BIOCHEMICAL SOCIETY TRANSACTIONS

448 collection under resting (unstimulated) conditions, a vitamin C solution (10 mg/ml) was applied to the tongue tip at 30 s intervals and saliva production was timed. Samples were centrifuged for 30 s at 12 300 g in a Microfuge, the supernatants were immediately frozen and stored at -70°C before analysis. Electrolyte concentrations were determined as follows: Na+ via ion-selective electrodes (Beckman Electrolyte 2 Analyzer); CI - by thiosulphate titration (Corning 925 Chloride Analyzer) and H C 0 , - colorimetrically, using phenolphthalein as indicator (Technicon SMAC I1 system). Results

Vitamin C solution maintained a constant stimulus with only slight variation in flow rate; no correlation of rate with age or sex was evident (data not shown). As shown in Table I , vitamin C increased the flow rate to the same extent in control and C F individuals (/’< 0.001 and P < 0.02, respectively, for differences from resting). However, C F individuals gave flow rates significantly lower than controls under both resting and stimulated conditions. Stimulation with vitamin C increased the Naf concentration of control saliva, but no effect on C F saliva was observed. Similarly, the HCO, - concentration was increased on stimulation in control but not C F individuals. Resting levels of H C 0 , - were significantly lower and resting levels of CI significantly higher in C F saliva than controls. Discussion

The data show abnormalities in the electrolyte content of CF submandibular saliva which are consistent with the anion transport defect observed in C F cells in vitro [3].Thus, the increased CI- and decreased H C 0 , - levels observed in C F

Table I . Flow rate and electrolyte content of suhmnndihulrir saliva from control and C’F individuals Values are expressed as means f S.D. for ti = 4 individuals, resting (unstimulated) saliva, and ti = 5 individuals. vitamin C-stimulated saliva. */’< 0.02, **/’< 0.0 1 for difference from control resting levels; t /’< 0.05, $/’< 0.001 for difference from corresponding control, by Student’s t-test. Na’ (mM)

Control Resting Stimulated CF Resting Stimulated

7f5 22 f 9* 5+3 6 31-

*

CI (mM)

1Ok3 I7 f 6 19k61’

I 6 f4

HCO,

Plow rate

(mM)

(ml/min)

5f3 I 7 f 5** I f It 2 k 2::

0.40f0.09 0.84 f0.06 0.l3fO.06 0.27 f 0.06

resting saliva suggest altered anion transport in salivary glands, possibly at the level of CI-/HCO,- exchange. In addition, a lack of stimulation of Na+ and H C 0 , - concentrations in C F submandibular saliva was demonstrated. This indicates a defect in regulation of submandibular secretion in C F in v i m , as has been observed in vitro [ I , 2 I. We thank the Wellcome Trust for financial wpport. 1. McPherson, M. A. & Dormer, R. L. ( 1988) 7i.rrich H i o c h w i . S c i . 13, 10-13 2. McPherson, M. A,, Dormer, R. L.. Bradhury, N. A,. Dodge, J. A. & Goodchild, M. C. ( 1986) Lancet ii, 1007- I008

3. Li, M., McCann, J. D., Liedtke, C. M., Nairn, A. C., Greengard, P. & Welsh, M. J. (1988)Nmirr (Loridon) 331, 358-360 4. McCarthy, D. & Bagg, J. ( 1987) Rr. 1)ent. J . 162, 148- I 5 0 Received 20 November I989

Intracellular Ca2 trigger mucin release from rat submandibular acini +

CHRIS LLOYD MILLS, ROBERT L. DORMER and MARGARET A. McPHERSON Deprirtment of Medical Biochemistry, University of Wales College of Medicine, Heath I’urk, Cardiff CF4 4XN, U.K. The main stimulus of mucin secretion from rat submandibular acini is P-adrenergic [ l ] . Although cyclic AMP is the most likely second messenger involved, our studies have shown dissociation between cyclic AMP increase and mucin secretion during incorporation of cyclic AMP phosphodiesterase into intact acini [2]. In addition, P-adrenergic stimulation is partially dependent on extracellular Ca?+ [ 11 suggesting a modulatory role for Ca” in regulating secretion. Results, however, showed that increases in intracellular free Ca” induced by the bivalent cation ionophore A23 187 did not trigger mucin secretion [3]. The present study was designed to directly investigate whether carbamylcholine stimulation of mucin secretion from rat submandibular acini is mediated by a rise in intracellular Ca’+ concentration. Thus, the actions of the C a 2 + chelator 1,2-bis(2-aminophenoxy)ethane- N, N, N’, ”-tetraacetic acid (Sigma) (BAPTA) incorporated into intact acini by hypotonic swelling, on carbamylcholine stimulation of mucin secretion were examined. Rat submandibular acini were prepared and mucins labelled with [3H]glucosamine as described [2]. Hypotonic swelling was carried out for 1 min at room temperature in

medium of approximately half-isotonicity [ 2 I. Where acini were swollen in the presence of BAPTA (Swollen acini + BAPTA), BAPTA was added at a concentration of 10 mM during the swelling procedure. After swelling, acini were resuspended in isotonic medium, washed and incubated for 15 min in Krebs-Henseleit buffer, pH 7.4, containing 0.01% (w/v) trypsin inhibitor and 2% (w/v)bovine serum albumin. They were then incubated in the presence or absence of carbamylcholine (carbachol) (10 L ( M ) for 30 min. Mucin secretion was determined by measuring trichloro-

Table 1. Chrhachol-stimulation of mucin secretiorr itr presence or absence of‘intracellulrr BAI’IA

Control acini (unswollen) or acini which had previously been subjected to hypotonic swelling in the prescncc o r abscncc of BAPTA (swollen acini) were incubated with or without carbachol ( 1 0 ,UM) for 30 min and mucin secretion was measured as described in the text. Results are expressed as a percentage of the basal secretion from similarly treated acini (no. of observations in parentheses). */’< 0.0 1 for difference from acini swollen in the absence of BAPTA, by Student’s t-test. Mucin secretion ( 7 ,hasal)

Unswollen acini Abbreviation used: BAPTA, I ,2-bi\(2-aminophenoxy)ethane N , N, N’, N’-tetra-aceticacid.

the

Swollen acini Swollen acini + BAPTA

* 106.2 * 2.6 (4)*

233.3

15.0 ( 3 ) 193.8 k 18.4 (4)

1990

033rd MEETING, LONDON

440

acetic acid/phosphotungstic acid-precipitable 'ti radioactivity in the medium. Table I shows that a maximally effective concentration o f carbachol (10 pM) increased m u c h secretion t o the same extent in acini not subjccted t o hypotonic swelling (unswollcn acini) ;is in acini swollen in the absence of BAPTA. In acini swollen in the presence o f BAPTA. howcvcr. the response t o carbachol was abolished. T h c data suggest that a rise in intracellular free Ca" is necessary for carbachol stimulation o f mucin secretion from rat submandibular acini. In this rcgard. submandibular acini respond in the same way a s rat pancreatic acini. where carbochol stimulation o f amylase release was shown t o he inhibited by intracellular BAPTA 141. Studies using A 2 3 187. however, indicated that, unlike in rat pancreatic acini, Cn' could not act a s a primary trigger o f mucin secretion from rat submandibular acini 131. Nevertheless, our present data show that an intracellular Ca' increase induced by a physiological secretagoguc can trigger mucin secretion. T h e hypotonic swelling method described can bc used to incorporatc impermeant macromolecules into intact cells 12 I. This technique will be invaluable for investigating how ;I +

+

mutation in the protein coded for by the cystic fibrosis gene 151 leads t o disturbances in autonomic function which are seen in this disease 16.71. We thank the Cystic Fihrosis Research Trust, U.K. lor financial wpport. 1 . McPherson, M. A . & Dormer, R. L. ( 19x1) Ilioc,/ic,rii. ./. 224.

473-48 1

2. Bradbury. N . A,. I>ormer. K. L. &i McPhcrson. M. A . ( I OHc)) H i c x h i ~ m H. i e ~ p l l i KCA. ~ . C ' c m i n i i i t i . 161. 66 1-67 I 3 . McPherson. M. A. Kr Dormer. R. L. ( 1984) Riochiwi. .Sot.. 7?ciils. 12. l ( l 9 I - l O Y 2 N . 1. Dormer. K. L. ( 1081)Niochcw1. Hiophys. Kc,.\. C ' ~ ~ H ~ I I I ~19. X 76 - 88 3 S. Riordan. J. R. 1" cil. ( 1989). k i c , r i c ~ ,245. 1060- 1073 0. Shori. D. K., L>ormer. K. L.. Goodchild, M. C. &i McPhcrson,

M.A.(lYXc)) I l i e > c ' k ~ r ~ ~263,613-610 ../. 7. McPherson. M. A. & Dormer, i K. L. ( I Y X X ) ' I r c ~ r i c kI ~ i e ~ / i e r r.Sci. i. 13, 10-13

Biochemical studies of dystrophic retinas in cats Nl(if:,l. ( i . flOl.MES and KO