CD36 Mediates the In Vitro Inhibitory Effects of Thrombospondin-1 on Endothelial Cells

Thrombospondin-1 (TSP-1) is a naturally occurring inhibitor of angiogenesis that is able to make normal endothelial cells unresponsive to a wide variety of inducers. Here we use both native TSP-1 and small antiangiogenic peptides derived from it to show that this inhibition is mediated by CD36, a transmembrane glycoprotein found on microvascular endothelial cells. Both IgG antibodies against CD36 and glutathione-S-transferase–CD36 fusion proteins that contain the TSP-1 binding site blocked the ability of intact TSP-1 and its active peptides to inhibit the migration of cultured microvascular endothelial cells. In addition, antiangiogenic TSP-1 peptides inhibited the binding of native TSP-1 to solid phase CD36 and its fusion proteins, as well as to CD36-expressing cells. Additional molecules known to bind CD36, including the IgM anti-CD36 antibody SM, oxidized (but not unoxidized) low density lipoprotein, and human collagen 1, mimicked TSP-1 by inhibiting the migration of human microvascular endothelial cells. Transfection of CD36-deficient human umbilical vein endothelial cells with a CD36 expression plasmid caused them to become sensitive to TSP-1 inhibition of their migration and tube formation. This work demonstrates that endothelial CD36, previously thought to be involved only in adhesion and scavenging activities, may be essential for the inhibition of angiogenesis by thrombospondin-1.     

Elevation of Basal Protein Kinase C Activity Increases Ethanol Sensitivity of GABAA Receptors in Rat Hippocampal CA1 Pyramidal Neurons

Abstract: The ability of ethanol to enhance GABA_A receptor function remains controversial; conflicting observations have been made even in the same brain region, and when using apparently similar methodologies. In this study we characterized a single protocol variable, the initial incubation temperature of brain slices, that had dramatic effects on the ethanol sensitivity of GABA_A inhibitory postsynaptic currents (IPSCs) recorded from rat hippocampal CA1 pyramidal neurons. Incubation of hippocampal slices at relatively low temperatures (11–15°C) immediately after slice preparation significantly affected a number of physiological and biochemical parameters. Such slices showed a decrease in extracellular inhibitory postsynaptic potential amplitude, a significant increase in the ethanol sensitivity of GABA_A IPSCs in CA1 pyramidal neurons, no change in pentobarbital or flunitrazepam potentiation of IPSCs, and an increase in basal protein kinase C (PKC) activity relative to slices incubated at 31–33°C. In addition, the increase in ethanol sensitivity of GABA_A IPSCs was blocked by chelerythrine, a selective inhibitor of PKC. These results suggest that differences in hippocampal slice incubation protocols may have contributed to the disparate results of previous investigations of ethanol modulation of GABA_A receptor-mediated synaptic transmission in the rat hippocampus. In addition, these findings provide further evidence that PKC activity positively modulates the interaction between ethanol and GABA_A receptors in the mammalian brain.     

Respiratory-related neurons of the fastigial nucleus in response to chemical and mechanical challenges

Xu, Fadi, and Donald T. Frazier. Respiratory-relatedneurons of the fastigial nucleus in response to chemical and mechanical challenges. J. Appl. Physiol. 82(4):1177-1184, 1997.Responses of cerebellar respiratory-relatedneurons (CRRNs) within the rostral fastigial nucleus and the phrenicneurogram to activation of respiratory mechano- and chemoreceptors wererecorded in anesthetized, paralyzed, and ventilated cats. Respiratorychallenges included the following: 1) cessation of the ventilator for asingle breath at the end of inspiration (lung inflation) or atfunctional residual capacity, 2)cessation of the ventilator for multiple breaths, and3) exposure to hypercapnia. NineteenCRRNs having spontaneous activity during control conditions werecharacterized as either independent (basic, n = 14) or dependent (pump,n = 5) on the ventilator movement. Thirteen recruited CRRNs showed no respiratory-related activity untilbreathing was stressed. Burst durations of expiratory CRRNs wereprolonged by sustained lung inflation but were inhibited when thevolume was sustained at functional residual capacity; it was vice versafor inspiratory CRRNs. Multiple-breath cessation of the ventilator andhypercapnia significantly increased the firing rate and/orburst duration concomitant with changes noted in the phrenic neurogram.We conclude that CRRNs respond to respiratory inputs fromCO₂ chemo- and pulmonarymechanoreceptors in the absence of skeletal muscle contraction.

     

pH-Dependent self-association of the Src homology 2 (SH2) domain of the Src homologous and collagen-like (SHC) protein.

The Src homologous and collagen-like (SHC) protein plays an essential role in signal transduction pathways in that it participates in the chain of events that leads to the activation of the protein Ras. The crystal structure of the SH2 domain of SHC has been determined using the method of multiple isomorphous replacement at a resolution of 2.5 A. The SH2 domain of SHC is similar in fold to other SH2 domains. The peptide-binding surfaces resemble that of the SH2 domain of Src in that a deep pocket is formed where the third amino acid C-terminal to the phosphotyrosine can insert. A novel feature of this structure is the observation of a disulfide bond and an extensive dimer interface between two symmetry-related molecules. Solution studies under reducing conditions using analytical centrifugation and PAGE suggest that the SH2 domain of SHC dimerizes in a pH-dependent manner where low pH conditions (approximately 4.5) are conducive to dimer formation. Dimerization of SHC may have important biological implications in that it may promote the assembly of large heteromultimeric signaling complexes.     

The role of the adrenal gland in control of H+ and NH4+ excretion by toad urinary bladder

The urinary bladder of Bufo marinus has been shown to excrete H+ and NH4+ and this excretion is increased by metabolic acidosis. The involvement of the adrenal gland and its steroid secretions in the adaptation for increased acid and ammonia excretion by the bladder was tested during the course of this study. Groups of toads were adrenalectomized and maintained in chronic NH4Cl-induced acidosis. Three other groups of toads were adrenalectomized and put in acidosis but repleted with 2.5 mg/day of either cortisol (CT), dexamethasone (Dexa), or deoxycorticosterone acetate (DOCA). All control groups were sham-operated. The bladders were excised after 3 days and mounted between 2-ml Lucite chambers. Net H+ and NH4+ fluxes into the mucosal media were measured and reported in units of nanomoles per 100 mg bladder per minute. In control acidotic toads H+ excretion was 20.1 +/- 2.0 and the adrenalectomized nonreplete group H+ excretion was 14.2 +/- 1.87 (P less than 0.04). For the same groups NH4+ excretion was 2.90 +/- 0.26 for the controls and 1.38 +/- 0.19 for the adrenalectomized (P less than 0.001). The H+ excretion in CT-, Dexa-, and DOCA-repleted toads was not significantly different from the control group. NH4+ excretion, however, showed a 55% decrease (P less than 0.001) in the CT group, and a 45% decrease (P less than 0.05) in the Dexa group. The NH4+ excretion in the DOCA repleted group was significantly different from the control group. Therefore, we conclude that the adrenal gland plays a role in the adaptive increase of H+ and NH4+ excretion by the urinary bladder in acidosis through the secretion of steroid hormones. The increase in NH4+ excretion appears to be a mineralocorticoid-stimulated process. We were not able to determine in this study if the steroid hormones had an exacting regulatory role or one of a permissive role over H+ and NH4+ excretion in the toad urinary bladder.     

Monoclonal antibodies that recognize calcium-dependent structures of human thrombospondin. Characterization and mapping of their epitopes

Monoclonal antibodies (mAbs) raised against reduced and alkylated thrombospondin (TSP) were screened for the ability to react with Ca2+-replete TSP versus EDTA-treated TSP. Two mAbs designated A6.1 and D4.6 were found to react much more strongly with TSP after EDTA treatment. The dissociation constants for these mAbs were measured in 5 mM EDTA and found to be 6 X 10(-10) M for A6.1 and 7 X 10(-9) M for D4.6. Binding to A6.1 was undetectable in the presence of 1 mM Ca2+ while binding of D4.6 occurred with about 100-fold lower affinity. The Ca2+ concentration dependence of A6.1 binding was broad with a midpoint near 50 microM free Ca2+ while that of D4.6 showed a sharp transition below 0.1 microM. Upon dialysis of EDTA-treated TSP into Ca2+ containing buffer, the binding of the mAbs was prevented or decreased, indicating reversibility of the conformational transition induced by the initial removal of Ca2+ . Mg2+ can compete with the Ca2+ binding sites involved in mAb binding, but TSP dialyzed from Ca2+ into Mg2+ binds the two mAbs as well as EDTA-treated TSP, indicating that Mg2+ cannot maintain the Ca2+-replete structure of TSP. The proteolytic fragments of TSP with which the two mAbs react were determined by probing Western blots of digests of TSP with the mAbs. A6.1 reacts with the 70-kDa fragment generated by chymotrypsin in EDTA which contains the interchain disulfide bonds of TSP and the binding site(s) for type V collagen (Mumby, S. M., Raugi, G. J., and Bornstein, P. (1984) J. Cell Biol. 98, 646-652). D4.6 reacts with fragments of 140 and 120 kDa found in digests of Ca2+-replete TSP which are absent from digests in EDTA. Electron microscopy of rotary shadowed, carbon-coated replicas of TSP mAb complexes confirms the Ca2+ sensitivity of mAb binding and has been used to localize the epitopes for both mAbs on the three-dimensional structure of TSP.     

Exploitation of marine turtles in the Indian Ocean

Marine turtles long have been of great value to peoples of the Indian Ocean, nutritionally, economically, and culturally. Once directed primarily toward subsistence, the hunting of marine turtles for international trade has increased; today their populations are often so depleted that they are not only insignificant as resources, but are endangered. An understanding of exploitation is imperative to guarantee future populations, yet available information is sketchy. Subsistence hunting is an ambiguous term, since the most intense exploitation is for export. Historically this has involved Chelonia and Eretmochelys, whose populations are now much reduced. Yet, newly discovered populations (Lepidochelys especially) are being exploited, under the stimulus of new foreign markets (e.g., leather), and their fates seem even less hopeful than those of long-exploited populations. Moreover subsistence hunting for immediate local consumption has led to depletion of nesting and feeding populations of turtles in areas where protein sources are in great demand and human population densities high. Neither the future nor the solution to this dilemma is clear, but it is obvious that economic considerations must be carefully considered, and ecological arguments alone are insufficient to manage these resources.