Platelet inhibition by low-dose aspirin but not by clopidogrel reduces the axon-reflex current-induced vasodilation in humans

We previously showed a prolonged inhibition of current-induced vasodilation (CIV) after a single oral high dose of aspirin. In this study, we tested the hypothesis of platelet involvement in CIV. Nine healthy volunteers took 75 mg aspirin/day, 98 mg of clopidogrel bisulfate/day, or placebo for 4 days. CIV was induced by two consecutive 1-min anodal current applications (0.08 mA/cm(2)) through deionized water with a 10-min interval. CIV was measured with laser Doppler flowmetry and expressed as a percentage of baseline cutaneous vascular conductance: %C(b). In a second experiment in 10 volunteers, aspirin and placebo were given as in experiment 1, but a 26-h delay from the last aspirin intake elapsed before ACh iontophoresis and postocclusive hyperemia were studied in parallel to CIV. In experiment 1, the means +/- SE amplitude of CIV was 822 +/- 314, 313 +/- 144, and 746 +/- 397%C(b) with placebo, aspirin (P < 0.05 from placebo and clopidogrel), and clopidogrel (NS from placebo), respectively. In experiment 2, CIV impairment with aspirin was confirmed: CIV amplitudes were 300 +/- 99, and 916 +/- 528%C(b) under aspirin and placebo, respectively (P < 0.05), whereas vasodilation to ACh iontophoresis (322 +/- 74 and 365 +/- 104%C(b)) and peak postocclusive hyperemia (491 +/- 137 and 661 +/- 248%C(b)) were not different between aspirin and placebo, respectively. Low-dose aspirin, even 26 h after oral administration, impairs CIV, while ACh-mediated vasodilation and postocclusive hyperemia are preserved. If platelets are involved in the neurovascular mechanism triggered by galvanic current application in humans, it is likely to occur through the cyclooxygenase but not the ADP pathway.     

Polyamine-mediated inhibition of in-vitro cell proliferation is not due to acrolein

The influence of acrolein or spermine on the viability and growth of phytohaemagglutinin-stimulated rat thymic lymphocytes in cultures supplemented with foetal calf serum have been investigated. Acrolein (greater than 20 microM) was cytotoxic; spermine had little effect on viability, but inhibited [3H]TdR incorporation at low concentrations (approximately 10 microM). Cells treated with greater than 8 microM acrolein 3 hr before stimulation exhibited irreversible inhibition of protein synthesis, whereas 50 microM spermine had no effect, even when cells were treated for 24 hr before stimulation. However, addition of 25 microM spermine after stimulation did inhibit both [3H]-uridine incorporation and protein synthesis: this was reversible if cells were freed of polyamine within 4 hr, but not if washed after 24 hr. These results show that, contrary to several previous reports, in-vitro inhibition of cell proliferation by spermine is not due to the formation and action of acrolein.     

Prolonged allograft survival in TNF receptor 1-deficient recipients is due to immunoregulatory effects, not to inhibition of direct antigraft cytotoxicity.

TNF-alpha and lymphotoxin (LT)alpha have been shown to be important mediators of allograft rejection. TNF-R1 is the principal receptor for both molecules. Mice with targeted genetic deletions of TNF-R1 demonstrate normal development of T and B lymphocytes but exhibit functional defects in immune responses. However, the role of TNF-R1-mediated signaling in solid organ transplant rejection has not been defined. To investigate this question, we performed vascularized heterotopic allogeneic cardiac transplants in TNF-R1-deficient (TNF-R1(-/-)) and wild-type mice. Because all allografts in our protocol expressed TNF-R1, direct antigraft effects of TNF-alpha and LTalpha were not prevented. However, immunoregulatory effects on recipient inflammatory cells by TNF-R1 engagement was eliminated in TNF-R1(-/-) recipients. In our study, cardiac allograft survival was significantly prolonged in TNF-R1(-/-) recipients. Despite this prolonged allograft survival, we detected increased levels of CD8 T cell markers in allografts from TNF-R1(-/-) recipients, suggesting that effector functions, but not T cell recruitment, were blocked. We also demonstrated the inhibition of multiple chemokines and cytokines in allografts from TNF-R1(-/-) recipients including RANTES, IFN-inducible protein-10, lymphotactin, and IL-1R antagonist, as well as altered levels of chemokine receptors. We correlated gene expression with the physiologic process of allograft rejection using self-organizing maps and identified distinct patterns of gene expression in allografts from TNF-R1(-/-) recipients. These findings indicate that in our experimental system TNF-alpha and LTalpha exert profound immunoregulatory effects through TNF-R1.     

The inhibition of capacitative calcium entry due to ATP depletion but not due to glucosamine is reversed by staurosporine.

The capacitative Ca2+ entry pathway in J774 macrophages is rapidly inhibited by the amino sugar glucosamine. This pathway is also inhibited by treatments such as 2-deoxy-D-glucose (2dGlc) or glucose deprivation that inhibit glycolysis and lead to significant decreases in cellular ATP and other trinucleotides. We sought to determine whether glucosamine's effect on capacitative Ca2+ entry was also due to ATP depletion, as has been suggested recently for its link to insulin resistance. In contrast to brief treatments with 2dGlc, there was no significant decrease in ATP following exposure to glucosamine. In addition, the 2dGlc-mediated inhibition of capacitative Ca2+ influx was reversed by staurosporine, a microbial alkaloid that inhibits a broad range of protein kinases. Staurosporine was also able to reverse the inhibition of capacitative Ca2+ entry seen following other treatments that decreased cellular ATP levels, including cytochalasin B and iodoacetic acid. Other inhibitors of protein kinase C, including bisindolylmaleimide, K252a, H-7, and calphostin C, were unable to mimic this effect of staurosporine. However, the inhibition of capacitative Ca2+ influx in the presence of glucosamine was not reversed by staurosporine. These data indicate that the inhibitory action on capacitative Ca2+ entry of glucosamine is distinct from that caused by ATP depletion.     

Geldanamycin-associated inhibition of intracellular trafficking is attributed to a co-purified activity

Geldanamycin, an ansamycin antibiotic that specifically inhibits heat-shock protein-90 (HSP90) and its endoplasmic reticulum homologue, glucose-regulated protein-94 (GRP94), accelerates the degradation of selected cellular proteins. We showed previously that geldanamycin inhibits maturation and transport of the epidermal growth factor receptor in addition to accelerating its degradation (Supino-Rosin, L., Yoshimura, A., Yarden, Y., Elazar, Z., and Neumann, D. (2000) J. Biol. Chem. 275, 21850-21855). Here we demonstrate that the additional activities of geldanamycin on intracellular transport and protein maturation are related to its supply source. By combining chemical separation of Streptomyces hygroscopicus var. geldanus extracts and biological screens, we show that the geldanamycin-associated effects on intracellular transport and protein maturation are not mediated by geldanamycin itself but are due to the presence of an additional component(s). Chromatography of S. hygroscopicus var. geldanus extracts on a silica-gel column allowed separation between the inhibition of intracellular trafficking and geldanamycin-mediated degradation. One fraction that was devoid of geldanamycin blocked secretion of a soluble form of the erythropoietin receptor, retarded maturation of the epidermal growth factor receptor without enhancing its degradation, and blocked anterograde transport of a temperature-sensitive mutant of the vesicular stomatitis virus G protein (VSVGtsO45) from the early Golgi cisternae. This fraction was enriched (>95%) in 17-demethylgeldanamycin. However, as synthetically derived 17-demethylgeldanamycin did not inhibit intracellular trafficking, we concluded that 17-demethylgeldanamycin is not the active component. We thus propose that a compound(s) that co-purifies with benzoquinone ansamycins inhibits intracellular transport. Taken together, our data demonstrate that the inhibitory effects on protein maturation and intracellular trafficking, previously attributed to geldanamycin, are mediated by another distinct moiety.     

Pulmonary vasodilation by acetazolamide during hypoxia is unrelated to carbonic anhydrase inhibition

Acute hypoxic pulmonary vasoconstriction can be inhibited by high doses of the carbonic anhydrase inhibitor acetazolamide. This study aimed to determine whether acetazolamide is effective at dosing relevant to human use at high altitude and to investigate whether its efficacy against hypoxic pulmonary vasoconstriction is dependent on carbonic anhydrase inhibition by testing other potent heterocyclic sulfonamide carbonic anhydrase inhibitors. Six conscious dogs were studied in five protocols: 1) controls, 2) low-dose intravenous acetazolamide (2 mg.kg(-1).h(-1)), 3) oral acetazolamide (5 mg/kg), 4) benzolamide, a membrane-impermeant inhibitor, and 5) ethoxzolamide, a membrane-permeant inhibitor. In all protocols, unanesthetized dogs breathed spontaneously during the first hour (normoxia) and then breathed 9-10% O(2) for the next 2 h. Arterial oxygen tension ranged between 35 and 39 mmHg during hypoxia in all protocols. In controls, mean pulmonary artery pressure increased by 8 mmHg and pulmonary vascular resistance by 200 dyn.s.cm(-5) (P <0.05). With intravenous acetazolamide, mean pulmonary artery pressure and pulmonary vascular resistance remained unchanged during hypoxia. With oral acetazolamide, mean pulmonary artery pressure increased by 5 mmHg (P < 0.05), but pulmonary vascular resistance did not change during hypoxia. With benzolamide and ethoxzolamide, mean pulmonary artery pressure increased by 6-7 mmHg and pulmonary vascular resistance by 150-200 dyn.s.cm(-5) during hypoxia (P < 0.05). Low-dose acetazolamide is effective against acute hypoxic pulmonary vasoconstriction in vivo. The lack of effect with two other potent carbonic anhydrase inhibitors suggests that carbonic anhydrase is not involved in the mediation of hypoxic pulmonary vasoconstriction and that acetazolamide acts on a different receptor or channel.     

Depletion of neural precursor cells after local brain irradiation is due to radiation dose to the parenchyma, not the vasculature

The underlying mechanisms associated with radiation-induced cognitive impairments remain elusive but may involve changes in hippocampal neural precursor cells. Proliferating neural precursor cells have been shown to be extremely sensitive to X rays, either from damage to the cells themselves and/or through microenvironmental factors, including the anatomical relationship with the microvasculature, which is altered by radiation. The neutron capture reaction in boron was used to determine whether the sensitivity of neural precursor cells was dominated by direct radiation effects or was mediated through changes in the microvasculature. Young adult rats were irradiated with X rays, neutrons only, or neutrons plus either mercapto-undecahydro-dodecaborane (BSH) or p-dihydroxyboryl-phenylalanine (BPA). BSH remains inside cerebral vessels, thereby limiting the neutron capture intravascularly; BPA readily passes into the parenchyma. One month after irradiation, cell proliferation and numbers of immature neurons were determined using immunohistochemistry. Results showed that (1) neural precursor cells and their progeny were decreased in a dose-dependent manner by mixed high- and low-LET radiation, and (2) selective irradiation of the microvasculature resulted in less loss of neural precursor cells than when the radiation dose was delivered uniformly to the parenchyma. This information, and in particular the approach of selectively irradiating the vasculature, may be useful in developing radioprotective compounds for use during therapeutic irradiation.     

Selective inhibition of endothelial cell proliferation by fumagillin is not due to differential expression of methionine aminopeptidases

The angiogenesis inhibitors fumagillin and TNP-470 selectively inhibit the proliferation of endothelial cells, as compared with most other cell types. The mechanism of this selective inhibition remains uncertain, although methionine aminopeptidase-2 (MetAP2) has recently been found to be a target for fumagillin or TNP-470, which inactivates MetAP2 enzyme activity through covalent modification. Primary cultures of human endothelial cells and six other non-endothelial cell types were treated with fumagillin to determine its effect on cell proliferation. Only the growth of endothelial cells was completely inhibited at low concentrations of fumagillin. MetAP1 and MetAP2 levels in these cells were investigated to determine whether differential enzyme expression plays a role in the selective action of fumagillin. Western blot analysis and RT-PCR data showed that MetAP1 and MetAP2 were both expressed in these different types of cells, thus, ruling out differential expression of MetAP1 and MetAP2 as an explanation for the cell specificity of fumagillin. Expression of MetAP2, but not of MetAP1, is regulated. Treatment of human microvascular endothelial cells (HMVEC) with fumagillin resulted in threefold increases of MetAP2 protein in the cells, while MetAP1 remained constant. Similar upregulation of MetAP2 by exposure to fumagillin was also observed in non-endothelial cells, eliminating this response as an explanation for cell specificity. Taken together, these results indicate that while MetAP2 plays a critical role in the effect of fumagillin on endothelial cell proliferation, differential endogenous expression or fumagillin-induced upregulation of methionine aminopeptidases is not responsible for this observed selective inhibition.     

Prolonged force increase following a high-frequency burst is not due to a sustained elevation of [Ca2+]i

A brief high-frequency burst of action potentials results in a sustained force increase in skeletal muscle. The present study investigates whether this force potentiation is the result of a sustained increase of the free myoplasmic [Ca2+] ([Ca2+]i). Single fibers from mouse flexor brevis muscles were stimulated with three impulses at 150 Hz (triplet) at the start of a 350-ms tetanus or in the middle of a 700-ms tetanus; the stimulation frequency of the rest of the tetanus ranged from 20 to 60 Hz. After the triplet, force was significantly (P < 0.05) increased between 17 and 20% when the triplet was given at the start of the tetanus and between 5 and 18% when the triplet was given in the middle (n = 7). However, during this potentiation, [Ca2+]i was not consistently increased. Hence, the increased force following a high-frequency burst is likely due to changes in the myofibrillar properties.     

Early vasodilator response to anodal current application in human is not impaired by cyclooxygenase-2 blockade

It is generally acknowledged that cutaneous vasodilatation in response to monopolar galvanic current application would result from an axon reflex in primary afferent fibers and the neurogenic inflammation resulting from neuropeptide release. Previous studies suggested participation of prostaglandin (PG) in anodal current-induced cutaneous vasodilatation. Thus the inducible cyclooxygenase (COX) isoform (COX-2), assumed to play a key role in inflammation, should be involved in the synthesis of the PG that is released. Skin blood flow (SkBF) variations induced by 5 min of 0.1-mA monopolar anodal current application were evaluated with laser-Doppler flowmetry on the forearm of healthy volunteers treated with indomethacin (COX-1 and COX-2 inhibitor), celecoxib (COX-2 inhibitor), or placebo. SkBF was indexed as cutaneous vascular conductance (CVC), expressed as percentage of heat-induced maximal CVC (%MVC). Urinalyses were performed to test celecoxib treatment efficiency. No difference was found in CVC values at rest: 14.3 +/- 4.0, 11.9 +/- 3.2, and 10.9 +/- 2.0% MVC after indomethacin, celecoxib, and placebo treatment, respectively. At 10 min after the onset of anodal current application, CVC values were 22.2 +/- 4.9% MVC (not significantly different from rest) with indomethacin, 85.7 +/- 15.3% MVC (P < 0.001 vs. rest) with celecoxib, and 70.4 +/- 13.1% MVC (P < 0.001 vs. rest) with placebo. Celecoxib significantly depressed the urinary prostacyclin metabolite 6-keto-PGF(1alpha) (P < 0.05 vs. placebo). Indomethacin, but not celecoxib, significantly inhibited the anodal current-induced vasodilatation. Thus, although they are assumed to result from an axon reflex in primary afferent fibers and neurogenic inflammation, these results suggest that the early anodal current-induced vasodilatation is mainly dependent on COX-1-induced PG synthesis.     

Pollen aperture heteromorphism is not due to unreduced gametophytes

Variations in ploidy level of the sporophyte are usually acknowledged as the main cause of polymorphism in pollen aperture number and pollen size. In heteromorphic species (two or more pollen morphs in all flowers of all plants) the different pollen types could thus be due to the presence of reduced and unreduced gametes. But data from the fossil record indicate that pollen aperture number has increased, suggesting that this trait may have evolved through selection. Our studies of Viola diversifolia have shown that the different aperture morphs have different selective values, also indicating that this character could have evolved and is not always due to ploidy level variations. To test this hypothesis, we measured DNA content of the reproductive nucleus as a test of gametophytic reduction in four heteromorphic species of Viola and in diploid and tetraploid individuals of a Petunia hybrida clone (which also served as a control to test for accuracy of the measurements). Our results show that aperture polymorphism is not due to gametophytic ploidy variations within plants and that variations in the proportions of the different pollen morphs are not correlated with variations of the amount of DNA in the sporophyte.     

The effect of dipicolinic acid on maize tissue culture growth is not solely due to inhibition of lysine biosynthesis

Dipicolinic acid, a known inhibitor of an enzyme (dihydrodipicolinic acid reductase) in the maize (Zea mays L.) lysine biosynthetic pathway, inhibits the growth of maize suspension and callus cultures. Inhibited cultures contain somewhat lower free lysine levels, but the inhibition of suspension culture growth was not reversible with simultaneous addition of L-lysine to the culture medium. It is concluded that dipicolinic acid does not act solely as an analog blocking lysine production. Dipicolinic acid thus appears to be unsuitable as a selection for maize tissue culture mutants with lysine overproduction.Abbreviations FW fresh weight - I₅₀ inhibitor concentration at which cell growth is inhibited by 50% - MS Murashige and Skoog (1962) culture medium - ZM Black Mexican Zea mays suspension culture of Chourey and Zurawski (1981)     

Thymidylate synthase inhibition in cells with arrested DNA synthesis is not due to an allosteric interaction in the replitase complex

Activity of thymidylate synthase was measured in situ in leukemia cells by tritium release from [5-3H]dUrd. Aphidicolin, an inhibitor of DNA polymerase alpha, but not thymidylate synthase, caused a time dependent inhibition of the enzyme when added to the cells after [5-3H]dUrd. Cells treated with hydroxyurea and aphidicolin in sequence before addition of [5-3H]dUrd had a high initial thymidylate synthase activity that decreased with time. This pattern indicates that thymidylate synthase activity is linked to DNA synthesis; however, its inhibition by drugs that inhibit DNA synthesis may be due to accumulation of thymidine nucleotide(s), rather than to an allosteric interaction in the replitase complex.     

Impaired endothelium-mediated vasodilation is not the principal cause of vasoconstriction in heart failure

The extent to which abnormal endothelium-dependent vasodilator mechanisms contribute to abnormal resting vasoconstriction and blunted reflex vasodilation seen in heart failure is unknown. The purpose of this study was to test the hypothesis that the resting and reflex abnormalities in vascular tone that characterize heart failure are mediated by abnormal endothelium-mediated mechanisms. Thirteen advanced heart-failure patients (New York Heart Association III-IV) and 13 age-matched normal controls were studied. Saline, acetylcholine (20 microg/min), or L-arginine (10 mg/min) was infused into the brachial artery, and forearm blood flow was measured by venous plethysmography at rest and during mental stress. At rest, acetylcholine decreased forearm vascular resistance in normal subjects, but this response was blunted in heart failure. During mental stress with intra-arterial acetylcholine or L-arginine, the decrease in forearm vascular resistance was not greater than during saline control in heart failure [saline control vs. acetylcholine (7 +/- 3 vs. 6 +/- 3, P = NS) or vs. L-arginine (9 +/- 2 units, P = NS)]. The increase in forearm blood flow was not greater than during saline control in heart failure [saline control vs. acetylcholine (1. 2 +/- 0.3 vs. 1.3 +/- 0.3, P = NS), or vs. L-arginine (1.2 +/- 0.2 ml x min(-1) x 100 ml(-1), P = NS)]. Furthermore, during mental stress with nitroprusside, the decrease in forearm vascular resistance was not greater than during saline control [saline control vs. nitroprusside (7 +/- 3 vs. 5 +/- 4 ml x min(-1) x 100 g(-1), P = NS)], and the increase in forearm blood flow was not greater than during saline control [saline control vs. nitroprusside (1.2 +/- 0.3 vs. 1.3 +/- 0.5 ml x min(-1) x 100 g(-1), P = NS)]. Because the endothelial-independent agent nitroprusside was unable to restore resting and reflex vasodilation to normal in heart failure, we conclude that impaired endothelium-mediated vasodilation with acetylholine-nitric oxide cannot be the principal cause of the attenuated resting- or reflex-mediated vasodilation in heart failure.     

Antiaflatoxigenic activity of eugenol is due to inhibition of lipid peroxidation

Eugenol inhibited aflatoxin production by Aspergillus parasiticus NRRL 2999 in a dose-dependent manner up to a concentration of 0.75 mmol l-1 without inhibiting growth. When the mould was grown for 3 d in the presence of 0.45 mmol l-1 eugenol (concentration inhibiting aflatoxin production by 50%), in vivo activities of components of polysubstrate monooxygenase were decreased at idiophase, concomitant with decreased activities of enzymes involved in free radical scavenging, lipid peroxidation and maintenance of redox potential. These results indicate that antiaflatoxigenic actions of eugenol may be related to inhibition of the ternary steps of aflatoxin biosynthesis involving lipid peroxidation and oxygenation.     

Pulsatile secretion of prolactin in the male rat after pimozide administration is not due to pulsatile inhibition of PIF secretion.

Sequential blood samples were taken every 2 min from intact male rats implanted with a permanent indwelling right atrial cannula. The relationship between pimozide dose and prolactin secreation was established by administering graded doses of pimozide (30–3000 μg/kg) as a single bolus injection through the indwelling cannula. The maximum response of prolactin secretion was achieved with 300 μg/kg pimozide. Higher doses of pimozide did not raise further the circulating prolactin concentration suggesting that the receptors for the presumed prolactin inhibiting factor (PIF) were blocked completely at this dose. Marked pulsatile fluctuations in circulating prolactin concentration were observed after administration of pimozide, at all dosages, or of another ‘specific’ dopaminergic receptor blocking agent, d-butaclamol. Since we assume that PIF receptors are completely blocked by the higher doses of pimozide, we conclude that this pulsatile secretion of prolactin cannot be due to the inhibition of PIF secretion but may be due either to the stimulation of prolactin releasing factor (PRF) secretion, or to an inherent rhythmicity in the prolactin secreting cells.     

The inhibition of prolactin secretion due to intrahypothalamic pituitary grafts is reversed by estradiol benzoate but not by progesterone

Anterior pituitary (AP) tissue grafted into the hypothalamus inhibited the luteotrophic response to mating and prevented pseudopregnancy (PSP) and pregnancy. All normal rats given 10 micrograms estradiol benzoate (EB) on estrus became PSP (15 days) while the same treatment caused 10-day PSPs in 20/21 grafted rats. Doses of 30 micrograms EB or 10 micrograms EB plus reserpine (1 mg/kg) resulted in 15-day PSP in grafted rats. By contrast progesterone (P; 10 mg on estrus) did not prolong cycles in rats with hypothalamic grafts though it did in 50% of normals. Earlier studies showed that PSP or pregnancy was restored in the grafted rats by blockade of dopamine (DA) secretion. The results above show that EB was similarly effective in restoring PSP while P was not, suggesting that EB both raised prolactin and lowered DA while P was unable to lower DA in rats with AP grafts in the hypothalamus.