Decreasing Order Of Potency Research Articles

Stimulation of Acidic Reduction of Nitrite to Nitric Oxide by Soybean Phenolics: Possible Relevance to Gastrointestinal Host Defense

This study aimed to evaluate the potential of soybean-promoted acidic nitrite reduction and to correlate this activity with the content of phenolics and with the bactericidal activity against Escherichia coli O157:H7. Extracts of embrionary axes and cotyledons enriched in phenolics increased •NO formation at acidic pH at values that were 7.1 and 4.5 times higher, respectively, when compared to the reduction of the nonenriched extracts. Among the various phenolics accumulated in the soybean extracts, five stimulated nitrite reduction in the following decreasing order of potency: epicatechin gallate, chlorogenic acid, caffeic acid, galic acid and p-coumaric acid. Extracts of embrionary axes presented higher contents of epicatechin gallate and caffeic acid, compared to that of cotyledons, indicating a positive correlation between activity of the extracts and content of phenolics with regard to nitrite reducing activity. Soybean extracts enriched in phenolics interacted synergistically with acidified nitrite to prevent E. coli O157:H7 growth. The results suggest that soybean phenolics may interfere with the metabolism of •NO in an acidic environment by accelerating the reduction of nitrite, with a potential antimicrobial effect in the stomach.

Effects of 5-hydroxytryptamine and adenosine on the canine isolated cerebral resistance vessels

We investigated the effects of 5-hydroxytryptamine (5-HT), adenosine, and adenine nucleotides on the canine isolated cerebral resistance arteries ranging from 300 to 500 µm in diameter. Addition of 5-HT and 5-carboxami-dotryptamine (5-CT) produced concentration-dependent contractions in the resistance arteries. Methiothepin (10−8 M-10−6 M) caused a parallel shift to the right of the dose-response curves for 5-HT and 5-CT. Neither ketanserin (10−7 M, 10−6 M) nor [(1αH, 5αH)-8-methyl-8 azabicyclo [3,2,1] oct-3α-y1]1H-indole-3-carboxylate hydrochloride (ICS 205–930 10−7M, 10−6M) caused any significant shift of the dose-response curve for 5-HT. Adenosine, ATP, ADP, and AMP caused concentration-dependent relaxations in the resistance arteries following contraction with PGF2α. The adenosine analogs also caused a dose-dependent relaxation of the arterial segments contracted by PGF2α. The decreasing order of potency of the agonists was 5′-N-ethylcarboxamido adenosine (NECA)>adenosine ≥L-diasteroisomers of phenylisopropyl adenosine (L-PIA). The relaxant response to NECA was competitively antagonized by 8-phenyltheophylline. The pretreatment with 3×10−5 M NG-monomethyl-L-arginine (L-NMMA) or 5×10−5 M aspirin caused no significant effect on the adenosine-induced relaxation in the resistance arteries. These results suggest that 5-HT produces contraction of the canine isolated cerebral resistance arteries, which is mainly mediated via 5-HT1-like receptors. Adenosine causes an endothelium-independent relaxation of the resistance arteries through the activation of adenosine A2-receptors.

In vitro anthelmintic activity of three medicinal plants against Haemonchus contortus

The development of anthelmintic resistance and the high cost of conventional anthelmintic drugs led to the evaluation of medicinal plants as an alternative source of anthelmintics. In the current study, in-vitro experiments were conducted to determine the possible anthelmintic effects of crude aqueous and hydroalcoholic extracts of the leaves of Chenopodium ambrosioides, Lawsonia inermis and seeds of Jatropha curcas, on eggs and adult Haemonchus contortus. Both extracts of C. ambrosioides and J. curcas inhibited the hatching of eggs at a concentration less than or equal to 2 mg/ml, while the effect of L. inermis was not dose-dependent and did not inhibit the hatching of eggs of H. contortus, significantly, at all tested concentrations. Based on their ED50, the two most potent extracts using egg hatch assay were the hydroalcoholic extract of C. ambrosioides (0.09 mg/ml) and the aqueous extract of J. curcas (0.1 mg/ml) in a decreasing order of potency. With regard to the effect of extracts on the survival of adult parasites, extracts from C. ambrosioides have shown a moderate effect, while J. curcas and L. inermis have shown no statistically significant effect on the survival of adult parasites at the concentrations tested, and the few mortality cases recorded were not dose-dependent (P < 0.05). The overall findings of the present study have shown that C. ambrosioides and J. curcas contain possible anthelmintic compounds and further evaluation of these plants should be carried out. Key words: Anthelmintic activity, Chenopodium ambrosioides, Haemonchus contortus, Jatropha curcas, Lawsonia inermis

N‐Coumaroyldopamine and N‐caffeoyldopamine increase cAMP via beta 2‐adrenoceptors in myelocytic U937 cells

N-caffeoyldopamine is a phytochemical found in various plants, including cocoa (Theobroma cacao L.). N-caffeoyldopamine and its natural analogs (N-cinnamoyldopamine, N-coumaroyldopamine, N-feruloyldopamine, and N-sinapoyldopamine) were synthesized and investigated to determine their potency as beta-adrenoceptor agonists, because they have chemical structural moieties found in beta-adrenoceptor agonists. Among the compounds tested in this study, N-coumaroyldopamine and N-caffeoyldopamine were the two most potent compounds, able to increase cAMP at the concentrations < 0.05 microM in U937 cells. The decreasing order of potency was N-coumaroyldopamine > N-caffeoyldopamine > N-feruloyldopamine > N-sinapoyldopamine > N-cinnamoyldopamine. Using beta2-specific antagonists (butoxamine and ICI 118551), N-coumaroyldopamine and N-caffeoyldopamine were found to increase cAMP via beta2-adrenoceptors in U937 cells. In producing cAMP in U937 cells, N-coumaroyldopamine and N-caffeoyldopamine were as potent as several well-known beta2-adrenoceptor agonists (salbutamol, procaterol, and fenoterol). These results indicate that N-coumaroyldopamine and N-caffeoyldopamine are potent compounds able to increase cAMP via beta2-adrenoceptors in U937 cells, and may have potential effects on human health.

Tryptophan?NAD+ pathway metabolites as putative biomarkers and predictors of peroxisome proliferation

The present study was designed to provide further information about the relevance of raised urinary levels of N-methylnicotinamide (NMN), and/or its metabolites N-methyl-4-pyridone-3-carboxamide (4PY) and N-methyl-2-pyridone-3-carboxamide (2PY), to peroxisome proliferation by dosing rats with known peroxisome proliferator-activated receptor alpha (PPARalpha) ligands [fenofibrate, diethylhexylphthalate (DEHP) and long-chain fatty acids (LCFA)] and other compounds believed to modulate lipid metabolism via PPARalpha-independent mechanisms (simvastatin, hydrazine and chlorpromazine). Urinary NMN was correlated with standard markers of peroxisome proliferation and serum lipid parameters with the aim of establishing whether urinary NMN could be used as a biomarker for peroxisome proliferation in the rat. Data from this study were also used to validate a previously constructed multivariate statistical model of peroxisome proliferation (PP) in the rat. The predictive model, based on 1H nuclear magnetic resonance (NMR) spectroscopy of urine, uses spectral patterns of NMN, 4PY and other endogenous metabolites to predict hepatocellular peroxisome count. Each treatment induced pharmacological (serum lipid) effects characteristic of their class, but only fenofibrate, DEHP and simvastatin increased peroxisome number and raised urinary NMN, 2PY and 4PY, with simvastatin having only a transient effect on the latter. These compounds also reduced mRNA expression for aminocarboxymuconate-semialdehyde decarboxylase (ACMSDase, EC 4.1.1.45), the enzyme believed to be involved in modulating the flux of tryptophan through this pathway, with decreasing order of potency, fenofibrate (-10.39-fold) >DEHP (-3.09-fold) >simvastatin (-1.84-fold). Of the other treatments, only LCFA influenced mRNA expression of ACMSDase (-3.62-fold reduction) and quinolinate phosphoribosyltransferase (QAPRTase, EC 2.4.2.19) (-2.42-fold) without any change in urinary NMN excretion. Although there were no correlations between urinary NMN concentration and serum lipid parameters, NMN did correlate with peroxisome count (r2=0.63) and acyl-CoA oxidase activity (r2=0.61). These correlations were biased by the large response to fenofibrate compared to the other treatments; nevertheless the data do indicate a relationship between the tryptophan-NAD+ pathway and PPARalpha-dependent pathways, making this metabolite a potentially useful biomarker to detect PP. In order to strengthen the observed link between the metabolites associated with the tryptophan-NAD+ pathway and more accurately predict PP, other urinary metabolites were included in a predictive statistical model. This statistical model was found to predict the observed PP in 26/27 instances using a pre-determined threshold of 2-fold mean control peroxisome count. The model also predicted a time-dependent increase in peroxisome count for the fenofibrate group, which is important when considering the use of such modelling to predict the onset and progression of PP prior to its observation in samples taken at autopsy.

Development of pressurized retinal resistance-sized arteriolar preparation with special reference to acetylcholine-induced nitric-oxide-mediated vasodilatation.

We examined the responses of pressurized bovine retinal functional arterioles (97-185 microm in diameter and approximately 3 mm long) to vasoactive substances and the mode of action of acetylcholine (ACh) on the pressurized arterioles. The retinal arterioles were cannulated at both ends with glass micropipettes and perfused at a constant pressure of 60 mmHg. Vasoconstrictions of the retinal arterioles were induced by prostaglandin F(2 alpha) (PG F(2 alpha)), U46,619, noradrenaline (NA), and 5-hydroxytryptamine (5-HT) in a dose-dependent manner. The decreasing order of potency (pD(2) value) in the constrictive responses was as follows: 5-HT = U46,619 > NA > PG F(2 alpha). On the other hand, sodium nitroprusside (SNP), isocarbacyclin (a stable prostaglandin I(2) analog), ACh, and isoproterenol (ISP) caused dose-dependent vasodilatation in the pressurized retinal arterioles preconstricted with high-potassium solution (40 mM K+). The decreasing order of potency in the vasodilative responses was as follows: isocarbacyclin > SNP > ACh. The ACh-induced vasodilatation was suppressed significantly by pretreatment with N omega-nitro-L-arginine methyl ester (L-NAME) (3 x 10(-5) M). A treatment with L-arginine (10(-3) M) in the presence of 3 x 10(-5) M L-NAME reversed completely the L-NAME-induced reduction of the vasodilatation. These results suggest that ACh causes the production and release of endogenous nitric oxide or its related compounds, which results in vasodilatation of the pressurized bovine retinal functional arterioles.

Influence of culture conditions on the DNA-damaging effect of benzene and its metabolites in human peripheral blood mononuclear cells.

The DNA-damaging ability of benzene and its metabolites on peripheral blood mononuclear cells (PBMC) has been investigated by using the alkaline comet assay. The PBMC were incubated with different compounds in two different media for 2 and 24 hr at concentrations that did not affect cell viability and the DNA damage was quantified by a computerized image analysis system. Benzene and phenol (5 mM) did not show any genotoxic activity after 2 hr of incubation in the two media tested, phosphate-buffered saline (PBS) and RPMI containing 5% of heat-inactivated fetal calf serum (RPMI + 5% FCS), whereas phenol was genotoxic and cytotoxic at 10 mM after 24 hr of incubation in RPMI + 5% FCS. All other benzene metabolites were genotoxic at micromolar concentrations when incubated in PBS with the following decreasing order of potency: benzenetriol, catechol, hydroquinone, and benzoquinone. When the PBMC were incubated in RPMI + 5% FCS, the effect of catechol (200-600 microM) and benzenetriol (10 microM) was reduced, whereas the genotoxicity of benzenetriol at high concentrations (50-100 microM) and hydroquinone (150-2500 microM) was not affected. In contrast, the effect of benzoquinone at 5 and 10 microM was greatly enhanced when the cells were incubated in RPMI + 5% FCS. This effect resulted mainly from the presence of serum in the medium and it was almost completely inhibited by boiling the serum (100 degrees C, 5 min) and was partially reduced by extensive dialysis. Benzoquinone was the most damaging compound when tested under more physiological conditions, thereby supporting the general observation that it is the most myelotoxic benzene metabolite.

Effects of eicosanoids and nitric oxide on the noradrenaline-induced contractions in the rat mesenteric bed.

1. The effects of the inhibition of the metabolism of arachidonic acid (AA) on the constrictor responses to noradrenaline (NA) were studied in the rat perfused mesenteric bed. The inhibitor of all the pathways of AA metabolism, 10 microM eicosatetraynoic acid (ETYA), reduced the constrictor responses to all the concentrations of NA assayed. 2. The constrictor responses to NA were also reduced by the cyclooxygenase (COX) inhibitor, indomethacin (10 microM), as well as by the lipoxygenase inhibitor, nordihidroguaiaretic acid (1 microM; NDGA), whereas they were unmodified by the cytochrome P450 monooxigenase inhibitors, clotrimazole (10 microM), metyrapone (10 microM) and proadifen (10 microM). 3. The reduction in NA contractility induced by indomethacin was reverted with a decreasing order of potency by the thromboxane A2 analogue, U-46619 > prostaglandin (PG) E2 > PGF2alpha. The exposure of the mesenteric bed to NA increased the production of PGF2alpha, whereas it did not modify the production of the remaining AA metabolites. 4. The increase in the NA-induced contractions caused by endothelium removal, as well as by the inhibition of nitric oxide synthase (NOs) with NG-nitro-L-arginine methyl ester (400 microM; L-NAME), was suppressed by indomethacin but not by NDGA. These observations suggest that the lipoxygenase-derived metabolites are formed in the endothelium, whereas the COX-derived metabolites are formed in the vascular smooth muscle. 5. The TP receptor antagonist, SQ29548, did not modify the NA-induced contractions, either in the presence or in the absence of the endothelium. 6. Contractions elicited by KCI (60-100 mM) were unmodified by the AA metabolism inhibitors, ETYA, NDGA and indomethacin. 7. In summary, these results show that metabolites of AA, through both the COX and the lipoxygenase pathways, are involved in the NA-induced contractions in the rat mesenteric bed. The lipoxygenase metabolites are likely to be formed in the vascular endothelium, whereas the COX metabolite, which could be PGF2alpha, is apparently formed within the vascular smooth muscle.

Characterization and tissue location of the neural adenosine receptor in the rat ileum.

1. The aim of the present investigation was to characterize and determine the tissue location of the adenosine receptors present in the rat ileum using a method that detects drug action on the cholinergic nerves innervating the longitudinal and circular muscles. 2. The non-selective adenosine agonist, NECA (10 and 100 nM) caused significant concentration-related reductions in the circular muscle responses to transmural stimulation over the frequency range of 2.5-40 Hz, but did not affect the responses of the longitudinal muscle, nor did it reduce the muscle responses of the guinea-pig ileum. 3. The affinity order of antagonists at inhibiting the effect of NECA on the circular muscle was: CPDPX>8-PT>DMPX with apparent pA2 values of 9.31, 7.54 and 5.63 respectively. CPDPX (10-100 nM) caused parallel displacements of the concentration-effect curves to CPA with a pKb value of 9.15 and Schild slope of 1.03. 4. The agonists previously tested in the rat jejunum peristaltic reflex preparation were also shown to inhibit responses of the rat ileum in the following decreasing order of potency: CPA>NECA>2-CADO>R-PIA>S-PIA>>PAA. In addition, CHA and CCPA were also potent agonists. NECA (100 nM) and CPA (32 nM) did not inhibit carbachol (1 microM)-induced tone of tissues pre-treated with TTX (1 microM). 5. In conclusion, the rat ileum contains inhibitory A1 adenosine receptors situated on cholinergic nerve endings innervating the circular muscle.

NO·–releasing substances that induce growth elongation in maize root segments

Root segments of maize were incubated in different solutions containing substances that non-enzymatically release nitric oxide, such as sodium nitrite (SN), sodium nitroprusside (SNP), nitrosoglutathione (NGLU) and nitrosocysteine (NCYS). We found that all of these substances induced root tip expansion in a dose-dependent manner. The decreasing order of potency for root-induced elongation was: 10 -7 M SN, pH 4.5; 10 -11 M NCYS, 10 -10 M SNP, 10 -9 M NGLU and 10 -7 M SN, pH 7.0. Nitric oxide scavenger such as methylene blue prevented the elongation induced by NO·–releasing substances, but had no effect on indole-3-acetic acid (IAA)-induced cell expansion. Our results suggest that nitric oxide is the putative elongation inducer and that IAA and NO·–releasing substances conceivably share common steps in the signal transduction pathway, since both elicited the same plant response. Vanadate, a plasmamembrane ATPase inhibitor, significantly reversed IAA-induced elongation when supplied at 10 μM concentration. IAA-induced elongation was strongly enhanced by 10 nM BAY K 8644, an agonist of voltage dependent Ca2+ channels. Promotion of root elongation in the absence of IAA occurred only at higher concentrations of BAY K. Vanadate and BAY K had no influence on the NCYS-induced elongation suggesting that the common steps in the signalling of IAA and NCYS are not at the level of the plasmamembrane.

Assessment of the antiexudative and antiproliferative activities of non-steroidal anti-inflammatory drugs in inflammatory models developed in rats by subcutaneous implantation of bacterial cell walls from the dental plaque

A purified bacterial cell walls suspension from human dental plaque were biochemically prepared to serve as flogogenous agent in producing experimental inflammatory models in rats. In the vascular permeability inhibition assay (edemogenic test), the subcutaneous implantation of the flogogenous agent elicited an acute inflammatory reaction highly susceptible to the effects of the non-steroidal anti-inflammatory drugs (NSAIDs). The intradermal injection of the flogogenous agent in the dorsum of rats developed experimental granulomas also susceptible to the anti-inflammatory effects of the NSAIDs. Otherwise, the antimitotic effect of drugs was carried out in the model of cellular proliferation of duodenal mucosa of rats by incorporation of tritiated thymidine (3H TdR) in the DNA. These models of acute and chronic inflammation, and the antimitotic model permitted us to evaluate the anti-inflammatory and antimitotic effects of sulindac, ibuprofen, naproxen and glucametacin. In the antiexudative activity, evaluated by the edemogenic test, naproxen was the more effective drug followed by sulindac, ibuprofen and glucametacin (in a decreasing order of potency) to inhibit the exudative response induced by the bacterial cell walls suspension, in all experimental periods. In the chronic anti-inflammatory activity, evaluated by the granuloma inhibition assay, all drugs were capable to demonstrate effectiveness against the development of the experimental granulomas induced by an intradermal injection of the flogogenous agent. In the model of cellular proliferation, all tested drugs demonstrated antimitotic activity in all experimental periods (4, 6 and 8 days), also. Sulindac induced the higher antimitotic effect, in all experimental periods, followed by ibuprofen, naproxen and glucametacin in a decreasing order of efficacy. There was a positive correlation between the antiexudative, anti-proliferative, and antimitotic effects.

Binding Properties of (±)[3H]Benidipine Hydrochloride to Rat Heart Membranes

Benidipine is a newly developed slow-onset and long-lasting dihydropyridine calcium antagonist. The kinetics of specific binding of (+/-)[3H]benidipine to the dihydropyridine receptor sites in rat heart membranes were assessed. The dissociation constant (Kd) and the maximal number of binding sites were 0.078 +/- 0.029 nM and 286 +/- 6 fmol/mg of protein, respectively. Association and dissociation rate constants of (+/-)[3H]benidipine binding were 4 and 50 times smaller, respectively, than those of (+/-)[3H]nitrendipine binding. In displacement studies, the decreasing order of potency was benidipine, nisoldipine, nicardipine, nitrendipine, nifedipine, and Bay K 8644. A high stereoselectivity was observed, with Ki values of 0.028 nM in the S-S-(+) isomer and 4.4 nM in the R-R-(-) isomer of this drug. Benidipine had a high affinity for specific binding, with an inhibition constant of 0.084 nM, which was in good agreement with the Kd value stated above. Among the stimulants and blockers of alpha- and beta-adrenergic, cholinergic, histaminergic, dopaminergic, serotonergic, or GABAergic receptors, no drug inhibited or enhanced specific (+/-)[3H]benidipine binding. These binding properties of (+/-)[3H]benidipine may explain its unique slow onset and long-lasting antihypertensive activities.

Effects of aortic pressure and vasoactive agents on the vascular resistance of the vasa vasorum in canine isolated thoracic aorta.

1. We have developed a new preparation for continuously measuring changes in vascular resistance of the vasa vasorum of the canine isolated thoracic aorta perfused at a constant flow rate with Krebs-bicarbonate solution. 2. An increase of more than 150 mmHg in aortic pressure caused a significant increase in the vascular resistance of the vasa vasorum. 3. 5-Hydroxytryptamine (5-HT), noradrenaline (NA), adrenaline and dopamine caused dose-dependent increases in the vascular resistance of the vasa vasorum. The decreasing order of potency in the vasoconstrictor responses was as follows: 5-HT >> NA = adrenaline >> dopamine. The 5-HT- and adrenaline-induced vasoconstrictor responses were inhibited by methysergide and by phentolamine plus propranolol, respectively. 4. Acetylcholine (ACh), isoprenaline (ISP), histamine (His), ATP, ADP and adenosine produced dose-related decreases in the vascular resistance of aortic vasa vasorum perfused with the Krebs solution containing 10(-5) M-NA. The decreasing order of potency in the response was as follows: ACh = ISP > His >> adenosine = ATP = ADP. The ACh-, ISP- and His-induced vasodilator responses were antagonized by atropine, propranolol and famotidine, respectively. 5. The results suggest that the preparation described is useful for studying the regulation of vascular resistance of aortic vasa vasorum and that aortic pressure and vasoactive compounds may directly regulate the vascular resistance of the vasa vasorum in canine isolated thoracic aorta.

Contractile and relaxant responses of the canine isolated spinal artery to vasoactive substances

1. Effects of vasoactive substances were investigated in the canine isolated spinal branch of the intercostal artery (SBICA). 2. Addition of angiotensin II (AII), vasopressin, noradrenaline (NA), adrenaline, 5-hydroxytryptamine (5-HT), and dopamine each produced concentration-dependent contraction in the SBICA, whereas prostaglandin F2 alpha, histamine, and tyramine caused only slight contraction. The decreasing order of the potency of contractile agents was AII much greater than vasopressin = NA greater than 5-HT greater than adrenaline much greater than dopamine. 3. Although the pD2 value for phenylephrine (5.31 +/- 0.36) was smaller than that for NA (6.48 +/- 0.13), there was no significant difference in Emax value between these two agonists in the SBICA. On the other hand, xylazine produced only a slight contraction, the pD2 value being 3.59 +/- 0.08. Phentolamine (10(-8)-10(-6) M) and prazosin (10(-8)-10(-6) M) competitively inhibited the NA-induced contraction, while yohimbine (10(-8)-10(-6) M) did not. 4. Acetylcholine (ACh), sodium nitroprusside (SNP), ATP, ADP, and adenosine caused concentration-dependent relaxations in SBICA following contraction with NA. On the other hand, isoprenaline up to 10(-4) M did not produce any relaxation. The decreasing order of potency of the relaxant agents was ACh greater than SNP much greater than ATP = ADP = adenosine. 5. The ACh-induced relaxation was competitively inhibited by atropine and was abolished by mechanical removal of the endothelium. Aspirin (5 x 10(-5) M) did not affect the relaxant response to ACh, while oxyhaemoglobin (10(-5) M) and methylene blue (10(-5) M) produced significant attenuation. 6. These results suggest that NA produces contraction of the isolated canine SBICA which is mainly mediated via alpha 1-adrenoceptors and that ACh causes a relaxation of the SBICA due to release of endothelium-derived relaxing factor (EDRF) from the endothelial cells.

Properties of vasoactive‐intestinal‐peptide receptors and β‐adrenoceptors in the murine radiation leukemia‐virus‐induced lymphoma cell line BL/VL3

1. Based on radioligand binding and adenylate cyclase activation, functional receptors to vasoactive intestinal peptide(VIP)/helodermin, were shown to coexist with beta 2-adrenoceptors and prostaglandin receptors in membranes from a cultured cloned BL/VL3 cell line of murine T-cell lymphoma induced by a radiation leukemia virus. 2. The relative potency of VIP-related peptides to stimulate adenylate cyclase activity was: helodermin greater than VIP greater than peptide histidine isoleucinamide. Five VIP analogs inhibited 125I-iodo-VIP binding and stimulated adenylate cyclase activity, their decreasing order of potency being: VIP greater than [D-Asp3]VIP greater than [D-Ser2]VIP greater than [D-Ala4]VIP = [D-His1]VIP = [D-Phe2]VIP. [D-Phe2]VIP acted as a partial agonist (with an intrinsic activity of 0.1 as compared to that of VIP = 1.0) and competitively inhibited helodermin- and VIP-stimulated adenylate cyclase activity with a similar Ki (0.07-0.10 microM). These data suggest the existence, in this murine T-cell lymphoma, of VIP receptors of the 'helodermin-preferring' subtype that are coupled to adenylate cyclase.

Acute effect of thyroid hormone on the heart: An extranuclear increase in sugar uptake

The thyroid hromone 3,5,3′-triiodo-L-thyronine (T3) produced a rapid increase in [ 3H]2-deoxyglucose (2-DG) uptake by freshly isolated rat heart slices in vitro, an effect that was evident after 1 min of pre-incubation with the hormone. This stimulatory effect of T3 was dose-related: the lowest effective concentration was 1 p m and maximal effect of about 80% above control was seen at 1 n m. Studies with several thyroid hormone analogues revealed that l-T3 was the most effective analogue which was followed in a decreasing order of potency by l-T4 = d-T3 > d-T4 > 3,5- l-T2 > rT3 > dL-thyronine. Further, the T3-induced increase in 2-DG uptake was independent of new protein synthesis because it was not blocked by the protein synthesis inhibitor cycloheximide under conditions in which [ 3H] leucine incorporation was inhibited by approximately 95%. Evaluation of the mechanism through which T3 exerts this action revealed that the uptake of 2-DG and 3-0-methyl- d-glucose (30MG) by heart slices was saturable, but that of l-glucose was not, and that T3 produced a similar increase in the uptake of both 2-DG and 30MG but failed to change l-glucose uptake. Saturation curve analysis of 2-DG and 30 MG uptake revealed that T3 increased V max values but had no effect on K m values. Moreover, T3, which promoted total 2-DG uptake rate, had no effect on the proportionate phosphorylation rate of 2-DG to 2-DG-6-phosphate by hexokinase. From this study it is concluded that thyroid hormone produces a direct and acute effect on the heart. This prompt effect of T3 to increase sugar uptake by heart slices, owing to the increase in the V max of the sugar transport system, is extranuclear in nature, is thyroid hormone specific, and has a physiologic relevance.

Action of polychlorocycloalkane insecticides on binding of [ 35S] t-butylbicyclophosphorothionate to Torpedo electric organ membranes and stereospecificity of the binding site

Binding of [ 35S] t-butylbicyclophosphorothionate ([ 35S]TBPS) to Torpedo electric organ membranes was characterized. A dose- and pH-dependent binding (100.8 pmol/mg protein) was detected with a single affinity ( K d of 0.9 μ m) in the presence of 150 m m KCl at pH 6.8. Other anions such as Br − and I − also increased binding affinity, but to a lower degree than Cl −, which increased the affinity by two- to threefold. In presence of 150 m m KCl, [ 35S]TBPS binding was inhibited noncompetitively by Zn 2+ and by 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) (IC50 of 9 μ m). The γ-isomer of hexachlorocyclohexane (BHC) was much more potent in inhibiting this [ 35S]TBPS binding and the inhibition was competitive ( K i = 40 n m). Like binding of [ 35S]TBPS to the γ-aminobutyric acid (GABA) receptor, its binding to Torpedo membranes was inhibited by pentobarbital, mephobarbital, and hexobarbital (IC50 of 85, 225, and 300 μ m, respectively), but not by phenobarbital. Binding was not inhibited by diazepam, GABA, bicuculline, or avermectin B 1a, ligands that bind to the GABA A receptor. [ 35S]TBPS binding was inhibited by BHC isomers with the following decreasing order of potency α = γ > σ > β, and by cyclodiene insecticides. Endrin was more potent than dieldrin, but endosulfan I and II had similar effects. The data suggest that the binding site for polychlorocycloalkane insecticides on this protein is much less stereoselective than that of the Cl − channel of the GABA A receptor. Also, even though this Torpedo protein has higher affinity for insecticides, such as γ-BHC, than does the GABA A receptor, it is the latter whose specificity correlates best with polychlorocycloalkane toxicity. Nevertheless, because of its high affinity for γ-BHC such a protein in muscles or brain may be an important target for the action of this insecticide.

In vitro inactivation of plasma alpha 1-proteinase inhibitor by epoxides and 1,2-dihaloethanes.

The inactivating effects of three epoxides and two dihaloethanes on the proteinase inhibitory activities of α1-proteinase inhibitor, as well as those of the plasma itself, were investigated. All of the three epoxides (styrene oxide, ethylene oxide and propylene oxide), individually, inactivated the elastase inhibitory or trypsin inhibitory activity of proteinase inhibitor, and that of whole plasma. Styrene oxide is the most effective inactivator of either purified α1-proteinase inhibitor or plasma, followed by ethylene oxide and propylene oxide in a decreasing order of potency. The dihaloethanes, 1,2-dichloroethane and 1,2-dibromoethane, also inactivated the proteinase inhibitory activities of α1-proteinase inhibitor with the former being somewhat more effective than the latter. Concomitant modification of the amino group was also observed with these chemicals.

Biochemical interactions of carbamates and ecothiophate with the activated conformation of nicotinic acetylcholine receptor.

Purified Torpedo nobiliana electric organ acetylcholine receptor (AChR) was reconstituted into membranes containing natural phospholipids supplemented with cholesterol (25% w/w). The reconstituted system facilitates the study of the effects of drugs on the regulation of the AChR channel complex under both resting and carbachol (carb)-stimulated conditions. Neostigmine (Neo) was the only carbamate to induce activation of [3-H]-phencyclidine ([3-H]-PCP) binding to the channel sites, acting as a weak agonist. The activation of [3-H]-PCP binding is dependent upon the nature of the reconstituted systems, with carb/Neo activation ratios of 8, 3, and 1 for the intact purified AChR vesicles fraction (PVF), the PVF reconstituted in phospholipid/cholesterol (CRPVF), and the PVF reconstituted in phospholipid (RPVF), respectively. The carbamates Neo, physostigmine (Physo), and pyridostigmine (Pyrido) inhibited carb-activated [3-H]-PCP binding with Ki values of 10, 20, and 1,600 microM, respectively. The inhibition was mixed competitive-noncompetitive in nature. The characteristic response of CRPVF to carb-stimulated [22-Na] influx was inhibited by the three carbamates, with IC-50 values of 6, 50, and 1,000 microM for Neo, Physo, and Pyrido, respectively. The quaternary ammonium organophosphate ecothiophate (Eco) inhibited carb-stimulated [22-Na] influx with potency similar to that of Neo. Preincubation of AChR preparation with the carbamates and ecothiophate caused a reduction in the binding of [125-I]-alpha-bungarotoxin ([125-I]-alpha-BGT) with the following decreasing order of potency: Neo less than Physo less than Eco less than Pyrido. Calcium has a direct modulatory role on the time-course inhibition of [125-I]-alpha-BGT binding by these drugs. While we observed a high potency of Neo and Physo in inhibiting [125-I]-alpha-BGT binding, it was undetectable for the carbamate insecticide 2-methyl-2-(methylthio)propionaldehyde-O-(methylcarbamoyl)oxime (aldicarb). These data suggest that the potent anticholinesterase carbamate agents interact differently with the AChR and its ionic channel. Their interactions with the nicotinic AChR channel system can be described as (a) weakly agonist, (b) directly acting on the open conformation of the channel, and (c) blocking the AChR-binding sites.

Effects of extracellular anions on cell growth of Chinese hamster V-79 cells.

The effects of extracellular anions (10-150 mM, added as Na salts to normal growth medium) on the growth of Chinese hamster V-79 cells were examined. Additions of NaCl and NaNO3 at concentrations greater than 60 mM reduced the growth rate dose-dependently. Several other anions also inhibited cell growth in the decreasing order of potency, SCN- greater than NO2- greater than NO3- greater than Br- greater than Cl- greater than gluconate- glutamate- greater than Mes-. When the added anions were removed, the growth rate was restored to the control rate. Cell survival was markedly reduced by the addition of SCN-, but was less affected by other anions (Cl-,NO3- and NO2-) of comparable potency. The respective syntheses of cellular DNA and protein, as estimated from the incorporation of [3H]-thymidine and [14C]leucine, also decreased with the increase in the concentration (60-120 mM) of anions added, the order of potency being SCN- greater than NO2- greater than NO3- greater than Cl-. After anion-treatment, the cellular Na+ concentration increased and the cellular Cl- concentration decreased in the order of SCN- greater than NO2- greater than NO3-, Cl-, but, the cellular K+ concentration did not change significantly. These data suggest that changes in extracellular anions affect cell growth and survival, probably through changes in the intracellular Na+ or Cl- concentration and in the rates of protein and/or DNA synthesis.