Planning Benchmark Study for Stereotactic Body Radiation Therapy of Pancreas Carcinomas with Simultaneously Integrated Boost and Protection: Results of the DEGRO/DGMP Working Group on Stereotactic Radiation Therapy and Radiosurgery.

PURPOSE/OBJECTIVE: The proximity or overlap of PTV and OAR poses a major challenge in SBRT of pancreatic cancer (PACA). This international treatment planning benchmark study investigates whether Simultaneously Integrated Boost (SIB) and Protection (SIP) concepts in PACA SBRT can lead to improved and harmonized plan quality. MATERIALS/METHODS: A multiparametric specification of desired target doses (GTVD50%, GTVD99%, PTVD95%, PTV0.5cc) with two prescription doses of GTVD50%=5×9.2Gy (46Gy) and GTVD50%=8×8.25Gy (66Gy) and OAR limits were distributed with planning CT and contours from 3 PACA patients. In phase 1, plans were ranked using a scoring system for comparison of trade-offs between GTV/PTV and OAR. In phase 2, re-planning was performed for the most challenging case and prescription with dedicated SIB and SIP contours provided for optimization after group discussion. RESULTS: For all 3 cases and both phases combined, 292 plans were generated from 42 institutions in 5 countries using commonly available treatment planning systems. The GTVD50% prescription was performed by only 76% and 74% of planners within 2% for 5 and 8 fractions, respectively. The GTVD99% goal was mostly reached, while the balance between OAR and target dose showed initial SIB/SIP-like optimization strategies in about 50% of plans. For plan ranking, 149 and 217 score penalties were given for 5 and 8 fractions, pointing to improvement possibilities. For phase 2, the GTVD50% prescription was performed by 95% of planners within 2% and GTVD99% as well as OAR doses were better harmonized with notable less score penalties. Fourteen of 19 planners improved their plan rank, 9 of them by at least 2 ranks. CONCLUSION: Dedicated SIB/SIP concepts in combination with multiparametric prescriptions and constraints can lead to overall harmonized and high treatment plan quality for PACA SBRT. Standardized SIB/SIP treatment planning in multicenter clinical trials appears feasible after group consensus and training.

Multi-center planning study of radiosurgery for intracranial metastases through Automation (MC-PRIMA) by crowdsourcing prior web-based plan challenge study.

BACKGROUND: Planning radiosurgery to multiple intracranial metastases is complex and shows large variability in dosimetric quality among planners and treatment planning systems (TPS). This project aimed to determine whether autoplanning using the Muliple Brain Mets (AutoMBM) software can improve plan quality and reduce inter-planner variability by crowdsourcing results from prior international planning study. METHODS: Twenty-four institutions autoplanned with AutoMBM on a five metastases case from a prior international planning competition from which population statistics (means and variances) of 23 dosimetric metrics and resulting composite plan score (maximum score = 150) of other TPS (Eclipse, Monaco, RayStation, iPlan, GammaPlan, MultiPlan) were crowdsourced. Plan results of AutoMBM and each of the other TPS were compared using two sample t-tests for means and Levene's tests for variances. Plan quality of AutoMBM was correlated with the planner' experience and compared between academic and non-academic centers. RESULTS: AutoMBM produced plans with comparable composite plan score to GammaPlan, MultiPlan, Eclipse and iPlan (127.6 vs. 131.7 vs. 127.3 vs. 127.3 and 126.7; all p > 0.05) and superior to Monaco and RayStation (118.3 and 108.6; both p < 0.05). Inter-planner variability of overall plan quality was lowest for AutoMBM among all TPS (all p < 0.05). AutoMBM's plan quality did not differ between academic and non-academic centers and uncorrelated with planning experience (all p > 0.05). CONCLUSIONS: By plan crowdsourcing prior international plan challenge, AutoMBM produces high and consistent plan quality independent of the planning experience and the institution that is crucial to addressing the technical bottleneck of SRS to intracranial metastases.

[Hemolytic kidney failure and transient ischemic attack in a 32-year-old female]. / Hämolytisches Nierenversagen mit transitorisch ischämischer Attacke bei einer 32-Jährigen.

We report on the case of a 32-year-old female patient who initially presented with oliguric acute renal failure, hemolytic anemia with moderate thrombocytopenia and subsequently developed a transient ischemic attack in the cerebellum. The kidney biopsy revealed clinically suspected atypical hemolytic-uremic syndrome (aHUS), which was confirmed by intraglomerular thrombotic microangiopathy (TMA). Treatment with plasmapheresis and sustained administration of the C5 inhibitor eculizumab resulted in hematological remission but without improvement of kidney function. Further etiological investigations led to reduced plasma levels of inhibitory complement factor I on the basis of a heterozygous CFI mutation. In patients with aHUS molecular genetic investigations are indicated in order to determine the underlying cause, to regulate the therapeutic regimen and to allow prognostic statements with respect to a potential kidney transplantation.

A Multiple Regression Analysis of Postoperative Complications After Body-Contouring Surgery: a Retrospective Analysis of 205 Patients : Regression Analysis of Complications.

BACKGROUND: As bariatric surgery becomes ever more popular, so does body-contouring surgery to eliminate excess skin after radical weight loss. To date, the literature has described a number of risk factors affecting the postoperative outcome. Our study aimed to define those factors more closely, focusing on abdominoplasty ("tummy tuck") patients who suffered intra- and postoperative complications. METHODS: The study collective included 205 patients over 5 years (2001-2006) who underwent dermolipectomy at our department. The mean follow-up was 5.94 years. Every abdominoplasty was performed under general anesthesia with intraoperative one-dose antibiotic. The analysis included a complete review of all medical records. Statistical analysis was performed with the R-2.5.0 Software for Windows. RESULTS: The overall rate for major complications that required operative revision and/or antibiotics was 10.2 %, including 2.9 % cases of infections. Forty-one percent had minor complications, such as seromas, hematomas, wound healing problems, and wound dehiscences. The logistic regression models demonstrated that smoking combined with the age, a BMI higher than 30 kg/m(2), and the amount of removed tissue (measured in g) lead to significantly more wound healing problems in nearly all age groups. The probability of infections correlated with later drain removal. CONCLUSIONS: Regardless of the amount of tissue removed, no main risk factor for complications could be identified. A complication-free course and good outcome can be best achieved with careful patient selection and preoperative planning.

Conformational flexibility of the agonist binding jaw of the human P2X3 receptor is a prerequisite for channel opening.

BACKGROUND AND PURPOSE: It is assumed that ATP induces closure of the binding jaw of ligand-gated P2X receptors, which eventually results in the opening of the membrane channel and the flux of cations. Immobilization by cysteine mutagenesis of the binding jaw inhibited ATP-induced current responses, but did not allow discrimination between disturbances of binding, gating, subunit assembly or trafficking to the plasma membrane. EXPERIMENTAL APPROACH: A molecular model of the pain-relevant human (h)P2X3 receptor was used to identify amino acid pairs, which were located at the lips of the binding jaw and did not participate in agonist binding but strongly approached each other even in the absence of ATP. KEY RESULTS: A series of cysteine double mutant hP2X3 receptors, expressed in HEK293 cells or Xenopus laevis oocytes, exhibited depressed current responses to α,ß-methylene ATP (α,ß-meATP) due to the formation of spontaneous inter-subunit disulfide bonds. Reducing these bonds with dithiothreitol reversed the blockade of the α,ß-meATP transmembrane current. Amino-reactive fluorescence labelling of the His-tagged hP2X3 receptor and its mutants expressed in HEK293 or X. laevis oocytes demonstrated the formation of inter-subunit cross links in cysteine double mutants and, in addition, confirmed their correct trimeric assembly and cell surface expression. CONCLUSIONS AND IMPLICATIONS: In conclusion, spontaneous tightening of the binding jaw of the hP2X3 receptor by inter-subunit cross-linking of cysteine residues substituted at positions not directly involved in agonist binding inhibited agonist-evoked currents without interfering with binding, subunit assembly or trafficking.

Increase of intracellular Ca2+ by P2X and P2Y receptor-subtypes in cultured cortical astroglia of the rat.

Astrocytes express purinergic receptors that are involved in glial-neuronal cell communication. Experiments were conducted to characterize the expression of functional P2X/P2Y nucleotide receptors in glial cells of mixed cortical cell cultures of the rat. The vast majority of these cells was immunopositive for glial fibrillary acidic protein (GFAP) and was considered therefore astrocyte-like; for the sake of simplicity they were termed "astroglia" throughout. Astroglia expressed predominantly P2X(4,6,7) as well as P2Y(1,2) receptor-subtypes. Less intensive immunostaining was also found for P2X(5) and P2Y(4,6,13,14) receptors. Pressure application of ATP and a range of agonists selective for certain P2X or P2Y receptor-subtypes caused a concentration-dependent increase of intracellular Ca(2+) ([Ca(2+)](i)). Of the agonists tested, only the P2X(1,3) receptor-selective alpha,beta-methylene ATP was ineffective. Experiments with Ca(2+)-free solution and cyclopiazonic acid, an inhibitor of the endoplasmic Ca(2+)-ATPase, indicated that the [Ca(2+)](i) response to most nucleotides, except for ATP and 2',3'-O-(benzoyl-4-benzoyl)-ATP, was due primarily to the release of Ca(2+) from intracellular stores. A Gprotein-mediated release of Ca(2+) is the typical signaling mechanism of various P2Y receptor-subtypes, whose presence was confirmed also by cross-desensitization experiments and by using selective antagonists. Thus, our results provide direct evidence that astroglia in mixed cortical cell cultures express functional P2Y (P2Y(1,2,6,14) and probably also P2Y(4)) receptors. Several unidentified P2X receptors, including P2X(7), may also be present, although they appear to only moderately participate in the regulation of [Ca(2+)](i). The rise of [Ca(2+)](i) is due in this case to the transmembrane flux of Ca(2+) via the P2X receptor-channel. In conclusion, P2Y rather than P2X receptor-subtypes are involved in modulating [Ca(2+)](i) of cultured astroglia and thereby may play an important role in cell-to-cell signaling.

Prolactin acts as a potent survival factor against C2-ceramide-induced apoptosis in human granulosa cells.

BACKGROUND: The role of prolactin in the regulation of ovarian folliculogenesis and corpus luteal function and in particular its relationship to atresia in these structures is as yet unclear. We established a model of apoptosis in which to examine the actions of prolactin. METHOD: Granulosa cells collected from IVF-flush were cultured at 0.1-0.3 x 10(6) cells/well in growth media for 48 h, placed into serum-free media for 24 h prior to dosing for 24 h. Dose responses to C2-ceramide and prolactin were performed. Cells were then treated with an apoptotic dose of C2-ceramide alone, prolactin (100 ng/ml) alone or a combination of the two. Cell death was assessed by Trypan Blue cell counting and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl Blue] assay and apoptosis confirmed by morphological assessment and flow cytometry. RESULTS: C2-ceramide (0-40 micro mol/l) induced a dose-dependent increase in cell death (63.8% increase at 40 micro mol/l) and, morphologically, cells exhibited classical features of apoptosis. Prolactin alone had no effect on metabolic activity or total cell number. On co- incubation, prolactin alone had no effect on cell death, whereas C2-ceramide induced an approximately 62.6% increase in apoptosis, which was inhibited in the presence of prolactin. CONCLUSIONS: Prolactin may contribute significantly to early corpus luteum formation and survival by acting as a potent antiapoptotic factor for human granulosa cells.

The effect of oligonucleotides to c-myb on vascular smooth muscle cell proliferation and neointima formation after porcine coronary angioplasty.

Proto-oncogenes, including c-myb, are expressed early after vascular injury. The application of antisense oligodeoxynucleotides (AS-ODNs) against these genes inhibits cell proliferation and neointima formation in small animals and in peripheral arteries. The aim of this study was to investigate the specificity of action of AS-ODN-c-myb in vitro and to assess its effect, when delivered locally, on neointima formation after percutaneous transluminal coronary angioplasty (PTCA) in porcine coronary arteries. AS-ODN-c-myb inhibited the proliferation of vascular smooth muscle cells (VSMCs) in vitro in a dose-dependent manner. There was a corresponding reduction in steady state levels of c-myb mRNA and protein. Expression of another early gene, c-fos, was unaffected. S1 nuclease analysis demonstrated intact full-length AS-ODN-c-myb retrieved from VSMCs in culture after 12 hours. A range of ODNs, related and unrelated to c-myb, with and without a GGGG sequence, inhibited VSMC proliferation. Phosphorothioated AS-ODN-c-myb was 30 times less potent than unphosphorothioated AS-ODN-c-myb. PTCA induced porcine coronary artery neointima formation. c-myb mRNA was maximally induced 18 hours after injury. Unmodified AS-ODN-c-myb, sense-ODN-c-myb, saline, or nothing was delivered immediately after balloon dilatation via a double-skinned porous balloon (Transport, SciMed). Fluorescence-labeled AS-ODN-c-myb was deposited throughout the vessel wall. Mean maximum intima/media cross-sectional area 4 weeks after PTCA was reduced with AS-ODN-c-myb by 79% compared with saline (P < .05), 82% compared with sense-ODN-c-myb, and 63% compared with nothing (P < .10). Conclusions are as follows: (1) c-myb is expressed in VSMCs after vascular injury. (2) AS-ODN-c-myb is retained intact in VSMCs, reducing their proliferation in vitro in dose-dependent fashion, with reduction in c-myb mRNA and protein, whereas sense-ODN-c-myb is not. (3) A range of ODNs can reduce VSMC proliferation by a non-sequence-specific mechanism. (4) Phosphorothioate protection of antisense molecules may reduce their efficacy. (5) Local delivery of unmodified AS-ODN-c-myb via the Transport catheter reduces neointima formation after porcine PTCA. (6) Local delivery of fluid may exacerbate neointimal thickening.

Lack of differences between the neuronal and extraneuronal handling of 3H-7- and 3H-ring-2,5,6-(-)noradrenaline.

The neuronal disposition of the side chain-labelled 3H-7-(-)noradrenaline and the ring-labelled 3H-2,5,6-(-)noradrenaline of relatively high specific activity was compared in the (incubated) rabbit aorta, the extraneuronal one in the (perfused) rat heart. Furthermore, the metabolism by monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT) of the labelled amines was studied in rat heart homogenates. In addition, some of the experiments were also carried out with the side chain-labelled 3H-7,8-(-)noradrenaline that is an inferior substrate of MAO. 1. In agreement with several other studies, the present results revealed the known differences between the two side chain-labelled amines brought about by the poorer deamination of 3H-7,8-(-)noradrenaline. This finding also indicated that the 3H-7-(-)noradrenaline used in this study was of the desired quality, i.e. was labelled exclusively in position 7. 2. In contrast to earlier findings, no differences between the side chain-labelled 3H-7-(-)noradrenaline and the ring-labelled 3H-2,5,6-(-)noradrenaline were observed with regard to neuronal and extraneuronal amine handling (intact tissues) and metabolism by MAO and COMT in rat heart homogenates. Hence, there is no reason to avoid the ring-labelled amine with high specific activity and, consequently, with slightly more than one tritium substituent per catecholamine molecule in experiments designed to analyse the neuronal and extraneuronal fate of (-)noradrenaline.

The uptake and O-methylation of 3H-(+/-)-isoprenaline in rat cerebral cortex slices.

The O-methylation and accumulation of 3H-isoprenaline in slices of the rat cerebral cortex were studied before and after inhibition of COMT. 1. Inhibition of COMT by 30 mumol/l U-0521 virtually abolished the O-methylation and increased the accumulation of 3H-isoprenaline; hence, there is evidence for the existence of a central O-methylating system (with a transport mechanism and intracellular COMT). 2. Experiments were carried out with selective uptake inhibitors for uptake1 (cocaine and desipramine) or uptake2 (corticosterone and OMI), with phenoxybenzamine (known to inhibit both carriers) and with changes in the ionic composition of the incubation medium. They revealed that the central carrier differed from both, uptake1 and uptake2, although exhibiting some resemblance with uptake2 (lack of dependence on Na+ and Cl-, sensitivity to K+ and phenoxybenzamine, ability to transport 3H-isoprenaline). 3. Although the central carrier was rather sensitive to inhibition by beta-adrenoceptor antagonists (propranolol, carteolol), the effect of propranolol was not stereoselective; hence, beta-adrenoceptors do not seem to be involved. 4. Virtually identical IC30-values were obtained for inhibitors, when determined with or without inhibition of COMT. Only OMI was found to inhibit COMT as well as the central transport system; hence it was more potent in inhibiting the O-methylation than the accumulation of 3H-isoprenaline. 5. IC50-values (against initial rates of accumulation of 3H-isoprenaline; COMT inhibited) were determined for various substrates and inhibitors of peripheral uptake2. There was no correlation with the IC50-values determined earlier for uptake2 in rat heart (Grohmann and Trendelenburg 1984).(ABSTRACT TRUNCATED AT 250 WORDS)

The acceleration of the extraneuronal efflux of 3H-(+/-)-isoprenaline induced by high extracellular potassium.

After loading of the extraneuronal tissues of the perfused rat heart with 3H-isoprenaline the elevation of extracellular K+ concentration (from 2.7 to 15 mmol/l) in the perfusion solution about doubled the rate constant for efflux of the 3H-amine. As this increase was not seen in the presence of 100 mumol/l 3-O-methyl-isoprenaline (OMI, a potent inhibitor of the uptake2-carrier), it is concluded that the change in the concentration of K+ modulates OMI-sensitive outward transport of 3H-isoprenaline by uptake2, not the diffusional efflux of the amine.

The handling of five amines by the extraneuronal deaminating system of the rat heart.

The handling of five amines by the extraneuronal deaminating system was studied in perfused hearts of rats (pretreated with reserpine; COMT and neuronal uptake inhibited). Hearts were perfused with 50 nmol/l 3H-noradrenaline for 30 min, in the presence of increasing concentrations of unlabelled (-)-adrenaline, (-)-noradrenaline, dopamine, tyramine and 5-HT. IC50's were determined as those concentrations of unlabelled amines which halved the steady-state rate of deamination of 3H-noradrenaline. After correction for changes in the tissue/medium ratio for 3H-noradrenaline, "half-saturating outside concentrations" were obtained. They increased in the order (-)-adrenaline (15 mumol/l) - tyramine - dopamine - noradrenaline - 5-HT (53 mumol/l). The Vmax for extraneuronal deamination was determined for 3H-(-)-adrenaline, 3H-(-)-noradrenaline and 3H-dopamine, as well as (by HPLC and electrochemical detection) for tyramine and 5-HT. It was low for (-)-adrenaline, intermediate for (-)-noradrenaline, dopamine and 5-HT, high for tyramine. For the three catecholamines the half-saturating outside concentrations of the extraneuronal deaminating system clearly exceeded those for the extraneuronal O-methylating system of the same organ (see Grohmann and Trendelenburg 1985), although the two enzymes appear to co-exist in the same cells, so that the same transport system is involved.

The activity of the neuronal and extraneuronal catecholamine-metabolizing enzymes of the perfused rat heart.

In a comparative study the neuronal and extraneuronal metabolism of several 3H-catecholamines (all of which were tritiated in the C-7 position of the side chain only) was determined in isolated rat hearts perfused at a concentration of the 3H-amines of 50 nmol/l. While the neuronal MAO activity was determined after inhibition of extraneuronal uptake (100 mumol/l OMI) and COMT (10 mumol/l U-0521), the extraneuronal MAO activity was estimated after inhibition of neuronal uptake (30 mumol/l cocaine) and COMT. The extraneuronal COMT activity was determined under conditions of inhibition of both neuronal uptake and MAO (pretreatment with pargyline). Hearts were perfused with the 3H-catecholamines until the rate of appearance of the various 3H-metabolites in the venous effluent has reached a steady state. From these rates (vst-st) and the steady-state content of the unchanged 3H-catecholamines in the tissue (Si), the rate constants (Vmax/Km) for the unsaturated intracellular enzymes COMT (kCOMT) and MAO (kMAO) were calculated. The kCOMT values for all four catecholamines, (-)-noradrenaline, dopamine, (-)-adrenaline and (+/-)-isoprenaline exhibit a range from 0.24 to 0.78 min-1; the metabolism of the catecholamines by the COMT differs: (-)-noradrenaline = dopamine less than (-)-adrenaline less than (+/-)-isoprenaline. The extraneuronal MAO activity was low for all three catecholamines, (-)-adrenaline, (-)-noradrenaline and dopamine (range of kMAO from 0.05 to 0.28 min-1) and declined in the order.(ABSTRACT TRUNCATED AT 250 WORDS)

The functional coupling of neuronal and extraneuronal transport with intracellular monoamine oxidase.

"Metabolizing systems" are responsible for the quick inactivation of noradrenaline released from adrenergic nerve endings: a transport mechanism (uptake1 or uptake2) is arranged in series with the intracellular enzyme (monoamine oxidase, MAO; catechol-O-methyltransferase, COMT). In the perfused rat heart, kenzyme-values were determined, i.e., those rate constants which characterize the unsaturated intracellular enzymes. In the extraneuronal metabolizing system kcomt greater than kmao for noradrenaline and adrenaline, while rather similar rate constants were obtained for dopamine. However, for the neuronal deaminating system, kmao is considerably higher than kmao for the extraneuronal system. Second, in the rat vas deferens it is demonstrated that inhibition of neuronal MAO leads to very pronounced rises of the axoplasmic noradrenaline concentration--and this is again a reflection of the high activity of neuronal MAO. In a third series of experiments (with the rat vas deferens), the evidence indicates that the neuronal inward transport of substrates of MAO fails to saturate the enzyme. This is the functional consequence of the high activity of neuronal MAO. It is concluded that a) neuronal MAO activity is very high, and--as a consequence--b) axoplasmic noradrenaline levels are very low.

Inhibition by K+ of uptake2 of 3H-(+/-)-isoprenaline in the perfused rat heart.

The kinetics of the inhibitory effect of extracellular K+ on uptake2 of 3H-(+/-)-isoprenaline were determined in isolated hearts obtained from reserpine-pretreated rats; catechol-O-methyl transferase was inhibited. Initial rates of uptake2 of a very low concentration of 3H-(+/-)-isoprenaline (10 nmol/l) were determined in the presence of various extracellular concentrations of K+ (2.7 to 60 mmol/l). The inhibitory effect of K+ was concentration-dependent with an IC50 of about 20 mmol/l. - In these experiments KCl was added to the perfusion solution, and some hypertonicity resulted. In some experiments NaCl was added to a solution containing 5 mmol/l K+ to result in the same degree of hypertonicity as that obtained for 60 mmol/l K+; hypertonicity increased the initial rate of uptake2 of 3H-(+/-)-isoprenaline. Thus, the inhibitory effect of K+ had been slightly underestimated. In subsequent experiments the increase of the concentration of K+ in the perfusion fluid to 30 mmol/l was compensated for by a corresponding reduction of Na+. Initial rates of uptake2 of 10 nmol/l 3H-(+/-)-isoprenaline were determined in the absence and presence of various concentrations of unlabelled (+/-)-isoprenaline. At 30 mmol/l K+ the IC50 (= Km for uptake2) did not significantly differ from that determined in an earlier study at 2.7 mmol/l K+ (Grohmann and Trendelenburg 1984). Finally, the Vmax for uptake2 of 3H-(+/-)-isoprenaline was determined at either 2.7 or 30 mmol/l K+. At 30 mmol/l K+ the Vmax was only about 1/4 of that observed at 2.7 mmol/l K+.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of partial inhibition of monoamine oxidase on the steady-state rate of deamination of 3H-catecholamines in two metabolizing systems.

Two different "deaminating systems" were compared (i.e., intact tissues in which an uptake process translocates the 3H-catecholamine from the extracellular space to the intracellular MAO): the adrenergic nerve endings of the rat vas deferens exposed to 10 nmol/l 3H-(-)-noradrenaline, and the extraneuronal deaminating system of the rat heart perfused with 50 nmol/l 3H-(-)-adrenaline. Vesicular uptake and COMT were inhibited. In both systems MAO was partially inhibited by pargyline, and the steady-state tissue content of the 3H-catecholamine was determined as well as the steady-state rate of deamination. Rat vas deferens (preincubated with 10-40 nmol/l pargyline for 30 min). Inhibition of neuronal MAO caused not more than a moderate decrease of the steady-state rate of deamination of 3H-(-)-noradrenaline, but the steady-state tissue content was greatly increased. Determinations of the activity of MAO in homogenates of vasa deferentia showed that preincubation with 10 and 20 nmol/l pargyline inhibited the enzyme by 80 to 95%. Rat heart (of animals pretreated with 1 to 30 mg/kg pargyline). Inhibition of extraneuronal MAO caused a steep decline of the steady-state rate of deamination of 3H-(-)-adrenaline, but only a small rise in the steady-state tissue content. The decisive difference between the two deaminating systems lies in the fact that the ratio "kmao/kout" (where the two k-values characterize the activity of the unsaturated intracellular MAO and the ability of the 3H-catecholamine to leave the relevant cells, respectively) is much higher for the neuronal deaminating system exposed to 3H-(-)-noradrenaline than for the extraneuronal deaminating system exposed to 3H-(-)-adrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)

Errors introduced by a tritium label in position 8 of catecholamines.

The neuronal and extraneuronal disposition of 3H-7,8- and 3H-7-labelled (-)-noradrenaline and dopamine was compared in in vitro studies. In agreement with earlier studies, the present results show that the presence of a tritium label in position 8 (i.e., on the alpha-carbon) has two consequences: a) the rate of deamination declines and b) part of the deamination results in the formation of an unlabelled aldehyde plus tritium water; tritium water is recovered from the OMDA-fraction of the column chromatographic procedure of Graefe et al. (1973). Whenever the deamination of a 3H-catecholamine is reduced (by tritium in position 8), the intraneuronal 3H-catecholamine concentration is increased. This increase, in turn, partly masks the decline in neuronal deamination (rat vas deferens). Irrespective of whether one determines the spontaneous efflux, the release of 3H-noradrenaline by nerve stimulation or the release of 3H-(-)-noradrenaline by the reserpine-like compound Ro 4-1284, the presence of tritium in position 8 distorts the results (experiments with rat vasa deferentia and/or rabbit aorta). In the extraneuronal system of the rat heart, two intracellular enzymes inactivate 3H-(-)-noradrenaline and 3H-dopamine: catechol-O-methyl transferase (COMT) and monoamine oxidase (MAO). Any hindrance of deamination (by tritium in position 8, COMT intact) leads to a shift of the metabolism of the 3H-catecholamines from the exclusively deaminated to the exclusively O-methylated metabolites. No differences between 3H-7,8- and 3H-7-labelled catecholamines were found after inhibition of MAO and COMT (extraneuronal accumulation and rate constant for efflux from the extraneuronal compartment III of the rat heart).(ABSTRACT TRUNCATED AT 250 WORDS)

The handling of five catecholamines by the extraneuronal O-methylating system of the rat heart.

In a comparative study, the handling of five catecholamines by the extraneuronal O-methylating system of the rat heart was determined; all rats were pretreated with reserpine, monoamine oxidase and neuronal uptake were inhibited in all experiments. Hearts were perfused for 7 min with a tracer concentration of 3H-(+/-)-isoprenaline, either in the absence or in the presence of unlabelled catecholamines (which reduced the O-methylation of the tracer amine). IC50's were determined for unlabelled catecholamines and then converted to "half-saturating outside concentrations", i.e., to those concentrations in the perfusion fluid that half-saturate the intracellular catechol-O-methyl transferase (COMT). The values for the (-)-isomers of dobutamine, isoprenaline, adrenaline and noradrenaline and that for dopamine were low and rather similar (between 0.67 and 2.7 mumol/l). Stereoselectivity for isoprenaline probably reflected the preference of uptake2 for the (-)-isomer. The effects of (-)- and (+)-dobutamine indicated that both isomers are a) transported by uptake2 and b) good substrates of COMT. The Vmax for O-methylation [determined for 3H-(+/-)-isoprenaline, 3H-(+/-)-adrenaline, 3H-(+/-)-noradrenaline and 3H-dopamine] was rather similar for all four catecholamines. It is concluded that the extraneuronal O-methylating system of the rat heart handles the five catecholamines in a similar manner, although the Km for uptake2 had been found to increase substantially in the order: dobutamine less than isoprenaline less than adrenaline less than noradrenaline less than dopamine (Grohmann and Trendelenburg 1984b).

The substrate specificity of uptake2 in the rat heart.

Experiments were carried out with hearts isolated from reserpine- and pargyline-pretreated rats; both noradrenaline-metabolizing enzymes and uptake1 were inhibited. Initial rates of extraneuronal uptake were measured after perfusion lasting for 2 min, either in the absence or in the presence of 100 mumol/l O-methyl-isoprenaline, a potent inhibitor of uptake2. The ID50 (i.e., the concentration of unlabelled substance that halves the rate of uptake of a tracer concentration of 3H-(+/-)-isoprenaline) was determined for a variety of agents. Two types of stereoselective preference of (-)-isomers were observed: for isoprenaline and adrenaline (but not for noradrenaline)--and also for dobutamine. The stereoselective preference for the (-)-isomers of isoprenaline and adrenaline is also evident from fluorimetric determination of initial rates of uptake of unlabelled isomers. Experiments with various tritiated compounds indicate that uptake2 has a broad substrate spectrum: uptake2 is not restricted to 3H-catecholamines and 3H-phenethylamines, but extends to resorcinols (3H-orciprenaline), imidazoline derivatives (3H-clonidine), 3H-histamine and 3H-5-hydroxytryptamine (3H-5-HT). Determinations of the Vmax of uptake2 revealed a correlation between the ID50 and the Vmax: the higher the ID50, the higher the Vmax. These results indicate that uptake2 is a carrier-mediated process.