Transfection of the CD20 cell surface molecule into ectopic cell types generates a Ca2+ conductance found constitutively in B lymphocytes.

CD20 is a plasma membrane phosphoprotein expressed exclusively by B lymphocytes. mAb binding to CD20 alters cell cycle progression and differentiation, indicating that CD20 plays an essential role in B lymphocyte function. Whole-cell patch clamp and fluorescence microscopy measurements of plasma membrane ionic conductance and cytosolic-free Ca2+ activity, respectively, were used to directly examine CD20 function. Transfection of human T and mouse pre-B lymphoblastoid cell lines with CD20 cDNA and subsequent stable expression of CD20 specifically increased transmembrane Ca2+ conductance. Transfection of CD20 cDNA and subsequent expression of CD20 in nonlymphoid cells (human K562 erythroleukemia cells and mouse NIH-3T3 fibroblasts) also induced the expression of an identical transmembrane Ca2+ conductance. The binding of a CD20-specific mAb to CD20+ lymphoblastoid cells also enhanced the transmembrane Ca2+ conductance. The mAb-enhanced Ca2+ currents had the same conductance characteristics as the CD20-associated Ca2+ currents in CD20 cDNA-transfected cells. C20 is structurally similar to several ion channels; each CD20 monomer possesses four membrane spanning domains, and both the amino and carboxy termini reside within the cytoplasm. Biochemical cross-linking of cell-surface molecules with subsequent immunoprecipitation analysis of CD20 suggests that CD20 may be present as a multimeric oligomer within the membrane, as occurs with several known membrane channels. Taken together, these findings indicate that CD20 directly regulates transmembrane Ca2+ conductance in B lymphocytes, and suggest that multimeric complexes of CD20 may form Ca2+ conductive ion channels in the plasma membrane of B lymphoid cells.

Cytoplasmic calcium in the mediation of macula densa tubulo-glomerular feedback responses.

Within each nephron of the mammalian kidney, a feedback mechanism operating between the macula densa segment of the distal tubule and the afferent arteriole participates in the regulation of glomerular filtration rate. Retrograde microperfusion studies in rats were conducted to test the hypothesis that activation of macula densa cytoplasmic calcium is involved in the transmission of feedback signals to the vascular elements. Perfusion into distal tubules with a hypotonic solution (70 milliosmolar) elicited moderate decreases in glomerular pressure of 6 +/- 0.8 millimeters of mercury. With the addition of a calcium ionophore (A23187) glomerular pressure decreased by 16 +/- 1.1 millimeters of mercury. When a solution devoid of calcium but containing A23187 was used, the feedback response was inhibited. Thus, cytoplasmic calcium within the receptor cells may participate in the transmission of feedback signals to the contractile cells.

Basic properties and potential regulators of the apical K+ channel in macula densa cells.

These studies examine the properties of an apical potassium (K+) channel in macula densa cells, a specialized group of cells involved in tubuloglomerular feedback signal transmission. To this end, individual glomeruli with thick ascending limbs (TAL) and macula densa cells were dissected from rabbit kidney and the TAL covering macula densa cells was removed. Using patch clamp techniques, we found a high density (up to 54 channels per patch) of K+ channels in the apical membrane of macula densa cells. An inward conductance of 41.1 +/- 4.8 pS was obtained in cell-attached patches (patch pipette, 140 mM K+). In inside-out patches (patch pipette, 140 mM; bath, 5 mM K+), inward currents of 1.1 +/- 0.1 pA (n = 11) were observed at 0 mV and single channel current reversed at a pipette potential of -84 mV giving a permeability ratio (PK/PNa) of over 100. In cell-attached patches, mean channel open probability (N,Po, where N is number of channels in the patch and Po is single channel open probability) was unaffected by bumetanide, but was reduced from 11.3 +/- 2.7 to 1.6 +/- 1.3 (n = 5, p < 0.02) by removal of bath sodium (Na+). Simultaneous removal of bath Na+ and calcium (Ca2+) prevented the Na(+)-induced decrease in N.Po indicating that the effect of Na+ removal on N.Po was probably mediated by stimulation of Ca2+ entry. This interpretation was supported by studies where ionomycin, which directly increases intracellular Ca2+, produced a fall in N.Po from 17.8 +/- 4.0 to 5.9 +/- 4.1 (n = 7, p < 0.02). In inside-out patches, the apical K+ channel was not sensitive to ATP but was directly blocked by 2 mM Ca2+ and by lowering bath pH from 7.4 to 6.8. These studies constitute the first single channel observations on macula densa cells and establish some of the characteristics and regulators of this apical K+ channel. This channel is likely to be involved in macula densa transepithelial Cl- transport and perhaps in the tubuloglomerular feedback signaling process.

Glomerular and renal hemodynamics during converting enzyme inhibition (SQ20,881) in the dog.

It has been suggested that intrarenal levels of angiotensin II may preferentially control efferent arteriolar resistance or may influence the glomerular filtration coefficient (Kf). To examine these possibilities, micropuncture and clearance experiments were performed on nine anesthetized dogs evaluating renal and glomerular hemodynamics before and during the administration of an angiotensin converting enzyme inhibitor (SQ20,881). During the micropuncture measurements, renal arterial pressure was reduced to range of 85 to 90 mm Hg in order to maximize renin secretion and intrarenal formation of angiotensin II. Also, this procedure minimizes potential errors in the determination of single nephron glomerular filtration rate (SNGFR) and of glomerular pressure when estimated by techniques that require complete blockade of proximal tubule fluid flow. During the administration of SQ20,881, a converting enzyme inhibitor (CEI), renal blood flow increased significantly by 13%, but GFR was not altered. There were no significant alterations in SNGFR, proximal tubule pressure, peritubular capillary pressure or estimated glomerular pressure. By using the micropressure measurements in combination with the whole kidney hemodynamic data, it was estimated that afferent resistance was reduced 23%. Although significant decreases in efferent resistance could not be documented, there was a tendency for this variable to decrease also. Neither Kf nor effective filtration pressure were altered significantly by CEI. These results do not support the contention that intrarenal effects of angiotensin II are exerted predominantly on the efferent arteriolar resistance segments; rather, they suggest that angiotensin may exert a modest tonic effect on both pre- and postglomerular resistance elements in the anesthetized hydropenic dog.

Influence of converting enzyme inhibition on renal hemodynamics and glomerular dynamics in sodium-restricted dogs.

Clearance and micropuncture experiments were performed to evaluate the influence of converting enzyme inhibition (CEI) (SQ 14,225) on renal hemodynamics, glomerular filtration rate (GFR), segmental vascular resistances, and superficial nephron function in anesthetized sodium restricted dogs. In one series (n = 8), renal blood flow (RBF) and GFR exhibited a high degree of autoregulatory efficiency when renal arterial pressure (RAP) was reduced from 126 +/- 5 to 86 +/- 1 mm Hg. With RAP maintained at the reduced level, CEI elicited increases in RBF (3.9 +/- 0.3 to 5.8 +/- 0.5 ml/min per g kw) and GFR (0.81 +/- 0.03 to 0.94 +/- 0.04 ml/min per g kw). With return of RAP to spontaneous levels during continued CEI, RBF and GFR autoregulatory efficiency was maintained, and was similar to that observed in control dogs subjected to the same procedures (n = 5). In the micropuncture experiments (n = 12), RAP was maintained at the reduced level (87.5 +/- .9 mm Hg), and measurements were made before and during CEI. Proximal tubule pressure, peritubular capillary pressure, stop flow pressure, and single nephron GFR (SNGFR) increased significantly. Regression analysis suggested that the increases in SNGFR were associated with small increases in the filtration coefficient. CEI reduced preglomerular resistance by 29% to 35% and efferent arteriolar resistance by 24% to 32%. These results indicate that the increased activity of the renin-angiotensin system that occurs during salt restriction exerts approximately equivalent vasoconstrictor influences on both preglomerular and postglomerular vascular resistance elements.

Luminal and cellular mechanisms for the mediation of tubuloglomerular feedback responses.

The results of our studies indicate that feedback-mediated changes in SFP are most likely the result of receptor cell detection of luminal fluid solute concentration. No specificity for any particular ion in mediating feedback responses could be identified. The detection of luminal fluid solute concentration may involve an intracellular cytoplasmic calcium system. The existence of a receptor cell intracellular calcium messenger system could serve as the link between changes in distal tubular fluid concentration and the alteration in glomerular vascular resistance.

Ionic transport in macula densa cells.

Recent work has provided substantial insights into functional characteristics of macula densa (MD) cells. Microelectrode and patch-clamp experiments on the rabbit isolated thick ascending limb (TAL)/glomerulus preparation have shown that MD cells possess a furosemide-sensitive Na:K:2Cl cotransporter, an apical 41-pS K+ channel, and a dominant basolateral Cl- conductance. Increasing luminal fluid [NaCl] ([NaCl]L) results in furosemide-sensitive cell depolarization due to a rise in intracellular [Cl-] that stimulates basolateral electrogenic Cl- efflux. Intracellular pH (pHi) measurements show the presence of an apical Na:H exchanger that couples transepithelial Na+ transport to pHi. Experimental results and thermodynamic considerations allow estimation of intracellular [Na+] and [Cl-] ([Na+]i, [Cl-]i) under different conditions. When the Na:K:2Cl cotransporter is equilibrated (or in the presence of furosemide), [Na+]i and [Cl-]i are low (approximately 6 to 7 mM), whereas when the cotransporter is fully activated, [Na+]i and [Cl-]i increase substantially to approximately 70 and 20 mM, respectively. Finally, luminal addition of NH4+ produces cell acidification that depends on NH4+ apical transport rate through the Na:K:2Cl. Using a simple transport model for NH4+, the initial NH4+ influx rate in MD cells is comparable to the corresponding flux in TAL. This challenges the idea that MD cells have a low transport activity but supports our findings about large changes in intracellular concentrations as a function of [NaCl]L.

Role of a macula densa feedback mechanism as a mediator of renal autoregulation.

The phenomenon of renal autoregulation demonstrates the presence of a sensitive intrarenal mechanism capable of maintaining GFR stable even during extrinsic disturbances that would be expected to alter renal hemodynamics. Substantial evidence has accumulated indicating that autoregulatory capability is dependent on the integrity of normal distal tubule flow dynamics and an intact distal tubuloglomerular feedback mechanism. Several whole-kidney and micropuncture studies have shown that interruption of volume delivery to the distal nephron interferes with autoregulation of renal blood flow (RBF) and GFR. The autoregulatory adjustments are probably localized at the afferent arterioles because the pressure in the larger arterioles does not exhibit autoregulation in response to decreases in renal perfusion pressure. It remains uncertain if the distal tubuloglomerular feedback mechanism is entirely responsible for autoregulatory responses. Data obtained in dog experiments indicate that under conditions of interrupted delivery to the distal nephron, SNGFR responses to decreases in arterial pressure are approximately those expected of a passive system where proximal tubule pressure is allowed to adjust to new steady-state levels with regard to the rapidity with which signals are transmitted to the distal nephron. Whole-kidney experiments indicate that, under conditions of a mild osmotic diuresis, the changes in urine flow following an increase in arterial pressure occur within 1 sec of the initiation of autoregulatory adjustments in vascular resistance. These experiments are consistent with the view that the major fraction of renal autoregulatory adjustments in resistance is mediated by the distal tubuloglomerular feedback mechanism that responds to some component of distal tubular flow and transmits signals to the afferent arteriolar segment of the same nephron.

A non-radioactive fluorescent method for measuring protein kinase C activity.

The purpose of this study is to report the development of a non-radioactive fluorescent peptide assay for measuring protein kinase C activity (PKC). The assay is based on a glycogen synthase derived fluorescent peptide that is phosphorylated by PKC. Phosphorylation causes the peptide to migrate toward the anode while the non-phosphorylated peptide migrates toward the cathode during agarose gel electrophoresis. Quantitation of PKC activity can be accomplished by excision of the appropriate bands and measuring their relative fluorescence. Using this assay, PKC activity was measured in whole cell homogenates from cultured renal mesangial cells. The enzyme(s)-substrate system followed Michaelis-Menten kinetics under limited conditions and, therefore, Lineweaver-Burk plots were used to obtain Michaelis constant and maximum velocity values. An apparent KM value of 40 microM was obtained for the fluorescent peptide substrate with a control Vmax value of 300 pmol/min. Addition of phorbol 12-myristate 13-acetate increased Vmax to 380 pmol/min.

Equity in the diagnosis of chest pain: race and gender.

OBJECTIVE: To explore gender and racial equity in emergency room treatment of chest pain. METHODS: Three hundred seventy-nine patient records were analyzed, taking into account effects of age, clinic, comorbid status, and insurance status. RESULTS: Analysis of covariance and logistic regression revealed statistically significant differences between races but not between genders for time to first EKG and percent of patients receiving cardiac catheterization and echocardiography. Blacks waited longer than whites for an EKG and were less likely to receive cardiac catheterizations but more likely to receive echocardiography. CONCLUSION: This study demonstrates a lack of equity by race in treatment of chest pain emergencies.

Cellular mechanisms of WNK4-mediated regulation of ion transport proteins in the distal tubule.

Gene mutations in WNK4 kinase cause a genetic form of hypertension by affecting multiple ion transport pathways through different mechanisms. Cai et al. report that the inhibitory effect of WNK4 on the thiazide-sensitive sodium-chloride cotransporter occurs through the lysosomal degradation pathway in mammalian cells.

Unraveling the relationship between macula densa cell volume and luminal solute concentration/osmolality.

At the macula densa, flow-dependent changes in luminal composition lead to tubuloglomerular feedback and renin release. Apical entry of sodium chloride in both macula densa and cortical thick ascending limb (cTAL) cells occurs via furosemide-sensitive sodium-chloride-potassium cotransport. In macula densa, apical entry of sodium chloride leads to changes in cell volume, although there are conflicting data regarding the directional change in macula densa cell volume with increases in luminal sodium chloride concentration. To further assess volume changes in macula densa cells, cTAL-glomerular preparations were isolated and perfused from rabbits, and macula densa cells were loaded with fluorescent dyes calcein and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate. Cell volume was determined with wide-field and multiphoton fluorescence microscopy. Increases in luminal sodium chloride concentration from 0 to 80 mmol/l at constant osmolality led to cell swelling in macula densa and cTAL cells, an effect that was blocked by luminal application of furosemide. However, increases in luminal sodium chloride concentration from 0 to 80 mmol/l with concomitant increases in osmolality caused sustained decreases in macula densa cell volume but transient increases in cTAL cell volume. Increases in luminal osmolality with urea also resulted in macula densa cell shrinkage. These studies suggest that, under physiologically relevant conditions of concurrent increases in luminal sodium chloride concentration and osmolality, there is macula densa cell shrinkage, which may play a role in the macula densa cell signaling process.

Standards and the integrated electronic health care record.

The goal of creating an integrated electronic health care record is within our reach. It will depend chiefly on the creation and adoption of standards for health care data. This article explains why standards development is important, gives examples of the different types of standards relevant to health care, offers examples of data sets used in health care, and, finally, presents examples of standards development organizations that health care supervisors should be familiar with.

Calcium antagonists and intrarenal regulation of glomerular filtration rate.

The intrarenal tubuloglomerular feedback mechanism operates at the site of contact between the thick ascending limb and glomerulus where specialized macula densa cells detect changes in tubular fluid composition and transmit information to the smooth muscle cells of the afferent arteriole. Increases in tubular fluid osmolality result in the transmission of vasoconstrictor signals and decreases in the rate of filtrate formation. Recent studies have identified two sites at which cytosolic calcium may play important roles. First, studies indicate that tubuloglomerular mediated vasoconstriction involves calcium mediated excitation contraction coupling of smooth muscle cells of the afferent arteriole. This calcium mediated event is sensitive to calcium channel blockade. Second, recent studies suggest that the macula densa cells may detect changes in tubular fluid osmolality through a cytosolic calcium system. The use of intracellular calcium antagonists further suggests that intracellular calcium mobilization is the primary mechanism responsible for increases in macula densa cytosolic calcium with increases in tubular fluid osmolality. Calmodulin and cyclic AMP may serve as modulators of this cytosolic calcium system. These studies suggest that calcium plays important roles in the regulation of renal hemodynamics.

Tubuloglomerular feedback responses in the rat during calmodulin inhibition.

Previously, we have suggested that a cytosolic calcium system participates in the transmission of signals between distal tubular fluid and the glomerular vascular elements. Since the Ca-binding protein calmodulin has been implicated in the intracellular actions of cytosolic Ca, we evaluated the effects of the calmodulin antagonists trifluoperazine (TFP) and calmidazolium (R24571) on tubuloglomerular feedback responses. In the rat, stop-flow pressure (SFP) was measured during retrograde perfusion into the distal tubule at 15 nl/min for 5 min. Perfusion with an isotonic Ringer solution resulted in a decrease in SFP of 12 +/- 1.1 mmHg (n = 34). With the addition of 50-75 microM TFP, SFP was decreased by 11 +/- 1.0 mmHg (n = 24); with 500 microM TFP, SFP feedback responses were 10 +/- 1.2 mmHg (n = 7). During perfusion with 20 microM R24571, SFP decreased by an average of 12 +/- 1.1 mmHg (n = 18). Single-nephron glomerular filtration rate feedback responses during retrograde microperfusion were also unaltered by R24571 (delta 15 +/- 2.5 vs. delta 14 +/- 2.6 nl/min). In the SFP experiments it was observed that the recovery of SFP toward preinfusion values after cessation of perfusion was significantly prolonged in tubules perfused with the calmodulin antagonists. SFP recovery times averaged 65 +/- 5.5 s (control), 104 +/- 9.9 s (50-75 microM TFP), 154 +/- 9.6 s (500 microM TFP), and 116 +/- 9.9 s (20 microM R24571). Accordingly, these results suggest that activation of calmodulin is not required for the transmission of tubuloglomerular feedback signals.(ABSTRACT TRUNCATED AT 250 WORDS)