Carbachol Induces a Rapid and Sustained Hydrolysis of Polyphosphoinositide in Bovine Tracheal Smooth Muscle Measurements of the Mass of Polyphosphoinositides, 1,Z-Diacylglycerol, and Phosphatidic Acid*

The effects of carbachol on polyphosphoinositides and 1,2-diacylglycerol metabolism were investigated in bovine tracheal smooth muscle by measuring both lipid mass and the turnover of [SH]inositol-labeled phosphoinositides. Carbachol induces a rapid reduction in the mass of phosphatidylinositol 4,tbbisphosphate and phosphatidylinositol 4-monophosphate and a rapid increase in the mass of 1,2-diacylglycerol and phos- phatidic acid. These changes in lipid mass are sustained for at least 60 min. The level of phosphatidylinositol shows a delayed and progressive decrease during a 60-min period of carbachol stimulation. The addition of atropine reverses these responses completely. Car- bachol stimulates a rapid loss in ['H]inositol radioactivity from phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate associated with production of ['Hlinositol trisphosphate. The car- bachol-induced change in the mass of phosphoinosi- tides and phosphatidic acid is not affected by removal of extracellular Ca2+ and does not appear to be second- ary to an increase in intracellular Ca2+. These results indicate that polyphosphoinositide and a the These results strongly suggest that carbachol-induced contraction is mediated by the hydrolysis of polyphos- phoinositides with the resulting generation of two messengers: inositol 1,4,5-trisphosphate and 1,Z-diacyl- glycerol.

The effects of carbachol on polyphosphoinositides and 1,2-diacylglycerol metabolism were investigated in bovine tracheal smooth muscle by measuring both lipid mass and the turnover of [SH]inositol-labeled phosphoinositides. Carbachol induces a rapid reduction in the mass of phosphatidylinositol 4,tbbisphosphate and phosphatidylinositol 4-monophosphate and a rapid increase in the mass of 1,2-diacylglycerol and phosphatidic acid. These changes in lipid mass are sustained for at least 60 min. The level of phosphatidylinositol shows a delayed and progressive decrease during a 60min period of carbachol stimulation. The addition of atropine reverses these responses completely. Carbachol stimulates a rapid loss in ['H]inositol radioactivity from phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate associated with production of ['Hlinositol trisphosphate. The carbachol-induced change in the mass of phosphoinositides and phosphatidic acid is not affected by removal of extracellular Ca2+ and does not appear to be secondary to an increase in intracellular Ca2+. These results indicate that carbachol causes phospholipase C-mediated polyphosphoinositide breakdown, resulting in the production of inositol trisphosphate and a sustained increase in the actual content of 1,2-diacylglycerol. These results strongly suggest that carbachol-induced contraction is mediated by the hydrolysis of polyphosphoinositides with the resulting generation of two messengers: inositol 1,4,5-trisphosphate and 1,Z-diacylglycerol.
The intracellular events involved in the regulation of smooth muscle contraction remain a matter of controversy. It has been proposed that an increase in intracellular free ea2+ concentration activates myosin light chain kinase, a ea2+-calmodulin-dependent enzyme, leading to the phosphorylation of myosin light chain, and that this molecular event induces contraction through an increased interaction of myosin with actin (1)(2)(3)(4)(5)(6)(7). This model assumes that during the sustained phase of contraction, the intracellular free ea2+ concentration and the amount of phosphorylated myosin light chain remain elevated. However, recent works done by Morand National Institutes of Health Grant HL 35849. The costs of * This work was supported by the Muscular Dystrophy Association publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ To whom correspondence should be addressed.
gan and Morgan (8,9) show that, when aequorin is employed as an intracellular calcium indicator, addition of either phenylephrine or angiotensin to vascular smooth muscle leads to a transient, rather than sustained, increase in intracellular free Caz+, but a sustained contractile response. Likewise, Silver and Stull (lo), and Aksoy et al. (11) have shown that the amount of phosphorylated myosin light chain rapidly rises after agonist addition to either tracheal or vascular smooth muscles and then gradually returns toward the base-line value during the sustained phase of contraction. These studies indicate that the mechanisms by which Ca2+ acts may be more complex than previously thought. They have led to the postulate that a second calcium-dependent mechanism operates during the sustained phase of smooth muscle contraction (10)(11)(12)58).
In recent years it has been shown that the interaction of Ca*+-mobilizing hormones with their receptors activates a specific phospholipase C which catalyzes the hydrolysis of PtdIns-4,5-P2' in a variety of tissues or cells (13)(14)(15)(16)(17). This results in production of Ins-P3 and 1,2-diacylglycerol. It is currently believed that Ins-P3 causes the release of Ca2+ from an intracellular pool (presumably endoplasmic reticulum) and produces the initial intracellular eaZ+ transient (14,18), thereby activating Ca2+-calmodulin-dependent enzymes. On the other hand, an increase in the 1,2-diacylglycerol content of the plasma membrane is thought to activate the Caz+activated, phospholipid-dependent protein kinase (C-kinase) (17,19). Based on results obtained from the use of agents which bypass receptor-mediated events and directly activate Ca2+-calmodulin-dependent kinases and the C-kinase in smooth muscle, we have proposed that in smooth muscle contraction as well as secretory responses in many tissues, the calmodulin branch of the Ca2+ messenger system is transiently activated by a transient rise in cytosolic free Ca'+ concentration and is largely responsible for initiating cellular response; the C-kinase branch which is activated by both the sustained increase in plasma membrane Ca2+ influx rate and the increase in the 1,2-diacylglycerol content of the plasma membrane is responsible for sustaining the response (20,21,(23)(24)(25)(26)(27). In this model a sustained increase in intracellular free Ca2+ is not a prerequisite for the sustained phase of smooth muscle contraction.
The present study was undertaken to determine whether in bovine tracheal smooth muscle an agonist, carbachol, causes polyphosphoinositide breakdown and generates the two mes-  sengers, Ins-P3 and 1,2-diacylglycerol. Our results show that carbachol stimulation causes a rapid decrease in the mass of the phosphoinositides and a small but significant increase in the mass of 1,2-diacylglycerol and phosphatidic acid. Furthermore, these changes in the lipid mass are sustained during a 1-h period of hormone action.

RESULTS
Effect of Carbachol on the Turnover of fH]Inositol-labeled Phosphoinositides-As shown in Fig. 1, the addition of carbachol to tracheal muscle strips prelabeled with [3H]in~sitol causes a rapid decrease in radioactivity from both the PtdIns-4,5-P2 and PtdIns-4-P pools. A loss of 28% of 3H radioactivity from PtdIns-4,5-P2 is detected at 30 s and is maximal at 2 min (a loss of 34%). The change in radioactivity of PtdIns-4-P follows a similar time course to that of PtdIns-4,5-P2. The radioactivity in both the PtdIns-4,5-Pz and PtdIns-4-P pool remains reduced for the initial 10 min. In contrast, the radioactivity of phosphatidylinositol shows no significant change during this period.
Effect of Carbachol on Inositol Phosphate Production-Carbachol stimulates the production of Ins-P, Ins-PZ, and Ins-P3 in [3H]inositol-prelabeled muscle strips (Fig. 2). Both Ins-P, and Ins-P3 increase rapidly following carbachol addition and reach peaks at 1 min (870% in Ins-P2 and 800% in Ins-P, of each control value). Then the values slightly fall but still remain 6-to 7-fold higher than the control values during 10 min. Ins-P rises less rapidly and reaches a peak (190% of control value) at 5 min and stays at that level. The rapid decrease in radioactivity of PtdIns-4,5-P2 and the concomitant accumulation of Ins-P, is consistent with carbachol- Effect of Carbachol on the Absolute Mass of Phosphoinositides and Phosphatidic Acid-The time course of the changes in the mass of phosphoinositides and phosphatidic acid is shown in Fig. 3. The resting content of PtdIns-4,5-P2 is about 0.4% of total phospholipid on a molar basis and also onetenth of the mass of phosphatidylinositol (about 4%). Upon carbachol stimulation, the content of PtdIns-4,5-P2 rapidly decreases and reaches a nadir (50% of the resting value) at 1 min. Then the content of PtdIns-4,5-P2 slightly recovers, but still remains appreciably lower than the resting value for at least 60 min. During a similar period of time, muscle strips not treated with carbachol show no significant change in the PtdIns-4,5-Pz content. The content of PtdIns-4-P also declines rapidly from the resting level of 0.4% and remains reduced during the carbachol stimulation of 60 min. Again, muscles not stimulated with carbachol show no significant change in the content of this phosphoinositide. These changes in PtdIns-4,5-Pp and PtdIns-4-P during the first 5 min are apparently similar to the changes in radioactivity of polyphosphoinositide in [3H]inositol-labeled muscles (Fig. l ) , but the change in the mass of PtdIns-4,5-P2 is quantitatively larger than that in the labeling experiments (50% uersus 34% as a maximal decrease). The mass of phosphatidylinositol shows a delayed decrease. The content of phosphatidylinositol shows no significant change at 30 s and then declines slowly and progressively for 60 min. The content of phosphatidylinositol at 60 min is reduced to 44% of the resting content. In contrast, the mass of phosphatidic acid rises rapidly and progressively from a resting value of about 0.2% of total phospholipid mass to reach a plateau of about 0.9% of this mass at 30 min.
As shown in Fig. 4, the changes in the mass of PtdIns-4,5-P p and phosphatidic acid are dose-dependent and become  greater with increasing doses of carbachol. The ED,o for the carbachol-induced changes in PtdIns-4,5-P2 and phosphatidic acid is 5 and 3 p~, respectively. The values are 15-to 30-fold higher than the ED5, for the carbachol-induced tension development (28). One possible interpretation of these data is the existence of large numbers of spare receptors. A second is that there is an amplification step between messenger generation and physiological responses.
The changes in the mass of polyphosphoinositides and phosphatidic acid are reversed by atropine, an antagonist of muscarinic-type receptor (Fig. 5). The content of PtdIns-4,5-P, rapidly rises to its base-line value within 2 min of atropine addition. However, the reversal of this change in PtdIns-4-P and phosphatidic acid mass is slower and takes 25 min before complete recovery is seen.
To see if these changes in the mass of phospholipids are a specific response to the agonist carbachol, the effect of 80 mM K' in the extracellular fluid on polyphosphoinositide metabolism was examined. Eighty mM K+, a concentration which causes a maximal contraction, did not elicit any changes in the mass of polyphosphoinositides or phosphatidic acid in tracheal muscle (data not shown). These results also indicate that the phospholipase C activation resulting from carbachol stimulation is not a consequence of an increase in an intracellular Ca2+ concentration.
To determine the dependency of carbachol-mediated breakdown of polyphosphoinositides on extracellular calcium, the effect of carbachol on polyphosphoinositide metabolism was compared in the presence or absence of extracellular Ca2+. The carbachol-stimulated breakdown of polyphosphoinositides and the production of phosphatidic acid were similar in the presence or absence of extracellular Ca2+ (data not shown). Thus, carbachol-stimulated breakdown of polyphosphoinositides is not affected by removal of extracellular Ca2+.
Effect of Carbachol on 1,2-Diacylglycerol Production-The results shown in Figs. 1-3 clearly indicate that carbachol stimulates phospholipase C-mediated hydrolysis of polyphosphoinositides. Therefore, the change in the level of 1,2-diacylglycerol, the other product of phospholipase C-mediated hydrolysis of polyphosphoinositides, was examined. Initially, the quenching method of adding ice-cold chloroform/metha-no1 (1:2, v/v) to muscle strips was employed. In experiments employing this method, no significant increase in the 1,2diacylglycerol level in terms of changes in the absolute amount, or of radioactivity in [3H]glycerolor [3H]arachidonic acid-labeled 1,2-diacylglycerol were found (data not shown). In experiments in which [3H]arachidonic acid-labeling was employed, a decrease in PtdIns-4,5-P2 and an increase in phosphatidic acid were found (Fig. 6). However, when another method for terminating the reaction, freeze-clamping the muscle, was employed, changes in 1,Z-diacylglycerol content were found. As shown in Fig. 7, the mass of 1,2-diacylglycerol in freeze-clamped muscle rises rapidly and reaches a peak at 2 min after the addition of 2 p M carbachol. It then falls slightly to remain for 60 min at a value significantly above the control value. The plateau value is about 130% of the resting value. An experiment in which a higher concentration of carbachol (0.1 mM) was employed gives a similar result (data not shown). Thus, these data indicate that the choice of quenching method is critically important for the measurement of 1,2-diacylglycerol content of this tissue.

DISCUSSION
The present results demonstrate that carbachol, a muscarinic agonist, causes a rapid reduction in the mass of PtdIns-4,5-P2 in bovine tracheal smooth muscle (Fig. 3). Because the change is associated with concomitant increases in [3H]Ins-P1 production (Fig. 2) and in the contents of 1,2-diacylglycerol ( Fig. 7) and phosphatidic acid (Figs. 3 and 6), these data indicate that a carbachol-induced decrease in the mass of PtdIns-4,5-Pz is caused by a stimulation of the hydrolysis of PtdIns-4,5-P2 catalyzed by phospholipase C. The present work is the first to report the measurement of polyphosphoinositide breakdown based upon measurement of lipid mass as well as radioisotopic labeling in a smooth muscle experiment.
Several investigators have shown that various agonists cause polyphosphoinositide breakdown in smooth muscle tissues employing radioactive tracer methods (13, [42][43][44][45][46][47]60). These previous studies demonstrated agonist-stimulated changes in radioactivity of phosphoinositides and/or production of [3H]inositol phosphates in smooth muscle prelabeled with 3' P or ['HH]inositol. When a freshly isolated tissue is employed for labeling experiments, it is unlikely that a true isotopic equilibrium is reached because of short periods of labeling. Therefore, it is not possible to accurately estimate a change in the lipid mass based upon experiments of this type. In addition, it is not easy to know from such labeling experiments whether or not polyphosphoinositide breakdown is sustained in response to agonists. The direct measurement of lipid mass gives one a means of overcoming these difficulties.
Recently a number of hormones and neurotransmitters have been shown to cause polyphosphoinositide breakdown in their target tissues. The resulting products, Ins-P, and 1,2diacylglycerol, have been shown to function as intracellular messengers in the action of the particular agonist (14)(15)(16)(17). One of the unanswered questions concerning polyphosphoinositide breakdown is whether polyphosphoinositide breakdown is sustained during the sustained phase of the hormonal response. The present study shows that the mass of PtdIns-4,5-Pz and PtdIns-4-P remain lower than their resting values even after 60 min of continuous exposure to carbachol, and that the level of phosphatidylinositol continues to fall progressively during this period. In contrast, the mass of phosphatidic acid and 1,2-diacylglycerol remain higher than their resting values. These results clearly indicate that polyphosphoinositide breakdown is sustained and continues to generate messengers during the sustained response to carbachol. These results give support to the notion that polyphosphoinositide breakdown plays a messenger role during the tonic as well as the acute phase of carbachol-induced contraction in bovine tracheal smooth muscle. It is our postulate that the major intracellular pathway involved in the sustained phase of carbachol-induced contraction is the C-kinase pathway and that the C-kinase is maintained in its Ca2+-sensitive state during the sustained phase (20,21,23,27,28). Since 1,2diacylglycerol in the plasma membrane is believed to be a physiological factor which activates the C-kinase in situ (17,19), our data showing a sustained increase in the 1,2-diacylglycerol content of muscle strips strongly suggest that the Ckinase is actually maintained in its Ca2+-sensitive state during the sustained phase of carbachol-induced contraction.
To our knowledge, the only previous report in which long term effects of agonists on the mass change in phosphoinositides are reported is from the work of Farese et al. (48) in rat adrenal subcapsular cells. These workers showed that angi-otensin 11, a typical agonist causing phosphoinositide breakdown in this tissue (29,61), induces increases in the mass of PtdIns-4,5-Pz, PtdIns-4-P, phosphatidylinositol, and phosphatidic acid at 60 min. Since agonists causing phosphoinositide breakdown stimulate both breakdown and resynthesis of phosphoinositides, it is possible that if the resynthesis is greater than the breakdown of phosphoinositides, one might see net increases in the mass of these lipids. In fact, a rapid increase in the mass of PtdIns-4,5-P2 after the initial decrease has been reported to occur at an early time point (30 s ) after thrombin addition in human platelets (49,50). Thus, a balance between breakdown and resynthesis of phosphoinositides following agonist stimulation may determine the net change in the mass of phosphoinositides in a given tissue.
The decrease in the mass of PtdIns-4,5-P2, seen in carbachol-stimulated tracheal muscle, is associated with an equally rapid loss of mass in the PtdIns-4-P pool (Fig. 3). The time course of Ins-P, production is also similarly as rapid as that of Ins-P3 (Fig. 2). However, the reversal of PtdIns-4-P level toward the resting value after atropine addition takes a longer time than that of PtdIns-4,5-P2 (Fig. 5). Therefore, a loss of the mass of PtdIns-4-P after carbachol addition may not be explained solely by phospholipase C-mediated breakdown of PtdIns-4-P. Accelerated conversion of PtdIns-4-P to PtdIns-4,5-Pz is likely to contribute to a reduction in the mass of PtdIns-4-P. Similarly, the time course of a delayed decrease in the mass of phosphatidylinositol and the relatively small increase in Ins-P compared to the much larger decrease in the mass of phosphatidylinositol (Figs. 2 and 3) suggest that the bulk of the decrease in the mass of phosphatidylinositol occurs via phosphorylation to PtdIns-4-P and PtdIns-4,5-P2.
In the present study, some differences in the data on phosphoinositide breakdown are noted between the [3H]inositol-labeling experiment (Fig. 1) and the measurement of the lipid mass (Fig. 3). In the labeling experiment the radioactivity in phosphatidylinositol shows no significant change for at least 10 min (Fig. I), while the mass of phosphatidylinositol shows a decrease of 11% at 1 min and 27% at 5 min (Fig. 3). Moreover, the extent of decrease in PtdIns-4,5-P2 after carbachol addition is larger in absolute amount than that estimated by radioactivity measurements. Because muscles were labeled for 3 h with [3H]inositol and presumably true isotopic equilibrium was not reached in the present experiment (51,52), stimulated incorporation of [3H]inositol into phosphoinositides associated with enhanced resynthesis of these lipids may account for the smaller relative changes observed in the labeling experiment. It is also possible that [3H]inositol is incorporated into only a particular pool of each phosphoinositide (53) and changes in radioactivity do not represent overall changes in the mass of these lipids.
The changes in the mass of phosphoinositides and phosphatidic acid are carbachol-specific events because the application of 80 mM K+ leads to no significant change in the mass of these lipids. These data also indicate that simply raising intracellular Ca2+ concentration with high extracellular K' is not sufficient to activate the phospholipase C-mediated breakdown of the phosphoinositides. Furthermore, carbachol-me-. diated breakdown of phosphoinositides is not dependent on extracellular Ca2+. These data are similar to previous reports in a variety of cells or tissues (14,15,52).
In the present study, the quenching method of adding icecold chloroform/methanol(1:2, v/v) to the incubation medium was initially employed for the measurement of 1,2-diacylglycerol mass. However, the experiments using this quenching method were unsuccessful in detecting a significant increase in 1,2-diacylglycerol content. Likewise, a significant change in [3H]arachidonic acid-or [3H]glycerol-labeled 1,Z-diacylglycerol was not observed using this approach. In contrast, in freeze-clamped muscle an increase in the mass of 1,2-diacylglycerol is seen (Fig. 7). The base-line value of the 1,2diacylglycerol content in the freeze-clamped muscle is oneseventh of that determined using the quenching method. These results suggest that the use of such a method as freeze clamping, which allows biochemical reactions to be terminated immediately, is critically important for the determination of the 1,2-diacylglycerol content of solid tissues like tracheal smooth muscle. Otherwise a small change in 1,2diacylglycerol mass might be masked by an artificial increase in the mass of this lipid derived from the degradation of phospholipids or triacylglycerol during the solvent-based quenching procedure.
The increase in the actual 1,2-diacylglycerol content appears to be small compared to the larger mass changes of phosphatidic acid and polyphosphoinositides (Fig. 3) (37). Using the present methods, we could show a substantial increase (2-fold) in the mass of 1,2-diacylglycerol in Swiss 3T3 fibroblasts upon bombesin stim~lation.~ Hence, it is unlikely that the method used for the quantitation of the mass of 1,2-diacylglycerol is inadequate to detect an increase in 1,2-diacylglycerol. The difference between the results in fibroblasts and smooth muscle indicate that the steady state concentration of 1,2-diacylglycerol is determined both by its rate of formation and its rate of further metabolism.
In contrast to the small increase in the mass of 1,2-diacylglycerol, the content of phosphatidic acid, a phosphorylated product of 1,2-diacylglycerol kinase, shows a striking increase after carbachol addition (Fig. 3). These data suggest that the 1,2-diacylglycerol formed as a result of PtdIns-4,5-P2 hydrolysis is rapidly converted to phosphatidic acid by relatively high activities of diacylglycerol kinase in carbachol-stimulated tracheal smooth muscle, resulting in a net accumulation of only a small amount of 1,2-diacylglycerol. However, it is possible that in some domains of the plasma membranes there is a larger increase in 1,2-diacylglycerol concentration than is indicated by its total mass change. Furthermore, the possibility that another mechanism is operating for activation of the C-kinase is not excluded. Recently, a lipoxygenaseproduct of arachidonic acid has been proposed as a C-kinase activator (54). Because arachidonic acid is known to be mobilized from diacylglycerol and phospholipids in smooth muscle tissue during agonist stimulation (55-57), the possibility that such arachidonic acid metabolites are involved in the regulation of C-kinase activity is an especially interesting avenue to be explored.