Modulation of cardiac beta-adrenergic receptors by dopamine beta-hydroxylase.

Incubation of cardiac sarcolemma in the presence of dopamine beta-hydroxylase (DBH), a catecholamine biosynthetic enzyme, increased beta-adrenergic receptor density by 68% as measured by [3H]dihydroalprenolol (DHA) binding. The addition of DBH to plasma membranes isolated from brain, kidney, skeletal muscle, liver and intestine did not alter [3H]DHA binding. Cardiac alpha-receptors were unaffected under similar conditions. Since DBH is coreleased with norepinephrine, these results indicate that a functional coupling of the putative beta-adrenergic receptor with DBH may exist in cardiac muscle.

Use of the University of Wisconsin solution for the preservation of cell organelle activities in rat heart and lungs.

BACKGROUND: The University of Wisconsin storage solution has been successful in some model systems in extending the storage period of heart-lung grafts for transplantation. METHODS: In this study an aerated preparation of rat heart and lung was stored for 24 hours at 4 degrees C in either University of Wisconsin or St. Thomas' Hospital solution. Cell organelles (reticular, mitochondrial, and cell membrane fractions) were isolated from the stored hearts and lungs. Protein yield and enzyme activities were assayed for each cell organelle (reticular fractions: Ca(2+)-ATPase, NADPH-cytochrome C reductase; mitochondria: Ca(2+)-ATPase, cytochrome C oxidase; cell membrane fraction: Na+,K(+)-ATPase, p-nitrophenylphosphatase) as a measure of the recovery of function. RESULTS: Only the cell membrane fraction of heart and lung was not affected by storage in either St. Thomas' Hospital or University of Wisconsin solution with respect to protein yield (milligrams per gram of homogenate) or enzyme activities (nanomole per milligram per minute). The reticular fraction was the most sensitive to storage, with both protein yield and enzyme activities being significantly reduced in both the heart and the lung stored in University of Wisconsin of St. Thomas' Hospital solution (p < 0.05). CONCLUSION: The mitochondrial fraction was not preserved in lung in either St. Thomas' Hospital or University of Wisconsin solution but was preserved in the heart stored in St. Thomas' Hospital solution. These criteria provide preliminary screening for a superior solution that may then be used in more complicated transplantation models to more fully assess cardiac and pulmonary function.

Preservation of cell organelles during storage of human atrial tissue in the University of Wisconsin solution.

AIM OF THE STUDY: The University of Wisconsin storage solution (UW) (E.I. du Pont de Nemours, Wilmington, DE) has been successful in extending the storage period using some model systems of donor heart preservation for cardiac transplantation. The ability of UW to preserve human cardiac cell organelle (sarcoplasmic reticulum, mitochondria and sarcolemmal) membrane composition (enzyme activity, protein, cholesterol and phospholipid content) was compared to St. Thomas's Hospital Solution (ST) and saline. METHODS: Human atrial appendages were stored at 4 degrees C for 24 h in saline, ST or UW or not stored (controls) and the cell organelles isolated. Each fraction was assayed for enzyme activity (mitochondria: azide sensitive Ca2+ ATPase, cytochrome C oxidase; sarcolemmal membrane: Na+K+ ATPase, p-nitrophenylphosphatase; sarcoplasmic reticulum: CA2+ uptake, Ca2+ ATPase, NADPH cytochrome C reductase), protein, cholesterol and phospholipid content. RESULTS: "Protein yield" proved to be the most sensitive marker for cell organelle preservation. Only the sarcolemmal membrane showed no decrease in either enzyme activities of "protein yield" after storage in saline, ST or UW. Mitochondria showed no decrease in enzyme activities but a decrease in "protein yield" after storage in all 3 solutions. The "protein yield" of sarcoplasmic reticulum was significantly reduced after storage in UW, saline and ST. No correlation could be drawn between cholesterol and phospholipid content and the preservation of cell organelle function. CONCLUSIONS: It is possible to distinguish between the ability of solutions to preserve the membrane composition of human cardiac tissue during hypothermic storage. Using simple assays to assess preservation provides preliminary screening for a superior solution which can then be used in more complicated transplantation models to more fully assess cardiac function.

The effect of phospholipids on the biodegradation of polyurethanes by lysosomal enzymes.

Although biodegradation of model poly(ester-urethane)s and poly(ether-urethane)s has been demonstrated using a single enzyme system (cholesterol esterase (CE) in vitro, in vivo biodegradation most likely involves many processes acting together. In this study, the physical (film vs textured surface) and chemical (poly(urethane)s containing polycaprolactone (PCL) vs poly(tetramethylene oxide) (PTMO)) nature of the materials as well as the products of enzymatic reactions known to occur during the inflammatory response (CE and phospholipase A2 (PLA)) were assessed for their effects on poly(urethane) (PU) biodegradation in vitro. A mixed micelle (phosphatidylcholine (PC):lysoPC (LPC):oleic acid (OA): 2:1:1) significantly increased the release of radiolabelled products from a C-labelled poly(ester-urethane) (TDI/PCL/ED) caused by CE. This effect was further enhanced when this material was cast as a textured surface. A model poly(ether-urethane) showed no significant enhancement of CE-mediated hydrolysis in the presence of phospholipids and their breakdown products whether cast as a film or a textured surface. PLA caused a small but significant release of radiolabel from TDI/PCL/ED which was enhanced in the presence of its substrate, PC, and a mixture of PC with its breakdown products, LPC and OA. Based on the results of this study, it may be possible to hypothesize that during the inflammatory response when PLA is activated, enhancement of the biodegradation of a PU could occur by direct action of PLA on the poly(ester-urethane) and by stimulation of CE due to the formation of LPC and OA occurring when PLA hydrolyses PC, its natural substrate

The importance of sampling site in the measurement of whole-blood platelet flow cytometry.

PURPOSE: Flow cytometry is an emerging technology that may be of use in clarifying the defects of platelet function after cardiopulmonary bypass. However, the technique used for platelet sampling may affect results. The objective of this study was to evaluate the influence of the sampling site on the degree of expression of a variety of platelet-associated proteins. METHODS: Whole-blood flow cytometric assays for the detection of platelet glycoprotein (GP) Ib, guanosine monophosphate (GMP)-140, thrombospondin, activated GPIIb/IIIa, and platelet-associated factor (FXIIIa) were developed. These markers were then measured in samples taken simultaneously from a peripheral vein, radial artery, and the side port of the central venous catheter, in eight patients about to undergo surgery. RESULTS: When multiple samples from individual patients were assessed, the degree of activation with all of the activation assays (GMP-140, thrombospondin, activated GPIIb/IIIa, FXIIIa) was significantly greater in samples taken from the arterial catheter (p < 0.05) compared with the central venous catheter or the peripheral vein. The mean difference between sample sites was calculated in the study patients. Percent activation of FXIIIa from arterial blood was significantly greater than from the central vein and the peripheral vein (arterial-peripheral venous, 18.7 +/- 8.6; central venous-peripheral venous, 3.7 +/- 3.6; p = 0.005). There was no site-related difference in detected expression of platelet GPIb. CONCLUSION: The site of platelet sampling significantly affects the degree of activation detected by flow cytometry. To approximate results that would be obtained from peripheral blood, samples should be taken from the side port of the central venous catheter and not from the arterial catheter in patients studied during surgery.