Stopped-flow kinetic studies of actinomycin binding to DNAs.

Stopped-flow kinetic studies of the association of actinomycins with narural and synthetic DNA duplexes are presented. The actinomycins examined were D (C1), D lactam (in which the pentapeptide rings are closed by lactam instead of lactone linkages), X2, XObeta, and actinomine. The DNAs used included claf-thymus DNA, PM2, DNA, and two synthetic d(A-T)-lide copolymers containing 2,6-diaminopurine (DAP) in place of adenine residues, poly[d(DAP-T)]-poly[d(DAP-T)] and poly[d(DAP-A-T]-poly[d(DAP-A-T)]. Apparent equilibrium constants indicate that the DAP-containing polynucleotides bind actinomycin strongly. Comples formation of actinomycins D, D lactam, X2 and XObeta with these DNAs can be deconvoluted into five rate processes. These steps do not necessarily proceed to completion. The rates of two of these steps display a firstorder dependence on DNA concentration. The large negative entropies of activation of these steps suggest a high degree of restriction to freedom of motion on the respective transition states. The rates of the remaining three steps are independent of DNA concentration. Kinetic parameters of actinimycin binding to DNAs are presented and suggestions are made about some of the molecular evente believed to be responsible for the appearance of the five rate processes. For example, for DNA, poly[d(DAP-A-T)], and poly[d(DAP-T)], the observed order of apparent second-order rate constants, normalized to the concentration of actinomycin binding sites, suggests that binding of the antibiotic occurs most rapidly at binding sites (G-C of d DAT-T) near d(A-T) base pairs, where weakening of the double-helical conformation requires the least energy. Results obtained from studies of actinomycin D binding to heat-denatured poly[d(DAP-A-T)] and of actinomine and actinomycin D lactam binding to DNA suggest that the slow rate processes are related to an actinomycyl-pentapeptide-induced unwinding of the sugar-phosphate backbone of DNA accompanying insertion of the cyclic peptides into DNA.     

CD of homopolymer DNA-RNA hybrid duplexes and triplexes containing A-T or A-U base pairs.

CD spectra and difference-CD spectra of (a) two DNA X RNA hybrid duplexes (poly[r(A) X d(U)] and poly[r(A) X d(T)]) and (b) three hybrid triplexes (poly-[d(T) X r(A) X d(T)], poly[r(U) X d(A) X r(U)], and poly[r(T) X d(A) X r(T)]) were obtained and compared with CD spectra of six A X U- and A X T-containing duplex and triplex RNAs and DNAs. We found that the CD spectra of the homopolymer duplexes above 260 nm were correlated with the type of base pair present (A-U or A-T) and could be interpreted as the sum of the CD contributions of the single strands plus a contribution due to base pairing. The spectra of the duplexes below 235 nm were related to the polypurine strands present (poly-[r(A)] or poly[d(A)]). We interpret the CD intensity in the intermediate 255-235 nm region of these spectra to be mainly due to stacking of the constituent polypurine strands. Three of the five hybrids (poly[r(A) X d(U)], poly[r(A) X d(T)], and poly[d(T) X r(A) X d(T)]) were found to have heteronomous conformations, while poly[r(U) X d(A) X r(U)] was found to be the most A-like and poly[r(T) X d(A) X r(T)], the least A-like.     

Large scale chemical synthesis, purification and crystallization of RNA-DNA chimeras.

RNA-DNA chimeras, in which both DNA and RNA monomers are site-specifically substituted in the same strand, may be prepared only by chemical synthesis. Biochemical studies have revealed a number of surprising and subtle effects resulting from the insertion of either a ribonucleotide into a DNA strand or a deoxyribonucleotide into an RNA strand. The availability of large quantities of these chimeras allows for their crystallization and subsequent x-ray structure determination. We describe a flexible and efficient method for the large-scale preparation of these compounds, their purification, and their crystallization. The methodology is based on a combination of existing DNA phosphoramidite synthons and those recently introduced for the preparation of biochemically active RNA1. We demonstrate that these two different synthons are compatible, produce large quantities of nucleic acid needed for physical studies, and that high resolution diffraction quality crystals may be grown from these chimeras. Of the duplex chimeras synthesized and crystallized, [r(G)d(CGTATACGC)]2, [d(GCGT)r(A)d(TACGC)]2 and [r(GCG)d(TATACCC) + d(GGGTATACGC)] form A-helices and d(CG)r(CG)d(CG)]2 forms a left-handed Z-helix.     

Mercury-induced transitions between right-handed and putative left-handed forms of poly[d(A-T).d(A-T)] and poly[d(G-C).d(G-C)]

Poly[d(A-T).d(A-T)] and poly[d(G-C).d(G-C)], each dissolved in 0.1 M NaClO4, 5 mM cacodylic acid buffer, pH 6.8, experience inversion of their circular dichroism (CD) spectrum subsequent to the addition of Hg(ClO4)2. Let r identical to [Hg(ClO4)2]added/[DNA-P]. The spectrum of the right-handed form of poly[d(A-T).d(A-T)] turns into that of a seemingly left-handed structure at r greater than or equal to 0.05 while a similar transition is noted with poly[d(G-C).(G-C)] at r greater than or equal to 0.12. The spectral changes are highly cooperative in the long-wavelength region above 250 nm. At r = 1.0, the spectra of the two polymers are more or less mirror images of their CD at r = 0. While most CD bands experience red-shifts upon the addition of Hg(ClO4)2, there are some that are blue-shifted. The CD changes are totally reversible when Hg(II) is removed from the nucleic acids by the addition of a strong complexing agent such as NaCN. This demonstrates that mercury keeps all base pairs in register.     

Syntheses of cyclic prodrugs of RGD peptidomimetics with various macrocyclic ring sizes: evaluation of physicochemical, transport and antithrombic properties.

The objective of this work was to synthesize cyclic prodrugs 1a-d of RGD peptidomimetics 2a-d with various ring sizes (n[CH2] = 1, 3, 5 and 7) and to evaluate the effect of ring size on their transport, physicochemical, enzymatic stability, and antithrombic properties. The syntheses of cyclic prodrugs 1a-d were achieved by converging two key intermediates, Boc-Phe-O-CH2-OCO-OpNP (5) and H2N-(CH2)n-CO-Asp(OBzl)-OTce (8a-d), to give linear precursors Boc-Phe-O-CH2-OCO-HN-(CH2)n-CO-Asp(OBzl)-OTce (9a-d). The N- and C-terminus protecting groups were removed from 9a-d to give 10a-d. Linear precursors 10a-d were cyclized, and the remaining Bzl-protecting group was removed to produce cyclic prodrugs 1a-d in around 20% overall yield. The linear RGD peptidomimetics (2a-d) were synthesized using standard Boc-amino acid chemistry by solution-phase method. Increasing the ring size by adding methylene groups also increases the hydrophobicity of the cyclic prodrugs and parent RGD peptidomimetics. The transport properties of cyclic prodrugs 1c and 1d were 2.6- and 4.4-fold better than those of parent compounds 2c and 2d, respectively. These results suggest that increasing the hydrophobicity of the cyclic prodrugs and parent RGD peptidomimetics enhanced their transport properties. The hydrodynamic radii of the cyclic prodrugs were also smaller than those of their respective parent compounds, suggesting that the change in size may contribute to their transport properties. The chemical stability of the cyclic prodrugs was affected by the ring size, and the cyclic prodrug with the larger ring size (i.e. 1d) was more stable than the smaller one (i.e. 1a). All the cyclic prodrugs were more stable at pH 4 than at pH 7 and 10. Prodrug-to-drug conversion could be induced by isolated esterase as well as esterase found in human plasma. An increase in the length of methylene group (n[CH2] = 1, 3, 5, 7) enhanced the antithrombic activity of the prodrugs and the parent compounds. In summary, the ring size of cyclic prodrugs affected their transport, physicochemical, and antithrombic properties.     

Analytic computation of the expectation of the linkage disequilibrium coefficient r2

The squared correlation coefficient r(2) (sometimes denoted Delta(2)) is a measure of linkage disequilibrium that is widely used, but computing its expectation E[r(2)] in the population has remained an intriguing open problem. The expectation E[r(2)] is often approximated by the standard linkage deviation sigma(d)(2), which is a ratio of two expectations amenable to analytic computation. In this paper, a method of computing the population-wide E[r(2)] is introduced for a model with recurrent mutation, genetic drift and recombination. The approach is algebraic and is based on the diffusion process approximation. In the limit as the population-scaled recombination rate rho approaches infinity, it is shown rigorously that the asymptotic behavior of E[r(2)] is given by 1/rho+O(rho(-2)), which, incidentally, is the same as that of sigma(d)(2). A computer software that computes E[r(2)] numerically is available upon request.     

Conformation and complexation with metal ions of cyclic hexapeptides: cyclo (L-Leu-L-Phe-L-Pro)2 and cyclo [L-Cys(Acm)-L-Phe-L-Pro]2

Cyclic hexapeptides, cyclo (L-Leu-L-Phe-L-Pro)2 and cyclo[L-Cys(Acm)-L-Phe-L-Pro]2, in which Acm represents an acetoamide-methyl group, were synthesized, and the conformation and complexation with metal ions were investigated. Cooperation of the carbonyl groups of the Cys(Acm) side chains with those of the cyclic skeleton in complexation was especially examined. Cyclo(L-Leu-L-Phe-L-Pro)2, which possesses no functional groups on side chains, was taken as the reference compound. 13C- and two-dimensional n.m.r. measurements revealed that cyclo(L-Leu-L-Phe-L-Pro)2 and cyclo[L-Cys(Acm)-L-Phe-L-Pro]2 took a C2-symmetric conformation containing cis L-Phe-L-Pro bonds in chloroform and acetonitrile. Both cyclic hexapeptides were found to complex selectively with Ba2+ and Ca2+ in acetonitrile. On complexation the conformation of either cyclic hexapeptide changed into a similar one. However, the binding constant of cyclo[L-Cys(Acm)-L-Phe-L-Pro]2 was higher than that of cyclo(L-Leu-L-Phe-L-Pro)2. The n.m.r. measurements showed that the amide carbonyl groups of Cys(Acm) side chains as well as those of cyclic skeleton in cyclo[L-Cys(Acm)-L-Phe-L-Pro]2 cooperatively bound the cations.     

Design, synthesis, and testing of potential antisickling agents. 9. Cyclic tetrapeptide homologs as mimics of the mutation site of hemoglobin S

As part of our continuing search for new agents which might be useful for the treatment of sickle-cell anemia, we have synthesized two cyclic tetrapeptide homologs, cyclo(-Val-Glu[-Thr-Pro-]-OH) (1a) and cyclo(-Phe-Glu[-Thr-Pro-]-OH (1b), and a tetrapeptide lactone homolog cyclo(H-Thr-Pro-Val-Glu-OH) (2). The intent was that these peptides would mimic a tetrapeptide region around the mutation site of HbS and thus be able to bind at the acceptor site of HbS and thereby inhibit polymerization. The synthesis of the linear peptides was accomplished in solution using both the polymeric reagent (PHBT) and DCC/HOBT methods; cyclization was accomplished by an improved method. 13C-n.m.r. studies were performed which allowed us to assign the conformation about the Thr-Pro bond in 1a and 2 as trans. The cyclic peptides were tested for their ability to increase the solubility of HbS under deoxygenating conditions, but only 1a had any antigelling activity, albeit low.     

On the protein residues that control the yield and kinetics of O(630) in the photocycle of bacteriorhodopsin

The effects of pH on the yield (phi(r)), and on the apparent rise and decay constants (k(r), k(d)), of the O(630) intermediate are important features of the bacteriorhodopsin (bR) photocycle. The effects are associated with three titration-like transitions: 1) A drop in k(r), k(d), and phi(r) at high pH [pK(a)(1) approximately 8]; 2) A rise in phi(r) at low pH [pK(a)(2) approximately 4.5]; and 3) A drop in k(r) and k(d) at low pH [pK(a)(3) approximately 4. 5]. (pK(a) values are for native bR in 100 mM NaCl). Clarification of these effects is approached by studying the pH dependence of phi(r), k(r), and k(d) in native and acetylated bR, and in its D96N and R82Q mutants. The D96N experiments were carried out in the presence of small amounts of the weak acids, azide, nitrite, and thiocyanate. Analysis of the mutant's data leads to the identification of the protein residue (R(1)) whose state of protonation controls the magnitude of phi(r), k(r), and k(d) at high pH, as Asp-96. Acetylation of bR modifies the Lys-129 residue, which is known to affect the pK(a) of the group (XH), which releases the proton to the membrane exterior during the photocycle. The effects of acetylation on the O(630) parameters reveal that the low-pH titrations should be ascribed to two additional protein residues R(2) and R(3). R(2) affects the rise of phi(r) at low pH, whereas the state of protonation of R(3) affects both k(r) and k(d). Our data confirm a previous suggestion that R(3) should be identified as the proton release moiety (XH). A clear identification of R(2), including its possible identity with R(3), remains open.     

Interaction of porphyrin with G-quadruplex structures

Isothermal titration calorimetry (ITC) is a sensitive technique for probing bimolecular processes and can provide direct information about the binding affinity and stoichiometry and the key thermodynamic parameters involved. ITC has been used to investigate the interaction of the ligand H2TMPyP to the two DNA quadruplexes, [d(AGGGT)]4 and [d(TGGGGT)]. Analysis of the ITC data reveals that porphyrin/quadruplex binding stoichiometry under saturating conditions is 1:2 for [d(AGGGT)]4 and 2:1 for [d(TGGGGT)], respectively.     

4 beta-Phorbol 12-myristate 13-acetate attenuates the glucagon-induced increase in cytoplasmic free Ca2+ concentration in isolated rat hepatocytes.

Hepatocytes were isolated from rats and then loaded with the fluorescent Ca2+ indicator quin2. Glucagon caused a sustained increase (at least 5 min) in the fluorescence of the quin2-loaded cells; the increase was much greater than that observed with control, non-quin2-loaded, cells. These observations indicate that glucagon caused an increase in cytoplasmic free Ca2+ concentration [( Ca2+]c). The effects of glucagon were mimicked if forskolin (to activate adenylate cyclase), dibutyryl cyclic AMP or bromo cyclic AMP were added directly to the cells. Thus an increase in cyclic AMP concentration may mediate the effect of glucagon on [Ca2+]c. If 4 beta-phorbol 12-myristate 13-acetate (PMA; an activator of protein kinase C) was added to the cells before glucagon, the magnitude of the increase in [Ca2+]c was greatly diminished. If PMA was added after glucagon it caused a lowering of [Ca2+]c. These effects of PMA on the glucagon-induced increase in [Ca2+]c could not be mimicked if [Ca2+]c was increased by the Ca2+-ionophore ionomycin. Thus an event involved in the mechanism by which glucagon increases [Ca2+]c appears to be required for the action of PMA. If [Ca2+]c was increased by forskolin, dibutyryl cyclic AMP or bromo cyclic AMP, the effect of PMA on [Ca2+]c was similar to that observed when glucagon was used to elevate [Ca2+]c. When [Ca2+]c was raised by dibutyryl cyclic AMP the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine did not prevent the subsequent addition of PMA from causing [Ca2+]c to decrease. These observations suggest that PMA can inhibit the cyclic AMP-induced increase in [Ca2+]c independently of any changes in cyclic AMP concentration. Glucagon appears to increase [Ca2+]c by releasing intracellular stores of Ca2+ and stimulating net influx of Ca2+ into the cell; PMA greatly diminishes both of these effects.     

Vacuum UV CD spectra of homopolymer duplexes and triplexes containing A.T or A.U base pairs.

Vacuum UV circular dichroism (CD) spectra were measured down to 174 nm for five homopolymers, five duplexes, and four triplexes containing adenine, uracil, and thymine. Near 190 nm, the CD bands of poly[d(A)] and poly[r(A)] were larger than the CD bands of the polypyrimidines, poly[d(T)], poly[d(U)], and poly[r(U)]. Little change was observed in the 190 nm region upon formation of the duplexes (poly[d(A).d(T)], poly[d(A).d(U)], poly[r(A).d(T)], poly[r(A).d(U)], and poly[r(A).r(U)]) or upon formation of two of the triplexes (poly[d(T).d(A).d(T)] and poly[d(U).d(A).d(U)]). This showed that the purine strand had the same or a similar structure in these duplexes and triplexes as when free in solution. Both A.U and A.T base pairing induced positive bands at 177 and 202 nm. For three triplexes containing poly[d(A)], the formation of a triplex from a duplex and a free pyrimidine strand induced a negative band centered between 210 and 215 nm. The induction of a band between 210 and 215 nm indicated that these triplexes had aspects of the A conformation.     

The DNA sequence at echinomycin binding sites determines the structural changes induced by drug binding: NMR studies of echinomycin binding to [d(ACGTACGT)]2 and [d(TCGATCGA)]2

The complexes formed between the cyclic octadepsipeptide antibiotic echinomycin and the two DNA octamers [d(ACGTACGT)]2 and [d(TCGATCGA)]2 have been investigated by using one- and two-dimensional proton NMR spectroscopy techniques. The results obtained for the two complexes are compared to each other, to the crystal structures of related DNA-echinomycin complexes, and to enzymatic and chemical footprinting results. In the saturated complexes, two echinomycin molecules bind to each octamer by bisintercalation of the quinoxaline moieties on either side of each CpG step. Binding of echinomycin to the octamer [d(ACGTACGT)]2 is cooperative so that only the two-drug complex is observed at lower drug-DNA ratios, but binding to [d(TCGATCGA)]2 is not cooperative. At low temperatures, both the internal and terminal A.T base pairs adjacent to the binding site in the [d(ACGTACGT)]2-2 echinomycin complex are Hoogsteen base paired (Gilbert et al., 1989) as observed in related crystal structures. However, as the temperature is raised, the internal A.T Hoogsteen base pairs are destabilized and are observed to be exchanging between the Hoogsteen base-paired and an open (or Watson-Crick base-paired) state. In contrast, in the [d(TCGATCGA)]2-2 echinomycin complex, no A.T Hoogsteen base pairs are observed, the internal A.T base pairs appear to be stabilized by drug binding, and the structure of the complex does not change significantly from 0 to 45 degrees C. Thus, the structure and stability of the DNA in echinomycin-DNA complexes depends on the sequence at and adjacent to the binding site. While we conclude that no single structural change in the DNA can explain all of the footprinting results, unwinding of the DNA helix in the drug-DNA complexes appears to be an important factor while Hoogsteen base pair formation does not.     

Structure of the alpha-form of poly[d(A)].poly[d(T)] and related polynucleotide duplexes

The alpha-form of poly[d(A)].poly[d(T)], observed in fibers at high (greater than 80%) relative humidity, is a 10-fold double-helical structure of pitch 3.2 nm. This new X-ray analysis shows that the two strands of the double helix are of the same kind conformationally and both B-like in containing C-2'-endo-puckered deoxyribose rings. Nevertheless, the two strands are different enough for the overall morphology of the duplex to resemble that of the heteromerous model for the drier (beta) form of poly[d(A)].poly[d(T)] in which one strand has C-2'-endo rings and the other C-3'-endo. Since the orientations of the bases in poly[d(A)].poly[d(T)] are persistently different from those of classical B-DNA it is likely that there will be local bending (about 10 degrees) at the junctions between general sequence tracts and the oligo[d(A)].oligo[d(T)] tracts that occur in some native DNAs. The conclusions about the structure of alpha-poly[d(A)].poly[d(T)] are reinforced by independent analyses of similar X-ray diffraction patterns from poly[d(A)].poly[d(U)] and poly[d(A-I)].poly[d(C-T)].     

Conformational properties of tripeptides having cyclic dipeptide backbone and their catalytic properties for enantiomer-selective hydrolysis

Conformation in aqueous solution at pH 6.95 of tripeptides having cyclic dipeptide backbones, cyclo[l-Glu(l-Leu-OBzl)-l-His] and cyclo[l-Glu(l-Leu-OH)-l-His], was investigated by u.v., c.d. and n.m.r. spectroscopy and by the lanthanide probe method. In the major conformation of cyclo[l-Glu(l-Leu-OBzl)-l-His], the cyclic dipeptide backbone takes a flagpole-boat conformation in which the sidechain of the l-His residue is nearly parallel with the backbone plane and the sidechain of the l-Glu residue protrudes outside the backbone plane. In the major conformation of cyclo[l-Glu(l-Leu-OH)-l-His], the cyclic dipeptide backbone takes a flagpole-boat conformation in which the sidechains of the l-His and l-Glu residues are accommodated in the same side of the backbone plane so that the imidazolyl sidechain of l-His residue is twisted slightly. Tripeptides were not found to change the conformation when metal salts or ammonium salts such as Cl^?H₃N^?(CH₂)₁₁ COOEt, Gly-OEt-HCl, dl-Val-OEt-HCl and l-Leu-OEt-HCl were added, but a significant conformation change occurred upon adding d-Leu-OEt·HCl. If the same situation holds with the addition of α-amino acid p-nitrophenyl ester hydrochlorides, the previously reported enantiomer-selective catalysis by the tripeptides which hydrolysed d-Leu-OPh(NO₂·HCl faster than l-Leu-OPh(NO₂)·HCl can be explained; that is, the tripeptides change the conformation only when d-Leu-OPh(NO₂)·HCl is bound and consequently the intramolecular reaction is facilitated. This phenomenon may be compared with that of ‘induced fit’ in enzyme catalysis.     

Raman spectra of single crystals of r(GCG)d(CGC) and d(CCCCGGGG) as models for A DNA, their structure transitions in aqueous solution, and comparison with double-helical poly(dG).poly(dC)

The self-complementary oligonucleotides [r(CGC)d(CGC)]2 and [d(CCCCGGGG)]2 in single-crystal and solution forms have been investigated by Raman spectroscopy. Comparison of the Raman spectra with results of single-crystal X-ray diffraction and with data from polynucleotides permits the identification of a number of Raman frequencies diagnostic of the A-helix structure for GC sequences. The guanine ring frequency characteristic of C3'-endo pucker and anti base orientation is assigned at 668 +/- 2 cm-1 for both dG and rG residues of the DNA/RNA hybrid [r(GCG)d(CGC)]2. The A-helix backbone of crystalline [r(GCG)d(CGC)]2 is altered slightly in the aqueous structure, consistent with the conversion of at least two residues to the C2'-endo/anti conformation. For crystalline [d(CCCCGGGG)]2, the Raman and X-ray data indicate nucleosides of alternating 2'-endo-3'-endo pucker sandwiched between terminal and penultimate pairs of C3'-endo pucker. The A-A-B-A-B-A-A-A backbone of the crystalline octamer is converted completely to a B-DNA fragment in aqueous solution with Raman markers characteristic of C2'-endo/anti-G (682 +/- 2) and the B backbone (826 +/- 2 cm-1). In the case of poly(dG).poly(dC), considerable structural variability is detected. A 4% solution of the duplex is largely A DNA, but a 2% solution is predominantly B DNA. On the other hand, an oriented fiber drawn at 75% relative humidity reveals Raman markers characteristic of both A DNA and a modified B DNA, not unlike the [d-(CCCCGGGG)]2 crystal. A comparison of Raman and CD spectra of the aqueous [d(CCCCGGGG)]2 and poly(dG).poly(dC) structures suggests the need for caution in the interpretation of CD data from G clusters in DNA.     

Characterization of the cyclic AMP-dependent protein kinase from rat pancreas, further purification of the catalytic subunit, substrate specificity, effect of the pancreatic heat stable inhibitor.

In order to investigate the sequence of events triggered by cyclic AMP and cyclic GMP in exocrine pancreatic cells, the identification of the various protein kinases possibly present in this tissue is of major interest. Further analysis of the two cyclic AMP-dependent protein kinases previously reported [11] suggests that KI is a degraded form of KII. It is therefore likely that a single holoenzyme is present in exocrine cells. In addition no protein kinase, specifically stimulated by cyclic GMP, has been detected in any fraction obtained in the course of purification of the cyclic AMP-dependent protein kinase. A faster and more efficient method than the one previously described [11] allows the purification (5000 times) of the protein kinase catalytic subunit. Analysis of the subunit by sodium dodecyl sulphate polyacrylamide gel electrophoresis indicates a molecular weight of 40 000 +/- 1 000. The enzyme phosphorylates specifically histone H2B (Vm = 236 min(-1), Km = 1.15 10(-5) M) and to a lesser extent H2A, H5 and H1 (Vm = 55--77 min(-1), Km 5--25 10(-5) M). Histones H3 and H4 are not phosphorylated. The effect of the heat stable inhibitor, extracted from rat pancreas, on the phosphorylation of H2B has been investigated. The inhibition is of the non competitive type with respect to ATP. The inhibition at various histone concentrations cannot be described by the Michaelis-Menten equation.     

Regulation of Spontaneous Activity of the δ-Opioid Receptor: Studies of Inverse Agonism in Intact Cells

Abstract: Adenylyl cyclase activity was measured following labelling of the cellular ATP pool with [³H]adenine in intact Rat-1 fibroblasts that had been stably transfected to express the murine δ-opioid receptor (clone D2). Basal [³H]cyclic AMP accumulation was low and was increased substantially by the addition of the diterpene forskolin. The synthetic enkephalin d -Ala², d -Leu⁵ enkephalin (DADLE) produced strong inhibition of forskolin-amplified [³H]cyclic AMP production, whereas the δ-opioid ligand ICI174864 augmented forskolin-amplified adenylyl cyclase activity. Naloxone was unable to mimic the effects of ICI174864, and coincubation of the cells with these two ligands attenuated the effect of ICI174864. The EC₅₀ (9.4 ± 0.6 × 10⁻⁸ M ) for ICI174864 augmentation of forskolin-stimulated adenylyl cyclase was equal to its estimated K _i. Pertussis toxin pretreatment of clone D2 cells prevented both this effect of ICI174864 and the inhibition produced by DADLE. Use of a Cytosensor microphysiometer demonstrated that treatment of clone D2 cells with DADLE increased and that with ICI174864 decreased the basal rate of cellular proton extrusion. By using these two distinct experimental strategies, ICI174864 was shown to function in a manner anticipated for an inverse agonist, demonstrating that such effects can be observed in intact cells and are not restricted to assays performed on membrane preparations.