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Carbachol changes spike-generation properties of GH3 pituitary cells
Brain Research, 402 (1987) 311 317
31 1
Elsevier BRE 12312
Carbachol changes spike-generation properties of GH 3 pituitary cells Britta H e d l u n d 1 and Jeffery L. Barker 2 1Department qf Biochemistry, Arrhenius Laboratory, University of Stockholrn, Stockholm (Sweden) and 2Laboratory Neurophysiology, N. 1.H.-N. 1.N. C. D. S., Bethesda MD 20893 (U. S. A.)
(Accepted 17 June 1986) Key words: Pituitary cell', Muscarinic response; Action potential; Potassium channel
Membrane effects of carbachol (50 pM) on GH 3 pituitary cells were studied using whole-cell type electrophysiological techniques. Carbachol (50 uM) decreased the threshold for generation of spontaneous and current-induced action potentials and prolonged their duration. It is suggested that carbachol blocks potassium channels in GH 3 pituitary cells, and that the effect is mediated via activation of muscarinic cholinergic receptors.
INTRODUCTION Muscarinic cholinergic binding sites have been identified in the pituitary gland 1~'19and are also present on G H 3 pituitary cells iT. G H 3 clonal pituitary cells synthesize and secrete prolactin in a manner that is inhibited by muscarinic cholinergic receptors 19. The mechanism by which acetylcholine inhibits prolactin production has not been elucidated. Acetylcholine stimulates phosphatidylinositol turnover associated with an increased calcium effiux in the pituitary gland 22. Acetylcholine also reduces the ability of other agents such as vasoactive intestinal polypeptide (VIP) (which is known to stimulate prolactin secretion) to stimulate adenosine-Y,5'-monophosphate (cAMP) synthesis 14"17. Both increased cellular levels of c A M P and Ca 2+ have been suggested to be intermediate steps in the stimulation of prolactin secretion 3"9. GH 3 pituitary cells are both chemically and electrically excitable 256 and the action potential is blocked by substances that block calcium ion conductance s,ls. A variety of hormones alter the excitability of these
cells together with a change in the rate of prolactin productionl; perhaps the best studies of these is thyrotropin releasing hormone ( T R H ) 7'8. As yet, there are no reports on effects of muscarinic ligands on the electrical membrane properties of GH3 cells. The aim of this study was to investigate whether carbachol, a muscarinic cholinergic agonist, has direct membrane effects on passive and/or active membrane properties of G H 3 cells. The data presented here indicate that carbachol (50 pM) decreases the threshold for generation of action potentials. MATERIALS AND METHODS G H 3 pituitary cells, passages 16-18 and 26-28, were used in these experiments. They were grown at 37 °C under controlled humidity at 5% CO2 in a medium composed of H a m ' s F-10 medium with 12.5% horse serum, 2.5% fetal calf serum, 2 mM glutamine, 2 mM sodium-pyruvate with 0.6 g/500 ml N a H C O 3 added. Eight to 12 days before the experiments the cells were subcultivated on 35 mm Petri dishes. The medium was then changed every 4 days.
('orrespondence: B. Hedlund, Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, S-106 91 Stockholm,
Sweden. 11006-8993/87/$03.50 (~) 1987 Elsevier Science Publishers B.V. (Biomedical Division)
At the day of e x p e r i m e n t the dish was placed on the stage of an inverted microscope and viewed under phase contrast optics. All experiments were carried out at room t e m p e r a t u r e (22-25 ° C ) As a recording m e d i u m a solution c o m p o s e d of 123 mM NaCI, 3 mM KCI, 1 m M MgCI> 10 m M CaCI> 5 mM H E P E S , p H 7 . 1 - 7 . 2 , 5.6 mM glucose. 310-32() m O s m , was used. W h o l e cell patch-clamp recordings were m a d e by the m e t h o d of Hamilt et al.II using a Dagan amplifier. Electrodes were fabricated from W P I - I n s t r u m e n t s TW-150 glass and had resistances of 5 - 1 0 M Q . They were filled with a solution con]posed of t40 m M potassium-gluconate, 2 m M MgCI:, 5 mM H E P E S , p H 7 . 1 - 7 . 2 , 280-290 mOsm. Data were recorded on a p a p e r - c h a r t r e c o r d e r and on a RT-11 c o m p u t e r for further analysis. Drugs were applied from pipettes with a tip d i a m e t e r of 3 - 4 urn. 20 /tin from the cell using pressure ( 1 0 - 2 0 kPa).
lasted a p p r o x i m a t e b 1 rain and could not be mmaicked by recording medium onlv ~data not shown
Effects on current-induced electrical respon~ses Several other types of pharmacological responses to carbachol, indicating a change m the abilitx ot the cell to generate action potentials. ~ e r c also observed (Figs. 2-5). tn the examples sho\~n m Fi~zs. 2 - 5 51) uM carbachol significantly decreased the threshold for action potential g e n e r a n o n and the amount of current required to obtain threshold, the rheobase. 1"his was not accompanied by a sl,.zniticant change in m e m b r a n e resistance either at the steady slate or non-steady state (l(t ms after onset of the currenl pulse) (Fig. 3). An increased frequency or action potential activlt~ (Fig. 5l. with the spike t r e q u e n c \ measured as the inverse of the first mterspike interval. was found {Figs. 4 and 5).
RESULTS
Pirenzepine blocks carbachol's prolongation ~# actio~z potential
A total of 45 cells were r e c o r d e d from, none of which had resting m e m b r a n e potentials of less than - 5 0 mV. All cells were s p o n t a n e o u s l y active with action potentials at least 40 m V in amplitude. Carbachol had m e m b r a n e effects on 32 of the cells
To answer the question whether the observed effects of carbachol were m e d i a t e d via muscarlmc cholinergic receptors, e x p e r i m e n t s were carried out to tesl whether muscarinic antagonists could block the carbachol-induced change in the electrical excitability of GH3 cells. This was difficult to test. however, since muscarmic antagonists were also found to have direct excitatory effects (data not shown). This phenomenon has been observed ill other cell phenotypes ~>13. H o w e v e r in some cells the effects of the muscarlnlC antagonists were less p r o n o u n c e d or nonexistent. F u r t h e r m o r e , carbachol was also found to prolong action potentials. As can be observed in Fig. 6. carbachol (50 u M ) prolonged the action potential in a m a n n e r that was blocked by the muscarinic antagonist pirenzepine (100 nM]. The effect of carbachol and pirenzepine a p p l i e d t o g e t h e r was equal to the effect of pirenzepine a d d e d alone. The conclusion was therefore drawn that carbachol affects the duration of action potentials in G[-t 3 cells in a m a n n e r that is blocked by a muscarinic cholinergic antagonist. The m e m b r a n e effects induced by "antagonists' themselves are a m a t t e r for further investigation.
recorded.
Effects on spontaneous action potential activi O, Application of carbachol ( 5 0 / t M ) increased the frequency of action potentials in cells (Fig. 1). These cells were held slightly below threshold for generation of s p o n t a n e o u s action potentials. The effect
application of carbachol (0,05 mM)
V I
-50 mV holding potential
40 mV [0.1 nA 20 sec
Fig, 1. Application of carbachol (50/tM) on a GH 3 cell, and recording of the observed change in firing frequency of the celt. The cell was held at -50 mV, which was 15 mV above the resting membrane potential and slightly below threshold for generation of spontaneous action potentials.
DISCUSSION
It is evident from these data that carbachol de-
313
CONTROL
"
'l'
i,
!
II,
J!'l ii I,II I t~"~ ,
lit
I~
CARBACHOL
RECOVERY
.
! '\
-
.
.
.
.
.
IN
I
I
|111
,~. . . .
J
20 mV 0 . 2 nA
100 msec
Fig. 2. Responses to depolarizing and hyperpolarizing current pulses by a GH~ cell at a -6() mV membrane potential in the absence and presence of carbachol (50 uM).
314 STEADY STATE I-V
8 ~
INSTANTANEOUS I-V olKnA) -o3
-01
o~i I(nA)
~-4.8C
==
CONTROL • CARBACHOL • RECOVERY
Y- 2 I.k 2O V(mV)
-120
V(mV)
O~0
CONTROL • CARBACHOL • RECOVERY
-= -
-
"
60 current
that carbachol effectively stimulated increase in voltage-gated Ca 2÷ conductance, since GH3 cells possesses calcium-dependent action potentials j°'ls. MUScarinic receptor-mediated Ca 2* influx has also been observed in other cellular systems 2i. The most likely explanation for these findings, however, is that carbachol blocks potassium conductances in GH3 cells.
CARBACHOL
t"
RECOVERY
J
,,,/
I
~ 30 pA i n j e c t e d
uM).
creases the threshold for acuon potential generation in GH 3 pituitary cells and increases their duration (Figs. 1, 2, 4 and 6). One possibility would be that carbachol decreases the threshold for spike generation by acting on transient-type K* conductance mechanisms known to regulate threshold 2° and to be expressed in GH 3 cells. Another possibility would be
!
•
Fig 5. The firing frequency, i.e. the inverse of the time interval between the first and second spike plotted versus the amount of injected current, in the absence and presence of carbachol f50
Fig. 3. Current-voltage relationships for the data shown in Fig. 2 at steady state and at non-steady state with the voltage values measured after 10 ms.
CONTROL
-
f
I
•
/
///"-- ......
fj/r i /
.I ,
Fig. 4. Responses to increasing depolarizing current pulses in the absence and presence of carbachol (50#M).
120 mV L?,;1 n A lOOmsee
315
CONTROL
-
r
CARBACHOL
RECOVERY
PIRENZEPINE
~
F
PIRENZEPINE +
CARBACHOL
RECOVERY
30 mV 0 . 4 nA 1 0 0 msec
Fig. 6. Responses observed in a GH 3 cell to depolarizing current pulses at a -80 mV membrane potential in the absence and presence of carbachol (50ruM) and/or pirenzepine (100 riM).
316 Transient-type K + conductances are k n o w n to regulate excitability in cultured hippocampal and spinal
well as increase the firing frequcnc~ of Gt-I? cells ~" The stimulation of action potentiN generation by
neurons 2°. Regulation of repetitive firing has been
T R H without detectable depolarization appears t{~
shown in several studies to be regulated via K* channels 4'm'13. Changes in the activation and/or inactiva-
be mediated via blocking of transient-type K ~ chan-
tion of K + conductances would help to explain the
associated with either the initial burs! phase or tttc
mechanism with which carbachol increases the firing
later sustained phase of prolactin release. From these data we conclude that carbach{}l may inhibit prolac-
frequency of these cells and prolongs the duration ot individual action potentials. Voltage-clamp analysis
nels ~. All of these events have been suggested to be
tin release independently bv its effects on firing fre-
and K + would resolve the pharmacological mecha-
quency or action potential duration, fhese changes in excitability per sc may not induce hormone re-
nism. One question is whether these effects of carbachol
creases in intracetlular ('a ~" tlmt occur throm, h
of the underlying m e m b r a n e currents carried bv Ca-' ~
are mediated via activation of muscarinic cholinergtc receptors present on G H 3 cells. Attempts to block the carbachol-induced excitation with muscarmtc cholinergic antagonists were rendered difficult by the fact that the antagonists had m e m b r a n e effects of their own, which also has been reported in other svsterns t2,t3. From the data shown in Fig. 6. however, tt is evident that pirenzepine, despite having membrane effects of its own. blocks the carbachol-induced prolongation of the action potential. The conclusion was drawn, therefore, that a c o m m o n binding
lease. Rather. a more likely suggestion is thal mmechanisms other than those delecled electrically and/or cAMP synthesis arc the primary events in prolactin release In fact, carbachol has been ~hown to inhibit VIP-stimulated cAMP svnthesis via activauon of muscarinic receptors w. which has been proposed as a mechanism for the muscarinic inhibition of prolactin secretiot~ ~" In summary, electrophysiological experiments reported here indicate that carbachol triggers action potential activity in GH3 cells apparently by lowering spike threshold and prolongs their duration, the lat-
site for carbachol and pirenzepine, through which
ter in a pirenzepine-sensitive man her
carbachol exerts its effects on action potential duration, is a muscarinic cholinergic receptor It is vet not
ACKNOWLE1)( ;EMENfS
clear what the relationship between the action potenThis stud~ was supported by grants from the
tial-inducing effects and the actions on duration are. A n o t h e r question concerns the functional basis of
N. I. N. C. D.S. to J.L.B. and by a grant from the Swe-
these findings. T R H , known to stimulate prolactin release IS, has been shown to stimulate calcium efflux
dish Medical Research Council to B.H. We thank Ms. J o A n n Mazetta and Ms. Veronica Smallwood
and influx3, c A M P synthesis ~, active K" channels ~. a~
for help with the tissue culture.
REFERENCES 1 Barker, J.L., Dufy, B., Owen, D.G. and Segal. M., Excitable membrane properties of cultured central nervous system neurons and clonal pituitary cells In 48:h Cold Spring Harbour Symposium on Quantitative Biology, Cold Spring Harbour Press, 1983, pp. 259-268. 2 Biales, B., Dichter, M.A and Tischter. A., Sodium and calcium action potential in pituitary cells. Nature f London~, 267 (1977) 172-174. 3 Brostrom, M.A., Brostrom. C.O.. Brotman, L.A. and Green, S.S., Regulation of Ca2. dependent cyclic AMP accumulation and Ca~-+ metabolism in intact pituitary tumor cells by modulators of prolactin production. Mol. Pharmacol., 23 (1983) 399-408. 4 Connor, J.A. and Stevens. C.F.. Prediction ot repetinve
firing behaviour lrom voltage clamp data on an isolated neurone somata. J. Physiol. rLondon~ 213 ( 1971 3l -53. 5 Douglas. W.W.. Stimulus-secret on coupling: concepts and clues from chromaffin and other cells Br, J. Pharmacol.. 34 11968) 451-474 C~Dufy, B. and Barker, J.L., Calcium-activated and voltagedependent potassium conductanees in clonal pituitary ceils. Life Sci.. 30 / 1982) 1933-1941. Duly. B., Dupuy, B.. Georgescauld, D. and Barker, J i . Calcium mediated inactivation of calcium conductance in a prolactin secreting cell line. In R M. MacLeod, M.O Thorner and U. Scapagnini IEds./. Prolactin: Basic and Clinical Correlates. Liviana Pres~ Padova, 1985. pp 177-183. b Duly. B.. Vincent. ,I.D.. F|eurv. H,. DuPasqmer. D . Gourdii, D. and Tixier-Vidal, A , Membrane-effects of -
317
9
l/)
11
12
13
14
15
thyrotropin releasing hormone and estrogen shown by intracellular recording from pituitary cells, Science, 204 (1979) 509-511. Gourdji, D., Bataille, D., Vauclin, N., Grouselle, D., Rossclin, G. and Tixier-Vidal, A., Vasoactive intestinal peptide (VIP) stimulates prolactin (PRL) release and cyclic AMP production in a rat pituitary cell line (GH3/B0). Additive effects of VIP and TRH on PRL release, FEBS Letr, 104 (1979) 165-168. Gustafsson, B., Afterhyperpolarization and the control of repetitive firing in spinal neurones of the cat, Acta Physiol. Stand.. Suppl. 416. (1974). Hamill, O.B., Marty, A., Neher, E., Sakmann, B. and Sigworth, F,J., Improved patch-clamp techniques for high resolution current recording from cells and cell free membrane patches, Pfliigers Arch.. 391 (1981) 85-100. Hcdlund, B., Owen, D.G. and Barker, J.L., Non-muscarinic effects of atropine on calcium conductances in cultured mouse spinal cord neurons, Neurosci. Letr, 55 (1985) 355-359. ltedlund, B., Arhem, P., korentz, M. and Sydbom, A., Dircct effects of scopolamine on N1E-I15 neuroblastoma cells, ,4eta Physiol. Stand.. 27 (1986) 127-132. Hcislcr, S., Larose, L. and Morisset, J., Muscarinic cholinergic inhibition of cyclic AMP formation and adrenocorticotropin secretion in mouse pituitary tumor cells, Biochem. Biophys. Res. Commun., 114 (1983) 289-295. Kernell, D. and SjOholm. H., Repetitive impulse firing: Comparisons between neurone models based on 'voltage clamp equations" anti spinal motoneurones, Acta Physiol.
Scand., 87 (1973) 40-56. 16 Mukherjee, A., Snyder, G.D. and McCann, S.M., Characterization of muscarinic cholinergic receptors on intact rat anterior pituitary cells, Life Sci., 27 (1980) 475-482. 17 Onali, P., Carola, E., Olianas, M.C., Scawartz, J.P. and Costa, E., In GH 3 pituitary cells acetylcholine and vasoactive intestinal peptide antagonistically modulate adenylate cyclase, cyclic AMP content and prolactin secretion, MoL PharmacoL, 24 (1983) 189-194. 18 Ozawa, S. and Kimura, N., Membrane potential changes caused by thyrotropin-releasing hormone in the clonal GH 3 cell and their relationship to secretion of pituitary hormone, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 6(]17-6020. 19 Rudnick, M.S. and Dannies, P.S., Muscarinic inhibition of prolactin production in cultures of rat pituitary cells, Biochem. Biophys. Res. Commun., 101 (1981) 689-696. 20 Segal, M., Rogawski, M.A. and Barker, J.L., A transient potassium conductance regulates the excitability of cultured hippocampal and spinal neurons, J. Neurosci., 4 (1984) 604-609. 21 Study, R.E., Breakefield, X.O., Bartfai, T. and Greengard, P., Voltage-sensitive calcium channels regulate guanosine 3',5' cyclic monosphosphate levels in neuroblastoma cells, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 6295-6299. 22 Young, P.W., Bicknell, R.J. and Schofield, J.G., Acetylcholine stimulates growth hormone secretion, phosphatidyl inositol labeling, calcium-45 ion efflux and cyclic GMP accumulation in bovine anterior pituitary gland, J. Endocrinol., 80 (1979) 203-213.
31 1
Elsevier BRE 12312
Carbachol changes spike-generation properties of GH 3 pituitary cells Britta H e d l u n d 1 and Jeffery L. Barker 2 1Department qf Biochemistry, Arrhenius Laboratory, University of Stockholrn, Stockholm (Sweden) and 2Laboratory Neurophysiology, N. 1.H.-N. 1.N. C. D. S., Bethesda MD 20893 (U. S. A.)
(Accepted 17 June 1986) Key words: Pituitary cell', Muscarinic response; Action potential; Potassium channel
Membrane effects of carbachol (50 pM) on GH 3 pituitary cells were studied using whole-cell type electrophysiological techniques. Carbachol (50 uM) decreased the threshold for generation of spontaneous and current-induced action potentials and prolonged their duration. It is suggested that carbachol blocks potassium channels in GH 3 pituitary cells, and that the effect is mediated via activation of muscarinic cholinergic receptors.
INTRODUCTION Muscarinic cholinergic binding sites have been identified in the pituitary gland 1~'19and are also present on G H 3 pituitary cells iT. G H 3 clonal pituitary cells synthesize and secrete prolactin in a manner that is inhibited by muscarinic cholinergic receptors 19. The mechanism by which acetylcholine inhibits prolactin production has not been elucidated. Acetylcholine stimulates phosphatidylinositol turnover associated with an increased calcium effiux in the pituitary gland 22. Acetylcholine also reduces the ability of other agents such as vasoactive intestinal polypeptide (VIP) (which is known to stimulate prolactin secretion) to stimulate adenosine-Y,5'-monophosphate (cAMP) synthesis 14"17. Both increased cellular levels of c A M P and Ca 2+ have been suggested to be intermediate steps in the stimulation of prolactin secretion 3"9. GH 3 pituitary cells are both chemically and electrically excitable 256 and the action potential is blocked by substances that block calcium ion conductance s,ls. A variety of hormones alter the excitability of these
cells together with a change in the rate of prolactin productionl; perhaps the best studies of these is thyrotropin releasing hormone ( T R H ) 7'8. As yet, there are no reports on effects of muscarinic ligands on the electrical membrane properties of GH3 cells. The aim of this study was to investigate whether carbachol, a muscarinic cholinergic agonist, has direct membrane effects on passive and/or active membrane properties of G H 3 cells. The data presented here indicate that carbachol (50 pM) decreases the threshold for generation of action potentials. MATERIALS AND METHODS G H 3 pituitary cells, passages 16-18 and 26-28, were used in these experiments. They were grown at 37 °C under controlled humidity at 5% CO2 in a medium composed of H a m ' s F-10 medium with 12.5% horse serum, 2.5% fetal calf serum, 2 mM glutamine, 2 mM sodium-pyruvate with 0.6 g/500 ml N a H C O 3 added. Eight to 12 days before the experiments the cells were subcultivated on 35 mm Petri dishes. The medium was then changed every 4 days.
('orrespondence: B. Hedlund, Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, S-106 91 Stockholm,
Sweden. 11006-8993/87/$03.50 (~) 1987 Elsevier Science Publishers B.V. (Biomedical Division)
At the day of e x p e r i m e n t the dish was placed on the stage of an inverted microscope and viewed under phase contrast optics. All experiments were carried out at room t e m p e r a t u r e (22-25 ° C ) As a recording m e d i u m a solution c o m p o s e d of 123 mM NaCI, 3 mM KCI, 1 m M MgCI> 10 m M CaCI> 5 mM H E P E S , p H 7 . 1 - 7 . 2 , 5.6 mM glucose. 310-32() m O s m , was used. W h o l e cell patch-clamp recordings were m a d e by the m e t h o d of Hamilt et al.II using a Dagan amplifier. Electrodes were fabricated from W P I - I n s t r u m e n t s TW-150 glass and had resistances of 5 - 1 0 M Q . They were filled with a solution con]posed of t40 m M potassium-gluconate, 2 m M MgCI:, 5 mM H E P E S , p H 7 . 1 - 7 . 2 , 280-290 mOsm. Data were recorded on a p a p e r - c h a r t r e c o r d e r and on a RT-11 c o m p u t e r for further analysis. Drugs were applied from pipettes with a tip d i a m e t e r of 3 - 4 urn. 20 /tin from the cell using pressure ( 1 0 - 2 0 kPa).
lasted a p p r o x i m a t e b 1 rain and could not be mmaicked by recording medium onlv ~data not shown
Effects on current-induced electrical respon~ses Several other types of pharmacological responses to carbachol, indicating a change m the abilitx ot the cell to generate action potentials. ~ e r c also observed (Figs. 2-5). tn the examples sho\~n m Fi~zs. 2 - 5 51) uM carbachol significantly decreased the threshold for action potential g e n e r a n o n and the amount of current required to obtain threshold, the rheobase. 1"his was not accompanied by a sl,.zniticant change in m e m b r a n e resistance either at the steady slate or non-steady state (l(t ms after onset of the currenl pulse) (Fig. 3). An increased frequency or action potential activlt~ (Fig. 5l. with the spike t r e q u e n c \ measured as the inverse of the first mterspike interval. was found {Figs. 4 and 5).
RESULTS
Pirenzepine blocks carbachol's prolongation ~# actio~z potential
A total of 45 cells were r e c o r d e d from, none of which had resting m e m b r a n e potentials of less than - 5 0 mV. All cells were s p o n t a n e o u s l y active with action potentials at least 40 m V in amplitude. Carbachol had m e m b r a n e effects on 32 of the cells
To answer the question whether the observed effects of carbachol were m e d i a t e d via muscarlmc cholinergic receptors, e x p e r i m e n t s were carried out to tesl whether muscarinic antagonists could block the carbachol-induced change in the electrical excitability of GH3 cells. This was difficult to test. however, since muscarmic antagonists were also found to have direct excitatory effects (data not shown). This phenomenon has been observed ill other cell phenotypes ~>13. H o w e v e r in some cells the effects of the muscarlnlC antagonists were less p r o n o u n c e d or nonexistent. F u r t h e r m o r e , carbachol was also found to prolong action potentials. As can be observed in Fig. 6. carbachol (50 u M ) prolonged the action potential in a m a n n e r that was blocked by the muscarinic antagonist pirenzepine (100 nM]. The effect of carbachol and pirenzepine a p p l i e d t o g e t h e r was equal to the effect of pirenzepine a d d e d alone. The conclusion was therefore drawn that carbachol affects the duration of action potentials in G[-t 3 cells in a m a n n e r that is blocked by a muscarinic cholinergic antagonist. The m e m b r a n e effects induced by "antagonists' themselves are a m a t t e r for further investigation.
recorded.
Effects on spontaneous action potential activi O, Application of carbachol ( 5 0 / t M ) increased the frequency of action potentials in cells (Fig. 1). These cells were held slightly below threshold for generation of s p o n t a n e o u s action potentials. The effect
application of carbachol (0,05 mM)
V I
-50 mV holding potential
40 mV [0.1 nA 20 sec
Fig, 1. Application of carbachol (50/tM) on a GH 3 cell, and recording of the observed change in firing frequency of the celt. The cell was held at -50 mV, which was 15 mV above the resting membrane potential and slightly below threshold for generation of spontaneous action potentials.
DISCUSSION
It is evident from these data that carbachol de-
313
CONTROL
"
'l'
i,
!
II,
J!'l ii I,II I t~"~ ,
lit
I~
CARBACHOL
RECOVERY
.
! '\
-
.
.
.
.
.
IN
I
I
|111
,~. . . .
J
20 mV 0 . 2 nA
100 msec
Fig. 2. Responses to depolarizing and hyperpolarizing current pulses by a GH~ cell at a -6() mV membrane potential in the absence and presence of carbachol (50 uM).
314 STEADY STATE I-V
8 ~
INSTANTANEOUS I-V olKnA) -o3
-01
o~i I(nA)
~-4.8C
==
CONTROL • CARBACHOL • RECOVERY
Y- 2 I.k 2O V(mV)
-120
V(mV)
O~0
CONTROL • CARBACHOL • RECOVERY
-= -
-
"
60 current
that carbachol effectively stimulated increase in voltage-gated Ca 2÷ conductance, since GH3 cells possesses calcium-dependent action potentials j°'ls. MUScarinic receptor-mediated Ca 2* influx has also been observed in other cellular systems 2i. The most likely explanation for these findings, however, is that carbachol blocks potassium conductances in GH3 cells.
CARBACHOL
t"
RECOVERY
J
,,,/
I
~ 30 pA i n j e c t e d
uM).
creases the threshold for acuon potential generation in GH 3 pituitary cells and increases their duration (Figs. 1, 2, 4 and 6). One possibility would be that carbachol decreases the threshold for spike generation by acting on transient-type K* conductance mechanisms known to regulate threshold 2° and to be expressed in GH 3 cells. Another possibility would be
!
•
Fig 5. The firing frequency, i.e. the inverse of the time interval between the first and second spike plotted versus the amount of injected current, in the absence and presence of carbachol f50
Fig. 3. Current-voltage relationships for the data shown in Fig. 2 at steady state and at non-steady state with the voltage values measured after 10 ms.
CONTROL
-
f
I
•
/
///"-- ......
fj/r i /
.I ,
Fig. 4. Responses to increasing depolarizing current pulses in the absence and presence of carbachol (50#M).
120 mV L?,;1 n A lOOmsee
315
CONTROL
-
r
CARBACHOL
RECOVERY
PIRENZEPINE
~
F
PIRENZEPINE +
CARBACHOL
RECOVERY
30 mV 0 . 4 nA 1 0 0 msec
Fig. 6. Responses observed in a GH 3 cell to depolarizing current pulses at a -80 mV membrane potential in the absence and presence of carbachol (50ruM) and/or pirenzepine (100 riM).
316 Transient-type K + conductances are k n o w n to regulate excitability in cultured hippocampal and spinal
well as increase the firing frequcnc~ of Gt-I? cells ~" The stimulation of action potentiN generation by
neurons 2°. Regulation of repetitive firing has been
T R H without detectable depolarization appears t{~
shown in several studies to be regulated via K* channels 4'm'13. Changes in the activation and/or inactiva-
be mediated via blocking of transient-type K ~ chan-
tion of K + conductances would help to explain the
associated with either the initial burs! phase or tttc
mechanism with which carbachol increases the firing
later sustained phase of prolactin release. From these data we conclude that carbach{}l may inhibit prolac-
frequency of these cells and prolongs the duration ot individual action potentials. Voltage-clamp analysis
nels ~. All of these events have been suggested to be
tin release independently bv its effects on firing fre-
and K + would resolve the pharmacological mecha-
quency or action potential duration, fhese changes in excitability per sc may not induce hormone re-
nism. One question is whether these effects of carbachol
creases in intracetlular ('a ~" tlmt occur throm, h
of the underlying m e m b r a n e currents carried bv Ca-' ~
are mediated via activation of muscarinic cholinergtc receptors present on G H 3 cells. Attempts to block the carbachol-induced excitation with muscarmtc cholinergic antagonists were rendered difficult by the fact that the antagonists had m e m b r a n e effects of their own, which also has been reported in other svsterns t2,t3. From the data shown in Fig. 6. however, tt is evident that pirenzepine, despite having membrane effects of its own. blocks the carbachol-induced prolongation of the action potential. The conclusion was drawn, therefore, that a c o m m o n binding
lease. Rather. a more likely suggestion is thal mmechanisms other than those delecled electrically and/or cAMP synthesis arc the primary events in prolactin release In fact, carbachol has been ~hown to inhibit VIP-stimulated cAMP svnthesis via activauon of muscarinic receptors w. which has been proposed as a mechanism for the muscarinic inhibition of prolactin secretiot~ ~" In summary, electrophysiological experiments reported here indicate that carbachol triggers action potential activity in GH3 cells apparently by lowering spike threshold and prolongs their duration, the lat-
site for carbachol and pirenzepine, through which
ter in a pirenzepine-sensitive man her
carbachol exerts its effects on action potential duration, is a muscarinic cholinergic receptor It is vet not
ACKNOWLE1)( ;EMENfS
clear what the relationship between the action potenThis stud~ was supported by grants from the
tial-inducing effects and the actions on duration are. A n o t h e r question concerns the functional basis of
N. I. N. C. D.S. to J.L.B. and by a grant from the Swe-
these findings. T R H , known to stimulate prolactin release IS, has been shown to stimulate calcium efflux
dish Medical Research Council to B.H. We thank Ms. J o A n n Mazetta and Ms. Veronica Smallwood
and influx3, c A M P synthesis ~, active K" channels ~. a~
for help with the tissue culture.
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