Hemodynamic effects of eating and prolonged supine position in healthy subjects studied under clinical-pharmacological test conditions.

The influences of both being in a supine position for a prolonged period and food intake on cardiovascular variables were studied under clinical-pharmacological test conditions. In a randomized crossover design study without drug or placebo administration, 6 healthy male volunteers received a light standard meal before and during test A and fasted in test B. In both tests, while they were continuously supine for more than 8 h, a synchronous recording of cardiovascular variables was done at 24, 26 and 28 min after starting the supine position (first recordings) and 25 times from 2 to 480 min after the first recordings. Using a multifactorial statistical analysis, each parameter was evaluated regarding the factors eating and time of supine recording. Eating led to a significant decrease in diastolic and mean blood pressure, PQ time and QS2 time, a downward trend in systemic vascular resistance and an upward trend in systolic blood pressure and cardiac output. When the subjects remained in a supine position for prolonged periods, significant increases in systolic, diastolic, mean blood pressure and systemic vascular resistance were noted as well as significant decreases in cardiac output and QS2 time. Thus, eating and remaining in a supine position for prolonged periods should be considered as sources of bias in clinical-pharmacological studies on cardiovascular drug effects and accompanying placebo controls.

Effect of locally applied trapidil on norepinephrine- and dinoprost-evoked hand vein constriction in healthy men.

OBJECTIVE: In this study the effect of locally administered trapidil on human hand veins was examined. SUBJECTS: 10 healthy male volunteers aged 20 - 30 years were included. METHOD: The dorsal hand vein compliance technique was used. In a crossover design the influence of locally infused trapidil (mainly 5 - 400 microg/min) on hand veins preconstricted with either norepinephrine (adrenoceptor agonist) or dinoprost (prostaglandin F2alpha) was investigated. Preconstriction reduced the vein diameter by about 80% with continuous local infusion of individually determined doses of norepinephrine in the range 11 - 1,000 ng/min and dinoprost in the range 90 - 5,600 ng/min. Blood pressure, cardiac function (electrocardiogram) and skin temperature of the hand infused were monitored. RESULTS: Locally applied trapidil produced a dose-dependent dilation of hand veins preconstricted with norepinephrine and dinoprost. The corresponding ED50 values of trapidil did not differ significantly on an intraindividual comparison. Clinically important side effects with the drugs used were not observed. CONCLUSIONS: The results indicate that trapidil has a direct dilating action on superficial veins in humans. This effect is apparently achieved without involvement of adrenoceptors or prostanoid receptors in venous smooth muscle.

Rapid protein tyrosine phosphorylation in the cytoskeleton of stimulated human platelets.

Upon activation platelets show elevated protein tyrosine phosphorylation, and translocation of the protein tyrosine kinase pp60c-src from the plasma membrane to the cytoskeleton occurs. We therefore investigated whether tyrosine phosphorylation also increases in the cytoskeletal compartment. Here we show that almost identical patterns of phosphotyrosine-containing proteins are detectable in the cytoskeleton after platelet stimulation with compounds that directly (phorbol 12-myristate, 13-acetate) or indirectly (thrombin, vasopressin, collagen, ADP) activate protein kinase C. The apparent molecular masses of the proteins phosphorylated at tyrosine residues are 145, 130, 100, 85, 80, 60, 56, 54 and 38 kDa. Elevation of cyclic AMP by prostaglandin E1 had no effect. Concentrations of thrombin as low as 0.01 units per ml are able to cause tyrosine phosphorylation of multiple proteins. The time course of protein tyrosine phosphorylation for thrombin- and vasopressin-stimulated platelets revealed a rapid increase in the cytoskeleton within 5 to 20 s following activation consistent with a role in early events of platelet function.

High-yield purification of a pp60c-src related protein-tyrosine kinase from human platelets.

A protein-tyrosine kinase (PTK, EC 2.7.1.112) from human platelets was purified with high yield. Purification of the enzyme involved sequential chromatography on casein-agarose, tyrosine-agarose, heparin-Sepharose and hydroxylapatite. The procedure resulted in substantially enriched 54/52 kDa polypeptides on SDS-polyacrylamide gel electrophoresis and a yield of about 25% in PTK activity. About 250 micrograms of purified protein could be obtained from 1 g of cell protein. The purification factor varied between 1000 and 1500. Determination of the molecular mass of the purified PTK under nondenaturating conditions by molecular sieve chromatography revealed that the enzyme is a monomer of about 50 kDa. Among various protein substrates tested, casein was most prominently phosphorylated. All substrates were exclusively phosphorylated at tyrosine residues. Autophosphorylation at tyrosine residues of the 54/52 kDa proteins was observed in the presence of Mg2+ or Mn2+. At each purification step, the 54/52 kDa proteins were precipitated by sera from tumor-bearing rabbits immunoprecipitating pp60src, but not by control sera. The amount of the immunoprecipitated purified 54/52 kDa phosphoproteins was directly proportional to the amount of antiserum used. Partial peptide mapping by V8 proteinase showed a 26 kDa tyrosine-phosphorylated fragment for the 54 and the 52 kDa proteins as well as for the pp60c-src molecules of intact platelets. All these data indicated that purified PTK is closely related to pp60c-src of human platelets. Using casein as a substrate for the purified enzyme, the Km for ATP was 4 microM and the Vmax for the reaction was 2.0 nmol/min per mg.

Botulinum C2 toxin ADP-ribosylates actin and disorganizes the microfilament network in intact cells.

Botulinum C2 toxin ADP-ribosylates actin in [32P]orthophosphate-labelled intact chick embryo cells (CEC). The toxin-induced rounding up of CEC is correlated with ADP-ribosylation of actin in intact cells in a time and concentration-dependent manner. Both, rounding up of cells and actin ADP-ribosylation, depend on the presence of both components of botulinum C2 toxin (components I and II) and are independent of the ability of CEC to divide. Treatment of CEC with botulinum C2 toxin induced a time-dependent disorganization of the typical architecture of the microfilament network as shown by fluorescein-phalloidin staining. Botulinum C2 toxin decreased the amount of Triton X-100 insoluble actin, while the fraction of Triton soluble actin was increased. Actin, which was 32P-labelled by botulinum C2 toxin in intact CEC, was recovered in the Triton soluble but not in the Triton insoluble actin fraction. It is suggested that in intact CEC botulinum C2 toxin causes ADP-ribosylation of G-actin but not of F-actin thereby leading to an accumulation in the pool of monomeric actin.

Zn2+ enhances protein tyrosine kinase activity of human platelet membranes.

In human platelet membranes enhanced tyrosine phosphorylation of certain proteins was observed when Zn2+ instead of Mg2+ or Mn2+ was used as a divalent cation for the kinase reaction. An enhanced level of phosphate incorporation into tyrosine residues occurred into a 68 kDa polypeptide besides the 45 kDa and 105 kDa proteins. Preincubation of platelet membranes with TBR-IgG showed a concentration-dependent inhibition of the phosphorylation of the 45, 68 and 105 kDa proteins. Moreover, pp60c-src, representing the major protein tyrosine kinase activity in platelets, was found to be stimulated by Zn2+. The data, thus, support the assumption that pp60c-src kinase is responsible for Zn2+ stimulated tyrosine phosphorylation.

Pyruvate kinase type M2 is phosphorylated at tyrosine residues in cells transformed by Rous sarcoma virus.

Chicken embryo cells (CECs) contain pyruvate kinase (PK) type M2 (M2-PK). Transformation of CECs by Rous sarcoma virus (RSV) leads to a reduction in the affinity of PK for the substrate phosphoenolpyruvate. In vitro, M2-PK can be phosphorylated at tyrosine residues by pp60v-src, the transforming protein of RSV. To study tyrosine phosphorylation of M2-PK in intact RSV-transformed cells, the protein was immunoprecipitated from 32P-labeled normal and RSV-SR-A-transformed CECs. Phosphoamino acid analysis of immunoprecipitated M2-PK revealed that M2-PK of both normal and transformed CECs contained phosphoserine and small amounts of phosphothreonine. Only M2-PK of transformed CECs contained phosphotyrosine in addition. For enzyme kinetic studies M2-PK was partially purified by chromatography upon DEAE-Sephacel and hydroxyapatite. A decreased affinity for phosphoenolpyruvate was observed 3 h after the onset of transformation using the temperature-sensitive mutant of RSV, ts-NY 68. The kinetic changes were correlated with tyrosine phosphorylation of M2-PK, but there is no direct evidence that they are caused by post-translational modification of the enzyme.

Platelet membrane glycoproteins IIb and IIIa are substrates of purified pp60c-src protein tyrosine kinase.

Human platelet glycoproteins IIb and IIIa form the receptor for fibrinogen, von Willebrand factor and fibronectin. Isolated human glycoproteins IIb-IIIa are phosphorylated by purified pp60c-src protein tyrosine kinase. Analysis of the phosphorylated proteins on SDS-PAGE showed that under reducing conditions both phosphoproteins change their relative molecular masses from 135 to 120 kDa and from 97 to 105 kDa, which are characteristic properties of glycoproteins IIb-IIIa. Phosphorylated proteins could be immunoprecipitated with an antiserum against glycoproteins IIb-IIIa but not by control serum. Some kinetic properties of the glycoprotein phosphorylations are also investigated. How the glycoprotein IIb-IIIa complex acquires its receptor activity in stimulated platelets is unknown; however, phosphorylation could be an important mechanism.

Heparin-associated thrombocytopenia: the effects of various intravenous IgG preparations on antibody mediated platelet activation--a possible new indication for high dose i.v. IgG.

The immunologic type of heparin-associated thrombocytopenia (HAT) is caused by antibodies which activate platelets via the Fc-receptor in the presence of polysulfated oligosaccharides. The antigen is formed by a releasable platelet protein (in many cases PF4) complexed to heparin. Since the role of GP IIb/IIIa in platelet activation by HAT antibodies is controversial, we investigated platelet activation by antibodies related to HAT. We used normal platelets and platelets from a patient with Glanzmann's thrombasthenia (GT) lacking GP IIb/IIIa. Heparin and sera from patients with HAT stimulated GT platelets in the same manner as determined by 14C-serotonin release and the changes in phosphorylation of p20 and p47. Platelet activation could be inhibited by an anti FcRII monoclonal antibody (IV. 3, Fab-fragments), and by Fc-fragments, but not by F(ab')2-fragments of human IgG. The effect of four different, commercially available preparations of intact i.v. IgG on the platelet activation by six HAT sera was investigated by 14C-serotonin release. The inhibitory effect was strongly dependent upon the manufacturing process. At a concentration of 20 mg/ml only IgG that had been subjected to low pH and traces of pepsin sufficiently inhibited platelet activation. IgG treated with polyethylenglycol or sulfitolysis was less effective, whereas beta-propiolactone-treated IgG almost completely lost the ability to inhibit platelet activation by antibodies related to HAT. We conclude that inhibition of GP IIb/IIIa-fibrinogen interaction is insufficient for preventing platelet activation in HAT.(ABSTRACT TRUNCATED AT 250 WORDS)

Secreted ADP plays a central role in thrombin-induced phospholipase D activation in human platelets.

Thrombin and other agonists that induce secretion and aggregation in human platelets also activate phospholipase D (PLD), but the signaling cascade leading to activation of PLD in human platelets is not yet clear. We have determined that apyrase, which scavenges ADP secreted during platelet activation, is able to block or reduce the PLD activation stimulated by low (0.1 U/ml or less) or high (0.3- 1.0 U/ml) concentrations of thrombin, respectively. Neither ADP (up to 100 microM) nor its more potent analogue 2-methylthio-ADP (up to 100 microM), however, are able to stimulate PLD alone, and even the addition of fibrinogen, which results in platelet aggregation, is not sufficient for PLD activation. In contrast, ADP is able to stimulate PLD in the presence of low concentrations of thrombin that alone have little or no effect, suggesting ADP may play an amplifying role in platelet PLD activation. This hypothesis is supported by the finding that the purinergic receptor antagonist ARL 66096, an ATP analogue, reduces in a concentration-dependent fashion the PLD response to thrombin (IC50=28 nM with 0.1 U/ml thrombin). ARL 66096 also abolishes the PLD activation by ADP observed in the presence of low concentrations of thrombin, confirming that the antagonist inhibits an ADP-dependent component of the response. In addition, the thromboxane A2 receptor agonist U46619 activates PLD, and this response is markedly reduced by ARL 66096. Concomitantly, phosphorylation of the protein kinase C substrate pleckstrin in response to thrombin or U46619 is partially or totally inhibited by ARL 66096, respectively, consistent with ADP stimulation of protein kinase C being involved in the PLD response to these agonists. Based on these findings, we conclude that ADP secretion and activation of purinergic ADP receptors is an important amplification mechanism in the signal transduction pathways leading to PLD activation in human platelets.

Amiloride inhibits the protein tyrosine kinases associated with the cellular and the transforming src-gene products.

Amiloride inhibits a protein tyrosine kinase from rat brain extracts. The kinase activity is characterized by an anti-serum (TBR-serum) which immunoprecipitates pp60c-src, the cellular counterpart of the transforming protein pp60v-src of Rous sarcoma virus. In immunocomplexes, TBR-IgG serves as an artificial but specific phosphate acceptor. The phosphate incorporation into TBR-IgG is a time- and temperature-dependent process. In the presence of amiloride the TBR-IgG phosphorylation is reduced. The drug does not influence the immunocomplexes formed by TBR-IgG and pp60src and no amiloride-activated protein tyrosine phosphatase can be detected in the immunocomplex system. Half-maximal inhibition of the tyrosine kinase occurs at 300 microM amiloride and is competitive with respect to ATP. Viral pp60src kinase of transformed cells is more sensitive to amiloride (IC50: 50-100 microM). Furthermore, normal cellular tyrosine kinases are to a lesser extent inhibited by amiloride as compared to the transforming viral pp60src kinase. These results may indicate different amiloride-sensitive forms of cellular pp60src kinases.

Cation sensitivity of [125I]heat binding to alpha 1-adrenoceptors in rat cerebral cortex membranes.

The alpha 1-adrenoceptor antagonist 2-[beta-(4-hydroxyphenyl)-ethylaminomethyl] tetralone (HEAT) was radiolabeled with 125I and purified to the maximal specific radioactivity of 2200 Ci/mmol. [125 I]HEAT selectively labels alpha 1-adrenoceptors (KD approximately 0.1 nM at 30 degrees C) in rat cerebral cortex membranes. Divalent cations stimulated [125 I]HEAT binding to the alpha 1-adrenoceptors. The rank order of potencies was Ni2+ greater than Mn2+ greater than Mg2+ greater than Ca2+ approximately Sr2+. The effect of Mg2+ was mainly on the Bmax of the alpha I-adrenoceptors. Monovalent cations also stimulated [125I]HEAT binding. Maximal stimulation (up to 3-fold) was seen with Na+ (optimal concentration at approximately 150 mM). Li+ and NH4+ were less effective whereas K+ was ineffective. The stimulation of [125I]HEAT binding to alpha I-adrenoceptors by Na+ was temperature-dependent.

Alpha adrenoceptors in rat brain: direct identification with prazosin.

Tritiated prazosin was used to characterize high affinity binding sites with characteristics similar to alpha 1 adrenoceptors in rat brain membranes. These sites were compared with alpha 2 adrenoceptors labeled with tritiated clonidine. The prazosin sites had an association constant of 2 nM-1 and bound to ligand optimal around pH 7.0. The density of the sites was 300 fmoles per mg of protein; the half time of dissociation of prazosin was 7 min at 30 degrees C. The order or potencies of agonists, determined from binding-inhibition experiments with labeled prazosin, was: naphazoline greater than clonidine greater than adrenaline greater than noradrenaline greater than phenylephrine greater than alpha-methylnoradrenaline greater than dopamine. The order of potencies of antagonists was: prazosin greater than phenoxybenzamine greater than phentolamine greater than clozapine greater than yohimbine. Sodium ions and divalent cations as well as guanyl nucleotides have little or no effect on the binding of the labeled antagonist. This is in contrast to the binding of the labeled agonist clonidine (Glossmann and Presek, 1979a, 1979b). Labeled prazosin may be a useful tool to characterize alpha 1 adrenoceptors.

Quercetin inhibits tyrosine phosphorylation by the cyclic nucleotide-independent, transforming protein kinase, pp60src.

The bioflavonoid quercetin is a potent inhibitor of a cyclic nucleotide-independent, tumor virus-coded protein kinase which phosphorylates tyrosine residues and acts as a cellular transforming protein. Half-maximal inhibition of the protein kinase occurred at 3-4 microM quercetin whereas rutin was much less effective. The finding, that quercetin inhibits a cyclic nucleotide-independent protein kinase activity, may provide clues to the diverse pharmacological effects of the bioflavonoids.

Botulinum C2 toxin treatment increases the G-actin pool in intact chicken cells: a model for the cytopathic action of actin-ADP-ribosylating toxins.

Botulinum C2 toxin ADP-ribosylates actin in intact chicken embryo cells in a concentration-dependent manner. This effect correlates with an enhancement in the inhibitory potency of the respective cell lysates on DNAse I activity, indicating an increase in the cellular G-actin content of toxin-treated cells. The data support our view, that ADP-ribosylation of cellular actin with subsequent depolymerization of cytoskeleton-associated F-actin to monomeric G-actin is involved in the cytotoxic effects of botulinum C2 toxin. A model of the cytopathic action of actin-ADP-ribosylating toxins is presented.

Medication adherence, self-care behaviour and knowledge on heart failure in urban South Africa: the Heart of Soweto study.

BACKGROUND: There is a paucity of data on treatment adherence in patients with chronic heart failure (CHF) in Africa. METHODS: We examined the pattern of treatment adherence, self-care behaviour and treatment knowledge in 200 consecutive patients with CHF attending the Chris Hani Baragwanath Hospital, Soweto, South Africa via a combination of questionnaire (100%, n = 200) and pill count (41%, n = 82). RESULTS: Mean age was 56 +/- 14 years, 157 were black African (79%) and 109 (55%) were male. CHF-specific treatment included loop diuretics (93%), beta-blockers (84%), ACE inhibitors (74%), spironolactone (64%) and cardiac glycosides (24%); mean number of medications was 6 +/- 2. Overall, 71% (58 of 82) adhered to their prescribed CHF regimen and individual medication adherence ranged from 64 to 79%. Behavioural adherence varied from 2.5 to 98%. Patient treatment knowledge was poor; 56% could not name medication effects or side effects. However, an average knowledge score of 69% was achieved on 10 questions concerning CHF management. CONCLUSION: As in other regions of the world, non-adherence to complex CHF treatment is a substantial problem in Soweto. Our data confirm the need for a dedicated CHF management programme to optimise CHF-related outcomes in a low-resource environment.

[Differential influence of immune therapy on relapses and progression in multiple sclerosis: interpretation and therapeutic consequences]. / Unterschiedliche Wirkung der Immuntherapie auf Schübe und schleichende Progression bei Multipler Sklerose: Deutung und Konsequenzen für die Therapie.

It is well established that relapses can be suppressed by different substances in patients with relapsing-remitting multiple sclerosis (MS). In contrast, patients with progressive forms of MS do hardly respond to immune therapy. Therefore, start of immune therapy after the first relapse has been proposed, especially in order to prevent degeneration and disability. This view is challenged in the present review. Actually no evidence exists in support of a retardation or an attenuation of secondary progression by early immune therapy. Widespread degeneration occurs early and progresses independently from inflammatory plaques. Therefore, autoimmunity per se is no adequate paradigm to explain MS-pathogenesis entirely. A virus/superantigen-dualism is proposed to explain the different parts of MS, instead. It is concluded that evidence-based immune therapy should be adapted to the actual inflammatory activity of the disease. A suitable parameter for this purpose is the interval between 2 relapses.