Failure of 6-thioGMP to inhibit guanylate kinase in intact cells.

Experiments have been performed that make necessary a modification of the hypothesis that 6-thioguanine produces cytotoxicity by a sequential blockade of the enzymes, ribosylamine-5-phos-phate: pyrophosphate phosphoribosyltransferase (PRPP-amidotransferase), inosinate dehydrogenase and guanylate kinase. L5178Y murine leukemia cells (in vivo and in vitro) were pretreated with 6-thio-guanine under conditions known to produce significant accumulations of the analog nucleoside 5'-monophosphate, 6-thioGMP, inhibition of purine de novo biosynthesis, and marked cytotoxicity. When these cells were incubated with [8-¹⁴C]guanine, no inhibition in the formation of guanine nucleotides was observed in comparison with control cells not treated with 6-thioguanine. Furthermore, measurement of changes in the intracellular concentrations of GMP, GDP and GTP did not provide evidence for the occurrence of a ‘cross-over’' between GMP and GDP in the presence of 6-thioGMP. Thus, the predicted accumulation of GMP resulting from the postulated blockade of guanylate kinase by 6-thioGMP did not occur. L5178Y cells incubated with guanine for periods of 1 hr or less accumulated concentrations of GDP and GTP that approximated the intracellular levels of ADP and ATP. Time studies were performed with human erythrocytes in which the rate of formation of nucleotides was compared in cells incubated with guanosine or 6-thioguanosine. With guanosine, the rapid formation of guanine nucleotides was observed with the ratio of GTP:GDP:GMP approximating the ratios of the adenine nucleotides of the erythrocytes, i.e. no accumulation of GMP was observed at any time period up to 6 hr. In contrast, with 6-thioguanosine, a rapid initial formation of 6-thioGMP was observed with a gradually accelerating formation of 6-thioGTP. After 2 hr, the concentration of 6-thioGMP decreased whereas the formation of 6-thioGTP achieved a velocity of about 0.007 μmole/ min/ml of erythrocytes. This velocity is about 2 per cent of that expected with saturating levels of GMP, if one assumes the intraerythrocytic activity of guanylate kinase to be 0.3 enzyme unit/ml of cells (enzyme unit: 1 unit catalyzes the conversion of 1 μmole substrate into product/min). These findings are in general agreement with the results of studies with purified guanylate kinase preparations described in the accompanying publication [R.L. Miller, D.L. Adamczyk, T. Spector, K.C. Agarwal, R.P. Miech and R.E. Parks Jr., Biochem. Pharmac. 26, 1573 (1977)], which indicate that the K_m value and V_max value of 6-thioGMP are approximately 2.0 mM and about 3 per cent of the V_max with GMP respectively. Therefore, it may be concluded that administration of 6-thioguanine does not cause significant inhibition of guanylate kinase. However, the poor reactivity of 6-thioGMP with guanylate kinase probably causes the marked intracellular accumulation of this analog nucleotide after administration of 6-thioguanine or its derivatives.     

Effect of exogenous phosphorylated monosaccharides on hormone-sensitive lipolysis in rat adipose tissue.

The activity of glucose-1-phosphate, glucose-6-phosphate, fructose, fructose-1-phosphate, fructose-6-phosphate, and fructose-1,6-diphosphate was tested on rat adipose tissue. The only active one was fructose-1,6-diphosphate (FDP). It slightly increased basal lipolysis and strongly potentiated the lipolytic effect of noradrenaline and theophylline, but not that of dibutyryl cAMP. Furthermore FDP increased basal ATP levels and potentiated cAMP accumulation induced by noradrenaline. Phosphate potentiated the lipolytic effect of noradrenaline but did not increase ATP level. The activity of FDP on lipolysis seems not due to its hydrolytic products and may be related to an increase of ATP availability.     

Studies on the biochemical actions of 6-selenoguanine and 6-selenoguanosine

Survival studies were performed in mice bearing Sarcoma 180 ascites tumor treated with 6-thio and 6-seleno analogs of guanine and guanosine. The selenium-containing analogs were somewhat superior to the sulfur-containing compounds in antitumor activity and therapeutic index. The formation of 6-SeGMP from 6-seleno-guanine (6-SeG) was demonstrated in Sarcoma 180 ascites cells. Guanine, 6-thioguanine (6-TG) and 6-SeG show comparable substrate activity whereas 8-azaguanine is a much poorer substrate for hypoxanthine-guanine phosphoribosyl transferase from Sarcoma 180 cells. Both 6-TG and 6-SeG are good substrates for purine nucleoside phosphorylase from Sarcoma 180 cells. Chemically and enzymatically synthesized 6-SeGMP behaved as a competitive inhibitor (K_i 1 × 10^?4 M) of erythrocytic and Sarcoma 180 guanylate kinases. Weak substrate activity was demonstrated in the presence of large amounts of erythrocytic guanylate kinase.     

Inhibitors of orotate phosphoribosyl-transferase and orotidine-5'-phosphate decarboxylase from mouse Ehrlich ascites cells: a procedure for analyzing the inhibition of a multi-enzyme complex

Orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase, the last two enzymes in the pathway for de novo pyrimidine biosynthesis, exist as a multi-enzyme complex in mammalian cells. Because the two enzymes are not separable, assays for the phosphoribosyltransferase are normally dependent on the decarboxylase, and this has in the past produced misinterpretations in evaluating the effects of inhibitors of either enzyme activity. We have developed a procedure for calculating each enzyme activity independently of the other activity in the complex. Instead of 2 separate assays, one reaction vessel is used, (starting with orotate and phosphoribosyl pyrophosphate). The two products (i.e. orotidylate (OMP), the product of the transferase, and UMP, the product of the decarboxylase) are recovered separately, and the amounts recovered were used to calculate both enzyme activities. Purine or pyrimidine bases did not inhibit the decarboxylase activity, but the transferase activity is inhibited by uracil and uracil analogs, barbiturate, and most effectively by 5-F orotate. Both purine and pyrimidine nucleotides inhibit the decarboxylase activity, although di- and tri-phos-phonucleosides were much less effective than monophosphonucleosides. XMP, UMP, and AMP were the most effective natural inhibitors of the decarboxylase, but the most potent inhibitors were 6-AzaUMP and 1-ribosylallopurinol-5'-phosphate. High concentrations of nucleotides inhibit both enzyme activities.     

Structural analogs of 5'-methylthioadenosine as substrates and inhibitors of 5'-methylthioadenosine phosphorylase and as inhibitors of human lymphocyte transformation

5'-Deoxy-5'-methylthioadenosine (MTA) phosphorylase was purified 13.4-fold from human peripheral lymphocytes. The enzyme demonstrated normal Michaelis-Menten kinetics with Km values of 26 microM and 7.5 mM for the two substrates, MTA and phosphate, respectively. The rate of MTA degradation was temperature dependent, 47 degrees being the optimum temperature. Five structural analogs served as alternative substrates with Km values ranging from 31 to 53 microM while two compounds, 5'-deoxy-5'-methylthiotubercidin (MTT) (Ki = 31 microM) and adenine (Ki = 172 microM), were inhibitory. These same analogs were examined as inhibitors of mitogen-induced human lymphocyte blastogenesis. MTT was found to be the most effective inhibitor of lymphocyte transformation with an I50 of 80 microM.     

Adenosine regulation of canine cardiac adenylate cyclase

Adenosine has a biphasic, [Mg²⁺]-dependent effect on the catalytic activity of dog heart adenylate cyclase. In the presence of 0.5 mM Mg²⁺, adenosine stimulated cyclic AMP formation, but when the cyclase was activated with 4 mM Mg²⁺ plus 0.5 mM Mn²⁺, adenosine inhibited catalytic activity in a dose-dependent fashion. Adenine, 3'-deoxyadenosine and selected purine-modified adenosine analogs stimulated the enzyme, whereas 2'-deoxyadenosine, 5'-deoxyadenosine and adenine-α-l-lyxofuranoside mimicked the inhibitory effect of adenosine on the Mg²⁺ plus Mn²⁺ stimulated enzyme. These results are consistent with the ‘two receptor’ model of Londos and Wolff [C. Londos and J. Wolff, Proc. natn. Acad. Sci. U.S.A.74, 5482 (1978)], but they raise the possibility of subtle organ and species differences in the chemical determinants of adenosine binding. Adenosine in both intracellular and extracellular compartments may modulate adenylate cyclase activity in the beating heart, in addition to its putative role in the regulation of coronary vascular resistance.     

5-Benzylacyclouridine and 5-benzyloxybenzylacyclouridine,potent inhibitors of uridine phosphorylase

Various pyrimidine acyclonucleosides (l-(2'-hydroxyethoxymethyl)uracils) are specific inhibitors of uridine phosphorylase [Niedzwicki et al., Biochem. Pharmac.30, 2097 (1981)]. 5-Benzyluracils have also been shown to inhibit this enzyme [Baker and Kelley. J. med. Chem.13, 461 (1970); Woodman et al., Biochem. Pharmac. 29,1059 (1980)]. We have synthesized the acyclonucleoside analogs of 5-benzyluracil (BU) and 5-benzyloxybenzyluracil (BBU). These compounds, 5-benzyl-1-(2'-hydroxy-ethoxymethyl) uracil (BAU) and 5-(m-benzyloxybenzyl)-1-(2'-hydroxyethoxymethyl)uracil (BBAU), are potent inhibitors of uridine phosphorylase. K_i values of 98 and 32 nM were estimated for BAU and BBAU respectively. These compounds are better inhibitors of uridine phosphorylase than BU (K_i = 1575 nM), BBU (K_i = 270 nM), and all other compounds previously tested, and they have no effect on thymidine phosphorylase, uridine-cytidine kinase, or thymidine kinase. Potential chemotherapeutic applications of BAU and BBAU are discussed.     

6-(Methylsulfinyl)hexyl isothiocyanate suppresses inducible nitric oxide synthase expression through the inhibition of Janus kinase 2-mediated JNK pathway in lipopolysaccharide-activated murine macrophages

6-(Methylsulfinyl)hexyl isothiocyanate (6-MITC) is an active ingredient of Wasabi (Wasabia japonica (Miq.) Matsumura), which is a very popular pungent spice in Japan. To clarify the cellular signaling mechanism underlying the anti-inflammatory action of 6-MITC, we investigated the effects of 6-MITC on the expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-activated murine macrophage RAW264 cells. 6-MITC showed a dose-dependent inhibition of LPS-induced nitric oxide (NO), iNOS mRNA and protein. LPS caused the c-Jun phosphorylation (a major component of AP-1) and IkappaB-alpha degradation. 6-MITC suppressed LPS-induced c-Jun phosphorylation, but did not inhibit IkappaB-alpha degradation. Cellular signaling analysis using MAPK-(U0126 for MEK1/2, SB203580 for p38 kinase and SP600125 for JNK) and Jak2-specific (AG490) inhibitors demonstrated that LPS stimulated iNOS expression via activating Jak2-mediated JNK, but not ERK and p38, pathway. 6-MITC suppressed iNOS expression through the inhibition of Jak2-mediated JNK signaling cascade with the attendant to AP-1 activation. In addition, the structure-activity study revealed that the inhibitory potency of methylsulfinyl isothiocyanates (MITCs) depended on the methyl chain length. These findings provide the molecular basis for the first time that 6-MITC is an effective agent to attenuate iNOS production.     

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.     

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.     

Protein kinase C isoforms as specific targets for modulation of vascular smooth muscle function in hypertension

Vascular contraction is an important determinant of the peripheral vascular resistance and blood pressure. The mechanisms underlying vascular smooth muscle (VSM) contraction and the pathological changes that occur in hypertension have been the subject of numerous studies and interpretations. Activation of VSM by vasoconstrictor stimuli at the cell surface causes an increase in [Ca(2+)](i), Ca(2+)-dependent activation of myosin light chain (MLC) kinase, MLC phosphorylation, actin-myosin interaction and VSM contraction. Additional signaling pathways involving Rho-kinase and protein kinase C (PKC) may increase the myofilament force sensitivity to [Ca(2+)](i) and MLC phosphorylation, and thereby maintain vascular contraction. PKC is a particularly intriguing protein kinase as it comprises a family of Ca(2+)-dependent and Ca(2+)-independent isoforms, which have different tissue and subcellular distribution, and undergo differential translocation during cell activation. PKC translocation to the cell surface may trigger a cascade of protein kinases, such as mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK) that ultimately interact with the contractile myofilaments and cause VSM contraction. Also, PKC translocation to the nucleus may promote VSM growth and proliferation. Increased PKC expression and activity have been identified in several forms of hypertension. The subcellular location of PKC may determine the state of VSM activity, and may be useful in the diagnosis/prognosis of hypertension. Vascular PKC isoforms may represent specific targets for modulation of VSM hyperactivity, and isoform-specific PKC inhibitors may be useful in treatment of Ca(2+) antagonist-resistant forms of hypertension.     

Synthesis and pharmacological evaluation of novel beta-nitrostyrene derivatives as tyrosine kinase inhibitors with potent antiplatelet activity

Protein tyrosine kinases have been known to be involved in regulation of platelet aggregation, suggesting a potential target for antiplatelet therapy. Our previous study showed that 3,4-methylenedioxy-beta-nitrostyrene (MNS) prevented platelet aggregation caused by various stimulators, and this action was accompanied by inhibition of tyrosine kinases. In the present study, in order to examine the structural determinants required for the actions of MNS and to develop more potent tyrosine kinase inhibitors and antiplatelet agents, a new series of beta-nitrostyrene derivatives were synthesized and pharmacologically characterized. The beta-nitrostyrene derivatives inhibited thrombin- or collagen-induced human platelet aggregation, ATP secretion, GPIIb/IIIa activation and protein tyrosine phosphorylation. In recombinant enzyme assay, some beta-nitrostyrene derivatives also demonstrated potent inhibition of Src and/or Syk kinase activity. Furthermore, there was a good correlation between the inhibitory potency of these compounds on tyrosine kinases and on platelet activation/aggregation. Among them, a benzoyl ester derivative (compound 10) possess up to 8-fold greater potency than MNS and over two orders of magnitude greater potency than genistein or tyrphostin A47 in inhibiting platelet responses to thrombin. Our data suggest that beta-nitrostyrenes may represent a new class of tyrosine kinase inhibitors with potent antiplatelet activity.     

Activation of deoxycytidine kinase in lymphocytes is calcium dependent and involves a conformational change detectable by native immunostaining

Deoxycytidine kinase (dCK), the principal deoxynucleoside salvage enzyme, plays a seminal role in the bioactivation of a wide array of cytotoxic nucleoside analogues. Recently, activation of dCK has been considered as a protective cellular response to a number of DNA-damaging agents in lymphocytes. Regarding the molecular mechanism of the enzyme activation, a post-translational modification by protein phosphorylation has been suggested. Here we provide evidence that both the activation process and the maintenance of the activated state require free cytosolic calcium. BAPTA-AM, a cell-permeable calcium chelator selectively inhibited the activation of dCK in a time- and concentration-dependent manner while extracellular calcium depletion had no effect. On the other hand, elevation of cytoplasmic calcium levels by thapsigargin did not potentiate the enzyme, referring to the permissive function of calcium in the activation process. Denaturing Western blots of extracts from lymphocytes incubated with 2-chlorodeoxyadenosine, aphidicolin and/or BAPTA-AM clearly demonstrated that dCK protein levels were unchanged during these treatments. However, a striking correlation was found between enzyme activity and the intensity of dCK-specific signals in native Western blots. Extracts from CdA-treated cells were much better recognized by the antibody raised against the C-terminal peptide of dCK than the BAPTA-AM-treated samples. These results indicate that the calcium-dependent activation of dCK is accompanied by a conformational change that renders the C-terminal epitope more accessible to the antibody.     

Effects of antifolates on the binding of 5-fluoro-2'-deoxyuridine monophosphate to thymidylate synthase

Folate based inhibitors of thymidylate synthase (TS) might facilitate binding of 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) to TS similar to the natural reduced folate 5,10-methylenetetrahydrofolate (CH(2)-H(4)-folate). We studied the lipophilic, non-polyglutamatable antifolates Nolatrexed (NTX) and AG331 and antifolates, that can have a polyglutamate side chain like the natural folate CH(2)-H(4)-folate; GW1843U89, Raltitrexed (RTX) and Multi-targetted antifolate (MTA) and pentaglutamates (RTX-Glu(5) and MTA-Glu(5)). The capacity of these compounds to facilitate the binding of [(3)H]FdUMP to Lactobacillus casei TS and an ammoniumsulphate precipitate of human TS was investigated. Only NTX, RTX-Glu(5) and MTA-Glu(5) facilitated FdUMP binding to L. casei TS and their dissociation constant K(d) (0.2-0.7 microM) was low compared to CH(2)-H(4)-folate (2.0 microM). The small lipophilic molecule NTX was favorable to the larger AG331. Polyglutamylation, as indicated by the difference in effect of RTX vs. RTX-Glu(5) and MTA vs. MTA-Glu(5), seems to be important for a classical antifolate to facilitate binding of FdUMP to bacterial TS. Effects of antifolates on FdUMP binding to human TS were different. At a low concentration (0.05 microM) NTX, RTX-Glu(5) and MTA-Glu(5) facilitated 3-5 times higher binding of [(3)H]FdUMP to TS than CH(2)-H(4)-folate. At higher concentrations (0.3-5 microM) of NTX, RTX-Glu(5) and MTA-Glu(5) the FdUMP binding decreased. The complex remained stable in the absence of (anti)folate for at least 24hr. The K(d) values of the antifolates for human TS varied from 19 to 387 nM, while the K(d) of CH(2)-H(4)-folate for human TS was 351 nM. The Hill coefficients, which indicated the type of cooperativity of the antifolates in the binding of FdUMP to TS were positive (0.58-0.99) at low concentrations (<0.3 microM) and negative (-0.35 to -0.81) at concentrations >0.3 microM except for GW1843U89, which only showed negative cooperativity (-1.70). It was shown with [(14)C]NTX that when the binding of FdUMP decreased at high NTX concentrations, the binding of NTX to TS still increased. This also held for the natural substrate dUMP. The negative cooperativity of the antifolates was clearly concentration dependent. The difference between human and L. casei TS in the FdUMP binding assays with antifolates can possibly be explained by interaction of the two subunits of human TS, which was absent in L. casei TS. The binding of antifolates to one of the two subunits induced a conformational change of the other subunit. This change no longer allowed the binding of FdUMP or dUMP at the active site. In conclusion this study showed that antifolates enhanced the binding of FdUMP to TS, especially at low antifolate concentrations, that are also clinically achievable, e.g. in human plasma.     

Stimulation of early gene induction and cell proliferation by lysophosphatidic acid in human amnion-derived WISH cells: role of phospholipase D-mediated pathway

Human-amniotic WISH cells express the lysophosphatidic acid (LPA) receptor, LPA(1), LPA(2) but not LPA(3). When WISH cells were stimulated with LPA, phospholipase D (PLD) activation was dramatically induced via a cytosolic calcium increase and protein kinase C activation. We also found that LPA stimulated two kinds of mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) and p38 kinase via PLD-dependent signaling pathways in WISH cells. In terms of the LPA-mediated functional modulation of WISH cells, we observed that LPA stimulates the induction of two early genes (c-Jun and c-Fos) and cellular proliferation in WISH cells. We examined the signaling pathways involved in LPA-mediated cellular responses. LPA-induced early gene induction was completely blocked by normal butanol (n-butanol) but not by t-butanol, suggesting that PLD activity is essentially required for the process. PD98059 (2'-amino-3'-methoxyflavone) but not SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole) also significantly blocked LPA-induced early gene induction, suggesting a crucial role for ERK. Pertussis toxin (PTX) did not affect on the LPA-induced early gene induction and ERK activation, ruling out the role of Gi/o protein(s) in the process. The cellular proliferation of WISH cells was also dramatically inhibited by n-butanol or PD98059. This study demonstrates the physiological role of LPA on the modulation of early gene induction and on WISH cell proliferation, and the crucial role played by PLD in the process.     

Comparative pharmacological analysis of Rho-kinase inhibitors and identification of molecular components of Ca2+ sensitization in the rat lower urinary tract

We aimed to compare the expression and function of molecular components of the RhoA/Rho-kinase signaling pathway in the contractile responses of detrusor, trigonal and urethral smooth muscle, using selective Rho-kinase inhibitors. Contractility studies and molecular approaches were employed to demonstrate the expression patterns and functional activity of the RhoA/Rho-kinase signaling pathway in the lower urinary tract. Frequency-response curves (1-32 Hz) and concentration-response curves (CRC) to carbachol (CCh, 0.01-30 microM), phenylephrine (PE, 0.01-300 microM) and endothelin-1 (ET-1, 0.01-100 nM) were significantly attenuated (p<0.01) following incubation with the Rho-kinase inhibitors H-1152 (0.1-1 microM), Y-27632 (1-10 microM) or HA-1077 (10 microM). Addition of Rho-kinase inhibitors also markedly reduced (p<0.01) the contractions evoked by either KCl (80 mM) or alpha,beta-methylene ATP (alpha,beta-mATP, 10 microM). Among the Rho-kinase inhibitors tested, H-1152 was approximately 9-16 times more potent than Y-27632 or HA-1077. In addition, basal tone of detrusor and trigonal strips was reduced following addition of Y-27632 (10 microM), H-1152 (1 microM) and HA-1077 (10 microM). The expression of RhoA, RhoGDI, leukemia-associated RhoGEF (LARG) and p115RhoGEF was similar among the detrusor, trigone and urethra, whereas Rho-kinase alpha, Rho-kinase beta and PDZ-RhoGEF protein levels were significantly lower in the urethra. Components of the RhoA/Rho-kinase signaling are expressed in detrusor, trigonal and urethral smooth muscle and dynamically regulate contraction and tone. Manipulation of RhoGEF expression may provide further understanding of mechanisms involving Ca(2+) sensitization in the lower urinary tract.     

Regulation of death and survival in astrocytes by ADP activating P2Y1 and P2Y12 receptors

ADP is the endogenous agonist for both P2Y(1) and P2Y(12) receptors, which are important therapeutic targets. It was previously demonstrated that ADP and a synthetic agonist, 2-methylthioadenosine 5'-diphosphate (2MeSADP), can induce apoptosis by activating the human P2Y(1) receptor heterologously expressed in astrocytoma cells. However, it was not known whether the P2Y(12) receptor behaved similarly. We demonstrated here that, unlike with the G(q)-coupled P2Y(1) receptor, activation of the G(i)-coupled P2Y(12) receptor does not induce apoptosis. Furthermore, activation of the P2Y(12) receptor by either ADP or 2MeSADP significantly attenuates the tumor necrosis factor alpha (TNFalpha)-induced apoptosis in 1321N1 human astrocytoma cells. This protective effect was blocked by the P2Y(12) receptor antagonist 2-methylthioAMP and by inhibitors of phospholipase C (U73122) and protein kinase C (chelerythrin), but not by the P2Y(1) receptor antagonist MRS2179. Toward a greater mechanistic understanding, we showed that hP2Y(12) receptor activation by 10nM 2MeSADP, activates Erk1/2, Akt, and JNK by phosphorylation. However, at a lower protective concentration of 100pM 2MeSADP, activation of the hP2Y(12) receptor involves only phosphorylated Erk1/2, but not Akt or JNK. This activation is hypothesized as the major mechanism for the protective effect induced by P2Y(12) receptor activation. Apyrase did not affect the ability of TNFalpha to induce apoptosis in hP2Y(12)-1321N1 cells, suggesting that the endogenous nucleotides are not involved. These results may have important implications for understanding the signaling cascades that follow activation of P2Y(1) and P2Y(12) receptors and their opposing effects on cell death pathways.     

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.     

Analysis of conjugated steroid androgens: Deconjugation,derivatisation and associated issues

Gas chromatography/mass spectrometry (GC/MS) is the preferred technique for the detection of urinary steroid androgens for drug testing in athletics. Excreted in either the glucuronide or sulfated conjugated form, steroids must first undergo deconjugation followed by derivatisation to render them suitable for GC analysis. Discussed herein are the deconjugation and the derivatisation preparative options. The analytical challenges surrounding these preparatory approaches, in particular the inability to cleave the sulfate moiety have led to a focus on testing protocols that reply on glucuronide conjugates. Other approaches which alleviate the need for deconjugation and derivatisation are also highlighted.