Inositol phospholipids in stimulated smooth muscles

Cell Calcium 3:

359-368, 1982

INOSITOL PHOSPHOLIPIDS IN STIMULATED SMOOTH MUSCLES Tadaomi Takenawa Institute of Basic Medical Sciences, University of Tsukuba Niihari-gun, Ibaraki-ken, 305 Japan ABSTRACT A variety of physiological stimulants, such as hormones and neurotransmity+rs, which exert physiological responses by increasing intracellevel, enhance phosphatidylinositol turnover in target organs. lular Ca This reaction is characterized by an initial breakdown of phosphatidylinositol followed by a compensatory increase in its resynthesis. In many studies, experimental conditions adopted to demonstrate receptor-mediated increase of phosphatidylinositol turnover as the results of physiological stimuli, however, seem to be either unphysiological or inadequate. In some, only the secondary resynthesis of phosphatidylinositol was measured. In others, although the primary breakdown of phosphatidylinositol was measured, unphysiologically high concentration of stimulants or extended period of reaction was used. It is necessary to use physiological stimuli and methods to study physiological roles of "phosphatidylinositol response". Therefore, in this review, discussions are focussed on relationships between physiological stimuli to smooth muscle tissues, alterations of phosphatidylinositol metabolism and their physiological roles. It is likely that there are two systems to activate phosphatidy$+ inositol turnover in smooth2puscles. One is dependent on exogenous Ca The other is not dependent on and can be2pimicked by Ca -ionophore. exogenous Ca and linked to receptor-mediated activation. On the other hand, several laboratories have found that phosphatidate accumulation as a result of the induced degradation of phosphatidylinositol is closely correlated with physiological functi;p+ (contraction) and that this phospholipid can behave as an endogenous Ca ionophore. These fac$zj suggest that phosphatidate itself plays an important role in opening Ca gate in plasma membrane in response to receptor activation. Thus, the following scheme could be proposed: Ex9genous independent Ca Agonists+receptor ~3 Phosphatidylinositol breakdown .-b Exogenous Ca2+-

Phosphatidate formation e

/ 2+ ' Ca influx--,Physiological 359

responses

INTRODUCTION It is known that when various tissues and cells are exposed to physiological stimulation, phosphatidylinositol turnover is enhanced (1). This enhancement has been attributed to a stimulation in phosphatidylinositol degradation, concomitant with a secondary increase in the resynthesis of the phospholipids. There now seems to be strong circumstantial evidence that this hydrolysis of phosphatidylinositol is an early event of receptor activation by hormoyf, neurotransmitters or mitogens which exert their functions through Ca as second messenger. With regard to the role of stimulated phosphatidylinositol degradation, Michell (1,2) proposed the hypothesis that activatifp of phosphatidylinositol turnover is associated wis$ the opening of a Ca 2$ate, resulting in an elevation of i tracellular 9+ Ca which activated the Ca -dependent processes, especially Ca -dependent protein kinase, and exerts the hormonal function. Subsequently, many investigators have been #tracted to the study on the role of phosphatidylinositol turnover in Ca influx. On the other hand, Cockcroft -et al (3) and Hawthorne (4) have opposed this hypothesis and considered that stimulated phosp&tidylinositol breakdown is initiated by an elevation of intracellular Ca as the res?$t of receptor activation but is not a cause of a rise in intracellular Ca , because there are several 9ystems in which phygphatidylinositol breakdown is diminished by removing Ca in medium and ionophore causes the breakdown. Thus, the exact relationship between ;:2+ and phosphatidylinositol turnover still remains to be solved. In smooth muscle tissues, the enhancement of phosphatidylinositol turnover was first reported in vas deferens (5) when an a-adrenergic stimulant was applied. Thereafter, the similar phenomena have been found to be induced by the application of cholinergic agonists (6-lo), histamine (8,10), adrenergic agonists (6,8,10-12) 5-hydroxytryptamine (6,lO) and high concentration of K (13) solution in various smooth muscle tissues. It is evident from these observations that there is a close correlation between this phenomenon and contraction of smooth muscle. In many cases so far, however, experiments on phosphatidylinositol turnover have been carried out under unphysiological conditions: namely (1) phosphatidylinositol turnover was determined at lo-60 min after the application of stimulants notwithstanding smooth muscle contraction occurs around l-3 min after the addition; (2) much higher doses of agonists than those needed for contraction of smooth muscle were used for measuring phosphatidylinositol turnover. To clarify the correlation between phosphatidylinositol turnover and physiological functions, it is necessary to reconsider the phenomenon by using such data that were obtained under physiological conditions. In this review, physiological significance of phosphatidylinosit2& metabolism in smooth muscle tissues, especially relationship between Ca and enhanced phosphatidylinositol metabolism, is discussed. PHOSHPATIDYLINOSITOL RESPONSE IN SMOOTH MUSCLES Alterations of inositol phospholipid metabolism in stimulated smooth muscles have been investigated mainly using vas deferens, ileum smooth muscle, iris and aorta. Results obtained so far are summarized in Table 1. Vas Deferens Canessa de Scarnati and Lapet&a (5) found that a-adrenergic agents stimulated the incorporation of [ P]Pi into phosphatidylinositol in rat 360

Table 1 Alterations of Phospholipid Metabolism in Stimulated Smooth Muscle

Tissue

Stimulant Method

Vas Nor Deferens

Epi Ach

Ileum

Ach Carbachol

Aorta

Reference

30-120 min PtdIns labeling t 3 min PtdIns labeling + labeling t PtdA Increase PtdA content PtdIns content 15 min Decrease Decrease PsdIns content 30 min l-20 min Decrease [ H]PtdIns 32 P-labeling

5-HT High[K+] Ach

32 P-labeling

5-30 min

Nor

5-30 min

5-HT

30 min

Dopa

30 min

Epi

30 min

His

30 min

Nor

Result

32 P-labeling

30 min 5-60 min dins content 30-120 min 35 P-labeling 30 min dins content 30 min "P-labeling 30 min 30 min

His

Iris

Incubation Time

32 P-labeling

2 h lh

Epi

lh

Met

l-2 h

PtdIns labeling PtdIns labeling Decrease PtdIns labeling Decrease PtdIns labeling PtdIns labeling

9 t + t q

(5) (15) (15) (15) (8) (9) (9) (7) (7) (7) (10) (8) (10) (13)

PtdIns labeling 1(6,16,25) PtdA labeling t(6,16,25) PtdIns4,5P21abeling &(16,25) PtdIns labeling t (6) PtdA labeling + (6) PtdIns labeling + (6) PtdA labeling + (6) PtdIns labeling + (6) PtdA labeling + (6) PtdIns labeling + (6) PtdA labeling t (6) PtdIns labeling + (6) PtdA labeling + (6) PtdIns PtdIns PtdA PtdIns PtdA PtdIns PtdA

labeling t labeling t labeling 4 labeling 9 labeling t labeling + labeling t

(11) (12) (12) (12) (12) (11,12) (11,12)

Abbreviations used are: PtdIns, phosphatidylinositol; PtdA, phosphatidic acid; PtdIns4,5P2, phosphatidylinositol 4,5-diphosphate; Nor, norepinephrine; Epi, epinephrine; Ach, acetylcholine; His, histamine; 5-HT, 5-hydroxytryptamine; Dopa, dopamine; Met, methoxamine.

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vas deferens as observed in other non-muscle tissues. This stimulation was considered to be a-receptor linked phenomenon since a-blocker could inhibit the incorporation. This stimulated incorporation, however, was demonstrable only at a concentration (100 MM) of norepinephrine 10 times higher than that needed for contraction. Moreover, the stimulated incorporation was not significant until 1 h after the application of norepinephrine. On the other hand, Egawa -et al (9) found that acetylcholine (10 uM) enhanced the degradation of phosphatidylinositol very rapidly in rabbit vas deferens. This change was detectable already within 1 min, whereas it took 1 h or more for stimulation of phosphatidylinositol labeling to become evident. These results suggest that phosphatidylinositol breakdown is a trigger reaction in "phosphatidylinositol response" as proposed by Michell (14). The acetylcholine-induced decrease in phosphatidylinositol content led to the compensatory synthesis of phosphatidylinositol. This synthesis was inhibited by acetylcholine, while the degradation was blocked by the muscarinic antagonist atropine. Because of the inhibitory effect of acetylcholine, the appearance of stimulation of phosphatidylinositol labeling was delayed compared to other tissues. More recently, it was found in rat vas deferens that norepinephrine induced the immediate stimulation of labeling of phosphatidate, which is a substance produced as a result of phosphatidylinositol degradation, and the increase in its content (15). This phenomenon appears to be closely related to the contraction in the view of dose response and time course studies. Details are discussed later in Role of Phosphatidate. Ileum Smooth Muscle Jafferji and Michell (7) investigated the effect of cholinergic agents on the metabolism of phosphatidylinositol and phsophatidate in longitudinal smooth muscle of 3guinea pig ileum and found that they stimulated the incorporation of [ P]Pi into phosphatidylinositol and phosphatidate. This effect appeared to be mediated through the activation of muscarinic The increased receptors since atropine inhibited the incorporation. phosphatidylinositol labeling was clearly observed within 5 min of application of carbamoylcholine at a high concentration (1 mM). At 100 HIM, however, the stimulation could be detected after 10 min. They also demonstrated that cholinergic stimulation provoked a decrease in phosphatidylinositol content in this tissue. Contraction, the physiological response of ileum smooth muscle, could be induced by several agonists, including histamine, 5_hydroxytryptamine, angiotensin II and norepinephr#e. These agonists also triggered the increased incorporation of [ P]Pi into phosphatidylinositol (10). When this ileum smooth pscle was incubated for 30 min in high K+ medium the incorporation of [ P]Pi into phosphatidylinositol was increased several fold (13). But in accompanying experiments, they showed that this high K medium did not have effect on the incorporation in pancreas although s onded to carbamoylcholine and displayed the increased incorporation ,i:;?Wp P]Pi into phosphatidylinositol. These findipgs are consistent with ous+knowledge that depolarization induced by high K is capable to increase influx in ileum smooth muscle but not in pancreas. They suggested th2f: Ca there was a functional association between phosphatidylinositol and Ca gating rather than between phosphatidylinositol metabolism and a receptoragonist interaction. However, if we assume that phosphagdylinositol degradation is enhanced by an elevation of intracellular Ca , which is

362

cqused by depolarization, increased phosphatidylifpsitol breakdown in high K med$$m appears to be a result of a rise in Ca influx but not a cause of Ca gating. Indeed, it was demonstrated that phosphatidylinositol degfadation in rabbit vas deferens was initiat?$ by increasing iq$racellular or by usp of Ca -ionophore concentration by increasing exogenous Ca Ca on phos&atidyl(9). Therefore, experiments on the effect of high K influx inositol metabolism does not afford a clue to elucidate which of Ca and phosphatidylinositol degradation is more directly connected to receptormediated activation. Iris Smooth Muscle When rabbit iris muscle was stimulated by norepinephri% or acetylchoP labeling of line (6), there also took place a marked increase in phosphatidylinositol and phosphatidate. This increase was proved to be dependent on time and concentration of the agonists. In this experiment, usually 1 mM norepinephrine for time course study and 30 min incubation for dose response study were adopted. Under these conditions, phenoxybenzamine blocked the stimulatory effect of norepinephrine. Acetylcholine (>50 JIM) also stimulated the incorporation during 30 min incubation, and atropine blocked the stimulatory effect completely. Dopamine, 5_hydroxytryptamine, epinephrine or histamine also increased the labeling of phosphatidylinostiol and phosphatidate. Recently, it was demons*ted (16) that 50 UM acetylP from phosphatidylinositol choline induced t% significant loss of 4,5-diphosphate in 32P-labeled iris mucle, which accompanied by a signifiP-labeling of phosphatidylinositol and phosphatidate cant increase in during 5 min incubation. Details about the effect on phosphatidylinositol 4,5-diphosphate metabolism are discussed later. The stimulated labeling of phosphatidylinositol and phosphatidate b+y norepinephrine or acetylc$oline was dependent on extracellular Na . Ouabain, which blocks the Na pump, inhibited t$e stimulated labeling of the phospholipids (17.2JqIt was speculated that Na plays an important role in delivering of Ca to the vtoplasm and muscarinic receptor activation is channel which depolarizes $+he membrane and thought to actiya+te the Na activates the Ca could activate channel. The resultant increase in Ca the breakdown of phosphoinositides to diacylglycerol and enhanced the labeling of phosphatidylinositol and phosphatidate. According to Putney (18), however, there is no+compelling evidence which tells that the initial events are mediated by Na notwithstanding the fact that the labelfng of phosphatidylinositol and phosphatidate was dependent on exogenous Na . He pointed out that net decrease in phosphatidylinositol content as an initial reaction of a receptor activation was not prevented by decreasing extracellular Na+ (19). Aorta Smooth Muscle 32 It was reported that a-adrenergic agents stimulated P-labeling of phosphatidylinositol in cat aorta (11) in a dose-dependent manner during inc$a tion for 1 - 2 h. In this experiment, further characterization, such P incorporation into other phospholipids and time course study was not Z&e. More recently another report on effect of a-adrenergic agents on phosphatidylinositol metabolism was appeared (12). It was demonstrated that labeling of phosphatidylinositol and phosphatidate was stimulated by

363

a-adrenergic agents In this experiment, of the labeling of than that required determined after 1 h

in a dose-dependent manner and blocked by a -blockers. however, the dose of agonists required for &timulation phosphatidylinositol was approximately 10 times higher for contraction of the muscle, and the labeling was incubation.

ROLE OF Ca2+ IN PHOSPHATIDYLINOSITOL METABOLISM Phosphatidylinositol levels and the turnover are directly regulated by two enzymes. One is CDP-diacylglycerol inositol 3-phosphatidyl transferase which catalyzes the final step of -de novo synthesis of phosphatidylinositol (20,21). The other enzyme is phospholipase C, which catalyzes the degradation of phosphatidylinositol to diacylglycerol. CDP-diacylglycerol inositol 3-@osphati$yltransferase is present in microsomal membranes2,and requires (22). The or Mn for its activity (20), and is inhibited by Ca Mg enzymes in smoo muscle tissues, such as vas deferens and aorta are very t& and 90% of t3+ activity is inhibited by 10 HM CaCl . sensitive to Ca Furthermore, when endogenous Ca was removed with2+ethyleneglycol-b1s ionophore A23187, (o-aminoethyl ether) N,N'-tetraacetic acid (EGTA) and Ca a marked increase in phosphatidylinositol labeling was observed (23). This may be the reason why rapid labeling of phosphatidylinositol is not able to be observed in smooth muscle tissues. On the other hand, phospholipase C, whslh is mainly located in cytosol, requires fairly high concentrations of Ca for its activity (24). But by the addition of unsaturated fatty acid, concentration required for the activation could be reduced to the These results imply a possibility that some $:iological level (22) endogenous factor is playing an important role in the regulation ?f phospholipase C activity. It is possible to think that an increase in Ca causes the inhibition of phosphatidylinositol synthesis as well as the enhancement of phosphatidylinositol breakdown, resulting in the decrease & phosphatidylinositol content. Actually, an increase in intracellular Ca concentration caused the phosphatidylinositol degradation in rabbit vas deferens (9). Likewise, when the homogenate of vas g+eferensy+s incubated in media containing various concentrations of Ca dependent a Ca decrease in phosphatidylinositol was observed. Phosphat:dylinositol content in rabbit vas deferens was 66.7 f 1.8 ug of phosphatidylinosito$+phosphorus/ mg of total lipid 2qhosphorus. After incubation in the Ca -containsyg buffer plus 10 PM Ca ionophore A23187, which increases intracellular Ca , the phosphatidyl$$ositol content decreased to 50.0 f 1.8. In co2frast, when intracellular Ca ionophore was removed by incubating EGTA and 10 PM Ca A23187, the content of phosphatidylinositol was increased to 75.0 + 1.7. l

2+ The enhancement of C," influx caused by depolarization of32cell membrane provoked by high K , which stimulated the incorporation of [ PlPi into phosphatidylinositol in ileum smooth muscle (13), did not alter phosphatidylinositol content nor its degradation in vas deferens (9) although it decreased the labeling of _&osphatidylinositol. This discrepancy may be due sensitivity of phosphatidylinosito12+synthesis to the difference in Ca system in tissues. However, even in the absence of exogenous Ca , acetylcholine could stimulate the degradation of phosphatidylinositol in vas to the action of deferens (9). Considering these observations witbAregard LI acetylcholine in the presence and absence of Ca , we can speculate t@t there are two systems of phosphatidylinosito12+degradation. One is Ca dependent degradation and the other is Ca -independent and receptqf activation-dependent degradation. Concerning the effect of exogenous Ca 364

on iris smooth muscle, Akhtar and Abdel-Latif (25) also reported that a removal of Ca2+ from medium inhibited the labeling of phosphatidate partially but not that of phosphatidylinositol. Tb$_ phosphatidylinositol labeling was not blocked even by adding EGTA to Ca -free medium. On the contrary, phosp&tidylinositol 4,5-diphosphate degradation was completely inhibited by Ca removal from medium. These results also suggesLfthat at least a part of "phosphatidylinositol response" is resistant to Ca removal from medium. ROLE OF PHOSPHATIDATE It is known that in consequence of receptor-mediated breakdown of phosphatidylinositol, phosphatidate is formed by diacylglycerol kinase. In smooth muscle, a significant increase in phosphatidate accumulation by receptor-activation was observed (15). This accumulation was transient and reached maximum at 3 min after the application of norepinephrine and levelled off with further incubation in rat vas deferens. Furthermore, such new formation of phosphatidate occurred very rapidly and could be detected within 10 set after the stimul?$ion. Recently, it has been shown that ionophore (26). If phosphatidate could phosphatidT$e can behave as a Ca act as Ca ionophore in plasma membranes, it is favorab&z to explain the influx. Salmon hypothesis that phosphatidylinositol breakdown triggers Ca and Honeyman (27) demonstrated that exogenously applied phosphatidate could mimic the acetylcholine effect on cultured smooth muscle. A similar effect of phosphatidate has been observed in other tissues (28). Putney -et al (29) also found that exogenous phosphatidate caused a similar function as observed by receptor-stimulation in parotid gland. Based on these findings, they proposed that stimulated phosphatidylinosi$yl degradation and the resultant phosph?$idate accumulation open the Ca gate and increase the level. This hypothesis was further supported by an intracellular Ca experiment on supersensitivity in vas deferens (15). Rat vas deferens shows supersensitivity to agonists 7 to 10 days after denervation. Denervated vas deferens could respond to lower concentration of noradrenaline and maximal response (contraction) becomes stronger. Using these tissues, alterations of phospholipid metabolism in response to norepinephrine were studied. Phosphatidate labeling was specifically increased by norepinephrine in both control and denervated vasa deferentia in a dose-dependent manner. Labeling of other phospholipids was not changed by norepinephrine in either tissue. Norepinephrine-induced labeling of phosphatidate was significantly accelerated by denervation. Two points relative to this are important. First, in comparison with control, phosphatidate labeling was stimulated by lower concentrations of norepinephrine in denervated tissue. Secondly, the maximum response to norepinephrine was increased by denervation, a phenomenon being also detected in the contractile response. The dose range of norepinephrine to increase phosphatidate labeling is virtually identical to that necessary for producing contractile response in either control or denervated tissue. Furthermore, the absolute amount of phosphatidate was also increased by norepinephrine and this increase was exaggerated by denervation. These results also lend support for the hypothesis presented by Michell (1). In smooth muscle, it seems to be mos$+ likely that phosphatsate plays an important role for ionophore. In this respect, the activation of Ca influx just like a Ca fact that the accumulation of phosphatidate occurred only transiently in

365

response to norepinephrine is quite favorable for possible participation of phosphatidate in physiological function of smooth muscle, ie contraction and relaxation. But unfortunately exogenously added phosphatidate did not contract vas deferens (unpublished observation). Since phosphatidate is an amphiphilic material and forms micelles in aqueous solution, it may be hard, as suggested by Putney (18), to peneterate into the plasma membranes of smooth muscle tissues in which each cell is tightly connected with connective tissues. That may be the reason why exogenously applied phosphatidate did not contract vas deferens, while it could contract single cells such as cultured smooth muscle (27).

RECEPTOR-MEDIATED ALTERATION OF PHOSPHATIDYLINOSITOL 4-MONOPHOSPHATE AND PHOSPHATIDYLINOSITOL 4,5_DIPHOSPHATE. Abdel-Latif et al (16) reported that addition of acetylcholine to 32P labeled rabbit iris smooth muscle increases significantly the breakdown of phosphatidylinositol 4,5-diphosphate and that these stimulatory effects are blocked by *opine. This phenomenon was accompanied with a significant increase in labeling of phosphatidylinositol and phosphatidate. With respect to Ca2$ effect, such acetyicholine-stimulated breakdown of phosphatidylinositol 4,5-diphosphate and 2+P-labeling of p%phatidate was found to f'labeling of phosph2; be dependent on the exogenous Ca (25), whereas tidylinositol was insensitive to the removal of Ca . Furthermore, Ca ionophore A23187 increased the breakdown of phosphatidylinositol 4,5diphosphate and labeling of phosphatidate (25). On the contrary, changes in labeling of phosphatidylinositol in response to ionophore were negligible. This ionophore-induced phosphal2i+dylinositol 4,5-diphosphate breakdown was also dependent on exogenous Ca . Thus, it was concluded that musffrinic receptor activation led to an elevation of intracellular Ca and subsequently to the increase in phosphatidylinositol 4,5-diphosphate breakdown and phosphatidate formation. However, as described above, th?Y also reported that phosphatidylinositol labeling was not affected by Ca depletion. Since they did not measure the degradation of phosphatidys; seems to be difficult to judge from these data whether Ca inositol, it influx preceded phosphatidylinositol degradation or resulted from it even breakdown though it was clear that phosphatidylinositol 4,5@phosphate was caused by an elevation of intracellular Ca . We measured the norepinephrine-induced alteration of phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5_diphosphate in both control and denervated vasa deferentia (15). In this case, the breakdown of both lipids was accelerated by norepinephrine, with phosphatidylinositol 4-monophosphate markedly. The influence of denervation on the norepinephrine-induced breakdown of these lipids was not detectable. Thus, it is likely that receptor-linked activation correlates more closely with an increased formation of phosphatidate rather than with the breakdown of these lipids. ACKNOWLEDGMENT The author is sincerely grateful to Dr. discussions and suggestions for this review.

366

K. Egawa for his useful

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Received 28.9.82 Revised version received and accepted 25.10.82

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