Diversity of Kir channel subunit mRNA expressed by retinal glial cells of the guinea-pig

One of the main functions of Müller glial cells is the performance of retinal K+ homeostasis which is thought to be primarily mediated by K+ fluxes through inwardly rectifying K+ (Kir) channels expressed in Müller cell membranes. Until now, there is limited knowledge about the types of Kir channel subunits expressed by Müller cells. Using RT-PCR, we investigated the expression of mRNA encoding different Kir channel subunits in the retina of the guinea pig. In order to verify expression by Müller cells, primary cultures of guinea pig Müller cells were also investigated. Both retinae and cultured Müller cells express mRNA for a diversity of Kir channel subtypes which include members of at least four channel subfamilies: Kir2.1, Kir2.2, Kir2.4, Kir3.1, Kir 3.2, Kir4.1, Kir6.1, and Kir6.2. mRNAs for the following Kir channel subtypes were not detected in Müller cells: Kir1.1, Kir2.3, Kir3.3, Kir3.4, Kir4.2, and Kir5.1. It is concluded that the spatial buffering of extracellular K+ by Müller cells may be mediated by cooperation of different subtypes of Kir channels, and that the distinct Kir channel types involved in this function may change depending on the physiological or metabolic state of the retina.     

Hypertension in the Elderly

In this review article, the authors cover the pathophysiology of elevated blood pressure in the elderly. Isolated systolic hypertension and pseudo-hypertension are noted. Some of the large treatment trials are discussed. Nonpharmacologic and pharmacologic management are covered. Neurologic aspects of hypertension in this population are discussed in some detail. Additional considerations regarding medication choices in the last part are noted.     

Rat nicotinic ACh receptor α7 and β2 subunits co-assemble to form functional heteromeric nicotinic receptor channels

Rat hippocampal interneurons express diverse subtypes of functional nicotinic acetylcholine receptors (nAChRs), including α7-containing receptors that have properties unlike those expected for homomeric α7 nAChRs. We previously reported a strong correlation between expression of the α7 and of the β2 subunits in individual neurons. To explore whether co-assembly of the α7 and β2 subunits might occur, these subunits were co-expressed in Xenopus oocytes and the functional properties of heterologously expressed nAChRs were characterized by two-electrode voltage clamp. Co-expression of the β2 subunit, both wild-type and mutant forms, with the α7 subunit significantly slowed the rate of nAChR desensitization and altered the pharmacological properties. Whereas ACh, carbachol and choline were full or near-full agonists for homomeric α7 receptor channels, both carbachol and choline were only partial agonists in oocytes expressing both α7 and β2 subunits. In addition the EC₅₀ values for all three agonists significantly increased when the β2 subunit was co-expressed with the α7 subunit. Co-expression with the β2 subunit did not result in any significant change in the current-voltage curve. Biochemical evidence for the co-assembly of the α7 and β2 subunits was obtained by co-immunoprecipitation of these subunits from transiently transfected human embryonic kidney (TSA201) cells. These data provide direct biophysical and molecular evidence that the nAChR α7 and β2 subunits co-assemble to form a functional heteromeric nAChR with functional and pharmacological properties different from those of homomeric α7 channels. This co-assembly may help to explain nAChR channel diversity in rat hippocampal interneurons, and perhaps in other areas of the nervous system.     

The effect of preconditioning on the Cu, Zn superoxide dismutase expression and enzyme activity in rat brain at the early period after severe hypobaric hypoxia

Severe hypoxia results in functional and structural injury of the brain. A preconditioning with repetitive episodes of mild hypoxia considerably ameliorates neuronal resistance to subsequent severe hypoxia. Activation of endogenous antioxidants including Cu, Zn-depending superoxide dismutase (Cu, Zn-SOD) (EC.1.15.1.1) is one of the main cell defense mechanisms against oxidative stress induced by hypoxia. Alterations of expression and enzyme activity of Cu, Zn-SOD 3 and 24h after severe hypobaric hypoxia in forebrain structures of preconditioned and non-preconditioned rats were investigated. We found that hypoxia without preconditioning suppressed the Cu, Zn-SOD enzyme activity at 3h time-point but preconditioning essentially modified the reaction to severe hypoxia by increasing the expression and activity of Cu, Zn-SOD during early stages of reoxygenation crucial for apoptosis initiation.     

Effects of 4-Aminopyridine on Cloned hERG Channels Expressed in Mammalian Cells

Introduction.  Non-clinical evaluation of a medication's potential to induce cardiac toxicity is recommended by regulatory agencies. 4-Aminopyridine (fampridine) is a potassium channel blocker with the demonstrated ability to improve walking ability in patients with multiple sclerosis. We evaluated the in vitro effects of 4-aminopyridine on the human ether-à-go-go -related gene (hERG) channel current, since hERG current inhibition is associated with QT interval prolongation—a precursor to torsade de pointes (TdP).
Methods.  4-Aminopyridine was evaluated in concentrations ranging from 0.1 mM to 30 mM in human embryonic kidney 293 cells stably transfected with the hERG gene; terfenadine 60 nM was used as a positive control.
Results and Discussion.  We observed concentration-dependent inhibition of hERG current with 4-aminopyridine doses between 0.3 and 30 mM. The concentration of 3.8 mM resulting in 50% inhibition (IC₅₀) is approximately three orders of magnitude higher than expected therapeutic plasma concentrations, suggesting 4-aminopyridine has low potential for prolonging QT interval or inducing TdP.     

Deferoxamine improves early postresuscitation reperfusion after prolonged cardiac arrest in rats.

The no-reflow phenomenon and delayed hypoperfusion after transient cardiac arrest (CA) impede postischemic recovery. Activation of lipid peroxidation (LPO) after ischemia and reperfusion is considered one of the mechanisms responsible for such abnormalities. The present study investigates the influence of iron-dependent LPO inhibitor deferoxamine (DFO) on the cerebral perfusion after prolonged CA and resuscitation. Fourteen male Sprague-Dawley rats were subjected to 17 minutes of CA, induced by esmolol (an ultrashort-acting beta-blocker) and apnea, followed by resuscitation by retrograde intraaortic infusion of oxygenated donor blood mixed with a resuscitation cocktail inside a vertical-bore 9.4-T magnetic resonance imaging (MRI) magnet. Animals were randomized double-blindly into two groups to receive DFO or saline, respectively. Cerebral perfusion was measured by MRI continuously using the arterial spin-labeling method before, during, and after CA. All animals were successfully resuscitated in 1.36 +/- 0.04 minutes with well-controlled arrest time (17.99 +/- 0.03 minutes) in both groups. Deferoxamine significantly increased cerebral perfusion in hippocampus, thalamus, hypothalamus, and amygdala, but not in cortex, during the first 20 minutes of reperfusion. In the DFO-treated group, the neurologic deficit score was significantly better (400 +/- 30 vs. 250 +/- 47, out of 500 as the best, P < 0.05) and weight loss was significantly less (33 +/- 6 vs. 71 +/- 19 g, P < 0.05) 5 d after arrest. The finding supports the notion that early reperfusion immediately after resuscitation is important for long-term outcome and that LPO may be involved in microvascular disorders during the reperfusion, particularly in the brain after prolonged cardiac arrest and resuscitation.     

Enhancement of growth and survival and alterations in Bcl-family proteins in beta-thalassemic erythroid progenitors by novel short-chain fatty acid derivatives

Accelerated apoptosis of erythroid progenitors is a characteristic of beta-thalassemia which presents a significant barrier to definitive therapeutic approaches utilizing induction of endogenous fetal globin gene expression. gamma-globin gene expression may not be inducible in, or may not be able to rescue, erythroid cells in which programmed cell death is initiated early in erythroblast development. In this report, short-chain fatty acid derivatives (SCFADs) which induce fetal globin gene expression were tested for their ability to promote proliferation and survival of erythroid progenitors cultured from beta-thalassemic subjects, and of cytokine-dependent erythroid cell lines. Certain SCFADs promoted thalassemic Bfu-e growth and cytokine-independent growth and survival of erythroid cell lines. A 40-80% increase in erythroid Bfu-e colony number was observed in cultures established with any of five mitogenic SCFADs, compared to control or butyrate-treated cultures from the same subjects. Immunoblot analysis demonstrated that these same SCFADs also regulated the expression of specific protein inhibitors of apoptosis. Anti-apoptotic ratios of the proteins Bcl-xL/Bcl-xS in thalassemic Bfu-e were increased by 30-120% with exposure to the SCFDs, compared to the ratios in the same cells cultured under control conditions. Similar anti-apoptotic increases in Mcl-1L/Mcl-1S ratios were induced by the SCFADs. These findings suggest that select fetal globin-inducing SCFADs which enhance proliferation of beta-thalassemia progenitors may enhance survival of these progenitors by altering levels of Bcl-family protein members. This combination of effects should enhance erythroid cell survival in the beta-thalassemia syndromes, allowing fetal globin gene expression to be induced more effectively than currently available, growth-suppressing, fetal globin-inducing agents, such as the butyrates or chemotherapeutic agents.     

Cortical and limbic excitability in rats with absence epilepsy

The classical cortico-reticular theory on absence epilepsy suggests that a hyperexcitable cortex is a precondition for the occurrence of absence seizures. In the present experiment seizure thresholds and characteristics of cortical and limbic epileptic afterdischarges (AD) were determined in a comparative cortical stimulation study in young and old adult genetically epileptic WAG/Rij, congenic ACI and Wistar rats. Fifteen-second series of 8Hz stimulation of the sensory-motor cortex were applied in 80- and 180-day-old rats with implanted electrodes. Strain differences were found for the threshold for movements directly induced by stimulation, low frequency spike-and-wave AD, maximal clonic intensity of seizures accompanying direct stimulation, and frequency characteristics of low frequency AD. None of these results agreed with a higher cortical excitability exclusively in WAG/Rij rats. However, WAG/Rij rats had the longest duration of the low frequency AD, and the lowest threshold for the transition to the limbic type of AD. The decrease of this threshold correlated with the increase of the incidence and total duration of spontaneous SWDs in WAG/Rij rats. It is concluded that the elevated excitability of the limbic system or pathways mediating the spread of the epileptic activity into this system can be attributed to the development of genetic epileptic phenotype in WAG/Rij rats.