565th MEETING, STIRLING
1003
FUNCTIONAL DIFFERENTIATION OF THE MAMMALIAN PLASMA MEMBRANE : a Colloquium organized on behalf of the Membrane Group by W. H. Evans (London) The Flow and Turnover of Plasma Membrane Z. A. COHN Rockefeller University, New York, N. Y. 10021, U.S.A.
Role of the Plasma Membrane in Endocytosis in Fibroblasts P. TULKENS, Y. J. SCHNEIDER and T. TROUET
International Institute of Cellular and Molecular Pathology, Brussels, Belgium
The Mechanism of Catecholamine Secretion:a Hypothesis for NeurotransmitterRelease JOHN H. PHILLIPS
Department of Biochemistry, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, Scotland, U.K. It is part of neurophysiological ‘dogma’ that neurotransmitters are released from nerve terminals in quanta or pulses containing roughly similar numbers of transmitter molecules. This is well established for acetylcholine release (Katz, 1971) and the concept is generally extended to noradrenaline release from sympathetic neurons. Since the discovery that most of the acetylcholine of the cerebral cortex is stored in synaptic vesicles (Whittaker et al., 1964), biochemists have devoted considerable energy to trying to answer the question of whether a quantum is to be identified with the complete content of a vesicle. The systems most accessibleto biochemists have been adrenergic, and work has been greatly aided by the similarity between the storage and release of noradrenaline in sympathetic nerves and of catecholamines in the adrenal medulla, a compact tissue that lends itself to subcellular-fractionationstudies and biochemical analysis. Banks & Helle (1965) showed that release from the gland was by exocytosis, a fusion of storage-granule membrane with plasma membrane in a calcium-dependent process (Douglas, 1968). A large body of evidence was then accumulated showing that similar mechanisms operate in adrenergic neurons (reviewed by Smith & Winkler, 1972). Exocytosis is generally thought of as an ‘all-or-none’ phenomenon. Membrane fusion is followed by expulsion of the soluble contents of the storage granule or synaptic vesicle to the extracellular space, as in the release of pancreatic zymogens (Palade, 1975) or the discharge of Tetrahymena mucocysts (Satir et al., 1973). Careful analysis has revealed a paradox, however. First, there is suggestiveevidence for both cholinergicand adrenergic neurons that the content of transmitter molecules in a quantum is considerably less than that in a vesicle (reviewed by S t j b e , 1975), perhaps of the order of 10%. Secondly, Marchbanks (1975) has stressed that, for cholinergic neurons, turnover studies support the idea that the transmitter is released from a pool only loosely bound to vesicles, rather than from inside the vesicle itself. A major argument in favour of transmitter release by exocytosis has always been that protein components of adrenergic vesicles [chromogranin A, probably involved in catecholamine storage, and the biosynthetic
VOl. 4
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BIOCHEMICAL SOCIETY TRANSACTIONS
enzyme dopamine (3,4-dihydroxyphenethylamine) b-hydroxylase] are released concomitantly with noradrenaline and ATP, the small molecules present in the vesicles. Recent studies with perfused cat adrenal glands suggest, however, that stoicheiometry between these components may not be maintained in the case of glands perfused in situ and stimulated via the splanchnic nerve (30 Hz) or by low acetylcholine concentrations: very little dopamine 8-hydroxylase was released compared with catecholamine (Dixon et al., 1975). These results, suggesting a preferential release of small molecules, contrast with the results of Viveros et al. (1971); they showed that 4 h of insulin treatment decreased the amount of catecholamine and dopamine b-hydroxylase in rabbit adrenal chromaffin granules, but did not alter their ratio, and this was interpreted in terms of an ‘all-or-none’ release. If total vesicle contents are released in neurotransmission, subsequent recovery of vesicle membranes from sites of exocytosis will produce ‘ghosts’, capable of accumulating only relatively low concentrations of transmitter for further cycles of release. Chromaffin granules, for example, contain catecholamines at a concentration estimated as 0.6 M (Hillarp, 1959), and this is presumably only possible because small molecules are bound in an osmotically inactive form to protein (in a complex with ATP, reviewed by Pletscher et al., 1974), which would be released during exocytosis. It is supposed that similar storage mechanisms occur in cholinergic vesicles : Torpedo vesicles, for example, contain the protein vesiculin, which may play a role similar to that of chromogranin (Whittaker, 1974). Much evidence suggests, however, that during low-frequency stimulation of a cholinergic neuron, vesicles fuse with the plasma membrane (and can then take upextracellular tracers), are recovered and can be re-utilized without decrease of quanta1 content (Hurlbut & Ceccarelli, 1974). These conflicting ideas can be reconciled by postulating that, while transmitter release does occur from vesicles, the time-course of the membrane fusion event is an important parameter. Calcium entry accompanying plasma-membrane depolarization is a rapid process (Katz & MiIedi, 1967), mainly utilizing a potential-dependent calcium channel (Baker, 1972). With a free Cazf-ion-concentration gradient of about lo4 ( 2 m extracel~ Marly) the concentration of ions on the inside of the plasma membrane will be transitorily high (Baker et al., 1971) and this may lead to adhesion of vesicles to the membrane, perhaps within 1ms. By contrast, exocytosis is probably a slow process; complete membrane fusion and release of contents may take about lOOms (Hksch, 1962) or much longer (Rohlich et al., 1971). The complete exocytotic process presumably accounts for zymogen release from the pancreas and the parotid gland; in both these cases, granules can also fuse with the membranes of other granules that have already fused with the plasma membrane. A fair amount is known, or can be guessed, about the molecular organization of chromaffin granules and of sympathetic nerve vesicles. (This is not the case with cholinergic synaptic vesicles, although by implication I suggest that the argument can be extended to these.) Catecholamines are present in two pools: as a storage complex with ATP and protein, and as molecules free in solution within the granules; the latter are maintained in equilibrium, exchanging slowly with the stored complex (Klein & Harden, 1975) and being lost by leakage to the cytoplasm, to be pumped back inside the granule in an ATP-requiring process (Kirshner, 1962).I suggest that, when adhering to the plasma membrane in the presence of Ca2+ions, catecholamines may diffuse not only across the granule membrane, but across the plasma membrane as well: it is this process that provides a ‘quantum’ of transmitter. Rapid removal of cytoplasmic Caz+ ions may reverse the adhesion process; but if, on the other hand, granules or vesicles remain attached to the plasma membrane for a sufficiently long time, membrane reorganization and total exocytosis occur. The key event is thus seen to be the calcium-recovery process, or perhaps the time-course of calcium entry. The hypothesis suggests that elevated intracelluIar Cat+ ion concentrations are extremely transient in nerve terminals, but more prolonged in, for example, an exocrine cell; the adrenal medulla may occupy an intermediate position. Alternatively, but less likely, the process might be regulated by the actual intracellular Ca2+ ion concentration that is reached. 1976
565th MEETING, STIRLING
1005
Chromafin granules as an experimental model It is difficult to devise adequate biochemical tests of very transient events. I have used the effect of Caz+ions on isolated chromaffin granules as a model, albeit rather an unsatisfactory one. Although highly purified preparations of granules are easily made (Smith & Winkler, 1967), the adrenal-medulla plasma membrane is not so accessible. Edwards et al. (1974) showed, however, that isolated chromaffin granules undergo a remarkable morphological change in the presence of high concentrations of bivalent cations. [The concentrations used were sufficient to bind Caz+ ions to at least half the possible binding sites (Dean & Matthews, 1974) and were chosen in order to maximize the effects observed by electron microscopy.] In the presence of 5 mM-CaC1, the granules not only aggregated, but deformed, part of their cores of protein and catecholamine becoming electron-translucent. In many cases the membranes between the granules disappeared, a process resembling that occurring in exocytosis. On removing CaZCwith a chelating agent, however, granules either lysed or reverted to their normal form, even though all present had been affected by the calcium treatment used. This suggests that granules alone may be a model for the release process; in this case high CaZ+ion concentrations produce osmotically stabIe aggregates. Interactions between granules and the plasma membrane would be similar, but would occur at a lower Ca2+concentration, with a smaller area of contact. This would be unstable and would lead eventually to membrane rupture and exocytotic release, following an increase in the osmotic pressure of the granule (Edwards et al., 1974). Taking this experiment further, we may ask whether lower concentrations of Caz+ ions, insufficient to lead to extensive rupture of granule membranes on their withdrawal, will permit small molecules to be transferred between granules, in the absence of large holes being made in the granule membranes. This experiment can be performed since, in addition to granules, it is possible to prepare resealed ‘ghosts’, osmotically active empty granule membranes (Phillips, 1974), and to separate these from intact granules on density gradients. Fig. 1 shows the results of an experiment to test this. Chromaffin granules, labelled with 32Pin their membranes (Phillips, 1973), were loaded with (-)[3H]noradrenaline. The radioactive granules were separated from the incubation medium on a small column of Sephadex G-50, and were then incubated with purified non-radioactive resealed ‘ghosts’ in the presence of 1.5 m ~ - E D T A(control), or of I mMCaCl2, in an iso-osmotic sucrose medium. After 15min at 20°C, 1. S ~ M - E D T A was added to the latter sample, and granules and ‘ghosts’ were separated on an iso-osmotic metrizamide gradient (S. J. Morris & I. Schovanka, personal communication). Fig. 1 shows particle-bound radioactivity through the gradients. If granules bind to ‘ghosts’ and their membranes rupture, removal of CaZ+by EDTA leads to loss of radioactive noradrenaline to the medium, and the formation of a radioactive (”P) ‘ghost’ from the granule membrane. Only in cases where the two adhering membranes remain intact during calcium treatment will the addition of EDTA lead to formation of a non-radioactive ‘ghost’ containing [3H]noradrenaline, which bands in the middle of the gradient (fraction 6 in this experiment). Fig. 1 suggests that, under the conditions used, there is little granule lysis, but a small proportion of noradrenaline is transferred to the ‘ghosts’. This is, in fact, dependent on the calcium concentration used, the noradrenaline found with the ‘ghosts’ is but at low concentrations (below 0.1 m) small compared with the background on the gradient; presumably few contacts are made and there is little transfer. In conclusion, I suggest that in nerve terminals CaZ+ion influx induces transient contact between vesicles and plasma membrane. Release of the intravesicular soluble pool of transmitter commences, normally rapidly terminated by Caz+ ion removal. Prolonged contact, however, leads to a greater degree of membrane reorganization, presumably permitting loss of protein molecules and uptake of tracers. Complete rupture leads to total loss of contents (exocytosis). As yet there are no clues about molecular mechanisms, although it is known that the chromaffin-granule interaction is accompanied by movement of membrane particles
Vol. 4
1006
BIOCHEMICAL SOCIETY TRANSACTIONS
I
I
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Fraction number Fig. 1. Density-gradientseparation of radioactivegranules incubated with non-radioactive ‘ghosts’ in thepresence of (a)EDTA and (6)1mM-CaClzfollowed by I S ~ M - E D T A Radioactive granules were prepared by incubation with [y-32P]ATP(Phillips, 1973) followed by (-)-[3H]noradrenaline (Phillips, 1974). They were separated from this incubation medium on a column (7cmx0.7cm) of Sephadex G-50 equilibrated with 0 . 3 ~ sucrose containing 10m-Hepes, pH7.0. They were then incubated (approx. O.Smg/ml) with purified non-radioactive ‘ghosts’ (Phillips, 1974) (approx. 0.3 mg/ml) in buffered sucrose containing (a) 1.5mM-EDTA and (6) l.Orn~-CaCl,. After 15min at 20”C, 1.5mM-EDTA was added to (b) and both samples were centrifuged (80min at 200000g at 4°C) through sucrose/metrizamide gradients. These contained mixtures of sucrose and metrizamide at approx. 0 . 3 5 ~with , a relative density of 1.15 (fraction 1, bottom of tube) to 1.05 (fraction 9). In this system mitochondria are found as a pellet at the bottom of the tube. Twelve fractions were collected, diluted with 0.3~-sucroseand filtered through 0.45pm Millipore filters which were counted for radioactivity in a scintillation counter. 0, 32P;0 , 3H.
away from the adhesion region (R. Schober & S. J. Morris, personal communication), as for mucocyst discharge (Satir et al., 1973) and cell fusion (Ahkong et al., 1975). It has also been suggested that rearrangement of charged lipids under the influence of Caz+ions may increase the permeability of the chromaffin-granulemembrane to small molecules (Neumann & Rosenheck, 1972), but the possibility still remains that calciumsensitive membrane proteins are required for channel formation. 1976
565th MEETING, STIRLING
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This work was supported by a grant from the Medical Research Council. Ahkong, Q. F., Fisher, D., Tampion, W. & Lucy, J. A. (1975)Nature (London) 253,194-195 Baker, P. F. (1972)Prog. Biophys. Mol. Biol. 24,177-223 Baker, P. F., Hodgkin, A. L. & Ridgway, E. B. (1971)J. Physiol. (London) 218,709-755 Banks,P. & Helle, K. (1965)Biochem.J. 97,40c-41 c Dean, P. M. & Matthews, E. K. (1974)Biochem.J. 142,637-640 Dixon, W. R., Garcia, A. G. & Kirpekar, S. M. (1975)J. Physiol. (London) 244,805-824 Douglas, W. W. (1968)Br. J. Phurmacol.34,451474 Edwards, W., Phillips, J. H. & Moms, S.J. (1974)Biochim. Biophys. Acru 356,164-173 Hillarp, N.-A. (1959)ActaPhysiol. S c d . 47,271-279 Hirsch, J. 0.(1962)J. Exp. Med. 116,827-834 Hurlbut, W . P. & Ceccarelli, B. (1974)Adv. Cytophurmacol.2,141-154 Katz, B. (1971)Science 173,123-126 Katz, B. & Miledi, R. (1967)J. Physiol. (London) 189,535-544 Kirshner, N.(1962)J.Biol. Chem. 237,2311-2317 Klein, R. L. &Harden, T.K. (1975)Life Sci. 16,315-322 Marchbanks, R. M. (1975)Znt. J. Biochem. 6,303-312 Neumann, E.& Rosenheck, K. (1972)J. Membr. Biol. 10,279-290 Palade, G . (1975)Science 189,347-358 Phillips, J. H. (1973)Biochem.J. 136,579-587 Phillips, J. H. (1974)Biochem.J. 144,311-318 Pletscher, A.,Da Prada, M., Berneis, K. H., Steffen, H., Lutold, B. & Weder, H. G. (1974)Adu. Cytopharmacol.2,257-264 Rohlich, P., Anderson, P. & Uvnb, B. (1971)J. Cell Biol. 51,465483 Satir, B., Schooley, C. & Satir, P. (1973)J. Cell Biol.56, 153-176 Smith, A. D. & Winkler, H. (1967)Biochem.J. 103,480-482 Smith, A. D.& Winkler, H. (1972)H d .Exp. Pharmacol. 33,538-617 S t j h e , L. (1975)H d .Psychopharmacol.6,179-233 Viveros, 0.H., Arqueros, L. & Kirshner, N. (1971)Mol. Pharmacol. 7,444-454 Whittaker, V. P. (1974)A h . Cytopharmacol.2,311-318 Whittaker, V. P., Michaelson, I. A. & Kirkland, R. J. A. (1964)Biochem.J. 90,293-303
The Liver Plasma Membrane as a Functional Mosaic W. HOWARD EVANS National Institute for Medical Research, The Ridgeway, Mill Hill, London
NW7 lAA, U.K.
The plasma membrane of the mammalian liver parenchymal cell may be pictured as a mosaic of three functionally differentiated domains or regions merged into a single physical entity. A conglomeration of functions takes place at each of these three sides of the hepatocyte and these are detailed in Table 1. Not only d o these plasma-membrane regions interface at different external environments, but also this tangential or regional differentiation of the plasma membrane is probably dictated by the extensive intracellular metabolic compartmentation of the hepatocyte, and this argues that the cytoplasmic environment of the regions is also likely to vary. To study the biochemical properties and synthesis of this multifunctional surface membrane, and especially to explain in the context of modern views of membrane organization how a membrane is regionally adapted to carry out radically different physiological functions, plasmamembrane fractions originating from blood sinusoidal, bile canalicular and contiguous (lateral) regions of the hepatocyte were prepared. Identification of the plasma-membrane fractions
Six plasma-membrane subfractions were purified from low- and high-speed pellets of rat livers dispersed in hypo- and iso-osmotic media and those fractions enriched in contiguous canalicular and sinusoidal plasma-membrane fragments were identified by VOl. 4
1003
FUNCTIONAL DIFFERENTIATION OF THE MAMMALIAN PLASMA MEMBRANE : a Colloquium organized on behalf of the Membrane Group by W. H. Evans (London) The Flow and Turnover of Plasma Membrane Z. A. COHN Rockefeller University, New York, N. Y. 10021, U.S.A.
Role of the Plasma Membrane in Endocytosis in Fibroblasts P. TULKENS, Y. J. SCHNEIDER and T. TROUET
International Institute of Cellular and Molecular Pathology, Brussels, Belgium
The Mechanism of Catecholamine Secretion:a Hypothesis for NeurotransmitterRelease JOHN H. PHILLIPS
Department of Biochemistry, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, Scotland, U.K. It is part of neurophysiological ‘dogma’ that neurotransmitters are released from nerve terminals in quanta or pulses containing roughly similar numbers of transmitter molecules. This is well established for acetylcholine release (Katz, 1971) and the concept is generally extended to noradrenaline release from sympathetic neurons. Since the discovery that most of the acetylcholine of the cerebral cortex is stored in synaptic vesicles (Whittaker et al., 1964), biochemists have devoted considerable energy to trying to answer the question of whether a quantum is to be identified with the complete content of a vesicle. The systems most accessibleto biochemists have been adrenergic, and work has been greatly aided by the similarity between the storage and release of noradrenaline in sympathetic nerves and of catecholamines in the adrenal medulla, a compact tissue that lends itself to subcellular-fractionationstudies and biochemical analysis. Banks & Helle (1965) showed that release from the gland was by exocytosis, a fusion of storage-granule membrane with plasma membrane in a calcium-dependent process (Douglas, 1968). A large body of evidence was then accumulated showing that similar mechanisms operate in adrenergic neurons (reviewed by Smith & Winkler, 1972). Exocytosis is generally thought of as an ‘all-or-none’ phenomenon. Membrane fusion is followed by expulsion of the soluble contents of the storage granule or synaptic vesicle to the extracellular space, as in the release of pancreatic zymogens (Palade, 1975) or the discharge of Tetrahymena mucocysts (Satir et al., 1973). Careful analysis has revealed a paradox, however. First, there is suggestiveevidence for both cholinergicand adrenergic neurons that the content of transmitter molecules in a quantum is considerably less than that in a vesicle (reviewed by S t j b e , 1975), perhaps of the order of 10%. Secondly, Marchbanks (1975) has stressed that, for cholinergic neurons, turnover studies support the idea that the transmitter is released from a pool only loosely bound to vesicles, rather than from inside the vesicle itself. A major argument in favour of transmitter release by exocytosis has always been that protein components of adrenergic vesicles [chromogranin A, probably involved in catecholamine storage, and the biosynthetic
VOl. 4
1004
BIOCHEMICAL SOCIETY TRANSACTIONS
enzyme dopamine (3,4-dihydroxyphenethylamine) b-hydroxylase] are released concomitantly with noradrenaline and ATP, the small molecules present in the vesicles. Recent studies with perfused cat adrenal glands suggest, however, that stoicheiometry between these components may not be maintained in the case of glands perfused in situ and stimulated via the splanchnic nerve (30 Hz) or by low acetylcholine concentrations: very little dopamine 8-hydroxylase was released compared with catecholamine (Dixon et al., 1975). These results, suggesting a preferential release of small molecules, contrast with the results of Viveros et al. (1971); they showed that 4 h of insulin treatment decreased the amount of catecholamine and dopamine b-hydroxylase in rabbit adrenal chromaffin granules, but did not alter their ratio, and this was interpreted in terms of an ‘all-or-none’ release. If total vesicle contents are released in neurotransmission, subsequent recovery of vesicle membranes from sites of exocytosis will produce ‘ghosts’, capable of accumulating only relatively low concentrations of transmitter for further cycles of release. Chromaffin granules, for example, contain catecholamines at a concentration estimated as 0.6 M (Hillarp, 1959), and this is presumably only possible because small molecules are bound in an osmotically inactive form to protein (in a complex with ATP, reviewed by Pletscher et al., 1974), which would be released during exocytosis. It is supposed that similar storage mechanisms occur in cholinergic vesicles : Torpedo vesicles, for example, contain the protein vesiculin, which may play a role similar to that of chromogranin (Whittaker, 1974). Much evidence suggests, however, that during low-frequency stimulation of a cholinergic neuron, vesicles fuse with the plasma membrane (and can then take upextracellular tracers), are recovered and can be re-utilized without decrease of quanta1 content (Hurlbut & Ceccarelli, 1974). These conflicting ideas can be reconciled by postulating that, while transmitter release does occur from vesicles, the time-course of the membrane fusion event is an important parameter. Calcium entry accompanying plasma-membrane depolarization is a rapid process (Katz & MiIedi, 1967), mainly utilizing a potential-dependent calcium channel (Baker, 1972). With a free Cazf-ion-concentration gradient of about lo4 ( 2 m extracel~ Marly) the concentration of ions on the inside of the plasma membrane will be transitorily high (Baker et al., 1971) and this may lead to adhesion of vesicles to the membrane, perhaps within 1ms. By contrast, exocytosis is probably a slow process; complete membrane fusion and release of contents may take about lOOms (Hksch, 1962) or much longer (Rohlich et al., 1971). The complete exocytotic process presumably accounts for zymogen release from the pancreas and the parotid gland; in both these cases, granules can also fuse with the membranes of other granules that have already fused with the plasma membrane. A fair amount is known, or can be guessed, about the molecular organization of chromaffin granules and of sympathetic nerve vesicles. (This is not the case with cholinergic synaptic vesicles, although by implication I suggest that the argument can be extended to these.) Catecholamines are present in two pools: as a storage complex with ATP and protein, and as molecules free in solution within the granules; the latter are maintained in equilibrium, exchanging slowly with the stored complex (Klein & Harden, 1975) and being lost by leakage to the cytoplasm, to be pumped back inside the granule in an ATP-requiring process (Kirshner, 1962).I suggest that, when adhering to the plasma membrane in the presence of Ca2+ions, catecholamines may diffuse not only across the granule membrane, but across the plasma membrane as well: it is this process that provides a ‘quantum’ of transmitter. Rapid removal of cytoplasmic Caz+ ions may reverse the adhesion process; but if, on the other hand, granules or vesicles remain attached to the plasma membrane for a sufficiently long time, membrane reorganization and total exocytosis occur. The key event is thus seen to be the calcium-recovery process, or perhaps the time-course of calcium entry. The hypothesis suggests that elevated intracelluIar Cat+ ion concentrations are extremely transient in nerve terminals, but more prolonged in, for example, an exocrine cell; the adrenal medulla may occupy an intermediate position. Alternatively, but less likely, the process might be regulated by the actual intracellular Ca2+ ion concentration that is reached. 1976
565th MEETING, STIRLING
1005
Chromafin granules as an experimental model It is difficult to devise adequate biochemical tests of very transient events. I have used the effect of Caz+ions on isolated chromaffin granules as a model, albeit rather an unsatisfactory one. Although highly purified preparations of granules are easily made (Smith & Winkler, 1967), the adrenal-medulla plasma membrane is not so accessible. Edwards et al. (1974) showed, however, that isolated chromaffin granules undergo a remarkable morphological change in the presence of high concentrations of bivalent cations. [The concentrations used were sufficient to bind Caz+ ions to at least half the possible binding sites (Dean & Matthews, 1974) and were chosen in order to maximize the effects observed by electron microscopy.] In the presence of 5 mM-CaC1, the granules not only aggregated, but deformed, part of their cores of protein and catecholamine becoming electron-translucent. In many cases the membranes between the granules disappeared, a process resembling that occurring in exocytosis. On removing CaZCwith a chelating agent, however, granules either lysed or reverted to their normal form, even though all present had been affected by the calcium treatment used. This suggests that granules alone may be a model for the release process; in this case high CaZ+ion concentrations produce osmotically stabIe aggregates. Interactions between granules and the plasma membrane would be similar, but would occur at a lower Ca2+concentration, with a smaller area of contact. This would be unstable and would lead eventually to membrane rupture and exocytotic release, following an increase in the osmotic pressure of the granule (Edwards et al., 1974). Taking this experiment further, we may ask whether lower concentrations of Caz+ ions, insufficient to lead to extensive rupture of granule membranes on their withdrawal, will permit small molecules to be transferred between granules, in the absence of large holes being made in the granule membranes. This experiment can be performed since, in addition to granules, it is possible to prepare resealed ‘ghosts’, osmotically active empty granule membranes (Phillips, 1974), and to separate these from intact granules on density gradients. Fig. 1 shows the results of an experiment to test this. Chromaffin granules, labelled with 32Pin their membranes (Phillips, 1973), were loaded with (-)[3H]noradrenaline. The radioactive granules were separated from the incubation medium on a small column of Sephadex G-50, and were then incubated with purified non-radioactive resealed ‘ghosts’ in the presence of 1.5 m ~ - E D T A(control), or of I mMCaCl2, in an iso-osmotic sucrose medium. After 15min at 20°C, 1. S ~ M - E D T A was added to the latter sample, and granules and ‘ghosts’ were separated on an iso-osmotic metrizamide gradient (S. J. Morris & I. Schovanka, personal communication). Fig. 1 shows particle-bound radioactivity through the gradients. If granules bind to ‘ghosts’ and their membranes rupture, removal of CaZ+by EDTA leads to loss of radioactive noradrenaline to the medium, and the formation of a radioactive (”P) ‘ghost’ from the granule membrane. Only in cases where the two adhering membranes remain intact during calcium treatment will the addition of EDTA lead to formation of a non-radioactive ‘ghost’ containing [3H]noradrenaline, which bands in the middle of the gradient (fraction 6 in this experiment). Fig. 1 suggests that, under the conditions used, there is little granule lysis, but a small proportion of noradrenaline is transferred to the ‘ghosts’. This is, in fact, dependent on the calcium concentration used, the noradrenaline found with the ‘ghosts’ is but at low concentrations (below 0.1 m) small compared with the background on the gradient; presumably few contacts are made and there is little transfer. In conclusion, I suggest that in nerve terminals CaZ+ion influx induces transient contact between vesicles and plasma membrane. Release of the intravesicular soluble pool of transmitter commences, normally rapidly terminated by Caz+ ion removal. Prolonged contact, however, leads to a greater degree of membrane reorganization, presumably permitting loss of protein molecules and uptake of tracers. Complete rupture leads to total loss of contents (exocytosis). As yet there are no clues about molecular mechanisms, although it is known that the chromaffin-granule interaction is accompanied by movement of membrane particles
Vol. 4
1006
BIOCHEMICAL SOCIETY TRANSACTIONS
I
I
I
I
I
Fraction number Fig. 1. Density-gradientseparation of radioactivegranules incubated with non-radioactive ‘ghosts’ in thepresence of (a)EDTA and (6)1mM-CaClzfollowed by I S ~ M - E D T A Radioactive granules were prepared by incubation with [y-32P]ATP(Phillips, 1973) followed by (-)-[3H]noradrenaline (Phillips, 1974). They were separated from this incubation medium on a column (7cmx0.7cm) of Sephadex G-50 equilibrated with 0 . 3 ~ sucrose containing 10m-Hepes, pH7.0. They were then incubated (approx. O.Smg/ml) with purified non-radioactive ‘ghosts’ (Phillips, 1974) (approx. 0.3 mg/ml) in buffered sucrose containing (a) 1.5mM-EDTA and (6) l.Orn~-CaCl,. After 15min at 20”C, 1.5mM-EDTA was added to (b) and both samples were centrifuged (80min at 200000g at 4°C) through sucrose/metrizamide gradients. These contained mixtures of sucrose and metrizamide at approx. 0 . 3 5 ~with , a relative density of 1.15 (fraction 1, bottom of tube) to 1.05 (fraction 9). In this system mitochondria are found as a pellet at the bottom of the tube. Twelve fractions were collected, diluted with 0.3~-sucroseand filtered through 0.45pm Millipore filters which were counted for radioactivity in a scintillation counter. 0, 32P;0 , 3H.
away from the adhesion region (R. Schober & S. J. Morris, personal communication), as for mucocyst discharge (Satir et al., 1973) and cell fusion (Ahkong et al., 1975). It has also been suggested that rearrangement of charged lipids under the influence of Caz+ions may increase the permeability of the chromaffin-granulemembrane to small molecules (Neumann & Rosenheck, 1972), but the possibility still remains that calciumsensitive membrane proteins are required for channel formation. 1976
565th MEETING, STIRLING
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The Liver Plasma Membrane as a Functional Mosaic W. HOWARD EVANS National Institute for Medical Research, The Ridgeway, Mill Hill, London
NW7 lAA, U.K.
The plasma membrane of the mammalian liver parenchymal cell may be pictured as a mosaic of three functionally differentiated domains or regions merged into a single physical entity. A conglomeration of functions takes place at each of these three sides of the hepatocyte and these are detailed in Table 1. Not only d o these plasma-membrane regions interface at different external environments, but also this tangential or regional differentiation of the plasma membrane is probably dictated by the extensive intracellular metabolic compartmentation of the hepatocyte, and this argues that the cytoplasmic environment of the regions is also likely to vary. To study the biochemical properties and synthesis of this multifunctional surface membrane, and especially to explain in the context of modern views of membrane organization how a membrane is regionally adapted to carry out radically different physiological functions, plasmamembrane fractions originating from blood sinusoidal, bile canalicular and contiguous (lateral) regions of the hepatocyte were prepared. Identification of the plasma-membrane fractions
Six plasma-membrane subfractions were purified from low- and high-speed pellets of rat livers dispersed in hypo- and iso-osmotic media and those fractions enriched in contiguous canalicular and sinusoidal plasma-membrane fragments were identified by VOl. 4
