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Twenty-four-hour rhythmicity in carbonic anhydrase activities of choroid plexuses and pineal gland.

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Twenty-four-hour Rhythmicity in Carbonic Anhydrase
Activities of Choroid Plexuses and Pineal Gland '
Department of Zoology, University o f California,
Berkeley, California 94720
Carbonic anhydrase (CA, carbonate hydro-lyase, EC
activity of choroid plexuses (ventricles I I1 and IV) and pineal glands of adult
male rats was determined by microtitration. Autopsies at precise times in relation to a daily photoperiod 14 hours long allowed in replicate series evaluation
of 24-hour rhythmicity. A slightly lower choroid CA activity during the light
phase was variable and marginal in significance. A highly significant and reproducible daily fall i n pineal CA activity near the onset of light was paralleled by,
and probably originated from, a fall in pineal content of erythrocytes. Low pineal
CA activity is consistent with its endocrine nature. Its morning changes in hemodynamics are likely to be due to local changes in content and release of norepinephrine and other vasoactive agents.
Since the discovery of high amplitude
24-hour rhythmicity in rat pineal content
of 5-hydroxytryptamine (Quay, '63) many
other chemical and metabolic characteristics of this organ have been shown to
follow distinctive and consistent circadian
or 24-hour cycles (Ebadi et al., '70; Klein
and Weller, '70; Quay, '70; Axelrod, '71;
Nir et al., '71; NovAkovA et al., '71; and
others). Pineal rhythmicity has excited research in relation to two physiological
topics. One of these centers on hypotheses
that the mammalian pineal organ, as a
neuroendocrine gland, has a role in the
mediation of certain daily and seasonally
timed physiological responses to environmental cues (Milin et al., '69; Quay, '69;
Reiter and Fraschini, '69; Wurtman and
Anton-Tay, '69). The second physiological
topic is the cellular and molecular regulation of neurotransmitters and their effects
in the nervous system. Our understanding
of some of the regulatory mechanisms has
been furthered by use of the mammalian
pineal organ as a model neural tissue system. This is appropriate because of: (1)
the embryological origin of major pineal
constituents from central and peripheral
nervous systems, ( 2 ) the relatively high
concentrations of certain neurotransmitters
and neurohumors within pineal tissue, and
( 3 ) the relatively great amplitude and conANAT.REC., 174: 279-288
sistency of 24-hour rhythms in these compounds in pineal as compared with other
tissues and brain regions.
Pineal constituents that have been
studied heretofore for 24-hour rhythmicity
pertain primarily to pinealocytes and sympathetic nerve fibers and their endings.
Possible occurrence of 24-hour rhythmicity
in pineal neuroglia has been neglected.
This deficiency is worthy of attention since
pineal glia or their derivatives could have
significance in the support or control of
metabolic rhythms within the organ. This
possibility is reasonable if one is willing to
postulate a parallel between the pineal's
glia-pinealocyte metabolic relationship and
those kinds of metabolic interactions found
in the central nervous system's glia-neuron
relationship (HydCn and Pigon, '60; HydBn
and Lange, '64; Pevzner, '71).
Neuroglia, especially astroglia, have
been accepted as one of the cell types generally present within mammalian pineal
organs (Hortega, '32; Quay, '65). At least
within the neuropil of higher vertebrate
central nervous systems astroglia are histochemically distinguishable from neurons
and other tissue elements on the basis of
Received May 8, '72. Accepted July 14. '72.
1 Supported in part by U.S . Public Health Service
research grant NS-06296 and the Miller Institute for
Basic Research in Science, University of California,
their higher level of carbonic anhydrase activity (Pesetsky, ’69). Carbonic anhydrase
(carbonate hydro-lyase, EC ) has
been studied in individual cells from rat
brains by the Cartesian diver technique
(Giacobini, ’61, ’62). In this instance also,
glia, choroid cells and erythrocytes had
relatively high and neurons relatively low
enzyme activity.
The present study serves to show that
pineal carbonic anhydrase activity is relatively much lower than that of choroid
cells and that its marked 24-hour rhythmicity can be attributed at least largely to a
rhythm in its vascular content of erythrocytes rather than to specific activities in
other cell types.
All studies used male laboratory rats
(Berkeley S, strain) born, raised and maintained within the same suite of windowless, temperature- and light-controlled
rooms. A complete diet and water were
available ad libitum. In all studies littermates were distributed through the different autopsy groups (times of analysis)
to allow for pairwise statistical evaluation
of significance of differences between
group means (Walker and Lev, ’53). Three
experiments were performed.
Experiment 1 . Thirty animals were
placed in individual suspended cages in
each of two rooms having daily photoperiods of white light 14 hours in length
(200 watt incandescent bulbs; room H :
lights on 5:OO AM - 7 : O O PM, room L:
lights on 8 :00 PM - 10:00 AM Pacific
Standard Time). Weak red lights (25 watts)
were on continuously in each room. Following a ten-day minimum interval of adaptation, pairs of animals were killed by rapid
decapitation at each of three times in each
room and on each of five different days
(January 24, 26, 31; March 1, 9). The
times are noted in figure 1, which can serve
to show also how the rooms ( H and L)
with staggered timing of daily photoperiod
were employed in order to obtain the equivalent of a 24-hour day. At the time of
autopsy, the animals were seven and one
half to eight and one half months old and
the choroid plexuses of the lateral ventricles (C.P. I
11) and the fourth ventricle
(C.P. IV) and the pineal gland were im-
mediately and totally removed, weighed to
the nearest milligram with a Cahn Electrobalance, and then quickly frozen in screwcapped 5 ml glass homogenizer tubes embedded in solid CO,. The choroid plexus
samples were weighed without draining or
blotting. The pineals were quickly dissected
free of blood clots and films and from attached pieces of dorsal sac, pineal stalk,
and dural sinuses. Rapidity and standardization of tissue processing and weighing
prevented significant variation due to sample drying.
Experiment 2. Forty animals were
placed in individual suspended cages in
room H under the same conditions as those
in experiment 1. Animals in pairs (8-9
months old) were rapidly decapitated at
each of five times (2:45, 4:00, 5 : 0 0 , 6:OO
and 7 : 30 A M ) on each of four days (March
14, 16, 20, 21). The samples at 5:OO AM
were taken within five to ten minutes after
the onset of white light. The autopsy procedures were the same as those used in experiment 1 with the exception that in addition to choroid plexuses and pineal gland,
hypothalamus (with arbitrary boundaries
as previously figured (Quay, ’68) ) was also
removed, weighed and frozen in a homogenizer tube.
Experiment 3 . Fifty animals were placed
in individual suspended cages in Room H
under the same conditions as those in experiments 1 and 2. On April 1, when they
were three and one half to five and one
half months old, the animals were decapitated in groups of 12 at each of five times
( 3 : 0 0 , 5:00, 6:00, 7:OO AM and 12:OO
noon). The pineal glands were removed
and dissected as previously but were then
fixed in 10% neutral formalin, 0.85%
sodium chloride for 24 hours. They were
then dehydrated, cleared, infiltrated with
wax (Paraplast) and cut with a rotary
microtome to provide serial sections seven
microns in thickness. The complete series
of affixed sections were stained with chromotrope 2R (C.I. No. 29, Nat. Aniline Div.)
according to the procedure of Crossmon
(’40) and with the purpose of selectively
staining erythrocytes. Total counts of erythrocytes were then made within the central
(“medulla”) and peripheral (lateral “cortex” capsule) portions of each gland using an ocular field 106 X 111 microns in
TIME --?-
28 1
Fig. 1 Graphs of mean carbonic anhydrase activities of whole choroid plexuses and
pineal glands of adult male rats (experiment 1 ) . Vertical lines extend one standard error
on each side of the means ( N zz 10 for each mean).
area. Erythrocytes were counted within ten
placements of the ocular field for each
region of each gland and employing two
or more tissue sections having minimal
artifacts and near an equatorial plane.
From these counts estimates were calculated for the number of erythrocytes per
mm3 x
of tissue representing the two
arbitrarily selected regions of the gland.
These estimates were intended to serve
only as evaluations of relative differences
in erythrocyte concentrations of pineal
glands such as those processed for carbonic
anhydrase determinations. Without other
kinds of supporting analyses the erythro-
cyte concentrations estimated here may not
be precisely indicative of concentrations in
vivo, for various technical reasons.
Measurement of carbonic anhydrase.
The microtitration method of Maren ('60)
utilizing phenosulfonphthalien (Phenol
Red, Chroma Gesellshaft) as indicator was
used, with modifications of Nair and Bau
('69 ) . Tissues were homogenized in demineralized distilled water while thawing
to ice bath temperature and immediately
before being added to the incubation tube
kept at a constant temperature (4" C ) by
a circulating cold water bath. Each incubation contained: ( 1 ) 500 pl of indicator
solution (6.25 mg Phenol Red, 1.30 ml
Na,HC03, 498.70 ml demineralized distilled
water); ( 2 ) 200 ,1 of water (= blanks) or
of tissue homogenate (= samples) or of
freshly prepared solution of crystalline carbonic anhydrase (= standard) (derived
from bovine erythrocytes and containing
2100 units/mg, Worthington Biochemical
Corporation, '72); and ( 3 ) 100 ,
(30.0 ml 1 M Na2C03,20.6 ml l~ NaHC03
and 49.4 ml water). Carbon dioxide was
bubbled from the bottom of the incubation
tube at a constant rate and timing of the
titration by an electrical timer (Precision
Scientific Co.) started simultaneously with
the addition of buffer. Where enzyme activity and tissue quantities allowed (choroid
plexuses and hypothalamus) replicate titrations were performed. Tissue enzyme activity was read from plots of net titration
times versus concentrations of standard.
Blank and standard solutions were titrated
at intervals throughout the analysis
Results from experiment 1 (fig. 1, table 1 ) suggested that carbonic anhydrase
activity in the choroid plexuses was higher
during the dark phase than during the light
phase of the daily photoperiod cycle. Furthermore, a suggested fall in pineal carbonic anhydrase near the time of the onset
of light (fig. 1) was difficult to support
statistically due to the relatively low enzyme activity of the pineal tissue and the
relatively great variability (table 1 ). Experiment 2 was designed to examine more
closely the enzyme activities during that
part of the day during which the greatest
changes were suggested by the data from
experiment 1. In this second experiment
lower choroid carbonic anhydrase activity
during the daily light phase was less apparent and the fall in pineal activity near
the daily onset of light was confirmed with
improved statistical support (fig. 2, table 2). Hypothalamic activity was examined
for comparative purposes only, and showed
slight change of but marginal ( P < 0.05)
or doubtful significance (fig. 2, table 2).
The first two experiments demonstrated
therefore, a reproducible and high amplitude ( 2 about 40% ) 24-hour rhythmicity
in whole pineal carbonic anhydrase activity, and at most only slight change in
that of the other tissues studied. However,
even this pineal rhythmicity, as far as its
peak and trough are concerned, can be accounted for largely if not entirely on the
basis of changes in total content of erythrocytes. The relatively low absolute unit enzyme activity of the whole pineal homogenates in the previous experiments
suggested by analogy with other tissues
having low activity (Maren, '67) that erythrocytes could constitute the major site of
pineal carbonic anhydrase. Experiment 3
succeeded in showing a morning fall in
pineal erythrocyte content comparable to
that in carbonic anhydrase activity (fig. 3 ) .
For purposes of computational reference
the 5 : 00 AM values in both experiments 2
(fig. 2 ) and 3 (fig. 3 ) were set as the base
line for dark phase and light phase comparisons. The percent differences of the
Carbonic anhydrase activities ( u n i t s / m g f r e s h tis.sue) o f choroid plexuses (C.P.)
and pineal glands i n experiment I , i n relation to autopsy time ( c f . , fig. 1 )
- I + I1
C.P.- IV
3 : 0 0 ~ ~
(8 hours dark)
9.11 f 0.59 (10)
(2hours light)
7.4120.51 (10)
Dark phase
8.90k0.31 (30)
Light phase
8.06f0.35 (30)
< 0.05
10.89 f 0.93 (10)
0.33k 0.06 ( 10)
9.942 0.71 ( 10)
< 0.02
10.93 & 0.38 (30)
< 0.02
10.04f0.44 (30)
0.50k0.10 (10)
(N) P
0.51f0.06 (29)
0.42f 0.04 (29)
Abbreviations: mean; Se, standard error of the mean; (N),number of individuals; P, probability based on
littermate comparisons and Student-Fisher t; n.s., not statistically significant.
Fig. 2 Graphs of mean carbonic anhydrase activities of whole choroid plexuses, hypothalami and pineal glands of adult male rats (experiment 2 ) . Vertical lines extend one
standard error on each side of the means ( N = 8 for each mean).
3:OO (+61%) and 6:OO AM (-25%) mean
erythrocyte contents from the 5 :OOAM base
line (fig. 3 ) do not exactly match those
of the 2:45 (+41%) and 6:OO AM (-42%)
mean pineal carbonic anhydrase activities
(fig. 2). However, calculations based on
the available data suggest that the trends
in morning whole pineal enzyme activity
and erythrocyte content are essentially
equivalent and that the pineal’s intrinsic
(non-erythrocyte) carbonic anhydrase ac-
tivity is probably negligible. Thus, letting
x = 5:OO AM enzyme units/mg for pineal
exclusive of erythrocytes, and y = 5 :00 AM
units/mg for erythrocytes, the equations
for whole pineal enzyme activity at 2 :45 3 : 0 0 ~ ~ a n d 6 : 0 0 ~ ~ a 1.61y=0.48
and x
~ 0.23 respectively. These
are based on the assumption that all enzyme activity changes are due to changes
in pineal content of erythrocytes. The values 1 . 6 1 ~and 0 . 7 5 ~are calculated from
t - u )
8 8
Carbonic hydrase activities of whole tissue homogenates in this study were measured in relation to activity of a purified
bovine erythrocyte carbonic anhydrase
standard. This had the purpose of allowing
comparability of tissue determinations on
different days and the correction of possible
technical differences on different occasions.
On the basis of relative activities of brain
regions, choroid plexuses and erythrocytes,
the results presented here are not out of
line with previously published data (Ashby,
'43, '44; Maren, '67; Nair and Bau, '69).
However, technical circumstances (Meldrum and Roughton, '33; Maren, '67) limit
the comparability of absolute tissue enzyme
activity determinations obtained by different laboratories. This is especially notable in the case of mammalian erythrocytes
which may have a two to four-fold variation in activity in individuals of the same
species and be modified by laking and
other events during processing. For these
reasons, attempts at more precise determination of the possibly present but small
intrinsic pineal carbonic anhydrase activity
are apt to be unrewarding.
The relatively low intrinsic pineal carbonic anhydrase activity remaining after
exclusion of probable erythrocyte activity
is consistent with the low or trace levels
found in other endocrine glands. It has
been questioned whether this enzyme could
have an obvious role in any of the endocrine glands, where in addition it would
impede the evolution of metabolic CO, and
thereby be more of a handicap than an aid
(Maren, '67). Nishimura et al. ('63) reported very low carbonic anhydrase in human pineals, where the activity was less
than that in all brain regions with the
possible exception of heavily myelinated
structures such as the corpus callosum and
the pyramid of the medulla oblongata.
This study demonstrates the importance
of daily changes in tissue blood content for
the interpretation of 24-hour chemical
2 s
u ! ?
" 8$1
u )
2 2
figure 3 (experiment 3 ) and the values
0.48 and 0.23 are taken from table 2 (experiment 2). Solution of the equations gives
values of x and y of 0.01 and 0.29 units/
mg for pineal tissue and erythrocytes
.sm yg)
- N
T I M E (AM)
Fig. 3 Graphs of mean pineal regional erythrocyte contents (intravascular) in adult male
rats (experiment 3 ) under conditions identical to those in experiment 2 (fig. 2). Vertical
lines extend one standard error on each side of the means (N= 12 for each mean).
rhythms apparent in some tissues. Daily
changes or 24-hour rhythms in hemal perfusion of particular vascular beds must be
kept in mind as potential contributors.
This is of the greatest significance when
the measured constituent is in high concentration in erythrocytes. Unfortunately,
little is known of a precise nature about localized vascular 24-hour rhythmicity in the
diverse specialized tissue regions within
the body.
Additional studies are needed in order
to reveal the nature and control of the
pineal‘s 24-hour vascular rhythmicity. It
was shown previously that the vascular
blood content of the rat pineal “cortex”
varies from 1.2 to 2.0 times that of the
pineal “medulla” and that the net changes
produced in the two regions by various experimental treatments are about the same
(Quay, ’58). Although pineal “cortex” and
“medulla” are terms not generally used and
are not sharply differentiated histologically
or cytologically, they are distinct in some
species and show quantitative differences
in cytological responses to light (Quay, ’65).
These could possibly be the results of differences in vascular content, perfusion or
timing of rhythmic changes.
The mechanism of the rapid morning
fall in pineal blood content (fig. 3 ) is not
clear, although neurotransmitter and neurohumoral components in the mechanism
can be suggested. Experimental manipulation of rat pineal blood content in vivo during the daily period of light failed to show
any marked and general pineal vascular
constriction, but norepinephrine, even in
small doses, inhibited pineal vasodilation
(Quay, '58). Norepinephrine content of the
rat pineal gland is maximum at the end
of the daily dark period and falls continuously during the subsequent light period
(Wurtman and Axelrod, '66). Release of
norepinephrine during the light phase and
interaction in effects with 5-hydroxytryptamine (Zweig and Axelrod, '69; Black and
Axelrod, '70) could be parts of a vascular
control mechanism.
I am grateful to Mr. Joseph D. Wong for
animal technical aid, Mrs. Emily Reid for
the final rendition of the illustrations and
Miss Robin Quate for office assistance.
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