Smooth muscle length-dependent PI(4,5)P2 synthesis and paxillin tyrosine phosphorylation.

We studied effects of increasing the length of porcine trachealis muscle on 5.5 microM carbachol (CCh)-evoked phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] synthesis and other parameters of phosphatidylinositol (PI) turnover. PI(4,5)P2 resynthesis rates in muscle held at 1.0 optimal length (L(o)), measured over the first 6 min of CCh stimulation, were 140 +/- 12 and 227 +/- 14% of values found in muscle held at 0.5 L(o) and in free-floating muscle, respectively. Time-dependent changes in cellular masses of PI(4,5)P2, PI, and phosphatidic acid, and PI resynthesis rates, were also altered by the muscle length at which contraction occurred. In free-floating muscle, CCh did not evoke increases in tyrosine-phosphorylated paxillin (PTyr-paxillin), an index of beta1-integrin signaling; however, there were progressive increases in PTyr-paxillin in muscle held at 0.5 and 1.0 L(o) during contraction, which correlated with increases in PI(4,5)P2 synthesis rates. These data indicate that PI(4,5)P2 synthesis rates and other parameters of CCh-stimulated inositol phospholipid turnover are muscle length-dependent and provide evidence that supports the hypothesis that length-dependent beta1-integrin signals may exert control on CCh-activated PI(4,5)P2 synthesis.

Aldolase A Ins(1,4,5)P3-binding domains as determined by site-directed mutagenesis.

We substituted neutral amino acids for some positively charged residues (R42, K107, K146, R148 and K229) that line the active site of aldolase A in an effort to determine binding sites for inositol 1, 4,5-trisphosphate. In addition, D33 (involved in carbon-carbon bond cleavage) was mutated. K229A and D33S aldolases showed almost no catalytic activity, but Ins(1,4,5)P(3) binding was similar to that determined with the use of wild-type aldolase A. R42A, K107A, K146R and R148A had markedly decreased affinities for Ins(1,4,5)P(3) binding, increased EC(50) values for Fru(1,6)P(2)-evoked release of bound Ins(1,4,5)P(3) and increased K(i) values for Ins(1,4, 5)P(3)-evoked inhibition of aldolase activity. K146Q (positive charge removal) had essentially no catalytic activity and could not bind Ins(1,4,5)P(3). Computer-simulated docking of Ins(1,4,5)P(3) in the aldolase A structure was consistent with electrostatic binding of Ins(1,4,5)P(3) to K107, K146, R148, R42, R303 and backbone nitrogens, as has been reported for Fru(1,6)P(2) binding. Results indicate that Ins(1,4,5)P(3) binding occurs at the active site and is not dependent on having a catalytically active enzyme; they also suggest that there is competition between Ins(1,4,5)P(3) and Fru(1, 6)P(2) for binding. Although Ins(1,4,5)P(3) binding to aldolase involved electrostatic interactions, the aldolase A Ins(1,4, 5)P(3)-binding domain did not show other similarities to pleckstrin homology domains or phosphotyrosine-binding domains known to bind Ins(1,4,5)P(3) in other proteins.

Effects of polyamines and calcium and sodium ions on smooth muscle cytoskeleton-associated phosphatidylinositol (4)-phosphate 5-kinase.

In many different cell types, including smooth muscle cells (Baron et al., 1989, Am. J. Physiol., 256: C375-383; Baron et al., J. Pharmacol. Exp. Ther. 266: 8-15), phosphatidylinositol (4)-phosphate 5-kinase plays a critical role in the regulation of membrane concentrations of phosphatidylinositol (4,5)-bisphosphate and formation of inositol (1,4,5)-trisphosphate. In unstimulated porcine trachealis smooth muscle, 70% of total cellular phosphatidylinositol (4)-phosphate 5-kinase activity was associated with cytoskeletal proteins and only trace activity was detectable in isolated sarcolemma. Using two different preparations, we studied cytoskeleton-associated phosphatidyl inositol (4)-phosphate 5-kinase under conditions that attempted to mimic the ionic and thermal cytoplasmic environment of living cells. The cytoskeleton-associated enzyme, studied using phosphatidylinositol (4)-phosphate substrate concentrations that produced phosphatidylinositol 4,5-bisphosphate at about 10% of the maximal rate, was sensitive to free [Mg2+], had an absolute requirement for phosphatidylserine, phosphatidic acid, or phosphatidylinositol, and included type I isoforms. At 0.5 mM free [Mg2+], physiological spermine concentrations, 0.2-0.4 mM, increased phosphatidylinositol (4)-phosphate 5-kinase activity two to four times compared to controls run without spermine. The EC50 for spermine-evoked increases in activity was 0.17 +/- 0.02 mM. Spermine-evoked enzyme activity was a function of both free [Mg2+] and substrate concentration. Cytoskeleton-associated phosphatidylinositol (4)-phosphate 5-kinase was inhibited by free [Ca2+] over a physiological range for cytoplasm--10(-8) to 10(-5) M, an effect independent of the presence of calmodulin. Na+ over the range 20 to 50 mM also inhibited this enzyme activated by 5 mM Mg2+ but had no effect on spermine-activated enzyme. Na+, Ca2+, and spermine appear to be physiological modulators of smooth muscle cytoskeleton-bound phosphatidylinositol (4)-phosphate 5-kinase.

Smooth muscle aldolase C-bound inositol 1,4,5-trisphosphate studied in vitro under physiological conditions.

Our goal was to quantitate inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding to aldolase C tetramer (aldolase4) and its displacement by inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) under conditions which approximated the in vivo state. Anions were found to have major effects. Decreasing [KCl] from 100 to 10mM, at 0 degrees C and pH 7.0, increased maximal Ins(1,4,5)P3 binding to 1.0 to 2.4mol per mol aldolase4. At 10 and 30mEq/l [Cl-], an additional high affinity site was detected (Kds = 0.43 and 0.86 microM, respectively). Increasing concentrations of other anions (SO42-, propanoate-, HCO3-, acetate-) also inhibited binding, but effects would be minimal at concentrations of these anions present in the cytoplasm of living cells. Ins(1,3,4)P3 displacement of aldolase C-bound Ins(1,4,5)P3 was sensitive to [Cl-]; at 30mEq/l [Cl-] and 37 degrees C, Ins(1,3,4)P3 released 20% of bound Ins(1,4,5)P3 at concentrations of 100nM. Changing temperature from 0 to 37 degrees C increased Kds for Ins(1,4,5)P3 binding. Changes in free [Ca2+], [Mg2+], [Na+] and [K+] and changes in osmolality had no effect on Ins(1,4,5)P3 binding to aldolase C. In vivo Ins(1,4,5)P3-aldolase4 binding at 30mEq/l [Cl-] and 37 degrees C were calculated for different [Ins(1,4,5)P3]free over the range 0.2 to 1.0 microM. For different cytoplasmic [Ins(1,4,5)P3]free. Ins(1,4,5)P3 binding to aldolase4 was sufficient, if acutely released, to nearly double cytoplasmic [Ins(1,4,5)P3]free. We proposed a schema whereby release of aldolase C-bound Ins(1,4,5)P3 evoked by Ins(1,3,4)P3 amplifies effects of phospholipase C-formed Ins(1,4,5)P3.

Smooth muscle sarcolemma-associated phospholipase C-beta 2; agonist-evoked translocation.

About 25% of the total cellular PLC beta 2 content was found to be associated with a sarcolemmal fraction (SARC) isolated from unstimulated porcine trachealis smooth muscle. SARC-associated PLC beta 2 was located within two compartments, a detergent-extractable compartment and a nondetergent extractable compartment. SARC PLC beta 2 was measured after extraction with 0.6 M KCI; therefore, PLC beta 2 was not bound solely by electrostatic forces within either of these compartments. PLC beta 2 was shown to translocate from cytosol to SARC during a 20-sec activation of intact muscle with a muscarinic agonist, carbachol (CARB); i.e., cytosolic total PLC beta 2 content decreased significantly to 73 +/- 7% of control and SARC total PLC beta 2 content increased to 180 +/- 15% of control value. This translocation was maintained at 5 min of CARB. CARB-evoked translocation occurred into the detergent-extractable SARC fraction, and PLC beta 2 content in this fraction increased 300% compared with that in unstimulated muscle. After CARB, SARC PLC beta 2 content accounted for > 50% of total cellular PLC beta 2 content. CARB-evoked increase in PLC activity in SARC paralleled the increase in PLC beta 2 content. CARB-induced translocations of PLC beta 2 from the cytosol to SARC were of a similar magnitude as occurred with phorbol ester-induced translocations of PKC alpha.

Inositol 1,4,5-trisphosphate binding to porcine tracheal smooth muscle aldolase.

A cytoskeletal fraction of porcine tracheal smooth muscle (PTSM) was found to contain > 90% of total cellular aldolase (fructose 1,6-bisphosphate aldolase, EC 4.1.2.13) activity. PTSM aldolase was purified by DEAE and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) affinity chromatography and found to react with an antibody directed against human aldolase C, but not anti-aldolase A and B. The molecular mass of native aldolase was about 138 kDa (on Sephacryl S-300); SDS-denatured enzyme was 35 kDa (comigrated with rabbit skeletal muscle aldolase). Total cellular aldolase tetramer (aldolase4) content was 34.5 pmol/100 nmol lipid P(i). Ins(1,4,5)P3) binding activity coeluted with aldolase during Sephacryl 300, DEAE, and Ins(1,4,5)P3 affinity chromatography. Ins(1,4,5)P3 bound to purified aldolase (at 0 degree C) in a dose-dependent manner over the range [Ins(1,4,5)P3] 20 nM to 20 microM, with maximal binding of 1 mol of Ins(1,4,5)P3/mol aldolase4 and a Kd of 12-14 microM. Fru(1,6)P2 and Fru(2,6)P2 displaced bound Ins(1,4,5)P3) with a 50% inhibition at 30 and 170 microM, respectively. Ins(1,3,4)P3 (20 microM) and glyceraldehyde 3-phosphate (2 mM) were also potent inhibitors of Ins(1,4,5)P3 binding, but not inositol 4-phosphate or inositol 1,4-bisphosphate (20 microM each). Aldolase-bound Ins(1,4,5)P3 may play a role in phospholipase C-independent increases in free [Ins(1,4,5)P3].

Smooth muscle stretch-activated phospholipase C activity.

Rabbit aortic muscles were stretched from a holding length of 0.6 maximum length (Lmax) to lengths as great as 1.0 Lmax and the new length maintained. When muscles were stretched to 1.0 Lmax, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,4-bisphosphate [Ins(1,4)P2] contents were increased at 375 ms (uncorrected for freezing time) poststretch to 209 +/- 27 and 139.8 +/- 12% (SE), respectively, of control values. Increases in Ins(1,4,5)P3 and Ins(1,4)P2 contents reached an apparent maximum at approximately 500 ms, i.e., to 243.7 +/- 15.8 and 180.9 +/- 16.2% of control, and were decreased to near control levels at 1,700 ms poststretch. The stretch threshold for phospholipase C (PLC) activation was 0.85 Lmax. The latency to onset of PLC activation, correcting for the time for freezing, was 275 to 375 ms. Maximal PLC activity was 91 pmol.s-1.100 nmol total lipid P(i)-1, which corresponded to 10% of total phosphatidylinositol bisphosphate being hydrolyzed per second. The mechanism of stretch-activated PLC activity involved influx of Ca2+ via gadolinium-sensitive ion channels, but not via nifedipine-sensitive ion channels.

Oxygen sensors in vascular smooth muscle.

Inhibition or activation of cellular function due to acute decreases in PO2 can be considered in terms of two different processes: 1) a sensor that monitors PO2 decreases and 2) transduction systems directed from the O2 sensor to reactions that control cellular function. We used the norepinephrine-contracted aortic smooth muscle model to study the nature of the O2 sensor and transduction system during decreased PO2-evoked relaxations. The phosphorylation potential, a measurement of kinetic energy required for ATP hydrolysis, was decreased to 30% of control at the onset of relaxation and progressively fell as muscle relaxed. The free inorganic phosphate intracellular concentration ([Pi]) was experimentally increased approximately 0.6 mM during transients that followed a rapid decrease in PO2. Relaxations to 80% of maximal force were more rapid under conditions of an augmented [Pi] than in control rings, and they occurred at a higher phosphocreatine concentration and phosphocreatine-to-free creatine ratio but at the same phosphorylation potential. Results support the operation of a cytochrome aa3 O2 sensor in the mechanism of decreased PO2-evoked relaxations and implicate an increase in [Pi] and a decrease in kinetic energy in the transduction mechanism directed at rate-limiting reactions that control force.

Inositol trisphosphate and energy-force coupling in rabbit aorta.

Five-minute exposures of aortic smooth muscle to 15 microM norepinephrine (NOR) under normoxia resulted in significant increases in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] (152%), inositol 1,4-bisphosphate [Ins(1,4)P2] (171%) and inositol 4-phosphate [Ins(4)P] (203%) contents compared with values measured in unstimulated muscle but no changes in inositol 1,3,4-trisphosphate [Ins(1,3,4)P3], inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] inositol 1/3-phosphate [Ins(1/3)P] or inositol 1,3/3,4-bisphosphate [Ins(1,3/3,4)P2] contents. Increases in Ins (1,4,5)P3 content and the contents of its by-products persisted or increased for at least 20 min of NOR exposure during normoxia. After a rapid decrease in Po2 at 5 min of NOR exposure, there were parallel decreases in Ins(1,4,5)P3 and Ins(1,4)P2 contents and in force. Ins(1,4,5)P3 and Ins(1,4)P2 contents were significantly decreased to 85 +/- 4.3% and 70 +/- 9.4% of control, respectively, at a time just after onset of relaxation. At near-maximal relaxation, Ins(1,4,5)P3 and Ins(1,4)P2 contents were decreased to 36.5 +/- 5.8% and 59.2 +/- 16.8%, respectively, of control normoxia values (P < .05). After readmission of O2 to the bubbling gas, Ins(1,4,5)P3 and Ins(1,4)P2 contents rapidly increased. Comparing decreased PO2-evoked [PCr] and [ATP] (phosphocreatine and adenosine triphosphate) decrements (measured previously; Coburn et al., 1992) with our present data, threshold [PCr] and [ATP] for a decrease in Ins(1,4,5)P3 content were shown to be 0.5 and 0.8 mM, respectively, and [PCr] and [ATP] at the time of 50% decrease in Ins(1,4,5)P3 content were 0.3 and 0.7 mM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Common phosphatidylinositol 4,5-bisphosphate pools are involved in carbachol and serotonin activation of tracheal smooth muscle.

We examined the rate and extent of labeling of total cellular phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol (PI) in canine tracheal smooth muscle in response to maximum levels of two different contractile agonists, carbachol (5.5 microM) and serotonin (5-hydroxytryptamine, 5-HT) (47 microM) and when a second agonist was given during a maximal contraction evoked by the first agonist. Unstimulated tracheal smooth muscle strips were incubated with [3H]-myo-inositol (MI) to label tissue MI without much labeling of inositol phospholipids. With carbachol, there was a 20-fold increase in the [3H]-MI incorporation rate into inositol phospholipids, decreases in PI and PIP2 contents, and increases in phosphatidic acid and diacylglycerol contents. PI and PIP2 specific radioactivities reached plateaus, 0.90 +/- 0.03 and 0.80 +/- 0.04, respectively, compared with [3H]-MI specific radioactivity. 5-HT at 47 microM evoked smaller changes including force development, [3H]-MI incorporation rate and lipid mass changes. However, the plateau of PI and PIP2 labeling reached levels similar to that determined during carbachol-evoked force, 0.90 +/- 0.06 and 0.82 +/- 0.04, respectively. Carbachol (55 microM) addition after incubation with 5-HT did not significantly alter the plateau levels of the specific radioactivities of PI or PIP2, although force and lipid mass changes were significantly changed. We conclude that 5-HT and muscarinic receptor coupling mechanisms utilize the same pool of PIP2 as a substrate for phospholipase C activation and the same PI pool for conversion to PIP and PIP2.

Smooth muscle guanine nucleotides and receptor-effector coupling following inhibition of oxidative energy production.

We measured total concentrations of guanosine triphosphate (GTP) and guanosine diphosphate (GDP) in rabbit aortic smooth muscle under several different conditions. We computed free [GDP] and free GTP/GDP (using a Keq of 1.0 for the nucleoside diphosphate kinase reduction) under these conditions. At a time when muscle was contracted by 15 microM norepinephrine (NE) for 5 min under normoxia, [GTP], free [GDP], and free GTP/GDP were 0.29 +/- 0.03 mM, 3.5 microM, and 82, respectively. Following rapid inhibition of oxidative energy production during NE-evoked maintained force, which is associated with slow decreases in mean tissue [PCr], and [ATP] and force relaxation, [GTP] and free GTP/GDP were decreased at relaxation threshold to 0.22 +/- 0.02 (SE) mM and 43, respectively, and progressively fell further, paralleling decreases in force and [ATP] and [PCr]. There were marked decreases in the sum of GTP + GDP contents under conditions where muscle energy stores were decreased (i.e., low [PCr] + [ATP]). Similar data were obtained during a 50 mM KCl-evoked contracture. Free [GDP] increased from normoxic values of 3.5 microM to values as great as 6.0 microM at low energy store states. Free GDP was equivalent to 6% of total GDP under normoxia and increased to 16-21% of total GDP under conditions of low energy stores. Evidence was obtained that decreases in [GTP] or free GTP/GDP seen under conditions of low total energy stores were not sufficient to inhibit heterotrimeric G protein function and uncouple receptor-effector coupling.

Rate-limiting energy-dependent steps controlling oxidative metabolism-contraction coupling in rabbit aorta.

1. We tested the hypotheses that coupling between oxidative metabolism and force in noradrenaline (NOR)-activated rabbit aorta is controlled (a) by an energy-dependent step or steps in receptor-operated coupling mechanisms upstream to myosin light chain (MLC) kinase, or (b) by energy limitation of MLC kinase-mediated phosphorylation of the MLC or actin-activated myosin ATPase. 2. Oxidative energy production was rapidly inhibited by decreasing organ bath PO2 to less than 30 mmHg. There was no difference, comparing KCl- or NOR-induced force, in the rates of decrease of [PCr] (phosphocreatine) or [ATP] following inhibition of oxidative energy production. (In this report we use the term [PCr] and [ATP] to indicate mean tissue values). Initial rates of decrease in [PCr] and [ATP] following inhibition of oxidative energy production were 0.05 mM/min and 0.06 mM/min, respectively. 3. Despite similar decreases in mean tissue [PCr] and [ATP], relaxations of KCl-induced contractions following inhibition of oxidative energy production were markedly delayed and were blunted compared to relaxations seen during NOR-induced contractions. The threshold mean tissue [PCr] and [ATP] for relaxation during KCl stimulation were 0.25 and 0.60 to 0.80 mM, respectively. During NOR stimulation, threshold values of [PCr] and [ATP] were 0.50 mM and 0.80 mM, respectively. Mean tissue [PCr] and [ATP] levels at 50% relaxation of KCl-induced force were less than 0.1 mM and 0.1 mM, respectively. Fifty per cent relaxation of NOR-induced force occurred at [PCr] and [ATP] values of 0.35 mM and 0.65 mM, respectively. 4. MLC phosphorylation levels decreased during relaxation of NOR force evoked by inhibition of oxidative energy production. There was no change in the level of MLC phosphorylation following inhibition of oxidative energy production in KCl-contracted muscle even at mean tissue [PCr] and [ATP] lower than values associated with decreases in MLC phosphorylation during relaxations of NOR-induced force. 5. Oxygen-induced redevelopment of force during NOR exposure was not dependent on extracellular Ca2+. Mean tissue [PCr] increased prior to onset of O2-evoked force redevelopment. Increases in MLC phosphorylation were seen at the time of onset of force redevelopment. 6. Oxidative metabolism-contraction coupling during NOR-stimulation seems not to be due to energy limitation of the MLC kinase reaction or actin-activated myosin ATPase. Data suggest the rate-limiting step is an energy-dependent reaction in receptor-operated coupling mechanisms upstream to MLC kinase.(ABSTRACT TRUNCATED AT 400 WORDS)

Inositol 1,4,5-trisphosphate compartmentalization in tracheal smooth muscle.

Pool sizes of inositol phosphate species in myo-[3H]inositol-labeled porcine tracheal smooth muscle were determined under three conditions: (a) unstimulated; (b) stimulated with carbachol; (c) atropine-relaxed from a carbachol contraction. In unstimulated muscle, the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) content was 14 pmol/100 nmol lipid P1. This is equivalent to a mean [Ins(1,4,5)P3] of about 3 microM (in total cellular water), a level about 30-fold in excess of that required for Ca2+ release from Ins(1,4,5)P3-sensitive sarcoplasmic reticulum (SR). Pool sizes of breakdown products of Ins(1,4,5)P3 were relatively small or absent in unstimulated muscle, suggesting that, under this condition, Ins(1,4,5)P3 was sequestered and had limited access to Ins(1,4,5)P3 5-phosphatase and/or 3-kinase. During carbachol stimulation, the Ins(1,4,5)P3 pool did not increase while those of other mono-, di-, and trisphosphate isomers increased over 10-fold. Subsequent atropine-induced relaxation resulted in a partial depletion (40%) of total tissue Ins(1,4,5)P3. Decreases in Ins(1,4,5)P3 were paralleled by decreases in Ins(1,4)P2 and Ins(1,3,4)P3. During contraction a portion of total tissue Ins(1,4,5)P3 has access to Ins(1,4,5)P3 3-kinase and 5-phosphatase and to Ins(1,4,5)P3-sensitive SR, though during antagonist-induced relaxation access to Ins(1,4,5)P3-sensitive SR for Ca2+ release is restricted. Data are consistent with a mechanism by which a large pool of Ins(1,4,5)P3 present in the unstimulated state in a sequestered compartment can contribute in activated muscle to increases in [Ins(1,4,5)P3] in a nonsequestered compartment, controlling SR Ca2+ release.

Inositol 1,4,5-trisphosphate, inositide flux rates and pool sizes during smooth muscle relaxation.

Decreases in D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] content and changes in inositol phospholipid contents occurred during the time of atropine-induced relaxation of swine tracheal smooth muscle contracted with 55 microM carbachol. Decrease in Ins(1,4,5)P3 occurred in a pool which makes up 40% of the total content of this inositol phosphate and which has access to Ins(1,4,5)P3 5-phosphatase and/or 3-kinase. A 50% decrease in this pool occurred at 16 s after addition of atropine and within 6-10 s after inhibition of phospholipase C (PLC). The maximal fall in Ins(1,4,5)P3 occurred at a time when force had only decreased 30% of the maximal response. A phosphatidylinositol 4-phosphate (PIP) pool linked to muscarinic receptor-activation increased 160% after addition of atropine, the maximal response occurring at a time when relaxation was 80% complete. The mechanisms for this increase were the maintained formation of PIP and phosphatidylinositol 4,5-bisphosphate (PIP2) even though PIP2 hydrolysis was inhibited and the apparent chemical equilibrium between PIP and PIP2.

Mechanical and biochemical events during hypoxia-induced relaxations of rabbit aorta.

Hypoxic relaxation of norepinephrine contractions of isolated rabbit aorta is rapid, whereas relaxation of KCl contractions is slower and blunted. The data given here suggest that with receptor-evoked contractions of rabbit aorta, the energy-limitation of ATP-dependent K+ channels and other sarcolemmal channels, myosin light chain kinase, and actin-activated myosin ATPase are probably not involved in oxidative energy-contraction coupling. The data strongly support the hypothesis that the rate limiting, energy-dependent step is upstream to myosin light chain kinase, which is 50% inhibited at an ATP concentration of about 0.5 mM. This energy-dependent step may be in the inositol phospholipid transduction system, as we have previously postulated (Coburn et al., 1988). In contrast the energy-limited reaction during KCl contractions appears to be the actin-activated myosin ATPase which is 50% inhibited at a mean ATP concentration of about 0.1 mM.

Coupling mechanisms in airway smooth muscle.

This review documents available information about coupling mechanisms involved in airway smooth muscle force development and maintenance and relaxation of force. Basic concepts, obtained from experiments performed on many different mammalian cell types, are in place regarding coupling between surface membrane receptors and cell function; these concepts are considered as a framework for understanding coupling between receptors and contractile proteins in smooth muscles and in airway smooth muscles. We have divided various components of coupling mechanisms into those dependent on changes in the surface membrane potential (electromechanical coupling) and those independent of the surface membrane potential (pharmacomechanical coupling). We have, to some degree, emphasized modulation of coupling mechanisms by intrasurface membrane microprocessing or by second messengers. A challenge for the future is to obtain a better understanding of how coupling mechanisms are altered or modulated during different phases of contractions evoked by a single agonist and under conditions of multiple agonist exposure to airway smooth muscle cells.

Phorbol ester effects on coupling mechanisms during cholinergic contraction of swine tracheal smooth muscle.

1. We studied effects of the phorbol ester, phorbol 12,13-dibutyrate (PDB), on carbachol-induced contractions of swine trachealis muscle. PDB (1-10 microM) markedly inhibited 5.5 microM-carbachol-induced inositol phosphate synthesis allowing us to study (a) whether the membrane potential-independent component of force (pharmacomechanical coupling component) developed in carbachol-stimulated trachealis muscle is dependent on activation of inositol phospholipid metabolism, and (b) whether carbachol-induced membrane depolarization and contraction are altered in muscle where second messenger signals generated by inositol phospholipid metabolism are inhibited and activation of protein kinase C (PKC) is already maximal. 2. Application of PDB (10 microM) to unstimulated trachealis muscle resulted in a small slowly developing contraction associated with a 10 m V membrane depolarization. PDB-evoked contractions were not influenced by Na+ or Cl- ion substitutions, or administration of amiloride, all of which inhibited PDB-evoked membrane depolarization. 3. Pre-treatment with PDB had no effect on [K+]-force, or [K+]-membrane potential relationships, over a range of extracellular [K+] from 40 to 70 mM. Pretreatment with PDB had no effect on extracellular [Ca2+]-force relationships during 40 mM-K+. 4. Carbachol-evoked contractions of muscle treated with PDB became similar to K+ contractions in regard to effects of organic Ca2+ antagonist drugs or decrease in bathing solution [Ca2+]. At low carbachol concentrations, verapamil plus PDB completely inhibited force development. With 5.5 microM-carbachol, over 90% of total carbachol-induced force was inhibited by verapamil, or nifedipine, plus PDB. 5. Control carbachol-evoked contractions were associated with 20-25 mV membrane depolarizations. In PDB-treated muscle, carbachol-evoked contraction occurred with a blunted depolarization, i.e. about 5 mV. 6. Force controlled by pharmacomechanical coupling mechanisms operating during maintained carbachol-evoked contractions was inhibited by treatment with PDB. Carbachol-induced force dependent on pharmacomechanical coupling mechanisms could be explained by signals generated via inositol phospholipid metabolism. 7. Electromechanical coupling mechanisms were augmented during carbachol in PDB-treated muscle. This appears to be due primarily to changes in the properties or number of surface membrane voltage-gated Ca2+ channels. 8. Data suggest an important role of PKC-mediated phosphorylations for control of both pharmacomechanical coupling mechanisms mediated by activation of inositol phospholipid metabolism and electromechanical coupling mechanisms mediated by effects on operation of surface membrane ion channels.

Inositol lipid turnover and compartmentation in canine trachealis smooth muscle.

We established conditions for the study of metabolism and compartmentation of inositol phospholipids in canine trachealis muscle. Unstimulated muscle was incubated with myo-[3H]inositol for 30 min at 37 degrees C which resulted in labeling of the tissue free myo-inositol pool, whereas only a small amount of radioactivity was incorporated into inositol phospholipids or inositol phosphates. After addition of 5.5 microM carbachol, phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2), specific radioactivities increased exponentially, reaching apparent constant values in 180-240 min. Initial rates of increases in PI, PIP, and PIP2 specific radioactivities were 39, 32, and 66 times that measured in unstimulated muscle. Metabolic flux rates (nmol.100 nmol total lipid Pi-1.min-1) during development of force averaged 0.42 +/- 0.09 and during force maintenance averaged 0.14 +/- 0.01. Fractions of total PI, PIP, and PIP2 pools that were linked to muscarinic cholinergic activation were estimated to be 0.97, 0.85, and 0.65, respectively. Initial rates of increase in specific radioactivities and specific radioactivities during carbachol activation were similar in PI, PIP, and PIP2 fast active compartments, suggesting metabolic flux from PI to PIP to PIP2 was in near chemical equilibrium. Turnover times for PI, PIP, and PIP2 fast active compartments were estimated to be 21, 1.6, and 4.0 min, respectively.

Pharmacomechanical coupling in smooth muscle may involve phosphatidylinositol metabolism.

Cholinergic contraction of canine trachealis muscle, a contraction that primarily utilizes membrane potential-independent mechanisms for activating contractile proteins (pharmacomechanical coupling), is associated with a decline in the phosphatidylinositol pool, an increase in the phosphatidic acid and diacylglycerol pools, and an increased incorporation of 32PO4 into phosphatidylinositol. We found that these changes occur during development of the contraction and during maintenance of tension and are independent of membrane depolarization or increases in cytosolic Ca2+ concentration. These findings suggest that phosphatidylinositol turnover may be part of a receptor transduction process controlling receptor-operated Ca2+ channels or other membrane potential-independent mechanisms involved in pharmacomechanical coupling in smooth muscle.

Binding of influenza virus to a reconstituted receptor complex containing glycophorin.

Membrane particles possessing receptor activity for influenza virions have been reconstituted following solubilization of human erythrocyte membranes with octylglucoside (OG) and fractionation on a DEAE-cellulose column. Fractions that eluted with 1.5 M NaCl yielded, after removal of OG, reconstituted membrane particles (RMP) which could bind virus and inhibit hemagglutination. RMP contained essentially two membrane proteins (glycophorin and a nonglycosylated protein of molecular weight 29,000), two phospholipids (sphingomyelin and phosphatidylcholine), cholesterol, and gangliosides. Incubation of influenza virus with RMP at 4 degrees resulted in the formation of a virus-RMP complex (liposomes). The specificity of the receptor was demonstrated by inhibition of viral attachment when RMP were treated with neuraminidase or preincubated with rabbit anti-M or anti-N antiserum, suggesting that both the carbohydrates and peptide moiety may play a role in attachment. Calculations suggest that there are 6 X 10(3) N-acetyl neuraminic acid residues per attached virion. This system provides a simple and gentle means of reconstituting membrane components to study receptor activity.