Rosa Caroppo

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.

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+.

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.

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.

Is resting state HCO3- secretion in frog gastric fundus mucosa mediated by apical Cl(-)-HCO3- exchange?

1. We have tested the widely accepted hypothesis that resting‐state bicarbonate secretion of gastric fundus mucosa is mediated by Cl(‐)‐HCO3‐ exchange in the apical membrane of surface epithelial cells (SECs). To this end, SECs of isolated fundus mucosa of Rana esculenta were punctured with double‐barrelled microelectrodes to measure intracellular pH (pHi). 2. No significant pHi changes were observed in response to changing luminal HCO3‐ and/or Cl‐ concentrations. The change in pHi (delta pHi) in response to luminal chloride substitution averaged 0.00 +/‐ 0.01 pH units (mean +/‐ S.E.M.; n = 48), and did not change after blocking putative basolateral acid/base transporters which could have masked the pHi response. 3. On the other hand, pHi responded readily and reversibly to luminal perfusion with either low‐pH (pH 2.5) solution (delta pHi = ‐0.36 +/‐ 0.05; n = 4; P < 0.01) or CO2‐free HCO3‐ Ringer solution (delta pHi = +0.10 +/‐ 0.01; n = 29; P < 0.001). These observations demonstrate that the solution change was effective and complete within 1 min and show that the apical membrane of SECs is permeable to CO2. 4. The apical membrane of frog SECs could not be stained with an antibody against the C‐terminal end of the mouse Cl(‐)‐HCO3‐ exchanger isoform AE2, although this antibody readily stained the basolateral membrane of the oxyntopeptic cells (OCs). 5. In conclusion, the presence of a Cl(‐)‐HCO3‐ exchanger in the apical membrane of SECs of frog gastric fundus mucosa in the resting state could not be confirmed, but other models of HCO3‐ secretion cannot be fully excluded. Observations from electrical measurements, favouring a model of conductive HCO3‐ secretion, point to the OCs rather than the SECs as a site of origin of HCO3‐ secretion.

Real time measurements of water flow in amphibian gastric glands: modulation via the extracellular Ca2+-sensing receptor.

The mechanisms for the formation of the osmotic gradient driving water movements in the gastric gland and its modulation via the extracellular Ca2+-sensing receptor (CaR) were investigated. Real time measurements of net water flux in the lumen of single gastric glands of the intact amphibian stomach were performed using ion-selective double-barreled microelectrodes. Water movement was measured by recording changes in the concentration of impermeant TEA+ ions ([TEA+]gl) with TEA+-sensitive microelectrodes inserted in the lumen of individual gastric glands. Glandular K+ (K+gl) and H+ (pHgl) were also measured by using K+- and H+-sensitive microelectrodes, respectively. Stimulation with histamine significantly decreased [TEA]gl, indicating net water flow toward the gland lumen. This response was inhibited by the H+/K+-ATPase inhibitor, SCH 28080. Histamine also elicited a significant and reversible increase in [K+]gl that was blocked by chromanol 293B, a blocker of KCQN1 K+ channels. Histamine failed to induce net water flow in the presence of chromanol 293B. In the “resting state,” stimulation of CaR with diverse agonists resulted in significant increase in [TEA]gl. CaR activation also significantly reduced histamine-induced water secretion and apical K+ transport. Our data validate the strong link between histamine-stimulated acid secretion and water transport. We also show that cAMP-dependent [K+]gl elevation prior to the onset of acid secretion generates the osmotic gradient initially driving water into the gastric glands and that CaR activation inhibits this process, probably through reduction of intracellular cAMP levels.

Ca2+-dependent K+ efflux regulates deoxycholate-induced apoptosis of BHK-21 and Caco-2 cells.

BACKGROUND & AIMS Deoxycholate (DC) has proapoptotic and tumorigenic effects in different cell types of the gastrointestinal tract. Exposure of BHK-21 (stromal) cells to DC induces Ca(2+) entry at the plasma membrane, which affects intracellular Ca(2+) signaling. We assessed whether DC-induced increases in [Ca(2+)] can impinge on plasma membrane properties (eg, ionic conductances) involved in cell apoptosis. METHODS Single- and double-barreled microelectrodes were used to measure membrane potential (V(m)) and extracellular [K(+)] in BHK-21 fibroblasts and Caco-2 colon carcinoma cells. Apoptosis was assessed by Hoechst labeling, propidium iodide staining, and caspase-3 and caspase-7 assays. RESULTS DC-induced cell membrane hyperpolarization was directly measured with intracellular microelectrodes in both cell lines. Diverse Ca(2+) mobilizing agents, such as membrane receptor agonists, an inhibitor of the sarco/endoplasmic reticulum Ca(2+) adenosine triphosphatase and a Ca(2+) ionophore, also induced increases in V(m). Removal of extracellular Ca(2+) reduced the agonist- and DC-induced membrane hyperpolarization by approximately 15% and 60%, respectively. These findings indicate a prominent role for Ca(2+) entry at the plasma membrane in the action of this bile salt. Blockade of Ca(2+)-activated K(+) conductances by charybdotoxin and apamin reduced DC-induced hyperpolarization by 75% and 64% in BHK-21 and Caco-2 cells, respectively. These inhibitors also reduced the DC-induced increase in extracellular [K(+)] by 75% and cell apoptosis by approximately 50% in both cell lines. CONCLUSIONS Ca(2+)-dependent K(+) conductance is an important regulator of DC-induced apoptosis in stromal and colon cancer cells.

Cadmium inhibits acid secretion in stimulated frog gastric mucosa

Cadmium, a toxic environmental pollutant, affects the function of different organs such as lungs, liver and kidney. Less is known about its toxic effects on the gastric mucosa. The aim of this study was to investigate the mechanisms by which cadmium impacts on the physiology of gastric mucosa. To this end, intact amphibian mucosae were mounted in Ussing chambers and the rate of acid secretion, short circuit current (I(sc)), transepithelial potential (V(t)) and resistance (R(t)) were recorded in the continuous presence of cadmium. Addition of cadmium (20 microM to 1mM) on the serosal but not luminal side of the mucosae resulted in inhibition of acid secretion and increase in NPPB-sensitive, chloride-dependent short circuit current. Remarkably, cadmium exerted its effects only on histamine-stimulated tissues. Experiments with TPEN, a cell-permeant chelator for heavy metals, showed that cadmium acts from the intracellular side of the acid secreting cells. Furthermore, cadmium-induced inhibition of acid secretion and increase in I(sc) cannot be explained by an action on: 1) H(2) histamine receptor, 2) Ca(2+) signalling 3) adenylyl cyclase or 4) carbonic anhydrase. Conversely, cadmium was ineffective in the presence of the H(+)/K(+)-ATPase blocker omeprazole suggesting that the two compounds likely act on the same target. Our findings suggest that cadmium affects the functionality of histamine-stimulated gastric mucosa by inhibiting the H(+)/K(+)-ATPase from the intracellular side. These data shed new light on the toxic effect of this dangerous environmental pollutant and may result in new avenues for therapeutic intervention in acute and chronic intoxication.

Gastric bicarbonate secretion

Gastric mucosa does not only secrete hydrochloric acid but it is also capable of secreting alkali, probably as part of a defence mechanism against acid-induced ulceration. Alkali secretion can be observed in gastric fundus mucosa if HC1 secretion is inhibited by blockers of histamine receptors (Flemstrom G. 1977, Takeuchi K. 1982). Which cells secrete alkali is still unknown. Amphibian gastric fundus mucosa consists of two major cell types: the oxyntopeptic cells (OC) which form the gastric glands and are known to secrete acid and the surface epithelial cells (SEC) which face the gastric lumen and have been assumed to secrete alkali possibly via an apically or basolaterally located CIVHCO3- exchanger (Flemstrom G. 1977, Takeuchi K. 1982). However, thus far no firm evidence for the localization and mechanism of alkali secretion is available.