ARE PROSTAGLANDINSINTRACELLULAR,TRANSCELLULAROR EXTRACELLULARAUTOCOIDS?
Recent experiments have shown that E and F PGs are excluded from the intracellular volume of rabbit erythrocytes. This demonstrates that the plasma membrane is impermeable to these PGs, and most likely, to PGs in general (Bito and Baroody, 1975). We must therefore examine the question whether the synthesis and release, as well as the action and metabolism of PGs, can be accounted for without the postulation of passage through cell membranes , or on the basis of facilitation of such passage by carrier-mediated PG transport processes. Anggard et al. (1972) have shown that PGs are synthesized predominantly by a subcellular fraction of renal papilla, which consisted of both cell membranes and membranes of the endoplasmic reticulum. These authors concluded that after their formation by these membranes, PGs are released into the cytoplasm. Wore recent experiments by Crowshaw (1973) showed, however, that endogeneously syntheti ed PGs do not accumulate in renal medulla slices, but rather all the 12C labeled PGF20 and PGE2 formed from incorporated [14C]arachidonic acid were found in the incubation medium. Crowshaw suggested that this is due to active transport of PGs across the plasma membrane into the extracellular fluids. Although active PG transport has been demonstrated in several in vitro s stems, these, in all cases, could be associated with active -[ H]PG ac umulation, while slices of rabbit renal medulla were shown to i; exclude [sB]PGs -in vitro (Bito, 1972); this exclusion was not prevented by saturating concentrations of PGs or by cold. Thus there is no direct evidence that this particular tissue has any PG transport function. Since the PG synthetase system is membrane-bound, postulation of release of PGs toward the cytoplasm followed by an active transport across these membranes is unwarranted. A much simpler interpretation of Crowshaw's finding is provided by the consideration that cellular membranes are not symmetrical, thus any membrane-bound enzyme system can be expected to show a polarity. An orientation of the last step of the PC synthesis process toward the outer surface of membranes would result in a preferential release of PGs toward the "outside", i.e. into the extracellular space (or the lumen of the endoplasmic reticulum) rather than into the cytoplasm (see Figure 1). Such an externally oriented PG synthetase system would not only account for Crowshaw's finding but may also explain how the lung can release large amounts of metabolically unaltered PGs in spite of the fact that it has very profound 15-hydroxy-PG-dehydrogenase activity. According to the concept proposed here, freshly synthetized PGs (or PG precursors, such as endoperoxides) do not have to pass through the cytoplasm before their release into the extracellular fluids, thus they would not be directly exposed to dehydrogenases or other cytoplasmic or mitochondrial enzyme systems.
PROSTAGLANDINS JUNE 1975
VOL. 9 NO. 6
851
PROSTAGLANDINS
INSIDE II MEMBRANE i OUTSIDE
m= PG m
Cdrrier
= Synthetase
PGp = Precursor
01c=+
STIMULUS
Figure 1. Membrane model of PG synthesis, unidirectional release, action and transport. Appropriate stimulation activates a phospholipase moiety (A), liberating a PG precursor (PGP1) which may be transferred directly to the synthetase moiety (B) of a membrane-bound enzyme complex. The last enzymatic site of the synthetase complex is postulated to be located at the outer surface of the membrane, thus PGs or active PG precursors will be released only toward the extracellular space or into the lumen of the endoplasmic reticulum. The final step of release may be mediated by special carrier molecules (C). Alternately, the precursor could diffuse within the membrane to a synthetase complex at the outer membrane surface (D). In some cells transmembrane PG transport must exist to deliver PGs to their site of metabolism. Such transmembrane transport can be facilitated by carrier-mediated (C -+ C') processes.
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PROSTAGLANDINS
PGM = PG Meta boli te
Figure 2. Passive diffusionaland facilitated movements of PGs. In most peripheralorgans PGs (or their initial metabolites)can enter the blood stream by passive diffusionor by bulk flow through fenestrated capillaries (A). In the case of some specializedepithelia such as the ciliary processesand the choroid plexus (B) or non-fenestrated(tightjunctional)capillariesof the brain and the retina (C) passage of PGs across cellularmembranesmust require carrier-mediatedprocesses (a). Intracellularmetabolismof circulatingPGs by the lung and the excretion of PGs, and their metabolites,by the kidney is also consideredto require facilitatedtransmembranePG transportprocesses.
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PROSTAGLANDINS
Since the microsomal fractionprepared by Anggard et al. was shown to contain a mixture of endoplasmicreticulumand plasma membrane fragments, it is not possible to tell which of these membraneshave PG aynthetasecapacity. We can assume that both do. Productionof PGs is, however, dependenton the liberationof membrane-boundPG precursors. Typical stimuli for gross PG release, such as mechanicalor chemical stimulation,is unlikely to affect the deeper portions of the endoplasmic reticulum. Thus, even if the PG synthetasesystem of all membrane fractionshad similar enzymatic activity,such stimuli could be expected to result in PG release primarily from the plasma membrane and contiguousregions of the endoplasmicreticulum. It is quite clear that the action of PG does not typicallyrequire the penetrationof cell membranes by endogenouslyreleased or exogenous PGs. In cases where the mechanism of action of PGs has been established PG effects were shown to be mediated by the phosphonucleotidecyclase/ cyclic nucleotidesystems. Activationof these systems does not require the entry of the mediator into the cell, since the receptormoieties of the phosphonucleotidecyclases are located on the external surface of cells (Figure1). This, of course, does not rule out the possibility that in some tissues, such as the eye, brain or female reproductive system, the access of PGs to their target site requires their transfer across cellularbarriers. Such transfer across specializedcellular membranes could be achieved by facilitatedor active PG transportprocesses (see Figures 1 and 2). It has already been shown, for example, that the absorptionof PGs from the rabbit vagina is saturableand thereforemust be carrier-mediated(Bito and Spellane,1974). The existenceof active PG transportprocesseshas now been demonstrated unequivocallyin one biologicalsystem (Bito, 1975). In contrast to the apparent orientationof the PG synthetasesystem and the putative "PG receptors"toward the exterior of the cell, metabolism of PGs is clearly intracellular. Thus, the rapid conversionof E and F PGs to their 15-keto derivativesby the lung must, for example, require the essentiallyquantitativetransferof PGs from the pulmonary circulationinto the cells during a single passage of blood through the lung. Such rapid transportcan be achieved by carrier-mediatedprocesses. Some preliminaryevidence has already been presented indicating that the lung, liver and the kidney cortex, tissueswhich are involved with PG metabolismand excretion,have such PG transportcapacity (Bito, 1972). The fact that endogenouslyproduced PGs can be released from the lung in an unmetabolizedform may be explainedon the basis that during the course of massive PG release, the local PG concentrationat the cell membrane exceeds the transportcapacity of the carriers. Alternately, we may consider the possibilitythat a PG precursor such as an endoperoxide,which may not be a substrate for the PG transportprocess, is released from the membrane and convertedto a PG extracellularly,or actually intravascularly,past the region of pulmonary PG carriers. Although more direct experimentswill clearly be required,it is reasonable to assume that intracellularmetabolismof PGs is facilitatedby, and depend on, an initial step of transmembranePG transport (Fig. 2).
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PROSTAGLANDINS
Final disposition of PGs and/or initial PG metabolites must also require their transfer from their site of release into the systemic circulation. Transfer of PGs across the fenestrated capillaries of most organ systems does not require transmembrane permeation. In the case of the brain or the eye, however, passage of any substance across specialized permeability barriers must require transmembrane transfer processes (Figure 2). The existence of such facilitated, saturable, and thus presumably carrier-mediated PG transport processes across the bloodocular fluid (Bito and Salvador, 1972) and blood-brain (Bito and Davson, 1974) barriers has already been demonstrated. Currently available evidence is therefore consistent with the concept that freshly synthetized PGs, or PG precursors such as PG endoperoxides, are released directly into the extracellular fluids of tissues, act on receptors located on the outer surface of cells and enter cells only to be metabolized, excreted or transported across specialized barrier structures. We must conclude, therefore, that the typical mechanism of PG release and action does not involve the accumulation of PGs within cells or the movement of PGs across cellular membranes, thus these autocoids must be regarded as extracellular mediators. Laszlo 2. Bito Department of Ophthalmology College of Physicians and Surgeons Columbia University, N.Y., NY 10032
REFERENCES Anggard, E., S. 0. Bohman, J. E. Griffin III, C. Larsson, A. B. Maunsbach (1972) Subcellular localization of the prostaglandin system in the rabbit renal papilla. Acta Physiol. Stand. 84: 231. Bito, L. Z. (1972) Accumulation and apparent active transport of prostaglandins by some rabbit tissues in vitro. J. Physiol. (Lond.) 221: 371. Bito, L. Z. (1975) Prostaglandin fransport: Saturable, energy-dependent transmembrane transport of [ Hlprostaglandins against a concentration gradient. Submitted to Nature. Bito, L. Z. and R. A. Baroody (1975) Exclusion of E and F prostaglandins from the intracellular volume of rabbit red blood cells (RBCs). Fed. Proc. 34: 791. Bito, L. Z., H. Davson (1974) Carrier-mediated reumval of prostaglandins from cerebrospinal fluid. J. Physiol. (Lond.) 236: 39P. Bito, L. Z., E. V. Salvador (1972) Intraocular fluid dynamics. III. The site and mechanism of prostaglandin transfer across the blood intraocular fluid barriers. Exp. Eye Res. 14: 233. Bito, L. Z., P. J. Spellane (1974) Saturable, "carrier-mediated", from the -in vivo rabbit vagina and absorption of prostaglandin F its inhibition by prostagland zE F 28' Pixstaglandins 8: 345. Crowshaw, K. (1973) The incorporation o [l- Clarachidonic acid into the lipids of rabbit renal slices and conversion to prostaglandins E2 and F20. Prostaglandins 3: 607.
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855
Recent experiments have shown that E and F PGs are excluded from the intracellular volume of rabbit erythrocytes. This demonstrates that the plasma membrane is impermeable to these PGs, and most likely, to PGs in general (Bito and Baroody, 1975). We must therefore examine the question whether the synthesis and release, as well as the action and metabolism of PGs, can be accounted for without the postulation of passage through cell membranes , or on the basis of facilitation of such passage by carrier-mediated PG transport processes. Anggard et al. (1972) have shown that PGs are synthesized predominantly by a subcellular fraction of renal papilla, which consisted of both cell membranes and membranes of the endoplasmic reticulum. These authors concluded that after their formation by these membranes, PGs are released into the cytoplasm. Wore recent experiments by Crowshaw (1973) showed, however, that endogeneously syntheti ed PGs do not accumulate in renal medulla slices, but rather all the 12C labeled PGF20 and PGE2 formed from incorporated [14C]arachidonic acid were found in the incubation medium. Crowshaw suggested that this is due to active transport of PGs across the plasma membrane into the extracellular fluids. Although active PG transport has been demonstrated in several in vitro s stems, these, in all cases, could be associated with active -[ H]PG ac umulation, while slices of rabbit renal medulla were shown to i; exclude [sB]PGs -in vitro (Bito, 1972); this exclusion was not prevented by saturating concentrations of PGs or by cold. Thus there is no direct evidence that this particular tissue has any PG transport function. Since the PG synthetase system is membrane-bound, postulation of release of PGs toward the cytoplasm followed by an active transport across these membranes is unwarranted. A much simpler interpretation of Crowshaw's finding is provided by the consideration that cellular membranes are not symmetrical, thus any membrane-bound enzyme system can be expected to show a polarity. An orientation of the last step of the PC synthesis process toward the outer surface of membranes would result in a preferential release of PGs toward the "outside", i.e. into the extracellular space (or the lumen of the endoplasmic reticulum) rather than into the cytoplasm (see Figure 1). Such an externally oriented PG synthetase system would not only account for Crowshaw's finding but may also explain how the lung can release large amounts of metabolically unaltered PGs in spite of the fact that it has very profound 15-hydroxy-PG-dehydrogenase activity. According to the concept proposed here, freshly synthetized PGs (or PG precursors, such as endoperoxides) do not have to pass through the cytoplasm before their release into the extracellular fluids, thus they would not be directly exposed to dehydrogenases or other cytoplasmic or mitochondrial enzyme systems.
PROSTAGLANDINS JUNE 1975
VOL. 9 NO. 6
851
PROSTAGLANDINS
INSIDE II MEMBRANE i OUTSIDE
m= PG m
Cdrrier
= Synthetase
PGp = Precursor
01c=+
STIMULUS
Figure 1. Membrane model of PG synthesis, unidirectional release, action and transport. Appropriate stimulation activates a phospholipase moiety (A), liberating a PG precursor (PGP1) which may be transferred directly to the synthetase moiety (B) of a membrane-bound enzyme complex. The last enzymatic site of the synthetase complex is postulated to be located at the outer surface of the membrane, thus PGs or active PG precursors will be released only toward the extracellular space or into the lumen of the endoplasmic reticulum. The final step of release may be mediated by special carrier molecules (C). Alternately, the precursor could diffuse within the membrane to a synthetase complex at the outer membrane surface (D). In some cells transmembrane PG transport must exist to deliver PGs to their site of metabolism. Such transmembrane transport can be facilitated by carrier-mediated (C -+ C') processes.
852
JUNE 1975
VOL. 9 NO. 6
PROSTAGLANDINS
PGM = PG Meta boli te
Figure 2. Passive diffusionaland facilitated movements of PGs. In most peripheralorgans PGs (or their initial metabolites)can enter the blood stream by passive diffusionor by bulk flow through fenestrated capillaries (A). In the case of some specializedepithelia such as the ciliary processesand the choroid plexus (B) or non-fenestrated(tightjunctional)capillariesof the brain and the retina (C) passage of PGs across cellularmembranesmust require carrier-mediatedprocesses (a). Intracellularmetabolismof circulatingPGs by the lung and the excretion of PGs, and their metabolites,by the kidney is also consideredto require facilitatedtransmembranePG transportprocesses.
JUNE 1975
VOL. 9 NO. 6
853
PROSTAGLANDINS
Since the microsomal fractionprepared by Anggard et al. was shown to contain a mixture of endoplasmicreticulumand plasma membrane fragments, it is not possible to tell which of these membraneshave PG aynthetasecapacity. We can assume that both do. Productionof PGs is, however, dependenton the liberationof membrane-boundPG precursors. Typical stimuli for gross PG release, such as mechanicalor chemical stimulation,is unlikely to affect the deeper portions of the endoplasmic reticulum. Thus, even if the PG synthetasesystem of all membrane fractionshad similar enzymatic activity,such stimuli could be expected to result in PG release primarily from the plasma membrane and contiguousregions of the endoplasmicreticulum. It is quite clear that the action of PG does not typicallyrequire the penetrationof cell membranes by endogenouslyreleased or exogenous PGs. In cases where the mechanism of action of PGs has been established PG effects were shown to be mediated by the phosphonucleotidecyclase/ cyclic nucleotidesystems. Activationof these systems does not require the entry of the mediator into the cell, since the receptormoieties of the phosphonucleotidecyclases are located on the external surface of cells (Figure1). This, of course, does not rule out the possibility that in some tissues, such as the eye, brain or female reproductive system, the access of PGs to their target site requires their transfer across cellularbarriers. Such transfer across specializedcellular membranes could be achieved by facilitatedor active PG transportprocesses (see Figures 1 and 2). It has already been shown, for example, that the absorptionof PGs from the rabbit vagina is saturableand thereforemust be carrier-mediated(Bito and Spellane,1974). The existenceof active PG transportprocesseshas now been demonstrated unequivocallyin one biologicalsystem (Bito, 1975). In contrast to the apparent orientationof the PG synthetasesystem and the putative "PG receptors"toward the exterior of the cell, metabolism of PGs is clearly intracellular. Thus, the rapid conversionof E and F PGs to their 15-keto derivativesby the lung must, for example, require the essentiallyquantitativetransferof PGs from the pulmonary circulationinto the cells during a single passage of blood through the lung. Such rapid transportcan be achieved by carrier-mediatedprocesses. Some preliminaryevidence has already been presented indicating that the lung, liver and the kidney cortex, tissueswhich are involved with PG metabolismand excretion,have such PG transportcapacity (Bito, 1972). The fact that endogenouslyproduced PGs can be released from the lung in an unmetabolizedform may be explainedon the basis that during the course of massive PG release, the local PG concentrationat the cell membrane exceeds the transportcapacity of the carriers. Alternately, we may consider the possibilitythat a PG precursor such as an endoperoxide,which may not be a substrate for the PG transportprocess, is released from the membrane and convertedto a PG extracellularly,or actually intravascularly,past the region of pulmonary PG carriers. Although more direct experimentswill clearly be required,it is reasonable to assume that intracellularmetabolismof PGs is facilitatedby, and depend on, an initial step of transmembranePG transport (Fig. 2).
854
JUNE 1975
VOL. 9 NO. 6
PROSTAGLANDINS
Final disposition of PGs and/or initial PG metabolites must also require their transfer from their site of release into the systemic circulation. Transfer of PGs across the fenestrated capillaries of most organ systems does not require transmembrane permeation. In the case of the brain or the eye, however, passage of any substance across specialized permeability barriers must require transmembrane transfer processes (Figure 2). The existence of such facilitated, saturable, and thus presumably carrier-mediated PG transport processes across the bloodocular fluid (Bito and Salvador, 1972) and blood-brain (Bito and Davson, 1974) barriers has already been demonstrated. Currently available evidence is therefore consistent with the concept that freshly synthetized PGs, or PG precursors such as PG endoperoxides, are released directly into the extracellular fluids of tissues, act on receptors located on the outer surface of cells and enter cells only to be metabolized, excreted or transported across specialized barrier structures. We must conclude, therefore, that the typical mechanism of PG release and action does not involve the accumulation of PGs within cells or the movement of PGs across cellular membranes, thus these autocoids must be regarded as extracellular mediators. Laszlo 2. Bito Department of Ophthalmology College of Physicians and Surgeons Columbia University, N.Y., NY 10032
REFERENCES Anggard, E., S. 0. Bohman, J. E. Griffin III, C. Larsson, A. B. Maunsbach (1972) Subcellular localization of the prostaglandin system in the rabbit renal papilla. Acta Physiol. Stand. 84: 231. Bito, L. Z. (1972) Accumulation and apparent active transport of prostaglandins by some rabbit tissues in vitro. J. Physiol. (Lond.) 221: 371. Bito, L. Z. (1975) Prostaglandin fransport: Saturable, energy-dependent transmembrane transport of [ Hlprostaglandins against a concentration gradient. Submitted to Nature. Bito, L. Z. and R. A. Baroody (1975) Exclusion of E and F prostaglandins from the intracellular volume of rabbit red blood cells (RBCs). Fed. Proc. 34: 791. Bito, L. Z., H. Davson (1974) Carrier-mediated reumval of prostaglandins from cerebrospinal fluid. J. Physiol. (Lond.) 236: 39P. Bito, L. Z., E. V. Salvador (1972) Intraocular fluid dynamics. III. The site and mechanism of prostaglandin transfer across the blood intraocular fluid barriers. Exp. Eye Res. 14: 233. Bito, L. Z., P. J. Spellane (1974) Saturable, "carrier-mediated", from the -in vivo rabbit vagina and absorption of prostaglandin F its inhibition by prostagland zE F 28' Pixstaglandins 8: 345. Crowshaw, K. (1973) The incorporation o [l- Clarachidonic acid into the lipids of rabbit renal slices and conversion to prostaglandins E2 and F20. Prostaglandins 3: 607.
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855
