Paul W. Fletcher

Time course of thrombin-induced increase in endothelial permeability: relationship to Ca2+i and inositol polyphosphates

The temporal relationship between the alpha-thrombin-induced increase in transendothelial permeability and the alpha-thrombin-mediated changes in several key transmembrane signaling events was examined in confluent monolayers of bovine pulmonary artery endothelial cells (BPAEC). The time courses of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] generation, changes in cytosolic [Ca2+] ([Ca2+]i), and reorganization of cytoskeletal F-actin were determined to assess the relationship between these events and the onset of the alpha-thrombin-induced increase in endothelial permeability. alpha-Thrombin (10(-7) M) increased the transendothelial 125I-albumin clearance rate half-maximally by approximately 1 min and maximally by approximately 2 min (160% over control level). The increase in permeability occurred concomitantly with reorganization of F-actin cytoskeleton (i.e., loss of peripheral band and increased stress fiber density) and increased actin polymerization. Stimulation of fura-2-loaded BPAEC with 10(-7) M alpha-thrombin produced a typical biphasic rise in [Ca2+]i. The initial rapid increase in [Ca2+]i peaked by approximately 16 s after thrombin challenge and the [Ca2+]i response showed a slow decrease to half-maximal within 50 s. The alpha-thrombin-induced increase in permeability as well as the increase in [Ca2+]i were consistently preceded by increased Ins(1,4,5)P3 generation detectable within 10 s after thrombin challenge. These results indicate that alpha-thrombin triggers a cascade of events (i.e., Ins(1,4,5)P3 generation and the ensuing rise in [Ca2+]i), which may comprise the second messengers that mediate F-actin reorganization and the increase in endothelial permeability.

Bradykinin- and thrombin-induced increases in endothelial permeability occur independently of phospholipase C but require protein kinase C activation.

We determined whether activation of phosphatidylinositol‐specific phospholipase C (PI‐PLC) and a subsequent increase in cytosolic calcium concentration ([Ca2+]i) was an obligatory signaling event mediating the increase in transendothelial permeability induced by bradykinin (BK) and α‐thrombin (α‐T). Both BK and α‐T (each at a concentration range of 0.01–1 μM) caused dose‐dependent increases in transendothelial 125I‐albumin permeability in cultured bovine pulmonary artery endothelial cell monolayers. Both agonists also produced a rise in inositol (1,4,5)‐trisphosphate [Ins(1,4,5)P3] by 10 sec that was followed by a prolonged increase in [Ca2+]i. Pretreatment of endothelial cells with the PLC inhibitor, 1‐(6‐((17β‐3‐methoxyestra‐1,3,5(10)‐trien‐17‐yl)amino)hexyl)‐1H‐pyrrole‐2,5‐dion [(U73122) at 10 μM for 15 min], prevented the increases in Ins(1,4,5)P3 and [Ca2+]i induced by both BK and α‐T. However, inhibition of PLC with U73122 or another PLC inhibitor, neomycin, did not prevent the increase in endothelial permeability induced by either agonist. In contrast, depletion of cellular protein kinase C (PKC) with phorbol‐12‐myristate 13‐acetate (0.01 μM for 20 hr) increased both BK‐ and α‐T‐induced phosphoinositide turnover but inhibited the agonist‐induced increase in permeability. A PKC inhibitor, staurosporine (5 μM) likewise inhibited the BK‐induced increase in endothelial cell permeability to albumin. We conclude that increases in endothelial permeability induced by the inflammatory mediators, BK and thrombin, can occur independently of PLC activation and increased [Ca2+]i but that a PKC‐dependent pathway is required for the permeability response. J. Cell. Physiol. 173:387–396, 1997.

Cellular processing of follicle-stimulating hormone by Sertoli cells in serum-free culture

Specific binding of radioiodinated human follicle-stimulating hormone [( 125I]hFSH) to receptors on the cell surface of immature rat Sertoli cells in serum-free culture (4-7 days) was concentration-, time- and temperature-dependent with an apparent steady state of binding stained after 2 h at 4, 18 or 32 degrees C. Analysis of [125I]hFSH binding data indicated an apparent Ka of 3 X 10(9) M-1 with approximately 670 fmoles binding sites/mg protein. The radioiodinated hFSH (60-70 microCi/micrograms) bound to receptor and stimulated estradiol production, thus confirming its biological activity and Sertoli cell viability. Approximately 50% of the specifically bound radioactivity was lost from the cell surface after 0.25 h at 32 degrees C, 1 h at 18 degrees C or 4 h at 4 degrees C. Most (greater than 90%) of the receptor-bound hormone was removed from the cell surface and degraded to [125I]mono- and diiodotyrosine within 4 h at 32 degrees C. FSH degradation was inhibited at low temperatures (4 degrees C) and by the lysosomotropic agents chloroquine, NH4Cl or concanavalin A. Bacitracin and methylamine, inhibitors of internalization, were effective in blocking degradation but not loss of membrane-bound [125I]hFSH. Thus it appears that, as with small peptide and simple protein hormones, complex glycoprotein hormone-receptor complexes, such as FSH Sertoli cell receptors, are endocytosed by normal target cells and subsequently degraded, most probably within the lysosomes.

Inhibition of FSH action on granulosa cells by low molecular weight components of follicular fluid.

Immature female rats were injected with a single dose (10 IU) of pregnant mare serum gonadotropin to induce growth and maturation of ovarian follicles. Using such ovaries as a model, we tested the effects of low molecular weight subfractions of charcoal-absorbed bovine follicular fluid (FF-c) on (a) radioiodinated human FSH (125I-hFSH) binding to ovarian homogenates, (b) ovine FSH-stimulated adenylate cyclase activity in granulosa cell homogenates and (c) cAMP production by intact granulosa cells. The follicular fluid was fractionated by ultrafiltration through membranes of differing pore-sized into molecular weight components of 1000-5000 (passing Amicon H1P-5 hollow fibers but retained by Amicon UM-2 membrane) and 500-1000 (passing Amicon UM-2 membrane but retained by Amicon Um-05 membrane). These low molecular weight fractions inhibited 125I-hFSH binding to ovarian receptors, FSH-stimulated cAMP production by rat granulosa cells and FSH-stimulated, as well as fluoride-ion-stimulated adenylate cyclase activity in granulosa cell homogenates. Inhibition of FSH-stimulated adenylate cyclase activity by the FF subfractions was non-competitive as determined by double reciprocal plot analysis. Our results suggest that modulation of FSH effects on granulosa cells may be mediated by low molecular weight constituents of follicular fluid.

The recombinant third domain of human alpha-fetoprotein retains the antiestrotrophic activity found in the full-length molecule

Previous studies have shown that alpha-fetoprotein (AFP) interferes with estrogen (E2)-stimulated growth, including E2-stimulated breast cancer growth. In an effort to localize the antiestrotrophic portion of the molecule, the C-terminal one-third (200 amino acids) of human AFP, known as Domain III, was produced in a baculovirus expression system as a fusion protein containing an amino terminal histidine tag. The histidine tag was included to facilitate purification by metal ion affinity chromatography. The purified recombinant Domain III fusion protein was functionally similar to full-length natural AFP isolated from human cord sera or from cultured human hepatoma cells (HepG2) in that they all produced significant and quantitatively similar inhibition of E2-stimulated growth of immature mouse uterus. Furthermore, the dose-response profiles of the recombinant Domain III AFP and natural full-length AFP were similar. Preincubation of either protein in a molar excess of E2 lowered the minimally effective antiestrotrophic dose and produced a difference spectrum consistent with a change in conformation. These findings indicate that the antiestrotrophic activity of AFP is contained within the third domain of the molecule, and they have obvious implications for the production of biologically active peptides derived from this portion of the AFP molecule.

Lead increases inositol 1,4,5-triphosphate levels but does not interfere with calcium transients in primary rat astrocytes

Alteration of receptor-mediated signal transduction pathways by inorganic lead (Pb) has been postulated to contribute to the neurotoxicity of this environmental toxicant, some of these effects involving astrocytes. As Pb is known to mimic Ca2+ in various biological systems or alter Ca(2+)-mediated cellular processes, we analyzed the effect of Pb exposure on alpha 1 receptor activated astrocytic phosphoinositide metabolism and Ca2+ responses in primary astrocyte cultures prepared from cerebral cortex of 1-day-old rats. Exposure to norepinephrine (NE; 10-100 microM) resulted in a significant increase in astrocytic inositol 1,4,5-trisphosphate levels, concomitant with an increase in intracellular Ca2+ levels. Fifteen minute exposure to Pb (10 microM lead acetate) significantly increased inositol 1,4,5-trisphosphate generation compared with controls, both in the presence and absence of NE. However, the inositol 1,4,5-trisphosphate-mediated Ca2+ transients following NE stimulation was unaltered in the presence of Pb (1-100 microM). NE-evoked intracellular Ca2+ responses, both in the presence and absence of extracellular Ca2+ did not differ between control and Pb-treated astrocytes. Additional studies failed to demonstrate the occurrence of Pb influx into astrocytes within the first 12 min of exposure such that Ca2+ responses would be directly affected. It therefore appears unlikely that astrotoxic effects of Pb are mediated via direct changes in intracellular Ca2+ transients.

Properties of Follicle Stimulating Hormone Binding Inhibitors Found in Physiological Fluids

Our interest in follicle stimulating hormone binding inhibitors (FSH-BI) stemmed from earlier studies on the presence of a low molecular weight testicular factor(s) in buffer or water extracts of mature rat testis which inhibited binding of 125I-FSH to gonadal receptors (Reichert and Abou-Issa, 1977). Shortly thereafter, we described the presence of similar factors in human serum (Reichert et al., 1979) and in bovine follicular fluid (Darga and Reichert, 1978). Most recently, low molecular weight components with FSH-BI activity have been detected in human seminal plasma (Dias et al., 1981). Our initial studies on these factors have been summarized in several reviews (Reichert, 1978; Reichert et al., 1981) and will not be discussed further at this time. Rather, we will present in this report a description of our latest efforts to characterize the FSH binding inhibitors found in human plasma and bovine follicular fluid.

BIOCHEMICAL PROPERTIES OF THE TESTICULAR FOLLITROPIN‐RECEPTOR SYSTEM*

The interaction of FSH with membrane receptors from rat and calf testis has been studied in some detail. The FSH receptor has been solubilized through use of the nonionic detergent Triton X-100 and highly purified by affinity chromatography on Affigel-10 coupled to ovine FSH. Hormone binding activity of the solubilized receptor has been preserved for extended periods through use of the structure-stabilizing agent glycerol. Other components of the FSH testes receptor system including the guanyl nucleotide binding protein and adenylate cyclase have been solubilized by nonionic detergents and also found to be stabilized by glycerol. FSH binding activity has been observed in testes cytosol and represents a putative class of receptors prepared from testes in the absence of detergent. The concentration of this buffer-soluble component decreased with age and increased concomitantly with loss of membrane receptors consequent to their down-regulation after administration of exogenous FSH. Phospholipids seem involved in the interaction of FSH with membrane-bound, detergent-solubilized, and buffer-soluble FSH binding activity. Phospholipids may maintain or stabilize a particular receptor conformation necessary for interaction with the hormone. A specific role for GTP seems indicated in regulation of FSH-stimulated adenylate cyclase activity in immature rat testis. Follitropin binding to testes receptor appears modulated by a variety of factors present in serum, testes extracts, follicular fluid, and seminal plasma, which are poorly understood at present. Inhibition of FSH binding by seminal plasma best-fit by a model proposing two hormone binding sites per receptor molecule, where binding to one site decreases the affinity of the other site for FSH. As a result of studies in this and other laboratories, the molecular endocrinology of FSH interaction with testis receptors is becoming increasingly understood.