Pflfigers Archiv
Pflfigers Arch. 380, 277-281 (~979)
EuropeanJournal of Physiology 9 by Springer-Verlag 1979
Instruments and Techniques Direct Visualization of Cell to Cell Coupling: Transfer of Fluorescent Probes in Living Mammalian Pancreatic Aeini Noriyuki Iwatsuki* and Ole Holger Petersen Department of Physiology, The University, Dundee DD1 4HN, Great Britain
Abstract. A technique whereby it is possible directly to observe the movement of organic molecules from cell to cell in living mammalian exocrine glands is described. Thin translucent segments of mouse pancreas are mounted in a superfusion bath. Fluorescent probes are injected intracellularly via fine micropipettes and fluorescence observed. Both fluorescein (mol. wt. 332) and procion yellow (tool. wt. 697) are shown to be transferred from the injection cell to neighbouring acinar cells. This shows directly the existence of intercellular communicating pathways. Key words: Fluorescent probes - Pancreatic acini Cell to cell coupling - Intracellular injection Electrical communication.
Introduction It has been known for some time that cells generally are connected to their neighbours by low resistance pathways (Loewenstein, 1966; Bennett, 1966). In the giant Chironomus salivary gland cells it has been possible to demonstrate direct cell to cell transfer of various fluorescent probes (Loewenstein and Kanno, 1964; Loewenstein and Rose, 1978). M a m m a l i a n gland cells are of course very much smaller than those of the Chironomus salivary glands, making experimental results much harder to obtain and less information is therefore available. However recently, quantitative and semiquantitative descriptions of the electrical communication network in the mouse liver and pancreas have been given ( G r a f a n d Petersen, 1978; Iwatsuki and Petersen, 1978c). Electrical intercellular communication has also been demonstrated in m a m m a l i a n Send offprint requests to O. H. Petersen at the above address
* MRC Research Fellow
salivary glands (Roberts et al., 1978; H a m m e r and Sheridan, 1978; Kater and Galvin, 1978) and lacrimal glands (Iwatsuki and Petersen, 1978b). Intercellular electrical communication can be modulated by: changes in intracellular [Ca 2+] (Loewenstein and Rose, 1978), changes in intracellular p H (Turin and Warner, 1977) and action of transmitters and peptide hormones (Iwatsuki and Petersen, 1978a, c). It would be very useful to have a method allowing assessment of the influence of hormones on cell to cell passage of organic molecules in living mammalian epithelia. The methods so far applied to demonstrate transfer of organic molecules from cell to cell ( H a m m e r and Sheridan, 1978; Kater and Galvin, 1978) in mammalian preparations, are not entirely satisfactory since they largely depend on observations made on fixed sectioned material and do not allow simultaneous fluorescence microscopy and electrophysiology. We are now describing a technique enabling us to observe directly the transfer of various fluorescent probes from cell to cell in living acini from superfused segments of mouse pancreas. We believe that this method will be useful for studies on the control and importance of intercellular channels. A live demonstration of our method was made at a recent meeting of The Physiological Society (Iwatsuki, 1978).
Materials and Methods A general outline of the set-up is shown in Fig. l. A thin translucent segment (2 • 2mm) of mouse pancreas was placed on a perspex platform in a bath. The bath was placed on the stage ofa Zeiss (Jena) (Technival) microscope in such a way that a light beam from below was focussed on to the perspex platform with the pancreatic tissue. The bath (t6ml) was perfused with a physiological salt solution (20ml/min) prewarmed to 37~ The solution contained (raM): NaC1, 103; KCI, 4.7; CaC12, 2.6; MgC12, 1.1; NaHCO3, 25; NaH2PO4, 1.2; D-glucose,2.8; Na pyruvate, 4.9; Na fumarate, 2.7; Na glutamate, 4.9; and was gassed with 95~ 02, 5~ CO2. The
0031-6768/79/0380/0277/S 01.00
278
Pfliigers Arch. 380 (1979)
microscope was fitted with a catadioptric objective (40 x , 2 cm working distance) (Zeiss0 Jena, 302355:001.26) and the field under examination viewed through eyepieces of 16 x or 8 x magnification. A camera could be attached to the microscope (Fig. l). Photographs of bright field images were taken using an automatic exposure unit. The microscope was also attached to a lighting unit (Zeiss, Jena, HBO 200) projecting a high intensity of incident ultraviolet light (Hgvapour lamp, BG 12 filter). In the cases of fluorescence microscopy the field was viewed through a yellow filter (OG 1). The lighting arrangement allowed rapid shifts between incident blue light and transmitted ordinary light or an appropriate mixing of the two, permitting alternate observation in dark field (fluorescence picture) and bright field, or a mixture of both. Photographs during dark field microscopy were taken using an exposure time of 1 min. Under visual control one, two or three glass microelectrodes could be inserted into acinar cells using Leitz micromanipulators. Glass micropipettes were pulled on a Palmer microelectrode puller and filled with 3mol - 1-1 KCI, 100mmol - l 1 Na-fluoresceinate (BDH) in 0.5 m o l 1-1 KC1 or procion yellow MX-4R (ICI) 4 % w/v) by the fibre glass method. The 3 m o t . 1-~ KCI microelectrodes had resistances of about 50 - 60 Mr2 immediately after filling and were subsequently beveled on a Sutter Instrument Co. beveler (Brown and Flaming, 1974) to attain final resistances of 3 0 - 5 0 M f L Procion yellow or fluorescein were injected into the cells by passing hyperpolarizing current pulses (100-500ms, 2 - 2 0 h A ) at a rate of l/s through the electrodes. Membrane potential and resistance were
Camera Hg-vapourlamp BG12
I ~Ocu[ar
I...............;
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fig. 1. A diagram showing some of the main features of the set-up
measured as previously described (Iwatsuki and Petersen, 1978a) using W P M 701 and 750 electrometer amplifiers. Potentials were displayed on a Tektronix double-beam storage oscilloscope and a Devices MX2 pen recorder.
Results
Visualization of Acinar Cells Figure2 shows a photograph of the edge of the pancreatic preparation in the bath. Individual cells can clearly be distinguished. The presence of numerous zymogen granules in one part of the cells (luminal) is also seen. The best image was always obtained at the edges of the preparation. During a prolonged period (1 h) of stimulation (ace@choline, 10 -6 M) these granules gradually disappeared. Figure 2 demonstrates the unique property of the mouse pancreas; no other mammalian exocrine gland preparation is thin enough to allow such easy identification of individual acinar cells.
Intracellular Injection of Fluorescein Insertion of a micropipette filled with fluorescein (tool. wt. 332) into an acinar cell was followed almost immediately by an intense fluorescence from that cell (Fig. 3). This fluorescence could be abolished by applying a depolarizing current (retaining current). Within the first minute of applying fluorescein fluorescence was also apparent in a neighbouring cell (Fig. 3b) and within the next few minutes spread to several neighbouring cells could be observed (Fig.3c). After stopping the fluorescein injection the fluorescence in all cells gradually disappeared. In many experiments several microelectrodes were inserted into neighbouring cells and electrical coupling could also be assessed. In all cases where fluorescein spread was observed electrical coupling with high coupling coefficients (>0.7) (Iwatsuki and Petersen, 1978c) were obtained.
Fig. 2 Photograph of edge of pancreatic preparation in the bath, Length of bar: 20 ~tm
N. Iwatsuki and O. H. Petersen: Cell to Cell Coupling
279 neighbouring acinar cells, but the fluorescence was not reversible within the period of the experiment (several hours). Also the outline of individual cells was not so clear as in the case of true intracellular deposition (Fig. 3).
Intracellular Injection of Procion Yellow
Fig.3. a Injection of fluorescein into an acinar cell through a microelectrode. Spread of fluorescein to one neighbouring cell. Mixture of bright and dark field. Photograph taken 1.5 min after start of fluorescein iontophoresis (300 ms pulses, 5 nA, l/s). Bar: 20 itm. x 750. b Same fieId as in a, this time viewed in dark field. This photograph was taken 3 min after start of fluorescein iontophoresis. Fluorescence has now spread to two further cells. Bar: 20 ram. x 750. e Same field as in a and b, 5 min after start of fluorescein injection. Fluorescence detectable in several neighbouring cells. Bar: 20gin. x 750
The correct intracellular location of the injection pipette was found to be crucial for meaningful results. While fluorescein ejected from the micropipette with the tip in a free extracellular position did not cause any observable fluorescence, very intense fluorescence could be obtained when the tip of the fuorescein micropipette just touched the cell membrane. In such cases fluorescence spread over the surface of m a n y
Figure 4 shows the result of an experiment in which one microelectrode filled with procion yellow (mol. wt. 697) was inserted into a superficial mouse pancreatic acinus. lontophoresis ofprocion yellow into the impaled acinar cell was signalled by an intense fluorescence in the injection cell followed by the appearance of fluorescence in two neighbouring cells (Fig. 4b, c). In contrast to fluorescein procion yellow only moved in one direction and always in a direction opposite to that in which the inserted microelectrode was pointing. Insertion of a microelectrode always causes an elevation of [Ca2+]i mainly localized to the tip of the electrode (B. Rose, personal communication) and it is likely that this may block the passage of particularly higher molecular weight compounds (Loewenstein and Rose, 1978). It is also possible that the microelectrode tip could influence the permeability of gap junctional membranes by a direct physical effect (pressure, stretch). Fluorescence spread was distinctly slower than for fluorescein and could therefore easily be quantified. Fluorescence in a neighbouring cell could be detected 1 - 4 m i n [mean: 2.4 + 1.1 (s.d.) min, n = 15] after start of procion yellow injections. Transfer of fuorescence was observed in 75 ~ of attempted injections. In 7 cases in which procion yellow transfer to neighbouring cells was observed a second microelectrode was inserted into a neighbouring cell. Electrotonic potential changes set up by current passage through the dye-containing microelectrode (dye injection) were in all these experiments transmitted to the microelectrode in the neighbouring cells. The precise coupling ratio could, however, not be measured since the tip resistance of the procion yellow-containing microelectrode was high and furthermore fluctuated considerably, making accurate compensation of tip resistance (balancing) impossible. After cessation of dye injection (reversal of the polarity of current injection) the fluorescence in all cells gradually diminished and after several minutes had disappeared.
Discussion This work demonstrates the feasibility of studying directly the movement of fluorescent probe molecules in a living m a m m a l i a n gland tissue. Figures 3 and 4 represent the first clear demonstration of cell to cell
280
Pflfigers Arch. 380 (1979)
movement of fluorescent tracers in mammalian exocrine glands. Two previous studies (Hammer and Sheridan, 1978; Kater and Galvin, 1978) on mammalian salivary glands have been reported. The documentation in the papers of Hammer and Sheridan (1978) and Kater and Galvin (1978) is, however, almost exclusively based on examination of fixed sectioned material. We feel that success in clearly demonstrating cell to cell movement of fluorescent probe molecules depends on: 1. the ability to dearly visualize individual cells in the living preparation (Figs. 2, 3, 4), 2. the ability to switch rapidly from bright to dark field images without the need to move the specimen or change the whole set-up of microscopes, 3. a high magnification objective with a long working distance permitting easy access with several microelectrodes, 4. correct intracellular localization of injection micropipettes. The technique outlined in this paper should be useful in probing the effects of various experimental conditions designed to change the resistance of the intercellular junctions on the movement of fluorescent probe molecules. This is important since marked changes in junctional conductance can take place without any change in electrical coupling coefficient (Socolar, 1977). We believe that the mouse pancreas is an ideal tissue for this purpose, since it is thin enough to allow good cellular visualization, the electrophysiology has been explored in more detail than in any other mammalian gland preparation (Petersen, 1979) and we have more information on the cellular stimulus-secretion coupling mechanisms than in other gland tissues (Case, 1978; Petersen and Iwatsuki, 1978; Schulz and Ullrich, 1979). Acknowledgement. This work was supported by grants from the MRC and the Cystic Fibrosis Research Trust. We thank Mr D. Williams (Zeiss Ikon) and Mr D. Doig for extensive and invaluable help with the selection of parts for the fluorescence microscope. Mr D. Doig also gave crucial assistance with the photography.
References Fig.4. a Photograph taken through phase-contrast microscope (bright field) of the edge of mouse pancreas fragment placed in the perspex bath. A microelectrode filled with procion yellow is inserted into a cell situated in the uppermost acinus. Bar: 20 klm. • 830. b Same field as in a, but this time viewed in a mixture of bright field (transmitted ordinary light) and dark field (incident blue light). Photograph taken 5 min after start of procion yellow iontophoresis (10 hA, l/s). The major part of the fluorescence from the injected acinus is orange yellow (procion yellow) whereas the other light spots are yellow green. Bar: 20 ~am. x 830. e Same field as in a and b, but now viewed in dark field. The procion yellow has clearly spread from the injected cell (extreme right) to two cells to the left. Fluorescence was detected in the neighbouring cell (middle cell) 2 min after start of injection. Five minutes after start of injection fluorescence was also detected from a third cell (extreme left). This photograph was taken 6 min after start of procion yellow injection. Bar : 20 I~m. x 830
Bennett, M. V. L. : Physiology of electrotonic junctions. Ann. N.Y. Acad. Sci. 137, 509-539 (1966) Brown, K. T., Flaming, D. G. : Beveling of fine micropipette electrodes by a rapid precision method. Science 185, 693-695 (1974) Case, R. M.: Synthesis, intracellular transport and discharge of exportable proteins in the pancreatic acinar cell and other cells. Biol. Rev. 53, 211-354 (1978) Graf, J., Petersen, O. H. : Cell membrane potential and resistance in liver. J. Physiol. (Lond.) 284, 105-126 (1978) Hammer, M. G., Sheridan, J. D. : Electrical coupling and dye transfer between acinar cells in rat salivary glands. J. Physiol. (Lond.) 275, 495-505 (1978) Iwatsuki, N. : Direct demonstration of cell-to-cell communication in mammalian pancreatic acini: transfer of fluorescent probe molecules. J. Physiol. (Lond.) 285, 1 - 2 P (1978)
N. Iwatsuki and O. H. Petersen: Cell to Cell Coupling Iwatsuki, N., Petersen, O. H. : Pancreatic acinar cells: ace@cholineevoked electrical uncoupling and its ionic dependency. J. Physiol. (Loud.) 274, 8 1 - 9 6 (1978a) Iwatsuki, N., Petersen, O. H. : Membrane potential, resistance and intercellular communication in the lacrimal gland: effects of acetylcholine and adrenaline. J. Physiol. (Lond.) 275, 507 520 (1978b) Iwatsuki, N., Petersen, O. H. : Electrical coupling and uncoupling of exocrine acinar cells. J. Cell Biol. 79, 533-545 (1978c) Kater, S. B., Galvin, N. J. : Physiological and morphological evidence for coupling in mouse salivary gland acinar cells. J. Cell Biol. 79, 2 0 - 2 6 (1978) Loewenstein, W. R. : Permeability of membrane junctions. Ann. N.Y. Acad. Sci. 137, 441-472 (1966) Loewenstein, W. R., Kanno, Y. : Studies on an epithelial (gland) cell junction. I. Modifications of surface membrane permeability. J. Cell Biol. 22, 565-586 (t964) Loewenstein, W. R., Rose, B. : Calcium in (junctional) intercellular communication and a thought on its behaviour in intracellular communication. Ann. N.Y. Acad. Sci. 307, 285-307 (1978)
281 Petersen, O. H. : Electrophysiology of gland cells. Monograph of the physiological society. London, New York: Academic Press (in press, 1979) Petersen, O. H., Iwatsuki, N. : The role of calcium in pancreatic acinar cell stimulus-secretion coupling: an electrophysiological approach. Ann. N.Y. Acad. Sci. 307, 599-615 (1978) Roberts, M. L., Iwatsuki, N., Petersen, O. H. : Parotid acinar cells : Ionic dependence of acetylchotine-evoked membrane potential changes. Pflfigers Arch. 376, 1 5 9 - 167 (1978) Schulz, I., Ullrich, K. J.: Transport processes in the exocrine pancreas. In: Membrane transport in biology, Vol. IV (G. Giebisch, D. J. Tosteson, and H. H. Ussing, eds.). Berlin, Heidelberg, New York: Springer (in press, 1979) Socolar, S. : The coupling coefficient as an index of junctional conductance. J. Membr. Biol. 34, 2 9 - 3 7 (1977) Turin, L., Warner, A. : Carbon dioxide reversibly abolishes ionic communication between cells of early amphibian embryo. Nature 270, 5 6 - 57 (1977)
Received February 13, 1979
Pflfigers Arch. 380, 277-281 (~979)
EuropeanJournal of Physiology 9 by Springer-Verlag 1979
Instruments and Techniques Direct Visualization of Cell to Cell Coupling: Transfer of Fluorescent Probes in Living Mammalian Pancreatic Aeini Noriyuki Iwatsuki* and Ole Holger Petersen Department of Physiology, The University, Dundee DD1 4HN, Great Britain
Abstract. A technique whereby it is possible directly to observe the movement of organic molecules from cell to cell in living mammalian exocrine glands is described. Thin translucent segments of mouse pancreas are mounted in a superfusion bath. Fluorescent probes are injected intracellularly via fine micropipettes and fluorescence observed. Both fluorescein (mol. wt. 332) and procion yellow (tool. wt. 697) are shown to be transferred from the injection cell to neighbouring acinar cells. This shows directly the existence of intercellular communicating pathways. Key words: Fluorescent probes - Pancreatic acini Cell to cell coupling - Intracellular injection Electrical communication.
Introduction It has been known for some time that cells generally are connected to their neighbours by low resistance pathways (Loewenstein, 1966; Bennett, 1966). In the giant Chironomus salivary gland cells it has been possible to demonstrate direct cell to cell transfer of various fluorescent probes (Loewenstein and Kanno, 1964; Loewenstein and Rose, 1978). M a m m a l i a n gland cells are of course very much smaller than those of the Chironomus salivary glands, making experimental results much harder to obtain and less information is therefore available. However recently, quantitative and semiquantitative descriptions of the electrical communication network in the mouse liver and pancreas have been given ( G r a f a n d Petersen, 1978; Iwatsuki and Petersen, 1978c). Electrical intercellular communication has also been demonstrated in m a m m a l i a n Send offprint requests to O. H. Petersen at the above address
* MRC Research Fellow
salivary glands (Roberts et al., 1978; H a m m e r and Sheridan, 1978; Kater and Galvin, 1978) and lacrimal glands (Iwatsuki and Petersen, 1978b). Intercellular electrical communication can be modulated by: changes in intracellular [Ca 2+] (Loewenstein and Rose, 1978), changes in intracellular p H (Turin and Warner, 1977) and action of transmitters and peptide hormones (Iwatsuki and Petersen, 1978a, c). It would be very useful to have a method allowing assessment of the influence of hormones on cell to cell passage of organic molecules in living mammalian epithelia. The methods so far applied to demonstrate transfer of organic molecules from cell to cell ( H a m m e r and Sheridan, 1978; Kater and Galvin, 1978) in mammalian preparations, are not entirely satisfactory since they largely depend on observations made on fixed sectioned material and do not allow simultaneous fluorescence microscopy and electrophysiology. We are now describing a technique enabling us to observe directly the transfer of various fluorescent probes from cell to cell in living acini from superfused segments of mouse pancreas. We believe that this method will be useful for studies on the control and importance of intercellular channels. A live demonstration of our method was made at a recent meeting of The Physiological Society (Iwatsuki, 1978).
Materials and Methods A general outline of the set-up is shown in Fig. l. A thin translucent segment (2 • 2mm) of mouse pancreas was placed on a perspex platform in a bath. The bath was placed on the stage ofa Zeiss (Jena) (Technival) microscope in such a way that a light beam from below was focussed on to the perspex platform with the pancreatic tissue. The bath (t6ml) was perfused with a physiological salt solution (20ml/min) prewarmed to 37~ The solution contained (raM): NaC1, 103; KCI, 4.7; CaC12, 2.6; MgC12, 1.1; NaHCO3, 25; NaH2PO4, 1.2; D-glucose,2.8; Na pyruvate, 4.9; Na fumarate, 2.7; Na glutamate, 4.9; and was gassed with 95~ 02, 5~ CO2. The
0031-6768/79/0380/0277/S 01.00
278
Pfliigers Arch. 380 (1979)
microscope was fitted with a catadioptric objective (40 x , 2 cm working distance) (Zeiss0 Jena, 302355:001.26) and the field under examination viewed through eyepieces of 16 x or 8 x magnification. A camera could be attached to the microscope (Fig. l). Photographs of bright field images were taken using an automatic exposure unit. The microscope was also attached to a lighting unit (Zeiss, Jena, HBO 200) projecting a high intensity of incident ultraviolet light (Hgvapour lamp, BG 12 filter). In the cases of fluorescence microscopy the field was viewed through a yellow filter (OG 1). The lighting arrangement allowed rapid shifts between incident blue light and transmitted ordinary light or an appropriate mixing of the two, permitting alternate observation in dark field (fluorescence picture) and bright field, or a mixture of both. Photographs during dark field microscopy were taken using an exposure time of 1 min. Under visual control one, two or three glass microelectrodes could be inserted into acinar cells using Leitz micromanipulators. Glass micropipettes were pulled on a Palmer microelectrode puller and filled with 3mol - 1-1 KCI, 100mmol - l 1 Na-fluoresceinate (BDH) in 0.5 m o l 1-1 KC1 or procion yellow MX-4R (ICI) 4 % w/v) by the fibre glass method. The 3 m o t . 1-~ KCI microelectrodes had resistances of about 50 - 60 Mr2 immediately after filling and were subsequently beveled on a Sutter Instrument Co. beveler (Brown and Flaming, 1974) to attain final resistances of 3 0 - 5 0 M f L Procion yellow or fluorescein were injected into the cells by passing hyperpolarizing current pulses (100-500ms, 2 - 2 0 h A ) at a rate of l/s through the electrodes. Membrane potential and resistance were
Camera Hg-vapourlamp BG12
I ~Ocu[ar
I...............;
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fig. 1. A diagram showing some of the main features of the set-up
measured as previously described (Iwatsuki and Petersen, 1978a) using W P M 701 and 750 electrometer amplifiers. Potentials were displayed on a Tektronix double-beam storage oscilloscope and a Devices MX2 pen recorder.
Results
Visualization of Acinar Cells Figure2 shows a photograph of the edge of the pancreatic preparation in the bath. Individual cells can clearly be distinguished. The presence of numerous zymogen granules in one part of the cells (luminal) is also seen. The best image was always obtained at the edges of the preparation. During a prolonged period (1 h) of stimulation (ace@choline, 10 -6 M) these granules gradually disappeared. Figure 2 demonstrates the unique property of the mouse pancreas; no other mammalian exocrine gland preparation is thin enough to allow such easy identification of individual acinar cells.
Intracellular Injection of Fluorescein Insertion of a micropipette filled with fluorescein (tool. wt. 332) into an acinar cell was followed almost immediately by an intense fluorescence from that cell (Fig. 3). This fluorescence could be abolished by applying a depolarizing current (retaining current). Within the first minute of applying fluorescein fluorescence was also apparent in a neighbouring cell (Fig. 3b) and within the next few minutes spread to several neighbouring cells could be observed (Fig.3c). After stopping the fluorescein injection the fluorescence in all cells gradually disappeared. In many experiments several microelectrodes were inserted into neighbouring cells and electrical coupling could also be assessed. In all cases where fluorescein spread was observed electrical coupling with high coupling coefficients (>0.7) (Iwatsuki and Petersen, 1978c) were obtained.
Fig. 2 Photograph of edge of pancreatic preparation in the bath, Length of bar: 20 ~tm
N. Iwatsuki and O. H. Petersen: Cell to Cell Coupling
279 neighbouring acinar cells, but the fluorescence was not reversible within the period of the experiment (several hours). Also the outline of individual cells was not so clear as in the case of true intracellular deposition (Fig. 3).
Intracellular Injection of Procion Yellow
Fig.3. a Injection of fluorescein into an acinar cell through a microelectrode. Spread of fluorescein to one neighbouring cell. Mixture of bright and dark field. Photograph taken 1.5 min after start of fluorescein iontophoresis (300 ms pulses, 5 nA, l/s). Bar: 20 itm. x 750. b Same fieId as in a, this time viewed in dark field. This photograph was taken 3 min after start of fluorescein iontophoresis. Fluorescence has now spread to two further cells. Bar: 20 ram. x 750. e Same field as in a and b, 5 min after start of fluorescein injection. Fluorescence detectable in several neighbouring cells. Bar: 20gin. x 750
The correct intracellular location of the injection pipette was found to be crucial for meaningful results. While fluorescein ejected from the micropipette with the tip in a free extracellular position did not cause any observable fluorescence, very intense fluorescence could be obtained when the tip of the fuorescein micropipette just touched the cell membrane. In such cases fluorescence spread over the surface of m a n y
Figure 4 shows the result of an experiment in which one microelectrode filled with procion yellow (mol. wt. 697) was inserted into a superficial mouse pancreatic acinus. lontophoresis ofprocion yellow into the impaled acinar cell was signalled by an intense fluorescence in the injection cell followed by the appearance of fluorescence in two neighbouring cells (Fig. 4b, c). In contrast to fluorescein procion yellow only moved in one direction and always in a direction opposite to that in which the inserted microelectrode was pointing. Insertion of a microelectrode always causes an elevation of [Ca2+]i mainly localized to the tip of the electrode (B. Rose, personal communication) and it is likely that this may block the passage of particularly higher molecular weight compounds (Loewenstein and Rose, 1978). It is also possible that the microelectrode tip could influence the permeability of gap junctional membranes by a direct physical effect (pressure, stretch). Fluorescence spread was distinctly slower than for fluorescein and could therefore easily be quantified. Fluorescence in a neighbouring cell could be detected 1 - 4 m i n [mean: 2.4 + 1.1 (s.d.) min, n = 15] after start of procion yellow injections. Transfer of fuorescence was observed in 75 ~ of attempted injections. In 7 cases in which procion yellow transfer to neighbouring cells was observed a second microelectrode was inserted into a neighbouring cell. Electrotonic potential changes set up by current passage through the dye-containing microelectrode (dye injection) were in all these experiments transmitted to the microelectrode in the neighbouring cells. The precise coupling ratio could, however, not be measured since the tip resistance of the procion yellow-containing microelectrode was high and furthermore fluctuated considerably, making accurate compensation of tip resistance (balancing) impossible. After cessation of dye injection (reversal of the polarity of current injection) the fluorescence in all cells gradually diminished and after several minutes had disappeared.
Discussion This work demonstrates the feasibility of studying directly the movement of fluorescent probe molecules in a living m a m m a l i a n gland tissue. Figures 3 and 4 represent the first clear demonstration of cell to cell
280
Pflfigers Arch. 380 (1979)
movement of fluorescent tracers in mammalian exocrine glands. Two previous studies (Hammer and Sheridan, 1978; Kater and Galvin, 1978) on mammalian salivary glands have been reported. The documentation in the papers of Hammer and Sheridan (1978) and Kater and Galvin (1978) is, however, almost exclusively based on examination of fixed sectioned material. We feel that success in clearly demonstrating cell to cell movement of fluorescent probe molecules depends on: 1. the ability to dearly visualize individual cells in the living preparation (Figs. 2, 3, 4), 2. the ability to switch rapidly from bright to dark field images without the need to move the specimen or change the whole set-up of microscopes, 3. a high magnification objective with a long working distance permitting easy access with several microelectrodes, 4. correct intracellular localization of injection micropipettes. The technique outlined in this paper should be useful in probing the effects of various experimental conditions designed to change the resistance of the intercellular junctions on the movement of fluorescent probe molecules. This is important since marked changes in junctional conductance can take place without any change in electrical coupling coefficient (Socolar, 1977). We believe that the mouse pancreas is an ideal tissue for this purpose, since it is thin enough to allow good cellular visualization, the electrophysiology has been explored in more detail than in any other mammalian gland preparation (Petersen, 1979) and we have more information on the cellular stimulus-secretion coupling mechanisms than in other gland tissues (Case, 1978; Petersen and Iwatsuki, 1978; Schulz and Ullrich, 1979). Acknowledgement. This work was supported by grants from the MRC and the Cystic Fibrosis Research Trust. We thank Mr D. Williams (Zeiss Ikon) and Mr D. Doig for extensive and invaluable help with the selection of parts for the fluorescence microscope. Mr D. Doig also gave crucial assistance with the photography.
References Fig.4. a Photograph taken through phase-contrast microscope (bright field) of the edge of mouse pancreas fragment placed in the perspex bath. A microelectrode filled with procion yellow is inserted into a cell situated in the uppermost acinus. Bar: 20 klm. • 830. b Same field as in a, but this time viewed in a mixture of bright field (transmitted ordinary light) and dark field (incident blue light). Photograph taken 5 min after start of procion yellow iontophoresis (10 hA, l/s). The major part of the fluorescence from the injected acinus is orange yellow (procion yellow) whereas the other light spots are yellow green. Bar: 20 ~am. x 830. e Same field as in a and b, but now viewed in dark field. The procion yellow has clearly spread from the injected cell (extreme right) to two cells to the left. Fluorescence was detected in the neighbouring cell (middle cell) 2 min after start of injection. Five minutes after start of injection fluorescence was also detected from a third cell (extreme left). This photograph was taken 6 min after start of procion yellow injection. Bar : 20 I~m. x 830
Bennett, M. V. L. : Physiology of electrotonic junctions. Ann. N.Y. Acad. Sci. 137, 509-539 (1966) Brown, K. T., Flaming, D. G. : Beveling of fine micropipette electrodes by a rapid precision method. Science 185, 693-695 (1974) Case, R. M.: Synthesis, intracellular transport and discharge of exportable proteins in the pancreatic acinar cell and other cells. Biol. Rev. 53, 211-354 (1978) Graf, J., Petersen, O. H. : Cell membrane potential and resistance in liver. J. Physiol. (Lond.) 284, 105-126 (1978) Hammer, M. G., Sheridan, J. D. : Electrical coupling and dye transfer between acinar cells in rat salivary glands. J. Physiol. (Lond.) 275, 495-505 (1978) Iwatsuki, N. : Direct demonstration of cell-to-cell communication in mammalian pancreatic acini: transfer of fluorescent probe molecules. J. Physiol. (Lond.) 285, 1 - 2 P (1978)
N. Iwatsuki and O. H. Petersen: Cell to Cell Coupling Iwatsuki, N., Petersen, O. H. : Pancreatic acinar cells: ace@cholineevoked electrical uncoupling and its ionic dependency. J. Physiol. (Loud.) 274, 8 1 - 9 6 (1978a) Iwatsuki, N., Petersen, O. H. : Membrane potential, resistance and intercellular communication in the lacrimal gland: effects of acetylcholine and adrenaline. J. Physiol. (Lond.) 275, 507 520 (1978b) Iwatsuki, N., Petersen, O. H. : Electrical coupling and uncoupling of exocrine acinar cells. J. Cell Biol. 79, 533-545 (1978c) Kater, S. B., Galvin, N. J. : Physiological and morphological evidence for coupling in mouse salivary gland acinar cells. J. Cell Biol. 79, 2 0 - 2 6 (1978) Loewenstein, W. R. : Permeability of membrane junctions. Ann. N.Y. Acad. Sci. 137, 441-472 (1966) Loewenstein, W. R., Kanno, Y. : Studies on an epithelial (gland) cell junction. I. Modifications of surface membrane permeability. J. Cell Biol. 22, 565-586 (t964) Loewenstein, W. R., Rose, B. : Calcium in (junctional) intercellular communication and a thought on its behaviour in intracellular communication. Ann. N.Y. Acad. Sci. 307, 285-307 (1978)
281 Petersen, O. H. : Electrophysiology of gland cells. Monograph of the physiological society. London, New York: Academic Press (in press, 1979) Petersen, O. H., Iwatsuki, N. : The role of calcium in pancreatic acinar cell stimulus-secretion coupling: an electrophysiological approach. Ann. N.Y. Acad. Sci. 307, 599-615 (1978) Roberts, M. L., Iwatsuki, N., Petersen, O. H. : Parotid acinar cells : Ionic dependence of acetylchotine-evoked membrane potential changes. Pflfigers Arch. 376, 1 5 9 - 167 (1978) Schulz, I., Ullrich, K. J.: Transport processes in the exocrine pancreas. In: Membrane transport in biology, Vol. IV (G. Giebisch, D. J. Tosteson, and H. H. Ussing, eds.). Berlin, Heidelberg, New York: Springer (in press, 1979) Socolar, S. : The coupling coefficient as an index of junctional conductance. J. Membr. Biol. 34, 2 9 - 3 7 (1977) Turin, L., Warner, A. : Carbon dioxide reversibly abolishes ionic communication between cells of early amphibian embryo. Nature 270, 5 6 - 57 (1977)
Received February 13, 1979
