Biochemical characterisation of ketoconazole inhibitory action on Aspergillus fumigatus

Abstract The effect of ketoconazole on growth, sterol composition, in vitro sterol biosynthesis and P450-CO complex formation and its interaction with microsomal P450 was determined. On solid medium and in liquid medium ketoconazole inhibited Aspergillus fumigatus growth completely at 5 × 10⁻⁵ M and 50% of the growth at 1.3 × 10⁻⁵ M and 2.1 × 10⁻⁵ M respectively. A close relationship between accumulation of 14α-methyl sterols (eburicol, obtusifoliol and 14α-methyl fecosterol) and depletion of ergosterol with growth arrest was observed in ketoconazole treated cultures. The half inhibitory concentration for in vitro ergosterol biosynthesis and half saturating concentration for type II binding spectrum of ketoconazole were calculated as 73.8 ± 6.3 nM and 0.13 ± 0.04 μM respectively. CO displacement studies revealed inhibition of CO-P450 complex formation by ketoconazole.     

Heterogeneous Na+ Sensitivity of Na+,K+-ATPase Isoenzymes in Whole Brain Membranes

Abstract: The Na⁺ sensitivity of whole brain membrane Na⁺,K⁺-ATPase isoenzymes was studied using the differential inhibitory effect of ouabain (α1, low affinity for ouabain; α2, high affinity; and α3, very high affinity). At 100 m M Na⁺, we found that the proportion of isoforms with low, high, and very high ouabain affinity was 21, 38, and 41%, respectively. Using two ouabain concentrations (10⁻⁵ and 10⁻⁷ M ), we were able to discriminate Na⁺ sensitivity of Na⁺, K⁺-ATPase isoenzymes using nonlinear regression. The ouabain low-affinity isoform, α1, exhibited high Na⁺ sensitivity [ K _a of 3.88 ± 0.25 m M Na⁺ and a Hill coefficient ( n ) of 1.98 ± 0.13]; the ouabain high-affinity isoform, α2, had two Na⁺ sensitivities, a high ( K _a of 4.98 ± 0.2 m M Na⁺ and n of 1.34 ± 0.10) and a low ( K _a of 28 ± 0.5 m M Na⁺ and an n of 1.92 ± 0.18) Na⁺ sensitivity activated above a thresh old (22 ± 0.3 m M Na⁺); and the ouabain very-high-affinity isoform, α3, was resolved by two processes and appears to have two Na⁺ sensitivities (apparent K _a values of 3.5 and 20 m M Na⁺). We show that Na⁺ dependence in the absence of ouabain is the result of at least of five Na⁺ reactivities. This molecular functional characteristic of isoenzymes in membranes could explain the diversity of physiological roles attributed to isoenzymes.     

Pituitary Adenylate Cyclase-Activating Polypeptide: Control of Rat Pineal Cyclic AMP and Melatonin but Not Cyclic GMP

Abstract: In this study, the effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on cyclic nucleotide accumulation and melatonin (MT) production in dispersed rat pinealocytes were measured. Treatment with PACAP (10⁻⁷ M ) increased MT production 2.5-fold. PACAP (10⁻⁷ M ) also increased cyclic AMP accumulation four- to fivefold; this effect was potentiated two- to three-fold by α₁-adrenergic activation. This potentiation appears to involve protein kinase C (PKC) because α₁-adrenergic activation is known to translocate PKC and the PACAP-stimulated cyclic AMP accumulation was potentiated ninefold by a PKC activator, 4β-phorbol 12-myristate 13-acetate (PMA). Phenylephrine and PMA also potentiated the PACAP-stimulated MT accumulation. These results indicate that cyclic AMP is one second messenger of PACAP in the pineal gland and that the effects of PACAP on cyclic AMP and MT production can be potentiated by an α₁-adrenergic → PKC mechanism. In addition to these findings, it was observed that PACAP treatment with or without phenylephrine or PMA did not alter cyclic GMP accumulation. This indicates that PACAP is the first ligand identified that increases cyclic AMP accumulation in the pineal gland without increasing cyclic GMP accumulation. That PACAP fails to activate the vasoactive intestinal peptide/cyclic GMP pathway suggests that the vasoactive intestinal peptide receptors present in the pineal may be distinct from the type II PACAP receptors.     

ACCUMULATION OF ADENOSINE 3'',5''-MONOPHOSPHATE IN BRAIN SLICES: INTERACTION OF LOCAL ANAESTHETICS AND DEPOLARIZING AGENTS

Abstract— Membrane depolarizing agents such as veratridine, ouabain and high concentrations of potassium ions elicit a remarkable accumulation of cyclic AMP in brain slices incubated in vitro , and this accumulation, but not that elicited by biogenic amines, is prevented by a membrane stabilizer, cocaine. The effect of various local anaesthetics (compounds which are known to stabilize the membrane of peripheral sensory nerves) on the accumulation of cyclic AMP elicited by depolarizing agents in incubated slices of guinea pig brain has now been examined. At optimal concentrations the anaesthetics inhibited by more than 95 per cent the accumulation of cyclic AMP elicited with veratridine, ouabain, and high concentrations of potassium ions. The order of the inhibitory potency vs. veratridine was: dibucaine (ED₅₀= 9.5 ± 10⁻⁶ M) > tetracaine > cocaine (ED₅₀= 1·3 ± 10⁻⁴ M) > lidocaine > procaine (ED₅₀= 1.7 ± 10⁻³M). This order is consistent with the order of their local anaesthetic potency, but is not consonant with the order of the relative toxicity of these agents when used as spinal anaesthetics.     

The δ-Opioid Receptor in SK-N-BE Human Neuroblastoma Cell Line Undergoes Heterologous Desensitization

Abstract: The human neuroblastoma cell line SK-N-BE expresses δ-opioid receptors negatively coupled to adenylyl cyclase. Prolonged treatment (2 h) of the cells with 100 n M etorphine leads to an almost complete desensitization (8.2 ± 5.9 vs. 45.8 ± 8.7% for the control). Other receptors negatively coupled to adenylyl cyclase, namely, D2-dopaminergic, α₂-adrenergic, and m2/m4-muscarinic, were identified by screening of these cells, and it was shown that prolonged treatment (2 h) with 1 µ M 2-bromo-α-ergocryptine or 1 µ M arterenol resulted in a marked desensitization of D2-dopaminergic and α₂-adrenergic receptors, respectively. Cross-desensitization experiments revealed that pretreatment with etorphine desensitized with the same efficiency the δ-opioid receptor and the D2-dopaminergic receptor, and pretreatment with 2-bromo-α-ergocryptine also desensitized both receptors. In contrast, pretreatment with etorphine desensitized only partly the α₂-adrenergic receptor response, whereas pretreatment with 1 µ M arterenol partly desensitized the δ-opioid receptor response. It is concluded that the δ-opioid receptor-mediated inhibitory response of adenylyl cyclase undergoes heterologous desensitization, and it is suggested that δ-opioid and D2-dopaminergic receptors are coupled to adenylyl cyclase via a G_i2 protein, whereas α₂-adrenergic receptor could be coupled to the enzyme via two G proteins, G_i2 and another member of the G_i/G_o family.     

Effects of phenolic acids on ammonia oxidation by terrestrial autotrophic nitrifying microorganisms

Abstract It has been hypothesized that vegetation in certain ecosystems inhibits nitrification in soil by producing phenolic compounds that inhibit oxidation of ammonia by nitrifying microorganisms. This hypothesis is based largely on a report that very low concentrations (10⁻⁶ M–10⁻⁸ M) of several phenolic acids (notably ferulic acid) completely inhibited NO₂⁻ production in an aqueous suspension of soil treated with (NH₄)₂SO₄ and a nutrient solution suitable for growth of Nitrosomonas and other autotrophic nitrifying microorganisms. To evaluate this hypothesis, we determined the effects of three ohenolic acids (ferulic acid, caffeic acid, and p -coumaric on nitrite production by representatives of three genera of terrestrial autotrophic nitrifying microorganisms ( Nitrosospira, Nitrosomonas , or Nitrosolubos ) grown on a defined medium containing NH₄⁺. We found that nitrite production by the Nitrososspira was not inhibited by ferulic acid, caffeic acid, or p -coumaric acid at concentrations of 10⁻⁶ or 10⁻⁵ M and was only slightly inhibited when these acids were at a concentration of 10⁻⁴ M. We also found that ferulic acid did not markedly inhibit nitrite production by the three genera of nitrifying microorganisms studied, even when its concentration was as high as 10⁻³ M. These observations invalidate the hypothesis tested because the phenolic acids studied did not significantly retard ammonia oxidation by autotrophic microorganisms even when their concentration in cultures of these microorganisms greatly exceeded their concentrations in soils.     

Water-relation parameters of giant and normal cells of Capsicum annuum pericarp

Abstract. Measurements of the water-relation parameters of the giant subepidermal cells (volume, V = 0.119 to 1.658 mm³; = 0.53±0.35 mm³, SD, n = 23) and the smaller mesocarp parenchyma cells ( V = 0.10 to 0.79×10⁻³ mm³; = 0.36±0.27×10⁻³ mm³, SD, n = 6) of the inner pericarp surface of Capsicum annuum L. were made using the Jülich pressure probe. The volumetric elastic modulus ɛ for the large cells was between 1.5 and 27 MPa for a pressure range of 0.09 to 0.41 MPa. For the small cells ɛ was 0.1 to 0.6 MPa for a pressure range of 0.22 to 0.39 MPa. The turgor pressure P , the half-time of water exchange T _1/2, and the hydraulic conductivity L _p were as follows, with SD and number of replicates: large cells, P = 0.27±0.06 MPa (23), T _1/2=2.7±2.2 s (46), L _p=5.8±3.7 pm s⁻¹ Pa⁻ (46); small cells, P = 0.33±0.07 MPa (6), T _1/2= 33±10s (12), L _p=0.21±0.07 pm s⁻¹ Pa⁻¹ (12). The determination of these basic water-relation parameters is considered as a prerequisite for future ecotoxicological and phytopathological studies. The differences between the large and the small cells are discussed in relation to a desirable biophysical definition of succulence. Further, for the large cells a pressure and volume dependence of ɛ was demonstrated.     

Acute toxicity of copper, zinc and manganese in single and mixed salt solutions to juvenile longfin dace, Agosia chrysogaster

The acute toxicity of copper, zinc and manganese and copper-zinc and copper-manganese mixtures were determined for juvenile longfin dace, Agosia chrysogaster in hard water bioassays (mean=218 mg 1⁻¹ CaCO₃). Copper-zinc was the most lethal toxicant (96-h L.c.₅₀= 0.21 mg 1⁻¹ copper and 0.28 mg 1⁻¹ zinc) and exhibited a more than additive toxicity which was in contrast to the additive toxicity of copper-manganese mixtures (96-h L.c.₅₀= 0.45 mg 1⁻¹ copper and 64.0 mg 1⁻¹ manganese). The toxicity of copper (96-h L.c.₅₀= 0.86 mg 1⁻¹) and zinc (96-h L.c.₅₀= 0.79 mg 1⁻¹) to the fish was similar but both were considerably more lethal than manganese (96-h L.c.₅₀= 130 mg 1⁻¹).     

VIP down-regulates the inflammatory potential and promotes survival of dying (neural crest-derived) corneal endothelial cells ex vivo: necrosis to apoptosis switch and up-regulation of Bcl-2 and N-cadherin

The neuropeptide vasoactive intestinal peptide (VIP) is anti-inflammatory and protective in the immune and nervous systems, respectively. This study demonstrated in corneal endothelial (CE) cells injured by severe oxidative stress (1.4 mM H₂O₂) in bovine corneal organ cultures that VIP pre-treatment (0, 10⁻¹⁰, 10⁻⁸, and 10⁻⁶ M; 15 min), in a VIP concentration-dependent manner, switched the inflammation-causing necrosis to inflammation-neutral apoptosis (showing annexin V-binding, chromatin condensation, and DNA fragmentation) and upheld ATP levels in a VIP antagonist (SN)VIPhyb-sensitive manner, while up-regulated mRNA levels of the anti-apoptotic Bcl-2 and the differentiation marker N-cadherin in a kinase A inhibitor-sensitive manner. As a result, VIP, in a concentration-dependent and VIP antagonist-sensitive manners, promoted long-term CE cell survival. ATP levels, a determining factor in the choice of apoptosis versus necrosis, measured after VIP pre-treatment and 0.5 min post-H₂O₂ were 39.6 ± 3.3, 50.8 ± 6.2, 60.1 ± 4.8, and 53.6 ± 5.3 pmoles/μg protein (mean ± SEM), respectively ( p  < 0.05, anova ). VIP treatment alone concentration-dependently increased levels of N-cadherin ( Koh et al. 2008 ), the phosphorylated cAMP-responsive-element binding protein and Bcl-2, while10⁻⁸ M VIP, in a VIP antagonist (SN)VIPhyb-sensitive manner, increased ATP level by 38% ( p  < 0.02) and decreased glycogen level by 32% ( p  < 0.02). VPAC1 (not VPAC2) receptor was expressed in CE cells. Thus, CE cell VIP/VPAC1 signaling is both anti-inflammatory and protective in the corneal endothelium.     

Responses of Bergmann Glia and Granule Neurons In Situ to N-Methyl-d-Aspartate, Norepinephrine, and High Potassium

Abstract: To gain insight into neuronal-glial signaling in brain, cerebellar Bergmann glia and granule neurons were studied in acutely isolated slices with the aid of laser scanning confocal microscopy. Both Bergmann glia and granule neurons responded to N -methyl- d -aspartate (NMDA) with a rise in [Ca²⁺]_i. However, the glial NMDA response was frequently inhibited by tetrodotoxin, suggesting that the response depended on neuronal action potentials, rather than on direct activation of NMDA receptors on the Bergmann glia. Further experiments demonstrated that the NMDA response in Bergmann glia was not inhibited by a combination of non-NMDA glutamate receptor blockers 6-cyano-7-nitroquinoxaline-2,3-dione and α-methyl-4-carboxyphenylglycine. Bergmann glia also responded to norepinephrine and high K⁺, and the responses were not inhibited by tetrodotoxin. The glial norepinephrine response was blocked by phentolamine but not by the removal of external Ca²⁺, indicating a direct activation of α₁-adrenergic receptors that mediated release of Ca²⁺ from intracellular stores. The KCI-induced response in both neurons and glia was dependent on external Ca²⁺ and was blocked by verapamil or nifedipine. In summary, our data indicate that Bergmann glia in situ recognize a signal(s) released from neurons during neuronal activity.     

Uptake and metabolism of taurine in the green alga Chlorella fusca

Taurine entered the alga Chlorella fusca Shihira et Krauss strain 21l-8b via a pH and energy-dependent system ("permease"). Transport followed triphasic kinetics from 10⁻⁶ to 10⁻² M with K_m values for taurine of 5.4 × 10⁻⁵, 4.1 × l0⁻⁴ and l.5 × 10⁻³ M. This uptake system was specific for sulfonic acids and showed no affinity for α- and β -amino acids or Na⁺; thus the permease of C. fusca is different from all known taurine transport systems with respect to structural specificity and lack of Na⁺ -dependence. Uptake was not observed in sulfate-grown algae but developed as a response to sulfate limitation within 2 h. Sulfate addition caused a rapid decline in taurine transport capacity. Labeled taurine was rapidly metabolized in C. fusca to sulfate and ethanolamine, suggesting oxidative hydrolysis as the mechanism of C-S bond cleavage. Further incorporation of these catabolic products in C - and S -metabolism was demonstrated. Taurine catabolism was also detected in other green algae and some cyanobacteria.     

Sodium Transport from Blood to Brain: Inhibition by Furosemide and Amiloride

Abstract: Brain sodium uptake in vivo was studied using a modified intracarotid bolus injection technique in which the uptake of ²²Na ⁺ was compared with that of the relatively impermeable molecule, [³H]l-glucose. At a Na ⁺ concentration of 1.4 m M , Na ⁺ uptake was 1.74 ± 0.07 times greater than l -glucose uptake. This decreased to 1.34 ± 0.04 at 140 m M Na ⁺, indicating saturable Na ⁺ uptake. Relative Na ⁺ extraction was not affected by pH but was inhibited by amiloride ( K _i= 3 ± 10⁻⁷ M ) and by 1 m M furosemide. The effects of these two inhibitors were additive. Brain uptake of ⁸⁶Rb ⁺, a K ⁺ analogue, was measured to study interaction of K ⁺ with Na ⁺ transport systems. Relative ⁸⁶Rb ⁺ extraction was also inhibited by amiloride; however, it was not inhibited by furosemide. The results suggest the presence of two distinct transport systems that allow Na ⁺ to cross the luminal membrane of the brain capillary endothelial cell. These transport systems could play an important role in the movement of Na ⁺ from blood to brain.     

Multisite Interactions Between Pb2+ and Protein Kinase C and Its Role in Norepinephrine Release from Bovine Adrenal Chromaffin Cells

Abstract: We investigated the interaction between Pb²⁺ and protein kinase C (PKC) in the Pb²⁺-induced release of norepinephrine (NE) from permeabilized adrenal chromaffin cells. Our analysis of endogenous PKC activity in permeabilized cells suggests that Pb²⁺ interacts with the adrenal enzyme at multiple sites. Pb²⁺ activates the enzyme through high-affinity ( K _A(Pb) = 2.4 × 10⁻¹² M ) interactions and inhibits the enzyme by competitive and noncompetitive interactions with nanomolar-( K _i = 7.1 × 10⁻⁹ M ) and micromolar- ( K '_i = 2.8 × 10⁻⁷ M ) affinity sites, respectively. Activation of PKC by 12- O -tetradecanoylphorbol 13-acetate (TPA) in Ca²⁺-deficient, Pb²⁺-containing medium, enhances the Pb²⁺-induced NE release from permeabilized chromaffin cells by lowering the concentration of Pb²⁺ required for half-maximal activation of the secretory response from 7.5 × 10⁻¹⁰ to 5.7 × 10⁻¹¹ M . The PKC inhibitors staurosporine and pseudosubstrate PKC (19–36) abolish the effect of TPA without affecting the Pb²⁺-induced secretion in the absence of TPA. These results indicate that (a) Pb²⁺ is a partial agonist of PKC, capable of both activating and inhibiting the enzyme and (b) synergistic activation of PKC by TPA and Pb²⁺ results in increased sensitivity of exocytosis to Pb²⁺ but is not obligatory for Pb²⁺-triggered secretion.     

IL-17 is involved in bone resorption in mouse periapical lesions

Periapical lesions are induced by bacterial infection of the dental pulp and result in destruction of the surrounding alveolar bone. Although various immunological studies concerning periapical bone resorption have been reported, the role of cytokines in the formation of periapical lesions remains unclear. In this study, the role of IL-17A in periapical lesions in mice was investigated. Normal C57BL/6, IFN-γ^−/−, TNF-α^−/−, and IL-17A^−/− mice were subjected to pulp exposure and infected with Prevotella intermedia (ATCC25611) and Porphyromonas gingivalis (ATCC33277) in the mandibular first molar. Periapical lesions were determined by μCT on day 21 after infection, and 3D visual construction was performed using 3D picture quantification software. The expression of IL-17A mRNA in periapical lesions was determined by the RT-PCR and real-time RT-PCR method. Periapical lesions developed in wild-type, IFN-γ^−/−, and TNF-α^−/− mice after infection with P. intermedia and P. gingivalis . However, periapical lesions were not observed in IL-17A^−/− mice. The expression of IL-17A mRNA was significantly induced in periapical lesions of wild-type mice after infection. These results suggest that IL-17A, but not IFN-γ or TNF-α, plays an important role in the formation of periapical lesions.     

Taste responses of the facial and glossopharyngeal nerves to amino acids in the rainbow trout

No significant differences were noted between responses of rainbow trout Oncorhynchus mykiss facial and glossopharyngeal nerves to 15 amino acids. Nine of these amino acids tested at 10⁻² M were stimulatory, whereas only two tested at 10⁻³ M were effective gustatory stimuli. For both nerve systems, ≤10⁻³ M L-proline was the most stimulatory amino acid, with an estimated threshold of 10⁻⁷ M; however, L-α-amino-β-guanidino-propionic acid (estimated threshold of 3×10⁻³ M), was the most potent compound at 10⁻² M. These results indicate that the same amino acids activate taste buds innervated by facial and glossopharyngeal nerves, respectively, and suggest that the same amino acids can be important in chemosensory feeding behaviour in the rainbow trout.     

The rooting ability of shoots raised 'in vitro' from the apple rootstock A2 in juvenile and in adult growth phase

The apple rootstock A2 can be readily propagated in vitro both in the juvenile and in the adult growth phase. Shoots were produced by meristem tip culture from the apple rootstock A2 in different growth phases. The influence of growth phases and different concentrations of PG and IBA was investigated as to rooting percentage, survival percentage, number of roots per rooted shoot, root length, shoot length and formation of callus. IBA at 15 μ M without PG gave a significantly lower rooting percentage than 5 and 10 μ M IBA. PG together with IBA stimulated rooting, the optimum concentrations of PG being, however, not the same for the different growth phases. For the adult growth phase, 10⁻⁴ M PG promoted rooting, whereas 10⁻³ M PG markedly inhibited rooting. In the juvenile growth phases, both 10⁻⁴ and 10⁻³ M PG stimulated rooting. PG at 10⁻⁴ M also increased the number of roots. The longest roots were obtained at 10⁻³ M PG and 5 μ M IBA. PG at 10⁻³ M reduced callus formation at all IBA concentrations used. Neither shoot length nor root length influenced the survival percentage.     

Bioelectrical reactions of Nitellopsis obtusa induced by indole-3-acetic acid

The complex of bioelectrical paramenters (membrane potential, membrane resistance and capacitance) of internodal cells of Nitellopsis obtusa was measured over a wide range of IAA concentration (10⁻¹⁰ to 10⁻⁴ M ) with two intracellular microelectrodes. Primary effects of IAA at a concentration as low as 10⁻¹⁰ M were observed. The optimum range of IAA action was from 10⁻⁹ to 10⁻⁶ M . The type of IAA-induced electroresponse depended on the initial level of membrane potential, which characterized the energetic state of the plasmalemma. In the energized state (ca −200 mV) N. obtusa cells appeared to have 3 typical reactions: hyperpolarization (membrane potential less than K⁺-equilibrium potential), depolarization (membrane potential higher than K⁺-potential) and absence of response at K⁺-electrochemical equilibrium. Membrane capacitance was found constant at 0.74 ± 0.05 μF cm⁻², but membrane resistance increased up to 50% independently of the sign of the electrogenic reaction. Increase of membrance capacitance and decrease of the membrane resistance was a feature of the de-energized state (ca −135 mV) and may be explained by lower viscosity of membrane lipids, which interacted with IAA. The complex of parameter, including cytoplasmic steaming taken as an indicator of energy supply, is discussed as indicating slow IAA penetration combined with a primary action of IAA on the plasmalemma receptor sites.     

Permeability of the Chara cell membrane for ethylene glycol, glycerol, meso-erythritol, xylitol and mannitol

The permeability of internodal cells of Chara australis R. Brown for polyol molecules was determined by using a turgor balance to measure the increase in the osmotic pressure of an internodal cell incubated in artificial pond water containing one of the polyol compounds tested. The permeabilities for ethylene glycol, glycerol, meso -erythritol, xylitol and mannitol were (4. 39 ± 0. 52) × 10⁻⁹, (1. 49 ± 0. 40) × 10¹⁰, (4. 92 ± 0. 27) × 10⁻¹⁰ (9. 9 ± 3. 4) × 10¹¹ and (7. 6 ± 4. 8) × 10⁻¹² m s⁻³, respectively. The permeability for glycerol was slightly smaller than that for meso -erythritol, whose molecular weight is larger than that of glycerol in this homologous series: but the reason for this is not clear.     

An extracellular α-L-arabinofuranosidase secreted from cell suspension cultures of carrot

Carrot ( Daucus carota L. cv. Kintoki) cell cultures secrete an α-L-arabinofuranosidase (α-L-AFase, EC 3.2.1.55) into their culture medium during growth. The extracellular α-L-AFase (α-L-AFase-II) was purified to electrophoretic homogeneity from the concentrated medium using ammonium sulfate precipitation, chromatography on DEAE-Sepharose CL-6B, CM-Sepharose CL-6B, Sephacryl S-200HR and Concanavalin A-Sepharose, and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 84 kDa by Sephacryl S-200HR gel-permeation, and 80 kDa by SDS-PAGE under denaturing conditions. The enzyme contained carbohydrate and protein in a ratio of 1:5 (w/w), and was analyzed for amino acid composition and the sequence of the first 21 amino acids of the N-terminus. The isoelectric point was pH 5.6, the pH optimum 3.8, and the temperature optimum 55°C. The activity was inhibited by Zn²⁺, Ag²⁺, Cu²⁺, Hg²⁺ and p -chloromercuribenzoate. The K_m and V_max values for p -nitrophenyl-α-L-arabinofuranoside were 0.22 m M and 0.11 mmol (mg protein)⁻¹ h⁻¹, respectively. The enzyme acted on beet arabinan in an exo-fashion, and was capable of hydrolysing arabinose-rich polymers purified from pectic polysaccha-rides of carrot cell cultures. However, even after an exhaustive reaction, the enzyme had little or no effect on cell walls from carrot cell cultures.     

Stimulation of Cyclic GMP Accumulation by Sodium Nitroprusside Is Potentiated via a Gs Mechanism in Intact Pinealocytes

Abstract: Cyclic GMP accumulation in pinealocytes is elevated>100-fold by norepinephrine (NE) through a mechanism involving conjoint activation of α₁- and β₁-adrenergic receptors. Little or no stimulation occurs if either α₁- or β₁-adrenergic receptors alone are activated. It appears that α₁-adrenergic effects are mediated by Ca²⁺ acting in part through nitric oxide (NO), and β₁-adrenergic effects are mediated by G_s. In the study presented here we investigated effects of adrenergic agonists or related postreceptor-active agents on stimulation of pineal cyclic GMP accumulation by the NO generator sodium nitroprusside (NP). The cyclic GMP response to NP (1 m M ) was potentiated by NE and isoproterenol (ISO) but not by phenylephrine, indicating that activation of β₁-adrenergic receptors potentiates the effects of NP. Similarly, vasoactive intestinal peptide (VIP), cholera toxin (CTX), and forskolin, all of which are known to mimic the effects of ISO in this system, also potentiated the effects of NP. In contrast, neither dibutyryl cyclic AMP nor agents that elevate intracellular Ca²⁺ levels caused marked potentiation of the effects of NP on pineal cyclic GMP. Depletion (90%) of G_sα by 21-h treatment with CTX reduced β-adrenergic potentiation of NP. These findings indicate that β-adrenergic agonists and VIP potentiate the effects of NP through a mechanism involving G_s. The molecular basis of this action may be an increase in guanylyl cyclase responsiveness to NO.