Interferon inhibition of thymidine incorporation into DNA through effects on thymidine transport and uptake

Replenishment of medium after 72 hr of growth of HeLa-S3 cells in dense suspension cultures increased [³H]-thymidine uptake into cells and incorporation into DNA, with the levels reaching a peak ～ 12 hr following medium change; β interferon inhibits the enhanced uptake of [³H]-thymidine and labeling of DNA in a dose-dependent manner. Some reduction in these processes is observed at a concentration as low as 1 u/ml, and ～ 75% inhibition at 640 u/ml. Kinetic analysis has revealed that the rate of labeling of the acid-soluble pool with [³H]-thymidine, measured either at 22°C, or 37°C, is reduced in interferon-treated (640 u/ml, 24 hr) HeLa-S3 cells. At 22°C, the initial rate of thymidine transport at a high (500 μM) thymidine concentration, determined within the first 30 sec of [³H]-thymidine addition was depressed by 44% in interferon-treated HeLa cells. At 37°C, labeled precursors accumulate in acid-soluble material for ～ 8 min after the addition of [³H]-thymidine, after which an apparent equilibrium level is attained. At this temperature, the rate of thymidine uptake and the apparent equilibrium level attained were depressed by 70% in interferon-treated HeLa cells. The reduced incorporation of [³H]-thymidine into DNA in interferon-treated HeLa-S3 cells can be largely explained by interferon inhibition of thymidine transport and phosphorylation.     

Evidence for a 5' leads to 3' direction of hydrolysis by a 5' mononucleotide-producing exoribonuclease from Saccharomyces cerevisiae.

[³H]rRNA labeled at the 5′ terminus with ³²P and [³H]rRNA labeled at the 3′ end with [¹⁴C] (pA)_n have been degraded at 0° with a highly purified exoribonuclease from $\text{Saccharomyces cerevisiae}$. The results show that with the [³²P, ³H] substrate, the ³²P label is rendered acid-soluble at a much faster rate than the ³H label. Both acid-soluble labels are found in 5′ mononucleotide. With the [¹⁴C, ³H]rRNA, the ³H label is hydrolyzed at a faster rate than the ¹⁴C label. The exoribonuclease hydrolyzes in the 5′ → 3′ direction.     

Preparation, properties, and uses of two fluorogenic substrates for the detection of 5'-(venom) and 3'-(spleen) nucleotide phosphodiesterases

A new method for ³H-labeling of native collagen and a specific microassay for collagenase activity are presented. Acid-soluble type I collagen derived from rat tail tendons was reacted with pyridoxal phosphate and then reduced with NaB³H₄ to yield [³H]collagen with a specific activity of more than 10 μCi/mg. With respect to rate of hydrolysis, trypsin susceptibility, and gelling properties this collagen compares favorably with biosynthetically labeled preparations. It was shown that chemical labeling procedures such as this, or N-acetylation with acetic anhydride, do not adversely affect properties of collagen which are important for its use as substrate in specific assays. The microassay employs 50-μl [³H]collagen gels (1 mg/ml) dispensed in microtest plates. At 36°C this assay combines rapid rate of hydrolysis with low trypsin susceptibility. As little as 1 ng of clostridial collagenase activity can be measured reproducibly. The high specific activity of the [³H]collagen allowed us to explore microassay conditions employing minute quantities of substrate in solution. These studies indicated that native type I collagen whether labeled or not, is cleaved in the helical region by trypsin at subdenaturation temperatures. It was concluded that, in order to remain specific, collagenase assays with collagen in solution as with collagen in fibrils must be performed at 10–12°C below the denaturation temperature, i.e., at 35–37°C with collagen gels and 27–29°C with collagen in solution.     

Migratory patterns of B lymphocytes. IV. Effect of anti-Ig on follicular localization of radioactively labeled murine spleen and bone marrow cells.

A study was made of the localization of nylon-wool-adherent (AD) and nonadherent (NA) murine spleen cells in lymphoid tissue of irradiated syngeneic recipients. Cells were labeled in vitro with [³H]uridine or ⁵¹Cr and injected intravenously. Localization in recipient tissues was expressed as percent of injected radioactivity. NA and AD [³H]uridine labeled cells gave spleen to lymph node (S:LN) ratios of 1.0 and 2.7, respectively. After treatment of AD cells with rabbit anti-mouse Fab + C at 37 °C, localization in S decreased markedly.NA cells primarily localized in LN paracortex and splenic periarteriolar sheaths. Untreated and NRS-treated AD cells localized in lymphoid follicles, whereas anti-Fab-treated AD cells did not. When ⁵¹Cr-labeled AD cells were treated with anti-Fab at 4 °C without C, there was a transient decrease in splenic localization at 24 hr followed by a recovery to the normal pattern at 48 hr after transfer. [³H]uridine-labeled bone marrow (BM) cells showed less localization in lymphoid tissue than did S cells. Some BM cells were seen in LN follicles, particularly at 48 hr after transfer, but this localization was not affected by prior treatment with anti-Fab + C. The possible role of surface Ig in the determination of follicular localization of B lymphocytes is discussed.     

Binding of [3H]batrachotoxinin A-20-α-benzoate and [3h]saxitoxin to receptor sites associated with sodium channels in trout brain synaptoneurosomes

1. [³H]Batrachotoxinin A-20-α-benzoate ([³H]BTX-b) and [³H]saxitoxin ([³H]STX), radioligands that bind to distinct sites on the voltage-sensitive sodium channel, were bound specifically to saturable sites in rainbow trout (Oncorhynchus mykiss) brain synaptoneurosomes.2. Specific [³H]BTX-B binding was temperature dependent with highest levels of specific [³H]BTX-B binding observed at 7°C. Specific binding was inversely correlated with assay temperature at temperatures above 7°C.3. Saturating concentrations of scorpion (Leiurus quinquestriatus) venom (ScV) stimulated specific [³H]BTX-B binding at 27°C, but not at 7°C. The dihydropyrazole insecticide RH 3421 inhibited specific [³H]BTX-B binding at 7°C but had no effect on specific binding at 27°C. The sodium channel activators veratridine and aconitine and the local anesthetic dibucaine inhibited specific [³H]BTX-B binding at both 7°C and 27°C.4. Displacement experiments in the presence of ScV at 27°C gave an equilibrium dissociation constant (K_d) for [³H]BTX-B of 710 nM and a maximal binding capacity (B_max) of 11.3 pmol/mg protein. Kinetic experiments established the rates of association (1.17 × 10⁵min⁻¹ nM⁻¹) and dissociation (0.0514min⁻¹) of the ligand-receptor complex.5. The binding of [³H]STX reached apparent saturation at 7.5 nM. Scatchard analysis of the saturation data indicated a K_d of 3.8nM and a B_max of 1.9 pmol/mg protein.6. These studies provide evidence for high affinity, saturable binding sites for [³H]BTX-B and [³H]STX in trout brain preparations. Whereas certain neurotoxins modified the specific binding of [³H]BTX-B in trout brain synaptoneurosomes in a predictable fashion, other compounds known to affect specific [³H]BTX-B binding in mammalian brain preparations had no effect on specific [³H]BTX-B binding in the trout.     

Thyroliberin is rapidly transferred to the nucleus of GH3 pituitary cells at both 4°C and 37°C

The time-course of thyroliberin transfer to the nucleus of GH3/B6 rat pituitary prolactin cells was studied by both autoradiography and cell fractionation of intact cells exposed to [³H]thyroliberin at 4°C or 37°C. It was previously shown that thyroliberin is not degraded in these conditions. It is found by autoradiography that [³H]-thyroliberin is transferred to the nucleus of GH3/B6 cells within 5 min at least at both 37° C and 4°C. Consistent results are obtained by fractionation of cells exposed to [³H]thyroliberin at 37°C. However after binding at 4°C 50% of the cell radioactivity is extractible by glutaraldehyde and after fractionation the isolated nuclei retain only 1–1.5% of the cell radioactivity. This suggests the existence of both tightly bound and loosely bound internalized thyroliberin molecules.     

Modulation of GABA binding sites by CNS depressants and CNS convulsants

[³H]Muscimol binding at 23°C and muscimol stimulated [³H]flunitrazepam binding at 37°C to membranes of rat cerebral cortex have been investigated. In washed membrane preparations, 2 apparent populations of [³H]muscimol binding sites can be observed. At 23°C [³H]muscimol binding is more sensitive to inhibition by NaCl and by other salts than at 0°C. The CNS depressants etazolate and pentobarbital reversibly enhance [³H]muscimol binding and they increase the affinity of muscimol as a stimulator of [³H]flunitrazepam binding. Conversely the CNS convulsants picrotoxin, picrotoxinin and isopropylbicyclophosphate (IPTBO) reversibly interfere with [³H]muscimol binding when NaCl is present and these drugs antagonize the effects of etazolate. In the presence of NaCl, picrotoxin, picrotoxinin and IPTBO also decrease the apparent affinity of muscimol or GABA as stimulator of [³H]flunitrazepam binding. Binding of [³H]muscimol to GABA recognition sites of rat cerebral cortex is enhanced by Ag⁺, Hg⁺ and Cu²⁺ in μM concentrations, Ag⁺ being most potent. The effects of 100 μM AgNO₃ persist after repeated washing of the membranes. When membranes are pretreated with AgNO₃ only one apparent population of [³H]muscimol binding sites with high affinity (K_d: 6–8 nM) is found. In AgNO₃ pretreated membranes, the affinity of muscimol as stimulator of [³H]flunitrazepam binding is increased 18 times (EC₅₀ 14 nM) when compared to control membranes, (EC₅₀ 253 nM). In AgNO₃ pretreated membranes, etazolate, pentobarbital and IPTBO fail to perturb either [³H]muscimol binding or baseline and muscimol stimulated [³H]flunitrazepam binding. The results demonstrate that the apparent sensitivity of GABA binding sites of the GABA-benzodiazepine-picrotoxin receptor complex can be increased by etazolate and pentobarbital and decreased by picrotoxin and IPTBO. These drugs have in common that they interfere with [³H]dihydropicrotoxinin binding.     

Dopamine Uptake by Rat Striatal Synaptosomes: Time-and Temperature-Dependent Decay and Protection by Dithiothreitol and Dopamine

The uptake of [³H]dopamine (DA) into rat striatal synaptosomes in the presence of a monoamine oxidase inhibitor was studied using a filtration technique. After a 10-min preincubation period, a fast initial uptake of [³H] DA was seen. Uptake reached a maximum after 4 min of incubation. If incubation was continued for more than 7 min, a gradual decrease in synaptosomal [³H]DA levels was found. Uptake was dependent on preincubation time; initial uptake velocity and maximal uptake decreased irreversibly with increasing preincubation periods. Moreover, the capacity of the synaptosomes to retain the [³H]DA during longer incubation times was progressively affected. The decrease in initial uptake activity was due to a decrease in the V_max of the transport system. Dithiothreitol (2.8 mM) protected synaptosomal uptake activity against deterioration at 37°C. Also, DA itself (10^-7M) stabilized the uptake mechanism if added to the suspension before preincubation was started. Since [³H]DA uptake observed after loading the synaptosomes with labeled DA was similar to the uptake seen if the synaptosomes were not previously loaded with DA, it was concluded that under these conditions synaptosomal DA is completely exchangeable with exogenous substrate. Prolonged storage of the synaptosomes at 0°C also resulted in a time-dependent decrease in uptake activity (t_1/2= 116 min). The addition of unlabeled DA or dithiothreitol to the suspension did not affect instability at 0°C.     

Benzodiazepine receptors: temperature dependence of [3H]flunitrazepam binding.

The characteristics of [³H]flunitrazepam binding to brain specific benzodiazepine receptors were determined at varying temperatures. The rates at which [³H]flunitrazepam associated with and dissociated from benzodiazepine receptors increased with increasing temperatures. The dissociation constant (K_D) also increased with increases in temperature. The (K_D) determined by Scatchard analyses of saturation isotherms showed a similar change with changes in temperature. The maximal binding capacity (Bmax) did not change with changes in temperature. The inhibitory constants of several benzodiazepines to inhibit [³H]flunitrazepam binding to brain were also higher at 37°C than at 0°C, suggesting that the binding affinity of all benzodiazepines to brain benzodiazepine receptors is lower at 37°C than at 0°C. Van't Hoff analysis of [³H]flunitrazepam binding to brain at different temperatures reveals two linear components to this relationship.     

Secreted proteins from rat Sertoli cells.

Sertoli cell cultures were prepared from the testes of 20-day-old rats. The proteins which were secreted by the cells into the culture medium were labeled with [³H]leucine or l-[³H]fucose. The proteins were concentrated by ultrafiltration and analysed by polyacrylamide slab gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). Autofluorography of the gels at ?70 °C showed that the rat Sertoli cells synthesized and secreted at least 7 major polypeptides. The polypeptides had molecular weights ranging from 16 000 to 140 000 D. Proteins which were secreted from cultures of testicular fibroblasts and myoid cells had electrophoretic properties on SDS-PAGE which were different from Sertoli cell secreted proteins. Addition of FSH and testosterone to the Sertoli cell cultures increased the total synthesis and secretion of [³H]leucine-labeled proteins. No qualitative changes in the proteins as a result of hormone application could be detected. However, the synthesis of a polypeptide of molecular weight 48 000 was increased relative to the other secreted peptides if the cells were maintained in FSH and testosterone. The Sertoli cell secreted proteins were shown to be glycoproteins which can bind to ConA-Sepharose and can be labeled with [³H]fucose. Tunicamycin, a specific inhibitor of N-glycosylation, inhibited the secretion of [³H]proteins by 50% but had little effect on the intracellular protein synthesis.     

Characterization of large mammalian DNA species sedimented in a reorienting zonal rotor

The characteristics of a Beckman-designed slow acceleration unit for the reorientation of alkaline sucrose gradients in a Ti-15 zonal rotor are described. The large DNA species (> 250S) obtained from cultured rat brain tumor cells with this system sediment linearly with time, have virtually no [³H]leucinelabeled or covalently bonded [³H]choline-labeled material sedimenting with them, sediment independently of smaller single-stranded DNA molecules (? 165S) and are 60–80% degraded by the single-strand-specific S1 nuclease. Therefore, it is postulated that these species are collapsed, partially denatured DNA molecules or a collapsed form of single-stranded DNA. When cells were labeled with [¹⁴C]TdR, then frozen and stored at ? 79°C, this system could detect radiation-induced DNA damage from decay of the incorporated label at accumulated doses as small as 18–126 rads.     

DNA crosslinks,single-strand breaks and effects on bacteriophage T4 survival from tritium decay of [2-3H]adenine, [8-3H]adenine and [8-3H]guanine

Escherichia coli and bacteriophage T4 DNA containing [2-³H]adenine accumulated crosslinks between the complementary strands. For T4 DNA stored in frozen solution there were 0.41 to 0.54 crosslinks formed per tritium decay. The crosslinks were demonstrated both by an increased DNA sedimentation rate in alkaline sucrose gradients and by an increasing amount of DNA that renatured quickly after denaturation by heat or alkali. Single-strand breaks were also formed with an efficiency of 0.08 to 0.50 breaks per tritium decay. DNA containing both [8-³H]adenine and [8-³H]guanine showed no crosslinking but did undergo single-strand breaks at a rate of 0.08 per tritium decay. T4 bacteriophage containing [2-³H]adenine lost plaque-forming ability when stored at 4 °C, with 0.34 lethal hits per tritium decay, whereas the same phage labeled with a mixture of [8-³H]adenine and [8-³H]guanine sustained only 0.12 lethal hits per tritium decay. The loss of plaque-forming ability in the latter case is probably due to a radiation effect from the emitted beta particle; the high lethal efficiency for tritium decay at 2-adenine is probably caused either by crosslinks between complementary strands or from some undetected lesion produced in the DNA.     

Developmental regulation of phospholipid methylation in rat brain synaptosomes

Phospholipid methylation was studied in cortical synaptosomes prepared from 7 and 14 day and adult rat brain. Using varying concentrations of [³H] S-adenosylmethionine, Km and Vmax values were determined for the formation of [³H] phosphatidylmonomethylethanolamine (PME), [³H] phosphatidyl-dimethylethanolamine and [³H] phosphatidylcholine (PC). At 25°C, the Km values for the formation of all three products, significantly decreased with development. Increasing the temperature to 37°C increased the Km values in the 14 day and adult but not the 7 day preparation. The Vmax values at 25°C were highest at 7 and 14 days, depending on the product and then decreased in the adult. At 37°C, the Vmax values were highest in the 14 day preparation. The overall results are discussed in terms of the developmentally regulated decrease in both synaptic membrane PC and membrane fluidity.     

Two subpopulations of α1-adrenergic receptors with high and low affinity for agonists: Short-term exposure to agonists reduced the high-affinity sites

Short-term receptor regulation by agonists is a well-known phenomenon for a number of receptors, including β-adrenergic receptors, and has been associated with receptor changes revealed by radioligand binding. In the present study, we investigated the rapid changes in α₁-adrenergic receptors induced by agonists. α₁-receptors were studied on DDT₁ MF-2 smooth muscle cells (DDT₁-MF-2 cells) by specific [³H]prazosin binding. In competition binding on membranes and on intact cells at 4°C or at 37°C in 1-min assays, agonists competed for a single class of sites with relatively high affinity. By contrast, in equilibrium binding at 37°C on intact cells agonists competed with two receptor forms (high- and low-affinity). We quantified the receptors in the high-affinity form by measuring the [³H]prazosin binding inhibited by 20 μM norepinephrine (this concentration selectively saturated the high-affinity sites). The low-affinity sites were measured by subtracting the binding of [³H]prazosin to the high-affinity sites from the total specific binding. High-affinity receptors were 85% of the total sites in binding experiments at 4°C, but only 30% at 37°C. On DDT₁-MF-2 cells preequilibrated with [³H]prazosin at 4°C, and then shifted to 37°C for a few minutes, norepinephrine selectively reduced the high-affinity sites by 30%. We suggest that at 4°C it is the native form of α₁-receptors that is measured, with most of the sites in the high-affinity form, while during incubation at 37°C the norepinephrine present in the binding assay converts most of the receptors to an apparent low-affinity form, so that they are no longer recognized by 20 μM norepinephrine. The nature of this low-affinity form was further investigated. On DDT₁-MF-2 cells preincubated with the agonist and then extensively washed at 4°C (to maintain the receptor changes induced by the agonist) the number of receptors recognized by [³H]prazosin at 4°C was reduced by 38%. After fragmentation of the cells, the number of receptors measured at 4°C was the same in control and norepinephrine-treated cells, suggesting that the disruption of cellular integrity might expose the receptors which are probably sequestered after agonist treatment. In conclusion, the appearance of the low affinity for agonists at 37°C may be due to the agonist-induced sequestration of α₁-adrenergic receptors, resulting in a limited accessibility to hydrophilic ligands.     

Binding of [3H] γ-Aminobutyric Acid and [3H]Muscimol in Purified Rat Brain Synaptic Plasma Membranes and the Effects of Bicuculline

Abstract: The binding of [³H] γ-aminobutyric acid ([³H]GABA) and [³H]muscimol has been studied in purified synaptic plasma membrane (SPM) preparations from rat brain. Scatchard analysis of specific binding (defined as that displaced by 100 μMγ-aminobutyrate) indicated that the binding of both radiolabelled ligands was best described by a two component Langmuir adsorption isotherm. The apparent K_D and B_max values for [³H]GABA at 4°C were K_D1, 20 nM; K_D2,165 nM; B_max1, 0.48 pmol;B_max2, 6.0 pmol. mg^?1; for [³H]muscimol at 4°C they were: K_D1, 1.75 nM; K_D2, 17.5 nM; B_maxl, 0.84 pmol. mg^?1; B_max2, 4.8 pmol.mg^?1; and for [³H]muscimol at 37°C they were: K_D1, 7.0 nM; K_m, 60 nM; B_max], 0.5 pmol-mg^?1; B_max2, 7.2 pmol-mg¹. Under the experimental conditions used, the similar B_milx values for [³H]GABA and [³H]muscimol binding to the SPM preparations suggests that the high- and low-affinity components for the two radiolabeled ligands are identical. The effects of the GAB A antagonist bicuculline on the binding of [³H]muscimol at 4^CC and 37°C were studied. At 4°C, antagonism of muscimol binding appeared to be competitive at the high-affinity site but noncompetitive at the low-affinity site. At 37°C, antagonism was again competitive at the high-affinity site but was of a mixed competitive/noncompetitive nature at the low-affinity site. Assuming that binding to the high-affinity site is associated with the pharmacological actions of bicuculline, the apparent K_D values obtained suggest a pA₂ value of 5.3 against [³H]muscimol at 4°C and 37°C. This figure is in good agreement with several estimates of the potency of bicuculline based on pharmacological measurements. Results from displacement studies using [³H]GABA and [³H]muscimol suggest that [³H]GABA might be a more satisfactory ligand than [³H]muscimol in GABA radioreceptor assays.     

Regulation of hormone biosynthesis in cultured islet cells from anglerfish

Summary The effects of glucose and arginine on islet hormone biosynthesis were investigated using primary cell cultures prepared from islets of the anglerfish (Lophius americanus). After dispersion under sterile conditions, islet cells were maintained at 23° C in medium containing RPMI 1640 with Hanks' buffer, pH 7.5, modified by the adjustment of glucose (to 0.56 or 5.6 mM) and arginine (to 0.1, 1.15, or 10 mM) with the addition of 10% fetal bovine serum (dialyzed, heat inactivated) and penicillin/streptomycin. After 48 h, media were replaced by incorporation media containing [¹⁴C]isoleucine and [³H]tryptophan and incubated for an additional 8 h under otherwise identical conditions. Culture samples (cells plus media) were extracted, desalted, and gel filtered to identify and quantitate [¹⁴C]insulin, [³H]glucagon(s) plus [³H]somatostatin-28, and [³H]somatostatin-14 were In some experiments, [¹⁴C]insulin, [³H]glucagon(s), [³H]somatostatin-28, and [³H]somatostatin-14 were separated by high performance liquid chromatography. Raising the medium glucose from 0.56 (control) to 5.6 mM resulted in an augmentation in incorporation of [¹⁴C]isoleucine into insulin and an augmentation of [³H]tryptophan into glucagon(s) and somatostatin-14, but no change in incorporation of [³H]tryptophan into somatostatin-28. Raising the concentration of arginine from 0.1 to 1.15 or 10 mM resulted in a dose-dependent inhibition of labeled amino acid incorporation into all hormones except somatostatin-28. The results demonstrate the usefulness of the culture system for studying the modulation of hormone biosynthesis in anglerfish islet cells. This work was supported by Grants AM 16921 and AM 26378 from the National Institutes of Health, Bethesda, MD.     

THE UPTAKE OF BIOGENIC MONOAMINES BY THE ISOLATED AURICLE OF THE SNAIL (HELIX POMATIA)

—The uptake of [³H]5HT, [³H]dopamine, [³H]noradrenaline and [³H]octopamine into the auricle of Helix pomatia was studied. When tissues were incubated at 25°C in media containing radioactive amines, tissue:medium ratios of about 49:1, 14:1 and 5:1 for 5-HT, dopamine, noradrenaline, and octopamine respectively were obtained after a 20–30 min incubation time. Tissues incubated at 25°C in media containing radioactive amines for 20–30 mins showed that almost all (96%) the radioactivity was present as unchanged [³H]5-HT, [³H]dopamine, [³H]octopamine or [³H]noradrenaline. The high tissue:medium ratios for 5-HT and dopamine, but not for noradrenaline and octopamine, showed saturation kinetics which were dependent upon temperature and sodium ions. From the Lineweaver–Burk plots, two uptake mechanisms for 5-HT at 25°C were resolved; the high affinity uptake process having a K_m1 value of 6.0 ± 10^?8m and a V_m1 value of 0.115 nmol/g/min while the lower affinity process had a K_m2 value of 1.04 ± 10^?6m and a V_m2 value of 0.66nmol/g/min. At 0°C a single uptake mechanism for 5-HT occurred which gave a K_m value of 5.02 ± 10^?8m and a V_m value of 0.0165 nmol/g/min. In the case of dopamine, the Lineweaver–Burk plot at 25°C showed a single uptake process with values for K_m and V_m of 1.55 ± 10^?7m and 0.086 nmol/g/min respectively. This process did not function at 0°C. The effect of various agents and ions upon the accumulation processes for all amines was also studied, and the data indicate that the same neurons probably accumulate more than one amine type. It is concluded that 5-HT and dopamine uptake in the auricle is a mechanism for inactivating these substances at 25°C and that an uptake mechanism for 5-HT also functions at 0°C. The results are discussed from the point of view of 5-HT's being the cardioexcitatory substance in the snail heart.     

Substance P-Induced Reduction in the Initial Accumulation of Cytosolic myo-[3H]Inositol in Rat Parotid Acinar Cells Mediated by the NK1 Tachykinin Receptor

Abstract: Stimulation of rat parotid acinar cells by the tachykinin neurokinin (NK) 1 receptor agonist substance P (SP) resulted in a significant reduction in the initial accumulation of cytosolic myo-[³H]inositol. This effect was rapid, because a reduction of ～15% could be seen already at 30 s, with the maximal effect (～45%) being observed at 15 min. The response to SP stimulation Was temperature dependent, because at 4°C no reduction was found, jln addition, at 4°C, cytosolic myo-[³H]inositol represented only 10% of the labeled inositol accumulated at 37°C. The SP-induced reduct on in cytosolic ravo[³H]inositol accumulation was concentration dependent; the EC₅₀ obtained for SP was 5.8 ± 2.5 nM. Spantide [N Arg¹, D-Trp⁷⁹, Leu]SP), a SP antagonist, used at a concentration oif 10⁵ A/, gave a competitive shift of the dose-response curve to SP. Various tachykinins and their analogs were evaluated for their ability to reduce cytosolic mvo-[³H]inositol. [L-Pro⁹]SP and SP methyl ester, two highly selective agonists of NK1 receptors, reduced the initial accumulation of myo-H]inositol with EQo values of 2.3 and 67.0 nM, respectively. Long SP C-terminal fragments were more potent than shorter ones. SP N-terminal fragments and SP free acid were -without effect. [Pro⁷]NKB, a selective NKB analog, had no effect. The rank order of potency of mammalian tachykinins was SP > NKA > NKB. These findings and the close correlation between EC50 values and IC₅₀ values obtained in binding studies implicate the NK 1 receptor. In addition, stimulation of muscarinic receptors by carbachol alscp resulted in a reduction in level of cytosolic mjw-[³H]inositol, with this effect being reversed by atropine. Moreover, atropine was unable tjo alter the SP-induced reduction in cytosolic myo-[³H]inositol accumulation. Other neurotransmitters, such as glutamic acid, serotonin, chplecystokinin, neurotensin, bradykinin, and neuropeptide Y, were without effect on initial cytosolic myo-[³H]inositol accumulation. In conclusion, NK1 and muscarinic receptors seem to regulate the membrane transport of inositol in acinar cells of the rat parotid gland. Measurement of the initial accumulation of cytosolic myo-[³H]inositol in this tissue could profitably be adopted as a very simple, rapid, [sensitive, and specific biochemical procedure for screening the activity of potential agonists and antagonists at NK1 receptors.     

The temperature-mediated metabolism of 1-acyl-lysophosphatidyl glycerol in cerulenin-treated cultures of Bacillus megaterium.

When [U-¹⁴C]palmitate was added to a culture of $\text{B. megaterium}$ that had been grown at 35°, transferred to 20° and treated with cerulenin, label was initially incorporated into lysophosphatidyl glycerol. The labeled lyso derivative, in turn, was converted to phosphatidyl glycerol, apparently by esterification of the 2-position with endogenous acyl groups. Labeled lysophosphatidyl glycerol synthesis at 20° was observed only when a culture was treated with cerulenin prior to the addition of [U-¹⁴C]palmitate. When [U-¹⁴C]palmitate was added before cerulenin, labeled lysophosphatidyl glycerol formation was not detected. When chloramphenicol was added with cerulenin at the time of culture transfer from 35° to 20°, the synthesis of lysophosphatidyl glycerol was unaffected but the rate of its esterification to phosphatidyl glycerol was significantly retarded. Transfer of such a culture back to 35° resulted in a marked acceleration in the rate of conversion of lysophosphatidyl glycerol to phosphatidyl glycerol.     

In vitro activation of rat cardiac glucocorticoid antagonist-versus agonist-receptor complexes

The synthetic antiglucocorticoid RU 38486 interacts with cardiac cytoplasmic glucocorticoid receptors and competes for in vitro binding with the potent agonist triamcinolone acetonide. In addition to binding to receptors with high affinity, RU 38486 also facilitates the in vitro conformational change in the receptor which is a consequence of the physiologically relevant activation step during which the receptor is converted from a non DNA- to a DNA-binding form. This ability of RU 38486 to promote receptor activation is reflected by both the appropriate shift in the elution profile of [³H]RU 38486-receptor complexes from DEAE-cellulose as well as by an increased binding of these complexes to DNA-cellulose. Although less effective than triamcinolone acetonide, RU 38486 promotes in vitro receptor activation under a variety of experimental conditions, including incubation of labeled cardiac cytosols at 25°C for 30 min or at 15°C for 30 min in the presence of 5 mM pyridoxal 5′-phosphate. Once thermally activated, the cardiac [³H]triamcinolone acetonide and [³H]RU 38486-receptor complexes bind to nonspecific DNA-cellulose with the same relative affinities, as evidenced by the fact that 50% of both activated complexes are eluted at approx. 215–250 mM NaCl. Thus, this pure antiglucocorticoid does promote, at least to some extent, many of the crucial in vitro events including high-affinity binding, activation, and DNA binding which have been shown to be required to elicit a physiological response in vivo.