Using amperometric techniques, electrochemical events associated with vesicular transmitter

release were recorded from isolated rat type I carotid body cells when exposed to a solution

containing 50mm K+. Events were enhanced in amplitude by preloading cells with the

catecholamine precursor, L-b-3,4-dihydroxyphenylalanine (L-DOPA). K+-evoked secretion was

abolished by the non-selective Ca2, channel blocker Cd2+ (100 ,/M) and markedly reduced by the

L-type Ca2+ channel blocker nifedipine (5 µM). Our results indicate that secretion from isolated rat

type I cells can be monitored electrochemically and we demonstrate a major role for L-type Ca2+

channels in mediating K+-evoked secretion

Hatton, C.J.

Peers, C.

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Electrochemical detection of K+-evoked quantal secretory events from isolated rat type I carotid body cells

Experimental Physiology (1997), 82, 415-418Printed in Great BritainELECTROCHEMICAL DETECTION OF K+-EVOKEDQUANTAL SECRETORY EVENTS FROM ISOLATEDRAT TYPE I CAROTID BODY CELLSC. J. HATTON AND C. PEERS*Institute for Cardiovascular Research, Leeds University, Leeds L52 9JT, UK(MANUSCRIPT RECEIVED 14 NOVEMBER 1996, ACCEPTED 16 DECEMBER 1996)SUMMARYUsing amperometric techniques, electrochemical events associated with vesicular transmitterrelease were recorded from isolated rat type I carotid body cells when exposed to a solutioncontaining 50mm K+. Events were enhanced in amplitude by preloading cells with thecatecholamine precursor, L-,8-3,4-dihydroxyphenylalanine (L-DOPA). K+-evoked secretion wasabolished by the non-selective Ca2, channel blocker Cd2+ (100 ,/M) and markedly reduced by theL-type Ca2+ channel blocker nifedipine (5 #UM). Our results indicate that secretion from isolated rattype I cells can be monitored electrochemically and we demonstrate a major role for L-type Ca2+channels in mediating K+-evoked secretion.INTRODUCTIONExcitation of the carotid body involves stimulus-evoked release of transmitters (predominantlydopamine) from type I cells (Gonzalez, Almaraz, Obeso & Rigual, 1994). Transmitter releasedepends on a rise of cytosolic [Ca2+] and most evidence suggests this rise is due to Ca2+ influxvia voltage-gated Ca2+ channels (for discussion see Gonzalez et al. 1994; Peers & Buckler,1995). Most studies to date have monitored transmitter release from intact carotid bodies, butrecent advances in electrochemical detection methods (Chow & Von Ruden, 1995) haveallowed detection of quantal events arising from release of transmitters from individualvesicles in isolated cells. This technique has recently been successfully applied to rabbit type Icells (Urefia, Fernandez-Chac6n, Benot, Alvarez de Toledo & L6pez-Barneo, 1994) but not tocells of another commonly used species, the rat. Potentially important differences existbetween the biophysical properties of rabbit and rat type I cells (Peers & Buckler, 1995). Wehave therefore investigated whether neurosecretion can be monitored from individual rattype I cells and examined the importance of L-type Ca2+ channels in mediating K+-evokedneurosecretion.METHODSCarotid bodies were removed from 10-day-old rats anaesthetized by inhalation of 5 % halothane-95 % 02through a face mask. Donor animals were killed by decapitation whilst still deeply anaesthetized. Type Icells were isolated by 15-20 min incubation at 37 'C in phosphate-buflered saline ([Ca2+] ca. 50,UM)containing 0-05 % collagenase (Lorne Laboratories Ltd, Twyford, UK) and 0 025 % trypsin (Sigma,Poole, UK), followed by trituration and resuspension in Ham's F-12 culture medium (Gibco, Paisley,UK) containing IO0% fetal calf' serum, insulin (80 U J-), penicillin (100 i.u.ml-') and streptomycin(100 ,ug ml l). Isolated cells were plated onto poly-L-lysine-coated glass coverslips overnight and usedwithin 36 h of isolation. For electrochemical recordings, fragments of coverslip were transferred to aTo whom correspondence should be addressed.RC334C. J. HATTON AND C. PEERSAI- ,,E1, L,i,i,,I.l . It, IIL.. C15 pA20 msBv lum ,,, ,, 11]lhf....nfil 1 .1---1. 1.l1A..-LL. 1. i..II I-..1 ie ,x |. .A4j-~50 pAlOsI 0slLD10 pA20 msFig. 1. Amperometric traces obtained from individual type I cells without (A) or with (B-E, difFerent cells) pre-exposure toL-DOPA-containing solution. All traces obtained at a potential of +800 mV. Arrows in A, B and E indicate time atwhich solution was exchanged for one containing 50 mMi K+ (downward arrow), then back to control solution (upwardarrow). Scale bar applies to all 3 traces. C, example of a 'foot' (arrowed) preceding a secretory event. D, example of a'flickering' event (arrowed) preceding a secretory event. ln E, the cell was initially exposed to 100 /,M Cd2t along with50 mM K+ for the period indicated by the horizontal bar.continuously perfused (1 -2 ml min-1) recording chamber (volume ca. 80 1ul). The perfusate wascomposed of (mM): NaCl, 135; KCl, 5; MgSO,, 1 2; CaCl2, 2.5; Hepes, 5; and glucose, 10 (pH 7-4,21-24 °C). To evoke secretion, the [K+] was raised to 50 mm (Na+ substitution). For most studies, cellswere pre-incubated for 30-60 min at 37 °C in the perfusate containing 1O /LM L-f-3,4-dihydroxy-phenylalanine (L-DOPA), 0.2 mM pargyline and 0.2 mm ascorbic acid to enrich the catecholaminecontent of vesicles. Each carbon fibre microelectrode (proCFE, Axon Instruments, diameter 5 ,um) waspolarized to +800 mV and placed so that its exposed face lightly touched an isolated type I cell.Amperometric current was recorded continuously using an Axopatch 200A amplifier (Axon Instruments),filtered at 1 kHz and digitized at 2 kHz for subsequent analysis (pCLAMP software, Axon Instruments).Spike-like quantal events (rapid, transient upward deflections > 3 times peak-to-peak amplitude ofbaseline noise) were binned every 5 s for menasurement of exocytotic events.RESULTSAll cells examined (n = 39) exhibited spike-like electrochemical events when exposed to50 mM K+-containing solution (e.g. Fig. 1A), as described for numerous other secretory cells(see Chow & Von Ruden, 1995 and references therein). No events were detected when cellswere bathed in control solution, or if the electrode was not polarized. Release was rapid inonset and declined gradually, but not completely, during 60 s exposures to 50 mM K+. Spikeamplitudes were variable, reflecting variations in quantal content and diffusional distancesbetween release sites and the electrode surface, and many events were barely detectable abovebaseline noise (mean (+ S.E.M.) amplitude, 6.8 + 0 8 pA; n = 300 events from 5 cells). Toenhance spike amplitude, cells were pre-incubated with L-DOPA, the precursor forcatecholamine synthesis. Pre-incubation increased average spike amplitude to 13 9 + 0 6 pAE416SECRETION FROM TYPE I CAROTID BODY CELLS30.2 20-010z0 10 20 30 40 50 60Time (s)Fig. 2. Plot of electrochemically detected quantal events recorded continuously for 60 s following exposure of type I cells toa solution containing 50 mm K+. Each point is the mean (± S.E.M.) number of events detected per 5 s following solutionexchange measured in the absence (0, n = 19 cells) and presence (0, n = 7) of 5 uM nifedipine.(1278 spikes from 8 cells, e.g. Fig. 1 B), and the spike events could be well resolved, revealingother exocytotic features such as 'foot'-like (in 9 6 % of events, e.g. Fig. 1 C) and 'flickering'(in 104 % of events, e.g. Fig. 1D) events thought to be associated with the initial eventspreceding full vesicle-plasma membrane fusion (see Chow & Von Ruden, 1995). Neuro-secretion from type I cells in response to high [K+] solutions is believed to involve Ca2, influxthrough voltage-gated Ca2+ channels. To test this, we exposed cells to 50 mM K+ in thepresence of 100 /UM Cd2 , a non-selective Ca2+ channel blocker. In the presence of Cd2 ,50 mM K+ was unable to evoke electrochemical events (n = 4 cells), but when removed, spike-like quantal events were rapidly observed (e.g. Fig. 1 E). Type I cells of the rat carotid bodyare known to possess L-type and other (including N-type) voltage-gated Ca2, channels. Toassess the importance of L-type Ca2, channels mediating K+-evoked neurosecretion, weexposed cells to 5 ,tM nifedipine 20 s before, and during, exposure of cells to 50 mM K+solutions. Figure 2 plots the number of detected quantal events per 5 s evoked by 50 mM K+ inthe absence and presence of 5 /LM nifedipine. Over the 1 min period of application,157 + 27 events (mean + S.E.M., n = 19) were detected in control cells; in the presence ofnifedipine this was reduced by approximately 80 %, to 31 + 15 events (n = 7).DISCUSSIONAlthough neurosecretion from type I cells has long been known to be fundamental to theoverall responses of the carotid body to various stimuli, this has only recently been directlydemonstrated in isolated rabbit type I cells (Urefia et al. 1994). Here we show that such eventsare detectable in rat type I cells, which show numerous biophysical differences compared withrabbit type I cells (Peers & Buckler, 1995). Furthermore, we have shown (although not in thesame single cell) that electrochemical events corresponding to release of electroactivetransmitters from individual vesicles can be enhanced in amplitude following pre-exposure ofcells to the catecholamine precursor L-DOPA.417C. J. HATTON AND C. PEERSSuch treatment allowed resolution of 'foot' and 'flicker' events, believed to be due to leak oftransmitter from a small pore which arises immediately before full fusion of vesicles with theplasma membrane (Chow & Von Ruden, 1995). Thus, type I cells of the rat carotid bodydisplay features associated with secretory events which are common to many neurosecretorycell types thus far studied with single cell amperometric techniques.L-type Ca2+ channels are known to be present in type I carotid body cells, but do notaccount for all voltage-gated Ca2+ channels in these cells: whole-cell patch clamp recordingshave shown that nifedipine maximally inhibits Ca2+ channel currents by ca. 50 %, with markedcell-to-cell variability (Peers, Carpenter, Hatton, Wyatt & Bee, 1996). Similarly, L-typechannel blockade reduced (but never abolished) K+-evoked rises of [Ca2+]i (Buckler &Vaughan-Jones, 1994). These studies, however, did not address the importance of L-typechannels in mediating stimulus-evoked neurosecretion. Here we have shown that K+-evokedtransmitter release was reduced by approximately 80 % following L-type Ca2+ channel blockade,suggesting that Ca2+ influx via these channels is of particular importance for evoking release.Influx via other channels could be of lesser importance if the channels are not located nearrelease sites, or such influx is buffered more effectively.In summary, the present study has demonstrated that vesicular neurosecretion can bedetected in rat type I cells, can be enhanced by pretreatment of cells with L-DOPA, and canbe greatly attenuated by nifedipine, indicating that L-type Ca2+ channels are the major routefor depolarization-mediated neurosecretion in rat type I carotid body cells.This work was supported by The British Heart Foundation. C.J.H. holds a British Heart Foundation PhDStudentship.REFERENCESBUCKLER, K. J. & VAUGHAN-JONES, R. D. (1994). Effects of hypercapnia on membrane potential andintracellular calcium in rat carotid body type I cells. Journal ofPhysiology 478, 157-171.CHOW, R. H. & VON RUDEN, L. (1995). Electrochemical detection of secretion from single cells. In SingleChannel Recording, 2nd edn, ed. SAKMANN, B. & NEHER, E., pp. 245-275. Plenum Press, New York.GONZALEZ, C., ALMARAZ, L., OBESO, A. & RIGUAL, R. (1994). Carotid body chemoreceptors: fromnatural stimuli to sensory discharges. Physiological Reviews 74, 829-898.PEERS, C. & BUCKLER, K. J. (1995). Transduction of chemostimuli by the type I carotid body cell.Journal ofMembrane Biology 144, 1-9.PEERS, C., CARPENTER, E., HATTON, C. J., WYATr, C. N. & BEE, D. (1996). Ca21 channel currents in type Icarotid body cells of normoxic and chronically hypoxic neonatal rats. Brain Research 739, 251-257.URENA, J., FERNANDEZ-CHAC6N, R., BENOT, A. R., ALVAREZ DE TOLEDO, G. & L6PEZ-BARNEO, J. (1994).Hypoxia induces voltage-dependent Ca2' entry and quantal dopamine secretion in carotid body glomuscells. Proceedings of the National Acadamy of Sciences of the USA 91, 10208-1021 1.418

Electrochemical detection of K+-evoked quantal secretory events from isolated rat type I carotid body cells

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