Functional connectivity alterations in patients with chronic hepatitis C virus infection: A multimodal MRI study.

Chronic hepatitis C virus (HCV) infection is associated with fatigue and depression. Cognitive impairments are also reported in a smaller number of HCV-positive patients. Recent studies linked HCV to low-grade inflammation in brain. Here, we test the hypothesis that chronic HCV is associated with 3T-neuroimaging-derived grey matter volume (GMV) and functional connectivity alterations in a sample of chronic HCV (1b), without severe liver disease. Regional GMV and resting-state fMRI-derived eigenvector centrality (EC) were compared between 19 HCV-positive patients and 23 healthy controls (all females, 50-69 and 52-64 years, respectively), controlling for white matter hyperintensities and age. Standard tests were used to assess fatigue, depression and cognitive performance. Also, liver fibrosis stage and viral load were quantified among patients. In comparison with controls, HCV-positive patients had higher scores in fatigue and depression, and worse alertness scores. The groups performed similarly in other cognitive domains. We report higher EC in a cluster in the right anterior superior parietal lobule in patients, while no differences are found in GMV. Post hoc functional connectivity analysis showed increased connectivity of this cluster with primary and secondary somatosensory cortex, and temporal and occipital lobes in patients. Higher mean EC in the superior parietal cluster, adjusted for mean framewise displacement, was associated with better memory and attention performance, but not with fatigue, depression, viral load or level of liver fibrosis, among patients. These results suggest a compensatory mechanism in chronic hepatitis C and explain equivocal results in the literature about cognitive deficits in infected persons. Further studies should define the relation of these connectivity changes to the brain's inflammatory activity.

Mesoderm-specific transcript (MEST) is a negative regulator of human adipocyte differentiation.

BACKGROUND: A growing body of evidence suggests that many downstream pathologies of obesity are amplified or even initiated by molecular changes within the white adipose tissue (WAT). Such changes are the result of an excessive expansion of individual white adipocytes and could potentially be ameliorated via an increase in de novo adipocyte recruitment (adipogenesis). Mesoderm-specific transcript (MEST) is a protein with a putative yet unidentified enzymatic function and has previously been shown to correlate with adiposity and adipocyte size in mouse. OBJECTIVES: This study analysed WAT samples and employed a cell model of adipogenesis to characterise MEST expression and function in human. METHODS AND RESULTS: MEST mRNA and protein levels increased during adipocyte differentiation of human multipotent adipose-derived stem cells. Further, obese individuals displayed significantly higher MEST levels in WAT compared with normal-weight subjects, and MEST was significantly correlated with adipocyte volume. In striking contrast to previous mouse studies, knockdown of MEST enhanced human adipocyte differentiation, most likely via a significant promotion of peroxisome proliferator-activated receptor signalling, glycolysis and fatty acid biosynthesis pathways at early stages. Correspondingly, overexpression of MEST impaired adipogenesis. We further found that silencing of MEST fully substitutes for the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) as an inducer of adipogenesis. Accordingly, phosphorylation of the pro-adipogenic transcription factors cyclic AMP responsive element binding protein (CREB) and activating transcription factor 1 (ATF1) were highly increased on MEST knockdown. CONCLUSIONS: Although we found a similar association between MEST and adiposity as previously described for mouse, our functional analyses suggest that MEST acts as an inhibitor of human adipogenesis, contrary to previous murine studies. We have further established a novel link between MEST and CREB/ATF1 that could be of general relevance in regulation of metabolism, in particular obesity-associated diseases.

High-throughput typing of Staphylococcus aureus by amplified fragment length polymorphism (AFLP) or multi-locus variable number of tandem repeat analysis (MLVA) reveals consistent strain relatedness.

This study investigates aspects of the general assumption that, in bacteria, genetic variation in functionally-constrained genomic regions accumulates at a lower rate than in regions of hypermutability such as DNA repeat loci. We compared whole genome polymorphism (using high-throughput amplified fragment length polymorphism [ht-AFLP]) as well as short sequence repeat length variation (using multi-locus variable number of tandem repeat analysis [MLVA]) for 994 Staphylococcus aureus strains isolated from both healthy carriers and invasive infections. MLVA and ht-AFLP minimum spanning trees (MSTs) were similar in their identification of totally different types of genetic variants. This suggests that, despite the enhanced inherent variability of repeats, clusters of strains remain traceable. Finally, no specific molecular marker of epidemicity or virulence was identified in this large strain collection by the MLVA approach. We demonstrate that there is a difference in the rates of cross-genome mutation versus regional repeat variability in the clonal bacterial pathogen S. aureus. Despite these dynamic differences, a conservation of type assignments as based upon these two inherently different typing techniques was observed.

Diet-dependent function of the extracellular matrix proteoglycan Lumican in obesity and glucose homeostasis.

OBJECTIVE: Extracellular matrix remodeling is required for adipose expansion under increased caloric intake. In turn, inhibited expandability due to aberrant collagen deposition promotes insulin resistance and progression towards the metabolic syndrome. An emerging role for the small leucine-rich proteoglycan Lumican in metabolically driven nonalcoholic fatty liver disease sparks an interest in further understanding its role in diet-induced obesity and metabolic complications. METHODS: Whole body ablation of Lumican (Lum-/-) gene and adeno-associated virus-mediated over-expression were used in combination with control or high fat diet to assess energy balance, glucose homeostasis as well as adipose tissue health and remodeling. RESULTS: Lumican was found to be particularly enriched in the stromal cells isolated from murine gonadal white adipose tissue. Likewise murine and human visceral fat showed a robust increase in Lumican as compared to fat from the subcutaneous depot. Lumican null female mice exhibited moderately increased fat mass, decreased insulin sensitivity and increased liver triglycerides in a diet-dependent manner. These changes coincided with inflammation in adipose tissue and no overt effects in adipose expandability, i.e. adipocyte formation and hypertrophy. Lumican over-expression in visceral fat and liver resulted in improved insulin sensitivity and glucose clearance. CONCLUSIONS: These data indicate that Lumican may represent a functional link between the extracellular matrix, glucose homeostasis, and features of the metabolic syndrome.

Analysis of gene expression patterns in small amounts of human ventricular myocardium by a multiplex RNase protection assay.

End-stage human heart failure is associated with changes in expression of steady-state messenger RNA (mRNA) levels. These changes correspond to alterations in protein levels and myocardial function and may have clinical implications regarding etiology, clinical state, or prognosis. However, analysis of mRNA levels in endomyocardial biopsies can be accomplished only by the quantitative polymerase chain reaction, which is difficult to standardize. The aim of the study was to evaluate whether the RNase protection assay is applicable to measure mRNAs of multiple genes simultaneously in small amounts of ventricular myocardium comparable to myocardial biopsies. Total RNA was prepared from left ventricular myocardium from terminally failing hearts with idiopathic (n=9) or ischemic cardiomyopathy (n=7) and from nonfailing control hearts (n=10). mRNA was measured by an optimized RNase protection assay for the beta1-adrenoceptor, the stimulatory G protein alpha-subunit (Gsalpha), phospholamban, the calcium ATPase of the sarcoplasmic reticulum (SERCA), beta-myosin heavy chain (beta-MHC), and the atrial natriuretic peptide (ANP). We extracted 10.7+/-2.1 microg total RNA from three myocardial biopsies taken in vitro. All of the six genes were measurable in duplicate in a total of 7 microg RNA. mRNAs of beta1-adrenoceptor, phospholamban, and SERCA were lower in failing than in nonfailing myocardium by 50%, 33%, and 42% respectively, whereas beta-MHC and Gsalpha mRNAs were unchanged. mRNA of ANP was expressed at high levels only in the failing myocardium, providing a highly specific and sensitive marker for discriminating nonfailing and failing hearts. A direct comparison with ANP and Gsalpha levels obtained by Northern blot analysis with 7.5 microg total RNA showed a good correlation between the two methods. The RNase protection assay is thus a suitable method for simultaneous measurements of multiple mRNA levels in human myocardial biopsies. Changes in mRNA levels closely reflected those identified by other methods using larger amounts of RNA. Increased myocardial ANP mRNA levels determined by the RNase protection assay may serve as a molecular marker of heart failure.

Tissue-specific transcriptional activity of a pancreatic islet cell-specific enhancer sequence/Pax6-binding site determined in normal adult tissues in vivo using transgenic mice.

A pancreatic islet cell-specific enhancer sequence (PISCES) shared by the rat insulin-I, glucagon, and somatostatin genes binds the paired domain-containing transcription factor Pax6 and confers strong transcriptional activity in pancreatic islet cell lines. It was found recently that Pax6 plays a major role in islet development. In the present study, transgenic mice were used to investigate PISCES-mediated transcription in normal adult islets in vivo. In several independent mouse lines expressing a PISCES-luciferase reporter transgene, the PISCES motif directed gene expression in the adult eye, cerebellum, and discrete brain areas, consistent with the tissue distribution of Pax6. These tissues contain two Pax6 isoforms caused by alternative splicing, only one of which was found to bind the PISCES motif in electrophoretic mobility shift assays. No reporter gene expression was detected in adult pancreatic islets or in any other peripheral organ tested. RT-PCR analysis confirmed that Pax6 mRNA is present in adult islets. These results demonstrate that the PISCES motif is sufficient to direct highly tissue-specific gene expression in whole animals. The lack of PISCES-mediated transcription in adult islets indicates that the Pax6 protein(s) expressed in adult pancreatic islets function differently from the ones in the eye and cerebellum.

Single-channel properties of L-type calcium channels from failing human ventricle.

OBJECTIVE: The aim of our study was to analyse the single-channel properties of L-type calcium channels from failing human heart and to compare them to the respective animal data. Furthermore, we intended to evaluate the feasibility of future single-channel studies on the role of calcium channels in the pathophysiology of heart failure. METHODS: Single L-type calcium channels were recorded in ventricular myocytes from explanted failing human heart, using the cell-attached configuration of the patch-clamp technique. RESULTS: One or more successful registrations of calcium channels could be obtained in 11 of 19 cell isolations. Determination of single-channel conductance yielded a mean value of 16.6 +/- 1.2 pS (70 mM Ba2+ as the charge carrier) under control conditions and 23.7 +/- 2.8 pS in presence of the calcium-channel agonist FPL 64176. The rapid gating process could be described by a C<-->C<-->O gating scheme. Slow gating analysis revealed a highly significant clustering of active and non-active sweeps. CONCLUSION: Single-channel measurements of L-type calcium channels in human failing ventricle are feasible and reproducible despite the varying patient characteristics. Their channel properties are qualitatively comparable to those found in other mammals. Whether there are quantitative differences due to the underlying heart failure can be elucidated in further studies.

Vasorelaxation by new hybrid compounds containing dihydropyridine and pinacidil-like moieties.

The synthesis and pharmacological properties of a novel type of vasorelaxant hybrid compounds are described. The investigated compounds originate from fluorinated 4-aryl-1,4-dihydropyridines, which are known calcium channel blockers, and/or from fluorinated analogues of pinacidil, which is an opener of ATP-sensitive potassium channels. In particular, we studied the most potent hybrid, 2,6-dimethyl-3,5-dicarbomethoxy-4-(2-difluoromethoxy-5-N-(N' '-cyano-N'-1,2,2-trimethyl-propylguanidyl)-phenyl)-1, 4-dihydropyridine (4a), together with its parent compounds, the dihydropyridine 1b and the pinacidil analogue 3. In isolated rat mesenteric arteries, micromolar concentrations of 4a relaxed contractions exerted by K(+)-depolarization or by norepinephrine. The latter effect was sensitive to the potassium channel blocker glibenclamide. Micromolar 4a also inhibited [(3)H](+)-isradipine and [(3)H]P1075 binding to rat cardiac membranes, and it blocked L-type calcium channels expressed in a mammalian cell line. The respective parent compounds 1b and 3 were always more potent and more selective regarding calcium channel or potassium channel interaction, respectively. In contrast, 4a combined both effects within the same concentration range, indicating that it may represent a lead structure for a novel class of pharmacological hybrid compounds.

The binding of (+)-isradipine by guinea-pig intact atria.

1. The accumulation of [3H]-(+)-isradipine (PN 200-110) was measured in quiescent guinea-pig left atria with normal (K+ 2.7 mM) or lowered (K+ 40 mM) membrane potential. 2. Under control conditions (2.7 mM K+) a high affinity binding of (+)-isradipine could not be detected. If, however, the atria were partially depolarized to about -30 mV by 40 mM K+, high affinity binding became evident displaying a dissociation constant of 4.2 x 10(-11) M and a capacity of 9.7 nmol kg-1 wet wt. 3. The depolarization-induced binding was reversible upon repolarization of the atria although isradipine was still present in the medium. This indicates that the high affinity binding sites disappear as soon as the cell membranes become polarized. 4. Isradipine belongs to the less hydrophobic dihydropyridines, but nevertheless the unsaturable binding led to an accumulation of about 84 fold. At a concentration of 2 x 10(-8) M (+)-isradipine, which reduces the contractile force by 50%, the cellular concentration will rise to more than 10(-6) M.

Sodium load and high affinity ouabain binding in rat and guinea-pig cardiac tissue.

An estimation of the actual Na/K-ATPase transport activity in intact cardiac cells was made by measuring the binding of [3H]-ouabain to rat and guinea-pig ventricular strips. At the low [3H]-ouabain concentration of 1 nM equilibrium binding was hardly obtained after an incubation time of five hours. Different procedures known to alter the sodium load of the cardiac preparations influenced [3H]-ouabain binding: the sodium ionophore monensin enhanced [3H]-ouabain binding, the local anaesthetic dibucaine and a reduction of external sodium ion concentration diminished [3H]-ouabain binding; [3H]-ouabain binding was similarly affected by these procedures in the rat and guinea-pig. Since [3H]-ouabain binding occurred predominantly at the high-affinity binding sites of rat myocardium under the applied experimental conditions, it was concluded that these binding sites represent Na/K-ATPase molecules involved in sodium ion transport.

Action of ouabain on rat heart: comparison with its effect on guinea-pig heart.

The inotropic dose-response curve of ouabain in rat cardiac ventricular strips exceeded a concentration range of two decades (1 X 10(-7) M to 3 X 10(-5) M) displaying an intermediate plateau phase. In guinea-pig ventricular strips the inotropic ouabain concentrations spanned only one decade (1 X 10(-7) M-1 X 10(-6) M). Ouabain-intoxication in guinea-pig ventricular strips occurring at 3 X 10(-6) M consisted of arrhythmia and contracture, while in rat ventricular strips at the toxic concentration of 1 X 10(-4) M only a progressive increase in diastolic tension was observed. By means of atomic absorption spectroscopy the ouabain-induced loss of cellular potassium and gain of sodium in rat ventricular strips was detected only at concentrations of ouabain higher than 10(-4) M. Ouabain reduced the activity of Na/K-ATPase prepared from rat and guinea-pig cardiac ventricles to half of its maximum at 6.5 X 10(-5) M in rat and 1.0 X 10(-6) M in guinea-pig, rat heart Na/K-ATPase thus being about 60 fold less sensitive towards ouabain. Specific [3H]-ouabain binding to membrane suspensions prepared from rat and guinea-pig ventricles was characterized by a similar affinity in rat (KD = 4 X 10(-8) M) and guinea-pig (KD = 13 X 10(-8) M). The number of ouabain binding sites in rat membranes was only about 10% of the number found in guinea-pig membranes. In rat the presence of additional ouabain-binding with low affinity and high capacity seemed possible, but could not be verified for methodological reasons. In the light of the biochemical results and binding data, the wider range of ouabain concentration exerting a positive inotropic effect in the rat may be attributed to the existence in the latter of two populations of receptors with different affinities for ouabain and different capacities. In contrast, in the guinea-pig, there is a single population. Nevertheless it is probable that all the receptors in both species are part of the Na/K-ATPase complex and mediate a positive inotropic effect after ouabain-binding in an identical manner.

Murine ventricular L-type Ca(2+) current is enhanced by zinterol via beta(1)-adrenoceptors, and is reduced in TG4 mice overexpressing the human beta(2)-adrenoceptor.

1. The functional coupling of beta(2)-adrenoceptors (beta(2)-ARs) to murine L-type Ca(2+) current (I(Ca(L))) was investigated with two different approaches. The beta(2)-AR signalling cascade was activated either with the beta(2)-AR selective agonist zinterol (myocytes from wild-type mice), or by spontaneously active, unoccupied beta(2)-ARs (myocytes from TG4 mice with 435 fold overexpression of human beta(2)-ARs). Ca(2+) and Ba(2+) currents were recorded in the whole-cell and cell-attached configuration of the patch-clamp technique, respectively. 2. Zinterol (10 microM) significantly increased I(Ca(L)) amplitude of wild-type myocytes by 19+/-5%, and this effect was markedly enhanced after inactivation of Gi-proteins with pertussis-toxin (PTX; 76+/-13% increase). However, the effect of zinterol was entirely mediated by the beta(1)-AR subtype, since it was blocked by the beta(1)-AR selective antagonist CGP 20712A (300 nM). The beta(2)-AR selective antagonist ICI 118,551 (50 nM) did not affect the response of I(Ca(L)) to zinterol. 3. In myocytes with beta(2)-AR overexpression I(Ca(L)) was not stimulated by the activated signalling cascade. On the contrary, I(Ca(L)) was lower in TG4 myocytes and a significant reduction of single-channel activity was identified as a reason for the lower whole-cell I(Ca(L)). The beta(2)-AR inverse agonist ICI 118,551 did not further decrease I(Ca(L)). PTX-treatment increased current amplitude to values found in control myocytes. 4. In conclusion, there is no evidence for beta(2)-AR mediated increases of I(Ca(L)) in wild-type mouse ventricular myocytes. Inactivation of Gi-proteins does not unmask beta(2)-AR responses to zinterol, but augments beta(1)-AR mediated increases of I(Ca(L)). In the mouse model of beta(2)-AR overexpression I(Ca(L)) is reduced due to tonic activation of Gi-proteins.

Negative chronotropic and inotropic effects exerted by diadenosine hexaphosphate (AP6A) via A1-adenosine receptors.

1. Diadenosine hexaphosphate (AP6A) exerts vasoconstrictive effects. The purpose of this study was to investigate whether AP6A has any effect on cardiac function. 2. The effects of AP6A (0.1-100 microM) on cardiac contractility and frequency were studied in guinea-pig and human isolated cardiac preparations. Furthermore, the effects of AP6A on the amplitude of the L-type calcium current, on the adenosine 3':5'-cyclic monophosphate (cyclic AMP) content and on the phosphorylation of regulatory phosphoproteins, i.e. phospholamban and troponin inhibitor, were investigated in guinea-pig isolated ventricular myocytes. 3. In isolated spontaneously beating right atria of the guinea-pig AP6A exerted a negative chronotropic effect and reduced the rate of contraction maximally by 35% (IC20 = 35 microM). 4. In isolated electrically driven left atria of the guinea-pig AP6A exerted a negative inotropic effect and reduced force of contraction maximally by 23% (IC20 = 70 microM). 5. In isolated electrically driven papillary muscles of the guinea-pig AP6A alone was ineffective, but attenuated isoprenaline-stimulated force of contraction maximally by 23% (IC20 = 60 microM). Furthermore, AP6A attenuated the relaxant effect of isoprenaline. 6. In human isolated electrically driven ventricular preparations AP6A alone was ineffective, but attenuated isoprenaline-stimulated force of contraction by maximally 42% (IC20 = 18 microM). Moreover, AP6A attenuated the relaxant effect of isoprenaline. 7. All these effects of AP6A were abolished by the selective A1-adenosine receptor antagonist 1,3-dipropyl-cyclopentyl-xanthine (DPCPX, 0.3 microM), whereas the M-cholinoceptor antagonist atropine (10 microM) and the P2-purinoceptor antagonist suramin (300 microM) failed to abolish the effects of AP6A. 8. AP6A 100 microM had no effect on the amplitude of the L-type calcium current, but attenuated isoprenaline-stimulated L-type calcium current. The maximum of the current-voltage relationship (I-V curve) was shifted to the left by isoprenaline and additional application of AP6A shifted the I-V curve back to the right to the control value. The phosphorylation state of phospholamban and the troponin inhibitor was unchanged by AP6A alone, but was markedly attenuated by AP6A in the presence of isoprenaline. Cyclic AMP levels remained unchanged by AP6A, even after stimulation with isoprenaline. 9. In summary, AP6A exerts negative chronotropic and inotropic effects in guinea-pig and human cardiac preparations. These effects are mediated via A1-adenosine receptors as all effects were sensitive to the selective A1-adenosine receptor antagonist DPCPX. Furthermore, the effects of AP6A on cyclic AMP levels, protein phosphorylation and the L-type calcium current are in accordance with stimulation of A1-adenosine receptors.

N-type calcium channel blockers - tools for modulation of cerebral functional units?

According to in vitro and in vivo studies, the direct application of N-type calcium channel blockers as for instance omega-conotoxin GVIA (omega-ctx) potently inhibits the release of neurotransmitters like dopamine. To find out whether this effect could be used for modulation of neurological functions, omega-ctx was used for continuous infusion into the functionally well characterized rat striatum. Over the 2-week time course of intrastriatal application, rats developed a decrease in spontaneous motor activity, spontaneous rotational asymmetry towards the side of application, and behavioral supersensitivity to apomorphine. After the end of infusion period, all functional deficits showed reversibility. The pattern of spontaneous neurological deficits - in particular supersensitivity to apomorphine - points to a substantial unilateral alteration of dopaminergic transmission due to omega-ctx, which is suggested also by an increase in dopamine receptor protein expression within the ipsilateral striatum. Time course and reversibility of neurological deficits caused by omega-ctx, as well as a lack of dopamine depletion contrast findings after selective destruction of dopaminergic neurons and support a functional modulation of dopaminergic transmission. The present study suggests that omega-ctx is an effective potent tool for the unilateral and reversible intracerebral modulation of neuronal circuits. Intracerebral application of omega-ctx could possibly open the way to therapeutic interventions.

Ca2+ channel activation by CGP 48506, a new positive inotropic benzodiazocine derivative.

Effects on L-type Ca2+ channels of a new positive inotropic compound, the active (+)-enantiomer of the Ca2+ sensitizer 5-methyl-6-phenyl-1,3,5,6,-tetrahydro-3,6,-methano-1,5-benzodiazocine -2,4-dione (CGP 48506), were studied in guinea-pig cardiomyocytes. Whole-cell currents (physiological solutions, 2 mM Ca2+) were enhanced approximately 1.8-fold (10(-4) M, n = 7). Slowing of (de)activation kinetics became apparent under conditions where K+ currents were fully eliminated and Ca(2+)-dependent inactivation was minimized (n = 7). Single-channel current (70 mM Ba2+) and mean open time were increased approximately 2.5-fold (10(-4) M, n = 5), because the drug specifically enhanced sweeps containing long openings (mode 2). Therefore, CGP 48506 stimulates Ca2+ channels in a manner reminiscent of, but not identical to chemically distinct activators like Bay K 8644.

Stereoselectivity of Ca2+ channel block by dihydropyridines: no modulation by the voltage protocol.

The L-type Ca2+ current inhibition by the enantiomers of the dihydropyridine niguldipine was investigated at various holding potentials (-40 to -120 mV) and stimulus frequencies (0.1-1 Hz), using guinea-pig ventricular myocytes. Block of whole-cell current is both voltage- and concentration-dependent. (S)-Niguldipine is more potent than its (R)-enantiomer. However, the extent of enantioselectivity is rather small (< or = x 4.4). Importantly, this value does not increase when stimulus conditions favour the inactivated channel state, although this leads to more potent block. This is in contrast to our expectation based on modulated receptor hypothesis, and to the high enantioselectivity of niguldipine binding found in guinea-pig heart membranes (x 40). We conclude that the common modulated receptor hypothesis had to be refined to explain the effects of niguldipine enantiomers.

Non-synergistic relaxant effects of vasoactive intestinal polypeptide and SIN-1 in human isolated cavernous artery and corpus cavernosum.

Since vasoactive intestinal peptide (VIP) and nitric oxide (NO) are considered to be non-adrenergic, non-cholinergic (NANC) inhibitory mediators in human penile erectile tissue, the goal of this study was to discover possible synergistic effects of exogeneous VIP and the NO donor 3-morpholino-sydnonimine (SIN-1) in human isolated cavernous arteries and cavernosal smooth muscle. In contrast to VIP, SIN-1 elicited complete and reproducible relaxant actions. Combined administration of VIP and SIN-1 revealed non-synergistic, independent relaxant effects in both investigated tissues. The results do not favour a combined administration of VIP and SIN-1 as a new therapeutic approach in the treatment of erectile dysfunction.

Acrihellin, a cardioactive steroid escaping from the organ-bath.

The concentration of acrihellin rapidly declines in oxygenated Tyrode-solution, because the compound escapes from the organ-bath being enriched in droplets sprayed from the surface of the bubbled solution. As checked by radiochromatography, acrihellin remains chemically unaltered during this process. Hellebrin and hellebrigenin persist in gassed Tyrode-solution, suggesting that the 3 beta-substituent dimethylacrylic acid endows acrihellin with amphiphilic properties, thus promoting its enrichment at gas-water interphases. Measurements of the inotropic effects in guinea pig left atria performed at concentrations of acrihellin kept constant yielded a dose-response curve, which closely resembles that of the conventional cardioactive steroid ouabain.

Potentiation of cardiodepressive action among calcium antagonists from different classes: evidence for a mechanism at the single calcium channel level.

The ability of calcium antagonists and antiarrhythmic agents to potentiate the negative inotropic effects of calcium antagonists was investigated in guinea-pig left atria. The potency of nitrendipine was enhanced by several amphiphilic agents by one order of magnitude or more (by pretreatment with quinidine or bepridil). The effect of preincubation with bepridil was investigated for a larger number of dihydropyridines. Only some of them were potentiated like nitrendipine. There was no potentiation between any two members of the same chemical group, i.e. between two dihydropyridines or two catamphiphilic calcium antagonists. The interaction between bepridil and nitrendipine was studied in more detail. In atria, its extent was influenced by several conditions, such as the stimulus frequency, the incubation temperature, or the extracellular K+ concentration. In measurements of whole-cell calcium currents in guinea-pig myocytes, the interaction was found to take place in a quantitatively similar manner. At the single channel level, an enhancement of the effects could also be demonstrated. It appears here that both drugs interact by binding to the same channel molecule. We conclude that the interaction may be due to 1.: an amphiphilic drug (like bepridil) binding to the channel very transiently and thus briefly favouring the inactivated channel state, which means that 2.: the other drug (like nitrendipine) has a higher chance to be bound because of its high affinity towards inactivated channels. Alternative explanations are also discussed.