Biomedical subjects
Publications and source records attributed to R Rubin.
A case report of a heretofore undocumented entity was presented. A brief literature review of associated lesions was included. The authors suggest that pedal varix may be a more common occurrence than previously documented.
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The change in distribution of cardiac output in septic shock was examined by radionuclide imaging with thallium 201 thallous chloride (201Tl) which allows noninvasive evaluation of relative blood flow to various organs except for the brain. Pseudomonas aeruginosa (1 X 10(9)-2 X 10(10) organisms) were inoculated into the thigh of rats 18-24 hr before the study. The mean arterial pressure was measured with an intracarotid catheter. Fractional blood flow to the heart, kidneys, and liver was evaluated as organ uptake of 201Tl. Those with zero or less than 5% kidney uptake (n = 8) had a high heart uptake and all died within 3 hr even if their pressure was maintained. In contrast, 20 out of 24 rats with kidney uptake greater than 5% survived for more than 6 hr. Those results suggest that the kidney uptake, representing fractional renal blood flow, is an excellent indicator of short-term prognosis in septic shock.
Phosphatidylethanol formation was examined in (3H)arachidonic acid-labeled human platelets. In the presence of ethanol, thrombin induced the formation of (3H)phosphatidylethanol in a time- and concentration-dependent manner. The appearance of phosphatidylethanol did not involve de novo synthesis since no 32P radioactivity was incorporated into phosphatidylethanol in 32P-labeled platelets or in platelets that were permeabilized with saponin in the presence of 32P-ATP. The data provide evidence for the direct activation by thrombin of phospholipase D in human platelets.
In previous studies we have demonstrated that ethanol activates hormone-sensitive phospholipase C in intact human platelets, resulting in the mobilization of intracellular Ca2+ and platelet shape change. The present study aims to localize further this effect of ethanol by examining its interaction with the regulation of phospholipase C in a permeabilized cell system. In platelets permeabilized with a minimal concentration (18 micrograms/ml) of saponin, ethanol by itself did not activate phospholipase C. However, ethanol potentiated the activation of phospholipase C in response to the non-hydrolysable GTP analogue GTP[S] (guanosine 5'-[gamma-thio]triphosphate), an effect similar to that observed with thrombin. Ethanol also potentiated the response to fluoride, which acts directly on G-proteins. Other short-chain alcohols also stimulated phospholipase C in a synergistic manner with GTP[S]. The ability of specific alcohols to stimulate phospholipase C was directly related to their respective lipid-solubilities, as determined by their partition coefficients. Moreover, the potencies of each alcohol correlated with their ability to elicit Ca2+ mobilization and shape change in intact platelets. These effects of ethanol were eliminated by a disruption of receptor-phospholipase C coupling induced by the addition of higher concentrations of saponin. These data indicate that the activation of phospholipase C by ethanol may occur by affecting protein-protein interactions in the signal-transduction complex involving GTP-binding regulatory proteins.
Ethanol has been shown to mobilize intracellular calcium in isolated rat hepatocytes by activation of phosphoinositide-specific phospholipase C. However, addition of ethanol to 32P-labeled hepatocytes resulted in a rapid increase in the level of [32P]phosphatidylinositol 4-phosphate over a period of 2 min, concomitant with a small decrease in [32P]phosphatidylinositol 4,5-bisphosphate and an increase in [32P]phosphatidic acid levels. These results indicate that polyphosphatidylinositol metabolism was stimulated by ethanol simultaneously with the activation of phospholipase C. Ethanol also caused a transient increase in the influx of extracellular calcium into quin 2-loaded hepatocytes over a similar period of time. The results demonstrate that ethanol, in common with calcium-mobilizing hormones, directly or indirectly stimulated polyphosphoinositide regeneration and allowed for increased movement of calcium across the hepatocyte plasma membrane.
Ethanol causes a transient activation of the phosphoinositide-specific phospholipase C in intact hepatocytes and mimics the action of receptor-mediated agonists [Hoek, Thomas, Rubin & Rubin (1987) J. Biol. Chem. 262, 682-691]. Preincubation of the hepatocytes with phorbol esters which activate protein kinase C prevented this effect of ethanol: phorbol ester treatment inhibited the ethanol-induced phosphorylase activation, the increase in intracellular free Ca2+ concentrations measured in quin 2-loaded hepatocytes, and the changes in concentrations of inositol phosphates, phosphoinositides and phosphatidic acid. Several lines of evidence indicate that these effects were mediated by protein kinase C. Phorbol esters acted in a concentration range where they activate protein kinase C; phorbol esters that do not activate protein kinase C were not effective in inhibiting the effects of ethanol. The permeant diacylglycerol oleoyl-acetylglycerol also inhibited the effects of ethanol, but other diacylglycerols were not effective in the intact cells. The inhibition of ethanol-induced Ca2+ mobilization by phorbol esters was prevented by preincubating the cells with the protein kinase C inhibitors 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H7) and sphingosine. H7 also enhanced the Ca2+ mobilization induced by ethanol in cells that were not pretreated with phorbol esters, indicating that the transient nature of the ethanol-induced Ca2+ mobilization may be due to an activation of protein kinase C caused by the accumulation of diacylglycerol. These data support a model whereby ethanol activates the phosphoinositide-specific phospholipase C, possibly by affecting receptor-G-protein-phospholipase C interactions in the membrane.
Administration of ethanol to human platelets resulted in a rapid shape change which was maximal within 30 s. Ethanol did not cause aggregation or secretion of ATP at any time and inhibited aggregation induced by collagen. In platelets that were loaded with the intracellular calcium indicator fura2, ethanol induced a rapid mobilization of calcium from internal, thrombin-sensitive pools. Cytosolic calcium increased to a maximum within 5 s and decreased slowly over the ensuing 5 min to near basal levels. The mobilization of calcium by ethanol coincided with the rapid formation of phosphatidic acid and a decrease in the level of phosphatidylinositol 4,5-bisphosphate, as measured in 32P-labeled platelets. In platelets labeled with myo-[2-3H]inositol, ethanol caused a 20-30% increase in the levels of inositol (1,4,5)-trisphosphate and inositol bisphosphate within 10 s. Ethanol also induced the transient phosphorylation of myosin light chain (20 kDa) and a 40 kDa protein, a known substrate for protein kinase C. The results indicate that ethanol activates phosphoinositide-specific phospholipase C in human platelets. The subsequent mobilization of intracellular calcium and activation of protein kinase C can account for the shape change induced by ethanol.
The short-term effects of ethanol on calcium homeostasis were studied in isolated hepatocytes. Ethanol caused a rapid transient activation of phosphorylase not associated with changes in cAMP levels which peaked after 20-30 s and declined slowly over a period of 5-10 min. Maximal activation was found with 200 mM ethanol, and a significant effect was observed at 25 mM ethanol. Similar effects were induced by other organic solvents and by halothane, with more hydrophobic agents being effective at lower concentrations. In hepatocytes loaded with the intracellular calcium indicator quin2, the addition of ethanol caused a transient increase in cytosolic free calcium, with a kinetic pattern compatible with its involvement in the activation of phosphorylase. Pretreatment of the hepatocytes with phenylephrine or vasopressin to deplete the hormone-sensitive calcium pools in the cells prevented the ethanol-induced calcium mobilization. In 32P-labeled hepatocytes addition of ethanol caused a small (5-7%) decrease in the level of [32P]phosphatidylinositol 4,5-bisphosphate and a 10-15% increase in [32P]phosphatidylinositol 4-phosphate and [32P]phosphatidic acid. In hepatocytes labeled with myo-[3H]inositol, ethanol induced a 50-100% increase in the levels of inositol 1,4,5-trisphosphate, inositol 1,3,4-trisphosphate, and inositol bisphosphate. The changes in the inositol 1,4,5-trisphosphate level due to ethanol paralleled the time course of the elevation of cytosolic free calcium levels and activation of phosphorylase a. The effects of ethanol were comparable to those of a physiologic (1 nM) dose of vasopressin; however, unlike with vasopressin, the inositol phosphates and cytosolic calcium levels declined to basal levels 2 min after the addition of ethanol. These results indicate that ethanol, in common with calcium-mobilizing hormones, activates hormone-sensitive phosphoinositide-specific phospholipase C. The resulting changes in inositol 1,4,5-trisphosphate can account for the mobilization of intracellular calcium and the consequent activation of phosphorylase by ethanol.
Guanine nucleotides are thought to mediate the interaction of the receptors for calcium-mobilizing hormones and phosphoinositide-specific phospholipase C. In the present study the characteristics of guanine nucleotide-dependent phospholipase C activation were studied in [3H]inositol-labeled permeabilized hepatocytes. The nonhydrolyzable GTP analogs guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and guanyl-5'-yl imidodiphosphate stimulated the production of inositol phosphates by phospholipase C. The effect was concentration-dependent with half-maximal and maximal stimulation occurring with 0.6 and 10 microM GTP gamma S, respectively. The guanine nucleotide-induced stimulation of phosphoinositide breakdown was selective for phosphatidylinositol (4,5)-bisphosphate over phosphatidylinositol (4)-phosphate. The individual inositol phosphates formed after maximal GTP gamma S exposure were analyzed by high-performance liquid chromatography. Inositol 1,4,5-trisphosphate was rapidly produced, followed by the formation of inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4-trisphosphate. Ethanol is known to activate hormone-sensitive phospholipase C in intact rat hepatocytes. Ethanol (0.3 M) was ineffective in altering the characteristics of GTP gamma S-stimulated phospholipase C activation, in both digitonin-treated and sonicated hepatocytes. The metabolism of the various inositol phosphate isomers was unaffected by ethanol. The findings demonstrate the potential for the use of permeabilized hepatocytes in the analysis of phospholipase C activation by guanine nucleotides. Ethanol does not activate phospholipase C by altering this process.
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We have isolated an 84-fold adriamycin resistant subline, P388/R84, from mouse leukemia P388 cells by serial cultivation in methylcellulose in the presence of increasing drug concentrations. Electrophoresis of detergent soluble fractions of radiolabeled sensitive and resistant cells suggested marked alterations in the protein fractions of 160, 100, 60, 45, and 30 kd. In resistant clones labeled with 125I an increase in 160 and 100 kd proteins was accompanied by concomitant reduction in the 60, 45, and 30 kd proteins. In 35S methionine-labeled resistant cells, similar increases in the 160 and 100 kd components were observed but in contrast to 125I-labeled cells the 30 kd component was also higher. Alterations in surface proteins were confirmed in experiments where the cell extracts were adsorbed to concanavalin A polymers and extracted with 0.26 M methyl-alpha-D-mannopyranoside. Our data confirm earlier reported observations on cell-surface protein changes in cells resistant to anthracyclines and alkaloids.
Binding sites for inositol trisphosphate (IP3) have been identified in bovine adrenal cortex, employing [32P]IP3 prepared from human erythrocytes radiolabeled with [32P]ATP. IP3 was bound to adrenal microsomes with high affinity (Kd = 5 nM) and low capacity (186 fmol/mg protein). During kinetic studies, half-maximal binding was reached in less than one min at 4 degrees C, and dissociation was even more rapid with t1/2 of about 10 sec. [32P]IP2 showed no binding to the microsomal sites, which represent putative receptors at which IP3 acts to elevate intracellular calcium concentration during the actions of peptide hormones such as angiotensin II.
The influence of extracellular Ca2+ on hormone-mediated increases of cytosolic free Ca2+ [( Ca2+]i) and phosphorylase activity was studied in isolated hepatocytes. In the presence of 1.3 mM extracellular Ca2+, the stimulation of phosphorylase activity produced by vasopressin or phenylephrine was maintained for 20-30 min. In contrast, the change in [Ca2+]i under these conditions was more transient and declined within 3-4 min to steady state values only 70 +/- 8 nM above the resting [Ca2+]i. Removal of the hormone from its receptor with specific antagonists caused a decline in [Ca2+]i back to the original resting values. Subsequent addition of a second hormone elicited a further Ca2+ transient. If the antagonist was omitted, the second hormone addition did not increase [Ca2+]i indicating that the labile intracellular Ca2+ pool remains depleted during receptor occupation. When extracellular Ca2+ was omitted, both the changes of [Ca2+]i and phosphorylase a caused by vasopressin were transient and returned exactly to resting values within 3-4 min. The subsequent readdition of Ca2+ to these cells produced a further increase of [Ca2+]i and phosphorylase activity which was larger than the changes observed upon Ca2+ addition to untreated cells. This reactivation of phosphorylase showed saturation kinetics with respect to extracellular [Ca2+], was maximally stimulated within 1 min of vasopressin addition and was inhibited by high concentration of diltiazem. We conclude that entry of extracellular Ca2+ into the cell is required in order to obtain a sustained hormonal stimulation of phosphorylase activity and is responsible for the maintenance of a small steady state elevation of [Ca2+]i.
Leukocyte distribution to arteriolar branches in the hamster cheek pouch microvasculature was studied by rendering the cells fluorescent via a constant intravenous infusion of acridine orange. A principal objective was to determine the major factors influencing the partitioning of the cellular flux between daughter branches at arteriolar branch points with internal diameters permitting single-file leukocyte flow. Leukocyte flux determinations (cells/sec) and individual cell velocities were made for over 8000 cells at 13 branch sites in 10 animals. The results indicate that for the branch flow ratios of 0.3 to 0.7 encountered in the present experiments, the predominant factor influencing the average leukocyte flux is the branch blood flow. Branch fractional flux dependence on fractional blood flow was analyzed using linear and nonlinear models. A linear characterization provided an excellent fit and description of the functional relationship but a slight nonlinear component was also predicted by the nonlinear sigmoidal model which also fit the data well. Because of the small degree of predicted nonlinearity and the overlap of the standard deviations of the parameter estimates, we were not able to statistically decide on which model, and hence which functional relationship, is operative. Thus, the answer to the question regarding the presence of any preferential distribution is inconclusive. However, the data and analyses suggest that if preferential distribution is operative, its magnitude over the flow range and conditions here studied is small, and that for many purposes the relationship between the in vivo average fractional cell flux and blood flow may be viewed as linear.
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The BAL is one of the essential methods which are performed as a routine diagnostic procedure in interstitial lung disease. Differentiation of cellular components and proteins by this method enables a characterization of alveolitis which is considered to be at the beginning of every disease of the structural elements of the lung. A notable feature is that via an assessment of the intensity of alveolitis it is also possible to arrive at a prognosis. Lavage can also supply important pointers to suitable therapeutic measures. Referring to the literature and own investigations this paper tries to give a review of technique of BAL as well as essential findings and interpretation of this subject.