Fenamates Research Articles

ChemInform Abstract: Use of N,N‐Dimethylformamide as Solvent in the Synthesis of N‐ Phenylanthranilic Acids.

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HISTAMINE‐STIMULATED PHOSPHOLIPASE C SIGNALLING IN THE ADRENAL CHROMAFFIN CELL: EFFECTS ON INOSITOL PHOSPHOLIPID METABOLISM AND TYROSINE HYDROXYLASE PHOSPHORYLATION

1. The present report gives a detailed account of histamine-stimulated phospholipase C (PLC) activity in bovine adrenal chromaffin cells. 2. Histamine activation of H1 receptors stimulates PLC with a biphasic sensitivity to extracellular Ca2+. The initial response (the first 15 s stimulation) was not reduced by the removal of extracellular Ca2+, whereas the maintenance of PLC activity beyond this time required Ca2+ influx. 3. Phospholipase C activity in response to a 10 min incubation with histamine was inhibited by La3+ (3 mmol/L) or SKF96365 (10 mumol/L). Nifedipine (10 mumol/L), but not omega-agatoxin IVA (100 nmol/L) or omega-conotoxin GVIA (300 nmol/L), produced a partial inhibition of PLC activity. The response was also partially inhibited by a reduction in the extracellular Cl- concentration (40 mmol/L) or by the inclusion of the Cl- channel blocker N-phenylanthranilic acid (300 mumol/L). 4. Kinetic analysis of the rate of turnover of the various inositol phosphate isomers in response to histamine suggested that the inositol monophosphates were being produced from a source in addition to inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) metabolism. This conclusion was supported by the differential action of pertussis toxin and neomycin on Ins(1,4,5)P3 formation compared with inositol monophosphate formation. 5. We have attempted to identify a defined role for the intracellular Ca2+ mobilized in these cells in response to histamine. After short incubations (up to 3 min), histamine was able to regulate the site-specific phosphorylation of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis. This observation has important implications for a possible role for the PLC signalling pathway in controlling the rate of catecholamine biosynthesis.

Bovine pancreatic duct cells express cAMP- and Ca(2+)-activated apical membrane Cl- conductances.

Secretion of salt and water by the epithelial cells that line pancreatic ducts depends on activation of apical membrane Cl- conductance. In the present study, we characterized two types of Cl- conductances present in the apical cell membrane of bovine pancreatic duct epithelial cells. Primary cultures of bovine main pancreatic duct epithelium and an immortalized cell line (BPD1) derived from primary cultures were used. Elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) or Ca2+ in intact monolayers of duct epithelium induced sustained anion secretion. Agonist-induced changes in plasma membrane Cl- permeability were accessed by 36 Cl- efflux, whole cell current recording, and measurements of transepithelial Cl- current across permeabilized epithelial monolayers. Elevation of intracellular cAMP elicited a sustained increase in Cl- permeability, whereas elevation of intracellular Ca2+ induced only a transient increase in Cl- permeability. Ca(2+)- but not cAMP-induced increases in Cl- permeability were abolished by preincubation of cells with the Ca2+ buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl) ester (BAPTA-AM). N-phenylanthranilic acid (DPC; 1 mM) and glibenclamide (100 microM), but not 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 500 microM), inhibited the cAMP-induced increase in Cl- permeability. In contrast, DPC and DIDS, but not glibenclamide, inhibited the Ca(2+)-induced increase in Cl- permeability. We conclude from these experiments that bovine pancreatic duct epithelial cells express at least two types of Cl- channels, cAMP and Ca2+ activated, in the apical cell membrane. Because the Ca(2+)-activated increase in Cl- permeability is transient, the extent to which this pathway contributes to sustained anion secretion by the ductal epithelium remains to be determined.

Use of N,N-dimethylformamide as solvent in the synthesis of N-phenylanthranilic acids

It is known that N-phenylanthranilic acids can be synthesized by Ullmann-Goldberg condensation in different conditions. This paper reports some parameters which influence the condensation and reports a general procedure for this reaction using N,N-Dimethylformamide as solvent.

Properties of chloride-conductive pathways in rat kidney cortical and outer-medulla brush-border membranes--inhibition by anti-(cystic fibrosis transmembrane regulator) mAbs.

The activity of the Cl(-)-conductive pathways, their regulation by protein kinase A (PKA) and their relationship to the cystic fibrosis transmembrane regulator (CFTR) protein were assessed in rat kidney cortical brush-border-membrane vesicles (cBBMV) and outer medullary vesicles (OMV) by measuring the rate of valinomycin-induced microsomal swelling by light scattering in the presence of an inward Cl- gradient. Valinomycin increased the rate of swelling of cBBMV and OMV, which is consistent with the presence of a Cl(-)-conductive pathway. PKA further increased these rates. This effect was blocked by the inhibitor of protein kinase A, suggesting that phosphorylation by PKA activates these pathways. Four anion-transport inhibitors were tested ¿N-phenylanthranilic acid (PhNHPhCOOH), 5-nitro-2-(3-phenylpropylamino)benzoic acid [N(PhPrNH2)BzOH], glybenclamide and 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid¿. Ph2COOH and 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid inhibited the basal Cl(-)-conductive pathways, while PKA-treated microsomes were sensitive also to N(PhPrNH2)BzOH and glybenclamide, suggesting that additional Cl- pathways were activated by phosphorylation. The pharmacological properties of these pathways were similar to those of the CFTR Cl- channel. Two anti-CFTR mAbs inhibited PKA-activated valinomycin-induced swelling in cBBMV and OMV, while immunoblot analysis of the corresponding proteins with the same antibodies indicated the presence of a 170-kDa protein. The results thus indicate the presence of a PKA-activated Cl(-)-conductive pathway in cBBMV and OMV, and suggest that CFTR protein is involved in PKA-activated Cl- fluxes in these vesicles.

Inhibition of thyroid hormone uptake by calcium antagonists of the dihydropyridine class.

A series of substituted 4-phenyl-1,4-dihydropyridines 2a-m was tested for their inhibitory effects on L-triiodothyronine (L-T3) uptake by human HepG2 hepatoma cells. The most potent compounds were the nitro-substituted derivatives 2,6-dimethyl-4-(4'-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid 3-ethyl ester 5-methyl ester (2m) and the well-known calcium antagonists nitrendipine (2k) and nifedipine (2j) with an uptake inhibition between 80.5 and 85.8% at an application dose of 10(-5) M. On the basis of a theoretical conformational analysis (ab initio MO theory, molecular mechanics, molecular dynamics) of the dihydropyridine derivatives, a unifying stereochemical concept was derived postulating an angular arrangement of the two rings where the phenyl ring of the calcium antagonists, which corresponds to the outer phenyl ring of the thyroid hormones, is bisecting the dihydropyridine ring as a prerequisite for inhibitory potency. This model includes also inhibitors of the N-phenylanthranilic acid type. The interaction of the calcium antagonists with transthyretin (TTR) is discussed in relation to thyroid hormones. The influence of hydrophobicity was estimated by the experimental determination of the 1-octanol/water partition coefficients.

Most basal I(SC) in Calu-3 human airway cells is bicarbonate-dependent Cl- secretion.

Serous cells secrete antibiotic-rich fluid, but secretion is impaired in cystic fibrosis. We are investigating Calu-3 cells as a serous cell model. Basal short-circuit current (I[SC]) in Calu-3 cells grown at air interface had a basal I(SC) approximately six times larger than submerged cultures (69 +/- 22 vs. 11 +/- 10 microA/cm2). Basal I(SC) in either condition was reduced only 7 +/- 5% by bumetanide and was unaffected by apical amiloride, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), or calixarene but was reduced 77 +/- 18% by N-phenylanthranilic acid. Three transport mechanisms accounted for almost all basal I(SC). The largest component is HCO3(-)-dependent Cl- secretion. Replacement of Krebs-Henseleit solution with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered solution and changing gassing from 95% O2-5% CO2 to air reduced the basal I(SC) by 61 +/- 10%. Acetazolamide decreased basal I(SC) by 33 +/- 6%, whereas acetazolamide + basolateral DNDS eliminated 42-58% of the bumetanide-insensitive basal I(SC). Neither DNDS nor acetazolamide had any effect when applied in HCO3(-)-free solution. Apical phlorizin, a blocker of Na+-glucose cotransport, eliminated one-half of the remaining I(SC). Cl- replacement with gluconate eliminated all I(SC) except the phlorizin-sensitive component. Unlike basal I(SC), 80 +/- 24% of stimulated I(SC) was inhibited by bumetanide. Thus basal and stimulated secretions are mediated by different mechanisms.

CAMP and genistein stimulate HCO3- conductance through CFTR in human airway epithelia.

We studied the role of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel as an HCO3- conductor during adenosine 3',5'-cyclic monophosphate (cAMP)-dependent regulation in human airway epithelial cell lines. HCO3- or Cl- currents across the apical membrane were measured in the presence of an HCO3- or Cl- gradient under short-circuit conditions in intact and alpha-toxin-permeabilized monolayers, which allowed manipulation of the intracellular regulators cAMP and ATP. CFTR as the current carrier for HCO3- was identified by 1) stimulation by cAMP, 2) ATP dependence, 3) blocker sensitivity, 4) stimulation by genistein, and 5) lack of stimulation in CF epithelia bearing mutated delta F508 CFTR. In pulmonary alpha-toxin-permeabilized Calu-3 monolayers, cytosolic addition of 100 microM cAMP stimulated apical HCO3- currents from -9.4 +/- 1.6 to -31.1 +/- 3.9 microA/cm2 (n = 18), and apical Cl- currents increased from -54.1 +/- 7.1 to -203.2 +/- 15.4 microA/cm2 (n = 27). Average relative permselectivity for HCO3- vs. Cl- was approximately 15%. Absence of cytosolic ATP resulted in loss of cAMP stimulation of HCO3- and Cl- currents. Genistein (50 microM), which has been proposed to inhibit phosphatases controlling apical CFTR, as well as the alkaline phosphatase inhibitor (-)-p-bromotetramisole (1 mM) further activated cAMP-stimulated HCO3- and Cl- currents. Activated currents remained stimulated on removal of cAMP, suggesting inhibition of a protein phosphatase by genistein and bromotetramisole. The Cl- channel blockers glibenclamide (300 microM) and N-phenylanthranilic acid (5 mM), but not 4,4'-dinitro-2,2'-stilbenedisulfonic acid (100 microM), inhibited cAMP- and genistein-stimulated HCO3- and Cl- currents. Blocker effects were absent in human CF tracheal cells homozygous for the delta F508 mutation of CFTR (CFT1); Cl- and HCO3- currents were rescued in CFT1 cells recombinantly expressing wild-type CFTR. Thus CFTR functions as a HCO3- and Cl- conductor, and genistein and bromotetramisole maximize CFTR activity in airway epithelial cells.

Electrogenic hyperpolarization-elicited chloride transporter current in blue cones of zebrafish retinal slices.

Voltage-activated currents in blue cones of the retinal slice of zebrafish were characterized using whole cell recording techniques. Depolarizing-elicited currents were recorded: an outward tetraethylammonium (TEA)-sensitive K+ current (IKx), an outward Ca(2+)-activated Cl- current (ICl(Ca)), from which we inferred an inward Ca2+ current (ICa) as well as a hyperpolarizing-elicited nonselective inward cation current (Ih). In addition, hyperpolarizing steps elicited an outward current (Iout-h) in about one-third of the blue cones. Iout-h seems to be carried by inward transported Cl- because it was abolished by equimolar substitution of bath Cl- with acetate; equimolar substitution of Na+ with choline or TEA had no effect; it was not affected by Cl- channel blockers, anthracene-9-carboxylic acid, 4,4'-diisothiocyanostilbene-2.2'-disulfonic acid, N-phenylanthranilic acid (DPC), niflumic acid, and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid but was suppressed by Cl- transporter blockers acetalzolamide, bumetanide, N-ethylmaleimide, furosemide, and vanadate, and no reversal potential was found. In addition, this current was suppressed by ouabains but unrelated to their Na(+)-K(+)-ATPase inhibitory effect, was not suppressed by Co2+ or nifedipine, was not affected by the gap junction decoupler, 2-octanol, was increased by bath application of Cs+, presumably due to suppression of Ih, which was masked by Iout-h, and was suppressed by intensive light. Similar current also was found in the short cones and double cones. As Iout-h operates over the same voltage range, and with similar magnitude and time course as Ih, we suggest that Iout-h contributes to the modulation of the photoresponse of cones.

Non-steroidal anti-inflammatory drugs (NSAIDs). A predisposing factor for streptococcal bacteraemia?

Many non-steroidal anti-inflammatory drugs (NSAIDs) are now available, including a wide variety of individual compounds within the general groups of carboxylic acids (salicylic, propionic, pyranocarboxylic, acetic and fenamic acids and alkanones) and enolic acids (benzotriazines, oxicams and pyrazolones). A recent estimate suggested that 20 million prescriptions are written annually in the UK and 70 million in the USA; additionally large amounts of these drugs are purchased for self-medication. The modes of action vary somewhat between the different classes of drugs but in general result in profound inhibitory effects on the acute inflammatory reaction with enhanced cytokine production and dose-dependent inhibition of white cell functions1, 5. These effects, and the delay in presentation brought about by symptom relief, are likely to be particularly significant for infection with streptococci, which have the facility to spread quickly in the tissues if unhindered.

Floctafenine: a Valid Alternative in Patients with Adverse Reactions to Nonsteroidal Anti-inflammatory Drugs

Choosing an alternative anti-inflammatory agent for a patient who has suffered adverse reactions to nonsteroidal anti-inflammatory drugs is a common problem in clinical practice, and it is complicated by the possibility of non-immunologic cross reactivity among the various members of this drug class. We performed a retrospective study based on oral challenge results in 150 patients with histories of adverse reactions to one or more nonsteroidal anti-inflammatory drugs. The alternative drugs tested included floctafenine, an analgesic of the fenamic acid group, nimesulide, or paracetamol (acetaminophen). One hundred fifty patients with histories of adverse reactions to nonsteroidal anti-inflammatory drugs received oral challenges with floctafenine; 101 were also challenged with nimesulide and paracetamol. The oral challenges were administered under single-blinded conditions. One-fourth of the therapeutic dose (floctafenine, 200 mg; nimesulide, 100 mg; and paracetamol, 500 mg) was administered initially; the remaining 3/4 was given one hour later if no symptoms had developed with the initial administration. In patients with histories of rhinitis and/or bronchial asthma unrelated to drug use and those whose adverse reactions had included bronchospastic or rhinitic symptoms, the FEV1 was measured. The response to the challenge was considered positive if any of the following symptoms developed: erythema, pruritus accompanied by erythema, urticaria/ angioedema, rhinorrhea, nasal obstruction, sneezing, dyspnea or cough associated with a decrease of at least 20% in the FEV1, hypotension. Floctafenine challenges were positive in 13/150 patients (8.7%). Nimesulide and paracetamol provoked reactions in 9/101 patients (8.9%). All positive reactions occurred within 24 hours of the challenge. None of the patients reacted to more than one of the drugs tested. Our findings confirm those of other investigators regarding the use of nimesulide and paracetamol in patients who do not tolerate aspirin or other nonsteroidal anti-inflammatory drugs, although the percentage of reactions to nimesulide observed in the present study is somewhat higher than that reported by some other groups. The study also indicates that floctafenine, a relatively weak cyclooxygenase inhibitor, can be a valid alternative for many patients who react adversely to other nonsteroidal anti-inflammatory drugs.

Use of Pyridine as Cocatalyst for the Synthesis of N-Phenylanthranilic Acids

Abstract It is reported the use of pyridine as cocatalyst for the synthesis of N-phenylantranilic acids by the Ullmann-Goldberg condensation in water or amyl alcohol as solvents, with good yield.

Synthesis, properties, and biological activity of β-aroylpyruvoyl hydrazides oF N-methyl- and N-phenylanthranilic acid

Synthesis, properties, and biological activity of β-aroylpyruvoyl hydrazides oF N-methyl- and N-phenylanthranilic acid

Cyclic AMP Modulation of Active Ion Transport in the Pigmented Rabbit Conjunctiva

This study was conducted to determine whether active ion transport in the pigmented rabbit conjunctiva was subjected to cyclic AMP modulation. 8-BrcAMP and compounds that may affect intracellular cAMP levels were tested for their effects on the conjunctival short-circuit current (Isc) following mucosal application. Conjunctival Isc was increased by 1 mM 8-BrcAMP, 0.001-20 microM forskolin, 0.01-2 mM theophylline, 2 microM 1-epinephrine, and 20 microM terbutaline in a Cl(-)-containing but not in a Cl(-)-free medium. The increase in Isc induced by 1 mM 8-BrcAMP was reversed by 1 mM N-phenylanthranilic acid, suggesting enhancement of Cl- secretion by cAMP. Similarly, the increase in Isc induced by 20 microM terbutaline was blunted by 1 microM timolol, suggesting a role for beta 2 adrenergic modulation of Cl- secretion. Collectively, these results are consistent with the notion of cAMP modulation of conjunctival active ion transport.

Methodology for assaying iodide conductance in proteoliposomes: specific induction by thyroid membrane protein

A sensitive assay is developed to assess the existence of an iodide channel in a fraction of solubilized membrane proteins. This step is critical when considering various procedures for purification of this channel. Sodium cholate is used as a detergent as it does not denature the iodide channel. A simple and rapid method involving gel-filtration chromatography is used simultaneously to remove the detergent and to adjust the buffer composition, before protein insertion into liposomes. The presence of an iodide channel is investigated by measuring the iodide conductance of these proteoliposomes at 4 degrees C. An outward iodide gradient is set up across the proteoliposomal membrane by anion-exchange chromatography, allowing uptake of radiolabelled iodide. This uptake is conductive as it is abolished by valinomycin in the presence of potassium. It is specifically mediated by a thyroid plasma-membrane protein inserted into liposomes, as its denaturation before insertion totally abolished uptake. It was observed only within a well-defined fraction of thyroid membrane proteins collected by size-exclusion chromatography (molecular mass between 100 and 200 kDa). Furthermore, it was not observed with other membrane proteins such as ileal brush-border-membrane proteins or bacteriorhodopsin. Like many anion channels, this conductance was also inhibited by N-phenylanthranilic acid. Optimization of the assay is described, validating the measurement of conductive iodide uptake at 30 s by proteoliposomes reconstituted in a ratio of 10 micrograms of protein to 90 micrograms of lipid, with an outward iodide gradient (KI 15 mM inside and 1 microM outside). This assay provides a test of the biological activity of the iodide channel at each step of the purification; it can be applied to any anionic channel.

Basolateral membrane chloride permeability of A6 cells: implication in cell volume regulation

The permeability to Cl- of the basolateral membrane (blm) was investigated in renal (A6) epithelial cells, assessing their role in transepithelial ion transport under steady-state conditions (isoosmotic) and following a hypoosmotic shock (i.e. in a regulatory volume decrease, RVD). Three different complementary studies were made by measuring: (1) the Cl- transport rates (delta F/Fo s-1 (x10(-3))), where F is the fluorescence of N-(6-methoxyquinoyl) acetoethyl ester, MQAE, and Fo the maximal fluorescence (x10(-3)) of both membranes by following the intracellular Cl- activities (ai Cl-, measured with MQAE) after extracellular Cl- substitution (2) the blm 86Rb and 36Cl uptakes and (3) the cellular potential and Cl- current using the whole-cell patch-clamp technique to differentiate between the different Cl- transport mechanisms. The permeability of the blm to Cl- was found to be much greater than that of the apical membranes under resting conditions: aiCl- changes were 5.3 +/- 0.7 mM and 25.5 +/- 1.05 mM (n = 79) when Cl- was substituted by NO3(-) in the media bathing apical and basolateral membranes. The Cl- transport rate of the blm was blocked by bumetanide (100 microM) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 50 microM) but not by N-phenylanthranilic acid (DPC, 100 microM). 86Rb and 36Cl uptake experiments confirmed the presence of a bumetanide- and a NPPB-sensitive Cl- pathway, the latter being approximately three times more important than the former (Na/K/2Cl cotransporter). Appli-cation of a hypoosmotic medium to the serosal side of the cell increased delta F/Fo s-1 (x10(-3)) after extracellular Cl- substitution (1.03 +/- 0.10 and 2.45 +/- 0.17 arbitrary fluorescent units s-1 for isoosmotic and hypoosmotic conditions respectively, n = 11); this delta F/Fo s-1 (x10(-3)) increase was totally blocked by serosal NPPB application; on the other hand, cotransporter activity was decreased by the hypoosmotic shock. Cellular Ca2+ depletion had no effect on delta F/Fo s-1 (x10(-3)) under isoosmotic conditions, but blocked the delta F/Fo s-1 (x10(-3)) increase induced by a hypoosmotic stress. Under isotonic conditions the measured cellular potential at rest was -37.2 +/- 4.0 mV but reached a maximal and transient depolarization of -25.1 +/- 3.7 mV (n = 9) under hypoosmotic conditions. The cellular current at a patch-clamping cellular potential of -85 mV (close to the Nernst equilibrium potential for K+) was blocked by NPPB and transiently increased by hypoosmotic shock (≈50% maximum increase). This study demonstrates that the major component of Cl- transport through the blm of the A6 monolayer is a conductive pathway (NPPB-sensitive Cl- channels) and not a Na/K/2Cl cotransporter. These channels could play a role in transepithelial Cl- absorption and cell volume regulation. The increase in the blm Cl- conductance, inducing a depolarization of these membranes, is proposed as one of the early events responsible for the stimulation of the 86Rb efflux involved in cell volume regulation.

Cell-swelling induced amino acid efflux from human placental tissue

Cells of the retinal pigment epithelium (RPE) were isolated from neonatal rats. The perforated-patch clamp technique, using amphotericin-B, revealed a chloride current, which was detected as a 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS)-sensitive component.A variety of chloride-channel inhibitors, other than DIDS, also blocked the chloride current, including 9-anthracenecarboxylic acid (9-AC), niflumic acid, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and N-phenylanthranilic acid (DPC). DIDS at 1 mmol l-1 and DPC at 2 mmol l-1 were both more potent than niflumic acid at 200 μmol l-1.The hyperosmotic condition (+ 50 mosmol) did not affect the chloride current. The hypo-osmotic condition (- 80 mosmol), however, led to an increase of total membrane conductance, during which a chloride conductance increase occurred. The membrane-conductance increase, evoked by the hypo-osmotic condition, ran up and then ran down by itself. As with the steady-state chloride conductance, 1 mmol l-1 DIDS and 2 mmol l-1 DPC were more potent blockers than 200 μmol l-1 niflumic acid. DIDS at 1 mmol l-1 was more effective in blocking the outward component than 2 mmol l-1 DPC, while the inward component was blocked oppositely.When the intracellular calcium concentration was presumed to have increased, using the calcium ionophore, ionomycin, membrane conductance abruptly increased, quickly ran up and then ran down by itself. This included an elevation of a DIDS-sensitive current, and was largely carried by chloride. Chloride currents of rat RPE cells did not appear to be regulated by cyclic AMP.

Cholinergic and VIP-ergic pathways mediate histamine H2 receptor-induced cyclical secretion in the guinea pig colon.

Previous studies demonstrated neurally mediated recurrent increases in short-circuit current (Isc) suggestive of anion secretion in guinea pig distal colon. To determine the neural pathways involved, segments of distal colon from guinea pigs were mounted in flux chambers. In muscle-stripped or whole thickness preparations, serosal addition of the histamine H2 receptor agonist, dimaprit, caused cyclical increases in Isc, which were reduced by the chloride channel blocker, N-phenylanthranilic acid, but not by the sodium channel blocker amiloride. Dimaprit stimulated release of [3H]acetylcholine and vasoactive intestinal polypeptide (VIP) from submucosal/mucosal sheets. Dimaprit caused recurrent increases in Isc, which were significantly decreased by mecamylamine, a nicotinic receptor antagonist, and nearly abolished by the muscarinic antagonist, atropine (M3 > M1 = M2). The muscarinic antagonist, 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP, M3 > M1), was more potent than pirenzepine (M1 > M3) in reducing recurrent increases in Isc. Dimaprit- and electrically evoked secretion were inhibited by the VIP antagonists [4Cl-D-Phe6, Leu17]VIP and VIP hybrid. The results suggest the involvement of VIP-ergic and cholinergic neurons utilizing nicotinic and muscarinic synapses in mediating secretion.

Chloride transport activation by plasma osmolarity during rapid adaptation to high salinity of Fundulus heteroclitus.

Transition from low salt water to sea water of the euryhaline fish, Fundulus heteroclitus, involves a rapid signal that induces salt secretion by the gill chloride cells. An increase of 65 mOsm in plasma osmolarity was found during the transition. The isolated, chloride-cell-rich opercular epithelium of sea-water-adapted Fundulus exposed to 50 mOsm mannitol on the basolateral side showed a 100% increase in chloride secretion, which was inhibited by bumetanide 10(-4) M and 10(-4) M DPC (N-Phenylanthranilic acid). No effect of these drugs was found on apical side exposure. A Na+/H+ exchanger, demonstrated by NH4Cl exposure, was inhibited by amiloride and its analogues and stimulated by IBMX, phorbol esters, and epithelial growth factor (EGF). Inhibition of the Na+/H+ exchanger blocks the chloride secretion increase due to basolateral hypertonicity. A Cl-/HCO3- exchanger was also found in the chloride cells, inhibited by 10(-4) M DIDS but not involved in the hyperosmotic response. Ca2+ concentration in the medium was critical for the stimulation of Cl- secretion to occur. Chloride cell volume shrinks in response to hypertonicity of the basolateral side in sea-water-adapted operculi; no effect was found on the apical side. Fresh-water-adapted fish chloride cells show increased water permeability of the apical side. It is concluded that the rapid signal for adaptation to higher salinities is an increased tonicity of the plasma that induces chloride cell shrinkage, increased chloride secretion with activation of the Na+K+2Cl- cotransporter, the Na+/H+ exchanger and opening of Cl- channels.

Modulation of cytosolic Ca 2+ concentration by thapsigargin and cyclopiazonic acid in human aortic endothelial cells

To clarify the agonist-induced Ca 2+ entry mechanism, effects of thapsigargin and cyclopiazonic acid, selective inhibitors of endoplasmic reticulum Ca 2+-ATPase, on intracellular free Ca 2+ concentration ([Ca 2+] i) were studied in cultured human aortic endothelial cells loaded with the fluorescent Ca 2+ indicator fura-2. Thapsigargin (1–1000 nM) and cyclopiazonic acid (0.1–100 μM) produced a biphasic change in [Ca 2+] i, which consisted of a transient peak elevation followed by a long-lasting decline of [Ca 2+] i in a concentration-dependent manner. In the presence of thapsigargin or cyclopiazonic acid, the rapid transient elevation of [Ca 2+] i elicited by histamine was attenuated in a time-dependent manner. The slow declining phase of the response to thapsigargin and cyclopiazonic acid was completely eliminated by removal of extracellular Ca 2+, and it was also prevented by reduction of the extracellular Cl − concentration to 40 mM or by the Cl − channel blocker N-phenylanthranilic acid. These findings suggest that the initial transient rising phase and the slow declining phase of [Ca 2+] i in response to thapsigargin and cyclopiazonic acid reflect a blockade of Ca 2+ uptake into the endoplasmic reticulum and the Cl −-sensitive Ca 2+ entry activated by the depletion of agonist-sensitive intracellular Ca 2+ stores, respectively, in human aortic endothelial cells.