The phosphatidate-phosphoinositide cycle: An intracellular messenger system in the action of hormones and neurotransmitters

REVIEW ARTICLE

The Phosphatidate-Phosphoinositide Cycle: An Intracellular Messenger System in the Action of Hormones and Neurotransmitters Robert V. Farese Many hormones and neurotransmitters provoke rapid and striking changes in the metabolism of phospholipids in the phosphatidate-inositide cycle. These changes appear to occur before and after the generation of other accepted “second messengers” (e.g., Ca++ and CAMP), and seem to be important intracellular effector substances for the elicitation of biological effects. The two major mechanisms for perturbing the phosphatidate-inositide cycle are phosphatidylinositol hydrolysis and de novo phosphatidate-inositide synthesis. Phosphatidylinositol hydrolysis occurs in the action of all agents which operate via Gaff and appears to be provoked both by Ca++-dependent and Ca”-independent mechanisms. Ca++-independent phosphatidylinositol hydrolysis may be triggered directly by receptor activation and may control Caf+ release or entry into the cytosol. Ca++ -dependent phosphatidylinositol hydrolysis may be important for further changes in synthesis effect cellular Ca+ + distribution and membrane fusion during exocytosis. The de novo phosphatidate-inositide has been observed in the action of most steroidogenic agents (ACTH. luteinizing hormone, angiotensin II, K+, serotonin), parathyroid hormone and insulin. The de novo effect is inhibited by cycloheximide, requires Ca++, and appears to serve as a post-second messenger mechanism to alter membrane structure and the function of membrane associated substances. Considerable evidence suggests that the de novo effect is important in the control of steroidogenesis by the abovementioned agents; it may also be important in the action of insulin in adipose tissue.

U

NTIL RECENTLY, most endocrinologists have considered phospholipids as nondescript, complex biochemical substances, which have terribly long names that were not worth remembering. Even with continued development of the lipid bilayer and fluidmosaic models, phospholipids have frequently been viewed as inert, “structural” substances, whose major reason for existence was, for the anatomist, to provide recognizable boundries for cells and organelles on electron micrographs, or, for the biochemist or physiologist, to provide a convenient matrix to support more important biochemical substances, such as membranebound enzymes, transporters or receptors. During recent years, however, it has been realized that phospholipids are neither “inert” nor simply “structural.” Many phospholipids have very high rates of turnover, and can profoundly alter membrane function. Moreover, most hormones and neurotransmitters have now been shown to provoke rapid, and, frankly, dramatic changes in phospholipid metabolism. These changes in phospholipids have been correlated to changes in cellular functions, and it is becoming increasingly clear that phospholipids serve as impor-

From the Veterans Administration Hospital and the Department of Medicine. University of South Florida College of Medicine. Tampa, Florida. Receivedforpublication October 18, 1982. Supported in part by funds from the National Institutes of Health (Grant No: i ROi HL28290-01) and the Research Service of the Veterans Administration. Address reprint requests to Robert V. Farese, M.D.. Veterans Administration Hospital and the Department of Medicine, Universiy of South Florida College of Medicine. Tampa, Florida 33612. 0 I983 by Grune & Stratton, Inc. 0026-0495/83/3206-0015f02.00/0

620

tant mediators which function both before and after the generation of the “second messengers”, Ca++ and cylcic nucleotides. GENERAL CONSIDERATIONS CONCERNING THE PHOSPHATIDATE-INOSITIDE

CYCLE

One area of phospholipid metabolism that has received considerable attention recently is the phosphatidate-inositide cycle. As shown in Fig. 1, phosphatidic acid is synthesized de novo from fatty acyl-CoA and glycerol-3-PO,, and is then converted to CDPdiglyceride, which, in turn, can combine with inositol (which is ultimately derived from glucose-6-PO, and actively concentrated within cells) to yield phosphatidylinositol, or with glycerol-3’-PO, (a glycolytic derivative) to yield phosphatidylglycerol-PO,, phosphatidylglycerol, and cardiolipin. Phosphatidylinositol can either be converted to the polyphosphoinositides, diand triphosphoinositide, or the headgroup of phosphatidylinositol can be removed by phospholipase-C to yield diglyceride. Diglyceride can be converted back to phosphatidic acid, or to substances with are extrinsic to the cycle, viz., triglyceride, phosphatidylcholine, phosphatidylethanolamine or phsophatidylserine. It may be noted that phosphatidic acid can be synthesized from two sources, either de novo or from diglyceride. The structural formula of phosphatidylinositol is shown in Fig. 2. As is apparent, the molecule contains the phosphatidate (phosphatidyl) moiety covalently coupled to inositol by a phosphoester bond. Phosphatidylinositol can be further phosphorylated at the 4’ and 5’ positions of the inositol ring to yield diphosphoinositide and triphosphoinositide, respectively. The structure of other phospholipids may be deduced by exchanging the inositol of phosphatidylinositol for Metabolism, Vol. 32, No. 6 IJune), 1983

PHOSPHOLIPIDS

629

AND HORMONE ACTION

GLYCEROL-3'-PO,, FATTY ACVLc

CoA CO++

AGONIST

PHOSPHATIDVLCHOLINE "OTEIN

PHOSPHATlDYLETHANOLAlNE

SYNTHESIS

[~~~~~lf~~~~~TO~~"]

I'

PHOSPHATIDVLSERINE

PHOSPHATIDIC ACID \ATy TRIACVLGLYCEROL w

\ DIACVLGLVCEROL

CDP-DIACYLGLYCEROL

\ PHOSPHATIDYLINOSITOL

AGONIST

!

/

DlPHOSPNOlNOSlTIDE

/

PNOSPHATIDYLGLYCEROL-POI,

PHOSPHITIDYLGLYCEROL CARDIOLIPIN

r-

I 0=-c

I

0

I

H-C------C-C-H

SEROTONIN. VASOPRESSIN A-II. K+, INSULIN,

1

SECRETIN, VIP, GIP, THROMBIN

i” c=o

I /

0

HO---P-O

1

O\ FREQUENTLYARACHIDONATE

L

i\r

y/bH

c-c I H

Fig. 2.

Structural

the mass of phosphatidic acid4 and diglyceride,’ and the concept emerged that the primary change was phosphlipase C-mediated phosphatidylinositol hydrolysis (also see Fig. 3 for a schematic summary of hydrolytic reactions involving substances in the phospholipid-lipid pathway), leading to an increase in diglyceride, which, in turn, combines with ATP to produce phosphatidic acid and, ultimately, phosphatidylinositol. This mechanism has been extensively studied in recent years and is the subject of several recent reviews.’ ’ ’ The second major mechanism for perturbing the cycle is to increase de novo synthesis of phosphatidic acid. This mechanism was recognized only recently in studies of ACTH action. 12-‘*In this mechanism, there are increases in the mass of most phospholipids in the phosphatidate-inositide cycle. The phosphatidate-inositide cycle can be perturbed by several other mechanisms including: (1) deacylation and reacylation at the sn-2-position of the glycerol moiety in phosphatidylinositol, phosphatidylcholine. phosphatidylethanolamine, phosphatidic acid or diglyceride, (2) generation of diglyceride from sources other than phosphatidylinositol, and subsequent conversion of diglyceride to phosphatidic acid, (3) interconversions of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, and (4) interconversions of the mono- and polyphosphoinositides. These ancillary mechanisms will not be discussed in detail in the present review, but it is important to mention: firstly, that deacylation of phosphatidylinositol can decrease levels of the latter substantially, and this may be confused with phospholipase C-mediated phosphatidylinositol hydrolysis; and, secondly, that diglyceride generation from any source may be

,%a;\.

C l

TRIPHOSPHOINOSITIDE

ACETVLCHOLINE, CCK

other substances, e.g., glycerol-PO,, glycerol, serine, ethanolamine and choline, which are also coupled to the phosphatidate moiety by a phosphoester bond. There are two major mechanisms whereby hormones and neurotransmitters may perturb the phosphatidate-inositide cycle (see Fig. 1). The first mechanism was discovered nearly 30 yr ago by the Hokins,lm3 who observed that acetylcholine in the pancreas provoked a rapid increase in P3*-incorporation into phosphatidic acid and phosphatidylinositol. This increase in labeling was, in some cases, associated with a decrease in the mass of phosphatidylinositol and an increase in Rl

t

a - ADRENERGIC AGENTS

Fig. 1. Major perturbations of the phosphatidate-inositide cycle. Shown in brackets are examples of agonists which provoke the two depicted major phospholipid responses, i.e., phosphatidylinositol hydrolysis and de nova phosphatidate synthesis.

formula of phosphatidylinositol.

I

H

630

ROBERT V. FARESE

(4’ FATTY ACID +

FWOSFWATIDIC ACID f/

-

t

LYSOPHOSPHATlDIC ACID

\-

CDP-DIACYLGLYCEROL

DIPHOSPHOINOSITIDE

Fig. 3. cycle.

LYSOPHOSPHATIDYLINOSITOL

(1)

PHOSPHATIDATE PHOSPHATASE

(2)

PHOSPHATIDYLINOSITOL PHOSPHODIESTERASE (PHOSPHOLIPASE C)

(3)

PHOSPHOLIPASE D

(4)

PHOSPHOLIPASE A2 (? SPECIFICITY)

(5)

DIPHOSPHOINOSITIDE PHOSPHC#WNOESTERASE

(6)

TRIPHOSPHOINOSITIDE PHW'HONESTERASE

(7)

DIPHDSlWOINOSITIDE FltOSFliODIESTERASE (PHOSPHOLIPASE C)

(8)

TRIPHOSPHOIMOSITIDE PHDSPHODIESTEAASE MCWHDLIPASE

(9)

DIGLYCERIDE LIPASE

C)

Hydrolitic enqmes in the phosphatidate-inositide

attended by an increase in [“P]Pi incorporation into phosphatidic acid and phosphatidylinositol. Hence, the P3’-labeling response is not specific for the phosphatidylinositol hydrolysis effect, and can also occur with de novo synthesis effect, as well as diglyceride generation from a variety of sources.

From the above considerations, it is clear that the phosphlipase C-mediated phosphatidylinositol hydrolysis effect can only be fully proven if there is a demonstrable decrease in the mass of phosphatidylinositol or radioactivity of prelabeled phosphatidylinositol, coupled with a release of inositol-phosphate or generation of diglyceride (or phosphatidic acid if phospholipase D action can be disregarded). In addition, the de novo synthesis effect can only be fully documented by an increase in the mass of the inositide phospholipids. Note that increases in glycerol or other precursor incorporation are not specific for the de novo effect and may be seen in cases (e.g., see 19-22) where only the phosphatidylinositol hydrolysis effect has been found to be operative; this may reflect a “trapping” of phosphatidic acid synthesized de novo by that generated from diglyceride; alternatively, it may reflect “compensatory” de novo synthesis secondary to phosphatidylinositol hydrolysis, but, if this is the case, this de novo synthesis is clearly separable from the de novo phosphatidate synthesis effect discussed elsewhere in this paper. The general metabolic consequences of perturbing the phosphatidate-inositide cycle by the two major mechanisms, i.e., phosphatidylinositol hydrolysis and de novo phosphatidate synthesis, are summarized in Fig. 4. Firstly, increases in phosphatidic acid, by either mechanism, may facilitate transmembraneous Ca++ movement, since phosphatidic acid is a natural Ca++ ionophore.23-27Secondly, increases in phosphatidic acid B. De NOVO PA SYNTHESIS EFFECT _-

A. PI HYDROLYSIS EFFECT

TRANSMEMBRANEOUSCo++ f4OVERENT

TRANSHEHBRANEOUSCo++ MOVERENT

of

tetc,)

M-ffi

I ACTIVATES

,o

ACTIVATES

t CiP-DG ..

+

Co*-DEPENDENT PROTEINKINASE

1

@I

Co* DEPENDENT +

t

tCDP-DG I \

Mj

PROTEINKINASE

(ALSOt BY PS)

(ALSO.+BY PS)

z

RELEASES Ca++

I PI

1

0 Fig. 4.

iDPI iTP1

. BINDS Co++

1

0

tDP1

t TPI

General metabolic consequences of perturbing the phosphetidate-inositide cycle.

?

PHOSPHOLIPIDS

AND

HORMONE

ACTION

631

and diglyceride by either mechanism may provide substrate for phospholipases and lipases to generate arachidonic acid, prostaglandins, and other related substances;28*29 the importantce of this mechanism for arachidonate generation, relative to phospholipase A*mediated deacylation of phosphatidylinositol or other phosphlipids, is presently uncertain. Thirdly, increases in diglyceride by either mechanism may activate a Ca ++-dependent protein kinase, which is also activated by phosphatidylinositol and phosphatidylserine.3” Fourthly, anionic phospholipids, particularly tri- and diphosphoinositide,3’.32 bind Ca ++, and decreases in inositides may release Ca’ +, while increases in inositides may increase Ca* ’ binding to membranes; along these lines, it may be noted that decreases in phosphatidylinositol may be associated with decreases in the polyphosphoinositides.” Finally, changes in phospholipids may influence the activity of membrane-bound enzymes, receptors or transporters, either directly or indirectly by changes in membrane fluidity or ion binding. THE PHOSPHATIDYLINOSITOL

HYDROLYSIS EFFECT

True phosphatidylinositol hydrolysis, determined directly as a decrease in labeled or unlabeled phosphatidylinositol, and suspected phosphatidylinositol hydrolysis, suggested by an increase in the labeling of phosphatidic acid and phosphatidylinositol, has been observed in many tissues. In some cases, as shown in Table 1, the phosphatidylinositol hydroIysis’“.34~-38 or the “P-labeling response3Y 43 does not appear to be dependent on Ca’ _, and this is of great interest, since these listed agonists employ Ca’ L as their major

Table

1.

Cat ‘-Independent

TlSSU3 Rat Parotid

Changes

Agomst

Bowne

Adrenal

Guinea

Pig Ileum

Smooth

Muscle

20

37

+

_

35.36

t

-t

22,

Angiotensin-II

+

+

22,34

Vasopressin

+

Acetylcholme

t

t _+

38

+

+

39,

Acetylcholine

?

+

40

Histamine

7

+

40

+

t

41.42,

7

+

43

Angiotensin-II

?

+

102

Anglotensin-II

7

+

SubmItted

Rat Pituitary

Cells

Thyrotropin

Rat Adipose

Tissue

a-Adrenergic

Beef Adrenal

Glomerulosa

References

+

Acetylcholine,

Medulla

(With [“P]Pi)

t

a-Adrenergic

Rat Vas Deferens

Pi Labelmg Effect

Effect

t

P

Serotonin

Gland

PI Hydrolysis

+

Substance

Rat Hepatocytes

Metabolism

t

Agents

Acetylcholine

BlowflySalivary

in Phosphatidylinositol

+

n-Adrenergic

Gland

intracellular “second messenger”. Moreover, otherwise effective doses of Cat+ ionophores may not provoke a similar phospholipid response in these tissues. Michell and coworkers6~8.20,22~34.37,44.45 have therefore postulated that phosphatidylinositol hydrolysis is directly triggered by receptor activation, and, moreover, is the cause, rather than the result of, increases in Ca + ‘. In support of this hypothesis, Berridge and Fain”‘.‘” have shown that Ca +* fluxes seem to be dependent on phosphatidylinositol in the blowfly salivary gland, and Wallace et al.46 have recently reported that vasopressin. via its receptor, directly increases phosphatidylinositol hydrolysis in a rat liver plasma membrane preparation. However, there are some difficulties with the Michell hypothesis. Firstly, in many instances, as shown in Table 2. true4’ 57 or suspected’“~“.SX~“5 phosphatidylinositol hydrolysis appears to be dependent on Ca' ‘. and, in most cases (e.g., see 50-56). phosphatidate levels or labeling increase, and it appears that phospholipase C action is involved. Secondly, in many cases, Ca +’ ionophores. A231 87 or ionomycin, can stimulate phosphatidylinositol hydrolysis3X.47 “J and/ h0.63h5 lonophore-induced decreases or labeling. z”~2’~42.Fx in phosphatidylinositol levels may also be partly due to phospholipase A, activation (e.g., see 66,67), but Ca’ directly activates cytosolic and membrane-bound phosphlipase C,JX.hX-7” and the latter seems likely to be at least partly involved in certain actions of ionophores, as evidenced by concurrent increases in phosphatidate labeling or synthesis (e.g., see 50). How can these discrepant observations be reconciled? One explanation is that experimental artefacts

Agents

Carbachol

Releasing Agents

Hormone

20

22,

34.

62

34,

62

124

121

(Cells) Rat Adrenal

Capsules

(Zona

+

= yes;

*Labeled

~- = no; ? = not reported mymositol

for

Publication

Glomerulosa)

was

used instead

of 32P.

632

ROBERT V. FAR&E

Table 2. Gaff-Dependent

or Ca++- lonophore-Induced

Tissue Rat Pancreas

Aaonwt

Changes in Phorphatidylinositol

-

PI Hydrolysis

Carbachol

+

Cholecystokinin

+

Secretin

+

Vasoactive Intestinal Peptide

+

Metabolism

PI Labeling Effect (Wkh [“P]Pi)

References

l

51,52

l

51

_

51

_

51

Insulin

+

Dibutyryl CAMP

+

+

A23187

L

l

Rat Adrenal Glomerulosa Cells

Angiotensin-II

Human Platelets

Thrombin

+ + +

_

51,54 53 5 1, 52 (& unpublished)

K’

+ +

A23187 Rat Islets of Langerhans

Glucose

+ +

+w +m +

55 (81 unpublished) 55 (& unpublished) 47-49 47-49 122, 123 (& Clements. R.S., Jr., personal communication)

Rabbit Neutrophils

+ + _

Carbachol

+ + + +

l

56

A23 187

+

_

56

fMet-Leu-Phe lonomycin

Rabbit Vas Deferens Rat Submaxilary Gland

A23187

50 50 38

Bovine Adrenal Medulla (Cultured Cells) Synaptosomes

Acetylcholine Acetylcholine A23187

Rat Parotid Gland

A23 187

Human Erythrocytes

A23187

Pig Lymphocytes

Phytohaemagglutinin A23187

Rat Corpus Luteum

A23187

Rat Pituitary Cells

A23187

Rat Adipocytes

A23187

Rat Hepatocytes

a-Adrenergic Agents

+ + +

21

+(3 ? ? ? ? ? ?

+(?) + + + + + +

20

60,61 60 58 59 59 64 42 43

7

+**

62

?

+**

62

= PA-PI labeling effect present but not dependent on Ca+’

l

l

? ? ?

* = PA-PI labeling effect inhibited with “severe” Ca*+ deficiency.

+ 0) = PA-PI labeling effect present, but dependency on Ca++ not reported.

+

=yes

-

= no

? = not reported

are responsible for seeming dependence or independence of Ca++, and only one mechanism occurs. In support of this possibility, the degree of Ca++ deficiency has been shown to determine whether or not Ca++-dependency is apparent in vasopressin effects on phosphatidylinositol hydrolysis in rat hepatocytes.62 Alternatively, there may be multiple mechanisms for increasing phosphatidylinositol hydrolysis and labeling, and we51357and others3’ have obtained evidence which supports this possibility. In the multiple mechanism scheme (Fig. 5), receptor activation in the plasma membrane may directly activate phospholipase-C, causing phosphatidylinositol hydrolysis and generation of diglyceride and phosphatidic acid. Phosphatidic acid may then mobilize Ca++ from internal and external sources, and Ca++ may activate a cytosolic phos-

pholipase-C, which enhances phosphatidylinositol hydrolysis in the cell interior. This, too, may mobilize more Ca++, and a cascade may propagate. Finally, the increase in cytosolic Ca++ and changes in phospholipids may evoke a variety of biological responses, depending upon the tissue. Two such biological responses that appear to ubiquitously involve Ca’+ and the phosphatidylinositol hydrolysis response are exocytotic secretion and contraction of smooth muscle. In support of the possibility that there are multiple mechanisms for phospholipase C-mediated phosphatidylinositol hydrolysis, we have recently reported that two mechanisms may, in fact, operate concurrently during carbachol action in the rat pancreas” and submaxillary gland.56 In both tissues, the rapid P32labeling of phosphatidic acid and phosphatidylinositol,

PHOSPHOLIPIDSAND

HORMONE

ACTION

633

RECEPTOR

fPHOSPHOLIPASE-C

Fig. 5. Concurrent operation of Ca++-independent and Ca’ +-dependent hydrolysis of phosphatidylinositol. This dual mechanism scheme is derived from studies in the rat pancreas and submaxillary gland (see text). Phosphatidylinositol hydrolysis is shown as being independent of Cat+ in the plasma membrane, but dependent on Cat+ in the cell interior. Cycloheximide inhibits phosphatidate synthesis which results from phosphatidylinositol hydrolysis occurring in the cell interior, but has no effect on phosphatidate synthesis which results from phosphatidylinositol hydrolysis occurring in the plasma membrane.

-;:-Ql-

PI

DG

t PA

*

BIOLOGICAL EFFECTS CYCLOHEXIMIDE INHIBITS

possibly resulting from phosphatidylinositol hydrolysis in a small but highly active pool in the plasma membrane, was insensitive to Ca++ and cycloheximide; on the other hand, the large-scale changes in phosphatidylinositol (decrease) and phosphatidic acid (increase) mass, presumably occurring in the cell interior, required Ca++, and the increase in phosphatidate mass was inhibited by cycloheximide. Although the hypothesis of Michell remains to be proved, even if it is ultimately shown that the initial increase in Ca++ is provoked by a non-phospholipid mechanism, the presently described phospholipid effects are occurring, and, in all likelihood, at least and other biologiserve to amplify Ca + + mobilization cal responses. THE DE NDVO PHOSPHATIDATE SYNTHESIS EFFECT

The second major effect of hormones and neurotransmitters on phospholipid metabolism, namely, an increase in de novo synthesis of phosphatidic acid, has been most fully characterized in studies of ACTH action in the adrenal cortex. However, similar effects have been observed in the action of: luteinizing hormone (LH) in the gonads; parathyroid hormone (PTH) in the kidney; serotonin, K’ and angiotensin in the adrenal glomerulosa; insulin in adipose tissue; and luteinizing hormone-releasing hormone (LHRH) in rat ovary granulosa cells. Awareness of the de novo phospholipid effect resulted from studies of the mechanisms whereby ACTH controls steroidogenesis. As had been shown previously, steroidogenesis is largely controlled by two

(e.g., exocytosls)

factors, viz., free cholesterol availability7’ and a steroidogenic factor which enhances the binding of cholesterol to a mitochondrial cytochrome P450, subsequent cholesterol side chain cleavage and, thus, pregnenolone synthesis.72-74 Protein synthesis75-77 and Ca’+78 are also known to be required for ACTH-induced increases in the steroidogenic factor, and it was logically thought that the factor may itself be a protein. In an attempt to identify the steroidogenic factor, I had observed that the cytosolic fraction from ACTH-stimulated adrenals contained a substance that stimulated steroidogenesis when added to adrenal mitochondria.79 Initial efforts to characterize this factor were unrewarding, but several years ago we noted that phospholipids were present in the stimulatory cytosolic fraction,” and we therefore examined the effects of phospholipids on cholesterol side chain cleavage. To our surprise, addition of triphosphoinositide, diphosphoinositide and cardiolipin to adrenal mitochondria provoked large increases in pregnenolone synthesis,” the rate-limiting step in steroidogenesis. Phosphatidylglycerol also stimulated this reaction slightly, but phosphatidylcholine, phosphatidylethanolamine, and a wide variety of other phospholipids had no effect or were inhibitory. In other optical spectrum studies,*’ we also observed that the stimulatory phospholipids enhanced the binding of cholesterol to mitochondrial cytochrome P450, and these “steroidogenic” phospholipids, in effect, could reproduce all known effects of ACTH on steroidogenesis. After observing that phospholipids having polyphosphorylated head groups could stimulate steroidogene-

634

sis, the next question was whether ACTH increased the levels of these steroidogenic phospholipids. In initial studies,‘2”3 adrenal diphosphoinositide and triphosphoinositide concentrations were found to increase rapidly after in vivo ACTH treatment; moreover, there was a striking direct correlation between changes in these phospholipids and changes in adrenal corticosterone production during: (1) the upswing and downswing of a short-lived ACTH pulse, (2) with increasing doses of ACTH in vivo, (3) with ether stress and endogenous ACTH release, and (4) with inhibition by cycloheximide treatment in vivo or in vitro. Cardiolipin, on the other hand, did not change with ACTH treatment, and this is in keeping with the impression that this phospholipid is metabolically quite stable. Since di- and triphosphoinositide are derived directly from phosphatidylinositol, and ultimately from phosphatidic acid, and since Laychock et a1.82 had found that ACTH increased phosphatidylinositol levels in cat adrenal cells, we examined the effects of ACTH on all measurable phospholipids in the phosphatidate-polyphosphoinositide pathway. In in vivo experiments,” ACTH treatment caused rat adrenal levels of corticosterone and polyphosphoinositides to increase in parallel during a 15 minute treatment period; phosphatidylinositol and phosphatidylglycerol increased to maximal levels even more rapidly, and the most rapid changes were observed in phosphatidic acid, which was maximal within 1-2 min of intraperitoneal ACTH injection. Similar effects of ACTH were observed in vitro during incubation of rat adrenal sections:14 phosphatidic acid levels reached a maximum rapidly, and this was followed sequentially by increases in phosphatidylinositol, di- and triphosphoinositide and, finally, corticosterone. Control levels of the phospholipids, on the other hand, did not change during the incubation. The same sequence of events was also observed with CAMP as the stimulatory agent, and this suggested that these phospholipid changes occurred subsequent to CAMP generation during ACTH action. The fact that physiological concentrations of ACTH provoke this phospholipid effect has been shown most clearly in recent studies (unpublished); upon incubation of rat adrenal cells with increasing doses of ACTH, there were graded increases in corticosterone production, and cellular concentrations of phosphatidylinositol and cyclic AMP. The maximal increase in phosphatidylinositol was observed at lo-l2 M ACTH, and this occurred in conjunction with a relatively small, but significant, increase in CAMP and a lessthan-maximal increase in steroidogenesis. In addition to showing the remarkable sensitivity of the de novo

ROBERT V. FARESE

phospholipid effect to ACTH, these findings suggest, firstly, that there are spare CAMP receptors for provoking the de novo phospholipid effect, and, secondly, that, in addition to phosphlipids, other ACTH-induced factors, e.g., free cholesterol, must also contribute importantly to steroidogenesis. Along the latter lines, we have also recently foundE3 that rX-MSH, which increases free cholesterol availability by activating cholesterol esterase,“4 enhances steroidogenesis only in conjunction with other agents, e.g., ACTH, which increase adrenal phospholipids in the phosphatidateinositide cycle: thus, we believe that steroidogenesis requires, and may be the resultant of, two factors, viz., free cholesterol availability and “steroidogenic” phospholipids. The fact that all effects of ACTH in vivo on adrenal phospholipids could be blocked by a short 10 minute pre-treatment with cycloheximide’3-‘5 is of great interest, since protein synthesis is known to be required for ACTH effects on steroidogenesis.“-” Even more dramatically, cycloheximide treatment in vivo rapidly reversed steady-state ACTH-induced increases in rat adrenal phospholipids,” as well as steroidogenesis.‘5,77 In these in vivo experiments,15 steroidogenesis fully returned to basal levels within 20 min of cycloheximide treatment (T ‘/2 = 3.5 min), and this was preceded by decreases in di- and triphosphoinositide (T ‘/z = 1.7 min), and by yet more rapid decreases in phosphatidylinositol (T ‘/z = 1 min) and phosphatidic acid concentrations (T ‘/z = 0.15). Very rapid reversal of ACTH effects on steroidogenesis and phospholipid levels has also been observed in vitro in incubations of rat adrenal cells.” In contrast, in rat adrenal sections,“5 ACTHinduced increases in steroidogenesis and phospholipids were found to return to basal levels much more slowly (T ‘/2= 28 min. for both processes) during cycloheximide treatment (note-puromycin, another protein synthesis inhibitor, also inhibits ACTH effects on phospholipids). The identical half-lives for steroidogenesis and phospholipids in adrenal sections provides strong evidence for a linkage of these two processes. The reason for the greater stability of the putative labile protein in adrenal sections in vitro is presently uncertain. All of the above findings, which indicated that ACTH and cycloheximide rapidly influence adrenal phospholipids, were derived from measurement of the phosphorus content of purified phospholipids. Diglyceride levels were also found to be increased by ACTH treatment in vivo,16 and, more recently (unpublished), we have found that lipid-inositol levels are increased by ACTH treatment and blocked by cycloheximide pretreatment. In addition, several phospholipid-synthesizing enzymes are rapidly altered by ACTH and cyclo-

PHOSPHOLIPIDS

AND HORMONE ACTION

heximide treatment; these include diglyceride kinase,16 phosphatidylinositol kinase,13,14 and, more recently (unpublished) glycerol-3-PO, acyltransferase, which catalyzes the de novo synthesis of phosphatidic acid. In the latter experiments, the acyltransferase was extracted from adrenal membranes by 3 M KC], dialyzed, and assayed kinetically with saturating amounts of fatty acyl-CoA and increasing amounts of labeled glycerol-PO,. After a 15 min ACTH treatment period in vivo, the acyltransferase activity of adrenal extracts was increased approximately twofold, and this increase was blocked by administering cycloheximide (10 mg intraperitoneally) 10 min before ACTH; from analysis of Lineweaver-Burk plots, ACTH appeared to of the extracted enzyme. increase the V,,, ACTH treatment in vivo also provoked a rapid increase in phosphatidate synthesis from diglyceride and ATP,16 and diphosphoinositide from phosphatidylinositol and ATP,13.14 as measured by incubation of rat adrenal homogenates in diglyceride kinase and phosphatidylinositol kinase assay systems, respecitvely. These effects of ACTH were only observed when these assays were conducted with endogenous substrate; if exogenous diglyceride or phosphatidylinositol substrates were added to the respective assays, the ACTH stimulatory effects were lost. On the other hand, the inhibitory effect of cycloheximide remained apparent in the diglyceride kinase assaysI despite the addition of exogenous substrate. These findings suggest, firstly, that ACTH increases phosphatidate and diphosphoinositide synthesis in these assay systems by increasing substrate availability, and, secondly, that cycloheximide diminishes the V,,, of diglyceride kinase. Although ACTH did not affect the apparent V,,, of these kinases, increased formation of phosphatidic acid and diphosphoinositide from endogenous diglyceride and phosphatidylinositol nevertheless provides additional independent indicators of the rapid activation of the phosphatidate-inositide pathway. Similarly, the rapid decrease in the activity of diglyceride kinase after cycloheximide treatment, viz., 0.17 min,lh attests to the rapidity with which this mechanism can be deactivated, and provides a second independent means to verify the apparent extreme lability of the putative labile protein (note-this half-life was nearly identical to that which was derived from the rate of decrease in phosphatidic acid following cycloheximide treatment-see above and Ref 15). Our findings have led us to a scheme of ACTH action in which cyclic-AMP may increase phosphatidate synthesis by three possible mechanisms. (Fig. 6). The first mechanism is to increase the availability of glycerol-PO, by stimulation of glycogenolysis,86 and possibly glucose uptake and glycolysis8’ as well. The

635

ACTH

TRIGLYCERIDE

l

REQUlRES PROTEIN SYNTHESIS

.t POTENTIALLY "STEROIWGENIC"

21 Ii PS

PGP**

'PG 1 DPI** 1 TPI

l

\ CL** *

Fig. 6. Proposed mechanism of action of ACTH. ACTH. via CAMP. increases I1 I glycogenolysis, (2) lipolysis and 131glycerol3’-PO, acyltransferase activity. As a result, there is a rapid increase in the synthesis of phosphatidic acid (PAL CDP-diglyceride (CDP-DG), phosphatidylglycerol-PO, (PGP), phosphatidylglycerol (PG). phosphatidylinositol (PI), diphosphoinositide (DPI), triphosphoinositide ITPI) and diglyceride IDG), and concentrations of these “lesser” phospholipids and lipids are increased by approximately 166%. Phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PSI concentrations also increased by approximately 25% (note-absolute increases in these phospholipids are. in fact, as large or larger than the increases in phospholipids in the cycle). Cardiolipin (CL) levels do not change with ACTH treatment. The glycerol-3’-PO, acyltransferase and diglyceride kinase appear to require a labile protein (‘1 for maximal activity. Phospholipkls having 2 or 3 phosphate radicals in their polar head group f”) have been found to stimulate cholesterol side chain cleavge and thus steroidogenesis in isolated adrenal mitochondria, but it is not certain that these phospholipids are directly responsible for stimulating steroidogenesis in situ.

second mechanism is to increase fatty acid and diglyceride availability through lipolysis.88 The third mechanism is to activate the glycerol-PO, acyltransferase. Ca++ is required for ACTHand CAMP-induced increases in phospholipids,” but the reason for this requirement is unknown. Protein synthesis appears to be required both for the diglyceride kinase and the acyltransferase, but the reason for this requirement is also presently uncertain. The ACTH-induced increase in phosphatidate synthesis first saturates the phosphatidate-inositide cycle causing a rapid doubling of these phospholipids. We have also found that phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine concentrations increase by approximately 25% after ACTH treatment in vivo,‘5,‘8 and these increases also seem to require Ca++ and protein synthesis” and presumably are secondary to the de novo phosphatidate effect. Steroidogenesis may be increased by phos-

636

pholipids having 2 or 3 phosphates in their head group (di- and triphosphoinositide and phosphatidylglycerolPO,), or perhaps by other undefined phospholipids. These “steroidogenic” phospholipids may increase cholesterol transport from the outer mitochondrial membrane to the cytochrome P-450 complex in the inner membrane, or increase the activity of cytochrome P-450, either directly, as suggested by Lambeth et a1.,89m9’ or by changes in mitochondrial Ca++, as suggested by Hanukoglu and coworkers.‘* Details of the precise molecular events which underlie the increase in steroidogensis remain to be determined. In support of the possibility that phospholipids may be important to steroidogenesis, we have also found that direct addition of diphosphoinositide and phosphatidylinositol to adrenal cells increases corticosterone production.“,‘4 Dr. John Davis and coworkers (persona1 communication) have noted a comparable stimulation of steriodogenesis by adding phosphatidic acid and phosphatidylinositol to bovine corpus luteum cells. Interestingly, the DPI-induced increase in steroidogenesis is not blocked by cyc10heximide,‘35’4 and this suggests that the putative labile protein is required before, but not after, the de novo phospholipid effect. Further support for involvement of the de novo phospholipid effect in steroidogenesis is derived from studies in other steroidogenic tissues. In purified rat Leydig cells,93 LH increased the levels of triphosphoinositide, diphosphoinositide, phosphatidylinositol and phosphatidic acid, nearly 1.5-2-fold, while phosphatidylcholine and phosphatidylethanolamine levels were increased only slightly (approx 20%). Cyclic-AMP provoked similar phospholipid effects, while stimulating testosterone production, and all effects of LH were inhibited by cycloheximide. These results are strikingly similar to those obtained with ACTH in the adrenal cortex. Although initial studies did not suggest that LH provoked changes in phospholipid metabolism in ovarian tissues,94*95more recent studies have shown that LH enhances [32P]Pi incorporation into phosphatidic acid, phosphatidylinositol and polyphosphoinositides in bovine corpus luteum cells96 and rat granulosa cells,97 while stimulating steroidogenesis therein. Small increases in the mass of phosphatidylinositol have also been observed in these cells, and this suggests that the de novo effect is in operation; however, against the latter, [‘*PIPi incorporation appears to be insensitive to cycloheximide (Davis, J.S., personal communication). LHRH and CAMP also increase [32P]Pi incorporation into the above phospholipids in rat ovary granulosa cells (Davis, J.S., personal communication). These findings suggest that the phosphatidate-inositide cycle is activated during the stimulation of steroidogenesis

ROBERT V. FARESE

by gonadotrophins in the ovary, but further studies are needed to more fully characterize these phospholipid effects, i.e., to determine whether the phosphatidylinositol hydrolysis or the de novo effect (or both) is (are) activated by LH and LHRH. In studies of the adrenal zona glomerulosa, every tested steroidogenic agent, viz., angiotensin and K+,55,98and serotonin, ACTH, and cyclic-AMP (submitted for publication) increased levels of phosphatidylinositol, while stimulating corticosterone synthesis during incubations of rat adrenal capsules. These results are of great interest not only for providing further evidence for linking steroidogenesis and phospholipid metabolism, but also because angiotensin and K’ apparently utilize Ca++, rather than cyclic-AMP, as their major “second messenger.“99 It thus seems likely that, at least in some cases, the de novo phospholipid effect can be provoked by Ca++, as well as cyclic-AMP. This may reflect the fact that both Ca++ and CAMP activate or deactivate common enzymes or processes, or that some CAMP effects may be elicited by Cat+ mobilization. Along these lines, one process which is common to the action of both Ca+’ and CAMP is g1ycogenolysis,86*‘W and this may provide glycerol-3-PO, for de novo phosphatidate synthesis. In other studies, we have found that angiotensins5 and K+ (submitted for publication) not only increase the levels of phosphatidylinositol in the adrenal zona glomerulosa, but simultaneously increase the rate of phosphatidylinositol hydrolysis. This is quite different from ACTH, which only increases de novo synthesis, without increasing phosphatidylinositol hydrolysis by phospholipase C action.“’ In accordance with Michell’s hypothesis (see above), the phosphatidylinositol hydrolysis effect conceivably may trigger or amplify the increases in Cat+ which are provoked by angiotensin and K+, and this is supported by failure of steroidogenesisor glycogenolysis-blocking concentrations of EGTA to inhibit the effects of angiotensin II on [32P]Pi incorporation into phosphatidylinositol in rat hepatocytes,34 bovine glomerulosa cells”* or rat adrenal capsules (unpublished observations). However, angiotensinand K+-induced increases in phosphatidylinositol hydrolysis and [32P]Pi incorporation in rat glomerulosa cells are inhibited by EGTA (submitted for publication), and this probably reflects a greater degree of Ca + + deficiency achieved therein. Further studies are obviously needed to determine whether Ca++ is required “permissively” or as a “mediator” for the phosphatidylinositol hydrolysis and labeling responses. Further studies are also needed to determine why angiotensin II only elicits a phosphatidylinositol breakdown response in the rat liver, without increasing de novo phosphatidate synthesis,34 as in the

PHOSPHOLIPIDS AND HORMONE ACTION

637

adrenal glomerulosa5s~98 (note-this could be explained by angiotensin-induced inhibition of glycolysis in the liver, but not in the adrenal). As with other steroidogenie agents, we believe that increases in polyphosphoinositides or other steroidogenic phosphoiipids via the de novo phosphatidate effect may be important for increasing aldosterone production, since this is the only phospholipid effect which is common to the action of all aldosterone-stimulating agents. Insulin is another polypeptide hormone which stimulates the de novo pathway, at least in rat adipose tissue [but not in rat pancreatic tissue where phosphatidylinositol hydrolysis occurss4]. As recently reported,‘03 insulin treatment in vivo provoked rapid increases (SO%-100%) in the concentrations of phosphatidic acid, phosphatidylinositol and polyphosphoinositides in rat adipose tissue, while phosphatidylcholine and phosphatidylethanolamine concentrations changed slightly, if at all. Similar effects were observed with physiological concentrations of insulin added to incubations of adipocytes in vitro,‘03 and more recently (unpublished observations), we have found that puromycin and cycloheximide inhibit the insulininduced increases in the mass of, and [32P]Pi incorporation into, phosphatidylinositol (other workers have also observed that insulin increases [32P]Pi incorporation into phosphatidylinositol in rat adipose tissuee.g., Ref 63). The “second messenger” which mediates this effect of insulin, and the full significance of these phospholipid changes in adipose tissue are unknown. However, Macaulay et a1.‘04 and Kiechle”’ have recently shown that phosphatidylserine and phosphatidic acid can activate low K, phosphodiesterase and pyruvate dehydrogenase. Since insulin increases these phospholipids (we have also observed increases in phosphatidylserine after insulin treatment-unpublished observations), they may serve as “second” or “third messengers” in the action of insulin on these and other enzymes. In addition, since phosphatidate synthesis is the first step in triglyceride synthesis, much of what has been discussed in relation to the action of Table 3. Summary

of Well-Characterized

Effects of Hormones

insulin and other polypeptide hormones on phosphatidate synthesis may also apply to insulin’s action on triglyceride synthesis, and thus fat deposition. One interesting possibility that emanates from these considerations is that insulin may use changes in phospholipids to couple triglyceride synthesis with the activation of pyruvate dehydrogenase, the latter being important for fatty acid synthesis from carbohydrates or amino acids. A final hormone that will be considered is PTH. Earlier studies had shown that PTH increases f3*P]Pi incorporation into phosphatidic acid and phosphatidylinositol in cat renal slices in vitro.‘06 To further characterize this effect, we found’07-‘09 that addition of synthetic, N-terminal PTH peptide to incubations of rabbit kidney cortical tubules provoked rapid, doserelated increases in triphosphoinositide, diphosphoinositide, phosphatidic acid, and phosphatidylinositol, and these increases were blocked by cycloheximide, and required Ca++. Furthermore, PTH rapidly provoked similar effects in vivo,“” and PTH-induced increases in phospholipids have been confirmed by Meltzer et al.“’ Dibutyryl CAMP elicited similar effects in vitro,‘07-‘09 and CAMP may therefore serve as the intracellular mediator for these PTH effects on phospholipids. The functional significance of these changes in renal phospholipids is unknown, but effects of PTH or CAMP on phosphaturia,“’ amino acid transport”2 and vitamin D hydroxylation”3 are inhibited by cycloheximide, and it would not be surprising if these and other membranerelated events require, or are mediated by, this phospholipid effect of PTH. CONCLUSION

Listed in Table 3 are those hormones and neurotransmitters whose effects on the phosphatidate-inositide pathway seem to be reasonably well characterized. The list is probably not complete. Indeed, many other hormones stimulate “P-labeling of phosphatidic acid and phosphatidylinositol [e.g., thyrotropin,“4.“5 LHRH,“&” corticotropin-releasing factor,“8 nerve and Neutrotransmitters

on the Phosphatidate-lnositide

Cycle

PI Hydrolysis ca+ +-Independent

Ca+ +-Dependent

De Nova-PA-P-PPI

Effect

cr-Adrenergic Agents (Lwer, Parotid)

Acetylcholine (Pancreas, Salivary Gland)

Serotonin (Blowfly Salivary Gland)

CCK (Pancreas) Secretin (Pancreas)

Vasopressin (Liver)

VIP (Pancreas)

K+ (Adrenal Glomerulosa)

Angiotensin-Ii (Liver, Adrenal Medulla)

GiP (Pancreas)

Serotonin (Adrenal Glomerulosa)

ACTH (Adrenal) LH (Gonads) Angiotensin-II (Adrenal Glomerulosa)

Insulin (Pancreas) TRF (Pituitary)

Angiotensin II (Adrenal Glomerulosa)

Insulin (Fat) PTH (Kidney Cortex)

KC (Adrenal Glomerulosa) Glucose (Islets)

LHRH (Ovary)

638

ROBERT V. FARESE

growth factor,“’ but labeling is non-specific. and it is not certain if the labeling reflects phosphatidylinositol hydrolysis, de novo phosphatidate synthesis, or another change in phospholipid metabolism (e.g., diglyceride generation from triglyceride, phosphatidylcholine, etc). In addition, it should be recognized that some hormones and neurotransmitters provoke changes in phospholipid metabolism which are extrinsic to the phosphatidate-inositide pathway (see 120). Presently, it seems likely that the first two mechanisms, viz., Ca”-independent and Ca++-dependent phosphatidylinositol hydrolysis, are important for the coupling of receptor activation with Ca’ T mobilization and subsequent secretory responses and possibly smooth muscle contraction. The third mechanism, de novo phosphatidate synthesis, seems to be important in steroidogenesis and possibly fat synthesis, and is probably involved in other processes as well. With respect to directions for future research in this area, there are many important questions that need to be answered. Chief amongst these are the following: (I ) do activated hormone receptors directly provoke phosphatidylinositol hydrolysis in the plasma membrane, and is this what triggers the opening of Ca++ channels or the mobilization of Ca++ from internal

stores, (2) how do CAMP and Ca++ provoke increases in phosphatidylinositol hydrolysis and de novo phosphatate synthesis in the cell interior, (3) why does the same agent, e.g., insulin or angiotensin, provoke different phospholipid effects in different target tissues, (4) is a labile protein truly required for de nova phosphatidate synthesis, and, if so, is this protein a key regulatory factor in the action of all hormones that provoke the de novo phospholipid effect, (5) is the cycloheximide-sensitive and Ca + +-requiring de novo effect also activated in cases of large-scale phosphatidylinositol hydrolysis, (6) what metabolic processes are controlled by what phospholipid effects, and (7) can specific inhibitors of phospholipid metabolism be developed, not only to serve as experimental probes, but also as forerunners for a new class of therapeutic agents? Clearly, the answers to these and many other important related questions should provide considerable insight into our understanding of the mechanisms of action of many hormones and neurotransmitters. What is most encouraging is that we now are not only more cognizant of the questions to ask, but, at this point, many of the answers to these questions seem to be within reach.

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