The contribution of gut bacterial metabolites in the human immune signaling pathway of non-communicable diseases.

The interaction disorder between gut microbiota and its host has been documented in different non-communicable diseases (NCDs) such as metabolic syndrome, neurodegenerative disease, and autoimmune disease. The majority of these altered interactions arise through metabolic cross-talk between gut microbiota and host immune system, inducing a low-grade chronic inflammation that characterizes all NCDs. In this review, we discuss the contribution of bacterial metabolites to immune signaling pathways involved in NCDs. We then review recent advances that aid to rationally design microbial therapeutics. A deeper understanding of these intersections between host and gut microbiota metabolism using metabolomics-based system biology platform promises to reveal the fundamental mechanisms that drive metabolic predispositions to disease and suggest new avenues to use microbial therapeutic opportunities for NCDs treatment and prevention. Abbreviations: NCDs: non-communicable disease, IBD: inflammatory bowel disease, IL: interleukin, T2D: type 2 diabetes, SCFAs: short-chain fatty acids, HDAC: histone deacetylases, GPCR: G-protein coupled receptors, 5-HT: 5-hydroxytryptamine receptor signaling, DCs: dendritic cells, IECs: intestinal epithelial cells, T-reg: T regulatory cell, NF-κB: nuclear factor κB, TNF-α: tumor necrosis factor alpha, Th: T helper cell, CNS: central nervous system, ECs: enterochromaffin cells, NSAIDs: non-steroidal anti-inflammatory drugs, AhR: aryl hydrocarbon receptor, IDO: indoleamine 2,3-dioxygenase, QUIN: quinolinic acid, PC: phosphatidylcholine, TMA: trimethylamine, TMAO: trimethylamine N-oxide, CVD: cardiovascular disease, NASH: nonalcoholic steatohepatitis, BAs: bile acids, FXR: farnesoid X receptor, CDCA: chenodeoxycholic acid, DCA: deoxycholic acid, LCA: lithocholic acid, UDCA: ursodeoxycholic acid, CB: cannabinoid receptor, COBRA: constraint-based reconstruction and analysis.

Laser Wakefield Driven Generation of Isolated Carrier-Envelope-Phase Tunable Intense Subcycle Pulses.

Sources of intense, ultrashort electromagnetic pulses enable applications such as attosecond pulse generation, control of electron motion in solids, and the observation of reaction dynamics at the electronic level. For such applications, both high intensity and carrier-envelope-phase (CEP) tunability are beneficial, yet hard to obtain with current methods. In this Letter, we present a new scheme for generation of isolated CEP tunable intense subcycle pulses with central frequencies that range from the midinfrared to the ultraviolet. It utilizes an intense laser pulse that drives a wake in a plasma, copropagating with a long-wavelength seed pulse. The moving electron density spike of the wake amplifies the seed and forms a subcycle pulse. Controlling the CEP of the seed pulse or the delay between driver and seed leads to CEP tunability, while frequency tunability can be achieved by adjusting the laser and plasma parameters. Our 2D and 3D particle-in-cell simulations predict laser-to-subcycle-pulse conversion efficiencies up to 1%, resulting in relativistically intense subcycle pulses.

Optical Bloch modeling of femtosecond-laser-induced electron dynamics in dielectrics.

A model based on optical Bloch equations is developed to describe the interaction of femtosecond laser pulses with dielectric solids, accounting for optical-cycle-resolved electron dynamics. It includes the main physical processes at play: photoionization, impact ionization, direct and collisional laser heating, and recombination. By using an electron band structure, this approach also accounts for material optical properties as nonlinear polarization response. Various studies are performed, shedding light on the contribution of various processes to the full electron dynamics depending on laser intensity and wavelength. In particular, the standard influence of the impact ionization process is retrieved.

Gain of electron orbital angular momentum in a direct laser acceleration process.

Three-dimensional "particle in cell" simulations show that a quasistatic magnetic field can be generated in a plasma irradiated by a linearly polarized Laguerre-Gauss beam with a nonzero orbital angular momentum (OAM). Perturbative analysis of the electron dynamics in the low intensity limit and detailed numerical analysis predict a laser to electrons OAM transfer. Plasma electrons gain angular velocity thanks to the dephasing process induced by the combined action of the ponderomotive force and the laser induced-radial oscillation. Similar to the "direct laser acceleration," where Gaussian laser beams transmit part of its axial momentum to electrons, Laguerre-Gaussian beams transfer a part of their orbital angular momentum to electrons through the dephasing process.

Electron Beam Driven Generation of Frequency-Tunable Isolated Relativistic Subcycle Pulses.

We propose a novel scheme for frequency-tunable subcycle electromagnetic pulse generation. To this end a pump electron beam is injected into an electromagnetic seed pulse as the latter is reflected by a mirror. The electron beam is shown to be able to amplify the field of the seed pulse while upshifting its central frequency and reducing its number of cycles. We demonstrate the amplification by means of 1D and 2D particle-in-cell simulations. In order to explain and optimize the process, a model based on fluid theory is proposed. We estimate that using currently available electron beams and terahertz pulse sources, our scheme is able to produce millijoule-strong midinfrared subcycle pulses.

Wavelength scaling of terahertz pulse energies delivered by two-color air plasmas.

We address the long-standing problem of anomalous growth observed in the terahertz (THz) energy yield from air plasmas created by two-color laser pulses, as the fundamental wavelength λ0 is increased. Using two distinct optical parametric amplifiers (OPAs), we report THz energies scaling like λ0α with large exponents 5.6≤α≤14.3, which departs from the growth in λ02 expected from photocurrent theory. By means of comprehensive 3D simulations, we demonstrate that the changes in the laser beam size, pulse duration, and phase-matching conditions in the second-harmonic generation process when tuning the OPA's carrier wavelength can lead to these high scaling powers. The value of the phase angle between the two colors reached at the exit of the doubling crystal turns out to be crucial and even explains non-monotonic behaviors in the measurements.

Spectral dynamics of THz pulses generated by two-color laser filaments in air: the role of Kerr nonlinearities and pump wavelength.

We theoretically and numerically study the influence of both instantaneous and Raman-delayed Kerr nonlinearities as well as a long-wavelength pump in the terahertz (THz) emissions produced by two-color femtosecond filaments in air. Although the Raman-delayed nonlinearity induced by air molecules weakens THz generation, four-wave mixing is found to impact the THz spectra accumulated upon propagation via self-, cross-phase modulations and self-steepening. Besides, using the local current theory, we show that the scaling of laser-to-THz conversion efficiency with the fundamental laser wavelength strongly depends on the relative phase between the two colors, the pulse duration and shape, rendering a universal scaling law impossible. Scaling laws in powers of the pump wavelength may only provide a rough estimate of the increase in the THz yield. We confront these results with comprehensive numerical simulations of strongly focused pulses and of filaments propagating over meter-range distances.

Theory of terahertz emission from femtosecond-laser-induced microplasmas.

We present a theoretical investigation of terahertz (THz) generation in laser-induced gas plasmas. The work is strongly motivated by recent experimental results on microplasmas, but our general findings are not limited to such a configuration. The electrons and ions are created by tunnel ionization of neutral atoms, and the resulting plasma is heated by collisions. Electrons are driven by electromagnetic, convective, and diffusive sources and produce a macroscopic current which is responsible for THz emission. The model naturally includes both ionization current and transition-Cherenkov mechanisms for THz emission, which are usually investigated separately in the literature. The latter mechanism is shown to dominate for single-color multicycle laser pulses, where the observed THz radiation originates from longitudinal electron currents. However, we find that the often discussed oscillations at the plasma frequency do not contribute to the THz emission spectrum. In order to predict the scaling of the conversion efficiency with pulse energy and focusing conditions, we propose a simplified description that is in excellent agreement with rigorous particle-in-cell simulations.

Mass conserved elementary kinetics is sufficient for the existence of a non-equilibrium steady state concentration.

Living systems are forced away from thermodynamic equilibrium by exchange of mass and energy with their environment. In order to model a biochemical reaction network in a non-equilibrium state one requires a mathematical formulation to mimic this forcing. We provide a general formulation to force an arbitrary large kinetic model in a manner that is still consistent with the existence of a non-equilibrium steady state. We can guarantee the existence of a non-equilibrium steady state assuming only two conditions; that every reaction is mass balanced and that continuous kinetic reaction rate laws never lead to a negative molecule concentration. These conditions can be verified in polynomial time and are flexible enough to permit one to force a system away from equilibrium. With expository biochemical examples we show how reversible, mass balanced perpetual reaction(s), with thermodynamically infeasible kinetic parameters, can be used to perpetually force various kinetic models in a manner consistent with the existence of a steady state. Easily testable existence conditions are foundational for efforts to reliably compute non-equilibrium steady states in genome-scale biochemical kinetic models.

Quantitative assignment of reaction directionality in a multicompartmental human metabolic reconstruction.

Reaction directionality is a key constraint in the modeling of genome-scale metabolic networks. We thermodynamically constrained reaction directionality in a multicompartmental genome-scale model of human metabolism, Recon 1, by calculating, in vivo, standard transformed reaction Gibbs energy as a function of compartment-specific pH, electrical potential, and ionic strength. We show that compartmental pH is an important determinant of thermodynamically determined reaction directionality. The effects of pH on transport reaction thermodynamics are only seen to their full extent when metabolites are represented as pseudoisomer groups of multiple protonated species. We accurately predict the irreversibility of 387 reactions, with detailed propagation of uncertainty in input data, and manually curate the literature to resolve conflicting directionality assignments. In at least half of all cases, a prediction of a reversible reaction directionality is due to the paucity of compartment-specific quantitative metabolomic data, with remaining cases due to uncertainty in estimation of standard reaction Gibbs energy. This study points to the pressing need for 1), quantitative metabolomic data, and 2), experimental measurement of thermochemical properties for human metabolites.

Efficacy of topically delivered moxifloxacin against wound infection by Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus.

Wound infection is a common risk for patients with chronic nonhealing wounds, causing high morbidity and mortality. Currently, systemic antibiotic treatment is the therapy of choice, despite often leading to several side effects and the risk of an insufficient tissue penetration due to impaired blood supply. If systemically delivered, moxifloxacin penetrates well into inflammatory blister fluid, muscle, and subcutaneous adipose tissues and might therefore be a possible option for the topical treatment of skin and infected skin wounds. In this study, topical application of moxifloxacin was investigated in comparison to mupirocin, linezolid, and gentamicin using a porcine wound infection and a rat burn infection model. Both animal models were performed either by an inoculation with methicillin-resistant Staphylococcus aureus (MRSA) or Pseudomonas aeruginosa. Wound fluid, tissue, and blood samples were taken, and bacterial counts as well as the moxifloxacin concentration were determined for a 14-day follow-up. A histological comparison of the rat burn wound tissues was performed. Both strains were susceptible to moxifloxacin and gentamicin, whereas mupirocin and linezolid were effective only against MRSA. All antibiotics showed efficient reduction of bacterial counts, and except with MRSA, infected burn wounds reached bacterial counts below 10(5) CFU/g tissue. Additionally, moxifloxacin was observed to promote wound healing as determined by histologic analysis, while no induction of bacterial resistance was observed during the treatment period. The use of topical antibiotics for the treatment of infected wounds confers many benefits. Moxifloxacin is therefore an ideal candidate, due to its broad antibacterial spectrum, its high efficiency, and its potential to promote wound healing.

Integrated stoichiometric, thermodynamic and kinetic modelling of steady state metabolism.

The quantitative analysis of biochemical reactions and metabolites is at frontier of biological sciences. The recent availability of high-throughput technology data sets in biology has paved the way for new modelling approaches at various levels of complexity including the metabolome of a cell or an organism. Understanding the metabolism of a single cell and multi-cell organism will provide the knowledge for the rational design of growth conditions to produce commercially valuable reagents in biotechnology. Here, we demonstrate how equations representing steady state mass conservation, energy conservation, the second law of thermodynamics, and reversible enzyme kinetics can be formulated as a single system of linear equalities and inequalities, in addition to linear equalities on exponential variables. Even though the feasible set is non-convex, the reformulation is exact and amenable to large-scale numerical analysis, a prerequisite for computationally feasible genome scale modelling. Integrating flux, concentration and kinetic variables in a unified constraint-based formulation is aimed at increasing the quantitative predictive capacity of flux balance analysis. Incorporation of experimental and theoretical bounds on thermodynamic and kinetic variables ensures that the predicted steady state fluxes are both thermodynamically and biochemically feasible. The resulting in silico predictions are tested against fluxomic data for central metabolism in Escherichia coli and compare favourably with in silico prediction by flux balance analysis.

Quantitative assignment of reaction directionality in constraint-based models of metabolism: application to Escherichia coli.

Constraint-based modeling is an approach for quantitative prediction of net reaction flux in genome-scale biochemical networks. In vivo, the second law of thermodynamics requires that net macroscopic flux be forward, when the transformed reaction Gibbs energy is negative. We calculate the latter by using (i) group contribution estimates of metabolite species Gibbs energy, combined with (ii) experimentally measured equilibrium constants. In an application to a genome-scale stoichiometric model of Escherichia coli metabolism, iAF1260, we demonstrate that quantitative prediction of reaction directionality is increased in scope and accuracy by integration of both data sources, transformed appropriately to in vivo pH, temperature and ionic strength. Comparison of quantitative versus qualitative assignment of reaction directionality in iAF1260, assuming an accommodating reactant concentration range of 0.02-20mM, revealed that quantitative assignment leads to a low false positive, but high false negative, prediction of effectively irreversible reactions. The latter is partly due to the uncertainty associated with group contribution estimates. We also uncovered evidence that the high intracellular concentration of glutamate in E. coli may be essential to direct otherwise thermodynamically unfavorable essential reactions, such as the leucine transaminase reaction, in an anabolic direction.

Characterisation of the rat SK4/IK1 K(+) channel.

BACKGROUND AND AIMS: The Ca(2+)-activated K(+) channel rSK4 is the rat homologue of the human SK4/IK1 (KCNN4) channel. In colonic mucosa rSK4 plays a key role during acetylcholin-induced secretion. This study was aimed to characterize the properties of the rat SK4 channel. METHODS: Electrophysiological measurements were performed on rSK4 expressing Xenopus laevis oocytes and rat colonic crypts. Intracellular Ca(2+) activity was assessed by Oregon Green fluorescence measurements. RESULTS: The 10 pS rSK4 expressed in oocytes was Ca(2+)-sensitive and inhibited by calmodulin antagonists. 1-ethyl-2-benzimidazolinone (1-EBIO), a known activator of SK4/IK1 channels, also activated rSK4. 1-EBIO affected the current neither at saturating Ca(2+) activities nor under Ca(2+)-free conditions, but increased the Ca(2+) sensitivity of rSK4. rSK4 was strongly activated by cytosolic ATP. However, PKA itself, PKA inhibitors and mutation of the PKA phosphorylation site (S332A) did not affect channel activity. The PKC activator 1,2-dioctanoyl-sn-glycerol and the PKC inhibitor bisindolylmaleimide also failed to influence rSK4. CONCLUSION: The Ca(2+)-sensitive rSK4 is activated by 1-EBIO probably via facilitation of the Ca(2+)-calmodulin-rSK4 interaction. The strong ATP-activation of rSK4 is likely to be caused by phosphorylation via a yet unknown kinase and might involve additional subunits.

The cellular mechanisms of Cl- secretion induced by C-type natriuretic peptide (CNP). Experiments on isolated in vitro perfused rectal gland tubules of Squalus acanthias.

We have examined the mechanism whereby C-type natriuretic peptide (CNP), an agonist acting through the second messenger cGMP, enhances NaCl secretion in the rectal gland of Squalus acanthias. Single rectal gland tubules (RGT) were dissected manually, perfused in vitro and equivalent short-circuit current [Isc=transepithelial voltage/transepithelial resistance (Rte)] as well as basolateral membrane voltage (Vbl) were measured. CNP was added to luminal and basolateral perfusates at concentrations between 1 and 1000 nmol/l and its effects on the above parameters were compared to those of a "stimulation cocktail" (Stim, containing dibutyryl cAMP, adenosine and forskolin) that maximally enhances cytosolic cAMP, and other agonists and hormones such as guanylin, vasoactive intestinal peptide (VIP), and adenosine. CNP had no effect from the luminal side (n=6). Its effects from the basolateral side consisted of a substantial increase in Isc (-31.6+/-7.7 to -316+/-82.2 microA/cm2, n=15). CNP significantly depolarized the luminal membrane from -87. 4+/-1.0 to -82.3+/-2.6 mV (n=12). Vbl was not changed (n=12) but the fractional conductance for K+ was increased (n=3). These effects were qualitatively and even quantitatively comparable to those of other agonists acting via cytosolic cAMP, but were less marked than those caused by Stim (n=64). The effects of VIP and CNP on Isc were not additive (n=5). The cytosolic Ca2+ concentration ([Ca2+]i) was monitored using the fura-2 fluorescence ratio (FFR 340/380 nm) and it was found that CNP, like agonists acting via cAMP, enhances FFR significantly from 1.02+/-0.05 to 1.32+/-0.05 (n=8) with a time constant in the 1-2 min in range. Our data suggest that CNP, acting via the second messenger cGMP, induces a marked increase in Isc in the rectal gland. The concomitant fall in Rte corresponds to increases in the luminal membrane Cl- conductance and in the basolateral membrane K+ conductance. The latter effect is probably due to an increase in [Ca2+]i.

Osmotically induced conductance and capacitance changes in in vitro perfused rectal gland tubules of Squalus acanthias.

The rectal gland of Squalus acanthias is critically involved in the homeostasis of NaCl and water metabolism and hence in overall osmoregulation. In the present study, we have examined the acute responses of rectal gland slices and in vitro perfused rectal gland tubule (RGT) cells to the exposure to dilute and hypertonic peritubule solutions. Five series were performed. (i) With changes in osmolality, Western blots to monitor tyrosine, threonine and serine phosphorylation in rectal gland slices did not reveal clear-cut changes in phosphorylation patterns. All other series were performed in in vitro perfused RGT. (ii) Relative cell volume was estimated by fura-2 fluorescence using the emission at the isosbestic excitation wavelength of 360 nm. Hypotonic solution (-100 mmol/l NaCl) reduced fura-2 fluorescence by 16% and hypertonic solution (+100 mmol/l NaCl) had the opposite effect (+12%). (iii) Transepithelial resistance was increased markedly by hypotonic solution, probably by cell swelling, and the opposite was seen with hypertonic solutions. (iv) Whole-cell patch clamp experiments indicated that hypotonic solution hyperpolarized the cells, and increased membrane conductance and membrane capacitance. The latter two changes correlated significantly with each other. Hypertonic solution had the opposite effect. (v) Measurements of the fura-2 fluorescence ratio (340/380 nm) revealed that hypotonic solution (-NaCl) increased cytosolic Ca2+ activtiy ([Ca2+]i). Hypertonic solution had no detectable effect on [Ca2+]i. These data indicate that RGT cells are swollen by removal of NaCl from the bath solution. This causes an increase in [Ca2+]i and a predominant increase in K+ conductance and hyperpolarization. Urea apparently permeates these cells quite well and its addition (+U) or its removal (-U) had only moderate osmotic effects. The removal of urea and replacement by mannitol produced effects similar to those seen with hypertonic NaCl solution.

Regulation of the Na+2Cl­K+ cotransporter in in vitro perfused rectal gland tubules of Squalus acanthias.

Previously it has been shown that the Na+2Cl­K+ cotransporter accepts NH4 + at its K+ binding site. This property can be used to estimate its transport rates by adding NH4 + to the bath and measuring the initial furosemide-dependent rates of change in BCECF fluorescence. We have utilized this technique to determine the regulation of the furosemide-inhibitable Na+2Cl­K+ cotransporter in in vitroperfused rectal gland tubules (RGT) of Squalus acanthias. Addition of NH4 + to the bath (20 mmol/l) led to an initial alkalinization, corresponding to NH3 uptake. This was followed by an acidification, corresponding to NH4 + uptake. The rate of this uptake was quantified by exponential curve fitting and is given in arbitrary units (Δfluorescence/time). This acidification could be completely inhibited by furosemide. In the absence of any secretagogue preincubation of RGT in a low Cl­ solution (6 mmol/l, low Cl­) for 10 min enhanced the uptake rate significantly from 4.04±0.51 to 12.7±1.30 (n=5). The addition of urea (200 mmol/l) was without effect, but the addition of 300 mmol/l mannitol (+300 mannitol) enhanced the rate significantly from 7.24±1.33 to 14.7±4.6 (n=6). Stimulation of NaCl secretion by a solution maximizing the cytosolic cAMP concentration (Stim) led to a significant increase in NH4 + uptake rate from 5.00±1.33 to 13.3±1.54 (n=6). Similar results were obtained in the additional presence of Ba2+ (1 mmol/l): the uptake rate was increased significantly from 4.23±0.34 to 15.1±1.86 (n=16). In the presence of Stim low Cl­ had no additional effect on the uptake rate: 15.1±3.1 versus 15.2±2.8 in high Cl­ (n=6). The uptake rate in Stim containing additional +300 mannitol (22.3±4.0, n=5) was not significantly different from that obtained with Stim or +300 mannitol alone. By whatever mechanism the NH4 + uptake rate was increased furosemide (500 µmol/l) always reduced this rate to control values. Hence three manoeuvres enhanced furosemide-inhibitable uptake rates of the Na+2Cl­K+ cotransporter probably independently: (1) lowering of cytosolic Cl­ concentration; (2) cell shrinkage; and (3) activation by cAMP.

Does stimulation of NaCl secretion in in vitro perfused rectal gland tubules of Squalus acanthias increase membrane capacitance?

NaCl secretion in rectal gland tubules (RGT) of Squalus acanthias requires the activation of Cl­ channels in the luminal membrane. The RGT and its mechanism of activation are an early evolutionary paradigm of exocrine secretion. The respective Cl­ channels probably resemble the shark equivalent of the cystic fibrosis transmembrane conductance regulator (CFTR). Activation of these Cl­ channels occurs via cAMP. It has been hypothesized that the activation of CFTR occurs via exocytosis or inhibited endocytosis. To examine this question directly by electrical measurements we have performed whole-cell patch-clamp analyses of in vitro perfused RGT. NaCl secretion was stimulated by a solution (Stim) containing forskolin (10 µmol/l), dibutyryl-cAMP (0.5 mmol/l) and adenosine (0.5 mmol/l). This led to the expected strong depolarization and an increase in membrane conductance (G m). The membrane capacitance (C m) was measured by a newly devised two-frequency synchronous detector method. It was increased by Stim significantly from 5.00±0.22 to 5.17±0.21 pF (n=50). The increase in C m correlated with the increase in G m with a slope of 51 fF/nS. Next the effect of furosemide (500 µmol/l) was examined in previously stimulated RGT. Furosemide was supposed to inhibit coupled Na+2Cl­K+ uptake and to reduce cell volume but not membrane trafficking of Cl­ channels. Furosemide reduced G m slightly (due to the fall in cytosolic Cl­ concentration) and C m to the same extent by which Stim had increased it. Both changes were statistically significant, and the slope of ΔC m/ΔG m was similar to that caused by Stim. Inhibitors of microtubules or actin (colchicine, phalloidin and cytochalasin D added at 10 µmol/l to the pipette solution and dialysed for >10 min) did not alter cell voltage, G m or C m, nor did these inhibitors abolish the stimulatory effect of cAMP. These data suggest that the small C m changes observed with Stim reflect a minor cell volume increase and an "unfolding" of the plasma membrane. The present data do not support the exocytosis/endocytosis hypothesis of cAMP-mediated activation of Cl­ channels in these cells.

The role of cytosolic Ca2+ in the secretion of NaCl in isolated in vitro perfused rectal gland tubules of Squalus acanthias.

In many exocrine glands cytosolic Ca2+ ([Ca2+]i) plays a pivotal role in stimulation-secretion coupling. In the rectal gland of the dogfish Squalus acanthias this appears not to be the case and it is believed that secretion is mainly controlled by the Cl- conductance of the luminal membrane. We have examined this question in a study of isolated in vitro perfused rectal gland tubules (RGT). Three types of measurements were performed: (1) measurements of [Ca2+]i by the fura-2 technique; (2) measurements of transepithelial electrical parameters, i.e. transepithelial voltage (Vte), transepithelial resistance (Rte), the equivalent short-circuit current (Isc) and the voltage across the basolateral membrane (Vbl), and (3) whole-cell patch-clamp measurements of cellular voltage (Vm), conductance (Gm) and membrane capacitance (Cm). The data indicates that carbachol (CCH) increases [Ca2+]i by increasing store release and Ca2+ influx. Other agonists, producing cytosolic cAMP, also increased [Ca2+] by enhancing Ca2+ influx. CCH hyperpolarized these cells and enhanced Gm significantly. The effect of CCH on Vte and Isc was most marked under control conditions and disappeared in RGT otherwise stimulated by agonists that lead to cAMP production. It is concluded that [Ca2+]i plays a major role in the stimulation of NaCl secretion in RGT by enhancing the basolateral K+ conductance. cAMP-producing agonists enhance [Ca2+]i by increased Ca2+ influx. CCH releases Ca2+ from respective stores. CCH, unlike the cAMP-producing agonists, only increases basolateral K+ conductance. It modulates secretion especially under conditions in which the cAMP pathway is not fully activated.

pH-regulatory mechanisms in in vitro perfused rectal gland tubules of Squalus acanthias.

Isolated in vitro perfused rectal gland tubules (RGT) were preincubated with the pH-sensitive dye 2', 7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and pH-regulatory mechanisms were studied. A reduction of bath Cl- concentration from 269 to 6 mmol/l increased the fluorescence ratio 488/436 [corresponding to cytosolic pH (pHi)] slightly but significantly (n=10). Depolarization by Ba2+ (1 mmol/l) or a bath solution containing 30 mmol/l K+ (n=4-6) increased the fluorescence ratio (pHi). These data suggest that HCO3- uptake and/or H+ extrusion is dependent on Cl- and/or voltage. A reduction of bath Na+ from 278 to 5 mmol/l reduced the ratio significantly (n=3). Addition of trimethylamine (Trima+, 20 mmol/l) alkalinized cytosolic pH (n=7). Similarly, addition of NH4+ (20 mmol/l) led to an initial alkalinization and a strong acidification when NH4+ was removed (n=59). The initial pHi-recovery rates after NH4+ removal were quantified and the responsible H+ extrusion and/or HCO3- import systems were examined. The recovery was almost completely abolished when the extracellular Na+ concentration was reduced to 5 mmol/l. In the presence of normal Na+, recovery was slower in the absence as compared to the presence of HCO3- (n=5). It was inhibited by 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) (0.5 mmol/l, n=11) in the presence of HCO3- and in the absence of HCO3- by the Na+/H+-exchange blocker HOE694 (0.5 mmol/l, n=6). These data suggest that acid extrusion probably occurs by basolateral Na+-2HCO3-/Cl- exchange in the presence of HCO3- and by basolateral Na+/H+ exchange in the absence of HCO3-. Luminal perfusion with a solution containing a low Cl- concentration (6 mmol/l) increased the fluorescence ratio (pHi) (n=5). The ratio (pHi) was further increased and pH recovery further delayed by basolateral addition of Trima+ (20 mmol/l, n=3). These data suggest that the HCO3-/Cl- exchanger is present in the luminal membrane. Luminal HCO3-/Cl- exchange and basolateral Na+-2HCO3-/Cl- exchange may work in tandem to secrete HCO3- and exchange it for luminal Cl-.