Elevation of intracellular cAMP by noradrenaline and vasoactive intestinal peptide in striated ducts isolated from the rabbit mandibular salivary gland.

Salivary gland intralobular ducts are responsible for the modification of the electrolyte composition of the primary fluid secreted by the acini. However, the intracellular messengers that regulate this and other intralobular duct cell processes have not been fully characterized. To investigate the possibility that cAMP-mobilizing agonists may be involved, intralobular (striated) ducts were isolated from the rabbit mandibular salivary gland by tissue dissociation and microdissection and maintained in tissue culture overnight. Individual duct fragments were stimulated with the secretory agonists noradrenaline, vasoactive intestinal peptide (VIP) and substance P and their cAMP content measured by acetylated radioimmunoassay. Both noradrenaline and VIP elevated intracellular cAMP content concentration dependently, but substance P did not. The response to noradrenaline was blocked by the beta-adrenoceptor antagonist propranolol, but not by the alpha-adrenoceptor antagonist prazosin. Application of the VIP analogue [D-p-Cl-Phe6, Leu17]-VIP decreased the VIP-induced cAMP response. These results demonstrate that striated intralobular duct cells possess beta-adrenoceptors and peptidergic receptors that are coupled to adenylate cyclase and activated by noradrenaline and VIP, respectively. By elevating ductal cAMP content, these agonists may regulate both the electrolyte content of the primary saliva and the secretion of protein(s) from the ducts.

An ammonium pulse inhibits fluid secretion.

Exposing perfused mandibular salivary glands to a 5-min pulse of NH4Cl almost totally inhibited fluid secretion evoked by acetylcholine. Recovery from exposure to 0.5 mM NH4Cl occurred within 10 min, but after exposure to 20 mM NH4Cl recovery was not complete even after 30 min. The inhibition could not be explained by changes in intracellular pH as there was no acidosis measurable after the 0.5 mM NH4Cl pulse. NH4Cl did not inhibit intracellular calcium release by acetylcholine so that receptor inhibition could not be the explanation for the inhibition of secretion. These observations indicate that data obtained from ammonium pulse experiments should be interpreted with caution.

Intracellular Cl- concentration in striated intralobular ducts from rabbit mandibular salivary glands.

Intralobular striated ducts have been isolated from rabbit mandibular salivary glands and maintained in primary culture for up to 2 days. Such ducts were loaded with the Cl(-)-sensitive fluorescent dye N-(ethoxycarbonylmethyl)-(6-methoxyquinolinium bromide) (MQAE) and intracellular Cl- concentration ([Cl-]i) monitored using a fluorescence microscope. Intracellular Cl- could be rapidly and reversibly emptied from striated duct cells by replacing Cl- in the superfusing solution with NO(3)-. [Cl-]i could be lowered by removal of external Na+, exposure to 10 microM amiloride or to 10 microM 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). Both amiloride and DIDS were able to inhibit the recovery of [Cl-]i after an initial exposure to Na(+)- or Cl(-)-free solution. The amiloride derivatives, benzamil (2 microM) and N-isobutyl-N-methylamiloride (MIBA), (10 microM) also lowered [Cl-]i by similar amounts as 10 microM amiloride. Varying external K+ concentration ([K+]o) also affected [Cl-]i. Increasing [K+]o increased [Cl-]i, but decreasing [K+]o did not decrease [Cl-]i. Instead, [Cl-]i was also increased when [K+]o was lowered below the control value. Bumetanide (0.1 mM) lowered [Cl-]i by only a small amount, while ouabain (1 mM) had no significant effect on [Cl-]i. These data are consistent with current models of electrolyte transport in salivary ducts which include Cl- channels, Na+ channels, and Na+/H+ exchangers in the apical membrane. The effects of low [K+]o can be interpreted in terms of a K(+)-dependent exit mechanism for Cl-.

Structural and functional characterization of striated ducts isolated from the rabbit mandibular salivary gland.

Ductal elements within salivary glands are responsible for modifying the electrolyte composition of primary saliva secreted by the acini. To study the mechanism and regulation of the transport processes involved requires a suitable preparation of functional ducts. To this end we have isolated intralobular ducts from rabbit mandibular salivary glands using the technique of tissue dissociation and microdissection. Light and electron microscopy demonstrated that the ducts corresponded ultrastructurally to striated intralobular ducts of the intact gland. Ducts could be maintained in tissue culture on polycarbonate filter rafts for up to 36 h, during which time the ends of the ducts did not usually seal. The overall resting content of ductal adenosine 3',5'-cyclic monophosphate (cyclic AMP) was 16.0 +/- 3.0 fmol mm-1 and increased dose dependently in response to stimulation with the beta-adrenoceptor agonist isoprenaline (10(-9)-10(-4) M; concentration required to produce a half-maximal response, K0.5 = 2.1 x 10(-6) M). The response to isoprenaline was blocked by the antagonist propranolol. Intracellular cyclic AMP content was also raised by the adenylate cyclase activator forskolin and by prostaglandin E2. Acetylcholine (3 x 10(-8)-10(-5) M) caused a dose-dependent and maintained rise in [Ca2+]i (K0.5 = 2.5 x 10(-7) M). This increase in [Ca2+]i could be reversed by the muscarinic antagonist atropine and appeared to result from a combination of mobilization of intracellular Ca2+ stores and entry of Ca2+ from the extracellular fluid. Noradrenaline induced only a very small, mainly transient rise in [Ca2+]i while phenylephrine failed to increase [Ca2+]i at all. Vasoactive intestinal peptide (5 x 10(-7) M) also produced a marginal, maintained rise in [Ca2+]i. Substance P, bombesin, isoprenaline, and prostaglandin E2 did not elevate [Ca2+]i. Application of the calcium ionophore ionomycin induced a substantial maintained rise in [Ca2+]i. Taken together, these results indicate that isolated and cultured striated ducts (i) possess intact beta-adrenoceptors coupled to adenylate cyclase, putative receptors for prostaglandin E2 and muscarinic receptors, and (ii) represent a viable preparation for the study of the transport mechanisms involved in the ductal modification of salivary fluid composition.

The effects of Na+ replacement on intracellular pH and [Ca2+] in rabbit salivary gland acinar cells.

1. The role of Na(+)-dependent mechanisms in regulating the intracellular pH (pHi) and free calcium concentration ([Ca2+]i) in acinar cells of the rabbit mandibular salivary gland was examined. The fluorescent dyes BCECF and Fura-2 were used to measure pHi and [Ca2+]i respectively in suspensions of isolated acini. 2. Replacement of all the extracellular Na+ with N-methyl-D-glucamine (NMDG) decreased resting pHi from a control value of 7.1-7.2 to 6.8-6.9. Re-addition of Na+ or Li+ caused a recovery of pHi towards control values. This recovery was blocked by 10-50 microM-ethylisopropylamiloride (EIPA), suggesting that it was mediated by Na(+)-H+ exchange. The rate of recovery of pHi when Na+ was re-introduced increased with Na+ concentration with an apparent Km for Na+ of around 30 mM. 3. Replacement of all of the extracellular Na+ with Li+ caused only a small decrease in resting pHi. 4. Stimulation of acini with 1 microM-acetylcholine (ACh) evoked an intracellular acidosis both under control conditions and when acini were bathed in Na(+)-free media. Following the acidosis pHi recovered in acini bathed in either control medium or Na(+)-free (Li+) medium, but not in acini bathed in Na(+)-free (NMDG) medium or in control medium containing EIPA. 5. Stimulation of acini bathed in Na(+)-free, HCO(3-)-free medium with ACh did not cause any change in pHi. 6. Re-addition of Na+ to acini bathed in Na(+)-free, HCO(3-)-free medium evoked the same rate of alkalinization whether or not the acini had been stimulated with ACh, suggesting that receptor stimulation per se did not lead to an activation of acid extrusion. 7. Resting [Ca2+]i was elevated in acini bathed in Na(+)-free (NMDG) medium, but not in acini bathed in Na(+)-free (Li+) medium. 8. ACh evoked a maintained rise in [Ca2+]i in acini bathed in control medium and in Na(+)-free media with either NMDG or Li+ as the Na+ substitute. 9. Experiments in which external Ca2+ was reduced to low levels (by the addition of EGTA) just prior to addition of ACh showed that ACh released intracellular Ca2+ stores under both control and Na(+)-free conditions. 10. In acini bathed in Na(+)-free (NMDG) solution and stimulated with ACh, re-addition of either Na+ or Li+ reduced [Ca2+]i. The reduction of [Ca2+]i on Na+ re-addition was blocked by EIPA. [Ca2+]i could also be reduced under these conditions by alkalinizing the cytosol using the weak base trimethylamine.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of bumetanide, amiloride and Ba2+ on fluid and electrolyte secretion in rabbit salivary gland.

1. In order to distinguish between models of anion secretion, the effects of transport inhibitors on saliva flow rate and electrolyte composition were studied during the plateau phase of secretion in rabbit mandibular salivary glands. 2. Bumetanide, an inhibitor of Na+,K+,2Cl- co-transport, inhibited flow rate (by 60%) and reduced Cl- concentration. K+ and HCO3- concentrations were increased. Forskolin, an adenylate cyclase activator which inhibits ductal transport, did not significantly affect this pattern of changes. 3. Amiloride, used at concentrations that would inhibit Na(+)-H+ exchange, inhibited flow rate (by 30%). Cl- concentration was initially increased before subsequently decreasing at the same time as HCO3- concentration increased. These concentration changes can probably be attributed to ductal transport. When amiloride was applied to glands perfused with nominally HCO3- -free solutions, inhibition of flow rate was rapid and almost complete. 4. When amiloride and bumetanide were both present in the perfusate, flow rate was inhibited by 92%. The pattern of electrolyte changes was not significantly different from that observed in the presence of bumetanide alone. 5. Inhibition of K+ channel activity using Ba2+ also inhibited flow rate. Cl- concentration was increased as was K+ concentration. HCO3- concentration was not increased. 6. The anion exchange inhibitor DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) had no effect on either flow rate or electrolyte concentration. It did, however, elicit secretion in the absence of acetylcholine. 7. The data suggest that Na(+)-H+ and Cl- -HCO3- exchangers are unlikely to be involved in fluid and electrolyte secretion in these glands as suggested by some authors. Most of the data can be explained by postulating the existence of non-specific anion channels in the apical membranes of the acinar cells.

Indirect evidence for the presence of non-specific anion channels in rabbit mandibular salivary gland acinar cells.

1. Intracellular pH (pHi) was measured using the fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein in acini isolated from the rabbit mandibular salivary gland. 2. Stimulation of the acinar cells with acetylcholine (ACh) evoked an intracellular acidosis, the size of which was dependent on the HCO3-concentration in the bathing medium. A half-maximal acidosis was observed at approximately 10 mM-HCO3-. ACh also evoked an acidosis in HCO3(-)-free solutions containing acetate; a half-maximal acidosis was observed at about 10 mM-acetate. 3. Propionate, lactate and butyrate were also able to support the ACh-evoked acidosis to varying extents. In contrast, formate, pyruvate and salicylate did not support the ACh-induced acidosis to any great extent. 4. Acetazolamide greatly reduced the size of the acidosis in HCO3(-)-buffered medium, but had no effect in acetate-buffered medium, suggesting that the inhibitory effect of acetazolamide was due to a specific inhibition of carbonic anhydrase activity. 5. The Cl- channel blockers diphenylamine-2-carboxylic acid (DPC, 1 mM) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (0.5 mM) abolished the ACh-evoked acidosis in both HCO3(-) -and acetate-buffered media. 6. The data are consistent with the presence in the acinar cell of relatively non-specific anion channels sensitive to DPC and its derivatives. Such channels, activated on stimulation with ACh, would allow HCO3- and other weak acid ions to leave the cell, leading to the observed acidosis. The existence of such channels, located in the apical membrane, could explain why HCO3- or acetate can sustain fluid secretion in the intact perfused rabbit mandibular gland in the absence of Cl-.

Acetylcholine-induced intracellular acidosis in rabbit salivary gland acinar cells.

Intracellular pH (pHi) was measured in acini isolated from rabbit mandibular salivary glands using the fluorescent pH-sensitive probe 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Resting pHi was estimated to be 7.13 +/- 0.01 (mean +/- SE of 29 experiments). Stimulation with acetylcholine (ACh) caused an intracellular acidosis followed by a return of pHi toward the control value with a half time of approximately 3 min. The intracellular acidosis was dose dependent and could be abolished by pretreatment of the acini with atropine (10 microM), suggesting that it was due to a receptor-mediated event. Incubation of the acini in HCO3- -free solutions or treatment of the acini with the carbonic anhydrase inhibitor acetazolamide (1 mM) abolished the acidosis, suggesting that the acidosis might be caused by loss of HCO3- from the cell. The acidosis was not affected by either 1) pretreatment of the acini with the anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), or 2) equilibration of the acini in Cl- -free solution (Cl- substituted with glucuronate). These results suggest that the postulated HCO3- efflux does not occur by Cl- -HCO3- exchange. However, Cl- -HCO3- exchange did appear to be present because replacement of Cl- caused a large DIDS-sensitive alkalinization of pHi, presumably caused by HCO3- uptake in exchange for Cl-. The recovery of pHi after the initial acidosis on stimulation with ACh could be blocked by 1 mM amiloride, suggesting that the recovery phase was mediated by Na+-H+ exchange.

Evidence for apical chloride channels in rabbit mandibular salivary glands. A chloride-selective microelectrode study.

Double-barrelled, chloride-selective microelectrodes were used to study mandibular gland acinar cells at rest and during cholinergic stimulation. At rest, intracellular chloride activity was five times the expected equilibrium activity. During sustained stimulation with acetylcholine, chloride activity fell to three times the expected equilibrium activity. Thus, the gradient for chloride exit was reduced in the stimulated cell. These results lead to the conclusion that stimulation increases the permeability of the acinar cell to chloride. Experiments in which extracellular chloride was removed provided evidence that the permeability increase was due to opening of chloride channels located principally in the apical membrane of the acinar cell.

Effect of hypertonicity on the increase in basolateral conductance of Necturus small intestine in response to Na+-sugar cotransport.

Exposure of Necturus small intestine to a galactose-containing perfusate that is 20% hypertonic compared to the galactose-free (control) perfusate results in a rapid depolarization of the electrical potential difference across the apical membrane, psi mc, and a decrease in the ratio of the resistance of the apical membrane to that of the basolateral membrane, (rm/rs); however, the slow repolarization of psi mc and increase in (rm/rs), observed under isotonic conditions, is blocked. These findings are consistent with the notion that the increase in the conductance of the basolateral membrane in response to Na+-coupled sugar (or amino acid) transport across the apical membrane may be a 'volume regulatory response' to cell swelling.

Seasonal variations in the fine structure of the Necturus maculosus urinary bladder epithelium: low transporters and high transporters.

Although the urinary bladder of Necturus maculosus provides an important model system for studying the mechanisms of active Na absorption, little critical attention has been paid to the fine structure of its epithelium. Moreover, two distinct groups of urinary bladders, low and high Na transporters, have been described based on short-circuit current or transepithelial potential difference. In the present study, over an 11-month period, stable electrical parameters (short-circuit current, transepithelial potential difference, and resistance) were recorded from 63 chamber-mounted bladders. Analysis of these parameters revealed a highly significant difference between two groups (low transporters and high transporters) occurring at different times of the year. Consistent with these data, in urine collected from the bladders, the Na concentration in low transporters was significantly higher than that in high transporters. A subpopulation of these bladders was subsequently fixed and examined at the light and/or electron microscopic level. Low-transporting bladders were characterized unequivocally by a thin, stratified squamous epithelium only 6-15 micron thick. High-transporting bladders were composed predominantly of columnar-shaped granular cells up to 70 micron in height, with ciliated, mitochondria-rich, and basal cells present in small numbers. There is thus a correlation between transport activity, as measured by electrophysiological techniques and urine sodium analysis, and the structure of the tissue. Moreover, these parameters exhibit significant seasonal variation, the underlying mechanisms of which remain obscure.

Cell swelling increases a barium-inhibitable potassium conductance in the basolateral membrane of Necturus small intestine.

Previous studies have shown that, immediately after the addition of galactose or alanine to the solution bathing the mucosal surface of Necturus small intestine, there is a rapid depolarization of the electrical potential difference across the mucosal membrane (psi mc). This is followed by a repolarization of psi mc that is paralleled by an increase in the ratio of the effective resistance of the mucosal membrane to that of the basolateral membrane (rm/rs); the latter was shown to be, at least in part, due to a marked increase in the conductance of the basolateral membrane. We now report the following. (i) Exposure of this epithelium to a 12% hypotonic solution results in a hyperpolarization of psi mc and an increase in rm/rs. These effects are blocked by metabolic inhibitors and by the presence of 5 mM Ba2+ in the bathing solution; indeed, in the presence of Ba2+, psi mc depolarizes and rm/rs decreases to low values. (ii) Addition of 15 mM galactose to the mucosal solution when the serosal solution alone contains 5 mM Ba2+ results in a depolarization of psi mc but no subsequent repolarization of psi mc or increase in rm/rs; however, psi mc repolarizes and rm/rs increases when Ba2+ is subsequently removed from the serosal bathing solution. We conclude that (i) the basolateral membrane normally possesses a Ba2+-inhibitable K conductance, which appears to be reduced in the presence of metabolic inhibitors; (ii) after exposure of the tissue to a hypotonic solution or the addition of galactose to the mucosal solution, this conductance increases; and (iii) these responses can be blocked by metabolic inhibitors. These findings suggest that the delayed response of this tissue to the addition of sugars or amino acids to the mucosal solution may be the result of cell swelling resulting from the intracellular accumulation of these solutes in osmotically active forms.

Thiocyanate transport across fish intestine (Pleuronectes platessa).

When bathed on both sides with identical chloride-containing salines the in vitro preparation of the plaice intestine maintains a negative (serosa to mucosa) short-circuit current of 107 +/- 11 muA/cm2, a transepithelial potential difference of 5.5 +/- 0.6 mV (serosa negative), and a mean mucosal membrane potential of -45.4 +/- 0.6 mV. Under these conditions the intracellular chloride activity is 32 mM. If chloride in the bathing media is partially, or completely substituted by thiocyanate the measured electrical parameters do not change but transepithelial flux determinations show a reduction in chloride fluxes and the presence of a significant thiocyanate flux. The addition of piretanide (10-4M) reduced the short-circuit current and the mucosa-to-serosa fluxes of chloride and thiocyanate; this inhibition is similar to the effect of piretanide on chloride transport in this tissue. The results indicate that thiocyanate is transported in this tissue via the piretanide-sensitive "chloride" pathway and are compared with the effects of thiocyanate on other tissues reported in the literature.