pol mutations conferring zidovudine and didanosine resistance with different effects in vitro yield multiply resistant human immunodeficiency virus type 1 isolates in vivo.

Specific mutations in the human immunodeficiency virus type 1 (HIV-1) pol gene that cause zidovudine (3'-azido-2',3'-dideoxythymidine; AZT) and didanosine (2',3'-dideoxyinosine; ddI) resistance were studied. The 50% inhibitory concentrations (IC50s) of nucleosides for cloned viruses containing these mutations were compared with the IC50s of the corresponding triphosphate analogs for mutant recombinant-expressed reverse transcriptases (RTs). Changes in ddATP inhibition of RNA-dependent DNA polymerase activity fully accounted for the ddI resistance of the virus caused by a Leu-74-->Val substitution in RT, including an augmentation by the AZT-selected substitutions Thr-215-->Tyr and Lys-219-->Gln in RT. In contrast, the AZT-selected substitutions studied did not cause as great a change in the IC50 of AZT-triphosphate (AZT-TP) for polymerase as they did in the IC50 of AZT for mutant virus. In addition, the mutation at codon 74 suppressed AZT resistance in the virus caused by the mutations at codons 215 and 219 but did not suppress the AZT-TP resistance of enzyme containing these same mutations in RT. The mutation at codon 74 was found in clinical isolates whether or not the patient had received AZT prior to starting ddI therapy. AZT resistance coexisted with ddI resistance following acquisition of Leu-74-->Val in three clinical isolates, indicating that the suppressive effect of Val-74 on the AZT resistance of the virus does not occur in all genetic contexts. When this suppression of AZT resistance was seen in the virus, Val-74 did not appear to cause mutually exclusive changes in AZT-TP and ddATP binding to RT in vitro. The results of the in vitro experiments and characterization of clinical isolates suggest that there are differences in the functional effects of these AZT and ddI resistance mutations.

Use of evolutionary limitations of HIV-1 multidrug resistance to optimize therapy.

Wild-type reverse transcriptase has evolved for the survival of human immunodeficiency virus type 1 (HIV-1) by natural selection. In contrast, therapy relying on inhibitors of reverse transcriptase by nucleosides like zidovudine (AZT) or dideoxyinosine (ddI), and by non-nucleosides like pyridinones or nevirapine, may exert different selection pressures on this enzyme. Therefore the acquisition of resistance to reverse transcriptase inhibitors by selection of mutations in the pol gene may require compromises in enzyme function that affect viral replication. As single mutations are unlikely to confer broad resistance when combinations of reverse transcriptase inhibitors are used, multiple mutations may occur that result in further compromises. Certain drug combinations may prevent the co-existence of adequate reverse transcription function and multi-drug resistance (MDR). Unlike bacterial or eukaryotic drug resistance, retroviral drug resistance is conferred only by mutations in its own genome and is limited by genome size. Combining drugs directed against the same essential viral protein may thus prevent HIV-1 MDR, whereas the conventional approach of targeting different HIV-1 proteins for combination therapy may not, because genomes with resistance mutations in different HIV-1 genes might recombine to develop MDR. Here we show that several mutations in the HIV-1 reverse transcriptase gene that confer resistance to inhibitors of this enzyme can attenuate viral replication. We tested whether combinations of mutations giving rise to single-agent resistance might further compromise or even abolish viral replication, and if multidrug-resistant viruses could be constructed. Certain combinations of mutations conferring resistance to AZT, ddI and pyridinone are incompatible with viral replication. These results indicate that evolutionary limitations exist to restrict development of MDR. Furthermore, a therapeutic strategy exploiting these limitations by using selected multidrug regimens directed against the same target may prevent development of MDR. This approach, which we call convergent combination therapy, eliminated HIV-1 replication and virus breakthrough in vitro, and may be applicable to other viral targets. Moreover, elimination of reverse transcription by convergent combination therapy may also limit MDR.

Phospholipase C modulates automaticity of canine cardiac Purkinje fibers.

Alpha-1 adrenergic agonists increase cardiac Purkinje fiber automaticity and elevate D-myo-inositol-trisphosphate (IP3) levels. To learn about the relationship between phosphoinositide metabolism and the modulation of cardiac rhythm, we used phospholipase C to activate phosphoinositide hydrolysis in an alpha-1 receptor-independent fashion and determined whether this intervention modulated automaticity. We used standard microelectrode techniques to study automaticity in adult Purkinje fiber bundles, fluorescence microscopy to study fura-2 fluorescence in isolated Purkinje and ventricular myocytes and standard biochemical techniques to measure inositol phosphate production in ventricular myocytes. Phospholipase C increased Purkinje fiber automaticity, a process that was enhanced by 10 mM lithium (which had no effect alone) and suppressed by verapamil or ryanodine (both 10 microM). Superfusion with 12-O-tetradecanoyl-phorbol-13-acetate phorbol ester, phospholipase D and A2, as well as L-alpha-phosphatidic acid, trypsin and D-myo-inositol-1-phosphate, D-myo-inositol-1,4-bisphosphate, IP3 and D-myo-inositol-1,4,5,6-tetrakisphosphate did not affect automatic rate or transmembrane potentials. Biochemical studies of ventricular myocytes demonstrated a phospholipase C-induced increase in intracellular and extracellular IP3, D-myo-inositol-1,4-bisphosphate and D-myo-inositol-1-phosphate at 3 min, with the extracellular increase persisting thereafter. Fluorescence microscopy with fura-2 revealed that phospholipase C increased systolic-free calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuronal expression of a newly identified Drosophila melanogaster G protein alpha 0 subunit.

Guanine nucleotide-binding proteins (G proteins) mediate signals between activated cell-surface receptors and cellular effectors. A bovine G-protein alpha-subunit cDNA has been used to isolate similar sequences from Drosophila genomic and cDNA libraries. One class, which we call DG alpha 0, hybridized to position 47A on the second chromosome of Drosophila. The nucleotide sequence of the protein coding region of one cDNA has been determined, revealing an alpha subunit that is 81% identical with rat alpha 0. The cDNA hybridizes strongly to a 3.8 kb mRNA and weakly with a 5.3 kb message. Antibodies raised against a trp-E-DG alpha 0 fusion protein recognized a 39,000 Da protein in Drosophila extracts. In situ hybridization to adult Drosophila sections combined with immunohistochemical studies revealed expression throughout the optic lobes and central brain and in the thoracic and abdominal ganglia. DG alpha 0 message and protein were also detected in the antennae, oocytes, and ovarian nurse cells. The neuronal expression of this gene is similar to mammalian alpha 0, which is most abundantly expressed in the brain.

Reduced lymphocyte stimulatory guanine nucleotide regulatory protein and beta-adrenergic receptors in congestive heart failure and reversal with angiotensin converting enzyme inhibitor therapy.

Adrenergic hyporesponsiveness in congestive heart failure has been understood previously in terms of a reduction in beta-adrenergic receptors. We have examined another hypothesis, one that states the stimulatory guanine nucleotide regulatory protein (Gs) that couples the beta-adrenergic receptor to adenylate cyclase activity is also decreased in congestive heart failure. In addition to the 40% decrease in lymphocyte beta-adrenergic receptors in patients in congestive heart failure (5.9 +/- 0.7 vs. 9.7 +/- 1.4 fmol/mg, p less than 0.05), we found an 80% decrease in levels of Gs compared with age- and sex-matched healthy control subjects (72.5 +/- 19 vs. 376 +/- 73 fmol/mg, p less than 0.05). Myocardial Gs levels correlated significantly with lymphocyte Gs levels. We also assessed the hypothesis that reductions in beta-adrenergic receptors and in Gs are reversible after successful therapy with angiotensin converting enzyme inhibitors. Treatment with either captopril or lisinopril was associated with clinical improvement, an increase in beta-adrenergic receptor density (from 5.5 +/- 0.7 to 8.7 +/- 1.5 fmol/mg), and a twofold increase in Gs levels (p less than 0.05). Thus, the data are compatible with Gs serving as an adaptable and reversible regulator of the adrenergic response in congestive heart failure. In view of the fact that Gs is a transducing element common to all hormones that stimulate cyclic adenosine 5'-monophosphate production, the observations could extend to other abnormal neurohumoral mechanisms in congestive heart failure.

Role of a pertussis toxin-sensitive protein in the modulation of canine Purkinje fiber automaticity.

We previously have shown that alpha-adrenergic stimulation of canine Purkinje fibers and rat ventricle decreases automaticity. Experiments on rat ventricular myocytes in tissue culture have suggested that the decrease in automaticity induced by alpha-adrenergic stimulation depends on the development of sympathetic innervation and the presence of a pertussis toxin-sensitive, 41-kDa guanosine triphosphate (GTP)-regulatory protein. In the present study, microelectrode and biochemical techniques were used to test the role of the pertussis toxin-sensitive protein and sympathetic innervation in modulating automaticity of adult canine Purkinje fibers. Fibers were incubated in Tyrode's solution alone or in Tyrode's solution plus pertussis toxin (0.1-0.5 microgram/ml) for 24 hours and were then superfused with phenylephrine. Phenylephrine in the 5 x 10(-9)-5 x 10(-8) M range induced a decrease in automaticity in 63% of the 16 fibers not treated with pertussis toxin and an increase in automaticity in 37%. The former group had a higher level of pertussis toxin-sensitive substrate by the [32P]nicotinamide adenine dinucleotide adenosine diphosphate (ADP)-ribosylation assay than the latter. In contrast, all fibers treated with pertussis toxin (0.5 microgram/ml) showed increased automaticity in response to phenylephrine and had no detectable pertussis toxin-sensitive substrate. Over the range of pertussis toxin concentrations studied, there was a smooth concentration-response relation between the substrate levels measured and the automatic response to phenylephrine. As ADP-ribosylatable substrate levels decreased, the percent of fibers showing an increase in automaticity increased.(ABSTRACT TRUNCATED AT 250 WORDS)

A pertussis toxin substrate regulates alpha 1-adrenergic dependent phosphatidylinositol hydrolysis in cultured rat myocytes.

The chronotropic response of the heart to alpha 1-adrenergic catecholamines influenced by pertussis toxin under certain conditions. In view of the fact that alpha 1-adrenergic action is mediated by the phosphatidylinositol pathway of hormone action in many cells, we examined the hypothesis that alpha-adrenergic agonists stimulate phosphatidylinositol hydrolysis in cardiomyocytes and that this effect is sensitive to pertussis toxin. Addition of norepinephrine to cultured rat ventricular myocytes prelabeled with myo-[2-3H]inositol resulted in rapid and significant accumulation of inositol phosphate (IP1) and inositol biphosphate. Norepinephrine-stimulated IP1 formation was not inhibited by propranolol, but was inhibited by alpha-adrenergic antagonists with an order of potency indicating alpha 1-adrenergic receptor subselectivity: prazosin (alpha 1; 3 nM) greater than yohimbine (alpha 2; 10 microM). The effect of norepinephrine to enhance IP1 formation was markedly attenuated in cells pretreated with pertussis toxin. Pertussis toxin also induced the transfer of ADP-ribose from NAD to a 41,000-dalton membrane protein in these cells. The concentration of pertussis toxin resulting in maximal inhibition of norepinephrine-stimulated IP1 formation correlated well with the concentration of pertussis toxin necessary to completely ADP-ribosylate a 41,000-dalton membrane protein (1 ng/ml). The range over which pertussis toxin inhibited norepinephrine-dependent IP1 formation and ADP-ribosylated the 41,000-dalton substrate was virtually identical. These observations establish a role for a 41,000-dalton pertussis toxin substrate in coupling the alpha 1-adrenergic receptor to phosphoinositol hydrolysis in myocardial cells.

Stimulatory and inhibitory regulation of myocardial adenylate cyclase by 5'-guanylyl-imidodiphosphate.

Particulate and soluble rat myocardial adenylate cyclase enzymes were characterized with respect to their stimulatory and inhibitory regulation by Gpp(NH)p. Gpp(NH)p (60 microM) stimulated Mg2+- and Mn2+-dependent adenylate cyclase. High concentrations of Gpp(NH)p (600 microM) attenuated the maximal stimulatory response to Gpp(NH)p but only at low cation concentrations. The attenuating effects of 600 microM Gpp(NH)p resulted predominantly from the introduction of a prolonged lag in the kinetics of activation of adenylate cyclase. Steady-state rates of adenylate cyclase activities were similar with either 60 or 600 microM Gpp(NH)p. At any concentration of Gpp(NH)p, the lag was eliminated by Mg ions or isoproterenol. No antihysteretic property for free Mn ions was evident. Forskolin-sensitive particulate adenylate cyclase was not stimulated further by Gpp(NH)p. A 600 microM concentration of Gpp(NH)p inhibited particulate forskolin-sensitive adenylate cyclase at low Mg ion concentrations. In contrast, Gpp(NH)p at 60 microM consistently activated forskolin-sensitive adenylate cyclase after solubilization. The early transient inhibitory properties of 600 microM Gpp(NH)p which resulted in attenuation of adenylate cyclase by 600 microM Gpp(NH)p were diminished by detergent extraction, resulting in only a minor effect of 600 microM Gpp(NH)p to inhibit solubilized adenylate cyclase. These findings indicate that guanine nucleotides exert both stimulatory and inhibitory control upon the myocardial adenylate cyclase enzyme; that solubilization shifts the balance between the stimulatory and inhibitory properties of Gpp(NH)p to allow more dominant expression of the stimulatory response; and that Mg ions critically modify the nature of the myocardial adenylate cyclase response to Gpp(NH)p.

Regulation of rat heart membrane adenylate cyclase by magnesium and manganese.

Rat myocardial adenylate cyclase activity was characterized with respect to its regulation by Mg and Mn. Myocardial adenylate cyclase is activated by both free divalent cations at concentrations greater than that required to form the active cation-ATP substrate. Six separate lines of evidence indicate that Mg and Mn are distinguishable from each other as regulators of adenylate cyclase: 1) After solubilization, adenylate cyclase is preferentially stimulated by Mn. 2) High concentrations of Mn (greater than 1 mM)--but not Mg--have an additional effect on particulate adenylate cyclase to attenuate basal adenylate cyclase activity and to render the membranes refractory to stimulation by isoproterenol and guanyl-5'-yl-imidodiphosphate [Gpp(NH)p]. This inhibitory property of Mn is lost after solubilization. 3) Isoproterenol enhances the affinity of adenylate cyclase for Mg but not for Mn. 4) Stimulation of adenylate cyclase by Gpp(NH)p is consistent with a simple single-site interaction of Gpp(NH)p with the adenylate cyclase complex in the presence of Mg. In contrast, Mn exposes a broad, 3-log-order, stimulatory response to Gpp(NH)p, 0.1 to 100 microM. After solubilization, Mn allows Gpp(NH)p to stimulate adenylate cyclase by simple Michaelis-Menten kinetics as shown for Mg in particulate and solubilized preparations. 5) The lag associated with inhibition of forskolin-sensitive adenylate cyclase is diminished by Mg but not by Mn. 6) Activation of membrane-bound adenylate cyclase by forskolin is consistent with an action at a single, high-affinity site. After solubilization, activation by forskolin is biphasic, with both high- and low-affinity components becoming apparent. The adenylate cyclase response to forskolin at the low-affinity site is greater with Mn than with Mg.(ABSTRACT TRUNCATED AT 250 WORDS)