Effects of extracellular ATP on ion transport systems and [Ca2+]i in rat parotid acinar cells. Comparison with the muscarinic agonist carbachol.

The effects of extracellular ATP on ion fluxes and the intracellular free Ca2+ concentration ([Ca2+]i) were examined using a suspension of rat parotid acinar cells and were contrasted with the effects of the muscarinic agonist carbachol. Although ATP and carbachol both rapidly increased [Ca2+]i about threefold above the resting level (200-250 nM), the effect of ATP was due primarily to an influx of Ca2+ across the plasma membrane, while the initial response to carbachol was due to a release of Ca2+ from intracellular stores. Within 10 s, ATP (1 mM) and carbachol (20 microM) reduced the cellular Cl- content by 39-50% and cell volume by 15-25%. Both stimuli reduced the cytosolic K+ content by 57-65%, but there were marked differences in the rate and pattern of net K+ movement as well as the effects of K+ channel inhibitors on the effluxes initiated by the two stimuli. The maximum rate of the ATP-stimulated K+ efflux (approximately 2,200 nmol K+/mg protein per min) was about two-thirds that of the carbachol-initiated efflux rate, and was reduced by approximately 30% (vs. 60% for the carbachol-stimulated K+ efflux) by TEA (tetraethylammonium), an inhibitor of the large conductance (BK) K+ channel. Charybdotoxin, another K+ channel blocker, was markedly more effective than TEA on the effects of both agonists, and reduced the rate of K+ efflux initiated by both ATP and carbachol by approximately 80%. The removal of extracellular Ca2+ reduced the ATP- and the carbachol-stimulated rates of K+ efflux by 55 and 17%, respectively. The rate of K+ efflux initiated by either agonist was reduced by 78-95% in cells that were loaded with BAPTA to slow the elevation of [Ca2+]i. These results indicated that ATP and carbachol stimulated the efflux of K+ through multiple types of K(+)-permeable channels, and demonstrated that the relative proportion of efflux through the different pathways was different for the two stimuli. ATP and carbachol also stimulated the rapid entry of Na+ into the parotid cell, and elevated the intracellular Na+ content to 4.4 and 2.6 times the normal level, respectively. The rate of Na+ entry through Na(+)-K(+)-2Cl- cotransport and Na(+)-H+ exchange was similar whether stimulated by ATP, carbachol, or ionomycin, and uptake through these two carrier-mediated transporters accounted for 50% of the ATP-promoted Na+ influx. The remainder may be due to a nonselective cation channel and an ATP-gated cation channel that is also permeable to Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of muscarinic, alpha-adrenergic, and substance P agonists and ionomycin on ion transport mechanisms in the rat parotid acinar cell. The dependence of ion transport on intracellular calcium.

The relationship between receptor-mediated increases in the intracellular free calcium concentration [( Ca]i) and the stimulation of ion fluxes involved in fluid secretion was examined in the rat parotid acinar cell. Agonist-induced increases in [Ca]i caused the rapid net loss of up to 50-60% of the total content of intracellular chloride (Cli) and potassium (Ki), which is consistent with the activation of calcium-sensitive chloride and potassium channels. These ion movements were accompanied by a 25% reduction in the intracellular volume. The relative magnitudes of the losses of Ki and the net potassium fluxes promoted by carbachol (a muscarinic agonist), phenylephrine (an alpha-adrenergic agonist), and substance P were very similar to their characteristic effects on elevating [Ca]i. Carbachol stimulated the loss of Ki through multiple efflux pathways, including the large-conductance Ca-activated K channel. Carbachol and substance P increased the levels of intracellular sodium (Nai) to more than 2.5 times the normal level by stimulating the net uptake of sodium through multiple pathways; Na-K-2Cl cotransport accounted for greater than 50% of the influx, and approximately 20% was via Na-H exchange, which led to a net alkalinization of the cells. Ionomycin stimulated similar fluxes through these two pathways, but also promoted sodium influx through an additional pathway which was nearly equivalent in magnitude to the combined uptake through the other two pathways. The carbachol-induced increase in Nai and decrease in Ki stimulated the activity of the sodium pump, measured by the ouabain-sensitive rate of oxygen consumption, to nearly maximal levels. In the absence of extracellular calcium or in cells loaded with the calcium chelator BAPTA (bis[o-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid) the magnitudes of agonist- or ionomycin-stimulated ion fluxes were greatly reduced. The parotid cells displayed a marked desensitization to substance P; within 10 min the elevation of [Ca]i and alterations in Ki, Nai, and cell volume spontaneously returned to near baseline levels. In addition to quantitating the activation of various ion flux pathways in the rat parotid acinar cell, these results demonstrate that the activation of ion transport systems responsible for fluid secretion in this tissue is closely linked to the elevation of [Ca]i.

New structure, the "olfactory pit," in human olfactory mucosa.

A whole-mount immunocytochemical method was devised to study the olfactory receptor neurons on the surface of the human olfactory mucosal sheet. Antibodies to neuron-specific tubulin and/or microtubule-associated protein 5 and phosphorylated neurofilament protein were used. Specimens taken at autopsy from 56 patients ranging in age from 2 days to 92 years revealed a structure not previously described, an olfactory pit. Round or oval openings with a diameter of 50 to 500 microns were observed on the surface of the olfactory epithelium in the whole-mount specimen. The morphology, number, and distribution of these openings varied among the different individuals. A detailed analysis of these structures was carried out by rehydrating and sectioning the whole-mount specimens. The olfactory pit (OP) is a blind pouch lined with olfactory epithelium (OE), which appears as an invagination of OE into the connective tissue, with a depth varying between 150 and 200 microns. In some sections through an OP, a thick axon bundle emerging from the bottom of the pouch was visible. The extension and termination of this axon bundle in the central nervous system has not been explored. We have found OPs in monkey olfactory mucosa, but none in rodents. The function of the pit specialization is unclear, but it appears to be a feature of normal, young epithelium. The configuration of the blind pouch may prolong odorant association with the olfactory receptor neurons, or the OP may contain specialized neurons that have not yet been recognized by morphological, biochemical, or functional techniques.

Two distinct cytosolic calcium responses to extracellular ATP in rat parotid acinar cells.

1. Increasing concentrations of ATP (0.5 microM-300 microM) produced a biphasic increase in intracellular calcium concentration [Ca]i in rat parotid acinar cells, reflecting two distinct Cai responses to extracellular ATP. 2. In the absence of Mg2+ (with 3 mM CaCl2 in the buffer solution), the more sensitive response was maximal at 3-5 microM and was not further increased by 30 microM ATP. This response to ATP was not well maintained and was blocked by ADP (0.5 mM). A second, much larger increase in Cai was observed on addition of 300 microM ATP. This larger effect, which we have described previously, appears to be mediated by ATP4-, and was selectively reversed by 4,4'-di-isothiocyanato-dihydrostilbene-2,2'-disulphonate as well as by high concentrations of alpha,beta-methylene ATP. 3. Among ATP analogues, only the putative P2Z agonist, 3'-0-(4-benzoyl)benzoyl-ATP distinguished between the two responses. This analogue was at least 10 fold more potent than ATP in stimulating the ATP(4-)-response, but did not evoke the more sensitive response. The agonist potency series for both responses to ATP was identical for other analogues examined (ATP > ATP gamma S = 2-methylthio ATP (a P2y-selective agonist) >> ADP, ITP and alpha,beta-methylene ATP (a P2x-selective agonist)). 4. Although the effect of ATP4- could best be characterized as a P2z-type purinoceptor response, this effect was strongly and selectively blocked by reactive blue 2, a putative P2y-purinoceptor antagonist. Reactive blue 2 may bind to and block P2z purinoceptors since [gamma 32P]-ATP binding to parotid cells was inhibited by this compound. 5. In contrast to the response to ATP4-, the more sensitive response to ATP was potentiated by 2+ reactive blue 2 and was less affected by increases in external Mg2+ and Ca2+.6. Parasympathetic denervation selectively increased the more sensitive response, suggesting that it maybe physiologically regulated.

Blockade of ATP binding site of P2 purinoceptors in rat parotid acinar cells by isothiocyanate compounds.

Extracellular ATP activates a P2Z-type purinergic receptor (purinoceptor) in rat parotid acinar cells that increases the intracellular free Ca2+ concentration via the entry of extracellular Ca2+ through an ATP-sensitive cation channel (Soltoff et al., Am J Physiol 262: C934-C940, 1992). To learn more about the ATP binding site of the purinoceptor, we examined the effects of several stilbene isothiocyanate analogs of DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), which block the binding of [32P]ATP to intact parotid cells (McMillian et al., Biochem J 255:291-300, 1988) and blocked the activation of the P2Z purinoceptor. The ATP-stimulated 45Ca2+ uptake was blocked by DIDS, H2DIDS (dihydro-DIDS; 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid), and SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid), but not by DNDS (4,4'-dinitrostilbene-2,2'-disulfonic acid), a stilbene disulfonate compound lacking isothiocyanate (SCN-) groups, or by KSCN. The potency of the stilbene disulfonates was related to the number of isothiocyanate groups on each compound. Under the experimental conditions, the IC50 value of DIDS (approximately 35 microM), which has two SCN-groups, was much lower than that of SITS (approximately 125 microM), which has only one SCN-group. The inhibitory effects of DIDS appeared to be much more potent than those of SITS due to the kinetics of their binding to the purinoceptors. Eosin-5-isothiocyanate (EITC) and fluorescein-5-isothiocyanate (FITC), non-stilbene isothiocyanate compounds with single SCN-groups, also blocked the response to ATP and were less potent than DIDS. Trinitrophenyl-ATP (TNP-ATP), an ATP derivative that is not an effective agonist of the parotid P2Z receptor, blocked the covalent binding of DIDS to the plasma membrane, suggesting that ATP and DIDS bind to the same site. Reactive Blue 2 (Cibacron Blue 3GA), an anthraquinone-sulfonic acid derivative that is a noncovalent purinergic antagonist, also blocked the covalent binding of DIDS to the plasma membrane. These results suggest that isothiocyanate compounds interact with the ATP binding site of this P2 purinoceptor, and that isothiocyanate groups make an important contribution in determining the effectiveness of the stilbene disulfonate compounds in blocking the binding of nucleotide agonists to this purinoceptor.

Elevation of [Ca2+]i and the activation of ion channels and fluxes by extracellular ATP and phospholipase C-linked agonists in rat parotid acinar cells.

Extracellular ATP initiates a variety of changes in the parotid acinar cell. The initial effect appears to be the entry of Ca2+ (and perhaps Na+), and a series of ion transport events result from the subsequent elevation of [Ca2+]i. Agonists of phospholipase C-linked receptors elevate [Ca2+]i by a different pathway, involving the generation of inositol polyphosphate compounds, but share in the subsequent initiation of the ion transport events. Although the maintenance of the physiological changes may depend on specific inositol polyphosphate intermediates, the critical initiating factor is the elevation of [Ca2+]i. Fluid secretion by the parotid gland is triggered by the action of neurotransmitters, which alter the membrane permeability of the acinar cell. The similarities between the two receptor-mediated activation pathways suggests that ATP may act as a neurotransmitter and play a role in the control of fluid secretion. Basing our analysis on the purinoceptor characteristics outlined by Gordon, we suggest that the parotid receptor belongs to the P2Z class, which is highly sensitive to ATP4-. Basing his analysis on the earlier report by Gallacher of the effects of ATP on mouse parotid cells, Gordon placed the parotid purinoceptor in a different P2 subclass (P2Y). However, our findings of an increased potency of ATP in the absence of Mg2+, as well as the potency order of different nucleotides, indicate that the P2Z class is a more appropriate category.

Pathologic changes in olfactory neurons in Alzheimer's disease.

Olfactory deficits and degenerative changes in central olfactory pathways are prominent in patients with Alzheimer's disease (AD). We hypothesized that peripheral olfactory neurons that reside in the nasal epithelium would show degenerative changes similar to the characteristic pathologic features of AD brain. Immunohistochemical studies of nasal tissue taken at autopsy reveal extensive degeneration in the sensory epithelium as well as abnormal neurites that share immunoreactive epitopes with dystrophic neurites and neurofibrillary tangles of the AD brain. The neuritic masses are stained with well-characterized monoclonal antibodies that do not normally stain olfactory neurons but which are very reactive with dystrophic neuritic structures and neurofibrillary tangles in AD brain. These include antibodies to phosphorylated and nonphosphorylated neurofilament subunits, tau, and also ALZ50, which is characteristically reactive with AD but not with normal brains. Such changes are present in 81% of AD patients. Similar accumulations of ectopic neurites are found in the olfactory epithelium of about 22% of non-demented patients. Preliminary statistical analysis fails to reveal any age-linked association. It has been proposed that the aged monkey is a good model for AD inasmuch as amyloid accumulations similar to those of humans are found in monkey brain. We examined a series of 13 rhesus monkeys, including aged animals with behavioral deficits. Although the olfactory epithelium was very similar to that of humans, no abnormal olfactory structures were observed. Aged rhesus monkeys do not appear to be a good model for the neuritic abnormalities of AD.

Parasympathetic denervation increases responses to VIP in isolated rat parotid acini.

Vasoactive intestinal peptide (VIP) is a putative neurotransmitter found in the salivary glands of many species, including the rat parotid gland. Parasympathetic denervation has been reported to deplete VIP in the rat parotid gland and to lead to supersensitivity to this peptide in vivo. We have compared the effects of VIP on acini isolated from parasympathetically denervated and unoperated parotid glands to examine possible supersensitivity to the peptide in vitro. VIP normally produced responses similar to those obtained with a low concentration of the beta adrenergic agonist isoproterenol (ISO), but strikingly different from the effects obtained with the muscarinic agonist carbachol (CARB). In parotid membrane preparations, VIP stimulated adenylate cyclase activity. Dissociated acini treated with VIP showed increases in cAMP accumulation and amylase release which were potentiated by forskolin and also by inhibition of phosphodiesterase. After parasympathetic denervation, maximal effects of VIP on adenylate cyclase, cAMP accumulation and amylase release in intact cells were increased two- to five-fold over contralateral control (or unoperated) parotid responses. The increase in adenylate cyclase-mediated responses after denervation was specific to VIP; there was no increased response nor increased sensitivity of any of these responses to ISO. Specific [125I]VIP binding to parotid acini increased two-fold per gland and three-fold per mg of protein after denervation; this probably explains the observed increases in the response to VIP.

Lack of desensitization to neurokinin A due to partial agonist property of this peptide at parotid substance P receptors.

Effects of substance P (SP) and neurokinin A (NKA) on intracellular free calcium concentration (Cai) were compared in Fura2-loaded parotid acinar cells. SP produced a pronounced increase in Cai which rapidly desensitized. In contrast, NKA produced a smaller increase which was well maintained. Cells desensitized to SP were cross-desensitized to NKA. The absence of NKA-induced desensitization reflects the inability of NKA to fully activate parotid SP receptors; NKA is a partial agonist in the parotid. Similarly, the action of NKA as a partial agonist in other systems may explain findings that SP responses desensitize while NKA responses do not.

Sphingosine, W-7, and trifluoperazine inhibit the elevation in cytosolic calcium induced by high K+ depolarization in synaptosomes.

A possible role for protein kinases in the regulation of free cytosolic Ca2+ levels in nerve endings was investigated by testing the effect of several kinase inhibitors on the increase in cytosolic Ca2+ (monitored with the Ca(2+)-sensitive dye fura-2) induced by depolarization with 15 or 30 mM K+. The ability of various drugs to inhibit the cytosolic Ca2+ response appeared to correlate with their reported mechanism of action in inhibiting protein kinases. W-7 and trifluoperazine, drugs reported to inhibit calmodulin-dependent events, were effective inhibitors of the increase in cytosolic Ca2+ induced by high K+ depolarization, as was sphingosine, a drug that inhibits protein kinase C by binding to the regulatory site, but which also inhibits calcium/calmodulin kinase. On the other hand, drugs that inhibit protein kinases by binding to the catalytic site, such as H-7 (1 mM), staurosporine (1 microM), and K252a (1 microM), were ineffective. Activation of protein kinase C, which is blocked by each of these drugs, does not appear to be essential to the maintenance of elevated cytosolic Ca2+ in depolarized synaptosomes. All of the drugs, including sphingosine, that functionally inhibit the depolarization-induced elevation in cytosolic Ca2+ have in common the ability to bind to calmodulin. Because the drugs that inhibit protein kinases by competing with ATP binding at the active catalytic site did not block the response in this system, we suggest that a calmodulin or a calmodulin-like binding site participates in the regulation of Ca2+ increases after depolarization.

Independent regulation of beta-adrenergic receptor and nucleotide binding proteins of adenylate cyclase. Developmental and denervation-dependent responses in rat parotid.

Rat parotid gland secretion cannot be activated through beta-adrenergic stimulation of adenylate cyclase until after 2 weeks postnatal. We have studied the relationship of the levels of putative guanine nucleotide-binding regulatory components (G/F) of the cyclase system to the onset of hormone responsiveness. The effect of sympathetic denervation on the components of this system during development of secretory function also has been examined. Nucleotide-dependent, hormone-stimulated, and fluoride-stimulated adenylate cyclase activities in parotid membranes are present at low levels at birth and increase 2-fold between 14 and 18 days postnatal while beta-adrenergic receptor levels remain constant. G/F proteins, regulatory for adenylate cyclase activation, were quantitated by ADP-ribosylation in the presence of cholera toxin. Labeling of two cholera toxin-specific substrates occurs at low levels in neonatal rats and increases sharply at the critical 14-18-day period. This provides a plausible explanation for the increase in adenylate cyclase sensitivity at this time, although increases in cyclase catalytic units and/or coupling efficiency of receptor and cyclase may also be involved. In previous studies we found that animals chemically sympathectomized with 6-hydroxydopamine at birth developed elevated levels of membrane-bound beta-adrenergic receptors. The functional consequence is that treated animals show a shift (1.7-fold) toward increased sensitivity in the dose-response curve for adenylate cyclase activation by isoproterenol. However, the levels of maximal hormone- and fluoride-stimulated adenylate cyclase activities do not change, suggesting that some component distal to the receptor is limiting under both control and treated conditions, or that there are deficiencies in coupling of the receptor pool.

Modulation of extracellular ATP-induced Ca2+ responses: role of protein kinases.

Evidence for the modulation of the P2z-purinoceptor for extracellular ATP in dissociated rat parotid cells is presented in studies using compounds that inhibit protein kinases. Preincubation of acinar cells with the protein kinase catalytic-site inhibitors K-252a and staurosporine, as well as with the regulatory-domain inhibitor sphingosine, specifically potentiates the elevation in cytosolic Ca2+ concentration ([Ca2+]i) mediated by extracellular ATP, but has no effect on the [Ca2+]i elevation mediated by muscarinic receptors through phospholipase C activation. Phorbol dibutyrate (PDBu), which activates protein kinase C (PKC), has no modulatory effect on ATP-mediated [Ca2+]i elevation. Further, pretreatment with PDBu does not reverse or block the effects of K-252a or sphinogosine, arguing against the involvement of PKC. Other pharmacological manipulations indicate that neither calmodulin-dependent nor cyclic-AMP-dependent kinases are involved. Neither the peak intracellular Ca2+ mobilization nor the sustained Ca2+ entry in response to carbachol or to a Ca2+ ionophore (4-bromo-A23187) is altered by the kinase inhibitors that potentiate the [Ca2+]i response to ATP, indicating that effects on the ATP response are not due to non-specific permeability changes, nor to decreased Ca2+ removal from the cytosol. ATP-mediated influx of Mn2+ as well as ATP-induced membrane depolarization are potentiated in cells preincubated with K-252a, directly demonstrating that cation influx is enhanced through a P2z-specific route. These results show that P2z responses (or purinoceptors) can be modulated and suggest that phosphorylation events are involved.

Octopamine release at two points along lobster nerve trunks.

Nerve cells in the proximal regions of second thoracic roots in lobsters have been injected with the fluorescent dye Procion Yellow. Examination of the roots reveals an elaborate array of cell branches in a superficial layer of the root in the vicinity of the cell bodies. Large varicosities, up to 10 mum in diameter, are seen lined up along fine nerve branches. 2. In these same regions, electron microscopic examination shows the presence of large profiles filled with 0-1-0-2 mum dense cored granules, and having the appearance of nerve endings. These profiles probably correspond to the varicosities seen in the Procion Yellow injections. The dense cored granules within the endings have a crystalline substructure. All the endings are found within 7 mum of the surface of the root and no obvious physiological target tissue exists in their surroundings. Endings have not been traced directly to root cell bodies.However, granules of similar dimensions to those seen in endings are found in cell bodies, axon-hillock regions and numerous axonal profiles in the superficial root regions near cell bodies. The morphological studies suggest that the root neurones have the typical appearance of neurosecretory cells. 3. Octopamine pools in cell body regions of second thoracic roots can be isotopically labelled by incubation with either [3H]tyramine or [3H]-tyrosine. After labelling, pulsing with 100 mM potassium causes an increase in the rate of release of radioactive material. Upon return to normal media background rates of release are re-established. The enhanced efflux has the following properties: (a) repeated pulses of potassium release less radio-active material each time; (b) a prolonged potassium pulse produces first a peak of release, then a decline to a plateau, and the plateau level of release is maintained for the duration of the potassium pulse; (c) release is dependent on the presence of calcium ions in the bathing fluid and 40 mM cobalt prevents release; (d) release is selective for octopamine. With tyrosine as a precursor compound, as much radioactive tyrosine as octopamine is found in tissues after incubation, yet pulsing with potassium causes an enhanced efflux only of octopamine from preparations. 4. Release of octopamine also can be demonstrated from pericardial organs near the ends of lateral branches of the roots and the properties of the release are identical to those seen with cell body regions. 5. Physiological studies, in which root cells are antidromically activated while recording from cell bodies, suggest that the distal endings of at least some of the root cells are at the pericardial organs. 6. The results suggest that root cell neurones are neurosecretory cells capable of releasing octopamine at two points: one near cell bodies, the other at the pericardial organs near the distal ends of the roots...

P2X(7) receptors in rat parotid acinar cells: formation of large pores.

1. Permeabilization of cells mediated by P2X(7) receptors occurs to varied degrees in native and heterologous expression systems. Previous studies on P2X(7) receptors in parotid acinar cells suggested that ATP does not permeabilize these cells. 2. Modification of the assay conditions showed that ATP permeabilizes freshly dissociated rat parotid acinar cells to the fluorescent dye YOPRO-1. 3. The pharmacological and physiological properties of this effect indicate that permeabilization is mediated by the P2X(7) receptor. Adenosine 5'-triphosphate (ATP) and 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (BzBzATP) were effective agonists with EC(50) values of 49.3 and 0.6 microM, respectively. 4. Permeabilization was best observed in low divalent cation concentrations and at physiological temperatures. Previous studies failed to detect permeabilization because of the sensitivity of this effect to temperature and divalent cations. 5. An important consideration in understanding the effect of divalent cations is that the fluorescence of YOPRO-1/nucleic acid complexes is directly quenched by addition of divalent cations. This must be considered if quantitative study of the interaction of divalent cations with P2X(7) receptors is carried out using fluorescent DNA-binding dyes. 6. In summary, our data show that P2X(7) receptors in parotid acinar cells can form large pores in the plasma membrane. This property likely contributes to signalling and may be cytotoxic and have particular significance in damaged or inflamed salivary glands.

Expression and trans-synaptic regulation of P2x4 and P2z receptors for extracellular ATP in parotid acinar cells. Effects of parasympathetic denervation.

Trans-synaptic regulation of muscarinic, peptidergic, and purinergic responses after denervation has been reported previously in rat parotid acinar cells (McMillian, M. K., Soltoff, S. P., Cantley, L. C., Rudel, R., and Talamo, B. R. (1993) Br. J. Pharmacol. 108, 453-461). Characteristics of the ATP-mediated responses and the effects of parasympathetic denervation were further analyzed through assay of Ca2+ influx, using fluorescence ratio imaging methods, and by analysis of P2x receptor expression. ATP activates both a high affinity and a low affinity response with properties corresponding to the recently described P2x4 and the P2z (P2x7)-type purinoceptors, respectively. Reverse transcription-polymerase chain reaction analysis reveals mRNA for P2x4 as well as P2x7 subtypes but not P2x1, P2x2, P2x3, P2x5, or P2x6. P2x4 protein also is detected by Western blotting. Distribution of the two types of ATP receptor responses on individual cells was stochastic, with both high and low affinity responses on some cells, and only a single type of response on others. Sensitivity to P2x4-type activation also varied even among cells responsive to low concentrations of ATP. Parasympathetic denervation greatly enhanced responses, tripling the proportion of acinar cells with a P2x4-type response and increasing the fraction of highly sensitive cells by 7-fold. Moreover, P2x4 mRNA is significantly increased following parasympathetic denervation. These data indicate that sensitivity to ATP is modulated by neurotransmission at parasympathetic synapses, at least in part through increased expression of P2x4 mRNA, and suggest that similar regulation may occur at other sites in the nervous system where P2x4 receptors are widely expressed.