Biomedical subjects

R J Ho

Publications and source records attributed to R J Ho.

At least 55 records · Page 3Linked to original sources

PubMed1989

Antigen-presenting liposomes are effective in treatment of recurrent herpes simplex virus genitalis in guinea pigs.

The therapeutic and immunologic effects of a liposome preparation containing both a macrophage activator, muramyl-tripeptide-phosphatidylethanolamine, and a recombinant antigen, glycoprotein D of herpes simplex virus type 1, have been investigated. This preparation was tested in vitro for the ability to stimulate peripheral blood lymphocytes and in vivo for the control of recurrent herpes genitalis in guinea pigs. Our results show that the liposome-antigen-adjuvant preparation is capable of enhancing antigen-specific lymphocyte stimulation, which may be related to the observed 75% suppression of the frequency and severity of reactivation of recurrent herpes simplex virus type 2 genitalis compared with that of placebo controls.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed1988

Kinetic and ultrastructural studies of interactions of target-sensitive immunoliposomes with herpes simplex virus.

The bilayer phase of dioleoylphosphatidylethanolamine (PE) can be stabilized with palmitoyl-IgG monoclonal antibody to the glycoprotein gD of the herpes simplex virus (HSV). Interactions of PE immunoliposomes with the target virions were characterized by analyzing the kinetics of lipid mixing, by liposomal content release, and by ultrastructural studies. As revealed by a resonance energy transfer assay, lipid mixing between PE immunoliposomes and virions was very rapid, with a second-order rate constant (kapp) of 0.173 (min)-1 (microgram/mL virus)-1. In comparison, content release from PE immunoliposomes was much slower and exhibited multiple-phase, mixed-order kinetics, indicating that liposome destabilization involved fusion of liposomes with HSV. The extent and the apparent rate of liposome destabilization were strongly dependent on liposome concentration. This was evident by the fact that only one to two liposomes were destabilized by each virus particle at low liposome concentration (0.1 microM). For higher liposome concentrations (1-10 microM), this value was 35-104. This finding implies that collision among the virus-bound liposomes is essential for the eventual collapse of PE immunoliposomes to form the hexagonal (HII) equilibrium phase which was observed using freeze-fracture electron microscopy. Studies employing soluble gD, immobilized on latex beads, indicated that a multivalent antigen source is essential for PE immunoliposome destabilization. Immediately after liposome-virus binding, fusion of liposome with the viral membrane then follows. Upon growth of the fusion complexes, which increase to 35-104 liposomes for each virus, an eventual collapse of the structure results, driving PE to its equilibrium structure of HII phase.

Antibodies, Monoclonal↗

PubMed1987

Target-sensitive immunoliposomes as an efficient drug carrier for antiviral activity.

We have shown previously that target-sensitive immunoliposomes composed of palmitoyl antibody stabilized phosphatidylethanolamine bilayers could be destabilized by binding to the target cells (Ho, R. J. Y., Rouse, B. T., and Huang, L., Biochemistry (1986) 25, 5500-5506). Target-sensitive immunoliposome-encapsulated and free cytotoxic drugs of nucleoside analogs cytosine-beta-D-arabinoside (AraC) or acycloguanosine (acyclovir, ACV) were compared for their antiviral efficacy and cell cytotoxicity. Target-insensitive immunoliposomes and nontargeted liposomes were also investigated. When the mouse fibroblast L929 cells were infected at low multiplicity with herpes simplex virus, AraC encapsulated in target-sensitive immunoliposomes composed of transphosphatidylated egg phosphatidylethanolamine effectively inhibited virus replication and had far less cell cytotoxicity than free drug. As a measure of cytotoxicity, the drug concentration required to inhibit 50% of [3H]thymidine incorporation from 6 to 42 h (CD50) was determined. For free AraC, this value was 0.3 ng/ml, whereas for target-sensitive immunoliposome-encapsulated AraC, the CD50 exceeded 1 microgram/ml. However, target-sensitive immunoliposome-encapsulated AraC was virus inhibitory (50% effective dose = ED50) at 1.8 ng/ml. A free drug concentration of at least 1000-fold greater was required for comparable antiviral activity. A similar phenomenon was observed when ACV was administered via target-sensitive immunoliposomes. The CD50 values of the free and target-sensitive immunoliposome-encapsulated ACV were 12.5 ng/ml and 1.4 micrograms/ml, respectively, whereas the ED50 values of the free and target-sensitive immunoliposome-encapsulated ACV were 1.1 and 125 ng/ml, respectively. Consequently, our results indicated the superiority of target-sensitive immunoliposomes at drug delivery, especially when drugs were cytotoxic to cells. The use of liposomes of the target-insensitive variety provided some enhancement of activity, but this was several-fold less than that observed with target-sensitive immunoliposomes. In addition, the nucleoside transport inhibitors, p-nitrothiobenzylinosine and dipyridamole, were shown to inhibit the liposome-mediated antiviral activity of AraC. This finding indicated that site-specific cytosolic delivery of nucleoside analogs by target-sensitive immunoliposomes involved a cellular nucleoside transport system. A mechanism of action is proposed.

Animals↗

PubMed1987

Interactions of target-sensitive immunoliposomes with herpes simplex virus. The foundation of a sensitive immunoliposome assay for the virus.

Interactions between target-sensitive (TS) immunoliposomes and herpes simplex virus (HSV) were investigated. Target sensitivity of phosphatidylethanolamine (PE) immunoliposomes is a result of the ability of acylated monoclonal anti-HSV glycoprotein D (gD) to stabilize the bilayer phase of PE, whereas by itself, PE does not form stable liposomes (Ho, R. J. Y., Rouse, B. T., and Huang, L. (1986) Biochemistry 25, 5500-5506). Upon binding of these immunoliposomes to HSV antigen-containing gD, destabilization of PE immunoliposomes was observed. By encapsulating either a self-quenching fluorescent dye, calcein, or alkaline phosphatase inside the liposomal compartment, the HSV-induced destabilization of TS immunoliposomes was shown to be target-specific. Neither Sendai, Semliki Forest, nor Sindbis virus could significantly destabilize the TS immunoliposomes. Moreover, HSV-induced liposome destabilization could be inhibited by free anti-gD (the same antibody used in TS immunoliposomes) but not by monoclonal anti-HSV glycoprotein B, indicating that the interaction was antigen-specific. Destabilization could also be induced by binding to truncated gD (tgD), but only when in a multivalent form immobilized on latex beads. Truncated gD is a cloned, 312-amino acid fragment of HSV-gD that lacks the transmembrane segment. Preincubation of soluble tgD with the TS immunoliposomes failed to induce destabilization and, in addition, abolished the tgD-bead-induced destabilization. This finding strongly indicated that multivalent binding is essential for TS immunoliposome destabilization. Using alkaline phosphatase encapsulated in the liposomes, TS immunoliposomes could be used to detect HSV in fluid phase with 50% signal recorded at 5 microliters of 3.2 x 10(3) pfu/ml; at least 10-fold more sensitive than the standard double-antibody sandwich enzyme-linked immunosorbent assay. The interactions described here may be useful in designing a homogeneous and sensitive immunoliposome assay.

Animals↗

PubMed1986

Trypsin induced destabilization of liposomes composed of dioleoylphosphatidylethanolamine and glycophorin.

Destabilization of liposomes composed of phosphatidylethanolamine (PE) and purified glycophorin of human erythrocytes was studied with the release of an entrapped fluorescent dye, calcein. Proteolytic cleavage of liposomes by trypsin induced a rapid increase of turbidity and the leakage of calcein from the liposomes. Kinetic experiments indicated that the destabilization was a second order reaction, i.e. it required liposome collision. Using N-(7-nitro-2,1,3-benzoxadiazol-4-yl) PE as a fluorescent probe for the formation of hexagonal phase of PE, tryptic digestion of the liposomes resulted in a higher tendency of the PE bilayer to transform into the hexagonal phase. We propose that hexagonal (or inverted micellar) structures are involved in the trypsin induced liposome destabilization.

Erythrocytes↗

PubMed1986

A dose-response study of forskolin, stimulatory hormone, and guanosine triphosphate analog on adenylate cyclase from several sources.

We have described relationships involving forskolin stimulation of adenylate cyclase (AC) from a variety of sources and the potentiation of forskolin effects by stimulatory hormones (glucagon, ACTH, and epinephrine) and beta, gamma-imidoguanosine 5'-triphosphate (Gpp(NH)p). The effects on AC were examined using membrane preparations of rabbit adipocytes, rat adipocytes, rat erythrocytes, and rat liver. Also examined was the AC of liver membranes of rat pretreated with pertussis toxin as well as that solubilized from rat liver membranes. Maximal forskolin stimulation of AC in all preparations studied revealed a consistent 10-fold increase in AC activity. The EC50 for forskolin was 10 microM for rat liver, 15 microM for rabbit and rat adipocytes and 17 microM for rat erythrocyte AC stimulation. In all cases the AC activity attained by forskolin stimulation was further enhanced by stimulatory hormones in a dose-dependent manner. Furthermore, a combination of all three activators (forskolin, stimulatory hormone, and Gpp(NH)p) resulted in an even greater overall stimulation to levels ranging from 25- to 30-fold over unstimulated activity levels. In the presence of saturating levels of each stimulatory hormone and Gpp(NH)p, the EC50 for forskolin diminished markedly to the range of 0.5 to 4.0 microM. In the absence of any apparent tissue specificity for forskolin stimulation, the general pattern of these results further implicates the catalytic site of the AC complex as the site of forskolin activation. Furthermore, activation of additional components of the complex by Gpp(NH)p and tissue specific hormones may further influence the AC activity and thereby potentiate the stimulation by forskolin.

Adenylyl Cyclases↗

PubMed1986

Conditional inhibition of forskolin-activated adenylate cyclase by guanosine diphosphate and its analog.

Forskolin-activated adenylate cyclases (AC) in intact membranes, solubilized with Lubrol or eluted following adsorption on a forskolin-Sepharose column, were examined for inhibition by GDP and GDP beta S. AC in intact membranes of rat or rabbit adipocytes was activated by 100 microM forskolin and further potentiated by 10 microM Gpp(NH)p in combination with either 230 microM epinephrine or 50 mU X ml-1 ACTH. GDP (0-1 mM) or GDP beta S (0-500 microM) inhibited activation in a dose-dependent manner to a level similar to or slightly below that produced by 100 microM forskolin alone. Forskolin at 100 microM stimulated solubilized AC of rabbit adipocytes and rat liver membranes for 10 +/- 4 to 160 +/- 10 and from 26 +/- 2 to 274 +/- 21 pmol(mg X min)-1, respectively, in the absence of GDP beta S; forskolin-activated activity decreased from 160 +/- 10 to 157 +/- 6 and from 274 +/- 21 to 238 +/- 14 pmol(mg X min)-1 in the presence of 500 microM GDP beta S. Forskolin-activated solubilized enzyme was further potentiated by 10 microM Gpp(NH)p from 160 +/- 10 to 289 +/- 52 and from 274 +/- 21 to 702 +/- 50 pmol(mg X min)-1. GDP beta S at 500 microM inhibited 93 and 103% of the Gpp(NH)p-potentiated activity. AC of rat adipocytes eluted from forskolin-Sepharose affinity column with 500 mM NaCl and 100 microM forskolin was not significantly activated by Gpp(NH)p nor inhibited by GDP beta S. However, it was activated by forskolin. The lack of inhibition of unmodified forskolin-activated activity by GDP or GDP beta S in contrast to the inhibition of Gpp(NH)p-activated enzyme or Gpp(NH)p-potentiated forskolin-activated enzyme may be a general phenomenon descriptive of the action of forskolin on AC. Furthermore, inhibition of forskolin-activated AC by GDP and its analog may be a useful index in analyzing the degree of guanine nucleotide potentiation of this enzyme.

Adenylyl Cyclases↗

PubMed1986

Forms of adenylate cyclase, activation and/or potentiation by forskolin.

Activation of different forms of adenylate cyclases (AC) by forskolin and displacement of [14,15-3H]dihydroforskolin binding from membranes by forskolin in the absence or presence of specific stimulatory hormone and beta, gamma-imidoguanosine 5'-triphosphate (Gpp(NH)p) have been studied. These conditions have been used to generate forskolin dose-response curves of AC activation. A plot of enzyme activation versus apparent forskolin-binding showed a linear and a nonlinear relationship, respectively, in the absence or presence of the other two stimulators. The latter relationship can be fitted by two linear regression lines with a defined intercept, the slopes of which represent two distinct binding-activation (B-A) effects. The B-A effects of forskolin for rat adipocyte and liver membranes in the absence of stimulatory hormone and Gpp(NH)p were 10 and 8 (pmol X min-1) X (pmol)-1, respectively. The B-A effects for the same membranes in the presence of the other two stimulators were 69 (high) and 13 (low) (pmol X min-1) X (pmol)-1 for adipocyte membrane, and 83 (high) and 9 (low) (pmol X min-1) X (pmol)-1 for liver membrane. The ratio of potentiation of forskolin-activated enzyme activity to the unmodified forskolin-stimulated activity (P-A ratio) was determined without the binding data. At 3 microM forskolin, with and without 230 epinephrine and 10 microM Gpp(NH)p, the P-A ratio was 3.7, decreasing to 1.1 with the addition 100 microM forskolin. The line representing a high B-A effect and a resulting high P-A ratio appears to describe the interactions between forskolin and the AC stimulated by epinephrine and Gpp(NH)p. The line of low B-A effect may represent the interaction between forskolin and the basal AC. Two peaks of AC activity were eluted from forskolin-Sepharose column. They have apparent differences in sensitivity to Gpp(NH)p and affinity for forskolin. Based on the results available thus far, with consideration for known limitations of the methodology, a working model has been proposed for forskolin activation of AC.

Adenylyl Cyclases↗

PubMed1986

Target-sensitive immunoliposomes: preparation and characterization.

A novel target-sensitive immunoliposome was prepared and characterized. In this design, target-specific binding of antibody-coated liposomes was sufficient to induce bilayer destabilization, resulting in a site-specific release of liposome contents. Unilamellar liposomes were prepared by using a small quantity of palmitoyl-immunoglobulin G (pIgG) to stabilize the bilayer phase of the unsaturated dioleoylphosphatidylethanolamine (PE) which by itself does not form stable liposomes. A mouse monoclonal IgG antibody to the glycoprotein D of Herpes simplex virus (HSV) and PE were used in this study. A minimal coupling stoichiometry of 2.2 palmitic acids per IgG was essential for the stabilization activity of pIgG. In addition, the minimal pIgG to PE molar ratio for stable liposomes was 2.5 X 10(-4). PE immunoliposomes bound with HSV-infected mouse L929 cells with an apparent Kd of 1.00 X 10(-8) M which was approximately the same as that of the native antibody. When 50 mM calcein was encapsulated in the PE immunoliposomes as an aqueous marker, binding of the liposomes to HSV-infected cells resulted in a cell concentration dependent lysis of the liposomes as detected by the release of the encapsulated calcein. Neither uninfected nor Sendai virus infected cells caused a significant amount of calcein release. Therefore, the release of calcein from PE immunoliposomes was target specific. Dioleoylphosphatidylcholine immunoliposomes were not lysed upon contact with infected cells under the same conditions, indicating that PE was essential for the target-specific liposome destabilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Preparation of rat liver plasma membrane with respect to glucagon-sensitive adenylate cyclase.

Further modification of Neville's method of preparation of rat liver plasma membrane has been made in order to study glucagon-sensitive adenylate cyclase. This modified method introduces two additional steps to the Neville procedures. One involves saving an intermediate layer above the pellet following 1500g centrifugation. The other adds a centrifugation step at 20,000g. The improved method increases the yield of membrane protein by 5-fold and increases forskolin, glucagon, and 5'-guanyl imidodiphosphate stimulated activity of adenylate cyclase by 2.8-fold. A 13-fold increase in the yield of total adenylate cyclase activity above the current method was obtained. The membrane adenylate cyclase and its hormone sensitivity was stable in liquid nitrogen for at least 8 months. This modified method appears to be useful in preparing a better yield and quality of rat liver plasma membrane from given starting hepatic tissue for studies of glucagon-sensitive adenylate cyclase.

Adenylyl Cyclases↗

PubMed1986

Destabilization of target-sensitive immunoliposomes by antigen binding--a rapid assay for virus.

Interactions of antibody stabilized phosphatidylethanolamine (PE) immunoliposomes with Herpes Simplex virus (HSV) and virus infected cells were studied by detecting the immune-dependent lysis of liposomes. Employing PE immunoliposomes bearing anti-HSV glycoprotein D (gD) IgG, immune-specificity of these liposomes were documented by the sole ability of HSV and the HSV-infected L cells to induce immunoliposome lysis. In addition, inhibition of PE immunoliposome lysis by free anti-gD IgG, but not anti-HSV glycoprotein B IgG, indicated the target antigen specificity of these immunoliposomes. Based on these observations, alkaline phosphate encapsulated PE liposomes were used to directly detect HSV in fluid phase. This immunoliposome assay which does not require washing was shown to be very rapid and sensitive: 35pfu of HSV-1 in 5ul could be detected within 1.5hr.

Animals↗

PubMed1985

Interactions of antigen-sensitized liposomes with immobilized antibody: a homogeneous solid-phase immunoliposome assay.

Dioleoyl phosphatidylethanolamine (DOPE) does not form stable bilayer liposomes at room temperature and neutral pH. However, stable unilamellar liposomes could be prepared by mixing DOPE with a minimum of 12% of a haptenated lipid, N-(dinitrophenylaminocaproyl)-phosphatidylethanolamine (DNP-cap-PE). When the liposomes bound to rabbit anti-DNP IgG that had been adsorbed on a glass surface, lysis of the liposome occurred with the release of the contents into the medium as judged by the fluorescence enhancement of an entrapped self-quenching dye, calcein. On the other hand, incubation of the same liposomes with glass surfaces coated with normal rabbit IgG had little effect. In addition, free anti-DNP IgG induced aggregation of the liposomes but did not cause any dye release. Liposomes composed of dioleoyl phosphatidylcholine (DOPC) and DNP-cap-PE did not lyse when added to the glass surfaces coated with either anti-DNP IgG or normal IgG. A likely mechanism for liposome lysis is that the DNP-cap-PE laterally diffuse to the contact area between the liposome and the glass. Binding of the haptenated lipid with the immobilized and multivalent antibody trap the haptenated lipids in the contact area. As a result of lateral phase separation, lipids may undergo the bilayer to hexagonal phase transition, leading to the leakage of the entrapped dye. Because both the free hapten and the free antibody inhibited the liposome leakage, this process could be used to assay for the free hapten or antibody. We have shown that inhibition assays performed by using this principle can easily detect 10 pmol of free DNP-glycine in 40 microliter. Furthermore, by substituting human glycophorin A, a transmembrane glycoprotein, for the lipid hapten, we have demonstrated that this assay system is also applicable to detect protein antigen with a sensitivity of sub-nanogram level.

Animals↗

PubMed1984

Evidence for a single forskolin-binding site in rat adipocyte membrane. Studies of [14,15-3H]dihydroforskolin binding and adenylate cyclase activation.

[14,15-3H] Dihydroforskolin has been used as a tracer in the study of forskolin binding to adipocyte plasma membrane and the subsequent activation of adenylate cyclase (EC 4.6.1.1) of this membrane. The specific binding of radioactivity to the membrane was rapid, temperature-dependent, saturable, and readily reversible. The equilibrium dissociation reaction constant (KD) for the binding was 13 microM, with a maximum binding (Bmax) of 61 pmol of forskolin per mg of membrane protein. The Hill coefficient was 1.0. The bound [14,15-3H] dihydroforskolin was displaced by forskolin with rate constants of 0.07 X 10(6) M-1 min-1 and 1.2 min-1 for the association and dissociation reactions, respectively (30 degrees C). The equilibrium dissociation constant (KD) was approximately the same as the concentration that produced half-maximum activation (EC50) of the adenylate cyclase of rat adipocyte plasma membrane. There was a linear correlation between forskolin binding and adenylate cyclase activation. The results are consistent with the concept of a single class of binding site which binds forskolin. [14,15-3H] Dihydroforskolin appears to be a potentially useful tracer in the study of the mechanism of activation of the catalytic unit of adipocyte adenylate cyclase.

Adenylyl Cyclases↗

PubMed1983

Cyclic AMP-lowering mediator of insulin.

What appears to be a mediator of insulin action has been successfully produced in rat adipocytes plasma membrane upon its treatment with insulin at concentrations of 50-200 microunits/ml. This apparent mediator, when isolated and presented to adipocyte cells, mimics insulin action in the lowering of hormonally stimulated cAMP levels as well as in stimulating lipogenesis and antilipolysis. The cAMP-lowering activity of such a mediator can be quantitated as insulin-activity equivalents. Insulin at 200 microunits/ml causes, in terms of insulin-activity equivalents, a generation of as much as 50 times more of insulin mediator. The magnitude of amplification is even greater when the amount of insulin bound to its receptor is taken into account in this calculation. The action of insulin in the generation of cAMP-lowering mediator is abolished by the insulin antibody. Inactive insulin analogs do not effectively generate such a mediator activity. On the other hand, while the cAMP-lowering action of insulin shown in the bioassay system is completely inhibited by the insulin antibody, the action of such a mediator is only slightly inhibited by the same antibody. The mediator has a low molecular weight and attributes that resemble a peptide. It can be separated from insulin in a Sephadex G-25 column and has a molecular size smaller than the insulin A-chain but larger than ATP. The molecular weight of this mediator is similar to the insulin mediators isolated by other investigators. In view of the fact that it is small in size and mimics several actions of insulin when used in extracellular situations, its theoretical, as well as practical, implications are substantial.

Adipose Tissue↗

PubMed1983

Forskolin as a probe of adenylate cyclase of adipocytes.

New information regarding the relationship between the agonist receptor and the regulatory and catalytic components of adenylate cyclase has been obtained by applying forskolin in combination with several different activators of adenylate cyclase. Adipocyte adenylate cyclase cannot be fully activated by forskolin; maximal stimulation by forskolin can be further enhanced by Gpp(NH)p. The effect of Gpp(NH)p on membrane enzyme differs between rat and rabbit and between enzymes of membrane-bound and solubilized preparations of rat adipocytes. Conditions that produce maximal activation of the membrane adenylate cyclase have been found. Gpp(NH)p and NaF are both activators of adenylate cyclase but mutually oppose each other. These findings indicate multiple activatable forms of catalytic components and multiple activatable forms of regulatory components of adenylate cyclase.

Adenylyl Cyclases↗

PubMed1981

Response of white adipocyte of mouse and rabbit to catecholamines and ACTH. 1. Correlation of cyclic AMP levels and initial rates of lipolysis.

Hormone stimulated lipolysis of mouse and rabbit adipocytes as measured by both free fatty acid and glycerol release, is proportionally elevated with increase in the adipocyte cAMP level up to 1 nmol/g. The correlation coefficients are 0.94 and 0.97 for FFA/cAMP and glycerol/cAMP respectively. Increments in cAMP greater than 1 nmol/g show no correlation with increase in lipolysis. The release of lipolytic products, glycerol and free fatty acids, from white adipocytes in response to ACTH, epinephrine or norepinephrine was measured using radiochemical assays in short term incubation systems, with cAMP levels measured at the same time and from the same cell sample. Under the conditions studied, epinephrine is a more effective lipolytic hormone than ACTh in mouse adipocyte, and ACTH is more effective than epinephrine in rabbit adipocyte. The effect of catecholamines on the rabbit adipocyte is not modified by phentolamine (10 microM), but it is potentiated by 1-methyl-3-isobutyl xanthine (0.1 mM). The results suggest that cAMP mediates the action of these lipolytic hormones in white adipocytes of mouse and rabbit.

1-Methyl-3-isobutylxanthine↗

PubMed1981

Response of white adipocyte of mouse and rabbit to catecholamines and ACTH. 2. Stability and restoration of activity of hormone-sensitive adenylate cyclase of adipocyte plasma membrane.

The isolated intact white adipocyte of the Swiss mouse responds to both ACTH and catecholamines by an elevation of cAMP levels and an increase in lipolysis. However, in the isolated plasma membrane of the mouse adipocyte, adenylate cyclase loses its responsiveness to ACTH but retains its ability to respond to catecholamines. This lack of responsiveness to ACTH by adenylate cyclase of mouse adipocyte plasma membrane can be overcome, at least partially, by addition of GPP (NH)p, an analog of GTP, to the assay medium. The data on mouse adipocyte membrane suggests that the coupling of ACTH receptor to adenylate cyclase is dependent on GTP and that catecholamine-activation of adenylate cyclase is less dependent on this nucleotide. The isolated intact white adipocyte of adult New Zealand rabbit responds to ACTH, but does not (or only weakly) respond to catecholamines. In contrast to the mouse plasma membrane preparation, adenylate cyclase of adipocyte membrane of the rabbit responds to ACTH. And the addition of GPP(NH)P is not required to demonstrate the CTH: sensitive adenylate cyclase activity. The difference between mouse and rabbit adipocyte membrane in the requirement for GPP(NH)P in ACTH action is not readily explained. The lack of catecholamine sensitivity of rabbit membrane enzyme cannot be reversed by addition of GPP(NH)P or adenosine deaminase. These two adenylate cyclase model systems using mouse and rabbit adipocyte plasma membrane may be useful tools for the study of the specificity and mechanism of action of lipolytic hormones such as ACTH and catecholamines.

Adenylyl Cyclases↗

PubMed1981

Response of white adipocyte of mouse and rabbit to catecholamines and ACTH. 3. Modified binding properties of beta-adrenoreceptor and its decreased coupling efficiency for adenylate cyclase.

Using (-)-[3H]-dihydroalprenolol ([3H]-DHA) and [3H]-norepinephrine ([3H]-NE) as probes, adrenoreceptors in mouse and rabbit adipocyte plasma membranes were studied and compared. The binding of either radioligand can be displaced by propranolol, alprenolol, isoproterenol or norepinephrine. [3H]-Norepinephrine bound to rabbit plasma membrane can be displaced by phentolamine. Based on displacement of radioligand by unlabelled stereoisomers, the binding is stereospecific for the (-) stereoisomer. Quantitative binding determinations of catecholamine radioligand as well as competitive inhibitory and displacement studies with Scatchard plots where appropriate, allowed calculation of binding parameters including Bmax and Kd for both radioligands and for the compounds listed above. Kinetics of displacement were also followed. Based on these binding parameters and kinetics, together with the activity of hormone and NaF sensitive adenylate cyclase, we conclude that both the population of beta-adrenoreceptor and its coupling efficiency for adenylate cyclase of the rabbit plasma membrane appear to be low as compared to that of mouse and rat.

Adenylyl Cyclases↗