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The role of the actin cytoskeleton in endothelial barrier function has been debated for nearly four decades. Our previous investigation revealed spontaneous local lamellipodia in confluent endothelial monolayers that appear to increase overlap at intercellular junctions. We tested the hypothesis that the barrier-disrupting agent histamine would reduce local lamellipodia protrusions and investigated the potential involvement of p38 mitogen-activated protein (MAP) kinase activation and actin stress fiber formation. Confluent monolayers of human umbilical vein endothelial cells (HUVEC) expressing green fluorescent protein-actin were studied using time-lapse fluorescence microscopy. The protrusion and withdrawal characteristics of local lamellipodia were assessed before and after addition of histamine. Changes in barrier function were determined using electrical cell-substrate impedance sensing. Histamine initially decreased barrier function, lamellipodia protrusion frequency, and lamellipodia protrusion distance. A longer time for lamellipodia withdrawal and reduced withdrawal distance and velocity accompanied barrier recovery. After barrier recovery, a significant number of cortical fibers migrated centrally, eventually resembling actin stress fibers. The p38 MAP kinase inhibitor SB203580 attenuated the histamine-induced decreases in barrier function and lamellipodia protrusion frequency. SB203580 also inhibited the histamine-induced decreases in withdrawal distance and velocity, and the subsequent actin fiber migration. These data suggest that histamine can reduce local lamellipodia protrusion activity through activation of p38 MAP kinase. The findings also suggest that local lamellipodia have a role in maintaining endothelial barrier integrity. Furthermore, we provide evidence that actin stress fiber formation may be a reaction to, rather than a cause of, reduced endothelial barrier integrity., (Copyright © 2015 the American Physiological Society.)

Methods for the isolation and quantitation of cholesterol involve time-consuming chromatographic procedures coupled with the development and measurement of color complexes. The intensity of most color complexes varies with time. This study was undertaken to develop a UV spectrophotometric method to measure cholesterol concentration in plasma lipoprotein fractions. The developed method compared favorably with other methods (chemical and enzymatic) and was then used to investigate the effects of diabetes and hypothyroidism on the lipoprotein cholesterol in rats. Lipoprotein fractions from fasted rats were obtained by gradient ultracentrifugation. Aliquots of the very low-density lipoprotein (VLDL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) layers were extracted into chloroform-methanol-water. Following hydrolysis of lipid extracts, the solvent was evaporated and the residue was dissolved in ethanol and read in the UV recording spectrophotometer. At 203 nm, a linear relationship between optical density and concentration of cholesterol was obtained. The results obtained with the new method were compared with those obtained using chemical and enzymatic methods and the statistical difference among the methods was insignificant (p>0.05). From the results of our comparative studies of the methods, it was concluded that the UV method is valid for measuring cholesterol in lipoprotein fractions. The method has the advantage being rapid, nondestructive and cost-effective.

The incidence of traumatic injury, frequently associated with hemorrhagic shock, is higher in the alcohol-intoxicated individual. The outcome, as it pertains to both morbidity and mortality of this population, is partly dependent on duration of alcohol exposure and levels of blood alcohol at time of injury. In previous studies, we demonstrated that prolonged alcohol intoxication (15-h duration) produces marked hemodynamic instability and exacerbated early lung proinflammatory cytokine expression after hemorrhagic shock. The present study examines whether a shorter and more modest period of alcohol intoxication is sufficient to alter hemodynamic and proinflammatory responses to hemorrhagic shock. Chronically instrumented, conscious male Sprague-Dawley rats (250-300 g) received a single intragastric bolus of alcohol (1.75 g/kg) 30 min before the administration of fixed-volume (50%) hemorrhagic shock, followed by fluid resuscitation with Ringer lactate. Time-matched controls were administered on isocaloric dextrose bolus (3 g/kg). Alcohol (blood alcohol concentration, 152 +/- 10 mg/dL) produced a 14% decrease in basal mean arterial blood pressure and a more profound hypotensive response to equal blood loss. The 2-fold rise in circulating norepinephrine levels was similar in alcohol- and dextrose-treated hemorrhaged animals despite greater hypotension in alcohol-treated animals. Significant upregulation in lung and spleen interleukin (IL) 1, IL-6, IL-10, and tumor necrosis factor alpha expression was observed immediately after hemorrhage and fluid resuscitation, as previously reported. Only the hemorrhage-induced rise in lung IL-6 and tumor necrosis factor alpha was prevented by alcohol administration. In contrast, spleen cytokine responses to hemorrhage were not altered by alcohol administration. These results indicate that moderate acute alcohol intoxication results in significant modulation of hemodynamic and neuroendocrine responses to hemorrhagic shock.

This is a retrospective study, in which we investigated the impact of regular alcohol use on the clinical management of non-insulin dependent diabetes mellitus (NIDDM) patients from the outpatient clinic of the VA Medical Center in New Orleans, Louisiana. The study population included randomly selected NIDDM patients of which 40% used alcohol regularly. The fasting blood sugar (FBS) in non-users of alcohol stayed in the "normal" (< or = 140 mg/dl) and "acceptable " (< or = 175 mg/dl) range and that of regular users of alcohol remained at the "fair" (< or = 235 mg/dl) and "poor" (> 235 mg/dl) range. NIDDM patients who were regular users of alcohol had a higher frequency of dose adjustments than that of non-users of alcohol (96% vs 4%, respectively). The treatment failure was significantly higher among patients who regularly used alcohol than among those who abstained (90 vs 10%, respectively). On the basis of our findings, it was recommended that attending physician should routinely identify the regular alcohol users and monitor blood alcohol levels of ambulatory NIDDM patients during their follow-up visits. Also, complete cessation of alcohol consumption should be established prior to making dosage adjustment in situations where the oral hypoglycemic agent fails.

We examined how acute diabetes mellitus and acute ethanol intoxication modulate factors that mediate immune responses as a basis for explaining the increased susceptibility to infection in these two conditions. Our working hypothesis is that ethanol intoxication in diabetes compromises host defense mechanisms to a greater extent than observed in each condition alone. Male and female rats were made diabetic with streptozotocin (65 mg/kg, i.p.). Forty-eight hours after administration of streptozotocin, rats either received no treatment (control group) or were treated with (1) ethanol (bolus injection of 1.75 g/kg, followed by a 3-h infusion at the rate of 300 mg/kg/h), (2) lipopolysaccharide [(LPS); 0.9 mg/kg], or (3) a combination of LPS+ethanol. At the end of 3 h, rats were killed, and the livers were digested by perfusion with collagenase-containing Hanks' balanced salt solution to isolate hepatocytes and Kupffer cells. To measure chemokine generation, hepatocytes (2.5x10(5) cells per well) and Kupffer cells (1x10(6) cells per well) were cultured for 20 h, and the supernatant was used to measure cytokine-induced neutrophil chemoattractant (CINC) and regulated on activation, normal T-cell expressed and secreted (RANTES) chemokines. Phagocytosis by Kupffer cells was measured by flow cytometry and expressed as mean channel fluorescence intensity (MCF). Induction of diabetes as well as treatment of nondiabetic rats with LPS, ethanol, or LPS+ethanol caused depression of MCF values of Kupffer cells. However, treatment of the diabetic male and female rats with LPS and LPS+ethanol increased the MCF values relative to those of Kupffer cells obtained from untreated diabetic rats, but administration of ethanol to diabetic rats did not have a similar effect. The induction of diabetes caused an increase in CINC generation by Kupffer cells obtained from male rats, but not from female rats. This diabetes-induced elevation of chemoattractant factor was decreased when diabetic animals were treated with LPS, ethanol, or LPS+ethanol, and the sex difference was obliterated. Thus, the induction of diabetes as well as treatment with LPS, ethanol, or LPS+ethanol in nondiabetic rats depressed the phagocytic capability of Kupffer cells, whereas the presence of endotoxemia (administration of the endotoxin LPS) or administration of LPS+ethanol reversed the diabetic effect, but ethanol intoxication did not. These findings seem to indicate a persistence of depression of host defense capacity in the ethanol-intoxicated diabetic condition. This is further reinforced by the depression of the diabetes-induced enhancement of chemotaxis when the diabetic rats became intoxicated.

In order to investigate the effects of transportation stress on metabolic activities, we measured the changes in plasma lipoprotein and catecholamine levels in those rats that had just arrived in our Animal Facility and age-matched rats which had acclimatized in the Facility for at least 21 days. The acclimatized rats were considered as control, and the values from the newly arrived rats was done within 4-6 days of their arrival in the Facility. The cholesterol levels in very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) were higher (71-84%) than the control levels. Also, the stressed animals had higher levels of norepinephrine (4.5-fold) and epinephrine (3-fold) than the control levels. However, dopamine levels was 34-fold lower than that of control. On the basis of the data, we concluded that the change in plasma levels of lipoprotein and catecholamines in response to the transportation stress is significant and may require at least three weeks after the transportation to establish a stable baseline for investigations in which the plasma levels of lipoproteins and catecholamines is a critical factor.

The distribution of 1,2-diacylglycerol (DAG) in the muscularis muscle of Bufo marinus has been determined using autoradiographic techniques. Strips of the muscle from the body of the stomach were fixed in 4% paraformaldehyde/2.5% glutaraldehyde, postfixed, washed and longitudinal sections (15 microns) were cut on a cryotome and placed on gelatinized slides. The sections were then incubated in the presence and absence of agonist, acetylcholine (ACh) 10(-5) M or carbachol (CCh) 10(-5) M plus Li+ (10 mM) in the medium which causes a marked potentiation of agonist-stimulated formation of cytidine diphosphate-DAG (CDP-DAG). The sections were processed through an autoradiographic technique using [3H]-cytidine, which binds to 1,2-diacylglycerol within the tissue to form CDP-DAG. Analysis of the developed tissue slides indicated that the dose of ACh (10(-5) M) which had been predetermined to elicit automaticity of the preparation in vitro increased the density of CDP-DAG grains. When the muscle strips were pretreated with ACh (10(-5) M) and CCh (10(-5) M) in the presence of LiCl (10 mM), the density of the CDP-DAG grains were further enhanced. CDP-DAG grains were localized throughout the muscle fibers. The increase in the density of the grains which was induced by the conditions eliciting automaticity suggest that DAG is one of the second messengers in the manifestation of automaticity of the muscularis muscle of Bufo marinus.

The possibility that the antihypertensive adrenoceptor antagonists (propranolol, phentolamine and metoprolol) may alter hepatic lipid metabolism was examined in freshly dispersed rat hepatocytes with [1-14C]oleate. Propranolol (1.8 x 10(-4) M) and phentolamine (1.4 x 10(-4) M) increased incorporation of [1-14C]oleate into cholesteryl esters by 51 and 92%, respectively, and decreased ketogenesis by 46 and 62%, respectively. While neither drug affected incorporation into total phospholipid, propranolol decreased triglyceride synthesis by 37%. These effects of propranolol and phentolamine may not occur through beta- or alpha-receptor inhibition since neither epinephrine nor norepinephrine reversed the effects of the adrenoceptor antagonists. Although epinephrine and norepinephrine per se did not alter the incorporation of [1-14C]oleate into triglyceride, phospholipid, cholesteryl esters or ketone bodies, they stimulated the production of 14CO2 (control 5.6 +/- 1.3; epinephrine 7.6 +/- 1.1; norepinephrine 9.1 +/- 0.2 nmol oleate incorporated/mg protein), and these effects were reversed by phentolamine and propranolol. The data suggest that adrenoceptor antagonists exert direct effects on hepatic metabolism of oleate.

In our search for therapeutic agents from natural sources with potential for the treatment of opportunistic infections in patients afflicted with acquired immunodeficiency syndrome (AIDS), we investigated antibacterial and antifungal activities of water extracts of Cassia alata (C. alata). The extracts are traditionally used in Ivory Coast, West Africa to treat bacterial infections caused by Escherichia coli (E. coli), and fungal infections caused by Candida albicans (C. albicans) and dermatophytes. Our working hypothesis was that the extract contains active ingredient(s) which can be isolated, identified and developed into useful antibacterial/antifungal agents for the treatment of opportunistic infections in patients with AIDS. We used the broth dilution and agar dilution methods. Specifically, we focused on E. coli and C. albicans and the effectiveness of the extracts was evaluated relative to those of standard antibacterial agent chloramphenicol and antifungal agent amphotericin B. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for the water extract of C. alata against E. coli were 1.6 mg/ml and 60 mg/ml, respectively; corresponding data for chloramphenicol were 2 micrograms/ml and 10 micrograms/ml. Similarly, the MIC and minimum fungicidal concentration (MFC) for the extract against C. albicans were 0.39 mg/ml and 60 mg/ml in contrast to 0.58 micrograms/ml and 0.98 micrograms/ml for amphotericin B. From the dose-response curve plots, the extract had an IC50 of 31 mg/ml for E. coli and 28 mg/ml for C. albicans. The data suggest that C. alata extracts contain agent(s) which have therapeutic potential and might be useful if isolated and developed for the treatment of opportunistic infections of AIDS patients.

In our search for therapeutic agents from natural sources with potential for the treatment of opportunistic infections in patients afflicted with acquired immunodeficiency syndrome (AIDS), we investigated antibacterial and antifungal activities of water extracts of Cassia alata (C. alata). The extracts are traditionally used in Ivory Coast, West Africa to treat bacterial infections caused by Escherichia coli (E. coli), and fungal infections caused by Candida albicans (C. albicans) and dermatophytes. Our working hypothesis was that the extract contains active ingredient(s) which can be isolated, identified and developed into useful antimicrobial/antifungal agents for the treatment of opportunistic infections in patients with AIDS. We used the broth dilution and agar dilution methods. Specifically, we focused on E. coli and C. albicans and the effectiveness of the extracts was evaluated relative to those of standard antibacterial agent chloramphenicol and antifungal agent amphotericin B. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for the water extract of C. alata against E. coli were 1.6 mg/ml and 60 mg/ml respectively; corresponding data for chloramphenicol were 2 ug/ml. Similarly, the MIC and minimum fungicidal concentration (MFC) for the extract against C. albicans were 0.39 mg/ml and 60 mg/ml in contrast to 0.58 ug/ml and 0.98 ug/ml for amphotericin B. From the dose-response curve plots, the extract had an IC50 of 31 mg/ml for E. coli and 28 mg/ml for C. albicans. The data suggest that C. alata extracts contain agent(s) which have therapeutic potential and might be useful if isolated and developed for the treatment of opportunistic infections of AIDS patients.

We examined the effects of infusion of alanine on hepatic concentration of glycero-3-phosphate (glycero-3-P) and output of triacylglycerol (TG) by isolated perfused livers from triiodothyronine (T3)-treated rats. It was expected that because of its gluconeogenic and antiketogenic properties, alanine might stimulate accumulation of glycero-3-P, which in turn might result in enhanced TG production by the hyperthyroid livers. The hepatic concentration of glycero-3-P is lower in livers from T3-treated rats than in euthyroid rats. Infusion of 1.83 and 4.23 mmol alanine/4 h did not alter the hepatic concentration of glycero-3-P and output of triacylglycerol by livers from T3-treated rats. However in these livers, the two concentrations of infused alanine increased the output of glucose and decreased the output of ketone bodies. In livers from euthyroid animals, the infusion of 1.83 mmol alanine/4 h had no effect, whereas 4.23 mmol/4 h decreased hepatic concentration of glycero-3-P. These concentrations of alanine did not alter the output of ketone bodies or TG, but progressively decreased the output of glucose by the euthyroid livers. Our data suggested that the decreased availability of glycero-3-P in livers from T3-treated rats is rate-limiting for TG production and correlated with the diminished output of TG, whereas in livers from euthyroid rats, the glycero-3-P concentration is sufficient to maintain maximal synthesis and secretion of TG. Furthermore, the effects of alanine on the output of ketone bodies or glucose appear to be independent of its effects on hepatic glycero-3-P.

Isolated villus from human term placenta contains about 167 nmol/g of acetylcholine (ACh). It was incubated in a muscle bath containing Krebs-Ringer bicarbonate buffer (pH 7.2-7.4) at 37 degrees C and ACh released into the medium was analyzed by pyrolysis-gas chromatography. The spontaneous release of ACh into the medium was linear with time and was 35 pmol/g/min. ACh was not released when Ca++ was absent in the medium. Raising the Ca++ concentration in the Krebs-Ringer bicarbonate buffer from 2.34 to 4.64 mM, or adding L-nicotine (58 muM) to the bath, increased the rate of release of ACh to 53 and 47 pmol/min respectively. Nicotine did not exhibit any effect on ACh release in the absence of Ca++ in the medium. Both the rate of the spontaneous release of ACh and the nicotine-induced increase in the release of ACh was decreased by atropine (152 muM). They were not influenced by d-tubocurarine (30 muM). Depolarizing concentrations of potassium (16-63 mM) in the medium increased the rate of release of ACh. Cocaine, a known Ca antagonist, decreased the rate of spontaneous release of ACh as well as nicotine-induced release of ACh. These observations indicate that 1) Ca++ ions in the external medium are required for release of ACh, 2) Ca++ ions in the external medium are required for release of ACh, 2) Ca++ ions act as a link between the stimulation of ACh release and the final release of ACh and 3) The effect of nicotine on placental release of ACh may be classified as a muscarinic type.

Hepatocytes from T3-treated rats synthesized less triglyceride and more ketone bodies from [1-14C]oleate at all concentrations from 0-2 mM, than did hepatocytes from euthyroid animals; addition of 1.0 mM glycerol increased triglyceride synthesis and reduced ketogenesis in hepatocytes from T3-treated rats to the rates observed in euthyroid hepatocytes in the absence of added glycerol. Glycerol did not alter triglyceride synthesis, but reduced ketogenesis genesis by euthyroid hepatocytes. It is probable from these and other data (J. Biol. Chem. 259, 8857-8862 (1985)) that, in the hyperthyroid rat, glycero-3-P, and not fatty acid, is rate limiting for synthesis of triglyceride, and, secondarily for reducing rates of ketogenesis in the hepatocyte.

The influence of calcium antagonists on hepatic lipid metabolism was investigated in freshly dispersed rat hepatocytes incubated with [1-14C]oleate and verapamil or 8-N,N-diethylamino-octyl-3,4,5-trimethoxybenzoate (TMB-8). Synthesis of triglyceride was calculated from the specific radioactivity of [1-14C]oleate in extracted total lipid, after separation of each lipid class by thin-layer chromatography. Ketogenesis was measured enzymatically or as the amount of radioactivity incorporated into neutralized acid-soluble extracts. Neither verapamil nor TMB-8 affected triglyceride synthesis. In contrast, TMB-8 and verapamil exerted a concentration-dependent inhibition of ketogenesis, with TMB-8 being more potent than verapamil (inhibition by 50 microM TMB-8 was 73 +/- 9% versus 38 +/- 2% inhibition by 50 microM verapamil). Increasing the concentrations of calcium (0 to 4.2 mM) or oleate (0 to 2.0 mM) increased the rate of ketogenesis but did not alter the antiketogenic potency of TMB-8 or verapamil, indicating that inhibition of ketogenesis by these drugs was not calcium dependent. Since the calcium antagonists did not affect ketogenesis from octanoic acid, and since carnitine stimulated ketogenesis from [1-14C]oleate by 25% and reversed the antiketogenic effects of TMB-8 and verapamil, it appeared that the two calcium antagonists inhibited ketogenesis by interfering with the activity of the outer mitochondrial carnitine palmitoyltransferase. In assays of the outer carnitine palmitoyltransferase in isolated mitochondria, both TMB-8 and verapamil were inhibitory. TMB-8 was the more potent inhibitor of this enzyme, and carnitine was able to overcome inhibition by each of the inhibitors. These results suggest that verapamil and TMB-8 may inhibit ketogenesis by mechanisms independent of their well known effects on cellular calcium concentrations, and that inhibition may be competitive with respect to carnitine concentration. However, direct inhibition of carnitine palmitoyltransferase may not explain completely the inhibition of ketogenesis by these drugs, since concentrations required for enzyme inhibition were greater than those required for inhibition of ketogenesis in isolated hepatocytes.

This article reviews our understanding of effects of thyroid hormone excess and deficiency on hepatic metabolism of FFA, and consequent effects on production, secretion, and metabolism of plasma lipoproteins. In the hyperthyroid state the following alterations are observed. Fatty acid oxidation and ketogenesis are stimulated simultaneously with a paradoxical stimulation of fatty acid synthesis, which may be linked by virtue of a blunted response of mitochondrial carnitine palmitoyltransferase I (CPT-I) to malonyl coenzyme A (CoA). Esterification of fatty acid to triglyceride (TG) is reduced, as is the secretion of the very low density lipoprotein (VLDL) (including VLDL TG, cholesterol, and apoprotein); this may be due, in part, to decreased concentrations of glycerol-3-phosphate (G3P) in the hepatic cell. In the intact animal or patient, however, serum TG concentration is variable, which may reflect increased adipose tissue lipolysis and elevated concentrations of plasma FFA, which would tend to drive VLDL secretion by the liver. Clearance of the VLDL and its metabolic product, the low density lipoprotein (LDL), is increased, resulting in decreased plasma total and LDL cholesterol. Although high density lipoprotein (HDL) cholesterol may also be reduced, the ratio of LDL/HDL cholesterol is further decreased. The regulatory role of the lipoprotein apoproteins is less clear, but hepatic apolipoprotein (apo) B secretion (required for VLDL) is diminished, while apo-AI secretion (required for HDL) is stimulated, perhaps both reflecting rates of synthesis. Plasma concentrations of apo-AI are variable, dependent on relative rates of secretion and clearance. In the hypothyroid, many of these effects are reversed, which results in hyperlipoproteinemias and greater risk for the development of atherosclerotic cardiovascular disease.

This retrospective study describes the prescription medication profile in an outpatient pediatric population (n = 510) retrieved from a hospital pharmacy computer file. The survey covers a three-month period. The study population included 281 male and 229 female patients divided according to age into three groups: infant (age 0-12 months); children (age 1-12 years); and adolescents (age 13-18 years). Medications prescribed were classified according to their pharmacotherapeutic properties as described in the American Hospital Formulary Service Drug Information 87. The findings pointed out that three pharmacotherapeutic categories (the antiinfective/chemotherapeutic, central nervous system (CNS), and respiratory agents) constituted 78.1 percent of the 1402 prescribed medications. The most frequently prescribed agents in each of these categories were, respectively, amoxicillin, aminophylline, and acetaminophen. These agents represent recent advances in drug usage because they became most frequently used only within the past ten years. The age-dependent medication profile indicated that there was a higher prescription rate of antiinfectives and respiratory disorder agents in the younger age groups; in the adolescent group CNS agents were more often prescribed.

According to our studies, human term placenta contains about 112 nmol/g of ACh. In the present studies, isolated placental villi were suspended in Kreb's bicarbonate buffer (pH 7.4) at 37 degrees and acetylcholine (ACh) released into the medium was analyzed by gas chromatography. The spontaneous release of ACh into the medium was linear with time and was 35 pmol/g/min. Cocaine in doses of 280 and 56o micrometer decreased the spontaneous release of ACh to 23, and 17 pmol/g/min, respectively. Raising the Ca2+ concentration in the Kreb's bicarbonate buffer from 2.34 to 4.64 mM, or the addition of nicotine (58 micrometer) to the bath increased the rate of release of ACh to 53 and 47 pmol/g/min, respectively. Cocaine decreased the rate of release of ACh even in the presence of Ca2+ or nicotine. ACh was not released when Ca2+ was absent in the medium. Nicotine did not stimulate the release of ACh in the Ca2+ free medium. High concentrations of K+ increased ACh release in the presence of Ca2+ and it did not have any effect on ACh release in the absence of Ca2+. These observations indicate that external Ca2+ is required for the release of ACh and cocaine acts possibly by preventing the entry of external Ca2+ into the syncytiotrophoblast, and thereby decreases ACh release from the placental villus.