Darcy permeability of agarose-glycosaminoglycan gels analyzed using fiber-mixture and donnan models

Agarose-glycosaminoglycan (GAG) membranes were synthesized to provide a model system in which the factors controlling the Darcy (or hydraulic) permeability could be assessed in composite gels of biological relevance. The membranes contained a GAG (chondroitin sulfate) that was covalently bound to agarose via terminal amine groups, and the variables examined were GAG concentration and solution ionic strength. The addition of even small amounts of GAG (0.4 vol/vol %) resulted in a twofold reduction in the Darcy permeability of 3 vol/vol % agarose gels. Electrokinetic coupling, caused by the negative charge of the GAG, resulted in an additional twofold reduction in the open-circuit permeability when the ionic strength was decreased from 1 M to 0.01 M. A microstructural hydrodynamic model was developed, based on a mixture of neutral, coarse fibers (agarose fibrils), and fine, charged fibers (GAG chains). Heterogeneity within agarose gels was modeled by assuming that fiber-rich, spherical inclusions were distributed throughout a fiber-poor matrix. That model accurately predicted the Darcy permeability when the ionic strength was high enough to suppress the effects of charge, but underestimated the influence of ionic strength. A more macroscopic approach, based on Donnan equilibria, better captured the reductions in Darcy permeability caused by GAG charge.     

Modulation of [3H]Muscimol Binding in Rat Cerebellar and Cerebral Cortical Membranes by Picrotoxin, Pentobarbitone, and Etomidate

Modulation of [3H]muscimol binding by picrotoxin, pentobarbitone, and etomidate was investigated in rat cerebellar and cerebral cortical membranes. In cerebellum, at 37 degrees C in the presence of chloride ions (150 mM), picrotoxin and picrotoxinin decreased specific [3H]muscimol binding to 43 +/- 3% of control, with an EC50 of 1.2 +/- 0.1 microM. [3H]Muscimol saturation experiments in the presence and absence of picrotoxin indicated that the picrotoxin effect was primarily due to a loss of high-affinity muscimol sites with KD approximately equal to 10 nM. Pentobarbitone enhanced specific [3H]muscimol binding to 259 +/- 3% of control, with EC50 = 292 +/- 37 microM, and etomidate increased binding to 298 +/- 18%, with EC50 = 7.1 +/- 1.0 microM. The influence of temperature and chloride ion concentration on these effects was investigated by comparing experiments at 37 and 0 degrees C in the presence or absence of chloride at constant ionic strength. The results indicate that studies at 0 degrees C underestimate the coupling between GABA receptors and barbiturate sites and that they greatly overestimate the importance of chloride ions in this phenomenon. In cerebral cortical membranes (37 degrees C, 150 mM Cl-), the effect of picrotoxin was similar to that observed in cerebellum, whereas the effects of pentobarbitone and etomidate were greater, but occurred at higher concentrations.     

Chromatographic Analyses of the Serotonin 5-HT1A Receptor Solubilized from the Rat Hippocampus

Serotonin 5-HT1A receptors in rat hippocampal membranes were solubilized by 10 mM 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and chromatographed on various gels in an attempt to design a relevant protocol for their (partial) purification. In particular, an affinity gel made of the 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) derivative 8-methoxy-2-[(N-propyl, N-butylamino)amino]tetralin (8-MeO-N-PBAT) coupled to Affigel 202 was specially developed for this purpose. First, studies of the effects of various compounds (detergents, lipids, reducing agents, sugars, etc.) on the specific binding of [3H]8-OH-DPAT and on the rate of heat-induced inactivation of solubilized 5-HT1A sites led to a buffer composed of 50 mM Tris-HCl, 50 microM dithiothreitol, 1 mM CHAPS, 10% glycerol, 0.1 mM MnCl2, and 50 micrograms/ml of cholesteryl hemisuccinate, pH 7.4, ensuring a high degree of stability of solubilized 5-HT1A sites, compatible with chromatographic analyses for 2-4 days at 4 degrees C. Adsorption and subsequent elution of [3H]8-OH-DPAT specific binding sites were found with several chromatographic gels, including wheat germ agglutinin-agarose, phenyl-Sepharose, hydroxylapatite-Ultrogel, diethylaminoethyl (DEAE)-Sepharose, and DEAE-Sephacel. Similarly, 8-MeO-N-PBAT-Affigel 202 allowed the adsorption and subsequent elution (by 1 mM 5-HT) of active 5-HT1A binding sites solubilized from rat hippocampal membranes. The two-step chromatography using 8-MeO-N-PBAT-Affigel 202 followed by wheat germ agglutinin-agarose gave a fraction enriched (by at least 400-fold) in 5-HT1A sites. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this partially purified fraction revealed a major protein band with Mr close to 60,000.     

delta-Opioid receptors exhibit high efficiency when activating trimeric G proteins in membrane domains

Low-density membrane fragments (domains) were separated from the bulk of plasma membranes of human embryonic kidney (HEK)293 cells expressing a delta-opioid (DOP) receptor-Gi1alpha fusion protein by drastic homogenization and flotation on equilibrium sucrose density gradients. The functional activity of trimeric G proteins and capacity of the DOP receptor to stimulate both the fusion protein-linked Gi1alpha and endogenous pertussis-toxin sensitive G proteins was measured as d-Ala2, d-Leu5-enkephalin stimulated high-affinity GTPase or guanosine-5'-[gamma-35S]triphosphate ([35S]GTPgammaS) binding. The maximum d-Ala2-d-Leu5 enkephalin (DADLE)-stimulated GTPase was two times higher in low-density membrane fragments than in bulk of plasma membranes; 58 and 27 pmol/mg/min, respectively. The same difference was obtained for [35S]GTPgammaS binding. Contrarily, the low-density domains contained no more than half the DOP receptor binding sites (Bmax = 6.6 pmol/mg versus 13.6 pmol/mg). Thus, when corrected for expression levels of the receptor, low-density domains exhibited four times higher agonist-stimulated GTPase and [35S]GTPgammaS binding than the bulk plasma membranes. The regulator of G protein signaling RGS1, enhanced further the G protein functional activity but did not remove the difference between domain-bound and plasma membrane pools of G protein. The potency of the agonist in functional studies and the affinity of specific [3H]DADLE binding to the receptor were, however, the same in both types of membranes - EC50 = 4.5 +/- 0.1 x 10(-8) and 3.2 +/- 1.4 x 10(-8) m for GTPase; Kd = 1.2 +/- 0.1 and 1.3 +/- 0.1 nm for [3H]DADLE radioligand binding assay. Similar results were obtained when sodium bicarbonate was used for alkaline isolation of membrane domains. By contrast, detergent-insensitive membrane domains isolated following treatment of cells with Triton X100 exhibited no DADLE-stimulated GTPase or GTPgammaS binding. Functional coupling between the DOP receptor and cognate G proteins was also blocked by high-energy ultrasound and repeated freezing-thawing. Our data indicate, for the first time, that membrane domains isolated using 'detergent-free' procedures exhibit higher efficiency of coupling between a G protein-coupled receptor and its corresponding G protein(s) than bulk plasma membranes. Detergent-extraction diminishes these interactions, even when the receptor and G proteins are physically tethered together.     

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.     

The formylpeptide chemotactic receptor on rabbit peritoneal neutrophils. I. Evidence for two binding sites with different affinities

F Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe binding to rabbit peritoneal neutrophils and purified membranes were measured at 4 degrees C silicone oil centrifugation assays, and the results were analyzed by the LIGAND computer program, which permits analysis of ligand binding to multiple classes of binding sites. LIGAND analysis of peptide binding to intact neutrophil indicated that both f Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe detected two population of binding sites. The apparent Kd values for f Met-Leu-[3H]Phe binding were 1.6 +/- 1.0 X 10(-9) M and 2.2 +/- 0.9 X 10(-8) M, respectively, and 3.1 +/- 0.2 X 10(-9) M and 1.2 +/- 0.6 X 10(-7) M for f Nle-Leu-[3H]Phe. Furthermore, the higher affinity sites detected on whole cells comprised approximately 15 to 30% of the total sites. Two populations of binding sites were also detected on purified neutrophil plasma membranes by both radiolabeled chemotactic peptides. LIGAND analysis of peptide binding to purified membranes yielded apparent Kd values of 5.0 +/- 2.5 X 10(-10) M and 4.8 +/- 0.6 X 10(-8) M for f Met-Leu-[3H]Phe binding, and 4.7 +/- 4.2 X 10(-10) M and 3.0 +/- 1.3 X 10(-8) M for f Nle-Leu-[3H]Phe. The percentage of higher affinity sites detected by f Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe on purified membranes were 1 to 5% of the total sites detected. These data are consistent either with the existence of two independent binding sites for formylpeptides on rabbit neutrophils or receptor negative cooperativity.     

Characterization of Opioid Receptors in Cultured Neurons

The appearance of mu-, delta-, and kappa-opioid receptors was examined in primary cultures of embryonic rat brain. Membranes prepared from striatal, hippocampal, and hypothalamic neurons grown in dissociated cell culture each exhibited high-affinity opioid binding sites as determined by equilibrium binding of the universal opioid ligand (-)-[3H]bremazocine. The highest density of binding sites (per mg of protein) was found in membranes prepared from cultured striatal neurons (Bmax = 210 +/- 40 fmol/mg protein); this density is approximately two-thirds that of adult striatal membranes. By contrast, membranes of cultured cerebellar neurons and cultured astrocytes were devoid of opioid binding sites. The opioid receptor types expressed in cultured striatal neurons were characterized by equilibrium binding of highly selective radioligands. Scatchard analysis of binding of the mu-specific ligand [3H]D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin to embryonic striatal cell membranes revealed an apparent single class of sites with an affinity (KD) of 0.4 +/- 0.1 nM and a density (Bmax) of 160 +/- 20 fmol/mg of protein. Specific binding of (-)-[3H]bremazocine under conditions in which mu- and delta-receptor binding was suppressed (kappa-receptor labeling conditions) occurred to an apparent single class of sites (KD = 2 +/- 1 nM; Bmax = 40 +/- 15 fmol/mg of protein). There was no detectable binding of the selective delta-ligand [3H]D-Pen2,D-Pen5-enkephalin. Thus, cultured striatal neurons expressed mu- and kappa-receptor sites at densities comparable to those found in vivo for embryonic rat brain, but not delta-receptors.     

Prolactin binding in the developing rat fetal liver

The binding of prolactin by fetal rat liver cell membrane fractions from 17 to 21 days gestation was studied. Particulate liver membranes were prepared in Dulbecco's Phosphate Buffered Saline (PBS) by ultracentrifugation and incubated at 22 degrees C for 16 hours with [125I] iodo-human growth hormone (hGH). Non-specific binding was assessed by parallel incubations in the presence of a 2000-fold excess ovine prolactin. Specific prolactin binding sites were detected only at 21 days gestation (2932 +/- 401 cpm/mg protein) in freshly prepared membranes. On freezing at -20 degrees C for 24 to 48 hours, the membranes of 20 days gestation animals were able to specifically bind prolactin (1295 +/- 239 cpm/mg protein). Freezing led to a 45 +/- 7% increase (4270 +/- 701 cpm/mg protein) in prolactin binding at 21 days gestation. No hormonal binding was detected from 17 through 19 days gestation in either fresh or freeze-thawed membranes. Scatchard analysis revealed a high affinity binding site with a Ka of approximately 1.4 X 10(8)M-1 in both fresh and freeze-thawed membrane preparations. The data show that 1) prolactin receptors appear in liver only during late fetal life and that 2) freezing of membranes may unmask binding sites that are initially unavailable to specifically bind prolactin.     

Agmatine oxidation by copper amine oxidase.

The product of agmatine oxidation catalyzed by Pisum sativum L. copper amine oxidase has been identified by means of one- and two-dimensional (1)H-NMR spectroscopy to be N-amidino-2-hydroxypyrrolidine. This compound inhibits competitively rat nitric oxide synthase type I and type II (NOS-I and NOS-II, respectively) and bovine trypsin (trypsin) activity, values of Ki being (1.1 +/- 0.1) x 10(-5) m (at pH 7.5 and 37.0 degrees C), (2.1 +/- 0.1) x 10(-5) m (at pH 7.5 and 37.0 degrees C), and (8.9 +/- 0.4) x 10(-5) m (at pH 6.8 and 21.0 degrees C), respectively. Remarkably, the affinity of N-amidino-2-hydroxypyrrolidine for NOS-I, NOS-II and trypsin is significantly higher than that observed for agmatine and clonidine binding. Furthermore, N-amidino-2-hydroxypyrrolidine and agmatine are more efficient than clonidine in displacing [(3)H]clonidine (= 1.0 x 10(-8) m) from specific binding sites in heart rat membranes, values of IC50 being (1.3 +/- 0.4) x 10(-9) m and (2.2 +/- 0.4) x 10(-8) m, respectively (at pH 7.4 and 37.0 degrees C).     

High affinity binders for cyclic adenosine 3', 5'-monophosphate on plasma membranes isolated from rat liver and ascites hepatomas.

Plasma membranes from rat liver were found to contain at least two types of specific binding sites for cyclic [3H] adenosine 3', 5'-monophosphate (c[3H]AMP) with apparent dissociation constants of 0.51 +/- 0.14 and 2.9 +/- 0.6 nM (O degrees), respectively. The levels of these binding sites in liver plasma membranes were about 0.60 +/- 0.20 and 1.3 +/- 0.5 pmole/mg protein. The highest affinity binders for c[3H]AMP were found to be reduced in amount in plasma membranes of ascites hepatomas to 1/3 to 1/4 as compared with liver membranes in the cases of AH-130 and AH-7974 and to an almost undetectable level in the case of AH-130F(N). No difference in the endogenous phosphorylation of plasma membranes by (gamma-32P])ATP was, however, detected among liver and hepatoma plasma membranes. Addition of cAMP or cGMP at various concentrations did not affect the endogenous phosphorylation of plasma membranes of these cells.     

A characterization of beta-adrenergic receptors on cellular and perigranular membranes of rat peritoneal mast cells

Beta-adrenergic receptors were characterized by measuring the specific binding of 3H-dihydroalprenolol (DHA) on intact isolated rat peritoneal mast cells (RPMC) and on perigranular membranes derived from purified RPMC granules. The specific binding of 3H-DHA reaches an equilibrium within 30 min at 5 degrees C and is linear with cell number. Scatchard analysis reveals two populations of binding sites on intact cells: with KD = 10.6 +/- 2.6 and 129 +/- 4.7 nM and Bmax of 186 +/- 38 and 1200 +/- 415 fmol/10(6) cells, respectively. Each cell contains 120 X 10(3) high-affinity binding sites and 720 X 10(3) low-affinity binding sites. There appears to be neither alpha-adrenergic nor muscarinic cholinergic receptors on the RPMC. Specific binding of 3H-DHA also occurred to isolated granules with perigranular membranes. The binding was saturable with a single population of binding sites with an affinity (KD) of 7.0 +/- 0.45 nM. Maximum binding (Bmax) was calculated at 56.6 +/- 1.9 fmol/10(9) granules. Subfractionation of granule components demonstrated that the specific binding sites appear to be localized exclusively on the perigranular membrane.     

Direct assessment of beta-adrenergic receptors in intact rat adipocytes by binding of [3H]CGP 12177. Evidence for agonist high-affinity binding complex and for beta 1 and beta 2 receptor subtypes

The characteristics of the binding of the hydrophilic beta-adrenergic antagonist [3H]CGP 12177 to intact rat adipocytes were studied at 37 degrees C and 6 degrees C. At both temperatures and at 90% saturation, the non-specific binding was less than 30% of the total binding. At 37 degrees C, specific [3H]CGP 12177 binding was rapid, reversible of high affinity (1.8 +/- 0.4 nM) and saturable. The number of specific binding sites per adipocyte increased with the fat cell size (about 35 000 and 115 000 sites per cell in adipocytes with diameters of 60 microns and 88 microns, respectively) but remained constant when expressed per unit fat cell surface area. Displacement of [3H]CGP 12177 bound to adipocytes by unselective and selective beta-antagonists was stereospecific, had the same characteristics as those found in adipocyte membranes and showed a heterogeneous specificity for beta 1 and beta 2 adrenergic subtypes. In contrast, beta agonist competition curves, which modeled to two affinity-states of binding, showed high-affinity-state Kd values for agonists 10-25-times higher than those found in membranes under the same experimental conditions. At 6 degrees C, although the number and affinity of the specific binding sites for [3H]CGP 12177 were the same as those found at 37 degrees C, the Kd value for (-)-isoproterenol binding to the high affinity state of these sites (3.0 +/- 0.5 nM) was 25-times lower than at 37 degrees C and similar to the value found in membrane preparations (1.5-4 nM). These results show that the [3H]CGP 12177 specific binding sites detected on intact adipocytes represent the physiological beta-adrenergic receptors. Moreover, this study extends to the adipocyte the validity of the model recently proposed for other cell lines, according to which in intact cells, but not in membranes, agonist-binding promotes a rapid and temperature-dependent conformational change of the beta-adrenergic receptors, leading to a progressive loss of capacity of agonists to form a high-affinity complex.     

Galanin binding sites in rat gastric and jejunal smooth muscle membrane preparations.

Receptors for galanin in membranes from the rat gastric and jejunal smooth muscle were studied using [125I] radioiodinated synthetic porcine galanin. Specific binding was time and temperature dependent. At 32 degrees C radioligand was degraded in the presence of smooth muscle membranes in a time-dependent manner. At optimal experimental conditions, the equilibrium binding analyses showed the presence of a single population of high affinity binding sites in both the rat stomach and jejunum (Kd value of 2.77 +/- 0.78 nM and 4.93 +/- 1.74 nM for stomach and jejunal smooth muscle membranes, respectively). The concentration of the high affinity binding sites was 58.19 +/- 11.04 and 32.36 +/- 5.68 fmol/mg protein, for gastric and jejunal preparations, respectively. Specific binding was completely inhibited by 10(-6) M of nonradioactive galanin; was 75% blocked by 1 microM of galanin(9-29); it was 10% blocked by 1 microM of galanin(15-29). Galanin(1-15) at a concentration of 1 microM was ineffective for inhibiting [125I]galanin binding. Deletion of four C-terminal amino acid residues from galanin(9-29) to give galanin(9-25) also resulted in almost complete loss of affinity. Radioiodinated galanin and N-terminally deleted fragments had receptor binding potency in the following order: galanin(1-29) greater than galanin(9-29) greater than galanin(15-29). We conclude that the C-terminal part of the galanin chain is important for the rat gastric and jejunal smooth muscle membrane receptor recognition and binding and that N-terminal amino acid sequences are probably not so important, since galanin(1-15) was not active but galanin(9-29) retained most of the receptor binding activity.     

Acetylcholine released from cholinergic nerves contributes to cutaneous vasodilation during heat stress.

Nitric oxide (NO) contributes to active cutaneous vasodilation during a heat stress in humans. Given that acetylcholine is released from cholinergic nerves during whole body heating, coupled with evidence that acetylcholine causes vasodilation via NO mechanisms, it is possible that release of acetylcholine in the dermal space contributes to cutaneous vasodilation during a heat stress. To test this hypothesis, in seven subjects skin blood flow (SkBF) and sweat rate were simultaneously monitored over three microdialysis membranes placed in the dermal space of dorsal forearm skin. One membrane was perfused with the acetylcholinesterase inhibitor neostigmine (10 microM), the second membrane was perfused with the NO synthase inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME; 10 mM) dissolved in the aforementioned neostigmine solution (l-NAME(Neo)), and the third membrane was perfused with Ringer solution as a control site. Each subject was exposed to approximately 20 min of whole body heating via a water-perfused suit, which increased mean body temperature from 36.4 +/- 0.1 to 37.5 +/- 0.1 degrees C (P < 0.05). After the heat stress, SkBF at each site was normalized to its maximum value, identified by administration of 28 mM sodium nitroprusside. Mean body temperature threshold for cutaneous vasodilation was significantly lower at the neostigmine-treated site relative to the other sites (neostigmine: 36.6 +/- 0.1 degrees C, l-NAME(Neo): 37.1 +/- 0.1 degrees C, control: 36.9 +/- 0.1 degrees C), whereas no significant threshold difference was observed between the l-NAME(Neo)-treated and control sites. At the end of the heat stress, SkBF was not different between the neostigmine-treated and control sites, whereas SkBF at the l-NAME(Neo)-treated site was significantly lower than the other sites. These results suggest that acetylcholine released from cholinergic nerves is capable of modulating cutaneous vasodilation via NO synthase mechanisms early in the heat stress but not after substantial cutaneous vasodilation.     

Characterization of the substance P receptor in rat brain cortex membranes and the inhibition of radioligand binding by guanine nucleotides

Rat brain cortex membranes bind to a conjugate of substance P and 125I-labeled Bolton-Hunter reagent, and this binding can be inhibited by a low concentration of substance P (Kd = 1.2 +/- 0.4 X 10(-8) M). This binding is reversible and saturable (0.5 +/- 0.1 pmol of binding sites/mg of protein). Fragments of substance P as small as the carboxyl-terminal hexapeptide can inhibit the binding although their potency decreases with the decrease in the length of the peptides. The binding affinities of smaller peptides or peptides in which the carboxyl-terminal amide or amino acids are removed are drastically reduced. Biologically active analogs of substance P, physalaemin, eledoisin, substance P methyl ester, [D-Ala0]hepta(5-11)substance P, kassinin, and the eledoisin-related hexapeptide also can inhibit the binding. However, the binding is not inhibited by polypeptides structurally unrelated to substance P or by amine hormones/neurotransmitters. The binding affinities of biologically active peptides to rat brain cortex membranes are almost identical with their affinities for rat parotid cells which we previously determined. Furthermore, the recently described substance P antagonist, [D-Pro, D-Trp]substance P, inhibits the binding of the 125I-labeled substance P derivative to brain cortex membranes and to parotid cells equally well. These results suggest that the substance P receptors in the brain cortex and the parotid gland are similar. The brain cortex membrane binding of the 125I-labeled substance P derivative can be inhibited by micromolar concentrations of GTP, GDP, and their analogs. ITP and IDP were less active. Adenine and pyridine nucleotides were inactive.     

Subsensitivity of adenylate cyclase and decreased beta-adrenergic receptor binding after chronic exposure to (minus)-isoproterenol in vitro.

In vitro incubation of frog erythrocytes with (minus)-isoproterenol, 0.1 mM, at 23 degrees for 10 to 24 hours caused a 63% decline (rho less than 0.001) in the maximum (minus)-isoproterenol-stimulated adenylate cyclase activity in the erythrocyte membranes. Affinity for (minus)-isoproterenol as judged by the concentration which half-maximally stimulated the enzyme was not markedly altered. Basal enzyme activity and stimulation by fluoride or prostaglandin E1 remained unaltered. The number of beta-adrenergic receptor binding sites, assessed by binding studies with the beta-adrenergic antagonist (minus)-[3-H] alprenolol, declined by 50% (rho less than 0.005) in the (minus)-isoproterenol-treated cells. The binding affinity of the sites was not changed. Regulation of the concentration of functionally active beta-adrenergic receptors in membranes may be one of the mechanisms by which chronic exposure to catecholamines desensitizes tissues to beta-adrenergic stimulation.     

Differences in unwinding of supercoiled DNA induced by the two enantiomers of anti-benzo[a]pyrene diol epoxide.

The unwinding of supercoiled phi X174 RFI DNA induced by the tumorigenic (+) and non-tumorigenic (-) enantiomers of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) has been investigated by agarose slab-gel and ethidium titration tube gel electrophoresis. The differences in adduct conformations were verified by flow linear dichroism techniques. Both enantiomers cause a reversible unwinding by the formation of noncovalent intercalative complexes. The effects of covalently bound BPDE residues on the electrophoretic mobilities of the RF I DNA form in agarose gels were investigated in detail in the range of binding ratios rb approximately 0.0-0.06 (covalently bound BPDE residues/nucleotide). In this range of rb values, there is a striking difference in the mobilities of (+)-BPDE- and (-)-BPDE-adducted phi X174 DNA in agarose slab-gels, the covalently bound (+)-BPDE residues causing a significantly greater retardation than (-)-BPDE residues. Increasing the level of covalent adducts beyond rb approximately 0.06 in the case of the (+)-BPDE enantiomer, leads to further unwinding and a minimum in the mobilities (corresponding to comigration of the nicked form and the covalently closed relaxed modified form) at rb 0.10 +/- 0.01; at still higher rb values, rewinding of the modified DNA in the opposite sense is observed. From the minimum in the mobility, a mean unwinding angle (per BPDE residue) of theta = 12 +/- 1.5 degrees is determined, which is in good agreement the value of theta = 11 +/- 1.8 degrees obtained by the tube gel titration method. Using this latter method, values of theta = 6.8 +/- 1.7 degrees for (-)-BPDE-phi X174 adducts are observed. It is concluded that agarose slab gel techniques are not suitable for determining unwinding angles for (-)-BPDE-modified phi X174 DNA because the alterations in the tertiary structures for rb < 0.06 are too small to cause sufficiently large changes in the electrophoretic mobilities. The major trans (+)-BPDE-N2-guanosine covalent adduct is situated at external binding sites and the mechanisms of unwinding are therefore different from those relevant to noncovalent intercalative BPDE-DNA complexes or to classical intercalating drug molecules; a flexible hinge joint and a widening of the minor groove at the site of the lesion may account for the observed unwinding effects. The more heterogeneous (-)-BPDE-nucleoside adducts (involving cis and trans N2-guanosine, and adenosine adducts) are less effective in causing unwinding of supercoiled DNA for reasons which remain to be elucidated.     

α2-Adrenergic and Muscarinic Cholinergic Receptors Have Oposing Actions on Cyclic AMP Levels in SK-N-SH Human Neuroblastoma Cells

Forskolin and vasoactive intestinal polypeptide (VIP) were shown to increase cyclic AMP accumulation in a human neuroblastoma cell line, SK-N-SH cells. The alpha 2-adrenergic agonist UK 14304 decreased forskolin-stimulated cyclic AMP levels by 40 +/- 2%, with an EC50 of 83 +/- 20 nM. This response was blocked by pretreatment with pertussis toxin (PT) (EC50 = 1 ng/ml) or by the alpha 2-antagonists yohimbine, idazoxan, and phentolamine. Antagonist IC50 values were 0.3 +/- 0.1, 2.2 +/- 0.3, and 1.4 +/- 0.1 microM, respectively. This finding suggests the presence of normal inhibitory coupling of SK-N-SH cell alpha 2-adrenergic receptors to adenylate cyclase via the inhibitory GTP-binding protein species, Gi. Muscarinic receptors in many target cell types are coupled to inhibition of adenylate cyclase. However, in SK-N-SH cells, muscarinic agonists synergistically increased (67-95%) the level of cyclic AMP accumulation elicited by forskolin or VIP. EC50 values for carbamylcholine (CCh) and oxotremorine facilitation of the forskolin response were 1.2 +/- 0.2 and 0.3 +/- 0.1 microM, respectively. Pharmacological studies using the muscarinic receptor subtype-preferring antagonists 4-diphenylacetoxy-N-methylpiperidine, pirenzepine, and AF-DX 116 indicated mediation of this response by the M3 subtype. IC50 values were 14 +/- 1, 16,857 +/- 757, and 148,043 +/- 16,209 nM, respectively. CCh-elicited responses were unaffected by PT pretreatment. Muscarinic agonist binding affinity was indirectly measured by the ability of CCh to compete for [3H]quinuclidinyl benzilate binding sites on SK-N-SH cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mapping of DNA gyrase cleavage sites in vivo oxolinic acid induced cleavages in plasmid pBR322

We have developed a procedure which permits the mapping of DNA gyrase cleavage sites in vivo. Addition of oxolinic acid, an inhibitor of DNA gyrase, to growing cells of Escherichia coli containing the plasmid pBR322 resulted in double-strand cleavage of DNA, and allowed the isolation of significant quantities of linearized plasmid DNA after lysis of treated cells with sodium dodecyl sulfate. Initially the linear product was purified from agarose gels, cleaved by restriction endonucleases, and then subjected to Southern hybridization analysis using defined DNA probes. A number of distinct cleavage sites, used with varying degrees of efficiency, were identified within pBR322 using this simple procedure. To achieve greater resolution and to improve sensitivity, we then employed an electroblotting procedure to transfer DNA fragments from acrylamide gels onto nylon membranes. This alternative method does not require the isolation of the linearized product before performing the mapping procedure. The improved resolution obtained from acrylamide gels and the superior binding properties of the nylon membranes have allowed us to accurately map 74 distinct oxolinic acid-induced cleavage sites within pBR322. The significance of these findings in light of previously reported studies in vitro, as well as the possible role of such sites during illegitimate recombination, are discussed.     

Galanin receptor and its ligands in the rat hippocampus

Receptors for the 29-amino-acid peptide, galanin, in membranes from the rat ventral hippocampus were examined using chloramine-T-iodinated porcine galanin as ligand. The equilibrium binding of 125I-galanin showed the presence of a high-affinity binding site (Kd = 1.91 +/- 0.40 nM). The concentration of the high-affinity-binding sites was 107 +/- 15 fmol/mg membrane protein. The on rate constant was estimated to be 2.6 +/- 0.1 M-1 min-1 at 37 degrees C. The affinity of rat galanin (differing in three amino acid residues from the porcine protein) was equal to that of porcine galanin. The 125I--galanin-binding site is a trypsin-sensitive membrane protein, which is heat-denaturated at 60 degrees C within 5 min. The effect of GTP and its analogs and of pertussis-toxin-catalyzed ADP-ribosylation on the binding of 125I-galanin suggest that the galanin receptor is coupled to an inhibitory G protein (Gi protein). 127I-galanin was shown to be a ligand with affinity equal to that of galanin in displacing 125I-galanin. The 125I-galanin-binding site in the ventral hippocampus recognizes as a ligand the tryptic fragments 1-20 and 21-29 of rat galanin and the synthetic fragments 12-29, 18-29 and 21-29 of porcine galanin. None of these afforded full inhibition of the binding of fragment 1-29 of 125I-galanin at a concentration of 1 microM.