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Journal of Physiology (1990), 422, pp. 321-331 Wtith 4 figures P'rintted( in (reat Britainl

ACTIVATION OF NEUROHYPOPHYSIAL VASOPRESSIN RELEASE BY Ca2+ INFLUX AND INTRACELLULAR Ca2+ ACCUMULATION IN THE RAT By KATSIUEI SHIBUKI* From the Department of Physiology, Jichi Medical School, Minamikawachi-machi. Tochigi-ken 329-04, Japan

(Received 3 April 1989) SUMMARY

1. Isolated rat neurohypophvses were stimulated electrically in an in vitro perifusion system. A Ca2"-sensitive microelectrode was placed in the centre of each neurohypophysis and [Ca2`]o decrease evoked by the stimulation were determined. 2. In neurohypophyses injected with Fura-2 AM (acetoxymethyl ester), increases and decreases in fluorescence excited at 340 and 380 nm, respectively, were evoked by stimulation. The time (ourse of the fluorescence changes was similar to that of [Ca2 ]. decreases, suggesting that the [Ca2+]0 changes mirrored [Ca2+]i increases. 3. Calcium influx into neurosecretory axons and terminals was estimated as the difference in [Ca2+ ] decrease rates immediately before and after train pulse stimulation. 4. Vasopressin release from the neurohypophysis. measured by specific radioimmunoassay, was facilitated by stimulation in parallel wvith a parameter of [Ca2+]o decrease multiplied by Ca2+ influx. 5. The 02 eonsumption rate. estimated as rate of PO decrease in the tissue, was facilitated by stimulation in parallel with [Ca2+]0 decreases. 6. Possible calcium-dependent mechanisms of vasopressin release, and the energydlependent step of the release by Caa2+, are discussed. INTRODUCTION

Neurohypophysial vasopressin release is activated by Ca2+ entry and the amount of hormone release is correlated with the amount of Ca2+ influx during high K+ stimulation (Douglas & Poisner, 1964a, b). During repetitive electrical stimulation of neurosecretorv axons and terminals. vasopressin release from the neurohypophysis is not maintained longer than 10-20 s (Bicknell. Brow,n, Chapman, Haneock & Leng. 1984; Cazalis Davanithi & Nordmann, 1985); this 'fatigue' has been attributed to changes in Ca2+ entry into axons and terminals during stimulation. No direct data showing changes in Ca2+ influx within the 10-20 s of stimnulation, however, are * 'resent address anid address for correspondence: Frontier Research Program oni Brain Mechanisms of Mind and Behaviour. Rikeni, 2-1 Hirosawa, WVako-shi. Saitania-keni. 3.51-01 .Japan. .IS 7,(24

II

I'H Y 422

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At. vSHIBtIK

available at present. In the neurohypophvsis, we have alrea(dy recordle(d 1(Ca2] decreases during electrical stimulation by' Ca2-sensitiNe microelectrodes (iLeng Shibuki & Wav, 1988). Although the [Ca25]0 changes are nlot (lirectly related to Ca2+ flux changes, recording of [Ca2+]0 is quantitative and reproducible in niature. The aim of the present work was to estimate Ca2+ influx changes frotn the [Ca2+]0 changes under simple assumptions and to determine whether changes in vasopressinl release during repetitive stimulation are explained by changes in Ca2+ influx or whether other factors such as rapid metabolic changes (for example. Shibuki. 1989) mnust be taken into account. In the neurohypophysis, transient increases in 02 consumption are evoked by electrical stimulation, and it is suggested that these increases may supply energy for hormone release because the 02 consumption is dependent on extracellular calcium and is not blocked by ouabain (Shibuki, 1989). The 'fatigue' of vasopressin release during electrical stimulation mav be attributed to insufficiencv of energy supply rather than to change in Ca2+ entry. To test this possibility. rapid changes in 02 consumption rate were estimated from J)o decrease rate in the neurohypophysis and were compared with vasopressin release. In presynaptic axon endings, transmitter release is evoke(d by Ca25 entry during each action potential and the release is facilitated during repetitive activation (for example, Kijima & Tanabe. 1988). The frequency facilitation of release is also observed in the neurohypophysis: low frequency stimulation (< 3 Hz) evokes little vasopressin release (for example, Bicknell, 1988). This frequency facilitation mav be attributed to intracellular accumulation of free Ca2+ during repetitive stimulation because it is blocked by a membrane-permeable of Ca2+ chelator, BAPTA (bis(o-aminophenoxy)ethane-N,N,N'.N'-tetraacetic acid) (Kijima & Tanabe. 1988). To explain changes in vasopressin release by changes in Ca25+ dynamics. therefore. not only Ca2+ influx but also accumulation of intracellular free Ca2+ withini neurosecretory axons and terminals must be considered. In the present study. these two parameters were separately estimated from [Ca2+]0 decrease and a simple model based on these two parameters was proposed to explain the complex changes in vasopressin release during stimulation at v-arious different frequencies. METHODS

Preparations Mlale Wistar rats (150-250 g) were used. Each animnal was stunnlle(l and decapitate(l. The neurohypophysis was isolated and perifused with oxygenated medium (in mM: NaCl. 142; KCl, 5; CaCl2, 2; MgCl2, 2; glucose. 10; HEPES, 10; adjusted to pH 7-4 with NaOH) warmed to 37 'C. To evoke various responses, the tissue was stimulated by biphasic train1pulses (intensity of each pulse: +0 5 mA, total width: 2 ms) through a bipolar electrode.

[Ca,2+] measurement Following the methodology of Lenig et al. (1988). [Ca 2 ]0 was measured in the neurohypophysis using a Ca2+-sensitive microelectrode. Briefly. the (a2+ electrode was made from a glass micropipette which was filled at the tip with a solution of a neutral Ca2+ carrier. ETH1001 (Fluka 21192). A second glass micropipette filled with saline was used as a reference electrode. The voltage difference between the pipettes was recorded by a differenitial am)lifier with a low-cut filter (time constant: 100 ms). The output was digitized at 20( ms intervals by an analysing recorder (Yokogawa-Hokushin Denki, model :3655) and stored by a computer (I)ata General. MlW-200) for

ca2+ ACTIVATION OF VASOPRESSIN RELEASE

323

later analysis. A sudden change in calcium concentration from 2 to 1 mM was followed by the recording system with a time constant of 137 + 11 ms (mean ± S.E.M., n = 6). The voltage swing was 8-8-9-4 mV and was close to the value of 9-2 mV expected from the Nernst equation. Fura-2 experiments Initial attempts to load Fura-2 into neurosecretory axons and terminals by incubating the neurohypophysis in medium containing Fura-2 AM1 (acetoxymethyl ester) (Molecular Probes. Eugene, OR. USA) were unsuccessf'ul. probably because the lipophilic derivative of' Fura-2 was bound to the outside of the tissue and did not penetrate into the centre. Accordingly 20-30 pul of' 10 /tM-Fura-2 AM was injected by pressure directly into the tissue through a glass micropipette. I)uring the injection, which lasted for 10-15 min, vehicle was perfused through the tissue while a yellow spot of Fura-2 AM was gradually deposited at the injection site. After incubation in normal medium for 60 min to allow intracellular accumulation of Fura-2, the tissue was placed between two pieces of silver wire which constituted a bipolar stimulating electrode in the chamber of a spectrophotometer (Hitachi, type 100-10) and continuously perfused with oxygenated. warmed medium. The tissue was alternately exposed to light at 340 and 380 nm and the fluorescence passing through a glass colour filter (Corning. No. 3-72) was recorded by the spectrophotometer.

Measurement of vasopressin release Each neurohypophysis was perifused with medium containing 0-1 %/0 bovine serum albumin at a flow rate of 04-05 ml min-'. After incubation for 1 h. the tissue was stimulated five times at 30 min intervals by trains of pulses at 10. 20 or 50 Hz which lasted for 2. 5 or 10 s. Fractions of the perifusate were collected at 10 min intervals and duplicate samples of each fraction were assayed f'or vasopressin by a specific radioimmunoassay which was about 100 times more sensitive to vasopressin than to oxytocin. The intra- and interassay variances were 9 and 14%. respectively. The amount of vasopressin collected between 10 and 20 min after stimulation was not markedly different from that between 20 and 30 min after stimulation. Therefore, almost all the release evoked by stimulation probably diffused into the medium during the first 10 min and the amount released was estimated from the difference in hormone content of the samples collected before and after stimulation. To check the stability of the release measurement, the vasopressin release evoked by the first and the last stimulus trains at 20 Hz for 5 s were compared: the first release was 1120+ 120 pg gland-1 (mean+S. E. M. n = 18) and the last was 90 +44% of the first. The rest of the data were normalized to the mean of these two values to reduce the effect of tissue variation on the result. Stimulation at 5 Hz for 10 s evoked almost no vasopressin release (less than 30 pg gland-'). Estimation of vasopressin relea,se pattern during repetitiv.,e stimulation It is not possible to measure directly bv radioimmunoassay of perifusate the vasopressin release pattern during repetitive stimulation because the diffusion of v-asopressin between medium and the extracellular space is rather slow. Instead, the amount of release evoked by a particular part of train pulse stimulation was estimated. For example, the release evoked by stimulation for 5 s can be regarded as the sum of release evoked by stimulation for the first 2 s and by the remaining 3 s. Therefore the amount of release evoked between 2 and 5 s after stimulus initiation mav be estimated as the difference between the release evoked by stimulation for 2 s and stimulation for 5 s. Such an estimation is justified provided conditions remain fairly stable for the duration of the experiment. This appeared to be the case since the amount of release evoked by stimulation at 20 Hz for 5 s decreased only by 10 % to 90 + 4% after 2 h. To minimize the effect, of the deterioration of the tissue on the averaged data. the sequence of the different pulse trains was altered so that the second, third and fourth release were evoked by stimulation for 2, 5 anid 10 s (n = 3 at each frequency) or 10. 5 and 2 s (n = 3). respectively. The amount and rate of release exvoked by pulses 0-2, 2-5 and 5-10 s after stimulus initiationi were calculated firom these (lata.

)02 measurement To estimate the 02 consumption rate.

PO in the neurohypophysis

was

measured by

a

miniatuire

Clark-type 02 electrode as described previously (Shibuki, 1989). Briefly, a glass micropipette was filled with an electrolyte solution (KCI, 3 M; KOH, 1 mm) and sealed with a thin rubber membrane. An anode and cathode of wire insulated except at the cut end were inserted into the pipette. The anode, located as close as possible to the rubber membrane, was kept at, -0-6 V relative to the 11-2

324

K. SHIBUKI

cathode. The current passing between the anode and cathode was proportional to PO at the tip of the 02 electrode. The electrodes used had tip diameters of less than 100 ,im and a 90'S,7 response time of less than 3 s (Fig. 4A). RESIULTS

Relation between [Ca2+]0 and Fura-2 fluorescence Repetitive stimulation evoked marked [Ca2+]0 decreases in the neurohypophysis (Figs 1B, 2A, and 4G-J). Although these [Ca2+]0 decreases are primarily caused by A 2.0

\

_ ~~~~~1-9 19E 18

o

40 s

_1-7 B

\sC

04

20 Hz, 10 s

20 Hz, 10 s

Fig. 1. [Ca2+]o and Fura-2 fluorescence in the isolated rat neurohvpophvsis. A. [Ca2+]0 in the middle of the tissue following a step decrease in the medium calcium concenltrationi from 2 to 1 mm (arrow-head). B. a [C'a2J]O decrease (upward defle(tion in the trace) evoked by stimulation at 20 Hz for 10 s. The ordinate is a linearize(d scale. (, stimulus-evoked changes in fluorescence alternately excited at 340 nim (traces of' upward deflection) and at 380 nm (downward deflection) in the neurohypophysis loaded with Fura-2 AM. Intenisity is in arbitrary units. Stimulation clearly evoked [Caa2+ ] decreases (B) and Fura-2 fluorescence changes (C) of similar pattern.

movement of calcium between intra- and extracellular compartments (Leng et al.

1988), it may be affected by diffusion of Ca2' between the tissue and perifusing medium. To observe the diffusion process, [Ca21] in the medium ([Ca2+]m) was altered rapidly from 2 to 1 mm or vice versa. The [Ca2+]O recorded in the middle of the tissue followed the step [Ca2+]m changes with a time lag of 20-40 s (Fig. 1A). In the middle of the tissue, therefore, [Ca21]. is unlikely to be affected by Caa2' diffusion into the tissue from the surrounding medium within 20-40 s after the steady state of Ca2+ distribution between compartments is altered by stimulation. The shift from calcium from extra- to intracellular compartments, as reflected by the [Ca2+]O decreases, may not necessarily reflect [Ca2+]i increases because cytosolic

(ya2+ ACTIVA TIO)N OF VASOPRE`SSIN

RELEASE325 325

free Ca2+ represents only a mninor component of the total intracellular calcium in the neurohypophvsis (Russell & Thorn, 197)5; Nordmann & Chevallier. 1981). To study the relationship between [Caa2+]O decreases and ['a2+]i increases. Fura-2 fluorescence was recor(led in the neurohypophysis during stimulation and compared with [Ca21]. changes (Fig. LB, and C). During 20 Hz stimulation, [Ca2+]0 decreases reached a plateau level within a few seconds and the plateau was maintained during stimulation for at least 10 s (Fig. 1 B). The fluorescence excited at 340 nm (or at 380 nm) was augmented (or reduced) by stimulation and showed similar plateau levels during stimulation (n = 4, Fig. 1 ). This similarity suggests that [Ca2+]0 decreases mirror [(a2+]i increases.

Estimation of Ca2+ influx Because [Ca2+] l (lecreases were likely to be the result of Ca2+ shift from the extrato intracellular space. Ca2+ net flux was able to be estimated from the [Ca2+]0 decrease rate or -

d(C:a 2+])/dt = Ca2+ net flux

= Ca2+ influx-Ca2+ efflux.

(1)

To separate Ca2+ influx from Ca2+ efflux, which was revealed as a [Ca2+]0 change toward the pre-stirnulus level after the cessation of stimulation (for example, Fig. 1B). the following assumptions were made: firstly that Ca2+ influx stops immediately following the last of the train of pulses or

Ca2+ influxt

= 0,

(2)

and secondlv that Caa21 efflux immediatelv before and after the stimulus cessation are equal or Ca effuxt=t,,dt = Ca effluxt=to+dt, (3) where to is the time when the last pulse was given to the tissue and dt is a fraction of time (dt