Aging decreases the contribution of MaxiK channel in regulating vascular tone in mesenteric artery by unparallel downregulation of α- and β1-subunit expression

Vascular disease increases in incidence with age and is the commonest cause of morbidity and mortality among elderly people. Large-conductance Ca²⁺-activated K⁺(MaxiK) channel, with pore-forming α-subunit and modulatory β1-subunit, is a key regulator of vascular tone. This study explored functional and molecular evidence of MaxiK alteration with aging in the mesenteric artery(MA). Young, Middle-aged, and Old male Wistar rats were used. Selective MaxiK channel blocker (Iberiotoxin) induced a significant increase of vascular tension in MA in all three age groups. However, these effects were greatly decreased in Old animals. The amplitude and frequency of spontaneous transient outward currents were significantly decreased with aging. Single channel recording revealed that aging induced a decrease of the open probability and the mean open time, but an increase of the mean closed time of MaxiK channel. The Ca²⁺/voltage sensitivity of MaxiK was also decreased. Western blotting showed that the protein expression of MaxiK β1- and α-subunit was significantly reduced with aging, and the suppression of β1 subunits was larger than that of α subunits. These data suggest that aging decreases capability of MaxiK channel in regulating vascular tone in the MA, which may be partially mediated by unparallel downregulation of α- and β1-subunit expression.     

In vitro evidence that endothelium-dependent vasodilatation induced by clozapine is mediated by an ATP-sensitive potassium channel

BackgroundThere is a definite association between antipsychotic drugs and arterial hypertension. However, endothelium functions are scarcely considered. This investigation was carried out to study the mechanisms involved in clozapine endothelium-dependent vascular reactivity.MethodsThe experimental animals were male Wistar rats with a mean age of 70–90 days (250–300 g). The endothelium-dependent vascular reactivity was studied by measuring the isometric force and then constructing clozapine concentration-response curves. The force registrations were obtained in the aorta rings with and without the endothelium precontracted with phenylephrine (PE10^?6M) treatment; this followed incubation for 30 min in “organ chambers” with different inhibitors: l- NAME (nitric oxide/cGMP); indomethacin (PGI2/cAMP); tetraethylammonium (TEA), and specific hyperpolarization blockers (paxillin, apamin, glibenclamide). The data were presented as the mean ± standard error of the mean (SEM) and were compared by one-way ANOVA or two-way ANOVA followed by the Bonferroni post-test.ResultsThe primary outcomes were: 1) Clozapine-induced endothelium-dependent relaxation was not inhibited by indomethacin, l-NAME, ODQ, and methylene blue (MB); 2) The combination of l-NAME + indomethacin partially prevented the relaxation; 3) Clozapine did not induce relaxation in vessels contracted with KCl; 4) TEA did not block the clozapine-induced relaxation in vessels precontracted with PE (10^?6 M); 5) The potassium channel blockers paxillin and apamin did not prevent relaxation but glibenclamide did.ConclusionConcerning the mechanisms involved in clozapine endothelium-dependent vascular reactivity, the present study suggests that there is synergistic participation that probably occurs through a crosstalk mechanism of the cAMP, cGMPpathways and hyperpolarization.     

Phencyclidine blocks two potassium currents in spinal neurons in cell culture

We studied the effects of phencyclidine (PCP) on the transient and delayed outward K+ currents recorded from spinal cord neurons grown (10-20 days) in cell culture. Sodium channels were blocked with tetrodotoxin (1 microM) and solutions containing low calcium concentrations in the presence of Mg2+ or Co2+ (5 mM) were used to reduce Ca2+ currents. PCP decreased the amplitude and prolonged the decay phase of the action potentials recorded at a holding potential of -70 mV. PCP (0.1-0.5 mM) was more effective than tetraethylammonium (TEA) or 4-aminopyridine (4-AP) in reducing both transient and delayed currents. The amplitude of the transient current during control experiments was always larger than that of the delayed current. It appeared that 4-AP (5 mM) was more potent in blocking the transient current, while TEA (10 mM) modified the delayed current more effectively. Both currents were also reduced by about 10% when the cell soma was perfused with Co2+. This suggested that a small fraction of the total outward current is a Ca2+-activated K+ current. The PCP-induced blockade of K+ currents in central neurons coupled with the profound synaptic effects of the drug may provide the basis for explaining the psychopathology of this hallucinogenic agent.     

Isolation and kinetic analysis of inward currents in neuroblastoma cells

The suction pipette method for combined voltage clamp and intracellular dialysis was applied to isolate the two components of voltage-gated inward current across membranes of NIE-115 neuroblastoma cells. In order to analyze the kinetic behavior of the Na+ and Ca2+ channels responsible for generating these components, current through K+ channels was effectively blocked by substituting impermeant Cs+ for internal and external K+. Block was confirmed independently by examining the effects of the application of external tetraethylammonium or Cd2+; and comparing the time course of Ca2+ tail currents with the decay of current during a maintained depolarization. Na+ currents studied at 8-10 degrees C, developed as a fourth order process giving a maximum e-fold conductance change for a 3 mV depolarization, with half activation occurring at -10 mV. The instantaneous current-voltage relationship was linear. Time constants of the activation parameter (m) varied from 0.5 ms (-50 mV) to 3-4 ms (-10 to -40 mV) at 10 degrees C. Inactivation (h) was a first order process having a time constant between 4 ms (+10 to +60 mV) and 225 ms (-60 mV). Steady-state inactivation for Na+ channels attained a value of 0.5 at -50 mV. A slow inactivation process, however, also is involved in gating of Na+ channels, and has a time course at least two orders of magnitude slower than that for h. The temperature sensitivity of Na+ currents was found to be similar to that found for other preparations. Ca2+ currents were studied at 24-29 degrees C in the presence of internal ethyleneglycolbis-(aminoethylether)-tetra-acetate (EGTA) and an external Ca2+ concentration of 20 mM. Ca2+ channel activation could also be described by a fourth order process giving an e-fold conductance change for a 5-6 mV change in potential and the half activation potential of -13 mV. Internal EGTA (20 mM) did not abolish inactivation of Ca2+ currents and no recovery from inactivation caused by a prepulse could be measured as the prepulse potential approached the null potential for Ca2+ influx. Time constants of both activation and inactivation of Ca2+ channels were measured between -20 and +50 mV. Currents through K+ channels could be completely eliminated by substitution of K+ with Cs+, although a residual non-linear leakage current remained, in addition to currents through the Na+ and Ca2+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Calcium-dependent potassium conductance studied on internally dialysed nerve cells

The Ca-dependent tetraethylammonium-resistant potassium conductance was studied in the membrane of internally dialysed isolated snail neurones. In these conditions the tetraethylammoniumresistant noninactivating outward potassium current decreased when the transmembrane influx of Ca²⁺ was suppressed either by blocking the calcium channels with external Cd²⁺ or internal F^? or by substituting Mg²⁺ for Ca²⁺ in the external solution. Elevation of intracellular Ca²⁺ concentration by dialysing the cell with solution containing Ca-EGTA buffer resulted in an increase of potassium outward current. The effect could be measured with Ca²⁺ concentrations as low as 10^?7 M. Much higher concentrations of Sr²⁺ or Ba²⁺ inside the cell did not potentiate the potassium current. The corresponding ionic channels are shown to be less selective for potassium ions as compared to the Ca-insensitive voltage-dependent channels.A conclusion is made that the changes in Ca²⁺ concentration near the inner surface of the membrane are the key factor which makes these channels ready for activation.     

Prevalence of head deformities in preterm infants at term equivalent age

Introduction

Due to a rising number of head deformities in healthy newborns, there has been an increasing interest in nonsynostotic head deformities in children over recent years. Although preterm infants are more likely to have anomalous head shapes than term newborns, there is limited data available on early prevalence of head deformities in preterm infants.

Aims

The purposes of the present study were to acquire quantitative data on head shape of preterm infants at Term Equivalent Age (TEA), to determine the prevalence of symmetrical and asymmetrical head deformities and to identify possible risk factors.

Methods

In a cross-sectional study design, Cranial Vault Asymmetry Index (CVAI) and Cranial Index (CI) calculated from routine head-scans with a non-invasive laser shape digitizer were recorded and categorized in type and severity of deformation for three different groups of gestational age. Perinatal and postnatal patient data was tested for possible associations.

Results

Scans of 195 infants were included in the study. CVAI at TEA was higher in very preterm (4.1%) compared to term and late preterm infants. Prevalence of deformational plagiocephaly was 38% in very preterm infants. CI was lower in very (71.4%) and late (77.2%) preterm infants compared to term infants (80.0%). Compared to term babies (11%), a large number of very (73%) and late (28%) preterm infants exhibited dolichocephaly at TEA.

Discussion

Prevalence of symmetrical and asymmetrical head deformities in preterm infants is high at TEA. Interventions are required to prevent head deformities in preterm infants during the initial hospital stay.     

Synergistic toxicity of carbon tetrachloride and several aromatic organohalide compounds

Subacute (20 days) oral administration of hexachlorobenzene (HCB) or the organohalide mixtures polybrominated biphenyls (PBB) or polychlorinated biphenyls (PCB) greatly increased th susceptibility of male rats to the toxic effects of carbon tetrachloride (CCl4). CCl4-induced acute growth retardation, renal tubular functional impairment and hepatocellular necrosis were quantitatively greater in rats pretreated with the aromatic organohalides than in naive rats. Pretreatment with HCB, PBB or PCB also reduced survival after i.p. administration of CCl4 and increased the severity of morphological liver injury. Despite functional impairment, only minor histological alterations attributable to CCl4 were detected in the kidney. CCl4 as well as HCB, PBB or PCB increased the lipid content of the liver, but not the kidney. CCl4 administration depressed energy-dependent accumulation of organic ions by renal cortical slices in vitro in a dose-dependent manner and increased the basal rate of respiration of renal cortical tissue in vitro. It is concluded from these results that exposure to certain commercial aromatic organohalides can greatly alter organ response to toxicants such as CCl4.     

Inhibition of phosphoenolpyruvate carboxykinase by streptonigrin

Streptonigrin, an antibiotic with antineoplastic activity, inhibited rat liver phosphoenolpyruvate carboxykinase with an I₅₀ of 0.3 μM when excess FeCl₂ was present. No inhibition occurred in the absence of added metal ion. Inhibition was partial and noncompetitive versus ITP and oxalacetic acid. The enzyme was more susceptible to inhibition by Streptonigrin in the absence of substrates. Fe²⁺ supported inhibition by Streptonigrin to a greater extent than did Fe³⁺, while Mn²⁺ activated the enzyme in the presence of Streptonigrin. For maximum inhibition, at least a 3-fold molar excess of iron over Streptonigrin was required. The methyl ester of Streptonigrin was also an inhibitor (I₅₀ = 4 μM) while the fragment containing the C and D rings was not, indicating that inhibition did not depend solely on the presence of the picolinic acid moiety. When oxalacetate synthesis was measured, Streptonigrin plus iron had no more effect on enzymatic activity than iron alone, and Mn²⁺ was capable of stimulating the streptonigrin-Fe²⁺ inhibited enzyme.     

Nephrotoxicity of bromobenzene in mice

I.p. administration of bromobenzene to male mice at doses ranging from 0 to 9.4 mmol/kg resulted in a dose-dependent increase in blood urea nitrogen (BUN) and serum glutamic-pyruvic transaminase (SGPT) activity and a decrease in renal cortical accumulation of para-aminohippurate (PAH) and tetraethylammonium (TEA). Induction of renal and hepatic mixed-function oxidases by beta-naphthoflavone (BNF) did not result in any alterations in the hepatotoxic or nephrotoxic response to bromobenzene. Renal and hepatic non-protein sulfhydryl (NPSH) concentrations were decreased significantly 1 h after administration of bromobenzene (7.5 mmol/kg) and were maximally depleted in both organs to 18% of control after 7 h. Depletion of renal NPSH by bromobenzene was dose-dependent up to 9.4 mmol/kg. Treatment of mice with diethyl maleate (DEM) (0.6 ml/kg) 60 min prior to bromobenzene administration resulted in greater hepatotoxicity, evidenced by increased SGPT, while renal toxicity was unchanged. These data demonstrate that large doses of bromobenzene produce functional alterations in the kidney.