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Long Distance Communication between Muscarinic Receptors and Ca2+ Release Channels Revealed by Carbachol Uncaging in Cell-attached Patch Pipette

2003; Elsevier BV; Volume: 278; Issue: 23 Linguagem: Inglês

10.1074/jbc.m302599200

1083-351X

Michael C. Ashby, Cristina Camello‐Almaraz, Oleg V. Gerasimenko, Ole H. Petersen, Alexei V. Tepikin,

Neuroscience and Neuropharmacology Research

We have investigated the characteristics of cytosolic Ca2+ signals induced by muscarinic receptor activation of pancreatic acinar cells that reside within intact pancreatic tissue. We show that these cells exhibit global Ca2+ waves and local apical Ca2+ spikes. This is the first evidence for local Ca2+ signaling in undissociated pancreatic tissue. The mechanism of formation of localized Ca2+ signals was examined using a novel approach involving photolysis of caged carbachol inside a patch pipette attached to the basal surface of an acinar unit. This local activation of basal muscarinic receptors elicited local cytosolic Ca2+ spikes in the apical pole more than 15 μm away from the site of stimulation. In some experiments, local basal receptor activation elicited a Ca2+ wave that started in the apical pole and then spread toward the base. Currently, there are two competing hypotheses for preferential apical Ca2+ signaling. One invokes the need for structural proximity of the cholinergic receptors and the Ca2+ release channels in the apical pole, whereas the other postulates long distance communication between basal receptors and the channels. Our intrapipette uncaging experiments provide definitive evidence for long distance communication between basal muscarinic receptors and apical Ca2+ release channels. We have investigated the characteristics of cytosolic Ca2+ signals induced by muscarinic receptor activation of pancreatic acinar cells that reside within intact pancreatic tissue. We show that these cells exhibit global Ca2+ waves and local apical Ca2+ spikes. This is the first evidence for local Ca2+ signaling in undissociated pancreatic tissue. The mechanism of formation of localized Ca2+ signals was examined using a novel approach involving photolysis of caged carbachol inside a patch pipette attached to the basal surface of an acinar unit. This local activation of basal muscarinic receptors elicited local cytosolic Ca2+ spikes in the apical pole more than 15 μm away from the site of stimulation. In some experiments, local basal receptor activation elicited a Ca2+ wave that started in the apical pole and then spread toward the base. Currently, there are two competing hypotheses for preferential apical Ca2+ signaling. One invokes the need for structural proximity of the cholinergic receptors and the Ca2+ release channels in the apical pole, whereas the other postulates long distance communication between basal receptors and the channels. Our intrapipette uncaging experiments provide definitive evidence for long distance communication between basal muscarinic receptors and apical Ca2+ release channels. In response to agonist stimulation, many cell types produce transient elevations in the cytosolic Ca2+ concentration ([Ca2+]i), which remain localized to a specific subcellular domain (1Bootman M.D. Lipp P. Berridge M.J. J. Cell Sci. 2001; : 2213-2222PubMed Google Scholar). In isolated pancreatic acinar cells, a low concentration of acetylcholine (ACh) 1The abbreviations used are: ACh, acetylcholine; IP3, inositol 1,4,5-trisphosphate; CCh, carbachol; AM, acetoxymethyl ester. can evoke repetitive local cytosolic Ca2+ spikes, which are confined to the granule containing apical pole (2Thorn P. Lawrie A.M. Smith P.M. Gallacher D.V. Petersen O.H. Cell. 1993; 74: 661-668Abstract Full Text PDF PubMed Scopus (430) Google Scholar). These local Ca2+ spikes can activate exocytosis through the apical plasma membrane (3Maruyama Y. Inooka G. Li Y.X. Miyashita Y. Kasai H. EMBO J. 1993; 12: 3017-3022Crossref PubMed Scopus (98) Google Scholar) and open Ca2+-dependent Cl-channels present exclusively in the apical plasma membrane, thereby regulating acinar fluid secretion (4Park M.K. Lomax R.B. Tepikin A.V. Petersen O.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10948-10953Crossref PubMed Scopus (94) Google Scholar). Agonist-receptor interaction causes activation of phospholipase C and the generation of inositol 1,4,5-trisphosphate (IP3), which in turn opens Ca2+ release channels in the endoplasmic reticulum membrane (5Streb H. Irvine R.F. Berridge M.J. Schulz I. Nature. 1983; 306: 67-69Crossref PubMed Scopus (1797) Google Scholar), thereby explaining the liberation of Ca2+ from this intracellular store (6Nielsen S.P. Petersen O.H. J. Physiol. (Lond.). 1972; 223: 685-697Crossref Scopus (63) Google Scholar). Because the G-protein coupled receptors for cholecystokinin are localized in the basolateral plasma membrane as demonstrated in early autoradiographic studies on intact pancreas (7Rosenzweig S.A. Miller L.J. Jamieson J.D. J. Cell Biol. 1983; 96: 1288-1297Crossref PubMed Scopus (85) Google Scholar), the classical view of pancreatic acinar cell stimulation involves the binding of agonist to plasma membrane receptors located at the base. It is this end of the cell that is close to blood vessels and pancreatic nerve terminals from where physiologically released agonists approach the cell (8Motta P.M. Macchiarelli G. Nottola S.A. Correr S. Microsc. Res. Technol. 1997; 37: 384-398Crossref PubMed Scopus (50) Google Scholar). If agonist-receptor interaction occurs at the base and Ca2+ release occurs at the opposite (apical) pole, there is a need for a long distance Ca2+-releasing intracellular messenger. The established Ca2+-releasing messenger IP3 (9Bootman M.D. Collins T.J. Peppiatt C.M. Prothero L.S. MacKenzie L. De Smet P. Travers M. Tovey S.C. Seo J.T. Berridge M.J. Ciccolini F. Lipp P. Semin. Cell Dev. Biol. 2001; 12: 3-10Crossref PubMed Scopus (388) Google Scholar, 10Meldolesi J. Prog. Neurobiol. 2001; 65: 309-338Crossref PubMed Scopus (92) Google Scholar) as well as the more recently discovered Ca2+-liberating agents cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate each elicit repetitive cytosolic Ca2+ spikes in the apical pole (2Thorn P. Lawrie A.M. Smith P.M. Gallacher D.V. Petersen O.H. Cell. 1993; 74: 661-668Abstract Full Text PDF PubMed Scopus (430) Google Scholar, 11Cancela J.M. Gerasimenko O.V. Gerasimenko J.V. Tepikin A.V. Petersen O.H. EMBO J. 2000; 19: 2549-2557Crossref PubMed Scopus (166) Google Scholar, 12Cancela J.M. Annu. Rev. Physiol. 2001; 63: 99-117Crossref PubMed Scopus (92) Google Scholar). Therefore, it has been proposed that messenger(s) generated at the basal membrane in response to agonist stimulation easily diffuse across the cell and release Ca2+ primarily in the apical pole because of the high density of Ca2+ release channels in this part of the cell (2Thorn P. Lawrie A.M. Smith P.M. Gallacher D.V. Petersen O.H. Cell. 1993; 74: 661-668Abstract Full Text PDF PubMed Scopus (430) Google Scholar, 13Petersen O.H. Petersen C.C. Kasai H. Annu. Rev. Physiol. 1994; 56: 297-319Crossref PubMed Scopus (258) Google Scholar, 14Cancela J.M. Van Copponolle F. Galione A. Tepikin A.V. Petersen O.H. EMBO J. 2002; 21: 909-919Crossref PubMed Scopus (159) Google Scholar, 15Lee M.G. Xu X. Zeng W. Diaz J. Wojcikiewicz R.J. Kuo T.H. Wuytack F. Racymaekers L. Muallem S. J. Biol. Chem. 1997; 272: 15765-15770Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 16Straub S.V. Giovannucci D.R. Yule D.I. J. Gen. Physiol. 2000; 116: 547-560Crossref PubMed Scopus (161) Google Scholar, 17Ashby M.C. Craske M. Park M.K. Gerasimenko O.V. Burgoyne R.D. Petersen O.H. Tepikin A.V. J. Cell Biol. 2002; 158: 283-292Crossref PubMed Scopus (69) Google Scholar). The view that the primary site of the neurotransmitter- or hormone-elicited intracellular Ca2+ release is determined not by the site of the neurotransmitter or hormone-receptor interaction but by the localization of the Ca2+ release channels (13Petersen O.H. Petersen C.C. Kasai H. Annu. Rev. Physiol. 1994; 56: 297-319Crossref PubMed Scopus (258) Google Scholar) has been challenged in a recent study (18Shin D.M. Luo X. Wilkie T.M. Miller L.J. Peck A.B. Humphreys-Beher M.G. Muallem S. J. Biol. Chem. 2001; 276: 44146-44156Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), which used immunofluorescence to localize G-protein coupled receptors in isolated pancreatic acinar cell clusters. This study shows localization of receptors close to the apical end of the cell in regions near to the tight junctions. The authors suggest that apical Ca2+ signals are preferentially produced in that region as a result of nearby production of intracellular messengers. The apparent contradiction between this study (18Shin D.M. Luo X. Wilkie T.M. Miller L.J. Peck A.B. Humphreys-Beher M.G. Muallem S. J. Biol. Chem. 2001; 276: 44146-44156Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) and the previously held view of agonist-evoked local Ca2+ signal generation (13Petersen O.H. Petersen C.C. Kasai H. Annu. Rev. Physiol. 1994; 56: 297-319Crossref PubMed Scopus (258) Google Scholar) lead us to test the ability of pancreatic acinar cells to produce apical Ca2+ release in response to basal stimulation. Because all previous Ca2+ signaling studies in the exocrine pancreas were carried out on isolated acinar cells or cell clusters, we first demonstrated that in intact undissociated pancreatic tissue, ACh stimulation preferentially produces [Ca2+]i elevations in the apical region of the acinar cells. Using intrapipette uncaging of carbachol (CCh), we then show that activation of muscarinic receptors in a very small restricted area of the basal plasma membrane is able to produce localized Ca2+ responses at the opposite end of the cell in the apical pole. Cell Preparation, Solutions, Dye Loading, and Chemicals—All of the experiments were performed at room temperature (22–24 °C). The pancreas was removed from mice killed by cervical dislocation. The isolated pancreas was immediately placed into extracellular solution, which contained (in mm): 140 NaCl, 4.7 KCl, 1.13 MgCl2, 10 HEPES, 10 glucose, 1 mm CaCl2, pH 7.2 (adjusted by NaOH). Freshly isolated mouse pancreatic clusters of acinar cells were prepared as described previously (2Thorn P. Lawrie A.M. Smith P.M. Gallacher D.V. Petersen O.H. Cell. 1993; 74: 661-668Abstract Full Text PDF PubMed Scopus (430) Google Scholar) and used within 4 h. All of the experiments were performed during continuous superperfusion of extracellular solution. Agonists and antagonists were added to the extracellular solution at the stated concentrations. Fluo-4 AM, Fura Red AM, caged fluorescein dextran-10K, α-carboxy-2-nitrobenzyl-caged carbachol, Mitotracker Green FM, and Lysotracker Red were from Molecular Probes. Hoechst 33342 was from Calbiochem. Loading/staining of cells with fluorescent compounds was achieved by incubation of cells/tissue with the particular dye in darkness at room temperature (except when specifically mentioned) under the following conditions: Fluo-4 AM (2.5 μm for 25 min); Fura Red AM (10 μm for 30 min); Mitotracker Green FM (500 nm for 25 min at 37 °C); Lysotracker Red (200 nm for 5 min); Hoechst 33342 (100 μg/ml for 2 min). Collagenase was obtained from Worthington, and all other chemicals were purchased from Sigma. Confocal Imaging—Fluorescence images were obtained using either a Zeiss LSM 510 confocal microscope or a Leica SP2 confocal microscope with ×40 oil-immersion or ×63 water-immersion objective. During the experiments, intact pancreatic tissue was immobilized on a glass coverslip using either a needle through the tissue or a slice hold-down (Warner Instruments). Intact tissue or cells adhered to poly-l-lysine-coated slides were imaged on the stage of an inverted microscope (Axiovert 100M, Zeiss, or DMIRBE from Leica). Fluo-4 AM, Mitotracker Green FM, and caged fluorescein dextran-10K were excited by a 488 nm laser line, and emission was collected through a bandpass filter of 505–550 nm. Fura Red was excited at 488 nm, and emitted light was collected through a long pass 560-nm filter or bandpass 580–680-nm filter. Excitation of Lysotracker Red was by a 543-nm laser line, and emission was collected through a long pass 570-nm filter. Hoechst 33342 was excited by UV light (364 nm), and emission was collected between 400 and 450 nm. Intrapipette Uncaging—Standard patch clamp technique was adapted using the EPC-8 amplifier and Pulse software (HEKA). We used pipettes having resistances of 2–4 megohms. Pipettes were back-filled with the extracellular solution containing, in addition, either 5 mg/ml caged fluorescein dextran-10K or 50 μm-5 mm caged CCh. Seals of 5–10 giga-ohm resistance were obtained between the pipette and the basal plasma membrane of individual acinar cells in small clusters. Uncaging was achieved during the scanning procedure by exposure of pre-defined regions to UV laser light at 364 and 351 nm. The regions were selected to surround the pipette but not expose the cell. Subcellular Localized Calcium Signaling in Intact Epithelial Tissue—Confocal fluorescence microscopy was used to image the Ca2+-sensitive dye, Fluo-4, in the cytoplasm of cells that were stimulated by bath perfusion of physiological agonists. We used an undissociated pancreatic preparation to image large areas of intact pancreatic tissue (see Fig. 1A) and showed that application of ACh triggered repetitive [Ca2+]i elevations in nearly all of the cells (n = 25 experiments). An analysis of the fluorescence changes in individual cells revealed the spatiotemporal profile of these Ca2+ oscillations (Fig. 1A). In some cells, lower levels of stimulation caused [Ca2+]i oscillations that were restricted entirely to the secretory granule region at the apical end of the cell (Fig. 1A, images 1–3 (n = 9)). Increasing the ACh concentration changed the profile of each transient from local to global. At higher doses, the [Ca2+]i transient became a wave, which was initiated at the apical pole and spread toward the basal plasma membrane (Fig. 1A, images 4–7 (n = 22)). As the intensity of stimulation increased, the oscillations became superimposed on an elevated base line (Fig. 1A). These observations demonstrate that pancreatic acinar cells in their native environment can produce apically localized [Ca2+]i elevations and global Ca2+ waves initiated in the apical region. Specific patterns of Ca2+ signaling are translated into the responses of cellular organelles such as exocytosis of secretory granules (19Burgoyne R.D. Morgan A. Trends Neurosci. 1995; 18: 191-196Abstract Full Text PDF PubMed Scopus (182) Google Scholar), changes of mitochondrial metabolism (20Voronina S. Sukhomlin T. Johnson P.R. Erdemli G. Petersen O.H. Tepikin A. J. Physiol. (Lond.). 2002; 539: 41-52Crossref Scopus (95) Google Scholar, 21RobbGaspers L.D. Burnett P. Rutter G.A. Denton R.M. Rizzuto R. Thomas A.P. EMBO J. 1998; 17: 4987-5000Crossref PubMed Scopus (323) Google Scholar), and nuclear gene expression (22Dolmetsch R.E. Xu K. Lewis R.S. Nature. 1998; 392: 933-936Crossref PubMed Scopus (1683) Google Scholar). Also, the positioning of intracellular organelles can have profound implications for patterns of [Ca2+]i signaling (23Tinel H. Cancela J.M. Mogami H. Gerasimenko J.V. Gerasimenko O.V. Tepikin A.V. Petersen O.H. EMBO J. 1999; 18: 4999-5008Crossref PubMed Scopus (322) Google Scholar, 24Park M.K. Ashby M.C. Erdemli G. Petersen O.H. Tepikin A.V. EMBO J. 2001; 20: 1863-1874Crossref PubMed Scopus (272) Google Scholar). We assessed the intracellular position of the major intracellular organelles in live cells within intact pancreatic tissue. The secretory (zymogen) granules were labeled using Lysotracker Red (based upon the affinity of the dye for acidic compartments), nuclei were stained using Hoechst 33342, and mitochondria were stained using Mitotracker Green FM. The majority of acidic compartments (mostly representing zymogen granules) were found near the apical (luminal) membrane (Fig. 1B). The acinar lumen was often visible as an unstained region between the cells. The nucleus or nuclei were always found outside the zymogen granule region toward the base (Fig. 1B). In isolated pancreatic acinar cells, several studies have shown that mitochondria have distinct and specific intracellular locations. The majority of the mitochondria are positioned in a perigranular belt, which limits the spread of apically localized Ca2+ signals (23Tinel H. Cancela J.M. Mogami H. Gerasimenko J.V. Gerasimenko O.V. Tepikin A.V. Petersen O.H. EMBO J. 1999; 18: 4999-5008Crossref PubMed Scopus (322) Google Scholar). This positioning was also apparent in the intact tissue, because the strongest mitochondrial staining was in the perigranular region (Fig. 1B). In addition, Mitotracker Green staining also revealed the perinuclear and subplasmalemmal mitochondria that have been identified in isolated cells (Fig. 1B) (24Park M.K. Ashby M.C. Erdemli G. Petersen O.H. Tepikin A.V. EMBO J. 2001; 20: 1863-1874Crossref PubMed Scopus (272) Google Scholar). The intracellular positioning of mitochondria is thought to have important functional interactions with nearby Ca2+ signaling pathways (24Park M.K. Ashby M.C. Erdemli G. Petersen O.H. Tepikin A.V. EMBO J. 2001; 20: 1863-1874Crossref PubMed Scopus (272) Google Scholar, 25Glitsch M.D. Bakowski D. Parekh A.B. EMBO J. 2002; 21: 6744-6754Crossref PubMed Scopus (180) Google Scholar, 26Montero M. Alonso M.T. Carnicero E. Cuchillo-Ibanez I. Albillos A. Garcia A.G. Garcia-Sancho J. Alvarez J. Nat. Cell Biol. 2000; 2: 57-61Crossref PubMed Scopus (416) Google Scholar). Our observations in the intact pancreas highlight the relevance of the polarity of organelles and Ca2+ signaling in cells within their native environment. Long Distance Signaling Demonstrated by Intrapipette Uncaging of Carbachol—Acinar cells within the intact pancreas can produce local apical [Ca2+]i spikes when ACh is applied to the basal side of the epithelium. This highlights the need for understanding the mechanism by which the local apical Ca2+ signals are generated. Here we have studied the patterns of Ca2+ signaling induced by exclusive stimulation of basally located receptors. To specifically excite basal receptors, we have combined electrophysiological and photolytic (uncaging) techniques. A patch pipette containing caged agonist in standard extracellular solution was used to isolate a small area of the basal membrane of a single acinar cell within a cluster of cells (3–10 cells). We used isolated cell clusters because the patching of cells within the intact pancreas was impossible because of the capsule surrounding the tissue. During continuous superfusion of the cell clusters, a high resistance (5–10 giga-ohms) seal was formed between the basal cell membrane and the glass pipette. Such a configuration could then be used to photolyse caged carbachol by UV laser light exposure of pre-defined regions (shown as dashed orange lines throughout) surrounding the pipette but avoiding the cells. To test this experimental configuration, we used caged fluorescein dextran-10K in the pipette and loaded the cytoplasm of the cells with the Ca2+-sensitive dye, Fura Red. This allowed us to characterize the intrapipette uncaging and test whether UV exposure itself would cause any [Ca2+]i elevation (Fig. 2). As expected, each UV exposure caused an increase in the fluorescence of fluorescein inside the pipette as the compound was uncaged (Fig. 2, n = 4). The increase in fluorescence peaked at the first frame obtained after uncaging and then declined, initially rapidly and then more slowly. This decline presumably reflects dilution of the product of uncaging in the solution in the rest of the pipette, which is much larger than the volume exposed to UV light. The uncaging itself did not cause any change in [Ca2+]I, whereas subsequent application of ACh to the bath triggered a large rapidly evolving [Ca2+]i transient (Fig. 2). These experiments showed that we can selectively expose a small region of basal plasma membrane to a photolytically activated compound. This technique is equally applicable to studies using site-specific stimulation of many other cell types including neurons, which produce polarized responses to stimulation. The rapid time course and range of UV exposure makes intrapipette uncaging faster, more sensitive, and better regulated than other local stimulation techniques (2Thorn P. Lawrie A.M. Smith P.M. Gallacher D.V. Petersen O.H. Cell. 1993; 74: 661-668Abstract Full Text PDF PubMed Scopus (430) Google Scholar). We tested the ability of CCh to trigger changes in [Ca2+]i measured by Fluo-4 in the cytoplasm of the cells when uncaged inside a cell-attached pipette. We found that intrapipette uncaging of carbachol at the basal pole was often able to trigger short lasting [Ca2+]I elevations, which were restricted to the extreme apex of the cell (Fig. 3A, n = 11 cells). The distance between the basal membrane under the pipette and the point of Ca2+ release was always over 15 μm and up to a distance of 25 μm. Approximately 60% of the cells responded to uncaging by producing Ca2+ signals, and each uncaging event usually only triggered a single Ca2+ spike. Intriguingly, in the other experiments, it was not possible to trigger Ca2+ release even with very strong and/or long lasting UV exposure. This inability of even strong uncagings to trigger Ca2+ signals in 40% of the cells could be explained either by insufficient production of second messengers or by absence of receptors under the patch pipette because of receptor clustering in other regions of the basal membrane. The clustering of transmembrane receptors has been reported previously (27Thomason P.A. Wolanin P.M. Stock J.B. Curr. Biol. 2002; 12: R399-R401Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) and is proposed to underlie high sensitivity areas of membrane that are able to amplify low level extracellular stimuli. We compared the size of the [Ca2+]i rises triggered by intrapipette CCh uncaging to those caused by bath application of ACh (10 nm) (Fig. 3A). Short lasting uncaging events (UV exposure of 100–500 ms) consistently generated smaller responses than ACh in the same cell (n = 4). For these durations of uncaging, the [Ca2+]i signals were localized to the apical part of the cells. To be certain that only muscarinic receptors under the patch pipette would be responsible for generating the intracellular Ca2+ release, we carried out experiments in which we made sure that even in the unlikely event that some of the uncaged CCh could escape the pipette, no activation of receptors outside the area of the plasma membrane covered by the patch pipette would occur. We blocked all muscarinic receptors outside the pipette by adding atropine (100 μm) after the gigaseal formation. This atropine concentration always blocked [Ca2+]i elevations triggered by ACh (1 μm) applied to the bath (n = 12). Under these conditions, short intrapipette uncaging was still able to trigger a localized [Ca2+]i elevation at the extreme apical pole of the cell (Fig. 3B, n = 6 cells). This confirms that the pipette does indeed form an impenetrable seal with the membrane (otherwise the extremely high atropine concentration in the bath would have prevented the uncaged CCh in the pipette from interacting with the muscarinic receptors under the patch pipette) and that only receptors under the pipette can be activated following uncaging. Global Ca2+Waves Triggered by Basal Intrapipette CCh Uncaging—The time course and size of the [Ca2+]i elevations described above correlate with a low level of ACh activation in studies on isolated cells (2Thorn P. Lawrie A.M. Smith P.M. Gallacher D.V. Petersen O.H. Cell. 1993; 74: 661-668Abstract Full Text PDF PubMed Scopus (430) Google Scholar). Therefore, we tested whether stronger activation of receptors in a small area of basal plasma membrane could generate larger global [Ca2+]i rises. Intrapipette uncaging of longer duration (800 ms-1.5 s) or repetitive short uncagings were able to trigger global Ca2+ waves (Fig. 4, n = 4). Such waves were always initiated in the apical region and propagated toward the basal plasma membrane, demonstrating that activation of a small area of basal plasma membrane is able to produce significant activation of intracellular Ca2+ signaling mechanisms. In some of our intrapipette uncaging experiments (n = 4), we could trigger a Ca2+ wave that propagated from the stimulated cell to other cells in the same cluster. This suggests that the level of second messenger produced by intrapipette uncaging is sufficient to support the spreading of the [Ca2+]I rise via gap junctions. The long distance signaling demonstrated directly in this study must rely on the production of some diffusible intracellular messenger following receptor activation. IP3, cyclic ADP-ribose, and nicotinic acid adenine dinucleotide phosphate are all able to release Ca2+ from intracellular stores in pancreatic acinar cells and are involved in agonist-evoked responses (11Cancela J.M. Gerasimenko O.V. Gerasimenko J.V. Tepikin A.V. Petersen O.H. EMBO J. 2000; 19: 2549-2557Crossref PubMed Scopus (166) Google Scholar, 28Wakui M. Osipchuk Y.V. Petersen O.H. Cell. 1990; 63: 1025-1032Abstract Full Text PDF PubMed Scopus (211) Google Scholar, 29Thorn P. Gerasimenko O. Petersen O.H. EMBO J. 1994; 13: 2038-2043Crossref PubMed Scopus (188) Google Scholar, 30Cancela J.M. Churchill G.C. Galione A. Nature. 1999; 398: 74-76Crossref PubMed Scopus (327) Google Scholar). Such small water soluble messengers are highly diffusible in the cytoplasm (13Petersen O.H. Petersen C.C. Kasai H. Annu. Rev. Physiol. 1994; 56: 297-319Crossref PubMed Scopus (258) Google Scholar), and it seems likely that one or more of them transmits the Ca2+ release signal from the activated basal receptor to the Ca2+ channels in the apical part of the endoplasmic reticulum store (31Ashby M.C. Tepikin A.V. Physiol. Rev. 2002; 82: 701-734Crossref PubMed Scopus (103) Google Scholar). In the majority of our experiments, it was possible to trigger large [Ca2+]i transients despite the fact that only a small area of plasma membrane underwent stimulation. We estimate that the area of plasma membrane covered by the patch pipette is at most 2% of the total surface area (based upon the Ω-shaped membrane inside the pipette tip and the total cell surface estimated from our confocal images). If the distribution of the muscarinic receptors were reasonably even, this would suggest that occupancy of a relatively small proportion of the receptors is sufficient to trigger not only local but also global and even intercellular [Ca2+]i signals. The ACh-detecting system in the basal membrane must therefore be highly sensitive.