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Receptor for ATP in the membrane of mammalian sensory neurones
Neuroscience Letters, 35 (1983) 41-45
41
Elsevier Scientific Publishers Ireland Ltd.
R E C E P T O R FOR A T P IN T H E M E M B R A N E OF M A M M A L I A N S E N S O R Y NEURONES
O.A. KRISHTAL, S.M. MARCHENKO and V.I. P I D O P L I C H K O
A.A. Bogomoletz Institute o f Physiology, Ukranian Academy o f Sciences, Bogomoletz str. 4, Kiev 24, 252601 GSP (U.S.S.R.). (Received November 22nd, 1982; Revised version received and accepted December 8th, 1982)
Key words: sensory neurons - ATP - receptors - membrane conductance - intracellular perfusion desensitization
ATP-activated conductance has been found in a large number of neurones isolated from various sensory ganglia of the rat and cat. The inward current produced by ATP (Ka = 5.10 -6 M) is carried by cations and demonstrates rapid activation and slow desensitization. The sequence of agonists ( A T P > A D P with AMP and adenosine ineffective) is different from those previously described for purinergic receptors P~ and P2.
Two types of conductance-activating chemoreceptors have been found so far in the m e m b r a n e of m a m m a l i a n sensory neurones: the receptors for G A B A [3] and for protons [5]. The present paper demonstrates an ATP-activated conductance in a variety of rat sensory neurones. Experiments were mostly performed on neurones isolated from nodose, vestibular, trigeminal and spinal ganglia obtained from 4-60-day-old rats. Some experiments were performed on the neurones from the same ganglia of 5-10-day-old cats. The neurones were enzymatically isolated [4] and investigated with the intracellular perfusion and voltage clamp technique. The set-up for rapid application of external solutions [5] was modified to enable their equally rapid removal, with each change taking less than 50 ms ('square pulse' application technique, Fig. 1A). The normal external solution contained 152 mM NaC1, 2.2 m M CaCI2, 1.1. mM MgC12, 10 m M H E P E S neutralized by N a O H to p H 7.4. The internal solution contained 130 m M KF and 30 mM Tris F (pH 7.2). Experiments were performed at r o o m temperature at about 22°C. Many sensory neurones clamped at a constant holding potential ( - 9 0 mV) responded with an inward current to the application of A T P added to the normal external saline. These responses were similar when recorded from rat or cat neurones and so the further investigation was carried out on the rat neurones, with only a few control experiments on the neurones from cat. The ATP-activated current elicited by prolonged application of A T P in different concentrations is 0304-3940/83/0000-0000/$ 03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd.
42
A
B ,L
C .....
. . . . .
2 nA
!
!
200ms
-3 1 nA
Fig. 1. A: the 'square pulse' application technique. The tip of the micropipette with the cell (a) is inserted into a plastic tube. The lower end of this tube can be exposed to different external solutions or to air. The suction applied to the upper end of the tube (indicated by arrow) is controlled by an electromagnetic valve (b). A preprogrammed sequence of current pulses applied to the valve allows a column of testing solution to form in the tube separated from the normal solution by airbubbles. Another sequence of pulses exposes the cell to the testing solution for the desired period of time by rapid displacement of the column along the tube. The electrical recording is unharmed since there is a thin layer of saline between the airbubbles and the walls of the tube. An invagination in the tip of the micropipette prevents the cell from damage. B: ATP-activated inward current (ordinate) elicited by the rapid but prolonged application of ATP in concentrations of 10-6 M (upper trace), 10-5 M (middle trace) and 10 -4 M (lowes trace). Holding potential 90 mV. C: inward current (ordinate) elicited by a short ('square pulse') application of 5 × 10 - 6 M ATP. The times at which ATP was applied and washed out are indicated by arrows. Holding potential - 9 0 mV.
demonstrated
i n Fig. l B . T h e d e c l i n e o f c u r r e n t r e f l e c t s t h e d e s e n s i t i z a t i o n o f t h e
receptor, the rate of which considerably increased with the increase in ATP concentration from 10-7 to 10-5 M and with the negative shift of the holding potential ( - 90 t o - 120 m V ) . A t a h o l d i n g p o t e n t i a l o f - 9 0 m V a n d a n A T P c o n c e n t r a t i o n o f 1 0 - 5 M t h e t i m e c o n s t a n t o f d e s e n s i t i z a t i o n w a s a b o u t 2.5 s. D e s e n s i t i z a t i o n w a s c o m p l e t e i n a b o u t 8 s. Its r e m o v a l w a s v e r y s l o w a n d l a s t e d f o r 5 - 1 5 m i n d e p e n d i n g on the ATP
concentration.
The latter property
made
it n e c e s s a r y t o a p p l y t h e
'square pulse' technique in order to obtain reliable information agonist dependence of peak ATP-activated
on the voltage and
c u r r e n t (Fig. 1C).
The dose-response r e l a t i o n s h i p f o r t h e p e a k s o f A T P - a c t i v a t e d c u r r e n t , as d e m o n s t r a t e d i n t h e H i l l p l o t o f Fig. 2 A , s u g g e s t s t h a t e a c h r e c e p t o r is a c t i v a t e d b y a single ATP molecule. The
Ka f o r t h e A T P - a c t i v a t e d c u r r e n t is 5 × 1 0 - 6 M . W h e n
43
A 19
B I/Imox.
o-
-100
0 (mV)
- mox.
2.
-50
i
Y
-5"
-10-
-15. -1.
-2.
I
I
-7
-6
•
I
-S
I
-4 19 [ATP]x~,"
-20-
I m (nA)
Fig. 2. A: the 'dose-response' relationship for the peaks of ATP-activated current (Hill plot). Unity slope is marked by a solid line. Dissociation constant Kd = 5 X 10- 6 M is indicated with a triangle on abscissa. Holding potential - 9 0 mV. B: voltage-dependence of the peaks of ATP-activated current. ATP (5 x 10-6 M) was applied in normal saline (circles) and in an external solution in which Na was substituted with TEA (triangles). Solid lines were drawn by eye to fit the data.
a n e u r o n e was i d e n t i f i e d to be A T P - s e n s i t i v e the f o l l o w i n g substances were a p p l i e d at c o n c e n t r a t i o n s b e t w e e n 5 x 1 0 - 4 a n d 1 0 - 2 M: A D P , A M P , c A M P , a d e n o s i n e , a d e n i n e a n d ~ , 3 , - m e t h y l e n e - A T P . O n l y A D P a n d ~ , ~ , - m e t h y l e n e - A T P were f o u n d to be w e a k a g o n i s t s o f the A T P r e c e p t o r (Ka = 5 x 1 0 - 4 for A D P ) . T h e rate o f desensitization, h o w e v e r , was m u c h slower w h e n / 3 , ~ - m e t h y l e n e - A T P was a p p l i e d . A p a r t f r o m A D P a n d ~ , 3 , - m e t h y l e n e - A T P n o n e o f the tested substances a f f e c t e d the A T P sensitive receptors. In a d d i t i o n , o u a b a i n in c o n c e n t r a t i o n s as high a s 10 - 4 M did n o t a p p e a r to influence the r e c e p t o r response. T h e v o l t a g e d e p e n d e n c e o f A T P - i n d u c e d c u r r e n t was n o n - l i n e a r , d e m o n s t r a t i n g i n w a r d - g o i n g r e c t i f i c a t i o n (Fig. 2B). T h e i n w a r d c u r r e n t was s t r o n g l y r e d u c e d b u t d i d n o t d i s a p p e a r when e x t e r n a l N a was s u b s t i t u t e d with Tris, choline, a c e t y l c h o l i n e a n d T E A (see Fig. 2B). L a r g e r m o n o v a l e n t cations like t e t r a e t h y l b e n z y l a m m o n i u m ( T E B A ) failed to c a r r y the c u r r e n t t h r o u g h these channels. S u b s t i t u t i o n o f o n l y 50°7o o f the external N a b y T E B A d i d n o t a b o l i s h the current, i n d i c a t i n g that T E B A does not affect the r e c e p t o r . A p e r m e a b i l i t y to d i v a l e n t cations was n o t detected when the c o n c e n t r a t i o n o f C a or M g were increased to 10 m M in the external s o l u t i o n c o n t a i n i n g T E B A . H o w e v e r , 10 m M o f N a or K, when a d d e d to this solution, c a r r i e d m e a s u r a b l e i n w a r d currents with similar a m p l i t u d e s . S u b s t i t u t i o n o f internal f l u o r i d e with larger a n i o n s , as well as s u b s t i t u t i o n o f ext e r n a l NaC1 with N a - H E P E S , d i d n o t affect the A T P - a c t i v a t e d current. It m a y be
44 concluded that the channels linked to the A T P receptors allow monovalent cations to pass with low selectivity. Strong inward-going rectification of these channels produces depolarization of the cell membrane in a wide range of negative membrane voltages. Purinergic receptors have been described in the autonomic nervous system and in a number of other tissues [1, 2]. Two types of receptors have been distinguished, namely Pt and P2 [1]. In our experiments the blockers of these receptors, apamin [2, 7] (5 × 10- 6 M) and theophylline [2] (10- 3 M) were ineffective. The action of quinidine and phentolamine, known to block the P2 receptors [2], was non-specific: these substances, when applied in concentrations of 10- 4 _ 10- 3 M were more effective in blocking the voltage-activated Na and K channels rather than ATPactivated channels. The sequence of agonists for P~ receptors (adenosine /> A M P > A D P /> ATP) is qualitatively different from the sequence observed in our experiments. Our data fit only partially into the sequence of agonists for P2 receptors (ATP ~> ADP > A M P /> adenosine). While A D P is (100 times) less effective than ATP, A M P and adenosine fail to open the channels. Consequently, at present it appears to be impossible to relate the properties of previously described purinergic receptors to ATPactivated conductance in the sensory neurones. ATP-sensitive neurones have been found in all of the investigated sensory ganglia, independent of the age of the animals. For example, 60 out of 124 trigeminal neurones responded to ATP. The receptors for GABA, protons and ATP appeared to be distributed in all possible combinations in spinal and trigeminal neurones. Cells could be found which demonstrated responses either to one of these substances or to all of them. In the latter case, the desensitization of any of these receptors did not affect the specific sensitivity of the others, proving their independence. It seems relevant to mention that in the vestibular ganglia all the cells tested were only sensitive to A T P and not to GABA or protons. Since the vestibular ganglia are probably monofunctional, it might be suggested that the type of receptors present at sensory neurones depends on the functional role of this neurone (see also ref. 6). It also appears possible that sensory neurones possessing two or more receptors are polymodal. We would propose that the discovered A T P receptors might also be present in the membrane of the primary afferent terminals of the sensory neurones and participate in the presumed purinergic transmission of signals from the primary receptor cells to such terminals. The authors thank Prof. P.G. Kostyuk for his encouragement and support and Prof. Fr.-K. Pierau for valuable comments on the manuscript. I Burnstock, G., A basis for distinguishing two types of purinergic receptors. In R.W. Straub and L. Bolis (Eds.), Cell Membrane Receptors for Drugs and Hormones: A Multidisciplinary Approach, Raven Press, New York, 1978, pp. 107 118.
45 2 Burnstock, G., Neurotransmitters and trophic factors in the autonomic nervous system, J. Physiol. (Lond.), 313 (1981) 1-35. 3 Debroat, W.C., GABA--depolarization of a sensory ganglion: antagonism by picrotoxin and bicuculline, Brain Res., 38 (1972) 429-432. 4 Kostyuk, P.G., Krishtal, O.A. and Pidoplichko, V.I., lntracellular perfusion, J. Neurosci. Meth., 4 (1981) 201-210. 5 Krishtal, O.A. and Pidoplichko, V.I., A receptor for protons in the nerve cell membrane, Neuroscience, 5 (1980) 2325-2327. 6 Krishtal, O.A. and Pidoplichko, V.|., A 'receptor' for protons in small neurons of trigeminal ganglia: possible role in nociception, Neurosci. Lett., 24 (1981) 243-246. 7 Vladimirova, I.A. and Shuba, M.F., The effect of strychnine, hydrastin and apamin on synaptic transmission in smooth muscle cells, Neurophysiology, 10 (1978) 295-299.
41
Elsevier Scientific Publishers Ireland Ltd.
R E C E P T O R FOR A T P IN T H E M E M B R A N E OF M A M M A L I A N S E N S O R Y NEURONES
O.A. KRISHTAL, S.M. MARCHENKO and V.I. P I D O P L I C H K O
A.A. Bogomoletz Institute o f Physiology, Ukranian Academy o f Sciences, Bogomoletz str. 4, Kiev 24, 252601 GSP (U.S.S.R.). (Received November 22nd, 1982; Revised version received and accepted December 8th, 1982)
Key words: sensory neurons - ATP - receptors - membrane conductance - intracellular perfusion desensitization
ATP-activated conductance has been found in a large number of neurones isolated from various sensory ganglia of the rat and cat. The inward current produced by ATP (Ka = 5.10 -6 M) is carried by cations and demonstrates rapid activation and slow desensitization. The sequence of agonists ( A T P > A D P with AMP and adenosine ineffective) is different from those previously described for purinergic receptors P~ and P2.
Two types of conductance-activating chemoreceptors have been found so far in the m e m b r a n e of m a m m a l i a n sensory neurones: the receptors for G A B A [3] and for protons [5]. The present paper demonstrates an ATP-activated conductance in a variety of rat sensory neurones. Experiments were mostly performed on neurones isolated from nodose, vestibular, trigeminal and spinal ganglia obtained from 4-60-day-old rats. Some experiments were performed on the neurones from the same ganglia of 5-10-day-old cats. The neurones were enzymatically isolated [4] and investigated with the intracellular perfusion and voltage clamp technique. The set-up for rapid application of external solutions [5] was modified to enable their equally rapid removal, with each change taking less than 50 ms ('square pulse' application technique, Fig. 1A). The normal external solution contained 152 mM NaC1, 2.2 m M CaCI2, 1.1. mM MgC12, 10 m M H E P E S neutralized by N a O H to p H 7.4. The internal solution contained 130 m M KF and 30 mM Tris F (pH 7.2). Experiments were performed at r o o m temperature at about 22°C. Many sensory neurones clamped at a constant holding potential ( - 9 0 mV) responded with an inward current to the application of A T P added to the normal external saline. These responses were similar when recorded from rat or cat neurones and so the further investigation was carried out on the rat neurones, with only a few control experiments on the neurones from cat. The ATP-activated current elicited by prolonged application of A T P in different concentrations is 0304-3940/83/0000-0000/$ 03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd.
42
A
B ,L
C .....
. . . . .
2 nA
!
!
200ms
-3 1 nA
Fig. 1. A: the 'square pulse' application technique. The tip of the micropipette with the cell (a) is inserted into a plastic tube. The lower end of this tube can be exposed to different external solutions or to air. The suction applied to the upper end of the tube (indicated by arrow) is controlled by an electromagnetic valve (b). A preprogrammed sequence of current pulses applied to the valve allows a column of testing solution to form in the tube separated from the normal solution by airbubbles. Another sequence of pulses exposes the cell to the testing solution for the desired period of time by rapid displacement of the column along the tube. The electrical recording is unharmed since there is a thin layer of saline between the airbubbles and the walls of the tube. An invagination in the tip of the micropipette prevents the cell from damage. B: ATP-activated inward current (ordinate) elicited by the rapid but prolonged application of ATP in concentrations of 10-6 M (upper trace), 10-5 M (middle trace) and 10 -4 M (lowes trace). Holding potential 90 mV. C: inward current (ordinate) elicited by a short ('square pulse') application of 5 × 10 - 6 M ATP. The times at which ATP was applied and washed out are indicated by arrows. Holding potential - 9 0 mV.
demonstrated
i n Fig. l B . T h e d e c l i n e o f c u r r e n t r e f l e c t s t h e d e s e n s i t i z a t i o n o f t h e
receptor, the rate of which considerably increased with the increase in ATP concentration from 10-7 to 10-5 M and with the negative shift of the holding potential ( - 90 t o - 120 m V ) . A t a h o l d i n g p o t e n t i a l o f - 9 0 m V a n d a n A T P c o n c e n t r a t i o n o f 1 0 - 5 M t h e t i m e c o n s t a n t o f d e s e n s i t i z a t i o n w a s a b o u t 2.5 s. D e s e n s i t i z a t i o n w a s c o m p l e t e i n a b o u t 8 s. Its r e m o v a l w a s v e r y s l o w a n d l a s t e d f o r 5 - 1 5 m i n d e p e n d i n g on the ATP
concentration.
The latter property
made
it n e c e s s a r y t o a p p l y t h e
'square pulse' technique in order to obtain reliable information agonist dependence of peak ATP-activated
on the voltage and
c u r r e n t (Fig. 1C).
The dose-response r e l a t i o n s h i p f o r t h e p e a k s o f A T P - a c t i v a t e d c u r r e n t , as d e m o n s t r a t e d i n t h e H i l l p l o t o f Fig. 2 A , s u g g e s t s t h a t e a c h r e c e p t o r is a c t i v a t e d b y a single ATP molecule. The
Ka f o r t h e A T P - a c t i v a t e d c u r r e n t is 5 × 1 0 - 6 M . W h e n
43
A 19
B I/Imox.
o-
-100
0 (mV)
- mox.
2.
-50
i
Y
-5"
-10-
-15. -1.
-2.
I
I
-7
-6
•
I
-S
I
-4 19 [ATP]x~,"
-20-
I m (nA)
Fig. 2. A: the 'dose-response' relationship for the peaks of ATP-activated current (Hill plot). Unity slope is marked by a solid line. Dissociation constant Kd = 5 X 10- 6 M is indicated with a triangle on abscissa. Holding potential - 9 0 mV. B: voltage-dependence of the peaks of ATP-activated current. ATP (5 x 10-6 M) was applied in normal saline (circles) and in an external solution in which Na was substituted with TEA (triangles). Solid lines were drawn by eye to fit the data.
a n e u r o n e was i d e n t i f i e d to be A T P - s e n s i t i v e the f o l l o w i n g substances were a p p l i e d at c o n c e n t r a t i o n s b e t w e e n 5 x 1 0 - 4 a n d 1 0 - 2 M: A D P , A M P , c A M P , a d e n o s i n e , a d e n i n e a n d ~ , 3 , - m e t h y l e n e - A T P . O n l y A D P a n d ~ , ~ , - m e t h y l e n e - A T P were f o u n d to be w e a k a g o n i s t s o f the A T P r e c e p t o r (Ka = 5 x 1 0 - 4 for A D P ) . T h e rate o f desensitization, h o w e v e r , was m u c h slower w h e n / 3 , ~ - m e t h y l e n e - A T P was a p p l i e d . A p a r t f r o m A D P a n d ~ , 3 , - m e t h y l e n e - A T P n o n e o f the tested substances a f f e c t e d the A T P sensitive receptors. In a d d i t i o n , o u a b a i n in c o n c e n t r a t i o n s as high a s 10 - 4 M did n o t a p p e a r to influence the r e c e p t o r response. T h e v o l t a g e d e p e n d e n c e o f A T P - i n d u c e d c u r r e n t was n o n - l i n e a r , d e m o n s t r a t i n g i n w a r d - g o i n g r e c t i f i c a t i o n (Fig. 2B). T h e i n w a r d c u r r e n t was s t r o n g l y r e d u c e d b u t d i d n o t d i s a p p e a r when e x t e r n a l N a was s u b s t i t u t e d with Tris, choline, a c e t y l c h o l i n e a n d T E A (see Fig. 2B). L a r g e r m o n o v a l e n t cations like t e t r a e t h y l b e n z y l a m m o n i u m ( T E B A ) failed to c a r r y the c u r r e n t t h r o u g h these channels. S u b s t i t u t i o n o f o n l y 50°7o o f the external N a b y T E B A d i d n o t a b o l i s h the current, i n d i c a t i n g that T E B A does not affect the r e c e p t o r . A p e r m e a b i l i t y to d i v a l e n t cations was n o t detected when the c o n c e n t r a t i o n o f C a or M g were increased to 10 m M in the external s o l u t i o n c o n t a i n i n g T E B A . H o w e v e r , 10 m M o f N a or K, when a d d e d to this solution, c a r r i e d m e a s u r a b l e i n w a r d currents with similar a m p l i t u d e s . S u b s t i t u t i o n o f internal f l u o r i d e with larger a n i o n s , as well as s u b s t i t u t i o n o f ext e r n a l NaC1 with N a - H E P E S , d i d n o t affect the A T P - a c t i v a t e d current. It m a y be
44 concluded that the channels linked to the A T P receptors allow monovalent cations to pass with low selectivity. Strong inward-going rectification of these channels produces depolarization of the cell membrane in a wide range of negative membrane voltages. Purinergic receptors have been described in the autonomic nervous system and in a number of other tissues [1, 2]. Two types of receptors have been distinguished, namely Pt and P2 [1]. In our experiments the blockers of these receptors, apamin [2, 7] (5 × 10- 6 M) and theophylline [2] (10- 3 M) were ineffective. The action of quinidine and phentolamine, known to block the P2 receptors [2], was non-specific: these substances, when applied in concentrations of 10- 4 _ 10- 3 M were more effective in blocking the voltage-activated Na and K channels rather than ATPactivated channels. The sequence of agonists for P~ receptors (adenosine /> A M P > A D P /> ATP) is qualitatively different from the sequence observed in our experiments. Our data fit only partially into the sequence of agonists for P2 receptors (ATP ~> ADP > A M P /> adenosine). While A D P is (100 times) less effective than ATP, A M P and adenosine fail to open the channels. Consequently, at present it appears to be impossible to relate the properties of previously described purinergic receptors to ATPactivated conductance in the sensory neurones. ATP-sensitive neurones have been found in all of the investigated sensory ganglia, independent of the age of the animals. For example, 60 out of 124 trigeminal neurones responded to ATP. The receptors for GABA, protons and ATP appeared to be distributed in all possible combinations in spinal and trigeminal neurones. Cells could be found which demonstrated responses either to one of these substances or to all of them. In the latter case, the desensitization of any of these receptors did not affect the specific sensitivity of the others, proving their independence. It seems relevant to mention that in the vestibular ganglia all the cells tested were only sensitive to A T P and not to GABA or protons. Since the vestibular ganglia are probably monofunctional, it might be suggested that the type of receptors present at sensory neurones depends on the functional role of this neurone (see also ref. 6). It also appears possible that sensory neurones possessing two or more receptors are polymodal. We would propose that the discovered A T P receptors might also be present in the membrane of the primary afferent terminals of the sensory neurones and participate in the presumed purinergic transmission of signals from the primary receptor cells to such terminals. The authors thank Prof. P.G. Kostyuk for his encouragement and support and Prof. Fr.-K. Pierau for valuable comments on the manuscript. I Burnstock, G., A basis for distinguishing two types of purinergic receptors. In R.W. Straub and L. Bolis (Eds.), Cell Membrane Receptors for Drugs and Hormones: A Multidisciplinary Approach, Raven Press, New York, 1978, pp. 107 118.
45 2 Burnstock, G., Neurotransmitters and trophic factors in the autonomic nervous system, J. Physiol. (Lond.), 313 (1981) 1-35. 3 Debroat, W.C., GABA--depolarization of a sensory ganglion: antagonism by picrotoxin and bicuculline, Brain Res., 38 (1972) 429-432. 4 Kostyuk, P.G., Krishtal, O.A. and Pidoplichko, V.I., lntracellular perfusion, J. Neurosci. Meth., 4 (1981) 201-210. 5 Krishtal, O.A. and Pidoplichko, V.I., A receptor for protons in the nerve cell membrane, Neuroscience, 5 (1980) 2325-2327. 6 Krishtal, O.A. and Pidoplichko, V.|., A 'receptor' for protons in small neurons of trigeminal ganglia: possible role in nociception, Neurosci. Lett., 24 (1981) 243-246. 7 Vladimirova, I.A. and Shuba, M.F., The effect of strychnine, hydrastin and apamin on synaptic transmission in smooth muscle cells, Neurophysiology, 10 (1978) 295-299.