Retinoic acid prevents mesenteric lymph node dendritic cells from inducing IL-13-producing inflammatory Th2 cells.

The vitamin A (VA) metabolite retinoic acid (RA) affects the properties of T cells and dendritic cells (DCs). In VA-deficient mice, we observed that mesenteric lymph node (MLN)-DCs induce a distinct inflammatory T helper type 2 (Th2)-cell subset that particularly produces high levels of interleukin (IL)-13 and tumor necrosis factor-α (TNF-α). This subset expressed homing receptors for skin and inflammatory sites, and was mainly induced by B220(-)CD8α(-)CD11b(+)CD103(-) MLN-DCs in an IL-6- and OX40 ligand-dependent manner, whereas RA inhibited this induction. The corresponding MLN-DC subset of VA-sufficient mice induced a similar T-cell subset in the presence of RA receptor antagonists. IL-6 induced this subset differentiation from naive CD4(+) T cells upon activation with antibodies against CD3 and CD28. Transforming growth factor-ß inhibited this induction, and reciprocally enhanced Th17 induction. Treatment with an agonistic anti-OX40 antibody and normal MLN-DCs enhanced the induction of general inflammatory Th2 cells. In VA-deficient mice, proximal colon epithelial cells produced TNF-α that may have enhanced OX40 ligand expression in MLN-DCs. The repeated oral administrations of a T cell-dependent antigen primed VA-deficient mice for IL-13-dependent strong immunoglobulin G1 (IgG1) responses and IgE responses that caused skin allergy. These results suggest that RA inhibits allergic responses to oral antigens by preventing MLN-DCs from inducing IL-13-producing inflammatory Th2 cells.

Effects of kaempferol on the mechanisms of drug resistance in the human glioblastoma cell line T98G.

Food contains components that may either increase or decrease the bioavailability of a drug. In particular, it is known that grapefruit juice and St. John's Wort induce drug interactions via an effect on the drug-metabolizing enzyme cytochrome P450 (CYP). However, interactions with membrane transporters, such as P-glycoprotein and multidrug resistance-related protein (MRP), may also influence drug bioavailability. The objective of the present study was to investigate the effects of kaempferol, a flavonoid present in food, on the cytotoxicity of anticancer drugs and the mechanisms of drug resistance in the human glioblastoma cell line T98G. Acute exposure to kaempferol inhibited the efflux of calcein, a substrate of MRP; however, chronic exposure caused no apparent effect on calcein efflux. The cytotoxicity of doxorubicin was not influenced by chronic exposure of cells to kaempferol, although that of cisplatin was significantly reduced. Multidrug resistance is often associated with increased levels of MRP1, glutathione S-transferase (GST) and activity by chronic exposure to kaempferol, although MRP2 protein levels are decreased. Accordingly, we hypothesized that the cytotoxicity of anticancer drugs that conjugate with glutathione and the substrate of MRPs may be influenced by long-term intake of drugs such as kaempferol, which are substrates of MRPs and GST.

Adrenergic nerves mediate acetylcholine-induced endothelium-independent vasodilation in the rat mesenteric resistance artery.

Mechanisms underlying acetylcholine-induced endothelium-independent vasodilation were studied in the rat mesenteric vascular bed isolated from Wistar rats. In preparations without endothelium, and contracted by perfusion with Krebs solution containing methoxamine (2-7 microM), perfusion of acetylcholine (1-100 microM) for 1 min produced a concentration-dependent vasodilation. Denervation of denuded preparations by cold storage (4 degrees C for 72 h) abolished the acetylcholine-induced vasodilation; 10 and 100 nM atropine abolished 1 and 10 microM acetylcholine-induced vasodilation, but it inhibited only 20% of vasodilation by 100 microM acetylcholine. The acetylcholine-induced atropine-resistant vasodilation was inhibited by 10 and 100 microM hexamethonium, 5 microM guanethidine, 50 microM bretylium, in vitro 6-hydroxydopamine (2 mM for 20 min, twice), 1 microM capsaicin and 0.5 microM calcitonin gene-related peptide (CGRP)-(8-37) (CGRP receptor antagonist). These findings suggest that the acetylcholine-induced endothelium-independent nicotinic vasodilation requires the presence of intact adrenergic nerves, and is mediated by endogenous CGRP released from CGRP-containing nerves.

Triphasic vascular responses to bradykinin in the mesenteric resistance artery of the rat.

The vascular effects of bradykinin were studied in rat perfused mesenteric vascular beds with active tone. Bolus injections of bradykinin (1-1000 pmol) but not des-Arg(9)-bradykinin (bradykinin B(1) receptor agonist) induced triphasic vascular responses: the initial sharp vasodilation followed by transient vasoconstriction and subsequent gradual vasodilation. The triphasic vascular responses to bradykinin were abolished by FR 172357 (3-bromo-8-[2,6-dichloro-3-[N-[(E)-4-(N,N-dimethylcarbamoyl) cinnamidoacetyl]-N-methylamino]benzyloxy]-2-metylimidazo[1,2-a]pyridine) (bradykinin B(2) receptor antagonist, 0.1 microM). Endothelium removal with sodium deoxycholate and N(w)-nitro-L-arginine (300 microM) abolished the bradykinin-induced initial sharp vasodilation. Indomethacin (0.5 microM) and seratrodast (thromboxane A(2) receptor antagonist, 0.5 and 5 microM) abolished the bradykinin-induced second vasoconstriction. The bradykinin-induced third vasodilation was abolished by capsaicin (1 microM) and calcitonin gene-related peptide (CGRP)-(8-37) (CGRP receptor antagonist, 0.5 microM). These findings suggest that the bradykinin-induced initial sharp vasodilation is endothelium dependent, that endogenous thromboxane A(2) is involved in the second vasoconstriction, and that the third slow vasodilation is produced by activation of capsaicin-sensitive CGRP-containing nerves.

Effects of long-term treatment with calcium antagonists on periarterial nerve function in the mesenteric artery of spontaneously hypertensive rats.

The effect of long-term treatment with dihydropyridine calcium antagonists (amlodipine, pranidipine, nicardipine) on the periarterial nerve function was investigated in the perfused mesenteric vascular bed isolated from spontaneously hypertensive rat (SHR). Male 8-week-old SHR received amlodipine (0.01% and 0.02%) and nicardipine (0.1%) in drinking water and pranidipine (0.0035% and 0.035%) in rat chow for 7 weeks. Mean blood pressure in SHR was significantly lowered by long-term treatment with each calcium antagonist. In mesenteric vascular preparations treated with each calcium antagonist, vasoconstriction induced by periarterial nerve stimulation (PNS; 4, 8 and 12 Hz) was significantly smaller than that in non-treated SHR. The PNS (8 Hz)-evoked norepinephrine (NE) overflow in the perfusate was significantly decreased by amlodipine and pranidipine treatment, whereas nicardipine-treatment significantly enhanced the overflow of NE. In preparations with active tone produced by methoxamine and guanethidine, the PNS-induced vasodilation mediated by calcitonin gene-related peptide (CGRP)-containing (CGRPergic) vasodilator nerves was not affected by these drugs. These results suggest that long-term treatment of SHR with long-acting drugs, amlodipine and pranidipine, reduces sympathetic adrenergic nerve function but calcium antagonists have no effect on CGRPergic nerve function.

Endogenous calcitonin gene-related peptide (CGRP) mediates adrenergic-dependent vasodilation induced by nicotine in mesenteric resistance arteries of the rat.

1. The mechanisms underlying vasodilator effect of nicotine on mesenteric resistance blood vessels and the role of calcitonin gene-related peptide (CGRP)-containing (CGRPergic) vasodilator nerves were studied in the rat. 2. Mesenteric vascular beds isolated from Wistar rats were perfused with Krebs solution, and perfusion pressure was measured with a pressure transducer. 3. In preparations with intact endothelium and contracted by perfusion with Krebs solution containing methoxamine, perfusion of nicotine (1 - 100 microM) for 1 min caused a concentration-dependent vasodilator response without vasoconstriction. 4. The nicotine-induced vasodilation was markedly inhibited by hexamethonium (nicotinic cholinoceptor antagonist, 10 microM) and blocked by guanethidine (adrenergic neuron blocker, 5 microM). 5. Either denervation by cold storage (4 degrees C for 72 h) or adrenergic denervation by 6-hydroxydopamine (toxin for adrenergic neurons, 2 mM for 20 min incubation, twice) blocked the nicotine-induced vasodilation. 6. Neither endothelium removal with perfusion of sodium deoxycholate (1.80 mg ml(-1), for 30 s) nor treatment with N(omega)-nitro-L-arginine (nitric oxide synthase inhibitor, 100 microM), atropine (muscarinic cholinoceptor antagonist, 10 nM) or propranolol (beta-adrenoceptor antagonist, 100 nM) affected the nicotine-induced vasodilation. 7. In preparations without endothelium, treatment with capsaicin (depleting CGRP-containing sensory nerves, 1 microM) or human CGRP[8 - 37] (CGRP receptor antagonist, 0.5 microM) markedly inhibited the nicotine-induced vasodilation. 8. These results suggest that, in the mesenteric resistanc artery of the rat, nicotine induces vasodilation, which is independent of the function of the endothelium and is involved in activation of CGRPergic nerves. It is also suggested that nicotine stimulates presynaptic nicotinic cholinoceptors on adrenergic nerves to release adrenergic neurotransmitters, which then act on CGRPergic nerves to release endogenous CGRP from the nerve.

The effects of exposure to cigarette smoke on the pharmacokinetics and pharmacodynamics of zonisamide in rats.

The effects of exposure to cigarette smoke on the pharmacokinetics and pharmacodynamics of zonisamide, an antiepileptic drug, were investigated in rats. Absorption of oral zonisamide was significantly inhibited by exposure to cigarette smoke. The Cmax, T1/2 and the area under the plasma concentration-time curve 0-24 values in the cigarette smoke exposure group were significantly lower than those in the control group. Although tonic extension (TE) induced by maximal electroshock was completely blocked by the administration of zonisamide in the control group, 50% of rats showed TE in the cigarette smoke exposure group. Exposure to cigarette smoke influences both the pharmacokinetics and antiepileptic effects of zonisamide. The effects of smoking on epileptic patients using zonisamide warrants further attention.

Long-term treatment with angiotensin converting enzyme inhibitor restores reduced calcitonin gene-related peptide-containing vasodilator nerve function in mesenteric artery of spontaneously hypertensive rats.

Effects of long-term treatment with angiotensin converting enzyme (ACE) inhibitor on decreased function of calcitonin gene-related peptide (CGRP)-containing vasodilator nerves (CGRP nerves) in mesenteric resistance artery were investigated in spontaneously hypertensive rats (SHR). Eight-week-old SHR were treated for 7 weeks with 0.1% captopril, 0.01% temocapril, 0.05% pindolol or 0.005% hydralazine in drinking water. Long-term treatment with each drug significantly lowered mean blood pressure of SHR. In isolated and perfused mesenteric vascular beds with active tone, periarterial nerve stimulation (PNS) (0.5 to 8 Hz) produced frequency-dependent vasodilations, which were abolished by CGRP(8-37) (CGRP-receptor antagonist) and significantly smaller in SHR than in normotensive Wistar Kyoto rats. Treatment of SHR with captopril and temocapril but not with pindolol and hydralazine resulted in significantly greater PNS-induced vasodilation than in non-treated SHR, but ACE-inhibitor treatment did not affect vasodilation induced by exogenous CGRP. In captopril-treated SHR preparations, PNS evoked significantly larger CGRP-like immunoreactive release than in non-treated SHR. In non-treated 15-week-old SHR preparations, direct perfusion of captopril or temocapril (0.1 microM and 1 microM) did not modify frequency-dependent vasodilation in response to PNS. These results suggest that long-term ACE inhibitor treatment prevents or restores CGRP nerve function reduction in SHR.

Involvement of histone phosphorylation in thymocyte apoptosis by protein phosphatase inhibitors.

Incubation of rat thymocytes with the inhibitors of protein phosphatase such as calyculin A and okadaic acid resulted in an increase in DNA fragmentation. These effects were dependent on the concentration of the inhibitors and the incubation time. Analyses of the fragmented DNA revealed the production of approximately 50 kbp of DNA and a 180 bp DNA ladder. In addition, a laser scanning-microscopic analysis showed that these compounds caused nuclear condensation. Thus, these results demonstrated that protein phosphatase inhibitors induced thymocyte apoptosis. The inhibitors of protein phosphatase increased the phosphorylation of proteins of approximately 15 kDa. The phosphorylation of proteins preceded the DNA fragmentation induced by these inhibitors. Judging from acetic acid-urea-Triton X-100 gel electrophoresis, the phosphorylated proteins were histone H1 and H2A/H3. Therefore, these results suggest that phosphorylation of histones triggers the DNA fragmentation of thymocytes undergoing apoptosis.

Involvement of calcitonin gene-related peptide (CGRP) receptors in insulin-induced vasodilatation in mesenteric resistance blood vessels of rats.

1. The vascular effect of insulin in the mesenteric resistance blood vessel and the role of calcitonin generelated peptide (CGRP)-receptor in insulin-induced vascular responsiveness were investigated in rats. 2. The mesenteric vascular beds isolated from Wistar rats were perfused with Krebs solution, and perfusion pressure was measured with a pressure transducer. In preparations contracted by perfusion with Krebs solution containing methoxamine in the presence of guanethidine, the perfusion of insulin (from 0.1 to 3000 nM) caused a concentration-dependent decrease in perfusion pressure due to vasodilatation. The pD2 value and maximum relaxation (%) were 6.94+/-0.22 and 43.9+/-5.2, respectively. 3. This vasodilator response to insulin was unaffected by 100 nM propranolol (beta-adrenoceptor antagonist) plus 100 nM atropine (muscarinic cholinoceptor antagonist), 100 microM L-NG-nitroarginine (nitric oxide synthase inhibitor), 1 microM ouabain (Na+-K+ ATPase inhibitor), or 1 microM glibenclamide (ATP sensitive K+-channel inhibitor). 4. In preparations without endothelium, perfusion of insulin produced a marked vasodilatation. The pD2 value and maximum relaxation (%) were 7.62+/-0.21 and 81.0+/-4.6, respectively, significantly greater than in preparations with intact endothelium. 5. The vasodilator responses to insulin in the preparations without endothelium were significantly inhibited by CGRP[8 37], a CGRP receptor antagonist, whereas pretreatment with capsaisin, a toxin for CGRP-containing nerves, did not affect insulin-induced vasodilatation. 6. These results suggest that insulin induces non-adrenergic, non-cholinergic and endothelium-independent vasodilatation, which is partially mediated by CGRP receptors.