Shin-Hee Yoon

α-Synuclein Interacts with Phospholipase D Isozymes and Inhibits Pervanadate-induced Phospholipase D Activation in Human Embryonic Kidney-293 Cells

α-Synuclein has been implicated in the pathogenesis of many neurodegenerative diseases, including Parkinsons disease and Alzheimers disease. Although the function of α-synuclein remains largely unknown, recent studies have demonstrated that this protein can interact with phospholipids. To address the role of α-synuclein in neurodegenerative disease, we have investigated whether it binds phospholipase D (PLD) and affects PLD activity in human embryonic kidney (HEK)-293 cells overexpressing wild type α-synuclein or the mutant forms of α-synuclein (A53T, A30P) associated with Parkinsons disease. Tyrosine phosphorylation of α-synuclein appears to play a modulatory role in the inhibition of PLD, because mutation of Tyr125 to Phe slightly increases inhibitory effect of α-synuclein on PLD activity. Treatment with pervanadate or phorbol myristate acetate inhibits PLD more in HEK 293 cells overexpressing α-synuclein than in control cells. Binding of α-synuclein to PLD requires phox and pleckstrin homology domain of PLD and the amphipathic repeat region and non-Aβ component of α-synuclein. Although biologically important, co-transfection studies indicate that the interaction of α-synuclein with PLD does not influence the tendency of α-synuclein to form pathological inclusions. These results suggest that the association of α-synuclein with PLD, and modulation of PLD activity, is biologically important, but PLD does not appear to play an essential role in the pathophysiology of α-synuclein.

Open Channel Block of A-Type, Kv4.3, and Delayed Rectifier K+ Channels, Kv1.3 and Kv3.1, by Sibutramine

The effects of sibutramine on voltage-gated K+ channel (Kv)4.3, Kv1.3, and Kv3.1, stably expressed in Chinese hamster ovary cells, were investigated using the whole-cell patch-clamp technique. Sibutramine did not significantly decrease the peak Kv4.3 currents, but it accelerated the rate of decay of current inactivation in a concentration-dependent manner. This phenomenon was effectively characterized by integrating the total current over the duration of a depolarizing pulse to +40 mV. The IC50 value for the sibutramine block of Kv4.3 was 17.3 μM. Under control conditions, the inactivation of Kv4.3 currents could be fit to a biexponential function, and the time constants for the fast and slow components were significantly decreased after the application of sibutramine. The association (k+1) and dissociation (k–1) rate constants for the sibutramine block of Kv 4.3 were 1.51 μM–1s–1 and 27.35 s–1, respectively. The theoretical KD value, derived from k–1/k+1, yielded a value of 18.11 μM. The block of Kv4.3 by sibutramine displayed a weak voltage dependence, increasing at more positive potentials, and it was use-dependent at 2 Hz. Sibutramine did not affect the time course for the deactivating tail currents. Neither steady-state activation and inactivation nor the recovery from inactivation was affected by sibutramine. Sibutramine caused the concentration-dependent block of the Kv1.3 and Kv3.1 currents with an IC50 value of 3.7 and 32.7 μM, respectively. In addition, sibutramine reduced the tail current amplitude and slowed the deactivation of the tail currents of Kv1.3 and Kv3.1, resulting in a crossover phenomenon. These results indicate that sibutramine acts on Kv4.3, Kv1.3, and Kv3.1 as an open channel blocker.

Effect of ethanol on spontaneous phasic contractions of cat gastric smooth muscle.

Background: Ethanol is generally believed to inhibit extracellular Ca 2+ influx, thereby inhibiting gastric muscle contraction. Recently, we observed that verapamil inhibited only the amplitude of spontaneous phasic contractions, whereas ethanol inhibited both amplitude and frequency. In our objective to investigate the mechanism of ethanols inhibition of gastric motility, the involvement of various protein kinases in ethanol-inhibited spontaneous phasic contractions of the stomach muscle strips was tested. Methods: Circular muscle strips (2.0 × 0.2 cm) were prepared from the corpus of cat stomach in order to measure isometric contraction in a chamber filled with Krebs-Ringer solution (pH 7.4, temperature 36 °C) bubbled with 5% CO 2 in O 2. Results: Spontaneous phasic contraction was not affected by various receptor antagonists (1 μM atropine, 1 μM hexamethonium, 1 μM phentolamine and 1 μM propranolol) or 1 μM tetrodotoxin. EGTA and verapamil dose-dependently inhibited only the amplitude of spontaneous phasic contractions and not the frequency. Ethanol dose-dependently inhibited both the amplitude and frequency of phasic contractions. The amplitude and frequency of spontaneous phasic contractions were significantly inhibited by protein kinase C and tyrosine kinase inhibitors. However, neither protein kinase C activator nor various phosphatase inhibitors blocked the inhibitory effect of ethanol. Conclusions: Ethanol appears to inhibit spontaneous phasic contractions by a mechanism other than the inhibition of protein kinase C or tyrosine kinase or the inhibition of extracellular Ca 2+ influx.

Interaction of Riluzole with the Closed Inactivated State of Kv4.3 Channels

The effect of riluzole on Kv4.3 was examined using the whole-cell patch-clamp technique. Riluzole inhibited the peak amplitude of Kv4.3 in a reversible, concentration-dependent manner with an IC50 of 115.6 μM. Under control conditions, a good fit for the inactivation of Kv4.3 currents to a double exponential function, with the time constants of the fast component (τf) and the slow component (τs), was obtained. τf was not altered by riluzole at concentrations up to 100 μM, but τs became slower with increasing riluzole concentration, resulting in the crossover of the currents. The inhibition increased steeply with increasing channel activation at more positive potentials. In the full activation voltage range positive to +30 mV, however, no voltage-dependent inhibition was found. Riluzole shifted the voltage dependence of the steady-state inactivation of Kv4.3 in the hyperpolarizing direction in a concentration-dependent manner. However, the slope factor was not affected by riluzole. The Ki for riluzole for interacting with the inactivated state of Kv4.3 was estimated from the concentration-dependent shift in the steady-state inactivation curve and was determined to be 1.2 μM. Under control conditions, closed state inactivation was fitted to a single exponential function. Riluzole caused a substantial acceleration in the closed state inactivation. In the presence of riluzole, the recovery from inactivation was slower than under control conditions. Riluzole induced a significant use-dependent inhibition of Kv4.3. These results suggest that riluzole inhibits Kv4.3 by binding to the closed inactivated state of the channels and that the unbinding of riluzole occurs from the closed state during depolarization.

Distributional patterns of phospholipase C isozymes in rat pancreas.

Phospholipase C (PLC) isozymes are believed to play a role in regulating pancreatic exocrine and endocrine secretion. In an attempt to investigate the role of PLC, we examined the distribution patterns of PLC isozymes in the normal rat pancreas by Western blot analysis and immunohistochemistry. Western blot analysis was performed on pancreatic acinar tissues and the islet of Langerhans, which were separated from each other. PLC-&bgr; isozymes (&bgr;1, &bgr;2, &bgr;3, and &bgr;4), &dgr;1, and &dgr;2 were detected in both acinar and islet cells, whereas PLC-&ggr;1 and &ggr;2 were observed only in acinar tissues. On immunohistochemistry, the immunoreactivities of PLC isozymes except for PLC-&ggr;1 were observed as follows: PLC-&bgr;1, in both the exocrine and endocrine tissues; PLC-&bgr;2, mainly in the periphery of the islet and acinar cells; PLC-&bgr;3, in the periphery of the islet and in some ductal epithelium; PLC-&bgr;4, through the islet of Langerhans and ductal epithelium; PLC-&ggr;1, not detected in pancreatic tissue; PLC-&ggr;2, mainly in acinar cells; PLC-&dgr;1 and &dgr;2, in the islet and in ductal epithelium. These results suggest that the intrapancreatic site-specific existence of PLC isozymes may modulate pancreatic exocrine and endocrine functions through a PLC-mediated signal transduction.

Stimulatory role of the dorsal motor nucleus of the vagus in gastrointestinal motility through myoelectromechanical coordination in cats.

This study was undertaken to investigate the effect of stimulation of the dorsal motor nucleus of the vagus (DMV) on myoelectric activity and motility of the gastric antrum and duodenum in normal and in vagotomized cats. 37 cats were starved for 24 h and then anesthetized with alpha-chloralose (70-80 mg/kg, iv). Electrical stimulation (0.1 mA, 0.2 ms, 50 Hz) of the left DMV was performed through a stereotaxically inserted electrode in 19 of the cats. The remaining 18 cats were injected in the left DMV with a glutamate solution (1 M, 200 nl) through an inserted 3-barreled micropipette. The myoelectric activity (slow wave) and the motility of the gastric antrum (2 cm proximal to the pylorus) and duodenum (3 cm distal to the pylorus) were measured using serosal bipolar electrodes and intraluminal balloons. Both the electrical and the glutamate stimulations of the DMV markedly increased the occurrence of spike potentials on the antral and duodenal myoelectric activity; however, the stimulations significantly decreased the frequency of the antral slow wave. The stimulations also produced increases in the motility of the antrum and duodenum which corresponded to the changes in the myoelectric activity. All the changes in the myoelectric activity and the motility were not observed after the ipsilateral vagotomy. Thus, these results strongly suggest that the dorsal motor nucleus of the vagus has a stimulatory influence on antral and duodenal motility through myoelectromechanical coordination via the vagus nerve in cats.

Effect of somatostatin on cholecystokinin-induced amylase release in rat pancreatic acini.

The effect of somatostatin on cholecystokinin-induced amylase release was investigated in isolated rat pancreatic acini. Acini were isolated by enzymatic digestion and incubated in a HEPES buffered Ringers solution with testing reagents for 30 minutes at 37°C. The activity of released amylase, cAMP, and inositol phosphate formation were measured. Intracellular calcium concentration ([Ca2+]i) was also checked. Somatostatin 14 and octreotide, a somatostatin analog, inhibited CCK-stimulated amylase release in a concentration-dependent manner. The inhibitory effect of octreotide on CCK-induced amylase release was not shown when the acini were treated with 8-Br-cAMP, irrespective of the presence of IBMX. Forskolin potentiated CCK-induced amylase release and this effect was blocked by octreotide treatment; although CCK-8 (3 x 10−11 M) failed to stimulate cAMP formation, octreotide significantly inhibited basal cAMP formation in the acini. The increase of [Ca2+]i in response to CCK was inhibited by octreotide. However, CCK-induced inositol phosphate formation was not changed by 10−9M octreotide. Octreotide had no effect on CCK-stimulated tyrosine phosphorylation, and tyrosine phosphatase inhibitors (NaF and Na2WO4) did not influence the effect of octreotide on CCK-induced amylase release. From these results, we conclude that octreotide inhibits CCK-induced amylase release by inhibiting basal cAMP formation and decreasing the [Ca2+]i stimulated by CCK.

H1 Receptor Mediates Inositol Phosphates Response to Histamine in Gastric Smooth Muscle of Guinea Pigs

The effect of histamine on [3H]-inositol phosphates (IPs) formation was investigated with [3H]-inositol-labeled gastric smooth-muscle cells in guinea pigs. Histamine (10(-5) M) increased the formation of [3H]-IPs in the muscle cells. The increase was significantly inhibited by pyrilamine (10(-5) M) but not by cimetidine (10(-5) M). The contractile response to histamine was also completely inhibited by pyrilamine but not by cimetidine. Phorbol ester 12-myristate 13-acetate (10 microM) significantly inhibited histamine-stimulated [3H]-IPs formation by 56%, whereas forskolin (10 microM) decreased it by 18%. This study demonstrates that the response of [3H]-IPs formation and contraction to histamine is mediated through H1 receptor, and the formation of [3H]-IPs is negatively regulated by protein kinase C in gastric smooth muscle of guinea pigs.

Regulation of protein kinases in steady-state contraction of cat gastric smooth muscle

Cat gastric smooth muscle strips were used to investigate the involvement of protein kinases in the steady-state contraction induced by 1 microM acetylcholine or 20 mM KCI. The steady-state contraction induced by acetylcholine or KCl was inhibited by EGTA dose dependently. Voltage-dependent Ca2+ channel antagonists dose dependently inhibited the contractions induced by KCI as well as by acetylcholine. Inhibitory effects of voltage-dependent Ca2+ channel antagonists were significantly more prominent on KCI-induced contractions than on acetylcholine-induced contractions. The acetylcholine-induced contraction was dose dependently inhibited by 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8, a blocker of intracellular Ca2+ release), but the KCl-induced contraction was not inhibited at all. Therefore both intracellular Ca2+ release and extracellular Ca2+ influx seem to be necessary for the acetylcholine-induced contraction, but intracellular Ca2+ release is not necessary for the KCl-induced contraction. Protein kinase C inhibitors, 10 microM 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine 2HCl (H-7) and 1 microM staurosporine, significantly inhibited the contraction induced by acetylcholine or KCl. Calmodulin antagonists, 30 microM trifluoperazine and 50 microM N-(6-aminohexyl)-5-chloro-2-naphthalenesulfonamide HCI (W-7), however, significantly inhibited the contraction induced by acetylcholine but not by KCl. A tyrosine kinase inhibitor, 50 microM genistein, did not affect the acetylcholine-induced contraction but significantly inhibited the KCl-induced contraction. These results strongly suggest that the involvement of protein kinases in regulation of the steady-state contraction may be agonist-dependent.

The Action of Histamine on the Isolated Stomach Muscle of the Cat

The effect of histamine on contractile and electric activity was studied in the isolated stomach muscle strips of 138 cats. Histamine dose-dependently produced tonic and phasic contractions of the muscle preparations from the fundus and the corpus but only phasic contraction of the antral muscle preparations. The frequency of gastric slow waves (SWs) was also increased dose-dependently by histamine. The responses of muscle contractions and gastric SW frequency to histamine were completely blocked by pretreatment with pyrilamine (10(-6) M) and were significantly inhibited by atropine (10(-5) M) but not by cimetidine (10(-5) M), hexamethonium (10(-5) M), phentolamine (10(-5) M), or propranolol (10(-5) M). The inhibition by pyrilamine was competitive. Although atropine inhibited the effect of histamine significantly, it could not completely block the effect of histamine even at a high concentration (3 x 10(-5) M). It is concluded that histamine may participate in the regulation of gastric motility in the cat by acting on the H1 receptor to cause the release of acetylcholine and also other contractile substance(s).