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P R Girard

Publications and source records attributed to P R Girard.

At least 19 recordsLinked to original sources
PubMed1997

Amidative peptide processing and vascular function.

Substance P (SP), an amidated peptide present in many sensory nerves, is known to affect cardiovascular function, and exogenously supplied SP has been shown to activate nitric oxide synthase (NOS) in endothelial cells. We now report that SP-Gly, the glycine-extended biosynthetic precursor of SP (which is enzymatically processed to the mature amidated SP), causes relaxation of rat aortic strips with an efficacy and potency comparable to that of SP itself. Pretreatment of the aortic strips with 4-phenyl-3-butenoic acid (PBA), an irreversible amidating enzyme inactivator, results in marked inhibition of the vasodilation activity induced by SP-Gly but not of that induced by SP itself. Isolated endothelial cell basal NOS activity is also decreased by pretreatment with PBA, with no evidence of cell death or direct action of PBA on NOS activity. Both bifunctional and monofunctional forms of amidating enzymes are present in endothelial cells, as evidenced by affinity chromatography and Western blot analysis. These results provide evidence for a link between amidative peptide processing, NOS activation in endothelial cells, and vasodilation and suggest that a product of amidative processing provides intrinsic basal activation of NOS in endothelial cells.

Amino Acid Sequence↗
PubMed1995

Shear stress modulates endothelial cell morphology and F-actin organization through the regulation of focal adhesion-associated proteins.

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.

Actins↗
PubMed1995

Elongation of confluent endothelial cells in culture: the importance of fields of force in the associated alterations of their cytoskeletal structure.

Studies using either animal models or in vitro flow systems have shown that the shape of large-vessel endothelial cells (ECs) was sensitive to the amplitude of the flow imposed on them. In order to better understand the morphological changes experienced by ECs when exposed to physical forces such as shear stress, the mechanical integrity of confluent bovine aortic ECs (BAECs) was anisotropically perturbed using the five following types of experiments: (i) slicing and partial scraping of BAEC monolayers; (ii) culture of BAECs on narrow strips of adhesive plastic; (iii) incubation of confluent BAECs with media containing low Ca2+ concentrations; (iv) culture of ECs on top of rectangular collagen gels; and (v) exposure of BAECs to laminar steady shear stress. In all five experimental systems, BAECs exhibited an elongated morphology and aligned their major axes in specific directions. In addition, a preferential alignment of actin microfilaments, vimentin intermediate filaments, and streaks of vinculin with the major axes of the cells often occurred concomitantly with BAEC elongation. In all five systems, the elongation of ECs was analyzed in terms of a mechanical deformation borne by the cytoskeleton, and possibly caused by anisotropic distribution of the forces experienced by the cell structure. In addition, the strain-stress and stiffness-stress relationships characterizing the elongation of BAECs exposed to steady flow were qualitatively similar to those computed for the uniaxial deformation of a spherical geodesic. Our findings suggest that the cytoskeleton of ECs plays an important role in the transduction of those forces which cause an elongation of ECs.

Animals↗
PubMed1995

Oscillatory shear stress and hydrostatic pressure modulate cell-matrix attachment proteins in cultured endothelial cells.

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.

Actins↗
PubMed1995

Regulation of vascular tone.

The intimal surface of the blood vessel in vivo is subject to shear stress resulting from blood flow, which in most of the circulation, at least at rest, is laminar. Turbulence can occur at bifurcations, especially those of the large arteries, and where vessels curve significantly. Shear stress is a frictional tangential force exerted at the fluid-intimal interface in the long axis of the vessel. It is now known that hemodynamic shear stress can influence a large variety of biological processes in endothelial cells, which vary from those with a short response time, just a few milliseconds, such as the opening of ion channels, to those that change over a period of minutes to several hours, for example, endocytosis and cytoskeleton rearrangement, and those features that alter much more slowly, such as cell shape and stiffness. In addition to these types of changes, there are suggestions that flow acting through shear stress may be responsible for several basic attributes of the vasculature, including the relative size and diameter of the components of a branching vascular system. In this symposium on the flow regulation of the blood vessel, the first presentation dealt with optimality principles that appear to govern the dimensions of the vasculature, in particular the geometry of the arterial branching and the role of shear stress. An optimally designed system is one that requires the least metabolic work to perform its function.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Isolation and culture of bovine intracranial arterial endothelial cells.

We report a simple explant technique to isolate and propagate endothelial cells from bovine cerebral arteries. The endothelial nature of the cells was confirmed by the presence of Factor VIII/von Willebrand antigen, the ability to phagocytize low-density lipoprotein, and the ability to be induced to express E-selectin. The lack of expression of the CD11c antigen and the absence of smooth muscle alpha-actin immunofluorescence suggested that the cultures were not contaminated with macrophages or smooth muscle cells, respectively. This technique yields pure cerebral arterial endothelial cell cultures, which will be of value for in vitro investigation of cerebrovascular physiology and disease processes.

Animals↗
PubMed1995

Changes in organization and composition of the extracellular matrix underlying cultured endothelial cells exposed to laminar steady shear stress.

BACKGROUND: In blood vessels, the extracellular matrix (ECM) underlying the endothelium supports endothelial cell (EC) attachment, spreading, migration, and proliferation. The structure and composition of the ECM may be modulated by hemodynamic shear stress, which may play a role in the pathogenesis of vascular diseases such as atherosclerosis. EXPERIMENTAL DESIGN: In this study, in vitro effects of fluid shear stress on the ECM of EC were investigated. Cultured bovine aortic EC (BAEC) were exposed to a steady laminar shear stress of 30 dyn/cm2 from 3 to 48 hours, using a parallel-plate flow chamber. Parallel control cultures were maintained under static conditions. The organization of fibronectin (Fn), laminin (Ln), collagen type IV (Col IV), and vitronectin (Vn) was analyzed by immunofluorescence microscopy. Changes in the profile of proteins present in the deoxycholate-insoluble ECM fraction of EC were determined using two-dimensional gel electrophoresis, and the levels of Fn, Ln, and Vn were determined by Western blotting. RESULTS: Fn, Ln, and Col IV exhibited both a granular pattern in cell perinuclear areas and a fibrillar pattern localized underneath EC. On exposure of bovine aortic EC to shear stress, Fn fibrils grouped into thicker tracts of fibrils, and there was a tendency for some of these tracks of fibrils to align with the direction of flow. Ln and Col IV also grouped into thicker fibers, which, in contrast to Fn, were randomly oriented. Vn exhibited a diffuse granular pattern, which did not change in response to shear stress. Consistent increases in the levels of four unidentified acidic proteins (mol wt/pI = 52/4.9, 70/4.7, 70/5.5, and 110/4.4) were observed after 3 to 6 hours of exposure to flow. The level of Fn present in the ECM was decreased twofold 12 hours after exposure of the cell monolayer to flow, and then increased after 24 and 48 hours. The level of Ln showed a twofold increase after 24 and 48 hours of flow, whereas the level of Vn was not altered by shear stress. CONCLUSIONS: These changes in organization and composition observed in the ECM of cultured EC may play a significant role in shear stress-induced morphologic alterations in EC and may represent relevant events in the initiation of atherosclerotic lesions by influencing both EC and smooth muscle cell function.

Amino Acid Sequence↗
PubMed1993

Endothelial cell signaling and cytoskeletal changes in response to shear stress.

Endothelial cells exposed to a fluid shear stress both in vivo and in vitro show an alignment with the direction of flow and an elongation of cells with a concomitant reorganization of their F-actin microfilament network. Specialized regions of the plasma membrane known as focal contacts are sites of transmembrane linkages between the actin microfilament bundles and the extracellular matrix (ECM) where cytoskeletal organization and, hence, cellular morphological changes may be modulated. Focal contact-associated proteins such as fibronectin receptors, vitronectin receptors and vinculin were shown in this study to play specific roles in the modulation of the cytoskeletal and cell shape changes. Data is also presented suggesting that protein kinase C is part of the cellular signaling system involved in shear stress-induced cytoskeletal reorganization.

Animals↗
PubMed1992

Peptide amidating enzymes are present in cultured endothelial cells.

Carboxy-terminal amidation is a prevalent post-translational modification necessary for the bioactivity of many peptides. We now report that the two enzymes essential for amidation, peptidylglycine alpha-monooxygenase (PAM) and peptidylamidoglycolate lyase (PGL), are present in both the cytosol and membrane fractions of cultured bovine aortic endothelial cells. Endothelial PAM exhibits ascorbate-dependent turnover and is inactivated by the mechanism-based inactivator, 4-phenyl-3-butenoic acid (PBA), whereas PGL activity is independent of ascorbate and is not affected by PBA. These enzymological characteristics correspond to those of amidating enzymes from other tissues. These results suggest a heretofore unrecognized role for alpha-amidated peptides in cardiovascular function.

Amidine-Lyases↗
PubMed1990

Protein kinase C and its 80-kilodalton substrate protein in neuroblastoma cell neurite outgrowth.

A potential role of the protein kinase C (PKC) system in differentiation of human neuroblastoma cell line LA-N-5 was investigated. It was found that neurite outgrowth induced by 12-O-tetradecanoylphorbol 13-acetate (TPA, 81 nM) was associated with a down-regulation of PKC as determined independently by immunocytochemistry, immunoblot, and enzyme activity assay. Down-regulation of PKC in cells induced to differentiate by retinoic acid (1 microM) was less pronounced, whereas it was undetected in cells induced to differentiate by nerve growth factor (100 ng/ml). The in vitro phosphorylation of an 80-kilodalton protein present in control LA-N-5 cells or in cells treated with TPA, retinoic acid, or nerve growth factor for 1 day decreased to various extents at days 4 or 7 concomitant with neuritogenesis. Pretreatment of LA-N-5 cells with a high concentration (1 microM) of TPA to deplete cellular PKC rendered the cells unresponsive to the differentiating effect of the agents. It was observed that CHP-100 cells, another human neuroblastoma line shown to be resistant to differentiation induced by the agents, had a reduced PKC level and the amount of in vitro phosphorylation of the 80-kilodalton protein was greatly reduced in control cells and remained relatively unchanged when the cells were treated with the agents for up to 7 days. The present studies suggested that PKC and its 80-kilodalton substrate protein were likely involved in initiation and/or progression of LA-N-5 cell differentiation induced by TPA and that separate PKC-independent pathways might also be involved in the differentiating effect of retinoic acid or nerve growth factor.

Axons↗
PubMed1990

Hemodynamic influences on vascular endothelial biology.

The vascular endothelium resides in a unique biomechanical stress environment resulting from the hemodynamics of the system. In vivo studies indicate that there are regional differences in endothelial biology and that this may be due to the influence of the local hemodynamic environment. To investigate this further, cell culture studies have been conducted using well-defined mechanical stress environments. To study flow effects, we have employed a parallel plate chamber in which endothelial cell monolayers are exposed to laminar flow. In such experiments and concomitant with changes in morphology, there are a variety of other alterations in cell function, including a decrease in the rate of cell proliferation for subconfluent monolayers. Changes in cell behavior due to the direct effect of pressure and in cultured cells which are in a cyclical stress field also have been observed. In the recognition/transduction of such a mechanical signal, the pathway may possibly include a membrane event linked to the control of intracellular calcium. It may be that the same signaling mechanisms are involved both in cytoskeletal/shape changes and in the control of the cell's growth program and, in exercising such an influence, hemodynamics may have an important role in the response of the arterial wall to injury and the resulting repair and/or disease processes.

Animals↗
PubMed1989

Effects of gold coordination complexes on neutrophil function are mediated via inhibition of protein kinase C.

Previous studies have shown that the gold compounds auranofin (AUR) and gold sodium thiomalate (GST) inhibit responses of various cells and tissues. We found that superoxide anion generation induced in human neutrophils by the chemotactic tripeptide fmet-leu-phe (1 microM), fluoride (18 mM), or phorbol myristate acetate (PMA, 100 nM) was inhibited by pretreatment of cells with 5-100 microM AUR. The extent of inhibition was dependent on AUR concentration and duration of the preincubation. GST was much less potent, inasmuch as only weak effects were observed at 5 times higher concentrations. The ineffectiveness of GST was attributed to its slower rate of penetration into cells, compared with AUR. The finding that mobilization of internal Ca2+ stores was not blocked in AUR-treated cells suggests that phospholipase C-mediated hydrolysis of polyphosphoinositides to inositol 1,4,5-trisphosphate was not inhibited by the drug. Because PMA is known to mimic the action of diacylglycerol in activating protein kinase C (PKC), we investigated the possibility that gold compounds might be interfering with signal transduction at this level. Enzymatic assays indicated that both gold compounds reduced the level of PKC activity associated with the cytosol; however, translocation of PKC to the plasma membrane was not found. Immunoblot analyses carried out with polyclonal anti-PKC antisera revealed that the gold compounds did not cause degradation of PKC or increase translocation to the membrane. Further studies indicated that enhanced endogenous protein phosphorylation resulting from PMA stimulation was attenuated in cells co-treated with AUR. Finally, in vitro enzymatic assays showed that both AUR and GST inhibited partially purified PKC in a concentration-dependent manner. It is suggested that modulation of PKC represents a mechanism of action of gold coordination complexes at the cellular level.

Auranofin↗
PubMed1988

Distribution of protein kinase C immunoreactivity in rat retina.

A polyclonal antiserum to protein kinase C has been used to study the distribution of the enzyme antigenic sites in rat retina. The results indicate that the kinase is concentrated in photoreceptor outer segments as well as in the outer and inner plexiform layers. In identified components of retinal neuronal circuits, the kinase immunoreactivity is present in photoreceptor presynaptic terminals, in bipolar cell dendrites and axons, and probably in bipolar cell presynaptic terminals impinging on retinal ganglion cell dendrites. Thus, protein kinase C is positioned to play a role in specialized compartments of photoreceptor membrane and at both pre- and postsynaptic levels in the function of retinal neuronal circuits. Label in the nucleus is observed in retinal ganglion cells, but not bipolar or horizontal cells and probably not in amacrine cells. A role for protein kinase C in neuronal function at the level of the cell nucleus is therefore not likely to be universal, but to be determined by the particular properties of individual neuronal types.

Animals↗
PubMed1988

Immunocytochemical localization of protein kinase C in developing brain tissue and in primary neuronal cultures.

Antisera to protein kinase C (PKC) have been used to examine the presence and distribution of the enzyme in developing cerebellar cortex of postnatal rat and in cultures of rat sympathetic ganglia. In the cerebellar cortex of 2-,4-, and 6-day old rats, immunostaining was observed in areas of early-forming presynaptic terminals and growth cones. No staining was evident in the cortical proliferative zone. Beginning at 10 days postnatal, nuclear staining, not apparent at earlier stages, was prominent in Purkinje cells. In neuronal cultures of dissociated rat sympathetic ganglia, PKC was immunolocalized in cell bodies and bundles of neuronal processes. Immunoreactivity was particularly striking in growth cones of extending neurites and in axonal varicosities. These results suggest a role for PKC in neuronal growth following cell proliferation and in synaptic function. The appearance of nuclear staining in later developmental stages suggests that the enzyme may be involved in the promotion and maintenance of the differentiated state of neurons.

Animals↗
PubMed1987

Effects of phorbol ester on translocation and down-regulation of protein kinase C and phosphorylation of endogenous proteins in human acute myeloid leukemia cell line KG-1 and its phorbol ester-resistant subline KG-1a.

12-O-Tetradecanoylphorbol-13-acetate (TPA) induced decreases in the catalytic activity and immunoreactivity of protein kinase C (PK-C) in the soluble fraction, accompanied by increases in their activities in the particulate fraction, of a human myeloid leukemia cell line KG-1. TPA also caused a similar down-regulation and translocation of PK-C in KG-1a, a cloned subline shown to be resistant to the differentiating effect of TPA. The activity levels of enzyme in the soluble and particulate fractions from KG-1 cells, however, were about three times higher than those from KG-1a cells. Immunocytochemical studies showed that, when KG-1 cells were treated with 10 nM TPA for 30 min, PK-C was translocated to the plasma membrane in the adherent subpopulation of cells, whereas the enzyme remained largely in the cytoplasm and perinuclear area of the nonadherent cells. TPA, in contrast, caused a PK-C translocation primarily to the perinuclear region in KG-1a cells. Phosphorylation patterns of PK-C substrate proteins in the two cell lines were similar, except that phosphorylation of the Mr 33,000 and 97,000 proteins were predominant in KG-1 and KG-1a cells, respectively. The present findings showed existence of certain differential effects of TPA on the PK-C system in the two leukemia cell lines, suggesting a molecular basis for the selective resistance of KG-1a cells to the differentiating action of TPA.

Biological Transport↗
PubMed1987

New signal transduction mechanisms of atrial natriuretic factor: inhibition of phosphorylation of protein kinase C and A 240 kDa protein in adrenal cortical plasma membrane by cGMP dependent and independent mechanisms.

The effects of synthetic atrial natriuretic factor (ANF) on the state of protein phosphorylation in plasma membranes of bovine adrenal cortex have been studied in vitro. ANF (1x10(-8)M - 1x10(-7)M) specifically inhibited the phosphorylation of two distinct proteins of 78 kDa and 240 kDa. Immunoblotting with specific antiserum to protein kinase C produced evidence that 78 kDa protein is most likely the protein kinase C whose phosphorylation is inhibited by both ANF and cGMP. However, cGMP did not affect the phosphorylation of 240 kDa protein, indicating a new cGMP-independent mechanism of ANF action in the adrenal, which is compatible with the lack of action of cGMP and its analogs in ANF-induced inhibition of aldosterone secretion from adrenal cortex. The inhibition of phosphorylation of putative protein kinase C by ANF or cGMP indicates a hitherto unknown signal transduction mechanism of ANF.

Adrenal Cortex↗
PubMed1987

Comparative effects of polymyxin B, phorbol ester and bryostatin on protein phosphorylation, protein kinase C translocation, phospholipid metabolism and differentiation of HL60 cells.

The effects of protein kinase C (PKC) inhibitor polymyxin B (PMB) and PKC activators 12-O-tetradecanoylphorbol-13-acetate (TPA) and bryostatin on intact HL60 cells were examined. It was found that each of the three agents exhibited similar effects on phosphorylation of certain endogenous proteins, PKC translocation from cytoplasm to plasma membrane and formation of CDP-choline. TPA, however, was the only agent that stimulated phosphatidylcholine formation. Differentiation of HL60 cells was potently induced by TPA; in comparison bryostatin was a relatively weaker inducer and PMB was without effect. The data indicated that the effects of the PKC inhibitor PMB on intact cells could not be predicted by its in vitro activity, and that certain TPA-dependent but PKC-independent reactions might be crucial in HL60 cell differentiation.

Bryostatins↗