Biomedical subjects

D L Tillotson

Publications and source records attributed to D L Tillotson.

15 recordsLinked to original sources

PubMed1992

Calcium channels and control of cytosolic calcium in rat and bovine zona glomerulosa cells.

Rat and bovine adrenal zona glomerulosa (ZG) cells possess a low-threshold, voltage-dependent Ca2+ current that was characterized using whole cell voltage clamp techniques. Activation of this current is observed at membrane potentials above -80 mV with maximal peak Ca2+ current elicited near -30 mV. Inactivation of the Ca2+ current was half-maximal between -74 and -58 mV, depending on the external Ca2+ concentration and was nearly complete at -40 mV. The voltage dependency of the current indicates that a calcium current could be sustained at membrane potentials between -80 and -40 mV and thereby elevates cytosolic calcium (Cai) levels. Under basal conditions, Cai is stable in single rat ZG cells, whereas more than half of the bovine ZG cells produce repeated Cai transients. These Cai transients, which are blocked by removal of external Ca2+ or addition of Ni2+, are likely due to repetitive electrical activity in bovine ZG cells. Cai responses can be elicited by small increases in external K+ concentration (5-10 mM) in both rat and bovine ZG cells, indicating the opening of low-threshold Ca2+ channels. However, these Cai changes remain robust at high external K+ concentrations (20-40 mM). In experiments combining Cai measurements and whole cell voltage clamp, a steep dependence of Cai on membrane potential was revealed beginning at depolarizing voltages near a holding membrane potential of -80 mV. A maximal increase in Cai occurred near -30 mV (equivalent to an external K+ concentration of 40 mM), a membrane voltage at which sustained current through low-threshold Ca2+ channels should be negligible. These data raise the possibility of additional voltage-dependent pathways for Ca2+ influx.

4-Aminopyridine↗

PubMed1992

K+ channels in adrenal zona glomerulosa cells. I. Characterization of distinct channel types.

Four distinct types of K+ channels were identified in rat and bovine adrenal zona glomerulosa (ZG) cells and characterized using single-channel recording techniques. Inward rectifier channels were the most frequently observed K+ channel types in the membrane patches of both rat and bovine ZG cells. The slope conductance of the inward current was 42 pS with an extracellular K+ concentration of 150 mM. The probability of the open state of these channels increased with depolarization. With the use of inside-out membrane patches with symmetric 150 mM K+ solutions, the rectifying behavior was found to require Mg2+ on the intracellular side of the membrane. Delayed rectifier K+ channels with conductances of 27 and 48 pS were found with rat ZG cells. These channels persisted with prolonged positive voltage steps and showed long mean open times with increasing depolarization. Transient outward currents with a conductance of 28 pS were observed only in bovine ZG cells. These channels showed substantial inactivation during positive voltage steps of 250 ms duration. Ca(2+)-activated K+ channels with a large conductance (228 pS) were identified in rat and bovine ZG cells. These different classes of K+ channels may be important for the control of resting membrane potential and the generation of action potentials, thus participating in the regulation of Ca2+ influx and aldosterone secretion in ZG cells.

Animals↗

PubMed1992

Single K+ channels in adrenal zona glomerulosa cells. II. Inhibition by angiotensin II.

The effects of angiotensin II (ANG II) on single K+ channels were studied in rat and bovine adrenal zona glomerulosa (ZG) cells, using the patch-clamp technique. ANG II (0.1-10 nM) induced substantial inhibition of inward rectifier and delayed rectifier K+ channel activities in rat and bovine ZG cells. Analysis of single-channel activities showed that the ANG II-induced channel-blocking effect involved reductions in the probability of the open state (Po) and the mean open time. The changes in these channel parameters occurred at all test voltages, indicating that the effect of ANG II was voltage independent. ANG II could not interact directly with the extracellular sides of the membranes in these experiments using cell-attached patches. Therefore, the effect of ANG II on K+ channels must occur through an indirect cytosolic transduction pathway. The ANG II-induced block of K+ channels will result in membrane depolarization, which may activate voltage-dependent Ca2+ channels, thereby increasing cytosolic free Ca2+ and stimulating aldosterone secretion. These channel-modulating actions of ANG II may be an important step in the initial sequence of events underlying its transduction mechanism.

Angiotensin II↗

PubMed1992

Ion channels in human erythroblasts. Modulation by erythropoietin.

To investigate the mechanism of intracellular Ca2+ ([Cai]) increase in human burst-forming unit-erythroid-derived erythroblasts by erythropoietin, we measured [Cai] with digital video imaging, cellular phosphoinositides with high performance liquid chromatography, and plasma membrane potential and currents with whole cell patch clamp. Chelation of extracellular free Ca2+ abolished [Cai] increase induced by erythropoietin. In addition, the levels of inositol-1,4,5-trisphosphate did not increase in erythropoietin-treated erythroblasts. These results indicate that in erythropoietin-stimulated cells, Ca2+ influx rather than intracellular Ca2+ mobilization was responsible for [Cai] rise. Both Ni2+ and moderately high doses of nifedipine blocked [Cai] increase, suggesting involvement of ion channels. Resting membrane potential in human erythroblasts was -10.9 +/- 1.0 mV and was not affected by erythropoietin, suggesting erythropoietin modulated a voltage-independent ion channel permeable to Ca2+. No voltage-dependent ion channel but a Ca(2+)-activated K+ channel was detected in human erythroblasts. The magnitude of erythropoietin-induced [Cai] increase, however, was insufficient to open Ca(2+)-activated K+ channels. Our data suggest erythropoietin modulated a voltage-independent ion channel permeable to Ca2+, resulting in sustained increases in [Cai].

Adult↗

PubMed1991

ANG II blocks potassium currents in zona glomerulosa cells from rat, bovine, and human adrenals.

Angiotensin II (ANG II) is a principal secretagogue of adrenal zona glomerulosa (ZG) cells. The transduction process includes a depolarization of the plasma membrane and the activation of calcium influx. The ANG II-induced depolarization is associated with an increase in total membrane resistance. To directly address the mechanism underlying these observations, we examined the effect of ANG II on K+ currents of rat, bovine, and human ZG cells, using whole cell patch clamp. Although some differences were seen in the characteristics of K+ currents between species, ANG II consistently blocked outward currents in ZG cells [rat: 47.1 +/- 4.5% (SE), n = 17; bovine: 38.6 +/- 3.3%, n = 21; and human: 13-63%, n = 3]. With the use of the cell-attached mode, single-channel recordings in bovine ZG cells demonstrated K+ channels that were reversibly blocked when ANG II was added to the bath solution. This indicates that the block of K+ channels by ANG II involves a diffusible intracellular messenger rather than a direct receptor-channel interaction. The decreased conductance of K+ can account for the ANG II-induced membrane depolarization.

Angiotensin II↗

PubMed1991

Nuclear calcium gradients in cultured rat hepatocytes.

Ca2+ concentrations ([Ca2+]) in cytosol and nucleus in fura-2-loaded cultured rat hepatocytes were determined by three-dimensional (3-D) optical-sectioning microscopy. After determining the empirical 3-D point spread function of the fluorescence microscope-coupled digital video imaging system, contaminating light arising from optical planes above and below the plane of interest was removed by deconvolution using the nearest-neighboring approach (NNA) algorithm. Although deconvolution resulted in substantial improvement in accuracy of fluorescence intensity determinations in single-wavelength excitation images as well as sharper delineation of boundaries between cellular compartments, the complicated mathematical process did not significantly enhance the precision of [Ca2+] values derived from ratiometric (ratio of dual-wavelength excitation) images. In resting hepatocytes, cytosolic Ca2+ (210 +/- 15 nM) was 1.6- to 2-fold higher than nuclear Ca2+ (128 +/- 12 nM). This difference in Ca2+ between the two compartments was detected both in raw ratiometric images and in those processed with NNA algorithm. Addition of arginine vasopressin or epidermal growth factor resulted in significant increases (2- to 3-fold) in both cytosolic and nuclear Ca2+; however, the nuclear-to-cytosolic Ca2+ gradient was preserved in hepatocytes stimulated with mitogens. We conclude that the hepatocyte nuclear membrane contains Ca2+ permeability barriers and Ca2+ transport mechanisms that may be hormonally sensitive. We postulate that the increase in nuclear Ca2+ may be important in regulation of cell proliferation induced by mitogens, possibly by activating Ca(2+)-dependent endonucleases, nuclear calmodulin, or nuclear protein kinase C.

Algorithms↗

PubMed1991

Kinetics of cytosolic calcium and aldosterone responses in rat adrenal glomerulosa cells.

To evaluate the relationship between cytosolic calcium (Cai) and aldosterone production, rat adrenal zona glomerulosa (ZG) cells were studied during long-term stimulation by different secretagogues. Cai was measured in single ZG cells using microspectrofluorimetry, and aldosterone was determined in cell populations using a superfusion system. For external potassium (K+), Cai increases are sustained, with only a slight decrement over time, a feature shared by aldosterone production. The relationship between aldosterone output and Cai is nonlinear, with a Cai value for half-maximal stimulation of approximately 500 nM. Furthermore, the sustained changes in Cai with external K+ indicate that ZG cells can use an amplitude-based Cai signal to stimulate aldosterone production. Cai changes stimulated by angiotensin-II (Ang-II) show a complex dose-response pattern, with high concentrations (greater than or equal to 1 nM) of Ang-II eliciting a peak-plateau signal and lower doses (0.1 nM to 10 pM) producing repeated Cai oscillations. The peak amplitude of the Cai response in individual cells is not dose dependent, with the ZG cell experiencing peak levels repeatedly at the lowest Ang-II concentrations. However, the Cai transients are more frequent with increasing Ang-II concentrations between 0.1 nM and 10 pM. When integrated over time, the mean Cai signal also shows only modest dose-dependency during the sustained phase of Ang-II stimulation. Unlike the integrated Cai signal, aldosterone production increases steeply between 10 pM and 0.1 nM Ang-II, indicating that the Cai signal is likely to be frequency-based. Conversely, the steroid response to high Ang-II closely mirrors the kinetics of the more sustained Cai signals, including the diminished Cai and aldosterone levels during sustained stimulation with the highest Ang-II doses. Arginine vasopressin stimulated Cai and aldosterone responses, which closely resemble those elicited by 0.1 nM Ang-II, except that both Cai and aldosterone return to basal values within 20 min of continuous presentation of arginine vasopressin. Each ZG secretagogue produces a distinct pattern of Cai and aldosterone response. In addition, Cai response patterns can be divided into two general classes: a sustained Cai response, which appears to modulate cell activation by the amplitude of the Cai signal, and an oscillating Cai response, which uses the frequency of the Cai transients to control the magnitude of stimulation.

Aldosterone↗

PubMed1990

Three-dimensional intracellular calcium gradients in single human burst-forming units-erythroid-derived erythroblasts induced by erythropoietin.

We have previously shown that the intracellular free Ca2+ increase induced by erythropoietin is likely related to differentiation rather than proliferation in human BFU-E-derived erythroblasts (1989. Blood. 73:1188-1194). Since cell differentiation involves transcription of specific regions of the genome, and since nuclear endonucleases responsible for single strand DNA breaks observed in cells undergoing differentiation are Ca2+ dependent, we investigated whether the erythropoietin-induced calcium signal is transmitted from cytosol to nucleus in this study. To elucidate subcellular Ca2+ gradients, the technique of optical sectioning microscopy was used. After determining the empirical three-dimensional point spread function of the video imaging system, contaminating light signals from optical planes above and below the focal plane of interest were removed by deconvolution using the nearest neighboring approach. Processed images did not reveal any discernible subcellular Ca2+ gradients in unstimulated erythroblasts. By contrast, with erythropoietin stimulation, there was a two- to threefold higher Ca2+ concentration in the nucleus compared to the surrounding cytoplasm. We suggest that the rise in nuclear Ca2+ may activate Ca2(+)-dependent endonucleases and initiate differentiation. The approach described here offers the opportunity to follow subcellular Ca2+ changes in response to a wide range of stimuli, allowing new insights into the role of regional Ca2+ changes in regulation of cell function.

Calcium↗

PubMed1990

Cytosolic calcium and aldosterone response patterns of rat adrenal glomerulosa cells stimulated by vasopressin: comparison with angiotensin II.

Cytosolic calcium (Cai) responses to arginine vasopressin (AVP) and angiotensin-II (Ang II) were examined in single rat adrenal zona glomerulosa (ZG) cells by monitoring fura-2 fluorescence with microspectrofluorimetry. ZG cells displayed dose-dependent Cai responses to a wide range of AVP and Ang II concentrations, starting from a threshold of 1 nM for AVP and less than 5 pM for Ang II. A dose-dependent delay of the onset of the Cai response was observed with both hormones. The response delay for Ang II was consistently briefer than that for the same concentration of AVP, showing a 2-3 log unit separation in the dose-response relations. After the delay, cells typically responded with an abrupt increase in Cai, which peaked within 15 sec. The amplitude of the peak Cai rise showed little dependency on AVP or Ang II concentration. At most AVP concentrations, the response consisted of Cai oscillations, with apparent fusion of these Cai oscillations at the highest AVP concentrations (1-0.1 microM). Similar oscillatory behavior was found with stimulations by much lower Ang II concentrations (0.5 nM to 5 pM). There appeared to be a 2-3 log unit shift in the sensitivity toward AVP and Ang II when Cai responses were compared. Sixty percent of ZG cells were responsive to AVP, while more than 90% displayed an elevation of Cai with Ang II. The Cai and steroid responses to 100 nM AVP and 100 pM Ang II were compared, since these two doses are reported to stimulate the phosphoinositide system to a similar extent. Individual ZG cells tested with both hormones responded with equivalent peak Cai changes, but a slightly longer response delay for AVP. The mean Cai response and aldosterone production for each secretagogue displayed parallel kinetics during 30-min stimulations. After initial oscillations, the Cai response returned to control values within 15 min of 100 nM AVP application. Likewise, the steroid output was transient. In contrast, 100 pM Ang II produced maintained Cai oscillations as well as a sustained and substantially greater aldosterone production for the same period of application. In conclusion, the disparate steroidogenic effects of AVP and Ang II appear to result from distinctly different Cai responses elicited during maintained secretagogue stimulation.

Aldosterone↗

PubMed1989

Cytosolic free calcium concentration in individual cardiac myocytes in primary culture.

Cytosolic free Ca2+ concentration, [Ca2+]i, of single isolated Ca2+-tolerant rat ventricular myocytes in primary culture was determined by digital video imaging of intracellular fura-2 fluorescence. In deenergized myocytes in which contractile elements were uncoupled by 2,3-butanedione monoxime, the maximum and minimum fluorescence intensity ratio values of fura-2 in the cell were similar when compared with those of fura-2 solutions observed in the microscope. Through the use of in vitro calibration, [Ca2+]i in quiescent, rod-shaped myocytes was 90 +/- 6 nM. There was no detectable spatial heterogeneity in [Ca2+]i in resting myocytes. Localized regions of [Ca2+]i elevation were observed in cells undergoing spontaneous rhythmic contractions or when subjected to mild depolarization by KCl. Additions of gramicidin or veratridine resulted in massive increases in [Ca2+]i (greater than 1 microM) and immediate cell hypercontracture. Ruthenium red elicited a modest increase in [Ca2+]i but extracellular ATP or epinephrine had no effect. We conclude the following: 1) digital video imaging of resting cardiac cells did not reveal any subcellular Ca2+ gradients; 2) the fluorescence properties of intracellular fura-2 were similar to that in free solution; and 3) subcellular heterogeneity of [Ca2+]i in isolated myocytes was observed in cells undergoing spontaneous rhythmic contraction.

Adenosine Triphosphate↗

PubMed1989

Erythropoietin stimulates a rise in intracellular-free calcium concentration in single BFU-E derived erythroblasts at specific stages of differentiation.

Human cord blood progenitor-derived erythroblasts have recently been shown to respond to erythropoietin (Epo) or granulocyte-macrophage colony-stimulating factor (GM-CSF) with a transient increase in intracellular free calcium concentration [Cac]. However, the importance of [Cac] changes in mediating cell proliferation and/or differentiation is undefined. In the present study, the response of erythroid precursors at different stages of differentiation to Epo was examined. Erythroblasts were derived from adult blood erythroid progenitors (BFU-E) at day 7 or day 10 of culture. [Cac] was measured in individual Fura-2 loaded cells with fluorescence microscopy coupled digital video imaging. The dynamic range (Rmax/Rmin) of intracellular Fura-2 was similar to that measured in free solution, suggesting insignificant amounts of intracellular Ca insensitive forms of Fura-2. Baseline [Cac] of erythroid cells calculated with an in vitro calibration method was 44 +/- 4 nmol/L and with an in vivo method was 46 +/- 4 nmol/L. Treatment of day 7 BFU-E derived erythroblasts with Epo resulted in no significant increase in [Cac]. In contrast, in more mature erythroblasts (day 10 of culture), Epo stimulated a large increase in [Cac] from 49 +/- 11 nmol/L at baseline to 279 +/- 47 nmol/L. This [Cac] increase occurred in phosphate buffered saline (PBS) containing no added calcium. The increase in [Cac] persisted for 18 minutes and was dose dependent. Day 7 and day 10 control cells treated with either insulin or media showed no significant change in [Cac] during 18 minutes of observation. Our data demonstrate that early (day 7) and late (day 10) erythroblasts display different responses to Epo, at least in terms of intracellular Ca++ fluxes. The differential [Cac] response observed in early and late erythroid precursors to growth factor stimulation suggests that [Cac] may be an important signal in cell differentiation.

Adult↗

PubMed1988

Calcium oscillations in single adrenal glomerulosa cells stimulated by angiotensin II.

The cytosolic calcium (Ca2+i) response to angiotensin II (Ang II) was examined in single rat zona glomerulosa cells by monitoring fura-2 fluorescence with microspectrofluorimetry. Ang II concentrations ranged from 5 X 10(-12) to 5 X 10(-8) M. The mean peak Ca2+i increase was similar at all Ang II concentrations (205 +/- 11 nM), with a significant difference (P less than 0.05) found only between 5 X 10(-12) M (151 +/- 16 nM) and 5 X 10(-9) M (236 +/- 24 nM). Striking differences over the range of Ang II concentrations were found in the Ca2+i response kinetics. A dose-dependent delay of the onset of the Ca2+i response was observed ranging from 2.6 +/- 0.3 sec at 5 X 10(-8) M to 181 +/- 27 sec at 5 X 10(-12) M Ang II. After the delay, cells typically responded with an abrupt increase in Ca2+i, complete within 15 sec. At low Ang II concentrations (5 X 10(-11) and 5 X 10(-12) M), a complex response was often observed consisting of Ca2+i oscillations. Higher Ang II concentrations gave some evidence of Ca2+i oscillation, especially at 5 X 10(-10) M where oscillations appeared fused. Above 5 X 10(-10) M Ang II, the initial Ca2+i increase decayed to an apparent steady-state value 38-40% of the peak response within 5 min; 5 X 10(-10) M Ang II produced a smaller decline to 63% of the initial Ca2+i increase. In contrast to cell population studies, assessment of individual glomerulosa cells demonstrates (i) a dose-dependent delay prior to a rapid increase in Ca2+i; (ii) a similar peak increase at most Ang II concentrations; (iii) greater sensitivity of the Ca2+i response; and (iv) a complex oscillating Ca2+i response in the physiological range of Ang II.

Adrenal Cortex↗

PubMed1988

Calcium response of single adrenal glomerulosa cells to external potassium.

The cytosolic calcium (Cai2+) response to external potassium (K+) was examined in single rat zona glomerulosa (ZG) cells by monitoring fura-2-fluorescence with microspectrofluorometry and digital imaging microscopy. The majority (68%) of morphologically identified ZG cells demonstrated an increase in Cai2+ during K+ stimulation. Cai2+ rose monotonically from a mean basal level of 232 +/- 15 to 285 +/- 37 nM at 5 mM and 680 +/- 60 nM at 10 mM K+ for responsive ZG cells. The Cai2+ response was largely (greater than 90%) inhibited by nominal zero calcium or 1 mM cadmium and substantially modified in the presence of 10(-5) M nifedipine. The response kinetics were characterized by a rising phase that depended on the size of the Cai2+ change, with larger increases associated with a faster onset. Cai2+ approached a plateau level that was sustained for the duration of K+ stimulation from 1 to 5 min. Cai2+ appeared to be more uniformly distributed across the cell under resting conditions than during stimulation. Assessment of the Cai2+ response in single ZG cells documents 1) a majority, but not all, of ZG cells respond to K+, 2) simple kinetics consisting of a rapid onset and sustained plateau Cai2+ level, 3) a dose-dependent Cai2+ increase in the physiological range of K+, and 4) inhibition by calcium channel blockers and sensitivity to small increases in K+ consistent with activation of low-threshold calcium channels.

Animals↗

PubMed1988

Erythropoietin stimulates a rise in intracellular free calcium concentration in single early human erythroid precursors.

Erythropoietin and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulate the differentiation and proliferation of erythroid cells. To determine the cellular mechanism of action of these growth factors, we measured changes in intracellular free calcium concentration [( Cac]) in single human erythroid precursors in response to recombinant erythropoietin and GM-CSF. [Cac] in immature erythroblasts derived from cultured human cord blood erythroid progenitors was measured with fluorescence microscopy digital video imaging. When stimulated with erythropoietin, [Cac] in the majority of erythroblasts increased within 3 min, peaked at 5 min, and returned toward baseline at 10 min. The percentage of cells that responded to erythropoietin stimulation increased in a dose-dependent manner. Additional stimulation with GM-CSF in cells previously exposed to erythropoietin resulted in a second [Cac] increase. Immature erythroblasts treated with GM-CSF followed by erythropoietin responded similarly to each factor with a rise in [Cac]. The source of transient calcium is intracellular since erythroblasts were incubated in medium devoid of extracellular calcium. Our observations suggest that changes in [Cac] may be an intracellular signal that mediates the proliferative/differentiating effect of hematopoietic growth factors.

Body Fluids↗

PubMedDate not supplied

Ion selective microelectrodes: computer-controlled calibration, plotting, and data analysis.

Calibration of ion-selective microelectrodes involves delivering and measuring the potential of several different solutions both before and after intracellular measurement. A microprocessor-controlled system is described which completely automates that tedious process. The microcomputer controls the delivery of the calibration solutions and prints out the calibration value along with the solution number. A second program plots the calibration curve using a modified Nicholsky equation and a third program converts intracellular ion signals into ion concentration. Specific examples based on Ca-selective microelectrodes are shown.

Autoanalysis↗