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Meiosis-specific protein SYCP3 is a major structural component of synaptonemal complex (SC) lateral elements. SYCP3 is rather well conserved in vertebrates. However, some differences in SYCP3 expression have been shown among mammals. To clarifiy these differences, we have investigated SYCP3 expression with the aid of bioinformatic, biochemical and cell biological methods. Here we show that, in contrast to other vertebrates, rat and mouse SYCP3 exist in 2 isoforms. The short isoform is conserved among vertebrates. However, the longer isoform, which represents an N-terminal extension of the shorter one, most likely appeared about 15 million years ago in a common ancestor of rat and mouse and after separation from the hamster branch.

Pristionchus pacificus is a nematode model organism whose genome has recently been sequenced. To refine the genome annotation we performed transcriptome and proteome analysis and gathered comprehensive experimental information on gene expression. Transcriptome analysis on a 454 Life Sciences (Roche) FLX platform generated >700,000 expressed sequence tags (ESTs) from two normalized EST libraries, whereas proteome analysis on an LTQ-Orbitrap mass spectrometer detected >27,000 nonredundant peptide sequences from more than 4000 proteins at sub-parts-per-million (ppm) mass accuracy and a false discovery rate of P. pacificus proteome contains a high proportion of small proteins with no known homologs in other species (“pioneer” proteins). Some of these proteins appear to be products of highly homologous genes, pointing to their common origin. We show that >50% of all pioneer genes are transcribed under standard culture conditions and that pioneer proteins significantly contribute to a unimodal distribution of predicted protein sizes in P. pacificus, which has an unusually low median size of 240 amino acids (26.8 kDa). In contrast, the predicted proteome of Caenorhabditis elegans follows a distinct bimodal protein size distribution, with significant functional differences between small and large protein populations. Combined, these results provide the first catalog of the expressed genome of P. pacificus, refinement of its genome annotation, and the first comparison of related nematode models at the proteome level.

Seminal roots are initiated at the scutellar node during maize (Zea mays L.) embryo development. The maize mutant rtcs (rootless concerning crown and seminal roots) does not initiate seminal roots while its wild-type siblings form on average 2.9 seminal roots per seedling. In this study, proteome profiles of 25-day-old immature maize embryos were compared between wild-type and rtcs plants via two-dimensional electrophoresis (2-DE). Electrospray ionization tandem mass spectrometry (ESI-MS/MS) identified 23 proteins encoded by 21 different genes that were differentially accumulated between wild-type and rtcs embryos (Fc> or =2; FDR

BackgroundFreshwater planktonic crustaceans of the genusDaphniashow a remarkable plasticity to cope with environmental changes in oxygen concentration and temperature. One of the key proteins of adaptive gene control inDaphnia pulexunder hypoxia is hemoglobin (Hb), which increases in hemolymph concentration by an order of magnitude and shows an enhanced oxygen affinity due to changes in subunit composition. To explore the full spectrum of adaptive protein expression in response to low-oxygen conditions, two-dimensional gel electrophoresis and mass spectrometry were used to analyze the proteome composition of animals acclimated to normoxia (oxygen partial pressure [Po2]: 20 kPa) and hypoxia (Po2: 3 kPa), respectively.ResultsThe comparative proteome analysis showed an up-regulation of more than 50 protein spots under hypoxia. Identification of a major share of these spots revealed acclimatory changes for Hb, glycolytic enzymes (enolase), and enzymes involved in the degradation of storage and structural carbohydrates (e.g. cellubiohydrolase). Proteolytic enzymes remained constitutively expressed on a high level.ConclusionAcclimatory adjustments of theD. pulexproteome to hypoxia included a strong induction of Hb and carbohydrate-degrading enzymes. The scenario of adaptive protein expression under environmental hypoxia can be interpreted as a process to improve oxygen transport and carbohydrate provision for the maintenance of ATP production, even during short episodes of tissue hypoxia requiring support from anaerobic metabolism.

In a recent study it was shown that callus cell cultures of Arabidopsis thaliana respond to changes in gravitational field strengths by changes in protein expression. Using ESI-MS/MS for proteins with differential abundance after separation by 2D-PAGE, 28 spots which changed reproducibly and significantly in amount (P

1. The adenosine receptor (P1-purinoceptor) agonists N6-cyclopentyladenosine and N-5'-ethyl-carboxamidoadenosine at concentrations up to 10 mumols 1(-1) affected neither basal, nor noradrenaline- and angiotensin II-stimulated formation of inositol-1-phosphate, inositol-1,4-bisphosphate, and inositol-1,4,5-trisphosphate in slices of rat renal cortex. 2. In contrast, adenine nucleotides (P2-purinoceptor agonists) markedly stimulated inositol phosphate formation. The observed rank order of potency adenosine-5'-O-(2-thiodiphosphate) (EC50 39 mumols 1(-1] greater than adenosine-5'-O-(3-thiotriphosphate) (587) greater than or equal to 5'-adenylylimidodiphosphate (App(NH)p, 899) greater than adenylyl-(beta, gamma-methylene)-diphosphate (4,181) was consistent with the interaction of the compounds with the P2Y-subtype of P2-purinoceptors. AMP and the ADP analogue (alpha, beta-methylene)-adenosine-5'-diphosphate were ineffective. ATP and ADP (less than or equal to 10 mmol 1(-1] did not produce a consistent increase, owing to their hydrolytic degradation in the incubation medium. 3. Whereas the inositol phosphate response to App(NH)p was linear only up to 5 min incubation, the time-dependent stimulation of noradrenaline declined at a slower rate. Following pre-exposure of the renal cortical slices to App(NH)p, renewed addition of App(NH)p caused no further enhancement in the accumulation of inositol phosphates, whilst noradrenaline was still capable of eliciting a response. This suggests that the apparent loss of responsiveness to App(NH)p is not due to substrate depletion or enzymatic inactivation, but most likely attributable to homologous desensitization of the purinoceptor. 4. Pretreatment of the animals with pertussis toxin caused a substantial reduction of functional Gi-protein, as indicated by the lack of [32P]-NAD incorporation in a membrane preparation of the renal cortex. Nevertheless, the increase in inositol phosphate formation induced by noradrenaline, angiotensin II, and App(NH)p was not significantly impaired. 5. We conclude that P2 gamma-purinoceptors are present in the renal cortex; these receptors stimulate formation of inositol phosphates via a pertussis toxin-insensitive pathway and undergo homologous desensitization. On the other hand, our results suggest that renal A,-adenosine receptors do not use stimulation of phosphoinositide breakdown as a transmembrane signalling system.

The regulatory effects of pindolol and celiprolol on cardiac β-adrenoceptor density were studied in vivo in order to assess the subtype selectivity of their partial agonistic activity (PAA). The substances were continuously administered to rats for 1 week by means of implanted osmotic minipumps. The density of β-adrenoceptor subtypes were estimated from ICYP saturation binding experiments performed on cardiac ventricular plasma membranes in the presence of a highly selective antagonist (CGP 20172 A or ICI 118,551). Both antagonists were employed at concentrations as high as to block one subtype only without affecting the complementary subtype. For control purposes, rats were also treated with isoprenaline (0.4 mg/kg/h) and propranolol (0.15 mg/kg/h), or vehicle. Pindolol (0.036 mg/kg/h) and celiprolol (0.36 mg/kg/h) reduced the density of ventricular β2-adrenoceptors by 46% and 23%, respectively, which — in the case of pindolol — was significant when compared to the non-treated controls. Both compounds, however, produced a small, but distinct increase in the number of β1-adrenoceptors by approximately 26%. This finding is in contrast to the propranolol-induced upregulation of both β1- and β2-adrenoceptors by approximately 80%. Since supramaximal doses of each drug were administered, a significant smaller increase of β1-adrenoceptors by pindolol and celiprolol —as compared to the increase produced by propranolol — can be interpreted as evidence for a PAA of pindolol and celiprolol on β1-adrenoceptors as well. Isoprenaline as a full agonist caused a marked loss of of both β-adrenoceptor subtypes. Although it exhibits equal affinity at both subtypes the decrease amounted to 80% of the β2- but only to 54% of the β1-adrenoceptors density. This indicates that the down-regulation of cardiac β-adrenoceptors in general seems to be more pronounced at the β2-than at the β1-adrenoceptors population. We conclude that the subtype desensitization pattern of agents with intrinsic activity precludes the determination of subtype-selectivity, since β1- and β2-adrenoceptors appear to differ in their sensitivity presumably as a result of subtype specific baseline desensitization produced by endogenous catecholamines.

The mammalian lamin B2 gene codes for two proteins, the somatic lamin B2 and the germ line-specific lamin B3. Lamin B3 lacks the N-terminus and a part of the alpha-helical rod domain present in lamin B2. These domains are substituted by 84 amino acids unique for lamin B3. When ectopically expressed in somatic cells, lamin B3 causes severe deformation of nuclei which adopt a hook-like configuration. Accordingly, it was proposed that lamin B3 provides the germ line cells with a more flexible nuclear periphery that facilitates spermatogenesis-specific nuclear reorganization events. Here we investigated which protein domains of lamin B3 are responsible for nuclear deformation in transfected cells, and how stable is the nuclear periphery of these cells. Expression of wild-type and mutant lamins evidenced that nuclear deformations are due to the shortened rod domain of lamin B3. Cell fractionation experiments revealed that lamin B3 can be solubilized more easily than lamin B2. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) analyses of transfected cells showed that lamin B3 has an increased mobility compared to B2. Our results lead to the conclusion that lamin B3 reduces the stability of the nuclear periphery. They are also consistent with the notion that lamin B3 is relevant to specific properties of the nuclear envelope during spermiogenesis.

Seed weight is a crucial plant life history trait, determining establishment success and dispersal ability. Especially in stressful environments, larger seeds may be selected at the expense of seed number, because larger seeds have a better chance of giving rise to an established offspring. We tested the hypotheses that between related species-pairs and among populations of single species a similar trend for increasing seed weight with increasing altitude should be present. Firstly, we measured seed weights from 29 species-pairs, with one species occurring in lowland areas and a congeneric species from high altitudes. Seeds of the alpine species were 28+/-8% larger than seeds from lowland species (P > 0.01). Compared to the related lowland species, 55% of the alpine species had heavier seeds, 3% (one species) had lighter, and 41% had seeds of approximately equal weight. Secondly, we compared seed weights among populations of four species from different habitats and with different life histories. Seeds from between 11 and 34 populations per species were sampled along altitudinal gradients of 800-1,500 m (ca. 800 m in Scabiosa lucida, ca. 1,000 m in Saxifraga oppositifolia, ca. 1,000 m in Epilobium fleischeri, and ca. 1,500 m in Carex flacca). In all the four species, we found no indication for heavier seeds at higher altitudes. Our results indicate a selection pressure for species with heavier seeds at higher altitude, but the trend does not seem to operate across all cases. Phylogenetic constraints may limit the correlation among altitude and seed weight, operating particularly against selection for larger seed size, the closer populations and species are related to each other.

Lamins are members of a multigene family of structural nuclear envelope (NE) proteins. Differentiated mammalian somatic cells express lamins A, C, B1, and B2. The composition and organization of the nuclear lamina of mammalian spermatogenic cells differ significantly from that of somatic cells as they express lamin B1 as well as two short germ line-specific isoforms, namely lamins B3 and C2. Here we describe in detail the expression pattern and localization of lamin B3 during mouse spermatogenesis. By combining RT-PCR, immunoblotting, and immunofluorescence microscopy, we show that lamin B3 is selectively expressed during spermiogenesis (i.e., postmeiotic stages of spermatogenesis). In round spermatids, lamin B3 is distributed in the nuclear periphery and, notably, also in the nucleoplasm. In the course of spermiogenesis, lamin B3 becomes redistributed as it concentrates progressively to the posterior pole of spermatid nuclei. Our results show that during mammalian spermiogenesis the nuclear lamina is composed of B-type isoforms only, namely the ubiquitous lamin B1 and the germline-specific lamin B3. Lamin B3 is the first example of a mammalian lamin that is selectively expressed during postmeiotic stages of spermatogenesis.

Recently, we reported that mutation A1529D in the domain (D) IV P-loop of the rat skeletal muscle Na(+) channel mu(1) (DIV-A1529D) enhanced entry to an inactivated state from which the channels recovered with an abnormally slow time constant on the order of approximately 100 s. Transition to this "ultra-slow" inactivated state (USI) was substantially reduced by binding to the outer pore of a mutant mu-conotoxin GIIIA. This indicated that USI reflected a structural rearrangement of the outer channel vestibule and that binding to the pore of a peptide could stabilize the pore structure (Hilber, K., Sandtner, W., Kudlacek, O., Glaaser, I. W., Weisz, E., Kyle, J. W., French, R. J., Fozzard, H. A., Dudley, S. C., and Todt, H. (2001) J. Biol. Chem. 276, 27831-27839). Here, we tested the hypothesis that occlusion of the inner vestibule of the Na(+) channel by the fast inactivation gate inhibits ultra-slow inactivation. Stabilization of the fast inactivated state (FI) by coexpression of the rat brain beta(1) subunit in Xenopus oocytes significantly prolonged the time course of entry to the USI. A reduction in USI was also observed when the FI was stabilized in the absence of the beta(1) subunit, suggesting a causal relation between the occurrence of the FI and inhibition of USI. This finding was further confirmed in experiments where the FI was destabilized by introducing the mutations I1303Q/F1304Q/M1305Q. In DIV-A1529D + I1303Q/F1304Q/M1305Q channels, occurrence of USI was enhanced at strongly depolarized potentials and could not be prevented by coexpression of the beta(1) subunit. These results strongly suggest that FI inhibits USI in DIV-A1529D channels. Binding to the inner pore of the fast inactivation gate may stabilize the channel structure and thereby prevent USI. Some of the data have been published previously in abstract form (Hilber, K., Sandtner, W., Kudlacek, O., Singer, E., and Todt, H. (2002) Soc. Neurosci. Abstr. 27, program number 46.12).

On 13 March 1998, we commemorated the 60th anniversary of the Anschluss, the annexation of Austria by Nazi Germany and her integration into the Third Reich. Already the night of 11–12 March had marked the beginning of an unprecedented era of terror against the Jewish citizens of Austria—e.g., Nobel Laureate Otto Loewi was arrested during this first night [1]—which swiftly resulted in inconceivable acts of humiliation, in the deprivation of Jewish citizens’ basic civil rights, the dismissal from professional positions, forced expulsion from their homes, theft of their private property, and their eventual exile. Those unable to escape were incarcerated in concentration camps, and more than one third of the prewar Jewish population of Austria perished or was exterminated in the Holocaust. It is with shame 60 years later that we remember the brutal expulsion of a major part of the faculty members from their positions at the University of Vienna, a process which commenced immediately after the takeover by the Nazis. The medical faculty was particularly severely affected. More than 50 percent of the senior staff were dismissed, mainly for racial, but in some cases for political reasons, inflicting irreparable damage to the Viennese Medical School, which was one of the largest in Europe and held a leading place in the hierarchy of medical excellence. Not only four Nobel Laureates originated from within the faculty—namely Robert Baranyi, Karl Landsteiner, Julius Wagner-Jauregg, and Otto Loewi—but also a series of scientists and physicians of world-wide renown: among many others one might name Anton von Eiselsberg, Adolf Lorenz, Clemens von Pirquet, Sigmund Freud, and Alfred Adler. However, attention should not only be drawn to the famous names, but also to the innumerable nameless doctors

Alterations in G-protein-controlled signalling pathways (primarily pathways controlled by Gs and Gi) have been reported to occur in animal models of diabetes mellitus. We have therefore studied the effect of a long-term exposure of human umbilical vein endothelial cells to elevated concentrations of glucose on expression and function of G-protein subunits and endothelial NO synthase. Long-term incubation in high glucose (30 mM for 15 days) did not affect the levels of Gialpha-2, Gqalpha, the splice variants (long and short form) of Gsalpha, and the G-protein beta-subunits or adenylate cyclase activity; basal, as well as isoprenaline-, forskolin- and guanosine 5'-[gamma-thio]triphosphate-stimulated enzyme activities were comparable in high- and low-glucose-treated cells, thus ruling out any functional changes in the stimulatory pathway. Pretreatment of endothelial cells with pertussis toxin blocked a substantial fraction (50%) of the mitogenic response to serum factor(s) which depend(s) of functional Gi2. The sensitivity of cells cultured in high glucose was comparable with that of the paired controls maintained in normal glucose (EC50 = 3.1 +/- 0.5 and 3.3 +/- 0.4 ng/ml respectively). Similarly, we failed to detect any differences in endothelial NO synthase expression, or intracellular distribution and basal activity of the enzyme in endothelial cells cultured in high glucose. Stimulation of NO synthase in intact cells revealed a comparable response to the calcium ionophore ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187). In contrast, stimulation with histamine (which acts via H1-receptors predominantly coupled to Gq) resulted in a significantly increased response in the cells maintained in high glucose. These data are suggestive of an altered H1-histamine receptor-Gq-phospholipase C pathway in endothelial cells cultured in high glucose concentrations, but rule out any glucose-induced functional changes in Gs- and Gi-controlled signalling pathways.

The mechanism of the beneficial effect of angiotensin converting enzyme inhibitors in patients with myocardial infarction is not clear. Recent in vitro data indicate that angiotensin II (AII) stimulates the expression of adhesion molecules and thus activates leukocyte endothelial interactions. In eight healthy volunteers we investigated the influence of exogenous AII on circulating adhesion molecules and on the blood leukocyte count. A systemically effective and a systemically ineffective dose of AII were administered intravenously over 4 h in a single blind crossover design. Examination of the time course of circulating intercellular and vascular adhesion molecules and E-selectin (cICAM-1, cVCAM-1, cE-selectin-1) in response to the AII infusion revealed increases of up to 11% (especially in cVCAM-1 levels) at single time points, but no significant sustained elevation or trend that can be interpreted as a drug-induced effect. The systemically effective dose, which induced an increase in mean arterial blood pressure from 80 to 108 mmHg (1 mmHg = 133.3 Pa), resulted in a significant increase in blood leukocytes, from 4.8 +/- 0.3 to 5.5 +/- 0.3 g/L (p < 0.05); the systemically ineffective AII dose did not alter blood leukocyte count significantly. Although we did not find an influence of AII on circulating adhesion molecules in vivo, we observed an increase in the blood leukocyte count; thus it may be intriguing to assess whether renin-angiotensin system modulating drugs might exert their favorable effect in ischemic diseases at least in part via an influence on leukocytes.

Abstract NO is a potent inhibitor of in vitro platelet aggregation and adhesion. In view of possible future widespread use of NO in pulmonary and cardiovascular diseases, we investigated the role of NO in hemostatic system activation in vivo in humans. Sixteen healthy male volunteers (age range, 22 to 33 years) received either NO by inhalation (50 ppm over 30 minutes; n=8) or the NO synthase inhibitor N G -monomethyl l -arginine ( l -NMMA 3 mg/kg body weight IV over 5 minutes; n=8). β-Thromboglobulin (β-TG), an indicator of platelet activity; prothrombin fragment 1+2 (F 1+2 ), an index of coagulation activation; and thromboxane B 2 (TxB 2 ), a measure of platelet prostaglandin synthesis, were determined in blood samples obtained from bleeding-time incisions (“shed blood”) at baseline and after administration of the respective drug. In addition, β-TG and F 1+2 were also determined in venous blood. To verify the systemic effects of the drugs, methemoglobin and plasma nitrites/nitrates were measured in the NO group, and cardiac output and exhaled NO were measured in the l -NMMA group. Compared with baseline, methemoglobin and plasma nitrates increased by 73±12% ( P =.006) and 60±9% ( P l -NMMA infusion resulted in decreases in both cardiac output (by 16±2%; P P l -NMMA infusion had no significant effect on β-TG, F 1+2 , and TxB 2 levels in shed blood. No significant changes in platelet counts and levels of coagulation activation markers were found in venous blood after drug administration. In contrast to the in vitro data, neither inhalation of NO nor inhibition of NO synthesis by infusion of l -NMMA had a major impact on hemostatic system activation in vivo in healthy individuals.

A long-term study to identify age-dependent alterations in vascular reactivity in obese Zucker rats, a model for non-insulin-dependent diabetes mellitus, was carried out. On aortic rings of 12-week-old obese Zucker rats, but not in older animals (36 and 52 weeks), the following different effects in comparison to the lean rat control group were observed: (i) a significantly enhanced maximal relaxation to acetylcholine and A23187, which was abolished by the nitric oxide-synthase inhibitor L-nitro-arginine methyl ester (L-NAME); relaxation of aortic rings to the endothelium-independent vasodilator nitroglycerin was similar; (ii) more pronounced maximal 5-hydroxytryptamine-induced-contractions in the presence of L-NAME, and (iii) a more pronounced reduction in phenylephrine-induced contractions by verapamil. These results are suggestive of an altered calcium metabolism in the first weeks of development in the obese rat strain, which is probably responsible for the hypotension seen in this early time period.

1. Adenosine is known to stimulate capillary outgrowth and endothelial cell proliferation, but the underlying mechanism has not been identified. In order to identify the receptor subtype involved, the effects of adenosine receptor agonists and antagonists on human umbilical vein endothelial cell (HUVEC) proliferation were investigated. 2. Raising intracellular adenosine levels by use of the adenosine transport inhibitor, 4-nitrobenzylthioinosine (NBMPR) did not affect cell growth. This observation suggests that stimulation of an extracellular adenosine receptor generates the mitogenic signal. 3. In the presence of adenosine deaminase (ADA), which was used to remove adenosine present in the culture medium, the adenosine receptor agonists N-ethylcarboxamidoadenosine (NECA, non-selective) and CGS21680 (A2A-receptor-selective) stimulated [3H]-thymidine incorporation with a half-maximum effect at about 10 nM, while N6-cyclopentyladenosine (CPA, A1-selective) was about 100 fold less potent. The adenosine receptor antagonist, xanthine amine congener (XAC) produced a concentration-dependent decrease in endothelial cell proliferation with a half-maximum effect at about 10 nM. Hence, stimulation of an endothelial A2A-adenosine receptor seems responsible for the mitogenic signal. 4. In the presence of ADA, isoprenaline is also able to stimulate [3H]-thymidine incorporation with a half maximal effect of about 3 nM, an effect, which is reversed by the highly beta 2-selective antagonist, ICI 118,551. In the absence of ADA, isoprenaline exerts only a minor stimulatory effect. Combination of A2A adenosine and beta 2-adrenoceptor agonists did not further enhance [3H]-thymidine incorporation when compared to the sole addition of each agonist. We therefore conclude that both receptors stimulate endothelial cell proliferation via a common signal transduction pathway. 5. Both receptors are coupled to stimulation of adenylyl cyclase via the stimulatory G protein G8.However, direct activation of downstream effectors in the cyclic AMP-signalling cascade (G8 with cholera toxin, adenylyl cyclase with forskolin, protein kinase A with 8Br-cyclic AMP) not only failed to mimic the action of receptor-activation, but even reduced cell proliferation.6. Similarly, pertussis toxin-treatment which inactivated the Gi 2 protein present in HUVEC and thus inhibited cell proliferation per se, did not impair the ability of A2A-receptor agonists to stimulate cell proliferation. This suggests that the A2A-adenosine and beta2-adrenoceptor-mediated stimulation of endothelial cell proliferation occurs via a mechanism that is independent of G8 and Gi.

Population biology is concerned with spatio-temporalvariation in numbers and sizes of individuals (Harper1977). Populations represent a level of biological inte-gration intermediate between the level of the individu-al and the genetic level on the one hand, and the levelsof the metapopulation and of the community on theother. By many interactions populations are linked tothese other levels. Reflecting such interactions, mod-ern population biology not only comprises demo-graphic studies, but increasingly integrates and takesadvantage of disciplines such as evolutionary ecology,population and conservation genetics, community andlandscape ecology, and systematics. Clearly, todaypopulation biology is best understood in a multidisci-plinary context. Since J.L. Harper’s book “Populationbiology of plants” (1977), which may be regarded asthe starting point of plant population biology, this hasalso become apparent in a widening of the scope ofpopulation biological publications.Plant population biology is the topic of a very activesection of the “Gesellschaft fur Okologie”, whose ac-tivities mirror the development and comprehensivescope of population biology (first documented inSchmid & Stocklin 1990). Hosted by the Institute ofBotany of the University of Agricultural Sciences, Vi-enna, this section held its 14

Class III antiarrhythmic drugs alter the relation between cycle length and the QT interval. However, the relation between cycle length and the QT interval varies, whether determined for single test stimuli ("restitution") or for sustained rhythm ("steady state"). In anesthetized guinea pigs (n = 6), we assessed QT prolongation by the selective class III antiarrhythmic agent dofetilide (10 micrograms/kg intravenously) by atrial pacing during steady state and during restitution. Spontaneous sinus rhythm was abolished by electrical ablation of the sinus node area. Dofetilide prolonged QT intervals at all cycle lengths (CL), and the changes were more pronounced at long than at short CL. This reverse rate-dependent effect was significantly greater during steady state than during restitution. Thus, at a cycle length of 200 ms, the difference between QT interval during steady state and QT interval during restitution was -10.0 +/- 1.7 ms at baseline and -11.1 +/- 3.5 ms after dofetilide (p = 0.8). At a CL of 500 ms, the respective values were 11.7 +/- 1.3 and 19.1 +/- 1.9 ms (p = 0.01). Dofetilide produces substantially more reverse rate-dependent increase in QT duration during steady state than during restitution. Clinically, these findings indicate that excessive drug-induced QT prolongation by dofetilide is more likely to develop during prolonged periods of bradycardia than after single long coupled extra-beats. Such excessive QT prolongation may in turn predispose to torsade de pointes arrhythmias.

In the Cardiac Arrhythmia Suppression Trial antiarrhythmic drug therapy with slow kinetic sodium channel blockers (class Ic antiarrhythmic drugs) was associated with excess mortality, presumably due to drug induced proarrhythmia. It has been suggested that the degree of rate-dependent conduction slowing produced by agents that have sodium channel blocking properties may be related to the proarrhythmic propensity of these agents. In the present study, rate-dependent conduction slowing by the antidepressants amitriptyline and maprotiline was investigated in anesthetized guinea pigs. After electrical ablation of the sinus node the left atrium was stimulated at cycle lengths between 200 ms and 500 ms. His bundle electrograms were registered by means of an epicardial electrode. Drugs were administered by i.v. infusion of 0.2 mg kg-1 min-1 for 30 min followed by 0.1 mg kg-1 min-1 for up to 30 min. Both drugs produced substantial rate-dependent conduction slowing within the His-Purkinje-system. The relationship between pacing rate and conduction slowing was well fitted by linear regression. The steepness of the regression line was significantly greater for amitriptyline than for maprotiline (slope factors: 9.10 x 10(-4) +/- 7.85 x 10(-5), n = 6, vs. 6.29 x 10(-4) +/- 2.97 x 10(-5), n = 6, P0.001), indicating that conduction slowing by amitriptyline exhibits a greater degree of rate-dependence than conduction slowing by maprotiline.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological effects observed with an antagonist are usually interpreted as the result of its ability to block receptor activation produced by an endogenous agonist. In this Principles article, Wolfgang Schutz and Michael Freissmuth show how considerable evidence has now been accumulated for G protein-coupled receptors that antagonists not only bind to the receptor, but also induce a conformational change that favours uncoupling of the receptor from its G protein. The spontaneous activity of the unliganded receptor (i.e. the receptor not occupied by any ligand) is a well-established phenomenon in reconstituted systems with purified components. However, its physiological relevance needs to be verified in a more physiological environment before biological effect of antagonists can be primarily ascribed to negative intrinsic activity.

Dopamine-stimulated adenylyl cyclase activity was measured in striatal homogenates of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated rhesus monkeys and humans with idiopathic Parkinson's disease and compared with the activity in control tissue. No differences between parkinsonian and control tissue were found in the presence of 20 mM NaCl. However, when 120 mM NaCl was included in the assay medium, a significantly higher increase in the Vmax of dopamine-stimulated adenylyl cyclase activity was observed in the caudate of MPTP-parkinsonian rhesus monkeys and the putamen of patients with idiopathic Parkinson's disease. No such sensitization was seen in the MPTP-treated rhesus putamen or human Parkinson's disease caudate tissue. A role of D2 receptors in this sensitization could be ruled out by the concomitant use of the D2 antagonist l-sulpiride and by [3H]spiperone saturation analysis of the D2 receptor density, which was found at control level in the caudate tissue of MPTP-treated rhesus monkeys. Similarly, on the basis of saturation binding with the D1 selective ligand 125I-SCH 23982, there was no difference in caudate nucleus D1 receptor densities between control and MPTP-treated monkeys. Our results point to a region-specific functional sensitization of D1 receptors as a consequence of severe dopaminergic denervation of the striatum and suggest the possibility of a therapeutic potential of a D1 agonist with full intrinsic activity in Parkinson's disease.

When expressed in Escherichia coli, the human beta 1- and beta 2-adrenergic receptors retain their ligand binding specificity. Their functional integrity was investigated by analyzing receptor-guanine nucleotide-binding regulatory (G) protein coupling by using two splice variants of the alpha subunit of the stimulatory G protein Gs synthesized in E. coli (rGs alpha-S and rGs alpha-L) and the beta gamma subunits of G protein purified from bovine brain. In competition binding experiments with (-)-[125I]iodocyanopindolol and (-)-isoproterenol, rGs alpha-S.beta gamma and rGs alpha-L.beta gamma reconstituted guanine nucleotide-sensitive high-affinity agonist binding with comparable affinities, whereas rGs alpha PT, a mutant of rGs alpha-L with an altered carboxyl terminus, and a recombinant subtype of the alpha subunit of the inhibitory G protein, rGi alpha-1, were approximately 20- and approximately 200-fold less potent, respectively. A comparison of the beta 1- and beta 2-adrenergic receptor expressed in E. coli with the beta 2-receptor in S49 murine lymphoma cyc- cell membranes revealed a similar affinity of rGs alpha-S and rGs alpha-L for the recombinant and native receptors. After stable incorporation of rGs alpha-S.beta gamma into E. coli membranes, receptor-G protein coupling was also verified by determining the isoproterenol-mediated acceleration of the rate for guanine 5'-[gamma-[35S]thio]triphosphate binding. These results show that (i) receptor-G protein coupling can be reconstituted in E. coli using recombinant components and that (ii) such an approach may be more generally used to evaluate coupling preferences between defined molecular species of receptors and G-protein subunits.

The ability of the bovine brain A1-adenosine receptor to discriminate between different G protein subtypes was tested using G protein alpha-subunits synthesized in Escherichia coli (rG alpha-subunits). When combined with a 3-fold molar excess of beta gamma-subunit purified from bovine brain and used at high concentrations, all three subtypes of rGi alpha (rGi alpha-1, rGi alpha-2, and rGi alpha-3) and rGo alpha were capable of reconstituting guanine nucleotide-sensitive high-affinity binding of the agonist radioligand (-)-N6-3-[125I] (iodo-4-hydroxyphenylisopropyl) adenosine ([125I]HPIA) to the purified A1-adenosine receptor (Kd approximately 1.2 nM). Titration of the A1-adenosine receptor with increasing amounts of rG alpha revealed a approximately 10-fold higher affinity for rGi alpha-3 compared with rGi alpha-1, rGi alpha-2, and rGo alpha. This selectivity was also observed in the absence of beta gamma. Other alpha-subunits (rGs alpha-s, rGs alpha-L, rGs alpha PT, and rGz alpha) did not promote [125I]HPIA binding to the purified receptor. In N-ethylmaleimide-treated bovine brain membranes, rGi alpha-3 was the only rG alpha-subunit capable of reconstituting high-affinity agonist binding. Similarly, rGi alpha-3 competed potently with rGo alpha for activation by the agonist-liganded A1-adenosine receptor, whereas a approximately 50-fold molar excess of rGo alpha was required to quench the receptor-mediated release of [alpha-32P]GDP from rGi alpha-3. Hence, in spite of the extensive homology between alpha-subunits belonging to the Gi/Go group, the A1-adenosine receptor appears to discriminate between the subtypes. This specificity is likely to govern transmembrane signaling pathways in vivo.

The bovine brain A1-adenosine receptor was purified 8000-fold by affinity chromatography on xanthine-amine-congener (XAC)-Sepharose. Addition of a 120-fold molar excess of a purified bovine brain G-protein preparation (Go,i a mixture of Go and Gi, containing predominantly Go) decreases the Bmax of the binding of the antagonist radioligand [3H]XAC to the receptor. This decrease is observed not only after insertion into phospholipid vesicles but also in detergent solution, and is reversed by GTP analogues. In the presence of Go,i, about 20 and 40% of the receptors display guanine-nucleotide-sensitive high-affinity binding of the agonist radioligand (-)-N6-3-([125I]iodo-4-hydroxyphenylisopropyl)adenosine after reconstitution into lipid vesicles and in detergent solution, respectively. The ability of Go,i to enhance agonist binding and decrease antagonist binding is concentration-dependent, with a half-maximal effect occurring at approximately 10-fold molar excess of G-proteins over A1-adenosine receptors. In the presence of the receptor, the rate of guanosine 5′-[gamma-[35S]thio]triphosphate (GTP[35S]) binding to Go,i is accelerated. This rate is further enhanced if the receptor is activated by the agonist (-)(R)-N6-phenylisopropyladenosine, whereas the antagonist XAC decreases the association rate of GTP[35S] to levels observed in the absence of receptor. These results show (1) that detergent removal is not a prerequisite for the observation of coupling between the A1-adenosine receptor and Go,i, and (2) that the regulatory effect of G-proteins on antagonist binding to the A1-adenosine receptor can be reconstituted by using purified components.

The effects of adenosine receptor agonists and antagonists on field-stimulated release of radioactivity from superfused guinea-pig papillary muscles preincubated with [3H]noradrenaline were studied. N 6-cyclopentyladenosine (CPA), N 6-(R-phenylisopropyl)-adenosine, and 5′-N-ethylcarboxamidoadenosine caused concentration-dependent inhibition of evoked overflow with a rank order of potency typical for interaction of the compounds with the A1-subtype of adenosine receptors. Maximum inhibition was 80%. The A1-selective antagonist 8-cyclopentyl-1,3-dipropyl-xanthine (DPCPX) induced a rightward shift of the concentration-response curve for CPA with a pA 2 of 8.35. However, DPCPX per se had no effect on stimulation-evoked tritium overflow. On the other hand, in the presence of 4-nitrobenzylthioinosine (2 μmol/l) and deoxycoformycin (1 μmol/l), inhibitors of adenosine uptake and deamination, respectively, DPCPX produced a concentration-dependent increase in overflow with a pD 2 of 8.1. Pretreatment of the animals with pertussis toxin caused a substantial reduction in the activity of toxin-sensitive G proteins, as indicated by a lack of [32P]ADP ribosylation in a ventricular membrane preparation. Nevertheless, the inhibitory effect of the adenosine receptor agonists on stimulus-evoked overflow remained unaffected. These results are compatible with the existence of inhibitory prejunctional adenosine receptors in guinea-pig papillary muscle, which appear to be coupled to a pertussis toxin-insensitive G protein. The role of endogenous adenosine in occupying these receptors seems minimal under basal conditions.

Two formulations of the drug combination furosemide (20 mg)-spironolactone (100 mg) were tested for bioequivalence in a randomized crossover trial in 12 healthy volunteers. By comparing the AUC values derived from drug serum concentrations, bioequivalence was only achieved for canrenone, the main metabolite of spironolactone, but not for furosemide. A significant difference in the tmax values indicates sustained release of furosemide from one of the formulations. By contrast, bioequivalence was achieved if pharmacodynamic criteria, such as urine volume and Na+ and Cl- excretion over a period of 12 hours, were used. Fractional measurements of urinary volume and electrolyte excretion (0 to 3 h, 3 to 6 h, 6 to 12 h) correlated with the different tmax values for both formulations. These data indicate that bioequivalence is more conclusively verified on the basis of pharmacodynamic parameters than on the basis of pharmacokinetic parameters. These considerations are applicable in particular to drugs displaying large inter-individual variations in serum levels and/or a poor correlation between serum levels and effect.

In Reply. —The medical faculty of the University of Vienna has produced negative headlines in recent years because of limited identification with the events occurring during and after the annexation (Anschluss) of Austria by Nazi Germany.1,2Serious reproaches are the alleged existence still today within our medical school of organ preparations derived from victims of Nazi persecution and the unanswered question of whether the corpses depicted in Pernkopf's atlas are of the same origin. We deeply regret that no relevant investigations were initiated during the half century that has elapsed since World War II to shed light on all aspects concerning the university's role under Nazi dictatorship. Furthermore, it is deplored that the events associated with Nazi terror were systematically suppressed, forgotten, and sometimes even denied. Current staff of the University of Vienna condemn this policy of sweeping unpalatable facts under the carpet. It is only recently, on the occasion of

We compared the germination of seeds of Carex canescens from populations in four regions: northern and southern Sweden, and northern and southern Germany. Germination behaviour was expected to differ between regions owing to adaptations to the local climates. We compared three or four populations from each region in 31 germination experiments (different pre-treatments, different temperature regimes, light vs darkness). In a parallel experiment, we recorded seedling emergence under outdoor conditions from seeds sown in the late summer in northern Germany. Although there were differences in the level of dormancy between the 13 populations and four regions, we were unable to detect geographic patterns which could be explained in terms of differential adaptation to the local climates. Our data did not support a proposed hypothesis that populations experiencing more severe winter conditions would require a longer cold-stratification period. We compared the results from the outdoor experiment with germination data from the laboratory experiments. The latter could not be used to predict differences in the timing of seedling emergence, the time-span during which seedlings emerged, or the number of seedlings emerging. We conclude that the relevance of detected differences in dormancy level among species or populations must be interpreted with great care. First, the detection of geographical patterns requires extensive sampling, second, the ecological consequences of such differences should be established.

Summary An in vitro system is described in which ethylene formation from the amino acid, 1-aminocyclopropane- 1-carboxylic acid (ACC), is catalyzed by horseradish peroxidase (HRP) in the presence of Mn 2+ and p-hydroxyacetophenone (pHAP) as cofactors. The monophenol can be substituted for by indole-3-acetic acid or indole-3-methanol. Oxidative ACC breakdown is accelerated at acidic pH whereas ethylene formation in the presence of IAA is stimulated by pHAP with a pH optimum at 7.8. ACC oxidation is inhibited by propylgallate and ascorbic acid by 100%, whereas OH scavengers such as mannitol or formate have no effect.

The present study was undertaken to investigate beta-adrenoceptor-adenylate cyclase coupling in a myocardial ventricular membrane preparation and in isolated renal glomeruli of cellophane perinephritis hypertensive rats. Adenylate cyclase activity and [125I]iodocyanopindolol binding were differentially affected in these preparations. In isolated glomeruli of hypertensive rats a reduced intrinsic activity of isoproterenol was associated with an apparent loss of the guanine nucleotide-sensitive high-affinity state of the beta-adrenoceptors, whereas their absolute number was unchanged when compared with the normotensive control rats. On the other hand, in the sarcolemmal preparation of hypertensive rats adenylate cyclase activity, the relative amount of high-affinity states, and the density of beta-adrenoceptors were not different from the respective values in normotensive controls. Experiments performed on isolated glomeruli of rats with unilateral cellophane perinephritis that developed only moderate hypertension provide evidence that the apparent loss of the high-affinity state is a consequence of hypertension, since no difference was observed in glomeruli from the wrapped, as compared with the intact kidney.

The study was carried out in 13 mongrel dogs. Regional myocardial blood flow was measured using radiolabelled microspheres. Lactate, H+-ions, and O2 content were measured in arterial and local venous blood. Partial occlusion of the ramus circumflexus (LCX) led to a decrease in left ventricular dp/dt max, systemic arterial pressure, and a marked release of lactate from the underperfused myocardium. With DHE (2 micrograms/kg infused within 5 min, 8 dogs), a slight decrease in heart rate, left ventricular dp/dt max, LCX-resistance and a small increase in femoral and total peripheral resistance as well as in arterial and left and right atrial pressure was observed. In the normally perfused and in the underperfused part of the left ventricle microsphere assessed blood flow was unchanged or slightly elevated with DHE. Lactate release from the underperfused myocardium was markedly reduced. None of the DHE-induced effects on hemodynamic and metabolic parameters were observed in control experiments with infusion of 0.9% NaCl (5 ml). The results indicate that DHE leads to an amelioration of impaired metabolism in the underperfused myocardium and exerts no vasoconstriction in this area.

The investigations were carried out on 24 mongrel dogs which were anaesthetized with chloralose or sodium pentobarbital. Ouabain was added to the coronary blood (50, 100 and 200 ng/ml coronary blood or 0.7, 1.4 and 2.8 X 10(-7) M) over periods of 30 or 60 min by intracoronary infusion of the glycoside. Under chloralose anaesthesia, ouabain at concentrations of 100 and 200 ng/ml coronary blood augmented left ventricular dp/dt significantly. No significant changes were observed in coronary resting flow and flow per beat at the same time. Likewise, the maximum reactive hyperaemic blood flow remained constant, indicating the absence of any changes in the tone of the large extramural arteries. Under sodium pentobarbital anaesthesia small decreases in heart rate and increases in left ventricular dp/dt were seen only at the highest ouabain concentration (200 ng/ml). All flow parameters remained unchanged. These experiments provide evidence that concentrations of ouabain which 100- and 200-fold exceed therapeutic maintenance levels and 10- and 20-fold exceed the concentrations that produce constriction of helically cut pig and rabbit coronary arteries in vitro, do not diminish the coronary blood supply of the anaesthetized dog in vivo.

A decrease in isoproterenol-stimulated adenylate cyclase activity has been shown in various tissues of hypertensive rats, a finding often associated with decreased beta-adrenoceptor number. The present study was undertaken to investigate whether adenylate cyclase stimulation by other hormones is similarly affected. Adenylate cyclase activity in cerebral microvessels under control conditions and following stimulation by isoproterenol, prostaglandin E1, and the adenosine analog 5'-(N-ethylcarboxamide)-adenosine (NECA) was significantly diminished in deoxycorticosterone acetate (DOCA)-hypertensive rats as compared with control rats, as was adenylate cyclase stimulation by GTP, fluoride, and forskolin. Similar results were obtained in cardiac ventricular membranes, apart from the fact that NECA did not influence adenylate cyclase activity in the heart, either in normotensive or in hypertensive rats. In both the heart preparation and the microvessel preparation, beta-adrenoceptor numbers were reduced in DOCA-hypertensive rats. Because the adenylate cyclase data also pointed to functional alteration at the guanine nucleotide regulatory site of the adenylate cyclase complex, the influence of DOCA hypertension on receptor-adenylate cyclase coupling was investigated by competition studies for beta-adrenoceptor binding. In ventricular membranes prepared from DOCA-hypertensive rats, the isoproterenol competition curve for [125I]iodocyanopindolol binding was only slightly altered in the presence of guanylyl imidodiphosphate (50 microM), in contrast to normotensive control rats, in which it was shifted to the right and became significantly steeper. These results are suggestive of decreased guanine nucleotide sensitivity of adenylate cyclase-coupled receptors in DOCA hypertension.

During indoleacetic acid (IAA) oxidation by horseradish peroxidase the water soluble model polyene, crocin, is bleached. IAA-oxidation and crocin bleaching are stimulated at acidic pH as well as by the monophenol p-hydroxyacetophenone. IAA oxidation and crocin bleaching are neither influenced by catalase or superoxide dismutase nor by different OH-radical scavengers, whereas both ascorbate and propylgallate are inhibitory.

Several studies indicate that myocardial ischemia causes a redistribution of beta-adrenergic receptors from a presumably intracellular compartment to the cell surface. However, a decreased adenylate cyclase and contractile responsiveness to beta-adrenergic stimuli has also been reported. The aim of the present study was to investigate possible ischemia-induced changes in myocardial beta-adrenoceptor coupling to adenylate cyclase. Myocardial ischemia was induced by hydraulic occlusion of the LAD in mongrel dogs anesthetized with isoflurane. After 90 min of ischemia, tissue samples were removed from the ischemic and nonischemic regions for tissue catecholamine determinations and for the preparation of particulate fractions from tissue homogenates. Saturation experiments on microsomal fractions obtained from the ischemic and control areas did not reveal any significant changes in the calculated dissociation constant for (-)[125I]iodocyanopindolol binding nor in the calculated receptor density. Likewise, the relative numbers of beta 2-adrenergic receptors were comparable in both preparations (approximately 20%). On the other hand, the proportion of beta-adrenoceptors stabilized in the high-affinity state by (-)isoproterenol was significantly reduced in the ischemic region when compared with the control myocardium (17 +/- 5 vs. 41 +/- 4%). This change was accompanied by a significant decrease in the intrinsic activity of (-)isoproterenol in stimulating adenylate cyclase activity. We propose that the initial uncoupling of the beta-adrenoceptor from its effector is a physiologically important, protective mechanism which guards the ischemic myocardium against the deleterious effect of excessive sympathetic stimulation.

A possible interaction of the antihypertensive drug, urapidil with beta-adrenoceptors was investigated in a guinea-pig ventricular membrane preparation. Urapidil at concentrations above 1 microM antagonized the isoproterenol-induced stimulation of adenylate cyclase activity. Urapidil 10 microM shifted the concentration-response curve for isoproterenol in a parallel manner to the right by a factor of 16. Urapidil competed with specific beta-adrenoceptor binding of [125I]iodocyanopindolol with an IC50 of 12 microM. Since the affinity of urapidil to beta-adrenoceptors appeared rather low, any beta-adrenoceptor blocking property may be of relevance in vivo at high dosages only.

It was investigated whether adenosine affects the adenylate cyclase system of microvessels, isolated from the rat cerebral cortex, via an external receptor recently classified as R-site receptor. Several adenosine analogs caused GTP-dependent stimulation of adenylate cyclase. The rank order of potency: NECA (5'-(N-ethylcarboxamido)-adenosine, EC50 0.2 microM) greater than adenosine (0.71 microM) = 2-chloroadenosine (0.72 microM) greater than L-PIA (N6-(L-phenylisopropyl)-adenosine, 1.03 microM) greater than D-PIA (N6-(D-phenylisopropyl)-adenosine, 5.27 microM) is consistent with that for agonism at activatory (Ra-site) adenosine receptors in other tissues. Adenylate cyclase stimulation by PIA displayed stereoselectivity. The action of NECA was competitively antagonized by 8-phenyltheophylline. These findings provide functional evidence for Ra-site adenosine receptors in rat brain microvessels. However, direct identification of these receptors by binding studies was not possible. Binding of [3H]NECA to rat brain microvessels displayed rapid on-off kinetics and saturability, but equivocal specificity.

The influence of dihydroergotamine on adenosine-induced and reactive vasodilation after long and short periods of coronary artery occlusion was investigated in thoracotomized dogs. Adenosine-induced vasodilation (intracoronary administration) and vasodilation after long periods of coronary artery occlusion (25-35 beats) were similarly influenced, i.e. decreased by the i.v. administration of 10 mug/kg dihydroergotamine. By contrast vasodilation after short periods of coronary artery occlusion (4-7 beats) tended to be increased. This difference in response is thought to arise from two distinct mechanisms of coronary vasodilation after coronary artery occlusion depending on the duration of the occlusion period. The vasodilation after short periods of coronary artery occlusion possibly corresponds to physiological autoregulation. With longer periods of coronary artery occlusion an additional, consecutive mechanism is called into action.

The effects of ouabain on intact and experimentally constricted coronary arteries were investigated in anaesthetized, thoracotomized dogs. Ouabain decreased the blood flow in the intact artery without changing the flow per beat values and increased flow in the constricted artery. Since ouabain decreased left ventricular enddiastolic pressure, it is proposed that the drug-induced augmentation of flow in the constricted artery is related to an elevation of driving pressure to the endocardial layers of the myocardium which are supplied by the constricted artery.

Experiments were carried out in anaesthetized, thoracotomized dogs. The dose dependent positive inotropic and chronotropic effects of intracoronary (i.cor.) infusions or bolus injections of isoproterenol (ISO) were neither antagonized by adenosine (0.46, 1.0, and 2.91 x 10(-5) mol/l) nor by the adenosine analogue N6-phenylisopropyl-adenosine (PIA, 3.23 x 10(-7) mol/l). The results indicate, that adenosine and PIA do not have any antagonistic effects on isoproterenol induced positive inotropy and chronotropy in the intact dog heart. Consequently it can be assumed that an adenosine-catecholamine antagonism is of no physiological relevance.

In mammalian heart tissue beta 2-adrenoceptors are known to coexist with beta 1-adrenoceptors. In the present study, evidence that beta 2-adrenoceptors in guinea-pig and rat ventricles are primarily localized on the coronary endothelium is provided by competition binding studies with the subtype-selective beta-adrenoceptor antagonists ICI 89.406 (beta 1-selective) and ICI 118.551 (beta 2-selective) on four different plasma membrane preparations. (1) Following density gradient centrifugation of cardiac ventricular microsomes from rats or guinea-pigs, endothelial plasma membranes migrated at slightly higher density than the sarcolemmal membranes, as verified by endothelial (angiotensin converting enzyme) and sarcolemmal markers (adenylate cyclase, [3H]ouabain binding). At the activity peak of angiotensin converting enzyme, the relative amount of beta 2-adrenoceptors in guinea-pigs and rats was 25% and 65%, respectively. (2) On sarcolemmal membranes corresponding to the activity peak of adenylate cyclase, beta-adrenoceptors consisted of the beta 1-type exclusively (guinea-pig), or to at least 90% (rat). (3) Cultures of coronary endothelial cells derived from guinea-pigs revealed only beta 2-adrenoceptors. (4) Isolated guinea-pig cardiomyocytes contained only beta 1-adrenoceptors, a finding recently established in rat myocytes as well.

Inhibition of receptor-coupled adenylate cyclase by hormones is proposed to be associated with GTP hydrolysis. Since adenosine inhibits cerebral-cortical adenylate cyclase via A1 adenosine receptors, the present study attempts to verify this mechanism for A1-selective adenosine derivatives. In guinea-pig cortical membranes N6-(phenylisopropyl)adenosine (PIA) increased the Vmax. of the low-Km GTPase, with an EC50 (concentration causing 50% of maximal stimulation) of about 0.1 microM, and the stimulatory effect was competitively antagonized by 5 microM-8-phenyltheophylline. The rank order of potency of the stereoisomers of PIA and of 5-(N-ethylcarboxamido)adenosine (NECA) to stimulate GTPase correlated with their ability to inhibit adenylate cyclase activity (R-PIA greater than NECA greater than S-PIA). Competition binding studies with (-)-N6- ([125I]iodo-4-hydroxyphenylisopropyl)adenosine suggest that adenylyl imidodiphosphate (p[NH]ppA), an essential component of the GTPase assay system, is a more potent A1-receptor agonist than ATP, with an IC50 (concentration giving half-maximal displacement of radioligand binding) of 7.9 microM. On the basis of the p[NH]ppA concentration used in the GTPase assay (1.25 mM), enzyme stimulation by adenosine seems to be highly underestimated. Nevertheless, adenosine-induced GTP hydrolysis reflects an increased turnover of guanine nucleotides at the Ni regulatory site and appears to be a crucial step in the sequence of events processing the inhibitory signal to adenylate cyclase.

In cellular systems provided with activatory (Ra-site) receptors for adenosine, such as rat cerebral microvessels and rat liver plasma membranes, the adenosine-receptor antagonist 8-phenyltheophylline (10 microM) significantly decreased adenylate cyclase activity if ATP was the substrate and only if GTP was present. With dATP as substrate, adenylate cyclase activities in both preparations remained unaffected by 8-phenyltheophylline. In rat cerebral-cortical membranes, with inhibitory (Ri-site) receptors for adenosine, 8-phenyltheophylline significantly enhanced adenylate cyclase activity only in the presence of GTP and if ATP was the substrate. In rat cardiac ventricular membranes, which are devoid of any adenylate cyclase-coupled adenosine receptor, the methylxanthine had no GTP-dependent effect, irrespective of the substrate used. All assay systems contained sufficiently high amounts of adenosine deaminase (2.5 units/ml), since no endogenous adenosine, formed from ATP, was found chromatographically. In order to demonstrate a direct influence of phosphorylated adenosine derivatives on adenylate cyclase activity, we investigated AMP in a dATP assay system. AMP was verified chromatographically to remain reasonably stable under the adenylate cyclase assay conditions. In the microvessels, AMP increased enzyme activity in the range 0.03-1.0 mM, an effect competitively antagonized by 8-phenyltheophylline. In the cortical membranes, 0.1 mM-AMP inhibited adenylate cyclase, which was partially reversed by the methylxanthine. The presence of GTP was again necessary for all observations. In the ventricular membranes, AMP had no effect. Since the efficacy of adenosine-receptor agonists and, probably, that of other hormones on adenylate cyclase activity can be more efficiently measured with dATP as the enzyme substrate, this nucleotide seems preferable for adenylate cyclase measurements in systems susceptible to modulation by adenosine.

The effects of an intracoronary aminophylline infusion, adjusted to give a constant concentration of 25 microgram.cm-3 coronary blood, on the reactive hyperaemic responses following coronary occlusion for 4, 10, and 25 heart beats were investigated in anaesthetised, open-chest dogs. The vasodilator effect of intracoronarily-administered adenosine and the hyperaemic response after coronary occlusion for 10 and 25 heart beats were both significantly diminished under the influence of aminophylline. However, the decrease in the coronary dilator effect of adenosine amounted to 80%, whereas the hyperaemic response was diminished by only 20%. The hyperaemic response following a coronary occlusion for only 4 heart beats remained unchanged. The present results obtained with aminophylline suggest at least a partial involvement of adenosine in mediating reactive hyperaemia after sufficiently long periods of coronary artery occlusion.