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Concentrative accumulation of prostaglandins in vitro at 7 and 37°C by tissues of normothermic and hibernating woodchucks (Marmota monax)
CONCENTRATIVE ACCUMULATION OF PROSTAGLANDINS IN VITRO AT 7 AND 37°C BY TISSUES OF NORMOTHERMIC AND HIBERNATING WOODCHUCKS (MARMOTA MONAX) ERICA V. SALVADOR. JANE C. ROBERTSand LASZLO Z. BITO Research
Division.
Department of Ophthalmology, College of Physicians Columbia University. New York. NY 10032, U.S.A. (Receircld 31 Map
and Surgeons
1977)
Eleven tissues from normothermic and hibernating woodchucks were incubated at 37 or 7 C with [3H]prostaglandins (PC&) A,. E,, F,,. C3H]arachidonic acid and/or [‘4C]sucrose. 2. Choroid plexus. anterior uvea and to a lesser extent, kidney cortex. liver. lung and brown fat showed concentrative PC accumulation at 37°C. White fat and spleen appeared to exclude PGs from their intracellular compartments. 3. With the exception of choroid plexus and kidney cortex of normothermic woodchucks. concentrative C3H]PGE, accumulation was significantly less at 7’C. The choroid plexus showed several-fold PG accumulation even at 7°C indicating that PG removal from the brain can continue during hibernation. 4. Concentrative accumulation of PGA, and PGE, by brown fat suggests that circulating PGs may be involved in the metabolic control of this tissue and hence may play a role in arousal. Abstract--l.
INTRODUCTION It has been shown (Bito & Roberts, 1974) that in spite of low body temperature, the chemical composition of the cerebrospinal and intraocular fluids of hibernating woodchucks is distinctly different from that of plasma ultrafiltrate. In fact, the concentrations of several chemical constituents which are known to be transported into or out of these fluid compartments remain remarkably normal even after several days of hibernation. These findings suggest that some transport activities across the blood-brain and blood-ocular fluid barriers must effectively continue at a body temperature of 7-10°C. In vitro accumulation of certain substances. such as iodide and iodopyracet, by the choroid plexus or anterior uvea has been used extensively as an index of in ho transport functions across the blood-cerebrospinal or blood-ocular fluid barriers (cf. Cserr. 1971; Becker, 1962; Kinsey, 1971). Recently, it was shown that prostaglandins (PGs) which are produced in both the eye and the brain, but are not locally metabolized. are accumulated by both the choroid plexus and the anterior uvea it? vitro (Bito. 1972a.h) and are removed from the extracellular fluids of the eye and the brain by facilitated transport processes in ko (Bito & Salvador, 1972; Bito et al., 1976). Some evidence has been presented indicating that the facilitated removal of PGs from the extracellular fluid of these organs has a physiological function (Bito & Wallenstein, 1977). These investigations were undertaken to determine which tissues of the woodchuck show concentrative PG accumulation in citro. whether this concentrative activity is maintained at an incubation temperature comparable to the body temperature of the hibernating animal, and whether there is a shift in the tem-
perature optimum of these apparent cesses during hibernation.
transport
pro-
MATERIALS AND METHODS Tissues were obtained from hibernating and from anesthetized, normothermic woodchucks. following the collection of samples used for other experiments (Bito & Roberts, 1974; Bito et al., 1973). Tissue pieces or slices weighing not more than IOOmg each were incubated for 0.5 or 1 hr at 7 or 37.‘C in oxygenated tissue culture medium (Basal Medium Eagle, Microbiologlcal Associates. Bethesda. Md.) containing a [3H]labelied prostaglandin E,. A,. F1, or arachidonic acid, and in some cases also [‘4C]sucrose to measure extracellular space. After removal from the medium. the tissue pieces were lightly blotted on moist filter paper. weighed on an electrobalance. placed in scintillation solvent solution (Aquasol; New England Nuclear, Boston. MA.) and eluted with continuous shaking overnight. In one experiment. half of the tissue pieces were prepared for isotope counting by a Packard Model 306 automatic tissue oxidizer. Aliquots (100 ~1) of the incubation medium were made up in the same scintillation solvent or oxidized by the same technique. All samples were counted for a minimum of 10,000 counts in a Packard Tricarb scintillation counter. Values are reported as the tissue-to-medium (T!M) accumulation ratio: (-‘H activity per lOOmg of tissue);(jH actlvitj per 100~1 of medium). In addition to the T/M ratio. the values given in Table 1 include ratios corrected for tissue solid content (apparent ‘H concentration in tissue water. Tw:M) and ratios corrected for both solid content and extracellular fluid volume (as determined by the sucrose space). expressed as the apparent ‘H concentration in intracellular water (Iw/M). A more detailed description of the technique and calculation was previously published (Bito. 1972~).
RESULTS The largest the choroid 173
accumulation plexus, the
ratios were observed in iris--ciliary body complex
174 Tahlc
I. The
[‘H]PGE,
apparent
Tissues
accumulation cxprcsscd on the has\ and intracellular water (1~1
T.!M ‘H
(n)
OI tot,)1 II\\LI~
LI;II~‘~
I 1-uI*
TW ‘M
Tw!M “H
I.y‘
0.78* 0.03 I .03 + 0 I I 0.30f 0.01 0.1’ f O.OI
Brown fat White fat Kidney cortex Spleen Llterus
0.x
f
0.0~
and [ “(‘jwcrosc: wiki pieces were incubated for I hr in medium containing [3H]PGF, was determined by dryng overnight at 75 <‘ in partial vacuum ,?c‘I unit \OI 01 total tlssuC *T = “H activity per unit weight of tissue; Tw = ‘H or ‘?’ actnIl> per unll ~01 of ~ncuhawater. Iw = apparent “H activity in intracellular water: M = ‘H or ‘Y’ actl\lt! tion medium. + Iw,M cannot be computed \~nce there 15 no apparent Intracellular Rued voi sn ahllc Ial (1.~’ Tu M for r14clsucr0sc IS c I). Tissue
content
uvea) and the kidney cortex (Fig. I ). For all tissues that showed accumulation ratios greater than unity, this ratio was larger following incubation at 37°C than at 7’C. This temperature effect was most pronounced in the case of the anterior uvea. With the exception of choroid plexus. kidney cortex and uterus. the 3H accumulation by tissues taken from hibernating animals was similar to that of tissues taken from non-hibernating woodchucks (Fig. 1). In this series of experiments on temperature effects. a OS-hr incubation period was used to estimate the initial rate of uptake. The values presented in Fig. I represent uncorrected tissue-to-medium accumulation ratios. In a few selected tissues (Table 1). water content. [“‘Clsucrose space. and C3H]PGE, accumulation were measured (anterior
6r
I
on each piece of tissue to allow the calculation of the apparent 3H activity in the intracellular water relative to that of the medium (Iw:M). In this series of experiments. a I-hr incubation period was used to rstimate the steady-state “H and 14C distribution ratios. The relative extent of accumulation of 3 diKerent PCs and a prostaglandin precursor (arachidonic acid) was compared on 5 tissues taken from active animals (Fig. 3). In brown fat. kidney cortex and lung tissues. all of which showed significant net ‘H accumulation (T,‘M > I). the largest “H accumulatton was observed when incubated with L3H]PGA, with the order of apparent accumulatmn being PGA, z PGE, D PGF,,. White fat did not show a net accumulation (T/M < I) of any of these substances.
6
I
5t
T jiij
I
m
- 37’6
I*
7°C
3 :: UJ 6 %?m, b I p_, L : t a ,=
,=
2 ,
0
---------
A
H
BRAIN
A
Ii
KIDNEY CORTEX
A Ii CHOROID PLEXUS
A
H
LIVER
h H IRIS
A
H
LUNG
A H BROWN FAT
A
H
SPLEEN
A H WHITE FAT
A
A H AORTA
H
UTERUS
accumulation hj tissues of normothermic and hibernatmg woodchucks Incubated at 7 and 37 C for 0.5 hr in tissue culture medium containing c3H]PGE,. Each column represents the mean T.iM ratio obtained on 69 pieces of tissues taken from two or more animals. The limits given are + _ I S.E. of the mean. l--~g. I. ‘H
Prostaglandin
a
13kfI
PGE,
mC3HI
PGA,
mt3H3
PGF/,
~L-kl-Amchtdomc
m=
ooBROWN
FAT
175
accumulation by tissues of a heterotherm
Aod
WHITE FAT
KIDNEY CORTEX
LUNG
UTERUS
Fig. 2. The specificity of PC accumulation by some tissues of non-hibernating woodchucks. Two or 3 pieces of tissues obtained from normothermic woodchucks were incubated at 37°C for I hr in culture medium containing tritium-labelled PGE,. A,, F,, or arachidonic acid. See also legend to Fig. 1.
DLSCUSSION
The very small effect of temperature on C3H]PGE1 accumulation by in vitro choroid plexus supports the concept that transport processes across the bloodbrain barrier can be maintained during hibernation in spite of the low body temperature (Bito & Roberts, 1974). In contrast, C3H]PGE, accumulation by the anterior uvea showed a large temperature effect which suggests that absorptive transport of PC’s across the blood-intraocular fluid barrier may be seriously hindered during hibernation. The difference between the ability of the choroid plexus and the anterior uvea to accumulate, and presumably to transport, PCs in the cold may be correlated with the differences in the functional requirements of these 2 tissues. Some parts of the brain remain electrically active during hibernation and hence continue to release PGs into the extracellular fluids. Therefore, maintenance of PC removal across the choroid plexus would be necessary to prevent the accumulation of these potent autacoids in the extracellular fluids of the brain during hibernation. The need for such a process to remove PGs from the brain is emphasized by the observation of Nakano & Prancan (1971) that. unlike most other tissues, homogenates of rat brain do not effectively metabolize PGE,, and was demonstrated directly by showing that known inhibitors of PG transport greatly enhance the adverse effects of exogenous PGs on the brain (Bito & Wallenstein, 1977). In contrast to the brain, the retina is electrically inactive in hibernating animals (Biewald, 1967) thus it is most unlikely that there is significant PC release into the extracellular fluid of the posterior segment of the eye during hibernation The existence of a hibernation-associated “adaptive change” in the temperature optimum of the PC accumulative capacity is not indicated by the present results. If the optimum temperature of this process were functionally variable, tissues of the hibernating animal would be expected to exhibit a greater concentrative PG accumulative capacity at 7 than at 37’C, or, at least, tissues taken from hibernating animals would be expected to show greater PG accumulation
at 7°C than tissues of active animals.
This was clearly not the case. In the case of the brown fat. in citro C3H]PG accumulation may reflect the ability of some cells or cellular processes to concentrate locally-released or circulating PGs and thus enhance the effects of these autacoids on this tissue. These considerations suggest that PG’s may have an important role in the regulation of the metabolic activity of brown fat, and hence may play a role in arousal. SUMMARY
1. Slices or pieces of tissues, not exceeding 1OOmg. were taken from normothermic and hibernating woodchucks (Marmota monax) and were incubated for 0.5 or 1 hr at 37 and 7’C in tissue culture medium containing tritiated prostaglandins (C3H]PGs) A,, E,. Fr, or arachidonic acid (and in some experiments also [‘4C]sucrose for the determination of the extracellular space). 2. Of the 11 tissues studied. the largest 3H accumulation was observed in the choroid plexus and anterior uvea following incubation at 37°C in medium containing [‘HIPGE,. Significant concentrative PC accumulation (T/M > 1) was also observed when kidney cortex, liver, lung, uterus or brown fat were incubated for 0.5 or I hr with C3H]PGs. White fat and spleen appear to exclude all PGs from much of their intracellular compartments. 3. In general, the results obtained on tissues taken from hibernating and from normothermic animals were similar. Small differences were observed. but the direction of these differences was not consistent with an adaptive change in the temperature optimum of the PG accumulative process by tissues of hibernating vs normothermic woodchucks, 4. These in Crro results suggest that PG transport by the choroid plexus, but not by the anterior uvea. and hence removal of PG’s from the extracellular fluids of the brain, but not of the eye. continues etfectively during hibernation. Concentrative PGA, accumulation by brown fat suggests that circulating PGs may be involved in the metabolic control of this tissue and hence may play a role in arousal.
EKICA \‘. SAL\ AIWK VI [Ii
17h
Ms Ann Zaragora and Mr David Turansky for their skilful assistance: Mr M. Rodak. N.Y. State Denartmcnt of Environmental Consrrcation. New Paltr. New York and Mr M. Kellison of Svkcsville. Maryland. for supplying the v+oodchucki used -in these studxs. This lnvestigatlon was supported bv Research Grant EY 00332 from the Natmnal Institutes &I’ Health. l1.S. Pubhc Health Scrvicc.
REFEREYCES Br.cartz B. (1962) The transport of orgamc anions by the rabbit eye. I. In rim iodopyracet (diodrast) accumulation by ciliary body -iris preparations. 4~1. .I. Ophrhd so, X6’- X67 BIFWALD G. A. (1967) Das Elektrorctlnogramm beim wnterschlafenden und hypothermischen Feldhamster. I+‘;.\\ %. Murrirl-Luthrr-C;,li~. Hullr, Wirrch. XVI, 299 314 BITO L. 2. (1972~) Accumulation and apparent acttvc transport of prostaglandins hq \omc rabbit tissues ,U f+rro. .I. Phy.siol.. Lad. 221. !7 I 3X7 BITO L. Z. (197%) Comparatikc \tud! of conccntratl\c prostaglandin accumulation by \armus tissues of mammals and marine vertebrates and Invcrtchratcs. (‘or~/~. B~ochcn~. Physiol. 43A. 65-81. BITCI L. Z. & ROBFRTS J. c. (1974) The cll’ccts of hlhernation on the chemical composition of cercbrospinal and intraocular fluids. hlood plasma and brain tissue of the
47A. 173 193. BITO L. Z.. Rosr RTS J. c‘. & SAKAI S (1972) Malntcniinci of normal cornea1 thickness in the cold 1,~ riw (hibermlion) as opposed to 111rum. ./. Ph~~uol.. Lrd. 231. ‘1 Xh. BIT0 L. Z. & SALVAI)OK E. v. (1972) Intraocular fluId d> ,);Imics. III. The site .~ntl mrchanlm of prostaglnndln transfer across the blood Intraocular fluid harrier\ E\;ll CL,? Kc? 14. 233 241. Bi-ro L. L. & W~II.FNS~II~ M. ( 11977) Tr-ansport 01 and blood prostaglandins across the blood bram aqueous harriers and the physlologlcal significance 01 these ahsorptlve transport proceswa. In 7./7r OU~/IU u,I~/ Cwc~hro.\/xn~~/ Fluids. Fogart! Intcrnntlonal C‘enter S) mposium (Edited b> BITO I. Z.. I)A\SOY H. B FI XSII I(\IA(‘tiI K J D.). E\[‘/. I:,<’ RL,\. 25. Suppl pp. 229 143. BIIO L. Z.. DAVSO\ H. & SAI.VAI)OH E \/. (1976) lnhtbltion of i,r r,rro concentratiic prostaglandln accumulation h! prostaglandins. prostaglandin analogues and h! some II~hibitors of organic anjon transport. .I Ph\,\roi I,cd 256. 257 171. (‘SI.RK H F. (1971 I Phj\mlogc of the chorold plezu\ P/II,\ro/. Rec.. 51, 273 3 I I KIN%> V. E. (lY71) Ion movement In cihaq procesws. In .%l~w~hru~w~urrd /on Trtrqwr (Edited bk BIT~AK tE.I Vol. 3. pp. IX5 209. Wiley. Nen York. NAKAI\O J. & PRAIU’C’AX !Z. V. (1971) Metabolic degredation of prostaglandin E, III the rat plasma and in rat brain. heart. lung. kidney and testlcle homogenates. .I. Pkrrr~w Phurrnc~. 23. 231 232.
Division.
Department of Ophthalmology, College of Physicians Columbia University. New York. NY 10032, U.S.A. (Receircld 31 Map
and Surgeons
1977)
Eleven tissues from normothermic and hibernating woodchucks were incubated at 37 or 7 C with [3H]prostaglandins (PC&) A,. E,, F,,. C3H]arachidonic acid and/or [‘4C]sucrose. 2. Choroid plexus. anterior uvea and to a lesser extent, kidney cortex. liver. lung and brown fat showed concentrative PC accumulation at 37°C. White fat and spleen appeared to exclude PGs from their intracellular compartments. 3. With the exception of choroid plexus and kidney cortex of normothermic woodchucks. concentrative C3H]PGE, accumulation was significantly less at 7’C. The choroid plexus showed several-fold PG accumulation even at 7°C indicating that PG removal from the brain can continue during hibernation. 4. Concentrative accumulation of PGA, and PGE, by brown fat suggests that circulating PGs may be involved in the metabolic control of this tissue and hence may play a role in arousal. Abstract--l.
INTRODUCTION It has been shown (Bito & Roberts, 1974) that in spite of low body temperature, the chemical composition of the cerebrospinal and intraocular fluids of hibernating woodchucks is distinctly different from that of plasma ultrafiltrate. In fact, the concentrations of several chemical constituents which are known to be transported into or out of these fluid compartments remain remarkably normal even after several days of hibernation. These findings suggest that some transport activities across the blood-brain and blood-ocular fluid barriers must effectively continue at a body temperature of 7-10°C. In vitro accumulation of certain substances. such as iodide and iodopyracet, by the choroid plexus or anterior uvea has been used extensively as an index of in ho transport functions across the blood-cerebrospinal or blood-ocular fluid barriers (cf. Cserr. 1971; Becker, 1962; Kinsey, 1971). Recently, it was shown that prostaglandins (PGs) which are produced in both the eye and the brain, but are not locally metabolized. are accumulated by both the choroid plexus and the anterior uvea it? vitro (Bito. 1972a.h) and are removed from the extracellular fluids of the eye and the brain by facilitated transport processes in ko (Bito & Salvador, 1972; Bito et al., 1976). Some evidence has been presented indicating that the facilitated removal of PGs from the extracellular fluid of these organs has a physiological function (Bito & Wallenstein, 1977). These investigations were undertaken to determine which tissues of the woodchuck show concentrative PG accumulation in citro. whether this concentrative activity is maintained at an incubation temperature comparable to the body temperature of the hibernating animal, and whether there is a shift in the tem-
perature optimum of these apparent cesses during hibernation.
transport
pro-
MATERIALS AND METHODS Tissues were obtained from hibernating and from anesthetized, normothermic woodchucks. following the collection of samples used for other experiments (Bito & Roberts, 1974; Bito et al., 1973). Tissue pieces or slices weighing not more than IOOmg each were incubated for 0.5 or 1 hr at 7 or 37.‘C in oxygenated tissue culture medium (Basal Medium Eagle, Microbiologlcal Associates. Bethesda. Md.) containing a [3H]labelied prostaglandin E,. A,. F1, or arachidonic acid, and in some cases also [‘4C]sucrose to measure extracellular space. After removal from the medium. the tissue pieces were lightly blotted on moist filter paper. weighed on an electrobalance. placed in scintillation solvent solution (Aquasol; New England Nuclear, Boston. MA.) and eluted with continuous shaking overnight. In one experiment. half of the tissue pieces were prepared for isotope counting by a Packard Model 306 automatic tissue oxidizer. Aliquots (100 ~1) of the incubation medium were made up in the same scintillation solvent or oxidized by the same technique. All samples were counted for a minimum of 10,000 counts in a Packard Tricarb scintillation counter. Values are reported as the tissue-to-medium (T!M) accumulation ratio: (-‘H activity per lOOmg of tissue);(jH actlvitj per 100~1 of medium). In addition to the T/M ratio. the values given in Table 1 include ratios corrected for tissue solid content (apparent ‘H concentration in tissue water. Tw:M) and ratios corrected for both solid content and extracellular fluid volume (as determined by the sucrose space). expressed as the apparent ‘H concentration in intracellular water (Iw/M). A more detailed description of the technique and calculation was previously published (Bito. 1972~).
RESULTS The largest the choroid 173
accumulation plexus, the
ratios were observed in iris--ciliary body complex
174 Tahlc
I. The
[‘H]PGE,
apparent
Tissues
accumulation cxprcsscd on the has\ and intracellular water (1~1
T.!M ‘H
(n)
OI tot,)1 II\\LI~
LI;II~‘~
I 1-uI*
TW ‘M
Tw!M “H
I.y‘
0.78* 0.03 I .03 + 0 I I 0.30f 0.01 0.1’ f O.OI
Brown fat White fat Kidney cortex Spleen Llterus
0.x
f
0.0~
and [ “(‘jwcrosc: wiki pieces were incubated for I hr in medium containing [3H]PGF, was determined by dryng overnight at 75 <‘ in partial vacuum ,?c‘I unit \OI 01 total tlssuC *T = “H activity per unit weight of tissue; Tw = ‘H or ‘?’ actnIl> per unll ~01 of ~ncuhawater. Iw = apparent “H activity in intracellular water: M = ‘H or ‘Y’ actl\lt! tion medium. + Iw,M cannot be computed \~nce there 15 no apparent Intracellular Rued voi sn ahllc Ial (1.~’ Tu M for r14clsucr0sc IS c I). Tissue
content
uvea) and the kidney cortex (Fig. I ). For all tissues that showed accumulation ratios greater than unity, this ratio was larger following incubation at 37°C than at 7’C. This temperature effect was most pronounced in the case of the anterior uvea. With the exception of choroid plexus. kidney cortex and uterus. the 3H accumulation by tissues taken from hibernating animals was similar to that of tissues taken from non-hibernating woodchucks (Fig. 1). In this series of experiments on temperature effects. a OS-hr incubation period was used to estimate the initial rate of uptake. The values presented in Fig. I represent uncorrected tissue-to-medium accumulation ratios. In a few selected tissues (Table 1). water content. [“‘Clsucrose space. and C3H]PGE, accumulation were measured (anterior
6r
I
on each piece of tissue to allow the calculation of the apparent 3H activity in the intracellular water relative to that of the medium (Iw:M). In this series of experiments. a I-hr incubation period was used to rstimate the steady-state “H and 14C distribution ratios. The relative extent of accumulation of 3 diKerent PCs and a prostaglandin precursor (arachidonic acid) was compared on 5 tissues taken from active animals (Fig. 3). In brown fat. kidney cortex and lung tissues. all of which showed significant net ‘H accumulation (T,‘M > I). the largest “H accumulatton was observed when incubated with L3H]PGA, with the order of apparent accumulatmn being PGA, z PGE, D PGF,,. White fat did not show a net accumulation (T/M < I) of any of these substances.
6
I
5t
T jiij
I
m
- 37’6
I*
7°C
3 :: UJ 6 %?m, b I p_, L : t a ,=
,=
2 ,
0
---------
A
H
BRAIN
A
Ii
KIDNEY CORTEX
A Ii CHOROID PLEXUS
A
H
LIVER
h H IRIS
A
H
LUNG
A H BROWN FAT
A
H
SPLEEN
A H WHITE FAT
A
A H AORTA
H
UTERUS
accumulation hj tissues of normothermic and hibernatmg woodchucks Incubated at 7 and 37 C for 0.5 hr in tissue culture medium containing c3H]PGE,. Each column represents the mean T.iM ratio obtained on 69 pieces of tissues taken from two or more animals. The limits given are + _ I S.E. of the mean. l--~g. I. ‘H
Prostaglandin
a
13kfI
PGE,
mC3HI
PGA,
mt3H3
PGF/,
~L-kl-Amchtdomc
m=
ooBROWN
FAT
175
accumulation by tissues of a heterotherm
Aod
WHITE FAT
KIDNEY CORTEX
LUNG
UTERUS
Fig. 2. The specificity of PC accumulation by some tissues of non-hibernating woodchucks. Two or 3 pieces of tissues obtained from normothermic woodchucks were incubated at 37°C for I hr in culture medium containing tritium-labelled PGE,. A,, F,, or arachidonic acid. See also legend to Fig. 1.
DLSCUSSION
The very small effect of temperature on C3H]PGE1 accumulation by in vitro choroid plexus supports the concept that transport processes across the bloodbrain barrier can be maintained during hibernation in spite of the low body temperature (Bito & Roberts, 1974). In contrast, C3H]PGE, accumulation by the anterior uvea showed a large temperature effect which suggests that absorptive transport of PC’s across the blood-intraocular fluid barrier may be seriously hindered during hibernation. The difference between the ability of the choroid plexus and the anterior uvea to accumulate, and presumably to transport, PCs in the cold may be correlated with the differences in the functional requirements of these 2 tissues. Some parts of the brain remain electrically active during hibernation and hence continue to release PGs into the extracellular fluids. Therefore, maintenance of PC removal across the choroid plexus would be necessary to prevent the accumulation of these potent autacoids in the extracellular fluids of the brain during hibernation. The need for such a process to remove PGs from the brain is emphasized by the observation of Nakano & Prancan (1971) that. unlike most other tissues, homogenates of rat brain do not effectively metabolize PGE,, and was demonstrated directly by showing that known inhibitors of PG transport greatly enhance the adverse effects of exogenous PGs on the brain (Bito & Wallenstein, 1977). In contrast to the brain, the retina is electrically inactive in hibernating animals (Biewald, 1967) thus it is most unlikely that there is significant PC release into the extracellular fluid of the posterior segment of the eye during hibernation The existence of a hibernation-associated “adaptive change” in the temperature optimum of the PC accumulative capacity is not indicated by the present results. If the optimum temperature of this process were functionally variable, tissues of the hibernating animal would be expected to exhibit a greater concentrative PG accumulative capacity at 7 than at 37’C, or, at least, tissues taken from hibernating animals would be expected to show greater PG accumulation
at 7°C than tissues of active animals.
This was clearly not the case. In the case of the brown fat. in citro C3H]PG accumulation may reflect the ability of some cells or cellular processes to concentrate locally-released or circulating PGs and thus enhance the effects of these autacoids on this tissue. These considerations suggest that PG’s may have an important role in the regulation of the metabolic activity of brown fat, and hence may play a role in arousal. SUMMARY
1. Slices or pieces of tissues, not exceeding 1OOmg. were taken from normothermic and hibernating woodchucks (Marmota monax) and were incubated for 0.5 or 1 hr at 37 and 7’C in tissue culture medium containing tritiated prostaglandins (C3H]PGs) A,, E,. Fr, or arachidonic acid (and in some experiments also [‘4C]sucrose for the determination of the extracellular space). 2. Of the 11 tissues studied. the largest 3H accumulation was observed in the choroid plexus and anterior uvea following incubation at 37°C in medium containing [‘HIPGE,. Significant concentrative PC accumulation (T/M > 1) was also observed when kidney cortex, liver, lung, uterus or brown fat were incubated for 0.5 or I hr with C3H]PGs. White fat and spleen appear to exclude all PGs from much of their intracellular compartments. 3. In general, the results obtained on tissues taken from hibernating and from normothermic animals were similar. Small differences were observed. but the direction of these differences was not consistent with an adaptive change in the temperature optimum of the PG accumulative process by tissues of hibernating vs normothermic woodchucks, 4. These in Crro results suggest that PG transport by the choroid plexus, but not by the anterior uvea. and hence removal of PG’s from the extracellular fluids of the brain, but not of the eye. continues etfectively during hibernation. Concentrative PGA, accumulation by brown fat suggests that circulating PGs may be involved in the metabolic control of this tissue and hence may play a role in arousal.
EKICA \‘. SAL\ AIWK VI [Ii
17h
Ms Ann Zaragora and Mr David Turansky for their skilful assistance: Mr M. Rodak. N.Y. State Denartmcnt of Environmental Consrrcation. New Paltr. New York and Mr M. Kellison of Svkcsville. Maryland. for supplying the v+oodchucki used -in these studxs. This lnvestigatlon was supported bv Research Grant EY 00332 from the Natmnal Institutes &I’ Health. l1.S. Pubhc Health Scrvicc.
REFEREYCES Br.cartz B. (1962) The transport of orgamc anions by the rabbit eye. I. In rim iodopyracet (diodrast) accumulation by ciliary body -iris preparations. 4~1. .I. Ophrhd so, X6’- X67 BIFWALD G. A. (1967) Das Elektrorctlnogramm beim wnterschlafenden und hypothermischen Feldhamster. I+‘;.\\ %. Murrirl-Luthrr-C;,li~. Hullr, Wirrch. XVI, 299 314 BITO L. 2. (1972~) Accumulation and apparent acttvc transport of prostaglandins hq \omc rabbit tissues ,U f+rro. .I. Phy.siol.. Lad. 221. !7 I 3X7 BITO L. Z. (197%) Comparatikc \tud! of conccntratl\c prostaglandin accumulation by \armus tissues of mammals and marine vertebrates and Invcrtchratcs. (‘or~/~. B~ochcn~. Physiol. 43A. 65-81. BITCI L. Z. & ROBFRTS J. c. (1974) The cll’ccts of hlhernation on the chemical composition of cercbrospinal and intraocular fluids. hlood plasma and brain tissue of the
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