Lucantonio Debellis

Free [Ca2+] dynamics measured in agonist‐sensitive stores of single living intact cells: a new look at the refilling process

Free [Ca2+] in agonist‐sensitive internal stores of single intact cells was measured in situ in order to examine the role of [Ca2+] in modulating the store refilling process. BHK‐21 fibroblasts were loaded with the low‐affinity fluorescent calcium indicator mag‐fura‐2‐AM such that >80% of the dye was trapped in organelles, where it reported [Ca2+] changes solely in an agonist‐ and thapsigargin‐sensitive internal store. The rates of store reloading following stimulation by 100 nM bradykinin were essentially unchanged when cytosolic [Ca2+] was clamped to resting values with BAPTA‐AM. In control cells, recharging of stores totally depended on the presence of external Ca2+, but pre‐loading the cells with BAPTA‐AM permitted efficient refilling in Ca2+‐free, EGTA‐containing external medium. Our results show: (i) Ca2+ stores normally are recharged by Ca2+ which must first transit the cytoplasm; (ii) an elevation in cytoplasmic [Ca2+] is not required to replenish Ca2+ stores; (iii) the activation of the plasma membrane Ca2+ pump during the Ca2+ spike ordinarily results in complete extrusion of released Ca2+; and (iv) the buffering capacity of the cytoplasm is an essential component of the store refilling process. An interesting finding was that acute treatment of cells with BAPTA‐AM activated capacitative Ca2+ entry at the plasma membrane, due to its efficient hydrolysis in the stores, and the ensuing decrease in the endoplasmic reticulum [Ca2+].

Asymmetrical, agonist‐induced fluctuations in local extracellular [Ca2+] in intact polarized epithelia

We recently proposed that extracellular Ca2+ ions participate in a novel form of intercellular communication involving the extracellular Ca2+‐sensing receptor (CaR). Here, using Ca2+‐selective microelectrodes, we directly measured the profile of agonist‐induced [Ca2+]ext changes in restricted domains near the basolateral or luminal membranes of polarized gastric acid‐secreting cells. The Ca2+‐mobilizing agonist carbachol elicited a transient, La3+‐sensitive decrease in basolateral [Ca2+] (average ≈250 μM, but as large as 530 μM). Conversely, carbachol evoked an HgCl2‐sensitive increase in [Ca2+] (average ≈400 μM, but as large as 520 μM) in the lumen of single gastric glands. Both responses were significantly reduced by pre‐treatment with sarco‐endoplasmic reticulum Ca2+ ATPase (SERCA) pump inhibitors or with the intracellular Ca2+ chelator BAPTA‐AM. Immunofluores cence experiments demonstrated an asymmetric localization of plasma membrane Ca2+ ATPase (PMCA), which appeared to be partially co‐localized with CaR and the gastric H+/K+‐ATPase in the apical membrane of the acid‐secreting cells. Our data indicate that agonist stimulation results in local fluctuations in [Ca2+]ext that would be sufficient to modulate the activity of the CaR on neighboring cells.

A Reassessment of the Effects of Luminal [Ca2+] on Inositol 1,4,5-Trisphosphate-induced Ca2+ Release from Internal Stores

Inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ release from intracellular stores displays complex kinetic behavior. While it well established that cytosolic [Ca2+] can modulate release by acting on the InsP3 receptor directly, the role of the filling state of internal Ca2+stores in modulating Ca2+ release remains unclear. Here we have reevaluated this topic using a technique that permits rapid and reversible changes in free [Ca2+] in internal stores of living intact cells without altering cytoplasmic [Ca2+], InsP3 receptors, or sarcoendoplasmic reticulum Ca2+ ATPases (SERCAs). N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylene diamine (TPEN), a membrane-permeant, low affinity Ca2+ chelator was used to manipulate [Ca2+] in intracellular stores, while [Ca2+] changes within the store were monitored directly with the low-affinity Ca2+ indicator, mag-fura-2, in intact BHK-21 cells. 200 μm TPEN caused a rapid drop in luminal free [Ca2+] and significantly reduced the extent of the response to stimulation with 100 nm bradykinin, a calcium-mobilizing agonist. The same effect was observed when intact cells were pretreated with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid(acetoxymethyl ester) (BAPTA-AM) to buffer cytoplasmic [Ca2+] changes. Although inhibition of Ca2+ uptake using the SERCA inhibitor tBHQ permitted significantly larger release of Ca2+ from stores, TPEN still attenuated the release in the presence of tBHQ in BAPTA-AM-loaded cells. These results demonstrate that the filling state of stores modulates the magnitude of InsP3-induced Ca2+release by additional mechanism(s) that are independent of regulation by cytoplasmic [Ca2+] or effects on SERCA pumps.

The Vibrio cholerae cytolysin promotes chloride secretion from intact human intestinal mucosa.

Background The pathogenicity of the Vibrio cholerae strains belonging to serogroup O1 and O139 is due to the production of virulence factors such as cholera toxin (CT) and the toxin-coregulated pilus (TCP). The remaining serogroups, which mostly lack CT and TCP, are more frequently isolated from aquatic environmental sources than from clinical samples; nevertheless, these strains have been reported to cause human disease, such as sporadic outbreaks of watery diarrhoea and inflammatory enterocolitis. This evidence suggested the possibility that other virulence factor(s) than cholera toxin might be crucial in the pathogenesis of Vibrio cholerae-induced diarrhoea, but their nature remains unknown. VCC, the hemolysin produced by virtually all Vibrio cholerae strains, has been proposed as a possible candidate, though a clear-cut demonstration attesting VCC as crucial in the pathogenesis of Vibrio cholerae-induced diarrhoea is still lacking. Methodology/Principal Findings Electrophysiological parameters and paracellular permeability of stripped human healthy colon tissues, obtained at subtotal colectomy, mounted in Ussing chamber were studied in the presence or absence of VCC purified from culture supernatants of V. cholerae O1 El Tor strain. Short circuit current (ISC) and transepithelial resistance (RT) were measured by a computerized voltage clamp system. The exposure of sigmoid colon specimens to 1 nM VCC resulted in an increase of ISC by 20.7%, with respect to the basal values, while RT was reduced by 12.3%. Moreover, increase in ISC was abolished by bilateral Cl− reduction. Conclusion/Significance Our results demonstrate that VCC, by forming anion channels on the apical membrane of enterocytes, triggers an outward transcellular flux of chloride. Such an ion movement, associated with the outward movement of Na+ and water, might be responsible for the diarrhoea caused by the non-toxigenic strains of Vibrio cholerae.

Extracellular calcium acts as a “third messenger” to regulate enzyme and alkaline secretion

It is generally assumed that the functional consequences of stimulation with Ca2+-mobilizing agonists are derived exclusively from the second messenger action of intracellular Ca2+, acting on targets inside the cells. However, during Ca2+ signaling events, Ca2+ moves in and out of the cell, causing changes not only in intracellular Ca2+, but also in local extracellular Ca2+. The fact that numerous cell types possess an extracellular Ca2+ “sensor” raises the question of whether these dynamic changes in external [Ca2+] may serve some sort of messenger function. We found that in intact gastric mucosa, the changes in extracellular [Ca2+] secondary to carbachol-induced increases in intracellular [Ca2+] were sufficient and necessary to elicit alkaline secretion and pepsinogen secretion, independent of intracellular [Ca2+] changes. These findings suggest that extracellular Ca2+ can act as a “third messenger” via Ca2+ sensor(s) to regulate specific subsets of tissue function previously assumed to be under the direct control of intracellular Ca2+.

Basolateral Ca2+-dependent K+-channels play a key role in Cl-secretion induced by taurodeoxycholate from colon mucosa

Abstract The diarrhea associated with malabsorption of bile salts such as the secondary hydrophobic taurodeoxycholate (TDC) may be partly explained by the TDC‐induced increase in colon Cl− secretion. We, therefore, investigated the effects of TDC (0.5–8 mM) on electrical parameters and electrolyte transport of rat proximal colon mucosa mounted in Ussing chambers. Colonic secretion, measured as short circuit current (I SC), progressively increased on mucosal incubation with TDC ranging 0.5–2 mM; up to TDC 2 mM, a spontaneous recovery toward control values with no changes in epithelial resistance (Rt), and lactate dehydrogenase (LDH) release was observed. In contrast, for TDC > 2 mM, I SC increased further and the effect was progressive and associated with a significant decrease in the Rt and increased LDH release, implying a cytolytic effect. Mucosal preincubation with the Cl− channel inhibitor 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB), fully prevented the precytolytic effect of TDC on I SC. Serosal preincubation with furosemide, a Na+/K+/2Cl− cotransporter inhibitor, significantly reduced TDC‐induced increase in I SC. Inhibition of the basolateral Ca2+‐dependent K+ channel—rSK4—with serosal clotrimazole or incubation with mucosal Ca2+‐free (EGTA) buffer completely prevented precytolytic TDC‐induced increase in I SC. In conclusion, Cl− secretion is activated in colon mucosa by TDC low concentrations; while at higher concentrations, a detergent cytotoxic effect intervenes. Activation of the Ca2+‐dependent basolateral K+ pathway, through TDC‐induced apical Ca2+ influx, provides the Na+/K+/2Cl− basolateral activation, thereby the driving force for the apical exit of Cl− ions. These findings further enhance the knowledge of the pathogenic mechanisms of diarrhea associated with bile salt malabsorption.

Alkaline secretion by frog gastric glands measured with pH microelectrodes in the gland lumen

1 In the present work we have measured the pH of the secreted fluid within the gland lumen of isolated but intact gastric mucosa of Ranaesculenta. Tissues were mounted in a double chamber allowing continuous perfusion of the mucosal and serosal compartment, and the measurements were made with double‐barrelled pH glass microelectrodes inserted into the glands from the serosal surface under microscopic inspection. 2 During inhibition of H+ secretion by cimetidine (100 μm) the luminal gland pH (pHgl) averaged 7.60 ± 0.05 pH units (mean ±s.e.m.; n= 35), a value significantly higher than bath solution pH (7.45 ± 0.02; P < 0.001) and also higher than intracellular pH of oxyntopeptic cells (pHi), which averaged 7.53 ± 0.06 (n= 18). 3 Stimulation of acid secretion with histamine (500 μm) reversibly decreased pHgl to values which could be as low as 2.5. Together with electrophysiological criteria this response was routinely used to verify the proper location of the microelectrode tip within the gland lumen. 4 Stimulation with carbachol (100 μm) or pentagastrin (50 μm) in the presence of cimetidine rapidly and reversibly increased pHgl by 0.10 ± 0.01 pH units (n= 24; P < 0.001) and 0.09 ± 0.02 pH units (n= 6; P < 0.05), respectively. 5 The observation that gastric gland fluid is more alkaline than the bath solutions and that carbachol or pentagastrin further alkalinize it strongly suggests that oxyntopeptic cells participate in gastric alkaline secretion at least under cholinergic stimulation.

Model of bicarbonate secretion by resting frog stomach fundus mucosa. I. Transepithelial measurements.

In the present in vitro experiments on gastric fundus mucosa of Rana esculenta we try to define the mechanism of alkaline secretion that is observed in summer frogs in the resting stomach (blockage of HCl secretion by ranitidine, 10−5 mol/l). The transepithelial voltage and the rate of alkalinization (ASR) of an unbuffered gastric lumen perfusate was measured as a function of serosal (and mucosal) fluid composition. ASR was high (0.88±S.E. 0.09 μEq·cm−2·h−1, n=11) during serosal bath perfusion with HCO3−-Ringer solution, decreased slightly to 0.50±0.07 μEq·cm−2·h−1 (n=6) in HCO3−-free HEPES-buffered Ringer solution of the same pH, and decreased to approximately 20% when carbonic anhydrase was inhibited by acetazolamide. While replacement of mucosal or serosal Cl− did not — within 1 h — significantly alter ASR, replacement of serosal Na+ in the presence or absence of HCO3− strongly reduced ASR, and a similar reduction was observed after serosal application of the anion transport inhibitor DIDS (4,4-diisomiocyanatostilbene-2,2-disulphonate, 2·10−4 mol/l), the metabolic poison rotenone (10−5 mol/l), the uncoupler dinitrophenol (10−4 mol/l), and the Na+ pump inhibitor ouabain (10−4 mol/l), while serosal amiloride (10−4 mol/l) had no effect. These data can be accounted for by a model of alkaline secretion that consists of basolateral HCO3− uptake from the serosal fluid into the cell via a DIDS-inhibitable Na+(HCO3−)n-cotransporter and HCO3− secretion from the cell to the gastric lumen via an anionic conductance pathway. Microelectrode experiments on oxyntopeptic cells reported in the subsequent paper suggest that these cells may also be involved in the resting state alkaline secretion.

Microelectrode determination of oxyntic cell pH in intact frog gastric mucosa. Effect of histamine

Intracellular pH (pHi) of acid-secreting cells was measured in intact gastric fundus mucosa of Rana esculenta with double-barrelled pH microelectrodes. Tissues were mounted, serosal side up, between two half chambers and individual cells were impaled after microsurgical removal of the serosal muscle layer. Transepithelial potential difference (Vt) and resistance (Rt) as well as serosal cell membrane potential (Vs) and pHi were continuously recorded at rest (0.1 mmol/l cimetidine) or during stimulation (0.5 mmol/l histamine). During chamber perfusion with HCO33−/CO2-buffered Ringer solution of pHo=7.36, Vt and Rt were −21.7, SD±6.0 mV and 229±83 Ω cm2(n=17) while Vs and pHi averaged −7.3±6.9 mV and 7.4±0.11 (n=25). The latter value is considerably more alkaline than all recent pHi measurements obtained with microspectrofluorometric techniques on isolated cells, glands or intact tissue. The difference may in part be explained by use of HCO3−-free solutions in most of the previous studies because we observed that such solutions decrease pHi to 6.89±0.18 (n=4). Again, in contrast to recent literature, application of histamine in HCO3−/CO2-buffered solution led to further transient alkalinization by 0.12±0.05 pH unit (n=8). Since in accidental punctures of the gastric gland lumen we noticed that H+ secretion only began approximately 5 min after histamine application, we conclude that the histamine-induced initial alkalinization does not reflect stimulation of the H+/K+ ATPase pump. Alternatively, it may result from histamine-induced activation or inactivation of other ion transporters, one possibility being activation of basolateral Na+/H+ and Cl−/HCO3−exchangers.

Glucose increases extracellular [Ca2+] in rat insulinoma (INS-1E) pseudoislets as measured with Ca2+-sensitive microelectrodes

Secretory granules of pancreatic β-cells contain high concentrations of Ca2+ ions that are co-released with insulin in the extracellular milieu upon activation of exocytosis. As a consequence, an increase in the extracellular Ca2+ concentration ([Ca2+]ext) in the microenvironment immediately surrounding β-cells should be expected following the exocytotic event. Using Ca2+-selective microelectrodes we show here that both high glucose and non-nutrient insulinotropic agents elicit a reversible increase of [Ca2+]ext within rat insulinoma (INS-1E) β-cells pseudoislets. The glucose-induced increases in [Ca2+]ext are blocked by pretreatment with different Ca2+ channel blockers. Physiological agonists acting as positive or negative modulators of the insulin secretion and drugs known to intersect the secretory machinery at different levels also induce [Ca2+]ext changes as predicted on the basis of their described action on insulin secretion. Finally, the glucose-induced [Ca2+]ext increase is strongly inhibited after disruption of the actin web, indicating that the dynamic [Ca2+]ext changes recorded in INS-1E pseudoislets by Ca2+-selective microelectrodes occur mainly as a consequence of exocytosis of Ca2+-rich granules. In conclusion, our data directly demonstrate that the extracellular spaces surrounding β-cells constitute a restricted domain where Ca2+ is co-released during insulin exocytosis, creating the basis for an autocrine/paracrine cell-to-cell communication system via extracellular Ca2+ sensors.