Rustu Onur

Persistent Defect in Transmitter Release and Synapsin Phosphorylation in Cerebral Cortex After Transient Moderate Ischemic Injury

Background and Purpose— Synaptic transmission is highly vulnerable to metabolic perturbations. However, the long-term consequences of transient metabolic perturbations on synapses are not clear. We studied the long-lasting changes in synaptic transmission and phosphorylation of presynaptic proteins in penumbral cortical neurons after transient moderate ischemia. Methods— Rats were subjected to 1 hour of middle cerebral artery occlusion. After reperfusion, electric activity of neurons in the peri-infarct region was recorded intracellularly and extracellularly in situ. Phosphorylation of synapsin-I and tyrosine residues was studied by immunohistochemistry. Results— Neurons in the penumbra displayed no postsynaptic potentials 1 to 3 hours after recirculation. However, these cells were able to generate action potentials and were responsive to glutamate, suggesting that postsynaptic excitability was preserved but the synaptic transmission was blocked because of a presynaptic defect. The synaptic transmission was still depressed 24 hours after recirculation in neurons in the peri-infarct area that survived ischemia. The amount of immunoreactive synapsin-I, synaptophysin, and synaptotagmin was not appreciably changed for 72 hours after reperfusion. However, phosphorylation of synapsin-l was significantly decreased, whereas phosphotyrosine immunoreactivity was increased, suggesting a selective defect in synapsin-I phosphorylation. Conclusions— These data demonstrate that synaptic transmission may be permanently impaired after transient moderate brain injury. Since postsynaptic excitability is preserved, the transmission failure is likely to be caused by presynaptic mechanisms, one of which may be impaired phosphorylation of presynaptic proteins.

Glycine is required for NMDA receptor activation: electrophysiological evidence from intact rat hippocampus.

Fimbrial/commissural stimulation evokes a prolonged negative field potential in stratum radiatum of CA1 region of the rat hippocampus, in situ, upon activation of N-methyl-D-aspartate (NMDA) receptors. This activity can be induced by iontophoresis of NMDLA (50 nA) or glycine (50-100 nA) during low-frequency stimulation. 7-Cl-Kynurenate (10-30 nA) fully antagonized the NMDA receptor-mediated negative wave induced not only by glycine (N = 3) but also by NMDLA (N = 9), suggesting that activation of NMDA receptors is not possible without glycine binding. 7-Cl-Kynurenate also depressed the extracellular negative d.c. potential shifts appearing during iontophoresis of NMDLA. Stimulation with brief, high-frequency trains evoked a negative wave of 2.1 +/- 0.2 mV and 176 +/- 4 ms (N = 20) on the hippocampal field response following the last stimulus. Ketamine (100-200 nA, N = 6) and MK-801 (50-200 nA, N = 7) blocked the negative wave by 74 +/- 13 and 62 +/- 8%, respectively, while glycine (100 nA) potentiated it by 35 +/- 2% (N = 6), indicating that it had a component mediated by NMDA receptors. 7-Cl-Kynurenate (100 nA) antagonized this activity at a comparable rate to the NMDA receptor antagonists (67 +/- 8%, N = 4). A similar negative wave of 0.9 +/- 0.2 mV and 41 +/- 3 ms (N = 12) was evoked in hippocampal slices by high-frequency orthodromic stimulation. Potentiation of this activity upon lowering Mg2+ in ACSF from 1.3 to 0.5 mM further supported that it had an NMDA-mediated component.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural and functional characterization of simvastatin-induced myotoxicity in different skeletal muscles.

BACKGROUND Statins are the most commonly used drugs for the treatment of hypercholesterolemia. Their most frequent side effect is myotoxicity. To date, it remains unclear whether statins preferentially induce myotoxicity in fast- or in slow-twitch muscles. Therefore, we investigated these effects on fast- (extensor digitorum longus; EDL), slow- (soleus; SOL), and mixed-twitch muscles (diaphragm; DIA) in rats by comparing their contractile and molecular structural properties. METHODS Simvastatin-induced functional changes were determined by muscle contraction measurements, and drug-induced molecular changes were investigated using Fourier transform infrared (FTIR) and attenuated total reflectance (ATR) FTIR spectroscopy. RESULTS With simvastatin administration (30 days, 50mg/kg), a depression in the force-frequency curves in all muscles was observed, indicating the impairment of muscle contractility; however, the EDL and DIA muscles were affected more severely than the SOL muscle. Spectroscopic findings also showed a decrease in protein, glycogen, nucleic acid, lipid content and an increase in lipid order and lipid dynamics in the simvastatin-treated muscles. The lipid order and dynamics directly affect membrane thickness. Therefore, the kinetics and functions of membrane ion channels were also affected, contributing to the statin-induced impairment of muscle contractility. Furthermore, a reduction in α-helix and β-sheet and an increase in random coil, aggregated and antiparallel β-sheet were observed, indicating the protein denaturation. Spectral studies showed that the extent of molecular structural alterations in the muscles following simvastatin administration was in the order EDL>DIA>SOL. CONCLUSIONS Simvastatin-induced structural and functional alterations are more profound in the fast-twitch than in the slow-twitch muscles. GENERAL SIGNIFICANCE Myotoxic effects of simvastatin are primarily observed in the fast-twitch muscles.

Low dose simvastatin induces compositional, structural and dynamic changes in rat skeletal extensor digitorum longus muscle tissue

Statins are commonly used drugs in the treatment of hypercholesterolaemia. There are many adverse effects of statins on skeletal muscle, but the underlying mechanisms remain unclear. In the present study, the effects of low dose (20 mg/kg) simvastatin, a lipophilic statin, on rat EDL muscle (extensor digitorum longus muscle) were investigated at the molecular level using FTIR (Fourier-transform infrared) spectroscopy. FTIR spectroscopy allows us rapid and sensitive determination of functional groups belonging to proteins, lipids, carbohydrates and nucleic acids simultaneously. The results revealed that simvastatin treatment induces a significant decrease in lipid, nucleic acid, protein and glycogen content. A significant increase in the lipid/protein and nucleic acid/protein ratios was also obtained with simvastatin treatment. Furthermore, an increase in lipid order and membrane fluidity was detected. A decrease in the bandwidth of the amide I band and shifting of the position of this band to higher frequency values in treated muscle indicates structural changes in proteins. Detailed secondary structure analysis of the amide I band revealed a significant increase in antiparallel and aggregated beta-sheet, random coil structure and a significant decrease in beta-sheet structure, which indicates protein denaturation.

Contractility and electrophysiological parameters of cremaster muscles of boys with a hernia or undescended testis

BACKGROUND/PURPOSE The cremaster muscle (CM) has been considered to participate in regulation of blood flow and temperature of the testis. Its contribution to testicular descent has been suggested. However, there is limited information about the CM in physiological and pathological states. Therefore, an experimental study has been conducted to evaluate and compare the contractile and electrophysiological properties of CM in boys with descended or undescended testes. METHODS Identical CM strips were obtained from eight boys who underwent orchidopexy with a mean age of 3+/-2.2 years and from eight boys who underwent herniorrhaphy with a mean age of 4+/-1.3 years. Muscle strips of 3 x 8 mm were vertically attached to an isometric force displacement transducer, and direct muscle contractions were elicited by rectangular electrical pulses. Direct isometric muscle contractions were recorded in an organ bath containing mammalian Ringers solution. In electrophysiological experiments, conventional microelectrode techniques were used. RESULTS Direct electrical stimulation of CM strips obtained from patients with descended and undescended testes elicited muscle twitches and frequency-dependent contractile responses. Tetanic contractions of undescended testes at 100 Hz were 67% greater in amplitude than that of descended testes (P< .002). Muscle strips of both groups exhibited increased twitch amplitudes by 105%+/-37% when the temperature of the bathing solution was increased from 22 degrees to 37 degrees C (P< .001). The electrophysiological findings were similar. CONCLUSIONS Contrary to other striated muscles, elevated temperature increases the contractility of CM. If the increased contractility by an increase in temperature is a property unique for CM, it should reflect the attempts at regulating testicular blood flow or temperature. The increased amplitude of contractions encountered among the CM of boys with undescended testis suggests the CM to have a role on the location of the testis.

Lack of carbachol response indicates the absence of cholinergic receptors in sacs associated with undescended testis

BACKGROUND/PURPOSE The mechanism of testicular descent remains controversial. The processus vaginalis (PV) contains smooth muscle and should have contractile activity that may contribute to descent. This study was designed to evaluate the smooth muscle of PVs associated with incomplete obliteration for spontaneous activities and responses to various stimuli, to determine if differences exist according to sex, diagnostic source, or location of the testis. MATERIALS Peritoneal samples (n = 4); sacs from girls (n = 8) and boys with inguinal hernia (n = 12); and sacs from boys with hydrocele (n = 3), hydrocele of the cord (n = 2), or undescended testis (n = 7) were used for the current study. Tissues were attached to the isometric force displacement transducer in an organ bath containing mammalian Ringers solution at 37 degrees C. Spontaneous mechanical activity and contractile responses of tissues to the electrical field stimulation, phenylephrine, carbachol, and serotonin were recorded. The values obtained from boys and girls with inguinal hernia and from boys with either undescended or descended testis were compared through Fishers Exact test. RESULTS There were no statistically significant differences in patient age between groups. Among the parameters studied, only the carbachol response of the sacs associated with undescended testis showed a significant difference compared with the others (P = .001). None of the sacs associated with undescended testis responded to carbachol, whereas all of the sacs from boys and girls with inguinal hernia responded to carbachol. CONCLUSIONS Lack of carbachol response suggests the absence of cholinergic receptors within the sacs associated with undescended testis. The lack of cholinergic receptors may play a role in the failure of the process of testicular descent by hindering either PV elongation into the scrotum or a possible propulsive activity of the PV on the testis.

Facilitatory effects of dexamethasone on neuromuscular transmission

The beneficial effects of glucocorticoids in myasthenia gravis are attributed to their immunosuppressive actions. There are also studies reporting direct facilitatory as well as depressant effects of glucocorticoids on neuromuscular transmission. The effects of dexamethasone on neuromuscular transmission were studied by intracellular and extracellular microelectrode recording techniques in the mouse phrenic nerve-diaphragm preparation. Creatinine had to be added to the bathing media to prevent precipitation of the glucocorticoid with Ca2+ and Mg2+; creatinine had no effect. One hour of perfusion with dexamethasone (10(-4) to 10(-3) M) increased the frequency of miniature end-plate potentials (MEPPs), as well as the amplitude and quantum content of end-plate potentials (EPPs), but did not change MEPP amplitude, suggesting an increase in acetylcholine release. Dexamethasone also enhanced presynaptic facilitation and potentiation during repetitive stimulation. It had no effect on muscle resting membrane potential but increased the amplitude, overshoot, and rate of rise of muscle action potentials. The amplitudes of nerve terminal action potentials were also enhanced by dexamethasone. These findings suggest that glucocorticoids have a direct facilitatory action on neuromuscular transmission by a presynaptic action.

Potentiation of the morphine-induced respiratory rate depression by captopril.

The effects of morphine and captopril, an angiotensin-converting enzyme inhibitor, on respiratory frequency have been investigated in non-anesthetized mice. Morphine (10.0 mg/kg) reduced the respiratory frequency which gradually returned to the control level 2 h after the injection. The same dose of morphine when given together with captopril (0.1 and 1.0 mg/kg), caused a similar reduction in respiratory rate. This respiratory depression however, persisted until the end of the observation period. Similar results were obtained with the same dose of morphine in mice pretreated with captopril. The minimal dose of morphine reducing respiratory frequency (3.0 mg/kg), when given to the mice pretreated with captopril (0.1 mg/kg) caused a significant reduction in respiratory frequency and this effect was equal to that obtained with 10.0 mg/kg morphine alone. The results are discussed from the point of the possible inhibitory effect of captopril on the enkephalin degrading enzyme(s).

Differential Contractile Impairment of Fast- and Slow-Twitch Skeletal Muscles in a Rat Model of Doxorubicin-Induced Congestive Heart Failure

Congestive heart failure (CHF) is associated with exercise intolerance that cannot be entirely explained by hypoperfusion of the skeletal muscles. We studied the contractile properties of fast-twitch (extensor digitorum longus; EDL) and slow-twitch (soleus; SOL) skeletal muscles in doxorubicin-induced CHF in rats, and evaluated the defective steps of excitation-contraction coupling. Both types of muscles-obtained from CHF rats displayed significant reduction in twitch and tetanic contractions. Twitch half-relaxation times of CHF SOL muscles were prolonged while there was no significant difference in EDL muscles. High K+ application induced lower contracture amplitudes in CHF muscles. Caffeine-induced contractures were significantly diminished in CHF SOL. Verapamil application depressed tetanic contractions in all preparations while depression was more pronounced in CHF SOL. Immunohistochemistry revealed reduced expression of sarcoplasmic reticulum Ca2+-ATPase-1 and -2 in CHF EDL and in CHF SOL, respectively. Sarcolemmal excitability and spontaneous neurotransmitter release were unaffected since resting membrane potential, action potential and miniature end-plate potentials were unaltered in CHF muscles. We conclude that CHF induces contractile impairment that occurs predominantly in rat slow-twitch skeletal muscles. Our results suggest that this muscle-type-specific effect of CHF is related to the defective intracellular Ca2+ release and uptake mechanisms and reduced sarcolemmal-dihydropyridine-sensitive Ca2+ channel activity.

Monitoring cellular edema at single-neuron level by electrical resistance measurements

Electrical resistance measurements have been used for investigating extracellular volume fraction (EVF) of brain tissue. Conventional techniques using multiple metal electrodes are limited in their spatial resolution, and thus not suitable for detecting local EVF changes at cellular level. We used a multibarrelled glass microelectrode to monitor cellular swelling locally at single-neuron level. The microelectrode was placed in CA1 region of the rat hippocampus, in situ. A constant current pulse was applied between one of the barrels and a reference electrode placed in the neck. The resultant voltage drop, which was directly proportional to the resistance of the immediate environment surrounding the tip of the microelectrode, was recorded through another barrel. A third barrel was used for iontophoretic injection of N-methyl-D-aspartate (NMDA) for inducing local cellular edema. The effect of diffuse edema induced by bilateral carotid artery ligation on EVF was also investigated. NMDA application increased the local tissue resistance by 2.0-, and ischemia, by 3.4-folds. We conclude that the method described can detect changes in EVF of minute volumes of brain tissue, and is suitable for monitoring very local effects of drugs or changes in the metabolism on cell volume.