Guanine Nucleotide Analogs Research Articles

Determination of Guanine Nucleotide Sensitivity of [<sup>125</sup>I]-Neuropeptide Y Binding in the Rat Pituitary Gland by Quantitative Autoradiography

[125I]-neuropeptide Y receptors were characterized in the rat pituitary gland using quantitative autoradiography. Scatchard analysis of saturation isotherms showed high affinity (Kd 8.5 x 10(-11) M) and single class of binding sites (Bmax 0.22 pmol/mg of protein) in the posterior lobe of the pituitary gland, with no specific binding in the intermediate or anterior lobe. Increasing concentrations of guanine nucleotide analogues and peptide YY inhibited neuropeptide Y binding. In addition, neuropeptide Y binding was pertussis-toxin-sensitive. These results add additional information about a possible neuronal (through neuropeptide Y) and hormonal (through peptide YY) regulation of posterior pituitary function, which is most probably GTP-dependent.

Link between D1 and D2 dopamine receptors is reduced in schizophrenia and Huntington diseased brain.

Dopamine receptor types D1 and D2 can oppose or enhance each other's actions for electrical, biochemical, and psychomotor effects. We report a D1-D2 interaction in homogenized tissue as revealed by ligand binding. D2 agonists lowered the binding of [3H]raclopride to D2 receptors in striatal and anterior pituitary tissues. Pretreating the tissue with the D1-selective antagonist SCH 23390 prevented the agonist-induced decrease in [3H]raclopride binding to D2 sites in the striatum but not in the anterior pituitary, which has no D1 receptors. Conversely, a dopamine-induced reduction in the binding of [3H]SCH 23390 to D1 receptors could be prevented by the D2-selective antagonist eticlopride. Receptor photolabeling experiments confirmed both these D1-D2 interactions. The blocking effect by SCH 23390 was similar to that produced by a nonhydrolyzable guanine nucleotide analogue, and SCH 23390 reduced the number of agonist-labeled D2 receptors in the high-affinity state. Thus, the D1-D2 link may be mediated by guanine nucleotide-binding protein components. The link may underlie D1-D2 interactions influencing behavior, since the link was missing in over half the postmortem striata from patients with schizophrenia and Huntington disease (both diseases that show some hyperdopamine signs) but was present in human control, Alzheimer, and Parkinson striata.

The Biphasic Calcium Dose-Response Curve for Parathyroid Hormone Secretion in Electropermeabilized Adult Bovine Parathyroid Cells

We have used the method of electropermeabilization to measure the dependence of PTH secretion on internal calcium concentration in adult bovine parathyroid cells. The dose-response curve is biphasic, with a peak at 10(-7) M calcium. This result differs significantly from the dose-response curves that have been determined by this method for many other secretory systems, where secretion requires much more calcium and the dependence of secretion on calcium is monotonic. Guanine nucleotide analogs did not modify the calcium dose-response curve of PTH secretion in the electropermeabilized parathyroid cells. The unique properties of adult parathyroid cell secretion are analogous to the unique properties of parathyroid calcium-activated potassium channels, which differ from calcium-activated potassium channels of other cells in that they open at unusually low calcium concentrations and have a peak open probability at about 10(-7) M calcium. We suggest that the opening of these channels in secretory vesicles is required for secretion.

Selective changes in mu opioid receptor properties induced by chronic morphine exposure.

Chronic infusion of morphine to guinea pigs produced selective changes in mu agonist binding properties in cerebrocortical membrane preparations. Employing the mu-selective opioid agonist [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAMGO) in direct binding studies and in competition of labeled antagonist binding, we found that the major changes were a decrease in the number of sites with high affinity for agonist, a small reduction in total receptor number, and a loss in the ability of guanosine 5'-[gamma-thio]triphosphate to regulate binding. A fraction of high-affinity mu receptors appeared to retain their high affinity for agonist and their sensitivity to guanine nucleotide analogue after the induction of morphine tolerance, possibly because the morphine concentrations achieved in brain were insufficient to uncouple all mu receptors from associated guanine nucleotide-binding regulatory proteins. Some membrane preparations were treated with pertussis toxin, which has been shown to functionally uncouple mu opioid receptors from their effector systems. In these preparations, a single agonist-affinity state of the receptor was observed. The apparent dissociation constant for this affinity state in pertussis toxin-treated membranes was similar to the lower-affinity state observed in preparations from morphine-tolerant animals. In contrast to the changes observed at mu opioid binding sites, no significant changes in agonist affinity or binding density were observed for selective delta or kappa agonists, consistent with the development of selective tolerance at mu receptors.

Kinetics of cell-free activation of neutrophil NADPH oxidase. Effects of neomycin and guanine nucleotides.

The effects of neomycin, fluoride and the non-hydrolysable guanine nucleotide analogue GTP gamma S on the kinetics of cell-free activation of NADPH oxidase in membranes of resting human neutrophils were investigated. Arachidonate-mediated activation of the oxidase followed a first-order reaction course (kobs. = 0.39 min-1 at 26 degrees C). In the presence of NaF during the activation process, activity was enhanced while the activation rate was slightly reduced (kobs. = 0.25 min-1 at 26 degrees C). Neomycin blocked activation (half-maximal effect at 25 microM) without affecting rates of superoxide release by preactivated enzyme in vitro or in vivo. In spite of reduced specific activity neither the first-order rate constant of the activation nor the Km of the oxidase were altered by neomycin. Oxidase activated in the presence of GTP gamma S exhibited increased specific activity and unchanged Km; the course of the reaction deviated from first-order kinetics. Kinetic evidence is presented for two separate activation reactions: a GTP gamma S-independent, basal, first-order process and a GTP gamma S-dependent sigmoid activation process. The results are compatible with the existence in neutrophil membranes of two separate pools of dormant oxidase. An alternative scheme of the formation of two active forms of NADPH oxidase is also presented.

Guanine nucleotides stimulate hydrolysis of phosphatidyl inositol bis phosphate in human myelin membranes

Phosphodiesterase activity was stimulated in myelin membranes in the presence of guanine nucleotide analogues. This activity was reduced in myelin membranes which had been adenosine diphosphate ribosylated in the presence of cholera toxin which ADP-ribosylated three proteins of Mr 46,000, 43,000 and 18,500. Aluminum fluoride treatment of myelin had the same stimulatory effects on phosphodiesterase activity as did the guanine nucleotides.

Guanine nucleotide-mediated inhibition of opioid agonist binding: Modulatory effects of ions and of receptor occupancy

We have analysed the potency of GTP, GDP and their analogues in reducing [ 3H]DADLE binding to opioid receptors in NG 108-15 cell membranes. Under conditions where non-specific hydrolysis and transphosphorylation is inhibited, the following rank order of potency was found: GDP ≥ GTPγS > GTP / GDPβS ≥ GDPNH 2 > GppNHp / GMP. Remarkably, the slopes for the inhibition curves of GTP, GDP and their thiosubstituted analogues, but not of GDPNH 2 and GppNHp, were extremely shallow, indicating either negative cooperativity or the existence of two states for the guanine nucleotide binding proteins, that both can mediate the effect of nucleotides on agonist receptor binding. The potencies of the different guanine nucleotide analogues, except that of GppNHp, were increased by the presence of sodium or chloride ions in the assay medium. Magnesium also affected GTP-mediated inhibition of opioid agonist binding since it decreased the ic 50 of the nucleotide and steepened the slope of the inhibition curve. The ic 50s of nucleotides and the slopes of their inhibition curves were also dependent on the extent of receptor occupancy by the agonist. From these data we conclude that (1) either diphospho- or triphosphonucleotides can regulate agonist binding. (2) Magnesium, sodium and chloride, by acting at different components of the receptorJG protein complex produce similar effects on nucleotide mediated regulation of agonist binding. (3) A mutual influence exists between receptor occupancy by agonists and G protein-mediated guanine nucleotide effect on the receptor.

Different requirements for protein kinase C activation and Ca2+-independent insulin secretion in response to guanine nucleotides: Endogenously generated diacylglycerol requires elevated Ca2+ for kinase C insertion into membranes

Abstract Electrically permeabilized RINm5F cells were used to assess the factors required for activation of protein kinase C (PKC) and insulin secretion. PKC was activated either by phorbol 12-myristate 13-acetate (PMA) or by the generation of endogenous diacylglycerol in response to the nonhydrolyzable guanine nucleotide analog guanosine 5'-O-(thiotriphosphate) (GTP gamma S). As shown previously, both PMA and GTP gamma S elicit Ca2+-independent insulin secretion. This effect was mimicked by guanyl-5'-yl imidodiphosphate (Gpp(NH)p) but not by guanosine 5'-O-(3-fluorotriphosphate) and guanosine 5'-O-(3-phenyltriphosphate) possessing only one negative charge in the gamma-phosphate group. The action of PMA was mediated by PKC, since the agent caused both phosphorylation of specific protein substrates and association of the enzyme with cellular membranes. This translocation was independent of the Ca2+ concentration employed. In contrast, GTP gamma S only promoted association of PKC with membranes at 10(-6) and 10(-5) M Ca2+ and failed to alter significantly protein phosphorylation in the absence of Ca2+. Neither Gpp(NH)p, which stimulates insulin release, nor the other two GTP analogs, increased the proportion of PKC associated with membranes. To verify that the Ca2+-dependent effect of GTP gamma S on PKC is due to activation of phospholipase C, we measured the generation of diacylglycerol. GTP gamma S indeed stimulated diacylglycerol production in the leaky cells by about 50% at Ca2+ concentrations between 10(-7) and 10(-5) M, an effect which was almost abolished in the absence of Ca2+. Thus, at 10(-7) M Ca2+, the concentration found in resting intact cells, the generated diacylglycerol was not sufficient to cause PKC insertion into the membrane, demonstrating that both elevated Ca2+ and diacylglycerol are necessary for translocation to occur. It is concluded that while PKC activation by PMA elicits Ca2+-independent insulin secretion, the kinase seems not to mediate the stimulatory action of GTP analogs in the absence of Ca2+.

An investigation into the mechanisms of inhibition of calcium channel currents in cultured sensory neurones of the rat by guanine nucleotide analogues and (−)− baclofen

1. The mechanism of inhibition of calcium channel currents by the guanine nucleotide analogue guanosine 5'-O-3 thiotriphosphate (GTP-gamma-S) and by the GABAB agonist (-)-baclofen has been studied in cultured dorsal root ganglion neurones of the rat. The inhibition by GTP-gamma-S is particularly characterized by an abolition of the transient component of calcium channel currents carried either by Ba2+ (IBa) or by Ca2+ (ICa). 2. The effect of agents increasing intracellular cyclic AMP levels has been examined. Neither internal cyclic AMP nor forskolin prevented the inhibition of IBa by baclofen. Neither forskolin nor pretreatment of cells with cholera toxin prevented the inhibition of the transient component of IBa by GTP-gamma-S. However, both these treatments increased the amplitude of the sustained IBa in the presence of GTP-gamma-S. The ATP analogue adenosine imido-diphosphate which inhibits many ATP requiring enzymes did not prevent the effect of GTP-gamma-S although it reduced the amplitude of IBa. 3. Baclofen (100 microM) produced a 22 +/- 2% increase in inositol phosphate production in 30 s, whereas the increase produced by bradykinin (1 microM) was 70 +/- 14%. However, unlike baclofen, bradykinin did not inhibit IBa or ICa in these cells. 4. The effect of protein kinase C inhibitors was examined. Polymixin B (20 microM in patch pipette) had no effect on the inhibition of IBa by baclofen or GTP-gamma-S. A higher concentration (100 microM) alone inhibited IBa and no further inhibition by baclofen was observed. Neither H7 (50 microM) nor staurosporine (100 nM), applied extracellularly, prevented the response to GTP-gamma-S. 5. The protein kinase C activator di-octanoyl glycerol (20 microM) did not inhibit IBa. Arachidonic acid (100 microM) also produced no inhibition of IBa. 6. In conclusion we have obtained no evidence that a second messenger system mediates the inhibition of calcium channel currents by GTP-gamma-S or baclofen in dorsal root ganglion neurones. These results support the hypothesis that GABAB receptors are directly coupled to calcium channels by G proteins.

Homologous desensitization of ovarian luteinizing hormone/human chorionic gonadotropin-responsive adenylyl cyclase is dependent upon GTP.

Homologous desensitization of ovarian LH/hCG-responsive adenylyl cyclase in cell-free systems has been reported to be dependent upon either ATP or GTP. We investigated LH/hCG-dependent desensitization of adenylyl cyclase in rabbit or pig ovarian follicular membranes and included adenyl-5'-yl imidodiphosphate to prevent nucleotide triphosphate degradation. It was found that GTP supported LH/hCG-induced desensitization with an apparent Km of approximately 0.1 microM in rabbit or pig ovarian membranes. Other nucleotide triphosphates were 100-1000 times less potent than GTP in supporting desensitization. Several nonhydrolyzable GTP analogs and the GDP analog guanosine-5'-O-(2-thiodiphosphate) would not support hCG-induced desensitization of ovarian adenylyl cyclase. Instead, these guanine nucleotide analogs were all inhibitors of GTP-supported hormone-dependent desensitization. Cholera toxin had no effect on LH-dependent desensitization. These results establish that GTP is the preferred nucleotide for homologous desensitization of the LH/hCG-sensitive adenylyl cyclase and that the GTP-dependent mechanism differs from that typically associated with guanine nucleotide-binding proteins. The GTP-dependent mechanism of desensitization of the LH receptor distinguishes it from the ATP-dependent desensitization of the beta-adrenergic receptor.

Properties of calcium stores and transient outward currents in single smooth muscle cells of rabbit intestine.

1. Single dispersed cells obtained by collagenase treatment of longitudinal muscle of rabbit small intestine were voltage clamped with low-resistance patch pipettes and membrane current was measured. 2. In cells held at -20 or -30 mV, a discharge of spontaneous transient outward currents (STOCs) was usually seen; these are believed to represent the sporadic release of calcium from storage sites in the cell in relation to TEA-sensitive, 4 AP-resistant, calcium-activated potassium channels. 3. Caffeine (20 mM) externally applied, accelerated and then abolished STOCs; carbachol (0.1 mM) had similar effects; the initial burst of STOCs was often carried on a large, temporary, outward current which could occur alone. This was suggested to be caused by the rapid release of stored calcium in relation to calcium-activated potassium channels. 4. If STOCs were abolished by caffeine (or carbachol) then carbachol (or caffeine) did not evoke outward current indicating that these drugs act on the same calcium store but by different pathways. Inclusion of ryanodine (10(-8)-10(-4) M) in the patch pipette abolished STOCs soon after establishing whole-cell recording mode; afterwards, outward current to caffeine or to carbachol could not be evoked. 5. STOCs were quickly abolished in cells patched with pipettes filled with GTP gamma S (0.1-1 mM) or Gpp(NH)p (0.1-1 mM) but were large or normal in size in cells where GDP beta S (0.1-1 mM) was included in the pipette. GTP gamma S or Gpp(NH)p in the cell abolished outward current to caffeine or to carbachol, but had no effect on calcium-activated potassium channel activity in isolated patches or on a TEA-sensitive, 4-AP-resistant, outward potassium current evoked in single cells by stepping positively from a -20 mV holding potential. These results suggest that the effect of guanine nucleotide analogues are on the calcium store rather than on calcium-activated potassium channels. 6. The effects of GTP gamma S or Gpp(NH)p could be explained if they depleted calcium stores via a G-protein mechanism; this effect may involve activation of phospholipase C enzyme (PLC) and D-myo-inositol 1,4,5-trisphosphate (IP3) production as well as a direct effect on stores. However a separate G-protein-independent pathway of activation of PLC by muscarinic receptor activation may exist as calcium release by carbachol was large or normal in cells filled with GDP beta S.

G regulatory proteins and muscarinic receptor signal transduction in mucous acini of rat submandibular gland

The involvement of G regulatory proteins in muscarinic receptor signal transduction was examined in electrically permeabilized rat submandibular acinar cells. The guanine nucleotide analog, GTPγS, caused the dose dependent hydrolysis of membrane phosphatidylinositol 4, 5-bisphosphate to release IP 3. This response was insensitive to pertussis toxin treatment and was duplicated by NaF but not GDPβS. Enhanced IP 3 synthesis was observed with a combination of GTPγS and carbachol. Exogenous IP 3, as well as carbachol and GTPγS, provoked the release of sequestered 45Ca 2+ from non-mitochondrial stores. In intact cells, carbachol significantly reduced the level of cyclic AMP induced by the β-adrenergic agonist, isoproterenol, to 69% of its normal value. Pertussis toxin abolished this inhibitory action of carbachol on cyclic nucleotide levels. These results suggest that muscarinic receptors are coupled to two separate G regulatory proteins in submandibular mucous acini - the pertussis toxin - insensitive G p of the phosphoinositide transduction pathway associated with elevated cytosolic calcium levels, and the pertussis toxin - sensitive G i inhibitory protein of the adenylate cyclase complex.

Somatostatin receptors on rat cerebrocortical membranes. Structural characterization of somatostatin-14 and somatostatin-28 receptors and comparison with pancreatic type receptors.

Somatostatin binding and cross-linking to its receptors on rat cerebrocortical membranes were characterized with [125I-Tyr1]somatostatin-14 and [125I-Leu8, D-Trp22, Tyr25]somatostatin-28. When [125I-Tyr1]somatostatin-14 was cross-linked to its receptors with the photoreactive cross-linker, N-(5-azido-2-nitrobenzoyloxy)succinimide, the hormone was specifically associated with a Mr = 72,000 protein band in the presence or absence of reducing agents. Affinity labeling of the Mr = 72,000 protein band was decreased with increasing concentrations of unlabeled somatostatin-14 and nonhydrolyzable guanine nucleotide analog, guanyl-5'-yl imidodiphosphate (Gpp(NH)p). Pretreatment of cerebrocortical membranes with islet-activating protein resulted in a decrease in subsequent labeled somatostatin-14 binding and affinity-labeling of the protein and abolished an inhibitory effect of somatostatin-14 on vasoactive intestinal peptide-stimulated increase in adenylate cyclase activity. When the affinity-labeled protein was solubilized with Zwittergent 3-12 and adsorbed to wheat germ agglutinin-agarose, it was eluted by N-acetylglucosamine. [125I-Leu8, D-Trp22, Tyr25]somatostatin-28 cross-linking to cerebrocortical and pancreatic membranes with the same photoreactive agent revealed specifically labeled protein bands of a Mr = 74,000 in cerebrocortical membranes and a Mr = 94,000 in pancreatic membranes, respectively. These results suggest that: 1) somatostatin receptor on cerebrocortical membranes is a monomeric glycoprotein with a Mr = 70,000 binding subunit, coupled to guanine nucleotide regulatory protein, and 2) the Mr = 70,000 protein may be a common receptor for somatostatin-28 and somatostatin-14 and is distinct from a common pancreatic type receptor.

Reconstitution of partially purified opioid receptors with a GTP-binding protein

Partially purified opioid receptors, obtained from rat brains using an affinity resin, AF-Amino Toyopearl with [D-Ala2, Leu5]enkephalin, were reconstituted with an inhibitory GTP-binding protein (Gi). In the reconstituted system, the displacement curve for the binding of a delta-agonist, [D-Ala2, D-Leu5]enkephalin, showed two states, high and low affinity binding ones, with different affinities for the agonist. The high affinity binding was eliminated by the addition of a guanine nucleotide analog to the system. These results directly showed that opioid receptors, at least the delta-type, could interact with Gi.

G-proteins, the inositol lipid signalling pathway, and secretion.

The formation of the second messenger cyclic AMP (cAMP) is known to be coupled to its receptor via a guanine nucleotide regulatory protein, GS. Ca2+-mobilizing receptors stimulate the hydrolysis of phosphatidylinositol bisphosphate (PtdIns(4,5)P2), which generates two intracellular signals Ins(1,4,5)P3 and diacylglycerol. We review the evidence that this signalling system is also composed of three types of proteins: receptor, G-protein and effector. The G-protein that couples to the effector, polyphosphoinositide phosphodiesterase (PPI-PDE), is a novel G-protein, GP, which is a substrate for pertussis toxin in some cells (e.g. neutrophils and platelets) but not others (e.g. pancreatic acinar cells and GH3 cells). This implies that GP is not a single G-protein but encompasses a family of proteins that can activate PPI-PDE. We have also identified a role for another G-protein, GE, which is involved in the secretory process in mast cells and neutrophils. In this case, neither the receptor nor effector has been identified and the main evidence for proposing this second G-protein is based on the ability of guanine nucleotide analogues (e.g. GTP gamma S) to stimulate secretion independently of PPI-PDE activation.

ADP-ribosylation with pertussis toxin modulates the GTP-sensitive opioid ligand binding in digitonin-soluble extracts of rat brain membranes.

Pertussis toxin-catalyzed ADP-ribosylation of the guanine nucleotide-binding proteins Gi and Go is shown to proceed in Mg2+-digitonin extracts from rat brain; the Mr 41,000 and Mr 39,000 peptides are labelled there as in the membranes. The ADP-ribosylation in detergent solution retains the differential sensitivity to guanine nucleotide analogues. This reaction also removes the partial inhibition by the guanine nucleotides of the binding of opioid agonists, as does the same treatment in the membranes. The partial inhibition of agonist binding by Na+, however, is left unchanged. The binding of the antagonist naloxone is little affected by Na+ or by guanine nucleotides in the treated membranes, but the treated soluble receptors show an enhanced binding in high-Na+ medium, although still guanine nucleotide insensitive. The data suggest that the toxin reaction in the absence of guanine nucleotides and agonist stabilizes the opioid receptor in a receptor-G-protein coupled state which is no longer sensitive to guanine nucleotides but retains its sensitivity to the Na+ ions.

Reconstitution of SEC gene product-dependent intercompartmental protein transport

Transport of α-factor precursor from the endoplasmic reticulum to the Golgi apparatus has been reconstituted in gently lysed yeast spheroplasts. Transport is measured through the coupled addition of outer-chain carbohydrate to [35S]methionine-labeled α-factor precursor translocated into the endoplasmic reticulum of broken spheroplasts. The reaction is absolutely dependent on ATP, stimulated 6-fold by cytosol, and occurs between physically separable sealed compartments. Transport is inhibited by the guanine nucleotide analog GTPγS. sec23 mutant cells have a temperature-sensitive defect in endoplasmic reticulum-to-Golgi transport in vivo. This defect has been reproduced in vitro using sec23 membranes and cytosol. Transport at 30°C with sec23 membranes requires addition of cytosol containing the SEC23 (wild-type) gene product. This demonstrates that an in vitro interorganelle transport reaction depends on a factor required for transport in vivo. Complementation of sec mutations in vitro provides a functional assay for the purification of individual intercompartmental transport factors.

Photoactivation of intracellular guanosine triphosphate analogues reduces the amplitude and slows the kinetics of voltage-activated calcium channel currents in sensory neurones

The influence of guanine nucleotide analogues on calcium channel currents in cultured rat dorsal root ganglion neurones has been studied using a technique in which the rate of diffusion of the analogues to their site of action is by-passed by photochemical release of the analogues within the neurones. The 1(2-nitrophenyl)ethyl P3-ester derivatives of guanosine 5'-0(3-thio)triphosphate (caged GTP-gamma-S) and 5'-guanylylimidodiphosphate (caged GMP-PNP) were synthesised and found to be completely photolysable by light, yielding free GTP-gamma-S and GMP-PNP. Calcium channel currents were recorded using the whole cell patch technique and either caged GTP-gamma-S or caged GMP-PNP (2 mM) were included in the patch pipette. Stable currents were recorded for 5-10 min, and a single pulse of 300-350 nm irradiation was directed using a liquid light guide onto the recording dish. Calcium channel currents were then recorded every 30-120 s following photochemical release of approximately 20 microM GTP-gamma-S. The peak calcium channel current was reduced by about 70% with a slow time course [t1/2 1.5 +/- 0.2 min (mean +/- SEM); n = 5]. The transient component of the peak current was usually completely abolished, whereas the sustained current measured at the end of the 100 ms depolarising pulse was less affected. Qualitatively similar effects were observed on photolysis of caged GMP-PNP.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic influences on agonist binding to cardiac β-receptors

Regulation of β-adrenoceptor-agonist function in the Maudsley Reactive (MR/Har) and the Maudsley Non-Reactive (MNRA/Har) rat strains was assessed by comparison of isoproterenol competition for [ 125I]iodocyanopindolol (ICYP) binding sites in crude left ventricular homogenate preparations. Non-linear, least-squares analysis of isoproterenol competition for ICYP binding in the absence of guanine nucleotide revealed different proportions of high- and low-affinity receptors in the two strains; MR/Har rats (59±3.3%) had a significantly greater proportion of receptors in the high-affinity state than the MNRA/Har rats (41±4.5%). Addition of the non-hydrolyzable guanine nucleotide analog guanylylimidodiphosphate (Gpp(NH)p) converted receptors to the low-affinity state. Analysis of Gpp(NH)p concentration-response curves in left ventricular homogenates of the two strains revealed that the MR/Har strain had a significantly (P<0.02) lower EC 50 for guanyl nucleotide inhibition of isoproterenol competition for ICYP binding than the MNRA/Har. Confirming previous experimental results, a significantly (P<0.04) greater density of ventricular β-receptors was found in MR/Har rats (13.16±0.92 fmol/mg protein) than in MNRA/Har rats (10.81±0.63 fmol/mg protein). Left ventricular catecholamine levels were found to be correlated inversely with β-adrenoceptor density in the two strains; norepinephrine (NE) and epinephrine (EPI) concentrations (ng/mg protein) in left ventricle were 12.19±0.94 for NE and 0.165±0.038 for EPI in MNRA/Har, and 8.73±0.95 and 0.018±0.018, respectively, in MR/Har. All other parameters of agonist interactions with the cardiac β-adrenoceptor for the MR/Har and MNRA/Har rat strains were similar [the IC 50 for displacement of ICYP binding by isoproterenol, the accompanying Hill coefficients in the Gpp(NH)p present and absent condition, the K d of the high- and low-affinity states in the absence of Gpp(NH)p, and the K d of the uniform low-affinity state in the presence of Gpp(NH)p]. We hypothesize that the strain-dependent differences in high-affinity state formation reported here may account for some of the in vivo differences in cardiovascular function previously demonstrated in the Maudsley rats.

Stimulation of arachidonic acid release by guanine nucleotide in saponin-permeabilized neutrophils: Evidence for involvement of GTP-binding protein in phospholipase A 2 activation

Addition of a guanine nucleotide analog, guanosine 5′- O-(thiotriphosphate) (GTPγS)(1–100 μ m) induced release of [ 3H]arachidonic acid from [ 3H]arachindonateprelabeled rabbit neutrophils permeabilized with saponin. The chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced arachidonate release was enhanced by GTPγS, Ca 2+, or their combination. Ca 2+ alone (up to 100 μ m) did not effectively stimulate lipid turnover. However, the combination of fMLP plus GTPγS elicited greater than additional effects in the presence of resting level of free Ca 2+. The addition of 100 μ m of GTPγS reduced the Ca 2+ requirement for arachidonic acid liberation induced by fMLP. Pretreatment of neutrophils with pertussis toxin resulted in the abolition of arachidonate release and diacylglycerol formation. Neomycin (1 m m) caused no significant reduction of arachidonate release. In contrast, about 40% of GTPγS-induced arachidonate release was inhibited by a diacylglycerol lipase inhibitor, RHC 80267 (30 μ m). These observations indicate that liberation of arachidonic acid is mediated by phospholipase A 2 and also by phospholipase C/diacylglycerol lipase pathways. Fluoride, which bypasses the receptor and directly activates G proteins, induced arachidonic acid release and diacylglycerol formation. The fluoride-induced arachidonate release also appeared to be mediated by these two pathways. The loss of [ 3H]arachidonate was seen in phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine. These data indicate that a G protein is involved between the binding of fMLP to its receptor and activation of phospholipase A 2, and also that the arachidonic acid release is mediated by both phospholipase A 2 and phospholipase C/diacylglycerol lipase.