angiotensin II binding showed higher affinity with the exposed unopposed AT2-receptor subtype than with the receptor pool, which could be due to alterations in the AT2-receptor structure and configuration. This increase in the binding affinity of angiotensin II with the AT2-receptor subtype may be categorized under the direct effect of the AT1-antagonist modality in producing cardioprotective effects.

Transport of prostaglandins through normal and diabetic rat hepatocytes.

Transport of alprostadil (prostaglandin E1) and dinoprost (prostaglandin F2 alpha) was studied in enzymatically dispersed normal and streptozocin-treated rat hepatocytes prepared by collagenase perfusion. Cell suspensions incubated at 37 degrees were sampled at time intervals for a period of 5 min and the supernatant analyzed for prostaglandins after centrifugation. The data analysis employed a theory and a model for solute transfer at the cell membrane-water interphase. Biophysical parameters such as the effective partition and the apparent permeability constants were used to define the transport mechanism. The apparent permeability coefficient of alprostadil and dinoprost transfer through normal hepatocytes was calculated to be 5 X 10(-3) and 3 X 10(-3) cm/sec with a mean partition coefficient of 1345 and 764 for both solutes, respectively. The permeability coefficient of alprostadil and dinoprost transfer through diabetic hepatocytes were 3 X 10(-3) and 2 X 10(-3) cm/sec with partition coefficient of 572 and 206, respectively. The results showed differences in prostaglandin transport between normal and diabetic hepatocytes, resulting from morphological and lipid alteration in the cytoplasmic membrane.

Angiotensin II binding and extracellular matrix remodelling in a rat model of myocardial infarction.

Clinical evidence points to a role for angiotensin II (Ang II) in the post-infarction remodelling of cardiac hypertrophy. The present study was designed to investigate the remodelling process in an animal model of myocardial infarction (MI) using the following criteria: 1) histological studies to examine the re-vascularisation process and collagen deposition in different regions of the myocardium; 2) histological evidence to investigate the cell type distribution using cell-specific markers; 3) histological and Western blot analysis to localise Ang II receptor subtypes (AT(1)-receptor and AT(2)-receptor) and to study their regulation; 4) kinetics of the binding of Ang II to its receptors in a heart perfusion model; and 5) to assess the effect of the Ang II antagonist (losartan) on these parameters. MI was induced by ligation of the left anterior descending coronary artery of Sprague-Dawley rats. Four different animal groups were established: 1) sham-operated, non-treated; 2) sham-operated, treated with losartan; 3) myocardial infarct, non-treated; and 4) myocardial infarct, treated with losartan. In infarcted rat hearts, fibroblasts and collagen types I and III increased in the remnant viable region of the left ventricle compared with sham-operated rats. One month of losartan treatment in myocardial infarcted rats revealed insignificant changes in fibroblasts and collagen types I and III compared with sham controls. Also, myocardial infarction increased AT(1)-receptor protein levels compared with sham-operated controls, as judged by Western blotting. In losartan-treated myocardial infarct animals, no changes were detected at the level of AT(1)-receptor expression compared with non-treated myocardial infarct rats. Binding studies of Ang II on endothelial cell lining and directly on myocytes in sham-operated and infarcted perfused rat hearts revealed that, in myocardial infarcted-animals, Ang II binding affinity increased both in the endothelium and in myofibres. This may be considered a major putative effect of the peptide in potentiating the pharmacodynamics of hypertrophy. In losartan-treated myocardial infarcted-animals, a marked increase in the binding affinities of Ang II for the AT(2)-receptor subtype was observed. Hence, potential cardioprotective effects of the AT(1)-receptor antagonist are proposed.

Characterization of insulin-resistance: role of receptor alteration in insulin-dependent diabetes mellitus, essential hypertension and cardiac hypertrophy.

Insulin-resistance is associated with a number of disease states such as diabetes, syndrome X, and hypertension. These situations may be coupled to insulin-resistance through the insulin signaling system as a common pathway. The purpose of this study was to investigate the receptor binding alterations in streptozotocin-induced diabetic rats, spontaneously hypertensive rats and aortocaval shunted rats (eccentric cardiac hypertrophy). A physical model describing a 1:1 stoichiometry of ligand binding with its receptor is proposed describing reversible binding of [(125)I]insulin or [(125)I]IGF-1 at the microvascular endothelial as well as with the cardiac myocytes after CHAPS-treatment. Analysis of the collected effluents are curve-fitted with a conservation equation and a first-order Bessel function which allowed the calculation of the forward binding constants (k(n)), the reversible constants (k(-n)), the dissociation constants (k(d)) and the residency time constants (tau). The results showed that streptozotocin-induced diabetic rats showed insulin-resistance through alterations in the kinetics of insulin receptor binding. The normotensive controls of the spontaneously hypertension rats (SHR) carry themselves insulin-resistant receptors whose binding to insulin worsens in the hypertensive SHR. Negative cooperativity between insulin-like growth factor IGF-1 and insulin receptors could be a causative factor predisposing for insulin-resistance in the aortocaval shunted rats to insulin resistance. The defects may be occurring at the receptor level in insulin-dependent diabetes mellitus, Wistar-Kyoto rats and spontaneously hypertensive rats. In conclusion, alterations in the kinetics of insulin binding to its receptor seem to play a central role for the initiation of insulin-resistance during the various pathophysiological states.

Comparative nephrotoxic effects of cis-platinum (II), cis-palladium (II), and cis-rhodium (III) metal coordination compounds in rat kidneys.

A Sprague-Dawley rat kidney perfusion technique was used in situ to study the effects of cis-dichloro-diamine platinum, PdCl2 (2,6-diaminopyridine), and RhCl3 (2,6-diaminopyridine) on sodium and calcium retention in the whole kidney. The technique involves perfusion of both kidneys via the abdominal aorta and then through the right and left renal arteries and dorsal aorta. Compared to controls, kidneys perfused independently with the three coordination compounds showed approximately equal to 45% decrease and approximately equal to 117% increase in Na+ and Ca2+ retention, respectively. Perfusates containing the coordination compounds in addition to 15 mM ouabain showed approximately equal to 76% decrease in Na+ and insignificant increase in renal Ca2+ retention. Hence, one can rule out the presence of voltage-gated Ca(2+)-channels at the basolateral side due to membrane depolarization. These results suggest that the three metal coordination compounds showed identical nephrotoxic effects on the handling of Na+ and Ca2+ ions by inhibiting both the Na(+)-Ca(2+)-anti-porter and the Na(+)-H(+)-exchanger with laxing effects on nonvoltage-gated Ca(2+)-channels at the basolateral side. However, their effects on the Na(+)-K(+)-ATPase and the Na(+)-Ca2+ symporter was insignificant.

Insulin-receptor binding characteristics in perfused SHR and WKY rat hearts.

This work uses a new heart-perfusion technique to measure 125I-insulin binding on capillary endothelium and myofiber cell membranes in Wistar-Kyoto and spontaneously hypertensive rats. Ringer-Lock buffer was infused at a rate of 1 ml min-1 in the presence of 20 meq l-1 K+ and 125I-insulin through an aortic cannula. The effluent was collected through a catheter introduced into the right atrium. The capillary endothelial lining was removed by detergent treatment to expose the cardiac myocyte surfaces. A physical model describing a 1:1 binding stoichiometry of 125I-insulin with its receptors is proposed and the derived mathematical equations allow for the calculation of binding constants (kn), unbinding constants (k-n), dissociation constants (kd), and residency time constants (tau). The results showed that in the spontaneously hypertensive rats' hearts significant alterations were not noticed in the kinetics of insulin binding with its receptor at the capillary endothelial site compared to hearts of the normotensive control Wistar-Kyoto rats. However, at the myocyte site and in the spontaneously hypertensive rats, steric, configurational, and/or structural modifications for insulin binding with the receptor were observed as indicated by changes in insulin affinity for its receptor. Hence, alterations in insulin binding rather than reduction in insulin receptor number due to hyperinsulinemia, can be considered among the peculiarities of insulin resistance in the spontaneously hypertensive rats. Hyperinsulinemia, therefore, may be considered an upregulatory process as a consequence of insulin-resistance. The results support the hypothesis that insulin-resistance on the myocytes could be a pathophysiologic defect in insulin-receptor structure, function and affinity, and therefore myocardial function.