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R M Henderson

Publications and source records attributed to R M Henderson.

At least 37 records · Page 2Linked to original sources
PubMed1996

Insulin restores the calcium-activated chloride conductance in dedifferentiated renal glomerular mesangial cells from the H-2KB-TSA58 [corrected] transgenic mouse.

The effects of duration in culture were assessed in mesangial cells from the H-2Kb-tsA58 transgenic mouse using the whole-cell configuration of the patch clamp technique. The whole-cell potassium conductance remained constant in cells from passages 8-17 while the chloride conductance was found to decrease in cells after passage 15. This reduction of the chloride conductance, indicating cell dedifferentiation, was the result of complete loss of the calcium-activated component of the conductance and loss of part of the calcium-insensitive component. Insulin, which increases the calcium-sensitivity of the calcium-activated component of the chloride conductance in early passage cells, restored part of the calcium-sensitive component of the chloride conductance in dedifferentiated cells. Vasoactive agonists such as arginine vasopressin and angiotensin II stimulate a chloride-dependent depolarization in mesangial cells that initiates cell contraction. In view of the importance of the chloride flux it is possible that the loss of a significant magnitude of chloride conductance may underlie mesangial cell pathology to some degree. This is supported by data that show insulin can reverse many of the early renal changes observed in diabetic nephropathy and, as shown in this study, that insulin enhances the chloride conductance in dedifferentiated, cultured mesangial cells.

Animals↗
PubMed1996

Inhibition by P1075 and pinacidil of a calcium-independent chloride conductance in conditionally-immortal renal glomerular mesangial cells.

1. Depolarization of mesangial cells has been shown to occur following an outward movement of chloride ions from the cell. We have shown previously that mesangial cells from the H-2Kb-tsA58 transgenic mouse possess a significant whole-cell chloride conductance and consequently are a suitable preparation for the study of potential chloride channel inhibitors. 2. The effects on the whole-cell chloride conductance of the chloride channel inhibitor, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and the potassium channel openers, (KCOs) P1075 and pinacidil were investigated in mesangial cells from the H-2Kb-tsA58 transgenic mouse cultured in permissive conditions (at 33 degrees C in the presence of 50 u ml-1 murine gamma-interferon). 3. In symmetrical solutions of 140 mM tetramethylammonium chloride (TMAC1) the whole-cell chloride conductance was 1.08 +/- 0.05 nS (n = 63) and this could be reversibly inhibited by 5 x 10(-5) M NPPB. 4. Both P1075 and pinacidil inhibited the whole-cell chloride conductance. This inhibition was not reversible after drug washout and was demonstrated only when drugs were applied to the extracellular surface of the cells. Very low concentrations of the drugs were found to reduce the chloride conductance after 16 h incubation but under no circumstances studied was the conductance totally inhibited, leaving a mean residual current of 0.33 +/- 0.03 nS (n = 12). 5. The effects of different peptide calcium concentrations on the magnitude of the residual current in the presence of the drugs were investigated. The residual current was reduced with 10(-8) M calcium in the pipette and increased with 10(-3) M pipette calcium. Therefore, these data suggest that P1075 and pinacidil selectively inhibit a calcium-independent chloride conductance in mesangial cells from the H-2Kb-tsA58 transgenic mouse.

Animals↗
PubMed1996

Viewing the renal epithelium with the atomic force microscope.

The atomic force microscope (AFM) was invented in 1986. It created the possibility to study the surfaces of biological material at the molecular level. The AFM is not only a microscope with atomic resolution (at least when applied to suitable samples) but is also an instrument with a broad variety of other applications. We present five examples demonstating the application of the AFM technique in different ways, mainly in renal epithelium. First, it was used as a high-resolution near-field microscope which generated images of canine kidney chromosomes. Second, dynamic changes of the plasma membrane of canine kidney cells were visualized with a resolution at least 10-fold greater than that obtainable by light microscopy. Third, the AFM was used as a tool which interacted directly with the plasma membrane of canine kidney cells. Fourth, due to its excellent resolution in the third dimension (namely height), continuous measurements of changes in 'height' of multimers of cloned plasma membrane potassium channel proteins of rat kidney (ROMK1) in response to ATP were performed with a time resolution of about 100 ms in physiological buffer. Fifth, the AFM continuously followed dissociation processes of multimeric macromolecules (such as transcription factors) under physiological conditions and, on the basis of individual molecular volume measurements, allowed estimation of the number of monomers in a multimeric complex. Taken together, the AFM offers a wide spectrum of novel approaches for the experimental nephrologist.

Animals↗
PubMed1995

A characterization of the chloride conductance in mesangial cells from the H-2Kb-tsA58 transgenic mouse.

The chloride conductance of conditionally immortalized mesangial cells isolated from the H-2Kb-tsA58 transgenic mouse was studied in cells grown in permissive and non-permissive culture conditions. No differences were found in the magnitude of the chloride conductance in 140 mM tetramethyl ammonium chloride between cells grown in permissive and non-permissive culture conditions (1.08 +/- 0.05 nS and 1.02 +/- 0.05 nS). Selectivity sequences were defined as 1.8I- > 1.2Br- > lCl- and 1.3I- > 1.1Br- > lCl- respectively and both the anion channel inhibitors niflumic acid and ochratoxin A inhibited the chloride conductance in a dose-dependent manner. The chloride conductance was made up from calcium-dependent and calcium-independent components and in the presence of 1 microM free calcium in the pipette this could be increased by the presence of 100 U ml-1 insulin added to the incubation medium. This appeared to increase the sensitivity of the conductance to levels of intracellular calcium as no differences were observed in the presence of either 1 mM or 100 nM pipette calcium. These data indicate that there is a significant chloride conductance in mesangial cells from the H-2Kb-tsA58 transgenic mouse and minimal changes are observed with changes from permissive to non-permissive culture conditions.

Animals↗
PubMed1995

The hepatocyte growth factor/scatter factor (HGF/SF) receptor, met, transduces a morphogenetic signal in renal glomerular fibromuscular mesangial cells.

Previous studies have demonstrated that hepatocyte growth factor/scatter factor (HGF/SF) is secreted by mesenchymal cells and that it elicits motility, morphogenesis and proliferation of epithelia expressing the met receptor. We now report that HGF/SF may act as an autocrine factor in fibromuscular renal mesangial cells. These cells mechanically support glomerular endothelia, control the rate of plasma ultrafiltration and are implicated in the pathogenesis of a variety of chronic renal diseases. We detected met protein in the vascular stalk of metanephric glomeruli and in the mature mesangium. Mesangial lines from a mouse transgenic for a temperature-sensitive simian virus 40 T antigen expressed met mRNA and protein, and recombinant HGF/SF phosphorylated the met receptor tyrosine kinase. Cells were immortal in the permissive condition and HGF/SF enhanced proliferation in a defined medium. In the absence of the immortalising protein, division ceased and recombinant HGF/SF caused multipolar cells to become bipolar. The factor diminished stress fibres, their focal contacts and immunostaining for extracellular fibronectin, hence suggesting reduced substratum adhesion and enhanced motility. Mesangial lines also expressed HGF/SF mRNA and secreted bioactive factor; immunocytochemistry showed both ligand and receptor in individual cells. HGF/SF blocking antibody aggregated the cells, suggesting that mesangial-derived factor affects basal cell conformation in an autocrine manner. We conclude that mesangial cells express both HGF/SF and met, and the factor induces morphogenesis of cultured mesangial cells. Therefore HGF/SF may have an autocrine role in mesangial biology but further studies are now required to investigate the potential importance of the factor in vivo.

Animals↗
PubMed1993

Activation of ion channels by lysylbradykinin in the HCA-7 colony 29 human adenocarcinoma cell line.

1. The patch-clamp technique, both cell attached and inside-out patches, was used to examine the effects of lysylbradykinin (LBK) and A23187 on ion channels in cultured Colony 29 epithelial cells derived from a human adenocarcinoma. 2. LBK and A23187 applied directly to the intact cell stimulated the opening of a number of types of ion channel including Ca(2+)-activated K+ channels. 3. By use of inside-out patches, anion channels could be stimulated to open by application of protein kinase A and ATP to the cytosolic surface. Ca(2+)-activated K+ channels were also identified in isolated membrane patches. 4. The results suggest that the anion secretion which is stimulated by LBK is a complex event, involving the activation of a number of different types of ion channel, and that part of the response is the result of hyperpolarization of the cell by activation of Ca(2+)-activated K+ channels. From the data presented in this and the accompanying papers it appears that the Ca(2+)-sensitive K+ channels would be equally effective in either the apical or basolateral membranes.

Adenocarcinoma↗
PubMed1992

Ion channel formation by duramycin.

The formation of ion channels by the nonadecapeptide antibiotic duramycin was examined using black lipid membranes and using the patch-clamp technique. In black lipid membranes made from glyceryl monooleate or a phosphatidylcholine/phosphatidylethanolamine mixture, duramycin induced complex fluctuations in membrane conductance, some step-like and some which were incapable of being resolved into discrete conductance states. Both conductance and largest step size increased with time. A similar time-dependent increase in conductance was seen in patch-clamp experiments with HCA-7 Colony 29 human colonic epithelial cell. The channels displayed weak anion selectivity and the smaller channels formed in patches from epithelial cells showed weak inward-rectification. Channel formation by duramycin was achieved at lower concentrations when the black lipid membrane was made with phospholipid rather than with glyceryl monooleate. Lower concentrations were effective in generating conductances in epithelial cells than in bilayers. It is concluded that duramycin forms ion channels in both artificial and biological membranes. Accumulation of duramycin and coalescence of initially small channels into larger ones is considered to be responsible for the recorded behaviour and to final disruption of membranes.

Anti-Bacterial Agents↗
PubMed1992

EEG in liver transplantation: visual and computerized analysis.

We prospectively evaluated 40 liver transplant candidates (including 28 recipients) with visually scored or computerized EEG. As expected, EEG frequencies rose significantly after successful transplant (P < 0.01). For all subjects, but especially for the transplant recipients, higher frequencies on computerized EEG at baseline had a strong association with survival at 18 months (P < 0.001). A logistic regression model allowed estimation of the odds for survival and indicated less than 50% long-term survival with central-occipital mean frequencies below 7.6 c/sec. Visual EEG scores closely paralleled the quantitative results, but only the latter were amenable to formal statistical analysis. EEG had much stronger predictive value for survival than serum albumin, although albumin improved more significantly in the months after transplantation.

Adult↗
PubMed1992

Chloride channels and anion fluxes in a human colonic epithelium (HCA-7).

1. Colonic epithelial cells, derived from a human adenocarcinoma (HCA-7), were examined by the patch clamp technique. 2. Outwardly rectifying anion (Cl-) channels were identified in the apical membrane. The conductance was g(in) approximately 26 pS, g(out) approximately 40 pS. The open state probability of the channels increased with depolarization and the selectivity for Cl- over K+ (PCl/PK) was approximately 7.5. 3. The channels were sensitive to intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP, 0.1 mM), but not to Ca2+ (at concentrations up to 1 mM). At depolarized potentials the channels were blocked by pirentanide (1-5 microM) applied intracellularly. 4. HCA-7 monolayers loaded with 125I- (as a marker for Cl-) were used to measure I- efflux and converted to instantaneous rate constants. 5. The rate constant for I- efflux was increased by forskolin and lysylbradykinin (LBK). The effects of forskolin were not effected by BAPTA (an intracellular calcium chelator). The effects of LBK were inhibited by BAPTA and by Ba2+, indicating that LBK raised intracellular Ca2+ (Cai) which activates Ca(2+)-sensitive K-channels, the latter being blocked by Ba2+. 6. Although it cannot be conclusively proved that the outwardly rectifying chloride channels described here are solely or partially responsible for the increased anion efflux or transepithelial chloride secretion, the channels are likely to be more relevant for cyclic AMP-requiring rather than Ca(2+)-requiring secretagogues.

Adenocarcinoma↗
PubMed1992

Anion channels in rat liver canalicular plasma membranes reconstituted into planar lipid bilayers.

Previous studies from this laboratory have demonstrated a Cl(-)-HCO3- exchanger and have provided evidence for a Cl- conductance in rat liver canalicular plasma membrane vesicles. To further investigate the apical Cl- conductance, we performed single-channel analysis after incorporation of canalicular liver plasma membrane vesicles into planar lipid bilayers. This was necessary, because the canalicular membrane is not accessible for the patch-clamp technique. Two types of anion channels could be identified (30- and 90-pS conductance) corresponding to the class of small and intermediate channels, respectively. The kinetics of the small channel were found to be voltage dependent with a maximum for the open probability at -20 mV. In contrast, intermediate channel kinetics were voltage independent. The anion channels described above could allow electrogenic Cl- efflux, to compensate Cl- influx via the electroneutral Cl(-)-HCO3- exchanger. Further studies will be required to prove their functional importance in bile formation.

Animals↗
PubMed1991

An outward-rectifying potassium channel in primary cultures of sweat glands from cystic fibrosis subjects.

We have previously described a high conductance calcium-activated 'maxi K' channel in primary cultures of human eccrine sweat gland cells both from normal subjects and those with cystic fibrosis. In further studies we have now identified a potassium-selective channel of much lower conductance which shows outward-rectification and which is present in sweat glands isolated from cystic fibrosis subjects. In experiments with inside-out patches using symmetrical pipette and bath solutions containing 140 mM K+ the channel showed an outward slope conductance (at +50 mV) of approximately 26 pS and an inward conductance (at -50 mV) of approximately 11 pS. When K+ in the bath was replaced by Na+ the reversal potential shifts to reveal a permeability ratio PK/PNa approximately 40 Unlike the maxi-K+ channel, the outward-rectifying channel does not show sensitivity to Ca2+. Channels were found in cells cultured from the glands of four out of five cystic fibrosis subjects. In cells cultured from 30 subjects who did not have cystic fibrosis, an outward-rectifying potassium channel was seen in only one out of approximately 3000 patches.

Calcium↗
PubMed1991

A high-conductance Ca(2+)-activated K+ channel in cultured human eccrine sweat gland cells.

Recordings have been made of a potassium-selective ion channel in primary cultures of cells derived from explants of human eccrine sweat glands obtained from normal subjects and from subjects suffering from cystic fibrosis. There appears to be no functional difference between potassium channels derived from normal subjects and those from cystic fibrosis subjects. The channel falls into the group generally known as "Maxi-K" channels, and has a slope conductance, with symmetrical solutions in bath and pipette containing 140 mM K+, of approximately 230 pS. It is calcium-activated, pH-sensitive and can be blocked by barium and quinine. The channel appears only very rarely in patches derived from confluent cells (in approximately 1 in 30 patches containing channels), but it is more frequently observed in younger cultures of dividing cells derived from recently explanted (within the previous 48-72 h) glands. It is possible that this channel is normally located on the basolateral membrane of the cell, and is responsible for the calcium-dependent secretory and absorptive events seen in the intact sweat gland.

Barium↗
PubMed1989

Mechanism of aldosterone-induced increase of K+ conductance in early distal renal tubule cells of the frog.

Isolated early distal tubule cells (EDC) of frog kidney were incubated for 20-28 hr in the presence of aldosterone and then whole-cell K+ currents were measured at constant intracellular pH by the whole-cell voltage-clamp technique. Aldosterone increased barium-inhibitable whole-cell K+ conductance (gK+) threefold. This effect was reduced by amiloride and totally abolished by ouabain. However, aldosterone could still raise gK- in ouabain-treated cells in the presence of furosemide. We tested whether changes in intracellular pH (pHi) could be a signal for cells to regulate gK+. After removal of aldosterone, the increase in gK+ was preserved by subsequent incubation for 8 hr at pH 7.6 but abolished at pH 6.6. In the complete absence of aldosterone, incubation of cells at pH 8.0 for 20-28 hr raised pHi and doubled gK+. Using the patch-clamp technique, three types of K+-selective channels were identified, which had conductances of 24, 45 and 59 pS. Aldosterone had no effect on the conductance or open probability (Po) of any of the three types of channels. However, the incidence of observing type II channels was increased from 4 to 22%. Type II channels were also found to be pH sensitive, Po was increased by raising pH. These results indicate that prolonged aldosterone treatment raises pHi and increases gK+ by promoting insertion of K+ channels into the cell membrane. Channel insertion is itself triggered by raising both pHi and increasing the activity of the Na+/K+ pump in early distal cells of frog kidney.

Aldosterone↗
PubMed1989

Inward-rectifying potassium channels in rat hepatocytes.

The patch-clamp technique has been used to investigate single-channel and whole cell conductances in freshly isolated rat hepatocytes. Whole cell experiments, with high (144 mM) intracellular and extracellular potassium as the principal conductive species, show some variation between cells in the current-voltage relationship (mean whole-cell conductance at physiological potentials being 2.7 nS). This may suggest functional heterogeneity of cells. The most common finding is that the current-voltage relationship shows inward rectification. This is reflected in cell-attached single-channel recordings in which channels displaying strong inward rectification and K+ selectivity are seen. The channels show a mean inward conductance (with 144 mM potassium in the pipette) of 44 pS and an outward conductance of 23 pS. The open probability is not voltage dependent, and the channels do not exhibit calcium dependence. The channels are quite different from others described in hepatocytes, but they show marked similarities to channels recently described in renal epithelial cells. Current-voltage relationships in the whole cell mode exhibit an increase in slope conductance at large hyperpolarizing and depolarizing potentials.

Animals↗
PubMed1988

Whole-cell potassium currents in single early distal tubule cells.

The apical membrane potassium conductance of amphibian early distal tubules is sensitive to changes in the intracellular pH, with cellular acidification causing a decreased conductance. With the whole-cell patch-clamp technique, we have measured the total potassium conductance of single isolated early distal tubule cells of the frog. With symmetrical potassium gluconate solutions, the whole-cell current was found to be strongly rectifying, with an inward conductance of 12.9 nS (at intracellular pH between 7.6 and 8.0) and an outward conductance of 1.0 nS. The inward current was almost totally inhibited by the addition of 10 mM Ba2+ to the bath solution. The use of pipette solutions with pH between 7.0 and 8.0 showed a positive correlation between intracellular pH and conductance. In contrast, acidification of the extracellular solution caused no significant change in conductance.

Animals↗
PubMed1988

Differences in morphology, growth rate, and protein synthesis between cultured arterial and venous endothelial cells.

Intrinsic differences between aortic and venous endothelial cells were demonstrated by studies of morphology, growth rates, and protein synthesis. Endothelial cells from bovine thoracic aortas (AECs) and inferior vena caval (VECs) were harvested, maintained in cell culture, and characterized. VECs were consistently larger (mean cell diameter 14.3 micron vs 12.7 micron; p less than 0.001) and more pleomorphic than were AECs. Scanning electron microscopy revealed that VECs were thinner than AECs and that VECs had numerous fine, cellular processes that were much less abundant on the AECs. Enzymatic disaggregation of confluent cells resulted in a reduced generation time by the AECs compared with VECs. Moreover, VECs responded to disaggregation by a significantly larger increase in cell size than the AECs. Protein synthesis was quantitated by computer analysis of autoradiograph of two-dimensional gel eletrophoresis and separation of 998 35S-methionine-labeled proteins from cell lysates. Synthesis of 257 proteins was significantly different (p less than 0.05); synthesis of 239 proteins by VECs was either decreased (219) or undetectable (20) compared with those of AECs. In contrast, only 18 proteins were produced in significantly greater quantity by VECs than by AECs. The cytoskeletal proteins actin and alpha- and beta-tubulins were produced in significantly greater quantity by AECs than VECs. These results indicate that cultured endothelial cells of arterial origin are substantially different from those of the venous circulation. These phenotypic differences are maintained in vitro despite eliminating variability in hemodynamic stress and interaction with other cellular elements of the vessel wall.

Animals↗
PubMed1987

Cell membrane and transepithelial voltages and resistances in isolated rat hepatocyte couplets.

The basic electrical properties of an isolated rat hepatocyte couplet (IRHC) system have been analyzed using classical techniques of epithelial electrophysiology, including measurement of electric potentials, resistances and intracellular ion activities. Applications of these techniques are discussed with respect to their limitations in small isolated cells. Mean intracellular and intracanalicular membrane potentials ranged from -23.7 to -46.7 and -4.3 to -5.9 mV, respectively. Membrane resistances were determined using an equivalent circuit analysis modified according to the geometry of the IRHC system. Resistances of the sinusoidal (basolateral) and canalicular (luminal) cell membranes and tight junctions averaged 0.15 and 0.78 G omega and 25 m omega, respectively. The cells are electrically coupled via low resistance intercellular communications (approximately 58 M omega). Intracellular ion activities for Na+, K+ and Cl- averaged 12.2, 88.1 and 17.7 mmol/liter, respectively. The basolateral membrane potential reveals a permeability sequence of PK greater than PCl greater than PNa. The luminal potential showed minimal dependence on changes in transjunctional ion gradients, indicating a poor ion selectivity of the paracellular pathway. The electrogenic (Na+-K+)-ATPase contributes little to the luminal and cellular negative electric potential. Therefore, the luminal potential probably results from the secretion of impermeant ions and a Donnan distribution of permeant ions, a mechanism which provides the osmotic driving force for bile formation. By providing the unique opportunity to measure luminal potentials, this isolated hepatocyte system permits study of secretory mechanisms for the first time in a mammalian gland using electrophysiologic techniques.

Animals↗