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There is emerging consensus that P2X₄ and P2X₇ ionotropic purinoceptors (P2X₄R and P2X₇R) are critical players in regulating [Ca²⁺]i dynamics and fluid secretion in the salivary gland. In contrast, details regarding their compartmentalization and selective activation, contributions to the spatiotemporal properties of intracellular signals and roles in regulating protein exocytosis and ion channel activity have remained largely undefined. To address these concerns, we profiled mouse parotid acinar cells using live-cell imaging to follow the spatial and temporal features of ATP-evoked Ca²⁺ dynamics and exocytotic activity. Selective activation of P2X7Rs revealed an apical-to-basal [Ca²⁺]i signal that initiated at the sub-luminal border and propagated with a wave speed estimated at 17.3 ± 4.3 μm s⁻¹ (n =6). The evoked Ca²⁺ spike consisted of Ca²⁺ influx and Ca²⁺-induced Ca²⁺ release from intracellular Ca²⁺ channels. In contrast, selective activation of P2X₄Rs induced a Ca²⁺ signal that initiated basally and propagated toward the lumen with a wave speed of 4.3 ± 0.2 μm s⁻¹ (n =8) that was largely independent of intracellular Ca²⁺ channel blockade. Consistent with these observations, P2X₇R expression was enriched in the sub-luminal regions of acinar cells while P2X₄R appeared localized to basal areas. In addition, we showed that P2X₄R and P2X₇R activation evokes exocytosis in parotid acinar cells. Our studies also demonstrate that the P2X₄R-mediated [Ca²⁺]i rise and subsequent protein exocytosis was enhanced by ivermectin (IVR). Thus, in addition to furthering our understanding of salivary gland physiology, this study identifies P2X₄R as a potential target for treatment of salivary hypofunction diseases.

It has been shown that the voltage (V(m)) dependence of ClC Cl(-) channels is conferred by interaction of the protopore gate with H(+) ions. However, in this paper we present evidence which indicates that permeant Cl(-) ions contribute to V(m)-dependent gating of the broadly distributed ClC-2 Cl() channel. The apparent open probability (P(A)) of ClC-2 was enhanced either by changing the [Cl(-)](i) from 10 to 200 mM or by keeping the [Cl(-)](i) low (10 mM) and then raising [Cl(-)](o) from 10 to 140 mM. Additionally, these changes in [Cl(-)] slowed down channel closing at positive V(m) suggesting that high [Cl(-)] increased pore occupancy thus hindering closing of the protopore gate. The identity of the permeant anion was also important since the P(A)(V(m)) curves were nearly identical with Cl(-) or Br(-) but shifted to negative voltages in the presence of SCN() ions. In addition, gating, closing rate and reversal potential displayed anomalous mole fraction behaviour in a SCN(-)/Cl() mixture in agreement with the idea that pore occupancy by different permeant anions modifies the V(m) dependence ClC-2 gating. Based on the ec1-ClC anion pathway, we hypothesized that opening of the protopore gate is facilitated when Cl(-) ions dwell in the central binding site. In contrast, when Cl(-) ions dwell in the external binding site they prevent the gate from closing. Finally, this Cl(-)-dependent gating in ClC-2 channels is of physiological relevance since an increase in [Cl(-)](o) enhances channel opening when the [Cl(-)](i) is in the physiological range.

Mouse parotid acinar cells express P2X4 and P2X7 receptors (mP2X4R and mP2X7R) whose physiological function remains undetermined. Here we show that mP2X4R expressed in HEK-293 cells do not allow the passage of tetraethylammonium (TEA+) and promote little, if any, ethidium bromide (EtBr) uptake when stimulated with ATP or BzATP. In contrast, mP2X7R generates slowly decaying TEA+ current, sustained Na+ current and promotes robust EtBr uptake. However, ATP-activated TEA+ current from acinar cells was unlike that generated by mP2X7R or mP2X4R. Functional interactions between mP2X4R and mP2X7R were investigated in HEK cells co-transfected with different mP2X4 : mP2X7 cDNA ratios and using solutions containing either TEA+ or Na+ ions. Co-expressed channels generated a TEA+ current that displayed faster decay during ATP stimulation than mP2X7R alone. Moreover, cells transfected with a 2 : 1 cDNA ratio displayed decaying kinetics similar to those observed in acinar cells. Concentration-response curves in Na+-containing solutions were constructed for heterologously expressed mP2X4R, mP2X7R and mP2X4R:mP2X7R co-expressions as well as acinar cells. The EC50 values determined were 11, 220, 434 and 442 microM, respectively. Na+ currents generated by expressing mP2X4R or mP2X7R alone were potentiated by ivermectin (IVM). In contrast, IVM potentiation in acinar cells and HEK cells co-expressing P2X4 and P2X7 (1 : 1 or 2 : 1 cDNA ratios) was seen only when the ATP concentration was lowered from 5 to 0.03 mM. Taken together our observations indicate a functional interaction between murine P2X7 and P2X4 receptors. Such interaction might occur in acinar cells to shape the response to extracellular ATP in salivary epithelia.

Using patch-clamp and molecular biological techniques, we identified and characterized membrane currents most likely generated by an electrogenic Na+-HCO3- cotransporter (NBCe) in acutely dissociated bovine parotid acinar (BPA) cells. When BPA cells were dialysed with a N-methyl-D-glucamine (NMDG)-glutamate-rich pipette solution, switching a Na-glutamate-rich, nominally HCO3--free bath solution to the one containing 25 mM HCO3-, but not Cl-, elicited a whole-cell current with a linear current-voltage relation. The HCO3- evoked current was abolished by total replacement of extracellular Na+ (Na+o) with NMDG or by 0.5 mM 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS), and was only partially supported by Li+o, but not by K+o, Cs+o, and cholineo. The reversal potential shift of DIDS (0.5 mM)-sensitive current induced by a change of [Na+]o corresponded to an apparent coupling ratio of HCO3- to Na+ of 2. RT-PCR analysis showed the presence of transcripts of NBCe1-B, but not NBCe1-A in BPA cells. Electrophysiological and pharmacological properties of whole-cell currents recorded from HEK293 cells transfected with the NBCe1-B, which was cloned from BPA cells resembled those of the native currents. Non-invasive measurements of membrane potential changes in the cell-attached patch configuration indicated that an NBCe activity is present in intact unstimulated BPA cells bathed in a 25 mM HCO3--containing solution. Collectively, these results not only suggest that an NBCe is present, functional and may be mediated, at least in part, by NBCe1-B in BPA cells, but also provide the first electrophysiological characterization of transport properties of NBCe expressed in native exocrine glands.

Using electrophysiological and molecular techniques, we investigated the molecular nature of an inwardly rectifying K+ channel in bovine parotid acinar (BPA) cells and examined its role in setting resting membrane potential. In whole-cell recordings from freshly isolated BPA cells, a predominant current was a K+ current rectified strongly in the inward direction. An inward conductance of the inwardly rectifying K+ (Kir) current was proportional to [K+]o(0.57). The selectivity sequence based on permeability ratios was K+ (1.00) > Rb+ (0.63) >> Li+ (0.04) = Na+ (0.02) and the sequence based on conductance ratios was K+ (1.00) >> Rb+ (0.03) = Li+ (0.03) = Na+ (0.02). The current was blocked by extracellular Ba2+ and Cs+ in a voltage- and a concentration-dependent manner, with a Kd at 0 mV of 11.6 microM and 121 mM, respectively. Cell-attached patch measurements identified 27 pS K+ channels as being the most likely to mediate whole-cell Kir currents. Addition of Ba2+ (100 microM) to the bathing solution reversibly depolarized the resting membrane potential in intact unstimulated cells. RT-PCR of RNA from bovine parotid cells revealed transcripts of bovine Kir2.1 (bKir2.1). HEK293 cells stably expressing bKir2.1 cloned from bovine parotid exhibited whole-cell and single channel Kir currents, of which electrophysiological characteristics were quantitatively similar to those of native Kir currents. Immunohistochemical studies showed a bKir2.1 immunoreactivity in BPA cells. Collectively, these results suggest that Kir2.1 may mediate native Kir currents responsible for setting resting membrane potential in BPA cells and might be, at least in part, involved in spontaneous secretion in ruminant parotid glands.

1. Isolated segments of mouse parotid gland were superfused with a physiological saline solution. Membrane potential and input resistance were measured with intracellular micro-electrodes. Amylase secretion was monitored using an automated fluorometric assay. The parotid gland was stimulated by exposure to isoprenaline (10(-6)M), ACh (10(-6) M), dibutyryl or monobutyryl cyclic AMP (10(-3)M). 2. ACh evoked a sharp decrease in input resistance (from about 3-6 M omega to 1-2 M omega ) accompanied by a modest (two-fold) increase in amylase output. Both effects were fully reversible. 3. Isoprenaline evoked a marked increase in amylase output (five to ten-fold) reaching maximum after 20-30 min of stimulation. Throughout the period of intensive secretion the input resistance remained at the control level. Short pulses of ACh stimulation dramatically reduced input resistance in this period. 4. Dibutyryl cyclic AMP and monobutyryl cyclic AMP caused marked increases in amylase output (five to ten-fold) peaking 40-50 min after start of the continuous stimulation. This increase in amylase secretion was not accompanied by any change in input resistance. Short pulses of ACh stimulation sharply and reversibly reduced input resistance both in control and test periods. 5. It is concluded that the ACh-evoked reduction in input resistance is not a consequence of the secretory event and that secretion does not itself cause a decrease in input resistance.

1. We previously reported that parotid gland secretion is decreased in rats deprived of vitamin D (Glijer, Peterfy & Tenenhouse, 1985). In the present study we examine whether this effect is a direct result of the absence of vitamin D or due to the secondary systemic effects of vitamin D deficiency. 2. Offspring of rats maintained on a calcium-supplemented (1.2%), vitamin-D-deficient diet were weaned onto the same diet and examined after 8 weeks. Using this method it was possible to maintain serum calcium and parathyroid hormone concentrations within normal limits. Serum 25-hydroxyvitamin D (25(OH)D3) was not detectable, but 1,25-dihydroxyvitamin D (1,25(OH)2D3) concentrations were normal. 3. Pilocarpine-stimulated flow of parotid saliva was reduced 57% in vitamin-D-deprived animals, but amylase secretion was unchanged. Treatment with vitamin D3 returned flow rates to normal. 4. The concentration of calcium in parotid saliva was normal in vitamin-D-deprived rats, although total parotid calcium output was reduced 57%. 5. Pilocarpine-stimulated salivary flow from submandibular gland, a tissue which does not possess 1,25(OH)2D3 receptors, was normal in vitamin-D-deprived rats. 6. Heart rate and arterial blood pressure changes in response to I.V. pilocarpine administration were identical in normal and vitamin-D-deficient rats. 7. Auriculotemporal nerve-stimulated flow of parotid saliva was also reduced by 50% and administration of vitamin D3 to these rats corrected this abnormality. 8. It is concluded that fluid and electrolyte secretion from parotid gland is directly dependent on vitamin D; abnormal parotid gland function seen in vitamin-D-deficient rats is not due to secondary hypocalcaemia or hyperparathyroidism, nor can it be explained by haemodynamic changes evoked during systemic administration of pilocarpine. We further conclude that the metabolite of vitamin D responsible for this effect is not 1,25(OH)2D3.

1. Intracellular micro-electrode recordings of membrane potential and input resistance were made from surface acini of mouse parotid glands placed in a Perspex tissue bath through which oxygenated physiological saline solutions were circulated. The acinar cells were stimulated by microionophoresis of both acetylcholine (ACh) and adrenaline (Ad) from extracellular micropipettes, and by electrical field stimulation via a pair of platinum electrodes. 2. The acinar cells had a mean resting membrane potential of -64.9 mV +/- 0.6 S.E. The input resistance of the unstimulated cell was 4.63 M omega +/- 0.19 S.E. In a number of cells spontaneous miniature depolarizations were observed, associated with synchronous reductions in input resistance. 3. The responses to ionophoresis of both ACh and Ad and the response to supra-maximal field stimulation were identical. Stimulation always evoked a marked decrease in input resistance associated with an initial potential change, generally followed by a delayed hyperpolarization during which the input resistance returned to normal. 4. Field-stimulation responses could be evoked to single shock (1-2 msec) and to low frequency (1-4 Hz) stimulation. The latency for this response was 245 msec +/- 12 S.E. 5. The field-stimulation response was shown to be susceptible to blockade of nerve conduction in sodium-free or tetrodotoxin- (TTX-) containing media; and to blockade of neurotransmitter release in calcium-free media. 6. The field-stimulation and ACh responses were recorded at different levels of membrane potential within the same cells by applying either hyperpolarizing or depolarizing direct current through the recording electrode. The membrane potential at which the initial potential change undergoes reversal, i.e. changes from a depolarization to a hyperpolarization, is known as the equilibrium or reversal potential, EFS and EACh respectively. The field-stimulation (FS) and ACh responses underwent simultaneous reversal at about -60 mV, i.e. EFS = EACh. Equilibrium potentials were also determined indirectly by analysis of the responses evoked by each stimulus in the manner described by Trautwein & Dudel (1958). Using this technique the equilibrium potentials of the responses to all three stimuli, field stimulation, ACh and Ad, were again about -60mV, i.e. EFS = EACh = EAd. 7. Both the field-stimulation and ACh responses were abolished by atrophine (10(-6) M) while the response to Ad persisted. Atropine also abolished all spontaneous activity. The alpha-adrenergic blocker phentolamine (10(-5) M) abolished the response to Ad but left the field-stimulation response unaffected. 8. Electrical field stimulation of isolated segments of salivary gland evoked release of endogenous neurotransmitter as a consequence of neural excitation. The technique of field stimulation thus makes it possible to investigate the functional innervation of a gland using the in vitro preparation. In the mouse parotid gland the field stimulus response was mediated by ACh released from parasympathetic nerve endings.

1. Adjacent acinar cells in isolated rat parotid and submaxillary glands were found to be electrically coupled in greater than 90% of the pairs tested. 2. Cells injected with fluorescein or procion yellow showed transfer of the dyes to their coupled neighbours. While not all coupled cells exchanged dye, exchange occurred only between coupled cells. 3. In experiments using three micro-electrodes, coupled acinar cells from parotid gland were found to have a mean coupling coefficient of 0.69 +/- 0.04. This value is higher than those reported for most other vertebrate epithelial systems. 4. Membrane damage sufficient to reduce the occurrence of coupling between cells by 97% lowered the transmembrane potential by only 13%. This would indicate that in this system membrane potential may not be the most sensitive indicator of cell damage. 5. The significance of the presence of electrical coupling and cell-to-cell transfer of small tracer molecules is discussed in relation to salivary gland structure and possible functional consequences.

1. The effects of stimulating the postganglionic parasympathetic nerve to the parotid gland, the auriculo-temporal nerve, continuously at 40 Hz for 80 min have been assessed on the flow of saliva and its amylase content during each 10 min period and on the glandular morphology at the end of the stimulation, in female Sprague-Dawley rats under chloralose anaesthesia. Adrenergic responses were blocked by prior administration of dihydroergotamine (1 mg/kg I.P.) and propranolol (1 mg/kg I.P.). The contralateral unstimulated parotid gland from each animal was used for morphological control purposes. 2. In normal animals there was a copious flow of saliva reaching 3 ml or more in 80 min. It was well maintained after a gradual small decline and the flow in the last 10 min period was still 60% of that in the initial period. Amylase output was relatively high in the initial period but then declined more rapidly than the flow. The acini were densely packed with granules on the control side and showed a moderate amount of degranulation on the parasympathetically stimulated side. 3. After atropine (2 mg/kg I.V.), normal animals initially showed a moderate flow of saliva, being 41% of the non-atropinized animals, but there was a rapid decline and the total flow over 80 min was only 15% of that in the non-atropinized animals. The amylase secreted was also high initially and showed a rapid decline; the total amount secreted was similar to that in non-atropinized animals. The morphology was similar to that in non-atropinized animals and showed a similar loss of acinar granules on the stimulated side. 4. Chronic bilateral postganglionic sympathectomy (4-6 weeks duration) caused a drastic reduction in the capacity of the gland to secrete saliva in response to parasympathetic stimulation, reaching only one-third of that from normal animals. The initial output of amylase was greater than in normal animals but the total output was similar. The control unstimulated sympathectomized glands appeared similar morphologically to normal resting glands. However, on the parasympathetically stimulated side, besides the usual amount of acinar degranulation, there was also a conspicuous development of acinar vacuolation, not seen in the other groups of animals.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Rat parotid gland slices, incubated in a balanced, buffered salt solution, were found to be physiologically stable for up to 2 hr with respect to O2 consumption, water content, extracellular space and cation content. 2. The slices could be stimulated to secrete amylase by activation of alpha-adrenergic, beta-adrenergic or muscarinic cholinergic receptors. 3. The secretion elicited through all three receptors appeared to involve exocytosis as revealed by electron microscopy. 4. The beta-agonist, isoprenaline, increased tissue content of cyclic adenosine 3',5'-monophosphate (cyclic AMP); alpha-adrenergic and cholinergic agents had no effect on the level of this cyclic nucleotide. 5. Secretion via cholinergic or alpha-adrenergic mechanisms required extra-cellular calcium; the beta-adrenergic mechanism did not. 6. It was concluded that stimulation of rat parotid cells activates distinctly separate pathways leading ultimately to exocytosis, one pathway involving cyclic AMP, and the other, external Ca2+ ion.

1. In cats under chloralose anaesthesia micro-electrodes were inserted into parotid gland cells. 2. The average resting potential was found to be -35 . 6 +/- 4 . 7 (S.D.) mV. 3. Stimulation of the auriculo-temporal nerve caused hyperpolarizing, or occasionally depolarizing, secretory potentials of 5--10 mV, which were abolishable with atropine. 4. Stimulation of the cervical sympathetic trunk regularly caused, after long latency (several seconds), slow depolarizations of 15--20 mV, accompanied by a decrease in input resistance. They were antagonized by practolol and therefore assumed to be mediated by beta 1-adrenoceptors. Occasionally hyperpolarization, ascribed to an effect on alpha-adrenoceptors, was observed. 5. In many units the slow depolarization on sympathetic stimulation was preceded by short-lasting hyperpolarizing (sometimes depolarizing) transients. They resembled the evoked cholinergic responses but could be abolished not only by atropine but also by guanethidine.

The technique of electrical field stimulation (e.f.s.) was employed in conjunction with selective pharmacological antagonists to specifically investigate the role of endogenous neurotransmitter(s) in the activation of beta-adrenergic receptor mechanisms in isolated parotid gland segments of the rat. The field-stimulus-induced amylase release due to beta-adrenergic receptor activation was characterized as that persisting in the presence of atropine (10(-5) M) and phentolamine (10(-5) M) and susceptible to blockade by propranolol (5 X 10(-6) M), i.e. combined beta 1- and beta 2-receptor blockade. The selective beta 1-receptor antagonist metoprolol (10(-5) M) was as effective as propranolol in blocking the beta-mediated enzyme release. The selective beta 2-receptor antagonist, H35/25 (10(-5) M) did not significantly affect the field-stimulus-induced amylase release. In the absence of any phosphodiesterase inhibitor the levels of cyclic AMP in the tissues were close to the limit of detection. Field stimulation was however associated with a fourfold increase in cyclic AMP. By comparison isoprenaline (10(-5) M) gave rise to a tenfold increase in cyclic AMP. The changes in cyclic AMP metabolism, in response to both field stimulation and isoprenaline, were greatly enhanced in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). The field-stimulus-induced increase in cyclic AMP was abolished by the beta 1-adrenergic receptor antagonist, metoprolol, but persisted in the presence of the beta 2-adrenergic antagonist, H35/25. IBMX was found to have a potent direct effect on amylase release. IBMX (10(-3) M) also gave rise to a tenfold increase in cyclic AMP. IBMX is then as effective as 10(-5) M-isoprenaline in stimulating both enzyme secretion and cyclic AMP metabolism. The secretory response to IBMX was unaffected by beta-adrenergic blockade by propranolol, was independent of extracellular calcium and did not give rise to 86Rb+ efflux. Importantly, isoprenaline (10(-5) M) failed to evoke any significant increase in amylase release if introduced during sustained superfusion of IBMX, yet it is in such protocols that the greatest changes in cyclic AMP metabolism are seen. The study clearly demonstrates that the beta-adrenergic-receptor-regulated amylase release in response to nerve stimulation is mediated predominantly, if not exclusively, by the beta 1-receptor subtype.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The pressures in the ducts of the parotid and submaxillary glands were recorded in dogs under chloralose-urethane anaesthesia. A single stimulus applied to the parasympathetic nerve regularly caused a pressure rise of short duration. Usually it did not evoke secretion.2. In both glands a single stimulus applied to the vagosympathetic trunk usually caused a pressure response of short duration. It had no secretory effect.3. In the parotid gland, repetitive vagosympathetic stimulation caused a sustained increase of pressure in the duct, usually without inducing secretion.4. In the submaxillary gland, repetitive vagosympathetic stimulation caused a sustained pressure rise, even when the secretory response had been abolished by propranolol.5. It is concluded that the salivary myoepithelial cells of dogs are supplied with fibres from both sympathetic and parasympathetic divisions of the autonomic nervous system and both can cause the cells to contract.

1. Intracellular recording of membrane potential was made from acinar cells of the isolated mouse parotid gland superfused with physiological salt solutions.2. The mean acinar resting membrane potential was - 68.5 mV during superfusion with Krebs-Henseleit solution. Shift of the superfusion solution to one containing ACh or adrenaline (10(-5)M) always caused a transient hyperpolarization (about 10-15 mV).3. The membrane potential was mainly dependent on the extracellular K concentration ([K](o)). Increasing [K](o) tenfold decreased the membrane potential by 50 mV. This depolarization was not mediated by ACh release from depolarized nerve endings, since it was seen in the presence of atropine (1.4 x 10(-6)M) and not caused by the accompanying reduction in [Na](o) to 40 mM caused only a small depolarization (less than 10 mV).4. When the superfusion solution was shifted, during intracellular recording, from a normal Krebs-Henseleit solution ([K] = 4.7 mM) to a K-free solution, a hyperpolarization of about 8 mV was measured. Reintroduction of the normal K-containing solution after a longer period of K deprivation (30-70 min) resulted in a short-lasting pronounced hyperpolarization (about 20 mV) which could be blocked by Strophanthin-G (10(-3)M).5. In contrast to previous reports, the present findings indicate that the membrane potential of salivary acinar cells is similar, with respect to magnitude and K-dependence, to that of cells of more thoroughly investigated tissues, such as muscle and nerve, and that the membrane Na-K pump is electrogenic, at least when the cells have been loaded with Na.

1. Saliva flowed from the horse's parotid duct only during mastication.2. The surface-active local anaesthetic administered by mouth inhibited salivary secretion.3. Salivary secretion was stimulated by pilocarpine and inhibited by atropine.4. The volume and composition of saliva secreted in 24 hr from one parotid duct was determined.5. The concentration of sodium, potassium, calcium, chloride and bicarbonate depended upon the rate of flow. The highest concentrations of these electrolytes were observed during periods of high flow rates.6. Horse parotid saliva contained a high concentration of calcium.7. In the absence of a dietary supplement of sodium bicarbonate, the sodium concentration of the saliva fell after about 21 days. There was an associated increase in the potassium concentration. The addition of a sodium supplement restored the sodium concentration of the saliva within 24 hr.

1. The composition of human saliva secreted in response to sour lemon drops (S.L.D.), and pilocarpine, was studied.2. At a given flow rate, pilocarpine-stimulated submandibular and parotid saliva contained less sodium and potassium and an equivalent amount of inorganic phosphate, and parotid saliva also contained more calcium and protein than did the corresponding types of S.L.D.-stimulated saliva.3. Prolonged S.L.D. stimulation did not cause a depletion in the protein concentration of either parotid or submandibular saliva and neither this procedure nor pilocarpine stimulation altered the proportions of the different proteins secreted.4. Pilocarpine was judged to be an inadequate substitute for more physiological, gustatory stimuli.