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Extracellular spaces of the rat pars intermedia as outlined by lanthanum tracer.

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THE ANATOMICAL RECORD 196:355-361 (1980)
Extracellular Spaces of the Rat Pars Intermedia
as Outlined by Lanthanum Tracer
Department ofAnatomy, University of New Mexico School of Medicine,
Albuquerque, New Mexico 87131
The extracellular spaces of the rat pars intermedia were examined after perfusion with fixative containing lanthanum. The tracer reveals complex and extensive interdigitations among secretory cells. Spaces appear continuous with extracellular “channels” between the epithelial cells lining the hypophyseal cleft. Some suggestion for close contiguity between endocrine cells was
present as “narrowing” or discontinuous areas within lanthanum-filled spaces.
Extracellular regions surrounding nerve fibers and terminals appear continuous
with the spaces between secretory cells. Areas between cells are thought to provide
access to vascular and local neurotransmitter input, as well as to provide a n
extensive area for extrusion of peptide hormones and endogeneous opiates.
The pituitary intermediate lobe (pars intermedia) is a source of several different peptide
molecules, including beta-lipotropin, melanocyte-stimulating hormone (MSH), adrenocorticotrophic hormone (ACTH),and the opiate peptide beta-endorphin (Chretien and Lis, ’78).Although the endorphin content has received
much attention recently in terms of its precise
localization (Akil et al., ’78), little is known
about control of synthesis of the molecule
within the pituitary cell or the mode of its release into the circulation. The situation is further complicated by the observation of few or no
intrinsic blood vessels in the pars intermedia,
which is a state quite different from elsewhere
in the pituitary gland (Page et al., ’76).Recent
studies in amphibians have shown a complex
system of “channels” between pars intermedia
cells, with implications for connections t o
peripheral vascular supply (Perryman, ’76;
Perryman and Bagnara, ’78).The present study
was initiated to characterize the extracellular
spaces in rat pars intermedia using a lanthanum tracer, in order to improve the understanding of how mammalian endorphin1
corticotrophin cells may communicate with one
another and with adjacent vascular spaces.
Some of the information has been presented
previously in a n abstract (Saland, ’79).
Ten adult male Sprague-Dawley rats (300 g)
were used for lanthanum perfusions. Rats were
0 1980 ALAN R. LISS, INC.
anesthetized with ether or pentobarbital and
perfused through the heart with 2.5%glutaraldehyde in 0.075 M cacodylateiHC1buffer containing 1% lanthanum nitrate. All procedures
were done a t room temperature. The lanthanum-containing fixative was prepared according to the procedures of Revel and Karnovsky
(’67).Pituitary glands were removed from perfused animals and placed in fresh fixative containing lanthanum for an additional 1-2 hours.
Neurointermediate lobes were separated and
rinsed in cacodylate buffer, postfixed for one
hour in 1%osmium tetroxide in cacodylate buffer, then dehydrated in graded ethanols and
embedded in Epon. Thin sections were examined in a Philips EM 200 or Hitachi HS-7S
electron microscope.
Low magnification electron micrographs of
lanthanum perfused intermediate lobes show
close association and complex interdigitations
of secretory cells with one another (Fig. 1-3).
Tangential sections of plasmalemmae between
two cells (Fig. 4) exhibit infoldings which sometimes reach some distance into endocrine cells.
Note that there are no blood vessels among the
cells (even a t lower magnifications) (Fig. 1).
Closer examination illustrates the presence of
coated caveolae which are filled with lanthanum, and have the appearance of “inReceived July 10, 1979; accepted August 29, 1979.
Fig. 1. Lanthanum surroundsgranulated cells ofpars intermedia. Note absence ofblood vessels. x 4,400.
Fig. 2. Note dense outlining of infoldings of extracellular space between secretory cells. x 11,100
Fig. 3. Nerve fiber (no partially surroundedby tracer. Greater density found in tangentially-cut area of
extracellular space (arrow). x 14,200.
Fig. 4. Complex interdigitations outlined between intermediate lobe cells. x 18,900,
vaginating” into the secretorycells (Fig.7). The
dense lanthanum content of the caveola in Figure 7 illustrates the continuity of the contents
with that of the extracellular space. Secretory
granules are observed to be closely associated
with the plasma membrane (Figs. 8,9), but fusion of the granule membrane with the plasma
membrane was not observed nor was there any
structural indication of exocytosis.
Epithelial cells lining the hypophyseal cleft
(Figs. 5, 6, montage) do not contain secretory
granules and are considered to serve as a border
between endocrine cells and the cleft. The
extracellular space between “cleft cells” is ob-
Figs. 5 and 6. Montage of cells bordering hypophyseal cleft (hc) and secretory cells (sc) beneath. Lanthanum filling demonstrates continuity of spaces.Note increaseddensity of tracer along area of basal lamina
(arrows). x 11,800.
Fig. 7. Lanthanum fills a coated caveola extending into secretory cell. Note nearby secretory granules
(sg). x 80,000.
Fig. 8. Tracer delineates “narrowing” of extracellular space. Secretory granules (sg) about plasma
membrane. x 85,000.
Fig. 9. “Break” in lanthanum (small arrow) illustrates possible junction between intermedia cells. Note
lanthanum in extracellular space surrounding cilium (large arrow). x 50,000.
Fig. 10. Lanthanum partially outlines nerve fiber (nf)containing small clear vesicles and some densecored vesicles. Note continuity of tracer with spaces between cells and nerve fiber. x 41,300.
served to be continuous with the spaces between secretory cells, as illustrated by the continuity of the lanthanum tracer from one cell
area to the next.
Occasionally, higher magnification of lanthanum-filled spaces reveals a “narrowing” of
the extracellular space (Fig. 81, or an occasional
focal “break” in the continuity of lanthanum
(Fig. 9). The narrowing and discontinuities
may be suggestive of close contiguity between
secretory cells.
The direct innervation of the pars intermedia
is clearly outlined by lanthanum tracer (Figs.
3,lO). Although both clear and dense-coredvesicles are visible in many fibers, the lanthanum
remains external to the neurovesicles and to
the nerve “terminals” as a whole, with no evidence for endocytic activity.
An additional observation was the lanthanum outlining of a cilium found deep within a
secretory cell (Fig. 9). Cilia are normally observed a t the surfaces of the epithelium lining
the hypophyseal cleft, and lanthanum was also
seen in some specimens to surround those cilia.
The “deeper” cilium in Figure 9 is obviously
surrounded by extensions of the extracellular
space, but the extent of the structure and its
relationship to other cells a s well as its possible
functions are not clear. In general, the overall
picture observed with lanthanum as an extracellular tracer is of secretory cells which
interdigitate with one another in a complex
manner. Surfaces of the endocrine cells must be
fairly large, since there are numerous infoldings.
In many vertebrates, including rat and frog,
the intermediate lobe is an unusual endocrine
organ in that it has a poor or nearly absent
blood supply (Howe and Maxwell, ’68; Howe,
’73).Several investigators have examined cellto-cell contact areas in the pituitary gland, and
have noted the presence of gap junctions in the
anterior lobe (Fletcher et al., ’75) and tight as
well as gap junctions between follicular cells of
the lobe (Mira-Moseret al., ’75). Extracellular
spaces of the anterior lobe of the pituitary are
clearly continuous with perivascular spaces of
numerous sinusoidal vessels, and granule release occurs into the spaces under normal circumstances (Shiino et al., ’72). Some unusual
conditions of hypersecretion or tumor formation may causerelease of anterior lobe granules
directly into spaces between cells rather than
into spaces adjacent to blood vessels (Horvath
and Kovacs, ’74).Recent freeze fracture studies
in rats, after hyperactivity of the intermediate
lobe induced by reserpine, have illustrated exocytosis directly into the extracellular spaces
(Saland, ’78).Semoff and Hadley (’78)have also
observed exocytosis after freeze fracture
methodology in pars intermedia of dark
adapted frogs. The spaces between intermedia
cells, therefore, appear to have important functions in serving as locations for granule (hormone) release. Conversely, the presence of
endocytosis, or caveolae which appear to be invaginating, in this investigation, and in studies
in amphibians (Perryman and Bagnara, ’78),
also illustrates that the rat intermediate lobe
cells may take up materials from extracellular
sources via the extensive spaces outlined by
Earlier studies on anuran amphibian intermediate lobes (Perryman, ’76) suggested that
stellate (nongranular, fusiform) cells which
ramify among secretory cells might function to
transport materials through the lobe. More recently, peroxidase injected to the vascular system illustrated the tortuous extensions of the
perivascular spaces in the frog intermediate
lobe (Perryman and Bagnara, ’78). The latter
authors noted that the spaces enlarged at the
“convergence of stellate cell processes,” and
suggested that both the extracellular spaces
and stellate cells might function in the transfer
of materials in the intermedia. The present investigation, using lanthanum as an outlining
tracer in the rat pituitary, demonstrates that a
mammalian species is similar to the frog, and
exhibits complex extracellular spaces between
secretory cells. Only a few “stellate” type cells
are present in the rat (Howe and Maxwell, ’68;
Howe, ’74),and these do not seem to be of major
importance, based upon morphologic observations, for transport of chemical or granular information. During the course of this study, it
was learned that DeBold and Kraicer (’79)
(abstracts of the Federation Meetings, Dallas,
Texas) had used peroxidase to study extracellular spaces in rat intermediate lobe, with findings similar to those observed in this study with
lanthanum. Here, there appears to be morphologic evidence for the entry of materials into the
cell, although the lanthanum tracer does not
provide additional evidence for extrusion of
The fact that the nerve terminals of the
intermediate lobe are surrounded by spaces
continuous with those around essentially all of
the secretory cells supports the concept of putative modulation by neurotransmitters directly
onto many different areas throughout the
gland. The number of nerve fibers varies from
one vertebrate species to another, with the rat
having relatively few nerve fibers, and animals
such a s t h e cat having larger numbers
(Bargmann et al., '67). Since the nerve fibers
are thought to be a major source of inhibition of
hormone release (Tilders et al., '791, transmitter molecules might be effective over a larger
area of cells via the extensive extracellular
spaces. Moreover, the apparent continuity of
the extracellular spaces (or channels, as they
are termed for the frog by Perryman and Bagnara, '781, with more peripheral regions, including cells adjacent to the hypophyseal cleft,
makes it easier to understand reported inhibitory or stimulatory effects of hypothalamic or
other molecules, such as the tripeptide, proleuglycinamide, also known as melanocytestimulating hormone release inhibiting factor
(MIF) (Kastinetal.,'71; Celiset al.,'73). Effects
of any hypothalamic factors on the pars intermedia have been open t o question because of
the absence of intrinsic blood vessels.
The implications of a complex, communicating extracellular space in the pars intermedia
are important for the understanding of the synthesis and release of the peptides contained in
the endocrine cells. Secretions may enter the
spaces and be carried to the general circulation
or through the purported backflow to the brain
(Page et al., '78; Mezey et al., '79). The intermediate lobe contains significant amounts of
beta-endorphin, an endogenous opiate peptide
(Bloom et al., '78). Synthesis and release of
pituitary endorphin appears closely tied with
release of other molecules, such as ACTH,
which are cleavage products of a larger peptide,
beta lipotropin (Guillemin e t al., '77). The intricate channels connecting intermedia cells with
one another are most likely the areas into
which pituitary endorphin is released. Those
areas may also contain molecules of hypothalamic or local neuronal origin which regulate production and extrusion of the opiate peptide.
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This study was supported by NIDA grant Saland,
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DA-02269-01 and in part by research funds
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from the University of New Mexico School of
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My thanks to Ms. Judi DeLongo for her excel(abstr.).
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intermedia as demonstrated by freeze-etching. J. Cell
Newel1 for typing the manuscript.
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