Flow-related shear stress has been shown to modulate endothelial cell structure and function including F-actin microfilament organization. Focal adhesion-associated proteins such as vinculin, talin, and specific integrins may play a role in the modulation of these cytoskeletal and morphological changes. Double-label immunofluorescence studies indicated that, in static culture, alpha 5 beta 1 fibronectin receptors (alpha 5 beta 1 FNRs) and alpha v beta 3 vitronectin receptors (alpha v beta 3 VNRs) were found predominantly in the peripheral regions of bovine aortic endothelial cells (BAECs) corresponding to the localization of vinculin, talin, and actin microfilament terminations. In response to shear stress, concomitant with cell elongation and the appearance of stress fibers aligned with the direction of flow, there was a prominent localization of vinculin and alpha v beta 3 VNRs as the "upstream" end of the cells. Stress fiber terminations were clearly evident at these concentrations of focal adhesion-associated proteins. These data suggest that the upstream concentration of these proteins may direct shear stress-induced stress fiber formation and may function in the alignment of the fibers in the direction of flow. Levels of surface alpha v beta 3 VNRs were found to decrease in response to flow, possibly reflecting the decrease in numbers of "downstream" receptors. Unlike the arrangement of vinculin and alpha v beta 3 VNRs observed following exposure to flow, talin and alpha 5 beta 1 FNRs, in addition to being localized at the upstream end of the cell, were also evenly distributed throughout the rest of the cell. Surface levels of alpha 5 beta 1 FNRs increased in response to shear stress, perhaps providing an increased adherence of BAECs to the extracellular matrix through these receptors. These data suggest that focal adhesion-associated proteins play specific roles in the response of BAECs to shear stress.

Antisera against phospholipid/Ca2+-dependent protein kinase (protein kinase C) were raised in rabbits. Immunospecificity of the polyclonal antibodies, as determined by immunoblot and ELISA, was shown by their reactivity to the enzyme but not to other protein kinases or any of many other proteins tested. Immunocytochemical localization of the kinase in rat brains revealed that although the enzyme was distributed broadly in different brain regions, it was highly restricted to the periphery of the nucleus of neurons in cerebral cortex and to axons and cells strongly resembling oligodendroglia in white-matter regions. Initial electron microscopy of cerebral cortex revealed that the enzyme was highly concentrated in the presynaptic terminals, and only rarely were labeled postsynaptic specialization elements seen. It is suggested that the discrete localization of the enzyme, which is distinct from that of the calmodulin/Ca2+-dependent system, may be related to certain biological and functional aspects of Phospholipid/Ca2+-dependent protein kinase (PL/Ca-PK, or protein kinase C) is a major class of protein-phosphorylating enzyme first found in brain (1) and subsequently shown to occur widely in tissues and phyla of the animal kingdom (2). The biological significance of this Ca2+ effector system is further suggested by the presence of its numerous but specific substrate proteins in tissues (for reviews, see refs. 3 and 4), such as myelin basic protein in brain (5, 6), and its key role in transduction of receptor-mediated signals (7,8), such as in the platelet activation induced by thrombin, platelet-activating factor, phorbol ester, diacylglycerol, and the Ca2+ ionophore A23187 (9). Some significant progress has been made during the last five years on the molecular, pharmacological, and regulatory aspects of this novel protein-phosphorylation system (3, 4). Immunological characterization of the enzyme, however, has not been possible. The major reasons for the lack of progress are the difficulties in the purification of the enzyme in large quantity for immunization, due to its instability, and the low antigenicity of the enzyme, probably because of its common occurrence in tissues. In this laboratory, monoclonal antibodies against the enzyme have been developed recently (10); unfortunately, they are of the IgM class of immunoglobulins and were found to be unsuitable for the intended immunological studies of the enzyme system. We now report the development of polyclonal antibodies against pig brain PL/ Ca-PK exhibiting a high immunospecificity toward the antigen and describe their use in immunocytochemical localization of the enzyme in rat brain. METHODSPurification of PL/Ca-PK. The enzyme from the crude extracts of pig brain (1 kg) was purified initially by DEAEcellulose (11) and Affi-Gel Blue chromatography (12), as previously described. The final step of the purification was affinity chromatography on polyacrylamide on which cholesterol and phosphatidylserine were immobilized, a procedure recently described by ...

Endothelial cells (ECs) may behave as hemodynamic sensors, translating mechanical information from the blood flow into biochemical signals, which may then be transmitted to underlying smooth muscle cells. The extracellular matrix (ECM), which provides adherence and integrity for the endothelium, may serve an important signaling function in vascular diseases such as atherogenesis, which has been shown to be promoted by low and oscillating shear stresses. In this study, confluent bovine aortic ECs (BAECs), were exposed to an oscillatory shear stress or to a hydrostatic pressure of 40 mmHg for time periods of 12 to 48 h. Parallel control cultures were maintained in static condition. Although ECs exposed to hydrostatic pressure or to oscillatory flow had a polygonal morphology similar to that of control cultures, these cells possessed more numerous central stress fibers and exhibited a partial loss of peripheral bands of actin, in comparison to static cells. In EC cultures exposed to oscillatory flow or hydrostatic pressure, extracellular fibronectin (Fn) fibrils were more numerous than in static cultures. Concomitantly, a dramatic clustering of alpha 5 beta 1 Fn receptors and of the focal contact-associated proteins vinculin and talin occurred. Laminin (Ln) and collagen type IV formed a network of thin fibrils in static cultures, which condensed into thicker fibers when BAECs were exposed to oscillatory shear stress or hydrostatic pressure. The ECM-associated levels of Fn and Ln were found to be from 1.5- to 5-fold greater in cultures exposed to oscillatory shear stress or pressure for 12 and 48 h, than in static cultures. The changes in the organization and composition of ECM and focal contacts reported here suggest that ECs exposed to oscillatory shear stress or hydrostatic pressure may have different functional characteristics from cells in static culture, even though ECs in either environment exhibit a similar morphology.

Polyclonal antisera to the phospholipid/Ca2+-dependent protein kinase have been used to study the distribution of the enzyme in identified neurons of several brain regions. The results indicated that the enzyme was concentrated in synaptic terminals of mossy fibers, Golgi II neurons and Purkinje neurons in the cerebellum, and in granule cell terminals in the hippocampus. These synapses have different physiological properties and utilize different neurotransmitters. Electron microscopic results indicated that the enzyme was concentrated in presynaptic terminals. Thus, the protein kinase may play a broad role in Ca2+-related events of the presynaptic terminal during neurotransmission. Light- and electron-microscopic immunocytochemical analysis also indicated that the enzyme was inside the nucleus concentrated in a region adjacent to the inner nuclear membrane, where it may play a role in the regulation of neuronal function.

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