Prostaglandins block a Ca2+-dependent slow spike afterhyperpolarization independent of effects on Ca2+ influx in visceral afferent neurons

Brain Research, 345 (1985) 345-349 Elsevier

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Prostaglandins block a Ca2+-dependent slow spike afterhyperpolarization independent of effects on Ca 2+ influx in visceral afferent neurons J. C. FOWLER, W. F. WONDERLIN and D. WEINREICH

Department of Pharmacology and Experimental Therapeutics, Universitv of Maryland School of Medicine, Baltimore, MD 21201 (U.S.A.) (Accepted May 21st, 1985)

Key words: nodose ganglia - - prostaglandin - - Ca2+-activated K+-dependent afterhyperpolarization - Ca2*-dependent spike --Ca 2* ionophore

The blockade of a slow Ca2+-activated K+-dependent afterhyperpolarization (AHP 0 in rabbit visceral sensory neurons by the prostaglandins, PGEj and PGD 2, was investigated to determine whether the blockade was indirectly due to a reduction in Ca 2+ influx. The prostaglandins (PGs) could block the AHP s in the absence of any change in Ca2+-dependent spikes elicited in the presence of tetrodotoxin and tetraethylammonium bromide. A PG-induced decrease in Ca2+-dependent spike width observed in some neurons was temporally dissociated from the PG-induced block of the AHP~. In addition, a slow afterhyperpolarization produced by the application of the Ca 2~ ionophore, A23187, was blocked by the PGs. It is concluded that a reduction in Ca 2+ influx is not responsible for the PG-induced blockade of the AHP~.

Prostaglandins (PGs) play an i m p o r t a n t n e u r o m o dulatory role in both the m o t o r and sensory limbs of the autonomic nervous system. PGs of the E series (El and E2) facilitate basal and stimulated release of acetylcholine ( A C h ) from p a r a s y m p a t h e t i c nerve terminalslS, =. On the o t h e r hand, P G E 1 inhibits A C h release from sympathetic preganglionic nerve endings and n o r e p i n e p h r i n e release from postganglionic endings 2,12. In the sensory limb, E series PGs activate and/or sensitize primary visceral afferents 5,6. The diverse effects of PGs on autonomic cholinergic and noradrenergic synaptic function a p p e a r related to their control of calcium homeostasis at autonomic nerve terminals 12,13. H o w PGs modify visceral afferent excitability remains largely unexplained. We have r e p o r t e d that PGE~ selectively abolishes a slow spike afterhyperpolarization ( A H P 0 in a subpopulation of C-type neurons in the n o d o s e ganglion of the rabbit s-9. This long-lasting AHPs, which persists for many seconds after a single spike, is p r o d u c e d by a Ca2+-dependent increase in K + conductance 8.9,14, and its presence in vagal afferents provides a mecha-

nism for influencing afferent neuronal excitability (ref. 14 and unpublished observations). A striking feature of P G E I is its ability to block the AHPs without affecting a Ca2+-sensitive fast A H P (AHPt) present in a s u b p o p u l a t i o n of C-type nodose neuronsS,9. This result is consistent with a PG-induced blockade of the Ca2+-sensitive AHPs in some manner other than by a reduction in Ca 2+ influx during an action potential. In the present series of experiments we e m p l o y e d ' C a 2+ spikes' (i.e. action potentials evoked in t e t r o d o t o x i n - t e t r a e t h y l a m m o n i u m bromide ( T I ' X - T E A ) - t r e a t e d p r e p a r a t i o n s ) and exogenous application of the Ca2+ i o n o p h o r e (A23187) to examine m o r e closely the relation between Ca 2+ influx and P G - i n d u c e d b l o c k a d e of the Ca2+-sensi tive A H P s. W e included P G D 2 in our study because degranulation of mast cells, which has been implicated in the stimulation of visceral afferents m,21, provides an abundant source of this P G ~7,20. O u r results show that low concentrations of P G E L (1.5/~M) or PGD2 (30 nM) reduce the AHP~ i n d e p e n d e n t l y of changes in Ca2+ influx during the action potential.

Correspondence: D. Weinreich, Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A.

346 Nodose ganglia were dissected from sodium pentobarbital-anesthetized New Zealand rabbits (2-2.5 kg), desheathed and pinned to the floor of a 100-kd recording chamber. Standard intraceitular recording was performed with glass micropipettes filled with 1 M KAc, D C resistance ranging from 40 to t00 Mff2. Responses were recorded on FM tape (frequency response, D C to 1250 Hz) for later recall and analysis on a P D P 11/23 computer and for display on a H-P 7470 X-Y plotter. The Locke solution was composed of (mM): NaC1, 136; KC1, 5.6; MgC1, 1.2; CaC12, 2.2; NaH3PO 4, 1.2; NaHCO3, 14.3; and dextrose, 11; equilibrated with 95% 02/5% CO2 throughout the experiment (pH 7.2-7.4). T E A (5 mM), T T X (1 ~M) and NiCI (1 mM) were added to the Locke solution. The prostaglandins, P G D 2 and PGE1, were dissolved in ethanol at 1 mg/ml (3 mM) then diluted daily in fresh Locke solution to a final concentration ranging from 0.5 nM to 1.5/~M. Ethanol, at concentrations as high as 10/A/ml, did not reduce the slow afterhyperpolarization or the Ca 2+ spike. All chemicals were purchased from Sigma (St. Louis, MO). Pressure ejection of the Ca 2+ ionophore A23187 was accomplished with a pressure ejection system similar to that described by Choi and Fischback 4. The ionophore was dissolved in chloroform (2.5 mg/ml) and diluted with Locke solution to a final concentration of 10 ktM. The ionophore was pressure applied (0,5-3 s duration) from glass capillary pipettes with tip diameters of about 10-20 ktm. The same amount of chloroform diluted in Locke solution did not produce membrane hyperpolarizations. In normal Locke solution, both P G E 1 and P G D 2 reversibly reduced the amplitude of the AHP~ in a dose-dependent fashion. P G D 2 was more potent than PGE1, having threshold effects at concentrations ranging from 0.5 to 1 nM; detectable depression with P G E 1 occurred around 30 nM. Bath application of 30 nM P G D 2 or 1.5 ~ M P G E 1 resulted in 80% or greater depression of the A H P amplitudes (n = 25). In the presence of T T X and T E A the overshooting action potentials recorded from many C-type nodose neurons are Ca2+-dependent and reflect the state of Ca2+ influxlS,16. In the presence of 1/~M T F X and 5 mM T E A P G D 2 and P G E 1 exhibited qualitatively similar blocking effects on the A H P s. The A H P s amplitude was reduced 94 + 6.5% (S.E.M.; n = 4) by

1.5 ktM P G E 1 and 62 + 7.7% (S.E.M., n = 4) by 30 nM P G D 2. While the reduction of the A H P s by the PGs was consistent for a given concentration, their effects on the CaZ+-dependent spikes were quite variable. In some cells (see Fig. 1), there was no measurable change in the spike width, while in others the spike width (measured at half-maximum amplitude) decreased transiently (Fig. 2). In 9 cells the correlation coefficient between magnitude of AHP~ reduction and change in the Ca2+-dependent spike width was 0.427, which was not statistically significant (P > 0.2). In the neuron illustrated in Fig. 1, addition of Ni 2+ ions to the T F X - T E A solution resulted in complete abolishment of the AHP~ and an accompanying reduction in spike width. The reduction of spike width may signal a diminution of the inward Ca -,+ current because this divalent metal is known to block calcium channels 1. The remaining active current may reflect incomplete block of the Ca 2+ current or TTXresistant Na + current present in some sensory neurons 3. L

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Fig. 1. PG-induced blockade of the AHP~ without a reduction in the Ca2+-dependent spike. A: the CaZ+-dependent spike and the associated AHP~. The spike in TTX (t /*M) and TEA (5 mM) was elicited by a brief 3-ms current pulse. B: upon the addition of PGD z (30 nM) the AHP~ amplitude was greatly reduced 57% while spike width was slightly increasedby 6%. C: when Ni2+ (1 mM) was added in the presence of PGD 2 the TTX-TEA spike was substantially reduced. The residual AHP~ was also blocked. The remaining active response required a longer duration current pulse (5 ms). Transient interruption in bottom right-hand trace is a tape recorder artifact. Sampling frequency was 2 kHz for the spike and 100 Hz for the AHP<.

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the spike. W e further dissociated the P G - i n d u c e d blockade of the Ca2+-activated K + conductance from any effect on Ca 2+ influx by using the Ca 2+ i o n o p h o r e A23187 rather than an action potential to trigger Ca 2+ influx. Bath application of this Ca 2+ i o n o p h o r e

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Fig. 2. Time course of block of the AHP s and reduction in Ca 2+dependent spike width by PGE 1 (1.5 #M). Data are presented as percent of control (predrug values). Although the onset of the effects on the AHP S and the TTX-TEA spike followed a similar time course, the reduction in spike width recovered much more quickly than the AHP s with wash in normal Locke. In 3 of 4 experiments, the AHP remained completely blocked after the spike width had completely recovered, demonstrating a temporal dissociation of the effects on the AHP~ and the Ca2+-dependent spike width.

Since PGs could reduce the width of the Ca2+-de p e n d e n t spike and, p r e s u m a b l y , Ca 2+ influx during the spike in some neurons, we e x a m i n e d further the nature of the relation b e t w e e n Ca2+-dependent spike and the A H P s. In 5 cells in which PGs completely abolished the A H P S, the Ca2+-dependent spike width was reduced. H o w e v e r , c o m p l e t e recovery of spike width occurred rapidly upon r e m o v a l of PGs, well before the A H P s r e t u r n e d to control values. In Fig. 2, the A H P amplitude and c o r r e s p o n d i n g spike width at half maximal amplitude are plotted before, during and after b a t h - a p p l i e d P G E 1. In two additional e x p e r i m e n t s we assessed the effects of Locke solutions containing one-half normal Ca 2+ on the relationship between the Ca 2+ spike and the AHP~ prior to the introduction of PGs. R e d u c t i o n of the Ca 2+ spike width by 6% resulted in about a 31% decrease in the AHP~. A f t e r returning to normal Ca 2+, addition of P G E 1 (1.5 a M ) completely abolished the AHP~ and r e d u c e d the Ca 2+ spike width 4%. Similar results were o b s e r v e d with P G D 2. Thus, the reduction in the A H P by PGs cannot be at-

rizing response that was similar in magnitude, duration, conductance increase and reversal potential to the A H P s. A m o n g different nodose neurons, the distribution of the hyperpolarizing response to the ionophore exactly paralleled the distribution of the A H P s. Twenty-six of 26 neurons with a d e m o n s t r a b l e A H P s revealed a slow hyperpolarizing response to the i o n o p h o r e , while 6 of 6 cells lacking a m e a s u r a b l e A H P s also lacked the hyperpolarizing response to the ionophore. In one cell where the A H P s spontaneously d i s a p p e a r e d the hyperpolarizing response simultaneously disappeared. Thus, puffer application of the Ca 2+ i o n o p h o r e provides an alternate m e t h o d of activation of the Ca2+-dependent K + conductance mechanism underlying the A H P s without requiring Ca 2+ influx through voltage-gated Ca 2+ channels. PGD2 (30 nM) was equally effective in reducing the A H P s and the hyperpolarizing response to the Ca 2+ i o n o p h o r e (n = 6). A n example of the responsiveness of the i o n o p h o r e - i n d u c e d P G D 2 b l o c k a d e is illustrated in Fig. 3. Fig. 3 shows the A H P s following 4 spikes and the hyperpolarizing response in the same cell to a 1-s puffer application of the i o n o p h o r e . Following superfusion with P G D 2 (30 nM) both the AHP~ and the hyperpolarizing response to Ca 2+ ionophore are blocked (Fig. 3B). Both responses recovered with wash (Fig. 3C) to similar extents. The present study demonstrates that PGs ( P G E 1 and PGD2) block the AHP~ in nodose ganglion C-type neurons i n d e p e n d e n t l y of effects on Ca 2+ influx. This conclusion is based on 5 observations: (1) the Ca2+-dependent A H P f (present in some nodose neurons) that precedes the AHP~ is not affected by PGs at concentrations that abolish the AHP~S,9; (2) PGs can, in some cells, block the A H P s without affecting the T T X - T E A spike duration; (3) the PG-induced changes of the Ca2+-dependent spike, when observed, can be t e m p o r a l l y dissociated from the

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(5) similar concentrations of PGs block the Ca2+-ion ophore-induced slow hyperpolarization u n d e r conditions which would be expected to provide constant Ca 2+ influx. The mechanism of P G - i n d u c e d blockade of the Ca2+-activated K+ conductance remains unex-

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plained. Our data indicate that PGs act at a step subsequent to the influx of Ca 2+ during the spike. Thus. PGs could directlv block a population of K ÷ channels distinct from those associated with the Ca2+-dependent AHPfS, 9, or they might interfere with an intracel-

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lular Ca2+-dependent process. Histamine 11 and norepinephrine 18 both reduce a slow Ca2÷-activated K conductance in hippocampal CA1 neurons without producing changes in the T T X - T E A spikes. These amine-induced effects have been attributed to increased cyclic A M P because stimulation of adenylate

Fig 3. Blockade of the AHPs and the Ca2+-ionophore-induced hyperpolarization. A: the AHPs following 4 spikes (left set of records) and the hyperpolarization produced by a 1-s puffer application of the Ca2+ ionophore (right set of records). B: following application of PGD 2 (30 nM), both the AHPs and the ionophore response were blocked. C: both responses recovered following wash-out of the PGD2. Pre-stimulus membrane potential was -50 mV and is depicted by horizontal line. Sampling frequency was 50 Hz and the signal was filtered at 25 Hz. Downward deflections are electrotonic voltage transients produced by 100 pA constant-current pulses. The increase in membrane conductance during the AHP s or the ionophore-induced response are not due to membrane rectification because polarizmg the membrane to similar values did not result in changes of conductance.

cyclase by forskolin or application of exogenous cyclic A M P reduces the A H P S in CA1 n e u r o n s TM. We suspect that PGs block the A H P s in nodose C-fiber neurons in a m a n n e r analogous to the effects of biogenic amines in hippocampal neurons, namely, by raising intracellular cyclic AMP. PGs of the E series elevate neuronal cyclic A M P 21. and exogenous application of forskolin blocks the AHPs in rabbit nodose neurons (unpublished). The authors wish to thank Drs. Terry Pellmar and Terry Sejnowski for their constructive comments on an earlier draft of this manuscript and Ms. E. Elizabeth for her excellent typing. We also thank the Department of Pharmacology, University of Maryland

block of the AHPs; (4) the P G - i n d u c e d reduction in spike width ts associated with a greater reduction of the A H P s than is associated with a c o m p a r a b l e reduction in spike width due to decreased Ca 2÷ influx: and

ing the rabbits used in this study. This work was supported by a N.I.H. G r a n t (NS-22069) to D . W . and a

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Dental School. and A n a q u e s t - B O C group for supply-

predoctoral training award (ES-07094) to W.F.W.

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