calcium distribution in islets of langerhans: a study of ... - CiteSeerX

J. Cell Sci. 19, 395-409 (i97S) Printed in Great Britain

395

CALCIUM DISTRIBUTION IN ISLETS OF LANGERHANS: A STUDY OF CALCIUM CONCENTRATIONS AND OF CALCIUM ACCUMULATION IN B CELL ORGANELLES S. L. HOWELL, W. MONTAGUE AND MARGARET TYHURST Biochemistry Laboratory, School of Biological Sciences, University of Sussex, Fainter, Brighton, Sussex, England

SUMMARY Calcium concentrations of various pancreatic B cell organelles have been determined by X-ray microanalysis of areas of frozen sections of unfixed rat islets of Langerhans. Highest concentrations were detected in storage granules and in mitochondria, although calcium was also present in nuclei, in areas of endoplasmic reticulum and of cytoplasm. Accumulation of 45Ca by isolated organelles has been studied in homogenates and isolated subcellular fractions of rat islets of Langerhans. In the presence of a permeant anion (oxalate or phosphate), accumulation of 4tCa into mitochondria and microsomes was strongly stimulated by ATP. This net uptake was diminished during incubation of homogenates or of a mitochondria-plus storage granule-rich fraction in the presence of cyclic AMP, dibutyryl cyclic GMP, 2:4-dinitrophenol or of ruthenium red. Investigations of the characteristics of 45Ca accumulation by homogenates prepared from storage granule-depleted islets showed no differences from those of normal islets, suggesting that the granules do not represent an important labile pool of calcium. With the exception of cyclic AMP and cyclic GMP none of the insulin secretagogues tested (glucose, leucine, arginine, adrenalin, noradrenalin, theophylline, glibenclamide) altered calcium accumulation by islet homogenates. On the basis of absolute calcium levels and of 46Ca uptake studies it is concluded that islet B cells contain a readily exchangeable mitochondrial calcium pool, and an endoplasmic reticulum pool containing a lower concentration of calcium which is also readily exchangeable. The storage granules, despite their high calcium content, do not appear to constitute a labile pool. It seems likely that the labile mitochondria and endoplasmic reticulum pools play a predominant role in the regulation of cytoplasmic free calcium levels, which may in turn be important in the regulation of rates of insulin secretion.

INTRODUCTION There is evidence to suggest that calcium may be the link between stimulation and secretion in a variety of endocrine tissues including the pancreatic B cell, and that intracellular calcium concentrations may play an important role in the regulation of rates of insulin secretion (Malaisse, 1973). Extensive studies have been made of the influx and efflux of 45Ca in isolated islets of Langerhans during stimulation of insulin secretion in response to a variety of agents, as reviewed by Malaisse (1973). However, there have so far been few investigations of the localization of calcium within B cell organelles or of factors which may alter its distribution. The problems involved, both in preparing subcellular fractions which contain the various B cell organelles in

396

S. L. Howell, W. Montague and M. Tyhurst

purified form and in avoiding diffusion of calcium out of and between fractions during the separation procedure, make it very difficult to determine the exact subcellular distribution either of labelled calcium or of total cellular calcium amongst the various organelles. We have attempted to resolve some of these problems by determining the distribution of total calcium in frozen sections of unfixed islets by X-ray microanalysis. In addition this paper presents results of a study of the accumulation of ^Ca by particulate components of homogenates and subcellular fractions of rat islets of Langerhans, and of its regulation by nucleotides, insulin secretagogues and other agents. A brief report of some aspects of this work has already appeared (Howell & Montague, 1975)MATERIALS AND METHODS Reagents 46

CaCl2 of specific radioactivity 28 /tCi//tg, 3i//tg Ca/ml, was obtained from the Radiochemical Centre, Amersham, Bucks. ATP, cyclic AMP, dibutyryl cyclic AMP, cyclic GMP, and dibutyryl cyclic GMP, were obtained from Boehringer Corp., Uxbridge, Middlesex, U.K. ADP, AMP, adenosine, ruthenium red and dinitrophenol were obtained from Sigma Chemicals, Kingston-upon-Thames, U.K. Other reagents were of 'Analar' grade or of the purest grade which was commercially available. Millipore membranes were obtained from the Millipore Corp., London, N.W.10, U.K. Methods Tissue preparation. Islets of Langerhans were isolated by collagenase digestion of pancreatic tissue taken from female rats (200-240 g) (Howell & Taylor, 1966), using a bicarbonatebuffered medium (Gey & Gey, 1936) containing 5-5 nun glucose and 2 mM CaCl2- Final rinsing and separation of the islets was performed in medium from which calcium was omitted. The islets were collected in a Kontes all-glass homogenizer, the medium was removed and the islets were homogenized in 0-25 M sucrose buffered with 10 mM Tris-HCl (pH 72). In some experiments subcellular fractions were obtained from the homogenate by centrifugation at 4 °C as follows: 600 g for 5 min to give a pellet of nuclei + debris; the supernatant was centrifuged at 24000 g for 10 min to give a pellet of mitochondria + storage granules and this supernatant was in turn centrifuged at 105000 g for 60 min to give a microsomal pellet and final supernatant (Howell, Fink & Lacy, 1969). All pellets were resuspended in 0-25 M sucrose— 10 mM Tris-HCl (pH 72) and assayed simultaneously at the end of the centrifugation. Assay of 16Ca accumulation. Accumulation of 45Ca by homogenates or isolated subcellular fractions was estimated by incubation of 5o-/tl aliquots of homogenate or subcellular fractions resuspended in sucrose (025 M), with 50 /tl of a solution which was prepared to give final concentrations of 0125 M sucrose, 35 mM KC1, 10 mM Tris-HCl, 4 mM MgCl2, 4 mM KjHPO4, 10 mM sodium succinate, 05 mM 3-isobutyl-i-methylxanthine, 20 /IM CaCla and 4tCaCla (5 /tCi/ml) pH 72. Nucleotides and other agents were added at the concentrations indicated in individual experiments. After 20 min incubation at 23 °C the particulate-bound calcium was separated from the calcium by filtration through Millipore membranes (450 nm, HAWP or 100 nm, VCWP), each membrane being rinsed 3 times with 025 M sucrose in 10 mM Tris-HCl (pH 7-2) to remove unbound radioactivity. Radioactivity remaining on the filters was determined in a liquid scintillation spectrometer (Beckman LS 233) using Instagel (Packard Instruments) as scintillant. Cryo-ultramicrotomy technique. Islets were incubated at 37 °C until immediately before immersion in liquid nitrogen. The procedures for freezing of individual islets and for sectioning were as described previously (Howell & Tyhurst, 1974), except that no trough liquid was used;

Calcium distribution in B cell organelles

397

sections were transferred directly from the dry knife surface to (Formvar + carbon)-coated nickel grids located on the knife holder or on the knife itself, by means of an eyelash probe. Both specimen and knife temperatures were maintained at —80 °C. After collection of sections the grids were transferred to a Petri dish in the bottom of the Cryokit chamber and allowed to reach room temperature over a period of 3 h. Dririte was placed in the chamber in order to maintain a dry atmosphere. The sections were stored in a dry atmosphere at room temperature piior to analysis. X-ray microanalysis. Sections were analysed in an AEI EMMA-4 analytical electron microscope using crystal spectrometers. The minimum probe size used was 02 /im and the microscope was operated at 60 kV. Count rates were assessed for 100 s at the peak for calcium and then for 100 s with the settings displaced to one side of the peak. A white count was recorded to give an assessment of the continuum, radiation in the area analysed and to allow for correction for variable thickness of the specimens, and the count rate recorded was assessed according to the formula c = (P — b)/(W — Wb) where P is the peak counts obtained, b the counts obtained from the same area with the detector offset from the peak, W is the white count in the same area and Wb is the white count obtained from an area away from the specimens, in this case the Formvar film. In order to obtain a valid sample, sections were prepared from several different islets on separate occasions. Protein determination. Protein concentrations were determined by the method of Lowry, Rosebrough, Farr & Randall (1951) using crystalline bovine albumin as standard. Calcium accumulation was expressed as nmol Ca accumulated per mg protein per 20-min incubation period; mean values ± S.E.M. are shown throughout. Electron microscopy. After incubation in the conditions described above, subcellular fractions were repelleted by centrifugation before fixation with 3 % glutaraldehyde, postfixation with OsO4, dehydration and embedding by standard procedures. Sections were examined in an AEI EM 6B electron microscope. RESULTS Calcium distribution in B cells determined by X-ray microanalysis The following organelles, which are identifiable in frozen sections of unfixed islets (Howell & Tyhurst, 1974), and are of a size (0-2 /im or greater) which can be located with the probe diameter available on the EMMA-4 instrument, were analysed for Table 1. Mass ratio of calcium in various B cell organelles determined by X-ray microanalysis Organelle

io J x Mass ratio

No. of observations

18 Mitochondria 57 ± 11 16 64 ± 14 Storage granules Nuclei 35 ± 7 15 41 ± 8 Rough-surfaced endoplasmic reticulum 5 3° ± 8 Other cytoplasmic areas 7 Results are given as means + S.E.M. of the number of observations shown.

their calcium content: nuclei, mitochondria, storage granules, areas of rough-surfaced endoplasmic reticulum, and areas of cytoplasmic ground substrate. The results shown in Table 1 are presented as the mass ratio of calcium found in each organelle; this method of expression includes a factor (the white count) which corrects for possible differences in mass (including section thickness) between the various areas which are

398

»S. L. Howell, W. Montague and M. Tyhurst

analysed. All of the areas examined contain detectable concentrations of calcium, the values falling into 2 groups: those areas of relatively low concentration (mass ratio 30-41) which include the nuclei, endoplasmic reticulum and areas of cytoplasmic ground substance, and a second group of significantly (P < 0-05) higher concentration (mass ratio 57—64) which comprise the mitochondria and storage granules. The mean concentration of calcium in the granules appears to be greater than that in the mitochondria although the differences were not statistically significant; similarly, differences in mass ratio between nuclei, rough-surfaced endoplasmic reticulum and cytoplasmic ground substance were not significant. accumulation by homogenates and subcellular fractions Some of the characteristics of calcium accumulation by particulate components of islet homogenates have been described in an earlier communication (Howell & Montague, 1975). Briefly, optimal binding occurred at an initial pH of the incubation 20 r Oxalate

w O 1Q.

Phosphate

00 •5

10

u

25

10 [Anion], mM

Fig. 1. Accumulation of 46 Ca in islet homogenates during incubation in the presence of 1-25 mM ATP and the concentrations of anion shown. Incubations were performed for 20 min at 23 °C.

medium of 7-2. Accumulation was a very rapid process, reaching an equilibrium within 2-5 min at 23 °C, and was maintained for at least 30 min; the extent of uptake was dependent on the temperature of incubation. Calcium uptake was dramatically increased by addition of ATP to the medium, the maximal effect being obtained at a final concentration of 1-25 mM ATP. Omission of a permeant ion (phosphate or


oxalate) reduced the binding by homogenates to very low levels, either in the absence or presence of ATP; phosphate or oxalate were satisfactory but oxalate was more effective in this respect than was phosphate (Fig. 1). Heating of the homogenate to 80 °C for 3 min before incubation completely abolished the calcium accumulation.

5-0

r

.£ 40 30

i:§ 20 o i

c

r+i

10 Homogenate

Nuclei

Mitochondria +granulej

Microtomes

Supernatant

Fig. 2. Effects of ATP (125 ITIM) and cyclic AMP (1 mM) in the presence of ATP, on accumulation of 48Ca by subcellular fractions of rat islets of Langerhans. Incubations were performed for 20 min at 23 °C. ^ , no addition; Q, with A T P ; ^ , with ATP + cAMP.

Table 2. Effect of cyclic AMP and related agents on calcium accumulation by islet homogenates % of control accumulation Control Cyclic AMP i mM at start o-i mM at start after $ min after io min after 15 min after 19 min Dibutyryl cyclic AMP 1 mM Dibutyryl cyclic GMP 1 mM AMP 1 mM Theophylline 5 mM 3-Isobutyl-i-methylxanthine 0-5 mM Caffeine 10 mM

100

46 ± 72 ± 74 ± 77 ± 80 ± 92 ± 67 ± 73 ± 79 ± 96 ± 102 ±

12* 7* 8* 6* 7* 8 9* 7* 5* 8 7

95 ± 8

Homogenates of islets of Langerhans were incubated for 20 min at 23 CC in buffer containing 1 25 mM ATP and the additions shown. Values were expressed as the percentage mean and standard error of the mean value of the calcium accumulated in control (no addition) incubations, which was 3 7 nmol Ca/mg protein/20 min. • Denotes values significantly different from the control (P < 005).

ATP-stimulated accumulation was inhibited by addition of cyclic AMP in the concentration range IO~ 6 -IO~ 3 M (Howell & Montague, 1975); it was found that cyclic AMP was almost as effective when added 5, 10, or 15 min after initiation of a 20-min incubation period (Table 2), as when added at the start of the incubation as in the

400


previous experiments. Comparison of the abilities of impure subcellular fractions (nuclei + debris, mitochondria + granule or microsome-rich fraction, prepared by differential centrifugation) to accumulate Ca showed that the effect of cyclic AMP was exerted principally on the fraction containing mitochondria + storage granules, there being much smaller effects on nuclear and microsomal fractions (Fig. 2). Table 3. Effect of inhibitors on calcium accumulation by islet homogenates % of control accumulation Control (i-25 mM ATP) Ouabain o-i mM Fluoride 10 mM Ruthenium red 10 /IM o-i mM

Oligomycin 2 fig/fd 2:4-dinitrophenol 0-2 mM

100

95 ± 6 #

135 ± 8 60 ± 1 0 * 49 ± ' 4 * 71 ± 8*

57 ± 7 # Homogenates of islets of Langerhans were incubated for 20 min at 23 °C in buffer containing 1-25 mM ATP and the additions shown. Values were expressed as the percentage mean and standard error of the mean value of the calcium accumulated in control (no addition) incubations, which was 3-3 nmol Ca/mg protein/20 min. • Denotes values significantly different from the control (P < 0-05).

Other nucleotides were also examined for their effectiveness in diminishing calcium accumulation. Dibutyryl cyclic AMP and dibutyryl cyclic GMP were less effective then cyclic AMP at the same concentration (1 mM). Similarly AMP was partially effective when used at high concentrations (Table 2). The cyclic nucleotide phosphodiesterase inhibitors, 3-isobutyl-i-methylxanthine, theophylline and caffeine did not alter calcium accumulation when added to homogenates, and 3-isobutyl-imethylxanthine was included routinely in the experiments utilizing cyclic nucleotides. ATP-dependent uptake of calcium in homogenates was inhibited 45 % by o-1 mM dinitrophenol, an uncoupler of oxidative phosphorylation, or by the respiratory inhibitor oligomycin (2/(g/ml). Accumulation was also inhibited by ruthenium red which is reported (Moore, 1971) to be a specific inhibitor of mitochondrial calcium uptake (Table 3). In subcellular fractionation experiments these agents were found to be effective principally on the mitochondria + storage granule fraction (results not shown). Sodium fluoride, an inhibitor of ATPase activity, stimulated calcium uptake by homogenates in similar conditions, while ouabain (an inhibitor of Na-K ATPase) had no significant effect on calcium accumulation. The role of the insulin storage granules in promoting calcium uptake was studied in two ways. First, filters were used of pore size which would either permit the passage of the granules (450 nm pore size) or retain them (100 nm pore size). In these experiments there was no significant difference in the basal or ATP-stimulated uptake of calcium or in the effectiveness of cyclic AMP in inhibiting calcium uptake, by the organelles retained by the two types of filter (Fig. 3). Secondly, calcium accumulation in homogenates of normal islets on 100-nm filters was compared with that in homogenates of islets from rats which had previously been treated with glibenclamide


S o

401

30

00

•5

20

"S 10

u •450

100

Pore diameter of filters, nm

Fig. 3. Accumulation of 45 Ca by paniculate components of islet homogenates in which separation was performed on 100-nm pore filters (which should retain most particulate components), or on 450-nm pore filters, which should allow passage of storage granules and vesicles of comparable size. The resultant accumulation of calcium whether basal, stimulated by ATP, or stimulated by ATP in the presence of cyclic AMP, is very similar regardless of the pore diameter of the filter used. E?, no addition; D, with A T P ; £?, with ATP + cAMP.

5

I4 Q.

I

o

I 3

U

Control

Gllbenclamlde injected

Fig. 4. Effect of granule depletion on the accumulation of 46Ca by homogenates of normal islets or of islets isolated from rats which had been treated with glibenclamide (1 mg/kg) 1, 12 and 24 h before sacrifice in order to deplete the storage granule store. Basal, ATP-stimulated uptake and the effect of cyclic AMP on ATP-stimulated uptake are shown, g?, no addition; Q, with A T P ; g£, with ATP + cAMP.

(intraperitoneal injections of 1 mg/kg at 24, 12 and 1 h before sacrifice) in order to deplete the insulin storage granule store. There was no significant difference in degree of calcium accumulation between normal and storage granule-depleted islets (Fig. 4), despite a dramatic reduction in the number of granules present in the B cells of glibenclamide-treated animals (Kern, Kern, Schmidt & Stork, 1969). Survival of the storage granules during incubation in the conditions of these experiments was

402


confirmed by electron-microscopic examination of the pellets at the end of the incubation period (Fig. 5). The effects of various insulin secretagogues on calcium accumulation by islet homogenates were studied in a further series of experiments (Table 4). Glucose (20 mM) had a small but consistent effect in diminishing calcium accumulation by islet homogenates, but an exactly similar effect was shown by galactose at the same concentration. Leucine and arginine, both stimulators of insulin secretion, had no Table 4. Effect of insulin secretagogues on ibCa accumulation by homogenates of rat islets of Langerhans % of control accumulation Control (1-25 mM ATP only) Glucose 20 mM Galactose 20 mM Glibenclamide 1 /tg//«l 10 /