ATPergic signaling disruption in human sepsis as a potential source of biomarkers for clinical use.

Sepsis is a life-threatening organ dysfunction caused by a dysregulated inflammatory response to infection. To date, there is no specific treatment established for sepsis. In the extracellular compartment, purines such as adenosine triphosphate (ATP) and adenosine play essential roles in the immune/inflammatory responses during sepsis and septic shock. The balance of extracellular levels among ATP and adenosine is intimately involved in the signals related to immune stimulation/immunosuppression balance. Specialized enzymes, including CD39, CD73, and adenosine deaminase (ADA), are responsible to metabolize ATP to adenosine which will further sensitize the P2 and P1 purinoceptors, respectively. Disruption of the purinergic pathway had been described in the sepsis pathophysiology. Although purinergic signaling has been suggested as a potential target for sepsis treatment, the majority of data available were obtained using pre-clinical approaches. We hypothesized that, as a reflection of deregulation on purinergic signaling, septic patients exhibit differential measurements of serum, neutrophils and monocytes purinergic pathway markers when compared to two types of controls (healthy and ward). It was observed that ATP and ADP serum levels were increased in septic patients, as well as the A2a mRNA expression in neutrophils and monocytes. Both ATPase/ADPase activities were increased during sepsis. Serum ATP and ADP levels, and both ATPase and ADPase activities were associated with the diagnosis of sepsis, representing potential biomarkers candidates. In conclusion, our results advance the translation of purinergic signaling from pre-clinical models into the clinical setting opening opportunities for so much needed new strategies for sepsis and septic shock diagnostics and treatment.

Quantification of fluorescent dyes in organ tissue samples via HPLC analysis.

The determination of regional blood flow via the accumulation of fluorescent microspheres is a concept regularly used in medical research. Typically, the microbeads get extracted from the tissue of interest and are then quantified by measuring the absorption or fluorescence of the incorporated dyes without further separation from the medium. However, in that case the absorption spectra of different dyes can overlap when used simultaneously, leading to an overestimation of the concentration. Additionally, background absorption from the medium can be problematic. Therefore, a high performance liquid chromatography method for the simultaneous detection of four dyes (orange, crimson, yellow-green and red) incorporated in different microbeads in samples from biological media such as organ tissue (brain, heart and kidneys) was developed. Since for biological samples often a large sample size is required for sufficient statistics, the method was optimized to yield very short run times. With this method it was possible to detect very low concentrations of only one microsphere per gram of organ tissue. By applying this sensitive quantification technique, it was demonstrated that the application of microbeads for perfusion measurements might not be reliable due to different organ distributions in each animal.

Superagonism at G protein-coupled receptors and beyond.

Ligands targeting GPCRs can be categorized according to their intrinsic efficacy to trigger a specific, receptor-mediated response. A ligand endowed with the same level of efficacy as the endogenous agonist can be classified as a full agonist, whereas a compound that displays greater efficacy, that is, higher receptor signalling output than the endogenous agonist, can be called a superagonist. Subsequent to GPCR activation, an intracellular signalling cascade is set in motion, which may generate substantial amplification of the signal. This may obscure superagonism in pharmacological assays and, therefore, the definition of superagonism necessitates a combination of operational approaches, reduction of spare receptors or estimation of receptor activation close to the receptor level to quantify relative agonist efficacies in a particular system. The first part of this review will compare GPCR superagonism with superagonism in the field of immunology, where this term is well established. In the second part, known GPCR superagonists will be reviewed. Then, the experimental and analytical challenges in the deconvolution of GPCR superagonism will be addressed. Finally, the potential benefit of superagonism is discussed. The molecular mechanisms behind GPCR superagonism are not completely understood. However, crystallography shows that agonist binding alone is not sufficient for a fully active receptor state and that binding of the G protein is at least equally important. Accordingly, the emerging number of reported superagonists implies that ligand-induced receptor conformations more active than the ones stabilized by the endogenous agonist are indeed feasible. Superagonists may have therapeutic potential when receptor function is impaired or to induce negative feedback mechanisms. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc.

Complementary analysis of the hard and soft protein corona: sample preparation critically effects corona composition.

Here we demonstrate how a complementary analysis of nanocapsule-protein interactions with and without application media allows gaining insights into the so called hard and soft protein corona. We have investigated how both human plasma and individual proteins (human serum albumin (HSA), apolipoprotein A-I (ApoA-I)) adsorb and interact with hydroxyethyl starch (HES) nanocapsules possessing different functionalities. To analyse the hard protein corona we used sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and a protein quantitation assay. No significant differences were observed with regards to the hard protein corona. For analysis of the soft protein corona we characterized the nanocapsule-protein interaction with isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). DLS and ITC measurements revealed that a high amount of plasma proteins were adsorbed onto the capsules' surface. Although HSA was not detected in the hard protein corona, ITC measurements indicated the adsorption of an HSA amount similar to plasma with a low binding affinity and reaction heat. In contrast, only small amounts of ApoA-I protein adsorb to the capsules with high binding affinities. Through a comparison of these methods we have identified ApoA-I to be a component of the hard protein corona and HSA as a component of the soft corona. We demonstrate a pronounced difference in the protein corona observed depending on the type of characterization technique applied. As the biological identity of a particle is given by the protein corona it is crucial to use complementary characterization techniques to analyse different aspects of the protein corona.

Pro-fibrotic processes in human lung fibroblasts are driven by an autocrine/paracrine endothelinergic system.

BACKGROUND AND PURPOSE: Since endothelin (ET) may act as pro-fibrotic mediator, expression and release of ET isoforms, their receptors and potential pro-fibrotic ET effects were studied in human lung fibroblasts. EXPERIMENTAL APPROACH: MRC-5 and primary human lung fibroblasts (phLFb) were cultured. Expression of prepro-ET isoforms was determined by qPCR and release of ET-1 by elisa. ET receptor function was analysed by real-time measurement of dynamic mass redistribution (DMR). Incorporation of [(3) H]-thymidine was determined as measure of proliferation and that of [(3) H]-proline for collagen synthesis. Phospho-p42/44 MAP kinase was determined by Western blot. KEY RESULTS: ET-1 is the predominant ET in human lung fibroblasts (hLF), and TGF-ß caused a further, selective and sustained up-regulation of ET-1 resulting in increased extracellular ET-1 accumulation. hLFb express mRNA encoding ET-A and ET-B receptors. Expression of both receptors was confirmed at protein level. ET-1 induced marked DMR signals, an effect that involved ET-A and ET-B receptors. Stimulatory effects of ET-1 on hLFb proliferation and collagen synthesis were mediated exclusively via ET-A receptors. ET-1, again via ET-A receptors, induced rapid activation of ERK MAPK, shown to be a crucial cellular signal in ET-1-induced collagen synthesis. ET-1-induced activation of ERK and collagen synthesis was, in contrast to corresponding effect of a muscarinic agonist, largely insensitive to pertussis toxin. CONCLUSIONS AND IMPLICATIONS: hLFb are endowed with all elements necessary to build a functional autocrine/paracrine endothelinergic system, which appears to drive pro-fibrotic airway and lung remodelling processes, effects for which only ET-A, but not ET-B receptors appear to be of significance.

Agonists with supraphysiological efficacy at the muscarinic M2 ACh receptor.

BACKGROUND AND PURPOSE: Artificial agonists may have higher efficacy for receptor activation than the physiological agonist. Until now, such 'superagonism' has rarely been reported for GPCRs. Iperoxo is an extremely potent muscarinic receptor agonist. We hypothesized that iperoxo is a 'superagonist'. EXPERIMENTAL APPROACH: Signalling of iperoxo and newly synthesized structural analogues was compared with that of ACh at label-free M2 muscarinic receptors applying whole cell dynamic mass redistribution, measurement of G-protein activation, evaluation of cell surface agonist binding and computation of operational efficacies. KEY RESULTS: In CHO-hM2 cells, iperoxo significantly exceeds ACh in Gi /Gs signalling competence. In the orthosteric loss-of-function mutant M2 -Y104(3.33) A, the maximum effect of iperoxo is hardly compromised in contrast to ACh. 'Superagonism' is preserved in the physiological cellular context of MRC-5 human lung fibroblasts. Structure-signalling relationships including iperoxo derivatives with either modified positively charged head group or altered tail suggest that 'superagonism' of iperoxo is mechanistically based on parallel activation of the receptor protein via two orthosteric interaction points. CONCLUSION AND IMPLICATIONS: Supraphysiological agonist efficacy at muscarinic M2 ACh receptors is demonstrated for the first time. In addition, a possible underlying molecular mechanism of GPCR 'superagonism' is provided. We suggest that iperoxo-like orthosteric GPCR activation is a new avenue towards a novel class of receptor activators.

The ß2-subtype of adrenoceptors mediates inhibition of pro-fibrotic events in human lung fibroblasts.

Fibrosis is part of airway remodelling observed in bronchial asthma and COPD. Pro-fibrotic activity of lung fibroblasts may be suppressed by ß-adrenoceptor activation. We aimed, first, to characterise the expression pattern of ß-adrenoceptor subtypes in human lung fibroblasts and, second, to probe ß-adrenoceptor signalling with an emphasis on anti-fibrotic actions. Using reverse transcription PCR, messenger RNA (mRNA) encoding ß(2)-adrenoceptors was detected in MRC-5, HEL-299 and primary human lung fibroblasts, whereas transcripts for ß(1)- and ß(3)-adrenoceptors were not found. Real-time measurement of dynamic mass redistribution in MRC-5 cells revealed ß-agonist-induced G(s)-signalling. Proliferation of MRC-5 cells (determined by [(3)H]-thymidine incorporation) was significantly inhibited by ß-agonists including the ß(2)-selective agonist formoterol (-logIC(50), 10.2) and olodaterol (-logIC(50), 10.6). Formoterol's effect was insensitive to ß(1)-antagonism (GCP 20712, 3 µM), but sensitive to ß(2)-antagonism (ICI 118,551; apparent, pA (2), 9.6). Collagen synthesis in MRC-5 cells (determined by [(3)H]-proline incorporation) was inhibited by ß-agonists including formoterol (-logIC(50), 10.0) and olodaterol (-logIC(50), 10.3) in a ß(2)-blocker-sensitive manner. α-Smooth muscle actin, a marker of myo-fibroblast differentiation, was down-regulated at the mRNA and the protein level by about 50% following 24 and 48 h exposure to 1 nM formoterol, a maximally active concentration. In conclusion, human lung fibroblasts exclusively express ß(2)-adrenoceptors and these mediate inhibition of various markers of pro-fibrotic cellular activity. Under clinical conditions, anti-fibrotic actions may accompany the therapeutic effect of long-term ß(2)-agonist treatment of bronchial asthma and COPD.

Pascal's law and the dynamics of compression therapy: a study on healthy volunteers.

AIM: The aim of this paper was to determine whether Pascal's law can be used to explain the dynamics of compression therapy. METHODS: Sub-bandage pressures were recorded at three different levels to investigate the transmission of applied pressure on the legs of 12 healthy volunteers and 216 applications. RESULTS: The experimental model revealed that when pressure is increased at a certain area in a compressed leg, the pressure is transmitted within the compressed area. CONCLUSION: The dynamics of effective compression therapy are explained by Pascal's Law, which states that when pressure is applied on a fluid (a muscle or muscle group) in a closed container (fascia muscularis and compression bandage), there is an equal increase at every other point in the container.

[Cinacalcet - an allosteric enhancer at the Ca2+-receptor]. / Cinacalcet - ein allosterischer Verstärker am Ca2+-Rezeptor.

Cinacalcet (trade name: Mimpara) enhances allosterically the action of Ca (2+)-ions at the parathyroid gland Ca (2+)-receptor which belongs to the superfamily of G protein-coupled receptors. As a consequence blood levels of Ca (2+) and parathyroid hormone decline. Cinacalcet is orally administered and approved for a) the treatment of secondary hyperparathyroidism in patients with end stage renal disease receiving hemodialysis and b) to lower hypercalcemia in patients with parathyroid carcinoma. Therapeutic monitoring includes measurement of blood levels of Ca (2+) and parathyroid hormone. The stable suppression of parathyroid hormone levels under chronic treatment was shown in clinical trials. Clinically relevant outcome parameters, such as bone mass and fracture risk, remain to be evaluated.

In vivo investigations on the cholinesterase-inhibiting effects of tricyclic quinazolinimines: scopolamine-induced cognitive impairments in rats are attenuated at low dosage and reinforced at higher dosage.

Tricyclic quinazolinimines as a novel class of potent inhibitors of cholinesterases in vitro are micro- and sub-micromolar inhibitors with activities at both acetyl- (AChE) and butyrylcholinesterase (BChE) or at BChE only. To further establish the antiamnesic properties of this class of compounds, an in vivo test system has been established. Cognitive impairment in rats was reversibly induced by scopolamine (0.05 mg/100 g body weight) and evaluated in an eight-arm radial maze. A representative quinazolinimine (MD212) showed attenuation of cognitive deficits at a low dosage (0.01 mg/100 g body weight), whereas at a high dosage (>0.1 mg/100 g body weight) the effect of scopolamine is markedly reinforced. As MD212 applied alone does not influence rat's cognition at all, the reinforcement of scopolamine effect has to be due to the amplification of scopolamine action possibly by (1) inhibition of scopolamine metabolism, (2) influence of scopolamine on MD212 metabolism or (3) allosteric modulation of mACh receptors. Receptor-binding studies proved hypothesis (3): MD212 stabilizes [3H]N-methylscopolamine binding to muscarinic receptors allosterically.

Complications of transpedicular screw fixation in the cervical spine.

Today, posterior stabilization of the cervical spine is most frequently performed by lateral mass screws or spinous process wiring. These techniques do not always provide sufficient stability, and anterior fusion procedures are added secondarily. Recently, transpedicular screw fixation of the cervical spine has been introduced to provide a one-stage stable posterior fixation. The aim of the present prospective study is to examine if cervical pedicle screw fixation can be done by low risk and to identify potential risk factors associated with this technique. All patients stabilized by cervical transpedicular screw fixation between 1999 and 2002 were included. Cervical disorders included multisegmental degenerative instability with cervical myelopathy in 16 patients, segmental instability caused by rheumatoid arthritis in three, trauma in five and instability caused by infection in two patients. In most cases additional decompression of the spinal cord and bone graft placement were performed. Pre-operative and post-operative CT-scans (2-mm cuts) and plain X-rays served to determine changes in alignment and the position of the screws. Clinical outcome was assessed in all cases. Ninety-four cervical pedicle screws were implanted in 26 patients, most frequently at the C3 (26 screws) and C4 levels (19 screws). Radiologically 66 screws (70%) were placed correctly (maximal breach 1 mm) whereas 20 screws (21%) were misplaced with reduction of mechanical strength, slight narrowing of the vertebral artery canal (<25%) or the lateral recess without compression of neural structures. However, these misplacements were asymptomatic in all cases. Another eight screws (9%) had a critical breach. Four of them showed a narrowing of the vertebral artery canal of more then 25%, in all cases without vascular problems. Three screws passed through the intervertebral foramen, causing temporary paresis in one case and a new sensory loss in another. In the latter patient revision surgery was performed. The screw was loosened and had to be corrected. The only statistically significant risk factor was the level of surgery: all critical breaches were seen from C3 to C5. Percutaneous application of the screws reduced the risk for misplacement, although this finding was not statistically significant. There was also a remarkable learning curve. Instrumentation with cervical transpedicular screws results in very stable fixation. However, with the use of new techniques like percutaneous screw application or computerized image guidance there remains a risk for damaging nerve roots or the vertebral artery. This technique should be reserved for highly selected patients with clear indications and to highly experienced spine surgeons.

[Results of simple fragment excision in lumbar disc surgery]. / Ergebnisse der alleinigen Sequestrektomie bei lumbaler Bandscheibenoperation.

BACKGROUND: While performing microsurgical disc excision, usually the sequestrated disc fragments as well as loosened or degenerated parts of the nucleus pulposus are removed. It is controversial whether this strategy is always necessary. The aim of this study was to examine this question based on clinical results. MATERIAL AND METHODS: Prospectively all relevant data from 149 consecutive patients after sequestrectomy were collected including the clinical course of the patients with a mean follow-up of 2.3 years. A detailed analysis of the actual pain status, the functional capacity and possible additional spinal operations was performed. RESULTS: During early follow-up, there was one superficial wound infection, which was treated conservatively. The success rate, as measured by patient self-assessment, was 62% for excellent and good and 25% for fair results; 13% of the patients treated declared a poor result having no benefits from surgery. The average FFbH score during follow-up was 74% (100% means no functional restriction). Radicular pain and low back pain had the same intensity on the average, in contrast to some other investigations, where low back pain was lower than radicular pain [14]. Of the 149 patients, 4 underwent a second spine surgery at the same level, 2 of which were recurrent disc herniations (=1.3%). CONCLUSION: Simple fragment excision revealed similar results compared to standard microdiscectomy. There was an especially low number of recurrences in contrast to former reports [16]. This was probably caused by the conscientious selection of patients for sequestrectomy according to well-defined criteria. Whether simple sequestrectomy can effectively treat an additional low back pain component must be clarified by further prospective studies.

Structure/activity relationships of M2 muscarinic allosteric modulators.

Allosteric modulation of G protein-coupled receptors has been intensively studied at muscarinic acetylcholine receptors. Findings made with archetypal allosteric agents such as gallamine, alcuronium, and bis(ammonio)alkane-type agents revealed that binding of orthosteric ligands that attach to the acetylcholine site can be allosterically decreased or increased or left unaltered in a subtype-selective fashion. Analyses of structure/activity relationships (SARs) help to elucidate the molecular events underlying the allosteric action and they may pilot the development of new allosteric agents with improved properties and therapeutic perspectives. With a focus on SARs, this review illustrates the principles of muscarinic allosteric interactions, gives an overview of SARs in congeners of archetypal allosteric agents, and considers the topology of M(2) muscarinic allosteric interactions that are characterized by divergent binding modes.

Residue formation of fumonisin B1 in porcine tissues.

The residues derived from the uptake of fumonisin B1, a toxic metabolite of Fusarium verticillioides frequently occurring in corn and corn products, were determined in growing pigs. After oral administration of 100 mg FB(1)/animal/day for 5-11 days, serum, bile, lung, liver, kidney, brain, spleen, pancreas, heart, muscle, eye, and fat samples were collected immediately and analysed by LC-MS. The highest values were measured in kidney (833 +/- 1329 microg kg(-1), mean +/- SD), liver (231 +/- 163 microg kg(-1)), lung (170 +/- 311 microg kg(-1)) and spleen (854 +/- 2212 microg kg(-1)). Muscle contained 26 +/- 41 microg kg(-1), while in fat only 2 +/- 3 microg kg(-1) were traceable. Despite the potential accumulation over extended feeding periods as well as the large variations in the residue formation of FB(1), a carry-over in edible tissues from swine was considered not to be of toxicological relevance.

[Management of obstetrical brachial plexus palsy--own experience with the primary operative technique]. / Management bei geburtstraumatischen Läsionen des Plexus brachialis - Eigene Erfahrungen mit der primären operativen Behandlung -

Obstetrical brachial plexus palsy occurs at a frequency of 0.6 to 2.5 per 1000 births. 80 to 95% of these lesions recover spontaneously. If spontaneous recovery does not occur within the first six months of life, investigations like electrophysiology, and CT-myelography and surgical exploration of the brachial plexus are recommended. During the last ten years 73 children with obstetrical brachial plexus lesions were examined in our department. 29 newborns underwent surgery on the brachial plexus. In 20 out of 29 children nerve root avulsions were diagnosed preoperatively. We performed in 16 cases nerve grafting after neuroma excision, in four cases nerve grafting combined with neurotization, in seven cases external or internal neurolysis, and in the remaining two cases neurotization and plexo-plexal transfer, respectively. The children were followed up between 18 and 50 months (range 27 months) in 18 cases. Elbow function according to Gilbert scale achieved one half of the patients four to five points and the other half two to three points. We found shoulder function with abduction between 45 degrees to 128 degrees and external rotation in 61% (Grade II to V, according to Gilbert scale). In 31% hand function showed Grade III and IV (Gilbert and Raimondi scale). We recommend decision for surgery at the age of six months. Operation should be planned between six to nine months of life. In addition, physical therapy and options including muscle transfers and orthopaedic procedures must be available to ensure the optimal outcome for these children.

Bisquaternary dimers of strychnine and brucine. A new class of potent enhancers of antagonist binding to muscarinic M2 receptors.

Bisquaternary dimers of strychnine and brucine were synthesized and their allosteric effect on muscarinic acetylcholine M(2) receptors was examined. The compounds retarded the dissociation of the antagonist [(3)H]N-methylscopolamine ([(3)H]NMS) from porcine cardiac cholinoceptors. This action indicated ternary complex formation. All compounds exhibited higher affinity to the allosteric site of [(3)H]NMS-occupied M(2) receptors than the monomeric strychnine and brucine, while the positive cooperativity with NMS was fully maintained. SAR studies revealed the unchanged strychnine ring as an important structural feature for high allosteric potency.

Muscarinic allosteric modulation: M2/M3 subtype selectivity of gallamine is independent of G-protein coupling specificity.

Among the five subtypes of muscarinic acetylcholine receptors, the sensitivity towards allosteric modulation is generally higher in M2 and M4 receptors that preferentially couple to inhibitory G-proteins of the Gi/o type than in M1, M3, and M5 that preferentially couple to stimulatory G-proteins such as Gq/11. We aimed to check whether the high allosteric sensitivity of the M2 receptor compared to M3 is related to the differential G-protein coupling preference. As the third intracellular loop (i3) is known to be the major determinant in receptor G-protein coupling specificity, we used wild-type M2 and M3 receptors and the related chimeric constructs with exchanged i3-loops, i.e., M2 containing M3-i3 (M2/M3-i3) and M3 containing M2-i3 (M3/M2-i3). The allosteric effect of the archetypal modulator gallamine on the dissociation and the equilibrium binding of [3H]N-methylscopolamine ([3H]NMS) was measured in membranes of mouse A9L cells stably expressing the wild-type and the chimeric receptors (4 mM Na2HPO4, 1 mM KH2PO4, pH 7.4, 23 degrees C). The dissociation of [3H]NMS was monophasic under all conditions studied. Control values of t 1/2 were (means +/- SEM, n = 4-7): M2: 3.8 +/- 0.2 min, M2/M3-i3: 4.8 +/- 0.3 min, M3:43.3 +/- 4.2 min, M3/M2-i3: 41.1 +/- 3.6 min. At M2 receptors, 0.2 microM gallamine allosterically reduced the apparent rate constant of dissociation k-1 to 51 +/- 5% of the control value (n = 5). At M2/M3-i3 the allosteric potency of gallamine was not significantly changed (0.2 microM gallamine --> k-1 = 61 +/- 4%, n = 7). At M3, a 20-fold higher concentration was required for an equieffective allosteric action (10 microM gallamine --> k-1 = 51 +/- 5%, n = 5). The potency of gallamine at M3/M2-i3 was not increased compared with M3 receptors (10 microM gallamine --> k-1 = 73 +/- 2%, n = 4) but even significantly diminished. [3H]NMS equilibrium binding experiments revealed that neither the binding constants of gallamine at free receptor subtypes (pKA,M2: 7.57 +/- 0.04, n = 4; pKA,M3: 5.56 +/- 0.13, n = 3) nor the factors of negative cooperativity with [3H]NMS (alphaM2 = 31 +/- 1, alphaM3 = 3 +/- 0.4) were affected by the exchanged i3-loops (pKA,M2/M3-i3: 7.65 +/- 0.03, pKA,M3/M2-i2: 5.35 +/- 0.24, alphaM2/M3-i3= 30 +/- 2, alphaM3/M2-i2 = 3 +/- 0.7). In conclusion, the different sensitivities of M2 and M3 receptors towards allosteric modulation by gallamine are not related to the G-protein coupling specificity of the receptors.

Lipophilicity and membrane interactions of cationic-amphiphilic compounds: syntheses and structure-property relationships.

This study was performed to elucidate the relationship between steric factors, lipophilicity, and the potency of cationic-amphiphilic compounds to displace calcium ions from phosphatidylserine monolayers. The latter property is considered to be a substance/phospholipid affinity measure. A series of cationic-amphiphilic 3-phenyl-N,N-dimethylpropylamine derivatives with systematic structural variations was synthesized. Lipophilicity values were determined by chromatographic (RP-HPLC, log D(7.4)), shake-flask (log P), and theoretical (CLOGP) techniques. The potency of the compounds to displace calcium ions from phosphatidylserine monolayers was determined using a radiotracer technique, employing the isotope (45)Ca(2+). The experimental lipophilicity values of several isomeric biphenyl- and diphenyl-congeners differ more than could be expected from the CLOGP-calculations and show a good correlation to the calculated molecular surface areas. Although the affinity of the substances to the phospholipid monolayer tends to increase with lipophilicity, no general interrelation between the two properties could be found. Surprisingly, the assay system (a phospholipid monolayer) was quite sensitive towards small steric changes at the 'ligand' molecules. Stereochemical factors have a considerable influence on the interaction of solutes with phospholipid membranes. It must be questioned whether lipophilicity measures alone, without taking other molecular features into account, can meaningfully be used to explain or predict the influence of solutes on membrane-related processes and properties.

Hexamethonium-type allosteric modulators of the muscarinic receptors bearing lateral dibenzazepine moieties.

Alkane-bisammonium compounds carrying lateral phthalimido substituents are known to have a high affinity for the allosteric binding site of the acetylcholine M2 receptor. The purpose of this study was to replace the lateral phthalimido moieties with rigid tricyclic skeletons of a large volume in order to learn more about the function of the lateral heterocycles. In addition, methyl groups were introduced into the lateral connecting chains. Allosteric inhibition of the dissociation of [3H]N-methylscopolamine from the M2 receptors in porcine cardiac homogenates served to indicate binding of the test compounds to the allosteric site. The phthalimido groups could be replaced with dibenzazepine moieties without any loss in potency. Interestingly, the additional methyl group in the lateral spacer seems to have a significant influence on the allosteric behaviour.