Electrophoresis gel image processing and analysis using the KODAK 1D software.

The present article reports on the performance of the KODAK 1D Image Analysis Software for the acquisition of information from electrophoresis experiments and highlights the utility of several mathematical functions for subsequent image processing, analysis, and presentation. Digital images of Coomassie-stained polyacrylamide protein gels containing molecular weight standards and ethidium bromide stained agarose gels containing DNA mass standards are acquired using the KODAK Electrophoresis Documentation and Analysis System 290 (EDAS 290). The KODAK 1D software is used to optimize lane and band identification using features such as isomolecular weight lines. Mathematical functions for mass standard representation are presented, and two methods for estimation of unknown band mass are compared. Given the progressive transition of electrophoresis data acquisition and daily reporting in peer-reviewed journals to digital formats ranging from 8-bit systems such as EDAS 290 to more expensive 16-bit systems, the utility of algorithms such as Gaussian modeling, which can correct geometric aberrations such as clipping due to signal saturation common at lower bit depth levels, is discussed. Finally, image-processing tools that can facilitate image preparation for presentation are demonstrated.

Effects of cell volume fraction changes on apparent diffusion in human cells.

Diffusion-weighted imaging was used to study the relationship between apparent diffusion coefficient (ADC) and cell volume fraction in cell suspensions and packed arrays. Cell volume fraction was varied by changing extracellular fluid osmolarity (for human glial cells) and by changing cell density (for human glial and red blood cells). In packed arrays of glial cells, ADC increased 10% when cells shrank and decreased 13% when cells swelled. ADC decreased 34% as cell density increased from 0 to 72%. In erythrocyte suspensions, ADC decreased 90% as the cell density increased from 0 to 89%. These results agree with theoretical predictions.

Immunochemical characterization of Na+/H+ exchanger isoform NHE4.

Mammalian Na+/H+ exchangers (NHEs) are a family of transport proteins (NHE1-NHE5). To date, the cellular and subcellular localization of NHE4 has not been characterized using immunochemical techniques. We purified a fusion protein containing a portion of rat NHE4 (amino acids 565-675) to use as immunogen. A monoclonal antibody (11H11) was selected by ELISA. It reacted specifically with both the fusion protein and to a 60- to 65-kDa polypeptide expressed in NHE4-transfected LAP1 cells. By Western blot analysis, NHE4 was identified as a 65- to 70-kDa protein that was expressed most abundantly in stomach and in multiple additional epithelial and nonepithelial rat tissues including skeletal muscle, heart, kidney, uterus, and liver. Subcellular localization of NHE4 in the kidney was evaluated by Western blot analysis of membrane fractions isolated by Percoll gradient centrifugation. NHE4 was found to cofractionate with the basolateral markers NHE1 and Na+-K+-ATPase rather than the luminal marker gamma-glutamyl transferase. In stomach, NHE4 was detected by immunoperoxidase labeling on the basolateral membrane of cells at the base of the gastric gland. We conclude that NHE4 is a 65- to 70-kDa protein with a broad tissue distribution. In two types of epithelial cells, kidney and stomach, NHE4 is localized to the basolateral membrane.

Chemosensitivity of rat medullary raphe neurones in primary tissue culture.

1. The medullary raphe, within the ventromedial medulla (VMM), contains putative central respiratory chemoreceptors. To study the mechanisms of chemosensitivity in the raphe, rat VMM neurones were maintained in primary dissociated tissue culture, and studied using perforated patch-clamp recordings. Baseline electrophysiological properties were similar to raphe neurones in brain slices and in vivo. 2. Neurones were exposed to changes in CO2 from 5% to 3 or 9% while maintaining a constant [NaHCO3]. Fifty-one per cent of neurones (n = 210) did not change their firing rate by more than 20% in response to hypercapnic acidosis. However, 22% of neurones responded to 9% CO2 with an increase in firing rate ('stimulated'), and 27% of neurones responded with a decrease in firing rate ('inhibited'). 3. Chemosensitivity has often been considered an all-or-none property. Instead, a method was developed to quantify the degree of chemosensitivity. Stimulated neurones had a mean increase in firing rate to 298 +/- 215% of control when pH decreased from 7.40 to 7.19. Inhibited neurones had a mean increase in firing rate to 232 +/- 265% of control when pH increased from 7. 38 to 7.57. 4. Neurones were also exposed to isocapnic acidosis. All CO2-stimulated neurones tested (n = 15) were also stimulated by isocapnic acidosis, and all CO2-inhibited neurones tested (n = 19) were inhibited by isocapnic acidosis. Neurones with no response to hypercapnic acidosis also had no response to isocapnic acidosis (n = 12). Thus, the effects of CO2 on these neurones were mediated in part via changes in pH. 5. In stimulated neurones, acidosis induced a small increase in the after-hyperpolarization level of 1.38 +/- 1. 15 mV per -0.2 pH units, which was dependent on the level of tonic depolarizing current injection. In voltage clamp mode at a holding potential near resting potential, there were small and inconsistent changes in whole-cell conductance and holding current in both stimulated and inhibited neurones. These results suggest that pH modulates a conductance in stimulated neurones that is activated during repetitive firing, with a reversal potential close to resting potential. 6. The two subtypes of chemosensitive VMM neurones could be distinguished by characteristics other than their response to acidosis. Stimulated neurones had a large multipolar soma, whereas inhibited neurones had a small fusiform soma. Stimulated neurones were more likely than inhibited neurones to fire with the highly regular pattern typical of serotonergic raphe neurones in vivo. 7. Within the medullary raphe, chemosensitivity is a specialization of two distinct neuronal phenotypes. The response of these neurones to physiologically relevant changes in pH is of the magnitude that suggests that this chemosensitivity plays a functional role. Elucidating their mechanisms in vitro may help to define the cellular mechanisms of central chemoreception in vivo.

Monoclonal antibodies for high-resolution localization of NHE3 in adult and neonatal rat kidney.

Previous immunochemical studies have shown that NHE3 is an apical Na+/H+ exchanger in some renal epithelia. The purpose of the present study was to develop high-affinity, isoform-specific monoclonal antibodies (MAbs) that would be useful for carrying out high-resolution immunocytochemical studies of NHE3 in the adult and neonatal mammalian kidney. Three MAbs were developed to a fusion protein containing amino acids 702-832 of rabbit NHE3. Specificity was established by immunoblotting membranes from NHE-deficient LAP cells that had been transfected with either NHE1,-2, -3, or -4. With the use of high-resolution immunocytochemical techniques, NHE3 was found in vesicles in the apical cytoplasm of proximal tubule cells, as well as in the apical plasma membrane of the proximal tubule, and in both the thin and thick limbs of the loop of Henle. When localized in the 1-day-old rat kidney, NHE3 was first detected in the late stages of the S-shaped body. In later stages of nephron development, the pattern of NHE3 staining was similar to that seen in the adult. This study demonstrates 1) the specificity of three MAbs for Na+/H+ exchanger isoform NHE3; 2) NHE3 is present in an intracellular vesicular compartment in cells of the proximal tubule, consistent with possible regulation by membrane recycling; and 3) NHE3 is expressed on the apical membrane in early stages of the developing nephron.

Sodium-hydrogen exchange isoform expression in blood cells: implications for studies in diabetes mellitus.

There have been many reports of increased Na-H exchange (NHE) activity in the peripheral blood cells (erythrocytes, lymphocytes and platelets) of patients with diabetes mellitus compared to nondiabetic controls. This raised NHE activity has been hypothesized to reflect increased NHE activity in kidney and vascular smooth muscle. Raised NHE activity in these tissues could play a pathophysiological role in mediating hypertension, vascular smooth muscle cell proliferation and progressive renal impairment. It is now known that there are at least five NHE isoforms, but a specific study examining expression of NHE isoforms in peripheral blood cells has not been reported. This study used specific antisera to NHE isoforms 1, 3 and 4 to examine NHE expression by immunoblot analysis. Erythrocyte, lymphocyte and platelet membranes from both rabbit and rat were separated by standard methods. A monoclonal antibody to NHE-1 reacted with a 100-110 kDa band in rabbit and rat platelets and lymphocytes (identical to that observed in basolateral-enriched renal cortical vesicles) and a 100 kDa band in rabbit and rat erythrocytes. In both species, the intensity of the staining was greatest in platelet membranes. A polyclonal antibody to NHE-3, the isoform present on the apical membranes of renal proximal tubule, showed no evidence of staining in any of the peripheral blood cell preparations. Similarly there was no evidence of expression of NHE-4 in the peripheral blood cell preparations. Peripheral blood cells express NHE-1, which likely accounts for amiloride-sensitive Na-H exchange in these cells, playing a role in cell volume and pH regulation. However, there is no evidence that there is expression of NHE-3 or NHE-4 in peripheral blood cells. These data have implications for studies in hypertension and diabetes mellitus which measure peripheral blood cell Na-H exchange and hypothesize regarding a direct pathophysiological role for this increased activity.

Expression of Na(+)-H+ exchanger isoforms NHE1 and NHE3 in kidney and blood cells of rabbit and rat.

Increased peripheral blood cell Na-H exchange (NHE) and erythrocyte Na-Li countertransport activity have been reported in hypertension and diabetic nephropathy and correlated with increased activity of the renal brush border Na-H exchanger. A relationship between cation exchange activities of blood cells and renal brush border membranes might exist if both were mediated by the same NHE isoform. We generated isoform-specific antibodies to compare the expression of NHE1 and NHE3 in peripheral blood cell membranes and renal cortical membrane vesicles. An NHE1-specific monoclonal antibody reacted with a 199- to 110-kD protein in basolateral membrane fractions isolated from rabbit and rat kidney. NHE1 protein expression was also detected in erythrocytes, platelets, and lymphocytes isolated from rabbit and rat. Two polyclonal antisera generated against nonoverlapping portions of NHE3 reacted with proteins of 82 and 85 kD in brush border membrane fractions isolated from rabbit and rat kidney, respectively, but failed to detect NHE3 expression in blood cells. These data do not support the hypothesis that Na-H exchanger of Na-Li countertransport in blood cells takes place via the renal brush border membrane NHE isoform, namely NHE3.

Characterization of Na+/H+ exchange activity in cultured rat hippocampal astrocytes.

Astrocytes actively maintain their intracellular pH (pHi) more alkaline than expected by passive distribution of H+. Acid extruding transporters such as the amiloride-sensitive Na+/H+ exchanger (NHE) are necessary for pH regulation. Currently, four mammalian NHEs (NHE1-NHE4) have been cloned, with a fifth (NHE5) partially cloned. We attempted to determine which isoform(s) of NHE was present in cultured hippocampal astrocytes using amiloride sensitivity and immunospecificity as criteria. In the absence of HCO3-, amiloride blocked pHi recovery after an acid load with an IC50 of approximately 3.18 microM, similar to values reported for the amiloride-sensitive isoforms NHE1 and NHE2. Immunoblotting with a highly specific antibody for NHE1 identified a 100 kDa protein, indicating the presence of NHE1 in whole brain, hippocampus, and cultured hippocampal astrocytes. Further probing for an additional amiloride-sensitive NHE failed to detect evidence of the presence of NHE4. Surprisingly, application of the potent analog of amiloride, ethylisopropylamiloride (EIPA), caused a reversible alkalinization of pHi, suggesting the presence of an additional acid/base transport mechanism that is EIPA-sensitive.

NHE3: a Na+/H+ exchanger isoform of renal brush border.

Na+/H+ exchangers in the brush-border (luminal, apical) membrane of renal proximal tubules are responsible for active, transcellular reabsorption of NaHCO3 and NaCl. Although well characterized kinetically, the protein that mediates Na+/H+ exchange in the renal brush border has not been identified. Several Na+/H+ exchanger genes, including NHE1, NHE2, NHE3, and NHE4, are expressed in the kidney. To identify the NHE3 gene product and to determine its cellular and subcellular localization in the rabbit kidney, an NHE3-isoform-specific antibody was prepared. Guinea pigs were immunized with purified fusion protein containing the carboxy-terminal 40 amino acids of NHE3 (fpNHE3-C40). After affinity purification, immune sera demonstrated specific reactivity to the NHE3 sequence within the fusion protein as well as to an 80-kDa polypeptide expressed in NHE3-transfected LAP1 cells. Western blot analysis showed that anti-fpNHE3-C40 specifically reacted with an 80-kDa protein that is relatively enriched in renal brush-border membrane compared with basolateral membrane. Immunocytochemical studies confirmed that the Na+/H+ exchanger isoform NHE3 is expressed along the microvillar membrane of the brush border of proximal tubule cells in the rabbit kidney.

Cloning, sequence, and tissue distribution of a rabbit renal Na+/H+ exchanger transcript.

Rabbit kidney expresses a transcript that is similar to the human growth-factor-activatable Na+/H+ exchanger. PCR and library screening were used to clone overlapping 2.5 kb, 1.4 kb, and 1.8 kb cDNAs that together contain the entire coding region (2448 bp) and 5' untranslated region (726 bp) and part of the 3' untranslated region (128 bp) of a rabbit renal Na+/H+ exchanger transcript. The nucleotide and inferred amino acid sequences are highly conserved between rabbit and human (88% nucleotide identity, 95% amino acid identity). In rabbit, the transcript is expressed in both epithelial and non-epithelial tissues, with highest expression in stomach, brain, kidney, lung and ileum, and minimal expression in liver and skeletal muscle.

Immunomagnetic separation, primary culture, and characterization of cortical thick ascending limb plus distal convoluted tubule cells from mouse kidney.

Renal cortical thick ascending limbs of Henle's loop (CAL) and distal convoluted tubules (DCT) represent sites at which much of the final regulation of urinary ionic composition, particularly that of calcium, is accomplished in both humans and in rodents. We sought in the present work to develop an efficient means for isolating parathyroid hormone (PTH)-sensitive cells from these nephron segments and to grow them in primary culture. [CAL+DCT] cells were isolated from mouse kidney using an antiserum against the Tamm-Horsfall glycoprotein which, in the renal cortex, is produced exclusively by these cells. A second antibody conjugated to coated ferrous particles permitted magnetic separation of [CAL+DCT] cells from Tamm-Horsfall negative renal cortical cells. Approximately 3 X 10(6) cells per kidney with a trypan blue exclusion greater than 94% were isolated by these procedures. Experiments were performed to characterize the cells after 7 to 10 days in primary culture. PTH and isoproterenol, but neither calcitonin nor vasopressin, stimulated cyclic AMP (cAMP) formation in [CAL+DCT] cells, consistent with the pattern of hormone-activated cAMP synthesis found in freshly isolated CAL and DCT segments. Alkaline phosphatase, an enzyme present dominantly in proximal tubule brush border membranes, was virtually absent from [CAL+DCT] cells but was present in Tamm-Horsfall negative cells. Similarly, Na-glucose cotransport was absent in [CAL+DCT] cells but present in Tamm-Horsfall negative renal cortical cells. Finally, transport-related oxygen consumption in [CAL+DCT] cells was blocked by bumetanide and by chlorothiazide, diuretics that inhibit sodium transport in CAL and DCT nephron segments. These results demonstrate that PTH-sensitive [CAL+DCT] cells can be isolated in relatively high yield and viability and grown in cell culture. Primary cultures of these cells exhibit a phenotype appropriate to their site of origin in the nephron.