Biomedical subjects
V M Steen
Publications and source records attributed to V M Steen.
Lack of genetic variation in the coding region of the myo-inositol monophosphatase gene in lithium-treated patients with manic depressive illness.
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Nomenclature for human CYP2D6 alleles.
To standardize CYP2D6 allele nomenclature, and to conform with international human gene nomenclature guidelines, an alternative to the current arbitrary system is described. Based on recommendations for human genome nomenclature, we propose that alleles be designated by CYP2D6 followed by an asterisk and a combination of roman letters and arabic numerals distinct for each allele with the number specifying the key mutation and, where appropriate, a letter specifying additional mutations. Criteria for classification as a separate allele and protein nomenclature are also presented.
Characterization and PCR-based detection of two different hybrid CYP2D7P/CYP2D6 alleles associated with the poor metabolizer phenotype.
The majority of humans deficient in the cytochrome P450 CYP2D6 enzyme, so-called poor metabolizers (PMs), can now be identified by genotyping for several different PM-associated mutations. However, additional null alleles remain to be identified as demonstrated by subjects with the PM phenotype in the absence of a corresponding genotype. The rare 11 kb band on Xba I RFLP analysis, which is distinct from the 13 kb CYP2D6D (CYP2D6*5) allele, has been proposed to constitute such a unique non-functional allele. Here we demonstrate that the 11 kb band represents at least two different nine exon CYP2D7P/CYP2D6 hybrids generated by large deletions in the CYP2D gene cluster due to unequal cross-over or looping-out mechanisms. The total allele frequency was approximately 0.001-0.01 in European and North American Caucasians. The most common variant (CYP2D6*16) had breakpoints lying between the end of exon 7 and the start of exon 9 of the respective genes. The "CYP2D7-like' part of the gene was most homologous to the previously described CYP2D7AP and CYP2D7 (44/11.5) sequences. The other chimeric allele consisted of exon 1 of CYP2D7 and exons 2-9 from CYP2D6, and may be similar to a hybrid gene termed CYP2D6*13 recently described in a French individual. Two different routine PCR assays were developed for rapid and sensitive detection of these alleles, namely amplification of a 8 kb fragment from both CYP2D6*13 and CYP2D6*16, together with a CYP2D6*16-specific method which gave a 1.4 kb PCR product. The 8 kb assay for the CYP2D6*13 and CYP2D6*16 alleles also produced a 9.5 kb fragment in samples positive for the 13 kb CYP2D6*5 allele. Therefore, it is now possible to screen for the large CYP2D gene deletions by a single long PCR method.
A retroviral element in the human CYP2D gene cluster.
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Homologous unequal cross-over involving a 2.8 kb direct repeat as a mechanism for the generation of allelic variants of human cytochrome P450 CYP2D6 gene.
The cytochrome P450 enzyme debrisoquine 4-hydroxylase (CYP2D6) metabolizes many different classes of commonly used drugs. In Caucasian populations, 5-10% are classified as poor metabolizers (PM) due to autosomal recessive inheritance of two mutant CYP2D6 null alleles. In contrast, up to 5% may demonstrate ultrarapid metabolism (UM) of debrisoquine caused by inherited amplification of functional CYP2D6 genes in the CYP2D6 locus. Poor metabolizer subjects may develop toxic plasma concentrations and adverse drug reactions, whereas UMs may suffer from therapeutic failure. Moreover, mutant CYP2D6 alleles have been implicated as a predictor of susceptibility for diseases such as cancer and neurological disorders. The break points and molecular mechanisms involved in the generation in the PM-associated CYP2D6(D) gene deletion allele and the UM-related CYP2D6 amplification have not been clarified. Here we demonstrate the presence of a 2.8 kb repeated region (CYP-REP) which flanks the active CYP2D6 gene in the wild type allele. The CYP-REP unit may be itself predispose to homologous unequal cross-over and contains the Alu element and a tandem 10 bp direct repeat, which could both serve as hotspots for recombination. The break points of the CYP2D6(D) deletion allele are present within the repeated 2.8 kb region, but the exact positions are non-determinable due to perfect recombination of the misaligned, homologous CYP-REP elements. We also propose that the alleles with multiple copies of CYP2D6, which represent the first example of inherited amplification of an active gene in man, can be explained by unequal cross-over events involving the CYP-REP units. In our model, the CYP2D6 deletion and amplification alleles are reciprocal to each other, generated through homologous unequal recombination of no-allelic CYP-REP elements.
Detection of the poor metabolizer-associated CYP2D6(D) gene deletion allele by long-PCR technology.
The cytochrome P450 enzyme debrisoquine 4-hydroxylase metabolizes many different classes of commonly used drugs, such as antidepressants and neuroleptics. Deficient hydroxylation of debrisoquine, known as the poor metabolizer (PM) phenotype, affects 5-10% of Caucasians and may lead to adverse reactions upon administration of drugs in standard doses. This autosomal recessive metabolic deficiency is caused by the possession of two PM-associated mutations in the human CYP2D6 gene locus coding for the enzyme. These mutations include at least four different single base mutations and two different large gene deletion alleles. The single base mutations can be rapidly detected by PCR methods. In contrast, the large gene deletions have so far only been directly identified by RFLP analysis. By the use of sequence data previously published by others, we report here an alignment of different CYP2D alleles to focus on the presence of almost completely identical sequences immediately downstream of both CYP2D7 and CYP2D6 which may seriously complicate and interfere with PCR-based detection of the gene deletion. Based on this analysis, we have developed a rapid assay which, for the first time, detects the 13kb (also called 11.5 kb) Xba I gene deletion allele by the use of long-PCR technology. The primers were designed to amplify a 3.5 kb PCR product in the presence of this D6(D) allele. We have evaluated the method on 23 different DNA samples heterozygous (n = 22) or homozygous (n = 1) for the 13 kb gene deletion allele (previously typed by RFLP analyses). All samples were correctly identified by the assay. The PCR method did not detect the rare 11 kb Xba I gene deletion allele (n = 5), and there was no false positive amplification from deletion negative DNA samples (n = 47). This sensitive and specific PCR-based assay for detection of the D6(D) allele will improve the scientific and clinical use of CYP2D6 genotyping.
The platelet-stimulating effect of adrenaline through alpha 2-adrenergic receptors requires simultaneous activation by a true stimulatory platelet agonist. Evidence that adrenaline per se does not induce human platelet activation in vitro.
The stimulating effect of adrenaline on human platelet phospholipase C (PLC) activation and responses in vitro (shape change, aggregation and dense granule secretion) was investigated with respect to its dependence on exogenously added agonists. All experiments were performed with human gel-filtered platelets pretreated with acetylsalicylic acid to prevent endogenous stimulation by the arachidonate pathway. (1) Preliminary experiments demonstrated the presence of trace amounts of extracellular ADP (0.05-0.58 microM) in non-stimulated platelet suspensions; ADP was effectively converted to ATP by the enzyme system creatine phosphate (CP)/creatine phosphokinase (CPK). (2) The adrenaline-induced optical aggregation and single particle (platelet) disappearance in the presence of trace amounts of ADP were almost abolished by the ADP-scavenger system CP/CPK. (3) The response of CP/CPK-treated thrombin- or platelet-activating factor (PAF)-stimulated platelets was markedly increased by a subsequent addition of adrenaline. When hirudin or BN 50726 was added just prior to adrenaline to terminate the activation by thrombin or PAF, respectively, the stimulating effect of adrenaline was also abolished. (4) CP/CPK-treated, PAF-stimulated platelets rapidly developed decreased responsiveness to a subsequent addition of PAF. When adrenaline was added instead of a second addition of PAF, the stimulating effect of adrenaline was gradually decreased and prevented in parallel with the homologous desensitization of PAF. (5) The weak platelet agonist serotonin by itself induced only shape change in CP/CPK-treated platelets. Adrenaline failed to enhance the extent of this serotonin-induced platelet activation. (6) These results strongly suggest that adrenaline per se is not a platelet agonist in vitro but acts to enhance the stimulation induced by true agonists.
Neomycin does not interfere with the inositol phospholipid metabolism, but blocks binding of alpha-thrombin to intact human platelets.
Neomycin was demonstrated to inhibit the binding of thrombin to intact human platelets. The effects of neomycin on both thrombin binding and thrombin-induced changes in inositol phospholipid metabolism could be reproduced by the thrombin antagonist hirudin. We propose that neomycin inhibits thrombin-induced platelet activation by interference with the cellular receptor.
Stimulation of human platelets with low concentrations of thrombin: evidence for equimolar accumulation of inositol trisphosphates and phosphatidic acid.
1. Gel-filtered human platelets prelabeled with [32P]Pi or [3H]glycerol were exposed to 0-0.3 U/ml of thrombin and analyzed for radioactivities and masses in the phosphoinositides, inositol trisphosphates (IP3), phosphatidic acid (PA) and diacylglycerol (DAG) at 15 and 180 sec of stimulation. 2. At thrombin concentrations below 0.1 U/ml, PA and IP3 accumulated in equimolar amounts. 3. The production and disappearance of the metabolites of the polyphosphoinositide cycle was balanced during 180 sec of stimulation with 0.03-0.1 U/ml of thrombin. 4. Under these conditions no increase in [3H]DAG or [3H]monoacylglycerol could be detected. 5. The data indicate that all DAG is converted to PA and support our conclusion that phosphatidylinositol 4,5-bisphosphate represents the major source for production of DAG upon stimulation of human platelets with low concentrations of thrombin.
Evidence that chlorpromazine and prostaglandin E1 but not neomycin interfere with the inositol phospholipid metabolism in intact human platelets.
Human platelets that had been prelabelled with [32P]Pi were stimulated with trombin in the presence or absence of neomycin, prostaglandin E1 (PGE1) or chlorpromazine. The content of [32P]Pi in phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate and phosphatidic acid (PA) were determined. The data demonstrate that PGE1 and chlorpromazine but not neomycin interfere with the tight metabolic relationship that exists between the inositol phospholipids and PA in thrombin-stimulated platelets [(1989) Biochem. J. 263, 621-624]. Our results therefore indicate that neomycin does not inhibit signal transduction in intact platelets at the level of the inositol phospholipid metabolism.
Evidence for tight metabolic control of the receptor-activated polyphosphoinositide cycle in human platelets.
The [32P]PIP2/[32P]PA and the [32P]PIP/[32P]PA relationships were demonstrated to be remarkably similar after stimulation of [32P]Pi-prelabelled platelets for 90 s with various combinations and concentrations of agonists and inhibitors. Thus the activity of the PI and PIP kinases with the corresponding phosphomonoesterases may be tightly controlled during receptor-mediated platelet stimulation involving phospholipase C activation.
Potentiation by adrenaline of thrombin-induced elevation of pHi is not essential for synergistic activation of human platelets.
Gel-filtrated human platelets were stimulated with thrombin in the absence and presence of adrenaline. Adrenaline markedly enhanced the thrombin-induced increase in cytoplasmic pH (pHi) in BCECF-loaded platelets. This rise in pHi was strongly inhibited by the Na+/H+ exchange blocker EIPA. The potentiation by adrenaline of thrombin-induced PLC activation measured as [32P]PA formation and final platelet responses was, however, not blocked by EIPA, even at low concentrations of thrombin. These results indicate that the enhancement by adrenaline of thrombin-induced cytoplasmic alkalinization may be a secondary effect which is not essential for the potentiation by adrenaline of platelet activation by thrombin.
Neomycin inhibits platelet functions and inositol phospholipid metabolism upon stimulation with thrombin, but not with ionomycin or 12-O-tetradecanoyl-phorbol 13-acetate.
Gel-filtered human platelets that had been pre-labelled with [32P]Pi were stimulated with thrombin, ionomycin or the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA). The effect of the hexacationic aminoglycoside antibiotic, neomycin, on platelet physiological responses, such as aggregation and secretion, as well as changes in phosphoinositide metabolism was studied. Neomycin strongly inhibited thrombin-induced aggregation and secretion whereas the antibiotic had no effect on ionomycin- or TPA-induced platelet functions. The thrombin-induced enhancement of inositol phospholipid metabolism was strongly inhibited by the presence of neomycin whereas the TPA- or ionomycin-induced increase in inositol [32P]polyphospholipids remained unaffected. The inhibitory effect of some other aminoglycoside antibiotics was compared to that of neomycin and the data demonstrate that the inhibition of platelet secretion and phosphatidic acid production was dependent on the cationic charge of the antibiotic. It is suggested that neomycin inhibits signal transduction in platelets at a level prior to the inositol-phospholipid-specific phosphodiesterase.
Synergism between thrombin and adrenaline (epinephrine) in human platelets. Marked potentiation of inositol phospholipid metabolism.
We have studied synergism between adrenaline (epinephrine) and low concentrations of thrombin in gel-filtered human platelets prelabelled with [32P]Pi. Suspensions of platelets, which did not contain added fibrinogen, were incubated at 37 degrees C to measure changes in the levels of 32P-labelled phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and phosphatidate (PA), aggregation and dense-granule secretion after stimulation. Adrenaline alone (3.5-4.0 microM) did not cause a change in any parameter (phosphoinositide metabolism, aggregation and dense-granule secretion), but markedly enhanced the thrombin-induced responses over a narrow range of thrombin concentrations (0.03-0.08 units/ml). The thrombin-induced hydrolysis of inositol phospholipids by phospholipase C, which was measured as the formation of [32P]PA, was potentiated by adrenaline, as was the increase in the levels of [32P]PIP2 and [32P]PIP. The presence of adrenaline caused a shift to the left for the thrombin-induced changes in the phosphoinositide metabolism, without affecting the maximal levels of 32P-labelled compounds obtained. A similar shift by adrenaline in the dose-response relationship was previously demonstrated for thrombin-induced aggregation and dense-granule secretion. Also, the narrow range of concentrations of thrombin over which adrenaline potentiates thrombin-induced platelet responses is the same for changes in phosphoinositide metabolism and physiological responses (aggregation and dense-granule secretion). Our observations clearly indicate that adrenaline directly or indirectly influences thrombin-induced changes in phosphoinositide metabolism.
Current aspects on human platelet activation and responses.
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Synergism between thrombin and epinephrine in human platelets: different dose-response relationships for aggregation and dense granule secretion.
Synergism between epinephrine and thrombin in human gel-filtered platelets was studied. Suspensions of platelets, which did not contain added fibrinogen, were incubated at 37 degrees C to measure aggregation and dense granule secretion after stimulation. Both aggregation and secretion induced with 0.03-0.3 U/ml of thrombin were markedly potentiated by epinephrine (0.5-4 microM), which alone was without effect. Addition of epinephrine before stimulation with thrombin resulted in a marked decrease in the concentration of thrombin required for half-maximal aggregation, without affecting the maximal aggregation response. The dose-response relationship for dense granule secretion was affected in a similar way with regard to the half-maximal response; in addition, epinephrine slightly increased maximal secretion response. However, the concentration range for thrombin at which synergism occurred was distinctly lower for aggregation than for secretion. Our observations suggest that aggregation and dense granule secretion are separate steps in the sequence of stimulus-response coupling, and suggest distinct thresholds of intracellular messengers for these different responses.
Inhibition of ADP-induced responses in human platelets by agents elevating the cyclic AMP level: comparison of aggregation and shape change.
The inhibitory effect of cAMP-elevating agents on shape change and aggregation in human platelets was studied to improve the understanding of the sequential relationship between these two responses. Human platelet-rich plasma was preincubated for 2 min at 37 degrees C with prostaglandin E1 or adenosine, agents known to elevate the intracellular level of cAMP. Their inhibitory effects on ADP-induced shape change and aggregation were determined both separately and simultaneously. The dose-inhibition patterns for shape change and aggregation were similar for both PGE1 and adenosine. There was no distinct difference between the inhibitory action of these two inhibitors. These observations suggest that elevation of the intracellular concentration of cAMP interferes with an early step in the stimulus-response coupling that is common for aggregation and shape change.