J. Phyaiol. (1978), 285, pp. 171-184 With 1 plate and 6 text-ftguree Printed in Great Britain

171

ACTION POTENTIALS IN GLAND CELLS OF RAT PITUITARY PARS INTERMEDIA: INHIBITION BY DOPAMINE, AN INHIBITOR OF MSH SECRETION

BY W. W. DOUGLAS AND P. S. TARASKEVICH From the Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510, U.S.A.

(Received 29 March 1978) SUMMARY

1. Cells were dissociated from rat pituitary pars intermedia, maintained in culture, and subjected to electrophysiological study. 2. Recorded membrane potentials varied widely (range - 18 to about -80 mV). They were relatively insensitive to changes in external Na but were rapidly and reversibly lowered by excess K. 3. Action potentials were elicited by passing current through intracellular recording electrodes. They were reversibly blocked by tetrodotoxin (TTX, 2 x 10-6 M) or by removal of Na from the recording solution and thus appeared to be Na spikes. Cells yielding action potentials in response to depolarization had relatively high membrane potentials (about -65 mV) which may be representative of the true resting membrane potential of pars intermedia cells. 4. Spontaneous action potentials were recorded extracellularly from nearly all the many isolated pars intermedia cells studied with a microsuction electrode. Their amplitude was reduced by TTX (0-1-2 x 10-6 M). Electron microscopic examination of cells producing action potentials showed them to be hormone-containing parenchymal (gland) cells. 5. Dopamine (10-6 M), a presumed physiological inhibitor of secretion of melanocyte stimulating hormone (MSH) from pars intermedia cells, decreased the frequency of spontaneous action potentials but not their amplitude. Similar effects were seen with noradrenaline (10-6 M), another inhibitor of MSH secretion, whereas isoprenaline and 5-hydroxytryptamine (5-HT), which do not inhibit MSH secretion, had no effect. 6. Action potentials may be involved in stimulus-secretion coupling in pars intermedia cells. INTRODUCTION

We recently reported the occurrence of action potentials in cells of the normal mammalian pituitary pars distalis and showed that in some cells such spikes could be initiated or their frequency increased by the hypothalamic peptide thyrotrophin releasing hormone (TRH) which is known to stimulate secretion from thyrotrophs and mammotrophs (Taraskevich & Douglas, 1977). The inference that action potentials may be involved in stimulus-secretion coupling in the adenohypophysis has prompted us to extend our investigation to the mammalian pituitary pars intermedia. Here the cells (whose classic secretary product is the melanocyte stimulating

W. W. DOUGLAS AND P. S. TARASKEVICH hormone, MSH) are also under central nervous control, but this control shows two features that together set pars intermedia apart from pars distalis and lend it a unique interest. In the first place, secretion from pars intermedia is under tonic inhibitory control. Secondly, pars intermedia is innervated; and the dopaminergic fibres involved are considered responsible, at least in large measure, for this tonic inhibition (see Howe, 1973; Hadley & Bagnara, 1975; Martin, Reichlin & Brown, 1977; Tilders & Smelik, 197 7). Pars intermedia thus seemed an especially appropriate preparation on which to pursue study of electrical phenomena in adenohypophyseal function. It also afforded an opportunity to assess electrical events in an endocrine system supplied by secreto-inhibitor nerves. We have chosen the rat for our experiments because this is the animal we used in our experiments on pars distalis. This choice thus allows comparison of the behaviour of the two adenohypophysial systems within a single species. The rat has the additional advantage of being the mammal in which the physiological control of pars intermedia has been most extensively studied, and where the evidence for an inhibitory dopaminergic control of MSH secretion is best established. In outline this evidence is as follows: first, the pars intermedia shows exalted secretary activity when removed from central control by hypothalamic lesions, stalk section, or transplantation (see Howe, 1973; Martin et al. 1977; Tilders & Smelik, 1977). Secondly, high levels of dopamine have been found in pars intermedia (Iwata & Ishii, 1969; Bjorklund, Falck, Hromek, Owman & West, 1970; Saavedra, Palkovits Kizer, Brownstein & Zivin, 1975) and histofluorescence has revealed a system of dopaminergic neurones with cell bodies in the medio-basal hypothalamus projecting to pars intermedia to form a fine network investing the gland cells (Baugmarten, Bjorklund, Holstein & Nobin, 1972; Bj6rklund, Moore, Nobin & Stenevi, 1973; Bjorklund, Falck, Nobin & Stenevi, 1974). Thirdly, dopamine inhibits MSH secretion when applied to neuro-intermediate lobes in vitro and manipulations that release endogenous dopamine have a similar effect (Bower, Hadley & Hruby, 1974; Tilders, Mulder & Smelik. 1975; Tilders & Smelik, 1977). Our experiments reveal that cells of the mammalian pars intermedia removed from the inhibitory influence of the brain and maintained in culture produce spontaneous action potentials and show further that these are inhibited by dopamine. The discovery of such behaviour not only extends the evidence implicating action potentials in the secretary function of the adenohypophysis but may offer a model for secretoinhibitor control of endocrine function. Some of the results have been communicated to the Physiological Society (Douglas & Taraskevich, 1978). 172

METHODS

Isolation and culture of pars internedia cells To prepare each batch of pars intermedia cells, neuro-intermediate lobes from ten to twenty male Sprague-Dawley rats (400-525 g), freshly decapitated, were carefully separated from the pars distalis with the aid of a dissecting microscope (16 x ). The lobes were pooled in about 25 ml. Ham's F-10 nutrient mixture (GIBCO) supplemented with 12-5 % horse serum, 2-5 % fetal calf serum, penicillin 100 u./ml., streptomycin 100 4ag/ml., and gassed with 95 % air 5 % CO2 at room temperature (21-24 TC). After all the lobes had been collected, they were transferred to a conical centrifuge tube containing 2 ml. of the above medium. Pars intermedia cells were dispersed mechanically by shearing: the lobes were repeatedly aspirated into and expelled from

ACTION POTENTIALS IN PARS INTERMEDIA CELLS

173

a Pasteur pipette with a flame-polished tip of inside diameter about 1-0 mm. After about 5 min of this treatment the lobes were allowed to settle and the milky solution containing the dispersed cells was decanted into another centrifuge tube. An additional 2 ml. of medium was added to the lobe-containing tube and shearing was continued more vigorously with pipettes having progressively smaller tips until it appeared that most of the pars intermedia cells were removed from the still intact pars nervosa. The lobes were again allowed to settle and this second sample of cell-containing medium was added to the first and centrifuged at 100 g for 10 min. The pelleted cells were resuspended in 2 ml. Hank's Balanced Salt Solution (HBSS, GIBCO) containing 0-1 % bovine serum albumin (BSA). To remove debris, the cell suspension was layered on 5 ml. HBSS containing 4 % BSA and the cells were spun through BSA at 100 g for 10 min. The BSA solution was decanted and the cells were resuspended in 2 ml. of the serumsupplemented Ham's F-10 with antibiotics. Portions containing 2-5 x 105 cells (approximately the equivalent of two rats) were maintained in individual Falcon dishes (35 mm diameter) in 2 ml. of the serum-supplemented Ham's F-10 medium with antibiotics in a 5 % C02/air atmosphere at 37 0C. All glassware used in the dissociation procedure was siliconized.

Cell ob8ervation and manipulation Falcon dishes containing the cultured cells were placed on the stage of an inverted microscope allowing phase contrast observation of the cells, microelectrodes and drug delivery pipettes at 800 x magnification ( x 40 long working distance water immersion objective). The microscope and the attached micromanipulators were mounted on a massive table supported by air cushions to damp vibration. Before recording (done 1-10 days after dissociating the cells) culture medium was removed and the cells were bathed in the appropriate recording solution (see Solutions, below). Changing the medium was simple when the cells were well attached as they usually were after 3-4 days. At shorter intervals, when many cells tended to float, the dislodged cells were harvested by centrifuging and returned to the Falcon dish. Such unattached cells could be readily recorded from with the microsuction electrode but they were difficult to record from with intracellular micro-electrodes. Most intracellular records were thus obtained from cells that had been cultured for 3-10 days whereas extracellular recordings were obtained anywhere from 1-10 days. The cells selected for recording were rounded, refractile cells easily distinguished from flattened fibroblasts. When examined by electron microscopy they had the typical appearance of pars intermedia gland cells (see P1. 1 and Kurosumi & Fujita, 1974). Extracellular recording

Extracellular recording was done with microsuction electrodes drawn from capillary glass and having a flame-polished tip of about 3-6 #Am inside diameter (Brandt, Hagiwara, Kidokoro, & Miyazaki, 1976; Taraskevich & Douglas, 1977). The fluid-filled shaft of the electrode was connected to an air-filled syringe (2 ml.) by a polyethylene tube. A screw-operated plunger in the syringe provided sufficiently fine pressure control to allow a portion of the cell to be gently drawn into the electrode lumen. The electrodes were connected via an Ag-AgCl wire to an amplifier of high input impedence (M4A, W. P. Instruments, Connecticut), which could also be used as a bridge circuit to pass current through the electrode. Potentials were displayed on the screen of a storage oscilloscope (RM564, Tektronix, AC coupled) and a chart recorder (7402A, HewlettPackard). Upward deflexions indicate that the potential at the recording electrode is positive with respect to the reference electrode, an Ag-AgCl wire in the bath. Intracellular recording Intracellular recording and current passing were done with single microelectrodes filled with 3 M-KCl (40-100 M0) by use of the amplifier with bridge circuit mentioned above. Solutions The bathing solution used during extracellular recording contained (mM): NaCl, 150; KC1, 5; CaCl2, 2; glucose, 5-5; and Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, 5 (pH 7.0-7-4); with the addition of bovine serum albumin, 1 mg/ml. For intracellular recording the CaCl2 concentration was increased to 10 mM (NaCl decreased to 142 mM). This seemed to stabilize recordings from these small cells. Na-free solutions were prepared by replacing NaCl

W. W. DOUGLAS AND P. S. TARASKEVICH

174

and Hepes with an equiosmotic amount of Tris (2-amino-2-(hydroxymethyl)-1,3-propanediol). All recording was done at room temperature (21-25 'C). Test solutions of TTX 0 1-2 x 10-6M, dopamine 1O04M, noradrenaline 1O0 M, 5-hydroxytryptamine 1O4-10-4M, and isoprenaline 1O-6-10 M, were made up in the appropriate recording solution and applied to cells by gravity flow from a delivery pipette, whose tip diameter was 10-15 ,um (Taraskevich & Douglas, 1977). Control experiments in which the delivery pipette contained recording solution only served to demonstrate that the effects were due to the drugs and not to any artefact arising from this mode of drug application. RESULTS

Intracellular recording Resting potentials recorded from 83 pars intermedia cells ranged from - 18 to about - 80 mV. The lower values were usually from cells in which a rapid decrease in potential had occurred from an initially higher level. Probably these came from cells injured by impalement. In contrast, the higher values (membrane potentials greater then about -50 mV) were often obtained from cells where there was a gradual increase in the potential recorded just after penetration. Probably these potentials came from less injured cells, and they may therefore more closely reflect the true level of membrane potential in pars intermedia cells. This possibility is consistent with results, to be described shortly, obtained from cells in response to depolarizing and hyperpolarizing current pulses. Membrane potential was sensitive to the concentration of K in the solution. The pars intermedia cells rapidly depolarized when bathed with a high K solution flowing from an adjacent 'drug delivery' pipette (Text-fig. 1). The depolarization was maintained throughout the exposure to excess K and potential recovered promptly when

Kit

_

;-

2 sec _~~~~~~~~~~~~~~~~~~2 m\0V

1 0 sec

K* Text-fig. 1. Intracellular records from pars intermedia cells showing K-induced depolarizations. Cells were flooded with excess K solution (100 mM-K replacing 100 mm-Na) from a delivery pipette during the periods indicated by the filled bars. A, depolarization in response to a single application of excess K (resting potential immediately before excess K = -72 mV). B, slower sweep showing two successive K-induced depolarizations elicited from a second cell (restingpotential before first exposureto excessK = -68 mV). Note rapidity of onset and offset of responses. Besides illustrating the effect of K, the records provide a rough indication of the time course of drug application by the delivery pipette method that is of value in interpreting the responses to catecholamines and TTX shown in Text-fig. 5. Kid

ACTION POTENTIALS IN PARS INTERMEDIA CELLS 175 the pipette containing excess K was removed. Such K-induced depolarizations could be repeatedly elicited from the same cell. Action potentials could be evoked from the pars intermedia cells by passing current through the recording electrode (Text-fig. 2). In many cells, the action potential appeared shortly after the onset of an outward (depolarizing) current pulse (Textfig. 2a). In these cells membrane potential was relatively high. In a second group of cells, such depolarization was ineffective but an action potential could be evoked by terminating an inward hyperpolarizing current pulse. In these cells, membrane potential was relatively low as it also was in a third group of cells which failed to produce action potentials in response to either procedure. Cells in these second and third categories may well have been damaged: the occurrence of anodal break responses would be consistent with this. 1 nA

J 2;mV

20 msec

Text-fig. 2. Action potentials recorded intracellularly from pars intermedia cells. A, an action potential produced during the second of two pulses of outward (depolarizing) current passed through the recording electrode. The first pulse of current produced only a subthreshold depolarization. B, an action potential, in a second cell, elicited by the termination of an inward pulse of current which hyperpolarized the cell to -78 mV. Top trace in each record indicates the current passed and the zero level of membrane potential.

Besides demonstrating the ability of pars intermedia cells to produce action potentials, these results may provide another clue to the true resting membrane potential. Thus Brandt et al. (1976) have argued, from their observations on chromaffin cells, that the resting potentials of cells yielding spikes only in response to depolarization more closely represent the true value of the membrane potential than does the mean potential from all cells. The membrane potential of the pars intermedia cells that yielded action potentials only in response to depolarization was -66+ 2*8 mV (mean + S.E., n = 17). The action potentials were rather uniform in shape. They were of short duration (time to peak about 2 msec) and showed after-spike hyperpolarization. They were abolished by TTX (2 x 10-6 M) applied by micropipette (Text-fig. 3A) or by replacing Na in the solution with Tris and these inhibitory effects were rapidly reversible (Text-fig. 3B). Seemingly the action potentials are Na spikes. In adrenal chromaffin cells, where the major portion of the action potential is sodium dependent (Biales, Dichter & Tischler, 1976; Brandt et al. 1976) a small Ca component has also been detected. We therefore sought a Ca component to the action potential in pars intermedia cells by recording from cells in Na-free solution

176 W. W. DOUGLAS AND P. S. TARASKEVICH with the addition of tetraethylammonium (TEA), 10 mM. In certain other tissues such conditions have revealed the contribution of Ca by suppressing the Na component and reducing delayed rectification (see Hagiwara, 1975). In pars intermedia cells, however, this procedure failed to reveal any residual electrical response that might suggest a Ca component (Text-fig. 3B, centre record). When Na was reintroduced by flooding the cell with the normal recording solution with the addition of TEA (10 mM) an action potential promptly reappeared when outward current was passed thereby showing that the inhibitory effect of Na deprivation was reversible (Textfig. 3B, right hand record). The spike duration was then somewhat longer than A

| 1 nA

--/ T - TTX _/

| t-\

_

2O mV

20 msec B

Na-free

10 msec

Text-fig. 3. Suppression of action potentials by TTX or Na-free conditions. A, intracellular records of responses elicited by pulses of outward (depolarizing) current before (left-hand record), during (centre record) and after (right-hand record) application of TTX (2 x 106 M) by delivery pipette. Centre record obtained about 15 see after exposure to TTX and right-hand record about 45 sec after removing TTX. Top traces indicate current ejected through recording electrode and zero level of membrane potential. B, intracellular records of responses to outward current in a second cell showing the dependence of the action potential on Na. In this experiment the solution in the dish of cells was Na-free (Tris replacing Na) but the individual cell recorded from was bathed by means of a delivery pipette with Na (142 mM) containing solution during the first (left-hand) record and last (right-hand) record. For the centre record, obtained in the interval, the Na-containing pipette was removed. In the absence of Na no action potential was evident in response to the same, or to a greater, depolarization. In this experiment TEA (10 mM) was present throughout in an attempt to facilitate demonstration of a Ca component. None was evident, but the action potential was prolonged. Note, however, the different time bases in A and B.

usual, however, and after-spike hyperpolarization was reduced. Both these effects were probably due to the familiar action of TEA in reducing K conductance. Several experiments of this sort on cells with resting potentials greater than -50 mV gave similar results. Extracellular recording The introduction of recording with microsuction electrodes has greatly facilitated the study of spontaneous electrical activity in small endocrine cells which are easily

177 ACTION POTENTIALS IN PARS INTERMEDIA CELLS damaged by impalement with intracellular electrodes. The technique commonly allows observation of action potential activity in these cells for prolonged periods and expedites study of the effects of hormones and chemical transmitters on such activity (Brandt et al. 1976; Taraskevich & Douglas, 1977). With this method, we found spontaneous action potential activity in almost all of the many pars intermedia cells studied. As shown in P1. 1, action potentials were produced by cells with electron microscopical appearances typical of the MSHsecreting gland cells. The frequency of spontaneous action potentials varied widely from cell to cell but often remained stable in individual cells for several minutes. In

A~~~~~~~~~~~~~~~~~~~~ 05

B~~i~~ n

llt

S

A

L

I

d

|O~~~~~0~mV

1 0 sec

Text-fig. 4. Spontaneous action potentials in pars intermedia cells. Extracellular records from two cells demonstrating different patterns of activity. A, upper record shows spontaneous action potentials occurring at random intervals. Below it (in the middle record) is a faster sweep from the same cell displaying the form of a single action potential. B, spontaneous activity in a second cell showing a bursting pattern of action potential discharge.

a selected group of nineteen cells in which recordings were made over continuous periods of 100-400 see during which no tests with drugs were made, the range was 0 01-3 Hz with a mean (+ s.ix.) of 1.6 + 0-2 Hz. In most cells action potentials occurred randomly (Text-fig. 4A) but in a few the spikes came in bursts at irregular intervals (Text-fig. 4B). Passage of outward current through the electrode generally increased the frequency of action potential discharge. The response appeared within a few milliseconds and did not outlast the current pulse. Inward current usually failed to evoke spikes, but in two cells which neither showed spontaneous activity nor responded to outward current, spikes appeared when a pulse of inward current was terminated. By analogy with intracellular recording (see above) such anodal break responses may indicate that these cells were damaged and required a conditioning hyperpolarization before they could respond. TTX (0 1-2 x 10-6 M) reduced the amplitude of the spontaneous action potentials within seconds (Text-fig. 5E) primarily as a result of a reduction of the positive phase which presumably represents activity in the unprotected portion of the cell

sec

W. W. DOUGLAS AND P. S. TARASKEVICH membrane outside the electrode lumen (see Brandt et al. 1976). Upon removal of the TTX-delivery pipette, spike amplitude gradually returned toward the control level. Dopamine and related substances The effect of dopamine (10-6M), a probable physiological inhibitor of MSH secretion, was to reduce the frequency of action potential discharge without reduction in action potential amplitude (Text-fig. 5B). Spiking was often completely abolished by dopamine (Text-fig. 5A). The inhibitory effect of dopamine could be repeatedly demonstrated in each cell tested. Noradrenaline, which is also known to inhibit MSH secretion (Bower et al. 1974) had a similar effect (Text-fig. 5C and D). Unlike tetrodotoxin, both dopamine and noradrenaline did not reduce spike amplitude. 178

A

C

DA J

B

E

NA

Du

DA

NA

|1 mV TTX 10 sec Text-fig. 5. Inhibition of spontaneous action potentials in pars intermedia cells by dopamine (DA), noradrenaline (NA) and TTX. Drugs were applied by delivery pipette for the periods indicated by the bars under each of the (extracellular) records. A and B, arrest and slowing respectively of spontaneous action potential discharge by dopamine (DA, 10-6 M) in two different cells. Note, in the latter, that action potential amplitude is not reduced by dopamine. C and D, comparable responses, arrest and slowing respectively, of spontaneous action potential discharge in two other cells exposed to noradrenaline (NA, 10-6 M). E, reduction of spontaneous action potential amplitude by TTX (2 x 10-6 M).

In contrast to dopamine and noradrenaline, isoprenaline and 5-HT are without inhibitory effect on MSH secretion (Bower et al. 1974). Isoprenaline and 5-HT (in concentrations of 10-6 or 10-5 M) had only small and inconsistent effects on spike frequency. DISCUSSION

The present experiments provide the first evidence on the electrical properties of the parenchymal (gland) cells of the pars intermedia of the mammalian pituitary and show that these cells are electrically excitable and display spontaneous action potentials whose frequency is lowered by exposure to dopamine, a substance believed (see Introduction) to be an important physiological mediator of the tonic inhibitory control exerted by the brain. From our experiments that revealed the

179 ACTION POTENTIALS IN PARS INTERMEDIA CELLS occurrence of action potentials in normal cells of the pars distalis and the stimulant effects, on action potential frequency, of the hypothalamic peptide TRH we concluded that 'it may be by initiating or modulating action potentials that the brain, through the hypophysiotrophic hormones, regulates secretion in the anterior pituitary' (Taraskevich & Douglas, 1977). The findings reported here support this view by showing, in another population of adenohypophysial cells of different hormonal function, that a decrease in spike frequency occurs in response to a known inhibitor of secretion. Observations on pars intermedia cells of a lizard also suggest that action potentials and secretion are related (Douglas & Taraskevich, 1978). That action potentials regulate secretion of posterior pituitary hormones from the neurohypophysis is now well established (for review see Douglas, 1974), but there the cells involved are simply specialized neurones (the neurosecretory fibres of the hypothalamo-neurohypophysial tract) and the participation of action potentials in stimulus-secretion coupling is readily understandable. By contrast, the cells of pars intermedia like those of pars distalis have none of the morphological features characteristic of neurones, and the occurrence there of action potentials may, on first view, seem surprising. On embryological, biochemical, and histochemical grounds, however, Pearse (1977) has argued that adenohypophysial and many other endocrine cells share a common parentage with neurones, arise from 'neuroendocrineprogrammed ectoblasts', and 'constitute the third, endocrine division of the nervous system'. And it is noteworthy that action potentials have also been detected in some other endocrine cells that Pearse (1977) includes in this category: the chromaffin cells of the adrenal medulla (Biales et al. 1976; Brandt et al. 1976) and the fi cells of the endocrine pancreas (Dean & Matthews, 1970a, b; Pace & Price, 1974; Meissner & Schmelz, 1974; Matthews & Sakamoto, 1975). There would now appear to be sufficient grounds for suspecting that the ability to generate action potentials may be wide-

spread in the normal endocrine system and reflects a common origin of endocrine cells and neurones. Evidence of spike production in various neoplastic cells of supposed endocrine origin also points in this direction, although interpretations drawn from such tumor cells must obviously be guarded (Kidokoro, 1975; Tischler, Dichter, Biales, DeLellis & Wolf, 1976; Biales, Dichter & Tischler, 1977; Douglas & Taraskevich, 1977). Although the list of endocrine cells shown to generate action potentials is short, it is already apparent that these action potentials show differences in duration and ionic dependence. In their brief time course the action potentials of pars intermedia cells resemble more closely those of pars distalis cells (Taraskevich & Douglas, 1977) and chromaffin cells (Biales et al. 1976; Brandt et al. 1976) than those of the pancreatic fi cells which are many times longer (Dean & Matthews, 1970a, b; Meissner & Schmelz, 1974; Matthews & Sakamoto, 1975). And in their requirement for Na to sustain spiking, the pars intermedia cells again resemble adrenal chromaffin cells (Biales et al. 1976; Brandt et at. 1976) and contrast with the ,6 cells of the endocrine pancreas. Such ionic dependence is somewhat surprising since our experiments on pars distalis indicate that spikes, at least in many cells there, are not dependent on Na and can be sustained by Ca as is true also in pancreatic , cells. That cells in pars distalis and pars intermedia of the same species should display ionically different spiking mechanisms appears curious considering the fact that they arise from the

W. W. DOUGLAS AND P. S. TARASKEVICH 180 same placodal region, the invagination of buccal ectoderm known as Rathke's pouch (Wingstrand, 1966). Since information on the embryological development of spiking mechanisms of different ionic basis is as yet scarce (see Hagiwara, 1975) it may be of value to point out that Rathke's pouch may provide useful material for studying the problem. Perhaps it is relevant that the particular region of Rathke's pouch that acquires the more neurone-like dependence on Na (pars intermedia) is that which shows an early and most intimate association with the brain, coming as it does to abut and adhere to the floor of the diencephalon (saccus infundibularis). Experimental intervention to prevent this intimate association is known to inhibit the normal differentiation of morphological and functional characteristics of pars intermedia (Blount, 1945). Although there are no previous records of electrical activity in mammalian pars intermedia, several authors have recorded such activity with extracellular electrodes placed in frog pars intermedia. In some instances (Oshima & Gorbman, 1969; Dawson & Ralph, 1974) the spikes were of neuronal origin and the interest of the authors was in the nervous paths controlling MSH secretion. In the experiments of Davis & Hadley (1976) on the other hand, 'potential oscillations' were observed after denervation and were therefore suspected of arising from gland or stellate cells (see also Hadley, Davis & Morgan, 1977). In the light of the present findings it seems probable that these oscillations were indeed extracellularly recorded action potentials from gland cells. But this interpretation must, for the moment, be guarded. Neither dopamine nor any other demonstrated or supposed regulator of MSH secretion was tested. Moreover, these oscillations, in contrast to the action potentials from the isolated rat pars intermedia gland cells, increased in frequency in response to a brief electrical pulse only after a latency of half to several seconds and the increased frequency lasted for as long as a minute. Furthermore the oscillations persisted in the presence of TTX. The fact that we encountered spontaneous action potentials in almost every pars intermedia cell tested whereas only about one pars distalis cell in five showed such activity (Taraskevich & Douglas, 1977), might be explained by the different patterns of secretary activity in the two cell populations. Pars intermedia cells are normally under tonic inhibitory control in vivo and hypersecrete in vitro whereas most pars distalis cells, being subjected to preponderant stimulant influence in vivo, are relatively quiescent in vitro and only the prolactin cells (the mammotrophs) show comparable disinhibition (Geschwind, 1971; Nicoll, 1971; Meites, Lu, Wuttke, Nagasawa & Quadri, 1972; Neill,t974; MacLeod, 1976; Martin etal. 1977). The inference that spontaneous action potentials may be related to spontaneous secretary activity in pars intermedia gains support from the inhibitory effects of dopamine and noradrenaline on spike frequency and the lack of such effects with isoprenaline and 5-HT. Both dopamine and noradrenaline inhibit MSH output from rat pituitary preparations in vitro, whereas isoprenaline and 5-HT do not (Bower, 1974; Bower et al. 1974). How dopamine (or noradrenaline) reduces the frequency of spiking in pars intermedia cells or abolishes spiking altogether remains to be determined. Our experiments so far have shown only that the mechanism of action of these drugs is distinct from that of TTX in that it does not involve a reduction in spike amplitude.

181 ACTION POTENTIALS IN PARS INTERMEDIA CELLS Presumably dopamine and noradrenaline depress the spike triggering mechanism or prevent the cells from reaching threshold. It is, however, evident that once again there is a parallel with nerve cells for dopamine and noradrenaline are familiar inhibitors of action potential discharge in both vertebrate (Krnjevid, 1974) and invertebrate (Kehoe & Marder, 1976) neurones. In invertebrate neurones where the inhibitory effects of monoamines have been studied most extensively, inhibition occurs by a variety of mechanisms (Gerschenfeld, 1973; Kehoe & Marder, 1976). Whatever the details in pars intermedia cells, the salient fact remains that our experiments provide novel evidence that in an endocrine system receiving a seemingly direct 'secreto-inhibitor' innervation, suppression of secretion has an electrophysiological correlate, namely reduction of spontaneous action potential discharge. It is conceivable that a similar mechanism may operate in other endocrine systems. For example, prolactin secretion from mammotrophs in the pars distalis, as just noted, is also under tonic inhibitory control and the evidence suggests that inhibition is again mediated, at least in part, by dopaminergic nerves in the medio-basal hypothalamus, dopamine being delivered to the mammotrophs by the portal system (see Bjorklund et al. 1974; Martin et al. 19771). The involvement of Ca in secretary activity of a wide spectrum of gland cells is now well established (see Rubin, 1974; Douglas, 1978) and a substantial body of evidence supports the view that Ca acts as a common mediator in stimulus-secretion coupling and serves to induce exocytosis (Douglas, 1968). Moreover the suspicion has been voiced that the pars intermedia gland cells may conform to this pattern for the secretion of MSH is calcium dependent (Hopkins, 1971; Bower & Hadley, 1972). It might therefore be suggested that the function of the action potentials observed in pars intermedia cells may be to promote secretion by increasing intracellular free Ca. Conceivably the action potentials could do this by opening potential-dependent Ca channels in the plasma membrane as occurs in neurones (see Katz, 1969; Baker & Glitsch, 1975; Llinas, Steinberg & Walton, 1976). Such a function for Na-dependent depolarization has long been speculated on for chromaffin cells (Douglas, Kanno & Sampson, 1967) and has received fresh impetus from the recent discovery of Na dependent action potentials in chromaffin cells (Biales et al. 1976; Brandt et al. 1976). This interpretation must, however, be considered tentative. It remains to be shown that the action potential activity in pars intermedia cells involves or results in inward movement of Ca. Our limited experiments on this have so far failed to demonstrate any calcium component to the action potentials. Moreover excess K which we have shown depolarizes pars intermedia cells has been reported not to stimulate MSH secretion (Bower & Hadley, 1972) although it is the commonly successful manoeuvre in eliciting secretion from neurones and endocrine cells that are known or believed to possess potential dependent calcium channels. It is conceivable that

K fails to elicit demonstrable secretion because secretion in disinhibited cells is already maximal or because the potential dependent Ca channels inactivate (see Baker & Rink, 1975) so rapidly that increased MSH output is transient and goes undetected with the prolonged hormone collection periods that were used. These are concerns that must be resolved, but the discovery of action potentials that are inhibited by dopamine, a known inhibitor of secretion, certainly introduces a new element into the problem of stimulus-secretion coupling in pars intermedia.

182

W. W. DOUGLAS AND P. S. TARASKEVICH

This work was supported by USPHS grant NS-09137. We thank Dr Teiichi Betchaku for his skilful electron microscopic work permitting identification of specified cells in a culture. REFERENCES BAKER, P. F. & GLITSCIr, H. G. (1975). Voltage-dependent changes in the permeability of nerve membranes to calcium and other divalent cations. Phil. Trans. R. Soc. 270, 389-409. BAKER, P. F. & RiNK, T. J. (1975). Catecholamine release from bovine adrenal medulla in response to maintained depolarization. J. Phy-iol. 253, 593-620. BAUMGARTEN, H. G., BJORKLUND, A., HoLswIN, A. F. & NOBIN, A. (1972). Organization and ultrastructural identification of the catecholamine nerve terminals in the neural lobe and pars intermedia of the rat pituitary. Z. Zellforsch. mikro8k. Anat. 126, 483-517. BTATIS, B., DICEi=R, M. & TIscWIxR, A. (1976). Electrical excitability of cultured adrenal chromaffin cells. J. Phy8iol. 262, 743-753. BTALES, B., DICHTER, M. A. & TIsc IER, A. (1977). Sodium and calcium action potential in pituitary cells. Nature, Lond. 267, 172-174. BJORKLUND, A., FALCK, B., HROMEK, F., OwmAN, CH. & WEST, K. A. (1970). Identification and terminal distribution of the tubero-hypophyseal monoamine fibre systems in the rat by means of stereotaxic and microspectroflurimetric techniques. Brain Res. 17, 1-23. BJORKLIJND, A., FALCK, B., NOBIN, A. & STENEvI, U. (1974). Organization of the dopamine and noradrenaline innervations of the median eminence-pituitary region in the rat. In Neuro8ecretion: The Final Neuroendocrine Pathway, ed. KNOWLES, F. & VoLLRATH, L., pp. 209-222. New York: Springer Verlag. BJ6RKLUND, A., MooRE, R. Y., NOBIN, A. STENEvI, U. (1973). The organization of tuberohypophyseal and reticulo-infundibular catecholamine neurone systems in the rat brain. Brain Res. 51, 171-191. BLOUNT, R. F. (1945). The interrelationship of the parts of the hypophysis in development. J. exp. Zool. 100, 79-102. BOWER, A. (1974). Control of melanocyte stimulating hormone MSH release by cholinergic and indoleamine receptors. Am. Zool. 14, 1304. BOWER, A. & HADLEY, M. E. (1972). Ionic requirements for melanophore-stimulating hormone (MSH) release. Gen. comp. Endocr. 19, 147-158. BOWER, A., HADLEY, M. E. & HRUBY, V. J. (1974). Biogenic amines and the control of melanophore stimulating hormone release. Science, N.Y. 184, 70-72. BRANDT, B. L., HAaIwARA, S., KIDOKORO, Y. & MYAZAKI, S. (1976). Action potentials in the rat chromaffin cell and effects of acetylcholine. J. Phygiol. 263, 417-439. DAWSON, D. C. & RALPH, C. L. (1974). Light-evoked neural activity in the intermediate lobe of the pituitary of Rana pipiens: electrophysiological evidence for a neural pathway linking the eyes with the pars intermedia. Neuroendocrinolegy 15, 267-280. DAvis, M. D. & HADLEY, M. E. (1976). Spontaneous electrical potentials and pituitary hormone (MSH) secretion. Nature, Lond. 261, 422-423. DEAN, P. M. & MATTUEws, E. K. (1970a). Glucose induced electrical activity in pancreatic islet cells. J. Physiol. 210, 255-264. DEAN, P. M. & MATr1'iws, E. K. (1970b). Electrical activity in pancreatic islet cells: effect of ions. J. Physiol. 210, 265-275. DOUGLAS, W. W. (1968) Stimulus-secretion coupling: the concept and clues from chromaffin and other cells (The First Gaddum Memorial Lecture). Br. J. Pharmac. 34, 451-474. DOUGLAS, W. W. (1974). Mechanisms of release of neurohypophyseal hormones: stimulussecretion coupling. In Handbook of Phy8iology, section 7, Endocrinology, vol. IV, The pituitary gland and its neuroendocrine control, part 1, ed. KNOBIL, E. & SAWYER, W. H., pp. 191-224. Washington: Am. Physiol. Soc. DouGLAs, W. W. (1978). Stimulus-secretion coupling: variations on the theme of calciumactivated exocytosis involving cellular and extracellular sources of calcium. Ciba Fdn Symp. 54, 61-90. DOUGLAS, W. W., KANNo, T. & SAMPSON, S. R. (1967). Effects of acetylcholine and other medullary secretagogues and antagonists on the membrane potential of adrenal chromaffin cells: an analysis employing techniques of tissue culture. J. Phy8iol. 188, 107-120.

ACTION POTENTIALS IN PARS INTERMEDIA CELLS

183

DOUGLAS, W. W. & TARASKEVICH, P. S. (1977). Action potentials (probably calcium spikes) in normal and adenomatous cells of the anterior pituitary and the stimulant effect of thyrotropin releasing hormone. J. Physiol. 272, 41-43P. DouGLAS, W. W. & TAR.AsKEvIcu, P. S. (1978). Action potentials in rat and lizard pars intermedia cells: inhibition by dopamine and stimulation by 5-hydroxytryptamine, two suspected regulators of MSH secretion. J. Physiol. 280, 13P. GERCscENFELD, H. M. (1973). Chemical transmission in invertebrate central nervous systems and neuromuscular junctions. Phy8iol. Rev. 53, 1-119. GEscuwuD, I. I. (1971). Mechanisms of release of anterior pituitary hormones: studies in vitro. Mem. Soc. Endocr. 19, 221-229. HALEY, M. E. & BAGNARA, J. T. (1975). Regulation of release and mechanism of action of MSH. Am. Zool. 15, suppl. 1, 81-104 HADLEY, M. E., DAVIs, M. D. & MORGAN, C. M. (1977). Cellular control of melanocyte stimulating hormone secretion. In Melanocyte Stimulating Hormone: Control, Chemistry and Effects, ed. TIKERs, F. J. H., SwAAB, D. F., VAN WIMERSmA GREDANus, T. B. Frontiers of Hormone Research, vol. 4, pp. 94-104. Basel: S. Karger. HAGIWARA, S. (1975). Ca-dependent action potential. In Membranes, A Series of Advances, vol. 3, ed. EIsEiMAN, G., pp. 359-381. New York: Dekker. Hoprws, C. R. (1970). Studies on secretary activity in pars intermedia of Xenopus laevis. 3. The synthesis and release of melanocyte stimulating hormone (MSH) in vitro. Tissue & Cell 2, 83-98. HowE, A. (1973). The mammalian pars intermedia: a review of its structure and function. J. Endocr. 59, 385-409. IWATA, T. & Isun, S. (1969). Chemical isolation and determination of catecholamines in the median eminence and pars nervosa of the rat and mouse. Neuroendocrinology 5, 140-148. KATZ, B. (1969). The Release of Neural Transmitter Substances. Illinois: Thomas. KEROE, J. & MARDER, E. (1976). Identification and effects of neural transmitters in invertebrates. A. Rev. Pharmac. Toxicol. 16, 245-268. KiDoKoKo, Y. (1975). Spontaneous calcium action potentials in a clonal pituitary cell line and their relationship to prolactin secretion. Nature, Lond. 258, 741-742. KRNJEVI6, K. (1974). Chemical nature of synaptic transmission in vertebrates. Physiol. Rev. 54, 418-540. KuRostmn, K. & FUJITA, H. (1974). An Atla of Electron Micrographs Functional Morphology of Endocrine Glands. Tokyo: Igaku Shoin. LLINAS, R., STEINBERG, I. Z. & WALTON, K. (1976). Presynaptic calcium currents and their relation to synaptic transmission: voltage-clamp study in squid giant synapse and theoretical model for the calcium gate. Proc. natn. Acad. Sci. U.S.A. 73, 2918-2922. MAcLEOD, R. M. (1976). Regulation of prolactin secretion. In Frontiers in Neuroendocrinology, vol. 4, ed. MARTY, L. & GANONG, W. F., pp. 169-194. New York: Raven. MARTN, J. B., REIcaumIN, S. & BROWN, G. M. (1977). Clinical Neuroendocrinology, pp. 129-145. Philadelphia: Davis. MATniEws, E. K. & SAXAMOTO, Y. (1975). Electrical characteristics of pancreatic islet cells. J. Physiol. 246, 421-437. MEIssNER, H. P. & ScHmULz, H. (1974). Membrane potential of Beta-cells in pancreatic islets. Pflugers Arch. 351, 195-206. MErrEs, J., Lu, K. H., WurKE, W., NAGASAWA, H. & QUADRI, S. K. (1972). Recent studies on functions and control of prolactin secretion in rats. Recent Prog. Horm. Res. 28, 471-516. NEnLL, J. D. (1974). Prolactin: its secretion and control. In Handbook of Physiology, section 7, Endocrinology, vol. IV, The pituitary gland and its neuroendocrine control, part 2, ed. KNOBIL, E. & SAWYER, W. H., pp. 469-488. Washington D.C.: American Physiological Soc. NICOLL, C. S. (1971). Aspects of the neural control of prolactin secretion. In Frontiers in Neuroendocrinology, ed. MARTm, L. & GANONG, W. F., pp. 291-330. New York: Oxford. OSHIMA, K. & GORBMAN, A. (1969). Pars intermedia: unitary electrical activity regulated by light. Science, N.Y. 163, 195-197. PACE, C. S. & PRICE, S. (1974). Bioelectrical effects of hexoses on pancreatic islet cells. Endocrinology 94, 142-147. PEARSE, A. G. E. (1977). The diffuse neuroendocrine system and the "common peptides". In Molecular Endocrinology, ed. MAcINTYRE, I. & SzEur , M., pp. 309-323. New York: Elsevier.

184

W. W. DOUGLAS AND P. S. TARASKEVICH

RUBIN, R. P. (1974). Calcium and The Secretory Proce88. New York: Plenum. SAAVEDRA, J. M., PALTKOVITS, M., KIZER, J. S., BROWNSTEIN, M. & ZIvIN, J. A. (1975). Distribution of biogenic amines and related enzymes in the rat pituitary gland. J. Neurochem. 25, 257-260. TARASKEVICu, P. S. & DOUGLAS, W. W. (1977). Action potentials occur in cells of the normal anterior pituitary gland and are stimulated by the hypophysiotropic peptide thyrotropinreleasing hormone. Proc. natn. Acad. Sci. U.S.A. 74, 4064-4067. TIUDERs, F. J. H., MULDER, A. H. & SMELIK, P. G. (1975). On the presence of a MSH-release inhibiting system in the rat neurointermediate lobe. Neuroendocrinology 18, 125-130. TiLDERs, F. J. H. & SMELaI, P. G. (1977). Direct neural control of MSH secretion in mammals: the involvement of dopaminergic tubero-hypophysial neurons. In Melanocyte Stimulating Hormone: Control, Chemietry and Effects, ed. TiDEuRS, F. J. H., SWAAB, D. F. & VAN WIMERSMA GREIDANUS, TJ. B. Frontier of Hormone Research, pp. 80-93. Basel: S. Karger. TiscuiLR, A. S., DIcHTER, M. A., BIALEs, B., DE LELLIS, R. A. & WOLFE, H. (1976). Neural properties of cultured human endocrine tumor cells of proposed neural crest origin. Science, N.Y. 192, 902-904. WINGSTRAND, K. G. (1966). Comparative anatomy and evolution of the hypophysis. In The Pituitary Gland, vol. 1, ed. HARRIS, G. W. & DONOVAN, B. T., pp. 58-126. Berkeley: University of California Press. EXPLANATION OF PLATE

Electron micrograph of pars intermedia preparation in culture and action potentials recorded therefrom. Two gland cells are present. The extracellular recording was made from the gland cell to the right. To permit this correlation a phase contrast micrograph was taken after the electrical activity had been registered and the micrograph was later used to locate the target cell after it had been fixed and embedded (electron microscopic procedures were performed by Dr Teiichi Betchaku).

The Journal of Physiology, Vol. 285

A..M_rw',5num_b*.go

Plate 1

: