Twenty-four-hour rhythmicity in carbonic anhydrase activities of choroid plexuses and pineal gland.код для вставкиСкачать
Twenty-four-hour Rhythmicity in Carbonic Anhydrase Activities of Choroid Plexuses and Pineal Gland ' W. B . QUAY Department of Zoology, University o f California, Berkeley, California 94720 ABSTRACT Carbonic anhydrase (CA, carbonate hydro-lyase, EC 126.96.36.199) 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, Berkeley. 279 280 W. B. QUAY their higher level of carbonic anhydrase activity (Pesetsky, ’69). Carbonic anhydrase (carbonate hydro-lyase, EC 188.8.131.52 ) 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. MATERIALS AND METHODS 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 + RHYTHMICITY IN CARBONIC ANHYDRASE +STANOARD TIME --?- 28 1 Room 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 282 W. B. QUAY 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 , , oflbuffer (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 sessions. OBSERVATIONS 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 TABLE 1 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 C.P. Time x&Se 3 : 0 0 ~ ~ (8 hours dark) (N) 9.11 f 0.59 (10) 7:OOA M (2hours light) 7.4120.51 (10) Dark phase total 8.90k0.31 (30) Light phase total 8.06f0.35 (30) P < 0.05 FeSe 10.89 f 0.93 (10) n.s. 0.33k 0.06 ( 10) 9.942 0.71 ( 10) < 0.02 10.93 & 0.38 (30) < 0.02 10.04f0.44 (30) ~ P 0.50k0.10 (10) n.s. ~ z, Pineal x&Se (N) - (N) P 0.51f0.06 (29) ns. 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. RHYTHMICITY I N CARBONIC ANHYDRASE 283 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 rex+ and x 0.75= ~ 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 + 284 W. B. QUAY ol 8 V V n h v z a 8 t - u ) tl tI m m f 2 v v 8 8 0 m m a 0.1 c9 0 $1 t- '9 2 m tl DISCUSSION 2 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 m V V m ol u 0.1 8$1 2 s m '9 0 v ol 1 pf 0 0 tI m I co cFI ti Q) ? m cc r \ Z u ! ? 0 $1 9 " 8$1 tl v 2tI h v 0 01 Z 2 +I u ) 2 2 8tI 2 n i a0: 0 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 respectively. rl 2 0 +I n 2 v h 2 v W W 0 +I 0 +I c? c? z+ $4 .sm yg) ;j .I .$I Jo PO <lo - N RHYTHMICITY IN CARBONIC ANHYDRASE - STANDARD T I M E (AM) 285 + 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 286 W. B. QUAY 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. ACKNOWLEDGMENTS 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. LITERATURE CITED Ashby, W. 1943 Carbonic anhydrase in mammalian tissue. J. Biol. Chem., 151: 521-527. 1944 On the quantitative incidence of carbonic anhydrase in the central nervous system. 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