Min Of Drug Application Research Articles

Slo1 Channel Desensitization to Ethanol is Coupled to Calcium-Sensing Structures in the Channel Cytosolic Domain

Data from native BK (Dopico et al., 1996; Pietrzykowski et al., 2004) and recombinant slo1 channels (Dopico et al., 1998; Yuan et al., 2008) consistently show that ethanol-induced increase in channel steady-state activity (NPo) disappears within several minutes of continuous alcohol exposure. Ionotropic receptor desensitization is usually a monotonic function of activating ligand (Dopico & Lovinger, 2009), and slo1 channels enter a low activity mode in presence of high levels of activating ligand (i.e., Ca2+i) (Rothberg et al., 1996). Moreover, Ca2+i is required for ethanol to activate slo1 channels (Liu et al., 2008). In this work, we began to examine the Ca2+i-dependence and structural bases of slo1 channel desensitization to protracted ethanol exposure. Following BK channel-forming protein expression in membrane patches from Xenopus laevis oocytes, exposure to 50 mM ethanol increased NPo×1.5-2 fold within 1-2 min of drug application, a response that totally disappeared within 5 min of alcohol exposure (n=7). Experimental conditions rule out a major role of genetic adaptation, change in channel protein composition, channel internalization, cytosolic signaling and ethanol metabolism in slo1 channel desensitization to alcohol. Rather, the phenomenon must be attributed to ligand interaction(s) with the slo1 subunit and/or its immediate lipid environment. Desensitization to ethanol was observed whether the slo1 channel was studied at 0.3, 1 or 10 μM Ca2+i, indicating that slo1 channel desensitization to ethanol is not significantly modified within Ca2+i levels that correspond to the physiological range. However, two constructs containing amino acid substitutions in the channel cytosolic domain that alter Ca2+i-sensing by slo1 (5D5N in calcium bowl; D362A/D367A in RCK1) displayed a significant delay in channel desensitization to ethanol. Thus, Ca2+i-sensing structures in the slo1 cytosolic domain are functionally coupled to ethanol-desensitization processes.Support: R37-AA011560 (AMD).

Tissue PO 2 and the effects of hypoxia on the generation of locomotor-like activity in the in vitro spinal cord of the neonatal mouse

The neonatal mouse en bloc spinal cord-brainstem preparation used in combination with advances in mouse genomics provides a novel strategy for studying the spinal control of locomotion. How well the mouse en bloc preparation is oxygenated however, is unknown. This is an important consideration given that (a) other superfused mammalian en bloc preparations have anoxic cores and (b) hypoxia can have profound effects on neuronal activity. Here we measure the level of tissue oxygenation in the mouse preparation and determine how neuronal activity within the spinal cord is influenced by poor superfusion and/or low oxygen. To measure tissue oxygenation, oxygen depth profiles were obtained (P0–1 and P2–3; Swiss Webster mice). At P0–1, spinal cords were oxygenated throughout under resting conditions. When fictive locomotor activity was evoked (5-HT 10 μM, dopamine 50 μM, NMA 5 μM), there was a substantial reduction in tissue PO 2 starting within 5 min of drug application. Following washout, the PO 2 slowly returned to control levels over a period of 30 min. The experiments described above were repeated using P2–3 preparations. In this older age group, the spinal cord preparations had a hypoxic/anoxic core that was exacerbated during metabolically demanding tasks such as drug-evoked rhythmic activity. To examine how an anoxic core affects neuronal activity within the spinal cord we either altered the flow-rate or manipulated superfusate PO 2. When the flow-rate was reduced a transient disruption in the rhythmicity of drug-induced locomotion occurred during the first 15 min (P0–1 preparations). However, the motor output adapted and stabilized. During prolonged superfusion with hypoxic artificial cerebrospinal fluid on the other hand, both the motor bursts in spinal nerves and the activity of most neurons near the center of the tissue were abolished. Overall, this study suggests that while oxygenation of P0–P1 preparations is adequate for studies of locomotor function, oxygenation of older preparations is more problematic. Our data also show that neonatal spinal neurons require oxygen to maintain activity; and the spinal locomotor rhythm generator continues to function providing the peripheral tissue of the cord is oxygenated. Together, these results are consistent with the results of a previous study which suggest that the locomotor pattern generator is located close to the surface of the spinal cord.

Ability of 5-HT4 receptor ligands to modulate rat striatal dopamine release in vitro and in vivo.

1. The ability of 5-HT4 (5-hydroxytryptamine4) receptor ligands to modify dopamine release from rat striatal slices in vitro and in the striatum of freely moving rats was assessed by the microdialysis technique. 2. The release of dopamine from slices of rat striatum continually perfused with Krebs buffer was enhanced by 5-HT4 receptor agonists; 5-HT (10 microM), 5-methoxytryptamine (5-MeOT; 10 microM), renzapride (10 microM) and (S)-zacopride (10 microM) maximally increased dopamine release by 133 +/- 5, 214 +/- 25, 232 +/- 29 and 264 +/- 69%, respectively (mean +/- s.e.mean, n = 3-8). The drug-induced responses were maximal within the first 2 min of drug application, and subsequently declined. The non-selective 5-HT3/5-HT4 receptor antagonist, SDZ205-557 (10 microM), failed to modify basal dopamine release from striatal slices but completely antagonized the (S)-zacopride (10 microM)-induced increase in dopamine release. 3. To allow faster drug application, the modulation of dopamine release from rat striatal slices in a static release preparation was also investigated. The 5-HT4 receptor agonist, renzapride (10 microM) also enhanced dopamine release in this preparation (maximal increase = 214 +/- 35%, mean +/- s.e.mean, n = 14), whilst a lower concentration of renzapride (3 microM) was less effective. The renzapride-induced response was maximal within the first 2 min of drug application, before declining. In this preparation, the stimulation of dopamine release by renzapride (10 microM), was completely antagonized by the selective 5-HT4 receptor antagonist, GR113808 (100 nM). In addition, both the Na+ channel blocker, tetrodotoxin (100 nM) and the non-selective protein kinase A inhibitor, H7 (100 nM) completely prevented the stimulation of dopamine release induced by renzapride (10 microM). 4. In vivo microdialysis studies demonstrated that the 5-HT4 receptor agonists, 5-MeOT (10 microM), renzapride (100 microM) and (S)-zacopride (100 microM) maximally elevated extracellular levels of dopamine in the striatum by 220 +/- 20, 161 +/- 10 and 189 +/- 53%, respectively (mean +/- s.e.mean, n = 5-9). A lower concentration of renzapride (10 microM) was less effective. The elevation of extracellular striatal dopamine levels induced by either renzapride (100 microM) or (S)-zacopride (100 microM) were completely antagonized by the non-selective 5-HT4 receptor antagonist, SDZ205-557 (100 microM). In addition, the elevation of extracellular levels of dopamine induced by either 5-MeOT (10 microM) or renzapride (100 microM) was completely prevented by the selective 5-HT4 receptor antagonist, GR113808 (1 microM) and the renzapride (100 microM)-induced response was also completely prevented by the non-selective protein kinase A inhibitor, H7 (1 microM). In this in vivo preparation, both GR113808 (1 microM) and H7 (1 microM), when perfused alone, reduced extracellular levels of dopamine. 5. In conclusion, the present study provides evidence that the 5-HT4 receptor facilitates rat striatal dopamine release in vitro and in vivo.

An iontophoretic analysis of the pharmacologic mechanisms responsible for trigeminal motoneuronal discharge during masticatory-like activity in the guinea pig

1. The effects of iontophoretic application of the excitatory amino acid antagonists kynurenic acid (KYN) and DL-2-amino-5-phosphonovaleric acid (APV), as well as the monoamines serotonin (5-HT) and norepinephrine (NE), on extracellularly recorded jaw opener motoneuron [digastric motoneuron (DIG)] discharge during cortically induced rhythmical masticatory-like activity (RMA) were examined in the anesthetized guinea pig. 2. Iontophoretic application of KYN, a broad-spectrum amino acid antagonist, suppressed the motoneuronal discharge evoked by short pulse train stimulation of the cortex for most cells tested. In contrast, iontophoretic application of APV, a specific N-methyl-D-aspartate (NMDA) antagonist, was usually without effect on the motoneuronal discharge evoked by short pulse train stimulation. 3. During RMA evoked by repetitive cortical stimulation, both KYN and APV suppressed rhythmical DIG motoneuronal discharge in many cells tested. 4. These data suggest that excitatory amino acid receptors on jaw opener motoneurons are involved in activation of RMA. It is proposed that the short-latency rapid excitation of jaw opener motoneurons, which occurs during both short pulse train cortical stimulation and RMA induced by repetitive cortical stimulation, is mediated, at least in part, by non-NMDA receptors. It is further suggested that the large-amplitude, long-duration slow rhythmical oscillations, which occur in the membrane potential of jaw opener motoneurons during RMA induced by repetitive cortical stimulation, are mediated, at least in part, by NMDA receptors. 5. Iontophoretic application of NE or 5-HT with low currents (less than 20 nA) produced a facilitation of digastric motoneuronal discharge during cycle-triggered glutamate application, short pulse train cortical stimulation, and RMA evoked by repetitive cortical stimulation. These facilitatory effects on motoneuronal discharge started within 1 min of drug application, reached a peak at approximately 3 min that persisted for several minutes after the application period, and recovered to control levels within 10-15 min. Direct application of NE or 5-HT, in the absence of chemical or synaptic activation, failed to activate these motoneurons. However, iontophoretic application of either monoamine could facilitate and bring to threshold rhythmical motoneuronal discharges during subthreshold repetitive cortical stimulation. 6. Iontophoretic application of methysergide, a 5-HT antagonist, and phentolamine, an alpha adrenoreceptor blocker, both produced a selective and reversible blockade of the facilitatory effects of 5-HT and NE, respectively, on motoneuronal discharge during cortically induced RMA. In contrast, iontophoretic application of sotalol, a beta adrenoreceptor blocker, had no effect on the NE-induced facilitation of RMA.(ABSTRACT TRUNCATED AT 400 WORDS)