ADP-ribosylation factor 6 regulates actin cytoskeleton remodeling in coordination with Rac1 and RhoA.

In this study, we have documented an essential role for ADP-ribosylation factor 6 (ARF6) in cell surface remodeling in response to physiological stimulus and in the down regulation of stress fiber formation. We demonstrate that the G-protein-coupled receptor agonist bombesin triggers the redistribution of ARF6- and Rac1-containing endosomal vesicles to the cell surface. This membrane redistribution was accompanied by cortical actin rearrangements and was inhibited by dominant negative ARF6, implying that bombesin is a physiological trigger of ARF6 activation. Furthermore, these studies provide a new model for bombesin-induced Rac1 activation that involves ARF6-regulated endosomal recycling. The bombesin-elicited translocation of vesicular ARF6 was mimicked by activated Galphaq and was partially inhibited by expression of RGS2, which down regulates Gq function. This suggests that Gq functions as an upstream regulator of ARF6 activation. The ARF6-induced peripheral cytoskeletal rearrangements were accompanied by a depletion of stress fibers. Moreover, cells expressing activated ARF6 resisted the formation of stress fibers induced by lysophosphatidic acid. We show that the ARF6-dependent inhibition of stress fiber formation was due to an inhibition of RhoA activation and was overcome by expression of a constitutively active RhoA mutant. The latter observations demonstrate that activation of ARF6 down regulates Rho signaling. Our findings underscore the potential roles of ARF6, Rac1, and RhoA in the coordinated regulation of cytoskeletal remodeling.

A role for POR1, a Rac1-interacting protein, in ARF6-mediated cytoskeletal rearrangements.

The ARF6 GTPase, the least conserved member of the ADP ribosylation factor (ARF) family, associates with the plasma membrane and intracellular endosome vesicles. Mutants of ARF6 defective in GTP binding and hydrolysis have a marked effect on endocytic trafficking and the gross morphology of the peripheral membrane system. Here we report that expression of the GTPase-defective mutant of ARF6, ARF6(Q67L), remodels the actin cytoskeleton by inducing actin polymerization at the cell periphery. This cytoskeletal rearrangement was inhibited by co-expression of ARF6(Q67L) with deletion mutants of POR1, a Rac1-interacting protein involved in membrane ruffling, but not with the dominant-negative mutant of Rac1, Rac1(S17N). A synergistic effect between POR1 and ARF6 for the induction of actin polymerization was detected. Furthermore, we observed that ARF6 interacts directly with POR1 and that this interaction was GTP dependent. These findings indicate that ARF6 and Rac1 function on distinct signaling pathways to mediate cytoskeletal reorganization, and suggest a role for POR1 as an important regulatory element in orchestrating cytoskeletal rearrangements at the cell periphery induced by ARF6 and Rac1.

Monensin inhibits recycling of macrophage mannose-glycoprotein receptors and ligand delivery to lysosomes.

Binding studies with cells that had been permeabilized with saponin indicate that alveolar macrophages have an intracellular pool of mannose-specific binding sites which is about 4-fold greater than the cell surface pool. Monensin, a carboxylic ionophore which mediates proton movement across membranes, has no effect on binding of ligand to macrophages but blocks receptor-mediated uptake of 125I-labelled beta-glucuronidase. Inhibition of uptake was concentration- and time-dependent. Internalization of receptor-bound ligand, after warming to 37 degrees C, was unaffected by monensin. Moreover, internalization of ligand in the presence of monensin resulted in an intracellular accumulation of receptor-ligand complexes. The monensin effect was not dependent on the presence of ligand, since incubation of macrophages with monensin at 37 degrees C without ligand resulted in a substantial decrease in cell-surface binding activity. However, total binding activity, measured in the presence of saponin, was much less affected by monensin treatment. Removal of monensin followed by a brief incubation at pH 6.0 and 37 degrees C, restored both cell-surface binding and uptake activity. Fractionation experiments indicate that ligands enter a low-density (endosomal) fraction within the first few minutes of uptake, and within 20 min transfer to the lysosomal fraction has occurred. Monensin blocks the transfer from endosomal to lysosomal fraction. Lysosomal pH, as measured by the fluorescein-dextran method, was increased by monensin in the same concentration range that blocked ligand uptake. The results indicate that monensin blockade of receptor-mediated endocytosis of mannose-terminated ligands by macrophages is due to entrapment of receptor-ligand complexes and probably receptors in the pre-lysosomal compartment. The inhibition is linked with an increase in the pH of acid intracellular vesicles.

Quantitative histochemical studies of the hypothalamus: dehydrogenase enzymes following androgen sterilization.

Four enzymes, selected as representative of major metabolic pathways (malic dehydrogenase, of the citric acid cycle, lactic dehydrogenase, of glycolysis, glucose-6-phosphate dehydrogenase, of the pentose pathway and glutamic dehydrogenase, of glutamate metabolism), were measured by quantitative histochemical methods in individual hypothalamic nuclei of adult neonatally androgenized female rats. Malic dehydrogenase (MDH) was significantly reduced in nuclei of the anterior hypothalamus: the suprachiasmatic, supraoptic and anterior. Lactic dehydrogenase (LDH) increased significantly in the lateral preoptic area. Glucose-6-phosphate dehydrogenase G-6-PDH was also significantly elevated in anterior hypothalamic nuclei: medial preoptic, lateral preoptic, supraoptic and paraventricular. Glutamic dehydrogenase (GDH) was generally elevated throughout the hypothalamus with significant increases of activity occurring in the paraventricular, lateral ventromedial, arcuate, medial mamillary and posterior nuclei.

PIP: As part of a detailed analysis of the specific enzyme metabolism in individual hypothalamic nuclei during different endocrinological and behavioral states, quantitative distribution of a group of enzymes representative of major metabolic pathways was examined. Malic dehydrogenase (MDH), representative of the citric acid cycle, lactic dehydrogenase (LDH), of glycolysis, glutamic dehydrogenase (GDH), of glutamate metabolism, and glucoseo-6-phosphate dehydrogenase (G-6-PDH), of the pentose pathway, were measured in 11 hypothalamic nuclei, the cerebral cortex, and the cerebellum of adult female rats neonatally treated with testosterone propionate (TP). Several significant metabolic changes occurred in specific hypothalamic nuclei following neonatal TP (1 mg) treatment. MDH activity was significantly reduced in the suprachiasmatic (11%), supraoptic (13%), and anterior (9%) nuclei. No statistically significant changes occurred in nuclei of the middle or posterior hypothalamus. LDH was significantly elevated only in the lateral preoptic areas (23%). Several significant increases of G-6-PDH activity occurred in the following nuclei of the anterior hypothalamus: medial preoptic (32%), lateral preoptic (33%), supraoptic (13%), and paraventricular (23%). No statistically significant changes occurred in nuclei of the middle or posterior hypothalamus; these results were similar to those for MDH and LDH. GDH activity was generally elevated in all of the hypothalamic nuclei examined, except in the anterior nucleus. Significant increases of enzyme level were found in each of the major divisions of the hypothalamus. In the anterior hypothalamus, GDH activity in the paraventricular nucleus rose significantly (16%); in the middle hypothalamus, lateral ventromedial and arcuate nuclear levels were elevated (14 and 17%), and medial and posterior nuclear levels were higher than control values (32 and 36%) in the posterior hypothalamus.

Quantitative histochemical studies of the hypothalamus. Control point enzymes following androgen sterilization.

Three control point enzymes of the Embden-Meyerhof pathway, hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK) were measured by quantitative histochemical methods in individual hypothalamic nuclei of adult neonatally androgenized female rats. HK activity was significantly increased in anterior hypothalamic nuclei: medial preoptic, lateral preoptic, and suprachiasmatic. PK was significantly elevated in the lateral preoptic and suprachiasmatic nuclei of the anterior hypothalamus and also in the medial mamillary nucleus and median eminence. No significant changes occurred in PFK activity.

Quantitative histochemical studies of the hypothalamus. Control point enzymes during the estrous cycle.

Three control point enzymes of the Embden-Meyerhof pathway, hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK), have been measured in individual hypothalamic nuclei during the 5-day estrous cycle of adult rats by quantitative histochemical methods. PK levels, low during proestrus, rise to maximum activity during estrus; this rise is significantly greater than on all other days of the cycle in the lateral preoptic area (LP), ventromedial pars medialis (VMM) and pars lateralis (VML) and posterior hypothalamic (Post) nuclei. HK activity also rises from low proestrous levels during the cycle but, in contrast to PK, reaches maximum activity during diestrus-1 (D-1) or diestrus-2 (D-2). PFK showed variable changes during the estrous cycle with peaks occurring during estrus in some nuclei and during diestrus in others, but these changes were not significantly different. These metabolic changes occur in specific hypothalamic nuclei which have been shown by electrical stimulation, lesion production, stereotaxic hormone implantation and localization of luteinizing hormone-releasing factor experiments to have an important role in reproductive physiology and sexual behavior.

Quantitative histochemical studies of the hypothalamus. Dehydrogenase enzymes during the estrous cycle.

Three enzymes selected as representative of major metabolic pathways (malic dehydrogenase, of the citric acid cycle, lactic dehydrogenase, of glycolysis and glucose-6-phosphate dehydrogenase, of the pentose pathway) were measured by quantitative histochemical methods in individual hypothalamic nuclei during the 5-day estrous cycle of adult rats. Malic dehydrogenase increases significantly from low proestrous levels to a peak at estrus and then declines during diestrus in the following nuclei and areas of the anterior hypothalamus: medial and lateral preoptic, suprachiasmatic, supraoptic, and anterior. Significant peaks of lactic dehydrogenase occur more often during diestrus-3 in hypothalamic nuclei of the middle and posterior hypothalamus, Glucose-6-phosphate dehydrogenase has a biphasic pattern with peaks usually occurring during the diestrous period.