- Email: [email protected]
Calcium signaling and secretion in pituitary cells
tions in pituitary gonadotrophs. Biophys J 69:785-795. Mollard E Schlegel W: 1996. Why are endocrine pituitary cells excitable? Trends Endocrinol Metab 7:361-365. Ortmann O, Vassmann D, Stojilkovic SS, Cart KJ, Schultz KD, Emons G: 1993. Ovarian steroids modulate endothelin-induced luteinizing hormone secretion from cultured rat pituitary cells. Endocrinology 133:2632-2638. Ortmann O, Merelli E Stojilkovic SS, Schultz KD, Emons G, Catt KJ: 1994. Modulation of calcium signaling and LH secretion by progesterone in pituitary gonadotrophs and clonal pituitary cells. J Steroid Biochem Mol Biol 48:47-54. Osipchuk YV, Wakui M, Yale DI, Gallacher DV, Petersen OH: 1990. Cytoplasmic Ca2+ oscillations evoked by receptor stimulation, Gprotein activation, internal application of inositol trisphosphate or Ca2÷: simultaneous microfluorimetry and Ca2+ dependent C1current recording in single pancreatic acinar cells. EMBO J 9:697-704. Oxford GS, Tse A: 1993. Modulation of ion channels underlying excitation-secretion coupling in identified lactotrophs and gonadotrophs. Biol Reprod 48:1-7. Rawlings SR: 1996. Pituitary adenylate cyclaseactivating polypeptide [Ca2+]i and electrical activity in pituitary cells through cell typespecific mechanisms. Trends Endocrinol Metab 7:374-378. Shangold GA, Murphy SN, Miller RJ: 1988. Gonadotropin-releasing hormone-induced Ca2+ transients in single identified gonadotropes require both intracellular Ca2+ mobilization and Ca2+ influx. Proc Natl Acad Sci USA 85:6566-6570. Stojilkovic SS, Iida T, Merelli E Torsello A, Krsmanovic LZ, Catt KJ: 1991. Interactions between calcium and protein kinase C in the control of signaling and secretion in pituitary gonadotrophs. J Biol Chem 266:10,37710,384. Stojilkovic SS, Kukuljan M, Iida T, Rojas E, Catt KJ: 1992. Integration of cytoplasmic calcium and membrane potential oscillations maintains calcium signaling in pituitary gonadotrophs. Proc Nat Acad SCi USA 89: 4081-4085. Stojilkovic SS, Kukuljan M, Tomic M, Rojas E, Catt KJ: 1993. Mechanism of agonist-induced [Ca2+]~ oscillations in pituitary gonadotrophs. J Biol Chem 268:7713-7720. Stojilkovic SS, Reinhart J, Cart KJ: 1994a. GnRH receptors: structure and signal transduction pathways. Endocr Rev 15:462-499. Stojilkovic SS, Tomic M, Knkuljan M, Cart KJ: 1994b. Control of calcium spiking frequency in pituitary gonadotrophs by a single-pool cytoplasmic oscillator. Mol Pharmacol 45: 1013-1021. 384
Total6 M, Cesnjaj M, Catt KJ, Stojilkovic SS: 1994. Developmental and physiological aspects of Ca2+ signaling in agonist-stimulated pituitary gnnadotrophs. Endocrinology 135: 1762-1771. Tse A, Hille B: 1992. GnRH-induced Ca2. oscillatiolas and rhythmic hyperpolarizations of pituitm'y gonadotropes. Science 255:462-464. Tse A, Tse F~, Hille B: 1994a. Calcium homeostasis in identified rat gonadotrophs. J Physiol (Lond) 477:511-525. Tse FW, Tse A, Hille B: 1994b. Cyclic Ca2+ changes in intracellular stores of gonado-
tropes during gonadotropin-releasing hormone-stimulated Ca2+ oscillations. Proc Natl :Acad Sci USA 91:9750-9754. Vergara L, Stojilkovic SS, Rojas E: 1995. GnRH induced cytosolic calcium oscillations in pituitary gonadotrophs: phase resetting by membrane depolarization. Biophys J 69: 1606-1614. Zheng L, Paik W-Y,Cesnjaj M, et al.: 1995.Effects of the phospholipase-C inhibitor, U73122, on signaling and secretion in pituitary gonadotrophs. Endocrinology 136:1079--1088. TEM
Calcium Signaling and Secretion in Pituitary Cells Robert Zorec
An important trigger of hormone secretion from pituitary cells is a rise in cytosolic Ca 2+ ([Ca2+].~. Pituitary cells may modulate [Ca2+]i by an increased membrane flux from the extracellular space and~or by a release from intracellular stores. Both mechanisms can support exocytosis, although in different pituitary cell types one or the other mechanism may predominate. Molecular events transducing a rise in [Ca2+]i into hormone secretion are still poorly understood. Here, the exocytotic machinery in pituitary cells is briefly reviewed in terms of the spatial organization of [Ca2+]i elevation relative to the Ca2+ sensor(s). © 1996, Elsevier Science Inc. (Trends Endocrinol Metab 1996;7:384-388).
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C a l c i u m Delivery to t h e
Exocytotic Machinery The role of [Ca2+]i in secretion of single anterior pituitary cells was demonstrated by Sikdar et al. (1989). They m e a s u r e d changes in m e m b r a n e capacitance (C~), a readout of the cell membrane surface area (Neher and Marty 1982), which increases u p o n exocytosis [see Zupan6id et al. (1994)], while cell cytosol was dialyzed with a high Ca 2÷containing solution. Figure 1 shows that such a treatment results in a rise in C m,
Robert Zorec is at the Laboratory of Neuroendocrinology, Institute of Pathophysiology, School of Medicine, University of Ljubljana, 1105 Ljubljana, Slovenia.
confirming a role of [Ca2+]i in dynamic changes of plasma m e m b r a n e area. As anterior pituitary cells are excitable and possess voltage-gated calcium channels (VGCCs), it has been suggested that control of the firing frequency of Ca 2+dependent action potentials plays a role in basal and stimulated secretion (Ozawa and Sand 1986). In addition, in the absence of spontaneous electrical activity, a steady-state entry of Ca 2 +, possibly also t h r o u g h VGCCs (Mollard et al. 1994), was shown to support secretory activity (Zorec et al. 1991). A direct role of VGCCs in secretory activity of melanotrophs was confirmed by T h o m a s et al. (1990), who showed that a rise in [Ca2+]i, elicited by activation of VGCCs, induced a rise in Cn~, indicating a direct
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A
]
B
5 pF •
. . . . . . 5.6 pF
°
5.5 pF I
". " . . . . . .
I
300 s
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3s Figure 1. Changes in membrane capacitance (Cm) recorded in single bovine lactotrophs with use of the whole cell patch clamp technique. Effect of cytosol dialysis with pipette filling solution containing (A)I pM and (B) 0 pM [Caz*] on Cm recorded in two lactotrophs. Numbers adjacent to records represent resting Cm. Arrows indicate the start of cytosol dialysis. (C) Short episode of membrane capacitance signal recorded at high gain. Discrete steps in Cm are due to single exocytotic and endocytotic events [see Zorec et al. (1991), Zupan~i6 et al. (1994)]. role of VGCCs in stimulus-secretion coupling in pituitary cells. In contrast to neurons, where a tight coupling between the activation of VGCCs and release from presynaptic sites persists typically at room temperature, pituitary cells secrete robustly only near physiological temperatures (Thomas et al. 1990, Fomina and Levitan 1995). This may be due to differences in the molecular makeup of exocytotic machinery, VGCCs and/or coupling between VGCCs and exocytotic machinery in the two cell types. Which VGCCs are present in pituitary cells? Electrophysiological studies allowed VGCCs to be classified into two groups: low-voltage activated (LVA, molecular structure not determined) and high-voltage activated (HVA) voltagegated Ca 2+ currents, which consist of a~, ¢t2-$, [3, and ~ subunits, where the tx1 subunit functions as the channel. Cloning has revealed that the cq subunit is coded by six genes, whereas the [3 subunit is coded by four genes (Perez-Reyes and Schneider 1995). Most pituitary cells possess LVA and HVA Ca 2+ currents [Armstrong and Matteson (1985), Cota TEM Vol. 7, No. 10, 1996
(1986), see Stojilkovi~ and Catt (1992), see Mollard and Schlegel (1996)], but in some cell types, depending on gender, animal species, or age, only one Ca 2+ current is present (see Corrette et al. 1995). With the use of specific pharmacological agents, it was possible, for example, to determine the diversity of HVA currents in melanotrophs of various species as L-, P-, Q-, and N-types (see Corrette et al. 1995, Ciranna et al. 1996). Interestingly, the LVA current was found to be sensitive to dihydropyridines, suggesting this current permeates through a subtype or modified L-type channel (Kocmur and Zorec 1993). By molecular biology approaches, mRNA of OtlA, O~IB, alC, and OtlD subunits were studied in clonal pituitary cells (Li6vano et al. 1994), revealing that all subunits were present in AtT-20 cells, whereas all but the ctm subunit were detected in GH 3 cells. In situ hybridization studies confirmed that anterior pituitary cells express tXlA, tx,B, ~1c, and a~D subunits, although the mRNA level for eqc and Ct1D subunits appeared to be more abundant (Tanaka et al. 1995). This may explain
the untight depolarization-secretion coupling at low temperature in pituitary cells (Fomina and Levitan 1995), as the tX1A and txm subunits have been shown to interact with SNARE molecules of exocytotic machinery (Bezprozvanny et al. 1995, Sheng et al. 1996) (Appendix). An increase in [Ca2+]i may also be caused by release from intracellular Ca 2+ stores, such as that generated by inositol 1,4,5-trisphosphate (IP3). It was shown that the addition of GnRH induces oscillations of [Ca2+]i in gonadotrophs, which are independent of extracellular Ca 2÷ (Shangold et al. 1988) and can be mimicked by cytosol dialysis with IP 3 (Tse and Hille 1992). These oscillations are sufficient to support secretory activity at the cellular level (Tse et al. 1993). By employing high temporal resolution Cm measurements to monitor simulaneously exocytosis and [Ca2+]i in identified gonadotrophs of adult male rats, it was shown that each cycle of increase in [Ca2÷]i, owing to release from intracellular stores, can trigger secretion (Tse et al. 1993). In gonadotrophs, VGCCs may only play a role to replenish intracellular Ca 2÷ stores and to deliver Ca 2÷ acting as a coactivator in the stimulation of Ca 2÷ release from the IPa-sensitive stores (Tse and Hille 1993). Interestingly, an application of thyrotropin-releasing hormone (TRH), an IP 3liberating agent, increases secretory activity in single lactotrophs, as well (Fomina and Levitan 1995). In this cell type, both Ca 2+ delivery systems are competent in supporting secretory activity. A question to be studied in the future is which of the two mechanisms is more efficient in inducing secretory activity.
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What D e t e r m i n e s the S p e e d o f S e c r e t i o n f r o m Pituitary Cells?
In comparison to neurons, pituitary cells secrete at a significantly slower rate. Hormone delivery to the target is limited by the transport in blood circulation, whereas in neurons the speed of exocytosis can be critical, as signal transduction in a synapse can occur in less than a millisecond (Katz 1969). There are several hypotheses as to why neuroendocrine secretion is slower than neural secretion: (a) that the VGCCs and secretory vesicles (granules) are not molecularly colocalized, (b) that the exocytotic machinery is intrinsically slower, or that
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the granules are not normally docked, but positioned distant from the plane of the membrane, separated by a cytoskeletal network, which must be disassembled upon a rise in [Ca2+]i [see Trifar6 and Vitale (1993)]. The third hypothesis contrasts with results of Parsons et al. (1995), which show that a significant number of granules are docked in pituitary and chromaffin cells. Moreover, it is unlikely that the exocytotic machinery of neuroendocrine cells is intrinsically slow, as the maximal rate of exocytosis recorded in chromaffin cells (Heinem a n n et al. 1994) and bipolar neurons (Heidelberger et al. 1994) is remarkably similar. Thus, it is likely that neuroendocrine secretion is slower than neural secretion because VGCCs and granules are not molecularly colocalized. This possibility is supported by the absence of morphological structures resembling active zones in pituitary cells, where channels and vesicles are colocalized in synapses (Robitaille et al. 1990). The shorter the distance between a Ca 2+ source and secretory vesicles, the faster the secretion. Furthermore, the distance between Ca 2+ sources (VGCCs and intracellular stores) and secretory vesicles determines the biochemical requirements of Ca 2+ receptor(s) that trigger exocytosis. Within a few nanometers of a Ca 2+ source, [Ca2+]i has been estimated to reach near millimolar levels, whereas at about 1 micrometer away, the concentration is expected to be in the low micromolar range (Smith and Augustine 1988) (Figure 2). Thus, near VGCCs, a low-affinity Ca 2÷ receptor(s) would be adequate to trigger secretion, whereas at greater distance the Ca 2+ receptor(s) would have to have a higher affinity. In agreement with this, in bipolar neurons the threshold for secretion is around 50 pM [Ca2+]i (Gersdorff and Matthews 1994), whereas pituitary cells start secreting at submicromolar [Ca2+]i (Rupnlk and Zorec 1995). The sensitivity of exocytotic machinery for Ca 2+ appears to be associated with the size of secretory vesicles (Figure 2). With larger secretory vesicles, the Ca 2+ receptor(s) appears to be positioned away from the Ca 2+ source. The results of Chow et al. (1994) indicate that the Ca 2÷ receptor(s) is quite a distance away from the Ca 2÷ source (Figure 2), as [Ca2+]i sensed by the secretory machinery during a short depolarization was estimated to be around 5 pM (Chow et al. 1994). This may be be386
I
150 n m
-0.3
LDCG
# M [ C a 2+]
~ ' I S "
.......
IS
- 5 #M [Ca 2+] -100#M[Ca
2+] SV
R.~ ".. R ' "~ii" "
VGCC Figure 2. Synaptic vesicle (SV, 50 nm in diameter) size is compared with the size of a secretory granule (large dense core granule, LDCG, 300 nm in diameter). Dashed lines indicate the steady-state [Ca2+]i during opening of a voltage-gated calcium channel (VGCC), axially from the channel mouth (Smith and Augustine 1988). Note that the dimensions of an SV span a narrow range of [Ca2+, whereas the dimensions of an LDCG span a much wider range of [Ca2+] upon opening ofa VGCC. An alternative Ca2÷ source may be a release from intracellular stores (IS), which appears to be functionally equally distant in comparison with VGCCs from Ca2+ receptors (R) that trigger exocytosis. Thus, it is appealing to think that one type of Ca2+ receptor triggering exocytosis is shared by different Ca2+ sources (VGCCs, intracellular stores) in pituitary cells. cause granules and VGCCs may not be molecularly colocalized in the plane of the membrane (Chow et al. 1994). In contrast, we may also consider that in pituitary cells, the Ca 2+ receptor(s) is positioned deeper in the cytoplasm. An advantage of such a distantly positioned Ca z+ receptor(s) is that it could sense Ca 2+ release from an intracellular store and from VGCCs and transduce the signal from both Ca 2+ sources into exocytosis of docked granules. •
Ca z+ R e c e p t o r ( s ) and E x o c y t o t i c Machinery
A major candidate for a vesicle-bound low-affinity Ca 2+ receptor in synapses is synaptotagmin (Brose et al. 1992), also present in the pituitary (Marqu6ze et al. 1995), although its role in exocytosis is controversial [compare Shoji-Kasai et al. 1992, Geppert et al. (1994)]. One reason for this is that multiple isoforms of synaptotagmin (Li et al. 1995) may be coexpressed in a cell. In contrast, a candidate for a cytosolic high-affinity Ca 2+ receptor(s) in pituitary cells m a y be p145 (Walent et al. 1992), a soluble pro-
tein that was purified based on its ability to stimulate Ca2+-triggered secretion from permeabilized PC 12 cells. Another candidate is annexin II, a m e m b e r of a family of Ca 2+ and phospholipid-binding proteins (Ali et al. 1989). In addition to Ca 2+, activation of GTP-binding proteins can trigger exocytosis. Both monomeric (Lledo et al. 1993, Rupnik et al. 1995) and heterotrimeric (Rupnik and Zorec 1995) GTP-binding proteins appear to be involved in regulated secretion of pituitary cells. The key question, pertinent not only to pituitary secretion, is how molecular events transduce activation of Ca 2÷ receptors into exocytosis. There are several views as to how exocytosis might be controlled by a Ca 2+ receptor(s). One possibility is by affecting cytoskeleton around granules, which is thought to present a barrier to exocytosis (Trifar6 and Vitale 1993). Another model predicts a "scaffold," a macromolecular structure that bridges the gap between the plasma and granule membrane, and that can direct and regulate curvature and promote conditions that are favorable for membrane fusion (Monck and
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Fernandez 1994). The number of proteins i m p l i c a t e d in r e g u l a t e d exocytosis has been growing in the last decade, a n d it was p r o p o s e d that t h e i r interactions controlling exocytosis functionally res e m b l e the activity of m o l e c u l a r chaperones (Burgoyne a n d M o r g a n 1995), w h i c h regulates p r o t e i n folding a n d the a s s e m b l y a n d d i s a s s e m b l y of multiprotein complexes. Currently, p r o t e i n s f o r m i n g a c o m p l e x t e r m e d "the S N A R E complex" are t h o u g h t to p l a y a universal role in vesicular traffic a n d in regulated exocytosis (S6llner a n d R o t h m a n 1994, see appendix), but there is no direct evidence that these p r o t e i n s play a role in exocytosis o f p i t u i t a r y cells. In a d d i t i o n to proteins, a s y m m e t r i c lipid composition of m e m b r a n e s a p p e a r s to play a role in exocytosis, as well ( C h e r n o m o r d i k et al. 1995). In particular, p h o s p h o i n o s i tides a p p e a r as key regulators of vesicular traffic (see De Camilli et al. 1996). Although the u n d e r s t a n d i n g of molecu l a r m e c h a n i s m s of exocytosis in pituitary cells is in its infancy, we have presented a discussion o f a biophysical perspective that predicts that in p i t u i t a r y cells, different C a Z + s o u r c e s - - s u c h as VGCCs or i n t r a c e l l u l a r s t o r e s - - m a y share a Ca 2+ receptor(s) with s i m i l a r b i o c h e m i c a l properties,
*
Aekowledgments
R.Z. is s u p p o r t e d by the Ministry of Sciences a n d Technology o f Slovenia. S. Grilc is a c k n o w l e d g e d for providing cell cultures, a n d R.H. Chow, M. Shipston, G. Z u p a n 6 i 6 , M. Rupnik, a n d L. Kocmur-Bobanovid a r e a c k n o w l e d g e d for their c o m m e n t s on the m a n u s c r i p t .
References All SS, Geisow MJ, Burgoyne RD: 1989. A role for calpactin in calcium dependent exocytosis in adrenal chromaffin cells. Nature 340: 313-315. Armstrong CM, Matteson DR: 1985. Two distinct populations of calcium channels in a clonal line of pituitary cells. Science 227:65-67. Bezprozvanny I. Scheller RH, Tsien RW: 1995. Functional impact of syntaxin on gating of N-type and Q-type calcium channels. Nature 378:623--626. Brose N, Petrenko AG, Sfidhof TC, Jahn R: 1992. Synaptotagrnin: a Ca2÷ sensor on the synaptic vesicle surface. Science 256:10211025.
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Burgoyne RD, Morgan A: 1995. Ca2÷ and secretory-vesicle dynamics. Trends Neurosci 18: 191-196. Chemomordik L, Kozlov MM, Zimmerberg J: 1995. Lipids in biological membrane fusion. J Membr Biol 146:1-14. Chow RFI, Klingauf J, Neher E: 1994. Tmae course of Ca2+ concentration triggering exocytosis in neuroendocrine ceils. Proc Natl Acad Sci USA 91:12,765-12,769. Ciranna L, Feltz P, Sehlichter R: 1996. Selective inhibition of high voltage-activated L-type and Q-type Ca2+ ctu~nts by serotonin in rat melanotrophs. J Physiol (Lond) 490.3:595-609. Corrette BJ, Bauer CK, Schwarz JR: 1995. Electrophysiology of anterior pituitary cells. /n Scherubl H, Hescheler J, eds. The Electrophysiology of Neuroendocrine Cells. Boca Raton, FL, CRC Press, pp 102-143. Cota G: 1986, Calcium channel currents in pars intermedia cells of the rat pituitary gland. J Gen Physiol 88:83-105. DeCarnilli P, Emr SD, McPerson PS, Novick P: 1996. Phosphoinositides as regulators in membrane traffic. Science 271:1533-1539. Fomina AF, Levitan ES: 1995. Three phases of TRH-induced facititation of exocytosis by single lactotrophs. J Neurosci 15:4982-4991. Geppert M, Goda Y, Hammer RE, et al.: 1994. Synaptotagmin I: a major Ca2÷ sensor for transmitter release at a central synapse. Cell 79:717-727.
Marqu~ze B, Boudier JA, Mizuta M, Inagaki N, Seino S, Seagar M: 1995. Cellular localization of synaptotagmin I, II, and III mRNAs in the central nervous system and pituitary and adrenal glands of the rat. J Neurosci 15:49064917.
Gersdorff H, Matthews G: 1994. Dynamics of synaptic vesicle fusion and membrane retrieval in synaptic terminals. Nature 367:735739.
Perez-Reyes E, Schneider T: 1995. Molecular biology of calcium channels. Kidney Int 48: 1111-1124.
Heidelberger R, Heinemann C, Neher E, Matthews G: 1994. Calcium dependence of the rate of exocytosis in a synaptic terminal. Nature 371:513-515. Heinemann C, Chow RH, Neher E, Zucker RS: 1994. Kinetics of the secretory response in bovine chrornaffin cells following flash photolysis of caged Ca2÷. Biophys J 67:2546--2557. Katz B: 1966. Nerve, Muscle and Synapse. New York, McGraw-Hill, p 115. Kocmur L, Zorec R: 1993. A new approach to separation of voltage-activated Ca currents in rat melanotropbs. Pfltigers Arch 425:172-174. Li C, Ullrich B, Zhang J~, Anderson RGW, Brose N, Sfidhof TC: 1995. Ca2+-dependent and -independent activities of neural and non-nenral synaptotagmins. Nature 375:594-599. Li6vano A, Bolden A, Horn R: 1994. Calcium channels in excitable cells: divergent genotypic and phenotypic expression of al-subunits. Am J Physiol 267:C411-C424. Lledo P-M, Vernier P, Didier J-D, Mason WT, Zorec R: 1993. Inhibition of Rab 3B expression attenuates Ca2÷-dependent exocytosis in rat anterior pituitary cells. Nature 364:543544.
Mollard E Schlegel W: 1996. Why are endocrine pituitary ceils excitable? Trends Endocrinol Metab 7:361-365. Mollard P, Theler J-M, Guerineau N, Vacher P, Chiavaroli C, Schlegel W: 1944. Cytosolic Ca2+ of excitable pituitary cells at resting potentials is controlled by steady state Ca2+ currents sensitive to dihydropyridines. J Biol Chem 269:25,158-25,164. Monck JR, Fernandez JM: 1994. The exocytotic fusion pore and neurotransmitter release. Neuron 12:707-716. Morgan A, Burgoyne RD: 1995. Is NSF a fusion protein? Trends Cell Biol 5:335-339. Neher E, Mm-ty A: 1982. Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin ceils. Proc Nail Acad Sci USA 79:6712-6716. Ozawa S, Sand O: 1986. Electrophysiology of excitable endocrine cells. Physiol Rev 66:887951. Parsons T, Coorssen JR, Horstmann H, Almers W: 1995. Docked granules, the exocytotic burst, and the need for ATP hydrolysis in endocrine cells. Neuron 15:1085-1096.
Robitaille R, Adler EM, Charlton MP: 1990. Strategic localization of calcium channels at transmitter release sites of frog neuromuscular synapses. Neuron 5:773-779. Rupnik M, Law GJ, Northrop AJ, Mason WT, Zorec R: 1995. Brefeldin A and a synthetic peptide to ADP-ribosylation factor inhibit regulated exocytosis in melanotrophs. Neuroreport 6:853-856. Rupnik M, Zorec R: 1995. Intraceilular chloride modulates Ca2+-induced exocytosis from rat melanotrophs through GTP-binding proteins. Pflfigers Arch 431:76-83. Shangold GA, Murphy SN, Miller RJ: 1988. Gonadotropin-releasing hormone-induced Ca2÷ transients in single identified gonadotropes require both intracellular Ca2. mobilization and Ca 2+ influx. Proc Natl Acad Sci USA 86:6566-6570. Sheng Z-H, Rettig J, Cook T, Catterall WA: 1996. Calcium-dependent interaction of N-type calcium channels with the synaptic core complex. Nature 379:451-454. Shoji-Kasai Y, Yoshida A, Sato K, et al.: 1992. Neurotransmitter release from synaptotagmin-deficient clonal variants of PC 12 ceils. Science 256:1820-1823.
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Sikdar SK, Zorec R, Brown D, Mason WT: 1989. Dual effects of G-protein activation on Cadependent exocytosis in bovine lactotrophs. FEBS Lett 253:88-92. Smith SJ, Augustine GJ: 1988. Calcium ions, active zones and synaptic transmitter release. Trends Neurosci 11:458--464. S611ner T, Rothman JE: 1994. Neurotransmission: harnessing fusion machinery at the synapse. Trends Neurosci 17:344-348. Stojilkovi6 SS, Catt KJ: 1992. Calcium oscillations in anterior pituitary cells. Endocr Rev 13:256-280.
SNARE hypothesis (S611ner and Rothman 1994). This hypothesis engages several proteins in the regulation of exocytosis. N-ethylmaleimide-sensitive fusion protein (NSF) is an ATPase and binds to membranes in the presence of soluble NSF attachment proteins (SNAPs). The model postulates that receptors (SNAREs) exist for SNAP proteins and proposes that docking specificity is maintained when a transport vesicle-associated SNARE (v-SNARE) pairs with a cognate target-associated SNARE (tSNARE) in a unique match. The v-SNARE would be synaptobrevin or a hornologue, whereas t-SNARE would be SNAP 25 (synaptosome associated protein 25) and syntaxin or holomogues. SNAREs are targets of neurotoxins, allowing functional assignment of these
proteins in exocytosis. The complex of NSF/SNAPs/SNAREs (SNARE complex) dissasembles upon ATP hydrolysis by NSF, which is thought to power fusion of two membranes. Current evidence, however, supports an earlier prefusion role of NSF (Morgan and Burgoyne 1995), which is consistent with results of studies on pituitary ceils (Parsons et al. 1995). There are additional regulatory components associated with the SNARE complex, such as synaptotagmin and VGCCs (S611ner and Rothman 1994, Sheng et al. 1996), which appear to inhibit the constitutive pathway. This block is relieved upon a rise in [CaZ+]i. One of the challenges for the future is to test whether the SNARE hypothesis explains regulated exocytosis in pituitary cells.
Tanaka O, Sakagami H, Kondo H: 1995. Localization of mRNAs of voltage-dependent Ca2+-channels: four subtypes of a t- and subunits in developingand mature rat brain. Mol Brain Res 30:1-16. Thomas P, Surprenant A, Almers W: 1990. Cytosolic Ca2+, exocytosis and endocytosis in single melanotrophs of the rat pituitary. Neuron 5:723-733. Trifar6 JM, Vitale ML: 1993. Cytoskeleton dynamics during neurotransmitter release. Trends Neurosci 16:466--472. Tse A, Hiile B: 1992. GnRH-induced Ca2÷ oscillations and rhythmic hyperpolarizations of pituitary gonadotropes. Science 255:462464. Tse A, Hille B: 1993. Role o[ vohage-gated Naand Ca2+ channels in gonadotropin-releasing hormone-induced membrane potential i n changes in identified rat gonadotropes. En- John Rinzel, Joel Keizer, and Yue-Xian Li docrinology 132:1475-1481. Tse A, Tse FW, Almers W, Hille B: 1993. Rhythmic exocytosis stimulated by GnRH-induced calcium oscillations in rat gonadotropes. Sci- The response of gonadotrophs to secretagogues involves dose-depenence 260:82-84. dent, complex dynamic patterns of electrical activity and inositol 1,4,5Walent JH, Porter BW, Martin TFJ: 1992. A novel 145 kd brain cytosolicprotein reconsti- trisphosphate (InsP3)-induced Ca2+ mobilization, including pulsatility tutes Ca2+-regulated secretion in permeable and oscillations on multiple time scales from milliseconds to minutes. neuroendocrine cells. Cell 70:765-775. Detailed in vitro experiments have enabled the identification of key Zorec R, Sikdar SK, Mason wr: 1991. Inmechanisms that underlie the plasma membrane (PM) electrical excitcreased cytosoliccalcium stimulates exocytoability and endoplasmic reticulum (ER) calcium excitability. We sumsis in bovine lactotrophs: direct evidence from changes in membrane capacitance. J marize these findings and review computer simulations of a biophysiGen Physiol 97:473--497. cal model that resynthesizes and couples these components and that Zupan~i6 G, Kocmur L, Verani6 P, Grilc S, reproduces quantitatively the observed time courses and dose-response Korda M, Zorec R: 1994. The separation of exocytosis from endocytosis in rat melan- characteristics, as well as effects of various pharamacological manipotroph membrane capacitance records. J ulations. The theory suggests that cytosolic calcium is the primary Physiol (Lond) 480:539-552. TEM
Modeling Plasma Membrane and Endoplasmic Reticulum Excitability Pituitary Cells
messenger in coordinating the PM and ER regenerative behaviors during ER depletion and refilling. © 1996, Elsevier Science Inc. (Trends
•
APPENDIX
E n d o c r i n o l M e t a b 1996;7:388-393).
The Snare Hypothesis In eukaryotic cells, several steps in the secretory pathway, including synaptic transmission, have been shown to depend on closely related proteins, which led Rothman et al. to propose a general mechanism of docking and fusion, the 388
John Rinzel is at the Mathematical Research Branch, NIDDK, National Institutes of Health, Bethesda, MD 20892; and Joel Keizer and Yue-Xian Li are at the Institute of Theoretical Dynamics (with Keizer also at the Section on Neurobiology, Physiology, and Behavior at the University of California, Davis, CA 95616, USA.
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Total6 M, Cesnjaj M, Catt KJ, Stojilkovic SS: 1994. Developmental and physiological aspects of Ca2+ signaling in agonist-stimulated pituitary gnnadotrophs. Endocrinology 135: 1762-1771. Tse A, Hille B: 1992. GnRH-induced Ca2. oscillatiolas and rhythmic hyperpolarizations of pituitm'y gonadotropes. Science 255:462-464. Tse A, Tse F~, Hille B: 1994a. Calcium homeostasis in identified rat gonadotrophs. J Physiol (Lond) 477:511-525. Tse FW, Tse A, Hille B: 1994b. Cyclic Ca2+ changes in intracellular stores of gonado-
tropes during gonadotropin-releasing hormone-stimulated Ca2+ oscillations. Proc Natl :Acad Sci USA 91:9750-9754. Vergara L, Stojilkovic SS, Rojas E: 1995. GnRH induced cytosolic calcium oscillations in pituitary gonadotrophs: phase resetting by membrane depolarization. Biophys J 69: 1606-1614. Zheng L, Paik W-Y,Cesnjaj M, et al.: 1995.Effects of the phospholipase-C inhibitor, U73122, on signaling and secretion in pituitary gonadotrophs. Endocrinology 136:1079--1088. TEM
Calcium Signaling and Secretion in Pituitary Cells Robert Zorec
An important trigger of hormone secretion from pituitary cells is a rise in cytosolic Ca 2+ ([Ca2+].~. Pituitary cells may modulate [Ca2+]i by an increased membrane flux from the extracellular space and~or by a release from intracellular stores. Both mechanisms can support exocytosis, although in different pituitary cell types one or the other mechanism may predominate. Molecular events transducing a rise in [Ca2+]i into hormone secretion are still poorly understood. Here, the exocytotic machinery in pituitary cells is briefly reviewed in terms of the spatial organization of [Ca2+]i elevation relative to the Ca2+ sensor(s). © 1996, Elsevier Science Inc. (Trends Endocrinol Metab 1996;7:384-388).
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C a l c i u m Delivery to t h e
Exocytotic Machinery The role of [Ca2+]i in secretion of single anterior pituitary cells was demonstrated by Sikdar et al. (1989). They m e a s u r e d changes in m e m b r a n e capacitance (C~), a readout of the cell membrane surface area (Neher and Marty 1982), which increases u p o n exocytosis [see Zupan6id et al. (1994)], while cell cytosol was dialyzed with a high Ca 2÷containing solution. Figure 1 shows that such a treatment results in a rise in C m,
Robert Zorec is at the Laboratory of Neuroendocrinology, Institute of Pathophysiology, School of Medicine, University of Ljubljana, 1105 Ljubljana, Slovenia.
confirming a role of [Ca2+]i in dynamic changes of plasma m e m b r a n e area. As anterior pituitary cells are excitable and possess voltage-gated calcium channels (VGCCs), it has been suggested that control of the firing frequency of Ca 2+dependent action potentials plays a role in basal and stimulated secretion (Ozawa and Sand 1986). In addition, in the absence of spontaneous electrical activity, a steady-state entry of Ca 2 +, possibly also t h r o u g h VGCCs (Mollard et al. 1994), was shown to support secretory activity (Zorec et al. 1991). A direct role of VGCCs in secretory activity of melanotrophs was confirmed by T h o m a s et al. (1990), who showed that a rise in [Ca2+]i, elicited by activation of VGCCs, induced a rise in Cn~, indicating a direct
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A
]
B
5 pF •
. . . . . . 5.6 pF
°
5.5 pF I
". " . . . . . .
I
300 s
C 10 fF
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,!
3s Figure 1. Changes in membrane capacitance (Cm) recorded in single bovine lactotrophs with use of the whole cell patch clamp technique. Effect of cytosol dialysis with pipette filling solution containing (A)I pM and (B) 0 pM [Caz*] on Cm recorded in two lactotrophs. Numbers adjacent to records represent resting Cm. Arrows indicate the start of cytosol dialysis. (C) Short episode of membrane capacitance signal recorded at high gain. Discrete steps in Cm are due to single exocytotic and endocytotic events [see Zorec et al. (1991), Zupan~i6 et al. (1994)]. role of VGCCs in stimulus-secretion coupling in pituitary cells. In contrast to neurons, where a tight coupling between the activation of VGCCs and release from presynaptic sites persists typically at room temperature, pituitary cells secrete robustly only near physiological temperatures (Thomas et al. 1990, Fomina and Levitan 1995). This may be due to differences in the molecular makeup of exocytotic machinery, VGCCs and/or coupling between VGCCs and exocytotic machinery in the two cell types. Which VGCCs are present in pituitary cells? Electrophysiological studies allowed VGCCs to be classified into two groups: low-voltage activated (LVA, molecular structure not determined) and high-voltage activated (HVA) voltagegated Ca 2+ currents, which consist of a~, ¢t2-$, [3, and ~ subunits, where the tx1 subunit functions as the channel. Cloning has revealed that the cq subunit is coded by six genes, whereas the [3 subunit is coded by four genes (Perez-Reyes and Schneider 1995). Most pituitary cells possess LVA and HVA Ca 2+ currents [Armstrong and Matteson (1985), Cota TEM Vol. 7, No. 10, 1996
(1986), see Stojilkovi~ and Catt (1992), see Mollard and Schlegel (1996)], but in some cell types, depending on gender, animal species, or age, only one Ca 2+ current is present (see Corrette et al. 1995). With the use of specific pharmacological agents, it was possible, for example, to determine the diversity of HVA currents in melanotrophs of various species as L-, P-, Q-, and N-types (see Corrette et al. 1995, Ciranna et al. 1996). Interestingly, the LVA current was found to be sensitive to dihydropyridines, suggesting this current permeates through a subtype or modified L-type channel (Kocmur and Zorec 1993). By molecular biology approaches, mRNA of OtlA, O~IB, alC, and OtlD subunits were studied in clonal pituitary cells (Li6vano et al. 1994), revealing that all subunits were present in AtT-20 cells, whereas all but the ctm subunit were detected in GH 3 cells. In situ hybridization studies confirmed that anterior pituitary cells express tXlA, tx,B, ~1c, and a~D subunits, although the mRNA level for eqc and Ct1D subunits appeared to be more abundant (Tanaka et al. 1995). This may explain
the untight depolarization-secretion coupling at low temperature in pituitary cells (Fomina and Levitan 1995), as the tX1A and txm subunits have been shown to interact with SNARE molecules of exocytotic machinery (Bezprozvanny et al. 1995, Sheng et al. 1996) (Appendix). An increase in [Ca2+]i may also be caused by release from intracellular Ca 2+ stores, such as that generated by inositol 1,4,5-trisphosphate (IP3). It was shown that the addition of GnRH induces oscillations of [Ca2+]i in gonadotrophs, which are independent of extracellular Ca 2÷ (Shangold et al. 1988) and can be mimicked by cytosol dialysis with IP 3 (Tse and Hille 1992). These oscillations are sufficient to support secretory activity at the cellular level (Tse et al. 1993). By employing high temporal resolution Cm measurements to monitor simulaneously exocytosis and [Ca2+]i in identified gonadotrophs of adult male rats, it was shown that each cycle of increase in [Ca2÷]i, owing to release from intracellular stores, can trigger secretion (Tse et al. 1993). In gonadotrophs, VGCCs may only play a role to replenish intracellular Ca 2÷ stores and to deliver Ca 2÷ acting as a coactivator in the stimulation of Ca 2÷ release from the IPa-sensitive stores (Tse and Hille 1993). Interestingly, an application of thyrotropin-releasing hormone (TRH), an IP 3liberating agent, increases secretory activity in single lactotrophs, as well (Fomina and Levitan 1995). In this cell type, both Ca 2+ delivery systems are competent in supporting secretory activity. A question to be studied in the future is which of the two mechanisms is more efficient in inducing secretory activity.
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What D e t e r m i n e s the S p e e d o f S e c r e t i o n f r o m Pituitary Cells?
In comparison to neurons, pituitary cells secrete at a significantly slower rate. Hormone delivery to the target is limited by the transport in blood circulation, whereas in neurons the speed of exocytosis can be critical, as signal transduction in a synapse can occur in less than a millisecond (Katz 1969). There are several hypotheses as to why neuroendocrine secretion is slower than neural secretion: (a) that the VGCCs and secretory vesicles (granules) are not molecularly colocalized, (b) that the exocytotic machinery is intrinsically slower, or that
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the granules are not normally docked, but positioned distant from the plane of the membrane, separated by a cytoskeletal network, which must be disassembled upon a rise in [Ca2+]i [see Trifar6 and Vitale (1993)]. The third hypothesis contrasts with results of Parsons et al. (1995), which show that a significant number of granules are docked in pituitary and chromaffin cells. Moreover, it is unlikely that the exocytotic machinery of neuroendocrine cells is intrinsically slow, as the maximal rate of exocytosis recorded in chromaffin cells (Heinem a n n et al. 1994) and bipolar neurons (Heidelberger et al. 1994) is remarkably similar. Thus, it is likely that neuroendocrine secretion is slower than neural secretion because VGCCs and granules are not molecularly colocalized. This possibility is supported by the absence of morphological structures resembling active zones in pituitary cells, where channels and vesicles are colocalized in synapses (Robitaille et al. 1990). The shorter the distance between a Ca 2+ source and secretory vesicles, the faster the secretion. Furthermore, the distance between Ca 2+ sources (VGCCs and intracellular stores) and secretory vesicles determines the biochemical requirements of Ca 2+ receptor(s) that trigger exocytosis. Within a few nanometers of a Ca 2+ source, [Ca2+]i has been estimated to reach near millimolar levels, whereas at about 1 micrometer away, the concentration is expected to be in the low micromolar range (Smith and Augustine 1988) (Figure 2). Thus, near VGCCs, a low-affinity Ca 2÷ receptor(s) would be adequate to trigger secretion, whereas at greater distance the Ca 2+ receptor(s) would have to have a higher affinity. In agreement with this, in bipolar neurons the threshold for secretion is around 50 pM [Ca2+]i (Gersdorff and Matthews 1994), whereas pituitary cells start secreting at submicromolar [Ca2+]i (Rupnlk and Zorec 1995). The sensitivity of exocytotic machinery for Ca 2+ appears to be associated with the size of secretory vesicles (Figure 2). With larger secretory vesicles, the Ca 2+ receptor(s) appears to be positioned away from the Ca 2+ source. The results of Chow et al. (1994) indicate that the Ca 2÷ receptor(s) is quite a distance away from the Ca 2÷ source (Figure 2), as [Ca2+]i sensed by the secretory machinery during a short depolarization was estimated to be around 5 pM (Chow et al. 1994). This may be be386
I
150 n m
-0.3
LDCG
# M [ C a 2+]
~ ' I S "
.......
IS
- 5 #M [Ca 2+] -100#M[Ca
2+] SV
R.~ ".. R ' "~ii" "
VGCC Figure 2. Synaptic vesicle (SV, 50 nm in diameter) size is compared with the size of a secretory granule (large dense core granule, LDCG, 300 nm in diameter). Dashed lines indicate the steady-state [Ca2+]i during opening of a voltage-gated calcium channel (VGCC), axially from the channel mouth (Smith and Augustine 1988). Note that the dimensions of an SV span a narrow range of [Ca2+, whereas the dimensions of an LDCG span a much wider range of [Ca2+] upon opening ofa VGCC. An alternative Ca2÷ source may be a release from intracellular stores (IS), which appears to be functionally equally distant in comparison with VGCCs from Ca2+ receptors (R) that trigger exocytosis. Thus, it is appealing to think that one type of Ca2+ receptor triggering exocytosis is shared by different Ca2+ sources (VGCCs, intracellular stores) in pituitary cells. cause granules and VGCCs may not be molecularly colocalized in the plane of the membrane (Chow et al. 1994). In contrast, we may also consider that in pituitary cells, the Ca 2+ receptor(s) is positioned deeper in the cytoplasm. An advantage of such a distantly positioned Ca z+ receptor(s) is that it could sense Ca 2+ release from an intracellular store and from VGCCs and transduce the signal from both Ca 2+ sources into exocytosis of docked granules. •
Ca z+ R e c e p t o r ( s ) and E x o c y t o t i c Machinery
A major candidate for a vesicle-bound low-affinity Ca 2+ receptor in synapses is synaptotagmin (Brose et al. 1992), also present in the pituitary (Marqu6ze et al. 1995), although its role in exocytosis is controversial [compare Shoji-Kasai et al. 1992, Geppert et al. (1994)]. One reason for this is that multiple isoforms of synaptotagmin (Li et al. 1995) may be coexpressed in a cell. In contrast, a candidate for a cytosolic high-affinity Ca 2+ receptor(s) in pituitary cells m a y be p145 (Walent et al. 1992), a soluble pro-
tein that was purified based on its ability to stimulate Ca2+-triggered secretion from permeabilized PC 12 cells. Another candidate is annexin II, a m e m b e r of a family of Ca 2+ and phospholipid-binding proteins (Ali et al. 1989). In addition to Ca 2+, activation of GTP-binding proteins can trigger exocytosis. Both monomeric (Lledo et al. 1993, Rupnik et al. 1995) and heterotrimeric (Rupnik and Zorec 1995) GTP-binding proteins appear to be involved in regulated secretion of pituitary cells. The key question, pertinent not only to pituitary secretion, is how molecular events transduce activation of Ca 2÷ receptors into exocytosis. There are several views as to how exocytosis might be controlled by a Ca 2+ receptor(s). One possibility is by affecting cytoskeleton around granules, which is thought to present a barrier to exocytosis (Trifar6 and Vitale 1993). Another model predicts a "scaffold," a macromolecular structure that bridges the gap between the plasma and granule membrane, and that can direct and regulate curvature and promote conditions that are favorable for membrane fusion (Monck and
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Fernandez 1994). The number of proteins i m p l i c a t e d in r e g u l a t e d exocytosis has been growing in the last decade, a n d it was p r o p o s e d that t h e i r interactions controlling exocytosis functionally res e m b l e the activity of m o l e c u l a r chaperones (Burgoyne a n d M o r g a n 1995), w h i c h regulates p r o t e i n folding a n d the a s s e m b l y a n d d i s a s s e m b l y of multiprotein complexes. Currently, p r o t e i n s f o r m i n g a c o m p l e x t e r m e d "the S N A R E complex" are t h o u g h t to p l a y a universal role in vesicular traffic a n d in regulated exocytosis (S6llner a n d R o t h m a n 1994, see appendix), but there is no direct evidence that these p r o t e i n s play a role in exocytosis o f p i t u i t a r y cells. In a d d i t i o n to proteins, a s y m m e t r i c lipid composition of m e m b r a n e s a p p e a r s to play a role in exocytosis, as well ( C h e r n o m o r d i k et al. 1995). In particular, p h o s p h o i n o s i tides a p p e a r as key regulators of vesicular traffic (see De Camilli et al. 1996). Although the u n d e r s t a n d i n g of molecu l a r m e c h a n i s m s of exocytosis in pituitary cells is in its infancy, we have presented a discussion o f a biophysical perspective that predicts that in p i t u i t a r y cells, different C a Z + s o u r c e s - - s u c h as VGCCs or i n t r a c e l l u l a r s t o r e s - - m a y share a Ca 2+ receptor(s) with s i m i l a r b i o c h e m i c a l properties,
*
Aekowledgments
R.Z. is s u p p o r t e d by the Ministry of Sciences a n d Technology o f Slovenia. S. Grilc is a c k n o w l e d g e d for providing cell cultures, a n d R.H. Chow, M. Shipston, G. Z u p a n 6 i 6 , M. Rupnik, a n d L. Kocmur-Bobanovid a r e a c k n o w l e d g e d for their c o m m e n t s on the m a n u s c r i p t .
References All SS, Geisow MJ, Burgoyne RD: 1989. A role for calpactin in calcium dependent exocytosis in adrenal chromaffin cells. Nature 340: 313-315. Armstrong CM, Matteson DR: 1985. Two distinct populations of calcium channels in a clonal line of pituitary cells. Science 227:65-67. Bezprozvanny I. Scheller RH, Tsien RW: 1995. Functional impact of syntaxin on gating of N-type and Q-type calcium channels. Nature 378:623--626. Brose N, Petrenko AG, Sfidhof TC, Jahn R: 1992. Synaptotagrnin: a Ca2÷ sensor on the synaptic vesicle surface. Science 256:10211025.
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Burgoyne RD, Morgan A: 1995. Ca2÷ and secretory-vesicle dynamics. Trends Neurosci 18: 191-196. Chemomordik L, Kozlov MM, Zimmerberg J: 1995. Lipids in biological membrane fusion. J Membr Biol 146:1-14. Chow RFI, Klingauf J, Neher E: 1994. Tmae course of Ca2+ concentration triggering exocytosis in neuroendocrine ceils. Proc Natl Acad Sci USA 91:12,765-12,769. Ciranna L, Feltz P, Sehlichter R: 1996. Selective inhibition of high voltage-activated L-type and Q-type Ca2+ ctu~nts by serotonin in rat melanotrophs. J Physiol (Lond) 490.3:595-609. Corrette BJ, Bauer CK, Schwarz JR: 1995. Electrophysiology of anterior pituitary cells. /n Scherubl H, Hescheler J, eds. The Electrophysiology of Neuroendocrine Cells. Boca Raton, FL, CRC Press, pp 102-143. Cota G: 1986, Calcium channel currents in pars intermedia cells of the rat pituitary gland. J Gen Physiol 88:83-105. DeCarnilli P, Emr SD, McPerson PS, Novick P: 1996. Phosphoinositides as regulators in membrane traffic. Science 271:1533-1539. Fomina AF, Levitan ES: 1995. Three phases of TRH-induced facititation of exocytosis by single lactotrophs. J Neurosci 15:4982-4991. Geppert M, Goda Y, Hammer RE, et al.: 1994. Synaptotagmin I: a major Ca2÷ sensor for transmitter release at a central synapse. Cell 79:717-727.
Marqu~ze B, Boudier JA, Mizuta M, Inagaki N, Seino S, Seagar M: 1995. Cellular localization of synaptotagmin I, II, and III mRNAs in the central nervous system and pituitary and adrenal glands of the rat. J Neurosci 15:49064917.
Gersdorff H, Matthews G: 1994. Dynamics of synaptic vesicle fusion and membrane retrieval in synaptic terminals. Nature 367:735739.
Perez-Reyes E, Schneider T: 1995. Molecular biology of calcium channels. Kidney Int 48: 1111-1124.
Heidelberger R, Heinemann C, Neher E, Matthews G: 1994. Calcium dependence of the rate of exocytosis in a synaptic terminal. Nature 371:513-515. Heinemann C, Chow RH, Neher E, Zucker RS: 1994. Kinetics of the secretory response in bovine chrornaffin cells following flash photolysis of caged Ca2÷. Biophys J 67:2546--2557. Katz B: 1966. Nerve, Muscle and Synapse. New York, McGraw-Hill, p 115. Kocmur L, Zorec R: 1993. A new approach to separation of voltage-activated Ca currents in rat melanotropbs. Pfltigers Arch 425:172-174. Li C, Ullrich B, Zhang J~, Anderson RGW, Brose N, Sfidhof TC: 1995. Ca2+-dependent and -independent activities of neural and non-nenral synaptotagmins. Nature 375:594-599. Li6vano A, Bolden A, Horn R: 1994. Calcium channels in excitable cells: divergent genotypic and phenotypic expression of al-subunits. Am J Physiol 267:C411-C424. Lledo P-M, Vernier P, Didier J-D, Mason WT, Zorec R: 1993. Inhibition of Rab 3B expression attenuates Ca2÷-dependent exocytosis in rat anterior pituitary cells. Nature 364:543544.
Mollard E Schlegel W: 1996. Why are endocrine pituitary ceils excitable? Trends Endocrinol Metab 7:361-365. Mollard P, Theler J-M, Guerineau N, Vacher P, Chiavaroli C, Schlegel W: 1944. Cytosolic Ca2+ of excitable pituitary cells at resting potentials is controlled by steady state Ca2+ currents sensitive to dihydropyridines. J Biol Chem 269:25,158-25,164. Monck JR, Fernandez JM: 1994. The exocytotic fusion pore and neurotransmitter release. Neuron 12:707-716. Morgan A, Burgoyne RD: 1995. Is NSF a fusion protein? Trends Cell Biol 5:335-339. Neher E, Mm-ty A: 1982. Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin ceils. Proc Nail Acad Sci USA 79:6712-6716. Ozawa S, Sand O: 1986. Electrophysiology of excitable endocrine cells. Physiol Rev 66:887951. Parsons T, Coorssen JR, Horstmann H, Almers W: 1995. Docked granules, the exocytotic burst, and the need for ATP hydrolysis in endocrine cells. Neuron 15:1085-1096.
Robitaille R, Adler EM, Charlton MP: 1990. Strategic localization of calcium channels at transmitter release sites of frog neuromuscular synapses. Neuron 5:773-779. Rupnik M, Law GJ, Northrop AJ, Mason WT, Zorec R: 1995. Brefeldin A and a synthetic peptide to ADP-ribosylation factor inhibit regulated exocytosis in melanotrophs. Neuroreport 6:853-856. Rupnik M, Zorec R: 1995. Intraceilular chloride modulates Ca2+-induced exocytosis from rat melanotrophs through GTP-binding proteins. Pflfigers Arch 431:76-83. Shangold GA, Murphy SN, Miller RJ: 1988. Gonadotropin-releasing hormone-induced Ca2÷ transients in single identified gonadotropes require both intracellular Ca2. mobilization and Ca 2+ influx. Proc Natl Acad Sci USA 86:6566-6570. Sheng Z-H, Rettig J, Cook T, Catterall WA: 1996. Calcium-dependent interaction of N-type calcium channels with the synaptic core complex. Nature 379:451-454. Shoji-Kasai Y, Yoshida A, Sato K, et al.: 1992. Neurotransmitter release from synaptotagmin-deficient clonal variants of PC 12 ceils. Science 256:1820-1823.
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Sikdar SK, Zorec R, Brown D, Mason WT: 1989. Dual effects of G-protein activation on Cadependent exocytosis in bovine lactotrophs. FEBS Lett 253:88-92. Smith SJ, Augustine GJ: 1988. Calcium ions, active zones and synaptic transmitter release. Trends Neurosci 11:458--464. S611ner T, Rothman JE: 1994. Neurotransmission: harnessing fusion machinery at the synapse. Trends Neurosci 17:344-348. Stojilkovi6 SS, Catt KJ: 1992. Calcium oscillations in anterior pituitary cells. Endocr Rev 13:256-280.
SNARE hypothesis (S611ner and Rothman 1994). This hypothesis engages several proteins in the regulation of exocytosis. N-ethylmaleimide-sensitive fusion protein (NSF) is an ATPase and binds to membranes in the presence of soluble NSF attachment proteins (SNAPs). The model postulates that receptors (SNAREs) exist for SNAP proteins and proposes that docking specificity is maintained when a transport vesicle-associated SNARE (v-SNARE) pairs with a cognate target-associated SNARE (tSNARE) in a unique match. The v-SNARE would be synaptobrevin or a hornologue, whereas t-SNARE would be SNAP 25 (synaptosome associated protein 25) and syntaxin or holomogues. SNAREs are targets of neurotoxins, allowing functional assignment of these
proteins in exocytosis. The complex of NSF/SNAPs/SNAREs (SNARE complex) dissasembles upon ATP hydrolysis by NSF, which is thought to power fusion of two membranes. Current evidence, however, supports an earlier prefusion role of NSF (Morgan and Burgoyne 1995), which is consistent with results of studies on pituitary ceils (Parsons et al. 1995). There are additional regulatory components associated with the SNARE complex, such as synaptotagmin and VGCCs (S611ner and Rothman 1994, Sheng et al. 1996), which appear to inhibit the constitutive pathway. This block is relieved upon a rise in [CaZ+]i. One of the challenges for the future is to test whether the SNARE hypothesis explains regulated exocytosis in pituitary cells.
Tanaka O, Sakagami H, Kondo H: 1995. Localization of mRNAs of voltage-dependent Ca2+-channels: four subtypes of a t- and subunits in developingand mature rat brain. Mol Brain Res 30:1-16. Thomas P, Surprenant A, Almers W: 1990. Cytosolic Ca2+, exocytosis and endocytosis in single melanotrophs of the rat pituitary. Neuron 5:723-733. Trifar6 JM, Vitale ML: 1993. Cytoskeleton dynamics during neurotransmitter release. Trends Neurosci 16:466--472. Tse A, Hiile B: 1992. GnRH-induced Ca2÷ oscillations and rhythmic hyperpolarizations of pituitary gonadotropes. Science 255:462464. Tse A, Hille B: 1993. Role o[ vohage-gated Naand Ca2+ channels in gonadotropin-releasing hormone-induced membrane potential i n changes in identified rat gonadotropes. En- John Rinzel, Joel Keizer, and Yue-Xian Li docrinology 132:1475-1481. Tse A, Tse FW, Almers W, Hille B: 1993. Rhythmic exocytosis stimulated by GnRH-induced calcium oscillations in rat gonadotropes. Sci- The response of gonadotrophs to secretagogues involves dose-depenence 260:82-84. dent, complex dynamic patterns of electrical activity and inositol 1,4,5Walent JH, Porter BW, Martin TFJ: 1992. A novel 145 kd brain cytosolicprotein reconsti- trisphosphate (InsP3)-induced Ca2+ mobilization, including pulsatility tutes Ca2+-regulated secretion in permeable and oscillations on multiple time scales from milliseconds to minutes. neuroendocrine cells. Cell 70:765-775. Detailed in vitro experiments have enabled the identification of key Zorec R, Sikdar SK, Mason wr: 1991. Inmechanisms that underlie the plasma membrane (PM) electrical excitcreased cytosoliccalcium stimulates exocytoability and endoplasmic reticulum (ER) calcium excitability. We sumsis in bovine lactotrophs: direct evidence from changes in membrane capacitance. J marize these findings and review computer simulations of a biophysiGen Physiol 97:473--497. cal model that resynthesizes and couples these components and that Zupan~i6 G, Kocmur L, Verani6 P, Grilc S, reproduces quantitatively the observed time courses and dose-response Korda M, Zorec R: 1994. The separation of exocytosis from endocytosis in rat melan- characteristics, as well as effects of various pharamacological manipotroph membrane capacitance records. J ulations. The theory suggests that cytosolic calcium is the primary Physiol (Lond) 480:539-552. TEM
Modeling Plasma Membrane and Endoplasmic Reticulum Excitability Pituitary Cells
messenger in coordinating the PM and ER regenerative behaviors during ER depletion and refilling. © 1996, Elsevier Science Inc. (Trends
•
APPENDIX
E n d o c r i n o l M e t a b 1996;7:388-393).
The Snare Hypothesis In eukaryotic cells, several steps in the secretory pathway, including synaptic transmission, have been shown to depend on closely related proteins, which led Rothman et al. to propose a general mechanism of docking and fusion, the 388
John Rinzel is at the Mathematical Research Branch, NIDDK, National Institutes of Health, Bethesda, MD 20892; and Joel Keizer and Yue-Xian Li are at the Institute of Theoretical Dynamics (with Keizer also at the Section on Neurobiology, Physiology, and Behavior at the University of California, Davis, CA 95616, USA.
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