Role of AMPA receptor endocytosis in synaptic plasticity.

Activity-mediated changes in the strength of synaptic communication are important for the establishment of proper neuronal connections during development and for the experience-dependent modification of neural circuitry that is believed to underlie all forms of behavioural plasticity. Owing to the wide-ranging significance of synaptic plasticity, considerable efforts have been made to identify the mechanisms by which synaptic changes are triggered and expressed. New evidence indicates that one important expression mechanism of several long-lasting forms of synaptic plasticity might involve the physical transport of AMPA-type glutamate receptors in and out of the synaptic membrane. Here, we focus on the rapidly accumulating evidence that AMPA receptors undergo regulated endocytosis, which is important for long-term depression.

Regulation of AMPA receptor endocytosis by a signaling mechanism shared with LTD.

The endocytosis of AMPA receptors is thought to be important in the expression of long-term depression (LTD) triggered by NMDA receptor activation. Although signaling pathways necessary for LTD induction have been identified, those responsible for the regulated internalization of AMPA receptors are unknown. Here we show that activation of NMDA receptors alone can trigger AMPA receptor endocytosis through calcium influx and activation of the calcium-dependent protein phosphatase calcineurin. A distinct signaling mechanism mediates the AMPA receptor endocytosis stimulated by insulin. These results demonstrate that although multiple signaling pathways can induce AMPA receptor internalization, NMDA receptor activation enhances AMPA receptor endocytosis via a signaling mechanism required for the induction of LTD.

NGF signals through TrkA to increase clathrin at the plasma membrane and enhance clathrin-mediated membrane trafficking.

Neurotrophin (NT) signals may be moved from axon terminals to neuron cell bodies via signaling endosomes-organelles in which NTs continue to be bound to their activated receptors. Suggesting that clathrin-coated membranes serve as one source of signaling endosomes, in earlier studies we showed that nerve growth factor (NGF) treatment increased clathrin at the plasma membrane and resulted in colocalization of clathrin with TrkA, the receptor tyrosine kinase for NGF. Strikingly, however, we also noted that most clathrin puncta at the surface of NGF-treated cells did not colocalize with TrkA, raising the possibility that NGF induces a general increase in clathrin-coated membrane formation. To explore this possibility further, we examined the distribution of clathrin in NGF- and BDNF-treated cells. NGF signaling in PC12 cells robustly redistributed the adaptor protein AP2 and the clathrin heavy chain (CHC) to surface membranes. Using confocal and epifluorescence microscopy, as well as biochemical assays, we showed the redistribution of clathrin to be attributable to the activation of TrkA. Significantly, NGF signaled through TrkA to induce an increase in clathrin-mediated membrane trafficking, as revealed in the increased endocytosis of transferrin. In that BDNF treatment increased AP2 and clathrin at the surface membranes of hippocampal neurons, these findings may represent a physiologically significant response to NTs. We conclude that NT signaling increases clathrin-coated membrane formation and clathrin-mediated membrane trafficking and speculate that this effect contributes to their trophic actions via the increased internalization of receptors and other proteins that are present in clathrin-coated membranes.

Gene transfer of endothelial nitric oxide synthase but not Cu/Zn superoxide dismutase restores nitric oxide availability in the SHRSP.

OBJECTIVE: Previous studies from our group have shown a deficit in nitric oxide (NO) bioavailability and an excess production of the superoxide anion (O(2)(-)) in the stroke prone spontaneously hypertensive rat (SHRSP) compared to the normotensive Wistar Kyoto (WKY) strain. This present study has investigated whether adenoviral-mediated gene transfer of human eNOS or Cu/ZnSOD can alter the NO/O(2)(-) balance, thereby improving endothelial function. METHODS: A recombinant adenovirus, Ad/Hu/eNOS, containing the human eNOS cDNA fragment was generated by homologous recombination in 293 cells. Ad/Hu/eNOS or Ad/Cu/ZnSOD was delivered into SHRSP carotid arteries in vivo, using a titre of 2x10(9)-2x10(10) plaque forming units (pfu)/ml, and the effect on gene expression was observed 24 h later. RESULTS: Western blotting confirmed increased enzyme levels of eNOS and Cu/ZnSOD in the viral-infused vessels. Ex vivo, the pressor response to phenylephrine (PE) in the presence of L-NAME was increased in the eNOS-infused arteries relative to the contralateral controls, indicating restoration of basal NO availability to that observed in untreated control WKY rats. Infusion of the SOD virus produced a statistically insignificant increase in NO bioavailability. CONCLUSIONS: Our results support our previous findings obtained using a bovine eNOS recombinant adenovirus, that recombinant adenoviral gene transfer of human eNOS has a significant effect on NO bioavailability. In contrast, AdCu/ZnSOD gene transfer does not elicit an effect in our model. These results indicate that short-term overexpression of a recombinant eNOS, but not Cu/ZnSOD gene, in carotid arteries of the SHRSP is an effective means of locally increasing NO bioavailability to improve endothelial function.

Role of AMPA receptor cycling in synaptic transmission and plasticity.

Compounds known to disrupt exocytosis or endocytosis were introduced into CA1 pyramidal cells while monitoring excitatory postsynaptic currents (EPSCs). Disrupting exocytosis or the interaction of GluR2 with NSF caused a gradual reduction in the AMPAR EPSC, while inhibition of endocytosis caused a gradual increase in the AMPAR EPSC. These manipulations had no effect on the NMDAR EPSC but prevented the subsequent induction of LTD. These results suggest that AMPARs, but not NMDARs, cycle into and out of the synaptic membrane at a rapid rate and that certain forms of synaptic plasticity may utilize this dynamic process.

Dynamin-dependent endocytosis of ionotropic glutamate receptors.

Little is known about the mechanisms that regulate the number of ionotropic glutamate receptors present at excitatory synapses. Herein, we show that GluR1-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) are removed from the postsynaptic plasma membrane of cultured hippocampal neurons by rapid, ligand-induced endocytosis. Although endocytosis of AMPARs can be induced by high concentrations of AMPA without concomitant activation of N-methyl-D-aspartate (NMDA) receptors (NMDARs), NMDAR activation is required for detectable endocytosis induced by synaptically released glutamate. Activated AMPARs colocalize with AP2, a marker of endocytic coated pits, and endocytosis of AMPARs is blocked by biochemical inhibition of clathrin-coated pit function or overexpression of a dominant-negative mutant form of dynamin. These results establish that ionotropic receptors are regulated by dynamin-dependent endocytosis and suggest an important role of endocytic membrane trafficking in the postsynaptic modulation of neurotransmission.

EGF receptor signaling stimulates SRC kinase phosphorylation of clathrin, influencing clathrin redistribution and EGF uptake.

Epidermal growth factor (EGF) binding to its receptor causes rapid phosphorylation of the clathrin heavy chain at tyrosine 1477, which lies in a domain controlling clathrin assembly. EGF-mediated clathrin phosphorylation is followed by clathrin redistribution to the cell periphery and is the product of downstream activation of SRC kinase by EGF receptor (EGFR) signaling. In cells lacking SRC kinase, or cells treated with a specific SRC family kinase inhibitor, EGF stimulation of clathrin phosphorylation and redistribution does not occur, and EGF endocytosis is delayed. These observations demonstrate a role for SRC kinase in modification and recruitment of clathrin during ligand-induced EGFR endocytosis and thereby define a novel effector mechanism for regulation of endocytosis by receptor signaling.

Endocytosis of activated TrkA: evidence that nerve growth factor induces formation of signaling endosomes.

The survival, differentiation, and maintenance of responsive neurons are regulated by nerve growth factor (NGF), which is secreted by the target and interacts with receptors on the axon tip. It is uncertain how the NGF signal is communicated retrogradely from distal axons to neuron cell bodies. Retrograde transport of activated receptors in endocytic vesicles could convey the signal. However, little is known about endocytosis of NGF receptors, and there is no evidence that NGF receptors continue to signal after endocytosis. We have examined early events in the membrane traffic of NGF and its receptor, gp140(TrkA) (TrkA), in PC12 cells. NGF induced rapid and extensive endocytosis of TrkA in these cells, and the receptor subsequently moved into small organelles located near the plasma membrane. Some of these organelles contained clathrin and alpha-adaptin, which implies that TrkA is internalized by clathrin-mediated endocytosis. Using mechanical permeabilization and fractionation, intracellular organelles derived from endocytosis were separated from the plasma membrane. After NGF treatment, NGF was bound to TrkA in endocytic organelles, and TrkA was tyrosine-phosphorylated and bound to PLC-gamma1, suggesting that these receptors were competent to initiate signal transduction. These studies raise the possibility that NGF induces formation of signaling endosomes containing activated TrkA. They are an important first step in elucidating the molecular mechanism of NGF retrograde signaling.

Persistent hypertension following inhibition of nitric oxide formation in the young Wistar rat: role of renin and vascular hypertrophy.

OBJECTIVE: To determine whether induction of arterial hypertension in young normotensive Wistar rats by chronic inhibition of nitric oxide production with NG-nitro-L-arginine methyl ester (L-NAME) produced a form of self-sustained hypertension, and to investigate the role of the renin-angiotensin system and vascular hypertrophy in the hypertensive process. METHODS: Three-week-old Wistar rats were given 100 or 40 mg/kg per day L-NAME or 40 mg/kg per day L-NAME plus 100 mg/kg per day captopril in their drinking water for between 4 and 7 weeks. Systolic blood pressure was measured by tail-cuff plethysmography both during treatment and after the treatment had been stopped. The effect of treatment on plasma renin was measured and the effect of treatment on mesenteric resistance artery structure was determined using a small-vessel myograph. RESULTS: L-NAME produced a progressive and marked increase in blood pressure during the period of treatment. Hypertension was sustained for 14 weeks after stopping treatment. L-NAME resulted in a fourfold increase in plasma renin which remained elevated after treatment was stopped. Blood pressure was correlated with plasma renin levels. Treatment with L-NAME plus captopril markedly attenuated the rise in blood pressure and captopril also produced a marked fall in blood pressure in rats that developed persistent hypertension. Rats with self-sustained hypertension exhibited both cardiac and mesenteric resistance vessel hypertrophy. The induction of vascular hypertrophy with low-dose L-NAME did not result in the development of self-sustained hypertension. CONCLUSIONS: Chronic L-NAME treatment in young rats can produce a form of persistent hypertension which is renin-dependent and which does not seem to involve a vascular amplifier mechanism.

Angiotensin II receptor antagonist losartan has persistent effects on blood pressure in the young spontaneously hypertensive rat: lack of relation to vascular structure.

The persistent effects on blood pressure of the angiotensin II receptor antagonist losartan and the converting enzyme inhibitor captopril were compared in the young spontaneously hypertensive rat (SHR). Losartan (DuP753/MK954, 15 mg/kg/day) and captopril (100 mg/kg/day) were given in the drinking water of 3-week-old SHRs for 4- and 10-week durations. Blood pressure was measured during treatment and after treatment was stopped until the age of 30 weeks. Both losartan and captopril given for 4 and 10 weeks prevented the development of hypertension during treatment and redevelopment of hypertension after treatment was stopped. Treatment for 10 weeks was more effective than for 4 weeks in lowering long-term pressure. Four weeks of treatment did not affect the mesenteric resistance artery media/lumen (m1/l1) ratio. In contrast, both losartan and captopril given for 10 weeks resulted in large and significant reductions in m1/l1 [5.3 +/- 0.8 and 5.63 +/- 0.8 vs 7.7 +/- 0.8 x 10(-2) (SD), p less than 0.001]. In losartan-treated rats, plasma renin and angiotensin II concentration were increased between 4- and 7-fold at the end of both treatment periods. These findings show losartan to be an effective antihypertensive agent and support data implicating angiotensin II in the early events leading to hypertension in this model. The abilities of losartan and captopril to affect blood pressure without affecting vascular structure suggest that the latter is a poor predictor of long-term hypertensive levels in the SHR.

Structural changes during the early onset of experimental hypertension: trophic influences of the renin-angiotensin system.

All forms of established hypertension are characterised by hypertrophy of the heart and blood vessels. Although these structural alterations are a normal adaptation to raised blood pressure, it has been suggested that hypertrophy may develop by a mechanism independent of pressure. Some investigators claim that in the young spontaneously hypertensive rat, hypertrophy of the blood vessels and heart is present at a time before the rise in pressure. Evidence also exists to support the role of angiotensin II in directly influencing growth within the vasculature. The present study confirms that hypertrophy of the mesenteric resistance vessels is present in the 3-week old spontaneously hypertensive rat. However, enhanced vascular structure was also associated with a higher mean arterial blood pressure. No increase in activity of the renin-angiotensin system could be detected. These observations raise doubts as to whether hypertrophy of the blood vessels can develop independently of raised pressure.

Vascular hypertrophy, renin and blood pressure in the young spontaneously hypertensive rat.

1. Cardiovascular reactivity, blood vessel morphology, blood pressure and the activity of the renin-angiotensin system were determined in the 3-week-old spontaneously hypertensive (SHR), Wistar-Kyoto (WKY) and outbred Wistar (WIS) rat. 2. In an isolated perfused mesenteric artery preparation the SHR had a significantly increased maximum response to KCl and noradrenaline (P less than 0.02) compared with the WKY. Using a myograph, vascular structure was measured over a range of resistance arteries and showed a significant correlation between lumen diameter and both media cross-sectional area and thickness, with the regression line for the SHR shifted upwards indicating both increased media area and thickness. This was associated with a slight, but significant, narrowing of the lumen (P less than 0.01) and an increased media/lumen ratio (0.049 +/- 0.01, 0.034 +/- 0.007, 0.036 +/- 0.008 for SHR, WKY and WIS, respectively, means +/- SD P less than 0.001). The SHR had a greater heart/body weight ratio than either the WKY or the WIS (P less than 0.001). 3. Both mesenteric artery and membrane protein content were higher in the SHR, indicating an increase in cell size or number. 4. Plasma renin activity (means +/- SD) was lower in the SHR (1.0 +/- 0.7 pmol of angiotensin I h-1 ml-1) than in the WKY (2.2 +/- 1.2 pmol of angiotensin I h-1 ml-1, P less than 0.001) but not different from that in the WIS (1.2 +/- 0.8 pmol of angiotensin I h-1 ml-1). Mesenteric artery vascular renin concentration was also lower in the SHR (P = 0.06).(ABSTRACT TRUNCATED AT 250 WORDS)

Hormone and electrolyte changes in post-deoxycorticosterone salt hypertension in rats.

Male Sprague-Dawley rats were uninephrectomized and given either deoxycorticosterone (DOC) pivalate (12.5 mg three times weekly) and 1% NaCl/0.2% KCl to drink for 4 weeks (DOC-treated), after which DOC was stopped and tap water substituted (post-DOC), or tap water to drink throughout (controls), DOC treatment increased blood pressure, serum sodium, plasma atrial natriuretic peptide (P-ANP) and plasma deoxycorticosterone (P-DOC) (P less than 0.05), while serum potassium, plasma renin and plasma angiotensin II were lower (P less than 0.05) than in control animals. Plasma vasopressin (P-AVP) was also raised but not significantly. These changes persisted for up to 4 weeks post-DOC and, in the case of plasma renin, plasma angiotensin II, P-AVP and P-ANP, for up to 12 weeks. Total body sodium was also increased at 2 weeks post-DOC (P less than 0.05). Rats which were adrenalectomized after 4 weeks of DOC treatment in which DOC injections were stopped, then drank either NaCl/KCl or tap water; blood pressure and P-DOC remained elevated while plasma renin remained suppressed. There were more deaths in rats given NaCl/KCl (five of six) than in the group given water (one of six). Rats treated with a subcutaneous DOC silastic implant had a comparable rise in blood pressure to rats given DOC injections. However, after removal of the implant, while blood pressure remained elevated, P-DOC levels were not raised and plasma renin rose to control levels after 4 weeks. These findings indicate that, in rats given DOC injections, post-DOC hypertension results from sodium and fluid retention as a consequence of chronic hangover of exogenously administered DOC.

Cleavage of amyloid beta peptide during constitutive processing of its precursor.

The amyloid beta peptide (A beta P) is a small fragment of the much larger, broadly distributed amyloid precursor protein (APP). Abundant A beta P deposition in the brains of patients with Alzheimer's disease suggests that altered APP processing may represent a key pathogenic event. Direct protein structural analyses showed that constitutive processing in human embryonic kidney 293 cells cleaves APP in the interior of the A beta P, thus preventing A beta P deposition. A deficiency of this processing event may ultimately prove to be the etiological event in Alzheimer's disease that gives rise to senile plaque formation.

The Alzheimer amyloid precursor protein. Identification of a stable intermediate in the biosynthetic/degradative pathway.

The amyloid forming beta-peptide of Alzheimer's disease is synthesized as part of a larger integral membrane precursor protein (beta APP) of which three alternatively spliced versions of 695, 751, and 770 amino acids have been described. A fourth beta APP form of 563 amino acids does not contain the beta-peptide region. Recent experiments using transient expression in HeLa cells (Weidemann, A., Konig, G., Bunke, D., Fischer, P., Salbaum, J.M., Masters, C.L., and Beyreuther, K. (1989) Cell 57, 115-126) indicate that the beta APP undergoes several posttranslational modifications including the cleavage and secretion of a large portion of its extracellular domain. The nature and fate of the fragment that remains cell-associated following this cleavage has not heretofore been described. The metabolism of this fragment may have particular significance in Alzheimer's disease since it must contain at least part of the beta-peptide. To study the metabolic fate of this fragment, we have established cell lines overexpressing the 695- and 751-amino acid versions of beta APP. Pulse-chase studies show that this system is similar to the HeLa cell system in that both proteins are synthesized first as membrane-bound proteins of approximately 98 and 108 kDa carrying asparagine-linked sugar side chains and are subsequently processed into higher molecular mass forms by the attachment of sulfate, phosphate, and further sugar groups including sialic acid, adding approximately 20 kDa in apparent molecular mass. The mature form of beta APP is cleaved and rapidly secreted, leaving an 11.5-kDa fragment with the transmembrane region and the cytoplasmic domain behind in the cell. This fragment is stable with a half-life of at least 4 h.

Inhibitors of rat renin and their use in experimental hypertension.

Hydroxy-ethylene dipeptide analogues (Leu[CH(OH)-CH2]Leu and Leu[CH(OH)-CH2]Val) of human substrate peptides are potent in vitro inhibitors of rat renin with IC50 values as low as 0.8 nmol/l. When given to renal hypertensive rats they lower blood pressure and suppress both plasma renin and angiotensin II. There was a divergence between the rapid rebound of renin and blood pressure which remained suppressed.

The secreted form of the Alzheimer's amyloid precursor protein with the Kunitz domain is protease nexin-II.

The A4 protein (or beta-protein) is a 42- or 43-amino-acid peptide present in the extracellular neuritic plaques in Alzheimer's disease and is derived from a membrane-bound amyloid protein precursor (APP). Three forms of APP have been described and are referred to as APP695, APP751 and APP770, reflecting the number of amino acids encoded for by their respective complementary DNAs. The two larger APPs contain a 57-amino-acid insert with striking homology to the Kunitz family of protease inhibitors. Here we report that the deduced amino-terminal sequence of APP is identical to the sequence of a cell-secreted protease inhibitor, protease nexin-II (PN-II). To confirm this finding, APP751 and APP695 cDNAs were over-expressed in the human 293 cell line, and the secreted N-terminal extracellular domains of these APPs were purified to near homogeneity from the tissue-culture medium. The relative molecular mass and high-affinity binding to dextran sulphate of secreted APP751 were consistent with that of PN-II. Functionally, secreted APP751 formed stable, non-covalent, inhibitory complexes with trypsin. Secreted APP695 did not form complexes with trypsin. We conclude that the secreted form of APP with the Kunitz protease inhibitor domain is PN-II.

Atrial natriuretic peptide vascular receptor: regulation of low-affinity binding by calcium.

1. In the presence of calcium, both high- [dissociation equilibrium constant (Kd) 5.8 x 10(-11) mol/l] and low- (Kd 1.2 x 10(-8) mol/l) affinity binding of atrial natriuretic peptide was detected on plasma membranes prepared from sodium-depleted rats. 2. In contrast, under calcium-free conditions only high-affinity binding (Kd 6.9 x 10(-11) mol/l) was detected. 3. The requirement for calcium for low-affinity binding suggests a different mechanism of action than that for the high-affinity receptor.