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Hyposmolar medium and ethanol in isosmotic solution induce the release of thyrotropin-releasing hormone (TRH) by isolated rat pancreatic islets
Life Sciences, Vol. 60, No. 11, pp. 845-872,1997 copyright 0 1997 Fbevicr science Inc. Printed in the USA. All rights resend 0024-3205/w $17.00 + .oo
ELSEVIER
PII s0024-3205(97)ooo15-5
HYPOSMOLAB MEDIUM AND ETHANOL IN ISOSMOTIC SOLUTION INDUCE THE RELEASE OF THYBOTBOPIN-RELEASING HORMONE (TBH) BY ISOLATED BAT PANCREATIC ISLETS
J. Benicky, M.A. Greer #, V. strbak Institute of Experimental Endocrinology, Slovak Academy of Sciences, Brat&lava, Slovakia, # Division of Endocrinology, Oregon Health Sciences University, Portland, Oregon (Received in final form December
28,1996)
Summary Cell swelling induced by hypotonic medium or small isotonic permeant molecules results in an immediate secretory response in various types of cells. We have expanded exploration of this phenomenon by examining the effect of either isotonic ethanol or hyposmotic medium on the release of TRH by freshly isolated islets of Langerhans in static incubation and peritusion. Ethanol (40, 80 or 160 mM in isotonic solution) dose-dependently evoked the release of TRH by statically incubated islets. The dynamics of TRH release induced by 80 mM isotonic ethanol or 30% hypotonic medium were similar to those induced by 50 mM KCl, with the highest secretion rate during the first 5 min of incubation irrespective of the duration of stimulation. Ca” depletion of the incubation medium abolished the response to 50 mM KC1 but did not diminish the response to 80 mM isotonic ethanol. We conclude that osmotic stimuli known to induce cell swelling also induce release of TRH by isolated pancreatic islets. i&y Words: thyrotropin-releasing
hormone,
pancreatic islets, ethanol
hyposmolarity,
cell swelling
Cell volume changes may play an important role in the intracellular regulation of metabolism and function (for review see 1). Permeant molecules (ethanol, urea, glycerol) when added in hypoor isosmotic medium diffuse rapidly across the cell membrane down their concentration gradient (2). An increase of their concentration inside the cell results promptly in cell swelling due to secondary water transport (this effect is suppressed in a hyperosmotic environment). Cell swelling induced in vitro by either medium hyposmolarity or isotonic per-meant molecules stimulates prompt high amplitude hormone secretion from various types of cells (3-6). Hyperosmolarity, on the contrary, causes cell shrinkage and depresses secretion (7-8). The secretory process induced by hyposmolarity or per-meant molecules is similar to that induced by specific secretagogues in many respects, in&ding response dynamics (a rapid high-amplitude secretory burst reaching a peak at l-2 min) and refractory periods (7). This process does not, however, require extracelhtlar calcium and microtubules and microtilaments and changes in sodium flux across the plasma membrane are not apparently involved (5). Immunoreactive thyrotropin-releasing hormone (TRH) has been detected in the rat pancreas (9) and immunocytochemically localized mainly in the insulin-producing p-cells of the islets of Langerhans ( 1O-l 1) where it is probably colocahzed with insulin in the same secretory granules ( 12).
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However, the role of TRH in this localization is unclear till present and remains to be explored. In the present work isolated pancreatic islets, providing a useful model for in vitro study of the endocrine pancreas, were used to evaluate the effect of osmotic forces on the secretory response of pancreatic TRH. A stimulatory effect of hyposmolarity on insulin secretion by perifused islets independent of extracellular Ca2’ concentration has been reported (3). This effect is presumably mediated by cell swelling. However, no data are available concerning pancreatic TRH. Methods Materials: The basal incubation medium - Krebs-Ringer bicarbonate (pH 7.4,290 mOsmo1) contained 118 mM NaCl, 4 mM KU, 1.2 mM KH2P0,, 1.2 mM MgSO,, 2.5 mM CaC&, 25 mM NaHCO,, 3 mM D-glucose, 0.3 mg/ml bacitracin, 0.25% bovine serum albumin in 10 mM Hepes. 30 % hyposmotic medium was prepared by dilution of basal medium with deionized distilled water(70 : 30). In isosmotic ethanol medium the concentration of NaCl was reduced (40 mM ethanol-98 mM NaCl, 80 mM ethanol-78 mM NaCl and 160 mM ethanol-38 mM NaCl) to maintain the osmolarity unchanged. The composition of the other components was the same as for basal medium. High potassium medium contained 50 mM KC1 and 72 mM NaCl without any changes in the concentration of the other components. Each medium was prepared also in Ca2+-free form in which Ca” was omitted without any other changes. Media were gassed 30 min before and throughout the experiment with a mixture of 0, and CO, (95:5). Preparation of the islets: Islets were isolated by collagenase digestion method according to Lacy & Kostianovsky (13). Briefly, male Wistar rats (250-3008) were anesthetized with pentobarbital and pancreases were distended by intraductal injection of Hanks balanced salt solution (HBSS, pH 7,4). Pancreases were then dissected out, minced and digested with collagenase (type XI, Sigma, 3 mg/ml) for 15 min at 37°C. Islets were separated from remaining acinar tissue manually with the aid of a dissecting microscope and collected 50 per tube for static incubation and 1000 per column for perifusion. Static incubation: Prior to incubation the islets were preincubated in basal medium for 30 min at 37°C and then incubated in the presence or absence of secretagogues in a total volume of 100 ~1. At the end of incubation the medium was saved and stored at -20°C until RIA. Perifusion exneriment: The dynamics of TRH secretion were analyzed by column per&ion. The flow rate was 0.25 ml/mm and fractions were collected every 2 min. Two parallel columns were simultaneously perifused; one starting with 2.5 mM Ca2’ and other with Ca2’-free medium. Samples were frozen at -20°C until analysis for TRH by RIA as described below. Radioimmunoassay: TRH was measured by radioirmmmoassay (RIA). The specific rabbit TRH antibody developed in our laboratory recognizes neither TRH-degradation products (such as TRH-OH, cyclo(His-Pro) or amino acids) nor such putative TRH precursor peptides as TRH-Gly, Gln-His-Pro-Gly-Lys-Arg or Lys-Arg-Gln-His-Pro-Gly-Arg-Arg (cross reactivity less than 1.O%). Synthetic TRH (a gift fiorn Prof. Kasafirek, Research Institute of Pharmacology and Biochemistry, Prague) was labelled with Na’2SI using the chloramine-T method and purified on a Sephadex G- 15 (Pharmacia) column (60 x 1 cm). All assays were performed in a total volume of 400 ul as previously described (14). Statistics: The results are expressed as mean & SE (n=5-8). performed using the unpaired Student’s t-test.
Statistical analysis was
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Results Effect of isosmotic ethanol on the release of TRH during static incubation of islets is illustrated in Fip 1,30 min exposure to ethanol (40, 80 and 160 mM in isotonic solution) resulted in significant dose-dependent induction of the release of TRH. TRH release induced by 80 mM isotonic ethanol was comparable to that induced by 30% hypotonic medium or 50 mM KC1 (m). This ethanol concentration was utilized as a stimulus in subsequent experiments.
4hM
SOmM
16Chni~l
I
Fig.1. Effect of increasing concentrations of ethanol in isosmotic (290 mosmol) medium on the release of TRH. Islets (50 per tube) were incubated for 30 min in basal (Control -C, open bar) or isosmotic ethanol (40.80 and 160 mM, cross-hatched bars) in medium containing 2.5 mM Ca”. At the end of incubation the medium was removed and analyzed for TRH. Results are expressed in pg TRW50 islets (mean f SE, n = 6-8 for each bar). * PcO.05, ** P
30 3 z d ; E
*
**
2520I5
m m mIn
30 % hyposmdic me&m 80 mM isosmotic EtOH 5omMKcl
Fig.2. Release of TFW in response to stimulation by 30% hyposmotic medium, 80 mM isosmotic ethanol or 50 mM KCI. Islets (50 per tube) were incubated for 10 min in the respective medium, each containing 2.5 mM Ca”. At the end of incubation the medium was removed and analyzed for TRH. Results are expressed in pg TRW1 0 islets (mean f SE, n = 5-6 for each bar). * PcO.01, ** P
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The dynamics of TRH release by statically incubated islets in response to 80 mM isotonic ethanol, 30% hyposmotic and high potassium medium is illustrated in Fia3a. After any stimulation, the major Inaction of TRH (approximately 80%) was secreted during the first 5 min. The dynamics of this osmotically induced release was similar to that induced by high potassium concentration. Fin.3b shows the secretion rate (pg TRH/min) in the same experiment.
-IB-
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-
B
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B
~C~DME~OH B
-
-
30%H,O
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50mMKCl
30% H,O
B
5mnMKCl
Fis3.A. Time course ofbasal and stimulated TRH release. Islets (50 per tube) were divided into four groups and incubated for eight subsequent periods inte.rrupted by medium exchange in the following order: preincubation (30 min) -basal (5, 10, 15 and 30 min)- I.interincubation (30 min)- 8OmM isosmotic ethanol (5, 10, 15 and 3Omin)- IIinterincubation (3Omin)- 30% hyposmotic (5, lo,15 and 3Omin)- III.interincubation (3Omin)- 50mM KC1 (5,10,15 and 30min). Release of TRH during 30 min basal interincubations (B) is illustrated by one bar which represents the mean + SE from all samples incubated. Groups of four neighbouring bars represent the release of TRH during 5, 10, 15, and 30 min, respectively, as indicated in the legend. Results are expressed in pg TRW50 islets (mean f SE, n=5 for each time interval). E The same results expressed as secretion rate (pg TRW50 isletsmin) for each collection period.
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Calcium dependency of the ethanol-induced TRH release was assessed using medium repleted or depleted of Ca” (FM). Eighty mh4 isotonic ethanol induced TRH release independently of the presence of extracellular Ca”. In contrast, extracellular Ca” was essential for 50 mM KCI-induced TRH release.
A. 80 -
T
2.5 mM Ca”
0
Basal
80 MEtOH
=
SOmMKCl
1
B. 80 c
T
2.5 u&l Ca*+
Fk.4.A. Effect of medium Ca” on the ethanol- and KCl-stimulated release of TRH. Islets (50 per tube) were incubated for eight subsequent 15-min periods interrupted by medium exchange in the following order: basal, 80 mM isosmotic ethanol, basal, 50 mM KCl, basal, 80 mh4 isosmotic ethanol, basal, 5Omh4 KCl. The first four incubations were performed in the presence of 2.5 mM Ca”, then the medium was changed to a Ca2’-free one. At the end of each incubation the medium was saved and analyzed for TRH. Results are expressed in pg TRW50 islets (mean f SE, n=8). B, The same experimental design as in Panel A, with the order reversed to start the first set of incubations with Ca2’-free medium.
Perifusion of the islets (m) confirmed the significant stimulation of the release of TRH by 80 mM isotonic ethanol. This effect, like that observed during static incubation, was independent of extracellular Ca2+ concentration.
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A. 80 -
2.5 mM Cal’ 0
B.
2.5 mM Ca”
20
40
80
80
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Fraction # (2miu collection) u The effect of isosmotic 80 mM ethanol on the release of TRH by perifused pancreatic islets in two parallel simultaneous columns, one starting with Ca2”-rep&d and the other with Ca”- depleted medium. Islets (1000 per column) were perifused at a flow rate of 0.25 ml/min; samples were collected every 2 min. Application of 80 mM isosmotic ethanol is indicated by black bars, the presence of Ca2’ in the medium is marked by horizontal line below the collection fractions.
Discussion Cell swelling produced by medium hyposmolarity or permeant molecules is a potent nontoxic inducer of hormone secretion (3-6). In the present study isotonic ethanol stimulated secretion of TRH by statically incubated pancreatic islets. The islets are, however, composed of several cell types which produce a number of biologically active substances. It is therefore necessary to take into account possible para- and/or autocrine influences which might lead to indirect effects and misinterpretations. As a second experimental approach we therefore used a perimsion system in which the secretory products are washed out, minimizing such indirect effects. The islet perifusion experiments confirmed the data obtained using static incubation, indicating the effect of isotonic
Vol. 60, No. 11,1997
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ethanol on TRH-secreting cells is direct. Although we do not have direct evidence that hyposmolar media or the addition of isosmolar permeant molecules causes cell swelling in isolated islets, cell swellinghas been directly observed to result from these conditions in both normal and tumor-derived pituitq cells (15-l 7). We assume that the islet cells would have similar volume changes in response to the osmotic challenges. In normal rat pituitary cells, Ca” influx has a negative influence on secretion induced by cell swelling (5,16). Depletion of medium Ca” also enhances osmotically induced hormone secretion in several other normal cell types (2-3,18- 19). Ca” influx through the plasmalemma is important in the regulatory volume decrease which begins l-2 min a&r initial swelling (2). It is possible that negative modulation of cell swelling-induced secretion is due to the greater degree of cell swelling produced when Ca” intlux is prevented since the regulatory volume decrease following cell swelling is Ca”-dependent. Unlike K+-induced TRH release, removal of medium Ca” did not reduce ethanol-induced TRH release by tieshly isolated islets. This is consistent with data obtained using other secretions from normal pancreas (3) or pituitary cells as in vitro models. Earlier reports demonstrated an important direct stimulation of endocrine and exocrine secretion by peritused pancreatic islets following exposure to extracellular hyposmolarity (3,20). Customary inhibitors of insulin secretion did not depress the insulin response to hyposmotic stimulation.Removal of medium Ca” resulted in an increased secretory response to a hyposmolar stimulus.Na’ transport and the microtubular and microtilamentous system of the B-cells apparently do not participate in osmotically induced release (3). These characteristics are similar to those in anterior pituitary cells (5), indicating a common mechanism for both cell types. On the other hand, Singh et al. (21) reported inhibitionby 65 mM ethanol on glucose- and tolbutamide-induced insulin release from perifused pancreatic islets. However, in their experiments the medium osmolarity was not adjusted for ethanol addition, resulting in a hyperosmolar medium which per se can inhibit exocytosis (7-8,22). Sir@ et al. (21) also reported inhibition of glucose- and tolbutamide-induced in vitro insulinrelease after in vivo intraperitoneal or oral administration of ethanol 1 hour prior to sacrifice of rats. Ethanol exposure in vivo may have diierent effects than those elicited from the acute in vitro exposure employed in the present study. To our knowledge, this is the first report concerning the effect of isosmotic ethanol on the release of pancreatic TRH. Although cell swelling is a potent stimulus for hormone secretion, it is uncertain how important a role it plays in normal in vivo physiology. Our data nevertheless emphasize that it is necessary to take into account changes in medium osmolarity during in vitro experiments since they may dramatically influence the secretory response of the cell. Acknowledgements.
The present work was supported by U.S.-Slovak Science and Technology Program, Project N” 93053 and by grants VEGA 211291196and 95153051239.The authors wish to thank to Susan E. Greer and AKrupkova for help and technical assistance.
1. 2. 3. 4.
F. LANG, G.L. BUSCH, H. VGLKL and D. IIAUSSINGER N.I.P.S. lQ 18-22 (1995). S.M.E. WONG and H.S. CHASE JR, Am. J. Physio1.m C841-852 (1986). W.G. BLACKARD, M. KIKUCHI, A. RABINOVITCH and A.E. RENOLD, Am. J. Physio1.m 706-713 (1975). M.A. GREER, S.E. GREER, Z. OPSAHL, L. McCAFFERTY and S. MARUTA,
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5.
6. 7. 8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
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Endocrinologym 1531-1533 (1983). M.A. GREER, S.E. GREER and S. MARUTA, Proc. Sot. Exp. Biol. Med. 193 203-209 (1990). X. WANG, N. SATO, M.A. GREER, S.E. GREER and S. McADAMS, Biochem. Biophys. Res. Commun. 163 471-475 (1989). M.A. GREER, S.E. GREEK, Z QPSAHL and S. MARUT_4, Proc. Sot. Eup. Biol. Med. 178 24-28 (1985). N. SATO, X. WANG and M.A. GREER, Mol. Cell Endocrino1.x 193-198 (1991). J.E. MORLEY, J.J. GARVIN, A.E. PEKARY and J.H. HERSHMANN, Biochem. Biophys. Res. Commun. 79 314-318 (1977). S. ARATAN-SPIRE, B. WOLF, B. PORTHA, D. BAILBE and P. CZERNICHOW, Endocrinology 114 2369-2373 (1984). P. LEDUQUE, S. ARATAN-SPIRE, B. WOLF, P.M. DUBOIS and P. CZERNICHOW, Cell Tissue Res. 248 89-94 (1987 ). P. LEDUQUE, S. ARATAN-SPIRE, R. SCHARFMANN, A. BASMACIOGULLARl, P. CZERNICHOW and P.M. DUBOIS, Biol. Cell @ 291-296 (1989). P.E. LACY and M. KOSTIANOVSKY, Diabetes _l&35-39 (1967). R.M. BASSIRI and R.U. UTIGER, Methods qfHormone Radioimmmloassqv, B.M Jaffe and H.R. Behrman (Eds), 37-44, Academic Press, New York and London, (1974). N. SATO, X. WANG, M.A. GREER, S.E. GREER and S. M&DAMS. Endocrinology 127 3079-3086 (1990). N. SATO, X. WANG and M.A. GREER, Am. J. Physiol. 360 E946-E950 (1991). N. SATO, M. MURAKAMI, X. WANG and M.A. GREER, Endocrinology 179 2541-2546 (1991). 0. SKOTT, Pfluegers. Arch. 406485-491 (1986). J.E. GREENWALD, M. APKON, K.A. HRUSKA and P. NEEDLEMAN, J. Clin. Invest. g 1061-1065, (1989). P.E. LUND, A. BERTS and B. HELLMAN, Mol. Cell. Biochem. 109 77-81 (1992 ). S.P. SINGH, D.G. PATEL and A.K.SNYDER, Acta Endocrinol. 93 61-66 (1980). X. WANG, N. SAT0 and M.A. GREER, Mol. Cell Endocrinol.83 79-84 (1992).
ELSEVIER
PII s0024-3205(97)ooo15-5
HYPOSMOLAB MEDIUM AND ETHANOL IN ISOSMOTIC SOLUTION INDUCE THE RELEASE OF THYBOTBOPIN-RELEASING HORMONE (TBH) BY ISOLATED BAT PANCREATIC ISLETS
J. Benicky, M.A. Greer #, V. strbak Institute of Experimental Endocrinology, Slovak Academy of Sciences, Brat&lava, Slovakia, # Division of Endocrinology, Oregon Health Sciences University, Portland, Oregon (Received in final form December
28,1996)
Summary Cell swelling induced by hypotonic medium or small isotonic permeant molecules results in an immediate secretory response in various types of cells. We have expanded exploration of this phenomenon by examining the effect of either isotonic ethanol or hyposmotic medium on the release of TRH by freshly isolated islets of Langerhans in static incubation and peritusion. Ethanol (40, 80 or 160 mM in isotonic solution) dose-dependently evoked the release of TRH by statically incubated islets. The dynamics of TRH release induced by 80 mM isotonic ethanol or 30% hypotonic medium were similar to those induced by 50 mM KCl, with the highest secretion rate during the first 5 min of incubation irrespective of the duration of stimulation. Ca” depletion of the incubation medium abolished the response to 50 mM KC1 but did not diminish the response to 80 mM isotonic ethanol. We conclude that osmotic stimuli known to induce cell swelling also induce release of TRH by isolated pancreatic islets. i&y Words: thyrotropin-releasing
hormone,
pancreatic islets, ethanol
hyposmolarity,
cell swelling
Cell volume changes may play an important role in the intracellular regulation of metabolism and function (for review see 1). Permeant molecules (ethanol, urea, glycerol) when added in hypoor isosmotic medium diffuse rapidly across the cell membrane down their concentration gradient (2). An increase of their concentration inside the cell results promptly in cell swelling due to secondary water transport (this effect is suppressed in a hyperosmotic environment). Cell swelling induced in vitro by either medium hyposmolarity or isotonic per-meant molecules stimulates prompt high amplitude hormone secretion from various types of cells (3-6). Hyperosmolarity, on the contrary, causes cell shrinkage and depresses secretion (7-8). The secretory process induced by hyposmolarity or per-meant molecules is similar to that induced by specific secretagogues in many respects, in&ding response dynamics (a rapid high-amplitude secretory burst reaching a peak at l-2 min) and refractory periods (7). This process does not, however, require extracelhtlar calcium and microtubules and microtilaments and changes in sodium flux across the plasma membrane are not apparently involved (5). Immunoreactive thyrotropin-releasing hormone (TRH) has been detected in the rat pancreas (9) and immunocytochemically localized mainly in the insulin-producing p-cells of the islets of Langerhans ( 1O-l 1) where it is probably colocahzed with insulin in the same secretory granules ( 12).
866
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However, the role of TRH in this localization is unclear till present and remains to be explored. In the present work isolated pancreatic islets, providing a useful model for in vitro study of the endocrine pancreas, were used to evaluate the effect of osmotic forces on the secretory response of pancreatic TRH. A stimulatory effect of hyposmolarity on insulin secretion by perifused islets independent of extracellular Ca2’ concentration has been reported (3). This effect is presumably mediated by cell swelling. However, no data are available concerning pancreatic TRH. Methods Materials: The basal incubation medium - Krebs-Ringer bicarbonate (pH 7.4,290 mOsmo1) contained 118 mM NaCl, 4 mM KU, 1.2 mM KH2P0,, 1.2 mM MgSO,, 2.5 mM CaC&, 25 mM NaHCO,, 3 mM D-glucose, 0.3 mg/ml bacitracin, 0.25% bovine serum albumin in 10 mM Hepes. 30 % hyposmotic medium was prepared by dilution of basal medium with deionized distilled water(70 : 30). In isosmotic ethanol medium the concentration of NaCl was reduced (40 mM ethanol-98 mM NaCl, 80 mM ethanol-78 mM NaCl and 160 mM ethanol-38 mM NaCl) to maintain the osmolarity unchanged. The composition of the other components was the same as for basal medium. High potassium medium contained 50 mM KC1 and 72 mM NaCl without any changes in the concentration of the other components. Each medium was prepared also in Ca2+-free form in which Ca” was omitted without any other changes. Media were gassed 30 min before and throughout the experiment with a mixture of 0, and CO, (95:5). Preparation of the islets: Islets were isolated by collagenase digestion method according to Lacy & Kostianovsky (13). Briefly, male Wistar rats (250-3008) were anesthetized with pentobarbital and pancreases were distended by intraductal injection of Hanks balanced salt solution (HBSS, pH 7,4). Pancreases were then dissected out, minced and digested with collagenase (type XI, Sigma, 3 mg/ml) for 15 min at 37°C. Islets were separated from remaining acinar tissue manually with the aid of a dissecting microscope and collected 50 per tube for static incubation and 1000 per column for perifusion. Static incubation: Prior to incubation the islets were preincubated in basal medium for 30 min at 37°C and then incubated in the presence or absence of secretagogues in a total volume of 100 ~1. At the end of incubation the medium was saved and stored at -20°C until RIA. Perifusion exneriment: The dynamics of TRH secretion were analyzed by column per&ion. The flow rate was 0.25 ml/mm and fractions were collected every 2 min. Two parallel columns were simultaneously perifused; one starting with 2.5 mM Ca2’ and other with Ca2’-free medium. Samples were frozen at -20°C until analysis for TRH by RIA as described below. Radioimmunoassay: TRH was measured by radioirmmmoassay (RIA). The specific rabbit TRH antibody developed in our laboratory recognizes neither TRH-degradation products (such as TRH-OH, cyclo(His-Pro) or amino acids) nor such putative TRH precursor peptides as TRH-Gly, Gln-His-Pro-Gly-Lys-Arg or Lys-Arg-Gln-His-Pro-Gly-Arg-Arg (cross reactivity less than 1.O%). Synthetic TRH (a gift fiorn Prof. Kasafirek, Research Institute of Pharmacology and Biochemistry, Prague) was labelled with Na’2SI using the chloramine-T method and purified on a Sephadex G- 15 (Pharmacia) column (60 x 1 cm). All assays were performed in a total volume of 400 ul as previously described (14). Statistics: The results are expressed as mean & SE (n=5-8). performed using the unpaired Student’s t-test.
Statistical analysis was
Vol. 60, No. 11, 1997
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Results Effect of isosmotic ethanol on the release of TRH during static incubation of islets is illustrated in Fip 1,30 min exposure to ethanol (40, 80 and 160 mM in isotonic solution) resulted in significant dose-dependent induction of the release of TRH. TRH release induced by 80 mM isotonic ethanol was comparable to that induced by 30% hypotonic medium or 50 mM KC1 (m). This ethanol concentration was utilized as a stimulus in subsequent experiments.
4hM
SOmM
16Chni~l
I
Fig.1. Effect of increasing concentrations of ethanol in isosmotic (290 mosmol) medium on the release of TRH. Islets (50 per tube) were incubated for 30 min in basal (Control -C, open bar) or isosmotic ethanol (40.80 and 160 mM, cross-hatched bars) in medium containing 2.5 mM Ca”. At the end of incubation the medium was removed and analyzed for TRH. Results are expressed in pg TRW50 islets (mean f SE, n = 6-8 for each bar). * PcO.05, ** P
30 3 z d ; E
*
**
2520I5
m m mIn
30 % hyposmdic me&m 80 mM isosmotic EtOH 5omMKcl
Fig.2. Release of TFW in response to stimulation by 30% hyposmotic medium, 80 mM isosmotic ethanol or 50 mM KCI. Islets (50 per tube) were incubated for 10 min in the respective medium, each containing 2.5 mM Ca”. At the end of incubation the medium was removed and analyzed for TRH. Results are expressed in pg TRW1 0 islets (mean f SE, n = 5-6 for each bar). * PcO.01, ** P
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The dynamics of TRH release by statically incubated islets in response to 80 mM isotonic ethanol, 30% hyposmotic and high potassium medium is illustrated in Fia3a. After any stimulation, the major Inaction of TRH (approximately 80%) was secreted during the first 5 min. The dynamics of this osmotically induced release was similar to that induced by high potassium concentration. Fin.3b shows the secretion rate (pg TRH/min) in the same experiment.
-IB-
BS9l
-
B
ftOmMEtOH B
B
~C~DME~OH B
-
-
30%H,O
B
50mMKCl
30% H,O
B
5mnMKCl
Fis3.A. Time course ofbasal and stimulated TRH release. Islets (50 per tube) were divided into four groups and incubated for eight subsequent periods inte.rrupted by medium exchange in the following order: preincubation (30 min) -basal (5, 10, 15 and 30 min)- I.interincubation (30 min)- 8OmM isosmotic ethanol (5, 10, 15 and 3Omin)- IIinterincubation (3Omin)- 30% hyposmotic (5, lo,15 and 3Omin)- III.interincubation (3Omin)- 50mM KC1 (5,10,15 and 30min). Release of TRH during 30 min basal interincubations (B) is illustrated by one bar which represents the mean + SE from all samples incubated. Groups of four neighbouring bars represent the release of TRH during 5, 10, 15, and 30 min, respectively, as indicated in the legend. Results are expressed in pg TRW50 islets (mean f SE, n=5 for each time interval). E The same results expressed as secretion rate (pg TRW50 isletsmin) for each collection period.
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Calcium dependency of the ethanol-induced TRH release was assessed using medium repleted or depleted of Ca” (FM). Eighty mh4 isotonic ethanol induced TRH release independently of the presence of extracellular Ca”. In contrast, extracellular Ca” was essential for 50 mM KCI-induced TRH release.
A. 80 -
T
2.5 mM Ca”
0
Basal
80 MEtOH
=
SOmMKCl
1
B. 80 c
T
2.5 u&l Ca*+
Fk.4.A. Effect of medium Ca” on the ethanol- and KCl-stimulated release of TRH. Islets (50 per tube) were incubated for eight subsequent 15-min periods interrupted by medium exchange in the following order: basal, 80 mM isosmotic ethanol, basal, 50 mM KCl, basal, 80 mh4 isosmotic ethanol, basal, 5Omh4 KCl. The first four incubations were performed in the presence of 2.5 mM Ca”, then the medium was changed to a Ca2’-free one. At the end of each incubation the medium was saved and analyzed for TRH. Results are expressed in pg TRW50 islets (mean f SE, n=8). B, The same experimental design as in Panel A, with the order reversed to start the first set of incubations with Ca2’-free medium.
Perifusion of the islets (m) confirmed the significant stimulation of the release of TRH by 80 mM isotonic ethanol. This effect, like that observed during static incubation, was independent of extracellular Ca2+ concentration.
870
Cell Swelling and Pancreatic TRH
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80
80
Fraction # (2miu collection) u The effect of isosmotic 80 mM ethanol on the release of TRH by perifused pancreatic islets in two parallel simultaneous columns, one starting with Ca2”-rep&d and the other with Ca”- depleted medium. Islets (1000 per column) were perifused at a flow rate of 0.25 ml/min; samples were collected every 2 min. Application of 80 mM isosmotic ethanol is indicated by black bars, the presence of Ca2’ in the medium is marked by horizontal line below the collection fractions.
Discussion Cell swelling produced by medium hyposmolarity or permeant molecules is a potent nontoxic inducer of hormone secretion (3-6). In the present study isotonic ethanol stimulated secretion of TRH by statically incubated pancreatic islets. The islets are, however, composed of several cell types which produce a number of biologically active substances. It is therefore necessary to take into account possible para- and/or autocrine influences which might lead to indirect effects and misinterpretations. As a second experimental approach we therefore used a perimsion system in which the secretory products are washed out, minimizing such indirect effects. The islet perifusion experiments confirmed the data obtained using static incubation, indicating the effect of isotonic
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ethanol on TRH-secreting cells is direct. Although we do not have direct evidence that hyposmolar media or the addition of isosmolar permeant molecules causes cell swelling in isolated islets, cell swellinghas been directly observed to result from these conditions in both normal and tumor-derived pituitq cells (15-l 7). We assume that the islet cells would have similar volume changes in response to the osmotic challenges. In normal rat pituitary cells, Ca” influx has a negative influence on secretion induced by cell swelling (5,16). Depletion of medium Ca” also enhances osmotically induced hormone secretion in several other normal cell types (2-3,18- 19). Ca” influx through the plasmalemma is important in the regulatory volume decrease which begins l-2 min a&r initial swelling (2). It is possible that negative modulation of cell swelling-induced secretion is due to the greater degree of cell swelling produced when Ca” intlux is prevented since the regulatory volume decrease following cell swelling is Ca”-dependent. Unlike K+-induced TRH release, removal of medium Ca” did not reduce ethanol-induced TRH release by tieshly isolated islets. This is consistent with data obtained using other secretions from normal pancreas (3) or pituitary cells as in vitro models. Earlier reports demonstrated an important direct stimulation of endocrine and exocrine secretion by peritused pancreatic islets following exposure to extracellular hyposmolarity (3,20). Customary inhibitors of insulin secretion did not depress the insulin response to hyposmotic stimulation.Removal of medium Ca” resulted in an increased secretory response to a hyposmolar stimulus.Na’ transport and the microtubular and microtilamentous system of the B-cells apparently do not participate in osmotically induced release (3). These characteristics are similar to those in anterior pituitary cells (5), indicating a common mechanism for both cell types. On the other hand, Singh et al. (21) reported inhibitionby 65 mM ethanol on glucose- and tolbutamide-induced insulin release from perifused pancreatic islets. However, in their experiments the medium osmolarity was not adjusted for ethanol addition, resulting in a hyperosmolar medium which per se can inhibit exocytosis (7-8,22). Sir@ et al. (21) also reported inhibition of glucose- and tolbutamide-induced in vitro insulinrelease after in vivo intraperitoneal or oral administration of ethanol 1 hour prior to sacrifice of rats. Ethanol exposure in vivo may have diierent effects than those elicited from the acute in vitro exposure employed in the present study. To our knowledge, this is the first report concerning the effect of isosmotic ethanol on the release of pancreatic TRH. Although cell swelling is a potent stimulus for hormone secretion, it is uncertain how important a role it plays in normal in vivo physiology. Our data nevertheless emphasize that it is necessary to take into account changes in medium osmolarity during in vitro experiments since they may dramatically influence the secretory response of the cell. Acknowledgements.
The present work was supported by U.S.-Slovak Science and Technology Program, Project N” 93053 and by grants VEGA 211291196and 95153051239.The authors wish to thank to Susan E. Greer and AKrupkova for help and technical assistance.
1. 2. 3. 4.
F. LANG, G.L. BUSCH, H. VGLKL and D. IIAUSSINGER N.I.P.S. lQ 18-22 (1995). S.M.E. WONG and H.S. CHASE JR, Am. J. Physio1.m C841-852 (1986). W.G. BLACKARD, M. KIKUCHI, A. RABINOVITCH and A.E. RENOLD, Am. J. Physio1.m 706-713 (1975). M.A. GREER, S.E. GREER, Z. OPSAHL, L. McCAFFERTY and S. MARUTA,
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5.
6. 7. 8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
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Endocrinologym 1531-1533 (1983). M.A. GREER, S.E. GREER and S. MARUTA, Proc. Sot. Exp. Biol. Med. 193 203-209 (1990). X. WANG, N. SATO, M.A. GREER, S.E. GREER and S. McADAMS, Biochem. Biophys. Res. Commun. 163 471-475 (1989). M.A. GREER, S.E. GREEK, Z QPSAHL and S. MARUT_4, Proc. Sot. Eup. Biol. Med. 178 24-28 (1985). N. SATO, X. WANG and M.A. GREER, Mol. Cell Endocrino1.x 193-198 (1991). J.E. MORLEY, J.J. GARVIN, A.E. PEKARY and J.H. HERSHMANN, Biochem. Biophys. Res. Commun. 79 314-318 (1977). S. ARATAN-SPIRE, B. WOLF, B. PORTHA, D. BAILBE and P. CZERNICHOW, Endocrinology 114 2369-2373 (1984). P. LEDUQUE, S. ARATAN-SPIRE, B. WOLF, P.M. DUBOIS and P. CZERNICHOW, Cell Tissue Res. 248 89-94 (1987 ). P. LEDUQUE, S. ARATAN-SPIRE, R. SCHARFMANN, A. BASMACIOGULLARl, P. CZERNICHOW and P.M. DUBOIS, Biol. Cell @ 291-296 (1989). P.E. LACY and M. KOSTIANOVSKY, Diabetes _l&35-39 (1967). R.M. BASSIRI and R.U. UTIGER, Methods qfHormone Radioimmmloassqv, B.M Jaffe and H.R. Behrman (Eds), 37-44, Academic Press, New York and London, (1974). N. SATO, X. WANG, M.A. GREER, S.E. GREER and S. M&DAMS. Endocrinology 127 3079-3086 (1990). N. SATO, X. WANG and M.A. GREER, Am. J. Physiol. 360 E946-E950 (1991). N. SATO, M. MURAKAMI, X. WANG and M.A. GREER, Endocrinology 179 2541-2546 (1991). 0. SKOTT, Pfluegers. Arch. 406485-491 (1986). J.E. GREENWALD, M. APKON, K.A. HRUSKA and P. NEEDLEMAN, J. Clin. Invest. g 1061-1065, (1989). P.E. LUND, A. BERTS and B. HELLMAN, Mol. Cell. Biochem. 109 77-81 (1992 ). S.P. SINGH, D.G. PATEL and A.K.SNYDER, Acta Endocrinol. 93 61-66 (1980). X. WANG, N. SAT0 and M.A. GREER, Mol. Cell Endocrinol.83 79-84 (1992).