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Palmatine, a protoberberine alkaloid, inhibits both Ca(2+)- and cAMP-activated Cl(-) secretion in isolated rat distal colon.

ABSTRACT: BACKGROUND AND PURPOSE: The protoberberine alkaloid berberine has been reported to inhibit colonic Cl(-) secretion. However, it is not known if other protoberberine alkaloids share these effects. We have therefore selected another protoberberine alkaloid, palmatine, to assess its effects on active ion transport across rat colonic epithelium. EXPERIMENTAL APPROACH: Rat colonic mucosa was mounted in Ussing chambers and short circuit current (I (SC)), apical Cl(-) current and basolateral K(+) current were recorded. Intracellular cAMP content was determined by an enzyme immunoassay. Intracellular Ca(2+) concentration was measured with Fura-2 AM. KEY RESULTS: Palmatine inhibited carbachol-induced Ca(2+)-activated Cl(-) secretion and the carbachol-induced increase of intracellular Ca(2+) concentration. Palmatine also inhibited cAMP-activated Cl(-) secretion induced by prostaglandin E(2) (PGE(2)) or forskolin. Palmatine prevented the elevation of intracellular cAMP by forskolin. Determination of apical Cl(-) currents showed that palmatine suppressed the forskolin-stimulated, apical cAMP-activated Cl(-) current but not the carbachol-stimulated apical Ca(2+)-activated Cl(-) current. Following permeabilization of apical membranes with nystatin, we found that palmatine inhibited a carbachol-stimulated basolateral K(+) current that was sensitive to charybdotoxin and resistant to chromanol 293B. However, the forskolin-stimulated basolateral K(+) current inhibited by palmatine was specifically blocked by chromanol 293B and not by charybdotoxin. CONCLUSIONS AND IMPLICATIONS: Palmatine attenuated Ca(2+)-activated Cl(-) secretion through inhibiting basolateral charybdotoxin-sensitive, SK4 K(+) channels, whereas it inhibited cAMP-activated Cl(-) secretion by inhibiting apical CFTR Cl(-) channels and basolateral chromanol 293B-sensitive, KvLQT1 K(+) channels.

SUBMITTER: Wu DZ

PROVIDER: S-EPMC2275457 | biostudies-other | 2008 Mar

REPOSITORIES: biostudies-other

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Project description:The vestibular system controls the ion composition of its luminal fluid through several epithelial cell transport mechanisms under hormonal regulation. The semicircular canal duct (SCCD) epithelium has been shown to secrete Cl- under ?2-adrenergic stimulation. In the current study, we sought to determine the ion transporters involved in Cl- secretion and whether secretion is regulated by PKA and glucocorticoids.Short circuit current (Isc) from rat SCCD epithelia demonstrated stimulation by forskolin (EC50: 0.8 ?M), 8-Br-cAMP (EC50: 180 ?M), 8-pCPT-cAMP (100 ?M), IBMX (250 ?M), and RO-20-1724 (100 ?M). The PKA activator N6-BNZ-cAMP (0.1, 0.3 & 1 mM) also stimulated Isc. Partial inhibition of stimulated Isc individually by bumetanide (10 & 50 ?M), and [(dihydroindenyl)oxy]alkanoic acid (DIOA, 100 ?M) were additive and complete. Stimulated Isc was also partially inhibited by CFTRinh-172 (5 & 30 ?M), flufenamic acid (5 ?M) and diphenylamine-2,2'-dicarboxylic acid (DPC; 1 mM). Native canals of CFTR+/- mice showed a stimulation of Isc from isoproterenol and forskolin+IBMX but not in the presence of both bumetanide and DIOA, while canals from CFTR-/- mice had no responses. Nonetheless, CFTR-/- mice showed no difference from CFTR+/- mice in their ability to balance (rota-rod). Stimulated Isc was greater after chronic incubation (24 hr) with the glucocorticoids dexamethasone (0.1 & 0.3 ?M), prednisolone (0.3, 1 & 3 ?M), hydrocortisone (0.01, 0.1 & 1 ?M), and corticosterone (0.1 & 1 ?M) and mineralocorticoid aldosterone (1 ?M). Steroid action was blocked by mifepristone but not by spironolactone, indicating all the steroids activated the glucocorticoid, but not mineralocorticoid, receptor. Expression of transcripts for CFTR; for KCC1, KCC3a, KCC3b and KCC4, but not KCC2; for NKCC1 but not NKCC2 and for WNK1 but only very low WNK4 was determined.These results are consistent with a model of Cl- secretion whereby Cl- is taken up across the basolateral membrane by a Na+-K+-2Cl- cotransporter (NKCC) and potentially another transporter, is secreted across the apical membrane via a Cl- channel, likely CFTR, and demonstrate the regulation of Cl- secretion by protein kinase A and glucocorticoids.

Project description:1. Nine bis-quinolinyl and bis-quinolinium compounds related to dequalinium, and previously shown to block apamin-sensitive small conductance Ca(2+)-activated K(+) channels (SK(Ca)), have been tested for their inhibitory effects on actions mediated by intermediate conductance Ca(2+)-activated K(+) channels (IK(Ca)) in rabbit blood cells. 2. In most experiments, a K(+)-sensitive electrode was employed to monitor the IK(Ca)-mediated net loss of cell K(+) that followed the addition of the Ca(2+) ionophore A23187 (2 microM) to red cells suspended at an haematocrit of 1% in a low K(+) (0.12 - 0.17 mM) solution. The remainder used an optical method based on measuring the reduction in light transmission that occurred on applying A23187 (0.4 or 2 microM) to a very dilute suspension of red cells (haematocrit 0.02%). 3. Of the compounds tested, the most potent IK(Ca) blocker was 1,12 bis[(2-methylquinolin-4-yl)amino]dodecane (UCL 1407) which had an IC(50) of 0.85+/-0.06 microM (mean+/-s.d. mean). 4. The inhibitory action of UCL 1407 and its three most active congeners was characterized by (i) a Hill slope greater than unity, (ii) sensitivity to an increase in external [K(+)], and (iii) a time course of onset that suggested use-dependence. Also, the potency of the nonquaternary compounds tested increased with their predicted lipophilicity. These findings suggested that the IK(Ca) blocking action resembles that of cetiedil rather than of clotrimazole. 5. Some quaternized members of the series were also active. The most potent was the monoquaternary UCL 1440 ((1-[N-[1-(3, 5-dimethoxybenzyl)-2-methylquinolinium-4-yl]amino]-10-[N'-(2-me thylqu inolinium-4yl)amino] decane (trifluoroacetate) which had an IC(50) of 1.8+/-0.1 microM. The corresponding bisquaternary UCL 1438 (1, 10-bis[N-[1-(3,5-dimethoxybenzyl)-2-methylquinolinium-4-yl]amino] decane bis(trifluoroacetate) was almost as active (IC(50) 2.7+/-0.3 microM). 6. A bis-aminoquinolium cyclophane (UCL 1684) had little IK(Ca) blocking action despite its great potency at SK(Ca) channels (IC(50) 4.1+/-0.2 nM). 7. The main outcome is the identification of new intermediate-conductance Ca(2+)-activated K(+) channel blockers with a wide range of IK(Ca)/SK(Ca) selectivities.

Project description:BackgroundTransmembrane protein 16A (TMEM16A) Ca2+-activated Cl- channels have diverse physiological functions, such as epithelial secretion of Cl- and fluid and sensation of pain. Recent studies have demonstrated that TMEM16A contributes to the pathogenesis of infectious and non-infectious inflammatory diseases. However, the role of TMEM16A in inflammation has not been clearly elucidated.Aim of reviewIn this review, we aimed to provide comprehensive information regarding the roles of TMEM16A in inflammation by summarizing the mechanisms underlying TMEM16A expression and activation under inflammatory conditions, in addition to exploring the diverse inflammatory signaling pathways activated by TMEM16A. This review attempts to develop the idea that TMEM16A plays a diverse role in inflammatory processes and contributes to inflammatory diseases in a cellular environment-dependent manner.Key scientific concepts of reviewMultiple inflammatory mediators, including cytokines (e.g., interleukin (IL)-4, IL-13, IL-6), histamine, bradykinin, and ATP/UTP, as well as bacterial and viral infections, promote TMEM16A expression and/or activity under inflammatory conditions. In addition, TMEM16A activates diverse inflammatory signaling pathways, including the IP3R-mediated Ca2+ signaling pathway, the NF-κB signaling pathway, and the ERK signaling pathway, and contributes to the pathogenesis of many inflammatory diseases. These diseases include airway inflammatory diseases, lipopolysaccharide-induced intestinal epithelial barrier dysfunction, acute pancreatitis, and steatohepatitis. TMEM16A also plays multiple roles in inflammatory processes by increasing vascular permeability and leukocyte adhesion, promoting inflammatory cytokine release, and sensing inflammation-induced pain. Furthermore, TMEM16A plays its diverse pathological roles in different inflammatory diseases depending on the disease severity, proliferating status of the cells, and its interacting partners. We herein propose cellular environment-dependent mechanisms that explain the diverse roles of TMEM16A in inflammation.

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Palmatine, a protoberberine alkaloid, inhibits both Ca(2+)- and cAMP-activated Cl(-) secretion in isolated rat distal colon.

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