Brain Research Bulle/in, Vol. 5, pp. 375-381. Printed in the U.S.A.

Relationships Between a-MSH Levels inBlood and in Cerebrospinal Fluid

A. A. DE RaTTE, H. J. BOUMAN AND Tj.B. VAN WIMERSMA GREIDANUSI

Rudolf Magnus Institute for Pharmacology, Medical Faculty, University of UtrechtVondellaan 6, 3521 GD Utrecht, The Netherlands

Received 13 March 1980

DE ROITE, A. A., H. J. BOUMAN AND Tj.B VAN WIMERSMA GREIDANUS. Relationships between a-MSH levels inblood and in cerebrospinal fluid. BRAIN RES. BULL. 5(4) 375-381, 1980.-Using a new cannulation technique of thecisterna magna of the rat, CSF was obtained in which a-MSH levels were determined under various conditions and werecompared with a-MSH levels in plasma. Basal levels of 66 ± 9 pg a-MSHlml were found in the CSF and of 179 ± 13 pga-MSHlml in the plasma. A rapid flow of CSF from the lateral ventricles to the cisterna magna could be established asdetected by elevated a-MSH levels in cisternal CSF 2 min after injection of a large quantity of a-MSH into the ventricularsystem. A half-time disappearance of a-MSH from the CSF of 33 min was calculated. The possibility of a contribution ofperipheral a-MSH to central melanotropic activity was suggested by the finding of elevated levels of a-MSH in the CSF,following artificially induced high a-MSH concentrations in the blood. Additionally, the movement of a-MSH from theCSF to the blood was demonstrated. Both these movements were shown to be independent of the pituitary gland, sincehypophysectomized animals exhibited the same rate of a-MSH transport as intact animals.

a-MSH Cerebrospinal fluid B1ood-CSF barrier

CEREBROSPINAL fluid (CSF) is no longer considered tobe an aqueous medium in which the brain floats and whichprevents it from damages, but has gained much in impor­tance among the body fluids. The pattern of flow and pro­duction of CSF have long been subjects of extensive re­search [2, 4, 15, 37], but recently interest has, among otherthings, been directed towards the presence in the CSF ofpeptides originating from the hypothalamus and the pituitary[14]. The hormones of hypothalamic origin, such as vaso­pressin and oxytocin seem to enter the CSF by a direct route[24], but the way of entry of peptides originating from theanterior and intermediate lobe of the pituitary, has not yetbeen established.

a-Melanocyte-Stimulating-Hormone (a-MSH), a frag­ment of the so-called 31 K ACTH/endorphin precursormolecule or pro-opiocortin, is mainly formed in the inter­mediate lobe of the pituitary, but also in the arcuate nucleusand several other nuclei of the hypothalamus in the brain[6,30]. a-MSH, which contains the ACTH I_ 13 fragment, hasbeen shown to be a modulator of brain functions related tobehavior [11, 31, 35]. Reduction of the bioavailability ofa-MSH and related peptides in the brain by central adminis­tration of antibodies, induces impaired behavior, especiallyWhen retrieval of recently stored information is involved[36]. Hypophysectomy also causes seriously impaired be­havior, which can be corrected by peripheral injection ofa-MSH or related peptides [35]. These observations, to­gether with the finding that hypophysectomy alters neitherthe immunoassayable a-MSH content of the CSF [28], northat of the arcuate nucleus of the hypothalamus [5], but on

the contrary reduces the a-MSH content of the whole brain[20,28] and of discrete brain regions [5, 19, 29], led to theconcept that a-MSH present in the CSF and the brain, mightbe partially of pituitary and partially of central nervous sys­tem (CNS) origin (see Fig. 7, left).

The present study was designed to obtain information onthe relationship between the presence ofa-MSH in the bloodand the CSF under various conditions, and to obtain infor­mation about the origin of a-MSH in these two body fluids.

METHOD

Animals

Male rats of an inbred Wistar strain (T.N .0., Zeist, TheNetherlands), weighing 250-300 g were used. The animalswere single housed and handled daily for at least three daysbefore starting the experiment. They had ad lib access tofood and water.

a-MSH was injected intravenously (IV) in a vehicle of0.25 ml saline into the tail vain, or intracerebroventricularly(lCV) in a vehicle of 2 fLl artificial CSF through a permanentpolyethylene cannula. This cannula had been placed underether anaesthesia into one of the lateral ventricles of the ratbrain at least one week before experimentation. Localizationof the tip of the cannula was determined at the end of theexperiment by ICV injection of Evans blue and macroscopi­cal inspection of the staining of the rat's brain-ventricularsystem.

Hypophysectomy was performed by the transauricularapproach and confirmed at autopsy by examination of the

'This study was partly supported by a grant of the Dutch Organization for the Advancement of Pure Research (ZWO-FUNGO).

Copyright <0 1980 ANKHO International Inc.-0361-9230/80/040375-07$01.40/0

376

sella turcica. Animals were used for experiments 3 days afterhypophysectomy, unless stated otherwise.

Removal of CSF

A cannulating technique for repeated sampling of CSF inunanaesthetized freely moving rats, as described recently[3], was used with slight modifications. A permanent stain­less steel cannula, constructed in such a way that no loss ofCSF could occur, was placed into the rat's cisterna magnaand fixed to the skull by anchoring screws and dental ce­ment. A special CSF outflow opening of the cannula wasconnected to polyethylene tubing for CSF sampling.Amounts of 50-150 p.1 CSF could be collected repeatedlywithout any sign of disturbing the animal. The CSF sampleswhich were obtained, were stored in plastic tubes at -20°Cuntil assayed.

Plasma Samples

Systemic blood was collected by decapitation, in chilled,heparinized polystyrene tubes and centrifugated at 4°C. Theplasma was then separated and stored at - 20°C until as­sayed.

Experiments

To determine whether the CSF, withdrawn from the cis­terna magna by the permanent cannula was representativefor the CSF in the ventricular space, a-MSH in doses rangingfrom 1-100 ng was injected into the lateral ventricle of thebrain. The a-MSH was measured in the cisternal CSF tenmin after injection. A time relationship for the transportationthrough the ventricular system to the cisterna magna wasdetermined by injecting 5.0 ng a-MSH ICV and measuringthe a-MSH levels in CSF withdrawn from the cisterna magnaat times ranging from 2-90 min.

The appearance of a-MSH in the blood after ICV injec­tion of 0.5 p.ga-MSH was measured at various time intervalsranging from 5-120 min, in intact and hypophysectomizedanimals.

Intravenous (IV) administration of 5.0 p.g a-MSH andmeasurement of the CSF-a-MSH levels 2-40 min after injec­tion, was performed in intact and in hypophysectomizedanimals in order to study the effect of high plasma-a-MSHlevels in the CSF.

Additionally, doses of 1.0, 5.0 and 10.0 p.g a-MSH wereinjected IV and the a-MSH levels in CSF determined after 20min, in order to study the dose-effect relationship oUV in­jected a-MSH and CSF-a-MSH levels.

Radioimmunoassay (RIA) of a-MSH

The RIA-procedure of Penny and Thody [27] with slightmodifications [20,22] was used to determine the a-MSHlevels in CSF and plasma. Briefly, the procedure was asfollows. As a diluent buffer a 0.05 M phosphate buffer pH 7.4containing 0.2% human serum albumin and 0.5%,B-mercapto-ethanol was used. Ten p.l I2:;I-a-MSH ( ± 2000cprn, specific activity approximately 320 p.Cilp.g) and 50 p.ldiluted rabbit antiserum to a-MSH (final dilution: 1:30.000,50% initial binding) were added to 150 p.l doubling dilutionsof standard a-MSH or appropriately diluted samples. Incu­bation time was 24 hr at 4°C. Separation of free and boundtracer was achieved by charcoal precipitation. The sensitiv­ity of the assay was 2 pg a-MSH/tube. Crossreactivity was

DE ROTTE ET AL.

minimal «0.03%) with ACTH I_ 1o, ACTH 4_ l o , ACTH 4_ 1 ,

ACTH 1_ l o , y-MSH, (0.07%) ACTH I _ 24 , (0.5%) ACTH 1_ 1 9

and ,B-MSH, whereas ACTH 1_ 1,NH2 was found to havemaximal cross-activity. The a-MSH antiserum appears to bemainly specific for the Lys-Pro- Val-NH 2 sequence (C­terminal of a-MSH). The measured a-MSH in biologicalmaterial yielded displacement curves which ran parallel tothe standard curve. Extraction of a-MSH from plasma wasnot necessary.

Calculations

The radioimmunoassay results were calculated on a Hew­lett Packard 104 calculator, which was programmed with alegit-curve fitting program [22]. The statistical processing ofresults was done with the Students t-test for calculating thedifferences between a treatment group and the correspond­ing control group. A combination of an analysis of varianceand an individual Students t-test was used in case dose de­pendency or a time course was subject of study. Each hor­mone level is reported as mean ± SEM for a number ofanimals (n).

Regression analysis was done with an exponential curvefitting program on the Hewlett Packard 104, equipped with aSTATS-ROM statistical unit.

The amount of a-MSH transported from blood to CSFand vice versa was estimated by assuming the CSF and ex­tracellular volume to be 0.5 ml and a blood volume of 10 rnlin rats weighing 250-300 g. From the peak values in a-MSHlevels the amount of a-MSH transported was then calculatedas a percentage of the initially injected quantities of a-MSH.

RESULTS

Basic a-MSH Levels in CSF and Plasma

The basal level of a-MSH in the CSF was determined inCSF, which was collected two days after cannulation of thecisterna magna. By then the food intake of the animals hasreturned to normal. The a-MSH level in CSF obtained from20 normal adult male rats was 66 ± 9 pg/ml (range 20-142pg/rnl) and the level in plasma, obtained from 16 rats was179 ± 13 pg/rnl (range 100-320 pg/rnl). The a-MSH concen­tration in plasma of rats I week after hypophysectomy was127;±: 17 pg/rnl (n=lO) (range 21-190 pg/rnl), which was sig­nificantly lower (p<0.05) as compared to that of intact orsham operated animals (196 ± IS pg/rnl). The CSF ofhypophysectomized animals contained 92 ± 24 pg/rnl (n=4)(range 35-136 pg/rnl), which tended to be higher, althoughnot significantly, as compared to the a-MSH levels in intactanimals. The withdrawal of CSF had no significant effect onthe a-MSH levels in the CSF at the time intervals used (2,3112 and 5 hours) (Table I).

Transport of a-MSH in CSF

a-MSH levels were measured in the cisternal CSF 10 minafter injection of various doses of a-MSH into one of thelateral ventricles of the brain (Fig. IA). Differences in thea-MSH content of cisternal CSF were found, these wererelated, although not proportionally, to the injected doses.

A marked increase in cisternal a-MSH level was observedwithin two min after injection of 5.0 ng of the peptide into alateral ventricle, followed by a gradual decrease in a-MSHlevel (Fig. lB).

By non-linear regression analysis an equation for theoverall disappearance of radioimmunoassayable a-MSH

a-MSH AND CSF 377

TABLE 1THE EFFECT OF WITHDRAWAL OF CSF

ON THE a-MSH LEVEL IN CSF

Apg/ml

11000

r--'--

a-MSH (pg/rnl)

Co t (hr) C, N P

65 ± 7* 2 58 ± 7* 5 NS67 ± 5 3'12 62 ± 15 5 NS72 ± 6 5 67 ± 3 5 NS

*mean ± SEM.N: number of animals; NS: not significant; Co: a·MSH levels at

time t=O (basal levels); C,: a-MSH levels at time 1.

from the cisternal CSF could be composed: y=3901 exp-0.0208 x, in which y=a-MSH level in the CSF (pg/rnl) andx=time in min (correlation coefficient: 0.89). Using this for­mula a half-time of overall disappearance of 33 min forradioimmunoassayable a-MSH from the CSF was calcu­lated.

1..LU1U-'oCL.ClJ

+-.~uc

9000

7000

5000

3000

FIG. I. (A) a-MSH levels (mean ± SEM) in cisternal CSF, 10 minafter ICV injection of different doses of a·MSH (number of animalsin parentheses). All a-MSH treated groups showed effects signifi­cantly different (P<0.05) from the control group , (8) a-MSH levels(mean ± SEM) in cisternal CSF at various times after ICV injec­tions of 5 ng a-MSH in vehicle of 2 JlI artifici al CSF (number ofanimals in parentheses). At all times after injection. the a -MSHtreated groups showed a significant difference (P<0.05) from theirappropriate controls.

artificial lOng lOng

CSF C(-MSH ex,·MSH

pg/ml B

1000

100 min

100ng

lX.·MSH

(3)

(51

(6)

80

(5)

60

(3)

(5)

(4)

....... artificial CSF

/,0

(3)

(7)

(15) (7)

o 20. t.InJ

0--0 5ng d,-MSH

2000

3000

1.000 (6) (5)

i5cL­ID

~u

.SIU'l~ 1000

I

lS

DISCUSSION

The present results confirm the previously reported pres­ence of immunoreactive a-MSH in the CSF of the rat [28].The levels were consistingly lower than those in plasma;66 :±: 9 pg/rnl and 179 :±: 13 pg/ml respectively, i.e, a ratio of1:3.

Although the a -MSH levels in plasma of hypophyscc-

Influence of CSF-a-MS/l 011 a-MSH Levels ill Blood

Injection of 0.5 Jlg a-MSH into the ventricular system ofthe brain, resulted in a significant rise of the plasma a-MSHlevel within 5 min. The highest concentration in plasma(870 :±: 191 pg/ml) was reached within 15 min after injection(Fig. 2). Approximately 2% of the originally intraventricu­larly injected dose was found in the circulation.

In hypophysectomized animals an a-MSH level in plasmaof 824 :±: 184 pg/rnl was found 15 min after ICV injection of0.5 Jlg a -MSH . This was not significantly different from thea -MSH concentration in plasma of sham operated or intactanimals treated likewise, while plasma of hypophysec­tomized animals treated with artificial CSF contained ana -MSH concentration of 160 :±: 12 pg/rnl (Fig. 3). ·

Influence of Plasma·a-MSH 011 the a-MSH Level ill CSF

Intravenous (IV) injection of 0.5 Jlg a-MSH resulted in asignificant rise in a-MSH levels in cisternal CSF, with amaximum around 10 min after injection: SIS:±: 101 pg/rnl,This is approximately 0.01% of the initially injected dose inthe blood (Fig. 4).

In hypophysectomized animals an a -MSH level of674 :±: 112 pg/rnl could be measured in CSF 20 min after IVinjection of 5.0 Jlg a-MSH. This was not sign ificantly differ­ent from that of sham operated animals treated likewise,which displayed a level of 387 :±: 71 pg/ml (Fig. 5).

The dose-effect relationship, determined at 20 min afterinjection is illustrated in Fig. 6. The effect on the a-MSHcontent in cisternal CSF appeared to be proportional to thedoses injected into the blood, but never exceeded 0.01% ofthe dose injected into the venous system.

40min

(6)

(6)

*..

o 20

(tj) 5) (4)

200

400

600

800

--'oCL­OJ

4J\J\U

C

u,(f)U

:r:(f)

zI

"d

DE ROTTE ET AL.

pg/ml

(L)...

(10)

• p'D.05

100 120mino 20 40 60 80t

inj0-0 0.5~g Ci(-MSH .-. artificial CSF

378

pg/ml (15)(12)• •

1000

800(7)•

0

E\J\

6000

c...~

IVI LOOLif

200(6)

FIG. 2. a-MSH levels (mean ie SEM) in plasma at various limesafter ICV injection of either 0.5 J1.g a-MSH in a vehicle of 2 J1.1artificial CSF or of 2 J1.1 artificial CSF as control (number of animalsin parentheses). Asterisks indicate differences between a-MSHtreated groups and the appropriate control groups who received aplacebo during the same time interval.

pg/ml

1000

.--. soline 0-0 5J.1g Cl-MSH

if p~o.05 .... p-O, 01 • -ft. p<0.001

800

artificial 0.51.19 O.5JJgCSF Ol-MSH ~-MSHo hypophysectomized [] shorn operated

.... p<.O.01

FIG. 3. a-MSH levels (mean ± SEM) in plasma of hypophysec­tomized or sham operated rats, IS min after injection of either 2 J1.lartificial CSF (vehicle), or 0.5 J1.g a-MSH (number of animals inparentheses). Asterisks indicate differences between a-MSH andplacebo treated groups. The two a-MSH treated groups were notsignificantly different from each other.

tomized rats were significantly lower than that in intactanimals, still a considerable amount of a-MSH was presentin plasma one week after hypophysectomy. The persistanceof apparently normal a-MSH levels in some cases after hy­pophysectomy, has been reported previously by Thody et al.[22]. They may be explained by a subtotal hypophysectomy,which was not recognizable by macroscopical inspection ofthe sella turnica, However, the persistance of detectablea-MSH levels after removal of the pituitary may be the resultof a transport of a-MSH-Iike-immunoreactivity (a-MSHLI)from the CNS through the blood-CSF barrier into the blood,as is illustrated in Fig. 7 (Right).

The fact that the a-MSHLI in plasma of hypophysec­tomized rats gave parallel displacement curves to the stand­ard curve in our radioimmunoassay, suggests that the meas­ured a-MSHLI closely resembles the peptide used as astandard. Further characterization of the a-MSHLI inplasma of hypophysectomized rats is in progress.

The cannulating system used to withdraw the CSF [3],appeared to be an ideal method for repeated sampling of CSFwithout influencing the a-MSH level in the CSF by the with-

FIG. 4. a-MSH levels (mean z; SEM) in cisternal CSF after IV in­jection of 5 p.g a-MSH in a vehicle of 0.25 ml saline, or of 0.25 mlsaline as control (number of animals in parentheses). Asterisks indi­cate differences between a-MSH treated groups and the appropriatecontrol groups who received a placebo during the same time interval.

(7)(9)

600

400

200

oEVIo-'o,

DE ROTTE ET AL. 379

LLIf)u

pg/ml

800

600

pg/ml

800 ••It

soline 5JJg 5JJg0(- MSH Q(-MSH

Dhypophysectomized []I shorn operated

.-it p(,O.01 .... p<O.01

(51

10.0J.,l9

(5)

•••

5.0J.,lg

..... p<O.001

lOugsoline

400

200

600

(5)(4)

400

200

~

oCL.OJ

iiiuc:r:If)z;.'is

FIG. 5. a -MSH levels in cisternal CSF (mean ± SEM) ofhypophysectomized or sham operated rats (number of animals inparentheses) 15min after IV injection of 0.25 ml saline (vehicle) or 5JLg a -MSH. Asterisks indicate differences between a-MSH andplacebo treated groups.

FIG. 6. a-MSH levels (mean ± SEM) in cisternal CSF at 20 minafter IV injection of different doses of a·MSH in a vehicle of 0.25 mlsaline. Controls received 0.25 ml saline (number of animals in par­entheses). Asterisks indicate differences between a-MSH andplacebo treated groups. The two a-MSH treated groups were notsignificantly different from each other.

drawaI. Animals used for two to three weeks showed no signof regression in their condition as a result of the cannulation.

The eSF from the cisterna magna was shown to be con­nected with the ventricular eSF by a rap idly flowing system,as was illustrated by the appearance of ventricularly ad­ministered high doses of a-MSH in the cisternal CSF withintwo minutes after injection. This flow of eSF is assumed tobe a pulsating flow directed from the lateral ventriclestowards the fourth ventricle, through the foramina ofLuschka and Magendi into the subarachnoid space [37J. Thepulsating component is probably provided by the arterialpulsations, and the overall flow by the formation of new CSFat a rate of 2.2 IlVmin [4] in the choroid plexus of the rat[2,15].

A half-time disappearance of 33 min could be calculatedfor a -MSH in the CSF. This is about ten times higher thanthe half-life in plasma, which is less than three min fora-MSH and other peptide hormones [8, 12, I3]. This longhalf-time disappearance in the eSF is in agreement with theresults obtained by Kastin et al . [13], who reported appar­ently unchanged 3H-a-MSH in the brain of rats 30 min afterinjection of this labeled peptide into the carotid artery. Thatsystemic injected a-MSH can exhibit an effect of severalhours duration on behavior [35J, may therefore among othersbe explained by the long half-life of this hormone in CSF andbrain tissue.

From the differences in concentrations and half-lives ofa-MSH in blood and eSF, it can be expected that the trans­port of a-MSH from the blood to the eSF is a regulatedsystem, which maintains different blood and eSF levels of

a -MSH, rather than an uncontrolled diffusion processthrough the blood-CSF barrier. In testing the permeability ofthe blood-CSf barrier, an artificial rise in the a-MSH level ofthe blood was induced, which caused the a-MSH level ofcisternal CSF to rise rapidly. This increase in a-MSH level ofthe eSF was independent of the pituitary, since hypophysec­tomized animals exhibited the same rise in eSF a-MSH con­centration as could be detected in intact or sham operatedanimals. The a·MSH content of the CSF did not exceed0.01% of the initially injected systemic dose both in intactand hypophysectomized rats.

This effect, which seems to be the result of a movement ofa-MSH acro ss the blood -CSF barrier, rimy be explained by adirect a-MSH transport across the blood-CSF barrier whichprobably takes place at the choroid plexu s (Fig. 7, Left) . Thefact that a-MSH increases the permeability of the bloodCSF barrier for proteins [7,26], assumably mediated bycyclic-AMP [18,25,33] would provide the underlying mech­anism for this positively auto-regulated transport.

Although only 0.01% of the IV injected a-MSH seems toreach the eSF and can thus act on the eNS, the long half-lifeof a-MSH in the eSF, combined with the relatively lowdoses necessary in the CNS to perform behavioral effects[32}, may account for the observed influence ofMSH/ACTH-like neuropeptides on adaptive behavior [11,31,35,36J.

When a-MSH was administered into the brain ventricularsystem, a rapid increase of the a-MSH content of the blood

380

CNS

DE ROTTE ET AL.

CNS

•IIII

CSF/ECV CSF I ECV

BLOOD BLOOD

FIG. 7 (Left) Flow system for a-MSH in intact animals . Large arrows indicate main direction of a-MSH passage, small arrows indicate otherpossible directions for a-MSH movements which have been established. Interrupted arrows indicate additional possibilities for a-MSHtransports. (Right) Flow system for a-MSH in hypophysectomized animals. Arrows indicate established a·MSH movements. Larger arrowspoint to the main direction of a-MSH transport.

was observed, with a maximum of2.()% of the intraventricu­larly injected dose in the CSF. This peptide movement isprobably the result of the flow ofCSF through the arachnoidvilli into the blood of the dural sinuses, at the rate of forma­tion of new CSF in the ventricular system [37]. As reportedby Gispen et al. [9], ICV injection of a-MSH can induceexcessive grooming or elicit the known stretching and yawn­ing syndrome. However, these effects were not observed inthe present study, probably as a result of the lower dosesused (0.5 ug vs 1.5 p,g).

The removal of the pituitary gland did not influence thispassage of a-MSH significantly as was demonstrated, whichis in favour of this proposed route for a-MSH from the CSFinto the blood (Fig. 7, Right).

Whether the pituitary can contribute to the increase ina-MSH levels after injecting this peptide into the ventricularsystem of intact animals can not be completely excluded.Indeed Wiegant et al. [34] have demonstrated an increase ofcorticosterone in plasma of intact rats and not in that ofhypophysectomized animals after ICV injection of ACTH1-16-NH2 , whereas peripheral injection of this analog has noeffect on the adrenal cortex.

However, it may also be that a-MSH found in plasma andCSF are of different origin and that the release of a-MSHinto the blood is regulated differently from the release ofa-MSH into the CSF. The.source of CSF-a-MSH may be thecentral nervous system itself. That melanotropic peptides ofcentral origin indeed inhibit the CSF, was demonstrated bythe persistingly high levels of radio-immunoassayablea-MSH in the CSF of rats after hypophysectomy, which is inagreement with the findings of Thody et al. [28]. Moreover,hypophysectomy does not alter the a-MSH content of thearcuate nucleus of the hypothalamus. .

However, in relation to the CNS, a-MSH of pituitaryorigin may not be disregarded, since it has been shown toplay an important role in brain functioning as was demon­strated by the impairment in behavior after hypophysec­tomy, which could be corrected by systemic administrationof a-MSH or related peptides (29J. Additionally, extirpationof the pituitary has been shown to reduce the radioirn­munoassayable a-MSH content of the whole brain [20,28J,the thalamus, the cerebellum and the brain stem [19], as wellas of the hypothalamus [29J.

a-MSH AND CSF

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