The synthesis of CCK-4 (H-Trp-Met-Asp-Phe-NH2) by using enzymes exclusively was described. As protection group for the amino group we used the Phenylacetyl group (Phac) which had been cleaved at the end of the synthesis with Penicillin G Amidase (PGA) without affecting the peptide bonds. Thus, beginning with Phac-Trp-OH we had successfully synthesized the target peptide with following 4 enzymes, alpha-Chymotrypsin, Papain, Thermolysin and PGA in four reaction steps. All reactions were carried out in aqueous buffer in reasonable yields (> 65%). FAB-MS or FD-MS verified the correct molecular mass of all peptides.
Chymotrypsin ; Enzymes, Immobilized ; Papain ; Peptides ; Tetragastrin ; chemical synthesis ; Thermolysin

The synthesis of CCK-4 (H-Trp-Met-Asp-Phe-NH2) by using enzymes exclusively was described. As protection group for the amino group we used the Phenylacetyl group (Phac) which had been cleaved at the end of the synthesis with Penicillin G Amidase (PGA) without affecting the peptide bonds. Thus, beginning with Phac-Trp-OH we had successfully synthesized the target peptide with following 4 enzymes, alpha-Chymotrypsin, Papain, Thermolysin and PGA in four reaction steps. All reactions were carried out in aqueous buffer in reasonable yields (> 65%). FAB-MS or FD-MS verified the correct molecular mass of all peptides.
Chymotrypsin ; Enzymes, Immobilized ; Papain ; Peptides ; Tetragastrin/*chemical synthesis ; Thermolysin

Medial vestibular nucleus (MVN) neurons are found to have spontaneous electrical activity in the absence of any detectable synaptic input. To investigate the contributions of intrinsic mechanisms to the spontaneous activity of type B MVN neurons, we examined the effects of various channel blockers on spontaneous firing by means of patch clamp recordings. Coronal slice (400 micrometer) of the vestibular nucleus region was sequentially treated with pronase 0.2 mg/ml and thermolysin 0.2 mg/ml, then single neurons were mechanically dissociated. MVN neurons recorded in neonatal rat were shown to have either a single deep afterhyperpolarization (AHP; type A cells), or an early fast and a delayed slow AHP (type B cells). In 300 nM TTX, spontaneous firing was blocked in type B cells tested. In 8 of 11 cells, underlying fluctuation or oscillations in membrane potential was not remained, and hyperpolarization did not produce rebound low-threshold calcium spikes. Although type B MVN neurons possessed hyperpolarization activated cation current (Ih), cesium had no effect on firing rates. The spike AHP is calcium dependent. When Ca2+ influx was blocked in external Ca2+ free solution, repetitive firing was abolished and the cell rested at depolarized membrane potentials. Application of apamin (300 nM) caused a profound reduction in the amplitude of the AHP and produced rhythmic burst firing. These findings suggest that the spontaneous activity of type B MVN neurons is regulated by interactions between the membrane depolarization mainly due to a persistent sodium conductances and hyperpolarization due to the calcium-activated potassium conductances.
Animals ; Apamin ; B-Lymphocytes ; Calcium ; Calcium Signaling ; Cesium ; Fires* ; Membrane Potentials ; Membranes ; Neurons* ; Potassium ; Pronase ; Rats ; Sodium ; Thermolysin ; Vestibular Nuclei*

thermolysin 0.2mg/ml, then single neurons were mechanically dissociated.Voltage-dependent sodium currents showed that the half-maximum activation potential was -41.8 +/- 1.8mV and half-maximum inactivation potential was -62.4 +/- 3.0mV.And the currents were blocked totally by application of 100nM tetrodotoxin.In a Ca2+ free solution,low-threshold transient (IA )and high-threshold sustained (IK )currents were recorded.The half-maximum activation and inactivation potential of IK were 2.5 +/- 1.9mV and -37.1 +/- 2.3mV,respectively.IA was activated and inactivated more rapidly than IK. The half-maximum acti-vation and inactivation potential were -21.6 +/- 6.3mV and -84.5 +/- 5.0mV,respectively. When a 4-aminopy-ridine of 5mM was applied, IA was almost totally blocked. These results reveal that MDH neurons express a variety of voltage-dependent ionic currents with distinct physiological and pharmacological properties,and they play an essential role in the transmission and modulation of sensation, especially pain, from trigeminal region.]]>
Animals ; Horns ; Myelin Sheath ; Neurons ; Patch-Clamp Techniques ; Potassium ; Pronase ; Rats ; Sensation ; Sodium ; Thermolysin ; Trigeminal Nucleus, Spinal

BACKGROUND AND OBJECTIVES: Medial vestibular nucleus (MVN) neurons are second-order afferent neurons that are involved in the reflex control of the head and eyes. Results from several studies utilizing the intracellular microelectrode recording techniques suggest the presence of several ionic conductances contributes to the regulation of the MVN neuron excitability in rats. In this study, the types and characteristics of voltage-dependent potassium currents were investigated in acutely isolated MVN neurons of postnatal rats. Material and Methods: Electrophysiological recordings were performed by means of the whole cell patch clamp techniques. Coronal slice (400 nm) of the vestibular nucleus region was sequentially treated with pronase 0.2 mg/ml and thermolysin 0.2 mg/ml, then single neurons were mechanically dissociated RESULTS: In a Ca2+ -free solution, low-threshold transient (IA) and high-threshold sustained (IK) currents were recorded. IK was activated (gamma=4.0-12.4 ms at 10 mV) and inactivated (gamma=180-720 ms at 10 mV) more slowly than IA. The half-maximum activation and inactivation potential were -3.1+/-3.4 mV and -38.8+/-3.6 mV, respectively. IA was activated rapidly (gamma=1.0-2.3 ms at 10 mV) and inactivated in 10-60 ms. The half-maximum activation and inactivation potentials were -22.3+/-4.5 mV and -58.4+/-3.8 mV, respectively. When a 4-aminopyridine of 10 mM was applied, IA was almost totally blocked. In a solution with 2 mM Ca2+, calcium dependent potassium currents were identified by application of a Ca2+ free solution and consisted of a transient and a sustained components. Exposure to 0.3 nM apamin induced a reversible reduction of a sustained components. CONCLUSION: These results suggest that MVN neurons express a variety of voltage-dependent potassium currents which are responsible for proper membrane excitability and firing of MVN neurons.
4-Aminopyridine ; Animals ; Apamin ; Calcium ; Fires ; Head ; Membranes ; Microelectrodes ; Neurons* ; Neurons, Afferent ; Patch-Clamp Techniques ; Potassium* ; Pronase ; Rats* ; Reflex ; Thermolysin ; Vestibular Nuclei*

This study was designed to investigate the effects an 8-Br-cGMP on the neuronal activity of rat vestibular nuclear cells. Sprague-Dawley rats aged 14 to 16 days were decapitated under ether anesthesia. After treatment with pronase and thermolysin, the dissociated vestibular nuclear cells were transferred into a chamber on an inverted microscope. Spontaneous action potentials and potassium currents were recorded by standard patch-clamp techniques under current and voltage-clamp modes. Twelve vestibular nuclear cells revealed excitatory responses to 1-5 microM of 8-Br-cGMP, and 3 neurons did not respond to 8-Br-cGMP. Whole potassium currents of vestibular nuclear cells were decreased by 8-Br-cGMP (n=12). After calcium-dependent potassium currents were blocked by tetraethylammonium, the potassium currents were not decreased by 8-Br-cGMP. These experimental results suggest that 8-Br-cGMP changes the neuronal activity of vestibular nuclear cells by blocking the calcium-dependent potassium currents that underlie the afterhyperpolarization.
Action Potentials ; Aged ; Anesthesia ; Animals ; Ether, Ethyl ; Humans ; Neurons ; Nucleotides, Cyclic ; Patch-Clamp Techniques ; Potassium ; Pronase ; Rats ; Rats, Sprague-Dawley ; Tetraethylammonium ; Thermolysin

Recent studies have implicated reactive oxygen species (ROS) as determinants of the pathological pain caused by the activation of peripheral neurons. It has not been elucidated, however, how ROS activate the primary sensory neurons in the pain pathway. In this study, calcium imaging was performed to investigate the effects of NaOCl, a ROS donor, on the intracellular calcium concentration ([Ca2+]i) in acutely dissociated dorsal root ganglion (DRG) neurons. DRG was sequentially treated with 0.2 mg/ml of both protease and thermolysin, and single neurons were then obtained by mechanical dissociation. The administration of NaOCl then caused a reversible increase in the [Ca2+]i, which was inhibited by pretreatment with phenyl-N-tert-buthylnitrone (PBN) and isoascorbate, both ROS scavengers. The NaOCl-induced [Ca2+]i increase was suppressed both in a calcium free solution and after depletion of the intracellular Ca2+ pool by thapsigargin. Additionally, this increase was predominantly blocked by pretreatment with the transient receptor potential (TRP) antagonists, ruthenium red (50 microM) and capsazepine (10 microM). Collectively, these results suggest that an increase in the intracellular calcium concentration is produced from both extracellular fluid and the intracellular calcium store, and that TRP might be involved in the sensation of pain induced by ROS.
Animals ; Calcium ; Capsaicin ; Diagnosis-Related Groups ; Dissociative Disorders ; Extracellular Fluid ; Ganglia, Spinal ; Humans ; Neurons ; Rats ; Reactive Oxygen Species ; Ruthenium Red ; Sensation ; Sensory Receptor Cells ; Spinal Nerve Roots ; Thapsigargin ; Thermolysin ; Tissue Donors