Biomedical subjects

H Bito

Publications and source records attributed to H Bito.

At least 19 recordsLinked to original sources

PubMed2003

Effect of the neuronal nitric oxide synthase inhibitor 7-nitroindazole on the righting reflex ED50 and minimum alveolar concentration during sevoflurane anaesthesia in rats.

BACKGROUND AND OBJECTIVE: The aim was to determine the effect of acute and chronic administration of 7-nitroindazole, a selective neuronal nitric oxide synthase inhibitor, on the righting reflex ED50 and the minimum alveolar concentration during sevoflurane anaesthesia in rats. METHODS: 7-Nitroindazole was acutely (0, 50 and 100 mg kg(-1)) and chronically (0 and 150 mg kg(-1) day(-1), 4 days) administered to rats. After the preparation, the minimum alveolar concentration and the righting reflex ED50 were measured. The concentration of cGMP in the brain, cerebellum and spinal cord was also measured. RESULTS: Acute administration reduced the minimum alveolar concentration (50 mg kg(-1), 58.8% (95% CI: 50.3-67.3%) of the baseline value, P < 0.01; 100 mg kg(-1), 55.8 (46.9-64.7), P < 0.01) and the righting reflex ED50 (50 mg kg(-1), 27.2 (17.2-37.2), P < 0.01; 100 mg kg(-1), 14.3 (6.6-22.0), P < 0.01). Chronic administration did not reduce the minimum alveolar concentration; however, it reduced the righting reflex ED50 (65.3 (52.9-77.7), P < 0.01). Overall, the reduction in minimum alveolar concentration in the acute and chronic protocol did not correlate with that of the righting reflex ED50. 7-Nitroindazole (100 mg kg(-1), acute) reduced the cGMP concentration within the cerebellum by 55.4%; however, it did not decrease concentrations in the brain or spinal cord. CONCLUSIONS: Different mechanisms are responsible for the observed alterations to the minimum alveolar concentration and the righting reflex ED50 following treatment with 7-nitroindazole. The nitric oxide-cGMP pathway might play a less important role in the determination of minimum alveolar concentration than the righting reflex ED50.

Anesthetics, Inhalation↗

PubMed2002

Vasopressin and epinephrine are equally effective for CPR in a rat asphyxia model.

Epinephrine has been administered as a drug essential for cardiopulmonary resuscitation (CPR). Recently, vasopressin has been reported to be more effective than epinephrine for CPR in a ventricular fibrillation (VF) model. As a different myocardial pathology is speculated to exist between the VF model and the asphyxia model, we investigated whether vasopressin is also effective in a rat asphyxia model. Twenty-one Sprague-Dawley male rats were divided into three groups: vasopressin 0.8 U/kg (Vaso-Gr), epinephrine 0.05 mg/kg (Epi-Gr), and saline same volume as the other two drugs (Sal-Gr). Five minutes after suffocation induced by obstruction of the tracheal tube, CPR was performed using each drug. Although only one animal survived (17%) in the Sal-Gr, 6/7 (85%) survived in both Vaso-Gr and Epi-Gr (P<0.01). Vasopressin is as effective as epinephrine for CPR in asphyxia-induced rats.

Animals↗

PubMed2002

Vasopressin with delayed combination of nitroglycerin increases survival rate in asphyxia rat model.

Recently, vasopressin has been reported as a more effective drug than epinephrine (adrenaline) for cardiopulmonary resuscitation (CPR). However, vasopressin decreases myocardial blood flow (MBF) because of its strong vasoconstriction, to maintain better coronary perfusion pressure (CPP) compared with epinephrine. Nitroglycerin is well known to be able to maintain MBF and increase survival rate. In a VF model, vasopressin combined with nitroglycerin maintained CPP; however, low survival rates were observed compared with vasopressin alone. We investigated the effectiveness of the delayed use of nitroglycerin combined with vasopressin in a severe asphyxia model. Fourteen Sprague-Dawley male rats were divided into two groups: vasopressin 0.8 U/kg alone (V-Gr.), and nitroglycerin 0.3 microg/kg 45 s after the administration of 0.8 U/kg vasopressin (VN-Gr.). Six min after asphyxia induced by obstructing the tracheal tube, CPR was performed in two ways. Three animals resuscitated in the V-Gr. (42%) and six/seven (84%) in the VN-Gr. (P<0.05). In the 6 min of asphyxia rat model, vasopressin combined with delayed nitroglycerin is more effective than vasopressin alone.

Animals↗

PubMed2001

Phosphatidylinositol 4,5-bisphosphate induces actin stress-fiber formation and inhibits membrane ruffling in CV1 cells.

Phosphatidylinositol 4,5 bisphosphate (PIP(2)) is widely implicated in cytoskeleton regulation, but the mechanisms by which PIP(2) effect cytoskeletal changes are not defined. We used recombinant adenovirus to infect CV1 cells with the mouse type I phosphatidylinositol phosphate 5-kinase alpha (PIP5KI), and identified the players that modulate the cytoskeleton in response to PIP(2) signaling. PIP5KI overexpression increased PIP(2) and reduced phosphatidylinositol 4 phosphate (PI4P) levels. It promoted robust stress-fiber formation in CV1 cells and blocked PDGF-induced membrane ruffling and nucleated actin assembly. Y-27632, a Rho-dependent serine/threonine protein kinase (ROCK) inhibitor, blocked stress-fiber formation and inhibited PIP(2) and PI4P synthesis in cells. However, Y-27632 had no effect on PIP(2) synthesis in lysates, although it inhibited PI4P synthesis. Thus, ROCK may regulate PIP(2) synthesis by controlling PI4P availability. PIP5KI overexpression decreased gelsolin, profilin, and capping protein binding to actin and increased that of ezrin. These changes can potentially account for the increased stress fiber and nonruffling phenotype. Our results establish the physiological role of PIP(2) in cytoskeletal regulation, clarify the relation between Rho, ROCK, and PIP(2) in the activation of stress-fiber formation, and identify the key players that modulate the actin cytoskeleton in response to PIP(2).

ADP Ribose Transferases↗

PubMed2001

Calcium/calmodulin-dependent protein kinase type IV (CaMKIV) inhibits apoptosis induced by potassium deprivation in cerebellar granule neurons.

The neuroprotective mechanisms of the Ca2+/calmodulin kinase (CaMK) signaling pathway were studied in primary cerebellar neurons in vitro. When switched from depolarizing culture conditions HK (extracellular K+ 30 mM) to LK (K+ 5 mM), these neurons rapidly undergo nuclear fragmentation, a typical feature of apoptosis. We present evidence that blockade of L-type Ca2+ channels (nifedipine sensitive) but not N/P/Q-type Ca2+ channels (omega-conotoxin MVIIC sensitive) triggered apoptosis and CPP32/caspase-3-like activity. The entry into apoptosis was associated with a progressive caspase-3-dependent cleavage of CaMKIV, but not of CaMKII. CaMKIV function in neuronal apoptosis was further investigated by overexpression of CaMKIV mutants by gene transfer. A dominant-active CaMKIV mutant inhibited LK-induced apoptosis whereas a dominant-negative form induced apoptosis in HK, suggesting that CaMKIV exerts neuroprotective effects. The transcription factor CREB is a well-described nuclear target of CaMKIV in neurons. When switched to LK, the level of phosphorylation of CREB, after an initial drop, further declined progressively with kinetics comparable to those of CaMKIV degradation. This decrease was abolished by caspase-3 inhibitor. These data are compatible with a model where Ca2+ influx via L-type Ca2+ channels prevents caspase-dependent cleavage of CaMKIV and promotes neuronal survival by maintaining a constitutive level of CaMKIV/CREB-dependent gene expression.

Animals↗

PubMed2001

Pax6 regulates granule cell polarization during parallel fiber formation in the developing cerebellum.

The molecular mechanisms that govern the coordinated programs of axonogenesis and cell body migration of the cerebellar granule cell are not well understood. In Pax6 mutant rats (rSey2/rSey2), granule cells in the external germinal layer (EGL) fail to form parallel fiber axons and to migrate tangentially along these fibers despite normal expression of differentiation markers. In culture, mutant cells sprout multiple neurites with enlarged growth cones, suggesting that the absence of Pax6 function perturbs cytoskeletal organization. Some of these alterations are cell-autonomous and rescuable by ectopic expression of Pax6 but not by co-culture with wild-type EGL cells. Cell-autonomous control of cytoskeletal dynamics by Pax6 is independent of the ROCK-mediated Rho small GTPase pathway. We propose that in addition to its roles during early patterning of the CNS, Pax6 is involved in a novel regulatory step of cytoskeletal organization during polarization and migration of CNS neurons.

Animals↗

PubMed2000

Citron, a Rho target that affects contractility during cytokinesis.

The small GTPase Rho, which regulates cell shape, is thought to contribute to cytokinesis. Recently, Citron was characterized as a Rho target. This large protein contains a Ser/Thr kinase domain related to that of ROCK, another Rho effector. Both endogenous Citron and recombinant Citron localize to the cleavage furrow in dividing cells and to the midbody in post-mitotic cells. Moreover, overexpression of Citron deleted from its C-terminal sequence caused abnormal contractions specifically during cytokinesis, resulting in the formation of multinucleated cells. Cell shape, F-actin, intermediate filaments, and microtubules appeared essentially normal in these cells during interphase. Thus, Citron is a Rho effector that appears to function during cytokinesis, modulating its contractile process. In brain, however, Citron is highly expressed in a subset of neurons as a brain-specific isoform that lacks a kinase domain, Citron-N. This protein accumulates in synapses and associates to the NMDA receptor via interaction with the adaptor protein PSD95, suggesting that the function of Citron is specialized in the neurons.

Animals↗

PubMed2000

Critical dependence of cAMP response element-binding protein phosphorylation on L-type calcium channels supports a selective response to EPSPs in preference to action potentials.

Activity-dependent gene expression in neurons shows a remarkable ability to differentiate between different types of stimulation: orthodromic inputs that engage synaptic transmission are much more effective than antidromic stimuli that do not. We have studied the basis of such selectivity in cultured hippocampal neurons in which nuclear cAMP response element-binding protein (CREB) phosphorylation is induced by synaptic activity but not by action potential (AP) stimulation in the absence of EPSPs, although spikes by themselves generate large elevations in intracellular Ca(2+). Previous work has shown that Ca(2+) entry through L-type Ca(2+) channels plays a dominant role in triggering calmodulin mobilization and activation of calmodulin-dependent kinases that phosphorylate CREB, raising the possibility that L-type channels contribute to the selective response to EPSPs rather than APs. Accordingly, we performed voltage-clamp experiments to compare the currents carried by L-type channels during depolarizing waveforms that approximated APs or dendritic EPSPs. The integrated current generated by L-type channels was significantly less after mock APs than with EPSP-like depolarizations. The difference was traced to two distinct factors. Compared with other channels, L-type channels activated at relatively negative potentials, favoring their opening with EPSP stimulation; they also exhibited relatively slow activation kinetics, weighing against their contribution during an AP. The relative ineffectiveness of APs as a stimulus for CREB phosphorylation could be overcome by exposure to the agonist Bay K8644, which potentiated the AP-induced influx through L-type channels by approximately 10-fold. Under normal conditions, the unique biophysical properties of L-type channels allow them to act as a kinetic filter to support spike-EPSP discrimination.

2-Amino-5-phosphonovalerate↗

PubMed2000

Quantification of the degradation products of sevoflurane using four brands of CO2 absorbent in a standard anesthetic circuit.

PURPOSE: CO(2) absorbents convert sevoflurane to fluoromethyl-2,2-difluoro-1-(trifluoromethyl) vinyl ether (compound A), whose toxicity in rats raises concern regarding the safety of sevoflurane in a low-flow system. The type of CO(2) absorbent is one of factors that affect compound A concentration in the anesthetic circuit. The aim of the present study was to investigate the concentration of compound A in an anesthetic model circuit following the use of different brands of soda lime and Baralyme. METHODS: We measured the concentrations of compound A in four different brands of CO(2) absorbent using a low-flow (1 l.min(-1) fresh gas) model circuit in which 2% sevoflurane was circulated. Sodasorb II, Baralyme, Sofnolime and Wakolime-A were used as CO(2) absorbents. The concentration of compound A was measured hourly, and the temperature of the CO(2) absorbent was monitored. RESULTS: The maximum concentration of compound A in the circuit was highest for Baralyme (25.5 +/- 0.6 ppm) (mean +/- SD), followed by Sodasorb II (18.9 +/- 1.6 ppm), Wakolime-A (16.1 +/- 0.7 ppm), and Sofnolime (15.8 +/- 1.4 ppm). The maximum temperature was 50.8 +/- 1.3 degrees C for Baralyme, 48.8 +/- 1.3 degrees C for Wakolime-A, 47.0 +/- 1.4 degrees C for Sodasorb II, and 43.5 +/- 3.9 degrees C for Sofnolime. CONCLUSION: The relative concentrations of compound A in the low-flow circuit were Baralyme > Sodasorb II > Wakolime-A = Sofnolime.

Journal Article↗

PubMed2000

A critical role for a Rho-associated kinase, p160ROCK, in determining axon outgrowth in mammalian CNS neurons.

We tested the contribution of the small GTPase Rho and its downstream target p160ROCK during the early stages of axon formation in cultured cerebellar granule neurons. p160ROCK inhibition, presumably by reducing the stability of the cortical actin network, triggered immediate outgrowth of membrane ruffles and filopodia, followed by the generation of initial growth cone-ike membrane domains from which axonal processes arose. Furthermore, a potentiation in both the size and the motility of growth cones was evident, though the overall axon elongation rate remained stable. Conversely, overexpression of dominant active forms of Rho or ROCK was suggested to prevent initiation of axon outgrowth. Taken together, our data indicate a novel role for the Rho/ROCK pathway as a gate critical for the initiation of axon outgrowth and the control of growth cone dynamics.

Actins↗

PubMed2000

Evaluation of the PhysioFlex closed-circuit anaesthesia machine.

The concentrations of nitrous oxide, sevoflurane and oxygen in the circle system of a closed-circuit anaesthesia machine, the PhysioFlex, were measured in seven patients. During anaesthesia, the settings for each gas were changed and their concentrations recorded. At the induction of anaesthesia, it took 80-510s (median 190s) for the end-tidal sevoflurane concentration to reach 2.0%, and 920-2640s (median 1500s) for the oxygen in the breathing circuit to reach 30%. At this time, the nitrous oxide concentration was 60+/-3% (mean+/-SD). During anaesthesia, it took 90-480s (median 140s) for the end-tidal sevoflurane concentration setting to decrease from 3.0 to 1.0%, and 90-400s (median 110s) to return from 1.0 to 3.0%. When the inspired oxygen was increased from 30 to 50%, circuit concentrations reached equilibrium in 40-60s (median 40s), and when decreased from 50% back to 30%, equilibrium took 310-470s (median 450s). During recovery from anaesthesia, inspiratory sevoflurane concentration took 40-70s (median 50s) to decrease to 0.2%. The PhysioFlex provided adequate control of sevoflurane and oxygen concentrations, but not of increasing nitrous oxide concentrations.

Adult↗

PubMed2000

Fentanyl augments block of sympathetic responses to skin incision during sevoflurane anaesthesia in children.

We studied 61 healthy ASA 1 patients (aged 2-6 yr) to determine if fentanyl affects the minimum alveolar concentration which blocks adrenergic responses to skin incision (MAC-BAR) in 50% of children in the presence of 60% nitrous oxide. Patients were allocated randomly to one of three fentanyl groups to receive 0, 2 or 4 micrograms kg-1. Patients also received sevoflurane at a preselected end-tidal concentration according to an 'up-and-down' design. After a steady-state sevoflurane concentration had been maintained for at least 15 min, fentanyl was given i.v. Skin incision was performed 5 min after administration of fentanyl. The response was considered positive if heart rate (HR) or mean arterial pressure (MAP) increased by 15% or more. The MAC-BAR of sevoflurane was 1.45 MAC (95% confidence intervals 1.25-1.65 MAC), and this was reduced markedly to 0.63 MAC and 0.38 MAC by addition of fentanyl 2 and 4 micrograms kg-1, respectively. A ceiling effect was not observed and there was a significant difference between the 2 and 4 micrograms kg-1 groups.

Adjuvants, Anesthesia↗

PubMed2000

Comparison of compound A concentrations with sevoflurane anaesthesia using a closed system with a PhysioFlex anaesthesia machine vs a low-flow system with a conventional anaesthesia machine.

Sevoflurane anaesthesia was conducted using a totally closed circuit PhysioFlex anaesthesia machine (PhysioFlex group) or with a standard Modulus CD anaesthesia machine (Modulus group) (n = 8 in each group). The PhysioFlex was used under closed system conditions and the Modulus was used under low-flow system conditions (flow rate 1 litre min-1). Concentrations of sevoflurane degradation products and the temperature of soda lime were compared. Degradation products in the circuit were measured hourly, and the temperature of soda lime was monitored. The only degradation product detected was CF2 = C(CF3)-O-CH2F (compound A). Maximum concentrations of compound A were significantly lower (median 8.5 (range 5.4-15.9) ppm) in the PhysioFlex than in the Modulus group (21.2 (16.5-27.4) ppm) (P < 0.05). The maximum temperature of soda lime was also significantly lower in the PhysioFlex group (35.3 (32.1-36.3) degrees C vs 44.6 (43.0-47.1) degrees C, respectively) (P < 0.05). Hourly compound A concentrations were lower in the PhysioFlex group than in the Modulus group. End-tidal sevoflurane concentrations during measurement of degradation products were not different between groups. Therefore, use of the totally closed PhysioFlex system may significantly reduce compound A concentrations compared with low-flow anaesthesia using a standard anaesthesia machine.

Adult↗

PubMed2000

S-Nitroso-N-acetylpenicillamine (SNAP) during hemorrhagic shock improves mortality as a result of recovery from vascular hyporeactivity.

UNLABELLED: Nitric oxide donors are protective against hemorrhagic shock (HS). However, no detailed investigation has been performed. We investigated this mechanism using S-nitroso-N-acetylpenicillamine (SNAP). HS (mean arterial pressure: 40 mm Hg) was induced in 20 dogs. Sixty min after HS, the animals were treated with saline (Cont-Gr: n = 7) or SNAP; 5 microg. kg(-1). 10 min(-1) followed by 5 microg. kg(-1). h(-1) (SNAP-Gr: n = 7). After another 60 min, the shed blood was reinfused. Reactivities to noradrenalin (NA), changes in hemodynamics, the plasma catecholamines, and nitric oxide derivatives were determined. In Cont-Gr, 3 dogs died at 90, 98, and 102 min after HS. In Cont-Gr, % changes of systolic arterial blood pressure to 1 and 2.5 microg/kg of NA after the recovery from HS decreased from 23.7% +/- 4.1% (before HS) to 6.5% +/- 0.6% and from 50.1% +/- 7.7% (before HS) to 14.5% +/- 2.6%, respectively (P < 0. 01). In SNAP-Gr, reactivity to NA was maintained. At 120 min after HS, mean arterial pressure and cardiac output in SNAP-Gr increased but not in Cont-Gr. Plasma catecholamine levels in SNAP-Gr were suppressed compared with those of Cont-Gr. In conclusion, a small dose of SNAP during HS decreased the mortality of the dogs. This might have been caused in part by residual vascular hyporeactivity. IMPLICATIONS: The administration of a small dose of S-nitroso-N-acetylpenicillamine (a nitric oxide donor), a dose which did not exert a significant vasodilator effect, was administered during hemorrhagic shock in dogs. S-nitroso-N-acetylpenicillamine improved the vascular hyporeactivity to noradrenaline and decreased the mortality rate.

Animals↗

PubMed2000

The effects of prolonged low-flow sevoflurane anesthesia on renal and hepatic function.

UNLABELLED: We assessed the effects of prolonged low-flow sevoflurane anesthesia on renal and hepatic functions by comparing high-flow sevoflurane with low-flow isoflurane anesthesia. Thirty patients scheduled for surgery of > or =10 h in duration randomly received either low-flow (1 L/min) sevoflurane anesthesia (n = 10), high-flow (6-10 L/min) sevoflurane anesthesia (n = 10), or low-flow (1 L/min) isoflurane anesthesia (n = 10). We measured the circuit concentrations of Compound A and serum fluoride. Renal function was assessed by blood urea nitrogen, serum creatinine, creatinine clearance, and urinary excretion of glucose, albumin, protein, and N:-acetyl-beta-D-glucosaminidase. The hepatic function was assessed by serum aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, and total bilirubin. Compound A exposure was 277 +/- 120 (135-478) ppm-h (mean +/- SD [range]) in the low-flow sevoflurane anesthesia. The maximum concentration of serum fluoride was 53.6 +/- 5.3 (43.4-59.3) micromol/L for the low-flow sevoflurane anesthesia, 47.1 +/- 21.2 (21.4-82.3) micromol/L for the high-flow sevoflurane anesthesia, and 7.4 +/- 3.2 (3.2-14.0) micromol/L for the low-flow isoflurane anesthesia. Blood urea nitrogen and serum creatinine were within the normal range, and creatinine clearance did not decrease throughout the study period in any group. Urinary excretion of glucose, albumin, protein, and N:-acetyl-beta-D-glucosaminidase increased after anesthesia in all groups, but no significant differences were seen among the three groups at any time point after anesthesia. Lactate dehydrogenase and alkaline phosphatase on postanesthesia Day 1 were higher in the high-flow sevoflurane group than in the low-flow sevoflurane group. However, there were no significant differences in any other hepatic function tests among the groups. We conclude that prolonged low-flow sevoflurane anesthesia has the same effect on renal and hepatic functions as high-flow sevoflurane and low-flow isoflurane anesthesia. IMPLICATIONS: During low-flow sevoflurane anesthesia, intake of Compound A reached 277 +/- 120 ppm-h, but the effect on the kidney and the liver was the same in high-flow sevoflurane and low-flow isoflurane anesthesia.

Acetylglucosaminidase↗

PubMed2000

Influence of age on hypnotic requirement, bispectral index, and 95% spectral edge frequency associated with sedation induced by sevoflurane.

BACKGROUND: Aging is associated with a reduction in anesthetic requirements. The effects of age on the electroencephalographic response to inhalational anesthesia have not been well documented. The objective of the present study was to determine the influence of age on hypnotic requirement and electroencephalographic derivatives such as bispectral index and 95% spectral edge frequency associated with sedation induced by sevoflurane. METHODS: Ninety-six patients were randomly allocated into one of three age groups A, B, and C, ranging in age from 18-39 yr, 40-64 yr, and 65-85 yr, respectively. Patients in each group were sedated with sevoflurane at two predetermined concentrations ranging between 0.45% and 0.85%. The relationship between sevoflurane concentration and response to a verbal command, as well as the relationships between response and bispectral index and 95% spectral edge frequency, was determined. RESULTS: Multiple regression analysis showed that end-tidal sevoflurane concentration and age significantly affected both bispectral index and 95% spectral edge frequency. ED50 values of sevoflurane concentration for loss of consciousness, defined as no response to verbal command, were different between groups A and C: 0.72 (95% confidence interval: 0.68-0.75) versus 0.59 (95% confidence interval: 0.56-0.62). However, the same effective values of bispectral index and 95% spectral edge frequency at this same clinical end point did not differ. CONCLUSIONS: Increasing age reduced sevoflurane requirements to suppress responses to a verbal command but did not change bispectral index and 95% spectral edge frequency associated with this end point, and in a population with a wide age range, bispectral index would predict depth of sedation better than end-tidal sevoflurane concentration.

Adult↗

PubMed2000

[The compound A concentration in a low-flow anesthesia circuit using the new CO2 absorbent SPHERASORB].

CO2 absorbents convert sevoflurane to fluoromethyl-2,2-difluoro-1-(trifluoromethyl) vinyl ether (compound A), whose toxicity in rats raises concern regarding the safety of sevoflurane in a low-flow system. SPHERASORB is a new CO2 absorbent which does not contain KOH. However, the reaction between SPHERASORB and sevoflurane has not been examined. We compared compound A concentration in a model circuit using SPHERASORB and the commonly used Sodasorb II. The anesthesia circuit was circulated with fresh gas flow at a rate of 1 l.min-1 containing 2% sevoflurane. Compound A concentration was measured hourly and the temperature of CO2 absorbent was monitored. The maximum concentration of compound A in the circuit was 12.2 +/- 1.4 ppm for SPHERASORB and 18.6 +/- 0.4 ppm for Sodasorb II (P < 0.05). The maximum temperature of SPHERASORB was lower than that of Sodasorb II (P < 0.05). SPHERASORB can reduce compound A formation, compared to Sodasorb II.

Absorption↗

PubMed1999

Cupidin, an isoform of Homer/Vesl, interacts with the actin cytoskeleton and activated rho family small GTPases and is expressed in developing mouse cerebellar granule cells.

A developmentally regulated Homer/Vesl isoform, Cupidin (Homer 2a/Vesl-2Delta11), was isolated from postnatal mouse cerebellum using a fluorescent differential display strategy. The strongest expression of Cupidin was detected in the cerebellar granule cells at approximately postnatal day 7. Cupidin was enriched in the postsynaptic density fraction, and its immunoreactivity was concentrated at glomeruli of the inner granular layer when active synaptogenesis occurred. Cupidin protein could be divided into two functional domains: the N-terminal portion, which was highly conserved among Homer/Vesl family proteins, and the C-terminal portion, which consisted of a putative coiled-coil structure, including several leucine zipper motifs. The N-terminal fragment of Cupidin, which was able to associate with metabotropic glutamate receptor 1 (mGluR1), also interacted with F-actin in vitro. In keeping with this, F-actin immunocytochemically colocalized with Cupidin in cultured cerebellar granule cells, and a Cupidin-mGluR1-actin complex was immunoprecipitated from crude cerebellar lysates using an anti-Cupidin antibody. On the other hand, the C-terminal portion of Cupidin bound to Cdc42, a member of Rho family small GTPases, in a GTP-dependent manner in vitro, and Cupidin functionally interacted with activated-Cdc42 in a heterologous expression system. Together, our findings indicate that Cupidin may serve as a postsynaptic scaffold protein that links mGluR signaling with actin cytoskeleton and Rho family proteins, perhaps during the dynamic phase of morphological changes that occur during synapse formation in cerebellar granule cells.

Actins↗