Control by protein phosphorylation and G proteins of calcium oscillations, and pituitary hormone secretion

Thesis

Reference

Control by protein phosphorylation and G proteins of calcium oscillations, and pituitary hormone secretion

CHIAVAROLI, Carlo

Abstract

Le corps humain est composé d'environ 1250 g de calcium, la quasi totalité se trouvant dans les os sous forme d'hydroxyapatite [Ca10(PO4)6(OH)2], assurant la rigidité de notre squelette. En plus de ce rôle constitutionnel, le calcium sous sa forme ionisée ou libre (quelques grammes du calcium total présent dans le corps) est un élément dynamique de première importance pour le bon fonctionnement de l'organisme: c'est un messager secondaire. La concentration de calcium libre dans l'espace extracellulaire, [Ca2+]e, est 10000 fois plus élevée que la concentration intracellulaire cytosolique, [Ca2+]i, (environ 1mM et 100 nM respectivement). Lorsqu'un signal externe (le messager primaire: une hormone par exemple) parvient à sa cellule cible et reconnaît son récepteur il induit dans de nombreux systèmes cellulaires une modification transitoire de la [Ca2+]i. Il est connu depuis longtemps que le calcium est un élément essentiel pour la sécrétion des cellules endocrines. Le développement de sondes fluorescentes sensibles aux ions Ca2+, telles que le fura-2, a permis d'effectuer des études temporelles et [...]

CHIAVAROLI, Carlo. Control by protein phosphorylation and G proteins of calcium oscillations, and pituitary hormone secretion. Thèse de doctorat : Univ. Genève, 1992, no.

Sc. 2545

DOI : 10.13097/archive-ouverte/unige:102346

Available at:

http://archive-ouverte.unige.ch/unige:102346

Disclaimer: layout of this document may differ from the published version.

l!

i

j

io

UNIYERSITE DE GENEVE FACULTE DE MEDECINE

Fondation pour Recherches

Médicales

^{Docteur W. Schlegel}

FACULTE

DES

SCIENCES

Département de Biochimie Professeur M. Balllvet

CONTROL BY PROTEIN PHOSPHORYLATION AND G PROTEINS OF CALCIUM OSCILLATIONS,

AND PITUITARY HORMONE SECRETION

THESE

présentée à ta Faculté des Sclences de I'Unlversité de Genève

pour obtenlr te grailede Docteur ès'Sclencest mention blochimlque

Carlo CHIAVAROLI

de Montegrlmano (Italte)

thèse No 2545

pâr

UNIVERSITE DE GENEVE FACULTE DE MEDECINE

Fondation pour Recherches

Médicales

Docteur W. Schlegel

FACULTE

DES

SCIENCES

Département de Biochimie Professeur M. Ballivet

CONTROL BY PROTEIN PHOSPHORYLATION AND G PROTEINS OF CALCIUM OSCILLATIONS,

AND PITUITARY HORMONE SECRETION

THESE

présentée à ta Faculté des Sciences de I'Université de Genève

pour obtenir Ie grade _de Docteur ès Sciences, mention biochimique

Carlo CHIAVAROLI

de Montegrimano (Italie)

Thèse No 2545

GENEVE par

UNIVERSITÉ DE

^{G E}

^NÈVT

FACULTÉ ^{DES SCIENCES}

Doclorat ès ^sclences menlion biochimiclue

Thèse de ltlonsieur ^Carlo CHIAVAROLI

intitulée

^:

''CONTROL BY PROTEIN PHOSPHORYLATION ^AND G PROTEINS OF CALCTUM OSCILLATIONS, ^AND

PITUITARY HORMONE SECRETION.''

La Faculté des Sciences, sur le ^préavis de Messieurs W.

^SCHLEGEL'

maître

d'enseignement

et de recherche (Facutté de Médecine -

^Fondation

pour recherches médicales) et M. ^BALLIVET, professeur ordinaire

(Département.

de biochimie) codirecteurs de thèse et B. ^DUFY,

^docteur

(Université de Bordeaux, France), autorise I'impression de la

^présente

thèse,

sans

exprimer d'opinion sur les propositions qui y sont

énoncées.

Genève,

le

6

juillet

1992

Thèse - ²⁵⁴⁵

Le Doyen, ^Pierre

^BURI

A Ctaire

Felix ^qui potuit rerum cognoscere causas

Remerciements

Je tiens à remercier tout particulièrement et très chaleureusement le Dr. Werner Schlegel pour sa disponibilité et sa gentillesse naturelle, ainsi que pour la qualité de ses nombreux conseils

qu'il

m'a prodigués tout au long de cette thèse. Pour moi

il

sera toujours un modèle et un ami.

J'exprime également toute ma reconnaissance ^au Dr. Gaston Zahndpour

m'avoir

accueilli ^{à la} Tulipe, ainsi qu'aux Profs. Claes Wollheim, Dermot

Cooper, Bob Clark et Daniel [,ew pour leurs

judicieux

_conseils.

J'adresse de même mes remerciements au Prof. Marc

Ballivet

et au Dr.

Bernard

Dufy

pour

avoir

accepté de corriger ce travail et de fonctionner comme jurés officiels.

J'aimerais également remercier les Drs. Nicolas Demaurex, Pierre Vacher, Yvan Lagnaux,

Karl-Heinz

Krause, Patrice

Mollard,

Jean-Marc Theler,

Ro semary Murray-Whelan, ^Mari sa Jaconi, Françoi se A ^s simacopoulo s, Stephen Rawlings et

William

Pralong pour les entretiens bénéfiques dont

j'ubénéficiê

durant cette thèse.

Toute ma gratitude va aussi à Isabelle Piuz pour son assistance

impeccable et sa

disponibilité

^{ainsi qu'à} Jacqueline Derrien, Nathalie Guérineau et Mardjan Hezarehpour leur aide efficace, sans oublier bien sûr toutes les personnes, qui de près ou de

loin m'ont

assisté durant ces

quelçes

années.

Pour terminer

je

_tiens à

féliciter

ma femme Nathalie pour moavoir supporté durant ^ces longues années, ainsi que mes parents, Ernesto et Marina, dont les sacrifices, acceptés avec le sourire sont aujourd'hui finalement

récompensés.

Content

I. Reader's guide

II. Preface

III. Introduction

1. Adenopiruitary cells, an overview

1.1 Clonal cells derived

from

the adenopituitary

2. Stimulus-secretion coupling: the concept

2.I.

The

stimuli:

hypothalamic ^factors

2.l.L

Activators

of

secretion

2.I.2. Inhibitors of

secretion 2.2. Cell surface receptors

3. Heterotrimeric ^guanine nucleotide-binding ^proteins 3.1 Mechanism

of

G proteins activation

3.2. Sub-classes of heterotrimeric G proteins

3.2.t.

The

G.

sub-class 3.2.2.

Th"

Gi sub-class 3.2.3. The Gt sub-class 3.2.4. The Go sub-class

3.2.5. The other sub-classes

of

G proteins

4. Two important effectors coupled to G proteins 4.1. Phospholipase C

4.2.

Adenylyl cyclase

p.7

p.8

p.9 p.9 p.12

p.12 p.

13

p.

13

p.17 p.

18

p.20 p.22 p.25 p.25 p.25

p. z7

p.27 p.28

p.29

p.29

p.30

5. Calcium ions as cytosolic messenger

5. 1. Cytosolic calcium-binding ^proteins

5.2.Proteins that transport

C**

through membranes

5.2.L.The

Ca2+-AIPase

5.2.2. The Na+/C

**

^{exchanger of} the plasma membrane

5 .2.3 . Calcium channels 5.3. Intracellular stores

5.3. 1. Endoplasmic

reticulum

5 .3.2. Sarcoplasmic

reticulum

5.3.3.

Mitochondria

5.4. Examples

of lC**limodification in

^clonal

^pituitary

^cells

5.4.L

Action

potential and spontaneous [Ca2*]i oscillations 5.4.2. _[Ca2*]i modification by an hormone

6. Some other second messengers 6.1.

cAMP

and protein kinase

A

6.2.

Inositol

^phosphates

6.2.I.

Role of

Ins(l,4,5)Ps

and

Ins(l ,3,4,5)P4in

[Ca2*]i homeostasis

6.3.

Diacylglycerol

and protein kinase C

7. Protein phosphatases

8. Secretion

p.32

p. ₃₅

p.36

p. 36

p.37 p.37

p.

4r p.41 p.43 p.45 p.46 p.46

p. 47

p.50 p.50

p. 53 p. 55

p.56

p. 58

p.59

IV. Experimental Approaches

1. Fluorescent dyes

l.I

Fwa-Z excitation spectrum 2. Setup

for

_[Ca2+Ji measurements

V. Publications Publication I:

Modulation

of calcium

influx

by protein

phosphorylation

in

single intact clonal

pituitary

cells.

p.62 p.62 p.65 p.67

p.69 p.70

Publication II: ^p.95

Simultaneous monitoring

of

cytosolic

free calcium and exocytosis at the single cell level.

Publication III: P.120

Spontaneous [Ca2+1i oscillations and G. expression are inversely correlated

with

secretory granule content in

pituitary

cells.

VI. Synthesis ^p.

¹⁵⁰

VII. ^References

^{p. 155}

p. r93

VIIL Résumé de thèse

I. Reader's ^guide

This

work

deals

with

the

regulation

of endocrine secretion.

It

was performed on adenopituitary cells, which control many functions such as growth, metabolism, lactation, stress, sexual development, etc.

In

the

introduction,

we

will

first recall the physiological context

of pituitary

hormone secretion and

its

control by the hypothalamus. Travelling along the pathways

for

"stimulus-secretion coupling" we

will

describe the nature of the hypothalamic agents which influence

pituitary

cells, some plasma

membrane receptors, the proteins (G proteins) that transduce the ^message

in

the plasma membrane after the occupancy of a receptor, some of the effectors

activated, and the second messengers generated. The elements involved in the regularion

of

the inrracellular level

of C*+

^([Ca2*]i)

will

be largely discussed.

Then we

will contnue

^our

trip looking

at protein kinases and phosphatases, which

modify

upon activation, the release of pituitary hormones.

A brief "Experimental Approaches"

section

will follow,

explaining essentially how to measure the level

of

_;Ca2+1,

in

single cells.

Three

pubtications produced during this

thesis

form

the core of

this work.

Each contains Introduction, Materials and Methods, Results, and

Discussion sections.

A

short

final

synthesis, dealing

with

the regulation

of

_[Ca2+]l oscillations

II. Preface

There is an analogy between

politics

and the endocrine system.

Usually

in

a stable democratic government, tasks need to be defined precisely

in

order to avoid anarchy. The same is true

in

biology.

The endocrine system is composed

of

several glands (pituitary, thyroid, parathyroid, adrenal, ^gonads, pancreas, etc.) located in different areas of the body that produce hormones

with

different functions. These glands, which are

richly

vascularized,release their secretory products directly into the bloodstream.

The anterior

pituitary

is under the control of the hypothalamus.

If

we compare the hypothalamus to the President of the endocrine system, the adeno-

pituitary

can be viewed as the Prime Minister. This gland produces major

hormones (laws) which regulate the physiology of the other glands ^(the regional governments). Once secreted

into

the blood the pituitary hormones bind to the surface of target cells, which

in

turn increase the synthesis and the secretion

of

peripheral hormones (regional laws).

A

democratic government should be attentive to the reaction of the citizen of the counffy. People can manifest

if

they do not agree

with

the

policy

chosen by their leaders. The endocrine system is also regulated by feed-back reactrons.

We

will

now describe

briefly

the laws ordered by the Prime Minister,

following

a request of the President, and see what

will

happen

in

the counffy.

How

will

these laws be applied? How

will

the people react?

Later on, and this is the main part of this work, we

will

be interested

in

how the Prime

Minister

did get the request from the President, and how he managed,

with

the help

of

a battery of lawyers to promulgate the laws.

III. Introduction

L. Adenopituitary cells, an overview

The

pituitary ^(from

600 to 900 mg

in

the man) is a composed of two lobes, the anterior (the adenopituitary) and the posterior ^(the neuropituitary). The adenopituitary representsT5Vo of the total mass. Adenopituitary cells are under the control of hypothalamic agents which act either by stimulating or

inhibiting

secretion. These epithelial cells can secrete 6 major different hormones. Once secreted

into

the blood ttre

pituitary

hormones bind to the surface of target cells, which

in

turn increase the synthesis and the secretion of peripheral hormones, leading to the activation or

inhibition

of many cellular functions

(fig.

1).

Prolactin

(PRL, 198 aa), was

originally

thought of only for its role in the

initiation

and maintenance of lactation.

It

has now been found to have a wide range of physiological activities. Prolactin by enhancing the release of prolactin-

inhibiting

factors controls its own secretion ^(see Lamberts and

Macleod,

1990)

for

a complete review of the regulation of PRL secretion).

Growth hormone

^(GH, somatotropin, 1"91 aa) stimulates the

liver

to produce somatomedin-l (also called

insulin-like

growth

factor-l),

which

in

turn controls the growth of body tissues such as bone, cartilage, muscle, etc.

Thyroid-stimulating hormone

^(TSH,

c-chain

92 aa, B-chain ^{115 aa),} stimulates the

thyroid

gland to produce thyroid hormones and

fatty

acid release

Fotlicte-stimulating hormone

^{(FSH, a-chain} 92aa, B-chain ^{118 aa), acts} on ovarian

follicles

to grow and secrete esffadiol and stimulates also spennato- genesis

in

testis.

Luteinizing-hormone ^(LH,

cr-chain 92 aa, B-chain ¹¹⁵ aa), stimulates oocyte maturation and ovulation ^as

well

as progesterone secretion from ovary, and stimulates testis to produce testosterone.

Adrenocorticotropic hormone (ACTH,39

aa), increases the production

of

cortisol by the adrenal cortex and

fatty

acid release from fat cells. This

hormone is the key component

in

the

conffol

of the stress response.

It

has been thought

for

many years that a single adenopituitary cell could secrete only one hormone type. This led to ttre concept of ^66one cell type, one hormonett. However, recent evidences, based on reverse

hemolytic

^plaque as- say experiments (RHPA), have revealed that a single pituitary cell can secrete both GH and PRL (Frawley and al., 1985).

This technique is based on the

following:

a monolayer

of

cells is

surrounded by erythrocytes manipulated to bind the hormone secreted by the cells.

To this end the erythrocytes are coated

with

specific antibodies against the secreted hormone. Upon stimulation by a secretagogue, an immune complex is produced around the erythrocytes

in

the

proximity

of secreting cells. In presence of the complement system the erythrocytes are lysed, and a radial zone is then clearly

visible

around the secreting cells.

PITUITARY HYPOTHALAMUS

THYROID

BREAgT

TSH

, ACTH GH

PRL LH

FSH Growth

Factors GROWTH

I

Fig.

^1..

Hormones

^secreted by

the adenopituitary.

L.L. Clonal cells derived from the adenopituitary

Due to the large heterogeneity of the adenopituitary cells,

in

most of the work

pituitary

cell lines derived from rat adenopituitary tumors were used, such as the clones GH3B6 and

GHaCI

(Thshjian et al., 1968). These cells produce and secrete spontaneowly in

vitro

only PRL and

GH

(Tashjian, 1979). They have preserved their

ability

to respond to several hypothalamic or steroid hormones

in

a manner analogous to normal anterior

pituitary

cells. Using reverse hemolytic plaque assay

it

was shown that a single clonal cell can secrete either just _{one type} of hormone (PRL, GH) or both hormones (Boocfor and Schwartz, 1988).

2. Stimulus-secretion coupling: ^{the concept}

The concept of excitation-conffaction coupling (Sandow 1952),

developed for muscle, has found its endocrine countelpart, the stimulus-secretion coupling, some years later (Douglas, 1968).

This term

refers

to

a chain

of biochemical

mechanisms

linking

an

initial

event, the

stimulus, to

endocrine secretion. Adenopituitary cells are an excellent model

for

the study of the stimulus-secretion coupling. Cells communicate

with

each other by a variety

of

signal molecules, such as neurotransmitters, hormones and growth factors, that are detected by specific receptors on the plasma membrane

of

the target cell.

Important

stimuli

acting on the

pituitary

are mostly from hypothalamic origin.

The hypothalamus communicates directly

with

the adenopituitary

pituitary via

the portal blood system.

2.1".

The stimuli: hypothalamic factors 2.LJ. Activators of ^secretion

Thyrotropin-Releasing-Honnone

^(TRH) is a tripeptide exffacted

from

the hypothalamus

(Nair etal.,1970).

This oligopeptide is a powerful stimulator of TSH and PRL secretion ^(see Lamberts and

Macleod for

review 1990).

TRH

binds to a single class of receptors. The binding affrnity is compatible

with

its secretagogue effect (Gershengorn, 1982). The binding of TRH to its receptor is the

first

step of the signalling cascade which leads to secretion (fig. _2). This pathway includes the activation of a G protein which activates in turn the phospholipase C (PLC). This lead to the cleavage of phospholipid

phosphatidylinositol4,5-bisphosphate

(PIP/

and to the production of

inositol I,4,5

trisphosphate (Ins( 1,4,5)P3) and L-2-dracylglycerol (DG). The former diffrrses

in

the cytosol and promotes

Cah mobilization frominternal

stores, and DG stays

in

the

lipid

bilayer and activates protein kinase C (PKC), an

important stimulator of many cellular functions, which can be

itself

activated by

C**

(Nishizuka,1984). PKC is able to phosphorylate voltage-dependent calcium channels of the

L-type (L-VDCC),

and promotes Ca2*

influx. In

pituitary

cells

in

primary structure, TRH was found to increase secretion of PRL

within

4 s. The secretory pattern apparent

in

a cell population is

biphasic

^(Bj6ro et al., 1990).

A

similar pattern

of

secretion is found

in

clonal cells (Aizawa _and

Hinkle,

1985).

+ +

@ ^TRII

_R

_TRH

GTP ^GDP

lCa

2+

IP3

2+

Ca

1

/

2+

I

i

/

Ca

L. ^VDCC

Many cellular

^responses

including PRL

secretion

@-t'@...

Fig.2.

Mechanisms of

TRII

action. See text

for

explanations.

Vasoactive

Intestinal

Peptide

(VIP)

is a 28-amino acid residue and was

originally

extracted from pork intestine ^(Said and Porter, 1979), where

it

^{plays a} role

in

the regulation of bicarbonate secretion (Flemstrom et al., 1985).

It

was also detected

in

^great amounts

in

the hypothalamus (Besson et

a1.,I979).In pituitary

cells

it

stimulates the secretion of PRL, GH and

ACTH

(Gourdji _{et al.,}

1979; Enjalbert et al, 1980; Matsushita et a7., 1981; White et al., 1982, Abe et al., 1985).

Uponbinding

to its receptor (49 Kd), VIP activates the guanine nucleotide binding protein G. which

in

turn stimulates adenylyl cyclase and hence increases

cAMP

accumulation

within

the cells (Gourdji et a1.,1979). This leads to the activation of protein kinase

A (PKA),

a

key

eîzyme of the metabolism.

PKA

then phosphorylates some voltage-dependent calcium channels and promotes

C** ^{entry (fig.}

^{3, Chang}

^et

^al.,

I99I).In

contrast to TRH, the secretory pattern obtained after

VIP

stimulation is monophasic, secretion starts after a a lag time of 45-60 s (BjOro et al., 1990).

Some observations suggest that

VIP

could act on the same cells that produce and release

it,

as an autocrine factor. Indeed

VIP

is produced in the

pituitary

gland (Arnaout et al., 1986), where the peptide might have some autore- gulatory role in prolactin secretion (I.{agy et al., 1988).

@ ^VIP

_R

_VIP

GTP GDP ^ATP

^CAMP

lCa

2+¹

I

_Ca ²⁺

I

\ L. VDCC

Many cellular

responses

including PRL

secretion

@tt@...

Fig.

3. Mechanisms of

VIP action.

See text for explanation

2.L.2. Inhibitors of ^secretion

Dopamine

^is a powerful

inhibitor

of PRL secretion (e.g. Luque et al., 1986).

It

is produced by the hypothalamus. Its action may be modulated through both the adenylyl cyclase and

the

Ca2* mobilization/phosphoinositide pathways.

Dopamine

inhibits

adenylyl cyclase (Stoof and Kebabian, 1984) and also

C**

mobilization induced by

TRH

(Schofield, _{1983), a} potent activator of PRL secretion.

It

appears that they are two classes

of

dopamine receptors, the one present

in

the

pituitary

gland is the D-2rcceptor. Interest

in

D-2 receptors stems largely

from

the involvement

in

the pathology of neurological and

psychiaric

disorders such as parkinsonism (Kebabian and Calne, 1979). This receptor is also ttre target

of

drugs used to alleviate the main symptoms

of

schizophrenia (See- man, 1980).

The neurotransmitter

y-aminobutyric

acid

(GABA)

inhibits PRL secretion by activating voltage-dependent Cl- channels (Inenaga and Mason,

I987aand b). As a consequence the cell is hyperpolari zed, Ca2+ can not enter through voltage-dependent calcium channels of the L-type and the secretion is impeded.

Somatostatin

^(SRIF) is a teffadecapeptide secreted by hypothalamic neurons.

It inhibits

both basal and stimulated PRL, TSH,

ACTH

and GH release

in pituitary

cells (Vale et a1., 1974; Patel and Srikant, 1986). SRIF acrs by

The presence of

esradiol

is essential

for

somatostatin to

inhibit

PRL secretion.

In

fact, estradiol directly regulates the number

of

somatostatin receptors

in

lactotrophs (Kimura et d..,1986).

Pituitary

cells

in

culture usually respond to somatostatin even

in

the absence

of

any added esffadiol. This is explained by the presence

in

the culture medium of phenol red, a pH indicator, which can mimic the effects of esffadiol (Hofland et

al.,

1987).

Adenosine is produced and released by most animal cells, and acts as a

modulator

of

cellular activity. In the CNS adenosine acts via A1 receptors to

inhibit

adenylyl cyclase (Londos et al., 1980) and hence neurotransmitters release.

In

clonal

pituitary

cells, adenosine inhibits PRL and GH release

(Dorflinger _and Schonbrunn, 1985) by abolishingCa2+ transients linked to action potentials (Cooper et

al.,

^1989;

Mollard

et a1.,1991).

2.2. Cell surface receptors

The first step

in

stimulus-secretion coupling is the activation

of

one receptor by a specific ligand. Fischer's theory of the

"lock

and key" has evolved and

it

is clear today that receptors are proteins showing an intrinsic high

malleability of

structure (the Koshland's "induced-fit model"). This allows the discrimination between an extraordinary variety of chemical signals. The binding of the ligand induces a subtle change

in

the conformation of the receptor, and the signalling cascade is switched on. Most cell-surface receptors belong to one

of

three classes, which are defined by the ffansduction mechanism used.

Channel-linked

^receptors (ex: the acetylcholine receptor) are

ffansmitter-gated

ion

channels involved for rapid signalling

in

excitable cells principally. Once activated by a neuroffansmitter they may open or close, therefore changing the permeability of the plasma membrane.

Catalytic

receptors ^(ex: the EGF receptor) when activated act directly as

enzymes. They are transmembrane proteins

with

an intracellular domain acting

as tyrosine protein kinase.

During the last 5 years, more than 100 different G

protein-coupled

receptors (ex: adrenergic, TRH,

VIP

receptors, etc.) have been cloned and sequenced (Birnbaumer et al., L990; Savarese and Fraser,1992). Current models for the secondary and tertiary sffucture of G protein-linked receptors are based

in

large part on the known

folding

^pattern

of

an ancient retinal-linked protein, bacteriorhodopsin, a proton pump, that is found

within

the purple membrane

of

Halobacterium halobium. This pigment, which is not bound to any G protein, has seven s-helices which can ffansverse the membrane.

All

the G protein-coupled receptors belong to a superfamily of proteins that also span the plasma

membrane

seven times as judged by hydropathy analyses. They all have an extracellular amino terminus and an intracellular carboxy terminus.

Not

surprisingly the sequence homology between some different receptors is rather low, excepted

in

their transmembrane spanning regions. The extremities show

little

homology. This is

likely

the reason to the variable ligand and G protein specificity.

3. Heterotrimeric ^guanine nucleotide-binding proteins

The guanine nucleotide-binding proteins (G proteins) act as a

switch

that controls and transmits ttre information

from

receptors

to

a variety

of

intracellular effectors, as suggested first by Rodbell and coworkers

in I97I.

At

least 40 different hormones and neurotransmitters act through the activation

of

G proteins.

A

single signal can activate different receptor types, which

in

turn can activate

multiple

G protein molecules, thus amplifying the ligand binding event. Moreover, one activated G protein may be capable

of

interacting

with

many effector proteins that further

amplify

the signal. The known effectors whose

activity

is modulated by G proteins include adenylyl cyclase,

cyclic

GMP phosphodiesterase of photoreceptor cells, phospholipases C and A2, as

well

as some K+, Ca2+ and Cl- channels ^(see Birnbaumer et al., 1990 for review). Each effector modulates the concentration of different intracellular messengers.

In

addition each messenger can modulate the activity of other inffac- ellular messengers. Because a single receptor subtype can be coupled to multiple effectors and

multiple

receptor subtypes can activate a single effector, the G pro- tein-coupled interactions form complicated

networks

^(fig. 4).

Signal ffansducing G proteins are part of the GTPase superfamily wittr variable functions which have

in

common their

ability

to bind and

hydrolyze

GTP.

After

solubilization from membranes

with

appropriate detergents G proteins seem to behave

like

a monomer

of

approximately 100 kD. Each monomer is composed of

three distinct subunits,

cr, F and T separable by sodium dodecyl sulphate-polyacrylamide gel elecffophoresis (SDS-PAGE)

if

a reducing agent is used.

A1 A2 A3 Agonists

]*\*L,,','.41

liiiiffiE#

l:iii:i.11!iiiiii!!!3:l

IiiÊijjiÈIilljiiill

ffiÆ ffiffi

_lÊgry

^Receptors

G proteins

Effectors

SMI Izl <+ SM2€ ^SM3 Second Messengers

Fig.

4. The

signalling network.

For

clarity

only positive effects are shown. See text

for

explanation.

The atpha subunit (varying

from

39 to 52 kD) is the essential

part

of the G protein ^(see Stryer 1986; Spiegel L987;Neer and Clapham, 1988; Taylor 1990;

Birnbaumer et al., 1990;

Dolphin, l99l;

Simon

etal.,l99l

^for review).

It

hydrolyzes guanine nucleotides and interact

with

both receptors and effectors.

At

present, the G protein

family

is known to contain at least 16

different

^genes

that

encode

s subunits, four

that encode _B subunits (35-36

kD)

and multiple ^genes encoding ysubunits ^(8-11

kD).

Different G proteins are distinguished by their cx

subunits, though there are also subtle sffuctural and functional differences

in

some _{F atrd} y subunits. G proteins have been divided

in

sub-classes ^(see below).

3.L. Mechanism of G protein activation.

G proteins cycle between an inactive GDP-bound and an active GTP- bound state. When GDP is bound,

c

associates

with

p/y subunits to form an inactive Gopy complex that is membrane bound. In the ^presence of the signal molecule, the receptor is occupied and a subtle conformational change of the

s

subunit occurs. GTP takes the place

of

GDP and the alpha

subunit

^is

dissociated from the complex and activates the effector. The activation stops when GTP is

hydrolyzedto

GDP by the subunit

a

itself, then the

s

^subunit

reassociates

with

_Bly, and the cycle is completed (fig. _5).

Inac:tivc statc

GDP

Pi

H GTP

Receptor activation

GTP GDP

+

I

t

I

Activation of effectors t(

Fig.

5. Mechanisms

of

G

protein activation.

See text

for

explanation.

Activc state

Usually, the activation of the effector is mediated by the dissociated subunit cr. However, in a controversial series of experiments,

it

was observed that Biy subunits were capable of opening a muscarinic K+ channel (Logothetis et al.,

L987), by activating PLA2

(Kim

and Clapham, 1989;

Kim

et al., 1989) and producing arachidonic acid, whose metabolites affect K+ channels function. This observation indicates that the role of the B/ycomplex may be not resfficted to anchoring the cr subunit to the plasma membrane ^as first described. Moreover, G proteins can be precisely modified by

fatty

acids (polyisoprenylation,

myristylation,

farnesylation, ^see e.g. Linder et

al.,

I99L). These post-

transcriptional modifications ^{are believed} to anchor the ^cx subunits to the plasma membraneo and hence to facilitate the formation of the heterofrimer.

The use of nonhydrolyzable

GTP

analogues is often required to assess

if

a given

biological

event proceeds through the activation

of

a G protein.

Another important test can be performed by adding to the cells the anion AlF4-, which sffucturally mimics the anion PO+-3 and thereby can associate wittr GDP and

form

a complex reminiscent

of

^{GTP. Moreover,} some G proteins are the target of toxins which modulate their function ^(see below).

3.2. Sub-classes of heterotrimeric G proteins 3.2.1. The G, sub-class

G., the ubiquitously distributed ^guanine binding protein which activates

adenylyl

cyclase ^has been intensively studied.'Western blots after SDS-PAGE have indicated the presence

of

2 bands, one of 45

kD

and one of 52

kD.

Four

different

cDNA

were found, ^the four different

G.a

being generated by alternative splicing

of

a single gene (Kozasa et al., 1988).

Many different receptors ^(see Birnbaumer et al., 1990) are coupled to G., including receptors which bind

VIP,

B-adrenergic,

D-1

dopaminergic,

M-2

muscarinic, A-2 adenosine, S-2 serotonin, H-histamine,

LH,

FSH, TSH, MSH, CRF, GRF, chorionic gonadotropin, cholecystokinin, PTH, calcitonin, some prostacyclins, secretin, glucagon, and

ACTH. Activation of

G, leads to an increase of the intracellular

cAMP

level.

In

addition to stimulating adenylyl cyclase, G. also

directly

activates voltage-dependent

calcium

channels of the

L-type

(Mattera et al., 1989).

G. is a subsffate

for

cholera

toxin,

which ADP-ribosylates the ^q, subunit and reduces its

intrinsic

GTPase activity, so the G, protein is continuously

activated (Cassel and Selingeg 1977).

3.2.2. The Gi sub-class

G1 appears to be as ubiquitously distributed as G..

Three different

^G1

coded by a different gene (Itoh et al., 1988). Gircl and G13û are 94Vo identical

in

their amino acid sequence and they are 88 and857o identical, respectively to

Gizo'.

G12 proteins can be activated by adrenergic og,

dopaminergicD-2,

muscarinic

'dZ, GABAB, S-1 serotonin, opioid,

LH,

somatostatin, angiotensin

II,

thrombin, bradykinine, neuropeptide Y, neurotensin, platelet activating factor, some prostaglandins,

V-l

vasopressin, and adrenergic cr-1 receptors. The

existence of

multiple

forms

of

oq raised the question of which protein regulates which effector function.

In

addition of its

inhibitory

effect on adenylyl cyclase, Gir_t can also,activate potassium channels (VanDongen et al., 1988; Yatani et al.,

1988). Interestingly Gis (but not Gir and Giz) is also found on the Golgi apparatus where

it inhibits

the budding

of

secretory vesicles (Burgoyne, L992).

Gl proteins also affects adenyl cyclase, but

in

conffast to G, the result

of

the activation

of

Gi is an

inhibition of adenylyl

cyclase. Gi proteins are

subsffates

for

pertussis

toxin (Ui,

1984) which, by ADP-ribosylation uncouples G' from

inhibitory

receptors. As a consequence, cAMP levels are increased, as

in

the case of the ADP-ribosylation

of

G, by cholera toxin.

While

Gi and G. G subunits clearly differ, the _{F and} Tsubunits of purified G. and G' appear to be identical.

It

has been shown that B/yis ^{even a better}

inhibitor

of adenylyl cyclase than G1c (Katada et a1., 1984).

It

has therefore been suggested that

inhibition of

adenylyl cyclase is due to a direct effect on G, of the Biy complex released

from

activation of Gi (Gilman, 1984). However this is

still

matter to debate.

3.2.3. The G, sub-class

The G protein involved in visual transduction has been called

transducin

(Stryer, 1986).

Q (lg ^kD)

^{is the}

^link

between the

light

receptor (rhodopsin) and

cGMP

phosphodiesterase ^(the effector) exclusively in rod and cones cells.

Cyclic GMP maintains plasma membrane sodium channels of rod and cone cells

in

an open state, thereby regulating synaptic transmission. Thus cGMP

hydrolysis is the crucial event

in

visual transduction.

Two different ffansducins exist: one (Gtr) is present in rod cells, the other

(Gd in

cone cells (Grunwald et al., 1986). Transducin can be ADP-ribosylated by both

cholera

^(which activates

it)

and pertussis (which uncouples

it from

rhodopsin) toxins.

3.2.4. The Go sub-class

This G protein is expressed in high levels in mammalian brain (Sternweis and Robishaw, 1984). Goc represents a 39

kD

species that is the major pertussis

toxin

targetin brain tissue, where

it

may comprise as much as l7o of total membrane protein. Its Biycomplex is indistinguishable from that of

Q.

^Its function

in

signal ffansduction has not yet been clearly clarified ^(The

"o"

^{of Go}

stands

for other). It

may be coupled

with

the muscarinic acetylcholine receptor,

a-2

adrenergic, S-2 serotonin, the y-aminobutyric acid receptor, neuropeptide

I

and the opioid receptor.

It

activates K+ channels and

inhibits Cah

channels

phosphoinositides metabohsm.

It

has been proven that two different mRNAs (and hence

two

Go proteins, Go1 and Go2) generated by alternative splicing of a single ^gene exist (Tsukamoto et al., 1991).

3.2.5. The other subclasses of G proteins

Gop ^(Jones et al., 1989) is expressed exclusively

in

olfactory neuro- epithelial cells where

it

stimulates adenylyl cyclase.

It

has 9OVo

anino

acid

identity with

G, and

is

atarget

for

cholera toxin.

Gn,

often

called Gn, can activate

PLC

(Smrcka et al., 1991). PLC catalyses the hydrolysis of phosphatidylinositol bisphosphate

(PIP)

to inositol 1,4,5-trisphosphate (Ins(1,4,5)P:) plus diacylglycerol (DG) as described

in

fr9.2.

PLA2 stimulates the production

of

arachidonic acid (Axelrod _et al., 1988).

A

Gn response via the activation of PLC is observed in neutrophils stimulated with the chemoattractant fMLP. This response is clearly pertussis

toxin

sensitive

(Nakamura and

Ui,

1985). However,

in

other cell types (e.g., pinritary cells stimulated

with

TRH,

Martin

et al., 1986), the response is pertussis

toxin

insensitive.

To complete this enumeration

it

is convenient to mention that some other G proteins, each encoded by a different gene, have been recently discovered.

They were named

G"

^(Fong et al., 1988),

Gtt,

Gtz,

G6,

Grc, G15 and G16.

Their exact function has

still

to be clariûed. However G11 and G14 are also believed to activate PLC. They belong to the Gn class.

4. IWo important effectors coupled to G proteins 4.L. Phospholipase C

The generation

of

second messengers from phosphatidylinositol phosphates ^(see below)

is

catalyzed by enzymes from the phospholipase C

family

^(see Meldrum et

al., I99I for

review).

PLCs

are phosphodiesterases which hydrolyze the glycerophosphate bond of intact phospholipids to generate DG and aqueous inositol phosphates, including Ins(1r4r5)Ps. Four types of PLCs

in

mammalian tissues have been characterized(u, _F,

Y-d

^ô).

^All

^{are produced}

by a different ^gene and share

little

sequence homology (Ryu et al., 1987; Rhee et al., 1989), however three of them contain two

highly

conserved sequences that may represent common catalytic and/or regulatory domains.

All

four enzymes

(M.

between62 and 154

Kd)

are able to hydrolyze three coûlmon phospho- inositides, phosphatidylinositol ^(PI), phosphatidylinositol4-phosphate ^(PIP) and phosphatidylinositol 4, 5-bisphosphate (PIPz).

They

are Ca2* -dependent

proteins

and exist both as soluble and membrane associated proteins. Recently four new

PlC-related

cDNAs were cloned and sequenced (see Rhee, 1991

for

review),

with

high similarities

with

the previously discovered types. They were named _{P2, ^(2,} ô2 and ô3.

A

PLC gene found

in

drosophila, norp A, closely resembles the PLC-B type (Bloomquist et al., 1988).

PLCs can be regulated by phosphorylation, either

in

a negative way or

in

a positive fashion. Some groups have observed that PLCs can be phosphorylated by PKC, which decreases the agonist-induced PIP2 hydrolysis and

C**

phosphorylation of PLC

itself

or of the G protein coupled to

it.

cAMP ^{may also} exert an

inhibitory

effect on the formation of

inositol

phosphates.

In

addition,

it

has been shown that cells treated

with

growth factors

like

EGF and PDGF increase both the tyrosine and serine phosphate content of PLC-y1 exclusively, and that tyrosine phosphorylation correlates

well with

the increased turnover

of PPz(Huckle

eta1.,1990). These results suggest ttrat PLC-y1 is the isoenzyme

of

PLC ttrat mediates PDGF and EGF phospholipid hydrolysis. The PLC isozyme activated by

TRH,

a major activator of PRL secretion in pinritary cells seems to be PLCp, which is

itself

activated by Gq and/or G11 (Bernstein et al., 1992).

4.2. Atdenylyl cyclase

This enzyme is found

in all

mammalian tissues.

It

is coupled to two G proteins

with

opposite effects: G, stimulates the enzyme whereas Gl inhibits

it.

The

utilization

of the diterpene

forskolin,

a direct activator of adenylyl cyclase (Seamon et a1., 1986) that can be covalently coupled to an agarose maffix, has allowed the purif,cation of the protein.

B oth

calmodulin-sensitive

and

calmodulin-insensitive forms of

adenylyl cyclase have been identified in brain and

in

other tissues (Rosenberg and Storm ,1987; Minocherhomje ^et al., 1988; Gao and Gilman, 1991).

A

soluble

form

stimulated by calmodulin but not coupled to any G protein (Gross et al.,

1981) has also been reported. So far four mammalian adenylyl cyclases have been cloned and were named

I, II, m

and IV. Only type I is calmodulin sensitive

(Thng

et

al., 1991). This form is inhibited by the ply _subunits

of

G proteins.

However, type

tr

and

IV

are activated

by Biyif

dissociated Gro is also present (Tang and

Gilman,l99l;

Federman et

al.,

1992). Thus the effects

of

_Biydepends on the type of adenylyl cyclase under study. This seems an elegant way in which the cell transforms an

inhibitor

of

cAMP

production

(Gd

into an activator.

The different adenylyl cyclases are thought to contain two domains that span the

lipid

bilayer, each domain consists

of

six transmembrane helices. The sffucture is reminiscent

of

those that have been proposed

for

several ffansporters and channels

(Krupinski

et al., 1989). The estimated molecular masses of these forms

fall in

the range

of

120-150 Kd.

Stimulation of

AC,

mediated by

C*+

^{acting via} calmodulin, is the longest established example of potential

positive 'crosstalk'

between the

C**

and

cAMP-signalling

systems (MacNeil et a1., 1985). However, a growing number of ^reports claim that physiological concenffations

of C** ^inhibit

adenylyl cyclase

in pituitary

cells, NCB20 cells and cardiac sarcolemma (Cooper,

IggD.

This implies that

C**

can control cAMP production bidirectionally, depending on the type

of

adenylyl cyclase ^present.

We now continue our

trip

down the secretion pathway and discuss some important second messengers, such as ca2*, cAMP, inositolphosphates and DG.

5. Calcium ^{ions as cytosolic messenger}

Among the second messengers ^know n,

C**

is the most

widely

studied.

A MEDLINE

search of the year L99I reveals the existence

of

8546 papers on the subject, whereas

cAMP

was studied o'only" L336 times, and Ins(1,4,5)P3 was practically ignored

with

143 publications. These numbers can explain by

themselves why we focused on such

ahot

subject. But this is not the only reason.

[Ca2*]i homeostasis is

finely

and carefully regulated by a battery of cellular elements ^(see below), each being exffemely important for

life. lC**li

homeostasis influences

all

the cellular events

critical for

the functions of our body (secretion, protein synthesis, protein degradation, mitosis, ffanscription, energetic metabolism, etc.).

The first insights showing that extracellular calcium is necessary for the liberation of

pituitary

hormones were provided by the

work

of Vale and

Guillemin _,

in

1967 . They could obtain TSH secretion by increasing the external concentration of K+ ions. Similar results were then obtained for the liberation

of

the other pituitary hormones. These observations suggested that calcium entry is a key component

in

the control

of

secretion

in pituitary

cells. The secretory response to K+ is due to high permeability of the membrane to this ion

(Milligan

and Kraicer, L97A). This leads to a depolarization of the membrane and hence to an entry of calcium which ^is potential dependent. Further studies have then revealed that

inhibitors

of calcium entry (such as the ion cobalt or verapamil) can block the exit of

pituitary

hormones (Thshjian et al., 1978; Ozawa and Kimura,

L982, Tan and Tashjian, L984).

In

the

majority of

the tissues tested ^so far, the basal [Ca2*]i level ^(the

in- facellular

concentration of free calcium) is

around

100

nM.

However, the extra- cellular

[Ca2*].level

^is

around I ^mM.

^This

^gradient

presents cells

with

an opportunity: the cytosolic concentration can be abruptly raised for

signalling

purposes by transiently opening calcium channels

in

the plasma membrane or

in

an inffacellular membrane. As a consequence,

C*+

^{rushes into} the cytosol, dra- matically increasin

glc**liand

activating Ca2*-sensitive responses

in

the cell.

For this signalling mechanism to work, [Ca2*]i

under

basal conditions must be

kept

low, and this is achieved

in

several ways ^(see below).

If

[Ca2+]i is elevated

for

a prolonged time

in

the cells, this could result

in

disruption

of

the cytoskeleton,

DNA

fragmentation (Onenius _et al., 1989), and the calcium ion

will

precipitate as hydroxyapatite, due to the presence of high amounts of phosphate esters

in

the cytosol.

lC*\iincreases

^{are essential}

^for

many cellular functions as already mentioned, however the increase should not be too high (no more than a

few pM)

and not be prolonged too much. The major regulatory systems

of

_[Ca2+]i are illusffated

in

fig. 6. Cells keep their _[Ca2*]i

low

by three essential mechanisms (Carafoli, 1987):

1) by reversibly

complexingc**

^to cytosolic ^proteins.

2)by

extruding actively

C**

^from the cytosol to the external medium.

3) by sequesteri

ngC**into

speci ahzedorganelles.

Ca2+ channels

ER

(calciosome)

mitochondria

Fig.

6. Elements

involved in the regulation of

[Ca2+]; homeostasis.

See text

for

explanations.

rca2l _e ^1mM

li - ^{0.1 pM}

lca ²⁺

5.L. Cytosolic Ca2*-binding proteins

The role of the soluble

Cah-binding ^proteins

^{is not only to}

buffer

the

lc**li,but

also to feel the changes

in

_[Ca2*]i. Parvalbumin is very abundant

in

muscle cells (up to 2

mM)

and has no apparent function other than to buffer

C**.

On the other hand e.g calmodulin and troponin C process the signal by changing their conformation. These proteins bind

(with

high

affinity)

usually a few

C**

ions (2 to 4). These

C**

modulated proteins contain repeat domains that bind

C**

selecrively and

with high

afflnity. Each domain consist of a loop

of

10-12 amino acid residues, flanked by two c-helices perpendicular to each other.

C**

binds

into

the loop.

This

structural pattern, first seen

in

the crystal sffucture

of

parvalbumin, has been called the

"E-F"

hand. The number of proteins which possess such domains for

C*+

binding is not far

from

200.

Calmodulin

(17Kd), is the most important sensor

of

[Ca2+]; (Manalan and

Klee,

1984; Cox, 1983).

It

is present in relatively high amounts in eukaryotic cells (10

pM),

and interacts

with

a variety

of

cellular enzymes, regulating their activity. The human genome contains at least three divergent genes coding an identical calmodulin protein (Fisher et al., 1988). This suggest that calmodulin is crucial for

life

and is under

high

selective pressure to avoid any mutation of its sequence. Among the proteins activated by calmodulin we find adenylyl

cyclase,

cAMP

phosphodiesterase, phosphorylase b kinase, Ca2*-ATPase of the plasma membrane, etc.

5.2. Proteins that transport C*+ through membranes

The

transport

of

C*+

is achieved by membrane bound proteins. Those

C**-tansporting

systems ^are present

in

the plasma membrane, mitochondria, sarcoplasmic reticulum, endoplasmic reticulum,

Golgi

vesicles and also in the lysosomes.

The plasma mernbrane contains three major systems able to modulate

lc**li,namely

^{1) a} specific Ca2*-ATPase, 2) aNa+/Ca2* exchanger ^{and 3)} many different

C**

^{channels (fig. 6).}

5.2.t The Ca2*-ATPase

All

eukaryotic cells have aCa2+-XlPase

in

their plasma membrane that uses the energy of

AIP

hydrolysys to pump

C**

out of the cytosol (Schatzmann, L982). This

pump

(a single polypeptide

of

128

Kd)

can bind calcium wittt high specificity and high affinity, its Ko being near 1

pM.

When [Ca2+]i increases,

C**

binds to calmodulin which is an activator of the AIPase, and the ion ^is

extruded from

the cell.

A

Ca2+-ATPase pump,

different

from the one present

in

the plasma membrane is also found

in

the membrane of the specialized

intracellular compartment

^and also plays an important part

in

keeping [Ca2*]i low. This pump is active even

if

_[Ca2+]i is around basal level and enables the compartment to take up large amounts

of C*+

^from the cytosol.

In

the lumen of the

inffacellular

compartment,

C**

^is loosely bound to

low-affinity/high

capacity Ca2+-binding proteins similar to e.g. calsequestrin, calreticulin, etc.

5.2.2.The Na+/C*+ ^exchanger of the plasma membrane

To reduce [Ca2*]i, muscle ^and nerve cells have an additional element

in

their plasma membrane that couples ^the efflux of

C*+

to the

influx

of Na+ (Vigne et al., 1988). This exchanger, sensitive to amiloride and to phosphorylaton by PKC, has a

low affinity for C*+

and therefore begins to operate efficiently only when _[Ca2*]i reaches values significantly higher than 100 nM. Its structure remains largely unknown.

5 .2.3 .

Calcium channels

Many calcium

channels have

already

^been described. Channel proteins allow the passive

diffusion

of ions, which is governed by electro- chemical gradients. They ^are transmernbrane proteins ^and

formpores

in the

lipid

bilayer through which ions can travel. The mechanism

of

channel opening

depends of the type of the channel considered.

Calcium channels can be voltage-dependent or voltage-independent.

The members of this latter

family

are predominately found

in

non-excitable tissues and are

poorly

defined

yel

They are activated by receptor occupancy and have therefore have been called ooreceptor-operated channel". This denomination is rather confusing because voltage-dependent calcium channels can also be opened by receptor occupancy.

In

the field of this introduction we

will

not be interested

in

the voltage-independent channels (however ^see Hallam and Rink,

1989;

Rink,

1990; Futney, 1990; Demaurex et ^a1., L992), but we focus on the channels that are largely present in pinritary cells, the voltage-dependent calcium

Voltage-dependent

calcium

channels ^(see

Miller,1992 for

a recent review) are predominantly found

in

excitable tissues, such as nerve and muscles.

They are opened by

depolanzationof

the plasma membrane. They are characterizedby high

ion

selectivity and voltage sensitivity. Four classes

of

voltage-dependent calcium channels have been caracterized due to their

pharmacological and electrophysiological properties: L-type, T:type, N-type and P-type.

L-type

voltage-dependent

calcium

channels

(L-VDCC).

L-VDCC

^(see Campbell et al., 1988; Catterall, 1988) are the target

of dihydropyridine ^(DHP)

drugs which can open or close the channel. DHP- sensitive calcium channels are also inhibited by phenylalkylamines and benzothiazepines.

It

has been demonstrated that DHP stereoisomers which selectively enhance or decrease Ca2* enffy also modulate the release and

production of

pituitary

hormones (e.g. Enyeart et a1.,1985; Enyeart et al., 1987;

Day and Maurer, 1990).

Most

information

on this channel has emerged from work on skeletal muscle, a

rich

source of

L-VDCC. It

is composed of

distinct subunits:

ul

⁽¹⁷⁰

Kd), a2lô

⁽¹⁷⁵

Kd),

_P ⁽⁵²

Kd)

and

y(32

Kd).

All

these subunits have now been purified and sequenced (Tsien et al., 1991). The most

important

subunit

^is crl., which is

highly

homologous to the

o

subunit of the sodium channel. Indeed, some point mutations at precise sites confer

C**

channel properties to a Na+ channel (Heinemann et a1.,1992). The

cl

^subunits

of

L-VDCC

is the

targetof

^DHPs, phenylalkylamines and benzothiazepines. This subunit is also the voltage-sensor of the channel and

it

has been shown that assemblies of pure cr subunits can

form

a functional channel.

In

pituitary

cells, the

biological

system used

in

our laboratory L-VDCCs are activated

if

^{the cells are} depolanzed (by an agonist or during patch-clamp experiments) at values beyond -20

mV

the resting potential being near -55 mV.

Under nonstimulated conditions, those channels ^are usually closed in pinritary cells. Once opened, their conductance is near 25 pS. Their activation is rather slow, and the duration

of

the aperture is quite long (some ms), compared to the other VDCCs ^(the

'L

of

L-VDCC

stands for Long lasting). They are blocked

by mM

concenffations

of

^Cd2+.

In

addition to their regulation by the plasma membrane

potential,

L-VDCCs

are regulated by the

protein _G,

(Yatani et al., 1987) and the channel is a subsffate for

PKA

and

PKC

(Chang et al., 1991). The channel is inactivated by

C*+ itself

^(feedback regulation).

In

clonal

pituitary

cells,

C**

enffy during the action potential ^(see below) occurs

principally

through

L-VDCC.

However those cells also contain calcium channels of the T:type (T-VDCC, Armstrong ^{and Matteson, 1985).}

T type

voltage-dependent

calcium

channels

(T'VDCC).

In

pituitary

cells, only one or two percent of those channels are opened at the resting potential.

Their

conductance is transient ^(less than 1 ms, the

"T" of T-VDCC

stands

for

Transient) and is smaller (10 pS) than that of the L-VDCCs.

Their sffucture is

still

unknown. They are insensitive to DHPs and Cd2+, but are

blocked by Coh

ions. They may be important

for

the beginning of the action potentials in

pituitary

cells.

N-type

voltage-dependent

calcium

channels

(N'VDCC).

N-channels are

widely

distributed

in

the Nervous system. They ^are

acrivared at high threshold, like

L-VDCC.

They are blocked by ro-conotoxin, but are insensitive to DHPs. They do not seem to be present

in

GH3B6 or GHaCI cells.

Little

is known about their sffucture.

P-type voltage-dependent

calcium

channels

(P-VDCC).

P-channels are insensitive to DHPs and co-conotoxin and are predominant in Purkinje cells. They are blocked by the venom of the

spiderA.

aperta

with

a

high

affinity (Kd <

10

nM).

Here again,

little

is known about the structure.

5.3. Intracellular ^stores

5.3. ¹ .

Endoplasmic reticulum

The endoplasmic reticulum (ER), is the main organelle able to buffer

lC**liin

non-muscle cells.

It

plays the role

of

a reserve

of C**.

This store is sensitive

to

Ins(1.r4r5)Pr (Benidge and

kvine,

1984). However some studies have shown that only part

of

the

C**

contained

in

the organelle is released by Ins(1,4,5)P3 (Burgess et

al.,

1984; Prentki et al., 1985; Biden et

al,

1986). Thus the ER comprises Ca2* storage elements

which

are Ins(1,4,5)P3 responsive and others which are Ins(l,4,S)P3unresponsive

with

^regard

n C**

ûberation. The Ins(1,4,5)P3 uffesponsive stores can be modulated by caffeine (Thayer et ^a1.,

1988) or

thapsigargin

(Thastrup et al., 1989).

The ER and the sarcoplasmic reticulum ^(SR), its counterpart

in

muscle cells, possess a specific Ca2*-ATPase (100 Kd), different from that of the plasma membrane, used

for

the uptake

of C*+.

The calcium is released back to the cytoplasmby a channel opened by

Ins(l

_,4,5)P3in the endoplasmic reticulum and sensitive to ryanodin

in

muscles.

These observations generated an increased interest for the purification

of

the part of the ER which is Ins(1,4,5)P: sensitive (Volpe et al., 1988; Krause et

aL., 1989; Rossier et al., 1989).

It

was suggested that this portion of the ER could

form

organelles. They received the sweet name

of

"calciosome". They comprise a specific combination

of

Ca2*-ATPases

with

second messenger Ins(1,4,5)P3- operated

C**

release channels, and

ahigh

capacity

C**-binding

protein similar

Krause et al., 1990).

In myeloid

cells this protein was identified as

calreticulin

(Treves et al., 1990).

The major

criticism

one could argue against the "calciosome" theory is that the assembly of molecular elements obtained

in microsomal fractions

^may not be

reliably

reporting the real functional and sffuctural context of the

Ins( 1,4,5)P3 responding store.

The intracellular receptor that mediates the effect of Ins(1,4,5)Pg was

first

purified, due to its

affinity

for heparin and concanavalin,

fromrat

cerebellum and found to correspond to a26O Kd phosphoglycoprotein (Supattapone et al.,

1988a). In the brain the Ins(1,4,5)P3 receptor is

highly

concenffated

in

cerebellar Purkinje cells and

in few

other types. Reconstitution snrdies have revealed that

this receptor

molecule includes the

ion

channel activated

by

Ins(1'4'5)P3

binding

^(Fenis et a1., 1989). The binding of Ins(1,4,5)P3 to the

tetramer

^is cooperative and requires at least the binding of three Ins(1,4,5)P3 molecules (Meyer

et

a1.,1983). The Ca2+ channel binds Ins(1,4,5)P3

Kd

^{in the nM range)}

and opens rapidly. The release of

C**

^{is a}

^quantal

^{process as seen in} intact cells (Muallem et al., 1989) and

in

vesicles reconstituted

with

Ins(1,4,5)P3 receptor (Ferris

et

aL.,1992).

Using immunological techniques, the receptor is found

primarily

on the smooth ER, but also to a lower extent on the rough ER. No specific labeling was detected

in

mitochondria, the nucleus or the plasma membrane (Satoh et al., 1990). The receptor is a major substrate

for cAMP-dependent