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Journal of Physiology (1992), 447, pp. 171-189 With 9 figures Printed in Great Britain

IMAGING OF INTRACELLULAR CALCIUM IN RAT ANTERIOR PITUITARY CELLS IN RESPONSE TO GROWTH HORMONE RELEASING FACTOR

BY M. KATO*, J. HOYLAND, S. K. SIKDAR AND W. T. MASON From the Department of Neuroendocrinology, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT and the *Department of Physiology, Institute of Endocrinology, Gunma University, Maebashi 371, Japan

(Received 29 April 1991) SUMMARY

1. Changes in intracellular ionized calcium [Ca2+]i induced by human growth hormone releasing factor (hGRF) were analysed by quantitative fluorescent microscopy using a dual-wavelength, ratiometric video imaging system and low light level charge-coupled device (CCD) camera visualizing Fura-2 in dispersed male rat anterior pituitary cells. 2. In cells responding to hGRF, spontaneous basal oscillations in [Ca2+]i were frequently observed, and these were usually characterized by a gradient of [Ca21] localized in the subplasmalemmal region of the cell. 3. Of the cells which responded to hGRF, the peptide evoked a rise in [Ca2+]i, especially in the region of the subplasmalemma. Continuous application of 10 nmhGRF produced several different temporal patterns of the [Ca2+]i response which were not attributable to spatial response profiles. A sustained rise in [Ca2+]i was the most common type of response to hGRF (44% of the cells examined). 4. One-third of the cells responding to 10 nM-hGRF showed spontaneous basal [Ca2+]i oscillations ranging from 100 to 500 nm. Mean values of basal and 10 nMhGRF-induced [Ca2+]i of these cells were 81+11 nM (mean+ S.E.M., n = 27) and 560 + 47 nM (n = 27) respectively. There was no significant correlation between basal [Ca2+]i and the hGRF-induced [Ca2+]i increase, nor was there any consistent correlation with regard to the spatial response profile. 5. Application of 2 mM-Co2+ abolished the hGRF-induced rise in [Ca2+]i. Quantitative analysis of this effect, performed by comparing the mean [Ca2+]i evoked during the application of hGRF with and without Co2+, respectively, also showed significant inhibition of the hGRF-induced rise in [Ca2+]i by the application of Co2+

(P < 0o001). 6. The hGRF-induced rise in [Ca2+]i was completely suppressed by replacing extracellular Na+ with impermeant molecules such as mannitol. The onset and offset of suppression was as rapid as that induced by Co2+. Quantitative analysis showed significant inhibition of the hGRF-induced rise in [Ca2+]i by Na+ replacement (P < 0 01). MS 9342

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M. KATO, J. HOYLAiND, S. K. SIKDAR ANAD W. T. MASON1

7. Tetrodotoxin, a potent blocker of voltage-sensitive Na' channels (5 and 20 aM), did not affect the hGRF-induced rise in [Ca2+]i. 8. Extracellular application of the membrane permeable dibutyryl cyclic AMP (DBcAMP) to elevate intracellular levels of cyclic AMP caused a large rise in [Ca2+]i, which was dependent on extracellular Na+ and was abolished by 2 mM-Co2+ applied in the bath. The DBcAMP-induced rise in [Ca2+]i thus had the same ionic requirement as that of hGRF, and showed an identical pattern of subplasmalemmal localization. 9. From these results, taken together with previous findings, we propose the possibility that hGRF activates tetrodotoxin-insensitive Na' (or non-selective cationic) channels via cyclic AMP, which in turn causes depolarization of the somatotroph leading to activation of Ca21 channels, Ca2" influx and exocytotic secretion of growth hormone. INTRODUCTION

Human growth hormone releasing factor (hGRF) is a potent and specific stimulator of growth hormone (GH) secretion both in vivo and in vitro in various species including the rat. Although the mechanism by which hGRF stimulates the secretion of growth hormone is not fully understood, adenosine 3', 5' cyclic monophosphate (cyclic AMP) is thought to be an intracellular mediator of GRF action in somatotrophs (Bilezikjian & Vale, 1983). The presence of extracellular Ca2+ is indispensable for hGRF-induced GH secretion (Kato & Suzuki, 1986). Verapamil and Co2+, known as potent blockers of voltagesensitive Ca2" channels, suppress hGRF-induced GH secretion without inhibiting the production of cyclic AMP (Bilezikjian & Vale, 1983; Kato & Suzuki, 1986). Action potentials in normal somatotrophs have been recorded (Israel, Denef & Vincent, 1983; Ozawa & Sand, 1986 for review) and the presence of voltage-sensitive Ca2+ channels has been described in rat somatotrophs (DeRiemer & Sakmann, 1986). Mason & Rawlings (1988) reported the presence of both voltage-sensitive Na+ and Ca2+ channels in bovine somatotrophs. In addition a close temporal relationship between action potentials and the oscillation of intracellular Ca2+ concentration ([Ca2+]i) was demonstrated in GH-secreting cells (Schlegel, Winiger, Mollard, Vacher, Wuarin, Zahnd, Wollheim & Dufy, 1987). Thorner, Holl & Leong (1988) showed in identified single somatotrophs that GRF produced a rise in [Ca2+]i which entirely depended on the presence of extracellular Ca2+, and they suggested that cyclic AMP generated in the somatotroph in response to GRF leads to phosphorylation of Ca2+ channels, which allows their opening and thereby increases Ca2+ influx. Thus the involvement of voltage-sensitive Ca2+ channels in GRF stimulation of GH secretion has been strongly suggested. However, it has not been fully determined how these Ca2+ channels are activated, although Thorner et al. (1988) proposed one possibility, as mentioned above. There are, however, at least two other possible mechanisms for the activation of voltage-sensitive Ca2+ channels. One is the blockade of resting K+ conductance as in the case of pancreatic cells (Asheroft, Harrison & Ashcroft, 1984). The other is the activation of Na+ channels. Both mechanisms could depolarize the cell in turn activating voltage-sensitive Ca2+ channels. In rat somatotrophs, both hGRF- and dibutyryl cyclic AMP (DBcAMP)-induced GH

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secretion is suppressed by replacing Na+ with impermeant molecules without affecting the excess-K+-evoked GH secretion (Kato, Hattori & Suzuki, 1988). Furthermore the photometric measurement of membrane potentials from a population of anterior pituitary cells revealed that hGRF depolarized anterior pituitary cells in a Na+-dependent but not in a Ca2+-dependent manner (Kato & Suzuki, 1989a). From these results, Kato et al. (1988) and Kato & Suzuki (1989a, b) proposed the following possible mechanism: GRF stimulates cyclic AMP production which in turn activates Na+- or non-selective cationic channels (but not Ca2+-permeable channels), thus depolarizing somatotrophs and activating voltage-sensitive Ca2+ channels. Ca2+ entry and GiH secretion are thereby promoted. The aim of this paper was to examine this hypothesis by studying the hGRF-induced rise in [Ca2+]i in single anterior pituitary cells. The entire population of anterior pituitary cells is composed of at least six different hormone-secreting cell types, hence the average response from populations of cells does not necessarily reflect the events at the level of the single somatotroph. In addition since somatotrophs are heterogeneous in their secretory capacities (Thorner et al. 1988), studies with populations of cells are likely to mask important observations in single somatotrophs. Furthermore, it is important to study the subeellular localization of [Ca2+], because exocytosis may be triggered by Ca2+ activation of the subplasmalemmal region of some secretory cells (Cheek, Jackson, O'Sullivan, Moreton, Berridge & Burgoyne, 1989). The technique of dynamic, realtime video imaging combined with quantitative image analysis enables study of intracellular ion concentration at the single-cell level in populations of pituitary cells. In the present experiments, we used dynamic intracellular Ca2+ imaging at the subcellular level and found that hGRF and DBcAMP elevated [Ca2+]i particularly in the subplasmalemmal region of the cells, an effect dependent on the presence of Ca2+ and Na+ in the extracellular space. METHODS

Perfusion media Normal perfusion medium had the following composition (mM): 137-5 NaCl, 5 KCl, 2-5 CaCl2, 0 8 MgCl2, 10 glucose, 20 N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid (HEPES; Sigma; pH 7 4 with NaOH), 0-25% bovine serum albumin (BSA; Fraction V, Sigma). Na'-free medium was prepared by replacing NaCl of the normal medium isosmotically with D-mannitol (BDH Chemicals, UK), or by replacing Na' with tris hydroxymethyl aminomethane (Tris+; Sigma) or with N-methyl-D-glucamine (Aldrich Chemicals, UK). Human growth hormone releasing factor (hGRF-1-44. Peninsula laboratories, USA), DBcAMP (Sigma), tetrodotoxin (TTX, Sigma), Fura2 AM (Molecular Probes, USA), poly-D-lysine (MW, 53000; Sigma) and Percoll (Pharmacia, Sweden) were also used. Cell isolation procedure Two or three anterior pituitaries from adult male Wistar rats (250-300 g body weight, killed by stunning and decapitation) were minced into ten to fifteen pieces in phosphate-buffered saline without divalent cations (PBS). The minced pituitaries were incubated in PBS containing 0-2% trypsin (type III, bovine pancreas, Sigma), 0-025% collagenase (from Clostridium histolyteum; Boeringer Mannheim, Germany) and 1 mM-CaCl2 at 37 °C for 10 min. After the incubation, the PBS was discarded and 20 ml PBS containing 10 ,ag deoxyribonuclease I (Sigma), 1 ,ug trypsin inhibitor (type II, Sigma) and 2 mM-EDTA (Fisons, UK) was added. After 5 min incubation at 37 °C, the pituitaries were mechanically dispersed by triturating with a 5 ml plastic pipette for 5 min. The cell

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M. KATO, J. HOYLAND, S. K. SIKDAR AND W. T. MASON

suspension was filtered with 60 1am nylon mesh and centrifuged at 400 g for 5 min. The supernatant was decanted and the cells were resuspended in 40 % Percoll and then placed on a 40, 45, and 80 % discontinuous Percoll density gradient. After centrifugation at 800 g for 15 min at room temperature, the interface between the 45 and 80 % Percoll was collected as a somatotrophenriched fraction. The cells were centrifuged (400 g for 5 min), washed three times with PBS and resuspended in 200 ,tl PBS. Cell viability was more than 90 % and cell yield was 5 x 105 cells/pituitary. Cells were plated on poly-D-lysine (0-01 %)-coated glass cover-slips with a suspension volume of 10-20 jl. After cells attached on the cover-slips, 2 ml Dulbecco's modified Eagle's medium (Gibco, UK) supplemented with 0-25% BSA was added and incubated at 37 °C until use (normally after 1-10 h). Cell viability was occasionally assessed by use of Trypan Blue, and more importantly by responsiveness to hGRF. In these cultures, the percentage of cells responding in a reversible fashion to hGRF at 10 nm varied typically between 25 and 40 %. It has been estimated that somatotrophs may comprise up to 50 % of the male rat anterior pituitary, and the remainder of the cells include the other anterior pituitary hormone-secreting cell types. hGRF is a highly specific stimulus for growth hormone secretion and fails to evoke measurable prolactin or gonadotrophin secretion in cultured cell preparations, and is thus a reliable indicator that responsive cells are somatotrophs. We cannot exclude that some viable hGRF non-responsive cells may also have been somatotrophs, however, but they were in any event of no interest for the present study. Digital imaging analysis of intracellular free Ca2" concentration Cells were loaded with 1 /LM-Fura-2 AM at 37 °C for 15 min. After the loading procedure, the cover-slip holding the cells was fixed into a temperature-controlled perfusion chamber (37 °C, 100 ,1u volume; Joyce Leobl Instruments, Gateshead, UK) mounted on the stage of an inverted Nikon Diaphot microscope and continuously perfused with normal medium (flow rate, 1 ml/min). The MagiCal system with TARDIS software (Joyce Loebl Instruments) was used for all dynamic video imaging and image processing (for review of methodology employed see Mason, Hoyland, Rawlings & Relf, 1990). Briefly, a Nikon quartz objective lens (CF-Fluor, x 40, oil immersion) was used. Excitation wavelengths (340 and 380 nm) were selected by means of a computer-controlled rotating filter wheel between a xenon lamp and the microscope. The emission light at 510 nm (10 nm half-bandwidth) was passed to an image-intensifying charge-coupled device (CCD) camera (Photonic Sciences, Robertsbridge, UK). The resulting image at each wavelength was averaged in real time (four or eight images), digitized at 8 bits accuracy to yield 256 grey levels, captured typically as a 256 x 256 pixel image, and stored in the 32 Mbyte dynamic random access memory of an image-processing unit under the software control of a PC/AT-compatible computer. Time resolution in most of these experiments was set at 3-6 s between ratio frames. The time points shown on some figures are the arithmetic average of the time at which the individual 340 and 380 nm frames were captured. The software also allowed for the capture of background 340 and 380 nm images at the start of an experiment to allow correction by subtraction of background fluorescence. The two background images (340 and 380 nm) were subtracted from their relevant experimental images at the same wavelengths at the completion of the experimental run. Care was taken to avoid overlap of samples with the limits of either the camera sensitivity or the analog-todigital converter, and this was carefully monitored during the experiment with an oscilloscope our the live camera output and post-experiment by graphical analysis. This eliminated any possible saturation of images which could have produced artifactually high or low [Ca2J]i concentrations. The ratio of emitted fluorescence at the two excitation wavelengths (340 and 3so nm) was calculated for each frame on a pixel-by-pixel basis and computed against a look-up-able to yield a ratio grey level representative of a calibrated [Ca2+]i concentration. The fluorescence ratios were converted to Ca2+ ion concentration by the following equation (Grynkiewvicz, Poenie & Tsien, 1985): [Ca2+]i = Kdfl[(R-Rmin)/(Rmax-R)], where Kd is the dissociation constant for Fura-2/Ca2+ (225 .M), R is the ratio at any pixel point, Rmin and Rmax are the ratio values of Fura-2 (at zero and saturating [Ca2]Ji respectively, and P is the ratio of fluorescence at 380 nm for the dye in saturating and zero [Ca2+]i. The calibration constants (Rmin, Rmax and /l) were empirically determined in the same experimental set-up using ionomycin permeabilization (1 /tM) of the cells in the presence of either 10 mM-EGTA or 10 mM-Ca2, in addition to the usual physiological salts, with measurement of the parameters on dye loaded in cells. Typical values of Rmin and Rmax were 0-2 and 6-5.

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Data analysis To produce a continuous trace of mean [Ca2+]i against time, an area around a cell was graphically defined with a light pen and the mean [Ca2J1i level within the area was computed. The computed [Ca2+]i was calculated on an individual pixel basis; the mean of all values in the pixel 'set' was then taken as the mean [Ca21]i. Individual data was exported to an ASCII file which was incorporated into a Lotus 123 spread sheet for further calculations. For quantitative analysis, the mean [Ca2+]i was calculated for the particular regions of interest. Duncan's multiple range test was used for statistical analysis. A probability of error less than 005 was considered to be significant and is indicated by asterisks in figures.

Technical comment Digital imaging is particularly prone to artifacts which may most commonly arise from incorrect adjustment of the camera sensitivity in relation to the imaging system's data acquisition hardware. This can under certain circumstances, for instance, give the false impression of intracellular calcium which is too high or too low because the ratio of grey levels at two different wavelengths is misleadingly high or low. In setting up the system used in this work, we took steps to use combinations of interference filters (340 and 380 nm) with variable half-bandwidths and quartz neutral density filters to ensure that typical light levels did not exhibit excursions outside approximately the top or bottom 10 % of the digitizing or averaging hardware sensitivity. In addition, the software digitally corrected for voltage offset typical of CCD video cameras, so that a full 256 grey levels were available for the working dynamic range of the system. Background of the dark camera due to ambient light was typically four to eight grey levels, and these were subtracted from individual raw images before ratios were calculated. The CCD camera used in this work had separate controls over gain of both the intensifier and video stages, and the single-pixel voltage output of the camera was monitored continuously on both an oscilloscope and initially using software options which ensured that overload of either the system analog-to-digital converters or hardware averagers did not take place. This was particularly important in the present experiments as the observation of calcium gradients within the single cells could have been an artifact due to either saturation of the system or too-low sensitivity of the camera. With dual-wavelength ratiometric experiments as performed here, these considerations are critical as the light measured at 510 nm as a result of excitation at 340 and 380 nm swings in opposite directions in response to a calcium change. To further eliminate any possible artifact, we routinely captured the raw 340 and 380 nm images and inspected post-experiment, at the single-pixel level, (using a light pen and software-based pixel profiling of grey mean levels) the pixel grey values in all regions of the experimental field following background subtraction. Areas of the cell showing particularly high rises in calcium were subject to special scrutiny. However, this inspection did not suggest that pixel saturation due to any of the above factors could have given rise to the observation of localized calcium changes near the plasmalemma. Finally, the observations of elevated calcium in the vicinity of the plasmalemma are almost certainly real - as opposed to artifact - simply because to date they have only been observed in this particular cell type. Other pituitary cells of similar dimensions to the rat somatotrophs, smooth muscle cells, lymphocytes, pancreatic cells, cultured neurones and oocytes examined on this system have failed to exhibit a similar feature. On the other hand, standing gradients and tides of calcium along the length of smooth muscle cells in response to thrombin (Neylon, Hoyland, Mason & Irvine, 1990) have been a common observation. A more comprehensive discussion of these points will be found in a recent review (Mason et al.

1990). RESULTS

Temporal and spatial patterns of response to hGRF Under both basal and hGRF-induced conditions, oscillations of [Ca2+]i were routinely observed. In most cells, the rise in [Ca2+]i was most prominent in the subplasmalemmal region (Figs 1 and 2). Figure 1 shows a typical spontaneous basal oscillation of [Ca21]i whereas Fig. 2 shows an identical oscillation when the

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M. KATO, J. HOYLAND, S. K. SIKDAR AND W. T. MASON continuous application of 10 nM-hGRF evoked a rise in [Ca2+]j, especially in the region of the subplasmalemma. The lowest concentration of hGRF which was found reliably to produce a maximal growth hormone secretory response and calcium signal

I A

11

Fig. 1. Image analysis of intracellular free Ca2+ concentration of a rat somatotroph pituitary cell under basal conditions during a spontaneous oscillation in intracellular [Ca2+]1. An identical spatial pattern or response was observed when DBcAMP was applied (data not shown). Approximately every 3-4 s, four frames with 340 nm excitation and four frames with 380 nm were captured within 0 2 s and processed, as described in Methods section, for calculated [Ca2+]1. Consecutive ratio frames are shown during a spontaneous oscillation in [Ca2+]i, where it can be seen that the [Ca2+]i fluctuation occurs repeatedly in the subplasmalemmal region and then falls again back to basal levels. The images are the representations of mean ratio grey level, determined against a calibration look-up table, with darker-shaded areas representing low [Ca2+]i and lighter-shaded areas representing high [Ca2+]i. Subsequent oscillations also appeared centred at the cell subplasmalemma. The horizontal ion calibration bar at the top of the figure is approximately equal to 15 ,tm.

I

was 10 nm-hGRF. hGRF was introduced into the perfusion medium at 3 min. There was about a 1 min delay before hGRF reached the cells. Figure 2 shows nine consecutive frames with a time interval of 3-4 s. At frame B just following hGRF

177 INTRACELLULAR CALCIUM IN RAT SOMATOTROPHS application, [Ca2+]i in the subplasmalemmal region suddenly increased from a basal value of about 100 to 500 nm or more. The subcellular distribution of [Ca2+]i showed a gradient from the subplasmalemmal region to the cell interior which was clearly

Fig. 2. Image analysis of intracellular free Ca2+ concentration of a rat somatotroph pituitary cell under basal conditions during a spontaneous oscillation in response to 10 nM-hGRF. Approximately every 3-4 s, four frames with 340 nm excitation and four frames with 380 nm were captured within 0-2 s and processed, as described in Methods section, for calculated [Ca2+]1. The figure shows a phenomenon similar to Fig. 1 during a different experiment where the cell was exposed continuously to 10 nM-hGRF. hGRF was applied from frame A. At frame B, the cell abruptly increased [Ca2+]1, especially in the subplasmalemmal region. In successive frames the [Ca2+]i was maintained and then fell back to basal levels as the oscillation of [Ca2+]1 fell back to basal levels. Details of representations and calibration are given in Fig. 1 legend.

observed from frame B onwards, and gradually declined to basal levels, but without the gradient in [Ca2+]i spreading throughout the cell. This pattern of response was typical of basal spontaneous oscillations in [Ca2+]i and was seen in over 90 % of the cells characterized by a response to hGRF.

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M. KATO, J. HOYLAND, S. K. SIKDAR AND IW T. MASON

The continuous application of 10 nM-hGRF produced several different temriporal patterns of [Ca2+]i response (Fig. 3). The most common pattern of response was a sustained rise in [Ca21]i in response to 10 nM-hGRF (44 % among forty-three cells examined). The second response type (28 % of cells) was characterized by cells attaining a peak value similar to that in sustained response cells, followed by a 10 nM-hGRF

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gradual decrease in [Ca2+]j. The third type of response showed a prominent peak with a relatively rapid fall to a lower sustained level in [Ca2+]j (23 % of cells). The fourth type showed a gradual and continuous increase in [Ca2+]i (5 % of cells). One-third of the cells that subsequently responded to 10 nM-hGRF with a rise in [Ca2+]i, showed spontaneous basal [Ca2+]i oscillations ranging from 100 to 500 nm.

179 INTRACELLULAR CALCIUM IN RAT SOMATOTROPHS The mean value of the basal [Ca2+]i of these cells was 81 + 11 nM (mean +S.E.M., n = 27). hGRF (10 nM) produced a sustained rise in [Ca2+]i as examined above. The mean value of the 10 nM-hGRF-induced rise in [Ca2+]i was 560+47 nM (n = 27). To investigate whether or not there was a relationship between basal [Ca2+]i and the hGRF-induced rise in [Ca2+]i, the correlation between these two values was 10 nM-hGRF

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calculated and plotted in Fig. 4B. There was no significant correlation (correlation r = 0 243, n = 27) between the basal [Ca 2+]i and the size of the hGRFinduced rise in [Ca'+]i. With respect to the different mean [Ca2+ ]i evoked by hGRF and the various temporal patterns of [Ca 2+]i response to hGRF described above, there was no discernible variation in the spatial pattern of the [Ca2+]i gradients observed. These temporal responses are thus probably due to factors other than localization of Ca2+ channels or stores.

coefficient,

M. KATO, J. HOYLAND, S. K. SIKDAR AND W. T. MASON

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Effect of Co2" and replacement of extracellular Na+ on the hGRF-induced rise in

[Ca2+]i The hGRF (10 nM)-induced rise in [Ca2+]i was abruptly abolished by the application of 2 mM-Co2+ (Fig. 5B). This inhibitory action of Co2+ was also observed A

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by the prior application of Co2+, which completely blocked the 10 nM-hGRF-induced rise in [Ca2+]i until Co2+ was removed from the perfusion medium (Fig. 5B). The effect of Co2+ was quantitatively analysed by the following procedure. The mean LCa2+]i was calculated during the application of hGRF with and without Co2+ respectively. The ratio between these two values was calculated and expressed as a

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percentage (Fig. 7). In control responses, the ratio between mean [Ca2+]i from 230 to 360 s (III) and from 420 to 540 s (II) was 61±9 %; while the application of Co2+ reduced the ratio to 7-8 + 1-5 % (n = 11, P < 0 001 vs. control). The total replacement of extracellular Na+ with the impermeant molecule

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M. KATO, J. HOYLAND, S. K. SIKDAR AND W. T. MASON

mannitol completely blocked the 10 nM-hGRF-induced rise in [Ca2+]i in all cells examined (n = 8, Fig. 6C and D). The hGRF-induced responses were also completely blocked by replacing 70 % of the Na+ with mannitol. However, 50 % Na+ replacement with mannitol produced a complete block of the hGRF-induced rise in [Ca2+]i in =

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Imaging of intracellular calcium in rat anterior pituitary cells in response to growth hormone releasing factor.

1. Changes in intracellular ionized calcium [Ca2+]i induced by human growth hormone releasing factor (hGRF) were analysed by quantitative fluorescent ...
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