Nitrone spin-traps block calcium channels and induce pulmonary artery relaxation independent of free radicals

Free radicals react with nitrones to form stable nitroxides which can be identified by ESR spectroscopy. Unfortunately, little is known regarding the pharmacological properties of these compounds. In this study, three commonly used nitrones, 5,5-dimethylpyrroline-N-oxide (DMPO), alpha-phenyl-tert-butylnitrone (PBN), and alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN), were found to induce relaxation of preconstricted isolated rat pulmonary artery rings. Additional experiments with PBN indicated that vasorelaxation could not be attributed to production of endothelial derived factors, prostaglandins, or free radicals. Patch-clamp techniques revealed reversible calcium channel blockade with PBN at a concentration below that needed to detect free radicals. Calcium channel blockade probably accounts for the vasorelaxation observed in the isolated ring preparations described here, and should be considered when using nitrone spin-traps both in in vivo and clinical studies.     

Pulmonary vasoconstrictor effects of prostacyclin in rats: potential role of thromboxane receptors

Endogenous prostacyclin (PGI2; epoprostenol) is a potent endothelium-derived pulmonary vasodilator. However, the effects of exogenous PGI2 on isolated arteries could be either relaxant or contractile, depending on the species and organ studied. The present study investigated the distal pathways involved in the PGI2-induced contraction in rat intrapulmonary artery (PA) and relaxation in lamb PA. When vessels were precontracted with 30 mM K+, PGI2 (1 microM) induced relaxation in lamb PA but caused contraction in rat PA. Use of 30 mM K+, phenylephrine, serotonin, angiotensin II, or hypoxia to precontract the vessels did not alter the contractile effect of PGI2 in rat PA. Nevertheless, PGI2 produced a mild relaxation in rat PA precontracted by U-46619, a thromboxane A2 (TxA2)-receptor agonist, whereas the TxA2-receptor blocker SQ-29548 (0.1-0.5 microM) abolished the contractile response in rat PA. These data suggest that PGI2-induced contraction is mediated by activation of TxA2 receptors. The PGI2-induced modest relaxation in rat PA, which was only observed when TxA2 receptors were blocked by SQ-29548, suggests that the PGI2-mediated vasorelaxant pathway is diminished in these vessels. Simultaneous application of forskolin, an adenylate cyclase activator, and rolipram, a phosphodiesterase inhibitor, caused similar relaxation in both rat and lamb PA. This suggests that the adenosine 3',5'-cyclic monophosphate-dependent relaxing pathway is intact in rat PA and is comparable to that in lamb PA. On the basis of these data, we conclude that the pathways responsible for the paradoxical effects of PGI2 on rat and lamb PA are located upstream of the adenosine 3',5'-cyclic monophosphate-dependent relaxing pathway and that a paucity of PGI2 receptors in rat PA may be responsible.     

Treatment of chronic allodynia in spinally injured rats: effects of intrathecal selective opioid receptor agonists

We examined the effects of intrathecal (i.t.) selective opioid receptor agonists in alleviating mechanical and cold allodynia in spinally injured rats. Both DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, a mu-opioid receptor agonist) and DPDPE ([D-Phe2,D-Phe5]-enkephalin, a delta-opioid receptor agonist) dose-dependently relieved the chronic allodynia-like behavior at doses selective for their respective receptors. The anti-allodynic effect of DAMGO and DPDPE was reversed by the selective mu- and delta-opioid receptor antagonists CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2) and naltrindole, respectively. In contrast, the selective kappa-opioid receptor agonist U50488H did not alleviate the allodynia-like behavior, but rather enhanced it. The anti-nociceptive and anti-allodynic effect of i.t. DAMGO was blocked by U50488H. Thus, activation of spinal mu- and delta-, but not kappa-opioid receptors produced anti-allodynic effect in this model of central pain. Drugs which act selectively on opioid receptor subtypes may be useful in managing chronic central pain of spinal cord origin.     

Reevaluation of Cl-/HCO3- exchange in cultured bovine corneal endothelial cells

PURPOSE: To determine the apical versus basolateral polarity of the putative anion exchanger in cultured bovine corneal endothelial cells (BCECs) and to examine the influence of Cl--dependent membrane potential (Em) changes on HCO3- transport. METHODS: BCECs grown on permeable supports were used for independent perfusion of apical and basolateral surfaces. Intracellular pH (pHi) was measured using the fluorescent dye BCECF. Relative changes in Em were measured using the fluorescent dye bis-oxonol. Western blot analysis was used to detect immunoreactivity against the anion exchanger (AE1 or AE2). RESULTS: Cl- removal from apical and basolateral surfaces produced cellular alkalinization (apical side, 0.07 pH units; basolateral side, 0.06 pH units; both sides, 0.20 pH units). Application of 100 microM H2-4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (DIDS), an anion exchange inhibitor, on the apical side produced an alkalinization (0.02 pH units) followed by acidification (-0.05 pH units), whereas basolateral H2DIDS caused a substantial acidification (-0.16 pH units). In the absence of Na+, Cl- removal from the apical side caused a transient alkalinization (0.03 pH units) followed by a return to baseline; Cl- removal from the basolateral side caused a small (-0.03) acidification. In Na+-free Ringer, apical H2DIDS produced a transient alkalinization (0.02 pH units), whereas basolateral exposure had no effect. 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), N-phenylanthranilic acid (DPC), and niflumic acid (50-200 microM), known Cl- channel blockers, produced cellular acidification in control Ringer. Niflumic acid hyperpolarized Em and inhibited depolarization after Cl- removal. Western blot analysis failed to detect AE2 expression in cultured BCECs. However, fresh BCECs produced a trace response. CONCLUSIONS: Physiological activity of an apical anion exchanger is weak in cultured BCECs. Cultured BCECs have significant Cl- conductance. Thus, cellular alkalinization after Cl- removal is caused primarily by depolarization of Em, which drives HCO3- influx through the basolateral electrogenic Na+:nHCO3- cotransporter. In contrast with cultured BCECs, AE2 may be present in fresh cells.     

Calcium coding and adaptive temporal computation in cortical pyramidal neurons

In this work, we present a quantitative theory of temporal spike-frequency adaptation in cortical pyramidal cells. Our model pyramidal neuron has two-compartments (a "soma" and a "dendrite") with a voltage-gated Ca2+ conductance (gCa) and a Ca2+-dependent K+ conductance (gAHP) located at the dendrite or at both compartments. Its frequency-current relations are comparable with data from cortical pyramidal cells, and the properties of spike-evoked intracellular [Ca2+] transients are matched with recent dendritic [Ca2+] imaging measurements. Spike-frequency adaptation in response to a current pulse is characterized by an adaptation time constant tauadap and percentage adaptation of spike frequency Fadap [% (peak - steady state)/peak]. We show how tauadap and Fadap can be derived in terms of the biophysical parameters of the neural membrane and [Ca2+] dynamics. Two simple, experimentally testable, relations between tauadap and Fadap are predicted. The dependence of tauadap and Fadap on current pulse intensity, electrotonic coupling between the two compartments, gAHP as well the [Ca2+] decay time constant tauCa, is assessed quantitatively. In addition, we demonstrate that the intracellular [Ca2+] signal can encode the instantaneous neuronal firing rate and that the conductance-based model can be reduced to a simple calcium-model of neuronal activity that faithfully predicts the neuronal firing output even when the input varies relatively rapidly in time (tens to hundreds of milliseconds). Extensive simulations have been carried out for the model neuron with random excitatory synaptic inputs mimicked by a Poisson process. Our findings include 1) the instantaneous firing frequency (averaged over trials) shows strong adaptation similar to the case with current pulses; 2) when the gAHP is blocked, the dendritic gCa could produce a hysteresis phenomenon where the neuron is driven to switch randomly between a quiescent state and a repetitive firing state. The firing pattern is very irregular with a large coefficient of variation of the interspike intervals (ISI CV > 1). The ISI distribution shows a long tail but is not bimodal. 3) By contrast, in an intrinsically bursting regime (with different parameter values), the model neuron displays a random temporal mixture of single action potentials and brief bursts of spikes. Its ISI distribution is often bimodal and its power spectrum has a peak. 4) The spike-adapting current IAHP, as delayed inhibition through intracellular Ca2+ accumulation, generates a "forward masking" effect, where a masking input dramatically reduces or completely suppresses the neuronal response to a subsequent test input. When two inputs are presented repetitively in time, this mechanism greatly enhances the ratio of the responses to the stronger and weaker inputs, fulfilling a cellular form of lateral inhibition in time. 5) The [Ca2+]-dependent IAHP provides a mechanism by which the neuron unceasingly adapts to the stochastic synaptic inputs, even in the stationary state following the input onset. This creates strong negative correlations between output ISIs in a frequency-dependent manner, while the Poisson input is totally uncorrelated in time. Possible functional implications of these results are discussed.     

The mitochondrial genome integrity gene, MGI1, of Kluyveromyces lactis encodes the beta-subunit of F1-ATPase

In a previous report, we found that mutations at the mitochondrial genome integrity locus, MGI1, can convert Kluyveromyces lactis into a petite-positive yeast. In this report, we describe the isolation of the MGI1 gene and show that it encodes the beta-subunit of the mitochondrial F1-ATPase. The site of mutation in four independently isolated mgi1 alleles is at Arg435, which has changed to Gly in three cases and Ile in the fourth isolate. Disruption of MGI1 does not lead to the production of mitochondrial genome deletion mutants, indicating that an assembled F1 complex is needed for the "gain-of-function" phenotype found in mgi1 point mutants. The location of Arg435 in the beta-subunit, as deduced from the three-dimensional structure of the bovine F1-ATPase, together with mutational sites in the previously identified mgi2 and mgi5 alleles, suggests that interaction of the beta- and alpha- (MGI2) subunits with the gamma-subunit (MGI5) is likely to be affected by the mutations.     

The putative alpha helix 2 of human immunodeficiency virus type 1 Vpr contains a determinant which is responsible for the nuclear translocation of proviral DNA in growth-arrested cells

Several viral determinants were shown to play a role in the ability of human immunodeficiency virus type 1 (HIV-1) to infect nondividing cells. In particular, Vpr and Gag matrix (MA) were recognized to be involved in the nuclear transport of the viral preintegration complex. The goal of the present study was to evaluate the ability of isogenic HIV-1 viruses harboring different vpr and gag genes to infect nondividing cells. Surprisingly, our results reveal that the introduction of mutations in the MA nuclear localization signal marginally affected the ability of proviruses to establish infection in growth-arrested HeLa or MT4 cells. In contrast, we show that in our experimental system, the absence of Vpr expression leads to a reduction in viral infectivity and production which correlates with a decrease in the synthesis and nuclear transport of proviral DNA as determined by PCR analysis. Moreover, our data demonstrate that this reduction of viral replication is also observed with proviruses containing different mutated Vpr alleles. In particular, the Vpr Q65E mutant, which contains a substitution in the second predicted amphipathic alpha-helical structure located in the central region of the protein, is associated with an impairment of the protein nuclear localization and a concomitant reduction of the nuclear transport of proviral DNA. The results of this study provide evidence that a putative amphipathic alpha-helical structure in the central region of Vpr contains a determinant involved in the nuclear translocation of the preintegration complex in nondividing cells.     

Submitochondrial distribution of three key steroidogenic proteins (steroidogenic acute regulatory protein and cytochrome p450scc and 3beta-hydroxysteroid dehydrogenase isomerase enzymes) upon stimulation by intracellular calcium in adrenal glomerulosa cells

In adrenal glomerulosa cells, angiotensin II (Ang II) and potassium stimulate aldosterone synthesis through activation of the calcium messenger system. The rate-limiting step in steroidogenesis is the transfer of cholesterol to the inner mitochondrial membrane. This transfer is believed to depend upon the presence of the steroidogenic acute regulatory (StAR) protein. The aim of this study was 1) to examine the effect of changes in cytosolic free calcium concentration and of Ang II on intramitochondrial cholesterol and 2) to study the distribution of StAR protein in submitochondrial fractions during activation by Ca2+ and Ang II. To this end, freshly prepared bovine zona glomerulosa cells were submitted to a high cytosolic Ca2+ clamp (600 nM) or stimulated with Ang II (10 nM) for 2 h. Mitochondria were isolated and subfractionated into outer membranes, inner membranes (IM), and contact sites (CS). Stimulation of intact cells with Ca2+ or Ang II led to a marked, cycloheximide-sensitive increase in cholesterol in CS (to 143 +/- 3. 2 and 151.1 +/- 18.1% of controls, respectively) and in IM (to 119 +/- 5.1 and 124.5 +/- 6.5% of controls, respectively). Western blot analysis revealed a cycloheximide-sensitive increase in StAR protein in mitochondrial extracts of Ca2+-clamped glomerulosa cells (to 159 +/- 23% of controls). In submitochondrial fractions, there was a selective accumulation of StAR protein in IM following stimulation with Ca2+ (228 +/- 50%). Similarly, Ang II increased StAR protein in IM, and this effect was prevented by cycloheximide. In contrast, neither Ca2+ nor Ang II had any effect on the submitochondrial distribution of cytochrome P450scc and 3beta-hydroxysteroid dehydrogenase isomerase. The intramitochondrial presence of the latter enzyme was further confirmed by immunogold staining in rat adrenal fasciculata cells and by immunoblot analysis in MA-10 mouse testicular Leydig cells. These findings demonstrate that under acute stimulation with Ca2+-mobilizing agents, newly synthesized StAR protein accumulates in IM after transiting through CS. Moreover, our results suggest that the import of StAR protein into IM may be associated with cholesterol transfer, thus promoting precursor supply to the two first enzymes of the steroidogenic cascade within the mitochondria and thereby activating mineralocorticoid synthesis.