Robert F. Highsmith

Endothelin receptors and calcium signaling.

Endothelin (ET) is a potent vasoactive peptide produced by endothelial cells that elicits prolonged constriction in most smooth muscle preparations and dilation in others. Of three isopeptides, ET‐1 is the only form constitutively released and may modulate vascular tone via binding to one of several receptor subtypes in smooth muscle. Activation of the ETA receptor is associated with pronounced vasoconstriction whereas ETB receptor occupation is linked to vasodilation. In addition, other subtypes of the ETB receptor exist, one mediating vasodilation (ETB1) and the other eliciting constriction (ETB2). An additional receptor subtype, ETc, has been identified although its physiological significance is uncertain. Distribution of these receptors varies between species and among tissue types, although it has been generally observed that ETA receptors predominate in arterial vessels whereas ETB receptors predominate on the low pressure side of the circulation. In vascular smooth muscle, an increase in intracellular Ca2+ is a common feature occurring after activation of all receptor subtypes. Upon binding of ET‐1 to ETA, phospholipase C is activated and inositol triphosphate is generated. Ca2+ is then released from intracellular stores accompanied by the influx of extracellular Ca2+ and activation of the contractile machinery. The precise mechanism by which ET‐1 affects intracellular Ca2+ regulation is not fully understood, but most likely involves multiple ion channels, protein kinases, and other intracellular mediators. The events coupled to non‐ETA receptor signaling are poorly understood.—Pollock, D. M., Keith, T. L., Highsmith, R. F. Endothelin receptors and calcium signaling. FASEB J. 9, 1196‐1204(1995)

Endothelial cell-derived vasoconstrictors: Mechanisms of action in vascular smooth muscle

Summary Intercellular signaling between the endothelial cell (EC) and vascular smooth muscle (VSM) is an important determinant of vasomotor tone. We evaluated mechanisms of action of EC-derived constrictors on VSM using conditioned medium from bovine aortic ECs in culture (EC-CM) or endothelin-1 (ET-1), and isolated coronary arteries or cultured VSM cells. EC-CM enhanced Ca2+ uptake into monolayers of rat aortic VSM and elicited sustained contractions in isolated coronary vessels in a time-and dose-dependent manner. The enhanced Ca2+ uptake and contractions were markedly attenuated by the Ca2+ channel antagonists bepridil, verapamil, and nitrendipine. EC-CM and ET-1 resulted in VSM membrane depolarization and increased excitability to electrical stimulation that was blocked by verapamil. ET-1 and EC-CM induced a dose-dependent increase in steady-state [Ca2+]i in Fura-2-loaded rat VSM cells. Most VSM responded with a rapid and transient increase in [Ca2+]i while others lacked only the transient phase. The elevated poststimulus [Ca2+]i level appeared to precede the influx of extracellular Ca2+ and contraction. EC-CM and ET-1 also resulted in time-and concentration-dependent increases in inositol monophosphate (IP) formation in rat aorta that paralleled the development of isometric force. We propose a biphasic mechanism in which the stable constrictors present in EC-CM elicit a rapid, phospholipase C-mediated mobilization of intracellular Ca2+ accompanied by or coupled to a sustained influx of extracellular Ca2+ through voltage-dependent channels.

Plasma proteolytic activity following burns

Because a number of metabolic events which are triggered by proteolysis in the bloodstream are activated following trauma, net proteolytic activity (P.A.) in the plasma of 37 pediatric burned patients was measured. The P.A. assay involved incubating plasma with a radioiodinated protein substrate and counting the isotopic activity of the hydrolyzed fragments in the acid-soluble fraction. In patients, plasma P.A. increased in direct proportion to the extent of burn injury. To examine additional trends in plasma P.A. suggested by the patient P.A. data, we measured plasma P.A. in a burned rat model: circulating P.A. was significantly elevated at 6 hours and until at least 2 weeks postburn; infection with Pseudomonas and use of the proteolytic debriding agent, Travase, each further elevated this activity; the plasma P.A. was not directly derived from the burn site. We postulate that this elevated circulating P.A. triggers some of the pathologic as well as some physiologic sequelae which follow burn trauma.

Effect of calcium channel modulators on isolated endothelial cells

Indirect evidence, using organic calcium channel modulators suggests that calcium channels exist in endothelial cells. Using freshly prepared and cultured bovine aortic endothelial cells, we have studied the effect of calcium channel modulators on Fura-2 fluorescence and have examined the binding of the dihydropyridine, (+)[3H]PN200-110. In both isolated primary and cultured cells, external calcium (0.5-2 mM) and bradykinin (10(-8) M) increased the intracellular calcium concentration. In cultured cells, the increase in calcium was not significantly attenuated by preincubation with nitrendipine (10(-8) M) or d-cis-diltiazem (10(-6) M). The calcium agonists (-)Bay k8644 and (+)202-791 had no effect on intracellular calcium concentration, but other agonists including ATP (10(-4) M) and thrombin (1.5 micrograms/ml) significantly increased the calcium concentration. Competition binding studies with (+)[3H]PN200-110 indicated specific binding of this ligand with a KD of 57 nM and a Bmax of 2.1 pmol/10(6) cells. While these data do not provide convincing evidence for the existence of calcium channels in cultured or fresh bovine aortic endothelial cells, explanations may yet reconcile our observations with the presence of calcium channels in these cells.

Quantitative Studies of Human Monokine-Induced Endothelial Cell Elongation

Leucocyte‐endothelial cell interactions are important in the inflammatory response. In this study, the effect of peripheral blood mononuclear cell (PBMC) products on endothelial cell (EC) shape was examined and quantified. PBMC were obtained from normal donors by Ficoll‐Hypaque separation of heparinized whole blood and cultured for 72 hr in media containing 10% fetal calf serum with and without concanavalin A (Con A). Media conditioned by PBMC or control, nonconditioned media were then added to preconfluent, first passage EC cultures derived from human umbilical veins. Conditioned media from Con A‐stimulated PBMC resulted in a dose‐dependent, marked elongation and whorling of cultured EC. The minimum effective concentration found to elicit a response was 1.25%, with a maximum response occurring at 10%. Quantitative morphometric analyses of treated EC indicated that the elongation was highly significant (p < 0.001) when compared to EC incubated with control, nonconditioned media. In addition, EC elongation was accompanied by a highly significant (p < 0.001) increase in cell area. Although less dramatic, conditioned media from unstimulated PBMC also elicited a similar, significant dose‐dependent change in EC shape. Significant changes in EC shape were evident within 6 hr and continued over the time course of the experiment (40 h). Cell shape changes were partially reversible at 18 h after removal of the PBMC‐conditioned media and replacement with control, nonconditioned media. The change in EC morphology induced by a PBMC‐derived factor(s) suggests a mechanism by which activated leucocytes may modulate cellular traffic at the blood‐vessel wall interface.

Streptokinase-dependent delayed activation of horse plasminogen.

Complete activation of purified horse plasminogen to plasmin was obtained with a 1:10 molar ratio of streptokinase to plasminogen after 5 min of incubation at 37 degrees C. At a 1:1 molar ratio, maximal activity did not appear until 15-30 min, while at a ratio of 6:1 complete activation was delayed for 120-180 min. Gel filtration studies of isotopically labeled streptokinase and horse plasminogen suggest that the delay was due to impaired formation of a streptokinase-plasminogen complex. The predominant streptokinase moiety within the streptokinase-plasmin complex which forms from the streptokinase-plasminogen complex had a molecular weight of about 25000. The streptokinase-horse plasmin complex activated bovine plasminogen and was relatively stable. Native streptokinase was rapidly modified by horse plasmin predominantly to a fragment with a molecular weight comparable to that of the streptokinase moiety within the horse streptokinase-plasmin complex, about 25000 daltons. Partial characterization of horse plasminogen revealed no striking differences from human plasminogen in terms of molecular weight, N-terminal analysis and amino acid composition. However, horse plasminogen did not react with antibodies to human plasminogen, and its isoenzymes were more acidic than those of the human. Further characterization of horse plasminogen will be required to ascertain whether activation by streptokinase can serve as a model for the altered kinetics which have recently been described for the activation of aberrant types of human plasminogen.

Kidney: Primary Source of Plasminogen after Acute Depletion in the Cat

The kidney was the primary source of plasminogen to restore normal plasma levels, after acute plasminogen depletion was produced by injection of streptokinase in cats. The concentration of plasminogen in the hepatic vein remained below that in the artery during the time when concentrations in the artery and renal vein were returning to normal.

Protease inhibitors and human plasmin: Interaction in a whole plasma system

Abstract Using trace amounts of [125I]-plasminogen and conventional biochemical techniques, the distribution of the labeled zymogen amongst the various protease inhibitors was studied in whole plasma before and after activation with urokinase and streptokinase. A small percentage of the labeled enzyme was bound to α2-macroglobulin while a majority was complexed to a component in plasma immunologically distinct from the well known human antiplasmins. The inhibitor was identified as α2-antiplasmin and confirmed the existence of this antiprotease recently described by others. These data also suggest that the other antiplasmins may play a minor, yet important role in the regulation of plasmin activity under different physiological conditions.

Building research administration applications for the academic health center: a case study.

The academic health center information environment is saturated with information of varying quality and overwhelming quantity. The most significant challenge is transforming data and information into knowledge. The University of Cincinnati Medical Centers (UCMC) focus is to develop an information architecture comprising data structures, Web services, and user interfaces that enable individuals to manage the information overload so that they can create new knowledge. UCMC has accomplished much of what is reported in this article with the help of a four-year Integrated Advanced Information Management Systems (IAIMS) operation grant awarded by the National Library of Medicine in 2003. In the UCMC vision for knowledge management, individuals have reliable, secure access to information that is filtered, organized, and highly relevant for specific tasks and personal needs. Current applications and tool sets will evolve to become the next generation knowledge management applications or smart digital services. When smart digital services are implemented, silo applications will disappear. A major focus of UCMCs IAIMS grant is research administration. Testing and building out existing and new research administration applications and digital services is underway. The authors review UCMCs progress and results in developing a software architecture, tools, and services for research administration. Included are sections on the evolution to full integration, the impact of the work at UCMC to date, lessons learned during this research and development process, and future plans and needs.

ALTERATIONS IN HUMAN ENDOTHELIAL CELL MORPHOLOGY, PROLIFERATION AND FUNCTION BY A MACROPHAGE-DERIVED FACTOR

Changes in endothelial cell (EC) morphology occur at sites of physiological lymphocyte traffic and in areas of chronic inflammation. Previous studies have shown that EC shape changes also occurin vitro following exposure of EC monolayers to peripheral blood mononuclear cell (PBMC)-derived conditioned media (CM). In the present study, quantitative image analysis is used to define the cell of origin of the elongating factor(s), to examine changes in EC proliferation and function accompanying PBMC-induced human EC elongation and to identify the active PBMC-derived products responsible for this elongation. By separating mononuclear cells into subpopulations (macrophages, B cells and T cells) and adding conditioned media derived from these subpopulations to cultured ECs, the macrophage (Mφ) is shown to be the primary cell of origin of the elongating factor(s). Furthermore, EC elongation is accompanied by both a dose-dependent decrease in cellular proliferation and an increase in prostacyclin production. These findings suggest that PBMC-induced changes in EC morphology may be associated with a shift from a proliferative state to a more secretory phase of the EC cycle. Finally, using recombinant factors it is shown that TNFoc acting in combination with IL-I may be the active PBMC-derived products which contribute to EC elongation.