Isolation of Zn2+ as an endogenous agonist of GPR39 from fetal bovine serum

We attempted to determine natural agonists of GPR39 in fetal bovine serum (FBS). FBS was conditioned to extract peptides and fractionated by two types of HPLC. The activity of each fraction was monitored by intracellular calcium mobilization. Then the purified active ingredient was analyzed by inductively coupled plasma mass spectrometry. In this fashion, Zn2+ ion was identified as an agonist of GPR39, though no peptidergic molecules were found. The calcium-mobilizing activity of Zn2+ was not abolished by pertussis toxin but was by a phospholipase C (PLC) inhibitor, U73122, indicating that the activity of GPR39 is mediated through the Gqalpha -PLC pathway. In addition, Zn2+ also activated mouse and rat GPR39, showing that the function of GPR39 as a Zn2+ receptor is conserved across species. This study is the first exploration of GPR39 agonists in FBS and indicates that GPR39 functions as a Gq-coupled Zn2+-sensing receptor.     

Novel Zn2+ Modulated GPR39 Receptor Agonists Do Not Drive Acute Insulin Secretion in Rodents

Type 2 diabetes (T2D) occurs when there is insufficient insulin release to control blood glucose, due to insulin resistance and impaired β-cell function. The GPR39 receptor is expressed in metabolic tissues including pancreatic β-cells and has been proposed as a T2D target. Specifically, GPR39 agonists might improve β-cell function leading to more adequate and sustained insulin release and glucose control. The present study aimed to test the hypothesis that GPR39 agonism would improve glucose stimulated insulin secretion in vivo. A high throughput screen, followed by a medicinal chemistry program, identified three novel potent Zn²⁺ modulated GPR39 agonists. These agonists were evaluated in acute rodent glucose tolerance tests. The results showed a lack of glucose lowering and insulinotropic effects not only in lean mice, but also in diet-induced obese (DIO) mice and Zucker fatty rats. It is concluded that Zn²⁺ modulated GPR39 agonists do not acutely stimulate insulin release in rodents.     

ZnR/GPR39 upregulation of K+/Cl−-cotransporter 3 in tamoxifen resistant breast cancer cells

Expression of the zinc receptor, ZnR/GPR39, is increased in higher grade breast cancer tumors and cells. Zinc, its ligand, is accumulated at larger concentrations in the tumor tissue and can therefore activate ZnR/GPR39-dependent Ca²⁺ signaling leading to tumor progression. The K⁺/Cl⁻ co-transporters (KCC), activated by intracellular signaling, enhance breast cancer cell migration and invasion. We asked if ZnR/GPR39 enhances breast cancer cell malignancy by activating KCC. Activation of ZnR/GPR39 by Zn²⁺ upregulated K⁺/Cl⁻ co-transport activity, measured using NH₄⁺ as a surrogate to K⁺ while monitoring intracellular pH. Upregulation of NH₄⁺ transport was monitored in tamoxifen resistant cells with functional ZnR/GPR39-dependent Ca²⁺ signaling but not in MCF-7 cells lacking this response. The NH₄⁺ transport was Na⁺-independent, and we therefore focused on KCC family members. Silencing of KCC3, but not KCC4, expression abolished Zn²⁺-dependent K⁺/Cl⁻ co-transport, suggesting that KCC3 is mediating upregulated NH₄⁺ transport. The ZnR/GPR39-dependent KCC3 activation accelerated scratch closure rate, which was abolished by inhibiting KCC transport with [(DihydroIndenyl) Oxy] Alkanoic acid (DIOA). Importantly, silencing of either ZnR/GPR39 or KCC3 attenuated Zn²⁺-dependent scratch closure. Thus, a novel link between KCC3 and Zn²⁺, via ZnR/GPR39, promotes breast cancer cell migration and proliferation.     

Zinc Released from Injured Cells Is Acting via the Zn2+-sensing Receptor,ZnR, to Trigger Signaling Leading to Epithelial Repair

A role for Zn²⁺ in accelerating wound healing is established, yet, the signaling pathways linking Zn²⁺ to tissue repair are not well known. We show that in the human HaCaT keratinocytes extracellular Zn²⁺ induces a metabotropic Ca²⁺ response that is abolished by silencing the expression of the G-protein-coupled receptor GPR39, suggesting that this Zn²⁺-sensing receptor, ZnR, is mediating the response. Keratinocytic-ZnR signaling is highly selective for Zn²⁺ and can be triggered by nanomolar concentrations of this ion. Interestingly, Zn²⁺ was also released following cellular injury, as monitored by a specific non-permeable fluorescent Zn²⁺ probe, ZnAF-2. Chelation of Zn²⁺ and scavenging of ATP from conditioned medium, collected from injured epithelial cultures, was sufficient to eliminate the metabotropic Ca²⁺ signaling. The signaling triggered by Zn²⁺, via ZnR, or by ATP further activated MAP kinase and induced up-regulation of the sodium/proton exchanger NHE1 activity. Finally, activation of ZnR/GPR39 signaling or application of ATP enhanced keratinocytes scratch closure in an in vitro model. Thus our results indicate that extracellular Zn²⁺, which is either applied or released following injury, activates ZnR/GPR39 to promote signaling leading to epithelial repair.     

GPR39 up-regulation after selective antidepressants

Recent studies indicated that zinc activates neural transmission via the GPR39 Zn²⁺-sensing receptor. Preclinical and clinical studies demonstrated the antidepressant properties of zinc. To investigate whether the GPR39 receptor is involved in the mechanism of antidepressant action, we measured the expression of the GPR39 receptor (Western Blot) in the frontal cortex of mice treated intraperitoneally with imipramine (30 mg/kg), escitalopram (4 mg/kg), reboxetine (10 mg/kg) or bupropion (15 mg/kg) for 14 days. The present study shows the up-regulation of the GPR39 receptor protein level after escitalopram (by 290%), reboxetine (by 816%) and bupropion (by 272%), but not imipramine treatment. This is the first report to indicate the involvement of the GPR39 Zn²⁺-sensing receptor in the antidepressant effect of selective monoamine reuptake inhibitors.     

Extracellular pH Regulates Zinc Signaling via an Asp Residue of the Zinc-sensing Receptor (ZnR/GPR39)

Zinc activates a specific Zn²⁺-sensing receptor, ZnR/GPR39, and thereby triggers cellular signaling leading to epithelial cell proliferation and survival. Epithelial cells that express ZnR, particularly colonocytes, face frequent changes in extracellular pH that are of physiological and pathological implication. Here we show that the ZnR/GPR39-dependent Ca²⁺ responses in HT29 colonocytes were maximal at pH 7.4 but were reduced by about 50% at pH 7.7 and by about 62% at pH 7.1 and were completely abolished at pH 6.5. Intracellular acidification did not attenuate ZnR/GPR39 activity, indicating that the pH sensor of this protein is located on an extracellular domain. ZnR/GPR39-dependent activation of extracellular-regulated kinase (ERK)1/2 or AKT pathways was abolished at acidic extracellular pH of 6.5. A similar inhibitory effect was monitored for the ZnR/GPR39-dependent up-regulation of Na⁺/H⁺ exchange activity at pH 6.5. Focusing on residues putatively facing the extracellular domain, we sought to identify the pH sensor of ZnR/GPR39. Replacing the histidine residues forming the Zn²⁺ binding site, His¹⁷ or His¹⁹, or other extracellular-facing histidines to alanine residues did not abolish the pH dependence of ZnR/GPR39. In contrast, replacing Asp³¹³ with alanine resulted in similar Ca²⁺ responses triggered by ZnR/GPR39 at pH 7.4 or 6.5. This mutant also showed similar activation of ERK1/2 and AKT pathways, and ZnR-dependent up-regulation of Na⁺/H⁺ exchange at pH 7.4 and pH 6.5. Substitution of Asp³¹³ to His or Glu residues restored pH sensitivity of the receptor. This indicates that Asp³¹³, which was shown to modulate Zn²⁺ binding, is an essential residue of the pH sensor of GPR39. In conclusion, ZnR/GPR39 is tuned to sense physiologically relevant changes in extracellular pH that thus regulate ZnR-dependent signaling and ion transport activity.     

Signaling pathways involved in human sperm hyperactivated motility stimulated by Zn2+

To fertilize the egg, sperm cells must reside in the female reproductive tract for several hours during which they undergo chemical and motility changes collectively called capacitation. During capacitation, the sperm develop a unique type of motility known as hyperactivated motility (HAM). The semen contains Zn²⁺ in millimolar concentrations, whereas in the female reproductive tract, the concentration is around 1 µM. In this study, we characterize the role of Zn²⁺ in human sperm capacitation focusing on its effect on HAM. Western blot analysis revealed the presence of GPR39‐type Zn‐receptor localized mainly in the sperm tail. Zn²⁺ at micromolar concentration stimulates HAM, which is mediated by a cascade involving GPR39–adenylyl cyclase (AC)–cyclic AMP (cAMP)–protein kinase A–tyrosine kinase Src (Src)–epidermal growth factor receptor and phospholipase C. Both the transmembrane AC and the soluble‐AC are involved in the stimulation of HAM by Zn²⁺. The development of HAM is precisely regulated by cAMP, in which relatively low concentration (5–10 µM) stimulated HAM, whereas at 30 µM no stimulation occurred. A similar response was seen when different concentrations of Zn²⁺ were added to the cells; low Zn²⁺ stimulated HAM, whereas at relatively high Zn²⁺, no effect was seen. We further demonstrate that the Ca²⁺‐channel CatSper involved in Zn²⁺‐stimulated HAM. These data support a role for extracellular Zn²⁺ acting via GPR39 to regulate signaling pathways in sperm capacitation, leading to HAM induction.     

Pharmacological doses of Zn<Superscript>2+</Superscript> induce a muscarinic cholinergic supersensitivity

The goal of this study was to evaluate the effect of chronic Zn²⁺ administration (1 mg/kg/day for 1 month) in Sprague-Dawley rats (n=11) on motility and rearing behaviors (number of events/10 min measured in motility cage), on memory (percentage of failures using a footshock double T maze), on the number of muscarinic receptors (using [³H]-QNB as a marker) and on the cholinacetyltransferase (Chat) activity (determined by Fonnun's method) in various brain areas (striatum, hippocampus and frontal cortex), as compared with saline-treated rats (n=10). Our results showed that Zn²⁺ induced a decrease in rearing (control: 24.6±3; Zn²⁺: 15.91±2.19) and in locomotor activity (control: 37±3.79; Zn²⁺: 25±4.37), a decrease in failures during memory trials (control: 26.12±5.6; Zn²⁺: 5.33±2.71) and an increase in muscarinic receptor density (fmol/mg) in the striatum (control: 539±6.18; Zn²⁺: 720±14.69), hippocampus (control: 396±7.41; Zn²⁺: 458±5.05) and frontal cortex (control: 506±10.28; Zn²⁺: 716±16.54). Chat activity (pmol/mg/min) was decreased only in the striatum (control: 4,240±158; Zn²⁺: 2,311±69). We conclude that Zn²⁺ induces a cholinergic functional supersensitivity which is related to receptor upregulation.     

Inhibition of Na+/K+-ATPase and Mg2+-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na⁺/K⁺-ATPase and Mg²⁺-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu²⁺, Zn²⁺, Fe²⁺ and.Co²⁺) and heavy metals (Hg²⁺and Pb²⁺) ions were studied. All investigated metals produced a larger maximum inhibition of Na⁺/K⁺-ATPase than Mg²⁺-ATPase activity. The free concentrations of the key species (inhibitor, MgATP^{2 ?} , MeATP^{2 ?} ) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu²⁺/Fe²⁺ or Hg²⁺/Pb²⁺caused additive inhibition, while Cu²⁺/Zn²⁺ or Fe²⁺/Zn²⁺ inhibited Na⁺/K⁺-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu²⁺/Fe²⁺ or Cu²⁺/Zn²⁺ inhibited Mg²⁺-ATPase activity synergistically, while Hg²⁺/Pb²⁺ or Fe²⁺/Zn²⁺ induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na⁺/K⁺-ATPase activity by reducing the maximum velocities (V_max) rather than the apparent affinity (K_m) for substrate MgATP^2-, implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg²⁺-ATPase activity by Zn²⁺, Fe²⁺ and Co²⁺ as well as kinetic analysis indicated two distinct Mg²⁺-ATPase subtypes activated in the presence of low and high MgATP^{2 ?} concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na⁺/K⁺-ATPase with various potencies. Furthermore, these ligands also reversed Na⁺/K⁺-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg²⁺-induced inhibition was not obtained.     

Differential tissue and ligand-dependent signaling of GPR109A receptor: Implications for anti-atherosclerotic therapeutic potential

Until recently, the anti-atherosclerotic effects of niacin were attributed primarily to its lipid modification properties mediated by adipocyte G-protein coupled receptor GPR109A, though recent studies have raised significant doubts about this mechanism. In fact, in rodents it has recently been demonstrated that niacin inhibits progression of atherosclerosis through actions on immune cells, particularly via macrophage-expressed GPR109A, independent of lipid-modifying properties. Here, we studied GPR109A signal transduction in human Langerhans cells, macrophages and adipocytes. We find that the consequences of receptor activation are profoundly influenced by cellular context and that ligand-biased signaling significantly impacts functionally relevant signaling. In Langerhans cells, niacin initiates GPR109A-mediated signaling pathways (Erk1/2 and Ca^2 +) responsible for the release of vasodilatory prostanoids, while the synthetic GPR109A agonist MK-0354 fails to elicit any signaling, providing a mechanistic basis for the latter compound's inability to cause flushing. While GPR109A mediates inhibition of cAMP in adipocytes, in macrophages GPR109A signaling via G_βγ subunits results in paradoxical augmentation of intracellular cAMP levels. Also, in macrophages niacin and GPR109A full agonists induce Erk1/2 and Ca^2 + signaling, release of prostanoids, upregulation of cholesterol transporters ABCA1 and ABCG1 and stimulation of reverse cholesterol transport in GPR109A dependent manner. A mechanism is presented in which signals from the autocrine action of released prostanoids and G_i protein mediated cAMP augmentation are integrated leading to modulation of reverse cholesterol transport regulatory components. These studies provide key insights into mechanisms by which GPR109A may influence cholesterol efflux in macrophages; a process that may be at least partially responsible for niacin's anti-atherosclerotic activity. MK-0354 does not induce niacin-like GPR109A signaling in macrophages, suggesting that biased agonists devoid of the flushing side-effect may also lack properties required for macrophage-mediated anti-atherosclerotic effects.     

The effect of Zn2+ on Pelodiscus sinensis creatine kinase: unfolding and aggregation studies

We studied the effects of Zn²⁺ on creatine kinase from the Chinese soft-shelled turtle, Pelodiscus sinensis (PSCK). Zn²⁺ inactivated the activity of PSCK (IC₅₀?=?.079?±?.004?mM) following first-order kinetics consistent with multiple phases. The spectrofluorimetry results showed that Zn²⁺ induced significant tertiary structural changes of PSCK with exposure to hydrophobic surfaces and that Zn²⁺ directly induced PSCK aggregation. The addition of osmolytes such as glycine, proline, and liquaemin successfully blocked PSCK aggregation, recovering the conformation and activity of PSCK. We measured the ORF gene sequence of PSCK by rapid amplification of cDNA end and simulated the 3D structure of PSCK. The results of molecular dynamics simulations showed that eight Zn²⁺ bind to PSCK and one Zn²⁺ is predicted to bind in a plausible active site of creatine and ATP. The interaction of Zn²⁺ with the active site could mostly block the activity of PSCK. Our study provides important insight into the action of Zn²⁺ on PSCK as well as more insights into the PSCK folding and ligand-binding mechanisms, which could provide important insight into the metabolic enzymes of P. sinensis.     

Regulation of Glial Na+/K+-ATPase by Serotonin: Identification of Participating Receptors

The purpose of the present study was the characterization of the receptors participating in the regulatory mechanism of glial Na⁺/K⁺-ATPase by serotonin (5-HT) in rat brain. The activity of the Na⁺ pump was measured in four brain regions after incubation with various concentrations of serotoninergic agonists or antagonists. A concentration-dependent increase in enzyme activity was observed with the 5-HT_1A agonist R (+)-2-dipropylamino-8-hydroxy-1,2,3, 4-tetrahydronaphthalene hydrobromide (8-OH-DPAT) in homogenates or in glial membrane enriched fractions from cerebral cortex and in hippocampus. Spiperone, a 5-HT_1A antagonist, completely inhibited the response to 8-OH-DPAT but had no effect on Na⁺/K⁺-ATPase activity in cerebellum where LSD, a 5-HT₆ agonist, elicited a dose-dependent response similar to that of 5-HT. In brainstem, a lack of reponse to 5-HT and other agonists was confirmed. Altogether, these results show that serotonin modulates glial Na⁺/K⁺-ATPase activity in the brain, apparently not through only one type of 5-HT receptor. It seems that the receptor system involved is different according to the brain region. In cerebral cortex, the response seems to be mediated by 5-HT_1A as well as in hippocampus but not in cerebellum where 5-HT₆ appears as the receptor system involved.     

Signaling pathways involved in human sperm hyperactivated motility stimulated by Zn2+

To fertilize the egg, sperm cells must reside in the female reproductive tract for several hours during which they undergo chemical and motility changes collectively called capacitation. During capacitation, the sperm develop a unique type of motility known as hyperactivated motility (HAM). The semen contains Zn²⁺ in millimolar concentrations, whereas in the female reproductive tract the concentration is around 1 µM. In this study, we characterize the role of Zn ²⁺ in human sperm capacitation focusing on its effect on HAM. Western blot analysis revealed the presence of G protein‐coupled receptor 39 (GPR39) type Zn‐receptor localized mainly in the sperm tail. Zn ²⁺ at micromolar concentration stimulates HAM, which is mediated by a cascade involving GPR39‐AC‐cAMP‐PKA‐Src‐EGFR and phospholipase C. Both the transmembrane adenylyl cyclase (AC) and the soluble‐AC are involved in the stimulation of HAM by Zn ²⁺. The development of HAM is precisely regulated by cyclic adenosine monophosphate, in which relatively low concentration (5–10 µM) stimulated HAM, whereas at 30 µM no stimulation occurred. A similar response was seen when different concentrations of Zn ²⁺ were added to the cells; low Zn ²⁺ stimulated HAM, whereas at relatively high Zn ²⁺, no effect was seen. We further demonstrate that the Ca ²⁺‐channel CatSper involved in Zn ²⁺‐stimulated HAM. These data support a role for extracellular Zn ²⁺ acting via GPR39 to regulate signaling pathways in sperm capacitation, leading to HAM induction.     

High affinity Zn2+ inhibitory site(s) for the trypsin-like peptidase of the 20S proteasome

The effect of Zn²⁺ on three major peptidase activities of the 20S proteasome purified from Xenopus oocytes was kinetically investigated. An extremely low concentration of Zn²⁺ (μM range) strongly inhibited the trypsin-like activity of the 20S proteasome which was fully recoverable by the addition of EDTA. The concentration of Zn²⁺ for half-maximum inhibition (K_0.5) was 0.60 μM which was at least 10 times lower than that of any other divalent cation tested and essentially the same as for proteasomes purified from various other organisms indicating that the inhibition is highly Zn²⁺-specific, reversible, and common to the proteasome regardless of its source. Zn²⁺ at concentrations below 100 μM instantaneously activated the chymotrypsin-like and PGPH activities, and the Zn²⁺ concentration for half-maximum activation was found to be 42-48 μM. These activities were time-dependently inactivated by submillimolar concentrations of Zn²⁺. The inactivation rates were dependent on the concentration of Zn²⁺ and reached a maximum of 1.60-2.40 min⁻¹ for the three peptidase activities under the conditions used. The Zn²⁺ concentration for half-maximum inactivation was found to be 0.70-1.23 mM. This time-dependent inactivation was not reversed by the addition of EDTA or DTT and might not be accompanied by the dissociation of subunits of the 20S proteasome indicating that all activities are inactivated by an identical phenomenon. These results reveal the three types of effects of Zn²⁺ on the 20S proteasome.     

The zinc sensing receptor,ZnR/GPR39, controls proliferation and differentiation of colonocytes and thereby tight junction formation in the colon

The intestinal epithelium is a renewable tissue that requires precise balance between proliferation and differentiation, an essential process for the formation of a tightly sealed barrier. Zinc deficiency impairs the integrity of the intestinal epithelial barrier and is associated with ulcerative and diarrheal pathologies, but the mechanisms underlying the role of Zn²⁺ are not well understood. Here, we determined a role of the colonocytic Zn²⁺ sensing receptor, ZnR/GPR39, in mediating Zn²⁺-dependent signaling and regulating the proliferation and differentiation of colonocytes. Silencing of ZnR/GPR39 expression attenuated Zn²⁺-dependent activation of ERK1/2 and AKT as well as downstream activation of mTOR/p70S6K, pathways that are linked with proliferation. Consistently, ZnR/GPR39 silencing inhibited HT29 and Caco-2 colonocyte proliferation, while not inducing caspase-3 cleavage. Remarkably, in differentiating HT29 colonocytes, silencing of ZnR/GPR39 expression inhibited alkaline phosphatase activity, a marker of differentiation. Furthermore, Caco-2 colonocytes showed elevated expression of ZnR/GPR39 during differentiation, whereas silencing of ZnR/GPR39 decreased monolayer transepithelial electrical resistance, suggesting compromised barrier formation. Indeed, silencing of ZnR/GPR39 or chelation of Zn²⁺ by the cell impermeable chelator CaEDTA was followed by impaired expression of the junctional proteins, that is, occludin, zonula-1 (ZO-1) and E-cadherin. Importantly, colon tissues of GPR39 knockout mice also showed a decrease in expression levels of ZO-1 and occludin compared with wildtype mice. Altogether, our results indicate that ZnR/GPR39 has a dual role in promoting proliferation of colonocytes and in controlling their differentiation. The latter is followed by ZnR/GPR39-dependent expression of tight junctional proteins, thereby leading to formation of a sealed intestinal epithelial barrier. Thus, ZnR/GPR39 may be a therapeutic target for promoting epithelial function and tight junction barrier integrity during ulcerative colon diseases.The intestinal epithelial barrier, located at the interface between the body and the digestive system lumen, facilitates electrolyte and nutrient absorption while protecting against permeation of antigenic, toxic or infectious materials.¹ Stressful conditions in the intestinal lumen require rapid and continuous renewal of the epithelial layer,² which occur through tightly regulated and balanced proliferation, migration and differentiation processes.³Zn²⁺ deficiency leads to a reduction of epithelial cell proliferation rate, thus limiting renewal of gastrointestinal mucosa, but it also impairs barrier function, increases permeability and enhances cell death.^{4, 5} Zn²⁺ supplementation reverses these processes and restores integrity of colon epithelium.^{5, 6, 7} Importantly, Zn²⁺ supplementation reduces diarrheal disease activity index^{8, 9} and is effective in decreasing severity of histological and clinical scores in in vivo models of ulcerative colitis.^{10, 11} Despite these observations, the signaling pathways linking Zn²⁺ to intestinal epithelial function or integrity are poorly understood.We identified a Zn²⁺ sensing receptor, ZnR, which links between changes in extracellular Zn²⁺ and major intracellular signaling pathways. Functional studies indicated that ZnR is a Gq-coupled receptor, which triggers inositol 1,4,5-trisphosphate (IP3)-dependent release of intracellular Ca²⁺,¹² leading to activation of mitogen-activated protein (MAP) and phosphoinositide 3 (PI3) kinase pathways.^{13, 14} Recent studies showed that the Zn²⁺-dependent Ca²⁺ rise is mediated by the G-protein-coupled receptor GPR39, in colonocytes.¹⁵ ZnR/GPR39 mediates recovery from acidic pH by upregulation of Na⁺/H⁺ exchange in colonocytic and keratinocytic cell lines as well as in native colon epithelial cells, demonstrating the important role of this receptor in epithelial physiology.^{13, 15, 16} Finally, ZnR/GPR39 enhances colonocytes survival from butyrate induced stress.¹⁵ Notably, GPR39 knockout (KO) mice show symptoms of Zn²⁺ deficiency: accelerated gastric emptying and increased fecal secretion.¹⁷ However how Zn²⁺ or ZnR/GPR39 signaling promote colon epithelial function is not well understood. Here we show that ZnR/GPR39 regulates extracellular Zn²⁺-dependent proliferation and differentiation processes in colonocytes. Furthermore, we show that ZnR/GPR39 is essential for expression and localization of tight junction proteins in vitro and in vivo, and thereby regulates the formation of the colon epithelial barrier.     

Zinc inhibition of chloride efflux from skeletal muscle ofRana pipiens and its modification by external pH and chloride activity

Summary Efflux of³⁶Cl^– from frog sartorius muscles equilibrated in depolarizing solutions was measured. Cl^– efflux consists of a component present at low pH and a pH-dependent component which increases as external pH increases. In depolarized muscles fromRana pipiens, the pH-dependent Cl^– efflux has an apparent pK _a near 6.4.The reduction of Cl^– efflux by external Zn²⁺ was determined at different external pHs and chloride activities. The effect of external chloride activity on the pH-dependent Cl^– efflux was also examined.At pH 6.5 and a membrane potential of –22 mV, increasing external Cl^– activity from 0.108 to 0.28m decreased inhibition of the pH-dependent Cl^– efflux at all activities of Zn²⁺. The Zn²⁺ activity needed to reduce Cl^– efflux by half increased from 0.39×10^–3 to 2.09×10^–3 m. By contrast, external Cl^– activity had no measurable effect on the apparent pK _a of the pH-dependent efflux.At constant Cl^– activity less than 0.21m, increasing external pH from 6.5 to 7.5 decreased inhibition by low Zn²⁺ activities with either a slight increase or no change in the Zn²⁺ activity producing half-inhibition. In other words, for relatively low Cl^– activities, protection against inhibition of Cl^– efflux by low Zn²⁺ activities was obtained by raising, not lowering, external pH; this is not what is expected if H⁺ and Zn²⁺ ions compete at the same site to produce inhibition of Cl^– efflux. We conclude that Zn²⁺ and low pH inhibit Cl^– efflux by separate and distinct mechanisms.By contrast, the protection against Zn²⁺ inhibition produced by high external Cl^– activity (0.28m) was partially reversed by raising external pH from 6.5 to 7.5 at all Zn²⁺ activities. The half-inhibition Zn²⁺ activity decreased from 2.09×10^–3 to 0.68×10^–3 m.The results can be simulated quantitatively by a model in which single Cl^– channel elements are in equilibrium with sextets of associated single-channel elements, each sextet having a conductance six times that of a single-channel element. The association into sextets is promoted by OH^– or Cl^– binding to a control site on the single-channel elements. Both the single Cl^– channel element and the sextet of Cl^– channel elements are closed when this same control site instead binds ZnOH⁺. The sextet has a much higher affinity for ZnOH⁺ than does the single Cl^– channel element.     

Protease-Activated Receptor 2 Activation Inhibits N-Type Ca2+ Currents in Rat Peripheral Sympathetic Neurons

The protease-activated receptor (PAR)-2 is highly expressed in endothelial cells and vascular smooth muscle cells. It plays a crucial role in regulating blood pressure via the modulation of peripheral vascular tone. Although several mechanisms have been suggested to explain PAR-2-induced hypotension, the precise mechanism remains to be elucidated. To investigate this possibility, we investigated the effects of PAR-2 activation on N-type Ca²⁺ currents (I_Ca-N) in isolated neurons of the celiac ganglion (CG), which is involved in the sympathetic regulation of mesenteric artery vascular tone. PAR-2 agonists irreversibly diminished voltage-gated Ca²⁺ currents (I_Ca), measured using the patch-clamp method, in rat CG neurons, whereas thrombin had little effect on I_Ca. This PAR-2-induced inhibition was almost completely prevented by ω-CgTx, a potent N-type Ca²⁺ channel blocker, suggesting the involvement of N-type Ca²⁺ channels in PAR-2-induced inhibition. In addition, PAR-2 agonists inhibited I_Ca–N in a voltage-independent manner in rat CG neurons. Moreover, PAR-2 agonists reduced action potential (AP) firing frequency as measured using the current-clamp method in rat CG neurons. This inhibition of AP firing induced by PAR-2 agonists was almost completely prevented by ω-CgTx, indicating that PAR-2 activation may regulate the membrane excitability of peripheral sympathetic neurons through modulation of N-type Ca²⁺ channels. In conclusion, the present findings demonstrate that the activation of PAR-2 suppresses peripheral sympathetic outflow by modulating N-type Ca²⁺ channel activity, which appears to be involved in PAR-2-induced hypotension, in peripheral sympathetic nerve terminals.     

Inhibition of GABAA ligand-gated Cl− channels by zinc in adult rat brain: A regional study

Zinc (Zn²⁺) was shown to invariably inhibit muscimol-stimulated³⁶Cl⁻ uptake by synaptoneurosomes in the cerebral cortex, hippocampus and cerebellum. The Zn²⁺ sensitivity of the GABA_A receptor-gated³⁶Cl⁻ uptake in the cerebral cortex was comparable to that in the hippocampus, whereas the uptake in the cerebellum was less sensitive to Zn²⁺. Although diazepam-potentiation of muscimol-stimulated³⁶Cl⁻ uptake was unaltered by 100 μM Zn²⁺ in the cerebellum. Zn²⁺ inhibited [³H]diazepam binding significantly at 1 mM in the cerebral cortex and cerebellum, whereas Ni²⁺ increased the binding in a concentration-dependent manner in both regions. Although lower concentrations of Zn²⁺ did not affect [³H]Ro 15-4513 binding to diazepam-sensitive sites, higher concentrations of Zn²⁺ increased the binding in both regions. Unlike the diazepam-sensitive sites the diazepam-insensitive [³H]Ro 15-4513 binding was not affected by Zn²⁺ or Ni²⁺ at any of the tested concentrations. These results suggest that the GABA_A ligand-gated Cl⁻ flux and its diazepam-potentiation are heterogeneously modulated in various brain regions. It is also suggested that cerebellar diazepam-insensitive [³H]Ro 15-4513 binding sites are insensitive to Zn²⁺ and Ni²⁺.     

Negative Regulation of CFTR Activity by Extracellular ATP Involves P2Y2 Receptors in CFTR-expressing CHO Cells

Extracellular nucleotides exert autocrine/paracrine effects on ion transport by activating P2 receptors. We studied the effects of extracellular ATP and UTP on the cystic fibrosis transmembrane conductance regulator (CFTR) channel stably expressed in Chinese Hamster Ovary cells (CHO-BQ1 cells). CFTR activity was measured using the (¹²⁵I) iodide efflux technique and whole-cell patch-clamp recording in response to either forskolin or xanthine derivatives. Using RT-PCR and intracellular calcium concentration ([Ca²⁺]_i) measurement, we showed that CHO-BQ1 cells express P2Y2 but not P2Y4 receptors. While ATP and UTP induced similar increases in [Ca²⁺]_i, pre-addition by one of these two agonists desensitized the response for the other, suggesting that ATP- and UTP-induced [Ca²⁺]_i increases were mediated by a common receptor, which was identified as the P2Y2 subtype. CFTR activity was reduced by ATP and UTP but not by ADP or adenosine applications. This inhibitory effect of ATP on CFTR activity was not due to a change in cAMP level. Furthermore, CFTR activation by forskolin or IBMX failed to promote [Ca²⁺]_i increase, suggesting that CFTR activation did not generate an ATP release large enough to stimulate P2Y2 receptors. Taken together, our results show that endogenous P2Y2 receptor activation downregulates CFTR activity in a cAMP-independent manner in CHO cells. B. Marcet and V. Chappe contributed equally to this work.