Download PDF

/ . Embryol. exp. Morph. Vol. 29, 3, pp. 681-696,1973
681
Printed in Great Britain
Gap junctions and electrotonic coupling in foetal
rabbit adrenal cortical cells
By T. JOSEPH, 1 C. SLACK2 AND R. P. GOULD 1
From the Departments of Anatomy and Biology as Applied to Medicine,
The Middlesex Hospital Medical School
SUMMARY
In an electron-microscope study of the developing rabbit adrenal gland numerous gap
junctions and desmosomes were identified between the cortical cells, particularly in the
juxtamedullary cortical tissue. An electrophysiological examination of the cortical cells
showed them to be electrotonically coupled. Such couplings were more frequently found in
the juxtamedullary cortical tissue than in the outer cortex.
INTRODUCTION
Electronic coupling between cells has been demonstrated in a wide variety
of adult (Furshpan, 1964; Barr, Dewey & Berger, 1965; Loewenstein, 1966)
and developing tissues (Potter, Furshpan & Lennox, 1966; Sheridan, 1966;
Ito & Loewenstein, 1969; Slack & Palmer, 1969; Warner, 1970). In several of
these systems gap or close junctions (nexuses) have been found between the
coupled cells (Trelstad, Revel & Hay, 1966; Trelstad, Hay & Revel, 1967;
Barr et al. 1965), and it has been suggested that they are the sites of low electrical
resistance through which ionic movement may occur.
Gap junctions have been described recently in the adrenal cortex of the adult
rat, mouse, hog and guinea-pig (Friend & Gilula, 1972) and observed in the
lemming and bat (Joseph & Gould, unpublished observations). Brief mention
has also been made of their presence in the human foetal adrenal gland (McNutt
& Jones, 1970).
This study describes the presence of gap junctions in both the adult and foetal
rabbit adrenal gland and reports on a correlated electrophysiological study of
the developing adrenal cortical cells.
1
Authors' address: Department of Anatomy, The Middlesex Hospital Medical School,
London W1P 6DB, U.K.
2
Author's address: Institute of Genetics, University of Glasgow, Glasgow, Scotland, U.K.
44-2
682
T. JOSEPH AND OTHERS
MATERIAL AND METHODS
(a) Animals
New Zealand large-white rabbits were used for both the morphological and
electrophysiological parts of the study. Non-pregnant and pregnant animals
were anaesthetized with intravenous Nembutal1 (0-5 ml/kg) administered via an
ear vein. Anaesthesia was maintained with a mixture of nitrous oxide and oxygen
delivered from a Boyle's anaesthetic machine through a mask applied to the
animal's face.
Nembutal was the anaesthetic of choice since barbiturates are known to block
stressful sensory stimuli (Royce & Sayers, 1958) which may otherwise induce
ACTH release and so initiate morphological alterations in the cortical cells.
(b) Tissue preparation for light and electron microscopy
Mature adrenals from four, 3-month-old virgin female and male rabbits
were studied. Post natal glands from two neonates and four 8- and 21-day-old
animals were also examined. The developing gland was studied in 22 foetuses
from day 10 (stage 7, Edwards, 1968) to birth. Foetuses were staged by noting
the times of coitus and delivery, since in rabbits mating initiates ovulation.
Foetuses were delivered one at a time from the uterus of the anaesthetized
animal, leaving the blood supply to the remaining uterus and foetuses intact
until they in turn were ready to be dissected out. The foetuses were decapitated
and the glands quickly dissected out into a modified ice-cold Karnovsky (1965)
fixative. The fixative consisted of 2 % formaldehyde prepared from paraformaldehyde, 2-5% gluteraldehyde and 0-05% calcium chloride in 0-1 M sodium
cacodylate buffer, pH 7-4.
The adrenals from foetuses day 19 to birth were dissected out from the dorsal
body wall, cut into halves or quarters and placed in fixative. Foetuses from day
10-12 were fixed whole after dissecting away the amniotic membrane and,
following embedding, were serially sectioned until the gland was identified.
In day 13-18 foetuses the heads and tails were sliced off with a razor blade and
the entire posterior abdominal wall placed in fixative.
The larger post-natal and mature glands were cut up into thin radial segments
so that each piece had its own area of capsule, cortex and medulla. This orientation later proved essential for identifying which region of the cortex was being
examined.
The blocks of tissue were left in the aldehyde fixative for a period varying
from 2 to 8 h, depending on block size. After rapid rinsing in 3-4 changes of
0-1 M sodium cacodylate buffer, pH 7-4, the tissue was postfixed for 2-4 h in
cacodylate buffered (pH 7-4) 1-0% OsO4.
The osmicated blocks were then rapidly dehydrated through a graded series
of cold ethanols into propylene oxide and finally embedded in Araldite.
1
Nembutal contains 60 mg sodium pentobarbitone per ml.
Gap junctions and electronic coupling
683
One-//m thick sections were cut on a Cambridge ultramicrotome and stained
with toluidine blue for light microscopy. Ultrathin sections were stained with
lead citrate (Reynolds, 1963) and examined in a Philips EM 300 electron
microscope.
Lanthanum staining was carried out on some blocks according to the method
of Goodenough & Revel (1970).
(c) Electrophysiological methods
Measurements were made on whole adrenal slices 1-2 mm thick prepared
from foetal glands obtained as in section (a) above. Foetuses 24 and 28 days old
were used in this study as well as 2- and 8-day-old postnatal animals. Some
15 foetal and postnatal adrenal glands were used altogether.
Micropins inserted through the adrenal capsule held the slice, with a cut
surface uppermost, to the base of a bath filled with 5 ml of bicarbonate-buffered
Krebs-Kenseleit-Ringer, gassed with 95% O2, 5% CO2 and maintained at
about 35 °C. The adrenal slices were superfused with this saline throughout
the experiments (3-4 h duration). The bath temperature was monitored by
a small thermistor positioned close to the gland and varied between 33 and
37 °C.
Glass microelectrodes filled with 0-8 M potassium citrate with resistances
greater than 50 MO were used to record membrane potentials and inject rectangular current pulses. On some occasions current-passing electrodes were
filled with 2 M potassium citrate to increase their conductivity.
Prior to 28 days gestation the small size of the adrenal cortical cells (6-8 /im)
made reliable measurements with microelectrodes difficult. Also the deposition
of interstitial connective tissue in the older glands placed severe limitations on
the success of experiments on animals more than 8 days old.
Electrical coupling between two adrenal cortical cells was recorded in the
following way. A micro-electrode was inserted into each cell; entry of the
electrode into the cell was marked by the appearance of a negative membrane
potential. Once both electrodes were satisfactorily inserted rectangular current
pulses (5 x 10~8 to 3 x 10~7 A) were injected into one cell and the resultant
voltage deflexion recorded in the other. The voltage-recording electrode was
then withdrawn from the cell until it lay in the extracellular space and the
voltage deflexion recorded again. Inter-electrode distances were measured using
a calibrated microscope eyepiece graticule. Conventional signal recording and
display methods were used.
RESULTS
(a) Morphological
It is perhaps most useful if first of all a brief outline is given of the early
development of the rabbit adrenal gland. The first stage in the development of
the adrenal cortical Anlage is seen at day 12. Mesothelial cells lining the coelomic
684
T. JOSEPH AND OTHERS
(A)
(B)
Fig. 1. (A) Diagram of transverse section through the trunk region of a 12/13-dayold rabbit foetus. Mesothelial cells lining the coelomic bay (c) between the mesonephros (k) and dorsal mesentery (dm) migrate to a position ventrolateral to the
aorta (a). Neural-crest precursors (nc) of abdominal chromaffin tissue move to
a position lateral to the aorta. (B) Diagram of a transverse section through the
trunk region of a 14- to 16-day-old rabbit foetus. Neural-crest precursors ( d ) of
the medullary chromaffin tissue have moved into the cortical Anlage (ag). Other
lettering as for (A).
bay between the mesonephros laterally and the dorsal mesentery medially
proliferate and migrate into the adjoining mesoderm lying ventrolateral to the
aorta (Fig. 1 A).
At day 13 a well-defined body of cells derived from the coelomic lining and
distinguishable from the surrounding mesoderm has formed and the neural-crest
precursors of the medullary and para-aortic chromaffin tissue have taken up
a position lateral and anterolateral to the aorta. Some of these latter neural
crest cells are seen to lie between the aorta and the adrenal cortical Anlage.
Between days 14 and 16 (Fig. IB) chromaflfin cells destined to become medullary
chromaffin tissue (Coupland &Weakley, 1968) migrate into the cortical Anlage
and by 19-20 days clumps of future medullary cells are seen scattered among
Gap junctions and electronic coupling
685
the cortical cells (Fig. 2). By day 24 the medullary cells have mostly reached
a central position in the gland, although some groups of chromaffin cells are
still seen to be scattered through the cortical tissue (Fig. 3). Between days 24 and
28 medullary cells have become consolidated in the gland's centre (Fig. 4).
Cytoplasmic differentiation of the early cortical cells into steroid type cells
occurs between days 19-24 (Joseph & Gould, unpublished observations).
In this present study we have found that throughout the whole of the early
formative period intercortical cell relationships show a trend which is one of
progessively closer cell contact, the cells eventually becoming separated from
each other by a 15-20 nm gap. From day 14-22 contacts between the cortical
cells are confined to small rudimentary desmosome-like attachments (Fig. 5).
At day 22, with the medullary chromaffin tissue approximately in its definitive position, a few gap junctions are found between the cortical cells lying
closest to the catecholamine secreting cells. By day 28 many more gap junctions
have appeared between the four or five layers of juxtamedullary cortical cells.
(Fig. 6). This distribution of gap junctions was maintained in the neonate, early
postnatal and adult glands examined in this study. Occasionally gap junctions
were found between the outer zona fasciculata cells. None was observed
between zona glomerulosa cells and it would seem that if they were present they
must be rare.
At low magnifications gap junctions may easily be seen as regions of increased
electron-density where two adjoining plasma membranes come very close
together and run a strictly parallel course over distances of 0-2-0-8 /im (Figs. 6,
7). The larger junctions are found closest to the medulla and run either a straight
or undulating course, although the latter appearance may be the result of tissue
shrinkage. No junctions were observed between cortical and medullary cells.
Although found singly between some cells, others had 2 or 3 junctions along
the same pair of lateral cells membranes (Fig. 7). Most gap junctions are bounded
at one end by a pronounced desmosome (Figs. 7, 8), although occasionally they
are seen sandwiched between two desmosomes, particularly where two cells
share more than one gap junction (Fig. 7). At the gap junctions the plasma
membranes are characteristically separated by a 2-5-3 nm space (Fig. 8) which
is clearly outlined in lanthanum-stained preparations (Fig. 9). No clear-cut
tight junctions were seen between cortical cells at any of the developmental
stages studied, nor were spot membrane fusions called focal tight junctions
(Trelstad et al. 1966, 1967) unequivocally identified.
(b) Electrophysiology
Individual cortical cells (average diameter 11 /tin) could not be readily resolved under a binocular microscope but microelectrode penetration was easily
recognized by the abrupt appearance of a negative resting potential. The
average initial membrane potential recorded during the course of the experi-
686
T. JOSEPH AND OTHERS
Gap junctions and electronic coupling
687
ments was — 56 ± 1-2 mV S.E.M. (n = 124), which is in fairly good agreement
with that reported previously (Matthews, 1967).
In situations where both current-passing and voltage-recording electrodes
were in an intracellular position, electrotonic coupling could be demonstrated
between cortical cells at all developmental stages studied. Fig. 10 shows records
obtained from cells situated close to the cut surface of a 28-day foetal adrenal
gland with an electrode separation of 50 /on. The upper trace (a) shows the
small deflexion seen on the voltage recording electrode when positioned extracellularly in response to the injection of a rectangular current pulse (lower
trace /). By contrast, when both microelectrodes were intracellular, a larger
electrotonic potential was observed, middle trace (b), suggesting that current
flows more easily between cells than across the cell membrane. In general, the
membrane potential of both cells declined during the course of maintained
electrode impalement. This was invariably accompanied by a decrease in the
electrotonic pulse height and probably reflects the damage inflicted on these
small cells by insertion of the micro-electrodes. Typical examples of intracellular
records from superficial cells in the cortex of older animals are shown in. Fig. 11.
The electrotonic pulse shown in (a) was recorded from cortical cells situated
close to the medullary boundary of a 2-day post-natal adrenal; that illustrated
in (b) came from the adrenal cortex of an 8-day-old rabbit where the impaled
cells were located more peripherally, i.e. in the inner part of the zona fasiculata.
In both cases the interelectrode distance was about 30 fim. Several experiments
were done using glands from 24-day foetal rabbits. Electrotonic coupling between cortical cells was only seen in one of these experiments.
A quantitative measure of the extent to which cells situated in different regions
of the developing cortex were coupled to each other was not possible because
this requires reliable measurements from many cell pairs in individual preparations. However, the accumulated data from our many experiments showed
FIGURES 2-5
Fig. 2. Light micrograph of a section, toluidine-blue stained, through a 19-day foetal
rabbit adrenal. The lighter stained islands of chromaffin cells (m) can be seen
scattered among the more deeply stained future cortical tissue (c). x 160.
Fig. 3. A light micrograph of part of a 24-day foetal rabbit adrenal. The lighterstained chromaffin cells are condensing in the gland's centre to form the medulla
(A/). Some chromaffin cells (m) are still found lying among the cortical tissue (c).
xl60.
Fig. 4. A light micrograph of part of a 28-day foetal rabbit adrenal. The chromaffin
cells are now mainly consolidated in the centre forming the medulla (M). The
cortex is now clearly differentiated into an outer zona glomerulosa (g) and an
inner fasciculata/reticularis (/). x 200.
Fig. 5. An electron micrograph showing parts of two differentiating cortical cells
(22-day foetal gland). Two rudimentary desmosomes (d) can be seen on their
opposed plasma membranes. The nucleus (n), mitochondria and proliferating
smooth endoplasmic reticulum (ser) are also shown, x 25000.
688
T. JOSEPH AND OTHERS
Gap junctions and electronic coupling
689
that there was no absolute restriction on cell-to-cell passage of current in any
region of the developing cortex, although coupling was generally less frequently
found between cells located in the peripheral areas of the tissue slice.
In favourable conditions (11 experiments) it was possible to demonstrate
cell-to-cell passage of injected current over interelectrode distances of at least
100/tm, i.e. across 9 or 10 cell diameters. Fig. 12 shows the voltage deflexions
recorded intracellularly at electrode separations of (a) 50 /*m and (b) 80 /un in
the same 28-day foetal cortex. These measurements were made close to the
cortico/medullary boundary. It can be seen that the size of the electrotonic pulse
is inversely related to the interelectrode distance. The relationship between
these two parameters was steep and significant voltage deflexions in response
to injected current pulses were rarely recorded at interelectrode distances of
more than 300 /im, suggesting that the space constant for the spread of ionic
current was short.
Cells situated close to the cut surface of the adrenal slice were coupled to
those at deeper levels within the tissue, which were also coupled to each other.
Current therefore flowed away from the polarizing micro-electrode in three
dimensions, making estimation of absolute values of surface membrane and
intercellular resistances impossible (Noble, 1962).
DISCUSSION
Our findings on the juxta-medullary foetal rabbit adrenal cells provide
another example of developing cells exhibiting both gap junctions and ionic
coupling. These features have previously been reported in chick embryos
(Sheridan, 1966; Trelstad et al. 1966) and in those of Fundulus (Lentz & Trinkaus, 1967; Bennett & Trinkaus, 1970). Electrotonic coupling has also been
found between cells in embryos of the squid (Potter et al. 1966), newt (Ito &
Loewenstein, 1969) and clawed toad (Slack & Palmer, 1969) but no correlated
ultrastructural studies were made.
Our electrophysiological results are most readily explained by supposing that
pathways of low electrical resistance exist between some, if not all, cells in the
developing adrenal cortex. A voltage drop in response to injected current
FIGURE 6
An electron micrograph showing parts of two longitudinally cut juxtamedullary
cortical cells (C) adjoining the medullary tissue (M). Their opposed lateral plasma
membranes show a small gap junction (arrow) sandwiched between two desmosomes (d). Elsewhere the plasma membranes are separated by an intercellular
space varying in width from 15 to 50 nm. The cortical cells from this 28-day foetal
gland show the typical features of a steroidogenic cell-extensive smooth endoplasmic reticulum (ser) and lipid droplets (Id) as well as the nucleus (n), Golgi
complex (g) and lysosomes (/). In the medullary cell (M) typical dense-cored
catecholamine containing granules (a) can be seen, x 22000.
690
T. JOSEPH AND OTHERS
Gap junctions and electronic coupling
691
would also occur if an additional high resistance were present between the
exterior of the cell membrane and the indifferent Ag/AgCl electrode. This seems
unlikely to be a cause of the observed coupling in view of the finding that the
electrotonic voltage was always extremely small unless both microelectrodes
were situated intracellularly. The possibility that coupling develops as a result
of damage to cells located close to the cut surface of the tissue is excluded by
the observation that cells situated 4-5 layers deep were also coupled.
Coupling was less frequently observed between the cells of the outer cortex,
particularly the differentiating glomerulosa, and may be correlated with the
morphological finding of fewer gap junctions. However, the glomerulosa cells
are smaller in size and therefore more susceptible to damage during experimental manipulation, and so uncoupling of the cells consequent upon damage,
as described in other cell types (Loewenstein, Nakas & Socolar, 1967), could
occur.
Gap junctions between adult adrenal cells have been found in a number of
different species (Friend & Gilula, 1972; and Joseph & Gould, unpublished
observations), and they have also been described in the human foetal gland
(McNutt & Jones, 1970) and figured for the human foetal adrenal (Johannisson,
1968). Friend & Gilula (1972) found gap junctions in all three zones of the rat
adrenal cortex but noticed they were preferentially distributed in the zona
glomerulosa and zona reticularis. Neither McNutt & Jones (1970) nor
Johannisson (1968) specify in which part of the zona fasciculata the junctions
they illustrate were found, nor do they mention their frequency.
Perhaps the most interesting of our observations on the foetal rabbit adrenal
gland are:
(1) That cortical gap junctions are first found at such time in the gland's
formation as the chromaffin tissue is moving into its definitive adult position,
when electrotonic coupling can be demonstrated between the cortical cells.
(2) That the gap junctions are mainly restricted to the layers of cortical cells
adjacent to the nascent medulla, and although electrotonic coupling is more
easily demonstrated between the cells in this region it also occurs more peripherally where gap junctions are rare.
FIGURES 7-9
Fig. 7. An electron micrograph illustrating parts of two adult zona reticularis
cells. Seven gap junctions (arrows) of varying length, sandwiched between several
desmosomes (d) can be seen as undulating, electron-dense membranes, x 25 600.
Fig. 8. This high-power electron micrograph illustrates a gap junction (arrow)
between two cortical cells. At the junction the two unit membranes are separated only
by a 3-5-4-5 nm gap. Elsewhere the intercellular space widens to 25 nm. x 110000.
Fig. 9. This high-power electron micrograph illustrates the lanthanum-filled intercellular space (arrow) between two 28-day foetal, adrenal cortical cells. At the gap
junction the space is seen to be only 3-5—4-5 nm wide, x 100000.
692
T. JOSEPH AND OTHERS
1
(a)
200 msec
Fig. 10. Electrotonic coupling recorded from a 28-day foetal rabbit adrenal cortex.
Upper trace (a): small voltage deflexion on extracellularly positioned voltagerecording electrode in response to a constant current pulse (lower trace /) passed
between the inside and outside of a cortical cell. Middle trace (b): electrotonic
pulse on voltage-recording electrode resulting from current pulse of same magnitude. In this case both micro-electrodes were intracellular and at approximately
50 /.tm separation.
electrotonic pulse height
Transfer resistance =
= 3-6 x 104 Q.
:
input current
The functional significance of these findings may be connected either with the
processes of adrenal organogenesis or with the gland's adult physiology, perhaps
with both.
One possible function of the inner cortical cells during development is that
in some way they may signal to the invading chromaffin tissue its final position
in the gland's centre, and the fact that they are coupled may enable them to
act in a unified way. It is therefore of interest that in reptile adrenals (Cayman)
where the small islands of chromaffin tissue remain distributed mainly in the
outer cortex and never consolidate to form a medulla, no gap junctions were
found (Wood & Gould unpublished observations) between the cells of the
adjoining cortical tissue. However, little is known about tissue signalling of this
kind, although in one thoroughly investigated system (the slime mould) cyclic
AMP has been demonstrated (Konijn, van de Meene, Bonner & Barkley,
1967) to be secreted by some cells and to act as an attractant in directive cell
migration and clumping.
Another function of the gap junctions and large desmosomes found between
the inner cortical cells is their possible role in contact inhibition. Tight junctions,
Gap junctions and electronic coupling
693
(a)
,
200 msec
(h)
^
T
•;[
200 msec
200 msec
Fig. 11
Fig. 12
Fig. 11. («) Electrical coupling between cells situated close to the medulla in a 2-day
rabbit adrenal cortex. Both micro-electrodes intracellular and at a separation of
approximately 30/mi.
Transfer resistance = v// = 6-4 x 104 Q.
(/>) Coupling between cells situated in the inner zona fasciculata of an 8-day rabbit
adrenal cortex. Both micro-electrodes intracellular at a separation of approximately
30 //m.
Transfer resistance = v/i = l-4x 105 £2.
Fig. 12. Electrotonic coupling as a function of inter-electrode distance in a 28-day
foetal rabbit adrenal cortex, (o) Electrotonic pulse recorded intracellularly at an
electrode separation of 50 /.im in response to constant current pulse, /. (b) Electrotonic pulse recorded intracellularly at an electrode separation of 80/<m in response
to constant current pulse, /. Transfer resistance = 2-4 x 104 O. Ratio vbjva = 053.
gap junctions, desmosomes and 15-20 nm junctions have all been shown to be
made by contact-inhibited cells (Flaxman, Revel & Hay, 1969; Harris, 1970;
Loewenstein, 1968; Martinez-Palamo, Braislovsky & Bernhard, 1969; McNutt
& Weinstein, 1969; Lentz & Trinkaus, 1967). Thus the presence of numerous
and quite extensive gap junctions and desmosomes between the juxtamedullary
cortical cells suggests that they are firmly contact inhibited.
Further, and here caution must be used when extrapolating in vitro results
to in vivo situation, it has been established that when contact-inhibited cells
approach or achieve confluency in culture their mitotic activity is greatly
diminished and contact inhibition of growth results (Stoker, 1969, 1972). Now,
it is known (Deane, 1962) that cortical mitoses are most numerous in the zona
694
T. JOSEPH AND OTHERS
glomerulosa and outer fasiculata in normal and stimulated glands, and our
own preliminary counts of mitoses in the 24- and 28-day foetal rabbit adrenal
cortex indicate that there are 4-8 times as many dividing cells in the outer
cortex compared with the juxtamedullary cells. This suggests that growth of
the inner cortex is significantly slower than its outer part.
These observations on gap junctions, desmosomes, ionic coupling and a low
mitotic count are therefore consistent with the view that these inner cells are
in a state of contact-inhibited growth and that this zone of cells surrounding the
medulla may prevent further inward growth of the cortex into the central
chromaffin tissue throughout its postnatal growth or when it is stimulated to
hypertrophy.
Another possible explanation for the number of gap junctions and desmosomes between the inner cortical cells is that they help resist osmotic (Brightman
& Reese, 1969) and/or mechanical (Goodenough & Revel, 1970) separation of
the cells, which may result from physiological alterations in blood flow through
the gland. Thus stress (Richards & Pruitt, 1957), increased circulating ACTH
(Sapirstein & Goldman, 1959) or raised adrenaline levels (Harrison, 1957) all
result in an augmented blood flow which in turn would lead to an increase in
diameter of the vascular channels, which are especially abundant and tortuous
in the zona reticularis (Pauly, 1957).
Finally, mention must be made of the well-established observations (Coupland, 1953; Pohorecky & Wurtman, 1971) that adrenal glucocorticoid hormones
are of primary importance in the synthesis of adrenaline because they are
necessary for the maintenance of a medullary enzyme, phenylethanolamine-A^methyl transferase (PNMT) (Kirshner & Goodall, 1957), which catalyses the
conversion of noradrenaline to adrenaline (Axelrod, 1962). In view of this
regulatory role of the glucocorticoid hormones on medullary function, the
adjacent zone of ionically coupled cortical cells and their output of corticosteroids may play an important part in the control of adrenaline production.
The authors would like to thank Mr C. Sym and Miss J. Hornidge for their photographic
and secretarial assistance, Mr H. S. Drury for drawing Fig. 1 and Professors E. W. Walls,
L. Wolpert and Dr A. Warner for reading the manuscript. One of us (C. S.) was supported by
the Nuffield Foundation during the course of this work.
REFERENCES
AXELROD, J.
(1962). Purification and properties of phenylethanolamine-Af-methyl transferase.
/. biol. Chem. 237, 1657-1660.
BARR, L., DEWEY, M. M. & BERGER, W. (1965). Propogation of action potentials and the
structure of the nexus in cardiac muscle. /. gen. Physiol. 48, 797-823.
BENNETT, M. V. L. & TRINKAUS, J. P. (1970). Electrical coupling between embryonic cells by
way of extracellular space and specialized junctions. /. Cell Biol. 44, 592-610.
BRIGHTMAN, M. W. & REESE, T. S. (1969). Junctions between intimately apposed cell membranes in the vertebrate brain. J. Cell Biol. 40, 648-677.
COUPLAND, R. E. (1953). On the morphology and adrenaline-noradrenaline content of
chromaffin tissue. /. Endocr. 9, 194-203.
Gap junctions and electronic coupling
695
R. E. & WEAKLEY, B. S. (1968). Developing chromaffin tissue in the rabbit: an
electron microscope study. /. Anat. 102, 425-455.
DEANE, H. (1962). The anatomy, chemistry and physiology of adrenal cortical tissue. In
Handbuch der Experiment ellen Pharmakologie (ed. O. Eichler & A. Farah), Band 14,
Teil 1, Chap. 1, pp. 1-185. Berlin: Springer-Verlag.
EDWARDS, J. A. (1968). The external development of the rabbit and rat embryo. Adv. Teratol.
3, 239-263.
FLAXMAN, B. A., REVEL, J. P. & HAY, E. D. (1969). Tight junctions between contact inhibited
cells in vitro. Expl Cell Res. 58, 438-442.
FRIEND, D. S. & GILULA, N. B. (1972). A distinctive cell contact in the rat adrenal cortex.
/. Cell Biol. 53, 148-163.
FURSHPAN, E. J. (1964). 'Electrical transmission' at an excitatory synapse in a vertebrate
brain. Science, N. Y. 144, 878-880.
GOODENOUGH, D. A. & REVEL, J. P. (1970). A fine structural analysis of intercellular junctions in the mouse liver. /. Cell Biol. 45, 272-290.
HARRIS, A. J. (1970). Lack of tight and gap junctions between 3T3 cells grown in monolayer
culture. Am. Zool. 10, 324.
HARRISON, R. G. (1957). The adrenal circulation in the rabbit. /. Endocr. 15, 64-71.
ITO, S. & LOEWENSTEIN, W. R. (1969). Ionic communication between early embryonic cells.
Devi Biol. 19, 228-243.
JOHANNISSON, E. (1968). The foetal adrenal cortex in the human. Its ultrastructure at different
stages of development and in different functional states. Acta endocr., Copenh. 58, suppl.
130, 7-107.
KARNOVSKY, M. J. (1965). A formaldehyde-gluteraldehyde fixative of high osmolality for
use in electron microscopy. /. Cell Biol. 27, 137 A.
KIRSHNER, N. & GOODALL, McC. (1957). The formation of adrenaline from noradrenaline.
Biochem. biophys. Acta 1A, 658-659.
KONIJN, T. M., VAN DE MEENE, J. G., BONNER, J. T. & BARKLEY, D. S. (1967). The acrasin
activity of adenosine-3',5'-cyclic phosphate. Proc. natn Acad. Sci. U.S.A. 58, 1152-1154.
LENTZ, T. L. & TRINKAUS, J. P. (1967). A fine structural study of cytodifferentiation during
cleavage, blastula and gastrula stages of Fundulus heteroclitus. J. Cell Biol. 32, 121-138.
LOEWENSTEIN, W. R. (1966). Permeability of membrane junctions. Ann. N.Y. Acad. Sci.
137, 441-472.
LOEWENSTEIN, W. R. (1968). The emergence of order in developing systems, Devi Biol.
Suppl. 2, 151-183.
LOEWENSTEIN, W. R., NAKAS, M. & SOCOLAR, S. J. (1967). Junctional membrane uncoupling.
Permeability transformations at a cell membrane junction. /. gen. Physiol. 50, 1865-1891.
MARTINEZ-PALOMO, A., BRAISLOVSKY, C. & BERNHARD, W. (1969). Ultrastructural modification of the cell surface and intercellular contacts of some transformed cell strains. Cancer
Res. 29, 925-935.
MATTHEWS, E. K. (1967). Membrane potential measurement in cells of the adrenal gland
J. Physiol., Lond. 189, 139-148.
MCNUTT, N. S. & WEINSTEIN, R. S. (1969). Carcinoma of the cervix: deficiency of nexus
intercellular junctions. Science, N.Y. 165, 597-599.
MCNUTT, N. S. & JONES, A. L. (1970). Observations on the ultrastructure of cytodifferentiation in the human foetal adrenal cortex. Lab. Invest. 22, 513-529.
NOBLE, D. (1962). The voltage dependence of the cardiac membrane conductance. Biophys. J.
2, 381-393.
PAULY, J. E. (1957). Morphological observations on the adrenal cortex of the laboratory rat.
Endocrinology 60, 247-268.
POHORECKY, L. A. & WURTMAN, R. J. (1971). Adrenocortical control of epinephrine synthesis. Pharmac. Rev. 23, 1-35.
POTTER, D. D., FURSHPAN, E. J. & LENNOX, E. S. (1966). Connections between cells of developing squid as revealed by electrophysiological methods. Proc. natn. Acad. Sci. U.S.A.
55, 328-336.
COUPLAND,
45
EMB 29
696
T. JOSEPH AND OTHERS
E. S. (1963). The use of lead separate citrate at high pH as an electron opaque
stain in electron microscopy. /. Cell Biot. 17, 208-212.
RICHARDS, J. B. & PRUITT, R. L. (1957). Hydrocortisone suppression of stress induced
adrenal 17-hydroxysteroid secretion. Endocrinology 60, 99-104.
ROYCE, P. C. & SAYERS, G. (1958). Blood ACTH: effects of ether, pentobarbital, epinephrine
and pain. Endocrinology 63, 794-800.
SAPIRSTEIN, L. A. & GOLDMAN, H. (1959). Adrenal blood flow in the albino rat. Am. J.
Physiol. 196, 159-162.
SHERIDAN, J. D. (1966). Electrophysiological study of special connections between cells in
the early chick embryo. /. Cell Biol. 31, Q - Q .
SLACK, C. & PALMER, J. F. (1969). The permeability of intercellular junctions in the early
embryo of Xenopus laevis, studied with a fluorescent tracer. Expl Cell Res. 55, 416-419.
STOKER, M. G. P. (1969). Homeostatic Regulators (ed. G. E. W. Wolstenholme & J. Knight),
pp. 264-275. London: Churchill.
STOKER, M. G. P. (1972). The Leeuwenhoeck Lecture. 1971. Tumour viruses and the sociology
of fibroblasts. Proc. R. Soc. Lond. B 181, 1-17.
TRELSTAD, R. L., REVEL, J. P. & HAY, E. D. (1966). Tight junctions between cells in the early
chick embryo, as visualized with the electron microscope. 7. Cell Biol. 31, Q.
TRELSTAD, R. L., HAY, E. D. & REVEL, J. P. (1967). Cell contact during early morphogenesis
in the chick embryo. Devi Biol. 16, 78-106.
WARNER, A. E. (1970). Electrical connexions between cells at neural stages of the axolotl.
/. Physiol., Lond. 210, 150P.
REYNOLDS,
{Received 9 October 1972)