Inhibition of acetylcholine-mediated effects by borneol

We previously reported that the aqueous extract from a medicinal plant Dryobalanops aromatica specifically inhibits the nicotinic acetylcholine receptor (nAChR) (Oh et al. Pharmacol Res 2000;42(6):559-64). Here, the effect of borneol, the main constituent of D. aromatica, on nAChR activity was investigated in bovine adrenal chromaffin cells. Borneol inhibited a nAChR agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP)-induced calcium increase with a half maximal inhibitory concentration (IC(50)) of 56+/-9 microM. In contrast, borneol did not affect the calcium increases induced by high K+, veratridine, and bradykinin. The sodium increase induced by DMPP was also inhibited by borneol with similar potency (49+/-12 microM), suggesting that the activity of nAChRs is inhibited by borneol. Borneol inhibited DMPP-induced secretion of [3H]norepinephrine with an IC(50) of 70+/-12 microM. Carbon-fiber amperometry also confirmed the inhibition of DMPP-induced exocytosis by borneol in single chromaffin cells. [3H]nicotine binding, however, was not affected by borneol. The inhibitory effect by borneol is more potent than the effect by lidocaine, a commonly used local anesthetic. The data suggest that borneol specifically inhibits the nAChR-mediated effects in a noncompetitive way.     

Chronic ethanol administration decreases the ligand binding properties and the cellular content of the mannose 6-phosphate/insulin-like growth factor II receptor in rat hepatocytes

We have shown previously that chronic ethanol administration impairs the maturation of lysosomal enzymes in rat hepatocytes. The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-IIR) is a protein that facilitates the transport of lysosomal enzymes into the lysosome. Therefore, we examined whether ethanol consumption altered the ligand binding properties and the cellular content of M6P/IGF-IIR. Rats were pair-fed liquid diets containing either ethanol (36% of calories) or isocaloric maltose-dextrin for either 1 week or 5-7 weeks. Hepatocytes prepared from these animals were examined for receptor-ligand binding and receptor content. One week of ethanol feeding had no significant effect on ligand [radioiodinated pentamannose phosphate conjugated to bovine serum albumin ((125)I-PMP-BSA)] binding to hepatocytes, but cells from rats fed ethanol for 5-7 weeks bound less (125)I-PMP-BSA than pair-fed controls. Scatchard plot analysis revealed that the number of (125)I-PMP-BSA binding sites in hepatocytes from ethanol-fed rats was 49% lower than that of controls. (125)I-PMP-BSA binding by perivenular (PV) and periportal (PP) hepatocytes from ethanol-fed rats was, respectively, 40 and 48% lower than their controls, but there was no significant difference between these two types of hepatocytes. Ligand blot analysis using (125)I-insulin-like growth factor II ((125)I-IGF-II) also showed that the receptor in lysates of hepatocytes from ethanol-fed rats bound 26-27% less ligand than controls. Similarly, immunoblot analysis of cell lysates from ethanol-fed rats revealed 62% lower levels of immunoreactive M6P/IGF-IIR than controls. Feeding rats a low carbohydrate-ethanol diet did not exacerbate the reduction in M6P/IGF-IIR-ligand binding nor did it reduce the levels of immunoreactive receptor. Our findings indicate that chronic ethanol consumption lowers M6P/IGF-IIR activity and content in hepatocytes. This reduction may account, in part, for the impaired processing and delivery of acid hydrolases to lysosomes previously observed in ethanol-fed rats.     

NMR and pattern recognition studies on liver extracts and intact livers from rats treated with alpha-naphthylisothiocyanate

The metabolite profiles from livers of toxin-treated rats were investigated using high resolution 1H NMR spectroscopy of aqueous (acetonitrile/water), lipidic (chloroform/methanol) extracts and magic angle spinning (MAS)-NMR spectroscopy of intact tissue. Rats were treated with the model cholestatic hepatotoxin, alpha-naphthylisothiocyanate (ANIT, 150 mg/kg) and NMR spectra of liver were analysed using principal components analysis (PCA) to extract novel toxicity biomarker information. 1H NMR spectra of control aqueous extracts showed signals from a range of organic acids and bases, amino acids, sugars, and glycogen. Chloroform/methanol extracts showed signals from a range of saturated and unsaturated triglycerides, phospholipids and cholesterol. The MAS 1H NMR spectra of livers showed a composite of signals found in both aqueous and lipophilic extracts. Following ANIT treatment, 1H NMR-PCA of aqueous extracts indicated a progressive reduction in glucose and glycogen, together with increases in bile acid, choline, and phosphocholine signals. 1H NMR-PCA of chloroform/methanol extracts showed elevated triglyceride levels. The 1H MAS-NMR-PCA analysis allowed direct detection of all of the ANIT-induced tissue perturbations revealed by 1H NMR of extracts, enabling metabolic characterisation of the lesion, which included steatosis, bile duct obstruction and altered glucose/glycogen metabolism. MAS-NMR spectroscopy requires minimal sample preparation and, unlike 1H NMR spectroscopy of tissue extracts, does not discriminate metabolites based on their solubility in a particular solvent and so this is a particularly useful exploratory tool in biochemical toxicology.     

Chronic ethanol consumption impairs receptor-mediated endocytosis of MAA-modified albumin by liver endothelial cells

Alcoholic liver disease has been associated with abnormalities in receptor-mediated endocytosis (RME) which results in abnormal degradation of metabolically altered proteins. Model systems using formaldehyde-modified albumin (f-Alb) have shown an impairment in RME following chronic alcohol consumption utilizing both in situ perfused rat livers and isolated rat liver endothelial cells (LECs). The discovery that alcohol metabolite derived aldehydes can modify proteins prompted a study to determine if malondialdehyde-acetaldehyde-modified albumin (MAA-Alb) would be degraded similar to that reported for f-Alb, and whether ethanol-fed rats would demonstrate an impaired RME with respect to this ligand which occurs as a consequence of chronic ethanol consumption. MAA-Alb was degraded slightly more than f-Alb in both in situ perfused livers and at the single cell level. This degradation was completely inhibited with 100x unlabeled f-Alb, which suggests the use of a similar receptor. Following alcohol consumption there was a 50-60% decrease in MAA-Alb degradation in whole livers and isolated LECs. Utilizing isolated LECs it was determined that impairment in internalization was the most likely mechanism for the decrease in the amount of MAA-Alb that was degraded. These data show that chronic alcohol consumption by rats does in fact impair RME of alcohol metabolite-derived adducted proteins, and this impairment is due to a defect in the post-internalization step rather than the binding or degradation of the modified protein.     

Cloning and characterization of a novel human 5-HT4 receptor variant that lacks the alternatively spliced carboxy terminal exon. RT-PCR distribution in human brain and periphery of multiple 5-HT4 receptor variants.

We have cloned a novel C-terminal splice variant of serotonin 5-HT4 receptors from human hippocampus. The deduced protein extends only one aminoacid past the splicing point. We propose to call the novel variant h5-HT4(n) since it contains none of the C-terminal exons alternatively spliced in other variants. The pharmacological profile of h5-HT4(n) stably expressed in HeLa cells is in agreement with other reported variants. Stably transfected cells showed increased basal levels of intracellular cAMP in absence of agonist, indicating constitutive activity of the expressed receptors. 5-HT induced robust increases of intracellular cAMP. The 5-HT4 receptor antagonist GR 113808 blocked the effects of 5-HT and brought intracellular cAMP below basal constitutive levels, indicating inverse agonism of this compound in this system. The RT-PCR distribution of all known human C-terminal splice variants in human brain regions and periphery showed complex patterns of variant expression, with the novel variant h5-HT4(n) being widely and abundantly expressed.     

Differentially altered cerebral metabolism in ischemic rats by alpha2-adrenoceptor blockade and its relation to improved limb-placing reactions.

The selective alpha2-adrenoreceptor antagonist, atipamezole, improves behavioural performance of rats subjected to focal cerebral ischemia. The aim of the present study was to investigate whether the facilitatory effect of atipamezole on behaviour is related to altered neuronal activity in specific brain areas. The right middle cerebral artery of rats was occluded for 120 min using the intraluminal filament method. Starting on day 2 after induction of ischemia, atipamezole (1mg/kg, s.c.) or 0.9% NaCl was administered to ischemic or sham-operated rats once a day 30 min before the limb-placing test. [14C]Deoxyglucose ([14C]DG) uptake was used to measure neuronal activity 30 min after atipamezole or 0.9% NaCl administration on day 6 after ischemia. Ischemia induced a significant decrease in [14C]DG uptake in several cortical areas ipsilateral and contralateral to the lesion, in the ipsilateral thalamus, and bilaterally in the cerebellum and spinal cord. Administration of atipamezole normalised [14C]DG uptake particularly in the cerebellum and spinal cord both in sham-operated and ischemic rats and to a lesser extent in the thalamus in sham-operated rats. The pattern of altered cerebral [14C]DG uptake following alpha2-adrenoceptor blockade suggests that plasticity in the cerebellum and spinal cord contributes to the improved performance of ischemic rats in tests assessing tactile/proprioceptive limb-placing reactions.     

Activation of deoxycytidine kinase by gamma-irradiation and inactivation by hyperosmotic shock in human lymphocytes

Deoxycytidine kinase (dCK) is a key enzyme in the intracellular metabolism of deoxynucleosides and their analogues, phosphorylating a wide range of drugs used in the chemotherapy of leukaemia and solid tumours. Previously, we found that activity of dCK can be enhanced by incubating primary cultures of lymphocytes with substrate analogues of the enzyme, as well as with various genotoxic agents. Here we present evidence that exposure of human lymphocytes to 0.5-2 Gy dosage of gamma-radiation as well as incubation of cells with calyculin A, a potent inhibitor of protein phosphatase 1 and 2A, both elevate dCK activity without changing the level of dCK protein. When cells were gamma-irradiated in the presence of calyculin A, a more pronounced activation of dCK was observed. In contrast, both basal and stimulated dCK activities were reduced by hyperosmotic treatment of the cells. DNA repair determined by the Comet assay and by thymidine incorporation was induced by irradiation. Complete repair of gamma-irradiated DNA was detected within 1 hr following the irradiation along with dCK activation, but the rate of repair was not accelerated by calyculin A. These data provide evidence for the activation of dCK upon DNA damage and repair that seems to be mediated by phosphorylation of the enzyme, suggesting the role of dCK in DNA repair processes.     

Further studies on the interaction of nonpolyglutamatable aminopterin analogs with dihydrofolate reductase and the reduced folate carrier as determinants of in vitro antitumor activity

Thirteen structural analogs of the potent nonpolyglutamatable dihydrofolate reductase inhibitor N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523) with modifications in the side chain, the para-aminobenzoyl moiety, or the 9,10-bridge were evaluated for the ability to inhibit human recombinant dihydrofolate reductase (DHFR), to utilize the reduced folate carrier (RFC) for influx, and to inhibit the growth of CCRF-CEM human leukemia cells in culture. In spectrophotometric assays of the kinetics of the reduction of dihydrofolate by DHFR in the presence of NADPH, these compounds had K(i) values ranging from 0.2 to 1.3pM, and thus were not greatly different in potency from the parent drug PT523. By comparison, the K(i) values of aminopterin (AMT), methotrexate (MTX), and 10-ethyl-10-deazaaminopterin (EDX) were 3.7, 4.8, and 11pM. In assays of competitive inhibition of [3H]MTX influx into CCRF-CEM cells, the K(i) values ranged from 0.21 to 7.3 micro M, as compared with 0.71, 5.4, and 1.1 micro M for PT523, AMT, and EDX. The K(t) for MTX was also re-analyzed and found to be 4.7 micro M, in better agreement with the literature than our previously reported value of 7.1 micro M. The IC(50) values of these compounds as inhibitors of the growth of CCRF-CEM cells after 72hr of drug exposure ranged from 0.53 to 55nM, and were qualitatively consistent with the other results.     

Effects of glycolipids from spinach on mammalian DNA polymerases

We purified the major glycolipids in the class of monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG) and sulfoquinovosyl diacylglycerol (SQDG) from a green vegetable, spinach (Spinacia oleracea L.). MGDG was an inhibitor of the growth of NUGC-3 human gastric cancer cells, but DGDG and SQDG had no such cytotoxic effect. Therefore, we studied MGDG and its monoacyglycerol-form, monogalactosyl monoacylglycerol (MGMG), in detail. MGMG with one fatty acid molecule was obtained from MGDG with two fatty acid molecules by hydrolyzing with a pancreatic lipase. MGMG was also found to prevent the cancer cell growth. MGDG was a potent inhibitor of replicative DNA polymerases such as alpha, delta and epsilon. MGMG inhibited the activities of all mammalian DNA polymerases including repair-related DNA polymerase beta with IC(50) values of 8.5-36 microg/mL, and the inhibition by MGMG was stronger than that by MGDG. Both MGDG and MGMG could halt the cell cycle at the G1 phase, and subsequently induced severe apoptosis. The relationship between the DNA polymerase inhibition and the cell growth effect by these glycolipids is discussed.     

Glucuronidation of fenamates: kinetic studies using human kidney cortical microsomes and recombinant UDP-glucuronosyltransferase (UGT) 1A9 and 2B7

Mefenamic acid, a non-steroidal anti-inflammatory drug (NSAID), is used commonly to treat menorrhagia. This study investigated the glucuronidation kinetics of flufenamic, mefenamic and niflumic acid using human kidney cortical microsomes (HKCM) and recombinant UGT1A9 and UGT2B7. Using HKCM Michaelis-Menten (MM) kinetics were observed for mefenamic (K(m)(app) 23 microM) and niflumic acid (K(m)(app) 123 microM) glucuronidation, while flufenamic acid exhibited non-hyperbolic (atypical) glucuronidation kinetics. Notably, the intrinsic renal clearance of mefenamic acid (CL(int) 17+/-5.5 microL/minmg protein) was fifteen fold higher than that of niflumic acid (CL(int) 1.1+/-0.8 microL/minmg protein). These data suggest that renal glucuronidation of mefenamic acid may result in high intrarenal exposure to mefenamic acyl-glucuronide and subsequent binding to renal proteins. Diverse kinetics were observed for fenamate glucuronidation by UGT2B7 and UGT1A9. Using UGT2B7 MM kinetics were observed for flufenamic (K(m)(app) 48 microM) and niflumic acid (K(m)(app) 135 microM) glucuronidation and atypical kinetics with mefenamic acid. Similarity in K(m)(app) between HKCM and UGT2B7 suggests that UGT2B7 may be the predominant renal UGT isoform catalysing niflumic acid glucuronidation. In contrast, UGT1A9 glucuronidation kinetics were characterised by negative cooperativity with mefenamic (S(50) 449 microM, h 0.4) and niflumic acid (S(50) 7344 microM, h 0.4) while atypical kinetics were observed with flufenamic acid. Additionally, potent inhibition of the renal glucuronidation of the UGT substrate 'probe' 4-methylumbelliferone by flufenamic, mefenamic and niflumic acid was observed. These data suggest that inhibitory metabolic interactions may occur between fenamates and other substrates metabolised by UGT2B7 and UGT1A9 in human kidney.     

Reversal of P-glycoprotein-mediated multidrug resistance by Alisol B 23-acetate

Herbal drugs were screened for their activity in reversing multidrug resistance (MDR) in P-glycoprotein (P-gp) over-expressing cancer cells. Through bio-assay guided fractionation an active compound was isolated from Rhizoma Alismatis, the underground part of Alisma orientale and the chemical structure of the isolate compound was confirmed by HPLC, LC-MS and NMR as Alisol B 23-acetate (ABA). ABA restored the sensitivity of MDR cell lines HepG2-DR and K562-DR to anti-tumor agents that have different modes of action but are all P-gp substrates. It restored the activity of vinblastine, a P-gp substrate, in causing G2/M arrest in MDR cells. In a dose-dependent manner, ABA increased doxorubicin accumulation and slowed down the efflux of rhodamin-123 from MDR cells. ABA inhibited the photoaffinity labeling of P-gp by [125I]iodoarylazidoprazosin and stimulated the ATPase activity of P-gp in a concentration-dependent manner, suggesting that it could be a transporter substrate for P-gp. In addition, ABA was also a partial non-competitive inhibitor of P-gp when verapamil was used as a substrate. Our results suggest that ABA may be a potential MDR reversal agent and could serve as a lead compound in the development of novel drugs.     

Selective activation of deoxycytidine kinase by thymidine-5'-thiosulphate and release by deoxycytidine in human lymphocytes

Deoxycytidine kinase (dCK) catalyses the rate-limiting step of the salvage of three natural deoxyribonucleosides as well as several therapeutic nucleoside analogues, which in turn can enhance its enzymatic activity [Biochem Pharmacol 56 (1998) 1175], improving the efficacy of the cytostatic therapy. Here, we measured the effect of the 5'-thiosulphate (5'-TS) derivatives of four deoxyribonucleosides (deoxyadenosine, deoxycytidine (dCyd), azidothymidine, thymidine) and two ribonucleosides (ribopurine, ribouridine (Urd)) on the activity of the two main salvage deoxynucleoside kinases, and on the salvage of dCyd and deoxythymidine (dThd). It turned out that only 2'-deoxythymidine-5'-thiosulphate (dThd-5'-TS) can potentiate the dCK activity, without influencing the thymidine kinase isoenzymes during short-time treatments of human peripheral blood and tonsillar lymphocytes. The enhancement of dCK activity by dThd-5'-TS can be reversed by dCyd, but dThd had no effect on the enzyme activation in cells. Neither dThd-5'-TS nor Urd-5'-TS had any effect on the dCK and thymidine kinase activities tested in cell-free extracts. The stimulation of dCK activity in cells was accompanied by an imbalance in the dThd and dCyd metabolism. The incorporation of 3H-dThd into DNA was suppressed by 90% in cells by dThd-5'-TS, while Urd-5'-TS only slightly influenced the same process. The 3H-dCyd incorporation into DNA was inhibited only to 50% of the control, while the 3H-dCyd labelling of the nucleotide fraction was enlarged in dThd-5'-TS-treated cells, as a consequence of the increased dCK activity. We suggest that the enhancement of dCK activity is a compensatory mechanism in cells that might be induced by different "inhibitors" of DNA synthesis leading to damage of DNA. The increased dCK activity is able to supply the repair of DNA with dNTPs in quiescent cells; this suggestion seems to be supported by the counteracting effect of extracellular dCyd, too.     

Important amino acids for the function of the human MT1 melatonin receptor

Models of G protein-coupled melatonin receptor structure suggest that ligand recognition occurs in a binding pocket formed by transmembrane helices III, V and VII. Constitutively active mutations in G protein-coupled receptors have revealed that transmembrane helix III/intracellular loop 2 interface and transmembrane domain VI are critical regions in receptor activation. In this study, nine site-directed mutants of the human MT1 melatonin receptor were created to test the importance of specific amino acids in these regions in ligand recognition and receptor activation events. We analyzed ligand binding, G protein activation and subcellular localization of MT1 receptors transiently expressed in COS-7 cells. Receptor ELISA was employed to study expression levels of N-terminally HA epitope tagged wild-type and mutant MT1 receptors. Mutations in histidine H195 (His(5.46)) in transmembrane domain V reduced receptor affinity for 2-[125I]iodomelatonin. Several other mutants had diminished expression on the plasma membrane. Amino acids M107 (Met(3.32)) in transmembrane domain III and S280 (Ser(7.46)) in transmembrane domain VII were found not to participate in ligand recognition in human MT1 receptor. Constitutive activity was not obtained with mutations in N124 (Asn(3.49)) or P253 (Pro(6.50)). These mutants failed to bind 2-[125I]iodomelatonin and had reduced expression levels. The need to upgrade current melatonin receptor models has become evident. Several important amino acids for the human MT1 melatonin receptor function were revealed in the current study, with effects of mutations ranging from slightly reduced affinity or efficacy to complete loss of function.     

Modulation of [3H] TBOB binding to the rodent GABAA receptor by simple disaccharides

We have recently reported evidence that a simple beta-linked alkylated mannose reversibly increased the magnitude of GABA(A) receptor currents evoked in cultured rat pyramidal neurons whilst concomitantly reducing the incidence of spontaneous synaptic activity. In this present study, the effects of the simple beta-linked disaccharide, lactose was investigated using a [3H] TBOB (t-[3H] butylbicycloorthobenzoate) binding assay in adult rat forebrain and cerebellum membranes. Lactose elicited a significant potentiation of [3H] TBOB binding to well-washed forebrain and cerebellar membranes (mean E(max) values=367 and 287%; mean EC(50) values=1.5 and 30 microM, respectively, N=4). The alpha-linked disaccharides, maltose and sucrose also potentiated [3H] TBOB binding, but with 100-600-fold higher EC(50) values than lactose. The lactose-mediated potentiation of [3H] TBOB in the forebrain and cerebellum was completely abolished in the presence of 0.3 microM GABA. Over the concentration range in which significant potentiation of [3H] TBOB binding was detected, lactose elicited no significant effect upon [3H] flunitrazepam binding. This study demonstrated that lactose can modulate the GABA(A) receptor channel, allosterically coupled to the agonist site, but independent of the benzodiazepine site. Furthermore, lactose displayed differential effects upon forebrain and cerebellar GABA(A) receptors indicating that it may be a novel subtype selective agent.     

Molecular pharmacology of the ovine melatonin receptor: comparison with recombinant human MT1 and MT2 receptors

The variations of the pharmacological properties of melatonin receptors between different mammalian species in transfected cell lines have been poorly investigated. In the present study, melatonin analogues have been used to characterize the pharmacology of the recombinant ovine melatonin receptor (oMT1) expressed in CHO cell lines and the native oMT1 from the pars tuberalis (PT). Studies with selective ligands on native and transfected oMT1 showed similar properties for binding affinities [r2(PT/CHO) = 0.85]. The affinities and the functional activities of these ligands were compared with the human receptors (hMT1 or hMT2) expressed in CHO cells as well. The oMT1 and hMT1 receptors had similar pharmacological profiles (r2=0.82). Nevertheless, some of the selective compounds at the human receptor presented a reduced affinity at the ovine receptor. Furthermore, some compounds showed marked different functional activities at oMT1 vs. hMT1 receptors. Our findings demonstrated differences in the pharmacological properties of melatonin receptors in ovine and human species.     

Modulation of A1 adenosine receptor signaling by peroxynitrite

Nitric oxide (NO) is a gaseous free radical involved in many pathophysiological processes. During oxidative stress, NO, its derivatives and adenosine are released. Considering adenosine neuroprotective role in the central nervous system (CNS) and toxicity of NO, we investigated the effect of a NO/peroxynitrite (ONOO(-)) donor, 3-morpholinosydnonimine (SIN-1), on A(1) adenosine receptor (A(1)AR) signaling pathway in rat cortical membranes. Membrane treatment with 0.5mM SIN-1 for various periods of time (0-240min) decreased specific binding of the radiolabeled A(1)AR agonist, [3H]N(6)-cyclohexyladenosine ([3H]CHA), in a time-dependent manner, reaching the steady state after 120min. The inhibitory effect of SIN-1 was concentration-dependent, with an EC(50) value of 0.60+/-0.30mM (N=3). Membrane pre-incubation with the superoxide anion (O(2)z.rad;(-)) scavenger superoxide dismutase (SOD) followed by SIN-1 addition, abolished SIN-1 inhibition of [3H]CHA binding. Membrane treatment with 0.5mM SIN-1 for 120min caused a significant 2-fold increase of the K(D) value for [3H]CHA without changing the B(max) value. Moreover, pre-incubation of membranes with A(1)AR agonists, CHA or N(6)-(2-phenylisopropyl)-adenosine (R-PIA) before SIN-1 addition increased the inhibitory effect while the selective A(1)AR antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) had no activity. Membrane treatment with SIN-1 decreased receptor-stimulated guanosine 5'-O-(gamma[35S]thio)triphosphate ([35S]GTPgammaS) binding in a concentration-dependent manner. This treatment influenced [35S]GTPgammaS binding affinity for A(1)AR activated G(i) proteins in cortical membranes. These findings suggest that ONOO(-) modulates A(1)AR signaling pathways by affecting receptor G(i) protein coupling.     

Modulation of the beta-adrenergic receptor system of vascular smooth muscle cells in vitro and in vivo by chronically elevated endothelin-1 levels

Endothelin-1 (ET-1) levels are chronically elevated in several cardiovascular diseases and correlate with an increased mortality. However, in contrast to acute biological activities such as vasoconstriction, little is known about long-term effects of ET-1. In this study we determined the effects of ET-1 on the beta(2)-adrenergic receptor (AR) system. Incubation of smooth muscle cells with ET-1 for 72 hr led to increased beta(2)AR density as determined by radioligand binding. Experiments with inhibitors of protein and RNA synthesis as well as RT-PCR revealed that beta(2)AR upregulation required de novo synthesis. In addition, protein kinase C but neither NO nor prostaglandin metabolism were involved in this effect. The enhanced expression of beta(2)AR was associated with an increased expression of its stimulatory G-protein and the receptor's ability to stimulate adenylyl cyclase. To study chronic effects of ET-1 in vivo, rats were infused with ET-1 for 3 weeks. Similarly as in cultured cells, prolonged ET-1 exposure led to increased betaAR expression in vivo. As a consequence, beta(2)AR-induced vasodilatation was increased in aortic rings from ET-1-treated animals. Our results therefore suggest that chronically elevated ET-1 levels in vitro and in vivo induce counterregulatory mechanisms by increasing betaARs that attenuate the vasoconstrictive effects of ET-1.     

Novel roles for palmitoylation of Ras in IL-1 beta-induced nitric oxide release and caspase 3 activation in insulin-secreting beta cells

We recently demonstrated that functional inactivation of H-Ras results in significant reduction in interleukin 1 beta (IL-1 beta)-mediated effects on isolated beta cells. Since palmitoylation of Ras has been implicated in its membrane targeting, we examined the contributory roles of palmitoylation of Ras in IL-1 beta-induced nitric oxide (NO) release and subsequent activation of caspases. Preincubation of HIT-T15 or INS-1 cells with cerulenin (CER, 134 microM; 3 hr), an inhibitor of protein palmitoylation, significantly reduced (-95%) IL-1 beta-induced NO release from these cells. 2-Bromopalmitate, a structurally distinct inhibitor of protein palmitoylation, but not 2-hydroxymyristic acid, an inhibitor of protein myristoylation, also reduced (-67%) IL-1 beta-induced NO release from HIT cells. IL-induced inducible nitric oxide synthase gene expression was markedly attenuated by CER. Further, CER markedly reduced incorporation of [3H]palmitate into H-Ras and caused significant accumulation of Ras in the cytosolic fraction. CER-treatment also prevented IL-1 beta-induced activation of caspase 3 in these cells. Moreover, N-monomethyl-L-arginine, a known inhibitor of inducible nitric oxide synthase, markedly inhibited IL-induced activation of caspase 3, thus establishing a link between IL-induced NO release and caspase 3 activation. Depletion of membrane-bound cholesterol using methyl-beta-cyclodextrin, which also disrupts caveolar organization within the plasma membrane, abolished IL-1 beta-induced NO release suggesting that IL-1 beta-mediated Ras-dependent signaling in these cells involves the intermediacy of caveolae and their key constituents (e.g. caveolin-1) in isolated beta cells. Confocal light microscopic evidence indicated significant colocalization of Ras with caveolin-1. Taken together, our data provide the first evidence to indicate that palmitoylation of Ras is essential for IL-1 beta-induced cytotoxic effects on the islet beta cell.     

Effect of heating on peroxynitrite scavenging capacity of garlic.

The ability to scavenge peroxynitrite (ONOO(-)) was studied in the following aqueous garlic extracts: (a) unheated extract of garlic powder (GP), (b) heated extract of garlic powder (HGP), (c) unheated extract of raw garlic (RG), (d) heated extract of raw garlic (HRG), (e) extract of boiled garlic cloves (BG), (f) extract of microwave-treated garlic cloves (MG), and (g) extract of pickled garlic (PG). All the extracts scavenged ONOO(-) in a concentration-dependent way. IC(50) (mg/mL) values for each extract were 0.30+/-0.02 and 0.35+/-0.04 for GP and HGP, respectively; and 0.84+/-0.08, 0.59+/-0.04, 0.76+/-0.09, 1.71+/-0.19, and 1.45+/-0.07 for RG, HRG, BG, MG, and PG, respectively. The ONOO(-) scavenging capacity (IC(50) values) was not decreased in HGP (vs. GP, p>0.05) and in HRG and BG (p>0.05 vs. RG). In contrast, the ONOO(-) scavenging capacity decreased in MG and PG (vs. RG, p<0.001). The IC(50) values for the reference compounds nordihydroguiaretic acid and penicillamine were 1.1 and 4.5mug/mL. The heating before or after garlic cutting was unable to eliminate the capacity of the extracts to scavenge ONOO(-); this capacity was significantly decreased in PG and MG.