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Inhibition by extracts of frog and rat brain of MSH release by frog pars intermedia
GENERAL
AND
COMPAR.4TIVE
Inhibition
7, 37&374
ENDOCRINOLOGY
by Extracts
of Frog
by Frog c. L. RALPH of Biology,
Department
(1966)
and
Rat Brain
of MSH
Release
Pars Intermedial SUMATHY
AND
Received
SAMPATH
of Pittsburgh,
University
February
Pittsburgh,
Pennsylvania
12, 1966
The secretion of melanin-dispersing substance from the adenohypophyses of frogs, Rana pipiens Schreber, as determined by bioassay with frog skin, was inhibited by saline extracts of frog and rat cerebrum and hypothalamus, particularly by the latter. Isolated pars intermedia was similarly inhibited. However, rat pituitaries under similar conditions were neither inhibited nor stimulated by such extracts. The inhibitor of secretion in the frog appears to be something other than the usually considered neurohumors.
In the last decade evidence has been accumulating about how the pituitary gland is regulated by the hypothalamus. While considerable numbers of investigators, have been concerned with the mechanism of control of the pars distalis of the adenohypophysis, relatively less attention has been paid to the control of the pars intermedia. For some time it has been speculated that, in the case of the amphibian pars intermedia, some kind of inhibition of MSH secretion was exerted by the hypothalamus (Etkin, 1962a; Dierst and Ralph, 1962). These ideas were based upon indirect evidence from studies with tadpoles (Etkin, tech1943) ) employing transplantation niques to alter intermedia-infundibula interrelationships, and with adult frogs (Etkin, 1962a), using various methods to interfere with normal brain-pituitary relations. Recently, Brinkley and Bercu (1965) reported the in vitro inhibition by brain extracts of the secretion of melanin-dispersing factors by the pars intermedia and distalis of the frog, Rana pipiens. However, their extracts of brain cortex appeared to be equally effective in exerting inhibition as ‘This investigation GB-51 and GB-4258 Foundation.
was from
supported by grants the National Science 370
those from hypothalamus. An “inhibitor” conforms to what prior studies had predicted, but inhibition has been considered to be an exclusive property of the hypothalamus (Etkin, 1962a). At the time these results were reported we had reached independently similar conclusions, but we also found that when the MSH-like activity inherent in the brain extracts (Ralph and Peyton, 1966) is taken into consideration, the hypothalamus is revealed to contain, more of the “inhibitor” than other parts of the brain. In addition, we were able to duplicate these results with frog pituitaries using extracts of rat brain (Ralph and Sampath, 1965). METHODS The obtaining and care of frogs (R. pipiens Schreber), and the method of hypophysectomy are described elsewhere (Ralph and Peyton, 1966). All frogs were light-adapted for 16-29 hours in white containers under a fluorescent lamp. Sacrifice by decapitation was done between 9 and 12 A.M. Skins. Two large pieces of skin were taken, one from each thigh of the hind leg, from the same frogs providing the brain tissues. The skins were washed in two changes of saline and mounted on aluminum frames 25 mm in diameter, similar to those described by Shizume et al. (1954). Bruin Homogenates. The brains with pituitary attached were dissected out and placed in a dish resting on ice. The pituitaries (see below) were
INHIBITION
OF
MSH
removed and the rostra1 end of the cerebrum and the hypothalamus (including the optic chiasma) were cut away from each brain. The hypothalamus, in some assays, was divided by a transverse cut into stalk-median eminence and anterior hypothalamus portions. The total wet weight of the pieces of cerebrum (about 175 mg per three pieces) equalled that of the hypothalamus in each assay. These were homogenized by sonication in 10 ml of a saline appropriate for the pituitaries used in the incubation. Rat brain homogenates were prepared in the same manner, except that the animals were not light adapted, but simply taken from their cages in the animal room prior to decapitation and die section of the equivalent parts of the brain, and the brains of two animals were homogenized in 10 ml of saline. Because of the larger brain size the amount of rat brain tissue was 1.5 times larger than that obtained from frogs. Pituitaries. The adenohypophyses of frogs were removed from the brains by fine forceps. The pars nervosa remains attached to the infundibulum when this is done. In most assays the adenohypophysis was separated under a microscope into pars distalis and pars intermedia, two of the latter being used in the assay. Incubation of frog pituitaries took place in brain extracts prepared in amphibian Ringer saline. In those instances where rat pituitary was used, whole pituitaries were split into right and left halves and three halves were incubated with the extracts, the latter being prepared in KrebsRinger bicarbonate solution. Bioassay. In the early experiments pituitaries were incubated on a gyratory shaker bath for 1 hour with 10 ml of extract from three brains (at 21-23°C for frog pituitary and 37°C for those of the rat) in a 50 ml beaker under an atmosphere of 95% 0% and 5% CO,. The pituitaries were then taken out of the medium and a mounted skin, which had just been read for reflectance (Photovolt reflectometer), was placed in each beaker. The skin was then incubated for 1 hour with the medium, which now contained melanocyte-stimulating hormone (MSH) (and probably ACTH also) released by the adenohypophysis, and again its reflectance was read. The change (decrease) in reflectance (AR) is an index of the melanindispersing activity in the medium. In later assays additional steps were introduced to permit compensation for MSH inherent in the extracts (Ralph and Peyton, 1956) and thereby allow a more accurate estimate of the amount of MSH released by the pituitaries. Six frog brains were used to make an homogenate in 20 ml of saline. Half of this was incubated with a skin to
RELEASE
IN
371
FROG
determine the amount of MSH-like activit.v in the homogenate, in terms of change in skin reiectance (A%). The aNther half was incubated simultaneously with two pars intermedia (to be more certain that the darkening agent was MSHl and not also ACTH of the pars distalis). The intermedia were t.hen removed from the homoaenate, and, in an incubation immediately after, a skin (which came from the other leg of the same frog supplying the skin for the first incubation) was exposed to the extract into which MSH had beet released. The difference between initial and final reflectance (AR?) values of this skin provided a measure of total MSH activity-that from brain plus intermedia. Therefore, in estimating the MSH activity actually released by the pars intermedia. the data are expressed as AR,AR,. Analysis of Data. All experiments were done as sets of two experimental tissues and a saline control. Each was replicated four to six times. The data are expressed as means with standard deviations. Student’s t test. with Bessel’s correetion for small samples, was used to determine significance of differences between the means of the experimental and saline control groups. I
/
RESULTS
Table 1 shows the results, in terms of decrease in skin reflectance (AR), of bathing frog and rat pituitaries in extracts of frog or rat brain extracts. It is Seen that frog adenohypophyses bathed in hypotbalTABLE
1
APPARENT EFFECT OF BRAIN EXTRACTS ON SECRETION OF MELANIN-DBPERSENG SUBSTANCE BY PITUITARPS in Vitm AS MEASURED BY CHANGE IN SKIN RJGFLECTAWJ~ AR Brain
extract
Frog pituitary
Rat pituitary
Bog Hypothalamus Cerebrum Saline
37.8 25.6 47.5
C 4.0G + 0.9” rl: 7.4
25.6 30.5 26.8
3~ 5.6 + 8.8 + 9.6
32.8 25.4 48.8
31 3.g6 f 5.2c + 6.9
25.8 31.6 29.1
f l.:t + 6.5 i 10.5
Rat Hypothalamus Cerebrum Saline Note. ap < 6p < cp <
n = 4. 0.05. 0.01. 0.001.
372
RALPH
AND
amic homogenates (of either rat or frog) appear to have released less darkening agent than those in saline, but those in cerebral homogenates released still less. The rat pituitaries, however, appear to have been unaffected by the rat or frog brain extracts. These results are deceptive, however, because the brain tissues contain an MSHlike substance (Ralph and Peyton, 1966). We, therefore, turned to using the modified assay as described above. When that procedure was used results such as those in Table 2 were obtained. The hypothalamus TABLE 2 RELATIVE AMOUNTS OF MSH RELEASED BY PARS INTERMEDIA INCUBATED IN BRAIN EXTRACTS Brain extract Intact
Hypothalamus
Note.
+ 4.5 & 7.1
H vs. S < 0.001 Hvs.C cvs.s
Frogs
Cerebrum Saline
9.9 + 4.6 19.8 37.4
13.3 25.3 33.2
k 9.6 &- 13.2 k 9.4
RELATIVE AMOUNTS PARS INTERMEDIA
H vs. S < 0.001 Hvs. C n.s. Cvs. S 11.s.
n = 6, n.s. = not significant.
is now seen to contain relatively more “inhibit’or” of MSH-release than the cerebrum. Frogs hypophysectomized 3 days prior to sacrifice were used as brain donors to see if absen.ce of the adenohypophysis would influence the inhibition effect. As can be seen also in Table 2 the brain content of “inhibitor” may have been somewhat less than normal, especially in the cerebrum, after hypophysectomy. As shown by the data of Table 3 the extracts of rat brain can influence the frog pars intermedia in the same relative manner as frog brain extracts. Table 4 shows that the “inhibitor” is in both the caudal and rostra1 parts of the hypothalamus of the frog, perhaps slightly more being present in the caudal (median eminence) portion. Several neurohormones commonly- found in hypothalamus were used in the assay in the same manner as brain extracts. They
TABLE 3 OF MSH RELEASED INCUBATED EXTRACTS
Brain extra&
IN RAT
BY FROG BRAIN
ARz - AR1
Hypothalamus
14.0
Cerebrum Saline
16.4 k 8.4&
+ 11.4a
32.7
I 10.4
Note. n = 6. a p < 0.001.
were: acetylcholine chloride (Nutritional Biochem., 100 pug/ml), epinephrine (adrenalin chloride, Parke Davis, 50 ,pg/ml), norepinephrine (dl-arterenol HCI, Calbiochem, creatinine sulfate 150 ~g/Illl) ) serotonin TABLE 4
P
Frogs
Hypothalamus Cerebrum Saline Hypox
ARz - AR1
SAMPATH
RELATIVE AMOUNTS OF MSH RELEASED BY PARS INTERMEDIA INCUBATED IN HYPOTHALAMIC EXTRACTS Extract
ARz - AR1
Anterior hypothalamus Stalk-median eminence Saline
18.0 11.8 38.0
i 9.3” + 7.3” i 2.6
Note. n = 6. a p < 0.001.
(Calbiochem, 30 ,pg/ml), histamine phosphate (Lilly, 100 pg/ml), vasopressin (Pitressin, Parke Davis, 20 units/ml), and oxytocin (Syntocinon, Sandoz, 1 unit/ml). None of these inhibited MSH release by pars intermedia. DISCUSSION
From our initial results with frog pituitaries we were led to the same conclusion as Brinkley and Bercu (1965)-namely, that cerebral and hypothalamic extracts of the frog appear equally effective in inhibiting the release of melanophore-darkening hormones. In addition, however, we have shown that, if account is taken of the MSH-like activity inherent in the brain extracts, the hypothalamus appears as a more important source of the “inhibitor.” We were able to produce the same result, on frog also, using rat brain extracts pituitaries.
INI-:IBITION
OF
MSH
are several points that argue for or against the idea that a substance in these extracts is an actual inhibitor of pars intermedia secretion. Favoring such an interpretation is the fact that the substance does cause inhibition of MSH release and therefore can be readily accommodated within current concepts of inhibition by the brain of MSH secretion (Etkin, 1962a) (i.e., if a “releaser” had been found it, would be difficult to explain its significance in the light, of prior information). Furthermore, if not confined to the hypothalamus (the apparent abode of all accepted hypophysiotropic factors), it. is a least more abundant there. On the other hand, the fact that rat brain also causes the same kind of inhibition suggests that the “inhibitor” may be, in fact, some common neurohumoral molecule (other t,han those tested). Likewise, because some inhibition was characteristic of the cerebral extracts, this molecule may have a general distribution in the brain. There is also the possibility that the functional inhibitor is only in the hypothalamus and the apparent inhibition by the cerebrum is due to another (and very common) molecule, that normally does not regulate the intermedia, and is revealed only by the artificial conditions of the bioassay. If the “inhibitor” should actually be a hypophysiotropic factor produced in the brain; then it will remain to be determined whether it is released in the median eminence, and then reaches the pars distalis via the blood, or it is delivered through neural processes directly to the intermediate lobe cells. Despite the presence of a vascular communication between the median eminence and the pars intermedia (Green, 1947), the evidence weighs in favor of the idea that inhibition is effected directly by neurons that penetrate the intermediate lobe (Etkin, 1962a). Both neurosecretory (Dawson, 1953; Et,kin, 1962b; Iturriza and Mestorino, 1965) and ordinary neurons (Enemar and Falck, 1965; Iturriza, 1964; Jorgensen and Larsen, 1963) have been described in the anuran pars intermedia, and both kinds of fibers have been proposed by different investigators as the mediator There
RELEASE
IN
FROG
373
of the well-documented inhibition by the hypotha!amus. If they are neurosecrctory, they apparently are not those of the preoptic magnocellular nuclei (Dierickx, 1965) and, as the preecnt study showy, do not exert, their effect through oxytocin or vasopressin. A direct neural inhibition would al!ow for use of a nonspecific chemicalt control being made specific by virtue of direct, delivery of that “neurohumor” to the intermedia. Although eplnephrine and norepinephrine had no effect on the reieaee af MSH in these experiments, some cireumstantial evidence from pharmacological studies of Dierst-Davies et al. (1966) suggest an adrenergic type neurohumor may be the final inhibitor of the int’ermediate lobe in R. pipiens. This possibility is strengthened by the discovery of a plexus of catecholamine containing nerve terminals in the pars intermedia of R. tempormaG by Enemar and Falck (1965) and the evidence of Iturriza (1966) that monoamines may be important in regulating t’he intermedia of Bufo arenarum. Accordingly, we will SWvey soon the various catecholamines to see if any act as an inhibitor in our assay. It is of interest to note that rat pituitaries were not affected by any of the extracts, suggesting that. only frog pituitaries are responsive to the “inhibitor,” the latter being common to both animal species. There is a report by Taleisnik and Qrias (1965) of in. ‘uivo “release” of MSR by stalk-median eminence extracts in rats. On the other hand, it was found by Kastin (1965) that when rat pituitaries were incubated for 96 hours with hypothalamic extract,s, in 6 of 9 incubations a 2%fold decrease in MSH activity of the medium was observed. These two reports appear to be conflicting, but it should be pointred out that because of difference in methods they are not comparable experiments. Our experiment,s are somewhat like Kastin’s in that in. vitro assays were used, but our incubations, being only 1 hour in duration, would not have detected the reduced MSR activity he found in his 4-day incubations. The short-term effect found in our study may arise from some mechanism different
374
RALPH AND SAMPATH
from both of the above-mentioned with rats.
studies
REFERENCES BRINKLEY, H. J., AND BERCU, B. B. (1965). Hypothalamic and cortical inhibitors of frog (Rana pipiend melanophore stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) secretion. Am. Zool. 5, 211-212. DAWSO’N, A. B. (1953). Evidence for the termination of neurosecretory fibers within the pars intermedia of the hypophysis of the frog, Rana pipiens. DIERICKX,
Anat.
Record
115,
6%69.
K. (1965). On the neurosecretory control of the pars intermedia of the hypophysis in the frog. Gen. Comp. Endocrinol. 2, 347-353. DIERST, K. E., AND RALPH, C. L. (1962). Effect of hypothalamic stimulation on melanophores in the frog. Gen. Comp. Endocrinol. 2, 347-353. DIERST-DAVIES, K. E., RALPH, C. L., AND PECHERSKY, J. (1966). Effects of pharmacological agents on the hypothalamus of Rana pipiens in relation to the control of skin melanophores. Gen. Comp. Endocrinol. 16, 40%419. ENEMA&, A., AND FALCK, B. (1965). On the presence of adrenergic nerves in the pars intermedia of the frog, Rana temporaria. Gen. Comp. ETKIN,
Endocrinol.
5,
577-583.
W. (1943). The developmental control of pars intermedia by brain. J. Exp, Zool. 92, 3147. ETKIN, W. (1962a). Hypothalamic inhibition of pars intermedia activity in the frog. Gen. Comp. Endocrinol., Suppl. 1, 14&159. ETKIN, W. (1962b). Neurosecretory control of the
pars intermedia. Gen. Comp. Endocrinol. ,2, 161-169. GREEN, J. D. (1947). Vessels and nerves of amphibian hypophyses. Anat. Record 99, 21-53. I~RRIZA, F. C. (1964). Electron-microscopic study of the pars intermedia of the toad Bufo arenarum. Gen. Comp. Endocrinol. 4, 492-508. ITYJRRIZA, F. C. (1966). Monoamines and control of the pars intermedia of the toad pituitary. Gen. Comp. Endocrinol. 16, 19-25. ITURRIZA, F. C., AND MESTORINO, M. (1965). Nervous fibers in the pars intermedia of the toad Bufo arenarum. Acta Anat. ISO, 398-405. JORGENSEN, C. B., AND LARSEN, L. 0. (1963). Nature of the nervous control of pars intermedia function in amphibians: rate of functiona recovery after denervation. Gen. Comp. Endocrinol. 3, 46&472. &WIN, A. J. (1965). Effect of hypothalamic extracts on release of MSH in vitro. Program of the 47th Meet. of the Endocrine Sot., p. 98. RALPH, C. L., AND PEYTON, S. C. (1966). MSH-like substance in the brain of the frog, Rana pipiens. Gen. Comp. Endocrinol. 7, 363-369. RALPH, C. L., AND SAMPATH, S. (1965). Inhibition 7 of melanin-dispersing hormone release from frog pituitaries by brain extracts of frog and rat.. Am. Zool. ,5, 671-672. SHIZUME, K., LERNER, A. B., AND FITZPATRICK, T.. B. (1954). In vitro bioassay for the melanocyte stimulating hormone. Endocrinology 54, 553-
560. S., AND ORIAS, R. (1965). A melanocyte-stimulating hormone releasing factor in hypothalamic extracts. Am. J. Physiol. 298, 293-296.
TALEISNIK,
AND
COMPAR.4TIVE
Inhibition
7, 37&374
ENDOCRINOLOGY
by Extracts
of Frog
by Frog c. L. RALPH of Biology,
Department
(1966)
and
Rat Brain
of MSH
Release
Pars Intermedial SUMATHY
AND
Received
SAMPATH
of Pittsburgh,
University
February
Pittsburgh,
Pennsylvania
12, 1966
The secretion of melanin-dispersing substance from the adenohypophyses of frogs, Rana pipiens Schreber, as determined by bioassay with frog skin, was inhibited by saline extracts of frog and rat cerebrum and hypothalamus, particularly by the latter. Isolated pars intermedia was similarly inhibited. However, rat pituitaries under similar conditions were neither inhibited nor stimulated by such extracts. The inhibitor of secretion in the frog appears to be something other than the usually considered neurohumors.
In the last decade evidence has been accumulating about how the pituitary gland is regulated by the hypothalamus. While considerable numbers of investigators, have been concerned with the mechanism of control of the pars distalis of the adenohypophysis, relatively less attention has been paid to the control of the pars intermedia. For some time it has been speculated that, in the case of the amphibian pars intermedia, some kind of inhibition of MSH secretion was exerted by the hypothalamus (Etkin, 1962a; Dierst and Ralph, 1962). These ideas were based upon indirect evidence from studies with tadpoles (Etkin, tech1943) ) employing transplantation niques to alter intermedia-infundibula interrelationships, and with adult frogs (Etkin, 1962a), using various methods to interfere with normal brain-pituitary relations. Recently, Brinkley and Bercu (1965) reported the in vitro inhibition by brain extracts of the secretion of melanin-dispersing factors by the pars intermedia and distalis of the frog, Rana pipiens. However, their extracts of brain cortex appeared to be equally effective in exerting inhibition as ‘This investigation GB-51 and GB-4258 Foundation.
was from
supported by grants the National Science 370
those from hypothalamus. An “inhibitor” conforms to what prior studies had predicted, but inhibition has been considered to be an exclusive property of the hypothalamus (Etkin, 1962a). At the time these results were reported we had reached independently similar conclusions, but we also found that when the MSH-like activity inherent in the brain extracts (Ralph and Peyton, 1966) is taken into consideration, the hypothalamus is revealed to contain, more of the “inhibitor” than other parts of the brain. In addition, we were able to duplicate these results with frog pituitaries using extracts of rat brain (Ralph and Sampath, 1965). METHODS The obtaining and care of frogs (R. pipiens Schreber), and the method of hypophysectomy are described elsewhere (Ralph and Peyton, 1966). All frogs were light-adapted for 16-29 hours in white containers under a fluorescent lamp. Sacrifice by decapitation was done between 9 and 12 A.M. Skins. Two large pieces of skin were taken, one from each thigh of the hind leg, from the same frogs providing the brain tissues. The skins were washed in two changes of saline and mounted on aluminum frames 25 mm in diameter, similar to those described by Shizume et al. (1954). Bruin Homogenates. The brains with pituitary attached were dissected out and placed in a dish resting on ice. The pituitaries (see below) were
INHIBITION
OF
MSH
removed and the rostra1 end of the cerebrum and the hypothalamus (including the optic chiasma) were cut away from each brain. The hypothalamus, in some assays, was divided by a transverse cut into stalk-median eminence and anterior hypothalamus portions. The total wet weight of the pieces of cerebrum (about 175 mg per three pieces) equalled that of the hypothalamus in each assay. These were homogenized by sonication in 10 ml of a saline appropriate for the pituitaries used in the incubation. Rat brain homogenates were prepared in the same manner, except that the animals were not light adapted, but simply taken from their cages in the animal room prior to decapitation and die section of the equivalent parts of the brain, and the brains of two animals were homogenized in 10 ml of saline. Because of the larger brain size the amount of rat brain tissue was 1.5 times larger than that obtained from frogs. Pituitaries. The adenohypophyses of frogs were removed from the brains by fine forceps. The pars nervosa remains attached to the infundibulum when this is done. In most assays the adenohypophysis was separated under a microscope into pars distalis and pars intermedia, two of the latter being used in the assay. Incubation of frog pituitaries took place in brain extracts prepared in amphibian Ringer saline. In those instances where rat pituitary was used, whole pituitaries were split into right and left halves and three halves were incubated with the extracts, the latter being prepared in KrebsRinger bicarbonate solution. Bioassay. In the early experiments pituitaries were incubated on a gyratory shaker bath for 1 hour with 10 ml of extract from three brains (at 21-23°C for frog pituitary and 37°C for those of the rat) in a 50 ml beaker under an atmosphere of 95% 0% and 5% CO,. The pituitaries were then taken out of the medium and a mounted skin, which had just been read for reflectance (Photovolt reflectometer), was placed in each beaker. The skin was then incubated for 1 hour with the medium, which now contained melanocyte-stimulating hormone (MSH) (and probably ACTH also) released by the adenohypophysis, and again its reflectance was read. The change (decrease) in reflectance (AR) is an index of the melanindispersing activity in the medium. In later assays additional steps were introduced to permit compensation for MSH inherent in the extracts (Ralph and Peyton, 1956) and thereby allow a more accurate estimate of the amount of MSH released by the pituitaries. Six frog brains were used to make an homogenate in 20 ml of saline. Half of this was incubated with a skin to
RELEASE
IN
371
FROG
determine the amount of MSH-like activit.v in the homogenate, in terms of change in skin reiectance (A%). The aNther half was incubated simultaneously with two pars intermedia (to be more certain that the darkening agent was MSHl and not also ACTH of the pars distalis). The intermedia were t.hen removed from the homoaenate, and, in an incubation immediately after, a skin (which came from the other leg of the same frog supplying the skin for the first incubation) was exposed to the extract into which MSH had beet released. The difference between initial and final reflectance (AR?) values of this skin provided a measure of total MSH activity-that from brain plus intermedia. Therefore, in estimating the MSH activity actually released by the pars intermedia. the data are expressed as AR,AR,. Analysis of Data. All experiments were done as sets of two experimental tissues and a saline control. Each was replicated four to six times. The data are expressed as means with standard deviations. Student’s t test. with Bessel’s correetion for small samples, was used to determine significance of differences between the means of the experimental and saline control groups. I
/
RESULTS
Table 1 shows the results, in terms of decrease in skin reflectance (AR), of bathing frog and rat pituitaries in extracts of frog or rat brain extracts. It is Seen that frog adenohypophyses bathed in hypotbalTABLE
1
APPARENT EFFECT OF BRAIN EXTRACTS ON SECRETION OF MELANIN-DBPERSENG SUBSTANCE BY PITUITARPS in Vitm AS MEASURED BY CHANGE IN SKIN RJGFLECTAWJ~ AR Brain
extract
Frog pituitary
Rat pituitary
Bog Hypothalamus Cerebrum Saline
37.8 25.6 47.5
C 4.0G + 0.9” rl: 7.4
25.6 30.5 26.8
3~ 5.6 + 8.8 + 9.6
32.8 25.4 48.8
31 3.g6 f 5.2c + 6.9
25.8 31.6 29.1
f l.:t + 6.5 i 10.5
Rat Hypothalamus Cerebrum Saline Note. ap < 6p < cp <
n = 4. 0.05. 0.01. 0.001.
372
RALPH
AND
amic homogenates (of either rat or frog) appear to have released less darkening agent than those in saline, but those in cerebral homogenates released still less. The rat pituitaries, however, appear to have been unaffected by the rat or frog brain extracts. These results are deceptive, however, because the brain tissues contain an MSHlike substance (Ralph and Peyton, 1966). We, therefore, turned to using the modified assay as described above. When that procedure was used results such as those in Table 2 were obtained. The hypothalamus TABLE 2 RELATIVE AMOUNTS OF MSH RELEASED BY PARS INTERMEDIA INCUBATED IN BRAIN EXTRACTS Brain extract Intact
Hypothalamus
Note.
+ 4.5 & 7.1
H vs. S < 0.001 Hvs.C cvs.s
Frogs
Cerebrum Saline
9.9 + 4.6 19.8 37.4
13.3 25.3 33.2
k 9.6 &- 13.2 k 9.4
RELATIVE AMOUNTS PARS INTERMEDIA
H vs. S < 0.001 Hvs. C n.s. Cvs. S 11.s.
n = 6, n.s. = not significant.
is now seen to contain relatively more “inhibit’or” of MSH-release than the cerebrum. Frogs hypophysectomized 3 days prior to sacrifice were used as brain donors to see if absen.ce of the adenohypophysis would influence the inhibition effect. As can be seen also in Table 2 the brain content of “inhibitor” may have been somewhat less than normal, especially in the cerebrum, after hypophysectomy. As shown by the data of Table 3 the extracts of rat brain can influence the frog pars intermedia in the same relative manner as frog brain extracts. Table 4 shows that the “inhibitor” is in both the caudal and rostra1 parts of the hypothalamus of the frog, perhaps slightly more being present in the caudal (median eminence) portion. Several neurohormones commonly- found in hypothalamus were used in the assay in the same manner as brain extracts. They
TABLE 3 OF MSH RELEASED INCUBATED EXTRACTS
Brain extra&
IN RAT
BY FROG BRAIN
ARz - AR1
Hypothalamus
14.0
Cerebrum Saline
16.4 k 8.4&
+ 11.4a
32.7
I 10.4
Note. n = 6. a p < 0.001.
were: acetylcholine chloride (Nutritional Biochem., 100 pug/ml), epinephrine (adrenalin chloride, Parke Davis, 50 ,pg/ml), norepinephrine (dl-arterenol HCI, Calbiochem, creatinine sulfate 150 ~g/Illl) ) serotonin TABLE 4
P
Frogs
Hypothalamus Cerebrum Saline Hypox
ARz - AR1
SAMPATH
RELATIVE AMOUNTS OF MSH RELEASED BY PARS INTERMEDIA INCUBATED IN HYPOTHALAMIC EXTRACTS Extract
ARz - AR1
Anterior hypothalamus Stalk-median eminence Saline
18.0 11.8 38.0
i 9.3” + 7.3” i 2.6
Note. n = 6. a p < 0.001.
(Calbiochem, 30 ,pg/ml), histamine phosphate (Lilly, 100 pg/ml), vasopressin (Pitressin, Parke Davis, 20 units/ml), and oxytocin (Syntocinon, Sandoz, 1 unit/ml). None of these inhibited MSH release by pars intermedia. DISCUSSION
From our initial results with frog pituitaries we were led to the same conclusion as Brinkley and Bercu (1965)-namely, that cerebral and hypothalamic extracts of the frog appear equally effective in inhibiting the release of melanophore-darkening hormones. In addition, however, we have shown that, if account is taken of the MSH-like activity inherent in the brain extracts, the hypothalamus appears as a more important source of the “inhibitor.” We were able to produce the same result, on frog also, using rat brain extracts pituitaries.
INI-:IBITION
OF
MSH
are several points that argue for or against the idea that a substance in these extracts is an actual inhibitor of pars intermedia secretion. Favoring such an interpretation is the fact that the substance does cause inhibition of MSH release and therefore can be readily accommodated within current concepts of inhibition by the brain of MSH secretion (Etkin, 1962a) (i.e., if a “releaser” had been found it, would be difficult to explain its significance in the light, of prior information). Furthermore, if not confined to the hypothalamus (the apparent abode of all accepted hypophysiotropic factors), it. is a least more abundant there. On the other hand, the fact that rat brain also causes the same kind of inhibition suggests that the “inhibitor” may be, in fact, some common neurohumoral molecule (other t,han those tested). Likewise, because some inhibition was characteristic of the cerebral extracts, this molecule may have a general distribution in the brain. There is also the possibility that the functional inhibitor is only in the hypothalamus and the apparent inhibition by the cerebrum is due to another (and very common) molecule, that normally does not regulate the intermedia, and is revealed only by the artificial conditions of the bioassay. If the “inhibitor” should actually be a hypophysiotropic factor produced in the brain; then it will remain to be determined whether it is released in the median eminence, and then reaches the pars distalis via the blood, or it is delivered through neural processes directly to the intermediate lobe cells. Despite the presence of a vascular communication between the median eminence and the pars intermedia (Green, 1947), the evidence weighs in favor of the idea that inhibition is effected directly by neurons that penetrate the intermediate lobe (Etkin, 1962a). Both neurosecretory (Dawson, 1953; Et,kin, 1962b; Iturriza and Mestorino, 1965) and ordinary neurons (Enemar and Falck, 1965; Iturriza, 1964; Jorgensen and Larsen, 1963) have been described in the anuran pars intermedia, and both kinds of fibers have been proposed by different investigators as the mediator There
RELEASE
IN
FROG
373
of the well-documented inhibition by the hypotha!amus. If they are neurosecrctory, they apparently are not those of the preoptic magnocellular nuclei (Dierickx, 1965) and, as the preecnt study showy, do not exert, their effect through oxytocin or vasopressin. A direct neural inhibition would al!ow for use of a nonspecific chemicalt control being made specific by virtue of direct, delivery of that “neurohumor” to the intermedia. Although eplnephrine and norepinephrine had no effect on the reieaee af MSH in these experiments, some cireumstantial evidence from pharmacological studies of Dierst-Davies et al. (1966) suggest an adrenergic type neurohumor may be the final inhibitor of the int’ermediate lobe in R. pipiens. This possibility is strengthened by the discovery of a plexus of catecholamine containing nerve terminals in the pars intermedia of R. tempormaG by Enemar and Falck (1965) and the evidence of Iturriza (1966) that monoamines may be important in regulating t’he intermedia of Bufo arenarum. Accordingly, we will SWvey soon the various catecholamines to see if any act as an inhibitor in our assay. It is of interest to note that rat pituitaries were not affected by any of the extracts, suggesting that. only frog pituitaries are responsive to the “inhibitor,” the latter being common to both animal species. There is a report by Taleisnik and Qrias (1965) of in. ‘uivo “release” of MSR by stalk-median eminence extracts in rats. On the other hand, it was found by Kastin (1965) that when rat pituitaries were incubated for 96 hours with hypothalamic extract,s, in 6 of 9 incubations a 2%fold decrease in MSH activity of the medium was observed. These two reports appear to be conflicting, but it should be pointred out that because of difference in methods they are not comparable experiments. Our experiment,s are somewhat like Kastin’s in that in. vitro assays were used, but our incubations, being only 1 hour in duration, would not have detected the reduced MSR activity he found in his 4-day incubations. The short-term effect found in our study may arise from some mechanism different
374
RALPH AND SAMPATH
from both of the above-mentioned with rats.
studies
REFERENCES BRINKLEY, H. J., AND BERCU, B. B. (1965). Hypothalamic and cortical inhibitors of frog (Rana pipiend melanophore stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) secretion. Am. Zool. 5, 211-212. DAWSO’N, A. B. (1953). Evidence for the termination of neurosecretory fibers within the pars intermedia of the hypophysis of the frog, Rana pipiens. DIERICKX,
Anat.
Record
115,
6%69.
K. (1965). On the neurosecretory control of the pars intermedia of the hypophysis in the frog. Gen. Comp. Endocrinol. 2, 347-353. DIERST, K. E., AND RALPH, C. L. (1962). Effect of hypothalamic stimulation on melanophores in the frog. Gen. Comp. Endocrinol. 2, 347-353. DIERST-DAVIES, K. E., RALPH, C. L., AND PECHERSKY, J. (1966). Effects of pharmacological agents on the hypothalamus of Rana pipiens in relation to the control of skin melanophores. Gen. Comp. Endocrinol. 16, 40%419. ENEMA&, A., AND FALCK, B. (1965). On the presence of adrenergic nerves in the pars intermedia of the frog, Rana temporaria. Gen. Comp. ETKIN,
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5,
577-583.
W. (1943). The developmental control of pars intermedia by brain. J. Exp, Zool. 92, 3147. ETKIN, W. (1962a). Hypothalamic inhibition of pars intermedia activity in the frog. Gen. Comp. Endocrinol., Suppl. 1, 14&159. ETKIN, W. (1962b). Neurosecretory control of the
pars intermedia. Gen. Comp. Endocrinol. ,2, 161-169. GREEN, J. D. (1947). Vessels and nerves of amphibian hypophyses. Anat. Record 99, 21-53. I~RRIZA, F. C. (1964). Electron-microscopic study of the pars intermedia of the toad Bufo arenarum. Gen. Comp. Endocrinol. 4, 492-508. ITYJRRIZA, F. C. (1966). Monoamines and control of the pars intermedia of the toad pituitary. Gen. Comp. Endocrinol. 16, 19-25. ITURRIZA, F. C., AND MESTORINO, M. (1965). Nervous fibers in the pars intermedia of the toad Bufo arenarum. Acta Anat. ISO, 398-405. JORGENSEN, C. B., AND LARSEN, L. 0. (1963). Nature of the nervous control of pars intermedia function in amphibians: rate of functiona recovery after denervation. Gen. Comp. Endocrinol. 3, 46&472. &WIN, A. J. (1965). Effect of hypothalamic extracts on release of MSH in vitro. Program of the 47th Meet. of the Endocrine Sot., p. 98. RALPH, C. L., AND PEYTON, S. C. (1966). MSH-like substance in the brain of the frog, Rana pipiens. Gen. Comp. Endocrinol. 7, 363-369. RALPH, C. L., AND SAMPATH, S. (1965). Inhibition 7 of melanin-dispersing hormone release from frog pituitaries by brain extracts of frog and rat.. Am. Zool. ,5, 671-672. SHIZUME, K., LERNER, A. B., AND FITZPATRICK, T.. B. (1954). In vitro bioassay for the melanocyte stimulating hormone. Endocrinology 54, 553-
560. S., AND ORIAS, R. (1965). A melanocyte-stimulating hormone releasing factor in hypothalamic extracts. Am. J. Physiol. 298, 293-296.
TALEISNIK,