Time-dependent inhibition of inositol-1,4,5-trisphosphate-5-phosphatase by calmidazolium chloride in rat GH3 cells

The calmodulin inhibitor calmidazolium chloride inhibited the activity of soluble and particulate Ins(1,4,5)P₃-5-phosphatase from GH₃ cells, with an IC₅₀ value of 100 μM following a 10-min preincubation with enzyme. The inhibition was time-dependent and could not be reversed by washing of the particulate fraction. It is concluded that although the inhibitory effect of calmidazolium chloride cannot be related per se to inhibition of calmodulin function, effects of this compound unrelated to actions upon calmodulin function may be found when concentrations that are only moderately supramaximal are used.     

Tissue distribution of GAP1 and GAP1: Two inositol 1,3,4,5-tetrakisphosphate-binding proteins

Two members of the GAP1 family, GAP1^IP4BP and GAP1^m, have been shown to bind the putative second messenger Ins(1,3,4,5)P₄ with high affinity and specificity, though other aspects of their behaviour suggest that in vivo, whereas GAP1^IP4BP may function as an Ins(1,3,4,5)P₄ receptor, GAP1^m may be a receptor for the lipid second messenger PtdIns(3,4,5)P₃. As a step towards clarifying their cellular roles, we describe here how we have raised and characterised antisera that are specific for the two proteins, and used these to undertake a comprehensive study of their tissue distribution. Both proteins are widely expressed, but there are several clear differences between them in the tissues that show the highest levels of expression.     

Estrogen effects on NADH oxidase and superoxide dismutase in prepubertal female rats

Thirty-four day old, ovariectomised rats were treated with increasing doses of estradiol, 2-hydroxyestradiol 2,3-dimethyl ether (23E₂), 4-hydroxyestradiol 3,4-dimethyl ether (34E₂) and 4-methoxy-estradiol (4ME₂) for five days by subcutaneous injection. Superoxide dismutase, phenol activated NADH oxidase and uterine dry weights were determined. Only estradiol was found to be uterotrophic and increased NADH oxidase activity in these experiments. Both 23E₂ and 34E₂ treatment reduced the enzyme activity significantly. Though 4ME₂ showed a decrease in NADH oxidase at 0.05μg/100gm body weight there was no further decrease at higher dose (5μg/100gm). The Superoxide dismutase (SOD) in uterus and liver was unaffected by estradiol, while 23E₂, 34E₂, and 4ME₂ significantly reduced SOD in both liver and uterus. These results indicate that 23E₂, 34E₂ and 4ME₂, in spite of their non-uterotrophic property, affect uterine metabolism. Furthermore, in view of the reports indicating the importance of SOD levels in various tumors and since catecholestrogens are observed to reduce SOD levels in liver and uterus, it is suggestive that catecholestrogens may play an important role in the pathophysiology of certain tumors.     

ERp44 C160S/C212S mutants regulate IP3R1 channel activity

Previous studies have indicated that ERp44 inhibits inositol 1,4,5-trisphosphate (IP₃)-induced Ca²⁺ release (IICR) via IP₃R₁, but the mechanism remains largely unexplored. Using extracellular ATP to induce intracellular calcium transient as an IICR model, Ca²⁺ image, pull down assay, and Western blotting experiments were carried out in the present study. We found that extracellular ATP induced calcium transient via IP₃Rs (IICR) and the IICR were markedly decreased in ERp44 over-expressed Hela cells. The inhibitory effect of C160S/C212S but not C29S/T396A/ΔT(331--377) mutants of ERp44 on IICR were significantly decreased compared with ERp44. However, the binding capacity of ERp44 to L3V domain of IP₃R₁ (1L3V) was enhanced by ERp44 C160S/C212S mutation. Taken together, these results suggest that the mutants of ERp44, C160/C212, can more tightly bind to IP₃R₁ but exhibit a weak inhibition of IP₃R₁ channel activity in Hela cells.     

Polyphosphates strongly inhibit the tRNA dependent synthesis of poly(A) catalyzed by poly(A) polymerase from Saccharomyces cerevisiae

Polyphosphates of different chain lengths (P₃, P₄, P₁₅, P₃₅), (1 μM) inhibited 10, 60, 90 and 100%, respectively, the primer (tRNA) dependent synthesis of poly(A) catalyzed poly(A) polymerase from Saccharomyces cerevisiae. The relative inhibition evoked by p₄A and P₄ (1 μM) was 40 and 60%, respectively, whereas 1 μM Ap₄A was not inhibitory. P₄ and P₁₅ were assayed as inhibitors of the enzyme in the presence of (a) saturating tRNA and variable concentrations of ATP and (b) saturating ATP and variable concentrations of tRNA. In (a), P₄ and P₁₅ behaved as competitive inhibitors, with K_i values of 0.5 μM and 0.2 μM, respectively. In addition, P₄ (at 1 μM) and P₁₅ (at 0.3 μM) changed the Hill coefficient (n_H) from 1 (control) to about 1.3 and 1.6, respectively. In (b), the inhibition by P₄ and P₁₅ decreased V and modified only slightly the K_m values of the enzyme towards tRNA.     

Ins(1,4,5)P3 induces Ca2+ release from brain microsomes loaded either by the Ca2+ ATPase or by the Na+/Ca2+ exchanger.

In this study we investigated the release of Ca²⁺ in brain microsomes after Ca²⁺ loading by the Ca²⁺-ATPase or by the Na⁺/Ca²⁺ exchanger. The results show that in microsomes loaded with Ca²⁺ by the Ca²⁺-ATPase, Ins(1,4,5)P₃ (5 μM) release 21±2% of the total Ca²⁺ accumulated, and that in the microsomes loaded with Ca²⁺ by the Na²⁺/Ca²⁺ exchanger, Ins(1,4,5)P₃ released 28±3% of the total Ca²⁺ accumulated. These results suggest that receptors of Ins(1,4,5)P₃ may be co-localized with the Na²⁺/Ca²⁺ exchanger in the endoplasmic reticulum membrane or that there are Ins(1,4,5)P₃ receptors in the plasma membrane where the Na²⁺/Ca²⁺ exchanger is normally present, or both. We also found that Ins(1,4,5)P₃ inhibited the Ca²⁺-ATPase by 33.7%, but that it had no significant effect on the Na²⁺/Ca²⁺ exchanger.     

Ca2+/Calmodulin-Mediated Regulation of Agonist-Induced Sequestration of Gq Protein-Coupled Histamine H1 Receptors in Human U373 MG Astrocytoma Cells

Abstract: We investigated the regulation by intracellular Ca²⁺ of agonist-induced sequestration of G_q protein-coupled histamine H₁ receptors in human U373 MG astrocytoma cells. Histamine-induced sequestration of H₁ receptors from the cell surface membrane was detected as the loss of [³H]mepyramine binding sites on intact cells accessible to the hydrophilic H₁-receptor antagonist pirdonium. The changes in the pirdonium-sensitive binding of [³H]mepyramine were mirrored by changes in the subcellular distribution of H₁ receptors detected by sucrose density gradient centrifugation. The histamine-induced sequestration of H₁ receptors did not occur in hypertonic medium, in which clathrin-mediated endocytosis is known to be inhibited, but was significantly accelerated in the absence of extracellular Ca²⁺ or in the presence of the calmodulin antagonists W-7 and calmidazolium. Inhibitors of protein kinase C (H-7 and GF109203X), Ca²⁺/calmodulin-dependent protein kinase II (KN-62), or protein phosphatase 2B (FK506) did not alter the time course of H₁-receptor sequestration. These results provide the first evidence that agonist-induced, clathrin-mediated sequestration of G_q protein-coupled receptors is transiently inhibited by Ca²⁺/calmodulin, with the result that receptors remain on the cell surface membrane during the early stage of agonist stimulation.     

The effect of temperature on the primary reaction of chloroplast Photosystem II Evidence for a temperature-dependent back reaction

The primary reaction of Photosystem II has been studied over the temperature range from −196 to −20 °C. The photooxidation of the reaction-center chlorophyll (P680) was followed by the free-radical electron paramagnetic resonance signal of P680⁺, and the photoreduction of the Photosystem II primary electron acceptor was monitored by the C-550 absorbance change.

At temperatures below −100 °C, the primary reaction of Photosystem II is irreversible. However, at temperatures between −100 and −20 °C a back reaction that is insensitive to 3-(3′,4′-dichlorophenyl)-1,1′-dimethylurea (DCMU) occurs between P680⁺ and the reduced acceptor.

The amount of reduced acceptor and P680⁺ present under steady-state illumination at temperatures between −100 and −20 °C is small unless high light intensity is used to overcome the competing back reaction. The amount of reduced acceptor present at low light intensity can be increased by adjusting the oxidation-reduction potential so that P680⁺ is reduced by a secondary electron donor (cytochrome b₅₅₉) before P680⁺ can reoxidize the reduced primary acceptor. The photooxidation of cytochrome b₅₅₉ and the accompanying photoreduction of C-550 are inhibited by DCMU. The inhibition of C-550 photoreduction by DCMU, the dependence of P680 photooxidation and C-550 photoreduction on light intensity, and the effect of the availability of reduced cytochrome b₅₅₉ on C-550 photoreduction are unique to the temperature range where the Photosystem II primary reaction is reversible and are not observed at lower temperatures.     

Binding of the inhibitor NH3 to the oxygen-evolving apparatus of spinach chloroplasts

Experiments are described on flash-induced luminescence of isolated spinach chloroplasts after addition of NH₄Cl. The results indicate a binding of NH₃, presumably in competition with water, in the oxidation states S₂ and S₃, i.e. the states reached upon illumination of dark-adapted material with one and two flashes, respectively. In the initial state S₁, no binding of NH₃ occurs. In state S₂ the binding of ammonia is rapid (half-time about 0.5 s) and rapidly reversible; in state S₃ the binding is slower (half-time about 10 s) and slowly reversible. NH₃ bound to S₄ prevents the oxidation of water. NH₃ bound to S₂ decreases the rate of the back reaction of reduced primary acceptor (Q⁻), indicating a charge stabilization, i.e. a decrease in the redox potential of S₂ due to interaction with ammonia. In Tris-washed chloroplasts, the stability of the positive charge generated in a flash is much smaller than in normal chloroplasts and not increased by NH₃. On the basis of these observations it is postulated that, in the absence of NH₃, states S₂ and S₃ are stabilized by manganese-coordinated, bound water.     

Regulation of synaptic vesicle accumulation and axon terminal remodeling during synapse formation by distinct Ca2+ signaling

The synaptic vesicle accumulation and subsequent morphological remodeling of axon terminals are characteristic features of presynaptic differentiation of zebrafish olfactory sensory neurons. The synaptic vesicle accumulation and axon terminal remodeling are regulated by protein kinase A and calcineurin signaling, respectively. To investigate upstream signals of presynaptic differentiation, we focused on Ca²⁺ signaling as Ca²⁺/calmodulin is required for the activation of both calcineurin and some adenylyl cyclases. We here showed that application of Ca²⁺/calmodulin inhibitor or olfactory sensory neuron-specific expression of calmodulin inhibitory peptide suppressed both synaptic vesicle accumulation and axon terminal remodeling. Thus, the trigger of presynaptic differentiation could be Ca²⁺ release from intracellular stores or Ca²⁺ influx. Application of a phospholipase C inhibitor or olfactory sensory neuron-specific expression of inositol 1,4,5-trisphosphate (IP₃) 5-phosphatase suppressed synaptic vesicle accumulation, but not morphological remodeling. In contrast, application of a voltage-gated Ca²⁺ channel blocker or expression of Kir2.1 inward rectifying potassium channel prevented the morphological remodeling. We also provided evidence that IP₃ signaling acted upstream of protein kinase A signaling. Our results suggest that IP₃-mediated Ca²⁺/calmodulin signaling stimulates synaptic vesicle accumulation and subsequent neuronal activity-dependent Ca²⁺/calmodulin signaling induces the morphological remodeling of axon terminals.     

The role of Ins(1,4,5)P(3) in signal transduction of the metabolic neuropeptide Mem-CC in the cetoniid beetle,Pachnoda sinuata

We have investigated the role of inositol triphosphate, Ins(1,4,5)P₃, in the transduction of the hypertrehalosaemic and hyperprolinaemic signal of the endogenous neuropeptide Mem-CC in the cetoniid beetle Pachnoda sinuata. Flight and injection of Mem-CC into the haemocoel of the beetle induce an increase of Ins(1,4,5)P₃ levels in the fat body of the beetle. When Mem-CC is co-injected with U 73122, which is an inhibitor of phospholipase C, this effect is abolished. Mem-CC also elevates Ins(1,4,5)P₃ concentration in fat body pieces in vitro. The increase in Ins(1,4,5)P₃ levels is tissue-specific and does not occur in brain and flight muscles. Elevation of the Ins(1,4,5)P₃ levels upon injection of Mem-CC is time- and dose-dependent: the maximum response is reached after 3 min and a dose of 10 pmol is needed. Compounds that mimic the action of cAMP (cpt-cAMP, forskolin) do not influence the concentration of Ins(1,4,5)P₃, while those that stimulate G-proteins (aluminium fluoride and cholera toxin) cause an increase of Ins(1,4,5)P₃ levels. The application (in vivo and in vitro) of F-Ins(1,4,5)P₃, an Ins(1,4,5)P₃ analogue that penetrates the cell membrane, causes a mobilisation of carbohydrate reserves via the activation of glycogen phosphorylase but does not stimulate proline synthesis. In addition, U 73122 abolishes the hypertrehalosaemic but not the hyperprolinaemic effect of Mem-CC. The results suggest that the hypertrehalosaemic signal of Mem-CC is mediated via an increase of Ins(1,4,5)P₃ levels in the fat body of P. sinuata.     

Effect of low temperature (−30 to −60°C) on the reoxidation of the Photosystem II primary electron acceptor in the presence and absence of 3(3,4-dichlorophenyl)-1,1-dimethylurea

The fluorescence yield has been measured on spinach chloroplasts at low temperature (−30 to −60°C) for various dark times following a short saturating flash. A decrease in the fluorescence yield linked to the reoxidation of the Photosystem II electron acceptor Q is still observed at −60°C. Two reactions participate in this reoxidation: a back reaction or charge recombination and the transfer of an electron from Q⁻ to Pool A. The relative competition between these two reactions at low temperature depends upon the oxidation state of the donor side of the Photosystem II center:

1. (1) In dark-adapted chloroplasts (i.e. in States S₀+S₁ according to Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475), Q, reduced by a flash at low temperature, is reoxidized by a secondary acceptor and the positive charge is stabilized on the Photosystem II donor Z. Although this reaction is strongly temperature dependent, it still occurs very slowly at −60°C.

2. (2) When chloroplasts are placed in the S₂+S₃ states by a two-flash preillumination at room temperature, the reoxidation of Q⁻ after a flash at low temperature is mainly due to a temperature-independent back reaction which occurs with non-exponential kinetics.

3. (3) Long continuous illumination of a frozen sample at −30°C causes 6–7 reducing equivalents to be transferred to the pool. Thus, a sufficient number of oxidizing equivalents should have been generated to produce at least one O₂ molecule.

4. (4) A study of the back reaction in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) shows the superposition of two distinct non-exponential reactions one temperature dependent, the other temperature independent.

The high-energy state of the thylakoid system as indicated by chlorophyll fluorescence and chloroplast shrinkage

Certain long-term fluorescence phenomena observed in intact leaves of higher plants and in isolated chloroplasts show a reverse relationship to light-induced absorbance changes at 535 nm (“chloroplast shrinkage”).

1. 1. In isolated chloroplasts with intact envelopes strong fluorescence quenching upon prolonged illumination with red light is accompanied by an absorbance increase. Both effects are reversed by uncoupling with cyclohexylammonium chloride.

2. 2. The fluorescence quenching is reversed in the dark with kinetics very similar to those of the dark decay of chloroplast shrinkage.

3. 3. In intact leaves under strong illumination with red light in CO₂-free air a low level of variable fluorescence and a strong shrinkage response are observed. Carbon dioxide was found to increase fluorescence and to inhibit shrinkage.

4. 4. Under nitrogen, CO₂ caused fluorescence quenching and shrinkage increase at low concentrations. At higher CO₂ levels fluorescence was increased and shrinkage decreased.

5. 5. In the presence of CO₂, the steady-state yield of fluorescence was lower under nitrogen than under air, whereas chloroplast shrinkage was stimulated in nitrogen and suppressed in air.

6. 6. These results demonstrate that the fluorescence yield does not only depend on the redox state of the quencher Q, but to a large degree also on the high-energy state of the thylakoid system associated with photophosphorylation.

PM2.5通过激活NLRP3/Caspse-1通路诱导大鼠子宫炎症反应

颗粒物(PM)对呼吸系统、心血管系统、神经系统和免疫系统均有损害,但目前关于吸入颗粒物对生殖损伤的研究较少。本研究旨在探讨细颗粒物(PM_2.5)短期暴露对大鼠子宫炎症损伤及其作用机制。PM_2.5暴露30 d后,高剂量组大鼠的子宫脏器系数、内膜上皮细胞厚度和腺上皮高度均明显高于对照组(P<0.05),抑制剂MCC950则能明显降低PM_2.5对子宫的影响。子宫组织免疫荧光双染色结果显示,PM_2.5暴露组子宫内CD45白细胞和CD11b巨噬细胞均明显增加(P<0.05)。Elisa法检测子宫组织和血清中白介素1β(IL-1β)和转化生长因子-β1(TGF-β1),暴露组子宫组织和血清中IL-1β和TGF-β1含量明显升高(P<0.05)。Western印迹法检测结果显示,PM_2.5上调核苷酸结合低聚体结构域样受体3 (NLRP3)、凋亡相关斑点样蛋白质(ASC)、pro-IL-1β、pro-Caspase-1和半胱氨酸天冬氨酸蛋白酶-1(Caspase-1)的蛋白质表达量(P<0.05)。与高剂量组相比,NLRP3抑制剂MCC950能明显降低NLRP3/Caspase-1通路中关键蛋白质表达水平(P<0.05)。综上,PM_2.5通过激活NLRP3/ Caspase-1信号,诱导大鼠子宫炎症反应,为PM_2.5生殖毒性预防和治疗提供理论基础。     

Functional involvement of calcium in the homologous up-regulation of the 1,25-dihydroxyvitamin D3 receptor in osteoblast-like cells

In several cell types 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) causes up-regulation of its receptor. The present study demonstrates that in the osteoblast-like cell line UMR 106 this up-regulation is inhibited by two different calcium channel blockers (nitrendipine, verapamil). Also with chelating extracellular calcium (EGTA) and by inhibition of calcium release from intracellular stores (TMB-8) comparable results were obtained. These findings indicate that calcium is functionally involved in this cellular response to the steroid hormone 1,25(OH)₂D₃. Moreover, data obtained with EGTA show that the 1,25(OH)₂D₃ receptor level is closely regulated by the extracellular calcium concentration.     

Characterization of [3H]CP-96,501 as a Selective Radioligand for the Serotonin 5-HT1B Receptor: Binding Studies in Rat Brain Membranes

Abstract: 3-(1,2,5,6-Tetrahydro-4-pyridyl)-5- n -propoxyindole (CP-96,501) was found to be a more selective ligand at the serotonin 5-HT_1B receptor than the commonly used 5-HT_1B agonist, 3-(1,2,5,6-tetrahydro-4-pyridyl)-5-methoxyindole (RU 24969). In rat brain membranes, the tritiated derivative, [³H]CP-96,501, was found to bind with a high affinity ( K _D, 0.21 n M ) to a single binding site ( n _H, 1.0). The receptor density of this site ( B _max, 72 fmol/mg of protein) matched that of the 5-HT_1B receptor determined with [³H]5-HT. Competition curves of 16 serotonergic compounds in [³H]CP-96,501 binding also indicated a single binding site. The rank order of their binding affinities with this new radioligand showed a high degree of correlation with their affinities at the 5-HT_1B receptor determined with [³H]5-HT or [¹²⁵I]iodocyanopindolol. Serotonergic compounds displayed competitive inhibition of [³H]CP-96,501 binding. In the presence of 5'-guanylylimidodiphosphate [Gpp(NH)p], [³H]CP-96,501 binding was reduced, while the potency of CP-96,501 to displace [¹²⁵I]iodocyanopindolol binding was also decreased. These findings are consistent with the agonist nature of CP-96,501. The results of this study suggest that [³H]CP-96,501 is a useful agonist radioligand for the 5-HT_1B receptor.     

Photosynthetic carbon metabolism in Panicum milioides, a C3-C4 intermediate species: Evidence for a limited C4 dicarboxylic acid pathway of photosynthesis

Panicum milioides, a naturally occurring species with C₄-like Kranz leaf anatomy, is intermediate between C₃ and C₄ plants with respect to photorespiration and the associated oxygen inhibition of photosynthesis. This paper presents direct evidence for a limited degree of C₄ photosynthesis in this C₃-C₄ intermediate species based on:

1. (a) the appearance of 24% of the total ¹⁴C fixed following 4 s photosynthesis in ¹⁴CO₂-air by excised leaves in malate and aspartate and the complete transfer of label from the C₄ acids to Calvin cycle intermediates within a 15 s chase in ¹²CO₂-air;

2. (b) pyruvate- or alanine-enhanced light-dependent CO₂ fixation and pyruvate stimulation of oxaloacetate- or 3-phosphoglycerate-dependent O₂ evolution by illuminated mesophyll protoplasts, but not bundle sheath strands; and

3. (c) NAD-malic enzyme-dependent decarboxylation of C₄ acids at the C-4 carboxyl position, C₄ acid-dependent O₂ evolution, and ¹⁴CO₂ donation from [4-¹⁴C]C₄ acids to Calvin cycle intermediates during photosynthesis by bundle sheath strands, but not mesophyll protoplasts.

However, P. milioides differs from C₄ plants in that the activity of the C₄ cycle enzymes is only 15 to 30% of a C₄ Panicum species and the Calvin cycle and phosphoenolpyruvate carboxylase are present in both cell types. From these and related studies (Rathnam, C.K.M. and Chollet, R. (1979) Arch. Biochem. Biophys. 193, 346–354; (1978) Biochem. Biophys. Res. Commun. 85, 801–808) we conclude that reduced photorespiration in P. milioides is due to a limited degree of NAD-malic enzyme-type C₄ photosynthesis permitting an increase in pCO₂ at the site of bundle sheath, but not mesophyll, ribulosebisphosphate carboxylase-oxygenase.     

Activation of D1 and D2 Dopamine Receptors Inhibits Protein Kinase C Activity in Striatal Synaptoneurosomes

Abstract: The effects of D₁ and D₂ dopamine ligands on protein kinase C (PKC) activity were examined in synaptoneurosomes. Incubation with D₁ agonists (SKF 38393, fenodopam), in the presence of calcium, decreased the soluble and increased the particulate PKC activity. These effects were reversed by SCH 23390, which by itself had the opposite effect of increasing the soluble and decreasing the particulate PKC activity. In contrast, incubation with the D₂ agonists [LY 171555, (+)-3-(3-hydroxyphenyl)- N - n -propylpiperidine, RU 24213] increased the soluble and decreased the particulate PKC activity. These effects were reversed by sulpiride. (−)-3-(3-Hydroxyphenyl)- N - n -propylpiperidine had a D₂ antagonist profile. Apomorphine showed a biphasic dose-response change; i.e., it decreased particulate PKC activity at the D₂ receptor at low concentrations (0.1 µ M ) and increased it at the D₁ receptor at higher concentrations (10 µ M ). Pretreatment with tetrodotoxin or omission of calcium in the incubation medium did not alter the responses of the D₂ agonists, but it reversed the changes in PKC activity induced by the D₁ agonists and converted the biphasic response of apomorphine to a monophasic inhibition. These results indicate that (1) D₁ and D₂ dopamine receptors are negatively coupled to PKC and (2) the increase in particulate PKC activity seen with the D₁ drugs in the presence of calcium is mediated indirectly via a transneuronal effect.     

Kinetics, Pharmacology, and Autoradiographic Distribution of l-[3H]Nitroarginine Binding Sites in Rat Cerebellum

Abstract: The kinetics and pharmacology of N ^G-nitro- l -[2,3,4,5-³H]arginine ( l -[³H]NOARG) binding to rat cerebellum were investigated using in vitro radioligand binding. Specific l -[³H]NOARG binding in cerebellum was of nanomolar affinity, reversible, saturable, and best fit to a single-site model. Specific binding was Ca²⁺ dependent and sensitive to pH (with an optimum of 5.5–7.0). Added calmodulin (1.5–40 µg/ml) had no influence on specific l -[³H]NOARG binding. However, the calmodulin antagonists W-5, W-13, and calmidazolium inhibited l -[³H]NOARG binding with IC₅₀ values in the micromolar range, and calmodulin (10 µg/ml) competitively reversed this inhibition. Nitric oxide synthase (NOS) inhibitors ( N ^G-nitro- l -arginine methyl ester and N ^G-monomethyl- l -arginine acetate) and l -arginine displaced l -[³H]NOARG binding with IC₅₀ values in the nanomolar range, whereas d -arginine and basic amino acids ( l -lysine and l -histidine) displaced l -[³H]NOARG binding with IC₅₀ values in the millimolar range. A comparison of the NOS functional assay with l -[³H]NOARG binding in rat cerebellum showed similar profiles of Ca²⁺ dependency and inhibitory kinetics. Quantitative autoradiographic distribution of l -[³H]NOARG binding sites was significantly higher in the molecular layer than in the granular layer of cerebellum. These studies confirm the potential use of l -[³H]NOARG binding to study the regional distribution and functional properties of NOS.     

Effect of calmidazolium and phorbol ester on catecholamine secretion from adrenal chromaffin cells

Carbamylcholine-stimulated catecholamine release from adrenal chromaffin cells was completely inhibited by pretreatment of the cells for 10 min with 1 μM calmidazolium. Catecholamine release due to 55 mM K⁺ and ionophore A23187 was also inhibited by calmidazolium but less effectively than release due to carbamylcholine. Inhibition of release appeared to be due to an effect of calmidazolium on a step distal to Ca²⁺ entry, since the carbamylcholine-stimulated rise in the concentration of intracellular free calcium, monitored using quin-2, was unaffected by calmidazolium. The possibility was considered that calmidazolium inhibited secretion through an effect on protein kinase C rather than calmodulin. However, the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), had no demonstrable effect on catecholamine release, arguing against a significant role for protein kinase C in secretion from adrenal chromaffin cells. These results give further support to the notion that calmodulin plays a role in the secretory process in chromaffin cells.