Particulate Preparations Research Articles

Enzymatic Synthesis and Purification of Uridine Diphosphate [14C] Galacturonic Acid: A Substrate for Pectin Biosynthesis

Pectins are complex polysaccharides that contain 1,4-linked α-D-galactosyluronic acid residues found in the primary wall of all higher plant cells. The pectic polysaccharides play critical roles in cell wall structure and in plant growth and development. As a first step in studying pectin biosynthesis a method was developed to routinely generate and purify UDP-[U-14C]galacturonic acid (UDP-[14C]GalA), the nucleotide sugar substrate for homogalacturonan biosynthesis. UDP-[14C]GalA was enzymatically synthesized by 4-epimerization of commercially available UDP-[U-14C]glucuronic acid (UDP-[14C]GlcA) using a particulate preparation from radish roots. The resulting mixture of UDP-[14C]GalA and UDP-[14C]GlcA was separated by high-performance anion-exchange chromatography using a Dionex CarboPac PA1 anion-exchange column. The UDP-sugars were detected by their absorbance at 262 nm or by pulsed amperometric detection following postcolumn addition of NaOH. The yield of UDP-[14C]GalA obtained using this procedure was 16% of the starting UDP-[14C]GlcA. Establishment of a reliable method to synthesize and purify UDP-[14C]GalA will facilitate the identification and purification of the galacturonosyltransferase(s) involved in pectin biosynthesis.

造粒散剤の薬剤学的特性と調剤との関係 調剤性の評価方法の検討

Formulations were established to evaluate the dispensing characteristics of fine granules.The formulations were prepared mainly according to “the Standard Formula for Fine Granules” established by the Particulate Preparation and Designing Group of the Society of Powder Technology of Japan. Granulation was performed either by agitating granulation, fluidized-bed granulation or extrusion granulation. The dispensing characteristics were comprehensively assessed on the basis of “average weight”, “coefficient of variation”, “weight loss”, and “time for dividing”, as well as “operativeness” based on a sensory test by pharmacists.As the results, it was observed that the dispensing characteristics of fine granules might greatly depend on the granulation method, the amount of binders, the size of bulk materials and the shape of the fine granules. In was also observed that content uniformity of a fine granule, i. e., relationship between its particle size and content, was closely related to the granulation procedure employed.

A1 adenosine receptor inhibition of cyclic AMP formation and radioligand binding in the guinea-pig cerebral cortex.

1. A1 adenosine receptors were investigated by radioligand binding and functional studies in slices and particulate preparations from guinea-pig cerebral cortex. 2. Binding of the adenosine receptor antagonist radioligand, 8-cyclopentyl-[3H]-1,3-dipropylxanthine (DPCPX) to guinea-pig cerebral cortical membranes exhibited high density (1410 +/- 241 fmol mg-1 protein) and high affinity (Kd 3.8 +/- 0.3 nM). 3. [3H]-DPCPX binding to guinea-pig cerebral cortical membranes was displaced in a monophasic manner by adenosine receptor antagonists with the rank order of affinity (Ki values, nM): DPCPX (6) < xanthine amine congener (XAC, 153) < PD 115,199 (308). 4. Agonist displacement of [3H]-DPCPX binding was biphasic and exhibited the following rank order at the low affinity site (Ki values): 2-chloro-N6-cyclopentyl-adenosine (CCPA, 513 nM) = N6-R-phenylisopropyladenosine (R-PIA, 526 nM) = N6-cyclopentyladenosine (CPA, 532 nM) < 2-chloroadenosine (2CA, 3.2 microM) = 5'-N-ethylcarboxamidoadenosine (NECA, 4.6 microM) < N6-S-phenylisopropyladenosine (S-PIA, 19.9 microM). 5. In cerebral cortical slices, [3H]-DPCPX binding was displaced by antagonists and agonists in an apparently monophasic manner with the rank order of affinity (Ki values, nM): DPCPX (14) < XAC (45) < R-PIA (266) < PD 115,199 (666) < S-PIA (21000). 6. Cyclic AMP accumulation stimulated by 30 microM forskolin in guinea-pig cerebral cortical slices was inhibited by R-PIA, CCPA and CPA up to 1 microM in a concentration-dependent fashion with IC50 values of 14, 18, and 22 nM, respectively. All three analogues inhibited the forskolin response to a similar extent (82-93% inhibition). NECA, S-PTA and 2CA failed to inhibit the forskolin response, but rather enhanced the accumulation of cyclic AMP at concentrations of 100 nM or greater, presumably through activation of A2b adenosine receptors coupled to stimulation of cyclic AMP accumulation in guinea-pig cerebral cortical slices.7. The inhibition of forskolin-stimulated cyclic AMP accumulation by CPA was antagonized with the rank order of affinity (Ki values, nM): DPCPX (6)<XAC (52)<PD 115,199 (505).8 Xanthine-based antagonists inhibited the adenosine receptor augmentation of histamine-induced phosphoinositide turnover in guinea-pig cerebral cortical slices with the rank order of affinity (Ki, nM):DPCPX (12)=XAC (17)<PD 155,199 (640).9 In summary, we observe a good correlation between antagonist affinity at A1 receptors defined by radioligand binding, inhibition of cyclic AMP generation or augmentation of histamine-evoked phosphoinositide turnover in guinea-pig cerebral cortex.

Diflubenzuron affects gamma-thioGTP stimulated Ca 2+ transport in vitro in intracellular vesicles from the integument of the newly molted american cockroach, Periplanet americana L.

To study the mechanism of action of diflubenzuron (DFB) and other benzoylphenylureas, we have initially hypothesized that their action may be related to exocytosis: to test the hypothesis, we obtained an intracellular vesicle preparation from the homogenate of integument of newly molted American cockroachs ( Periplaneta americana L.) in 10 mM MES buffer containing 250 mM sucrose (isotonic) and 2.5 mM MgSO 4, at pH 6.6. By studying DFB's effect on various ion transporting activities, we demonstrated that calcium uptake in this intracellular particulate preparation was significantly inhibited by DFB at low concentrations (e.g., 10 −8 M). Such an inhibitory effect of DFB on Ca 2+ uptake was eliminated by the addition of ionophores or membrane disruptors, as well as the sonication of vesicle preparation. On the other hand, oligomycin, protein phosphorylation modulators, Na +, and Li + did not affect the calcium uptake. Among ionophores, agents disrupting H + gradients (e.g. FCCP and NEM) totally eliminated 45Ca uptaking activity by vesicles as well as the inhibitory effect of DFB. Among calcium ion modulators, calmodulin inhibitors such as calmidazolium and trifluoperazine decreased the Ca 2+-uptake, whereas membrane calcium channel blocker, verapamil, did not. ATP and γ-S-GTP stimulated Ca 2+ uptake. However, the former increased only the DFB insensitive portion and the latter largely the DFB sensitive part of Ca 2+. Together these data support the hypothesis that the action site of DFB in this preparation is the GTP-dependent Ca 2+ transport process which is coupled to vacuolar type intracellular vesicles in the integument cells.

Inositol trisphosphate metabolism in Saccharomyces cerevisiae: identification, purification and properties of inositol 1,4,5-trisphosphate 6-kinase.

Ins(1,4,5)P3 metabolism was examined in Saccharomyces cerevisiae extracts. S. cerevisiae contains readily detectable Ins(1,4,5)P3 kinase activity that is predominantly soluble, but phosphomonoesterase activity acting on Ins(1,4,5)P3 was not detected in either soluble or particulate preparations from this organism. We have purified the kinase activity approximately 685-fold in a rapid four-step process, and obtained a stable preparation. The enzyme has an apparent native molecular mass of approximately 40 kDa, and displays Michaelis-Menten kinetics with respect to its two substrates, ATP and Ins(1,4,5)P3. The Km for ATP was 2.1 mM, and that for Ins(1,4,5)P3 was 7.1 microM. The enzyme appeared to be the first step in the conversion of Ins(1,4,5)P3 into an InsP5, and the partially purified preparation contained another activity that converted the InsP4 product into an InsP5. The InsP4 product of the partially purified kinase was not metabolized by human erythrocyte ghosts and co-chromatographed with an Ins(3,4,5,6)P4 [L-Ins(1,4,5,6)P4] standard, identifying it as D-Ins(1,4,5,6)P4. The yeast enzyme is thus an Ins(1,4,5)P3 6-kinase. This activity may be an important step in the production of inositol polyphosphates such as InsP5 and InsP6 in S. cerevisiae.

Lack of glibenclamide or TEA affinity for opioid receptors: further evidence for in vivo modulation of antinociception at K + channels

Radioligand binding of the opioid receptor subtype selective ligands, [ 3H][ d-Ala 2, N-Me-Phe 4,Gly-ol 5]-enkephalin (DAMGO)_ (μ), [ 3H][ d-Pen 2, d-Pen 5]-enkephalin (DPDPE) (δ) and [ 3H]U-69,593 (κ), to rat brain particulate preparations was virtually unaffected by 1–100 μM glibenclamide (Glib) or tetraethylammonium bromide (TEA). These results argue against opioid receptor antagonism by Glib or TEA and support the hypothesis that antagonism of opioid-induced antinociception by Glib or TEA occurs at the level of K + (possibly ATP-sensitive K ATP) channels.

Characterization of protein kinase C in rat and human prostates

The properties of protein kinase C (PKC) activity have been studied in cytosolic and membrane fractions from rat and human prostate. Ion exchange chromatography indicated the existence of different PKC isoforms, PKC from rat ventral prostate behaved as a classical Ca(2+)- and phospholipid-dependent enzyme and was activated by 1,2-diacylglycerol as well as by high concentrations of arachidonic acid. PKC activity in the cytosolic fraction was higher and presented different cofactor requirements than that in the membrane fraction. PKC from human benign hyperplastic prostate was also phospholipid dependent, activated by tumor-promotong phorbol esters, and appeared to belong to the group of PKC isozymes which lack Ca2+ sensitivity. Human prostatic PKC activity appeared to be of similar nature in both membrane and cytosolic fractions but the specific activity was higher in the particulate preparation which could be related to the stage of endogenous activation of the enzyme. These results extend previous observations in rat ventral prostate and present evidences on the human counterpart. Forthcoming experiments are needed to establish the exact nature of PKC isozymes and their physiological and pathophysiological role in this gland.

Identification and characterization of endothelin receptor subtype B in rat retina.

The presence of immunoreactive (IR) endothelin (ET)-1 and ET-1 receptors in rat retina has been studied by radioimmunoassay and receptor assay, respectively. The specific binding of 125I-ET-1 to rat retinal particulate preparations was saturable. Apparent equilibrium conditions were established within 120-140 min. Scatchard analysis of binding data indicated a single class of high-affinity binding sites with a KD of 35 +/- 11 pM and a Bmax of 168 +/- 60 fmol/mg of protein. 125I-ET-1 binding to retinal particulate preparations was not inhibited by 1 microM concentrations of somatostatin, atrial natriuretic factor, brain natriuretic peptide, thyroid-stimulating hormone, growth hormone, or insulin. The three endothelin isoforms, ET-1, -2, and -3, had similar affinity for the receptor. Cross-linking of 125I-ET-1 to retinal particulate preparations with disuccinimidyl suberate resulted in the labeling of two bands with apparent molecular masses of 52 and 34 kDa. We have established a highly sensitive and specific radioimmunoassay for ET-1. The concentration of IR-ET-1 in rat retina was 35 +/- 10 fmol/g wet weight. The demonstration of specific high-affinity ETB receptors and the presence of IR-ET-1 suggest that the peptide may act as a neurotransmitter or neuromodulator in the retina.

Proton Transport and Chloride Cells in the Gill of Rainbow Trout (Oncorhynchus mykiss)

Particulate preparations from freshwater rainbow trout (Oncorhynchus mykiss) gill homogenates contain an active magnesium ion activated ATPase that transports protons into the vesicles. Oligomycin at a concentration of 20 μg∙mL−1had little effect on the proton transport, which was completely inhibited by N-ethylmaleimide. This inhibition was partly counteracted by dithiothreitol. Proton transport in freshwater trout gill submitochondrial particles was completely inhibited by oligomycin. When freshwater trout were kept for 40 min in water equilibrated with air containing 5% carbon dioxide, their gill lamellar epithelium contained many cells densely covered with erect microvilli. After changing to water bubbled with room air, the cells rapidly lost their microvilli. Control fish that were killed immediately after removal from the storage tank also had microvilli, but less erect and fewer in number. Labelled latex microspheres and dextran were used as markers for external medium uptake into gill epithelial cells. The fish were pretreated by bubbling the water by air containing 5% carbon dioxide, and the uptake took place when the water was bubbled with room air.

Localization of β-glucan synthetase in membranes ofParacoccidioides brasiliensis

To localize β-1,3-glucan synthetase in membranes of Paracoccidioides brasiliensis, particulate preparations were obtained from both phases of the fungus and separated on sucrose gradients. The yeast glucan synthetase distributed between the 20% and 30% sucrose bands whereas the mycelial enzyme concentrated in the 40% band. In all cases, vanadate-sensitive, azide-insensitive ATPase was found together with glucan synthetase, suggesting that the latter is located in the cytoplasmic membrane.

Mannosyl transfer in Cryptococcus neoformans.

A particulate enzyme preparation from Cryptococcus neoformans transferred the mannosyl residue from GDP-mannose to an acceptor consisting of a commercial preparation of methyl 3-O-alpha-mannopyranosyl-alpha-mannopyranoside (containing 10% 2-O-alpha-mannopyranosyl-alpha-mannopyranoside). The configuration of the new bond was alpha by its susceptibility to alpha-mannosidase; the amount of product was dependent on the concentration of enzyme, of GDP-mannose, and of acceptor. The optimal temperature and pH were 37 degrees C and 7.0, respectively. Manganous ion was required for activity and acetyl coenzyme A was stimulatory. Studies suggested that dolichyl phosphate intermediates were not involved in this mannose transfer. The fact that none of the several acapsular mutants tested were deficient in this mannosyltransferase suggested that this enzyme was not involved in synthesis of backbone mannan linkages in capsular polysaccharide. NMR analysis of the methylmannotriose product showed only alpha(1-->2) linkages between sugar moieties. This mannosyltransferase evidently extends alpha(1-->2) mannan by adding another alpha(1-->2)-linked mannosyl residue. Its activity is appropriate for a role in synthesis of "high mannose" oligosaccharide moieties of glycoproteins.

Protein measurement of particulate and solubilized ovine liver membranes.

Protein concentration measurements are critical in biochemical work with cellular membranes, including the determination of cell surface receptor concentration in human malignant tissues obtained at surgery or after biopsy. In this study we compared the results of protein concentration measurements in ovine liver cellular membranes using either particulate preparations or membranes solubilized with four different detergents. In all cases protein was determined by two different indirect methods (Lowry's Folin phenol method and Bradford's Coomassie Brilliant Blue dye binding method) and compared to the direct biuret method. Our results indicate that the direct biuret method gives the highest protein concentrations followed by the method of Lowry. Maximal concentrations (approaching those obtained by the direct biuret method) were obtained after membrane solubilization with Triton X-100 (3-5%). It is suggested that either the direct biuret method (whenever protein concentrations permit it) or the method of Lowry after solubilization of membranes with Triton X-100 (3-5%) should be used preferentially for the determination of membrane protein samples.

Partial purification and characterization of the sodium-ion-coupled 5-hydroxytryptamine transporter of rat cerebral cortex.

A procedure for the extensive purification of the Na(+)-coupled 5-hydroxytryptamine transporter of rat cerebral cortex has been developed. The 5-hydroxytryptamine transporter was solubilized with the non-ionic detergent digitonin, and the detergent extracts were subjected to sequential affinity chromatography on a citalopram-based agarose support and wheat-germ-agglutinin-Sepharose. 5-Hydroxytryptamine transporters in the affinity-purified preparation were identified by using the selective 5-hydroxytryptamine-uptake inhibitor [3H]paroxetine, and were shown to display a similar pharmacological profile to those present in particulate preparations. An overall transporter purification of around 2000-fold was achieved with a 9% recovery. SDS/PAGE of affinity-chromatographed material starting from detergent extracts incubated in the presence or absence of 1 mM-citalopram indicated that a polypeptide of M(r) 73,000 corresponded to the 5-hydroxytryptamine-transporter protein.

Identification and initial characterization of a putative neuromedin B-type receptor from rat urinary bladder membranes

Receptor binding site(s) on the rat urinary bladder membranes were characterized using a biologically active analog of bombesin, [Tyr 4, Leu 14]bombesin, and a 50 000 × g total particulate preparation. The binding was specific, reversible, saturable, time- and concentration-dependent. A dissociation curve showed that both bombesin and neuromedin B equally displaced the radioligand in the first 10 min after saturation. From the rate constant of association k +1 = 7.60 × 10 9 M −1 min −1, and the rate constant of dissociation k −1 = 0.050 min −1, the apparent equilibrium dissociation constant K d = 6.57 ± 1.09 pH was determined. A linear Scatchard plot of the specific binding of 125I-[Tyr 4, Leu 14]bombesin to the membranes revealed that the radioligand bound with high affinity, K d = 6.38 ± 0.86 pM, to a single class of sites (B max = 2.3 fmol/mg protein). The Hill coefficient of the same binding data was 1.05 ± 0.21, indicating that the radioligand was binding to a single population of noninteracting bindings sites. Both bombesin and neuromedin B displaced the radioligand dose dependently (IC 50 = 0.3 nM). Neurokinin A and neurokinin B were less potent (IC 50 = 20 and 110 nM, respectively). Substance P, or the specific bombesin receptor antagonists [D-Phe 6]bombesin-(6–13) methyl ester, [D-F 5Phe 6,D-Ala 11]bombesin-(6–11) methyl ester, [D-Phe 6]bombesin-(6–13) propylamide, [D-Phe 6,Leu 13psi(CH 2NH)Leu 14]bombesin or [D-Cpa 6,Phe 14(psi13–14)]bombesin-(6–14) had an IC 50 > 1 μM. The results presented suggest the presence of neuromedin B receptor sites on the rat urinary bladder membranes that can be occupied also by some other peptides, notably bombesin, neurokinin A and neurokinin B. The presence of a non-specific bombesin receptor subtype cannot be excluded.

Cyclic adenosine monophosphate accumulation and β-adrenergic binding in unweighted and denervated rat soleus muscle

Unweighting, but not denervation, of muscle reportedly “spares” insulin receptors, increasing insulin sensitivity. Unweighting also increases β-adrenergic responses of carbohydrate metabolism. These differential characteristics were studied further by comparing cyclic adenosine monophosphate (cAMP) accumulation and β-adrenergic binding in normal and 3-day unweighted or denervated soleus muscle. Submaximal amounts of isoproterenol, a β-agonist, increased cAMP accumulation in vitro and in vivo (by intramuscular [IM] injection) to a greater degree ( P < .05) in unweighted muscles. Forskolin or maximal isoproterenol had similar in vitro effects in all muscles, suggesting increased β-adrenergic sensitivity following unweighting. Increased sensitivity was confirmed by a greater receptor density (B max) for [ 125I]iodo-(−)-pindolol in particulate preparations of unweighted (420 · 10 −18 mol/mg muscle) than of control or denervated muscles (285 · 10 −18 mol/mg muscle). The three dissociation constant ( K d ) values were similar (20.3 to 25.8 pmol/L). Total binding capacity (11.4 fmol/muscle) did not change during 3 days of unweighting, but diminished by 30% with denervation. This result illustrates the “sparing” and loss of receptors, respectively, in these two atrophy models. In diabetic animals, IM injection of insulin diminished cAMP accumulation in the presence of theophylline in unweighted muscle (−66% ± 2%) more than in controls (−42% ± 6%, P < .001). These results show that insulin affects cAMP formation in muscle, and support a greater in vivo insulin response following unweighting atrophy. These various data support a role for lysosomal proteolysis in denervation, but not in unweighting, atrophy.

Growth factor stimulation of phospholipase C-gamma 1 activity. Comparative properties of control and activated enzymes.

We demonstrated previously tyrosine phosphorylation-dependent modulation of phospholipase C-gamma 1 (PLC-gamma 1) catalytic activity (Nishibe, S., Wahl, M. I., Hernandez-Sotomayor, S. M. T., Tonks, N. K., Rhee, S. G., and Carpenter, G. (1990) Science 250, 1253-1256). The increase in PLC-gamma 1 catalytic activity in A-431 cells occurs rapidly, with maximal activation 5 min after epidermal growth factor (EGF) stimulation. Certain other growth factors (fibroblast growth factor, platelet-derived growth factor) also stimulate PLC-gamma 1 catalytic activity, whereas insulin does not. A similar increase in PLC-gamma 1 specific activity (2-3-fold) was observed in both soluble (cytosol) and particulate (membrane) preparations from EGF-treated cells. Tyrosine-phosphorylated PLC-gamma 1 was detected in both cytosol and membrane fractions in lysates from EGF-treated A-431 cells, but the proportion of tyrosine-phosphorylated PLC-gamma 1 was higher in the cytosol (approximately 50%) than in the membrane (approximately 20%). Because a micellar concentration of the non-ionic detergent Triton X-100 allows detection of the tyrosine phosphorylation-dependent increase in PLC-gamma 1 catalytic activity in this assay, we evaluated the kinetic properties of PLC-gamma 1, immunoprecipitated from cytosol of control or EGF-treated cells, using substrate, phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2), solubilized in Triton X-100 at various molar ratios. The behavior of the control enzyme differed from the EGF-activated enzyme with respect to both Ks and Km. The control enzyme has a 7.5-fold higher Ks value than the activated enzyme (1.5 mM as compared with 0.22 mM). Activation by EGF is also a positive allosteric modifier of PLC-gamma 1-catalyzed PtdIns 4,5-P2 hydrolysis, i.e. the activated enzyme displayed apparent Michalis-Menton kinetics, with a Km of 0.6 mol fraction PtdIns 4,5-P2, whereas the control enzyme displayed sigmoidal kinetics with respect to PtdIns 4,5-P2 hydrolysis. At low substrate mol fractions (e.g. 0.07), the reaction velocity of the control enzyme was 4-fold lower than the activated enzyme. However, at a high substrate mol fraction (e.g. 0.33), the estimated maximal reaction velocities (Vmax) for both forms of PLC-gamma 1 were equivalent. PLC-gamma 1 activity from both control and EGF-treated cells was stimulated by increasing nanomolar Ca2+ concentrations. Although the catalytic activity of PLC-gamma 1 from EGF-treated cells was greater than control PLC-gamma 1 at every Ca2+ concentration tested, the relative stimulation of activity was markedly greater at Ca2+ concentrations above approximately 300 nM.

Study on the stereoselectivity of inositol 1,4,5-trisphosphate recognition sites of bovine adrenal cortex.

Inositol 1,4,5-trisphosphate (InsP3) is an intracellular messenger generated from the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C in response to Ca(2+)-mobilizing stimuli. InsP3 interacts with a specific receptor responsible for the release of sequestered Ca2+ from an intracellular store. The purpose of the present study was to evaluate the relative affinities of the naturally occurring D-isomer of InsP3 and that of its L-stereoisomer for the InsP3 receptor and the InsP3 metabolizing enzymes from bovine adrenal cortex. The InsP3 receptor recognized D- and L-isomers with respective affinities of 4.8 nM and 7.3 microM. This high degree of selectivity was also reflected in the capacity of both isomers to mobilize Ca2+ from the microsomal preparation. The partially purified InsP3 kinase preparation was also able to discriminate between the two stereoisomers. The activity of the kinase was half-maximally inhibited in the presence of 11 microM L-InsP3, a value much higher than the Km of the kinase for D-InsP3 (0.4 microM). Both stereoisomers exhibited equipotent affinities (around 17 microM) for the particulate preparation of InsP3 phosphatase. The enzyme, however, appeared to hydrolyze L-InsP3 at a much slower rate. These results demonstrated that the different recognition sites for InsP3 were expressing distinct levels of stereoselectivity. This property, which is an important aspect of ligand-receptor interaction, could be exploited for the design of new selective drugs interfering with InsP3 action and metabolism.

Calmodulin independence of human duodenal adenylate cyclase.

The calmodulin and calcium dependence of human adenylate cyclase from the second part of the duodenum was assessed in washed particulate preparations of biopsy specimens by investigating (a) the concentration dependent effects of free [Ca2+] on enzyme activity, (b) the effects of exogenous calmodulin on enzyme activity in ethylene glycol bis (b-aminoethyl ether)N,N'-tetra-acetic acid (EGTA) washed particulate preparations, and (c) the effects of calmodulin antagonists on enzyme activity. Both basal (IC50 = 193.75 (57.5) nmol/l (mean (SEM)) and NaF stimulated (IC50 = 188.0 (44.0) nmol/l) adenylate cyclase activity was strongly inhibited by free [Ca2+] greater than 90 nmol/l. Free [Ca2+] less than 90 nmol/l had no effect on adenylate cyclase activity. NaF stimulated adenylate cyclase activity was inhibited by 50% at 2.5 mmol/l EGTA. This inhibition could not be reversed by free Ca2+. The addition of exogenous calmodulin to EGTA (5 mmol/l) washed particulate preparations failed to stimulate adenylate cyclase activity. Trifluoperazine and N-(8-aminohexyl)-5-IODO-1-naphthalene-sulphonamide (IODO 8) did not significantly inhibit basal and NaF stimulated adenylate cyclase activity when measured at concentrations of up to 100 mumol/l. These results suggest that human duodenal adenylate cyclase activity is calmodulin independent but is affected by changes in free [Ca2+].

Thermodynamic and kinetic aspects of agonist and antagonist binding to 1,4-dihydropyridine receptors

The kinetic and equilibrium binding properties of the 1,4-dihydropyridine activator [ 3H](−)-S-Bay K 8644 and the antagonist [ 3H](+)-PN 200-110 were determined in rat heart membrane particulate preparations at temperatures between 4 and 37°C. The binding of [ 3H](−)-S-Bay K 8644 was temperature-dependent with a single binding site with K D = 3.57 nM and B max = 330 fmol/mg · protein at 25°C. The association and dissociation rate constants were 3.4 × 10 7 min −1 M −1 and 0.095 min −1 respectively at 25°C and decreased slightly at lower temperatures. In contrast, [ 3H](+)-PN 200-110 bound to high K D(H) = 0.032 nM B max(H) = 316 fmol/mg · protein) and low affinity sites K D(L) = 27.6 nM and B max(L) = 6432 fmol/mg · protein) at 25°C in rat heart preparation. A similar two-site binding of [ 3H](+)-PN 200-110 was found in rat brain preparation, but only a single binding site was detected in rat skeletal muscle. Binding of [ 3H](+)-PN 200-110 to the high and low affinity sites in cardiac membranes was sensitive and insensitive respectively to temperature. Association and dissociation rates of [ 3H](+)-PN 200-110 at the high affinity binding sites were best fitted as mono-exponential functions. Association and dissociation rates of [ 3H](+)-PN 200-110 were 3.94 × 10 8 min −1 M −1 and 7.86 × 10 −3 min −1 at 25°C. The association rate varied only slightly (3-fold), but the rate of dissociation decreased significantly (200-fold) with temperature from 37 to 4°C. Thermodynamic analysis of equilibrium binding showed that the binding of activator was enthalpy driven, whereas the binding of antagonist to the high affinity site was both entropy- and enthalpy-driven and to the low affinity site was totally entropy-driven.

Porcine Adrenal Adrenocorticotropic Hormone Receptors: Characterization, Changes during Neonatal Development, and Response to a Stressor*

ACTH receptors were characterized in the porcine adrenal cortex using a biologically active [125I] Tyr23,Phe2,Nle4ACTH(1-38) analog, and a 149,000 X g(av) total particulate tissue preparation. Binding of this labeled ACTH analog to adrenocortical ACTH receptors was demonstrated to be time and temperature dependent, tissue and hormone specific, saturable, reversible, have a binding optimum at pH 7.5, and be dependent on the presence of Ca++. Scatchard analyses of saturation data using the computer program LIGAND indicated a single class of high affinity binding sites [association constant (Ka) = 2.00 +/- 0.25 10(9) M-1)]. Hill plots of saturation data had slopes (b = 0.83 +/- 0.09) that did not differ from 1, and hence also indicated a single class of binding sites. The rate constant of association (k1 = 3.72 +/- 1.19 X 10(8) min-1M-1) was compatible with the known rapidly occurring action of ACTH in pigs in vivo. Kinetic experiments also indicated the ACTH receptors to be of high affinity (4.18 +/- 0.54 X 10(10) M-1; n = 4). Receptor-associated degradation occurring to analog during incubations involved cleavage of the peptide into fragments rather than deiodination and could be partially prevented by use of 0.01% bacitracin. Subsequently, a study was conducted to determine changes in the ability of piglets to respond, in terms of plasma cortisol, to the physiological and psychological stressor of overnight maternal deprivation (MD) at various neonatal ages (days 3-31 of age); to measure age-related and stressor-associated changes in adrenal ACTH receptors; and to associate any changes in adrenal response with changes in ACTH receptors. Plasma cortisol obtained by venipuncture did not vary with age in either control or MD piglets but was elevated by MD (16.98 +/- 2.5 vs. 68.3 +/- 8.2 ng/ml). There was no effect of MD and associated hormonal perturbations on adrenal ACTH receptors. However, there were significant age-related differences in these receptors, with a 1.6-fold increase in concentrations (55.6 +/- 12.0 fmol/mg protein), and a 63% decrease (P less than 0.05) in affinity (4.19 +/- 0.86 x 10(8) M-1) evident at 17 days of age compared with 10 and 24 days of age. The significance of these age-related changes in ACTH receptors for adrenal function remains to be determined; however, it is evident that in neonatal pigs, there is no stress nonresponsive period.