Enteric neuroanatomy and smooth muscle activity in the western diamondback rattlesnake (Crotalus atrox).

BACKGROUND: Gastrointestinal (GI) functions are controlled by the enteric nervous system (ENS) in vertebrates, but data on snakes are scarce, as most studies were done in mammals. However, the feeding of many snakes, including Crotalus atrox, is in strong contrast with mammals, as it consumes an immense, intact prey that is forwarded, stored, and processed by the GI tract. We performed immunohistochemistry in different regions of the GI tract to assess the neuronal density and to quantify cholinergic, nitrergic, and VIPergic enteric neurons. We recorded motility patterns and determined the role of different neurotransmitters in the control of motility. Neuroimaging experiments complemented motility findings. RESULTS: A well-developed ganglionated myenteric plexus (MP) was found in the oesophagus, stomach, and small and large intestines. In the submucous plexus (SMP) most neurons were scattered individually without forming ganglia. The lowest number of neurons was present in the SMP of the proximal colon, while the highest was in the MP of the oesophagus. The total number of neurons in the ENS was estimated to be approx. 1.5 million. In all regions of the SMP except for the oesophagus more nitric oxide synthase+ than choline-acetyltransferase (ChAT)+ neurons were counted, while in the MP ChAT+ neurons dominated. In the SMP most nerve cells were VIP+, contrary to the MP, where numerous VIP+ nerve fibers but hardly any VIP+ neuronal cell bodies were seen. Regular contractions were observed in muscle strips from the distal stomach, but not from the proximal stomach or the colon. We identified acetylcholine as the main excitatory and nitric oxide as the main inhibitory neurotransmitter. Furthermore, 5-HT and dopamine stimulated, while VIP and the ß-receptor-agonist isoproterenol inhibited motility. ATP had only a minor inhibitory effect. Nerve-evoked contractile responses were sodium-dependent, insensitive to tetrodotoxin (TTX), but sensitive to lidocaine, supported by neuroimaging experiments. CONCLUSIONS: The structure of the ENS, and patterns of gastric and colonic contractile activity of Crotalus atrox are strikingly different from mammalian models. However, the main excitatory and inhibitory pathways appear to be conserved. Future studies have to explore how the observed differences are an adaptation to the particular feeding strategy of the snake.

Neural influences on human intestinal epithelium in vitro.

KEY POINTS: We present the first systematic and, up to now, most comprehensive evaluation of the basic features of epithelial functions, such as basal and nerve-evoked secretion, as well as tissue resistance, in over 2200 surgical specimens of human small and large intestine. We found no evidence for impaired nerve-evoked epithelial secretion or tissue resistance with age or disease pathologies (stomach, pancreas or colon cancer, polyps, diverticulitis, stoma reversal). This indicates the validity of future studies on epithelial secretion or resistance that are based on data from a variety of surgical specimens. ACh mainly mediated nerve-evoked and basal secretion in the small intestine, whereas vasoactive intestinal peptide and nitric oxide were the primary pro-secretory transmitters in the large intestine. The results of the present study revealed novel insights into regional differences in nerve-mediated secretion in the human intestine and comprise the basis by which to more specifically target impaired epithelial functions in the diseased gut. ABSTRACT: Knowledge on basic features of epithelial functions in the human intestine is scarce. We used Ussing chamber techniques to record basal tissue resistance (R-basal) and short circuit currents (ISC; secretion) under basal conditions (ISC-basal) and after electrical field stimulation (ISC-EFS) of nerves in 2221 resectates from 435 patients. ISC-EFS was TTX-sensitive and of comparable magnitude in the small and large intestine. ISC-EFS or R-basal were not influenced by the patients' age, sex or disease pathologies (cancer, polyps, diverticulitis). Ion substitution, bumetanide or adenylate cyclase inhibition studies suggested that ISC-EFS depended on epithelial cAMP-driven chloride and bicarbonate secretion but not on amiloride-sensitive sodium absorption. Although atropine-sensitive cholinergic components prevailed for ISC-EFS of the duodenum, jejunum and ileum, PG97-269-sensitive [vasoactive intestinal peptide (VIP) receptor 1 antagonist] VIPergic together with L-NAME-sensitive nitrergic components dominated the ISC-EFS in colonic preparations. Differences in numbers of cholinergic or VIPergic neurons, sensitivity of epithelial muscarinic or VIP receptors, or stimulus frequency-dependent transmitter release were not responsible for the region-specific transmitter contribution to ISC-EFS. Instead, the low atropine-sensitivity of ISC-EFS in the colon was the result of high cholinesterase activity because neostigmine revealed cholinergic components. Colonic ISC-EFS remained unchanged after tachykinin, P2X, P2Y or A1 and A2 receptor blockade. R-basal was smaller and ISC-basal was higher in the small intestine. TTX and bumetanide decreased ISC-basal in all regions, suggesting nerve-dependent secretory tone. ISC-basal was atropine-sensitive in the small intestine and PG97-269-sensitive in the large intestine. This comprehensive study reveals novel insights into region-specific nerve-mediated secretion in the human small and large intestine.

A distinct vagal anti-inflammatory pathway modulates intestinal muscularis resident macrophages independent of the spleen.

The cholinergic anti-inflammatory pathway (CAIP) has been proposed as a key mechanism by which the brain, through the vagus nerve, modulates the immune system in the spleen. Vagus nerve stimulation (VNS) reduces intestinal inflammation and improves postoperative ileus. We investigated the neural pathway involved and the cells mediating the anti-inflammatory effect of VNS in the gut. The effect of VNS on intestinal inflammation and transit was investigated in wild-type, splenic denervated and Rag-1 knockout mice. To define the possible role of α7 nicotinic acetylcholine receptor (α7nAChR), we used knockout and bone marrow chimaera mice. Anterograde tracing of vagal efferents, cell sorting and Ca(2+) imaging were used to reveal the intestinal cells targeted by the vagus nerve. VNS attenuates surgery-induced intestinal inflammation and improves postoperative intestinal transit in wild-type, splenic denervated and T-cell-deficient mice. In contrast, VNS is ineffective in α7nAChR knockout mice and α7nAChR-deficient bone marrow chimaera mice. Anterograde labelling fails to detect vagal efferents contacting resident macrophages, but shows close contacts between cholinergic myenteric neurons and resident macrophages expressing α7nAChR. Finally, α7nAChR activation modulates ATP-induced Ca(2+) response in small intestine resident macrophages. We show that the anti-inflammatory effect of the VNS in the intestine is independent of the spleen and T cells. Instead, the vagus nerve interacts with cholinergic myenteric neurons in close contact with the muscularis macrophages. Our data suggest that intestinal muscularis resident macrophages expressing α7nAChR are most likely the ultimate target of the gastrointestinal CAIP.

Mechanisms involved in the muscle relaxing effects of STW 5 in guinea pig stomach.

INTRODUCTION: The herbal preparation STW 5 ameliorates functional dyspepsia partly by relaxing smooth muscle of the proximal stomach, thus improving gastric accommodation. We explored the unknown pathways responsible for this effect by testing targets known to modulate gastric smooth muscle relaxation. METHODS: STW 5-induced relaxation of smooth muscle strips from guinea pig gastric corpus before and after pharmacological interventions were recorded with force transducers in an organ bath. ORAI1 mRNA expression was tested in the proximal stomach. KEY RESULTS: Blockade of Ca2+ -activated K+ and Cl- channels, voltage-gated L- or T-type Ca2+ channels, TRPA1-, TRPV1-, adenosine or 5-HT4 receptors, antagonizing ryanodine receptors, inhibiting cyclooxygenase or sarcoplasmic reticulum calcium ATPase did not affect STW 5-evoked relaxation. Likewise, protein-kinase A or G were not involved. However, the relaxation evoked by STW 5 was significantly reduced by phorbol-12-myristat-13-acetat, an activator of protein-kinase C, by 2- aminoethyldiphenylborinate, an inhibitor of the IP3 receptor-mediated Ca2+ release from the sarcoplasmic reticulum or by SKF-96365, a nonselective store-operated calcium entry (SOCE) blocker. Furthermore, the mixed TRPC3/SOCE inhibitor Pyr3, but not the selective TRPC3 blocker Pyr10, reduced the effect of STW 5. Finally, BTP2, a potent blocker of ORAI-coupled SOCE, almost abolished STW 5-evoked relaxation. Expression of ORAI1 could be demonstrated in the corpus/fundus. CONCLUSIONS & INFERENCES: STW 5 inhibited SOCE, most likely ORAI channels, which are modulated by IP3- and PKC-dependent mechanisms. Our findings impact on the design of drugs to induce muscle relaxation and help identify phytochemicals with similar modes of actions to treat gastrointestinal disturbances.

Distension evoked mucosal secretion in human and porcine colon in vitro.

It was suggested that intestinal mucosal secretion is enhanced during muscle relaxation and contraction. Mechanisms of mechanically induced secretion have been studied in rodent species. We used voltage clamp Ussing technique to investigate, in human and porcine colonic tissue, secretion evoked by serosal (Pser) or mucosal (Pmuc) pressure application (2-60 mmHg) to induce distension into the mucosal or serosal compartment, respectively. In both species, Pser or Pmuc caused secretion due to Cl- and, in human colon, also HCO3- fluxes. In the human colon, responses were larger in proximal than distal regions. In porcine colon, Pmuc evoked larger responses than Pser whereas the opposite was the case in human colon. In both species, piroxicam revealed a strong prostaglandin (PG) dependent component. Pser and Pmuc induced secretion was tetrodotoxin (TTX) sensitive in porcine colon. In human colon, a TTX sensitive component was only revealed after piroxicam. However, synaptic blockade by ω-conotoxin GVIA reduced the response to mechanical stimuli. Secretion was induced by tensile rather than compressive forces as preventing distension by a filter inhibited the secretion. In conclusion, in both species, distension induced secretion was predominantly mediated by PGs and a rather small nerve dependent response involving mechanosensitive somata and synapses.

Activity of protease-activated receptors in the human submucous plexus.

BACKGROUND & AIMS: Protease-activated receptors (PARs) are expressed in the enteric nervous system. Excessive release of proteases has been reported in functional and inflammatory bowel diseases. Studies in several animal models indicate the involvement of neural PARs. We studied the actions of different PAR-activating peptides (AP) in the human submucous plexus and performed comparative studies in guinea pig submucous neurons. METHODS: We used voltage- and calcium-sensitive dye recordings to study the effects of PAR1-AP, PAR2-AP, PAR4-AP, the PAR1 activator thrombin, and the PAR2 activator tryptase on neurons and glia in human and guinea pig submucous plexus. Human preparations were derived from surgical resections. Levels of mucosal secretion evoked by PAR-APs were measured in Ussing chambers. RESULTS: PAR1-AP and thrombin evoked a prominent spike discharge and intracellular Ca(2+) concentration ([Ca](i)) transients in most human submucous neurons and glia. PAR2-AP, tryptase, and PAR4-AP caused significantly weaker responses in a minor population. In contrast, PAR2-AP evoked much stronger responses in enteric neurons and glia of guinea pigs than did PAR1-AP or PAR4-AP. PAR1-AP, but not PAR2-AP or PAR4-AP, evoked a nerve-mediated secretion in human epithelium. The PAR1 antagonist SCH79797 inhibited the PAR1-AP, and thrombin evoked responses on neurons, glia, and epithelial secretion. In the submucous layer of human intestine, but not guinea pig intestine, PAR2-AP evoked [Ca](i) signals in CD68(+) macrophages. CONCLUSIONS: In the human submucous plexus, PAR1, rather than PAR2 or PAR4, activates nerves and glia. These findings indicate that PAR1 should be the focus of future studies on neural PAR-mediated actions in the human intestine; PAR1 might be developed as a therapeutic target for gastrointestinal disorders associated with increased levels of proteases.

Metabolic and transcriptomic phenotyping of inorganic carbon acclimation in the Cyanobacterium Synechococcus elongatus PCC 7942.

The amount of inorganic carbon is one of the main limiting environmental factors for photosynthetic organisms such as cyanobacteria. Using Synechococcus elongatus PCC 7942, we characterized metabolic and transcriptomic changes in cells that had been shifted from high to low CO(2) levels. Metabolic phenotyping indicated an activation of glycolysis, the oxidative pentose phosphate cycle, and glycolate metabolism at lowered CO(2) levels. The metabolic changes coincided with a general reprogramming of gene expression, which included not only increased transcription of inorganic carbon transporter genes but also genes for enzymes involved in glycolytic and photorespiratory metabolism. In contrast, the mRNA content for genes from nitrogen assimilatory pathways decreased. These observations indicated that cyanobacteria control the homeostasis of the carbon-nitrogen ratio. Therefore, results obtained from the wild type were compared with the MP2 mutant of Synechococcus 7942, which is defective for the carbon-nitrogen ratio-regulating PII protein. Metabolites and genes linked to nitrogen assimilation were differentially regulated, whereas the changes in metabolite concentrations and gene expression for processes related to central carbon metabolism were mostly similar in mutant and wild-type cells after shifts to low-CO(2) conditions. The PII signaling appears to down-regulate the nitrogen metabolism at lowered CO(2), whereas the specific shortage of inorganic carbon is recognized by different mechanisms.

How big is the little brain in the gut? Neuronal numbers in the enteric nervous system of mice, Guinea pig, and human.

BACKGROUND: Despite numerous studies on the enteric nervous system (ENS), we lack fundamental knowledge on neuronal densities or total neuron numbers in different species. There are more anecdotal than actual figures on nerve counts. METHODS: We used standardized preparation techniques and immunohistochemistry with validated panneuronal markers (human or mouse anti-HuD/C) to determine neuronal densities in specimen from the entire gastrointestinal tract of mice, guinea pig, and humans. In parallel, we measured the dimensions of the gastrointestinal regions in mouse and guinea pig. For humans, we had to rely on literature data. KEY RESULTS: The average neuronal densities along the gastrointestinal tract were 35,011 ± 25,017 1/cm2 for the myenteric and 16,685 ± 9098 1/cm2 for the submucous plexus in mice, 24,315 ± 16,627 and 11,850 ± 6122 1/cm2 for guinea pig myenteric and submucous plexus, respectively, and 21,698 ± 9492 and 16,367 ± 5655 1/cm2 for human myenteric and submucous plexus, respectively. The total number of neurons in the ENS was 2.6 million for mice, 14.6 million for guinea pig, and 168 million for human. CONCLUSIONS & INFERENCES: This study reports the first comprehensive nerve cell count in mice, guinea pig, and human ENS. Neuronal densities were comparable between the three species and the differences in the total numbers of enteric neurons are likely due to body size and intestinal length. The number of enteric neurons is comparable to the number of neurons in the spinal cord for all three species.

Metabotropic 5-HT receptor-mediated effects in the human submucous plexus.

BACKGROUND: Serotonin (5-HT) is an important mediator in the gastrointestinal tract, acting on different neuronal 5-HT receptors. The ionotropic 5-HT3 receptor mediates immediate but transient spike discharge in human enteric neurons. We studied the role of the metabotropic 5-HT1P , 5-HT4 , and 5-HT7 receptors to activate human submucous neurons. METHODS: Neuroimaging using the voltage sensitive dye Di-8-ANEPPS was performed in submucous plexus preparations from human surgical specimens of the small and large intestine. We synthesized a new, stable 5-HT1P agonist, 5-benzyloxyhydrazonoindalpine (5-BOHIP). KEY RESULTS: 5-HT evoked a fast and late-onset spike discharge in enteric neurons. The fast component was blocked by the 5-HT3 receptor antagonist cilansetron, while the remaining sustained response was significantly reduced by the 5-HT1P receptor antagonist 5-hydroxytryptophanyl-5-hydroxytryptophan amide (5-HTP-DP). The newly synthesized 5-HT1P agonist 5-BOHIP induced a slowly developing, long-lasting activation of submucous neurons, which was blocked by 5-HTP-DP. We could not demonstrate any 5-HT7 receptor-induced spike discharge based on the lack of response to 5-carboxamidotryptamine. Similarly, the 5-HT4 agonists 5-methoxytryptamine and prucalopride evoked no immediate or late-onset spike discharge. CONCLUSIONS & INFERENCES: Our work demonstrated for the first time the presence of functional 5-HT1P receptors on human submucous neurons. Furthermore, we found no evidence for a role of 5-HT4 or 5-HT7 receptors in the postsynaptic activation of human submucous neurons by 5-HT.

The beta3-adrenoceptor agonist GW427353 (Solabegron) decreases excitability of human enteric neurons via release of somatostatin.

BACKGROUND & AIMS: beta3 Adrenoceptor (beta3-AR) is expressed on adipocytes and enteric neurons. GW427353 is a human selective beta3-AR agonist with visceral analgesic effects. Some of its effects may involve release of somatostatin (SST) and actions on enteric neurons. The aim of this study was to investigate the mode of action of GW427353 in human submucous neurons. METHODS: Voltage sensitive dye imaging was used to record from human submucous neurons. SST release from human primary adipocytes was measured with enzyme-linked immunoabsorbent assay. Immunohistochemistry was used to detect adiponectin, beta3-AR, SST, SST2 receptors, tyrosine hydroxylase (TH), and protein gene product 9.5. RESULTS: Confocal imaging showed cytoplasmic beta3-AR labeling in somata of submucous neurons and nerve varicosities. GW427353 had no direct postsynaptic actions but decreased fast synaptic input to submucous neurons. Tissue perfusion with GW427353 reduced nicotine-evoked neuronal spike frequency, an effect prevented by the beta3-AR antagonist SR-59230 and the SST2-receptor antagonist CYN154806 and mimicked by the SST2 receptor agonist octreotide. Adipocytes expressed adiponectin, beta3-AR, and SST. TH-positive fibers were in close proximity to adipocytes. Submucous neurons expressed SST2 receptors. Human primary adipocytes released SST in response to GW427353 in a concentration-dependent manner, an effect abolished by SR-59230. CONCLUSIONS: Inhibitory action of GW427353 involves release of SST which stimulates inhibitory SST2 receptors on human submucous neurons. Adipocytes are a potential source for SST. beta3-AR activation may be a promising approach to reduce enteric neuron hyperexcitability. The action of GW427353 may be the neurophysiologic correlate of its beneficial effect in patients with irritable bowel syndrome.

New insights into the function of the iron deficiency-induced protein C from Synechococcus elongatus PCC 7942.

Iron limitation has a strong impact on electron transport reactions of the unicellular fresh water cyanobacterium Synechococcus elongatus PCC 7942 (thereafter referred to as S. elongatus). Among the various adaptational processes on different cellular levels, iron limitation induces a strongly enhanced expression of IdiC (iron-deficiency-induced protein C). In this article, we show that IdiC is loosely attached to the thylakoid and to the cytoplasmic membranes and that its expression is enhanced during conditions of iron starvation and during the late growth phase. The intracellular IdiC level was even more increased when additional iron was replenished in the late growth phase. On the basis of its amino acid sequence and of its absorbance spectrum, IdiC can be classified as a member of the family of thioredoxin (TRX)-like (2Fe-2S) ferredoxins. The presence of an iron cofactor in IdiC was detected by inductive coupled plasma optical emission spectrometry (ICP-OES). Comparative measurements of electron transport activities of S. elongatus wild type (WT) and an IdiC-merodiploid mutant called MuD, which contained a strongly reduced IdiC content under iron-sufficient as well as iron-deficient growth conditions, were performed. The results revealed that MuD had a strongly increased light sensitivity, especially under iron limitation. The measurements of photosystem II (PS II)-mediated electron transport rates in WT and MuD strain showed that PS II activity was significantly lower in MuD than in the WT strain. Moreover, P(700) (+) re-reduction rates provided evidence that the respiratory activities, which were very low in the MuD strain in the presence of iron, significantly increased in iron-starved cells. Thus, an increase in respiration may compensate for the drastic decrease of photosynthetic electron transport activity in MuD grown under iron starvation. Based on the similarity of the S. elongatus IdiC to the NuoE subunit of the NDH-1 complex in Escherichia coli, it is likely that IdiC has a function in the electron transport processes from NAD(P)H to the plastoquinone pool. This is in agreement with the up-regulation of IdiC in the late growth phase as well as under stress conditions when PS II is damaged. As absence or high reduction of the IdiC level would prevent or reduce the formation of functional NDH-1 complexes, under such conditions electron transport routes via alternative substrate dehydrogenases, donating electrons to the plastoquinone pool, can be assumed to be up-regulated.

Fast synaptic excitatory neurotransmission in the human submucosal plexus.

BACKGROUND: Acetylcholine is the main excitatory neurotransmitter in the enteric nervous system (ENS) in all animal models examined so far. However, data for the human ENS is scarce. METHODS: We used neuroimaging using voltage and calcium dyes, Ussing chamber, and immunohistochemistry to study fast synaptic neurotransmission in submucosal plexus neurons of the human gut. KEY RESULTS: Electrical stimulation of intraganglionic fiber tracts led to fast excitatory postsynaptic potentials (fEPSPs) in 29 submucosal neurons which were all blocked by the nicotinic antagonist hexamethonium. The nicotinic agonist DMPP mimicked the effects of electrical stimulation and had excitatory effects on 56 of 73 neurons. The unselective NMDA antagonist MK-801 blocked fEPSPs in 14 out of 22 neurons as well as nicotine evoked spike discharge. In contrast, the application of NMDA showed only weak effects on excitability or calcium transients. This agreed with the finding that the specific NMDA antagonist D-APV reduced fEPSPs in only 1 out of 40 neurons. Application of AMPA or kainite had no effect in 41 neurons or evoked spike discharge in only one out of 41 neurons, respectively. Immunohistochemistry showed that 98.7 ± 2.4% of all submucosal neurons (n = 6 preparations, 1003 neurons) stained positive for the nicotinic receptor (α1 , α2 or α3 -subunit). Hexamethonium (200 µM) reduced nerve-evoked chloride secretion by 34.3 ± 18.6% (n = 14 patients), whereas D-APV had no effect. CONCLUSION & INFERENCE: Acetylcholine is the most important mediator of fast excitatory postsynaptic transmission in human submucous plexus neurons whereas glutamatergic fEPSPs were rarely encountered.

Activation of human enteric neurons by supernatants of colonic biopsy specimens from patients with irritable bowel syndrome.

BACKGROUND & AIMS: Pathological features in irritable bowel syndrome (IBS) include alterations in mucosal cell content and mediator release that might alter signaling to nearby submucosal neurons. METHODS: Voltage sensitive dye imaging was used to record the effects of mediators, released from mucosal biopsies of IBS patients, on cell bodies of 1207 submucosal neurons from 76 human colonic tissue specimens. Supernatants, containing these mediators, were collected following incubation with colonic mucosal biopsies from 7 patients with diarrhea-predominant IBS (D-IBS), 4 with constipation-predominant IBS (C-IBS), and 4 healthy controls. Serotonin, histamine and tryptase concentrations in supernatants and lamina propria mast cell density were determined. RESULTS: In contrast to controls, IBS supernatants significantly increased the rate of spike discharge in 58% of human submucosal neurons. Neurons that responded to IBS supernatant had a median spike frequency of 2.4 Hz compared to 0 Hz for control supernatants. Supernatants from C-IBS and D-IBS evoked similar spike discharge. The activation induced by IBS supernatants was inhibited by histamine receptor (H1-H3) antagonists, 5-HT3 receptor antagonist, and protease inhibition. Serotonin, histamine and tryptase levels in supernatants correlated with the spike discharge induced by the supernatants. Mast cells density as well as histamine and tryptase levels in supernatants were higher in IBS than in controls. CONCLUSIONS: Mediators released from mucosal biopsies of IBS patients can activate human submucosal neurons. The activation required histamine, serotonin and proteases but was not associated with IBS subtype. Altered signaling between mucosa and the enteric nervous system might be involved in IBS pathogenesis.

Peppermint and caraway oils have muscle inhibitory and pro-secretory activity in the human intestine in vitro.

BACKGROUND: Herbal medicinal products with a broad activity spectrum may be promising alternatives to treat functional gastrointestinal disorders (FGD). Menthacarin® is a drug with a fixed combination of peppermint and caraway oils, which is clinically used to treat FGD-associated symptoms. MATERIALS: We studied the effects of peppermint and caraway oils on contractile and secretory activity in 255 human small and large intestinal preparations derived from surgical resections (73 patients). Motility was recorded in circular smooth muscle strips and secretion with the Ussing chamber-voltage clamp technique. Electrical field stimulation evoked nerve induced contractile responses. KEY RESULTS: Peppermint and caraway oil concentrations dependently inhibited muscle contractility as indicated by sustained muscle relaxation and decrease in phasic contractility. These effects occurred in small and large intestinal preparations with IC50 values ranging between 17 and 90 µg/mL for peppermint oil and between 7 and 127 µg/mL for caraway oil. Neither peppermint nor caraway oil influenced the nerve evoked contractile response. The inhibition of contractile activity, but not the muscle relaxation, was prevented by the L-type calcium channel activator Bay K8644 but not by the neurotoxin tetrodotoxin. Both peppermint oil and caraway oil increased epithelial secretion, which remained in tetrodotoxin. CONCLUSION & INTERFERENCE: The findings revealed a strong muscle inhibitory and pro-secretory action of peppermint and caraway oils at clinically relevant concentrations. Both actions were nerve-independent. The inhibition of contractility was mediated by inhibition of L-type calcium channels. The effects on muscle and epithelial activity may contribute to the beneficial effects observed in patients with FGD.

Compression and stretch sensitive submucosal neurons of the porcine and human colon.

The pig is commonly believed to be a relevant model for human gut functions-however, there are only a few comparative studies and none on neural control mechanisms. To address this lack we identified as one central aspect mechanosensitive enteric neurons (MEN) in porcine and human colon. We used neuroimaging techniques to record responses to tensile or compressive forces in submucous neurons. Compression and stretch caused Ca-transients and immediate spike discharge in 5-11% of porcine and 15-24% of human enteric neurons. The majority of these MEN exclusively responded to either stimulus quality but about 9% responded to both. Most of the MEN expressed choline acetyltransferase and substance P; nitric oxide synthase-positive MEN primarily occurred in distal colon. The findings reveal common features of MEN in human and pig colon which we interpret as a result of species-independent evolutionary conservation rather than a specific functional proximity between the two species.

Detection of an L-amino acid dehydrogenase activity in Synechocystis sp. PCC 6803.

The protein Slr0782 from Synechocystis sp. PCC 6803, which has similarity to L-amino acid oxidase from Synechococcus elongatus PCC 6301 and PCC 7942, has been characterized in part. Immunoblot blot analysis showed that Slr0782 is mainly thylakoid membrane-associated. Moreover, expression of slr0782 mRNA and Slr0782 protein were analyzed and an activity assay was developed. Utilizing toluene-permeabilized cells, an L-arginine-stimulated O(2) uptake became detectable in Synechocystis sp. PCC 6803. Besides oxidizing the basic L-amino acids L-arginine, L-lysine, L-ornithine, and L-histidine, a number of other L-amino acids were also substrates, while D-amino acids were not. The best substrate was L-cysteine, and the second best was L-arginine. The L-arginine-stimulated O(2) uptake was inhibited by cations. The inhibition by o-phenanthroline and salicylhydroxamic acid suggested the presence of a transition metal besides FAD in the enzyme. Moreover, it is shown that inhibitors of the respiratory electron transport chain, such as KCN and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, also inhibited the L-arginine-stimulated O(2) uptake, suggesting that Slr0782 functions as an L-arginine dehydrogenase, mediating electron transfer from L-arginine into the respiratory electron transport chain utilizing O(2) as electron acceptor via cytochrome oxidase. The results imply that Slr0782 is an additional substrate dehydrogenase being able to interact with the electron transport chain of the thylakoid membrane.

The hybrid histidine kinase Slr1759 of the cyanobacterium Synechocystis sp. PCC 6803 contains FAD at its PAS domain.

The cyanobacterium Synechocystis sp. PCC 6803 harbours 47 histidine kinases (Hiks). Among these are hybrid histidine kinases with one or two response regulator domains as well as numerous Hiks with several sensory domains. One example is the hybrid histidine kinase Slr1759 (Hik14) that has two PAS domains arranged in tandem linked to a predicted GAF domain. Here, we show that a Slr1759 derivative recombinantly expressed in Escherichia coli has a flavin cofactor. Using truncated Slr1759 variants, it is shown that the flavin associates with the first PAS domain. The cofactor reconstitutes the activity of D: -amino acid oxidase apoprotein from pig kidney, indicating that the flavin derivative is FAD. Furthermore, the Slr1759 histidine kinase domain indeed undergoes autophosphorylation in vitro. The phosphorylated product of a recombinant Slr1759 derivative is sensitive to acids, pointing to a histidine residue as the phosphate-accepting group.

Structural response of Photosystem 2 to iron deficiency: characterization of a new photosystem 2-IdiA complex from the cyanobacterium Thermosynechococcus elongatus BP-1.

Iron deficiency triggers various processes in cyanobacterial cells of which the synthesis of an additional antenna system (IsiA) around photosystem (PS) 1 is well documented [T.S. Bibby, J. Nield, J. Barber, Iron deficiency induces the formation of an antenna ring around trimeric photosystem I in cyanobacteria, Nature 412 (2001) 743-745, E.J. Boekema, A. Hifney, A.E. Yakushevska, M. Piotrowski, W. Keegstra, S. Berry, K.P. Michel, E.K. Pistorius, J. Kruip, A giant chlorophyll-protein complex induced by iron deficiency in cyanobacteria, Nature 412 (2001) 745-748]. Here we show that PS2 also undergoes prominent structural changes upon iron deficiency: Prerequisite is the isolation and purification of a PS2-IdiA complex which is exclusively synthesized under these conditions. Immunoblotting in combination with size exclusion chromatography shows that IdiA is only bound to dimeric PS2. Using single particle analysis of negatively stained specimens, IdiA can be localized in averaged electron micrographs on top of the CP43 subunit facing the cytoplasmic side in a model derived from the known 3D structure of PS2 [B. Loll, J. Kern, W. Saenger, A. Zouni, J. Biesiadka, Towards complete cofactor arrangement in the 3.0 A resolution structure of photosystem II, Nature 438 (2005) 1040-4]. The presence of IdiA as integral part of PS2 is the first example of a new PS2 protein being expressed under stress conditions, which is missing in highly purified PS2 complexes isolated from iron-sufficient cells.

Transcript profiling indicates that the absence of PsbO affects the coordination of C and N metabolism in Synechocystis sp. PCC 6803.

Transcript profiling of nitrate-grown Synechocystis sp. PCC 6803 PsbO-free mutant cells in comparison to wild-type (WT) detected substantial deviations. Because we had previously observed phenotypical differences between Synechocystis sp. PCC 6803 WT and its corresponding PsbO-free mutant when cultivated with l-arginine as sole N source and a light intensity of 200 mumol photons m(-2) s(-1), we also performed transcript profiling for both strains grown either with nitrate or with l-arginine as sole N source. We observed a total number of 520 differentially regulated transcripts in Synechocystis WT because of a shift from nitrate- to l-arginine-containing BG11 medium, while we detected only 13 differentially regulated transcripts for the PsbO-free mutant. Thus, the PsbO-free Synechocystis mutant had already undergone a preconditioning process for growth with l-arginine in comparison to WT. While Synechocystis WT suffered from growth with l-arginine at a light intensity of 200 mumol photons m(-2) s(-1), the PsbO-free mutant developed only a minor stress phenotype. In summary, our results suggest that the absence of PsbO in Synechocystis affects the coordination of photosynthesis/respiration and l-arginine metabolism through complex probably redox-mediated regulatory pathways. In addition, we show that a comparison of the transcriptomes of nitrate-grown Synechococcus elongatus PCC 7942 WT cells and its corresponding PsbO-free mutant cells resulted in only a few differentially regulated transcripts between both strains. The absence of the manganese/calcium-stabilizing PsbO protein of PSII with an assigned regulatory function for photosynthetic water oxidation causes bigger changes in the transcriptome of the permissive photoheterotrophically growing Synechocystis sp. PCC 6803 than in the transcriptome of the obligate photoautotrophically growing S. elongatus PCC 7942.