Search PubMed⌕ Search

Biomedical subjects

U Hopfer

Publications and source records attributed to U Hopfer.

At least 55 records · Page 3Linked to original sources
PubMed1995

Osmolar changes regulate the paracellular permeability of cultured human cervical epithelium.

Extracellular nucleotides induce a biphasic change in the transepithelial electrical conductance (GT) of human cervical cells grown on filters: a rapid increase (phase I) followed by a sustained decrease (phase II). To probe the involvement of the intercellular space, its magnitude was varied by manipulating cell volume through changes in extracellular osmolarity. Under baseline conditions [GT = 115 mS/cm2 (approximately 9 omega.cm2)] and during phase II, hypertonic challenges resulted in an increase in GT (0.98% .mosmol-1.l-1 and 0.73%.mosmol-1.l-1, respectively). However, a hypertonic challenge during phase I decreased GT (-0.16%.mosmol-1.l-1). Hypotonic challenges decreased GT during baseline, phase I, and phase II conditions by -1%.mosmol-1.l-1. Similar trends were observed with regard to pyranine permeability. Reduction of extracellular calcium increased GT, abrogated the phase II effect of extracellular ATP, and reversed the effect of a hypertonic challenge. The additive nature of the permeability changes in response to osmotic challenges and to ATP during phase II suggests that different sites are involved in each response, i.e., the resistance of the intercellular space changes with osmolarity and that of the tight junction during phase II.

Adenosine Triphosphate↗
PubMed1995

Stimulation of Cl- secretion by extracellular ATP does not depend on increased cytosolic Ca2+ in HT-29.cl16E.

Extracellular ATP and elevated cytosolic Ca2+ concentration ([Ca2+]i) are major secretagogues for Cl- in the goblet cell-like clone cl.16E derived from colonic HT-29 cells. The involvement of [Ca2+]i as a messenger for the purinergically stimulated Cl- secretion was investigated using whole cell patch-clamp and Ussing chamber techniques, as well as [Ca2+]i measurements using fura 2-loaded cells. Under voltage-clamp conditions, the whole cell current at +50 mV was 3 +/- 1 pA/pF under unstimulated conditions. Stimulation of purinergic receptors with 200 microM extracellular ATP increased the current at +50 mV to 41 +/- 10 pA/pF, with a half-maximal effective dose (ED50) of approximately 3 microM. The current was transient, usually lasting 1-2 min, and the current-voltage relationship was approximately linear between -70 and +50 mV. Evidence that the ATP-stimulated current was carried by Cl- included 1) the reversal potential of the current closely followed the Cl- equilibrium potential, and 2) the stimulated current was absent when Cl- was removed from both bath and pipette solutions. Exposure to ATP also increased [Ca2+]i, with an ED50 of approximately 1 microM and maximal changes (at 200 microM) from baseline (71 +/- 3 nM) to 459 +/- 50 nM. The ATP-dependent Cl- conductance increase was not diminished when [Ca2+]i was clamped at 100 nM using a Ca(2+)-1,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or Ca(2+)-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffering system. However, the ATP effect did require some basal level of Ca2+ because clamping [Ca2+]i at < 10 nM abolished activation of the Cl- conductance. The presence of the protein kinase A inhibitor H-89 or the protein kinase C inhibitor staurosprine did not change the ATP-activated Cl-conductance. These data demonstrate that the ATP-stimulated increase in Cl- current does not require an increase in [Ca2+]i, suggesting the involvement of either another signaling pathway or direct activation of Cl- channels by purinergic receptors.

Adenosine Triphosphate↗
PubMed1994

Cyclic AMP-induced mucin exocytosis is independent of Cl- movements in human colonic epithelial cells (HT29-Cl.16E).

The human colonic epithelial goblet cell line HT29-Cl.16E was used to test whether stimulated Cl- transport is involved in the mucin exocytotic response to an increase in intracellular cyclic AMP by measuring in parallel the short-circuit current (Isc) and mucin exocytosis. Addition of 50 microM forskolin to HT29-Cl.16E cells resulted in a 2-fold stimulation of mucin release and an increase in Isc by 20 microA/cm2. To evaluate the requirement for cosecretion of Cl-, the Cl- flux was altered by three different manipulations: (1) Cl- in the medium was replaced by the poorly transported anion gluconate; (2) basolateral Cl- influx through the Na(+)-K(+)-2Cl- cotransporter was inhibited by bumetanide; and (3) an inward Cl- flux through the apical plasma membrane was generated by reversing the Cl- gradient. These manipulations did not change the forskolin-stimulated mucin release and thereby provide evidence that Cl- movements are not required for fusion of mucin granules with the plasma membrane.

Amphotericin B↗
PubMed1994

Interferon-gamma modulates cAMP-induced mucin exocytosis without affecting mucin gene expression in a human colonic goblet cell line.

The regulation of intestinal mucin secretion by cytokines, soluble factors released by mucosal activated immune cells, is so far unknown. The aim of the present study was (1) to investigate the regulatory effects of interferon-gamma on baseline and stimulated mucin secretion elicited by an increase in intracellular cAMP, either a short-term increase (induced by vasoactive intestinal peptide or by forskolin) or a long-term increase (cholera toxin-induced), and (2) to attempt to delineate the site of action of interferon-gamma. The in vitro model used was the human colonic goblet cell line Cl.16E, which has already been shown to respond to physiological secretagogues in terms of mucin secretion. We examined the effects of interferon-gamma 1) on mucin exocytosis, measured as release of [3H]glucosamine-labeled macromolecules trapped at the stacking/running gel interface of polyacrylamide gels, and 2) on mucin biosynthesis, examined at the RNA level using a cDNA probe directed to the MUC2 mucin gene. We demonstrated that, while interferon-gamma did not alter baseline Cl.16E mucin secretion and MUC2 gene expression, it strongly inhibited the protein kinase A-dependent secretory response to VIP, forskolin, or cholera toxin. However, interferon-gamma had no effect on the protein kinase A-dependent MUC2 over-expression induced by cholera toxin. We thus concluded that the target for interferon-gamma inhibition of cAMP-stimulated Cl.16E mucin secretion is distal to protein kinase A and might be a component of the exocytotic machinery. Together, our results establish interferon-gamma as a pharmacologically powerful tool to specifically inhibit stimulated secretory processes without affecting baseline secretion.

Blotting, Northern↗
PubMed1994

ATP-stimulated electrolyte and mucin secretion in the human intestinal goblet cell line HT29-Cl.16E.

The response of confluent monolayers of HT29-Cl.16E cells to stimulation by extracellular ATP and ATP analogues was investigated in terms of mucin and electrolyte secretion. Mucin secretion was measured as release of glucosamine-labeled macromolecules trapped at the stacking/running gel interface of polyacrylamide gels and electrolyte secretion as short-circuit current (Isc). Luminal ATP stimulated a transient increase in the release of mucins and of Isc corresponding to a secretory Cl- current. Both secretions peaked at 3 to 5 min after addition of ATP. Maximal ATP-stimulated mucin secretion over 15 min was up to 18-fold above control with an apparent ED50 of approximately 40 microM. Maximal peak Isc after stimulation with ATP was approximately 35 microA/cm2 with an apparent ED50 of about 0.4 mM. ATP-dependent Isc was at least in part due to Cl- secretion since removal of Cl- from the medium reduced the peak Isc by 40% and the Isc integrated over 40 min by 80%. The secretory responses were not associated with cell damage as assessed by failure of ethidium bromide to enter into the cells, absence of release of lactate dehydrogenase, maintenance of monolayer conductance, viability, and responses to repeated applications of ATP. The order of efficacy of nucleotide agonists was similar for both processes with ATP > ADP > AMP > or = adenosine. Luminal ATP was much more effective than basolateral addition of this compound. These results suggest involvement of a luminal P2-type receptor which can initiate signaling pathways for granule fusion and mucin release as well as for activation of Cl- channels. P2-receptor-stimulated mucin and Isc release was strongly inhibited by a 30 min preincubation with the classical K+ channel blockers quinine (1 mM), quinidine (1 mM), and Ba2+ (3 mM). Experiments with amphotericin B to measure separately the conductance changes of either luminal or basolateral plasma membrane revealed that quinidine did not directly block the ATP-induced basolateral K+ or the luminal anion channels. The quinidine inhibition after preincubation is therefore most easily explained by interference with granule fusion and location of anion channels in granule membranes. Luminal P2 receptors may play a role in intestinal defense mechanisms with both fluid and mucin secretion aiding in the removal of noxious agents from the mucosal surface.

Adenosine↗
PubMed1994

Human uterine cervical epithelial cells grown on permeable support--a new model for the study of differentiation.

The purpose of the present study was to establish culture conditions for human uterine cervical epithelial cells on permeable support and to determine how it affects cervical cell differentiation. Human ectocervical epithelial cells (hECE), HPV-16 immortalized hECE cells (ECE16-1) and Caski cells were grown on collagen-coated filters. Culture conditions, density of cells in culture and expression of epithelial and cervical-cell phenotypic markers were determined and compared in cells grown on filter and on solid support. Compared with the latter, cultures on filter had a higher cell density, hECE cells stratified to 5-12 cell layers compared to 1-3 on solid support, and cells of all three types expressed intercellular tight junctions. The cytokeratin profiles revealed differences between the three cell types as well as differences within the same cell species when grown on filter, compared to solid support. Of particular importance was the finding of a higher expression of K-13 in hECE grown on filter compared to solid support; K-13 is a marker of ectocervical cell differentiation. The cytokeratin profiles of the cultured hECE, ECE16-1 and Caski cells resembled those of ectocervical, squamous metaplastic and endocervical epithelia, respectively. hECE and ECE16-1 expressed involucrin protein, the level of which in both was higher in cells grown on filter compared to solid support. Polarization of the cultures was determined by morphology (stratification of hECE cells, expression of pseudomicrovilli in the apical cell membrane), selective apical vs. basolateral secretion of [35S]methionine- and [35S]cysteine-, [3H]fucose- and [14C]glucosamine-labeled molecules, and positive short-circuit current (Isc) under voltage-clamp conditions. Confluency of the cultures was determined by measuring transepithelial unidirectional fluxes of inert molecules with different molecular weights (MWs) through the paracellular pathway, and by measuring transepithelial conductance. The results indicated transepithelial permeability of 7-22.10(-6) cm.sec-1, which was 5-100 fold smaller compared to blank inserts, with a cut-off MW of 40-70 kDa for hECE and Caski cells. Transepithelial conductance ranged 18.5 to 51.5 mS.cm-2, indicating a leaky but confluent epithelia. Collectively the results indicate the epithelial nature of the cells and their improved differentiation when grown on filter support; hECE is a model for ectocervical epithelium while ECE16-1 and Caski express phenotypic characteristics of squamous metaplastic cervical epithelium and endocervical epithelium respectively.

Carcinoma, Squamous Cell↗
PubMed1994

ATP decreases acutely and reversibly transport through the paracellular pathway in human cervical cells.

We studied the effect of ATP on transepithelial transport through the paracellular pathway in human cervical cells. Transepithelial conductance and transepithelial permeability (determined from the measurements of unidirectional flux of inert molecules) were measured in Caski cells grown on permeable support. Transepithelial conductance was 55.9 +/- 17.7 mS/cm2 and permeability was 12.5 +/- 2.7 x 10(-6) cm/s for a 0.51-kDa probe. Addition of ATP to the medium decreased acutely and reversibly the conductance and the permeability to probes between 0.18 and 10 kDa by 23-31% in a dose-related fashion; the 50% effective concentration was 1 microM, with a maximal effect at 5-10 microM extracellular ATP. The ATP effect was observed regardless of the pressure gradient across the epithelium. These results indicate that extracellular ATP in micromolar concentrations decreases acutely and reversibly the permeability through the paracellular pathway in cervical cells, possibly by affecting the permeability of the tight junctions and the resistance of the intercellular space. On the basis of these data, we speculate that ATP may play a role in the regulation of solutes and fluid transport across the cervical epithelium in vivo.

Adenosine Triphosphate↗
PubMed1993

Signal transduction mediated by angiotensin II receptor subtypes expressed in rat renal mesangial cells.

Recent evidence suggests that there are two classes of receptors for angiotensin II (AngII), AT1 which is sensitive to losartan (DuP753) and is G-protein coupled, and AT2 which is sensitive to both PD123319 and CGP42112A, and is non-G-protein coupled. In rat mesangial cells two subtypes of AT1 receptor could be distinguished, AT1A subtype is more sensitive to losartan whereas AT1B subtype is more sensitive to PD123319, but insensitive to CGP42112A. The present studies were designed to ascertain which receptor subtype mediates three AngII-induced physiologic functions in rat mesangial cells namely intracellular Ca2+ mobilization, adenylyl cyclase inhibition and protein synthesis as monitored via [3H]leucine incorporation. The rank order of potency for inhibition of AngII-induced [Ca(2+)]i mobilization and adenylyl cyclase regulation was PD123319 > or = losartan > CGP42112A. By contrast, losartan was quite effective at inhibiting protein synthesis (IC50 = 8 nM) while PD123319 was without effect. These findings are consistent with AngII mediated signal transduction through AT1A and AT1B sites for phospholipase C mediated [Ca(2+)]i mobilization and inhibition of adenylyl cyclase. On the other hand, AT1A receptors appear to exclusively mediate AngII-induced protein synthesis. These observations underscore the complexity of AngII mediated signal transduction in glomerular mesangium.

Adenylyl Cyclase Inhibitors↗
PubMed1992

ATP-sensitive K+ conductance in pancreatic zymogen granules: block by glyburide and activation by diazoxide.

The properties of transporters (or channels) for monovalent cations in the membrane of isolated pancreatic zymogen granules were characterized with an assay measuring bulk cation influx driven by a proton diffusion potential. The proton diffusion potential was generated by suspending granules in an isotonic monovalent cation/acetate solution and increasing the proton conductance of the membrane with a protonophore. Monovalent cation conductance had the sequence Rb+ > K+ > NA+ > Cs+ > LI+ > N-methyl glucamine+. The conductance could be inhibited by Ca2+, Mg2+, Ba2+, and pharmacological agents such as quinine, quinidine, glyburide and tolbutamide, but not by 5 mM tetra-ethyl ammonium or 5 mM 4-aminopyridine, when applied to the cytosolic surface of the granule membrane. Over 50% of K+ conductance could be inhibited by millimolar concentrations of ATP or MgATP. The inhibition by MgATP, but not by ATP itself, was reversed by the K+ channel opener diazoxide. The inhibitory effect is probably by a noncovalent interaction since it could be mimicked by nonhydrolyzable analogs of ATP and by ADP. The reversal of MgATP inhibition by diazoxide may be mediated by phosphorylation since it was not affected by dilution, and was blocked by the protein kinase inhibitor H7. The properties of the K+ conductance of pancreatic zymogen granule membranes are similar to those of ATP-sensitive K+ channels found in the plasma membrane of insulin-secreting islet cells, neurons, muscle, and renal cells.

Adenosine Triphosphate↗
PubMed1992

Large-scale purification of calf pancreatic zymogen granule membranes.

A protocol for isolating milligram quantities of highly purified zymogen granule membranes from calf pancreas was developed. The method provides a fivefold enriched zymogen granule fraction that is virtually free from major isodense contaminants, such as mitochondria and erythrocytes. Isolated granules are osmotically stable in isosmotic KCl buffers with half-lives between 90 and 120 min. They display specific ion permeabilities that can be demonstrated using ionophore probes to override intrinsic control mechanisms. A Cl- conductance, a Cl-/anion exchanger, and a K+ conductance are found in the zymogen granule membrane, as previously reported for rat pancreatic, rat parotid zymogen granules, and rabbit pepsinogen granules. Lysis of calf pancreatic secretory granules in hypotonic buffers and subsequent isolation of pure zymogen granule membranes yield about 5-10 mg membrane protein from approximately 1000 ml pancreas homogenate. The purified zymogen granule membranes are a putative candidate for the rapid identification and purification of epithelial Cl- channels and regulatory proteins, since they contain fewer proteins than plasma membranes.

Animals↗
PubMed1992

Development and characterization of rabbit proximal tubular epithelial cell lines.

We have isolated rabbit kidney proximal tubular epithelial cell lines. The selection was based on their ability to form confluent monolayers on porous supports and to maintain receptor-mediated signal transduction and ion transport, characteristic of the proximal tubule. The isolation method consisted of several steps: (1) superficial cortical proximal tubule segments were microdissected and cultured on a matrix-coated porous support until cells formed a confluent monolayer; (2) primary cultures showing hormone-regulated ion transport typical for the proximal tubule were selected and co-cultured with irradiated fibroblasts; and (3) the epithelial cells surviving after several passages were expanded and passaged on porous substrates. Most of the cell lines developed in this manner were obtained by co-culture with irradiated fibroblasts producing a recombinant retrovirus encoding SV40 large T antigen and G418 resistance. However, SV40 T antigen expression was not essential for immortalization, since neither T antigen nor G418 resistance was detected in the isolated cell lines and co-culture with non-producing 3T3 cells gave similar results. One cell line (vEPT) has been characterized in some detail with respect to morphological, biochemical, and ion transport properties. This line forms confluent monolayers with apical microvilli, tight junctions, and convolutions of the basolateral plasma membrane. Once confluent, monolayers maintain conductances of 25 to 32 mS/cm2 for several weeks in culture and possess phlorizin-sensitive short-circuit current (Isc) in glucose containing media, indicative of apical Na(+)-glucose co-transport. vEPT cells also retain receptor and signaling mechanisms for angiotensin II (Ang II). Apical and basal Ang II and 5,6-epoxy-eicosatrienoic acid (5,6-EET) modulate the Isc in a manner similar to primary cultures. The cell lines share with primary cultures expression of the cytokeratins K8, K10/K11, and K19 ("nomenclature" [21]). They also retain several receptor and signal transduction mechanisms. For example, Ang II, arachidonate, bradykinin, 5,6-EET, parathyroid hormone (residues 1 through 34), and purine nucleotides increase cytosolic Ca2+, PTH elevates cAMP levels, and Ang II enhances proximal tubule-specific arachidonic acid metabolism.

Animals↗
PubMed1991

An epoxygenase metabolite of arachidonic acid mediates angiotensin II-induced rises in cytosolic calcium in rabbit proximal tubule epithelial cells.

Previous studies from this and other laboratories have shown that angiotensin II (AII) induces [Ca2+]i transients in proximal tubular epithelium independent of phospholipase C. AII also stimulates formation of 5,6-epoxyeicosatrienoic acid (5,6-EET) from arachidonic acid by a cytochrome P450 epoxygenase and decreases Na+ transport in the same concentration range. Because 5,6-EET mimics AII with regard to Na+ transport, it effects on calcium mobilization were evaluated. [Ca2+]i was measured by video microscopy with the fluorescent indicator fura-2 employing cultured rabbit proximal tubule. AII-induced [Ca2+]i transients were enhanced by arachidonic acid and attenuated by ketoconazole, an inhibitor of cytochrome P450 epoxygenases. Arachidonic acid also elicited a [Ca2+]i transient that was attenuated by ketoconazole. 5,6-EET augmented [Ca2+]i similar to that seen with AII, but was unaffected by ketoconazole. By contrast, the other regioisomers (8,9-, 11,12-, and 14,15-EET) were much less potent. [Ca2+]i transients resulted from influx through verapamil- and nifedipine-sensitive channels. These results suggest a novel mechanism for AII-induced Ca mobilization in proximal tubule involving cytochrome P450-dependent arachidonic acid metabolism and Ca influx through voltage-sensitive channels.

8,11,14-Eicosatrienoic Acid↗
PubMed1991

An epoxygenase metabolite of arachidonic acid 5,6 epoxy-eicosatrienoic acid mediates angiotensin-induced natriuresis in proximal tubular epithelium.

Several laboratories have documented that angiotensin II (AII) induces natriuresis in proximal tubular epithelium by a mechanism that has as yet not been disclosed. The present studies were designed to test the hypothesis that cytochrome P450-dependent metabolites of arachidonic acid mediate this effect. In cultured rabbit proximal tubule a ketoconazole-sensitive product comigrating with 5,6-epoxy-eicosatrienoic acid (5,6-EET) on reverse phase and normal phase HPLC was stimulated two-fold by AII. We employed cultures of early S1 segments on a modified Ussing chamber as a bioassay to evaluate the effects of AII and 5,6-EET on unidirectional 22Na (apical to basolateral) flux (JNa). AII inhibited JNa, an effect that was abolished by ketoconazole. Furthermore, 5,6-EET decreased JNa in a manner analogous to AII, and the effect was potentiated by inhibition of endogenous 5,6-EET production. Employing the Ca2(+)-sensitive fluorescent probe, Fura 2, we observed a dose-dependent increase in [Ca2+]i with nM to microM 5,6-EET, effects that were abolished by depletion of extracellular Ca2+ and voltage-sensitive Ca-channel blockers. These observations support the hypothesis that 5,6-EET represents the second messenger that mediates AII-induced natriuresis via stimulation of Ca2+ influx through voltage-sensitive channels.

8,11,14-Eicosatrienoic Acid↗
PubMed1990

Dual modulation of chloride conductance by nucleotides in pancreatic and parotid zymogen granules.

The regulation of Cl- conductance by cytoplasmic nucleotides was investigated in pancreatic and parotid zymogen granules. Cl- conductance was assayed by measuring the rate of cation-ionophore-induced osmotic lysis of granules suspended in iso-osmotic salt solutions. Both inhibition and stimulation were observed, depending on the type and concentration of nucleotide. Under optimal conditions, the average inhibition measured in different preparations was 1.6-fold, whereas the average stimulation was 4.4-fold. ATP was inhibitory at 1-10 microM but stimulated Cl- conductance above 50 microM. Stimulation by ATP was more pronounced in granules with low endogenous Cl- conductance. The potency of nucleotides in terms of inhibition was ATP greater than adenosine 5'-[gamma-thio]triphosphate (ATP[S]) greater than UTP much greater than or equal to CTP much greater than or equal to GTP much greater than or equal to guanosine 5'-[gamma-thio]triphosphate (GTP[S]) much greater than or equal to ITP. The potency with respect to stimulation had the following order: adenosine 5'-[beta gamma-methylene]triphosphate (App[CH2]p) greater than ATP greater than guanosine 5'-[beta-thio]diphosphate (GDP[S]). Adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p) was also stimulatory, and was more potent than ATP in the parotid granules, but less potent in the pancreatic granules. Aluminium fluoride stimulated Cl- conductance maximally at 15-30 microM-Al3+ and 10-15 mM-F. F was less effective at higher concentrations. Protein phosphorylation by kinases was apparently not involved, since the nucleotide effects (1) could be mimicked by non-hydrolysable analogues of ATP and GTP, (2) showed reversibility, and (3) were not abolished by the protein kinase inhibitors 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) or staurosporine. The data suggest the presence of at least two binding sites for nucleotides, whereby occupancy of one induces inhibition and occupancy of the other induces stimulation.

Adenosine Triphosphate↗
PubMed1990

Regulation of chloride transport in parotid secretory granules by membrane fluidity.

Zymogen granule membranes contain Cl- conductance and Cl/anion exchange activities that become important for primary fluid production after fusion with the apical plasma membrane of the acinar cell. We have used steady-state fluorescence anisotropy of diphenylhexatriene derivatives and measurements of Cl- transport in isolated secretory granules to determine the contribution of membrane fluidity to the regulation of transport across the granule membrane. Secretory granules from several unstimulated glands (rat pancreas and parotid, rabbit gastric glands) were shown to have low membrane fluidity compared to plasma membranes. In addition, Cl- transport activity in different granule preparations showed a strong correlation to the membrane fluidity when measured with 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH), but not with 3-[p-(6-phenyl)-1,3,5-hexatrienyl)-phenyl]propionic acid (PA-DPH). These data suggest that TMA-DPH preferentially partitions into a specific lipid environment associated with, or which exerts an influence on, the Cl- transport proteins and that increases in the fluidity of this environment are associated with higher transport rates. Data from other types of plasma membranes indicate that TMA-DPH partitions much more than PA-DPH into the cytoplasmic leaflet, suggesting that this part of the granule membrane is involved in the observed fluidity changes. Furthermore, increasing the bulk membrane fluidity with the local anesthetics benzyl alcohol and n-alkanols increased the Cl- transport rates up to 10-fold. This increase was apparently through specific transporters as anion selectivity was maintained in spite of the higher absolute rates.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohols↗
PubMed1990

Proximal tubular epithelial cells possess a novel 42-kilodalton guanine nucleotide-binding regulatory protein.

The proximal tubule of the kidney represents an important location where adenylate cyclase regulates salt and water transport; yet a detailed characterization of the distribution and classification of guanine nucleotide-binding protein (G protein) and adenylate cyclase is lacking. We used purified brush border (20-fold) and basolateral membranes (14-fold) to characterize parathyroid hormone- and G protein-regulated adenylate cyclase and G-protein distribution. Adenylate cyclase was predominantly localized to basolateral membranes, while the 46-kDa alpha subunit of the stimulatory G protein (Gs) was 2-fold higher in brush border membranes than in basolateral membranes. The alpha subunit of the inhibitory G protein (Gi; 41 kDa) was equally distributed on immunoblotting but was 2-fold higher in brush border membranes than in basolateral membranes on radiolabeling with pertussis toxin. A 42-kDa cholera toxin substrate that cross-reacted with antisera to the common alpha subunit of G proteins and to Gs on immunoblotting and that was not immunoprecipitated with two Gi antisera was the most abundant alpha subunit and comprised approximately 1% of the total membrane proteins. These observations suggest that G proteins are important regulators of proximal tubular transport independent of adenylate cyclase.

Adenosine Diphosphate Ribose↗