Biomedical subjects

D L Small

Publications and source records attributed to D L Small.

27 records · Page 2Linked to original sources

PubMed1997

Antagonism of N-methyl-D-aspartate-evoked currents in rat cortical cultures by ARL 15896AR.

The purpose of this study was to characterize the kinetics and voltage-dependence of the block of N-methyl-D-aspartate (NMDA)-induced currents in primary cultures of rat cortical neurons by the neuroprotective, low-affinity, NMDA antagonist ARL 15896AR, using whole-cell voltage-clamp techniques. ARL 15896AR caused rapid and reversible inhibition of NMDA (50 microM)-evoked currents from neurons held at -60 mV, with an IC50 of 9.8 microM. The EC50 for NMDA was not significantly affected by 10 microM ARL 15896AR (P > .05), consistent with a noncompetitive mechanism of block. ARL 15896AR antagonism was use-dependent, because application of the drug 60 sec before NMDA did not attenuate the initial NMDA-evoked current, although the block developed rapidly thereafter. Once bound, ARL 15896AR remained trapped upon removal of NMDA until subsequent NMDA re-exposure, whereupon currents recovered rapidly. The forward and reverse binding rate constants were estimated to be 2.406 x 10(4) M(-1) sec(-1) and 0.722 sec(-1), respectively. Antagonism was strongly voltage-dependent; the K(D) values at 0 and -60 mV were 60 and 11 microM, respectively. Additionally, there was a component of the block by ARL 15896AR that was voltage-insensitive. This component of the block did not act at the ligand binding site, because it was not influenced by NMDA concentration, or at the polyamine site, because it was not affected by spermine. However, there was an interaction of ARL 15896AR with the glycine regulatory site. In contrast to many uncompetitive NMDA antagonists, like MK-801, ARL 15896AR exhibited rapid kinetics. This property may result in a large margin of safety while maintaining the efficacy associated with use-dependent NMDA antagonists, making this compound an excellent candidate for clinical trials.

Animals↗

PubMed1996

Mechanisms of 1S,3R-ACPD-induced neuroprotection in rat hippocampal slices subjected to oxygen and glucose deprivation.

The efficacy and mechanisms of 1-amino-cyclopentyl-1S,3R-dicarboxylate (1S,3R-ACPD)-induced neuroprotection were investigated in rat hippocampal slices subjected to 10 min of oxygen and glucose deprivation. Neuronal viability was assessed by measuring both the amplitude of evoked population spike in the CA1 pyramidale and by imaging CA1 neurons using a live/dead fluorescence assay with confocal microscopy. CA1 pyramidal neurons in oxygen-glucose deprived slices remained viable for up to 120 min following the insult but were dead by 240 min. Pretreatment with 1S,3R-ACPD significantly protected the oxygen-glucose deprived slices in a concentration-dependent fashion. Oxygen-glucose deprived slices pretreated for the same period with the protein kinase C (PKC) activation phorbol 12-myristate 13-acetate (PMA; 1 microM) were significantly protected whereas oxygen-glucose deprived slices treated with the adenylyl cyclase activator, forskolin (30 microM) were not. Oxygen-glucose deprivation induced a rapid and persistent decrease (approximately 50%) in PKC activity and a > 6 fold increase in cyclic adenosine monophosphate (cAMP) levels in whole hippocampal slices. While 1S,3R-ACPD did not stimulate PKC activity and had no effect on basal cAMP in whole slices, it significantly enhanced the rate of return of cAMP to basal levels following reperfusion. Consistent with this observation, the 1S,3R-ACPD-induced neuroprotection was inhibited by forskolin (30 microM). These results suggest that in vitro neuroprotection of CA1 neurons by 1S,3R-ACPD involves metabotropic glutamate receptors negatively linked to cAMP and possibly those which increase PKC activity.

Animals↗

PubMed1996

Mechanisms of cerebral ischemia: intracellular cascades and therapeutic interventions.

In order to gain insight into the pathophysiology of cerebral ischemia, the focus is on the discrete compartmentalization of neurons and the exquisite homeostasis of the neurochemical, ionic, and molecular environment within these compartments. This review looks at excitotoxic mechanisms of cerebral ischemia spatially by separating presynaptic and postsynaptic events as well as temporally by separating early and late events. Drugs that target these events in the excitotoxic cascade are presented and discussed as potential therapeutic interventions for cerebral ischemia. Despite a better understanding of the mechanisms of cerebral ischemia through a myriad of animal model studies with various "neuroprotective" compounds, the challenge remains to apply this knowledge to the development of compounds that demonstrate neuroprotective efficacy in terms of quality-of-life outcomes in humans.

Animals↗

PubMed1995

Neuroprotective effects of omega-Aga-IVA against in vitro ischaemia in the rat hippocampal slice.

Excessive accumulation of Ca2+ in neurones and glutamate release are involved in neuropathological processes, including ischaemia. We investigated the neuroprotective effects of the Ca2+ channel antagonist, omega-Aga-IVA, in CA1 pyramidal neurones in rat hippocampal slices following an in vitro hypoxic-hypoglycaemic insult. Following this insult, evoked post-synaptic response amplitudes decreased from 3.7 +/- 0.5 mV to 0.6 +/- 0.2 mV and the CA1 neurones appeared dead using a live/dead fluorescence assay with confocal microscopy. Slices treated with 200 nM omega-Aga-IVA had evoked response amplitudes not significantly different from control (3.3 +/- 0.5 mV) and the CA1 neurones appeared viable using the live/dead fluorescence assay. The neuroprotective efficacy of omega-Aga-IVA suggests that omega-Aga-IVA-sensitive Ca2+ channels participate in ischaemic neuronal death and constitute a potential target of therapeutic intervention.

Animals↗

PubMed1995

Pharmacology of stretch-activated K channels in Lymnaea neurones.

1. Single-channel recording was used to describe the pharmacology of stretch-activated K channels in Lymnaea neurones using channel blockers amiloride, tetraethylammonium (TEA), quinidine, gadolinium (Gd) and diltiazem. 2. Amiloride, TEA and quinidine applied to the outside face of the membrane all produced a fast flickery block of stretch-activated K channels. All of these agents were without effect when applied at the inside face at concentrations as high as 10, 200 and 10 mM respectively. Neither Gd nor diltiazem had any effect on stretch-activated K channels extracellularly (100 microM). 3. Amiloride, TEA and quinidine block were voltage-independent with IC50 values at positive (and negative membrane potentials of 2.3 (and 2.0) mM, 48 (and 54) mM and 0.8 (and 0.7) mM respectively. Woodhull plots for TEA and quinidine block confirmed the voltage independence of stretch-activated K channel block by these agents. 4. Hill plots of the amilorde, TEA and quinidine block yield Hill coefficients at positive (and negative) membrane potentials of 1.7 (and 1.5), 1.4 (and 1.2) and 1.5 (and 1.6 mM) respectively. 5. Ethanol (3%) had no apparent effect on stretch-activated K channel kinetics or conductance yet reduced the efficacy of quinidine block. 6. The above pharmacological fingerprint of the stretch-activated K channel is discussed with reference to other K-selective and stretch-activated channels.

Amiloride↗

PubMed1994

FMRFamide and membrane stretch as activators of the Aplysia S-channel.

The long-standing distinction between channels and transporters is becoming blurred, with one pump protein even able to convert reversibly to a channel in response to osmotic shock. In this light, it is plausible that stretch channels, membrane proteins whose physiological roles have been elusive, may be transporters exhibiting channel-like properties in response to mechanical stress. We recently described a case, however, where this seems an unlikely explanation. An Aplysia K channel whose physiological pedigree is well established (it is an excitability-modulating conductance mechanism) was found able to be activated by stretch. Here we establish more firmly the identity of this Aplysia conductance, the S-channel, as a stretch channel. We show that the permeation and fast kinetic properties of the stretch-activated channel and of the FMRFamide-activated S-channel are indistinguishable. We have also made progress in extending the kinetic analysis of the stretch channel to situations of multiple channel activity. This analysis implements a novel renewal theory approach and is therefore explained in some detail.

Animals↗

PubMed1994

Delayed activation of single mechanosensitive channels in Lymnaea neurons.

Some stretch-activated (SA) channels challenged with suction jumps exhibit adaptation, a dynamic behavior that can be overlooked because of its mechanical fragility. In previous studies of neuronal SA K channels, we detected no adaptation, but the protocols used were not designed to detect dynamics. Here, we reproduce the adaptation seen by others in Xenopus SA cationic (Cat) channels but show that, with the same protocol, no adaptation occurs with SA K channels. Instead, SA K channels exhibit a different dynamic behavior, delayed activation. Lymnaea SA K channels subjected to pressure jumps responded after a 1- to 4-s delay with a gradual, rather than abrupt, onset of activation. The delay was pressure dependent and was longer for patches from older cultured neurons. Delayed responses were fragile like SA Cat channel adaptation; they disappeared with repeated stimuli. Cytochalasin D decreased the delay and increased the stretch activation of SA K channels. Unlike SA Cat channel adaptation, which occurs only at hyperpolarized potentials, SA K channel delay was not voltage dependent. We note that once SA Cat and SA K channels are "stripped" of their fragile (cytoskeleton-dependent?) dynamics, however, their gating behaviors show little fundamental difference; both are stretch activatable and have a higher open probability at depolarized potentials.

Adaptation, Physiological↗

PubMed1992

Endothelium-independent potentiating effects of neuropeptide Y in the rat tail artery.

The role of the endothelium in the potentiating action of neuropeptide Y (NPY) to contraction induced by KCl, alpha, beta-methylene ATP (mATP), and noradrenaline (NA) was tested on rat tail arteries. Endothelium-intact and denuded ring segments and freshly isolated single smooth muscle cells were used in the study. Contraction responses to KCl and mATP were potentiated by NPY (50 nM) in both intact and denuded arteries. Contraction to NA was potentiated by NPY at 500 nM but not at 50 nM. The potentiation effect of NPY was antagonized by nifedipine. Similarly, the shortening of single smooth muscle cells in response to KCl and mATP was potentiated by NPY (50 nM). The noradrenaline response was potentiated by NPY at 500 nM but not at 50 nM. Our results suggest that the potentiating effect of NPY is more specific to contraction mediated by nifedipine-sensitive calcium channels and is not dependent on the presence of an intact endothelium.

Adenosine Triphosphate↗

PubMedDate not supplied

Biology of ischemic cerebral cell death.

With the approval of alteplase (tPA) therapy for stroke, it is likely that combination therapy with tPA to restore blood flow, and agents like glutamate receptor antagonists to halt or reverse the cascade of neuronal damage, will dominate the future of stroke care. The authors describe events and potential targets of therapeutic intervention that contribute to the excitotoxic cascade underlying cerebral ischemic cell death. The focal and global animal models of stroke are the basis for the identification of these events and therapeutic targets. The signalling pathways contributing to ischemic neuronal death are discussed based on their cellular localization. Cell surface signalling events include the activities of both voltage-gated K+, Na+, and Ca2+ channels and ligand-gated glutamate, gamma-aminobutyric acid and adenosine receptors and channels. Intracellular signalling events include alterations in cytosolic and subcellular Ca2+ dynamics, Ca2+ -dependent kinases and immediate early genes whereas intercellular mechanisms include free radical formation and the activation of the immune system. An understanding of the relative importance and temporal sequence of these processes may result in an effective stroke therapy targeting several points in the cascade. The overall goal is to reduce disability and enhance quality of life for stroke survivors.

Animals↗