AMERICAN JOURNAL OF PHYSIOLOGY Voj. 228, No. 2, February 1975. Printed in U.S..4,

Induction of a myogenic response in tonic airway

smooth muscle by tetraethylammonium

N. 1;. STEPHENS, E. A. KROEGER, AND U. KROMER Dqbartment of Physiology, Faculty of Medicine, University of Manitoba, Wk@eg, Manitoba, Canada R3E 0 IV3

STEPHENS, T\T. L., E. A. KROEGER, AND U. KROMER. Inductzbn

0j a myogenk resfionse in fonic airway smooth muscle by tetraethylam- mu&m. Am. J. Physiol. 228(Z) : 628-632. 1975.~In multi- unit tracheal smooth muscle (TSM), quick stretches applied at a velocity of 5 times the measured maximum velocity of isotonic shortening of the muscle, of a magnitude 3 times the measured extension of the series-elastic component when the muscle con- tracts maximally, and at optimal muscle length (L,) were un- able to elicit any myogenic response (MR). Experimental con- ditions such as hypoxia (Paz < 60 mmHg) and acidosis (pH = 6.8) or the presence of Ba2-i- (2 mM), acetylcholine ( 10e6 M), or high (K+)O (59 mM) were also unable to elicit the MR. How- ever, tetraethylammonium chloride (TEA, 0.4-67 mM) produces I) spontaneous phasic contractions and 2) a MR to quick stretch. The ionic basis for these changes was then investigated by study- ing the Ca and Mg dependence of the response to TEA. The dose-response relationship to TEA was shifted to the left by de- creasing external Mg 2+ from 2.5 to 0.5 mM. The ability of TSM to produce a MR was absolutely dependent on external Ca, but the threshold concentration required shifted from 2.5 X 10m5 M at normal external Mg (2.5 mM) to 5 X lo-” M at the reduced external Mg (0.5 mM). The effects of TEA on spontaneity and the MR were abolished by D-600. These results suggest that I) TEA functionally converts multiunit smooth muscle into a single unit one and leads to the development of a MR and 2) the MR results from a depolarization-activated mobilization of Ca and is inhibited by ionic conditions known to increase membrane permeability.

quick stretch of smooth muscle; mechanics of airway smooth muscle; tetraethylammonium chloride

THE REALIZATION that the tracheobronchial airways are not merely passive conduits but that by contraction of their smooth muscles they control the distribution of venti- lation to the alveoli has raised many questions as to the precise nature of this control (27, 32, 33). Since the airways may be subjected to considerable and sudden transmural forces, we considered it of interest to investigate the elects of sudden changes in length on the mechanics of airway smooth muscle: specifically, we were interested to know if a myogenic response (MR) could be elicited upon quick stretching of the preparation, using tracheal smooth muscle (TSM) as our model. Although, reasoning teleologically, a MR might be of value to this muscle (e.g., in autoregula- tion of airway caliber), it must be admitted that since IX4 is of the multiunit type (4) a priori we felt that a MR would not be elicited since other workers have shown that multiunit muscles do not possess the response (7).

In the case of single-unit smooth muscle, several investi- gators have shown that the MR is easily elicited (1, 3, 5, 9, 1245, 26, 27).

The mechanism for the production of a MR in smooth muscle appears to be dependent on the characteristics of the preparation with regard to automaticity. Cells from single-unit muscles demonstrate spontaneous membrane electrical activity with cyclic depolarization, development of action potentials, and a MR to quick stretch. These phenomena are not seen in vitro in multiunit smooth muscle (32, 35). Th us the MR appears to be correlated with the ability of a mechanical stress (quick stretch) to depolarize the membrane and increase the rate of discharge of action potentials (7). Of interest to us were reports (3, 18, 25) of agents such as norepinephrine, high (K+)O, and tetraethylammonium chloride (TEA) which converted multiunit arterial smooth muscles into functionally single- unit preparations exhibiting conducted action potentials and rhythmic contractions.

The aims of the present investigations were to determine I) whether a MR could be elicited in TSM in vitro nor- mally or under experimental conditions, 2) the effect of a membrane stabilizer such as Mg2+ on the production of the MR, and 3) the Ca compartment mobilized in producing the MR. The latter was of special interest as multiunit smooth muscles usually contain a large amount of se- questered Ca in comparison to single-unit preparations

W) The results suggest that 1) a MR cannot be elicited

normally in TSM, but that in the presence of TEA phasic electrical and mechanical activity is produced and a MR can be easily elicited; 2) the MR cannot be elicited in solutions in which the membrane permeability is in- creased; 3) the Ca mobilization that is associated with depolarization is responsible for the MR.

METHODS

Tracheal smooth muscle was obtained from the cervical tracheae of mongrel dogs anesthetized with 30 mg/kg pentobarbital administered intravenously. Parallel-fibered muscle strips about 1 cm x .I cm x .075 cm were dissected out, mounted, and equilibrated in a Krebs-Ringer bi- carbonate solution of the following composition, in milli- moles per liter: NaCl, 115; NaHCO3, 23.8; KHaPOa, 1.2; KCl, 4,7; MgCl2, 2.5; CaCls, 2.5; dextrose, 5.5. The solu- tion was equilibrated with a 95 7c, 0 3-5 % CO 2 gas mixture so as to maintain a measured bath Po2 of 600 mmHg,

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MYOGENIC RESPONSE IN MULTILJNIT SMOOTH MUSCLE 629

Pcoy of 40 mmHg, and pH 7.40 at a temperature of 37 & 1 “C. In preparing solutions containing TEA, TEA4 was substituted for NaCl on an equimolar basis. Low-Ca and low-Mg solutions were prepared by reducing, to the extent indicated, the amount of these respective salts added to the bathing solution.

Quick stretches for eliciting- the myogenic response were applied with a Levin-Wyman ergometer. The vertical muscle strip was fixed rigidly to a clamp below. The upper end was attached by a short segment of jeweller’s chain to a force transducer (RCA 5734) which was mounted firmly on the lever of the ergometer. The compliance of the system, including the force transducer and attachments to the muscle, was 2 pm/g wt. The amount of stretch applied was measured with a linear displacement transducer (Hewlett-Packard model 7DCDT-500), the displacement- measuring core of which rested on the lever of the ergome- ter. With this ergometer, quick stretches of given magni- tudes and velocities could be applied. The signals from the RCA 5734 valve and linear displacement transducers were recorded in parallel on a Brush Mark 280 oscillograph and Tektronix model 564 oscilloscope.

RESULTS

ExparirmnIs Using A-ormcil Kre bs-Ringer Bicar bonute So/u tion

Guinera: t.ig taeniu co/i (sinple-unit smooth muscle). In order to ascertain that our equipment could measure a T\/IR in a smooth muscle preparation which is known to demonstrate such a response, our initial experiments investigated the effect of quick stretch on the taenia coli of the guinea pig- Figure 1A shows an instantaneous rise and decay of tension at the t4me of application of the stretch followed after a short latency by an active contraction or myogenic re- sponse (MR). The time course of the ,MR is influenced by the initial length of the muscle as shown. In both instances

ACIDOSIS. In the presence of a bathing solution at pH

6.6-6.8, active responses to stimulation were impaired, but a myogenic response could not be elicited.

POTASSIUM. Treatment with 59 mM K (59 n&I XaC1 replaced with KCl), which resulted in a membrane de- pilarization from -51 to - 23 mV (unpublished obsel*-

-4 TEA+ 33 mM

-0 1 MIN

FIG. 1, Effect of quick stretch on guinea pig taenia coli (panel A) and canil-le trachealis (panels B-D). In panel A responses to stretches of 0.1 and 0.25 L, are shown. In panels B-D are shown effects of in- creasing initial muscle length from 0.75 to 1.25 L, (panel B) , increasing velocity of stretch from 0.5 to 5 times V,,,, (panel C), and increasing magnitude of stretch from 0.1 to 0.25 L, (panel 0).

the velocity of stretchin, m was more than 5 times the nlaxi- mum shortening velocity of slnooth muscle (33)-

These results indicate that the equipment employed can elicit and measure an 1,IR in a unitary smooth muscle such as taenia coli. Subsequent experiments were carried out on tracheal smooth muscle.

G-mine Tracheal Smooth Muscle (_Mdtiunit)

l?fect of initiul muscle length, r&e, and magnitude of stretch. The results of these studies (Fig. 1, B-D) showed that, although the velocity of quick stretching was up to 20 times that of the maximum shortening velocity of the muscle (33), a MR could not be elicited from this muscle within muscle lengths to 1.24 L, and magnitudes of stretch to 0.25 L, in normal solution. It should be noted that the series-elastic component of trachealis is 7.5 % of L, (34), considerably less than the magnitude of stretch imposed, and thus the quick stretch was not merely absorbed by the series elasticity.

Effect of experimental conditions and agents which he@ elicit a myogenic response. HYmxrA. Loofbourrow et al. (23) have demonstrated spontaneous rhythmic tracheal contractions induced by hypoxia in the intact dog. This observation coupled with our finding that severe hypoxia may cause a small increase in resting tension (22) and the finding of

others that a myogenic response is elicited more easily in a partially contracted muscle ied us to investigate the effect of hypoxia on the response of tracheal smooth muscle to stretch. Using appropriate gas mixtures, the Krebs-Ringer solution was equilibrated in a reservoir and then perfused into the bath. The Pas was 30 mmHg, Pcog 40 mmHg, and pH 7.40. We have previously shown (3 1) that a Paz of 60 mm impairs tension development by 40 76. However, the hypoxia used here did not elicit the myogenic response, even though contractile function was impaired.

FIG. 2. Effect of TEA (33 n-AI) on mechanica activity of TSM

and response to stretch. C&vr record: stretch applied by a Levin-

Wyman ergometer. Lower reco& muscle tension as a function of time.

Responses to TEA were recorded after 5 min exposure to this agent,

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630 STEPHENS, KROEGER, AND KROMER

vations) and the development of some mechanical tension, was unsuccessful in producing a myogenic response.

BARIUM. In the presence of solutions containing normal (Ca). and BaCl2 2 mM, basal tension was occasionally increased and active responses to stimulation were in- hibited, but a myogenic response to stretch could not be elicited.

ACETYLCH~LINE. When treated with acetylcholine ( 1 O+ to lOA M), the muscles responded with dose-dependent tonic contractions, but a myogenic response could not be elicited under these conditions.

TETRAETHYLAMM~NIUM CHLORIDE (TEA) g Shortly after the application of TEA (33 mM), TSM exhibited rhythmic bursts of partially fused twitches, and the basal tension be- tween contractions was usually increased (Fig. 2). The fre- quency of these spontaneous contractions and the increase of basal tension were widely variable between preparations A myogenic response to quick stretching was easily elicited. The MR was initiated 1.5-3 s after the application of the stretch, and its duration was typically 3-4 s and magnitude was about 10 % of P,. Atropine, lOA M, did not affect the mechanical activity of MR observed in the presence of TEA.

i5ffecl of (Mg2f) 012 resfionse to TEA. Since the phasic mechanical responses to TEA suggested the involvement of an action potential (Zl), we decided to investigate the effects of Mg2+, a membrane stabilizer (6) on the responses to TEA.

In measuring the dose-response relation to TEA in preliminary experiments, we noted that at subthreshold concentrations of TEA which did not produce an increase in tension exposure to a short electrical stimulation resulted in phasic mechanical activity following this stimulation. Since this effect is never seen in untreated TSM, this ob- servation indicated an effect of TEA. The dose-response relationship to TEA was thus determined at normal (2.5 mM) and reduced (0.5 mM) Mg2+ by stimulating the muscle electrically (60 Hz) for 2 s and then measuring the area under the myogram which followed (by planimetry). The response observed at a concentration of 67 mM TEA was arbitrarily assigned the value 100 %. Although higher concentrations of TEA yield slightly greater responses, these are difficult to interpret because of the deficiency of these soIutions in TL’a+ A IO-min rest was allowed between

101

FIG. 3. Effect of Mg on dose-response relation of TSM to TEA.

successive doses of TEA and no evidence of desensitization was seen. The results (Fig. 3) show a threshold for the effect of TEA at 8 mM. Of special interest was our finding that the sensitivity of the muscle to TEA was increased (about fourfold) by reducing the (Mg2”), to 0.5 mM. This effect was readily reversible. Decreasing (Mg”+). below 0.5 mM had little additional effect on the dose- response relationship.

E$ects of Ca, Mg, and D-600 on LVJ?. These studies were undertaken to elucidate 1) the role of the physiological divalent cations on the effect of TEA, and 2) the Ca pool utilized in the production of the MR. In order to interpret the effects of Ca and Mg on responses to TEA, it was neces- sary to consider their interrelated eff’ects on membrane permeability, the role of Ca in excitation-contraction coupling, and the apparent inhibition by Mg2+ of responses to TEA (documented above). We thus decided to investi- gate the effect of reducing (Mg2+)” from 2.5 to 0.5 mM on the (Ca2+), required for the MR. The results shown in Fig* 4 indicate that 1) the MR is quantitatively related to (Ca2+)o and has an absolute requirement for (Ca2+),,, 2) the Ca threshold for the MR is inversely dependent on

(MS”+> 03 and 3) Ca in concentrations above 0.5 mM can substitute for Mg in supporting the membrane permeability requirements for the MR.

Since the MR demonstrated a quantitative relation to (Ca”+), in these studies, we considered it of interest to de- fine in greater detail the mechanism of Ca mobilization which results in the MR. An increased intracellular free Ca might result from 1) a nonspecific increase in mem- brane permeability, 2) a specific depolarization-activated Ca influx, or 3) a mobilization of sequestered Ca by de- polarization (17, 30). I n order to rule out the first possi- bility, we used the substance D-600 which has been shown to inhibit selectively the depolarization-activated Ca in- flux without affecting the resting permeability of the mem- brane in both cardiac muscle (19) and uterine smooth muscle (20). The results (Fig. 5) show that D-600 inhibits the MR completely.

E’ect of Quick Stretch on TSM in Control and Test Conditions I

Any experimental condition that allows a transient in- crease in the ionic permeability of the membrane non- specifically in response to stretch should, because of the strong inwardly directed electrochemical gradient for Ca,

mM 0.5

0.4

0.3

0.2

0.1

0

- I

\ b

\ l - •

A Ca, WI

FIG, 4. Relation of (Ca2+). to myogenic response (MR). A: effect of (Mg2f)0 on concentration of Ca required to produce MR. B: effect of (Caz+), on magnitude of MR. These data are typical of 6 experiments in this series.

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MYOGENIC RESPONSE IN MULTIUNIT SMOOTH MUSCLE 631

4 D-600

FIG. 5. Effect of D-600, 10B5 M on of TEA, 33 mM.

I

5 g 0

myogenic response in presence

evoke a MR. The refractoriness of TSM to quick stretch even in the presence of various direct agonists is somewhat surprising. Thus it is of interest that TEA, which produces a relatively small change in basal tension, can produce such a fundamental change in its biophysical properties, producing spontaneity and a myogenic response to stretch. The mechanism whereby TEA effects this change probably involves two levels of the control of muscle function: I) its effect on membrane potassium conductance (Zl), and 2) the ionic requirements of excitation-contraction coupling. The present study focusses on the latter.

Interralafion of Ca and Mg in the MR

The complex interrelation of Ca and Mg in the eff‘ects of TEA and the MR are consistent with dual effects of both Ca and Mg on membrane permeability, sensitivity of the muscle to TEA, and excitation-contraction coupling. Thus Mg inhibits the effect of TEA as is shown in Fig. 3 but also helps to stabilize the permeability of the mem- brane in solutions deficient in Ca as has been shown by Bulbring and Tomita (6) in the taenia coli. A normal membrane permeability is apparently a prerequisite for the processes supporting the MR; an action potential with a low safety factor would be a likely candidate for this process (2 1). Calcium, which also stabilizes membrane permeability, couples excitation and contraction, and thus the observation that the MR is dependent on (Ca2+). within concentrations at which the electrochemical gradient for Ca is inward provides additional evidence that the MR is dependent on membrane phenomena. This is of special interest in that multiunit smooth muscle usually contains a relatively large store of sequestered Ca which is resistant to depletion and can be mobilized by drugs for contraction (10, 22). The reasons why it is not mobilized by a quick stretch are not clear, and one might speculate that I) there are no direct electrical connections between the mem- brane and these Ca stores, assuming that quick stretch does produce depolarization (7), or 2) that the mobilization of sequestered Ca by depolarization requires the release of

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Cc trigger Ca” which is subject to depletion in Ca-deficient solutions (17). Hinke ( 16) has also proposed that depolari- zation results in contraction by increasing Ca influx, whereas sequestered Ca is mobilized primarily by stimula- tory agonists.

Additional experiments were then designed to elucidate the mechanism whereby the stretch produced an increase in intracellular free Ca* Thus the Ca mobilized might result from I) a nonspecific increase in membrane per- meability which results in depolarization and Ca influx in parallel, 2) a specific activation of Ca channels by de- polarization which provides activator Ca for contraction, or 3) a release of trigger Ca which mobilizes sequestered Ca (17). The substance D-600, which has been shown to inhibit the activation of Ca influx by depolarization in other preparations (19, 20), promised to be a useful tool in differentiating among these possibilities if it could be shown that D-600 is selective in inhibiting only the de- polarization-activated Ca influx. In testing this assumption our preliminary experiments on both trachealis and myo- metrium have shown I) that D-600 inhibits potassium- induced contractions to a greater extent than it inhibits acetylcholine-induced contraction, and 2) that D-600 does not inhibit Ca-sensitive actomyosin ATPase activity (R. Bose, personal communication) or contractions in- duced by the Ca ionophore A23 187 (cf. 28). In other preparations, it has been shown that D-600 minimally affects Ca binding by isolated cardiac sarcoplasmic re- ticulum at 100 times the pharmacologically effective dose (11, 36) and that D-600 inhibits only the entry of the trigger Ca associated with action potentials in the taenia coli (24). Thus the evidence available indicates that the inhibitory effect of D-600 is exerted primarily through an inhibition of depolarization-activated Ca influx rather than through eflects on intracellular structures concerned with Ca me- tabolism and contraction (36). The observation that D-600 completely inhibited the MR thus argued against the possibility that a nonspecific increase in membrane per- meability might explain the MR. However, since the re- lationship between the relative roles of trigger Ca and the Ca current of depolarization in initiating contraction, as well as the effect of D-600 on the release of trigger Ca are not yet established, these experiments could not differ- entiate between the latter two possibilities.

In conclusion, one might speculate that the potential for spontaneity as unmasked by TEA in the normally multi- unit TSM might have some physiological significance such as the autoregulation of air flow in the smaller air- ways or possibly the stabilization of airways in conditions associated with the sudden development of large trans- mural pressures.

This investigation was supported by grants from the Canadian Heart Foundation and the Medical Research Council of Canada.

Keceived for publication 10 January 1974.

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