Addition of trans fat and alcohol has divergent effects on atherogenic diet-induced liver injury in rodent models of steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD) are common causes of chronic liver disease. The overlap between ALD and NAFLD suggests the existence of metabolic steatohepatitis. Development of in vivo models that reflect various aspects of human steatohepatitis is essential for drug discovery. We aimed to characterize several models of steatohepatitis (SH) and to investigate whether the pathology could be modulated. Sprague-Dawley rats were fed a high-fat diet (HFD) for 9 wk, followed by either a high-fat, high-cholesterol and cholate diet (HFC) or a HFC diet containing 13% trans fat (HFC-TF). A subset received 15% ethanol-water twice a week for 12 wk. Serum triglycerides, cholesterol, LDL, HDL, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and rodent NH2-terminal propeptide of type III collagen (rPRO-C3) were assessed. The liver was weighed and evaluated using modified Nonalcoholic Steatohepatitis Clinical Research Network histological score system criteria. All diets induced hepatomegaly, but only HFC-TF increased the size of visceral adipose tissue. Trans fat augmented HFC-induced dyslipidemia, and cholesterol was higher and HDL was lower in the HFC-TF groups. Alcohol lowered triglycerides in both dietary groups. HFC elevated ALT and AST, which were lowered by trans fat. All diets induced histological SH, addition of trans fat induced more steatosis but less inflammation. Inclusion of alcohol augmented the HFC-induced inflammation. All diets induced mild fibrosis. Inclusion of trans fat and alcohol significantly increased rPRO-C3. The addition of trans fat reduced the HFC-induced inflammation but augmented steatosis and dyslipidemia. Inclusion of alcohol induced a more inflammatory and fibrogenic phenotype.NEW & NOTEWORTHY Alcoholic liver disease and nonalcoholic liver disease share significant overlap, which suggests the existence of metabolic steatohepatitis. Trans fat has been implicated in steatohepatitis development. Here, we show that the addition of trans fat to an atherogenic diet results in a more steatotic but less inflammatory phenotype, whereas the addition of alcohol to an atherogenic diet augments the inflammatory and fibrogenic properties of the diet.

Comparison of the activation kinetics of the M3 acetylcholine receptor and a constitutively active mutant receptor in living cells.

Activation of G-protein-coupled receptors is the first step of the signaling cascade triggered by binding of an agonist. Here we compare the activation kinetics of the G(q)-coupled M(3) acetylcholine receptor (M(3)-AChR) with that of a constitutively active mutant receptor (M(3)-AChR-N514Y) using M(3)-AChR constructs that report receptor activation by changes in the fluorescence resonance energy transfer (FRET) signal. We observed a leftward shift in the concentration-dependent FRET response for acetylcholine and carbachol with M(3)-AChR-N514Y. Consistent with this result, at submaximal agonist concentrations, the activation kinetics of M(3)-AChR-N514Y were significantly faster, whereas at maximal agonist concentrations the kinetics of receptor activation were identical. Receptor deactivation was significantly faster with carbachol than with acetylcholine and was significantly delayed by the N514Y mutation. Receptor-G-protein interaction was measured by FRET between M(3)-AChR-yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)-Gγ(2). Agonist-induced receptor-G-protein coupling was of a time scale similar to that of receptor activation. As observed for receptor deactivation, receptor-G-protein dissociation was slower for acetylcholine than that for carbachol. Acetylcholine-stimulated increases in receptor-G-protein coupling of M(3)-AChR-N514Y reached only 12% of that of M(3)-AChR and thus cannot be kinetically analyzed. G-protein activation was measured using YFP-tagged Gα(q) and CFP-tagged Gγ(2). Activation of G(q) was significantly slower than receptor activation and indistinguishable for the two agonists. However, G(q) deactivation was significantly prolonged for acetylcholine compared with that for carbachol. Consistent with decreased agonist-stimulated coupling to G(q), agonist-stimulated G(q) activation by M(3)-AChR-N514Y was not detected. Taken together, these results indicate that the N514Y mutation produces constitutive activation of M(3)-AChR by decreasing the rate of receptor deactivation, while having minimal effect on receptor activation.

Dysregulation of bacterial proteolytic machinery by a new class of antibiotics.

Here we show that a new class of antibiotics-acyldepsipeptides-has antibacterial activity against Gram-positive bacteria in vitro and in several rodent models of bacterial infection. The acyldepsipeptides are active against isolates that are resistant to antibiotics in clinical application, implying a new target, which we identify as ClpP, the core unit of a major bacterial protease complex. ClpP is usually tightly regulated and strictly requires a member of the family of Clp-ATPases and often further accessory proteins for proteolytic activation. Binding of acyldepsipeptides to ClpP eliminates these safeguards. The acyldepsipeptide-activated ClpP core is capable of proteolytic degradation in the absence of the regulatory Clp-ATPases. Such uncontrolled proteolysis leads to inhibition of bacterial cell division and eventually cell death.

Contributions to the biodiversity of Vietnam - Results of VIETBIO inventory work and field training in Cuc Phuong National Park.

VIETBIO [Innovative approaches to biodiversity discovery and characterisation in Vietnam] is a bilateral German-Vietnamese research and capacity building project focusing on the development and transfer of new methods and technology towards an integrated biodiversity discovery and monitoring system for Vietnam. Dedicated field training and testing of innovative methodologies were undertaken in Cuc Phuong National Park as part and with support of the project, which led to the new biodiversity data and records made available in this article collection. VIETBIO is a collaboration between the Museum für Naturkunde Berlin - Leibniz Institute for Evolution and Biodiversity Science (MfN), the Botanic Garden and Botanical Museum, Freie Universität Berlin (BGBM) and the Vietnam National Museum of Nature (VNMN), the Institute of Ecology and Biological Resources (IEBR), the Southern Institute of Ecology (SIE), as well as the Institute of Tropical Biology (ITB); all Vietnamese institutions belong to the Vietnam Academy of Science and Technology (VAST). The article collection "VIETBIO" (https://doi.org/10.3897/bdj.coll.63) reports original results of recent biodiversity recording and survey work undertaken in Cuc Phuong National Park, northern Vietnam, under the framework of the VIETBIO project. The collection consist of this "main" cover paper - characterising the study area, the general project approaches and activities, while also giving an extensive overview on previous studies from this area - followed by individual papers for higher taxa as studied during the project. The main purpose is to make primary biodiversity records openly available, including several new and interesting findings for this biodiversity-rich conservation area. All individual data papers with their respective primary records are expected to provide useful baselines for further taxonomic, phylogenetic, ecological and conservation-related studies on the respective taxa and, thus, will be maintained as separate datasets, including separate GUIDs also for further updating.

Drug discovery and development in idiopathic pulmonary fibrosis: challenges and opportunities.

The pharmacological and adverse effect profiles of the two approved therapies for IPF make the development of new therapies challenging. Considering the similarity of the characteristics of drug candidates to Standard of Care is important in defining positioning and development strategies for this disease.

A Systematic Review of Inverse Agonism at Adrenoceptor Subtypes.

As many, if not most, ligands at G protein-coupled receptor antagonists are inverse agonists, we systematically reviewed inverse agonism at the nine adrenoceptor subtypes. Except for ß3-adrenoceptors, inverse agonism has been reported for each of the adrenoceptor subtypes, most often for ß2-adrenoceptors, including endogenously expressed receptors in human tissues. As with other receptors, the detection and degree of inverse agonism depend on the cells and tissues under investigation, i.e., they are greatest when the model has a high intrinsic tone/constitutive activity for the response being studied. Accordingly, they may differ between parts of a tissue, for instance, atria vs. ventricles of the heart, and within a cell type, between cellular responses. The basal tone of endogenously expressed receptors is often low, leading to less consistent detection and a lesser extent of observed inverse agonism. Extent inverse agonism depends on specific molecular properties of a compound, but inverse agonism appears to be more common in certain chemical classes. While inverse agonism is a fascinating facet in attempts to mechanistically understand observed drug effects, we are skeptical whether an a priori definition of the extent of inverse agonism in the target product profile of a developmental candidate is a meaningful option in drug discovery and development.

Validity of the cold pressor test and pain sensitivity questionnaire via online self-administration.

To determine the feasibility of complex home-based phenotyping, 1,876 research participants from the customer base of 23andMe completed an online version of a Pain Sensitivity Questionnaire (PSQ) as well as a cold pressor test (CPT) which is used in clinical assessments of pain. Overall our online version of the PSQ performed similarly to the original pen-and-paper version. Construct validity of the PSQ total was demonstrated by internal consistency and consistent discrimination between more and less painful items. Criterion validity was demonstrated by correlation with pain sensitivity as measured by the CPT. Within the same cohort we performed a cold pressor test using a layperson description and household equipment. Comparison with published reports from controlled studies revealed similar distributions of cold pain tolerance times (i.e., time elapsed before removing the hand from the water). Of those who elected to participate in the CPT, a large majority of participants did not report issues with the test procedure or noncompliance with the instructions (97%). We confirmed a large sex difference in CPT thresholds in line with published data, such that women removed their hands from the water at a median of 54.2 seconds, with men lasting for a median time of 82.7 seconds (Kruskal-Wallis statistic, p < 0.0001), but other factors like age or current pain treatment were at most weakly associated, and inconsistently between men and women. We introduce a new paradigm for performing pain testing, called testing@home, that, in the case of cold nociception, showed comparable results to studies conducted under controlled conditions and supervision of a health care professional.

Optical techniques to analyze real-time activation and signaling of G-protein-coupled receptors.

The activation of G-protein-coupled receptors (GPCRs) is traditionally measured either by monitoring downstream physiological events or by membrane-based biochemical assays. Neither of these approaches permits detailed kinetic or spatial analysis of receptor activation and signaling. Recently, several optical techniques have been developed to monitor receptor activation either by using purified reconstituted GPCRs or by observing GPCRs, G proteins and second messengers in intact cells. These techniques are providing, literally, new views on both the mechanistic basis of the signaling process and the kinetic and spatial properties of GPCR-mediated signals. They suggest that agonists can activate GPCRs within milliseconds, that different compounds can induce distinct active conformations of GPCRs, that G-protein activation is the rate-limiting step in GPCR signaling, and that cellular signals can be temporally and spatially confined. They are also raising controversial issues, such as whether or not receptors and G proteins are pre-coupled and whether G proteins dissociate during activation.

Arginine-pyrimidine conjugates with therapeutic and prophylactic activity in lethal bacterial infections.

Arginine-pyrimidine conjugates represent a novel class of compounds that exhibits therapeutic and prophylactic activity in lethal infections by Gram-positive and Gram-negative bacteria without showing antibacterial activity in vitro.

The future R&D landscape in non-alcoholic steatohepatitis (NASH).

Nonalcoholic steatohepatitis (NASH) is emerging as a major public health issue for the 21st century and is associated with significant liver-related morbidity and mortality. At present, there are no approved drug therapies for NASH. Consequently, NASH has become the focus of significant public and private research and development. In this review, we highlight the research and development (R&D) challenges and opportunities in this emerging therapeutic area. In particular, we consider the impact of the development of new biomarker strategies on clinical trial execution and design, and the positioning of single and combination therapies in future approaches to the treatment of NASH.

Biochemical and histological characterisation of an experimental rodent model of non-alcoholic steatohepatitis - Effects of a peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist and a glucagon-like peptide-1 analogue.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is a prevalent disease that is highly associated with the metabolic syndrome and type II diabetes. The development of in vivo models that reflect all nuances of the human NASH pathology is essential for drug discovery and development. We aimed to further characterise a dietary induced model of NASH both biochemically and histologically. In addition, we also investigated whether pioglitazone and liraglutide, drugs that have both been investigated as potential NASH treatments, could modulate the pathological changes induced by the NASH diet. Furthermore, to aid the translation of data from pre-clinical in vivo models, we aimed to adapt the NASH Clinical Research Network (CRN) histological score system for use in rodent studies. METHODS: Sprague Dawley rats were fed a high-fat diet (HFD) for 9 weeks, after which they were switched to a high fat, high cholesterol and cholate diet (HFCC) for 12 weeks. The rats were divided into treatment groups, receiving either 30 mg/kg pioglitazone p.o. SID or liraglutide s.c. 200 µg/kg BID or the respective vehicles. Serum levels of triglycerides (TG), cholesterol (Chol), LDL, HDL, AST and ALT, as well as body weight were assessed in all subjects. Upon termination, the liver was weighed and evaluated histologically using modified NASH-CRN criteria. RESULTS: HFCC feeding induced severe hepatic injury and hepatomegaly as indicated by significant increases in AST, ALT and an increased liver weight. Additionally, HFCC feeding induced dyslipidaemia, significant increases in circulating cholesterol and LDL were observed. No obesogenic effect of the HFCC diet was observed, though the diet did induce insulin resistance. Histological analysis showed that the HFCC diet induced several NASH like features, though it did not induce the development of severe fibrosis. However, microgranulomas were often prevalent in addition to lobular inflammatory foci. Pioglitazone showed little efficacy upon both biochemical and histological features. However, liraglutide induced weight loss, improved glycaemic control, reduced ALT and AST and showed some beneficial effects upon steatosis and lobular inflammation. CONCLUSION: Similar to previous reports we have shown that the atherogenic diet, HFCC, induces a phenotype akin to that seen in human NASH patients. Despite inducing all histological features of NASH, HFCC feeding does not promote the development of significant fibrosis within rodents. Pioglitazone and liraglutide have been investigated as potential NASH treatments. Within this model of NASH we have shown that pioglitazone has little efficacy, whereas liraglutide reduced the levels of circulating aminotransferases and had some beneficial effects upon NASH histological parameters.

Methods to assay inhibitors of tRNA synthetase activity.

Aminoacyl-tRNA synthetases (aa-RS) attracted interest as potential targets for new antibacterial compounds. Most organisms express 20 aa-RSs: one for each amino acid. Aa-RSs are essential proteins in all living organisms. When one aa-RS is inhibited, the corresponding tRNA is not charged and is therefore unavailable for translation. This leads to protein synthesis inhibition, which in turn causes cell growth arrest. Consequently, each compound that inhibits any of the aa-RS could be a potential antibacterial agent. Only one aa-RS inhibitor, the Ile-RS inhibitor mupirocin, is currently marketed as an antibacterial agent. We focused on phenylalanyl (Phe)-tRNA synthetase (Phe-RS), but the described methods are not restricted to Phe-RS and might be adapted to other aa-RS.

Signal transduction and regulation: are all alpha1-adrenergic receptor subtypes created equal?

The current manuscript reviews the evidence whether and how subtypes of alpha(1)-adrenergic receptors, i.e. alpha(1A)-, alpha(1B)- and alpha(1D)-adrenergic receptors, differentially couple to signal transduction pathways and exhibit differential susceptibility to regulation. In both regards studies in tissues or cells natively expressing the subtypes are hampered because the relative expression of the subtypes is poorly controlled and the observed effects may be cell-type specific. An alternative approach, i.e. transfection of multiple subtypes into the same host cell line overcomes this limitation, but it often remains unclear whether results in such artificial systems are representative for the physiological situation. The overall evidence suggests that indeed subtype-intrinsic and cell type-specific factors interact to direct alpha(1)-adrenergic receptor signaling and regulation. This may explain why so many apparently controversial findings have been reported from various tissues and cells. One of the few consistent themes is that alpha(1D)-adrenergic receptors signal less effectively upon agonist stimulation than the other subtypes, most likely because they exhibit spontaneous internalization.

Nociceptin/orphanin FQ receptor expression in clinical pain disorders and functional effects in cultured neurons.

The nociceptin/orphanin FQ peptide receptor (NOP), activated by its endogenous peptide ligand nociceptin/orphanin FQ (N/OFQ), exerts several effects including modulation of pain signalling. We have examined, for the first time, the tissue distribution of the NOP receptor in clinical visceral and somatic pain disorders by immunohistochemistry and assessed functional effects of NOP and µ-opioid receptor activation in cultured human and rat dorsal root ganglion (DRG) neurons. Quantification of NOP-positive nerve fibres within the bladder suburothelium revealed a remarkable several-fold increase in detrusor overactivity (P < 0.0001) and painful bladder syndrome patient specimens (P = 0.0014) compared with controls. In postmortem control human DRG, 75% to 80% of small/medium neurons (≤50 µm diameter) in the lumbar (somatic) and sacral (visceral) DRG were positive for NOP, and fewer large neurons; avulsion-injured cervical human DRG neurons showed similar numbers. NOP immunoreactivity was significantly decreased in injured peripheral nerves (P = 0.0004), and also in painful neuromas (P = 0.025). Calcium-imaging studies in cultured rat DRG neurons demonstrated dose-dependent inhibition of capsaicin responses in the presence of N/OFQ, with an IC50 of 8.6 pM. In cultured human DRG neurons, 32% inhibition of capsaicin responses was observed in the presence of 1 pM N/OFQ (P < 0.001). The maximum inhibition of capsaicin responses was greater with N/OFQ than µ-opioid receptor agonist DAMGO. Our findings highlight the potential of NOP agonists, particularly in urinary bladder overactivity and pain syndromes. The regulation of NOP expression in visceral and somatic sensory neurons by target-derived neurotrophic factors deserves further study, and the efficacy of NOP selective agonists in clinical trials.

Effects of ageing on muscarinic receptor subtypes and function in rat urinary bladder.

We compared the density and function of M2 and M3 muscarinic acetylcholine receptor subtypes in the urinary bladder of young adult (3 months) and old (23 months) male Wistar rats. Old rats had a reduced density of muscarinic receptors (96+/-10 vs. 156+/-21 fmol/mg protein), but competition experiments with the M3-selective darifenacin did not indicate alterations in the relative roles of M2 and M3 receptors, with the former being more abundant. The amount of immunodetectable alpha-subunits of various G-proteins potentially linked to muscarinic receptor function was unchanged. The potency of carbachol to contract bladder strips was also unaltered; its maximum effects as well as those of a single KCl concentration were unchanged if raw data or those corrected for strip length were analysed, but somewhat reduced when those corrected for strip weight were analysed. Antagonistic effects of atropine, the M2-selective Ro 320-6206 and the M3-selective darifenacin were unchanged. Agonistic effects of the M3-sparing agonist 4-(2-oxo-2,3-dihydro-benzoimidazol-1-yl)-[1,4']bipiperidinyl-1'-carboxylic acid ethyl ester were similarly poor in young and old rats. Additional experiments were concomitantly performed in submandibular glands from the same animals. While total muscarinic receptor density in submandibular glands was not significantly affected by age (56+/-5 vs. 61+/-4 fmol/mg protein), the relative contribution of M3 receptors significantly declined from 68+/-3% to 57+/-2% based upon darifenacin competition curves. We conclude that aged Wistar rats express fewer muscarinic receptors in their urinary bladder, but there is no change in the relative abundance of M2 and M3 receptors; this is accompanied by only minor if any alterations in receptor responsiveness. In contrast, submandibular gland expresses similar receptor numbers in young and old rats, but slightly fewer M3 receptors in old animals.

Comparison of signalling mechanisms involved in rat mesenteric microvessel contraction by noradrenaline and sphingosylphosphorylcholine.

1 We have compared the signalling mechanisms involved in the pertussis toxin-sensitive and -insensitive contraction of rat isolated mesenteric microvessels elicited by sphingosylphosphorylcholine (SPC) and noradrenaline (NA), respectively. 2 The phospholipase D inhibitor butan-1-ol (0.3%), the store-operated Ca(2+) channel inhibitor SK>F 96,365 (10 microM), the tyrosine kinase inhibitor genistein (10 microM), and the src inhibitor PP2 (10 microM) as well as the negative controls (0.3% butan-2-ol and 10 microM diadzein and PP3) had only little effect against either agonist. 3 Inhibitors of phosphatidylinositol-3-kinase (wortmannin and LY 294,002, 10 microM each) or of mitogen-activated protein kinase kinase (PD 98,059 and U 126, 10 microM each) did not consistently attenuate NA- and SPC-induced contraction as compared to their vehicles or negative controls (LY 303,511 or U 124). 4 The phospholipase C inhibitor U 73,122 (10 microM) markedly inhibited the SPC- and NA-induced contraction (70% and 88% inhibition of the response to the highest NA and SPC concentration, respectively), whereas its negative control U 73,343 (10 microM) caused only less than 30% inhibition. 5 The rho-kinase inhibitors Y 27,632 (10 microM) and fasudil (30 microM) caused a rightward-shift of the NA concentration-response curve by 0.7-0.8 log units and reduced the response to 10 microM SPC by 88% and 83%, respectively. 6 These data suggest that SPC and NA, while acting on different receptors coupling to different G-protein classes, elicit contraction of rat mesenteric microvessels by similar signalling pathways including phospholipase C and rho-kinase.

Gαi2- and Gαi3-specific regulation of voltage-dependent L-type calcium channels in cardiomyocytes.

BACKGROUND: Two pertussis toxin sensitive G(i) proteins, G(i2) and G(i3), are expressed in cardiomyocytes and upregulated in heart failure. It has been proposed that the highly homologous G(i) isoforms are functionally distinct. To test for isoform-specific functions of G(i) proteins, we examined their role in the regulation of cardiac L-type voltage-dependent calcium channels (L-VDCC). METHODS: Ventricular tissues and isolated myocytes were obtained from mice with targeted deletion of either Gα(i2) (Gα(i2) (-/-)) or Gα(i3) (Gα(i3) (-/-)). mRNA levels of Gα(i/o) isoforms and L-VDCC subunits were quantified by real-time PCR. Gα(i) and Ca(v)α(1) protein levels as well as protein kinase B/Akt and extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation levels were assessed by immunoblot analysis. L-VDCC function was assessed by whole-cell and single-channel current recordings. RESULTS: In cardiac tissue from Gα(i2) (-/-) mice, Gα(i3) mRNA and protein expression was upregulated to 187 ± 21% and 567 ± 59%, respectively. In Gα(i3) (-/-) mouse hearts, Gα(i2) mRNA (127 ± 5%) and protein (131 ± 10%) levels were slightly enhanced. Interestingly, L-VDCC current density in cardiomyocytes from Gα(i2) (-/-) mice was lowered (-7.9 ± 0.6 pA/pF, n = 11, p<0.05) compared to wild-type cells (-10.7 ± 0.5 pA/pF, n = 22), whereas it was increased in myocytes from Gα(i3) (-/-) mice (-14.3 ± 0.8 pA/pF, n = 14, p<0.05). Steady-state inactivation was shifted to negative potentials, and recovery kinetics slowed in the absence of Gα(i2) (but not of Gα(i3)) and following treatment with pertussis toxin in Gα(i3) (-/-). The pore forming Ca(v)α(1) protein level was unchanged in all mouse models analyzed, similar to mRNA levels of Ca(v)α(1) and Ca(v)ß(2) subunits. Interestingly, at the cellular signalling level, phosphorylation assays revealed abolished carbachol-triggered activation of ERK1/2 in mice lacking Gα(i2). CONCLUSION: Our data provide novel evidence for an isoform-specific modulation of L-VDCC by Gα(i) proteins. In particular, loss of Gα(i2) is reflected by alterations in channel kinetics and likely involves an impairment of the ERK1/2 signalling pathway.

Coupling mode of receptors and G proteins.

Signaling via G-protein-coupled receptors (GPCRs) is crucial to many physiological and pathophysiological processes in multicellular organisms, and GPCRs themselves are targets for important drugs. Classical cell supplementation experiments suggest a collision coupling model, in which receptors and G proteins diffuse randomly within the cell membrane and interact only if receptors are activated. This model is also backed by kinetic and live cell imaging data. According to the challenging theory, receptors and G proteins are precoupled--meaning they are forming stable complexes in the absence of agonist, which prevail during signaling. This model has been favored on the basis of copurification and coimmunoprecipitation of inactive receptors with G proteins and more recently by some approaches measuring energy transfer between labeled receptors and G proteins. This article reviews key findings regarding the receptor/G protein coupling mode, including most recent findings obtained by optical techniques.

Gs activation is time-limiting in initiating receptor-mediated signaling.

To analyze individual steps of G(S)-linked signaling in intact cells, we used fluorescence resonance energy transfer (FRET)-based assays for receptor-G protein interaction, G protein activation, and cAMP effector activation. To do so, we developed a FRET-based sensor to directly monitor G(S) activation in living cells. This was done by coexpressing a Galpha(s) mutant, in which a yellow fluorescent protein was inserted, together with cyan fluorescent protein-tagged Gbetagamma subunits and appropriate receptors in HEK293 cells. Together with assays for receptor activation and receptor-G protein interaction, it is possible to characterize large parts of the G(S) signaling cascade. When A(2A)-adenosine or beta(1)-adrenergic receptors are coexpressed with G(S) in HEK293T cells, the receptor-G(S) interaction was on the same time scale as A(2A) receptor activation with a time constant of <50 ms. G(S) activation was markedly slower and around 450 ms with similar kinetics following activation of A(2A)- or beta(1)-receptors. Taken together, our kinetic measurements demonstrate that the rate of G(S) activation limits initiation of G(S)-coupled receptor signaling.

A role for muscarinic receptors or rho-kinase in hypertension associated rat bladder dysfunction?

PURPOSE: Essential arterial hypertension is a frequent condition. Spontaneously hypertensive rats (SHRs) show bladder dysfunction similar to that seen in patients with overactive bladder. Since muscarinic receptors and rho-kinase have a key role in the regulation of bladder contractility, we determined whether alterations of either one might contribute to hypertension associated bladder dysfunction. MATERIALS AND METHODS: The bladders of SHRs and normotensive Wistar Kyoto rats (WKYs) were compared in in vitro radioligand binding and contractility studies. RESULTS: The mean total number of muscarinic receptors +/- SEM (181 +/- 14 vs 191 +/- 22 fmol/mg protein) and the relative roles of their subtypes were similar in SHRs and WKYs. Contractile responses to the muscarinic agonist carbachol (maximum effect 2.04 +/- 0.24 vs 2.05 +/- 0.14 mN/mm strip length and -log EC50 5.61 +/- 0.07 vs 5.64 +/- 0.04) and to KCl in a receptor independent manner were similar in the 2 strains. The M3 selective antagonist darifenacin inhibited carbachol responses much more potently than the M2 selective antagonist methoctramine but the potency of the 2 drugs was similar in each strain. The rho-kinase inhibitor Y27,632 attenuated carbachol induced contraction in a quantitatively similar manner in SHRs and WKYs. CONCLUSIONS: An altered function of muscarinic receptor subtypes or rho-kinase does not appear to contribute to bladder dysfunction in SHRs.