Biomedical subjects

Bruce D Schultz

Publications and source records attributed to Bruce D Schultz.

12 recordsLinked to original sources

PubMed2007

Lack of pendrin HCO3- transport elevates vestibular endolymphatic [Ca2+] by inhibition of acid-sensitive TRPV5 and TRPV6 channels.

The low Ca(2+) concentration ([Ca(2+)]) of mammalian endolymph in the inner ear is required for normal hearing and balance. We reported (Yamauchi et al., Biochem Biophys Res Commun 331: 1353-1357, 2005) that the epithelial Ca(2+) channels TRPV5 and TRPV6 (transient receptor potential types 5 and 6) are expressed in the vestibular system and that TRPV5 expression is stimulated by 1,25-dihydroxyvitamin D(3), as also reported in kidney. TRPV5/6 channels are known to be inhibited by extracellular acidic pH. Endolymphatic pH, [Ca(2+)], and transepithelial potential of the utricle were measured in Cl(-)/HCO(3)(-) exchanger pendrin (SLC26A4) knockout mice in vivo. Slc26a4(-/-) mice exhibit reduced pH and utricular endolymphatic potential and increased [Ca(2+)]. Monolayers of primary cultures of rat semicircular canal duct cells were grown on permeable supports, and cellular uptake of (45)Ca(2+) was measured individually from the apical and basolateral sides. Net uptake of (45)Ca(2+) was greater after incubation with 1,25-dihydroxyvitamin D(3). Net (45)Ca(2+) absorption was dramatically inhibited by low apical pH and was stimulated by apical alkaline pH. Gadolinium, lanthanum, and ruthenium red reduced apical uptake. These observations support the notion that one aspect of vestibular dysfunction in Pendred syndrome is a pathological elevation of endolymphatic [Ca(2+)] due to luminal acidification and consequent inhibition of TRPV5/6-mediated Ca(2+) absorption.

Absorption↗

PubMed2006

Apical electrolyte concentration modulates barrier function and tight junction protein localization in bovine mammary epithelium.

In vitro mammary epithelial cell models typically fail to form a consistently tight barrier that can effectively separate blood from milk. Our hypothesis was that mammary epithelial barrier function would be affected by changes in luminal ion concentration and inflammatory cytokines. Bovine mammary epithelial (BME-UV cell line) cells were grown to confluence on permeable supports with a standard basolateral medium and either high-electrolyte (H-elec) or low-electrolyte (L-elec) apical medium for 14 days. Apical media were changed to/from H-elec medium at predetermined times prior to assay. Transepithelial electrical resistance (R(te)) was highest in monolayers continuously exposed to apical L-elec. A time-dependent decline in R(te) began within 24 h of H-elec medium exposure. Change from H-elec medium to L-elec medium time-dependently increased R(te). Permeation by FITC-conjugated dextran was elevated across monolayers exposed to H-elec, suggesting compromise of a paracellular pathway. Significant alteration in occludin distribution was evident, concomitant with the changes in R(te), although total occludin was unchanged. Neither substitution of Na(+) with N-methyl-d-glucosamine (NMDG(+)) nor pharmacological inhibition of transcellular Na(+) transport pathways abrogated the effects of apical H-elec medium on R(te). Tumor necrosis factor alpha, but not interleukin-1beta nor interleukin-6, in the apical compartment caused a significant decrease in R(te) within 8 h. These results indicate that mammary epithelium is a dynamic barrier whose cell-cell contacts are acutely modulated by cytokines and luminal electrolyte environment. Results not only demonstrate that BME-UV cells are a model system representative of mammary epithelium but also provide critical information that can be applied to other mammary model systems to improve their physiological relevance.

Amiloride↗

PubMed2006

PVD9902, a porcine vas deferens epithelial cell line that exhibits neurotransmitter-stimulated anion secretion and expresses numerous HCO3(-) transporters.

Epithelial ion transport disorders, including cystic fibrosis, adversely affect male reproductive function by nonobstructive mechanisms and by obstruction of the distal duct. Continuous cell lines that could be used to define ion transport mechanisms in this tissue are not readily available. In the present study, porcine vas deferens epithelial cells were isolated by standard techniques, and the cells spontaneously immortalized to form a porcine vas deferens epithelial cell line that we have titled PVD9902. Cells were maintained in continuous culture for >4 yr and 200 passages in a typical growth medium. Frozen stocks were generated, and thawed cells exhibited growth characteristics indistinguishable from their nonfrozen counterparts. Molecular and immunocytochemical studies confirmed the origin and epithelial nature of these cells. When seeded on permeable supports, PVD9902 cells grew as electrically tight (>6,000 ohms x cm2), confluent monolayers that responded to forskolin with an increase in short-circuit current (I(sc); 8 +/- 1 microA/cm2) that required Cl-, HCO3(-), and Na+, and was partially sensitive to bumetanide. mRNA was expressed for a number of anion transporters, including CFTR, electrogenic Na+-HCO3(-) cotransporter 1b (NBCe1b), downregulated in adenoma, pendrin, and Cl-/formate exchanger. Both forskolin and isoproterenol caused an increase in cellular cAMP levels. In addition, PVD9902 cell monolayers responded to physiological (i.e., adenosine, norepinephrine) and pharmacological [i.e., 5'-(N-ethylcarboxamido)adenosine, isoproterenol] agonists with increases in I(sc). Unlike their freshly isolated counterparts, however, PVD9902 cells did not respond to glucocorticoid exposure with an increase in amiloride-sensitive I(sc). RT-PCR analysis revealed the presence of both glucocorticoid and mineralocorticoid receptor mRNA as well as mRNA for the alpha- and gamma-subunits of the epithelia Na+ channels (alpha- and gamma-ENaC), but not beta-ENaC. Nonetheless, PVD9902 cells recapitulated most observations in freshly isolated cells and thus represent a powerful new tool to characterize mechanisms that contribute to male reproductive function.

Animals↗

PubMed2005

Redesigning channel-forming peptides: amino acid substitutions that enhance rates of supramolecular self-assembly and raise ion transport activity.

Three series of 22-residue peptides derived from the transmembrane M2 segment of the glycine receptor alpha1-subunit (M2GlyR) have been designed, synthesized, and tested to determine the plasticity of a channel-forming sequence and to define whether channel pores with enhanced conductive properties could be created. Sixteen sequences were examined for aqueous solubility, solution-association tendency, secondary structure, and half-maximal concentration for supramolecular assembly, channel activity, and ion transport properties across epithelial monolayers. All peptides interact strongly with membranes: associating with, inserting across, and assembling to form homooligomeric bundles when in micromolar concentrations. Single and double amino acid replacements involving arginine and/or aromatic amino acids within the final five C-terminal residues of the peptide cause dramatic effects on the concentration dependence, yielding a range of K1/2 values from 36 +/- 5 to 390 +/- 220 microM for transport activity. New water/lipid interfacial boundaries were established for the transmembrane segment using charged or aromatic amino acids, thus limiting the peptides' ability to move perpendicularly to the plane of the bilayer. Formation of discrete water/lipid interfacial boundaries appears to be necessary for efficient supramolecular assembly and high anion transport activity. A peptide sequence is identified that may show efficacy in channel replacement therapy for channelopathies such as cystic fibrosis.

Amino Acid Sequence↗

PubMed2004

Transepithelial fluctuation analysis of chloride secretion.

Transepithelial fluctuation analysis (noise analysis) provides valuable information about the density and single-channel properties of ion channels in intact epithelia. Here we investigate cystic fibrosis transmembrane conductance regulator (CFTR)-dependent chloride (Cl-) secretion in T84 human colonic epithelia by inducing noise using the diarylsulfonylurea DASU-01, a low-affinity open-channel blocker of CFTR. Our data indicate that the apical membrane of maximally stimulated T84 epithelia has a very high Cl- conductance generated by approximately 7000 active CFTR channels per cell with open probability (Po) of approximately 0.4 and single-channel amplitude (i) of approximately 0.1 pA. Similar experiments might provide important information about how drugs regulate CFTR in intact epithelia.

Cell Membrane↗

PubMed2004

Activity and structural comparisons of solution associating and monomeric channel-forming peptides derived from the glycine receptor m2 segment.

A number of channel-forming peptides derived from the second transmembrane (TM) segment (M2) of the glycine receptor alpha(1) subunit (M2GlyR), including the 22-residue sequence NK(4)-M2GlyR p22 wild type (WT) (KKKKPARVGLGITTVLTMTTQS), induce anion permeation across epithelial cell monolayers. In vitro assays suggest that this peptide or related sequences might function as a candidate for ion channel replacement therapy in treating channelopathies such as cystic fibrosis (CF). The wild-type sequence forms soluble associations in water that diminish its efficacy. Introduction of a single substitution S22W at the C-terminus, NK(4)-M2GlyR p22 S22W, eliminates the formation of higher molecular weight associations in solution. The S22W peptide also reduces the concentration of peptide required for half-maximal anion transport induced across Madin-Darby canine kidney cells (MDCK) monolayers. A combination of 2D double quantum filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and rotating frame nuclear Overhauser effect spectroscopy (ROESY) data were recorded for both the associating WT and nonassociating S22W peptides and used to compare the primary structures and to assign the secondary structures. High-resolution structural studies were recorded in the solvent system (40% 2,2,2-Trifluoroethanol (TFE)/water), which gave the largest structural difference between the two peptides. Nuclear Overhauser effect crosspeak intensity provided interproton distances and the torsion angles were measured by spin-spin coupling constants. These constraints were put into the DYANA modeling program to generate a group of structures. These studies yielded energy-minimized structures for this mixed solvent environment. Structure for both peptides is confined to the 15-residue transmembrane segments. The energy-minimized structure for the WT peptide shows a partially helical extended structure. The S22W peptide adopts a bent conformation forming a hydrophobic pocket by hydrophobic interactions.

Amino Acid Sequence↗

PubMed2004

ClC-2 chloride secretion mediates prostaglandin-induced recovery of barrier function in ischemia-injured porcine ileum.

BACKGROUND & AIMS: Ischemia results in the breakdown of the intestinal barrier, predisposing patients to sepsis and multiple organ failure. Prostaglandins play a critical role in mediating recovery of barrier function in ischemia-injured intestine through a mechanism involving stimulation of Cl - secretion. In the present study, we investigated the contributory role of individual Cl - channels in the recovery of barrier function in ischemia-injured porcine ileum. METHODS: Ischemia-injured porcine ileal mucosa was mounted in Ussing chambers. Short-circuit current (Isc) and transepithelial resistance (TER) were measured in response to prostaglandin E 2 (PGE 2 ) and pharmacologic inhibitors of epithelial Cl - channels. Immunoassays were used to assess the expression and localization of ion channels. RESULTS: Application of PGE 2 to ischemia-injured ileal mucosa stimulated increases in Isc, an indicator of Cl - secretion, that was followed by marked increases in TER, an indicator of barrier function recovery. In vitro studies revealed that although PGE 2 induced Cl - secretion via at least 3 distinct secretory pathways, recovery of barrier function was initiated by Cl - secretion via ClC-2 Cl - channels co-expressed with occludin and localized to tight junctions within restituting epithelium. Intravenous administration of furosemide to pigs subjected to 1 hour of ileal ischemia impaired recovery of barrier function, as evidenced by decreased TER and increased mucosal-to-serosal 3 H-mannitol flux after a 2-hour reperfusion/recovery period, confirming an important role for Cl - secretory pathways in vivo. CONCLUSIONS: ClC-2-mediated intestinal Cl - secretion restores TER in ischemia-injured intestine. These data may provide the basis for targeted pharmacologic therapy for diseases associated with impaired barrier function.

Animals↗

PubMed2004

Channel-forming peptide modulates transepithelial electrical conductance and solute permeability.

NC-1059, a synthetic channel-forming peptide, transiently increases transepithelial electrical conductance (g(TE)) and ion transport (as indicated by short-circuit current) across Madin-Darby canine kidney (MDCK) cell monolayers in a time- and concentration-dependent manner when apically exposed. g(TE) increases from <2 to >40 mS/cm(2) over the low to middle micromolar range. Dextran polymer (9.5 but not 77 kDa) permeates the monolayer following apical NC-1059 exposure, suggesting that modulation of the paracellular pathway accounts for changes in g(TE). However, concomitant alterations in junctional protein localization (zonula occludens-1, occludin) and cellular morphology are not observed. Effects of NC-1059 on MDCK g(TE) occur in nominally Cl(-)- and Na(+)-free apical media, indicating that permeation by these ions is not required for effects on g(TE), although two-electrode voltage-clamp assays with Xenopus oocytes suggest that both Cl(-) and Na(+) permeate NC-1059 channels with a modest Cl(-) permselectivity (P(Cl):P(Na) = 1.3). MDCK monolayers can be exposed to multiple NC-1059 treatments over days to weeks without diminution of response, alteration in the time course, or loss of responsiveness to physiological and pharmacological secretagogues. Together, these results suggest that NC-1059 represents a valuable tool to investigate tight junction regulation and may be a lead compound for therapeutic interventions.

Animals↗

PubMed2003

Adenosine stimulates anion secretion across cultured and native adult human vas deferens epithelia.

Experiments were conducted to determine the responsiveness of human vas deferens epithelial cell monolayers to adenosine and related agonists. Human abdominal vas deferens epithelial cells have been isolated from adult tissues and grown to confluence on permeable supports. All cells exhibit intense ZO-1 and cytokeratin immunoreactivity. Cultured cell monolayers exhibit high electrical resistance with a lumen-negative potential difference and short circuit current (I(sc)) indicative of anion secretion and/or cation absorption. A portion of the basal I(sc) is inhibited by amiloride. Amiloride-sensitive I(sc) is enhanced by exposure to glucocorticoids and is Na(+) dependent, indicating the presence of epithelial sodium channel-mediated Na(+) absorption. Epithelial anion secretion and intracellular generation of cAMP are acutely stimulated by adenosine and the adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine (NECA), with these effects being fully blocked by 8-phenyltheophylline. Adenosine receptors are localized to the apical membrane of the epithelial cells, as basolateral adenosine is without effect. Freshly excised human vas deferens recapitulate observations made on cultured epithelia when evaluated with the self-referencing vibrating probe: amiloride inhibition of basal ion transport, stimulation by adenosine, and inhibition by 8-phenyltheophyline. These results demonstrate that adult human vas deferens epithelium actively transports ions to generate the luminal environment of the deferent duct. Thus, vas deferens epithelium likely plays an active role in male fertility, and interventions that modulate epithelial function might be exploited to treat male-factor infertility or in contraception.

Adenosine↗

PubMed2002

Distinct structural elements that direct solution aggregation and membrane assembly in the channel-forming peptide M2GlyR.

Restoration of chloride conductance via the introduction of an anion selective pore, formed by a channel-forming peptide, has been hypothesized as a novel treatment modality for patients with cystic fibrosis (CF). Delivery of these peptide sequences to airway cells from an aqueous environment in the absence of organic solvents is paramount. New highly soluble COOH- and NH(2)-terminal truncated peptides, derived from the second transmembrane segment of the glycine receptor alpha-subunit (M2GlyR), were generated, with decreasing numbers of amino acid residues. NH(2)-terminal lysyl-adducted truncated peptides with lengths of 22, 25, and 27 amino acid residues are equally able to stimulate short circuit current (I(SC)). Peptides with as few as 16 amino acid residues are able to stimulate I(SC), although to a lesser degree. In contrast, COOH-terminal truncated peptides show greatly reduced induced I(SC) values for all peptides fewer than 27 residues in length and show no measurable activity for peptides fewer than 21 residues in length. CD spectra for both the NH(2)- and COOH-truncated peptides have random structure in aqueous solution, and those sequences that stimulated the highest maximal I(SC) are predominantly helical in 40% trifluoroethanol. Peptides with a decreased propensity to form helical structures in TFE also failed to stimulate I(SC). Palindromic peptide sequences based on both the NH(2)- and COOH-terminal halves of M2GlyR were synthesized to test roles of the COOH- and NH(2)-terminal halves of the molecule in solution aggregation and channel forming ability. On the basis of the study presented here, there are distinct, nonoverlapping regions of the M2GlyR sequence that define solution aggregation and membrane channel assembly. Peptides that eliminate solution aggregation with complete retention of channel forming activity were generated.

Amino Acid Sequence↗

PubMed2002

Steroids modulate transepithelial resistance and Na(+) absorption across cultured porcine vas deferens epithelia.

Epithelial cells were isolated from adult porcine vas deferens and grown in the absence or presence of steroid hormones. Transepithelial resistance (R(te)), basal short circuit current (I(sc)), and the effects of selected ion transport modulators on these parameters were evaluated in modified Ussing chambers at three time points (5-8, 11-14, and 18-22 days postseeding). At the earliest time point, no significant differences were observed. At the middle time point, when compared with R(te) in untreated control monolayers, R(te) in monolayers exposed to 17beta-estradiol, aldosterone, cortisol, cortisone, prednisolone, prednisone, and dexamethasone was significantly lower; in contrast, R(te) in monolayers exposed to testosterone, dihydrotestosterone, or progesterone did not differ from that in control monolayers. Treatments with cortisol, prednisolone, and dexamethasone were associated with an elevated basal I(sc) that was amiloride sensitive, indicating ongoing Na(+) absorption by these monolayers. R(te) was increased by amiloride treatment in glucocorticoid-treated monolayers but remained significantly less than that of control monolayers. At the third time point, the postamiloride R(te) of glucocorticoid-treated monolayers was not different from that of control monolayers. Responses to ATP, forskolin, bumetanide, and DASU-02 were not affected by steroid treatment at any time point. Taken together, these results suggest that estrogens and corticosteroids can modulate epithelial function in the distal excurrent duct of the adult male reproductive system. At physiological or pharmacological concentrations, these hormones would be expected to modify the luminal environment (both the ionic composition and pH) to which sperm are exposed and thus affect male fertility.

Absorption↗

PubMed2002

Functional and molecular evidence for Na(+)-HCO cotransporter in porcine vas deferens epithelia.

This study focused on the role of sodium-bicarbonate cotransporter (NBC1) in cAMP-stimulated ion transport in porcine vas deferens epithelium. Ion substitution experiments in modified Ussing chambers revealed that cAMP-mediated stimulation was dependent on the presence of Na(+), HCO, and Cl(-) for a full response. HCO-dependent current was unaffected by acetazolamide, bumetanide, or amiloride but was inhibited by basolateral 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Na(+)-driven, HCO-dependent, stilbene-inhibitable anion flux was observed across the basolateral membrane of selectively permeabilized monolayers. Results of radiotracer flux studies suggest a 4,4'-dinitrostilbene-2,2'-disulfonate-sensitive stoichiometry of 2 base equivalents per Na(+). Antibodies raised against rat kidney NBC epitopes (rkNBC; amino acids 338-391 and 928-1035) identified a single band of ~145 kDa. RT-PCR detected NBC1 message in porcine vas deferens epithelia. These results demonstrate that vas deferens epithelial cells possess the proteins necessary for the vectoral transport of HCO and that these mechanisms are maintained in primary culture. Taken together, the results indicate that vas deferens epithelia play an active role in male fertility and have implications for our understanding of the relationship between cystic fibrosis and congenital bilateral absence of the vas deferens.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗