TRPV4 Stimulation Releases ATP via Pannexin Channels in Human Pulmonary Fibroblasts.

We previously described several ionic conductances in human pulmonary fibroblasts, including one activated by two structurally distinct TRPV4 (transient receptor potential, vanilloid-type, subtype 4)-channel agonists: 4αPDD (4α-phorbol-12,13-didecanoate) and GSK1016790A. However, the TRPV4-activated current exhibited peculiar properties: it developed slowly over many minutes, exhibited reversal potentials that could vary by tens of millivolts even within a given cell, and was not easily reversed by subsequent addition of two distinct TRPV4-selective blockers (RN-1734 and HC-067047). In this study, we characterized that conductance more carefully. We found that 4αPDD stimulated a delayed release of ATP into the extracellular space, which was reduced by genetic silencing of pannexin expression, and that the 4αPDD-evoked current could be blocked by apyrase (which rapidly degrades ATP) or by the P2Y purinergic receptor/channel blocker pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), and could be mimicked by exogenous addition of ATP. In addition, we found that the 4αPDD-evoked current was blocked by pretreatment with RN-1734 or HC-067047, by Gd3+ or La3+, or by two distinct blockers of pannexin channels (carbenoxolone and probenecid), but not by a blocker of connexin hemichannels (flufenamic acid). We also found expression of TRPV4- and pannexin-channel proteins. 4αPDD markedly increased calcium flashing in our cells. The latter was abrogated by the P2Y channel blocker PPADS, and the 4αPDD-evoked current was eliminated by loading the cytosol with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or by inhibiting Ca2+/calmodulin-sensitive kinase II using KN93. Altogether, we interpret these findings as suggesting that 4αPDD triggers the release of ATP via pannexin channels, which in turn acts in an autocrine and/or paracrine fashion to stimulate PPADS-sensitive purinergic receptors on human pulmonary fibroblasts.

Molecular analysis of amantadine-resistant influenza A (H1N1 pdm09) virus isolated from slum dwellers of Dhaka, Bangladesh.

Influenza is a highly contagious viral infection associated with excessive hospitalizations and deaths throughout the world. Continuous antigenic shift and drift is not only responsible for this devastating effect of influenza but also causes ineffectiveness of antiviral drugs and vaccines. In this study, we investigated the effectiveness of ribavirin, oseltamivir, and amantadine drugs in vitro against nine influenza A isolates collected during June 2012-August 2013 from different slums in Dhaka city. The effectiveness of these drugs was determined by measuring the inhibition of virus-induced cytopathic effect on MDCK cells through MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Our data showed that all nine influenza isolates (6 H1N1 pdm09 and 3 H3N2 subtypes) were completely susceptible to ribavirin (The 50% effective concentrations, EC50 3.0 µg/ml) and oseltamivir (EC50 0.35 µg/ml). When influenza A infection was challenged with amantadine drug, eight out of nine isolates (88%) demonstrated susceptibility to amantadine drug (EC50 0.30 µg/ml) while one H1N1 pdm09 isolate exhibited higher EC50 value (>10 µg/ml) beyond the cell tolerance level of drug (>5 µg/ml). Genetic analysis of transmembrane matrix protein 2 (M2), which is a target for the amantadine drug and vital for viral replication, showed a substitution of amino acid at position 31(S31 N) of that amantadine-resistant isolate indicating the possible reason of amantadine drug resistance.

Electrophysiological characterization of voltage-dependent calcium currents and TRPV4 currents in human pulmonary fibroblasts.

We have presented indirect evidence of a key role for voltage-dependent Ca(2+) currents in TGFß-induced synthetic function in human pulmonary fibroblast (HPF), as well as in bleomycin-induced pulmonary fibrosis in mice. Others, however, have provided indirect evidence for transient receptor potential vanilloid 4 (TRPV4) channels in both of those effects. Unfortunately, definitive electrophysiological descriptions of both currents in HPFs have been entirely lacking. In this study, we provide the first direct electrophysiological and pharmacological evidence of the currents in HPFs at rest and during overnight stimulation with TGFß. These currents include a Ca(2+)-dependent K(+) current, a TRPV4 current, a chloride current, and an L-type voltage-dependent Ca(2+) current. Evidence for the TRPV4 current include activation of a large-conductance change by two putatively TRPV4-selective agonists (4α-phorbol-12,13-didecanoate; GSK1016790A), with a reversal potential near 0 mV, partial sensitivity to two different TRPV4-selective blockers (RN1734; HC067047), and partial reduction following removal of external Na(+) Substantial reduction of the evoked current was seen following the coapplication of RN1734, DIDS, and niflumic acid, suggesting that a chloride current is also involved. The voltage-dependent Ca(2+) current is found to be "L-type" in nature, as indicated by the voltage and time dependence of its activation, deactivation, and inactivation properties, and by its pharmacology (sensitivity to replacement with barium and inhibition by nifedipine, verapamil, or mibefradil). We also found that overnight treatment with TGFß evoked a periodic current (inward at negative holding potentials, with reversal potential near 0 mV), which is sufficient to trigger the voltage-dependent Ca(2+) currents and, thereby, account for the rhythmic Ca(2+) oscillations, which we have described previously in these cells.

Mechanisms of VIP-induced inhibition of the lymphatic vessel pump.

Lymphatic vessels serve as a route by which interstitial fluid, protein and other macromolecules are returned to the blood circulation and immune cells and antigens gain access to lymph nodes. Lymph flow is an active process promoted by rhythmical contraction-relaxation events occurring in the collecting lymphatic vessels. This lymphatic pumping is an intrinsic property of the lymphatic muscles in the vessel wall and consequent to action potentials. Compromised lymphatic pumping may affect lymph and immune cell transport, an action which could be particularly detrimental during inflammation. Importantly, many inflammatory mediators alter lymphatic pumping. Vasoactive intestinal peptide (VIP) is a neuro- and immuno-modulator thought to be released by nerve terminals and immune cells in close proximity to lymphatic vessels. We demonstrated the presence of the peptide in lymphatic vessels and in the lymph and examined the effects of VIP on mesenteric collecting lymphatic vessels of the guinea pig using pharmacological bioassays, intracellular microelectrode electrophysiology, immunofluorescence and quantitative real-time PCR. We showed that VIP alters lymphatic pumping by decreasing the frequency of lymphatic contractions and hyperpolarizing the lymphatic muscle membrane potential in a concentration-dependent manner. Our data further suggest that these effects are mainly mediated by stimulation of the VIP receptor VPAC2 located on the lymphatic muscle and the downstream involvement of protein kinase A (PKA) and ATP-sensitive K⁺ (KATP) channels. Inhibition of lymphatic pumping by VIP may compromise lymph drainage, oedema resolution and immune cell trafficking to the draining lymph nodes.

Effect of Gene 33/Mig6/ERRFI1 on hexavalent chromium-induced transformation of human bronchial epithelial cells depends on the length of exposure.

Hexavalent chromium (Cr(VI)) compounds are environmental and occupational lung carcinogens. The present study followed the chronic effect of Cr(VI) on the neoplastic transformation of BEAS-2B lung bronchial epithelial cells with or without deletion of Gene 33 (Mig6, EFFRI1), a multifunctional adaptor protein. We find that Gene 33-deleted cells exhibit increased anchorage-independent growth compared to control cells after transformed by 8-week but not 24-week Cr(VI) exposure. Gene 33-deleted cells show a higher level of cell proliferation and are more resistant to acute Cr(VI) toxicity compared to control cells after transformed by 8-week but not 24-week Cr(VI) exposure, despite that 24-week-transformed cells have increased resistance to acute Cr(VI) toxicity. However, Gene 33-deleted cells show increased migration after transformed by both 8-week and 24-week Cr(VI) exposures. Furthermore, only cells transformed by 24 weeks of Cr(VI) exposure can form subcutaneous tumors in nude mice. Although no significant difference in the size of tumors formed by the two cell types, there is a marked difference in the histological manifestation and more MMP3 expression in tumors from Gene 33-deleted cells. Our results demonstrate progressive neoplastic transformation of BEAS-2B cells and the adaptation of these cells to Gene 33 deletion during chronic exposure to Cr(VI).

Stability of diagnostic rate in a cohort of 38,813 colorectal polyp specimens and implications for histomorphology and statistical process control.

This work sought to quantify pathologists' diagnostic bias over time in their evaluation of colorectal polyps to assess how this may impact the utility of statistical process control (SPC). All colorectal polyp specimens(CRPS) for 2011-2017 in a region were categorized using a validated free text string matching algorithm. Pathologist diagnostic rates (PDRs) for high grade dysplasia (HGD), tubular adenoma (TA_ad), villous morphology (TVA + VA), sessile serrated adenoma (SSA) and hyperplastic polyp (HP), were assessed (1) for each pathologist in yearly intervals with control charts (CCs), and (2) with a generalized linear model (GLM). The study included 64,115 CRPS. Fifteen pathologists each interpreted > 150 CRPS/year in all years and together diagnosed 38,813. The number of pathologists (of 15) with zero or one (p < 0.05) outlier in seven years, compared to their overall PDR, was 13, 9, 9, 5 and 9 for HGD, TVA + VA, TA_ad, HP and SSA respectively. The GLM confirmed, for the subset where pathologists/endoscopists saw > 600 CRPS each(total 52,760 CRPS), that pathologist, endoscopist, anatomical location and year were all strongly correlated (all p < 0.0001) with the diagnosis. The moderate PDR stability over time supports the hypothesis that diagnostic rates are amendable to calibration via SPC and outcome data.

Agonist function of the recombinant alpha 4 beta 3 delta GABAA receptor is dependent on the human and rat variants of the alpha 4-subunit.

1. It is known that the alpha(4)-subunit is likely to occur in the brain predominantly in alpha(4)beta(3)delta receptors at extrasynaptic sites. Recent studies have revealed that the alpha(1)-, alpha(4)-, gamma(2)- and delta-subunits may colocalize extrasynaptically in dentate granule cells of the hippocampus. In the present study, we characterized a series of recombinant GABA(A) receptors containing human (H) and rat (R) alpha(1)/alpha(4)-, beta(2)/beta(3)- and gamma(2S)/delta-subunits in Xenopus oocytes using the two-electrode voltage-clamp technique. 2. Both H alpha(1)beta(3)delta and H alpha(4)beta(3)gamma(2S) receptors were sensitive to activation by GABA and pentobarbital. Contrary to earlier findings that the alpha(4)beta(3)delta combination was more sensitive to agonist action than the alpha(4)beta(3)gamma(2S) receptor, we observed extremely small GABA- and pentobarbital-activated currents at the wild-type H alpha(4)beta(3)delta receptor. However, GABA and pentobarbital activated the wild-type R alpha(4)beta(3)delta receptor with high potency (EC(50) = 0.5 +/- 0.7 and 294 +/- 5 micromol/L, respectively). 3. Substituting the H alpha(4) subunit with R alpha(4) conferred a significant increase in activation on the GABA and pentobarbital site in terms of reduced EC(50) and increased I(max). When the H alpha(4) subunit was combined with the R beta(3) and R delta subunit in a heteropentameric form, the amplitude of GABA- and pentobarbital-activated currents increased significantly compared with the wild-type H alpha(4)beta(3)delta receptor. 4. Thus, the results indicate that the R alpha(4)beta(3)delta, H alpha(1)beta(3)delta and H alpha(4)beta(3)gamma(2S) combinations may contribute to functions of extrasynaptic GABA(A) receptors. The presence of the R alpha(4) subunit at recombinant GABA(A) receptors containing the delta-subunit is a strong determinant of agonist action. The recombinant H alpha(4)beta(3)delta receptor is a less sensitive subunit composition in terms of agonist activation.

Immunohistochemistry Use by Diagnostic Category and Pathologist in 4477 Prostate Core Biopsy Sets Assessed at Two Hospitals.

BACKGROUND: Immunohistochemistry (IHC) use in prostate cores is not routinely determined and its value assessed. METHODS: Pathology reports for cases accessioned 2011 to 2017 at two hospitals were retrieved. IHC orders by pathologist and hospital were extracted with a custom program and tabulated. The diagnostic category (and highest grade cancer if applicable) was obtained by a hierarchical (free text) string matching algorithm. RESULTS: The study period contained 4477 biopsy sets. Categorized by worst pathology (% stained), the cohort was: benign: 1184 cases (42%); prostatic intraepithelial neoplasia: 168 (68%); suspicious: 323 (93%); grade group 1 cancer (WHO1): 900 (78%); grade group two (WHO2): 840 (60%); WHO3 cancer: 451 (54%); WHO4 cancer: 363 (46%); WHO5 cancer: 215 (56%); cancer grade not specified: 33 (52%). The hospital was a predictor; site A(2716 biopsies) and site B(1761) accounted for 10,183 and 14,852 IHC, respectively. The cases with IHC decreased in the last 4 years (site A: 57->45%, site B: 79->73%). Thirty-five pathologists read >20 cases each and together interpreted 4418 (range, 21 to 415; median, 88). In total 24,766 IHCs were done on the 4,418 cases (5.6/case). The mean/median/SD/max/min IHCs/case for the 35 pathologists was 5.6/4.1/3.9/15.2/0.9. High IHC users (1st and 2nd quintile pathologists) called more suspicious for malignancy but not significantly more WHO1 than low IHC users. CONCLUSIONS: IHC use is most frequent at the benign/malignant interface, and dependent on the pathologist and hospital; however, it is independent of WHO1 cancer rate. Diagnostic rate information can inform and define appropriate and rational IHC use. We plan to follow IHC utilization retrospectively in relation to the diagnostic category going forward.

CINtec PLUS and cobas HPV testing for triaging Canadian women referred to colposcopy with a history of low-grade squamous intraepithelial lesion: Baseline findings.

OBJECTIVE AND METHODS: CINtec PLUS and cobas HPV tests were assessed for triaging women referred to colposcopy with a history of LSIL cytology. Both tests were performed at baseline using ThinPrep cervical specimens and biopsy confirmed cervical intraepithelial neoplasia grade 2 or worse (CIN2+) served as the clinical endpoint. RESULTS: In all ages, (19-76 years, n = 600), 44.3% (266/600) tested CINtec PLUS positive vs. 55.2% (331/600) HPV positive (p = 0.000). Based on 224 having biopsies, sensitivity to detect CIN2+ (n = 54) was 81.5% (44/54) for CINtec PLUS vs. 94.4% (51/54) for HPV testing (p = 0.039); specificities were, 52.4% (89/170) vs. 44.1% (75/170), respectively (p = 0.129). In women ≥30 years (n = 386), 41.2% (159/386) tested CINtec PLUS positive vs. 50.8% (196/386) HPV positive (p = 0.008). Based on 135 having biopsies, sensitivity to detect CIN2+ (n = 24) was 95.8% (23/24) for both CINtec PLUS and HPV tests; specificities were, 55.0% (61/111) vs. 50.5% (56/111), respectively (p = 0.503). CONCLUSIONS: For women referred to colposcopy with a history of LSIL cytology, CINtec PLUS or cobas HPV test could serve as a predictor of CIN2+ with high sensitivity, particularly in women ≥30 years. Either test can significantly reduce the number of women requiring further investigations and follow up in colposcopy clinics.

A comparison of the pharmacological properties of recombinant human and rat alpha(1)beta(2)gamma(2L) GABA(A) receptors in Xenopus oocytes.

In the present study, we compared the pharmacology, particularly neurosteroid modulation of the GABA(A) receptor, between human and rat alpha(1)beta(2)gamma(2)(L) GABA(A) receptors and between human receptors containing the long (L) and short (S) forms of the gamma(2)-subunit. We observed that maximum responses to GABA were significantly higher with the human alpha(1)beta(2)gamma(2)(L) receptor compared with the rat receptor. In terms of neurosteroid modulation, increases in the EC(15) response to GABA induced by 3alpha-OH-5beta-pregnan-20-one (3alpha5betaP), 5alpha-androstane-3alpha,17beta-diol (3alpha5alphaADL) and 5alpha-pregnane-3alpha,20beta-diol (3alpha5alpha-diol) were significantly greater for the rat compared with the human receptor. Responses to 30 micromol/L GABA were inhibited by 3beta-OH-5alpha-pregnan-20-one (UC1010) and 5beta-pregnan-3beta,20(R)-diol (UC1020) to a greater degree for human and rat receptors, respectively. Responses to GABA + 3alpha5alphaTHDOC were inhibited by 5alpha-pregnan-3beta,20(S)-diol (UC1019) and pregnenolone sulphate to a greater degree for human and rat receptors, respectively. The GABA dose-response curves for human alpha(1)beta(2)gamma(2)(S) and alpha(1)beta(2)gamma(2)(L) receptors were identical. However, the maximum GABA-evoked current, the direct gating effect of pentobarbital and the allosteric potentiation of the GABA EC(15) response by 3alpha5alphaTHDOC and 3alpha5betaP were significantly higher with alpha(1)beta(2)gamma(2)(S) than alpha(1)beta(2)gamma(2)(L) receptors. Inhibition of the response to 30 micromol/L GABA by UC1010 and UC1020 was greater for a(1)beta(2)gamma(2)(L) and alpha(1)beta(2)gamma(2)(S) receptors, respectively. Inhibition of responses to 3alpha5alphaTHDOC + GABA by UC1019 and UC1010 was significantly higher for alpha(1)beta(2)gamma(2)(L) receptors. In conclusion, the site of activation by GABA and neurosteroid modulation differ between human and rat alpha(1)beta(2)gamma(2)(L) receptors, as well as between human receptors containing the L and S splice variants of the gamma(2)-subunit.

3Beta-hydroxysteroids and pregnenolone sulfate inhibit recombinant rat GABA(A) receptor through different channel property.

3Beta-hydroxysteroids are pregnenolone sulfate-like GABA(A) receptor antagonists. The aim of the current study was to compare the functional differences between 3beta-hydroxysteroids and pregnenolone sulfate to inhibit GABA(A) receptors expressed in Xenopus oocytes. Recombinant rat GABA(A) receptors encoding wild type alpha1 beta2 gamma2L receptor, mutant alpha1V256S beta2 gamma2L and alpha1 beta2A252S gamma2L receptors were examined using a two-electrode voltage-clamp technique. A homologous mutation of the residue at 2'position closest to the cytoplasmic end of the M2 helix to serine on both alpha1 and beta2 subunit, alpha1V256S and beta2A252S, reduced the slow desensitization components of GABA-activated currents at saturating doses. Compared to the wild type receptor, the potency of GABA increased significantly in the alpha1V256S beta2 gamma2L receptor (P<0.05), whereas it decreased moderately in the alpha1 beta2A252S gamma2L receptor. We found that 5alpha-pregnan-3beta, 20(S)-diol (UC1019) and 5beta-pregnan-3beta, 20(R)-diol (UC1020) were the most effective blockers of maximal GABA responses among a panel of 3beta-hydroxysteroids. Pregnenolone sulfate, UC1019 and UC1020 were potent antagonists in the wild type receptor with calculated IC50s of 0.20+/-0.07 microM; 1.88+/-0.32 microM and 2.58+/-0.58 microM, respectively. The inhibitory effect of pregnenolone sulfate was significantly reduced in both mutants alpha1V256S beta2 gamma2L and alpha1 beta2A252S gamma2L receptors (P<0.05), whereas the inhibitory effects of UC1019 and UC1020 were reduced only in the mutant alpha1V256S beta2 gamma2L receptor. Pregnenolone sulfate promoted slow desensitization with prolonged GABA application in a dose-dependent manner in the wild type receptor, but not mutant receptors. On the contrary, UC1019 and UC1020 (< or = 20 microM) did not promote desensitization in both wild type and mutant receptors. In conclusion, the GABA(A) receptor inhibition by pregnenolone sulfate, but not 3beta-hydroxysteroids, was dependent on desensitization kinetics of the Cl- channels. A point mutation at M2 helix of the beta2-subunit (beta2A252S) can dramatically reduce the inhibitory effect of pregnenolone sulfate on the GABA(A) receptors without affecting the inhibitory properties of 3beta-hydroxysteroids. These results are consistent with the hypothesis that pregnenolone sulfate-inhibition does not share with 3beta-hydroxysteroids the coincident channel property at the GABA(A) receptor.

Neurosteroid modulation of recombinant rat alpha5beta2gamma2L and alpha1beta2gamma2L GABA(A) receptors in Xenopus oocyte.

GABA(A) receptors containing alpha(5)-subunit have an important role in cognitive function. As the agonistic effect of 3alpha-hydroxy ring-A reduced steroids depends on subunit combinations of the GABA(A) receptor, the antagonistic effect of pregnenolone sulfate and 3beta-hydroxypregnane steroids may vary between alpha(5)-subunit and alpha(1)-subunit containing receptors. We investigated the effect of agonist and antagonist steroids in the recombinant rat alpha(1)beta(2)gamma(2L) and alpha(5)beta(2)gamma(2L) receptors expressed in Xenopus oocytes using a two electrodes voltage-clamp technique. We did not find any significant difference in potency and efficacy of GABA response between alpha(1)beta(2)gamma(2L) and alpha(5)beta(2)gamma(2L) receptors. Compared to the alpha(1)beta(2)gamma(2L) receptor, a significantly lower degree of desensitization was observed in the alpha(5)beta(2)gamma(2L) receptor. In addition, the potencies of 3alpha-OH-5alpha-pregnan-20-one (3alpha5alphaP), 5alpha-pregnan-3alpha,21-diol-20-one (3alpha5alphaTHDOC) and 5alpha-androstane-3alpha,17beta-diol (3alpha5alphaADL) to enhance GABA response were significantly higher in the alpha(5)beta(2)gamma(2L) receptor, whereas their efficacies remained unchanged between two receptors. In either receptor, the efficacy of 3alpha5alphaTHDOC was significantly higher than 3alpha5alphaP and 3alpha5alphaADL. The efficacies of 5beta-pregnan-3beta,21-diol-20-one(UC1015) and 5alpha-pregnan-3beta,20alpha-diol(UC1019) to inhibit 30 microM GABA response, and the efficacies of 3beta-OH-5beta-pregnan-20-one (UC1014) and 5beta-pregnan-3beta, 20beta-diol (UC1020) to inhibit 3 microM 3alpha5alphaTHDOC+3 microM GABA response were higher in the alpha(5)beta(2)gamma(2L) receptor compared to the alpha(1)beta(2)gamma(2L) receptor. The potencies of pregnenolone sulfate and 3beta-hydroxypregnane steroids to inhibit the GABA response and the 3alpha5alphaTHDOC+GABA response did not vary between two receptors. Interestingly, the potencies and efficacies of pregnenolone sulfate and 3beta-hydroxypregnane steroids to inhibit the GABA response were positively correlated to their potencies and efficacies to inhibit the 3alpha5alphaTHDOC+GABA response. Results from the current study revealed a different modulation pattern by neurosteroids between the alpha(1)beta(2)gamma(2L) and alpha(5)beta(2)gamma(2L) receptor.

Protons inhibit Cl- conductance by direct or allosteric interaction with the GABA-binding site in the rat recombinant alpha1beta2gamma2L and alpha1beta2 GABAA receptor.

Functional roles of external pH on the Cl- conductance were examined on Xenopus oocytes expressing rat recombinant alpha1beta2gamma2L and alpha1beta2 GABAA receptors. Acidic pH inhibited GABA-response in a reversible and concentration-dependent manner, significantly increasing the EC50 without appreciably changing the slope or maximal currents induced by GABA in the alpha1beta2gamma2L and alpha1beta2 receptors. In contrast, protonation did not influence the pentobarbital-gated currents in the alpha1beta2gamma2L receptors, suggesting that protons do not modulate channel activity by directly affecting the channel gating process. Protons competitively inhibited the bicuculline-induced antagonism on GABA in the alpha1beta2gamma2L receptors. The data support the hypothesis that protons inhibit GABAA receptor function by direct or allosteric interaction with the GABA-binding site.

Spontaneous transient depolarizations in lymphatic vessels of the guinea pig mesentery: pharmacology and implication for spontaneous contractility.

Guinea pig mesenteric lymphatic vessels exhibit rhythmic constrictions induced by action potential (AP)-like spikes and initiated by entrainment of spontaneous transient depolarizations (STDs). To characterize STDs and the signaling mechanisms responsible for their occurrence, we used intracellular microelectrodes, Ca2+ imaging, and pharmacological agents. In our investigation of the role of intracellular Ca2+ released from Ca2+ stores, we observed that intracellular Ca2+ transients accompanied some STDs, although there were many exceptions where Ca2+ transients occurred without accompanying STDs. STD frequency and amplitude were markedly affected by activators/inhibitors of inositol 1,4,5-trisphosphate receptors (IP3Rs) but not by treatments known to alter Ca2+ release via ryanodine receptors. A role for Ca2+-activated Cl(-) (Cl(Ca)) channels was indicated, as STDs were dependent on the Cl(-) but not Na+ concentration of the superfusing solution and were inhibited by the Cl(Ca) channel blockers niflumic acid (NFA), anthracene 9-carboxylic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid but not by the volume-regulated Cl(-) blocker DIDS. Increases in STD frequency and amplitude induced by agonist stimulation were also inhibited by NFA. Nifedipine, the hyperpolarization-activated inward current blocker ZD-7288, and the nonselective cation/store-operated channel blockers SKF-96365, Gd3+, and Ni2+ had no or marginal effects on STD activity. However, nifedipine, 2-aminoethoxydiphenyl borate, NFA, SKF-96365, Gd3+, and Ni2+ altered the occurrence of spontaneous APs. Our findings support a role for Ca2+ release through IP3Rs and a resultant opening of Cl(Ca) channels in STD generation and confirm the importance of these events in the initiation of lymphatic spontaneous APs and subsequent contractions. The abolition of spontaneous APs by blockers of other excitatory ion channels suggests a contribution of these conductances to lymphatic pacemaking.