Lung Membranes Research Articles

Characterization of the guinea pig lung membrane leukotriene D4 receptor solubilized in an active form. Association and dissociation with an islet-activating protein-sensitive guanine nucleotide-binding protein.

Membrane fractions from the guinea pig lung had high- and low-affinity binding sites for LTD4 with Kd values of 0.016 and 9.1 nM, respectively. In the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or by prior treatment of the membrane with islet-activating protein (IAP), the high-affinity site shifted to a low-affinity state. Consistently, a 41-kDa protein was ADP-ribosylated by treatment of the lung membranes with IAP, and this event was inhibited by the addition of GTP gamma S. We solubilized the LTD4 receptor from the lung membranes in an active form with 5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and 10% glycerol. On a gel filtration column, the binding activity was eluted at the volume corresponding to a Mr of 70,000 or over 500,000 in the presence or absence of Mg2+ (5-20 mM), respectively, in solubilizing buffers. The Kd value of [3H]LTD4 binding to the 70-kDa protein was similar to the low-affinity binding constant of the membrane and was insensitive to GTP gamma S. The preparation solubilized in the absence of Mg2+ showed both high- and low-affinity binding sites for LTD4, and the addition of GTP gamma S shifted the high-affinity site to a low-affinity one. Thus, 1) the LTD4 receptor is coupled to an IAP-sensitive GTP-binding protein, 2) this GTP-binding protein is dissociable from the receptor by solubilizing the lung membrane with CHAPS and Mg2+, and 3) the receptor associated to or dissociated from a GTP-binding protein exhibited a high- or low-affinity state, respectively. These data provide an insight into the molecular mechanism of regulation of the LTD4 receptor signaling process by association and dissociation with an IAP-sensitive GTP-binding protein.

Binding sites of a novel neuropeptide pituitary‐adenylate‐cyclase‐activating polypeptide in the rat brain and lung

Pituitary-adenylate-cyclase-activating polypeptide (PACAP) is a novel 38-amino-acid neuropeptide isolated from ovine hypothalamic tissues based on its activity of stimulating adenylate cyclase of rat pituitary cells. Binding sites for PACAP were studied in rat tissue membranes using a 27-amino-acid N-terminal derivative of PACAP [PACAP(1-27)] labelled with 125I. Particularly high specific binding sites of 125I-PACAP(1-27) were noted in the hypothalamus, brain stem, cerebellum and lung. Specific binding sites are also present in the pituitary gland, but at a lower concentration, and mainly in the anterior lobe. Very low concentration of 125I-PACAP(1-27)-binding sites were found in the colon, aorta and kidney membranes and no binding sites were detected in the pancreas and testis. Maximal binding of 125I-PACAP(1-27) was observed at pH 7.4. Interaction of 125I-PACAP(1-27) with its binding site was rapid, specific and saturable as well as time, pH and temperature dependent. PACAP(1-27) is more potent than PACAP in displacing the binding of 125I-PACAP(1-27) with brain membranes [concentration that inhibits 50% of the binding (IC50) = 7.45 +/- 1.52 nM and 11.45 +/- 3.65 nM, respectively; mean +/- SEM, n = 4] and lung membranes (IC50 = 4.41 +/- 0.87 nM and 10.68 +/- 3.09 nM, respectively). Vasoactive intestinal peptide displaced the binding of 125I-PACAP(1-27) in lung membrane (IC50 = 16.88 +/- 5.14 nM) but not in brain membranes. The equilibrium binding of 125I-PACAP(1-27) at 4 degrees C was characterized by a single class of binding site for the brain membrane with a dissociation constant (Kd) of 2.46 +/- 0.53 nM and a maximal binding capacity (Bmax) of 8.44 +/- 3.13 pmol/mg protein, but there were two classes of binding site for lung membranes with Kd of 1.02 +/- 0.51 nM and 5.19 +/- 0.99 nM, and Bmax of 2.84 +/- 0.72 pmol/mg protein and 9.13 +/- 1.89 pmol/mg protein, respectively. These findings suggest that subtypes of PACAP-binding sites exist and PACAP may have a physiological role in the hypothalamus/pituitary axis as well as in other regions of the brain and lung.

Imidazoline receptors in rat liver cells: A novel receptor or a subtype of α2-adrenoceptors?

An imidazoline/guanidine receptor has been characterized in rat liver cells. Binding of [ 3H]idazoxan, a selective benzodioxan antagonist, to imidazoline receptor on intact fresh hepatocytes (B max = 801 ± 23 fmol/mg protein,K d = 11 ± 0.8 nM) and to liver membranes (B max = 400 ± 38 fmol/mg protein, K d = 10 ± 2 nM) was saturable at 4°C within 3.5 h and at 30°C within 30 min, respectively. Rat lung membranes had more imidazoline sites (B max = 578 ± 30 fmol/mg protein, K d = 14 ± 1.4 nM) than α 2-adrenoceptors (B max = 175.0 ± 20.0 fmol/mg protein,K d = 4.8 ± 2.0 nM). We also screened other tissues for imidazoline sites; labeled with [ 3H]idazoxan displaced by cirazoline was lower in rat lung compared to rat brain and human platelets. The imidazoline receptor has common pharmacological properties with α 2-adrenoceptors, although it is not a subtype of the adrenoceptor, since it bound neither the endogenous agonists norepinephrine and epinephrine, nor the selective α 2-antagonists yohimbine and phentolamine. All guanidine type α 2-adrenoceptor drugs (e.g. guanabenz, guanoxan) and imidazolines (e.g., UK-14,304, naphazoline) competed with high affinity for the liver imidazoline receptor. The lack of effect by Gpp(NH)p, a non-hydrolysable GTP analogue, on the affinity of guanidine- and imidazoline-type ligands for liver imidazoline receptors suggests that the mode of action of these drugs at imidazoline receptors is different than at conventional α 2-adrenoceptors. Ionic changes were considered as a possible mechanism underlying the α 2-adrenoceptor effects in various cells. Opening of K + channels by α 2-adrenoceptors agonists is a pathway which might be shared by imidazoline-type agonists at imidazoline sites. Indeed, 4-aminopyridine, a K + channel blocker, inhibited the specific binding of [ 3H]idazoxan to liver cells with an IC 50 of 0.34 ± 0.07 mM, a concentration which is effective in blocking K + channels in neuronal cells. Similarly, Cs + and NH 4 + effectively interfered with [ 3H]idazoxan binding, suggesting a possible coupling of imidazoline sites to K + gating. The endogenous ligand clonidine-displacing substance (CDS), which was isolated from bovine brain and which binds to α 2-adrenoceptors in brain membranes and human platelets competed with idazoxan at rat liver imidazoline receptors. Although the need of an endogenous ligand for imidazoline sites is obvious, it has yet to be determined whether CDS is the endogenous ligand for imidazoline receptors in general, and for liver cells in particular, and whether K + channels are involved in the mechanism of action of these sites.

Isolation of anti-endothelin receptor monoclonal antibodies for use in receptor characterization

Monoclonal antibodies reactive with endothelin (ET) receptors have been prepared by immunization of mice with rat lung membranes. Of four clones isolated, three clones preferentially recognized 32,000-dalton ET receptor and the other has a higher affinity for the 45,000-dalton receptor. The binding of 125I-ET-1 to detergent-solubilized ET receptors which were adsorbed to the antibodies was displaced by increasing concentrations of unlabeled ET isopeptides. These results demonstrate that the four clones specific for the receptor have the potential to be a useful tool in the characterization of ET receptors.

Structural requirements of ATP for activation of basal and atrial natriuretic factor-stimulated guanylate cyclase in rat lung membranes

ATP has been reported to increase basal and atrial natriuretic factor (ANF)-stimulated guanylate cyclase activity. The structural features of ATP involved in the activation of guanylate cyclase were examined by employing a variety of ATP analogs with modification either at the phosphate chain or at the ribose moiety. Among the natural adenme nucleotides, ATP and ADP were able to increase both basal and ANF-stimulated guanylate cyclase activities in rat lung membranes. AMP had no effect, ATP was more effective than AMPPCP (the non-hydrolyzable analog of ATP), and ADP was more effective than ADPβS and AMPCP (the hydrolysis-resistant analogs of ADP) to increase basal and ANF-stimulated guanylate cyclase activities. Removal of the oxygen atom from the ribose moiety of ATP or ADP significantly reduced their potency. Thus, the length of the phosphate chain and the hydroxyl groups at the ribose moiety are both determinants for nucleotide mediated guanylate cyclase activation.

The modulation of [3H]noradrenaline and [3H]serotonin release from rat brain synaptosomes is not mediated by the ?2B-adrenoceptor subtype

The present study aimed at relating the presynaptic alpha 2-adrenoceptors, known to modulate noradrenaline and serotonin release, with the recently described alpha 2A- and alpha 2B-adrenoceptor subtypes. The effects of the agonist oxymetazoline (selective for alpha 2A subtype) and of three adrenoceptor antagonists (idazoxan, 1-(2-pyrimidinyl)piperazine (PmP) and prazosin, the last one known to be alpha 2B selective) were evaluated on [3H]noradrenaline and [3H]serotonin release in superfused synaptosomes from rat brain cortex. These drugs were also tested in [3H]yohimbine binding to human platelet membranes (containing only alpha 2A receptors) and to neonatal rat lung membranes (containing only alpha 2B receptors). The affinity pattern of these compounds at alpha 2A-adrenoceptors in binding studies was oxymetazoline greater than = idazoxan greater than PmP greater than prazosin; at alpha 2B-adrenoceptors it was idazoxan greater than = prazosin greater than PmP = oxymetazoline. Oxymetazoline inhibited with high and similar potencies the K(+)-evoked [3H]noradrenaline and [3H]serotonin release, IC50 18 and 7 nM, respectively; in the same conditions, the IC50 values of noradrenaline were 42 and 168 nM, respectively. The antagonist affinity pattern (antagonism against noradrenaline) was idazoxan greater than PmP greater than prazosin, either on [3H]serotonin release. These results indicate that presynaptic alpha 2 auto- or heteroreceptors do not belong to the alpha 2B subtype and suggest that the modulation of noradrenaline and serotonin release may be mediated by the alpha 2A-adrenoceptor subtype.

Identification of heterogeneity of leukotriene receptors in membranes of human lung using a computerized modelling approach

Identification of heterogeneity of leukotriene receptors in membranes of human lung using a computerized modelling approach

Molecular mapping of pulmonary endothelial membrane glycoproteins of the intact rabbit lung

To study the biochemical characteristics of endothelium in vivo, we radioiodinated endothelial membrane proteins of the perfused rabbit lung using a water soluble analog of the Bolton-Hunter reagent, 125I-sulfosuccinimidyl (hydroxyphenyl) propionate (125I-s-SHPP). This technique led to a 10-fold increase in specific activity of radioiodinated lung membrane protein compared with our previously reported method using lactoperoxidase and glucose oxidase-catalyzed radioiodination. Tissue autoradiography confirmed that radioiodination was largely confined to the endothelium. Perfusion pressure, wet-to-dry weight ratios, and the morphological appearance of the lungs were within normal limits, indicating that the procedure does not cause apparent lung injury. Lectin binding to a crude membrane fraction of 125I-s-SHPP labeled lung led to isolation of several putative endothelial membrane proteins. Immunoprecipitation studies with appropriate antibodies enabled the identification of radioiodinated angiotensin-converting enzyme and beta 2-microglobulin associated major histocompatibility complex class I molecules in the membrane fraction. This technique will be useful for studying biochemical responses of the endothelium in vivo to a variety of pharmacological and physiology stimuli.

Rat PHI, PHI-GLY and PHV(1–42) stimulate adenylate cyclase in six rat tissue and cell membranes

PHI and the two C-terminally extended forms PHI-GLY and PHV(1–42) coexist in rat tissues. We compared the relative potency and efficacy of these three PHI forms and of VIP to stimulate adenylate cyclase activity and, when feasible, to occupy VIP receptors in six rat tissue and cell membranes. With the exception of lung membranes, all three PHI forms were markedly less potent than VIP but all were systematically as efficacious. PHI-GLY and PHV(1–42) were never more potent than PHI itself and their relative potencies revealed four spectra, depending on the membrane preparation tested: 1) PHI = PHI-GLY = PHV(1–42) in hepatic, pulmonary and pancreatic membranes; 2) PHI > PHV(1–42) = PHI-GLY in membranes from circulating lymphocytes; 3) PHI = PHV(1–42) > PHI-GLY in membranes from the thymocyte cell line 51E; and 4) PHI > PHI-GLY = PHV(1–42) in anterior pituitary membranes. These results indicate that the two naturally observed C-terminal extensions of rat PHI variously affected peptide potency on 6 rat membrane preparations.

Characterization of Receptors for Platelet-activating Factor in Guinea Pig Lung Membranes

Platelet-activating factor (PAF) is a potent contractile agonist in guinea pig peripheral lung strips. Whether PAF acts via specific receptor sites in the lung has not been fully established. To determine if specific receptor sites could be demonstrated in guinea pig peripheral lung tissue, we performed direct radioligand binding studies in tissue homogenates with [3H]C16-PAF (1-O-hexadecyl-sn-glyceryl-3-phosphorylcholine) and the PAF antagonists [3H]WEB 2086 and [3H]RP52770. These studies demonstrated binding sites for [3H]C16-PAF of high affinity (Kd of 2.6 nM from saturation isotherms and 0.9 nM from kinetic experiments) and a mean density of 200 fmol/mg protein. Binding was inhibited to the same degree with unlabeled C16-PAF, WEB 2086, and RP52770, all with pseudo-Hill slopes of unity. [3H]WEB 2086 binding demonstrated a receptor density similar to that of [3H]C16-PAF (226 fmol/mg protein) and was inhibited to the same degree by unlabeled C16-PAF, C18-PAF, WEB 2086, and RP52770. Although antagonist inhibition yielded pseudo-Hill slopes near unity, agonist inhibition slopes were shallow, suggesting two types or states for the PAF receptors. Direct binding studies with [3H]RP52770 revealed a much larger density of binding sites (1,200 fmol/mg protein), and this binding was not inhibited with C16-PAF, C18-PAF, WEB 2086, or lyso-PAF. These results indicate that guinea pig lung has specific binding sites for [3H]C16-PAF, that WEB 2086 is an effective antagonist of C16- and C18-PAF binding at these sites, and that RP52770 binds to the PAF site but, in addition, binds to another site with a much greater density.

Characterization of the soluble leukotriene B4 receptor from sheep lung membranes

Leukotriene B4 (LTB4) is an arachidonate metabolite which elicits a variety of pro-inflammatory responses by activation of a guanine-nucleotide-binding protein-coupled membrane receptor. As a prelude to receptor isolation and purification, we have established assay methods for LTB4 receptor solubilization and characterization from sheep lung membranes. [3H]LTB4 binding to the soluble receptor was saturable, specific, protein-concentration- and time-dependent and reversible. Binding of [3H]LTB4 was enhanced by divalent cations and inhibited by sodium ions in a manner analogous to its binding to the human leukocyte membrane receptor. Saturation binding yielded a dissociation constant (Kd) of 0.50 +/- 0.05 nM and a receptor density (Bmax) of 330 +/- 90 fmol/mg of protein for [3H]LTB4 binding to detergent-solubilized receptor. In competition experiments, the rank order of binding affinity was LTB4 greater than 20-OH-LTB4 greater than trans-homo-LTB4 greater than 6-trans-LTB4 greater than U-75302. Gel-filtration chromatography showed that the LTB4 receptor protein in the detergent micellar state has a molecular mass in the range 800-1000 kDa. These results demonstrate that the physiologically and pharmacologically important LTB4 receptor may be readily solubilized from sheep lung membranes without alteration in binding specificity and characteristics, suggesting that sheep lung membranes represent a rich source with which to pursue receptor isolation and purification.

Characterization of receptors for glucagon-like peptide-1(7–36) amide on rat lung membranes

Specific binding of 125I-labelled GLP-1(7–36)amide to rat lung membranes was dependent upon time and temperature and was proportional to membrane protein concentration. Binding was inhibited in a concentration-dependent manner by unlabelled GLP-l(7–36)amide consistent with the presence of a single class of binding sites with a dissociation constant ( K d) of 1.67 ± 0.29 nmol l . GLP-1(1–36)amide was 260 times less potent in inhibiting the binding of 125I-labelled GLP-1(7 36)amide to lung membranes ( k d of 448 ± 93 nmol l ). Vasoactive intestinal polypeptide and peptide-histidine-isolcucine also displaced 125I-labelled GLP-1(7–36)amide from the receptor concentration-dependently; the k d was 4.31 ± 0.8 and 7.93 ± 4.79 nmol l . respectively. Guaninc nucleotides (GTP-γ-S, GDP-β-S) decreased the binding of 125I-labelled GLP-1(7–36)amide to rat lung membranes as was found for GLP-1(7–36)amide receptors in RINm5F cells which were also shown to be coupled to the adenylate cyclase system.

Characterization and Distribution of Binding Sites for the Hypothalamic Peptide, Pituitary Adenylate Cyclase-Activating Polypeptide*

A novel bioactive peptide was recently isolated from ovine hypothalamus and was named PACAP (pituitary adenylate cyclase-activating polypeptide). PACAP was present in two bioactive, amidated forms, PACAP27 and PACAP38 (27 and 38 amino acids, respectively), and showed a 68% sequence homology with vasoactive intestinal peptide (VIP) in the N-terminal 28 residues. PACAP38 was at least 1000 times more potent than VIP in stimulating adenylate cyclase in pituitary cells, but both peptides exhibited comparable vasodepressor activity. Thus, we sought to determine whether PACAP acts on specific binding sites in the anterior pituitary or other tissues and whether these binding sites are different from those of VIP. Binding of [125I] PACAP27 to freshly prepared rat anterior pituitary membranes in the presence and absence of 212 nM unlabeled PACAP27 was specific, saturable, and more rapid at 22 C than at 4 C. Scatchard analysis of this binding site using increasing doses of unlabeled PACAP27 revealed a single high affinity site with a Kd of 446 +/- 141 pM and a maximum number of sites of 1312 +/- 182 fmol/mg protein. These results do not exclude the possibility of a second pituitary binding site with significantly lower affinity. Unlabeled PACAP38 and PACAP38OH exhibited significantly higher affinity binding (3- to 5-fold) than PACAP27 with a similar number of pituitary sites. A variable distribution of binding sites was observed between PACAP27 and VIP when binding to different tissue membranes was measured with 125I-labeled peptides. Very high specific binding of both PACAP27 and VIP was observed in lung membranes. An almost identical relative magnitude of binding was observed between PACAP27 and VIP in lung, liver, duodenum, ovary, and thymus. However, whereas PACAP27 binding to hypothalamic and pituitary membranes was great, VIP binding to these tissues was almost absent. To determine if VIP and PACAP might share a binding site in peripheral tissues, displacement curves were generated using [125I]PACAP27 binding to lung membranes and VIP, PACAP27, and PACAP38 as unlabeled ligands. VIP was highly potent in displacing [125I] PACAP27 binding in lung membrane, and the IC50 values for all three of these peptides were between 1-10 nM. These results suggest that 1) a saturable, high affinity binding site for PACAP is present on anterior pituitary membranes; 2) PACAP27 and PACAP38, but not VIP, share this binding site in the anterior pituitary and possibly the hypothalamus; and 3) PACAP27, PACAP38, and VIP share a similar or identical binding site on lung membranes and possibly other peripheral tissues.

TIME COURSE OF RECOVERY OF β‐ AND α1‐ADRENOCEPTORS IN EXPERIMENTAL ASTHMA

SUMMARY1. The time course of recovery of reduced β‐adrenoceptors caused by ovalbumin (OA) challenge was investigated using guinea‐pigs.2. The effects of prednisolone on the recovery time course were also evaluated.3. β‐ and α1‐receptor assays were performed using lung membranes. Adenylate cyclase activity was also measured.4. OA challenge reduced the number of β‐adrenoceptors by 35%, and a significant decrease (13%) persisted for 7 days. The number of β‐adrenoceptors recovered after 14 days.5. OA challenge elevated the number of α1‐adrenoceptors. A significant increase (24%) was observed after 7 days, and it took a further 7 days for the recovery.6. After OA challenge there was a significant decrease in adenylate cyclase activity after 7 days, which recovered after a further 7 days.7. Inhalation of prednisolone accelerated the recovery of β‐adrenergic responsiveness, though it did not affect the recovery of the number of α1‐adrenoceptors. Prednisolone inhalation also elevated β‐adrenergic responsiveness in non‐asthmatic subjects.8. It is concluded that reduced β‐adrenergic responsiveness caused by OA challenge persisted for 7 days and recovered after a further 7 days. Steroid hormone increased β‐adrenoceptors.

Inhibition by ibudilast of leukotriene D4‐induced formation of inositol phosphates in guinea‐pig lung

1. The effects of a novel anti-asthmatic drug, 3-isobutyryl-2-isopropylpyrazolo [1,5-a]pyridine (ibudilast, KC-404) on leukotriene D4 (LTD4)-induced formation of inositol phosphates were studied in slices of guinea-pig lung and hippocampus. 2. In guinea-pig lung, ibudilast inhibited LTD4 (0.01-1 microM)-induced formation of inositol monophosphate (IP1) in a concentration-dependent manner (IC50 = 10 microM) without affecting LTD4 receptor binding. 3. Ibudilast (10 microM) inhibited histamine (0.1-1 mM)-induced formation of IP1 in guinea-pig lung slices but not in hippocampal slices. 4. Inhibition of agonist-induced formation of IP1 by ibudilast was non-competitive. 5. Ibudilast had no effect on either GTP- or calcium-stimulated phosphatidylinositol specific-phospholipase C activity of lung membranes. 6. These results suggest that ibudilast has no direct effect on LTD4 receptors, GTP binding proteins (G proteins) or phospholipase C, but inhibits inositol phosphate formation, possibly by interfering with the coupling between receptors and G proteins.

Caged ATP potentiates guanylate cyclase activity stimulated by atrial natriuretic factor in rat lung membranes

ATP increases both basal and atrial natriuretic factor (ANF)-stimulated guanylate cyclase activities. The present studies were designed to compare the effect of adenosine-5′-triphosphate. P 3-1-(2-nitrophenyl)-ethyl ester (caged ATP), a photolabile derivative of ATP, with ATP. Caged ATP increases both basal and ANF-stimulated enzyme activities in a concentration-dependent manner, but is more potent than ATP. The effect of caged ATP significantly decreases after it is photo-converted to ATP by UV irradiation. These observations suggest that the caged group introduces a hydrophobic interaction thereby facilitating granylate cyclase cyclase.

The inhibition of [ 3H]leukotriene D 4 binding to guinea-pig lung membranes. The correlation of binding affinity with activity on the guinea-pig ileum

Guinea-pig lung membranes contain high affinity (K D = 0.8 nM) binding sites for [ 3H]leukotriene D 4 ([ 3H]LTD 4). The binding is inhibited by leukotriene antagonists, such as ICI 198615 and SK & F 104353, in a manner consistent with the Law of Mass Action at a single site. It is also inhibited by a range of leukotriene analogues in a dose-related manner. Inhibition by some of these e.g. LTC 4 suggests that the [ 3H]LTD 4 binding sites are heterogeneous. The binding affinity of the leukotriene analogues is significantly correlated (P < 0.001) to their spasmogenic activity on guinea-pig ileum but not on guinea-pig lung strip. The binding affinity of the leukotriene antagonists is also correlated to their antagonist activity on guinea-pig ileum (P < 0.05) but not on guinea-pig lung. These results indicate that the [ 3H]LTD 4 binding site in guinea-pig lung is similar to the leukotriene receptor activated by LTD 4 and LTE 4 on guinea-pig ileum. The contractile response of guinea-pig lung strips to leukotrienes, in the presence of indomethacin, is mediated by a distinct type of leukotriene receptor.

Carbonic anhydrase IV from human lung. Purification, characterization, and comparison with membrane carbonic anhydrase from human kidney.

We have purified carbonic anhydrase (CA) IV from human lung membranes to apparent homogeneity in a form which is catalytically active and stable to storage. It has an apparent molecular mass of 35 kDa, is insensitive to endoglycosidases, and seems to contain no N-linked or O-linked oligosaccharide chains. Reduction of disulfide linkages led to altered migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and loss of catalytic activity. CA IV resembles CA II in being a "high activity" isozyme, relatively resistant to inhibition by halide ions and sensitive to inhibition by sulfonamides. Application of this purification to human kidney membranes produced homogeneous enzyme with nearly identical properties. Amino acid compositions of both lung and kidney CA IV were similar, as were tryptic peptide patterns resolved on high performance liquid chromatography (HPLC). Amino-terminal sequences of native enzyme from lung and kidney were identical, as were amino-terminal sequences of the three major tryptic peptides resolved on reverse phase HPLC. Isoelectric focusing revealed microheterogeneity in enzyme from both sources. Antibody raised to human lung CA IV reacted equally strongly with CA IV from kidney, but very weakly or not at all with other CAs. Treatment of lung membranes and kidney membranes with phosphatidylinositol-specific phospholipase C released over half of the membrane-bound CA IV, suggesting that at least half of the CA IV in both organs is anchored to membranes by phosphatidylinositol-glycan linkages.

Competition of nucleoside transport inhibitors with binding of 6-[(4-nitrobenzyl)-mercapto]purine ribonucleoside to intact erythrocytes and ghost membranes from different species

The potency of nucleoside transport inhibitors, including 6-[(4-nitrobenzyl)-mercapto]purine ribonucleoside (NBMPR), dilazep, mioflazine and its derivatives soluflazine and R57974 as inhibitors of the binding of [3H(G)]NBMPR to intact erythrocytes and respective ghost membranes from human, mouse and hamster was determined. There was no close agreement between the ic50 profiles for the different inhibitors when comparing values obtained for intact cells and membranes from each species, and there was no consistent profile of differences when considering individual drugs and comparing their actions in the three species. Present data also were compared with potency values obtained previously with the same drugs directly in nucleoside transport inhibition studies with erythrocytes from the same species as well as with [3H(G)]NBMPR binding studies in isolated liver and lung membranes from hamster. The overall conclusion from this and previous studies is that the evaluation of relative potencies in screening of potential nucleoside transport inhibitors is best carried out at the level of actual nucleoside transport studies in intact cells, since [3H(G)]NBMPR binding studies yield discrepant data.

Characterization of ATP-stimulated guanylate cyclase activation in rat lung membranes

Many of the effects of ANP are mediated through the elevation of cellular cGMP levels by the activation of particulate guanylate cyclase. While the stimulation of this enzyme is receptor-mediated, the molecular mechanism of activation remains unknown. In this study we present evidence that ATP as well as its analogues adenosine-5′- O-(3-thiotriphosphate) (ATPγS) and adenylylimidophosphate (AMPPNP) activates guanylate cyclase from rat lung membranes and markedly potentiates the effect of ANP on the enzyme. The order of potency is ATP γS > ATP > AMPPNP. The enzyme activation by adenine nucleotide and ANP together is much more than the sum of the individual activations, suggesting that ATP may be the physiological component essential for the ANP-stimulated guanylate cyclase activation. The ATPγS-stimulated guanylate cyclase activity diminishes in the presence of various kinds of detergents, suggesting either that the conformation of an ATP binding site in guanylate cyclase is altered by detergents or that protein-protein interaction may be involved in the activation of guanylate cyclase by ATP. Guanylate cyclase from rat lung membranes is poorly activated by ANP and/or ATPγS after removing the cytosolic and weakly membrane-associated proteins or factors by centrifugation. Pre-incubation of the membranes with ATPγS retains enzyme activation after membrane washing. These results suggest either that ATPγS stabilizes the conformation of nucleotide binding site in guanylate cyclase from denaturation by membrane washing, or that the stimulatory effect of ATP on guanylate cyclase activity may be mediated by accessory proteins or non-protein cofactors which are lost during membrane washing, but remain bound to membranes by ATPγS pretreatment.