Exchange of reduced sulfur gases between lichens and the atmosphere

Fourteen lichens, 10 green algal lichens and four cyanolichens, as well as a cyanobacterium emitted significant quantities of H₂S (0.01–0.04 pmol g dw^–1 s^–1) and DMS (0.005–0.025 pmol g dw^–1 s^–1) but were sinks for COS (0.015–0.14 pmol g dw^–1 s^–1). In contrast, exchange of CH₃SH and CS₂ were sporatic and inconsistent. Although some interspecific variation occurred for the first three gases, exchange rates were relatively uniform and were not influenced by irradiance conditions. In contrast to DMS and H₂S emission, COS uptake was strongly influenced by degree of thallus hydration. Because lichen dominated systems cover extensive terrestrial habitats, COS uptake is potentially important in the world's sulfur budget.     

Endothelin-1 binding to endothelin receptors in the rat anterior pituitary gland: possible formation of an ETA-ETB receptor heterodimer

1. Interaction in the recognition of endothelin-1 (ET-1), a typical bivalent ET receptor-ligand, between ET_A and ET_B receptors was investigated in the rat anterior pituitary gland, using our quantitative receptor autoradiographic method with tissue sections preserving the cell-membrane structure and ET receptor-related compounds.2. In saturation binding studies with increasing concentrations (0.77–200 pM) of ¹²⁵I-ET-1 (nonselective bivalent radioligand), ¹²⁵I-ET-1 binding to the rat anterior pituitary gland was saturable and single with a K _D of 71 pM and a B _max of 120 fmol mg^–1. When 1.0 M BQ-123 (ET_A antagonist) was added to the incubation buffer, binding parameters were 8.3 pM of K _D and 8.0 fmol mg^–1 of B _max, whereas 10 nM sarafotoxin S6c (ET_B agonist) exerted little change in these binding parameters (K _D, 72 pM; B _max, 110 fmol mg^–1).3. Competition binding studies with a fixed amount (3.8 pM) of ¹²⁵I-ET-1 revealed that when 1.0 M BQ-123 was present in the incubation buffer, ET_B receptor-related compounds such as sarafotoxin S6c, ET-3, IRL1620 (ET_B agonist), and BQ-788 (ET_B antagonist) competitively inhibited ¹²⁵I-ET-1 binding with K _is of 140, 18, 350 pM, and 14 nM, respectively, however, these compounds were not significant competitors for ¹²⁵I-ET-1 binding in the case of absence of BQ-123.4. In cold-ligand saturation studies with a fixed amount (390 pM) of ¹²⁵I-IRL 1620 (ET_B radioligand), IRL1620 bound to a single population of the ET_B receptor, and no change was observed in binding characteristics in the presence of 1.0 M BQ-123. ¹²⁵I-IRL1620 binding was competitively inhibited by ET-1 and ET-3 in the absence of BQ-123, with K _is of 20 and 29 pM, respectively, the affinities being much the same as those of 29 nM, in the presence of 1.0 M BQ-123.5. Two nonbivalent ET_A antagonists, BQ-123 and PD151242, were highly sensitive and full competitors for ¹²⁵I-ET-1 binding (5.0 pM), in the presence of 10 nM sarafotoxin S6c.6. Taken together with the present finding that mRNAs encoding the rat ET_A and the ET_B receptors are expressed in the anterior pituitary gland, we tentatively conclude that although there are ET_A and ET_B receptors with a functional binding capability for ET receptor-ligands, the ET_B receptor does not independently recognize ET-1 without the aid of the ET_A receptor. If this thesis is tenable, then ET-1 can bridge between the two receptors to form an ET_A–ET_B receptor heterodimer.     

Carbon disulfide oxidation by a microbial consortium from a trickling filter

Biological oxidation rates of CS₂ with a mixed microbial culture obtained from a trickling filter were optimal with 3 mM CS₂, pH 7, 30°C and SO₄ ^2– below 25 g l^–1. Degradation rates were 3.4 mg CS₂/g_proteinmin and 13.8 mg H₂S/g_proteinmin. The concentrations of intermediates (H₂S, COS and S°) and the product (SO₄ ^2–) of CS₂ oxidation were measured. The biological oxidation was due principally to Gram negative bacteria.     

Variability in abundance and fluxes of dimethyl sulphide in the Indian Ocean

Dimethyl sulphide (DMS) is a biogenic gas of climatic significance on which limited information is available from the Indian Ocean. To fill this gap, we collected data on DMS and total dimethylsulphoniopropionate (DMSP_t) by participating in a dozen cruises. Here, we discuss the variability in DMS and DMSP_t in the north and central Indian Ocean in terms of their spatial and temporal variation. DMS and DMSP_t exhibited significant spatial and temporal variability. Apart from the concentration gradients in DMS within the Arabian Sea, Bay of Bengal and Central Indian Ocean basins, differences in average abundances were conspicuous between these basins. The Arabian Sea contained more DMS (mixed layer average was 7.8 nM) followed by the Bay of Bengal (2.8 nM) and the Central Indian Ocean (2.7 nM). The highest concentrations of DMS and DMSP_t (525 nM and 916 nM, respectively) were found in upwelling regimes along the west coast of India during the Southwest monsoon and fall intermonsoon seasons. Average surface DMS was the highest in the Arabian Sea. On the other hand observed sea-to-air fluxes of DMS were higher in the Bay of Bengal due to the prevalence of turbulent conditions. In the Arabian Sea wind speeds were low and hence the sea-to-air fluxes. The total diffusive flux of DMS from the study area to atmosphere is estimated to be about 1.02 × 10¹² g S y⁻¹, which contributes to 4.1–6.3% of the global DMS emission     

Soil core method for direct simultaneous determination of N2 and N2O emissions from forest soils

In order to quantify N₂-emissions from a spruce and a beech site at the Höglwald Forest, a new measuring system was developed, that allowed simultaneous, direct determination of N₂- and N₂O-emission with high accuracy (detection limit approx. 10 g N m^–2 h^–1 for N₂ and <1 g for N₂O) using a gas-flow core method. This method requires exchange of the soil atmosphere with an artificial atmosphere, that differs only in that N₂ is substituted by He. The measuring system, the methodology of measurements and validation experiments are described in detail. Due to the huge heterogeneity of denitrification activity in different soil cores taken from our forest sites, no general trends of N₂ and N₂O production in relation to soil moisture and temperature could be demonstrated. Based on reasonable number of measurements, this work gives for the first time an estimate of the magnitude of N₂-losses from temperate forest soils. Both the magnitude of N₂-emissions (spruce: 7.2±0.7 kg N₂-N ha^–1 yr^–1; beech: 12.4±3.1 kg N₂-N ha^–1 yr^–1), as well as the N₂O–N₂ ratio (spruce: 0.136±0.04; beech: 0.52±0.19) were significantly higher for soils from the beech sites as compared to soils from the spruce site. The results suggests that N₂-emissions from N-saturated forest soils, still receiving high loads of atmospheric N-deposition, are approx. 30% of atmospheric N-input at the spruce site, and approx. 50% at the beech site. Our results demonstrate that losses of nitrogen in the form of N₂ cannot be neglected in the context of calculating N-balances for given forest sites.     

Effect of tree distance on N2O and CH4-fluxes from soils in temperate forest ecosystems

Complete annual cycles of N₂O and CH₄ flux in forest soils at a beech and at a spruce site at the Höglwald Forest were followed in 1997 by use of fully automatic measuring systems. In order to test if on a microsite scale differences in the magnitude of trace gas exchange between e.g. areas in direct vicinity of stems and areas in the interstem region at both sites exist, tree chambers and gradient chambers were installed in addition to the already existing interstem chambers at our sites. N₂O fluxes were in a range of –4.6–473.3 g N₂O-N m^–2 h^–1 at the beech site and in a range of –3.7–167.2 g N₂O-N m^–2 h^–1 at the spruce site, respectively. Highest N₂O emissions were observed during and at the end of a prolonged frost period, thereby further supporting previous findings that frost periods are of crucial importance for controlling annual N₂O losses from temperate forests. Fluxes of CH₄ were in a range of +10.4––194.0 g CH₄ m^–2 h^–1 at the beech site and in a range of –4.4––83.5 g CH₄ m^–2 h^–1 at the spruce site. In general, both N₂O-fluxes as well as CH₄-fluxes were higher at the beech site. On a microsite scale, N₂O and CH₄ fluxes at the beech site were highest within the stem area (annual mean: 49.6±3.3 g N₂O-N m^–2 h^–1; –77.2±3.1 g CH₄ m^–2 h^–1), and significantly lower within interstem areas (18.5±1.4 g N₂O-N m^–2 h^–1; –60.2±1.8 g CH₄ m^–2 h^–1). Significantly higher values of total N, C and pH in the organic layer, as well as increased soil moisture, especially in spring, in the stem areas, are likely to contribute to the higher N₂O fluxes within the stem area of the beech. Also for the spruce site, such differences in trace gas fluxes could be demonstrated to exist (mean annual N₂O emission within (a) stem areas: 9.7±0.9 g N₂O-N m^–2 h^–1 and (b) interstem areas: 6.2±0.6 g N₂O-N m^–2 h^–1; mean annual CH₄ uptake within (a) stem areas: –26.1±0.6 g CH₄ m^–2 h^–1 and (b) interstem areas: –38.4±0.8 g CH₄ m^–2 h^–1), though they were not as pronounced as at the beech site.     

The neuropeptide bradykinin stimulates phosphoinositide turnover in HSDM1C1 cells: B2-antagonist-sensitive responses and receptor binding studies

Bradykinin (BK) and its analogs (1 nM-100 M) stimulated phosphoinositide (PI) turnover in murine fibrosarcoma (HSDM1C1) cells in a concentration-dependent manner. The relative potencies (EC₅₀) were: BK=48±4 nM; Lys-BK=39±3 nM; Met-Lys-BK=158±33 nM; Des-Arg⁹-BK=2617±598 nM (means±SEM, n=3–14). All these analogs were full agonists and they produced up to 5.4±0.4-fold stimulation of PI turnover at the highest concentration (10–100 M) of the peptides. In contrast, the analogs [D-Arg⁰-HYP³-Thienyl^5,8-D-Phe⁷]-BK (HYP³-antagonist), [D-Arg⁰-HYP³-Thienyl,^5,8-D-Phe⁷]-BK (Thienyl antagonist) and Des-Arg⁹-Leu⁸-BK were inactive, as agonists, at 0.1 nM-1 M in this system. These data suggested that BK-induced PI turnover in these cells was mediated via B₂-type of BK receptors. This was confirmed further by the fact that both the B₂-selective Hyp³- and Thienyl-antagonists inhibited BK-induced PI turnover with K_BS of 369±51 nM and 368±118 nM respectively while the B₁-selective antagonist, Des-Arg⁹-Leu⁸-BK, was inactive at 1 M. [³H]BK receptor binding studies revealed two binding sites, one with high affinity (K_d=0.24±0.06 nM; B_max=1.4±0.4 pmol/g tissue) and the other with low affinity (K_d=18.5±0.95 nM; B_max=25.1±0.52 pmol/g tissue), on HSDM1C1 cell homogenates. The rank order of affinity of BK analogs at inhibiting specific [³H]BK binding was similar to that found for PI turnover. Taken together, these data have provided evidence for the presence of two B₂-type BK binding sites on the HSDM1C1 cells. Based on the affinity parameters, the low-affinity component of [³H]BK binding in HSDM1C1 cells appears to be coupled to the phospholipase C-induced PI turnover mechanism. The high-affinity component has been previously shown to mediate the production of prostaglandins by activation of phospholipase A₂.     

Interaction between Duodenase and α1-Proteinase Inhibitor

The interaction between duodenase, a newly recognized serine proteinase belonging to the small group of Janusfaced proteinases, and ₁-proteinase inhibitor (₁-PI) from human serum was investigated. The stoichiometry of the inhibition was 1.2 mol/mol. The presence of a stable enzyme–inhibitor complex was shown by SDS-PAGE. The mechanism of interaction between duodenase and ₁-PI was shown to be of the suicide type. The equilibrium and inhibition constants are 13 ± 3 nM and (1.9 ± 0.3)·10⁵ M^–1·sec^–1, respectively. Based on the association rate constant of the enzyme–inhibitor complex and localization of duodenase and ₁-PI in identical compartments, ₁-PI is suggested to be a duodenase inhibitor in vivo.     

Regulation of the calcium slow channel by cyclic GMP dependent protein kinase in chick heart cells

In order to assess the interaction between the cAMP-dependent and the cGMP-dependent phosphorylation pathways on the slow Ca²⁺ current (I_Ca(L)), whole-cell voltage-clamp experiments were conducted on embryonic chick heart cells. Addition of 8Br-cGMP to the bath solution reduced the basal (unstimulated) I_Ca(L). Intracellular application of the catalytic subunit of PK-A (PK-A(cat); 1.5 M) via the patch pipette rapidly potentiated I_Ca(L) by 215±16% (n=4); subsequent addition of 1 mM 8Br-cGMP to the bath reduced the amplitude of I_Ca(L) towards the initial control values (123±29%). Intracellular application of PK-G (25 nM pre-activated by 10^–7 M cGMP), rapidly inhibited the basal I_Ca(L) by 64±6% (n=8). Heat-denatured PK-G was ineffective. Subsequent additions of relatively high concentrations of 8Br-cAMP (1 mM) or isoproterenol (ISO, 1–10 M) did not significantly remove the PK-G blockade of I_Ca(L). The results of the present study suggest that: (a) 8Br-cGMP can inhibit the basal or stimulated (by PK-A(cat)) I_Ca(L) in embryonic chick myocardial cells. (b) PK-G applied intracellularly inhibits the basal I_Ca(L).     

Interrelationships Between Nitrogen Supply and Photosynthetic Parameters in Vicia faba L.

We determined for Vicia faba L the influence of nitrogen uptake and accumulation on the values of photon saturated net photosynthetic rate (P _Nmax), quantum yield efficiency (), intercellular CO₂ concentration (C _i), and carboxylation efficiency (C _e). As leaf nitrogen content (N_L) increased, the converged onto a maximum asymptotic value of 0.0664±0.0049 mol(CO₂) mol(quantum)^–1. Also, as N_L increased the C _i value fell to an asymptotic minimum of 115.80±1.59 mol mol^–1, and C _e converged onto a maximum asymptotic value of 1.645±0.054 mol(CO₂) m^–2 s^–1 Pa^–1 and declined to zero at a N_L-intercept equal to 0.596±0.096 g(N) m^–2. fell to zero for an N_L-intercept of 0.660±0.052 g(N) m^–2. As N_L increased, the value of P _Nmax converged onto a maximum asymptotic value of 33.400±2.563 mol(CO₂) m^–2 s^–1. P _N fell to zero for an N_L-intercept of 0.710±0.035 g(N) m^–2. Under variable daily meteorological conditions the values for N_L, specific leaf area (_L), root mass fraction (R_f), P _Nmax, and remained constant for a given N supply. A monotonic decline in the steady-state value of R_f occurred with increasing N supply. _L increased with increasing N supply or with increasing N_L.     

Ethanol Inhibits the Binding of Substance P to Rat Brain Cortex NK1 Receptors

The binding of ¹²⁵I-labeled substance P (SP) to rat brain cortex membranes has been studied Under control conditions and in the presence of ethanol. The binding of SP at low concentrations (20–1000 pM) gave two components, one with a K _D value of 80 pM and another one with a K _D of 500 pM. The higher-affinity component is due to NK₁ receptors, as confirmed by the inhibition Of the SP binding by the rodent NK1 specific agonist [Sar₉ Met(O₂)₁₁]SP. Ethanol (1.7 mM) added to the binding assays inhibited by more than 50% the specific binding at a very low SP concentration (20 pM); however, it had no effect at SP concentrations ranging from 50 to 120 pM. This suggests a decrease by ethanol of the affinity of SP to the NK₁ receptors involved in this binding component. The ethanol effect disappeared at [EtOH] 0.17 mM.     

Activation and deactivation of sarcoplasmic reticulum calcium release channels: Molecular dissection of mechanisms via novel semi-synthetic ryanoids

The plant alkaloids ryanodine and dehydroryanodine are high affinity, biphasic modulators of the intracellularly located, calcium-regulated calcium release channels of a variety of cell types. To date, little is certain about the molecular basis of the interactions that prompt low concentrations of ryanodine (nanomolar to low micromolar) to activate (open) the channels and higher concentrations to deactivate (functionally close) the sarcoplasmic reticulum calcium release channel. In the present study, we approached this question using novel, semi-synthetic C₁₀–O_eq ester derivatives of ryanodine and dehydroryanodine as molecular probes of the ryanodine binding sites on the calcium release channel.Binding affinities of these C₁₀–O_eq ester derivatives of ryanodine and dehydroryanodine with acidic, basic and neutral side chains (K_d values> 53.9 nM, K_d values 0.3–0.7 nM and K_d values 1.3–20.4 nM, compared with 2.3 and 2.8 nM for ryanodine and dehydroryanodine, respectively) were evaluated for their ability to modulate, the patency of the sarcoplasmic reticulum calcium release channel. With the exception of only two derivatives tested to date, all the semi-synthetic C₁₀–O_eq esters selectivelyactivate the Ca²⁺ release channel. That is, they produce no functional closure of the sarcoplasmic reticulum calcium release channels at the highest concentration, that could be tested. Half-maximal concentrations for activation (EC_50act , values) ranged from 0.87–4.2, M, compared with an EC_50act of 1.3 M for ryanodine.     

Relation between the anion exchange protein in kidney medullary collecting duct cells and red cell band 3

Summary A membrane protein that is immunochemically similar to the red cell anion exchange protein, band 3, has been identified on the basolateral face of the outer medullary collecting duct (MCD) cells in rabbit kidney. In freshly prepared separated rabbit MCD cells, M.L. Zeidel, P. Silva and J.L. Seifter (J. Clin. Invest. 77:1682–1688, 1986) found that Cl^–/HCO ₃ ^- exchange was inhibited by the stilbene anion exchange inhibitor, DIDS (4,4-diisothiocyano-2,2-disulfonic stilbene), with aK ₁ similar to that for the red cell. We have measured the binding affinities of a fluorescent stilbene inhibitor, DBDS (4,4-dibenzamido-2,2-disulfonic stilbene), to MCD cells in 28.5 mM citrate and have characterized both a high-affinity site (K ₁ ^s =93±24 mM) and a lower affinity site (K ₂ ^s =430±260 nM), which are closely similar to values for the red cell of 110±51 nM for the high-affinity site and 980±200 nM for the lower affinity site (A.S. Verkman, J.A. Dix & A.K. Solomon,J. Gen. Physiol. 81:421–449, 1983). When Cl^– replaces citrate in the buffer, the two sites collapse into a single one withK ₁ ^s =1500±400 nM, similar to the singleK ₁ ^s =1200±200 nM in the red cell (J.A. Dix, A.S. Verkman & A.K. Solomon,J. Membrane Biol. 89:211–223, 1986). The kinetics of DBDS binding to MCD cells at 0.25 M^–1 are characterized by a fast process, =0.14±0.03 sec, similar to =0.12±0.03 sec in the red cell. These similarities show that the physical chemical characteristics of stilbene inhibitor binding to MCD cell band 3 closely resemble those for red cell band 3, which suggests that the molecular structure is highly conserved.     

Hydrography and characteristics of the phytoplankton in shelf and oceanic waters off southeastern Brazil during winter (July/August 1982) and summer (February/March 1984)

The physical and chemical environment, and the phytoplankton primary production of southeastern Brazil were studied in relation to the general oceanographic structure during two research cruises (winter and summer). In each cruise, a total of 91 stations were occupied. Data were collected on the spatial distribution of nutrients, phytoplankton biomass and photosynthetic capacity over the coastal, shelf and oceanic areas off São Paulo, Paraná and Santa Catarina States.During wintertime, the mixing processes between tropical warm waters of the Brazil Current and subantarctic waters of the Malvinas Current formed strong environmental gradients. The drainings of Rio de La Plata and Lagoa dos Patos are transported northwards by coastal currents, enriching the shelf waters off Santa Catarina State with inorganic nutrients and consequently increasing the chlorophyll a to the highest concentrations (> 3.5 mg m ^–3) measured during the two cruises. In slope waters chlorophyll values were always low (0.05–0.45 mg m ^–3). The chlorophyll within the euphotic layer varied from 8.8–36.7 and 1.2–18.5 mg m^–2 during winter and summer, respectively.The surface photosynthetic rates during winter and summer cruises ranged respectively from 0.21–9.17 and 0.66–19.60 mgC/mgChl.a/h. The mean rates were higher in nearshore waters and decreased seaward.The thermal structure of the water column affected the vertical distribution of chlorophyll a and photosynthesis within the euphotic zone; During unstratified periods (winter) they were uniformly distributed but the occurrence of subsurface peaks of chlorophyll and strong photosynthetic inhibition of low light adapted cells in deeper layers are associated to the seasonal thermocline. Occasionally, upwelling of deep waters from shelf break enriched the deeper euphotic layers in offshore areas. Intensive upwelling was observed off Paranagua Bay (Parana State) and the mechanisms of its formation are discussed.     

M1 Muscarinic Receptors Contribute to, whereas M4 Receptors Inhibit, Dopamine D1 Receptor-Induced [3H]-Cyclic AMP Accumulation in Rat Striatal Slices

In rat striatal slices labelled with [³H]-adenine and in the presence of 1 mM 3-isobutyl-1-methylxantine (IBMX), cyclic [³H]-AMP ([³H]-cAMP) accumulation induced by the dopamine D₁ receptor agonist SKF-81297 (1 μM; 177±13% of basal) was inhibited by the general muscarinic agonist carbachol (maximum inhibition 72±3%, IC₅₀ 0.30±0.06 μM). The muscarinic toxin 7 (MT-7), a selective antagonist at muscarinic M₁ receptors, reduced the effect of SKF-81297 by 40±7% (IC₅₀ 251±57 pM) and enhanced the inhibitory action of a submaximal (1 μM) concentration of carbachol (69±4% vs. 40±7% inhibition, IC₅₀ 386±105 pM). The toxin MT-1, agonist at M₁ receptors, stimulated [³H]-cAMP accumulation in a modest but significant manner (137±11% of basal at 400 nM), an action additive to that of D₁ receptor activation and blocked by MT-7 (10 nM). The effects of MT-7 on D₁ receptor-induced [³H]-cAMP accumulation and the carbachol inhibition were mimicked by the PKC inhibitors Ro-318220 (200 nM) and Gö-6976 (200 nM). Taken together our results indicate that in addition to the inhibitory role of M₄ receptors, in rat striatum acetylcholine stimulates cAMP formation through the activation of M₁ receptors and PKC stimulation.     

β-Adrenoreceptors of multiple affinities in a clonal capillary endothelial cell line and its functional implication

-Adrenoreceptor has been studied in a clonal capillary endothelial cell line established from the vascular bed of the bovine adrenal medulla. [³H]Dihydroalprenolol ([³H]DHA) binding to the isolated plasma membranes from these cells has demonstrated the presence of -adrenoreceptors with two different affinities. the dissociation constants (K_d) have been found to be 0.27±0.09×10^–9 M and 2.96±0.31×10^–9 M, respectively with the corresponding B_max of 5.1±0.05 and 70.0±0.2 pmol/mg protein, respectively. Inhibition of [³H]DHA binding to the -receptor by atenolol (a ₁-antagonist) and ICI 118,551 (a ₁-antagonist) has suggested that the IC_50cor (=K_i) for atenolol and ICI 118,551 for high affinity site are 0.08±0.03×10^–12 M and 0.25±0.08×10^–12 M, respectively. This, therefore, indicates that both atenolol and ICI 118,551 are able to displace the bound ligand effectively but the ₁-selective antagonist atenolol is 3 times more potent than its ₂ counterpart, ICI 118,551. Displacement of [³H]DHA binding to the endothelial cell plasma membrane by the agonists isoproterenol, epinephrine and norephinephrine has established a relative order of K_i for these agents as isoproterenol (0.56±0.19×10^–9 M)–9 M)>-norepinephrine (0.71±0.24×10^–9 M) for the high affinity site. The corresponding values for the low affinity site, however, are 4.62±0.64×10^–9 M, 6.21±0.86×10^–9 M and 5.90±0.82×10^–9 M, respectively for the same agonists. Increased intracellular cAMP accompanied with cellular proliferation in the presence of isoproterenol has suggested not only the coupling of -adrenoreceptors to the adenylate cyclase system but also its involvement in endothelial cell proliferation.Abbreviations DHA Dihydroalprenolol - cAMP 3:5 cyclic adenosine monophosphate - DTT dithiothreitol - MEM minimal essential medium - 8Br-cAMP 8-bromo-adenosine 3:5 cyclic monophosphate     

M3 muscarinic receptors on murine HSDM1C1 cells: Further functional,regulatory, and receptor binding studies

In the present studies, the pharmacology and regulation of the functional muscarinic receptors on HSDM1C1 cells were probed using phosphoinositide (PI) turnover assays. In addition, the receptor binding of the putative M₃-selective radioligand, [³H]4-DAMP, to cell homogenates was characterized. Carbachol (EC₅₀=9 M), (+)muscarine (EC₅₀=4.5 M) and cis-dioxolane (EC₅=0.72 M) were full agonists which stimulated PI turnover by 13.3±1.0 fold above basal values. The potencies of numerous agonists in this assay system were relatively similar to their affinities in receptor binding assays. Exposure of HSDM1C1 cells to 10 nM–10 M muscarine during the last 24h of [³H]myo-inositol-labeling resulted in a concentration-dependent reduction in the cisdioxolane affinity and maximal PI response induced by subsequent treatment with cis-dioxolane. pertussis toxin (5–2000 ng/ml) caused a partial reduction in the cis-dioxolane-induced PI turnover. Likewise, exposure of the HSDM1C1 cells to an active phorbol ester (TPA) resulted in a partial inhibition of the cis-dioxolane-induced (100 M) PI turnover. The half-maximal effect of TPA was produced at 1.8±0.3 nM. [³H]4-DAMP binding to cell homogenates was of high affinity (K_d=0.19±0.04 nM) and moderate capacity (B_max=201±22 fmol/mg protein). The pharmacological specificity (4-DAMP>p-FHHSiD>dicyclomine>pirenzepine>methoctramine>AFDX-116 >gallamine) resembled that for [³H]NMS binding and correlated well with that observed for inhibition of PI turnover. These studies further support the identification of M₃ receptors on HSDM1C1 cells. These receptors have been shown to be influenced by pertussis toxin, an active phorbol ester and to exhibit desensitization.     

Affinities of muscarinic drugs for [3H]N-methylscopolamine (NMS) and [3H]oxotremorine (OXO) binding to a mixture of M1−M4 muscarinic receptors: Use of NMS/OXO-M ratios to group compounds into potential agonist,partial agonist,and antagonist classes

The relative affinities of various muscarinic drugs in the antagonist ([³H]N-methyl scopolamine ([³H]NMS)) and agonist ([³H]Oxotremorine-m ([³H]OXO-M)) binding assays using a mixture of tissues containing M₁–M₄ receptor subtypes have been determined. [³H]NMS bound with high affinity (K_d=25±5.9 pM; n=3) and to a high density (B_max=11.8±0.025 nmol/g wet weight) of muscarinic receptors. [³H]OXO-M appeared to bind to two binding sites with differing affinities (K_d1=2.5±0.1 nM; K_d2=9.0±4.9 M; n=4) and to a different population of binding sites (B_max1=5.0±0.26 nmol/g wet weight; B_max2=130±60 nmol/g wet weight). Well known antagonists exhibited high affinity for [³H]NMS binding but a lower affinity for [³H]OXO-M binding. The opposite was true for acetylcholine and other known agonists. However, pilocarpine and McN-A-343 had similar affinities for sites labeled by both radioligands. Using the ratios of antagonist-to-agonist binding affinities, it was possible to group compounds into apparently distinct full agonist (ratios of 180–665; e.g. carbachol, muscarine, OXO-M, OXO-S and arecoline), partial agonist (ratios of 14–132; e.g. McN-A-343, pilocarpine, aceclidine, bethanechol, OXA-22 and acetylcholine) and antagonist (ratios of 0.22–1.9; e.g. atropine, NMS, pirenzepine, methoctramine, 4-DAMP and p-fluorohexahydrosialo-difenidol) classes. These data suggest that the NMS/OXO-M affinity ratios using a mixture of M₁–M₄ muscarinic receptors may be a useful way to screen and group a large number of compounds into apparent agonist, partial agonist, and antagonist classes of cholinergic agents.     

GABA-agonists induce the formation of low-affinity GABA-receptors on cultured cerebellar granule cells via preexisting high affinity GABA receptors

The kinetics of specific GABA-binding to membranes isolated from cerebellar granule cells, cultured for 12 days from dissociated cerebella of 7-day-old rats was studied using [³H]GABA as the ligand. The granule cells were cultured in the presence of the specific GABA receptor agonist 4, 5, 6, 7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 150 M) or THIP plus the antagonist bicuculline methobromide (150 M of each) or in the absence of the agonist or antagonist. Membranes isolated from granule cells cultured in a medium without the GABA agonist revealed a single binding site for GABA with a binding constant (K _D) of 7.9±0.4 nM and aB _max of 3.42±0.08 pmol×mg^–1 protein. Membranes from cells cultured in the presence of THIP had two binding sites for GABA withK _D-values of 6.8±0.9 nM and 476±311 nM, respectively. The correspondingB _max values were 4.41±0.42 pmol×mg^–1 and 5.81±1.20 pmol×mg^–1. The effect of culturing the cells in THIP was antagonized by the simultaneous presence of bicuculline in the culture media, i.e. no significant low-affinity binding for GABA was found on the membranes from granule cells cultured in both THIP and bicuculline. TheK _D value (14.3±1.4 nM) for the high affinity binding site was, however, slightly increased compared to the non-treated cells. These findings suggest that the ability of THIP to induce formation of low-affinity GABA receptors is mediated by preexisting high-affinity GABA-receptors on the granule cells.     

Purification and characterization of membrane-bound hydrogenase from Methanosarcina barkeri MS

Hydrogenase was solubilized from the membrane of acetate-grown Methanosarcina barkeri MS and purification was carried out under aerobic conditions. The enzyme was reactivated under reducing conditions in the presence of H₂. The enzyme showed a maximal activity of 120±40 mol H₂ oxidized · min^–1 · min^–1 with methyl viologen as an electron acceptor, a maximal hydrogen production rate of 45±4 mol H₂ · min^–1 · mg^–1 with methyl viologen as electron donor, and an apparent K _m for hydrogen oxidation of 5.6±1.7 M. The molecular weight estimated by gel filtration was 98,000. SDS-PAGE showed the enzyme to consist of two polypeptides of 57,000 and 35,000 present in a 1:1 ratio. The native protein contained 8±2 mol Fe, 8±2 mol S^2–, and 0.5 mol Ni/mol enzyme. Cytochrome b was reduced by hydrogen in a solubilized membrane preparation. The hydrogenase did not couple with autologous F₄₂₀ or ferredoxin, nor with FAD, FMN, or NAD(P)⁺. The physiological function of the membrane-bound hydrogenase in hydrogen consumption is discussed.Abbreviation CoM-S-S-HTP the heterodisulfide of 7-mercaptoheptanoylthrconine phosphate and coenzyme M (mercaptoethanesulfonic acid)