Faculty Bibliography

Patients with the acquired immunodeficiency syndrome (AIDS) commonly develop hematological abnormalities, including anemia, leukopenia, and thrombocytopenia. Heme synthesis and heme degradation are critical to the maintenance of cellular heme homeostasis and to hematopoietic differentiation. We examined heme oxygenase activity and expression of the heme oxygenase gene in adherent cells (monocytes-macrophages) obtained from the peripheral blood of AIDS patients and normal controls. Heme oxygenase activity in normal control cells was 43 +/- 16 pmol bilirubin formed/4 x 10(5) cells/hr as compared to 133 +/- 30 pmol bilirubin formed/4 x 10(5) cells/hr in the AIDS patients. Via blot hybridization analysis with human heme oxygenase cDNA, heme oxygenase mRNA levels in cells of the normal and the AIDS patients were compared. Total RNA from normal cells displayed only weak hybridization with the cDNA probe. In contrast, cells from peripheral blood of the AIDS patients displayed marked increases over normal levels in heme oxygenase mRNA. Heme oxygenase activity could be substantially suppressed by the competitive inhibitor of the enzyme, Sn-mesoporphyrin. Elevated heme oxygenase activity in cells of AIDS patients could produce a decrease in cellular heme needed for transductional signalling for the growth factor network, which regulates the hematopoietic microenvironment, and for other metabolic purposes. Suppression of heme catabolism by inhibitors of this enzyme may thus be useful in potentiating erythropoietic responses in this disorder

We studied the immediate and long-term effects of azidothymidine (AZT) and heme on murine hemopoietic and stromal progenitor cells in vivo and in vitro. Treatment of mice for 37 days with AZT produced anemia and leukopenia, whereas combined treatment with heme abrogated some of the toxic effects which were apparent even 2 weeks after cessation of treatment. Quantitation of spleen (CFU-S), erythroid (BFU-E) and myeloid (CFU-GM) colony formation from AZT-exposed animals revealed reductions in these progenitors, and this was partially reversed after heme treatment, especially when mice were allowed a 2-week recovery period. Long-term bone marrow cultures (LTBMC) of cells from treated groups revealed difficulty in establishing an adherent cell layer (ACL) by the first week in culture. Total cellularity, CFU-S, BFU-E and CFU-GM clonogenic potential of cultures remained depressed throughout 10 weeks of culture, whereas heme treatment overcame these depressions when AZT-exposed mice were allowed to recover for 14 days prior to culture of their cells in LTBMC. Interleukin-1 (IL-1) treatment to the same recovery group of AZT-exposed mice also resulted in an improvement of CFU-GM growth in LTBMC that was not seen in the nonrecovered group. Transplantation of cells from treated mice under the renal capsule of recipient mice revealed that AZT depressed the regeneration of osteogenic and hemopoietic cell growth within ectopic foci. These effects were reversed with heme treatment in vivo. In other experiments, heme was found to inhibit human immunodeficiency virus (HIV-1) reverse transcriptase and to potentiate the activity of AZT triphosphate against HIV-1 reverse transcriptase. In summary, these results demonstrate that AZT inhibits the growth and development of a variety of hemopoietic, stromal and adherent cells in vivo and in vitro. Treatment of animals with heme produced recovery to near normal levels and suggests possible therapeutic potential

(-)-Emopamil ((S)-emopamil, (2S)-2-isopropyl-5-(methylphenethylamino)- 2-phenylvaleronitrile hydrochloride) is a Ca(2+)-antagonistic phenylalkylamine which also blocks serotonin (5-HT2) receptors and has antiischemic properties. The (-)-[3H]emopamil tissue distribution profile of specific binding is in striking contrast to that observed for (+)-[3H]PN 200-110 or (-)-[3H]desmethoxyverapamil: (-)-[3H]emopamil labels membrane fractions from guinea-pig liver much greater than adrenal gland greater than kidney approximately lung approximately ductus deferens approximately brain approximately skeletal muscle. Binding to liver membrane was saturable (KD = 12.8 nM, Bmax = 35 pmol/mg of protein), stereoselective, reversible (K-1 = 0.22 min-1 at 25 degrees C) and inhibited by tetraethylammonium (IC50: 1.8 mM) greater than Li+ (IC50: 12.5 mM) approximately Na+ (IC50: 13.6 mM) and [NH4+] (IC50: 79.3 mM) but not by Rb+, Cs+ or K+. The high-affinity liver membrane binding sites have a pharmacological profile that is distinct from the phenylalkylamine receptor domain of the voltage-dependent L-type Ca2+ channel. Similar sites exist in brain and other tissues, albeit with a lower density. Amiodarone, butoprozine and amiloride derivatives bind with high affinity whereas 1,4-dihydropyridines do not interact at all. It is suggested that the novel phenylalkylamine site is linked to a sodium-dependent carrier or transport system

The alpha 1-subunit of the voltage-dependent L-type Ca2+ channel has distinct, allosterically coupled binding domains for drugs from different chemical classes (dihydropyridines, benzothiazepines, phenylalkylamines, diphenylbutylpiperidines). (-)-BM 20.1140 (ethyl-2,2-di-phenyl-4-(1-pyrrolidino)-5-(2-picolyl)- oxyvalerate) is a novel Ca2+ channel blocker which potently stimulates dihydropyridine binding (K0.5 = 2.98 nM) to brain membranes. This property is shared by (+)-cis-diltiazem, (+)-tetrandrine, fostedil and trans-diclofurime, but (-)-BM 20.1140 does not bind in a competitive manner to the sites labeled by (+)-cis-[3H]diltiazem. (+)-cis-Diltiazem and (-)-BM 20.1140 have differential effects on the rate constants of dihydropyridine binding. (+)-BM 20.1140 reverses the stimulation of the positive allosteric regulators (pA2 value for reversal of (-)-BM 20.1140 stimulation = 7.4, slope 0.72). The underlying molecular mechanism of the potentiation of dihydropyridine binding has been clarified. The K0.5 for free Ca2+ to stabilize a high affinity binding domain for dihydropyridines on purified L-type channels from rabbit skeletal muscle is 300 nM. (+)-Tetrandine (10 microM) increases the affinity 8-fold (K0.5 for free Ca2+ = 30.1 nM) and (+)-BM 20.114 (10 microM) inhibits the affinity increase (K0.5 for free Ca2+ = 251 nM). Similar results were obtained with membrane-bound Ca(2+)-channels from brain tissue which have higher affinity for free Ca2+ (K0.5 for free Ca2+ = 132 nM) and for dihydropyridines compared with skeletal muscle. It is postulated that the dihydropyridine and Ca(2+)-binding sites are interdependent on the alpha 1-subunit, that the different positive heterotropic allosteric regulators (by their differential effects on Ca2+ rate constants) optimize coordination for Ca2+ in the channel pore and, in turn, increase affinity for the dihydropyridines

The complete amino acid sequence of the L-type calcium channel alpha 1 subunit from the carp (Cyprinus carpio) white skeletal muscle was deduced by cDNA cloning and sequence analysis. The open reading frame encodes 1852 amino acids (Mr 210,060). A 155-amino acid COOH-terminal sequence (after the fourth internal repeat) is evolutionarily preserved (90% homology) and may represent an important functional domain of L-type calcium channels. The photolabeled, membrane-bound, and purified carp alpha 1 subunits have masses of 211 and 190 kDa. The purified channel could not be phosphorylated by cAMP-dependent protein kinase. Two glycoproteins (alpha 2 subunits) are associated with the alpha 1 subunit and change their apparent masses from 235 and 220 kDa to 159 kDa upon reduction of disulfide bonds. Nucleic acid hybridization with alpha 2 cDNA revealed an 8.0-kilobase transcript in carp skeletal muscle. Evidence for a copurification of subunits similar in size to mammalian beta or gamma subunits was not obtained

1. Piperazinylindoles (DPI 201-106, BDF 8784), drugs known to act on voltage-dependent Na(+)-channels, bind with very high affinity to a Ca2(+)-channel-associated phenylalkylamine receptor in Drosophila melanogaster head membranes. These compounds and (+)-tetrandrine, a naturally occurring Ca2(+)-antagonist, were the most selective inhibitors for phenylalkylamine-labelled Drosophila Ca2(+)-channels compared to mammalian L-type Ca2(+)-channels. 2. Replacement of the cyano group by a methyl group in (+)-DPI 201-106 ((+)-BDF 8784) increases the IC50 value for inhibition of phenylalkylamine labelling of Drosophila Ca2(+)-channels from 0.29 to 2.1 nM but decreases the IC50 value for inhibition of phenylalkylamine labelling of mammalian skeletal muscle Ca2(+)-channels from 3480 to 49 nM. 3. DPI 201-106 enantiomers completely block (at 0.1 microM) phenylalkylamine photolabelling of a 136 K polypeptide in Drosophila head membranes whereas 10 microM aconitine or lidocaine are without effect. 4. Assessment of the Ca2(+)-antagonist effects of the substituted DPI 201-106 enantiomers in K(+)-depolarized taenia strips from guinea-pig caecum yielded pA2 values of 6.33 +/- 0.07 for (-)-BDF 8784 and 6.99 +/- 0.17 for (+)-BDF 8784, respectively. 5. Piperazinylindoles, previously believed to act nonspecifically on voltage-dependent mammalian L-type Ca2(+)-channels, therefore have stereoselectivity for a novel binding site and chemical selectivity unrelated to local anaesthetic activity. 6. It is proposed that a very high affinity piperazinylindole-selective site is coupled to the phenylalkylamine receptor of Drosophila Ca2(+)-channels. These sites are still present on mammalian L-type Ca2(+)-channels but have lower affinity and/or are less tightly coupled to phenylalkylamine receptors on the alpha 1-subunit