Gen. Pharmac. Vol. 24, No. 5, pp. 1097-1100, 1993 Printed in Great Britain. All rights reserved
0306-3623/93 $6.00 + 0.00 Copyright © 1993 Pergamon Press Ltd
ATP 4- I N C R E A S E S THE I N T R A C E L L U L A R C A L C I U M C O N C E N T R A T I O N IN R A T S U B M A N D I B U L A R G L A N D S J. P. DEHAYE Department of General Biochemistry, Institute of Pharmacy, Universit6 libre de Bruxelles, Brussels, Belgium (Received 4 March 1993)
Abstract--1. The intracellular calcium concentration of a crude suspension from rat submandibular glands was increased by extracellular ATP. 2. The stimulatory effect of ATP was favored by removal of maguesium from the extracellular medium. ADP and adenosine had no effect. ATP did not modify the concentration of calcium in isolated rat pancreatic acini. 3. It is concluded that ATP is a potential neurotransmitter regulating the submandibular gland function.
INTRODUCTION The control of salivary glands is exerted for the most part by neurotransmitters. Parasympathetic stimulation of parotid or submandibular glands activate a polyphosphoinositide-specific phospholipase C leading to a secondary increase in intracellular calcium and diglycerides (Putney, 1986). The permeability to potassium of the basolateral membrane and to calcium of the apical membrane is increased and there is a massive loss of potassium chloride and a decrease in cell volume (Foskett and Melvin, 1989). Sympathetic stimulation activates adenylate cyclase and the increase in cyclic A M P removes the inhibition of a regulatory subunit on the catalytic subunit of a cyclic A M P dependent protein kinase. In response to protein phosphorylation, secretory granules migrate towards and fuse with the apical plasma membrane, the key step in exocytosis (Quissell et al., 1983). The composition of this primary saliva secreted by the acinar cells of the salivary glands is modified during its progression to the mouth through the ductal tree, specially at the level of intercalated and striated ducts. Neurotransmitters (adrenergic, peptidergic or muscarinic) also control the ductal functions (Denniss et al., 1978; Case et al., 1988; Dehaye and Turner, 1991; Valdez and Turner, 1991). But the control of various functions of the digestive tract seems to involve neurotransmitters distinct from muscarinic or adrenergic agents. Peptides like substance P is a well-known stimulant of salivary glands where it stimulates the inositoi pathway, at least in the acinar cells (Dehaye and Turner, 1991). It has been recently described that salivary glands also respond to vasoactive intestinal peptide which in-
creases their intracellular cyclic A M P concentration (Larsson et al., 1986; Turner and Camden, 1990). But another component of the non-adrenergic, nonmuscarinic stimulation probably involves purinergic agents (Gallacher, 1982). The purpose of this work was to test whether A T P could be a neurotransmitter for submandibular cells. We measured the intracellular calcium concentration in these cells in response to ATP. MATERIALS AND METHODS
Male Wistar rats (150-200 g) fed ad libitum and with free access to water were used for these experiments. One rat was used for each experiment. The animal was killed around 9 a.m. with ether. Its submandibular glands were excised and dissected. The two glands were finely minced in 0.3 ml of HEPES Buffer saline (HBS) containing (mM): NaC196, KCI 6, MgCI2 1, NaH2PO4 2.5, glucose I 1, Na pyruvate 5, Na glutamate 5, Na fumarate 5, HEPES 24.5 (pH 7.4 with NaOH), amino acid mixture (without glutamine) 1% (v/v), albumin 0.1% (w/v). The minced tissue was then incubated for 40 min under constant shaking (160 cycles/min) at 37°C in 5 ml of the same medium, but containing 0.8 mg collagenase (collagenase P 2.6 U/mg). After the first 20min of incubation, the tissue was aspirated 10 times in a 10 ml pipet. At the end of the incubation, the suspension was aspirated through a 10 ml, then a 5 ml and a 2 ml pipette and finally through a l-ml Pipetman blue tip mounted on a 2 ml pipette. This crude suspension was washed 4 times with the digestion medium (without collagenase). The final pellet was resuspended in 5 ml of this medium and kept in a cold room until use. Pancreatic acini were isolated as described previously (Grosfils et al., 1992). To measure the intracellular calcium concentration, 1 ml of either suspension was used and 1 ml of HBS with 1% albumin, 1% amino acid mixture and 0.5mM CaCI2 was added. Fura-2/acetoxymethyl ester (fura-2/AM) was stored at -80°C in pure DMSO at a 2 mM concentration, in 20 #1 aliquots. For each experiment, one aliquot was used and mixed with 3#1 of a 15% pluronic F-127. 4#1 of the fura-2/AM solution were then added to the cellular suspension (final concentration of fura-2/AM slightly less than
J.P. DEHAYE Z
(= (I) U C U
. . . . .
. . . . .
. . . . .
t 2 Time (minutes)
t 2 Time (minutes)
Fig. 1. Effect of carbachol and ATP on the intracellular calcium concentration in a crude suspension from submandibular glands. A crude cellular suspension from rat submandibular glands was prepared and its intracellular calcium concentration measured as described in Materials and Methods. After 1 min, the cells were exposed to 100/IM carbachol (A) or 1 mM ATP (B). The results are expressed as nm calcium and are the means + SEM of 19 (A) or 14 experiments (B). 4#M). With this protocol, 5ml of the initial cellular suspension could be loaded successively with one tube of fura-2/AM. The addition of pluronic acid to the dye was done daily. The tubes with the cells and the dye were incubated for 60 min at 25°C under constant shaking (60 cycles/min). At the end of the incubation, 1 ml was removed and washed with 12 ml of an isotonic solution. The pellet was resuspended in 2 ml medium B and the intracellular calcium assay was performed at room temperature as described previously (Dehaye and Turner, 1991). Collagenase P, bovine serum albumin and the ATP were from Boehringer (Mannheim, Germany). A fresh solution of collagenase and ATP were prepared daily. The ATP was put into solution at a 200mM concentration in a 245 mM HEPES buffer (pH 7.4). It was diluted at least 200-fold in the cuvette of the fluorimeter. Carbachol was from Sigma (St Louis, MO) and fura-2/AM and pluronic acid were from Molecular Probes (Eugene, OR).
c o n c e n t r a t i o n range (half-maximal c o n c e n t r a t i o n at 5 # M ) . The response to A T P was decreased when m a g n e s i u m chloride was included in the i n c u b a t i o n medium: for instance, at a 3 0 0 # M concentration, A T P increased calcium c o n c e n t r a t i o n from 34 to 6 6 n M in the absence o f m a g n e s i u m a n d to 52 n M in the presence o f the divalent cation. A D P or adenosine did not affect the basal calcium c o n c e n t r a t i o n or the response to A T P (data not shown). As shown in Fig. 3(B), A T P h a d a very weak, non-significant effect o n a suspension of crude pancreatic acini which was responsive to carbachol [Fig. 3(A) a n d (B)].
The basal c o n c e n t r a t i o n of calcium in the crude suspension was 3 6 . 3 + 3 . 4 n M (n = 19). It remained stable for at least 1 5 m i n suggesting t h a t in o u r conditions, the leak of the dye was minimal. C a r b a c h o l at a 100kLM c o n c e n t r a t i o n increased this c o n c e n t r a t i o n to 92.6 + 7.3 n M (n = 19) within 10see [Fig. I(A)]. After this peak value, the calcium c o n c e n t r a t i o n decreased steadily a n d reached 4 0 % o f the peak value after 2 min. A T P at a 1 m M c o n c e n t r a t i o n increased the calcium c o n c e n t r a t i o n from a n average 33 + 3.1 to 65.8 + 6.2 n M (n = 14) [Fig. I(B)]. This c o n c e n t r a t i o n remained stable for the next 2 m i n . The stimulatory effect of A T P was dose-dependent (Fig. 2). The half-maximal c o n c e n t r a t i o n was at 1 0 0 p M a n d a purinergic effect was consistently observed at c o n c e n t r a t i o n s in the 1 0 # M range. By comparison, c a r b a c h o l increased the calcium c o n c e n t r a t i o n in the 1-100/~M
kgonits concentration (Log N)
Fig. 2. Dose-response curve of carbachol and ATP on the intracellular calcium concentration in a crude suspension from submandibular glands. A crude cellular suspension from rat submandibular glands was prepared and its intracellular calcium concentration measured as described in Materials and Methods. After 1 min, the cells were exposed to various concentrations of carbachol (C) or ATP (A). Results are expressed as percent of maximal calcium increase. They are the means + S.E.M. of 3 to 5 experiments.
ATP and submandibular glands
t00.~... . .............
i 2 3 4 5 Time Minutes)
Fig. 3. Effect of carbachol and ATP on the intracellular calcium concentration in a crude suspension from the pancreas. A crude cellular suspension from rat pancreas was prepared and its intracellular calcium concentration measured as described in Materials and Methods. After 1 min, the ceils were exposed to 100/~M carbachol (A) or 1 mM ATP followed by I00/~M carbachol at 4 min (B). The results are expressed as nM calcium and are the means _ S.E.M. of 3 experiments.
D I S C U S S I O N
A new technique to separate a crude cellular suspension from rat submandibular glands and to prepare isolated ducts and acini from this suspension has been used. It is inspired from a method which had been published previously (Dehaye and Turner, 1991). Its major advantages are its rapidity and its low cost. The cells isolated by this method had a rather low intracellular calcium concentration (below 50nM) and the maximal response to stimulatory agents never exceeded 100nM. These differences with previous results (Morris et al., 1987; Brink and Hurley, 1989; Elliott et al., 1991; Melvin et al., 1991) might also be due to a different protocol to load the cells with the dye: fura-2 AM was mixed with pluronic acid which gave us a good dispersion of the dye. We also kept the cells during and after the loading at 25°C in order to avoid the diffusion of the dye in intracellular organelles or the leakage of the dye from the cells. And the final point was the fact that the cells were resuspended in a medium which contained a low (0.5mM) concentration of calcium. According to the results of Morris et al., (1987) and our previous results (Dehaye and Turner, 1991), this should indeed drastically decrease the intracellular calcium concentration. Using the crude suspension, we showed that in submandibular glands from rat, the intracellular calcium concentration increased in response to ATP. The fact that the omission of magnesium potentiated the purinergic response and that ADP and adenosine had no effect demonstrates that the agonist is ATP4- . This stimulatory effect of the nucleo-
tide triphosphate is not merely a detergent effect since ATP had no effect on a similar cellular suspension prepared from rat exocrine pancreas. These results rather suggest that rat submandibular cells have a purinergic receptor comparable to the receptor described on rat mast cells (Cockroft and Gomperts, 1980), and on rat parotid acini (McMillian et al., 1987), distinct from the receptor described in a cell line (the HSG-PA cells) derived from human submandibular ductai cells (Yu and Turner, 1991). The effector coupled to the purinergic in submandibular cells remains unknown, but considering the different time-course observed with carbachol and ATP, it is unlikely that ATP stimulates the phosphoinositidase C stimulated by carbachol. ATP could rather stimulate the uptake of extraeellular calcium via a non-specific cation channel similar to the channel described in lacrimal (Sasaki and Gallacher, 1990; Vincent, 1992) or parotid cells (Soltoff et al., 1992). In conclusion, ATP is a neurotransmitter likely to play an important role in salivary gland function since it can modulate the activity of the two major glands, the parotids (Gallacher, 1982; McMillian et al., 1987, 1988; Soltoff et al., 1992) and submandibular glands (this report). It remains to be established if ATP regulates the acinar and/or the ductal function of the submandibular gland and the nature of the effector coupled to these purinergic receptors. Acknowledgements--This work was supported by grant No. 3.4558.92 from the Fonds de la Recherche Scientifique M~dicale. The author wishes to thank P. O. Dehaye for his help in computing the results.
J.P. DEHAYE REFERENCES
Brinck R. W. and Hurley T. W. (1989) Regulation of cytosolic Ca-' + in resting and stimulated rat submandibular salivary gland acini. Archs Oral Biol. 34, 917-922. Case R. M., Howorth A. J. and Padfield P. J. (1988) Effects of acetylcholine, isoprenaline and forskolin on electrolyte and protein composition of rabbit mandibular saliva. J. Physiol. 406, 411~,30. Cockroft S. and Gomperts B. D. (1980) The ATP4receptor of rat mast cells. Biochem. J. 188, 789-798. Dehaye J. P. and Turner R. J. (1991) Isolation and characterization of rat submandibular intralobular ducts. Am. J. Physiol. 261, C49(~C496. Denniss A. R., Schneyer L. H., Sucanthapree C. and Young J. A. (1978) Actions of adrenergic agonists on isolated excretory ducts of submandibular glands. Am. J. Physiol. 235, F548 F556. Elliott A. C., Lau K. R. and Brown P. D. (1991) The effects of N a + replacement on intracellular pH and [Ca 2+] in rabbit salivary gland acinar cells. J. Physiol. 444, 419-439. Foskett J. K. and Melvin J. E. (1989) Activation of salivary secretion: coupling of cell volume and [Ca]l in single cells. Science 244, 1582-1585. Gallacher D. V. (1982) Are there purinergic receptors on parotid acinar cells? Nature (Lond.) 296, 83-86. Grosfils K., Gomez F. and Dehaye J. P. (1992) Inhibition by mepacrine of amylase secretion from intact and permeabilized rat pancreatic acini. Biochem. Biophys. Res. Commun. 184, 408-J,13. Larsson O., Dun6r-Engstr6m M., Lundberg J. M., Fredholm B. B. and Anggard A. (1986) Effects of VIP, PHM and substance P on blood vessels and secretory elements of the human submandibular gland. Regul. Peptides 13, 319-326. McMillian M. K., Soltoff S. P., Cantley L. C. and Talamo B. R. (1987) Extracellular ATP elevates intracellular free calcium in rat parotid acinar cells. Biochem. Biophys. Res. Commun. 149, 523 530. McMillian M. K., Soltoff S. P., Leichleiter J. D., Cantley
L. C. and Talamo B. R. (1988) Extracellular ATP increases free cytosolic calcium in rat parotid acinar cells. Biochem. J. 255, 291-300. Melvin J. E., O'Connell A. C., Koek L. and Bowen W. H. (1991) Agonist-induced Ca 2+ mobilization in the rat submandibular gland during aging and subsequent to chronic propranolol treatment. Mechanisms Ageing Dev. 61, 33-44. Morris A. P., Fuller C. M. and Gallacher D. V. (1987) Cholinergic receptors regulate a voltage-insensitive but Na+-dependent calcium influx pathway in salivary acinar cells. FEBS Lett. 311, 195-199. Putney J. R. (1986) Identification of cellular activation mechanisms associated with salivary secretion. A. Rev. Physiol. 48, 75-88. Quissell D. O., Deisher L. M. and Barzen K. A. (1983) Role of protein phosphorylation in regulating rat submandibular mucin secretion. Am. J. Physiol. 245, G44-G53. Sasaki T. and Gallacher D. V. (1990) Extracellular ATP activates receptor-activated cation channels in mouse lacrimal cells to promote calcium influx in the absence of phosphoinositide metabolism. FEBS Lett. 264, 130-134. Soltoff S. P., McMillian M. K. and Talamo B. R. (1992) ATP activates a cation-permeable pathway in rat parotid acinar cells. Am. J. Physiol. 262, C934-C940. Turner J. T. and Camden J. M. (1990) The influence of vasoactive intestinal peptide receptors in dispersed acini from rat submandibular gland on cyclic AMP production and mucin release. Archs Oral Biol. 35, 103-108. Valdez I. H. and Turner R. J. (1991) Microfluorimetric studies of the effects of secretagogues on cytosolic Ca 2÷ levels in rat submandibular granular ducts. Am. J. Physiol. 261, G359-G363. Vincent P. (1992) Cationic channels sensitive to extracellular ATP in rat lacrimal cells. J. Physiol. 449, 313-331. Yu H. and Turner J. T. (1991) Functional studies in the human submandibular duct cell line HSG-PA, suggest a second salivary gland receptor subtype for nucleotides. J. Pharmac. Exp. Ther. 259, 1344 1350.