L-carnitine supplementation for the treatment of fatigue and depressed mood in cancer patients with carnitine deficiency: a preliminary analysis.

Nutritional factors are among the postulated causes of fatigue, a highly prevalent symptom in the cancer population, with serious impact on patients' quality of life. Deficiency of the micronutrient carnitine may play a role by reducing energy production through fatty acid oxidation. We present preliminary data of an open-label, dose-finding study to determine safety and maximally tolerated dose (MTD) of 1 week of L-carnitine supplementation in cancer patients with fatigue and carnitine deficiency. Patients who met inclusion/exclusion criteria underwent carnitine level determination. Eighty-three percent of these patients (15/18) had carnitine deficiency. Preliminary data analysis of 13 patients showed that total carnitine increased from 30.0 +/- 6.9 to 41.0 +/- 12.1 (mean +/- SD) after 1 week of supplementation (P = 0.01), and free carnitine increased from 24.3 +/- 6.1 to 33.8 +/- 9.8 (P = 0.004). Outcome measures were fatigue (BFI score), depression (CES-D), sleep disruption (ESS), and performance status (Karnofsky). Median (min, max) BFI score at baseline was 73 (46, 82) versus 50 (3, 82) after 1-week supplementation (P = 0.009). CES-D score at baseline was 29 (16, 42) and 22 (8, 32) after 1 week (P = 0.028). ESS at baseline was 46.5 (0, 69) and 30.4 (0, 72) after 1 week (P = 0.015). Karnofsky score did not change significantly (P = 0.38). We are currently conducting a randomized, double-blind, placebo-controlled study to rigorously assess the role of L-carnitine for the treatment of fatigue and depression in cancer patients.

Duodenal obstruction following elective abdominal aortic aneurysm repair.

A case where duodenal obstruction complicated elective repair of abdominal aortic aneurysm is reported. A review of the literature shows that the incidence of this rare complication has probably been underestimated. Patients should be treated expectantly, regardless of the pathogenesis of the obstruction.

Presence in neuroblastoma cells of a mu 3 receptor with selectivity for opiate alkaloids but without affinity for opioid peptides.

Evidence is presented for the occurrence of a unique opiate alkaloid-selective, opioid peptide-insensitive binding site in N18TG2 mouse neuroblastoma cells and in late passage hybrid F-11 cells, derived from N18TG2 neuroblastoma cells and rat dorsal root ganglion cells. Those cells lacked classical opioid peptide-sensitive receptor subtypes, but contained [3H]morphine and [3H]diprenorphine binding sites with affinity for certain opiate alkaloids but not for any endogenously occurring opioid peptide or peptide analog tested, including D-ala2-D-leu5-enkephalin (DADLE), D-Ala2,N-Me-Phe4,Gly5-ol (DAGO) and dynorphin A(1-17). The binding site differed from hitherto described mu, delta and kappa neuronal opioid receptors not only on the basis of peptide insensitivity, but also on the basis of selectivity and affinities of alkaloids. Saturation experiments with [3H]morphine indicated the presence of a single site with Kd = 49 nM and Bmax = 1510 fmol/mg protein. This novel binding site was not present in F-11 hybrid cells at early passage. Instead the hybrid cells contained conventional opioid receptors (predominantly delta and also mu) capable of binding DADLE and other peptides as well as opiate alkaloids. With additional passage (cell divisions) of the hybrid cells, during which a limited change occurred in mouse chromosome number, the peptide-insensitive binding appeared and the opioid peptide-binding (delta and mu) receptors were lost reciprocally. Thus, expression of the peptide-insensitive binding normally may be repressed when conventional opioid receptors are expressed. The peptide-insensitive opiate binding site described here appears to correspond to the mu 3 receptor subtype, recently identified pharmacologically and functionally in several cell types of the immune system.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyl-L-carnitine treatment increases nerve growth factor levels and choline acetyltransferase activity in the central nervous system of aged rats.

The hypothesis that some neurodegenerative events associated with ageing of the central nervous system (CNS) may be due to a lack of neurotrophic support to neurons is suggestive of a possible reparative pharmacological strategy intended to enhance the activity of endogenous neurotrophic agents. Here we report that treatment with acetyl-l-carnitine (ALCAR), a substance which has been shown to prevent some impairments of the aged CNS in experimental animals as well as in patients, is able to increase the levels and utilization of nerve growth factor (NGF) in the CNS of old rats. The stimulation of NGF levels in the CNS can be attained when ALCAR is given either for long or short periods to senescent animals of various ages, thus indicating a direct effect of the substance on the NGF system which is independent of the actual degenerative stage of the neurons. Furthermore, long-term treatment with ALCAR completely prevents the loss of choline acetyltransferase (ChAT) activity in the CNS of aged rats, suggesting that ALCAR may rescue cholinergic pathways from age-associated degeneration due to lack of retrogradely transported NGF.

Cl- channels are randomly activated by continuous GABA secretion in cultured embryonic rat hippocampal neurons.

Throughout the adult vertebrate central nervous system (CNS) gamma-aminobutyric acid (GABA) mediates transient Cl- conductances commonly identified as fast, Cl(-)-dependent inhibitory synaptic signals [Prog. Neurobiol., 36 (1991) 35-92]. In the rat hippocampus Cl(-)-dependent excitatory transients mediated by GABA emerge during the first postnatal week superimposed on a steady-state baseline that is also Cl(-)- and GABA-dependent [Int. J. Dev. Neurosci., 8 (1990) 481-490]. Here we report that many embryonic rat hippocampal neurons cultured for hours to days exhibit random fluctuations in Cl- channel activity that are mediated by continuous secretion of GABA in the absence of transients. Thus, GABA is broadcast tonically before it is released transiently.

Characterization of the gamma-aminobutyric acidA receptor-channel complex composed of alpha 1 beta 2 and alpha 1 beta 3 subunits from rat brain.

The cloned alpha 1, beta 2 and beta 3 subunits of the gamma-aminobutyric acid (GABA)A receptor-channel complex from rat brain were coexpressed as alpha beta complexes in cultured Chinese hamster ovary cells. Electrophysiological characterization of alpha 1 beta 2 and alpha 1 beta 3 receptor subunit arrangements was performed utilizing patch electrodes in the whole-cell recording configuration. The reversal potential of the current activated by either GABA or muscimol corresponded to that expected for Cl- ions and was dependent on the Cl- gradient. The dose response to GABA for activation of Cl- currents by either subunit combination displayed similar potencies. Currents were partially blocked by the reversible antagonist bicuculline. (-)Pentobarbital was ineffective by itself, but potentiated responses to GABA. The steroid alphaxalone (3 alpha-hydroxy 5 alpha-pregnane 11,20-dione) produced just-detectable inward currents, but did not potentiate GABA-activated currents. Diazepam was completely ineffective. The kinetics and conductance of the Cl- ion channels were inferred from spectral analysis of agonist-induced current fluctuations. Both kinetics and conductance were dependent on agonist structure.

Direct coupling of opioid receptors to both stimulatory and inhibitory guanine nucleotide-binding proteins in F-11 neuroblastoma-sensory neuron hybrid cells.

Evidence is presented for linkage of opioid receptors directly to the stimulatory G protein (guanine nucleotide-binding protein), Gs, in addition to the generally accepted linkage to the inhibitory and "other" G proteins, gi and Go, in F-11 (neuroblastoma-dorsal root ganglion neuron) hybrid cells. Treatment of intact F-11 cells with cholera toxin decreased specific binding of the opioid agonist [D-Ala2,D-Leu5]enkephalin to F-11 cell membranes by 35%, with the remaining binding retaining high affinity for agonist. Under these conditions cholera toxin influenced the alpha subunit of Gs (Gs alpha) but had no effect on the alpha subunit of Gi/o (Gi/o alpha), based on ADP-ribosylation studies. Pertussis toxin treatment decreased high-affinity opioid agonist binding by about 50%; remaining binding was also of high affinity, even though pertussis toxin had inactivated Gi/o alpha selectively and essentially completely. Simultaneous treatment with both toxins had an additive effect, reducing specific binding by about 80%. While opioid agonists inhibited forskolin-stimulated adenylate cyclase activity of F-11 cells as expected, opioids also stimulated basal adenylate cyclase activity, indicative of interaction with Gs as well as Gi. Cholera toxin treatment attenuated opioid-stimulation of basal adenylate cyclase, whereas pertussis toxin treatment enhanced stimulation. In contrast, inhibition by opioid of forskolin-stimulated activity was attenuated by pertussis toxin but not by cholera toxin. It is concluded that a subset of opioid receptors may be linked directly to Gs and thereby mediate stimulation of adenylate cyclase. This Gs-adenylate cyclase interaction is postulated to be responsible for the novel excitatory electrophysiologic responses to opioids found in our previous studies of sensory neurons and F-11 cells.

Characterization of the alpha subunit of the IFN-alpha receptor. Evidence of N- and O-linked glycosylation and association with other surface proteins.

We studied the association of the alpha subunit of the (IFN-alpha-receptor) to other receptor components in the human H-929 and U-266 myeloma cell lines. Immunoprecipitation performed with the IFNaR3 mAb showed that two proteins with molecular masses of 205 and 145 kDa are co-precipitated with the alpha subunit. These complexes may not bind IFN-alpha as shown by studies using the heterobifunctional cross-linking reagent Denny-Jaffe and by partial cleavage of the homobifunctional cross-linker dithio succinimidyl propionate. These studies also provided evidence that at least two subunits with molecular mass of 130 kDa (alpha subunit) and 110 kDa (including 20 kDa corresponding to IFN-alpha) contribute to the formation of the IFN-alpha-receptor complex. To further characterize the alpha subunit of the IFN-alpha-receptor, immunoprecipitates using the mAb IFNaR3 were sequentially treated with N-glycanase, neuraminidase and O-glycanase. These studies showed that the alpha subunit is heavily glycosylated and has a protein precursor with a molecular mass of 68 kDa. Binding studies provided evidence for high and low affinity binding sites for IFN-alpha 2. Affinity cross-linking experiments under low and high affinity conditions suggest that the high affinity binding site of the IFN-alpha-receptor is formed by a complex containing the alpha subunit, whereas the 110-kDa subunit may bind IFN-alpha 2 under low affinity conditions.

Magainin 2, a natural antibiotic from frog skin, forms ion channels in lipid bilayer membranes.

We have examined the ion channel forming properties of magainin 2 by incorporating the peptide into artificial lipid bilayers held under voltage clamp. Magainin 2 increased lipid bilayer conductance in a concentration dependent manner with a Hill coefficient of 1.7. The magainin 2 conductance was selective for monovalent cations over anions with a ratio of 5:1 and had both voltage-sensitive and -insensitive components. Two structurally related but antibiotically less potent analogues, magainin 1 and Z-12, also increased lipid bilayer conductance with a similar ion selectivity but these peptides were less potent than magainin 2. We propose that the weak cation selectivity of the magainin channels can be accounted for by the inclusion of negatively charged lipids in the channel complex and suggest two possible structures for such a channel. The ionophoric properties of these peptides are likely to be proximal to their antibiotic activities.

Multichain structure of the IFN-alpha receptor on hematopoietic cells.

The structure of IFN-alpha receptor was studied by 1) developing antibodies against the receptor, and 2) screening a number of cell lines by affinity cross-linking to identify cells that express different IFN-alpha 2 receptor structures. We report that two different patterns of IFN-alpha 2 receptor are observed in human cells of hematopoietic origin. The predominant form of the IFN-alpha receptor is a multichain structure in which IFN-alpha 2 forms complexes of 110 and 130 kDa (alpha-subunit). A high Mr complex of 210 kDa results from the association of alpha-subunit and other receptor components. In contrast, another form of the receptor has been identified in the IFN-alpha-resistant U-937 cell line and in some cases of acute leukemia. This form of the IFN-alpha receptor is characterized by the presence of the alpha- subunit, and the absence of the 110- and 210-kDa bands. Also a novel 180-kDa complex and a more prominent 75-kDa band are observed. Functional studies performed in U-937 cells showed that this cell line is not only partially resistant to the antiproliferative and antiviral effects of IFN-alpha, but also fails to down-regulate the alpha-subunit of the IFN-alpha receptor upon IFN-alpha binding.

Antibiotic magainins exert cytolytic activity against transformed cell lines through channel formation.

Magainins are an ionophoric class of vertebrate peptides with antibiotic activity against various microorganisms. Here we show that magainin 2 and synthetic analogues can rapidly and irreversibly lyse hematopoietic tumor and solid tumor target cells with a relative cytotoxic potency that parallels their antibacterial efficacy and at concentrations that are relatively nontoxic to well-differentiated cells. The cytotoxicity is prevented by cell depolarization. Magainins represent a natural cytolytic agent in vertebrates and may provide another therapeutic strategy for certain tumors.

Computer modeling of subtypes of kappa opioid receptors in adrenal medulla.

The existence of multiple subtypes of kappa opioid receptors in brain and adrenal medulla, has been controversial. We have characterized opioid receptors in frozen membranes from bovine adrenal medulla by use of objective mathematical modeling. [3H]Etorphine, [3H]ethylketocyclazocine (EKC) and [3H][D-Ala2,D-Leu5]enkephalin (DADL) were utilized as labeled ligands. Self- and cross-displacement curves were constructed using the three corresponding unlabeled ligands in the presence or absence of increasing concentrations of DADL. Results indicated: (1) each of the three ligands studied individually showed the presence of heterogeneity of binding sites; (2) sites labeled by etorphine were heterogeneous: 84% of etorphine binding was displaceable by 10(-4) M DADL, while the remaining 16% was DADL non-suppressible; (3) 75% of the binding of EKC was displaceable by DADL while 25% was non-suppressible; (4) mathematical modeling showed the presence of three subtypes of kappa binding sites (a) kappa 1, showing slight selectivity for EKC relative to etorphine; (b) kappa 2, with Kd approximately equal to 1 nM for etorphine, and sufficiently high affinity for DADL (Kd approximately equal to 150 nM) so that it is suppressible by 100 microM DADL; and (c) K3, with no measurable affinity for DADL and a 27-fold selectivity for etorphine relative to EKC. The three subtypes of kappa sites were present at concentrations of 7.4, 75, and 55 fmol/mg protein, respectively. The relative affinities of a series of kappa agonists for the etorphine-binding sites were characterized. The present studies confirm the existence of three subtypes of kappa opioid receptors in bovine adrenal medulla, and indicate the utility of mathematical modeling for characterization of complex receptor systems.

Functional coupling of GABAA receptors and benzodiazepine recognition site subtypes in the spinal cord of the rat.

The interaction between GABAA receptors and benzodiazepine (BZD) recognition site subtypes in the spinal cord of the rat was investigated. Computer analysis of displacement curves for [3H]flunitrazepam [( 3H]FNT) binding by 2-oxo-quazepam (2OXOQ) indicated the presence of two subtypes of BZD recognition sites in this region. Type I sites accounted for approximately 25% of the total number of BZD recognition sites, the remainder being Type II sites. A similar proportion of Type I and Type II sites was obtained by Scatchard analysis of the saturation curves for [3H]FNT, [3H]2OXOQ and [3H]ethyl-beta-carboline-3-carboxylate [( 3H]beta CCE) binding. The in vitro addition of GABA (10(-8)-10(-4) M) to spinal cord membrane preparations produced an increase in the binding of [3H]FNT and [3H]2OXOQ. The maximal enhancement produced by GABA was 50 and 82% above control values for [3H]FNT and [3H]2OXOQ, respectively. In contrast, GABA stimulated both [3H]FNT and [3H]2OXOQ binding in the cerebellum to a similar extent. We also evaluated the effects of different ligands for BZD recognition sites on the binding of [3H]GABA to spinal cord membranes, as compared with brain areas containing a higher proportion ( greater than 30%) of Type I sites. Diazepam, quazepam and the beta-carboline, ZK 93423, enhanced the specific binding of [3H]GABA in a concentration-dependent manner (10(-7)-10(-5) M) in the cerebral cortex and hippocampus but not in the spinal cord and cerebellum. These results indicate that there is a regional variation in the interaction between GABA and BZD recognition sites in the central nervous system.

Spontaneous polymerization of the antibiotic peptide magainin 2.

We describe here the ability of the magainin 2 peptide to assemble spontaneously into characteristic 13-nm diameter filaments having a 30 nm periodic helical substructure. Optimal conditions for extensive polymerization into filaments of several hundred microns required low pH and high ionic strength. Polymerization of the magainin 2 peptide may be involved in its recently described in vitro membrane-disrupting and antibiotic activities.

Quantitative autoradiographic localization of neuropeptide Y (NPY) binding sites in rat posterior pituitary lobe.

1. We have localized and quantified neuropeptide Y (NPY) binding sites in the rat pituitary gland after incubation of tissue sections in the presence of 125I-Bolton-Hunter NPY followed by autoradiography, computerized microdensitometry, and comparison to 125I-standards. 2. In the rat, NPY binding sites are localized exclusively to the part of the posterior pituitary lobe closer to the pituitary stalk. No NPY binding sites could be found in the intermediate or the anterior pituitary lobes. 3. Our results suggest a role for NPY in the regulation of pituitary function and, in particular, that of the neural lobe.

Localization of neuropeptide Y binding sites in the zona glomerulosa of the bovine adrenal gland.

We studied neuropeptide Y (NPY) binding sites in the bovine adrenal gland by incubating tissue sections with [125I]-Bolton Hunter NPY, and then by measuring the number and affinity of binding sites in the tissue with quantitative autoradiography. Specific NPY binding sites were localized exclusively in the zona glomerulosa. These binding sites have an apparent dissociation constant (Kd) of 0.45 +/- 0.06 nM and a binding capacity (Bmax of 134 +/- 15 fmol mg-1 protein. Our results suggest that NPY may directly affect the release of aldosterone in the zona glomerulosa of the adrenal gland.

Characterization of beta 1- and beta 2-adrenoceptor subtypes in the rat atrioventricular node by quantitative autoradiography.

We characterized beta-adrenoceptor subtypes in the atrioventricular node of the rat heart by quantitative autoradiography. Consecutive 16-microns-thick sections from single rat hearts containing the atrioventricular node were incubated with increasing concentrations of [125I]iodocyanopindolol. After exposure to [3H]Ultrofilm, optical densities corresponding to the atrioventricular node were determined by computerized densitometry after comparison with [125I]standards. The computer program LIGAND was used for analysis of receptor subtypes. Delineation of beta-adrenoceptor subtypes was achieved by incubating consecutive tissue sections with 50 pM [125I]iodocyanopindolol in the presence of increasing concentrations of the beta 1-selective antagonist atenolol or the beta 2-selective antagonist ICI 118,551. The atrioventricular node contains a higher concentration of beta-adrenoceptors than the adjacent interventricular septum. We estimated that the proportions of beta 1- and beta 2-adrenoceptors in the atrioventricular node were about 56% and 44% of the total binding capacity respectively.

Naloxonazine effects on the interaction of enkephalin analogs with mu-1, mu and delta opioid binding sites in rat brain membranes.

The authors have characterized the opioid receptors of rat brain membranes using self- and cross-displacement studies with both tritiated and unlabeled [D-Ala2, D-Leu5]-enkephalin and [D-Ala2, MePhe4, Gly-ol5]-enkephalin. Mathematical modeling demonstrated the presence of three classes of binding sites, corresponding to mu, delta and the putative mu-1 classes of site. Unlabeled naloxonazine shows high affinity for all three classes of sites, with highest affinity for the mu-1 sites. Membranes were preincubated with 50 nM naloxonazine or with controls (50 nM naloxone or buffer) for 30 min. Preincubation of membranes with 50 nM naloxonazine resulted in a dramatic, nearly 2-fold reduction in the binding of [3H][D-Ala2, D-Leu5]-enkephalin and [3H][D-Ala2, MePhe4, Gly-ol5]-enkephalin relative to the controls. Quantitative analyses using mathematical modeling with program "LIGAND" suggested that this effect was primarily "competitive," i.e., attributable to changes in affinity, with no apparent or detectable noncompetitive or irreversible effects on binding capacities for the three classes of sites.

Dopamine D1 receptor in rat brain: a quantitative autoradiographic study with 125I-SCH 23982.

We report the regional distribution and characteristics of 125I-SCH 23982 binding to D1 receptors in rat brain using a quantitative autoradiographic technique. The substantia nigra pars reticulata, the caudate putamen, the nucleus accumbens and the olfactory tubercle had a single class of high affinity binding sites for 125I-SCH 23982. Binding sites were also present in a discrete, continuous band connecting the caudate putamen with the substantia nigra.