Catechol blocks the fast outward potassium current in melanotrophs of the rat pituitary

136

Neurowtence Letters, 125 (1991) 136-138 ,,0', 1991 Elsewer Sclennfic Pubhshers Ireland Ltd 0304-3940/91/$ 03 50 ADONIS 0304394091001753

NSL 07693

Catechol blocks the fast outward potassium current in melanotrophs of the rat pituitary Steven J. K e h l Department of Phystology, Umverstty of Brtttsh Columbta, Vancouver, B C (Canada) (Recewed 30 November 1990, Revised version recewed 7 January 1991, Accepted 21 January 1991)

Key words Catechol, Block of fast p o t a s s m m current, Melanotroph, Rat pituitary The effect of catechol on the fast voltage-gated K + current (IK(f)) of acutely dlssocmted rat melanotrophs was investigated m whole-cell recordings Half-maximal inhibition of lK(f) occurred at an external concentratxon of l 7 m M and this effect was assocmted with a decrease of the rate of the current decay Internal catechol had no measurable effect on IK(f) Catechol appeared to be equally effectwe as a blocker of the slow voltage-gated K ÷ current (IK(s)) Despite this lack of specificity the blocking action of catechol was voltage- and frequency-independent and was rapidly reversible Catechol therefore represents a useful alternative to 4 - a m m o p y n d m e as a blocker of IK(I')

The release of ~-melanocyte stimulating hormone from melanotrophs of the rat imtermediate lobe is linked to the firmg of action potentials [7]. One of the membrane currents which Influences cell firing m melanotrophs is IK(f) [5], a voltage-gated K + current which activates and inactivates rapidly and in this regard is similar to the A-current expressed m mammalian neurones and a number of other excitable cell types [9] The pharmacological propemes of IK(f) are also similar to those normally attrtbuted to the A-current. For example, IK(f) lS not affected by 10o20 mM external TEA but is blocked by mllllmolar concentrations of 4-amlnopyridlne (4-AP) ' [6]. 4-AP is however of limited usefulness as a blocker of IK(f) not only because 4-AP affects other voltage-gated K ÷ currents but also because the block of IK(f) by 4-AP xn melanotrophs [6], as in some other cell types [4, 8, 10, 11], is voltage- and time-dependent and only slowly reversible Catechol (1,2-benzenedtol), like 4-AP, is a convulsant and facihtates transmission at the neuromuscular junction [3]_ Reports that catechol selectively blocks the A-type current in invertebrate [1] and amphibian [2] neurones stimulated this inquiry into its effect on IK(f) in acutely dissociated melanotrophs of the rat pituitary gland Melanotrophs were obtained from the pars mtermedm of male Wistar rats (2000300 g) as described previously [6] Macroscopic currents were measured at the room temperature (20-25°C) using a LIST EPC-7 amphfier Correspondence S J Kehl, Department of Physiology, Umverslty of Brmsh Columbia, Vancouver, B C , V6T IW5, Canada

and conventtonal patch clamp methods. Except where noted, the holdmg potential was - 7 0 mV. The membrane capacitance was read from the front panel settmg of the EPC-7 after adjusting the capacttance compensatton The external recording solution normally contained (m mM) 110--120 NaC1, 20 tetraethylammomum (TEA) chloride to block the slow voltage-gated K + current (IK(S)), 3.5 KC1, 10 HEPES, 2 CaCI2, 1 MgCI2 and 5 glucose. The pH was adjusted to 7 4 with N a O H Except where noted, 1 /~M tetrodotoxm (TTX) and 300 /zM Cd 2+ were present to block voltage-gated Na ÷ and Ca 2+ currents The pipette (internal) solutton contained (m mM). 120 KC1, 5 MgCI2, 10 EGTA, 1 Na2ATP, 10 HEPES and was tltrated to pH 7.4 with K O H Current signals were low-pass filtered ( - 3 dB at 2 kHz) and digitized (4 kHz) for analysts using BASIC-Fastlab (Indec, Sunnyvale, CA). Non-linear least squares curve fitting was done using programmes in BASIC-Fastlab or SYSTAT (Evanston, IL) Solutions contamlng catechol (Sigma) were prepared just before the start of the experiment by equtmolar substitution for NaC1 Catechol solutions were equally effective after 4-5 h at room temperature In the preliminary experiments the effect of 10 mM catechol was assessed The results of one such experiment are illustrated m Fig. 1A After changing to the catecholcontaining solution there was a rapid and nearly complete block of IK(f) which reversed after returning to control solution A second apphcatlon of catechol produced identical effects and there was no evidence for any desensiUzatlon with prolonged (10030 mm) apphcations

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Fig 1 A the onset of and recovery from the block of I~(D by catechol is rapid. Test currents were evoked at 0 mV once every 4 s Catecholcontaining medmm replaced the control solution at 100-200 and 400500 s. The arrows in&care the approximate time at which current traces a--c were obtained The reduction ofthe amphtude of the steadystate current may reflect the block by catechol of residual It(s) B the current-voltage curve m 0.1-8 0 mM catechol m&cates the lack of any apparent voltage-dependence of the blocking effect between - 2 0 and 40 mV

I n c r e a s m g o r decreasing the stimulus frequency in the range o f 1.TN).l H z h a d n o effect on the m h i b l t i o n o f IK(0 by c a t e c h o l (not shown) The g r a p h o f Fig. 1B plots the p e a k a m p l i t u d e oflK(f), m e a s u r e d as d e s c r i b e d below, in the presence o f different c o n c e n t r a t i o n s o f catechol, versus the m e m b r a n e p o t e n tial d u r i n g the d e p o l a r i z i n g c o m m a n d . A s i d e f r o m tnd~c a t l n g the d o s e - d e p e n d e n c e o f the block, the g r a p h shows t h a t the b l o c k i n g a c t m n is n o t v o l t a g e - d e p e n d e n t m the r a n g e tested. F o r example, 2 m M c a t e c h o l inhibits the c u r r e n t e v o k e d at - 2 0 , 0, 20 o r 40 m V by a p p r o x i m a t e l y 50%. F o r the g e n e r a t i o n o f a d o s e - r e s p o n s e curve (Fig. 2A) the p e a k a m p l i t u d e o f 1K(f) was c a l c u l a t e d as follows. The test pulse consisted o f a 300 ms d e p o l a r l z a t t o n to 0 inV. In s o m e trials the test pulse was p r e c e d e d by a 500 ms c o n d i t i o n i n g pulse to - 2 0 m V to e h m i n a t e IK(f) [5] a n d t h e r e b y u n c o v e r the leak c u r r e n t a n d a n y residual n o n - i n a c t i v a t i n g K + currents (e.g. IK(s)). The p e a k a m p l i t u d e o f IK(0 was then t a k e n f r o m the difference c u r r e n t o b t a i n e d by s u b t r a c t i n g the average o f 3 c o n & tioned test currents f r o m the average o f 3 u n c o n d i t i o n e d test currents. N o r m a l i z e d p e a k currents were then calculated as (Icatechol/((Icontrol+Irecovery)/2)) a n d p l o t t e d against the l o g a r i t h m o f the catechol c o n c e n t r a t i o n . The hne fitted b y n o n h n e a r regression analysis to the d a t a points o f Fig. 2 A represents the s o l u t i o n to the Hill equation, I = A (1 + ( [ c a t e c h o l ] / K d ) - " ) -

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Fig 2_ A the dose-response curve for the mlubmon of IK(f) at 0 mV by catechol The line fitted to the data points indicates that half-maximal block occurs at approx]mately 1 7 mM The number near the symbol (mean + S_E M ) m&cates the number of cells tested at that concentration_ B catechol blocks IK(S)as well as &(f) m TEA- and TTX-free medium The dechne of the tml current m catechol indicates that the reduced amphtude of the pulse current is not due to a depolanzmg shift of the EK The fast reward Na + current (arrow) persisted m the catechol C 0) m a cell perfused internally with 1 mM 4-AP and 20 mM catechol and without external TEA, a 405 ms depolanzatlon from - 70 to 0 mV elicited a small 1K(f) and a prominent &(s) (fi) In the same cell held at - 10 mV to relieve the voltage dependent block of It(t) by 4-AP, and following a 200 ms hyperpolanzatlon (not shown) to - 7 0 mV to remove Inactivation of IK(f) channels, a depolarization to 0 mV evoked a prominent l~(f) This indicates that internal catechol is not as effectwe, if at all, as external catechol as a blocker of IK(f). The reduced s]ze of IK(s) m (10 reflects its slow recovery from lnactwatlon [5]

c o n t r o l a n d t r e a t e d cells this d e c a y was well-fitted by a single exponential, however, whereas in c o n t r o l cells Zdecay at 0 m V was 3 6 . 2 + 1.5 m s ( m e a n + S.E M ) in 1.5 m M catechol rdecay was 70.6__+ 2.4 ms (e.g. mset traces o f Fig. 2A). A f t e r r e t u r n m g to catechol-free m e d m m Tdecay was 35.4-1-0,9 ms (n = 6 for each group). T o assess the specificity o f catechol as a channel b l o c k e r it was tested on cells in which I t ( s ) was n o t b l o c k e d wtth T E A Fig. 2B shows t h a t in a d d i t i o n to b l o c k i n g IK(f), c a t e c h o l c a u s e d a d o s e - d e p e n d e n t reduction o f IK(s). The same effect was o b s e r v e d in each o f the 3 cells tested. A l t h o u g h a d o s e - r e s p o n s e curve was n o t constructed, the parallel dechne o f the a m p l i t u d e o f 1K(f) a n d IK(s) (Fig. 2B) implies t h a t the c o n c e n t r a t i o n o f catechol required for h a l f - m a x i m a l block o f these currents is s]milar. The effect o f mternal p e r f u s i o n o f the d r u g was studied using 20 m M catechol, a c o n c e n t r a t i o n which ff a p p l i e d externally w o u l d virtually e l i m m a t e IK(f) (Fig. 2A). The p e a k a m p h t u d e o f IK(F) in three whole-cell r e c o r d i n g s with 20 m M catechol in the p a t c h ptpette was 1 1 6 + 16 p A / p F (versus 7 9 + 4 p A / p F , m e a n - t - S . E . M . , in 12 control cells) a n d d i d n o t decline over a 10 m m r e c o r d i n g p e r i o d (not shown) To d e m o n s t r a t e t h a t the lack o f an effect was n o t due to p o o r diffusion o f the catechol into

138 the cell, in two further experiments the m t e r n a l solution c o n t a i n e d b o t h 20 m M catechol a n d 1 m M 4 - A P U n d e r v o l t a g e - c l a m p c o n d i t i o n s which f a v o u r e d the b i n d i n g o f the 4 - A P to the channels subserving In(f) [6] (Fig 2C1) a d e p o l a r i z i n g c o m m a n d to 0 m V e v o k e d a small IK(f) a n d a p r o m m e n t Ii<(s). After c h a n g i n g to a stimulus p r o tocol whxch reheved the block o f the channels by 4-AP, a d e p o l a r i z i n g pulse to 0 mV u n c o v e r e d a r o b u s t I~:(f) (Fig, 2Cu). T h a t the typical b l o c k i n g effect o f internal 4A P was evtdent m d t c a t e d the effective perfuston o f the cell interior w~th the test drugs. Consequently, the simplest conclusion is that internal 20 m M catechol has httle or no effect on the gating o f IK(f), p r e s u m a b l y because there is no catechol b i n d i n g site on the internal face o f the channel_ The results show that catechol blocks IK(f) m m e l a n o t r o p h s H a l f - m a x i m a l b l o c k occurs at a p p r o x i m a t e l y 1 7 m M , a value similar to t h a t r e p o r t e d for the half-maximal b l o c k o f the A - t y p e c u r r e n t in frog sensory n e u r o n e s (0 5 r a M ) [2] a n d shad n e u r o n e s (5 r a M ) [1]. C a t e c h o l decreased the rate o f d e c a y o f h:(f) (see also ref. 1) b u t w i t h o u t single-channel r e c o r d i n g it is n o t possible to be certain o f the basis for this effect. The persistence o f IK(f) in 20 m M internal catechol is consistent w~th the conclusion o f I t o a n d M a e n o [2] t h a t catechol acts only from the o u t e r face o f the channel In th~s c o n n e c t i o n catechol is clearly distinguishable from 4 - A P which does b l o c k Ire(f) via an internal r e c e p t o r site [4, 6]_ U n f o r t u n a t e l y catechol a p p e a r s to be equally effectwe as a b l o c k e r o f IK(s) in m e l a n o t r o p h s which c o n t r a s t s w~th ItS selectivity m snail a n d frog n e u r o n e s [1, 2]. Nonetheless there was no voltage- or u s e - d e p e n d e n c e to the block and since there was also c o m p l e t e a n d r a p i d recovery, as n o t e d

previously [1, 2], catechol does have some a d v a n t a g e s over 4 - A P as a b l o c k e r o f IK(f)

S u p p o r t e d by a Scholarship f r o m the B C H e a l t h Care Research F o u n d a t i o n a n d a G r a n t f r o m the M R C (Canada)

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