Effect of estrogen on flow-induced dilation in NO deficiency: role of prostaglandins and EDHF.

To investigate the role of estrogen in flow-induced dilation (FiD) in nitric oxide (NO) deficiency, FiD was examined in isolated gracilis arterioles of ovariectomized (OVX) and OVX rats with estrogen replacement (OVE). Both groups of rats were treated chronically with N(omega)-nitro-L-arginine methyl ester. Plasma concentration of NO(2)/NO(3) was reduced in both groups. Plasma concentration of estradiol was lower in OVX than in OVE rats. FiD was similar in vessels of the two groups; calculated wall shear stress and basal tone were significantly greater in OVX vs. OVE rats. Indomethacin did not affect FiD in vessels from OVE rats but abolished dilation in vessels from OVX rats. Valeryl salicylate or NS-398 inhibited FiD by approximately 50%, whereas their simultaneous administration eliminated the response in arterioles from OVX rats. In vessels from OVE rats, miconazole or charybdotoxin eliminated FiD. Thus, in NO deficiency, prostaglandins derived from both cyclooxygenase isoforms mediate FiD in gracilis arterioles of OVX rats. Estrogen replacement switches the mediation, showing dependence on endothelium-derived hyperpolarizing factor in the arterioles of OVE rats.     

EDHF is not K+ but may be due to spread of current from the endothelium in guinea pig arterioles

Endothelium-derived hyperpolarizing factor (EDHF)-attributed hyperpolarizations and relaxations were recorded simultaneously from submucosal arterioles of guinea pigs with the use of intracellular microelectrodes and a video-based system, respectively. Membrane currents were recorded from electrically short segments of arterioles under single-electrode voltage clamp. Substance P evoked an outward current with a current-voltage relationship that was well described by the Goldman-Hodgkin-Katz equation for a K+ current, consistent with the involvement of intermediate- and small-conductance Ca2+-activated K+ channels. 1-Ethyl-2-benzimidazolinone relaxed the arterioles and evoked hyperpolarizations that were blocked by charybdotoxin, but not by iberiotoxin. Application of K+ induced depolarization under conditions in which EDHF evoked hyperpolarization. The Ba2+-sensitive component of the K+-induced current was inwardly rectifying, in contrast to the outwardly rectifying current evoked by substance P. EDHF-attributed hyperpolarizations in dye-identified smooth muscle cells were indistinguishable from those recorded from dye-identified endothelial cells in the same arterioles. These results provide evidence that EDHF is not K+ but may involve electrotonic spread of hyperpolarization from the endothelial cells to the smooth muscle cells.     

Heterogeneous function of ryanodine receptors, but not IP3 receptors, in hamster cremaster muscle feed arteries and arterioles

The roles played by ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP?Rs) in vascular smooth muscle in the microcirculation remain unclear. Therefore, the function of both RyRs and IP?Rs in Ca(2+) signals and myogenic tone in hamster cremaster muscle feed arteries and downstream arterioles were assessed using confocal imaging and pressure myography. Feed artery vascular smooth muscle displayed Ca(2+) sparks and Ca(2+) waves, which were inhibited by the RyR antagonists ryanodine (10 μM) or tetracaine (100 μM). Despite the inhibition of sparks and waves, ryanodine or tetracaine increased global intracellular Ca(2+) and constricted the arteries. The blockade of IP?Rs with xestospongin D (5 μM) or 2-aminoethoxydiphenyl borate (100 μM) or the inhibition of phospholipase C using U-73122 (10 μM) also attenuated Ca(2+) waves without affecting Ca(2+) sparks. Importantly, the IP?Rs and phospholipase C antagonists decreased global intracellular Ca(2+) and dilated the arteries. In contrast, cremaster arterioles displayed only Ca(2+) waves: Ca(2+) sparks were not observed, and neither ryanodine (10-50 μM) nor tetracaine (100 μM) affected either Ca(2+) signals or arteriolar tone despite the presence of functional RyRs as assessed by responses to the RyR agonist caffeine (10 mM). As in feed arteries, arteriolar Ca(2+) waves were attenuated by xestospongin D (5 μM), 2-aminoethoxydiphenyl borate (100 μM), and U-73122 (10 μM), accompanied by decreased global intracellular Ca(2+) and vasodilation. These findings highlight the contrasting roles played by RyRs and IP?Rs in Ca(2+) signals and myogenic tone in feed arteries and demonstrate important differences in the function of RyRs between feed arteries and downstream arterioles.     

Resistin impairs endothelium-dependent dilation to bradykinin, but not acetylcholine, in the coronary circulation

Elevated plasma levels of fat-derived signaling molecules are associated with obesity, vascular endothelial dysfunction, and coronary heart disease; however, little is known about their direct coronary vascular effects. Accordingly, we examined mechanisms by which one adipokine, resistin, affects coronary vascular tone and endothelial function. Studies were conducted in anesthetized dogs and isolated coronary artery rings. Resistin did not change coronary blood flow, mean arterial pressure, or heart rate. Resistin had no effect on acetylcholine-induced relaxation of artery rings; however, resistin did impair bradykinin-induced relaxation. Selective impairment was also observed in vivo, as resistin attenuated vasodilation to bradykinin but not to acetylcholine. Resistin had no effect on dihydroethidium fluorescence, an indicator of superoxide (O(2)(-)) production, and the inhibitory effect of resistin on bradykinin-induced relaxation persisted in the presence of Tempol, a superoxide dismutase mimetic. To determine whether resistin impaired production of and/or responses to nitric oxide (NO) or prostaglandins (e.g., prostacyclin; PGI(2)), we performed experiments with N(omega)-nitro-L-arginine methyl ester (L-NAME) and indomethacin. The effect of resistin to attenuate bradykinin-induced vasodilation persisted in the presence of L-NAME or indomethacin, suggesting resistin may act at a cell signaling point upstream of NO or PGI(2) production. Resistin-induced endothelial dysfunction is not generalized, and it is not consistent with effects mediated by O(2)(-) or interference with NO or PGI(2) signaling. The site of the resistin-induced impairment is unknown but may be at the bradykinin receptor or a closely associated signal transduction machinery proximal to NO synthase or cyclooxygenase.     

Nitric oxide in plants. To NO or not to NO

The current knowledge on the occurrence and activity of NO in plants is reviewed. The multiplicity of nitrogen monoxide species and implications for differentiated reactivity are indicated. Possible sources of NO are evaluated, and the evidence for the presence of nitric oxide synthase in plants is summarised. The regulatory role of NO. in plant development and in plant interactions with microorganisms, involving an interplay with other molecules, like ethylene or reactive oxygen species is demonstrated. Finally, some other suggestions on potential functions of NO. in plants are indicated.     

Heme oxygenase-mediated endothelial dysfunction in DOCA-salt, but not in spontaneously hypertensive, rat arterioles

Vascular heme oxygenase (HO) metabolizes heme to form carbon monoxide. Carbon monoxide inhibits nitric oxide synthase and promotes endothelium-dependent vasoconstriction. We reported HO-1-mediated endothelial dysfunction in Dahl salt-sensitive hypertension. Previous studies suggested that salt-sensitive hypertensive rats, but not spontaneously hypertensive rats (SHR), display endothelial dysfunction. This study examines the hypothesis that HO-1-mediated arteriolar endothelial dysfunction develops in deoxycorticosterone acetate (DOCA)-salt hypertensive (DOCA) rats, but not in SHR. Uninephrectomized (isoflurane anesthesia) male Sprague-Dawley rats received DOCA injections and saline drinking solution for 4 wk. Rats subjected to sham surgery received vehicle injections and tap water. Blood pressure was elevated in DOCA rats and SHR compared with sham and Wistar-Kyoto (WKY) groups. Aortic HO-1 expression and blood carboxyhemoglobin levels were elevated in the DOCA group, but not in SHR. In isolated gracilis muscle arterioles, ACh caused concentration-related vasodilation in all groups, with attenuated maximum responses in DOCA, but not in SHR, arterioles. Acute pretreatment with an inhibitor of HO, chromium mesoporphyrin, restored ACh-induced responses in DOCA arterioles to sham levels. ACh responses remained the same in SHR and WKY arterioles after chromium mesoporphyrin treatment. These data show that HO-1 levels and activity are increased and arteriolar responses to ACh are decreased in DOCA rats, but not in SHR. Furthermore, in DOCA arterioles, an inhibitor of HO restores ACh-induced vasodilation to sham levels. These results suggest that elevated HO-1 levels and activity, not resulting from hypertension per se, contribute to endothelial dysfunction in DOCA rats.     

Dicer-1, but not Loquacious, is critical for assembly of miRNA-induced silencing complexes

Double-stranded RNA-binding proteins (dsRBPs), such as R2D2 and Loquacious (Loqs), function in tandem with Dicer (Dcr) enzymes in RNA interference (RNAi). In Drosophila, Dcr-1/Loqs and Dcr-2/R2D2 complexes generate microRNAs (miRNAs) and small interfering RNAs (siRNAs), respectively. Although R2D2 does not regulate siRNA production, R2D2 and Dcr-2 coordinately bind siRNAs to promote assembly of the siRNA-induced silencing (siRISC) complexes. Conversely, Loqs enhances miRNA production. It is uncertain if Dcr-1 and Loqs facilitate miRNA loading onto the miRISC complexes. Here we used loqs knockout (KO) flies to characterize the physiological functions of Loqs in the miRNA pathway. Northern analysis revealed consistent accumulation of precursor (pre)-miRNAs in loqs(KO) flies. However, the lack of Loqs had differential effects on mature miRNAs: some are diminished, whereas others maintain wild-type levels. Importantly, the data suggest that miRNA production is not the rate-limiting step of the miRNA pathway. We show that Dcr-1, but not Loqs, is critical for assembly of miRISCs by using dcr-1 or loqs null egg extract. Consistent with this, recombinant Dcr-1 could efficiently interact with miRNA duplex in the absence of Loqs. Together, our results indicate that Loqs plays a prominent role in miRNA biogenesis, but is largely dispensable for miRISC assembly. Thus, Loqs and R2D2 represent two distinct functional modes for dsRBPs in the RNAi pathways.     

Photoperiodic modification of opiate but not beta-adrenergic or benzodiazepine binding sites in hamster brain

Exposure to short photoperiods induces gonadal regression in the golden hamster. This is accompanied by a fall in serum gonadotropins and changes in hypothalamic neurotransmitter turnover. We have attempted to correlate these changes with alterations in neurotransmitter receptor binding parameters. Compared to hamsters held in long photoperiods, opiate ([3H] naloxone) binding in hamsters exposed to short photoperiods is elevated in whole brain and in cerebral cortex but not in hypothalamus. However, beta-adrenergic ([3H] dihydroalprenolol) and benzodiazepine ([3H] flunitrazepam) binding are unaffected in whole brain, cerebral cortex or hypothalamus. Thus, changes in hypothalamic noradrenaline turnover rate previously reported may not be correlated with changes in beta-adrenergic binding.     

Matricaria chamomilla is not a hyperaccumulator, but tolerant to cadmium stress

The influence of low (3 μM) and high (60 and 120 μM) cadmium (Cd) concentrations were studied on selected aspects of metabolism in 4-week-old chamomile (Matricaria chamomilla L.) plants. After 10 days’ exposure, dry mass accumulation and nitrogen content were not significantly altered under any of the levels of Cd. However, there was a significant decline in chlorophyll and water content in the leaves. Among coumarin-related compounds, herniarin was not affected by Cd, while its precursors (Z)- and (E)-2-β-d-glucopyranosyloxy-4-methoxycinnamic acids (GMCAs) increased significantly at all the levels of Cd tested. Cd did not have any effect on umbelliferone, a stress metabolite of chamomile. Lipid peroxidation was also not affected by even 120 μM Cd. Cd accumulation was approximately seven- (60 μM Cd treatment) to eleven- (120 μM Cd treatment) fold higher in the roots than that in the leaves. At high concentrations, it stimulated potassium leakage from the roots, while at the lowest concentration it could stimulate potassium uptake. The results supported the hypothesis that metabolism was altered only slightly under high Cd stress, indicating that chamomile is tolerant to this metal. Preferential Cd accumulation in the roots indicated that chamomile could not be classified as a hyperaccumulator and, therefore, it is unsuitable for phytoremediation.     

Trait fitness is not a propensity,but fitness variation is

The propensity interpretation of fitness draws on the propensity interpretation of probability, but advocates of the former have not attended sufficiently to problems with the latter. The causal power of C to bring about E is not well-represented by the conditional probability Pr(E|C). Since the viability fitness of trait T is the conditional probability Pr(organism O survives to adulthood|O has T), the viability fitness of the trait does not represent the degree to which having the trait causally promotes surviving. The same point holds for fertility fitness. This failure of trait fitness to capture causal role can also be seen in the fact that coextensive traits must have the same fitness values even if one of them promotes survival and the other is neutral or deleterious. Although the fitness of a trait does not represent the trait’s causal power to promote survival and reproduction, variation in fitness in a population causally promotes change in trait frequencies; in this sense, fitness variation is a population-level propensity.     

Fame is a bubble,but not for some

Brilliant, acerbic, not given to suffer much of anybody gladly, let alone fools, Francis Crick had enormous influence that was due to his style and high scientific standards.     

Endothelium-derived NO, but not cyclic GMP, is required for hypoxic augmentation in isolated porcine coronary arteries

The present study investigated the mechanism underlying the transient potentiation of vasoconstriction by hypoxia in isolated porcine coronary arteries. Isometric tension was measured in rings with or without endothelium. Hypoxia (Po(2) <30 mmHg) caused a transient further increase in tension (hypoxic augmentation) in contracted (with U46619) preparations. The hypoxic response was endothelium dependent and abolished by inhibitors of nitric oxide synthase [N(ω)-nitro-L-arginine methyl ester (L-NAME)] or soluble guanylyl cyclase (ODQ and NS2028). The addition of DETA NONOate (nitric oxide donor) in the presence of L-NAME restored the hypoxic augmentation, suggesting the involvement of the nitric oxide pathway. However, the same was not observed after incubation with 8-bromo-cyclic GMP, atrial natriuretic peptide, or isoproterenol. Assay of the cyclic GMP content showed no change upon exposure to hypoxia in preparations with and without endothelium. Incubation with protein kinase G and protein kinase A inhibitors did not inhibit the hypoxic augmentation. Thus the hypoxic augmentation is dependent on nitric oxide and soluble guanylyl cyclase but independent of cyclic GMP. The hypoxic augmentation persisted in calcium-free buffer and in the presence of nifedipine, ruling out a role for extracellular calcium influx. Hypoxia did not alter the intracellular calcium concentration, as measured by confocal fluorescence microscopy. This observation and the findings that hypoxic augmentation is enhanced by thapsigargin (sarco/endoplasmic reticulum calcium ATPase inhibitor) and inhibited by HA1077 or Y27632 (Rho kinase inhibitors) demonstrate the involvement of calcium sensitization in the phenomenon.     

CaM-kinaseII-dependent commitment to microcystin-induced apoptosis is coupled to cell budding, but not to shrinkage or chromatin hypercondensation

The protein phosphatase inhibitor microcystin-LR (MC) induced hepatocyte apoptosis mediated by the calcium-calmodulin-dependent multifunctional protein kinase II (CaMKII). CaMKII antagonists were added at various times after MC to define for how long the cells depended on CaMKII activity to be committed to execute the various parameters of death. Shrinkage and nonpolarized budding were reversible and not coupled to commitment. A critical commitment step was observed 15-20 min after MC (0.5 microM) addition. After this, CaMKII inhibitors no longer protected against polarized budding, DNA fragmentation, lost protein synthesis capability, and cell disruption. Commitment to chromatin hypercondensation occurred 40 min after MC addition. In conclusion, irreversible death commitment was coupled to polarized budding, but not to shrinkage or chromatin condensation. Antioxidant prevented chromatin condensation when given after the CaMKII-dependent commitment point, suggesting that CaMKII had mediated the accumulation of a second messenger of reactive oxygen species nature.     

NMDA-, but not kainate- or quisqualate-dependent increases in cerebellar cGMP are dependent upon monoaminergic innervation.

As previously reported, intracerebellar injections of D-serine, quisqualate and kainate elevated mouse cerebellar cGMP levels. Similarly, activation of endogenous excitatory amino acid utilizing neurons with harmaline or pentylenetetrazole also increased cerebellar cGMP. We previously have demonstrated that the harmaline- and pentylenetetrazol-dependent cGMP increases are NMDA receptor mediated. In this report we further demonstrate that NMDA-dependent increases in cGMP are dependent upon monoaminergic innervation of the cerebellum, while kainate- and quisqualate-dependent cGMP increases are independent of such innervation.     

Phosphodiesterase-5 Gln817 is critical for cGMP, vardenafil, or sildenafil affinity: its orientation impacts cGMP but not cAMP affinity

The side group of an invariant Gln in cGMP- and cAMP-specific phosphodiesterases (PDE) is held in different orientations by bonds with other amino acids and purportedly discriminates between guanine and adenine in cGMP and cAMP. In cGMP-specific PDE5, Gln(775) constrains the orientation of the invariant Gln(817) side chain, which forms bidentate bonds with 5'-GMP, vardenafil, sildenafil, and 3-isobutyl-1-methylxanthine (IBMX) (Sung, B. J., Hwang, K. Y., Jeon, Y. H., Lee, J. I., Heo, Y. S., Kim, J. H., Moon, J., Yoon, J. M., Hyun, Y. L., Kim, E., Eum, S. J., Park, S. Y., Lee, J. O., Lee, T. G., Ro, S., and Cho, J. M. (2003) Nature 425, 98-102; Huai, Q., Liu, Y., Francis, S. H., Corbin, J. D., and Ke, H. (2004) J. Biol. Chem. 279, 13095-13101; Zhang, K. Y., Card, G. L., Suzuki, Y., Artis, D. R., Fong, D., Gillette, S., Hsieh, D., Neiman, J., West, B. L., Zhang, C., Milburn, M. V., Kim, S. H., Schlessinger, J., and Bollag, G. (2004) Mol. Cell 15, 279-286). PDE5(Q817A) and PDE5(Q775A) were generated to test the hypotheses that Gln(817) is critical for cyclic nucleotide or inhibitor affinity and that Gln(775) immobilizes the Gln(817) side chain to provide cGMP/cAMP selectivity. Allosteric cGMP binding and the molecular mass of the mutant proteins were unchanged compared with PDE5(WT). For PDE5(Q817A), K(m) for cGMP or cAMP was weakened 60- or 2-fold, respectively. For PDE5(Q775A), K(m) for cGMP was weakened approximately 20-fold but was unchanged for cAMP. For PDE5(Q817A), vardenafil, sildenafil, and IBMX inhibitory potencies were weakened 610-, 48-, and 60-fold, respectively, indicating that Gln(817) is a major determinant of potency, especially for vardenafil, and that binding of vardenafil and sildenafil differs substantially. Sildenafil and vardenafil affinity were not significantly affected in PDE5(Q775A). It is concluded that Gln(817) is a positive determinant for PDE5 affinity for cGMP and several inhibitors; Gln(775), which perhaps restricts rotation of Gln(817) side chain, is critical for cGMP affinity but has no measurable effect on affinity for cAMP, sildenafil, or vardenafil.     

Microvascular dilation in response to occlusion: a coordinating role for conducted vasomotor responses

In rat cremasteric microcirculation, mechanical occlusion of one branch of an arteriolar bifurcation causes an increase in flow and vasodilation of the unoccluded daughter branch. This dilation has been attributed to the operation of a shear stress-dependent mechanism in the microcirculation. Instead of or in addition to this, we hypothesized that the dilation observed during occlusion is the result of a conducted signal originating distal to the occlusion. To test this hypothesis, we blocked the ascending spread of conducted vasomotor responses by damaging the smooth muscle and endothelial cells in a 200-microm segment of second- or third-order arterioles. We found that a conduction blockade eliminated or diminished the occlusion-associated increase in flow through the unoccluded branch and abolished or strongly attenuated the vasodilatory response in both vessels at the branch. We also noted that vasodilations induced by ACh (10(-4) M, 0.6 s) spread to, but not beyond, the area of damage. Taken together, these data provide strong evidence that conducted vasomotor responses have an important role in coordinating blood flow in response to an arteriolar occlusion.     

Hyaloraphidium curvatum is not a green alga, but a lower fungus; Amoebidium parasiticum is not a fungus, but a member of the DRIPs

The unicellular heterotrophic protist Hyaloraphidium is classified with a family of green algae, the Ankistrodesmaceae. The only species that exists in pure culture and that is available for taxonomic studies is H. curvatum. Comparative 18S ribosomal RNA sequence analyses showed that H. curvatum belongs to the fungi rather than to the algae. Within the fungi, H. curvatum preferentially clustered with Chytridiomycetes. Unlike Chytridiomycetes, H. curvatum propagates by autosporulation, and the presence of flagella has never been reported. Transmission electron microscopy indicated that H. curvatum in some respects resembles Chytridiomycetes, but no elements of a flagellar apparatus were detected. The habitus of H. curvatum is unlike that of other fungi except the trichomycete Amoebidium parasiticum. The cell wall sugar composition of H. curvatum was unique, but to some extent resembled that of A. parasiticum. However, H. curvatum and A. parasiticum are not closely related to each other according to 18S rRNA sequence data. Moreover, A. parasiticum clustered with protistan animals, the Mesomycetozoa (DRIPs). Combined molecular, ultrastructural and chemical data do not allow assignment of H. curvatum to any recognized clade of fungi. This suggests that H. curvatum may represent an independent evolutionary lineage within the fungi.     

Drinking,but not feeding,is opiate-sensitive in hamsters

The long-lasting opiate antagonist, naltrexone (NTX), was examined for its effects on various types of consummatory behavior in male golden hamsters and rats. Rat, but not hamster, 24 hr food and water intakes were significantly decreased by four daily NTX (10.0 mg/kg) injections. Hamsters displayed a minimal night to day feeding ratio compared to rats. hamsters increased food intake following insulin (50 U/kg) administration, but not after 24 hr food deprivation (FD) or 2-deoxy-D-glucose (2-DG; 800 mg/kg) injections. NTX (1.0 and 10 mg/kg) had no effect on feeding, but markedly attenuated hamster drinking induced by 48 hr water deprivation or hypertonic saline injection. Dexamethasone (DEX), a glucocorticoid which depletes pituitary β-endorphin and produces anorexia in rats, had no effect on daily hamster intake. Since the normal feeding profile of the hamster is similar to that of naloxone and DEX-treated rats, hamsters appear to lack an opiate-sensitive feeding system. In contrast, stimulated drinking behavior of hamsters operates through an opiate-sensitive mechanism. Thus, there are marked species differences concerning the involvement of endogenous opioids is consummatory behavior.     

Intracellular iron, but not copper, plays a critical role in hydrogen peroxide-induced DNA damage

The role of intracellular iron, copper, and calcium in hydrogen peroxide-induced DNA damage was investigated using cultured Jurkat cells. The cells were exposed to low rates of continuously generated hydrogen peroxide by the glucose/glucose oxidase system, and the formation of single strand breaks in cellular DNA was evaluated by the sensitive method, single cell gel electrophoresis or "comet" assay. Pre-incubation with the specific ferric ion chelator desferrioxamine (0.1-5.0 mM) inhibited DNA damage in a time- and dose-dependent manner. On the other hand, diethylenetriaminepentaacetic acid (DTPA), a membrane impermeable iron chelator, was ineffective. The lipophilic ferrous ion chelator 1,10-phenanthroline also protected against DNA damage, while its nonchelating isomer 1,7-phenanthroline provided no protection. None of the above iron chelators produced DNA damage by themselves. In contrast, the specific cuprous ion chelator neocuproine (2,9-dimethyl-1,10-phenanthroline), as well as other copper-chelating agents, did not protect against H(2)O(2)-induced cellular DNA damage. In fact, membrane permeable copper-chelating agents induced DNA damage in the absence of H(2)O(2). These results indicate that, under normal conditions, intracellular redox-active iron, but not copper, participates in H(2)O(2)-induced single strand break formation in cellular DNA. Since BAPTA/AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester), an intracellular Ca(2+)-chelator, also protected against H(2)O(2)-induced DNA damage, it is likely that intracellular Ca(2+) changes are involved in this process as well. The exact role of Ca(2+) and its relation to intracellular transition metal ions, in particular iron, needs to be further investigated.